Objective: The leaves of kumis kucing (Orthosiphon stamineus [Benth.]) are used as folk medicine for lifestyle-related diseases such as hypertension,hyperlipidemia, and diabetes mellitus in Southeast Asia countries, especially Indonesia. Obesity is closely associated with lifestyle-related disease.The objective of this research was to evaluate the effect of kumis kucing leaves ethanol extract in high-fat diet-induced obese mice, and its ability toinhibit pancreatic lipase enzyme activity in vitro.Methods: Obese mice groups were fed a high-fat diet for 30 days and treated with kumis kucing leaves ethanol extract for 2 weeks. The measurementparameter includes body weight, organ weight, and visceral fat mass. Experiments were also carried out to clarify the effect of kumis kucing leavesethanol extract inhibited pancreatic lipase enzyme activity in vitro.Results: Kumis kucing-treated groups showed a significant decrease in body weight, organ weight, and visceral fat mass after 2 weeks treatmentcompared with untreated groups. Furthermore, kumis kucing leaves ethanol extract showed potency as the pancreatic lipase activity inhibitor in vitro.Conclusion: Kumis kucing leaves ethanol extract can inhibit the development of obesity in high-fat diet-induced obese mice. The effect appearsmediated by inhibiting the pancreatic lipase activity. The results from this study suggested that the kumis kucing leaves may have a considerableimpact as potential anti-obesity agents.Keywords: Kumis kucing, Extract, Obesity, High-fat diet

