Induction of nitric oxide synthase expression by

Withania somnifera in macrophages

Teresa Iuvone, Giuseppe Esposito, Francesco Capasso, Angelo A. Izzo*

Department of Experimental Pharmacology, University of Naples Federico II, via D. Montesano 49, 80131 Naples, Italy

Received 25 July 2002; accepted 23 October 2002

Abstract

Withania somnifera (ashwagandha, Indian ginseng) is an immunostimulant herbal medicine used to improve

overall health and prevent diseases, particularly in the elderly. However, the mechanisms underlying its

immunostimulant effect is poorly understood. To elucidate the mechanism of Withania somnifera, we investigated

the effect of a methanolic extract from the root ofWithania somnifera (WS) on nitric oxide (NO) production in J774

macrophages. We found that WS (1256 Ag/ml) produced a significant and concentration-dependent increase in NOproduction, an effect which was abolished by NGnitro-L-arginine methyl ester (L-NAME, 3300 AM), a non-selective inhibitor of NO synthase (NOS), dexamethasone (10 AM), an inhibitor of protein synthesis and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK, 0.0110 AM), an inhibitor of nuclear factor-nB (NF-nB) activation.Dexamethasone did not have any effect on NO production once NOS had been induced (i.e. 12 h after WS).

Moreover, western blot analysis showed that WS increased, in a concentration-dependent fashion, inducible NOS

protein expression. These results demonstrate that WS may induce the synthesis of inducible NOS expression likely

by acting at transcriptional level. The increased NO production by macrophages could account, at least in part, for the

immunostimulant properties of Withania somnifera.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Macrophages; Nitric oxide synthase; NF-nB; Ashwagandha; Withania somnifera; Immune system; Herbalmedicines; Phytotherapy

Introduction

Withania somnifera Dunal (ashwagandha, Indian ginseng, winter cherry) is a small or middle-sized

undershrub (Fam Solanaceae) which is widely used in Ayurvedic medicine, the traditional medical

0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0024-3205(02)02472-4

* Corresponding author. Tel.: +39-81-7486439; fax: +39-81-7486403.

E-mail address: aaizzo@unina.it (A.A. Izzo).

www.elsevier.com/locate/lifescie

Life Sciences 72 (2003) 16171625

system of India, and also marketed in Western countries [13]. It is generally used as general tonic

to increase energy, improve overall health and longevity, and prevent disease in the elderly [4,5].

Many pharmacological studies have been conducted to investigate the properties of Withania

somnifera in an attempt to authenticate its use as a multi-purpose medical agent. Experimental

studies have shown that Withania somnifera possesses anti-inflammatory [6], antitumour [7],

cardioprotective [8] and antioxidant [9] properties. It also appears to exert a positive influence on

the endocrine [10], urogenital [11] and central nervous systems [1214]. The biologically active

chemical constituents of Withania somnifera are alkaloids, steroidal lactones (including withanolides),

and terpenoids with a tetracyclic skeleton like cortisol; the root is the part of the plant used

medicinally [13].

The use of Withania somnifera as a general tonic to increase energy and prevent disease may be

partially related to its effect on the immune system [1519]. Preparations from Withania somnifera have

been shown to activate peritoneal macrophages and phagocytosis, to increase the activity of the

lysosomal enzymes and to exert a protective effect in cyclophosphamide-induced myelosuppression

in mice [15,19]. Furthermore, treatment with Withania somnifera produced an enhancement in the

circulating antibody titre and the number of plaque forming cells in the mouse spleen [16]; however, the

mechanism of the immunomodulatory effect of Withania somnifera is still unknown.

