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FONG, H arry Hong Sang, 1935— A PHYTOCHEMICAL INVESTIGATION OF THE NON-PHENOLIC TERTIARY AND QUATERNARY ALKALOIDS OF THALICTRUM ROCHEBRUN- IANUM, FRANC. AND SAV. (RANUNCULACEAE).
The Ohio State University, Ph.D., 1965 Health Sciences, pharmacy
University Microfilms, Inc., Ann Arbor, Michigan A PHOTOCHEMICAL INVESTIGATION OF THE NON-PHENOLIC TERTIARY AND
QUATERNARY ALKALOIDS OF THALICTRPM ROCHEBRUNIANUM.
FRANC. AND SAV. ( RANUNCULACEAE)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
Rv
Harry Hong Sang Fong
**********
The Ohio S ta te U n iv ersity 1965
Approved by ACKNOWLEDGMENT
The author wishes to express his sincerest thanks and appreciation to Dr. Jack L. Beal for his encouragement, patience, and guidance during this investigation and throughout the author's association with him.
The author also wishes to express his gratitude to Drs.
Michael P. Cava, Professor of Chemistry, and Raymond W. Doskotoh,
Assistant Professor of Pharmacy, for their invaluable suggestions and discussions of the chemistry of alkaloids during this study; the members of his committee, Drs. Raymond W. Doskotch, Jules
LaPidus, Arthur Tye, and the Graduate School Representatives, for their help and assistance.
He is especially grateful to Dr. Tosiaki Tomimatsu, Post
Doctoral Fellow at The Ohio State University, College of Pharmacy, and Assistant Professor of Pharmaceutical Chemistry, Tokushima
University, Tokushima, Japan, whose expert knowledge of the chemistry of the Thallctrum alkaloids and isolation techniques, which he so gracefully divulged to the author, were particularly helpful in overcoming many of the difficulties encountered during this investi gation, and for his efforts in elucidating the structure of alkaloid
TNG.
He is further indebted to Dr. David R. Dalton, Post Doctoral
Fellow at the Department of Chemistry for his determination and
Interpretation of the N.M.R. spectra of the alkaloids, as well as to
Dr. P. N. Patil, Mr. Richard Hahn, and Mr. Paul Schiff for their technical aid and assistance. ii VITA
June 30, 1935 Born - Tung Kal V illage, Kwangtung Province, China
1939 ...... B.S. in Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania
1959-1961.... Teaching Assistant, Department of Pharmacognosy, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania
1961 ...... M.S. The Graduate School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania
1961-1964 ... Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio
1965 ...... Research Assistant Professor of Pharmacognosy, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania
PUBLICATIONS
Studies on Vinca major (Apocynaceae). II. Phytochemical I n v e stig a tio n . J. Pharm. S c i. , J51:217-22^+, 1962.
FIELDS OF STUDY
Major Field: Pharmacognosy
Professor Jack L. Beal, Adviser
iii CONTENTS
Page
ACKNOWLEDGMENT...... i i
VITA, PUBLICATIONS, AND FIELDS OF STUDY...... i i i
TABLES...... viii
FIGURES...... l x
CHARTS...... x
I . INTRODUCTION AND STATEMENT OF PROBLEM ...... 1
I I . LITERATURE SURVEY...... 4
A. Taxonomic Description of Thai!ctrum Rochebrunianom. Franc, and Sav., (Ranunculaceae) ...... 4
B. The Use of ThaHct-rum Plants as Home Remedies...,. 7
C. Alkaloids of the Genus Thai let-rum...... 9
1. Types of alkaloids present....o.o.o.o.o.o...,. 9
2. Alkaloids isolated from ThajViot-rum species.... 15
I I I . EXPERIMENTAL...... 37
A. M a te r ia ls ...... 37
B. Preliminary Investigations...... 37
1. Quantitative estimation of total alkaloid co n te n t ...... 37 2. Extraction of alkaloids for chromatographic studies....»...... 39 3. Thin-layer chromatographic studies of the P-E, T-T and Qu-l fractions ...... 4 l
4. Methylene chloride extraction for berberine... 45
5. Fractionation of the tertiary alkaloids into the phenolic (TP) and non-phenolic (TN) fr a c tio n s ...... 46
6. Pharmacological screening of the TN, TP, and Qu-1 fractions...... 48
iv CONTENTS (Cont’d) Pag©
7. Solubility of TN and TP alkaloids in chloroform and benzene ...... 52
8. Chromatography of TN and TP alkaloids on multibuffered paper...... 5^
9. Gradient pH separation of the TN and TP alkaloids...... 55
a. TN alkaloids ...... 5&
b. TP alkaloids ...... 58
10. Preliminary column chromatographic separation of the quaternary alkaloids (Qu-l) on neutral alum ina ...... 58
C. General Extraction and Fractionation of Roohebrunianum Root Alkaloids ...... 6 l
1. Extraction of total alkaloids ...... 6 l
a. Compound »»20-x”...... 64
2. Fractionation of the total alkaloid extractive into the tertiary non-phenolic (TN)( tertiary p h en o lic (TP), and quaternary (Qu) a lk a lo id f r a c t io n s ...... 65
a. Total tertiary and quaternary alkaloid f r a c t io n s ...... 65
b. Fractionation of the tertiary alkaloids in to th e t e r t ia r y non -p h en olic (TN) and tertiary phenolic (TP) alkaloid fractions. 69
D. Separation and Isolation of the Tertiary Non- p h en o lic (TN) A lk a lo id s ...... 71
1. Separation of the TN alkaloids by gradient pH e x t r a c t io n ...... 71
2. The isolation of alkaloids TNA, TNB, TNC, and hernandezine from gradient pH extractives 75
a. Alkaloid TNA ...... 75
v CONTENTS (Cont’d) Page
b. Alkaloid TNC (The major tertiary non- phenolic alkaloid) ...... 80
c. Hemandezine (Alkaloid TNC-50)...... 82
d. Alkaloid TNB (The hypotensive alkaloid)... 90
3. Alkaloids of the gradient pH extractives at pH 6.0, 6.5, 7.3, and 8.8 ...... 97
The T ertia ry P h en olic (TP) Alkaloids...... 98
Chromatography and Isolation of the Quaternary A lk a lo id s ...... 99
1. Thin-layer chromatographic studies of the total alkaloid fraction ...... 99
2. Chromatographic separation of the quatemaiy alkaloids for isolation ...... 100
3. The isolation and identification of berberine, jatrorrhizine, and magnoflorine ...... 10 5
a. Berberine iodide ...... 106
b. Tetrahydroberberine ...... 108
c. Jatrorrhizine chloride and jatrorrhizine io d id e ...... 109
d. Magnoflorine iodide ...... 113
e. Magnoflorine chloride ...... 115
Methodology - Chemical and Physical Analyses...... 117
1. Thin-layer chromatography5 ...... 117
a. Thin-layer chromatography on silica gel G p la t e s ...... 117
b. Thin-layer chromatography on microcrystalline cellulose plates ...... 118
2. Benedict’s test for reducing sugars ...... 119
3. Labat test for methylene dioxy groups ...... 119 CONTENTS (Cont’d)
Page
**-. Ammon±acal~phosph oddolybdic acid test for hindered hydroxyl groups,« ...... 120
5. Claisen1s cryptophenol reagent for the detection of phenolic groups...... 120
6. Tomita* s test for dibenzo-p-dioxane groups.... 120
7. Melting point determination ...... 121
8. Optical rotation measurements ...... 121
9. Ultraviolet spectral analyses...... 121
10. Infrared spectropho tome trie analyses ...... 122
11. Nuclear magnetic resonance spectrophotometric a n a ly s e s ...... 122
12. Molecular weight determination...... 123
13. Microanalyses...... 123
14. Structural elucidation studies...... 123
15. Pharmacological screening for hypotensive A c tiv ity o... 00. ooooo,ooooooaa,..o....o...... 123
IV. DISCUSSION...... 125
V. SUMMARY OF FINDINGS...... 1^*0
APPENDIX A...... 1^2
APPENDIX B...... 1^6
APPENDIX C...... 15^
BIBLIOGRAPHY...... 158
vii TABLES
Table Page
1. Physical and Chemical Properties of the Thalictrum Series of Alkaloids ...... 27
2. Hypotensive Activity of the Alkaloid Fractions T e s t e d ...... 52
3. Paper Chromatographic R esu lts o f the Column Chromatographic Fractions of the Quaternary A lk aloid s ...... 60
4. Weights and JJj. Values of the Extractives from the Gradient pH Separation of the TN A lkaloids ...... 74
5. Chromatographic Separation of TNB Mother Liquors . 95
6. Results of the Chromatographic Separation of the Qu Alkaloids on Silicic Acid: Celite-5^5 (6:1) C olum n ...... 102
7. Absorption Maxima of the Ultraviolet Spectra of the Alkaloids of T. Rochebrunianum ...... 14-3
8. Nuclear Magnetic Resonance Data of the Alkaloids, TNA, TNC, TNC-50 (Hemandezine) , Tetandrine, O-methylrepandine, and Isotetandrine ...... 145
viii FIGURES
Figure Page
1. Thalictrum Rochebrunianum, Above Ground P a rts ...... 6
2. Thalictrum Rochebrunianum, Roots ...... 6
3. The Protoberberine Alkaloids of Thalictrum Species . . . 3^
The Aporphine Alkaloids of Thalictrum S p ecies ...... 3^
5. Bisbenzylisoquinoline Alkaloids of Thalictrum Specie s ...... 35
6. Aporphine-benzylisoquinoline and other Types of Alkaloids of Thalictrum Species ...... 36
7. Two-dimensional Thin-layer Chromatogram of F-E Alkaloids (200 pg) on Silica gel G ...... ^3
8. Two-dimensional Thin-layer Chromatogram of TT Alkaloids (hO pg) on Silica gel G ......
9. Thin-layer Chromatograms of the Methylene Chloride Extractive on Silica gel G plates ...... ^7
10. One-dimensional Thin-layer Chromatogram of TN and TP Fractions on Silica gel G ...... **9
11. Two-dimensional Chromatogram of TN Alkaloids (160 pg) on Silica gel G ...... 50
12. Two-dimensional Chromatography of TP Alkaloids (160 pg) on Silica gel G Thin-layer Plate ...... 51
13. One-dimensional Thin-layer Chromatogram of TN Alkaloids from Gradient pH Separation on Silica gel G P late ...... 57
1^-. "Cyclic-percolation" Apparatus ...... 63
15. One-dimensional Thin-layer Chromatogram of TN Alkaloids from Gradient pH Separation on Silica gel G plates ...... 73
16. Two-dimensional Thin-layer Chromatogram of TNA Alkaloid plus TNB Alkaloid (10 pg each)on Silica gel G p late ...... 7 6
ix. FIGURES “ (Cont’d)
Figure Page
17. Two-dimensional Thin-layer Chromatogram of TNC plus TNC-50 (Hemandezine) (5 Pg each) on Silica gel G plate ...... 83
18. I.R. Spectrum of Compound 20-x (Sucrose) ...... 1^7
19. I.R. Spectrum of TNA ...... 1 ^
20. I.R. Spectrum of TNC (in KBr pellet) ...... 1^9
21. I.R. Spectrum of TNC (in CHCl, film ) ...... 1^+9
22. I.R. Spectrum of TNC-50 (Hemandezine) in KBr pellet . 150
23. I.R. Spectrum of TNC-50 (Hemandezine) in CHCl^ film . 150
2k. N.H.R. Spectrum of TNA ...... 151
25. N.M.R. Spectrum o f TNC ...... 152
26. N.M.R. Spectrum of TNC-50 (Hemandezine) ...... 153
CHARTS
Chart Page
I. Flow Sheet for the Extraction of the Total A lk a lo id s ...... 155
II. Flow Sheet for the Fractionation of the Total Alkaloids ...... 15^
x INTRODUCTION AND STATEMENT OF PROBLEM
Supported by a grant from the Ohio State University Development
Fund in 1956, Dr. Jack L. Beal, this author’s adviser, initiated a phytochemical screening program in search of new medicinal compounds from plants. Since the phytochemicals known as alkaloids constitute the largest class of medicinal agents from higher plants (vis. morphine, codeine, atropine, reserpine, ergotamine, ephedrine, d- tubocurarine, leurocristine, quinine, and quinidine), emphasis wa3 placed on alkaloid-bearing plants, particularly those found in the state of Ohio which had not been previously investigated or had re ceived only a cursory examination. A thorough search of the litera ture was conducted to ascertain the most promising fam ilies or genera of plants that would serve to accomplish the purposes of this program.
Subsequently, specimens of the most promising plants were collected and tested for the presence of alkaloids.
Among th e fir s t plants to be screened in this manner were three species of the genus Thalictrum (Ranunculaceae), namely, T. revolutum,
T. dasycarpum, and T. dloicum. Alkaloids were shown to be present in all three species. Particularly heavy precipitation with Valser1 s reagent was observed from extracts of T. revolutum and T. dasycarpum.
A re-examination of the literature revealed that three other species in this genus had been evaluated for alkaloids, all giving positive tests. It was further noted that various species had been popularly employed as home remedies; yet, phytochemical investigations of these species were seldom reported. On this basis, eleven species of
1 Thalictrum plants were obtained and tested for alkaloids, followed by a general pharmacological screening of the total alkaloid fraction.
Thai^ nt.mm Rochebrunianum. Franc, and Sav. , a cultivated species in both Europe and the United States, was one of the plants screened.
It was found to be one of the highest alkaloid-containing Thalictrum species, in terms of total root alkaloid content, as evidenced by the density of precipitate formed by the addition of Valser* s reagent to an aqueous acid extract of the powdered root. In addition, the total tertiary alkaloid fraction, when injected intravenously in an anesthe tized, normotensive dog at a dose of two milligrams per kilogram, caused a substantial drop in the arterial blood pressure (1). In view of these findings, it was felt that a comprehensive study of the alkaloids from the roots of this plant might result in the isolation of the compound(s) responsible for this activity.
Certain preliminary investigations were conducted prior to initiating the actual alkaloid isolation studies. Employing a quanti tative procedure commonly used by workers in this laboratory, it was found that both the tertiary and quaternary alkaloid fractions were each composed of more than 0.1# alkaloid. The total tertiary alkaloid fraction was further divided into phenolic and non-phenolic alkaloid fractions. These fractions, in addition to the quaternary fraction, were evaluated pharmacologically to determine that particular fraction, or type of alkaloid present, mainly responsible for the hypotensive activity previously determined.
The non-phenolic alkaloid fraction was found to be the most active and had the longest duration (50-60 minutes), while both the phenolic and quaternary fractions gave only a transient hypotensive 3 activity of approximately two minutes. It was also noted that the alkaloid content of the non-phenolic fraction was almost four times that of the phenolic fraction, when compared on a dry weight basis. ^
On the basis of these findings, in addition to the fact that no phytochemical investigations were found reported in the literature, a phytochemical investigation of £. Rochebrunianum was undertaken as the subject of this dissertation.
The purpose of this investigation was to isolate, characterize, and identify as many alkaloids as possible from the non-phenolic tertiary alkaloid, the quaternary alkaloid fractions, and isolate, if possible, the alkaloid or alkaloids responsible for the hypoten sive activity observed in dogs.
^ The details of these findings are presented in the experimental section of this text, under "Preliminary Investigations" on pages 39~4 i , h-5-46. 4
I I . LITERATURE SURVEY
A. Taxonomic Description of Thalictrum Rochebrunianum, Franc, and Sav. (Ranunculaceae)
Thalictrum Rochebrunianum, Franc, and Sav., is one of numerous species of plants commonly known as "Meadow Rue" belonging to the
Ranunculaceae (Buttercup or Crowfoot family). These plants are perennial herbs mainly distributed in the temperate and cold regions of the earth (2).
A number of treatises have been published concerning the botanical description of the genus Thalictrum ( 3*^*5*6,7»8) sin ce
Lecoyer's I885 monograph in which he listed 69 species (9).
J. Rochebrunlanum. Franc, and Sav., is a glaucescent perennial species native to Japan. The stems of this plant appear glabrous and weakly striated, while the leaves are bipinnate to ternate, and have petioles associated with large, auriculated sheaths. Its auricles are sm a ll, s c a r io u s, and entire. Leaflets are fairly large and thick, with filiform petiolules; oval, round or cordate bases; entire or tridentate tips, which are more or less obtuse; dark green, glabrous, and lusterless upper surfaces; and bright green, glabrous lower sur faces with slightly prominent brownish veins. The inflorescence is p a n ic le ( compound racem e), w ith lavender-m auve hermaphrodite flo w e r s, and the pedicels on which these flowers are borne are 5-50 in length and have large, scarious, trilobed bracteoles. The rachis is sometimes terminated by one to three minute leaflets. Four oval sepals,
5-7 mm. by 2.5-3*0 mm in size, compose the ca ly x . Stamens are numer ous, 15-20 in number with elliptical prominent yellow anthers slightly 5 mucronulate, and attached to filiform filaments of 2-3 mm. in length.
P istils number 15-25, having subsessile ovaries, are connected to oval stigmas (0.5 mm.) by 0.5 mm. long styles and the stigmas are slightly recurved and incline when in bloom. The unripe achenes stipitate and are glabrous, fusiform in shape (3-^ nun* by 1-15 mm.) with indistinct sutures, provided with six longitudinally running veins. These achenes are distributed obliquely on the receptable and prolongated on small short, beak-like structures, which re sulted from withered stigmas (8,9,10,11,12,13).
» Features which distinguish T. Rochebrunianum from other species of
Thalictrum are the stipes of the achenes, the form of the stigmas, and the bracteoles of the inflorescence (9).
Figure 1 illustrates the above-ground portion of this plant, showing the flowers and its inflorescence, while the roots are shown in Figure 2. FIGURE 1 FIGURE 2
Thalictrum Rochebrunianum Thalictrum Rochebrunianum
Above-Ground Parts Roots Bo The Use of Thalictrum Plants as Home Remedies
Various species of the genus Thalictrum have, at one time or another, been household remedies for many types of ailments. T. flavum was used as an aperient and stomachic by the English in the eighteenth and nineteenth centuries, and by the Russians for hydro phobia (14)o In the early l800»s, this plant was employed in the
United States for its reputed effectiveness as a purgative, diuretic, and febrifuge (9)° Fomentations of T. minus were used in Russia for snake b it e and fe v e r (9), while the Chinese prescribed T. siense.for pectoral complaints (14)„ In India, T. follolosum, although known to be effective as a laxative and tonic, was chiefly employed in indigenous medicine as an inexpensive, but valuable, substitute for
C optis teata (Ranunculaceae) in the preparation of collyria for ophthalmic disorders (15). In addition, other species were employed by the Indians in indigenous medicine as a stomachic, bitter tonic, antiperiodic, alterative, diaphoretic, laxative, antipyretic, purga tive, blood purifier, and for snake bite, jaundice leprosy, vomiting of pregnancy, rheumatism, indolent ulcers, conjunctivitis, gastric and duodenal ulcers, and oriental sores (16,17). The Japanese have also employed Thalictrum species for various diseases (18). As cures fo r snake bite, the use of Thalictrum plants was not restricted only to the Old World, for it has been known that the natives of Canada and the American Indians had long used the roots of certain native species for this purpose (14). For gonorrhea and the common cold, the
American Indians of Nevada employed a tea prepared from T. fendleri,
Enge'lm, (19). In view of their world-wide acceptance as home remedies for the aforementioned ailments, it would not be out of perspective to assume that certain, if not all species, of the genus Thalictrum do possess some th e r a p e u tic a lly a c tiv e component or components. The recen t appearance of several reports on the pharmacological studies of alka loid fractions from Thalictrum plants substantiates, at least partially, this assumption.
Ovsepyan (20) studied the pharmacological effects of the total alkaloid hydrochlorides of T. minus, L., and hypotensive effects on frogs, cats and dogs were observed. Employing rabbits as test animals
Daleva and Sherkava reported that alkaloid fractions from Bulgarian
T. minus produced a significant but transient hypotension, and ex citation of the choline-reactive system, spasmolytic action on the intestine, and a slight diuretic effect ( 21). Patil et al. observed that one alkaloid fraction from T. revolutum, D.C. produced hypo tensive effects in dogs, cats, and rabbits, generalized CNS depression in rats, slowing of the isolated turtle heart, emesis in pigeons, and a positive rabbit head-drop test (22). Kupchan and Yokoyama reported the Isolation of an alkaloid from T. dasycarpum, Fisch. and L a ll., which they named thalicarpine, possessing a transient hypotensive effect in cats (23). In addition to these published results, lyophi- lized aqueous extracts of _T. rugosum and T. adiantifolium prepared in this laboratory have confirmed activity against Sarcoma 180 in fciee (1) 9
C. The alkaloids of the Genus Thalictrum
1. Types of alkaloids present
Alkaloids obtained from the genus Thalictrum to date are theoret
ically derivable from the benzylisoquinolines (I). The p rotob erb erin es
RO. RO [-H RO [-H RO
R0- to n
I I I 2 (II), the aporphines (III (a,b), and the dimeric benzylisoquinolines
having one or two ether linkages (IV) are the main types of nuclei
+ . 7
-CH, (7CH3 H C- OR R0' OR RO
'OR RO''
III IV possessed by these alkaloids. In addition, two novel types of nuclei have been reported, one being an alkaloid having a phenathrene nucleus
(V) (24), while the other type is composed of benzylisoquinoline and
an aporphine moiety (VI) (23, 25, 26, 27, 28).
2 a: Numbering system according to The i 960 Ring Index; b: numbering system from earlier editions of the Index. 10
I-CH. N-CH. CH0 '
OCH.
V VI
The protoberberine alkaloids (VTI, VIII) can theoretically be derived from the benzylisoquinolines (I) by condensation with formaldehyde. This has been achieved in vitro by Spath and Kruta (29), and also by Pictet and Chou (30). There are no known n a tu r a lly occurring protoberberines with less than four 0- substituents, but there are some with an additional hydroxyl group at position 1 or at position 13 (31)*
RO RO
RO RO OR
OR OR //
VIII
These alkaloids occur in a number of botanical families. In the order Rhoeadales of the Papaveraceae, they are usually in the form of tetrahydro bases, while in the order Polycarpicae of the Berberi- daceae, Ranunculaceae, Minispermaceae, Rutaceae, Anonaceae and
Lauraceae, they occur mainly as the quaternary bases (31)* 11
The aporphine alkaloids are derivable from the benzylisoquinolines by the abstraction of two hydrogens in such a manner as to result in the final product having a 9, 10 dihydrophenanthrene nucleus (III (b)).
According to biogenetic schemes, the benzylisoquinolines are derived from precursors having oxygen substituents in the 3 and k positions, e - g* 3t^-dihydroxphenylalanine, therefore, the aporphines can have the substituents only in positions 2, 3. 5» or 6. The majority of these alkaloids possess four oxygen substituents. Some.however, have less than four. For example, isothebaine (IX) has three oxygen atoms, while roemerine (X) has only two (32).
[-CH [-CH.
HO
H_C0
IX X
As in the case of the protoberberine alkaloids, the aporphine alkaloids are found in both the orders Polycarpicae and Roeadales.
Among the fam ilies in the former order in which these alkaloids have been found are the Anonaceae, Aristolochiaceae, Berberidaceae,
Lauraceae, Magnoliaceae, Menispermaceae, Monimiaceae, Nymphaeaceae,
Rutaceae, and Ranunculaceae. The nitrogen atom of their aporphine alkaloids may be fully methylated in the forms of tertiary and quaternary alkaloids or non-methylated secondary amines. In the latter order of the Papaveraceae, the aporphine alkaloids are usually N-methylated (32). 12
It is of interest to note that the Papaveraceae is the only family, among many in the order Rhoeadales, having the benzyliso quinoline alkaloids or their derivatives. For this reason, plus
other different characteristics from other families in this order,
Hegnauer has proposed a reclassification of the Papaveraceae into the order Polycarpicae (33)*
The bisbenzylisoquinoline or biscoclaurine alkaloids possess
two benzylisoquinoline nuclei joined by one , two, or three ether linkages.
The number and p o s itio n o f th e eth er lin k a g e s in the m olecule can
serve as criteria for the classification of these alkaloids into
subgroups. Faltis et al. (3^) considered all members of this class of alkaloids to be offsprings of the benzylisoquinoline compounds, coclaurine (XI) or norcoclaurine (XU).
N.
XI (R=Me) X II (R=H)
Enzymatic dehydrogenation of two molecules of XI or XII, fol lowed by methylation of some or all of the (OH) and/or (NH) groups in the plant gives rise to the bisbenzylisoquinoline alkaloids. The dehydrogenation occurs between a phenolic hydroxyl of one molecule of
XI or XII and a reactive nuclear hydrogen of another molecule. The simplest case is illustrated by the alkaloids having only one diphenyl ether linkage, where the oxygen bridge formation takes place between 13 the 12-hydroxyl of one molecule and the 11-hydrogen of another molecule
of XIX. Dauricine (XIII) is an example of this type alkaloid. The
oxyacanthine-berbamine (XIV and XV) type of alkaloids are those having
two diphenyl ether linkages. In these alkaloids, an additional ether
linkage is formed between the 7"-0H and 8’-H or the 8-H and 7,- 0H of the
OCH. I-H
CH HO
X III XIV
norcoclaurine (XU) skeletal structures. The two diphenyl ether
linkages between the two molecules of XU may be formed at different
positions from the positions of the oxyacanthine-berbamine series, and
give rise to alkaloids such as berbeerine (XVI) and isochondodendrine
(XVII) (35). -CH. HO I-CH. OH l-CH.