JUANDA, DADANG

KURNIA, IKA

PURWANI, HENI

SETIAWAN, FINNA

YUNIARTO, ARI

English

Innovare Academic Sciences: E-Journals

Vol 8, Issue 6, 2015 ISSN - 0974-2441KUMIS KUCING ORTHOSIPHON STAMINEUS BENTH.  LEAVES ETHANOL EXTRACT AS ANTI OBESITY AGENT IN HIGH FAT DIET INDUCED OBESE MICEARI YUNIARTO1*, HENI PURWANI1, DADANG JUANDA2, FINNA SETIAWAN3, IKA KURNIA11Pharmacology Research Group, Bandung School of Pharmacy, Bandung, Indonesia. 2Pharmaceutical Biology Research Group, Bandung School of Pharmacy, Bandung, Indonesia. 3Pharmacology and Clinical Pharmacy Research Group, Bandung Institute of Technology, Bandung, Indonesia. Email: ariyuniarto@yahoo.co.idReceived: 16 August 2015, Revised and Accepted: 30 September 2015ABSTRACTObjective: The leaves of kumis kucing (Orthosiphon stamineus [Benth.]) are used as folk medicine for lifestyle-related diseases such as hypertension, hyperlipidemia, and diabetes mellitus in Southeast Asia countries, especially Indonesia. Obesity is closely associated with lifestyle-related disease. The objective of this research was to evaluate the effect of kumis kucing leaves ethanol extract in high-fat diet-induced obese mice, and its ability to inhibit pancreatic lipase enzyme activity in vitro.Methods: Obese mice groups were fed a high-fat diet for 30 days and treated with kumis kucing leaves ethanol extract for 2 weeks. The measurement parameter includes body weight, organ weight, and visceral fat mass. Experiments were also carried out to clarify the effect of kumis kucing leaves ethanol extract inhibited pancreatic lipase enzyme activity in vitro.Results: Kumis kucing-treated groups showed a significant decrease in body weight, organ weight, and visceral fat mass after 2 weeks treatment compared with untreated groups. Furthermore, kumis kucing leaves ethanol extract showed potency as the pancreatic lipase activity inhibitor in vitro.Conclusion: Kumis kucing leaves ethanol extract can inhibit the development of obesity in high-fat diet-induced obese mice. The effect appears mediated by inhibiting the pancreatic lipase activity. The results from this study suggested that the kumis kucing leaves may have a considerable impact as potential anti-obesity agents.Keywords: Kumis kucing, Extract, Obesity, High-fat diet.INTRODUCTIONRecently, obesity has become an epidemic on a global scale and one of the world’s most serious public health problems [1]. Obesity condition increases the risk of morbidity and mortality. World Health Organization (WHO) defines obesity as abnormal or excessive fat accumulation which influences body health. In 2005, WHO gave estimation that obese adults in the world average 400 million and predicted that in 2015 it will increase to 700 million adults with obesity [2]. Obesity results from disequilibrium between energy intake and energy expenditure [3]. Therefore, inhibition of digestion and absorption of dietary fat can be useful in treatment of obesity. Furthermore, obesity is with various diseases, including Type 2 diabetes mellitus (DM), hypertension, dyslipidemia, cardiovascular diseases, atherosclerosis, and cancer [4,5].Diet modification and activity as obesity treatment have low effectiveness in long-term weight loss and maintenance. Several studies suggest that adjunctive therapy in combination with exercise and nutrition therapy increase efficacy. However, some of these medications have been correlated with serious adverse effects [6].Orlistat, is one of the two clinically approved drugs for obesity treatment, has been shown to act by inhibiting pancreatic lipase enzyme. The long-term effects of Orlistat administration are oily stools, oily spotting, and flatulence, among others. So, the potential of natural products for the treatment of obesity might be an excellent alternative strategy for the development of safe and effective anti-obesity drugs [7].Kumis kucing or java tea classified under the family Lamiaceae or Labiatae. Kumis kucing has some synonyms, such as Orthosiphon aristatus (Blume) Miq, Orthosiphon longiflorum Ham., Orthosiphon grandiflorum et aristatum Bl., Orthosiphon grandiflorus Bold., and Orthosiphon spiralis Merr. This plant can be found in Southeast Asia (Indonesia, Malaysia, Myanmar, and Vietnam) and Australia. Kumis kucing is one of the popular traditional plants in Southeast Asia, especially in Indonesia which used