1978, Vol. 33, pp. 63-69, O Hippokrates Verlag GmbH
Joumd d
rnedica Planti w hnt-
i
The Alkaloids of Mitragyna rubrostipulata (Schum.) Havil E. J. Shellard and P. K. Lala
Key Word Index: Mitragyna rubrostipulata; Rubiaceae; lndole and Oxindole Alkaloids; Antirotundifoline N-oxide; ~ h ~ n c h o ~ h ~ lN-oxide. line
Abstract
*
Hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, rotundifoline, isorotundifoline, mitraphylline, isomitraphylline, rhynchophylline N-oxide and anti rotundifoline N-oxide have been isolated from Mitragyna rubrostiAll were prepulata (SCHUM.)HAVIL. sent in the root bark but hirsutine and hirsuteine were not found in the leaves or stem bark. Comparative studies have been made of the distribution of these same alkaloids in related species of Mitragyna.
Introduction
Four species of Mitragyna are recognised in the tropical regions of Africa, M. stipulosa (D. C.), 0. KUNTZE,M. ciliata AUBR.et PELLEGR. and M. inermis (WILLD),0.KUNTZEin West Africa and M. rubrostipulata (SCHUM.)HAVIL. in
East Africa. The alkaloidal content of the leaves, stem-bark and root bark of the West African species have been fully reported [I-61, these reports citing references to earlier work undertaken on the three species concerned. Previously reported work on the alkaloids of Mitragyna rubrostipulata (SCHUM.)HAVIL. ( M . rubrostipulacea HAVIL.) (ADINA RUBROSTIPULATA Schum.) is given in Table I. A more detailed examination of the leaves, stem-bark and root bark, utilising chromatographic techniques for the separation, detection and isolation of the alkaloids and using modern physicochemical methods for their characterisation has shown the following alkaloids to be present. Hirsutine and hirsuteine (both from the root-bark only), rhynchophylline, isorhynchophylline, rotundifoline, isorotundifoline, mitraphylline, isomitraphylline, rhynchophylline N-oxide and anti rotundifoline N-oxide.
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Shellard, Lala
64
Table I Research on M. rubrostipulata up to 1963 Plant part
MICHIELS aod LEROUX
bark leaves DENIS bark RAYMOND HAMET bark leaves BADGER,COOKand. ONGLEY bark ONGLEY leaves SEATONand MARION leaves SEATON,TONDEURand MARION bark SEATON,MUIR,EDWARDS .and MARIONbark & SIMS bark HENDRICKSON
Alkaloids
Date
mitraphylline mitraphylline rubradinine (= mitraphylline) rubradinine (= mitraphylline) rubradinine (= mitraphylline) L-mitraphyllineo D-mitraphylline* rhynchophylline mitraphylline isorhynchophylline stipulatine (= rotundifoline)
" SHELLARD, E. J. and PILLIPSON, J. D . (Tetrahedron letters 11, 1113 (1966) gave a possible explanation for the reporterd optical isomers of -mitraphylline. Experimental
Column chromatography: The alumina used was Spence type H and the silica gel was Silica gel H (Merck). TLC: The hRf values were obtained on Silica gel G (Merck) 250 nm thick with the solvent systems (a) chloroform: acetone, 5:4, (b) chloroform:ethanol, ( c ) ethylacetate :isopropanol: 6:1, NH,OH, 70:25 :15 and (d) methanol. For preparative thin-layer chromatography Silica gel G (Merck) and Silica gel GF254 (Merck) - equal parts, 0.5 mm and 1.0 mm thick was used, the solvent systems being (i) h1oroform:ethanol, 95:5 followed by ether-ethylacetate 1:1, (ii) chloroform:ethanol95 :2 X 2 and (iii) methanol X 2 (for the N-oxides). Detection of the alkaloids was by means of ferric chloride (0.2 M) in 35 010 perchloric acid and heating gradually to 100° [7-91. The R t values were obtained on a Perkin-Elmer F 11 GLC with 1.5 OIo SE52 on Chromosorb HMDS
(80-100 mesh) using .F.I.D. a t temp. 240' and Np15 p.s.i. MS were determined on an AEI MS 962 high resolution mass spectrometer a t 70 eV with inlet temp between 220 and 240°. NMR spectra in CDC13 with TMS as internal reference at.60 MHz. The C D curves obtained with RousselJouan Dichograph in methanol solutions.
