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D. Mandal et al. / Phytochemistry 67 (2006) 1316–1321
ether (60–80 ꢁC) and then successively extracted with
MeOH (3 · 4 l) at ambient temperature. The combined
MeOH extract was concentrated under reduced pressure.
The concentrated extract was partitioned between n-
BuOH and H2O. The organic layer was further washed
with water for complete removal of inorganic impurities
and free sugars and the solvent removed under reduced
pressure to yield a dark-brown residue (42 g). The residue
was applied to a column of Diaion HP-20 (500 g) and,
washed with water followed by 30%, 50%, 80% and
100% of MeOH. Fraction eluted with 50% MeOH
(4.5 g) was chromatographed over silica gel (100 g).
Graded elution was carried out with chloroform followed
by various mixtures of CHCl3–MeOH (19:1, 9:1, 4:1 and
3:1). A total of 35 fractions (each 75 ml) were collected
and those giving similar spots on TLC were combined.
The more polar fractions eluted with CHCl3–MeOH
(4:1) were combined and rechromatographed over silica
gel. Earlier fractions eluted with CHCl3–MeOH (17:3)
was found to be a mixture of racemosides B and C
(120 mg), while later fractions furnished racemoside A
(75 mg). Racemosides B and C were successfully separated
by preparative TLC (mobile phase: CHCl3:MeOH:H2O;
60:23:3) to give racemoside B (39 mg) and racemoside C
(28 mg).
treated with Dowex 50 (H+) and the solution concentrated.
Boric acid was removed by co-distillation with MeOH and
the product acetylated with acetic anhydride–pyridine (1:1)
at 100 ꢁC for 1 h, diluted with water and extracted with
CHCl3. The alditol acetates of the monosaccharides
obtained after removal of the solvent were identified to be
glucose and rhamnose in the ratio 3:1 from GLC analysis.
3.6. Racemoside B (2)
Colourless crystals from MeOH, m.p. 240–242 ꢁC,
26
½aꢁD ꢀ 41:1ꢂ (c 0.81, MeOH); IR (KBr): mmax cmꢀ1; 3410,
1440, 1360, 1150, 1050, 978, 920 and 901 (absorption
920 > 901); ESI-TOFMS (positive): m/z 909.34 [M+Na]+;
1H NMR (C5D5N): 0.81 (s, H3-18), 0.85 (s, H3-19), 1.17
(d, J = 6.6 Hz, H3-21), 1.08 (d, J = 6.6 Hz, H3-27), 3.38
(d, J = 11.4 Hz, H-26), 4.07 (unresolved, H-26), 4.31 (unre-
solved, H-3), 4.57 (unresolved, H-16) and sugar moiety
(Table 2); 13C NMR (Table 1); (Found: C, 60.87; H,
8.46; C45H74O17 requires: C, 60.93; H, 8.41%).
3.7. Racemoside C (3)
Colourless needles from MeOH, m.p. 236–238 ꢁC,
26
½aꢁD ꢀ 55:4ꢂ (c 0.56, MeOH); IR (KBr): mmax cmꢀ1; 3430,
1445, 1365, 1155, 1050, 980, 920 and 900 (absorption
920 > 900); ESI-TOFMS (positive): m/z 893.14 [M+Na]+;
1H NMR (C5D5N): 0.82 (s, H3-19), 0.83 (s, H3-18), 1.17
(d, J = 7.2 Hz, H3-21), 1.09 (d, J = 7.2 Hz, H3-27), 3.40
(d, J = 11.4 Hz, H-26), 4.09 (d, J = 9.0 Hz, H-26), 4.32
(H-3), 4.61 (t-like, J = 7.2 Hz H-16) and sugar moiety
(Table 2); 13C NMR (Table 1); (Found: C, 61.97; H,
8.41; C45H74O16 requires C, 62.05; H, 8.56%).
3.4. Racemoside A (1)
Colourless needles from MeOH, m.p. 244–246 ꢁC;
26
½aꢁD ꢀ 34:9ꢂ (c 0.90, H2O); IR (KBr): mmax cmꢀ1 ; 3415,
1435, 1367, 1150, 1060, 986, 918, 900 and 851 (absorption
918 > 900); ESI-TOFMS (positive): m/z 1071.55
[M+Na]+; 1H NMR and 13C NMR (Tables 1 and 2);
(Found: C, 58.31; H, 8.12; C51H84O22 requires: C, 58.38;
H, 8.07%).
3.8. Acid hydrolysis of 2 and 3
3.5. Acid hydrolysis of 1
A solution of 2 and 3 (8 mg each) was subjected to acid
hydrolysis by the same procedure as described for 1. Usual
work up followed by chromatographic purification on a sil-
ica gel column furnished sarsasapogenin as aglycone for
both 2 and 3. The monosaccharides were identified as glu-
cose and rhamnose in the ratios 2:1 and 1:2 for 2 and 3,
respectively, by GLC analysis on comparison with authen-
tic samples.
Racemoside A (1) (15 mg) was heated with 1 mol/L HCl
in aqueous dioxane (1:1, v/v, 10 ml) at 80 ꢁC for 3 h, cooled,
and 5 ml of water was added. Dioxane was removed under
reduced pressure, and the solution was extracted with
EtOAc (5 ml · 3). The organic layer was washed with water
until free from acid and dried to give a white powder. Puri-
fication of the product over a silica gel column and subse-
quent crystallization from chloroform–methanol mixture
afforded the aglycone in fine needles, identified as sarsas-
apogenin by TLC comparison with an authentic sample
and 13C NMR chemical shifts (Agrawal et al., 1997). The
aqueous part of the acid hydrolysate was neutralized with
silver carbonate and filtered. The filtrate containing sugar
mixture was evaporated to dryness in vacuo. This was then
dissolved in water (10 ml) and divided into two parts. The
first part was examined for sugars by paper chromatogra-
phy, leading to the identification of glucose and rhamnose
using authentic specimens. The second part was reduced
with NaBH4 (200 mg) for 2 h. The reduced product was
Acknowledgements
We thank Dr. Debjani Basu, Scientist, Botanical Survey
of India, Howrah, West Bengal, India for identification of
the plant material and Shri Rajendra Mahato, Helper of
this Department for collection of the plant material. Dr.
N.P. Sahu is indebted to CSIR for the award of Emeritus
Scientist, and Mr. D. Mandal is thankful to CSIR for
financial assistance as SRF.