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 1

Background 1

Literature and Previous Mesquite Research 2

Long Term Proj ect Goals 5

CHAPTER II. EXPERIMENTAL 6

General Methods 6

Proton Magnetic Resonance

Spectroscopy 6

Infrared Spectroscopy 6

Ultraviolet Spectroscopy 7

High Pressure Liquid Chromatography 7

Column Chromatography 7

Gas Chromatography 7

Thin Layer Chromatography 9

Preparative Thin Layer

Chromatography 9 Extraction of Organic Compounds

from Mesquite Plants 9

Source of Mesquite Plants 9

Preparation of Segregated

Mesquite Plant Parts 10 Extraction of Different Parts of the Mesquite Plant n

iii

Page

Column Chromatography 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

Ether Derivative 19

Formation of Ester Derivative 20

Shift Reagnet Experiment 20

CHAPTER III. 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

Decomposition of Compound 1_ 35

Treatment of Compound 1 with Base 39

Structural Determination of Compound 1 40 IV

Page

Silylation of Compound 1_ 40

Esterification of Compound 1 42

Shift Reagent Experiment 44

Proposed Structure of Compound 1_ 45

CHAPTER IV. SUMMARY AND SUGGESTIONS FOR FURTHER RESEARCH 52

Summary 52

Suggestions for Future Research 54

LIST OF REFERENCES 55

APPENDIX A

B

D

HPLC Chromatograms 56

Proton Magnetic Resonance Spectra 75

Infrared Spectra.

Ultraviolet Spectra,

89

96

LIST OF TABLES

Page

1. Extraction of Honey Mesquite 2

2. Chromatographic Conditions for HPLC Analysis 8

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 36

7. Effect of Shift Reagent upon the Chemical Shifts of Absorptions in the PMR Spectrum of Acetylated _1 46

8. Shift in PMR Absorptions Cause by the Addition of Shift Reagent 47

VI

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. Gradient Solvent Separation 33

6. Trimethylsilyl Ether Derivative of (+)-Catechin and Its

PMR Spectrum 49

7. Proposed Structure of Compound 1 and Its Derivatives 50

8. Possible Stereo Structures of Compound 1^ 51

9. 3,3;4;7,8-Pentahydroxyflavan 53

10. HPLC-1, Crude Mesquite Heartwood Extract 57 11. HPLC-2, Fraction B from Gradient Solvent Column

Chromatography 58

12. HPLC-3, Fraction D from Gradient Solvent Column Chromatography 59

13. 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

21. HPLC-12, Compound 1^ after Irradiation with the Light Source for 24 Hours 68

vii

HPLC-13, Compound 1^ after Irradiation with the Light Source for 48 Hours 69

23. HPLC-14, Compound 1^ after Irradiation with the Light Source for 72 Hours 70

24. HPLC-15, Chromatogram of Sample 3 71

25. HPLC-16, Sample 3 after Being Kept in the Dark for 72 Hours 72

26. HPLC-17, Sample 3 after Irradiation with the Light Source for 72 Hours 73

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. PMR-7, Acetate Derivative of Compound 1_ 82 35. PMR-8, First Addition of Shift Reagent to Acetate

Derivative of Compound 1^ 83

36. PMR-9, Second Addition of Shift Reagent to Acetate Derivative of Compound 1^ 84

37. PMR-10, Third Addition of Shift Reagent to Acetate Derivative of Compound _1 85

38. PMR-11, Fourth Addition of Shift Reagent to Acetate Derivative of Compound 1^ 86

39. PMR-12, Fifth Addition of Shift Reagent to Acetate Derivative of Compound 1^ 87

40. PMR-13, Sixth Addition of Shift Reagent to Acetate Derivative of Compound _1 88

viii

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. IR-7, Acetate Derivative of Compound 1^ 95

47. UV-1, UV Spectrum of Compound 1. 97

48. UV-2, Compound 1^ after Irradiation with the Light Source for 24 Hours 98

49. UV-3, Compound 1 after Irradiation with the Light Source for 48 Hours " 99

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

Light Source for 72 Hours 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 I

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. However, potential uses of mesquite are many and

varied. Presently, mesquite is being used primarily as a source of

fuel. 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 consider

ably more attractive.

