COMPARATIVE STUDIES OF DATURA METEL L. AND DATURA METELOIDES DUNAL

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By EMMA P. MANIQUIS, B.S., M.S. The Ohio State University

1954

Approved by:

Adviser ACKNOWLEDGMENTS

The author wishes to express her sincere gratitude, to

her adviser, Dean Bernard V. Christensen, for the encourage­ ment he has given her to pursue graduate studies sufficient­

ly to attain the degree of Doctor of Philosophy; for his un­

tiring efforts in guiding and promoting this research.

A deep appreciation goes to Dr. Jack L. Beal, Assistant Professor in Pharmacognosy, whose sympathetic direction and

pertinent suggestions were invaluable in accomplishing this work. To the professors, who served on the advisory and read­

ing committees: Drs. E. P. Guth, F. W. Bope, J. W. Nelson, and R. II. Bohning, goes sincere thanks also for the help given in advancing this dissertation. To her colleagues, whose interest was manifested by

their stimulating questions and their continuous cooperation, much credit is due. To the American Association of University Women, whose fellowship has been enjoyed by the author, throughout the two years (1952-195^) of study, words of appreciation are inadequate unless they find expression in a dedication to the field of pharmaceutical education in the Philippines.

ii

\ 4 S 1 4 1 TABLE OP CONTENTS Page I. INTRODUCTION ...... 1 II. HISTORICAL BACKGROUND ...... > 3 III. REVIEW OP LITERATURE ...... 5

IV. EXPERIMENTAL ...... 13 Identification of the Plant Species ..... 13

A. Identification of Datura meteloides Dunal. 13

Morphological study ...... 15

B. Identification of Datura metei L ...... 17 Histological Studies ...... 18 A. Preparation of the materials ...... 18

B. Microscopical studies of Datura metel L. . 24

Palisade ratio ...... 27

Stomatal number ...... 35 Vein islet number ...... 37 C. Microscopical studies of Datura meteloides Dunal ...... 39 Identification of Constituents ...... 49

A. Paper chromatographic studies ...... 49

Experimental on Datura metel L. (oven dried) ...... 70

Official total alkaloidal assay .... 70

Individual alkaloidal assay ...... 72 Experimental on Datura meteloides Dunal (oven dried) ...... 76 Experimental on Datura meteloides Dunal (lyophilized) ...... jQ iii TABLE OF CONTENTS CONT'D. Page

B. Paper electrophoresis ...... 80

Apparatus and materials ...... 8l

Procedure ...... 82

V. DISCUSSION OF RESULTS ...... 87 A. Morphological and histological studies .. 87

B. Identification of constituents ...... 90 0 ' Paper-.-.electrophoresis ...... 96

VI. SUMMARY AND CONCLUSIONS ...... 99

BIBLIOGRAPHY ...... 102

AUTOBIOGRAPHY ...... 10 5

iv LIST OF FIGURES Figures Page

1 . D. meteloides leaf ...... 16

2. Fruits of D. meteloides Dunal...... 19

3. Fruits of D. metel L ...... 19 4. Trichomes found in D. metel L ...... 29

5. Transverse section of the midrib, D. metel L. .. 29

6 . Vein islets of D. metel L. with calcium oxalate crystals ...... 31

7. Trichomes of D. metel L ...... 31 8 . Transverse section of the upper portion of the stem of D. metel L ...... 33

9. Fibro vascular bundle, D. metel L ...... 33 10. Transverse section of the midrib, D. meteloides. 42

11. Trichomes found in the epidermis of the leaves, D. meteloides ...... 42 12. Vein islets of D. meteloides with calcium oxalate c;rystals ...... 44

13. Transverse section of the upper portion of the stem of D. meteloides ...... 44 14. Transverse section of the young stem of D. meteloides .. .;...... 46 15. Fibro vascular bundle of D. meteloides ...... 46 16. Descending chromatography apparatus ...... 54

17. Ascending chromatography apparatus ...... 5^

18. Strip chromatograms of pH 7.^ ...... 7^

19. Strip chromatograms at pH 6.8 ...... 20. Diagram of electrophoresis cell ...... 83

v LIST OP TABLES Table Page

I Series of Tertiary Butyl Alcohol Mixture (TBA) for Dehydration in the Paraffin Method ...... 22

II The Palisade Ratio of the Mature Leaves of Datura metel L ...... 35

III Values Obtained for the Stomatal Number of.the Matured Leaves of Datura metel L ...... 36

IV Values Obtained for the Vein Islet Number of the Matured Leaves for Datura metel L ...... 38

V The Palisade Ratio of the Matured Leaves of Datura meteloides Dunal ...... 47

VI Values Obtained for the Stomatal Number of the Matured Leaves of Datura meteloides Dunal ..... 48

VII Values Obtained for the Vein Islet Number of the Matured Leaves of Datura meteloides Dunal ..... 49

VIII The Rf Values Obtained by Descending Chromato­ graphy for Known Samples of Scopolamine, Hyoscyamine and Meteloidine ...... 59 IX Average Rf Values Obtained for Hyoscyamine, Scopolamine and Meteloidine at Different pH Values ...... 63 X Values of Scopolamine Standardization Vital!- Morin Colorimetric Assay ...... 66

XI Values of Hyoscyamine Standardization Vitali- Morln Colorimetric Assay ...... 66

XII Percentage Recovery of Alkaloids Hyoscyamine and Scopolamine from Elution Process ...... 70

XIII Official Assay of the Total Alkaloids of the Matured Leaves of Datura metel L. (oven Dried).. 71

XIV Quantitative Determination of the Individual Alka­ loids of the Leaves of Datura metel L. by Vitali- Morin Colorimetric Assay ...... 75

XV Offidal Assay of the Total Alkaloids of the Matured Leaves of Datura meteloides (oven dried) ...... 76 vi LIST OF TABLES CONT'D.

Table Page XVI Quantitative Determination of the Individual Alkaloids of Datura meteloides Leaves (oven dried) by the Vltall-Morin Colorimetric assay. 78

XVII Official Assay for the Total Alkaloids of Datura meteloides Leaves (lyophillzed)...... 78

XVIII Quantitative Determination of the Individual Alkaloid of Datura meteloides Leaves (lyophi­ lized) by the Vital!-Morin Colorimetric Assay. 79

XIX Results Obtained by Electrophoresis of the Alka­ loids at pH 4.2 Acetate Buffer with 460 Volts. 84

XX Results Obtained by Electrophoresis of Alkaloids at pH 9.5 Using 1 M Glycine and NaOH Buffer with 460 Volts ...... 85 XXI Results Obtained by Electrophoresis of Alkaloids at pH 10, Using 1 M Glycine and NaOH with 460 Volts ...... 85

XXII Results Obtained by Electrophoresis of Alkaloids at pH 10.5 Using 1 M Glycine and NaOH with 460 Volts ...... 86 XXIII Results Obtained by Electrophoresis of Alkaloids at pH 11 Using 1 M glycine and NaOH with 460 Volts ...... 86 XXIV Average Values of the Palisade Ratio, Vein Islet Number and Stomatal Number of Datura metel L. and Datura meteloides Dunal ...... 89

XXV Quantitative Alkaloidal Content of Datura metel L. and Datura meteloides Dunal ...... 98

vii COMPARATIVE STUDIES OF DATURA METEL L. AND DATURA METELOIDES DUNAL

1

INTRODUCTION

There has been much confusion regarding the nomencla­ ture and the alkaloidal content of Datura metel L. and

Datura meteloides Dunal in various localities, and also the method of separation of the individual alkaloids present in the plant, and it is the purpose of this investigation to furnish some data concerning these aspects.

Datura metel L. and Datura metelolds Dunal have not been widely investigated and have been quite often mislabeled as other species like Datura fastuosa L. and Datura innoxla

Miller, respectively. The morphological and histological characteristics of each species, D. metel L and D. meteloides

Dunal, were undertaken for identification.

According to some Investigators, the amount of total alkaloids present in Datura species varies with the change of environment, and it is one of the objectives of this in­ vestigation to ascertain the alkaloidal content of the samples of D. metel L. and D. meteloides Dunal that were obtained from the Ohio State Medicinal Plant Garden.

The most commonly occurring alkaloid in the official

Daturas, D. stramonium L. and D. tatula L., is hyoscyamine and in some specie^ like D. metel L. and D. meteloides Dunal 1 2 the predominating alkaloid is scopolamine. Some alkaloids from the plant may be separated individually by chromato­ graphy, electrophoresis and ion exchange methods. To check a simple, accurate method for the separation and quanti­ tative determination of the individual alkaloids of Datura metel L. and Datura meteloidesDunal, is one of the purposes of this study. t II

HISTORICAL BACKGROUND

Datura metel L. Datura metel L . belongs to the family Solanaceae and its natural habitat is tropical Asia and Africa (l); it is

also found naturalized in Mexico, Central America and South America. The word metel was an Arabic or East Indian vernacu­

lar name (2) and according to Bergner (3).. Chatterji (4) and

Safford (5), Datura metel L. was synonymous with Datura fastuosa L. According to Narayanswami (6), Datura fastuosa L. can be considered as a variety of Datura metel L. The other syn­ onyms for D. metel are D. fastuosa, D. cornucopia, D. aegyptica or Hindu Datura (7)- Timmerman (8) classified D. metel L . as another name

for D. fastuosa var. alba. Some authors for example have called the Mexican species of Datura innoxia Miller, by the name D. metel, but the true D. metel described by Linnaeus was a species based upon the Asiatic metel nut, which was used as a narcotic by theArabs, Persians, and Hindoos long before the discovery of America (9)*

The recommended revision of the British Pharmacopeial monograph for Dauura folia as a result of Timmerman's study of the nomenclature of Datura species, which appeared in the

1934 British Pharmacopeial Codex (9) was: Datura leaf con­

sists of the dried leaves and flowering tops of Datura metel L . 3 an annual plant indigenous to India, and also of Datura innox-

ia Miller, an annual plant indigenous to Mexico, now growing

freely in India and cultivated in England. The predominating

alkaloid in D. metel L. is scopolamine and occasionally a

small amount of hyoscyamine and nor-hyoscyamine (27).

Datura meteloides Dunal: Datura metelolds Dunal, which is sometimes called

Sacred Datura (7) was naturalized in the river valleys, dry hills and plains of Texas to California and Colorado, also on roadsides east to Florida (l).

According to Gerlach, (9) Datura innoxia Miller closely

resembles D. meteloides Dun., which was an American species used ceremonially by the Mexicans, Zunis, and California Indians. These species of Daturas are still found in the

southwestern portion of the United States and their use as

an intoxicant and hypnotic still prevails among the Indian tribes of Utah, Arizona and Mexico.

The name Datura was given to the genus by Linnaeus, in which he Latinized the East Indian name Dhatura. Whenever

found, the Daturas first appeared in symbolism and religious ceremonies, since they were recognized for their hypnotic properties and were employed as poisons and intoxicants (9). Ill

REVIEW OF LITERATURE

The alkaloids commonly found in the genus Datura are hyoscyamine, scopolamine and atropine. In some species, like

Datura meteloides, Datura ferox, and Datura myoporoides, a minor alkaloid called meteloidine is found (10). Carr and Reynolds (ll) reported that nor-hyoscyamine was found to occur in Datura metel, Scopolia .japonica,

Dubolsia myoporoides and Datura meteloides. Nor-hyoscyamine differs from hyoscyamine in that the former is devoid of the methyl group attached to the nitrogen atom.

Differences in climate and soil are known to produce considerable alteration in the constituents of the plant as stated by Andrews (12). He experimented on D. metel grown in

India and in Europe and found that those Daturas in India contain scopolamine and those in Europe contained only hyoscy­ amine. Schmidt (13) examined D. metel grown in France and found that scopolamine is present in all parts of the plant and unaccompanied by any notable quantity of the other mydriatic bases. Evans and Partridge (48) claimed that the amount of alkaloid present in the plant varies with the environment and also with the age of the plant. D. metel. in which 1 -scopolamine is the dominant al­ kaloidal constituent (1 5 ) is one of the major sources of this alkaloid. However, G'erlach (9) reported that Datura innoxia 5 6

Miller, is also a good source of scopolamine, and contains from 0.394 to O .713 per cent of 1-scopolamine base, depending on the method of analysis used.

