EXTRACTIOn of Organic Chemicals of Prosopis Juliflora

EXTRACTION OF ORGANIC CHEMICALS
FROM MESQUITE
by
SHOU-JEN R. CHEN, B . S .
A THESIS
IN
CHEMISTRY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
/Dekn of/ciJfe GrAfiuate School

December, 1981
)Jd>, l?'?'^
ACKNOWLEDGMENTS
I would like to express my special thanks to my research advisor

and committee chairman. Dr. Richard A. Bartsch, for his encouragement,
valuable guidance and assistance.
Without his generous help this
research would have been impossible.
I would also like to thank
Dr. John N. Marx for the helpful suggestions and advice during the
research period, and to Dr. John A. Anderson for serving as member
of my committee and spending time in analyzing and evaluating the
thesis.
I want to extend my thanks to Mr. Alan Croft for helping me
correct the manuscript.
Finally, I would like to express my sincerest
thanks to my wife Feng-Ying A. Chen for her patience, understanding

and most importantly her continued faith in me.
11
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
ii
LIST OF TABLES
vi
LIST OF FIGURES
vii
CHAPTER
I. GENERAL INTRODUCTION
CHAPTER II.
Background
Literature and Previous Mesquite Research
Long Term Proj ect Goals
EXPERIMENTAL
General Methods
Proton Magnetic Resonance

Spectroscopy
Infrared Spectroscopy
Ultraviolet Spectroscopy
High Pressure Liquid Chromatography
Column Chromatography
Gas Chromatography
Thin Layer Chromatography
Preparative Thin Layer

Chromatography
Extraction of Organic Compounds
from Mesquite Plants
Source of Mesquite Plants

Preparation of Segregated
Mesquite Plant Parts
Extraction of Different Parts
of the Mesquite Plant
iii
10
n
Page
12
Preliminary Separation
12
Gradient Solvent Separation
13
Final Separation
13
Properties of the Isolated Compound 1^
15
Decomposition of Compound 1_
15
Treatment of Compound _1 with Base
18
Structural Determination of Compound 1_
19
Formation of Trimethylsilyl
CHAPTER III.
Ether Derivative
19
Formation of Ester Derivative
20
Shift Reagnet Experiment
20
RESULT AND DISCUSSION
22
Extraction and Spectral Analysis

of the Crude Extracts
22
Heartwood
22
Seasonal Changes
26
Heartwood from Chemically

Defoliated Mesquite
27
Other Parts of the Mesquite Plant
28
Chromatography and Spectral Analysis

of the Separated Material
30
Properties of the Isolated Compound 1^
35
35
Treatment of Compound 1^ with Base
39
IV
Structural Determination of Compound 1
40
Page
CHAPTER IV.
Silylation of Compound 1_
40
Esterification of Compound 1^
42
Shift Reagent Experiment
44
Proposed Structure of Compound 1_
45
SUMMARY AND SUGGESTIONS FOR FURTHER RESEARCH
52
Summary
52
Suggestions for Future Research
54
LIST OF REFERENCES
APPENDIX A
B
55
HPLC Chromatograms
56
Proton Magnetic Resonance Spectra
75
Infrared Spectra.
89
Ultraviolet Spectra,
96
LIST OF TABLES
Page
1. Extraction of Honey Mesquite
2.
Chromatographic Conditions for HPLC Analysis
3.
Gradient Solvent System
14
4.
Extraction of Chipped Mesquite Heartwood
23
5.
Extraction of Chipped Mesquite Sapwood, Chipped Bark,

and Shredded Leaves
29
6.
Solubility of Compound 1_ in Various Solvents
7.
Effect of Shift Reagent upon the Chemical Shifts of

Absorptions in the PMR Spectrum of Acetylated _1
46
Shift in PMR Absorptions Cause by the Addition of

Shift Reagent
47
8.
VI
36
LIST OF FIGURES
Page
1.
Soxhlet Extraction Apparatus
11
2.
Flow Scheme A
16
3.
Flow Scheme B
17
4.
Flow Scheme C
18
5.
33
6.
Trimethylsilyl Ether Derivative of (+)-Catechin and Its

PMR Spectrum
49
7.
Proposed Structure of Compound 1^ and Its Derivatives
8.
Possible Stereo Structures of Compound 1^
51
9.
3,3;4;7,8-Pentahydroxyflavan
53
HPLC-1, Crude Mesquite Heartwood Extract

HPLC-2, Fraction B from Gradient Solvent Column
Chromatography
57
10.
11.
12.
50
58
HPLC-3, Fraction D from Gradient Solvent Column

Chromatography
59
HPLC-4, Major Portion of Fraction C from Gradient

Solvent Column Chromatography
60
14.
HPLC-5, HPLC Chromatogram of Compound 2^
61
15.
HPLC-6, Photodecomposition of Compound 3^(1)
62
16.
HPLC-7, Photodecomposition of Compound 2.(2)
63
17.
HPLC-8, Photodecomposition of Compound j^(3)
64
18.
HPLC-9, Photodecomposition of Compound ]^(4)
65
19.
HPLC-10, Photodecomposition of Compound 1^(5)
66
20.
HPLC-11, Compound 1^ before Irradiation with the

Light Source
67
13.
21.
HPLC-12, Compound 1^ after Irradiation with the

Light Source for 24 Hours
vii
68
Page
69

70
24.
HPLC-15, Chromatogram of Sample 3
71
25.
HPLC-16, Sample 3 after Being Kept in the Dark

for 72 Hours
72
HPLC-17, Sample 3 after Irradiation with the

73
22.
23.
26.
27.
HPLC-18, Sample 3 after Adding One Drop of

Concentrated HCl
74
28.
PMR-1, Crude Mesquite Heartwood Extract
76
29.
PMR-2, Non-Polar Fraction of Mesquite Heartwood Extract....
77
30.
PMR-3, Polar Fraction of Mesquite Heartwood Extract
78
31.
PMR-4, PMR Spectrum of Compound 1^
79
32.
PMR-5, PMR Spectrum of Mesquite Sapwood Extract
80
33.
PMR-6, Trimethylsilyl Ether Derivative of Compound 1_
81
34.
35.
PMR-7, Acetate Derivative of Compound 1_

PMR-8, First Addition of Shift Reagent to Acetate
Derivative of Compound 1^
82
83
PMR-9, Second Addition of Shift Reagent to Acetate

84
PMR-10, Third Addition of Shift Reagent to Acetate

Derivative of Compound _1
85
PMR-11, Fourth Addition of Shift Reagent to Acetate

86
PMR-12, Fifth Addition of Shift Reagent to Acetate

87
PMR-13, Sixth Addition of Shift Reagent to Acetate

Derivative of Compound _1
88
36.
37.
38.
39.
40.
viii
Page
41.
IR-1, Crude Mesquite Heartwood Extract

IR-2, Non-Polar Fraction of Mesquite Heartwood Extract
90
42.
IR-3, Polar Fraction of Mesquite Heartwood Extract
91
43.
IR-4, IR Spectrum of Compound J^
92
44.
IR-5, IR Spectrum of Mesquite Sapwood Extract
93
45.
IR-6, Trimethylsilyl Ether Derivative of Compound ]^
94
46.
47.
IR-7, Acetate Derivative of Compound 1^

UV-1, UV Spectrum of Compound 1.
95
97
48.
UV-2, Compound 1^ after Irradiation with the

98
UV-3, Compound 1^ after Irradiation with the

"
99
49.
50.
UV-4, Compound 1_ after Irradiation with the

100
51.
UV-5, Base and Acid Treatment of Sample 4
101
52.
UV-6, Acid Treatment of Sample 4
102
53.
UV-7, Base Treatment of Sample 4
103
IX
CHAPTER
GENERAL INTRODUCTION
Background
The control of mesquite proliferation is a major problem in the

West Texas area.
Destruction and removal of mesquite from rangeland
and farmland are expensive if the only objective is to destroy the

undesirable brush.
varied.
fuel.
However, potential uses of mesquite are many and
Presently, mesquite is being used primarily as a source of

Occasionally mesquite has been used for fenceposts, but there
does not appear to be any significant commercial utilization of

mesquite for this purpose at this time.
If economically valuable
products can be obtained from mesquite, its removal would be considerably more attractive.
An almost totally unexplored area of potential mesquite utilization involves the isolation and use of the organic compounds, other
than carbohydrates, that are present in the mesquite plant.
Although
mesquite contains a relatively high amount of material which may be

extracted with solvents.
little is known concerning the composition
of the organic compounds which make up such extracts.

Currently, the feasibility of utilizing treated mesquite for
animal feed is being evaluated by the Chemical Engineering Department
at Texas Tech University.
In connection with these investigations.
it would be very beneficial to identify compounds present in the

mesquite plant or produced by chemical treatment of mesquite which
may be potential digestion inhibitors.
Literature and Previous

Mesquite Research
The first report of mesquite wood extraction was published in

2
1922.
G. J. Ritter and L. C. Fleck
performed the extraction on un-
specified proportions of mesquite sapwood and heartwood.

tion results have been cited numerous
The extrac-
times between 1922 and 1972.
In 1972, I. S. Goldstein and A. Villarreal published a paper

entitled "Chemical Composition and Accessibility to Cellulose of
3
Mesquite Wood."
In this paper,
the relative amounts of chemicals
which may be extracted from mesquite sapwood and heartwood were

reported for the first time.
Table 1.
Extraction of Honey Mesquite
Extraction Yield, Percent of

Air-Dry Sample
From Sapwo od
Fnom Heartwood
Water
6.0%
5.8%
Benzene-ethanol
4.4%
12.2%
Benzene-ethanol-water
10.4%
18.0%
1% NaOH
20.5%
28.9%
Extraction Solvent
The mesquite wood was extracted according to the procedures in

4
Browning,, but the chemical composition of the extracts was not
determined.
"In 1975, the State of Texas appropriated funds to the College
of Agricultural Sciences at Texas Tech University to investigate
methods for commercial use of mesquite which had been harvested from
ranchlands."
For the potential use of mesquite as roughage in
animal feed, the Chemical Engineering Department at Texas Tech University investigated a number of thermochemical pretreatments with the
goal of improving the digestibility of harvested mesquite.
These
included the treatment of mesquite with sulfur dioxide, sulfuric acid,

elemental sulfur and methanol.
The sulfur dioxide treatment has
shown the most promise for increasing the J^ vitro digestibility of

mesquite wood.
This significant increase in the _in vitro digesti-
bility may allow treated mesquite wood to be used as a roughage substitute in animal feed.
Isolated reports of mesquite wood extraction using various
solvents demonstrate that appreciable amounts of organic compounds
2 3
may be removed. *
However, in no instance has the identity of the
extracted organic compounds been determined.

