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This is to certify that the

thesis entitled

SEASONAL VARIATION IN THE COMPOSITION OF BLACK LOCUST
(Robinia pseudoacacia L.) SAPWOOD EXTRACTS

Date

presented by

Shyong, Bao—Jen

has been accepted towards fulfillment

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SEASONAL VARIATION IN THE COMPOSITION OF BLACK LOCUST
(Robiniuseudoacacia L.) SAPWOOD EXTRACTS

Shyong, Bao-J en

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirement
for the degree of
MASTER OF SCIENCE
Department of Forestry

1989

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J. C...

b 0

ABSTRACT

SEASONAL VARIATION IN THE COMPOSITION OF BLACK LOCUST
(Robinia pseudoacacia L.) SAPWOOD EXTRACTS

W
Shyong, Bao-Jen

The heartwood of black locust is not only highly
resistant to decay but it also contains a high level of
flavonoids among its extractable chemicals. The overall
objective of this study was to determine where, and when
flavonoid.biosynthesis takes place in the wood.of'b1ack locust
(Robinia pseudoacacia L.).

In this study, the level of flavonoids and/or flavonoid
precursors in the cambium, sapwood, transition zone and
heartwood were investigated seasonally by quantitative and
qualitative analysis. The results show that the flavonoids
exist only in the heartwood and transition zone of black
locust while the simple phenolics are found in the sapwood of
black locust. Thus, biosynthesis of these flavonoids must
occur during the transition from sapwood to heartwood. There
are 3 possible precursors (compound a, b, d) of flavonoid
biosynthesis found in the sapwood of black locust. It is
necessary to accumlate more data and prove the hypothesis.

This is interesting because there are relatively few
cells in sapwood which are physiologically active. The
quantity of starch and soluble sugars in.a series of locations
from cambium to heartwood was also determined in an attempt

to understand the relationship between primary (starch,

soluble sugars) and secondary (flavonoids) metabolism. The
results suggest that primary metabolities could be an indirect

source of carbon for flavonoids biosynthesis.

ACKNOWLEDGEMENT

I would like to express my sincere gratitude to my
advisor Dr. James W. Hanover, for his guildance and support
during the course of my graduate studies. I am much obliged
to Dr. Rawel Hollingworth, for his inspiration, valued
advancement, and help in my study; I would also like to thank
the other member of my committee, Dr. Daniel E. Keathley, for
his participation.

Paul Bloese, Roy Prentice, and Paul Schultz are
responsible for sample collection, and assisting in sample
preparation and I appreciate their help. For their
friendship, assistance, and patience I thank my colleagues
Dr. Tesfai Mebrahtu, Peggy Payne, Sue Plesko, and Chishich
Chu.

I am indebted to my good friends; Charles and Mary
Campbell, Debora Gaudiello, Rose-Marie Muzika, and Dr. Andrew
and Dr. Sandy Smith, who have encouraged and helped me
immensely. Especially to Charles and Mary Campbell, and
Debora Gaudiello, they are not only helpful in proofreading
of this manuscript and my academic work but also in my daily
life.

Finally, I would like to thank my family for their

understanding; without it, this would not have been possible.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... viii
LIST OF FIGURES ................................................................................ ix
INTRODUCTION .................................................................................. 1
(A)BACKGROUND AND PROPOSED WORK. .......................... l
(B)CHARACTERISTICS OF BLACK LOCUST .......................... 3
(1)Species Characteristics ............................................................ 3
(2)Wood Characteristics ............................................................... 4
(3)Utilization of Black Locust. ................................................... 4
(C)DEFINITION OF TECHNICAL TERMS ............................... 5
(1)Sapwood and Heartwood ........................................... 5
(2)Sapwood/Heartwood Boundary ............................................. 7
(3)Flavonoids .................................................................................. 8
LITERATURE REVIEW ........................................................................ 9

(A) STUDIES RELATED TO THE EXTRACTABLE
MATERIALS 0F BLACK LOCUST ......................................... 9

(B) FLAVONOIDS BIOSYNTHESIS ............................................. 12

iv

(C) SAPWOOD-HEARTWOOD TRANSFORMATION ............. 16

MATERIALS AND METHODS .......................................................... 22
(A) SAMPLES DESCRIPTION AND PREPARTION ................. 22
(B)FLAVONOIDS OR FLAVONOID PRECURSORS ............... 24

(l)Qualitative Analysis ................................................................. 24
(a)Equipment. ............................................................................ 24
(b)Chemicals .............................................................................. 25
(c)Experimental Procedure ..................................................... 25

(2)Quantitaitve Analysis .............................................................. 27
(a)Equipment. ............................................................................ 27
(b)Chemicals .............................................................................. 27
(c)Prepartion of Standard Phenolics Curve ....................... 27
(d)Analysis of Black Locust Samples .................................. 28

(C)CARBOHYDRATES ..................................................................... 29

(1)Quantitative Analysis ............................................................... 29

(a)Soluble Sugars ..................................................................... 29
(i)Preparation of Standard Curve .................................... 29
(ii)Analysis of Black Locust Samples ............................. 30

(b)Starch ..................................................................................... 30

(i)Preparation of Standard Curve ................................... 30
(ii)Analysis of Black Locust Samples ............................. 31
RESULTS AND DISCUSSION ............................................................ 33
(A)FLAVONOIDS ............................................................................... 36
(1)Qualitative Analysis ................................................................. 36
(a)TLC Results .......................................................................... 36
(b)Instrument Analysis ............................................................ 39
(2)Quantitative Analysis ............................................................... 50
(a)Standard Reference Curve of Phenolics ......................... 50
(b)Variation of Samples .......................................................... 56
(B)CARBOHYDRATED ..................................................................... 62
(1)Quantitative Analysis ............................................................... 62
(a)Soluble Sugars ..................................................................... 62
(i)Standard Reference Curve of Soluble Sugars ........... 62
(ii)Variation of Samples ..................................................... 63
(b)Starch ................................................................................... 67
(i)Standard Reference Curve of Starch .......................... 67

vi

(ii)Variation of Samples ..................................................... 68

CONCLUSIONS ....................................................................................... 73
RECOMMENDATIONS FOR FURTHER STUDY .......................... 76
REFERENCES ......................................................................................... 77

vii

LIST OF TABLE

Table 1: The Concentration of Flavonoids in MeOH Extracts of

Black Locust (Roux and Paulus, 1962) ............................... 10
Table 2: The Harvest Date, Sample Number, and Moisture

Content of Black Locust. ........................................................ 23
Table 3: The Yield of Extractives in Black Locust. ......................... 26
Table 4: (a) ANOVA of Total Phenolics Test

(b) ANOVA of Total Phenolics Test

(c) ANOVA of Starch Test. .................................................... 34

viii

LIST OF FIGURE

Figure 1: The Cross Section of Black Locust. .................................. 6
Figure 2: The Labeling Pattern of Chalcone .................................. 13
Figure 3: Biosynthetic Pathway of Flavonoids from Primary

Metabolities .............................................................................. 15
Figure 4: The Structure of Flavonoid Products from Chalcone

Intermediate ............................................................................. 17
Figure 5: The TLC Results of EtOAc Extractives Before and After

Hydrolysis with Cellulase ...................................................... 37
Figure 6: The TLC Comparison of EtOAc Extractives from

Sapwood Before and After Hydrolysis with Cellulase

and 2N HCl:MeOH (1:1) ....................................................... 40
Figure 7: The GC-MS Comparison in Sapwood Before and After

Hydrolysis with Cellulase and 2N HCl:MeOH

(1:1) ............................................................................................ 42

Figure 8: Total Ion Chromatography (TIC) of GC-MS in the
Cambial Zone of Black Locust After Samples

Hydrolyzed with Cellulase ..................................................... 44

Figure 9: Total Ion Chromatography (TIC) of GC-MS in the
Sapwood of Black Locust After Samples Hydrolyzed
with Cellulase ........................................................................... 46
Figure 10: Total Ion Chromatography (TIC) of GC-MS in the
Transition Zone of Black Locust After Samples
Hydrolyzed with Cellulase ..................................................... 48
Figure 11: Total Ion Chromatography (TIC) of GC-MS in the
Sapwood of Black Locust Before Samples Hydrolyzed
with Cellulase ......................................................................... 51
Figure 12: Total Ion Chromatography (TIC) of GC-MS in the
Transition Zone of Black Locust Before Samples
Hydrolyzed with Cellulase ................................................... 53
Figure 13: The Mass Spectra of Unidentified compound (a), (b),
and (c) ...................................................................................... 55
Figure 14: The Seasonal Variation of Phenolics in Black

Locust ....................................................................................... 57

Figure 15: Sector of a Tree with Heartwood Showing Diagramm
Actically the Diffemt Zones Between Cambium and
Pith with Their Various Cytological and
Physiological Characteristics .............................................. 60

Figure 16: The Seasonal Variation of Soluble Sugars in Black
Locust. ................................................................................... 64

Figure 17: Average Seasonal Variation as Percent of Dry Weight
of Tissue in Reducing Sugars, Sucrose, Starch, and
Total Reserve Carbohydrates in the Living Bark

Tissue of robinia Trees During the Period from May

 

1949 to August 1950 ............................................................. 65

Figure 18: The Seasonal Variation of Starch in Black Locust.....69

xi

INTRODUCTION

(A)BACKGROUND AND PROPOSED WORK

Due to its valuable characteristics and many uses,
black locust has been extensively planted in the United States
and introduced into.Australia and.many countries in Europe and
Asia since the early seventeenth century. Black locust is one
of the most widely planted boardleaf species in the world
(Keresztesi,1981). The reasonsufor this widespread planting
of black locust are: a wide site tolerance, the ability to fix
nitrogen, abundant and frequent seed crops, fast growth,
excellent.sprouting'abilityy high timber yield, short.rotation
age, and relatively few damaging pests and diseases
(Keresztesi, 1988).

The results of in vitrg and 13 vivg durability studies
have suggested that the extractable chemicals from black
locust heartwood play an important role in the decay
resistance of wood. The methanol extracts from black locust
heartwood consist mainly of flavonoids (dihydrorobinetin and
robinetin) and a minor amount of tannins (Roux and Paulus,
1962: Shain, 1977; Smith et al., 1988). Preliminary
qualitative analysis indicates that the sapwood of black
locust does not contain the major flavonoids or flavonoid
glycosides found in the heartwood.

The durability* of Iblack locust. has received. much

attention and has been compared with that of poplars,

1

2

pedunculated oak and Norway spruce (Koloc, 1953). According
to reports, the decay resistance of black locust is found only
in the heartwood, but not in the sapwood. The durability of
black locust has been examined in this laboratory. These
studies indicated that the heartwood extracts are at least
partially responsible for the durability of the wood (Smith
et al.,1988). Additionally, Shain (1977), Freudenberg and
Hartmann (1954) and Erdtman (1953) studied the inhibition of
several types of fungus by flavonoids in vitro and suggested
that flavonoids, especially robinetin and dihydrorobinetin,
found in the heartwood extracts are able to inhibit fungal
growth.

These facts provide an interesting question about
how and where flavonoid biosynthesis takes place in black
locust. The objective of this study is to investigate this
process as follows:

1) To determine, by quantitative analysis, the levels of
flavonoids or flavonoid precursors in the cambium, sapwood,
transition zone and heartwood of black locust :

2) To investigate the quantity of starch in a series of
locations from cambium to pith to help in understanding the
relationship between primary (starch) and secondary
(flavonoid) metabolism;

3) To perform the above analyses seasonally on samples

which are collected at 1-2 month intervals over one year in

3
order to correlate flavonoid formation with the seasonal

changes.

(B)CHARACTERISTICS OF BLACK LOCUST

(1)Species Characteristics

The growth season of Black locust is from the beginning
of April to the end.of'Julyu The heartwood formation of black
locust is thought to start in July and end by the end of the
following March (Nobachi,T. et al., 1984b). Black locust
flowers in May and early June, about one month after flushing.

Black locust has less sensitivity to poor sites than
other species in the same family, but it requires strong light
for growth. Suitable drainage and soil will help the tree
grow and improve the quality of timber; however, black locust
is able to grow under conditions of low precipitation.

Black locust, like the other legumes, is able to improve
the soil through a symbiotic process with nitrogen-fixing
bacteria living in the nodules of the roots (Keresztesi,
1988).

Black locust is one of fastest growing species,
especially in youth; its growth averages 2 to 4 ft in height
per year on the better sites (Keresztesi, 1988). In closed
forest stands, it has a straight bole free of branches, but
in free growing areas it often develops crooked and twisted

stems with thick branches. In spite of the fact that black

4
locust seed hard germinates, the species is spread over a‘wide
range of the world because of the excellent rooting ability

of the seedling (Keresztesi, 1988).

(2)Wood Characteristics

Black locust is a ring porous hardwood with "shining"
annual rings and rays. 'The sapwood and the heartwood of black
locust can be distinguished by the difference in wood color
and by the fluorescence produced by illuminating the wood
under UV light (254nm). The normal color of heartwood is
brown to dark-brown and sapwood is light-yellow. The
heartwood fluoresces yellow and the sapwood blue. The wood
of black locust is hard and stiff (Koloc, 1953). Its density
is 0.73 g/cc and is classed as a medium-heavy wood by Koloc
(1953).

An outstanding property of black locust is its
durability. The durability of black locust has been examined
by several authors (Shain, 1977; Hart et al., 1977; Smith gt
gig; 1988). Their reports have shown that the extractable
chemicals (primarily flavonoids) of black locust are at least

partially responsible for the durability of the wood.

(3)Utilization of Black Locust
Traditional utilization of black locust wood has
emphasized timber products such as firewood and fencepost due
to the strength and durability of its heartwood (Funk, 1961:

Scheffer, 1966) . The current direction is to develop multiple

5

utilization of black locust due to its wide distribution and
potential. Potentially valuable chemicals could be extracted
from wood and used as anti-fungal treatments, dyes, instead
for dihydrrobinetin having partaial resistance for fungi
growth in aspen wood, and robinetin having golden yellow
color. Although black locust has a high level of extractable
chemicals in the heartwood, the pulp of this species also has
been tested with favorable results and considered for use in
the pulp industry (Menasha Corp., 0tsego, MI). The foliage
of black locust is thought to have potential as a fodder due
to a high level of protein, although it contains a high
concentration of glycoside flavonoids in leaves (Oehme, 1978) .
Baertsche, Yokoyama, and Hanover (1986) showed that the yield
of protein from the foliage of black locust was 30% of dry
weight for primary growth and 20% for regrowth, which was
significantly higher than other species compared.

(C)DEFINITION OF TECHNICAL TERMS

(1)Sapwood and Heartwood
Figure 1 shows a cross section of a tree. The phloem
of a tree is the tissue principally concerned with the
distribution.of organic substances. The cambial zone:is a'very
thin meristematic layer between the xylem and the inner bark:
it is a lateral meristem responsible for the formation of
xylem and phloem. The xylem of a tree is organized into

concentric annual growth rings which function as the principal

 

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7
strengthening and water-conducting tissue of the stem, roots
and leaves. Newly formed xylem cells not only serve in the
support and water-conduction function of the tree but also
provide a place for storage of the food reserve. The storage
activity in the xylem is carried out in the living parenchyma
cells which run through the xylem in both horizontal and
vertical directions. Sapwood is that portion of the xylem
which has parenchyma cells which are physiologically active.
After a period of years, the living cells in the xylem die.
This is accompanied by many secondary changes such as wood
coloration, metabolism rate, and structure feature, and then
the formation of the physiologically dead part of the xylem

called heartwood (Panshin and de Zeeuw, 1980).

(2)Sapwood/heartwood Boundary

The sapwood/heartwood boundary (intermediate zone)
occurs as a zone between the inner sapwood and the outer
heartwood (Frey-Wyssling, 1959; Nobuchi gt al., 1982). In a
number of species, an intermediate zone, a narrow zone
surrounding the outer heartwood, is called a transition zone.
It has been reported that the transition zone of Pinus Radiata
contains living parenchyma cells (Sandermann et al., 1967).
Hillis (1977) found that a transition zone usually contained
a smaller amount of extractives and lower moisture content
than the surrounding tissues. It was suggested that the

formation of heartwood extractives is initiated in this zone.

8

The apparent changes in the transition zone are not
always obvious. For’ example, the color change of the
transition zone in Acer saccharum.was very gradual preventing
the destinction of heartwood and sapwood. However, the
transition zone of sugi (CrytomeriaJagonica) is easily
observed because it appears paler than either sapwood or
heartwood (Nobuchi et al,, 1982: Sameshima gt a1,, 1967).
Thus, this boundary is frequently combined with either

heartwood or sapwood when the zone is not distinctly colored.

