. , ’ HEARTWOOD mmcnvrss 0F MACLURA POM‘IFERA
I AND THEIR ROLE IN DECAY RESISTANCE '

DISSERTATION FOR THE DUAL DEGREE 0F PH. D.
MICHIGAN STATE UNIVERSITY
SHIH-CHI WANG
1977

 

 

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This is to certify that the

thesis entitled

Heartwood Extractives of Maclura pomifera
and Their Role in Decay Resistance

presented by

Shih-chi Wang

has been accepted towards fulfillment
of the requirements for

Dual Ph.D. degree in Botany and Plant
Pathology
and Forestry

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5/20/77

Date

 

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ABSTRACT

HEARTWOOD EXTRACTIVES OF MACLURA POMIFERA

 

AND THEIR ROLE IN DECAY RESISTANCE

BY
Shih-chi Wang

The heartwood of osage-orange (Maclura pgmifera (Raf.)
Schneid.) is considered one of the most durable woods.

Hence, the mechanism of decay resistance and some of the
chemical properties of this wood were investigated.

Samples of ground heartwood were extracted with hexane,
ether, chloroform, Efbutanol, ethanol-benzene, acetone,
methanol, ethanol-water or distilled water. Polar solvents
removed the largest amount of extraneous material. The
decay resistance of the extracted ground wood was determined
by the oxygen consumption method. Decay resistance decreased
significantly following extraction with polar solvents,
particularly methanol and ethanol-water.

Extracted wood blocks were exposed to decay fungi to
investigate the structural mechanism of decay resistance.
After exhaustive extraction, the weight losses of heartwood
blocks increased from 0.4% to 16.2%, from 0.6% to 37.0%, from
0.2% to 2.5%, from 0.4% to 7.0% for Poria placenta, Coriolus

versivolor, Gloeophyllum trabeum, and Lentinus lepideus

 

 

 

respectively. These results revealed that the mechanism of

decay resistance of Maclura pomifera was mainly chemical.

 

Shih-chi Wang

However, the limited decay caused by Gloeophyllum trabeum

 

after exhaustive extraction suggested that a structural
mechanism may be partially involved.

To isolate and identify the active component in the wood,
ground heartwood was extracted sequentially with hexane,
chloroform and methanol. The dry methanol extract was
partitioned between water and ether to separate the water-
soluble materials from those soluable in ether. Further
separation was accomplished by liquid-liquid extraction of
the ether fraction with aqueous sodium bicarbonate and sodium
carbonate solutions to differentiate the phenolics from
organic acids and neutral materials. The phenolic components
were separated by paper chromatography using benzene-acetic
acid-water (125/72/3) as the developing solvent. The
separated components were examined quantitatively as well as
qualitatively. The fungal toxicities of various fractions
and compounds were determined by incorporating each material
into non-durable ground wood or wood-base material and then
inoculating the sample with one of several wood decay fungi.
Fungal growth in wood was measured by respirometry or visual
observation. The compound with the greatest inhibitory effect
on wood decay fungi, as determined by spectroscopic methods ,

appears to be a pentahydroxystilbene.

HEARTWOOD EXTRACTIVES OF MACLURA POMIFERA

 

AND THEIR ROLE IN DECAY RESISTANCE

BY

Shih-chi Wang

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

Department of Forestry

1977

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. John H.
Hart for his enthusiastic support, assistance and encourage-
ment throughout the period of my study at the Michigan
State University.

Thanks are extended to Dr. Eldon A. Behr for his counsel
and guidance for this work.

I highly appreciate the contributions of other members
of my guidance committee: Dr. Everett 8. Beneke, Dr. James
W. Hanover and Dr. Otto Suchsland.

Acknowledgments are also extended to Dr. Jacob B. Huffman,
Dr. Robert A. Schmidt and Dr. Ramon C. Littell at the
University of Florida for establishing my training and
attitudes on WOod Technology, Forest Pathology and Statistical
Methods during early years of my career. Their inputs are
gratefully remembered.

I also wish to express deep gratitude to my parents, Mr.
and Mrs. Yun-lan Wang, for their encouragement during many
years of my schooling and for their unfailing support of high
goals.

Thanks are also due to Dr. William H. Reusch and Mr. K. P.
Subrahamanian for their assistance with the spectroscopic
analysis.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES AND FIGURES ..... .............. . ....... iv
INTRODUCTION ................................ ..... .... 1
MATERIALS AND METHODS ... ......... . ................ ... 5

Determinations of Natural Resistance of the
wood and Its Mechanism ....................... 5
Extraction and Separation of the Extraneous
Components in the Wood ....................... 8
Investigation of the Fungistatic Properties
of Each Fraction and Subfraction of the
Extractives ............................. ..... 12
Examination of the Chemical Nature of the
Fungistatic Component ........................ 14

RESULTS AND DISCUSSION ............................... 16

The Natural Decay Resistance of WOod ... ........ . 16
The Decay Resistance Mechanism: Chemical

vs Structural ................................ 19
Extraction, Separation and Bioassay of the

Extraneous Components in the Heartwood ....... 23
Fractionation and Bioassay of the Methanol

Extract ...................................... 26
Chemical Nature of the Fungistatic Component .... 35

COWLUSIONS O O O C C O O O OOOOOOOOOOOOOOOOOOOOOOO O ......... O 42

LITEMTURECITED ......OOOOOOOOOOOOOOO......OOOOOOOOOO 43

APPENDIX 0 O O O .......... O ..... O OOOOOOOOOOOOOOOOOOOOOOOO 46

iii

LIST OF TABLES AND FIGURES

Page
Table

1. Weight losses (mean of 8 determinations) of
the heartwood blocks of Maclura pomifera caused
by Gloeophyllum trabeum, Poria placenta, Lentinus
lepideus and CorioIfis versicaior ................. 17

 

 

 

 

2. weight losses (mean of 6 determinations) of the
heartwood and sapwood of Maclura pomifera caused
by Gloeophyllum trabeum, Poria placenta, Lentinus

 

 

 

 

 

lepideus and CorioIfis versicolor ... ..... ......... 18
3. Solubilities of the wood of Maclura pomifera in
various solvents (mean of 6 determinations) ...... 20
4. Oxygen consumption, l/Hr (average of S-hour

testing), and growt rate of Coriolus versicolor
and Gloeophyllum trabeum on groundvheartwood of
Maclura pomifera extracted with various solvents.. 21

 

5. weight loses of exhaustively extracted blocks of
Maclura pomifera caused by Poria placenta,
Gloegphyllum trabeum, Coriolus vers1coIor and

 

 

 

 

 

Lentinus lepideus ........ ........ ........ ....... 22
6. Effect on each heartwood extract of Maclura