Plant Material Leaves, stem bark and root bark collected from trees growing in Uganda as follows: leaves; September 1973, June, July and August 1974, stem bark; June 1974 and root bark; June and July 1974. Authentication was undertaken by the botanists employed by the National Chemotherapeutics Research Laboratories, Kampala; Herbarium Specimens are held in the Pharmacognosy Research Laboratories, Chelsea College, University of London.
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Authors
Alkaloids of Mitragyna rubrostipulata
Coarsely powdered materials, leaves 250 g, stem bark 85 g, root bark 150 g, were moistened with dilute solution of ammonia, allowed to stand overnight and then macerated with ethyl acetate followed by methanol. The extractives were then concentrated to low bulk under reduced pressure and as the methanol and ethyl acetate extracts contained the same alkaloids as shown by TLC, they were bulked for each separate plant part and evaporated to dryness. Each dry extract was dissolved in chloroform (10 ml) and shaken with 2O/o sulphuric acid (2 X 25 ml). After separation the acid layer was made alkaline (pH 8.5) with strong solution of ammonia and extracted with chloroform (3 X 25 ml). The chloroform extractives
were washed with the same 5 ml water, reduced to low bulk and after drying with anhydrous sodium sulphate, the solvent was removed to yield crude ,alkaloidal fractions. The crude alkaloid was subjected to purification by column chromatography. 15 g deactivated silica gel containing ether was packed into a glass column (10 X 1 cm) and the crude alkaloid mixed with ether was placed on the cop of the column. The alkaloids we& then eluted by gradient elution using ether (150 ml) 'chloroform (150 ml) and methanol (250 ml) respectively, 10 ml fractions being collected and monitored by means of TLC. Similar eluates were bulked and evaporated to dryness under reduced pressure. (Table 11). Three distinct fractions wereobtained: (A) pale yellow solid containing 3 major
Table II Isolation of alkaloids from Mitragyna rubrostipulata Leaves
Crude drug Crude alkaloid . . Purified alkaloid Column obromutography Fraction A
,B C Preparative TLC Rotundifoline lsorctundifoline Rhynchophylline Isorhyn~hoph~lline Mitraphylline Isomitraphylline Oxindole N-oxides Hirsutine Hirsuteine
Stembark
Rootbark
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Isolation of the alkaloids
65
slightly polar alkaloids and traces N-oxide, were identified by comparison of more polar alkaloids, of the NMR, MS, Rt and hRf values (B) pale yellow solid containing 3 major on several TLC systems (Table 111) with polar alkaloids and traces of less authentic alkaloids isolated from other polar alkaloids, species of Mitragyna as follows:(C) dark brown solid with traces of poHirsutine and hirsuteine from Mitralar alkaloids and in that obtained gyna hirsuta [13], mitraphylline, isofrom root bark, a major indole al- mitraphylline, rotundifoline, isorotunkaloid and traces of a second indole difoline, rhynchophylline and isorhynalkaloid. chophylline from Mitragyna tubulosa Repeated preparative TLC using the [lo] and rhynchophyllineN-oxide from solvent system indicated enabled a num- Mitragyna inermis [14]. ber of alkaloids to be isolated (Table 11). The anti rotundifoline N-oxide isolated from the plant was identified by comparing the UV, NMR, MS, CD and Separation of the N-oxides hRf values on several TLC systems with The N-oxides obtained by column the sample of anti rotundifoline Nchromatography and preparative TLC oxide obtained by oxidation of rotundiwere subjected to analytial TLC using foline [12]. all four systems which indicated two alkaloids, one of which c6rmponded to rhynchophylline N-oxide [lo, 111. The two alkaloids were separated by preparativeTLC (System iii) and then treated with sulphurous acid (5 O/o) overnight, Table 111 made alkaline with NH,OH and ex- Rt and hRf values of isolated alkaloids tracted with chloroform. TLC of these Rt hRf values reduced products indicated rhynchob c d (mean) a phylline and rotundifoline. One of the 73 90 85 74 N-oxides was, therefore, considered, to ~otundifoline 13.0 be rhynchophylline N-oxide and was 58 85 82 62 Isorotundifoline identified as such. 35 55 61 47 Rhynmophylline In order to identify the second N3.75 oxide it was necessary to prepare rotun- Isorhynchophylline 58 89 83 71 46 60 45 47 difoline N-oxides in order to make a Mitraphylline 3.50 compari'son with the naturally occurrIsomitraphylline 57 67 48 57 ing alkaloid [12]. Hirsutine 6.50 8 4 7 21
Identification of the isolated alkaloids The isolated alkaloids, with the exception of the new anti rotundifoline
Hirsuteine Rhynchophylline N-oxide anti-rotundifoline N-oxide
6.75
15
11
11
26
0
9
10
15
0
10
24
57
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Shellard, Lala
66
Alkaloids of Mitragyna rubrostipulata
Six alkaloids in addition to those previously reported in this species were isolated but only the anti-rotundifoline Noxide had not previously been found in Mitragyna species. Rotundifoline is a C(9)-OH normal open E ring oxindole alkaloid (A 'series) but because of its configuration it is non-phenolic because of strong hydrogen bonding between the N(4) and the hydrogen atom of the hydroxy group at C(9). The identification of the alkaloid as the the anti-rotundifoline N-oxide was, therefore, somewhat surprising particularly as the other N-oxide was that of rhynchophylline (B series) and not that of isorhynchophylline (A series) which occurs naturally in Mitragyna tubulosa [lo]. The alkaloidal pattern conforms to the hypothesis regarding the possible biogenesis of oxindole alkaloids put for[8] ward by SHELLARDand HOUGHTON except that there was no evidence of the corynoxeines in the plant. However, they normally occur in very small quantities and are not detected in all samples, even if the mitraphyllines are. The general pattern, involving the normal-pseudo series of open E ring alkaloids, is similar to that which occurs in Mitragyna stipulosa [I, 2, 4, 61, Mit: ragyna ciliata [I], Mitragyna inermis [3,5] and Mitragyna tubulosa [lo], but there are a number of important differences. In M. rubrostipulata the alkaloids are identical with those in M. stipulosa but in the former the indole alkaloids are found.only in the roots, whereas in M. stipulosa they are found only
in the leaves (Fig 1, 2). The question thus arises as to whether the biogenetic sites differ in the two species.
ALKALOIDAL PATTERN IN M. RUEROSTIWLATA
-. Rhynchophylllne
I
ALKALOIDAL PATTERN IN
M
1
STWLOSA
The sequence of alkaloids differs from those in M. ciliata and M. inermis in as much as the C(9)-OCH, alkaloids ciliaphylline and rhynchociline are absent from M. rubrostipulata. In M. ciliata and M, inermis the indole alkaloids are present in the leaves and not in the roots
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Discussion
and they are the corresponding C(9)OCH, alkaloids, speciogynine and mitraciliatine (Figure 3) so that the biogenetic pathway in these two species is in the opposite direction to that in M. rubrostipulata. Sprclogynlne Loaves
1
Mltraclllotlno
RhynchocIIRe
7 41
CIlIaphylllno
Ir 1
Rotundlfollno
4
Isorotundliollno
Rhynchlphylllne
Jt
Isorhynchophylllno
Roots
ALKALOIDAL PATTERN IN M. CILIATA AND M. INERMIS
By way of interesting comparison M. tubulosa contains both the C(9)-H and C(9)-OCH, open E ring indole alkaloids and all corresponding oxindole alkaloids but the unsubstituted indoles are found only in the root and the substituted indoles only in the leaves (Figure 4).
*
.--
(Spoclogynine)
Leaves
I
ALKALOIDAL
Mit~aclllatlnO
CIIIaphylllne
/"
PATTERN IN M.'IUBUWSA
jr
Rhynchoelllno
I
There would appear to be a close taxonomic link between these five species of Mitragyna. However, Mitragyna inermis, which is a small bush - the others are tall trees - possesses in addition to a number of morphological differences, some pronounced biochemical differences [4]. It is interesting to note, therefore, that LE ROY [I 51 has suggested a close taxonomic link between the three African trees, M. stipulosa, M. ciliata and M . rubrostipulata based on a number of botanical features, particularly their monopodhl pattern of development, but has excluded M. tubulosa, the Asian tree because of differences shown by the electron microscopy of the pollen. From a phytochemical point of view there appears to be no reason why it should not also be included in the new genus he has named 'Hallea J. F: LE ROYafter Halle who specialised in itud y i n i the Rubiacae in Gabon.
Acknowledgements
We are grateful to Mr. N. K. MUBURU, Principal Research Officer, National Chemotherapeutics Research Laboratory, Kampala, Uganda, for arranging the collection, drying and transport of the plant material. We thank Mr. D. CARTER,School of Pharmacy, University of London for MS determinations, Dr. P. M. SCOPES, Chemistry Department, Westfeld College, University of London for C D determination and Mr. G. MCDONOUGH of this College for the NMR spectra. One of us (PKL) would like to thank the Vice-Chancellor, Jadavpur Univer-
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Shellard, Lala
68
sity, Calcutta, India, for extended leave of absence thus enabling him to study in the Pharmacognosy Research Laboratories, Chelsea College, University of London.
Literatur 1. Bedtett, A. H.,Shellard, E. J. and Tadtie, A. N.: J. Pharm. Pharmac. 15. 158 (1963). 2. Bedrett, A. H.,SheIlard, E. J. and Tackie, A. N.:ibid. 166 (1963). 3. Shellard, E. J. and Sarpong, K.: J. Pharm. Pharmac. 2 1 Suppl. 113 (1969). 4. Shellard, E. J. and Sarpong, K.: ibid. 22 Suppl. 34 (1970). 5. Shellard, E. J. and Sarpong, K.:ibid. 23 559 (1971). 6. Houghton, P. J., Lala, P. K., Shellard, E. J. and Sarpong, K.: J. Pharm. Pharmac. 28, 664 (1976). 7. Shellard, E. J. and Alam, M. 2.: J. Chromatogr. 33, 347 (1968). 8. Shellard, E. J. and Houghcon, J. P.: Planta Medica 25, 80 (1974). 9. Phillipson, J. D. and Hemingway, S. R.: J. Chromatogr. 105, 163 (1975). 10. Shellard, E. J. and Rungsijakul, D.: Planta Medica 23, 221 (1973). 11. Phillipson, J. D., Rungsijakul, D. and Shellard, E. J.: Phytochemistry 12, 2043 (1973).
12. Shellard, E. J., Hougthon, P. J, and Lala, P. K.: Phytochemistry 16, 1427 (1977). 13. Shellard, E. J., Tantivatana, P. and Beckett,, A. H. J. Pharm. Pharmac. 15, 366 (1967). 14. Shellard, E. J., Phillipson, J. D. and Sarpong, K.: Phytohemistry, 10, 2505 (1971). 15. LeRoy, J.-F.: Adansonia ser. 2, 15 (I), 6568 (1975). 17. Michels, L. and Leroux, M.: Bull. Acad. Med. Belg. 5 , 403 (1925). IS. Denis, P.: Bull. Acadm. Belg. Sci. 24, 403 (1938). 19. Raymond-Hamet: Bull. Soc. Pharm. 46, 327 (1939). 20. Badger, G. M.,Cook, J. W. and Ongley, P. A,: J. Chem. Soc. 867 (1950). 21. Ongley, P. A.: Ph. D. Thesis, Glasgow University (1950). 22. Seaton, J. C. and Marion, L.: Canad. J. Pharm. 35, 1103-1108 (1957). 23. Seaton, J. C., Tondeur, R. and Marion, L.: Canad. J. Pharm. 36, 1031 (1958). 24. Seaton, J. C.,Nair, M. D., Edwards, O.E. and Marion, L.: Canad. J. Pharm. 38, 1035 (1960). 25. Hendrickson, J. H.and Sims, J. J.: Tetrahedron Letters, 14, 929 (1963).
Adress: Prof. E. 1. Shellard Department of Pharmacy Chelsea College University o f London Manresa Road, London SW3 6LX
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Alkaloids of Mitragyna rubrostipulata
Joumd d
rnedica Planti w hnt-
i
The Alkaloids of Mitragyna rubrostipulata (Schum.) Havil E. J. Shellard and P. K. Lala
Key Word Index: Mitragyna rubrostipulata; Rubiaceae; lndole and Oxindole Alkaloids; Antirotundifoline N-oxide; ~ h ~ n c h o ~ h ~ lN-oxide. line
Abstract
*
Hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, rotundifoline, isorotundifoline, mitraphylline, isomitraphylline, rhynchophylline N-oxide and anti rotundifoline N-oxide have been isolated from Mitragyna rubrostiAll were prepulata (SCHUM.)HAVIL. sent in the root bark but hirsutine and hirsuteine were not found in the leaves or stem bark. Comparative studies have been made of the distribution of these same alkaloids in related species of Mitragyna.
Introduction
Four species of Mitragyna are recognised in the tropical regions of Africa, M. stipulosa (D. C.), 0. KUNTZE,M. ciliata AUBR.et PELLEGR. and M. inermis (WILLD),0.KUNTZEin West Africa and M. rubrostipulata (SCHUM.)HAVIL. in
East Africa. The alkaloidal content of the leaves, stem-bark and root bark of the West African species have been fully reported [I-61, these reports citing references to earlier work undertaken on the three species concerned. Previously reported work on the alkaloids of Mitragyna rubrostipulata (SCHUM.)HAVIL. ( M . rubrostipulacea HAVIL.) (ADINA RUBROSTIPULATA Schum.) is given in Table I. A more detailed examination of the leaves, stem-bark and root bark, utilising chromatographic techniques for the separation, detection and isolation of the alkaloids and using modern physicochemical methods for their characterisation has shown the following alkaloids to be present. Hirsutine and hirsuteine (both from the root-bark only), rhynchophylline, isorhynchophylline, rotundifoline, isorotundifoline, mitraphylline, isomitraphylline, rhynchophylline N-oxide and anti rotundifoline N-oxide.
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Shellard, Lala
64
Table I Research on M. rubrostipulata up to 1963 Plant part
MICHIELS aod LEROUX
bark leaves DENIS bark RAYMOND HAMET bark leaves BADGER,COOKand. ONGLEY bark ONGLEY leaves SEATONand MARION leaves SEATON,TONDEURand MARION bark SEATON,MUIR,EDWARDS .and MARIONbark & SIMS bark HENDRICKSON
Alkaloids
Date
mitraphylline mitraphylline rubradinine (= mitraphylline) rubradinine (= mitraphylline) rubradinine (= mitraphylline) L-mitraphyllineo D-mitraphylline* rhynchophylline mitraphylline isorhynchophylline stipulatine (= rotundifoline)
" SHELLARD, E. J. and PILLIPSON, J. D . (Tetrahedron letters 11, 1113 (1966) gave a possible explanation for the reporterd optical isomers of -mitraphylline. Experimental
Column chromatography: The alumina used was Spence type H and the silica gel was Silica gel H (Merck). TLC: The hRf values were obtained on Silica gel G (Merck) 250 nm thick with the solvent systems (a) chloroform: acetone, 5:4, (b) chloroform:ethanol, ( c ) ethylacetate :isopropanol: 6:1, NH,OH, 70:25 :15 and (d) methanol. For preparative thin-layer chromatography Silica gel G (Merck) and Silica gel GF254 (Merck) - equal parts, 0.5 mm and 1.0 mm thick was used, the solvent systems being (i) h1oroform:ethanol, 95:5 followed by ether-ethylacetate 1:1, (ii) chloroform:ethanol95 :2 X 2 and (iii) methanol X 2 (for the N-oxides). Detection of the alkaloids was by means of ferric chloride (0.2 M) in 35 010 perchloric acid and heating gradually to 100° [7-91. The R t values were obtained on a Perkin-Elmer F 11 GLC with 1.5 OIo SE52 on Chromosorb HMDS
(80-100 mesh) using .F.I.D. a t temp. 240' and Np15 p.s.i. MS were determined on an AEI MS 962 high resolution mass spectrometer a t 70 eV with inlet temp between 220 and 240°. NMR spectra in CDC13 with TMS as internal reference at.60 MHz. The C D curves obtained with RousselJouan Dichograph in methanol solutions.
Plant Material Leaves, stem bark and root bark collected from trees growing in Uganda as follows: leaves; September 1973, June, July and August 1974, stem bark; June 1974 and root bark; June and July 1974. Authentication was undertaken by the botanists employed by the National Chemotherapeutics Research Laboratories, Kampala; Herbarium Specimens are held in the Pharmacognosy Research Laboratories, Chelsea College, University of London.
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Authors
Alkaloids of Mitragyna rubrostipulata
Coarsely powdered materials, leaves 250 g, stem bark 85 g, root bark 150 g, were moistened with dilute solution of ammonia, allowed to stand overnight and then macerated with ethyl acetate followed by methanol. The extractives were then concentrated to low bulk under reduced pressure and as the methanol and ethyl acetate extracts contained the same alkaloids as shown by TLC, they were bulked for each separate plant part and evaporated to dryness. Each dry extract was dissolved in chloroform (10 ml) and shaken with 2O/o sulphuric acid (2 X 25 ml). After separation the acid layer was made alkaline (pH 8.5) with strong solution of ammonia and extracted with chloroform (3 X 25 ml). The chloroform extractives
were washed with the same 5 ml water, reduced to low bulk and after drying with anhydrous sodium sulphate, the solvent was removed to yield crude ,alkaloidal fractions. The crude alkaloid was subjected to purification by column chromatography. 15 g deactivated silica gel containing ether was packed into a glass column (10 X 1 cm) and the crude alkaloid mixed with ether was placed on the cop of the column. The alkaloids we& then eluted by gradient elution using ether (150 ml) 'chloroform (150 ml) and methanol (250 ml) respectively, 10 ml fractions being collected and monitored by means of TLC. Similar eluates were bulked and evaporated to dryness under reduced pressure. (Table 11). Three distinct fractions wereobtained: (A) pale yellow solid containing 3 major
Table II Isolation of alkaloids from Mitragyna rubrostipulata Leaves
Crude drug Crude alkaloid . . Purified alkaloid Column obromutography Fraction A
,B C Preparative TLC Rotundifoline lsorctundifoline Rhynchophylline Isorhyn~hoph~lline Mitraphylline Isomitraphylline Oxindole N-oxides Hirsutine Hirsuteine
Stembark
Rootbark
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Isolation of the alkaloids
65
slightly polar alkaloids and traces N-oxide, were identified by comparison of more polar alkaloids, of the NMR, MS, Rt and hRf values (B) pale yellow solid containing 3 major on several TLC systems (Table 111) with polar alkaloids and traces of less authentic alkaloids isolated from other polar alkaloids, species of Mitragyna as follows:(C) dark brown solid with traces of poHirsutine and hirsuteine from Mitralar alkaloids and in that obtained gyna hirsuta [13], mitraphylline, isofrom root bark, a major indole al- mitraphylline, rotundifoline, isorotunkaloid and traces of a second indole difoline, rhynchophylline and isorhynalkaloid. chophylline from Mitragyna tubulosa Repeated preparative TLC using the [lo] and rhynchophyllineN-oxide from solvent system indicated enabled a num- Mitragyna inermis [14]. ber of alkaloids to be isolated (Table 11). The anti rotundifoline N-oxide isolated from the plant was identified by comparing the UV, NMR, MS, CD and Separation of the N-oxides hRf values on several TLC systems with The N-oxides obtained by column the sample of anti rotundifoline Nchromatography and preparative TLC oxide obtained by oxidation of rotundiwere subjected to analytial TLC using foline [12]. all four systems which indicated two alkaloids, one of which c6rmponded to rhynchophylline N-oxide [lo, 111. The two alkaloids were separated by preparativeTLC (System iii) and then treated with sulphurous acid (5 O/o) overnight, Table 111 made alkaline with NH,OH and ex- Rt and hRf values of isolated alkaloids tracted with chloroform. TLC of these Rt hRf values reduced products indicated rhynchob c d (mean) a phylline and rotundifoline. One of the 73 90 85 74 N-oxides was, therefore, considered, to ~otundifoline 13.0 be rhynchophylline N-oxide and was 58 85 82 62 Isorotundifoline identified as such. 35 55 61 47 Rhynmophylline In order to identify the second N3.75 oxide it was necessary to prepare rotun- Isorhynchophylline 58 89 83 71 46 60 45 47 difoline N-oxides in order to make a Mitraphylline 3.50 compari'son with the naturally occurrIsomitraphylline 57 67 48 57 ing alkaloid [12]. Hirsutine 6.50 8 4 7 21
Identification of the isolated alkaloids The isolated alkaloids, with the exception of the new anti rotundifoline
Hirsuteine Rhynchophylline N-oxide anti-rotundifoline N-oxide
6.75
15
11
11
26
0
9
10
15
0
10
24
57
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Shellard, Lala
66
Alkaloids of Mitragyna rubrostipulata
Six alkaloids in addition to those previously reported in this species were isolated but only the anti-rotundifoline Noxide had not previously been found in Mitragyna species. Rotundifoline is a C(9)-OH normal open E ring oxindole alkaloid (A 'series) but because of its configuration it is non-phenolic because of strong hydrogen bonding between the N(4) and the hydrogen atom of the hydroxy group at C(9). The identification of the alkaloid as the the anti-rotundifoline N-oxide was, therefore, somewhat surprising particularly as the other N-oxide was that of rhynchophylline (B series) and not that of isorhynchophylline (A series) which occurs naturally in Mitragyna tubulosa [lo]. The alkaloidal pattern conforms to the hypothesis regarding the possible biogenesis of oxindole alkaloids put for[8] ward by SHELLARDand HOUGHTON except that there was no evidence of the corynoxeines in the plant. However, they normally occur in very small quantities and are not detected in all samples, even if the mitraphyllines are. The general pattern, involving the normal-pseudo series of open E ring alkaloids, is similar to that which occurs in Mitragyna stipulosa [I, 2, 4, 61, Mit: ragyna ciliata [I], Mitragyna inermis [3,5] and Mitragyna tubulosa [lo], but there are a number of important differences. In M. rubrostipulata the alkaloids are identical with those in M. stipulosa but in the former the indole alkaloids are found.only in the roots, whereas in M. stipulosa they are found only
in the leaves (Fig 1, 2). The question thus arises as to whether the biogenetic sites differ in the two species.