(3)Flavonoids

Flavonoids are one type of secondary metabolites which
are extractable from a variety of tissues. A C,-C,-C, unit
provides the basic building block of the flavonoids and this
basic unit is modified by hydroxylation and methylation to
form the different flavonoid compounds. Large numbers of
flavonoids have been found in the heartwood of many species.
Most flavonoids are hydroxylated at the C, position ; however,
flavonoids in the heartwood of the Anacardiaceae family and
some of the Leguminoseae lack a C, hydroxyl. The C, deoxy
pattern of flavonoids is unique by fluorescence under
ultraviolet (UV) light. This fluorescence allows for the

detection of heartwood.

LITERATURE REVIEW

(A)FREVIOUS STUDIES RELATED TO THE EXTRACTABLE MATERIAL OF
BLACK LOCUST

Roux and.Paulus (1961) have isolated 10 flavonoids from
the methanol extract of the heartwood and sapwood of black
locust (nginig pseugoagggia L.); they identified the
flavonoids using two-dimensional paper chromatography. Their
report indicated that the wax-free methanol extracts from the
sapwood of black locust contained 1% of flavonoids 60% of
which was (+)dihydrorobinetin, 22% robinetin and 10%
(4-)? , 3 ' , 4 ' , 5 ' -tetrahydroxyflavan -3 , 4-diol . The heartwood
contained 6% flavonoids of which 52% was (+)dihydrobinetin,
20% robinetin, and 13% (+)7,3',4',5'-tetrahydroxyflavan-3,4-
dial. ‘The flavonoids of the heartwood and sapwood of Robinia
pseudoacagia L. are shown in Table 1. Smith et a1, (1988)
obtained similar results from the heartwood, they found that
the methanol extract were 5.8% of the dry weight of the sample
and that it contained about 90% flavonoids. The flavonoids
and flavonoid glycosides of the leaves of black locust have
also been investigated (Farkas et a1.I 1976; Kubota and Base,
1966; Ebel gt al., 1970; and Freudenberg and Hartmann, 1954).
Flavonoid aglycones identified in leaves included kaempferol,
apigenin, acacetin, and quercetin. These authors also

determined that the flavonoid glycosides were not present in

10

Table 1: The concentration of flavonoids in MeOH extracts of
Black Locust (Roux and Paulus, 1962)

 

 

 

 

Flavonoid Sapwood1 Heartwood1
(+)dihydrorobinetin 2.3 20.0
Robinetin 0.8 8.0
(+)7,3',4',5'-tetrahydroxy- 0.4 6.2
flavan-3,4-diol
Leuco-robinetinidin 0.2' 1.0'
Robtin 0.05 1.5
Robtein absent 0.9
(+)7,3',4'-trihydroxy- absent ___
Flavan-3,4-diol
Fustin absent 0.5
Butin absent 0.5
Butein absent 0.4
Fisetin traces ___
2',4',4-trihydroxychalcone absent 0.01

 

1. The concentration is calculated based on total dry weight
of extracted material.
*: Values are with reference to (+)7,3',4',5'-

tetrahydroxyflavan-3,4-diol.

11
the heartwood of black locust (Freudenberg and Hartmann,
1953).

Roll and Poschenrieder (1975) studied the radial
distribution of lipid extracts in the xylem of black locust
wood from cambium to heartwood. They divided tissues into 5
areas by annual rings and titrated the quantity of lipids in
the extracts by measuring acid value, unsaponifiable material,
ester value, and iodine value and characterized the lipids
with paper chromatography and thin layer chromatography. It
was suggested that heartwood formation might be accompanied
by fatty acid turnover because the ester value, iodine value,
and unsaponifiable material were highest in the extracts from
the innermost heartwood. The lipid extracts from the
innermost sapwood of black locust had the maximum acid value,
therefore, the lipid extracts from this region consist of more
free fatty acids than extracts from other regions. According
to TLC results, the higher lipid concentration in the older
part of the trunk of black locust came partly from an
accumulation of more polar lipid classes but did not include
phospholipids. In general, energy rich triglycerides should
be deposited in a tissue which exhibited only dead cells.
However, H011 and Poschenrieder's study (1975) indicated that
the increase in lipid content of the interior part of black
locust is not due to an accumulation of triglycerides.

Tchorbajiev, Ivanov and Stefanov (1969) characterized

the composition of the hydrocarbons of Bulgarian black locust

12
flowers. Their work showed that the concrete of black locust
consisted of normal paraffins from C,,H,, to C3311... and the amount
of odd hydrocarbons (89.16% of the hydrocarbons) are
significantly higher than that of the even hydrocarbons
(10.97% of the hydrocarbons). The major hydrocarbons present

in the concrete are C2,, C2,, and C,, hydrocarbons.

(B) GENERAL CONCEPTS IN FLAVONOIDS BIOSYNTHESIS

It is recognized that the flavonoid biosynthetic pathway
incorporates precursors from both the "shikimate" and
"acetate-malonate" pathway (Hahlbrock and Grisebach, 1975;
Wong, 1976). Tracer experiments indicate that the B-ring and
the carbons at positions 2, 3, and 4 on the C-ring of
flavonoids come from a phenylpropanoid (C,-C,) unit while the
A-ring is made from three acetate (C2) units. The labeling
pattern obtained is shown as Figure 2. (Grisebach, 1952).
Hillis et al., (1963) pointed out that cultured cambial
tissues can utilize glucose to synthesize shikimic acid and
lignin. The shikimate pathway also leads to synthesis of the
phenylpropanoid compounds via L-phenylalanine. Cinnamic acid,
the starting compound for the biosynthesis of flavonoids, is
derived from phenylalanine via the phenylalanine cinnamic acid
pathway (Gross, 1985). Hence, shikimic acid is the common
precursor in the synthesis of lignin and flavonoids. An

outline of the relationship between different primary

13

Melony! CoA Acetyl CoA

 

H00" . Shikimic Acid
2 .
fo‘L-scu gale“ 1
;o .
,3” ”Mr/alanine
ace. 1

”ewe-@w

a - Coumerjc Acid

 

 

 

l,
"n
HYDothe tlcal
intermediate

 

ll

"Carboxyl group of acetate

 

iv .e Pheny/propane uni t
Fla vonoide

Figure 2: The labeling pattern of chalcone (Walker, 1975)

l4
metabolites with flavonoid biosynthetic pathway is drawn in
Figure 3. (Hillis, 1986).

The first flavonoid produced after the confluence of the
acetate and shikimic acid pathways is thought to be the
chalcone- 2',4,4',6'-tetrahydroxy-1,3-diphenyl-2-propen-1-one.
All other flavonoid are derived from this compound (Light and
Hahlbrock, 1980; Heller and Hahlbrock, 1980). Tracer
experiments and enzymatic studies have confirmed this (Wong,
1976). The key enzyme for synthesis of this chalcone
intermediate of flavonoid is chalcone synthase. Much evidence
from in vitro and in vivo experiments have suggested that 4-
hydroxy-cinnamoyl CoA ester is the most efficient substrate
of chalcone synthase (Grisebach, 1985; Hrazdina et al., 1976;
Stich and Forkmann, 1987). Chalcone synthase can also accept
another phenylpropanoid thioester, caffeic CoA, as a substrate
having an optimum activity at. pH =6.8 while 4-hydroxy-
cinnamoyl CoA having an optimum activity at pH =8.0 (Krenzaler
et al., 1979; Welle and Grisebach, 1987). However, Stich and
Forkmann (1987) found that the predominant product came from
the 4-hydroxy-cinnamoyl CoA when the two precursors of
chalcone synthase were incubated with the enzyme at pH =6.8.
Evidence indicates that chalcone synthase has a high substrate
specificity for 4-hydroxy-cinnamoyl-CoA ester and that the
further hydroxylation of the flavonoid B-ring takes place at
the Cu level (Grisebach, 1985).

It is not easy to isolate and incubate the chalcone

15

 

 

 

 

 

 

 

 

 

 

 

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“"lb' ’nflr‘ ~l'ueammwmn
'eteeey
MM “H. n
y up procaine Aene
15'.\I IanUUWD
roe Crete Aeem Gee 4 Fatty Aene
l
emery.
fereeeetee om we
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Fla vonoids

Figure 3: Biosynthetic pathway of flavonoids from primary
metabolitiee. (Hillis, 1987).

16

intermediate in flavonoid biosynthesis pathway because the
isomers (flavanones) are synthesized more rapidly by a very
active enzyme (chalcone isomerase) (Heller, 1986). After
flavanones are formed, the flavonoid biosynthesis pathway goes
in several directions and toward final products. These final
products include flavones, dihydroflavonols, flavanols,
anthocyanidins and condensed tannins. The relationship
between the chalcone intermediate and final products is shown
in Figure 4. These products are classified by different
oxidation pattern and stereochemistry at the C-ring. Numerous
enzymes are involved in the sequential procedures.