During the summer of 1979, Gaul and Bartsch
prepared a report
entitled "Survey of the Literature Pertaining to the Extraction of

Organic Chemicals from Mesquite".
Since information up to 1969 was

Q
contained in the book Literature on Mesquite
(edited by Joseph L.
Shuster), this survey treated only the post-1969 literature on the

subject.
The goal of the report was to summarize the existing data
4
concerning organic compounds which can be extracted from mesquite
(Prosopis juliflora).
A few references dealing with the isolation of certain compounds
or classes of compounds such as tannins, waxes, and flavonoids from
specific mesquite plant parts or unspecified mesquite sources were
located.
However, the data was found to be extremely fragmentary and
provided little basis for a judgement concerning whether or not

economically attractive non-carbohydrate organic chemicals could be
extracted from mesquite, in general, or, specifically, the heartwood
of mesquite.
Gaul and Bartsch also performed an exploratory study of the
extraction and identification of non-carbohydrate compounds from
mesquite.
In this initial effort, shredded whole mesquite plants
(Prosopis juliflora) from the same source utilized in the chemical

treatment of mesquite for animal feed were used.
With a varity of
solvents, organic materials were extracted from the shredded whole

mesquite and were shown to be mixtures of mostly waxes and carbohydrates by spectroscopic methods.
It was also found that the amount of extracted organic compound
was very sensitive to the weathering which the mesquite had experienced.
From this observation, it was concluded that the source of mesquite
used in these preliminary extraction studies was unsuitable because
potentially interesting organic compounds had probably disappeared
during the weathering process.
Long Term Project Goals
The specific objective of this research is to continue the

investigation of non-carbohydrate organic chemicals which may be
extracted from mesquite.
Attention is to be focused upon freshly
harvested mesquite plants.
In addition, separated plant parts
(particularly the heartwood) is to be used rather than the entire

mesquite plant.
Long term project goals are centered around two main subjects.
The first objective is an assessment of the feasibility of obtaining
useful non-carbohydrate organic compounds from mesquite.
If unique
or rare organic compounds are found to be present in reasonable

amounts, economical extraction methods will be explored.
The second
goal is a search for non-carbohydrate organic compounds in mesquite

which might have an adverse effects (ie. digestion inhibitors) upon
the use of untreated or treated mesquite as a source of roughage in
animal feed.
CHAPTER
II
EXPERIMENTAL
General Methods
Proton Magnetic Resonance Spectroscopy

Proton magnetic resonance (PMR) spectra were measured with a
Varian EM 360 nuclear magnetic resonance spectrometer.
Samples were
dissolved in various deuterated solvents:

Acetone-d, (CD^COCD-, Aldrich Chemical Co.)
Chloroform-d^ (CDC1-, Norell Chemical Co., Inc.)
Carbon tetrachloride (CCl,, Norell Chemical Co., Inc.)
All PMR spectral data are reported using the 5 scale (parts per
million, ppm) with tetramethylsilane (Norell Chemical Co., Inc.) as
an internal standard (0.0 ppm).
Infrared Spectroscopy
The samples were examined using a Perkin-Elmer Model 457 infrared
spectrophotometer or a Beckman Acculab 8 infrared spectrophotometer.
The majority of the samples were prepared as thin films between NaCl
plates.
In a few cases, solid samples were prepared as KBr pellets.
All infrared (IR) spectral data are reported in wavenumbers (cm ) .
Ultraviolet Spectroscopy
Ultraviolet spectra (UV) were recorded with a Gary Model 17
ultraviolet-visible spectrophotometer.
In all cases, methanol
(anhydrous, MCB, Omnisolv) was used as the solvent.
All UV spectral
data are reported in wavelength units (nm).

High Pressure Liquid Chromatography
High pressure liquid chromatography (HPLC) was performed on a
Waters Associates Model 244 high pressure liquid chromatograph
equipped with a Model 440 ultraviolet absorbance detector
( \= 254 nm) and a Waters Associates Model 660 solvent programmer.
HPLC analysis utilized a Waters Associates y-Bondapak C-18 column.
The chromatographic conditions are given in Table 2.
In all cases,
solutions of samples and standards were prepared using methanol

(anhydrous, MCB, Omnisolv) as the solvent.
All column chromatography was performed using silica gel
(60-200 Mesh, chromatographic grade, Sargent-Welch Scientific
Company) as the packing material.
Chromatographic solvents and
their sources were: methanol and methylene chloride (MCB, Omnisolv);

diethyl ether, carbon tetrachloride, benzene and acetone (distilled
reagent grade chemicals).
Gas Chromatography
Gas chromatographic (GC) cUialysis was conducted on an Antek
Model 400 thermal conductivity gas chromatograph, A 5 foot x 1/4
8
Table 2. Chromatographic Conditions
for HPLC Analysis
High pressure liquid
chromatograph
Waters Associates, Inc.,

Model 244
Detector

Model 440 ultraviolet
absorbance detector,
X = 254 nm.
Pumps

Pump A, Model M 6000A
Pump B, Model M-45 solvent
delivery system
Programmer

Model 600 solvent programmer
Column

U-Bondapak C-18 reverse
phase column
Mobile phase
Acetonitrile (Omnisolv, MCB)

in triply distilled water:
Initial: 10%
Final: 60%
Programmer curve: #11
Program time: 8 minutes
Flow rate: 1.5 ml/min
Temperature: ambient
Recorder
SOLTEC
Model 252A
Chart speed: 0.5 inch/min
or
Linear Instruments Co.
Model 252 INT
Chart speed: 1.0 cm/min
(absorbance versus
retention time)
inch aluminum tubing column packed with Chromosorb 104 (Applied

Science Laboratories, Inc.) was used.
The carrier gas was helium.
Thin Layer Chromatography

Thin layer chromatographic analysis (TLC) was performed using
precoated silica gel GF glass plates for TLC (Analtech Inc.).
In some cases, Eastman Chromagram Sheets for TLC (Eastman Kodak
Company) were used.
Preparative Thin Layer Chromatography
The adsorbent material used was GF 254, type 60 silica gel
(E. Merck & Co.).
In making the preparative thin layer chromato-
graphic plates, the adsorbent material was mixed with distilled

water in a two to one ratio (W/W) and stirred with a glass rod to
make it a well-mixed slurry.
The slurry was then spread evenly on
a clean glass plate (25 cm x 25 cm) and allowed to air dry for
three days.
Samples were applied to the plates using capillary
micropipets, and developed in a chromatographic chamber.
Extraction of Organic Compounds

from Mesquite Plants
Source of Mesquite Plants

Mesquite plants were obtained locally from the field which is
across the road from Texas Tech University School of Medicine.
The plants chosen were those having trunk diameters of at least
2 inches when measured at ground level. Segments about 2 feet long
10
were cut with a hand saw.
The chemically-defoliated mesquite was obtained from the farm
of Mr. D. E. Sosebee which is two miles east of Anson, Texas.
The trees had been sprayed on approximately July 1, 1980 with
Tordon 225 (1 gallon of Tordon 225/100 gallons of water, Tordon
225 is a 1:1 mixture of the triisopropanol amine salt of 4-amino-3,
5,6-trichloropicolinic acid and the propylene glycol butyl ether
esters of 2,4,5-trichlorophenoxyacetic acid).
The mesquite trees
were sprayed until the spray dripped from the leaves. The chemically-defoliated mesquite was harvested in early January of 1981.
Preparation of Segregated Mesquite
Plant Parts
The freshly-harvested mesquite plants were separated into
component parts (trunk, branches, and leaves) in the laboratory
using a hand saw. The trunk portion was cut further into 25 cm
long sections.
Samples which were not subjected to extraction
soon after harvesting were stored in polyethylene bags and kept in

the dark.
When being prepared for extraction, the trunk section was
separated into bark, sapwood, and heartwood constituents using a
razor knife.
The segregated materials were then chipped into thin
shavings (approximate size, 1/2 cm x 1/2 cm x 1/16 cm) with a

razor knife.
11
Extraction of Different Parts
of the Mesquite Plant
Segregated mesquite plant parts were extracted using a
Soxhlet extraction apparatus (Figure 1).
T-TUBE
>f
-r^ 1<DRYING TUBE
(fill with CaCI^)
COOLING WATER -
--V^COOLING WATER
THIMBLE
SOLVENT
Figure 1.
ROUND BOTTOMED
FLASK
Soxhlet Extraction Apparatus
12
The thin shavings of the separated plant part were weighed into
the cellulose extraction thimble (single thickness, 60 mm x 180 mm
or 43 mm X 123 mm or 20 mm x 100 mm, Whatman) of a continuous
Soxhlet extraction apparatus which was protected from moisture
absorption by a calcium chloride filled drying tube. Either one
pure solvent or a mixture of solvents was used for each extraction.
In most cases, the extraction period was 72 hours; but in few cases,
the extraction period was 24 hours. At the completion of the
extraction, the solution was transferred into a tared flask and
the solvent was evaporated in vacuo. A listing of the material
extracted, the solvent employed, the extraction yields is provided
in tabular form in the Results Section.
Preliminary Separation
The crude heartwood extracts were initially separated into
non-polar and polar portions using a 60 cm x 5 cm column of silica
gel.
The crude heartwood extract was dissolved in minimum quantity
of methanol and applied to the top of column.
The eluting solvents
were sequentially 100 ml each of carbon tetrachloride, benzene, and

methylene chloride.
The total eluent was combined and the solvents
were evaporated in vacuo.

spectroscopy.
The residue was analyzed by PMR and IR
The material remaining in the column was eluted with
200 ml of methanol.
The solvent was removed from the eluent i^
vacuo and the residue was examined by PMR and IR spectroscopy.
13
The quantities of non-polar and polar portions of the crude
heartwood extracts which were obtained are presented in the Results
Section.
PMR and IR spectra are given in Appendix Section.

