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 Kada

  

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

Diinduksi Potasium Oksonat

  

Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi

Faculty of Pharmacy , Universitas Muhammadiyah Surakarta

Jl.Ahmad Yani Tromol Pos I, Pabelan Kartasura Surakarta 57102

  

E-mail : 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 pengham-

batan 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 rancan-

gan post-test dengan kontrol. Dua puluh lima mencit jantan berusia 2-3 bulan dengan berat badan 20-40 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 menunjuk-

kan 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 pening-

katan 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.

_{of uric acid levels in Swiss male mice induced by potassium oxonate intraperitoneally. This study used post-test}

The aim of this study was to evaluate the effect of ethanolic extract of stem bark Sala plant on the inhibitory effect

with control design, Twenty five mice 2-3 month of age, weight 20-40 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

_{respectively. All treatment were administered orally. The mice induced hyperurecemia by additional food of the}

  

III-V were treated by extracts of Sala stem bark with dose of 250mg/kgBW, 500mg/kgBW, and 1000mg/kgBW,

chicken liver juice 10% and potassium oxonate intraperitoneally. Test results were analyzed by Analysis of vari-

ance (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 1.000 mg/kgBW could inhibit the increasing uric acid

_Keywords: levels equivalent to Allopurinol (p>0,05).

  Cynometra ramiflora, stem bark, gout, hyperuricemia

BY POTASSIUM OXONATE

  CMC Na orally 0.5 mL/20kgBW, 1 hour later were given 0.5% CMC Na intraperitoneally 0.5 mL/20kgBW.

  

Efek Ekstrak Etanol Kulit Batang Sala (Cynometra ramiflora Linn.) TerhadapPenghambatan Peningkatan Kadar Asam Urat Mencit Jantan Galur Swiss Yang

_{Diinduksi Potasium Oksonat}

  

EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK(Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED

  Twenty five mice were measured uric acid levels as baseline. Then mice were given chicken liver juice for 2 days, animal test di- vided into 5 groups (each group consist of 5 mice): Group I : given allopurinol 10 mg/kgBW orally, 1 hour later they were induced by potassium oxonate 250 mg/kg (0.5 mL/20gBW) intraperitoneally (posi- tive control)

  The main test

  ., 2005).

  b. Control hyperuricemia: Given dis tilled water orally 0.5 mL/20gBW, 1 hour later was induced by potassium oxonate 250 mg/kgBW intraperito- neally 0.5 mL/20gBW. Blood sampling was done at a time when lev- els of uric acid in the blood showed the highest levels at 2 hours after induction of potassium oxonate (Nguyen et al

  INTRODUCTION

  Uric acid is the end product of the deg- radation of purines (Shamley, 2005). High lev- els of uric acid in the blood (> 7 g/dL) cause great pain, consequently the quality of life of the patient decreased. Patients with hyperuri- cemia 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 num- ber 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

  Preliminary test

  ., 2010). Aziz and Iqbal (2013) stat- ed that these plants have several compounds such as tannins, saponins, flavonoids and ter- penoids.These research conducted to found the biological activity of Sala plants as antihy- peruricemia.

  used as medicine in communities to cure dis- eases such as hypertension, hyperuricemia, and hypercholesterolemia. However, the use of that plants only based on empirical data (Ab- dullah et al

  Cynometra ramiflora Linn plant, traditionally

  One of the heritage from Solo Palace is

MATERIALS AND METHOD

  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 al- headache, dizziness and hair loss (Tjay and Rahardja, 2002). In consequences research on medicinal plants have grow. In the past, tradi- tional medicine only based on empirical data and or heritage. Nowadays, lots of research conducted to prove the efficacy of plants, in- cluding efficacy as antihiperurisemia. Previ- ous result stated that Sida rhombifolia (Iswan- tini 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).

  al ., 2009).

  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

RESULTS AND DISCUSSION

  Group II : given CMC-Na 0.5 mL/20gBW orally, 1 hour later they were induced by potassium oxonate 250 mg/kg (0.5 mL/20gBW) intraperitoneally (nega- tive control)

  Group III : given the ethanolic extract of Sala plant stem bark dose of 250 mg/kg orally, 1 hour later they were induced 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 orally, 1 hour later they were induced 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 orally, 1 hour later they were induced 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

  enzymatic reaction using uric acid reagents kit FS*TBHBA. Serum was mixed homogeneous- ly 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 homo- geneous 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.

  Obviously, hyperuricemia condition of animal could reached by induction intraperi- tonially 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 re- search was modified as research conducted by Purwantinigsih et al (2010), which using high purine diet. As the previous research, the lev- els of uric acid was more than normal value.

  Preliminary study was done to get the model, and to determine the potential potas- sium 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 sol- uble. In hyperuricemia test, male mice induced orally with distilled water, one hour later in- duced by potassium oxonate dose of 250 mg/kg intraperitoneally. Two hours after the adminis- tration of potassium oxonate, blood was taken. The reason using interval 2 hours because the level of uric acid in the blood showed the high- est 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) Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi

  

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

_{Diinduksi Potasium Oksonat}

  In this research, the biological variation Evaluation of antihyperuricemia ef- must be controlled to eliminate the bias of data. fect of

  C.ramniflora stem bark extracts were compared with Allopurinol as positive control.

