a biochemical investigation of callus tissue...

A BIOCHEMICAL INVESTIGATION OF CALLUSTISSUE IN THE SAGUARO CACTUS (CARNEGIEA

GIGANTEA ((ENGELM.)) BRITT. & ROSE)

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Authors Caldwell, Roger L.

Publisher The University of Arizona.

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A BIOCHEMICAL INVESTIGATION OF CALLUS TISSUE IN THE SAGUARO

CACTUS (CARNEGIEA GIGANTEA ((ENGEIM.)) BRITT. & ROSE)

by

Roger Lee Caldwell

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF CHEMISTRY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 9 6 6

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by Roger Lee Caldwell

entitled A Biochemical Investigation of Callua Tissue in the

Saguaro Cactus (Carneglea gigantea fengelm/]) Britt. and Rose)

be accepted as fulfilling the dissertation requirement of the

degree of Doctor of Philosophy

/ mAuo Dissertation Director Date

Cornelius Steelink

After inspection of the dissertation, the following members

of the Final Examination Committee concur in its approval and

recommend its acceptance:*

5"- 5 /- & &

y-

• S - 3 i - 6 t

S-1. i

5 - 3 / - 6 C

*This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination.

I

STATEMENT BY AUTHOR

This dissertation has been submitted in; partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to Dr.

Cornelius Steelink, the director of this research, for his

continuing encouragement and numerous* suggestions through

out this work. Thanks are also due to Dr. Paul D. Keener

for helpful discussions concerning plant staining tech

niques, to Dr. Charles Lowe for a zoologist's viewpoint

of saguaro ecology, to Drs. Henry Kircher and Roy Keller

for several discussions concerning chromatography problems,

and to Mr. Hobart Koffer for chromatographic assistance in

the summer of 1963. Superintendents Monty Fitch and Paul

Judge of Saguaro National Monument were kind enough to

allow the collection of materials on monument grounds.

Mr. Frank Montford, park ranger, was particularly helpful

during sample collections. Special thanks are due to Dr.

Stanley Alcorn, to whom the author is greatly indebted

not only for help and advice on this research, but for

many challenging and stimulating discussions in all phases

of science.

The author was awarded a Teaching Associateship for

the year 1964-1965, and an Ethyl Corporation Fellowship for

the year 1965-1966.

iii

Financial support from the National Institutes of

Health and the American Cancer Society is gratefully

acknowledged.

TABLE OF CONTENTS

Page

LIST OF FIGURES viii

LIST OF TABLES xi

ABSTRACT xii

Chapter

I INTRODUCTION . 1

A. Historical Background 1

B. Statement of the Problem ........ &

C. Approach to the Problem &

D. Disease Responses in Plants 9

1. General Consideration 9

2. The Role of Phenols 10

3. The Role of Lignin 11

4. The Role of Melanin 12

E. Previous Chemical Work 17

II EXPERIMENTAL 21

A. Methods and Materials ..... 21

1. Apparatus 21

2. Procedures 22

a. Preparation of the Samples ... 22

b. Extraction of the Sample .... 23

c. Chromatography 24

v

i vi

Page

B. Results . 33

1. Initial Studies on Callus Tissue . . 33

a. Soxhlet Extractions 35

b. Comparison of Extracts of Three

Samples 35

c. Fractionation of the Extractives 37

d. Steam Distillation 37

e. Flavonoids 40

f. Phenolic Acids 44

g. Alkaloids 45

h. Column Chromatography 45

2 . Fresh Callus Analysis 52

a. Tannin Tests (Portion 1) .... 52

b. Phenolic Acids (Portion 2) ... 54

c. Alkaloids (Portion 2) ..... . 54

3. Analysis of Ribs for Alkaloids ... 55

4. Biological Activity 56

5. Wax Portion of Callus 61

6. Initial Pulp Analysis 6&

7. Fresh Pulp Analysis 69

3. Micrographs of Callus Tissue .... 7&

9. Decaying Saguaro Tissue Si

10. Observations on the Formation of

Callus 83

vii

Page

III DISCUSSION 90

A. Information Obtained During This Study . 90

1. Phenolic Acids 90

2. Flavonoids 93

3. Alkaloids 93

4. Phenolic Amines . 98

B. Mechanism of Callus Formation 99

INFRARED SPECTRA 106

APPENDIX A Cross Section of the Saguaro 108

APPENDIX B Data Sheet for Thin Layer Chromatograms 109

APPENDIX C Common Names of Chemicals Used in This

Work 110

LIST OF REFERENCES Ill

LIST OF FIGURES

Figure Page

1 Healthy Saguaro . 2

2 Mean Apical Growth in Saguaro of Various Heights (Saguaro National Monument, East) . 4

3 Height-Age Relationships in the Saguaro (Tumamoc Hill) 4

4 Diseased Saguaro 5

5 Partial Structure of Lignin 13

6 Postulated Biosynthesis of Lignin 14

7 Partial Structures of Melanin 15

8 Postulated Biosynthesis of Melanin .... 16

9 Cactus Alkaloids 16

10 Extraction Scheme for Callus 34

11 Results of Extraction Study 36

12 Extraction Scheme for P40X 38

13 Extraction Scheme for Native Lignin .... 39

14 Chromatogram of Callus Alkaloids 4#

15 Chromatography of Callus Alkaloids .... 49

" 16 "Apparatus for Column Chromatography .... 50

17 Extraction Scheme for Ribs/Callus 59

18 Extraction Scheme for Pulp 60

19 Chromatography of Wax Fraction (1) .... 63

20 Chromatogram of Wax Fraction (2) 64

viii

ix

Figure Page

21 Chromatography of Wax Fraction (3) 65

22 Chromatogram of Wax Fraction (4) 66

23 Standard Curve of Dopamine 72

24 Chromatogram of Seedling/Fresh Saguaro ... 75

25 Chromatogram of Saguaro Pulp (1) 75



28 Chromatogram of Saguaro Pulp (4) . . . ... 77


30 Cross Section of Callus/Pulp (1) 79

31 Cross Section of Callus/Pulp (2) 79

32 Cross Section of Callus (1) 80

33 Cross Section of Callus (2) 80

34 Extreme Callus on Saguaro 84

35 Cut Callus at Larvae Hole (1) 86

36 Cut Callus at Larvae Hole (2) 86

37 Callus Forming at Edge of Wound 87

38 Artificial Callus Formation 87

39 Postulated Biosynthesis of Phenolic Acids . 91

40 Biosynthesis of Quercetin 94

41 Postulated Biosynthesis of Carnegine and Related Compounds 96

42 Alkaloid Structure Comparisons 97

43 Biosynthesis and Metabolism of Dopamine . . 100

X

Figure Page

44 Melanin Formation Via Dopamine 101

45 Postulated Mechanism of Wound Response .... 105

LIST OF TABLES

Table Page

1 Extractives of the Saguaro 20

2 Thin Layer Absorbent Preparation Directions . 26

3 Chromatography Data for Flavonoids (Rf x 100) 42

4 Ultraviolet Maxima Shifts for Flavonoids (mil) 43

5 Melting Points of Pentaacetates 44

6 Chromatography Data for Phenolic Acids (Rf x 100) 46

7 Ultraviolet Maxima for Phenolic Acids (mn) . . 47

3 Column Chromatograph Elution Solvents .... 51

9 Chromatography Data and Ultraviolet Maxima of Column Eluents (Rf x 100) 53

10 Data for Alkaloids of Rib (Rf x 100) . • . . . 56

11 Biological Activity of Saguaro Extracts ... 57

12 Chromatography Data for Cinnamic Acids (Rf x 100) 69

13 Chromatography Data for Phenolic Amines (Rf x 100) 71

14 Effect of Wounding ..... 74

15 Chromatography Data of Decomposing Saguaro (Rf x 100) 32

16 Summary of Compounds Identified in the Saguaro &9

xi

ABSTRACT

The tallest cactus in the United States, the saguaro,

is restricted to southern Arizona in this country. When

the saguaro is wounded, either mechanically (knives, rocks)

or naturally (birds, wind), it responds by forming a hard

tissue layer (callus) around the injured area. Paper and

thin layer chromatographic analyses together with ultra

violet spectral analysis of saguaro callus extracts indi

cated the presence of several phenolic acids. Among these

were: 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid,

and 3-methoxy-if-hydroxybenzoic acid. Following the same

procedure, extracts of the pulpy cortex indicated the

presence of 4-hydroxycinnamic acid, 3,4-dihydroxycinnamic

acid, and 3-methoxy-4-hydroxycinnamic acid. One flavonoid,

quercetin, and a second unidentified flavonoid were found

to occur in the callus. There was apparently no flavonoid

content in the vascular elements of the cactus, the woody

ribs.

Although no structural identification of alkaloids

was attempted, chromatographic analysis of the callus,

the ribs, and the intermediate pulpy area indicated the

presence of at least five alkaloids. Previously, only one

alkaloid, carnegine, had been found in the saguaro.

xii

xiii

Chromatographic analysis of the intermediate pulpy

cortex indicated the presence of a glycoside of 4-hydroxy-

benzoic acid. This glycoside was also found in the "slime

flux" of a decomposing saguaro (decomposition due to bac

terial necrosis). In addition, dopamine (3,4-dihydroxy-

P-phenylethylamine) was shown to occur as the major

phenol in the pulpy cortex. The concentration of dopa

mine increased markedly in areas of the cactus that were

wounded.

The high lignin content (30.4$) of the callus may

result from hormonal influence of dopamine on early

lignin precursors. Dopamine as a melanin precursor may

also play an important role in the wound metabolism, as

a temporary response. A plausible mechanism for the for

mation of callus tissue is given.

I. INTRODUCTION

A. Historical Background

The saguaro cactus (Carneeiea gjgantea ((Engelm.))

Britt. & Rose) is a plant unique to the Sonoran Desert.

Its range within the United States is essentially limited

to the southwestern portion of Arizona, with the greatest

concentration occurring in the general vicinity of Tucson.

The saguaro may attain a maximum height of 25-35 feet"'"

(the tallest saguaro on record is 40 feet ), and may occa

sionally live to be 200 years old (Fig. 1).

The earliest recorded description of the saguaro

was by Pfefferkorn in 1794. Early scientific work on

the saguaro was carried out by botanists Shreve and

Mac Dougal while working at the Carnegie Foundation's

Desert Laboratory on Tumamoc Hill (now The University of 7 & Arizona Geochronology Laboratories). Shreve, Shantz,

q and Peebles have described the saguaro in detail. Within

recent years, personnel of The University of Arizona and

the United States Department of Agriculture have been

conducting several studies on the saguaro, which include

studies on pollination requirements, seedling establish-1 1 11 12 ment, bacterial necrosis, ' climatic factors, and

13 chemical constituents. J

1

Figure 1. Healthy Saguaro

3 5 In elaborating on a hypothesis by Shreve, Hastings

and Alcorn described the relationship of rainfall to

saguaro growth and established an approximate (climate

dependent) height-age correlation (Figs. 2, 3)« Hastings

12 and Turner have shown how climate may have an effect on

the saguaro population by comparing rephotographed areas

that had been originally photographed 60 years ago. These

authors also relate the possible role of cattle and rodents

to the changing environment of the saguaro. Niering et 15 al. have studied the influence of temperature, location,

grazing, and rodents on the saguaro population. The

results of the above studies emphasize that the saguaro

population is declining in some areas (e.g., Saguaro •t

National Monument, East ) while in others repopulation is

very effective (e.g., Ventana Cave"*" ).. Although differ

ences of opinion exist regarding the reasons for saguaro

decline, it appears that it is, in fact, a combination of

many factors. (For recent reviews see Refs. 14, 15).