The free alkaloidal base, 1-scopolamine is a viscous liquid soluble in ordinary organic solvents, but less readily 20 Q soluble in benzene. It has an optical rotation of [°^]D -l3 in ethyl alcohol. Scopolamine has an empirical formula of c 17H21°4N and has a structural formula of (39): H H

^ 9 N-CHoq — CH-O-C-C-CH2OH1 8 \ I j I C5H5 c ------c ------. ch2 H H

1 -Scopolamine, when treated with hydrobromic acid, readily forms the hydrobromide salt, which has a melting point of 193“194°C. and occurs as rhombic tablets, readily soluble in water or alcohol, sparingly soluble in chloroform and insoluble in ether. The matured leaves and flowering tops of the official

Daturas, D. stramonium and D. tatula contain chiefly, the alkaloid hyoscyamine. Atropine is the racemic mixture of d-hyoscyamine and 1 -hyoscyamine. 1-Hyoscyamine has a chemical formula of Cand has a structural formula (3 9 ) of: The free base, 1-hyoscyamine, which crystallizes in the form of silky needles, melts at 108-111°C. and has an 20 optical rotation of [oclp -22° in ethyl alcohol. Hyoscyamine base, when treated with gold chloride forms the aurichloride

which melts at l65°C. and is composed of golden yellow hexagonal crystals. Meteloidine, an alkaloid isolated by Pyman and Reynolds

from D. meteloides Dunal, has been reported to be present in

the amount of 0.07 per cent in the plant. Meteloidine was isolated by percolation of the dried, matured leaves of D. meteloides with 95 pex* cent ethyl alcohol and the alcoholic extract was concentrated to a semi-solid mass, from which the

alkaloid was removed by stiring with 1 per cent hydrochloric

acid. The aqueous liquor was rendered alkaline with ammonia and extracted with chloroform. Then the chloroform fractions were extracted with successive portions of dilute hydrobromic acid. The extracts were concentrated separately and allowed to crystallize. On cooling, meteloidine crystals appeared in the first fraction as the hydrobromide.

Meteloidine (17) is a crystalline base itfith an empiri­

cal formula of C13H21O4N and has a structural formula of (3 9 ): Meteloidine is freely soluble in alcohol, chloroform, and acetone but sparingly soluble in water, ether and benzene.

The base, meteloidine, when treated with hydrobromic acid, gold chloride and picric acid forms the corresponding salts, the hydrobromide, aurichloride and picrate.

King (l8 ) reported that meteloidine is optically inac­ tive and has no marked physiological activity. Hydrolysis of meteloidine will give a base called teloidine and tiglic acid.

C13H21O4N 4- H 2 O ------> C^Hg02 + CgH^O^N (meteloidine) (tiglic (teloidine) acid)

Meteloidine has two hydroxy groups attached to two car­ bon atoms in the cis configuration (10). The dihydro deriva­ tive of meteloidine has been synthesized by Sheehan and Bissell (10). The basic hydrolytic product teloidine, crystal llzes from moist acetone with one molecule of water of crystal ization. The hydrobromide of meteloidine crystallizes from water in hard clusters of chisel shaped needles and contains two molecules of water of crystallization and is readily soluble in water and absolute alcohol.

Intensive work .has been done on the separation and the quantitative determination of the individual tropane alkaloids. 9 Evans and Partridge (14,19) separated scopolamine and hyoscyamine by chromatography using a column of kieselguhr.

They stated that in adsorption chromatography, the process of adsorption involves the Van der Waals forces between the parts of the molecules, whereas in partition chromatography, the factors related to the whole molecule which control its solu­ bility properties are involved. For a good separation, most of their experiments were based upon the effects of pH value, the total alkaloid in the column and the proportion of the individual alkaloids. The application of chromatography to the separation and purification of alkaloids has been described (11 , 14, 2 0 , 2 1 ), usually with benzene as the solvent and alumina as the adsor­ bent .

Kuhn and Schofer (21) reported a quantitative separa­ tion of hyoscyamine and scopolamine based on their differences in basicity, in that scopolamine is a weaker base and is more soluble in ether than hyoscyamine or atropine. They used sodium bicarbonate to liberate scopolamine and then extracted the scopolamine with ether, leaving the hyoscyamine in the mother liquor. The extraction of scopolamine with ether us­ ing sodium bicarbonate to liberate the free base had to be done several times to obtain a pure alkaloidal sample.

Rowson (2 2 ) suggested that hyoscyamine, scopolamine and atropine can be separated by Kuhn and Schofer’s method, in which the individual alkaloid was determined by the opti­ 10 cal rotation. The alkaloidal free base was dissolved in alcohol and he reported that the specific rotation of 1 -hyo- scyamine is -22° and 1 -scopolamine is -18° and the amount of atropine was calculated from the difference of the total

■alkaloids. Trautner and Roberts (20) made a study of the separa­ tion of scopolamine and hyoscyamine using a silica gel column.

They used dimethylaminoazobenzene as a color indicator. In this method, the adsorbent was colored red except in the posi­ tions where the alkaloids were absorbed. The application of paper chromatography for the separa­ tion of the alkaloids has been investigated. Drey and Foster

(2 3 ) separated hyoscyamine and scopolamine quantitatively by paper chromatography. Strips of Whatman paper No. 1 were used and buffered with Sorensen's phosphate buffer at pH 7.4 before it was chromatographed. They used Dragendorff1s reagent to detect the position of the spots on the paper. Then the untreated paper strips carrying the individual alka­ loids were eluted with 95 per cent ethyl alcohol overnight and the amount of alkaloid was determined quantitatively by

Vitali Morin Colorimetric assay. Separation of the tropane alkaloids by partition paper chromatography was also studied by Brindle et al (24). They found by using filter paper Impregnated with Sorensen's phos­ phate buffer at pH 7.4-,. that scopolamine and hyoscyamine will give different Rf values. The size of the spot formed on the 11 paper gives an estimate of the amount of alkaloid present, but a quantitative separation was achieved by eluting with 95 per cent ethyl alcohol and assaying for the individual alkaloid by Vitali Morin Colorimetric Assay. They have re­ ported that a better reagent, which is 0.2 per cent iodine in potassium iodide solution, will identify the spot of alkaloid on the paper. The method they Used to develop the spots with this solution, was similar to photographic developing, in which the strips of paper containing the alkaloids were passed in the solution and the characteristic colors of the alkaloids were shown. Scopolamine gave a brownish red color and hyoscyamine a light bluish gray color. The intensity of the color was greatest after 30 minutes and then faded after an hour. However, the color could be reproduced by passing the strips again in a fresh iodine solution. The spots may be permanent on the paper when they are developed with Drag- endorff's reagent, but all the spots are the same color as the reagent, which is orange color. Concentration of the alkaloid in less than 100 microgram failed to show with the iodine solution. Scopolamine faded faster than hyoscyamine in the iodine solution. The paper chromatography of alkaloids has been done by Consden et al (25) and Munier (26), in which they described how the spot formation depends upon the solubility of the alkaloid and the pH of the solvent. Strong bases, (Kb great­ er than 10 ^ ) gave a round spot in neutral solvents, and 12 alkaloids which range in their constants between lO-^ to

10 10 gave elongated trailing spots in neutral solvents. The separation of atropine, 1-scopolamine and 1-hyo­ scyamine has been effected by the fractional crystallization of their aurichlorides. However, the solubility relations of these derivatives are dependent upon the impurities and the relative amounts of each present in the mixture (27). A method of separation of 1-hyoscyamlne and atropine was pro­ posed which employed fractional crystallization of their oxalates from acetone and ether in which the 1 -hyoscyamine derivative had the greater solubility (1 5 ). Gore (28) has reported the detection of alkaloids as applied to paper chromatograms using formic, acetic and pro­ pionic acids mixed with the solvent. The Rf values were cal­ culated after treating the strips with Dragendorff1s reagent.

Schlite (29) reported the separation of some solanaceous alkaloids in the form of their bases, using an ammoniacal solvent and he further studied the factors which influence the Rf values. Roberts and James (30) described a method of determin­ ing the total alkaloids using one gram samples and extracting the alkaloids with non-polar solvents, which were purified by adsoprtion in a double column of silica and alumina. IV

EXPERIMENTAL

Identification of the Plant Species

A. Identification of Datura meteloids Dunal. According to Bailey (2) and Small (l) the description of Datura meteloides Dunal includes the following:

Handsome herbaceous perennial, 1-3 ft., cultivated as annual, glaucesent and purulent, grayish; the slender branches forked; leaves unequally ovate, repand, or subentire, acuminate, the upper often in unequal pairs, the larger 2-21/4 inches long; flowers erect, singly, calyx, tubular, 3 inches long with long acute unequal teeth; corolla white or tinged with purple, sweet scented, about twice, as long as the calyx, with 5 or 10 slender-subulate teeth; capsule subglobose, 2 Inches in diameter, with long sharp spines, bursting irregularly; seeds narrowly margined. D. meteloides Dunal resembles very much another spec­ ies, D. innoxia Miller. According to the description based on Safford*s key (5)* D. meteloides Dun, has distinguishing characteristics from D. Innoxia Miller in the following: Plant softly pubescent, corolla white, 10 toothed.

Datura innoxia Miller Plant glaucescenlp, corolla usually suffused with pale

lavander, 5 toothed Datura meteloides Dun.

In general, D. innoxia has the indument of the young branch- lets and the petioles composed of obvious hairs 0.5 to 1 mm. long, whereas the corresponding hairs of D. meteloides Dunal are much shorter, usually 0.1 to 0.3 rom. long. 13 14

One of the differences between D. meteloides and D.

innoxia is that, the former has smaller leaves, acute at

both ends but neither cordate nor angular in outline (9 ). D. meteloides has been reported by Pyman and Reynolds

(1 7 ) to contain 0.13 per cent of scopolamine, 0.03 per cent of atropine, and 0.07 per cent of meteloidine. The total alkaloidal content of the whole plant is 0.4 per cent.

Cultivation: The seeds of D. meteloides were collect­

ed from plants growing on a farm in Kansas. A sample of the plant grown on this farm was sent to the Department of Botany,

at Kansas University and was identified as Datura meteloides

Dun. The seeds were planted, May, 1953 at The Ohio State Medicinal Plant Garden. When the leaves and fruits became mature, they were collected September J, 1953> some of which were preserved in a fixing solution of formalin-acetic acid- alcohol mixture (31), and the others were dried for total alkaloidal assay.

Preliminary investigation of the alkaloidal content of

D. meteloides did not show the presence of meteloidine, and

because this plant closely resembles another species, D. in­ noxia Miller, samples of the plant in question in the fixing

solution were sent to Smithsonian Institution, Washington, D.C. for further identification. According to their descrip­

tion in the letter, "these two species are certainly very

closely allied, and the identification does not seem very

dependable. It would be my conclusion, on the basis of our herbarium material, and lacking any positive contradictory 15 characters in Safford's work, that your plant represents

Datura meteloides." General external morphological features of Datura

meteloides. The plant was one to three feet high and the branch­ es were slender, forked and spreading. The stems and leaves were puberulent, or covered with soft, fine hairs. The leaves were unequally ovate, repand or bent backward, acumin­ ate and velvety in appearance due to the numerous hairs cover­ ing the entire leaf. They measured from 8 to 10 cm. long and about 7 cm. at the broadest part. The margin Was entire and the apex was acute. There were 5 to 7 prominent veins on the lower side of the leaf and the outer surface was darker green in color than the lower surface. The veins do not run all the way out to the leaf margin but instead stop about 2 to 3 mm. from the edge and were joined to one another by arches as shown in Fig. 1. The flowers were erect, singly, and white.

The calyx were tubular, 3 Inches long with long acute unequal teeth. The corolla was white, about 6 inches long, with 5 slender-subulate teeth. There were 5 stamens which were long and narrow. The capsules were subglobose, measuring about an inch and a half in diameter, with long sharp spines, bursting irregularly.

Fig. 2 shows the fruit capsule and the characteristic spines, with the broad,receptacle. 16

Datura meteloides leaf showing the veins along the margin joined to form an arc. "‘17 B. Identification of Datura metel L. Bailey (2) stated the following description for D. metel L.: Annual glabrous herb, 4-5 ft. high; leaves ovate, lanceolate, acuminate or acute, unequal at the base, sinuate-toothed or repand, glabrous on both sides, solitary or the upper in pairs with one larger than the other, the larger 7-8 inches long; flowers erect, 6 1/2 to 7 inches long; calyx purple, circumscissile, tubular andulate 2 inches long, 5 toothed, the teeth triangular-lanceolate, acumin­ ate; corolla white, the limb typically 5 but some- - times with 6 long cuspidate acute angles, stamen 5 -6 ; capsules subglobose, short spiny, 1 1/4 inches in diameter and indehiscent. The seeds of D. metel L. used in this investigation came from the University of Washington and were planted in the Ohio State University Medicinal Plant Garden in May, 1953. The leaves, stems and flowers when matured were collected for histological study. They were preserved in a fixing solution of acetic acid-formalin-alcohol mixture (31). Some of the leaves collected were dried for the alkaloidal assay.

General external morphological features: The plant is an annual herb, 3 “5 ft. high. The leaves were ovate, some were lanceolate or acuminate and unequal at the base. The full grown leaf measured from 8-18 cm. long and averaged

7-16 cm. at the broadest part. The margin is usually entire but sometimes sinuately toothed on both sides of the margin, however, not as distinct as D. stramonium. The leaves ap­ peared to the naked eye to be smooth and were rather thin in texture. The upper surface of the leaves are greener than the lower surface. The midrib was prominent on the lower 18 surface and the main veins extend along to the margin of the leaf, which are branched. The flowers were erect, about 6-7

inches long and five toothed. The corolla was white. There

xvere 5 long and narrow stamens. The calyx was tubular and 5

toothed. The fruit capsules were subglobose, short, spiny and

about an inch to an inch and half in diameter. Pig 3 shows

the fruit of D. metel L.

Preliminary investigation of D. metel L. showed scop­

olamine as the main alkaloid and a trace of hyoscyamine.