Column chromatography on silica gel with a gradient eluting
solvent system was used to separate the polar portion of heartwood
extract into various fractions.
Thirteen different mixtures of solvents were prepared (Table 3).
A glass column (60 cm x 3 cm) packed with silica gel was used.
The polar portion of the heartwood extract was dissolved in minimum
amount of methanol and applied to the top of the column. The
column was connected with a separatory funnel through which the
eluting solvents were sequentially added to the column to effect
the separation.
Ten ml fractions were collected in 2 dram, shell
cap vials. After two hundred and ten fractions had been collected,
a 500 ml round bottomed flask was used to collect the remaining
solution.
The solvent was allowed to evaporate from the fractions
at room temperature.
The results of this chromatography are pre-
sented in the Results Section.
Final Separation
For the final separation, a 60 cm x 2 cm glass column packed
with silica gel was used.
The sample was dissolved in 5 ml of
acetone and transferred from a vial to a 25 ml round bottomed flask

and 5.0 g of silica gel was added.
The solvent of the resulting
slurry was removed completely in vacuo.
Thus, the sample to be
14
Table 3.
Solvent Number
Gradient Solvent System
CH CI
CH OH
Percent
Total
(ml)
(ml)
^ ^^3^^
(ml)_
200
200
190
10
200
180
20
10
200
160
40
20
200
140
60
30
200
120
80
40
200
100
100
50
200
80
120
60
200
60
140
70
200
10
40
160
80
200
11
20
180
90
200
12
10
190
95
200
13
200
100
200
15
chromatographed was coated on a small portion of silica gel which
was then applied to the top of the column.
The eluting solvent
was a mixture of methanol-methylene chloride (1:9, V/V). Five ml

fractions were collected using 2 dram, shell cap vial.
The solvent
was allowed to evaporate from the fractions at room temperature.

All fractions were examined by HPLC.
The chromatographic results are presented in the Results
Section.
In Appendix A, some of the HPLC chromatograms are given.
Properties of the Isolated

Compound 1
Compound 1_ is the major component in mesquite heartwood polar
extract which was isolated by column chromatography.
A small quantity of Compound 1^ was dissolved in methanol
(anhydrous, MCB, Omnisolv) and kept in a 1/2 dram, screw cap vial.
In order to study the suspected photodecomposition, this sample was
examined by HPLC soon after being prepared and the vial was then
capped and left on the laboratory bench (exposed to overhead
fluorescent light and reflected sunlight) for three weeks before
being analyzed by HPLC again.
The sample was then returned to the
laboratory bench and was examined by HPLC every two weeks for a
period of two months.
A more detailed study of the photodecomposition of Compound 1
was performed according to Flow Scheme A in Figure 2.
16
Two samples of Compound 1 were prepared as methanol solutions
by the method given above.
Sample 1 was examined by UV spectroscopy,
while sample 2 was analyzed by the HPLC. Both samples were examined
immediately after preparation and were then irradiated by a 60 W
light bulb placed 10 cm from the screw cap vial.
Periodically
(every 24 hours), these samples were analyzed either by UV or HPLC

for a period of 72 hours.
Sample 1 (UV-1)
Sample 2
(HPLC-11)
Irradiated with
light source
(UV-2)^^
24 hours
-> (HPLC-12)
(UV-3) <-
48 hours
> (HPLC-13)
(UV-4) <-
72 hours
-> (HPLC-14)
Samples of Compound 1_ (prepared by the method given above) were

freshly prepared every 24 hours, and served as standards in all
UV spectra (curve A in UV-2, UV-3, and UV-4).
Sample 2 has higher concentration then Sample 1.
Figure 2. Flow Scheme A
17
A small amount of Compound 1_ was dissolved in methanol
(sample 3) and the solution was placed in three 2 dram, shell cap
vials.
The contents of each vial were analyzed by HPLC according
to Flow Scheme B in Figure 3.
Sample 3
Kept in the
dark for
72 hours
(HPLC-16)
(HPLC-15)
Irradiated with
light source
for 72 hours
(HPLC-17)
Add one drop of

concentrated
HCl^
(HPLC-18)
This sample had slightly decomposed.

The light source was a 60 W light bulb place 10 cm from the vials
Reagent grade concentrated HCl from MCB.
Figure 3.
Flow Scheme B
18
HPLC chromatograms from the photodecomposition study are presented in Appendix A.
UV spectra may be found in Appendix D.
The photodecomposition of Compound 1^ in methanol is discussed in

the Results and Discussion Section.
Treatment of Compound 1^ with Base
Another sample of Compound 1^ in methanol (sample 4) was prepared in the same manner as before and was examined by UV spectroscopy according to Flow Scheme C in Figure 4.
Sample 4 (UV-5)
\f
Add 5 drops of
NaOH solution
Add 3 drops of
HCl solution
Add 3 drops of
NaOH solution
Add 5 drops of
HCl solution
Add 10 drops of
HCl solution
Add an excess
(20 drops) of
NaOH solution
(UV-5)
(UV-6)
-2
The concentration of the NaOH solution was 2.9 x 10
M
b
2
The concentration of the HCl solution was 2.9 x 10
M
Figure 4.
Flow Scheme C
(UV-7)
19
The HPLC chromatograms and UV spectra which resulted from the
experiments conducted with samples 3 and 4 are given in the Appendix
Section.
The effects of acid and base treatment of a methanol
solution of Compound _1^ are discussed in the Results and Discussion

Section.
Structure Determination
of Compound 1_
Formation of Trimethylsilyl
Ether Derivative
A sample (0.328 g) of Compound 1_ was dissolved in 1.0 ml of
pyridine (solvent) in a 10 ml round bottomed flask and a reflux
condenser was attached.
A drying tube filled with calcium chloride
was connected to the reflux condenser to prevent moisture from

9
entering the system. Then 1.0 ml of hexamethyldisilanzane (HMDS,
Pierce Chemical Co.) and 0.5 ml of trimethylchlorosilane (TMCS,
Pierce Chemical Co.) were added and the solution was refluxed for
3.5 hr. At the end of reflux period, the reaction mixture was
allowed to cool for 0.5 hr.
removed in vacuo.
Solvent and excess reagents were then
The residue was subjected to hard vacuum for 4 hr
to remove the last traces of volatile materials.
The residue
(0.388 g) was a brown liquid, and show one spot in TLC.

The PMR and IR spectra of the resulting residue were obtained
and appear in Appendices B (PMR) and C (IR), respectively.
20
Formation of Ester Derivative
Compound 1^ (0.224 g) was refluxed for 0.5 hr with 1.0 ml of
pyridine (solvent) and 0.5 ml of acetic anhydride. At the end of
the reflux period, the reaction mixture was allowed to cool for
0.5 hr and was then transferred to a 60 ml separatory funnel.
A 10 ml quantity of distilled water, 20 ml of diethyl ether and
10 ml of chloroform were added and the reaction mixture was extracted
for 5 min.
After the layers separated, the aqueous layer was removed
and discarded.
The organic layer was than washed with three portions
of distilled water and then dried with MgSO, . The resulting organic
layer was transferred to a 50 ml round bottomed flask and the
solvents were evaporated in vacuo.
The residue (0.333 g) showed one
spot in TLC and was analyzed by PMR and IR spectroscopy.
The effec-
tiveness of this ester-forming reaction is discussed in the Results

and Discussion Section.
The PMR and IR spectra are given in Appen-
dices B (PMR) and C (IR).