Frequently mature plants are seen with soft dis

colored areas that often become the site of a black 16

exudate (Fig. 4). Lightle et al. first reported that

this soft rotting condition was caused by the bacterium

Erwinia carneeieana Standrig and proposed the term "bac

terial necrosis" for the rotting process. Boyle"'"''' has

since shown that the larvae of Cactobrosis fernaldialis

u cti a> >* (0 a> rC o G H

Xi •p

f-i O

' 2 4 6 6 10 12 1416 IS 20 22 24 26 2g 30 32 Height (feet)

Figure 2. Mean Apical Growth in Saguaro of Various , Heights (Saguaro National Monument, East)

40 -

m 30 u CtJ a> >h

a> oo

2 0 "

10-

—i 1 1—i 1 1 1—i 1 1 1 1 '— — 12 3 4 5 6 7 8 9 10 11 12 13 14 15

Height (feet) Figure 3• Height-Age Relationships in the Saguaro

(Tumamoc Hill)lif

5

i

•03m i A , -rl» • r • .

Figure 4. Diseased Saguaro

Hulst are vectors for this bacterium. Penetrating the

epidermis of the saguaro, these insects tunnel into the

plant, distributing the E„ carnegiena as they go. This

disturbance apparently sets off a mechanism for protec

tive callus* formation, resulting in the formation of a 17 layered tissue around the tunnel. Hemenway ' has given a

brief anatomical description of the callus tissue.

Boyle"®"" has opened infected cacti and followed the larvae

tunnels throughout the cactus. Tunnels were indeed found

throughout the cactus, indicating the following possibil

ities: 1) entry and exit through the same hole, 2) entry

with exit through a different hole, and 3) entry and

death within the cactus. In each of these cases, a piece

of the protective callus tissue could be found around 11 "I & 1Q 20

the tunnel. ' More recent findings indicate that

the bacteria apparently can be disseminated by a number of

insect vectors and that they can enter through holes in

the epidermis resulting from mechanical wounding (e.g., 21 5 birds, rocks, bullets, knives). Lightle and Shreve

have pointed out that decomposition by bacterial necrosis

may be quite dependent upon the season in which the wound

occurred, with total decomposition normally following if

* Callus may be defined as the hard yellow encrust ing material formed in response to any injury of the cactus and is composed of many layers of a highly ligni-ferous material.

wounding took place in May or June. This may be related

to high temperature, flowering of the saguaro, or to low

rainfall.

20 Graf has shown that Drosophila nigrospiracula

Patterson and Wheeler occur in and around the "slime flux"

or rotting portion of a decaying saguaro (Fig. 4). The

D. nigrospiracula were shown to contain E. carnegieana 22 both internally and externally. Burger has found that

Volucella isabellina Williston may be present throughout

the decaying tissue, and V. apicifera Townsend may be

found in the "slime flux" areas of the decaying saguaro.

Santana has studied the biology of Diptera associated

with the bacterial necrosis and concluded that certain

species are able to exist quite well in the "slime flux"

areas.

In summary, where the saguaro population is

declining, bacterial necrosis appears to be the prime

cause of loss among older plants. Extensive damage is

usually prevented by the formation of a protective callus

layer within the cactus. However, if the injury occurs

during May or June, the callus formation apparently does

not keep pace with the bacterial encroachment, and further

decomposition occurs. The factors that are responsible

for the bacterial necrosis and the low rate of seedling

establishment in some areas are not known exactly, but

a

probably include climatic change, cattle grazing, rodent

increases, and the spread of civilization and vandalism.

Statement of the Problem

The apparent decline of the saguaro population in

recent years has prompted many researchers to investigate

the cactus in detail. Lightle, ' Boyle and several 5 2 L others ' have noticed the relationship of the protec

tive callus tissue to the bacterial necrosis of the cac

tus. Early chemical work by Steelink et al. ' indica

ted that the callus tissue was somewhat unique in its

composition and location within the plant. The callus 11 17 tissue forms around wounded areas only, ' ' and presents

a sharp division with the normal pulpy cortex of the cac

tus. In view of the declining saguaro population due to

wounding (e.g., larvae entry, mechanical injury) and the

sharp division of the resulting callus tissue from the

rest of the cactus, the saguaro appears to be an excellent

model to study general wound responses in plants.

C. Approach to the Problem

The problem of callus formation will be approached

by comparing known wound responses of plants to that of the

saguaro. Studies on lignin, melanin, and phenolic com

pounds (previously known to exist in the cactus) will be

related to the wound response.

9

Paech and Tracy, Ruhland, and Robinson have

outlined many useful procedures for preparing plant sam

ples for chdtnical analysis. 23 29 Initially, paper chromatography ' was used to

study plant constituents, but more recently it has been

replaced by thin layer chromatography (TLC). ~ By

utilizing cellulose layers on TLC the vast reference sys-33-35 tem of paper chromatography Rf values may be used. "

Spectral techniques such as ultraviolet spectroscopy "

and infrared spectroscopy* are used extensively in

plant analysis.

D. Disease Responses in Plants

1. General Considerations

It has been long known that plants respond to

wounding or to disease by one or a combination of several

mechanisms: phytoalexin production, " precipitation of

invading enzymes, walling off of the affected area, ' 17 or formation of a corky, callus type covering. {For

recent reviews see Refs. 44-49)•

Indoleacetic acid (IAA) (1), kinetin (2), and

gibberellic acid (3) have all been strongly implicated in

an interdependent role in plant responses to disease or

to wounding. °~54 This interdependent role emphasizes

the hormonal aspects of wound response by the plant, and

10

HiCOOH

the need for metabolic regulation during the response

process. (For recent reviews, see Refs. 56-60).

2. The Role of Phenols

Because of their effect on auxin (hormone) metabo

lism, >62 enaymesj61-63 oxidation-reduction reactions,

65 and their accumulation in wounded areas, phenols are the

most important class of compounds known thus far to be

involved in plant response to infection. Among the com

pounds that accumulate in infected areas are mono- and

di-hydric phenols, phenolic glycosides, flavonoids,

coumarins, and aromatic amino acids. (For recent reviews

see Refs. 45, 49> 65-67).

Tannin, a polyphenol based on the flavonoid nucleus, 6$ £>Q

has long been known to precipitate proteins. ' ' y

11

This phenomenon of tannin may well play a role in the LQ

plant response to infection by precipitating enzymes.

Phenol polymerization is also involved in the formation of

lignin and melanin.

3 • The Role of Lignin

Koblitz ' has shown how growth stimulating

materials affect increased deposition of lignin at the

expense of polysaccharide (the total concentration of

lignin plus polysaccharide is constant). Isherwood, 71 72 Jacobs,' and Hepler and Newcomb' have also shown that

lignin synthesis is dependent on auxin levels. Simionescu 73 et al. have shown that the lignin content of tomatoes

and sunflowers infected with Agrobacterium tumefaciens

is greater, and the cellulose content less than that of

controls. Lignin content has also been shown to be

higher in plants that are resistant to decay (e.g., red-/LA rj i rt c

wood, cedar) than in susceptible types. >'*>'<> Ligni-

fied tissues of the saguaro remain essentially intact

long after the remainder of the cactus has decomposed

(with the callus tissue surviving longer than the woody

vascular elements) It is apparent, then, that lig

nin is somehow involved with the wound or disease response

of the plant.

The structure of lignin has yet to be definitely

resolved (a recent proposal of a partial structure is

1

12

illustrated in Fig. 5)* As lignin is the binding mater-

77 ial for the cell wall (Sachs'' has published an electron

micrograph of lignin within the cell), much effort has

been put forth regarding its analysis and biosynthe-0£» C*0

sis, ~ (A plausible biosynthesis is shown in Fig. SO Si 6). Recently, Freudenberg has simulated the biosyn

thesis of lignin by radical polymerization of coniferyl

alcohol(4) and has produced lignin-like substances.

ctfrzCH-cHjOH

HO*

(4)

4. The Role of Melanin

Melanin has also been implicated in the wound go

responses of plants. * * Melanin, a polyquinone derived

from the dopamine nucleus, has been shown to precipitate til

proteins through co-polymerization ? during melanin

synthesis. Thus, melanin may precipitate enzymes of

invading organisms as tannin does.

The structure of melanin is not known exactly,

but some recently proposed partial structures are

shown in Fig. 7« Structure d appears to be the most

plausible, ' resulting from co-polymerization of quinones

(dopaquinone, dopachrome) that may exist along the bio-

synthetic pathway (Fig. 8). An occasional protein may be

H.COH

H2C0H

HJ.OH

H.COH HCOH

HCO HfOH OMe OH

OMeHC

°H HC OMe H.COH

(OMe)o,S

HCOH HCOH

OMe H£OH CH [ 12 | I

HC 0 OMe ^ WjCOW

HCO(CtHJ).)nH

H.COH al CH

HCOH

HC—0 MeO^>v

MeO

Figure 5. Partial Structure of Lignin

CHt<-COOtt

0\-CU-CV\toH

Ctt,

CHrrCHCVAj.Ott

4-

^=CV^-cWtOV\

CM. \

Y*

CV\-C«-Ctt2.oH

ocM-

T

Lignin

SO Figure 6. Postulated Biosynthesis of Lignin

PROTEX N

Figure 7» Partial Structures of Melanin

fast

Melanin

Figure 8. Postulated Biosynthesis of Melanin o.

17

incorporated. Melanins are best defined as the substances

that are formed by the action of tyrosinase on tyrosine or

related compounds (e.g., DOPA, dopamine, epinephrine, tyra-

•\ 84>$6 mine). '

E. Previous Chemical Work

The first chemical investigation of the saguaro

was made by Heyl in 1923 who found that the alkaloid

carnegine (5) occurred to the extent of 0,7%. Unfortu

nately, it is unclear as to exactly which portion of the

saguaro he extracted. Spath^ confirmed Heyl's structure 90 by synthesizing carnegine. Recently, Hodgkins' found the

adrenaline analog (6) in the saguaro. The alkaloids of

the Cactaece are generally 0-phenylethylamines or iso-

ort

( 5 ) ( 6 )

quinolines,91-93 examples of which are given in Fig. 9.

Greene^ in 1936 analyzed the pulp and seeds for

their sugar, protein, ash, and acid content. He also

analyzed the saguaro wine "Tiswin" of the local Indians

(Papago) for alcoholic content, acids, protein, sugar,

ftor'deiiine

,e x N (CH,\

Cadicine

Octtg

Mescaline

MCtt,

N-methylmescaline

^-TiO

Anhalamine Anhalonidine

Figure 9. Cactus Alkaloids

Coryneine

cH,c

o-

>cvu

M-H °=W

N-acetylmescaline

C O.

"tjO-irx. ctt. Pellotine

Hydrocotarine Lophophorine Anhalonine

xcv\.

Carnegine (5) ((Pectenine))

Corypalline

cU, CH-

Hydrohydrastinine

Salsoline ((Not in Cactus))

Halostachine ((Not in Cactus)) Trichocereine

/ Vn-*,

Figure 9 (Cont.) Cactus Alkaloids

19

95 and ash. Kircher ^ has isolated P-sitosterol from the

pulpy cortex of the saguaro.

In an investigation of the lignin and carbohydrate

portions of the saguaro, Steelink et al."^'^ have found

that the lignin content of the callus (30.4$) was much

higher than that of the ribs (21.9$); the intermediate

area (pulp) was apparently void of lignin. Vanillin (7)5

4-hydroxybenzaldehyde (3), and syringaldehyde (9) were

shown to be products of nitrobenzene oxidation of the *p\

lignin. The presence of syringaldehyde indicates the

saguaro could be classified as a hardwood. The relative

CHO. QUO o\o

(7) (3) (9)

percentages of extractives in callus and rib were also

determined by Steelink et al.^ (Table 1). Ho^ found

(7)» (3), and (9) present in the acid hydrolysates of the

ethanol extracts of callus and rib tissue. Recently, 97 Steelink and Onore7' have found"Jthat the predominate sugar

in the saguaro pulp is galactose.

Table 1

Extractives of the Saguaroa

Solvents*3

Tissue Pet. Ether

Ethyl Ether Benzene Ethanol

Cold Water

Hot Water

1% NaOH

Steam Distillate

Holo-Cellulose Lignin0

Rib 0.16 0.11 0.05 2 .65 3.41 ,1.65 11.7 0.03 65.56 21.90

Callus 3.16 2.20 1.02 4.24 2.92 1.46 13.5 o.oa 53.30 30.40

a. All results in % of oven dry (105°C) unextracted samples

b. Successive extractions with petroleum ether, ethyl ether, 95% ethanol, cold

water, hot water, and 1% NaOH.

c. TAPPI standard T-13 m method.

II. EXPERIMENTAL

A. Methods and Materials

1. Apparatus

Melting points were determined in capillary tubes

with a Mel-Temp apparatus. Infrared spectra and ultra

violet spectra were obtained with a Perkin Elmer Infracord

Model 137B spectrophotometer and a Cary Model 11 recording

spectrophotometer, respectively. All temperatures are in

degrees centigrade.

Extractions were carried out by two methods: 1)

by grinding the sample in a Waring blender with subsequent

filtration, and 2) by the Soxhlet technique.

Paper chromatography was accomplished by the

descending technique (Research Specialties Co., Richmond, QCfc

California, Tank #101A, described and pictured by Macek).

The paper used for all chromatograms was Whatman No. 1,

except as noted.

Thin layer chromatography (TLC) was run by the

ascending method. The equipment (Research Specialties Co.)

is described and pictured by Bobbitt.^ Preparative

plates were made using a Research Specialties Co. aligning

tray and a Dasaga variable thickness spreader (Brinkmann

Instruments, Inc., Westbury, New York), described and

21

22

pictured by Bobbitt.''"^ Standard size (75 nun and 25 mm)

microscope slides were coated with the aid of a Kensco

apparatus (Kensington Scientific Corp., Oakland, Califor

nia) described and pictured by Bobbitt

Photographs of TLC plates were taken with an

Exacta Model VX camera using Eastman Kodak "Kodacolor X"

film, ASA 64. Photographs were taken in artificial blue

light on an apparatus designed for that purpose.

Micropipets (Misco Co., Berkeley, California,

MG0010, 10 X bent tip) were used for all spotting when a

quantitative determination was to be made. Drawn out cap

illary tubes (melting point) were used for qualitative

determinations. Drawn out transfer pipets were used for

applications to preparative plates.

Chromatographic spots were detected with Aerosol

propellent units (Nutritional Biochemicals Co., Cleveland,

Ohio) attached to a bottle of the required reagent.

2. Procedures

a. Preparation of the Sample

Callus, rib, and pulp tissue of the saguaro were

- obtained from the surrounding desert; the date and location

of the particular collection are described under each

analysis.

After collection the callus tissue was buffed with

a wire grinding wheel to remove the dark oxidized outer

23

layer. The resulting fragrant, yellow pieces were broken

into small sections and ground in a Wiley Mill (Arthur

Thomas Co. Model No. 2) to pass through a 2 mm screen.

Rib material collected from the field was broken

into small pieces and ground in a Wiley Mill to pass a

2 mm screen. For rib material from fresh cactus, the ribs

were removed, air dried (approximately 2 weeks), and ground

as above.

The pulp tissue was removed from the cactus, then

ground and extracted simultaneously with ethanol in a

Waring blender for approximately 2 minutes. After grind

ing, the resulting mash was filtered through a Buchner

funnel (15 cm), reground with fresh solvent, and again

filtered. The combined filtrates were then concentrated

and used for analysis.

b. Extraction of the Sample

The callus and rib material were both extracted

in the following manner. The ground sample was placed in

a Soxhlet extractor with a piece of glass wool placed over

the siphon tube to prevent loss of sample to the boiler

and a piece of filter paper placed on top of the sample to

spread the refluxing solvent. The Soxhlet was then fitted

with a 2-1. round bottom flask equipped with a Glas-Col

heating mantle. In order to vent solvent fumes an inver

ted 250 ml filter flask was placed on top of the extractor

with a hose leading to a hood.

24

The apparatus was filled with 1500 ml of solvent

and extracted for 5 days; the solvent was changed after

the first day. After extraction, the two portions were

combined and concentrated in a flash evaporator (Buchler

Instruments, New York, New York) nearly to dryness. This

procedure was successively repeated on each sample with

petroleum ether (66-75°)) ethyl ether, and ethanol/benzene

1/2 v/v. In order to remove traces of the previous sol

vent, air was drawn through the ground material by an

aspirator attached to the siphon tube.

The pulp extraction was accomplished simultane

ously with the preparation of the sample and is discussed

under that section.

Any variation in these procedures will be described

where necessary.

c. Chromatography

(1) Paper

(a) Preparation of the Paper

Standard 46 cm x 57 cm Whatman No. 1

chromatography papers were cut in. half, yielding a paper

23 cm x 57 cm* Light pencil lines were drawn 5 cm and 7

cm from the narrow (23 cm) end of the paper. The line at 7

cm was marked at intervals of 3 cm with a pencil, allowing

the application of 7 sample spots. The line at 5 cm was

subsequently folded and placed over the antisiphon rod

25

with the edge of the paper then lining the solvent trough.

A glass rod (10 mm diameter) bent at each end was placed

in the solvent trough over the paper to serve as an anchor.

(b) Preparation of the Chamber

The chromatographic tanks were saturated

with the developing solvent for a minimum of i+ hours before

use. To accomplish this, solvent was placed in the trough

and also in a crystallizing dish on the bottom of the tank.

In the case of immiscible solvents, the organic phase was

placed in the trough and the aqueous phase in the crystal

lizing dish. During equilibration the papers were nor

mally placed over the antisiphon rods, but did not dip

into the solvent.

(c) Development

The equilibrated chambers were disturbed

as little as possible when the paper was dipped into the

trough. This was accomplished by slowly sliding the tank

cover, rather than lifting it. After the solvent front

had progressed the necessary distance, the paper was

removed and the solvent front immediately marked with a

pencil. The papers were then allowed to air dry.

(2) Thin Layer

(a) Preparation of the Plates

For most analyses, 3 in x S in x l/S in

plate glass plates were used, although some 2 in x 3 in

26

were used occasionally. Standard microscope slides (M.S.)

were used for determining suitable solvent systems. Table

2 lists the directions for preparing layers.

Table 2-

Thin Layer Absorbent Preparation Directions

Layer Thickness No. plates Code*

Cellulose 0.250 mm 6 A

Silica Gel 1.00 mm 3 B

Silica Gel 0.250 mm 6 C

Silica Gel 0.012 in 24 (M.S.) D

*Code: A - 10 gm Cellulose, 52 ml water, 10 ml methanol.

B - 52 gm Silica Gel, 90 ml water, 10 ml methanol.

C - 23 gm Silica Gel, 45 ml water, 5 ml methanol.

D - 10 gm Silica Gel, 19 ml water, 5 ml methanol.

The Silica Gel initially used was Silica Gel G (Research

Specialties); subsequent determinations employed Silica

Gel G (Merck, Brinkmann Instruments, Westbury, New York).

The cellulose used was also Merck (Brinkmann Instruments).

Before spreading, the plates were thoroughly washed with

water followed by ethanol. The absorbent was mixed with

the necessary solvent mixture until it became a homo

geneous solution (approximately 1 minute), poured into the

spreader, and spread with a rapid motion of the hand.

27

After spreading, the plates were allowed to air dry approx

imately 1 hour and placed in a dessicator for storage.

Immediately before use, the Silica Gel plates were acti

vated by placing them in a 110° oven for at least 30 min

utes. The cellulose plates were not heated before use.

(b) Preparation of the Chamber

The chromatographic chambers were lined

with filter paper (10 cm x 19 cm) on the narrow ends of

the tank and the solvent equilibrated at least 30 minutes

before use.

(c) Development

The equilibrated chambers were disturbed

as little as possible when plates were introduced. The

racks supplied by the manufacturer were not used; instead,

the plates were placed directly on the bottom of the

chamber. After the solvent had progressed the required

distance (normally predetermined at 10 cm), the plate was

removed and air dried.

(3) Application of the Sample

Application of the sample was essentially

the same for both paper and thin layer. During spotting of

the paper, a heat gun (Master Appliance Co., Racine,

Wisconsin, Model No. 12200) was held below the paper so

that the solvent would dry as applied. When streaking

23

the paper necessary for preparative work, a pipet was

drawn across the starting line several times. During the

spotting of thin layer plates, a slow air stream was

directed at the plate to assist in drying the solvent.

When a preparative thin layer plate was required, a small

indentation was made across the plate where the solution

was to be applied, followed by repeated applications with

the drawn pipet.

(4) Detection

With the exception of iodine vapor, all

chromatograms were detected with spray reagents. Ini

tially, glass aspirators (Research Specialties) were used,

but it was later found more satisfactory to use the aero

sol propellent units.

(5) Detection Reagents

The following reagents and their abbrevia

tions were used throughout the course of this work:

Reagent Reference Abbreviation

Diazotized sulfanilic acid 23 (DASA)

Solution A: 0.9 gm sulfanilic acid (HgNC^H^SO^H.HgO)

in 9 ml conc. hydrochloric acid diluted to 100 ml with water.

Solution B: 5% w/v sodium nitrite in water. Mix 5 parts A with 25 parts B immediately before use. While the chromatogram is still damp, spray with 20$ w/v sodium carbonate.

29


Ninhydrin 23 (NIN)

Commercial indicator (aerosol bomb, Nutritional Bio-chemicals) "Ninspray," Q.5% ninhydrin in n-butanol.

Bromocresol Green 23 (BCG)

Commercial Indicator (Nutritional Biochemicals) 0.1 fo in n-butanol.

Erlichs Reagent 23 (Erlich)

1fo w/v p-dimethylaminobenzaldehyde in 1 N hydrochloric acid.

Ferric Chloride 102 (FeCl^)

fo w/v in 95fo ethanol.

Dragendorff Reagent 103 (Drag)

Solution A: 350 gm bismuth subnitrate, 10 ml gl. acetic acid, 40 ml water.

Solution B: 3 gm potassium iodide in 20 ml water. Mix 1 part A with 1 part B immediately before use.

2,4-dinitrophenylhydrazine 102 (2,4-D)

3 gm 2,4-D dissolved in 15 ml conc. sulfuric acid. This mixture added to 90 ml of a solution containing 70 ml 95f° ethanol and 20 ml water.

