Immunosuppressive and hepatoprotective potential of Sarcococca saligna and its biomarker components Hamid Ali a , Syed Ghulam Musharraf a,b , Naveed Iqbal b , Achyut Adhikari b , Omer Mohamed Abdalla a , M. Ahmed Mesaik a,c , Nurul Kabir d, ⁎ a Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan b HEJ, Research Institute of Chemistry, International Center for Chemical Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan c Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia d Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia abstract article info Article history: Received 2 December 2014 Received in revised form 11 May 2015 Accepted 5 June 2015 Available online 17 June 2015 Keywords: Histopathology T-cell proliferation Cytotoxicity IL-2 S. saligna Sarcococca saligna methanolic extract, fractions and isolated pure compounds saracocine (1), saracodine (2), pachyximine-A (3) and terminaline (4) were found to possess potent immunosuppressive activities. The frac- tions and compounds were tested in-vitro for their effects on human T-cell proliferation, and cytokine (IL-2) pro- duction. All the fractions, sub-fractions and purified compounds showed significant suppressive effect on IL-2 production in a dose-dependent manner. They also exhibited a suppressive effect on the phytohemagglutinin- stimulated T-cell proliferation. None of the extracts and purified compounds showed any cytotoxicity effects on the 3T3 mice fibroblast cell line. The crude extract, DCM fraction (pH 9), DCM fractions (pH 7) and one of the steroidal alkaloids (terminaline) were checked in-vivo for their hepato-protective potential against CCl 4 - induced liver injury. In in-vivo experiments, the basic and neutral DCM fractions and terminaline (4) significantly reduced inflammation in the liver. DCM fraction (pH 9), DCM fractions (pH 7) and compound 4 reduced the serum enzyme levels (ALT, AST, and ALP) down to control levels despite CCl 4 treatment. They also reduced the CCl 4 -induced damaged area to almost zero as assessed by histopathology. The pale necrotic areas and mixed inflammatory infiltrate which are seen after CCl 4 treatment were absent in the cases of basic, neutral fractions and terminaline treatment. These hepato-protective effects were better than the positive control silymarin. Our results suggest the therapeutic effect of S. saligna extract, fractions and bioactive steroidal alkaloids against CCl 4 -induced liver injury in vivo and their immunosuppressive function in vitro. © 2015 Elsevier B.V. All rights reserved. 1. Introduction The liver plays a vital role in the innate immune response, providing the first line of defense against microbes and toxins [18]. Inflammatory cytokines (such as TNF-α) are produced from macrophages that lead to various conditions such as inflammatory, allergic, or autoimmune diseases of different organs [35]. Acute inflammation of the liver or acute hepatitis is characterized by infiltration of inflammatory cells mostly consisting of macrophages, neutrophils and T-cells ([15]: [2]). Interleukin-2 is produced mainly by CD4 + T-cells in response to dif- ferent stimuli via T-cell activation of TCR and the major histocompatibil- ity complexes (MHC) I and II molecules of the APCs [22]. The circulating level of IL-2 in normal healthy subjects is almost undetectable; however, when a person is exposed to infection, the level of circulating IL-2 rises dramatically. Kupffer cells of the liver are critically involved in the rapid clearance of microorganisms from the systemic circulation [14,25]. Although Kupffer cells themselves are highly phagocytic and are able to remove microorganisms, they also facilitate the generation of the inflammatory response leading to the recruitment of inflammatory cells such as neutrophils, monocytes, T and B lymphocytes, as well as natural killer (NK) cells to the site of injury in the liver caused by viruses, bacteria or drug/chemicals. Upon infection, Kupffer cells generate neutrophilic inflammatory mediators including tumor necrosis factor-α (TNF-α), in- terleukins (IL-1, and IL-6), chemokines and reactive oxygen species (ROS) [34]. If the liver injury is persistent then these inflammatory mediators will activate neutrophils in the hepatic microvasculature leading to a variety of oxidative stress events culminating in hepatocel- lular death. For the treatment of such inflammatory diseases anti-inflammatory agents that would be harmless to the immune system are required. The development of effective anti-inflammatory and immunosuppressive agents over the last decades has made organ transplantation and the con- trol of autoimmune diseases possible. Most of the immunosuppressive International Immunopharmacology 28 (2015) 235–243 ⁎ Corresponding author. Tel.: +60 3 7967 4213; fax: +60 3 7967 4178. E-mail address: nurul.kabir@um.edu.my (N. Kabir). http://dx.doi.org/10.1016/j.intimp.2015.06.009 1567-5769/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp