New ursane glycoside from the roots of asparagus racemosus

Article abstract of DOI:10.14233/ajchem.2013.14837

One new compound urs-1,12,19- trien-3b-ol-28-oic acid-3b-D-glucopyranosyl (4'-1'')-b-D-glucopyranoside (1) along with five known compounds (2-6) have been isolated from the roots of Asparagus racemosus. Their structures have been elucidated with the help

Full text of DOI:10.14233/ajchem.2013.14837

Asian Journal of Chemistry; Vol. 25, No. 15 (2013), 8557-8560
http://dx.doi.org/10.14233/ajchem.2013.14837
New Ursane Glycoside from the Roots of Asparagus racemosus
1*
2
3
4
5
NASIR A. SIDDIQUI , MOHD ALI , ATEEQUE AHMAD , TAJDAR H. KHAN and AFTAB AHMAD
1Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia
2Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110 062, India
3Process chemistry and Product Development Department, Central Institute of Medicinal and Aromatic Plants, Lucknow-226 015, India
4Department of Pharmacology, College of Pharmacy, Salman Bin Abdul Aziz University, Al-Kharj, Riyadh, Kingdom of Saudi Arabia
5Health Information Technology Department, Jeddah Community College, KingAbdulaziz University, P.O. Box-80283, Jeddah-21589, Kingdom
of Saudi Arabia
*Corresponding author: Fax: +96 614 677245; Tel: +96 654 4016921; E-mail: nsiddiqui@ksu.edu.sa
(Received: 19 November 2012;
Accepted: 26 August 2013)
AJC-13995
One new compound urs-1,12,19- trien-3β-ol-28-oic acid-3β-D-glucopyranosyl (4'-1'')-β-D-glucopyranoside (1) along with five known
compounds (2-6) have been isolated from the roots of Asparagus racemosus. Their structures have been elucidated with the help of 500/
125 MHz NMR using 1D and 2D spectral methods viz: 1H and 13C NMR, ESIMS and aided by IR spectroscopy.
Key Words: Asparagus racemosus, Liliaceae, Ursane glycoside.
immunomodulatory effects. The drug is regularly used by local
Indian traditional medicine system practitioners and many other
benefits of these herbs are also recognized internationally.
The ethanol extract of the A. racemosus was separated by
a combination of column chromatography over silica gel and
obtained one new urs-1,12,19-trien-3β-ol-28 oic acid- 3β-D-
glucopyranosyl (4'-1'')-β-D-glucopyranoside (1) and five known
compounds n-octadecanyl hexadecanoate (2), n-hexacosanyl-
n-octadecanoate (3), β-sitosterol (4), 1-lauryl 3-arachidyl glyc-
erol-3-phosphate (5), stigmasterol (6), were recrystallized to
get the pure compound have been identified with 500/125 MHz
NMR using 1D and 2D spectral methods viz., 1H and 13C NMR,
ESI MS and FAB MS aided by IR spectroscopy.
INTRODUCTION
The genus Asparagus is usually an undershrubs or herbs,
distributed in the temperate and tropical parts of the world.
The genus was recently moved from the family Liliaceae (sub-
family: Asparagae) to a newly created family Asparagaceae1.
About 60 species are known to occur worldwide, while only
about 22 species are recorded in India. Asparagus racemosus
wild, a climbing under-shrub with numerous finger-like,
clustered tuberous roots is an important member of the genus
known for its adaptogenic, galactogogue and aphrodisiac
activities2-7. It is commonly sold as tonic and is an active
ingredient in several Ayurvedic and Unani medicinal prepara-
tions. Some constituents were reported from A. racemosus8-15
.
EXPERIMENTAL
It is also used for dry coughs and gastric ulcers. A. racemosus
was found to be effective for phagocytic activity and killing
capacity of macrophages16. It is also used successfully for
nervous disorders, inflammation, liver diseases and certain
infectious diseases. The juice of fresh roots of A. racemosus
has curative effect in patients with duodenal ulcers. Oral
administration of decoction of powered root enhances the
immuno-modulatory effect17. Also, recent research indicates
Shatavari enhances immune function, increases corticosteroid
production and promotes cell regeneration18. The selection of
this herbs was made as it is available in abundance in the local
vicinity of Western U.P. (Bareilly district), India and is well
known for different curative properties in addition to their
All the chemicals and reagents were obtained from SD
Fine Chemicals and were of analytical grade. Sodium sulphate
was used as drying agent for various solvents used to run the
column. All the weighings were done on a single pan metler
balance. Melting points were determined on microcontroller
based, digital, automatic-photosensing MEPA melting point
apparatus (LAB INDIA). Ultraviolet spectra were recorded on
Lambda bio 20 spectrometer in methanol. Infrared spectra were
recorded on Biored FT IR spectrophotometer using KBr pellets
and CCl4, νmax values are given in cm-1. 1H NMR spectra were
screened on advance dry 400, Brucker spectrospin 400 MHz,
instrument using CDCl3 and DMSO as solvent and TMS as an