An almost totally unexplored area of potential mesquite utili

zation 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. The extrac

tion results have been cited numerous 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 Solvent

Water

Benzene-ethanol

Benzene-ethanol-water

Extraction Yield, Percent of Air-Dry Sample

1% NaOH

From Sapwo

6.0%

4.4%

10.4%

20.5%

od Fn om Heartwood

5.8%

12.2%

18.0%

28.9%

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 Univer

sity 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 sub

stitute 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 carbohy

drates 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.

Column Chromatography

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 Waters Associates, Inc., Model 440 ultraviolet absorbance detector, X = 254 nm.

Pumps Waters Associates, Inc., Pump A, Model M 6000A Pump B, Model M-45 solvent delivery system

Programmer Waters Associates, Inc., Model 600 solvent programmer

Column Waters Associates, Inc., 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 chemical

ly-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

COOLING WATER •-

-r^ 1<—DRYING TUBE

SOLVENT

(f i l l with CaCI )

--V^COOLING WATER

THIMBLE

ROUND BOTTOMED FLASK

Figure 1. 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.

Column Chromatography

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. The residue was analyzed by PMR and IR

spectroscopy. 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.

Gradient Solvent Separation

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. Gradient Solvent System

Solvent Number CH CI CH OH Percent Total

(ml) (ml) °^ ^^3^^ (ml)_

200 0 0 200

190 10 5 200

180 20 10 200

160 40 20 200

140 60 30 200

120 80 40 200

100 100 50 200

8 80 120 60 200

60 140 70 200

10 40 160 80 200

11 20 180 90 200

12 10 190 95 200

13 0 200 100 200

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

Decomposition of 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)

(UV-2)^^

(UV-3) <-

(UV-4) <-

Sample 2 (HPLC-11)

Irradiated with light source

i 24 hours

I 48 hours

1 72 hours

-> (HPLC-12)

•> (HPLC-13)

-> (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.

Kept in the dark for 72 hours

(HPLC-16)

Sample 3 (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 pre

sented 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 pre

pared in the same manner as before and was examined by UV spectros

copy according to Flow Scheme C in Figure 4.

Sample 4

y \

(UV-5)

f V

Add 5 drops of NaOH solution

Add 5 drops of HCl solution

(UV-5)



(UV-6)

Add 3 drops of NaOH solution

Add an excess (20 drops) of NaOH solution

(UV-7)

a -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

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. Solvent and excess reagents were then

removed in vacuo. 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).

Shift Reagent Experiment

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. The weight

of each addition was calculated by difference. Six portions of

shift reagent were added during the experiment.

