- Email: [email protected]
Bacoside A3A triterpenoid saponin from Bacopa monniera
Pergamon
BACOSIDE
Phykxhtmirtry. Vol. 36, No. I, pp. 133 137. 1994 Copyririghl c 1994 Elsncr Sacna Ltd Prinkd in Great Britain All rights reserved 0031 9422i94 5600+0.00
A,-A SUBHA
TRITERPENOID
SAPONIN
FROM BACOPA
MONNIERA*
RASTOGI, RAGHWENDRA PAL and DINESH K. KULSHREsHTHAt
Medicinal Chemistry Division, Central Drug Research Institute, Lucknow, 226001, India (Received in reuisedfirm
Key Word Index-Bocopa
monniera;
Scrophulariaceae;
17 August 1993)
tritergene; saponin; bacoside A,.
Abstract-A
new triterpenoid saponin, bacoside A,, a constituent of bacosides, the saponin mixture of Bacopa was isolated and characterized. Its structure was established as 3-/i-[O-/l-D-glucopyranosyl( l-+3)-O-[a-Larabinofuranosy(1 +2)]-0-/?-D-glucopyranosyl)oxy] jujubogenin by chemical and spectral analyses. The cis-isomer of ebelin lactone was also obtained as one of the artefacts of the aglycone and its structure revised. monniera,
INTRODIJCllON
hexosyl unit from the [M-H] - ion, respectively. Thus, bawside A, had a terminal pentosyl and a hexosyl sugar unit, besides an inner hexosyl unit attached to the aglycone. Bacoside A, was subjected to acid hydrolysis. The aglycone portion, which consisted of one major spot on TLC corresponding to ebelin lactone, was purified by column chromatography over neutral alumina and finally by crystallization from methanol. It furnished needles, mp 173”, EIMS: m/z 454 CM]‘, UV: &,,.. 266, 276, 286 (open chain conjugated triene system). Its ‘HNMR spectrum exhibited signals for seven tertiary C-Megroups(6:0.72,0.81,0.93, 1.00, 1.72, 1.75and 1.78) a CHzOCO (two d, J = 10 Hz each, 64.23 and 4.35), the signals for olefinic protons H, (d, J = 10 Hz, 5 5. I6), H, (d, 5=15Hz,66.04),H,(dd,J=l1,15Hz,66.30)andH,(d, .I = 11 Hz, S5.78), and those for H, (m, 62.75) and H,/H, (two d, J= 18 Hz each, 62.05 and 2.40). The aglycone was thus identified as trons-ebelin lactone(Ia) by comparison of its data with those reported in the literature [ 121. The crystalline trans-ebelin lactone, as well as the mother liquor, were subjected to acetylation. The ‘H NMR spectrum of the acetylated product of the mother liquor exhibited two sets of signals, one set corresponding to trans-ebelin lactone monoacetate and the second set corresponding to its other isomer. Since ebelin lactone has a side chain consisting of a triene system, it can exist as four isomers depending upon the stereochemistry about the double bonds. However, the all tmns-configuration (la) is the preferred one. A cis-isomer of ebelin lactone (2) has also been reported earlier [ 133, in which the A**-double bond has been assigned a cis-configuration on the basis of UV and IR spectra. A difference UV spectrum obtained by subtracting the UV spectrum of the pure trans-acetyl ebelin lactone (d,_ 266, 276, 286 nm) from that of the acetylated mother liquor was therefore recorded. The difference spectrum showed nearly the same absorption maxima (A,,, 271,280 and 292 nm) as reported for the cis-acetyl ebelin lactone [13].
Bacopa monniera (Wettst) (Hindi-brahmi) is used as a reputed net-vine tonic in the traditional system of medicine [l]. Its alcoholic extract was found to improve the performance of rats in several learning tests as manifested by better acquisition, consolidation and retention of newly acquired behavioural responses C2-43. The activity has been attributed to the saponin mixture consisting of bacosides A, B and other saponins. On acid hydrolysis, bacosides yielded a mixture of aglycones, bacogenin A, [S, 63, A, [7J A, [8] and A, [9]. Bacogenin A, has been identified as ebelin lactone. Recently, a minor saponin, bacoside A, was isolated and characterized as 3-0-[a-~arabinofuranosyl( l-+3)-a+arabinopyranosylUujubogenin [lo]. The present communication reports the isolation and characterization of a new triterpenoid saponin, bacoside A,, from the mixture of bacosides obtained from the ethanolic extract of the plant according to the method described earlier [1 l]. RESULTS AND DISCUSSION
The saponin mixture on repeated chromatography involving silica gel and reverse phase C-18 flash column chromatography yielded bacoside A,, the main constituent of the saponin mixture. It was obtained as a white amorphous solid and gave a positive Fiegel’s test revealing its glycosidic nature. Its IR spectrum showed bands at 3400 (OH), 2920, 1630, 1550, 1370 and 1030cm-*. Its positive FAB mass spectrum exhibited two major peaks at m/z 951 [M+Na]+ and 455 [(aglycone - HzO) + H] +. The negative FAB mass spectrum gave a [M-H]peak at m/z 927. Other peaks were observed at m/z 795 [M-H132]- and 765 [M-H - 162]- which were due to the loss of a pentosyl and a
*CDRI communication no. 5141. tAuthor to whom correspondence. should be addressed. 133
