Pergamon Phykxhtmirtry. Vol. 36, No. I, pp. 133 137.

1994
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0031 9422i94 5600+0.00

BACOSIDE A,-A TRITERPENOID SAPONIN FROM BACOPA MONNIERA*

SUBHA RASTOGI, RAGHWENDRA PAL and DINESH K. KULSHREsHTHAt

Medicinal Chemistry Division, Central Drug Research Institute, Lucknow, 226001, India

(Received in reuisedfirm 17 August 1993)

Key Word Index-Bocopa monniera; Scrophulariaceae; tritergene; saponin; bacoside A,.

Abstract-A new triterpenoid saponin, bacoside A,, a constituent of bacosides, the saponin mixture of Bacopa
monniera, was isolated and characterized. Its structure was established as 3-/i-[O-/l-D-glucopyranosyl( l-+3)-O-[a-L-
arabinofuranosy(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.

INTRODIJCllON hexosyl unit from the [M-H] - ion, respectively. Thus,

Bacopa monniera (Wettst) (Hindi-brahmi) is used as a
reputed net-vine tonic in the traditional system of medi-
cine [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+arabinopyranosylUujubo-
genin [lo]. The present communication reports the isola-
tion 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 consti-
tuent of the saponin mixture. It was obtained as a white
amorphous solid and gave a positive Fiegel’s test re-
vealing 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 spec-
trum gave a [M-H]- peak at m/z 927. Other peaks were
observed at m/z 795 [M-H- 132]- 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.
bawside A, had a terminal pentosyl and a hexosyl sugar
unit, besides an inner hexosyl unit attached to the agly-
cone.
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 spec-
trum 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-config-
uration (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-configura-
tion 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].

133
134 S. RASTOGI et al.

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 cis-
configuration 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.
28 29
Thus, the structures of cis-ebelin lactone and its acetyl
la R=H derivative were revised to 3a and 3b, respectively, which
lb R=Ac were consistent with the spectral data.
* Hf/Hg Since ebelin lactone is known to be the transformation
product of the genuine sapogenin, jujubogenin (4), ob-
tained by acid catalysed transformation during hydroly-
Ha Hb sis [14], the genuine aglycone of bacoside A, was juju-
bogenin.
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 permethyl-
ation. The FAB mass spectrum (positive) of the permeth-
ylated 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
4 R=H
3b R=Ac
* Hf/Hg

To separate and identify the ‘HNMR signals of the

two isomers presentin 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
 Racoside A, from Bucopa monniero 135