to treat several diseases such as arthritis, DM, hypertension, kidney disorder, inflammation, and menstrual disorders [8].Several studies identified that kumis kucing contains important compounds including polyphenols, flavonoids, terpenoids, sterols, orthosiphols, saponins, caffeic acid, and oleanolic acid. The compounds were known to be associated with anti-obesity [9]. Therefore, the aim of the present study is to evaluate the effect of kumis kucing leaves ethanol extract as anti-obesity agents in high-fat diet-induced obese mice model.METHODSReagentsAll reagents in this study were of analytical grade. Porcine pancreatic lipase, oleic acid, and bovine serum albumin were obtained by Sigma-Aldrich. Orlistat (Xenical)® was purchased from Kimia Farma Pharmacy.Plant material and identificationKumis kucing leaves were obtained from The Centre for Research and Development of Medicinal Plants and Traditional Medicine, Tawangmangu, Central Java, Indonesia. Plant identification was done in Laboratory of Taxonomy, Faculty of Mathematics and Natural Sciences, Padjajaran University, Bandung, Indonesia.Preparation of extractThe leaves of kumis kucing were dried and then powdered. The powder of kumis kucing was extracted with 96% ethanol by maceration method and evaporated by Rotary Evaporator. This kumis kucing leaves ethanol extract was used for the in vivo and in vitro assays.Research ArticleAsian J Pharm Clin Res, Vol 8, Issue 6, 2015, 234-236 Yuniarto et al. 235Phytochemical screening of the kumis kucing extractPhytochemical screening was performed to monitor the presence of phytochemical constituents such as alkaloids, flavonoids, saponins, tannins, quinones, and steroids/triterpenoids.Pancreatic lipase enzyme in vitro assayPancreatic lipase enzyme activity was determined by measuring the release rate of oleic acid from sesame oil. This method is based on Han et al. [10] but with a slight modification. The substrate (5 ml in a 10 ml tube) was prepared through sonication process for 5 minutes. The substrate contained 15 mmol/l sesame oil, 1 mmol/l NaCl, 1 mmol/l CaCl2, 10 mg of bovine serum albumin/ml, and phosphate buffer solution with pH 8.0. After sonication, the substrate was incubated with 50 μl of porcine pancreatic lipase enzyme and kumis kucing extract for 30 minutes at 37°C with various concentrations. 30 minutes after incubation at 37°C, the substrate was added to 3 ml of a 1:1 (v/v) mixture of chloroform and n-heptane, extracted by shaking on the tube for 10 minutes in a shaker. The mixture was centrifuged at 2000 rpm for 10 minutes. The upper aqueous phase was removed, and the lower phase was added with a copper reagent with volume 0.5 ml. The tube was shaken again for 10 minutes, was centrifuged at 2000 rpm, and 0.5 ml of the upper phase (organic phase) was added with 0.5 ml diethyldithiocarbamate-sodium solution. The absorbance was then measured at 480 nm.The inhibitory activities of kumis kucing leaves ethanol extract and orlistat, a positive control of pancreatic lipase were measured at concentrations of 1000, 100, 10, 1, and 0.1 μg/ml. The measurements were performed in triplicate, and the IC50 value, the concentration of the extract that results in 50% inhibition of maximal activity was determined.Anti-obesity activity in vivo assayThe anti-obesity activity of kumis kucing leaves ethanol extract has been examined in male Swiss-Webster mice, and all animal experiments were conducted under institutional ethical guidelines. Animal study was conducted in the Laboratory of Pharmacology, Bandung School of Pharmacy, Indonesia. 25 Swiss-Webster mice, of 2-3 months, weighed about 25 and 30 g, were adapted under room temperature. The animals were fed with standard diet and drink ad libitum.Following adaptation period, the healthy mice were used in the experiments. The mice were randomly divided into five groups of five mice each. The experimental group consisted of the following groups control group, high-fat diet group, orlistat 15.6 mg/kg, kumis kucing leaves extract 100 mg/kg, and kumis kucing leaves extract 200 mg/kg. The control group was fed standard animal laboratory diet and other groups were fed high-fat diet for 30 days.The measurement parameter included body weight, organ weight, and visceral fat mass. Body weight was measured daily. At the end of the experiment, the mice were