A STUDY OF THE INFLUENCE OF

GIBBERELLIC ACID ON

DIGITALIS PURPUREA L. AND FAGOP1RUM ESCULENTUM MOEMCH

Dissertation

Presented in Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in

the Graduate School of the Ohio State

University

By

MAHMOUD DARWISH SATED, B. Ph.Ch., M. Pharm.

The Ohio State University

1958

Approved by

^7 Adviser College of Pharmacy ACKNOWLEDGMENTS

I wish to express my sincere appreciation and deep gratitude to Dr. Jack L. Beal, for his guidance, counsel and encouragement without which this work could not have been ful­ filled.

To the following persons who have served on the advisory committee or reading committee and/or given invaluable classroom instructions, I offer my gratitude and obligation: G.W. Blaydes,

R.H. Bohning, E.P. Guth, L.E. Harris, J.W. Nelson, L.M. Parks,

R.S. Piatt.

I wish also to acknowledge Dr. W.B. Mcintosh's assist­ ance in the statistical evaluation of the data in this dissertation.

To the friends and colleagues who have shown interest in this work and who have offered valuable suggestions, I express my thanks.

The Author

ii TABLE OF CONTENTS

PAGE ACKNOWLEDGMENT ii LIST OF TABLES v LIST OF FIGURES vii INTRODUCTION 1 Review of the Literature 1 Statement of the Problem 16 Chosen Experimental Plants 17 Purpose of the Research 17 EXPERIMENTAL Preparation of Gibberellic Acid Solution 19 Digitalis purpurea L. 19 Raising Digitalis Plants 19 Treatment of Digitalis Plants with Gibberellic Acid Solution 19 Harvesting and Sampling of Digitalis 19 Morphology and Histology of Treated and Control Plants 20 Influence on the Dry Weight of the Leaves 20 Evaluation: Preparation of Standard Curve of Digitalis Glycosides 33 Assay of Samples for Total Glycosidal Content 36 Determination of Total Sugar Contents 39 Determination of Digitoxose Sugar 40 Preparation of Standard Curve of Digitoxose Sugar 40 Determination of Crude Fiber Content 46 Determination of Chlorophyll 47 Fagopyrum esculentum Moench 54 Raising Buckwheat plants 54 Treatment of Buckwheat Plants with Gibberellic Acid Solution 54 Harvesting and Sampling of Buckwheat Samples 54 Influence of Gibberellic Acid on the Linear Growth and Dry Weight of the Plant 54 Evaluation: Preparation of Standard Curve of Rutin Glycoside 5& Assay of Buckwheat Samples for Rutin Content 57

iii TABLE OF CONTENTS (Continued)

PAGE Determination of Total Sugar Content 57 Separation of Rhamnose Sugar Using Paper Chromatography Technique 67 Localization of Rhamnose on Chromatograms 69 Preparation of Standard Curve of Rhamnose 71 Per Cent Recovery of Rhamnose from Chromatograms 72 Quantitative Determination of the Measurable Rhamnose in Treated and Control Samples of Buckwheat 75 Determination for Quercetin using Paper Chromatography - 77 Determination of Crude Fiber Content 79 Determination of Chlorophyll Content 83

DISCUSSION 84

SUMMARY 93

CONCLUSIONS 96

BIBLIOGRAPHY 98

AUTOBIOGRAPHY 105

iv LIST OF TABLES TABLE PAGE I Fresh and Dry Weight of Digitalis Samples 21 II Length of the Petioles and Dimensions of Treated Control Digitalis Leaves 31 III Surface Area of Treated and Control Digitalis Leaves 32 IV Palisade Ratio and Vein-islet Number of Treated

. and Control Digitalis Leaves :~ 33 V Data for. Standard Curve of Digitalis Glycosides —— 34 VI Total Glycosides in Digitalis Samples 36 VII Percentage of Total Sugar in Digitalis Samples 41 VIII Data for Standard Curve of Digitoxin 44 IX Percentage of Digitoxose in Digitalis Samples 46 X Percentage of Crude Fiber in Digitalis Samples 48 XI Percentage of Chlorophyll in Digitalis Samples 53 XII Data on Length of Stems, Dry Weights of Stems

and Leaves of Buckwheat Samples 55 XIII Data on Number of Leaves Per Plant 56 XIV Data for Rutin Standard Curve 58 XV Percentage of Rutin in Buckwheat Samples 60 XVI Per Cent of Total Sugar in Buckwheat Samples 63

XVII Rf and R„ Values of Rhamnose and Separated Sugars — 69 XVIII Data of Rhamnose Standard Curve 73 XIX Percentage Recovery of Rhamnose 75 XX Percentage of Rhamnose in. Buckwheat Samples 77 LIST OF TABLES (Continued) TABLE PAGE XXI R of Rutin and Quercetin 79 XXII Per Cent Crude Fiber in Buckwheat 81 XXIII Per Cent of Chlorophyll in Buckwheat Samples 83 XXIV Rutin Content Calculated on Dry Weight Basis of Leaves Per Plant 91 XXV Average Determination of Dry Weight, Measurements of Lamina and Surface Area of Digitalis Leaves 9^ XXVI Average Per Cent of Evaluation of Digitalis Samples 9k XXVII Average Per Cent of Evaluation of Buckwheat Samples 95

vi LIST OF FIGURES FIGURE PAGE 1 A Control Digitalis Plant 22 2 A Treated Bolting Digitalis Plant 22 3 A Treated Digitalis Plant 23

4 Diagram of Treated, Control Digitalis Leaf and Stem 24 5 Diagramatic Transverse Section of the Stem 26 6 Transverse Section in the Stem 27 7 Isolated Elements of the Stem 28 8 Standard Curve for Total Glycosides of Digitalis 35 9 Analysis of Total Glycosides of Digitalis Samples 37 10 Variation of Glycosidal Percentage of Digitalis Samples with Period of Harvest 38 11 Per Cent of Total Sugar in Digitalis Samples 42 12 Variation of Total Sugar Percentage of Digitalis Samples with Period of Harvest 43 13 Standard Curve Used for Determination of Digitoxose - 45 14 Per Cent of Crude Fiber in Digitalis Samples • 49 15 Standard Curve for Rutin 59 16 Per Cent of Rutin in Buckwheat Samples 61 17 Variation of Glycosidal Percentage of Buckwheat Samples with Period of Harvest 62 18 Per Cent of Total Sugar in Buckwheat Samples 64 19 Variation of Total Sugar Percentage of Buckwheat Samples with Period of Harvest 65 20, Illustrations of Rj of Glucose, Rhamnose and Sugars 24 Separated from Buckwheat Extract 70 vii LIST OF FIGURES (Continued)

FIGURE PAGE

25 Standard Curve for Rhamnose 74

2.6- Illustrations of Attempts of Separation of 30 Quercetin from Buckwheat Extract 80

31 Per Cent of Crude Fiber in Buckwheat Samples 82

viii INTRODUCTION

Review of the Literature

During the past thirty years a number of Japanese botanists have been working on a group of plant growth substances which were first shown to be produced by a rice disease fungus, Gibberella

Fu.jikuroi, by Kurosawa (1), a Japanese plant pathologist working in

Formosa. This group named Gibberellin after the fungus by investi­ gators at the University of Tokyo, notably Yabuta and Sumiki (2).

It was these men who were responsible for the isolation of the

Gibberillins and the first studies of their properties. Around 1952, their work came to the attention, apparently independently, of a group at the Imperial Chemical Industries in Britain, and another group at the United States Department of Agriculture. These two groups confirmed the Japanese work and contributed their own find­ ings, bringing their results to a much wider audience.

History and Identifi cat ion of the Fungus Producing the Gibberellins

Ito and Kimura (3) attributed the earliest known description of what is now called the "bakanae disease" to Konishi (4), a semi- literate farmer who dictated an agricultural book in 1809. The most characteristic symptom of the disease is the appearance of tall thin plants, markedly overgrowing their uninfected neighbors.

The first description of the disease with its causal fungus is attributed to Hori (5,7). The most complete monographs on this

1 2 disease are those of Ito and Kimura (3) which covers aspects of the history, symptoms, methods of infection, treatment and recom­ mended agricultural practices to reduce its incidence. The disease has since been described in India (8), in the Philippines (9)i in

China (10),in British Guiana, where it is known as "man rice" (11), and in Ceylon where it is called "Wanda peedema" (sterile ripen­ ing) (12).

Hori (5) identified the agent of the disease as an imper­ fect fungus, Fusarium heterosporum Nees. Later, Fujikuro discovered the perfect stage and this was described as Lisea Fujikuroi by

Sawada (13). Noting the similarities of this micro-organism to

Giberella moniliformis Winel, Ito (3) suggested Gibberella Fu.jikuroi as a more generic designation. Today, this is the most accepted nomenclature.

The agreement on the identify of the bakanae organism has left one question unexplained. Why does a broadly distributed fungus apparently cause its overgrowth effect naturally only in rice? No natural occurrence of an overgrowth in plants other than rice seems to have been reported, although artificial infections have led to overgrowth in mait^ '1^-17), barley (14-), sugar cane (16), wheat (17), and oats (17).

A conclusive answer to this paradox has not yet been given, but the following clues are available. Ito and Kimura (3) and later

Stol (18) reported that once effective strains maintained in artificial culture lost their bakanae-inducing ability after a few months, and 3 this could not be re-established by inoculation on rice and re- isolation. However, Stol did restore the overgrowth symptom when he grew the fungus on a rice grain medium. It is thus possible that some nutritional factor supplied by rice promotes the over­ growth. There is also some evidence of an effect of temperature on the infected plant. Many workers have shown that the optimum temper­ ature for the growth of the fungus is about 27°C, but for the infection, 30°C. is optimal (19), whereas Seto (20) stated that 35°C. is best for the bakanae effect. Although Seto (21) and Hemmi (22) have indicated that different strains were not identical in temper­ ature response, it seems clear that the optimum temperature for the increased linear growth of the plant was always about 5°C. above that which is optimum for the in vitro culture of the fungus. Another variable is the soil moisture. Sawada and Kurosawa (23) and Taka- hashi (2*0 reported less disease in well-watered soils.

The Production of Gibberellins

All research on the Gibberellins stems from the work of

Kurosawa (25). In 1926, after several failures, he succeeded in producing the bakanae effect in rice and maize seedlings solely by treating them with a culture medium in which G. Pujikuroi had been grown.

The Tokyo workers investigated the properties of the bakanae-inducing substance (26), (27) and reported that it is best adsorbed from the cultural filtrate on activated animal charcoal and eluted by basic methanol. It was given the name Gibberellin k

by Yabuta (28). With these facts in mind they devised a general

scheme of isolation (29)* which has been used with minor modifica­

tions by all workers since.

The culture filtrate is treated with activated animal

charcoal. The adsorbed substances are eluted with methanol ammonia, and the methanol extract is evaporated to dryness. The residue left is taken up in bicarbonate and extracted with ether, ethyl acetate

(30,31) or butyl acetate (32). The last step has varied from time to time. Some studies included lead acetate precipitation, and final separation was done using counter-current distribution.

The substance isolated by these Japanese workers was thoroughly investigated for its physical and chemical properties and was found to be nonenzymatic, since it survived three hours boiling (33). Ito and Shimada (33) at Hokkaido University confirmed the heat stability of the substance but found that its activity dis­ appeared on ashing, that it was not volatile, that it could be dial- ized, and that it was adsorbed on animal charcoal.

Kurosawa (3*0 substantiated those observations by showing that it was stable to autoclaving, freezing, sunlight, and short exposures to ultraviolet light.

Although the active principle is heat.stable, activity generally decreases by increasing temperature and markedly decreases on boiling (33). It was postulated that either part of the activity is heat labile or that an inhibitor is produced on heating.

The effect of temperature on the activity of Gibberellic acid was thoroughly investigated by Kurosawa (35)• He concluded 5 that maximum activity is obtained at 20°C. A British worker (31) reported that activity is best at 25° - 30°C. Further investiga­ tion on the effect of temperature on activity is worth while.

