ADVANCES in NATURAL and APPLIED SCIENCES

ADVANCES in NATURAL and APPLIED SCIENCES ISSN: 1995-0772 Published BYAENSI Publication EISSN: 1998-1090 http://www.aensiweb.com/anas 2017 July 11(9): pages 154-158 Open Access Journal Alkaloid immunomodulatory effects of sambiloto (andrographis paniculate l.) On the response of gamma interferon and t helper cell (cd4 + ) 1 Wurlina, 2 Dewa Ketut Meles, 3 Imam Mustofa, 4 Sunarni Zakaria, 5 I Dewa Putu Anom Adnyana 1,2,3 Veterinary Medicine Faculty of Universitas Airlangga 4 Medicine Faculty of Universitas Airlangga 5 Veterinary Medicine Faculty of Universitas Brawijaya Received 1April 2017; Accepted 18 June 2017; Available online 2 July 2017 Address For Correspondence: Wurlina, Veterinary Medicine Faculty of Universitas Airlangga Copyright 2017 by authors and American-Eurasian Network for Scientific Information (AENSI Publication). This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ ABSTRACT This research aims to demonstrate the immunomodulatory effects of alkaloid sambiloto on innate immunity and adaptive immunity in helping the body maintain and regulate the immune system due to low body resistance during transmission of infectious disease or climate change or extreme environments through the examination of Interferon Gamma (IFN) and T helper cells (CD4) which is a specific immune response. A total of 125 rats (Rattus norwegicus) were divided into five groups such as one control group (K), and four treatment groups. The treatment group (P0) and the groups that were intraperitoneallyinfected with Salmonella typhimuriumof 105 CFU (P1, P2 and P3) were administered with alkaloids in varying doses (0 mg/kg bw, 3.78 mg/200 g bw of 7.56 mg/200 g bw, and 11.34 mg/200 g bw) for 10 days. The parameters measured were the levels of IFNγ and the expression of T-helper cells (CD4 + ). The research data was analyzed using one-way ANOVA. The result concluded that the alkaloid sambilotois able to reduce the IFNγ activity and the expression of CD4 + towards normal after being infected with Salmonella typhimurium. KEYWORDS: Immunomodulator, Sambiloto alkaloid, Salmonella typhimurium,leucocyte, IFNγ INTRODUCTION Sambiloto which contains active substance, andrographolides are used to boost the body immunity. As an immunomodulator, sambiloto can be used as immunostimulatoryagent that boost the immune response when the immunity is reduced, and can also be an immunosuppressor i.e., lowering immune response when the immune system increases beyond normal body conditions. In addition, as an immunomodulator, sambiloto is able to normalize the body condition despite the presence of infection. The purpose of this research is to prove the immunomodulatory activity of alkaloid sambiloto against a specific immune response i.e., interferon γ (IFNγ) and T-helper cells (CD4+) after and before being infected with Salmonella typhimurium. ToCite ThisArticle: Wurlina, Dewa Ketut Meles Imam Mustofa,Sunarni Zakaria, I Dewa Putu Anom Adnyana., Alkaloid immunomodulatory effects of sambiloto (andrographis paniculate l.) On the response of gamma interferon and t helper cell (cd4+). Advances in Natural and Applied Sciences. 11(9); Pages: 154-158

155 Wurlina et al., 2017/Advances in Natural and Applied Sciences. 11(9) July 2017, Pages: 154-158 MATERIALS AND METHODS Fractionation of Alkaloid Sambiloto: Sambiloto extract is prepared through various steps that involvesprocesses such asmaceration, filtration, and evaporation. and the plant extraction process is as follows: A number of Sambiloto plants are dried in a closed room at room temperature and are not exposed to direct sunlight. Then it is weighed to determine the dry weight and the depreciation of weight iscalculatedafter drying. The dried plants are then made a fine powder using blender and sieving was done using a sieve with a size 00. The fine powder of the plant was macerated using n- hexane repeatedly to dissolve the fat and green substance (chlorophyll) present in the leaves. Once the filter looks clear, the dregs were collected to macerate with ethanol which is left for two days at room temperature. This was further filtered and the liquid was collected. The maceration procedure with ethanol was carried out repeatedly until the filtrate liquid looks clear. Subsequently, the liquid filtrate from each ethanol plant was vaporized using an evaporator machine at a temperature of 50-55 C, until it gets concentrated as a thick extract, otherwise called as ethanol extract of Sambiloto. Then was then extracted