S. RASTOGI et al.
134
28
29
la R=H lb R=Ac *
Hf/Hg
Ha
Hb
constants of the olefinic proton signals in both the isomers precluded the presence of cis-configuration of the A’*-double bond in the cis-isomer, as in that case the J HbeHc valuefor the cis-isomer would have been smaller than 15 Hz observed for the trans-isomer. Thus, a cisconfiguration about A 22-double bond was ruled out and the only other alternative was a cis-configuration about A”-double bond, in which H, and Me groups were cis. Thus, the structures of cis-ebelin lactone and its acetyl derivative were revised to 3a and 3b, respectively, which were consistent with the spectral data. Since ebelin lactone is known to be the transformation product of the genuine sapogenin, jujubogenin (4), obtained by acid catalysed transformation during hydrolysis [14], the genuine aglycone of bacoside A, was jujubogenin. PC and TLC of the aqueous portion of the hydrolysate showed that the sugar moieties present in it were glucose and arabinose. In order to determine the sequence of the sugar chain in bacoside A,, it was subjected to permethylation. The FAB mass spectrum (positive) of the permethylated bacoside A, showed peaks at m/z 1077 and 1055 corresponding to [M +Na]+ and [M+ H]+ ions, and indicated the presence of nine methoxy groups. Thus, the sugar chain contained nine free hydroxyl groups which
OH
ROT
RO
3a R=H 3b R=Ac * Hf/Hg
To separate and identify the ‘HNMR signals of the two isomers present in the mother liquor, the ‘H NMR signals of the all trns-acetyl ebelin lactone (lb) were subtracted from those of the mixture. Thus, a neat ‘H NMR spectrum of the other (&)-isomer was obtained which exhibited signals at 60.89,0.90,0.92, 1.08, 1.82, 1.84 and 1.88 (7 x tert. C-Me), 2.07 (s, MeCOO), 4.30,4.38 (2 x d, J = 10 Hz each, CH,OCO) and 4.47 (dd, J = 5,ll Hz, CHOAc); the chemical shift values of these signals being nearly the same as for the all trans-isomer. Other signals belonging to the side chain appeared at 65.06 (d, J = lOHz, HA, 66.36 (d, J= 15 Hz, H,,), 66.50 (dd, J = 11 Hz, 15 Hz, H,), 65.93 (d, J= 11 Hz, Hd), 62.93 (m, H,) and 62.15, 2.42 (2 x d, J = 18 Hz each, H,/HJ. On comparison, it was observed that the ‘H NMR signals of the trans-acetyl ebelin lactone and those of its cis-isomer had the same coupling constants (J values) with slight differences in the chemical shift values. Identical coupling
4
R=H
135
Racoside A, from Bucopa monniero were also indicative
of the presence
one pentosyl
attached
units,
of two hexosyl
and
to one another.