OH OH OCH. 'CH.
XV XVI xvn
A third group of bisbenzylisoquinoline alkaloids have, in their
structures, three oxygen bridges consisting of one diphenyl ether
linkage and a diphenylene dioxide ring. One may consider these alka
loids to be derived from the oxyacanthine-berbamineQdV and XV)series
of alkaloids, in which the third oxygen bridge is added between
the two isoquinoline portions of the molecule to form a diphenylene dioxide ring. Micranthine (XVXIl) and trilobine (XIX) are examples of this type of alkaloid (36).
I-R H„C- I-CH.
OH ICH. R^ or R^=CE^ ( other R^ or R^=CH^ ( oth er = H) XVIII XIX
The bisbenzylisoquinoline alkaloids have been found in the
Menispermaceae, Berberidaceae, Magnoliaceae, Monimiaceae, Anonaeeae,
Ranunculaceae, and Hernandiaceae (37).
Thalicthuberine (V) (24) is 1-dimethylaminoethyl-3,4-dlmethoxy-
6,7-methylenedioxyphenanthrene, which is identical with the a-methine from the Hoffman degradation of a corresponding aporphine alkaloid, nantenine (XX). I t is not inconceivable to consider this alkaloid
CH
XX to be either a naturally occurring a-methine from enzymatic degradation of natenine, or a precursor of an unknown quaternary base. Thus far, this is the only alkaloid of its kind to have been isolated from a ranunculaceous plant. 15
Thalicarpine (VI) (23, 25, 26) and thalmelatine (XXI) (27, 28)
are members of the novel aporphine-benzylisoquinoline types of alka
loids. The total synthesis of thalicarpine from 6»-bromo-(-)-
laudanosine and isocorydine (25, 26) strongly suggests the precusors
of these alkaloids as being laudanosoline-3’ -dimethylether (XXH)
and isocorydine (XXIII). Following the Faltis theory of bisbenzyllso-
quinoline formation (3^) » one can consider the formation of the
oxygen bridge of these alkaloids as involving the 6*-H of XXH and
the phenolic (OH) of XXIII. Subsequent methylation would then lead
to the formation of thalmelatine (XXI) and thalicarpine (VI).
OH H^O CE
OCH. fCH >CH
XXI XXII XXIII
To date the distribution of this type of alkaloid has been
found only in the genus Thalictrum. however, it is not inconceivable
that they may also be found in other ranunculaceous plants.
2. Alkaloids isolated from Thalictrum species
The first alkaloid isolated from a Thalictrum plant was thalio-
trine-A along with macrocarpin-A, a neutral yellow compound, from T. macrocarpum, Gren. by Henriot and Doassans in 1880 (38, 39. ^ 0).
Since then, the isolation of 22 different alkaloids from 12 species 16 and two varieties of Thalictrum has been reported in the literature.
To facilitate a review of the plant species and their contained alkaloids, alphabetical rather than the usual chronological order is used in this section of the literature survey.
Thalictrum acteaefolium. Sieb. and Succ., was found to contain the quaternary protoberberine alkaloid, berberine, by Matsul,
Tomimatsu, and Fujita in 1962. This alkaloid was isolated as the iodide salt (C^qH-^qO^NI) from the whole herb. From a sample of the roots growing in a different area of Japan, the same alkaloid was identified by derivation to its corresponding tertiary base, t e t r a - hydroberberine, by treating the amorphous iodide salt with zinc and a c e t ic a c id (^+1).
In a paper published in 1939, Lazur^vskii andSadykov reported detecting alkaloids in the whole plant of T. alpinum L., however, none were isolated (^2).
T. aquilegifollum L. is another species reported to contain alkaloids (^3).
While investigating the quaternary alkaloid fraction from the roots of T. dasycarpum var. hypoglaucum in 1961, Hogg, Beal, and
Cava succeeded in isolating berberine and magnoflorine as the iodide salts (^-4). In 1963» Kupchan et al. . in a series of papers, reported the isolation, structural and configurational determinations, and total synthesis of thalicarpine (C^-lH^qOq^ ) , a non-phenolic dimeric aporphine-benzylisoquinoline alkaloid, having a transient hypotensive effect in cats, from the roots of T. dasycarpum, Fisch. and Lall.
(23, 25, 26). 17
In addition to his investigation of T. macrocarpum, Gren.,
Doassans also studied T. flavum L. From the latter plant, he and
Moussett isolated berberine in the 1880’s (45). Amaudon also isolated this alkaloid from the roots of the same plant in 1891 (46)•
T. foetidua was investigated by Sargazakov in 1963 (47). From an ether extract of the above ground parts of this plant, he isolated fetidine (C^H^QOgNgHgO) a norv-phenolic bisbenaylisoquinoline alka loid having one dipehnyl ether linkage. He was able to elucidate the structure, except for the exact position of the oxygen bridge at one of the phenyl rings.
Three groups of Indian investigators studied the alkaloids of
T. fo lio lo su m D.C. Vashista and Siddiqui isolated berberine and thalictrine (C^H^O^M) from the powdered rhizome in 1941 (15).
This alkaloid, however, was not the same as Doussan* s thalictrine-A, for their physical properties were different. Chatterjee, Guha, and Chatterjee (48) in 1952 reported the isolation of berberine, jatrorrhizine (C^H^O^NI) , and palmatine (C^Hg^O^N) as the chloride, iodide, and tetrahydroanhydro forms respectively, from the rhizome.
They failed, however, to find the thalictrine of Vashista and Siddiqui.
Seven years later, Gopinath et al. re-investigated this plant and were able to isolate the thalictrine of Vashista and Siddiqui. By comparison of the melting points, mixture melting points, and infra red spectra of the iodide and picrate salts, they proved that thalic trine and magnoflorine were identical (49).
Hernandezine (C^H ^ jO^Nq) , a non-phenolic, bisbenzylisoquinoline alkaloid having two diphenylether linkages, was isolated from the roots 18 o f T. h e rn a n d ezll, Tausch., and the structure subsequently determined by Padilla and Herr£n in 1962 (50)-
Ismailov et al. reported the isolation of two alkaloids from
X* iBopyroides in 1959 (51)* A partial elucidation of one of the compounds was presented. In 1961 they reported these alkaloids to be isomers of the triphenylidine (XXIV) type. Talicopirine and talisopine (CP-|H?^O^N) were the names given to these alkaloids (52).
It was also stated in the paper that these two alkaloids were isomeric with thalmidine, an alkaloid previously isolated from T. minus L. (53)-
XXIV
When one examines the structures of the Thalictrum alkaloids isolated to date, one finds that they are theoretically derivable from the benzylisoquinolines, except the aforementioned alkaloids isolated by Ismailov et al. Chemotaxonomically, the proposed structures of talicopirine and its isomers are not compatible with all the other
Thalictrum alkaloids. It seems logical, in view of present data, th a t this chemotaxonomic imcompatibility would suggest that either Ismailov’s
isopyroldes is not a true Thalictrum species or that these tri phenylidine alkaloids, if the plant is indeed T. isopyroldes, are experimental artifacts. A further examination of this species might be of interest. 19
In addition to the thalmidine (C?-| Hp^O^N) Yunusov and Progressov isolated thalmine (C^Hg^O^N) from the tops of T. minus L. From the roots, they reported the isolation of three more alkaloids. One of these was an unknown orange base in minute quantities. Thalicmine
(Ca H 2^0^N) and thalicmidine (^£0^25^4^) were ^ e names given to the other two alkaloids (53) in 1951* Tw° years later, these same investigators postulated thalicmidine and thalicmine to be 2,3.5- trimethoxy-6-hydroxyaporphine and 3*4,7-trime thoxy-5,6-me thylene- dioxyaporphine-) respectively (5*0. Thalmine was proposed to be
3 , 5-dimethoxy-K-mathyl-11-hydroxyhexahydrotriphenylidine by Yunusov and Ismailov in 19 60 (55)• At th e same time, they revised the struc ture of thalicmidine by interchanging the positions fo the 5-m®thoxyl and 6-hydroxyl groups. The new chemical name became 2 ,3 ,6- trimethoxy-
5-hydroxyaporphine (56). Shamma, however, fe lt that the revised structure of thalicmidine was still incorrect. In 19^3* he pointed out that three of the four possible isomers of O-desmethylglaucine were known, namely, N-methyllaurotetanine, glaucentrine, and 0,N- dimethyllaurelliptine. Since none of these componds possesses a set of physical data attributed to thalicmidine, the only possible alterna tive would be 2,5.6-trimethoxy-3-hydroxyaporphine (57).
A year later, Shamma and Slusarchyk, in their "Review of the
Aporphine Alkaloids" (58)* further cited the isolation of coscarmine iodide from Cocculus sarmentosus by Tomita and Furukawa (59). S in ce
3 1,9,10- trime thoxy-2-hydroxyaporphine and 3*10,11- trimethoxy-1, 2-methylenedioxy aporphine, numbering system according to the i960 edition of the Ring Index. 20
coscarmine iodide is the quaternary salt of 1 ,2,9-trimethoxy-10-hydroxy-
aporphine (2,5,6-trimethoxy-3“hydroxyaporphine), the physical data for
this salt would be identical to those of thalicmidine methyliodide. Un
fortunately, this was not the case. It was the opinion of these authors
that a reconsideration of the experimental work on thalicmidine was in
order before settling the final structure of this alkaloid. Tschesche
££ (60), on the other hand, upheld Yunusov and Ismailov's structure
for thalicmidine as theoretically correct based on their recent study
of the structures of their alkaloid "A" obtained from Simplocos celas-
trinea and the four possible O-desmethylglaucine derivatives. By
comparison studies of the physical properties of their alkaloid and
those of its o-methyl derivative with the properties of isoboldine and
N-methyllaurelliptine and their respective O-methyl derivatives, they
were able to prove that these three dihydroxydimethylaporphines were
identical and their O-methyl derivatives were identified as 5”bydroxy-
2,3.6-trimethoxyaporphine. They were able to prove that their alkaloid
was identical to isoboldine and N-methyllaurelliptine and revised the
stru ctu re o f the l a s t compound from 3»6-dihydroxy-2,5~dim ethoxyaporphine
to 2,5-dihydroxy-3,6-dimethoxyaporphine isoboldine. In the process,
they establised the structures of O-methylisoboldine, O-methylboldine
and N-me thy'llaurotetanine as the 5“» 6-, and 2-0-desmethylglaucine
derivatives, and proposed that the structure of glaucentrine be
revised to 3“bydroxy-2,5*6“trimethoxyaporphine (3”0-desmethyl-
glaucine). At the same time, they noted that all of these four
possible O-desmethylglaucine derivatives were dextrorotatory,
while thalicmidine was determined to be levorotatory and that the 21 specific rotation and other physical properties of thalicmidine were in good agreement with those of O-methylisoboldine. Therefore, these authors felt that thalicmidine could be considered as (-)-5-hydroxy-2,
3,&-trimethoxyaporphine ((-)-2,3,6-trimethoxy-5-hydroxyaporphine) and that thalicmidine methyliodide could be considered as the 1-form of the quaternary aporphine iodide obtained from Fagara tinguassoiba (58). which had been demonstrated to have the same structural arrangement as N-methyllaurelliptine (O-methylisoboldine).
In 1963* Vernengo (6l) proved the structure of thalicmine, as
advanced by Yunusov and Ismailov, was incorrect. Based on data
obtained from U.V., N.M.R., and optical rotatory dispersion curve
analyses, he was able to show that thalicmine was identical to ocoteine,
an alkaloid isolated from Ocotea puberula by Iacobucci in 1951 (62).
According to a paper published in 1956, Yunusov and Ismailov
claimed that the epigenous parts of five species of Thalictrum contain primarily thalmine and thalmidine (63).
A second group of Russian investigators, who studied T. minus L. which was obtained from a different area of Russia than Yunusov's
source, isolated a new alkaloid, which they named thalictrimine
(C20H23O4N) (6 4 ).
Thalictrum minus var. elatum, widely employed as a home remedy for assorted ailments in Japan, was investigated in 19^5 by Nakajima.
He was able to obtain two ether soluble, tertiary alkaloids, the main
alkaloid being non-phenolic and having the molecular formula of
E la tr in e was th e name a ssig n ed to i t . The minor a lk a lo id was phenolic in nature (18). Recently, Mollov and his co-workers 22 isolated three alkaloids from the same speeies growing in Bulgaria.
The first alkaloid obtained was identified as thalicarpine, which was previously isolated by Kupchan et al. from T. dasycarpum. The second alkaloid has yet to be identified due to its limited avail, a b i l i t y . The th ird a lk a lo id is o la t e d from t h is p la n t was named thalmelatine, a phenolic aporphine-benzylisoquinoline alkaloid having a formula of ^ 2^30^8^2' Upon treatment with diazomethane, it was converted to thalicarpine. The hydroxyl group was determined to be at position 7 of the benzylisoquinoline moiety (27, 28).
Wali ct al. investigated T. pedunculatum var. Edgw. , obtained from the western Himalayas, where it is used for ophthalmia. From this plant, they obtained berberine and three tertiary alkaloids, two of which were not identified. The third tertiary alkaloid was identified as berbamine a known phenolic tertiary alka loid possessing two diphenylether linkages (65).
The only reported alkaloid investigation on T. revolutum, D.C. was in i 960. Magnoflorine and jatrorrhizine were isolated from the roots by Spiggle (66). Weilenmann, working on the same plant con currently in the same laboratory, isolated berberine (67).
Current literature failed to reveal any recent studies on the alkaloids of T. rugosum, Ait. (T.glaucum, Desf.), and T. rhynchocarpum,
D ill, and Rich. However, Rochebrune did report the presence of thalic trine and macrocarpine in the roots of Spanish T. glaucum, Ait. and
African T. rynchocarpum. D ill, and Rich., before the turn of the cen tury (**5) • 23
Thalictrinine, a tertiary base was isolated from T. simplex L.
by two groups of Russian investigators ten years apart, each group
presenting different data for this compound. In 1950» Norkina and
Pakhareva reported a molecular formula of **°r ^ is alkaloid
obtained from the leaves (68). In i960, Yunusov and Ismailov claimed
that the correct formula should be ^21^25®^ (69). The melting points were the same by the optical rotations were different. The former
group reported [oQ ^ -80.9° (chloroform), while the latter group re- ported£3^-65.0° (c.,3.08 in chloroform). Yunusov and Ismailov also
reported the isolation of thalcimine (C-^^^NO^) from the same plant
growing in a different region.
Between 1956 and 1963, Tomimatsu _et _al. published sixteen articles dealing with phytochemical studies of the alkaloids of Thalictrum thun-
bergii, D.C. (T. minus var. hypoglaucum, Miq.) (24, 71-84). Thus far,
eight alkaloids, six of which are new, have been reported to be
present in this species. Subsequently, the two known alkaloids were
characterized and identified, and the structures were established for
the unknown ones.
The first alkaloid isolated from this species in 1956 was identi
fied as the quaternary aporphine, magnoflorine. This was the first
reported existence of this alkaloid in the genus Thalictrum, although
Vashista and Siddiqui had previously isolated it as thalictrine in
1941 (15)• Their failure to identify this alkaloid correctly plus the fact that it was not until 1959 that Gopinath et al. (49) finally proved that Vashista's thalictrine was in reality magnoflorine, a fact
that justified Tomimatsu1s claim to be the first to report the iso- 2k lation of magnoflorine from a Thalictrum species.
The next seven papers of Tomimatsu et a l., appearing in 1959-
1960, were on the isolation, characterization, and structural deter mination of takatonine, O-methylthalicberlne, thalicthuberine, and thalicberine from T. thunbergii. Takatonine, C^Hg^O^N, was found to be a new quaternary benzylisoquinoline alkaloid. The chemical name g iven to t h is compound was 1 - (h _m ethoxybenzyl)-2-m eth yl-6, 7 ,8 - t r i - methoxyisoquinoline. O-methylthalicberine (C^gHj^OgN^) was obtained from the leaves and the stems. It was found to be a non-phenolic, bisbenzylisoquinoline alkaloid with two diphenylether linkages.
From the roots, thalicthuberine, a tertiary non-phenolic alkaloid possessing a phenanthrene nucleus and a formula of was isolated. At the same time, the phenolic parent alkaloid of 0- methylthalicberine, thalicberine ( C^H^OgNg) » was obtained from the leaves and stems (2h, 72-77).
Two phenolic bases, thalicrine, (C^H^gOg^) and homothalicrine
(C^H^OgNg) » were found in the roots of this plant in 1962 (78-80).
Both o f th ese compound are b isb e n z y liso q u in o lin e s jo in ed by two diphenylether linkages. They differ in the 12* position, where a phenolic hydroxyl group is attached in the thalicrine structure, In homothalicrine, this hydroxyl is replaced by a methoxyl group.
In a study of the effect of habitat on alkaloid production,
Tomimatsu et al. noted that the alkaloids present in T. thunbergii growing in one area of Japan differed from those growing in another area. In one case, magnoflorine was found in the roots and taka tonine in the leaves and stems, whereas in the second case, takatonine 25 was absent and the alkaloids present were magnoflorine and berberine
(8l). Additional information concerning the structures of thalic berine and O-methylthalicberine is contained in two papers published in I963 by these authors (82, 83).
In their most recent publication (84), Tomimatsu _et al. revised the structures of thalicrine and homothalicrine. As originally pro posed in 1982, basic structure XXV was assigned to these alkaloids.
A fu rth e r study o f th e sodium liq u id ammonia cleavage products o f thalicrine and aromoline, however, showed them to be identical on the basis of their infrared spectra and other physical data.
.OCH.
I-CH f-CH ,C-
RO RO
XXV XXVI
Therefore, structure XXVI was designated as the correct basic structure for these alkaloids.^
In a personal communication, Tomimatsu has indicated the isolation of two other tertiary non-phenolic alkaloids in the form of their nitrate salts from this species ( 85 ).
In the case of Thalictrum Rochebrunianum. Franc, and Sav., the subject of this dissertation, the literature revealed no previous phytochemical work with the exception of the recently completed
4 Thalicrine (aromoline), R=H{ homothalicrine ,R=CH^ alkaloid screening by thin-layer chromatography of thirteen species
of Thalictrum by Schiff in this laboratory (86). By comparison of
the values of available reference alkaloids and the alkaloids
present in the various fractions of this plant, Schiff reported the
possible occurrence of thalicthuberine, thalicrine, and thalicarpine
in the tertiary fraction. From a spectrophotometric analysis at the
quaternary fraction of the root, he further indicated that the quan
tity of berberine present was 0.37-0.40
The molecular formulae and physical properties of all the alka
loids and/or their salts that have been isolated from the afore mentioned Thalictrum species are summarized in Table 1.
The structures of those alkaloids that are known, or which
have been proposed are presented in Figures 3 to 6. TABLE 1
Physical and Chemical Properties of the Thalictrum Series of Alkaloids
(b) Specie PlantAlkaloid Empirical M elting rr7]t n Reference Part Formula Point (°C) k JH
1. acteaefolium r berberine I C^H^gO^NI d255 43
r tetrahydroberberine C^I^O^N 169=170 43
dasycarpum. F.& L. r thalicarpine ^4l®48®6^2 l 60- l 6l +8 9 , t25,C f 23. 25, 26 (c.0o88);+133 t25,M,(e.0.83)
1. dasycarpum b e rb e rin e io d id e CggH^gO^NI d 258 44 van hypoglaucum
magnoflorine iodide C^H^O^N d 248-249 44
1. flavum. L. r b erb erin e 45, 46
1 . foetidum ag 47 f e tid in e C14H50°8N2 m 132=135 +121.4, tl5, (EtOAc) M, ( c 2 .57)
ag fetidine* 2HCL d 228-230 -30.9, t20 47
ag fetidine* 2HN0^ d 200 47
ag fetidine* 2HBr m 225-230 47
ag fetidine sulfate d 215-218 47
fetidine* 2CHgI m 215-210 47 TABLE 1 (cont'd)
1 . foliolosum rh berberine I C^H-^gO^NI m 260 15
rh thalictrine C^H^OnN m 208 49 (m agnoflorine (Me0H/Et20 Pet/Et^O
rh th a lic tr in e . 3H20 C^H^O^N* 3H20 +308, t25 (1$ aq. sol.) 15
rh berberine chloride 48
rh tetrahydroanhydro- m 173 ^ berberine
rh berberine-acetone d 168 48 compound
rh tetrahydroanhydro- 021^25^4^ m 143 48 palm atine (MeOH)
rh tetrahydroanhydro- m 206-207 48 jatrorrhizine
rh jatrorrhizine iodide C^H^O^NI m 212 48
1. hemandezii r hernandezine c ^aHwjP 7N? m 192-193 +250, t 20* 50 (hexane) C, (c,0.2) m 157-158 (MeOH) m 122-124 fae2C0) 00 m 158-159 (Et^O) TABLE 1 (cont'd)
JJ. iso p y ro ld e s wp talisopine C9,H?£;Oi,N m 151-153 -104.9°, t20 5 1 , 52 ^ ° (H20:MeOH^1:3) (Me2C0) - 71. 0 2 , t20 C
■wp thalicopinine 51, 52 C21H25°5N wp talisopine HI m 235-237 51, 52
.£. macrocarpum r thalictrine-A 38, 39, 40
To minus t thalm ine C^H.^O^N m 253 53, 55 (EtOH-CHClo) t thalmine HCL d 147-15? J 53 (EtOH) t thalmine CH^I d 250 53 (EtOH) t thalmine HCIO^ d 238-241 53
t thalmidine CnH«-0,.N d 192-193 +252.2 C 53 1 5 (EtOH) t th alm id in e CH^I d 254-255 53
r thalicmine C9,H 9 ,0,N m 137-138 53, 5^, 60 5 (MeOH) r thalicmine HCL m 268-270 +255.3 (EtOH) 53
r th alicm in e HI d 223-224 53 (sealed tube) r thalicmine HBr m 258-260 53 (H pO ) thalicmine CH^I m 238-237 53 $ (sealed tube) TABLE 1 (cont'd)
thalicmine m 191-192 o p tica lly 53 (acetylated) inactive
thalicmidine -84° C20H25V m 192-193 53, 54, 56, (EtOH) (EtOH) 57, 58
thalicmidine d 239-240 53 tartrate (sealed tube)
thalicm idine* HI d 222-226 53 (sealed tube)
thalicmidine•CH^I m 217-217.5 53 (EtOH)
unknown orange base m 243 53
wp thalictrim ine ^20^23^4^ m 169.5-170 63 (alc.CHCl^)
wp thalictrimine sulfate m 208-210 63
•wp thalictrimine HC1 d 177-179 63
wp thalictrim ine CH^I m 182-183 63
wp thalictrimine NO, d 178-179 63 1 . minus var. elatum wp elatrin e ^+0H56°6N3 d 180-183 +228.94 (EtOH) 18 wp elatrine picrate d 174-175 18 ^ TABLE 1 (cont’d)
1 . minus v&r. ela-tqm wp elatrine picrolonate d 178-182 18
wp unknown phenolic base 18
ag thalmelatine chnHiiA0aN9 m 131-135 +110°, t2L 2?, 28 (EtOH) (c.l, EtOH) m 120-123 (Abs. EtOH)
ag thalicarpine C4lH48°8N2 2^f
1 . pedunculatum wp berbamine m 157 64 var. Edeew. wp berberine C20H19°5N m 64
1 . D.C. r magnoflorine d 256-258 65
r jatrorrhizine 65
r berberine 66
1. simplex ag thalictrinine C^H^OhN d 169-170 -65.0°, t20 69 ° C, (c 3.08
thalictrinine C^gH^O^Ng d 169-170 80.9°, t=?, C. 68
X. tjiiatogU r magnoflorine picrate d 230-231 70, 71
r magnoflorine iodide C^Ho^OkNI d 252 +214° M, (c 0.254) 70, 71 H r 0,0-dimethyl- d 243-244 +165 (7.8 mg./ 70, 71 magnoflorine® J® 5 ml. CH^OI TABLE 1 (cont’d)
1. thunbergii r 0,0-dimethyl- d 237-238 70,71 magnoflorine*5 Cl
r th ali cthube rine C21H23°4N m 126-12? 24 r th a lic r in e m 221-222 C36H38°6N2 +341.2°, t23 78, 79, 80, 84
r homothalicrine d 235-236 +425.3°, t21 C37H4o°6N2 78, 79, 80 r magnoflorine C20R27°4NI d 252 70, 71, 81 iod id e
r thalicberine m 161 C37H4o°6N2 75, 76, 73, 74, 77 , 82, 83
r 0-m ethyl- C38H42°6N2 d 186-187 72, 73, 74, thalicberine 76, 77, 82, 83 1 takatonine C21H2if0l l NI m 192-193 72 iodide (MeOH)
1 1 »2,3,^-tetrahydro- m 192-193 72 takatonine HC1 (EtOH- Pet Et20)
1 1*2,3,^tetrahydro- m 184-185 72 takatonine HBr TABLE 1 (cont'd)
I. thunbereii 1 1 , 2 , 3 ,^-tetrahydro- m 141.5-1^3 takatonine picrate
(a) r, roots; ag, above ground; rh, rhizomes; wp, whole plant; t, tops; 1 , le a v e s |
(b) t, temperature in °C; c, concentration (o/o); M, CH^OH; C, CHCl^ 34
•CH- OCR ICR OCR
B erb erin e Tetrahydroberberine
HO H CO
OCR CH. OCR •CH.
