Objective: This study was conducted to evaluate the immunomodulatory effects of ethanol extract of Curcuma mangga by in vivo study.Methods: The ethanol extract of C. mangga was comprised to carbon clearance method for its immunomodulatory potential. The extract wasadministered orally at doses of 100, 200, and 400 mg/kg BW to mice for 7 days. On day 8, carbon ink was injected, and the blood was collected formeasurement of elimination of carbon. Total leukocyte count was also determined.Results: The evaluation of immunomodulatory potential of ethanol extract of C. mangga revealed a dose-dependent increase in phagocytosis ability.The phagocytic index of ethanol extract of C. mangga was more than those of negative control, indicating the immunostimulatory activity of C. mangga.It showed low stimulation on total leukocyte count.Conclusion: The results indicate that ethanol extract of C. mangga rhizomes possesses immunomodulatory activity and has therapeutic potential forthe treatment of infectious diseases

Suwarso, Edy

Yuandani

English

Innovare Academic Sciences: E-Journals

Vol 10, Issue 9, 2017Online - 2455-3891 Print - 0974-2441IMMUNOMODULATORY EFFECTS OF ETHANOL EXTRACT OF CURCUMA MANGGA  RHIZOMES IN MICEYUANDANI*, EDY SUWARSODepartment of Pharmacology, Faculty of Pharmacy, Universitas Sumatera Utara, 5 Jalan Almamater, USU-Kampus, Medan 20155, Indonesia. Email: yuan_dani@yahoo.comReceived: 10 March 2017, Revised and Accepted: 24 May 2017ABSTRACTObjective: This study was conducted to evaluate the immunomodulatory effects of ethanol extract of Curcuma mangga by in vivo study.Methods: The ethanol extract of C. mangga was comprised to carbon clearance method for its immunomodulatory potential. The extract was administered orally at doses of 100, 200, and 400 mg/kg BW to mice for 7 days. On day 8, carbon ink was injected, and the blood was collected for measurement of elimination of carbon. Total leukocyte count was also determined.Results: The evaluation of immunomodulatory potential of ethanol extract of C. mangga revealed a dose-dependent increase in phagocytosis ability. The phagocytic index of ethanol extract of C. mangga was more than those of negative control, indicating the immunostimulatory activity of C. mangga. It showed low stimulation on total leukocyte count.Conclusion: The results indicate that ethanol extract of C. mangga rhizomes possesses immunomodulatory activity and has therapeutic potential for the treatment of infectious diseases.Keywords: Curcuma mangga, Phagocytosis, Total leukocyte count, Immunomodulatory.INTRODUCTIONImmune system is a complex system, but its components are interrelated act in a highly coordinated and specific manner. Thus, most infections caused by pathogens in most individuals are short-lived with minor permanent damage. Innate immunity is the most universal and the most rapidly acting, which is largely mediated by professional immunocytes (neutrophils, monocytes, and macrophage cells) [1]. Phagocytosis is an effective innate internal defense and in activating the adaptive immune response. At the site of infection, phagocytes engulf and destroy the foreign substances by microbicidal agents [2]. There are various toxic molecules for microorganisms generate from superoxide anion (O2−) such as hypochlorous acid (HOCl-), hydroxyl radicals (OH-), and singlet oxygen. In addition, nitric oxide (NO-) which is produced by macrophages during the respiratory burst reacts with O2- to form peroxynitrite, a strong antimicrobial [3,4].Several plant extracts and their isolates have been reported for their immunomodulatory activity [5-7]. The extracts of many herbs such as Panax ginseng, Tinospora cordifolia, Phyllanthus amarus, Centella asiatica, Trigonella foenum graecum, Pouteria cambodiana, Picrorhiza scrophulariiflora, Garcinia mangostana, Thymus guyonii, Salvia verbenaca, Capparis spinosa, and Stachys circinata were able to upregulate or downregulate both innate and adaptive arms of the immune response [8-11].The plants belonging to the genus Curcuma (family: Zingiberaceae) are widely distributed in most tropical countries [12]. Among them, Curcuma mangga is widely used in traditional medicine to treat various diseases such as stomach disorders, fever, and cancer-related diseases [13]. The variety of its organic compound of medicinal importance such as β-sitosterol, curcumin, demethoxycurcumin, and bisdemethoxycurcumin has