The ester derivative of Compound 2. (0.0646 g) was dissolved
in 0.3723 g of CDCl^ and analyzed by PMR.
The shift reagent was
Resolve-Al Eu(F0D)^(99 + %, Aldrich Chemical Co.).
The shift rea-
gent was added to the PMR sample tube in small quantities (10-20 mg
each time).
After each addition of the shift reagent, the PMR
sample tube was weighed and the PMR spectrum was taken.
of each addition was calculated by difference.
shift reagent were added during the experiment.
The weight
Six portions of
21
The PMR spectra are presented in Appendix B.
The influence of
the shift reagent upon the Pl-IR spectrum of Compound _1 is discussed

in the Results and Discussion Section.
CHAPTER III
RESULTS AND DISCUSSION
Extraction and Spectral Analysis

of the Crude Extracts
Heartwood
Chipped mesquite heartwood was extracted using a Soxhlet
extraction apparatus.
The yields of the extracted material (based
upon the original weight of the heartwood sample) and the extraction
conditions are reported in tabular form in Table 4.
Since the project goal was to obtain useful non-carbohydrate
organic compounds from mesquite, diethyl ether was used initially
as the extracting solvent.
It was thought that this solvent would
be most appropriate for extracting the non-carbohydrate organic

compounds from the heartwood while leaving the ether-insoluble
carbohydrates behind.
Using diethyl ether, it was found that maximum extraction
yields were obtained when the chipped heartwood was extracted
within a few days following the harvesting of the mesquite plant.
Thus, extraction of mesquite heartwood samples which had been stored
in plastic bags for four or eight weeks gave much lower extraction
yields than when a portion of the sample was extracted one week
after harvesting (compare Experiments 2 and 4 with Experiment 1
22
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25
in Table 4). According to this result, it appears that under the
conditions of extended storage, the organic chemicals in the chipped
heartwood are either lost by evaporation or converted into unextractable forms (possibly by air oxidation or decomposition).
Extraction periods were also compared.
The heartwood samples
in both Experiment 3A and Experiment 4 were obtained on the same

day and stored in the same place. Although three days elapsed
between the two extraction experiments, the yield change caused by
this variation of storage time was expected to be small. The yield
of extracted material in Experiment 4 (72 hour extraction) was
found to be two times larger than the yield of extracted material
in Experiment 3A (24 hour extraction).
Thus, extraction time is
demonstrated to play an important role in determining the extraction

yields.
In an effort to increase the yields of extracted material,
extractions were conducted with benzene, ethanol (95%), and mixtures
of these two solvents (Experiments 9-13 in Table 4). With ethanol,
benzene-ethanol (1:2), and benzene-ethanol (2:1), extraction yields
ranging from 10.6-13.1% were obtained which surpass the highest
yield of 4.1% (Experiment 1) obtained with diethyl ether as the
extraction solvent. When ethanol-containing solvent mixtures were
used, a higher ethanol concentration produced enhanced extraction
yields (compare Experiments 10 and 11 and Experiments 12 and 13).
Infrared and proton magnetic resonance spectra of the heartwood extracts obtained with diethyl ether, ethanol, benzene, and
mixtures of ethanol and benzene were found to be quite similar
26
(PMR-1, IR-1).
This indicates that the composition of the various
extracts remains invariant even though the extraction solvent was

changed.
The mesquite heartwood extracts obtained by diethyl ether
extraction of mesquite harvested at various times of the year were

also found to have very similar PMR and IR spectra.
The infrared spectrum of the mesquite heartwood extract (IR-1)
shows a broad band at 3600 cm~ -3000 cm""^ indicating that the
extracted material contains the OH group functionality.
weak absorption at 1750 cm
^C=0 functionality.
A rather
indicates the possible presence of the
The proton magnetic resonance spectrum (PMR-1)
showed several peaks in the region 6.0-7.0 ppm which suggests the
presence of aromatic hydrogens.
Strong PMR absorption at 1.0-1.5 ppm
indicated the presence of alkyl chains in the mesquite heartwood

extract.
Seasonal Changes
Even when chipped heartwood samples were extracted only a few
days after the mesquite had been harvested, the yields of extractable non-carbohydrate organic chemicals were found to vary (compare
Experiments 1, 5-8 in Table 4).
This variation may be ascribed
to seasonal changes in the amount of extractable organic chemicals

present in the mesquite heartwood.
Much higher extraction yields
were obtained from mesquite heartwood samples for which the mesquite
was harvested in the fall (Experiment 1) or spring (Experiment 8)
compared with the winter (Experiments 5-7).
Taking into account the seasonal variations in the amount of
27
material which may be extracted from the heartwood, extraction
yields were found to be reproducible.
Thus, diethyl ether extrac-
tion of two different heartwood samples gave yields of 4.0% and 4.1%
(Experiments 1 and 8). Similarly, extraction of two different
heartwood samples with benzene-ethanol (1:2) gave yields of 10.6%
and 11.7% (Experiments 9 and 11).
Heartwood from Chemically
Defoliated Mesquite
The heartwood samples used for Experiments 14 and 15 were
obtained from the same chemically-defoliated mesquite plant. The
extraction sample for Experiment 14 was heartwood that came from
the portion of the plant that was above the ground, while the extraction sample for Experiment 15 was the heartwood that was in the
portion of the plant that was below the ground.
The extraction yields were 7.9% and 12.5%, indicating that the
extractable organic chemicals in chemically-defoliated mesquite
heartwood were not affected greatly by the chemical spray (compare
the Experiments 14, 15 and 10 in Table 4).
Although 6 months
elapsed between the time the chemical was sprayed and the mesquite
was harvested, the yield of extracted material in Experiment 15 was
found to be similar to the yield of extracted material in Experiment
10.
The lower extraction yield in Experiment 14 was thought to
result from some decomposition of the above ground mesquite heartwood components after chemical defoliation.
PMR spectroscopic analysis of the extracts showed that both
extracts from the chemically-defoliated mesquite were quite similar
28
to the various extracts from freshly harvested mesquites.
Other Parts of Mesquite Plant
Limited data were also obtained from the extraction of chipped
sapwood, chipped bark, and shredded leaves with diethyl ether in a
Soxhlet extraction apparatus. Results are recorded in Table 5.
Comparison of extraction data for the various components of
mesquite obtained from the same harvesting reveals that with diethyl
ether, more material can be extracted from the heartwood (Experiment
1 in Table 4) than from the sapwood, bark, or leaves (Experiments 16,
18, and ,9 in Table 5).
Chromatographic and spectroscopic examina-
tion of the extracted materials (Experiments 16-19 in Table 5)

revealed considerable differences in gross compositions of the
extracts obtained from the different mesquite plant parts.
Since primary emphasis was placed upon the heartwood extracts,
very limited information was obtained concerning the nature of the
sapwood, bark, and leaf extracts. Visual examination of the leaf
extract (green and waxy material) indicated that a considerable
amount of chlorophyll and wax were present.
Infrared (IR-5) and proton magnetic resonance (PMR-5) spectra
of the sapwood extract (Experiment 16 in Table 5) showed the
absence of presumed phenolic compounds and the presence of mostly
non-aromatic hydrocarbons.
29
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30
Chromatography and Spectral Analysis
of the Separated Material
Prior to a preliminary separation of the heartwood extract,

thin layer chromatography was performed on silica gel plates to
select the most appropriate eluent for the preliminary column
chromatography.
The TLC results showed that using the series of
eluents of carbon tetrachloride, benzene, and methylene chloride,

the heartwood extract could be separated into non-polar and polar
components.
(The non-polar components migrated and the polar
material remained at the origin.)
Column chromatography of the
heartwood extract on silica gel with carbon tetrachloride, benzene,

and methylene chloride eluted the non-polar fraction (about 10% of
the original extract).
Subsequent rinsing of the column with
methanol eluted the polar fraction (about 90% of the original heartwood extract) from the column.
A proton magnetic resonance spectrum (PMR-2) of the non-polar
heartwood component showed only aliphatic proton absorptions at
0.9-2.2 ppm.
The infrared spectrum (IR-2) of the non-polar fraction
in heartwood extract showed strong absorption at 2950 and 2850 cm

which also indicates the presence of aliphatic C-H bonds.
bonyl absorption at 1740 cm
suggests that this non-polar fraction
is a mixture of waxes (general structure:

are long alkyl chains).
The car-
R-CO-OR', where R and R'
The physical appearance of this material
is also consistent with this hypothesis.

The polar component of the heartwood extract is the component
31
of interest.
Infrared (IR-3) and proton magnetic resonance (PMR-3)
spectra of this polar component showed that the non-polar component

had been successfully separated (no absorption at 0.9-2.0 ppm in
PMR spectrum, and the disappearance of 1750 cm" . carbonyl functionality in IR spectrum).
The poor resolution in "the finger print
region" and a broad OH strectching absorption at 3750 to 3000 cm"

in the IR spectrum are noted.
This suggests the presence of a
complex mixture of compounds of which some are probably phenolic

in nature (the PMR spectrum shows absorption of aromatic protons
at 6.4 and 6.8 ppm). Additional evidence for the presence of
phenolic compounds was obtained from ultraviolet spectral shifts
observed for the polar fraction of the heartwood extract upon addition of base.
In a qualitative experiment, the A^^^^ of the polar
heartwood component in ethanolic solution shifted to longer wavelength upon the addition of NaOH solution).
Separation of the heartwood extract polar components by preparative thin layer chromatography was also attempted.
Although
bands of compounds with similar retention characteristics could be

separated, individual components could not be obtained.
Attempts to use gas chromatography to separate and collect
individual components from the polar fraction of heartwood extract
were hampered because of the low volatility of the heartwood extract
polar components.
(A majority of the injected material remained in
the column inlet and subsequently decomposed at the high temperature

of the injection port.)
At this point, it was concluded that improved column chromato-
32
graphy would offer the best possibility for separating the polar
fraction of the heartwood extract into the individual components
in sufficient quantities to allow for their identification.
However,
an appropriate analytical technique was needed to monitor the effectiveness of the column chromatography in separating the individual
components of the heartwood extract polar fraction.
Using this
analytical technique, the success or failure of a particular column

chromatographic experiment could be monitored.
After expending considerable time and effort, a successful and
appropriate high pressure liquid chromatographic monitoring system
was developed.
This effort involved testing different columns,
mobile phases (eluting solvent and combinations of eluting solvents),

and solvent programs whereby the percentages of two solvents could
be varied during the high pressure liquid chromatographic analysis.
The optimum conditions for the analysis of the heartwood
extract components included utilizing a Waters Associates y-Bondapak
C-18 column with a total flow rate of 1.5 ml/min from pump A and
pump B combined and a solvent program from an acetonitrile-water
mixture of 10:90 to an acetonitrile-water mixture of 60:40 over an
8 minute period, using programmer curve 11 on a Waters Associates
Model 660 solvent programmer.
A high pressure liquid chromatogram of the polar fraction of
the heartwood extract is shown in HPLC-1.
In this chromatogram the
extract is shown to contain at least 12 components.

