892
Vol. 57, No. 8
Pradesh, India. A voucher specimen (PLP-11100) has been deposited in the
herbarium of IHBT, Palampur, India.
Extraction and Isolation Air dried powder of roots of A. racemosus
(2.75 kg) was subsequently extracted with methanol (3ꢇ4 l) and water :
methanol (7 : 3, 4ꢇ4 l) in a percolator at room temperature. Each combined
rhamnopyranosyl by analyzing NMR values and coupling
constants followed by acid hydrolysis of 2 and comparison
with reference sugars on TLC plate. The sugars were further
identified by GC-MS analysis of their alditol acetates with
reference sugars. The linkages between sugar moieties were percolation was dried under reduced pressure to yield 300 g and 700 g of
methanol and water : methanol extract, respectively. Methanol extract (300 g)
was subjected to a column chromatography over Diaion HP-20 (500 g) and
eluted with deionised water followed by 10, 20, 30, 50 and 100% ethanol in
water. Total 60 fractions (200 ml each) were collected. Fractions no. 51—60
determined on the basis of HMBC spectrum, mass fragmen-
tation pattern and shift values (a- and b-effects) in 13C-NMR
spectral data. The HMBC cross correlation between H-1ꢄ (d
4.78 d, Jꢂ7.3 Hz) of glc I and C-3 (d 75.3) of aglycon sug-
eluted with 50 and 100% ethanol in water were combined and dried (15 g).
The dried fraction (15 g) was further chromatographed over silica gel (60—
120 mesh) using a gradient elution in CHCl3 and mixture of CHCl3 :
MeOH : H2O (65 : 5 : 10, 65 : 10 : 10, 65 : 15 : 10, 65 : 20 : 10, 65 : 22 : 10,
65 : 25 : 10 and 65 : 30 : 10). Total 67 fractions (80 ml each) were collected.
Fractions no. 24—30 eluted with CHCl3 : MeOH : H2O (65 : 20 : 10) yielded
3 (85 mg) on crystallization with MeOH. The fractions no. 33—38 eluted in
the same polarity as above afforded 2 (42 mg) after crystallization in
methanol. The fractions no. 40—44 eluted with CHCl3 : MeOH : H2O
(65 : 22 : 10) yielded 1 (30 mg) after crystallization with methanol.
gested the direct linkage of glc I (inner sugar) to the aglycone
part. Similarly, the correlations between H-1ꢅ (d 4.76, d,
Jꢂ7.3 Hz) of glc II and C-2ꢄ (d 80.3) of glc I, H-1ꢆ (d 5.56,
br s) of rham and C-6ꢄ (d 68.0) of glc I and H-1ꢅꢅ (d 5.44, d,
Jꢂ6.8 Hz) of xyl and C-4ꢄ (d 79.0) of glc I indicated the di-
rect attachment of glc II, rham and xyl to C-2ꢄ, C-6ꢄ and C-
4ꢄ of glc I, respectively. The arrangement of sugar units was
further supported by mass fragmentation pattern.
Shatavaroside A (1): White amorphous powder; mp 187—192 °C, [a]D35
ꢁ31.4 (cꢂ0.06, DMSO); IR (KBr) cmꢁ1: 3433 (OH), 2929 (CH), 1049,
919, 900, 847 cmꢁ1; HR-ESI-QTOF-MS (positive) m/z: 857.4866 [MꢀH]ꢀ
(Calcd for C44H72O16, 857.4899); MS/MS m/z: 725 [MꢀHꢁ132]ꢀ, 711
[MꢀHꢁ146]ꢀ, 579 [MꢀHꢁ132ꢁ146]ꢀ, 441 [132ꢀ162ꢀ146ꢀH]ꢀ, 417
[MꢀHꢁ132ꢁ146ꢁ162]ꢀ, 399 [MꢀHꢁ132ꢁ146ꢁ162ꢁH2O]ꢀ, 272 [Mꢀ
Hꢁ132ꢁ146ꢁ162ꢁ144]ꢀ; The 1H- and 13C-NMR data (DMSO-d6): see
Table 1.
ESI-QTOF-MS/MS of 2 (m/z 1019) displayed the frag-
ment at m/z 887 [MꢀHꢁ132]ꢀ, 873 [MꢀHꢁ146]ꢀ and 857
[MꢀHꢁ162]ꢀ represented glycosidic cleavage by simultane-
ous loss of a pentose, deoxyhexose and a hexose sugar unit
from molecular ion peak, hence, suggested that the three
sugar moieties were present in terminal positions. The loss of
a deoxyhexose and/or pentose unit from m/z 887 and 873 re-
vealed the fragment at m/z 741 [MꢀHꢁ132ꢁ146]ꢀ. Simi-
larly the fragment at m/z 725 [MꢀHꢁ132ꢁ162]ꢀ indicated
the loss of a hexose and/or a pentose unit from m/z 887 and
857, respectively. The fragment at m/z 711 was observed due
to the loss of a hexose and/or a deoxyhexose sugar unit from
m/z 873 and 857, respectively. Other fragments at m/z 579
[aglyconeꢀHꢀglc I]ꢀ and 417 [aglyconeꢀH]ꢀ were ob-
served due to the sequential loss of other remaining sugar
units from m/z 741, 725 and 711. The mass ion at m/z 603
[pentoseꢀ2ꢇhexoseꢀdeoxyhexoseꢀH]ꢀ were due to the
cleavage of polysaccharide portion from aglycone part which
proved the presence of all sugar units in one group. The frag-
ments at m/z 435 and 273 were observed due to the subtrac-
tion of ring F (spirostane, m/z 144) from m/z 579 and 417, re-
spectively. Thus, on the basis of above evidences the struc-
ture of saponin 2 was assigned as 3-O-{[b-D-glucopyra-
nosyl(1→2)]-[b-D-xylopyranosyl-(1→4)]-[a-L-rhamnopyra-
nosyl-(1→6)]-b-D-glucopyranosyl}-(25S)-5b-spirostan-3b-ol
and designated as shatavaroside B (Fig. 1).