Investigators at Tokyo University (36) isolated an inhibitory material from the fungal extracts (from which Gibberellic acid is isolated), thus confirming the frequent suggestion that both a growth-stimulating and a growth-inhibiting material were produced.

This inhibitor was named "Fusaric acid" and recently given the name

Fusarinic acid (36). Chemically it vras shown to be 5 - n - butylpic- olinic acid (36). This compound is a strikingly potent growth inhib­ itor and wilting agent, being effective in some plants in concentra­ tions as low as 0.1 mg./L. Recent studies in Japan (37) and in Switz­ erland (38) have shown that its effectiveness seems to be due to its chelating properties, which make it a strong inhibitor of certain enzymes.

The Chemistry of Gibberellins

Yabuta and Sumiki (39) announced in 1933 the isolation of two crystalline biologically active principles from G, Fu.jukuroi, which they named Gibberellins "A" and nB." In a series of papers on the degradative chemistry of Gibberellins "A" and "B" (40-47), it was shown among other points that Gibberellin "A" is converted by heating at 50 - 70°C. in dilute acid into Gibberellin "B." Raising the temperature to boiling converted both "A" and "B" into another compound, Gibberic acid, which is not biologically active. A third biologically effective compound, Gibberellin "C," was also isolated by acid treatment, of Gibberellin "A" (41), (46), (47). 6

The first work on the Gibberellins in the United States was carried out by J.E. Mitchel and Angel (48), at Camp Detrick,

Maryland. They cultured the fungus and obtained the stimulated linear growth of beans by using the fungal culture medium. Their work prompted a group at the U.S. Department of Agriculture, headed by Stodola (49), to undertake the purification of the active principle of the cultured fungus. Apparently almost simultaneously workers at the Imperial Chemical Industries in Britain also began large- scale preparation of the material. The British investigators reported the isolation of one single compound which was later given the name

Gibberellic acid (50). The U.S.D.A. Chemists obtained a mixture of

Gibberellic acid and a compound similar to Gibberellin "A" of the

Tokyo group, which could be separated by partition chromatography (50).

In view of this work, Takahashi et al. (52) re-examined the University of Tokyo's Gibberellin "A" upon paper chromatography in ten solvents, partition chromatography in several systems, and by countercurrent distribution. Their material appeared homogenous. However, esterification followed by chromatography on alumina led to the isolation of three methyl esters. All three parent acids of these esters have biological activity and they were named Gibberellin "A"-,,

"A"2 and "A"o. Comparison of "A"-} with Gibberellic acid of the

British and American groups showed them to be identical, and the latter name was accepted by the Tokyo group.

All three Gibberellins have decomposition points falling within the range 232 - 237°C; their mixed decomposition points are 7

not significantly depressed; and their infrared spectra are closely

similar (32). It is not surprising, therefore, that their crystal­

line mixture had so long been considered homogenous. The compounds

can be differentiated by their methyl ester, their melting points,

their specific rotation, and their behavior to acid degradation.

Although the existence of these three distinct Gibberelllns

seems established, only Gibberellic acid has been thoroughly char­

acterized. It was first identified as 1, 7-dimethyl fluorene, and

then Cross et al. (53) proposed the following tentative structure.

This structure bears no close relation to any previously described

natural product, nor does it resemble any auxin. Chemical assays of

Gibberellic acid are in the process of development.

Physiological Action of Gibberellic Acid

The most typical and striking plant response to treatment

with Gibberellic acid is stem elongation. Hori's initial paper (5*0

cited stem measurement to be 150 per cent of normal. In general,

the effect of Gibberellic acid is mainly limited to younger tissues

which are still growing. However, the response of the plant varies widely, even within a species, and may be influenced by external

conditions and the age of the plant. 8

The anatomical basis of the increased growth has been the object of several studies. Sawada (55) and Kurosawa could not detect any cell length differences in infected rice plants and suggested an increased cell division. Later Kurosawa (56) showed that epidermal and parenchymatous cells of leaf and internode were longer and decreased slightly in their radial and tangential diam­ eter s.Recently Lang (57) et al. reported that a treated variety of

Hyoscyamus nipcer showed a striking increase in the number of cell divisions in the subapical region starting between 12 and 18 hours after treatment. Cell division in the apical region was unaffected.

The same author concluded that Gibberellic acid treatment had a directional influence in the subapical region, with longitudinal cell division being greater than transverse division.

In a study to determine the best concentration of

Gibberellic acid solution for treatment, Chardon (58) treated pine­ apple plants with solutions of the following concentrations: 50, 100,

200, and 500 parts per million. Growth averages taken at 1^1- and 28 days respectively were, 100, 122, 158, and 1^6 per cent over the control plants.

With sugar cane plants treated with 20 and 100 p.p.m. of

Gibberellic acid, the increase in growth over untreated plants was

60 and 108 per cent respectively. Kato and Yukio (59) reported that concentration of 100 p.p.m. accelerated the pollen germination and pollen-tube growth of Lilium longiflorum. However high concentrations of Gibberellic acid are growth inhibitory, but not always toxic (60). 9

In general, optimum concentration is not universal. What is true for one plant may not be true for another.

Leaf Expansion

It is apparent from several studies of diseased rice plants that the leaves increased in total area (6l). However, the leaves of treated tomato and three cucurbits showed marked reduction in surface area (62). The number of leaves remained about the same in tomato but slightly increased in cucumber. A group of investigators at Tokyo University reported a decrease in surface area of leaves of treated tobacco (63). The treated plants contained only one quarter as much nicotine per gram of dry weight as did the controls. When tea plants were sprayed with a solution of Gibberellic acid (100 p.p.m.) and harvested three weeks later, the total fresh weight and number of leaves were increased by 58 and 28 per cent respectively (6*0.

Two other experiments carried out late in the season after the second picking on another variety of tea showed a decrease in both these quantities, although the fresh weight per bud was higher.

In all experiments there are clear differences between the effect on the leaves on upper and lower parts of the stem. The upper leaves were always equal or less in weight than controls, whereas the lower leaves weighed more. The quality of the tea is stated to be unchanged although the younger leaves were yellowed and the older leaves wrinkled by the Gibberellic acid.

Sunflower and Soybean leaf area was also reduced in short- term experiments (65). 10

Curry and Wassink (65) reported that Gibberellic acid induced reduction in leaf number of treated plants.

Kurashi and Hashimoto (66) have found that kinetin pro­ moted leaf expansion similar to Gibberellic acid,and when they were applied to the plant together, both the fresh weight and the area increments were nearly the sum of the effect of either compound alone, amounting to nearly a 100 per cent increase.

Chlorosis

Chlorosis is a phenomenon resulting from inhibition of chloro­ phyll synthesis. It has been reported that some plants treated with

Gibberellic acid showed this condition as well as a decrease in the rate of chlorophyll synthesis compared with the control plants (61).

Chardon (58) reported yellowing of leaves of pineapple plants following treatment with Gibberellic acid solution. The yellowing was overcome by spraying the leaves with 2 per cent urea solution.

With sugar cane seeds dipped in 2 per cent urea solution for 5-15 hours and treated with 20 and 100 p.p.m. of Gibberellic acid solution, the increase in growth over untreated seed was respec­ tively 60 and 108 per cent. With the same Gibberellic acid concentra­ tion but without urea treatiaent, the growth increases were only *K) and

85 per cent. Leben et al. (67) recently reported that Crab grass

(Digitaria Sanguinalis L.) was sprayed with Gibberellic acid solution

(100 p.p.m.) on Sept. 10, 1956. By October 1, 1956, control plants assumed the usual autumnal red color, while treated plants remained 11 green, and had significantly elongated to a height of 26 cm. A third group treated by spraying with 10 p.p.m. remained green but did not elongate. The same investigators reported an experiment of treating Kentucky Blue Grass (Poa partensis L.) with Gibberellic acid. They stated that it was fertilized with a granulated fertilizer

(10-10-10) on October 23. 1956 and sprayed once with Gibberellic acid solution. The plants were in slow growth stage, common at this time of the year. Within **• days, the grass that had been treated with Gibberellic acid began to grow again as revealed by brightening of the green color and development of new shoots.

Flowering

In the first definitive description of the bakanae disease

Hori (35) noted that those infected plants which reached adult size fruited earlier.

The Tokyo investigators (63) tested Gibberellic acid on tobacco inflorescenses and found them to grow faster and larger. Lang

(68) reported that Gibberellic acid induced bolting and flowering in biennial Hyoscyamus niger. Plants treated daily with 2 meg. of

Gibberellic acid showed 100 per cent flower formation; 10 meg. daily induced 72 per cent flowering. This flower inducing effect of

Gibberellic acid occurred only with 18 - 20 hour days; treated plants under 9 hour days remained vegetative. Although this investigator initially reported that the effect was more marked in long days, he later reported that flower buds were also formed on short days (69) and so he concluded that Gibberellic acid apparently replaces the long day requirements for flower formation. 12 The extension of this phenomenon to other biennial plants is being actively investigated. Bunson and Harder (70) found that two species of Bryophyllum could be induced to flower in the first year with Gibberellic acid. Other biennial plants which were in­ duced to flowering by treatment with Gibberellic acid are carrots, beets, and cabbage (71).

Gibberellic acid not only influences flower formation but also flower size. Linstrom and Wittwer (72) sprayed a variety of

Pelargonium hortorium with an aqueous solution of Gibberellic acid

10 p.p.m. There was a definite increase in the size of the inflorescence. The same group sprayed a variety of Brick Red Irene, having well-developed buds, with 1, 5t 10» 25 and 100 p.p.m. of

Gibberellic acid, and in all cases the diameters of the inflorescenses were increased. At 10 p.p.m. and above, the pedicel and peduncle lengths were markedly increased such that the weight of the inflorescences broke or bent the peduncles.

The relationship of these results to light effects on flowering is being sought. Curry and Wassink (65) showed that

Gibberellic acid induced shoot growth and flower buds of Hyoscyamus in long days at all wavelengths tested; flowers failed to form only in green light. It was thus concluded that Gibberellic acid replaced the far red light required for flowering.

The varietal differences within a species to the flowering response are investigated by Wittwer and Bukovac (73)» They found that in beans and tomatoes flowering was only hastened in determinate varieties, although elongation was promoted in all plants. No 13 reduction in internode number resulted from earlier flowering, since growth of the treated plant was faster. Lettuce bolted and flowered as if normally induced, whereas cabbage formed a stalk but no flowers.

Marth (74) et al. noted that flowering was hastened in dahlia and salvia but retarded in geranium and peppers. In general the data suggested that Gibberellic acid acts primarily on elongation, incidentally releasing the flowering response in some cases rather than directly on flowering itself. Recently (75.76), substances with identical physiological activity to that of Gibberellic acid have been shown to occur also in flowering plants, which possibly suggests a basic role for Gibberellic acid in the flowering mechanism. It is likely that Gibberellic acid will strongly influence future work on flowering and may lead to practical application.

Seed Germination

It has been observed that seed germination time is reduced by Gibberellic acid treatment. Hayashi's data (77) show that barley and rice germinated more rapidly when treated with Gibberellic acid.

Helgeson and Green (78), working with seeds of wild oats, reported that seeds dipped in Gibberellic acid solution 50 p.p.m. for about

2 hours germinated faster than untreated seeds. Recently Wittwer and

Bukovac (79) reported that Gibberellic acid incorporated with a slurry seed protectant and applied to the seed coat of peas and beans promoted earlier germination in both greenhouse and field plantings.

Heights, lengths of hypocotyls, and intemodes of plants grown from treated seeds were directly related to the concentration of Gibber­ ellic acid in the slurry. Appropriate concentrations of Gibberellic 14 acid in a slurry for induction of earlier germination and production

of acceptable seedlings in peas and beans ranged from 500 to 1,000 p.p.m. Consistently favorable seedling growths was not achieved

at concentrations of 2,500 p.p.m. or higher.

Metabolism

Ito and Kimura (80) found that bakanae plants had increased in fresh weight, but dry weights were less. Haan (81) and Edward

(82) reported a definite increase in dry weights. Shimada (83) showed that elongation paralleled fresh weight increases in several plants.