 

pomifera on Coriolus versicolor and GIoeopHyllum
tra eum as determined in ground aspen wood by the

oxygen consumption method ...... ....... .......... 24
7. The calculated percentage (air-dried weight

basis) of each fraction of the methanol extract

in the heartwood of Maclura pomifera ............ 27

 

8. Effect of each fraction of the methanol extract
on the growth of Coriolus versicolor and
Gloeophyllum trabeum as measured in ground aspen
wood by respirometry ... ......... .......... ...... 30

 

iv

Page
Table

9. The calculated percentage (air-dried weight
basis) of each component of F6 in the heart-
wood of Maclura pomifera ........................ 33

 

10. Effect of each component of the fraction F6
on wood decay fungi as evaluated by fungal

growth on treated cellulose paper .......... ..... 34
11. The color reactions of the component F6-A ....... 36
Figure

1. Separation scheme for heartwood extractives
Of Maelura EQmifera 0000.00.00.00.........0....0. 9

2. Separation scheme for the methanol extract ...... 10

3. Diagrammatic representation of the paper
chromatographic separation of the fraction F6
from the heartwood extractives of Maclura
pomifera ........................................ 32

4. NMR spectrum of the component F6-A from the
heartwood extractives of Maclura pomifera ....... 37

 

5. Mass spectrum of the component F6-A from the
heartwood extractives of Maclura pomifera ....... 38

INTRODUCTION

The principal components of the wood cell wall - poly-

saccharides and lignin - are essentially similar in all
species of wood. In addition to these structural components,
all woods contain smaller amounts of minor or extraneous
components which are much more diverse in their chemical
nature. This diversity often accounts for the difference in
chemical features among various wood species. These
extraneous components are termed extractives because they
can, to a large extent, be extracted from the wood with
neutral solvents, without destroying the structure of the
wood. They include many different classes of organic compounds,
ranging in complexity from relatively simple molecules, such
as phenols and sugars, to highly complex coloring matters,
i.e. tannins, resin, etc. These components are of considerable
importance because a number of technically important properties
of wood are dependent upon the presence of extractives in the
wood. One of the most important of these properties is the
wood's natural durability. WOod extractives and their
significance have been reviewed by Hillis st 21 (17).

Factors contributing to decay resistance of wood have been

studied over a period of years and the presence of certain

fugistatic extractives in heartwood is known to be the

major mechanism for the decay resistance of wood. Hawley
and co-workers (1924) were the first to demonstrate that the
presence of extractives in heartwood is toxic to wood-
destroying fungi (15). Since then a large number of
heartwood constituents have been tested in zitrg for their
funcicidal or insecticidal properties. Most of the active
compounds have been found to be of a phenolic nature. This
chemical mechanism of wood durability -— the presence of
fungistatic extractives -— is by far the most commonly
accepted theory for the decay resistance of wood (26).
Nevertheless, there is some evidence (22) that wood structure
is also a factor contributing to the decay resistance.

The heartwood of osage-orange (Maclura pgmifera (Raf.)

 

Schneid.) has long been known as an exceptionally durable

wood (31), based on its extremely long service life under
conditions favorable to decay. When exposed to decay fungi
under controlled environmental conditions conducive to rot,

it is also very durable (13). However, little is known of

the mechanism of ecay resistance of osage-orange heartwood.
The only work done on relating the chemistry with the dura-
bility of this species is that by Barnes and Gerber (6). They
reported the presence of 2, 4, 3', 5'-tetrahydroxystilbene in
the heartwood and in gitgg tests revealed that it had

antifungal properties.
Stilbenes are diphenylethylenes which can be extracted

with acetone or alcohol. Generally they occur free in wood

and are only substituted by hydroxyl or methoxyl groups.
All of them exhibit an intense blue fluorescence under
ultraviolet light (18). Pinosylvin, a dihydroxy-stilbene,
and its monmethyl either occur in the pines, and are of
special significance in connection with the durability of
pine heartwood (10, 24, 25). Reservatrol, a trihydroxy-
stilbene, may play a role in the durability of eucalypts(l4l .
Thus, there are a number of reports suggesting that stilbenes
increase the resistance of wood to deterioration (26).
However, this hypothesis has recently been questioned (14,
20).

A recent study (14) has emphasized the difficulties in
relating the results of in_vit£g_testing of wood extractives
and their pure components with the durability of the wood
from which the extractives were obtained. The study conducted
by Barnes and Gerber (6) did not provide information concern-
ing the fungistatic values of the active compounds in wood
or wood-base materials. Their in vitrg testing determined
only the extent that the compound protected the liquid medium
against the test fungi. In addition, their research did not
employ wood-inhabiting fungi or the decay fungi commonly
recommended for such tests (3, 4). In view of these facts,
many facets of potentially useful information concerning the
durability of osage-orange heartwood remain to be developed.

The objectives of this study were to extend our present

knowledge of the decay resistance of osage-orange heartwood,

\[((l {I ‘II (I‘ ll‘ III"! E“ I
‘ ‘llll‘l (E. II lll‘II ' E. '1 'Il' I; III
I I1! I'.II II III

to determine what factors are responsible for the natural
decay resistance and to obtain more information on the

chemical properties of this wood.

MATERIALS AND METHODS

The test wood came from an osage-orange tree (Maclura
pomifera), collected at W. K. Kellogg Forest, Augusta,
Michigan, in September 1974, with a diameter inside bark of
15 cm. A portion of the stem, free from knots or defects,
was selected for the test material. The bark was removed
and the log was air-dried, cut into short logs and stored
in a cold room. To prepare samples for decay tests, the
heartwood was cut into blocks 2.5 x 2.5 x 0.9 cm (longitudi—
nal) and the sapwood into blocks 2.5 x 0.5 (radial) x 2.5 cm.
The remainder of the log was cut into 1 cm2 strips, with
heartwood and sapwood kept separate. These strips were
ground in a Wiley Mill with a 40-mesh screen and the ground

wood stored in plastic bags at 4°C.

Determinations of Natural Decay Resistance of the Wood and

 

Its Mechanism

 

Using the standard soil-block or agar-block method (4),
the wood blocks were exposed to a decay fungus -— Poria

placenta Murr (Madison 698), Coriolus versicolor L. ex Fr.

 

(Madison 697), Gloeophyllum trabeum Pers. ex Fr. (Madison

 

617) or Lentinus lepideus Fr. (Madison 534). The test

 

blocks were weighed before and after exposure, and the loss

5

in weight was used to determine the amount of decay. The
test was terminated when 60% weight loss was obtained in
non-durable aspen sapwood blocks.

To determine the possibility of the presence of
fungistatic extractives in the heartwood, samples of the
ground wood were extracted individually in Soxhlet apparati
for eight hours with hexane, ether, chloroform, E butanol,
ethanol-benzene (1/2, v/v), acetone, methanol, ethanol-water
(l/l, v/v) or distilled water. After extraction, the
solubility of the wood in each solvent was determined. The
procedures used for the extraction and solubility tests were
similar to those described in ASTM-D 1107 or ASTM-D 1108 (l,
2).

