Withanolides isolated from Withania somnifera with α-glucosidase inhibition
Transcript of Withanolides isolated from Withania somnifera with α-glucosidase inhibition
ORIGINAL RESEARCH
Withanolides isolated from Withania somnifera with a-glucosidaseinhibition
Murad Ali Khan • Haroon Khan • Tahir Ali
Received: 20 July 2013 / Accepted: 7 October 2013 / Published online: 17 October 2013
� Springer Science+Business Media New York 2013
Abstract Phytochemical investigations on the chloro-
form soluble fraction of the whole plant of Withania
somnifera led to the isolation of 20b hydroxy-1-oxo(22R)-
witha-2,5,24 trienolide 1, (20R, 22R-14a, 20a)-dihydroxy-
1-oxowitha-2,5,16,24 tetraenolide 2, and (20R, 22R)-1-
oxo-5a, 8b-dihydroxywitha-6a, 7b-epoxide-2,24-dienolide
(withasomilide) 3. The structures of these compounds were
confirmed through spectral studies in comparison with data
in the literature. These isolated compounds (1–3) exhibited
potent inhibition against a-glucosidase with IC50 values of
98.60, 38.20, and 40.65 lg/ml respectively.
Keywords Withania somnifera � Withanolides �a-Glucosidase inhibition
Introduction
Withania somnifera Dunal (family: Solanaceae, Vernacu-
lar: Sanskrit: Aswagandha; Telugu: Panneru; Pashto:
Khotilal; Trade name: Aswagandha) is widely distributed
in Pakistan, India, Sri Lanka, Mediterranean regions,
Canaries, S. Africa, Iraq, Iran, Syria, and Turkey (Thakur
et al., 1989). It is an evergreen shrub, 30–150 cm long. In
Ayurvedic and Unani systems, the leaves of the plant are
used for tumor and tubercular glands. The roots of the plant
are used in constipation, senile debility, rheumatism, in
cases of debility, nervous exhaustion, loss of memory, loss
of muscular energy, and spermatorrhoea. The decoction of
the root boiled with milk and vegetable oil is recommended
for curing sterility in women (Kirtika and Basu, 1991). The
methanolic extract and hexane fraction of the aerial parts of
W. somnifera showed significant antimicrobial activity
(Arora et al., 2004).
The anti-inflammatory and hepatoprotective effect
against CCl4-induced hepatotoxicity of the alcoholic
extract of leaves of W. somnifera have also been assessed
(Lakshmi-Chandra et al., 2000). The same was also found
to be effective in an animal model of Alzheimers disease
and perturbed central cholinergic markers of cognition in
rats. These findings supported the effect of W. somnifera as
‘‘medharasayan’’ (promoter of learning and memory) as
used in Ayurvedic medicines (Scarfiotti et al., 1997).
The genus Withania is distributed all over the world that
mainly contains withanolides, alkaloids, and other minor
chemical constituents. In the current research article, we
describe the isolation of three different withanolides from
the chloroform soluble fraction followed by their effect on
the inhibition of a-glucosidase.
Materials and methods
General techniques
Optical rotations were measured on a JASCO DIP-360
polarimeter. UV spectra were recorded on Hitachi U-3200
M. A. Khan
Department of Chemistry, Kohat University of Science and
Technology, Kohat, Pakistan
H. Khan (&)
Gandhara College of Pharmacy, Gandhara University, Peshawar,
Pakistan
e-mail: [email protected]; [email protected]
T. Ali
Department of Biology, College of Natural Sciences (RINS) and
Applied Life Science (BK 21), Gyeongsang National University,
Jinju 660-701, Republic of Korea
123
Med Chem Res (2014) 23:2386–2390
DOI 10.1007/s00044-013-0838-3
MEDICINALCHEMISTRYRESEARCH
spectrophotometer. IR spectra were recorded on FTIR-8900
Shimadzu spectrometer. The 1H, 13C-NMR, HMQC, and
HMBC spectra were recorded on Bruker spectrometers
operated at 400 MHz for 1H and 100 MHz for 13C, respec-
tively. The chemical shift values are reported in ppm (d)
units. MS and HR-MS were respectively obtained on a JMS-
HX-110 with a data system and on JMS-DA 500 mass
spectrometers. Aluminum sheets pre-coated with silica gel
60 F254 (20 9 20 cm2, 0.2 mm thick; E-Merck) were used
for TLC and flash silica gel (230–400 mesh) was used for
column chromatography. Visualization of the TLC plates
was carried out under UV at 254 and 366 nm and also by
spraying ceric sulfate reagent with heating.