The most detailed studies of the effect of Gibberellic acid on dry weights are those of the I.C.I, group (84) who showed that the total dry weight of wheat and peas is increased by Gibberellic acid treat­ ment. The increased dry weight was shown, in most cases, to be due, in part, to an increase in carbohydrates. It should be noted that this effect of Gibberellic acid is not directly related to photo­ synthesis, since bakanae effect occurs in the dark (62,63,85).

It was reported that enzymatic activity increases in treated plants proportionally to the applied Gibberellic acid concentration

(86).

Recently Hayashi et al. (87) have examined changes of enzymatic activity with time during the development of the seventh leaf of rice.

Activity of phosphatase, alkaline pyrophosphatase, acetyl esterase, maltase, B-glycosidase, -galactosidase, amylase, urease, ascorbic acid oxidase, and catalase increased significantly in treated 15 plants over that in control plants. However, peroxidase and invertase were found not to be influenced by Gibberellic acid. The same investigators concluded that Gibberellic acid does not influ­ ence any qualitative change in the activity of these enzymes, but possibly some quantitative changes may occur in treated plants.

Recently it has been reported that the activity of polyphenol oxidase was doubled in treated tomato plants over the controls, while ascorbic acid oxidase was not significantly affected (88).

Relationship to Auxins

Although Gibberellins promote cell elongation, it is apparent that they differ from Auxins in several respects. They do not, in short term tests, inhibit root growth; they inhibit rather than promote root initiation, and they are more potent than auxins

in inducing parthenocarpy (a condition of seedlessness). The designa­ tion "seedlessness" as applied in this connection refers to a lack of viable seeds; many parthenocarpic fruits contain partially developed

seeds which are empty, i.e., lacking an embryo.

Until the specific site of action of Gibberellic acid is

identified, any definition for this substance will remain in part

ambiguous. For the present it has been suggested that Gibberellins

be defined as that class of compounds which cause intemode elongation when applied to certain intact gentically dwarfed plants (89). 16

STATEMENT OF THE PROBLEM

The previously reviewed literature revealed that Gibber­

ellic acid has a definite influence on the growth of plants, as well

as the biosynthesis of some plant constituents, e.g., carbohydrates,

chlorophyll in some plants, and nicotine in some species of Nicotiana tabacum. Enzymatic activity, as that of phosphatase, maltase,

B-glycosidase, galactosidase, esterase and possibly others, is

affected by Gibberellic acid. However, the influence of Gibberellic

acid on glycoside biosynthesis has never been reported. The fact that carbohydrate was reported in many cases to be increased led to the idea that glycoside biosynthesis may be also affected, owing to the ultimate relation between the metabolism of both substances as

illustrated in the following diagram.

Sugar Fraction

m 4- w n Photo- ^ C^ (Hexose) Enzyme ^Glycoside ^ synthesis ^0 ? / ^0 ? / / °x< / Aglycone

For this purpose it was decided to study the influence of Gibberellic acid on two glycosidal containing plants, namely Digitalis purpurea L.

(Foxglove), Fam. Scropholureaceae and Fagopycum esculentum Moench

(Buckwheat), fam. Polygonaceae. These plants were chosen because -

1. They represent two different types of over-all growth.

Digitalis does not develop measurable internodes (the over-ground part consisting mainly of cluster of leaves), while Buckwheat has an erect herbacious stem. In addition, Digitalis is a biennial plant, while Buckwheat is an annual plant.

2. Their methods of assay are established.

3. They grow well in Ohio.

4. Their active constituents are recognized as thera­ peutic agents.

Purpose of the Research

The objective of this investigation is to study the influence of Gibberellic acid on -

1. Growth.

2. The morphology and histology of the plant.

3. The biosymthesis of glycosides in the experimental plants, as well as the rate of glycoside formation during the grow­ ing season.

k. Carbohydrates or plant constituents related to glycoside biosynthesis.

For items No. 1 and 2, the following steps were decided upon:

1. Periodic measurement of stems or other organs of the plant, if

necessary, as well as the dry weight 2. The morphological and histological study of organs that show

any response to Gibberellic acid

For items No. 2 and 3 the following determinations were decided to be carried out:

1. Periodic determination of the glycosidal content during the

growing season

2. Determination of total sugar content

3. Determination of aglycone and sugar fractions k. Determination of crude fiber (as a criterion for cellulose

content EXPERIMENTAL

Preparation of Gibberellic Acid Solution

Gibberellic acid used in this investigation was in the form of potassium salt.* One hundred milligrams of this salt were dis­ solved in distilled water in a liter volumetric flask and the solu­ tion completed to volume. This solution represents 100 p.p.m. and was used in spraying the treated plants.

Digitalis purpurea (Foxglove)

The Digitalis plants were started from seeds in a green­ house and then transplanted into pots in March, 1957. They were transplanted into the Medicinal Plant Garden of the Ohio State Univer­ sity on May 18, 1957• They were divided into treated and control rows which were arranged alternately. Spraying was delayed until the plants recovered from the transplantation into the Medicinal Plant

Garden; it was started on June 17 and was continued twice weekly until September 5*

Harvesting was done at approximately weekly intervals be­ tween July 17'and September 12. The leaves gathered from both the treated and control groups were separately pooled, and three samples were drawn at random from each group. The samples were dried at a temperature of 70°C, reduced to a No. 60 powder, and saved for evaluation.

* Gibberellic acid, as the potassium salt, was supplied through the courtesy of Merck and Co., Inc.

19 20

Fresh and Dry Weight Determination

Three samples each consisting of five leaves were drawn at random from each group. The fresh and dry weight (dried at 70°C. for 2k hours) were determined. The results obtained are given in

Table I.

Influence of Gibberellic Acid on the Morphology

Normally Digitalis purpurea does not develop a detectable stem during the first year of growth. However, some of the treated plants bolted forming a definite stem but they did not flower

(Figs. 1,2,3). The stem that was developed was subjected to detailed morphological and histological study.

The Stem

The stem developed by some of the treated plants, (Fig. k), is erect, woody, longitudinally wrinkled and very hairy. It measures

25-30 cm. in height and 1.5-2 cm. in diameter. In transverse section

(Fig. 5). it shows a relatively wide cortex bounded on the inside by a distinct endodermis and on the outside by a hairy epidermis. The vascular system consists of collateral bundles which are separated by narrow medullary rays, and. surround a large parenchymotous pith.

The wood is porous. The phloem consists of soft bast. The pericycle consists of parenchyma cells with distributed large isolated, or 2-4 groups of fibers.

Epidermis. The epidermal cells (Fig. 6) are rectangular or subrect angular with slightly wavy anticlinal walls. In size they vary 21

TABLE I

Fresh and Dry Weight of Digitalis Samples

Fresh Weight (5 leaves)GM Dry Weight (5 leaves) Sample Treated Control Treated Control

July 17 39.^ 4.4 6.9 10.0 32.5 37.4 6.4 8.3. 37.6 31.3 7.8 6.3 Average 33.1 37.5 7.03 8.2 July 24 18 4.2 3.6 11.0 19.3 3.2 3.9 7.9 14.2 29.7 3.1 7.8 Average 17.1 34.5 3.5 8.9

July 31 17.1 36.5 **.? 9.05 15.2 33.7 3.8 8.05 21.2 37.5 5.3 8.35 Average 17.8 35-9 4.6 8.48 August 9 '20.6 45.0 4.6 11.3 21.36 36.0 4.85 8.1 17.60 31.7 4.10 8.0 Average 19.86 37.5 4.51 9.1 August 23 19.2 30.1 4.4 7-3 19.55 29.2 4.4 6.2 20.0 27.4 4.7 6,6 Average 19.58 28.9 4.5 6.7 September 12 19.1 32.15 4.30 7*28 19.41 28.9 ^•37 6.45 20.30 26.45 4.50 6.00 Average 19.6 29.1 4.39 6.51 Fig. 1 - A Control Digitalis Plant

, '', |i#*¥tfc«M t

'** WW^iS**:' -^.^

5.=->^a

Fig. 2 - A Treated Bolting Digitalis Plant 23

«^jp M, s\r

&/$> »-"

Fig. 3 - A Treated Digitalis Plant Fig. k - Diagram of Treated, Control Digitalis Leaf and Stem x l/3.

T: Treated Leaf; C: Control Leaf; Sti Stem 25 from 32 u to 40 u in breadth and 12-16 u in height. The outer wall is covered with a thin smooth cuticle. In surface view

(Fig. 7) they appear polygonal with slightly wavy outline. Stomata

(Fig. 7) are few in number, each being surrounded by 3-4 cells of the Ranunculacious type. The covering trichomes are numerous. They consist of 3-7 cells. The average length of these hairs is from

330-^50 u.

The cortex (Fig. 6) varies in thickness from 10-13 layers of cells. The outermost layers are collenchymatously thickeneds gradually passing into thin-walled parenchyma.

The endodermis consists of tangentially elongated cells with slightly thickened radial walls.

The pericycle (Fig. 6) shows isolated or 3-4 grouped ligni- fied fibers separated by pericyclic parenchyma. The fibers are slight­ ly angular or circular in cross section with varying wide lumen. They are 30-^u-'indiameter and 220 to 250 u in length.

The phloem tissue is well developed and consists of sieve tubes accompanied with companion cells and intermixed with phloem parenchyma.

The wood (Fig. 6) consists mainly of scalariform and spiral vessels varying from 33-78 u in diameter. The vessels are intermixed with wood parenchyma, tracheids, and wood fibers. The wood fibers are more or less similar in shape to the pericyclic fibers.

The pith is composed of large polygonal parenchyma with distinct intercellular spaces and may show simple pits. 26

Fig. 5 - Diagramatic Transverse Section of the Stem x 60. Ep: epidermis; coll: collenchyma; End: endodermis; P.F.: pericyclic fibers; Ph: phloem; x: xylem; P: pith. 27 28

Fig. 6 - Transverse Section in the Stem x 120 Coll: collenchjrma; Par: parenchyma; End: endodermis; P.F.: pericyclic fibers; Ph: phloem; P: pith Fig. 7 - Isolated Elements of Stem x 60. H: nonglandular trichome; W. par: wood parenchyma; Ep: epidermis in surface view; Tr: tracheid; F: fiber: Ves: vessel v^o3 30

The Leaf

The leaves of the treated plants showed a marked morpho­ logical difference from the control (Fig. ty). Their petioles were much longer than those of the control, and their lamina were much more linear and almost lanceolate in shape. For the purpose of comparison, k5 leaves were harvested at random from each group. The length of their petioles and the dimensions of the lamina (length and breadth at the broadest part) were measured to the nearest mm.

The results obtained are given in Table II.

