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This is to certify that the
dissertation entitled

SEPARATION AND QUANTITATION OF LIMONOIDS
AND F LAVONOIDS IN JUICE AND BY-PRODUCTS OF
SWEET ORANGE (Citrus Sinensis)

presented by

Korada Sunthanont Saipetch

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Food Science

 

 

%a 47 @463“

Maj or Professor’s Signature

MSU is an Affirmative Action/Equal Opportunity Institution

-.—.-—-_.-._-

. -.-...._.-.—.-.-.—.-.- _.-.-.—2__ .. - -

 

 

 

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sari-“tam AND 0'

ME AND B?-

a

 

 

SEPARATION AND QUANTITATION OF LIMONOIDS AND FLAVONOIDS 1N
JUICE AND BY-PRODUCTS OF SWEET ORANGE (Citrus Sinensis)

By

Korada Sunthanont Saipetch

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

2004

 

 

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ABSTRACT

SEPARATION AND QUANTITATION OF LIMONOIDS AND F LAVONOIDS IN
JUICE AND BY-PRODUCTS OF SWEET ORANGE (Citrus Sinensis)

By

Korada Sunthanont Saipetch

Two major classes of citrus phytochemicals, limonoids and flavonoids, have
attracted considerable attention from science and industry because of their
pharmacological properties. Large production of by—products accompanying orange juice
production offers inexpensive starting materials for recovery of secondary metabolites
with potential anticarcinogenic and cardioprotective activities.

The objectives of this study were 1) to determine limonoid and flavonoid content
of orange juice and lay-products resulting from commercial orange juice processing, and
2) to determine the influence of lime treatment on these phytochemicals.

Limonoid and flavonoid content was determined in various by-products (seed,
peel, peel press cake, rag, and peel press liquid) and orange juice from commercially
grown orange varieties (Hamlin, Parson Brown, and Valencia). Twenty one compounds
in the categories limonoid aglycones, limonoid glucosides, flavanone glucosides, and
polymethoxylated flavones were analyzed.

Seeds had the highest content of limonoids, while peel and peel press cake had the
highest concentrations of flavonoids. Water removal by pressing extracted limonoid
glucosides and polymethoxylated flavones from the peel into peel press liquid, but
concentrated limonin in peel press cake. Average g/ 100g dry wt. of total contents in solid

fractions from three varieties were 7.1 (flavanone glucosides), 4.1 (limonoid glucosides),

 

 

l; limonoid a? 1‘ 1”:th " 2
not: it 15": ‘53“ Km
nonmethoxtiawd fie“ on:
net in edible Grant-‘6 lira?"
containing seeds at 90°C
_i‘fl5 and P563 W555
oohmetnonylateci ia‘t '1‘?
{hoohdes and pol-\Tni’lh'
Orangejnice is a good Sf
In the lime stut
oasuredhefore and afi-
nitonals. so that the i
hours. the samples \K'C‘
treated with (l.3"-o Cat
tontent oi limonooo‘
ttltmethoxylated fiat.
With limo trea
tll‘ht leached from pi
to: increased ohno‘h
, . t.
Amt treatment. In so:
“'0 offset on limonoi
All? treatment It‘su'

“millions.

1.2 (limonoid aglycones), and 0.26 (polymethoxylated flavones); and in liquid fractions
were 0.150 (flavanone glucosides), 0.072 (limonoid glucosides), 0.009
(polymethoxylated flavones), and 0.002 (limonoid aglycones). Limonoid glucosides are
rich in edible orange fraction and are extracted into the juice. The results show that rags
containing seeds are good sources for limonoid aglycones and limonoid glucosides, while
peels and. peel press cake are good sources for flavanone glucosides and
polymethoxylated flavones. Peel press liquid is a potential source for limonoid
glucosides and polymethoxylated flavones after evaporation to the molasses end-product.
Orange juice is a good source of limonoid glucosides.

In the lime study, limonoid and flavonoid content in waste products were
measured before and after lime treatment. CaO was added to peel and rag, primary waste
materials, so that the final concentration of CaO was 0.3% CaO (wet wt). After 48
hours, the samples were pressed to yield press cakes and press liquids. Seeds were
treated with 0.3% CaO (wet wt.) separately. These fractions were analyzed for the
content of limonoid aglycones, limonoid glucosides, flavanone glucosides and
polymethoxylated flavones.

With lime treatment, more limonoid aglycones (25%) and limonoid glucosides
(12%) leached from press cake into press liquid (rag and peel). Overall, there was a trend
for increased phytochemical content release from press cakes into press liquids due to
lime treatment. In seed, lime treatment resulted in losses of limonoid glucosides, but had
no effect on limonoid aglycones, flavanone glucosides or polymethoxylated flavones.
Lime treatment r esulted in increased p hytochemical c ontent in p ress liquids e specially

limonoids.

 

I’ .

Ectopic} lite t0 c
continued suPPO-E and g
margin, His t'tsrttn amt
scientist.
lo'oulci like it s
tut-arenug confidence tr
tuning in the lab. Ant
hateful that Dr. Bennrn‘u
Special thanks to
food Science and Hum
Pathology) for their valu
l uish to express
I.Qlllot'itling pilot pla
inatrial support for thi:
Thanks to scien'
lltl A. Berhoit chor

unable limonortis and

loam 10 thank

no tools. hi) hi

In

Sl

or.
km blends llth‘ 31

in“: i‘ -
ll ohom made rm

 

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Dr. Mark Uebersax for his
continued support and guidance throughout my graduate studies at Michigan State
University. His vision and. insight were invaluable to my development as a well-rounded
scientist.

I would like to especially thank Dr. Maurice Bennink for his guidance and
unwavering confidence in me. It is safe to say that without his efforts, I would still be
working in the lab. And. in the years that I have spent at this great University, I am
grateful that Dr. Bennink was not only my research advisor, but also my friend.

Special thanks to the members of my committee, Dr. Jerry Cash (Department of

Food Science and Human Nutrition) and Dr. Ray Hammerschmidt (Department of Plant

Pathology) for their valuable contribution throughout my graduate study.

I wish to express my appreciation to the Tropicana Products, Inc. (Bradenton, FL)
for providing pilot plant space for me to prepare my samples and for their partial

financial support for this research.

Thanks to scientists from the USDA [Dr. Gary D. Manners (Pasadena, CA), Dr.

Mark A. Berhow (Peoria, IL), and Dr. John A. Manthey (Winter Haven, FL)] for their

valuable limonoids and flavonoids standards.

I want to thank p. Mum, p. Yim and p. Kok for their warm support and yummy
Thai foods. My big sisters were always there and willing to help. I would like to thank
all my friends here at Michigan State University for their support and encouragement.

sill of whom made my being away from home a much better experience.

iv

 

 

 

 

new

hire} thanis .-
the pleasure of Spenéii'g 7
at hip Actually W: 5?“
oil always remember e ‘L
a graduate Students 2?- pr:
llfilOOdlt‘ sottps at I a".
You hendshrps. l tiff. ;
lttant to thanlt r.
a} dai-in-iatt tAnan S
encouragement. support.
morn tSuchada Sunthat
always been true git erg,
milling you taught r
or little sister {Kookt
When I was done
finally, 1 “mil.
mutt endless l0\ c 2

thrgpan oi mt succe'

Many thanks to my Lab 110 gang. In the years spent pursuing this degree, I had
the pleasure of spending it with four amazing friends: Abby, Kathy Lai Pui Kwan, Mar,
and Mo. Actually, we spent so much time in the lab I think we should have paid rent. I
will always remember even those occasional “little” moments that put our miserable lives
as graduate students in perspective; that one day we too will live “normal” lives as well—~
the noodle s oups at 2 am a nd w atching taped Friends and the B achelor with m y g ang.
Your friendships, I will always cherish.

I want to thank my entire family. Especially to my mom—in-law (Mary Saipetch),
my dad—in—law (Anan Saipetch), my brother-in-law (Poon) and n. Ohm for their love,
encouragement, support, and belief in me. I am forever thankful to be a daughter of my
mom (Suchada Sunthanont) and dad (Kampol Sunthanont). All my life, they have
always been true givers. Thank you mom and dad for your unbelievable sacrifice and for
everything you taught me. I would not have come to this far without you. Also thanks to
my little sister (Kook) for always being there for me and occasionally helping me up
when. I was down.

Finally, I would like to thank Auan, my husband and my best friend. Thank you
for your endless love and understanding. Your positive attitude and sense of humor were

a big part of my success.

 

 

liaoilaoies... .

its: oi Figures. . .

in of Abbreviations . ..

hooductton...... .
References... .

lzterature Ret'i ett ......
Citrus juice proces
Citrus lit-product:
limonoids in cup

Delayed h
dehittertn;

Biologrca
Flat'onoids in crt‘
Chemota'
Bitterncs
Biologic
Sample prep arat
Analyses of cttr
Citrus lt
Citrus f

Raf

tierences .. .

 

TABLE OF CONTENTS

Page number

List of Tables .................................................................................. x
List of Figures ................................................................................. xv
List of Abbreviations ......................................................................... xx

Introduction .................................................................................... 1

References ............................................................................. 3

Literature Review .............................................................................. 4

Citrus juice processing ............................................................... 4

Citrus by-products .................................................................... 6
Limonoids in citrus products ........................................................ 10
Delayed bitterness and glucosidation. (natural ............................ 14

debittering process)

Biological activities of limonoids ......................................... l6
Flavonoids in citrus products ....................................................... 20
Chemotaxonomic marking and authenticity ............................. 24
Bitterness and precipitation problems ..................................... 26
Biological activities of flavonoids ......................................... 27
Sample preparation in citrus phytochemistry .................................... 29
Analyses of citrus limonoids and flavonoids ..................................... 3]
Citrus limonoids ............................................................. 31
Citrus flavonoids ............................................................ 35
References ............................................................................. 40
vi

 

 

 

Suit 1; .finaltucai melted
' of Innontuos an: :

Pan 1: Screening it
Abstract .

3. lntroductior. .

3. Materials and:
4. Results and ti:

Conclusron

o. References. .

Pan ll: Optimizat

1. Abstract.

‘. lnuoductron.

3. Malenals ant

b

. Results and.
. Conclusion

to. References.
Suit ll; lsolati on and

Pan l: lsolatror
IDXGI and nor

.. Abstract

\
-. lnuoductio

7,)

 

Study 1: Analytical methodology suitable for isolation and. quantitation .............. 50
of limonoids and flavonoids in sweet orange

Part 1: Screening for major limonoids and flavonoids ........................... 50
1. Abstract ........................................................................... 50
2. Introduction ....................................................................... 50
3. Materials and methods .......................................................... 51
4. Results and discussion ........................................................... 55
5. Conclusion ........................................................................ 60
6. References ......................................................................... 66
Part II: Optimization of analytical methods ....................................... 67
1. Abstract ............................................................................ 67
2. Introduction ....................................................................... 67
3. Materials and methods ........................................................... 68
4. Results and discussion ........................................................... 77
5. Conclusion ........................................................................ 90
6. References ........................................................................ 91
Study 11: Isolation and identification of selected limonoids and flavonoids ........... 93
Part I: Isolation and identification of deacethylnomilin glucoside ............ 93

(DNG) and nomilin glucoside (NO)

1. Abstract ............................................................................ 93
2. Introduction ....................................................................... 93
3. Materials and methods ........................................................... 94
4. Results and discussion ........................................................... 97

vii

 

 

: (91111113101
( , rt rprl’ PIE
ll, R1161 ,,l>'-'-

hill: Isolation and toes:
itatone tliXt an: n

'1 Abstract

3. lntroductton .
3. Materials and?
1.. Results and 013
5. Conclusion .

ti. References.

‘{’
a
Q-
/'

. pill: Distributions c
fractions of so

1. Ahsnact ......
I. Introduction.
3. Materials an.
4. Results and.
. Conclusion.
6. References

in 4 - ‘- “
not i) . Eilect oi hut
in lit-produc

l Abstract...
‘ 1

-~ ntroducuo
Materials:
~ Results ant

 

 

5. Conclusion ........................................................................ 99
6. References ........................................................................ 104
Part II: Isolation and identification of 3,5,6,7,3’,4’-hexamethoxy ...................... 104
flavone (HX) and narirutin-4’-glucoside (NT-4’-G)
1. Abstract ............................................................................ 104
2. Introduction ....................................................................... 104
3. Materials and methods ........................................................... 105
4. Results and discussion ........................................................... 110
5. Conclusion ........................................................................ 119
6. References ........................................................................ 120
Study III: Distributions of limonoids and flavonoids in edible and inedible .......... 123

fractions of sweet oranges (Citrus sinensis)

1. Abstract ............................................................................ 123
2. Introduction ....................................................................... 124
3. Materials and methods ........................................................... 125
4. Results and discussion ........................................................... 132
5. Conclusion ........................................................................ 162
6. References ......................................................................... 168
Study IV: Effect of lime treatment on of limonoid and flavonoid content ............ 170

in by-products from orange juice process

Abstract ............................................................................ 170
Introduction ....................................................................... 1 70
Materials and methods ........................................................... 172
Results and discussion ........................................................... 178
Conclusion ........................................................................ l 96

viii

 

 

 

5 References
Research conclusror...
Recommendations in: fut-t
Attendees

Appendix 1: Screer
orange

Appendix ll: Punf

t.
tIlEi‘t‘.
5 .

Appendix lll. Fun

in gift

Appendix I)? Pur
WAT

Appendix \': Pres]

011 l
OIEIT.

Appendix \'1: to
ora

Appendix \ll: C
n‘
in.

Appendix \lll:

Appendix Vllll

Arte-non x1 (1

Attends .\‘t; 5

 

6. References ........................................................................ 201
Research conclusion .......................................................................... 204
Recommendations for future research ...................................................... 206
Appendices

Appendix I: Screening of limonoids and flavonoids in different .............. 208

orange fractions.

Appendix II: Purification of limonoid aglycones by preparative .............. 217
high performance liquid chromatography.

Appendix III: Purification of limonoid glucosides by preparative ............ 221
high performance liquid chromatography.

Appendix IV: Purification of polymethoxylated flavones by .................. 224
preparative high performance liquid chromatography.

Appendix V: Preliminary trials of mass spectrometric techniques ............ 227
on flavonoids and limoniods extracted from sweet

orange (C. sinensz’s).

Appendix VI: Limonoid and flavonoid content in rag and ..................... 241
orange juice prepared domestically.

Appendix VII: Chromatographic retention and UV spectra of ................. 245
minor citrus limonoids compared to limonin and

nomilin (common limonoids).

Appendix VIII: Oil content in studied sweet orange seeds ..................... 252

Appendix VIIII: Processing qualities for juice production ...................... 254
of studied orange varieties.

Appendix X: Calculations ........................................................... 256

Appendix XI: Summary of HPLC instrumentation diagnostics ................ 258

used during this research.

ix

 

 

’a'olel limonoid agiytort
pith and Without
latte 3: cent er} of 11m:
tetramethylether r
heatingtEZ‘C to:

little? Limonord glucos
extracted ht our:

Title is. Total limonoid
extracted bx ‘iti‘

~:ahle S: flat'anone glue-c
by different SOl\

Table 6: Total limonoid
extracted by dtf

Table ‘4 Recot‘ert‘ of ne
ht dimetht'l f on

fable 8: ANOVA oftot
and flavonoicls

Idle 9: A\‘0\' A of tor
and flat‘onoids

lahle lit: Total phttoc.

in solid tractt

:thre ll: Total photoc
in liquid frac
.aole II: ANOVA of

f

:‘p‘ 1. .
l 'l l - lutal hum.

of so eel orar

 

LIST OF TAB LES

Page

Tablel: Limonoid aglycone content in seed, peel, and peel juice extracts ............ 78
with and without heating (82°C for 30 min).

Table 2: Recovery of limonin (limonoid aglycone) and scutellarein ................... 78
tetramethylether (polymethoxylatedflavone) extracted under
heating (82°C for 30min).

Table 3: Limonoid glucoside content in sweet orange seeds ............................ 78
extracted by different solvent extraction conditions.

Table 4: Total limonoid glucoside content in sweet orange seeds ...................... 80
extracted by 70% methanol at different conditions.

Table 5: F lavanone glucosides in sweet orange peel extracted .......................... 80
by different solvent extractions.

Table 6: Total limonoid glucoside content in sweet orange seeds ...................... 82
extracted by different solvent extractions.

Table 7: Recovery of neohesperidin and hesperidin extracted .......................... 82
by dimethylformamide/methano1 (1:2).

Table 8: ANOVA of total phytochemical content (limonoids ........................... 133
and flavonoids) in solid fractions of sweet oranges.

Table 9: AN OVA of total phytochemical content (limonoids ........................... 133
and flavonoids) in liquid fractions of sweet oranges.

Table 10: Total phytochemical content (limonoids and flavonoids) .................... 134

in solid fractions of sweet oranges.
Table 11: Total phytochemical content (limonoids and flavonoids) .................... 134
in liquid fractions of sweet oranges.

Table 12: ANOVA of total limonoid aglycone content in solid fractions .............. 138
of sweet oranges.

Table 13: Total limonoid aglycone concentrations in solid fractions ................... 138

of sweet oranges.

 

 

n 1 -. ,: "I'vlf'fl
‘t- tt'iGua:
13011:” m0

’a't'rl‘ ANOVA of hint

sweet orangt‘i

‘iole lo: Tota‘: ltrnonoic 2

of so 66’. oran gtf

fable 1': Individual 111110!
of sneer oranges

Title ll: ANOVA of fun
oranges

Table 19: Total limonorc.
of so eet orange

Title Ill: lndit'rdual limc
fractions of so

Tahlell: ANOVA of In
of street orange

iahle 23: Total limonoit
fractions of so

tdhlf 33: lndit'idual lim
fractions of sv

this 34: ANOVA of p
of so eet oranc

" it. i'.
1th.). Total polptnc
fractions of s‘

i‘ta 1. . .
fractions of;

I 'i
tare “'- '
t . .Total potxim

ITak‘ll OTIS 0 ‘1 5

a
\
n

on -- " ‘
at .8. lllllhldllal It
tractions of:

 

Table 14: Individual limonoid aglycone concentrations in solid ........................ 140
fractions of sweet oranges.

Table 15: ANOVA of limonoid aglycones in liquid fractions of ....................... 142
sweet oranges.

Table 16: Total limonoid aglycone concentrations in liquid fi'actions ................. 142
of sweet oranges.

Table 17: Individual limonoid aglycone concentrations in liquid fractions ........... 144
of sweet oranges.

Table 18: ANOVA of limonoid glucosides in solid fractions of sweet ................ 145
oranges.
Table 19: Total limonoid glucoside concentrations in solid fractions ................. 145

of sweet oranges.

Table 20: Individual limonoid glucoside concentrations in solid ....................... 148
fractions of sweet oranges.

Table 21: ANOVA of limonoid glucosides in liquid fractions .......................... 149
of sweet oranges.

Table 22: Total limonoid glucoside concentrations in liquid ............................ 149
fractions of sweet oranges.

Table 23: Individual limonoid glucoside concentrations in liquid ...................... 151
fractions of sweet oranges.

Table 24: ANOVA of polymethoxylated flavones in solid fractions ................... 153
of sweet oranges.

Table 25: Total polymethoxylated flavone concentrations in solid ..................... 153
fractions of sweet oranges.

Table 26: ANOVA of polymethoxylated flavone concentrations in liquid ............ 155
fractions of sweet oranges.

Table 27: Total polymethoxylated flavone concentrations in liquid .................... 155
fractions of sweet oranges.

Table 28: Individual polymethoxylated flavone concentrations in solid .............. 158
fiactions of sweet oranges.

xi

 

 

. . ‘ - irr'flQUiVm
"late 39' lndlt lcuc ; "4,.
nacnonf 01 3“ “'

"a‘iie‘tt’t: ANOVA or 11232
0 street oranges.

Title 11: Total fiat'anone
factions or so ee

lah1e31;.ANO\'.A of f rat
of sneer orari ges

Title 33; Total fiat anone

ot street oranges

Title 34: lndtt'idual flat 2
fractions of so:

Table 35: lndit'idual flax
fractions of S\NE

T
t
.

the 3d: Moisture contt

lahle 3‘: Moisture cont:
process ttt'ith .

Title 38: pH and Bm \
lime treatm en

T I I - v a
are 39: limonoid ael

liquids (with

ltoletlt limonoid as
liquids north

I“; ~ ‘

art 41. Totar lmtonc
and without '

ll‘s t). '

.tt ~... limonoid cl
liquids ittttl

: he i‘.

at Limonoid

liquids [WI

‘1
s
Q

Li

is: ' ..
at +4.. Total hump
and trithout

 

Table 29: Individual polymethoxylated flavone concentrations in liquid .............. 159
fractions of sweet oranges.

Table 30: ANOVA of flavanone glucosides in solid fractions of ....................... 160
sweet oranges.

Table 31: Total flavanone glucoside concentrations in solid ............................ 160
fractions of sweet oranges.

Table 32: ANOVA of flavanone glucosides in liquid fractions ......................... 163
of sweet oranges.

Table 33: Total flavanone glucoside concentrations in liquid fractions ................ 163
of sweet oranges.

Table 34: Individual flavanone glucoside concentrations in solid ...................... 165
fractions of sweet oranges.

Table 35: Individual flavanone glucoside concentrations in liquid ..................... 166
fractions of sweet oranges.

Table 36: Moisture content of raw materials prior to pressing process ................ 180

Table 37: Moisture content of press cakes recovered from pressing ................... 180
process (with and without lime treatment).

Table 38: pH and Brix values of press liquids (with and without ....................... 181
Time treatment).

Table 39: Limonoid aglycone content in peel press cakes and press ................... 182
liquids (with and without lime treatment).

Table 40: Limonoid aglycone content in rag press cakes and press .................... 183
liquids (with and without lime treatment).

lTable 41: Total limonoid aglycone content1 in peels and rags (with ................... 185
. and without lime treatment).

Table 42: Limonoid glucoside content in peel press cakes and press .................. 186
liquids (with and Without lime treatment).

Table 43: Limonoid glucoside content in rag press cakes and press .................... 187
* liquids (with and without lime treatment).

Table 44: Total limonoid glucoside content in peels and rags (with .................... 189
and without lime treatment).

xii

 

 

 

 

 

a; D hum-TIE", ‘law
Tilllf 0 ii

DICSSI l1 011105 l. vs.

”role 46: Poltrneutoryiate
press liquids in?

Title titaianonf glue:
llQlllClS I“ if” (ill

I flatanonegl
hq tlidSl \tilfr c111

Title 5'}: Total flat mom
and without Tin

lthleil: limonoid aglp
lime treatment

iahleil: Limonoid glut
1 me trea merit

Tahle 53: Polimethoxxl
lime treatnten‘

T i .
reheat: flatanone alt
lime treatmen

not. to. Posrttte tons
positit'e TAB

lah‘o“ " '
.1. so: Positrte ions
positite TAE

Tarn: .. u

«at : \egalix e iot‘
negaux e TA

'2‘“ .‘I\
lx.‘ S.\QU gal“ C IOT

lls‘galite FA

5 V '
le.It Positrte ion
posititc TA

 

Table 45: Polymethoxylatedflavone content in peel press cakes and .................. 190
press liquids (with and without lime treatment).

Table 46: Polymethoxylatedflavone content in rag press cakes and .................... 191
press liquids (with and without lime treatment).

Table 47: Total polymethoxylated flavone content in peels and rags .................. 193
(with and without lime treatment).

Table 48: Flavanone glucoside content in peel press cakes and press ................. 194
liquids (with and without lime treatment).

Table 49: Flavanone glucoside content in rag press cakes and press ................... 195
liquids (with and without lime treatment).

Table 50: Total flavanone glucoside content in peels and rags (with ................... 197
and without lime treatment).

Table 51: Limonoid aglycone content in seeds (with and without ...................... 198
lime treatment).
Table 52: Limonoid glucoside content in seeds (with and without ..................... 198

lime treatment).

Table 53: Polymethoxylatedflavone content in seeds (with and without .............. 199
lime treatment).
Table 54: F lavanone glucoside content in seeds (with and without .................... 199

lime treatment).

Table 55: Positive ions produced from Valencia seed extract by ...................... 230
positive FABMS/ glycerol matrix.

Table 56: Positive ions produced from Valencia seed extract by ...................... 231
positive FABMS/m-nitrobenzyl alcohol matrix.

Table 57: Negative ions produced from Valencia seed extract by ..................... 232
negative FABMS/ glycerol matrix.

Fable 5 8: Negative ions produced from Valencia seed extract by ..................... 233
negativeFABMS/m-nitrobenzyl alcohol matrix.

Table 59: Positive ions produced from Valencia peel extract by ....................... 234
positive FABMS/ glycerol matrix.

xiii

 

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Tint Y D ID
3053136 lid): 5'

'H t ' '- TIC fil'
totem. Regains to..- _..

. .r n
rieaatite Halli-

his 62: Negatiie 109.5 2:
negatiie FAlel

Tableo?‘ Electron impact

spectrometr} ea

totem Electron impale
spectrometry d2

Table 65: Electrosprai io

specoometri a

Table to: Electrospra} 1'.”
spectrometr} d

.able 1": lntlit'idual lim
domesticallt p

:alle 68; lndii'idual lint
domesticalb t

ialle till: lndit'idual 90'.
tlomestiealb t

itote ”'0'. lnClthdllal tla
domesticall}

Tit
W'tc" . .
iii. 1.011 content 1;

i351: “- .

l

' f’)

‘ 'I
l.’ ‘ ‘1
lathe \'

 

Table 60: Positive ions produced from Valencia peel extract by ....................... 235
positive FABMS/m-nitrobenzyl alcohol matrix.

Table 61: Negative ions produced from Valencia peel extract by ...................... 236
negative F ABMS/ glycerol matrix.

Table 62: Negative ions produced from Valencia peel extract by ..................... 237
negative F ABMS/m—nitrobenzyl alcohol matrix.

Table 63: Electron impact ionization gas chromatography-mass ...................... 238
spectrometry data of crude seed extract (5111).

Table 64: Electron impact ionization gas chromatography-mass ....................... 238
spectrometry data of crude peel extract (5 pl).

Table 65: Electhpray ionization liquid chromatography-mass ........................ 239
spectrometry data for crude presscake extract (0.5 ul).

Table 66: Electrospray ionization liquid chromatography-mass ........................ 240
spectrometry data for crude seed extract (1 14]).

Table 67: Individual limonoid aglycone concentrations in .............................. 243
domestically prepared juice and rags of sweet oranges.

Table 68: Individual limonoid glucoside concentrations in .............................. 243
domestically prepared juice and rags of sweet oranges.

Table 69: Individual polymethoxylated flavone concentrations in ..................... 244
domestically prepared juice and rags of sweet oranges.

Table 70: Individual flavanone glucoside concentrations in ............................. 244
domestically prepared juice and rags of sweet oranges.

Table 71: Oil content in studied sweet orange seeds ..................................... 253
Table 72: Processing qualities of studied orange varieties .............................. 255
.‘able 73: Summarized HPLC trouble shootings .......................................... 259

xiv

 

:hjr: Orange lUlC-f pr:

Emir 2: Cross section c1

‘ -

Fame Ieeo llllli am. c

Figure 1'. Chemical struc‘.
Figure 5: Chemical struc‘
i. re a: Biosmthesrs ar
Figure ‘: Lactoriizatiori :

Figure 1: Chemical struc
lmtinosidesr.

Figure 9: Chemical SlTUt
meohesperidos

More 10: Chemical str
heme 11: Polar compo
in Rmhl phos
21 (1 um.

i. ,- ‘
.igtire 12. hoiioolar co
acetomtrile h

heir
~ l 134 Polar come

in 3111M pho
ll011m. and
limit I. ~ 1
~ l*-\Oltpoiarci
acetomtnlet
illicit .
"ll Stoctra
acid. and he

 

LIST OF FIGURES
Page

Figure 1: Orange juice production ........................................................... 7
Figure 2: Cross section of intact orange fruit .............................................. 8
Figure 3: Feed mill unit operation .......................................................... 11
Figure 4: Chemical structures of the major citrus limonoid aglycones ................ 12
Figure 5: Chemical structures of the major citrus limonoid glucosides ................ 13
Figure 6: Biosynthesis and accumulation of limonoids in citrus fruit .................. 15
Figure 7: Lactonization and glycosylation processes in citrus fruit .................... 17
Figure 8: Chemical structure of the major citrus flavanone glucoside ................. 21

(rutinosides).
Figure 9: Chemical structure of the major citrus flavanone glucoside ................. 22

(neohesperidosides).

Figure 10: Chemical structure of the major citrus polymethoxylated flavones ....... 23

Figure 11: Polar compounds (reconstituted with 10% acetonitrile ..................... 56
in 3mM phosphoric acid) in seed extract at 340 nm, 280 nm, and
210 nm.

iigure 12: Nonpolar compounds (reconstituted with 100% ...................................... 57
acetonitrile)in seed extract at 340 nm, 280 nm and 210 nm.

"igure 13: Polar compounds (reconstituted with 10% acetonitrile ..................... 58
in 3mM phosphoric acid) in peel extract at 340 nm,
280 nm, and 210 nm.

igure 14: Nonpolar compounds (reconstituted with 100% ...................................... 59
acetonitrile)in peel extract at 340 nm, 280 nm and 210 nm.

igure 15: UV spectra of deacetylnomilin, nomilin, deacetylnomilinic ............... 61

acid, and nomilinic acid glucoside standards obtained from photo
diode array detector.

XV

 

....-. .w.‘ “it
hurt» Li sperm ..

obtaineo trot:

r

Eitur 1' Cl spectra c.

7
‘

inure la: 1\ spectra o.

hare 19f 1V spectra c-:
.tandard ootazr

figure 30: Flop diagram.
llat'one extrac

Figure 31: Flon diagram
Figure 22: Pipe diagram

Figure 33: Separation of
pohmethoxi'l

hgurelt Separation of
in seed extrac

:7 1.. a ' ‘

hue -3. Separation o:

1: ~- . eparation o:
at 210 nm.

C a:

:ll all ‘. .

pm. .1. Hon drama;

llama to. ~ .

‘ k"'bellflmltott o
539d extract ‘
Volume-st

lltlli‘ "i- -~ .
. hipunnm lmjt

Tire}:- _
‘ ‘ sepflfalmn
iTOm “011m

 

Figure 16: UV spectra of unknown peaks at 58.6 and 62.7 minutes ................... 62
obtained from photodiode array detector.

Figure 17: UV spectra of unknown peaks at 103.2 minutes ............................. 63

Figure 18: UV spectra of published 3,5,6,7,3’,4’—hexamethoxyflavone ............... 64

Figure 19: UV spectra of unknown peaks at 33.1 minutes and narirutin .............. 65
standard obtained from photodiode array detector.

Figure 20: Flow diagram of limonoid aglycone and polymethoxylated ............... 70
flavone extraction

Figure 21: Flow diagram of limonoid glucoside extraction .............................. 73

Figure 22: Flow diagram of flavanone glucoside extraction ............................ 75

Figure 23: Separation of limonoid aglycones (210 nm) and ............................ 84

polymethoxylated flavones (340 nm) in peel extract.

Figure 24: Separation of polymethoxylated flavones at 340 nm ........................ 86
in seed extract.
Figure 25: Separation of limonoid aglycones at 210 nm in seed extract ............... 87
Figure 26: Separation of limonoid glucosides in seed and peel extracts ............... 88
at 210 nm.
Figure 27: F lavanone glucosides at 280 nm in peel extract .............................. 89
Figure 28: Flow diagram of limonoid isolation from orange seeds ..................... 96
igure 29: Separation of limonoid glucosides from polar fraction of .................. 98
seed extract by analytical HPLC (40 1.11 and 80 ul injection
volumes).
igure 30: Purified unknown 1 and unknown 2 from seed extract ..................... 100
igure 31: UV spectra of unknown 1 and unknown 2 obtained from .................. 101
photodiode array detector.
igure 32: Flow diagram of flavonoid isolation from orange peels .................... 107
igure 33: Separation of polymethoxylatedflavones (fraction 15) ..................... 111

from nonpolar fraction of peel extract

xvi

 

 

 

Fruit: 34‘. Separation or :5
and puntrec an

' .... ’P‘r'. rii
"‘mhffifll Spa-ac ..

array detector

cur: its: Separation .
r .t "Wat‘-
01' 06:1 5 an n

i1nre3':Puntied urtlanr

iitue 38: 1V spectra of
array detector

Figure 39: Phitochentrce
in SOllCl fractic

Figure 111: Phttochemic:
liquid fractio

Figure 41: limonoid an
ltgure4lzlimon01d as:
More 413: limonoid cl

donut limonoid ~1

lit ~ 4’.
"gm“?- l)(tllmethor
Sheet 013nm

figure to: Poltmethoi
Sheet Wane:

T
H'f
l ‘Edl.

t‘ 1'
lift it ‘
a .
3mM pltost'
340 unl‘ N QD
in ~ tr. -

 

 

 

Figure 34: Separation of polymethoxylated flavone standards .......................... 113
and purified unknown 3 at 340 nm.

Figure 35: UV spectra of unknown 3 obtained from photodiode ....................... 114
array detector.

Figure 36: Separation of compounds (fraction 2) from polar fraction ................. 1 16
of peel extract at 280 nm

Figure 37: Purified unknown 4 from peel extract ......................................... l 17

Figure 38: UV spectra of unknown 4 obtained from photodiode ....................... 118
array detector.

Figure 39: Phytochemical content (limonoids and flavonoids) .......................... 135

in solid fractions of sweet oranges.

Figure 40: Phytochemical content (limonoids and flavonoids) in ...................... 136
liquid fractions of sweet oranges.

Figure 41: Limonoid aglycones in solid fractions of sweet oranges ................... 139
Figure 42: Limonoid aglycones in liquid fractions of sweet oranges .................. 143
Figure 43: Limonoid glucosides in solid fractions of sweet oranges ................... 146
Figure 44: Limonoid glucosides in liquid fractions of sweet oranges .................. 150
Figure 45: Polymethoxylated flavones in solid fractions of ............................. 154
sweet oranges.
igure 46: Polymethoxylated flavones in liquid fractions of ........................... 156
sweet oranges.
igure 47: Flavanone glucosides in solid fractions of sweet oranges .................. 161
igure 48: F lavanone glucosides in liquid fractions of sweet oranges ................. 164
igure 49: Polar compounds (reconstituted in 10% acetonitrile in ..................... 209
3mM phosphoric acid) in peel press cake extract at
340 nm, 280 nm and 210 nm.
igure 50: Nonpolar compounds (reconstituted in 100% acetonitrile) ................ 210

in peel press cake extract at 340 nm, 280 nm and 210 nm.

xvii

 

 

' r 4 . v' | NJ:
rirur: : .. Polar cornpc l
i 1“ it'll Doreen?

3551;: mm. 2510 .U

True 53. Nonpolar corn;

in rae extract 2:

Pure 53: Polar compo:
3mM phosphot
341.?th :8": It:

Eicue .' '1: Nonpolar corn
in peer press ;:
111‘ _
830-1l'l'll‘i:

Figure 55: Polar compou
phosphoric are
3811 nm. and I

here so: Nonpolar cor
in orange jurc

v-r

figure 57: Flori diamar

1

orange seeds

hare SS: Separation o

t
1

1191639: Separation o

rirueoh; Separation c
HPLC

lim ' -

...ure o1. Flott diaura

o‘idomestie;

I'
‘. 1911.“:

"1.
~ “ ll~~ Chromatog
ltmonorc at
r. ._ . .
gun “’- lltrontato o
ohacunom

fittest- “—
l '- l llfomatog
8th] and l‘it‘

‘

Spur ‘~ '
too. Chromatoc

 

Figure 51: Polar compounds (reconstituted in 10% acetonitrile ........................ 21 l
in 3mM phosphoric acid) in rag extract at 340 nm,
280 nm, and 210 nm.

Figure 52: Nonpolar compounds (reconstituted in 100% acetonitrile) ................ 212

in rag extract at 340 nm, 280 nm and 210 nm.

Figure 53: Polar compounds (reconstituted in 10% acetonitrile in ..................... 213
3mM phosphoric acid) in peel press liquid extract at
340 nm, 280 nm and 210 nm.

Figure 54: Nonpolar compounds (reconstituted in 100% acetonitrile) ................ 214
in peel press liquid extract at 340 nm, 280 nm
and 210 nm.

Figure 55 : Polar compounds (reconstituted in 10% acetonitrile in 3mM ............. 215
phosphoric acid) in orange juice extract at 340 nm,
280 nm, and 210 nm.

Figure 56: Nonpolar compounds (reconstituted in 100% acetonitrile) ................. 216
in orange juice extract at 340 nm, 280 nm, and 210 nm.

Figure 57: Flow diagram for the isolation of limonoid aglycones from ............... 218
orange seeds.

Figure 58: Separation of limonoid aglycones on preparative HPLC ................... 220

Figure 59: Separation of limonoid glucosides on preparative HPLC .................. 223

Figure 60: Separation of polymethoxylated flavones on preparative .................. 226
HPLC

iigure 61: Flow diagram for the sample preparations and analyses ................... 242

of domestically prepared orange juice and rags.

figure 62: Chromatograms and UV spectra of 17, 19— didehydro- ..................... 247
Lirnonoic acid and deoxylimonin.

'igure 63: Chromatograms and UV spectra of deacetylnomilin and .................. 248
obacunoic acid.

igure 64: Chromatograms and UV spectra of l9-dehydrolimonoic .................. 249
acid and limolinic acid.

.gure 65 : Chromatograms and UV spectra of isoobcunoic acid and .................. 250
obacunone.

xviii

 

 

"~’\('Trrr

irate 6‘3: Gretna-.5. a.

,— f“ A“ (”arr
turret r1011 was.

’\ F .. rl‘ JV"
3 I "(I o . s
[L'a 0;; v 5}; LL

Figure 66: Chromatograms and UV spectra of limonin, and nomilin .................. 251

Figure 67: Flow diagram of orange seed oil extraction for estimation ................. 253
of oil content.

xix

 

tyCl; 311110513th {11in
Bill: outt'iatec? hydf'i". 2‘"
7C1car‘oono13' isotope
(E: capillary electropho
El: electron impact mon
131: electrosprat' iontra
~211le negatiie its:
at TAB: posture last
1111: didtmin

llhlli: deacetylnorm l‘
0111: deacetylnomilir
DNCr. deaceti'lnomtlir
llll: eriocitrin

ith: last atom bomb.

