Vaccine 27 (2009) 6080–6087 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of Th1 immunostimulatory chemical entity Sheema Khan a,1 , Fayaz Malik a,∗,1 , Krishan A. Suri b , Jaswant Singh a,∗ a Division of Pharmacology, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu-Tawi 180001, India b Natural Products Chemistry, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu-Tawi 180001, India article info Article history: Received 11 May 2009 Received in revised form 30 June 2009 Accepted 6 July 2009 Available online 21 July 2009 Keywords: Withania somnifera Freund’s adjuvant Th1/Th2 cytokines Immunoglobulin isotypes Co-stimulatory molecules Integrins abstract Withania somnifera, commonly called Ashwagandha in the Indian traditional system of medicine has been reported for several pharmacological activities. This study demonstrates, for the first time, the potential role of the chemically standardized leaf extract of W. somnifera (WSL) and it’s identified component in activating immune system. WSL enhanced Th1 cytokine IFN- expression in Con A primed splenocytes in vitro. When given orally for 2 weeks to BALB/c mice immunized with emulsion of OVA in Freund’s adjuvant (OVA-FCA), it caused dose-dependent proliferation of T cells and improved their ability to secrete IL-2 and IFN-, but moderately down-regulated Th2 cytokine IL-4. Flow cytometric analysis of lymphocyte surface markers of T cells CD3 + , CD4 + and CD8 + , and B cells CD19 + indicated prominent enhancement in proliferation and differentiation of lymphocytes. Further, the effect of WSL in immunized mice elicited up- regulation of -integrins LFA (CD11a) and Mac-1 (CD11b) in splenocytes. Co-stimulatory molecules CD80 and CD86 that are important secondary signals for the activation of immune system elicited remarkable enhanced expression when observed in spleen-derived macrophages isolated from WSL treated mice. Chemical standardization of WSL suggested that the withanolide 2,3 dihydro-3-sulphonile withanone is a major constituent of WSL responsible for skewing to Th1 immune polarization by stimulating the expression of IFN- and B cell switch over to secrete IgG2a while simultaneously enhancing the expression of co-stimulatory molecules and integrins. These studies demonstrate the possible usefulness of WSL and its major constituent WSL-2 as Th1 immune adjuvants for chronic infectious ailments where patients suffer from weakened Th1 immunity. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Withania somnifera, also known as Ashwagandha in the Indian traditional system of medicine, Ayurveda, has been in use since time immemorial for the treatment of various diseases. Several pharmacological activities of the plant have been attributed in general to roots [1,2] while the leaves contain significantly higher level of new generation anti-cancer withanolide withaferin A [3,4]. Our current understanding of W. somnifera chemical signature amply demonstrates that the medicinal properties of the plant are associated with certain steroidal alkaloids and steroidal lac- tones in a class of constituents called withanolides [1,4–6]. For instance, withanolide A isolated from roots of W. Somnifera appears ∗ Corresponding authors. Tel.: +91 191 2569000x09; fax: +91 191 2569333/2569019. E-mail addresses: malik fayaz@yahoo.com (F. Malik), jsishar1@yahoo.com (J. Singh). 1 Authors have contributed equally in the study. a good candidate against neurodegenerative diseases and potenti- ating humoral and cell mediated Th1 immunity [1,7]. Withaferin A mostly found in the leaves of W. Somnifera readily induces apoptosis through ROS generation mediating modulation of both intrinsic and extrinsic apoptosis signaling cascades together with abrogation of NF-kB functions [4] besides being strongly anti- angiogenic and anti-metastatic in nature [8]. We know there are at least 50 accessions of W. somnifera [3], each of which may differ inherently in its chemical profile that may reflect differ- ently in biological activities. We recently reported extensive studies on pro-apoptotic activities of withaferin A [4], enhanced Th1 immunity of root extract due to its withanolide [1] and immune up-regulation and anti-cancer activity of a constituted W. somnifera formulation prepared from roots and leaves extract of W. somnifera [6]. For all these studies we used a multi-locational and stably grown elite variety AGB002 with established consistent chemi- cal signature. During chemical standardization of the leaves using eleven withanolide markers, we came across a withanone WSL-2 which was found in a very high concentration followed by with- aferin A. We thus became interested in investigating the immune 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.07.011