When subjected to column chromatography on silica gel using a
gradient solvent system (Table 2 ) , the polar fraction was separated
33
into serveral different fractions (Fractions A, B, C, D in Figure 5).
WEIGHT
(mg)
200
100
200
VIAL NUMBER)
Figure 5.
Although it had been anticipated that the fractions resulting

from the column chromatographic separation described above would
be individual components which could be subsequently identified,
this was found not to be the case.
Thin layer chromatography and
high pressure liquid chromatography revealed that each fraction

contained more than one component.
Fraction A (vial numbers 1-10) contained too small of an amount
of the material to be analyzed by spectroscopic methods. Fraction B
(vial numbers 77-83, HPLC-2) was examined by PMR and IR spectroscopy.
It was found that this fraction contained mostly compounds that have
long alkyl chains.
Examination of Fraction C by PMR revealed several
absorption peaks in the aromatic hydrogen region (6.0-7.0 ppm).

The IR spectrum of Fraction C supports the presence of aromatic
-1
compounds with IR absorptions at 1610 cm
-1
and 1510 cm
''
. The
extreme broadening of the OH stretching absorption in the region

3500 to 3000 cm
in the IR spectrum suggests that the compounds
of interest might be contained in this fraction. Because of poor

solubility in most deuterated solvents, satisfactory spectral data
could not be obtained for Fraction D (HPLC-3).
Final separation of the polar heartwood extract components in
Fraction C by column chromatography was accomplished using silica
gel as the packing material and methanol-methylene chloride (1:9,
V/V) as the eluent.
The major portion of Fraction C (vial numbers

*
96-108, HPLC 4) (vide supra) was combined and applied to the column.
j^
Five ml fractions were collected and stored in 2 dram, shell cap

vials.
'I
This procedure allowed the separation of the major component

(Compound 1^) in Fraction C.
The effectiveness of the chromatographic
separation was evaluated by thin layer chromatographic and high

pressure liquid chromatographic analysis of each of the collected
fractions.
Some fractions were shown to be pure by these two analy-
tical methods (the TLC plate showed a single spot after development
and the HPLC chromatogram showed only a single peak, HPLC-5).
The major component (Compound 1) in the heartwood polar fraction was examined by infrared spectroscopy (IR-4), proton magnetic
resonance spectroscopy (PMR-4), and ultraviolet spectroscopy (UV-1).
The IR spectrum exhibits broad OH stretching absorption at 3750 to
3000 cm"
and possible aromatic hydrocarbon absorption at 1610 cm
The PMR spectrum gave a broad absorption at 7.0-8.0 ppm, probably
II
35
indicating the presence of OH groups. Integration of this broad
absorption seemed to indicate a multiplicity of OH groups. A doublet
at 6.8 ppm and sharp singlet at 6.4 ppm in a ratio of 3:2 lead to an
assumption of 5 (or a multiple of 5) aromatic hydrogens being present
in Compound 1_, The peaks around 2.9 ppm might be attributable to
methylene hydrogens adjacent to a carbonyl group (-CH^-COOH) or an
alcohol group (-CH2-OH).
The two absorptions in the region of
4.0-5.0 ppm probably are due to olefinic hydrogens or a methylene

unit in an ester group (-CO-O-CH^-).
The UV spectrum showed X
at 278 nm.
max
Lacking more information, the structure of Compound 1 could
not determined at this stage.
Properties of the Isolated

Compound 1
The major component (Compound 1_) isolated from the heartwood

polar extract was shown to be pure by TLC and HPLC.
Compound 1_ is
a light yellow-colored solid with a melting point of 108-110*^0

(Fisher-Johns melting point apparatus).
The elemental analysis of
Compound 1^ (Integral Microanalytical Laboratories, Raleigh, North

Carolina) showed C, 58.17%, H, 5.18%.
(?ERKIN-ELMER 141.Polarimeter).
MJ.
(methanol) = -51.0
The solubility characteristics of
Compound _1 in various solvents are summerized in Table 6.
Decomposition of Compound 1^
It was noted that when a methanolic solution of Compound 1
36
Table 6.
Solvent
Solubility of Compound _1
in Various Solvents
Insoluble
Partially Soluble
Soluble
CCl
CH2CI2
CHCl.
EtÔ
Mi
CH^COCH^
CHÔH
HÔ
w e r e examined b y h i g h p r e s s u r e liquid chromatography
immediately
II
after b e i n g p r e p a r e d , the c h r o m a t o g r a m showed a single peak (peak 8

in H P L C - 5 ) .
After this sample had been left on the laboratory
b e n c h (exposed to l a b o r a t o r y l i g h t i n g and reflected s u n l i g h t ) for

three w e e k s and w a s examined by H P L C a g a i n , the c h r o m a t o g r a m
indicated the p r e s e n c e of several c o m p o n e n t s .
(HPLC-6)
(When the r e t e n t i o n
t i m e s o f the n e w p e a k s w e r e compared w i t h H P L C - 1 , the n e w p e a k s

seem to m a t c h w i t h p e a k s numbered 2 , 6, 9, 1 1 , and 1 2 in H P L C - 1 . )
T h e s a m p l e w a s then returned to the laboratory b e n c h and w a s examined
by H P L C every two w e e k s for a period of two m o n t h s .
chromatograms
A l l the H P L C
(HPLC-7 to 1 0 ) show similar trends w i t h the o r i g i n a l
37
peak gradually decreasing and new peaks gradually increasing.
(Quantitative data could not be obtained due to a slow evaporation
of solvent from the sample which produced changes in concentration.
To determine whether the peaks areas were increasing or decreasing,
the relative intensities of peaks in a chromatogram were compared.)
Since the possibility that these changes arose from some type of
contamination appears to be small, it seems that a photodecomposition of this sample might have taken place.
A more detailed study of the photodecomposition of Compound 1_
was conducted following Flow Scheme A in Figure 2.
Before irradiation with the light source (a 60 W light bulb) ,
samples of the material were examined by either UV spectroscopy or
**
HPLC immediately after being prepared (UV-1 and HPLC-11).
"^
After
si
these samples had been irradiated by the light source for 24 hours
41
!
they were again examined by UV spectroscopy and by HPLC (curve B in

UV-2 and HPLC-12).
Some decomposition of the samples was apparent.
II
;
(The decomposition was confirmed by comparing the relative peak

intensities in HPLC-11 and HPLC-12 using the comparison method described
above.)
This trend became more pronounced after the samples
had been irradiated for 48 hours (curve C in UV-3, and HPLC-13).

By the end of 72 hours of irradiation, the original absorption peak
in the UV spectrum was almost unrecognizable (curve D in UV-4).
The HPLC chromatogram (HPLC-14) showed a significant decrease in
the relative area of the original peak.
As an alternative to photodecomposition, an acid-catalyzed
decomposition caused by the methanol solvent was also considered as
38
a possible source of the observed changes. A direct comparison of
photodecomposition and acid-catalyzed decomposition was performed
according to Flow Scheme B in Figure 3.
Although the sample (Sample 3) used for study had slightly
decomposed (HPLC-15), it could still be used as a standard.
Sample
3 was dissolved in methanol. A first portion (1/3) of the solution

was kept in the dark for 72 hours and then examined by HPLC. The
chromatogram did not show impressive changes (compare the relative
intersities of peaks numbered 8, 10, and 11 in HPLC-15 and HPLC-16).
A second portion (1/3) of original solution was irradiated with the
light source (a 60 W light bulb) for 72 hours. The chromatogram of
this irradiated solution (HPLC-17) showed dramatic changes in the

composition of the sample (compare the relative intensities of peaks
"'I
-^
m
numbered 2, 5, 8, 10, and 11 in HPLC-15 and HPLC-17).
A third
portion (1/3) of the original solution was examined by HPLC immediately after adding one drop of concentrated HCl.
If acid-catalyzed decomposition had been major factor in the
composition changes, the chromatogram should have changed greatly
upon the addition of concentrated acid.
However, the results
(HPLC-18) show that this is not the case.
(The relative intensities
of peaks numbered 8, 10, and 11 in HPLC-18 do not change markedly

if compared with HPLC-15.)
In UV-6, it is also demonstrated that
the presence of acid is not detrimental to a methanolic solution of

-2
the Compound 1_, Even when 10 drops of 2.9 x 10
M HCl were added,
the UV absorption did not changed significantly (curve H in UV-6).
Based on these chromatographic results, methanolic solutions of
It
II
39
the Compound 1^ are apparently very sensitive to light. When subjected to light irradiation, decomposition occurs.
Treatment of Compound 1 with Base
The PMR and IR spectra of Compound 1^ indicate that it might be
a phenolic compound.
If this were true, the A__ of this Compound 1
should shift to higher wavelengths upon adding base.