emphasized to evaluate its immunomodulatory activity [13,14]. The extracts and compounds isolated from C. mangga have revealed a wide spectrum of pharmacological activities, including analgesic, anti-inflammatory, antioxidant, anticancer, antifungal, and nitric oxide inhibitory activities [15-18]. In addition, the ethanol extract of C. mangga did not induce significant short-term toxicity as reported in our previous study [19]. However, there are little studies to validate the traditional use of C. mangga leaves to treat diseases related to the immune system.Previous in vitro study has indicated that the methanol extracts of C. mangga displayed strong immunomodulatory effects on polymorphonuclear neutrophils and macrophage cells. However, in vivo study is necessary to elaborate its immunomodulatory activity. This study was conducted to investigate the effects of ethanol extract of C. mangga on phagocytosis ability of mice leukocytes as well as total leukocyte count.METHODSChemicals and reagentsThe chemicals used in this study were ethanol (Smart Lab, Indonesia) and natrium carboxymethylcellulose (Na-carboxymethylcellulose [CMC]) (Sigma, USA), imboost® (Soho, Indonesia), China ink (pelican B-17), acetic acid (Smart Lab, Indonesia), and NaCl (Otsuka, Indonesia). A spectrophotometer (Shimadzu, Japan) was also used in this study.Plant materialsThe rhizomes of C. mangga were collected from Medan, Sumatera Utara, Indonesia. Then, the plant was authenticated in Herbarium Medanense, Universitas Sumatera Utara, Indonesia.Extraction procedureThe plant materials were allowed to dry under shade. 350 g of dried material of plant sample was ground and macerated in ethanol at the ratio of 1:10 (w/v). The extraction was repeated twice on the residue. The filtrates were combined, and the solvent was removed © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i9.18398Research Article149Asian J Pharm Clin Res, Vol 10, Issue 9, 2017, 148-150 Yuandani and Suwarso under reduced pressure to obtain an extract of C. mangga (38.4 g, 10.95% w/w).Phagocytosis responseThe phagocytosis ability was evaluated by carbon clearance method as described previously with slightly modification [20]. The animals were treated with ethanol extract of C. mangga at doses of 100, 200, and 400 mg/kg BW for 7 days. Meanwhile, the negative control group received Na CMC 0.5% as vehicle. Imboost® was used as positive control at a dose of 32.5 mg/kg BW. On day 8, all the animals received the treatment of an intravenous injection of (0.1 ml per 10 g) China ink dispersion via tail vein. Thereafter, 25 µL of blood samples were collected from each animal at an interval of 5, 10, 15, and 20 minutes after the injection of ink dispersion. Blood samples were added to 4 ml of 1% acetic acid to lyse the erythrocytes. Absorbance of the samples was measured at 640.5 nm using spectrophotometer. After 12 hrs of blood, collection animals were sacrificed and the livers and spleens were collected and weighed.Rate of carbon clearance (K), and phagocytic index (α) were calculated using following formula:Log OD5- log OD20Rate of  carbon clearance (K) = t2 - t11 3K  body wt of  animalPhagocytic index ( ) =Liver wt + spleen wt×αWhere, OD5 is the log absorbance of blood at 5 minutes; OD20 is log absorbance of blood at 20 minutes; t2 is the last time point of blood collection; t1 is the first time point of blood collection. The use of mice was approved by the Animal Research Ethics Committees of Universitas Sumatera Utara (approval number 599/KEPH-FMIPA/2016).Total leukocyte countAfter the treatment with extracts for 7 days and injection with carbon ink dispersion, the blood sample of all animals was also collected for determination of total leukocyte count.Statistical analysisThe data were analyzed using Statistical Package for Social Sciences (SPSS) version 15.0. Each sample was measured in triplicate and the data presented as mean±standard error of the mean. Data were analyzed using a one-way analysis of variance for multiple comparisons and followed by Tukey post hoc test. p<0.05 was considered to be different significantly.RESULTS AND DISCUSSIONPhagocytosis responsePhagocytosis is performed using pseudopodia which are extended to surround an organism or particle and followed by intracellular destruction [21]. The effect of ethanol extract on phagocytosis ability of mice leukocytes was determined by the removal of carbon from