This agreed with other reports (15) that the chief alkaloid of this plant is scopolamine.

Histological Studies A. Preparation of the Materials:

The matured leaves, stems, flowers and fruits of D. metel L. and D. meteloides Dunal were preserved in a fixing

solution of formalin-acetic acid-alcohol mixture (3 1 ) pre­ pared as follows: five ml. of formalin and 5 nil. of acetic

acid were added to 70 per cent ethyl alcohol to make a volume of 100 ml. then 0.2 per cent of copper sulfate was added to the mixture as preservative.

The specimens were kept separately in the fixing so­ lution for further histological studies. Permanent mounts were made of the leaves and stems of each species.

The rest of the leaves were collected and dried for the official assay of the total alkaloids and the determin- 19

D. Meteloides D.

P i g . 2 Fruits of D. meteloides Dunal

L>. .Motel I-..

Fig. 3 Fruits of' D. metel L. 20 ation of the individual alkaloids. The matured leaves of D. metel L. were oven dried for 48 hours at 6o°C. Then the dried leaves were passed through a Wiley Mill using a No. 40 sieve. The powdered leaves were kept in amber colored bottles. The matured leaves of D. meteloides Dun. were dried in two ways, oven drying and lyophilization. The oven dried leaves were subjected to the same procedure as for Datura metel. The lyophilized leaves were processed or dried by collecting the fresh leaves from the plant and putting them directly on a sufficient amount of dry ice in thermos con­ tainers. Then the leaves from the containers were trans­ ferred to the lyophilizer for drying. They were lyophilized for 24 hours and were powdered in a Wiley Mill using a sieve, size No. 40. The powdered leaves were kept in amber colored bottles for later .investigation of the alkaloidal content.

Preparation of the Slides

Preparation of the stem: The stems of D. metel L. and D. meteloides Dun. were cut by free-hand sectioning. The sections were stained with safranin and fast green in alcoholic solution. The safranin stain was a saturated solution in 95 per cent ethyl alcohol; the fast green stain was one Gram of fast green dissolved in

25 ml. of absolute alcohol and 75 ml. of clove oil.

The sectioned stems were placed for 15 minutes in a staining vessel to which was added 15 drops of the safranin 21 solution diluted with distilled water. The excess stain was washed off from the specimens with distilled water and then run through a series of alcohol dilutions of 5 0 , 6 0 , 7 0 , 8 0 ,

9 0 , and finally to 95 per cent till the desired color of the sections was obtained. Approximately 5 minutes v/as suffici­ ent time for"'each dilution. Then 5 drops of the fast green stain v/as added to the sectioned stem and allowed to stand for 15 minutes. Tne excess dye v/as removed v/ith 95 per cent ethyl alcohol and then followed with absolute alcohol. The tissues were dehydrated by passing through absolute alcohol, then the sections were rinsed with equal parts of absolute alcohol and xylol mixtures. Dehydration v/as complete when the solution v/as no longer turbid. Lastly, the sections were transfered to pure xylol and then mounted in a mounting media consisting of equal parts of xylol and Canada balsam. The slides were labeled and dried at ordinary temperature.

The leaf preparation: The material v/as made by the paraffin method as described by Johansen (31).

The matured leaf v/as cut into small pieces about 5 mm. long and 3 mm. wide. Because the leaves were left in the fix­ ing solution, the sections were washed with S> per cent ethyl alcohol and followed by a series of dilutions, increasing each by 10 per cent up to 70 per cent. The sections were kept for almost an hour in each dilution. From the 70 per cent ethyl alcohol, the material v/as transferred to a 50 per cent terti­ ary butyl alcohol mixture (TBA) and then through the succeed­ 22 ing series of dilutions as shown in Table I* The different series of alcohol strengths were made according to Johansen's method (3 1 ). Table I

Series of Tertiary Butyl Alcohol Mixtures (TBA) for Dehydration in the Paraffin Method (According to Johansen)

Approximate total of alcohol 50 70 85 95 100 Distilled Water 50 30 15

Ethyl alcohol 95$ 40 50 50 45 Tertiary Butyl alcohol 10 20 35 55 75 Absolute Ethyl alcohol 25

To give a better orientation in the microtoming, the

100 per cent TBA was tinged with enough erythrosin for the specimen to obtain a light pink color. The color facilitates in seeing the specimen clearly when it is embedded in the paraffin. The material should remain for at least two hours in each immersion of the different alcohol mixtures. From the 100 per cent TBA, the material was transferred to pure tertiary butyl alcohol and was allowed to stay for two hours.

Then the tertiary butyl alcohol was changed again and the specimen was set aside for another hour. Finally, the speci­ men was placed in an equal mixture of tertiary butyl alcohol and paraffin oil for an hour.

Infiltration:

A small bottle of about an ounce capacity was filled three-fourths full of melted paraffin. Then the paraffin was allowed to solidify and the material from the TBA paraffin mixture was transferred on top of the solidified paraffin.

Care being taken that a few ml. of the TBA paraffin mixture v/as added to prevent the drying of the specimen. The bot­

tles were placed in the oven at a temperature not exceeding 65°C. until all the paraffin had melted and the specimen had gone down to the bottom of the container. The TBA mixture should be completely removed by changing the paraffin-TBA mixture twice with pure melted paraffin. Then the material v/as poured into paper blocks. To avoid damaging of the tis­ sues, the paper v/as v/armed so that the paraffin would not

solidify before the specimens were placed in the proper order.

Care was taken not to move the specimens, once the paraffin had solidified. The paper blocks were cooled in ice v/ater. The paraffin blocks containing the specimens were cut with a

sharp razor blade leaving about 2 mm. of paraffin along either side to prevent injuring of tissues during microtoming.

The trimmed paraffin blocks were attached to a piece of wood, about 3 cm. long and one cm. thick, by heating gently one side of the paraffin block. Then the whole structure was allowed to cool in ice v/ater to facilitate sectioning. The rotary microtome was used and the sections were cut about 20 microns thick. Strips of paraffin containing 3“^ sections v/ere placed on clean slides previously covered with a thin film of Mayer’s Adhesive (31) which is composed of 50 ml. of egg albumin, 50 ml. of glycerin and one gram of sodium benzoate 24 as preservative, and filtered. The slides were warmed at

35°C. until the strips were flattened, and then allowed to

cool. The paraffin was removed from the specimen before staining by passing the slides through pure xylol and then

through a series of alcohol dilutions down to 50 per' cent.

The specimens affixed on the slides were stained for 15 minutes in a safranin stain, as made in the previous prepara­

tion of the stem. The excess stain was removed with 50 per

cent ethyl alcohol, then the specimens were passed through

different concentrations of ethyl alcohol, 6 0 , 70, So, and 90 per cent. Finally, they were passed through the 95 per

cent ethyl alcohol until only a slight tinge of red v/as shown

on the specimen. From the 95 per cent alcohol, the slides v/ere stained for 15 minutes v/ith fast green, as prepared in

the previous preparation of the stem. The excess stain was removed v/ith absolute alcohol until only a faint bluish green color v/as produced. Lastly, the slides v/ere dipped in

xylol-absolute alcohol mixtures of equal parts and then in

pure xylol. The specimens v/ere mounted in Canada balsam in

which a few ml. of xylol v/ere added. The slides were care­ fully labeled and allowed to dry at room temperature.

B. Microscopical Studies of Datura metel L .

The slides containing the sections of the stem and

leaf of Datura metel, as previously prepared, v/ere examined under the microscope for their structural characteristics. 25 Transverse section of the lamina. The lamina measured about 250-300 microns in thickness. A thin cuticle was pres­ ent on the outer epidermis. Belov/ the upper epidermis, v/as a single layer of palisade cells v/hich are elongated and occu­ pied about 180 microroof the thickness of the lamina. Below the palisade tissue was the spongy mesophyll which was com­ posed of rectangular parenchyma cells. Numerous calcium oxalate crystals, both in rosette and prism forms v/ere found in the thin, irregular parenchyma cells. The middle section consisted of spongy cells and intercellular spaces, inter­ sected v/ith vascular veins. The lower epidermis ivas composed of a single layer of cells v/ith numerous stomates. Uniserate trichomes v/ith three to four cells v/ere found in both the upper and lower epidermis. Occasional gladular hairs are sometimes located on the upper epidermis. Pig. 4.

Transverse section of the midrib. The lov/er surface of the midrib v/as round and measured about one to one and a half millimeter in diameter. The epidermis consisted of a single layer of cells, which are rectangular in shape and partially thickened at the end. The epidermal cells give rise to numerous uniserate three celled trichomes. Occasional glandular trichomes were also found on the epidermis. The cortex was composed of two layers, which are the hypodermis and the cortical parenchyma. The hypodermis, located just below the epidermis, is composed of 3 to 4 layers of collen- chyma cells, which are rounded and thick in outline. In the 26 cortical parenchyma v/ere found large and thin walled paren­ chyma cells which were almost colorless. Numerous crystals of calcium oxalate in aggregate rosettes and prisms v/ere scattered in the cortical layer. The xylem vessels v/ere arranged in an arc and opposite to each of them on the lower side were the phloem cells. The upper surface of the midrib v/as made up of a single layer of epidermal cells and two to three rows of collenchyma cells. The longitudinal ridge or the upper surface of the midrib v/as almost on a level v/ith the upper surface of the lamina as; shov/n in Fig. 5.

Surface preparation. Small fragments of the powdered leaves, preferably the larger particles v/ere macerated with chloral hydrate T.S, (19) in a beaker and heated on a v/ater bath for 10 minutes. Upon microscopical examination, calcium oxalate crystals v/ere found in abundance and the vein islets were very clear as shov/n in Fig. 6 . D. metel. L. had tv/o kinds of hairs, the glandular type which are short stalked and a very prominent gland at the tip, the hair usually measured from 27 to 100 microns long and the other type of hairs are the uniserate hairs composed of three or four cells which vary from 30 to 135 microns in length. The base of the uniserate trichomes was very characteristic and measured from

15 to 25 microns in diameter. The glandular hairs are most commonly found in D. metel and v/ith few non-glandular type of trichomes. Fig. 7- Transverse section of the stem. The outer surface of the stem consisted of a. thick celled epidermis on which numer- our trichomes were found, Pig. 8 . Belov/ the epidermis is the cortex composed of tv/o layers, the hypodermis and the corti­ cal parenchyma. The hypodermis v/as made up of 6 to 7 rows of thick walled collenchyma cells which were more or less rounded in shape. The cortical parenchyma v/as composed of 2 to 3 layers of thin walled rectangular cells and are sometimes irregular in outline. The endodermis consisted of elongated cells o r nearly equal cells and within this sheath v/as the pericycle. The cambium cells v/ere found in the inner layer of the xylem, which forms intraxylary phloem of the stem. Numerous calcium oxalate crystals v/ere scattered in the cor­ tex, usually in aggregate rosettes, v/ith occasional prisms.

Pig. 9 shov/s the xylem vessels with the medullary rays in be­ tween them. The pith comprised about a third of the diameter of the stem and v/as composed of thin walled parenchyma cells. The glandular hairs were short stalked and sometimes broader at the base, v/hile the uniserate type, were made up of three to four cells and were longer than the glandular hairs.

Determination of the palisade ratio of Datura metel L.

In several matured leaves of D. metel, pieces were cut about 3 to 5 millimeters square from the tip, middle and base. The segments v/ere cleared by boiling them with chloral hydrate

T.S. (l6 ), for 15 minutes in a test tube on a water bath, separately. A segment was mounted on a slide and examined micro- 28

Fig. 4 Trichomes found in D. metel L.

a. glandular hair b. uniserate type of hair

Fig. 5 Transverse section of the midrib D. metel

a. upper surface of the midrib showing the longititudinal ridge composed of collenchyma cells. b. transverse section of the lamina with the palisade cells. c . fibro vascular bundle with the xylem vessels and phloem tissues. 29

Pig. 4

rtt '

Pig. 5 30

Fig. 6 .

Vein Islets of D. metel L . v/ith calcium oxalate crystals.

a. leaf vein b. calcium oxalate crystals

Fig. 7 Trichomes of D. metel L.

a. glandular hair b. uniserate hair c. stornate s . * * Fig. i. 7 Fig. 6 h-"/ t i l f rwiil gnjuweyw ^i.Vv '\U«vr • *%. I^Tig.lVkv 31 32

Fig. 8 Transverse section of the outer portion of the stem, D. metel

a. Trichomes b. epidermis c . Collenchyma cells d. This rectangular parenchyma cells of the cortex.

Pig. 9 Transverse section of the inner portion of the stem of D. metel L showing the xylem and phloem regions a. phloem cells b. xylem vessels c . medullary rays 33

jjii ,*i.

Fig. 9 scopically. By means of a camera lucida, four epidermal cells were traced on the upper epidermis. Then the fine adjustment of the microscope was focused down to the palisade layer and again sufficient palisade cells were traced cover­ ing the tracings of the four epidermal cells. The total palisade cells were counted under the four epidermal cells and in making this count, those palisade cells, which were more than half within the area of the epidermal tracing-, were counted and when they were less than half, were not counted. The average number of palisade cells beneath a single upper epidermal cell was calculated and this figure corresponds to the palisade ratio. The different segments of the leaves were examined as mentioned above and the re­ sults are shown in Table II.