Willi: louri er trans

iCOl: T:

01611 C 011C 51‘

Li : gas chromatocra

,er
:2. deutenum

9D: hesperidin

I'D \ h i
q“ ‘ . 5 \ l c
“4u\.b‘ .333 4‘

at . .
lC . high pen'ont'

 

LIST OF ABBREVIATIONS

APCI: atmospheric chemical ionization
BHT: butylated hydroxytoluene

13C: carbon-13 isotope

CE: capillary electrophoresis

El: electron impact inonization

ESI: electrospray ionization

-eV FAB: negative fast atom bombardment
+eV FAB: positive fast atom bombardment
DD: didyrnin

DNAG: deacetylnomilinic acid glucoside
DNM: deacetylnomilin

DNG: deacetylnomilin glucoside

ERT: eriocitrin

FAB: fast atom bombardment

FTNMR: fourier transform nuclear magnetic resonance
F COJ : Frozen concentrate orange juice
GC: gas chromatography

1H: deuterium

HD: hesperidin

HP: 3,4,5,6,7,8,3 ’,4’-heptamethoxyflavone

HPLC: high performance liquid chromatography

XX

 

 

/ n 0.“ i 1 ' 'Y {J
"112:1. - .4 meta-.1.
r 5.,.,o-“ .-

.5.

We ‘4‘ 6 'iéflfilnept
ll. mired raoratior.

"; limonin

LC: liuuid chromatogra;
'13: limonin glucosrde

113: ms spectrometr;

llSllS: tandem mass st

1

116. nomilinic acid

(”'4'

1h: nomilin glucoside
1111: nobiletin
111D: neohespen din

111: not from content

111: nomilin glucoside
1 .. - ~
1.11. nomilin

llhl: nuclear magnet

llinarirutm
\T .‘ *. -
‘ ”l '11. 1131111111141
ll: obacunone

‘1‘.-
lo. ohacuuone cluco

.3) . . .
3Whl‘lt‘ledfi an:

as.
.1 paper imprinter

\

i, .- .
"- llneusetm

 

 

 

 

HX: 3,5,6,7,3 ’,4’-hexamethoxyflaovne
HP: 3,4,5,6,7,8,3 ’,4’-heptamethoxyflavone
IR: infrared radiation

L: limonin

LC: Liquid chromatography

LG: limonin glucoside

MS: mass spectrometry

MS/MS: tandem mass spectrometry
NAG: nomilinic acid glucoside

NG: nomilin glucoside

NBT: nobiletin

NHD: neohesperidin

NFC: not from concentrate

NG: nomilin glucoside

NM: nomilin

NMR: nuclear magnetic resonance
NT: narirutin

NT-4’-G: narirutin-4’-glucoside

O: obacunone

OG: obacunone glucoside

PDA: photodiode array

PC: paper chromatography

ST: sinensetin

xxi

 

 

 

 

n
‘V‘ 'p’lra
I 'vu. ,,

" f’ '0! ’ \'rpl'l
SM: sachet”, .

 

 

STME: scutellarein tetramethylether
TLC: thin layer chromatography
TT: tangeretin

UV: Ultra violet

 

xxii

 

 

 

OVertx'l'li‘lmmE C
of chromt disease. Pa“
Block 61 al. 1993 slot
iegewhles was 05?" 0“
hit there art COml‘OD'
reducecahcemsl: Ste
these biologically act
protectite effects art-
ha Show additional
ml. l993l. \K'lllCll n:

tonhihute to their 2
lsmoris. limes. and g
Vitamin C. lolate. an
Component is respon
Closes of phitochm
flal'lllml'k (Bernie
3011ll‘otmds also hax
m5 and motion:

l\D~\ X31
loduction b} a gh

'ttSt‘so

. 2003i

 

 

INTRODUCTION

Overwhelming evidence has indicated that a plant—based diet can reduce the risk
of chronic disease, particularly cancer. In 1992, a review of 200 epidemiological studies
(Block et al., 1992) showed that cancer risk in people consuming diets high in fruits and
vegetables was only one-half that in those consuming fewer of these foods. It is apparent
that there are components in a plant-based diet other than traditional nutrients that can
reduce cancer risk. Steinmetz and Potter (1991a) identified more than a dozen classes of
these biologically active plant chemicals, commonly termed “phytochemicals.” The
protective effects are commonly attributed to antioxidant activity, although recent work
has shown additional role of these polyphenolic components of the higher plants (Hertog
et al., 1993), which may act as antioxidants or agents of other complex mechanisms that
contribute to their anticarcinogenic or cardioprotective actions. Although oranges,
lemons, limes, and grapefruits are a principal source of such important nutrients such as
vitamin C, folate, and dietary fibers, Elegbede et al. (1993) have suggested that another
component is responsible for the anticancer activity. Citrus fruits are particularly high in
classes 0 f p hytochemicals known a s the limonoids (Hasegawa and M iyake, 1 996) and
flavonoids (Benavente-Garcia et al, 1997). Beside health-related properties, these
compounds also have shown possibility to functionally serve as antioxidants, insecticidal
agents and taxonomic tracers.

USDA National Agricultural Statistics Service has reported that the citrus
production by eighteen major countries is approximately 73 million metric tons in 2002

(FASfUSD, 2003). Among the total citrus agronomic production classes, sweet orange

 

 

 

[and mean: t 21:29.23
Brazil and the '1- noted 5'
tithe total as further a
produce and process or;
large amount of pro-tee
on 03515! oi these
countries (Grohmarui e‘
stashed pulp solids. ant
to almost 50“ (‘ oi the if
molasses. cold-press
lla‘onoids (Braddock.
incorporation (“add h
Additionally. there
adulteration of hi gh \;

health benefits are 1"

processing lll’idmer .

otteotne source of fat

The goal of t'

he quantihine the l

 

(Citrus sinensis) accounted for 68% of the total. Two major orange producing countries,
Brazil and the United States, have contributed to 60% of the world production with 85%
of the total as further processed products. Mediterranean countries have also started to
produce and process oranges in significant amounts. These data show that there is also a
large amount of processing by-product available. Approximately two million dry tons
(dry basis) of these residues are generated annually in those two major citrus-processing
countries (Grohmann et al., 1999). The major by-products include dried pulp, molasses,
washed pulp solids, and essential oil. The peel residue is the primary fraction, accounting
to almost 50% of the fresh fruit weight. This part of the fruit is the source of dried pulp,
molasses, cold-press oils, d-limonene, pectin, potential seed derived products, and
flavonoids (Braddock, 1995). Processing practices set minimum levels of by-product
incorporation (“add back”), because of an impact on flavor, texture or appearance.
Additionally, there are numerous regulatory standards to control for economic
adulteration of high value juices. As a result, the bulk of these components with potential
health benefits are processed into cattle feed for sale at 5-10% above the cost of
rocessing (Widmer and Montanari, 1996). Therefore, these by-products would be an

ffective source of functional food additives or pharmaceutical products.

The goal of this project is to utilize sensitive analytical methods for identifying
nd quantifying the limonoids and flavonoids in edible and inedible fractions of oranges
o enhance the potential utilization of phytochemical from citrus by-products obtained

om commercial orange juice production.

 

 

 

 

Relerences:

Braddock. R 1. E99: :5;

Rename-Garcia. O . f.
and properties

Bloch. E. 199:. I he c-r
organic chemist:

Elegbede. l. A. Malt:
anticarcinogenic
Carcinogenesis

IlSl’SDA. 3003. Sira

hrohmann. K. Manth:
citrus peel juice

llasegau'a S. and Mr
lirnonoids. Foot

llonog. M. G. L. Fesl
lggj‘ Dlt’lal")
Zutphen Elderl

”0.90

51mm. RA. and ‘
Cancer Causes

lllilmer. \l' ll. 3 nd Tl

hlI‘Em Ultiliou
ill-IIlSllOnQ D‘

 

References:

Braddock, R. J. 1995. By-products of citrus fruit. Food Technology. September: 74-77

Be‘navente—Garcia, 0., Castillo, J ., Marin, F. R., Ortuno, A., Del Rio, J. A. 1997. Uses
and properties of Citrus Flavonoids. J. Agric. Food Chem. 45(12): 4505-4515

Block, E. l 992. T he 0 rganosulfur c hernistry o f t he g enus A Ilium: Implications for the
organic chemistry of sulfur. Angew. Chem. Int. Edn. Engl. 31: 1135-1178

Elegbede, J. A., Maltzman, T. H., Elson, C. E., and Gould, MN. 1993. Effects of
anticarcinogenic monoterpenes on phase II hepatic metabolizing enzymes.

Carcinogenesis. 14: 1221-1223

PAS/USDA. 2003. Situation and outlook for orange juice. httgflwwwfasusdagov

 

Grohmann, K., Manthey, J.A., Cameron, R.G., and Buslig, BS. 1999. Purification of
citrus peel juice and molasses. J. AgricFood Chem. 47:4859-4867

Hasegawa, S. and Miyake, M. 1996. Biochemistry and biological fimctions of citrus
limonoids. Food Rev. Intl. 12: 413-435

Hortog, M. G. L., Feskens, E. J. M., Hollman, P. C. H. Katan, M. B., and Krumhout, D.
1993. Dietary antioxidant flavonoids and risk of coronary heart disease: The
Zutphen Elderly Study. The Lancet 342: 1007 -101 1

Stavric, B. 1994. Antimutagens and anticarcinogens in foods. Food Chem. Toxicol. 32:
79—90

Steinmetz, K.A. and Potter, J. D. 1991a. Vegetables, fruit and cancer 11. Mechanisms.
Cancer Causes Control 2: 427-442

Widmer, W .W. and M ontanari, A .M. 1 996. T he p otential for citrus p hytochemicals in
hypernutritious foods. In: Hypemutritious Foods. Edited by Finley, J .W.,
Armstrong, D.J., Nagy, S., and Robinson, S.F. Agscience, Inc., Florida, p. 75—89

 

 

 

 

 

Citrus juice ProccSSillg
DE‘t'ClOI‘men,‘ l.
responsible for the N
ha: ll 11mg- 1%“
hesh fruits such as 5
requirements for rat
important source oft
more important tc
promoting aspects 0'
and taste. make it ‘
technology of citrus
the product econont
Citrus trees ;
told. The treesc
Elma“ llemperatu‘
influence on the go
During 2(ttftj
trillion mm

C lOI'

contented into prc

tilt-lop

fi—Oa

are crtru

Mm and burn

S m
“.3“ orange ‘(fir'

 

 

LITERATURE REVIEW

Citrus juice processing

Development of the frozen concentrated orange juice (FCOJ) industry is
responsible for the most significant increase in citrus fruit consumption since World

War II (Ting, 1980). This innovation solved manyp roblems associated with citrus

 

fresh fruits such as storage diseases, susceptibility to physiological disorders and
requirements for rapid transportation. Citrus fruits and their products are an
important source of vitamin C in the American diet, and are becoming increasingly
more important to other developed and deveIOping countries. The health-
promoting aspects of citrus, together with its appealing color and delightful aroma
and taste, make it the most popular of the processed fruit products. Improved
technology of citrus production, processing, storage, and transportation have placed
the product economically within reach of more consumers.

Citrus trees are cultivated in tropical and subtropical regions throughout the

 
  
   
 
  
  
  
  
  

world. The trees can grow in a wide range of soil, yet growing conditions such as
climate (temperature and rainfall), types of soil and cultural practices, have a large

influence on the quality of fruit produced and juice extracted (Anonymous, 1998).

 

During 2001—2002, the world production of citrus fruit was approximately 73
million metric tons, of which 49% was marketed as fresh fruit and 42% was
converted into processed products USDA/PAS (2003). In that same time period,
the-top five citrus producing countries were Brazil, the United States, China,
Mexico, and Spain, together producing approximately 74% of world production.

Sweet orange (Citrus sinensis) has been the main citrus produced (68%), followed by

 

 

tangerines tttrru: res.
(and limonin to" Li 1

mattered as freer. per.

primarily orange or:

Ettracting equtpm'i’ 1‘
tutce quality. PW
lunher it is also ‘
maximize the profit
theiuice market (At
One of the
especially orange a
With astringent “at
cultitars and cultt
therefore. debitter
studies int'olred
tllcfolloch. 1950,
and Rouseff. 100:
lliimball. lQS‘t, 1'
Militia \tlttclt

“-

that these 1 ecltrttt

itrus. thel are

“4;.-
“loom step:

 

tangerines (Citrus reticulate) (18%), grapefruit (Citrus paradisz’i) (5%), and lemon
(Citrus limon) (6%). In the United States, of all sweet oranges produced, 15% were
marketed as fresh produce. The remaining 85% accounted for processed products,
primarily orange juice.

Cost efficiency and juice yield are very important to citrus juice processors.
Extracting equipment has been developed to increase juice yield while maintaining
juice quality. Process designs have been deveIOped to effectively use energy.
Further it is also very important to increase by-product applications to help
maximize the profit and minimize the waste produced from this growing sector of
the juice market (Anonymous, 1998).

One of the long-standing sensory problems in processed citrus products,
especially orange and grapefruit juices, has been bitterness, generally associated

with astringent “after taste”. The level of bitterness varies among the different

  
  
  
  
  
  
  
  
  
  

cultivars and cultural practices. Bitter juices have a much lower market value;
therefore, debittering of citrus juice has been investigated extensively. Primary

studies involved applications of adsorption and ion exchange techniques

 

(McColloch, 1950, Chandler et al., 1968, Nisperos and Robertson, 1982, Couture
and Rouseff, 1992). Other debittering methods include super critical carbon dioxide
(Kimball, 1987), immobilized naringinase (Gray and Olson, 1981), and immobilized
bacteria which metabolize limonoids (Hasegawa, 1987). N otwithstanding the fact
that these techniques have been able to effectively remove bitter compounds from
citrus, they are still not practical for commercial routine operation due to the

additional steps of cleaning up and regeneration, column clogging, and associated

 

 

 

 

 

 

r lpW:
rid-11am: pr:-
LJ A -' '

k

l ' ‘1 Q Iglr o‘:
“‘"tf'llfS‘ 10inch: .,
.i 5.1 .

ulti. auxin 0D in?
hmonoatea’s-ring 3'01
Post-hartest treatmf‘F
content in Sutsho-b a?
on: substantia'i i085
grapefruittblaier e".
ethod has been to
he most promising
debittering actiyity
thasegan‘a 3000i
orange juice produc
Citrus byproducts
Accompanie.
Emit residues. acco
llaste products it
‘mptured juice \‘es
{nit shorting eont‘
1astet‘ractiott b} (
SSSfilial oils. den 0
mall-slit has bee

’i’.’

PIN

 

offeflavor problems. In addition to juice debittering treatments, preventions of
bitterness formation were also studied at pre- and post-harvest levels. T reatment
with auxin on fruit-bearing plants showed reduction of limonin precursor
(limonoate~A-ring lactone) in Navel orange fruit by 10-23% (Hasegawa, 1988).
Post-harvest treatments of citrus fruits with ethylene showed no effect on naringin
content in Suisho-buntan (Citrus grandis [L] Osbeck) fruits (Nishikawa et al., 2002),
but substantial loss in limonoate-A-ring lactone in Navel orange, lemon, and
grapefruit (Maier et al., 1973). It should be noted that awidely used debittering
method has been to dilute the bitter juice with non-bitter juice (Hasegawa, 2000).
The most promising solution to this problem is to create a new variety with high
debittering activity and low aglycone concentration through genetic engineering
(Hasegawa, 2000). Figure 1 shows a diagram highlighting of a typical commercial
orangejuice production and illustrating the primary products and by-products.
Citrus by-products
Accompanied bythe increased p roduction of orange juice, large amount of

fruit residues, accounting for more than half of the fruit wet weight, are generated.

aste products from juice extractors consist of peel, internal membrane, rag
ruptured juice vesicle), and seed. Figure 2 presents a cross section of intact orange
ruit showing common fruit p arts and their terminology. The peel is the primary

aste fraction by both weight and volume. Added value by-products such as pectin,
ssential oils, flavonoids, molasses, are extracted from portions of these wastes. The
emainder has been used as cattle feed, both wet and dried forms (Maier, 1978) with

e marketing price lower than its production cost.

 

 

 

 

 

 

 

.llnitt products

1,—
\i’.

luice e7

Pulpit

ililf'C '

Clarii

‘

l

: l. '
.;ltgtt‘-Sll't‘llfl.‘?

p, .
bot tom concetttra
tune production

\

51013536 0

Rel‘mc‘essr'

hm 110nm t

Picking of fruit

Transport

Truck unloading

l

Prewash

Destemming

Pregrade

Sampling

Fruit storage

Surge in water flume

 

 

 

 

 

 

 

 

 

 

Main roducts l B - roducts
p Final fruit wash y p
\l/
\l/ Final grading
Juice extraction _____O_i{‘___> Peel oil recovery—-> Flavor manufacturing
Fulpy :- ------ 1:8?! ----- > Feed mill --------- > Pelletised animal feed
juice

A .
: Large pzeces of membrane and seed

gm 1‘ [I ll ' . .
139-"(3,3- wa_,_cfle_ni_> Pulp production --> Reconstrtuted
sac wall, core, seed . .

at juice packers

 

Clarification ------ Reign) Pulp wash - Wigi’flEE‘g Feed mill

production

Single-strength _ Evaporated : > Added to concentrate
\l/ QI orange juice or used

Not from concentrate Concentrate _____ as a base for juice drinks

juice production production .

V

Storage or bulk transport Essence recovery

--d--—--

 

 

Reprocessing and packaging

Figure 1: Orange juice production (after: Anonymous, 1998).

 

 

 

 

 

Core\

Segment or

luice rest

1‘
Ila 3.
ml?“ CmSS 3‘

N

Cor

   
 

Segment wall

Juice vesicles

Figure 2: Cross section of intact orange fruit.

 

 

 

 

 

 

 

1 . .. "‘9’—
lrteorettca. )1-

oere; juice l5.z ’1“ r I.
.rgf‘m at. 72° t. mots:
tglljo‘o at 8‘1 monster
ntraste heat etapora‘
separated from ey ape
0diot1993t n
the efficiency of cttru
point. it is the przrn
,roduces the main bj
A detailed re
”991 The operatic
hammer mills to be
the can cause 31, .
dryness and can c a
rs‘Sitlues are blend:
W to aClllfte 3 r
a: readily h l'dra les
hydroxide pg 3101i

lfli

lll. Tradition;

mlll‘d'tfg’

lll‘ls‘allv .

of ii)

fmraClot a

 

Theoretical yields (wet weight basis) of products from Valencia oranges
were: juice (55.74%), residue to feed mill (44.52% at 82% moisture), press cake
(19.78% at 72% moisture), water evaporated in dryer (13.74%), finished dried pulp
(6.02% at 8% moisture), press liquor (24.77% at 90% moisture), water evaporated
in waste heat evaporator (19.11%), concentrated press liquor (5.15%), and limonene
separated from evaporator condensate (0.28%) (Braddock et al., 1979).

Odio (1993) has described that the feed mill Operation is very important to
the efficiency of citrus processing plants, because it is the largest energy consuming
point, it is the primary pollution control point (especially liquid wastes), and it
produces the main by-products (cattle feed, molasses, d—limonene).

A detailed review of citrus feed mill operations was written by Braddock
(1999). The Operation begins with delivering the wet residue from the peel bin to the
hammer mills to be chopped to optimum size (0.6-2 cm). Particles of too fine a mesh
size can cause air pollution and yield lost, but too large pieces may not achieve
dryness and can cause mold or so-called “spontaneous combustion”. The chopped
residues are blended with 02—05% lime (wet wt. basis) (primarilycalcium oxide,
CaO) to achieve a more rapid dehydration process. CaO is commonly used, because
it readilyhydrates with water in the residue, liberating heat and forming calcium
hydroxide [Ca(OH)2]. Further, lime neutralizes the peel acidity and de~esterifies the
pectin. Traditionally, residence time from mixing and pressing ranges from 10-15
minutes.

Typically, during pressing processes, orange peel is fed on to the top Opening

Of the extractor and the peel is pressed by the rotating screw, pushing toward the

 

 

 

 

 

. .Xy
Put; 1': .9”

I‘"
“We ll,-
7 . ,h-
hi—l‘ b

r'. Y
o

W .’ r t

fmrmehm .
:ermea "PICSS liq”:
Braddock 1999i 1":
:epprted to be retain
An tnnco'atft
produces more horror:
presscahe moisture
and requires less 12
diagram oicitrus tie.
Limonoids in citrus
limonotds
hnranyes.presen‘
hmonlune orang
predominant limo
and oats reported '
WI More than
(TtHrOr With a
{Il'SIallographt l.
Silt)“ ghmmap Si!
According

metrical t‘orttts

h] . r
Tlltg latlonet.

 

exit die. Pulp is pushed out toward the side Opening, while peel juice is collected
from the bottom opening. The resulting pulp is termed “press cake” and the juice is
termed “press liquid”. Final pH of the press liquid is approximately 6.5-7.0
(Braddock, 1999). However, the pH of molasses (press liquid end product) was
reported to be relatively more acidic (5—6) (Hendrickson and Kesterson, 1971).

An innovative procedure enabling continuous lime addition and mixing
produces more homogenous end products. Enhanced efficient lime reaction lowers
presscake moisture, lowers power consumption, capital investment, maintenance,
and requires less labor cost (Braddock, 1999). Figure 3 shows the typical flow
diagram of citrus feed mill operations (Anonymous, 1998).

Limonoids in citrus products

Limonoids are a group of highly oxygenated, tetracyclic triterpene
derivatives, present in Citrus and its closely related genera that include fruits such as
lemon, lime, orange, and grapefruit (Hasegawa et al., 2000). Limonin, the most
predominant limonoids for all Citrus, was first discovered in 1841 (Bernay, 1841),
and was reported to be a principle bitter compound in navel orange juice (Emerson,
1949). More than 120 years later, its chemical composition was elucidated to be
C26H3008 with a molecular weight of 470 using chemical methods and X-ray
crystallography (Arigoni et al., 1960 and Bart on et al., 1961). Figure 4 and Figure 5
show chemical structure of major citrus limonoids and their glucosides.

According to Hasegawa (2000), limonoids are present in Citrus in three
chemical forms: 1) limonoid monolactones (Open D-ring aglycones such as limonoate

A-ring lactone), 2) limonoid dilactones (D-ring closed aglycones such as limonin),

 

 

lie: res-3a:

tpee'i. pulp. rag a

0i mill in plant it‘ast:

‘

l

J
l
l
I

!—> \b‘ast

bit-261:

p——_

\t
d-limonent

\t
Flaror ntanuiact:

\\
;'|““l a ‘
I:(J\ “a

bed mill

 

 

 

Orange

\1/
Wet residues é——— Juice extractor ——-——9 Juice
(peel, pulp, rag and seeds)

 

 

 

 

\l/ W \l/

d-Limonene Molasses (~40°Bx) Animal feed

v

Flavor manufacturing

 

Evaporated

l l

Molasses (~72°BX) Molasses (~40°Bx) ..............

 

Lime
9 v . 1
Hammer mtlls :
Reaction time .
\l/ :
Presses
Oil mill & plant waste :
Press liquid %— 7‘ Press cake 1
—9 Waste heat evaporator :- ----- > Dryer feed
Waste heat evaporators Dryer
i Pellets 1

Citrus alcohol fermentation

igure 3: Feed mill unit operations (after: Braddock, 1999).

11

 

 

 

\Omlllll

/ “:Z“‘\

 

Obacunonf

bulk

“gates: Chemica

 

Nomilin Limonin

 

Obacunone Deacetylnomilin

Figure 4: Chemical structures of the major citrus limonoid algycones.

12

 

 

 

\orn

 

0b

iii .3 ',
gm“ E . Chem}

  
 
  
 

   
  
 

O ‘ B ~d-glucose

)L O ~B-d-glucose
0

COOH COOH

0 — B-d-glucose 0 — B—d-glucose

COOH COOH

Obacunone glucoside Deacetylnomilin glucoside

Figure 5: Chemical structures of the major citrus limonoid glucosides.

l3

 

 

 

.n ,- . , r_!i« (F
are ‘l titttO-YJUtC :z“

. "_
L

{375:5 tiff depfr'tG'f» ’
as solubility. t‘li 937‘
tug estertsr‘re
narrated by H2155
biosynthesis of him
monolactones irorn
bring lactone hyd:
dilactones. cl l'DF
limonoid glucosides
and accumulation
Figure e lliasegao
limonoids
bitterness is terntc
bitterness in juice
treblem. because
Etc tasteless. 0r.
iot'ironntent. o h
'rltdilactone mg

nomilin. lintoi‘

‘0 \
AMP.

“llller ‘ir

“
..) ,

 

 

and 3) limonoid glycosides such as limonin-l7-B-D-glucopyranoside. These three
forms are dependently synthesized and have different chemical characteristics such
as solubility, pH stability, and taste perception.

An extensive review on biosynthesis and accumulation Of citrus limonoids
was written by Hasegawa (2000). There are three types of enzymes involved in the
biosynthesis of limonoids: a) enzymes involved in the biosynthesis of limonoid
monolactones from the precursor nomilinate A—ring lactone from stem, b) limonin
D-ring lactone hydrolase, which lactonizes open D-ring of monolactones to form
dilactones, c) UDP—D-glucose transferase which convert Open D—ring to form
limonoid glucosides during fruit maturation (Fong et al., 1993). The biosynthesis
and accumulation of these limonoids and enzymes involved are summarized in
Figure 6 (Hasegawa, 2000).

Delayed bitterness anchlucosidation (natural debittering process)

Limonoids make up one of the bitter principles in citrus juice. Limonoid
bitterness is termed “delayed bitterness”, characterized by gradual development of
bitterness in juice a few hours after its extraction. Fresh fruits do not posse this
problem, because when intact, limonoids are present in monolactone forms, which
are tasteless. Once fruit cells are ruptured, monolactones are exposed to an acidic
environment, which causes D-ring tO close, producing dilactone molecules. Some of
the dilactone molecules are bitter; these include the major limonoids, limonin and
nomilin. Limonin bitterness is a problem primarily in the early season to mid
season winter fruit, but not is not p resent in late season fruit. Asthe fruit ripen,

monolactone levels decrease (Hasegawa et al., 1991).

14

 

 

 

Seed
Dilactctnt
D C‘ E
p.,
l?
hlonolat
n er %—

lintortoui intone

i . l
p % r
a ,
l
} (Hucos
{Dd CG
l
y
blond
C
\

it. ..
3m°hmmd

 

Stern

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Acetate
9 Nomilin
Mevalonate biosynthetic
1 enzyme
\1/
Nomilinoate A—ring lactone
(Precursor)
Seed Fruit tissue
Dilactones Monolactones
D’ C’ B’ A’ A a B——> C —> D
, A Limonoid biosynthetic enz.
Limonoid \ /
D-ring [atone Limonoid
hydrolase glucosyl
transferase
Monolatones
D <- C <— B 6‘ A Glucosides
Limonoid biosynthetic enz. AG BG CG DG
Limonoid
glucosyl Monolactones
transferase % A a B __> C _> D
Limonoid biosynthetic enz.
Glucosides
DG CG BG AG
Leaf
“Germination”
Limonoid
glucoside
fl-glucosidase
Monolatones
D C B A

 

 

 

Figure 6: Biosynthesis and accumulation of limonoids

15

in Citrus (after: Hasegawa, 2000).

 

 

 

 

lt has beer. 0::
. '1 ‘f .ui 13:3:
"tucoStoesin tru.
". s -
or“:
maturation proas.

9““.

fruit tissue. srnc:

produced iron. the '.
ligure ’ shorts sh“:
see
ltt‘ettty-Ottt
tsolated and charat
glucose molecule a
linkage l02alci eta
bitterness percept
attached are terrn
tater soluble. oh:
but solubility in o
Preyious
C0Yttpounds tltro~
tunes and thus

to "
ttno repossess

Eli diet.

 

It has been described that monolactones are converted to their corresponding
glucosides in fruit tissues and seeds during the late stage of fruit growth. During the
maturation process, the glycosidation contributes to the reduction of bitterness in
fruit tissue, since monolactones are no longer available. However, in seed, both
glycosidation and lactonization occur simultaneously, therefore, dilactones
produced from the lactonization process can still cause bitter taste in mature seed.
Figure 7 shows glycosylation and lactonization processes in citrus fruit tissue and
seed.

Twenty—one limonoid glucosides from Citrus and its hybrids have been
isolated and characterized, in which one limonoid molecule is linked with one D-
glucose molecule at the 17-position of the Open limonoid D-ring by a B—glycosidic
linkage (Ozaki et al., 1995). The closed D-ring structure is a key requirement for
bitterness perception (Hasegawa et al., 2000). Molecules with no sugar moiety
attached are termed “aglycones”. Limonoid glycosides are almost tasteless and are
water soluble, whereas some limonoid aglycones are extremely bitter and have very
low solubility in water.

Biological activities of limonoids

Previous studies on limonoids focused on the removal of these bitter
compounds through various techniques aimed at improving taste quality of citrus
juices and thus enhance their commercial value. Recently, limonoids have been

found to possess beneficial biological activities and engender positive health benefits

in the diet.

 

 

' lactonization

limonoate
(X.

hftlyeosylation

limonin

tgare ~t Lactoni;
lafier' li

A. Lactonization

OH Acidic pH

L
fir

C00 Limonoid D-ring
lactone hydrolase o

 

Limonoate A-ring lactone Limonin
(Nonbitter) (Bitter)

B. Glycosylation

+ UDP-D- glucose + UDP

  
 

O "B-d-glucose
COOH

Limonoate A-ring lactone Limonin glucoside
(N onbitter) (N onbitter)

Figure 7: Lactonization (A) and glycosylation (B) processes in citrus fruit
(after: Hasegawa, 2000).

17

 

 

 

 

 

 

limonin are
such astnhtbtttng tit
and Hasegart a. 39359
al.1989r and red-ac
hare discussed the c
to chemical structttr
itrthe induction ct
conjugation of glut
resulting in less
lmponant structur
the A ring tthrc‘n
hasegarra 1939,,
at'ailable to lll'dl
addition. limonoid
Wild lliasegatt
der'elopment of D
211.. 300(1), Miller

carcinogenesis r'

in random or no

Ito triterpettes

‘\ ,, g ""
u signttteartth

Thi‘ I '
...s ending is

.rrs and are t"
,.

t

 

 

 

Limonin and nomilin were first shown to have chemo-preventive activities
such as inhibiting the development of neoplasiain the forestomach of mice (Lam
and Hasegawa, 1989), inhibiting tumors in the buccal pouch of hamsters (Miller et
al., 1989) and reduced skin cacinogenesis (Lam et al., 1994). Miller et al. (1994)
have discussed the cancer chemo preventive activity of citrus limonoids in relation
to chemical structure. The furan ring on a limonoid molecule is an important key
for the induction Of glutathione S-transferase (GST), an enzyme that catalyzes the
conjugation of glutathione with electrophiles that include activated carcinogens,
resulting in less reactivity, more water solubility, and facilitated excretion.
Important structural features for GST induction are furan moiety, triterpene, and
the A ring which is nonperpendicular to the plane of the molecule (Lam and
Hasegawa, 1989). Numerous bacteria are present in the intestinal flora and
available to hydrolyze limonoid glucosides and liberate limonoid aglycones, in
addition, limonoid glycosides themselves have been shown to have direct anticancer
activity (Hasegawa, 2000a). Limonoid glucoside has been found to inhibit the
development of DMBA-induced tumor for oral carcinogenesis in hamster (Miller et
al., 2000). Miller et al. (1992), in a study of the inhibition Of hamster buccal pouch
carcinogenesis, reported that the addition Of glucose and the Opening of the D-ring
in limonin or nomilin do not modify the cancer chemopreventive activity of these
two triterpenes. Limonoid glucosides, both. individual and mixed, have been found
to significantly inhibit human breast cancer cell proliferation (T ian et a., 2001).
This finding is very important because the glucosides are more abundant in the

fruits and are tasteless, whereas aglycones are concentrated in the seeds and some of

18

 

 

their; possess b11167
glucosides camp's“:
limonoids r.
insects including C<
rlepidoptera: Ior
rLepidoptera: Nozt
lanae rRubertcr
liolbc rSerit er a.
primary pests oath
practice is yery r
property oi lrrno
replacements for
limonoid strucrur
necessary 1 or fee.
bore: and that 1
“mt er al. rro
a:lainst insects

area or blOlOgtcg

hilt Shall'Srg

t‘otentral 1mm,

ilk!“ do - l
”Winter.

 

 

them possess bitter off-flavor. Thus, the general consumption of the limonoid
glucosides compared with the aglycones is relatively high.

Limonoids have also been shown to possess antifeedant activity against
insects including Colorado potato beetle (Bentley et al., 1988), spruce budworrn
(Lepidoptera: Tortricidae) (Alford and Bentley, 1986), fall armyworm
(Lepidoptera: Noctuidae) larvae (Mendel et al., 1993), and Spodoptera frugzperda
larvae (Ruberto et al., 2002); as well as the common termite, Reticulitermes speratus
Kolbe (Serit et al., 1991). These responses are very important because each is
primary pests with high economic impact. Appropriate pest control in agricultural
practice is very important indirectly to overall human health. This biological
property of limonoids suggests consideration of the compounds as potential
replacements for chemical insecticides. Mendel et al. (1993) investigated the
limonoid structures and concluded that the furan system and epoxide group are
necessary for feeding deterrence against fall armyworm (Lepidoptera: Noctuidae)
larvae; and that nomilin was the most active among the limonoids tested. However,
Murray et al. (1999) reported that limonoid glucosides have no antifeedant activities
against insects. Thus, the assessment of insecticidal properties remains an active
area Of biological research.

Distribution information of neutral limonoids in the citrus seed obtained by
HPLC analysis was found to be specific to species and cultivars. This implied the
potential importance of the limonoid profiles of seeds as a Chemotaxonomic tool in

the development of new Citrus cultivars (Manners and Hasegawa, 1999).

19

 

 

Flaronoids 'trr citrus ;
Fiayorrorr's a:
polyphenolic compo

considered to be 1:

1":
r_)
E»
C)-
(v
._,,
('D
0..
$3
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L'T"
y—a
.J"
'1‘

Flavonoids
anclosrng a heteroc
distinguished by tr.
groups. which are
include frat‘anorres
and third groups
diglucosides. o‘he:
Figure 9 short ch
lb shorts clten'

TeS'r‘efllVelr:

Most crtr

‘Eb’cosrlared 3.1 ,

r
.are been “i de':

D-glumsfl. tr l"

\
lr

ronrrrhmgS to .

l

 

 

 

Flavonoids in citrus products

Flavonoids are one of the most widely distributed and diverse groups Of
polyphenolic compounds in the plant kingdom (Harbome et al., 1975). They are
considered to be the most important natural plant pigments, particularly when

considered with the carotenoids and. the tetrapyrrole derivatives.

Flavonoids have a typical chemical structure consisting of two benzene rings
enclosing a heterocyclic six-member ring containing an oxygen atom. They are
distinguished by means of differences in the heterocyclic ring and added hydroxyl
groups, which are free, methylated, or bound to sugars. Flavonoids found in citrus
include flavanones, flavones, and flavonols, with much lower levels in the second
and third groups (Ooghe et al., 1994 a). Citrus flavanones occur mostly as
diglucosides, whereas methoxylated flavones occur as free aglycones. Figure 8 and
Figure 9 show chemical structures of major citrus flavanone glucosides and Figure
10 shows chemical structures of major citrus polymethoxylated flavones,

respectively.

Most citrus cultivars can be classified by the glycosylation patterns, which
Occur at the 7 position on the flavonoid skeleton, except for rutin which is
glycosylated at the 3 position. The two main flavonoid glycosylation patterns that
have been widely used are a) neohesperidosides (2-B-1-rhamnosyl-D-g1ucose), which
are found only in species related to pummelo, and b) rutinosides (6-B-1-rhamnosyl-
D-glucose), which are found in all species of citrus (USDA, 2002). The glycosylation
contributes to increased polarity Of the flavonoids, which is necessary for storage in

plant cell vacuoles (J ustesen et al., 1998). It is hypothesized that flavonoids in plants

 

OH HO O CH3

OHHfiy‘A/

Hesperidin

0H “HZQW
Narirutin
OH
OOHHO OH
% %O O O
OH HHO
Eriocitrin
OH O

Figure 8: Chemical structure of the major citrus flavanone glucoside (rutinosides).

21

 

 

OH
HO
0
H1590 0 . .
OH Nartngm
0 OH o

H

OH
O-CH3
HO
O
OH
HO OH O Neohesperidin
OH
OH
HO
%0 O o
HO
O
%h
HO OH O Neoeriocitrin

Figure 9: Chemical structure of the major citrus flavanone glucoside (neohesperidosides).

 

 

 

81116115an

\obile‘

' lrertr

 

 

 

 

OCH,

OCH,

 

3 ,5 ,6,7,3 ’,4’-Hexamethoxyflavone

OCH, OCH,

OCH,

 

Nobiletin 3 ,4,5 ,6,7,8,3 ’ ,4’ -Heptamethoxyflavone

OCH, OCH,

   

OCH, r) OCH, C)

Tetra-O-methylscutellarein Tangeretin

Figure 10: Chemical structures Of the major citrus polymethoxylated flavones

23

 

 

 

. ';- .. termite

' ‘ ‘ ; a . ‘1’.
lilCIUZIC Kill; :‘11’. torcr

Regarding :72

hatonoid content it.

concentr'tion as the
titular as a resuit
dendnchson. 1953
concentration and
housed and Dough:
a grapefruit iurce
conditions.
Flaronoids
lemons and pol
inE‘fltrim is Speci

boa , .
”\DUSEG10CaICG.

mots polynrethe

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serve as protective agents against UV radiation and microorganism infection

(Robard and Antolovich, 1997).

Regarding flavonoid content during fruit growth, it is understood that
flavonoid content in the whole fruit increases at the early stage Of fruit development
and then remains almost constant (Rouseff, 1980). Decreased flavonoid
concentration as the fruit matures is due to the absolute content and its gradual
dilution as a result of the increase in the size Of the fruit (Kesterson an and
Hendrickson, 1953). However, there is disagreement on juice flavonoid
concentration and whether changes occur as the fruit matures (Rouseff, 1980).
Rouseff and Dougherty (1979) Observed a small but consistent decrease Of naringin

in grapefruit juice as the fruit matures under strictly controlled experimental

conditions.