Acid hydrolysis of the permethylated bacoside A, yielded partially methylated sugars which were identified as 2,3,4,6tetra-0-methylglucos, 2,3,5-tri-O-methylarabinose and 4,6-di-0-methylglucose by the CC-MS analyses of their alditol acetates [lS]. The identified partially methylated sugars correspond to a 1,2,3-linked glucose, a terminal arabinofuranose and a terminal glucose. Further, in order to determine the exact linkage of the terminal sugar units, bacoside A, was subjected to Mannich hydrolysis [ 163 to yield a diglycoside. Only the arabinose unit was lost, as confirmed by the TLC of the aqueous portion of the hydrolysate and the positive FAB-mass spectrum of the diglycoside, which exhibited major peaks at m/z 817,801 and 779 corresponding to the presence of [M -Hz0 +K]+, [M-H,O+Na]+ and [M-H,O+H]+ ions, respectively. The diglycoside was subjected to permethylation followed by acid hydrolysis. The resulting partially methylated sugars were identified as 2,3,4,6-tetra-Omethylglucose and 2,4,6-tri-O-methylglucose by GC-MS analyses of their alditol acetates [ 151. The formation of 2,4,6-tri-0-methylglucose indicated the presence of a free -OH group at C-2 of the inner glucopyranosyl unit of the diglycoside, which was, however, absent in that of bacoside A,. The arabinose unit was therefore attached at C2 of the inner glucose unit. These methylation studies clearly indicated that the terminal glucopyranosyl unit and the terminal arabinofuranosyl unit were attached to the inner glucopyranosyl unit through l-+3 and l-+2 linkages, respectively, and the inner glucopyranosyl unit was attached to the aglycone through an 0-glycosidic linkage. The above inference was further confirmed by the 13C and ‘HNMR chemical shifts of bacoside A,. The 13CNMR spectrum established that the glucose units
Table 1. 13C NMR spectral data of bacoside A, (CD,OD) Aglycone part -_-C
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sugar part
39.9 21.3 90.7 40.5 57.5 19.2 37.1 38.5 54.2 38.0 22.5 29.2 38.3 54.6 36.9
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
C 111.4 54.4 19.2 16.8 69.4 29.6 45.4 69.7 126.3 136.7 25.8 18.4 28.3 16.8 66.9
Glc
Glc
Ara
1’ 2 3’ 4 5 6 1” 2” 3” 4” 5” 6” 1”’ 2”’ 3”’ 4” 5”’
104.2 78.2 83.6 IQ.3 78.1 62.6 105.5 75.5 77.2 71.7 71.6 62.7 109.9 84.1 80.0 87.4 62.1
existed in the pyranose form, whereas the arabinose unit was present in the furanose form. Assignments of the signals were made on the basis of broad band decoupled as well as DEPT “CNMR spectra [lfl. The chemical shift assignments were made straightaway by comparison with the reported values of O-methyl-a-L-arabinofuranoside and 0-methyl-/?-D-glucopyranoside, taking into consideration the a- and &eKects of glycosidation. *3C NMR chemical shifts of the aglycone portion including that of C-3 were also consistent with those assigned to the jujubogenin portion of the saponins isolated from Colubrina asiatico [18] (Table l), indicating that the sugar chain was linked to jujubogenin at C-3. In the ‘H NMR spectrum of bacoside A,, the anomeric proton signals for two /?-D-glucopyranosyl units ap peared at 64.31 (d, J = 7 Hz) and 4.35 (d. J = 7 Hz) and that for the arabinofuranosyl unit at 65.23 (bs). Thus, on the basis of the above evidence, the structure of bacoside A, was established as 3-B-[@B-D-glucopyranosyl( l-+3)-0-[rx-L-arabinofuranosyl(l -r2)]-O-B-~-glucopyranosyl)-oxyljujubogenin (5). FXPERIMENTAL Plant material. Bacopa monniera (whole plant) was procured from Calcutta market (India). A voucher specimen of the plant is preserved in the herbarium of Botany Division, CDRI. General. Mps: uncorr. IR: KBr disc. UV: EtOH. ‘H NMR: 400 MH& TMS as int. standard. 