The surface area per leaf was also determined. This was done as follows: Five leaves, drawn at random from each group, were separately traced on a sheet of paper. The paper leaf was accurately weighed in each case. By a determination of the accurate weight of one square centimeter of the same paper (the average weight of three determinations at different parts of the paper), the surface area of the paper leaf could be calculated, which corresponds to the surface area of the traced leaf in each case. The results obtained are given in Table III. On studying the histology of the leaves of treated and control plants, no difference could be observed in the type or arrangement of tissues. However, in determining the palisade ratio and vein-islet number, following the procedure described by

Youngken (90), some difference could be observed. The results obtained are given in Table IV. 31 TABLE II

Length of the Petioles and Dimensions of Treated and Control Digitalis Leaves

Length of Petiole (Cm) Dimensions of Leaf (Cm) Treated Control Treated Control

6. 2.5 6.5 x 2.5 4.5 x 3 6.5 1.5 5 x 2 5 x 3-5 5.5 3.0 8.5 x 2.5 9 x 6.5 8 2.5 8.5 x 2.5 5-5 x 4 4.5 3.0 5.5 x 2 7 x5.5 8.0 3.5 6 x 2 8 x 6.5 9.0 2.5 10 x 3 7 x 6 9.0 2.0 6 x 2.5 6 x 4.5 7.5 3.5 9 x 3 7 x 5.5 8.5 3.0 7 x 2 6 x 4.5 8.0 2.5 7 x 2 8 x 7 9.5 3.5 8 x 2.5 7.5 x 6 7.5 3.0 7 x 2.0 6 x 4.5 8.5 2.5 8 x 2.0 5 x 3 8.0 2.0 6 x 3.5 4.5 x 2.5 7.0 3.5 8 x 5 4.5 x 3 8.0 4.5 6 x 5.5 6 x 4.5 7.5 3.5 5 x 3 7 x5 9.0 4.0 9 x 3 6 x 3.5 7.0 2.5 9 x 5 7.5 x 5.0 6.5 1.5 6 x 5.5 6 x 4.5 7.0 2.5 5 x 3 7 x 5.5 8.0 2.0 8 x 4.5 6 x 3.5 6.0 1.5 8 x 5 7 x 3 9.0 2.0 7 x 3 8 x 6.5 8.0 3.5 9 x 6 7 x5 7.0 2.5 6 x 4 6 x 4.5 8.5 1.5 7 x 2 7 x 5 7.5 2.0 6 x 3-5 6 x 4 8.0 2.0 9 x 6 7 x 3.5 7.0 1.5 8 x.3.5 7 x 4 6.5 2.5 8 x 6 8 x 3.5 8.0 2.0 7 x 2 8 x 5>5 9.0 1.5 6.5 x3 7 x 6.0 8.5 2.5 8 x5 8 x 4.5 8.0 1.5 7 x 2.5 6 x 3 7.0 3.0 8 x5.5 7 x 4.5 8.5 2.0 9 x 6 8 x 5 7.5 1.0 6 x 3.5 7 x 3.5 9.0 3.0 7 x 3.0 8 x 4 8.0 2.5 7 x 4 7 x 4 32

TABLE II (Continued)

Length of Petiole (Cm) Dimensions of Leaf (Cm) Treated Control Treated Control

7.0 2.5 9 x 3.5 8 x 3.5 9.0 1.5 5 x 3 8 x 6 7.5 3.0 7 X 4 2.2 7 x5 6,5 7 x 3.5 6.5 x5 Avg. 7.5 2.5 7 x 3-4 6.6 x 4.5

TABLE III Surface Area of Treated and Control Digitalis Leaves

Control Treated Cm2 Cm2

109.2 43.3 110.6 45.0 108.9 42.1 109.3 47.0 109.8 42.7 Average 109.5 44.0 33 TABLE IV

Palisade Ratio and Vein-islet Number of Treated and Control Digitalis Leaves

'Treated Control

Palisade Ratio 4.2 2.8 5*5 3.6 6 4.0 Av. 5.2 3.4 Vein-islet Number 4 2.0 4 4.5 3.1 5.8 Av. 3.7 4.1

Determination of Total Glycosides in Digitalis Samples

Samples of Digitalis leaves were assayed for total glyco­ sides following the procedure described by Bell and Krantz (91).

The principle of this procedure is based upon spectrophotometry determination of the color developed by digitalis glycosides on the addition of alkaline picrate.

Preparation of a Standard Curve

Three grams of U.S.P. Digitalis Reference Standard were used in preparing the tincture according to the U.S.P. XV procedure.

The volume of menstruum was adjusted so that every 10 ml of final volume corresponded to 10 Reference Standard units.

Ten "milliliters of the tincture were transferred to a 25 ml. volumetric flask. Two milliliters of a 12.5 per cent w/v solution of neutral lead acetate were added to precipitate the tannins, phenols 3^ and other interfering substances. The mixture was shaken and made to volume with distilled water, thoroughly mixed, and filtered through a dry filter paper. In order to precipitate the excess lead, a 12.5 ml» aliquot of the filtrate was placed in a 25 ml. volumetric flask and a2ml. of a 4.7 per cent w/v solution of sodium diphosphate was added. The mixture was shaken and made to volume with distilled water, mixed thoroughly, and filtered through a dry filter paper.

The alkaline picrate solution was prepared by mixing 95 parts of a 1 per cent w/v aqueous solution of picric acid with 5 parts of a 10 per cent w/v aqueous solution of sodium hydroxide.

To develop the color reaction, a 12.5 ml. of the alkaline picrate was added to 12.5 ml* aliquot of the filtrate. The absorbancy was read on a Beckman Spectrophotometer Model D U. A standard curve was constructed Jay- using adequate dilutions of the tincture prepared from the Digitalis Reference Standard. The results obtained are given in Table V and illustrated in Fig. 8.

TABLE V

Data for Standard Curve of Digitalis Glycosides

ML of U.S.P. Reference Standard Tincture Absorbancy

0.2 0.026 0.5 0.066 0.75 0.100 1.0 0.132 1.5 0.200 2.0 0.271 35

0.5 1.0 1.5 2.0 2.5 3.0 Standard Reference Units Standard Curve for Total Glycosides of Digitalis Figure 8 36

Samples of harvested Digitalis leaves were assayed for total glycosides by preparing a tincture, using 3 Qm.. ±n each case, following exactly the same procedure described for preparing the standard curve. The results are expressed as per cent potency in terras of Reference Standard. Table VI and Figures 9 and 10 illus­ trate the results.

TABLE VI

Total Glycosides in Digitalis Sample

Per Cent Potency in Date of Harvest Terms of Reference Standard 'reated Control July 17 196 99 192 100.1 195 98 Average 194.3 99 July 24 170 140 175 137 173 139 Average 172.6 138.6

July 31 168 124 167 118 165 120 Average 166.6 120.6

August 9 136 120 136.5 120 138 125 Average 136.8 121.6

August 23 166 130 168 133 170 132 Average 168 131

Sept. 12 165 154 169 155 168 157 Average 167.3 155.3 "2 o 55

(0 £

o o

July 17 July 24 July 31 Aug. 9 Aug.23 Sept. 12 Samples Analysis of Total Glycosides of Digitalis Samples Figure 9 200- Treated °~

190 Control 180 170 160- 150- 140 130 120 110 1001-

July 17 July 24 July 31 Aug. 9 Aug. 23 Sept. 12 Date of Harvest Variation of Glycosida! Percentage of Digitalis Samples with Period of Harvest a>

Figure 10 39 Total Sugar

Total sugar was determined following the procedure of the A.O.A.C. 19^5 (92). Five grams of each sample were accurately- weighed and used for each determination. To the weighed sample about 60 ml. of previously neutralized 80 per cent alcohol were added and the mixture was heated on a steam bath for 30 minutes with continuous stirring. The alcohol extract was filtered. The insolu­ ble material was transferred to a beaker, covered with another por­ tion of 80 per cent alcohol, warmed on a steam bath for one hour and filtered. The residue was left to dry and then transferred to an extraction thimble and extracted with 80 per cent alcohol for

12 hours in a Soxhlet extractor. The total alcoholic extracts were transferred to a 200 ml. volumetric flask and completed to volume with 80 per cent alcohol. Twenty five milliliters of distilled water were added to the alcoholic extract and the alcohol evaporated on a steam bath. The aqueous concentrate was then transferred to a

250 ml. volumetric flask. The gummy precipitate was thoroughly washed into the flask. Ten milliliters of a saturated solution of neutral lead acetate were added to precipitate the tannins, phenols, and the possible interfering substances. The mixture was shaken and made to volume with distilled water, thoroughly mixed, and fil­ tered through a dry filter paper. The first 10 ml. of the filtrate were discarded because of possible adsorption of the sugars on the filter paper. In order to precipitate the excess lead acetate, solid anhydrous sodium carbonate was added to the filtrate. The mixture 40 was thoroughly mixed and filtered through a dry filter paper.

The first 10 ml. of filtrate was again discarded.

In order to bring about hydrolysis of the carbohydrates, a 50 ml* aliquot of the filtrate, 5 ml* of 36 per cent hydrochloric acid,and 25 ml. of distilled water were placed in a 10 ml. volum­ etric flask and hydrolyzed at room temperature for fifteen hours.

The mixture was neutralized with solid anhydrous sodium carbonate, diluted to volume, and thoroughly mixed by shaking.

The sugar determination was carried out on a 50 ml. aliquot portion of the neutralized sample according to the Munsen and Walker method (93) and calculated as dextrose. Table VII and Figures 11 and

12 illustrate the results.

Determination of Digitoxose Sugar

With a view toward getting an insight into the influence of Gibberellic acid on the biosynthesis of specific sugars of the glycoside molecule, the material was subjected to a quantitative

Keller-Kiliani determination to measure the digitoxose present.

The procedure followed was that of the U.S.P. XIII (94) method 11. This procedure is based on spectrophotometric measure­ ment of the color developed by digitoxose and ferric chloride in presence of acetic and sulfuric acids.

Preparation of a Standard Curve

By using U.S.P. Digitoxin Reference Standard, values for a standard curve were determined as follows: Ten milligrams of 41

VII

Percentage of Total in Digitalis Samples

' Date of Harvest $> of Total Sugar Treated Control

July 17 24.90 8.9 23.80 8.7 24.40 9.1 Av. 24.30 8.9

July 24 17.70 11.7 18.20 12 17.90 11.8 Av. 17.90 11.8

July 31 8.30 5.6 8.50 5.7 8.60 5.9 Av. 8.40 5.7

Aug. 9 13.20 10.12 13.80 10.40 14.01 10.00 Av. 13.60 10.1

Aug. 23 12.60 10.24 12.90 9.90 13.10 9.70 Av. 12.90 9.90

Sept. 12 19.40 7.8 20.0 8.1 19.6 8.2 Av. 19.6 8.0 Treated Control

July 17 July 24 July 31 Aug. 9 Aug. 23 Sept. 12 Samples Per Cent of Total Sugar in Digitalis Samples Figure II Treated o o Control « «

July 17 July 24 July 31 Aug.9 Aug.30 Sept. 12 Date of Harvest Variation of Total Sugar Percentage of Digitalis Samples with Period of Harvest 5 Figure 12 44

of U.S.P. Digitoxin Reference Standard was accurately weighed and dissolved.in sufficient alcohol to make 100 ml. The following por­ tions of this solution were transferred quantitatively to suitable

containers: 0.4, 1, 2, 3 and 4 ml., representing .04, 0.1, 0.2,

0.3 and 0,4 mgm. respectively, and dried at 100°C. for one hour.

To each portion was added 6 ml, of glacial acetic acid, 0,2 ml, of a freshly prepared 5 per cent w/v ferric chloride solution, and 0,5 ml, of sulfuric acid. Each solution was thoroughly mixed, and allowed to stand for 45 minutes protected from air and from direct sunlight. The absorbancy of each solution was determined on a

Beckman Spectrophotometer Model DU at a wavelength of 550 m.u. The results are given in Table VIII and illustrated in Fig. 13.

TABLE VIII

Data for Standard Curve of Digitoxin

Solution of Digitoxin Reference Standard mgm. of Digitoxin Absorbency

.4 ml .04 .025 1.0 0.1 0.075 2.0 0.2 0.150 3.0 0.3 0.251 4.0 0.4 0.375

Determination of Digitoxose was done on three harvests, notably July 17, July 24, and Aug. 23.

Three grams of Digitalis samples were extracted with 80 per cent alcohol following the same procedure of determination of sugar. ^5

u c o •O o CO <

0.2 03 0.4 0.5 Milligrams Digitoxin

Standard Curve Used for Determination of Digitoxose Figure 13 k6

To 10 ml. aliquot of the filtrate, each of the follow­ ing reagents was added: 6 ml. glacial acetic acid, .2 ml. of freshly prepared 5 per cent ferric chloride solution and 0.5 ml. of sulfuric acid. The mixture was left to stand for A-5 minutes and the absorbancy read on a Beckman Spectrophotometer Model DU at a wavelength of 550 m.u. against a blank of the same reagents and distilled water in place of the solution. The digitoxose con­ tent in each case was calculated from the digitoxin content ob­ tained by interpreting the absorbancy of each solution on the standard curve.

The results obtained are given in Table IX.