Sulfuric acid 30 (H2S<V

60fo v/v sulfuric acid in water.

Iodine vapor 30 (I2)

Place a few crystals of iodine in a clean chromatography tank and leit them equilibrate with their vapor. Immerse the chromatogram in the tank.

Potassium permanganate 30 (KMnO^)

1% w/v aqueous solution.

30


Ultraviolet light 30 (UV)

Scan chromatogram before spraying, marking fluorescent areas with a pencil or end of a capillary tube. The lamp was a short wave length (3660A) Mineral light

(6) Chromatographv Solvents

(a) Paper Solvent

S olvent Reference Number

Butanol/gl. acetic acid/water 28 1-1 4/1/5 v/v organic phase

Butanol/gl. acetic acid/water 28 1-2 11/3/8 v/v

t-butanol/2-butanone/formic acid/ water 35 1-3 40/30/15/15 v/v

Ethyl acetate/formic acid/water 35 1-4 7/2/1 v/v

Ethyl acetate/water 28 1-5 saturated organic phase

5% acetic acid 28 1-6

Phenol/water 28 1-7 3/1 w/v

2-butanone/acetone/formic 33 1-8 acid/water 40/2/1/6 v/v

Butanol/2$ ammonia 28 1-9 saturated organic phase

31

(b) Thin Layer

Solvent Solvent Reference Number

Butanol/gl. acetic acid/water 28 2-1 4/1/5 v/v organic phase

Butanol/2^ ammonia 28 2-2 saturated organic phase

Benzene/dioxane/gl. acetic acid 30 2-3 90/25A v/v

Ethyl acetate/formic acid/water 35 2-4 70/20/10 v/v (on cellulose)

Methanol/chloroform 30 2-5 1/9 v/v

Petroleum ether (66-75°)/ethyl 104 2-6 ether 4/6 v/v

Hexane/acetone 104 2-7 9/1 v/v

Chloroform/acetone/triethylamine 104 2-8 lloroform/acetone/triethylamine 5/4/1 v/v

Chloroform/ethyl acetate 104 2-9 3/2 v/v

Hexane/ethyl ether/gl. acetic acid 30 2-10 90/10/1 v/v

Hexane/ethyl ether 30 2-11 4/6 v/v

Methanol/chloroform 30 2-12 2/8 v/v

t-butanol/2-butanone/formic acid/ 35 2-13 wat er 40/30/15/15 v/v (on cellulose)

32

(7) Documentation

Paper chromatograms were stored for future use

after circling the spots and calculating Rf values. Thin

layer plates were documented in one of several ways: 1)

tracing directly into a notebook, 2) Xerox copy of the

plate, 3) Neatan preservation (Merck, Brinkmann, Inc.),

4) free hand sketching into a notebook, or 5) photograph

ing the plate. Photography was found to be far superior

to the other methods. Rf values were read off the TLC

plates with the aid of a plastic grid designed for that

purpose (The Forger Co., Tucson, Arizona). The grid is

marked in 1 cm squares and fits directly over the plate;

therefore, the Rf value is read directly if the solvent

front is 10 cm.

(a) Ultraviolet Spectra

Standard spectra were run in 95% ethanol.

When shift reagents were used, equal amounts were placed

in both the sample and reference cells. After chroma

tography, plant samples were eluted from the chromatogram

with 95% ethanol, concentrated, and placed in the sample

cell. An unspotted chromatogram was run with the same

solvent, extracted, concentrated, and placed in the refer

ence cell.

Samples were always scanned from high to low wave

length. Before running the sample spectrum, a base line

was run with the solvent in both cells in order to obtain

a true base line.

5. Results

1. Initial Studies on Callus Tissue

Callus material was obtained from an area approxi

mately 3 miles west of the Arizona-Sonora Desert Museum,

Tucson, Arizona, on 1 September 1962. Callus is the hard

material remaining on the ground after the living portion

of a cactus has fallen and decomposed. The callus was

picked up from areas around several cacti and combined.

The hard material at the base of the cactus, apparently

identical with callus, was termed bark; this was obtained

in the same collecting area as the callus and in the same

manner.

After cleaning and grinding, the samples were

divided as follows:

Extraction 1 333 gm Callus

Extraction 2 333 gm Callus/Bark l/l w/w

Extraction 3 333 gm Bark

A flow diagram is shown in Fig. 10; the extraction was as

previously described. An infrared spectrum of the

unextracted callus material is shown in IR-1.

34

Callus

Petroleum Ether

Meal ~1 Solution

Ether

r

Cool to Room Temp.

4T Meal

~1

Ether

Solid P22A Solution

Ethanol/ Benzene

Evaporate

Evaporate

Boiling Syrup Water

Meal

Waxes

Cool

Solution jEvaporat<

P27A

\y

Solid P102A

Figure 10. Extraction Scheme for Callus

35

a. Soxhlet Extractions

(1) Petroleum Ether Fraction

The solid obtained by cooling to room tempera

ture and filtering the petroleum ether fraction, P22A, had

a pleasant "vanilla-like" smell, quite similar to that of

the original callus. It was a very pale yellow solid. A

further description of this fraction will be given under

waxes.

(2) Ethyl Ether Fraction

The viscous material obtained after evapora

tion of the ether fraction had a red-brown color with a

very strong, irritating odor. By extracting this material

with boiling water followed by cooling the water, a yellow

precipitate was obtained. This solid was characterized

as quercetin and will be described under flavonoids.

(3) Ethanol/Benzene Fraction

The ethanol/benzene fraction contained the

greatest amount of extractives and was a black, odorless,

sticky mass after evaporation. This fraction, P27A, was

investigated further and is described under "Fractiona

tion of the Extractives."

b. Comparison of Extracts of Three Samples

The results of the various extractions are shown

in Fig. 11. It appears that the bark is richer in

extractives than the callus.

Petroleum Ether and Ethyl Ether {% Air Dried Sample)

H h-» H H H to N5 • • • • • • •

O N> O 00. o to i__ . -J 1 1 1 1 1

H* ?

fl>

PO (D cn £ c+ (0 o

tt a 2 o ct H-O 3 CO c+ £

N>_

• • 0

*0 W W ® ct ct c+ &• {3* 4 ft) O H 3 M O (I) W H

c+ \ P t3* W

(D (D W y ct N & (D at 4 » 4 P> tt>

O c+

4 H- 4 P O JB o 3 O c+ c+ H* H* O O

S

VjJ_

On (J> >3 >3 >3 • • • • • • ro o\ o -F~ oa ro

Ethanol/Benzene Fraction (% Air Dried Sample)

9C

Filmed as received

3' without page(s)

UNIVERSITY MICROFIIWS, INC.

38

Ethanol/Benzene Fraction

lj Ether 2) Filter

>T Residue

Solution

H20 pH 7

r Ether

5% NaHCO,

Ether

Solution

Evaporate

Solid

Ether

Water

1 Water

Water

Solid

1) HC1 2) Ether

Ether (P40X)

Figure 12. Extraction Scheme for P40X

39

Ethanol/Benzene Fraction

r~ Residue

1) Et 2) Fi

Ether Filter

Ethyl Acetate

r~ Residue

Ethyl Acetate

CHC1.

Residue Solution

Dioxane

Solution

1) Et 2) Fi

Ether Filter

Ether I

Residue P3SP

sK Ether

5% HC1

Water Ether

Evaporate

t

P3SC

Figure 13. Extraction Scheme for Native Lignin

I

40

the distillate, and it was re-extracted with 100 ml of

ether. The combined ether extracts were dried over mag

nesium sulfate, filtered, and concentrated on a flash

evaporator. Upon evaporation, a pale yellow oil with a

very pungent odor was obtained. It weighed 0.0010 gm

(0,003% of 30 gm sample), gave a red 2,4-dinitrophenyl-

hydrazine derivative melting at 137-139°> and had a boil

ing point of 110-111° (micro capillary method) Upon

repeating the steam distillation, the 2,4-dinitrophenyl-

hydrazone was found to melt at 133-134°. An ultraviolet

spectrum of the distillate showed no absorption maxima

between 220 and 400 mp.. An infrared spectrum gave sev

eral peaks indicative of a substituted cyclopentenone

(IR-3). Further identification of this compound was not

attempted.

e. Flavonoids

The P3&C fraction (Fig. 13) was isolated from the

ethanol/benzene extract. This pale yellow semisolid

(impure) reacted with ethanol/magnesium in hydrochloric 105 acid to produce a magenta color, becoming yellow again

upon addition of sodium hydroxide. With ethanol/zinc in

hydrochloric acid, P3SC gives a faint pink color. These 105 reactions are indicative of the flavonoid nucleus.

Upon reaction with cold nitric acid, a dark purple color

formed; reaction with conc. sulfuric acid produced an

41

immediate red-brown color, 1% ferric chloride gave a

green-purple solution. Several Rf values for P3&C are

shown in Table 3• Ultraviolet spectra are shown in Table

4. Further identification of this compound was not

attempted.

The P102A fraction (Fig. 10) was obtained by-

extracting the original ether residue with boiling water.

Upon cooling, a bright yellow compound precipitated. It

gave a cherry red color with ethanol/magnesium in hydro

chloric acid, an olive green color with 1% ferric chloride,

and a red-orange color with conc. sulfuric acid; 0.060 gm

were obtained, indicating that this material represented

0.09$ of the callus/bark batch from which it was derived.

Chromatography in several solvent systems (Table 3) and

ultraviolet spectral shift reagents10'5'(Table 4)

suggested that P102A was quercetin. Further proof for

the identity of P102A as quercetin came from the penta-

acetate derivative. A 0.027 gm sample of P102A, 1 ml of

acetic anhydride, and 1 ml of pyridine were mixed in a

10 ml pear-shaped flask and allowed to remain at room tem

perature for 3 hours.After the addition of 5 ml of

water, 5 ml of ether, and 1 ml of conc. hydrochloric acid,

the ether layer was separated. After washing With 2 ml of

5% sodium bicarbonate and 2 ml of water, followed by

evaporation, the remaining solid gave white plates upon

recrystallization from 95# ethanol. This same procedure

Table 3

Chromatography Data for Flavonoids (Rf x 100)

-- Solvent £

C ompound 1-1 1-9 1-6 1-7 1-2 1-5

Rutin 63 04 - 62 75 08

Rhamnetin 88 - - - - -

Myricetin 65 - - 30 - -

Querc imeritrin 46 - - - - -

Quercetin Si 10 02 51 86 93

P102A S3 - 02 51 86 93

P38C - 06 - - 80 92

P102A + Quercetin — - - 51 - -

* Solvents: described on page 30

Table 4

Ultraviolet Maxima Shifts for Flavonoids (mn)

Reagent

C ompouna v. Neutral Na0C2H5 AICI3 Na Acetate

H3BO3 Na Acetate

Quercetin 257,373 305a*

322,240a 430,351a 383,326 258,268a

388,305a 260

P102A 257,372 303a

320,242a 430 385,328 259,265a

389,261

P38C 370,292 255

319 425 373,330 253,273a

384,332 260

Rhamnetin 373,256 360,297 - 379,256 387,261

* a indicates shoulder.

44

was followed with quercetin. The comparison of melting

points is given in Table 5.