13C NMR: 100 MHz. CC: silica gel, bondapak C,, and neutral alumina. TLC: (i) silica gel coated plates using solvent system: (1) C,H,-MeOH (96:4); (2) EtOAc-MeOHHz0 (80: 10: 10); (3) EtOAc-MezCO-Hz0 (50:40: 10); (ii) reverse phase-C,, coated plates (E. Merck) using solvent system (4) MeCN-Hz0 (50:50); (iii) cellulose coated plates using solvent system (5) n-BuOHAcOH-H,O (40: 10:50). PC: Whatman No. 1 paper and solvent (6) n-BuOH satd with H,O. Silica gel TLC were visualized by spraying with 1% ceric sulphate in 1 M H,SO* followed by heating at 1lo”, and PC and cellulose TLC chromatograms by spraying with aniline phthalate reagent followed by heating at 110”. Isolation. T’he bacosides mixt. was obtained from the ethanolic extract of the plant according to the method described earlier [ll]. It was repeatedly chromatographed over silica gel by gradient elution with EtOAc satd with Hz0 containing increasing amounts of MezCO-Hz0 (40: 10) to yield a fr. rich in bacoside A,. This fr. (900 mg) was subjected to flash chromatography over a column of Bondapak Cl8 using MeCN-Hz0 (30: 70) as the eluent to yield pure bacoside A, (120 mg). Bacoside A,. Amorphous solid; Fiegel’s test for carbohydrates positive. IR eicm-‘: 3400 (OH), 2920, 1630 (C=C), 1550, 1370 and 1030. Positive FAB-MS m/z: 951 [M +Na]+, 455 [aglycone-H,O]‘. Negative FARMS m/z: 927 [M-H]-, 795 CM-H-1321 and 765 [M-H - 1621. ‘H NMR (400 MHz, DMSO-d,): 60.72, 0.75, 0.91,0.93, 1.00, 1.58, 1.66(each 3H,s, 7 x Me),4.31 (lH, d, 5=7Hz,GlcH-1’),4.35(1H,d,J=7Hz,GlcH-1”),5.23
Bacoside A, from Bucopa nwnniera
fluxed with 2 h4 HCl in 80% aq. EtOH (0.5 ml) for 4 hr. Hydrolysate was then diluted with H,O, freed of EtOH by evapn and further heated at 100” for 1 hr. It was then neutralized with Amberlite IR 400 (CO:-) resin, coned to 0.5 ml and stirred with NaBH., (15 mg) at room temp. After 2 hr Amberlite IR 120 (H+) resin was added to maintain the pH at 3.5 and the mixt. was filtered, evapd and co-distilled with 3 portions (5 ml each) of MeOH. The resulting mixt. of alditols was treated with Ac,O and pyridine (0.5 ml, each) for 2 hr at 100” and the reagents were removed by co-distillation with toluene. The residue containing the alditol acetates was subjected to GC-MS using a GLC column containing 3% of OV-1 at 160”. The results are summarized in Table 2. Pennethylation and preparation ofalditol acetates of the diglycoside, obtained by Mannich hydrolysis of bacoside A,. The diglycoside was permethylated, the product
hydrolysed, and the resulting partially methylated sugars were converted into their alditol acetates according to the procedure described above for bacoside A,. Results are summarized in Table 3. Acknowledgements-The
authors are grateful to Dr B. N. Mehrotra for identifying the plant material and to Mrs Preeti Rastogi for Technical Assistance. They also thank the staff of the Regional Sophisticated Instrumentation Centre, Lucknow for spectral data. REFERENCES
1. Chopra, R. N. and Chopra, I. C. (1956) in Glossary of Indian Medicinal Plants, p. 32. Counsil of Scientific and Industrial Research, New Delhi. 2. Singh, H. K. and Dhawan, B. N. (1978) fnd. J. Pharmacol. 10, 72.
PIWTO36:1-J
137
3. Singh, H. K. and Dhawan, B. N. (1982) J. Ethnopharmacol. 5, 205.
4. Singh, H. K., Rastogi, R. P., Srimal, R. C. and Dhawan, B. N. (1988) Phytother. Res. 2, 70. 5. Kulshreshtha, D. K. and Rastogi, R. P. (1973) Phytochemistry 12, 887.
6. Kawai, K., Itaka, Y., Shibata, S., Kulshreshtha, D. K. and Rastogi, R. P. (1973) Acta Cryst. 829, 2947. 7. Kulshreshtha, D. K. and Rastogi, R. P. (1974) Phytochemistry 13, 1205. 8. Chandel, R. S., Kulshreshtha, D. K. and Rastogi, R. P. (1977) Phytochemistry 16, 141. 9. Kulshreshtha, D. K. and Rastogi, R. P. (1973) Phytochemistry 12, 2074.
10. Jain, P. and Kulshreshtha,
D. K. (1993) Phyto-
chemistry 33, 449.
11. Chatterjee, N., Rastogi, R. P. and Dhar, M. L. (1963) Ind. J. Chem. 1, 212. 12. Kulshreshtha, M. J. and Rastogi, R. P. (1972) Ind. J. Chem. 10, 152. 13. Eade, R. A., Ellis, J., Shannon, J. S., Simes, H. V. and Simes, J. J. H. (1970) Aust. J. Chem. 23, 2085. 14. Kawai, K., Akiyama, T., Ogihara, Y. and Shibata, S. (1974) Phytochemistry 13, 2829. 15. Jansson, P. E., Kenne, L., Liedgren, H., Lindberg, B. and Longren, J. (1976) A Practical Guide to Methylation Analyses of Carbohydrates No. 8. Chem. Commun., Univ. Stockholm. 16. Mannich, C. and Siewert, G. (1942) Ber. dt. Chem. Ges. 75, 737. 17 Agrawal, P. K., Jain, D. C., Gupta, R. K. and Thakur, ’ R. S. (1985) Phytochemistry 24, 2479. 18. Wagner, H., Ott, S., Jurcic, K., Morton, J. and Neszmelyi, A. (1983) Planta Med. 48, 136.