8558 Siddiqui et al.
Asian J. Chem.
30
20
internal standard. Chemical shift values are given in δ (ppm)
scale and coupling constant (J) in Hz. Notations used through-
out as a s = singlet, d = doublet, dd = double doublet, t =
triplet, m = multiplet and br s = unresolved broad singlet. The
electrospray mass spectra were recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer. The samples
(dissolved in suitable solvents) were introduced into the ESI
source through a syringe pump at the rate of 5 µL/min.
The dried drug (Shatavari roots, 5 kg) was purchased from
Natural Drugs and Botanicals, Sahibabad, Ghaziabad (U.P.)
and was identified by Dr. M.P. Sharma, Reader and Taxonomist,
Department of Botany, Faculty of Science, Jamia Hamdard,
New Delhi.
29
19
21
22
12
26
18
17
11
9
28
COOH
25
13
14
1
4
16
2
8
HO
HO
10
15
27
3
6'
5'
7
5
HO
HO
O
4'
O
6
6''
5''
23
24
2'
OH
1'
O
4''
O
3'
1''
2''
OH
3''
OH
1
Extraction of drugs: The air-dried drug (2 kg) was
coarsely powdered and extracted in a Soxhlet apparatus with
ethanol (95 %) for 48 h. The combined extracts were then
concentrated on a water bath and dried under reduced pressure
to get a dark brown mass (250 g).
CH3(CH2)14COOCH2(CH2)16CH3
2
CH3(CH2)16CO-OCH2(CH2)24-CH3
The concentrated extract of the drug was taken in a China
dish and heated continuously on a water bath, gradually adding
methanol in small portions with constant stirring, till desired
consistency obtained. Silica gel (for column) was then added
(weighed quantity) slowly with continuous mixing with a steel
spatula until desired dry mixture is obtained. The larger lumps
were broken-up and finally passed through a sieve (No. 8) to
get a slurry of uniform particle size.
3
Isolation of the compounds from ethanol extract: The
viscous dark green mass was adsorbed on silica gel (60-120
mesh) for column after being dissolved in little quantity of
methanol for preparation of slurry. The slurry (150 g) was air
dried, packed and chromatographed over silica gel column
packed in petroleum ether. The column was eluted successively
with petroleum ether, mixture of petroleum ether and benzene
(90:10, 80:20, 70:30, 60:40, 50:50, 40;60, 50:50, 60:40, 70:30,
80:20.90;10), pure benzene and then mixture of benzene and
chloroform (90:10, 80:20, 70:30, 60:40, 50:50, 40;60, 50:50,
60:40, 70:30, 80:20.90;10), pure chloroform and finally the
mixture of chloroform and methanol (99:1, 98:2, 96:4, 95:5,
90:10).
HO
4
CH2-O-CO-(CH2)10-CH3
CH-O-CO-(CH2)18CH3
O
CH2-O-P-OH
OH
5
Various fractions were collected separately and matched
by TLC to check homogeneity. Similar fractions (having same
Rf values) were combined and crystallized. The isolated
compounds urs-1,12,19-trien-3B-ol-28 oic acid-3B-D-gluco-
pyranosyl (4'-1'')-B-D-glucopyranoside (1), n-octadecanyl
hexadecanoate (2), n-hexacosanyl-n-octadecanoate (3), β-
sitosterol (4), 1-lauryl 3-arachidyl glycerol-3-phosphate (5),
stigmasterol (6), were recrystallized to get the pure compound
(Fig. 1).
HO
Fig. 1. Compounds (1-6) isolated from ethanolic extract of A. racemosus
roots
6
(0.0077 % yield); Rf : 0.67 (benzene-chloroform-methanol :
3:4:1); m.p.: 315-316 ºC (decomposed); UV νmax (MeOH):
243 nm (log ε 5.6); IR (KBr, νmax, cm-1): 3447, 3380, 3260,
Homogeneity of the fractions: The fractions collected
were subjected to thin layer chromatography to check homo-
geneity of various fractions. Chromatographically identical
fractions (having same Rf values) were combined together and
concentrated. They were then crystallized with suitable solvent
system.
1
2922, 2845, 1698, 1636, 1457, 1396, 1260, 1019, 795; H
NMR (DMSO-d6) : δ 6.61 (1H, d, J = 6.8 Hz, H-1), 6.57 (1H,
dd, J = 6.8, 4.8 Hz, H-2), 5.65 (1H, dd, J = 8.0 Hz, H-12),
5.17 (1H, d, J = 3.6 Hz, H-1'), 4.78 (1H, brs, H-1''), 4.52 (1H,
m, H-5'), 4.50 (1H, m, H-5''), 4.36 (1H, m, H-2'), 4.33 (1H, m,
H-2''), 4.11 (1H, d, J = 4.8 Hz, H-3a), 4.09 (1H, m, H-3'), 4.06
(1H, m, H-3''), 3.63 (1H, m, H-4'), 3.57 (1H, m, H-4''), 3.12
(2H, brs, H2-6'), 3.04 (2H, brs, H2-6''), 2.92 (1H, d, J = 3.6
Urs-1, 12, 19- trien-3β-ol-28-oic acid-3β-D-glucopyra-
nosyl (4'-1'')-β-D-glucopyranoside (1): Elution of the
column with chloroform-methanol (4:1) mixture afforded
colourless crystals recrystallized from methanol, 137.7 mg