with 10% methanol tartaric acid. Tartaric acid methanol extract basified by the addition of ammonium hydroxide (NH 4OH), then extracted with chloroform to separate the alkaloids. Then the alkaloid content of Sambiloto was determined using Thin Layer Chromatography (TLC) and column chromatography, followed by High-Performance Liquid Chromatography (HPLC). Alkaloid Dose of Sambiloto: Based on the research conducted earlier, Sambiloto is used to improve body conditions when a person is suffering from common cold. Usually being consumed in powder form inserted in capsulewith a dose of 500 mg, it is used three times a day, or 1,500 mg/day (calculated on the basis of average weight of Indonesian people being 50 kg). A drug substance, derived from dried leaf powder to be made into extracts or alkaloid fractions to separate the active ingredients,is alleged as a medicine, in general found a number of + 10% of the dry powder (Meles, 2005). So the dose of alkaloid Sambiloto is 10% x 1500 mg = 150 mg/day. The dose of the drug used in humans (70 kg bw) is converted for rats (200 grams bw) which results in 0.018. So in treatment 1 (P1), Sambiloto alkaloid dose in rats with 200 grams = 70/50 mm x 150 mg x 0.018 = 3.78 mg/day, treatment 2 (P2) alkaloid Sambiloto dose was 2 x 3.78 mg = 7,56 mg/200 g/day and treatment 3 (P3) dose of alkaloid Sambiloto was 3 x 3.78 mg = 11.34 mg/200 g bw/day. Alkaloid Sambiloto as an immunomodulator administered to rats after infected with Salmonella typhimurium: The rats were infected with Salmonella typhimuriumat a concentration of 105 bacteria/0.5 ml intraperitoneally expecting that the immune response will decrease. As per the results from the research conducted by Gamashinta [6], after 4 days of intraperitoneal injection of Salmonella, the rats suffered from fever were used as treatments. In this research, 125 rats were divided into five groups so that each group had 25 animals. The groups consist of negative control consists of healthy rats which were orally administered only with the drug solvent, i.e, 0.5% solution of carboxymethylcellulose (CMC 0.05%) at a dose of 0.5 ml for 10 days. While the four treatmentrat groups were infected intraperitoneally with Salmonella typhimurium bacteria at concentrations of 105 at a dose of 0.5 ml per animal. The treatment groups consisted of oral group that were administered only with a solution of 0.5% CMC 0.5 ml/day/orally and the groups P1, P2 and P3 which were orally administered with alkaloid Sambiloto for 10 days with a dose of 3.78 mg/200 g bw/day, 7.56 mg/200 g bw/day and 11.34 mg/200 g bw/dayrespectively. Each group was divided into five times observations in the 2 nd, 4 th, 6 th, 8 th and tenth day towards the adaptive immune response in the form of levels of Interferon Gamma (IFNγ) using ELISA and the number of T-helper cells (CD4 + ) by immunohistochemical methods. RESULT AND DISCUSSION Alkaloids Sambiloto administration in rats after infected withsalmonella typhimurium toward the interferon gamma on 2 nd,4 th, 6 th, 8 th, and 10 th days: Based on the results of Optical Density on the IFNγ secretion by indirect ELISAtest, the data showed that on Sambiloto treatment with a dose of 3.78 mg/kg already showed a positive Optical Density, which is the secretion of IFNγ over the twice value of COV on the second day at 0.3876, the fourth day at 0.3904, the sixth day at 0.3952, the eighth day at 0.3956, and the tenth day at 0.3964. IFNγ secretion on several doses alkaloid Sambiloto can be seen in Table 1.

156 Wurlina et al., 2017/Advances in Natural and Applied Sciences. 11(9) July 2017, Pages: 154-158 Table 1: Average number of IFNγ in Rats AdministeredAlkaloids Sambiloto after Infected with Salmonella typhimurium Treatment Average IFNγ in Rats Day 2 Day 4 Day 6 Day 8 Day 10 K 0.1838 e ±0.005 0.1892 e ± 0.003 0.1896 e ± 0.004 0.1886 e ±0.003 0.1882 e ±0.005 P0 0.6176 a ± 0.101 0.6236 a ± 0.081 0.6198 a ±0.056 0.6248 a ± 0.066 0.6266 a ± 0.083 P1 0.5562 b ± 0.032 0.5548 b ±0.062 0.5474 b ±0.069 0.5412 b ± 0.058 0.5414 b ± 0.051 P2 0.4528 bc ± 0.097 0.4462 bc ± 0.034 0.4318 bc ±0.081 0.4264 bc ± 0.055 0.4354 bc ± 0.086 P3 0.3926 cd ± 0.006 0.3948 cd ±0.012 0.3976 cd ± 0,10 0.3926 cd ± 0,013 0.3974 cd ± 0,009 Different superscript in the same column refers to significant difference (p<0,05) The administration of alkaloid Sambiloto began at the second day after infecting with Salmonella typhimuriumand it was able to substitute the role of IFNγ in replacing the body's immune response in both enhancing the