bloodstream. The enhanced clearance rate of carbon particle from blood flow indicates the increment of phagocytosis activity of leukocytes. The rate of carbon clearance of the C. mangga extract at different doses (100, 200, and 400 mg/kg) was higher than the negative control (p<0.05), signifying that they were increasing the percentage of carbon ingestion and thus stimulating the phagocytic cells (Table 1). The stimulation on carbon engulfment of C. mangga was in a dose-dependent manner. The ethanol extract of C. mangga at a dose of 400 mg/kg BW demonstrated the strongest stimulant with phagocytic index of 6.71 which was comparable with those of positive control, Imboost® with phagocytic index of 6.82 (Fig. 1). Imboost® is a marketed drug to enhance an immune system which contained Echinacea purpurea 250 mg, black elderberry 400 mg, and zinc picolinate 10 mg. The previous study reported the ability of E. purpurea to stimulate cytokine release from macrophages [22].The enhancement of phagocytosis activity to eradicate pathogens is markedly increase the immune system to protect the body from bacterial infection [23]. Fig. 1 shows that the phagocytic index of ethanol extract of C. mangga was more than those of negative control, indicating the immunostimulatory effect of C. mangga by enhancing phagocytosis ability. The result was in agreement with the previous study which reported the ability of methanol extract of C. mangga to enhance the phagocytosis response of human neutrophils by in vitro study [24].Total leukocyte countCells of the immune system are generated from pluripotent hematopoietic stem cells in bone marrow. Thereafter, various immune cells are circulating in the blood stream, lymph, gastrointestinal system, and respiratory tract. The presence of pathogen-derived chemotactic factors attracts leukocytes to the site of infection [25]. Ethanol extract of C. mangga demonstrated low stimulation on the total leukocyte count as compared to negative control (p>0.05) (Table 2). The previous study was also reported the inhibition effect of C. mangga on the migration of neutrophils to the site of infection [26].Fig. 1: Effect of administration of ethanol extract of Curcuma mangga rhizomes on phagocytic index. Data are mean±standard error of the mean (n=5); *p<0.05 compared to the respective controlTable 1: Effect of ethanol extract of C. mangga rhizomes on rate of carbon clearanceS.No. Samples Rate of carbon clearance1 Na CMC 0.0059±0.00272 100 mg/kg BW 0.0283±0.0065*3 200 mg/kg BW 0.0301±0.0051*4 400 mg/kg BW 0.0411±0.0084*5 Imboost® 0.0494±0.0167*Data were analyzed by one-way ANOVA, and followed by Tukey post hoc test. *p<0.05 compared to the respective control, CMC: Carboxymethylcellulose, C. mangga: Curcuma manggaTable 2: Effect of C. mangga extract on total leukocyte count (mean±SEM, n=5)S.No. Samples Total leukocyte count (109/L)1 Na CMC 6.24±0.732 C. mangga extract 100 mg/kg 8.26±0.663 C. mangga extract 200 mg/kg 8.03±0.874 C. mangga extract 400 mg/kg 7.85±0.715 Imboost® 7.70±0.59Data were analyzed by one-way ANOVA, and followed by Tukey post hoc test. None of the sample showed p<0.05 . C. mangga: Curcuma mangga, CMC: Carboxymethylcellulose150Asian J Pharm Clin Res, Vol 10, Issue 9, 2017, 148-150 Yuandani and Suwarso CONCLUSIONThe ethanol extract of C. mangga was able to modulate the innate immune response especially phagocytosis ability of mice phagocytes. In addition, the plant extracts revealed low stimulation on the total leukocyte count. The highest phagocytic index was observed when ethanol extract was administered at a dose of 400 mg/kg. Hence, from the results obtained, it can be concluded that C. mangga has therapeutic potential and could be served as an effective immunomodulatory candidate. However, further studies are required to elucidate their activities on other mechanisms of immunomodulatory responses.ACKNOWLEDGMENTThis work was supported by University of Sumatera Utara through BPPTN research grant scheme in 2016.REFERENCES1. Elgert KD. Immunology Understanding the Immune System. 2nd ed. Virginia: A John Wiley & Sons, Inc.; 2009.2. 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IMMUNOMODULATORY EFFECTS OF ETHANOL EXTRACT OF CURCUMA MANGGA RHIZOMES IN MICE

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IMMUNOMODULATORY EFFECTS OF ETHANOL EXTRACT OF CURCUMA MANGGA RHIZOMES IN MICE

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