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

23

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m

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a (U 4J •H

cr CO (U

S na cu a a

•H

O

y-i

o c o

•H o CO u 4-1

X

CO H

o

s rH o >

c >

O CO

c cu >

O CO

tod 14-1

o H 4-1 o -i= o 60 ^

•H 4J (U M

12 CO o;

O •H 4J CD O -U CO CO U Q •U

W

o o o o o o

o o o o o o

o o o o o o

o o o o o o

o o CO

o o o o o in

CVJ CNI 4J

w

o CN

u w

o rg

4J

w

o eg

4J

a

O CN

u W

O c

4-1

w CNJ CM

W

00 CO

CTi CO

C^ CM 00

CN CM

CTi

O

<3^

O CO

CO

o 00 O

O 00 o

00 CO CM

o 00

CN

CO

o u w I

CM

CO

o 00

CM

CO

o 00

o 00

o CN

3 C

• H 4-1 c o o

a o 4J

w

K O •u W 1 vO

ffi vO

u

X — \

CM • «

f H ^-^

sd o i j , ^ W i H 1 vO CN

ffi ^ ^ SO

CJ

CN

• CN CO

00

o 00 <r o 00

o 00

o 00

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0^

sO O

O 00

in

CN < CO

o 00 i n

o 00

CN

CO

O 00

CM

CO

o 00

o 00

o 00

o 00

o 00

o 00

m vO 00 CJN CN

24

0) 3

•H

O

(U f H .a CO H

-d 5>5

•W i H O CO cd u u

K H W CO •H

«4-l g O

(U 4J

6 C 3 0)

'T! > O rH

> o CO

> fH O

CO

001

O •

•y o bO ^

•H 4J (U M

5 CO (U 3S

o • H 4J 0) O 4J CO CO U Q 4J

X

a <u o 4J - H

3 O 4J

c r <u CO CO rH Q <U iH S o

O CO

u C

V4 0) a. X

CM CM

O

CN

O o m

o o

33 O VO 4-1

o

00

CO CO

00 •

CO CO

o 00

o 00

o 00

o 00

00

so o

00

vO

o

(X3

CO o 00

ro

o

Xi liO

u JC

-<r «N

CO

JC u

•H JC 5

c •H

< CO

4J C (U g

• H V4 (U cu X u M o

tM

4J O. (U CJ X (U

TS o

•H u . CO

1 3 0) >

i H O >

c •H

CO

c o

•H u o CO V4 4J

X <u

rH rH <

• TJ <u N

•H rH •H 4-1 3

CO CO 5

-o O

•H U (U a. c o

•H 4.1 O CO

4J X (U

-o O O

4J J.4 CO 0)

<u i J •H 3

cr CO (U

4-1 CO

•H rH o

«4-4 (U

T3 1

rH rH CO CJ

• H

s 0) X o

 0) .c H

CO

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 unex-

tractable 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 heart-

wood 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. A rather

weak absorption at 1750 cm indicates the possible presence of the

^C=0 functionality. 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 extract-

able 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 extrac

tion 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 heart-

wood 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

^3 O O :5 a CO

CO

CU •u •H 3 cr CO (U S T3 (U Q. a.

•H j : : CJ

14-1

o c o •H 4-1

a CO u 4J X

Cd

• LO

0) • i

CO H

CO 0) > CO (U hJ

^3 a)

T3 T3 O CO u 4-1 X

s M

i H CO

•H >.i (U 4-1 CO

s

u Si

0\

0) B

•H

i H •

7-i

CN r

Cd H

C o

• H 4J 0) O 4-1 CO CO 4-1 X

W

C d) o 4-1 ' H • H 4J 3 O cr (U CO CO (U 2 o

CJ

rH Q

(Xl

CO

CS

e •H M

X Cd

CM

C3N

ON

vO

CN

CM

O

CO

CM CM CN

U-t GO

o A

4-1 0) Xi .H 00 O. •H e ( U CO IS CO

m •

r r

i H •

Csj CM

vO •

OS m

CO •

CO m

vO CN

ON

cn

in

CJN

ON

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CT\

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CT»

<3^

<T> o%

u CO

0^

4J

c CO y-i

X) o o 5 a CO

CO

13 O 0 :s a CO

CO

^ M CO

pq

CO (U > CO (U

1-1

0 0 OS

(U CO 3

X

S-i

0)

cu • H T3

O O O

4J •J CU M 4-1

X 0)

a

o ClH

CO

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 heart-

wood 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. The car-

bonyl absorption at 1740 cm suggests that this non-polar fraction

is a mixture of waxes (general structure: R-CO-OR', where R and R'

are long alkyl chains). 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 function

ality 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 addi

tion of base. In a qualitative experiment, the A ^ ^ of the polar

heartwood component in ethanolic solution shifted to longer wave

length upon the addition of NaOH solution).

Separation of the heartwood extract polar components by pre

parative 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 effec

tiveness 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. Gradient Solvent Separation

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 -1 ''

compounds with IR absorptions at 1610 cm 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.

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 II

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 frac

tion 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

'•I

I

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%. MJ. (methanol) = -51.0°

(?ERKIN-ELMER 141.Polarimeter). 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

Table 6. Solubility of Compound _1 in Various Solvents

36

Solvent Insoluble Partially Soluble Soluble

CCl

CH2CI2

CHCl.

X

X

EtÔ

CH^COCH^

CHÔH

HÔ

X

X

X

Mi

were examined by high pressure liquid chromatography immediately

after being prepared, the chromatogram showed a single peak (peak 8

in H P L C - 5 ) . After this sample had been left on the laboratory

bench (exposed to laboratory lighting and reflected sunlight) for

three weeks and was examined by HPLC again, the chromatogram (HPLC-6)

indicated the presence of several components. (When the retention

times of the new peaks were compared with HPLC-1, the new peaks

seem to match with peaks numbered 2, 6, 9, 11, and 12 in HPLC-1.)

