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).


[a] D25 ? 43 (c = 0.35, CHCl3). IR mmax Cm-1 3540, 3450,

1715, 1660. UV kmax (MeOH) nm: 224. HREIMS:


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).


[a] D25 ? 42 (c = 0.30, CHCl3). IR mmax Cm-1 3455, 2930,

1700, 1670. UV kmax (MeOH) nm: 227. HREIMS:


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

2388 Med Chem Res (2014) 23:2386–2390

123

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.

References

Ali M, Shuaib M, Ansari SH (1997) Withanolides from the stem bark

of Withania somnifera. Phytochemistry 44:1163–1168

Fig. 2 Dose-dependent inhibition of compound 1 against a-glucosi-

dase. Values are mean ± SEM of 3–4 independent assays





Med Chem Res (2014) 23:2386–2390 2389

123

Ali MS, Jahangir M, Hussan SS, Choudhary MI (2002) Inhibition of

a-glucosidase by oleanolic acid and its synthetic derivatives.

Phytochemistry 60:295–299

Arora S, Dhillon S, Rani G, Nagpal A (2004) The in vitro

antibacterial/synergistic activities of Withania somnifera

extracts. Fitoterapia 75:385–398

Ashraf R, khan RA, Ashraf I (2011) Garlic (Allium sativum) supple-

mentation with standard antidiabetic agent provides better diabetic

control in type 2 diabetes patients. Pak J Pharm Sci 24:565–570

Atta-ur-Rahman A, Dur-e-Shahwar NA, Choudhary MI (2003) Wit-

hanolides from Withania coagulans. Phytochemistry 63:387–390

Benalla W, Bellahcen S, Bnouham M (2010) Antidiabetic medicinal

plants as a source of alpha glucosidase inhibitors. Curr Diabetes

Rev 6:247–254

Iqbal K, Malik A, Mukhtar N, Anis I, Khan SN, Choudhary MI (2004)

a-Glucosidase inhibitory constituents from Duranta repens.

Chem Pharm Bull 52:785–789

Jong-Sang K, Chonk-Sok K, Son KH (2000) Inhibition of alpha

glucosidase and amylase by Luteolin, a flavonoid. Biosci

Biotechnol Biochem 64:2458–2461

Kirtika KR, Basu BD (1991) Indian medicinal plants, vol. 3. Shiva

Publishers, Dehradun, p 1783

Lakshmi-Chandra M, Sing BB, Dagenais S (2000) Scientific basis for

the therapeutic use of Withania somnifera (Ashwagandha): a

review. Altern Med Rev 5:334–346

Li W, Zheng H, Bukuru J, De Kimpe N (2004) Natural medicines

used in the traditional Chinese medical system for therapy of

diabetes mellitus. J Ethnopharmacol 92:1–21

Sabu MC, Kuttan R (2002) Anti-diabetic activity of medicinal plants

and its relationship with their antioxidant property. J Ethnophar-

macol 81:155–160

Scarfiotti C, Fabris F, Cestaro B, Giuliani A (1997) Free radicals,

atherosclerosis, ageing, and related dysmetabolic pathologies:

pathological and clinical aspects. Eur J Can Prev 6:S31–S36

Thakur RS, Puri HS, Hussain A (1989) Major medicinal plants of

India. Central Institute of Medicinal and Aromatic Plants,

Lucknow, p 37

Velde VV, Lavie DA (1982) D16-withanolide in Withania somnifera as a

possible precursor for a-side-chains. Phytochemistry 21:731–733

2390 Med Chem Res (2014) 23:2386–2390

123

Withanolides isolated from Withania somnifera with α-glucosidase inhibition

Documents

Transcript of Withanolides isolated from Withania somnifera with α-glucosidase inhibition