Faculty of Pharmacy, Universitas Muhammadiyah Surakarta Jl.Ahmad Yani Tromol Pos I, Pabelan Kartasura Surakarta

Transcription

1 EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK (Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED BY POTASSIUM OXONATE Efek Ekstrak Etanol Kulit Batang Sala (Cynometra ramiflora Linn.) Terhadap Penghambatan Peningkatan Kadar Asam Urat Mencit Jantan Galur Swiss Yang Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi Faculty of Pharmacy, Universitas Muhammadiyah Surakarta Jl.Ahmad Yani Tromol Pos I, Pabelan Kartasura Surakarta har254@ums.ac.id ABSTRAK Cynometra ramiflora Linn (Tanaman Sala) di masyarakat secara empiris digunakan untuk mengobati asam urat. Penelitian ini bertujuan untuk mengetahui pengaruh ekstrak etanol kulit batang tanaman Sala dalam penghambatan kadar asam urat dalam mencit galur Swiss yang diinduksi kalium oksonat dan dosis ekstrak yang dapat menghambat peningkatan kadar asam urat yang setara dengan Allopurinol. Penelitian ini menggunakan rancangan post-test dengan kontrol. Dua puluh lima mencit jantan berusia 2-3 bulan dengan berat badan gram dibagi 5 kelompok. Kelompok I diberi Allopurinol sebagai kontrol positif, kelompok II diberi CMC-Na sebagai kontrol negatif, kelompok III sampai V berturut-turut diberi ekstrak etanol kulit batang tumbuhan Sala dosis 250; 500 dan 1000mg/kgBB. Untuk menginduksi hiperurisemia mencit diberi diberi makanan tambahan berupa jus hati ayam dengan konsentrasi 10% dan diinduksi dengan kalium oksonat secara intraperitonial. Hasil penelitian menunjukkan bahwa ekstrak tumbuhan Salah dosis 250, 500 dan 1000 mg/kgbb dapat menghambat peningkatan kadar asam urat (P<0,05). Ekstrak kulit batang Sala dosis 500mg/kgBB dan 1000mg/kgBB mampu menghambat peningkatan kadar asam urat setara dengan Allopurinol (P>0,05) Kata kunci: Cynometra ramiflora Linn, kulit kayu, asam urat, strain swiss ABSTRACT Cynometra ramiflora Linn, (Plant Sala) has been used empirically in the treatment of uric acid by the community. The aim of this study was to evaluate the effect of ethanolic extract of stem bark Sala plant on the inhibitory effect of uric acid levels in Swiss male mice induced by potassium oxonate intraperitoneally. This study used post-test with control design, Twenty five mice 2-3 month of age, weight gram divided into five groups. Group I was treated Allopurinol 10 mg/kgbw as positive control, group II was given CMC Na 0,5 % as negative control, group III-V were treated by extracts of Sala stem bark with dose of 250mg/kgBW, 500mg/kgBW, and 1000mg/kgBW, respectively. All treatment were administered orally. The mice induced hyperurecemia by additional food of the chicken liver juice 10% and potassium oxonate intraperitoneally. Test results were analyzed by Analysis of variance (Anova) followed by Bonferoni test with level of confidence 95%. The results showed that the extract dose of 250, 500 and 1000 mg/kgbw could inhibit the increasing of uric acid levels compared with negative control (p<0.05). Extract of Sala stem bark with dose of 500 and mg/kgbw could inhibit the increasing uric acid levels equivalent to Allopurinol (p>0,05). Keywords: Cynometra ramiflora, stem bark, gout, hyperuricemia Volume 7, No.2, Desember

2 EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK(Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED BY POTASSIUM OXONATE Efek Ekstrak Etanol Kulit Batang Sala (Cynometra ramiflora Linn.) TerhadapPenghambatan Peningkatan Kadar Asam Urat Mencit Jantan Galur Swiss Yang INTRODUCTION Uric acid is the end product of the degradation of purines (Shamley, 2005). High levels of uric acid in the blood (> 7 g/dl) cause great pain, consequently the quality of life of the patient decreased. Patients with hyperuricemia at the age above 40 years can be quite serious because it can cause gout arthritis (Roddy and Doherty, 2010). In Indonesia, gout arthritis is the first number of a arthritis type, followed by osteoarthritis (Utomo, 2005). In developed and developing countries, the number of patients with hyperuricemia increasing from year to year. As in the United States the number of gout patients in 2002 increased from 2 million to more than 5 million people in 2006 (Yu, 2006). In the UK, prevalence of gout increased from 208 of 1000 people in 2005 to 213 of 1000 people in 2007 (Elliot et al., 2009). Treatment of uric acid can be carried out by inhibiting the synthesis of xanthine oxydase, as a result is uric acid levels can be decreased. Synthetic drugs to lower uric acid levels in the blood, has been widely used. However, these drugs can cause a variety of side effects, for example Allopurinol side effects are skin allergies, stomach and intestinal disturbances, headache, dizziness and hair loss (Tjay and Rahardja, 2002). In consequences research on medicinal plants have grow. In the past, traditional medicine only based on empirical data and or heritage. Nowadays, lots of research conducted to prove the efficacy of plants, including efficacy as antihiperurisemia. Previous result stated that Sida rhombifolia (Iswantini and Darusman, 2003), celery (Rhamadani, 2004) and ginger plants (Mudrikah, 2006) have antihyperuricemia activity. Chemical compounds that responsible to reduce uric acid levels in blood are flavonoids (Cos et al., 1998), terpenoids and quercetin (Mudrikah, 2006). One of the heritage from Solo Palace is Cynometra ramiflora Linn plant, traditionally used as medicine in communities to cure diseases such as hypertension, hyperuricemia, and hypercholesterolemia. However, the use of that plants only based on empirical data (Abdullah et al., 2010). Aziz and Iqbal (2013) stated that these plants have several compounds such as tannins, saponins, flavonoids and terpenoids.these research conducted to found the biological activity of Sala plants as antihyperuricemia. MATERIALS AND METHOD Preliminary test Six mice were measured uric acid levels as baseline. After that they were given chicken liver juice 10% for 2 days three times a day. Then 6 mice were weighed and divided into 2 groups, namely: a. Control CMC Na o,5% : Given CMC Na orally 0.5 ml/20kgbw, 1 hour later were given 0.5% CMC Na intraperitoneally 0.5 ml/20kgbw. b. Control hyperuricemia: Given dis tilled water orally 0.5 ml/20gbw, 1 hour later was induced by potassium oxonate 250 mg/kgbw intraperitoneally 0.5 ml/20gbw. Blood sampling was done at a time when levels of uric acid in the blood showed the highest levels at 2 hours after induction of potassium oxonate (Nguyen et al., 2005). The main test Twenty five mice were measured uric acid levels as baseline. Then mice were given chicken liver juice for 2 days, animal test divided into 5 groups (each group consist of 5 mice): Group I : given allopurinol 10 mg/kgbw by potassium oxonate 250 mg/kg (0.5 ml/20gbw) intraperitoneally (positive control) Volume 7, No.2, Desember

3 Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi Group II : given CMC-Na 0.5 ml/20gbw by potassium oxonate 250 mg/kg (0.5 ml/20gbw) intraperitoneally (negative control) Group III : given the ethanolic extract of Sala plant stem bark dose of 250 mg/kg by potassium oxonate 250 mg/kg (0.5 ml/20gbw) intraperitoneally Group IV : given the ethanolic extract of Sala plant stem bark dose of 500 mg/kg by potassium oxonate 250 mg/kg (0.5 ml/20gbw) intraperitoneally Group V : given the ethanolic extract of Sala plant stem bark dose of 1000 mg/kg by potassium oxonate 250 mg/kg (0.5 ml/20gbw) intraperitoneally Two hours after induced by potassium oxonate, aproximately 0,5 ml blood was drawn through the orbitalis