Jatrorrhizine P alm atin e
FIGURE 3
The Protoberberine Alkaloids of S p ec ie s.
■CH. HO CH: HO I-CR HO
H„CO * 0CH3 Magnoflorine Thalicmidine OCH
I-CH.
HoC0 OCH3 T halicm ine
FIGURE 4
The Aporphine Alkaloids of Thalictrum Species. 35
.OCH. l-CH 3 och' f-CH.
iCH. OR
Hernandezine O-methyl thalicberine (R=CH-^)
.OCH. CH~
RO OCH.
T h a lic rin e = arom oline (R=H) H om othalicrine (R^CH^) Berbamine
OCH. OCH. I-CH.H^C-
0 'CH.
F e tid in e
FIGURE 5
Bisbenzylisoquinoline Alkaloids of Thalictrum Specie s.
(a) Position of Oxygen linkage not fully determined. It may be at 2* o r 6 » position. 36
OCH OR f-CH. 0 JOCH 'CH. T h a lm e la tin e (R=H) T h a lic a rp in e (R^CH^)
OH
CH.
Thalicthuberine Thalm ine
-CH.
T ak ato n in e
FIGURE 6
Aporphine-benzylisoquinoline and Other types of Alkaloids of Thalictrum Species. I H . EXPERIMENTAL
A. M a teria ls
The plant material used in this investigation was the roots of
Thalictrum Rochebrunianum, Franc, and Sav. (Ranunculaceae), which was
cultivated and supplied by the Wayside Gardens Company of Mentor,
Ohio. Upon arrival in this laboratory, the roots were washed, dried, and ground to a fine particle size by means of a Wiley m ill.
B. Preliminary Investigations
In order to solve a problem, be it of phytochemical or any other nature, it is necessary to conduct preliminary studies to obtain in formation that can help the investigator to determine the methodology
or approach best suited to solve his particular problem. With this in mind, certain preliminary phytochemical studies of T. Rochebrunianum were conducted.
1. Quantitative estimation of total alkaloid content
Twenty grams of the finely ground roots of T. Rochebrunianum was placed in a 250 ml. round bottom flask. A volume of 150 ml. of U.S.P.
alcohol was added and the mixture refluxed for two hours. The content
of the flask was allowed to cool to room temperature, filtered, and
37 38 the filtrate was then evaporated to dryness in vacuo. To the residue was added 50 m l. o f fiv e p e r c e n t ammonium hydroxide and 50 ml. of chloroform. The mixture was stirred to dissolve as much of the resi due as possible in the two solvents. The content of the flask was then transferred to a separatory funnel and allowed to separate into two layers. The chloroform layer was collected and the alkaline mother liquor was extracted twice more with 25-ml. portions of chloroform.
The chloroform extractives were combined, dried over anhydrous sodium sulfate and filtered. The filtrate thus obtained was concentrated to a small volume and extracted with five m illiliters of five per cent hydrochloric acid. This aqueous acid extractive was designated as the total tertiary alkaloid fraction. The alkaline mother liquor, devoid of tertiary alkaloids, was acidified with hydrochloric acid and fil tered. The filtrate was adjusted to 50 ml. with two per cent hydro chloric acid and designated as the quaternary alkaloid fraction. Two m illiliters each of the tertiary and quaternary alkaloid fractions were placed in separate 10-m l.centrifuge tubes, and the volumes made up to five m illiliters each. Three drops of Valser’s reagent were added to each of the tubes, and the resulting precipitates centrifuged.
The heights of the precipitates in these tubes were then compared to the heights of reference alkaloid standards for the quantitative estimations. It was found that both the tertiary and quaternary alka loid fractions have alkaloid contents greater than 0 . 10$.
Brucine and berberine chloride were employed as the standards for the tertiary and quaternary alkaloid fractions, respectively.
The concentrations of these reference alkaloids and the interpretation 39 of the test results are as follows:
(a), ’’Tertiary" alkaloids - brucine, prepared in two per cent hydrochloric acid in the following concentrations:
0.4 mg./ml. = +1
1.3 mg./ml. = +2
4.0 mg./ml. = +3
(b). Quaternary ammonium alkaloids - berberine, prepared in two per cent hydrochloric acid in the following concentrations:
0.05 m g ./m l.= +1
0.17 mg./ml.= +2
0.50 mg./ml.= +3
(c). Interpretation of the results:
+1 indicates 10 kg. of dried plant w ill yield approximately one gram of alkaloid or 0.01$.
+2 indicates that 3 kg. of dried plant w ill yield approxi mately one gram of alkaloid or 0 . 033$«
+3 indicates that 1 kg. of dried plant w ill yield approxi mately one gram of alkaloid or 0.10$.
+4 indicates an alkaloid content greater than 0.10$.
2. Extraction of alkaloids for chromatographic studies
One and one-half kilograms of the dried, powdered roots were defatted with petroleum ether (B .P . 40-75° C.). The extract being found to be alkaloid positive, the petroleum ether fracton was designated as the "P_E" alkaloids. The marc was dried and repacked into the same Soxhlet type extraction apparatus and continously ex tracted with ethanol, fresh solvent being exchanged for the alcoholic extracts every two to three days. These extracts (alkaloid positive) were combined, designated as the "ET" fraction, and concentrated to a syrupy consistency. Next, the alkaloids in this fraction were ex tracted into a two per cent hydrochloric acid solution by mixing the
ET concentrate with the aqueous acid, followed by steam distillation, under reduced pressure, to remove the alcohol in the mixture. After cooling to room temperature, the resultant aqueous acid solution of the alkaloids was filterd in vacuo. The residue obtained was re dissolved in a small volume of alcohol and again extracted into two per cent hydrochloric acid, followed by cooling and filtration to obtain additional quantities of the ET alkaloids. This process was repeated several times until the residue no longer yielded alkaloidal material. The acidic filtrates were combined and concentrated to a workable volume, which was than made alkaline by the addition of ammonium h y d ro x id e (2 8 $ ). T his a lk a lin e s o lu tio n , along w ith i t s precipitates, was placed in a liquid-liquid extractor and continously extracted with methylene chloride. The methylene chloride extractives were combined, dried over anhydrous sodium sulfate, and evaporated to dryness in vacuo yielding 31.3 gm. of alkaloids as the total tertiary bases (T-T).
The aqueous mother liquor was then acidified and the non-alkaloidal precipitate collected was discarded. To the filtrate, containing the q u a te rn a ry a lk a l o id s , was slo w ly added a two p e r c e n t ammonium r e in - eckate solution (prepared in two per cent hydrochloric acid) with mechanical stirring until precipitation no lorger occurred. This mix ture was placed in the refrigerator overnight and filtered. Additional reineckate solution was added to the filtrate and a second crop of precipitate was collected. The precipitates were combined and washed first with small portions of water, then ether, and finally dried in a desiccator. The dried m aterial, weighing 23.662 gm. was next dissolved i n 500 ml. of acetone, filtered, and the non-alkaloidal residue dis carded. To the filtrate, a 0.06$ silver sulfate solution (1,355 ml.) was added slowly with mechanical stirring to completely precipitate the excess reineckate and convert the alkaloid reineckate to the sulfate.
The precipitates were collected on a filter and washed with five per cent acetone in water. An amount of barium chloride ( 623.3 ml. of a
1 . 0257$ aqueous solution) equivalent to the quantity of silver sulfate used was added to the filtrate to completely precipitate any excess silver ion as silver chloride and to convert the alkaloid sulfates to chlorides. The mixture was filtered with the aid of siliceous earth.
The residue was discarded and the filtrate was freed from acetone by means of vacuum distillation at 40° C. The aqueous solution thus obtained was again filtered and lyophilized, yielding 1 2 . 16? gm. o f quaternary alkaloid chlorides (Qu~l). The method just described was modified after Panouse ( 87 ) and Kapfhammer ( 88 ).
3. Thin-layer chromatographic studies of the P-E, T-T and Qu-1 alkaloid fractions
One-and two-dimensional thin-layer chromatography^ were employed to determine the number of alkaloids in the P-E, T-T, and Qu-1 fractions prepared above. The thin-layer plates were prepared with silica gel G
Thin-layer chromatography w ill henceforth be designated as "T-L-C." 42 by the method of Stahl (89).
For the one-dimensional chromatogram, 80 pg. of the P-E and 40 pg. of the T-T alkaloids were spotted 20 mm. apart on a 50 n®1* X 200 mm. silica gel G plate. After development in methanolj 28$ ammonium hydro x id e (99:1). the plate was sprayed with a modified Dragendorff's spray reagent. Alkaloid positive spots were found at R^. 0.72, 0.65, 0.49,
0.40, 0.33 and at the origin for both of these alkaloid fractions. In the case of the P-E alkaloids, an additional alkaloid positive spot at
0.26 was found. Two-dimensional chromatograms of these two fractions on 200 mm. X 200 mm. silica gel G plates were developed in chloroform: m ethanol (95*5) in the first direction and methanol: 28$ ammonium hydro xide (99:1) in the second direction. Again, it was found that the P-E alkaloid fraction contained a greater number of Dragendorff's reagent positive spots that the T-T fraction (Figures 7 and 8).
The quaternary alkaloid fraction, Qu-1, was shown to contain six alkaloids on one-dimensional silica gel G T-L-C plates developed in
^-butanol; acetic acid: water (4;l;l). Twenty micrograms of this alkaloidal fraction was spotted along with five micrograms each of berberine chloride, magnoflorine chloride, and jatrorrhizine chloride
(known quaternary alkaloids from Thalictrum species), 10 mm. apart on a 50 X 200 mm. plate. The Qu-1 alkaloid spots were detected by Dragendorff's spray reagent at R^»s 0.80, O.6 7 , 0.35. 0-30. 0.25 and 0 .1 3 .
The R^ 0.35 spot was identical to the reference berberine spot, in Rj. value, visible yellow color, and intense yellow fluorescence under u lt r a v io le t l i g h t . The R^. 0.30 spot was identified as ------wodmesoa Thi lyr hoaorm - Al oi (0 pg) (200 s id lo a lk A P-E f o Chromatogram -layer in h T ensional o-dim Tw Chloroform:methanol (95:5) — i i ndi e a ti aedrf i and n tio c a e r ragendorff D e iv it s o p a te a ic d in s e n i l lid o S he or i ty b; , ght +1 aeae +, nt. t in a f . , n + ta T, and average; e; 1, + lu b t t; h h ig ig r r b b BB, 2, + red; by; R, y it s n e t in r lo o c e th epnig ors b t y os C, ra; , ue; lu cor b e B, th and cream; ts o Cr, sp t n bols; e c sym s e r e o s e flu th e by th s r te lo a o c ic d in s e responding n i l Broken tao:amnu hdoie 8 (9;l) l ; (99 ) 28$ ( hydroxide ammonium ethanol: M Cr f » — — ' > "yf on Silica gel gel on Silica $ FIGURE 7 Q ______& 'Cr ( * s I r C O * / 1 *BB
^ x
~Cr~J 43 Methanol: ammonium hydroxide (28/6) (99 = 1)
FIGURE 8
Two-dimensional Thin-layer Chromatogram of T-T Alkaloids (40 pg) on Siliea Gel G
Solid lines indicate a positive Dragendorff reaction and the color intensity by: +2, bright; +1, average; +, faint. Broken lines indicate the fluorescent spots and the corresponding colors by these symbols: Cr, cream; B, blue; R, red; BB, bright blue. 45 jatrorrhizine by virtue of its value, light yellow fluorescence under ultraviolet light, and reddish-orange color when exposed to ammonia vapor. The sp ot a t 0.13 was tentatively identified as magnoflorine on the basis of the R^. value and bright, blue fluores cence under the ultraviolet light.
4. Methylene chloride extraction for berberine
The yellow fluorescent alkaloid, which was consistently ob served at the origin on the thin-layer chromatograms of the total tertiary alkaloid fraction, was found to be berberine by thin- layer charomatography with n-propanol: ammonium hydroxide: water
(2:1:1) as the developer. This observation is not surprising since berberine can readily form a pseudobase in the presence of alkali (31).
In an effort to eliminate this alkaloid from the T-T fraction, an experiment was conducted on the advice of a fellow graduate student,
■who had previously encountered the same problem and succeeded in removing berberine from an acid solution by extraction with methylene c h lo r id e .
A sample of the ground roots was defatted, extracted with ethanol and the alkaloids converted to the chloride salts as described under
”2. Extraction of alkaloids for chromatographic studies," pages 39-41.
The aqueous acid solution containing the alkaloids was then extracted with methylene chloride in a liquid-liquid extractor. After complete extraction (when the methylene chloride solution spotted on a filter paper no longer showed an orange spot when sprayed with Dragendorff’s reagent) the methylene chloride solubles were concentrated and subjected k6 to thin-layer chromatographic studies.
Contrary to the proposition that only berberine chloride would be soluble in methylene chloride, both one- and two-dimensional T-L-C studies showed that tertiary alkaloids were also present in the ex tractive (Figure 9).
5. Fractionation of the tertiary alkaloids into the phenolic (TP) and non-phenolie (TN) fractions
The tertiary alkaloid fraction, T-T, weighing 21.63 gm. was dissolved in 300 ml. of chloroform and placed in a separatory funnel.
Ten 150-ml. portions of five per cent sodium hydroxide solution (w/v) were used to completely extract the phenolic alkaloids from the chloro form solution. To the alkaline aqueous extract carbon dioxide gas was introduced to lower the pH from approximately 11. 5 to approximately
8.0, using Hydrion paper as an indicator. The precipitate formed at the lower pH was completely extracted with chloroform. The chloroform solution was dried over anhydrous sodium sulfate, filtered, and con centrated ifl vacuo at *K)° C to a powdery mass (3.02 gm.). T his material was designated as the phenolic tertiary alkaloid (TP) fraction.
The chloroform mother liquor remaining from the aqueous sodium hydroxide extractives was dried with anhydrous sodium sulfate, and
subsequently taken to dryness in vacuo at 40° C. A fluffy powder,
11.51 gm., designated as the non-phenolic tertiary (TN) fraction, was
o b tain ed .
One-dimensional T-L-C of the phenolic alkaloids on silica gel G
and developed in methanol: ammonium hydroxide (99 si)* gave two Methanol:ammonium hydroxide (28$) (99:1) a (b) (a) ______n-aye Crmtgas t Mehln Choie r tve on e ctiv tra x E hloride C ethylene M e th f o Chromatograms er y -la in h T Cr Cr , e; B bri bl ; n T tn; , oe l t. n e c s e r o flu none cor N, the ; and tan ts T, o sp and t n e; e c lu s b e r o t h flu ig r b e th BB, te a red; ic d R, in s e in l Broken i ndi e a ti aedrf i and n tio c a e r ragendorff D e iv it s by: o p y a it s n e te t a in ic d r in lo o c s e n i the l lid o S epnig ors b t e smbl: r cem B bl e; lu b B, cream; Cr, bols: sym se e th by s r lo o c responding a On-i ninl () w-i ensional Two-dim (b) ensional; ne-dim O (a) Y-Cr o o & Y-Cr 2 ^ e G at s te la P G Gel a c i l i S ehnlamnu hdoie 2$ H^9:T7 120$) hydroxide Methanol:ammonium FIGURE 9 +2, ght +1 aeae +, nt. t in a f , + average; 1, + t; h ig r b ______/Cr ,LB
k7 48
Dragendorff positive spots plus one- at the origin, while that of the non-phenolic alkaloids revealed five positive spots in addition to one at the origin (Figure 10). Two-dimensional chromatograms spotted with
160 pig. each of these alkaloid fractions and developed in the usual s o lv e n t system o f ch lo ro fo rm : m ethanol and m ethanol: ammonium hydroxide showed that the TN fraction contained five alkaloids plus one at the origin (Figure 11), while the TP fraction had four different alkaloids in addition to the positive spot at the origin (Figure 12).
6 . Pharmacological screening of the TN, TP, and Qu-1 alkaloid fractions for hypotensive activity^
A female dog weighing 8 kg. was employed as the test animal for this experiment. It was anesthetized with pentobarbital sodium (35 mg./kg.), and a tracheal cannula was inserted to insure free respiration.
Both common carotid arteries and both vagi were isolated, and a femoral vein was cannulated for injections. The left common carotid artery was then cannulated and connected by means of rubber tubing to a mercury manometer. The tubing and the arm of the manometer were filled with
10$ sodium citrate. The blood pressure (B.P.) was recorded by means of a writing pen and kymograph. The alkaloid fractions were then evaluated, the results being summarized in Table 2.
^ This experiment was performed by Dr. P. N. P atil, Post Doctoral Fellow, The Ohio State University, College of Pharmacy. Methanol:ammonium hydroxide (28$) (99:1) ^ ^ ______+1 edmesoa Thi l r hoaorm T ad P ato on s raction F TP and TN f o Chromatogram er y -la in h T ensional ne-dim O a y . - \ r [Cr \ Y-Cr Y-Cr |Cr i i ndi e a ti rgnof reacton and n tio c a e r Dragendorff e iv it s o p a te a ic d in s e lin lid o S kal ds ( T al oi (f) Bnee ol e P kal ds; id lo a lk a TP le b lu so Benzene ) f ( ; s id lo a t. lk a TP rescen o ) -flu (e n o n cox- s; N, id the and lo a and lk tan a ts o T, sp t e; n e lu c b s e r t o h ig r flu b BB, the te red; a ic R, d in s e lin Broken h col ntensi y +2 brght +1, vrg; , nt. t e; lu in b a f B, +, cream; Cr, average; , 1 symbols: + t; h ese th rig b by 2, s + r lo o by: c y it s n responding e t in r lo o c the c) ezn s ubl N kal ds; d Bnee nsol e TN le b lu o s in Benzene (d) ; s id lo a lk a TN le b lu so Benzene ) (c g Bnee nsol e T al oi . s id lo a lk a TP le b lu o s in Benzene (g) a Toa terti kal d f i () N kal ds; id lo a lk a TN (b) ; n tio c a fr id lo a lk a y r ia t r e t otal T (a) .Cr !LB Cr Y-Cr c ILB ’ Cr e G Gel a c i l i S FIGURE 10 rigin O ______+2 +2* r\ ’Cr Y-T e +2' 2 i+ JCr Y-T Y-T \
Cr
49
______w-i ninl hoaorm T al oi (6 jg. on .) jig (160 s id lo a lk a TN f o Chromatogram ensional Two-dim Chlo rofo rm:methanol (95:5) low. w ello y , e; B bri bl ; , a; B l le n Y, and blue t h g li LB, tan; T, e; lu b t h ig r b BB, red; R, he or i ty b: 2, ght + aeae +, nt. t in a f , + average; , +1 t; h ig r b , +2 by: y it s n e t in r lo o c e th i nes i cat posi ve Drgnof reacton and n tio c a e r ragendorff D e iv it s o p a te a ic d in s e in l lid o S epnig ors b t s smos C, ra; , ue; lu cor b B, the and cream; ts o Cr, sp t n e symbols: c s e r o ese flu th by the s r te lo a o c ic d in s responding e lin Broken Cr tao:moim yrxd (8) 99: ) :1 9 (9 (28$) hydroxide ethanol:ammonium M li e G Gel a ic il S FIGURE 11 ______\ ( Cr' ©
/ 1 ‘ /'•- ✓ T3&
50 r ,T &;» Methanol:ammonium hydroxide (28$) (99:1) BS'"' FIGURE 12
Two-dimensional Chromatogram of TP Alkaloids (160 jig.) on Silica ______Gel_ G. T h in -layer P la te ......
Solid lines indicate a positive Dragendorff reaction and the color intensity by: +2, bright; +1, average; +, faint. Broken lines indicate the fluorescent spots and the cor~ responding colors by these symbols; Cr, cream; B„ blue; R, red; BB, b r ig h t b lu e; T, tan ; and LB, li g h t b lu e. 52
TABLE 2
Hypotensive Activity of the Alkaloid Fractions Tested
Alkaloid fraction. Hasasa . Hypotensive Activity ______
TN 2 mg./kg. Maximum drop in B.P. of 60 mm. mercury •with a duration of 50 minutes.
TP 2 mg./kg. Transient drop in B.P. for 2.5 minutes.
Qu-1 2 mg./kg. Transient drop in B.P. for 2.5 minutes.
From these results, it would appear that the major hypotensive agent(s) belonged to the non-phenolic, tertiary class of alkaloids.
7. Solubility of TN and TP alkaloids in chloroform and benzene
In the course of preparing alkaloid solutions for chromatographic studies, chloroform and benzene were chosen as the vehicles for the TN and TP fractions. In the case of chloroform, materials from both the
TN and TP fractions were completely soluble. They were, however, in completely soluble in benzene. Whereas 20 mg. of each of these alka- loidal materials was completely soluble in one m illiliter of chloroform, they were found to be only sparingly soluble in five m illiliter portions of benzene. Even vigorous shaking, combined with heating on a steam bath failed to render these alkaloid materials soluble in benzene. In view of this, it was felt that there might be benzene soluble and benzene insoluble alkaloids in each fraction. Consequently, these suspensions were filtered and the benzene insolubles were dissolved in chloroform. These chloroform solutions were designated as "Benzene insoluble TN" and "Benzene insoluble TP" alkaloids, while the benzene filtrates were designated as "Benzene soluble TN" and "Benzene soluble
TP" alkaloids. All four solutions were spotted on a silica gel G plate, by means of cappillary tubes drawn to fine points, and developed in methanol: ammonium hydroxide (99:1). The results of this study are shown in Figure 10 (c, d, f, and g). It appeared that only one alka loid, aside from the Dragendorff positive origin, in each of these fractions was not completely soluble in benzene. Therefore, benzene could effectively be used to exclude some of the chloroform soluble non-alkaloidal materials from the tertiary alkaloid fractions. With this in mind, the available TN and TP alkaloids were further fraction ated into the "Benzene soluble" and Benzene insoluble" fractions. In each case, the particular alkaloid fraction (TN or TP) was reduced to a fine powder using mortar and pestle and extracted with benzene by trituration. The mixture was filtered and the benzene insolubles were triturated again with benzene and filtered. This procedure was repeated several additional times, or until the benzene filtrate failed to give a positive Dragendorff’s spray test, when spotted on a piece of filter paper. The benzene filtrates were then combined and taken to dryness ill vacuo at 40° C.
From these data, it is apparent that chloroform is a relatively non-selective solvent for the tertiary alkaloids of .J. Rochebrunianum and that benzene or ether should be employed in future extraction procedures for these alkaloids. 54
8. Chromatography of the TN and the TP alkaloids on multibuffe red paper
Like all bases, alkaloids can theoretically form salts with acids at defined pH levels depending on their pKf s. If these pH levels can be determined for a group of related alkaloids, then one can separate the individual alkaloids by extracting the bases, dissolved in an organic solvent, with aqueous solutions buffered at predetermined pH* s in a decreasing order.
Schmall £& al. (90) devised such a chromatographic procedure for the separation of some opium alkaloids on multibuffered paper. It seemed worthwhile to attempt the separation of the tertiary alkaloids of Rochebrunianum. according to this method, with the aim of deter mining the pH levels at which individual alkaloids w ill form salts, and ultimately separate them with buffered solutions. Since it is known that the Papaygf (opium), as well as the Thalictrum alkaloids, are benzylisoquinoline derivatives, the chance of success in separating the tertiary alkaloids of .J. Rochebrunianum by this method appeared better than average. Therefore, a number of experiments were conducted in an attempt to separate the TN alkaloids by multibuffered paper chromatography.
Whatman No. 1 paper strips were buffered with Mcllvain buffers
(double strength) (91) at two centimeter intervals in decreasing order, with the highest pH buffer being applied three centimeters from the area where the alkaloids were to be spotted. The papers were buffered with zones of ph 7.0, 6.8, 6.6, 6.4, 6.2, and 6.0; pH 6.2, 5.8, 5.6,
5.4, 5-2, and 5.0; pH 5-2, 4.8, 4.6, 4.4, 4.2, and 4.0; pH 4.2, 3.8 y
55
3*6, 3.4, 3.2 and 3.0; and pH 3.2, 2.8, 2.6, 2.4, 2.2, and an unbuf fered zone. TN and TP alkaloids in varying quantities from 40-500 jig. were applied to these chromatograms and developed by both descending and ascending procedures, employing either chlorofonn or benzene as the mobile phase in the presence of water vapor. After development, the paper strips were dried, viewed under U.V. ligh t, and sprayed with
Dragendorff’s reagent.
In almost a ll cases, the pH zones above 4.0 were alkaloid nega tive, while every one of those below 4.0 was found to be positive.
It was even observed that on some paper strips where the solvent front extended beyond the last buffered zone, Dragendorff positive areas were encountered. Because of these adverse results, this method did not appear to be applicable to the separation of these a lk a lo id s .
9. Gradient pH separation of the TN and TP alkaloids
A gradient pH technique for the separation of alkaloids from semi-purified fractions has been described by Svoboda (92), who has utilized it in the isolation of a number of Catharanthus alkaloids.