BACOSIDE
Phykxhtmirtry. Vol. 36, No. I, pp. 133 137. 1994 Copyririghl c 1994 Elsncr Sacna Ltd Prinkd in Great Britain All rights reserved 0031 9422i94 5600+0.00
A,-A SUBHA
TRITERPENOID
SAPONIN
FROM BACOPA
MONNIERA*
RASTOGI, RAGHWENDRA PAL and DINESH K. KULSHREsHTHAt
Medicinal Chemistry Division, Central Drug Research Institute, Lucknow, 226001, India (Received in reuisedfirm
Key Word Index-Bocopa
monniera;
Scrophulariaceae;
17 August 1993)
tritergene; saponin; bacoside A,.
Abstract-A
new triterpenoid saponin, bacoside A,, a constituent of bacosides, the saponin mixture of Bacopa was isolated and characterized. Its structure was established as 3-/i-[O-/l-D-glucopyranosyl( l-+3)-O-[a-Larabinofuranosy(1 +2)]-0-/?-D-glucopyranosyl)oxy] jujubogenin by chemical and spectral analyses. The cis-isomer of ebelin lactone was also obtained as one of the artefacts of the aglycone and its structure revised. monniera,
INTRODIJCllON
hexosyl unit from the [M-H] - ion, respectively. Thus, bawside A, had a terminal pentosyl and a hexosyl sugar unit, besides an inner hexosyl unit attached to the aglycone. Bacoside A, was subjected to acid hydrolysis. The aglycone portion, which consisted of one major spot on TLC corresponding to ebelin lactone, was purified by column chromatography over neutral alumina and finally by crystallization from methanol. It furnished needles, mp 173”, EIMS: m/z 454 CM]‘, UV: &,,.. 266, 276, 286 (open chain conjugated triene system). Its ‘HNMR spectrum exhibited signals for seven tertiary C-Megroups(6:0.72,0.81,0.93, 1.00, 1.72, 1.75and 1.78) a CHzOCO (two d, J = 10 Hz each, 64.23 and 4.35), the signals for olefinic protons H, (d, J = 10 Hz, 5 5. I6), H, (d, 5=15Hz,66.04),H,(dd,J=l1,15Hz,66.30)andH,(d, .I = 11 Hz, S5.78), and those for H, (m, 62.75) and H,/H, (two d, J= 18 Hz each, 62.05 and 2.40). The aglycone was thus identified as trons-ebelin lactone(Ia) by comparison of its data with those reported in the literature [ 121. The crystalline trans-ebelin lactone, as well as the mother liquor, were subjected to acetylation. The ‘H NMR spectrum of the acetylated product of the mother liquor exhibited two sets of signals, one set corresponding to trans-ebelin lactone monoacetate and the second set corresponding to its other isomer. Since ebelin lactone has a side chain consisting of a triene system, it can exist as four isomers depending upon the stereochemistry about the double bonds. However, the all tmns-configuration (la) is the preferred one. A cis-isomer of ebelin lactone (2) has also been reported earlier [ 133, in which the A**-double bond has been assigned a cis-configuration on the basis of UV and IR spectra. A difference UV spectrum obtained by subtracting the UV spectrum of the pure trans-acetyl ebelin lactone (d,_ 266, 276, 286 nm) from that of the acetylated mother liquor was therefore recorded. The difference spectrum showed nearly the same absorption maxima (A,,, 271,280 and 292 nm) as reported for the cis-acetyl ebelin lactone [13].
Bacopa monniera (Wettst) (Hindi-brahmi) is used as a reputed net-vine tonic in the traditional system of medicine [l]. Its alcoholic extract was found to improve the performance of rats in several learning tests as manifested by better acquisition, consolidation and retention of newly acquired behavioural responses C2-43. The activity has been attributed to the saponin mixture consisting of bacosides A, B and other saponins. On acid hydrolysis, bacosides yielded a mixture of aglycones, bacogenin A, [S, 63, A, [7J A, [8] and A, [9]. Bacogenin A, has been identified as ebelin lactone. Recently, a minor saponin, bacoside A, was isolated and characterized as 3-0-[a-~arabinofuranosyl( l-+3)-a+arabinopyranosylUujubogenin [lo]. The present communication reports the isolation and characterization of a new triterpenoid saponin, bacoside A,, from the mixture of bacosides obtained from the ethanolic extract of the plant according to the method described earlier [1 l]. RESULTS AND DISCUSSION
The saponin mixture on repeated chromatography involving silica gel and reverse phase C-18 flash column chromatography yielded bacoside A,, the main constituent of the saponin mixture. It was obtained as a white amorphous solid and gave a positive Fiegel’s test revealing its glycosidic nature. Its IR spectrum showed bands at 3400 (OH), 2920, 1630, 1550, 1370 and 1030cm-*. Its positive FAB mass spectrum exhibited two major peaks at m/z 951 [M+Na]+ and 455 [(aglycone - HzO) + H] +. The negative FAB mass spectrum gave a [M-H]peak at m/z 927. Other peaks were observed at m/z 795 [M-H132]- and 765 [M-H - 162]- which were due to the loss of a pentosyl and a
*CDRI communication no. 5141. tAuthor to whom correspondence. should be addressed. 133
