17p .
' _

"W1 \\

\\\\\\\\\\ll\g\§\ll\l§l;l§\l§ll§@ljlgl\\ L

This is to certify that the

thesis entitled

An Investigation of Capsaicinoids & Bioactive
Compounds In 'Scotch Bonnet' & Seven Other

Cultivars of Pepper
presented by

Jinpin Yao

has been accepted towards fulfillment
of the requirements for

MS Natural Products

degree in
Chemistry

 

 

 

 

 

Major professor

g/23[°13

Date

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

    
 

 

LIBRARY

Michigan State
University

 
 

 

  

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date duo.

 

DATE DUE DATE DUE DATE DUE I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mml

MSU I. An Afflrmdlvo Action/Equal Opportunity Institution
cmufl-M

 

 

.w—MW

AN INVESTIGATION OF CAPSAICINOIDS AND EIOACTIVE CONFOUNDS IN

'sco-rcn som'r' am: am can cumrms or upper:
(Wm)

BY
Jinpin Yao

A THESIS

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

MASTER OF SCIENCE

Bioactive Natural Products Laboratory
Department of Horticulture

1992

ABSTRACT

AN ruvnsrrcnrron or carsnrcruorbs AND sroncrrvn consensus IN
'scorcn nouns?! nun snvnx ownnn cunrrvnns or psppnn
(gsngisss_snnnun)
By

Jinpin Yao

The capsaicinoids and bioactive compounds in eight capsicum
annuum cultivars were investigated. Lyophilized peppers,
grown in the greenhouse, were extracted with supercritical
(Ky, hexane, ethyl acetate and methanol. Two major
capsaicinoids, capsaicin and dihydrocapsaicin, were isolated
and purified by column chromatography and preparative thin-
layer chromatography. Capsaicin was quantified by reverse-
phase high performance liquid chromatography and characterized
by nuclear magnetic resonance, mass spectrometry and
ultraviolet spectrophotometry. The biological activities of
the crude extracts from eight cultivars of pepper (C. annuum) ,
pure capsaicin and dihydrocapsaicin were studied on fungi,
bacteria, nematodes and mosquito larvae. The hexane extract
of 'Scotch Bonnet’, at 250 ppm, killed all mosquito larvae
(Aedes aegypti) in 30 min. Purification of this active
extract afforded bonnetenol, characterized by “L,‘%L.‘H - Hi
decoupling and DEPT NHR, and MS experiments. This compound

gave 100% mortality against Aedes aegypti at 0.1 ppm in 24 h.

Dedication

This thesis is dedicated with love, to my wife, Cen.

iii

ACIHOILEDGNENT

I wish to express my sincere gratitude to my major
professor, Dr. Huraleedharan Nair, for providing me with the
opportunity to study in his laboratory, and for his patience
and guidance in completing my research and this thesis. I
would also to thank Dr. Jack Kelly, Dr. James Miller and Dr.
Frank Dennis for participating as members of my guidance
committee.

To my fellow laboratory colleagues and friends, Deborah
Thorogood Williams, Amitabh Chandra, Marshall Elson and Yuchen
Chang, a special thanks for their friendship, support and
helpful suggestions.

A special word of appreciation to my wife, Cen Yang, for
her love, sacrifices, understanding, support and encouragement

during my studies.

iv

TABLE OF CONTENTS

PAGE
LISTOFFIGURESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Vii

CHAPTER 1 : LITERATURE REVIEW

General introduction .................................
Horticultural consideration of pepper . . . . . . . . . . . . . . . .
Chemistry of the pungent principles in peppers . . . . . . .
Sensory properties of capsaicin 1

mmNH

REFERENCESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 20

CHAPTER 2: INVESTIGATION OF CAPSAICINOIDS IN
CAPSICUM ANNUUM CULTIVARS

ABSTRACTOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 24
INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 25

MATERIALSANDMETHODS ................................. 29
General experimental 29
Plantmaterials 31
Separation of capsaicin and dihydrocapsaicin from
Aldrichsample 31
Standardcurve 33
Isolation of capsaicin and dihydrocapsaicin from
'ScotchBonnet’ 33
SFEextraction 4O
Solventextraction 40
Quantification....................................... 42

RESULTS AND DISCUSSION ................................ 42
Isolation of capsaicin and dihydrocapsaicin . . . . . . . . . . 42
Identification of capsaicin and dihydrocapsaicin . . . . . 43
Extraction of capsaicin 44
Quantification of capsaicin and dihydrocapsaicin . . . . . 45

REFERENCESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 48

CHAPTER 3: INVESTIGATION OF BIOACTIVE COMPOUNDS IN
CAPSICUM ANNUUM CULTIVARS

ABSTRACT O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 5 1
INTRODUCTI ON O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 5 2
MATERIALS AND METHODS O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O S 2

Generalexperimental 52
Plantmaterials 54

TABLE OF CONTENTS continued:

PAGE
SFE extraction of 'Scotch Bonnet’ 55
Solvent extraction of Capsicum annuum cultivars . . . . . . 55
Isolation of bioactive compounds from 'Scotch Bonnet' . 55

Microorganisms 60
Antifungal bioassay 60
Antibacterial bioassay 65
Insecticidal bioassay 65
Nematicidal bioassay ................... 65

RESULTSANDDISCUSSIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 66
Resultsofbioassay............. ...... 66
Characterization of bonnetenol 67

REFERENCESOO OOOOOOOO OOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO 71

vi

LIST OE EIOURES

FIGURE PACE

CHAPTER 1
1 Structures of piperine alkaloids . . .......... . . 7
2 Structures of isomers of piperine ............. 8
3 Structures of capsaicinoids 9
4 Synthetic scheme for dihydrocapsaicin . . . . . . . . . 12
5 Synthetic scheme for capsaicin . . . . . . . . . . . . . . . . 13
6 Stereospecific synthetic scheme for capsaicin. . 15

CHAPTER 2
1 Supercritical CO2 phase diagram . . . . . . . . . . . . . . . 28
2 ‘H NMR spectrum of Aldrich capsaicin . . . . . . . . . . 32
3 Standardcurveofcapsaicin................... 34
4 Isolation and purification of capsaicin

and dihydrocapsaicin from ' Scotch Bonnet' . . . . . . . 35
5 ‘H NMR spectrum of the mixture of capsaicin

and dihydrocapsaicin from 'Scotch Bonnet' . . . . . . 38
6 ‘H NMR spectrum of capsaicin from

'ScotchBonnet' 39
7 ‘H NMR spectrum of dihydrocapsaicin from

'ScotchBonnet' 41
8 Comparison of SFE and solvents extraction

of capsaicin from 'Scotch Bonnet' . . . . . . . . . . . . . . 46

CHAPTER 3
1 Isolation and purification of bonnetenol

from ’Scotch Bonnet' 56
2 UVspectrumof bonnetenol.................... 58
3a,b lIiINIKIRspectrum of bonnetenol 59
4 ‘H-‘H decoupling NER spectrum of bonnetenol . . . 61
5 13CNMRspectrumof bonnetenol................ 62
6 DEPT NMR spectrum of bonnetenol . . . . . . . . . . . . . . 63
7 Mass spectrumof bonnetenol.................. 64
8 MS fragmentation pattern of bonnetenol . . . . . . . 69
9 Structure of bonnetenol...................... 70

vii

CHAPTER 1

LITERATURE REVIE'

General Introduction: Peppers have long been an important
spice and flavoring in cuisines from one continent to another.
The archaeological record at Tehuancan, Mexico, southeast of
Mexico City, showed that wild peppers were eaten in
Mesoamerica as early as 7000 B.C. and were domesticated
probably by 2500 B.C. (Andrews, 1984). The fiery fruit of
pepper is discomforting to eat and causes mouth burns, body
perspiration and watery eyes. However, the flavor of pepper
and its unique role in food preparation tempt many people to
overlook the "heat" generated by the fruit.

Pepper, sometimes considered as the king of spices, is
one of the earliest spices known to man. Peppers are an
important vegetable commodity and are highly prized for their
flavor, color, vitamins A and C, and pungency (Groviadaraian,
1979). The products made from peppers are sold in a variety
of forms, ranging from whole fruits to ground powders. The
fruit itself has been used in different forms, ranging from
aid in child-birth to an instrument of torture. The Indians
in Panama trail pepper pods behind their dugouts to repel
sharks. Some city dwellers in the United States and Canada
use sprays made from chili peppers to ward off muggers
(Andrews, 1984).

In the international spice trade, peppers contribute

1

2

about 40% (77,000 MT) to the total world spice trade
(Rathnawathie and Buckle 1984). The 1990 world production of
peppers was 9.1 MT. Major areas of pepper production are
Asia, Europe, and Africa, with about 44, 24, and 19 percent,
respectively, of the total world production. Nations
producing large amounts of peppers are China, Nigeria,
Turkey, Mexico, the United States and Egypt (FAO, 1990). In
the United States, with about 3 - 4 % of the total world
production, the major producing states are California,
Colorado, Florida, New Mexico and Washington

(U.S.D.A. Agriculture statistics, 1990).

Horticultural considerations of Pepper: Pepper (Piper nigrum)
was the first oriental spice to reach Europe from Asia and
remains today the most widely used spice throughout the world.
In 1493, Columbus, while seeking a source of black pepper
(Piper nigrum), discovered chili instead, and named chili as
"pepper" (Andrews, 1984).

Pepper (chili) plants are shrubby perennials, but are
usually grown as herbaceous annuals in tropical, subtropical
and temperate regions (Andrews, 1984). Pepper has a perfect
flower, and is primarily self-pollinated, and to a lesser
extent cross—pollinated by insects. The plant is frost-
sensitive and grows best at warm temperatures, preferably
within a range of 25° C to 30° C. In fertile, fine-textured

soils, such as sandy loans, the optimum pH range for high

3

productivity is between 6.0 and 6.5 (Purseglove, et a1. 1981).
Applying a water-soluble fertilizer high in phosphorus, and
topdressing with high nitrogen fertilizer at bloom, will
enhance yield (Cochran, 1932). Peppers are one of the few
vegetables that germinate with very low soil moisture,
therefore overwatering should be avoided. The water should be
applied after emergence. Since growth of the pepper plants
from seed requires considerable time, transplanting is
commonly employed (Lafavore, 1983). Generally, a warm
environment is required for rapid germination and to prevent
damping off, and seeds ‘will not germinate if the soil
temperature is 15°C or lower. The optimum temperature range
reported for germination is 25° to 30°C. (Andrews, 1984).
Pepper plants are unable to withstand freezing (Kader, et a1.
1982) .

Peppers are affected by many of the same diseases and
insects as tomatoes, including bacterial spot, Fusarium.wilt,
aphids and pepper weevil. These problems can be reduced by
planting resistant cultivars or use of fungicides and
insecticides rotations (Boswell, 1964).

Peppers can be harvested at any stage, but quality is
generally best if left to maturity. For the pungent types,
the flavor reaches its peak only when fruits are mature. The
red mature fruit is reported to be higher in vitamins A and C
(Kitagawa, 1973). In order to maintain product quality and

avoid mold growth, peppers should be stored at near 15°C at

low humidity.

There are a number of varieties of ”peppers" , and not
all varieties or types are called pepper. Black pepper, for
example, is obtained from the corn or seed of the climbing
vine of Piper nigrum which is native to India (Govindorajan,
1977). Chili pepper, from plants of the genus Capsicum, has
many varieties which differ in pungency, color and flavor
(Andrews, 1984; Erwin, 1932). There are also other "peppers"
which belong to different genera. For example, Jamaican
pepper (pimentoi or’ allspice) belongs to .Pimenta dioica;
Japanese pepper, the black fruit from the tree Zanthoxylum
piperatum DC. , belongs to the family Rutaceae, and the African
or Negro pepper, a pod-like fruit of a shrubby tree,
Xylopiasethiopica Dunn, belongs to the family Anonaceae.
(Groviadarajan, 1977).

Nearly 200 varieties of domesticated peppers (Capsicum
annuum) are grown in North, Central and South America. The
name capsicum is derived either from the Greek " Kapso "
meaning to bite or the Latin " Capsa " referring to the fruit
pod or capsule (Domenici, 1983). Capsicums are members of the
Solanaceae or nightshade family, which includes tomatoes,
eggplants and potatoes, and probably evolved from an ancestral
form in the area of Bolivia or Peru (Heiser, 1976). Capsicums
are perennial in suitable climatic conditions but are often
cultivated as an annual. Geographical and climatic conditions

also cause variations in Capsicum species throughout the world

5
(Andrews, 1984) . The genus Capsicum includes 20 to 30
species native to the New World tropics and subtropics.
Within this genus there are five domesticated species -
Capsicum annuum, Capsicum frutescens, Capsicum pubescens,
Capsicum chinense, and Capsicum baccatum var pendulum. C.
annuum is the most widely cultivated species and includes
almost all of the varieties grown in the U.S. and Europe.
This plant, an annual in temperate climates, can be
distinguished by its white flowers. The fruits vary in length
and color when mature. C. frutescens, of which the tabasco
variety is a member, is perennial usually and has smaller
fruits. They are cultivated mainly in warm regions. Fruits
of wild forms of C. chinense are spherical in shape, but the
cultivated forms produce variable-shaped fruits. Plants have
large leaves and are grown mainly in tropical South America.
C. baccatum is widely distributed in South America and
cultivated to a very limited extent in the U.S. The climatic
requirements for C. baccatum are similar to those of C.
annuum. It can be identified by the yellow or brown spots on
its flowers, which form conical pungent fruits. C. pubescens
is found only at high elevations in the tropics or subtropics.
The plant is characteristically hirsute and requires a long
and cool growing season. C. annuum, C. frutescens and C.
chinense can be hybridized. However, no hybrids are known for
C. baccatum and C. pubescens. Therefore, relatively few

cultivars of C. pubescens and C. baccatum are commercially

6

significant (Smith, 1987; Andrews, 1984).

Chemistry of Pungent Principles In Peppers: The two different
classes of peppers, black pepper and chili pepper, contain
very different compounds responsible for their pungency and
flavor. Black pepper is one of the most important spices,
used in many dishes, meat products and sauces. Its pungency
is due to many non-volatile compounds such as piperine
alkaloid and its isomers, piperlonguminine, piperylin,
piperanine, piperine, piperettyline and.piperttine (Figure 1)
(Kenneth and Michael, 1988) . The most abundant and major
pungent principle in black pepper is piperine ( Genest, et a1.
1963) . The geometric isomers of piperine (I), i.e. , chavicine
(cis-cis)(II), isopiperine (cis-trans)(III) and iso-chavicine
(trans-cis)(IV) (Figure 2) also occur in black pepper, but in
very small amounts (Kulka, 1967).

The primary compounds responsible for the pungency of
capsicum fruits are capsaicinoids (N - vanillyl nonamides),
and the most pungent compound among them is capsaicin (Figure
3), which is 100 times more pungency than piperine
(Groviadaraian, 1979). Capsaicinoids are amino acids of
vanillylamine and.C5 - Cn branched-chain fatty acids. These
fatty acids are secreted from the cells of both pungent and
nonpungent cultivars of peppers.( Fujiwake, et al. 1979). The
vanillyl amides with varying side-chain lengths responsible

for the flavor and hotness of pepper, are capsaicin,

<.:./\/\/ C \N/Y Pipperlonguminine

e o .
<o:./\/\/a \NQ piperylin
< e ‘\ 3NO 1......

' \\-§\N I ‘Pirine
<0 0 p.

0
II
0

8\N > Piperettine
c

Figure 1: Structures of piperine alkaloids

 

Figure 2: Structures of isomers of piperine

O
H,CO 3 1 "I

M WW!

z/“\c/“\c/‘<: unevenness
z/“\¢/‘\/’\\ cmmmmmuma
/’\~/C\c/\\/’ Nmmnmmmmu=
/\/\/\/\ DecylvannIyllnilh

,/\\/’\c/“\r’\x . mmummhnmfidnfl

Figure 3: Structures of capsaicinoids .

10
dihydrocapsaicin, norcapsaicin, nordihydrocapsaicin,
homocapsaicin I, homocapsaicin II, homodihydrocapsaicin I,
homodihydrocapsaicin II, caprylvanillylamide,
decylvanillylamide and nonylvanillylamide (Figure 3)(Patric,
et a1. 1983). The most abundant capsaicinoids are capsaicin
and dihydrocapsaicin. Capsaicin is the most pungent compound
of all the capsaicinoids and is present mainly in the
placental tissues of the inner walls of the fruit. The hull
and seeds of chili pepper contain little or no capsaicin
(Hoffman, et al. 1978; Newman, 1953).
Capsaicin has a melting point of 65°C and boils at 210 -
220%L. It has a strong absorption at 280 nm in the
ultraviolet spectrum. Capsaicin is practically insoluble in
cold ‘water but freely soluble in, ethanol, methanol and
chloroform, and slightly soluble in carbon disulfide (Merck
Index, 10th edition, 1989).

Chemical synthesis of dihydrocapsaicin was achieved
first.by Nelson.and.Dawson (1923). The synthesis involved the
treatment of 4-methyl pentanoic acid with sodium ethoxide at
room temperature for 4 h. The resulting 4-methy1-pentyl
alcohol was refluxed with hydroiodic acid for 3 h to obtain 4-
methyl-pentyl iodide; this was condensed further with aceto-
acetic ester to produce 4-methyl-pentyl aceto-acetic ester.
The ester was saponified by sodium ethoxide for 4 h, and the
resulting product, ethyl-6-methyl-heptylate, was converted

into 6-methyl-heptyl alcohol by reacting with sodium ethoxide

11

in ethanol at room temperature. The alcohol thus obtained was
reacted with hydroiodic acid to form 6-methyl-heptyl iodide
followed by condensation with aceto-acetic ester. The 8-
Methyl-nonanoic acid, obtained by saponification of ethyl-8-
nonanoate, was further converted into its acid chloride by
thionyl chloride. The acid chloride finally was condensed
with vanillyl amine to form dihydrocapsaicin (Figure 4).

Newman (1953) reported the first synthesis of capsaicin
using isobutyl zinc iodide as the starting material. 8-
Methyl-nonan(6)on-(1) ic acid was prepared by reacting isobutyl
zinc iodide and monoethyladipylchloride in acetic acid-toluene
solution at room temperature and then reduced with sodium
powder in ethanol to form 8-methyl-nonan(6)on-(1)ic acid(6)-
ol. This was converted into 8-methyl-nonan(6)-Br-(1)ic
acid(6)-ol by heating with HBr at loo-105°C in a closed
ampule. 8-Methyl-non(6)en-(1)oic acid was produced by the
distillation of 8-methyl-nonan(6)-Br-(1)ic acid (6)-ol with
HBr. The capsaicin was obtained from the reaction of 8-
methyl-non(6)en-(1)oic acid with vanillyl amine at room
temperature (Figure 5).

Crombie et al. (1955) reported that Newman's synthesis
of capsaicin resulted in a mixture of isomers. Therefore,
Crombie et al. synthesized capsaicin using an unambiguous
stereo-specific method as follows: 2 , 3-dichlorotetrahydropyran
was slowly added, with stirring, to isopropylmagnesium bromid

in ether at 0°C, and the resulting 3-chlorotetrahydro-2-

12

(019,01 - CH, - CH2 - coon

1

(01920"! - CH2 - CH2 - (:11on

lm
(Grimm - CH:- CH. - CH2:
1 ma-g-mz-g-O-czm
C) C)
(GiQZQi-Cflz-Cflz-Cflz-O-C-CHz-C-CH3

1 EtONa

(CH3)ZCH-Giz-CHz-CI-Iz-CHZ-CHZ-CI-Iz-COOH
l 5002

C)
“
(CH3):CH ‘ (C1123 ' C ° C1

1 thflbdzmmnc

H3C0

HO CHz ' NH ° | (CH?)5 ' CH(CHa)2

'-
C)

Figure 4: Synthetic scheme for dihydrocapsaicin

13

C)
(013)20'1-0'12‘znl 4' Cl'g'(CI'Iz)4'COOQH5

o
(CH3),CH - cn2 - ('3' - (CH7); - coon

l

(013501-012- CH- (cum-coon

3m

1

(cn3hcn - CH2 - CH - (cnz)4 - coon

I

(Ilfimfifii-(II-CEI-«Jflgu-CIXNH
l ‘menfilmmkw

H3. 0

no 0 m,-Nn-c'5-(cn,),-cn=CH-CH<GI.)2

Figure 5: Synthetic scheme for capsaicin

14

isopropylpyran was converted to 6-methylhept-trans-4-en-1-ol
by adding 3-chlorotetrahydro-2-isopropylpyran slowly into
powdered sodium in anhydrous ether. 6—Methylhept-trans-4-en-
1-ol was reacted with phosphorus tribromide in.pyridine at 0%:
for 30 min., and refluxed with ethyl sodiomalonate for 2 h to
form 8-methylnona-trans-6-enoic acid. This compound was
hydrogenated in dioxan with the catalyst and the resulting
product was treated with thionyl chloride for 18 h to produce
8-methylnona-trans-6-enoic acid chloride. Finally, the trans-
capsaicin was prepared by adding 8-methylnona-trans-6-enoic
acid chloride to vanillyl amine suspended in ether and shaking
for 3 days (Figure 6).