Table 5

Melting Points of Pentaacetates

Compound Melting Point

Quercetin <• " 202-203°

P102A 200-201°

Mixture l/l 196-200°

By extracting a portion of ribs (250 gm) identi

cally with the P102A fraction above, and testing with mag-

nesium/ethanol and hydrochloric acid, no color was pro

duced. It was concluded that there apparently were no

flavonoids present in the ribs.

f. Phenolic Acids

The P40X fraction was isolated from the ethanol/

benzene fraction as shown in Fig. 12. This fraction was

dissolved in ethanol and chromatographed in several sol

vents. From the results, it was concluded that 4-hydroxy-

benzoic acid, 3,4-dihydroxybenzoic acid, and 3-methoxy-4-

hydroxybenzoic acid were present. To confirm these assign

ments, P40X was streaked on Whatman #3 paper and developed

in solvent 1-9. After development, the streaks corresponding

45

to the 3 acids were cut out, extracted with ethanol, and

rechromatographed with authentic samples of the acids

(Table 6). Further proof of the identity of these acids

was obtained by comparing their ultraviolet spectra with

those of authentic samples (Table 7)*

g. Alkaloids

A 5.90 gm sample of the crude ethanol/benzene

extract (bark/callus l/l) was mixed with 300 ml of ethanol

and stirred overnight. The solution was filtered through

a sintered glass funnel to yield a clear maroon solution

(0.70 gm solid). The ethanolic solution was evaporated

to dryness and a mixture of 100 ml of ether and 50 ml of

5$ sodium hydroxide was added and allowed to stir over

night. The apparatus was transformed into a liquid-

liquid extractor and extracted for two days. The ether

layer (A1223) was concentrated, dried over magnesium sul

fate, and analyzed for alkaloids chromatographically

(Figs. 14, 15).

h. Column Chromatography

A 90 gm portion of Baker-7.6 pH Alumina was placed

in a 2 cm x 40 cm column, as shown in Fig. 16, with the

aid of a petroleum ether (66-75°) slurry. A 2.13 gm sam

ple of the ethanol/benzene extract was mixed with 40 gm

of washed sand and added to the column. The sample was

\

46

Table 6

Chromatography Data for Phenolic Acids (Rf x 100)

"" Solvent

Compound — 1-1 1-9 1-6 2-3

DASA Color

3,4-dihydroxybenzoic acid 34 07 58 30 purple P40X: 79-1 85 03 57 26 purple 3-methoxy-4-hydroxybenzoic

60 63

purple

acid 95 14 60 63 orange P40X: 64-2 92 12 60 - orange 4-hydroxybenzoic acid 94 18 65 49 yellow P40X: 64-1 92 14 64 - yellow

4-hydroxycinnamic acid 95 13 45 45 1,

3,5-dimethoxy-4-hydroxy-56 benzoic acid 90 12 56 55 -

3,5-dimethoxy-4-hydroxy-cinnamic acid 90 13 35 - purple 2,4-dihydroxybenzoic acid 93 21 55 - -

3,4-dimethoxybenzoic acid 90 10 61 - -

3-hydroxybenzoic acid 94 27 68 - -

Chlorogenic acid - 10 - - -

2-hydroxycinnamic acid 95 48 52 - orange 4-hydroxyphenylpyruvic acid 91 05 89 - -

3,4,5-trihydroxybenzoic 76 69 acid 76 11 69 - -

Gentisic acid 90 31 64 - purple Homogentisic acid SO 05 78 - -

3-methoxy-4-hydroxy-84 54 cinnamic acid 84 09 39 54 pink

3,4-dihydroxycinnamic acid 79 38 — pink

* Solvents described on page 30.

Table 7

47

Ultraviolet Maxima for Phenolic Acids (mn)

— Reagent

Compound — Neutral NaOC Hjj

4-hydroxybenzoic acid

P40X: 64-1

255

255

2$0

3,4-dihydroxybenzoic acid

P40X: 79-1

294,257

292,257

239,249

3-methoxy-4-hydroxyben-zoic acid

P40X: 64-2

290,260,220

290,260,220

297,278a*

296,232a

3-methoxy-4-hydroxy-cinnamic acid 324 345

* a indicates shoulder.

4S

layer: silica gel

solvent: BAW 4/1/5 (2-1) detection: DRAG and UV

solvent front sLwi

0

i ' 0

1 1

1 2

1. A122B 2. papaverine

Figure 14. Chromatogram of Callus Alkaloids

layer: silica gel solvent: CHCl /acetone/triethyl amine 5/4/1 (2-8) •

detection: as indicated

solvent front

DRAG NIN UV DASA

o 0 0

0

0 0 0

O 0 0

0 0

0

0 0 0

[ 1 • •

1 1 » •

1 1 to

v r-—-1

1. A122B 2. papaverine

3. A122B

4. tryptophane

5. A122B 6. veratrole

7. A122B 8. tryptophane

8

Figure 15• Chromatography of Callus Alkaloids

50

reservoir > / / / / / / j j / n i I ' m u j i n

sand/sampl •m-nr

sintered glass

screw clamp

receiver

Figure 16. Apparatus for Column Chromatography

eluted as shown in Table 8. After elution, the fractions

were concentrated in a flash evaporator and placed on

watch glasses for final evaporation. The dried samples

were then placed in 1 dram vials for storage. Each frac

tion was tested with 2,4-D, FeCl , NaOH, and H SO using

capillary tubes as reaction vessels; those fractions with

similar results were then combined. The odor of fractions

37 and and only these, was very similar to that of a

decaying saguaro, noticeable in the air on a summer evening

Table 8

51

Column Chromatograph Elution Solvents

Solvent Fractions Volumn (each) ml

benzene 1-11 125

benzene 12-14 250

benzene/CHCl l/l 15-21 125

chloroform 22-24 250

CHCl /ethyl acetate l/l 25-27 250

ethyl acetate 28-30 250

ethyl acetate/acetone l/l 31-33 250

acetone 34-37 250

acetone/ethanol l/l 38-44 250

ethanol 45-48 250

ethanol/5% NaOH l/l 49-50 250

2.5$ sodium hydroxide 51-53 250

in the saguaro forest. Fraction 37 gives a negative test

with DASA, F Cl , 2,4-D, and magnesium/hydrochloric acid.

The ultraviolet maxima and preliminary chromatographic

data are shown in Table 9. Fraction 37 was streaked and

developed in solvent 1-1. Two bands were detected with

UV, cut out, and analyzed chromatographically (Table 9).

Approximately 0.08 gm each of fractions 37 and 38 were

obtained (3.7% of ethanol/benzene extract). These frac

tions were not investigated further.

2. Fresh Callus Analysis

Fresh callus material was obtained from an area

approximately 6 miles northwest of San Xavier Mission,

Tucson, Arizona, on 27 October 1963. The material was

removed with a hand axe from an area of the cactus that

was solid yellow callus (about 1 foot square). A 60 gm

sample of very fragrant yellow chips was obtained in this

manner. These chips were not ground but were divided

into two portions.

a. Tannin Tests (Portion 1)

A 14 gm sample of callus was boiled with 200 ml of

water for four hours, producing a yellow-brown solution.

This was then filtered and the solid boiled with a fresh

portion of water (200 ml). After filtration, the two

aqueous portions were combined and tested for tannic acid.

There was no color with FeClj and no precipitate with

Table 9

Chromatography Data and Ultraviolet Maxima of

Column Eluents (Rf x 100)

Solvent*

1-1 1-9 1-6 Ultraviolet Maxima (miu)

C omp ouncT\. 1-1 1-9 1-6 Neutral NaOCgH

Fraction 37 67, 7 34,42, 81

12,50 310a**, 340a 280

346,236, 240

Band 1 70 52 64,85 30£a,278 252a,231

No Shift

Band 2 86 06 56 320a,283 353


a indicates a shoulder.

54

gelatin. Commercial tannic acid was used as a control,

and gave positive reactions with both tests.

An 18 gm sample of the callus remaining after suc

cessively extracting the bark (described on page 23) was

boiled with water for 1 hour. After filtration, the mash

was reboiled with a fresh portion of water (200 ml).

After filtering again, the combined aqueous extracts were

tested with FeCl and gelatin; these tests, too, were nega

tive, using tannic acid as a control.

b. Phenolic Acids (Portion 2)

A 25 gm sample of the fresh callus vida infra was

ground with a mortar and pestle and was then placed in a

small Soxhlet extractor. The sample was successively

extracted with 200 ml of each of the-following solvents for

three days: petroleum ether (66-75°), ether, and ethanol/

benzene l/l. After evaporation, the ethanol/benzene extract

was found to contain 4-hydroxybenzoic acid, 3>4-dihydroxy-

benzoic acid, and 3-methoxy-4-hydroxybenzoic acid, as had

previously been found in the callus tissue (page 44).

c* Alkaloids (Portion 2 )

The residue from the ethanol/benzene extraction of

portion 2 (vida infra) was mixed with 100 ml of ethanol

and stirred for l/2 hour. After filtration through a sin

tered glass crucible, the filtrate was evaporated nearly

to dryness with a flash evaporator. The resulting solid

was mixed with 100 ml of ether and filtered. The ether

solution was washed with 4 - 50 ml portions of 5% hydro

chloric acid. A 25 ml portion of conc. aqueous ammonia

was added to the aqueous layer and it was again extracted

with ether. The ether layer was slightly yellow, so the

acid and ammonia process was repeated. The resulting clear

ether layer was dried over magnesium sulfate and concen

trated. Chromatography in solvent 2-1 indicated one

Dragendorff positive spot (Rf 0.1-0.2) and two fluorescent

spots.

3. Analysis of Ribs for Alkaloids

A 250 gm portion of ground ribs was obtained from

storage (work done 3 years previously) and extracted suc

cessively with petroleum ether (66-75°)) ether, and

ethanol/benzene l/Z for 5 days each. The yield of the

dried ethanol/benzene residue was 1.45 gm (0.5&$ of ribs

as compared with 6.2% for the callus extraction equiva

lent). The procedure described on page 45 for callus alka

loids was followed, producing a final ether solution of

alkaloids (A12&B). Chromatography data and detection

with DRAG suggest the presence of a major and a minor

alkaloid (Table 10).

Table 10

56

Data for Alkaloids of Rib (Rf x 100)

N,*s >Solvent:<c 2-1

Ultraviolet** mn

Compound 2-1

Neutral NaOC Hij

A128B 80,35 312a, 285

312 only

PapaTar.irie = 50


** (a) indicates a shoulder.

4. Biological Activity

Tests for biological activity were run against

three stains of E, carnegieana: 106, 72-1, and 1-12. The

results of this test are shown qualitatively in Table 11 108 and were obtained by the saturated disc technique. Two

discs were placed in a single plate and 2 plates of each

extract were run per bacterial strain.***

The saguaro was extracted as shown in Figs. 17

and 18, and the extracts were broken into three categories:

1) pulp (P), 2) callus (C), and 3) ribs (R). The cactus

*** I am indebted to Dr. Stanley Alcorn, Department of Plant Pathology, The University of Arizona, for performing the biological activity tests.

Table 11

Biological Activity of Saguaro Extracts

Extract letters refer to fractions described in Figs. 17 and 18. Disc techniques were used as described on page 56, with a diameter of 13 mm.