TABLE IX

Percentage of Digitoxose in Digitalis Samples

Treated Control

July 17 015 .013 015 .017 011+ .016 Av. 015 .015

July Zh om- .016 017 .01*)- 015 .015 Av. 015 .015 Aug. 23 017 .009 .014 .010 015 .008 Av. 015 .009

Determination of Crude Fiber Content

From a study of Table VII, it is observed that the per cent of total sugar is influenced by Gibberellic acid. It is deemed of 47 interest to study the crude fiber content (as a criterion for cellulose content) of both treated and control plants. The pro­ cedure followed is that described in U.S.P.,XV.

Three grams accurately weighed of the sample were exhausted with ether in a Soxhlet extractor. To the exhausted sample 200 ml. of boiling 1.25 per cent sulfuric acid are added in a 600 ml. container and the mixture refluxed for exactly 30 minutes.

It was then filtered through a linen filter. The residue was washed with boiling water until no longer acid. It was rinsed back into the container with 200 ml. of boiling 1.25 per cent sodium hydroxide solution. The mixture was heated again to boiling and kept boiling for exactly 30 minutes under the reflux condenser, then rapidly filtered through a prepared Gooch crucible. The residue was washed with boiling water until the last washing was neutral to litmus and dried at 100°C. to constant weight. The dried residue was incinerated, cooled in a desiccator, and the wash weighed. The difference between the weight obtained by drying at 110°C and that of the ash represents the weight of the crude fiber. The results obtained are given in Table X and illustrated in Fig. Ik.

Determination of Total Chlorophyll and the a and b Components

Chlorophylls a and b, because of their characteristic absorption spectra, comprise a system which is eminently suitable for the application of spectrophotometric methods of analysis. An accurate, rapid and relatively simple spectrophotometric method for the determination of the individual components, as well as total chlorophyll in green plant tissue based on the fundamental absorption 48

TABLE

Percentage of Crude Fil in Digitalis Samples

Date of Harvest Crude Fiber $ Treated Control

July 17 12.1 6.01 12.3 5.90 12.1 6.12 Av. 12.1 6.01

July 24 17.2 8.2 17.3 8.11 17.1 8.3 Av. 17.2 8.2

July 31 10.1 5.01 10.3 5.4 9.65 5.3 Av. 10.01 5.2

August 9 10.30 9.01 10.10 9.00 10.50 9.02 Av. 10.30 9.01

August 23 9.4 9.1 9.6 9.2 9.2 9.1 Av. 9'. 40 9.1

Sept. 12 9.31 9.15 9.50 9.30 9-45 9.20 Av. 9.^2 9.2 o CO <£ £ CNJ O CO C\J CVJ J9q|d apnjQ jo |uao jad 50 spectra of chlorophylls a and b is available (96,97). This method is recognized in the Methods of Analysis of the Associa­ tion of Agricultural Chemists (98).

Extraction of chlorophyll from plant tissue. Comar and

Zsheile (97) found it most convenient to extract chlorophyll from plant material with aqueous acetone in the presence of a small amount of CaCOo or MgCO- for the neutralization of plant acids.

In practice the extraction had been most satisfactorily carried out with a Waring Blendor and 85 per cent acetone. The method sug­ gested by these workers was therefore followed.

Determinations were done on three harvests; namely, July

17, July 31, and August 23. About 1 GM., accurately weighed, of each saraple of plant material was disintegrated in a Waring Blendor

cup containing 85 per cent acetone C.P. to which a small amount of

CaCOo (about 0.1 GM.) had been added. After the tissue was thor­ oughly disintegrated, the extract was filtered through a Buchner funnel. The residue was washed with 85 per cent acetone and re­ turned to the Waring Blendor cup for complete extraction with the

solvent. The extract was filtered again and washed, as directed previously, into the flask containing the first filtrate. This was repeated several times until the filtrate from the last extract

showed no more chlorophyll in it, as indicated by its clear color

and by showing at least 96 per cent transmission when read at

525 m.u. on a Beckman Spectrophotometer.

The filtrate was transferred to a 250 ml. volumetric flask

and made to volume with 85 per cent acetone. A 50 ml. aliquot was 51 pipetted into a separatory funnel containing about 50 ml. of

ether; then water was added until there were two distinct layers.

The aqueous layer was discarded. A second separatory funnel con­

taining about 200 ml. of water was arranged so that the stem of the first funnel containing the ether layer extended inside the

second funnel and beneath the surface of the water. The ether

layer from the first funnel was then slowly run into the second

funnel so as to wash the acetone out of the ether. The water layer

in the second funnel was then discarded. The procedure was repeated from five to seven times. Finally the ether layer was transferred to a 100 ml. volumetric flask and completed to volume with ether.

Spectrophotometry measurements. Approximately h Gm. of

anhydrous Sodium Sulfate C.P. were placed in a 250 ml. iodine flask,

and the ether solution of the pigments poured into it. The sodium

sulfate does not absorb any pigment from the solution during the

first Zk hours, but there is a considerable loss of the pigments

after this period of time (97)• All the spectrophotometric measure­ ments were, therefore, made within this period of time. 'When the

solution was optically clear, the spectrophotometric analysis for

chlorophyll and its components was made with a-Beckman Spectrophoto­

meter, Model D U. The analysis for the chlorophyll pigments was

made by pipetting some of the ether solution into glass stoppered

absorption cells and by measuring the absorbancy at wavelengths 660

and 6^-2.5 m.u. The readings at these two wavelengths should fall

between 0.2 and 0.8. In case they do not fall within this range, an 52 aliquot of the ether solution should be diluted with sufficient dry ether to cause the absorbancy to fall between 0.2 and 0.8.

In the samples tested in this investigation, dilution was not necessary, since the ether solution in each case complied the above specifications.

The amount of total chlorophyll, chlorophyll a, and chlorophyll b were solved from the following equations (63-65),

.(100-101):

(1) Total chlorophyll (Mg./L) o o = 7.2 As (at 6600 A) + 16.8 Ag (at 6*4-25 A). (2) Chlorophyll a (Mg./L) o o o = 9.93 As (at 6600 A) - 0.777 A (at 6^25 A) (3) Chlorophyll b (Mg./L) o o = 17.6 Ag (at 6425 A) - 2.81 As (at 6600 A).

The percentages of total chlorophyll, chlorophyll a, and chlorophyll b in the tested treated and control samples are given in Table XI. 53

TABLE XI Percentage of Chlorophyll in Digitalis Samples July 17 July 31 August 23 1Treate d Control Treated Control Treated Control

Total .281 .254 .414 .236 .381 .213 Chlorophyll .283 .253 .410 .225 .380 .216 .311 .251 .395 .241 .376 .213 Av. .295 .253 .403 .234 .379 .214

Chlorophyll .197 .193 .314 .181 .260 .163 a .196 .194 .310 .165 .261 .163 .202 .195 .320 .155 .260 .163 Av. .198 .194 .315 .167 .260 .163 Chlorophyll .083 .0597 .099 .054 .122 .051 b .083 .0582 .130 .051 .121 .052 .118 .0580 .115 .050 .118 .052 .094 .0586 .114 .051 .120 .051 FAGOFHtUM ESCULENTUM (BUCKWHEAT)

For investigating the influence of Gibberellic acid on

Buckwheat plant, the plants were propagated from seeds in the Ohio

State University Medicinal Plant Garden on May 25, 1957* Spray­ ing was started on June 17, after the true leaves were well de­ veloped, and was continued twice weekly until September 5«

Harvesting was done approximately at weekly intervals from June 28 until September 12. At each harvest 15 entire plants from each of the treated and control groups were gathered, and five plants were further drawn at random from each 15 plants.

These five plants were used for the determination of the height and dry weight of the stem and dry weight of the leaves. The drying was done at 100°G. for 24 hours. The results obtained are given in

Table XII.

The leaves of the remainder of the gathered plants were dried separately at 100°G. for 24 hours, and three samples were drawn at random from each group, and saved for evaluation.

When the plants attained maturity (fruiting stage),the number of leaves on fifteen plants from each group was counted. The results obtained are given in Table XIII.

Assay of Samples of Buckwheat for Rutin Content

For rutin estimation the spectrophotometric procedure of

Arthur (99) was followed. The principle of this procedure is based

54 55 TABLE XII

Data on Length of Stems, Dry Weights of Stems and Leaves of Buckwheat Samples

,Length of Stem Dry Weight of Stem Dry Weight of Leaves iDate of Cm. Cm. Cm. Harvest Treated Control Treated Control Treated Control

June 28 Av. of 15 50.8 34.1 5.3 2.5 4.8 3.4 plants July 10 Av. of 15 71.5 56 6.2 3.9 6.5 4.5 plants July 17 89.5 79 8.9 7.9 8.0 7.1 98 60 10.8 2.8 7.5 2.6 79 31 8.1 1.6 4.5 1.4 65 41 4.9 2.1 5.3 3.3 81 50 5*5 1.9 5.0 2.3 Av. 82./+ 52.2 7.4 3.2 6.06 3.3

July 24 90.5 5k 16.4 3.8 13.5 3.5 87 61 12.4 3.9 14.1 3.5 92 50 18.4 4.2 13.5 4.0 89 k9 7.8 3.3 6.9 3.0 78 62.0 11.8 4.0 6.9 2.8 Av. 87.2 55.2 13.4 3.8 10.69 3.3

July 31 96.5 kk 30.90 9.20 13.4 10.20 90.3 79.5 22.65 19.90 13.1 10.79 87.0 62 26.60 16.70 15.0 7.70 91 • 69.5 26.50 18.40 18.85 14.20 91 61.3 25.30 12.30 15.1 13.10 Av. 91.1 63.2 26.39 15.30 15.09 11.19

Aug. 23 89-0 49.3 22.1 5.5 10.5 5.8 78.0 kl.6 17.6 2.4 8.1 6.7 68.5 51.5 11.6 5.3 9.0 5.3 80.0 59.0 16.9 7.4 8.9 6.3 70.0 58.0 10.8 7.9 11.0 7.4 Av. 77.1 51.9 15.8 5.7 9.5 6.3

Sept. 12 82 51 20.2 5.4 11.9 4.8 74 kO 16.3 5.3 8.5 5.0 66 48 10.7 6.0 8.9 5.3 78 54 17.0 6.1 9.5 7.1 61 81 9.8 8.6 8.0 8.3 Av. 72.2 54.8 14.8 6.28 9.3 6.1 56

TABLE XIII

Data on Number of Leaves/Plant

Number of Leaves/Plant Treated Control

156 93 140 83 99 64 89 66 79 174 89 110 120 84 115 94 122 89 142 110 98 114 180 112 125 101 122 89 99 79

Av. 118.3 97.5

on spectrophotometric measurements of rutin aluminum chloride complex which has an absorption maximum at 416 m.u. This rutin complex is stable and the absorbancy remains unchanged for at least two hours.

Thus all determinations were done within this period.

Preparation of a Standard Curve of Rutin

One hundred milligrams of pure rutin were accurately weighed and dissolved in 100 ml, of IM aluminum chloride in a 100 ml. volumetric flask. Appropriate dilutions representing various concentrations were prepared so as to maintain the absorbancy between 0.2 and 0,8. Through this range the solutions follow Beer's 51

Law accurately. The absorbancy of each dilution was determined on a Beckman Spectrophotometer Model DU. A calibration curve was prepared. Table XIV and Fig. 15 show the results.

For the determination of rutin content in the samples of

Buckwheat, one gram of the sample was accurately weighed and ex­ tracted with about 50 ml. of absolute alcohol in a Soxhlet extractor for 6 hours. The alcoholic extract was quantitatively transformed to a 100 ml. volumetric flask and completed to volume with isoamyl alcohol. A twenty-five milliliter aliquot portion of the isoamyl alcohol solution was transferred to a separatory funnel and extracted with three successive portions each of 25 ml. of 0.1 M aluminum chloride. The combined aqueous extracts were collected in a 100 ml. volumetric flask and completed to volume with distilled water. The absorbancy of this solution was determined at a wavelength of M.6 m.u. on a Beckman Spectrophotometer Model DU. The percentage of rutin was calculated from the standard curve of rutin. The results ob­ tained are given in Table XV, and illustrated in Figures 16 and 17.