Bennet and Kirby (1968) concluded that both
phenylalanine and tyrosine are the precursors for the
biosynthesis of the aromatic portion of capsaicin. The
conversion of phenylalanine into capsaicin proceeded via
hydroxylated cinnamic acids. Bennet and Kirby (1968) also
reported that vanillylamine might be the most direct
precursor; whether the amino group of vanillylamine was
retained during the biosynthesis of capsaicin in the plant was
not established” In :most cases, natural and synthetic
capsaicin differ in the cis-trans arrangement at the double
bond in the fatty acid side-chain moiety; the trans or cis
arrangement may not be retained during chemical synthesis.
However, natural and synthetic capsaicin can be distinguished

by their infrared absorption spectra (Datta and Susi, 1961)

15

' a
(013)201-er + C1.Cn-dn-(Cn,),-.cn2

L__o___J
l

Cl
(mahw-m-ch-(cnaz-mz

l INa
(mmm-m-Cn-(anh-cnzon

‘ PBr3
(C11,)2cn-cn=cn-(Cn,),-3-Br

1 Malonatc
((143)201-G-hfl-I-(Cl-194-C00H (trans)

1 soc,
‘i
(043)201-012CH-(Qifl4-C-Q (trans)

‘ ‘menylanfimc

n
3 ‘ S?
liq—.1 O u 2-Nn-c-(CIi,),,-(:H=CH(CH;)2 (trans)

Figure 6: Stereospecific synthetic scheme for capsaicin

16
and also by evaluating the fatty acids associated with the
amide portion of capsaicin.(Todd.and.Perum, 1961). The latter
method was not accurate because of interference by various
fatty acids from the lipid fraction resulting from other
biosynthetic pathways (Masada, et al. 1971).

The capsaicinoids can be extracted from the fruits of
peppers by several methods. Roshchina et al. (1986) used
petroleum ether, methanol and hot water, respectively, as
solvents under different conditions. They found that methanol
extraction gave the highest yield of capsaicin, even though
hot water and petroleum ether could yield a purer extract of
capsaicin. Many published reports describe various solvent
systems under specific conditions for extracting capsaicinoids
from chili peppers (Mary, 1984; Hoffman, 1978 and Roshchina,

et al. 1986).

Sensory Properties 0: Capsaicin: The traditional method for
assessing pepper pungency is the Scoville procedure, in which
a sample of capsaicin is diluted until it becomes undetectable
by taste to a panel of judges (Nagy, 1982). An aliquot of an
ethanol extract of the capsicum product is diluted serially
with a 5% sucrose solution. The concentration at which 3 out
of 5 panelists perceive a slight burning sensation in the
throat upon swallowing 5 ml of the solution establishes the
numerical Scoville heat value or dilution factor (ASTA, 1968) .

This method has been criticized severely for

17
irreproducibility and the time required for the need for
extensive training of the panelists (Meilgaard, et al. 1987).

Lou (1984) suggested that HPLC is the best analytical
method for quantifying the capsaicin content and evaluating
the heat ‘value in. pepper and. pepper products.
Spectrophotometric methods employing the reactions of
capsaicinoids with several reagents to produce a colored
complex were reported by Rymal, et al. (1984). Reverse-phase
is preferred over normal-phase chromatography for the
purification of capsaicin in dehydrated Capsicum annuum var.
Separation of the capsaicinoids on normal-phase silica gel is
difficult because the only difference in the structures of
capsaicinoids is their fatty acid moiety (Chiang, 1986; Weaver
and Awde, 1986).

The savor for peppers consist mainly in their pungency
or 'heat' value. Ironically, the enjoyment of chili pepper is
actually due to the irritation to nerve endings by capsaicin.
The trigeminal nerves, which are distinct from the taste
receptors, are chemically irritated by capsaicin (Anonymous,
1986). These nerves also are responsible for sensing touch,
heat, cold, and pain in the mouth (Nagy, et al. 1982). The
contribution of trigeminal nerves to the flavor sensation of
foods often is overlooked. In the nose, the trigeminal nerves
respond to irritants in foods and beverages sudh as carbon
dioxide and.to a variety of aromatic flavors that normally are

considered "just smells". Therefore, the enormous number of

18
nerves making up the trigeminal system is very important for
the flavor sensation (Nagy, et al. 1982).

One can taste all the other food flavors through the
pepper burn. However, research in this area has produced
mixed results. To some degree, pungency seems to mask bitter
and sour tastes (Cowart, 1987). Overall, trigeminal
sensations interact very little with other flavors under the
influence of " hot " pepper.

Pepper also has a number of pharmacological functions
such as anaesthetic properties (Jancso, et al. 1977;
Anonymous, 1986). Endorphin, an opiate-like substance,
reportedly is secreted by the brain when capsaicin irritates
the trigeminal nervous system (Jessell, et a1. 1977). Some
studies indicate that peppers, especially hot peppers, are
cancer-producers and are not recommended for good health
(Virus, 1979).

The aromatic ring in capsaicinoids plays an important
role in the pungency of chili pepper in addition to the alkyl
side chain. The optimal chain length of alkyl group is 8 - 10
carbon atoms for the highest pungency; increasing or
decreasing the carbon chain length would reduces the ’heat'
value of capsaicinoids. The degree of unsaturation of the
alkyl side chain does not have much influence or at least is
not essential for the pungency produced by these compounds
(Nelson 1919) . However, the acylamide part exhibits the

relationship between chemical structure and pungency.

19
Replacing the nitrogen atom in capsaicin with oxygen.has very
little effect on. pungency. However, when. nitrogen 'was
replaced by elements or groups such as ethylene, pungency of

capsaicinoids was reduced significantly (Szolcsanyi, 1975).

REFERENCES

Agriculture Statistics. U.S.D.A. - 1990. Washington D.C. 1991.

Andrews, J. Peppers: the domesticated capsicums. University of
Texas Press, Austin. 1984.

Anonymous. Metabolism and toxity of capsaicin. Nutri. Rev.
1986 44(1), 20-22.

ASTAHAnalytical.methods. 2nd ed., method 21.0. Am. Spice'Trade
ASSOC. NY. 1968.

Bajaj, K.L. Colorimetric determination of capsaicin. J. Assoc.
Off. Anal. Chem. 1980, 63, 1314-1316.

Bennett, D.J. and Kirby, G.W. Constitution and biosynthesis of
capsaicin. J. Chem. Soc. 1968, 442-446.

Boswell, V.R Pepper production. US. Dept. Agr. Information
Bull. 1964. 276, 11-19.

Celin, R. Quinones - Seglie; Edward E. Burns and Benigno
Villalon. Tech. Leb. - Wisconsin University. 1989, 22, 196-
198.

Chiang, G.H. HPLC analysis of capsaicin and simultaneous
determination of capsaicin and piperine by HPLC-ECD and UV.
J. Food Sci. 1986, 51, 499-503

Cochran, H.L. Factorsraffecting flowering'and fruit.setting in
the pepper. The Pro. Amer. Soc. Hort. Sci. 1932, 29, 434-437.

Cowart, B.J. Oral chemical irritation: Does it reduce
perceived taste intensity. Chem. Senses. 1987, 12, 467-472.

Crombie, L., Dangdegaonker, S.H. and Simpson, K.B. Amides of
vegetable origin. 6. Synthesis of capsaicin. J. Amer. Chem.
SOC. 1955, 1025-1027.

Datta, P.R. and Susi, H. Quantitative differentiation between
natural capsaicin (8-methyl-N-vanillyl-6-nonenamide) and
vanillyl-N-nonanamide. Anal. Chem. 1961, 33, 148-153.

Domenici, P. The correct way to spell chili. Congressional
Record 129. 1983.

Eliezer, Z., Orana, S. and Dan, P. Ultrastructure of
capsaicinoid -secreting cells in pungent and nonpungent red
pepper (Capsicum annuum L.) cultivars. Bot. Gaz. 1987, 148 (1),
1-6.

20

21

Erwin, A.T. The Peppers. Iowa Agr. Exp. Station Bull. 1932.
293.

FAO. FAO Production Yearbook - 1990. Food and Agriculture
Organization of The United Nations. 1990, 44.

Fujiwake, H.; Suzuki, T. and Iwal, K. Intracellular
localization of capsaicin and its analogues in Capsicum
fruits. Plant Cell Physiol. 1979, 23, 1023-1030.

Genest, C., Smith, D.M. and Chapman, D.G. A critical study of
two procedures for the determination of piperine in black and
white pepper. J. Agr. Food Chem. 1963, 11, 508-512.

Gillette, M.H.; Appel, C.E. and Lego, M.C. A new method for
sensory evaluation of pepper heat. J. Food Sci. 1984, 49,
1028-1032.

Groviadarajan, V.S. Pepper - chemistry, technology, and
quality evaluation. CRC Criti. Rev. In Food Sci. And Nutri.
1977 , 9 , 115-125 .

Groviadarajan, V.S. Pungency: the stimuli and their
evaluation. Amer. Chem. Soc. Washington, D. C. 1979, 6, 78-
81.

Heiser, C.B. The evolution of crops and plants. Longman Press,
London. 1976. 87-93.

Hoffman, V.L., Schadle, E.R., Villalon, F. and Burns, E.E.
J. Food Sci. 1978, 43, 1809

Jancso, G., Kiraly, E. and Jancso-Gabor, A. Pharmacologically
induced selective degeneration of chemosensitive primary
sensory neurones. Nature (London). 1977, 270, 741-743.

Jessell, T.M., Wersen, L.L. and Cuello, A.C. Capsaicin -
induced depletion of substance P from primary sensory
neurones. Brain Research. 1978, 152, 183-187.

Kader,.A.A., Kasmire, R.F., Michell, F.G., Reid, M.S., Sommer,
N.F. and Thompson, J.F. Postharv Tech. Horti. crops.
University of California, Div. ANR Special Publication 3311.
1982 .

Kitagawa, Y. Distribution of vitamin C related to growth of
some fruits(2), Tomato, pimiento and strawberry. J. Jap. Soc.
Food and Nutrition. 1973, 26, 139-134.

Kulka, K. Aspects of functional groups and flavor. J. Agr.
Food Chem. 1967, 15, 48-57.

22

Lafavore, M. A pepper talk. Organic Gardening. 1983, 31(1),
34-38.

Lou, 8. HPLC quantifies heat levels in chili pepper products.
Food Process. 1984, 21, 70-75.

Mary, C.L. HPLC in flavor industry. Food Tech. 1984, 38, 84-
87.

Masada, Y., Hashimoto, K., Inoue, T. and Suzui, M. Analysis
pungent principles of Capsicum annuum by combined GC-MS. J.
Food Sci. 1971, 36, 858-861.

Meilgaard, M., Civille, G.V. and Carr, B.T. Sensory evaluation
techniques. CRC Press: Boca Rotan, FL, 1987, 1&2. 59-64.

Merck Index, 10th edition. 1989, 243-246

Nagy, J.I., Iverson, L., Iverson, S.D. and Snyder (editor).
Handbook of Psychopharmacology. 1982, 15, 185. Plenum. New
York.

Nelson, E.K. and Dawson, L.E. The constitution of capsaicin -
pungent principle of Capsicum. J. Amer. Chem. Soc. 1923, 45,
2179-2181.

Nelson, E.K. Vanillyl-acyl amides. J. Amer. Chem. Soc. 1919,
41, 2121-2126.

Newman,.A.A. Chemistry of capsaicin. Chem. Produc. Chem, News.
1953, 16, 413-418.

Patric, G.H. , Mary, C.L. and William, G.G. Separation and
quantification of red pepper major heat principles by reverse-
phase high-pressure liquid chromatography. J. Agr. Food Chem.
1983, 31(6), 1326-1330.

Purseglove, J.W., Brown, E.G., Green, C.L. and Robbins, S.R.J.
Spices. 1981, 1, 343. Longman.