. The decay resistance of the extracted samples was
determined by respirometry (7, ll, 28, 29, 30). Petri dishes
with a thin layer of malt-agar were inoculated with Q.
versicolor or Q. trabeum. After the fungal mycelium covered
the agar plate, a 25 ml sample, previously sterilized, was
evenly spread over the agar surface. Unextracted osage-orange
heartwood and unextracted aspen sapwood were used as decayé
resistant and decay-susceptible controls, respectively. Samples
were incubated for three weeks at 25°C and 90-95% relative
humidity. After incubation, fungal growth was evaluated by
visual examination, and rated on a scale from 1 to 5 with
each integer representing coverage of the wood surface of

0-20% to 80-100%, respectively. Incubated ground wood was

then removed from the plate and tightly packed in a vial

to obtain a 2 m1 volume. These samples were transferred to
Warburg flasks and changes in manometer levels were recorded
hourly for five hours. Differences in oxygen consumption
were used for estimating the rate of fungal growth in each
sample which was used to evaluate the loss of decay
resistance of the extracted ground wood.

Decay resistance due to wood structure was tested by
measuring the decay resistance of exhaustively extracted
blocks. To accomplish this objective, the extractives were
removed from the wood without destroying its structure.
Blocks of osage-orange heartwood were successively
extracted in Soxhlet apparati with ethanol-benzene (1/2, v/v),
ethanol-water (1/1, v/v), acetone and distilled water for
66 hours, 72 hours, 24 hours and 72 hours respectively.

Three transverse canals were drilled on each side of the test
block to facilitate extraction of soluable materials. Blocks
were weighed before and after extraction to measure the
amount of material removed. The extracted wood blocks were
then tested (4) for decay resistance against}; placenta,

g. versicolor, g. trabeum and E. lepideus. Sapwood blocks

extracted in the same manner were used as controls.

Extraction and Separation of the Extraneous Components in

the Wbod

Since the fungistatic extractive previously reported
(6) was of a phenolic nature, the chemical analysis was
focused on the isolation of phenolic substances. Several
extractions were conducted as shown in Figure 1. Ground
osage-orange heartwood (200 g) was extracted with each
solvent for ten days at room temperature in a glass column.
The column was fitted at the bottom with a stopcock to
control the flow of liquid and the solvent exit was covered
with a loose plug of glass wool. Preliminary extractions with
hexane and chloroform were conducted to remove most of the
nonpolar extractives. After the completion of each
extraction, the ground wood was removed from the column,
dried at room temperature and then replaced into the column
for the next extraction. The extract was collected five
times per day by draining the column to the level of the
wood surface, and than replacing fresh solvent. Each
extract was reduced in a roatary vacuum evaporator at less
than 40°C, air-dried at room temperature, and the dry weight
recorded. The air-dry weights of ground wood before and
after extraction were used for quantitative determinations.

To fractionate the extraneous materials, several partitions
were conducted as shown in Figure 2. The methanol extract

was first partitioned between ether and water to separate

OSAGE-ORANGE HEARTWOOD

 

 

 

 

 

 

 

Hexane
extraction
l ' l
OOEW-l EEXANE EXT.
Chloroforn
extraction
l ' l
CHLOEOFOEM EXT. ‘OOEW—Z
Acetone Methanol
extraction extraction
.1/ \L
f I l I
DORE-3 ACETONE EXT. METEANOL EXT. OOEW-G
ETOE-Hater ‘ Water
extraction extraction
I ' ’ I I ' I
ETOB-WATER EXT. GORE-4 WATER EXT.-2 OOEW-7
Water
extraction
I ’ ' i
DORE-5 HATER EXT.-l

Figure 1. Separation scheme for heartwood
extractives of Maclura pomifera.

10

METHANOL EXTRACT

Evaporation
DISSOLVED IN ETEER

I Filter

 

r ' 1
ETHEE SOLUBLE ETHEH INSOLUBLE

Partition flash with
(water/ether) water

 

 

 

r ’ I r’ ’ 1
WATER ETHER HATER WATER
SOLUBLE -SOLUBLE SOLUBLE INSOLUBLE

flash with ( P 1 ) Dissolved
ether in

methanol

r* ’ I
WATER ETHER ( F 2 )
SOLUBLE SOLUBLE

( F 3 ) ETEER SOLUBLE
l Partition

 

A L

 

 

(sodium bicarbonate solo/ether)

 

I ' l
AQUEOUS ETHER
PHASE PHASE

Wash with
ether

 

I ' I
AQUEOUS ETHEH
PHASE ' PHASE

Acidified ETEER SOLUBLE
Extract with l Partition

 

ethyl acetate (sodium carbonate soln/ether)
ETHYL ACETATE

SOLUBLE r’ - '
( P ‘ ) AQUEOUS ETHER

PHASE PHASE

Wash with
ether

 

 

 

I ’ I
AQUEOUS ETHEE
PHASE PHASE

Acidified truss SOLUBLE
Extract with ( F 8 )
ethyl acetate

ETHYL ACETATE
SOLUBLE

( P 6 )

 

Figure 2. Separation scheme for the methanol
extract ( see Figure 1. ).

11

those less polar substances from the highly polar materials.
The air-dried methanol extract was dissolved in ether and
filtered to remove insoluble materials. The ether-insoluble
materials were washed with distilled water to extract those
materials soluble in water. The remaining water-soluble
substances were redissolved in methanol. The ether solution
was then extracted with distilled water in a separatory
funnel. The water phase was removed and washed with ether
to extract entrained materials which were returned to the
main ether solution.

The second and the third partitions were accomplished
by utilizing the weakly acidic character of phenolic sub-
stances (9). These depend on the removal of strong acids
by liquid-liquid extraction into an aqueous phase ofia weak
alkali (sodium bicarbonate) followed by extraction of the
phenolics from the neutral materials by a strong aqueous
alkali (sodium carbonate). Thus, the ether-soluble fraction
was next partitioned between ether and saturated aqueous
sodium bicarbonate. The aqueous extract was washed with
ether to remove entrained materials and the washings returned
to the main ether fraction. The aqueous phase was then
neutralized to pH 6.5 with hydrochloric acid and the
neutralized aqueous solution was extracted with ethyl acetate.

The third partition was the extraction of the remaining

ether-soluble materials with saturated aqueous sodium

12

carbonate. The procedures used were similar to those used
during partitioning with sodium bicarbonate. Each fraction
was dried in a rotary vacuum evaporator and its weight
recorded for quantitative determinations.