Shemotaxonomic mark ingLand authenticity

Flavonoids are present in Citrus fruits in two major classes: glycosylated
lavanones and polymethoxylated flavones. They are found only in citrus and their
ngerprint is specific of each species (BoccO et al., 1998). F lavanone glucosides have
een used to categorize citrus and its hybrids (Tatum et al., 1974). The presence of
arious polymethoxylated flavones were also used to distinguish between nucellar
d zygotic seedlings from leave extracts, taxonomic classification (Tatum et al.,

78), including differentiation between common Species (Gaydou et al., 1987).

With respect to the citrus industry, there are two main reasons for the
ditions Of non-C. sinensis juices to orange juice: 1) addition of cheap j uice from

apefruit or sour orange to sweet orange juice to increase the financial profit

24

 

 

 

 

I" ‘r a 9;
T a a [1.-
rtpuSer‘. e. a... .
tangerine to orange

r. i F i r l.\ {v'
tongue. and D510 .

.tccordzng tr.
orange juice has e
sound. ripe orange:
juicemay contarr. 1
Food and Drug .
mandarin if. retrc
concentrated orar
Jttt‘arttitrnrl lRouse

not their any add

Flar‘onoids
because they are
Specific. cl multrt
their structural
Addition of sntal
detected by it
ueet oranges rt
also do not

It} .

‘Oisbe er al., j.

 

(Rouseff et al., 1987) and 2) addition of juice from expensive hybrids such as
tangerine to orange juice from early season oranges to improve the juice quality

(Ooghe, and Detavernier, 1997).

According to Codex Alimentarius (1992), “orange juice and concentrated
orange juice have to be Obtained by a mechanical process from the endocarp of
sound, ripe oranges (Citrus sinensis), preserved exclusively by physical means. The
juice may contain up to 10% (m/m) of mandarin juice (Citrus reticulata)”. The U. S.
Food and Drug Administration (FDA) permit the addition of 10% (m/m) of
mandarin (C. reticulate) or hybrids to pasteurized and canned orange juice. Frozen
concentrated orange juice also may contain up to 5% (m/m) sour orange (C.
aurantium) (Rouseff, 1988). However, most countries within the European Union do

not allow any addition of non—C. sinensis juices (Ooghe and Detavernier, 1999).

Flavonoids are promising as means for determination of juice authenticity,
because they are a) ubiquitous and present in measurable quantities, b) genetically
specific, 0) multiple and diverse, and d) mostly expensive to synthesize as a result of
their structural complexity (Rouseff et al., 1987, Schnull, 1990, and Wade, 1 992).
Addition of small amount of C. paradise, C. aurantium, and/or C. bergamia juice may
be detected by the presence of flavanone neohesperidosides, which are not present in
sweet oranges (Ooghe et al., 1994a). However, for tangerines and its hybrids, which
also do not contain these neohesperidosides, distribution patterns of

olymethoxylated flavone offer more sensitive mean to detect their contamination

(Ooghe et al., 1994b).

 

25

 

 

 

1 a(' .7
gmsrttess anti “7‘s”

 

Th6 imam

' I r. 9 r_ '
i'uttntodttrttor. c
fthflft’ldOSiGES an.

211le t

llatanorte -1
humour neohesp'
neohesperidosdes 2
grapefruit and r
ttltmetltoxylated t'
reltttt‘elt' lou. ther

the tutor 01 matte:

AA

Ditterentiat
fruit to be bitter

.‘ttttt. Btttet llth'
ttttt

Otangfl and l

H“ Um bf preS

:tttonotds are 1101
«1:115:1ij lien}

ESQ §a\
“mm mm

l‘ifltRouset‘t‘ to

“fireman

W‘\‘
\.'. a . “
rats. 15 the

 

Bitterness and precipitation problems

The flavonoids have significant influence on nearly every aspect of citrus
fruit production and processing. Two main impacts are bitter taste of f lavanone
hesperidosides and low solubility in aqueous solutions of hesperidin (Horowitz,

1961).

Flavanone glucosides are present in citrus in two structural isomers: a) bitter
flavanone neohesperidosides, and b) tasteless flavanone rutinosides. Major
neohesperidosides are naringin and neohesperidin, which are commonly found in
grapefruit and pummelo. Veldhuis et al., (1970) reported that some
polymethoxylated flavones were bitter, but their concentration in orange juice was
relatively low, therefore, they are considered not highly important contributors to

the flavor of orange juice.

Differentiated from limonoid bitterness, flavanoid bitterness causes intact
'ruit to be bitter and also imparts immediate bitterness to the freshly prepared
uice. Bitter flavonoids occur only in a. few Citrus species (grapefruit, pummelo,
our orange, and Ponderosa lemon), but limonin occurs in all Citrus, even though it
tay not be present in sufficient amounts to cause highly bitter taste. Since
avonoids are not evenly distributed through out the fruit, extraction pressure and
intact time between the juice and high flavonoid fractions (albedo, central core
td segment membrane) play an important role on their final concentration in the

ice (Rouseff, 1980).

Hesperidin, the most abundant flavonoid compound in lemons and sweet

tnges, is the most insoluble of all citrus flavonoids (Rouseff, 1980). In intact

26

 

 

 

trots. hesperidin o:
attraction, liberatzr. g
ritually preeiptta:
itssohed by form
totals are found at
grtces during storag
productior. of from
presence of these it:

appearance \thteh ;
Biological activities

Although. 7
quality juice. it h;

beneficial biologic

Bettatettte-
properties of eitrt
trri‘iotaseular jo‘
rarities; and it
their ability to at
troeess: damage
%rrttl

littfillmll

Aemrrtals. \l‘idi

an

11 the d‘

I»):
‘-\v\fo

.ertttgiriitt that

 

 

fruits, hesperidin occurs as a soluble complex, which is destroyed during juice
extraction, liberating free hesperidin (Horowitz and Gentili, 1977). Free hesperidin
gradually precipitates as fine, white, needle-shaped crystals, which can only be
dissolved by formamide, pyridine, or dilute alkali (Rouseff, 1980). Hesperidin
crystals are found in frost damaged oranges (Hume, 1957) and concentrated orange
juices during storage; and are found as a thin crust coating the evaporators used in
production of frozen concentrate orange juice (USDA, 1962). Even though the
presence of these hesperidin particles does not affect juice flavor, it results in visual

appearance which is considered a major quality defect (Rouseff, 1980).

 

Biological activities of flavonoids

Although, high accumulation of unfavorable flavonoids results in lower-
quality juice, it has been reported in many studies that these compounds possess

beneficial biological activities, especially health-promoting functionalities.

 
 
  
  
  
  
  
  
  
  
  

Benavente~Garcia et a1 (1997) systematically described. health-related
properties of citrus flavonoids. These properties include a) antioxidant activities; b)

cardiovascular properties; c) anti—inflammatory, d) antiallergic, and e) analgesic

 

activities; and f) antimicrobial activities. Due to their antioxidant properties and
their ability to absorb UV light, flavonoids may act in all stages of the carcinogenic
process: damage to the DNA (initiation), tumor growth (promotion), and invasion
(proliferation). Flavanone glycosides are not absorbed by humans or other
mammals. Widmer and Montanari (1996) concluded that intestinal floras in the gut
cleave off the disaccharides of hesperidin and naringin, producing hesperitin and

naringinin that were absorbed. Numerous studies on different chemically induced

27

 

 

 

 

‘o‘
.

cancers hate 75790"
33.356; Janette er a
5:21-199’bt Fiat
tit-induced DNA d
Polnnethox;
thannaeodynarnz:
note acute that;
llllt and exhibit
counterparts tAtt.
greater antr-adhes
glycosides. Rand
tangetetin on the
'11le The) it
and quereetin am
attu'itt' m} be d
nethoxylation o:
leteral of PM?
aunties. 31111-11'
ailfltltt reactions

l“ addttte

LEl‘t ‘ .

\3' »
“MEIR 3nd l

\x\“ ‘
“1135130361110
K

 

cancers have reported that hesperidin was found to inhibit chemically induced colon
cancer (Tanaka et al., 1997a and Miyagi et al., 2000) and esophageal cancer (Tanaka
et al., 1997b). Flavonoids were also reported to have the protective effect against

UV-induced DNA damage (Kooststra, 1994).

Polymethoxylated flavones (PMFs) have been found to possess
phannacodynamic properties (Ooghe et al., 1994). PMFs were found to be much
more active than naringin, hesperidin, or their flavanone aglycones (Wall et al.,
1988) and exhibit higher levels of biological activity than their hydroxylated
counterparts (Attaway, 1994). Robbins (1974) found that isolated PMFs had
greater anti-adhesive effects on red blood cells and platelets than did flavanone
glycosides. Kandaswami et al (1991) examined quercetin, taxifolin, nobiletin and
tangeretin on the in vitro growth of a human squamous cell, carcinoma cell line
(HTB43). They found that nobiletin and tangeretin markedly inhibited cell growth
and quercetin and taxifolin exhibited no significant inhibition. These differences in
activity maybe due to the relatively greater membrane uptake of the PMFs since
. ethoxylation of the phenolic groups decreases hydrophilicity of the flavonoid.
everal of PMFs have been demonstrated to possess antimicrobial, antiviral
ctivities, anti-inflammatory properties and inhibit histamine release to reduce
llergic reactions (Widmer et al., 1996).

In addition to the health potential roles of flavonoids mentioned above, some
avonoids may be suitable for industrial food ingredient sweetener application.
aringin and neohesperidin have been found to be able to convert into their

orresponding dihydrochalcones which are 100 and 15,000 times sweeter than

28

 

 

 

 

sucrose tllorou ttz.
neohesperidin ctr}:
natural sneetentng

anti stnthetre so een
Sample preparatior

Generally ;
ainoehernteais is 1
The critical goal
components of lI‘al

Illllt‘t.

Reeot'ert‘ 1
name aetit'it}. a
the lack of chem
l‘llllt. The eonsi:
and that of isola
seleetion. There:

audition. thernt

532' r
npla (Antolot

lsolanorl

onfibtuon “11}

let .
‘ ‘ p0llll‘it'tltm

ii, too0
'l- “hen

it“ ~ -
.inunesj()23,
, |

 

 

sucrose (Horowitz, 1986, Bar et al., 1990, and Borrego et al., 1991). Since
neohesperidin dihydrochalcone and naringin dihydrochalcone are non-saccharide
natural sweetening agents, they could be used to overcome the problems of sucrose

and synthetic sweeteners in selected product applications (Venkata et al., 2002).
Sample preparation in citrus phytochemistry

Generally, prerequisite to qualitative and quantitative analyses ofthe natural
phytochemicals is the extraction of these compounds from the plant tissue matrices.
The critical goal is to obtain a sample extract uniformly concentrated in all
components of interest and free from interfering components (Antolovich et al.,

2000).

Recovery is complicated as fruit constitutes a natural matrix with a high
enzyme activity, and therefore care must be taken to ensure correct extraction and
the lack of chemical modification, which will result in artifacts (Macheix et al.,
1990). The consistency of the relative compound profile between starting material
and that of isolated extract provides a theoretical basis for analytical technique

selection. T herefore, the conditions u sed should be as mild as p ossible to p revent

    
   
  
   
 

oxidation, thermal degradation, and other chemical and biochemical changes in the

samples (Antolovich et al., 2000).

Isolation of biological compounds is also complicated by their uneven
distribution within various fruit tissue fractions. For instance, citrus peel contains
igh polymethoxylated flavones (Gaydou et al., 1987, Morin et al., 1991, and Dugo et
1., 1996), where as citrus molasses (evaporated peel j uice) contains high limonoid

lycosides (Ozaki et al., 1995 and Hasegawa et al., 1996). Accumulation of soluble

29

 

. ‘ r I , I 4 ‘P'
nit’l'tl'llli'i 15 ‘elccte.
’ ,.5 L5 w

in: than in :ne 1:
I". 1P‘Ir"i€)fi-'fir
i‘DOlDULlhfiA .rr.» .4

compounds ma} er.

'1‘
(":1

eat oer r an
arrangement one;
neuron. the ear:

irfietent subcelluia

111th iett et
tith liquid sample
llilti heating may
illtl‘ldti at liquit
ertraetion. ln 1:
analyses. ln the
obtain complete ‘
attractions and r

ntero extractions

ll l5 Essen

life at .
filw sompot

“\.\.‘
t-?\ \e ,
1“ illok’i‘tluj

il'al ‘1 . .
kl ‘|a\01101d§

mo

1321.

 

 

phenolics is greater in the outer tissues (epiderm a1 and subepidermal layers) of the
fruit than in the inner tissues (mesocarp and pulp) (Bengoechea et al., 1997).
Hypothetical explanations at the subcellular level have been proposed that phenolic
compounds may exist in the vacuole or within the cell wall (Yamaki, 1984); they
may occur in soluble, suspended and/or colloidal forms and in covalent
arrangement with cell wall components (Lichtenthaler and Schweiger, 1998).
Therefore, the extractions of these phytochemicals may greatly influenced by these

different subcellular-level accumulations (Antolovich et al., 2000).

With few exceptions (hesperidin and naringin), it is less complicated to work
with liquid samples because the compounds present are most readily extractable.
Mild heating may be needed to achieve complete dissolution. Extraction techniques
include: a) liquid-liquid extraction, b) solid phase extraction and c) solvent
extraction. In limited circumstances, no sample treatment is needed prior to
analyses. In the case of solid samples, more extensive extraction is required to
obtain complete recovery. These conditions range from a sequence of exhaustive
extractions and preconcentration to supercritical fluid extractions and solid phase

micro extractions (Antolovich et al., 2000).

It is essential to recognize that sample manipulation to increase selectivity of
targeted compounds may result in a relative decrease of their sensitivity. The
precise procedure selected depends on both natures of analytes (e. g. total limonoids,
total flavonoids, glycosides, or aglycones) and sources of samples (e. g. peel, seeds,

'uice, or rag). The extraction procedure is simplified in analyses focusing only a

30

 

 

 

S't’Clilt‘ aornpounc. c

ti‘TO’dC range ct cor
tnalvses of citrus 11

Virus hrnonoads

lfhr “’
Anal\ 5:: c

absorb 1\' light.
complex matrix

prehrninarp stept

limonoids
glucosides are 5
ohms their cc
practice to spec
limonoid glucc
aglycones and
importantto u
in the complex
Dieter
determination
nuclear magi
Since then. t
identified, T

l“ EXPOSmE

specific compound, and the degree of analytical challenge increases when analyses of

a broad range of compounds are the goal.
Analyses of citrus limonoids and flavonoids

Citrus limonoids

 

Analyses of these particular compounds possess many technical problems
including: a) their existence in the samples in minute quantities, b) the low ability to
absorb UV light, 0) thelack ofcommercial standards, and (1) their presence in a
complex matrix. Consequently, the isolation of limonoids becomes an important

preliminary step essential to the quantitative analyses.

Limonoids occurs in both aglycone and glucoside forms. Limonoid
glucosides are soluble in aqueous solution, due to an added glucose molecule,
whereas their corresponding aglycones are much less nonsoluble. It is a common
practice to specifically analyze each group independently. Solvent extraction of
limonoid glucosides usually renders flavanone glucosides, likewise limonoid
aglycones and polymethoxylated flavones are usually co-extracted. Thus, it is
important to use analytical methods that effectively differentiate these compounds

in the complex mixture.

Dreyer (1965) was the initial contributor who developed quantitative
determination by thin layer chromatography (TLC), and structural elucidation. by
nuclear magnetic resonance to characterize the limonoid compounds in citrus.
Since then, there have been 38 limonoid aglycones and 20 limonoid glucosides
identified. The reddish-orange limonoid color developed with Ehrlich’s reagent and

by exposing it to hydrogen gas is specific enough to be readily differentiated.

31

 

 

 

 

 

 

 

 

main} control and
reproducibilit} prc
system to separa‘
glucoside as 2 tot
Berhow. 31111.1 t.
Recent on
hPIC-tl tFong
llansell. 199".
tllclntosh. Ztttfitt’jt
tlloodley et al.,
lot 111%. requ
partitioning, :5
HPLC i
lel‘mt‘lucible. a
dominant in tin.
tther compoun
such complex r
OlgPtlllt‘ (Kok-en.
Sa'il‘lfil tor
Citrus Seeds. it

”Dried Sillt

 

Subsequently developed TLC methods (Maier and Grant, 1970 and Tatum and
Berry, 1973) are more specific, sensitive, and precise. TLC is suitable for routine
quality control analyses because it is simple and rapid, however, there are analytical
reproducibility problems associated with this technique. Also, there is no solvent
system to separate limonoid glucosides; therefore TLC determines limonoid
glucoside as a total value rather than as the individual species (Hasegawa and
Berhow, 2000).

Recent quantitative techniques of limonoids include TLC (Ohta,1993),
HPLC-UV (Fong et al., 1993, Ozaki et al., 1995, Hsu et al., 1998, McIntosh and
Mansell, 1997, Hasegawa and Manners, 1999) radio immunoassay (RIA)
(McIntosh, 2000), HPLC-MS (Schoch et al., 2001), and capillary electrophoresis
(Moodley et al., 1995, Braddock and Bryan, 2001). Most of these methods, except
for RIA, require sample preparation such as organic solvent extraction,
partitioning, or solid phase extraction (Hasegawa et al., 2000).

HPLC is the most commonly used method, because it is accurate,
reproducible, and highly accessible. The application of reverse phase HPLC is
dominant in limonoid quantitative analyses not only for citrus limonoids but also for
other compounds in plant extracts, as it provides higher separating efficiency for
such complex mixtures through various mobile phase selections and it consumes less
organic solvent compared to normal phase HPLC. Reverse phase HPLC has been
developed for analytical determination of both limonoid aglycones and glucosides in
citrus seeds, juice, peel, fruit tissue, and by-products. These techniques utilized C18

onded silica columns with solvent mixtures of acetonitrile/water,

32

 

 

 

 

 

 

aetomtrtl’: 2103”“ “3
italff. Acetonitrilt
[lunar 1'1 cutoff
men'ere with the l
tact? pressure ptot
Rouseff ant
in analysis of lim
column with a te
system was abl
deoxylimonin. ll
binary mobile pl
The high selecti
separation of mt
Deterntir
later than 220
0f retention tirt'
alt‘anced idett
lTOduces t‘\‘
7.211"an 1111p:
ngllfl SPECifi
1here by the
til-r

tnttttcatton

 

acetonitrile/aqueous acid or mixtures of acetonitrile, methanol, tetrahydrofuran and
water. Acetonitrile has been the solvent of choice because it has low UV absorption
(190nm UV cutoff) and low viscosity (0.38 cP). Therefore, acetonitrile does not
interfere with the limonoid detection (typically at 210 nm) and does not cause high
back pressure problem to HPLC pump.

Rouseff and Fisher (1980) recently developed a normal phase HPLC system
for analysis of limonoid aglycones. They used a combination of a CN bonded silica
column with a ternary mobile phase (hexane/2-propanol/methanol). This HPLC
system was able to effectively separate obacunone, nomilin, limonin, and
deoxylimonin. Hasegawa and Manners (1999) used a spherical silica column with a
binary mobile phase (cyclohexane/tetrahydrofuran) to resolve limonoid aglycones.
The high selectivity achieved allows the option of commercial scale up for the
separation of minor limonoid aglycones.

Determination of limonoids was achieved by UV detection at wavelengths
lower than 220 nm. Identification of individual limonoids is based on a comparison
of retention times to those of standards. Photodiode array detection offers a more
advanced identification compared to UV-vis detection. This type of detector
produces UV spectral data for each resolved compounds, thus aiding to screen
flavonoid impurities that may be present and adjacent to limonoid compounds.
Higher specification of limonoid analyses was achieved by application of LC—MS,
where by the resolved compounds is immediately subjected to MS system. The
identification of selected compounds is therefore dependent on retention time,

structural. and/or molecular weight data.

33

 

 

sat enabler} increas
resolution. a large 1
and characterized.
techniques. nhtch '
aat‘eled to more
littttbt.

limonoid 5
mini ed studies.
be analtred by be
the iragrnentatior
addition of nonoc

t‘aoorize.

loo mart
spectrometry lit
llanners et al..
tSattabe et al..

25 l0\1‘ 35 p m
"‘ l

nascgatta 31 a1

.monoid 321m.
for 1‘
“‘6 analyst,

5’ x

 

The first comprehensive explanation of limonin structure determination
using 1H and 13C NMR was achieved by Dreyer (1965). Since the evolution ofNMR
has enabled increased magnetic field strengths with corresponding large increase in
resolution, a large number of limonoid aglycones and glucosides have been isolated
and characterized- The development of Fourier transform NMR (FTNMR)
techniques, which produce extensive intramolecular 1H-lH and 1H—‘3C correlations
haveled to more rapid structural assignments of the limonoids (Hasegawa et al.,
2000b).

Limonoid structural characterization by mass spectrometry has been found
in limited studies. Due to their nonvolatility, limonoids are not readily adaptable to
be analyzed by basic EI-GC-MS instrumentation combinations necessary to acquire
the fragmentation patterns. Derivatization of these high oxygenated molecules by
addition of nonpolar moieties may produce large molecules that are still difficult to

vaporize.

Two main mass spectrometric techniques are electrospray ionization mass
pectrometry liquid chromatography (ESI-LC—MS) (Schoch et al., 2001, and
anners et al., 2003), and fast atom bombardment mass Spectrometry (FABMS)
Sawabe et al., 1999). Among these techniques, LC-MS provides a high sensitivity
3 low as 42 picograms for analysis of citrus limonoids (Hasegawa et al., 2000).
asegawa et al. (2000) described that EI-LC-MS was useful only for the analysis of
'monoid aglycones, whereas ESI-LC-MS was found to be the most effective method
r the analysis of limonoid glucosides. In addition, an established normal phase

PLC condition suitable for limonoid aglycone analyses introduced by Manner and

34

 

 

 

. .’(. ‘
Y"arcane 11W" ‘
:A'b:

chemical ionization
They concluded 1h
lf-l’rSrnocie. Sat
usrng positire ant
establishing mole:

nomilinate l'-O-l‘

primarily used t
confirmation.

chromatographi

(itrus flat‘onoic

\

that their mole
This 1V absor

andhigher tle‘

Analts
it‘lltttethoxrl

soluble comp.

lillfia ., ,

“at alhalt
a,“ .
Lassol ”11011

“Oml‘s‘unds

Hasegawa (1999) was reported to be able to directly adapt to the atmospheric
chemical ionization—mass spectrometry system (APCI-MS) (Hasegawa et al., 2000b).
They concluded that the flow rates up to 2 ml/min were compatible with the APCI -
LC-MS mode. Sawabe et al. (1999) successfully identified citrus limonoid glucosides
using positive and negative FABMS. Both modes showed consistent results for

establishing molecular weight of nomilinic acid 1 7-0-B—D-glucopyranoside, methyl

nomilinate 17-0—B-D-glucopyranoside, and obacunone 17-O-B-D-gluc0pyraside.

In the field of limonoid structural analyses, mass spectrometry has been
primarily used to obtain molecular weight information for their identification and
confirmation. These results are supplemental to other techniques such as

chromatographic retention, UV spectra, and NMR spectra.
Citrus flavonoids

Citrus flavonoid are more readily analyzed than limonoids, because the fact
that their molecules contain potent chromophores, which highly absorb UV light.
This UV absorption property allows higher specification for the flavonoid detection

and higher flexibility for the mobile phase selections.

Analyses of citrus flavonoids generally deal with flavanone glucosides and
polymethoxylated flavones as major compounds. Most flavanone glucosides are
soluble compounds, except for hesperidin, which dissolve in formamide, pyridine, or
dilute alkali (Rouseff, 1980), and narigin, which requires heating for complete
dissolution in alcohol solutions. Polymethoxylated flavones are nonpolar

compounds, which exist in much lower levels than the flavanoid glucosides.

 

 

 

 

 

 

 

 

Earlier

oernonhorornetrac

technioue was the '
amount oi narrngar
then the juice s

iietht'lene glycol.

inexpensite tltng

Because c
analttical techni
al.. 19971. Arr
tStremple. 1993
et al.. 1999. h
radioimmunoas
electrophoresis
199‘. Robards
fut'clens and
I-thantnospli
thamnostl-l-
tatinosides;
disaccharide

 

Earlier studies on flavonoid quantitative analyses involved
spectrophotometric methods (Coustou and Babin, 1957), paper chromatography
(Edward et al., 1957, Toshio and Shintaro, 1959) and thin layer chromatography
(Tatum et al., 1957, Swift, 1967). Among those analyses, the most widely used
technique was the “Davis test” introduced by Davis (1947). This method determines
amount of naringin in grapefruit juice and total flavonoid in orange juice at 420 run
when the juice sample is incorporated with 4 N sodium hydroxide and 90%
diethylene glycol. It has been known to be nonspecific but is simple, rapid and

inexpensive (Ting and Rouseff, 1986).

Because of the number and. diversity of flavonoids in citrus juice, the
analytical techniques have been developed based on chromatography(Robards et
al., 1997). Available methods for citrus flavonoid determinations include GC
(Stremple, 1998), HPLC-UV/PDA (Ooghe et al., 1994a, Robards et al., 1997, Kawaii
et al., 1999, Mouly et al., 1999), HPLC—Fluorescence (Robards et al., 1997),
radioimmunoassay (Jourdan et al., 1982, and Barthe et al., 1988), capillary
electrophoresis (Takei et al., 1998), GC-MS (Stremple, 1998), LC—MS (He et al.,
1997, Robards et al., 1997, Ishii etal., 2000), and LC-PDA-MS (Baldi et al., 1995,
Cuyckens and Claeys, 2002). The developed radioimmunoassay is very specific to
2-rhamnosyl-1-glucopyranose at the C-7 position but not with the isomeric 6-
rhamnosyl-l-glucopyranose moiety. It appeared that this method was limited to
rutinosides; however, it can be used to identify the stereochemistry of this
disaccharide moiety at the C-7 position of flavonoids (Jourdan et al., 1982).

Advanced techniques such as GC—MS, LC-MS, and LC-PDA-MS allow

 

 

 

 

 

 

smtltarieous ouant
notifications are be
70.5. is coupieot. 1%
tan ttidelp‘ at at};

analyses,

The most cc
detector. Th6 Hl
nontolatile comp
glucosides anti
tentatization or
obtain both chro
data tabsorbanct
tool compared it
one 01 “to tree.
hat'onoids. HPl

of tlaronoids i

., ,p ‘
3‘llllti‘s.

lit reco
mi fla‘Ottotd

\

it
t‘ ,

tam? 0tdcr

simultaneous quantitative determination and structural identification. The
identifications are dependent on retention time, mass spectra, and UV spectra (when
PDA is coupled). However, these sophisticated and expensive systems are still not
very widely available and are not used for commercial juice quality control

analyses.

The most commonly used method has been HPLC coupled with UV or PDA
detector. The HPLC is a flexible tool for analysis of both polar and nonpolar
nonvolatile compounds; therefore, it is readily adaptable to both flavanone
glucosides and polymethoxylated flavones without the need for additional
derivatization or heating. Photodiode Array Detector (PDA) has been used to
obtain both chromatographic (absorbance as a function of time) data and spectral
data (absorbance as a function of wavelength). This type of detector is a powerful
tool compared to standard UV—vis detector, which produces Chromatograms at only
one or two wavelengths. Since UV spectra are relatively specific markers of
flavonoids, HPLC coupled with PDA can provide effective systematic determination
of flavonoids for both routine quality control analyses and method-deveIOpment

studies.

In recognition of the complexity of the flavonoids in most extracts, reverse
phase HPLC with gradient elution has been the method of choice for separation of
the flavonoids in citrus fruits. Under these conditions, the elution profile for
flavonoids containing equivalent substitution patterns is flavanone glycosides,
flavonol glycosides, flavone glycosides and subsequently the free aglycones in the

same order. However, the separations between glycosides and aglycones are not

37

 

 

 

vv'n‘rTF \al K
dfl:uufiuh 9
HP'

each troutl mm

tosses oi llat‘ODUTC'E‘

inttial

1}. flat
at least too of 11155
:rysta‘: character: 5‘
teteloptnent after
spectra. dt U s;
contributed an it
because they male
of carbons. numb
hltlll spectra. 3
structures. and c

iat'onoid struct

llabrt‘ et al. 119
Mass sp
ionization gas

haltmethotsla

.351 atom l‘or‘
:ratonoids.

‘ht; i5, -
:uTttltalttl’tlt'

adequate for the resolution of a complex mixture containing many compounds of
each group. Therefore, it has been a common practice to separate the various

classes of flavonoids in a preliminary extraction step (Robards et al., 1997).

Initially, flavonoid structures were established primarily on a combination of
at least two of these techniques: a) physicochemical data (such as melting point or
crystal characteristics) (Nishiura et al., 1971), b) chemical reactions (such as color
development after addition of NH3, NH4Cl, FeC13) (Nishiura et al., 1971), c) IR
spectra, d) UV spectra, e) TLC and f) PC. Development of NMR techniques has
contributed an important improvement in structural elucidation of flavonoids,
because they make possible the complete structural assignments including numbers
of carbons, number of hydrogens, and their specific arrangement shown directly in
NMR spectra. NMR can be used for elucidation and/or confirmation of chemical
structures, and/or purity analysis of isolated compounds. A systematic review of the
flavonoid structural identification using UV and NMR spectra was written by

Mabry et al. (1970).

Mass spectrometric applications in citrus flavonoids are electron impact
ionization gas chromatography mass spectrometry (EI-GC-MS) for analysis of
polymethoxylated flavones (Rizzi and Boeing and Berahia et al., 1994, Stremple,
1998, Chen et al., 1998), liquid chromatography mass spectrometry (LC-MS) for
analysis of flavanone glucosides (Baldi et al., 1995, He et al., 1997), or direct probe
fast atom bombardment mass spectrometry (FABMS) for analysis of both major
flavonoids. Fewer studies were found utilizing the FABMS, because prior

purification of individual compounds has been a necessary step to obtain simpler

38

 

 

 

 

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mass spectral data required for molecular weight assignment. Tandem mass
spectrometry instruments (MS/MS) offer higher specificity of the analysis with
second order fragmentations than that obtained from singular MS. This system was
demonstrated to produce consistent ion fragments that could be used as compound
“finger prints”. Trace amounts of naringin and its metabolites in human urine were
analyzed by electrospray ionization mass spectrometry (ESI—MS), tandem mass
spectrometry—mass spectrometry (MS/MS), and tandem mass spectrometry-mass
spectrometry-mass spectrometry (MS/MS/MS) technique in an absorption study of
flavonoids (Ishii et al., 2000). The absorptions of both pure naringin and hesperidin
and those in grapefruit and orange juice were identified by positive chemical
ionization-collisionally activated dissociation tandem mass spectrometry (PCI-CAD
MS/MS). In addition to high compound specificity, these studies demonstrated that
the mass spectrometric conditions used were very sensitive for the trace levels of

targeted flavonoids.

39

 

 

 

 

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46

 

 

 

 

 

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Pill-001115. R C. 9 , _,
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glucoside content
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Rousefi. R. 1.. Martin. :
naringin. hespert
1027-1030

Rouseff. R. E. 1980. Eia
Eds. 8.. Nagy at
D. C. pp 84-108

Rouseff. R. l. and Don.

Rouseff. R. E. and E
limonoids in cit‘
Chem. 53: 1228

Rubeno. 6.. Renda. A
limonoids and
Spodoptera frat
Agric. Eood C l'

Satt'abe. A. Morita.
Matsubara ll
limonoid glycc
(1-31: 142-14"

Schnull. ll.. Nets ana
Eluess. 0bst. i

Schoch. T.. Manners
trom Cirrus
Spectrometry

Sent. 11.. lsltida. M

trom Citrus
Relbe. Agrtc

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useff, R. L. 1988. Chapter 3: Differentiating citrus juices using flavanone
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Eds. Nagy, S., Attaway, J. A., Rhodes, M. E. Dekker, New York, pp. 49-65

tuseff, R. L., Martin, S. F., Youtsey, C. O. 1987. Quantitative survey of narirutin,

naringin, hesperidin, and neohesperidin in Ctitrus. J. Agric. Food Chem. 35:
1027-1030

ruseff, R. L. 1980. Flavonoids and citrus quality. In: Citrus Nutrition and Quality.

Eds. S., Nagy and J. A. Attaway. American Chemical Society, Washington,
D. C. pp 84-108

ouseff, R. L. and Dougherty, M. 1979. Unpublished data.

ouseff, R. L. and Fisher, J. F. 1980. Determination of limonin and related

limonoids in citrus juices by high performance liquid chromatography. Anal.
Chem. 52: 1228—1233

.uberto, G., Renda, A., Tringali, C., Napoli, E. M., Simmonds, M. S. J. 2002. Citrus
limonoids and their semisynthetic derivatives as antifeedant agents against

Spodoptera frugiperda larvae. A structure- activity relationship study. J.
Agric. Food Chem. 50(23):6766-6774

awabe, A., Morita, M., Kiso, T., Kishine, H., Ohtsubo, Y., Minematsu, T.,
Matsubara Y., and Okamoto, T. 1999. Isolation and characterization of new

limonoid glycosides from Citrus unshiu peels. Carbohydrate Research. 3 15
(1-2): 142-147

chnull, H., New analytical methods for determining the authenticity of fruit juices.
Fluess. Obst. 57: 28-42

hoch, T., Manners, G. H., and Hasegawa, S. 2001. Analysis of limonoid glucosides
from Citrus by electrospray ionization liquid chromatography-mass
spectrometry. J. Agric. Food Chem. 49(3): 1102-1108

rit, M., Ishida, M., Kim, M., Yamamoto, T., and Takahasi, S. 1991. Antifeedants

from Citrus natsudaidai Hayata against termite Reticulitermes speratus
Kelbe. Agric. Biol. Chem. 55(9): 2381-2385

temple, P. 1998. GC/MS analysis of polymethoxylated flavones in citrus oil. J.
High Resol. Chromatogr. 21 (11): 587-591

 

 

 

 

 

iot'ftl H. H90 TUTD
oriu’ttg6 PC" 1

Tales. 11.. Dhsone. M".
tlatanone glycoszc
using capillary ele
of the lapan Socre

lanalal- Makita. ll ..
A. Sumida. T.
azoxymethane-inc
ilatonoidsd rosin

Tanala. T .. Malcita. El ..
A. Sumida. T.. E
of X-methyl-X-a:
dietary ieedin g c
Carcinogenesis. '

Tatum] ...ll Ream. C

Tatum..1 1.11 and Berry
juices] EoodS

Tatum. l.li.. Berry. R.
separation of n
Proceedings of t

lion. 0.. Miller. E. G.
of human bre;
Cancer. «10131

ting. 8. V. 1980. Nut
Quality. Eds.
Washington. D.

hog. S. ‘1'. arid Rou

Citrus and Th
. Rouseff. M.

l\
l:thio \ and Shirt

\ellou ttt aria -

mdllClS 8(t 1

 

wift, L. J. 1967. TLC—spectrophotometric analysis for neutral fraction flavones in
orange peeljuice. J. Agric. Food Chem. 15(1): 99-101

'akei, H., Ohsone, M., Okamura, Y., and Yoshizaki, F. 1998. Separation of
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Tanaka, T., Makita, H., Kawabata, K., Mori, H., Kakumoto, M., Satoh, K., Hara,
A., Sumida, T., Tanaka, T., and Ogawa, H. 1997a. Chemoprevention of
azoxymethane-induced rat colon carcinogenesis by the naturally occurring
flavonoids, diosmin and hesperidin. Carcinogenesis. 18: 957-965

Canaka,T., Makita, H., Kawabata, K., Mori, H., Kakumoto, M., Satoh, K., Hara,
A., Sumida, T., Fukutani, K., Tanaka, T., and Ogawa, H.1997b. Modulation
of N-methyl-N-amylnitrosamine-induced rat oesophageal tumourigenesis by
dietary feeding of diosmin and hesperidin, both alone and in combination.
Carcinogenesis. 18: 761-769

Tatum, J. H., Hearn, C. J., and Berry, R. E. 1978. J. Am. Hort. Sci. 103: 492

Tatum, J. H. and Berry, R. E. 1973. Method for estimating limonin content of citrus
juices. J. Food Sci. 38: 1244-1246

Tatum, J.H., Berry, R. E. Hearn, J. C. 1975. Characterization of citrus cultivars and
separation of nucellar and zygotic seedlings by thin-layer chromatography.
Proceedings of the Florida State Horticultural Society. 87: 75-81

 

Tian, Q., Miller, E. G., Ahmad, H. Tang, L., Patil, B. S. 2001. Differential inhibition
of human breast cancer cell proliferation by citrus limonoids. Nutrition

Cancer. 40(2): 180-184

Ting, S. V. 1980. Nutrients and nutrition of citrus fruits. In: Citrus Nutrition and
Quality. Eds. S., Nagy and J. A. Attaway. American Chemical Society,

Washington, D. C. pp 84-108

Ting, S. V. and Rouseff, R. L. 1986. Chemical constituents affecting quality. In:
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L. Rouseff. Marcel Dekker, Inc., New York. pp.109

oshio, N. and Shintaro, K. 1959. Citrus flavonoids II. Substances which tumed
yellow in an alkaline condition in mandarin orange sirup. J. Utilization Agr.
Products. 6: 149-155

 

 

t'eldhuis. M. 14.. Strait.
Elorida orange .1 or

tenants. 8. D .. 8 risilarr.
from plants. 1. Mt

llade. R. l. 1993. Ken
adulteration. Flue

11all.M.lE.. Ryan. M .C
Walker. 1.. Meg
lNatProd. 5111'

l‘.8.D.A. D161. Agricu

CSDA. 2002. Elayon
phenolics conic,

lamali. 8. 108-1. Plant

 

 

Veldhuis, M. K., Swift, L. J ., Scott, W. C. 1970. Fully-methoxylated flavones in
Florida orangejuices. J. Agric. Food Chem. 18: 590-592

Venkata, S. D., Srisilam, K., and Veeresham, c.2002. Natural sweetening agents
from plants. J. Medicinal and Aromatic Plant Sciences. 24(2): 468-477

Wade, R. L. 1992. New analytical methods in the U. S. for detecting fruit juice
adulteration. Fluess. Obst. 59: 62-72

Wall, M.E., Wan, M.C., Manikumar, G., Graham, P.A., Taylor, H., Hughs, T.J.,
Walker, J ., Mcgivney, R.V. 1988. Plant antimutagenic agents: Flavonoids.
J.Nat.Prod. 51:1084-1091

U. S. DA. 1962. Agricultural handbook. 98: 44

USDA. 2002. Flavonoid composition of citrus http://www.ars.usda.gov/is/np/
phenolics /comp.htm.