The sample was then returned to the laboratory bench and was examined

by HPLC every two weeks for a period of two months. All the HPLC

chromatograms (HPLC-7 to 10) show similar trends with the original

II

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 photodecomposi

tion 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) , 2

samples of the material were examined by either UV spectroscopy or **

HPLC immediately after being prepared (UV-1 and HPLC-11). After " si 41

these samples had been irradiated by the light source for 24 hours «•!

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 de

scribed 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 H

this irradiated solution (HPLC-17) showed dramatic changes in the

"'I composition of the sample (compare the relative intensities of peaks -

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 immedi

ately 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 sub

jected 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 per

formed 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. H

It was found that the UV spectra of a methanolic solution of fi\ "•I

Compound 1 changes markedly when subjected to high concentrations .« .'•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 solu

tion completely to the original absorption which indicates a poss

ible 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

used for a model study in order to determine the best reaction con

ditions for silylation. The study involved the testing of different ••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 intergra-

tion ratio of phenyl ring and trimethylsilyl functional group protons

equal to 5:9.

Because of the poor solubility of Compound 1_ in the silylation

M)

m

41

reagent mixture, Compound 1 was dissolved in a small amount of pyri

dine 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 solu

tion. 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. New absorptions at 1260 cm (absorption of Si-CH^) and ^

1080 cm (absorption of Si-O-C), are found in the spectrum. 2

The PMR spectrum of the silylated material (PMR-6, CCl, as 3 solvent) gave additional information when compared with that of the SI

pure Compound 1_ (PMR-4). The broad absorption at 7.0-8.0 ppm in »«

the PMR spectrum of Compound 1 completely vanishes which suggests »i

~ II

that this absorption is due to phenolic hydrogens and that the sily

lation 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 pro

bably contains five OH groups, with one of the OH groups being in a

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 trime

thylsilyl 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 com

pound by 72 mass units and there are five trimethylsilyl groups.

Compound 1 should have a molecular weight of 290.

Esterification of Compound 1 III

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 ••' !i l\

esterification procedure. Optimum reaction conditions were estab- n

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

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 145-147°C.

The IR spectrum (IR-7) of this ester derivative shows the com-

pleted disappearance of the OH absorption of Compound 1 at 3750 to '^

-1 *• 3000 cm which indicates that the reaction had gone to completion. Ml

-1 "

A strong absorption at 1750 cm is assigned as the carbonyl stretch- T|

ing vibration of the acetyl functionality.

The PMR spectrum of the ester derivative (PMR-7) shows dramatic ?i

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 hydro

gens. 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

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).

Shift Reagent Experiment

The acetate derivative of Compound 1_ showed an entirely diffe-

rent PMR spectrum from that of the original Compound ^. This result jj[

tot is to be expected since substituting the hydroxyl groups with ace- *'

"I toxy groups should cause a strong deshielding effect to neighboring '^

«l hydrogens. 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 de-

shielding 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 m

of 14 hydrogens (or a multiple thereof) in Compound ] .

It was found that among the absorptions G, was the most sensi- dl •'• )i

tive to the shift reagent (compare PMR-8 and 13) . Relative sensi- ,!

tivity 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-

46

Table 7. Effect of Shift Reagent upon the Chemical Shift of Absorption in the PMR Spectrum of Acetylated 1

Shift a Reagent h ^2 ^3 ^ h "l ^ ^1 ^ 2 Added(mg) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Before .," ,. 7.20 7.20 7.20 6.80 5.27 5.27 2.88 2.23 1.98 Addition First Addition^ 7.52 7.38 7.32 6.89 5.73 5.52 3.07 2.35 2.35 (11.9 mg)

Second 5} Addition 7.82 7.50 7.40 6.96 6.12 5.77 3.23 2.44 2.60 i^j

(8.6 mg) g|

Third Addition 8.37 7.86 7.57 7.08 6.82 6.18 3.47 2.63 3.14 (20.1 mg)

^ 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

Fourth 'w Addit ion 8.83 8.10 7.70 7.17 7.37 6.55 3.67 2.78 3.58 J[ (12.9 mg) J,

II Fifth " Addition 9.11 8.20 7.80 7.23 7.73 6.78 3.84 2.81 3.84 (8.8 mg)

Sixth Addition 9.65 8.42 7.94 7.33 8.26 7.10 4.04 2.98 4.30 (16.0 mg)

47

Table 8. Shift^* in PMR Absorptions Cause by the Addition of Shift Reagent

Shift Reagent h ^2 ^3 ^ S S ^ ^1 ^2 Added(mg) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Before « « « ^ ^ Addition 0 0 0 0 0 0 0 0 0

First Addition 0.32 0.18 0.12 0.09 0.46 0.25 0.19 0.10 0.37 (11.9 mg)

Second . Addition 0.30 0.12 0.08 0.07 0.39 0.25 0.16 0.09 0.25 "•!