Further separation and analysis were accomplished by
using paper chromatography. Benzene-acetic acid-water
(125/72/3) (18) gave good resolution of the components in
F4, F6, and F8 fractions. Toluene-acetic acid-water (4/1/5)
(9) separated the components in F1, F2, and F3 fractions.

The quantitative determinations were accomplished by
weighing the various dry extracts and then calculating their
percentage of the original wood based on the total air-dried
weight of the wood from which each was obtained.

Investigation of the Fungistatic Properties of Each Fraction

and Subfraction of the Extractives

To identify the component responsible for the decay
resistance, a 10 ml aliquot of each fraction of subfraction
containing the amount of that material extractable from 10 g
of osage-orange heartwood was used to impregnate a 10 g
sample of ground aspen sapwood. The ground aspen sapwood
and the extractive solution were mixed until the solvent
was completely evaporated. To obtain even impregnation,
excess solvent was added and periodically stirred. The

treated samples and the untreated controls -— aspen sapwood,

l3

and sapwood and heartwood of osage-orange, were sterilized
in an autoclave. All samples were then exposed to Coriolus

versicolor or Gloeophyllum trabeum and fungal growth was

 

measured by respirometry as previously described.

Since final separation of the extractives was conducted
by paper chromatography, collection of enough of a compound
to impregnate a sample of ground aspen sapwood was difficult.
Hence the fraction containing the fungistatic extractive
was streaked on Whatman 3 MM chromatographic paper, and
eluted with benzene-acetic acid-water (125/72/3) in one
direction. The developed paper was cut into strips
corresponding to individual compounds and the impregnated
paper was used as a substrate to evaluate fungistatic
properties. The use of cellulose paper as a test substrate
has the advantage that the paper is a wood-base material.

Coriolus versicolor, a white-rot fungus, can readily degrade

 

cellulose paper without other wood constituents (16).

The amount of the extractive in the paper was equivalent
to the concentration of that chemical in wood. The paper
strips were cut approximately 5 cm%. placed in a Petri dish,
wetted with distilled water, and sterilized in an autoclave.
An equal amount of even-aged fungal inoculum was placed on the
center of each test paper. Dishes were then incubated for
three weeks at a relative humidity of 95-100% and 25°C.

After incubation, fungal growth was evaluated by visual

examination. Controls used were untreated paper and paper

14

impregnated with 5% pentachlorophenol solution.

Examination of the Chemical Nature of the Fungistatic

 

Component

 

The location of fluorescent compounds was determined on
developed chromatograms, both before and after exposure to
the funes of concentrated ammonium hydroxide. A large
number of color reactions with chromogenic sprays are
available for the detection of wood extractives by applying
these reagents to the developed chromatogram. Many of
these reactions exhibit considerable selectivity, and are
of value in examination of mixtures and estimation of
possible structures. The spray reagents used were similar
to those suggested for phenolic substances by Browning (9).
The resulting colors and the Rf values were recorded for
semiqualitative determinations.

The active compound was further characterized by NMR
and mass spectroscopic analysis. The fungistatic fraction
was separated into individual components by paper chroma-
tography. Components were eluted from the papervdxfiiacetone.
A 2 g portion of anhydrous sodium sulphate was added to the
solution which was stirred, allowed to settle for ten
minutes and filtered. The acetone was removed in a rotary
vacuum evaporator at less than 40°C and any residual
acetone allowed to evaporate in a hood. The dry compound

was dissolved in deuteron acetone and transferred into a

15

capillary tube for NMR spectroscopic analysis. For mass
spectroscopic analysis, the acetone was not completely
removed because the solvent used was acetone. The spectra
were examined and compared with several reference spectra

of similar compounds.

RESULTS AND DISCUSSION

The Natural Decay Resistance of Wbod

The weight losses caused by various decay fungi are
summarized in Table 1 and Table 2. The data revealed that
the heartwood was very decay resistant to the test fungi
and that the sapwood was not resistant. According to the
ASTM scale (4), the heartwood can be rated in the highly
resistant category. These results are similar to those
obtained by Hart and Johnson (13).

There was a difference between the soil-block method
and the agar-block method in the evaluations of decay
caused by Q. versicolor and g. placenta (Table l). The
difference was particularly pronounced with g. versicolor,
a white-rot fungus, which produced more decay when the
agar-block method was employed. The decay caused by'g.
versicolor might be limited by some environmental factor in
the soil-block method, e.g., this method might not have
allowed adequate wetting of samples for optimum fungal
growth. Hence subsequent decay tests were conducted by the
agar-block method which resulted in a higher moisture content

of test wood.

16

Table l .

17

Weight losses (mean of 8 determinations) of the

heartwood blocks of Maclura pomifera caused by
Gloeophyllum trabeum, PorIa placenta, Lentinus

 

 

 

lepideus and Coriolus ver§Icolor.

 

 

 

 

 

 

 

 

Weeks of Heartwood Aspen
Fungus Test Method. Decay' % Wt Lossa % Wt Lossb
g. trabemm Soil-block 11 0.2 51.6
P. placenta; Soil-block 9 0.5 64.8e
L, lepideus Soil-block 16 0.4 47.3
g. versicolor Soil-block l6 0 . l 35 . 5c
‘_. trabeum .Agar-block 11 0.2 48.2
g. placenta Agar-block 9 0.4 43.5f
E. lepideus .Agar-block 16 0.4 50.1
E. versicolor Agar-block l6 0 . 6 68 . 0d
a is significantly different from b at 0.05 level (T test).

C

e

is significantly different from d at 0.05 level (T test).

is significantly different from f at 0.05 level (T test).

18

Table 2. Weight losses (mean of 6 determinations) of the
heartwood and sapwood of‘Maclura pgmifera caused
by Gloeophyllum trabeum, Porii placenta, Lentinus

 

 

 

lepideus and CBrIqus‘veriicolor.

 

 

% Wt. Loss in 8 Weeksa

 

 

Fungus

Heartwoodc Sapwoodd
Q. trabeum. 0.2 38.9
g. placenta 0.3 32.3
E. lepideus 0.4 35.1
g. versicolor 0.4 37.5

 

 

aMeasured by Agar-block method.

c is significantly different from d at 0.05 level (T test).

19

The Decay Resistance Mechanism: Chemical vs Structural

The decay resistance of wood is commonly believed to be
due to the presence of extractives which inhibit fungal
growth activity. To examine the amount of various extrac-
tives and their effects on the decay resistance, samples of
ground heartwood were extracted (1, 2) with various solvents
and the extracted samples were bioassayed. The extractives
of M, pomifera were mainly located in the heartwood (Table 3)
and they were more soluble in the more polar solvents such
as methanol and ethanol-water. The results of the bioassays
accomplished by respirometry (Table 4) revealed that the
decay resistance of the ground heartwood was removed or
significantly reduced by extracting the wood with a polar
solvent, particularly methanol or ethanol-water. These
results indicated that certain extractives in the heartwood
were inhibitory to wood-destroying fungi and those
extractives were most soluble in methanol or ethanol-water
(l/l, v/v).