Determination of Total Sugar in Buckwheat

Total sugar was determined in terms of dextrose following the A.O.A.C. method previously described on page 39» The results obtained are given in Table XVI and illustrated in Figures 18 and 19.

From a study of Table XV and XVI, it can be observed that the percentage of rutin in treated plants is less, while the percentage of total sugar is more than the controls. It was 58

TABLE XIV

Data for Rutin Standard Curve

Dilution ml. Stock ml. Distilled Dilution Water Dilution mgm. Rutin Absorbancy Average

0.5 99.5 .5 0.163 0.162 0.163 0.162

1.0 99.0 1.0 0.315 0.312 0.310 0.312

1.5 98.5 1.5 0.495 0.496 0.495 .495

2.0 98.0 2.0 0.670 0.675 0.672 0.655

2.5 97.5 2.5 0.837 0.833 0.836 0.835

3.0 97.0 3.0 1.00 1.00 1.01 1.00 ' 59

1.0

.9

.8

.7

.6 —

.5

.4

.3

.2

.1 n / 1 1 I Milligram of Rutin

Standard Curve for Rutin Figure 15 TABLE XV

Percentage of Rutin in Buckwheat Samples

Percentage of Rutin Treated Control

June 28 4.15 4.35 4.10 4.40 4.15 4.30 Av. 4.10 4.30 July 10 3.84 4.30 3.90 4.85 3.75 5.00 Av. 3.83 4.88 July 17 3.4 4.80 3.43 4.40 3.41 4.35 Av. 3.41 4.4 July 24 3.80 4.30 3.75 4.27 3.70 4.25 Av. 3.71 4.27

July 31 2.30 3.60 2.401 3.63 2.35 3.65 Av. 2.34 3.60

August 23 2.70 '3.30 2.70 3.40 2.70 3-35 Av. 2.70 3-31 Sept. 12 2.65 3.20 2.72 3-31 2.68 3.28 Av. 2.68 3.26 Per Cent of Rutin

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Variation of Glycosidal Percentage of Buckwheat

Samples with Period of Harvest ON Figure 17 TABLE XVI

Per Cent of Total Sugar i Buckwheat Samples

Date of Harvest Percentage of Total Sugar Treated Control

June 28 3.1 3.08 3.3 2.94 3.25 2.90 Av. 3.21 2.97 July 10 6.70 3.10 6.55 2.90 6.80 3.12 Av. 6.68 3.06

July 17 6.70 3.30 6.55 3.40 6.64 3.34 Av. 6.63 3.34 July 24 6.50 3.80 6.40 3.51 6.48 3.50 Av. 6.46 3.49

July 31 5.60 4.01 5.43 4.21 5.51 4.20 Av. 5.51 4.14

Aug. 23 5.10 4.30 5.00 4.50 4.89 4.35 Av. 4.99 4.38 Sept. 12 4.78 4.01 4.99 4.31 4.98 4.25 Av. 4.91 4.19 . 64

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Figure 19 66 deemed pertinent, therefore, to study the influence of Gibber- ellic acid on the specific sugar rhamnose which is necessary for the biosynthesis of the glycoside.

Reviewing the current literature, no standard method could be traced for the determination of this sugar in any plant material. However, the use of paper chromatography as a means of gaining rapid and specific information regarding the saccharide composition of a plant extract provided a useful tool in plant analysis. In addition, this useful technique proved to be very applicable in quantitative determinations of sugar in plant extracts.

The separation of monosaccharides by paper chromatography was first described by Partridge (100), and since then, developments in this field have been unceasing. A large number of solvent com­ binations have been used for different purposes utilizing ascending, descending, and even horizontal techniques. Emphasis is always placed on the necessity of providing appropriate controls in paper chromatography. Spots of known sugars should always be included in runs where it is desired to deduce the nature of saccharides in

"unknown" systems. The inclusion of such control spots greatly de­ creases the chances of erroneous deductions arising from unforeseen temperature changes, etc.

Several reagents are cited in the current literature for locating sugars on paper, of which the following are examples:

1. Oxidizing agents (101). ammoniacal silver nitrate is a good

example of an oxiding agent. The developed chromatogram is 67

dried to remove the solvent and is sprayed with a mixture

of equal volumes of 0.1N AgNOo and 5N NEfyOH. The chromato­

gram is placed in the oven at 105°C. for 5-10 minutes, the

reducing sugars appear as brown spots on the paper.

2. Alkaline permanganate (101). The chromatogram, after drying,

is sprayed with a 1 per cent solution of potassium permanganate

containing 2 per cent sodium carbonate. The chromatogram is

heated for a few minutes at 100°C, and the sugars appear as

yellow spots on a purple background.

3. Aniline hydrogen phthalate (100). The reagent is made by adding

0.93 Gin., of aniline and 1.6 Gm. of phthalic acid to 100 ml. of

water saturated with n-butanol. The air-dried paper is sprayed

with this reagent and heated for 10 - 15 minutes at 105°G.;

pentoses and desoxy sugars appear as bright red, hexoses appear

brown or dark brown.

4. p-anisidine HCl (101). The dried chromatogram is sprayed with

a 3 per cent p-anisidine HCl solution in n-butanol and heated

at 100°C. for 3-10 minutes; pentoses appear red, hexoses

appear brown.

Separation of Rhamnose Sugar from Buckwheat by Paper Chromatography

The separation of rhamnose from Buckwheat involved the following steps:

1. Extraction of the plant tissue

2. Removal of interfering substances from the extract 68

3. Hydrolysis of rutin and present carbohydrates

k. Qualitative analysis of the hydrolysate by paper

chromat ography.

Extraction of Buckwheat was done with 80 per cent alcohol following the procedure of A.O.A.C. (92) (19^5). The tannins, phenols, and other interfering substances were removed by lead acetate. The excess lead salt was precipitated as the phosphate by disodium hydrogen phosphate. The mixture was filtered.

The clear filtrate was hydrolyzed following the N.F. (1955) procedure (102), which consists of refluxing the solution with about

50 ml. of hydrochloric acid (1:18) for 1 hour. The hydrolysate was cooled, filtered, and neutralized with sodium carbonate. About 200

Lambda of the neutral solution was then spotted on Whatman No. 1 filter paper. Alongside, the following chromatograms were run:

1. Neutralized hydrolysate plus rhamnose

2. Rhamnose

3- Glucose

4. Rhamnose plus glucose

The solvent system used was ethyl acetate, pyridine, water (8:2:1). The chromatograms were equilibrated for 15 hours in chambers, previously saturated with the solvent, and were run by using descending technique for 5 hours. They were air-dried and sprayed with aniline hydrogen phthalate reagent and then heated in an oven from 10 to 15 minutes. The separated rhamnose was identified 69 by the color reaction (red), by the Rf value compared with that

of pure rhamnose, as well as by mixed chromatograms (plant ex­ tract and rhamnose) run under identical conditions. Other sugars

appeared on the chromatograms prepared from the plant extract which were not identified, since none of them interfered with rhamnose.

The R«, and the R values (distance travelled by rhamnose/the -1- g distance travelled by glucose) are given in Table XVII. Figures

20 - 2k illustrate the results obtained.

TABLE XVII

Rf and Rg Values of Rhamnose and Separated Sugars

Sample Chromatogramed Glucose Rhamnose Rf Rg

Glucose .130 Glucose + Rhamnose .131 .50 3-3 Buckwheat Extract .132 .48 3.3

Quantitative Determination of Rhamnose by- Paper Chromatography

It was a logical sequence to the separation of rhamnose

from Buckwheat by the paper chromatography technique that attempts

were made to apply this technique to quantitative analysis.

The procedures commonly adopted for quantitative estima­

tion by the paper chromatography technique are -

1. Visual comparison. This procedure is based on the

fact that the intensity of color and the size of the spot vary with 70 u o w. ^» X a> W CO o ^ c CM o E a> a XZ .c

a P) a OJ o 1 S • o o o JC 3 o X o» UJ GO LL

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C\oJ a> CO 0) o w. ( o 3 . o 3 o> o 71 the quantity of the substance chromatogramed. The spot of an unknown sample is compared with a spot produced by a standard solution. Several techniques for this procedure are described in the current literature (103,10i(-). However this method is still of limited application because of unavoidable experimental error, and the multiple variants affecting the accuracy of the results.

2. Elution of the substance to be determined. The area of the chromatogram containing the substance to be estimated is cut out, eluted with a convenient solvent, and the substance deter­ mined in the elute by conventional methods. This procedure proved to be of sufficient accuracy (105) and was followed in the present investigation.

Spectrophotometric Estimation of Rhamnose

Rhamnose was estimated spectrophotometrically following the procedure described by Loewus (106), which is based on the spectrophotometric determination of the color produced by rhamnose on the addition of a 2 per cent anthrone solution in ethyl acetate

in presence of sulfuric acid.

Preparation of a Standard Curve of Rhamnose

Ten milligrams of pure rhamnose. were accurately weighed

and dissolved in 100 ml. of distilled water in a volumetric flask.

A series of dilutions representing 10, 15, 20, 25. 50 and 75 meg. of

rhamnose were prepared. To each dilution was added 0.5 ml. of a

solution of 2 per cent anthrone in ethyl acetate; then 5 ml. sulfuric 72 acid, were carefully layered on the solution. The container was gently swirled until the ethyl acetate has hydrolyzed as indi­

cated by the "floe" of anthrone which appears. More rapid swirl­ ing was done by thoroughly mixing the contents and dissolving the

anthrone. The developed color was read after 10 minutes at 620 m.u. on a Beckman spectrophotometer, Model D U against a blank of the reagent using distilled water instead of sugar solution. A cali­ bration curve relating the absorbancy with the concentration in the series of dilutions was plotted. Table XVIII and Figure 25 illustrate the results.

Per Cent Recovery of Rhamnose from Chromatograms

It has been reported in the literature that in quantita­ tive practice, compounds separated by paper chromatography are not recovered completely from the developed chromatograms. Whether this was also true for the rhamnose sugar was not known. It was, there­

fore, important in this quantitative work to find out how much of

the compound under determination could be recovered. Hence the

purpose of conducting the following experiment.

Ten milligrams of rhamnose was dissolved in 100 ml. of

distilled water in a volumetric flask. A 200 lambda volume (repre­

senting .02 mg. of rhamnose) was spotted on the chromatographic

strips and chromatographed descendingly for about 5 hours. Rhamnose was located on three of the chromatograms by spraying the air-dried

strips with aniline phthalic acid reagent. The unsprayed paper

strips were cut into separate parts carrying the rhamnose. These 73

>' TABLE XVIII Data of Rhamnose Standard Chirve micrograms rhamnose absorbancy

10 .053 .052 • 05 Av. .051 15 .058 .056 .054 Av. .056 20 .130 .110 .112 Av. .117 25 .140 .130 .140 Av. .136

50 .273 .270 .270 Av. .271 75 .41 .408 .400 Av. .402 10 20 30 40 50 60 70 80 90 100 Micrograms Rhamnose

Standard Curve for Rhamnose -p- Figure 25 75 strips werfe then eluted with 80 per cent alcohol by careful packing in a thimble and extracted in a Soxhlet extractor for k hours. Ex­ periments allowing extraction to proceed for about 5 hours did not show increase in the recovered sugar, indicating complete elution in 4 hours.

The eluants containing the rhamnose were completed to

25 ml. with 80 per cent alcohol. Two milliliter aliquot portions of each eluant were treated with anthrone and sulfuric acid reagent, and the absorbancy was determined spectrophotometrically on a Beck- man Spectrophotometer Model DU., and the concentration of rhamnose interpreted from the calibration curve prepared previously. Table

XIX shows the percentage recovery of rhamnose from the chromatograms.

TABLE XIX

Percentage Recovery of Rhamnose

Weight Placed on Weight Recovered from Per Cent Chromatogram (mgm). Chromatograins mg. Recovery

1. .02 .017 85.0 2. .02 .018 90.0 3. .02 .017 85.0

Avg. .02 .0175 87.5

Quantitative Determination of the Measurable Rhamnose in Treated and Control Samples of Buckwheat

Rhamnose determination was done on three harvests, notably

June 28, July 2k, and Aug. 23. Three Grams of the powdered sample 76 were extracted with 80 per cent alcohol in a Soxhlet extractor.

The alcoholic extract was completed to 100 ml. with 80 per cent

alcohol and treated as described on page 39* A 12.5 ml. aliquot portion of the clear filtrate (from the lead phosphate) was hydrolyzed by refluxing with HCl (1:18) as described on page 67.

The neutralized hydrolyzate was transferred to a 25 ml. volumetric flask and completed to volume with 80 per cent alcohol. One hundred lambda of this solution were spotted on paper strips and

chromatographed descendingly as previously described. After estab­

lishing the identity of the spots, the area containing rhamnose was

eluted with 80 per cent alcohol as previously described on page 72.

An aliquot portion of the eluate was treated with anthrone reagent,

and the absorbancy of the colored solution was read on a Beckman

spectrophotometer Model DU. The concentration was interpreted

from the standard curve, taking into consideration the percentage

of rhamnose recovered on elution. The results obtained are given

in Table XX. The table also gives the Student "t" value, which is used as a measure for significance.

The "t" value was determined, as proposed by Snedecor (107),

by pooling the variance of the treated plants with the variance of

control plants using h degrees of freedom and testing the hypothesis

that the means of rhamnose per cent in the two groups are the same.

A "t" value greater than 2.776 is considered significant, since

the chances of obtaining a higher "t" value at k degrees of freedom

is less than 5 per cent. The percentage of rhamnose was found to be significantly higher in control than in treated plants. 77

In order to have a more complete idea on the influence of Gibberellic acid on the biosynthesis of rutin glycoside, it was decided to carry on a comparative quantitative determination of quercetin which is the aglycone fraction of rutin in treated and control plants.

TABLE XX

Percentage of Rhamnose in Buckwheat Samples

Date of Harvest Per Cent Rhamnose Treated Control

June 28 1.4 1.51 1.25 1.56 1.35 1.47 Av. 1.3 "t" value -3.92

July 24 3.66 2.9 2.60 2.7 2.44 2.76 Av. 2.56 2.78 "t" value -2.77

Aug. 23 2.50 2.80 2.34 2.95 2.41 2.75 Av. 2.41 2.83 "t" value -5.4

No conclusive evidence could be found in the current literature of the presence of quercetin in the free state in Buck­ wheat. However, it was reported to occur in other plants belonging to the fam. Polygonaceae. Tryer, in his attempts to separate the flavonoids of Buckwheat by traditional solvent extraction methods 78 and by ion exchange, did not report free quercetin. However, the same author could detect very small quantities of four unidentified flavonoids at an early stage of the life history of the plant (and reported that none of them was quercetin).

With this in mind, it was decided to make an investigation of Buckwheat flavonoids particularly searching for quercetin. For this investigation paper chromatography technique was resorted to with a series of solvent systems selected from the current litera­ ture.

For this purpose about three Cm. samples of Buckwheat representing different stages of age of the plant, namely June 28,

July 31» Aug. 23, were extracted with about 100 ml. of absolute alcohol in a Soxhlet extractor. The alcoholic extract in each case was concentrated by spontaneous evaporation to about 10 ml. About two hundred lambda were spotted on strips of Whatman No. 1 filter paper. The following solvent systems were tried:

1. Chloroform - butanol - water (2:4:4) v/v.

2. Acetic acid - water (6:4) v/v.

3. Ethyl acetate - acetic acid - water (4:1:5) v/v.

4. Butanol, acetic acid - water (4:1:5) v/v.

5. Ethyl acetate saturated with water

In each solvent system, control chromatograms of pure rutin, pure quercetin, and a mixture of both pigments were run alongside with the Buckwheat extract. The chromatograms were equili­ brated with the particular solvent for fifteen hours and descendingly 79 run for 6 hours. Rutin and quercetin, when present, were identi­

fied by their R^ values, the color of their respective aluminum

chloride complex, and the fluorescence of this complex under the ultra violet light. The chromatograms were air-dried and sprayed with .1 M aluminum chloride solution, and examined under the ultra violet light. Rutin gives a yellow, while quercetin gives an

emerald green fluorescence. Table XXI lists the Rf values of rutin

and quercetin in the different solvent systems. Figures 26 - 30

illustrate the results obtained.

TABLE XXI

Rf of Rutin and Quercetin

Chloroform Acetic Ethyl Acetate Butanol Ethyl Butanol/HgO Acid/H20 Acetic Acid/HJ} Acetic/Water Acet/HgO

Quercetin .05 .82 .78 .75 .92 Rutin •52 .72 .11 .51 .11

Determination of Crude Fiber Content

To study the influence of Gibberellic acid on the cellulose

content of Buckwheat during the growing season, quantitative crude

fiber determination was carried on, following the procedure of the

U.S.P. XV described on page 35• The results obtained are given in

Table XXII and illustrated in Fig. 31. E-Extract R-Rutin Q-Quercetin

1 1 i E Q+R R 0 E ' Q+R R Q E Q+R R Q E Q+R R Q E Q+R R | ° - • 0 : 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 i 0 0 0 0 0 0 0

! i i

Chloroform- Isobutanol- Acetic Acid-Water Ethyl Acetate- Acetic Acid Butanol-Acetic Acid-Water Ethyl Acetate Saturated Water Water With Water

Figure 26 Figure 27 Figure 28 Figure 29 Figure 30

CD O 81

TABLE XXII Per Cent Crude Fiber in Buckwheat

Date of Harvest Percent Crude Fiber Treated Control June 28 1.30 O.90 l.*JO 0.80 1.13 O.73 Av. 1.27 0.81 July 10 .95 0.98 1.01 0.85 0.99 0.87 Av. 0.98 0.90 July 17 0.84- 0.90 0.90 0.94 O.79 0.82 Av. 0.81 0.88 July 2k 0.78 0.93 0.70 1.10 0.73 0.92 Av. O.73 O.98 July 31 0.8*4- 1.10 0.86 1.03 0.7*4- 0.98 Av. 0.81 1.03 Aug. 23 1.02 1.20 .99 .93 1.10 1.10 Av. 1.03 1.01 Sept. 12 1.10 1.21 1.02 .97 .89 -99 Av. 1.00 1.05 82

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J9qjj gpnjQ *U9Q J9d 83 Determination of Chlorophyll in Buckwheat

Three harvests of treated and control Buckwheat leaves were subjected to a quantitative chlorophyll determination, fol­ lowing the procedure described on page 50. The results obtained

are given in Table XXIII.

TABLE XXIII

Per Cent of Chlorophyll in Buckwheat Samples

Determination Sample June 28 July 24 Aug.23 Treated Control Treated Control Treated Control

Treated 3.81 2.73 2.36 2.17 2.96 2.70 Chlorophyll 3-49 2.48 2.38 2.24 2.94 2.72 3.51 2.51 2.40 2.31 3.20 2.74 Av. 3.6 2.57 2.38 2.24 3.03 2.72 Chlorophyll a 2.48 1.45 I.69 1.63 2.40 2.18 2.44 1.48 1.71 1.65 2.37 2.16 2.45 1.43 1.68 1.60 2.41 2.10 Av. 2.68 1.45 1.66 1.62 2.36 2.14

Chlorophyll b 1.31 .891 .66 .55 .88 .62 1.21 .90 .69 .59 .91 .61 1.27 .85* .67 .58 .87 .64 Av. 1.26 .88 .67 .54 .88 .62 DISCUSSION

Gibberellic acid was reported to stimulate linear growth, induce flowering, influence the morphology, as well as the bio­ synthesis of some plant constituents.

The objectives of this study were to determine the influ­ ence of Gibberellic acid on the growth, morphology, histology and the biosynthesis of the glycosides in two glycosidal containing plants; namely, Digitalis purpurea L. (Foxglove) and Fagopyrum esculentum

Moench, (Buckwheat).

In reviewing the literature, it was observed that most of the previous reports on the influence of Gibberellic acid on plant growth were short-term experiments, or at least nothing was reported on the periodic follow-up of the influence of Gibberellic acid on plant constituents in general, and glycosides in particular.

For the purpose of long-term experiments it was decided

to raise the experimental plants in the Ohio State College of

Pharmacy Medicinal Plant Garden. A number of plants were arranged

in test plots for the purpose of being treated with Gibberellic acid,

and similar plots were arranged to serve as controls. Harvesting was done approximately at weekly intervals; samples which were drawn

at random from each plot were used in the comparative qualitative

and quantitative study.

The morphological response of Buckwheat plants was observed

to be mainly an increase in linear growth, Table XII, and an increase

8k 85 in dry weight, Table XII. No observed difference could be detected in the shape or size of the leaves of the experimental plants. How­ ever, the number of leaves per plant was found to be greater in treated plants than in controls, Table XIII.

The fact that the increase in linear growth of treated plants was accompanied by an increase in their dry weight suggests that the response to Gibberellic acid is more than mere cell elonga­ tion, since the increase in dry weight was of such magnitude that it could not be accounted for by the increase in cell wall that results from mere cell elongation. However, increased cell division could bring about an increase in dry weight for two reasons:

1. Increase in cell wall formation

2. Increase in cell contents due to the presence of more cells

Histological studies by others (55) have demonstrated that the effect of Gibberellic acid is more likely to be that of increas­

ing cell division. The increase in dry weight measured in the present

investigation agrees favorably with this conclusion.

The now universally accepted principle that cells can arise

only from pre-existing cells was first demonstrated beyond any reason­

able doubt by Wageli (108) before the middle of the nineteenth century.

In higher plants cell division occurs chiefly in certain restricted

regions called "meristems." The formation of new cells involves not

only the division of pre-existing cells but the subsequent enlargement

and maturation of their cell progeny. The cell wall material of a

cell is mainly cellulosic. Thus a determination of the cellulose 86 content (crude fiber content) of treated and control plants at periodic intervals would show the influence of Gibberellic acid on both the rate and quantity of cellulose formation.

A study of Table XXII reveals an increase in cellulose content particularly in early harvests. This could possibly suggest that Gibberellic acid stimulates faster cell division, and thus cell maturation is attained earlier than control plants. By the time the control plants reach a comparable degree of maturity as the treated,

at later harvests, the cellulose content of both groups becomes al­ most the same. This suggestion agrees favorably with the statement

of Lang (57) concerning the fact that Gibberellic acid is most effec­ tive when applied to the growing points of the treated plants.

Although treated and control plants showed almost the same

cellulose content at the latest harvest, the dry weight of the former remained higher. This suggests that increased cell wall formation

owing to cell division in treated plants cannot solely be responsible

for the increased dry weight. Another factor should exist contribut­

ing to this increase, notably cell contents, among which carbohydrates

constitute a major part. Determination of total carbohydrate content,

Table XIV, revealed an increase in treated plants in all harvests, which could also mean an increased dry weight.

It has been reported that Gibberellic acid has no direct

effect on photosynthesis, since the bakanae effect occurs in the

dark. Moreover, Haber and Tolbert (109) using Cr*02 concluded that

Gibberellic acid did not enhance the rate of CO2 fixation per unit 87 area of leaf tissue, and did not alter the general pathways of short time metabolism of the newly fixed C-^02 in sugars, organic acids, and amino acid products. Thus the increase in total sugar induced by Gibberellic acid is most likely due to an indirect factor related to photosynthesis output. Studying Table XIII, .it is observed that there is a marked increase in the number of leaves per Buckwheat plant in the treated group, which means an increase in photosynthetic area. Since the carbohydrates formed during photosynthesis are translocated to all parts of the plants, it could be suggested that an increase in total carbohydrate per cent is to be expected in treated plants oxiring to the increased number of leaves.

Treated Digitalis plants, on the other hand, showed a marked decrease in surface area of the leaves, Table III; an in­

crease in length of the lamina and petioles, Table II; bolting of

some plants, as well as a marked decrease in dry weight. The total

carbohydrate determination, Table VII, however, revealed a higher

per cent in treated plants.