The theory and methodology of this technique are the reverse of that of the multi-buffered paper chromatography. In this case, the alkaloids are in their salt forms in a citric acid solution. Separation is ef fected by the dropwise addition of a dilute ammonium hydroxide solution
(10$) to elevate the pH of the alkaloid-citric acid solution to an appropriate level, where one or more alkaloids is (or are) converted to the base form, while others remain in solution as the salts. Then, by virtue of having a greater distribution coefficient in organic solvents,
the alkaloid base(s) in question can be removed by extraction of the
alkaloid-citric acid solution with organic solvents.
Even though the Thalictrum alkaloids are derived from the benzylisoquinolines and the Catharanthus alkaloids are of the indole
type, it was decided to employ this gradient pH separation technique
to the separation of the alkaloids of X- Rochebrunianum. Therefore,
experiments were carried out to determine the applicability of this method for the separation of TN and TP alkaloids.
a. XU alkaloids
A one-half gram sample of the "Benzene soluble" TN alkaloid fraction was completely dissolved in 50 ml* of benzene and 50 ml*
of 0.1M citric acid solution was added, followed by the removal of
the organic solvent at 40° C under reduced pressure. Any insoluble material was then removed by filtration. The aqueous acid filtrate was then extracted at the resulting pH (2.10) with two 50~ml« portions
of benzene. The pH of the aqueous phase was then adjusted with 10$
ammonium hydroxide solution (dropwise) to pH 3*00, 4.00, 5*00, 6.70
and 9. 50. two 50-ml. benzene extractions being made at each level.
The benzene extractives from the various pH levels were dried over
anhydrous sodium sulfate and concentrated to approximately three m illiliters each.
A thin-layer chromatogram on silica gel G and developed in methanol: ammonium hydroxide (99:1). was made of these extractives
(Figure 13). An inspection of this chromatogram revealed that 57
pH 2.1 3.0 4.0 5.0 6 .7 9 .5
f t
O n O n oo n (B CM 0) T3 ■H Cr O G © ^ C r - T e i R I f / •H \/ G o * Y-Cr o r\ ctiG ,C { / Cr -(->© /\ 33 ' y / | Y < JY t v 1 O rigin
FIGURE 13
One-dimensional Thin-layer Chromatogram of TN Alkaloids from Gradient ------Bfl Separation on Silica Gel G Plates ______
Solid lines indicate a positive. Dragendorff reaction and the color intensity by: +2, bright; +1, average; +, faint. Broken lines indicate the fluorescent spots and the cor responding colors by these symbols: Cr, cream; B, blue; R, red; T, tan; and Y, yellow. separation indeed had been achieved, although there was some overlap of alkaloids through the different pH levels. These overlaps could perhaps be reduced with some modifications in the number of pH units between increments, and an increase in the number of extractions with benzene at each of these pH levels. b. TP alkaloids
A one-half gram sample of the "Benzene soluble11 phenolic tertiary,
TP, alkaloids was treated as the TN alkaloids for gradient pH separa tion. Extractions were made at pH 2.15, 3-30, **.**0, 5*25, 6.75, and
9.10. Unlike the TN alkaloids, separation was not achieved.
It appeared, from these results, that the technique of gradient pH extraction as described by Svoboda, is applicable to the separation of the TN alkaloids, but not of the TP alkaloids.
10. Preliminary column chromatographic separation of the quaternary alkaloids (Qu-l) on neutral alumina
Ten grams of the quaternary alkaloid chlorides (Qu-l) realized from 1.5 kg. of ground T. Rochebrunianum roots was dissolved in a small volume of methanol and adsorbed on 50 gm. of Woelm ®, neutral, activity grade H alumina. After drying, the lumpy mixture was triturated in a glass mortar. The finely ground powder was then sifted through a 100 mesh wire screen to remove lumps and the resulting powder was then added to the top of a previously packed column.
The packing was accomplished by sifting 500 gm. of Woelm®, neutral, activity grade II alumina into a column containing benzene. 59
The column in question had an internal diameter of 3* 20 cm. and an adjustable outlet.
Solvents of increasing polarity were employed for the elution of the alkaloids. One liter fractions of the eluate were collected and examined by descending paper chromatography on Whatman No. 1 paper using a developer of n-propanol: ammonium hydroxide (28$): water
(2:1:1). The developed chromatograms were treated with Dragendorff*s spray reagent in the usual manner, and the values of the alkaloids noted. The individual eluates were then combined into larger fractions according to the R^ values of the alkaloid(s) in each. Paper chroma tographic results of these fractions are summarized in Table 3>
The presence of jatrorrhizine in Fractions 7 and 8 was evidenced by a sim ilarity of the R^. values and the type fluorescence, when com pared with a known sample, in addition to the color change of these spots from visible yellow to red on exposure to ammonia vapor. The isolation of this alkaloid, however, was not accomplished due to its low concentration in these fractions.
The bright blue fluorescent alkaloid at R^ 0.48-0.50 in fractions
6-9 was identified as magnoflorine by its R^ value and fluorescence.
The final proof of its identity was accomplished when 300 mg. of this alkaloid was isolated as the crystalline chloride salt from methanol, and its physical data were found to be identical with those of an authentic sample.
The highest R^ value (0.93) alkaloid was probably a tertiary alkaloid left in the quaternary fraction from incomplete removal by chloroform extraction prior to precipitating the aqueous mother liquor 60
TABLE 3
Paper Chromatographic Results of the Column Chromatographic Fractions of the Quaternary Alkaloids
Fractions Solvent Systems Values^ Fluorescences'm (b) '
1 . Benzene 200 ml. (“) blue
2. Benzene: CHC1- (1:1) 1,000 ml. 0 .9 3 blue CHCl^ (100$ 1*000 ml.
3. CHC1-:CH~0H(97:3) 2,000 ml. 0 .9 3 blue CHC1^:CH^0H(95.5) 2,000 m l.(1 s t)
CHCl^:CH^OH(95:5) M 0 0 ml. ( 2nd) 0 .5 0 bright blue
5. CHClyCH^OH (90:10) 3,000 m l.(lst) 0 .7 7 none
6 . CHCl^:CH^OH (90:10) 6,000 ml. (2nd) 0 .4 9 bright blue 0 .5 1 l ig h t blue
7. CHCl^:CH^0H(80:20) 8,000 m l.(1 s t) 0 .4 8 bright blue 0 .6 2 ta n -y ello w
8 . CHC1o:CH_0H(80:20) 2,000 m l.(2nd) 0.48 bright blue CHC1^:CH'0H(50:50) 5,000 ml. 0.60 tan-yellow
9. CH^OH (100# 4,000 ml. 0 .5 0 bright blue
Jatrorrhizine Chloride 0.60 tan-yellow
Magnoflorine Chloride 0 .5 0 bright blue
(a)TheRj, values were calculated from the Dragendorff * s reagent stained spots: (b) Viewed under U.V. prior to staining with Dragendorff’s reagen t. 61
■with ammonium reineckate. This assumption was made on the basis of its being eluted from the alumina column with benzene and the ease with which it migrated to the solvent front in the chromatograms.
Although separation was accomplished to some degree, it was not completely satisfactory because only four alkaloids were recovered, whereas in another experiment, six alkaloids were shown to be present in the total extractive (see pageh2). Another dissappointing feature of the separation was found in jatrorrhizine being eluted along with magnoflorine. Consequently, an attempt was made to separate the quaternary alkaloids on acid alumina. Unfortunately, separation was not achieved.
C. General Extraction and Fractionation of Thalictrum Ro chebrunianum Root Alkaloids
1. Extraction of total alkaloids
Twenty kilograms of the dried, finely ground root of Thalictrum
Rochebrunianum was extracted with ethanol by a cold "cyclie-percolation" method. In this process, the basic principle of percolation is em ployed. Unlike the usual process, however, the percolate is pumped back into the drug as menstruum for further percolation instead of being collected immediately. Collection is made when the percolate becomes "saturated”, or considerably more concentrated than the in itia l p e r c o la te .
For this purpose, a commercial earthen sanitary crock was converted into a percolator for large scale extraction. The crock had an internal diameter of J6 cm ., a h e ig h t o f k? cm. at the outer perimeter, where 62 it tapered at the bottom to the center at a 30 degree angle and where an opening with a diameter of 4.5 cm. was found. The distance from this opening to the entirely exposed top measured 5 6 cm. A mechanical valve for the regulation of solvent flow was fitted onto the small
center opening in the bottom. Extending from this valve was a copper pipe elbow and a piece of Tygon tubing, which extended to the inlet
o f a liq u id so lv en t pump?. From th e o u t le t o f t h is pump, and extending over the top of the crock, was another piece of Tygon tubing (Figure
1 4 ).
With the valve open, and the pump turned on, the solvent can be
recycled as often as desired. A layer of glass wool was then placed
at the bottom of the apparatus, which was covered with two sheets of
circular filter paper, and a layer of sand. The drug was then intro
duced and the menstruum added.
Since it was determined previously that the defatting petroleum
ether also extracted some of the alkaloids in the plant material, the usual defatting procedure was not followed. Ten gallons of ethanol was
added to the drug in the percolator and the mixture was allowed to
macerate overnight. At the end of this period, the solvent (percolate) was recycled for 24 hours before being collected. The percolate ob
tained was then concentrated to a syrypy consistency in a Precision
Tubular Concentrator . The recovered solvent was re-introduced into
the drug for another ncyclic-percolation” and concentration. This
process was repeated 12 additional times. All the percolates were
7° Cole-Parma Instrum ent and Equipment Company, C hicago, I l l i n o i s . g Precision Scientific Instruments Company, St. Louis, Missouri. y/ 38 cm
-M etal rod (support tubing) Earthen Crock 7 cm
Tygon Tubing
(Valve Metal Elbow 3**way Stopcock re-Pagaa Liquid Draining tube— Pump J
Metal clamp,^-^- " s
Support Rack
FIGURE 14
"Cyclic-percolation" Apparatus concentrated and combined, yielding a total volume of approximately six liters. It was noted at this stage that crystalline material had formed on the sides of the amber bottles in which the total alkaloid extractive was stored. These crystals were collected by vacuum filtration. The crude crystals were further purified by washing several times with ethanol, and the washings were combined with the filtrate and further concentrated for subsequent fractionation. This concentrate was then designated as the "Total Alkaloid Extractive."
The crude crystals obtained were labelled as "20-x" for further inve stigation. a. Compound "20-x"
The "20-x" crude crystals weighed 56-00 » after drying over calcium carbonate in a desiccator. They were re-crystallized three times from methanol to a constant melting point of 186.5-187.5° C, w ith ou t decom p osition , y ie ld 35.00 gm. (6 0 . 350* The compound gave a negative Valser1s test for alkaloids. Its infrared spectrum (in potassium bromide pellet) revealed a broad hydroxyl band at 3,600-
3,100 cm. 1 with the peak being at 3*425 cm.- '*' (Figure 18). Ultra violet spectral analysis of this compound in methanol showed a complete absence of absorption, indicating a saturated molecule.
Analysis: ^2**22®11; Calculated: C, 42-10; H, 6.44; 0, 51.46.
Found: C, 42.66; H, 6.70; 0, 50.64.
Because of these data, the possibility of this compound being a sugar was then investigated. A negative Benedict’s test for reducing sugars was obtained for this compound. However, the hydrochloric acid (10# w/v) hydrolysate gave a positive Benedict’s test, indicating the presence of reducing su gars.
Thin-layer chromatography of the hydrochloric acid hydrolysate of
”20-x” on microcrystalline cellulose (’’Avirin”^) according to the method of Wolfrom jal. (93) showed the presence of two sugars. The values and color spots of these two sugars corresponded to those of reference glucose and fructose, when developed in solvent systems of jl-butanol: acetic acid: water (3:1:1), and j^-butanol: acetic acid: water (4:1:1). These results strongly suggested that ”20-x’’ was su crose.
A comparison of the infrared spectra of known sucrose and the unknown compound gave proof th a t compound "20-x" i s su cro se, as th e se spectra were identical. This was substantiated by the melting point of sucrose, which has been reported as 185° C dec.(94).
2. Fractionation of the total alkaloid extractive into the tertiary non-phenolic (TN), tertiary phenolic (TP), and quaternary (Qu-l) alkaloid fractions a. Total tertiary and quaternary alkaloid fractions
The fractionation of the "Total Alkaloid Extractive" into the tertiary (20-TT) and the quaternary (Qu) alkaloid fractions was
Brand name product obtained from the Avicel Sales Division of American V iscose Company, Marcus Hook, P ennsylvania. 66
accomplished by immiscible solvent extractions.
Two liters of a five per cent hydrochloric acid solution was mixed with the concentrated ethanol extractive containing the total alkaloids, followed by the removal of alcohol at 40° C ia vacuo. The resulting aqueous acid solution was filtered and the filtrate set aside. The residue obtained was re-dissolved in ethanol, mixed with one and owfr* half liters of five per cent hydrochloric acid, and followed by the removal of alcohol and filtration of the resulting aqueous acidic
solution. This procedure was repeated several times until the alka loids were completely extracted into the aqueous acid solution. The aqueous acidic filtrates were then combined to give a total volume of about seven liters. Next, sufficient ice cubes were added to cool the acid extract, followed by the addition of ammonium hydroxide, in 10 m l.- portions with stirring, until the solution was alkaline to litmus paper. The resulting precipitate (formed upon the addition of the alkali) was collected on a large Blichner funnel and dried jjj vacuo.
This material, weighing 403.8 gm., constituted the major part of the crude total tertiary alkaloids (20-TT-S). The aqueous mother liquor was then extracted with chloroform until chloroform soluble alkaloids were no longer obtainable. These extractives were combined, dried over anhydrous sodium sulfate, and taken to dryness in vacuo, yielded 25.20 gm. of a solid material (20-C).
The post (20-C) alkaline mother liquor (approximately 10 liters) was freed from residual chloroform by vacuum evaporation at 40-45° C and then acidified with 10$ hydrochloric acid. This acidic solution was filtered and the non-alkaloidal material on the filter pad was 67 discarded. To the filtrate was added a two per cent ammonium reineckate solution, which precipitated the quaternary alkaloids as alkaloid reineckates. After collection, the alkaloid reineckate
(Qu-A) was placed in a vacuum desiccator for drying.
The crude total tertiary alkaloid fraction, 20-TT-S, was divided into three portions for further purification. Each portion was dis solved in a minimum volume of methanol and combined with one and one- half liters of five per cent hydrochloric acid. The mixture was refluxed for 30 minutes, followed by vacuum evaporation of the methanol and filtration of the aqueous acidic extract. The residue from the filtration was then refluxed with 500 ml. of five per cent hydrochloric acid for two hours, and the mixture subsequently filtered. The residue from this filtration was labelled as "20-R” and set aside. The two aqueous acid filtrates were combined, and boiled with 20 gm. of Hyflo
Supercel for 30 minutes. After cooling, this mixture was filtered and the celite residue obtained was washed with four 250-ml. portions of five per cent hydrochloric acid solution. The washings were combined with the acidic filtrate and extracted with ether.
The ethereal extractives, shown by T-L-C to contain the same alka loids as the aqueous mother liquor, was washed with several portions of five per cent hydrochloric acid. These acid washings were added to the aqueous mother liquor. Ice cubes and lh$ ammonium hydroxide solution were then added to adjust the pH of the combined acid solution to ap proximately 9.0, using Hydrion papers as an indicator.
The alkaloid precipitate formed at this pH was collected by vacuum filtration, and subsequently purified by placing the dried material into 68 a three liter round bottom flask, followed by refluxing with two liter portions of ether. Fresh solvent was exchanged every five to six hours for the extractive until a positive alkaloid test with Dragendorff’s reagent was no longer found. The ethereal extractives were then pooled, dried over anhydrous sodium sulfate, evaporated to dryness in vacuo a t 40° C, and coded as "20-TT-B11.
The post 20-TT-B residue, thought to be devoid of alkaloids, was found to be alkaloid positive with Valser1s test. It was therefore designated as the ether insoluble alkaloid fraction and coded as
"20-D" (12.98 gm.).
The approximately pH 9.0 filtrate was exhaustively extracted with ether in a lighter than water liquid-liquid extractor. The ethereal extractive was afforded the same treatment as the 20-TT-B alkaloids, and la b e lle d as "20-TT-A11. The mother liq u o r , now devoid o f t e r t ia r y alkaloids, still gave a positive Valser1s test. This was attributed to quaternary alkaloids being trapped in the original tertiary alkaloid
(20-TT-S) precipitates on collection. Consequently, this aqueous mother solution was warmed on a hot water bath to evaporate any ether present, and treated with 10$ hydrochloric acid and two per cent ammonium reineckate in the same manner as the post 20-C mother liquor.
Thin-layer chromatographic patterns of the 20-TT-B and the 20-TT-A alkaloids from each of the portions of 20-TT-S were identical to each other, and superimposable with that of the TT alkaloids on Figure 10.
Therefore, they were combined and designated as the semipurified total tertiary alkaloid fraction (20-TT), which weighed 86.30 gm. 69
Thin-layer chromatographic studios of fraction 20-C revealed it to contain alkaloids in very low concentration, as indicated by the inten sity of the Dragendorff positive colored spots, and that they were tertiary alkaloids. To obtain these alkaloids in semipure form, the ether extraction procedure employed to obtain the 20-TT-B alkaloids was applied. From 25.20 gm. of this material, one gram of semipurified total tertiary alkaloids was obtained. This was then added to the
20-TT alkaloids previously obtained, for a total of 87.30 gm. or
0.44$ of the starting root material.
The quaternary alkaloid reineckates obtained from the post 20-C and the post 20-TT-A aqueous mother liquors were dried and subsequently converted to the quaternary alkaloid chlorides by the method previously described on pages 39”4l. From a combined weight of 230.0 gm. of quaternary alkaloid reineckates, a yield of 53.50 gm. of quaternary chlorides (Qu) representing 0.26$ of the starting plant material was ob tain ed . b. Fractionation of the tertiary alkaloids into the tertiary n on -p h en olic (TN) and t e r t ia r y p h en o lic (TP) a lk a lo id fr a c tio n s
Eighty seven grams of the total tertiary alkaloids, 20-TT, was re-dissolved in ether by refluxing with 1,000-ml.portions of the solvent at 45-50° C. An exchange of fresh solvent was made every three to four hours until all the alkaloids were dissolved.
Each ether fraction, obtained above, was individually treated by extraction with a five per cent sodium hydroxide solution for the 70 separation of the phenolic alkaloids (TP). Four equal volumes of the alkali were required for the complete removal of the TP alkaloids from the ether solutions. A fifty per cent acetic acid solution was im mediately added to each sodium hydroxide extractive to lower the pH to approximately 6.0 in order to prevent degradation of the phenolic alka loids in the alkaline solvent. This aqueous solution was then placed under the vacuum to remove any r e sid u a l e th e r . F ilt r a t io n under vacuum was applied next, to remove any insoluble material, and the filtrate alkalinized with a 14$ ammonium hydroxide solution (ice cubes being added beforehand to insure flocculant precipitates). When precipita tion ceased, the flocculant suspension was completely extracted with ether. This ether solution was dried over anhydrous sodium sulfate, filtered, and the filtrate evaporated to a slightly resinous powder in vacuo at 40° C, yielding a brownish solid. It weighed 22.40 gm. (0.11$ of the starting plant material) and was designated as the phenolic (TP) a lk a lo id s.
The ethereal mother liquor le ft after the sodium hydroxide ex tractions, containing the alkali insoluble or tertiary non-phenolic
(TN) alkaloids, was dried over anhydrous sodium sulfate. After filtration, this ether solution was taken to dryness under vacuum at
40° C which resulted in a tan colored, fluffy powder weighing 56.80 gm. or 0.28$ of the starting material.
Flow sheets of the "Total Alkaloid Extraction'1 and the
"Fractionation" procedures previously described, are presented in
Charts I and I I . 71
D. Separation and Isolation of the Tertiary N on-phenolic (TN) A lk aloid s
It was shown from the preliminary investigations that the greatest hypotensive activity in dogs was due to the tertiaiy non-phenolic or
TN alkaloids, and that these alkaloids could be separated by gradient pH extraction (Figure 13). In addition, the number of alkaloids present was fixed at five, by both one and two dimensional thin-layer chromatograms (Figures 10 and 11). Taking these facts into considera tion, it was decided to place emphasis on a study of this alkaloid fraction. Another incentive for investigating this fraction was furnished when 56.80 gm. or 0.28$ of this fraction was obtained from
20 kg. of the plant material.
1. Separation of the TN alkaloids by gradient pH extraction
A modified version of the gradient pH separation technique described earlier (pages 55~58 ) was employed for the separation of the TN alkaloids. The modifications are as follows:
(1) A 0.20 M citric acid solution was employed instead of the recommended 0.10 M citric acid solution.
(2) Instead of one or two benzene extractions being made at each pH level, the number of extractions made was increased. The completeness of extraction, rather than the number of extractions was the criterion for changing the pH to the next level. Only when the concentrated benzene solution from the last extraction spotted on a piece of filter paper and sprayed with Dragendorff’s reagent, gave a negative test or a very faint orange color, was the extraction termed ”complete”.
(3) The benzene extractives from each pH level were concen trated and subjected to acid-base treatment as a purifi cation step. Forty-seven grams of the alkaloid bases, in two portions,were dissolved in chloroform. Each of these portions was extracted into a
liter of 0.20 M citric acid solution by first warming the mixture on
a steam bath with stirring. Then, by means of a rotary evaporator and
reduced pressure the organic solvent was removed from the mixture. The
alkaloid saturated citric acid solution remaining after this treatment was
then decolorized with a very small amount of charcoal by warming on a
steam bath for 10-15 minutes. After the mixture was cooled to room
temperature, it was filtered. The residue left on the filter was washed with fresh citric acid solution (0.20 M), and the washings were
added to the filtrate. The pH of the combined solution was determined
(pH 2.50) and the alkaloids were extracted to completion with a commer
cial grade benzene. The pH of the aqueous mother liquor was then
adjusted ia.th 10$ ammonium hydroxide solution, added dropwise, to a pH
of 3.00 , 3.50, ^.00, ^.50 , 5.00 , 5.50 , 6.00 , 6.50 , 7.30 and 8.80.
Complete extraction of alkaloids with benzene being made at each of
these levels. Each of these extractives was then dried over anhydrous
sodium sulfate, concentrated to a small volume, and extracted into 250
ml. of five per cent hydrochloric acid in a rotary evaporator in vacuo
at 40° C. The hydrochloric acid extractive was then basified with 14$
ammonium hydroxide, followed by extraction with chloroform. After
drying over anhydrous sodium sulfate, the chloroformic solution was
transferred to a tared 250 ml. round bottom flask and evaporated to
dryness, yielding a fluffy alkaloidal powder.
Thin-layer chromatography of the extractives, on silica gel G and
developed with methanol:ammonium hydroxide (99:1) (Figure 15), served Methanol;ammonium hydroxide (28$) (99:1) 3-5 .0 3 .5 2 pH edmesoa Ti-l hoaorm T Al li fo Gradient G from s aloid lk A TN f o Chromatogram r e y la Thin- ensional ne-dim O odn col b t s smos C, ra; , ue A, e; lu b B, cream; Cr, symbols: ese th by s r lo o c ponding rkn i ndi e te l s t ad h c rres co the and ots sp t n e c s e r o flu the te a ic d in s e lin Broken i i ndi e a tve Drgnof reacton ad the and n tio c a e r ragendorff D e itiv s o p a te a ic d in s e lin lid o S bobd ad , a . tan T, and absorbed; or i ty b: 2 brght +, vrg; fai . t in a f , + average; +1, t; h rig b +2, by: y it s n e t in r lo o c /+2 H eaain n e G at s te la P G Gel a c i l i S on Separation pH k.o FIGURE 15 ^•5
rigin O 5.0
||b 5-5 J ) T'Cr Cr G r 'jC T r )o r JJp
6.0 0 .5 6
73 7.3 i~',B
8.8 jCr to point out those consecutive fractions which could be combined on the basis of similar alkaloid composition.
The weights and values of the alkaloid extractives from the various pH levels are summarized in Table 4.
TABLE 4
Weights and Values of the Extractives from the Gradient
______pH Separation of the TN Alkaloids ______
pH F ra ctio n W eight (gm) Rf V a lu e s ^
2.50 3.667 0.60
3.00 0.8 2 3 0 .6 1
3.50 1.5 7 2 0 . 6 l ; 0 .4 l
4.00 4.056 0 .5 9 ;0 .4 1
4.50 9.256 0. 58;0.40
5.00 7.8 4 2 0. 58;0.39;0. 31
5.50 6.470 0. 39;0. 31;0. 24
6.00 3-192 0 . 3 9 ;0 . 32;0 . 25
6.50 1.884 0 . 3 2 ;0 . 25
7.30 1.605 0. 24
8.80 1.131 0 .2 5
(a) Dragendorff1 s reagent stained areas on silica gel G T-L-C plate shown in Figure 15. 75
2. The isolation of alkaloids TNA, TNB, TNC, and hernendezine from gradient pH extractives a. Alkaloid TNA
The alkaloids from the pH 2.5 and 3*0 fractions were found to have similar Rj. values on one dimension, and identical migration patterns on two-dimensional thin-layer chromatograms (Figures 15 and 16). There was, however, one difference noted on the one demension T-L-C plate. When viewed under U.V. light, the alkaloid from the lower pH level extractive had an absorbing effect, which was absent in the alkaloid from the higher pH level extractive. It was therefore felt that two different alkaloids, rather than one alkaloid as originally assumed, might be present. Consequently, the pH 2.5 extractive was coded as "TNA", and the non-absorbing, R^. 0.6l alkaloid from the pH 3*0 fraction was assigned the code name of "TNB.11 They were treated separately.