Yamaki, S. 1984. Plant Cell Physiol. 25: 151

49

 

 

 

Study 1: Analytical math
I and flasonoids I

pan128cre€nifl§ for ma
1, Abstract
Modified methan
Presscalc. 1385- Pefil F"
encoded gradient systft
ending nith 600 o acetor
no on (limonoid det:
polymethoxylated flay
15.3161 obtained allott-
their relatiye retenti ons
Compounds \yi'
obacunone (limonoid
nomilinic acid gluct
nobiletin. 3.4.5.6.“.5
tangeretin (polymetlt
lllal'anone glucosides
Results from
“115 tenses of polar
“ith similar chromat

l
.. lntrodltction

The l‘EES'cnc

among ll .
x 3‘ 1 a
i “I“ gone

I

 

Study 1: Analytical methodology suitable for isolation and quantitation of limonoids
and flavonoids in sweet orange

Part I: Screening for major limonoids and flavonoids
1. Abstract

Modified methanol extracts from orange fraction including: seeds, peels, peel
press c ake, rags, p eel p ress liquid and 0 range juice w ere analyzed b y H PLC u sing an
extended gradient system, starting with 10% acetonitrile in 3 mM phosphoric acid and
ending with 60% acetonitrile in 115 minutes. UV-visible absorbance was measured at
210 nm (limonoid detection), 280 nm (flavanone glucoside detection), and 340 nm
(polymethoxylated flavone detection). Chromatographic separation (Rs = 0.9/N =
95,216) obtained allowed screening of major limonoids and flavonoids and estimation of
their relative retentions.

Compounds within a detectable level were limonin, deacetylnomilin, nomilin, and
obacunone (limonoid algycones); limonin glucoside, deacetylnomilinic acid glucoside,
nomilinic acid glucoside, obacunone glucoside (limonoid glucosides); sinensitin,
nobiletin, 3,4,5,6,7,8,3’,4’-heptamethoxyflavone, scutellarein tetramethylether, and
tangeretin (polymethoxylated flavones); eriocitrin, narirutin, hesperidin, and didymin
(flavanone glucosides).

Results from this study suggested that to analyze various compounds possessing
wide ranges of polarity, an improved HPLC system was required to separate compounds
with similar chromatographic retentions.

2. Introduction
The presence of citrus limonoids and flavonoids has been known to be diverse

among tissues, genetically specific among species, and quantitatively varied ranging from

 

 

mm w W was. QI
do“, My with 1115
W115, flavonoids 1“
mmdology and 00mm“
Our research has
flavonoids that could b
inportant to condimt a 5:
amount present in divr
chromatographic retent
objectives are as follows
1) To select 1
subsequent c

2) To obtain r
orange, and

3) To select ta

3. Materials and me

3.1 Orange san

Orange sampli

take 4) I383, 5) Peel
Products Company (B

A single siren

stored a tefrigualed

M (Vmoem Co:

 

 

 

ppm to percent units. Quantitative analyses on limonoids in sweet orange have been
done primarily with the major compounds (limonin and nomilin). Compared to
limonoids, flavonoids have been analyzed more extensively due primarily to the
methodology and commercial standards available for their determination.

Our research has attempted to quantify the broad spectrum of limonoids and
flavonoids that could be consistently detected in sweet orange. Therefore, it was
important to conduct a screening study to select these compounds based on their relative
amount present in diverse tissues of sweet orange and to estimate their relative
chromatographic retention required for effective method optimization. Specific

objectives are as follows:
1) To select limonoids and flavonoids present in sweet orange for the

subsequent quantitative study,

 

 

2) To obtain relative retention of limonoids and flavonoids present in sweet

orange, and
3) To select raw materials for isolation of unknown limonoids and flavonoids.

1. Materials and methods

3.1 Orange samples

Orange samples of Valencia variety including: 1) seeds, 2) peels, 3) peel press
:e, 4) rags, 5) peel press liquid, and 6) orange juice were obtained from Tropicana
ducts Company (Bradenton, FL). Descriptions of samples are as follows:

A single strength orange juice was pasteurized (95°C/2 sec), vacuum-sealed, and

:l at refrigerated temperature. Peel press liquid was prepared using a Vincent screw

9 (Vincent Corporation, Tampa, FL). The pulp resulting from the pressing process

51

tanned “are“ wk“ “

mum”, Peel press W‘

mm W6 (1
Rags (containing

from (-20°C). The 5am}

Food Science and Human

32 Sample prepai

Upon arrival, sari

week before analyses. 1‘

completely thawed '1

combined and mixed fl:

and then stored at —20°(

Samples of rags

pass 1 mm screen using
20°C until analyzed

immature to remove

glass vials as described

3.3 Studied cor

Studied compo

dwosidw, and Polym

USDA, Dr. Gary D. i

John A. Maxim (W1
limonin glucoside (

 

 

 

 

 

is termed “press cake”. The liquid squeezed from pulp is termed “press liquid” or “press
liquor”. Peel press liquid was pasteurized (95°C/2 sec), vacuum—sealed and stored at
refrigerated temperature (2i1°C).

Rags (containing seeds), peels, and peel press cake were vacuum-sealed and
frozen (-20°C). The samples were shipped in Styrofoam containers to the Department of
Food Science and Human Nutrition, Michigan State University, E. Lansing, MI.

3.2 Sample preparation

Upon arrival, samples were immediately stored at «20°C for approximately one
week before analyses. Juice samples were held at refrigerated temperature (211°C) until
completely thawed. To ensure homogeneity, all containers of each sample were
combined and mixed thoroughly, sub-sampled, and collected into 100 ml glass bottles,
and then stored at —20°C until analyzed.

Samples of rags, peels, and peel press cake were freeze-dried, and then ground to
pass 1 mm screen using a UDY-Mill (Chicago, IL). The ground samples were stored at —
20°C until analyzed. Seeds were extracted twice with hexane (1 :4, W/V) at room
temperature to r emove orange oil b efore b eing milled with a U DY-Mill and stored in
glass vials as described above.

3.3 Studied compounds and standards

Studied compounds included limonoid glucosides, limonoid aglycones, flavanoid
glucosides, and polymethoxylated flavones. Standards, kindly donated by scientists from
USDA, Dr. Gary D. Manners (Pasadena, CA), Dr. Mark A. Berhow (Peoria, IL), and Dr.

John A. Manthey (Winter Haven, FL), included deacetylnomilin (DNM), obacunone (O),

limonin glucoside (LG), deacetylnomilinic acid glucoside (DNAG), nomilinic acid

52

 

 

 

W (NAG), W
(10A), dwxylinmi”
Mme acid (I
mum (NET), 3.4.5.6."J
Limonin (L), no
lNllDlihwpefifin (HT). .
from Siam Comrmy ('
(WE), nan'Iufin (NT
Extrasynthese, (Genay, l
3.4 Extraction
Gmlmd. freeze—
extracted twice with it
supernatants were ooml
40°C to 2-3 ml under \
(1000 mg), which wer
pair was washed with
Elnzte was evapomt
acetonitrile in 3mM
(10,000X g1 10 minuti
acetonitrile “ouch

leoninilein]

 

l__—__—’

glucoside (NAG), obacunone glucoside (0G), obacunoic acid. (0A), isoobacunoic acid
(IOA), deoxylimonin (DL), 17-19-didehydrolimonoic acid (DDHLA), l9-
dehydrolimonoic acid (DHLA), limolinic acid (LA), mtaevin (R), sinensetin (ST),
nobiletin (NBT), 3,4,5,6,7,8,3’,4’-heptamethoxyflavone (HP), and tangeretin (TT).
Limonin (L), nomilin (NM) hesperidin (HD), naringin (NG), neohesperidin
(NHD), hesperitin (HT), diosgenin (DN), coumarin (CM), quercetin (QT) were purchased
from Sigma Company (St. Louis, MO). Sinensetin (ST), scutellarein tetramethylether
(STME), narirutin (NT), didymin (DD), and eriocitrin (ERT) were purchased from
Extrasynthese, (Genay, France).
3.4 Extraction
Ground, freeze-dried orange parts (peel, peel press cake, and rag) (l g) was
extracted twice with 10 ml 70% methanol, and once with 10 ml 100% methanol; the
supematants were combined, and methanol was evaporated in the round bottom flasks at
40°C to 2—3 ml under vacuum. The evaporated extract was passed through C18 Sep-pak
(1000 mg), which were preconditioned with 3 ml methanol and 10 ml water. The Sep-
pak was washed with 10 ml water and the compounds were eluted with 10 ml methanol.
Eluate was evaporated at 50°C under vacuum and reconstituted with 2 ml 10%
acetronitrile in 3mM phosphoric acid (initial mobile phase). Additional c entrifugation
(10,000X g/ 10 minutes) was done due to remaining residues, which was dissolved in lml
acetronitrile. Therefore, there were two fractions analyzed a) fraction dissolved in 2 ml

10% acetronitrile in 3mM phosphoric acid, and b) fraction dissolved in 1 ml acetronitrile.

53

 

 

Juice and p061 1’m
mimic ml) was 1'
mm Me was I

3.5 High perfomfl
HPIJC system 00!
Pump Control Module), t
and Water 996 Photodior
400 nm and recorded 1
detection) and 340 nm 1
(Waters Company) was r
Mobile phase co
and acetonitrile (solver:
in 250mm x 4 .6m,
2763) with gradient run
BatllS minutes. Flov
Identification r
obnomone). limonoid
nomilinic acid glueos
mouth. hesperidin I
hcptarnerhoxyflavone.

mdmfirneUVsp

 

 

Juice and peel press liquid were thawed at room temperature. The juice or peel
press liquid (10 ml) was mixed with 23 ml methanol to obtain 70% methanol. The rest of
extraction procedure was the same as solid fractions.

3.5 High performance liquid chromatography (HPLC) analysis

HPLC system consisted of two pumps model Waters 510 (controlled by Waters
Pump Control Module), equipped with an injection system (Water 717 plus autosampler),
and Water 996 Photodiode Array Detector (PDA). Absorption was measured from 200-
400 nm and recorded at 210nm (limonoid detection), 280 nm (flavonoid glucoside
detection) and 340 nm (polymethoxylated flavone detection). Millennium 32 software
(Waters Company) was used. for data acquisition and processing.

Mobile phase consisted of 10% acetonitrile in 3 mM phosphoric acid (solvent A)
and acetonitrile (solvent B). Separation is achieved on C18 column (Alltima, Alltech:
5p, 2 50mm x 4.6mm, 16 % c arbon load, void time 2 .02 minutes, p acking lot number
2763) with gradient run starting with 0% B to 20% B in 20 minutes, and ending with 60%
B at 115 minutes. Flow rate was 1 ml/minute. Injection volume was 10 ul.

Identification of limonoid aglycones (limonin, deacetylnomilin, nomilin, and
obacunone), limonoid glucosides (limonin glucoside, deacetylnomilinic acid glucoside,
nomilinic acid glucoside, and obacunone glucoside), flavanone glucosides (eriocitrin,
narirutin, hesperidin, and didymin) and polymethoxylated flavones (sinensitin, nobiletin,
heptamethoxyflavone, scutellarein tetramethylether, and tangeretin) were based on

retention time, UV spectra and response factors of external standards.

54

 

4. W and W
Selection5 Of the ‘
mm mm high
pajamas, while seed l
selection of flavonoids (
seedwas used for the sell
Figures 11 to Fig
with10% acetonitrile in
with 100% acetonitrile)
respectively. Chromam
and juice are presente
Retention time: of th
(limonin glucoside), .
(hesperidin), 64.6 (n
(didymin), 98.3 (dean!
109.5 (3,4,5,6,7,8,3
(nomilin), 115.4 (tan!
coclution of lim
hqitamethoxyflavonc
The results sl

the similar region. a!

the enumamgraphi'

 

 

 

 

4. Results and discussion

Selections of the compounds (based on peak size) were determined from orange
fractions containing highest content of limonoids and flavonoids. Peel had the highest
flavonoids, while seed had the highest limonoids. Therefore, peel was used for the
selection of flavonoids (flavanone glucosides and polymethoxylated flavones), whereas
seed was used for the selection of limonoids.

Figures 11 to Figure 14 show Chromatograms of polar compounds (reconstituted
with 10% acetonitrile in 3mM phosphoric acid) and nonpolar compounds (reconstituted
with 100% acetonitrile) from Valencia peel and seed extracts at 210, 280, and 340 nm,
respectively. Chromatograms of other orange parts including: rag, press cake, peel juice,
and juice are presented in Appendix 1. Selected compounds are the labeled peaks.
Retention times of the selected compounds were as follows: 40.7 (eriocitrin), 42.0
(limonin glucoside), 48.0 (deacetylnomilinic acid glucoside), 53.0 (narirutin), 57.8
(hesperidin), 64.6 (nomilinic acid glucoside), 67.6 (obacunone glucoside), 74.2
(didymin), 98.3 (deacetylnomilin), 100.6 (sinensitin), 102.6 (limonin), 106.6 (nobiletin),
109.5 (3,4,5,6,7,8,3’,4’-heptamethoxyflavone/scutellarein tetramethylether), 109.7
(nomilin), 115.4 (tangeretin), and 117.2 (obacunone) minutes, respectively. There were
coelution of limonin/unknown at 102.6 minutes; and 3,4,5,6,7,8,3’,4’-
heptamethoxyflavone/scutellareintetramethylether at 109.5 minutes.

The results showed that flavonoid glucosides and limonoid glucosides eluted in
the similar region, and the same as limonoids and polymethoxylated flavones. Based on

the chromatographic retention alone, flavonoid glucosides could be simultaneously

55

 

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ended m a comparabk ‘
Based on them
ax'adable limonoid star.
Identified 2E potemiadij‘
Figure 16 shows L'\' s;
B256 on chrom
31.1997 and Sendra 6‘1
be narirutin-41 glucogj
W spectrum
355-73143hexamefl
Spemm of unknom

19').

3. Conclusion

The HPLC
mentions and selec‘
analyses. since 1h
Chromatographic rel

mhhmonoid Elm-cc

n.
dbmdhoxylaled 1‘

 

 

analyzed with limonoid glucosides; and limonoid aglycones could be simultaneously
analyzed with polymethoxylated flavones.

There were unknown peaks at 33.1, 53.8, 58.6, 62.7, and 103.2 minutes that
existed in a comparable range with other identifiable compounds.

Based on chromatographic retention (F ong et al., 1993), and UV spectra of other
available limonoid standards (Figure 15), unknown peaks at 58.6 and 62.7 min were
identified as potentially deacetylnomilin glucoside and nomilin glucoside, respectively.
Figure 16 shows UV spectra of unknown peaks at 58.6 and 62.7 minutes.

Base 0 n c hromatographic retention (Manthey and Grohmann, l 996, Robards et
al., 1997 and Sendra et al., 1988), unknown at 33.1 and 103.2 minutes had the potential to
be narirutin-4’—glucoside and 3,5,6,7,3’,4’-hexamethoxyflavone, respectively.

UV spectrum of unknown at 103.2 minutes (Figure 17) was similar to that of
3,5,6,7,3’,4’-hexamethoxyf1avone presented in Figure 18 (Sendra et al., 1988), while UV
spectrum of unknown at 33.1 minutes was similar to that of narirutin standard (Figure
19).

5. Conclusion

The HPLC gradient mobile phase used was suitable for estimation of relative
retentions and selection of compounds (based on peak height), but not for quantitative
analyses, since the achieved separation was relatively low. Based on the
chromatographic retention alone, flavonoid glucosides could be simultaneously analyzed

with limonoid glucosides; and limonoid aglycones could be simultaneously analyzed with

1301ymethoxylated flavones.

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6. References:

Pom; C. H. Hasegaxx 'a.
1993. hmonm
gromh and dex E

Hasegawa. 8.; Benneu.
and relalix'e con

Kawaii. 8.: Tomono, ‘
flaVonoid consr

Nhyazaxx'a M. Okur:
Antimutagemc
Agric. Food C}

Manthey. J. A. and G
peel flax'onoid
814

Mouly. P. P. Gaydox
orange juice:
Analysis. 37:

Mouly. P. P.; Arzow
of citrus juim
flmnone 91L

00gb; W. C; O:
Charamen'za‘
J-Agficfoc

OPEN WC .1 O.
Charactem:
Agfic. Food

0231;] \ AVanO

leonoid
Mahdann \W

RONNIS‘ K L] \

Chrommog

6. References:

Fong, C. H., Hasegawa, S., Miyake, M., Ozaki, Y., Coggins, Jr. C. W., and Atkin, D. R.
1993. Limonoids and their glucosides in Valencia orange seeds during fruit
growth and development. J. Agric. Food Chem. 41: 112-115

Hasegawa, S.; Bennett, R. D., and Verdon, C. P. 1980. Limonoids in citrus seeds: origin
and relative concentration. J. Agric. Food Chem. 28: 922-925

Kawaii, S.; Tomono, Y.; Katase, E.; Ogawa, K.; and Yano, M. 1999. Quantitation of
flavonoid constituents in Citrus Fruits. J. Agric. Food Chem. 47: 3565-3571

Miyazawa, M., Okuno, Y., Fukuyama, M., Nakamura, S., and Kosaka, H. 1999.
Antimutagenic activity of polymethoxyflavonoids from Citrus aurantz'um. J.
Agric. Food Chem. 47(12): 5239-5244

Manthey, J. A. and Grohamnn, K. 1996. Concentrations of hesperidin and other orange
peel flavonoids in citrus processing byproducts. J. Agric. Food Chem. 44: 811-
814

Mouly, P. P. Gaydou, E. M.; and Arzouyan, C. 1999. Separation and quantitation of
orange juices using liquid chromatography of polymethoxylated flavones.
Analysis. 27: 284-288

Mouly, P. P.; Arzouyan, C. R.; Gaydou, E. M.; and Estienne, J. M. 1994. Differentiation
of citrus juices by factorial discriminant analysis using liquid chromatography of
flavanone glucosides. J. Agric. Food Chem. 42: 70-79

Ooghe, W. C.; Ooghe, S. J .; Detavemier, C. M.; and Huyghebaert, A. 1994a.
Characterization of orange juice (Citrus sinensis) by polymethoxylated flavones.
J. Agric. Food Chem. 42: 2191—2195

Ooghe, W.C.; Ooghe, S.J.; Detavernier, C.M.; and Huyghebaert, A. 1994b.
Characterization of orange juice (Citrus sinensis) by flavanone glyc031des. J.
Agric. Food Chem. 42: 2183-2190

Ozaki, Y.; Ayano, S.; Inaba, N.; Miyake, M.; Berhow, M.A.; and Hasegawa, S. 1995.
Limonoid glucosides in fruit, juice and processing by-products of Satsuma
Mandarin (Citrus unshiu Marcov.)

Robards, K.; Li, X., Antolovich, M.; and Boyd, S. 1997. Characterization of citrus by
chromatographic analysis of flavonoids. J. Sci. Food Agnc. 75: 87-101

66

 

 

Panll: Optimization 01
1. Abstract
Extraction E

iglytcones'polymEmO-‘iti
mic optimized usrng c?
‘ngh temperature (nit
technique. The obtai:
acceptable recoyery t g
gradient systems. exc
tsocratic system. Sep
ll N = 33.538 \t'hicl

l. lntroductlon

Even though

are relatiyely exten
halved Separately
Oranges nag repon,

tt‘ell established an
3199] and Pong

and comparisons .

the Wt content
Analyses
done commonly

W DPSO ct

 

Part II: Optimization of analytical methods
1. Abstract
Extraction and chromatographic conditions for limonoid
aglycones/polymethoxylated flavones, limonoid glucosides, and flavanone glucosides
were optimized using extracts of seeds, peels, and peel press liquid. Solvent extraction at
high temperature (with additional steps for limonoid aglycones) was the primary
technique. The obtained extraction techniques were rapid, inexpensive, and allowed
acceptable recovery (greater than 90%). Reverse phase HPLC conditions were mainly
gradient systems, except for analyses of seed limonoid aglycones which employed an
isocratic system. Separations obtained were in the range of Rs = 0.6/N = 13,079 to Rs
l.l/N = 23,588 which was acceptable for these complex matrices.
2. Introduction
Even though quantitations of citrus limonoids and flavonoids in previous studies
are relatively extensive, these two different citrus major phytochemicals have been
analyzed separately. Distribution of limonoid aglycones and glucosides in Valencia
oranges was reported by Fong et al. (1993). The analytical method used in this study is
well established and have been used in numerous studies in their laboratory (Hasegawa et
al., 1991 and Fong et al., 1992). However, in this study the fresh samples were analyzed
and comparisons were based on the amount of individual limonoids per fruit, in which
the water content of these tissues can be greatly varied.
Analyses of flavanone glucosides and polymethoxylated flavones have been
done commonly in juice (Ooghe and Detavernier, 1999) and peel oil (Gaydou et al.,

1987, Dugo et al., 1996, Chen et al., 1997, and Stremple, 1.998). Extraction of

67

 

 

 

compounds from these 2

peel. seed and ragt beta

salient used in flayon'

howemlec'

used for detection of in

The objectiyes

mmhemrnpesoisz

'ene'ftts and dran'bacl
uhhns

lt lo obtain

ll To obtain

sunmnel

3. Materials andr

3i Orange 5

Ground. tie

it solid fractions

31Bmm

3.2.l Extr;

“Pam.

liquids “ET? that

m ”Pied to ro

Pl5httnstu

 

compounds from these liquid samples is much simpler than from solid samples (such as
peel, seed, and rag) because the compounds present are most readily extractable. Primary
solvent used in flavonoid extraction were dimethylsulfoxide and dimethylformamide.
They have high-UV cutoff, therefore producing large solvent front under wavelength
used for detection of limonoids.

The objectives of this study were to optimized extractions and HPLC conditions
suitable for types of samples and natures of compounds studied and thus incorporated the
benefits and drawbacks of previous studies in the analytical methods. Specific objectives
as follows:

1) To obtain a rapid extraction method with high selectivity and recovery,

2) To obtain a rapid chromatographic condition that allows separation resolution

suitable for quantitative determination.

3. Materials and methods

3.1 Orange samples

Ground, freeze—dried orange peel and seed were selected for method optimization
for solid fractions, while peel press liquid was selected for that for liquid fractions.

3.2 Extraction and analysis of limonoid aglycones and polymethoxylated flavones

3.2.1 Extraction

The extraction procedure of Fong et al. (1993) was modified from. Peel press
liquids were thawed at room temperature and heated in a water bath (82°C for 30 min),
then cooled to room temperature. Ten ml of peel press liquid was then mixed with 25 m1

of 0.5 M Tris buffer (pH 8) for 15 minutes and then acidified to pH 2 with 1 N HCl.

68

 

 

 

Ground. freeze—é
in: 15 minutes and their
tree mixed with 35 T7?-
acidified 10 pH 2 \t'iti. '.
seednere heated in a ti

Ethyl acetate 12
has added to all sar
‘ecanted. Ethyl aceta
combined eyaporatec
extract (0.4Su nylonl

lncorporation
modified front 111 etli
extraction \then he.
conditions but no hg
andpolymethoxylat
32.: H)’dl‘0
let mm o
Extracted by the in
Control “35 lllc‘ .
ililll‘een Spiked . .
at

almonds 1l.\‘drol\-

 

Ground, freeze-dried peel (l g) was mixed with 25 m1 of 0.5 M Tris buffer (pH 8)
for 15 minutes and then acidified to pH 2 with 1 N HCl. Ground, freeze-dried seed (1 g)
was mixed with 25 ml of 0.15 M Tris buffer (pH 8) overnight (20 hours), and then
acidified to pH 2 with l N HCl. The acidified mixtures of peel, peel press cake, rag and

seed were heated in a water bath (82°C for 30 min).

Ethyl acetate (25 ml) containing 200 ppm butylated hydroxytoluene (antioxidant)
was added to all samples, shaken for 15 minutes, and the ethyl acetate layer was
decanted. Ethyl acetate extraction was performed twice. The ethyl acetate layers were
combined, evaporated to dryness, and reconstituted to 10 ml with methanol. Filtered

extract (0.45u nylon) was analyzed by HPLC.

Incorporation of heat (82°C for 30 min) to limonoid aglycone extraction was
modified from m ethod of M clntosh and M ansell (1997). T o evaluate the recovery 0 f
extraction when heat was incorporated, a set of control was subjected to the same

conditions but no heating applied. Figure 20 shows a flow diagram of limonoid aglycone

and polymethoxylated flavone extraction.

3.2.2 Hydrolysis of limonoid glucosides

Ten ppm (mg/L) of limonin glucoside in 25 ml 0.15 M Tris buffer pH 8 was
extracted by the method described above when heat (82°C for 30 min) was incorporated.
Control was the buffer without limonin glucoside. Comparison of limonin content

between spiked and control samples indicated whether there was occurrence of limonin

glucoside hydrolysis.

69

 

 

 

 

Orange tissues
iltags. peels. and peel p1

la

i
ill
gm :0 HO“ (

 

 

Orange tissues Orange liquid samples Orange seeds
(Rags, peels, and peel press cake) (Juice and peel press liquid) 1g
1 g 10 ml
I
Heat at 82°C, 30 minutes
I
Sampled 10 ml
l

 

 

Mixed with 0.5M Tris pH8 Mixed with 0.15M Tris pH8
25ml, 15 minutes 25ml, 20 hours

|

Acidified with 1 N HCl
(pH 2.5)
Heat at 823C, 30 minutes
Extracted with dthyl acetate 25ml
Centrifuged at l10,000X g, 10 min
Extracted twice wilth ethyl acetate 25 ml
Combineld extracts
Evaporatiad at 50°C
Reconstituted to 1'0 ml with methanol
Filtered with 0|.45u nylon filter

|
Analyzed by HPLC

Figure 20: Flow diagram of limonoid algycone and polymethoxylatedflavone extraction.

7O

 

 

 

3.2.3 Rec-diff.“
Rec-0W1"! 01, m
the extraction With heat
limonin (13.5 WW a
man by compete
from spiked and contrr
3.2.4 HPLC

The mobile
acetonitrile lsolyent
ananiagad
ymSWoBatfilm
cdnnnlluna:(TlS.
mMMywmeo
polymethoxylated f
Since seeds

limonoid aglycone
limonoid aglycone
m°tmmm L
nmmmhmmr

has 10 ill.

ldennficar

”Width. and Obs

01 crtemal Stand

 

 

3.2.3 Recovery

Recovery of limonoid aglycones and polymethoxylated flavones obtained from
the extraction with heating (82°C for 30 min) was performed by spiking the samples with
limonin (12.5 ppm) and scutellarein tetramethylether (2.5 ppm). The recovery was
obtained by comparison of limonin and scutellarein tetramethylether content extracted
from spiked and control samples.

3.2.4 HPLC

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid aglycones and polymethoxylated flavones were
resolved with a gradient that started with 30% B, was 40% B in 20 minutes and ended
with 50% B at 50 minutes. Flow rate was lml/min. Separation was achieved on a C18
column (Luna: C18, Spa, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml).
Injection volume was 10 ul. Limonoid aglycones were detected at 210 nm, while
polymethoxylated flavones were detected at 340 nm.

Since seeds are rich in limonoid aglycones and low in polymethoxylated flavones,
limonoid aglycone analysis was carried out separately for seed extract. Separation of
limonoid aglycones was achieved on C18 column (Alltima: C18, Su, 250 mm x 4.6 mm,
16 % carbon load, void time 2.02 minutes) and an isocratic mobile phase
(acetonitrile/methanol/water, 10:41 :49). Flow rate was lml/minute and injection volume
was 10 ul.

Identification and quantitation of limonoid aglycones (limonin, deacetylnomilin,
nomilin, and obacunone), were based on retention time, UV spectra and response factors

of external standards. Identification of polymethoxylated flavones (sinensitin, nobiletin,

71

 

 

 

3.45.6283?Meme“
one based on retentrc
quantitations01PM“:if
for scutellarein tetrarrre
Effect of heat
hettyeen limonin cont
Analyses were condu
3.3 Extractior

3.3.1 Extractt
Ground. free

minutes. and heated
(10.00th g for 10 '
again with 70% m e‘

ml at 40°C under
10.4511 nylonl oer:-
Solyent ex

(tins bl Tris bui
ltoom. amp and

ill“ diagram of]
3.321139.
Refinery

hemmed 1“. SP

 

 

3,4,5,6,7,8,3’,4’-heptamethoxyflavone, scutellarein tetramethylether, and tangeretin)
were based on retention time and UV spectra obtained with external standards. The
quantitations of polymethoxylated flavones were based on the response factor determined
for scutellarein tetramethylether.

3.2.5 Data analysis

Effect of heat treatment was determined by significant difference (P S 0.05)
between limonin content extracted with and without heating using paired t test (Excel).
Analyses were conducted in triplicate.

3.3 Extraction and analysis of limonoid glucosides

3.3.1 Extraction

Ground, freeze-dried seed (1 g) were mixed with 25 ml. of 70% methanol for 15
minutes, and heated in a water bath (82°C for 5 min). The samples were centrifuged
(10,000X g for 10 min), and the supematants were decanted. The pellet was extracted
again with 70% methanol. Combined supematants were evaporated to approximately 2-3
ml at 40°C under vacuum, and reconstituted with 10 ml methanol. Filtered extracts
(0.45 p nylon) were analyzed by HPLC.

Solvent extraction conditions using 70% methanol was studied at two pH levels
(0.05 M Tris buffer pH 7.83 and purified water pH 4.4), three heating temperatures
(room, 60°C, and 82°C), and two heating times (5 and 15 minutes). Figure 21 shows a
flow diagram of limonoid glucoside extraction.

3.3.2 Recovery

Recovery of limonoid glucosides obtained from adjusted extraction was

performed by spiking the sample with limonin glucoside (10 ppm), while the control was

72

 

 

 

Orange 5:
(Rags. peels. pet;

I l
0

Mixed with 2:

1;...1.
r 1.].

Heat at 82°C

bone 21: Plots .

 

 

Orange solid fractions Orange liquid fractions

(Rags, peels, peel press cake, and seeds) (Juice and peel press liquid)
1g 10 m1
1 |
Mixed with 25 ml 70% methanol Heat at 82°C, 5 minutes
15 minutes

I Sample 10 m1
1

Heat at 82°C, 5 minutes Mixed with 23 m1 methanol
’ 15 minutes

Centrifuged at 10,000X g, 10 minutes
I

Transferred supernatant to round bottom flask
|
Mixed with 25 ml 70% methanol, 15 minutes
I
Centrifuged at 10,000X g, 10 minutes

Combined supernatant in round bottom flask

Evaporated to minimal amount at 5 0°C

Redissovled in 10 ml methanol

1
Filtered with 0.45 p nylon filter
I
Analyzed by HPLC

Figure 21: Flow diagram of limonoid glucoside extraction.

73

 

 

 

added with the W
comparison of limonin
3.3.3 High perit
The mobile p
aetonitrile tsolyent B
ttith 11.10013 and endtr
column tluna: C18. :
ttithl mlmin tlon r2
atllll nrn.
ldentiftcation
deacetylnomilinic ac
were based on reten
Standards.
3.3.4 Data ar
Different es
limonoid glucoside:
tonditionl (Excel).
3.4 Extraeti
3.4.1 Extrac
Ground. 1"“
dimly linclndino
ll sodium pm.

i ~ ‘
tttcthyltonnamn

 

 

 

 

added with the same amount of blank methanol. The recovery was obtained by
comparison of limonin glucoside contents extracted from spiked, and control samples.

3.3.3 High performance liquid chromatography (HPLC) analysis

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid glucosides were separated with linear gradient starting
with 10% B and ending with 26% B in 70 minutes. Separation was performed on C18
column (Luna: C18, Spa, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml)
with 1 ml/min flow rate and 10 ”1 injection volume. Limonoid glucosides were detected
at 210 nm.

Identification and quantitation of limonoid glucosides (limonin glucoside,
deacetylnomilinic acid glucoside, nomilinic acid glucoside, and obacunone glucoside)
were based on retention time, UV spectra, and response factors obtained with external
standards.

3.3.4 Data analysis

Different extraction conditions were compared for the highest recovery of
limonoid glucosides using analysis of variance (ANOVA) with single factor (extraction
condition) (Excel). Analyses were conducted in triplicate.

3.4 Extraction and analysis of flavanone glucosides

3.4.1 Extraction

Ground, freeze-dried peel (1 g) was mixed well with different modified solvents
(25ml) [including 70%, 80%, 90% methanol in water; 70%, 80%, 90% methanol in 0.01
M sodium phosphate buffer (pH 7); dimethylformamide/methanol (1:1); and

dimethylformamide/methanol (1:2)], heated (82°C for 5 min), and centrifuged at

74

 

 

 

 

Orange solid

lRags. peels. peel or

|
I

Mixed with 25 ml
1‘.-

lleat at 8H

Mi?

Billie :3. l:
.. ‘0“.

 

 

 

 

Orange solid fractions Orange liquid fractions

(Rags, peels, peel press cake, and seeds) (Juice and peel press liquid)
1 g 10 m1
1 1
Mixed with 25 ml dimethylformamide/methanol Heat at 82°C, 5 minutes
(1 :2)

Sample 10 ml
1
Heat at 82°C, 5 minutes Mixed 20 ml
Dimethylformamide/methanol
(1:2)

 

 

Centrifuged at 10,000X g, 10 minutes
I

Transferred supernatant to round bottom flask
|
Mixed again with 25 ml dimethylformamide/methanol (1:2)
15 minutes
I
Centrifuged at 10,000X g, 10 minutes
1

Combined supernatant in round bottom flask

l
Evaporated to minimal amount at 50°C

Redissovled in 25 ml methanol

l
Filtered. with 0.45u nylon filter

|
Analyzed by HPLC

Figure 22: Flow diagram of flavanone glucoside extraction.

75

 

 

 

(.1000); g 101 ‘10 min
amputated at 41.“ UT
tilted dirnetllfl {01mm
glucoside estraction.
3.4.2 Recot’er
Recoyery 01
performed by spikin
smile the control on
obtained by compar
and control sample:
meet orange. \ylie
insoluble llayanonc
3.4.3 High
The HPLC
119991. Flayanon-
14.6 ttmt. lb 00 r
Ml M potassiur
linear gradient 51
“'4‘ 1 ml min 1‘:
detected at 330
didlitttn \t'ere b

ertemal standar

 

 

 

10,000X g for 10 min. The pellet was extracted again and supematants were combined,

evaporated at 40°C under vacuum, and reconstituted with methanol to '10 ml (or 25 ml
when dimethylforrnamide was used). Figure 22 shows flow diagram of flavanone
glucoside extraction.

3.4.2 Recovery

Recovery of flavanone glucosides obtained from adjusted extraction was
performed by spiking the sample with neohesperidin (10 ppm) and hesperidin (5 ppm),
while the control was added with the same amount of blank methanol. The recovery was
obtained by comparison of neohesperidin and hesperidin contents extracted from spiked
and control samples. Neohesperidin was used because it is naturally absent from these
sweet orange, whereas hesperidin was used because it was the most concentrated and
insoluble flavanone glucoside in the sweet orange.

3.4.3 High performance liquid chromatography (HPLC) analysis

The HPLC analysis of flavanone glucoside was based on the method of Ooghe
(1999). Flavanone glucosides were separated on C18 column (Alltima: C18, 5p, 250 mm
x. 4.6 mm, 16 % carbon load, void time 2.02 minutes) with a mobile phase consisting of
0.01 M potassium phOSphate monobasic (solvent A) and acetonitrile (solvent B). A
linear gradient starting at 10%B and ending at 30% B in 60 minutes was used. Flow rate
was 1 ml/min flow rate and injection volume was 10 ul. Flavanone glucosides were
detected at 280 nm. Identification and quantitation of eriocitrin, narirutin, hesperidin,

didymin were based on retention time, UV spectra, and response factors obtained with

external standards.

 

 

 

 

 

 

34.4 Data aria-i
Different extra
glucosides using anal
tltcelt. Analyses. M
4. Results andr
Studied comp
chromatographic re‘.
limonoid glucosides
4.1Extractic
4.1.1 limor
We adjuster
Adjusted method
thrones. Limc
characteristics in t
lncorporat
ttas belieyed to it
Since the extract
liable ll 5110\t'e(
heating,
The hyd
:llt‘cosidic links
“‘4 hadrons,

limonoid 3.11“

 

 

 

3.4.4 Data analysis

Different extracting solvents were compared for the highest recovery of flavanone
glucosides using analysis of variance (ANOVA) with single factor (extracting solvent)
(Excel). Analyses were conducted in triplicate.

4. Results and discussion

Studied compounds were divided into three groups, based on their solubility and
chromatographic retention: a) limonoid aglycones and polymethoxylated flavones; b)
limonoid glucosides; and c) flavanone glucosides.

4.1 Extraction procedure

4.1.1 Limonoid aglycones and polymethoxylatedflavones

We adjusted Fong et al. (1993) method that was designed for limonoid aglycones.
Adjusted method was subsequently verified for the recovery of polymethoxylated
flavones. Limonoid aglycones and polymethoxylated flavones have common
characteristics in that they both are nonpolar and neutral (carrying no charge).

Incorporation of heat (82°C for 30 min) in the extraction was evaluated. Heating
was believed to improve dissolution of these nonpolar limonoids in freeze—dried samples,
since the extraction method was originally used for fresh orange tissues. The results
(Table 1) showed that there was a significant increase (P3005) in limonin content due to
heating.

The hydrolytic study was conducted to assure the absence of hydrolysis of B-
glycosidic linkage on the limonoid glucoside molecules due to heat (82°C for 30 min).
The hydrolysis would produce limonoid aglycones and result in the overestimation of

limonoid aglycones. Result showed no peak of limonin in limonin glucoside extract

 

 

 

 

 

 

Table l: Limonord 3gp
Without heating

Sample _

Peels

Seeds
3 = 2. Heating result
16511.