Plant material
The whole plant of W. somnifera Dunal was collected from
the tribal area (Khyber Agency) of Khyber Pakhtunkhawa,
Pakistan and was identified by Mr. Shahid Farooq of the
botany section of the Pakistan Council of Scientific and
Industrial Research (PCSIR) laboratory complex Peshawar.
A voucher specimen no 9741 (PES) was deposited in the
herbarium of PCSIR Peshawar.
Extraction and isolation
The shade-dried whole plant material (20 kg) was chopped
and extracted thrice with methanol (40 l) at room tem-
perature for 96 h. The methanolic extract was evaporated
under reduced pressure to afford a dark-greenish residue
which was suspended in water and successively extracted
with n-hexane, CHCl3, EtOAc, and n-BuOH. The CHCl3soluble fraction was subjected to column chromatography,
and eluted with n-hexane-CHCl3 in the increasing order of
polarity to provide five fractions. The fraction obtained
from n-hexane-CHCl3 (8:2) was further purified by column
chromatography on silica gel using n-hexane-CHCl3 (9:1)
as a solvent system to afford compound 1 (10 mg) as 20bhydroxy-1-oxo (22R)-witha-2,5,24 trienolide (Atta-ur-
Rahman et al., 2003). The fraction obtained from n-hex-
ane-CHCl3 (6:4) was re-chromatographed over flash silica
gel using n-hexane-CHCl3 (9:1–4:6) as a solvent system to
give two successive fractions. The first fraction provided
compound 2 (15 mg) characterized as (20R, 22R-14a,
20a)-dihydroxy-1-oxowitha-2, 5, 16, 24 tetraenolide (Vel-
de and Lavie, 1982) and the second fraction afforded
compound 3 (13 mg) as (20R, 22R)-1-oxo-5a, 8b-dihydr-
oxywitha-6a, 7b-epoxide-2,24-dienolide (withasomilide)
(Ali et al., 1997) through column chromatography over
silica gel using n-hexane-CHCl3 (8:2 and 7:3) as an elu-
ents, respectively.
Characterization of compound 1
[a] D25 ? 34 (c = 0.0053, CHCl3). IR (KBr) mmax cm-1:
3447, 1716, 1685. UV kmax (MeOH) nm: 219. HREIMS:
m/z 438.6070 (calcd. for C28H38O4; 438.6073). EIMS:
m/z (rel. int., %) 438 (9), 313 (22), 169 (47), 126 (100). 1H
NMR (CDCl3, 400 MHz) 5.86 (dd, H-2), 6.75 (m, H-3),
3.27 (dd, H-4a), 2.8 (dd, H-4b), 5.56 (d, H-6), 2.00 (m,
H-7a), 1.91(m, H-7b), 1.50 (m, H-8), 1.60 (m, H-9), 1.51
(m, H-11), 1.68 (m, H-12), 3.65 (m, H-14), 1.10 (m,
H-15b), 1.25 (m, H-15a), 2.00 (m, H-16), 1.60 (m, H-17),
0.90 (s, H-18), 1.23 (s, H-19), 1.28 (s, H-21), 4.20 (dd,
H-22), 2.38 (m, H-23a) 2.12 (m, H-23b), 1.89 (s, H-27),
1.95 (s, H-28).13C (CDCl3, 100 MHz) 203.8 (C-1), 128.1
(C-2), 145.5 (C-3), 33.4 (C-4), 135.8 (C-5), 124.8 (C-6),
31.5 (C-7), 40.0 (C-8), 40.2 (C-9), 49.8 (C-10), 21.8 (C-11)
23.6 (C-12), 49.7 (C-13), 54.7 (C-14), 29.7 (C-15), 42.9 (C-
16), 56.6 (C-17), 13.8 (C-18), 19.0 (C-19), 75.5 (C-20),
20.4 (C-21), 81.1 (C-22), 30.7 (C-23), 149.7 (C-24), 122.1
(C-25), 166.3 (C-26), 12.8 (C-27), 21.9 (C-28).