sacrificed, and the organs collected. Organs collected included liver, spleen, testicle, kidney, and visceral fat mass such as perirenal fat, perianal fat, retroperitoneal fat, and epididymal fat.Statistical analysisStatistical comparisons of the results of the anti-obesity assay were performed using the one-way Analysis of Variance method, coupled with the post-hoc least significant difference test. A value of p<0.05 was used to denote statistical significance. All data were expressed as mean ± standard deviation of the mean (SD) for each group.RESULTS AND DISCUSSIONComposition of experimental dietThe composition of the experimental diet is shown in Table 1 and its was composition based on Adnyana et al. [11]. By the end of 30 days acclimatization period, the body weight of the obese mice group (including high-fat diet group, orlistat group, and kumis kucing extract group) was approximately 20% higher than the negative control group.Result of phytochemical screening kumis kucing leaves ethanol extractThe percentage of yield of kumis kucing leaves ethanol extract was found to be 6.11%. Result of phytochemical screening as shown in Table 2. Phytochemical screening of kumis kucing leaves ethanol extract showed the presence of flavonoids, saponins, tannins, quinones, and steroid/triterpenoids. Iswantini et al. [12] showed that phytoconstituents, such as flavonoids, saponins, alkaloids, tannins, and steroids/triterpenoids, have effect on obesity and contribute to inhibit pancreatic lipase enzyme activity. The phytoconstituents which were derived from plants such as flavonoids, terpenoids, phenolic acids, and other groups exhibit potential effect as anti-obesity agents.Effect of kumis kucing leaves ethanol extract on pancreatic lipase activityThe inhibitory activities of kumis kucing leaves ethanol extract against porcine pancreatic lipase are shown in Fig. 1. The kumis kucing leaves ethanol extract inhibited pancreatic lipase activity by 38.55%, 40%, 44.26%, 57.5%, and 63.92% at concentrations 0.1, 1, 10, 100, and 1000 μg/ml in vitro, respectively. Orlistat, a pancreatic lipase inhibitor used as anti-obesity agent, inhibited the enzyme activity at the concentrations 0.1, 1, 10, and 100 μg/ml, respectively. The kumis kucing leaves ethanol extract and Orlistat had IC50 as 53.68 μg/ml and < 0.1 μg/ml, respectively.Effect of kumis kucing leaves ethanol extract on body weightAs shown in Fig. 2, the body weight gain was reduced by administration of kumis kucing leaves ethanol extract at the dose 100 mg/kg and 200 mg/kg compared with high-fat diet group. Subsequently, the mean body weight and the body weight gain in these groups were significantly lower than high-fat diet group. Body weight of the kumis kucing leaves ethanol extract group at the dose 100 mg/kg was significantly different to the control group but comparable with high-fat diet group.Fig. 1: Inhibition of pancreatic lipase activity by kumis kucing extract and orlistatTable 1: Composition of the experimental dietComposition Standard diet (g/kg) High-fat diet (g/kg)Corn starch 250 250Fish flour 160 160Bean flour 140 140Wheat flour 410 130Fat - 320Vegetable oil 40 -Table 2: Result of phytochemical screening of kumis kucing leaves ethanol extractGroup ResultsAlkaloids -Flavonoids +Saponins +Tannins +Quinones +Steroid/Triterpenoids +Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 234-236 Yuniarto et al. 236Effect of kumis kucing leaves ethanol extract on organ weightHigh-fat diet group showed significantly high liver and testicle weight in comparison to the control group, but not spleen and kidney weight (Table 3) (p<0.05). Conversely, groups treated with kumis kucing leaves ethanol extract with dose 100 mg/kg and 200 mg/kg didn’t effective to decrease liver weight caused by fat accumulation. Furthermore, kumis kucing extract at the dose of 100 mg/kg and 200 mg/kg showed a significant decrease in spleen weight compared to high-fat diet group. As shown in Table 3, kumis kucing leaves ethanol extract at the dose of 200 mg/kg showed a significantly lower kidney weight and testicle weight compared to the high-fat diet group (p<0.05). Effect of kumis kucing leaves ethanol extract on visceral fat massAs shown in Table 4, kumis kucing leaves ethanol extract at 100 mg/kg and 200 mg/kg showed a significantly lower retroperitoneal, perirenal, and perianal