The flavonoids from.black locust heartwood are 5-deoxy,
which have no hydroxyl group at C-5 and show yellow
fluorescence under UV light (254 nm). Although the
biosynthesis of 5-deoxy flavonoids has not been completely
described, Grisebach's studies (1988) found that the chalone
synthase and an "extra protein" are required for the formation
of 5-deoxy chalone. H011 and Lendzian (1973) have shown that
in black locust the maximum oxygen consumption is in the
oldest sapwood ring (perhaps at the sapwood/heartwood

boundary).

(C)SAPWOOD-HEARTWOOD TRANSFORMATION

It has been suggested by Hart (1968) that the
transformation of sapwood to heartwood is associated with a)

the death of the living cells in the sapwood, b) an abrupt

17

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18
decrease in the amount of starch and non-cell wall
carbohydrates, and c) an abrupt increase in the amount of
polyphenolics (tannin, lignin, flavonoid). Data from Hart's
study indicated that starch granules of black locust were
numerous in the ray cells of the sapwood but none were found
in the heartwood or discolored sapwood.

The seasonal variation in the reversable carbohydrates
of living bark of black locust and the relation to its frost
hardiness have been studied by Siminovitch et a1. (1953).
They suggeated that the carbohydrate fluctuation was related
to a precise temperature control mechanism. The storage
carbohydrates, sucroseiand starch, are used in the late spring
by the flush of new growth. The starch and sucrose level
decreased rapidly in the growth season. The sucrose level,
however, increased again after a short time and reached a
maximum in September and maintained that level during the cold
weather. In) contrast, the starch level accumulated until
September, then decreased again during the cold weather. In
the winter season, the reducing sugars reached.a:maximum level
of about 3% of dry weight. The levels of reducing sugars were
constant (0.8% to 1.1% of dry weight) in the other seasons of
a year.

Nobuchi et al, (1984b) have observed tissues from the
cambium to the heartwood of black locust from a cytological
point of view to investigate the season of heartwood

formation. The activity of ray parenchyma cells near sapwood/

19

heartwood boundary was determined by observing the changes in
the cell contents. Their report indicated that the nuclei of
parenchyma cells, starch granules, and lipid droplets of the
boundary appeared to change in the summer season. After
starch granules disappeared from the innermost annual ring of
the sapwood in late summer, the formation of colored
compounds associated with the heartwood formed. During
heartwood formation, the size of lipid droplets was shown to
be rapidly increasing and starch granules and nuclei of the
ray parenchyma cells were present in the heartwood. These
results observed in the heartwood conflict with the definition
of International Association of Wood Anatomy (IAWA) for
heartwood which states that there are no living cells and
starch granules. It was suggested that the boundary zone has
significant changes during heartwood formation and that the
living cells in the heartwood gradually produce the heartwood
substances and become dead cells before the next onset of
cambial activity. Frey-Wyssling and Bosshard (1959) got
similar results with several species of gymnosperms and
angiosperms. In this experiment, the activity of mitochondria
was determined an indicator of normal respiration.

Mitochondria in the living cells can accumulate the stain of
a dye (Janus green 8) and transform it into colorless leucodye
because reduction of Janus green 8 provides a proton which
enters the TCA cycle. The ability of a cell to reduce this

dye indicates active mitochondria. Frey-Wyssling and Bosshard

20
determined that mitochondria in wood lose their activity at
a very early age (a few rings inward from the cambium). The
heartwood phenolics of Sugi (Cmtomeria japonica D. Don) have
been examined by Nobuchi (1984a) . They found three main
heartwood phenolics and that each compound occurred in a
different radial position and in different types of ray
parenchyma cells. One exists only in the transition zone to

heartwood. The others are located in the outer heartwood.

Holl (1972 and 1973) has determined the radial changes
of enzymatic activity in black locust wood. There were two
active amylases in the outer sapwood, 4-glucano-hydrolase and
4-glucano-maltohydrolase while only 4-glucano-hydrolase was
found in the inner sapwood. The total amylase activity in
black locust was shown to increase toward the center of the
wood and the sapwood/heartwood boundary had the most intensive
activity of starch-decomposing enzymes. In contrast, the
enzymes involved in starch synthesis decreased in activity
from peripheral to central tissues. The amyloplast at the
boundary zone had an inhibitor compound which suppressed
starch synthesis in the peripheral annual rings of black
locust trunk.

Radioactivity studies aimed at identifying the
fluctuation of phenolic compounds found in the boundary zone
of Prugus yedoensis were undertaken by Hasegawa et al. (1966).

They assumed that the sucrose or other sugars were

21
translocated from leaves, and then the phenolic compounds were
synthesized in situ. l‘C-labeled sucrose was administered to
the cambium of Eggnus yedoensis for 3 hours in November and
the labeled tree cut down after 12 days. They fbund that
metabolism varied in the different tissues. The labeled
compounds were translocated into the sapwood/heartwood
boundary via ray cells after the labeled sucrose was
converted. The labeled shikimic acid in the outer sapwood
had a higher density than the inner sapwood and transition
zone. The labeled quinic acid and citric acid, however, were
present in higher radioactivity in the inner sapwood and
transition zone. The radioactivity of essential amino acids
was also determined and appeared that tyrosine had a higher
density in the inner sapwood and transition zone. The
radioactivity' of‘ basic components decreased at the
sapwood/heartwood boundary. Simple sugars converted from the
labeled sucrose were also examined. The radioactivity of
glucose and fructose decreased in the boundary when the

radiation of those sugars increased in the outer sapwood.

MATERIALS AND METHODS

(A)SAMPLES DESCRIPTION AND PREPARATION

Wood disks (2-3 inch thick) were collected from black
locust trees (Robinia pseudoacacig L.) at 1-2 month intervals
for one year. One tree per month was sampled unless noted
otherwise. The trees (average age 15-20 years old) were from
one of two Michigan State University locations: the Water
Quality research area and Kellogg Forest. Two disks were
taken from each tree at breast height. One was used to
measure the moisture content.and the other for the qualitative
and quantitative analyses. Detailed information about
individual trees is listed in Table 2.

After the disks were cut, the samples were immediately
placed into a cooler with ice to inhibit the activity of
amylase and brought back to the laboratory. One of two disks
was immediately dried in an oven at 100°C for 2 hours and then
dried to constant weight at 70°C to destroy the activity of
amylase. Each disk.was separated into 5 parts using a chisel
according to color differences in the fluorescence under UV
light at 254 nm and in the wood coloration as described
previously. The first section, cambium, was removed from the
inside of the barkn Next, the sapwood portion of each section
was removed. The intermediate zone included the edge of the

heartwood/sapwood boundary as determined by fluorescence and

22

23

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24
visual inspection and 0.1-0.3 cm of sapwood. After the
intermediate zone was removed, the heartwood was collected.
For each type of sample, the small pieces obtained were ground
in a Wiley mill to pass through a 40 mesh screen. The ground

samples were stored in glass bottles at room temperature.

(B) FLAVONOIDS OR FLAVONOID PRECURSORS

(1)Qualitative Analysis
(a)Equipment

The gas chromatograph-mass spectrophotometer (GC-
MS) utilized in this study was a HEWLET PACKARD (HP) 59708
mass selective detector with a 5890A gas chromatograph. The
column used.was a.J&W'SCIENTIFIC fused silica capillary column
(film thickness: 0.25 micrometer) with nonpolar DB-l
(methylsilicone) phase. The temperature program used was an
initial temperature of 200°C and held for 5 minutes, an
increased rate of temperature of 5°C/min, and the final
temperature of ZBOtrfor 30 minutes.

The gas chromatograph-Fourier transform infrared
spectrophotometer (GC-FTIR) utilized in this study was a BIO-
RAD Digilab Division FTS-40 Fourier transform infrared
spectroscopy system with a HEWLET PACKARD (HP) 5890A gas
chromatograph. The column used was a J&W SCIENTIFIC fused
silica capillary column (film thickness: 0.25 micrometer) with

nonpolar DB-l (methylsilicone) phase.

25
(b)Chemicals
The solvents (analyzed grade) used for extraction
were purchased from J.T. Baker and Aldrich. The silylation
reagent, bis(trimethylsilyl)trifluoroacetamide, BSTFA was

purchased from SUPELCO.