A structural mechanism has also been suggested (22) for
the durability of wood. The weight losses caused by various
decay fungi of heartwood blocks following exhaustive
extraction (Table 5) enabled the effect of wood structure on
decay resistance to be evaluated. By comparing these weight
losses with those obtained with the agar-block method (Table

1), it can be seen that decay increased from 0.4% to

20

Table 3. 'Solubilities of the wood of Maclura pgmifera in

various solvents (mean of 6 determinat ons .

 

Solubility %

 

 

Solvent Heartwood Sapwood
Hexane 2.1 2.1
Ether 2.4 1.3
Chloroform 0.7 0.1
5 Butanol s. 65 2. 6b
Ethanol-Benzene 19.6c 6.3d
Acetone ’ 14.7e 3.7f
Methanol 26 . 79 7 . 3h
Ethanol-water 30.91 9.05
Water 16.7," 10.71

 

a, c, e, g, i, k are significantly different from b, d, f,
h, j, 1 respectively at 0.05 level (T test).

21

Table 4. Oxygen consumption, /Hr (ave

rage of S—hour

testing), and growth rate of Coriolus versicolor

 

and Gloeophyllum trabeum on gr
Maclura pomifera extracted wit

 

 

ound heartwood of
h various solvents.

 

C. versicolor

 

 

g. trabeum

 

 

Extracting O consumpta Growth Rage 02 consumpt:a Growth Rate
Solvent ()Il/Hr) (1-—-5) (fl/Hr) (1----5)‘D
None 2.5c 1d 3.7C 1d
Hexane 2.0 1 3.4 l
Ether 6.2 1.3 3 3 1.1
Chloroform 4.2 1.3 3.7 1.4
p—Butanol 15.0e 1.3 2.1 1.5
Ethanol-Benzene 81.2e 4.3 136.0f 4.9
Acetone 35.1e 3.3 18.5f 3.1
Methanol ~ 90.2e 5 154.4f s
Ethanol-Water 94.3e 5 167.4f 5
Water 22.5e 2.1 44.0f 3.6
Aspen Control 40.0 4 100.6 4

 

aTest temperature: 30°C.

bEach integer represents coverage of the

of 80-100%.

cMean of 3 determinations.

dAverage of 4 estimations.

eValues are significantly different from
unextracted samples (2.5) at 0.05 level
fValues are significantly different from
unextracted samples (3.7) at 0.05 level

wood surface of 2-20%

the value for
(T test).

the value for
(T test).

22

Table 5. weight losses of exhaustively extracted blocks of
Maclura mifera caused by Poria placenta,

 

§Ioeopfiyl um trabeum, Coriolus versicoIOr and
Lentinus'Iepideus.

 

 

 

 

 

weeks % Weight Loss'3
Fungus of

Decay Heartwoodb Sapwoodc
g. placenta 9 15. 26 48 . 5(1
Q. trabeum. 11 2.5 24.4
9, versicolor 16 37.0 78.3
p. lepideus 16 7.0 55.8

 

3Determined by the Agar-block method.

bAverage % extractive removed: 25.

cAverage % extractive removed: 5.

dMean of 6 determinations.

Decay resistance of extracted heartwood blocks are
significantly different from those of unextracted

heartwood blocks (Table 1) at 0.05 level (T test).

23

to 16.2%, from 0.6% to 37.0%, from 0.2% to 2.5%, from 0.4%
to 7.0% (on oven-dried weight basis) for P, placenta, g.
versicolor, g. trabeum and g. lepideus, respectively. The
limited increase in decay caused by g. trabeum after
exhaustive extraction suggests that a structural mechanism

of decay resistance may be partially involved for this fungus.

Extraction, Separation and Bioassay of the Extraneous

Components in the Heartwood

The total amount of variOus extracts obtained from the
ground heartwood of M. Em‘ifera by successive extraction with
four solvents of increasing polarity (hexane, chloroform,
methanol and distilled water) was 23% (on air-dried weight
basis). The hexane, chloroform, methanol and water extracts'
comprised 1.5%, 0.5%, 16.2% and 4.8%, respectively.

The fungal toxicity of each extract, as determined by
the oxygen consumption method using ground aspen wood, is
presented in Table 6. The data revealed that fungistatic
extracts obtained from the first extraction series (hexane,
chloroform, acetOne, ethanol-water, and water) were the
acetone and ethanol-water extracts. In the second extraction
series (hexane, chloroform, methanol, and water), only the
methanol extract contained toxic materials. The hexane,

chloroform, and both water extracts of OOHW-4 and OOHW-6

24

Table 6. Effect on each heartwood extract of Maclura pgmifera
on Coriolus versicolor and Gloeophyllum trabeum as

 

 

 

determined in ground aspen wood by thé oxygen
consumption methodE.

 

 

 

 

 

 

=====
g. versicolor- g. trabeum
Extract b b
02 consumpt Visual exam 02 consumpt Visual exam
( f: 1/Hr) (1---5)c (f: 1/Hr) (1---5) ‘3
Hexane Ext 40.7f 39 80.0f 3.49
Hexaneh 33.0 3.3 68.6 3.9
Chloroform Ext 52.2 2.8 75.9 3.2
Chloroformh 46.3 3.5 68.4 3.2
Acetone Ext 14.4 1 0.7 1
Acetoneh 43.5 3.8 90.9 3.8
B§::“°1’”“°‘ 19.3 2 7.9 1.9
Ethanol-Waterh 26.9 3.5 52.8 3.6
Water Ext-1 76.1 3.8 58.3 4.8
oouwbs 93.3 4.5 122.6 5
Methanol Ext 10.5 1 1.8 l
Methanolh 28.0 4.5 76.6 4.8
Water Ext-2 40.6 3.3 41.5 4.5
oonw~7 72.1 4.3 104.6 5
Aspen Control 40.5 3.3 94.2 4.4
008wd 1.5 1 2.7 1
ooswe 211.1 5 178.7 3.6

 

aVarious extracts refer to Figure l and the retention of
treating chemicals in each ground aspen sample is equivalent
to their concentration in the wood.

25

Table 6, continued

bTest temperature: 30°C. lpl/Hr: Average of S-hour testing.

CEach integer represents coverage of the wood surface of
0-20% to 80-100%.

dGround heartwood of M, pomifera.
eGround sapwood of M, pomifera.
fMean of 3 determinations.
9Average of 4 estimations.
hSolvent only.

Value for acetone extract is significantly different from
the value for acetone or aspen control at 0.05 level

(T test).

Value for methanol extract is significantly different from

the value for methanol or aspen control at 0.05 level
(T test).