S. RASTOGI et al.
134
28
29
la R=H lb R=Ac *
Hf/Hg
Ha
Hb
constants of the olefinic proton signals in both the isomers precluded the presence of cis-configuration of the A’*-double bond in the cis-isomer, as in that case the J HbeHc valuefor the cis-isomer would have been smaller than 15 Hz observed for the trans-isomer. Thus, a cisconfiguration about A 22-double bond was ruled out and the only other alternative was a cis-configuration about A”-double bond, in which H, and Me groups were cis. Thus, the structures of cis-ebelin lactone and its acetyl derivative were revised to 3a and 3b, respectively, which were consistent with the spectral data. Since ebelin lactone is known to be the transformation product of the genuine sapogenin, jujubogenin (4), obtained by acid catalysed transformation during hydrolysis [14], the genuine aglycone of bacoside A, was jujubogenin. PC and TLC of the aqueous portion of the hydrolysate showed that the sugar moieties present in it were glucose and arabinose. In order to determine the sequence of the sugar chain in bacoside A,, it was subjected to permethylation. The FAB mass spectrum (positive) of the permethylated bacoside A, showed peaks at m/z 1077 and 1055 corresponding to [M +Na]+ and [M+ H]+ ions, and indicated the presence of nine methoxy groups. Thus, the sugar chain contained nine free hydroxyl groups which
OH
ROT
RO
3a R=H 3b R=Ac * Hf/Hg
To separate and identify the ‘HNMR signals of the two isomers present in the mother liquor, the ‘H NMR signals of the all trns-acetyl ebelin lactone (lb) were subtracted from those of the mixture. Thus, a neat ‘H NMR spectrum of the other (&)-isomer was obtained which exhibited signals at 60.89,0.90,0.92, 1.08, 1.82, 1.84 and 1.88 (7 x tert. C-Me), 2.07 (s, MeCOO), 4.30,4.38 (2 x d, J = 10 Hz each, CH,OCO) and 4.47 (dd, J = 5,ll Hz, CHOAc); the chemical shift values of these signals being nearly the same as for the all trans-isomer. Other signals belonging to the side chain appeared at 65.06 (d, J = lOHz, HA, 66.36 (d, J= 15 Hz, H,,), 66.50 (dd, J = 11 Hz, 15 Hz, H,), 65.93 (d, J= 11 Hz, Hd), 62.93 (m, H,) and 62.15, 2.42 (2 x d, J = 18 Hz each, H,/HJ. On comparison, it was observed that the ‘H NMR signals of the trans-acetyl ebelin lactone and those of its cis-isomer had the same coupling constants (J values) with slight differences in the chemical shift values. Identical coupling
4
R=H
135
Racoside A, from Bucopa monniero were also indicative
of the presence
one pentosyl
attached
units,
of two hexosyl
and
to one another.