Nitric oxide is synthesized by sequential oxidation of a terminal guanidino nitrogen of L-arginine, a

reaction catalyzed by the NO synthase (NOS) [20]. Three NOS isoforms have been described: two of

three NOSs isoforms (i.e. neuronal and endothelial NOS) are costitutively expressed in cells, and they

synthesize NO generally in response to increased Ca2 +; a third isoform, namely inducible NOS (iNOS)

is typically synthesized in response to inflammatory or proinflammatory mediators in a variety of

immune cells and its expression may be beneficial in host defense or in modulating the immune response

[21,22]. Indeed, the massive iNOS-derived production of NO by macrophages inhibits the growth of

many pathogens, including bacteria, fungi, viruses, and parasites [22].

In order to verify if NO production may mediate the immunostimulant properties of Withania

somnifera, in the present study we have evaluated the effect of Withania somnifera root methanolic

extract (WS) on NO production in J774 macrophages.

Material and methods

Plant material

Plant material was a kind gift from Laboratory Carlo Sessa (Milan Italy). A methanolic extract

from the roots of Withania somnifera (WS) was prepared as previously described [9]. Briefly, one

Kg of powdered plant material (Eur. Ph. Sieve No. 355) was mixed with 3:1 methanol Eur. Ph.,

stirred for 30 min and allow to stand overnight. After stirring for another 30 min, it was filtered

under vacuum through filter paper and the filtrate was evaporated to dryness in a rotavapor. The

extracted drug was treated as said two more times with 3 liters methanol, filtered and the filtrate

evaporated to dryness. The starting material of Withania somnifera were 3900 g; the extraction yield

was 213 g (5.46%). According to previous work [9], WS extract showed major absorption in the

200250 nm range. WS was dissolved in DMSO. DMSO (0.01%) had no effect on the response

under study.

T. Iuvone et al. / Life Sciences 72 (2003) 161716251618

Fig. 2. Inhibitory effect of the NOS inhibitor L-NAME (3300 AM) on the production of NO induced by Withania somniferaroot methanolic extract (WS, 256 Ag/ml) in J774 macrophages. NO production was determined by measuring the accumulationof nitrite in the culture medium. Each bar shows the mean F SEM. of 56 experiments. ***P < 0.001 vs. vehicle; ##P < 0.01and ###P < 0.001 vs. WS.

Fig. 1. Effect of Withania somnifera root methanolic extract (WS, 1256 Ag/ml) on NO production in J774 macrophages. NOproduction was determined by measuring the accumulation of nitrite in the culture medium. Each bar shows the mean F SEMof 56 experiments. *P < 0.05, **P < 0.01 and P < 0.001 vs. vehicle.

T. Iuvone et al. / Life Sciences 72 (2003) 16171625 1619

Cell culture

The monocyte/macrophage cell-line J774 was grown in Dulbecco Modified Eagles Medium (DMEM)

supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin at37 jC in 5% CO2/95% air. The cells were plated in 24-well culture plates at density of 2.5 105 cells/ml/well and allowed to adhere for 2 h at 37 jC. Thereafter the medium was replaced with fresh medium andcells were activated with WS (1256 Ag/ml). NG-nitro-L-arginine methyl ester (L-NAME, 330300AM), and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK, 0.010.1110 AM) were added to the cells10 min beforeWS. Dexamethasone (10 AM) was added to the cells 2 h before and 12 h after WS. The doseof dexamethasone was selected on the basis of previous published work [23].

Determination of nitrites production

Production of NO was assayed by measuring the amount of nitrite in the culture medium of J774 cells

24 h after WS addition, using a spectrophotometric assay based on Griess reaction [23].

Preparation of cytosolic fractions

Extracts of macrophages stimulated for 24 h with WS (1256 Ag/ml) were prepared as previouslydescribed [23]. Briefly, harvest cells (2 107 cells) were washed twice with ice-cold phosphate

Fig. 3. Inhibitory effect of TLCK (0.0110 AM), an inhibitor of NF-kB activation, on the production of NO induced byWithania somnifera root methanolic extract (WS, 256 Ag/ml) in J774 macrophages. NO production was determined bymeasuring the accumulation of nitrite in the culture medium. Each bar shows the mean F SEM of 56 experiments. ***P