A sample of 3-65 S®* ^he alkaloid fraction from a total of
3.67 gm. was dissolved in methanol. The methanolic solution was fil tered and the volume reduced to about 20 ml. on the steam bath. The side of the container (250 ml. Erlenmeyer flask) was scratched with a glass rod and placed in a refrigerator overnight. Crystallization did not take place, therefore the remainder of the solvent was removed in vacuo using the rotary evaporator. Absolute ethanol, followed by acetone, and finally ether, were then employed as crystallizing solvents.
As with methanol, negative results were obtained. It was noted during the attempted ether crystallization that a copious amount of grayish, non-alkaloidal material separated during refrigeration. Subsequently, ______w-i ninl n-aye Crmtga of N Al li u TNB lus P aloid lk TNA A f o Chromatogram er y -la in h T ensional Two-dim Chloroform: methanol (9 5 '5) Origin. i nes i cat posi ve Daedrf i and n tio c a r Dragendorff e iv it s o p a te a ic d in s e in l lid o S y hee ybl: , bobd ad B light bl e. lu b t h g i l LB, and absorbed; A, symbols: ese th by he .. l s s n te orsodn col s r lo o c corresponding the and ts o sp t n e c s e r o flu U.V. e th h c or ntensi bri . rkn i ndi te a ic d in s e lin Broken t. h ig r b , 2 + : y it s n e t in r lo co the kaod (0 g ec) n e G at s te la P G Gel a c i l i S on each) ng. (10 aloid lk A ehnlamnu hdoie 2$ (99:1) (28$) hydroxide Methanol:ammonium FIGURE 16 / +2
76
77 this mixture was filtered, the filtrate evaporated to dryness on a steam bath, and the residue obtained was subjected to purification by acid-base treatment in the Usual manner. The material was first dis solved in five per cent hydrochloric acid, then precipitated with ammonium hydroxide, and followed by extraction with chloroform.
Attempts to crystallize this purified material from methanol, abso lute ethanol, and ethyl acetate also gave negative results. As a last resort, an attempt was made to isolate the alkaloid as the hydriodide salt.
The amorphous powder recovered from the ethyl acetate solution was dissolved in a minimum volume of methanol and a 10$ aqueous hydriodic acid solution was added dropwise, swirling the flask continuously, until a gummy precipitate was formed. After being chilled overnight in a freezer, the content of the flask was filtered.
The filtrate was chilled overnight once more, and a second crop of precipitate was obtained. The two crops of gummy, alkaloidal precipi tate were then combined and crystallized from acetone containing about
10$ water. This was brought about by warming the alkaloidal material in the solvent mixture on a steam bath, followed by chilling in a fr e e z e r .
The reddish, crude, micro-crystalline material obtained in this manner was recrystal'lized three times from hot acetone to a yellowish orange crystalline material, m.p. 22*4-228° C, dec. (air dried), yield
0.252 gm. This alkaloid was given the code name of ’’TNA-HI", pending ide n tifi cation. TNA-HI was then placed in a drying pistol containing phosphorous pentoxide at 0.5 mm Hg. over boiling benzene. At the end of two hours, it was noticed that the color of this compound had changed from yel lowish-orange to reddish-orange. Since this could indicate decomposition of the alkaloid, the drying process was stopped. A melting point of this darkened compound was taken, and found to be 226-228° C, dec.
However, satisfactory infrared (I.R .) and ultraviolet (U.V.) spectra of this compound were not obtainable. A sample of TNA-HI was subsequently converted to its free base form. The sample of TNA-HI (100 mg.) was dissolved in approximately 100 ml. of 5# hydrochloric acid, filtered, and the filtrated alkalinized with 14$ ammonium hydroxide, followed by extraction of the liberated free base with ether. The ether extractive was evaporated and the residue subsequently chromatographed on Woelm ®, neutral, activity grade IV alumina, with chloroform as the eluent, to remove any liberated iodine that might be present.
The column (1.0 cm. X 20 cm.) was packed with 10 gm. of the adsorbent by the method previously described for the preliminary separation of quaternary alkaloid (Qu-l) on page 5®* The elution with chloroform was monitored using Dragendorff* s spray reagent on filte r paper spotted with a drop of the eluate. Approximately 50 mg. of an amorphous base was obtained, of which 20 mg. was dried in a phosphorus pentoxide-containing pistol over boiling water at 0.5 mm Hg. A portion of the dried sample was then subjected to nuclear magnetic resonance and optical rotation analyses. The remainder of the amorphous material was dissolved in U0S.P0 alcohol, filtered, and the volume of the filtrate evaporated on a rotary evaporator to a small volume (2-3 ml.) in a 50-ml. 79
Erlemeyer flask. This was stoppered with a cork and set aside in a refrigerator. Crystallization required approximately one week. The needle to rod-shaped crystals collected were recrystallized from ethanol: yield 10 mg.; m.p. 19*+-197° C; 1 (0.026 mg. /ml.) 332 mp. , F + OPT 233 mp* . and a slight inflection at 275“2?7 mp; 1 315 n*P* and 221 mp. It was also noted that the spectrum did not shift when 0.01-N alcoholic sodium hydroxide was employed as the solvent (Table 7).
An infrared spectrum of this compound, tentatively called ,,TNA,,, in a potassium bromide pellet, (Figure 19) showed an absence of carbonyl functions and probable absence of any alcohol groups.
Nuclear magnetic resonance spectral analysis of the dried amorphous m a teria l revealed one (N-CH-p and f iv e (0-CH^) groups. The v a lu e o f these groups and those of the aromatic protons, as well as the ring
(CH2) are summarized in Table 8, while the spectrum is shown in Figure
24.
Optical rotation was also determined using the amorphous powder.
2Q Q The value obtained was: [o]57-151.0w(c .f 0 .69 in chloroform).
Because of the small quantity of crystals available, elemental analysis was not determined. For the same reason, structural studies cannot be performed for TNA at this time.
From the scant physical data available, TNA appears to be a non- phenolic bisbenzylisoquinoline alkaloid with five methoxyl groups and a conjugated nitrogen in one of the benzylisoquinoline moieties and an unknown number of diphenyl ether linkages. 80 b. Alkaloid TTOC (The major tertiary non-phenolic alkaloid
The pH 3°5o 4.0 and 4.5 TN alkaloid fractions obtained from gradient pH separation, having been shown to contain alkaloids of the same R^, values (0.57 and 0.40), were combined for the purpose of iso lating these alkaloids (the total weight of these alkaloid fractions was 14.88 gm.). Methanol (A.R.) was added to the respective containers of the dried extractives to dissolve the material in order to facilitate handling during the combining process. As each extractive was being dissolved, a cream white amorphous material was observed to have sep arated from solution. This material was collected by vacuum filtration, washed with methanol, and found to weigh 7.23 gm. It gave a strong positive alkaloid reaction with Valser1s T.S. Thin-layer chromatography o f t h is compound on both one and two dim ensional chromatograms, d ev el oped in methanol:ammonium hydroxide (99:1), chloroform:methanol (95:5); and methanol:ammonium hydroxide (99:l)» respectively, revealed the presence of only one alkaloid. This alkaloid was identified as the
R^ 0.40-0.41 alkaloid illustrated in Figure 15 and assigned the code name o f TNC.
Additional quantities of this amorphous alkaloid (1.29 gm. total weight) were obtained from the methanol mother liquor by successively concentrating the volumes of the solvent.
Having established the presence of only one alkaloid, the amorphous material was dissolved in methanol and crystallized. The process em ployed involved dissolving the material by refluxing the mixture on a 81 steam bath. After dissolution, the hot methanolic solution was immediately filtered into a one liter Erlenmeyer flask. The flask was then fitted with a distilling head, connected to a water cooled con denser, and the content of the flask was reduced to about one-fourth of its original volume by distillation. The concentrated solution was then set aside to cool to room temperature. Crystallization from the cooled solution usually required from one-half to 24 hours. From
8.52 gm. of the starting amorphous material, 7.25 gm. of radiating rosette-like crystals were obtained, m.p. 168-172 0 C (air dried).
The crude TNC crystals were recrystallized four times from pQ methanol, m.p. 172-173° c* H d + 160.0° (c ., 0.90 in methanol),
[a] ^6+ 119.4° (c. , 0.68 in chloroform); X 281-282 mp., 205 mp. , E+OH with an inflection at X 240- 242 mp. ; X 263 mp. Measurements in 0.01 N alcoholic sodium hydroxide caused no shift in the U.V. spectrum. On the other hand, measurements in 0.01 N alcoholic- hydrochloric acid shifted the maximum absorption peak to X 289 mp.
(see Table 7).
Nuclear magnetic resonance spectroanalysis revealed the presence of five (O-CH-j) and two (N-CH^) groups (Figure 25 and Table 8), and a molecular weight of 642 t JZ was determined by the Osmometer technique fo r t h is compound.
Analysis; C^H^08N2;5-OCH^; Calculated: C, 69.82; H, 6.91;
K, 4.17; 0, 19.10; 0CII3, 23.18.
Found; C, 70.22 (70.16); H, 6.94 (7.02); N, 4.31 (4.21);
0, 18.53 (18.62); OCH^, 23.04. 82
An examination of these physical data led to the conclusion that
TNC is a bisbenzylisoquinoline alkaloid possessing five O-methyl groups, A check of the known bisbenzylisoquinoline alkaloids revealed that none possessed a set of physical properties corresponding to those of TNC, and therefore it was concluded that this was a new a lk a lo id , c. Hernandezine (A lk a lo id TNC-50)
The Rj. 0,39 alkaloid found in the gradient pH extractives at pH
5.0 and 5,5 (Table 4) was isolated as very fine crystalline needles.
It was shown to have the same value (0.4l) as the alkaloid, TNC, by one dimensional T-L-C on silica gel G, developed in methanol: ammonium hydroxide (99:1), and a two-dimensional chromatogram spotted with a mixture of these two alkaloids showed only one spot (Figure 17), thus these compounds were assumed to be identical, even though their crystalline structures and melting points were not in agreement. At first, the differences in the crystalline habits were thought to be caused by concentration differences in the mother liquors, and that the different melting points observed were due to these differences in crystalline structure. Later, however, divergent optical rotations and similar but not superimposable infrared spectra were obtained from these two crystalline compounds, and because of these differences, the alkaloid (needles) from the pH 5-00 fraction was labeled TNC-50. This compound was later identified as hernandezine, a non-phenolic, tertiary, bisbenzylisoquinoline alkaloid previously isolated from Thaiiotmm hernandezii. Tausch, by Padilla and Herrin (50). 83
rH O
O rigin ______M gthanol: aimrcnium hydroacl.de (28#) _ X22. :1).
FIGURE 17
Two-dimensional Thin-layer Chromatogram of TNC Plus TNC-50 (Hernandezine) (5 Jig each) on Silica gel G Plate
Solid line indicates a positive Dragendorff reaction and the color intensity by: +2, bright; N, non fluorescence under U. V. light. In the case of the pH 5*00 extractive, methanol was added to dissolve the dried material (7.84 gm.). Not unlike the pH 3*5» ^ .0 , and 4.5 gradient pH extractives discussed in the preceding section, a white semi-crystalline material weighing 3*17 gm- was observed to have separated from solution. This material was collected by means of vacuum filtration and chromatographed on silica gel G plates with the usual methanol:ammonium hydroxide solvent system. Three non- fluorescent, Dragendorff’s reagent positive spots were found at
0.60, 0.41, and 0.30 with the R^. 0.4l spot being the most intensely colored and the R^. 0. 30 spot the lea st colored. Subsequent concentration (sev eral time ^ of the methanol filtra te on a steam bath yielded additional crops of the same mixture. The combined weight of this alkaloid fraction was
4 ,0 5 gm. I t was la b eled as "5.0-TNB+TNC+TND."
The process described for the recrystallization of TNC, was then employed in an attempt to fractionally crystallize this mixture of alkaloids. For the purpose of identification, the R^ 0.60 alkaloidal spot was designated as "5.0-TNB", the R^ 0.4l spot as '^.O-TNC'’, and the R^. 0.30 spot as "5.0-TND".
Four grams of the sem i-crystalline mixture was dissolved in 800 ml. of methanol, filtered, and the filtrate concentrated to about 400 ml. followed by refrigerating for one-half hour. After filtration and washing with fresh solvent, 0.771 gm. of rosette and needle crystals, m.p. 154-l60O C, was obtained. The compound gave only one alkaloid spot, Rj. 0.40, on T-L-C using silica gel G and a solvent system of methanol:ammonium hydroxide (99:1), and was labeled "5.0-TNC”. 85
A second crop of the same material (0.538 gm.) was obtained by- concentrating the mother liquor. To aid in identification, the subscript n2" was added to 5.0-TNC (e.g. 5.0-TNC2).
The alkaloid, hernandezine, was obtained from the post 5-O-TNCg mother liquor by further concentrating the volume of the solution.
When the mixture had been cooled to room temperature, the alkaloid crystallized as fine needles, m.p. I58-I6O0 C (air dried), yield
0.216 gm. Although this compound was shown to have an identical valu e as 5«0-TNC by the same T-L-C technique d escrib ed fo r the l a t t e r alkaloid, it was designated as "TNC-50" because of a sharper melting point than that of 5»0-TNC. An analytically pure sample of this com pound was obtained by two rccrystallizations from methanol, m.p. 162-
163. 5° C; Jjofj p1 + 227 . 3° (c. , 0.^+9 in chloroform), [a] ^ + 228.0° (c. ,
0.20 in chloroform); X ^82 an8 205 mp, X ^in^ m^’ Absorption studies with 0.01 N alcoholic sodium hydroxide and 0.01 N alcoholic hydrocloric acid revealed no shift in X. (Table 7).
Originally TNC-50 was suspected to be identical with the alkaloid,
TNC, because of their values (4.0-4.1) in one-dimensional T-L-C and not separable on two-dimensional T-L-C. An examination of their respec tive melting points, optical rotations and infrared spectra (in potassium bromide pellets) (Figures 20 and 22), however, dispelled this assumption.
Discernable differences in th e ir infrared spectra (potassium bromide pellet) were discovered at wavelengths 6.15- 6. *.’0 p, 7.10-7* 30)1, 8. 20-8.40 p,
8,80-9.-10 >1 , 9’.80-10.00 p and 10. 50 p. These differences in their I.R. spectra were confirmed by analyses of the two compounds in chloroform liquid films (Figures 21 and 23). Additional evidence th a t TNC and 86
TNC-50 lie re indeed different compounds was furnished by a mixture melting point determination (50$ w/w) of these two alkaloids. Where the compounds, TNC and TNC-50, gave sharp melting points at 172-173°
C and 162-163.5° C, respectively, the mixture gave a melting range of
155-160° C.
Nuclear magnetic resonance spectrophotometric analysis of TNC-50 revealed five 0-methyl and two N-methyl groups (Figure 26 and Table 8).
An analysis of the physical data experimentally determined for TNC-50 and those reported by Padilla and Herrin (50) for hernandezine, the only known ThaH otrum alkaloid having five 0-methyl and two N-methyl groups, gave strong indications that these two compounds were identical.
Some of the physical data reported for hernandezine are: m.p. 157-15®°
C; k Max. 283 mp. and 209 mp. ; and K d° + 250° (c ., 0.20 in chloroform).
These values reported for hernandezine and those determined for TNC-50 were also found to be in agreement, when one takes into account such induced experimental errors as the degree of purity of the compounds, enviromental temperature, and the sensitivities of the instruments employed.
A comparison of the I.R. spectrum of TNC-50 prepared in a liquid film of chloroform (Figure 23), and the published I.R. spectrum of hernandezine (50) showed a very remarkable sim ilarity in their charac teristic peaks, although different instruments were employed, while the N.M.R. spectrum of TNC-50 and the published N. M.R. spectrum of hernandezine were shown to be id e n t ic a l. 87
The identity of this alkaloid as hernandezine was confirmed by comparative studies of a reference sample of hernandezine^ and
TNC-50. Mixed melting point determination showed no depression from the value of 162-163.5° 0 obtained for TNC-50. The lower melting point of 157-1580 C originally reported for hernandezine was due to methanol crystallization. This fact was made known in a communication from one of the authors, Professor J. Herrin, who stated that, "a somewhat higher melting point was achieved after successfully obtaining crystals freed of methanol." The infrared absorption spectra of both hernandezine and
TNC-50 in potassium bromide pellets analyzed in the same instrument were found to be superimposable. This was also true of their N.M.R. spectra.
Following the isolation of hernandezine, the mother liquor was again concentrated in an attempt to obtain an additional quantity of this alkaloid. Instead of obtaining a single compound as expected, however, a mixture of 5*0-TNB and the R^ 0.^0 alkaloid (detected by
T-L-C, silica gel G, methanol:ammonium hydroxide (99:1) solvent system) in very fine needles with an air-dried weight of 1.9^ gm. was obtained.
T his a lk a lo id m ixture was coded as "5.0-TNB+-C". The mother liq u o r was sim ilarly chromatographed and found to contain all three of the afore mentioned alkaloids (5.0-TNB, 5.0-TNC and 5.0-TND), however, further crystallization attempts failed to yield crystals.
The 5*0 TNB+C alkaloid mixture was then fractionally crystallized by successive reduction of the solvent volumes followed by filtration
Supplied by Prof. J. HerrSn, Instituto de Q^mica de la Universitad Nacional Aut<5nona de Mexico, Mexico City, Mexico. 88
of the cooled mixture and washings with fresh solvent, yielding a total
of 0.32 gm. of a crystalline material, m.p. 162-164° C; O.^-l (T-L-C
silica gel G, methanol:ammonium hydroxide 99:1 ); infrared spectrum (in potassium bromide pellet) identical to that of hernandezine. Because of these data, this alkaloid was also identified as hernandezine.
When this mother liquor ceased to yield the alkaloid, hernandizine, it was further concentrated to almost dryness and the container, a 125 ml. Erlenmeyer flask, was covered with a piece of aluminum fo il and
s e t a sid e on the bench. Upon c o o lin g to room tem perature, n e e d le -lik e
crystals began to form on the sides of the flask. This crystalline material (1.4^0 gm.) was found to be a mixture of the starting alka loids, 5-0-TNB+-C. Attempts to further fractionate this mixture using methanol, ethanol (absolute), ethanol (U.S.P.), acetone, ethyl-acetate, ether, and benzene were not successful.
The alkaloid samples designated as 5-0-TNC and 5.O-TNC2 (second crop) were thought to be mixtures of hernandezine and TNC because of the samples* broad melting range and their similar, but non-super- imposable infrared spectra with those of TNC and hernandezine.
The quantity of hernandezine isolated from the total crystalline mixture, 5*O-TNB+-TNC+TND, by the fractional crystallization method described above was 0.535 gm* This, however, may not be the true
amount of hernandezine present in the pH 5-00 gradient pH extractive because unknown quantities of this alkaloid are, undoubtedly, still p r e se n t in the 5*0-TNC, S^O-TNCg, 5*0-TNB+C c r y s ta llin e m ixtu res, and the post 5*0-TNBtTNC+TND methanolic mother liquor. In the isolation of hernandezine from the pH 5*5 gradient pH extractive, the material (6.^7 gm.) was in methanol. Following f il tration, the alkaloid negative residue was discarded and the filtrate concentrated ifl vacuo. The syrupy concentrate was added to 250 ml. of five per cent hydrochloric acid solution, mixed well by stirring with a glass rod, followed by the removal of methanol by evaporating Aa vacuo.
The residue obtained from filtration of the resulting acid solution was re-dissolved in methanol and again extracted into 50 ml. of five per cent hydrochloric acid as described above. This procedure was repeated once more and the filtrate was added to those previously obtained. The combined acid solution was then decolorized with activated charcoal by refluxing on a steam bath for 30 minutes. After the mixture had been cooled to room temperature, it was filtered and the residue washed twice with 25 ml. portions of five per cent hydrochloric acid, the washings being added to the filtrate. Ammonium hydroxide (lh ■$>) was then added to render the combined acid solution alkaline, followed by ether ex tractions of the resulting alkaloid precipitates. The ether solutions were pooled, dried over anhydrous sodium sulgate, and evaporated to dry n e ss.
The residue obtained from the removal of ether was dissolved in approximately 100 ml. of methanol, and the filtrate concentrated suc cessively by the method employed for the fractional crystallization of hernandezine from the pH 5*00 extractive. From these concentrated solutions, 0.35^ of a crystalline needle-shaped alkaloid, m.p. 160-
162° C, was obtained. This compound was identified as hernandezine by subsequent infrared spectrophotometric analysis and mixture melting 90 point determination with reference material.
The post hernandezine mother liquor was shown by thin-layer chromatography, silica gel G plates, methanol:ammonium hydroxide
(99:1) solvent system, to contain alkaloids at R^. 0.31 and 0.25, in addition to residual hernandezine. Further attempts to fractionally crystallize these alkaloids were unsuccessful. d. iUXalsld .TJffi (lha
The Rp 0.58-0.61 alkaloid, TNB, present in gradient pH extractives at pH levels of 3.0, 3« 5, ^-0 » 5 and 5.0 (Table b and Figure 15) was found to be the pharmacologically active principle responsible for the hypo tensive effect in dogs. The crude crystals isolated from these pH fractions totaled 0.229 gm.
Although the gradient pH 3*0 extractive weighed 0.823 gm. and contained only the 0.6l or TNB alkaloid, the isolation of this com pound proved to be most difficult.
Initial attempts to isolate TNB using methanol as the crystallizing solvent and the technique successfully employed for the isolation of
TNC and hernandezine, ended in failures. The same was true when the solvent was changed to ethanol (absolute), ethanol (U.S.P.), and finally, acetone. Attem pts to is o la t e th e compound as i t s h y d rio d id e, hydro chloride, oxalate and sulfate salts were also unsuccessful.
The alkaloid was then converted to its free base form from the sulfuric acid-methanol solution of the alkaloid sulfate derivation attempt by diluting the solution with distilled water, followed by removal of methanol ia vacuo and alkalinization of the resulting acid 91
solution with 14$ ammonium hydroxide. The liberated alkaloid base was removed by ether extractions.
The ethereal extractives thus obtained were combined, dried over anhydrous sodium sulfate, evaporated to dryness, and the residue was dissolved in 25 ml. methanol by warming on a steam bath. The resulting methanol solution was filtered, while hot, into a 50-ml* Erlenmeyer flask and the solution evaporated to near dryness on a steam bath. After
the sides of the flask had been scratched with a microspatula, the con tainer and its content were place in a refrigerator for two days. The long, slender, needle-like crystals which formed were collected by fil tration, and washed with a few drops of fresh methanol. A second crop of these crystals was obtained when the filtrate was refrigerated again for
twenty-four hours. These two crops of crystals totaled 0.093 gm. and was labeled as "TNB-1." The mother liquor yielded, after refrigera
tion for a week, only minute amount of crystals, which could not be
collected on the filter.
A second sample of this alkaloid was isolated from the combined pH 3.5, 4.0, and 4.5 extractives. The preparation and treatments
given to these extractives leading to the methanol solution labeled,
"TNC-mother liquor" were fully described in the discussion of the
isolation of the alkaloid, TNC.
Thin-layer chromatographic studies (silica gel G; methanol:ammonium
hydroxide (99:11) showed the presence of the alkaloids, TNB and TNC, in
the "TNC-mother liquor." Since it was not possible to fractionally
crystallize the alkaloids from this mixture, it was column chromato
graphed on a mixture of four parts of 100 mesh silicic acid and one part of Celite-5^5 activated at 200° C for 48 hours. Fifty grams of this material was packed into a column, l-l/8 n diameter by 15” length, employing the slurry method with chloroformjmethanol (98:2) as the vehicle. The "TNC-mother liquor," having been previously evaporated to dryness, was dissolved in 10 ml. of chloroform and introduced on top of the column by means of a pipet. The alkaloids were then eluted from the column with chloroform:methanol (98:2), with 50 ml. eluates being collected. Sharp separation was not achieved, but it was noted that the color of the Dragendorff positive spot at R^, 0.60 (TNB) was much brighter than that of the R^. 0.40 spots on the T-L-C plates of the first six eluates. This was interpreted as the concentration of TNB being greater than TNC and that the former could possibly be fraction ally crystallized. Therefore, these six eluates were combined and evaporated to dryness in vacuo at 40° C. The residue obtained was dissolved in methanol, filtered, and the filtrate concentrated from approximately 100 ml. to about 20 ml. followed by refrigeration for two days. However, crystallization did not occur and it was noticed that the solution had become very oily. At this point, it was felt that the materials in this solution must be purified at least once more prior to attempting any further crystallizations.
The "oily" methanol solution was then mixed with 50 ml. of five per cent hydrochloric acid, followed by the removal of methanol by vacuum evaporation. The acid solution was filtered and the filtrate was made alkaline with 14$ ammonium hydroxide, followed by ether extractions of the resulting alkaloid precipitates in the usual manner. The ethereal extractive was evaporated on a steam bath in 93 the hood to dryness, yielding a yellowish powder.