vein using capillary tube. Blood was sentrifused at a speed of 5000 rpm for 20 minutes and the supernatant was taken as serum. Determination of uric acid levels Uric acid levels are determined by an enzymatic reaction using uric acid reagents kit FS*TBHBA. Serum was mixed homogeneously with the uric acid reagent FS*TBHBA, then incubated for 10 min at 37 C. Solution of the sample, standard and blank were read using a spectrophotometer visible at a wavelength of 546 nm. Data analysis The data were analysed by Shapiro-Wilk test, and the homogenity were performed by Levene test. The data obtained were not homogeneous and not normal, to normalize the data then transformed using the formula (uric acid levels after treatment)/(initial uric acid levels) x 100%), after that the data proceed with one way ANOVA test with degree of freedom level at 95%) and followed by Bonferroni. RESULTS AND DISCUSSION Obviously, hyperuricemia condition of animal could reached by induction intraperitonially of potassium oxonate. However, based on the previous study the uric acid levels of animal induced by oxoante only less than 3.0 mg/dl which still normal value. Design of research was modified as research conducted by Purwantinigsih et al (2010), which using high purine diet. As the previous research, the levels of uric acid was more than normal value. Preliminary study was done to get the model, and to determine the potential potassium oxonate in increasing uric acid levels in test animals. Potassium oxonate is an uricase inhibitor enzym. Uricase enzyme in mice will convert uric acid to allantoin that is water soluble. In hyperuricemia test, male mice induced orally with distilled water, one hour later induced by potassium oxonate dose of 250 mg/kg intraperitoneally. Two hours after the administration of potassium oxonate, blood was taken. The reason using interval 2 hours because the level of uric acid in the blood showed the highest yield (Nguyen et al., 2005). The level of uric acid was measured by using reagents TBHBA. The mechanism of reaction described in figure 1, that involve enzyme (Schunack et al., 1990; Jacobs et al., 1990; Foster et al., 2011) Figure 1. Mechanism of formation quinoneimina (Schunack et al., 1990) Volume 7, No.2, Desember

4 EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK(Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED BY POTASSIUM OXONATE Efek Ekstrak Etanol Kulit Batang Sala (Cynometra ramiflora Linn.) TerhadapPenghambatan Peningkatan Kadar Asam Urat Mencit Jantan Galur Swiss Yang In this research, the biological variation must be controlled to eliminate the bias of data. Controlled variable in this study were gender, age, body weight, food and beverages provided. Male mice was chosen as animal model because they did not have the estrogen hormone which will effect on increasing the excretion of uric acid (Johnson and Rideout, 2004). The results of preliminary test (Table 1.) showed that potassium oxonate at dose 250 mg/kg was able to increase the levels of uric acid levels in the serum of mice. The dose of potassium oxonate as described by Ishibuchi (2001) and Osada (1993). Based on statistical tests of the results showed that potassium oxonate can increase uric acid levels 2.5 times compared to CMC-Na control (P<0,05). Evaluation of antihyperuricemia effect of C.ramniflora stem bark extracts were compared with Allopurinol as positive control. Allopurinol is a antihyperuricemia drug that works by compete with purine to interact with the enzyme xanthine oxidase, as a result, the uric acid levels decreased (Tjay and Rahardja, 2007). The ethanolic extracts of Sala stem bark suspended with CM-Na 0,5%, the solutions always freshly made due to experience that solutions which stored for several days affected to the