weight than that of the high-fat diet group (p<0.05), but less effective to decrease fat accumulation in epididymal. It can be concluded that kumis kucing leaves ethanol extract 100 mg/kg and 200 mg/kg might have anti-obesity effects by preventing visceral fat accumulation.CONCLUSIONThe anti-obesity effect of kumis kucing leaves ethanol extract in high-fat diet-induced obese mice might be mediated by inhibiting pancreatic lipase activity. The present study showed that kumis kucing leaves ethanol extract may have a considerable potential as anti-obesity agents because of its effect on body weight, visceral fat parameters, and organ parameters in obese mice.ACKNOWLEDGMENTThis research was supported by Young Lecturer Research Grant from Directorate General of Higher Education, Republic of Indonesia.REFERENCES1. Yun JW. P ossible anti-obesity therapeutics from nature – A review. Phytochemistry 2010;71(14-15):1625-41.2. Cowley P, Palmer A, Williams R. Obesity and its treatment. Drugs Future 2008;33(12):1077-82.3. Han LK, Zheng YN, Yoshikawa M, Okuda H, Kimura Y. Anti-obesity effects of chikusetsusaponins isolated from Panax japonicus rhizomes. BMC Complement Altern Med 2005;5:9.4. Redinger RN. The pathophysiology of obesity and its clinical manifestations. Gastroenterol Hepatol (N Y) 2007;3(11):856-63.5. Na HN, Nam JH. Proof-of-concept for a virus-induced obesity vaccine; vaccination against the obesity agent adenovirus 36. Int J Obes (Lond) 2014;38(11):1470-4.6. Hill JO, Hauptman J, Anderson JW, Fujioka K, O’Neil PM, Smith DK, et al. Orlistat, a lipase inhibitor, for weight maintenance after conventional dieting: A 1-y study. Am J Clin Nutr 1999;69(6):1108-16.7. Lee JK, Jang JH, Lee JT, Lee JS. Extraction and characteristics of anti-obesity lipase inhibitor from Phellinus linteus. Mycobiology 2010;38(1):52-7.8. Al-Yahya AA. Reproductive toxicity of Orthosiphon stamineus Benth (Java Tea) in Swiss albino mice. Br J Pharm Toxicol 2013;4(5):181-7.9. Son JY, Park SY, Kim JY, Won KC, Kim YD, Choi YJ, et al. Orthosiphon stamineus reduces appetite and visceral fat in rats. J Korean Soc Appl Biol Chem 2011;54(2):200-5.10. Han LK, Kimura Y, Okuda H. Reduction in fat storage during chitin-chitosan treatment in mice fed a high-fat diet. Int J Obes Relat Metab Disord 1999;23(2):174-9.11. Adnyana IK, Sukandar EY, Yuniarto A, Finna S. Anti-obesity effect of the pomegranate leaves ethanol extract (Punica granatum L.) in high-fat diet induced mice. Int J Pharm Pharm Sci 2014;6(4):626-31.12. Iswantini D, Silitonga RF, Martatilofa E, Darusman LK. Zingiber cassumunar, Guazuma ulmifolia, and Murraya paniculata extracts as antio-besity: In vitro inhibitory effect on pancreatic lipase. Hayati J Biol 2011;18(1):6-10.Fig. 2: Effect of the kumis kucing leaves ethanol extract on body weightTable 3: Result of kumis kucing leaves ethanol extract on organ weightGroup Organ weight (g)Liver Spleen Kidney TesticleControl group 1.47±0.04 0.12±0.01 0.51±0.02 0.14±0.02*High-fat diet group 1.91±0.26 0.11±0.03 0.49±0.04 0.31±0.03Orlistat 15.6 mg/kg 1.66±0.50 0.10±0.02 0.52±0.05 0.28±0.02Kumis kucing extract 100 mg/kg 1.76±0.19 0.07±0.01* 0.51±0.05 0.24±0.05Kumis kucing extract 200 mg/kg 1.50±0.48 0.04±0.02* 0.36±0.10* 0.21±0.04*(*) Data represent the mean±SD of observations from 5 mice, significantly different from high-fat diet group at p<0.05, SD: Standard deviationTable 4: Result of kumis kucing leaves ethanol extract on visceral fat massGroup Visceral fat mass (g)Retroperitoneal Perirenal Perianal EpidydymalControl group 1.66±0.17 0.21±0.02 0.29±0.17* 0.40±0.05High-fat diet group 3.23±0.17 0.41±0.10 1.15±0.03 0.94±0.16Orlistat 15.6 mg/kg 2.50±0.13 0.41±0.04 0.31±0.03 0.94±0.09Kumis kucing extract 100 mg/kg 1.33±0.25* 0.14±0.01* 0.08±0.01* 0.42±0.04Kumis kucing extract 200 mg/kg 0.37±0.22* 0.09±0.05* 0.07±0.01* 0.15±0.01(*) Data represent the mean±SD of observations from 5 mice, significantly different from high-fat diet group at p<0.05, SD: Standard deviation

KUMIS KUCING (ORTHOSIPHON STAMINEUS [BENTH.]) LEAVES ETHANOL EXTRACT AS ANTIâ€‘OBESITY AGENT IN HIGHâ€‘FAT DIETâ€‘INDUCED OBESE MICE

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KUMIS KUCING (ORTHOSIPHON STAMINEUS [BENTH.]) LEAVES ETHANOL EXTRACT AS ANTIâ€‘OBESITY AGENT IN HIGHâ€‘FAT DIETâ€‘INDUCED OBESE MICE

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