The study of base addition to Compound 1_ in methanol was performed according to Flow Scheme C in Figure 4. When Sample 4 was
treated with a small amount of NaOH solution, the A
of the UV
max
spectrum shifted from 278 nm (curve A in UV-5) to 290 nm (curve E
in UV-5).
This observation provides strong evidence that Compound 1^
is a phenolic compound.
It was found that the UV spectra of a methanolic solution of
Compound 1 changes markedly when subjected to high concentrations
^
^
H
fi\
"I
.'I
of base. After the sample solution was treated with base, it appears
that the composition of the sample might have changed (curve F in
UV-5).
After treatment of the sample solution with 5 drops of

-2
2.9 x 10
M NaOH solution, 5 drops of the same concentration HCl
did not reconvert the absorption of the base treated sample solution completely to the original absorption which indicates a possible irreversible reaction with base.
A significant change was observed upon addition of excess
_2
(more than 20 drops) of 2.9 x 10
M NaOH solution (curve J in
UV-7, where curve A' was the untreated sample absorption and I was
_2
treated with 3 drops of 2.9 x 10
M NaOH solution).
II
40
Structural Determination of
Compound 1^
Silylation of Compound 1^
The mass spectrum of Compound 1_ did not provide an identifiable
parent peak.
Therefore, the molecular weight of Compound l_ could
not be determined by this method.
Since Compound 1_ was thought to
be a phenolic compound, a trimethylsilyl ether derivative (obtained

by silylation of Compound 1^) should have enhanced volatility and
therefore be more useful in a mass spectral determination of the
molecular weight.
Before the silylation of Compound 1_ was attempted, phenol was
M)
used for a model study in order to determine the best reaction conditions for silylation.
The study involved the testing of different
m
I
silylation reagents, solvents, reaction temperatures, and refluxing

times.
Optimum reaction conditions were developed after a number
^
II
o f exp er iment s.
Using hexamethyldisilazane (HMDS) and trimethylchlorosilane
(TMCS, as a catalyst) and phenol,refluxing for two hours gave
complete conversion to the phenyl trimethylsilyl ether.
Completion
of this silylation reaction was demonstrated by the disappearance

of the OH signal in the PMR spectrum of phenol (5.72 ppm) and the
appearance of a trimethylsilyl signal at 0.1 ppm with the intergration ratio of phenyl ring and trimethylsilyl functional group protons
equal to 5:9.
Because of the poor solubility of Compound 1_ in the silylation
41
reagent mixture, Compound 1^ was dissolved in a small amount of pyridine prior to silylation.
A very fine white precipitate appeared
immediately after the HMDS and TMCS were added. During the reflux
period, all of the precipitate (probably NH.Cl) sublimed out of the
reaction flask onto the inner condenser surface leaving a clear solution.
At the completion of the reflux period, the solution was allow-
ed to cool.
The solvent and excess reagent were than removed in vacuo.
The IR spectrum (IR-6) of the residue showed that the broad OH

absorption of Compound 1_ at 3750 to 3000 cm" has disappeared com-1
"M
pletely.
N e w absorptions at 1260 cm
(absorption of Si-CH^) and
1 0 8 0 cm
(absorption of S i - O - C ) , a r e found in the spectrum.
^
2
The P M R spectrum of the silylated material (PMR-6, CCl, as

solvent) gave additional information when compared with that of the
3
SI
pure Compound 1_ (PMR-4).
T h e broad absorption at 7.0-8.0 ppm in
the PMR spectrum of Compound 1 completely vanishes which suggests

~
that this absorption is due to phenolic hydrogens and that the silylation reaction has gone to completion. New absorptions were found
at 0.34 ppm, 0.11 ppm, and -0.25 ppm in a ratio of 3:1:1. These
new signals apparently result from trimethylsilyl groups. The other
absorption signals in PMR-4 and PMR-6 were quite similar. This
indicates that Compound did not decompose during the silylation
reaction.
In PMR-6, the integration ratio of B: C: D: E: F: A = 3:
2: 1: 1: 2: 45. If it is assumed that all the OH groups in Compound

1 have been silylated, since each OH hydrogen was replaced by nine
hydrogens of the trimethylsilyl functional group. Compound 1^ probably contains five OH groups, with one of the OH groups being in a
i
II
42
very different chemical environment from the others (the one that
shows a -0,25 ppm signal after been silylated).
Owing to technical problems, the mass spectrum of this trimethylsilyl ether derivative of Compound 1^ was not reproducible.
Therefore the actual molecular weight is still doubtful.
According
to the best mass spectrum obtained, a molecular weight of 650 was

indicated.
Since each trimethylsilyl group substitution for a phe-
nolic hydrogen would increase the molecular weight of original compound by 72 mass units and there are five trimethylsilyl groups.
Compound 1^ should have a molecular weight of 290.
III
Esterification of Compound 1
An acetate derivative of Compound 1_ was prepared by reaction
^
A]
with acetic anhydride.
Si
I
Phenol was again used as a model compound for developing the
esterification procedure.
Optimum reaction conditions were estab-
lished after a period of trial and error.
It was found that when
phenol was refluxed with acetic anhydride and pyridine (which serves
as solvent and a proton acceptor) for 0.5 hr, work-up of the reaction
mixture revealed that all of the phenol had been converted to phenyl
acetate.
This was demonstrated by the PMR spectrum of the product.
The OH hydrogen signal at 5.72 ppm totally disappeared after the

reaction and the signal for the acetyl group hydrogens appeared at
2.23 ppm in a ratio of 3:5 when compared with the signal for the
aromatic hydrogens.
Esterification of Compound 1_ was performed using these optimum
reaction conditions.
At the end of the reflux period, the reaction
'
!i
l\
n
43
mixture was cooled and transferred to a separatory funnel for the
extraction.
It was noted that an insoluble material precipitated
immediately after the diethyl ether was added to the separatory

funnel.
Chloroform was therefore added to dissolve the insoluble
material.
After separation and drying, the organic layer was transferred
to a round bottomed flask and the solvents were reomoved in vacuo.
The residue was a light brown solid with melting point of 145-147C.
The IR spectrum (IR-7) of this ester derivative shows the completed disappearance of the OH absorption of Compound 1^ at 3750 to
'^
-1
3000 cm
which indicates that the reaction had gone to completion.
-1
*
Ml
"^
A strong absorption at 1750 cm
T|
is assigned as the carbonyl stretch-
ing vibration of the acetyl functionality.

The PMR spectrum of the ester derivative (PMR-7) shows dramatic
changes of the chemical shifts from those of the absorptions in
Compound 1^. With the exception of absorption F which remains at
2.93 ppm, all the absorptions in the spectrum were shifted.
The
integration of the absorption areas in PMR-7 gives:

123* ^ ^12* F : A^' : A^ = 3 : 2 : 2 : 2 : 12 : 3
where A'' and A" are thought to result from the acetyl group hydrogens.
Based on this assumption, two conclusions may be drawn:
a)
Five OH functionalities have been substituted in Compound 1^.
One of these OH groups is in a quite different chemical environment

from the others (the one that causes the A'J absorption at 1.98 ppm
in PMR-7 is different from those which cause the AV absorption at
?i
,|
44
2.23 ppm and A^ : A^ = 4 : 1) .
b) The integration ratio of PMR-7 shows a total hydrogen number
of 24 (or a multiple thereof) in the acetate derivative.
Since each
OH hydrogen has been replaced by three acetyl group hydrogens, this

indicates that Compound 1_ probably has 14 (or a multiple thereof)
hydrogens, which coincides with the number of hydrogens in Compound 1_
as indicated by the trimethylsilyl ether derivative (vide supra).
The acetate derivative of Compound 1_ showed an entirely different PMR spectrum from that of the original Compound ^.
This result
jj[
tot
is to be expected since substituting the hydroxyl groups with acetoxy groups should cause a strong deshielding effect to neighboring
hydrogens.
*^'
"I
'^
l
Thus, hydrogens that are in the magnetic field of the

I
'1
acetoxy groups will be deshielded and shift further downfield in

It
the PMR spectrum.
||
(
A shift reagent experiment was conducted to ascertain if the

completely changed chemical shift and splitting in PMR spectrum of
the acetate derivative of Compound 1^ was due to the postulated deshielding effect or due to other reasons.
It was thought that the
absorption changes could also have resulted from some side reactions
which might have decomposed Compound 1_ during the esterification.
In the shift reagent experiment, the acetate derivative of
Compound 1_ was dissolved in CDC1 and analyzed by PMR spectroscopy
(PMR-7).
Small quantities of the shift reagent Eu(FOD)^ (Aldrich)
were then added to the PMR sample tube. After each addition, the
45
PMR sample tube was weight and the PMR spectrum was taken (PMR-8 to
13).
The resulting hydrogen chemical shift data are presented in
Table 7.
Table 8 lists the changes in chemical shift caused by the
addition of the shift reagent.
It is immediately apparent that PMR
absorptions of the acetate derivative of Compound 1_ (PMR-7) are

affected by the shift reagent. Upon adding shift reagent to the
sample, every signal shifted further downfield.
As more shift
reagent was added, longer downfield shifts were observed.

After the fourth addition (PMR-IO) of the shift reagent, all the
absorptions that were previously suspected to be overlapping in PMR-7,
such as B^^^, G^^, and AV show clear separation into individual absorp-
^.
^
m
tions with the integration ratio: B^ : B^: B^: C: G^ : G^: F: A": A'' =
1 Z 3
i Z
z l
1:1:1:2:1:1:2:3:12 This ratio is again consistent with the presence
^
;?l
of 14 hydrogens (or a multiple thereof) in Compound ]^.

It was found that among the absorptions G, was the most sensi'
tive to the shift reagent (compare PMR-8 and 13) . Relative sensitivity to shift reagent for all the absorptions was:
G
> B
> A^ > G^ > B^ > F > A^ > B^ > C
Proposed Structure of Compound 1_
Many varieties of trees contain a number of phenolic substances.