26

did not inhibit the growth of either fungus in the ground
aspen wood.

The unextracted sapwood of M, pgmifera and both the
extracted woods, OOHW-S and OOHW-7 (see Figure l), were
more susceptible to fungal growth than the aspen controls.
For both fungi, the difference was particularly pronounced
with the unextracted sapwoods. This suggested that some
components in the sapwood promoted the growth of both fungi.
In addition, aspen wood may have contained certain materials

that were slightly inhibitory to fungal growth.

Fractionation and Bioassay of theAMethanol Extract

Since the methanol extractives contained the fungitoxic
compounds, this material was fractionated as shown in Figure 2.
The amount of each fraction (% air-dried weight basis) in
the heartwood was determined (Table 7).

Obtaining of a reliable quantitative information depends
greatly on the extraction and partition performed. The
nature of each extract was primarily determined by the solvent
used. Since solubility characteristics of most organic
compounds are determined chiefly by their polarity. NOn-polar
solvents or weakly polar compounds dissolve in non-polar
or weak polar solvents: highly polar compounds dissolve in
highly polar solvents (21). This rule of thumb, ”like dis-

solves like“, was the basic principle used for selecting

27

Table 7. The calculated percentage (air-dried weight basis)
of each fraction of the methanol extract in the
heartwood of Maclura pomiferafi

 

 

F1 2.1
F2 2.2
F3 1.3
F4 2.5
F6 7.4
F8 0.7

 

aMethanol extract refers to Figure 1.
bFraction codes refer to Figure 2.

cTheoretically maximum value.

28

solvent and designing sequence of extractions. Thus,

hexane and chloroform were used to remove non-polar compounds
and methanol or water were used to extract polar or ionic
compounds. The majority compounds in the methanol extract
were phenolic compounds which were detectable by chroma-
tography (9). Owing to the instability of some extractives
particularly at high temperature, hot extraction was not
suitable for the isolation of active compounds. To avoid
possible decomposition of the active components, the highly
efficient Soxhlet extraction was not employed.

Partitions with solvents follow the so-called distribution
law which states that at a given temperature, a solute
distributes itself between two immiscible solvents in such
a way that an equilibrium is reached (12). The equilibrium
ratio of the two concentrations of a given solute in two
given solvents at a given temperature is a constant (dis-
tribution coefficient). The effectiveness of liquid-liquid
extraction at given temperatures is evaluated by the follow-

ing formula (5):

V1 n
W1 '3 "0‘ )

 

Where: W68 weight of original solute.

W12 weight of solute remaining in solvent 1 after
n extractions

V1: volume of solvent 1.

V2= volume of solvent 2 (extracting solvent) used
in each extraction.

29

K = distribution coefficient of the solute.

number of extractions.

:3
II

The most efficient way to minimize W1 is to increase n,
the number of extractions. As it can be seen from the
above formula, the extraction is never complete, but it
can be made nearly so by repeated extractions with fresh
solvent. For this reason, each liquid extraction conducted
in this experiment was repeated with fresh solvent for five
times to enable a satisfactory separation.

The selection of developing solvents was essential to
achieve a successful separation in paper chromatograph.
Many solvent systems can be found in the literature to deal
with the majority of commonly occurring compounds. The
solvent system for optimum separation of components depends
on the nature of these components and the complexity of the
mixture. In examination of materials for which conditions
have not been well established, trial of several developers
may be necessary. Solvents of many compositions have been
tried for separating components in each extractive fraction.
Benzene-acetic acid-water (125/72/3) (18) was selected
because it gave a good separation of components in F4, F6,
and F8.

Evaluation of the fungistatic properties of each fraction
of the methanol extractives showed that fraction F6 was the

only fraction toxic to Q. versicolor (Table 8). Fractions

 

30

Table 8. Effect of each fraction of the methanol extract on
the growth of Coriolus versicolor and Gloeophyllum
trabeum as measured 15 ground aspen wood by

respIrometryQ

 

 

 

g. versicolpr g. trabeum

 

 

 

 

Extractive
Fraction 02 consumptb Visual exam 02 consumptb Visual exam
( [ll/Hr) (1---5)c ([11/an (1---5)°

51 31.4d 4.4e 28.3d 2.3c
F2 27.8 3.4 11.9 2

F3 33.5 4.9 50.9 4

F4 46.3 4.5 18.4 2

F6 11.6' 1.3 6.2 2
F8 41.5 4.5 37.0 2.8
Etherf 32.9 4.5 55.1 5
Ethyl Acetatef 25.5 4.5 60.8 5
Aspen Control 40.2 3.3 91.4 3.8

 

“Various fractions refer to Figure 2 and the retention of
treating chemicals in each ground aspen sample is equivalent
to their concentration in the wood.

bTest temperature: 30°C. /ul/Hr: Average of S-hour testing.

cEach integer represents coverage of the wood surface of
0-208 to 80-1008.

dMean of 3 determinations.
8Average of 4 estimations.
fSolvent only.

F6 is significantly different from the solvents or aspen
control at 0.05 level (T test).

31

F2, F4 and F6 reduced the growth of G. trabeum but F6,
based on the oxygen consumption data (Table 8), was the
most inhibitous.

Separation of components in fraction F6 was obtained by
paper chromatography (Figure 3). The amount of each of the
seven components in F6 in the heartwood (% air-dried weight
basis) is given in Table 9. The fungistatic properties of
each component in F6 were evaluated by the rate of fungal
growth on treated cellulose paper (Table 10). Cellulose
paper, a wood-base material, was used to substitute for the
ground aspen wood as the substrate of this bioassay. High
humidity was very critical to the growth of these fungi on
the cellulose paper. Several tests were necessary to find
out the optimum conditions required.

Many studies on the fungal toxicity of wood extractives
have been conducted by tests utilizing agar or nutrient
solution. The main objection of using agar or nutrient
solutions to test the toxicity is that the substrate is not
wood (8), because it determines the extent that the chemical
protects agar or nutrient solutions against fungi, and no
data are obtained as to its protective effect for wood or
wood component. Hence, the advantage of the paper bioassay
is that it allows for the testing of very small quantities
of a compound on a cellulose base. This may help to minimize
the dangers of misinterpretation regarding the causes of

decay resistance of wood.

FG-A
F6-B
FG-C
FG-D
FG-E

F6-F

0

F6-6

4—-—-(aumuaosaa) aim-010v sum-sumac

32

fol00

 

15
20
25
32

40

59

COLOR UNDER UV

 

Bright Blue

Rust
Blue
Green-Yellow

f Tan

Sky Blue

Violet Blue

Figure 3. Diagrammatic representation of the paper
chromatographic separation of the fraction F6 from
the heartwood extractives of Maclura pgmifera.