Table 2: Recoyery o:
tpolymethom

C ompour

limonir
Scutellarein tetran
Eh'=3

Table 3: limonoid
extraction c

Room temp- PH
HTdmmnm'
oWCSmmpH
mTlhmnni
oHClSnnnpl
RTSmmME
ETSmmpH
RTlhmnn
MTlhmnp
a=hmmndt
btmmmmn
thilnsbufier

Table 1: Limonoid aglycone content in seed, peel, and peel juice extracts with and

without heating (82°C for 30 min).

 

 

 

Sample Limonin (mg/Kg) :4 %CV1
Without heating With heating
Peels 180i2.9 3424115
Seeds 12301412 12031432
Peel press liquid 16414.3 35i1.5

 

jN = 2, Heating resulted in a significant increase (P S 0.05) in limonin content (paired t

test).

Table 2: Recovery of limonin (limonoid aglycone) and scutellarein tetramethylether
(polymethoxylatedflavone) extracted under heating (82°C for 30 min).

 

 

 

Compound Recovery (g/100g)li%CV
Peel Seed Peel press liquid Buffer
Limonin 9147.9 9445.7 9541.3 -
Scutellarein tetramethylether 111i3.3 - - 9442.2

 

lN=2

Table 3: Limonoid glucoside content in sweet orange seeds extracted by different solvent
extraction conditions.

 

 

 

Extraction conditions mg/Kg i %CVr
LG Potential NMG NAG OG
Room temp/water] 13052433 11999403 7314416 17157443
Room temp/pH 7.83 12295454 9744330 7920405 27730421
60°C/5 min/water 13121414 11839403 7524416 17282438
60°C/5 min/pH 7.8 13355421 4544179 8030408 27390416
60°C/15 min/water 13241407 12046403 7551406 17286405
60°C/15 min/pH 7.8 12858423 7564283 8041407 28618408
82°C/5 min/water 13287402 12169402 7527400 17308402
82°C/5 min/pH 7.8 12654464 56548.7 7943403 27950429
82°C/15 min/water 13196401 12141402 7415402 17618401;
__82°C/15 min/pH 7.8 12524401 59446.3 7821408 28036422

 

 

LG = limonin glucoside, NMG = nomilin glucoside, NAG = nomilinic acid glucoside,
0G = obacunone glucoside, 1N = 2, 270% methanol in water (pH 4.4), 370% methanol in
0.05 Tris buffer (pH 7.8)

78

 

 

which indicated thief: ’-
Pecoyenes of the ad?-
scutellarein tetrarnet‘n‘
4.1.2 lirnottO1
l'nlihe assofl
initial attempts f or 1
other neutral-polar
exchange extractior
glucosides.
Extractions
temperatures n ere :
are completely to:
Results in Table 3
glucoside and a of
heating leyels and
counted to obact
increase in ohacu
nomilin glucoside
Table 4 11‘.
ll 711% median
Slillllficant differ
3331ng tirttes.

is.) \i ‘ ‘
l-‘l‘duttbrlny \

 

 

which indicated that there was no hydrolysis of limonin glucoside under heating used.
Recoveries of the adjusted extraction were approximately 93% for limonin and 102% for
scutellarein tetramethylether (Table 2).

4.1.2 Limonoid glucosides

Unlike associated compounds, limonoid glucosides contain a carboxylated group.
Initial attempts for their extraction w ere to u se anion e xchange to s eparate them from
other neutral-polar compounds, primarily flavanone glucosides. However, anion
exchange extraction used produced high variations and low recoveries of limonoid
glucosides.

Extractions by 70% methanol at different pH, heating times, and heating
temperatures were studied. At pH ~ 6.5 to 7, the carboxylate group on these molecules
are completely ionized and more soluble, therefore higher recovery was expected.
Results in Table 3 showed that at pH 7.5, there were a 90% decrease in potential nomilin
glucoside and a 60% increase in obacunone glucoside compared to that at pH 4.4 at all
heating levels and heating times. According to Hasegawa (2000), nomilin glucoside was
converted to obacunone glucoside at pH 2: 8 and nomilinic acid glucoside at pH 5 3. The
increase in obacunone glucoside concentration could be contributed from the converted
nomilin glucoside. As such, extraction at pH 7.5 was not analyzed.

Table 4 presents total limonoid glucoside content in sweet orange seeds extracted
by 70% methanol at different heating temperature and heating time. There were no
significant differences of limonoid glucoside content due to heating temperature and
heating times. However, it was shown that when heating was applied extraction

reproducibility was improved. Extraction by 70% methanol at 82°C for 5 minutes was

79

 

 

 

Table 4: Total limono
methanol at c

Extracuon

Roorr: 1e

WC 5 :

oU‘C 15

ETC 5

82°C 11'
\311381395 of :
(ANOVA with sin gl

Table 5: Tlayanone
extractions

Extraction condi

90% methanol. n
80% methanol. n
10% methanol. v
90°omethanol
Shl‘omethanol.
111% methanol.
1)th "methanolt,
404-0 = nariru'
= didymin. DhlE

T
l
l

Table 4: Total limonoid glucoside content in sweet orange seeds extracted by 70%

methanol at different conditions.

 

 

Extraction conditions mg/Kg 4 %CVT
Room temp. 87 047a
60°C/5 min 87693a
60°C/15 min 88201a
82°C/5 min 88313a
82°C/15 min 88548a

 

TIN = 2, LSD(p50_()5) = 1300, Different superscripts indicate significant difference at P3005

(ANOVA with single factor)

Table 5: F lavanone glucosides in sweet orange peel extracted by different solvent

 

 

 

extractions.
Extraction conditions mg/Kgd:%CVl
potential ERT NT HD DD
NT-4'G
90% methanol, water 5194108 35443.2 1166432 4194462 33243.3
80% methanol, water 52243.9 37946.3 1170463 41574240 3174141
70% methanol, water 52240.7 38345.1 1158451 3586478 2934104
90% methanol, pH 72 50246.9 3614183 11634183 46704656 3604579
80% methanol, pH 72 53840.5 37140.0 1139400 3592475 29542.0
70% methanol, pH 72 52740.2 37043.9 1134439 36144145 3104147
DMF/methanol(1:1) 68740.8 61541.9 2057419 27381413 1371413
DMF/ methanol (1:2) 61340.4 58640.2 1958402 25915405 1301404

 

NT-4’-G = narirutin-4’-glucoside, ERT = eriocitrin, NT = narirutin, HD = hesperidin, DD
= didymin, DMF = dimethylformamide, 1N=2, 20.01sodiumphosphate (pH 7)

 

 

 

8O

 

 

selected sintfi ll “33 :
[add this extraction
has obtained.
413Hmww
Tlayanone g1
glucoside extraction
glucosides t’Kan'ati
19961haye bcfin T
111th l as extracting
The use 0
mmnmdmem
rmmmgndonerc
Table 5 sh
Mums hmh
extracted by diff
mmngdm
111616 Has no 81
extracted by dim
1mmhmnhme
glucosides in th
$4035 moot-err
inndhhhonna

h“Sllctidin and '

 

selected, since it was a short heating extraction which resulted in low variation (Table 3).
Under this extraction condition (70% methanol/ 82°C for 5 min), 90% (45.9) recovery
was obtained.

4.1.3 Flavanone glucosides

Flavanone glucosides are polar-neutral compounds. The difficulty of flavanone
glucoside extraction was insolubility of hesperidin. Quantitative studies on flavanone
glucosides (Kawaii et al., 1999; Ooghe and Detavernier, 1997; Manthey and Grohmann,
1996) have been primarily used dimethylsulfoxide (DMSO) and dimethylformamide
(DMF) as extracting solvents to enhance hesperidin solubility.

The use of dimethylformamide was initially not preferable because of the
toxicity and the high boiling point (153°C), which do not evaporate well and therefore
resulting in lower detection sensitivity.

Table 5 shows flavanone glucosides in sweet orange peel extracted by different
solvents. Table 6 shows total limonoid glucoside content in sweet orange seeds
extracted by different solvent extractions. Significantly higher flavanone glucoside
content (P S 0.05) was obtained when extracting solvent contained dimethylformamide.
There was no significant difference (P 2 0.05) between flavanone glucoside content
extracted by dimethylformamide/methanol (1 :1) and dimethylfonnamide/methanol (1 :2).
Therefore, d imethylformamide/methanol ( 1:2) w as s elected for extraction 0 f f lavanone
glucosides in the subsequent studies, since less dimethylformamide was used. Table 7
shows recovery of neohesperidin and hesperidin in peel and peel press liquid extracted by

dimethhylformamide/methanol (1:2). The recoveries were approximately 99% for

hesperidin and 90% for neohesperidin.

 

 

 

Table 6: Total limont‘
solt ent extra

Extractior

900.11. melt.

50" a met

71, 1°... metl

9001. 111611

80011 T131611

700 0 111611
Dimethylforrnan
Dimethylfonnan
$337.18ng.4115 =
difference at P E {It
= dimethylfonnamn

Table 3: Recoyery .
methanol

 

Compounr

Neohesperidi

M
y 4 i.

 

Table 6: Total limonoid glucoside content in sweet orange seeds extracted by different
solvent extractions.

 

 

Extraction conditions mg/Kg4%CVr
90% methanol, water 6565a
80% methanol, water 6545a
70% methanol, water 5942a
90% methanol, pH 72 70572!
80% methanol, pH 72 593621
70% methanol, pH 72 5956a
Dimethylformamide/methanol(1 : 1) 321 12b
Dimethylforrnamide / methanol (1:2) 30376b

 

IN = 2, LSD(PSO.OS) = 3145, LSD(p50,m)= 4576, Different superscripts indicate significant
difference at P s 0.01 (ANOVA with single factor), 20.01socliumphosphato (pH 7), DMF
= dimethylformamide

Table 7: Recovery of neohesperidin and hesperidin extracted by dimethylformamide
/methanol (1 :2)

 

 

 

Compound Recovery (g/100g)i%CVI
Peel Peel press liquid
Neohesperidin 9444.7 10441 .4
Hesperidin 9145.5 8942.9

 

 

1N=2

82

 

 

 

4.2HPL-C
Chromatogr.-ta;
at limonoid aglycort
tlaranone glucost es
Quantitation
because it accounts
Standards for each
minimize detector
system before analy
11 was obse
tt‘ere obtained usir
luna column (15,
1331101641 separa‘
Chromatograms it

used.

4.2.1 Lint

Since Ec
limonoid aglyc
polymethoxylate
llon‘er slope. “ o

Retentio

dimoninl :1 (

tetramethylethe

 

4.2 HPLC

Chromatographic conditions were separately adjusted for each compound group:
a) limonoid aglycones and polymethoxylated flavones, b) limonoid glucosides, and c)
flavanone glucosides.

Quantitation was based on “Peak height”, instead of more common “peak area”,
because it accounts only peaks of interest when baseline resolution is not achieved.
Standards for each group were analyzed before and after each series of samples to
minimize detector response variations. A blank methanol was run to equilibrate the
system before analyses.

It was observed that, for studied flavonoids and limonoids, improved separations
were obtained using Luna column compared to Alltima column. Higher carbon load in
Luna column (17.8%), compared that to Alltima column (16%), may contribute to this
improved separation. Therefore, when analyzed complex mixtures (where the

Chromatograms include numerous peaks that were closely retained, Luna column was

used.

4.2.1 Limonoid aglycones and polymethoxylatedflavones

Since Fong et al. (1993) condition was originally designed for separating
limonoid aglycones in fruit tissues. To separate limonoid aglycones and

polymethoxylated flavones, the mobile phase gradient was adjusted to be more extended
(lower slope, %/min). Separation at 210 nm was Rs 0.94/ N 12,000.

Retention times were 27 (sinensitin), 27 (deacetylnomilin), 31 (unknown), 32
(limonin), 33 (nobiletin), 35 (3,4,5,6,7,8,3’,4’—heptamethoxyflavone), 37.2 (scutellarein

tetramethylether), 39 (nomilin), 47 (obacunone), and 42 (tangeretin) minutes. Figure 23

 

 

 

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thrones in orange 5
flayone detection 1.
for analyses
Separation at 210 t
deacetylnomilin). I
other samples n‘hicl
and deacetylnomili
polymethoxylated 1
seed extract under '
4.2.2 Limo
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11111131011111. 0.3 1111
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figure y

hit. SEPartition

 

shows separation of limonoid aglycones and polymethoxylated flavones in orange peel
extract under the gradient system at 210 nm (limonoid aglycone detection) and at 340 nm
(polymethoxylated flavone detection). Figure 24 shows separation of polymethoxylated
flavones in orange seed extract under the gradient system at 340 nm (polymethoxylated
flavone detection).

For analyses of limonoid aglycone in orange seed, isocratic system was used.
Separation at 210 nm was Rs 1.3/ N 1,131. Retention times were 18 (limonin), 21
(deacetylnomilin), 3O (nomilin), and 53 (obacunone). This system was not suitable for
other samples which contained high flavonoid and low limonoid content, because limonin
and deacetylnomilin were not separated from impurities, and obacunone coeluted with
polymethoxylated flavones. Figure 25 shows separation of limonoid aglycones in orange
seed extract under the isocratic system at 210 nm (limonoid aglycone detection).

4.2.2 Limonoid glucosides

Fong et al., (1993) was modified. Separation obtained was Rs 1.1/N 23,588.
Retention time w ere 3 8 (limonin glucoside), 4 6 (deacetylnomilinic acid glucoside), 5 3
(unknown), 63 (unknown), 65 (nomilinic acid glucoside), and 68 (obacunone glucoside).
Under the chromatographic system used, an addition of a glucose molecule on limonoid
aglycones did not change the elution order of limonoids. Figure 26 shows separation of

limonoid glucosides in orange seed and peel extracts at 210 nm (limonoid glucoside
detection).

4.2.3 F lavanone glucosides

Figure 27 shows separation of flavanone glucosides in orange peel extract at 280

nm. Separation obtained. was Rs 0.6/N 13,079. Retention times of interested flavanone

85

 

 

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glucosides were 26 (potential narirutin-4’-glucoside), 34 (eriocitrin), 42 (narirutin), 46
(hesperidin), 61 (didymin) minutes.

All flavanone glucosides found in detectable levels were tasteless rutinosides,
which are found primarily in sweet oranges. Their relative retention is correlated to
number of hydroxyl and methoxyl groups on the B ring. Compounds with more hydroxyl
groups possess increased polarity (less retained in reverse phase) and those with more
methoxyl groups are more nonpolar (more retained in reverse phase).

5. Conclusion

It was necessary to categorize limonoids and flavonoids studied into groups with
similar chromatographic retentions: 1) polymethoxylated flavones and limonoid
aglycones, 2) limonoid glucosides, and 3) flavanone glucosides.

Extraction of polymethoxylated flavones and limonoid aglycones was improved
by heat (82°C for 30 min). The used of pH 7 resulted in structural instability of
suspected nomilin glucoside. Reproducibility of limonoid glucoside extraction was

improved by heating. Extraction of flavanone glucosides required dimethylformamide.

90

 

 

 

6. References:

Chen. 1.. Montana:
ilarones. a c
cold pressed

Dueo. P .. M ondellt
Myers. P. 1
supercntica‘

tong. C. ll. Haseg
1993. Lirnl
gmnhmm

long. C. H. Has
Contents (
Valencia c
1178-1181

Gmmene
dift‘erentiz
Chem. 35

Hamas:
Limonoic
Berhow,
Washing

Hasegawa 8.. t
1991. t
glUCOpy

Emma S-
and [’61:

immflx

Peelfl;
$14

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01‘ citr

 

6. References:

Chen, J., Montanari, A. M., and Widmer, W. W. 1997. Two new polymethoxylated
flavones, a class of compounds with potential anticancer activity, isolated from
cold pressed Dancy tangerine peel oil solids. J. Agric. Food Chem. 45: 364-368

Dugo, P ., Mondello, L., Dugo, G ., Heaton, D. M., B artle, K. D ., Clifford, A. A ., and
Myers, P. 1996. Rapid analysis of polymethoxylated flavones from citrus oils by
supercritical fluid chromatography. J. Agric. Food Chem. 44: 3900-3905

Fong, C. H., Hasegawa, S., Miyake, M., Ozaki, Y., Coggins, Jr. C. W., and Atkin, D. R.
1993. Limonoids and their glucosides in Valencia orange seeds during fruit
growth and development. J. Agric. Food Chem. 41: 112-115

Fong, C. H., Hasegawa, S., Coggins, Jr., C. W., Atkin, D. R., and Miyake, M. 1992.
Contents of limonoids and limonin 17-b-D-glucopyranoside in fruit tissue of
Valencia 0 range during fruit growth and m aturation. J. A gric. F ood C hem. 4 0.
1178-1181

Gaydou, E. M., Bianchini, J. and Randriamiharisoa. 1987. Orange and mandarin peel oils
differentiation using polymethoxylated flavone composition. J. Agric. Food
Chem. 35: 525-529

Hasegawa, S. 2000. Chapter 2: Biochemistry of Limonoids in Citrus. From Citrus
Limonoids: Functional Chemicals in Agriculture and Foods. Edited by Mark A.
Berhow, Shin Hasegawa, and Gary D. Manners. American Chemical Society,
Washington, DC. p. 21

Hasegawa, 8., Cu, P., Fong, C. H., Herman, Z., Coggins, Jr., C. W., and Atkin, D. R.
1991. Changes in the limonoate A-ring lactone and limonin 17-b-D-
glucopyranoside content of navel oranges during fruit growth and maturation. J.
Agric. Food Chem. 39: 262—265

Hasegawa, S., Bennett, RD, and Verdon, C. P. 1980. Limonoids in citrus seeds: Origin
and relative concentration. J. Agric. Food Chem. 28(5): 922-925

Manthey, J .A. and Grohmann, K. 1996. Concentrations of hesperidin and other orange
peel flavonoids in Citrus processing byproducts. J. Agric. Food Chem. 44(3): 811-
814

McIntosh, CA. and Mansell, KL. 1997. Three—dimensional distribution of limonin,
limonoate A— ring monolactone, and naringin in the fruit tissues of three varieties
of citrus paradise. J. Agric. Food Chem. 45: 2876-2883

 

 

 

 

 

 

' ‘. :

Mouly. P. P- arm
of cnrus tutc
flavanone gr

(Jodie. WC. and B
sinensis jute

Ooghe. WC. and I
and hybri ds

Ooghe. \V. C.. t
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Food Ch

 

 

 

 

 

 

 

Mouly, P. P., Arzouyan, C. R., Gaydou, E. M., and Estienne,J. M. 1994. Differentiation
of citrus juices by factorial discriminant analysis using liquid chromatography of
flavanone glycosides. J. Agric. Food Chem. 42: 70~79

Ooghe, WC. and Detavernier, CM. 1999. Flavonoids as authenticity markers for Citrus
sinensis juice. Fruit Processing. 9(8): 308-313

Ooghe, WC. and Detavernier, CM. 1997. Detection of the addition of Citrus reticulata
and hybrids to Citrus sinensis by flavonoids. J. Agric. Food Chem. 45: 1633-1637

Ooghe, W. C., Ooghe, S. J., Detavernier, C. M., and Huyghebaert, A. 1994.
Characterization of orange juice (Citrus sinensis) by polymethoxylated flavones.
J. Agric. Food Chem. 42: 2191-2195

Ooghe, W. C., Ooghe, S. J., Detavernier, C. M., and Huyghebaert, A. 1994a.
Characterization of orange juice by flavanone glycosides. J. Agric. Food Chem.
42: 2183-2190

Ooghe, W. C., Ooghe, S. J., Detavernier, C. M., and Huyghebaert, A. 1994b.
Characterization of orange juice by flavanone glycosides. J. Agric. Food Chem.

42: 2183-2190

Robards, K.; Li, X., Antolovich, M.; and Boyd, S. 1997. Characterization of citrus by
chromatographic analysis of flavonoids. J. Sci. Food Agric. 75: 87-101

Stremple,P. 1 998.GC/MS analysis 0 f p olymethoxylated f lavones in citrus oils. J. High
Resol. Chromatogr. 21(11): 587-591

Yusof, S., Ghazali, H. M., and King, G. S. 1990. Naringin content in local citrus fruits.
Food Chemistry.37: 113-121

92

 

 

 

Study 11: Isolation

Part 1: Isolation an
nomilin glut

1. Abstract

Tito unlmO‘
to those oi deaceth
for these two c om
material. Prelimtn
in a simpler liir
Separation (Rs = ‘1
sample load (4th
itiormation ohtai:
2. Introduction

Deacetylr
limonoid glucosi
limonoid glucos
Standards. Pong
glucosides; the
Pulitied in thet
pnmarih~ using
in the less eater

lC-llSl (Soho.

rs‘lllired l‘Or \

complex SW0

 

 

 

Study II: Isolation and identification of selected limonoids and flavonoids

Part I: Isolation and identification of deacethylnomilin glucoside (DNG) and
nomilin glucoside (N G)

1. Abstract
Two unknown peaks from seed extract having similar chromatographic retention
to those of deacetylnomilin glucoside (DNG) and nomilin glucoside (NG) were identified
for these two c ompounds. G round 3 eed from the V alencia v ariety w as u sed a s a raw
material. Preliminary cleaning using liquid-liquid and anion exchange extraction resulted
in a simpler limonoid glucoside mixture for the subsequent isolation by HPLC.
Separation (Rs = 0.75/N = 5,575) was performed on analytical scale HPLC using a large
sample load (40ul). Identification was confirmed based on their molecular weight
information obtained from negative F ABMS.
2. Introduction
Deacetylnomilin glucoside (DNG) and nomilin glucoside (NG) are among minor
limonoid glucosides detected in sweet orange (Citrus sinensis). Quantitative analyses of
limonoid glucosides were found in limited studies, due to the lack of the commercial
standards. Fong et al. (1993) quantitatively analyzed both limonoid aglycones and their
glucosides; the identification was based on the retention times of standard compounds
purified in their laboratory. Identification of limonoid glucosides have been done
primarily using nuclear magnetic resonance MR) established by Hasegawa (1989) and,
in the less extent, electrospray ionization liquid chromatography mass spectrometry (ESI-
LC-MS) (Schoch et al., 2001). Purified and concentrated (at least 5 mg/O.7ml) sample is
required for NMR analyses, which provide detailed structural information for these

complex structures. For ESl—LC-MS analyses, prior purification is not required and

93

 

 

 

 

 

much less concentr
protides both ch
bombardment mass
compared to those
sensitive comparet
llouerer. operati
llouerer. additior
required for the de
The two ‘
chromatographic
glucoside (NG).
techniques to ide
3. Materials at
3.1 Seed
Seed iror
under and ext
ltlllOVe orange .
llill and stored
3.2 San
Seed pt
bio'holllogeniz
The Ulleure \

hydroWOluen

 

 

 

much less concentrated sample can be used (as small as picogram unit). This technique
provides both chromatographic and molecular weight information. Fast atom
bombardment mass spectrometry (FABMS), found in lesser extent (Sawabe et al., 1999)
compared to those two techniques, since it requires preliminary purification step, it is less
sensitive compared to ESI—LC-MS, and less structurally informative compared to NMR.
However, operation of this instrument is very simple and relatively inexpensive.
However, additional information such as UV spectra and chromatographic retention are
required for the definitive conclusion.

The two unknowns present consistently in orange seed extracts had a similar
chromatographic retention to those of deacetylnomilin glucoside (DNG) and nomilin
glucoside (NG). We were interested to verify this assumption by using the appropriate
techniques to identify these unknown peaks.

3. Materials and methods

3.1 Seed

Seed from Valencia variety was used. Freeze-dried seed was ground using coffee
grinder and extracted with hexane extraction (1:4, W/V) twice at room temperature to

remove orange oil. The ground seed was ground again to pass 1 mm screen using UDY-

Mill and stored at —20°C until analyses.

3.2 Sample preparation and extraction

Seed powder was homogenized with 0.05 M Tris buffer pH 8 (1:10, WN) with
bio-homogenizer for 2-3 minutes. The mixture was acidified to pH ~ 2.5 with 1 N HCl.
The mixture was extracted twice using ethyl acetate (containing 200 ppm butyrated

hydroxytoluene). Nonpolar compounds partitioned into ethyl acetate fraction, which was

94

 

 

 

 

swarmed b‘f CEBU—l
imonoid aglycones
shorts tlou (313?? 31
3.3Prelirn1:

Io remore
fraction the mist
column was precc
iraction was app?
limonoidglucosid:
lo remort

me). which was
Washed With 10
residue was digsc

3.4 lsola
pe

Separatic

e' w
“ith -600 acetot

Litttliun tolum

53ml “'35 menti

Isolated

 

 

 

 

separated by centrifuge at 5000X g for 10 min. Ethyl acetate fraction was a source for
limonoid aglycones and buffer fraction was a source for limonoid glucosides. Figure 28
shows flow diagram of limonoid isolation from orange seed.

3.3 Preliminary purification for limonoid glucosides

To remove neutral impurities such as flavanone glucosides, sugars from the buffer
fraction, the mixture was passed through anion exchange (75 ml). Anion exchange
column was preconditioned with 50 m1 1 M acetic acid and 100 ml water. The buffer
fraction was applied on to the top of the column, washed with 100 ml water, and
limonoidglucosides were eluted with 50 m1 1 M sodium chloride.

To remove salts, each 10 ml of eluate was passed through the C18 Sep-Pak (1000
mg), which was preconditioned with 3 m1 methanol and 10 ml water; the column was
washed with 10 ml water and eluted with 6 ml. Methanol was evaporated; and the
residue was dissolved in minimal amount of water, stored at -20°C until use.

3.4 Isolation of deacetylnomilin glucoside and nomilin glucoside by high
performance liquid chromatography (HPLC)

Separations were done on C18 column (Luna column: C18, 5 pl, 250mm x 4.6
mm, 17.8% carbon load, Phenomenex). The linear gradient started at 15% and ended
with 26% acetonitril in 3mM phosphoric acid in 60 minutes. Flow rate was at 1 ml/min.
Injection volume was 40 tel. Limonoid glucosides were detected at 210 nm. The HPLC
setup was mentioned in Study I/Part I (3 .4).

Isolated fraction was evaporated at 40°C under vacuum and concentrated using
C18 cartridge (500mg). Eluted methanol from C 18 cartridge was evaporated at 37°C

under N2 gas and stored at refrigerator until analyzed by mass spectrometer.

95

 

 

 

 

Er

Xe

lune :s; it

 

 

Valencia orange seed powder
Homogenized in Tris buffelr (0.05M, pH 8), 1:10 W/V
Filter (paper No. 4)

Acidified to pH|3 with 1 N HCI
Extracted with ethyl acetate, 1:1 WV

|
Centrifuged at 5000X g, 10 min

 

 

 

 

J
l l
Ethyl acetate fraction Buffer fraction
I l
Nonpolar compounds Polar compounds
(Neutral limonoid aglycones) (Acidic limonoids, flavonoids

, acids, sugar, salts,. . .)

|

Anion exchange purification

(Acidic limonoids, acids, salts)
I

C18 solid phase purification

(Desalt and concentrate limonoid glucosides)
1
Filter (0.45 pt)
I
HPLC

 

 

 

Figure 28: Flow diagram of limonoid isolation from orange seeds.

96

 

3.5 Fast are:
FAB mass
spectrometer (JOE
produced by born
glycerol and m-nit
resolution was set
collected from m
ionization on a prt
3.6 Stande
Detail on
4. Results andt
Seed was
hiSheet limonoi
Purification of Ir
Different i nj em
Simple load, 5‘
81‘] Pl injection t
Huntues) are sht
tndh’tgp d m

maintained ade

Retentit
nomilinic acid

Testectirelt. l

 

 

 

3.5 Fast atom bombardment mass spectrometry

FAB mass spectra were obtained using a JEOL HX-110 double-focusing mass
spectrometer (JOEL USA, Peabody, MA) Operating in negative ion modes. Ions were
produced by bombardment with a beam of Xe atoms (6 keV). Matrixes used were
glycerol and m-nitrobenzyl alcohol (NBA). The accelerating voltage was 10 kV and the
resolution was set at 3000. The instrument was scanned from m/z 0 to 1500, data were
collected from m/z 50-1500. The sample was mixed with the matrix, which supported
ionization on a probe tip and was then inserted into the instrument.

3.6 Standards

Detail on limonoid standards were mentioned in Study l/Part I (3.5).

4. Results and discussion

Seed was used for the purification of limonoid glucosides because it contains
highest limonoid glucoside and low flavanone glucoside impurities. Preliminary
purification of limonoid glucosides resulted in a simpler mixture for separation by HPLC.
Different injection v olumes w ere studied ( 10, 2 0, 4 0, and 8 0 u 1) to discern m aximum
sample load. Separation of limonoid glucosides on C18 analytical column using 40 and
80 pl injection volumes (gradient system: 18% to 26 % acetonitrile in 3 mMH3P04 in 40
mintues) are shown in Figure 29. Resolution (between the most difficult pair, unknown 2
and NAG) decreased with increasing injection volumes. The largest sample load that still
maintained adequate resolution was 40 ul (Rs = 0.75/N = 5,575).

Retention times (RT) of limonin glucoside, deacetylnomilinic acid glucoside,
nomilinic acid glucoside, and obacunone glucoside were 28, 33, 55, and 62 minutes,

respectively. Based on published retention time (Fong et al., 1993), peak at 41 minutes

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U' spear;
are similar 10 It
deacetylnomilinic
Confirma‘t
were based on m
mecuomem' t—e‘
\x‘eighl of unknm
-e\' FABMS she
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5' Conclusiol
Pretimi
“mt‘ter hm

WP]? toad t

 

(unknown 1) and 54 minutes (unknown 2) were potentially deacetylnomilin glucoside
(DNG) and nomilin glucoside (NG). Figure 30 shows Chromatograms of purified
unknown 1 and unknown 2.

UV spectra of isolated unknown 1 (41 minutes) and 2 (54 minutes) (Figure 31)
are similar to the typical UV spectra of limonoid compounds (deacetylnomilin,
deacetylnomilinic acid glucoside, nomilin, and nomilinic acid glucosides) (Figure 15).

Confirmation o f d eacetylnomilin g lucoside (DNG) and nomilin glucoside ( NG)
were based on molecular weight, determined by negative fast atom bombardment mass
spectrometry (—eVFABMS) due to limited amount of purified compounds. Molecular
weight of unknown 1 was found to be 652 (corresponding to deacetylnomilin glucoside);
-eV FABMS showed a peak at m/z 651 [M-H]' in both glycerol and NBA matrices. The
molecular weight of unknown 2 was found to be 694 (corresponding to nomilin
glucoside); -eV FABMS showed a peak at m/z 693 [M-H]' in both glycerol and NBA
matrices.

It s hould b e n oted that there was an o ccurrence of n omilinic acid glucoside in
collected unknown 2 (nomilin glucoside) after 2 weeks storage at refrigerated
temperature. This may be due to the conversion of nomilin glucoside to nomilinic acid
glucoside at pH (~26) of mobile phase used. According to Hasegawa (2000), nomilin
glucoside may be converted to nomilinic acid glucoside at pH S 3.

5. Conclusion

Preliminary cleaning using liquid-liquid and anion exchange extraction resulted in

a simpler limonoid glucoside mixture for the subsequent isolation by HPLC. Large

sample load up to 40 ul can be used with optimized HPLC mobile phase to increase

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31:11. This mO‘Di‘
nomilinglucosidfi.
pseudo-moleculaf

nomilinglucoside

 

yield. This mobile phase system allowed isolation of deacetylnomilinglucoside and
nomilinglucoside. -eVFABMS on glycerol and NBA matrices effectively produced
pseudo-molecular ions for molecular weight assignment of deacetylnomilinglucoside and

nomilinglucoside.

102

 

6. References:

lone. C. H. H2156
1993. lirr
gomh ant

Hasegawa. 5.. 1:0
orange Inc

Hasegawa. 8.. B
glucoside:

Sawabe. A- Mor
Y- and O

glycoside

Sehoch. 114.. M
from Cit
J. Agric.

 

6. References:

Fong, C. H., Hasegawa, S., Miyake, M., Ozaki, Y., Coggins, Jr. C. W., and Atkin, D. R.
1993. Limonoids and their glucosides in Valencia orange seeds during fruit
growth and development. J. Agric. Food Chem. 41: 112-115

Hasegawa, S., Fong, C. H., Miyake, M., and Keithly, J. H. 1996. Limonoid glucosides in
orange molasses. J. Food Science. 61(3): 560-561

Hasegawa, S., Bennett, R. D., Herman, Z., Fong, C. H., and Cu, P. 1989. Limonoid
glucosides in Citrus. Phytochemistry. 28 (6): 1717—1720

Sawabe, A., Morita, M., Kiso, T., Kishine, H., Ohtsubo, Y., Minematsu, T., Matsubara
Y., and Okamoto, T. 1999. Isolation and characterization of new limonoid
glycosides from Citrus unshiu peels. Carbohydrate Research. 315 (1-2): 142-147

Schoch, T.K., Manners, G. D., and Hasegawa, S. 2001. Analysis of limonoid glucosides

from Citrus by electrospray ionization liquid chromatography-mass spectrometry.
J. Agric. Food Chem. 49 (3): 1120-1108

103

 

Part II: Isolation
narirutin

1. Abstract
loo unl'n
3.5.6.7.3'41hexa
narirutin repone1
Orange p
purification inch
and column chr
Final isolations '
hexamethoxiila'
separation cond'
=1.4T\‘ = 9791
were based on I
3. lntroducth
3.5.6.“,
minor throng.
110th tGa)‘d0
“W‘ailablg CC
Identit
thmmalOng
6131.. 199.1;
and Dam”

 

 

 

Part II: Isolation and identification of 3,5,6,7,3’,4’-hexamethoxyflavone (HX) and
narirutin-4’-glucoside (N T -4’-G)

1. Abstract

Two unknowns, having similar relative retention and UV spectra to those of
3,5,6,7,3’,4’-hexamethoxyflavone and narirutin (subsequently demonstrate not to be
narirutin) reported in previous study, were identified for these two compounds.

Orange peel from Valencia variety was used as a raw material. Preliminary
purification included soxhlet extraction used to separate nonpolar and polar compounds;
and column chromatography used to separate among the nonpolar/polar compounds.
Final isolations were carried out using analytical HPLC. Sample loads were 100 111 for
hexamethoxyflavone isolation and 20 ul for narirutin-4’—glucoside isolations. Both
separation conditions optimized for HX and NT-4’—G produced resolved peak for HX (Rs
= 1.4/N = 979) and well isolated single peak for NT-4’G, respectively. Identifications
were based on molecular weight information using -eVFABMS and NMR spectral data.

2. Introduction

3,5,6,7,3’,4’—Hexamethoxyflavone (HX) and narirutin-4’—glucoside (NT—4’-® are
minor flavonoids in sweet oranges. These two flavonoids have been reported in limited
works (Gaydou et al., 1987, Manthey and Grohmann, 1996, Hsu et al., 1998) due to the
unavailable commercial standards.

Identifications of flavonoids have been done through various spectrometric and
chromatographic techniques. For routine analyses HPLC-PDA is primarily used (Ooghe
et al., 1994a, Ooghe et al., 1994a, Ortuno et al., 1995, Bronner and Beecher, 1995, Ooghe
and Detavernier, 1997, Kawaii et al., 1999). NMR is (Castillo et al., 1993, Ortuno et al.,

1995, Miyake et al., 1997,Chen et al., 1997, Mitsuo et al., 1999) used when high

104

 

mam-e details
myimhbma
interpretation. E
distinctive U S
nation of purii
identification of
Mabry e1 al. (19'
lC-MS (Robard
et al.. 1994. Mi}
loo un
Ilaronoids haw
glucoside balsa
1o continn thi
Compounds.
3. Materials
3.1Pe
Peels
1355 1 mm 5

analyses.

'1)

I)
’fl

Grin:
loi 3'1 he

dlnlelln'ldic

 

 

informative details are needed and relatively concentrated sample is available (at least 5
mg/0.7 ml), b ut a series 0 f p urification steps p rior to analysis is required for a ccurate
interpretation. Highly conjugated structures of flavonoids which correspond to the
distinctive UV spectrum is considered. suitable identification tool when only small
quantity of purified compound is available. An extensive review for systematic
identification of flavonoids by application of UV and NMR techniques was written by
Mabry et al. (1970). Other techniques include GC-MS (He et al., 1997, Stremple, 1998),
LC-MS (Robards et al., 1997, Ishii et al., 2000, Dugo et al., 2000) and FABMS (Takashi
et al., 1994, Miyake et al., 1997).

Two unknown peaks detected in comparable quantity with other identified
flavonoids have demonstrated a potential to be hexamethoxyflavones and narirutin-4’-
glucoside based on their chromatographic retention and UV spectra. We were interested

to confirm this assumption by using additional techniques suitable to identify these
compounds.

3. Materials and methods

3.1 Peel

Peels from Valencia variety was used. The peel was freeze—dried, and ground to
pass 1 mm screen using UDY-Mill. The ground samples were stored at —20°C until

analyses.

3.2 Sample preparation and extraction

Ground peel (35 grams) was refluxed with dimethyldichloromethane (1 :50, W/V)
for 24 hours, then evaporated to minimal under vacuum at 40°C. This

dimethyldichloromethane fraction was a source of polymethoxylated flavones. Peel

105

 

 

 

residue was faith
eraporated to non
tlaranone glue-05:

until use.

3.3 Prelin
chrorr

The r€SlC
minimal l1€Xalle
silica gel C01
Chromatograph:
area 500-600
oretnight and S

Crudefi
of each solr
heuane'toluene
herane‘toluen:
toluene chlorc
chloroform.
chloroform et
or introdue
column. Tilt
pollmtlhOX)

Orange Peel.

 

 

residue was further refluxed with methanol (1:50, W/V) for another 24 hours, then
evaporated to minimal under vacuum at 40°C. This methanol fraction was a source of

flavanone glucosides. Both fractions were stored at refrigerated temperature (2i1°C)

until use.

3.3 Preliminary purification for polymethoxylated flavones by column
chromatography

The residue from dimethyldichloromethane extract (1 g) was reconstituted with

minimal hexane (20ml). To separate polymethoxylated flavones (nonpolar compounds),

silica gel column chromatography was used. Silica powder from Sorbsil

Chromatographic Media (Sorbsil C60 40/60H, synthetic amorphous silica, BET surface
area 500-600 mZ/g, pore diameter 0.72-0.82 ml/g) was preconditioned in hexane
overnight and slurry packed into glass columns (10 m1) .

