IMMUNOMODULATORY ACTIVITY OF AN ACETONE EXTRACT OF TERMINALIA BELLERICA ROXB FRUIT ON THE MOUSE IMMUNE RESPONSE IN VITRO Original Article AURASORN SARAPHANCHOTIWITTHAYA 1 , 2 * , KORNKANOK INGKANINAN 3 1 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand, 2 Pharmaceutical Biotechnology Research Unit, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand, 3 Received: 22 Aug 2014 Revised and Accepted: 25 Sep 2014 Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand. Email: aurasorns@nu.ac.th ABSTRACT Objectives: To investigate the immunomodulatory activity of an acetone extract of T. bellerica fruit. Methods: Mitogen induced-lymphocyte proliferation using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) technique, Th1- and Th2-related cytokine production by lymphocytes using ELISA and peritoneal macrophage function in ICR mice were assayed. Results: The results show that the extract had a mild inhibitory effect on the generation of oxidase enzyme (Phagocytic Index 0.8, 100 µg/ml) but did not influence acid phosphatase enzyme function during phagocytosis. The extract stimulated the proliferation of both T and B lymphocytes. The maximal activation (Stimulation Index 3.2, 100 µg/ml) was presented with concanavalin A induction, indicating a major effect on T lymphocyte proliferation. The extract reduced the production of IFN-γ (89%, 100 µg/ml) and IL-2 (98%, 100 µg/ml) but increased IL-10 secretion (231%, 100 µg/ml) compared to concanavalin A. Gallic acid, a pharmacological component contained in this plant, presented a similar effect as that of T. bellerica extract and may contribute to the immunomodulatory activity of T. bellerica fruits in cooperation with other phytocompounds. The decrease in the IFN-γ/IL-10 ratio indicated a shift in the Th1/Th2 balance towards a Th2-type response, which might lead to a treatment for Th1- mediated inflammatory immune diseases. Conclusion: Our investigations show that the acetone extract of T. bellerica fruit possesses immunomodulatory activity, which could be used to explain its folklore applications and provide a pharmacological basis for its usefulness in immune-related disorders. Keywords: Lymphocytes, Proliferation, Macrophages, Phagocytosis, Cytokines, Anti-inflammatory. INTRODUCTION Traditional, complementary, alternative, or non-conventional medicines are used by 70–95% of the global population, particularly in developing countries [1]. Traditional medicines primarily depend on the usage of plants, compared to other natural resources[2]. Herbs have been used for food and medicinal purposes for centuries. Nowadays, alternative medicine for the treatment of various diseases, including immunological disorders, is becoming more popular. Research interest has been focused on various herbs that possess immunomodulatory properties that may be useful in reducing the risk of various diseases and cancers [3]. Immune activation is an effective as well as protective approach against emerging infectious diseases [4]. Although the working mechanisms of some of the herbs are unclear and remain to be elucidated, they are worth studying further as new potential therapeutic agents for immunomodulation [3]. Terminalia bellerica Roxb (Combretaceae) is a perennial herb mainly distributed in the tropical regions and commonly found in South- East Asia, including Thailand [5]. The fruit of T. bellerica has been used in various ailments in the indigenous system of medicine to treat cough, asthma, colic, diarrhoea, dyspepsia, anaemia, cancer, fever and inflammation and to promote rejuvenation[5,6]. It is one of the ingredients in “triphala”, an ayurvedic formulation rich in antioxidants which is believed to promote health, immunity and longevity [7] and is used to treat many diseases such as anaemia, jaundice, constipation, asthma, fever and chronic ulcers [8]. Chemically, the fruit of T. bellerica had been found to contain gallic acid as an active component, as well as other phytochemical compounds such as ellagic acid, ethyl gallate, chebulagic acid and β- sitosterol [9,10]. The gums of the plant have been used for the formulation of a microencapsulated drug delivery system, and a polysaccharide isolated from it is used as a pharmaceutical excipient[11]. T. bellerica has been scientifically shown to possess antibacterial [12], antifungal[13], antioxidant[14] and hypotensive [15] effects. The combination of three lignans and a flavan from T. bellerica extract showed significant anti-HIV, anti-malarial and antifungal activity in vitro [16]. Moreover, T. bellerica seed extract has therapeutic potential for the treatment of gastrointestinal diseases, especially inflammatory bowel diseases [17]. Our preliminary study of T. bellerica fruit extracted using various solvents on the mouse immune response showed interesting results, especially with the methanolic and acetone extract. The effect of a methanolic extract on macrophage phagocytosis and mitogen-induced lymphocyte proliferation was previously reported [18]. In the current study, we demonstrate the immunomodulatory activity of an acetone extract of T. bellerica fruit in vitro; gallic acid may contribute to this activity. Mitogen-induced lymphocyte proliferation, Th1- and Th2- related cytokine production by lymphocytes and peritoneal macrophage function in ICR mice were investigated in this study. MATERIALS AND METHODS Plant material Dried fruits of T. bellerica were authenticated by Associate Professor Wongsatit Chuakul, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. A specimen was prepared and deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand. Chemicals 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitroblue tetrazolium (NBT) dye, p-nitrophenyl phosphate (p-NPP), phytohemagglutinin (PHA), concanavalin A (con A), lipopolysaccharide (LPS), pokeweed mitogen (PWM), dimethyl sulfoxide (DMSO), zymosan A, antibiotic-antimycotic solution (100 U penicillin, 100 µg streptomycin and 0.25 µg/ml amphotericin B), phosphate buffer saline (PBS) and gallic acid (3,4,5-trihydroxy International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 11, 2014 Innovare Academic Sciences