33

Table 9. The calculated percentage (air-dried weight basis)
of each component of F6 in the heartwood of

 

 

Maclura ‘ iff‘era“.
Component % in Woodb
F6-A 2.4
F6-B 0.8
F6-C 0.5
F6-D 0.6
F6-E 0.4
F6-F 1.3
F6-G 1.4

 

“various components refer to Figure 3.

bTheoreticallymaximum.value.

34

Table 10. Effect of each component of the fraction F6 on
wood decay fungi as evaluated by fungal growth
on treated cellulose paper“.

% Fungal Growthc
Based on Untreated Control

 

Fungus Controlb
Tuneumdonumemfl..h B C own F G

 

 

Q. versicolor 100 0 22 85 100 100 95 100
g. placenta 100 0 23 95 100 93 100 100
G. trabeum 100 0 27 88 100 100 100 100

 

aV’arious components refer to Figure 3 and the retention of
treating chemical in each sample is equivalent to its
concentration in the wood.

bControl sample was previously eluted with solvent.
cAverage of 4 estimations.

A is significantly different from the control at 0.05 level
(T test).

35

Chemical Nature of the Fungistatic Compgnent

The developed chromatogram of the active fraction-F6,
with the Rf values and the color of each component under
ultraviolet illumination is shown in Figure 3. The
important color reactions with chromogenic sprays are
given in Table 11. The NMR and mass spectra of the
fungistatic component, F6-A, are presented in Figure 4 and
Figure 5 respectively.

After spraying the developed chromatogram with ferric
chloride-potassium ferricyanide, the fungistatic component,
F6-A, gave an instantaneous deep blue spot which is the
positive reaction for phenolic substances (9). The color
reactions with diazotized p—nitroaniline, biodianzotized
benzidine, diazotized sulfanilic acid and the color under
ultraviolet were similar to those obtained from known
stilbenes (18). These color reactions suggest that F62A is
a phenolic compound, possibly a stilbene.

F6-A was further characterized by NMR spectroscopic
analysis and the spectrum was translated into a moleular
structure by comparing it with the spectra of several known
stilbenes including the previous reported 2, 4, 3', 5%-
tetrahydroxystilbene (6). Since the reactions of F6-A with
chemical indicators suggested it to be a phenolic compound,
an Ar-OH group was suspected. A strong singlet at 8 7 .0

36

Table 11. The color reactions of the component F6~AE

 

 

Reagent Color
Ferric chloride-potassium ferricyanide Deep Blue
Diazotized p-nitroaniline Yellow Brown
Diazotized sulfanilic acid Dark Tan
Bisdiazotized benzidine Dark Rust
Vanillin-toluene-p-sulfonic acid Violet Red
Ferric ammonium sulfate ---
Sodium.molybdate ---

 

“Code F6-A refers to Figure 3.

37

 

'c-II-I
1

 

 

 

 

 

 

Figure 4. NMR spectrum of the component F6-A from
the heartwood extractives of Maclura pgmifera.

38

 

231:2 6111605076 1153: 147-02306 13 34 45 £1 1000 5mm
:c-z-EC-TG scan I 21 m . 295414 ame- 3=18
1867.3 “184.
r
1
1

 

   

 

  

L

leleuve 1mm,

 

 

   

In].

Figure 5. Mass spectrum of the component F6-A from
the heartwood extractives of Maclura pomifera.

39

was identified to be the signal for phenolic protons and
this identification was confirmed by a D20 test-elimination
of this peak by shaking with D20 (27). The possibility of
a -CH=CH- group was confirmed by the olefinic proton
absorption in the NMR spectrum at about 5 6.0. The
spectrum of authentic 3, 4', 5-trihydroxystilbene gave a
similar absorption pattern. Thus, the doublet at 5 5.9 was
translated into the absorption of olefinic protons. The
remaining peaks down field are rather complex and are
considered to be the aromatic signals. The peaks at
about 8 1.5 were identified as the solvent signals. The
peaks at 5 2.1 and 5 0.7 were considered to be the signals
of impurities.

After a close examination of the nature of the chemical
shifts and splitting in the NMR spectrum of 3, 4', S-tri-
hydroxystilbene, the following information was obtained:

Kinds of proton Chemical shift 5

a 5.6 (e) (d) (C) (b)

H H H H on
b 5.8 - 5.9 I

HO C:C H (a)

c 6.3 '
d 6.6 s. 6.8 H H H H on

(e) (d) (c) (b)
e 6.1 a 6.2

This information suggests that F6-A is a trans-hydroxystilbene
containing a resorcinol group. The integration indicates

that the phenolic peak contains five or six protons. Hence

40

the likely structure for F6-A from information discussed

so far is:

H H OH

I

...—..-. .
30H
or I
40H H H OH

The parent peak at m/e 260 in the mass sepctrum of F6-A
suggests a molecular weight of 260. This would indicate that
F6-A is a pentahydroxystilbene. Pentahydroxystilbene is the
most fully substituted natural stilbene so far encountered.
The previous report concerning the pentahydroxystilbene
in wood was that of 3, 4, 5, 3', 5'-pentahydroxystilbene
which was found in the ether extract of Vouacapoua macropetala
(19). The fragmentation was generally similar to those of

authentic stilbenes with the following pattern:

\O c:§:c O”:

HO \ ’

Thus, presence of a fragment peak at m/e 138 is an additional

evidence in favor of a pentahydroxystilbene. No further

41

information was gained from the mass spectrum for character-
ization.

After being sprayed with vanillin-toluene-p-sulfonic
acid, flavonoid substances containing phloroglucinol nuclei
turned a strong violet-red (23). The reaction of F6-A with
vanillin-toluene-p-sulfonic acid was a strong violet-red
similar to that produced by authentic phloroglucinol.

These results suggest that F6-A contains a phloroglucinol
group. Hence, the molecular structure of F6-A, based on the

information available, appears to be:

H OH 11 H OH

However, additional information is needed before the exact
location of the various hydroxy groups on the rings is firmly

established.

CONCLUSIONS

The heartwood of Maclura ppmifera has a very high

decay resistance.

The chemical responsible for the decay resistance

appears to be a pentahydroxystilbene.

42

LITERATURE CITED

10.

LITERATURE CITED

A.S.T.M. 1975. Alcohol-benzene solubility of wood.
Standard D-llO7-56, A.S.T.M., Philadelphia.

A.S.T.M. 1975. Ether solubility of wood. Standard
D-1108-56, A.S.T.M., Philadelphia.

A.S.T.M. 1975. Standard method of testing wood
preservatives by laboratory soil-block cultures.
Standard D-l4l3-61, A.S.T.M., Philadelphia.

A.S.T.M. 1975. Standard method for accelerated
laboratory test of natural decay resistance of wood.
Standard D-2017-63, A.S.T.M., Philadelphia.

AYRES, G. H. 1958. Quantitative chemical analysis.
Harper and Brothers, New York, 726 p.