biological activity. The data from main tests showed that of uric acid levels on the groups of Sala bark extracts, positive control and negative control were varied (Table 2). The ethanolic extracts of Sala stem bark doses of 250; 500 and mg/kg could inhibit the uric acid levels compared with negative control. Table 1. Uric Acid Levels (mg/dl) of group of Hyperuricemia and CMC-Na Treatment Animal test baseline after treatment Potassium oxonate 250 mg/kg 1 1,1 3,8 2 1,0 4,0 3 1,2 5,0 x ±SD 1,1 ± 0,1 4,4 ± 0,6 1 1,3 1,8 CMC Na 0,5% 2 1,4 1,4 3 1,5 1,8 x ±SD 1,4 ± 0,1 1,7 ± 0,2 Groups Table 2. Uric Acid Levels of Baseline and After Treatment (n=5) Uric acid levels baseline after treatment (mg/dl) (mg/dl) Positive control 0,92 ± 0,41 0,74 ± 0,21 (allopurinol 10 mg/kgbw) Negative control given CMC 0,7 ± 0,16 3,22 ± 0,15 Na 0.5% Sala plant stem bark extract 0,38 ± 0,11 1,72 ± 0, mg/kg BW Sala plant stem bark extract 0,88 ± 0,19 1,46 ± 0, mg/kg BW Sala plant stem bark extract 1,24 ± 0,18 0,56 ± 0, mg/kg BW Volume 7, No.2, Desember

5 Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi The data were analysed by one way ANOVA to determine the significances of ability of the extract to inhibit uric acid levels compared to groups. The distribution of data showed normal but not homogeneous, due to the value of the normality of (p> 0.05) and homogeneity value of (p <0.05). Consequently, the data could not tested by one way ANOVA (Trihendradi, 2012). as the result its has potential to decrease uric acid levels by inhibiting the oxidation of xanthine (Cos et al., 1998 and Mo et al., 2007). Previous research of Haryoto et al. (2013) isolated apigenin from C. ramiflora, Cos et al. (1998) stated that apigenin could inhibits xanthine oxidase (XO) activity with a similar values with allopurinol. Table 3. Post hoc test of Uric Acid Levels after Treatment to Baseline Group ,000* 1,000 0,000* 0,000* 2 0,000* 1,000 1, ,000* 0,000* 4 0,328 5 Description: Group 1: Negative control (0.5% CMC Na) Group 2: Positive control (allopurinol 10 mg/kg) Group 3: Ethanolic extract of Sala stem bark dose of 250 mg/kg Group 4: Ethanolic extract of Sala stem bark dose of 500 mg/kg Group 5: Ethanolic extract of Sala stem bark dose of 1000 mg/kg Owing to get normal data, its transformed into a percentage of uric acid levels. The results of this statistical test were the data distribute normally and homogenous. One way ANOVA followed by post hoc statistical analysis showed that differences of groups extract with dose of 500 mg/kg and mg/kgbw compared to positive control was not significant (Table 3). It can be concluded that the extract of the Sala stem bark has equivalent potential on inhibiting hyperurecemia to Allopurinol. The antihyperuricemia activity of the sample might be based on the flavonoids compounds. Mo et al. (2007) and Mudrikah (2006) stated that flavonoids such as morin, kaemferol, apigenin, puerarin, quercetin and myrsetin could decreasing uric acid levels by inhibition the xanthine oxidase enzym. The others active compounds are terpenoids (Mudrikah, 2006). In the other hand, flavonoids which is phenolic compounds are powerful antioxidants CONCLUSION The ethanolic extract of Sala plant stem bark (Cynometra ramiflora Linn.) dose of 250; 500 and 1000 mg/kg BW could inhibit the increase uric acid levels in the Swiss male mice induced by potassium oxonate. The ethanolic extract of Sala plant stem bark (Cynometra ramiflora Linn.) dose of 500 and 1000 mg/kg BW have equivalent effect to Allopurinol. REFFERENCES Abdullah M., Dewi M., dan Talitha W Inventarisasi Jenis-Jenis Tumbuhan Berkhasiat Obat di Hutan Hujan Dataran Rendah Desa Nyamplung Pulau Karimunjawa. Biosaintifika, 2(2): Aziz AFA. dan Iqbal M. 2013, Antioxidant Activity and Phytochemical Composition of Cynometra cauliflora. J Exp Integr Med., 3(4): Cos P., Ying L., Calomme M., Cimanga JP., Van Poel K., Pieters BL., Vlietinck AJ. dan Berghe DV Structure-Activity Relationship and Classification of Flavonoid as Volume 7, No.2, Desember