(8.6 mg) j2

Third

Addition 0.55 0,36 0.17 0.12 0.70 0.41 0.24 0.19 0.54 vi (20.1 mg) :?l

m Fourth Addition 0.46 0.24 0.14 0.09 0.55 0.37 0.20 0.15 0.44 (12.9 mg)

All shifts are downfield.

The values were obtained by measured the position differences for each signal in two consecutive PMR spectra.

: I

CI ii II

Fifth .1 Addition 0.28 0.10 0.10 0.07 0.36 0.23 0.17 0.13 0.26 (8.8 mg)

Sixth Addition 0.54 0.22 0.14 0.09 0.53 0.32 0.20 0.17 0.46 (16.0 mg)

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 and

1800 to 1200 cm 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

12 concerning the study of flavonoid compounds was found. 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

5.9 ppm OR "Q'2pDm H H ^ ' )

V / 3.9 ppm

2.8 ppm

.8 ppm

CH-

R = -S i -CH3

CH.

f\ '^{

n m

'«! -'i <l ^1

II •I

25:6'

8 6

Jjiiiu IV

10 8 4 0 ppm

Figure 6. Trimethylsilyl Ether Derivative of (+)-Catechin and Its PMR Spectrum

50

COMPOUND 1:

R=H

DERIVATIVES:

R'= or

R'=

9 -C-CH

-Si-CHq I ^ CHo

Figure 7. Proposed Structure of Compound 1 and Its Derivatives

n

•n

:%l ill

strongly affected by the acetoxy group than that at position 5.

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. Calculation

for C, H,,0.: C, 62.07; H, 4.83. 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^-H^O, C, c-H, , 0^*2H^0,

and C,^H,,0,*3H„0. It was found that the values for C,^H..0,-H^O: 15 14 0 2 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.

II

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**.

13 Based on the report by King and co-workers 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.

OH

41

•I f\ %\ CI

II •!

B

Figure 8. Possible Stereo Structures of Compound _1

It is expected that in trans-2,3-flavan derivatives, the confor

mation in which both the 2- and the 3-substituent are quasi-equatorial

14 15 (structure A ) will be highly favored at room temperature. '

52

CHAPTER IV

SUMMARY AND SUGGESTIONS FOR FURTHER RESERACH

Summary

During the research period, attention has been focused pri

marily 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 i

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 various chromatographic

methods, such as: 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 Jj

41 fractions.

The isolated Compound 1_ was examined by infrared, proton magnetic 'H 1 «i

resonance, and ultraviolet spectroscopy. The trimethylsilyl ether ;||

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 pro

posed 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 spec

troscopic analysis of Compound 1 , its trimethylsilyl ether and ace

tate derivatives would provide most convincing evidence for the J

proposed structure. li

Compound _1 was found to be sensitive to both light and base. •••

%l The UV spectra of both the irradiated sample and the base treated z\

sample showed similar types of decomposition. Among these decom- -.i

'\\ position products, some appear to be the same as some components in ji

II

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. More

investigation of this possibility is necessary.

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. 1922, 1^, p. 1050.

