Study of Sungkai (Peronema canescens, Jack) Leaf Extract Activity as an Immunostimulators With In vivo and In vitro Methods

Abstract

Introduction: Sungkai (Peronema canescens, Jack.) contains polysaccharides, terpenoids, alkaloids, and polyphenols which have pharmacological activity as immunostimulants. Objective: This study aimed to see how the effect of Sungkai extract as an immunostimulant agent was carried out in vitro and in vivo. Materials and Methods: This study was conducted using two methods, namely in vivo and in vitro. In vivo research method was conducted to test the activity and phagocytic capacity of macrophage cells, the percentage of leukocytes, and the total number of leukocytes. This study used 30 male white mice as the test animals that were randomly divided into 5 treatment groups. Each group was consisting of 6 mice which were given different treatments. The negative control group was given with the 0.5% NaCMC suspension, the mice test substance group was given with the suspension of Sungkai ethanol extract with various doses of 800, 400, and 200 mg/kgBW, and lastly the comparison group was given with the Stimuno in a dose of 50 mg/kg orally for 7 days. On day 8, blood was taken from the mice's vein to count the number and percentage of its leukocytes, then followed by the intraperitoneal injection of a Staphylococcus aureus bacteria suspension. After 1 hour of administration of the bacterial suspension, the peritoneal fluid was taken to be observed for its activity and phagocytic capacity of macrophage cells. The in vitro research method was used to test the viability and immunostimulatory activity of RAW 264.7 cells with the Sungkai extraction at the concentration of 1, 10, 100 μg/mL. This cell viability test using the microtetrazolium (MTT) method aims to see whether the Sungkai sample used is safe and not toxic to RAW 264.7 cells by observing at the cell viability value that should exceed >90%. The concentration of Sungkai extraction at 1, 10, 100 μg/mL was found to be safe and non-toxic to RAW 264.7 cells with a viability value of >90%. Thus, this concentration of Sungkai extraction can be performed for its immunostimulatory activity test on LPS induced of RAW 264.7 cells by observing their levels of IL-6 and TNF-α. (proinflammatory cytokines) were compared with the LPS alone as a control using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method. Results: The observations were analyzed by one-way ANOVA and Duncan's follow-up test (significance was taken at p<0.05). The results showed that variations in concentration increased significantly (p<0.05) on the activity and phagocytic capacity of macrophage cells, along with the total leukocyte cells. The percentage of leukocytes showed that the cells had a significant increase (p<0.05). It was found that the Sungkai extraction on 1.10, 100 g/mL could significantly increase the concentration of TNF- and IL-6 (p<0.05) which were tested by one-way ANOVA and followed by Duncan's post hoc test. Conclusion: Sungkai leaf extract (Peronemacanescsens Jack.) in a dose of800, 400, and 200 mg/kgBW has an immunostimulant effect both in vivo and in vitro. Key words: Sungkai (Peronema canescens, Jack), phagocytosis, macrophages, total and percentage of leukocytes, MTT (Microtetrazolium), LPS (lipopolysaccharide), RAW 264.7 cells, cell viability, immunostimulant.