Characterization of compound 2
[a] D25 ? 43 (c = 0.35, CHCl3). IR mmax Cm-1 3540, 3450,
1715, 1660. UV kmax (MeOH) nm: 224. HREIMS:
m/z 452.5904 (calcd. for C28H38O5; 452.5908). EIMS:
m/z (rel. int., %) 452 [M?] (7), 434 (9), 416 (100), 309
(57), 251 (16), 125 (49). 1H NMR (CDCl3, 400 MHz) 1.15
(3H, Me-18), 1.22 (3H, Me-19), 1.30 (3H, Me-21), 1.86
(3H, Me-27), 1.97 (3H, Me-28), 2.85 (1H, dd, H-4eq), 3.25
(1H, br d, H-4ax), 4.50 (1H, dd, H-22), 5.61 (1H, H-6), 5.79
(1H, H-16), 5.85 (1H, dd, H-2), 6.75 (1H, dq, H-3), 1.95 (s,
H28). 13C (CDCl3, 100 MHz) 203.9 (C-1), 130.8 (C-2),
142.5 (C-3), 70.4 (C-4), 64.8 (C-5), 60.8 (C-6), 26.5 (C-7),
40.0 (C-8), 36.2 (C-9), 48.8 (C-10), 21.8 (C-11), 41.6 (C-
12), 45.7 (C-13), 84.7 (C-14), 78.7 (C-15), 36.9 (C-16),
51.6 (C-17), 14.8 (C-18), 15.2 (C-19), 38.5 (C-20), 17.4
(C-21), 78.1 (C-22), 31.7 (C-23), 152.7 (C-24), 122.1
(C-25), 167.3 (C-26), 12.8 (C-27), 20.9 (C-28).
Characterization of compound 3
[a] D25 ? 42 (c = 0.30, CHCl3). IR mmax Cm-1 3455, 2930,
1700, 1670. UV kmax (MeOH) nm: 227. HREIMS:
m/z 470.6058 (calcd. for C28H38O5; 470.6061). EIMS:
m/z (rel. int., %) 470 [M?] (4), 422 (3), 346 (6), 202 (6),
194 (10), 172 (15), 125 (100). 1H NMR (CDCl3, 400 MHz)
0.90 (3H, Me-18), 1.10 (3H, Me-19), 1.15 (3H, Me-21),
1.60 (3H, Me-20), 1.95 (6H, Me-27, 28), 3.07 (1H, d,
H-6a), 3.15 (1H, d, H-7a), 3.25 (1H, m, H-4), 4.45 (1H, m,
H-22), 5.73 (1H, d, H-2), 6.65 (1H, m, H-3). 13C (CDCl3,
100 MHz) 203.1 (C-1), 129.0 (C-2), 140.1 (C-3), 36.4
(C-4), 74.0 (C-5), 56.3 (C-6), 76.5 (C-7), 84.9 (C-8), 35.2
Med Chem Res (2014) 23:2386–2390 2387
123
(C-9), 49.8 (C-10), 21.7 (C-11), 36.6 (C-12), 43.5 (C-13),
44.9 (C-14), 22.7 (C-15), 33.0 (C-16), 49.1 (C-17), 10.4 (C-
18), 15.0 (C-19), 37.1 (C-20), 16.4 (C-21), 78.5 (C-22),
32.7 (C-23), 150.7 (C-24), 121.5 (C-25), 167.4 (C-26), 12.3
(C-27), 20.4 (C-28).
a-Glucosidase inhibitory assay
The a-glucosidase (E.C. 3.2.1.20) inhibitory activity of test
compounds (1–3) was estimated using an already established
method (Iqbal et al., 2004). The source of a-glucosidase
(E.C. 3.2.1.20) was saccharomyces sp purchased from Wako
Pure Chemical Industries Ltd. (Wako 076-02841). The
inhibition has been measured spectrophotometrically at pH
6.9 and at 37 �C using 1 mM p-nitrophenyl a-D-glucopy-
ranoside (PNP-G) as a substrate and 0.69 U/ml enzyme, in
50 mM sodium phosphate buffer containing 100 mM NaCl.