3. Goldstein, I. S., and Villarreal, A., Wood Science, 1972, 5, p. 15. ~

4. Browning, B. L., "Methods of Wood Chemistry," Vol. 1, Inter-science, 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. Jf

^t 7. Gaul, D. F., and Bartsch, R. A., "Survey of the Literature J[

Pertaining to Extraction of Organic Compounds from Mesquite," Mesquite Utilization Program, College of Agriculture, Texas J!j Tech University, 1979. ^

SI 8. Schuster, J. L., "Literature on the Mesquite (Prosopis L.) of

North America," Texas Tech University, 1969. 2 'l

9. Pierce, A. E., "Silylation of Organic Compounds." Pierce -• Chemical Co., 1968, p. 33-39, 72-154.

10. 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

II

APPENDIX A

*l n 0» m

High Pressure Liquid Chromatograms 5

II

II

56

57

P q M ^ ! f ^ # n iV Ii ' 'i i II W 1 ! I^MMhill itttwHt .100 ^ -^^-9o4-M-^-i-8a-Hf ^-^,7ci44^4 ^ 0 - ^

1 ' I

, ' [ • - -1 1 — 1 - ,

|_j X

:::::::+::5t:i::::::::::::::::::::±:: ^ r r rt-t- 1- -+-'1 1 1 1 rL i+ f t X ., i-a-.-TUxi-^^ :±::-: 4;-_^_.._ln:44:_,.,T._. r -4T

u: ^ ' ' ^ ! - 1 ' 1 2 - i ' • 1 1 S .-_± T--t-4-4-t-t-.--i tit- i - -1X4-- -iTLLj ..-X - . ; t - t -_4 : - -

.,„ , ^ ,L , ' i ' ' • 1 ' ' ' i

1 i : 1 i 11 1 -H Uj. ^ ^ ^ ^ ^ ^ _ _H _p

M l r : ' i 1 1 ' 1 1 1

T 1 ' '

- ..H-4--u-U.Li - t i l ' , U L-+.4--i L 1 1 ' 1 ' • ' ! ' '

1 1 i 1 I ! 1 1 1 1 ' 1 ' 1

_ _ i ! .--L-lrn •i4--.-J u--i-.-J u-aJ ' 1 i ! ' 1 1 ! 1 1 I ' 1 ! I l 's 'i 1 I 1 f M ' 1 •

1 1 IT-I I 1 1 M Hi ' ' ,=F • M l

T n ! rH 1 i t., iJ ; ! ' ' i ^ i 1 ' ' 1 ' 11 , : 1 ; 1 1 1 I i 1 1 ' ' 1 1 1 i

M r ' ^ i' r ' M 1 1 l l ! i 1 ! M 1 1 I ' 1 ! ' ' ' 1 1 ' ' 1 1 1

1 ' j 1 '• ^--r t - i " 1 -i L ' M 1 ^ T 1 T ^ j:-i---^j: r|r nx' xd: ± i ^zx-ix inn 1 'nn ' \ riol 1 1 i Inn i IrrJ

100- j - i - t -go - 1 i 1 ' 8 0 1 1 i /u • • • - rUCn ' L ' - ^ - t • : ' • ' i i • 1 1 1 ' 1 •• < : ' 11 ( 1 • •

1 1 • 1 i 1 M-J—\-' '• ^ '—' t 1 ' . 1 1 — 1 1 i 1 1 ; - - H

" "T t "4± ' "T ' i ' i t — x Ml nn ; M T !^ T TT :4::::4.,..- fr.xlr|-Xl±t_4±^_4. j^t\ _ . , 1 ' 1 M , ' ' • • ' 1 1 1 1 1 ' M ' '

1 1 i 1 1 ! i 1 ) 1 1 1 1 > 1 I I !

f f - -1 " t ' - ±-r '• 1 n T T M " " j T ' X t r u ) • \\' 11 ' • • i + t - n t f 1 r^ 1 1 1 1 . M i l t t ! H it"T;

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_ _ . J_L 1 : i • ' • ' 1

tii±x-±t y—-r-4-r-- i t ! ' i ' it^r-nH ::::±::::::::::::;-- | | jn | | / ; i i+H"—4"—i--^ |--L-4--U- ll IIIM 11 -j^JLL -|-ffpo 4(1-1- 3ol4i--i42(J4-4-|-4i>|fr4+r^o

^!ii||lilllllllllllll|j:ttltltt^^ - + - ^ - r t - - r ^ ^ ^ ^ ^ ^ : ^ = ^ = g ^ 1 II -l-Ll-L-L4-L.J-^ u ^

±::::::::::--t-^+—+4 4 i t t l r 'ill i i 11 ^ l ^ t ^ . t 1 1 r ! ' i ' 1 ^ ' l^' ' 1 1 ' 1 ^-4-11 j \i --\-^ jJJ^jj^.^-tii-L-^i±±-!^