+ « inhibits 13-25 mm (including disc) ++ = inhibits 26-35 mm (including disc) +++ = inhibits more than 40 mm (including disc) - 83 no inhibition

Sample 106 72-1 1-12

Petroleum ether - - -

Phenol - -

Ethanol - +

R1 +++ +++ +++

R2 +++ +++ +++

R3 +++ +++ +++

R4 + + ++

R5 + + +

R6 + + -

R7 ++ +

Rg -

CI + ++ ++

C2 + • + +

C3 ++ +++ ++

C4 +

Table 11 (cont.)

Sample 106 72-1 1-12

C5 + +

C6 ++ ++ +++

C7 + ++ +

C8 + + +

C9 +++ ++ ++

PI + +

P2 ++ ' +

P 3 . . .

P4 + + +

P5 + + +

P6 ++ ++

cross section of a saguaro (in place of a disc) + ++ +

J

59

Ethanol/Benzene Extract

1) Ether 2) Filter

J" Resin Ether

1) Hydrolyze 21 " Ether

Water Cn .Rn • • •

NH,

1 Ether

Ether Water

HCl/Ether

R '20

20

Na CO /Ether

I

Ether

HCl/Ether

Ether Water c7'R6

Water c8 ,r8

~7 Ether c6,R5

are petroleum ether extract of original callus.

C-,,R, are ethyl ether extract of original callus.

C,,R, are hydrolyzed hot water extract of solid ribs and callus remaining after ethanol

benzene extraction.

Figure 17, Extraction Scheme for Ribs/Callus

Pulp

Mesh

r Ether

Water

Hg Ether

Na2C03

Ether

Water *

Ether P5

60% Ethanol

I Solution

Evaporate

Ether/Water

Ether Water

"1 Water

Hydrolyze

Ether

Water Ether

P,

Na2C03 Ether

Ether P0

T,

Water

HC1

Ether P,

Water

Figure 18. Extraction Scheme for Pulp

/

61

sample was collected approximately 2 miles north of the

Arizona-Sonora Desert Museum on 16 June 1964. The ribs,

callus, and pulp all came from the same saguaro. A 754 gm

sample of pulp was extracted in a Waring blender with

2 - 200 ml portions of 60% ethanol containing 1/2 gm of

ascorbic acid/liter. The callus material was quite fra

grant and was ground on a Wiley Mill to pass through a 2

mm screen, producing 466 gm of sample. The ribs were cut

out, air dried 2 days, and ground in a Wiley Mill to pass

through a 2 mm screen, producing 164 gm of sample.

Hydrolyses were accomplished by adding enough

conc. hydrochloric acid to make a 3 N solution and

refluxed for 45 minutes. All extractions were continuous

liquid-liquid and were continued until the pot solution of

ether was colorless.

In summary, it has been shown that several frac

tions of the extracted saguaro are active against

E. carnegieana. Although no attempt was made to identify

the active fractions, it is evident that bactericidal com

pounds are present in the saguaro.

5. Wax Portion of Callus

The solid obtained (Fig. 10) by evaporating the

cold petroleum ether extract of the bark portion was

analyzed for lipid type material (the solid obtained by

cooling the petroleum ether fraction to room temperature;

62

P22A was different from this fraction). A 3*4 gm sample

of this yellow sticky substance was mixed with 100 ml of

ether and stirred until most of the solid was dissolved

(about 1/2 hour). The solution was filtered and the solid

stirred with a fresh 100 ml portion of ether. After fil

tration, the two filtrates were combined and extracted

with 3 x 100 ml of saturated sodium carbonate (final wash

was clear) yielding a deep yellow aqueous solution (the

ether layer was labeled A132B and the solid labeled

A131A). Concentrated hydrochloric acid was added to the

aqueous portion until a pH of 1 was reached (pH paper).

This was followed by extraction with ether (the ether

layer was labeled A132A). The fraction A131A was dis

solved in hot acetone and cooled successively to 25°, 0°,

and -63°, with filtering at each step. The solution

remaining after this was strongly fluorescent, even while

in the flask, and had ultraviolet maxima of 230 ran and

322 mn. Treatment with 5% sodium hydroxide produced

shifts to 275 mn and 355 mja. Some preliminary chromato-

grams are shown in Figs. 19-22. No further work was done

on this portion of the callus.

The solid obtained by cooling the petroleum ether

fraction to room temperature, P22A, was also investigated.

When P22A was partially dissolved in dioxane and chromato-

graphed in solvent 1-1, a yellow spot with Rf 0.25 was

noticed when sprayed with DASA. Chromatography in solvent

layer: silica gel

solvent: hexane/ether/acetic acid (2-10)

detection: iodine vapor

solvent front

1. wax, sol. at -63°

2. wax, insol. at 0°

3. A132A

4. A132B

5. P22A/ether 6. cholesterol

7. steric acid

8. A131A

Figure 19. Chromatography of Wax Fraction {1}

layer: silica gel

solvent: hexane/acetone 9/l (2-7)

detection: KMnO

solvent front

1. wax, sol. at -63°



4. wax, insol. at -6&°

5. A132A

6. A132B 7. oleic acid

B. vanillic acid

Figure 20. Chromatogram of Wax Fraction (2)

65 layer: silica gel

solvent: hexane/ether 4/6 (2-11)

detection: DASA and UV

solvent front

o o 0 o

o 0 o

0

o D

o O o

0 O

0 o

©

1. wax, insol. at -68°



4. A132A

3 4 5 6 7

5. A132B 6. P122A/ether

7. Cholesterol

8. A131A

Figure 21. Chromatography of Wax Fraction (3)

66 layer: TLC silica gel

solvent: petroleum ether/ether 4/6 (2-6) detection: DASA and UV

7 cm

solvent front

7

1. ferulic acid 6. scopoletin

2. 4-hydroxycinnamic 7. 4-hydroxybenzoic acid acid .

~ . " _ o. vanillic acid 3. fract. 1 wax _ .

(-60° sol.) 9. A85B 4. A132A methyl anthranilate

5. A131A

Figure 22. Chromatogram of Wax Fraction (4)

67

2-1 and detection with BCG indicated the presence of yel

low, "blue, and green spots. A portion of P22A (0.1 gm)

was oxidized with a mixture of 3 gm of sodium dichromate, 102 5 ml of water, and 6 ml of conc. sulfuric acid.

After refluxing one hour, the resulting product was extrac

ted with ether and evaporated. An infrared spectrum (IR-4)

of this oxidation product was nearly identical with that

obtained from the steam distillate (IR-3) of the callus.

The odor was also very similar to that of the steam dis

tillate. A 2,4-D derivative melted at 127-129° and was 102 an orange-red color. The boiling point (micro method)

was 95°; it gave a negative FeCl test and a positive conc.

sulfuric acid test.

Steam distillation of P22A produced no apparent

products, as the callus had done. No further work was

done on this fraction.

In summary, the wax portion of saguaro callus has

been shown to contain several compounds, some of which

gave positive BCG tests, indicating the presence of free

acids. The most intriguing part of this investigation is

the fact that the oxidation product of P22A has an infra

red spectrum and odor nea'rly identical with that of the

steam distillate (page 37) of the callus. The color of

the 2,4-D derivative indicates a conjugated carbonyl

compound; the infrared spectrum indicates hydroxyl,

63

conjugated double bond, strained carbonyl, and a C-methyl.

With the exception of the lack of an ultraviolet maximum,

the compound could possibly be similar to a structure such

as 4-hydroxy-5,5-dimethyl-2-pentene-l-one (biosyntheti-

cally possible from geranyl pyrophosphate). No further

work was done in an attempt to identify this compound.

6. Initial Pulp Analysis

A sample of saguaro pulp was obtained near the SUS

picnic area of Saguaro National Monument, east, approxi

mately one mile northeast of the Arizona-Sonora Desert

Museum, on 6 November 1963. A heavy wind had caused

several arms to fall during the previous week. The pulp

was collected by removing the epidermis, cutting out the

pulp, and dropping it into warm ethanol in a gallon jar.

After remaining in the ethanol 15 hours, the pulp was

ground in a Waring blender, filtered, reground with fresh

ethanol, and stored (total volume 3 liters). One liter of 109 this solution was hydrolyzed 7 with 100 ml of conc.

hydrochloric acid by heating for 22 hours. The ethanol

was evaporated and the aqueous portion extracted with ether

in a liquid-liquid extractor; this hydrolyzed solution,

A&5B, gave 6 fluorescent spots when chromatographed in sol

vent 2-1. Further chromatography and detection with DASA

(Table 12) indicated the possible presence of 4-hydroxy-

cinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid),

and 3-methoxy-4-hydroxycinnamic acid (ferulic acid).

69

The basic fraction, obtained by evaporating the

filtered solution (1-liter) vida infra, adding 6 N sodium

hydroxide and extracting with ether, was chromatographed

in solvent 2-9 indicating a number of 2,4-D, BCG, and UV

active components. No further work was done with these

fractions.

Table 12

Chromatography Data for Cinnamic Acids (Rf x 100)

•v Solvent*

Compound —v.

1-6 1-1 1-9

A85B 27,36,45 76,82,98 09,11

Caffeic Acid 24 79 -

Ferulic Acid 36 84 09

4-Hydroxycinnamic Acid 48 90 13


7. Fresh Pulp Analysis

Pulp was obtained from an arm brought into the

laboratory after removal from a healthy saguaro approxi

mately 1/4 mile east of Old Tucson, in the Tucson Moun

tains, on 9 September 1965. The epidermis was removed in

part and 165 gm of pulp was cut out and extracted with

1-liter of ethanol containing 1/4 gm of ascorbic acid per

70

liter. While cutting the cactus, it was noticed that in

areas where larvae holes had callused over, there appeared

a red color on the pulp upon exposure to air. A 15 gm

sample of this red colored pulp was cut out and extracted

with 100 ml of the above ethan l solution. These solu

tions were partially evaporated and made up to 50 ml with

ethanol; this gave 1:11 ratio for comparison of the two

samples. Chromatography of the solutions indicated that

one spot was more concentrated in the red sample than in

the normal pulp. Chromatography in several solvents

indicated that the spot in question was dopamine (Table

13). A standard curve of dopamine was made and the molar

absorptivity was found to be 2403 (Fig. 23).

The red solution was streaked on 2 Whatman #3

papers and developed in solvent 1-1. After cutting a

small section from each side of the paper and spraying

with DASA, the areas of the paper corresponding to dopa

mine and a yellow spot below it were cut out. Ultra

violet spectral analysis showed the dopamine spot to have

an ultraviolet maximum at 262 mn (neutral) which shifted

to 340 m|i in 5% sodium hydroxide. The ultraviolet spec

trum of commercial dopamine was identical to this. The

yellow spot gave a maximum at 275 m|i (neutral) with a very

small shift to 281 m|i in sodium hydroxide. (This yellow

spot was subsequently identified as a glycoside of 4-hydroxy-

benzoic acid and is further described under section 9.)

Table 13

Chromatography Data for Phenolic Amines (Rf x 100)

— Solvent s*

Compound 1-3 1-4 1-1 1-9 2-1 2-5 1-8

Dopamine 60 75 45 35 42 57 30

Saguaro (4) 60 75 45 35 40 58 29

Norepinephrine 49 59 26 16 31 51 30

Epinephrine 57 72 34 - 31 48 35

Tyramine 77 79 56 72 43 63 52

Dopa 32 49 21 62 28 53 24

Tyrosine 64 ; 73 35 08 27 - 35

Tryptophane 69 77 45 15 39 63 49

Chlorogenic Acid 62 62 62 05 43 67 72

* Solvents described on page 30. Detection was by DASA and NIK.