It might appear, offhand, that Digitalis plants responded

differently from Buckwheat to Gibberellic acid as far as the dry

weight is concerned. However, it has been suggested by Lang (57)

that Gibberellic acid has a directional influence on cell division.

In other words, it stimulates division in a certain direction which was concluded to be the radial longitudinal rather than the tan­

gential direction. If this is true, it could be a possible 88

explanation for the increased linear growth of treated Buckwheat;

increased length of the lamina, and petioles; and decreased breadth

of treated Digitalis. As a consequence of this change in the di­ mensions of treated Digitalis leaves, a marked decrease in the

surace area amounting to approximately one-third of that of the

controls did occur. Since the treated leaves had a higher per­

centage of total sugar, as well as a higher percentage of crude

fiber, the decreased dry weight per leaf would most likely be due

to the decreased surface area. However, if the dry weight is to be calculated per unit area it will be found higher in treated than

controls.

In summary of the previous discussion, it can be stated

that both the treated Buckwheat and Digitalis plants responded in

a similar way, morphologically; namely,

1. Faster linear growth as shown by the increased crude fiber

content particularly at early harvests

2. Increase in dry weight per unit area

To study the influence of Gibberellic acid on the biosyn­

thesis of Glycosides in Digitalis and Buckwheat plants, random

samples of the treated and control plants were periodically har­

vested (approximately at weekly intervals, and assayed for Glycosidal

content).

On studying Table VI, it is observed that treated Digitalis

had higher glycosidal per cent in all harvests. Figures 11 and 12

show that this increase is rather abrupt and reaches maximum at the 89 first harvest. Although the following harvests still showed higher glycosidal per cent in the treated than the control, yet the former never attained the initial level of the first harvest.

However, the controls showed a relatively low initial glycosidal content with a gradual increase as the plant continued to grow.

A decrease in glycosidal per cent was observed at the third and fourth harvests of both groups, followed by a recovery and consequent increase in the following harvests. Tsoa and

Youngken (110) indicated that the season of growth has a great influence on the glycoside formation in Digitalis. It could be suggested, therefore, that this decrease is attributed to environ­ mental conditions, since it was followed by a significant increase in both treated and control plants.

In general the biosynthesis of Glycosides in plants can be roughly illustrated as follows:

Aglycone + Sugar(s) Enzyme Glycoside

In order to interpret the effect of changes in the magnitude of any one of the various factors influencing a process, such as glycoside biosynthesis in plants, it is necessary to formulate certain guiding principles. In 1843 Leibig (111) proposed his well-known "Law of the Minimum," which was the first attempt at such a formulation, and was further elaborated by Blackman (111). The law was stated by its author as follows: "When a process is conditioned as to its rapidity by a number of separate factors, the rate of the process

is limited by the pace of the slowest factor." 90

Considering Digitalis glycosides, let us assume that the aglycone content and the enzymatic activity permitted the utiliza­ tion of one unit weight of sugar per hour to form one unit weight of glycoside. If only a half unit weight of sugar is available, the rate of biosynthesis of the glycoside is therefore limited by the sugar factor. The glycosidal yield will consequently increase t by increase in sugar fraction until a unit weight of the later is available. Any further increase in the supply of sugar will have no influence oh glycoside biosynthesis unless another factor be­ comes limiting.

In the present investigation, on studying Table IX for digitoxose estimation, it is observed that no significant difference occurs between treated and control plants. This could mean that digitoxose, which is the specific sugar for Digitalis glycosides, cannot be a limiting factor in glycosidal formation, as far as the influence of Gibberellic acid is concerned. Thus it could be postu­ lated that either the aglycone fraction or the specific enzyme or enzymes responsible for the glycoside formation, or both of them, are limiting factors.

It was not possible to determine the influence of Gibber­

ellic acid on the specific enzymes, although it has been reported that Gibberellic acid increased the activity of some enzymes includ­

ing B-glycosidase. However, the assay procedure used for the total

glycosides of Digitalis is based upon the aglycone fraction of the

glycoside molecule. Therefore an expression of an increase in 91 total glycosides is also an indication of an increase in aglycone, and it does not necessarily mean an increase in the sugar fraction.

This may suggest that the increase in glycosidal percentage in treated plants may be due to a higher aglycone percentage rather than to digitoxose.

In the case of Buckwheat plants, the glycosidal percentage of the controls reached a maximum on July 10 and then decreased. In treated plants, however, the glycosidal percentage was always rela­ tively lower, reaching a peak at a later date than controls (July

24). If the rutin content were to be calculated on dry weight basis of leaves per plant, the treated leaves would show a higher content than controls. This is due to the relative increase in the dry weight and number of leaves of treated plants (Table XII and

XIII). Table XXIV illustrates this fact.

TABLE XXIV

Rutin Content Calculated on Dry Weight Basis of Leaves per Plant

Date of Average Rutin Average Dry Weight of Rutin Content of Leaves Harvest Per Cent Leaves Per Plant Gm Per Plant Gm T* C* T C T C

June 28 4.1 4.3 4.8 3.4 0.19? 0.146 July 10 3.83 4.88 6,5 4.5 0.249 0.220 July 17 3.41 4.40 6.06 3.3 0.207 0.145 July 24 3.71 4.27 10.69 3.3 0.397 0.141 July 31 2.34 3.60 15.09 11.19 0.353 0.403 Aug. 23 2.70 3.31 9.50 6.30 0.257 0.208 Sept.12 2.68 3.26 9.30 6.10 0.249 0.220

* T = Treated C = Control 92

Studying the rhamnose percentage in treated and control plants,

Table XX, it is observed that the control plants had a higher rhamnose percentage than the treated plants. Thus the decrease in rutin content in treated plants could possibly be due to either of the following factors:

1. A retarding effect induced by Gibberellic acid on the

enzyme or enzymes responsible for linking the sugar

fraction with the aglycone fraction of the glycoside

2. Less available rhamnose sugar (which is the specific

sugar for rutin) in treated plants.

3. Less available aglycone (quercetin) in treated plants

which could be due to an influence of Gibberellic acid

at a stage in the aglycone biosynthesis.

If the influence of Gibberellic acid is a retarding effect on the activity of the enzyme or enzymes responsible for linking the

sugar fraction with the aglycone fraction, without influencing the rate of quercetin formation, the latter could have occurred, at least

in part, free in the plant. However, this was not the case, since

no free quercetin could be detected in the experimental plants,

Figures 26-30* This suggests that the influence of Gibberellic

acid is most probably at a stage of quercetin biosynthesis or on

rhamnose formation or both together. In other words, quercetin and

rhamnose both could be limiting factors in the rate of rutin bio­

synthesis in treated Buckwheat plants. SUMMARY

Gibberellic acid, a metabolite recently isolated from a fungus called "Gibberella Pujikuroi, was reported to stimulate linear growth, increase dry weight, induce flowering, influence biosynthesis of some constituents, in plants and other effects.

The influence of this substance on two glycosidal con­ taining plants, namely Digitalis purpurea L. and Fagopyrum esculentum Moench, was investigated.

Experimental plants were treated twice weekly with a solu­ tion of Gibberellic acid (100 p.p.m.). Harvesting was done approxi­ mately at weekly intervals.

Samples of treated and control plants were subjected to morphological, histological investigation as well as to a compara­ tive qualitative and quantitative study.

Digitalis purpurea L.

The average determinations of dry weight, of measurements of lamina, and of surface area of treated and control Digitalis samples are given in Table XXIV.

93 94

TABLE XXV

Average Determinations of Dry Weight, Measurements of Lamina and Surface Area of Digitalis Leaves

Dry Length of Measurement of Surface Weight Gm. Petioles (Cm.) Lamina (Cm.) Area (Cm.2) Treated Control Treated Control Treated Control Treated Control

4.74 7.99 7.5 2.5 7.2x3.** 6.6x^.5 44 109.5

No histological difference could be detected between treated and control leaves. However, the palisade ratio was higher, while vein islet number was lower in treated leaves.

Samples of treated and control Digitalis leaves were also subjected to the following quantitative determinations:

1. Total glycosides

2. Total sugar

3. Digitoxose

4. Crude fiber

5. Chlorophyll

The average results obtained are summarized in Table XXV.

TABLE XXVI

Average Per Cent of Evaluation of Digitalis Samples

Total Glycosides $ Total Sugar $ Digitoxose # Crude Fiber % Chlorophyll $ T*C*TC TCTC TC

167.6 127.68 16.1 9.06 0.015 0.013 11.4 7.7 0.359 0.233 * T = Treated C = Control 95 Fagopyrum esculentum Moench. (Buckwheat)

Samples of treated and control Buckwheat were similarly-

subjected to morphological investigation including determination

of linear growth and dry weight of stems as well as dry weight of

leaves. No difference could be observed between surface area of

treated and control leaves. However, an increase in the number of

leaves per plant was observed in treated plants.

The samples were also subjected to the following quantita­

tive determinations:

1. Rutin

2. Total Sugar

3. Rhamnose

k. Crude Fiber

5. Chlorophyll

The average results obtained are summarized in Table XXVI.

Ko free quercetin, the aglycone of rutin, could be detected in Buck­

wheat leaves.

TABLE XXVII

Average Per Cent of Evaluation of Buckwheat Samples

Rutin fo Total Sugar $ Rhamnose ft, Crude Fiber # Chlorophyll $ T* C TC TC TC TC

3.25 4.0 5.^8 3.65 2.09 2.36 0.94 0.95 3.0 2.51

* T = Treated C = Control CONCLUSIONS

From the previously discussed experiments one may conclude the following:

Plants of Digitalis purpurea L. treated with a solution of

Gibberellic acid (100 p.p.m.) showed the following responses:

1. The leaves were morphologically affected, developing longer petioles and less surface area than the controls. The shape of the treated leaves were also different in being more linear, al­ most lanceolate. The dry weight was markedly decreased.

2. Histologically no difference could be observed between both groups. However, the treated leaves had a higher palisade ratio and a lower vein-islet number than the controls.

3. A stem was developed by some of the treated plants.

k. An increase in glycoside and total sugar per cent, but no significant differences could be observed in digitoxose per cent.

5. Crude fiber was almost the same at maturity, however, the treated plants showed higher content at earlier ages.

6. The percentage of total chlorophyll as well as chloro­ phyll "a" and "b" was higher in treated plants.

Plants of Fagopyrum esculentum Moench. (Buckwheat), sub­ jected to the same type of treatment as Digitalis, showed the follow­ ing responses:

1. An increase in linear growth and dry weight of stems, as well as an increase in dry weight of the leaves

96 2. An increase m the number of leaves per plant

3. A decrease in glycosidal per cent

4. An increase in total sugar per cent

5. A decrease in rhamnose sugar content

6. Crude fiber was almost the same at maturity, however, the treated plants showed higher content at earlier ages

7. An increase in chlorophyll per cent (total "a" and

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AUTOBIOGRAPHY"

I, Mahmoud Darwish Sayed, was born in Cairo, Egypt,

December 5» 1920. I received my secondary school education in the public schools of Cairo, and my undergraduate training at

Cairo University, which granted me the Bachelor of Pharmacy and Chemistry in 19^2. From the same University, I received the degree Master of Science in Pharmacy in 1950. While in residence there, I was a lecturer of pharmacognosy in the Faculty of Pharmacy. In October, 1955. I was appointed University

Scholar at Philadelphia College of Pharmacy and Science. In

June 1956 I enrolled in the Graduate School of the Ohio State

University to pursue my graduate studies in Pharmacognosy toward the degree of Doctor of Philosophy. In January, 1958, I received an appointment as Graduate Assistant in the Ohio State University,

College of Pharmacy which I held while completing the require­ ments for the degree Doctor of Philosophy. In May 1958 I have been awarded the Edwin Leigh Newcomb Memorial Award of

Pharmac ognosy.

I am a member of the Egyptian Pharmaceutical Associa­ tion, and the Sigma Xi.