Among these phenolic compounds, flavonoids and tannins are the most
commonly observed.
Thus, a number of IR spectra of these two types
of compounds were selected and compared with the IR spcetrum for

Compound _1.
Although none of the IR spectra were found to be iden-
dl
)i
,!
46
Table 7. Effect of Shift Reagent upon the Chemical
Shift of Absorption in the PMR Spectrum
of Acetylated 1
Shift
Reagent
Added(mg)
Before
.," ,.
Addition
First
Addition^
(11.9 mg)
Second
Addition
a
h
^2
^3
^
h
"l
^
^1
^^2
(ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
7.20 7.20 7.20
6.80 5.27 5.27 2.88 2.23 1.98
7.52 7.38 7.32 6.89 5.73 5.52 3.07 2.35 2.35
7.82 7.50 7.40 6.96 6.12 5.77 3.23 2.44 2.60
(8.6 mg)
Third
Addition
(20.1 mg)
5}
i^j
g|
8.37 7.86 7.57 7.08 6.82 6.18 3.47
2.63 3.14
Fourth
Addition
(12.9 mg)
8.83
Fifth
Addition
(8.8 mg)
9.11 8.20 7.80 7.23 7.73 6.78 3.84 2.81 3.84
Sixth
Addition
(16.0 mg)
9.65 8.42 7.94 7.33 8.26 7.10 4.04 2.98 4.30
8.10
7.70
7.17
7.37
6.55
3.67
2.78
3.58
^ See PMR-4.
^ After each addition of shift reagent, the PMR sample tube was
shaken vigorously and the PMR spectrum was taken after all of the
shift reagent had dissolved.
?!
l
'w
J[
J,
II
"
47
Table 8.
Shift
Reagent
Added(mg)
Before
Addition
First
Addition
(11.9 mg)
Second
Addition
Shift^* in PMR Absorptions Cause by

the Addition of Shift Reagent
h
^2
^3
^
S
S
^
^1
^2
(ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
0
0.32
0.30
0
0.18
0.12
0
0.12
0.08
^
0
0.09
0.07
^
0
0.46
0.39
0
0.25
0.25
0
0.19
0.16
0
0.10
0.09
0
0.37
0.25
(8.6 mg)
.^
"!
j2
Third
Addition
(20.1 mg)
0.55
Fourth
Addition
(12.9 mg)
0.46
0,36
0.17
0.12
0.70
0.41
0.24
0.19
0.54
vi
:?l
0.24
0.14
0.09
0.55
0.37
0.20
0.15
0.44
: I
CI
ii
II
Fifth
Addition
(8.8 mg)
.1
0.28
0.10
0.10
0.07
0.36
0.23
0.17
0.13
0.26
Sixth
Addition
(16.0 mg)
0.54
0.22
0.14
0.09
0.53
0.32
0.20
0.17
0.46
All shifts are downfield.

The values were obtained by measured the position differences for
each signal in two consecutive PMR spectra.
48
tical with that of Compound 1^ some IR spectra of flavonoid compounds
showed types of absorption in the regions of 3750 to 2800 cm
1800 to 1200 cm
and
which were similar to those in the IR spectrum of
Compound ]^,
A number of literature references regarding flavonoid compounds
were then searched.
After substantial time and effort had been
expended, a report published in 1963 by A. C. Waiss and co-workers

concerning the study of flavonoid compounds was found.12
In this
study of various trimethylsilyl ethers of flavonoid compounds, some

PMR spectra of derivatives were presented.
It was noted immediately
^J
that the PMR spectrum of the trimethylsilyl ether derivative of
5l
(+)-catechin (Figure 6) showed great similarity to the PMR spectrum
"H
n
of the trimethylsilyl ether derivative of Compound 1^ (PMR-6) .
Jlj
The only difference observed between the PMR spectra of trimethyl-
silyl derivatives of (+)-catechin and Compound 1^ is that the signals
ai
II
()
at 6.1 ppm and 5.9 ppm shown in the PMR spectrum of the catechin
derivative are not present in the PMR spectrum of the derivative of
Compound 1^. Instead the latter shows a singlet at 6.33 ppm.
This
suggests that Compound 1^ and (+)-catechin are structurally quite

similar.
With all the spectral data in hand, a possible structure of
Compound _1 is proposed in Figure 7.
In the proposed structure, protons at positions 5, and 6 are
in similar chemical environments. Thus they are assigned to the
almost identical absorptions at 6.40 ppm in PMR-4; and 6.33 ppm in
PMR-6.
In the acetate derivative the proton at position 6 is more
49
^.Sppm
^.8 ppm
f\
5.9 ppm
OR
H H ^ ' ) "Q'2pDm
V/
3.9 ppm
'^{
CH-
2.8 ppm
n
m
R = -Si-CH3
CH.
'!
-'i
<l
^1
II
I
25:6'
8 6
Jjiiiu
10
Figure 6.
IV
Trimethylsilyl Ether Derivative of

(+)-Catechin and Its PMR Spectrum
0
ppm
50
COMPOUND 1:
R=H
DERIVATIVES:
R'= -C-CH
or
R'= -Si-CHq
I
CHo
n
Figure 7.
Proposed Structure of Compound 1^

and Its Derivatives
:%l
ill
strongly affected by the acetoxy group than that at position 5.
II
Therefore they show an AB pattern centered at 6.83 ppm.

The proposed structure has a molecular formula of C^-H^.O^.
15 14 6
Elemental analysis data were obtained for the compound.
for C, ^H,,0.:
C, 62.07; H, 4.83.
Calculation
Found: C, 58.17: H, 5.18.
Since
15 14 D
many flavonoid compounds have been reported to occur in the hydrated

form , percentages were calculated for C, ^.H^ , O^-HÔ, C, c-H, , 0^*2H^0,
and C,^H,,0,*3H0.
15 14 0
2
It was found that the values for C,^H..0,-HÔ:

15 14 D z
C, 58.44; H, 5.19, best reproduce the elemental analysis result.

Thus, Compound ]^ may well exist as the C^ j-H^ ,0^ monohydrate, with
molecular weight equal to 308.
An attempt to determine the stereochemistry at carbons 2 and 3
51
of the Compound 1_ was also made by measuring the coupling constant
for the hydrogens attached to these positions in the Compound 1_
(PMR-4) and trimethylsilyl ether derivative of Compound 1^ (PMR-6).
It was found that J^^ = 8 Hz.
Therefore, according to the Karplus
rule, the dihedral angle between 2H and 3H is either 180 or 0**.

Based on the report by King and co-workers
13
that 2H and 3H have
trans-configuration in (+)-catechin and have cis-configuration in

(-)-epicatechin (an isome of (+)-catechin, differing only with respect
to 2H and 3H), two possible structures _A and B^ have been proposed for
Compound 1.
41
I
f\
%\
CI
OH
II
!
B
Figure 8.
Possible Stereo Structures of Compound _1
It is expected that in trans-2,3-flavan derivatives, the conformation in which both the 2- and the 3-substituent are quasi-equatorial
(structure A^) will be highly favored at room temperature.
14 15
'
52
CHAPTER IV
SUMMARY AND SUGGESTIONS
FOR FURTHER RESERACH
Summary
During the research period, attention has been focused primarily upon the reddish-brown heartwood of mesquite, Prosopis
juliflora.
Effects of varying the extracting organic solvent upon
*!
the amount of material extracted from the heartwood and the overall
î
composition of the extracted material were assessed.
The amounts
,^
of non-carbohydrate organic chemicals which can be extracted from
the heartwood of mesquite were found to depend upon the solvent and
the season of the year when the mesquite was harvested.
['
ii
In order to separate the heartwood extract of mesquite into
individual components or fractions of components, portions of the
heartwood extract were submitted to
methods, such as:
various chromatographic
column chromatography, thin layer chromatography,
preparative thin layer chromatography, gas chromatography, and high

pressure liquid chromatography.
Among these chromatographic methods,
column chromatography proved to be the most effective for separating

the components of the heartwood extract in useful amounts.
By column chromatography, the heartwood extract was readily
separable into non-polar fraction (about 10%) and polar fraction
(about 90%). The non-polar fraction was shown to be a mixture of
53
at least twelve components.
Many of these components are probably
phenolic compounds,
A high pressure liquid chromatographic system was developed for
analyzing the individual components of the polar fraction of the
mesquite heartwood extract.
This analytical technique was used to
monitor the effectiveness of the column chromatographic separation of

individual compounds in amounts which allow for their identification.
The polar extract of mesquite heartwood was separated by column
chromatography into fractions of components.
The major component
of the polar extract. Compound 1_, was isolated from one of these
fractions.
Jj
41
The isolated Compound 1_ was examined by infrared, proton magnetic

resonance, and ultraviolet spectroscopy.
The trimethylsilyl ether
'H
1i
;||
and acetate derivatives of Compound 1 were prepared and examined by
spectroscopic methods.
.,
Shift reagent experiments were conducted
il
upon the acetate derivative of Compound 1^.
From the accumulated data, the structure of Compound 1^ is proposed to be:
(MONOHYDRATE)
Figure 9.
3,3,'4 J 7 ,8-Pentahydroxyf lavan
54
Suggestion for Future Research
It is believed that with a proper modification of the techniques

developed in this research, more components of the mesquite heartwood
may be separated.
Thus, the composition of the mesquite heartwood
extract may be more clearly identified.

Although all the spectral data obtained form Compound jL and its
derivatives supported the proposed structure, the actual mass spectroscopic analysis of Compound 1^, its trimethylsilyl ether and acetate derivatives would provide most convincing evidence for the
^J
proposed structure.
li
Compound _1 was found to be sensitive to both light and base.

The UV spectra of both the irradiated sample and the base treated
%l
z\
sample showed similar types of decomposition.
-.i
Among these decom-
position products, some appear to be the same as some components in

the polar fraction of the heartwood extract.
It would be useful if
the composition of the decomposed samples could be identified.