1.20-

1.10-

1.00-

.90-

.80-0) o

5 -70' CO 6

8 -60"

.50-

.40-

.30-

.20"

.10-

Oy

e = 2403 at 282 mn

0 1.0 2.0 3.0 4.0 5.0 6.0 Concentration (x 10 )

Figure 23. Standard Curve of Dopamine

7.0 8.0

73

From these results it was concluded that a comparison of

young, old, and wounded (red) plants should be conducted.

On 4 November 1965 a fresh arm was removed from an

area approximately 1/2 mile east of Old Tucson, in the

Tucson Mountains. At the same time a seedling (9 cm high)

was obtained from Dr. Stanley Alcorn (greenhouse plant).

The samples were extracted by two methods: 1) using 95%

ethanol, and 2) using 95% ethanol containing 1/2 gm of

ascorbic acid per liter. Pulp from the fresh arm (labeled

"fresh" in chromatograms) and pulp from the seedling (la

beled "seedling") were removed in the usual manner. The

red or wounded pulp (labeled "brown") was obtained by cut

ting out the pulp around a larvae hole that had callused

over and exposing the cut out section to air until the

pulp had turned red (about 10 min). Each sample was 30 gm

and was analyzed spectrophotometrically, indicating an

increase of dopamine content in the wounded cactus (Table

14)» Spectra were run on crude plant extracts and the

absorption was measured at 2&3 (chromatography indicated

the lack of interfering substances at this wavelength).

The chromatography of these 6 extracts is shown in

Fig. 24. Other chromatograms (Figs. 25-29) confirm the

existence of dopamine, and show the relative concentrations

of some of the pulp alkaloids.

Table 14

Effect of Wounding

Change of Dopamine Concentration upon Wounding

Sample Treatment* Sample % Dopamine Absorption (mn) Color Sample Number (Fresh Wt.) max min

Fresh a 1 1 0.30 233 251 Yellow

Seedling a 3 0.43 233 251 Yellow.

Wounded a 2 0.53 233 251 Red-brown 475 330

Fresh b 4 0.40 Yellow

Seedling b 6 0.51 Yellow

Wounded b 5 0.64 • Maroon

* Treatment:

a. Fresh tissue extracted with 95$ Ethanol.

b. Fresh tissue extracted with 95% Ethanol containing

0.5 g. Ascorbic acid/liter.

75

Figure 24* Chromatogram of Seedling/Fresh Saguaro

Figure 25. Chromatogram of Saguaro Pulp (1)

i

Figure 2S. Chromatogram of Saguaro Pulp (4)

%


7S

8. Micrographs of Callus Tissue*

A cross section of the junction of callus tissue

and pulp is shown in Fig. 30. It appears that the pulp

(lower left) and callus are separated by approximately 6

cells, denoting an abrupt change. The pulp on both sides

of the callus is shown in Fig. 31; the portion on the

upper right (outside) shows hypertrophy of the cells. A

view of the callus itself is shown in Fig. 32. The above-

mentioned slices were all stained with the Orseillin BB

reagent,** giving the callus a yellow color and the pulp

cell walls a reddish tinge. The hexagonal structure of

callus (stained with phloroglucinol/hydrochloric acid)"

is shown in Fig. 33> indicating that the callus is

highly ligniferous, while the surrounding pulp is void of

lignin.

Hemenway"*"''' and Morris and Mann have described

similar micrographs of plant wound tissue.

* I am indebted to Dr. Paul Keener, Department of Plant Pathology, The University of Arizona, for instruction and assistance in preparing these slides (freehand sections) .

** Orseillin BB Reagent: Solution A: 0.25$ Orseillin BB in yfo acetic acid. Solution B: 10 gm of phenol, 20 gm of lactic acid, 40 gm of glycerin, 20 gm of water. Mix 1 part A with 9 parts B.

Figure 30. Cross Section of Callus-Pulp (1)

Figure 31* Cross Section of Callus—Pulp (2)

Figure 32. Cross Section of Callus (1)

Figure 33• Cross Section of Callus (2)

81

9. Decaying Saguaro Tissue

The saguaro arm collected on 8 November 1963 near

the SUS picnic area, Saguaro National Monument (west) was

left in the laboratory after the necessary pulp had been

removed. On 22 November 1963, it was noticed that the

arm was exuding a black substance, apparently similar to

that present in the field through natural wounding. The

odor of this exudate was also similar to that of the

natural exudate and to that of fractions 37 and 38 of the

column chromatography experiment. Chromatography in sol

vent 2-1 indicated the presence of 3 fluorescent spots and

one DASA positive spot. A comparison of fraction 37 and

the exudate in the above solvent indicated they have at

least 2 fluorescent spots in common, none of which were

scopoletin or umbelliferone. No further work was done

with this exudate.

In March, 1966, Dr. Stanley Alcorn obtained some

exudate from a living cactus from Saguaro National Monu

ment, east, and it was chromatographed. A yellow spot

with DASA was found in the exudate, and was apparently

the same yellow spot previously found in the pulp.

Chromatographic analysis (Table 15) indicates that this

compound is a 4-0-glycoside of 4-hydroxybenzoic acid. The

yellow compound was isolated by streaking and development

in solvent 2-2.

82

Table 15

Chromatography Data of Decomposing Saguaro (Rf x 100)

•" Solvent

Compound 2-13 2-1 2-2 2-3

Sag 5 (yellow spot) 88 30 20 -

Eluted yellow - 26 - -

Hydrolyzed yellow 88 80 20

4-OH-benzoic acid 89 82 20 45

Ether solution of slime flux 88 83 20 47

Hydrolyzed slime flux - 80 20 46

Vanillic acid - 80 16 50

Slime flux - 30 - -


33

10. Observations on the Formation of Callus

During the course of this work, several items of

interest concerning callus formation were noticed.

Observation 1

Many saguaro in the field are missing a top por

tion of the main trunk which apparently cracked off due to

a resonant motion in response to the wind. One of these

cacti was examined shortly after loss of the upper trunk

(about 1 week had elapsed). It was noticed that a thin

dark substance was forming under a hardened carbohydrate

portion that was exposed to the air. The dark substance

appeared to come from the periphery of the cut portion,

moving toward the center, as the closing of a camera

aperature. This is apparently the early part of callus

formation.

Observation 2

A saguaro near Saguaro National Monument Head

quarters (west) was noticed with approximately 75% of its

surface covered with callus, apparently in good health

(Fig. 34).

Observation 2.

A saguaro arm was allowed to remain in the labora

tory after collecting the necessary pulp samples. Six

days later, the exposed carbohydrate laywer was hard and

Figure 34. Extreme Callus on Saguaro

35

turned light red when cut. Two days later, the area that

was red had turned to a dark resinous material. A thin

dark layer was beginning to form under the dried carbohy

drate layer in places, but most of the cactus had decom

posed by this time.

Observation

The arm collected as described on page 69 remained

in the laboratory after sample collection. When cutting

through the pulp across a callus area that had formed in

response to larval entry, the red color noticed earlier

appeared only in the area of the callus. This has been

observed many times since.

Observation £

The seedling obtained from the greenhouse (page

73) was completely cut off approximately 1/3 the distance

up from the base. In six days the entire tip of the cac

tus had a thin dark layer under the dried carbohydrate.

Just under the dark layer was a thin yellow layer. This

yellow portion had the flavor of vanillin and appeared

identical with fresh callus. See Figs. 35-37 for the

callus-pulp juncture.

Observation 6

In the laboratory, two holes were bored in an arm

(about 2 in deep) with a #3 cork borer. In one hole the

Figure 35# Cut Callus at Larvae Hole (1)

Figure 36. Cut Callus at Larvae Hole (2)

Figure 37- Callus Forming at Edge of Wound

Figure 3d. Artificial Callus Formation

88

center core was removed, and in the other it was allowed

to remain. One week later, the piece of pulp was sliced

across the holes and photographed after 3 hours exposure

to air. The red substance appeared only around the holes,

indicating that mechanical wounding apparently can also

initiate callus formation (Fig. 38). It would be interest

ing to study the change of dopamine concentration as a

function of distance from the wound during this process.

39

Table 16

Summary of Compounds Identified in the Saguaro

C ompound Location

Quercetin Callus 4-hydroxybenzoic acid Callus 3,4-dihydroxybenzoic acid Callus 3-methoxy-4-hydroxybenzoic acid Callus

4-hydroxycinnamic acid Pulp 3,4-dihydroxycinnamic acid Pulp 3-methoxy-4-hydroxycinnamic acid Pulp Dopamine Pulp 4-0-glycoside of 4-hydroxybenzoic acid Pulp

I?

III. DISCUSSION

A. Information Obtained During This Study

1. Phenolic Acids

The occurrence of free phenolic acids in the saguaro

callus may be related to its high lignin content. In the

biosynthesis of lignin (Fig. 6), coniferyl alcohol (4) was

shown to be a reasonable precursor. A slight variation on

this scheme (Fig. 39) indicates that the substituted cin-

namic acids (10), (11), and (12) which were tentatively

identified in the saguaro pulp could very easily arise from

a similar pathway. Further air oxidation of these cin-

namic acids would result in the formation of the substi

tuted benzoic acids (13), (14), and (15)» found in the

callus tissue. It is well known that the "active" metabo

lites of plants are present in low concentration due to

their high turnover rate; " components that are likely to

be found by conventional means are in the "inactive" areas

of metabolism. It would seem, then, that the formation of

the substituted benzoic acids would be due to the breakdown

of lignin, or of lignin precursors, rather than formation

specifically as a result of wounding.

90

jpr > CCft3

(4)

CooH

Shikimic Acid

4.

5> NH,

C t t — c o o H -

ocrt. <—

S'

coovV

oft (15)

CU=Ctt-COdU

J 00IV

> soW

ch=CH-COOH

.00H

Figure 39» Postulated Biosynthesis of Phenolic Acids 49

92

There have been many examples in the literature of

phenolic acids occurring before or after infection or

wounding ' > 5-67 these examples, however, were

within the living system and may not apply to the "dead"

callus tissue. The benzoic acids found in this work were

in the callus, but apparently not in the surrounding pulp.

If they were formed as a consequence of an injury, they

would probably be found in that area of active wound

metabolism, the adjacent pulp (approximately within a 4 cm

radius of the wound, see Fig. 33).

In the adjacent pulp, however, a 4-0-glycoside of

4-hydroxybenzoic acid was found to occur as the only

apparent phenolic acid in the wound area. Upon closer

investigation, the substituted cinnamic acids, (10), (11),

and (12), should also be found in the active wound area

(due to their role in lignin biosynthesis). This glycoside

occurred in the "slime flux" of a decomposing saguaro and

possibly in fraction 37 of the column chromatography exper

iment of callus tissue. In this case, the compound is

indeed in the active center of metabolism. It appeared

that the concentration of this glycoside (sugar not deter

mined) was related to that of dopamine found in the pulp

(Figs. 24, 25), but this was not fully investigated.