were also indicative of the presence of two hexosyl and existed in the pyranose form, whereas the arabinose unit
one pentosyl units, attached to one another. Acid hy- was present in the furanose form. Assignments of the
drolysis of the permethylated bacoside A, yielded par- signals were made on the basis of broad band decoupled
tially methylated sugars which were identified as 2,3,4,6- as well as DEPT “CNMR spectra [lfl. The chemical
tetra-0-methylglucos, 2,3,5-tri-O-methylarabinose and shift assignments were made straightaway by comparison
4,6-di-0-methylglucose by the CC-MS analyses of their with the reported values of O-methyl-a-L-arabinofurano-
alditol acetates [lS]. The identified partially methylated side and 0-methyl-/?-D-glucopyranoside, taking into con-
sugars correspond to a 1,2,3-linked glucose, a terminal sideration the a- and &eKects of glycosidation. *3C NMR
arabinofuranose and a terminal glucose. Further, in order chemical shifts of the aglycone portion including that of
to determine the exact linkage of the terminal sugar units, C-3 were also consistent with those assigned to the
bacoside A, was subjected to Mannich hydrolysis [ 163 to jujubogenin portion of the saponins isolated from Colu-
yield a diglycoside. Only the arabinose unit was lost, as brina asiatico [18] (Table l), indicating that the sugar
confirmed by the TLC of the aqueous portion of the chain was linked to jujubogenin at C-3.
hydrolysate and the positive FAB-mass spectrum of the In the ‘H NMR spectrum of bacoside A,, the anomeric
diglycoside, which exhibited major peaks at m/z 817,801 proton signals for two /?-D-glucopyranosyl units ap
and 779 corresponding to the presence of [M -Hz0 peared at 64.31 (d, J = 7 Hz) and 4.35 (d. J = 7 Hz) and
+K]+, [M-H,O+Na]+ and [M-H,O+H]+ ions, that for the arabinofuranosyl unit at 65.23 (bs).
respectively. The diglycoside was subjected to permethyl- Thus, on the basis of the above evidence, the structure
ation followed by acid hydrolysis. The resulting partially of bacoside A, was established as 3-B-[@B-D-glucopy-
methylated sugars were identified as 2,3,4,6-tetra-O- ranosyl( l-+3)-0-[rx-L-arabinofuranosyl(l -r2)]-O-B-~-glu-
methylglucose and 2,4,6-tri-O-methylglucose by GC-MS copyranosyl)-oxyljujubogenin (5).
analyses of their alditol acetates [ 151. The formation of
2,4,6-tri-0-methylglucose indicated the presence of a free
FXPERIMENTAL
-OH group at C-2 of the inner glucopyranosyl unit of the
diglycoside, which was, however, absent in that of bac- Plant material. Bacopa monniera (whole plant) was
oside A,. The arabinose unit was therefore attached at C- procured from Calcutta market (India). A voucher speci-
2 of the inner glucose unit. These methylation studies men of the plant is preserved in the herbarium of Botany
clearly indicated that the terminal glucopyranosyl unit Division, CDRI.
and the terminal arabinofuranosyl unit were attached to General. Mps: uncorr. IR: KBr disc. UV: EtOH.
the inner glucopyranosyl unit through l-+3 and l-+2 ‘H NMR: 400 MH& TMS as int. standard. 13C NMR:
linkages, respectively, and the inner glucopyranosyl unit 100 MHz. CC: silica gel, bondapak C,, and neutral
was attached to the aglycone through an 0-glycosidic alumina. TLC: (i) silica gel coated plates using solvent
linkage. system: (1) C,H,-MeOH (96:4); (2) EtOAc-MeOH-
The above inference was further confirmed by the 13C Hz0 (80: 10: 10); (3) EtOAc-MezCO-Hz0 (50:40: 10);
and ‘HNMR chemical shifts of bacoside A,. The (ii) reverse phase-C,, coated plates (E. Merck) using
13CNMR spectrum established that the glucose units solvent system (4) MeCN-Hz0 (50:50); (iii) cellulose
coated plates using solvent system (5) n-BuOH-
AcOH-H,O (40: 10:50). PC: Whatman No. 1 paper and
solvent (6) n-BuOH satd with H,O. Silica gel TLC were
Table 1. 13C NMR spectral data of bacoside A, (CD,OD)
visualized by spraying with 1% ceric sulphate in 1 M
Aglycone part Sugar part
H,SO* followed by heating at 1lo”, and PC and cellulose
-_-- TLC chromatograms by spraying with aniline phthalate
C C C reagent followed by heating at 110”.
Isolation. T’he bacosides mixt. was obtained from the
1 39.9 16 111.4 Glc 1’ 104.2 ethanolic extract of the plant according to the method
2 21.3 17 54.4 2 78.2 described earlier [ll]. It was repeatedly chromato-
3 90.7 18 19.2 3’ 83.6 graphed over silica gel by gradient elution with EtOAc
4 40.5 19 16.8 4 IQ.3
satd with Hz0 containing increasing amounts of
5 57.5 20 69.4 5 78.1
MezCO-Hz0 (40: 10) to yield a fr. rich in bacoside A,.
6 19.2 21 29.6 6 62.6
This fr. (900 mg) was subjected to flash chromatography
7 37.1 22 45.4 Glc 1” 105.5
8 38.5 23 69.7 2” 75.5 over a column of Bondapak Cl8 using MeCN-Hz0
9 54.2 24 126.3 3” 77.2 (30: 70) as the eluent to yield pure bacoside A, (120 mg).
10 38.0 25 136.7 4” 71.7 Bacoside A,. Amorphous solid; Fiegel’s test for carbo-
11 22.5 26 25.8 5” 71.6 hydrates positive. IR eicm-‘: 3400 (OH), 2920, 1630
12 29.2 27 18.4 6” 62.7 (C=C), 1550, 1370 and 1030. Positive FAB-MS m/z: 951
13 38.3 28 28.3 Ara 1”’ 109.9 [M +Na]+, 455 [aglycone-H,O]‘. Negative FARMS
14 54.6 29 16.8 2”’ 84.1 m/z: 927 [M-H]-, 795 CM-H-1321 and 765 [M-H
15 36.9 30 66.9 3”’ 80.0 - 1621. ‘H NMR (400 MHz, DMSO-d,): 60.72, 0.75,
4” 87.4
0.91,0.93, 1.00, 1.58, 1.66(each 3H,s, 7 x Me),4.31 (lH, d,
5”’ 62.1
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.
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