._ — 1 4_i ilL.XUiz^-f-1- , 111 1111 -LLi-L„. i-Li-->- ' • - 44+-

f—+-1 -^-|tp~tr"ill ^r M" n^t"^'"^' i t - •••• 1 1 1 1 t 1 I : • ! ' 1 ; ' ; i ' :

::::± :T : : 1 ' j '' | ' ' !• ^^vi,!;! - . - i . . . 11 ., 1 ,1 1 : i ; ! 1 1 1 ' • ! ' , 1 1 i >•

1 1 1 1 1 1 i i 1 I 1 ' i 1 1 1 1 1 . 1 1 ; : i ,' ; '

M • • 1 1 '

1 1 1 i 1 1 ! ' H - - I - ; ! ' - : • • • • i i 1 i i 1 1 1 1 1 ! i 1 I 1 , 1 1 ' 1 i : ' • 1 ' ' •

i t ! ' 1 ^ T 1

1 1 . ! i ' ' ' I ; ' ' ' 1 1 1 1 1 1 1 1 M ; : 1 : 1 ; , : 1 ;

I I 1 1 1 ! ! ; i ' I I I 1 i i i 1 i 1 ; ' ! ; 1 1 1 !

M . i . i i . ..!, .l iU . . - i . , . . L . . j I i 111 1 • ' ' ,', '. i I 1 1 1 1 1 i 1 ' ! i 1 i 1 i 1 ! 1 1 1 1 i 1 i

1 1 ' 1 1

• 1 ' • 1 1 '

1 1 1 1 1

t 1 t ' ! P " r I I I ' i l l 1 i •

1 I I ' 1 111 1 ' I I I 1 1 1 1 I ' l l 1 1 ' I I ' 1 1 i . ' .11 1 1 ' 1 M ' 1 ' ; 1 ! i /

' M ' 1 1 1 H i 1 ! ! ' ' ! 1 / I I I ' ' - 1 ' 1 1 1 ' 1 • I ' : / , M 1 1 , 1 1 1 ' 1 . 1 ' 1 t • 1 1 • • /

I i 1 '1 1 1 1 1 ; 1 1 : 1 1 I ; 1 H 1 1 i I j 1 i : ! ' 1 ' . '/ : 1 i 1 i 1 1 i i 1 1 i 1 1 1 1 1 1 i i ! 1 I 1 ' • 1 1 / ' 1

1 ' i 1 i l l ' i 1 ' I ' M 1 . •,' ' * o , * - ^ 1

1 otJ 1 1 1 AU ' ' t ' d u • I I /{J ^ ij y • ' : ' \j

. i II 1 I h i , | i i l i l )..!, . - . . . ; i . . .^ i f r r -L . Ja-i-^ - : rT- - i^^^^-4k=: t4=^r^7 lT- t [H- iT tT ill,Ml 'h ' ! "\i"''i ' • • r H 1 'MiM'^' ' ' •'U

M ' i l l ; ' • • 1 1 1 1 1 1 1 1 1 i 1 t t 1 , 1 ' , • i • • 7 ' ± t . n x . . ± t X 1 M i 1U i i 1 i : ] . i 1 __ l i t 4J^_ i.., ' • ( • 1 ' 1 ' i ! M 1 ii-Li^-[|t-|-l-V4!4-T-+—4i4J5--4 I f f -Mt--^- 1 1 1 1 1 ' I I 1 1 — 1 ; 1 I I I i 1 1 i • I I . 1 . T ' 1 1 1 1 1 1 ii>ii I I

1 ' 1 1 1 1 , i 1 ' i M l t i l I ' l l i • l i l t 1 . , "" 1 1 1 t ' i ' ' ; '

- t L 1 1 1 i 1 1 ' i 1 1 i ^ •' ' 1 ! 1 ! 1 1 1 • 1 ' 1 ' 1 1 ~r -"-rr ~n—r • t-* 11 t > i i |: I , Ml I 1 l 1 1 • . i : • • < • ' f

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extraction of organic chemicals of prosopis juliflora

Documents

isolated compound

decomposition of compound

treatment of compound

esterification of compound

solubility of compound

extraction of honey

previous mesquite research

defoliated mesquite