Rathnawathie, M. and Buckle, K.A. Effect of berry maturation
on some chemical constituents of black, green and white
pepper from three cultivars. J. Food Tech. 1984, 19(3), 361-
367.

Roshcina, V.V., Ruzieva, R.K.H. and Mukhin, E.N. Capsaicin
from the fruit of the red pepper-Capsicum annuum.
Appl.Biochem. Microbiol. 1986, 22, 335-340.

Rymal, K.S., Cosper, R.D. and Smith, D.A. Injection-extraction
procedure for rapid determination of relative pungency in
fresh Jalapeno peppers. J. Assoc. Off. Anal. Chem. 1984, 67,

23
658-659.

Saria, A., Lembeck, F. and Shofitshch, B. Determination of
capsaicin in tissues and separation of capsaicin analogues by
HPLC. J. Chromatogr. 1981, 208, 41-46.

Smith, P.G., Villalon, B. and.‘Villa, P.L. Horticultural
classification of peppers. Hortscience. 1987, 22(1), 11-13.

Sticher, 0., Soldati, F. and Joshi, R.K. HPLC separation and
quantitative determination of capsaicin. J. Chromatogr. 1978,
166, 221-231.

Szolcsanyi, J. and Jancso-Gabor, A. Sensory effects of
capsaicin congeners: Part 2. Relationship between chemical
structure and pain producing potency of capsaicin - type
compounds. Arzneim - Forsch. 1975, 26, 33-37.

Todd, P.H., Bensinger, M.G. and Bittftu, T. Determination of
pungency due to capsaicinoids by GC. J. Food Sci. 1977, 42,
660-665.

Todd, P.H. and Perum, C. GC analysis of Capsicum amides.
Food Technol. 1961, 15(5), 270-273.

Virus, R.M. and Gebhart, G.F. Peppers. Life Sci. 1979, 25,
1273-1277.

Weaver, K.M. and Awde, D.B. Rapid HPLC method for the
determination of very low capsaicin levels. J. Chromatogr.
1986, 10, 603-612.

CHAPTER THO

INVESTIGATION OF CAPSAICINOIDS IN CAPSICUM ANNUUM CULTIVARS
ABSTRACT

Capsaicin content in various capsicum annuum cultivars
were investigated. Supercritical carbon dioxide was used to
extract ’Scotch Bonnet' pepper at 50°C and 450 atm, 50°C and
600 atm for 30 min and 1 h, respectively. The SFE results
were compared with those obtained by solvent extraction using
hexane, ethyl acetate and methanol, respectively. Preparative
thin-layer and column chromatographic methods were employed
for the separation of capsaicin and dihdrocapsaicin. A
reverse-phase high performance liquid chromatographic method
was used to quantify capsaicin. The capsaicin and
dihydrocapsaicin were characterized by nuclear magnetic
resonance, mass spectrometric and ultraviolet spectroscopic
methods. The SFE extract of 'Scotch Bonnet' with the heat
value of 300,000 Scoville units, afforded 3.2% capsaicin /g
dry weight compared to the 0.5% capsaicin /g dry weight from
the total solvent extracts. Chili with heat value of 8,000,
Jalapeno with heat value of 5,000, Cayenne with heat value of
3,000 Scoville units and bell pepper with no heat value were
found to contain 0.09%, 0.07%, 0.03% and 0% capsaicin /g dry

weight, respectively.

24

INTRODUCTION

The chili pepper belongs to the family Solanaceae, which
also includes the eggplant and tomato and has about 90 genera
containing some 2,000 species of herbs, shrubs and.small.trees
(Heiser, 1976). The term " Capsicum " refers to the fruit of
numerous species of the genus capsicum comprising over 200
varieties. They range from the very hot ’Jalapeno' to
heatless ’Bell' peppers (Andrews, 1984). Their fruits vary
widely in size, shape, flavor and sensory heat. C. annuum is
the most widely cultivated pepper in the world and practically
all of the fresh, processed, dried and frozen peppers on
grocer's shelves belong to this species. Capsicum fruits are
used throughout the world because of their unique flavor and
pungency, and also have widespread use as a drug (Newman,
1953).

The primary pungent principle from C. annuum was first
purified and obtained in a crystalline form from the crude
extract by Thresh (1846), who named the compound capsaicin.
Nelson (1919) , determined its structure as the amide of
vanillyl amine and isodecenoic acid (Figure 3, Chapter 1).
Capsaicin and dihydrocapsaicin, which are responsible for 90%
of the total pungency (Iwai, et al. 1979) , are the most
abundant principles of hot pepper (Kosuge and Furata, 1970).
The C. annuum Scotch Bonnet, with a heat value of 250,000 -

300,000 Scoville unit, is considered to be the most pungent

25

26
pepper available on the market. It is grown widely in Jamaica
for its pungency and flavor, and presently cultivated in the
United States (Garrett, et al. 1991).

A variety of chemical and instrumental methods are
available to identify and quantify capsaicinoids in peppers.
Colorimetric and spectrophotometric analysis (Bajaj, 1980;
Ramos, 1979) , chromatographic methods such as thin layer
chromatography (TLC) (Pankar and Magar, 1977), high pressure
liquid chromatography (HPLC) (Attuquayefio and Buckle, 1987;
Hoffman, et al. 1983; Mary, 1984; Saria, et al. 1981; Weaver
and. Awde, 1986), gas chromatography (GC) (Krajewska and
Powers, 1987; Todd. and. Perum, 1977), gas chromatography
coupled with mass spectrometry(GC-MS) (Masada, et al. 1971)
are some of the most commonly used techniques for separation
and identification of capsaicinoids. However, HPLC is
considered to be the most reliable and rapid method. Also,
HPLC has the advantage over GC because derivatization is not
required for quantification.

During the past decade supercritical fluid extraction
has been employed widely for the extraction of variety of
compounds from different matrices in areas sudh as polymer
fractionation, monomer purification, spice extraction and
coffee decaffeination (Eldridge, et al. 1986; Friedrich and
List, 1982; Hawthron, et al. 1988; Johnston and Penninger,
1989; Krukonis and Kurnik, 1985; McHugh and Krukonis, 1986;

Mcnally and Wheeler, 1988; Stahl, et al. 1980).

27

A gas becomes a supercritical fluid at temperatures and
pressures above its critical point (Figure 1) . Carbon dioxide
, commonly used as a supercritical fluid, exhibits a critical
temperature and pressure of 31.1°C and 73.8 atm, respectively.
In the range above its critical temperature and pressure,
carbon dioxide is in the supercritical state and can dissolve
a variety of compounds. Supercritical fluid extraction (SFE)
exploits the unique properties of gaseous solvents above their
critical states to fractionate mixtures of compounds in a
single step (McHugh and Krukonis, 1986 ).. .At the critical
state, they have the dissolving powers of a liquid while
retaining such.properties.of'a gas as high diffusivity and low
viscosity. In other words, supercritical fluids possess a
liquid-like density, but also exhibits gas-like transport
properties of diffusivity and viscosity. Therefore,
supercritical fluids are able to dissolve a variety of
compounds, even those with high molecular mass and low
volatility (Gouw and Jentoft, 1972).

Carbon dioxide is probably the most widely used
supercritical fluid because its critical temperature
(Tc =31.1°C) makes it an ideal solvent for extracting materials
that are thermally labile. Also, CO2 is non-toxic, non-
flammable, and environmentally preferred over organic
solvents. The extraction conditions of 275 atm and 35°C were
generally chosen to achieve a density of 0.93 at its

supercritical state.

%

/m

/

///

77/

/

       

 

EEEEE

 

29

Supercritical extraction also can be performed at
varying conditions, e.g. , changing temperatures at constant
pressure or changing pressures at constant temperature. Other
supercritical fluids, such as NH3 and N20, can also be employed
depending on the nature of the compounds to be extracted
(Rizvi, et al. 1986).

The objective of this work was to compare the pungency
of 'Scotch Bonnet’ pepper vs. other peppers, including
Jalapeno, Chili, Cayenne, Bell Captain, Sweet Banana and Forti
F, peppers, to isolate and characterize capsaicin and
dihydrocapsaicin, and compare the extraction efficiency of

supercritical CO2 fluid with organic solvents.

HATERIALS AND METHODS

General experimental: Vacuum liquid chromatography (VLC) was
performed on the silica gel (Analtech silica gel 60 A pore
size, 35 - 75 micron particle size). Preparative thin layer
chromatography (Prep. TLC) was performed on silica gel
Uniplates (Analtech silica gel GF-254, 0.5 mm, 20 x 20 cm,
2000, 1500, 500, 250 microns). Column chromatography was
performed using a 3.3 cm X 33 on column containing silica gel
(Analtech silica gel 60 A pore size, 35 - 75 microns particle
size), and thin layer chromatography (TLC) on silica gel
(Aldrich silica gel G F-254, 0.250 mm layer, 2.25 1: mean

particle size). Unless specified, the developed plates were

30
viewed in an ultraviolet fluorescence analytical cabinet
(Spectroline Inc.) at 366 nm and 254 nm, respectively, or
sprayed with 50% H2804, then heated to charc the compound.

Low pressure C-18 column chromatography was performed
using an LC-SORB glass column (Chemco, 3.2 cm X 50 cm) packed
with C“ silica gel and equipped with a low-pressure pump
(Model 81-M-2, Chemco), rheodine injector (Chemco) and a
UV-IS 200 detector at 280 nm (Sanki Laboratories Inc.).

Supercritical fluid extraction (SFE) of peppers was
achieved on a Dionex - 703 SFE equipped with 8cm X 32 ml
extraction cells (Dionex Co. USA).

High performance liquid chromatography (HPLC) was
performed using an HPLC system consisting of a Waters
Automated Gradient Controller equipped with a Waters Model U6K
injector, Shodex Degasser, Waters Model 590 pump and two
Waters M-6000A pumps. The guard column was Nova-Pak (Waters
Associates) with removable C-18 cartridge. The column was
Waters Radial - Pak C18 cartridge, 4 u particle size, 5 X 10 mm
inserted in a Waters RCM 8 X 10 radial compression module
(RCM) . Detection was at 280 nm with a Waters 490 programmable
multiwavelength U.V. detector. Data acquisition was carried
out on a Waters 740 data module. The mobile phase was
acetonitrile : water (1:1). The flow rate was 0.75 ml/min.

Melting point was determined on a Kofler hot stage
melting point apparatus (Bristoline Co. USA) and was

uncorrected.

31

Proton (‘H)and carbon (13C) , ‘H - lH decoupling and DEPT
nuclear magnetic resonance spectra were obtained on a Varian
VXR - 300 spectrometer (Varian Co. USA), 300 Mhz for proton
and 75 Mhz for carbon, and a Varian VXR - 500 spectrometer,
500 Mhz for proton and 125 Mhz for carbon. Electron impact
mass spectra (EIMS) were obtained on a Jeol model JMAX 505
mass spectrometer at 70 eV. (Jeol JMS Co. USA). Ultraviolet
(UV) absorption analysis was conducted on a Shimadzu UV-

Visible model UV-260 spectrophotometer.