(c)Experimental Procedure

The ground wood meal was extracted with 100%
methanol followed by 50% aqueous acetone at room temperature
for one day. The extracts were filtered, concentrated to
dryness by rotary-evaporation, lyophilized and then weighed.
The yield of extractives is listed in Table 3. A sample of
the crude extract (generally 100 milligrams) was redissolved
in 10 ml H20 and extracted with 10 ml hexane (2x) to remove
wax from 'the extracts followed. by 10 ml ethyl acetate
(EtOAc)(2x) to isolate the neutral flavonoids. The residual
aqueous solution was hydrolyzed with cellulase for 24 hours
to break sugar-flavonoid bonds. The acid treatment of the
residual aqueous solution were also taken to determine whether
both hydydrolysis methods occurred properly. The flavonoid
aglycones were extracted from the aqueous hydrolysis mixture
with EtOAc (2x). Both ethyl acetate extracts were taken to
dryness by rotary-evaporation and redissolved into 400
microliters of acetonitrile. An aliquot (100 microliter) of
each sample was analyzed by thin-layer chromatography (silica

gel with fluorescent indicator) using chloroform: ethyl

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27
acetate: formic acid (5:5:1) as the mobile phase and
shortwave UV light (254nm) for detection of spots. A 50
microliter aliquot of the acetonitrile sample was silylated
with bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 280°C
for 1 houru The silylated compounds were then analyzed by gas
chromatography-mass spectrometry (GC-MS) and gas
chromatography-Fourier transform infrared spectroscopy (GC-

FTIR).

(2)Quantitative Analysis
(a)Equipment
All absorption spectra were determined with a PERKIN-
ELMER lambda 4B uv/vis spectrophotometer with tungsten-bromide
and deuterium lamps using 10 mm light-path absorption cells.
(b)Chemicals
For colormetric tests, the standard compounds (D-(+) -
glucose, starch, and gallic acid) were purchased from Sigma.
The Folin-Ciocalteu (FC) reagent of phenolic test was also

purchased from Sigma.

(c)Preparation of Standard Phenolics Curve
A series of standard gallic acid solution ( 1x10”,
2x10”, 5x10”, 1x10°, 2x10‘, 5x10°, 6x10‘, 8x10‘, 1x10°, and
5x10° g/ml) were prepared by dilution a 0.1g/100m1 solution
freshly prepared, and analyzed using a modification of the

phosphomolybdic-phosphotungstic acid method (Singleton and

28
Joseph A.Rossi, 1965) . A 1 ml of Folin-Ciocalteu (FC) reagent
was added and mixed with a 10 ml standard gallic acid solution
followed by adding 3 ml of 20% (w/v) sodium carbonate aqueous
solution; the procedure was completely finished within 8 mins.
Then, color was allowed to develop for 2 hours to obtain the
maximum absorption. The absorbance was determined at 765 nm.
A blank was also prepared. The standard curve of phenolics
was generated by plotted the absorption vs. concentration of
standard phenolic solutions. The linear range of standard
curve was determined by the correlation coefficient of the

linear regression.

(d)Ana1ysis of Black Locust Samples

For each ground sample ten replications (0.59) were
placed into a 50 ml graduated test tube and extracted with
hexane (5 mL) for 30 mins. in a sonicator followed by
centrifugation at 500 rpm for 3 mins. The hexane extracts
containing wax, were discarded. The residue was extracted
with 80% aqueous ethanol (15 mL) for 12 hours then centrifuged
at 500 rpm for 3 mins. It was determined that the amount of
total phenolics and soluble sugars extracted did not
significantly increase after 12 hours extraction. The
supernatant was removed from the tube and used in the analyses
of total phenolics and soluble sugars. The residue was dried
overnight and then used for starch analysis since starch was

not extractable in 80% aqueous ethanol. In order to fall into

29
the linear range of the standard curve, the ethanol extract
was diluted to 50 ml volume (solution I) with double distilled
water. A 10 ml aliquot from solution I was diluted to 50 ml
(solution II). In the transition zone and heartwood portion,
solution II was used for the phenolic test while solution I
was used in the cambium zone and sapwood samples. The total
phenolics in a portion of black locust was expressed as
equivalents of gallic acid by comparison with the standard
curve. The procedure for the quantitative determination of
total phenolics from black locust was the same as that
described for preparing the standard curve (Singleton and

Joseph A.Rossi, 1965).

(C)CARBOHYDRATES

(1)Quantitative Analysis
(a)Soluble sugars

(i)Preparation of Standard Curves: The sugar
standard used was D-(+)-glucose. The glucose standard
solutions were prepared by dilution of a 10mg/ml solution.
The absorption of a series (1x10‘, 2x10°, 4x10°, 5x10°, 1x10'
‘, 2x104, 4x10“, 5x10‘, and 1x10" g/ml) of D—(+)-glucose
solutions were analyzed to construct a standard curve using
the phenol-sulfuric acid colorimetric method (Hodge and
Hofreiter, 1962). 1 ml aliquots of the solutions were placed

into a 25 ml glass test tube with 16 to 20 mm diameter, 1ml

30
of 5% (w/v) aqueous phenol was added. Then, 5 ml of
concentrated sulfuric acid was added rapidly. The tubes were
allowed to stand 10 minutes, then they were shaken and placed
for 20 mins. in an ice bucket before the absorbance was taken.
Absorbance were measured at 490 nm. Water was used as a
reference. The standard curve of absorbance vs concentration
for glucose was generated by the linear regression. The
linear range of the curve was determinated with the

correlation coefficient.

(ii)Analysis of Black Locust Samples: Each ground

sample (0.5g) was extracted with hexane and followed with 80%
ethanol in which.the detail of this procedure was as described
for the total phenolics analysis in.black locust samples” The
ethanol extract was diluted to 50 ml (solution I). A 10 ml
aliquot of solution I was diluted to 50 ml (solution II). A
1 ml aliquot from solution II was the sample used to determine
the concentration of soluble sugars. The procedure for the
quantitative determination of soluble sugars from black locust
was the same as that described for preparing the standard
curve (Hodge and Hofreiter, 1962). Each portion in this

experiment was replicated ten times.

(b)Starch

(i)Preparation of Standard Curve: The absorption of

a series of starch standard solutions (1.6x10'3, 2.2x10",

5.5x10°, 9.1x10°, 1.06x10j, 2.25x1oj, 5.5x10“, 8.09x10Q,

31
1.065x10’1 g/50 ml) were prepared and analyzed by a modified
perchloric acid colorimetric method (Humphreys F.R. and Kelly
J., 1961). A weighed starch sample was placed into a 50ml
plastic graduated test tube, dispersed with 5 ml of 7.2M
perchloric acid and allowed to react for 20 mins. with
occasional stirring. The volume was then brought to 50 ml
(solution I) with double distilled water. The sample was
centrifuged at 500 rpm for 3 mins. A 10 ml aliquot from
solution I was placed into a 50 ml graduated test tube with
1 drop of phenolphthalein and made alkaline with 2N sodium
hydroxide. 2N acetic acid was added until the color
disappeared, 2.5 ml more acetic acid was added, followed by
20 drops of 10% (w/v) potassium iodide and 5 ml 0.01N
potassium iodate. The color was allowed to develop for 20
mins followed by dilution of the sample to 50 ml. The
absorbance was measured at 650nm. Water was used for
reference. The standard curve of starch was generated by
plotted the absorption vs. concentration of standard starch
solutions. The linear range of the curve was determinated by

the correlation coefficient of the linear regression.

(ii)Analysis of Black Lcmust Sample_s: The starch
content in 0.5 gram of ground wood meal was determined by the
procedure used for the standard curve of starch preparation.
After hydrolysis with procholoric acid, the samples were

diluted to 50 ml (solution I). Black locust contained higher

32
levels of starch than the standard starch samples so that
black locust samples were diluted and fell into the linear
range of the standard curve. A 10 ml aliquot from solution
I was diluted to 50 ml (solution II). A 10 ml aliquot from
solution II was used to determine the starch content of wood.
Ten replication of each portion in this experiment were

examined.

RESULTS AND DISCUSSION

All woody stems original from the cambial zone and
through aging processes for several years. The cambial zone,
parenchyma cells and ray cells are living tissues in a wood
stem. The cambial activity, cell division, enlargement, and
differentiation, is modified by environmental conditions such
as temperature, soil, light, or moisture and mediated by
internal processes such as the rate of photosynthesis and
hormone production. However, the factors affecting wood
structure do not influence the distribution of extractable
chemicals in the cell lumen. Individual cell types within a
species may differ in the distribution of extractable
chemicals. Multiple samples were collected in March from one
location and.the three quantitative procedures*were tested for
tree to tree ‘variabilityu The detailed information of
variation for each analysis is listed in Table 4. It appears
that the proportional variation from multiple samples is much
greater than the variation between trees even though both
sources of both variations are significant. In other words,
the tree variation in this study plays a minor part
indetermining the relative level of metabolic compounds.