Shatavaroside B (2): White amorphous powder; mp 217—219 °C; [a]D19
ꢁ67.7 (cꢂ0.53, py); IR (KBr) cmꢁ1 3429 (OH), 2932 (CH), 1069, 918, 900,
847 cmꢁ1; HR-ESI-QTOF-MS (positive) m/z: 1019.5410 [MꢀH]ꢀ (Calcd
for C50H20O21, 1019.5427); MS/MS m/z: 887 [MꢀHꢁ132]ꢀ, 873 [MꢀHꢁ
146], 857 [MꢀHꢁ162]ꢀ, 741 [MꢀHꢁ132ꢁ146]ꢀ, 725 [MꢀHꢁ132ꢁ
162]ꢀ, 711 [MꢀHꢁ146ꢁ162]ꢀ, 603 [132ꢀ162ꢀ146ꢀ162ꢀH]ꢀ, 579
[MꢀHꢁ132ꢁ146ꢁ162]ꢀ, 435 [MꢀHꢁ132ꢁ146ꢁ162ꢁ144]ꢀ, 417 [Mꢀ
Hꢁ132ꢁ146ꢁ2ꢇ162]ꢀ, 399 [MꢀHꢁ132ꢁ146ꢁ162ꢁ162ꢁH2O]ꢀ, 273
[MꢀHꢁ132ꢁ146ꢁ162ꢁ162ꢁ144]ꢀ; The 1H- and 13C-NMR data (C5D5N):
see Table 1.
Acid Hydrolysis of 1 and 2 The saponins (1, 2) (10 mg each) was hy-
drolyzed with 2 M HCl in 1,4-dioxane (1 : 1, 1.5 ml) and refluxed for 2 h,
cooled and 5 ml of water was added. Dioxane was removed under reduced
pressure and the solution was extracted with EtOAc (5 mlꢇ3). The organic
layer was washed with water until free from acid and dried to give a white
powder. Purification of the product over a silica gel column and subsequent
crystallization from chloroform–methanol mixture afforded the aglycone in
fine needles (5.7 mg from 1 and 4.9 mg from 2) and identified as sarsasa-
pogenin by comparing its 13C-NMR data with earlier report.25) The aqueous
part of the acid hydrolysate was neutralized with silver carbonate and fil-
tered. The filtrate containing sugar mixture was evaporated to dryness in
vacuum, dissolved in water (5 ml) and was examined by TLC [EtOAc–
MeOH–HOAc–H2O (6 : 2 : 1 : 1)] with reference sugars. Alditol acetates
were prepared by reduction of sugar part and reference sugars at room tem-
perature for 30 min using a solution of 0.25 M NaBH4 in NH4OH (1.5 ml)
and then quenched by a solution of 10% of acetic acid in MeOH (20 ml).
The reduced product was then acetylated with acetic anhydride–pyridine
(1 : 1) at 100 °C for 1 h. This mixture was diluted with water and extracted
with ethyl acetate and dried. The ethyl acetate extract was analyzed by GC-
MS and sugars were identified by comparing their retention times with au-
thentic sugars. GC-MS analyses were carried out on a Shimadzu QP 2010
GC-MS system equipped with a Carbowax BP20 capillary column (30 mꢇ
0.25 mm, 0.25 mm film thickness) from SGE, Australia; carrier gas, helium,
at a flow rate of 1.1 ml/min; ionization energy, 70 eV; ion source tempera-
ture, 230 °C; injector temperature, 220 °C. Oven temperature was pro-
grammed as follows: initially at 70 °C for 4 min, rising at 4 °C/min to 220 °C
and then held isothermal (4 min) at 220 °C. Peaks corresponding to L-rham-
nose, L-arabinose, D-xylose and D-glucose appeared at 2.65, 3.31, 3.45,
5.86 min, respectively. The alditol acetates of sugars were identified as D-
glucose, L-rhamnose and L-arabinose for saponin 1 and D-glucose, L-rahm-
nose and D-xylose for saponin 2.
Compound 3 was isolated as amorphous powder and was
identified as filiasparoside C by comparing its spectroscopic
data with reported values.22)
It is worth pointing out that the occurrence of steroidal
saponins is characteristic of Asparagus genus and is consid-
ered as a chemotaxonomic marker of the genus.
Experimental
General Methods Melting points were determined on a Barnstead
Electrothermal 9100 and were uncorrected. Optical rotation was determined
on Horiba Sepa-300 Polarimeter and IR spectra were recorded on a NICO-
LET-6700 FT-IR spectrometer with KBr disc. Mass spectra were recorded
on QTOF-Micro of Waters Micromass. NMR experiments were performed
on Bruker Avance-300 spectrometer. Diaion HP-20 was procured from Kit-
ten Enterprises Pvt. Ltd., Mumbai, India and Silica gel (60—120 mesh),
TLC silica gel 60 F254 plates and all other chemicals used were purchased
from Merck India Ltd. Standards glucose, xylose, rhamnose and arabinose
were purchased from Life Technologies (India) Pvt. Ltd.
Acknowledgement Authors are grateful to the Director, IHBT, Palam-
pur for providing necessary facilities. Thanks are also due to CSIR for
awarding junior research fellowship to U. Sharma.
Plant Material The plant material was collected in August 2006 from
Medicinal Plant Research Institute (ISM), Joginder Nagar, Himachal