According to Wallis (32) the palisade ratio may aid in the identification of the different species of a plant, but in the case of D. metel L., the palisade ratio did not give a very significant identification of the species. The values obtained differ in the parts of the leaf and it was also ob­ served that the age of the leaf might add to the difference in palisade ratio, because the palisade ratio of the young leaf was determined and the values obtained were lower than that for the matured leaf. The figures obtained In this investigation were very close to that found by Chaudhuri (33) for Datura metel L. 35 Table II The Palisade Ratio of the Matured Leaves of Datura metel L.

Leaf Apex Middle Base

1 5.75 5.25 5-5

5.5 6.5 6.75 2 6.75 6.25 6.5

6.5 5.5 6.5 Average 6.1 5.87 6.31

Determination of the stomatal number of D. metel L. The microscope was standardized with a stage micrometer,

in which the diameter of the field of view of the microscope using a 4 mm. objective and a 10 X ocular was measured and an

area of one square millimeter was calculated on the paper.

The upperepidermis was removed from a matured leaf by using a sharp blade, first, a small incision was made and by using the tip of two fingers, the loose portion v/as pulled off, taking only the epidermis. Then the sections were mounted in distilled v/ater and examined. This method was preferred rather than the clearing of the tissues with chloral hydrate, because it gives a more vivid view of the stomates and is al­ so a time-saving process. The area'of the microscopic field was calculated in millimeters by using a standard stage micro­ meter. The stomates v/ere counted in one microscopic field from the different segments of the leaf samples and those 36 stomates, which were more than half included within the micro­

scopic field v/ere counted and those stomates with less than

half v/ithin the field were excluded. The results of the

count in each field were recorded and the average number of

stomates per square millimeter area v/as calculated.

The lower epidermis of the leaf was examined in the same manner as the above procedure and the average stomatal

number was determined. Results are shown in Table III.

Table III

Values Obtained for the Stomatal Number of the Matured Leaves of Datura metel L.

Leaf Lov/er epidermis Upper epidermis

1 255 193 230 210

261 214

2 270 195 282 198

257 220 3 275 187 250 190

278 210 240 180 Average 259.8 199.7

In D. metel L., the stomatal number was more at the lov/er epidermis than the upper epidermis, but the difference was. not much as compared to other plants. According to Timmerman (8 ), the stomatal number may

serve as the basis of identifying one species from the other. Usually, the type of stomates may help in the identification,

as in the case of Daturas, the Cruciferous type, that is, the stomate is usually surrounded by three or more subsidiary cells, one of which is markedly smaller than the others.

In Timmerman's study of D. metel L. (8 ), she found some collapsed stomates, but none v/as observed in this plant investigated.

Determination of the Vein Islet Number of Datura metel L .

Small 5 mm. square sections of matured leaves from the apex, middle and base v/ere cut and then placed in a test tube. Three ml. of chloral hydrate T.S. (l6 ) was added to the test tube v/hich v/as then boiled for 30 minutes on a v/ater bath to clear the tissues. The microscope was standardized by using a stage micro­ meter. By means of a camera lucida, a 4 mm. objective and a

10 X ocular, a line exactly one mm. long v/as drawn on the paper on which a square v/as constructed. A piece of leaf, previously boiled in chloral hydrate T.S., v/as mounted on a slide and examined microscopically. The veins v/ere traced on the square constructed and the vein islets v/ere counted. The vein islet number represents the total veins completed with­ in one square millimeter area using a 4 mm. objective and 10 X 38 ocular. Pour different readings v/ere made by moving the

slide in four positions about one mm. each time, and the

average reading was taken. The results obtained are illus­ trated in Table IV. Table IV

Values Obtained for the Vein Islet Number of the Matured Leaves of Datura metel L.

Leaf Apex Middle Base

1 15 14 15 17 18 19 17 16 20 2 16 14 18

18 16 19 16 14 21

15 17 16

The figures obtained in this investigation were a little higher than those reported by Chaudhuri (33), for D. metel. Figure 6 shows a vein with the crystals of calcium oxalate scattered within the vein islets. The vein islet number according to some investigators

helps in the identification of the different species, but in

D. metel, the results obtained were similar to those of D. meteloides Dunal. 39 C. Miscroscopical studies of Datura meteloides The slides containing the sections of the leaves and

stems as previously prepared were examined under the micro- scope, for histological characteristics.

Transverse section of the lamina and the midrib. The

lamina measured from 200 to 300 microns in thickness. The

palisade cells were about 150 microns thick and were found

just below the upper epidermis. The spongy mesophyll which

comprised almost two fifths of the lamina was made up of irreg­ ular, large and thin walled parenchyma cells. Intercellular spaces v/ere located in the mesophyll with the vascular veins

intersecting the spongy parenchyma. Both on the upper and

the lower epidermis, numerous hairs were found of both the glandular and the uniserate types. Crystals of calcium oxal­ ate in rosette form were scattered in the spongy parenchyma.

A characteristic distinct layer of calcium oxalate crystals

was observed in the first row of cells below the palisade

tissue. The stomates were found both in the upper and lower

epidermis of the lamina, but more so in the lower epidermis.

The midrib is composed of a single layer of epidermal cells,

then followed by the cortex made up of collenchyma and paren­

chyma cells. The fibro vascular bundles are arranged in an

arc as shown in Fig. 10.

Surface preparation. The coarse powder of the leaves were treated v/ith chloral hydrate T.S. (16 ), and boiled on a water bath for 10 minutes. Then the segments were placed on 4o

a slide and examined v/ith the microscope. There were numer­

ous hairs of both types, the glandular and the uniserate, as shov/n in Fig. 11. In this plant, D. meteloides, the trichomes

v/ere mostly the short stalked, uniserate type which were tapering at the ends and a few glandular hairs. The glandu­

lar trichomes ranged from 25 to 150 microns in length, and the basal cell measured about 70 microns. Numerous crystals of calcium oxalate were found, in the rosette and prism aggre­

gates. The vein islets were clearly observed as shown in Fig. 12. Transverse section of the stem. The epidermis was com­ posed of a single layer of cells and numerous trichomes were scattered on the epidermis. The cortex v/as composed of tv/o

layers, the hypodermis and the cortical parenchyma as shov/n

In Fig. 13. In the hypodermis, there were 4 to 5 rows of collenchyma cells, which were more or less rounded in appear­ ance. In the young stem, the hypodermis v/as much thicker (Fig. 14). The cortical parenchyma was composed of rectangular thin walled parenchyma cells and some times irregular in out­ line. The xylem vessels, as shown in Fig. 15 were inter­

sected by the medullary rays and the phloem cells were ar­ ranged in bi-collateral bundles. The pith was composed of thin walled parenchyma cells and usually in the matured stem, the pith v/as hollow. Calcium oxalate crystals in rosette form were found in the collenchyma cells and the pericycle. 41 Fig. 10.

Transverse section of the midrib of D. meteloides

a. epidermis b. parenchyma cells of the cortex c. xylem tissue d. phloem tissue

Fig. 11 Trichomes found in the epidermis of the leaves of D. meteloides mostly of the uniserate type of hair. h ,V.--

Fig. 11 43

Fig* 12 Vein Islet of Datura meteloides with calcium oxalate crystals* a* leaf vein b* calcium oxalate crystals

Fig* 13 Transverse section of the o.u&er portion of the stem of Datura meteloides Bunal. a. Trichomes b. Epidermal cells c. Collenchyma cells d* Parenchyma cells 44

.. ■’ i -7 '(ki.*'*-' J \'' «t

Fig. 13 4p

Fig. 14

Transverse section of the young stem of D. meteloides .

a. trichomes b. epidermis c . hypodermis

Fig. Ip Transverse section of the inner portion of the stem of D. meteloides shov/ing the xylern and phloem region

a. phloem tissue b. xylern vessel c . medullary ray 46

Fig. 14

Fig. 15 47 Determination of the palisade ratio of Datura meteloides.

The matured leaves of D. meteloides were cut 3 to 5 millimeters square from the tip, middle and base. The seg­ ments were cleared with chloral hydrate T.S. in the same man­ ner as for D. metel, and the results are recorded in Table V.

Table V The Palisade Ratio of the Matured Leaves of Datura meteloides Dun.

Leaf Apex Middle Base

1 5.0 4.75 4.75

5.5 5.0 5.25 2 4.75 5.5 5.0 5.5 4.75 5.25 Average 5-19 5.0 5.08

The palisade ratio from the different parts of the plant did not show very much difference on the average read­ ing obtained. However, the results are a little lower than the results obtained for D. metel.

The palisade ratio of the young leaves was investigated and the values obtained were less than those of the matured leaves, the average was 3-25

Determination of the stomatal number of Datura meteloides.

The matured leaves of D. meteloides Dun. were deter­ mined in the same manner as that for D. metel. The values 48

obtained are illustrated on Table VI.

Table VI

Values Obtained for the Stomatal Number of the Matured Leaves of Datura meteloides Dunal

Leaf Lower epidermis Upper epidermis

1 190 190

235 160 225 170

2 195 159 199 174 227 172

3 230 145 250 154 210 168

215 162

Average 217.6 165.4

Determination of the vein islets of Datura meteloides.

Segments of 5 mm. square were cut from the matured leaves of D. meteloides from the apex, middle and base. The

vein islet number was determined In the same manner as for

D. metel. The results obtained are illustrated in Table VII. According to the figures obtained, the vein islets of

D. meteloides are very close to the results for D. metel. 49 However, the average readings obtained were different in segments taken from different areas of the leaf- apex, middle and base.

Table VII Values Obtained for the Vein Islet Number of the Matured Leaves of Datura meteloides Dunal

Leaf Apex . Middle Base

1 , 13 15 14

15 17 18

12 17 15

12 15 15 2 14 18 18

15 16 19

12 15 19

Average 13.3 16.1 16.8

Identification of Constituents A. Paper Chromatographic Studies_____ Introduction. The tropane alkaloids have been separ­ ated and determined quantitatively by paper chromatography (24, 28, 42, 44). Consden et al. (2 5 ) considered filter paper as an inert support of an aqueous stationary phase and ex­ plained the observed separations as a result of continuous partitions of the substances between the aqueous stationary phase and the water immiscible organic solvent flowing down 50 the paper. The identification of the spots are made by means of the Rf value, represented as,

Distance traveled by the solute _ Distance traveled by the solvent

The Rf values depend upon the partition coefficient and.the relative amounts of the two phases in contact, and are usual­ ly measured with reference to the liquid front. While for the identification by boiling or melting points, a pure compound is necessary, the Rf value is not influenced by the presence of many impurities. Further, only micro quantities are re­ quired for a paper chromatographic identification. To iden­ tify a substance, a known substance must be run on the same paper at the same time and its Rf value measured. However, it is not possible to identify an unknown substance by its Rf value alone. There are numerous mixtures of isomers, for CH2OH ?H2NH2 example CHNHo and CHOH , that produce only one spot in all 6h3 ch3 solvents examined (3^) and also in the case of atropine, a racemic mixture of d-hyoscyamine and 1 -hyoscyamine. Some of the solvents used in early chromatography were colloidine, phenol, benzyl alcohol and n-butanol. Addition of acids like acetic acid to the organic solvent increases the solubility of the solvent for water and the Rf values were generally raised in such a solvent (3^-) • The organic solvent vias usually saturated with water to form two liquid phases for the partition to take place. Paper chromatography requires a good technique and a 51 slight variation in some factors may not produce a good sep­

aration. Munier and Macheboeuf (2 6 ) observed the tailing of

spots with the alkaloids and weak bases instead of the usual

round spots. However, overloading of the amount of alkaloids

on the paper may also cause the trailing or the formation of comets.

Bate-Smith (35) studied the conditions necessary to ob­ tain accurate Rf values. He stated that the factors affect­

ing the Rf are temperature, time of running the chromatogram

should be constant, paper should be equilibrated with atmos­

phere in the chamber for 24 hours prior to irrigation with solvent, one batch of paper should be used for all determina­

tions and control substances should be run on every chromato­

gram.

A variation in pH has a considerable effect on some organic substances such as alkaloids and by suitable buffering

of the paper, an improvement in the separation may be achieved.

Different pH values give different Rf values with the same

substance on the paper. There are several other factors which

affect partition chromatography and Consden et al. (25)

showed that the Rf value changes with the temperature when the solubility of water in the solvent changes. Some inves­ tigators hence employed thermostatically controlled or insu­

lated chromatographic chambers.

Balston and Talbot (36) considered the suitability of

Whatman papers with special regard to flow rates. The common­ 52 ly used paper is Whatman No. 1 for better separation and spot formation. This type of paper is necessary in chromatography, to regulate the rate of movement of the solvent and also the degree of penetration or adsorption. According to these workers, papers which are thin and soaked in buffer solutions for a certain period of time are easily torn and the rate of flow of the solvent is very slow. Thick papers give a fast rate of flow and trailing of spots are more common. Paper chromatography may be worked by either the as­ cending or descending process and each one gives a good result, the type to be used depending upon the convenience of the investigator. Both processes were employed, but the ascending method was found to be better because it gives an even liquid front and an easier apparatus to handle.

Apparatus: The apparatus as shown in Pig. 16 was used in the pre­ liminary investigation. It is composed of a glass cylindri­ cal chamber, 24 inches high and 12 inches in diameter, which could be tightly closed with a glass circular plate. In the descending method, a glass trough supported by a metal stand was employed, and placed inside the chamber. In the ascending method, Pig. 17, instead of the glass trough used in descend­ ing method, five glass rods that were 18 cm. long and connect­ ed parallel to each other at a distance of 4 cm. apart, were used to support the paper strips. This structure was placed 53

Fig. 16 Descending Chromatography apparatus

a. Chromatographic chamber b. metal stands c. glass trough containing the solvent d. paper strips

Fig. IT Ascending Chromatography apparatus

a. Chromatographic chamber b. glass rods with rubber stoppers on four corners for paper support c. paper strips d. solvent Fig. 17 Fig. 16

VJl 55 inside the chamber by means of four rubber stoppers attached to the four ends of the glass rods, which fitted exactly the sides of the chamber.

Reagents:

(l) Sorensen Phosphate Buffer at pH 7.4 (23). Disodium Phosphate, anhydrous...... O .76 Gm. potassium Acid Phosphate, anhydrous ... 0.18 Gm. Distilled Water to make 100.0 ml.

(2) Dragendorff1s Stock Solution (23). Bismuth subnitrate, O .85 Gm. was added to a

mixture of 40 ml. of water and 10 ml. of

glacial acetic acid. Then 50 ml. of 50 per cent of potassium iodide solution was shaken with the liquid until all the solid materials had dissolved. The solution was stored in an

amber-colored bottle. (3 ) Dragendorff1s reagent (2 3 ). Dragendorff1s reagent was prepared by the addi­ tion of 10 ml. of the Stock Solution to 20 ml. of acetic acid and the volume adjusted to 100 ml.

with distilled water. This reagent had to be freshly prepared before use.

(4) Iodine Solution (19). Iodine crystals ...... 0.2 Gm. Potassium iodide ...... 0.4 Gm. Distilled water to make ...... 100.0 ml. 5 6

(5) n-Butanol solvent. The solvent was prepared by shaking the n-butan-

ol with distilled water in a separatory funnel. The mixture was allowed to stand until the two

layers separated. The upper layer, composed of

the water-saturated n-butanol, was separated to be used as the chromatographic solvent.

Preparation of Known Alkaloidal Samples: 1. Meteloidine* solution. One hundred fifty-five mg. of meteloidine base was transferred to a 10 ml.-volumetric flask and v/as brought up to the marked volume with chloroform.

The resultant solution therefore, had a concentration of

0.155 nig. per 10 lambda. 2. Scopolamine solution. Two hundred fifty mg. of scopolamine hydrobromide was weighed and dissolved in a few ml. of water. The solution was made alkaline with ammonium hydroxide T.S. and the scopolamine base was extracted with chloroform. The chloroform was evaporated to dryness and the alkaloidal residue was dissolved in exactly 10 ml. of chloroform. The resultant chloroform solution contained

0.172 mg. of scopolamine per 10 lambda.

3. Hyoscyamine solution. One hundred sixty-six mg. of hyoscyamine sulfate v/as weighed and dissolved in a few ml. of

* Courtesy of S. B. Penick and Company, New York. distilled water. The aqueous solution was made alkaline with ammonium hydroxide T.S., and the hyoscyamine base was extract ed with chloroform. Then the chloroform was evaporated to dryness and the alkaloidal residue was dissolved in exactly

10 ml. of chloroform. The resultant chloroform solution contained 0.134 mg. of hyoscyamine per 10 lambda.

Descending Paper chromatography: Strips of filter paper, Whatman N o . 1, 55 cm. long and three cm. wide, were soaked for 15 minutes in Sorensen’s phos phate buffer of pH J . b and dried at room temperature for not less than two and a half hours. A line was drawn five cm. from one end of each of the paper strips, and volumes ranging from 10 to 50 lambda were placed on the middle of the line.

The chloroform solution was allowed to dry after every addi­ tion of 10 lambda. Then the strips were hung on the trough from the tip closer to the alkaloidal spot. The trough was filled with water-saturated n-butanol solvent. The chroma­ tograms were developed for 15 hours at constant temperature. The strips were then removed from the chamber and dried at room temperature for not less than two and a half hours.

The strips were passed through the Dragendorff1s reagent to note the position of the alkaloids. An orange color spot was shown by the alkaloids, scopolamine and hyoscyamine, but in the case of meteloidine, there was no spot formation with

Dragendorff's reagent. ..To calculate the Rf values of scop- 58

olamine and hyoscyamine, the distance from the starting point

of the alkaloid, to the middle of each spot was measured and

divided by the distance traveled by the sqlvent, which was

measured from the same starting point of the alkaloid, to the

liquid front. According to Drey and Poster (23), some alkaloids give a characteristic color spots with iodine solution. However, they have reported .that Brindle et al. (24) were not success­

ful in getting the color spots by using a 0.5 per cent iodine

in potassium iodide solution. They found out that by using

just the right concentration of iodine, which was 0.2 per cent iodine in potassium iodide solution, the characteristic

color of each alkaloid was observed. Some of the strips were developed with 0.2 per cent

iodine solution, and hyoscyamine gave a bluish gray color,

scopolamine a brownish red and meteloidine gave a light yel­ low color. Because meteloidine does not show up with Drag-

endorff's reagent, a 0.2 per cent iodine solution was found

to be better to detect the alkaloidal spots on the paper.

The Rf values of scopolamine, hyoscyamine and metelo­

idine are given in Table VIII.

Two-dimensional paper chromatography:

The two dimensional chromatogram on paper was devised by Consden, Gordon and Martin (37) and was applied to the qual­

itative microanalysis of protein hydrolysates or other amino Table VIII The Rf Values Obtained, by Descending Chromatography for Known Samples of Scopolamine, Hyoscyamine and Meteloidine

Strip no. Scopolamine Hyoscyamine Meteloidine

1 .81 .74 .76 2 .88 .81 .76

3 .87 .80 .75

4 ■ .85 .78 .75 5 .85 .79 .76 6 .86 .80 .76

Average .853 .786 .756 acids. In this technique, the separation depends upon the difference in partition coefficient between the cellulose and the organic solvent, which constitute a combination of capil­ lary analysis with partition chromatography. Procedure: The apparatus used in this investigation was similar to that used in the ascending chromatography in Fig. 16. Whatman No. 1 filter paper was cut 45 cm. square and buffered in Sorensen's phosphate buffer at pH 6.8 for 15 minutes. The paper was allowed to dry for not less than two and a half hours at room temperature. A chloroform solution containing the alkaloid from the drug sample and corresponding to 100 micrograms of alkaloid was placed near a corner of the paper, 5 cm. from either edge. Then a cylinder was constructed 60 from the paper stapling the two ends together, and was hung on the glass rod by means of a wire, and placed inside the chamber. The chromatogram was developed with water-saturated n-butanol for 15 hours. Then the paper was taken out of the chamber and dried at room temperature for not less than two and a half hours. The paper was passed through a 0.2 per cent iodine in potassium iodide solution. After 20 minutes the characteris­ tic colors of hyoscyamine and scopolamine were observed in Datura metel L. and scopolamine in Datura meteloides Dun., as compared to the previous colors shown in the known samples of the alkaloids. No meteloidine spot showed on the paper, when it was developed with this solution. A spreading spot in­ stead appeared close to the front liquid, on both unknown plant samples, but does not correspond to the Rf value of meteloidine. The paper was dried after passing in the iodine solution, to reproduce the spots, but Dragendorfffe reagent was used to make the spots permanent on the paper. In D. metel, three spots were shown, two of which were identified as hyo­ scyamine and scopolamine by their Rf values and the charac­ teristic colors produced with 0.2 per cent iodine solution, and the third spot was unidentified. In D. meteloides. two spots were observed. One of them was scopolamine, based on its Rf value and the brownish red color produced when devel­ oped with 0.2 per cent iodine solution. The second spot was unidentified, but the Rf was greater than that of hyoscyamine 6l

and scopolamine.

Ascending Paper Chromatography:

The ascending chromatography was used mainly in this investigation. The bottom of the chromatographic chamber, as shown in Fig. 1 7 , was filled with water-saturated n-but­ anol solvent.

Strips of Whatman No. 1 filter paper, 55 cms. long and

3 cms. wide, were soaked in Sorensen phosphate buffer at pH

7.4 for 15 minutes. The strips were dried for not less than

2 1/2 hours at room temperature. Then a line was drawn two cms. from one end. of each strip. Twenty to fifty lambda of the previously prepared chloroform solution of the alkaloid were placed in the middle of the line. The strips contain­ ing the alkaloids were stapled on the glass rods, the end of the paper bearing the alkaloid the farthest to the rod. The strips were, placed inside the chamber which was then complete­ ly sealed to maintain the atmospheric equilibrium. The chrom­ atograms were developed for 15 hours. Then the strips were removed from the chamber and the front liquids were marked, and the papers were allowed to dry at room temperature for not less than 2 1/2 hours.

Several strips, after drying, were treated with Drag­ endorf f's reagent which made the orange-red spots of hyoscy­ amine and scopolamine visible. No spot for meteloidine was produced on the strip chromatograms by treating them with the 62 same reagent. The remaining strips were passed through the

0.2 per cent iodine solution and the characteristic color of

each individual alkaloids were shown, hyoscyamine, a bluish- gray color; scopolamine, a brownish-red and meteloidine, a

light yellow color. The greatest intensity of the color was

shown after 15 to 20 minutes and then slowly faded in an hour. However, the colors ivere reproduced when the strips were passed again in a fresh iodine solution. To obtain a perma­ nent spot of the alkaloids on the paper after passing in the iodine solution, the strips were dried, and developed with

Dragendorff1s reagent. Hyoscyamine and scopolamine were re­ produced but not meteloidine.

The Rf values were calculated in the same way as the Rf values in the ascending chromatography. Several strips soaked in Sorensen’s phosphate buffer from pH 5.4 to 8.6 were investigated with scopolamine, hyoscyamine and meteloidine. Table IX shows the different Rf values obtained at different pH values, maintained at constant temperature, and chromato­ graphed at the same number of hours. Care must be taken, to seal the chamber tightly because the slight variation in equilibrium inside the chamber will change the Rf value of the alkaloids.

The size of the spots on the chromatogram may be an es­ timate of the relative amount of alkaloids present, but a quantitative assay was determined by the Vital! Morin Color­ imetric assay. 63 Table IX

Average Rf Values Obtained for Hysocyamine, Scopolamine and Meteloidine at Different pH values

pH Scopolamine Hyoscyamine Meteloidine

5.4 .67 - -

6.0 .79 .34 -

6.3 .67 .30 ~ 6.8 . .76 • 50 .72 7.2 .80 .61 .76

7.4 .89 .65 .74 co -3"

8.0 • .61 .79 8.04 .90 .63 .83

8.1 .82 . 60 .77 8.6 .87 - .82

Vibali Morin Colorimetric Assay for Scopolamine: (3 8 ).

Preparation of the standard curve: Seven and two tenths mg. of scopolamine hydrobromide was accurately weighed and

dissolved in a small quantity of water. The solution was made alkaline with ammonium hydroxide T.S. and the free alkaloidal base was extracted with chloroform. The chloroform extract was evaporated and the residue dissolved in ml. of chloro­ form in a volumetric flask. The resultant solution contained

0.1 mg. per milliliter of pure scopolamine base. Appropriate

aliquot portions representing 0.05 nig., 0.1 mg., etc. as shown in Table X were used to make the curve. The aliquot portions were evaporated in an evaporating dish on a water bath. Then 0.2 ml. of fuming nitric acid was added immedi­

ately, making sure that the acid made contact with the whole alkaloidal residue. The mixture was evaporated to dryness and the residue was dissolved in acetone and the solution quantitatively transferred to a 25 ml. volumetric flask. The mixture was allowed to cool and the volume was adjusted to 25 ml. with acetone. A purple coloration was produced by the addition of 0.1 ml. of a 3 per cent methyl alcoholic potas­ sium hydroxide solution. The per cent transmittancy of the colored solution was determined at exactly seven minutes from the time the purple color was first produced using a Cenco Sheard Photelometer with a green filter and using acetone as the blank. The results are summarized in Table X. The read­ ings obtained were correlated with the amount of alkaloid present in the aliquot portions taken.

Vitali Morin Colorimetric assay for hyoscyamine; (3 8 ). A known amount of hyoscyamine sulfate, 6.1 mg. was weighed and dissolved in water. The free alkaloidal base was extracted with chloroform and evaporated. The residue was dissolved in a 50 ml. volumetric flask, with chloroform. The resultant solution contained 0.1 mg. per ml. of the solution.

The same procedure was followed as for the determination of the alkaloid, scopolamine. The readings obtained in the transimtta.ncy percent too .05 CPLMN STANDARDIZATION CURVE5COPOLAM1NE IA!S OO TS ASSAy TFST COLOR VITAL! S .1 ilgas f scopolamine of milligrams G r k 1 k. p L <

2 25 .5 m n a c 66 per cent transmifctancy were correlated to the amount of hyoscyamine present in the aliquot portions used in the assay.

