Protection against experimental visceral leishmaniasis by immunostimulation with herbal drugs derived from Withania somnifera and Asparagus racemosus Sukhbir Kaur, Kalpana Chauhan and Heena Sachdeva Correspondence Sukhbir Kaur puzoology@yahoo.com Received 7 January 2014 Accepted 29 July 2014 Department of Zoology, Panjab University, Chandigarh-160014, India Visceral leishmaniasis (VL) is a vector-borne parasitic disease targeting tissue macrophages. It is among the most neglected infectious diseases. As available therapeutics for treatment of this disease have many side effects, there is a need for safer alternatives. One of the immunopathological consequences of active visceral leishmaniasis is suppression of protective T-helper (Th)-1 cells and induction of disease-promoting Th-2 cells, and thus the treatment of VL relies on immunomodulation. In the current study, herbal drugs derived as whole-plant extracts of Asparagus racemosus and Withania somnifera were used to treat Leishmania donovani-infected BALB/c mice. Keeping the scenario of immunosuppression during VL in mind, the potential of these drugs in the restoration of murine Th-1-type protective immune responses was evaluated. To investigate the propensity of these drugs to treat VL, liver parasite load, delayed-type hypersensitivity responses and parasite-specific immunoglobulin levels were studied. Various biochemical and haematological tests were also carried out. A positive-control group used the standard drug treatment of sodium stibogluconate. Treatment of infected mice with A. racemosus and W. somnifera in combination at the higher dose of 200 mg (kg body weight) ”1 not only resulted in a successful reduction in parasite load but also generated protective Th1-type immune responses with normalization of biochemical and haematological parameters, suggesting their potential as potent anti-leishmanial agents. INTRODUCTION Visceral leishmaniasis (VL) or kala-azar is the most severe form of leishmaniasis and is responsible for most Leishmania-associated deaths. VL represents a serious public health problem that affects many countries (Paletta-Silva & Meyer-Fernandes, 2012). There are an estimated 500 000 new cases per year of VL globally (Moore & Lockwood, 2010). The annual estimate for the incidence and prevalence of VL cases worldwide is 0.5 million and 2.5 million, respectively. Of these, 90 % of the confirmed cases occur in India, Nepal, Bangladesh and Sudan. In India, the disease is a serious problem in Bihar, West Bengal, Jharkhand and eastern Uttar Pradesh (Bora, 1999). In the absence of vaccines, pentavalent antimonials like sodium stibogluconate (SSG) and meglumine antimoniate have remained the first-line therapy for VL for over half a century, and are still in use in many parts of the world. However, the prolonged treatment requiring parenteral administration, toxicity and the emergence of significant resistance are all factors limiting the usefulness of this drug (Croft & Coombs, 2003). Although a few other options are available such as amphotericin B, miltefosine, paromomycin and lipid-conjugated formulations of amphotericin B, they also suffer from one or more limitations (Maltezou, 2010). Therefore, there is a need to develop newer drug therapies that are more efficacious and safer. There are many immunosup- pressive or immune-evasion mechanisms contributing to the pathogenesis of VL. Leishmania infection is classically associated with a depression of T-helper type 1 (Th1) cells and preferential expansion of Th2 cells (Awasthi et al., 2004). Therefore, recovery from leishmaniasis is generally affected by upregulation of cellular immune responses capable of activating host macrophages and involves activation of Th1 cells. Plant extracts or plant-derived compounds are likely to provide a valuable source of new medicinal agents, and the urgent need for alternative treatments has led to a programme to screen natural products for potential use in the therapy of leishmaniasis (de Carvalho & Ferreira, 2001). Medicinal plants are a good source of bioactive agents that are useful against many diseases, including leishmaniasis, and their active components have been shown to be an important Abbreviations: ALP, alkaline phosphatase; BUN, blood urea nitrogen; DTH, delayed-type hypersensitivity; Hb, haemoglobin; KAU, King– Armstrong units; LDU, Leishman–Donovan unit; p.i. post-infection; p.t., post-treatment; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate transaminase; SRBC, sheep red blood cells; SSG, sodium stibogluconate; TLC, total leukocyte count; Th, T- helper; VL, visceral leishmaniasis; WBC, white blood cell. Journal of Medical Microbiology (2014), 63, 1328–1338 DOI 10.1099/jmm.0.072694-0 1328 072694 G 2014 The Authors Printed in Great Britain