BARNES, R. A. and N. N. GERBER. 1955. The antifungal
agent from osage-orange wood. J. Am. Chem. Soc. 7:
3259-3262.

BEHR, E. A. 1972. Development of respirometry as a
method for evaluating wood preservatives. Forest
Prod. J. 22(4): 26-31.

BEHR, E. A. 1973. Decay test methods. In wood
deterioration and its prevention by preservative
treatment, Vol. 1, Chap. 6, 217-246 (Nicholas, D. D.,
Editor), Syracuse Univ. Press, Syracuse, 390 p.

BROWNING, B. L. 1967. Methods of wood chemistry. Vol. 1,
Interscience, New York. 384 p.

ERDTMAN, H. 1955. The chemistry of heartwood

constituents of conifers and their taxonomic importance.
Intern. Congr. Pure Appl. Chem., 14: 156-180.

43

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

44

HALABISKY, D. D. and G. IFJU. 1968. Use of respirometry
for fast and accurate evaluation of wood preservatives.
Proc., A.W.P.A. 64: 215-223.

HAMILTON, L. F. and S. G. SIMPSON. 1964. Quantitative
chemical analysis. 12th Ed. Macmillan, New York, 576 p.

HART, J. H. and K. C. JOHNSON. 1970. Production of
decay resistance sapwood in response to injury. wood
Science and Technology 4: 267-272.

HART, J. H. and W. E. HILLIS. 1974. Inhibition of
wood-rotting fungi by stilbenes and other polyphenols
in Eucalyptus‘sideroxylon. Phytopath. 64: 939-948.

HAWLEY, L. F., L. C. FLECK and C. A. RICHARDS. 1924.
The relation between durability and chemical composition
in wood. Industrial and Engineering Chemistry 16: 699-
700.

HIGHLEY, T. L. 1975. Can wood-rot fungi degrade
cellulose without other wood constituents? Forest Prod.
J. 25(7): 38-39.

HILLIS, W. E. 1962. wood extractives and their
significance to the pulp and paper industries. Academic
Press, New Yerk and London, 513 p.

HILLIS, W. E. and ISHIKURA. 1968. The chromatographic
and spectral properties of stilbene derivatives. J.
Chromatog. 32: 323-336.

KING, E. E., T. J. KING, D. H. GODSON and L. C. MANNING.
1956. The chemistry of extractives from hardwoods.

Pt. XXVIII. The occurrence of 3, 4, 3',5'-tetrahydroxy
and 3,4,5,3',5'-pentahydroxyl-stilbene in Vouacapoua
species. J. Chem. Soc. 1956: 4477—4480.

LOMAN, A. A. 1970. Bioassays of fungi isolated from
Pinus contorta var. latifolia with pinosylvin, pino-
syIvIn monomethyl ether, pinobanksin, and pinochembrin.
Can. J. Bot. 48: 1303-1308.

MORRISON, R. T. and R. N. BOYD. 1973. Organic
chemistry. 3rd Ed. Allyn and Bacon, Boston, 1258 p.

PETERSON, C. A. and E. B. COWLING. 1964. Decay
resistance of extractive-free coniferous woods to white-
rot fungi. Phytopath. 54: 542-547.

23.

24.

25.

26.

27.

28.

29.

30.

31.

4S

ROUX, D. G. and A. E. MAIHS. 1960. Selective spray
reagents for the identification and estimation of
flavonoid compounds associated with condensed tannins.
J. Chromatog. 4: 65-74.

RUDMAN, P. 1963. The cause of natural durability in
timber. Pt. XI. Some tests on the fungi toxicity of
wood extractives and related compounds. Holzforschung

RUDMAN, P. 1965. The cause of natural durability in
timber. Pt. XVIII. Further notes on the fungi toxicity
of wood extractives. Holzforschung 19: 57-58.

SCHEFFER, T. C. and E. B. COWLING. 1966. Natural
resistance of wood to microbial deterioration. Am.
Rev. Phytopath. 4: 147-170.

SILVERSTEIN, R. M., G. C. BASSLER and T. C. MORRILL.
1974. Spectrometric identification of organic
compounds. 3rd Ed. John Wiley and Sons, New York,
340 p.

SMITH, R. S. 1969. WOod preservative toxicity
evaluation using wood weight loss and fungal respiration
methods. WOod Science 2(1): 44-63.

SMITH, R. S. 1975. Automatic respiration analysis of
the fungitoxic potential of wood preservatives, including
an oxathin. Forest Prod. J. 25(1): 48-53.

TOOLE, E. R. 1975. Oxygen utilization by decay fungi
for the evaluation of wood preservatives. Forest Prod.
Jo 25(7): 46-68e

U. S. Forest Products Laboratory. 1961. Comparative
decay resistance of heartwood of different native
species when used under conditions that favor decay.
Tech. NOte No. 229. Revised May 1961.

APPENDIX

46

 

 

 

 

 

 

 

 

' Ivr L' ‘fil ‘1 *‘J"'*fi' *J'rfil [a
I '—I ' I ' I ' +1 I

h - u - I. O.
«a-

1 1 1 A 1 1 1 1 A J

A 1 1 I .1 11:. 1111 If 1 .L-4+L+- A I QI
~‘ 0 I. I

 

Appendix A. NMR spectrum of cis stilbene ( Sample
was purchased from Pfaltz and Bauer, Inc. Flushing,

N. Y. ).

47

 

 

" L 'J'IT '1 T 'I‘fv I "'If' I'VT'J""Ifi
.‘ I v ‘ v - r v
D C - - l'. ‘ TI.
-
I

 

 

 

 

 

 

 

 

Appendix B. NMR spectrum of trans stilbene ( Sample
was purchased from Eastman Kodak Co. Rochester, N. Y. ).

48

 

 

-rv-w—

 

 

 

 

 

Appendix C. NMR spectrum of p-hydroxystilbene ( Sample
was purchased from Pfaltz and Bauer, Inc. Flushing, N. Y. )

49

 

 

 

 

 

 

 

 

 

Appendix D. NMR spectrum of 3,4',5-trihydroxystilbene
( Sample furnished courtesy Dr. W. E. Hillis, Forest
Products Laboratory, Division of Applied Chemistry,
CRISO, South.Melbourne, Australia ).

SO

 

 

 

 

I.
P-
I

 

—I
I
P
I
: n
P
iii
a IWF

- l .1 - 1
- - l 4.1.. 4. 1. 1r,., - - - I 1.... - - I ., - -
f as De le :0

Appendix E. MR spectrum of 3,5,2',4'-tetrahydroxy-
stilbene ( Sample furnished courtesy Dr. W. E. Hillis,
Forest Products Laboratory, Division of Applied Che-
mistry, CRISO, South Melbourne, Australia ).

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