The data are shown in Table XI.

Table X Values of Scopolamine Standardization Vital! Morin Colorimetric Assay

Sample mg. scopolamine per cent transmittancy

1 0.05 80 2 0.10 66

3 0.15 58

4 0.20 47

5 0.25 40 6 0.30 35

7 0.35 30

Table XI Values of Hyoscyamine Standardization Vital! Morin Colorimetric Assay

Sample mg. hysocyamine per cent transmittancy

1 0.05 74

2 0.10 63

3 0.15 52 4 0.20 44

5 0.25 36 6 0.30 30 transmittancy percent 100 VSyUU STANDARDIZATIONHVOSCyAUlUE CURVE ' IA!S OO TEST T S E T COLOR S VITAL! ilgas f hyosctjamine of milligrams Gv* a. p h. jl

ASSAy 6 8 Quantitative determination of known alkaloidal samples:

Strips of paper, Whatman No. 1, were soaked in Sorensen's phosphate buffer at pH 7.^- for 15 minutes. The excess buffer was drained off and the strips were dried in air for not less than 2 1/2 hours. By means of a micropipette, known amounts of alkaloidal samples were placed on the strips. Twenty lambda of each alkaloid, scopolamine, hyoscyamine and metelo­ idine representing 0.2 mg., 0.162 mg. and 0.2 mg. respective­ ly, were used on different strips. The strips were chromato­ graphed by the ascending method, as described in the previous experiment, for 15 hours. Some of the strips were treated either with Dragen­ dorf f's reagent or iodine solution, and the spots, so obtain­ ed, were an indication of the alkaloidal areas on the untreated strips. The untreated-paper strips were cut into parts carrying the individual alkaloids, which were then eluted with 95 per cent ethyl alcohol. The elution process was as follows: the apparatus used was composed of a desic­ cator, about 12 inches in diameter and a trough, similar to the descending chromatography. The trough was placed across the center and was raised slightly higher than the small beakers, which served as receptacles for the eluants. The small beakers were correctly labeled, corresponding to the alkaloid contained in the strips, and were placed along the sides of the trough. The paper strips were arranged in the trough, one end in trough and the other in a beaker in such 69 a manner that the paper never touches any of the solution in

the beaker. The trough was filled with 95 per cent ethyl alcohol and the elution was run overnight. To prove for com­ plete elution, the strips were dried and tested for the presence of alkaloid by treating with Dragendorff1s reagent.

The eluants containing the alkaloid were evaporated

and determined quantitatively by the Vital! Morin Colorimet­

ric Assay, in the same manner as the preparation of the stan­ dard curve of hyoscyamine and scopolamine.

According to Drey and Foster (23) the amount of alkaloid recovered from the elution was only 62 to 76 per cent

of hyoscyamine and 66 to 73 per cent of scopolamine, although

the strips did not give a positive test or spot when treated with Dragendorff1s reagent. However, this loss, was consid­

ered in determining the individual alkaloids present in the plant or unknow. Data shown in Table XII represented only

62 to 74 per cent of hyoscyamine and 62 to 68 per cent of

scopolamine, that were recovered from the elution with alco­ hol. Meteloidine did not give a positive Vitali Morin test. Table XII Percentage Recovery of Hyoscyamine and Scopolamine from Elution Process Percent Weight placed on Weight recovered age re- Assay Alkaloid______chromatograms______by assay______covery

1 hyoscyamine 0.162 mg. 0.110 mg. 67.9 scopolamine 0.20 mg. 0.135 mg. 67.5 2 hyoscyamine 0.162 mg. 0.12 mg 74.

scopolamine - 0.20 mg. 0.125 mg. 62.5 3 hyoscyamine 0 .1 62 'mg. 0.10 mg. 62 scopolamine 0.20 mg. 0.13 mg. 65

I. Datura metel L. Alkaloidal Determination: A. Official alkaloidal assay: The procedure in Nation al Formulary IX Edition {40) was followed. About 10 Gms. of the oven dried, matured and powdered leaves of D. metal L was weighed and macerated overnight with 3 ml. of ammonia water,

10 ml. of ethyl alcohol and 20 ml. of ether. The alkaloids were extracted with ether in a Soxhlet extractor for seven hours. The ether extracts were treated successively with ap­ proximately 10 ml. portions of 0.5 N sulfuric acid, until the acidulated portion did not give a positive test with Valser's reagent. Then the acidulated portions were made alkaline with ammonium hydroxide T.S. and the free alkaloidal base was extracted with chloroform. The chloroform was evaporated and allowed to dry on the water bath for 15 minutes more. Then 71 another few ml. of chloroform was added and evaporated again, to volatilize the other amines present in the sample. Fif­ teen ml. of a known standard acid, (o.02011 N) was added to the alkaHdal residue and the excess acid was titrated with a standard base (0.0197 N). The total alkaloid was calculat­ ed as scopolamine, because scopolamine was the predominating alkaloid present in the plant. The number of ml. of 0.02 N acid was multiplied by the scopolamine factor, which is 6 .0 6 , gave the number of milligrams of scopolamine present in the sample.

Data in Table XII shows the percentage of the total alkaloids calculated on moisture free basis. The solution containing the alkaloids after the titra­ tion was saved for the quantitative determination of each individual alkaloid by paper chromatography.

Table XIII Official Assay of the Total Alkaloids of the Matured Leaves of Datura metel L. (oven dried)

Wt. of sample Acid used Base fo Total alkaloid in Gms. (0.02011 N) (0.0197 N) (moisture free) 10.0342 15 cc . 7.66 cc. 0.504

9.9923 15 c c . 7.46 cc. 0.501

9.9955 15 cc. 7.52 cc. 0.494 10.0068 15 cc. 7.58 cc. 0.498

Average 0.499 72 B. Quantitative determination of the Individual alka­ loids in D. metel L. (oven dried). Each solution from the previous titration of the total alkaloid determination was made acidic with approximately

0.5 N sulfuric acid, and the color due to the methyl red in­ dicator, was removed by extracting it with chloroform. The acidulated portion was made alkaline with ammonium hydroxide

T.S. and free alkaloidal base was re-extracted with chloro­ form. The chloroform was evaporated to dryness and the residue was dissolved in exactly five milliliter of chloro­ form. These chloroform solutions, which contained the re­ extracted alkaloids, were used in paper chromatography for the separation and quantitative determination of the individ­ ual alkaloids. Thirty lambda of the above solution was assayed by the

Vitali-Morin Colorimetric Assay, because the individual alka­ loids were assayed by the same process and therefore, a com­ parative investigation was made on the same basis. The total alkaloidal assay by the Vitali Morin method was found to be less than the official assay by titration with a difference of 0.067 bo 0.112 per cent. Thirty lambda of the same chloroform solution was chro­ matographed by the ascending method using Whatman paper No. 1 and previously buffered with Sorensen's phosphate buffer at different pH values. ' A good separation was observed at pH

7.4, although separation was also possible by pH value 6 .8 . 7 3 The positions of the spots were detected by developing with

either Dragendorff1s reagent or 0.2 per cent iodine in potas­

sium iodide solution. Three spots were noticed on the paper

as shown in Fig. 18. From the color of the spot and the Rf

value calculated, the two spots were identified as scopolamine

and hyoscyamine. However, the third spot does not correspond

to the Rf value of meteloidine, as compared to the value ob­

tained from the known sample of the alkaloid. The spot v/as light yellow with iodine solution and orange-pink with

Dragendorff1s reagent. The Rf value ranges from 0.95 to 0.97* which was almost near the front liquid.

The untreated papers were cut into parts carrying the individual alkaloid and were eluted with 95 per cent ethyl alcohol, as described in the previous process. The eluants

were assayed by the Vitali Morin Colorimetric Assay. The

amount of alkaloids obtained were calculated from the standard

curve prepared in the previous chapter, as shown in Graphs I and II. A transmittancy of 42 to 48 per cent v/as observed

for scopolamine and 43 to 52 per cent transmittancy for hyo­ scyamine. The moisture content of the drug and the loss

during elution were considered in calculating for the amount

of the individual alkaloids present. From the data obtained,

it v/as found that more scopolamine is present than hyoscyamine.

Table XIV shows the amount of scopolamine and hyoscya­ mine present in the matured leaves of Datura metel L.

The unknown spot was further studied, in the hopes of -.M ' ’ l|t It *-! h ' s * * *-k 1

- 1

g , , y g y .

Fig. 18 Fig. 19 Strip chromatograms at pH 7A Strip chromatograms at pH 6.8 S- scopolamine S- scopolamine M- meteloidine (marked H - hyoscyamine only to show position of alkaloid) H- hyoscyamine -t- 75 Table XIV Quantitative Determination of the Individual Alkaloids of the Leaves of Datura metel L. by Vitali-Morin Colorimetric Assay

cf/O Total Alkaloids % Individual Alkaloid Ratio Sample Vitali Morin* Scopolamine Hyoscyamine S/H

1 0.428 0.225 0.198 1.14 0.417 0.213 0.190 1.12 2 0.427 0.231 0.204 1.13 0.413 0.210 0.204 1.03 3 0.394 ' 0.207 0.141 1.47 0.389 0.215 0.147 1.46 4 0.403 0.225 0.175 1.34

Average 0.410 0.218 0.179 1.24

* Based on the scopolamine curve. gaining additional information, that would lead to its identity.

Several untreated strip chromatograms, carrying the unknown, were cut from the original strips and eluted with 95 per cent ethyl alcohol. A Vitali-Morin test was made and no purple colorhtion was produced upon the addition of 0.1 ml. of 3 per cent methanolic potassium hydroxide.

Several alkaloidal reagents like Kraut's T.S., Val- ser's T.S., Wagners T.S., and phosphotungstic acid T.S. were tried on the eluted portions to test for the presence of alkaloids. All the above mentioned reagents gave a positive test.

An attempt to make the alkaloidal picrate of the un­ known from the eluants collected in several chromatograms was done. The picrate did not form. 76 II Datura meteloides Dunal (oven dried)

Official total alkaloidal assay. The official assay for the total alkaloid was conduct­ ed in the same manner as for D. metel L. following N.F. IX.

The percentage for D. metel L. of the total alkaloids ex­ pressed on a moisture free basis in the plant is illustrated in Table XV.

TABLE XV Official Assay of the Total Alkaloids of the Matured Leaves of Datura meteloides (Oven dried)

Wt. of samples Acid Base % Total alkaloid in Gms. (0.02011 N) (0.0197 N) (moisture free)

10.002 15 cc, 9.94 0.356

10.007 15 cc, ' 9.92 0.356

10.032 15 cc. 10.02 0.350

10.020 15 cc. 9.84 0.362

10.052 15 cc. 10.06 0.354

Average ____ 0.354

Individual alkaloidal determination.

The individual alkaloids were separated by paper chro­ matography and determined quantitatively by the Vitali-Morin

Assay, in the same manner as for D, metel L. Two spots were observed on the paper, and one of them was identified as scopolamine, based on the Rf value compared to a known sample and the characteristic brownish color produced by passing 77 through the iodine solution. The second spot was un-identi-

fied, but the Rf value was calculated to be from 0.9^ to

0.95, which was within the range of the Rf value of the unknown compound that was found in D. metel L. Further in­ vestigation was attempted on this unknown spot by making the

alkaloidal picrate which was unsuccessful. This spot was tested with several alkaloidal precipitants and a positive test was obtained with Valser's T.S., Kraut's T.S., Wagner's

T.S. and phosphotungstic acid T.S. The unknown compound did not give a positive Vitali-Morin test. Some of the untreated paper chromatograms carrying the alkaloidal spot of scopolamine were cut corresponding to the spot that showed when the strips were treated with Dragen­ dorf f's reagent. Then the alkaloid was eluted with 95 per cent ethyl alcohol and determined quantitatively by the

Vitali-Morin Assay. The per cent transmittancy ranged from 42 to 48 and the amount of scopolamine was calculated from the standard curved of scopolamine in Graph I. The amount of alkaloid present in D. meteloides Dun. is given in Table XVI, and the amount of moisture and loss in elution were consid­ ered. 78 Table XVI Quantitative Determination of the Individual Alkaloids of Datura meteloides D. leaves (oven dried) by the Vitali-Morin Colorimetric Assay

p(rf Total Alkaloids fj Individual alkaloids Sample Vit all-Morin* Scopolamine* Difference**

1 0.243 0.204 0.039 2 0.211 0.191 0.020

3 0.195 0.196 0.000 4 0.0235 0.219 0.016 Average 0.221 0.202 0.019 ^ Average reading from 3 to 4 strip chromatograms ** Total alkaloid minus scopolamine. Ill Datura meteloides Dunal (lyophilized)

Official total alkaloidal assay. The total alkaloidal assay of the matured leaves of

D. meteloides Dunal was made in the same manner as D. metel.

The results are given in Table XVII. Table XVII

Official Assay for theTotal Alkaloids of Datura meteloides Leaves (lyophilized*)

VJt. of samples Acid Base

10.086 15 cc. 9.8 cc. 0.345 9.490 15 cc. 9.14 cc. 0.411

9.910 15 cc. 8.84 cc. 0.412

10.096 15 CC . 8.7 cc. 0.415 Average 0.396 79 Quantitative individual alkaloidal determination.