Crude extract was applied to the top of the silica gel column followed by 50 ml
of each solvent with increasing polarity: 100%hexane, hexane/toluene (8:2),
hexane/toluene (6:4), hexane/toluene (5:5), hexane/toluene (4:6), hexane/toluene (3:7),
hexane/toluene (2:8), hexane/toluene (1:9), 100% toluene, toluene/chloroform (8:2),
toluene/chloroform (6:4), toluene/chloroform (4:6), toluene/chloroform (2:8), 100%
chloroform, chloroform/ethylacetate (8:2), chlorofonn/ethylacetate (6:4),
chlorofonn/ethylacetate (4:6), chloroform/ethylacetate (2:8), respectively. Nitrogen gas
was introduced at the top of the column to speed up the flow of eluates through the

column. These eighteen 50 ml fractions were collected and analyzed for the presence of

polymethoxylated flavones. Figure 32 shows a flow diagram of flavonoid isolation from

orange peel.

106

 

 

Reflux W

Traction

. IA
1'1 N
I I {I 1

-.F

Figure \‘t.

 

 

Valencia peel powder 35 g
l
l l

Reflux with dichloromethane Refluxed with methanol

 

 

 

Fraction Preparative silica column Fraction Preparative C18 column
I l
1 100% hexane 1 10% methanol
2 hexane/toluene (8:2) 2 20% methanol (first)
3 hexane/toluene (6:4) 3 20% methanol (second)
4 hexane/toluene (5:5) 4 30% methanol (first)
5 hexane/toluene (4:6) 5 30% methanol (second)
6 hexane/toluene (3:7) 6 40% methanol
7 hexane/toluene (2:8) 7 50% methanol
8 hexane/toluene (1:9) 8 75% methanol
9 100% toluene 9 100% methanol
10 toluene/chloroform (8:2)
1.1 toluene/chloroform (6:4)
12 toluene/chloroform (4:6)
13 toluene/chloroform (2:8)
14 100% chloroform
15 chloroform/ethylacetate (8:2)
16 chloroform/ethylacetate (6:4)
17 chloroform/ethylacetate (4:6)
18 chloroform/ethylacetate (2:8)
I
chloroform/ethylacetate (2:8) 20% methanol (first)
|
Hexamethoxyflavone Narirutin-4’-glucoside
isolated by HPLC isolated by HPLC

Figure 32: Flow diagram of flavonoid isolation from orange peels.

107

 

 

3.4 Prelirnr
Residue it
n11 and sonicatec
separation was ca
C18 (SE1
Company) panir
packed on to '5
uith 150 ml me
Crude e
followed by 11'
311° 11. 30° 11. 30'
collected anc‘

chromatograp'

3.5 Pu

HPLC
(1811 Aumma

SR1 lnc.

Sep.

C18-5 til.

Pollttietlit

 

 

3.4 Preliminary purification for narirutin-4’-glucoside by column chromatography

Residue from m ethanol fraction ( 1 g) w as r econstituted with minimal w ater (25
ml) and sonicated for 20 minutes. To separate flavanone glucoside (polar compound),
separation was carried out on C18 column chromatography.

C18 (50 um irregular-shaped silica, 60A porosity, 6% carbon load, Alltech
Company) particles (10 g) were soaked in purified water for 15 minutes and slurry
packed on to 75-ml column (Alltech Company). The packed column was conditioned
with 150 m1 methanol and washed with 250 m1 of purified water.

Crude extract was applied on the top of the column, washed with 250 ml water
followed by 150 ml of each solvent with increasing methanol percentage: 10%, 20%,
20%, 30%, 30%, 40%, 50%, 75%, and 100%, respectively. Nine 150 ml fractions were
collected and analyzed for the presence of narirutin-4’-glucoside (based on

chromatographic retention and UV spectrum).

3.5 Purification of 3,5,6,7,3’,4’-hexamethoxyflavone by high performance liquid
chromatography (HPLC)

HPLC setting consisted of two-pump model Waters 515 (controlled by Waters
680 Automated Gradient Controller), connected with Waters 486 Tunable Absorbance
Detector, and manual injection unit. Integration software was PEAKW32, version 2.08,
SRI Inc.

Separation of polymethoxylated flavones was conducted on C18 column (Luna:
C 18, 5 til, 250mm x 4.6 mm, 17.8% carbon. load, Phenomenex ) with isocratic mobile
phase consisted of 1:1 [solvent A (water/acetonitrile/ propanol/acetic acid,
81:15:321):solvent B(water/acetonitrile/propanol/acetic acid, 40:56:3:1)] at 0.8 ml/min.

Polymethoxylated flavones were detected at 340 nm. The collected eluate was

 

 

 

 

etaporated (WC
analtses.

3.6 Purifi
cl

HPLC s

neinnethonfla‘

'rsn

ild min. 1
starting with 11
11111111111116. An
lheHPlC setc
mentioned abo
smdanmg
Pas

300 ill
X1 gas. Metlt
accelerating tl
Weights using
OlDXMG 91111

‘
J

”/9

X1
\th

l‘uirersit)‘.

nanometer

is”ll‘ttature

 

 

 

evaporated (50°C) to minimal amount and stored at refrigerated temperature until further

analyses.

3.6 Purification of narirutin-4’-glucoside by high performance liquid
chromatography (HPLC)

HPLC setting was the same as that for purification of 3,5,6,7,3’,4’-
hexamethoxyflavone. Separation was achieved on C18 column (Luna: C18, 5 pl, 250mm
x 4.6 mm, 17.8% carbon load, Phenomenex). Mobile phase used was gradient system
starting with 15% acetonitrile and ending with 26% acetonitrile in 40 minutes at 1
ml/minute. An injection volume was 20 ul. The resolved peaks were detected at 280 nm.
The HPLC setup was the same as that for 3,5,6,7,3’,4’-hexamethoxyflavone purification
mentioned above. The collected eluate was evaporated (50°C) to minimal amount and
stored at refrigerated temperature until further analyses.

3.7 Fast atom bombardment mass spectrometry (MS)

200 pl of purified unknowns collected were evaporated to dryness at 37°C under
N2 gas. Methanol was added to the collected fraction to decrease its boiling point, and
accelerating the evaporation. Unknown compounds were analyzed for their molecular

weights using -eVFABMS. Conditions used were previously mentioned in identification

of DNMG and NMG.

3.8 Nuclear magnetic resonance (NMR)

NMR analyses were conducted at Max T. Rogers NMR Facility, Michigan State
University, E. Lansing. NMR spectra were acquired on a Varian VXR-SOOS
spectrometer. The spectra were recorded in deuteriorated methanol (CD3OD) at a

temperature of 25°C. The 1H spectral width of 12ppm was acquired with a recycle time

109

 

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of 4 seconds. Data were fourier transformed to 65k points and referenced relative to
tetramethylsilane (TMS).

To ensure removal of solvent containing proton, samples were subjected to
vacuum drying (room temperature/15 min). Residue was reconstituted with 0.7 m1
deuteriorated methanol (CD 30D) and transferred to NMR tube.

3.9 Standards

Detail on limonoid standards were mentioned in Study I/Part I (3.5).
4. Results and discussion

4.1 Hexamethoxyflavone (HX)

Separation of compounds in dimethyldichloromethane fraction using silica
column chromatography resulted in isolated polymethoxylatedflavones in fraction 15
(ethylacetate/chloroform, 2:8).

Subsequent separation of fraction 15 using analytical HPLC with up to 100111
sample load was successful (Rs 1.4 [N = 979). Application of excess sample load, where
common injection volumes for analytical HPLC is 10 or 20 ul, resulted in peak
broadening. However, obtained separation allowed resolved peak of unknown 3 from
sinensitin and nobiletin. Figure 33 shows separation of polymethoxylated flavones in
fraction 15. Retention times (RT) were 21.4 (sinensitin), 26.6 (unknown 3), 30.9
(nobiletin), 34.8 (3 ,4,5,6,7,8,3 ’,4’—heptamethoxyflavone), 3 8.1 (scutellarein
tetramethylether), and 49.7 (tangeretin) minutes.

This separation condition demonstrated the advantages of analytical HPLC
application for purification. The advantages of using analytical HPLC compared to

preparative HPLC include 1) higher separation resolution (smaller packing materials, at

 

 

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least 2-time, greatly improves separation quality), 2) lower solvent consumption and
organic waste generation (lower flow rate, at least 5 times, reduce the use of organic
solvent, particularly important during method development), 3) shorter conditioning time
(5—time column volume is ideally required for adequate conditioning), 4) minimized
pump work.

Figure 34 shows polymethoxylated flavone standards and isolated unknown 3. It
is shown that this separation allowed isolation of unknown 3 without other interfering
polymethoxylated flavones. Both relative retention and UV spectrum of unknown 3
(Figure 35) were matched with those reported by Sendra et al.(1988) (Figure 18).
Identical UV spectra taken from three different positions of the unknown-3 peak ensured
that the isolated peak was relatively pure.

The molecular weight of unknown 3 was found to be 402 by FABMS (which
corresponds to molecular weight of hexamethoxyflavone). Negative FABMS showed a
peak at m/z 402 [M'] and 494 [M‘+Gly] in glycerol matrix. There was no peak at m/z
402 in NBA matrix.

The obtained 1H NMR (500MHz, CD3OD) spectrum was 6 3.86, 3.88, 3.92, 3.94
(each 3H, s, OMe), 4.00 (6H, 3, 2x OMe), 7.08 (1H, s, H-8), 7.12 (2H, d, J=9 Hz, H-S’),

7.75 (1H, d, J=2 Hz, H-2’), 7.79 (1H, dd, J=2 Hz,H-6’). The resulted NMR spectra was
consistent to hexamethoxyflavone structure, based on Miyazawa et al.(1999) who
identified three polymethoxylated flavones (tetra-O-methylscutellarein, sinensitin, and

nobiletin) extracted from C. aurantium by EI-MS, 1H- and ”(j—NMR.

112

 

 

 

 

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4.2 Narirutin—4’-glucoside (NT-4’-G)

Preliminary isolation of methanol extract of Valencia peel by reverse phase
column chromatography showed that fraction 2 (first 150 ml 20%methanol) contained an
unknown 4 (potential narirutin—4’-glucoside).

The separation on reverse phase HPLC was achieved in 20 min. Injection volume
of 20111 appeared to be the largest sample load to maintain adequate separation of
unknown 4 in the fraction 2 (first 150 ml 20%methanol) (Figure 36). The increase of
sample load may become less flexible when compounds to be separated are minimally
retained, for example, separation of high polar compounds on highly nonpolar stationary
phase such as C18 column.

Figure 37 shows chromatogram of purified unknown 4 (on C18 column using
linear gradient starting from 15% to 26 % acetonitrile in 60 minutes). The HPLC
condition used allowed. isolation of unknown 4 with relatively high purification.

Figure 38 shows UV spectra of unknown 4 obtained from photo diode array
detector. Identical UV spectra taken from three different positions of the unknown-4
peak indicate that the isolated peak was relatively pure.

The molecular weight of unknown 4 was found to be 743 (corresponding to
narirutin—4’-glucoside molecular weight). -eVFABMS spectral data showed a peak at
m/z 765 [M—H+Na]+ and fragment ions at m/z 579 [M-H-gluccscr, and m/z 625 [M-H-
glucoseJrZNa]+ in NBA matrice. The m/z 625 [M-H—glucose + 2Na]+ was also found in

glycerol. The presence of m/z 765 [M—H+Na]+ and 787 [M-H+2Na]+ was confirmed by

Kumamoto et al. (1986) who purified and identified narirutin-4’-glucoside from Unshiu

orange peels using -eVFAB and NMR.

 

 

 

 

 

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118

 

 

 

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The result of 1H-NMR (500MHz, CD3OD) spectrum was 5 1.20 (3H, d, J=6 Hz,
rhamnose-Me), 3.00 ( 1H, dd, J=3, 17 Hz, H-3), 4.65 [1H, d, J=1 Hz, or—rhamnose (H-l”)],
5.50 (1H, dd, J=3, 12 Hz, H-2), 6.20 (2H, s, H-6, H-8), 7.04 (2H, d, J=8 Hz, H-2’, H-6’).
This NMR spectrum confirmed the presence of narirutin-4’-glucoside based on that
reported by Kumamoto et al. (1986).

5. Conclusion

Preliminary purification including soxhlet extraction to separate nonpolar and
polar compounds; and column chromatography to separate among the nonpolar/polar
compounds resulted in a simpler polymethoxylated flavone and flavanone glucosides
mixture for the subsequent HPLC isolation. Large sample load up to 100 ml can be use
in 3,5,6,7,3’,4’-hexamethoxyflavone isolation, but only up to 20 ml was allowed for
narirutin-4’-glucoside isolation. Based on data from UV, -eVFABMS, and 1H—NMR
spectra, these two unknowns were 3,5,6,7,3’,4’-hexamethoxyflavone and narirutin-4’-

glucoside.

119

 

 

6, References:

Broflflff V! . E.
flaronor

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Chen. 1.. Mom
thrones
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Dugo. P- 1101.
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1993.
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Food(

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and re

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11:11

 

 

 

 

6. References:

Bronner, W. E. and Beecher, G. R. 1995. Extraction and measurement of prominent
flavonoids in orange and grapefi'uit juice concentrates. J. Chromatogr. A.
705(2):247-256

Chen, J., Montanari, A. M., and Widmer, W. W. 1997. Two new polymethoxylated
flavones, a class of compounds with potential anticancer activity, isolated from
cold pressed Dancy Tangerine peel oil solids. J. Agric. Food. Chem. 45: 364-368

Dugo, P., Mondello, L., Dugo, L., Stancanelli, R., Dugo, G. 2000. LC-MS for the
identification of oxygen heterocyclic compounds in citrus essential oils. J.
Pharrnaceu. Biomed. Anal. 24(1): 147-154

Fong, C. H., Hasegawa, S., Miyake, M., Ozaki, Y., Coggins, Jr. C. W., and Atkin, D. R.
1993. Limonoids and their glucosides in Valencia orange seeds during fruit
growth and development. J. Agric. Food Chem. 41: 112-115

Gaydou, E. M., Bianchini, J ., and Randriamiharisoa, R. P. 1987. Orange and mandarin
peel oils differentiation using polymethoxylated flavone composition. J. Agric.
Food Chem. 35: 525-529

Hasegawa, S. 2000. Chapter 2: Biochemistry of limonoids in Citrus. In Citrus Limonoids:
Functional chemicals in Agriculture and Foods. Edited by Mark A. Berhow, Shin
Hasegawa, and Gary D. Manners. American chemical society, DC, p.9-30

Hasegawa, S., Bennett, RD, and Verdon, GP. 1980. Limonoids in citrus seeds: Origin
and relative concentration. J. Agric. Food Chem. 28 (5): 922-925

Hasegawa, S.; Fong, C.; Miyake, M.; Keithly, J.H. 1996. Limonoid glucosides in orange
molasses. J. Food Science. 61(3): 560-561

He, X., Lian, L., Lin, L., Bernart, M. W. 1997. High—performance liquid
chromatography-electrospray mass spectrometry in phytochemical analysis of
sour orange (Citrus aurantz'um L.). J. Chromatogr. A. 791( 1+2): 127-134

Hsu, W., Berhow, M., Robertson, G. H., and Hasegawa, S. 1998. Limonoids and
flavonoids in juice of Oroblanco and Melogold grapefruit hybrids. J. Food Sci. 63
(1): 57-60

Ishii, K., Furuta, T., Kasuya, Y. 2000. Mass Spectrometric identification and high
performance liquid chromatographic determination of a flavonoid glucoside
naringin in human urine. J. Agric. Food Chem. 48(1): 56-59

Kawaii, S., Tomono, Y., Katase, E., Ogawa, K., and Yano, M. 1999. Quantitation of
flavonoid constituents in Citrus fruits. J. Agric. Food Chem. 47:3565-3571

120

 

 

 

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and it}?
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Lambert. 1. B.
10111221111
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Kumamoto, H., Yoshiharu, M., Yoshitomi,I., Kozo, O., and Katsumi, Y. 1986. Structures
and hypotensive effect of flavonoid glycosides in unshiu peel. H. Studies on
physiologically active substances in citrus peel. Part VII. 35(5): 379-381

Lambert, J. B.; Shurvell, H. F.; Lightner, DA; and Cooks, KG. 1998. Chapter 13:
Ionization and mass analysis. In Organic Structural Spectroscopy. Published by
Prentice-Hall, Inc. NJ 07458. p. 346-391

Mabry, T. J ., Markham, K. R., and. Thomas, M. B. 1970. The systematic identification of
flavonoids. Springer—Verlag, New York

Manners, G.D.°, Hasegawa, S.; Bennett, RD. and Wong, KY. 2000. LC-MS and NMR
techniques for the analysis and characterization of Citrus limonoids. In Citrus
Limonoids: Functional chemicals in Agriculture and Foods. ACS Symposium
series 758. Edited by Mark A. Berhow, Shin Hasegawa, and Gary D. Manners.
American Chemical Society, Washington, DC. p. 43-45

Manthey, J. A. and Grohmann, K. 1996. Concentrations of hesperidin and other orange
peel flavonoids in citrus processing byproducts. J .Agric. Food. Chem. 44(3): 811-
814

Miyazawa, M., Okuno, Y., Fukuyama, M., Nakamura, S., and Kosaka, H. 1999.
Antimutagenic activity of polymethoxyflavonoids from Citrus aurantium. J.
Agric. Food Chem. 47( 12): 5239-5244

Miyake, Y., Yamamoto, K., Morimitsu, Y., and Osawa, T. 1997. Isolation of C-
glycosylflavone from lemon peel and antioxidative activity of flavonoid
compounds in lemon fruit. J. Agric. Food Chem. 45(12): 4619-4623

Ooghe, W. and Detavernier, C. M. 1997. Detection of the addition of Citrus reticulata
and hybrids to Citrus sinensis by flavonoids. J. Agric. Food Chem. 45(5): 1633-
1637

Ooghe, W. C., Ooghe, S. J., Detavernier, C. M., and Huyghebaert, A. 1994a.
Characterization of orange juice by flavanone glycosides. J. Agric. Food Chem.
42: 2183-2190

Ooghe, W. C., Ooghe, S. J., Detavernier, C. M., and Huyghebaert, A. 1994b.
Characterization of orange juice by flavanone glycosides. J. Agric. Food Chem.
42: 2183-2190

Ortuno, A., Garcia-Puig, D., Fuster, M. D., Perez, M. L., Sabater, F., Porras, 1., Garcia-

Lindon, A., and Del Rio, J. A. 1995 . Flavanone and Nootkatone levels in different
varieties of grapefiuit and pummelo. J. Agric. Food Chem. 43(1): 1-5

121

 

 

 

Ozaki 1,?0ng
1991. Li

Robards. K. i.
chroma‘t

Sawabe. .511 Mi
1.: am
glycosi

Sendra. 1. M-
periorr
metho:

Snemple. P. ‘.
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lakashi. K-
Trans;
alkalo

glyco:

 

 

 

Ozaki Y.; Fong, C.; Herman, Z.; Maeda, H., Miyake M.; Ifuku, Y.; and Hasegawa, S.
1991. Limonoid glucosides in Citrus seeds. Agric. Biol. Chem. 55(1): 137-141

Robards, K., Li, X., Antolovich, M., andBoyd, S. 1997. Characterization of citrus by
chromatographic analysis of flavonoids. J. Sci. Food Agric. 75(1): 87-101

Sawabe, A; Morita, M.; Kiso, T.; Kishine, H.; Ohtsubo, Y.; Minematsu, T.; Matsubara,
Y.; and Okamoto, T. 1999. Isolation and characterization of new limonoid
glycosides from Citrus unshiu peels. Carbohydrate Research. 315: 142—147

Sendra, J. M., Navarro, J. L., and Izquierdo, L. 1988. C18 solid-phase isolation and high
performance liquid chromatography/ultraviolet diode array determination of fully
methoxylated flavones in citrus juices. J. Chromatogr. Sci. 26: 443-448

Stremple, P. 1998. GC/MS analysis of polymethoxylatedflavones in citrus oils. J. High
Resol. Chromatogr. 21 (11): 587-591

Takashi, K., Yoshinobu, T., Takashisa, N., Hiroshi, T., Shigetaka, O. 1994.
Transglycosylation to hesperidin by cyclodextrin glucanotransferase from an
alkalophilic Bacillus species in alkaline pH and properties of hesperidin
glycosides. Biosci. Biotech. Biochem. 58(11):1990-4

122

 

 

 

Stud? [1]: D1511
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1. Abstract
Quantitz
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Study 111: Distributions of limonoids and flavonoids in edible and inedible fractions
of sweet oranges (Citrus sinensis)

1. Abstract
Quantitative analyses of limonoids and flavonoids were determined on different
fruit fractions including 1) seed, 2) peel, 3) peel press cake, 4) rag, 5) orange juice, and 5)
peel press liquid from three commercial orange varieties (Hamlin, Parson Brown, and
Valencia). Compounds analyzed were limonoid aglycones (limonin, nomilin,
deacetylnomilin, and obacunone), limonoid glucosides (limonin glucoside,
deacetylnomilinic acid glucoside, deacetylnomilin glucoside, nomilin glucoside,
nomilinic acid glucoside, and obacunone glucoside), flavanone glucosides (narirutin-4’-
glucoside, eriocitrin, narirutin, hesperidin, and didymin), polymethoxylated flavones
(sinensitin, 3,5,6,7,3’,4’-hexamethoxyflavone, nobiletin, scutellareintetramethylether,
3,4,5,6,7,8,3’,4’-heptamethoxyflavone, and tangeretin).

Seeds h ad the highest 0 oncentrations o f b 0th limonoid a glycones and limonoid
glucosides, and contained very low flavonoid levels. Peel and peel press cake had the
highest concentration of polymethoxylated flavones and flavanone glucosides. Peel press
liquid contained higher phytochemical content than juice with an exception of limonoid
glucosides, suggesting that limonoid glucosides were highly extractable through
commercial juice extraction. Water removal by pressing process in feed mill operation
extracted limonoid glucosides and polymethoxylated flavones from the peel into peel
press liquid, but concentrated limonoid aglycones in the peel press cake.

Flavanone glucosides were the predominant phytochemicals, followed by

limonoid glucosides, limonoid aglycones, and polymethoxylated flavones. Valencia

123

 

 

raj-jay had the
Hamlin had the
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variety had the highest content of limonoids and polymethoxylated flavones, while
Hamlin had the highest content of flavanone glucosides.
2. Introduction

There has been an increased interest in citrus secondary metabolites, since the
discovery of pharmacological properties of certain compounds found exclusively in citrus
products. The two major classes of secondary metabolites are limonoids and flavonoids.
Among 36 citrus limonoids identified, 13 of them were detected in sweet oranges.
Limonin and nomilin, previously described as the primary bitter compounds in orange
juices exhibited anti-cancer properties (Hasegawa et al., 1994). It was reported that
addition of glucose to limonoid glucosides does not modify the chemopreventive activity
of l imonoids (Miller et al., 1 992). This is important b ecause limonoid glucosides are
more abundant and they are tasteless, thus there is higher consumption of these forms.
Diets rich in citrus limonoids may prevent or deter the development of certain types of
cancers (Lam and Hasegawa, 1989, Miller et al., 1992, Wattenberg and Coccia, 1991,
Gould, 1993, Hasegawa et al., 1994, Lam et al., 1994, Miller et al., 1994, Miyagi et al.,
2000)

Flavonoids are widely found in the plant kingdom, but there are some groups that
are specific to Citrus, such as polymethoxylated flavones and several flavanone
glucosides. Flavonoids have been reported to act as antioxidants, anti-inflamatory,
antimicrobials, free radicals scavengers, antiallergic, and analgesic agents (Benavente-
Garcia et al., 1997). Due to their antioxidant properties and their ability to absorb UV
light, flavonoids may act in all stages of the carcinogenic process (Kandaswami et al.,

1991). Epidemiological studies have suggested that flavonoid consumption is associated

124

 

 

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with a reduced risk of cancer (Kawaii et al., 1999) and heart disease (Benavente-Garcia et
al., 1997).

During 2001—2002, the world production of citrus fruit was approximately 73
million metric tons, of which 49% was marketed as fresh fruit and 42% was converted
into p rocessed products (FAS/USDA, 2 003). T hirty m etric tons o f p rocessed o ranges
produce a large amount of residue. From the citrus industry standpoint, it is important to
increase the utilization of byproducts to help maximize profits and minimize wastes.
Since limonoids and flavonoids have many potential beneficial properties, many fields
such as nutritional science, phytochemistry, chemistry, food science, and the citrus
industry need to know the distribution and concentration of these compounds in both
edible parts and waste materials. The purpose of this study was to investigate the
distribution and to determine the concentrations of these compounds in three commercial
sweet orange varieties.

3. Materials and methods

3.1 Orange samples

Hamlin, Parson Brown, and Valencia varieties were studied. These three
varieties, having different processing characteristics were selected to provide more
complete results on phytochemical distribution in commercial sweet oranges. Table 72
(Appendix XI) presents processing qualities of orange varieties used in this research.
Orange fractions including 1) seeds, 2) peels, 3) peel press cake, 4) rags, 5) peel press
liquid, and 6) orange juice from these three varieties were obtained from Tr0picana

Products Company (Bradenton, FL).

125

 

 

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A single strength orange juice was pasteurized (95°C/2 sec), vacuum-sealed, and
stored at refrigerated temperature. Peel press liquid was prepared using a Vincent screw
press®, (Vincent Corporation, Tampa, FL). The pulp resulting from the pressing process
is termed “press cake”. The liquid squeezed from pulp is termed “press liquid” or “press
liquor”. Peel press liquid was pasteurized (95°C/2 sec), vacuum-sealed and stored at
refrigerated temperature (2-l_-1°C).

Rags (containing seeds), peels, and peel press cake were vacuum—sealed and
frozen (—20°C). The samples were shipped in Styrofoam containers to the Department of
Food Science and Human Nutrition, Michigan State University, E. Lansing, MI.

3.2 Sample preparation

Upon arrival, samples were immediately stored at —20°C for approximately one
week before analyses. Juice samples were held at refrigerated temperature (2i1°C) until
completely thawed. To ensure homogeneity, all containers of each sample were
combined and mixed thoroughly, sub-sampled, and collected into 100 ml glass bottles,
and then stored at —20°C until analyzed.

Samples of rags, peels, and peel press cake were freeze—dried, and then ground to
pass 1 mm screen using a UDY—Mill, Chicago, IL. The ground samples were stored at —
20°C until analyzed. Seeds were extracted twice with hexane (1 :4, W/V) at room
temperature to r emove orange 0 il b efore b eing milled w ith a UDY-Mill and stored in
glass vials as described above.

3.3 Studied compounds and standards

Studied compounds included limonoid glucosides, limonoid aglycones, flavanoid

glucosides, and polymethoxylated flavones. Standards, kindly donated by scientists from

 

 

 

 

l'SDA Dr. C
lohn A. Man
limonin glut
glucoside lb
llOAl, dee
dehydrolimc
nobiletin (N
limonin tl.
hesperitin ll
Sigma Cor:
(Sllr'lE). n.
Extrastmhg
3.4
3.4.
The
press liquj.
mint then
mixed uitl
With 1 x p
GT1
“hit 35 Ill
Hfl, Gro‘

uH Sl m

 

USDA, Dr. Gary D. Manners (Pasadena, CA), Dr. Mark A. Berhow (Peoria, IL), and Dr.
John A. Manthey (Winter Haven, FL), included deacetylnomilin (DNM), obacunone (O),
limonin glucoside (LG), deacetylnomilinic acid glucoside (DNAG), nomilinic acid
glucoside (NAG), obacunone glucoside (OG), obacunoic acid (OA), isoobacunoic acid
(10A), deoxylimonin (DL), 17—19—didehydrolimonoic acid (DDHLA), 19-
dehydrolimonoic acid (DHLA), limolinic acid (LA), rutaevin (R), sinensetin (ST),
nobiletin WBT), 3,4,5,6,7,8,3’,4’-heptamethoxyflavone (HP), and tangeretin (TT).
Limonin (L), nomilin (NM) hesperidin (HD), naringin (NG), neohesperidin (NHD),
hesperitin (HT), diosgenin (DN), coumarin (CM), quercetin (QT) were purchased from
Sigma Company (St. Louis, MO). Sinensetin (ST), scutellarein tetramethylether
(STME), narirutin (NT), didymin (DD), and eriocitrin (ERT) were purchased from
Extrasynthese, (Genay, F rance).

3.4 Extraction and analysis of limonoid aglycones and polymethoxylated flavones

3.4.1 Extraction

The extraction procedure was modified from Fong et a1. (1993). Juice and peel
press liquid were thawed at room temperature and heated in a water bath (82°C for 30
min), then cooled to room temperature. The juice or peel press liquid (10 ml) was then
mixed with 25 m1 of 0.5 M Tris buffer (pH 8) for 15 minutes and then acidified to pH 2
with 1 N HCl.

Ground, freeze-dried orange parts (peel, peel press cake, and rag) (1 g) was mixed
with 25 ml of 0.5 M Tris buffer (pH 8) for 15 minutes and then acidified to pH 2 with 1 N
HCl. Ground, freeze-dried orange seed (1 g) was mixed with 25 ml of 0.15 M Tris buffer

(pH 8) overnight (20 hours), and then acidified to pH 2 with 1 N HCl. The acidified

127

 

 

 

mixtures of p
flliIll.

Ethyl
was added I
decanted. E
combined. e‘
(Mitt nylo

poltmethor

The
acetonitrile
resulted \\"
uitb 50% I
Column (1-
lnjection r
puhmethc

Sir
limonoid

limonoid

 

 

mixtures of peel, peel press cake, rag and seed were heated in a water bath (82°C for 30
min).

Ethyl acetate (25 ml) containing 200 ppm butyrate hydroxytoluene (antioxidant)
was added to all samples, shaken for 15 minutes, and the ethyl acetate layer was
decanted. Ethyl acetate extraction was performed twice. The ethyl acetate layers were
combined, evaporated to dryness, and reconstituted with 10 ml methanol. Filtered extract
(0.45u nylon) was analyzed by HPLC. A flow diagram of limonoid aglycone and
polymethoxylated flavone extraction is presented in Figure 20 (Study I/part II).

3.4.2 High performance liquid chromatography (HPLC) analysis

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid aglycones and polymethoxylated flavones were
resolved with a gradient that started with 30% B, was 40% B in 20 minutes and ended
with 50% B at 50 minutes. Flow rate was lml/min. Separation was achieved on a C18
column (Luna: C18, 5n, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml).
Injection volume was 10 ul. Limonoid aglycones were detected at 210 nm, while
polymethoxylated flavones were detected at 340 nm.

Since seeds are rich in limonoid aglycones and low in polymethoxylated flavones,
limonoid aglycones analysis was carried out separately for seed extract. Separation of
limonoid aglycones was achieved on C18 column (Alltima: C18, 5n, 250 mm x 4.6 mm,
16 % carbon load, void time 2.02 minutes) and an isocratic mobile phase
(acetonitrile/methanol/water, 10:41:49). Flow rate was lml/minute and injection volume

was 10 ul. The HPLC system was described in Study I/Part I (3.4).

128

 

 

 

ldentit'
nomilin. and t
of external st
3.4.5.6183
were based .

3.5.6.7 .43

-
(J.,

polunethoxi
spectrometr)
study ll’par

response Tar

'J)
i)

'2)
(JI

.luic
l33°C for 5
ml) l\‘as mi
parts (peel.

.

10! l5 min

lh

we dtt;
“Penna,

recOustitu
HPLC a

1llartlll

 

 

 

Identification and quantitation of limonoid aglycones (limonin, deacetylnomilin,

nomilin, and obacunone), were based on retention time, UV spectra and response factors
of external standards. Identification of polymethoxylated flavones (sinensitin, nobiletin,
3,4,5,6,7,8,3’,4’-heptamethoxyflavone, scutellarein tetramethylether, and tangeretin)
were based on retention time and UV spectra obtained with external standards. For
3,5,6,7,3’,4’-hexamethoxyflaovne, identification was based on retention relative to other
polymethoxylated flavones, which was verified by negative fast atom bombardment mass
spectrometry (-eVFABMS) and nuclear magnetic resonance spectroscopy (NMR) in
study II/part II. The quantitations of polymethoxylated flavones were based on the
response factor determined for scutellarein tetramethylether.

3.5 Extraction and analysis of limonoid glucosides

3.5.1 Extraction

Juice and peel press liquid were thawed at room temperature, heated in water bath

(82°C for 5 min), and cooled to room temperature. The juice and peel press liquid (10
ml) was mixed with 25 m1 of 70% methanol for 15 minutes. Ground, freeze-dried orange
parts (peel, peel press cake, rag, and seed) (1 g) were mixed with 25 m1 of 70% methanol
for 15 minutes, and heated in a water bath (82°C for 5 min).

The samples were centrifuged (10,000X g for 10 minutes), and the supernatants
were decanted. The pellet was extracted again with 70% methanol. Combined
supematants were evaporated to approximately 2-3 ml at 40°C under vacuum, and
reconstituted with 10 ml methanol. Filtered extracts (0.45rt nylon) were analyzed by
HPLC. A flow diagram of limonoid glucoside extraction is presented in Figure 21 (Study

l/part II)

129

 

 

 

l 2 )
\J\
is.)
“—4

acetonitrile is
with 10‘? t. B .
column llur.
pith l mlr‘rn:
at El 0 run. '.
ldent
deacetylnon
were based
standards.
identificatit
previously
deacetylno:

deacetylng

response f;

 

 

3.5.2 High performance liquid chromatography (HPLC) analysis

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid glucosides were separated with linear gradient starting
with 10% B and ending with 26% B in 70 minutes. Separation was performed on C18

column (Luna: C18, Spa, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml)

with 1 ml/min flow rate and 10 ul injection volume. Limonoid glucosides were detected
at 210 nm. The HPLC system was described in Study I/Part I (3.4).

Identification and quantitation of limonoid glucosides (limonin glucoside,
deacetylnomilinic acid glucoside, nomilinic acid glucoside, and obacunone glucoside)
were based on retention time, UV spectra, and response factors obtained with external
standards. For deacetylnomilin acid glycoside and nomilin acid glucoside, the
identifications were based on retention relative to other limonoid glucosides which were
previously verified by —eVFABMS in study II/part II. The quantitation of
deacetylnomilin glucoside was based on the response factor determined for
deacetylnomilinic acid glucoside, while that of nomilin glucoside was based on the
response factor determined for nomilinic acid glucoside.

3.6 Extraction and analysis of flavanone glucosides

3.6.1 Extraction

Juice or peel press liquid was thawed at room temperature, heated in water bath
(82°C for 5 min), and cooled to room temperature. The juice or peel press liquid (10 ml)
was then mixed with 25 ml dimethylformamide/methanol (1:2) for 15 minutes. Ground

freeze-dried orange parts (peel, peel press cake, rag, and seed) (1 g) was mixed with 25

130

 

 

rnl dimethylit
til‘f iorSrn
The s;
decanted.
Combined su
and reconstit‘
HPLC. A t
iSlUdy'l’pan
3.6.2
The '

ll999l Flat

 

 

 

ml dimethylformamide/methanol (1:2) for 15 minutes, and then heated in a water bath
(82°C for 5 min).

The samples were centrifuged (10,000X g for 10 min), and the supernatant was
decanted. Extractions with dimethylformamide/methanol (1 :2) were done twice.
Combined supematants were evaporated to approximately 15 ml at 50°C under vacuum,
and reconstituted to 25 ml with. methanol. Filtered extract (0.4514 nylon) was analyzed by
HPLC. A flow diagram of flavonoid glucoside extraction is presented in Figure 22
(Study I/part II).

3.6.2 High performance liquid chromatography (HPLC) analysis

The HPLC analysis of flavanone glucoside was based on the method of Ooghe
(1999). Flavanone glucosides were separated on C18 column (Alltima: C18, 5 it, 250 mm
x 4.6 mm, 16 % carbon load, void time 2.02 minutes) with a mobile phase consisting of
0.01 M potassium phosphate monobasic (solvent A) and acetonitrile (solvent B). A
linear gradient starting at 10%B and ending at 30% B in 60 minutes was used. Flow rate
was 1 ml/min flow rate and injection volume was 10 n1. Flavanone glucosides were
detected at 280 nm. The HPLC system was described in Study I/Part I (3.4).

Identification and quantitation of eriocitrin, narirutin, hesperidin, didymin were
based on retention time, UV spectra, and response factors obtained with external
standards. For narirutin—4’-glucoside, the identification was based on retention relative to

other flavanone glucosides, which were previously confirmed by -eVFABMS and NMR
in study II/part II. The quantitation of narirutin-4’-glucoside was based on the response

factor determined for narirutin.

131

 

 

 

 

 

 

37 D.

The e

33 obseryatit
using I-u'ay
4. Results :
Anal
llaronoidsl '.
and their int
Tota
Mmmh
phytochemi
considering
much lone
lBraddock.

liquid 53m

limonoids
ltflimts sr
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liquid prt‘

C0merit n1

 

 

3.7 Data analysis

The experimental design had two main effects (orange varieties and fruit parts),
22 observations (22 compounds), and 2 replications. Statistical analyses were conducted
using 2-way analysis of variance (ANOVA) (Excel).

4. Results and discussion

Analysis of variance (ANOVA) of total phytochemical contents (limonoids and
flavonoids) in Table 8 and Table 9 show significant differences among varieties, fractions
and their interaction in both solid and liquid fractions (P E 0.01).

Total phytochemical content of sweet orange solid and liquid fractions were
shown in Table 10 and Table 11 and Figure 39 and Figure 40. Seeds had the highest total
phytochemical c ontent; followed b y p eels, p eel press c ake, a nd r ags. H owever, w hen
considering total orange waste produced, seed contributes to phytochemical content at a
much lower level than peel, since it accounts for only 0.5—1% of the fruit (wet wt.)
(Braddock, 199%), while peel accounts for almost 50% (wet wt.) (Braddock, 1995). For
liquid samples, peel press liquid contained higher phytochemical content than orange
juice.

Significant difference among varieties indicated that distribution patterns of these
limonoids and flavonoids were specific even though they were in the same species
(Citrus sinensis). In solid by-products, Valencia and Hamlin varieties contained
Significantly higher phytochemical content compared to Parson Brown variety, and in
liquid products (juice and peel press liquid) Hamlin contained highest phytochemical

content among three varieties.