Vol. 25, No. 15 (2013)
New Ursane Glycoside from the Roots of Asparagus racemosus 8559
-C
H
O
12 22 11
m.w.342)
(
m/z 434
-Me
419
COOH
COOH
O
+
O
Glu
Glu
452
O
Glu
Glu
C
H
16 22 2
m/z 246
-Me
C
H
O
26 44 11
m/z 532
O
231
C
H
O
14 21
m/z 205
Fig. 2. Mass fragmentation pattern of compound (1)
Hz, H-18 a), 2.61 (2H, brs, H2-11), 2.37 (2H, m, H2-21), 2.02
(1H, m, H-5 a), 1.96 (3H, brs, Me-29), 1.82 (2H, brs, H2-22),
1.68 (3H, brs, Me-30), 1.68 (2H, m, H2-6), 1.63 (2H, m, H2-
7), 1.39 (2H, m, H2-15), 1.17 (6H, brs, Me-25, Me-23), 1.15
(3H, brs, Me-26), 1.07 (3H, brs, Me-27), 0.95 (2H, m, H2-
16), 0.85 (3H, brs, Me-24); 13C NMR (DMSO-d6): δ 138.96
(C-1),, 109.16 (C-1'), 103.81 (C-1''), 164.27 (C-2), 74.85 (C-
2'), 74.85 (C-2''), 73.89 (C-3), 64.18 (C-3'), 66.21 (C-3''), 42.61
(C-4), 65.07 (C-4'), 63.89 (C-4''), 53.29 (C-5), 81.73 (C-5'),
82.08 (C-5''), 19.71 (C-6), 62.11 (C-6'), 62.77 (C-6''), 32.25
(C-7), 41.33 (C-8), 47.81 (C-9), 37.23 (C-10), 23.19 (C-11),
129.75 (C-12), 139.38 (C-13), 42.11 (C-14), 29.16 (C-15),
20.17 (C-16), 48.21 (C-17), 53.83 (C-18),142.13 (C-19),
143.41 (C-20), 36.22 (C-21), 34.18 (C-22), 27.37 (C-23), 19.28
(C-24), 16.51 (C-25), 21.35 (C-26), 22.89 (C-27), 183.12 (C-
28), 25.16 (C-29), 21.36 (C-30); +ve ion ESI MS m/z (rel.
int.): 648 [M]+, (C42H64O13) (7.5), 295 (8.1), 183 (8.3).
Acid hydrolysis of 1: Compound 1 was refluxed with
2 mL of 1 mol/L hydrochloric acid: dioxane (1:1, V:V) in
awater water bath for 4 h. The reaction mixture was evaportaed
to dryness and partitioned with chloroform and water four
times, extyract concentrated. The chloroform extract contained
the aglycone portion, while the water extract contained β-D
glucose.
the pentacyclic carbon framework, three in the vinylic linkages,
one in the carboxylic group and the remaining two in the glyco-
sidic moieties. The retro-Diels-Alder fragmentation of 1
generated ion peaks at m/z 242, 205 [M-246-C12H21O10]+ and
231 [246-Me]+ indicating that the molecule contained one each
vinylic linkage in rings A, C and E. The other prominent ions
arose at m/z 434 [M-C12H22O11, m.u. 342]+ and 419 [434 - Me]+
indicating that a diglycoside moiety was attached to the
molecule. The fragmentation pattern of 1 is shown in Fig. 2.
Its1H NMR spectrum showed two one-proton doublets at
δ 6.61 (J = 6.8 Hz) and δ 5.17 (J = 3.6 Hz) assigned to vinylic
H-1 and anomeric H-1', respectively and two one-proton
double doublets at δ 6.57 (J = 6.8, 4.8 Hz) at δ 6.61 (J = 6.8
Hz) and d 5.17 (J = 3.6 Hz H-2 and H-12, respectively. A one-
proton broad singlet appeared at δ 4.78 accounted to anomeric
H-1''. Four one-proton multiplets appeared at δ 4.52, 4.50,
4.36 and 4.33 attributed to hydroxy methine protons H-5', H-
5'', H-2' and H-2'', respectively.A one-proton doublet appeared
at δ 4.11 (J = 4.8 Hz) was ascribed to H-3α. The remaining
carbinol protons of the sugar moiety appeared between δ 4.09-
3.57. Two two-proton broad signal appeared at δ 3.12 and
3.04 accounted for hydroxy methylene protons H2-6' and H2-
6'' respectively. A one-proton doublet appeared at δ 2.92 (J =
3.6 Hz) ascribed to H-18α. A two-proton broad singlet at δ
2.61 accounted for H2-11 adjacent to the vinylic linkage. A
two-proton multiplet at δ 2.37 was assigned to H2-21. A one
proton multiplet appeared at δ 2.02 assigned to H-5α. Two
three proton broad signals appeared at δ 1.96 and 1.68 attri-
buted to Me-29 and Me-30, attached to the vinylic carbons,
respectively. A six proton broad signal appeared at δ 1.17 was
ascribed to tertiary methyl Me-25, Me-23 protons. Three broad
signals at δ 1.15, 1.07 and 0.85, integrated for three protons
each, were accounted correspondingly to tertiary C-26, C-27
and C-24 methyl protons. The remaining methylene protons
resonated between δ 1.82 - 0.95. The 13C NMR spectrum of 1
showed important signals for C-28 carboxylic carbon at δ
183.12, vinylic carbons at δ 138.96 (C-1), 164.27 (C-2), 129.75
RESULTS AND DISCUSSION
Compound 1, named asparagusursenyl glycoside, was
obtained as colourless crystals from chloroform-methanol (4:1)
eluants. It responded positively to triterpenic glycosides. It
showed UV absorption maxima at 243 nm which is a charac-
teristic of highly conjugated system. Its IR spectrum exhibited
absorption band for hydroxyl group (3447, 3380 and 3260
cm-1), carboxylic group (1698 cm-1) and unsaturation (1638
cm-1) in the molecule. Its ESIMS spectrum displayed mole-
cular ion peak at m/z 776 corresponding to molecular formula
of pentacyclic triterpenic diglycoside, C42H64O13. It indicated
eleven double bond equivalents; five of them were adjusted in