ALKALOIDAL PATTERN IN M. RUEROSTIWLATA
-. Rhynchophylllne
I
ALKALOIDAL PATTERN IN
M
1
STWLOSA
The sequence of alkaloids differs from those in M. ciliata and M. inermis in as much as the C(9)-OCH, alkaloids ciliaphylline and rhynchociline are absent from M. rubrostipulata. In M. ciliata and M, inermis the indole alkaloids are present in the leaves and not in the roots
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Discussion
and they are the corresponding C(9)OCH, alkaloids, speciogynine and mitraciliatine (Figure 3) so that the biogenetic pathway in these two species is in the opposite direction to that in M. rubrostipulata. Sprclogynlne Loaves
1
Mltraclllotlno
RhynchocIIRe
7 41
CIlIaphylllno
Ir 1
Rotundlfollno
4
Isorotundliollno
Rhynchlphylllne
Jt
Isorhynchophylllno
Roots
ALKALOIDAL PATTERN IN M. CILIATA AND M. INERMIS
By way of interesting comparison M. tubulosa contains both the C(9)-H and C(9)-OCH, open E ring indole alkaloids and all corresponding oxindole alkaloids but the unsubstituted indoles are found only in the root and the substituted indoles only in the leaves (Figure 4).
*
.--
(Spoclogynine)
Leaves
I
ALKALOIDAL
Mit~aclllatlnO
CIIIaphylllne
/"
PATTERN IN M.'IUBUWSA
jr
Rhynchoelllno
I
There would appear to be a close taxonomic link between these five species of Mitragyna. However, Mitragyna inermis, which is a small bush - the others are tall trees - possesses in addition to a number of morphological differences, some pronounced biochemical differences [4]. It is interesting to note, therefore, that LE ROY [I 51 has suggested a close taxonomic link between the three African trees, M. stipulosa, M. ciliata and M . rubrostipulata based on a number of botanical features, particularly their monopodhl pattern of development, but has excluded M. tubulosa, the Asian tree because of differences shown by the electron microscopy of the pollen. From a phytochemical point of view there appears to be no reason why it should not also be included in the new genus he has named 'Hallea J. F: LE ROYafter Halle who specialised in itud y i n i the Rubiacae in Gabon.
Acknowledgements
We are grateful to Mr. N. K. MUBURU, Principal Research Officer, National Chemotherapeutics Research Laboratory, Kampala, Uganda, for arranging the collection, drying and transport of the plant material. We thank Mr. D. CARTER,School of Pharmacy, University of London for MS determinations, Dr. P. M. SCOPES, Chemistry Department, Westfeld College, University of London for C D determination and Mr. G. MCDONOUGH of this College for the NMR spectra. One of us (PKL) would like to thank the Vice-Chancellor, Jadavpur Univer-
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Shellard, Lala
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sity, Calcutta, India, for extended leave of absence thus enabling him to study in the Pharmacognosy Research Laboratories, Chelsea College, University of London.
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Adress: Prof. E. 1. Shellard Department of Pharmacy Chelsea College University o f London Manresa Road, London SW3 6LX
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Alkaloids of Mitragyna rubrostipulata