Plant material: The matured fruits of C. annuum Scotch
Bonnet, Mayata F1, Hybrid Bell Captain, Cuba and Sweet Banana
were collected from pepper plants grown in the greenhouse of
the Department of Horticulture, Michigan State university,
East lansing, Michigan. Other types of C. annuum pepper used
were Chili purchased from Meijer Inc., Lansing, Michigan,
Cayenne purchased from Kroger Co., Okemos, Michigan, Jalapeno
purchased from.Horrock's Farm Market, Lansing, Michigan. All
peppers were lyophilized by a DURA-DRY FTS - Tray Lyophilizer
(FTS SYSTEM Inc. USA), and stored in sealed plastic bags at -

20°C prior to extraction.

Separation of capsaicin and dihydrocapsaicin from Aldrich
sample: The 1H NMR spectrum of capsaicin (Aldrich Chemical
Co.) indicated that it was a :mixture of capsaicin and

dihydrocapsaicin (Figure 2). The capsaicin (190.4 mg)

32

 

 

 

 

 

 

.-
II
.-
U
N
.2

Figure 2: 'H NMR spectrum of Aldrich capsaicin

 

33
(Aldrich Chemical Co.) was dissolved in 1 ml of acetonitrile
and injected onto the LC-SORB glass column (Chemco, 32 mmlx 50
cm) packed with C18 silica gel and eluted with
acetonitrile/water (1./ 1). 'The 10 fractions, fraction I (200
ml), II (150 ml), III (20 ml), IV (15 ml), v (40 ml), VI (130
ml), VII (150 ml), VIII (200 ml), Ix (210 ml) and x (250 ml)
collected were analyzed by HPLC, and those having a peak with
the retention time of capsaicin (fraction III, IV and V) or
dihydrocapsaicin (fraction VIII) were combined separately.
Removal of solvent in vacuo afforded the pure capsaicin (29.5

mg) and dihydrocapsaicin (4.9 mg), respectively.

standard Curve: A stock solution of capsaicin (1,6000 ug/ml)
was prepared by dissolving pure capsaicin (Aldrich Chemical
Co.) in acetonitrile. Serial dilutions were made to obtain
standard solutions containing 400, 200, 100, 80, 40, 20, 10,
8, 4, 2, 1, 0.8, 0.4, 0.2, 0.1 ug/ml of capsaicin,
respectively. A standard curve was generated by plotting an
HPLC peak area of capsaicin against the concentration of

capsaicin (Figure 3).

Isolation of capsaicin and dihyrocapsaicin from, 'Scotch
Bonnet' pepper: The procedure for the isolation and
purification of capsaicin and dihydrocapsaicin from the
lyophilized 'Scotch Bonnet' pepper is shown in figure 4. The

dried tissue (192 g) was blended with hexane (1L, 2 min.) and

34

 

 

10.0-1 /
J /
/
/.
/
/
/
m d
if 51) //
/
/
‘ /
J -/
//
. ,,/
0.0 / ' . f r fi r T
0.0 5.0

Capsaicin ( ug )

Figure 3: Standard curve of capsaicin

35

 

Lycphilized 'Scotch Bonnet' tissue
(192 9)

Sequentially extracted with —
hexane, chloroform and methanol

Hexane extract I [Chlorcform extract

 

 

 

Methanol extract

(10.6 g) kept aside kept aside

 

 

 

.Purificaticn
by‘nc

 

 

I Fractions 11 6 12
| .2...

Purification by prep.TLc,
hexane:acetone (4:1)

 

 

Band I (Rf-0.11)
265 mg

 

Repeat purification by prep.TLc,
chloroform:methanol-8:l

 

Band II(Rf-0.56)
(mixture of capsaicin and dihydrocapsaicin)
192 mg

 

Separation
by c" column chromatography

 

 

 

Dihydrocapsaicin

21.6 mg

Capsaicin
50.5 mg

 

 

 

Figure 4: Isolation and purification of capsaicin and
dihydrocapsaicin from ’Scotch Bonnet'

36

the slurry was poured into a glass column (33 mm X 33 cm) and
percolated with hexane (5 L, 24 h). The extraction was
continued with chloroform (500 ml X 4, 28 h) and methanol (500
ml X 5, 26 h), respectively. All extracts were evaporated in
vacuo and afforded reddish-brown (19.1 g), brownish-red (1.5
g) and dark yellow (30.7 g) oily products from hexane,
chloroform and methanol extracts, respectively. The hexane
extract, containing most of capsaicin and dihydrocapsaicin
among these extracts, was purified initially using vacuum
liquid chromatography. A slurry of column silica (fine, 4.5 -

5.0 pm) in hexane was packed under vacuum in a sintered glass
filter and washed with hexane. The hexane extract (10.6 9),
dissolved in hexane (60 ml), was applied on the silica under
vacuum and eluted with hexane (fraction 1 to 7, 1870 ml),
hexane : acetone (4 : 1, fraction 8 to 12, 950 ml),
chloroform : methanol (3 : 1, fraction 13, 500 ml) and
methanol (fraction 14, 280 ml), respectively. Fractions 11
and 12 containing mostly capsaicin and dihydrocapsaicin upon
TLC and taste analysis, were combined and evaporated to
dryness (320 mg), then further purified by preparative TLC
using hexane : acetone (4:1). The band I (R, = 0.11),
containing capsaicin and dihydrocapsaicin, was eluted with
chloroform : methanol (4 : 1) and the removal of solvent in
vacuo afforded a low melting-point creamy solid (265 mg).
This was purified further by preparative TLC using chloroform

: methanol (8 : 1) and the band II at Rf 0.56, containing

37
capsaicin and dihydrocapsaicin was removed and eluted with
chloroform : methanol (4 : 1). 1H NMR spectrum of this
mixture is showed in figure 5.

The separation of capsaicin and dihydrocapsaicin from
the mixture was achieved by C18 column chromatography on a LC-
SORB glass column (Chemco, 32 mm X 50 cm) packed with C18
silica gel and eluted with acetonitrile :iwater (50: 50, v/v).
The mixture of capsaicin and dihydrocapsaicin (0.192 g) was
applied on the column and 16 fractions, fraction I (150 ml),
II (70 ml), III (10 ml), IV (30 ml), V (20 ml), VI (40 ml),
VII (10 ml), VIII (20 ml), IX (80 ml), X (150 ml), XI (60 ml),
XII (120 ml), XIII (50 ml), XIV (150 ml), xv (120 ml) and XVI
(140 ml), were analyzed by HPLC. Those having a single peak
with retention time 5.9 min for capsaicin (fractions III, IV,
V, VI, VII and VIII) or 8.8 min for dihydrocapsaicin
(fractions XII, XIII and XIV), respectively, were combined
separately. The pure capsaicin (50.5 mg) and dihydrocapsaicin
(21.6 mg) were obtained.after the removal of solvent in vacuo,
and had the following characteristics:

Capsaicin C18H27NO3; mp, 65 - 66°C; ‘H NMR (CDC13): 6 0.94 (6H,
d, J=6.7Hz, 8’- and 9'-cn,), 1.47 (6H, m, 2', 3'and 4'-cn,),
1.97 (1H, m, J=7Hz, 10'-CH), 2.19 (2H, t, J=7.8Hz, 1'-Cn,),
3.84 (3H, s, -OCHQ, 4.34 (ZHQ d, J=5.9Hz, benzylic CHfl, 5.33
(2H, m, 5'and 6’- vinyl CH), 5.59 (1H, 8, NH), 5.63 (1H, s,
phenolic -OH), 6.77 (3H, m, 3, 5 and 6- aromatic protons)

(Figure 6). 13c NMR (cools): 6 172.81 (C=0), 146.72, 145.14

38

 

 

 

 

 

Figure 5: 1H NMR spectrum of the mixture of capsaicin and
dihydrocapsaicin from 'Scotch Bonnet'

39

 

 

 

 

 

Figure 6: 'H NMR spectrum of capsaicin from 'Scotch Bonnet’

40

(2xc-o aryl), 55.92 (OMe), 43.53 (CH2-N), 22.63 (211011,). EI-
ns: m/z(% int.) 305 (14*, c,,n,,no,,16.4), 195 (Cmn,,o,, 8.1), 168
(C,n,,No, 4.5), 152 (c,n,,,No,, 12.9), 137(c,n,o,, 100).

Dihydrocapsaicin C,,n,,N0,; 1H NMR (c0c13): 5 0.84 (6H, d,
J=5.6Hz, 8'and 9'-CH3), 1.37 (10H, m, 2’, 3’, 4’, 5'and 6'-
cn,), 1.58 (1H, m, 7’-CH), 2.18 (2H, t, J=7.8Hz, 1'-cn,), 3.86
(3H, s, -ocn3), 4.35 (2H, d, J=5.6Hz, benzylic CH2), 5.61 (1H,
s, NH ), 5.63 (1H, s, phenolic -OH), 6.80 (3H, m, 3, 5 and 6-
aromatic protons) (Figure 7). ”C NMR (CDCl3): 6 174.6 (C=0),
145.12, 146.68 (2XC-o aryl), 55.94 (One), 43.54 (RCHz-N),
22.68 (2xcn3). EI-MS: m/z(% int.) 307(n+, c,,n,,N03, 6.1), 195
(ownnoy 8.1), 168 (C,n,,No, 4.5), 152 (c,n,ono,, 12.9),

137 (0,115.02, 100) .

Supercritical co, fluid extraction: The lyophilized 'Scotch
Bonnet’ pepper was milled and 2.4 g of the powdered tissue was
packed in the 32 ml stainless steel extraction cell (1.5 cm X
20 cm). The extraction was carried out at 50°C and 450 atm,
and 50°C and 600 atm for 30 min and 1 h, respectively. In
each experiment, the extracts were collected in chloroform and
the solvent was evaporated in vacuo to dryness prior to

analysis.

Solvent extraction of peppers: The milled peppers (10 g,
each) were extracted sequentially with hexane, ethyl acetate

and methanol (500 ml each, 24h per solvent). The

41

' f
l l ‘ 7
I I i E
l l f t
l l
. l t
' 9
I i
i
i
I
!

.__.. .. .._. _.—.
—

 

 

 

 

 

 

 

 

..... 1......11- .1 ' ”UL.

TillrrllllllerlTlllITI‘ITTFIIITIITT

7 6 5 4 3 2 pp.

 

Figure 7: 'H NMR Spectrum of dihydrocapsaicin from ’Scotch Bonnet'

42
extracts were evaporated in vacuo to dryness and stored at

20°C prior to the analysis.

Quantification of capsaicin in Q;_angggm cultivars: 10.0 mg
each of the crude extract were dissolved in 5 ml of
acetonitrile and filtered through the MILLEX-FGS filter (0.2
pm, Millipore Inc., Bedford, MA). The solutions (50 ul each)
were analyzed by HPLC. The peak area for capsaicin was
recorded and the percentage of capsaicin to dry weight in each

C. annuum cultivars was calculated.

RESULTS AND DISCUSSION

Preliminary HPLC analysis of commercially available
capsaicin indicated that acetonitrile/water (50/50, v/v)
produced the best separation of capsaicin and
dihydrocapsaicin. The HPLC chromatogram of purified capsaicin
and dihydrocapsaicin from both ’Scotch Bonnet’ and Aldrich
capsaicin showed only single peaks at 5.9 min for capsaicin
and 8.8 min for dihydrocapsaicin, respectively.