The yellowish purified, powdery residue was dissolved in approx imately 50 ml. of methanol with the aid of heat from a steam bath. The solution was filtered, and the filtrate reduced to one half its volume in an open 125 ml. Erlenmeyer flask on a steam bath, followed by refri geration. Tan colored needles of TNB contaminated with minute quantities of TNC alkaloid was obtained by filtration the next day. The mother liquor was concentrated twice more in the same manner to yield two additional crops of these crystals. The three crops of impure TNB crys tals had a combined weight of O.I89 gm. Recrystallization from ethanol yielded 0.104 gm. of TNB alkaloid crystals free from TNC contamination.
This sample o f c r y s ta ls was la b e le d as "TNB-2” .
The post n5°0-TNB+TNC+DM mother liquor obtained from the pH 5*0 gradient pH extractive (see page 87) yielded a third sample of TNB crystals. This was brought about when the methanolic solution was set aside in a darkened locker for about two weeks. The tan colored crystalline needles which formed on the sides of the flask were collected on a filter, and the product was chromatographically identified to be the alkaloid, TNB. The crystalline material, weighing 0.032 gm., was labeled as TNB-3* Unfortunately, the mother liquor resisted all attempts to isolate more of this compound.
The alkaloid samples TNB-1, TNB-2, and TNB-3. isolated from the various fractions, were combined to give a total weight of 0.229 gm.
In a routine pharmacological test for hypotensive activity, this alkaloid was found to cause a pronounced and prolonged drop in the blood pressure of a normotensive dog. For this test, the procedure 94 employed to screen the various alkaloid fractions described on page 48 was utilised. A dose of 2.5 mg./kg. was injected intravenously into a 9*1 kg. male dog. An immediate drop in blood pressure to 70 mm. mercury from the norm of 130 mm. mercury for this dog was recorded.
This drop in blood pressure was continued for more than two hours, with the maximum drop to 36 nun. mercury being recorded at 12 min. from the time of injection. In view of this result and the fact that the alkaloids, TNA, TNC and hernandezine, elicited only very transient and statistically insignificant drops in the blood pressure of the test animals in similar studies, it seemed quite logical to designate
TNB as the major hypotensive alkaloid.
Meanwhile, all but a few milligrams of the TNB crystals available were dissolved in methanol and recrystallized three times by the pro cedure employed for TNC and hernandezine. The crystals recovered were still slightly colored, and found to become softened on heating to 142-
145° C, and finally melting into a yellow liquid at I62-I650 C. Be cause of the coloration and melting range, the crystals were redissolved in methanol for further recrystallization. Instead of becoming purer, the crystals were found to be more intensely colored, and the quantity less on each succeeding recrystallization, until finally, only a few crystals remained. Thin-layer chromatography of the mother liquor revealed the presence of several Dragendorff positive spots, indicating the compound had been degraded in the r e c r y s t a lliz a t io n p ro cess.
Attempts were then made to obtain sufficient quantity of this alka loid from the various mother liquors for characterization studies. In these efforts, the TNB-2, and TNB-3 mother liquors were subjected to 95 adsorption column chromatography with adsorbents of different activities in an increasing order. Unfortunately, separation of TNB from the other alkaloids present, e.g. TNC, hernandezine, and TND, was not achieved.
The adsorbents utilized, the methods of packing the columns, the eluting solvents employed, and the results obtained are summarized in Table 5*
TABLE 5
Chromatographic Separation of TNB Mother Liquors
Order of Adsorbent Packing Eluent Alkaloid use method Separation
1. Silicic AcidtCelite-545 Slu rry CHClo sCH.,OH None (6 :1 ) (9 8 : 2) 3
2. Silicic Acid:Celite-545 Slu rry CHC13 (10058) None (8 :1 )
3. N eutral Woelm, Grade IV S if t in g benzene (100^) None
4. Neutral Woelm, Grade II S if t in g benzene (100$) None benzene:CHClo (1 :1 ) 3
5. Neutral Woelm, Grade I Sifting benzene:CHC1- None (1 :1 ) 3
In a final effort to separated this active compound, the TNB-2 mother liquor recovered from the column chromatographic separation attempts was subjected to gradient pH separation. This experiment was conducted on the basis that during the original gradient pH separation of the tertiary, non-phenolic fraction (TN), this alkaloid was extracted from the citric acid-ammonium hydroxide solution at pH 3 .0 (se e Table 4 and F igure 1 5 ). The m eth anolic TNB-2 mother liq u o r was extracted into 200 ml. of 0.2M citric acid solution by the method presented earlier for the TN alkaloids. The resulting aqueous solution was adjusted to pH 2.8-3.0 with 10 $ ammonium hydroxide solution and
exhaustively extracted with benzene. After drying over anhydrous sodium
sulfate, the benzene extractive was taken to dryness in vacuo. and the
residue subjected to the usual acid-base treatment for purification.
The resulting amorphous material, thin-layer chromatographically demonstrated to contain only the TNB alkaloid, was dissolved in methanol and filtered into a 50 ml. Erlenmeyer flask. The filtrate was evaporated to near dryness in vacuo at bO° C, the sides of the flask scratched with a microspatula, and the flask placed in the refrigerator for 2k hours. Crystallization did occur, although the quantity was very small. Recrystallizing by concentrating the methanol
solution in vacuo at *+0° C. instead of the steam bath gave pure crys talline material, 0.018 gm. , m.p. I62-I650 C; [cQ ^+103.2° (c. ,0.10 in chloroform); \ 273 mu. with an inflection at 297 mu. ; \ Min^
265 mu. This spectrum was not altered in the presence of alkali.
Satisfactory spectra were not obtainable from the infrared and nuclear magnetic resonance studies due to the small quantities of the
crystalline material available.
Because of the lack of sufficient physical data, no definite
conclusions could be drawn concerning the structure of this alkaloid. 97
3. Alkaloids of the gradient pH extractives at pH 6.0, 6.5, 7.3. and 8.8
The alkaloids present in the pH 6.0 extractive were determined by thin-layer chromatography to be three, having the same Rf values (0.39.
0.32 and 0,25) as those in the pH 5*5 extractive (Table 4 and Figure 15).
It can be seen that the R^ 0.39 alkaloid, corresponding to hernandezine, was of low concentration when compared to the other alkaloids on the basis of the intensity of the Dragencorff's reagent positive color spots.
The 0.32 alkaloid, previously shown to be present in the pH 5 .0 as
t well as the pH 5*5 fractions, was designated as "TND," while the R^
0.24-0.25 alkaloid was designated as "TNE". The same chromatogram revealed the presence of two alkaloids in the pH 6.5 fraction at R^.
0.32 (TND) and R^. O.25 (TNE), with the lower R^. being dominant. It could also be seen that only the alkaloid, TNE (R^ 0.24-0.25), was pre sent in the pH 7.3 and pH 8.8 fractions.
Attempts to isolate crystalline alkaloids from these pH fractions by fractional crystallization from methanol, ethanol (absolute and
U.S.P.), acetone, ethyl acetate, benzene, ether, mixtures of methanol- petroleum ether, and methanol-water were not successful. In addition, attempts to isolate the R^ 0.24-0.25 alkaloid as its hydriodide, oxalate, and sulfate salts from the pH 6.5, 7.3 and 8.8 fractions were to no avail. Perhaps, if the alkaloids in the pH 6.5 and 6.0 extractives were individually separated by adsorption or partition chromatography, or an additional gradient pH separation prior to the crystallization attempts, more fruitful results might be obtained. The same may be true if the pH 7*3 and 8.8 alkaloid were first purified by chromatography or immiscible solvent extractions. Due to the emphasis being placed on the studies of the alkaloids TNC, TNB, and hernandezine, however, these experiments were not performed.
E. The T ertia ry P h en olic (TP) A lk a lo id s
As indicated by the introductory statement of the problem, it was not within the scope of the present investigation to conduct any ex tensive phytochemical studies of the tertiary phenolic (TP) alkaloids.
Therefore, other than the experiments performed during the preliminary investigations, the only studies of note on the 22.40 gm. of TP alka loids obtained from the 20 kg. of plant materials were some thin-layer chromatographic determinations of the number of alkaloids present.
Thinr-layer chromatographic studies of this alkaloid fraction were conducted in the usual manner. Five Dragendorff reagent positive spots having R^ values Of 0.68, 0.62, 0.48, 0.42 and 0.35 were detected on the one-dimensional chromatogram spotted with 100 jig of the TP alkaloid in methanol on a 50 X 200 mm. silica gel G plate and developed in methanol:ammonium hydroxide (99:1). The intensity of the R^. 0.42 and
0.35 orange Dragendorff spots was much greater than the other three.
In fact, the color of the R^ 0.68, 0.62 and 0.48 spots disappeared when the chromatogram was le ft standing overnight, -which would indicate their concentration was very low. This finding was confirmed by a two- dimensional chromatogram of the same adsorbent on a 200 mm. X 200 mm. plate spotted with 160 jig of the alkaloid solution and developed in the first direction with chloroformrmethanol (95:5)t and the second 99 direction with methanol:ammonium hydroxide (99:1).
F. Chromatography and Isolation of the Quaternary Alkaloids
1. Thin-layer chromatographic studies of the total alkaloid fraction
The presence of six alkaloids in the quaternary alkaloid fraction of £. Rochebrunianum was determined earlier in a preliminary thin-layer chromatographic study on silica gel G plates spotted with 20 ug. of the alkaloids and developed in jj-butanol: acetic acidrwater (4:1:1). This finding was later confirmed by developing a similar chromatogram in propanol:ammonium hydroxide:water (2:1:1). In the second case, the alkaloid spots detected by the Dragendorff* s spray reagent were found to have R^ values of 0.97. O.85, 0.74, 0.70, 0.65, and O.63. It was also noted that the fluorescence and numerical values of the R^
0.74, 0.70, and O.63 alkaloid spots correspond to reference samples of berberine, jatrorrhizine and magnoflorine, respectively, on the same chromatogram. In addition, the coloration of the R^ 0.97 and
O.65 spots were very light, and become completely faded on standing, indicating their concentration to be very low.
In another experiment, microcrystalline cellulose thin-layer plates, previously described for the study of sucrose (Compound ,,20-x11) hydrolysates, were employed for the chromatographic studies of these alkaloids. The chromatograms, after development in jj-propanol: ammonium hydroxide:water (2:1:1), showed five alkaloid spots at
0.99. O.89, 0.75 (berberine), 0.68 (jatrorrhizine) and 0.6l 100
(magnoflorine). Although the separation appeared very good, these alkaloids were not detectable in those chromatograms spotted with less than 500 pg. of the alkaloid mixture. In view of the need of applying large quantities of alkaloids, the microcrystalline cellulose plates would seem to be better suited for preparative purposes, rather than for qualitative detection of the quaternary alkaloids.
2. Chromatographic separation of the quaternary alkaloids for isolation
In the preliminary column chromatographic separation of the total quaternary a lk a lo id fr a c tio n (QU-l) on n eu tra l WoelnfP a c t iv it y grade
II alumina, separation was achieved, although the results were not entirely satisfactory because of the overlapping of jatrorrhizine and magnoflorine in the eluates and the length of time (eight weeks) re quired to develop the column.
In an effort to overcome these disadvantages, other adsorbents or combinations of adsorbents, among which was a mixture of silicic acid of 100 mesh size and Celite-5^5 a stx to one ratio, were tried for the separation of these alkaloids. This adsorbent mixture was found to give a more satisfactory separation of the alkaloids and required a shorter time period (two to three weeks) than the aforementioned alumina. Because of these findings, this adsorbent mixture was used for the chromatographic separation of the quaternary alkaloids for
subsequent isolations.
A 4-5.00 gm. sample of the 53.50 gm. of quaternary alkaloid chlorides obtained from 20 kg. of ground roots was dissolved in methanol and ad
sorbed on 4-5.00 gm. of silicic acid:Cellte-5^5 (6:1) mixture. This 101 was accomplished by adding the alkaloid solution to the adsorbent to form a slurry in a 1000-ml. round bottom flask. The mixture was then evaporated to dryness in vacuo, transferred to a glass mortar, and finely powdered. After sifting through an 80 mesh wire sieve, the powder was packed on top of a packed column, previously prepared by
sifting 1000 gm. of the adsorbent into a 6.50 cm. diameter column filled with chloroform, to a height of 63.00 cm. The alkaloid-ad-
sorbent mixture was placed on the column in the same way. The column was then developed by a gradient elution-type technique with the eluates
being ml. each in volume. Thin-layer chromatography of the eluates,
on silica gel G and developed with jj-propanol:ammonium hydroxide:water
(2:1:1), served to point out those eluates which could be combined on
the basis of similar alkaloid composition.
The eluting solvents in their order of employment, and the results
of this experiment, are presented in Table 6.
On the basis of their thin-layer chromatographic Rj. values,
fluorescence under ultraviolet ligh t, and behaviors when exposed to
ammonia vapor prior to being sprayed with the Dragendorff reagent, as
presented in Table 6, it can be assumed that berberine, jatrorrhizine,
and magnoflorine are present as single entities in fractions D, E and
F, and I and J, respectively. Also, it can be said that these alkaloids
are present with other alkaloids in fractions C, G, and H, in the same
order. In fraction B, the orange color of the Dragendorff positive,
blue fluorescent alkaloid at R^. 0.97 was seen only momentarily. Once
the Dragendorff!s reagent sprayed plate became dried, the colored spot
disappeared, probably due to this alkaloid being present in a very TABLE 6
Results of the Chromatographic Separation of the Qu Alkaloids on S ilicic Acid:Celite—545 ______(6:1) Column______
Eluent Eluates (500 ml. each) Qu Fractions (Combined E luates) U.V. Fluorescence Alkaloid Rj. Values ^a^& ^ Composition Number on Column U.V.Fluorescence on T-L-C Code Weight chci3 (1 0 0 0 1 Cream A 0.16 gm. CHC13 :CH30H (95:5) 1-7 L ight blue o CD 8-9 tXJ 0.97 Blue B 0.09 gm. 0 .85 Tan
10-11 Cream O.85 Cream C 0.0 8 gm. 0.75 Yellow
14-17 Bright yellow^) 0.75 Bright yellow ^ D 0 .7 4 gm. CHC13:CH30H (9 0 :10) 1-7 Bright yellow ^ 0.75 Bright yellow^
8-16 Light yellow^ 0.71 Greenish-yellow^ E 1.46 gm.
CHClyC&jOH (80:20) 1-5 Dull yellow^ 0.68 Dull yellow^ F 2.64 gm.
6-8 Light yellow O.69 Dull yellow ^ G 0.60 None 1 .7 1 gm.
9-13 Tellovrish-green 0.66 Greenish-yellow 102 0.64 Light green H 3.22 gm. 0 .6 l None TABLE 6 (Gont’d)
CHClyCH^OH (50:50) 1-5 Greenish-blue 0.64 Bright blue 9 .0 8 gm.
6-10 Blue 0.62 Bright blue 20.11 gm. CH^OH (1 0 0 # 1-3 Blue O.63 Bright blue
Berberine 0.?4 Bright yellow
Jatrorrhizine 0.70 Dull yellow
Magnoflorine O.63 Bright blue
(a) R^. values calculated for the Dragendorff ’ s reagent stained spots.
(b) Thin-layer chromatography on silica gel G with rj-propanol: ammonium hydroxide:water (2:1:1) as the developer. Plates examined for fluorescence under U.V. light prior to staining with reagent.
(c) Visible red band on column in addition to the fluorescence under U.V. light.
(d) Visible yellow band on column in addition to fluorescence under U.V. light.
(e) Visible yellow spots in addition to fluorescence under U.V. light.
(f) Visible spots turn red on exposure to NH-, vapor, prior to staining with Dragendorff’s reagent. ^ 104 minute quantity.
During the T-I/-C studies, an interesting chance observation was made concerning the color changes of berberine and Jatrorrhizine in the presence of ammonia vapor, before and after spraying the thin-layer plate with Dragendorff1s reagent.
It has been known that Jatrorrhizine takes on a yellow color in the presenoe of aoids and a dark orange to red color in the presence of alkali suoh as ammonium hydroxide (95). Because of this fact, paper and thin-layer chromatograms of quaternary alkaloids suspected to oontain this protoberberine compound have been routinely exposed to hydrochloric acid and ammonia vapors to detect its presence or absence prior to being sprayed with Dragendorff* s reagent in this laboratory. Naturally, a reference sample of Jatrorrhizine is applied to each of these chromatograms for comparision studies of the values and c o lo r changes o f th e known and unknown a lk a lo id s .
In the T-L-C studies of the Qu eluates, the chromatograms were viewed under an ultraviolet light to determine the fluorescence of their constituents, then exposed to ammonia vapor to detect the prescence of Jatrorrhizine, and finally, stained with Dragendorff»s reagent for the determination of the number and R^ values of the alka loids. After having been sprayed with Dragendorff1s reagent, the reference berberine and Jatrorrhizine on one of these chromatograms momentarily appeared orange in color, then, instantaneously changed
to purple and y e llo w , r e s p e c tiv e ly . This phenomenum was h e r eto fo re unknown. Upon investigation, the only difference found between this and other thin-layer chromatographic studies was that in this particular occasion, the bottle of ammonium hydroxide used for the detection of jatrorrhizine was le ft in the hood. The thought then occurred that these color changes may have been caused by escaping ammonia vapor from the bottle. To test this theory, samples of berberine and jatrorrhizine were sp otted on f i l t e r papers and exposed to ammonia vapor, p r io r to and im mediately after being sprayed with Dragendorff’s reagent. Indeed, the previously observed color changes for berberine and jatrorrhizine were again evident. In addition, a mixture of these two alkaloids were similar ly treated and the orange Dragendorff spot was found to have quickly changed to a brown color. In another experiment, the observed color changes in these alkaloid spots did not take place when the Dragendorff’s reagent stained papers were allowed to be dried prior to being exposed to the ammonia vapor and that they take place only when the spotted papers were still moist with the alkaloid spray reagent. The possibility of utilizing these color changes as microchemical tests for the detection of these alkaloids was proved to be feasible when the Qu fractions D (R^
0.75) • E (Rj.0.71), and F (Rf 0.70) were spotted on filte r papers and treated in this manner. The berberine color change (orange to purple) was detected in fraction D and jatrorrhizine color change (orange to yellow) in fractions
E and F. Final proof was obtained when berberine was later isolated from fraction D and jatrorrhizine from fractions E and F.
3. The isolation and identification of berberine, jatrorrhizine and magnoflorine
The protoberberine quaternary alkaloids, berberine and jatrorr hizine, and the aporphine quaternary alkaloid, magnoflorine, were isolated as their respective iodide salts from chromatographically 1 0 6 separated Qu fractions (Table 6 ). Berberine iodide was obtained from fraction D ( 0 . 534- gm.), jatrorrhizine iodide from fractions E and F
(1.039 gm.}* and magnoflorine iodide from fractions I and J ( 2 33^0 . gm.). i Due to the presence of mixtures of alkaloids in the Qu fractions
B, C, G, and H, which would render increased difficulties in the crys tallization of these quaternary amines, the isolation of the alkaloids from these fractions was not attempted in the present investigation. a. Bertrerire
Berberine iodide was isolated from the quaternary alkaloid frac tions "Qu Fraction D" (Table 6) and "20-D" (Charts I and II). Qu
Fraction D, contained the bright yellow fluorescent alkaloid, R^.
0.75. The fraction, weighing 0.74- gm., was dissolved in $0 ml. of hot methanol and decolorized with charcoal by heating on a steam bath for
15 minutes. The mixture was vacuum filtered and the residue washed three times with 20-ml. portions of hot methanol. These washings were added to the filtrate, which was subsequently concentrated to approx imately 50 ml. by distillation on a steam bath. To the hot concentrated solution was added, dropwise, a hot saturated aqueous solution of potas sium iodide until the solution was slightly cloudy. Following refriger ation for two hours, filtration and washing, 0.408 gm. of a crude, crystalline, yellow-needle product was obtained. Farther concentration and refrigeration of the filtrate gave an additional 0.126 gm. of the same crystals for a total yield of 0 . 534- gm.
This crude yellow alkaloid iodide was recrystallized three times from hot methanol to obtain 0.178 gm. of a pale yellow alkaloid iodide, 107 m.p. 259~26l° C, dec. (air dried), which was used for analytical purposes. Additional quantities of the same alkaloid iodide was obtained by further concentration of the combined mother liquors.
After drying over boiling Skelly Solve-F at 0.5 mm. mercury, the pale yellow, crystalline alkaloid iodide gave m.p. 261-262° C, dec.
The melting point of berberine iodide had been reported by different investigators to be 258° C, dec. (44); 260° C, dec. (15); 255° C, dec.
(81); and 162° C, dec. (98). Ultraviolet spectral analyses of this compound with 95$ alcohol, 0.01-N alcoholic sodium hydroxide, and 0.01-
N alcoholic hydrochloric acid gave spectra (Table 7) which correspond to th ose obtained by Reed (96) fo r berberin e io d id e . The compound was found to be optically inactive, which corresponds to the optical activity of berberine iodide, and its infrared spectrum (potassium bromide pellet) was superimposable with that of an authentic sample of berberine iodide.
A d d itio n a l evidence fo r the id e n t if ic a t io n o f t h is compound to be berberine iodide was obtained by preparing the tetrahydro derivative.
The isolation of berberine iodide from fraction "20-D" was accom plished by the procedure just described. From 12.98 gm. of the residue,
9.66 gm. of crude berberine iodide crystals was obtained. A small sample of this material was recrystallized three times from methanol for analytical purposes.
The compound was id e n t if ie d to be berberine io d id e on the b a s is of no depression in mixture melting point determination and super- imposable infrared spectra (potassium bromide pellets) with authentic berberine iodide. 108
The total yield of 10.194 gm. of berberine iodide from these two fractions represents approximately 0.05$ of "the plant material. b. Tetrahvdroberberine
To further prove that the alkaloid isolated as the iodide from fractions, Qu fraction D and **20—0**, was berberine, the tetrahydro derivative was made.
Three grams of the crude berberine iodide obtained from fraction
,120-D" was dissolved in 50 ml. of a 50$ acetic acid solution by warming on a steam bath, followed by the addition of three grams of zinc powder. The resulting mixture was refluxed on a steam bath, after affixing an air-cooled condenser to the flask. When the dark yellow solution had changed to a light green color at the end of one hour, an additional gram of zinc powder was added to the mixture.
After the mixture had been refluxed for 12 hours, accompanied by occasional shaking, it was cooled, filtered, and the residue discarded.
The filtrate was diluted with 50 ml. of distilled water, followed by the addition of ice cubes and finally, rendered alkaline with 14$ ammonium hydroxide solution. The flocculent alkaloid precipitate formed was extracted with four 500-ml. portions of ether. After drying over anhydrous sodium sulfate, the ethereal extractive was concentrated to 100 ml., yielding 1.86 gm. of a light yellow crystalline alkaloid.
A 1.50 gm. sample of this product was recrystallized twice from m ethanol to ob tain 5 ^ mg. o f pure compound as c o lo r le s s , slen d er r o d s, m.p. 170-171° C; X 284 mu. (log E 3.89). Tetrahydroberberine (dl- canadine) was reported in the literature to have a melting point of 109
169-170° C (41); 173° C (48); and 171° C (97). A mixture melting point determination with authentic sample of tetrahydroberberine gave no depression.
In frared sp ectra o f t h is compound and known tetrahyd rob erberine were shown to be identical.
The identity of the tetrahydro derivative was therefore proved to be tetrahydroberberine. This, in turn, gave further proof to the identity of the alkaloid isolated from Qu fraction D and fraction
*'20-D" as being berberine. c . Jataarrhlains, ,chloride anti ■1a~trorrfalalne iodide
Jatrorrhizine was isolated from Qu fraction E as its chloride and iodide salts by the method described by Reed (96). Fraction E, 1.46 gm., was dissolved in 100 ml. of boiling water and immediately vacuum fil tered through a pad of celite on a Buchner funnel. The resinous material collected on the filter was washed twice with 20-ml. portions of fresh boiling water and the washings were added to the filtrate.
After having scratched the sides of the container with a glass rod and the solution cooled to room temperature, crystallization occurred in the form of very fine yellow needles. The product, 0.086 gm., was harvested by vacuum filtration and was labeled as "W-E + Cl (meaning
Fraction E alkaloid chloride obtained from water).
The post W-E + Cl aqueous filtrate was concentrated to 50 ml. in vacuo a t 50° C, warmed on a steam b a th , and im m ediately f i l t e r e d through a freshly prepared celite pad on a Buchner funnel by vacuum. This 1 1 0 filtrate was placed on a steam bath and a hot, saturated, aqueous potassium Iodide solution (3.S.K .I.) was added dropwise, while con tinually shaking the flask, until slight turbidity resulted. It was then filtered using a Buchner funnel. Upon cooling the filtrate to room temperature, the alkaloid iodide crystallized as fine yellow needles. A yield of 0.234 gm. was obtained which was recrystallized by dissolving in 50 °f boiling water. The resulting solution was im m ediately f il t e r e d and allow ed to c o o l to room tem perature. From the cold filtrate, 0.04-3 gm. of needle crystals was obtained, m.p. 196-
198° C, dec. The code name of "W-E + l ” " was given to this product.
Meanwhile, the residue remaining on the filter paper from the filtration of the turbid, aqueous solution was found to be alkaloid positive by testing with Valser*s T.S. This alkaloidal residue was dissolved in hot U.S.P. alcohol (25 m l.), which was subsequently
concentrated in vacuo to approximately five m illiliters, followed by warming on a steam bath and filtering. The filtrate, after cooling
to room temperature, yielded 0.018 gm. of yellow needle crystals of
alkaloid iodide +1" »), m.p. 211-212° C, dec; A 428 mp
(log E 3-77), 352 mp (4.48), 265 mp (4.50), 222 mp (4.62).
The melting point of this compound was in good agreement with
those reported in the literature of 210° C, dec. (98); 208-210° C,
dec. (99); 212° C, dec. (48); and 213-215° C, dec. (95) for
jatrorrhizine iodide. A mixture melting point determination
with an authentic sample of jatrorrhizine showed no depression.
Ultraviolet spectra of this compound were also determined in 0.01-N
alcoholic sodium hydroxide and 0.01-N alcoholic hydrochloric acid Ill
(Table 7) and found to correspond to those reported for jatrorrhizine iodide by Reed (96 ) and Hussein (95)•
The compound was found to be o p t ic a lly in a c tiv e . J a tr o r rh izin e iodide is optically inactive. An infrared spectrum (potassium bromide pellet) was superimposable with that of reference jatrorrhizine iodide, providing further proof for the identity of the compound to be jatrorr hizine iodide.
The alkaloid iodide, W-E + I ~ , was also identified to be jatrorr hizine by infrared spectral analysis and T-L-C studies, silica gel G and n-propanol:ammonium hydroxide:water (2:1:1) as the developer.
The alkaloid chloride, W-E +CI ”, originally crystallized from water, was also recrystallized from 95$ ethanol by the procedure em ployed for the jatrorrhizine iodide (E-E + I “ ) crystals. From 0.086 gm. of the crude product 0.020 gm. of analytically pure alkaloid chloride tf'E-E + Cl “ ") was obtained, m.p. 20*4—206° C, dec. , with the remainder of the starting material being recovered as such from the mother liquor. The melting point of jatrorrhizine chloride has been reported as
206° C, dec. (98 ) and 205-205.5° C, dec. (96 ). A mixture melting point determination of this compound with an authentic sample of jatrorrhizine chloride showed no depression.
The ultraviolet spectra of this compound taken in 95$ ethanol,
0.01 -N alcoholic hydrochloric acid, and 0.01-N alcoholic sodium hydro xide (Table 7) corresponded to those reported by Reed (96 ); the infrared spectrum (potassium bromide pellets) was superimposable with that of 112 referen ce ja tr o r r h iz in e c h lo r id e ; and the compound was shown to be optically inactive.
The identity of the alkaloid, E-E + Cl ” , was thus proven by
these physical data to be jatrorrhizine chloride.
As previously stated, jatrorrhizine was also isolated from Qu
fraction F. Unlike Qu fraction E, the procedure utilized for the
isolation of berberine iodide rather than Reed’s method for jatrorr hizine iodide was used for the isolation of jatrorrhizine iodide from
this fraction because of the low yield of the alkaloid from the
former fraction by Reed’s method.
The dried fraction, 2.6^ gm., was dissolved in 100 ml. of 95$
alcohol by heating on a steam bath. A small amount of activated char
coal was added and the mixture refluxed for one-half hour. At the end
of this period, the mixture was filtered and the charcoal residue was
washed with three 10-ml. portions of fresh solvent. These washings
were added to the filtrate, which was warmed on a steam bath and a hot,
saturated, aqueous solution of potassium iodide was added dropwise until
the solution became cloudy. After cooling to room temperature, the
alkaloid iodide crystallized as yellow needles, yield 0.716 gm. The
compound was found to resemble jatrorrhizine in its microchemical test
c o lo r (red) when exposed to ammonia vapor.
Concentrating the mother liquor to one third of its original volume
in vacuo, followed by warming on a steam bath (to redissolve any pre
cipitated material while concentrating) and subsequent cooling to room
temperature, yielded an additional 0.2^2 gm. of the same crystals. 113
The two crops of crude crystalline material were combined (0.958 gm. total weight), washed with about five m illiliters of water to remove any potassium iodide that might be present, and taken up in boiling ethanol for recrystallization. The pure alkaloid iodide crystallized as yellow needles, m.p. 210° C, dec., yield 0.710 gm. It was identified as jatrorrhizine iodide by mixture melting point and infrared spectral comparison with an authentic sample.
From the total of 4.100 gm. of Qu Fractions E and F, 1.192 gm. of crude jatrorrhizine iodide and 0.860 gm of pure jatrorrhizine chloride was obtained using different crystallization methods. The theoretical yield of jatrorrhizine as the iodide is (2.26 gm)X gm* = 2*687 gm., or 0.0134$ of the starting plant material. This figure, however, is not the true alkaloid content in the plant since jatrorrhizine was also detected in Qu fraction G and possible in Qu fraction H, but not is o la t e d . d. Magnoflorine iodide
The quaternary aporphine alkaloid, magnoflorine, was isolated from the Qu fractions I and J in the form of its iodide salt. Its identifi cation was made on the basis of physical evidence obtained from its iodide and chloride derivatives. Although both of these fractions
seemingly contained the same alkaloid, they were kept separated because
Weight of total Qu fraction; 12. Weight of Qu fraction chromatographically separated for alkaloid isolation. 1 1 4 of the slight difference of their fluorescence on the eluting column,
■where fraction I showed a greenish-blue fluorescence and J showed a bright-blue fluorescence (Table 6).
Magnoflorine iodide was isolated and recrystallized from these fractions by the procedures described for the isolation and recrys tallization of jatrorrhizine iodide from Qu fraction F. From 9-08 gm. of the Qu fraction I and 20.11 gm. of the Qu fraction J, respective yields of 7*52 gm. and 15.82 gm., or a total of 23.3^ g®* » of crude magnoflorine iodide was obtained. The theoretical yield of this alkaloid iodide from the starting plant material is 27.77 gm.^ or
0.1389# 14.
Twelve grams of the crude alkaloid iodide was recrystallized once from ethanol, yield 9«*K) gm. of colorless crystals, m.p. 248-249° C, dec. The melting point of magnoflorine iodide has been reported to be:
252° C, dec. (70); 256-258° C, dec. (65); and 248-249° C, dec. (44, 58).
A mixture melting point determination of an authentic sample of magno f lo r in e io d id e and t h is compound showed no d ep ressio n o f the m eltin g point of the latter sample. 28 5 c Optical rotation for this compound was found to be: [a]jj * + 204.3
(c ., 0.20 in methanol). The literature reported varying values for magnoflorine iodide:[ a ] + 214° (c., 0.254 in methanol) (70); [cQ^ +
(total weight of 13 23.34 gm. (magnoflorine iodide isolated) X ' fractJjMreJfitoma- tograpnically separated for is o la tio n ) 14 20 ,ouo gm. X 100 = O*1# # . 115 17 197.50° ( c * » 0. 244in methanol) (81) ; [a] ^ + 203. 2° (c. , 0. 25? in methanol) (8l) ; [cQd5 + 220' 10 (methflmo1) C58)' An ultraviolet spectrum of this compound, detemined in 95$ alcohol, corresponded well to that reported by Tomimatsu (70, 71, 81) for magno- florine iodide, despite the fact that water and methanol were alternately employed as the solvent by the author. In addition to 95$ ethanol, the spectrum of the newly isolated, blue fluorescent, 0.62-0.64 alkaloid was determined in 0.01-N alcoholic sodium hydroxide and 0.01-N alcoholic hydrochloric acid. These data are presented in Table 7.
Infrared spectra (potassium bromide pellets) of reference magno- f lo r in e io d id e and t h is compound were id e n t ic a l.
Based on the physical data presented above, the alkaloid iodide isolated from Qu fractions E and F was identified as magnoflorine in the form of its iodide salt. Additional evidence of the identity of this alkaloid as magnoflorine was furnished by converting a sample of the crude alkaloid iodide to its corresponding chloride salt, followed by physical analyses of the latter compound. c. Magnoflorine chloride
Magnoflorine chloride was obtained from a one gram sample of crude magnoflorine iodide by the application of the principle of ion-exchange.
For this purpose, an exchange column was prepared with 200 gm. of activated IRA-410 ion-exchange resin of the commercial type.
The activation and preparation of the ion-exchange resin column was accomplished by washing the resin with 10$ hydrochloric acid six times, followed by washing with distilled water until the washings 1 1 6 were pH 7.0 as indicated by Hydrion paper. The resin was then packed into a chromatographic column (4.5 cnu diameter X 48 cm. length) by the slurry method using methanol as the vehicle.
After washing the column of resin with methanol several times, the sample of magnoflorine iodide, dissolved in a minimum quantity of meth anol, was passed through the column at the rate of 10 ml. per minute.
Additional volumes of solvent were employed to completely elute the ion- exchanged alkaloid from the column. The absence of the typical bright blue fluorescence of magnoflorine under ultraviolet light was taken as the end point.
The methanol eluates were combined and vacuum evaporated to dryness, yielding an amorphous residue. This material was then dis solved in 50 ml. of a hot solvent mixture of methanol and ethanol in equal volum es. Upon c o o lin g the so lu tio n to room tem perature, magno florine chloride crystallized from the solvent as fluffy needles. After these crystals were collected, the mother liquor was concentrated to 20 ml. which re stilted in a second crop of the alkaloid. The combine weight of the magnoflorine chloride crystals obtained was found to be 0.345 gm., m.p. 236- 237° C, dec.; (a]^ + 215° (c ., 0.204 in methanol); EtOH \ Max. 327 dijx (log E 3.82), 2?8 mp (4.19), and 230 mp (4.57). The melting point of magnoflorine chloride was reported to be 238-240° C, dec. by Hussein (95) and 242-243° C, dec. by Winek (100). A mixture melting point determination of this compound and a reference sample of magnoflorine chloride showed no depression. The optical rotation of this compound was in good agreement with those of 213° (methanol) and 212° (methanol) reported by Winek (100) and Hussein (95)» 11?
respectively, for magnoflorine chloride.
The ultraviolet spectra of this compound, determined in 95$
ethanol, 0.01 N-alcoholic hydrochloric acid and 0.01-N alcoholic
sodium hydroxide (Table 7), corresponded to those reported by the
aforementioned workers for magnoflorine chloride (95• 100).
Infrared sp ectra (potassium bromide p e lle t s ) o f t h is compound and
that of reference magnoflorine chloride were superimposable.
There is no doubt, from the above data, that this ion-exchanged
alkaloid was magnoflorine chloride. This, then, is further proof that
the alkaloid iodide isolated from the Qu fractions, I and J, was magnoflorine iodide.
G. Methodology-Chemical and Physical Analyses
1. Thin-layer chromatography
a. Thin-laver chromatography on silica eel G plates
Except in a few isolated situations, the chromatographic studies
of the alkaloids were performed throughout this investigation utilizing
the thin-layer technique of Stahl (88) with silica gel G^ as the ab
sorbent. After development, the chromatograms were viewed tinder a com
bination of long and short wave ultraviolet light to note the color of
the fluorescense(s) of the alkaloid(s), if any, prior to being stained
^ Silica gel G acc. to Stahl for thin-layer chromatography, E. Merck Ag., Darmstadt, Germany, Distributor: Brinkman Instruments Inc., 115 Cutter M ill Rd., Great Neck, L .I., New York. 118
■with Munier and Machebouef's Modified Dragendorff' s spray reagent (101). b. Thin-laver chromatography on microcrvstalline cellulose plates
The qualitative thin-layer chromatography of sugars and quaternary alkaloids was performed on plates coated with microcrystalline cellu lose, commercially known as "Avirin", according to the method described by Wolfrom ^1. (93)• In this method, the principles of thin-layer chromatography and paper-partition chromatography are involved, since the preparation and development of the chromatograms are those of the former, while the developers are solvent systems that have been successfully used in paper-partition chromatography.
In the preparation of the chromatoplates, 100 gm„ of microcrys talline cellulose is blended in a Waring blendor for 30-^5 seconds with k2Q ml. of distilled water and 10 ml. of methanol. The glass plates of
200 mm. X 200 mm. size, having first been thoroughly cleaned with soap, water, methanol and then acetone, were coated with a 1.0 mm. thick layer by means of a Desaga applicator , which was moved slowly across the plates at an even rate. The poured plates, after being dried over night at room temperature, were stacked together in a locker until use with no special storage containers being required.
For the sugar analysis, the developed chromatograms were lightly sprayed with the diphenylamine-aniline reagent devised by Bailey and
Bourne (102) and placed in a pre-heated oven at 80° C for five minutes.
Brinkman Instruments Inc., Great Neck, L .I., New York. 119
After removing the chromatograms from the oven, the characteristic colors and Rj. values of the spots were noted. Examples of the colors of sugars are greenish-blue for D-glucose and yellow-brown for fructose. The formula for this reagent is as followss
Diphenylamine 4 .0 gm. A n ilin e 4 .0 ml. 80 $ Orthophosphoric Acid 20.0 ml. Acetone 200.0 ml.
The detection of quaternary ammonium alkaloids (Qu) was performed in the usual manner of viewing under ultraviolet light, followed by staining with modified Dragendorff’s spray reagent.
2. Benedict’s test for reducing sugars
In the a n a ly s is o f compound ”20-x” (s u c r o se ), a few m illigram s o f the crystals were dissolved in five m illiliters of distilled water in a test tube. Two drops of a 0.20 N sodium hydroxide solution was then added to render the solution alkaline, followed by the addition of two drops of Benedict’s T.S (103) and boiling the contents for three minutes.
A positive test for reducing sugars was indicated by the presence of a red precipitate at the bottom of the test tube.
The h y d roch loric a cid h yd rolysate o f t h is compound was s im ila r ly tr e a te d .
3. Labat test for methylene dioxy groups (104)
A few crystals of the alkaloid were dissolved in two m illiliters of concentrated sulfuric acid in an evaporating dish. To this mixture 120 was added 0.10 ml. of a solution of five per cent gallic acid in 95$ ethanol. The resulting mixture was warmed on a steam bath. A positive test was recorded if the solution changed from its original yellow- brown color to an emerald green, and then to a blue color. h-. Ammoniacal-phosphomolybdic acid test for hindered hydroxyl groups ( 105)
A few crystals of the alkaloid were dissolved in 0.10 ml. of ethanol in a spot plate. A drop of phosphomolybdic acid was added, followed by a drop of 28$ ammonium hydroxide solution. A positive
test is indicated by a color change in the solution (any color other
than the original color).
5. Claisen*s cryptophenol reagent for the detection of phenolic groups (106)
To two m illiliters of a 50$ potasium hydroxide solution was
added an equal volume of methanol, followed by a few crystals of the
material being tested. Phenolic compounds are readily soluble in this
strong alkali, while non-phenolic compounds are insoluble.
6. Tomita1s test for dibenzo- p-dioxane groups (107)
A few crystals of the sample were dissolved in one m illiliter of
concentrated sulfuric acid. Two or three potassium nitrate crystals
were then added and stirred with a small glass rod. A positive test
is indicated by a blue color „ 121
7. Melting point determination
The melting points of the isolated compounds were determined using either a Fisher-Johns melting point apparatus^ or a Thomas-
Hoover capillary melting point apparatus with the former being o used for compounds melting below 200 C and the latter for those compounds that melt or decompose at temperatures greater than 200°
C and for mixture melting point determinations. Each apparatus was standardized with U .S.P.^ reference compounds and the melting points reported for the isolated compounds were corrected accordingly.
8. Optical rotation measurements
The optical rotation of each alkaloid was measured in a Carl-
Zeiss polarimeter using a 0.5 decimeter sample tube. The solvents employed were either chloroform or methanol.
9. Ultraviolet spectral analyses
The ultraviolet absorption spectra were determined using a Cary 20 Model 15 Recording Spectrophotometer . With the exceptions of tetrahydroberberine and sucrose (compound 20-x ), which were determined
F ish e r S c ie n t i f ic Company, P ittsb u r g h , P ennsylvania. “I Q Arthur H. Thomas Company, P h ila d e lp h ia , Pennsylvania.
^ Distributed by the Board of Trustees of the United States Pharma- copoeial Convention, Inc. 20 Allied Physics Corp., Monrovia, California. 122 o n ly in 95$ ethanol, all compounds were analyzed in 0.01-N alcoholic sodium hydroxide and 0.01-N alcoholic hydrochloric acid, in addition to the 95$ ethanol.
In the preparation of the sample solutions for analysis, stock
solutions of known concentrations were prepared in 95$ ethanol. For the neutral spectra determinations, one m illiliter of the stock solu tion was added to nine m illiliters of 95$ ethanol. Where acid or alka line spectra were desired, one m illiliter of 0.1 N hydrochloric acid in
95$ ethanol or 0.1 N sodium hydroxide in 95$ ethanol and one m illiliter of the sample stock solution were placed in a volumetric flask and the volume adjusted to 10.00 ml. with 95$ ethanol.
10. Infrared spectrophotometric analyses
The infrared absorption spectra were determined as suspensions in potassium bromide pellets or in chloroform liquid films as indicated, using a Perkin Elmer Model 237 Grating Infrared Spectrophotometer. 21
11. Nuclear magnetic resonance spectrophotmetric analyses
The nuclear magnetic resonance spectra determinations and inter
pretations were made by Dr. David R. Dalton, Post Doctoral Fellow,
Department of Chemistry, The Ohio State University, using a Varian op A-60 Nuclear Magnetic Resonance Spectrometer with tetramethylsilane
as an internal standard at - 1 0 .0 0 .
pi Perkin-Elmer Corp., Norwalk, Connecticut.
^ Varian Associates, Paloalto, California. 123
12. Molecular weight determination
The molecular weight determination of the alkaloid, TNC, using a
Mechrolab Model 301-A Osmometer2-^, was conducted by Mr. Charles Root,
Department of Chemistry, The Ohio State University.
13. Microanalyses
Microanalysis of TNC for C, H, and N was made by the Midwest
XT. Microlab, Inc., 6000 East 46— Street, Indianapolis, Indiana.
Analyses of the sucrose (compound 20-x) composition and per cent of (OCH^) in the alkaloid, TNC, were performed by Alfred Berhart,
Mikroanalytisch.es Laboratorium im Max-Planck Institut fur Kohlen-
If forsch u n g, Hohenweg 1 7 , 433 Mulheim (Ruhr), Germany.
14'. Structural elucidation studies
The structural analysis of the major alkaloid, TNC, is being
2Ll conducted by Dr. Tosiaki Tomimatsu, Post Doctoral Fellow in the
College of Pharmacy, Department of Pharmacognosy, Tine Ohio State
University.
15. Pharmacological screening for hypotensive activity
The hypotensive studies of the total alkaloid extractive, the
•'tertiary" non-phenolic alkaloid fraction, the "tertiary" phenolic
2^ Mechrolab, Inc., Mountain View, California. 24- Assistant Professor of Pharmaceutical Chemistry, Tokushima University, Tokushima, Japan. 12k alkaloid fraction, and the quaternary alkaloid fraction of Roche- brunianum were conducted by Dr. Popat N. Patil, Post Doctoral Fellow,
The Ohio State University, College of Pharmacy, Department of Pharma co lo g y .
The hypotensive activities of the alkaloids, TNA, TNB, TOC, and hemandezine were determined by Mr. Richard Hahn of the College of
Pharmacy, The Ohio State University. IV. DISCUSSION
The isolation of sucrose and seven alkaloids from Thaii ntrum
Rochebrunlanum. (Ranunculaoeae) during this investigation marked the first recorded isolation and characterization of chemical constituents from this plant. Four of these alkaloids have been identified as compounds previously isolated from other species of
Thalictrnnit three are yet -unidentified.
The four known alkaloids were shown to be jatrorrhizine, ber- berine, magnoflorine, and hernandezine. It can be seen from the structures illustrated in Figures No. 3 to 6, that Jatrorrhizine and berberine, are of the quaternary protoberberine series and that magno florine belongs to the quaternary aporphine group, while hemandezine is a tertiary, non-phenolic, bisbenzylisoquinoline alkaloid. The iso lation of these known alkaloids from 1. Rochebrunianum and other species of the same genus is not surprising from a chemotaxonomic view, for it is a well known fact that related plant species, genera, or even fam ilie s of the same order do produce similar and/or identical chemical compounds.
Two of the three unidentified alkaloids, TNA, and TNC, can definitely be stated to be tertiary, non-phenolic alkaloids of the bisbenzylisoquinoline type on the basis of their characteristic physical properties, which, while similar, are not identical to those of any of
125 12 6 the known alkaloids of this series.
TNC is the major tertiary alkaloid present in this plant. This
conclusion was based on the fact that 7.25 gm. of this alkaloid was
isolated as compared with the quantities of the other tertiary alkaloids
(0.89 gm. of hemandezine, 0.252 of TNA.HI, and 0.229 gm. of TNB) iso lated from the same plant, in addition to the fact that the quantities
of the tertiary alkaloids remaining to be isolated were estimated by
the intensity of their Dragendorff’s reagent stained T-L-C spots, to be very small.
Although the N.M.R., I.R. and U.V. spectra strongly suggest TNC
to be nonrphenolic, the possibilities of a hindered hydroxy or other
functions such as a dibenzo-prdioxane group could not be excluded.
These possibilities were brought about by the theoretical formula of
C39H46°8N2 ^or TNC, while the formula of hemandezine (50) » the only
known dimeric benzylisoquinoline alkaloid containing five 0-methyl
groups, was established as C^H^O^Ng. To ascertain the presence or
absence of these functional groups, a number of microchemical tests were performed. The Labat test for methylene dioxy groups (106), the
ammoniacal-phosphomolybdic acid test for hindered hydroxyl groups (107)»
the Claisen’s cryptophenol reagent test (108) for phenolic compounds
and Tomita* s test for dibenzo-p-dioxane functions (109) gave negative
r e s u lt s .
In view of these negative microchemical tests, and the fact that
its N.M.R., I.R ., and U.V. spectra resembled those of hemandezine, the
only alternative left to account for the elemental analytical data of
TNC was that one molecule of water of crystallization was present in 1 2 7 the sample. Were this true, then the formula could be rewritten as
C39%14.O7N2.H2O. To test the validity of this water of crystallization th eo ry , a second sample o f th e compound was d ried over b o ilin g w ater at 0.30 mm. mercury for eight hours, with phosphorous pentoxide as the desicant, and analyzed for its composition. It was found that even at this elevated temperature and low pressure, the result was about the same as those obtained from the original analyses, for wich the sam ples were dried at 0.5 nnn. mercury and heated over boiling benzene.
The TNC composition from this analysis was found to be: C, 70.22;
H, 6.72; N, 4.35; and 0, 18.71 (by difference).
Because of the difficulties encountered at this point, the exact molecular weight of this compound could not be determined and additional physical and chemical analyses must be conducted.
The s tr ik in g s im ila r it ie s o f the U .V ., I.R . and N.M.R. sp ectra o f
TNC and those of hernandezine, plus the fact that it was not possible to chromatographically separate them, resulted in the assumption that these two alkaloids are either isomers, or that they possess very
similar structures.
It can be seen from the N.M.R. data in Table 8 that the chemical
shifts in terms of the -f values, of the methoxyl groups of TNC and hernandezine correspond to each other, but not of their respective methylimino resonances. When one compares these data to those found by Bick jjl. (108) for tetandrine (XXVII) and O-methylrepandine
(XXVIII)(see Table 8), it would seem logical that the hernandezine
structure would resemble tetandrine and that TNC should resemble 0- methylrepandine. 128
3 h3<> f-CH. 3
xxvn XXVIII
Indeed, Padilla and HerrSn (50) presented the structure of her
nandezine patterned after tetandrine -with the extra methoxyl group,
having a 'j*' value of 6.17, fixed in position 8*. Following the same
line of reasoning, one could postulate structure XXIX for TNC,
patterned after O-methylrepandine with th e q" = 6.19 methoxyl in the 8»
position. This, however, is not possible, in view of position 8’
being occupied by an aromatic proton as seen in the low field of the
N.M.R. spectrum and the fact that when TNC was cleaved with sodium in
liquid ammonia, followed by methylation of the resulting phenolic 2*5 compounds with diazomethane, the products XXX and XXXI were obtained .
5 Degradative studies of TNC were performed by Drs. T. Tomimatsu and D. R. Dalton, Post Doctoral Fellows in the College of Pharmacy and Department of Organic Chemistry, respectively. Compound XXX, m.p. 62.0 ° Cl EDS' + 82.3° (c ., 0.66 in chloroform) was identified as d-O, 0, N-trimethylcoclaurine or l-(4 ,-methoxybenzyl)-2-methyl- 6,7-dimethoxy-1,2,3, tetrahydroisoquinoline by mixed melting point, and comparisons of its I.R. and N.M.R. spectra with those o f a known a u th en tica ted sample. The stru ctu re fo r compound XXXI was deduced from i t s N.M.R. spectrum. 129
OCtt OCE f-CH.
XXIX XXX
If the fifth methoxyl group (T = 6.10) of TNC is located at position 8* of O-methylrepandine, as in structure XXIX, then theoreti c a lly m eth ylation o f the cleavage products o f t h is compound would y ie ld compound XXXII in add tion to d -0 ,0 ,N -tr im e th y lc o c la u r in e . But compound XXXII was n o t o b tain ed , th e r e fo r e , one can elim in a te stru ctu re
XXIX as being that of TNC. On the other hand, one can assume that TNC is derived from compounds XXX and XXXI.