8560 Siddiqui et al.
Asian J. Chem.
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(C-12), 139.38 (C-13), 142.13 (C-19) and 143.41 (C-20),
carbinol carbon at δ 73.89 (C-3), anomeric carbons at δ 109.16
(C-1') and 103.81 (C-1''), hydroxyl methylene carbons at δ
62.11 (C-6'' and 62.77 (C-6''), hydroxymethine carbons
between δ 82.00-63.89 and the remaining methine and meth-
ylene carbons in the range δ 53.89-19.71. The methyl carbon
appeared at δ 27.37 (C-23), 19.28 (C-24), 16.51 (C-25), 21.35
(C-26), 22.89 (C-27), 25.16 (C-29) and 21.36 (C-30). Acid
hydrolysis of 1 yielded β-D-glucose (TLC comparable). On
the basis of spectral data analysis and chemical reactions the
structure of 1 has been formulated as urs-1, 12, 19- trien-3β-
ol-28-oic acid-3β-D-glucopyranosyl (4-1'')-β-D-glucopyrano-
side. This is a new ursane-type triterpenic diglycoside isolated
from a natural source for the first time.
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Conclusion
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Cancer Lett., 104, 3 (1996).
One new compound as Urs-1, 12, 19- trien-3β-ol-28-oic
acid-3β-D-glucopyranosyl (4'-1'')-β-D-glucopyranoside along
with five known compounds have been isolated.
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