Due to the known anti-bacterial properties of phenolic compounds,
it is thought that the digestion inhibitors in freshly harvested
mesquite may be compounds such as Compound 1_ in the heartwood.
investigation of this possibility is necessary.
More
'\\
ji
II
LIST OF REFERENCES
1.
Mesquite: Growth and Development, Management, Economics,

Control, Uses." Texas A & M University, The Texas Agricultural
Experiment Station, Research Monograph 1, Nov. 1973, p. 20.
2.
Ritter, G. J., Fleck, L. C , Ind. Eng. Chem.
3.
Goldstein, I. S., and Villarreal, A., Wood Science, 1972, 5,

p. 15.
~
4.
Browning, B. L., "Methods of Wood Chemistry," Vol. 1, Interscience, New York, 1967, p. 79-82, 87.
5.
Fahle, D. W., "Processing Mesquite as a Cattle Feed," Texas Tech

University, Lubbock, Texas. 1978, p. 1.
6.
Vernor, T. E., "Processing of Mesquite for Cattle Feeding,"

Texas Tech University, Lubbock, Texas. 1977.
7.
8.
9.
10.
1922, 1^, p. 1050.
Gaul, D. F., and Bartsch, R. A., "Survey of the Literature

Pertaining to Extraction of Organic Compounds from Mesquite,"
Mesquite Utilization Program, College of Agriculture, Texas
Tech University, 1979.
Schuster, J. L., "Literature on the Mesquite (Prosopis L.) of
North America," Texas Tech University, 1969.
Pierce, A. E., "Silylation of Organic Compounds." Pierce
Chemical Co., 1968, p. 33-39, 72-154.
Geissman, T. A., "The Chemistry of Flavonoid Compounds."
The Mamillan Company. 1962, p. 70.
11.
Richard, J. H., and Hendeichson, J. B., "The Biosynthesis of

Steroids, Terpenes, and Acetogenins." W. A. Benjamin, INc,
1964, p. 50-61.
12.
Waiss, A. C. Jr., Lundin, R. E., and Stern, D. J., Tetrahedron

Letter, 1964, p. 513.
13.
King, F. E., Clark-Lewis, J. W., and Forbes, W. F., J. Chem. Soc.

1955, p. 1338.
14.
Clark-Lewis, J. W., and Jackman, L. M,, Proceedings Chemical

Society, 1961, p. 165.
15.
Weinges, K., and Paulus, E., Liebigs Ann. Chem., 1965, 681,
p. 154.
55
Jf
^t
J[
J!j
^
SI
2
'^l
-II
APPENDIX A
*l
m
High Pressure Liquid Chromatograms
II
II
56
57
1 1 1 1 ' 1 1 1 ' ' 1 1 1 1 M 1 1 ! 1 1 ' 1 1 1 ' 1 1 ' 1 t ' 1 1 1 M 1 r I I P 1 r 11 1 1 l l 'i 1 '7! 1
::::=:5g:=l3i||sS:::::M||^
:i::::x::::::W:::::::Sffi:::::::::ffi+!i:
:::t--::::::::x::-V--+i^-iiif+i tX
'
J _
1_
tiix-t
J_L
'
'
y-r-4-r--it!
' i ' it^r-nH
| | jn | | / ; IT)
|--L-4--U- ll IIIM 11 -j^JLL
^4(1-13ol4i--i42(J4-4-|-4i>|fr4+rô
::::::::::::::::;-P q M ^ ! f ^ # n iV Ii ' 'i i II

W 1 ! I^MMhill itttwHt
ii+H"4"i--^
.100 ^ -^^-9o4-M-^-i-8a-Hf ^-^,7ci44^4 ^ 0 - ^-|-ffpo
o
(0
u
u
^!ii||lilllllllllllll|j:ttltltt^^
-+ - ^ - r t - - r ^ ^ ^ ^ ^ ^ : ^ = ^ = g ^
1 II -l-Ll-L-L4-L.J-^
u^
O
O
1
,
' [
::::::::::--t-^++4
4 i t t l r 'ill' 1i i^ 1' 1l^' ^' l1^1 t' ^
.
t
1 1 r !' i
' I
-
|_j
'1
1 1
.,
u:
^
2
''
! i
1 '
' 1
i
t -
X :::-: ::::
:T::
-4T
^,L
' i ' '
.-i...
1
'
' '
1 i : 1
-H
Uj. ^ ^ ^ ^ ^ ^
r
: '
1 '1
1
11 .,
1
1 1
_' _ i1!
I ! 1
Il's
'i'
M
f
1 1 IT-I
1 1
'
L-+.4--i
'
-i
'
'
1!
' ' ' 1
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1'
100-
i - t -go - 1
i 1 ' 80 1
'
-^-t
i i 1
11
1i 1
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j-
: ' '
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M-J\-' ' ^ ''
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t 1
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1 ' 1 M
1
1
, '
i 1
" " ij +Tt '-Xn tt fr 1ur^

) 1 1\\'1
'
) 1 1
'
tI
i1
'
1
II
' M
11
I I
M
^zx-ix
i
IrrJ
i/u -
<
' . 11
: ' 11
11i
1 1
1 .11 M i l t
1 1 '
1 >
'
1 I : !'
''
; ! 11
1'
i i 1 I 1 ' i 1 1
'
rUCn
111 !
'
111
I
I
1
'
1
1I
1
'
'
1
'
1 1!
1 I
, 1
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0)
at
cr
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'
1' 1i
1
CO
(U
-I-;!
^
S
u
'-:
: ' 1
' '
T1
CO
iH
I; '' , :
'1 ;
1 ;
1
!
!
;
i
'
i 1 ;
' !;1 1 1 !
1
1
1
1
'
I
CJ
1-3
PU
in
'
' l ! Pl
,', '.
1 1 1 i1 i
1
1
1
1
" r
111
1 '
111
III
'
, 1
1 1 1 1
M'
H
'
1
I'll
1 1 '
1 '
'
'
1
1.
,'
II
/{J
1 1
.
I I '
1
! !'
1 ' 1
1' 1t
1 i
'
I
11
. ' .11
!i /
!1 /
' : / ,
/
1i 1i 111 i1 ;11 1
: 1 1 I ; 1 H 1 1 i I j 1 i :1 !/ ' '1 1 ' .
1 11 11 i i !1 I 1' 1
otJ 1
i l l '
1 1 AU
i II 1 I h
'
i 1
'
' ' t
I ' M
' du
i,|i i l i l
)..!, . -
'
*o
^ ij
'/ :
' : ' \j
. . . ; i . . . ^ i f r r - L . Ja-i-^
-: rT--i^^^^-4k=:t4=^r^7lT-t[H-iTtT
- - H ill,Ml ' h ' ! "\i"''i
' r H 1 'MiM'^' ' ' 'U
t . nM x' . . i tl Xl 1
;'
11
M i 1U
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EXTRACTIOn of Organic Chemicals of Prosopis Juliflora

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EXTRACTIOn of Organic Chemicals of Prosopis Juliflora

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EXTRACTION OF ORGANIC CHEMICALS

/Dekn of/ciJfe GrAfiuate School

I would like to express my special thanks to my research advisor

Without his generous help this

research would have been impossible.

I would also like to thank

Finally, I would like to express my sincerest

thanks to my wife Feng-Ying A. Chen for her patience, understanding

Literature and Previous Mesquite Research

Long Term Proj ect Goals

Proton Magnetic Resonance

High Pressure Liquid Chromatography

Thin Layer Chromatography

Preparative Thin Layer

from Mesquite Plants

Source of Mesquite Plants

Gradient Solvent Separation

Properties of the Isolated Compound 1^

Treatment of Compound _1 with Base

Structural Determination of Compound 1_

Formation of Ester Derivative

Shift Reagnet Experiment

RESULT AND DISCUSSION

Extraction and Spectral Analysis

Heartwood from Chemically

Other Parts of the Mesquite Plant

Chromatography and Spectral Analysis

Properties of the Isolated Compound 1^

Treatment of Compound 1^ with Base

Structural Determination of Compound 1

Shift Reagent Experiment

Proposed Structure of Compound 1_

SUMMARY AND SUGGESTIONS FOR FURTHER RESEARCH

Suggestions for Future Research

Proton Magnetic Resonance Spectra

Chromatographic Conditions for HPLC Analysis

Gradient Solvent System

Extraction of Chipped Mesquite Heartwood

Extraction of Chipped Mesquite Sapwood, Chipped Bark,

Solubility of Compound 1_ in Various Solvents

Effect of Shift Reagent upon the Chemical Shifts of

Shift in PMR Absorptions Cause by the Addition of

Soxhlet Extraction Apparatus

Gradient Solvent Separation

Trimethylsilyl Ether Derivative of (+)-Catechin and Its

Proposed Structure of Compound 1^ and Its Derivatives

Possible Stereo Structures of Compound 1^

HPLC-1, Crude Mesquite Heartwood Extract

HPLC-3, Fraction D from Gradient Solvent Column

HPLC-4, Major Portion of Fraction C from Gradient

HPLC-5, HPLC Chromatogram of Compound 2^

HPLC-6, Photodecomposition of Compound 3^(1)

HPLC-7, Photodecomposition of Compound 2.(2)

HPLC-8, Photodecomposition of Compound j^(3)

HPLC-9, Photodecomposition of Compound ]^(4)

HPLC-10, Photodecomposition of Compound 1^(5)

HPLC-11, Compound 1^ before Irradiation with the

HPLC-12, Compound 1^ after Irradiation with the

HPLC-14, Compound 1^ after Irradiation with the

HPLC-15, Chromatogram of Sample 3

HPLC-16, Sample 3 after Being Kept in the Dark

HPLC-17, Sample 3 after Irradiation with the

HPLC-18, Sample 3 after Adding One Drop of

PMR-1, Crude Mesquite Heartwood Extract