Capsaicin and dihydrocapsaicin were isolated and
purified from both 'Scotch Bonnet' and Aldrich capsaicin. The
mixture of capsaicin and dihydrocapsaicin obtained from the
hexane extracts of 'Scotch Bonnet' was purified by C18 silica

column chromatography, using the method used for purification

43
of Aldrich capsaicin, and gave pure capsaicin and
dihydrocapsaicin (Figure 5, 6 and 7).

The quantification of capsaicin and dihydrocapsaicin
from ’Scotch Bonnet' and Aldrich capsaicin were carried out by
HPLC and the characterization by NMR and mass spectral data.

1H and ”C NMR spectra of capsaicin and dihydrocapsaicin
from ’Scotch Bonnet' and Aldrich capsaicin were found to be
identical. In the 1H NMR spectra of capsaicin and
dihydrocapsaicin from 'Scotch Bonnet' (Figure 6 and 7)
revealed three protons at 6.8 ppm, indicative of a benzene
ring with three substituents. The multiplet indicates that
two of the aromatic protons are ortho coupled to each other
and the third is coupled to one of the ortho coupled protons.
The singlets at 5.59 and 5.63 ppm support the phenolic -OH and
-NH protons in the molecule. The singlet at 3.84 ppm was
assigned to an aromatic -OCH3. The l3C NMR spectra of
capsaicin from Scotch Bonnet, shows the signal at 172.81 ppm
for the carbonyl carbon. The 146.72 and 145.14 ppm were
assigned for two arylic carbons. The peak at 55.92 ppm was
methoxyl carbon (One). The signal at 43.53 ppm was assigned
toich-NH.

The mass spectra of both capsaicin and dihydrocapsaicin
of 'Scotch Bonnet' clearly indicated a molecular ion at m/z
305 and 307, respectively. Both compounds gave base peaks at
m/z 137, for the fragment C3H902, and peaks at m/z 195 for

CanO3, at m/z 168 for CusNO, and at m/z 152 for CgHwNOz.

44

Extraction of the lyophilized, milled fruits of 'Scotch
Bonnet' using supercritical CO2 and organic solvents afforded
red oily residues in all cases. HPLC analysis indicated that
the capsaicin content in the organic extract was 0.514% d.w.
in ’Scotch Bonnet’, 0.088% d.w. in 'Chile’, 0.068% d.w. in
'Jalapeno', 0.034% d.w. in ’Cayenne', 0.047% d.w. in ’Forti
F1', and zero in ’Bell Captain’, 'Mayata and Sweet Banana’,
respectively. The capsaicin content (% d.w.) of peppers in
supercritical fluid extract was 3.2% in 'Scotch Bonnet'. On
the basis of the above results, the supercritical fluid
extraction of capsaicin was superior to the conventional
organic solvent extraction.

The capsaicinoids can be distinguished individually by
their fatty acid moieties. Sticher et al. (1978) reported
that the elution order of capsaicinoids on HPLC were related
to their fatty acid chain length.and the degree of saturation.
Therefore, only a reverse-phase HPLC system can separate these
compounds.

An authentic sample of capsaicin purchased from Aldrich
Chemical Co. contained 67% of capsaicin and 33% of
dihydrocapsaicin (Figure 2). Purification of this sample by
C", column chromatography yielded the pure capsaicin and
dihydrocapsaicin. 'The pure capsaicin ‘was used for the
quantification of capsaicin in all the pepper extracts
investigated.

Hexane, ethyl acetate and. methanol were used

45
sequentially to extract dried fruits of C. annuum cultivars
and.the capsaicin content in each pepper was analyzed by HPLC.

Supercritical fluid extractions of peppers also were
investigated. The supercritical fluids are unique in their
dissolving capabilities and hence have been used widely in the
extraction of natural products from plant and microbial
tissues.

Eight pepper cultivars with different capsaicin contents
were extracted with solvents. Supercritical CO2 was used to
extract ’Scotch Bonnet’ pepper and the result was compared
with the solvent extraction of ’Scotch Bonnet’ . Figure 8
illustrates the results of capsaicin content by both
supercritical CO2 and solvent extractions. The most pungent
pepper, ’Scotch Bonnet’, gave the highest level of capsaicin
among these peppers. The SFE extraction of ’Scotch Bonnet’
gave 3.2% capsaicin /g dry weight which is about 32 times
higher than that from total solvent extraction of ’Scotch
Bonnet’ (0.51%). ’Chile’, ’Jalapeno’ and ’Cayenne’ peppers
were found.totcontain 0.09%, 0.07% and 0.03%, respectively, by
using solvent extraction. Capsaicin was not detected in ’ Bell
Captain’ , ’ Cuba’ , ’Mayata F1’ and ’Sweet Banana’ peppers.
These results indicate that the pungency of pepper is related
to the amount of capsaicin present in the pepper and is in
agreement with the published results (Mary, 1984; Todd et al.
1977).

Identification of capsaicin and dihydrocapsaicin from

46

CAPSAICIN CONTENT 74 lg DRY WEIGHT
IN SCOTCH BONNET

 

3.2

a; \

 

0.61

°':i__- 1

- Solvent cxtractlon \\\\‘ SFE extractlcn

a. Z-O->UJ'U>O
N

 

 

 

 

 

 

 

 

 

Figure 8: Comparison of SFE and solvents extraction of Capsaicin
from ’Scotch Bonnet’

47

C. annuum cultivars were achieved by comparing the retention
times of the purified capsaicin and dihydrocapsaicin from
Aldrich capsaicin. Only two capsaicinoids, capsaicin and
dihydrocapsaicin were isolated and purified from the pepper
cultivars studied. The purified capsaicin and
dihydrocapsaicin from ’Scotch Bonnet’ gave identical spectral
data to the capsaicin and dihydrocapsaicin from Aldrich
sample.

Capsaicin and dihydrocapsaicin can be distinguished
clearly from therr‘HiNMR spectra. The signal at 5.3 ppm for
two olefiniijrotons in capsaicin (Figure 6) was absent in the
lH'NMR spectra of dihydrocapsaicin (Figure 7). Also, based on
the 1H NMR spectra of the capsaicinoids mixture obtained from
’Scotch Bonnet’, the relative ratio of capsaicin and
dihydrocapsaicin in the mixture was 82% and 18%, respectively
(Figure 5). There were no detectable levels of other
capsaicinoids in these peppers. This is the first report of
the isolation, purification and characterization of capsaicin

and dihydrocapsaicin from ’Scotch Bonnet’ pepper.

REFERENCES

Andrews, J. Peppers: the domesticated capsicums. University of
Texas Press, Austin. 1984.

Attuquayefio, V.K. and Buckle, K.A. Rapid sample preparation
method for HPLC analysis of capsaicinoids in capsicum fruits
and oleoresin. J. Agric. Food Chem. 1987, 35, 777-779.

Bajaj, K.L. Colorimetic determination of capsaicin in capsicum
fruits. J. Assoc. Off. Anal.Chem. 1980, 63, 1314-1316.

Eldridge, A.C., Friedrich, J.P., Warner, K. and Kwolek, W.F.
Preparation and evaluation of supercritical carbon dioxide
defatted soybean flakes. J. Food Sci. 1986, 51, 584-587.

Friedrich, J.P.and List, G.R. Characterization of soybean oil
extracted by supercritical carbon dioxide and hexane. J. Agr.
Food Chem. 1982, 30, 192-198.

Garrett, L., Dyremple, B. and Wollo, W. Production of Scotch
Bonnet pepper in Missouri: an economic evaluation. 88th ASHS
Abstract. July 19-24, 1991.

Gouw, T.H. and Jentoft, R.E. Supercritical fluid
chromatography. J. Chromatogr. 1972, 68, 303-323.

Hawthron, S.B., Kreiger, M.S. and Miller, D.J. Analysis of
flavor and fragrance compounds using supercritical fluid
extraction coupled with gas chromatography. Anal. Chem. 1988,
60, 472-477.

Heiser, C.B. Peppers capsicum (Solanaceae). Longman press,
London. 1979.

Hoffman, P.G., Lego, M.C. and Galetto, W.G. Separation and
quantitation method of red pepper major heat principles by
reverse-phase HPLC. J. Agr. Food Chem. 1983, 31, 1326-1330.

Iwai, K. , Suzuki, T. and Fuj iwake, H. Simultaneous
microdetermination of capsaicin and its four analogues by HPLC
and GC/MS. J. Chromatogr. 1979, 172, 303-311.

Johnston, K.P. and Penninger, J .M.L. Supercritical fluid
science and technology. Amer. Chem. Soc., Washington, D.C.
1989.

Krukonis, V.J and. Kurnik, R.T. Solubility of the solid
aromatic isomers in carbon dioxide. J. Chem. Eng. Data. 1985,
30, 247-253.

Kosuge, S. and Furata, M. Studies on the pungent principle of

48

49

Capsicum. Part XIV: chemical constitution of the pungent
principle. Agr. Biol. Chem. 1970, 34(2), 248-256.

Krajewska, A.M. and Powers, JuJ. Gas chromatographic
determination of capsaicinoids in green capsicum fruits. J.
Assoc. Off Anal. Chem. 1987, 70, 926-928.

Mary, C. L. HPLC in the flavor industry. Food Technol. 1984,
38, 84-87.

Masada, Y., Hashimoto, K., Inoue, T. and Suzuki, M. Analysis
of the pungent principles of capsicum annuum by continued gas
chromatography-mass spectrometry. J. Food Sci. 1971, 36, 850-
860.

McHugh, M.A. and Krukonis, V.L. Supercritical fluid extraction
: principles and practice. Butterworth Stoneham Press, MA.
1986. 23-76.

Mcnally, M.E.P.and. ‘Wheeler, J.R. Supercritical fluid
extraction coupled with supercritical fluid chromatography for
the separation of sulfonylurea herbicides and their
metabolites from complex matrices. J. Chromotogr. 1988, 435,
63-71.

Newman, A.A. Chemistry of capsaicin - the pungent principle of
the Capsicum peppers. Chem. Prod. Chem. News. 1953, 16, 413-
418.

Nelson, E.K. Vanillyl-acylamides. J. Amer. Chem. Soc. 1919,
41, 2121-2130.

Panker, D. S. and Magar, N. G. New method for the
determination of capsaicin by using multiband thin-layer
chromatography. J. Chromatogr. 1977, 144, 149-152.

Ramos, P.J.J. Further study of the spectrophotometric
determination of capsaicin. J. Assoc. Off. Off. Chem. 1979,
62, 1168-1170.

Saria, A., Lembeck, F. and Shofitsheh, B. Determination of
capsaicin in tissues and separation of capsaicin analogues by
HPLC. J. Chromatogr. 1981, 208, 41-46.