OCH 0-CH.
0-CH.
XXXI xxxn
Of course, it is not possible to write the absolute structure of this new compound when the structural elucidation studies are incomplete.
One can, however, propose a possible structure from available informa tion such as the reported structures and properties of compounds of the same chemical class from other species of the same genus or family of plants in which X, Rochebrunianum belongs, the physical and chemical 130 properties of the ■unknown compound, and the results of the preliminary degradative studies. It therefore follows that any and all of the structures proposed in this manner are purely conjectural. In this case, one could propose entity XXXIH as a theoretically possible structure for TNC.
dCi'zqme^.]
xxxm (XXX) + (XXXI)
This structure appears quite plausible on the basis of the theoretical degradative sequences indicated by the above equation.
The a lk a lo id , TNA, was shown by i t s N.M.R. spectrum to be composed of five methoxyl groups in addition to one N-methyl group
(Table 8). This would indicate that the compound is either a des-N- methyl bisbenzylisoquinoline or a monomeric alkaloid such as those of the aporphine series, when the known alkaloids isolated from this plant and other species of TfraTlctrmn are taken into consideration. Usually, the question of whether a compound is monomeric or dimeric can readily be resolved by a molecular weight determination and an analysis of its chemical composition. Unfortunately, the small quantity of this alka loid available was not sufficient for such an analysis, and resolution of this question is therefore dependent on physical data.
The possibility of TNA being an aporphine alkaloid was feasible in view of the recently synthesized 1,2,8,9,10-pentamethoxyaporphine by 131
H irai (1 0 9 ). The f a c t th a t the U.V. ab sorp tion maxima o f t h is synthetic aporphine at 290-310 mp and 280 mp do not correspond to those of TNA (Table ?), and the fact that the N.M.R. spectrum of
TNA (Table 8) indicated the presence of more than two aromatic protons, the maximum number present in a pentame thoxy aporphine, eliminated this possibility.
The fact that a protoberberine alkaloid with a methyl group attached to its nitrogen atom, would by necessity be a quaternary alkaloid, of course eliminated it from consideration.
By a process of elimination, TNA must, therefore, belong to the bisbenzylisoquinoline series of alkaloids. The nuclear magnetic resonance (Table 8) of four of the five methoxyl groups in TNA cor respond to those of isotetandrine, a diastereoisomer of tetandrine
(XXXIV) and hemandezine, gave added support to this theory. Further-
E + Q t l more, A. 332 mp of TNA could be interpreted to mean that one of the nitrogen atoms is an internally bounded imino function, and the hypothetical structures XXXV and XXXVI can be written for TNA. If this hypothesis is true, then one can also assume this alkaloid to
C.H3
XXXIV XXXV XXXVI be the immediate parent compound of hernandezine. All these assumptions and theories, of course, can not be proven until a sufficient quantity 132 of this alkaloid is obtained and its structure fully studied.
The third unidentified alkaloid isolated from this plant is the hypotensive, tertiary, non-phenolic alkaloid designated as "TUB."
Because it was decomposed during recrystallization, physical and/or chemical characterization of this compound was not possible. Although it was detected to be present in the post TNC and hernandezine methanolic mother liquors, originally obtained from the gradient pH extractives at pH 3»5» *K0, 4 .5, and 5*0* attempts to isolate this alkaloid by adsorption column chromatography and fractional crystalli zation were to no avail, possibly due to the presence of other alkaloids and impurities. A few milligrams were obtained, however, when one of these fractions was subjected to an additional gradient pH extraction at pH 2.8- 3.O. It was noted at this point that when the citric acid solu tion of the alkaloids had been extracted five or six times With benzene, the pH of the acid solution dropped approximately 0. 2 units and further extractions yielded no additional alkaloid in the organic solvent,
although TNB was detected to be s till present in the aqueous mother
liquor along with other alkaloids. This change of pH may be explained
on the basis that the amount of this alkaloid base formed at this
particular pH level was completely extracted by benzene, and thereby
caused a lowering of the pH. The presence of this alkaloid in the
mother liquor was probably due to an insufficient addition of the
quantity of ammonium hydroxide solution required to convert a ll of
this alkaloid to its free base form. Therefore, not being extracted
by the organic solvent, it eventually 11 spilled-over11 into subsequent
extractions at higher pH levels. If this theory is true, then a 133 further modification of the pH gradient technique might be made to give
a more complete separation of each alkaloid at the proper pH level with a minimum amount of "spill-over” into the next pH level. One
such modified procedure that might accomplish these goals is as fo llo w s t
(1) Dissolve the total alkaloid extractive in chloroform and filter to remove apy non-alkaloidal precipitate.
(2) Extract the alkaloids from the chloroform solution into 0.2 or 0.3 M citric acid by the usual method of intimately mixing the two immiscible solvents, followed by vacuum evaporation of the organic solvent.
(3) After complete removal of chloroform, filter the aqueous solution, record the pH of the filtrate (usually about 2.4— 2.5 at this point) and then exhaustively extract with benzene to remove the alkaloid base formed at this pH (e.g. TNA).
(4-) Adjust the pH of the aqueous mother liquor to pH 2.8-3.0 with 10$ ammonium hydroxide solution followed by successive extractions with benzene to remove the second alkaloid (e.g. TUB) until a lowering of the pH of the aqueous solution is noted. At this point, additional ammonium hydroxide solution is added to raise the pH of the aqueous solution back to 2.8- 3.0 and again extract with benzene. Note: do not exceed pH 3.0, as the third alkaloid (e.g. TNC) w ill be formed at a pH slightly higher than 3.0 and therefore, be removed along with the second alkaloid. If necessary, repeat this procedure a number of times until most, if not all, of this particular alkaloid has been removed from the aqueous solution.
(5) The mother liquor should then be successively raised to pH 3.5# *K0, 4-. 5, 5.0, 5.5, 6.0, 6.5 and 7.5-8.5- With the procedure outlined in Step No. 3 being employed for the com plete extraction of the alkaloids at each of these pH levels.
(6) The benzene extractives from each pH level should be im mediately dried over anhydrous sodium sulfate or calcium carbonate to remove residual water from the extractive, filtered, and the filtrate concentrated to a small volume. Follow by extraction of the concentrate into five per cent hydrochloric acid, which is then alkalinized with ammonium hydroxide and the alkaloid(s) recovered by extraction with eth er. 13^
This proposed method, while logical in theory, must still be sub jected to actual experimental determinations as to its applicability for the separation of these alkaloids.
The discussion of the alkaloid TNB would not be complete without discussing the procedures used, or which could possibly be used, for its crystallization. As indicated earlier, this alkaloid is very un stable to heat. Normally, compounds of this nature are made into salts such as hydrochlorides, oxalates, hydroiodides, or even sulfates to overcome this undesirable effect. But all attempts to make these derivatives with TNB ended in failure. This phenomenon may be due to the alkaloid being a very weak base, or to a lack of skill on the part of this investigator; at any rate, this alkaloid could only be obtained as the free base. Since the method of concentrating the crystallization solvent by distillation on a steam bath, so successfully used throughout this investigation, could not be employed in this case, vacuum concen tration must then be used in future studies with this compound.
Perhaps the crystallization of this alkaloid could be achieved more easily if, prior to the initial crystallization step, the impure alkaloid extractive is dissolved in ether and the resulting solution is chilled in a dry iee-acetone bath to obtain a crop of relatively pure amorphous material, which could then be crystallized from either methanol or absolute alcohol. The most important factors to keep in mind during the crystallization process are the reduction of the volume of the solution in vacuo and the use of minimal heat to re dissolve any precipitates in the concentrate prior to placing it in the refrigerator. 135
The isolation of hernandezine is a lesson in the fallacy of placing too great an emphasis on one single physical property as the criterion for the identification of a compound. At first, TNC and hernandezine
(TNC-50) were thought to be th e same compound because o f th e ir id e n t i cal T-L-C (one-dimensional) values and an inability to resolve them by two-dimensional T-L-C. The fact that they were eventually proved to be different alkaloids illustrates the point of how one should not have too much '‘faith" in a usually reliable T-L-C identification method, which theoretically w ill separate two closely related compounds on two- dimensional, if not on one-dimensional chromatograms.
The chemotaxonomic significances of the quaternary alkaloids, jatrorrhizine, magnoflorine, and berberine, in establishing the plant investigated as being a Thalictrum species, have already been d isc u sse d . The q u estio n o f which o f th ese compound i s th e major quaternary alkaloid, however, was not discussed. The quantitative percentage of jatrorrhizine isolated from this plant, 0.0134$ ex pressed in terms of its iodide salt, as compared to 0.14$ of magno florine (iodide), of course, eliminated its being considered as the major alkaloid. Similarly, the quantity of berberine iodide (0. 05%) would also exclude it from consideration. Therefore, magnoflorine would have to be the major compound by a process of elimination.
Berberine and jatrorrhizine were the only quaternary alkaloids in X* Rochebrunianum. whose Dragendorff positive orange spots were observed to change colors (purple and yellow, respectively) when the freshly sprayed chromatograms were immediately exposed to ammonia vapor. Perhaps, advantage can be taken of this observation for the 136 development of a microchemical color test for jatrorrhizine and berberine. However, time was not available to test a series of quaternary alkaloids of related and dissim ilar structure s to determine the specificity and mechanism of action for this reaction. Without such information, it is not possible to state definitely that these color changes can be used for characterization work.
The ”cyclic-percolation” method employed for the extraction of the total alkaloids, summarized in Charts I and U , appeared to be an efficient method on the strength of 0.44$ (w/w) of total tertiary and
0.28$ (w/w) of quaternary alkaloids being extracted from 20 kg. of ground roots. In reality, this process was very tedious, both in the number of steps, and the length of time required to achieve these
separations. The main undesirable feature of this method is the length of time required for the alcohol extractions, which was
approximately three months. During this period, a search for better
extraction methods was conducted since the extraction of the 20 kg.
of powdered roots by ”cyclic-percolation” was well on its way.
Among the experimental methods utilized was the petroleum ether
extraction procedure by which the author’s adviser was able to isolate
thalicarpine from a Thallctrum species (l). This method involved
percolating the powdered plant material, previously moistened with a
20$ ammonium hydroxide solution, with petroleum ether, B.P. 30“60° C.
Applying this technique to a 200 gm. sample of £. Rochebrunianum.
0.832 gm. or a 0.42$ yield of total tertiary alkaloids of comparable
purity as the semi-purified total tertiary alkaloids, 20-TT, from the
’’cyclic-percolation” process was obtained. The quantities of these 137 two total tertiary alkaloid fractions were comparable (2Q-TT=0.44$ of
20 kg.; petroleum ether extracted alkaloid = 0.42$ of 200 gm.). A comparative chromatographic study indicated the alkaloids in each fraction to be similar. Carrying the experiment further, the petroleum ether alkaloid fraction was fractionated into the phenolic and non- phenolic fractions via the same method used for the TN and TP fractions from 20-TT. Again, the chromatographic results of the corresponding fractions (e.g. TP from 20-TT and the phenolic fraction from the petroleum ether extraction) appeared to be identical. This indicates that the tertiary alkaloids of X* Rochebrunianum can be extracted from the plant material in one step by the petroleum ether extraction method, whereas the cyclic-percolation process required a number of steps.
Aside from the advantage of a one-step extraction, the best feature of this new method is that only one week was needed for complete extraction of the tertiary alkaloids.
It could be argued that this method may be good for extracting the tertiary alkaloids, but what about the sugars (sucrose, etc.) and the quaternary alkaloids? If an investigator is interested in the isolation of sugars, or in glycosides other than sucrose, then this extraction process is not applicable. As for the quaternary alkaloids, the petroleum ether method is still good. This was proven when the petroleum ether exhausted marc was percolated first with benzene and then ethanol. Negligible amounts of tertiary alkaloids were ex tracted by the benzene menstruum, indicating almost 100$ extraction by the petroleum ether percolations. This is not surprising in view of these alkaloids being very weak bases, as indicated by their being extractable from highly acidic aqueous solutions by organic solvents 138
and the difficulties encountered in attempts to form salts. Meanwhile,
the alkaloids extracted by the ethanol percolations were identified by
T-L-C to be the quaternary alkaloids also found in the Qu fraction of
the "cyclic percolation11 extractives.
The shorter length of time and fewer number of steps required by
the method of percolating an ammonium hydroxide moistened plant material with petroleum ether for the complete extraction and frac
tionation of the total alkaloid, certainly would seem to be more
advantageous than the "cyclic-percolation" method for obtaining the
alkaloids from 1. Rochebrunianum. and possibly other Thalictrum
species. Indeed, the procedure as outlined below would have been
followed were this method discovered prior to the extraction of the
plant material by the "cyclic-percolation" method.
A. E x tra ctio n P rocedure:
1. Moisten the ground roots with 20$ ammonium hydroxide so lu tio n .
2. Macerate the moist powder with petroleum ether overnight, followed by percolation with fresh menstruum until the tertiary alkaloids are completely extracted.
3. Percolate the petroleum ether exhausted marc with benzene to remove any residual tertiary alkaloids and combine these w ith 2.
Extract the marc with ethanol to remove the quaternary a lk a lo id s .
B. Fractionation Procedures.
1. Fractionate the petroleum ether extracted tertiary alka loids into the phenolic and non-phenolic fractions as for the TN and TP fractions on pages 69-70.
2. Separate the non-phenolic alkaloids by the proposed gradient pH technique as outlined on pages 71-72. Separate the quaternary alkaloids by chromatography with silicic acid-celite-5^5 (6:1) as described for the Qu alkaloids on pages 100-101. V. SUMMARY OF FINDINGS
A complete literature survey regarding the alkaloids of the genus Thalictrum and the taxonomy of 1. RftghstermUnMBl have been presented. Preliminary investigations of the alkaloids of the roots of Thalictrum Rochebrunianum. Franc, and Sav., were made and the results of the quantitative estimation of alkaloid content; extraction and fractionation of the alkaloids into the tertiary phenolic (TP), tertiary non-phenolic (TN), and quaternary (Qu-l) fractions; thin-layer chromatographic studies; pharmacological screening of the different fractions for hypotensive activities; solubility studies of the TN and TP alkaloids in chloroform and benzene; multibuffered paper chromatography and gradient pH separation of the TN and TP alkaloids; and column chromatographic separation of the quaternary alkaloids are reported. A large scale extraction of Thalictrum Rochebrunianum root alka loids from a 20 kg. sample by a "cyclic-percolation" process has been described. Concentrating the volume of the alcoholic percolate vacuo resulted in the isolation and identification of sucrose. Thin- layer chromatographic results, a microchemical test, and analytic and physical data fo r sucrose have been reported. The fractionation of the total crude alkaloids into the tertiary non-phenolic (TN), tertiary phenolic (TP) and quaternary (Qu) fractions is described and the weight and per cent yield of each fraction reported. The separation of the TN alkaloids by gradient pH extraction resulted in the isolation of the alkaloids, TNA, TNB, TNC, and hemandezine (TNC-50). Several physical constants have been reported for these alkaloids, except TNB. Analytical data and a partial structure were determined and reported for TNC, the major alkaloid. In addition, the identification of TNC-50 as hernandezine, and the hypotensive activity of TNB are reported. Thin-layer chromatographic studies of the tertiary phenolic (TP) alkaloids were performed and the results reported.
The chromatographic sep a ra tio n o f the quaternary (Qu) a lk a lo id s on silicic acid-celite (6:1) column resulted in the isolation and identification of jatrorrhlzine, magnoflorine and berberine iodides. Physical constants of these alkaloid iodides, as well as the chloride salts of magnoflorine and jatrorrhizine, and tetrahydroberberine have been determined and reported. In addition, magnoflorine was designated as the major quaternary alkaloid. APPENDIX A TABLE 7
Absorption Maxima of the Ultraviolet Spectra of the Alkaloids of RjOcfrebrpj^ajyp. ( a ) A lkaloid Solvent X. max. Mp (log E)
TNA EtOH 332; 233; 210; inflection 275-277 0.0 1 N HCl-EtOH 332; 251; 233; inflection 275“2?7 0.01 K NaOH-EtOH 332; 233; inflection 275-277
TNB EtOH 273; inflection 297 0.01 N NaOH-EtOH 273; inflection 297
TNC EtOH 281-282; 206; inflection 240-242 0.01 N HCl-EtOH 289-206 0.01 N NaOH-EtOH 281-282
TNC-50 (Hemandezine) EtOH 282 (3.95); 205 (5.10) 0.01 N HCl-EtOH 283 (3.92); 206 (4.98) 0.01 N NaOH-EtOH 282 (3.88 );
Jatrorrhizine Iodide EtOH 428 (3.77);352 (4.48); 265 (4 . 50); 222 (4 . 62) 0.01 N HCl-EtOH **28 (3.77);3**8 ( 4 . 53); 266 (4 . 53); 222 (4.59) 0.01 N NaOH-EtOH 380 (4.21);327-331 M 6); 2 7 2 (4 . 54); 2 4 5 (4 . 38 ); 219 (4.57) Jatrorrhizine Chloride EtOH 430 (3.75); 348 (**.47); 266 (**.**9); 228 (**.51) 0.0 1 N HCl-EtOH 432 (3.7*0: 348 (**.39); 265 (**.**7); 228 (**.33) 0.01 N NaOE-StOH 382 (**. 2*0 ; 328 (**.23); 272 (**.59); 245 (4.42)
Magnoflorine Iodide EtOH 307 (3-91); 269 (**.37); 223 (**.73) 0 .01 N HCl-EtOH 305 (3.85); 267 (**.15); 223 (4.70) 0 .01 N NaOH-EtOH 311 (3.72); 271 (**.17); 227 (4.66)
£ VjO TABLE 7 (Cont»d)
Magnoflorine Chloride EtOH 32? (3.82); 278 (4.19); 230 (*•57) 1 0.01 N HCl-EtOH 302 (3.88); 267 (*.15); 224 (*.65) 0.01 N NaOH-EtOH 328 (3.86); 279 (3-74); 231 (*•55) Berberine Iodide EtOH 351 (4.42); 265 (*.49); 225 (*•58) 0.01 N HCl-EtOH 351 (*.40); 266 (4.46); 225 (*.58) 0.01 N NaOH-EtOH 362 (4.41); 281 (4. 22)
Tetrahydroberberine EtOH 284 00
(a) Log E was not calculated for the alkaloids, TNA, TNB, and TNC, because the exact • molecular weights of these compounds have not been determined. TABLE 8 Nuclear Magnetic Resonance Data of the Alkaloids, TNA, TNC, TNC-50 (Hemandezine), Hemandezine , Tetandrine^) t Q-methylrepandine ^ , and Isotetandrlne^. ______
Alkaloids ______Functional Group Values inunits, TKS = IO.OOT ^ OCH, Ring CH2 nch3 Aromatic Protons )
TNA 6.09 6.1 2 6.20 6.39 6.70 7.21 7.52 2.67 2.92 3.11 3.45 3.90
TNC 6.14 6.18 6.22 6.66 6.80 7.2 2 7 M 7.54 2.2? 3.50 4.10 TNC-50 (Hemandezine) 6.08 6.19 6.21 6.65 6.76 7 .02- 7.20 7.37 7.70 2.75 3.11 3.42 3.97 Hernande zine 6.09 6.17 6.21 6.66 6.76 7.37 7.70 Tetrandrine^b' 6.10 6. 2? 6.65 6.82 7.00 7.41 7.70 0-me thylrepandine ( 6.05 6.25 6.60 6.95 7.22 7.45 7.45 Isotetrandrine^b ^ 6.05 6.22 6.37 6.82 7.0 7 7.40 7.72
From T. hernandezii by Padilla and HerrAh ( 50).
^ Data published by I.R.C. Bick e£ j l. (108 ). (c) Tetramethylsilane as the internal standard. APPENDIX B
146 TRANSMITTANCE (X) 100 003500 4000 . 30 4C IFN 5 1 70 MICRONS 8 0 7 0 0 6 1 50 MICFCNS 4C 5 3 3.0 2.5 00800 3000 tUNT1* > CJr«) FREQUENCY (CM- ) (C\JCro««) HtQUlNCT 1C*'> 501400 2500 I.R. Scoe i ftBr in (Sucrose) Pellet Spectrum Compound of 20-X 2000 FIGURE 18 1600 1200 1000 iq Q 1 1. 16.0 12.0 110 .Q 40 100 0 8 45 h TRANSMITTANCE (•/&, 100 60 80 4000 2.5 r 3500 3.0 3000 . 40 IRN 50 MICRONS 4.0 3.5 '•IC j N* C IN'* 2500 m . R..Spectrum I. TNA of in.KBr Pellet 2000 FIGURE 801400 1800 19 TNA (Fr*« 6.0 1600 . 80 IRN 1. 1. 12.0 11.0 10.0 MICR0NS 8.0 7.0 *i»Ou»NC* C m 1200 1000 800 16.0 60 100 80 £ 8 0 MICRONS 2.5 3.0 3.5 4 0 WICRONS j^o 6.0 7.0 10.0 11.0 12 16.0 100 100
4000 3500 3000 2500 2000 1B00 1600 1400 1200 1000 800 FIGURE 20 2.5 4 0 MICRONS 50 _____ I__ L_ 3.0 3.5 6.0 7.0 10.0 11.0 12.0 16.0 1 0 0 • • 100
Z 40
4000 3500 3000 2500 1800. 1600 lioo 1200 1000 800 FIGURE 21 PltO W M O i Cm 'I Fig. 20 (upper), I.R. Spectrum of TNC in KBr Pellet; Fig. 21 (lower), I.R. Spectrum ______of TNC in Chloroform 3.0 3.5 4.0 MICRONS SO 1 6.0 7.0 8.0 MICRONS jq.o 11.0 12.0 16.0 100
Z 40
4000 3500 30002500 2000 1800 1600 1400 1200 1000 800 FIGURE 22 TW-jO (»»rp«d«l— ) 40 m i c r o n s 5X) 8 0 MICRONS 2.5 3.0 3.5 6.0 7.0 10.0 11.0 12.0 16.0 100 100
r 80
60
Z 40
4000 3000 25002000 1800 1600 , 1400 1200 ’ 1000 800 3500 _aC-V> (««rMnto»l»»l FIGURE 23 (®CH! Fig. 22 (upper), I.R. Spectrum of TNC-50 (Hemandezine) in KBr Pellet; Fig. _23 (lower). I.R. Spectrum of TNC-50 (Hemandezine) in Chloroform 5 -rl * ‘i i-' uno|n 1 1 I i t I*!!* ]* •' i" i ft j i i" •' « Is 0*0 e X X e H-i £ §
N.M.R. Soectrum of TNA St S-t St ! ... pcrm TNC f o Spectrum N.M.R. FIGURE 25
Hill Kn N> H mm »1I»»
t m o*« Stm uwwmtf fcwdMr U«gn«f« Rdoitonci
FIGURE 26
M.M.R. Spectrum of TNC-50 (Hemandezine) H VJX »oJ APPENDIX C 155
CHART NO. I
Flow Sheet for the Extraction of the Total Alkaloids
20 Kg D ried, Ground Roots
P e r c o l a t e w i t h EtO H S e p a r a t e
EtO H M a re Concentrate s c a r d e d EtOH Concentrate
•'20-x" C rystals 1EtOH F iltrate 3 5 * 0 0 g® 5% HC1 EtOH W ashed Evaporate EtOH F i l t e r :tOH W ashings R e s i d u e
A q u e o u s EtO H HC1 Extract 558 HC1 Evaporate EtOH F i l t e r ______F i l t e r A q u e o u s HC1 20-TT-S P recipitates (Total Tertiary Alkaloids) F i l t r a t e ______4 0 3 . 8 gm ______CHC1 S e p a r a t e CHClo E xtract Aqueous soln. (Qu) E v a 3 0 r a t e NIfy. r e i n e c k a t e F i l t e r Q u a t e r n a r y F i l t r a t e l Alkaloid Reineckate (Q u -A ) D i s c a r d e d CHART NO. I I
Flow Sheet for the Fractionation of the Total Alkaloids
20-TT-S (iK)3.00 gm) (Qu) reineckate (Total Tertiary Alkaloids)
MeOH HC1 F ilte r Evaporate
20-C F ilte r Residue Aqueous Residue Acid Ext. MeOH Et'O 5$ HC1
Evaporate
F ilte r Aqueous Evaporate Acid Ext. ^ Aqueous Acid Ext 5# HC1
Separate
F ilte r E to0 5# HC1‘ F iltr a te
Evaporate
F ilte r Separate CHART NO. I I (Cont'd) i _ i 1 l ____ J___ (20 NAP) (20-D) E t,0 *5---- F iltr a te (20-TT-A)
Evaporate reineckate
m t e — L 1 Tertiary Alkaloid 20-TT Ho0 (Qu-B) - — ) (Qu) 1 .0 gm 87.3 gm Reineckate 230.00 gm
Discarded Ag2S04
E t ^ Ba 3Lo
F ilte r 2 | F iltrate | Precipitate 50# NaOH DiseaSrded Separate ly o p h iliz e 1 - — 1 NaOH EtoO (Qu) f 53.50 gm 50#HAcO Evaporate to JH 6.0 F iltr a te |
“ I Discarded pH 6.0 Soln. (TN) 56.8 gm 1 $ N%0H —Separate' E t20 TP Evaporate— Et20 22.40 gm BIBLIOGRAPHY
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