1-Deoxynojirimycin (0.425 mM) was used as a positive
control (Ali et al., 2002). The increment in the absorption at
400 nm due to the hydrolysis of PNP-G by a-glucosidase
was monitored continuously with the spectrophotometer
(Molecular Devices, USA).
Results
The chloroform soluble fraction afforded three com-
pounds 1–3 (Fig. 1), as described in the experimental
part. These compounds 1–3, when studied for a-glucosi-
dase inhibitory activity, they showed significant inhibition
in a concentration-dependent manner. Regarding the
results of compound 1, the inhibition was increased with
an increase in concentration with a maximum inhibition
(69.74 %) at 100 lg/ml (Fig. 2). The IC50 of compound 1
was calculated at 85.80 lg/ml. When compound 2 was
tested against a-glucosidase, it had marked inhibitory
action which was concentration dependent (Fig. 3). The
maximum inhibition (96.07 %) was observed at 100 lg/
ml, while the estimated IC50 for compound 2 was
38.20 lg/ml.
Similarly, compound 3 showed profound a-glucosidase
inhibitory activity at various concentrations (Fig. 4). The
maximum inhibition (94.33 %) was observed at the highest
tested concentration i.e. 100 lg/ml. The estimated IC50 for
compound 3 was 40.65 lg/ml. The standard drug, deoxy-
nojirimycin showed IC50 of 422.33 lM.
O
OHH3C
CH3 H
H3C
H H
HH
O
O
CH3
CH3
OH3C
CH3 H
H3C
OH
HO
O
CH3
CH3
OH
12
OH3C
CH3 OH
H3C
H
HO
O
CH3
CH3
OH
HOH
H
H
3
Fig. 1 Structures of isolated
compounds 1–3
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Discussion
The present study revealed the strong a-glucosidase
inhibitory activity of three different withanolides isolated
from the chloroform soluble fraction of whole plant of W.
somnifera in vitro.
Diabetes mellitus is a chronic metabolic syndrome which
has already affected the lifestyle of millions of people
throughout the world (Li et al., 2004). The clinicians are
facing the greatest challenge for the effective management
of diabetes in keeping the blood glucose close to normal
(Jong-Sang et al., 2000). As a result of ineffective glucose
control, diabetes late complications have been observed in
patients suffering from type 2 diabetes (non-insulin depen-
dent) such as retinopathy, neuropathy, neuropathy, and
cardiovascular complications (Sabu and Kuttan, 2002;
Ashraf et al., 2011).
a-glucosidase (AGH, EC 3.2.1.20) binds to the membrane
of the epithelium of small intestine that catalyzes the final step
in the cleavage of carbohydrates and thus interfered in the
absorption process of glucose. Hence, a-glucosidase inhibi-
tors could hinder the absorption of dietary carbohydrates and
monitor post-prindal hyperglycemia. a-glucosidase inhibitors
are therefore, symbolized as a rational therapeutic strategy for
management of hyperglycemia. Typical a-glucosidase
inhibitors include acarbose, miglitol, and voglibose (Benalla
et al., 2010). When studied in chronic BHV animal, experi-
mental findings have been shown antiviral activity of gluco-
sidase inhibitors by changing glycosylation. As the a-
glucosidase inhibitors act in the induction of misfold or
otherwise defective glycoproteins therefore such inhibitors
may be very effective in the treatment of infections caused by
them (Iqbal et al., 2004). Additionally, DNJ (deoxynojiri-
mycin), NB-DNJ, and a-Glucosidase inhibitors have shown
the potential inhibition of human immunodeficiency virus
(HIV) replication and HIV-mediated syncytium formation
(Ali et al., 2002).
In short, the isolated withanolides (1–3) from the whole
plant of W. sominfera demonstrated significant attenuation
of a-glucosidase. It is therefore assumed that these com-
pounds 1–3 may be useful therapeutic strategies for the
effective management of diabetes as well as patients suf-
fering from different viral infections. Thus detailed studies
of these compounds are most warrant to evaluate their
clinical potential.
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Fig. 3 Dose-dependent inhibition of compound 2 against a-glucosi-
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Fig. 4 Dose-dependent inhibition of compound 3 against a-glucosi-
dase. Values are mean ± SEM of 3–4 independent assays
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