  Controlled variable in this study were gender, age, body weight, food and beverages provid- Allopurinol is a antihyperuricemia drug that ed. Male mice was chosen as animal model be- works by compete with purine to interact with cause they did not have the estrogen hormone the enzyme xanthine oxidase, as a result, the which will effect on increasing the excretion of uric acid levels decreased (Tjay and Rahardja, uric acid (Johnson and Rideout, 2004). 2007). The ethanolic extracts of Sala stem bark

  The results of preliminary test (Table 1.) suspended with CM-Na 0,5%, the solutions al- ways freshly made due to experience that solu- showed that potassium oxonate at dose 250 mg/kg was able to increase the levels of uric tions which stored for several days affected to acid levels in the serum of mice. The dose of the biological activity. potassium oxonate as described by Ishibuchi The data from main tests showed that of uric acid levels on the groups of Sala bark (2001) and Osada (1993). Based on statisti- cal tests of the results showed that potassium extracts, positive control and negative control were varied (Table 2). The ethanolic extractS oxonate can increase uric acid levels 2.5 times compared to CMC-Na control (P<0,05). of Sala stem bark doses of 250; 500 and 1.000 mg/kg could inhibit the uric acid levels com- pared with negative control. _{Treatment Animal test baseline after treatment}

Table 1. Uric Acid Levels (mg/dL) of group of Hyperuricemia and CMC-Na

Potassium oxonate 250 mg/kg

  1 1,1 3,8 2 1,0 4,0 3 1,2 5,0 x̄±SD 1,1 ± 0,1 4,4 ± 0,6 1 1,3 1,8 3 1,5 1,8 _{1,4 ± 0,1 1,7 ± 0,2} x̄±SD

  

Table 2. Uric Acid Levels of Baseline and After Treatment (n=5)

Groups _{baseline after treatment} Uric acid levels (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 ± 0,51 250 mg/kg BW Sala plant stem bark extract 0,88 ± 0,19 1,46 ± 0,11 500 mg/kg BW Sala plant stem bark extract 1,24 ± 0,18 0,56 ± 0,39 1000 mg/kg BW

  Haryoto, Tanti Azizah, Zedny Norachuriya, Andi Suhendi, Muhtadi

  The data were analysed by one way as the result its has potential to decrease uric acid levels by inhibiting the oxidation of xan- ANOVA to determine the significances of abil- ity of the extract to inhibit uric acid levels thine (Cos et al., 1998 and Mo et al., 2007). Pre- compared to groups. The distribution of data vious research of Haryoto et al

  . (2013) isolated showed normal but not homogeneous, due to apigenin from

  C. ramiflora, Cos et al. (1998)

  stated that apigenin could inhibits xanthine the value of the normality of 0.113 (p> 0.05) oxidase (XO) activity with a similar values with and homogeneity value of 0.009 (p <0.05). allopurinol. Consequently, the data could not tested by one way ANOVA (Trihendradi, 2012).

  

Table 3. Post hoc test of Uric Acid Levels after Treatment to Baseline

Group

  1

   2

  

3

   4

   5 1 0,000* 1,000 0,000* 0,000*

  2 0,000* 1,000 1,000

  3 0,000* 0,000*

  4 0,328 _Description:

5 CONCLUSION

  Group 1: Negative control (0.5% CMC Na)

  The ethanolic extract of Sala plant stem

  Group 2: Positive control (allopurinol 10 mg/kg)

  bark (

  Group 3: Ethanolic extract of Sala stem bark dose of 250 mg/kg Cynometra ramiflora Linn.) dose of 250;

  Group 4: Ethanolic extract of Sala stem bark dose of 500 mg/kg

  500 and 1000 mg/kg BW could inhibit the in- crease uric acid levels in the Swiss male mice

  Group 5: Ethanolic extract of Sala stem bark dose of 1000 mg/kg induced by potassium oxonate.

  The ethanolic extract of Sala plant stem Owing to get normal data, its trans- bark (

  Cynometra ramiflora Linn.) dose of 500 formed into a percentage of uric acid levels.

  and 1000 mg/kg BW have equivalent effect to The results of this statistical test were the data Allopurinol. distribute normally and homogenous. One way ANOVA followed by post hoc statistical analysis

  REFFERENCES

  showed that differences of groups extract with dose of 500 mg/kg and 1.000 mg/kgBW com- Abdullah M., Dewi M., dan Talitha W. 2010. pared to positive control was not significant

  Inventarisasi Jenis-Jenis Tumbuhan (Table 3). It can be concluded that the extract

  Berkhasiat Obat di Hutan Hujan Dat- of the Sala stem bark has equivalent potential aran Rendah Desa Nyamplung Pulau on inhibiting hyperurecemia to Allopurinol.

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  Cynometra cauliflora. J Exp Integr

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EFFECTS OF ETHANOLIC EXTRACT OF SALA STEM BARK(Cynometra ramiflora Linn.) ON INHIBITING HYPERURICEMIA IN SWISS MALE MICE INDUCED

  

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