If it is true that the flavonoids (dihydrorobinetin and
robinetin) found in the black locust.heartwood are responsible

for the durability of heartwood, it would be interesting to

33

34

Table 4: (a) ANOVA of total phenol ics test for multiple
samples in March . (b) ANOVA of soluble sugars test for
multiple samples in March . (0) ANOVA of starch test for

multiple samples in March.

(a)

35

 

 

 

 

 

 

 

 

 

 

 

SOURCE OF (SUM)2 (MEAN)2 F-RATIO
TREE(A) 3 1.151744 .3839146 75.49
LOCATION(B) 3 57.66125 19.22042 3779.52

A x B 9 2.657956 .2953285 58.07
ERROR 144 .7322992 5.085E-03

TOTAL 159 62.20324

(b)

SOURCE DF (SUM)2 (MEAN)2 F-RATIO
TREE(A) 3 1.268E-03 4.228E-04 176.40
LOCATION(B) 3 1.264E-02 4.214E-03 1757.88
A x B 9 8.003E-04 8.893E-05 37.10
ERROR 144 3.452E-04 2.397E-06
TOTAL 159 .0150568
(C)

SOURCE DF (SUM)2 (MEAN)2 F-RATIO
TREE(A) 3 3.603E-04 1.201E-04 153.40
LOCATION(B) 3 2.286E-03 7.620E-04 973.11
A x B 9 2.467E-04 2.74lE-05 35.01
ERROR 144 1.127E-04 7.83lE-07
TOTAL 159 3.006E-03

 

36
determine the location of flavonoid biosynthesis. The
heartwood.of black locust.contains.a high level of flavonoids,
about 6% of dry weight. The sapwood, however, has a very low
level of flavonoids less than 1% of dry weight (Roux and
Paulus, 1962) . It has been suggested that flavonoids in
heartwood may be translocated from the cambial zone and/or the
sapwood into the heartwood with the amount increasing and
transformed in specific tissues such as the transition zone.
Therefore, in this study, the flavonoids in the heartwood of
black locust were analyzed qualitatively to confirm the
location of the flavonoids of black locust using the procedure
described previously. The total phenolics as gallic acid
equivalents, starch, and soluble sugars, from cambium to
heartwood, were analyzed quantitatively over a period of one

year to look for a flux of flavonoids into the heartwood.

(A)FLAVONOIDS

(1)Qualitative Analysis
(a)TLC Results
An ethyl acetate extract of the unhydrolyzed
heartwood was used as a reference. The EtOAc extracts of
samples, obtained as described previously, were separated by
TLC. Samples from cambial zone, sapwood, and transition zone
were separated both before and after hydrolysis with

cellulase. The results are presented in Figure 5. The TLC

37

Figure 5: (Top) The TLC results of EtOAc extractives before
hydrolysis with cellulase. (Bottom) The TLC result of EtoAc
extractives after hydrolysis with cellulase.

(C= Cambial zone, S= Sapwood, T= Transition zone, H=

Heartwood, and the numbers are presented the month)

.m
..
e

 

39
separations showed that the EtOAc extracts of the heartwood
and transition zone before hydrolysis contained neutral
flavonoids (dihydrorobinetin and robinetin) while the sapwood
and cambium contained only simple phenolic compounds.
However, the TLC results from EtOAc extracts of the transition
zone after hydrolysis suggested that the flavonoids in the
transition zone did not exist.as.glycosides or that glycosides
were present at a low level. It was necessary to use the more
precise analysis technique to monitor the particular
flavonoids (dihydrorobinetin and robinetin). The TLC
separation pattern from the EtoAc extracts of the sapwood and
cambial zone before hydrolysis were similar to that obtained
after hydrolysis. The results suggested that the compounds
form sapwood extracts had some compounds in common with the
cambial zone. The TLC comparison of sapwood before hydrolysis
and after hydrolysis with cellulase and 2N HCl:MeOH (1:1)
appears in Figure 6. The chromatography demonstrated that the
two hydrolysis methods lead to the formation of two different

set compounds.

(b)Instrumental Analyses
The EtOAc extractives before and after hydrolysis
with cellulase were analyzed with GC-MS in order to whether
the minor amounts of robinetin, dihydrorobinetin and/or other
flavonoids were present but not detectable by TLC. Samples

from December and February sapwood were also selected and

Rf

13

0.7

OJ

Figure 6: The TLC comparison of sapwood before and after

40

 

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41

hydyolyzed 2N HCl:MeOH (1:1) . The GC-MS comparison of sapwood
before hydrolysis and after hydrolysis with cellulase and 2N
HCl:MeOH (1:1) is presented in Figure 7. Both results from
the total ion chromatography and ion.current.profile suggested
that dihydrorobinetin and robinetin were not present in the
sapwood or the cambial zone of black locust. The
chromatograms, samples from each portion of black locust wood
hydrolyzed with cellulase, are presented in Figure 8, 9, and
10, respectively. Fatty acids, hexadecanoic acids (Cu), and
octadecanoic acid (Cw),‘were the major fatty acid found in.the
cambial zone, sapwood, and 'transition. zone. Only
dihydrorobinetin was found in the transition zone after
samples were hydrolyzed with cellulase. From biosynthesis
pointview, this result suggested that dihydrorobinetin was
first synthesized in the pathway. Some monosaccharides (D-
glucose and D-mannose) were found in the cambial zone and the
sapwood- The flavonoids were not found in the sapwood.through
a whole year. The compounds (a) and (b) were presented in
every tissue sample from the cambial zone to the heartwood.
The compound (a) had a retention time 25.390 min., the base
peak 239, and the molecular ion 372. The compound (b) had a
retention time 27.063 min., the base peak 387, and the
molecular ion 402. Beside compound (a), and (b), simple
phenolic compound, benzoic acid, was found in the extracts of
sapwood and cambial zone after acid hydrolysis.

The chromatograms of unhydrolysis samples from the

42

Figure 7: The Total ion chromatograph (TIC) comparison of
GC-MS in sapwood before and after hydrolysis with
cellulase and 2N HCl:MeOH (1:1)

(1) December sample before hydrolysis with
cellulase.

(2) December sample after hydrolysis with acid.

(3) December sample after hydrolysis with cellulase.

(1') February sample before hydrolysis with
cellulase.

(2') February sample after hydrolysis with acid.

(3') February sample after hydrolysis with
cellulase.

(a, and b are presented the unidentified compounds

which are the possible precursors of flavonoids)

 

 

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50
sapwood and transition zone are presented in Figure 11 and 12.
The compositions of samples in the transition included
flavonoids (dihydrorobinetin, robinetin, and robinetinidol),
saturated. and. unsaturated fatty’ acids (Cm, (La and. some
unidentified compounds (a) and (b). In comparison, the
composition of sapwood were only fatty acids and the
unidentified compounds (a), (b) and (d). The compound (d),
having a retention time 31.188 min., base paek 398, and
molecular ion 590 (?) was only presented in August sapwood and
the transition zone through entire year. The mass spectra of
compound (a), (b) and (d) apppears in Figure 13. The results
suggested that the flavonoids found in the heartwood began to
synthesize in the transition zone without the seasonal
changes. .According to the current data, compound (a), (b) and
(d) are possible precursors of flavonoid biosynthesis.
However, more evidences have to be accumulated to illustrate

the hypothesis.

(2)Quantitative Analysis
(a)Standard Reference Curve of Phenolics
A standard reference curve for phenolics was created
using gallic acid. Gallic acid solutions containing from 10'
7g/ml to 104g/m1 were prepared by dilution from a freshly
prepared 0.100g/100ml solution. Total phenolic concentration,
as gallic acid equivalents, was determined using the procedure

described previously. The concentration of the standard

51

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(b), and (c).

56
solutions was plotted against the absorption at 750nm. The
formulate of a reference curve obtained from the data was
Y=1.3533558-06 + 1.2902693-05 * x (Y= concentration of gallic
acid, X= Absorption), which had a correlation coefficient of
0.998. When the concentration of gallic acid was higher than

2x10ig,'the absorption was out of the range of the instrument.