The individual alkaloids were assayed quantitatively

in the same manner as D. metel L. using ascending paper chromatography, and the Vitali-Morin Colorimetric Assay.

Two spots were observed on the paper and one of them was identified as scopolamine by its Rf value and its charac­

teristic brownish red color produced when treated with iodine solution. The amount of scopolamine present in the matured

leaves of D. meteloides (lyophilized) is given in Table XVIII.

Table XVIII

Quantitative Determination of the Individual Alkaloid of Datura meteloides Leaves (lyophilized) by Vitali Morin Colorimetric Assay

fo Total alkaloids % Individual alkaloid Samples Vitali Morin* Scopolamine* Difference**

1 0.258 0.204 0.054

2 0.263 0.207 0.056

3 0.274 0.232 0.042 4 0.267 0.235 0.032 Average 0.265 0.219 0.046 * Average reading from 3 to 4 strip chromatograms ** Total alkaloids minus scopolamine. 80 B. Paper Electrophoresis

Introduction: Electrophoresis may be defined as the migration of colloidal particles under the Influence of an electrical field. The speed and direction of the movement of the parti­ cles Is dependent upon their nature, more specifically upon the free charges on the surface of the particles (46).

History: The early foundation of electrophoresis was in the nineteenth century. In 1807, the Russian physicist Reuss, observed the electro-osmosis of water through clay by the passage of an electric current. He also noted that the clay particles moved in the direction opposite that of water. The investigations on protein electrophoresis using egg albumin was reported in 1899. Hardy (46) reported that negatively charged particles of denatured egg albumin could be made positive by addition of acid and that with the change from alkaline to acid the particles migrated in the electric field in the opposite direction.

Tiselius (46) made an electrophoresis apparatus that permitted quantitative estimation of the mobilities of the various components in a mixed protein solution, such as plasma, and opened a new era in the study of proteins.

The future prospect of electrophoresis in the applica­ tion to several other compounds aside from proteins was very 8l

promising. Kariyone et al. (^7) made a microchemical study of plant constituents by paper electromigration and electro-

chromatography of organic bases. They have reported the migration of alkaloidal particles by using paper electro­

phoresis. They found that by the paper electromigration method, the distance of migration to the cathode is always

constant, whether the alkaloids are in free form or salt form.

Also, they have reported that the difficulty in paper chroma­ tography of alkaloids is due to the fluctuation of Rf value

depending on the kind of acid which is combined with the

alkaloids. They investigated the migration of hyoscyamine

and atropine on paper electrophoresis using 30 per cent

acetic acid as solvent at different voltage. Because of the fact that the molecules carry'electrical charges, the current

passed through the paper will cause the particles to move to a certain distance in a specified length of time.

Apparatus: The apparatus as shown in Pig. 18 is composed of a

glass box with two wires attached to a power kit, Heath-Kit model PS-2. The voltage used was around 460 volts. Carbon

electrodes were dipped in the electrolyte contained in two

enameled vessels. Glass rods with rubber stoppers at the

ends were used to support the paper during electrophoresis. Materials:

1. Strips of paper Whatman No. 1, 40 cm. long and 3 cm. wide. 82

2. Solvents. a. Buffer acetate solution at pH 4.2 prepared by mixing 20 ml. of 2 M.

sodium acetate and 33*7 nil. of 3-5 M. acetic acid and making the volume to 2 liters. b. Phosphate buffer at pH 6.2 prepared by mixing 9.2 ml. of 0.5 M Disodium phosphate solution and 6.6 ml. of 4 M sodium acid phosphate and making the volume of two liters, with dis­ tilled water. c. Buffer at pH 9*5 prepared by mixing

34.5 ml. of 1 M glycine and 1 M sodium chloride mixtures; and 2.7 ml. of 2 N sodium hydroxide, making up to two liters with distilled water. d. Buffer at pH 10.5 , prepared by mixing 23.2 ml. of 1 M glycine and 1 M sodium Chloride mixtures; and 8.4 ml. of 2 N sodium hydroxide making up to a vol­ ume of two liters, with distilled water. Procedure.

Eight cms. was measured at both ends of the filter paper and penciled, drawing a straight line. Another line was drawn,

10 cms. from one end of the strip, to mark the starting point D i ram ?f Vapzr Etecirophore$i$ Cell a S

PiG. 20

A Electrodes (carbon)

B Whatman jjaper I'lo. 1

0 Glass rod with, rubber stoppers

D Wires E Power kit

P Switch

G Voltmeter

II Electrolyte bufTer solutions

I Glass chamber 84 of the alkaloid. The papers were creased from the 8 cm. line to form an angle of 90°. Then appropriate amount of the

alkaloidal solution in chloroform as prepared in the known samples for paper chromatography, was placed on the strips

as marked.■ The strips were hung on the glass rods, the end

containing the alkaloidal spots, towards the positive prole. •

The elec.trolyte to be used was poured in the enameled vessels about half full, being sure that the two vessels con­ tained the same volume of solution. The electrophoresis was

run for several hours hanging from two to four hours. A good number of solvents were tried as mentioned above and the

results are shown in Tables XXX and XXIII. Table XIX Results Obtained by Electrophoresis of the Alkaloids at pH 4.2 Acetate Buffer with 460 Volts Time Alkaloids 2 Hours 3 Hours

Meteloidine 14.5 cms. 18.8 cms. Hyoscyamine 15.2 cms. 21.5 cms.

Scopolamine 14.9 cms. 19.2 cms.

There was no separation observed at this pH, although the alkaloids moved quite a distance. 85 Table XX Results Obtained by Electrophoresis of Alkaloids at pH 9-5 Using 1 M Clycine and NaOH Buffer with 460 Volts

Time Alkaloid 2 Hours 3 Hours 4 Hours

Meteloidine 6.7 cm. 8.2 cm. 8.5 cm.

Hyoscyamine 14.5 cm. 17.5 cm. 17.2 cm. Scopolamine 6.1 cm. 8.9 cm. 8.0 cm .

There was separation observed of meteloidine and hyos- cyamine and of scopolamine and hyoscyamine but not of metelo- dine and scopolamine mixtures.

Table XXI

Results Obtained by Electrophoresis of Alkaloids at pH 10, Using 1 M Clycine and NaOH with 460 volts

Time Alkaloid 2 Hours 3 Hours 4 Hours

Meteloidine 6.4 cms. 10.1 cm. 9.8 cm.

Hyoscyamine 15.0 cm. 17.2 cm. 18.3 cm. Scopolamine 6.5 cm. 11.3 cm. 8.1 cm.

There was separation observed at the above pH of meteloidine and hyoscyamine, also of scopolamine and hy­ oscyamine, but not of meteloidine and scopolamine. Table XXII Results Obtained by Electrophoresis of Alkaloids at pH 10.5 Using 1 M Glycine and NaOH with 460 Volts

Time Alkaloids 2 Hours 3 Hours 4 Hours

Meteloidine 6.2 cm. 11.2 cm. 10.2 cm.

Hyoscyamine 16.1 cm. 18.6 cm. 17.4 cm. Scopolamine 6.9 cm. 10.0 cm. 9.3 cm.

There was separation observed at Ph 10.5 with meteloi­ dine and hyoscyamine, also with scopolamine and hyoscyamine, but no separation of meteloidine and scopolamine.

Table XXIII Results Obtained by Electrophoresis of Alkaloids at pH 11.0 Using 1 M. Glycine and NaOH with 460 Volts

Alkaloids Time - 3 Hours

Meteloidine 9.5 cm. Hyoscyamine 18.2 cm. Scopolamine 10.1 cm.

There was a separation observed at pH 11, with metel­ oidine and hyoscyamine, also with scopolamine and hyoscyamine but hot of scopolamine and meteloidine. DISCUSSION OP RESULTS

A. Morphological and Histological Studies

Datura metel L. : Morphologically, D. metel L. was described as a herbaceous plant which attains the height of three to five feet and has a purple-colored stem. The leaves are entire or sometimes sinuately toothed on both sides of the margin. The flowers are white, singly and erect, with stamens of five to six. The fruit capsule has spines which are arranged irregularly.

Microscopically, the structures of the stems are com­ posed of the epidermis, which gives rise to numerous trichomes of both glandular and uniserate types of hairs, (Pig. 4). The cortex is made up of thick-walled collenchyma cells or the hypodermis, and the cortical parenchyma which are more or less irregular thin-walled parenchyma cells. Numerous cal­ cium oxalate crystals of rosette and prism aggregates were found in the cortical layer. Micro crystals are scattered along the cortex. The leaf structure is composed of the epidermis, which gives rise to numerous trichomes and usually the glandular type of hair was predominant rather than the uniserate type, (Fig. 7)- Stomates are more numerous on the lower surface than on the upper one. Calcium oxalate crystals are found in the palisade layer. The lower epidermis is com­ posed of a single layer of cells and also gives rise to sevo^ al trichomes. 88

The stomatal number, the vein-islet number and the palisade ratio did not furnish any satisfactory diagnostic

features in the identification and characterization of Datura metel L. It can be observed in Table XXIV that the average

vein islet number, stomatal number and palisade ratio of D. metel L. are similar to the values obtained for D. meteloides

Dunal. The wide range of Data (Table II-VII) from which these average values were calculated actually overlap in that,

some of the data for D. metel are the same for D. meteloides.

Datura meteloides Dunal. Morphologically, D. meteloides Dunal grows from one to three feet high and the stem is green with slight purple

coloration at the base of the petiole. The branches are slender, forked and spreading. The leaves are velvety due to the numerous trichomes covering the surface. The flowers are white, five-lobed and tubular in form. The fruit capsules are full of spines which are long and narrow, and more dense than those of D. metel L. (Pig. 2). Microscopically, the structures of the leaf and stem of D. meteloides Dunal are similar to those of D. metel. The two species could be distinguished by their characteristic hairs found on the leaves. In D. meteloides. the non-glandu- lar or uniserate type of hairs is very predominant, while in

D. metels the three to four-celled glandular trichomes, with their prominent hair bases, are more common (Pigs, f and 11). The vein islets number, palisade ratio and stomatal number determinations for D. meteloides gave close values to

that obtained for D. metel (Table XXXV). No basis of dis­

tinguishing these two species by these determinations exists

for the same reason as previously stated under D. metel L.

Table XXIV Average Values of the Palisade Ratio, Vein Islet Number and Stomatal Number of Datura metel L. and Datura meteloides Dunal A. Palisade Ratio

Base Middle Apex Average

D. metel 6.3 5.87 6.12 6.1

D. meteloides 5.06 5.0 5.19 5.08

B. Vein Islets Number

Base Middle Apex Average

D. metel 18.3 15.6 16.3 16.7

D. meteloides 16.8 16.1 13.3 15.4

C. Stomatal Number

Upper epidermis Lower epidam

D. metel 199.7 259.8 D. meteloides 165.4 217.6 90 B. Identification of Constituents

Paper Chromatography Datura metel L.: The alkaloids of D. metel L. were

extracted by the official method and the alkaloidal free base in chloroform was prepared. This solution was used in paper chromatography for the separation and quantitative de­ termination of the individual alkaloids. D. metel contained total alkaloids from 0.494 to 0.504 per cent (Table XIII) and the individual alkaloids detected were scopolamine and hyoscy­ amine. A quantitative determination of each alkaloid was calculated by Vitali-Morin Colorimetric Assay and it was found that the scopolamine content was 0.207 to 0.231 per cent and the hyoscyamine content was from 0.141 to 0.204 per cent (Table XIV). Besides hyoscyamine and scopolamine alkaloids, a third unknown spot was detected on the strip chromatograms, when developed with either Dragendorff*s reagent or 0.2 per cent iodine solution. The Rf value was calculated for this unknown spot and it gave values from 0.95 to 0.97. Further investigation of this spot was attempted by eluting them from the paper with 95 Per cent ethyl alcohol and the eluants were tested with several alkaloidal reagents like Kraut1s T.S.,

Wagner's T.S., Valser's T.S., and phosphotungstic acid T.S., and all gave positive tests. Formatinn of the picrate with picric acid was not successful. Datura meteloides Dunal (oven dried) The total alkaloids were determined for D. meteloides 91 "by the official method and it was found to contain from 0.346 to 0.362 per cent (Table XV). The alkaloids detected were scopolamine and an unidentified compound, which gave a posi­ tive test with several alkaloidal reagents, the same as in D. metel L. The Rf value was calculated and it gave the same

Rf as that of the unidentified compounds in D. metel L.

Scopolamine was present in the plant from 0.191 to 0.219 per cent, as determined by the Vitali-Morin Assay (Table XVI).