immune response, that are nonspecific in the form of increased responsiveness of leukocytes, especially the role of neutrophils, monocytes and lymphocytes in phagocyte and it also directly started working against infectious microorganisms that enter the body such as Salmonella typhimurium which is an infectious agent [13].The role of Sambiloto alkaloid in replacing the function of IFNγ gets increased according to the increased dosage of alkaloid Sambiloto administered on the rats after or before being infected with Salmonella typhimurium through the reduced secretion of IFNγ, denoting that the IFNγ role in maintaining the body's immune response and stabilizing the immune response is replaced by Sambiloto alkaloid. Being primarilyproduced by macrophages, IFNγ is also produced by B lymphocytes, T lymphocytes, and NK cells. The role of IFNγ, in addition to stimulating the role and function of macrophages,isincreasing the mechanism of phagocytes against infectious agents and also stimulatingthe mechanism of proliferation and differentiation of T-cells into T-Helper cells-1 (TH1), TH2, cytotoxic T cells, T-cell suppressor and memory T-cells in which each one has a specific function in the body's immune response [7,14]. Similarly, the role of IFNγ in differentiating the B cells, which are actually derived from B lymphocytes, into memory B cells and plasma cells to produce immunoglobulins that play a role in enhancing the humoral immune response. In acute disease conditions, the response and role of immunoglobulin M are more dominant in helping the body's immune response, whereas, in chronic diseases, immunoglobulin G has predominant role in helping the immune response [5]. In this research onsalmonella typhimurium infection, the effects were visible on the second-dayof the administration of alkaloid Sambiloto which is able to replace some IFNγ role in stabilizing the body's immune response. When the alkaloid Sambiloto were administered on the fourth, sixth, eighth and tenth day after being infected with Salmonella typhimurium, it was able to replace the role of IFNγ in substituting the body s immune response and improved the nonspecific immune responses in the form of increased responsiveness of leukocytes or the specific immune response to activate the macrophages to perform its role and function in phagocyting the infectious agents that enter the body [13]. Effect of SambilotoAlkaloids in rats after infected with Salmonella on the expression of T-Helper Cells (CD4 +): The results of examination of the expression of CD4 + on rats (Rattus norvegicus) administered alkaloid Sambiloto after infected with Salmonella can be seen in Table 2. Table 2:Average number of CD4 + in Rats AdministeredAlkaloids Sambiloto after Infected with Salmonella typhimuriumon day 2, 4, 6, 8, and 10 Treatment AverageCD4 + in Rats Administered Alkaloid Sambiloto after Infected with Salmonella Day 2 Day 4 Day 6 Day 8 Day 10 K 222.8 e ± 14,97 228.2 e ± 20,68 223.2 e ± 29,79 229.8 e ± 22,47 223.2 e ± 18,63 P0 401.2 a ± 25,57 412.8 a ± 15,51 417.6 a ± 11,65 425.4 a ± 20,69 444.6 a ± 23,50 P1 380.8 b ± 19,25 379.2 b ± 26,97 374.2 b ± 29,14 376.8 b ± 15,05 367.6 b ± 29,69 P2 354.8 c ± 15,54 348.2 c ± 28,89 343.4 c ±25,22 347.6 c ± 24,51 334.4 c ± 36,71 P3 295.6 d ± 24,14 276.8 d ± 31,30 261.6 d ± 29,43 249.2 d ± 19,70 237.2 d ± 38,48 Different superscript in the same column refers to significant difference (p<0,05) Control P0 P1

157 Wurlina et al., 2017/Advances in Natural and Applied Sciences. 11(9) July 2017, Pages: 154-158 P2 P3 Fig. 1: The Expression of CD4+ in Rats Administered Alkaloid Sambiloto after Infected with Salmonella typhimurium The higher dose of alkaloid Sambiloto was administered to rats on the second day after infected with Salmonella typhimuriumprovided results with reduced CD4 + expression. This indicates that the administration of alkaloid Sambiloto which was started on the second day after infection with Salmonella typhimurium,were able to replace the role of CD4 + in replacing the body's immune response in the form of a specific immune response.the role of Sambiloto alkaloid in replacing the function of CD4 + i.e higher the dose of alkaloid Sambiloto administered to rats infected with Salmonella typhimurium, lower the expression of CD4 +, i.e., CD4 + roles in maintaining the body's immune response are replaced by Sambiloto alkaloids. CD4 + is known for proliferation and differentiation of T-cellsinto T helper cells, T helper-1 cells (TH1) and T helper-2 cells (TH2). TH1 cells