(b)Variation of Total Phenolics

The seasonal variation of phenolics in black locust
is presented in Figure 14. The phenolic content was also
calculated based on the dry weight of tissues. These data
showed that the major location of phenolics is in the
transition zone and heartwood of black locust. The seasonal
variation of phenolics in the heartwood was roughly parallel
with that in the transition zone. The level of phenolics in
the heartwood was relatively constant during the whole year
except the heartwood formation season (from July to late
September). The phenolic concentration of July heartwood was
the highest and the concentration variation of phenolics in
heartwood formation season could result from which the
activity of enzymes in the outermost heartwood rapidly
increased. The phenolic level in the transition zone was
variable and may be a function of the size of the sapwood
portion of the sampling. The extractable flavonoids in the
heartwood of black locust were about 6% of dry weight (Roux

and Paulus, 1962; Smith, A.L. et al., 1987).

57

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58
The phenolic content of the cambial zone and sapwood was
relatively low compared with the heartwood and transition
zone. The level of phenolics detected in the cambial zone of
black locust in this study was higher than in the sapwood.
The fluctuation of phenolic in the cambial zone declined to
a valley (0.1 mg/g) on September, increased to a maximum value
(0.3 mg/g) in late fall, and remained at a constant level (0.3
mg/g) over the other months; until the next growth season the
phenolic level started to build up again to synthesize lignin.
The result from the cambial zone could be explained by
Nobuchi's study (1984b). Nobuchi (1984b) indicated that the
most.part.of’heartwood formation in black.locust.occurred from
July to late September. In addition, the GC-MS result
demonstrateed.that the flavonoidS‘were not found.in th cambial
zone. The level of phenolics in sapwood.was a constant ( less

than 0.1 mg/g) through one year.

It is generally accepted that heartwood formation is
related to the deposition of the phenolic substances into the
ray parenchyma cells of heartwood, which leads to the death
of ray parenchyma cells. However, the quantitative changes
of phenolics in heartwood formation varied among species
(Hillis, 1987). The relationship between.heartwood formation
and cytological structure has been widely studied by Frey-
Wyssling and Bosshard (1959), Fahn and Arnon (1963), and
Nobuchi (1984 a,b). It was thought that the living ray

parenchyma cells in the transition zone and the heartwood are

59

concerned with the formation of heartwood substances.
Nobuchi's study indicated that there were no living ray
parenchyma cells in the heartwood of black locust from April
to July, but the nuclei of the ray parenchyma cells existed
in the heartwood after July and through next spring.
Additionally, the nuclei of the living ray parenchyma cells
existed in the sapwood of black locust in the spring; the
transition zone had nuclei after July and extended into the
heartwood.

Frey-Wyssling et al. (1959) suggested that heartwood
formation occurred by a semi-anaerobic mechanism, in which the
oxidation of the phenols was stimulated by the hydrolysis of
starch in the transition zone under low concentration of
oxygen (Figure 15). However, Hillis (1972) and Roll (1973)
obtained results that indicated respiration in the transition
zone of Eucalyptus and black locust studies both in vivo and
in vitro had a higher respiration than sapwood.

It is known that the proportions of oxygen and carbon
dioxide inside the wood are very different from those in the
atmosphere. It might be expected that a higher concentration
of carbon dioxide within the wood would result in an increase
in the production of malonyl-CoA and phosphoenol pyruvate from
increasing respiration. These compounds are known to be
precursors of polyphenolics and flavonoids. If heartwood
formation and the death of ray parenchyma cells were due to

a reduction in the level of oxygen and/or an increase in the

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61
level of carbon.dioxide‘within.the wood, one would expect that
the production of heartwood substances (polyphenolics and
flavonoids) could be detected in vitro under a high
concentration of carbon dioxide.

Carrodus (1971) examined the effect of high
concentrations of carbon dioxide on the formation of
flavonoids in the heartwood of Acacia mearnsii. Production
of three out of four flavonoids in the heartwood. were
stimulated by carbon dioxide when the sapwood and transition
zone contained no heartwood.portion. In this experiment, those
flavonoids were absent when the sapwood and middle transition
zone were exposed to the presence of nitrogen. ‘Thus,
confirming the response was due to the presence of carbon
dioxide, not the lack of oxygen. Seasonal variation of this
reaction to the presence of carbon dioxide in the wood tissue
paralleled the formation flavonoids (late spring and summer).
It is suggested that polyphenolic formation in the heartwood
of A. mearnsii might be stimulated from June to September due
to a high level of carbon dioxide produced in the wood
tissues.

The results from quantitative and qualitative analysis
here both indicted that the flavonoids in the heartwood of
black locust might be transformed in the transition zone
(sapwood/heartwood boundary) in a very short period of time.
Thus, the seasonal variation of primary metabolites will be

one of possible methods to monitor a flux between secondary

62
and primary metabolites. The carbon source of flavonoids
couLd be from several directions: small phenolics, starch,
soluble sugars and degraded cellulose. In addition,
carbohydrates are: essential compounds for flavonoids
biosynthesis. The seasonal variation of soluble sugars and

starch was examined to determine the fluctuation.

(B)CARBOHYDRATES

(1)Quantitative Analysis
(a)Soluble Sugars

(i)Standard Reference Curve of Soluble Sugars: A
standard reference curve of soluble sugars was established
using a glucose standard solution purchased from Sigma
chemical Co. The standard glucose solutions ranged from 1x10'
6g/ml to 1x10'zg/ml and were prepared by dilution. The
absorption of standard glucose solutions was determined using
the "phenol-sulfuric acid" method described above. The
concentration of the standard.solutionuwaSjplotted against the
absorption at 490nm. The formulate of a reference curve
obtained from the data was Y=-2.923483-05 + 1.9137078-04 * x
(Y= concentration of glucose, X=absorption), the linear
regression revealed a correlation coefficient of 0.932. The
correlation coefficient of linear regression became to 0.989
when based on a concentration range of glucose standard

solutions from 1x10‘g/ml to 5x10‘g/m1. Therefore, it was

63
suggested that a linear relationship might not exist between
the absorption and glucose solutions at low and very high
concentrations. Black locust samples were prepared by dilution

so that their absorption fell into the linear range.

(ii)Variation of Soluble Sugars: The seasonal
variation of soluble sugars in black locust is depicted in
Figure 16. The amount of soluble sugars was calculated based
on the dry weight of the tissues and expressed as g/g. The
data from the cambial zone shows that the amount of soluble
sugars was higher than in the other tissues. The
concentration of soluble sugars in the cambial zone increased
during the cold weather seasons and decreased during the
growth season. This result agrees with the results of
previous investigations. .Jones and Bradlee (1933) studied the
seasonal changes of hexose sugars in the inner bark of sugar
maple. It was observed that the hexose sugars content
increased in the mid-autumn, reached a maximum point in
January, then decreased from this point during spring, and
remained a low concentration in the summer. Siminovitch et al.
(1953) examined carbohydrate fluctuation using Hassid's
oxidation with ferricyanide and ceric sulfate titration (1936)
and the results were expressed in Figure 17. Siminovitch's
study found that the reducing sugars of the living bark of
black locust stayed.at.a relatively constant, low amount (0.8%

to 1.1% of dry weight of tissues) over the year, reaching a

64

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maximum (3% of dry weight) in late winter. The results from
this study had a higher level of soluble sugars in the cambial
zone than the previous studies.

Generally speaking, the level of soluble sugars in the
sapwood was lower than that of cambial zone in this study.
The level of soluble sugars in the sapwood continuously
increased from September until January, reaching a maximum of
3.2% of dry weight, then decreased to 1.0% in February and
March. The soluble sugars content of the sapwood increased
to 2.1% of dry weight in April and then decreased to 1.0% and
maintained the level through the growth season. JOnes and
Bradlee's (1933) study also determined the seasonal variation
in hexose sugars of the outer wood (outer sapwood) of sugar
maple in northern Vermont. Their results suggested that the
content of hexose sugars reached a maximum in early winter,
began to decrease from there, and decreased rapidly to a low
content (less than 0.1% of dry weight) in spring season. The
changes of soluble sugars in sapwood of black locust were
similar to that of sugar maple in the fall and winter. During
the growth season the changes of soluble sugars in the sapwood
were different from those of sugar maple. In Hansen and
Grauslund's (1973) study, seasonal variation in the
concentrations of soluble sugars of wood from the trunk of
young apple trees showed similar results. The recent study
of Holl (1985) in the trunkwood of spruce (Bicea abies (L)

Karst) indicated that the concentration of soluble sugars was

 

67
higher during the cold period (from December until early
March) than in the spring and summer months.

The results from this study substantiated the
physiological roles of the various tissues. The level of
soluble sugars in the cambial zone is higher than that of
sapwood since the function of a cambial zone is to transport
nourish from leaves to roots. The level of soluble sugars in
the transition zone and heartwood remained a constant low
level, 1% and 0.78% of dry weight, respectively. The data
obtained in March were lower than an expected perhaps due to
site variations. Few attempts have been made to study the
soluble sugars content in ‘transition zone and. heartwood
because the two positions were thought to the physiologically
inactive. However, cytological differences in transition zone
and heartwood have been widely investigated in various
species. It is generally accepted that the outer most rings
of heartwood may have physiological living cells while the
inner heartwood is a completely dead tissue. In addition, the
function of soluble carbohydrates is to increase the osmotic
pressure of the cell sap. Thus, the level of soluble sugars

in heartwood and transition remained a constant and low level.

(b)Starch
(i)Standard Reference Curve of Starch: A standard
reference curve for starch was prepared using a Chem Service

analytical starch standard. Starch solutions ranging from

68
1.4x10‘3g/50ml to 1.196x10‘1g/50ml were obtained by dilution and
analyzed using the procedure described above. The
concentration of the standard solution was plotted against the
absorption at 650nm. The formula of a reference curve
obtained from the data was Y=-8.06373963-04 + 3.6336653-02 *
x (Y=concentration of starch, X=absorption). A correlation
coefficient of 0.962 was determined.by linear regression. 'The
correlation coefficient of linear regression became to 0.991
when the concentration range of starch standard solutions was
reduced to a range of 1.4x10'3g/50ml to 5.51x10'2g/50ml. Black
locust samples were prepared by dilution such that they fell

into the linear range.

(ii)Variation of Starch: The seasonal variation of
starch in black locust sections is shown in Figure 18. The
starch content was calculated based on the dry weight of the
tissues. It is clear that the highest concentration of the
starch is in the sapwood. This is consistent with a storage
function for sapwood. The starch content of sapwood decreased
from fall until February, reaching a minimum point of 0.7% of
dry weight, then began to increase in late winter and early
spring. The starch level of the sapwood dropped gradually
from early spring to late spring, reaching the other minimum
point of 0.7% of dry weight, then increased again after the
growth season. The starch concentration of the sapwood in

black locust varied in a manner similar to that of the outer

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sapwood of sugar maple in the growth season from the end of
march to August (Jones and Bradlee, 1933). Black locust
sapwood, however, had a higher overall maximum starch content
(about 9.8% of dry weight) than did sugar maple (about 3% of
dry weight). Investigation of starch-glucose in the outermost
5-7 xylem rings of spruce (Roll, 1985) revealed that the
starch contents were relatively constant from early autumn to
late winter, and increased in the spring and decreased in the
summer. In comparison, the amount of starch in the sapwood
of black locust rapidly dropped from fall to the mid—winter,
and then slowly increased in the growth season.

The starch level in the cambial zone decreased from
a maximum value (5.5%) in September to a minimum level (0.78%)
in February, then increased from early spring to May and
dropped on June and slightly increased again. The fluctuation
of starch in the cambial zone was cooperated with the sucrose
level in this tissue and the transformation of photosynthesis
products. The starch analysis of the cambial zone showed no
significant differences during winter and early spring
(average= 0.8% of dry weight). Siminovitch et al. (1953) also
found that the starch concentration of living bark in black
locust rapidly decreased from a maximum point (8% of dry
weight) in early autumn to reached a minimum value (0.1%) in
February, and remained at this level during June and July in
Figure 15. Their data also demonstrated that after the

sucrose level reached 5% or 6% of dry weight in early fall,

71
the starch began to accumulate. In the growing season an
increase in starch was coupled with a decrease in sucrose.
Thus, they suggested that the seasonal variation in the
carbohydrates of black locust was related to temperature and
reversible enzymes in the bark.

The seasonal changes of starch concentration in the
transition zone of black locust were similar to the trend
shown for the sapwood. The transition zone, however, had a
low level of starch (the maximum about 2% and the minimum less
than 0.1%). The concentration of starch in the heartwood of
black locust remained at a constant low level (less than 0.5%
of dry weight). Jones and Bradlee (1933) found that the inner
wood of sugar maple had a lower starch concentration than the
outer wood.

In most cases, cytological studies have show that
starch granules will not be present in transition zone and
heartwood. However, a few starch granules were found in the
transition zone and heartwood of black locust. These granules
existed in the ray parenchyma cells from late summer to late
winter (Nobuchi et al., 1984a).

The results from this study of total phenolics,
starch, and soluble sugars indicate that the phenol level of
the heartwood increases in the fall and winter while the
starch level of the sapwood decreases during these seasons.
0n the other hand, the level of soluble sugars increases in

these seasons. It has been agreed that soluble sugars

72
increase the osmotic pressure of the cell sap which is
considered to the tolerance of cold temperature. They also

serve as an important energy source for the numerous metabolic

activities (Smith, 1968: Alden et al., 1971; Krasnuk et al.,
1975). Starch is a storage form of carbohydrates. Starch

hydrolysis is precisely regulated by low temperature (Krasnuk
et al., 1976; Dear et 1.,1972: Marvin and Morselli, 1971;
Siminovitch et al., 1953). The soluble sugars produced from
starch hydrolysis might cause a rise in the level of energy-
releasing compounds or pentose sugars for nucleic acid
synthesis. In addition, flavonoid synthesis requires ATP as
energy and cofactor. Thus, it is suggested that soluble
sugars and starch could be utilized as indirect or direct
sources for flavonoids biosynthesis in the heartwood of black

locust.

CONCLUSIONS

The major flavonoids (dihydrorobinetin and robinetin)
found in the heartwood of black locust are thought to be
responsible for its durability. The results obtained in this
study suggest that flavonoid biosynthesis occurs at the
transition zone. The apparent site of synthesis does not
change with the season. No major flavonoids could be detected
by TLC and GC-MS in the sapwood portion even though the
samples were hydrolyzed with cellulase and 2N HClzueOI-I ( 1:1) .
Three possible processors of flavonoids (compound (a),(b),
and (d)) showed a fluorescence with varied Rf value under UV
light (254nm) have been detected. The unhydrolyzed samples
from the transition zone contained both major flavonoids while
the hydrolyzed samples showed only dihydrorobinetin. The
starch level of the transition zone was much less than that
of the sapwood while the total phenolics content of the
transition zone was greater than that of the sapwood. The
level of extractable chemicals found in the transition zone
was one third to one half of the amount found in the
heartwood. Flavonoids were the major extractable compounds
in both the transition zone and heartwood.

Quantitative analysis of the phenolic content of
sapwood from this study was inconsistent with the results of
previous studies (Roux and Paulus, 1962). The investigators
found that both dihydrorobinetin and robinetin were detected
in the sapwood and heartwood while we found that the

73

74
flavonoids were only detected in the heartwood and transition
zone. This discrepancy might be due to a difference in the
definition, in terms of wood coloration and physiological
activity, of transition zone and heartwood. The range of the
transition zone in this study included the edge of the
heartwood and 0.1-0.3 cm of sapwood. The data from TLC and
GC-MS analysis confirmed that the major flavonoids found in
the heartwood of black locust did not exist in the cambial
zone. However, an intermediate precursor (cinnamic acid) has
been detected in 50% acetone extractives of sapwood. (Cinnamic
acid is also a lignin precursor.

The variation in total phenolics of the wood from
sapwood to heartwood is the reverse of the starch variation.
Starch is hydrolyzed and then transformed to soluble sugars
so that the level of soluble sugars increases. It has been
suggested that the activity of starch hydrolysis increases in
the transition zone. After starch hydrolysis occurs, the
activity of enzymes in the parenchyma cells of the sapwood
will be changed. This change in activity may be transmitted
through the various zones by ray cells so that flavonoids are
rapidly synthesized in the sapwood/heartwood boundary. The
results from this study show that the level of starch in the
transition zone is less than that of the sapwood. The results
of these experiments, combined with the qualitative results,

suggest that the carbohydrate flux (soluble sugars, starch)

75
may be an indirect carbon source for flavonoids or may be the

energy source of flavonoid biosynthesis.

RECOMMENDATIONS FOR FURTHER STUDY

The recommendations for further work in this study are
as follows:
1) To look for the biochemical changes across the stem by
growth rings of stand trees using labeled compounds (sucrose,
glucose, or cinnamate),
2) Continue to identify the possible precursors (compound a,
b, and d) of flavonoids,
3) Isolate and characterize the enzymes which are related with
flavonoid biosynthesis in the sapwood or transition zone of
black locust,
4) To investigate the factors affecting flavonoid biosynthesis
in the parenchyma cells of the transition zone such as the

ratio of carbon dioxide/oxygen, pH value.

76

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