132

 

 

 

 

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4.1 Limonoid aglycones

Limonoid a glycones o ccur in citrus s eeds in two f orms: d ilactone (closed D -
ring) and monolactones (open D—ring). The predominant form in mature seeds is
dilactones, such as limonin, while in other orange fractions only monolactones, such as
limonoate A-ring lactone (LARL), occur (F ong et al., 1993). The analytical method used
in this study measured both forms.

ANOVA of total limonoid aglycone content in solid fractions (Table 12)
detected significant differences among varieties, fractions and their interaction (P S 0.01).
Table 13 and Figure 41 show total limonoid aglycone concentrations in solid fractions.
Seeds had the greatest concentrations of limonoid aglycones. Limonoid algycone content
in the seeds was at least 40—time higher than that of peel press cake (the second highest-
limonoid aglycone fraction). There have been limited quantitative studies on limonoid
aglycone found in juice and fruit tissues from sweet oranges. Most studies focus on
limonin due to its bitterness problem.

Higher limonin concentrations were found in peel press cake compared to peel.
The peel press cake is the pulp obtained after peel press liquid is pressed from the peel to
reduce water in this waste residue. The extraction of soluble solids especially sugars (the
primary constituents of peel, pulp, and rag dry solid reported by Braddock, 1999b) into
the peel press liquid may concentrate limonin and explain why peel press cakes contain
more limonin than peel.

Valencia contained the most total limonoid aglycones, followed by Parson
Brown, and then Hamlin. Table 14 shows individual limonoid aglycone concentrations

in solid fractions. Seeds were the only fraction containing measurable amounts of the

137

 

 

 

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minor limonoid aglycones (nomilin, deacetylnomilin, and obcunone). In seeds, limonin
was the predominant limonoid aglycone, followed by deacetylnomilin, nomilin, and
obacunone. According to Fong et al. (1993), nomilin, commonly known as a major
limonoid aglycone, does not accumulate to a measurable concentration until late harvest
season. Low nomilin levels in this study may indicate that the orange samples used were
not in their late harvest season.

ANOVA of total limonoid aglycone content in liquid fractions (Table 15) showed
significant differences among varieties, fractions and their interaction (P S 0.01). Table
'16 and Figure 42 show total limonoid aglycone concentrations in liquid fractions. Peel
press liquid contained higher limonoid aglycones than juice. Valencia contained highest
limonoid aglycones, followed by Hamlin and Parson. Brown.

Table 17 shows individual limonoid aglycone concentrations in liquid fractions.
Limonin was the only limonoid aglycone detected in measurable quantity. Estimated
levels of limonin in peel press liquid (10 to 35 ppm) are considered relatively low.
Therefore, peel press liquid is not judged to be a good source for limonin. Limonin levels
in juice were similar to those reported for Valencia orange juice (Widmer, 1993).

Estimated total limonin consumption for one serving (240 m1) of Valencia orange
juice was 2 mg. Consumption of one Valencia orange fruit would be equivalent to 3.8

mg of limonin.

4.2 Limonoid glucosides

ANOVA of total limonoid glucoside contents in solid fiactions (Table 18)
detected significant differences among varieties, fractions and their interaction (P S 0.01).

Table 19 and Figure 43 show total limonoid glucoside concentrations in solid. fractions.

141

 

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"cads had

glucosides

pad press
glucosides

Valencia

Hamlin.

fractions
glucosid
confirm
Deacet}

seeds.

dslecte
Peel p]
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COME!

Pred(

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Seeds had the highest content of limonoid glucosides. Rags contained more limonoid
glucosides than peel. This may be partly due to crushing of seeds during juice extraction.

Limonoid glucosides in peel were significantly higher (P S 0.01) than those in
peel press cake. The results suggest that water-soluble compounds like limonoid
glucosides were extracted from the peel into peel press liquid during pressing process.
Valencia contained highest limonoid glucoside contents, followed by Parson Brown and
Hamlin.

Table 20 shows the individual limonoid glucoside concentrations in solid
fractions. Nomilin glucoside was the predominant glucosides in seed, while limonin
glucoside was the predominant in other orange fractions including juice and peel juice,
confirming previous reports (Herman et al., 1990, Ozaki et al., 1991, Fong et al., 1993).
Deacetylnomilinic acid and obacunoic acid were found in detectable levels only in the
seeds.

ANOVA of total limonoid glucoside content in liquid fractions (Table 21)
detected significant differences among varieties, fractions and their interaction (P S 0.01).
Peel press liquid contained less limonoid glucosides than juice (Table 22 and Figure 44).
However, these levels would be expected to increase many times in orange molasses
(peel press liquid end product). Valencia contained highest total limonoid glucoside
content, followed by Hamlin and Parson Brown.

Table 23 shows individual limonoid glucoside concentrations in liquid fractions.

Predominant limonoid glucosides in liquid samples were limonin glucoside and nomilinic

acid glucoside.

 

 

 

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Valencia oran1é'3 _iui
eQuit'alent to l 00 IT

4.3P0l3m61

ANOVA
detected significan
Table 35 and Fig
fractions. P0131116
al.. 19871. The
poltmcthoxylated
significantly niOi
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Grohmann H996
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Hamlin and Pars
ANOVA
detected sign-fi-
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Table 2'
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llai‘nnes than it

 

 

Estimated total limonoid glucoside consumption for one serving (240 ml) of
Valencia orange juice was 96 mg. Consumption of one Valencia orange fruit would be
equivalent to 100 mg of limonoid glucoside.

4.3 Polymethoxylated flavones

ANOVA of total polymethoxylated flavones in solid fractions (Table 24)
detected significant differences among varieties, fractions and their interaction (P S 0.01).
Table 25 and Figure 45 show total polymethoxylated flavone concentrations in solid
fractions. Polymethoxylated flavones are found mainly in the peel of citrus (Gaydou et
al., 1987). The flavedo (external part of the citrus peel) is particularly rich in
polymethoxylated flavones (Mouly et al., 1999). Peel and peel press cake contained
significantly more polymethoxylated flavones than the edible parts of the fruit.
Polymethoxylated flavone concentrations in Valencia peel as reported by Manthey and
Grohmann (1996) were slightly lower than obtained in this study. Higher recovery
obtained in this study may be attributed to utilization of heat (82°C for 30 min) during
extraction. Valencia contained highest polymethoxylated flavone content, followed by
Hamlin and Parson Brown.

ANOVA of total polymethoxylated flavone content in liquid fractions (Table 26)
detected significant differences among varieties, fractions and their interactions (P S
0.01).

Table 2 7 and Figure 4 6 s how total p olymethoxylated f lavone c oncentrations in
liquid fractions. Peel press liquid had a higher concentration of polymethoxylated

flavones than juice. The levels of polymethoxylated flavones in juices in this study were

152

 

 

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156

Valencia

Parson Brown

Hamlin

of sweet oranges.

xylated flavones in liquid fractions

Figure 46: Polymetho

 

similar to those re
Valencia had the g

Table 28 a
in solid and liquid
were nobiletin an
pohme‘thoxylated

Ooghe (

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Table 3
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\l'lllle 536d C01
mp0” by Mar

similar to those reported by Mouly et. a1 (1999). Among the three varieties studied,
Valencia had the greatest polymethoxylated flavone content.

Table 28 and Table 29 show individual polymethoxylated flavone concentrations
in solid and liquid fractions. Primary polymethoxylated flavones in these sweet oranges
were nobiletin and sinensitin, which accounted for approximately 36 and 27 % of total
polymethoxylated flavones in both solid and liquid fractions.

Ooghe (1999) described criteria for authentic orange juice. Seven compounds
are consistently present in sweet orange juice - sinensitin, 3, 5, 6, 7, 3’, 4’-
hexamethoxyflavone, nobiletin, 3,4,5,6,7,8,3’,4’-heptamethoxyflavone, scutellarein
tetramethylether, tangeretin, and one unidentified minor compound. The results obtained
in this study are consistent with this criterion for all orange fractions.

Estimated total polymethoxylated flavone consumption for one serving (240
ml) 0 f V alencia 0 range juice w as 1 .7 m g. C onsumption o f o ne V alencia 0 range fruit
would be equivalent to 2.1 mg of total polymethoxylated flavones.

4.4 Flavanone glucosides

ANOVA of total flavanone glucoside contents in solid fractions (Table 30)
detected significant differences among varieties, fractions and their interactions (P S
0.01).

Table 3 1 and Figure 4 7 show total flavanone glucoside c oncentrations in s olid
fractions. Peel and peel press cake contained the highest levels of flavanone glucosides,
while seed contained the lowest levels. The results reported here are consistent with the
report by Manthey and Grohmann (1996) for peel and are higher than those reported by

Kawaii et a1. (1999) who quantitatively determined flavonoids in edible fruit parts

157

 

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uithout using hea
greatest flat'anone
ANOVA
detected Sigrtifica
Table 33 and Fig
Peel press liquid
Similar to the 5
compared among
Table 34
solid and liquid
compounds in tl‘
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hesperidin. F13
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without using heat in their extraction procedure. Among three varieties, Hamlin had the
greatest flavanone glucoside content in solid fractions.

ANOVA for total flavanone glucoside contents in liquid fractions (Table 32)
detected significant differences among varieties, fractions and their interaction (P S 0.01).
Table 33 and Figure 48 show total flavanone glucoside concentrations in liquid fractions.
Peel press liquid had higher flavanone glucoside content than juice by at least two times.
Similar to the solid fractions, Hamlin had the highest flavanone glucoside content
compared among three varieties.

Table 34 and Table 35 show individual flavanone glucoside concentrations in
solid and liquid fractions, respectively. Hesperidin and narirutin were the predominant
compounds in the sweet oranges studied with hesperidin having the greatest amounts.

Seed was the only fraction that had a higher concentration of narirutin than
hesperidin. F lavanone glucosides found in these three cultivars were all rutinosides, the
nonbitter forms. Rutinosides are found in all Citrus, while the bitter neohesperidosides
are found in species related to pummelo (Ooghe, 1999).

Estimated total flavanone glucoside consumption for one serving (240 ml) of
Valencia orange juice was 88 mg. Consumption of one Valencia orange fruit would be
equivalent to 236 mg of total flavanone glucosides.

5. Conclusion

Different orange fractions exhibited varying concentrations of limonoids and
flavonoids. Seeds had the greatest concentration of limonoids (aglycones and
glucosides), while peels and peel press cake had the highest concentrations of

polymethoxylated flavones and flavanone glucosides. However, it may not be effective

162

 

 

 

 

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166

 

to isolate seeds
waste. ln addil
such as isolatior
Peel pre
exception of li
extractable thro
extracted limor
press liquid. bu‘
High co
juice consumpti
\ialenci
and pol§rtietho
glucosides.
Flavant
liquid samples

followed by lit‘

 

to isolate seeds from the waste stream, since seeds account for a small part of the total
waste. In addition, the use of seed to isolate limonoids would require additional steps
such as isolation and grinding.

Peel press liquid contained higher phytochemical content than juice with the
exception of limonoid glucosides, suggesting that limonoid glucosides were highly
extractable through commercial juice extraction. Pressing process in feed mill operation
extracted limonoid glucosides and polymethoxylated flavones from the peel into peel
press liquid, but concentrating limonoid algycones in peel press cake.

High content of limonoid glucosides in juice indicated their intake through orange
juice consumption would be high.

Valencia had the greatest concentrations of limonoids (aglycones and glucosides)
and polymethoxylated flavones, and Hamlin had the greatest concentration of flavanone
glucosides.

Flavanone glucosides were found as the predominant group in both solid and
liquid samples, accounting for approximately 60% of total phytochemicals studied,

followed by limonoid glucosides, limonoid aglycones, and polymethoxylated flavones.

167

 

 

6. References:

Benavente-Garc:
and prop

Braddock. R. l9
produc15

Braddock R. Z
cornpone
lohnlh'il

Braddock. R. l
citrus b}

Braddock. R. l‘
processi

Braddock. R. l‘

Gould. M. N. l
using re
mdmd

Hasegawa. 8,.
their an
Anteric

Hasegawa. 8..
and rel:

HElman. Z" F
juices l

KaItdasn'anti.
Allllpl‘t
in vim”
Kawaii. 3, Tc
flmon

”8 l’SDA. :

Gal'dou. E. 3

peel c
F00d:

 

6. References:

Benavente-Garcia, 0., Castillo, J., Marin, F. R., Ortuno, A., Del Rio, J. A. 1997. Uses
and properties of Citrus Flavonoids. J. Agric. Food Chem. 45(12): 4505-4515

Braddock, R. 1999a . Chapter 15: Flavonoids and limonoids. In: Handbook of citrus by—
products and processing technology. JohnWiley & Sons, Inc. pp. 209-219

Braddock, R. 1999b . Chapter 3: Composition, properties, and evaluation of fruit
components. In: Handbook of citrus by-products and processing technology.

JohnWiley & Sons, Inc. pp. 28

Braddock, R. 1 9990 . C hapter 1 O: D ried p ulp, p ellets, and m olasses. In: H andbook o f
citrus by—products and processing technology. JohnWiley & Sons, Inc. pp. 146

Braddock, R. 1999d. Chapter 16: Seed products. In: Handbook of citrus by-products and
processing technology. JohnWiley & Sons, Inc. pp. 222

Braddock, R. 1995. By—products of citrus fruit. Food Tech. Sep: 74—7 7

Gould, M. N. 1993. The introduction of activated oncogenes to mammary cells In Vivo
using retroviral vectors: a new model for the Chemoprevention of premalignant
and malignant lesions of the breast. J. Cell. Biochem. 17G: 66-72

Hasegawa, S., Miyake, M ., and Ozaki, Y. 1994. Biochemistry of citrus limonoids and
their anticarcinogenic activity. In: Food Phytochemistry 1: Fruits and Vegetables.

American Chemical Society. pp. 198-219

Hasegawa, S., Bennett, R. D., and Verdon, C. P. 1980. Limonoids in citrus seeds: origin
and relative concentration. J. Agric. Food Chem. 28: 922-925

Herman, Z., Fong, C. H., Cu, B, Hasegawa, S. 1990. Limonoid glucosides in orange
juices by HPLC. J. Agric. Food Chem. 38: 1860-1861

Kandaswami, C., Perkins, E., Soloniuk, D. S., Drzewiecki, G., Middleton, E., Jr. 1991.
Antiproliferative effects of citrus flavonoids on a human squamous cell carcinoma

in vitro. Cancer Letters (Shannon, Ireland). 56(2): 147-52

Kawaii, S. Tomono, Y., Katase, E., Ogawa, K., and Yano, M. 1999. Quantitation of
flavonoid constituents in Citrus fruits. J. Agric. Food Chem. 47: 3565-3571

PAS/USDA. 2003. Situation and outlook for orange juice. http:// www. fas. usdagov

Gaydou, E. M., Bianchini, J ., and Randriamiharisoa, R. P. 1987. Orange and mandarin
peel oils differentiation using polymethoxylated flavone composition. J. Agric.
Food Chem. 35: 525-529

168

 

 

concentra
Food Ag:

Gil-Izquierdo, .5
industrial
Agric. PC

Lam. K. I. Zha
induced t

Lani. K. I. and
neoplasia

Manthey. .l. and
tlax‘onoit

Miller, E. (3.. G
and Ian
Food Tet

Miller. E. (3.. C
and I an
limonin

Miyagi. it. 0
Azoxtn‘.
229

Mouly. P.P.. C
orange
Analusi:

Ooghe. \ll 19
Processi

Olakj \ F011;
AfiuB

Wattenberg L
Plfidyl‘
Canine

l'€\'€l‘S€
1472-1.

 

Guadagni, D. G., Maier, V. P. Tumbaugh, J. G. 1974. Effect of subthreshold
concentrations of limonin, narigin, and sweeteners on bitterness perception. J. Sci.

Food Agric. 25: 1199-1205

Gil-Izquierdo, A.; Gil, I. M.; Ferreres, F. 2002. Effect of processing techniques at
industrial scale on orange juice antioxidant and beneficial health compounds. J.

Agric. Food Chem. 50(18): 5107-5114

Lam, K. T., Zhang, J ., and Hasegawa, S. 1994. Citrus limonoids reduction of chemically
induced tumorigenesis. Food Technology. 1994 (Nov): 104-108

Lam, K. T., and Hasegawa, S. 1989. Inhibition of Benzo[a]pyrene—induced forestomach
neoplasia in mice by citrus limonoids. Nutr. Cancer. 12: 43—47

Manthey, J. and Grohmann, K. 1996. Concentrations of hesperidin and other orange peel
flavonoids in citrus processing by-products. J. Agric. Food Chem. 44: 811-814

Miller, E. G., Gonzales-Sanders, A. P., Couvillon, A. M., Binnie, W. H., Hasegawa, S.,
and Lam, L. K. T. 1994. Citrus limonoids as inhibitors of oral carcinogenesis.

Food Technology. 1994(Nov):110-114

Miller, E. G., Gonzales-Sanders, A. P., Couvillon, A. M., Wright, J. M., Hasegawa, S.,
and Lam, L. K. T. 1992. Inhibition of hamster buccal pouch carcinogenesis by

limonin 17-B—D-glucopyranoside. Nutrition Cancer. 17(1): 1-7

 

Miyagi, Y., Om, A. S., Chee, K. M., and Bennink, M. R. 2000. Inhibition of
Azoxymethane-induced colon cancer by orange juice. Nutr. Cancer. 36(2): 224-
229

Mouly, P.P., Gaydou, E.M., and Arzouyan, C. 1999. Separation and quantitation of
orange juices using liquid chromatography of polymethoxylated flavones.

Analusis. 27: 284—288

Ooghe, W. 1999. Flavonoids as authenticity markers for Citrus sinensis juice. Fruit
processing. 9(8): 308-313

Ozaki, Y., Fong C. H., Herman, Z., Maeda, H., Miyake, M., Ifuku, Y., and Hasegawa, S.
Agric Biol. Chem. 55(1): 137-141

Wattenberg, L. W. and Coccia, J. B. 1991. Inhibition of 4-(methylnitrosamino)—1-(3-
pyridyl)-1—butanone carcinogenesis in mice by D-limonene and citrus fruit oils.

Carcinogenesis 12(1): 115-117

Widmer, W. W. 1993. Improvement in the quantitation of limonin in Citrus juice by
reverse-phase high performance liquid chromatography. J. Agric. Food Chem. 39:

1472-1476

169

 

 

Study IV: EffeCt
produ

1. Abstract
Waste 51:
materials to prod
other industries.
after line treatm
(net tit). presse
for the content
poljmethoxylate
With lin
((12%) leached f
shutting increa
liquids due to li
of limonoid glt
poljtnethoxyla'
increased phjtt‘
2. Introducti
During
million metric
ml“ Processet
MS process

mild? peel.

“Eight lAItor

 

Study IV: Effect of lime treatment on of limonoid and flavonoid content in by-
products from orange juice process

1. Abstract

Waste streams from orange juice manufacturing provide inexpensive raw
materials to produce value-added by-products for health, pharmaceutical, and a variety of
other industries. Limonoid and flavonoid in waste products were measured before and
after lime treatment. Peel and rag, primary waste materials, were treated with 0.3% CaO
(wet wt.), pressed to yield press cakes and press liquid. These fractions were analyzed
for the content of limonoid aglycones, limonoid glucosides, flavanone glucoside and
polymethoxylated flavones.

With lime treatment, more limonoid aglycones (25%) and limonoid glucosides

(12%) leached from press cake into press liquid (both in rag and peel). There was a trend

 

 

 

showing increased phytochemical content were released from press cakes into press
liquids due to lime treatment. In seed, lime treatment (0.3% CaO wet wt.) resulted in loss
of limonoid glucosides, but had no effect on limonoid aglycone, flavanone glucoside and
polymethoxylated flavone content. The results suggested that lime treatment resulted in
increased phytochemical content in press liquid especially limonoids.

2. Introduction

During 2001-2002, the world production of citrus fruit was approximately 73
million metric tons, of which 49% was marketed as fresh fi'uit and 42% was converted
into processed products (FAS/USDA, 2003). With such significant amounts of fruit
being processed, large quantities of waste materials are produced. Waste products
include peel, rag, core, seed, and pulp. These residues, accounting for 50% of the fruit

weight (Anonymous, 1998); have been used or converted into a variety of end products

170

 

lBraddoclc, 19991
for animal feeds.
the raw material 2
Direct lin
facilitate the de
materials (Bradd
are processed. li:
waste materials
reduction) and
necessary proce
Waste 1
(Ozaki et al.. 1
and flavonoids
sec0ndm~ ma
have Pharmac
flal'onoids int
Statengers‘ a
well as $1661
limonoids h;
1989. M iller
weeds“ 8

 

 

(Braddock, 1999). Most dried pulp (final form) of the remained waste materials is used
for animal feeds. These dried end products have lighter weight and longer shelf life than
the raw material and thus enable stable shipment and storage prior to use.

Direct lime treatment has been used widely in fruit and vegetable processing to
facilitate the dehydration of pulp, clarification of juice, and for other pectinacious
materials (Braddock, 1999). During feed mill operations, where orange waste materials
are processed, lime is used to aid the dewatering process of waste materials. Lime treated
waste materials are subsequently pressed to remove water (approximately 10% moisture
reduction) and then dried to about 10% final moisture content. Lime treatment is a
necessary processing aid to reduce energy consumption and to increase drying rate.

Waste materials from orange juice processing are rich sources of limonoids
(Ozaki et al., 1995, Hasegawa et al., 1996, Braddock, 1999, Braddock and Bryan, 2001)
and flavonoids (Manthey and Grohmann, 1996, Braddock, 1999). These principal
secondary metabolites, specifically found in Citrus species, have been demonstrated to
have pharmaceutical and industrial applications. Claimed beneficial properties for
flavonoids include antioxidant, anticancer, anti-inflamatory, antimicrobial, free radical
scavengers, anti-allergic, and analgesic properties (Benavente—Garcia et al., 1997), as
well as sweetening agents (Horowitz, 1986, Bar et al., 1990, and Borrego et al., 1991).
Limonoids have been shown to have chemopreventive activities (Lam and Hasegawa,
1989, Miller et al., 1989, Lam et al., 1994, Miller et al., 2000, Tian et a., 2 001), and

antifeedant activities (Alford and Bentley, 1986, Bentley et al., 1988, Serit et al., 1991,

Mendel et al., 1993, Ruberto et al., 2002).

 

 

 

 

Citrus flat
aglycones are m
commercial citrus
was to investigat
major wasre mate
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Valencia) were
and rag with se.

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and Control;

To 5

 

Citrus flavonoids are more soluble (Di Mauro et al., 2000) and limonoid
aglycones are more stable (Miyake et al., 1993) under alkali conditions. Since
commercial citrus w aste s treams are generally lime-treated, the o bj ective o f this study
was to investigate influences of lime treatment on limonoid and flavonoid content in
major waste materials (peel, rag, and seed).

3. Materials and methods

3.1 Waste samples

Waste materials from three orange varieties (Hamlin, Parson Brown, and
Valencia) were obtained from the Tropicana Products Company (Bradenton, FL). Peel
and rag with seed were vacuum-sealed and shipped frozen to Michigan State University.
These waste samples were stored at -20°C until sample preparation.

3.2 Sample preparation

3.2.1 Lime treatment

Samples were thawed at room temperature. Seeds were manually separated from
rag. Half-cut peels were sliced. into approximately 12 mm-wide sections with a
mechanical slicer. Each sample was mixed thoroughly with 0.3% CaO (wet wt), which
prepared as slurry by addition of water (10 ml). For example, one kg of peel was added
with 3 g of CaO which was initially mixed with 10 ml of water. The control samples
were mixed with the same amount of water as used in the CaO slurry. Both lime-treated
and control samples were incubated for two days.

3.2.2 Pressing

To simulate the industrial pressing process that partially extracts liquid from the

residues, each sample :t lime treatment was processed with a domestic juice extractor

172

 

 

 

lluiceratOI). Liqu
a screen to CXPE
remaining pulp 1‘
analyzed. Thep
screen using a \V
3.3 Studi
Studied <
glucosides. and
USDA. Dr. Gar
lohn A. Manth:
limonin gluco:
glucoside (NA
110A). deox
dehydrolimon
nobiletin (NB
Limor

MD). hespt
from Sigma
(STEVIE). na
Exrrassnthes
3.4)

The

Samples 12

 

 

(J uicerator). Liquid was recovered by centrifugal force which pressed the residue against
a screen to expel the fluid juice. The liquid is termed “pressed liquid”, while the
remaining pulp is termed “pressed cake”. The press liquid was stored at -20°C until
analyzed. The press cake materials were freeze-dried, ground to pass through a 1mm
screen using a Wiley Mill, and stored in a desiccated chamber at -20°C until analyzed.

3.3 Studied compounds and standards

Studied compounds included. limonoid glucosides, limonoid aglycones, flavanoid
glucosides, and polymethoxylated flavones. Standards, kindly donated. by scientists from
USDA, Dr. Gary D. Manners (Pasadena, CA), Dr. Mark A. Berhow (Peoria, IL), and Dr.
John A. Manthey (Winter Haven, FL), included deacetylnomilin (DNM), obacunone (O),
limonin glucoside (LG), deacetylnomilinic acid glucoside (DNAG), nomilinic acid
glucoside (NAG), obacunone glucoside (0G), obacunoic acid (OA), isoobacunoic acid
(10A), deoxylimonin (DL), 17-19-didehydrolimonoic acid (DDHLA), l9-
dehydrolimonoic acid (DHLA), limolinic acid (LA), rutaevin (R), sinensetin (ST),
nobiletin (NBT), 3,4,5,6,7,8,3’,4’-heptamethoxyflavone (HP), and tangeretin (TT).

Limonin (L), nomilin (NM) hesperidin (HD), naringin (NG), neohesperidin
(NHD), hesperitin (HT), diosgenin (DN), coumarin (CM), quercetin (QT) were purchased
from Sigma Company (St. Louis, MO). Sinensetin (ST), scutellarein tetramethylether
(STME), narirutin (NT), didymin (DD), and eriocitrin (ERT) were purchased from
Extrasynthese, (Genay, France).

3.4 Moisture content analyses

The AOAC (1990) method for moisture in animal feed (7.003) was followed.

Samples (2 g) were dried (50°C) under vacuum condition for 12 hours.

 

 

 

35 Extract
3.5.1 Extrz
The extra
We thawed at I
cooled to room ti
Tris buffer (pH 5
Ground.
Tris buffer (pH
freeze-dried see
hours). and the
press calte. rag
Ethyl at
was added to
decanted. Eth
combined. eV:
extract (0.4511
polnrrethoxyl
3.5.2‘
The
acetonitrile
rcsoh‘ed wit
\t‘itlr 509-0 B

milllllll (Lu

 

 

3.5 Extraction and analysis of limonoid aglycones and polymethoxylated flavones

3.5.1 Extraction

The extraction procedure of Fong et al. (1993) was modified from. Press liquids
were thawed at room temperature and heated in a water bath (82°C for 30 min), then
cooled to room temperature. Ten ml of press liquid was then mixed with 25 ml of 0.5 M
Tris buffer (pH 8) for 15 minutes and then acidified to pH 2 with 1 N HCl.

Ground, freeze—dried press cake (peel or rag) (1 g) was mixed with 25 ml of 0.5 M
Tris buffer (pH 8) for 15 minutes and then acidified to pH 2 with 1 N HCl. Ground,
freeze-dried seed (1 g) was mixed with 25 ml of 0.15 M Tris buffer (pH 8) overnight (20
hours), and then acidified to pH 2 with 1 N HCl. The acidified mixtures of peel, peel
press cake, rag and seed were heated in a water bath (82°C for 30 min).

Ethyl acetate (25 ml) containing 200 ppm butylated hydroxytoluene (antioxidant)
was added to all samples, shaken for 15 minutes, and the ethyl acetate layer was
decanted. Ethyl acetate extraction was performed twice. The ethyl acetate layers were
combined, evaporated to dryness, and reconstituted to 10 ml with methanol. Filtered
extract (0.4511 nylon) was analyzed by HPLC. A flow diagram of limonoid aglycone and
polymethoxylated flavone extraction is presented in Figure 20 (Study I/part 11).

3.5.2 High performance liquid chromatography (HPLC) analysis

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid aglycones and polymethoxylated flavones were
resolved with a gradient that started with 30% B, was 40% B in 20 minutes and ended
with 50% B at 50 minutes. Flow rate was lml/min. Separation was achieved on a C18

column (Luna: C18, 511, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml).

 

 

lrrjection rolurne
pohmethoxylated
Since seed
limonoid aglycor
limonoid aglycor
16 ”at carbon
(acetonitrile met
waleul. The
ldentifrc
nomilin. and 01;
of external star
14.56.78.314
Were based or
3.5.6.7.3‘51‘1l
Pillmtethoxyl;
Spectrometry
Smd)’ llpan

resl’onse fact.

 

 

Injection volume was 10 1.11. Limonoid aglycones were detected at 210 nm, while
polymethoxylated flavones were detected at 340 nm.

Since seeds are rich in limonoid aglycones and low in polymethoxylated flavones,
limonoid aglycone analysis was carried out separately for seed extract. Separation of
limonoid aglycones was achieved on C18 column (Alltima: C18, 51.1, 250 mm x 4.6 mm,
16 % carbon load, void time 2.02 minutes) and an isocratic mobile phase
(acetonitrile/methanol/water, 10:41:49). Flow rate was lml/minute and injection volume
was 10 ul. The HPLC system was described in Study I/Part I (3.4).

Identification and quantitation of limonoid aglycones (limonin, deacetylnomilin,
nomilin, and obacunone), were based on retention time, UV spectra and response factors
of external standards. Identification of polymethoxylated flavones (sinensitin, nobiletin,
3,4,5,6,7,8,3’,4’—heptamethoxyflavone, scutellarein tetramethylether, and tangeretin)
were based on retention time and UV spectra obtained with external standards. For
3,5,6,7,3’,4’-hexamethoxyflaovne, identification was based on retention relative to other
polymethoxylated flavones and was verified. by negative fast atom bombardment mass
spectrometry (—eVFABMS) and nuclear magnetic resonance spectroscopy (NMR) in
study II/part II. The quantitations of polymethoxylated flavones were based on the
response factor determined for scutellarein tetramethylether.

3.6 Extraction and analysis of limonoid glucosides

3.6.1 Extraction

Press liquids were thawed at room temperature, heated in a water bath (82°C for 5

min), and cooled to room temperature. Ten ml of press liquid was mixed with 25 ml of

70% methanol for 15 minutes.

 

 

 

 

Ground: f“
Bl were mixed WT
133°C for 5 min ’-

The 521ij
were dCCEDICd-
supematants W61
reconstituted tO '
HPLC. A flout
llpartlll.

3.6.2 Hit

The mc
acetonitrile (sol
with 10% B an
column (Luna:
with 1 ml min
atllOnrn. Th

ldentif
deacetylnomil
were based 0
Standards.
identification
Subsequently

deacetylrrorn

 

 

Ground, freeze-dried solid fractions (peel press cake, rag press cake, and seed) (1
g) were mixed with 25 m1 of 70% methanol for 15 minutes, and heated in a water bath
(82°C for 5 min).

The samples were centrifuged (10,000X g for 10 minutes), and the supematants
were decanted. The pellet was extracted again with 70% methanol. Combined
supematants were evaporated to approximately 2-3 ml at 40°C under vacuum, and
reconstituted to 10 ml with methanol. Filtered extracts (0.4514 nylon) were analyzed by
HPLC. A flow diagram of limonoid glucoside extraction is presented in Figure 21 (Study
'l/part 11).

3.6.2 High performance liquid chromatography (HPLC) analysis

The mobile phases consisted of 3 mM phosphoric acid (solvent A) and
acetonitrile (solvent B). Limonoid glucosides were separated with linear gradient starting
with 10% B and ending with 26% B in 70 minutes. Separation was performed on C18

column (Luna: C18, 5pc, 250 mm x 4.6 mm, 17.8 % carbon load, void volume 2.5 ml)

with 1 ml/min flow rate and 10 ul injection volume. Limonoid glucosides were detected
at 210 nm. The HPLC system was described in Study I/Part I (3.4).

Identification. and quantitation of limonoid glucosides (limonin glucoside,
deacetylnomilinic acid glucoside, nomilinic acid glucoside, and obacunone glucoside)
were based on retention time, UV spectra, and. response factors obtained with external
standards. For deacetylnomilin acid glycoside and nomilin. acid glucoside, the
identifications were based on retention relative to other limonoid glucosides and
subsequently verified by —eVFABMS in study II/part II. The quantitation of

deacetylnomilin glucoside was based on the response factor determined for

176

 

 

 

deacetylnomilinic
response factor d
3.7 Extra

3.7.1 Ext

Press liq

min). and cooler
dimethylforrnan
Ground.

“'35 mixed “'1'
heated in a war
The sar
decanted.

E
Combined sup
and reconstitu
uric. .1 fl
(Study [Part ‘

3722‘

The }
119991 F11“.
”6 mm. 1
Olll M pot;

linear Slfidir

 

deacetylnomilinic acid glucoside, while that of nomilin glucoside was based on the
response factor determined for nomilinic acid glucoside.

3.7 Extraction and analysis of flavanone glucosides

3.7.1 Extraction

Press liquids were thawed at room temperature, heated in water bath (82°C for 5
min), and cooled to room temperature. Ten ml of press liquid was then mixed with 25 ml
dimethylformamide/methanol (1 :2) for 15 minutes.

Ground, freeze-dried solid parts (peel press cake, rag press cake, and seed) (1 g)
was mixed with 25 ml dimethylformamide/methanol (1 :2) for 15 minutes, and then
heated in a water bath (82°C for 5 min).

The samples were centrifuged (10,000X g for 10 min) and the supernatant was
decanted. Extractions with dimethylforrnamide/methanol (1 :2) were done twice.
Combined supematants were evaporated to approximately 15 ml at 50°C under vacuum,
and reconstituted to 25 ml with methanol. Filtered extract (0.4511 nylon) was analyzed by
HPLC. A flow diagram of flavonoid glucoside extraction is presented in Figure 22
(Study I/part 11).

3.7.2 High performance liquid chromatography (HPLC) analysis

The HPLC analysis of flavanone glucoside was based on the method of Ooghe
(1999). Flavanone glucosides were separated on C18 column (Alltima: C18, 5pc, 250 mm
x 4.6 rrnn, 16 % carbon load, void time 2.02 minutes) with a mobile phase consisting of
0.01 M potassium phosphate monobasic (solvent A) and acetonitrile (solvent B). A

linear gradient starting at 10%B and ending at 30% B in 60 minutes was used. Flow rate

177

 

 

n'asl ml'min ant
381mm. The HP:
ldentifica

based on retent
standards. For r.
other flavanone
study lllpart ll.
factor deternrin
3.8 Den

The p:
limonoid and
were a limit-
interaction be
duplicate. Q'
cake and pre

4. Results:
Cali

readily hydr
recommend
11Eattnentr

l999).

Th

ESiel'ifiCat

 

 

was 1 ml/min and injection volume was 10 pl. Flavanone glucosides were detected at
280 nm. The HPLC system was described in Study I/Part I (3.4).

Identification and quantitation of eriocitrin, narirutin, hesperidin, didymin were
based on retention time, UV spectra, and response factors obtained with external
standards. For narirutin-4’-glucoside, the identification was based on retention relative to
other flavanone glucosides and subsequently confirmed by -eVFABMS and NMR in
study II/part II. The quantitation of narirutin-4’-glucoside was based on the response
factor determined for narirutin.

3.8 Data analysis

The paired-t test was to determine the differences significant difference in
limonoid and flavonoid content between control and lime-treated samples. Since there
were a limited number of samples, potential variety differences and the potential
interaction between treatment and variety were not tested. Analyses were conducted in
duplicate. Quantitative comparisons of the limonoid and flavonoid content between press
cake and press liquid are based on the dried weight of raw materials.

4. Results and discussion

Calcium oxide (CaO, lime) is commonly used to treat citrus waste materials, as it
readily hydrates with water in the residues, and forms calcium hydroxide [Ca(OH)2]. The
recommended concentration of lime ranges between 02—05% (wet wt. basis). Lime
treatment reduces waste acidity and de-esterifies pectin in the waste materials (Braddock,
1999)

The pKa of pectin is between 3.55 and 4.10, depending on the degree of

esterification (Plaschina et al., 1978). Neutralization prevents protonation of carboxylate

 

 

 

 

 

 

groups on the pet
that is favorable i
pectic acids \t'hic
liberating water .
Moisture
79%. respective
for peels. comp
The raw mater
Water holding
contents estjm
64% in peel 31
in our study 1
Braddock. 1g
Content of pr
005i in rag 1
may be beca
treated pres'
hqulCiS sug.
molecules 1
Tab
Press liqui
C0mpound

trend (p

A

 

 

 

groups on the pectin molecule and prevents formation of hydrogen bonding, a condition
that is favorable for hydration. De-esterification of pectin under basic condition produces
pectic acids which react with the calcium ions (Ca2+) in lime to form calcium pectate salt,
liberating water and methanol during subsequent pressing (Braddock, 1999).

Moisture contents of peel and rag raw materials were approximately 66% and
79%, respectively (Table 36). These initial moisture values are relatively low, especially
for peels, compared to industrial data (SO-82%) (Anonymous, 1998 and Braddock, 1999).
The raw materials were frozen and stored before analyzed; therefore some reduction in
water holding capacity and/or direct moisture loss may have occurred. The moisture
contents estimated in press cake with and without lime treatment were approximately
64% in peel and 75% in rag. Moisture reduction by pressing (2 % in peel and 7% in rag)
in our study is relatively low compared to industrial data (10%) (Anonymous, 1998 and
Braddock, 1999). Lime treatment had no significant effect (P 2 0.05) on moisture
content of peel press cake but resulted in significantly decreased moisture content (P S
0.05) in rag press cake (Table 37). The small loss in water content due to lime treatment
may be because peels were drier than commercial peels. Further, the pH values of lime-
treated press liquids (Table 38), ranged from 5.07 to 5.71. The relatively acidic press
liquids suggest that less than optimal cross-linking between calcium ions and pectin
molecules were achieved and that minimal de-esterification occurred.

Table 39 and Table 40 show limonoid aglycone concentrations in press cakes and
press liquids of peel and rag with and without lime treatment. L imonin was the only
compound detected in measurable quantity in both peel and rag samples. There was a

trend (P S 0.05) for lime treatment to decrease limonin content in press cakes

 

 

 

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(approximately
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concentration 1
lnpeel. ln fee.
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Press liquids
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“llh limonin
Lime

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(approximately 14% in both peel and rag) and to increase limonin content in press liquids
(approximately 24% in peel and 22% in rag).

Table 38 presents the Brix (°Bx) values of press liquids from peel and rag. °Bx
measurement is a rapid method commonly used to measure orange juice and molasses
concentration (soluble solid content). °Bx values of press liquid were ~11 in rag and ~14
in peel. In feed mill operation, press liquids are evaporated to 72°Bx to produce molasses
(the press liquid end products). Microbial spoilage is prevented by the low water activity
in the molasses. The 72°Bx molasses is 5-6 times more concentrated than press liquids.

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in press liquids, press liquid may not be a direct source for limonin. However, the

limonin concentration would. be expected to be increased significantly in 72°Bx.

 

 

Comparison of the total limonin content from press cake and press liquid showed
that there was significant lower (P S 0.5) total limonin concentration in lime—treated
samples compared to controls (Table 41). Loss of total limonin was approximately 10%
in peel and approximately 11% in rag. Thus, it can be concluded that lime treatment
results in the degradation of limonin.

Table 42 and table 43 show limonoid glucoside concentrations in press cakes and
press liquids of peel and rag with and without lime treatment. Limonin glucoside,
nomilin glucoside, and nomilinic acid glucoside were detected in measurable quantity,
with limonin glucoside being the primary compound.

Lime treatment resulted in a significant decrease (P S 0.05) in limonoid glucoside
content in press cake and, a significant increase (P S 0.05) in press liquid from both rag

and peel samples. These results indicate that limonoid glucosides were released into

184

 

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press liquid due
groups dependrr
[based on those
pH 5.07-5.71, 5
group are ionic
Compar
liquid showed ‘
lime-treated so:
was approxime
treatment resul
Lewls
73°Bx molass
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stable throng}
glucoside 105
Conducted.
Table
Cakes and .
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nobiletin.
Iangeretiu. 3

Lime

decrease in l

 

press liquid due to lime-treatment. Limonoid glucosides possess one or two carboxylate
groups depending on the particular limonoid with approximate pKa] = 2.7 and pKa; = 4.7
[based on those reported for limonoic acid (USDA, 2003)]. Lime-treated samples had a
pH 5.07-5.71, so that all of first carboxyl group and a significant fraction of the second
group are ionic that results in increased compound solubility in aqueous solutions.

Comparison of the total limonoid glucoside content from press cake and press
liquid showed that there w as significant loss (P S O .5) o f total limonoid glucosides in
lime—treated samples compared to controls (Table 44). Loss of total limonoid glucosides
was approximately 5.2 % in peel and 8.6 % in rag. Thus, it can be concluded that lime
treatment results in a small degradation of limonoid glucoside compounds.

Levels of limonoid glucosides in press liquids may be increased up to 5 times in
72°Bx molasses. Hasegawa et a1. (1996) suggested that press liquids could be a good
source for limonoid glucosides. Even though limonoid glucosides are reported to be
stable through juice processing conditions (Hasegawa, 2000), evaluation of limonoid
glucoside losses due to heat evaporation during molasses production has not been
conducted.

Table 4 5 and T able 4 6 show p olymethoxylated flavone c oncentrations in p ress
cakes and press liquids of peel and rag with and without lime treatment.
Polymethoxylated flavones detected were sinensitin, 3,5,6,7,3’,4’-hexamethoxyflavones,
nobiletin, 3,4,5,6,7,8,3’,4’—heptamethoxyflavone, scutellareintetramethylether, and
tangeretin. Sinensitin and nobiletin were the principal compounds.

Lime treatment effect on polymethoxylated flavone content was limited. A

decrease in polymethoxylated flavone content (approximately 2.6%) in peel press cake

188

 

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and a s mall increase in polymethoxylated f lavone c ontent (approximately 13%) in rag
press liquid were statistically detected (P _<_ 0.05). Similar to limonoid aglycones, these
polymethoxylated flavones have a tendency to remain in press cake since they are not
water soluble. The polymethoxylated flavones were minimally leached into press liquid
during pressing process. The concentration of polymethoxylatedflavones would be
expected to be much greater in molasses. There was a small degradation (P 5 0.5) of
total polymethoxylated flavones due to lime treatment (Table 47). Loss of total
polymethoxylated flavones was approximately 2.4 % in peel and 3.7 % in rag.

Table 48 and Table 49 show flavanone glucoside concentrations in press cakes
and press liquids of peel and rag with and without lime treatment. Flavanone glucosides
detected in rag and peel samples included narirutin-4’-glucoside, eriocitrin, narirutin,
hesperidin, and didymin, with hesperidin being the major compound.

The effect of lime treatment on flavanone glucoside content was minimal. A
significant increase (P S 0.05) in flavanone glucoside content due to lime treatment was
observed only in rag press liquid (approximately 19%).

Feel is a better source for flavanone glucosides compared to rag and seed, and
press cake contained higher flavanone glucoside content than press liquid. It was
observed that hesperidin was approximately 48 times higher in peel press cake than in
peel press liquid. Its concentration may be greatly increased in molasses. However
hesperidin has a limited solubility and with concentration of the press liquid to produce
molasses, hesperidin would become saturated and the heSperidin would be expected to

precipitate.

192

 

 

 

 

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Tablef
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Table 50 presents the total flavanone glucoside content from press cake and press
liquid in peel and rag residues with and without lime treatment. There were no
significant effects of lime treatment (P _<_ 0.5) on the total flavonoid glucosides in either
peel or rag residues. Degradation of flavanone glucosides due to lime treatment is not
significant.

Table 51 Table 54 show distributions of limonoids and flavonoids in orange seeds
with and without lime treatment. Since seed is a part of waste residue and may not be
homogeneously distributed within the rag fraction, seeds were studied independently.
Influence of lime treatment was studied without pressing process, as it already had
relatively low moisture content. Lime treatment resulted in a significant decrease (P s
0.05) in limonoid glucoside content in seed, while no effects (P 2 0.05) were observed on
the concentrations of other compounds (limonoid aglycones, polymethoxylated flavones,
and flavanone glucosides).

It should be noted that seeds are a particularly rich source of limonoid aglycones
and glucosides. The data presented in Table 51 and Table 52 are expressed as g/lOOg
while the data in most tables are expressed as mg/Kg. However, when considering total
orange waste produced, seeds account for only 0.5-1% of the fruit (wet wt.) (Braddock,
1999), while peels account for almost 50% (wet wt.) (Braddock, 1995).

5. Conclusion

Analyses of limonoids and flavonoids in press cake and press liquid (peel and rag)
demonstrated that lime treatment did not appreciably alter their content in citrus waste.
However, when. the extract from both rag and peel were analyzed, there were significant

losses of limonoid aglycones (~ll% in peel and rag), limonoid glucosides (~5% in peel

 

 

 

 

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and~1 ”/0 in
lime treatmen
Lime
demonstrated
glucosides fn
Lime
treatment di
glucoside of
The
pressing pn

physical ch:

 

 

and ~l3% in rag), and polymethoxylated flavones (~9°/o in peel and ~3% in rag) due to

lime treatment.

Lime treatment had the greatest effect on limonoid glucosides. It was

demonstrated that lime treatment resulted in a significant migration of limonoid
glucosides from solid wastes in to press liquids.

Lime treatment resulted in loss of limonoid glucosides in seeds, but lime
treatment did not change limonoid aglycone, polymethoxylated flavone, flavonoid
glucoside of seeds.

The extent of compound leaching from pressed solids to pressed liquids during

pressing process depends on the nature of the compounds (such as solubility) and

physical characteristics of raw materials (such as surface area).

200

 

 

 

 

6, Reference

AOAC. 1990
chemis

Anomrnous.
Proces

Alford. A. R.
sprucl
38

Bar. A. Bo
dihyc

Benavente-C
and l

Borrego. F
p0te
lngr

Braddock.
ana‘.
Che

Braddock.
citr

Braddock.

Di Mauro

pn
res

PAS 1‘s]

Hasega“
F
d
\

 

 

6. References:

AOAC. 1990. Official methods of analysis of the association of official analytical
chemists. 4th Edition. Edited by Williams, S. AOAC, Inc., Virginia.

Anonymous. 1998. Chapter 5.9: Feed mill Operations. In: The Orange Book. Tetra Pak
Processing Systems AB, Lund, Sweden. pp. 81-82

Alford, A. R., and Bentley, M. D. 1986. Citrus limonoids as potential antifeedants for the

spruce budworm (Lepidoptera: Tortricidae). J. Economic Entomology. 79(1): 35-
38

Bar, A., Borrego, F. Benavente, O. Castillo, J., Del Rio, J. A. 1990. Neohesperidin
dihydrochalcone: properties and applications. Food Sci. Technol. 23: 371-376

Benavente-Garcia, 0., Castillo, J., Marin, F. R., Ortuno, A., Del Rio, J. A. 1997. Uses
and properties of Citrus Flavonoids. J. Agric. Food Chem. 45(12): 4505-4515

Borrego, F., Castillo, J., Benavente—Garcia, 0., Del Rio, J. A. 1991. Applications

potential of the citrus origin sweetener neohesperidin dihydrochalcone. Int. Food
Ingredients. 2: 23—26

Braddock, R. and Bryan, C. 2001. Extraction parameters and capillary electrOphoresis

analysis of limonin glucoside and phlorinin citrus byproducts. J. Agric. Food
Chem. 49: 5982-5988

Braddock, R. 1999. Chapter 10: Dried pulp, pellets, and molasses. In: Handbook of
citrus by—products and processing technology. John Wiley & Sons, Inc. p. 136

Braddock, R. 1995. By-products of citrus fruit. Food Tech. Sep: 74-77

Di Mauro, A., Fallico, B., Passerini, A., Maccarone, E. 2000. Waste water from citrus
processing as a source of hesperidin by concentration on styrene-divinylbenzene

resin. Istituto di Industrie Agrarie, Universita di Catania, Italy. J. Agric. Food
Chem. 48(6): 2291-5.

Fong, C. H., Hasegawa, S., Miyake, M., Ozaki, Y., Coggins, Jr. C. W., and Atkin, D. R.

1993. Limonoids and their glucosides in Valencia orange seeds during fruit
growth and development. J. Agric. Food Chem. 41: 112—1 15

PAS/USDA. 2003. Situation and outlook for orange juice. http://www.fas.usda. gov
Hasegawa, S. 2000. Biochemistry of limonoids in Citrus. In: Citrus Limonoids:

Functional chemicals in Agriculture and Foods. ACS Symposium series 758. Eds.

M. A. Berhow, S. Hasegawa, and G. D. Manners. American Chemical Society,
Washington, DC. ppZI

201

 

 

 

 

Hasega\\'&_ S
orang

Horowitz. R
Medi
Eds.
l 75

Lam. L. K
chen
546

Lam, L. K.
neoi

Manthey. .l
pee?
814

Mendel. N.
citr
(Le

Miller. E.
Qlu
Plij

Miller. is.
K.
C Ell

Miyake. l

P1‘éBcliiii;

 

 

Hasegawa, S., Fong, C. H., Miyake, M., and Keithly, J. H. 1996. Limonoid glucosides in
orange molasses. J. Agric. Food Chem. 61(3):560-56l

Horowitz, R. M. 1986. Taste effects of flavonoids. In: Plant Flavonoids in Biology and

Medicine: Biochemical, Pharmacological, and Structure-activity relationships.
Eds. Cody, V., Middleton, E., Jr., and Harborne, J. B. Liss, New York. pp. 163-

I75

Lam, L. K. T., Zhang, J., Hasegawa, S., and Schut, H. A. J. 1994. Inhibition of
chemically induced carcinogenesis by citrus limonoids. ACS Symposium Series.

546 (Food phytochemicals for cancer prevention 1): 209-219

Lam, L. K. T. and Hasegawa, S. 1989. Inhibition of benzo(a)pyrene-induced forestomach
neOplasia in mice by citrus limonoids. Nutrition and Cancer. 12(1): 43-47

Manthey, J. A. and Grohmann, K. 1996. Concentration of hesperidin and other orange
peel flavonoids in citrus processing byproducts. J. Agric. Food Chem. 44(3): 811-

814

Mendel, M. J., Alford, A. R., Rajab, M. S., and Bentley, M. D. 1993. Relationship of
limonoid structure to feeding deterrence against fall armyworm

citrus
(Lepidoptera: Noctuidae) larvae. Environmental Entomology. 22(1): 167-173

Miller, E. (3., Record, M. T., Binnie, W. H., and Hasegawa, S. 2000. Limonoid
glucosides: systemic effects on oral carcinogenesis. Phytochemicals and

Phytopharmaceuticals. 2000: 95-105

Miller, E. G., Fanous, R., Rivera-Hidalgo, F ., Binnie, W. H., Hasegawa, S., and Lam, L.
1989. The effect of citrus limonoids on hamster buccal pouch

K. T.
carcinogenesis. Carcinogenesis. 10(8): 1535-1537

Miyake, M., Ibana,N., Ayano, S., Ozaki, Y., Maeda, H., Ifuku, Y., Hasegawa, S. 1993.
Recovery of seeds from processing waste of natsudaidai (Citrus natsudaidai

Hayata). Nihon Shokuhin K’ogy'o Gakkaishi (J. Food Science and Technology).
40(11): 807-813

Ozaki, Y., Ayano, S., Inaba, N., Miyake, M., Berhow, M. A., and Hasegawa, S. 1995.
Limonoid glucosides in fruit, juice, and processing by—products of Satsuma

mandarin (Citrus unshiu Marcov.). J. Agric. Food Chem. 60(1): 186-190

Plaschina, I.G., E.E. Braudo, and VB. Tolstoguzov. 1978. Circular dichroism studies of
pectin solutions. Carbohydrate Res. 60: 108.

202

 

 

 

 

Ruberto. G .. l
limom
Spodo
Food'

Seni.ll..lsh
Citrus
Biol.

Tian, Q, Mi
hum:
40(2

USDA Sam

Whistler. R
Fem

 

 

Ruberto, G ., Renda, A ., Tringali, C ., Napoli, E. M ., Simmonds, M. S. J. 2 002. Citrus
limonoids and their semisynthetic derivatives as antifeedant agents against
Sp0d0ptera f rugiperda larvae. A s tructure- a ctivity r elationship study. J. A gric.
Food Chem. 50(23):6766-6774

Serit, M., Ishida, M., Kim, M., Yamamoto, T., and Takahasi, S. 1991. Antifeedants from
Citrus n atsudaidai H ayata a gainst termite R eticulitermes speratus Kelbe. Agric.
Biol. Chem. 55(9): 2381-2385

Tian, Q., Miller, E. G., Ahmad, H. Tang, L., Patil, B. S. 2001. Differential inhibition of
human breast cancer cell proliferation by citrus limonoids. Nutrition Cancer.
40(2): 180-184

USDA Sample prOposal. Oct. 2003. Membrane-based process for debittering citrus juice.
htgfl/sbtdc.org/pdf/sbirianrgegrrmosalsmdf

Whistler, R. L., and Daniel, J. R. 1985. Carbohydrates. In: Food Chemistry. Ed. 0. R.
Fennema. Marcel Dekker, Inc. New York. pp. 124-125

 

 

 

203

 

Studr l: Anahtice
llavonoid

o limonoid
retention.
0 Limonoid
retention.
' Screening
“ith pot:

glucosid<

Extractic

by heatir

70% me

Extracti.
Smdl’ H: lsolati
' Prelimii
Simpler
StaleH

° Based
dearer}

3‘5\6~

 

RESEARCH CONCLUSION

Study 1: Analytical methodology suitable for isolation and quantitation of limonoids and

flavonoids in sweet orange

Limonoid aglycones and polymethoxylated flavones had similar chromatographic

retention.

Limonoid glucosides and flavanone glucosides had similar chromatographic

retention.

Screenings of compounds from different orange fractions found four unknowns
with potential to be deacetyl nomilin glucoside, nomilin glucoside, narirutin-4’-
glucoside, and 3,5,6,7,3’,4’-hexamethoxyflavone.

Extraction of polymethoxylated flavones and limonoid aglycones was improved
by heating (82°C for 30 min).

70% methanol in water was suitable for limonoid glucoside extraction.

Extraction of flavanone glucosides required dimethylformamide.

Study II: Isolation and identification of selected limonoids and flavonoids

Preliminary extractions and purifications of orange seeds and peels provided a
simpler and more concentrated extract for purification of unknowns by analytical-
scale HPLC.

Based on UV, MS, and NMR spectra, four unknowns were identified to be
deacetyl nomilin glucoside, nomilin glucoside, narirutin—4’-glucoside, and

3,5,6,7,3 ’,4’-hexamethoxyflavone

204

 

Study lll: Distribi
sweet

0 Flaranont

glucoside

o Valencia

content v

o The resr

aglycone
sources

0 Peel p
polyrne'

0 Orange

Study 1V: Efie
from“.

' With 1

 

 

 

Study III: Distributions of limonoids and flavonoids in edible and inedible fractions of

sweet oranges (Citrus sinensis)
F lavanone glucoses were the predominant phytochemicals, followed by limonoid
glucosides, limonoid aglycones, and polymethoxylated flavones.
Valencia had highest phytochemical content, except for flavanone glucoside
content which was highest in Hamlin.
The results show that rags containing seeds are a good source for limonoid
aglycones and limonoid glucosides, while peel and peel press cake are good
sources for flavanone glucosides and polymethoxylated flavones.
Peel press liquid is a potential source for limonoid glucosides and
polymethoxylated flavones after evaporation to the molasses end-product.

Orange juice is a good source of limonoid glucosides.

Study IV: Effect of lime treatment on of limonoid and flavonoid content in by—products

from orange juice process
With lime treatment, more limonoid aglycones (25%) and limonoid glucosides
(12%) leached from press cake into press liquid (both in rag and peel).
There was a trend showing increased phytochemical content were released from
press cakes into press liquids due to lime treatment.
In seed, lime treatment (0.3% CaO wet wt.) resulted in loss of limonoid
glucosides, but had no effect on limonoid aglycone, flavanone glucoside and
polymethoxylated flavone content.
The results suggested that lime treatment resulted in increased phytochemical

content in press liquid especially limonoids.

205

 

 

 

0 In this re
varieties
phitoche
treated 5
statistic:
blockst
effects
apparer
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interac

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andt

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com

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per

al‘l

RECOMMENDATIONS FOR FUTURE RESEARCH

In this research the lime treatment study was conducted using three sweet orange
varieties to represent the whole orange population and lime treatment. Effects on
phytochemical content were analyzed using paired t-test to compare between lime—
treated samples and controls, since limited samples were available. It would be more
statistically meaningful to employ either 1) randomize complete block design [(3
blocks (3 varieties) and 2 factor (lime treatment and orange fractions] to remove the
effects of variety in order to make the effects of lime and orange fraction more
apparent, or 2) factorial design (3 varieties X 2 lime treatments X 2 waste fractions)
to obtained the influence of variety, lime treatment, waste fraction, and their
interaction.

Addition of lime to citrus waste is limited, because the lime—treated waste is primarily
used for the animal feed. However to improve recovery of limonoids and flavonoids
in press liquid, higher lime concentrations, more effective mixing between lime and
waste materials (continuous mixing and application of smaller size of waste material)
and different types of lime could be studied.

Orange-byproducts are rich sources of flavonoids and limonoids but isolation of these
compounds are very expensive. It may be warranted to conduct the absorption study
(in vitro or in vivo) using orange by-products directly such as freeze-dried peel
powder, freeze—dried rag-with-seed powder, or spray-dried orange molasses. Direct

application of these wastes would be greatly profitable to citrus industry.

206

 

 

APPENDICES

207

 

 

APPENDIX I

Screening of limonoids and flavonoids in different orange fractions.

208

 

 

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216

APPENDIX 11

Purification of limonoid aglycones by
preparative high performance liquid chromatography.

217

Seed
Homogenized in Tris buffelr (0.05M, pH 8), 1:10 W/V
Filter (paper No. 4)
Acidified to le3 with 1 N HCl
Extracted with ethyl acetate, 1:1 V/V
Centrifuged at 4'000 rpm, 10 min
I

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| i
Nonpolar compounds Polar compounds
(Neutral limonoid aglycones) (Acidic limonoids, limonod

l glucosides, flavonoids,
acids, sugars, salts,. . .)

Evaporated to minimal volume
(50°C/vacuum condition)

Reconstituted with methanol

|
Filter (0.45 u)
|
Preparative HPLC

Figure 5 7: Flow diagram for the isolation of limonoid aglycones from orange seeds.

218

 

HPLC condition

 

Column: Econosphere (C18, lOu, 250mmx22mm, Alltech)

Mobile phase: acetonitrile/methanol/water (10:41:49), Fong et a1. (1993)

Flow rate: 10ml/min
Injection volume: 100 u]
Detection: 210 nm

Identification: based on retention time of standards

219

 

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220

APPENDIX III

Purification of limonoid glucosides by
preparative high performance liquid chromatography.

221

Extraction of limonoid glucosides

Flow diagram for the limonoid glucoside isolation from orange seeds is present in

Figure 28 (Study II/part 1).

HPLC condition

 

Column: Econosphere (C18, lOu, 250mmx22mm, Alltech)

Mobile phase: 17.5 % acetonitrile in 3 mM phosphoric acid, 10 ml/min
Injection volume: 100 u]

Detection: 210 nm

Identification: based on retention time of standards

222

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223

APPENDIX IV

Purification of polymethoxylated flavones by
preparative high performance liquid chromatography.

224

Extraction of polymethoxylated flavones
Flow diagram for the polymethoxylated flavone isolation from orange peels is
present in Figure 32 (Study II/part II).

HPLC Conditions

 

Column: Econosphere (C18, lOu, 250mmx22mm, Alltech)

Mobile phase: 65% B

Where, A = water/acetonitrile/ propanol/acetic acid (81 : 15:3:1)
B = water/acetonitrile/propanol/acetic acid (40:56:321)

Injection volume: 100 pl

Flow rate: 8 ml/min

Detection: 340 nm

225

 

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APPENDIX V

Preliminary trials of mass spectrometric techniques on flavonoids
and limoniods extracted from sweet orange (C. sinensis).

227

Sample preparations
Seed (limonoid source) and peel (flavonoid source)
70% methanol extract

Evaporated at 40°C/vacuum condition

1

 

Reconstituted in Reconstituted in
10% acetonitrile in 100% acetonitrile
3 mM phosphoric acid (Nonpolar compounds)

(Polar compounds)

I l
l l 7 l l 1

FAB GCMS LCMS FAB GCMS LCMS
(-eV, +eV) (-eV, +eV)

Direct probe fast atom bombardment mass spectrometry (FABMS)

The mass spectra were obtained using a JEOL HX-llO double-focusing mass
spectrometer (JOEL USA, Peabody, MA) operating in the both positive and negative ion
modes. Ions were produced by bombardment with a beam of Xe atoms (6 keV).
Matrixes used were glycerol and m-nitrobenzyl alcohol (NBA). The accelerating voltage
was 10 kV and the resolution was set at 3000. The instrument was scanned from m/z 0 to
1500, data were collected from m/z 50-1500. The sample was mixed with the matrix,
which supported ionization on a probe tip and was then inserted into the instrument.
Electron impact ionization gas chromatography mass spectrometry (EI-GC-MS)

Gas chromatography column were a) 3/18: 30m DB1 0.32mm ID, 0.25um film
b) 4/ 10: 30m DB5 0.32mm ID, 0.25um film. Mobile phase was helium gas. Program

started with 50°C for 10 minutes, then 320°C for 3 minutes for 3/ 18 column; and started

228

 

with 50°C for 10 minutes, then 320°C for 30 minutes for 4/ 10 column. Flow rate was set
at 1 nil/minutes. Injection volume was 5 ul.

The mass spectrometry condition was consisted of JEOL-AX—SOSH double
focusing mass spectrometer coupled to a Hewlett-Packard 5890] gas chromatograph via a
heated interface approximately 280°C, ion source temperature 220°C, electron energy
70eV, and m/z range 45-750.

Electrosprav liquid chromatography mass spectrometry (ESI-LC-MS)

Liquid chromatography column used was self-packed Vydac, C18 reverse-phase
capillary column with 75 u ID and approximately 5-8 cm in length. Mobile phases were
0.1% formic acid in 2% acetonitrile (solvent A) and 0.1% formic acid in 95% acetonitrile
(solvent B). Program started from 20% to 60%B in 30 minutes, then 95%B in 5 minutes,
then down to 2%B and equilibrating for 20 minutes at 200 nl/min. Injection volume was
0.5 ul for peel and 1 ul for seed extracts.

The mass spectrometry program used was a 60 minute run with triple play to
acquire a full scan, a zoom scan (to determine the charge state), and MS/MS spectrum (to

look at fragmentation at 35% nonnalized collision energy). The settings for the MS (tune

file) were: spray voltage 2.4, and capillary temperature of 93°C.

229

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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240

APPENDIX VI

Limonoid and flavonoid content in rag and orange juice
prepared domestically.

241

Frozen whole oranges of Hamlin, Parson Brown, and Valencia varieties
Thawed at room temperature
Cut in halves

Squeezed by domestic orange juice extractor

I 1

Orange juice Rag (ruptured juice vesicle retained
after squeezing)

 

Collected in 100-ml, glass bottles Freeze—dried

Stored at -20°C Ground to pass 1 mm screen

Stored at -20°C

I

Analyzed for the content of limonoid aglycones,
limonoid glucosides, flavanone glucosides,
and polymethoxylated flavones using

procedure described in Study 1H and IV

Figure 61: Flow diagram for the sample preparations and analyses of domestically
prepared orange juice and rags.

242

 

Table 67: Individual limonoid aglycone concentrations in domestically prepared juice and
rags of sweet oranges.

 

 

 

 

 

Variety Sample ppm (mg/Kg or mg/L) 0 %CVr
L NM DNM O
Hamlin Rags 7004.5 T2 T T
Juice 4905.1 T T T
Parson Brown Rags 10805.1 T T T
Juice 4500.4 T T T
Valencia Rags 17708.8 T T T
Juice 9.0044 T T T

 

L = limonin, NM = nomilin, DNM = deacetylnomilin, O = obacunone, IN = 2, 2Trace

Table 68: Individual limonoid glucoside concentrations in domestically prepared juice
and rags of sweet oranges.

 

 

 

Variety Sample ppm (mg/Kg or mg/L) 0 %CVT
LG DNAG DNG NG NAG
Hamlin Rags 2153029 T2 10505.6 97800.6 74403.8

Juice 36702.2 1100.1 8702.8 21702.6

 

Juice 26403.5 8.8042 8800.7 19302.4

 

T

Parson Brown Rags 1792016 T 7507.0 1024020 70800.8
T
T

Valencia Rags 214101.1 60006.2 84704.0 55400.0
Juice 32603.7 T 9.00123 3701.4 17800.9

0
>-l-1'-l>-l’-l*-lm

 

LG = limonin glucoside, DNAG = deacetylnomilinic acid glucoside, DNG =
deacetylnomilin glucoside, NG = nomilin glucoside, NAG = nomilinic acid glucoside,
OG = obacunone glucoside, 1N=2, 2Trace

243

 

 

Table 69: Individual polymethoxylated flavone concentrations in domestically prepared
juice and rags of sweet oranges.

 

 

 

Variety Sample ppm (mg/Kg or mg/L) 0 %CV1
ST HX NBT HP STME TT
Hamlin Rags 2801.2 5.0051 4300.8 1200.5 1301.0 3704.0

Juice 06014.2 01014.6 1.0090 03014.0 0.5069 02012.2
Parson Brown Rags 19.6008 4.5024 21.3008 6.1019 7.4026 1.7044

Juice 0.7044 0201.6 0704.0 0201.8 0301.7 00704.2

Valencia Rags 3002.8 6.9042 3203.3 1105.2 1104.5 3509.1

Juice 1905.8 0408.3 2.00109 06019.6 06014.5 0.20377
ST = sinensitin, HX = 3,5 ,6,7,3’,4’—hexamethoxyflavone, NBT = nobiletin, HP =
3,4,5,6,7,8,3’,4’-heptamethoxyflavone, STME = scutellarein tetramethylether, TT =
tangeretin, 1N=2

Table 70: Individual flavanone glucoside concentrations in domestically prepared juice
and rags of sweet oranges.

 

 

 

Variety Sample ppm (mg/Kg or mg/L) 0 %CV]
NT-4'—G ERT NT HD DD
Hamlin Rags 72200.4 26101.3 1608010 9615015 83200.5

Juice 8101.5 3700.8 14900.1 48200.0 3700.8

 

Parson Brown Rags 62901.4 26001.3 1635000 9502013 779007
Juice 2602.2 1005 .6 6803 .4 40304.0 2004.0

 

Valencia Rags 47301.2 17102.8 1485010 8791001 63200.3
Juice 2106.1 8000.4 5400.3 37201.6 1601.5

 

NT-4’-G = narirutin-4’-glucoside, ERT = eriocitrin, NT = narirutin, HD = hesperidin, DD
= didymin, 1N=2

244

 

APPENDIX VII

Chromatographic retention and UV spectra of minor citrus limonoids compared to
limonin and nomilin (common limonoids).

245

HPLC condition

Column: C18 (Alltima, Alltech: Sit, 250mmx4.6mm, 16 % carbon load)

Mobile phase:
Flow (ml/min) Minute % Acetonitrile
1 0 30
l 40 50
1 50 50

Injection volume: 10 ul
Detection: 210 nm

Identification: based on retention time of standards

246

 

 

 

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APPENDIX VIII

Oil content in studied sweet orange seeds.

252

Freeze-dried orange seeds

Ground roughly using coffee grinder

Ground seeds

Wrapped by filter paper No. 4
Soxhlet extraction using hexane (12 hours)

Hexane was evaporated to minimal under vacuum

Transferred to pre-weighed beaker (50 ml)

Air dried
l

Oven dil'ied (60 oC)
Cooled at room temperature in desicator
|

Weighed the extracted seed oil

Figure 67: Flow diagram of orange seed oil extraction for estimation of oil content.

Table 71: Oil content in studied sweet orange seeds.

 

 

Variety % Seed oil 0 Std'
Hamlin 32.14 0 2.42
Parson Brown 35.23 0 0.36
Valencia 31.64 0 0.32

 

IN=3

253

 

APPENDIX VIIII

Processing qualities for juice production of studied orange varieties.

254

Table 72: Processing qualities of studied orange varieties.

 

 

Characteristics Hamlin Valencia Parson Brown
Harvest season Oct-J an Feb-Jun Oct-J an
Early mature. Latest maturing of

all oranges.

 

 

Quality of orange Larger and less Medium to large, Seedy (15 seeds
seedy than Parson well color and thin per fruit), small,
Brown, dual rind, high juice smooth thin and
purpose variety, content, well color rind,
high juice content, 2-4 seeds per fruit, high juice
thin rind, more cold excellent shipping content
tolerant fi'uit tree and storing quality
Quality of processed Light color, Good color, Not good color,
orange juice Sweet but light good flavor Poor quality
flavor

 

Blending variety for
both NFCOJ and
FCOJ

Processing application

Main variety for
both NFCOJ' and
FCOJ

For FCOJ2 only

 

1 . .
Not from concentrate orange julce
2 Frozen concentrate orange juice

255

 

APPENDIX X

Calculations.

256

 

Plate No.

N = 16[(t/W)2]

t. . .Retention time

W. . .Bandwidth
0 Resolution (Rs)
Rs = 2 t2-t1
W1+W2

t1 and t2. . .Retention times of the first and second adjacent bands
W1 and W2. . .Baseline bandwidths

Concentration of a compound (%) in an extract analyzed by HPLC:

 

Compound = (Rs)(CF)(Vtotal) X 100
(Vinj)(Amount of sample)
Rs ........ Detector response value for the test sample

Vtotal. . .Total volume of solution (ml)
Vinj . . ....Volume of unknown injected solution (ml)
CF ....... Calibration factor from the slope of standard calibration

curve (g/AU or HU)

Standard deviation (S)

s = 12: (u-fi)2/(n-1)
n. . .number of observations

u. . .observation
1'1. . .mean of observation

Coefficient of variation (%CV)

CV = Standard deviation X 100
Mean

%Moisture content

% = 1- Wt. of sample after drying X 100
Wt. of sample before drying

257

 

 

 

APPENDIX XI

Summary of HPLC instrumentation diagnostics used during this research.

258

Table 73: Summarized HPLC trouble shootings.

 

Problem

Cause

Solution

 

No peaks or very small
peaks

Inconsistent retention time

Detector off

Broken connection between
detector

No flow

No sample/wrong sample

Wrong setting on recorder
or detector
Leak

Change in mobile phase
composition
Air bubble in the pump

Temperature fluctuations

Sample overloading
Sample dissolved in a
solvent that is incompatible
with the mobile phase

Check detector
Check connection

Check flow

Check sample

Be sure it is not degraded
Check the setting.

Check fittings, pump, and
seals for leaks
Check. Make new

Check flow

Prime pump

Check and change seals
Be sure the mobile phase is
degassed

Stabilize column temp
Use column oven
Dilute sample

Dissolve sample in the
mobile phase whenever
possible. Adjust

 

Change of separation or loss
of resolution

Leak

Obstructed guard or column

259

Contamination of the
mobile phase. Prepare new
one

If guard column is
obstructed, change the filter
or repack it.

If the analytical column is
obstructed reverse it and
flushes disconnected from
the detector or replace the
column.

 

 

Table 73: Summarized HPLC trouble shootings.

 

Problem

Cause

Solution

 

Peak splitting

Peak tailing

Contamination of column or
guard

Plugged column

Plugged inlet frit

Active sites within the
column

Wrong pH

Wrong column

Void volume at inlet
Wrong sample solvent

Remove guard column. If
the problem is solved
replace it.

If not go next.

If guard column is
obstructed, change the filter
or repack it.

If the analytical column is
obstructed reverse it and
flushes disconnected from
the detector or replace the
column.

Replace. If the problem
persists discard column
Test with standard test
mixture. If 0k, add
competing base or acid
modifier.

Correct.

Change.

May need repacking.
Dissolve sample in mobile
phase

 

Peak fronting

Column overload

Wrong pH

Sample solvent
incompatible with mobile
phase

Dilute the sample.
Correct

Dissolve sample in mobile
phase

 

Void volume at inlet May need repacking
Wrong sample solvent Dissolve sample in mobile
phase
Rounded peaks Detector outside linear Reduce sample
dynamic range
Gain too low Adjust
Column overloaded Dilute the sample.
Time constants (detector, Reduce

recorder) too high

Wrong sample solvent

260

Dissolve sample in mobile
Phase.

 

 

 

Table 73: Summarized HPLC trouble shootings.

 

Problem

Cause

 

Solution

 

Base line drift

Base line noise

Fluctuation of column temp.

Contamination of mobile
phase

Air bubble in the detector
cell

Air bubble in the detector

Plugged detector outlet line
Default mixing

Plugged detector outlet line
Strongly retained materials

Un-optimized detection
Air bubbles

 

Stabilize. Use column oven.
Use HPLC grade solvent.
Degas.

Use HPLC grade solvent.
Degas.

Clean cell. If necessary use
a pressure restrictor at
outlet.

Replace

Check mixer unit. Check
flow rate and composition
Replace.

Flush column with strong
solvent.

Optimized detector.

F lush system, prime pumps,
degas mobile phase.

 

Pump pulse Use a pulse damper.

Incomplete mixing Promote complete mixing

Electronic Check electronic equipment
in the same line.

Leak Check fitting, pump, and
seals for leaks.

Broad peaks Altered mobile phase Make new.
Low flow rate Increase.
Leak Check fittings, pump, and

Incorrect detector settings
Column overload

Void volume. Tubing too

long or to wide

Low buffer concentration
Column or guard column

contamination

Void volume at inlet

261

seals for leaks.

Check and correct

Dilute sample.

Use 0.010" tubing. Shorten
path.

Increase

Replace.

Repack

 

Table 73: Summarized HPLC trouble shootings.

 

 

 

 

 

Problem Cause Solution
Change in peak height Sample deterioration Use fresh sample
Leak Check fittings, pump, and
seals for leaks
Non-reproducible sample Ensure loop is completely
volume filled
Low detector response Check detector settings and
operating conditions
Negative peaks Recorder connections Check polarity
Refractive index of mobile Change mobile phase
phase higher than that of
solute
Vacancy peaks Originate from great
difference in composition
between sample solvent and
mobile phase. Dissolve
sample in mobile phase
Mobile phase more Use mobile phase that is
absorptive than sample transparent at the
components wavelength used
Ghost peaks Contamination of injector Always flush injector after
or column each injection.
Retained compound from Flush column with strong
previous injection solvent after operation to
remove late eluting
compounds
No flow Pump off Start pump

Flow interrupted

Leak

Air bubble in the system

262

Check reservoirs for
position of the inlet tubing
Check loop for air bubble
Check degassing of mobile
phase

Check compatibility of the
mobile phase components
Check fittings, pump, and
seals for leaks

Disconnect column and
prime pump

Flush system with 100%
methanol or isopropanol

 

Table 73: Summarized HPLC trouble shootings.

 

Problem

Cause

Solution

 

Low pressure

Leak

Flow interrupted

Air bubble in pump

Worn pump seals

Check fittings, pump, and
seals for leaks

Check reservoirs for
position of the inlet tubing
Check loop for air bubble
Check degassing of mobile
phase

Check compatibility of the
mobile phase components
Disconnect column and
prime pump

Flush system with 100%
methanol or isopropanol
Replace seals

Check pistons and replace if
necessary

 

High pressure

Pump, injector, tubing

Obstructed column or guard
column

After: http://www.dq.fct.unl.pt/qof/hplctsl .html

263

Disconnect column, run
pump at 25 ml/min: Is the
pressure minimal? Go to
next step

If pressure still high check
systematically from detector
to pump for obstruction.

If guard column is
obstructed, change the filter
or repack it.

Ifthe analytical column is
obstructed reverse it and
flush disconnected from the
detector or replace the
column.

LIBRARIES

‘-

MICHIGAN STATE UNIVEFIDlT
1293 0249

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