Datura meteloides Dunal (lyophilized) The matured leaves of D. meteloides were lyophilized and the total alkaloidal content was determined by the offi­ cial assay, which gave values from 0.345 to 0.415 per cent (Table XVII). The individual alkaloid found was scopolamine, in the amount of 0.204 to 0..235 per cent (Table XVIII) . Meteloidine was not identified from the matured leaves bf D. meteloides grown at the Ohio State Medicinal Plant Garden. There are several reasons which might contribute to the non-identification of meteloidine from D. meteloides and one of them is the environmental effects. According to some investigators, environment affects the alkaloidal content of some plants and it might be that the soil and climate where this plant was grown were not suitable. For example, Andrews

(12) experimented on D. metel grown in India and in Europe and he found that those Daturas in India contain scopolamine and those in Europe contain only hyoscyamine. Schmidt (13)* also investigated grown in France and he found that 92 scopolamine is present in all parts of the plant and unac­ companied by any notable quantity of other mydriatic bases. Another reason that might be considered is that, only 0.07 per cent of meteloidine was reported by Pyman and Reynolds to be present in the plant and by the use of paper chromato­ graphy, it may not be sensitive enough to detect the alka­ loidal spot on the paper with 10.2 per cent iodine solution.

The ascending paper method of chromatography was found to be more efficient than the descending method, because at a constant temperature, the front liquids of the strip chro­ matograms traveled an equal distance and as a result, the Rf values are the same for each alkaloid. Another advantage of ascending method, is that, the solvent does not travel too fast to go beyond the length- of the strip during developing for a certain period of time, while that of the descending method, the solvent sometimes, does go beyond the strip. A good separation of the alkaloids with paper chroma­ tography may be based on several factors, one of which is pH. In the case of the alkaloids, hyoscyamine, scopolamine and meteloidine, a good separation was obtained at pH al­ though, separation was observed at pH 6.8 at constant temper­ ature, using water-saturated n-butanol as the solvent. At different pH values, 5.^ to 6.3 meteloidine does not separate from scopolamine and hyoscyamine, whereas, at pH 8.6, hyoscy­ amine did not separate from scopolamine and meteloidine. Consistant Rf values were noticed, when the glass cham- 93 ber was sealed to maintain the atmospheric equilibrium inside

the chamber. The previously buffered strips were handled

carefully to prevent the change in the rate of flow of the

solvent, and minimize the source of experimental error.

The total alkaloids present in Datura metel L. and Datura meteloides Dunal were determined by two different methods. One method was the Official Assay for Stramonium as given in N.F. IX (40). The other method was the Vitali-

Morin Colorimetric Assay (3 8 ). It has been shown by other investigators (3 8 ) that the Vitali-Morin Colorimetric Assay gives results that are about 0.6 per cent less than the cor­

responding results of the Official Assay method. With this

information on hand, it was thought that perhaps the plant

samples should be assayed for the total alkaloid using the official method. This procedure served a twofold purpose: first, it provided data for an Official Assay; and secondly, it served as a check, showing that the samples assayed repre­ sented the entire sample.

In the separation of the individual alkaloids by chro­ matography, it was necessary to use an assay method that measures minute amounts of alkaloid. The Vitali-Morin Color­ imetric Assay is capable of measuring 0.05 mg. of hyoscyamine and scopolamine and therefore was chosen as the assay pro­ cedure for the individual alkaloids. Because of the fact that the individual alkaloids were assayed by the Vitali-Morin

Assay method, it was felt that the total alkaloids should be 94 determined by the same procedure. For this reason, the

amount of total alkaloids in the samples were determined by the Vitali-Morin Colorimetric Assay, although this procedure

did give values which were less than the Official Assay. The percentage of total alkaloids present in D. metel

L. as determined by the Vitali-Morin Colorimetric Assay was

0.409 per cent, while in the Official Assay it was 0.499 per cent. Thus the Vitali-Morin Assay gave a value which was 18

per cent less than the Official Assay, In the case of D. meteloldes Dunal (oven dried), the

Vitali-Morin Assay measured 0.221 per cent of total alkaloid

and the Official Assay measured 0.354 per cent. The Vitali-

Morin Assay value was 37 per cent less than the Official Assay. With D. meteloides (lyophilized), the Vitali-Morin

Assay gave 0.265 per cent total alkaloid and the Official

Assay was 0.330 per cent, which the Vitali-Morin Assay was

30 per cent less than the Official Assay.

The following are possible explanations for the above discrepancy. First, on each chromatographic strip for D. metel and D. meteloides there was a spot of an unknown com­

pound. This unknown compound, on further investigation,

gave positive tests with several alkaloidal reagents but did

not give a positive Vitali-Morin test. It is entirely possi­ ble that this compound is basic in nature and thus would be' measured along with the alkaloids in the Official Assay. Since

the unknown compound does not give a positive Vitali-Morin test its presence would not affect the Vitali-Morin Colori­

metric Assay. As a result, the Official Assay would give a

greater value than the Vitali-Morin Assay. Judging by the

size of the spot of the unknown compound as it appeared on

the chromatrgraphic strips,it seemed to be present in a great­

er amount in D. meteloides than in D. metel. This substanti­ ates the above explanation for, should it be true, D. metel­

oides would show a greater difference in the values of the two different assay procedures, since it contains a greater

amount of the unknown compound. It so happens, that the dif­

ference between the two assay values for D. meteloides Dunal was greater than the values for D. metel L. (Table XXV)

The second possible explanation for the discrepancy lies within the experimental procedure. The Vitali-Morin

Colorimetric Assay for the total alkaloids and the individual alkaloids was made on aliquot portions of the same alkaloidal material that was titrated in the Official Assay,

The alkaloidal materials that were assayed by the

Vitali-Morin Colorimetric Assay was obtained by re-extracting the alkaloids from the titrated alkaloidal solutions of the

Official Assay. There may have been some loss of alkaloids in the re-extraction process, in spite of the fact that the solutions were checked for total extraction of the alkaloid. Hydrolysis of some of the alkaloids could have taken place in the titrated solutions before the alkaloids were re-extract­ ed. A loss in this manner could be minimized if the re- 96 extraction of the alkaloids from the titrated solutions were

made soon after the titration. The indicator used in the titration may have absorbed some of the alkaloids. A better

experimental procedure would have been, to have used separ­

ate samples for the Vitali-Morin Colorimetric Assays instead of using the same samples that were used for the Official

Assay.

C. Paper Electrophoresis:

One advantage of using paper electrophoresis in the separation of alkaloids is that it is a less time consuming process. In partition paper chromatography, it usually takes from 22-25 hours while in paper electrophoresis, it could be completed in 2 to 4 hours. Another advantage is the regular­ ity of the distance traveled by the alkaloid in a certain buf­ fer solution, whereas in paper chromatography, a constant Rf value is difficult to obtain, which involves several factors like temperature, amount of alkaloid on the paper and the equilibrium between the solvent phase and the water vapor. The alkaloids, hyoscyamine, scopolamine and meteloidine are positively charged and thus move from the positive pole to the negative pole. The distance traveled by the alkaloid seems to depend on how heavily charged they are, and the amount of current used to pass through the electrolyte buffer solu­ tion.

As result of the investigation, it was found that hyo­ scyamine moved faster than scopolamine and meteloidine at pH 97 values 9*5 to 11. No separation of the three alkaloids was observed from pH values 4 to 9 and in the case of the metelo­ idine and scopolamine, there was no separation at all from pH values 4 to 11.

The alkaloidal spots of hyoscyamine and scopolamine showed on the paper by either treating with Dragendorff1s reagent or iodine solution, but meteloidine was identified only by passing the strip in 0.2 per cent iodine in potassium iodide solution. After eluting the alkaloids from the paper with ethyl alcohol, in a manner similar to paper chromato­ graphy, the quantitative determination of scopolamine and hyoscyamine could be done by the Vitali Morin Colorimetric

Assay. The distance traveled by the alkaloid was increased as the length of time during electrophoresis was increased, but at the end of four hours, there seemed to be a slight de­ crease. Hyoscyamine moved faster than scopolamine and meteloidine in electrophoresis both in the acid and alkaline media, Whereas, in paper chromatography, scopolamine moved faster than hyoscyamine. The reason for this difference might be that in paper electrophoresis, the amount of charge each individual alkaloid carries, or how heavily charged they are, has something to do with the rate of movement, whereas, in paper chromatography, it is the partition coefficient of the alkaloids in the solvent that affects the rate of movement.

Also, in paper electrophoresis, hyoscyamine was observed to 98 move about the same rate in both alkaline and aeid media. The reason for this observation has not yet been clearly understood. Table XXV Quantitative Alkaloidal Content of Datura metel L. and Datura meteloides Dunal

% Total Alka- Alkaloids °/o Individual Alka­ Plant sample loid Present Found loids Present

1. Datura 0.499 Scopolamine 0.218 metel L. leaves Hyoscyamine 0.179 Unidentified* 0 .102**

2. Datura 9.354 Scopolamine 0.202 meteloides (oven dried) Unidentified* 0 .152** leaves

3. Datura 0.396 Scopolamine 0.219 meteloides (lyophilized) Unidentified* O.177** leaves

* Not meteloidine. ** Difference from the total alkaloids. VII

SUMMARY AND CONCLUSIONS

A. Histological Studies

The leaves and stems of Datura metel L. and Datura meteloides Dunal were studied histologically. The vein is­ let number, stomatal number and palisade ratio were deter­ mined and the following conclusions are drawn: 1. The structures of the two species studied are sim­ ilar except that the glandular hairs of D. metel L. are more numerous than those of D. meteloides. The uniserate type of hairs, which are from three to four celled and with very characteristic hair bases are predominantly found in D. metel­ oides Dunal. 2. The palisade ratio, vein islet number and stomatal number are not valuable in the characterization and differen­ tiation of the two species investigated.

B. Identification of Constituents

The alkaloids of D. metel L. and D. meteloides Dun. were separated by paper chromatography using Whatman No. 1 and water-saturated n-butanol as the solvent. The results of the experiments conducted lead to the following conclusions: 1. The following Rf values were established at pH 7.U- Scopolamine; 0.89 Meteloidine: 0.74 Hyoscyamine: O .65

2. A procedure was developed for the separation of

99 100 hyoscyamine, scopolamine and meteloidine. This separation was best accomplished at pH 74. 3. a) The average total alkaloid content of D. metel L. was 0.499 per cent. b. The constituents detected in D. metel L. were the alkaloids scopolamine and hyoscyamine and an unidentified compound. c. The unidentified compound had an Rf value greater than that of the identified alkaloids. d. The average scopolamine content of D. metel L. was

0.218 per cent. e. The average hyoscyamine content of D. metel L. was

0.173 per cent. f. The average scopolamine and hyoscyamine ratio was

1.24. 4. a. The average total alkaloid content for D. meteloides (oven dried) was 0.354 per cent, D. meteloides (l.yophilized) was 0.398 per cent. b. The lyophilization process of drying is comparable to the oven drying process in respect to the amount of total alkaloids in the samples assayed. c. The constituents detected in D. meteloides was the alkaloid scopolamine, and an unidentified compound. d. Meteloidine was not detected in D. meteloides.

e. The unknown compound had the same Rf value as the unknown compound in D. metel L. The Rf valuer was different than that of meteloidine. f. The average scopolamine content of D. meteloides (oven dried) was 0.202 per cent; and D. meteloides (lyophilized) was 0.219 per cent.

C. Separation of Hyoscyamine, Scopolamine and Meteloidine by

Paper Electrophoresis. Known alkaloidal samples of hyoscyamine, scopolamine and meteloidine were investigated by paper electrophoresis using Whatman paper No. 1 different buffer solutions as the electrolyte, and a current of 460 volts. The experimental re­ sults lead to the following conclusions:

1. The alkaloids, hyoscyamine, scopolamine and metelo­ idine migrated from the positive pole to the negative pole. 2. Hyoscyamine traveled faster than acopolamine and meteloidine at pH values 9.5 to 11. 3. There was a separation of meteloidine from hyoscy­ amine, and scopolamine from hyoscyamine at pH values from 9.5 to 11. ^ 4. No separation of meteloidine and scopolamine was observed at pH values 4 to 11.

5. Hyoscyamine moved fastest on the paper strips in this method, while in paper chromatography, it moved the slow­ est .

6. Paper electrophoresis involves less time than paper chromatography in the separation of the alkaloids. 102 BIBLIOGRAPHY

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I, Emma Pascual Maniquis, was born in Manila, Philip­ pines, September 19, 1926. I received my secondary school education in Bulacan, Philippines in 1946, in which my schooling was interrupted for three years during the war in 194l. My college education was obtained in Centro Escolar

University, Manila, in 1950> from which I received the degree Bachelor of Science in Pharmacy. I passed the Philippine

Board of Pharmaceutical Examiners in the same year. In March, 1952 I received the Master of Science in Pharmacy, also in Centro Escolar University, Manila. My thesis was on the subject, "Histological Study of the Bark of Plumiera acutifolia Poir." I came to the United States in September,

1952 to continue graduate studies in Pharmacy with a major in Pharmacognosy, on a fellowship sponsored by the American

Association of University Women and a Fulbright Travel Grant.

I held the former fellowship till June, 1953, and I am now holding a partial fellowship from The Ohio State University (fees waived) and my maintenance from the American Associa­ tion of University Women, while completing the requirements for the degreb Doctor of Philosophy.