produce cytokines such as IFNγ, Interleukin 2 and TumorNecrosis Factor (TNF), whereas TH2 produce Interleukin 4 (IL4), Interleukin 5 (IL5) and IL 10. Interferon gamma (IFNγ) produced by TH1 cells would affect the role and function of macrophages in phagocyting the infectious agents that enter the body, while IL2 helps the proliferation of T-cell whereas IL12 has a role in the growth process of the TH1. All interleukins and IFNγ produced by TH1 cells play a role in enhancing the body's immune response. Interleukin 4 and Interleukin 5 produced by TH2 cells play a role in the mechanism of proliferation and differentiation of B cells into memory B cells and plasma cells that contribute in generating immunoglobulin, which is a humoral immune response [7,14]. CD4+ T cells act in stimulating the immune response i.e., both cellular and humoral immune responses. The role of IFNγ, in addition to stimulating the role and function of macrophages, is increasing the mechanisms of phagocyting infectious agents and also stimulating the mechanism of proliferation and differentiation of T cells into T helper-1 cells (TH1), TH2, cytotoxic T cells, T cell suppressor and memory T cells, each of which has a specific function in the body's immune response. In this research, it is known that Salmonella typhimurium infection on the second day of alkaloid Sambiloto administration could replace the role of a partly CD4 + in stabilizing the immunity response of the body. Adminiteringalkaloid Sambiloto on fourth, sixth, eighth and tenth day after infection with Salmonella typhimurium were able to replace the role of CD4 + [14]. Conclusion: From the results, it can be concluded that the alkaloid Sambiloto at a dose of 3.78 mg/200 g body weight up to 11.4 mg/200 g body weight after infected with Salmonella typhimurium, causing a decrease in CD4+ IFNγ and towards normal levels. REFERENCES 1. Belkaid, Y., B.T.Rouse,2005. Natural regulatory T Cells in infection disease. Nat Immunol., 6:353-60. 2. Cummings, 2005. Flic-specific CD4+ T cell Responses are Restricted by Bacterial Regulation of Antigen Expression. The Journal of Immunology, 174:7929-7938. 3. Criss, A.K., M. Silva, J.E. Casanova and B.A. McCormick, 2001. Regulation of Salmonella-Induced Neutrophil Transmigration by Epithelial ADP-Ribosylation Factor 6. J. Biol. Chem., 276:48431-48439. 4. Ealick, S.E., W.J.Cook, S.Vijay-Kumar, M.Carson, T.L.Nagabhushan, P.P.Trotta, C.E.Bugg,1991. Threedimensional structure of recombinant human interferon-gamma. Science, 252:698-702. 5. Folds, J.D., 2008. Overvi.ew of Immunology. Handbook of Human Immunology 2 nd Edition.Ed. O Gorman dan Donnenberg.CRC Press: Boca Raton. 6. Gamashinta, S., 2015. Effect of various concentrations of Salmonella typhimurium infection Against Macrophage Overview of activity Phagocytic Cells in Mice injected intraperitoneally 7. Mayer G., 2009. Immunology-Chapter One. Innate (Nonspecific) Immunity. http://pathmicro.med.sc.edu/book/immunol-sta.htm. 8. Mitruka, B., 1981. Clinical Biochemical and Hematological Reference Value in Normal Experimental Animal and Normal Humans. Second Edition. Printed in the United States of America. pp: 57-130.

158 Wurlina et al., 2017/Advances in Natural and Applied Sciences. 11(9) July 2017, Pages: 154-158 9. Shirwan, H., A.M. Hoyan, E.S.Yolcu, X.Y. Que and S. Ibrahim, 2003. Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is Associated with indirectrecognition by CD4+ T cells. TransplantImmunol., 11:179-185. 10. Sadir, R., E.Forest, H.Lortat-Jacob,1998. The heparin sulfate binding sequance of interferon-gamma increased the on rate of the interferon-gamma receptor complex formation 11. Schoenborn, J.R., C.B.Wilson,2007. Regulation of interferon-gamma during innate and adaptive immune responses. Adv. Immunol., 96: 41-101. 12. Vanhaverbeke, C., J.P.Simorre, R.Sadir, P.Gans, H.Lortat-Jacob,2004. NMR Characterization of the interaction between the C-terminal domain of Interferon-gamma and heparin-derived oligosaccharides. Biochem. J. 384(Pt 1): 93-9. 13. Wurlina, Mustofa, I., D.K.Meles, Niluh Suwasanti and I.D.P.Adnyana,2016. Potential Alkaloids Bitter (Andrographis paniculata L) of the total leukocyte and leukocyte count type on setelsh Mice infected with Salmonella typhimurium..vet Met. Juli 2016. ISSN 1979-1305;9(2): 173-184. 14. Xu, Y., 2009. Adaptive Immune Response-modifying and Antimicrobial Properti.es of Andrographis paniculate and Andrographolide Dissertation. The Department of Biological and Physical Sci.ence The University of Queensland.