Rizvi, S.S.H., Daniels, J.A., Benado, A.L. and Zollweg, J.A.
Supercritical fluid extraction: operating principles and food
applications. Food Tech. 1986, 40(7) 57-64.

Stahl, E., Schutz, E. and Mangold, H.K. Extraction of seed oil
with liquid and supercritical carbon dioxide. J. Agr. Food
Chem. 1980, 28, 1153-1158.

50

Sticher, 0., Soldati, F. and Joshi, R.K. HPLC separation and
quantitative determination of capsaicin. J. Chromatogr. 1978,
166, 221-226.

Thresh, L.T. Isolation of capsaicin. J. Pharm. 1846, 6, 941-
945.

Todd, P. H. and Perun, C. Gas-liquid chromatography analysis
of capsicum amides. Food Technol. 1977, 15, 270-273.

Weaver, R.M. and .Awde, D.B. Rapid HPLC :method for the
determination of very low capsaicin levels. J. Chromatogr.
1986, 367, 438-442.

CHAPTER THREE
INVESTIGATION OF BIOACTIVE COMPOUNDS IN CAPSICUM
ANNUUM CULTIVARS

ABSTRACT

The major compounds responsible for the pungency of capsicum
were capsaicin and its analogues. Supercritical fluid and
organic solvent extraction of Capsicum annuum cultivars
afforded extracts containing the pungent principles, capsaicin
and dihydrocapsaicin. Bioassays of extracts and pure
capsaicin and dihydrocapsaicin were carried out on fungi,
bacteria, nematodes and mosquito larvae. The hexane extracts
of fruits of Capsicum annuum Sweet Banana, Maya F1, Bell
Captain, Jalapeno, Chile, Cayenne and Scotch Bonnet were found
to be active against fungi, bacteria and mosquito larvae at
250 ppm. The hexane extract of ’Scotch Bonnet’ fruits showed
strong mosquitocidal activity on fourth instar Aedes aegypti
larvae at 50 ppm. Purification of this hexane extract
afforded a pure compound, bonnetenol, that gave 100% mortality
on A. aegypti larvae at 0.1 ppm in 24 h. Bonnetenol was
characterized by mass spectral, ‘H, 13C, 1H - lH decoupling and
DEPT nuclear magnetic resonance analyses. Bonnetenol was

confirmed to be z-hept-3-en-1-ol.

51

INTRODUCTION

The principal compounds responsible for the pungency of
Chile peppers are capsaicinoids, amino acids of vanillylamine
and C, - Cu branched fatty acids (Figure 3, Chapter 1) (
Hoffman, et al. 1983). Capsaicin and dihydrocapsaicin are the
predominant compounds in Chile peppers, contributing 90% or
more of the total heat value (Iwai, et al. 1979).

There are no reports on the biological activity of
compounds extracted from C. annuum cultivars, against fungi,
bacteria, nematodes and insects. Supercritical fluid and
organic solvents were used to extract capsaicinoids from C.
annuum cultivars (Chapter 2) . Capsaicin and dihydrocapsaicin
were isolated and purified by VLC, prep.TLC and a reverse -
phase C18 column chromatography (Chapter 2). All extracts of
peppers, pure capsaicin and dihydrocapsaicin were assayed
separately for nematicidal, fungicidal, bactericidal and

mosquitocidal activities.

MATERIALS AND METHODS

General experimental: Vacuum liquid chromatography (VLC) was
performed on the silica gel (Analtech silica gel 60 A pore
size, 35 - 75 micron particle size). Preparative thin layer

chromatography (Prep. TLC) was performed on silica gel

52

53

Uniplates (Analtech silica gel GF-254, 0.5 mm, 20 X 20 cm,
2000, 1500, 500, 250 microns). Column chromatography was
performed using a 3.3 cm X 33 cm column containing silica gel
(Analtech silica gel 60 A pore size, 35 - 75 microns particle
size), and thin layer chromatography (TLC) on silica gel
(Aldrich silica gel G F-254, 0.250 mm layer, 2.25 )1 mean
particle size). Unless specified, the developed plates were
viewed in an ultraviolet fluorescence analytical cabinet
(Spectroline Inc.) at 366 nm and 254 nm, respectively, or
sprayed with 50% H2804, then heated to charc the compound.

Low pressure C-18 column chromatography was performed
using an LC-SORB glass column (Chemco, 3.2 cm X 50 cm) packed
with C", silica gel and equipped with a low-pressure pump
(Model 81-M-2, Chemco), rhoeadine injector (Chemco) and a
UV-IS 200 detector at 280 nm (Sanki Laboratories Inc.).

Supercritical fluid extraction (SFE) of peppers was
achieved on a Dionex - 703 SFE equipped with 8cm X 32 ml
extraction cells (Dionex Co. USA).

High performance liquid chromatography (HPLC) was
performed using an HPLC system consisting of a Waters
automated gradient controller equipped with a Waters Model U6K
injector, Shodex Degasser, Waters Model 590 pump and two
Waters M-6000A pumps. The guard column was Nova-Pak (Water
Associates) with removable C-18 cartridge. The column was
Waters Radial - Pak C18 cartridge, 4 )1. particle size, 5 X 10 mm

inserted in a Waters RCM 8 X 10 radial compression module

54
(RCM) . Detection was at 280 nm with a Waters 490 programmable
multiwavelength U.V. detector. Data acquisition was carried
out on a Waters 740 data module. The mobile phase was
acetonitrile : water (1:1). The flow rate was 0.75 ml/min.

Melting point was determined on a Kofler hot stage
melting point apparatus (Bristoline Co. USA) and was
uncorrected.

Proton (‘H)and carbon (”C) , lH - 1H decoupling and DEPT
nuclear magnetic resonance spectra were obtained on a Varian
VXR - 300 spectrometer (Varian Co. USA), 300 Mhz for proton
and 75 Mhz for carbon, and a Varian VXR - 500 spectrometer,
500 Mhz for proton and 125 Mhz for carbon. Electron impact
mass spectra (EIMS) were obtained on a Jeol model JMAX 505
mass spectrometer at 70 Ev. (Jeol JMS Co. USA). Ultraviolet
(UV) absorption analysis was conducted on a Shimadzu UV-

Visible model UV-260 spectrophotometer.

Plant material: The matured fruits of C. annuum cultivars
Scotch Bonnet, Mayata F1, Hybrid Bell Captain, Cuba and Sweet
Banana were collected from pepper plants grown in the
greenhouse of the Department of Horticulture, Michigan State
University, East Lansing, Michigan. Other types of C. annuum
used were Chili purchased from Meijer Inc. , Lansing, Michigan,
Cayenne purchased from Kroger Co., Okemos, Michigan, Jalapeno
purchased from.Horrock’s Farm Market, Lansing, Michigan. All

peppers were lyophilized by a DURA-DRY FTS - Tray Lyophilizer

55
(FTS SYSTEM Inc. USA), and stored in sealed plastic bags at -

20°C prior to extraction.

Supercritical co, fluid extraction: The lyophilized Scotch
Bonnet pepper was milled and 2.4 g of the powdered tissue was
packed in.a 32 ml stainless steel extraction cell (1.5 cm.X 20
cm). The extraction was carried out at 50°C and 450 atm, and
50°C and 600 atm for 30 min and 1 h, respectively. In each
experiment, the extracts were collected in chloroform and the

solvent was evaporated in vacuo to dryness prior to analysis.

Solvent extraction of peppers: The milled peppers (10 g each)
were extracted sequentially with hexane, ethyl acetate and
methanol (500 ml each, 24h per solvent). The extracts were
evaporated in vacuo to dryness and stored at 20°C prior to the

analysis.

Isolation of bioactive compounds from ’Scotch Bonnet’:
Lyophilized and milled ’Scotch Bonnet’ tissue (139.9 g) was
packed in a glass column (20 mm X 37 cm) and extracted with
hexane (2L, 24 h) at room temperature. Removal of solvent in
vacuo afforded an oily dark red residue (11.1 g)(Figure 1).
Because only the hexane extract was active against mosquito
larvae, it was further purified by VLC. A slurry of column
silica (fine, 4.5 - 5.0 um) was packed under vacuum in a

sintered glass filter and washed with hexane. This hexane

56

 

l Lyophilized ’Scotch Bonnet’ tissue (139.9 g)]

 

Extracting with hexane

 

 

[ hexane extract (11.1 g)]
(active)

 

Purification by VLC

Fraction V
(inactive)

 

[Fraction III] Fraction IV]

Fraction I

(inactive) (inactive) (inactive)

(3.69)
(active)

 

 

 

[Fraction II

 

 

Fractionation by VLC

 

 

 

 

 

 

l
7 Fraction A Fraction 8 A
(inactive)] I (0.548 g)
~ (active)

 

 

Further purification
by prep.TLC

Bonnetenol (2.5 mg)l
(active)

 

Figure 1: Isolation and purification of bonnetenol from
’Scotch Bonnet’

57
extract (11.1 9) then was applied on silica as a hexane
solution and eluted sequentially with hexane (fraction I, 280
ml), hexane : acetone (9 : 1, fraction II, 700 ml), hexane :
acetone (4 : 1, fraction III, 450 ml), chloroform : methanol
(4 : 1, fraction IV, 200 ml) and methanol (fraction V, 400
ml). Only fraction II (3.6 9) showed mosquitocidal activity.
Further fractionation of this fraction by VLC afforded two
fractions, eluted with hexane : acetone (4 : 1, fraction A,
470 ml) and hexane : acetone (1 : 1, fraction B, 410 ml ),
respectively. Upon TLC analysis, fraction B (1.011 g, R,=
0.28) was active on mosquito larvae. Preparative TLC of this
fraction (0.548 g) (hexane : acetone, 6 : 1) gave three bands,
which were eluted with chloroform : methanol (4 : 1) . Band I
(233.8 mg) at R,=0.45 (light yellow) gave strong activity on
mosquito larvae and on further purification by preparative TLC
afforded pure bonnetenol. The chemical identification of
bonnetenol was achieved by 1H, 13C, 1H - lH decoupling and DEPT

NMR experiments, along with MS and UV analysis.