6 EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK(Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED BY POTASSIUM OXONATE Efek Ekstrak Etanol Kulit Batang Sala (Cynometra ramiflora Linn.) TerhadapPenghambatan Peningkatan Kadar Asam Urat Mencit Jantan Galur Swiss Yang Inhibitors of Xanthine Oxidase and Superoxide Scavengers. J Nat. Prod, 61: 71-7 Elliot AJ., Cross KW., Fleming DM Sea sonality and Trends in The Incidence and Prevalence of Gout in England And Wales Ann Rheum Dis, 68: Foster A., Barnes N., Speight R., Keane MA Identification, Functional Expression and Kinetic Analysis of Two Primary Amine Oxidases from Rhodococcus Opacus. Journal of Molecular Catalysis B: Enzymatic, 74: Haryoto,Suhendi,A.,Sujono, T. A.,& Yudha, P. I,. 2013, Pengembangan Potensi Herbal Medicine Dari Ekstrak Tumbuhan Sala (Cynometra ramiflora Linn.) Menjadi Obat Jerbal Terstandart: Uji Farmakologi, Toksisitas, dan Penyelidikan Kimia. Laporan Penelitian. PINPRU Ishibuchi S., Morimoto H., Oe T., Ikebe T.,Inoue H., Fukunari A., Kamezawa M., Yamada T., Naka Y Synthesis and Structure Activity Relationships of 1Phenylpyrazoles as Xanthine Oxidase Inhibitors, Bioorg. Med. Chem. Lett., 11: Iswantini D. dan Darusman LK. 2003,Effect of Sidaguri as an uric acid lowering agent on the activity of oxidase enzim. Proceeding of International Symposium on Biomedicine, 18-19th 2003, Biopharmacia Research Center, Bogor Agriculture University. Johnson RJ. and Rideout BA Uric acid and Diet-Insights Into the Epidemic of Cardiovascular Disease, NEJM 350(11) Mudrikah F Potensi Ekstrak Jahe Merah (Zingiber officinale Rosc.) dan Herba Suruhan Sebagai Antihiperurisemia Pada Tikus. Skripsi, Bogor: Institut Pertanian Bogor Murray RK., Granner DK. dan Rodwell VW Biokimia Harper. edisi 27. Jakarta : EGC Mo S., Zhou FLv., Hu YZ., Zhang QH., Kong DM Hypouricemic Actio of Selected Flavonoids in Mice : Structure-Activity Relationship, China : Nanjing University Nguyen MTT., Awale S., Tezuka Y., Shi L., Zaidi SFH., Ueda J., et al Hypouricemic Effects of Acacetin and 4,5-O-Dicaffeoylquinic Acid Methyl Ester on Serum Uric Acid Levels In Potassium Oxonate Pretreated Rats, Biol. Pharm. Bull., 28(12): Osada Y., Tsuchimoto M., Fukushima H., Taka hashi K., Kondo S., Hasegawa M., Komoriya K. Hypouricemic Effect of The Novel Xanthine Oxidase Inhibitor, TEI- 6720, in rodents, Eur. J. Pharmacol., 241: Purwatiningsih, Hakim, A. R., & Purwantini, I., Antihyperuricemic Activity Of The Kepel [Stelechocarpus burahol (Bl.) Hook. F. & Th.] Leaves Extract and Xanthine Oxidase Inhibitory Study, Int. J. Pharm. Pharm. Sci. Vol 2, 2, Rhamadani T. 2004, Isolasi dan Identifikasi Senyawa Bioaktif Seledri Dalam Menghambat Aktivitas Enzim XO, Skripsi, Bogor: Institut Pertanian Bogor Roddy E. dan Doherty M Epidemiology of Gout, Arthritis Research and Therapy, 12: 223 Schunack W., Mayer K. and Manfred H Senyawa Obat Kimia Farmasi, Yogyakarta: Gajah Mada University Press Shamley D Pathophysiology an Essential Text for the Allied Health Professions, USA: Elsevier Limited Tjay HT. dan Rahardja K Obat-Obat Penting, edisi 6, Jakarta: PT Elex Media Komputindo Trihendradi C Step by Step SPSS 20 Analisis Data Statistik, Yogyakarta : Andi Utomo P Apresiasi Penyakit Pengobatan Secara Tradisional dan Modern, Volume 7, No.2, Desember

7 Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi Jakarta: Asdi Mahasatya Yu KH Febuxostat : A Novel Nonpurinae Selective Inhibitor of Xanthin Oxidase for The Treatment of Hyperuricemia in Pirai, Inflamation and Alergy Drugs Discovery 1(1). Volume 7, No.2, Desember