93 " ~

2. Flavonoids

Geissman"*" has compared flavonoid content in nor

mal and infected cherry leaves. Normally, flavonoids are

used for taxonomic purposes and have not been strongly

implicated in disease metabolism (other than through their

general phenolic characteristics - i.e., capability of

polymerization into melanin- or tannin-like compounds). 112 De Ends has discussed the physiological properties of

flavonoid metabolism, particularly the "Vitamin P,T effect,

on the vascular system. Quercetin, one of the commonly 105 occurring flavonoids, has been found in the callus

tissue but not in the rib tissue. In this respect it is

of interest to note that in contrast to the pale yellow

color of the ribs, the callus is a deep yellow; this pos

sibly reflects the high quercetin content (0.09$) in the

callus. The hydroxylation patterns of quercetin suggest

its biosynthesis from compounds already tentatively iden

tified in the saguaro (Fig. 40). It was shown that there

was at least one additional flavonoid in the callus tis

sue, but this was not characterized.

3. Alkaloids

The metabolic use of alkaloids is not at all well

understood. They have been implicated in many processes,

such as: plant waste production, plant protective uses, 11*5

and reserve energy sources. J Jackson has shown that

oH

CoA

o 4

cri cc

c°l o ° >A ^ hocc*»4cCOA C rc^o** ^

OH O °T

Quercetin

Figure 40. Biosynthesis of Quercetin ; —49

vO -p-

95

the alkaloid content may vary with respect to time of day

and time of flowering, indicating that alkaloids may, in

fact, play an active role in plant metabolism. In the

saguaro, it appeared that there was a relationship between

the dopamine content and alkaloid content of the pulp as a

function of distance from the callus tissue; however, this

was not fully investigated. Using Heylfs structure for

carnegine (5), a plausible biosynthesis of it in the

saguaro is shown in Fig. 41 utilizing dopamine as the pre

cursor. Also shown in Fig. 41 are some possible structural

variations for the other saguaro alkaloids (the open chain

analogs of dopamine and adrenaline are also possible).

Although their structures were not determined, the

saguaro alkaloids were investigated chromatographically.

By analyzing with solvent 2-9 there appear to be at least

5 alkaloids* in the saguaro; their structures differ,

depending on their location in the plant (i.e., rib, callus,

or pulp). The callus and ribs have the major alkaloidal

content, with the callus containing the greater amount

(visual chromatographic estimation). There are 3 alkaloids

in the callus, 2 of which are also in the rib (Fig. 42).

The pulp (sag. 5 in Fig. 28) contains 2 additional alkaloids,

* The term alkaloid for purposes of this discussion is any chromatographic spot giving an orange color with DRAG spray; thereby eliminating primary amines (blue-black color) from consideration.

Figure 41. Postulated Biosynthesis of Carnegine and Related Compounds vO ON

chromatogram:

solvent:

detection:

TLC silica gel

CHC13/CH30H 8/2 2-12

DRAG

1 2

Figure 42.

solvent front

3 4 5 6 7 8

Alkaloid Structure Comparisons

1. Hordenine

2. nor-epinephrine

3. epinephrine

4. dopamine

5. papaverine

6. sag. fresh

7. a

8. R

20

20

increasing methyla-tion

vO -o

93

one in minor amount. The alkaloid content appears greater

in the fresh pulp than in the wounded pulp (Figs. 28, 29),

but this was not fully investigated.

4. Phenolic Amines

Recently, phenolic amines (catechol amines) have

become quite important in concepts of metabolic regulation.

Finkle " has pointed out the similarities in metabolic

activity, structure, biosynthesis, and regulatory func

tions of plant phenolic amines and phenolic acids with

those of animals. Daly has studied the animal phenolic

amines, adrenaline (16), noradrenaline (17), and serato-

nin (IS), indicating their similarities to plant phenolic

amines. Other workers have studied the effects of

phenolic amines on the central nervous system and their

general metabolic fate. Dopamine, the phenolic amine 119-121 found in saguaro pulp, has been found in many plants. 7

(16) (17)

99 121 Gardener has found the oxidation product of dopamine to

119 be toxic to Cercospora beticola. Buckley 7 has done con

siderable work with dopamine in relation to the blackening

reaction in bananas.

A plausible biosynthesis and some possible metabolic

pathways of dopamine are shown in Figs. 43> 44. Although

tyrosine was found to occur in the saguaro pulp, whether

dopamine was synthesized via tyramine (19) or DOPA (20) is

not known (both were chromatographically undectable). Con

sidering the structural similarities of dopamine to known

hormones, it would appear that in addition to functioning

as a precursor for alkaloids and melanin, it may also

exert a hormonal influence during callus formation.

B. Mechanism of Callus Formation

From this study, it is possible to postulate sev

eral feasible mechanisms for the formation of callus tis

sue: 1) oxidation of phenols to form a physical barrier

(lignin), 2) precipitation of tissue destructive enzymes

by polyphenols (melanin), 3) host production of bacteri

cidal compounds in response to injury, and 4) a combina

tion of all of these factors.

The presence of some saguaro extracts which are

able to inhibit E. carnegieana growth suggests that these

extracts may have a bactericidal effect on the invading

bacteria; however, this role is probably a minor one,

DOPA (20)

Melanin

Tyramine (19)

Dopamine

Enzyme Inhibition

Alkaloids

*

on

Norepinephrine

Stimulation of Lignin Precursors

Figure 43. Biosynthesis and Metabolism of Dopamine 122

O O

VV\

Melanin

Coi

.CoT

Dopamine Quinone

Dopachrome

21

Ascorbic Acid

Y r-\

«0>

(Trace Amount)

Figure 44« Melanin Formation Via Dopamine

102

considering the overall wound response. It was described

earlier (page90) how lignin may be formed from available

saguaro constituents. Several authors 5 have shown how

oxidation of phenolic compounds may result in the forma

tion of physical barriers within the plant. The physical

appearance of the callus tissue is that of such a barrier,

with the callus itself apparently acting as a stimulus

for further callus production. Possibly this is related to 1$* no 17

seasonal growth in the cactus, ' as photomicrographs '

(c.f., Figs. 30,32) indicate an alternating of ligniferous

material with carbohydrate within the callus. It thus

appears that if sufficient callus forms rapidly enough

around a wound or an initial site of infection, the estab

lishment or further spread of an infection could be

stopped. The callus may form in some areas in response to 17 constant exposure to the environment (e.g., wind, sand);

with such a gradual process the callus barrier may easily

build up rapidly enough to prevent any transmission of pos

sible infection. However, on other types of wounding,

melanin production may beneficially augment the callus for-123 mation by precipitating invading enzymes. Steelink has

investigated the red color produced when a wounded area of

the saguaro is exposed to air, concluding that red color

is due to dopachrome (21) production. Dopachrome is a

well-known intermediate " 'in melanin synthesis.

103

It appears possible that melanin formation could be a tem

porary response to injury, with lignin production result

ing in a more permanent response.

Dopamine is quite easily transformed into

melanin 'and with its previously-mentioned similarity

to hormonal compounds, it could well control the synthe

sis of lignin.

The compartmentalization theory''" f'"'" ~" of plant

response to injury could easily account for the initial

stimulus of dopamine production. This theory states, in

general, that two substances such as a substrate and its

enzyme are separated by a cell wall. When the cell wall

is ruptured (by action of a pathogen, mechanical injury,

or freezing temperatures) the substrate and enzyme come in

contact forming a product (in this case, dopamine). 126 S.-cndheimer has pointed out that when phenols

are functioning in a regulatory capacity, they are

affecting IAA metabolism. He has also shown how phenols

exert a synergistic effect with IAA in affecting cell

elongation; however, Sandheimer is quick to point out that

there are a large number of phenols that do not affect

regulatory systems.

Thus, it appears that dopamine may be a crucial

intermediate in callus formation. It is known that dihy-

dric phenols inhibit IAA oxidase and that IAA is related 53 to cell enlargement. Although the presence of J.AA was

104

not demonstrated in the saguaro (chromatographically), its 52 53 occurrence in other plants is well documented. 'The

127 phototropic effect of IAA has also been well documented, '

and may play a minor roll in callus formation. Zenk and

Muller have shown a relation between IAA concentration and i2d area of wounded surface in Helianthus. IAA accumulation

129 130 at the site of wounding is well documented. J Quite

possibly the increase in dopamine concentration as a result

of wounding could affect the IAA (and, therefore, gibber-

ellic acid and kinetin) in cell division.131-133 con

currently- dopamine may be stimulating the production of

lignin precursors, thus permitting the development of lig-

nin and new cells to occur in the same area (Fig.

In summary, it appears that callus formation is a

result of several processes, apparently interrelated through

dopamine.

bound phenol light

I feeding of

early precursors by enzymatic regulation

/

105

flavoprotein

activate IAA oxidase

wounding

*

/ /

> inhibit IAA oxidase

/ DOPAMINE kinetin \

lignin precursors I gibberel-lie acid

increase in IAA

melanin (temporary response)

y

lignin

«4/

cell division

callus

Figure 45. Postulated Mechanism of Wound Response

V

o o

WAVEKNGTH JMOONSI „ Q „

XR-1 Unextracted Callus Phase: KBr

4000 3000 2000 1500 1200 1000 CM'

900 800 700

IR-2 Native Lignin, P3<Sp Phase: KBr

M a; ?o 5 W a CO

m o •-3 to >

4000 3000 2000 1500 1200 1000 900 800 700

WAVEI r TH, r*T. 11 12 13 14

4000 3000 2000 1500

XR-3 Stean Distillate Phase: CHCJ_

IH-4 Oxidation Product, of P22A Phase: CHC1

yniifniiiiEs KfiSHU

H O ->3 4000 3000 1500 1000 900

APPENDIX A

Cross Section of the Saguaro

epidermis

pulpy cortex

ribs

callus tissue

spines

108

APPENDIX B

Data Sheet for Thin Layer Chromatograms

TLC # Operator_

Literature: Date

Solv. Syst..

Solv. Dist.

In

Time:

Quant. Appl.a

_Adsorption Layer_

cm.

Out

Detection:

Remarks: Yes_

Date

TLC #

No (over)

Type Recording

Phot o

N eatan

Other

Substance Notes

1

2

3

k

5

6

7

a

9

10

11

12

109

X

APPENDIX C

Common Names of Chemicals Used in This Work

Common Name Chemical Name

dopamine tyramine dopa epinephrine

adrenaline nor-epinephrine

nor-adrenaline protocatechuic acid chlorogenic acid vanillic acid syringaldehyde vanillin coniferyl alcohol

gentisic acid homogentisic acid ferulic acid caffeic acid veratrole seratonin papaverine

carnegine

3 >4-dihydroxy-P-phenylethylamine 4-hydroxy-0-phenylethylamine 3,4-dihydroxyphenylalanine N-methyl-a-hydroxy-3,4-dihydroxy-

0-phenylethylamine see epinephrine a-hydroxy-3,4-dihydroxy-0-phenyl

ethylamine see nor-epinephrine 3.4-dihydroxybenzoic acid quinic acid ester of caffeic acid 3-methoxy-4-hydroxybenzoic acid 3.5-dimethoxy-4-hydroxybenzaldehyde 3-methoxy-4-hydroxybenzoic acid 3 -methoxy-ii--hydroxycinnamyl

alcohol 2,5-dihydroxybenzoic acid 2,5-dihydroxyphenylacetic acid 3-methoxy-4-hydroxycinnamic acid 3,4-dihydroxycinnamic acid 1,2-dimethoxybenzene 5-hydroxytryptamine 1-(3 > k*-dimethoxybenzyl)-

6,7-dimethoxyisoquinoline 1,2-dimethyl-6,7-dimethoxy-

1,2,3,4-tetrahydroisoquinoline

110

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a biochemical investigation of callus tissue...

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