Bonnetenol: C7H,,O, showed UV absorption at 202 (6 = 3109) , 256
(6 = 1080) and 273 (6= 166) nm, respectively (Figure 2). 1H
NMR (CDCl,): 6 0.96 (3H, t, J=7.5Hz, 7-CH3), 1.26 (2H, m, 5-
CH2), 1.45 (2H, m, 6-CH2), 1.54 (1H, s, exchanged with D20, -
on), 1.72 (2H, m, 2-CH2), 4.31(t, J=6.7nz, 1-cn2), 7.52 (d,d,
J=3.4, 5.8Hz, olefinic H), 7.71 (d,d, J=3.4, 5.8Hz, olefinic

H) (Figure 3a and 3b); 'H-‘H decoupling NMR spectrum is showed

58

; ”9'0 um rzoz
009 0

 

 

 

 

I X...
5 $5 §
c o c

Figure 2 : UV spectrum of bonnetenol

59

 

 

 

 

LL- 1 A- L IA_J__

"_ '— ‘7

 

[IUTUITFVTIUTIIIllilllllIlVIIII—TVITIITT

8 7 6 5 4 3 2 1

Figure 3a: 1H NMR spectrum of bonnetenol with D20 exchange

 

 

ll - . 1 milling

11IIITUIITITIITI’IIII'TTrII[TIT]ITTTITTI

8 7 6 5 4 3 2 1

Figure 3b: 'H NMR spectrum of bonnetenol

60
in figure 4. ”C NMR (CDCl,): 6 13.71 (c-7), 19.17 (C-6),
29.68 (c-5), 30.56 (C-2), 65.56 (C-l), 128.83 (C-4), 130.89
(C-3)(Figure 5); 13C DEPT NMR spectrum is showed in figure 6.
El (DI) 113: m/z(% int.) 113 (c,n,,o, 14.7), 111 (c,n“o,
38.6), 97 (C,H,,, 62.6), 83 (gnu, 62.3), 69 (C511,, 72.3), 57

(CHE, 100) (Figure 7).

Microorganisms: Aspergillus flavus, Candida albicans,
FUsarium oxysporum, .Fusarium .monolinifbrme, Streptococcus
aureus, Staphylococcus epidermidis and Escherichia coli were
grown on plates containing 20 ml of Emmons agar (EM)(made by
dissolving 10g of neopeptone, 20g of glucose, 189 of agar in
1 liter' distilled. water). .Phomopsis occulta, Phomopsis
viticola, Aspergillus flavus, Botrytis sp., Rhizoctonia sp.
and Gloesporum sp. were grown on potato dextrose agar (PDA)
plates (made by dissolving 39g of potato dextrose agar in 1
liter distilled water). All media were autoclaved at 120%:

and 15 atm for 20 min.

Antifungal bioassay: Known amounts of the extracts, as well
as pure bonnetenol, capsaicin and dihydrocapsaicin were
dissolved in DMSO, to obtain 50 mg/ml stock solution. These
solutions, 5 pl each (250 pg), were applied on the plates that
were lawned with the test organisms and incubated at 26°C for
2-6 days. Inoculated plates without test compounds, but with

solvent DMSO, served as the control. A clear zone of

61

 

 

 

A ‘L‘ _ .14 ”A A A 4. AL A‘ h A “
w— ,— v' v v v . fi f v w ‘—
T v t I v T 1' Ij 7* v W—f ‘ v f v v ' ‘I rT v I f‘f m j—iq'W'

 

 

 

 

 

W'— rv—‘v

f1 T V— T Y r T V V 1 I

Figure 4: ‘H-‘H decoupling NMR spectrum of bonnetenol

62

Qggi 12!:
eat: :22:

 

 

f”

d"‘
a..-

 

frVVTT'VT'lY'V‘I' 'UVTI[‘1‘VjVVWV'VUYUT'OWf‘IVIUVlVWT'III r7 Tir
130 no :10 :00 90 00 70 60 50 40 N 20 00s
6
Figure 5:

13C NMR spectrum of bonnetenol

63

WW

,1. * )1 . .. ....I . ..
1111.1 Ilnuilt'arh'l [nil ll.) likUIl II.“ J.Inlhllllbi.wlu‘ti.fli ldllih'drillbjd. 1.1111.“I ML‘ul‘.l ii 1.11.11...“- A111)

no no no no no so so 7o so no so so pp.

Figure 6: DEPT NMR spectrum of bonnetenol

OODOQDCU’D "(*fiO—Ofl

64

 

 

 

 

 

 

 

 

 

 

 

 

 

’0? 5?
4 L
so L
4 69 D
‘ 55 71 33 .
4 L
68‘ 97 -
4 P
j 85 ’
‘9: 31 1:1 f
T b
. 8 .
29. '53 5 4 19 .
l } 73 1 13 10
4 so 79 919 '
1 51 ° 1 - 7 ME [’15 H
, .1. 1151*, .93 .1- _ 11.93]. 1 _ _ , . 7 1:1 , .111
so 60 70 so 98 ' 100 110 120
rvz

Figure 7:

Mass spectrum of bonnetenol

65
inhibition characterized by the absence of the fungal growth
around the test compound droplet was recorded after 2 - 6

days.

Antibacterial assay: The antibacterial activity of all test
extracts and standard capsaicin was evaluated by the same
procedure as in the antifungal assay experiments except that

the test organisms were bacteria instead of fungi.

Insecticidal assay: Insecticidal activity was evaluated with
4th instar mosquito larvae, Aedes aegypti, reared from eggs
(University of Davis California Straw) in degassed distilled
water. After emergence, the larvae were fed liver powder for
one week. The bioassy was conducted in glass test tubes (10
X 75 mm) each of which contained 6 - 7 mosquito larvae. DMSO
solutions of the extracts (25 pl each) and.pure capsaicin were
added to 975 pl of distilled water with 6 - 7 mosquito larvae.
The.experiments were carried out in three replications at room
temperature. ILarvae*were observed for mortality at 0.5, 1, 2,
4, 8, 10, 12, 24, 48 and 72 h intervals. Control tubes
containing 6 - 7 larvae received 25 pl of DMSO alone and the

mortality was recorded as in the case of test extracts.

Nematicidal activity: Nematicidal activity was tested on the
free-living nematode, Panagrilus redivivus, reared in NG media

for two weeks. 5 ml of sterile saline solution (8.5 g sodium

66
chloride in 1 liter distilled water) were used per plate to
prepare the nematode suspension, and transferred into a
sterile test tube. The nematode suspension (48 pl),
containing 40 - 60 nematodes at various developmental stages
was added to each well (0.7 on d X 1.0 cm h) of a 96-well
tissue culture plate. 2 pl of each extract and pure capsaicin
in DMSO*were added into each well. The control well contained
pure DMSO. The inoculated plates were held in a container at
ca. 100% humidity. Plates were observed with an inverted
microscope at 40X. Mortality was recorded as the mean of
three replications at each dose after 2, 4, 8, 24, 48 and 96

h.

RESULTS AND DISCUSSION

Hexane extracts of C. annuum cultivars showed good
activity on A. aegypti, C. albicans and S. epidermidis, and
SFE extracts of ’Scotch Bonnet’ were also active on
Rhizoctonia spp. However, ethyl acetate and methanol extracts
of most C. annuum cultivars., as well as pure capsaicin and
dihydrocapsaicin, were inactive against fungi, bacteria and
mosquito larvae. No nematicidal activity was observed with
any of the test compounds.

The most active compound, bonnetenol (0.0018% /g dry
weight) from ’Scotch Bonnet’ had a minimum inhibitory

concentration of 0.1 ppm against A. aegypti. It was extracted

67
from ’Scotch Bonnet’ using hexane and purified by means of VLC
and preparative TLC.

Isolation and purification of pure capsaicin and
dihydrocapsaicin from both Scotch Bonnet and Aldrich capsaicin
were achieved by VLC, preparative TLC and column
chromatographic methods. NMR and MS were used to characterize
capsaicin and dihydrocapsaicin. Although capsaicin and
dihydrocapsaicin are the most pungent compounds in peppers,
they did not show any biological activity against the test
microorganisms.

The 1H NMR signal at 6 0.96 for three protons with
J=7.5Hz was assigned to a CH3 group adjacent to a methylene
group. This was confirmed by 13C and DEPT NMR data which
indicated only one -CH3 functionality in the molecule. The
multiplet for two protons each at 6 1.26, 1.45 and 1.72, were
assigned to three methylene groups, respectively. A triplet,
which integrated for two protons at 6 4.31, was indicative of
a CHz-O functionality adjacent to a methylene group. A
singlet for one proton at 6 1.54, exchanged with D20,
supported the presence of a -OH group. Two doublets of
doublets at 6 7.52 and 7.71, respectively, were assigned to
two olefinic protons cis to each other as evident from their
coupling constant of 6.5Hz. The smaller coupling, J=3.4Hz,
was due to the cis coupling of the olefinic protons to one of
the adjacent methylene groups (Figure 3a).

The 1H - lH decoupling experiments provided additional

68

evidence for the assignments of the signals observed in the HI
NMR spectra for bonnetenol. Irradiation of the triplet at 6
4.31 collapsed the multiplet at 6 1.72 to a tight doublet of
doublets appearing as a triplet. The doublet of doublets at
6 7.52 and 7.71, respectively, were collapsed to a triplet
each when they were irradiated separately. Decoupling the
multiplet at 6 1.45 collapsed the -CH3 at 6 0.96 to a singlet
(Figure 4).

13'C NMR of bonnetenol gave only seven signals (Figure 5) .
The DEPT experiments indicated that two signals at 128.83 and
130.89 ppm, respectively, were olefinic carbons, one methyl
carbon at 13.71 ppm, and four methylene carbons at 19.17,
29.68, 30.56 and 65.56 ppm, respectively. The signal at 65.6
ppm was assigned to the carbon with a hydroxyl group (Figure
5) .

The EI (DI) mass spectrum of bonnetenol gave a single
peak in the total ion current chromatogram (TIC). The mass
peak at m/z 113 was attributed to the loss of one proton from
the parent compound, bonnetenol. A peak at m/z 97 was
characteristic of the loss of the only -OH from bonnetenol.
The peak at m/z 83 and 69, differing by 14 mass units from
the peaks m/z 97 and m/z 83, respectively, were due to the
loss of a CH2 fragments. The base peak at m/z 57 was assigned
to the fragment CHE (Figure 7 and 8). Therefore, based on the
spectral evidence, bonnetenol is confirmed to be Z-hept-3-en-

1-ol (Figure 9).

69

C33C32C32C3'CHC32C3103 (G.H.pm/z 114) [escapees-camor (C’rrHuOomlz 113)

[emancmca-cacnzcm“ renew/z 97) (ca.ca.cn.cn=caca.c-01* (enema/z 111)

4
[CH.CH.CH.CH=CHCH.J* (0.8",m/z 33)

[magmas-car (C,H,,m/s 69)

4
[omensmcmr (C.B..m/z 57)

Figure 8: MS fragmentation pattern of bonnetenol

70

7 6 5 4 3 2 1
CH3'CH2'CH2‘CH = CH‘CHz'CHz'OH

Figure 9: Structure of bonnetenol

71

This is the first report of the isolation and
characterization of a biologically active compound from
’Scotch Bonnet’. There are no previous reports on
mosquitocidal compounds in any of these Capsicum annuum
cultivars. Bonnetenol, Z-hept-3-en-1-ol, has potential
application as a mosquitocidal compound and may prove to be
effective for the control of diseases such as malaria. More
research must be conducted to evaluate the practical value of

bonnetenol.

REFERENCES

Hoffman, P. G., Lego, M. C. and Galetto, W. G. Separation and
quantitation of red pepper major heat principles by reverse-
phase HPLC. J. Agr. Food Chem. 1983, 31, 1326-1330.

Iwai, K., Suzuki, T. and Fujiwake, H. Simultaneous

microdetermination of capsaicin and its four analogues by HPLC
and GC/MS. J. Chromatogr. 1979, 172, 303-311.

72

"I1111111111111“