Acid hydrolysis of the permethylated bacoside A, yielded partially methylated sugars which were identified as 2,3,4,6tetra-0-methylglucos, 2,3,5-tri-O-methylarabinose and 4,6-di-0-methylglucose by the CC-MS analyses of their alditol acetates [lS]. The identified partially methylated sugars correspond to a 1,2,3-linked glucose, a terminal arabinofuranose and a terminal glucose. Further, in order to determine the exact linkage of the terminal sugar units, bacoside A, was subjected to Mannich hydrolysis [ 163 to yield a diglycoside. Only the arabinose unit was lost, as confirmed by the TLC of the aqueous portion of the hydrolysate and the positive FAB-mass spectrum of the diglycoside, which exhibited major peaks at m/z 817,801 and 779 corresponding to the presence of [M -Hz0 +K]+, [M-H,O+Na]+ and [M-H,O+H]+ ions, respectively. The diglycoside was subjected to permethylation followed by acid hydrolysis. The resulting partially methylated sugars were identified as 2,3,4,6-tetra-Omethylglucose and 2,4,6-tri-O-methylglucose by GC-MS analyses of their alditol acetates [ 151. The formation of 2,4,6-tri-0-methylglucose indicated the presence of a free -OH group at C-2 of the inner glucopyranosyl unit of the diglycoside, which was, however, absent in that of bacoside A,. The arabinose unit was therefore attached at C2 of the inner glucose unit. These methylation studies clearly indicated that the terminal glucopyranosyl unit and the terminal arabinofuranosyl unit were attached to the inner glucopyranosyl unit through l-+3 and l-+2 linkages, respectively, and the inner glucopyranosyl unit was attached to the aglycone through an 0-glycosidic linkage. The above inference was further confirmed by the 13C and ‘HNMR chemical shifts of bacoside A,. The 13CNMR spectrum established that the glucose units
Table 1. 13C NMR spectral data of bacoside A, (CD,OD) Aglycone part -_-C
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sugar part
39.9 21.3 90.7 40.5 57.5 19.2 37.1 38.5 54.2 38.0 22.5 29.2 38.3 54.6 36.9
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
C 111.4 54.4 19.2 16.8 69.4 29.6 45.4 69.7 126.3 136.7 25.8 18.4 28.3 16.8 66.9
Glc
Glc
Ara
1’ 2 3’ 4 5 6 1” 2” 3” 4” 5” 6” 1”’ 2”’ 3”’ 4” 5”’
104.2 78.2 83.6 IQ.3 78.1 62.6 105.5 75.5 77.2 71.7 71.6 62.7 109.9 84.1 80.0 87.4 62.1
existed in the pyranose form, whereas the arabinose unit was present in the furanose form. Assignments of the signals were made on the basis of broad band decoupled as well as DEPT “CNMR spectra [lfl. The chemical shift assignments were made straightaway by comparison with the reported values of O-methyl-a-L-arabinofuranoside and 0-methyl-/?-D-glucopyranoside, taking into consideration the a- and &eKects of glycosidation. *3C NMR chemical shifts of the aglycone portion including that of C-3 were also consistent with those assigned to the jujubogenin portion of the saponins isolated from Colubrina asiatico [18] (Table l), indicating that the sugar chain was linked to jujubogenin at C-3. In the ‘H NMR spectrum of bacoside A,, the anomeric proton signals for two /?-D-glucopyranosyl units ap peared at 64.31 (d, J = 7 Hz) and 4.35 (d. J = 7 Hz) and that for the arabinofuranosyl unit at 65.23 (bs). Thus, on the basis of the above evidence, the structure of bacoside A, was established as 3-B-[@B-D-glucopyranosyl( l-+3)-0-[rx-L-arabinofuranosyl(l -r2)]-O-B-~-glucopyranosyl)-oxyljujubogenin (5). FXPERIMENTAL Plant material. Bacopa monniera (whole plant) was procured from Calcutta market (India). A voucher specimen of the plant is preserved in the herbarium of Botany Division, CDRI. General. Mps: uncorr. IR: KBr disc. UV: EtOH. ‘H NMR: 400 MH& TMS as int. standard. 13C NMR: 100 MHz. CC: silica gel, bondapak C,, and neutral alumina. TLC: (i) silica gel coated plates using solvent system: (1) C,H,-MeOH (96:4); (2) EtOAc-MeOHHz0 (80: 10: 10); (3) EtOAc-MezCO-Hz0 (50:40: 10); (ii) reverse phase-C,, coated plates (E. Merck) using solvent system (4) MeCN-Hz0 (50:50); (iii) cellulose coated plates using solvent system (5) n-BuOHAcOH-H,O (40: 10:50). PC: Whatman No. 1 paper and solvent (6) n-BuOH satd with H,O. Silica gel TLC were visualized by spraying with 1% ceric sulphate in 1 M H,SO* followed by heating at 1lo”, and PC and cellulose TLC chromatograms by spraying with aniline phthalate reagent followed by heating at 110”. Isolation. T’he bacosides mixt. was obtained from the ethanolic extract of the plant according to the method described earlier [ll]. It was repeatedly chromatographed over silica gel by gradient elution with EtOAc satd with Hz0 containing increasing amounts of MezCO-Hz0 (40: 10) to yield a fr. rich in bacoside A,. This fr. (900 mg) was subjected to flash chromatography over a column of Bondapak Cl8 using MeCN-Hz0 (30: 70) as the eluent to yield pure bacoside A, (120 mg). Bacoside A,. Amorphous solid; Fiegel’s test for carbohydrates positive. IR eicm-‘: 3400 (OH), 2920, 1630 (C=C), 1550, 1370 and 1030. Positive FAB-MS m/z: 951 [M +Na]+, 455 [aglycone-H,O]‘. Negative FARMS m/z: 927 [M-H]-, 795 CM-H-1321 and 765 [M-H - 1621. ‘H NMR (400 MHz, DMSO-d,): 60.72, 0.75, 0.91,0.93, 1.00, 1.58, 1.66(each 3H,s, 7 x Me),4.31 (lH, d, 5=7Hz,GlcH-1’),4.35(1H,d,J=7Hz,GlcH-1”),5.23
Bacoside A, from Bucopa nwnniera
fluxed with 2 h4 HCl in 80% aq. EtOH (0.5 ml) for 4 hr. Hydrolysate was then diluted with H,O, freed of EtOH by evapn and further heated at 100” for 1 hr. It was then neutralized with Amberlite IR 400 (CO:-) resin, coned to 0.5 ml and stirred with NaBH., (15 mg) at room temp. After 2 hr Amberlite IR 120 (H+) resin was added to maintain the pH at 3.5 and the mixt. was filtered, evapd and co-distilled with 3 portions (5 ml each) of MeOH. The resulting mixt. of alditols was treated with Ac,O and pyridine (0.5 ml, each) for 2 hr at 100” and the reagents were removed by co-distillation with toluene. The residue containing the alditol acetates was subjected to GC-MS using a GLC column containing 3% of OV-1 at 160”. The results are summarized in Table 2. Pennethylation and preparation ofalditol acetates of the diglycoside, obtained by Mannich hydrolysis of bacoside A,. The diglycoside was permethylated, the product
hydrolysed, and the resulting partially methylated sugars were converted into their alditol acetates according to the procedure described above for bacoside A,. Results are summarized in Table 3. Acknowledgements-The
authors are grateful to Dr B. N. Mehrotra for identifying the plant material and to Mrs Preeti Rastogi for Technical Assistance. They also thank the staff of the Regional Sophisticated Instrumentation Centre, Lucknow for spectral data. REFERENCES
1. Chopra, R. N. and Chopra, I. C. (1956) in Glossary of Indian Medicinal Plants, p. 32. Counsil of Scientific and Industrial Research, New Delhi. 2. Singh, H. K. and Dhawan, B. N. (1978) fnd. J. Pharmacol. 10, 72.
PIWTO36:1-J
137
3. Singh, H. K. and Dhawan, B. N. (1982) J. Ethnopharmacol. 5, 205.
4. Singh, H. K., Rastogi, R. P., Srimal, R. C. and Dhawan, B. N. (1988) Phytother. Res. 2, 70. 5. Kulshreshtha, D. K. and Rastogi, R. P. (1973) Phytochemistry 12, 887.
6. Kawai, K., Itaka, Y., Shibata, S., Kulshreshtha, D. K. and Rastogi, R. P. (1973) Acta Cryst. 829, 2947. 7. Kulshreshtha, D. K. and Rastogi, R. P. (1974) Phytochemistry 13, 1205. 8. Chandel, R. S., Kulshreshtha, D. K. and Rastogi, R. P. (1977) Phytochemistry 16, 141. 9. Kulshreshtha, D. K. and Rastogi, R. P. (1973) Phytochemistry 12, 2074.
10. Jain, P. and Kulshreshtha,
D. K. (1993) Phyto-
chemistry 33, 449.
11. Chatterjee, N., Rastogi, R. P. and Dhar, M. L. (1963) Ind. J. Chem. 1, 212. 12. Kulshreshtha, M. J. and Rastogi, R. P. (1972) Ind. J. Chem. 10, 152. 13. Eade, R. A., Ellis, J., Shannon, J. S., Simes, H. V. and Simes, J. J. H. (1970) Aust. J. Chem. 23, 2085. 14. Kawai, K., Akiyama, T., Ogihara, Y. and Shibata, S. (1974) Phytochemistry 13, 2829. 15. Jansson, P. E., Kenne, L., Liedgren, H., Lindberg, B. and Longren, J. (1976) A Practical Guide to Methylation Analyses of Carbohydrates No. 8. Chem. Commun., Univ. Stockholm. 16. Mannich, C. and Siewert, G. (1942) Ber. dt. Chem. Ges. 75, 737. 17 Agrawal, P. K., Jain, D. C., Gupta, R. K. and Thakur, ’ R. S. (1985) Phytochemistry 24, 2479. 18. Wagner, H., Ott, S., Jurcic, K., Morton, J. and Neszmelyi, A. (1983) Planta Med. 48, 136.