MATERIALS AND METHODS

Plant materials: Sida rhombifolia L. plants were collected from its natural habitat in Bogor, East Java, Indonesia.

Preparation of flavonoid crude extract. The herb (stem and leaf) was cleaned, sun-dried, ground into powder and extracted using modified Markham method (Markham, 1988). Approximately 200 g sample was treated with hexane to remove the lipid constituent and macerated using methanol: water ( MeOH:H₂O 9:1 and 1:1) mixtures until the residue was free of flavonoids and then evaporated at 35°C. The extract was freeze-dried and tested qualitatively for flavonoids (Harborne, 1987). The toxicity assay was performed on Artemia salina (Finney, 1971).

The inhibitory assay on XO: The optimum conditions for the assay were obtained from earlier study by Tamta et al. (2005) with modifications. We measured the inhibitory effect of the crude extract in concentrations ranging from 100 to 800 mg L^-1, while for the fractioned flavonoids we employed concentrations based on the toxicity test results. Into each extract, potassium phosphate buffer 50 mM pH 7.5 was added until the volume reached 1.9 mL. One milliliter xanthine 2.1 mM and 0.1 mL XO of 0.1 unit mL^-1 were added. The solution was incubated for 45 min at 20°C and then 1 mL HCl 0.58 M was added to terminate the reaction. The absorbance of the mixed solution was measured at 262 nm.

The kinetics inhibition assay on XO: The assay was conducted on the first concentration of the flavonoids crude extract to exhibit an inhibition effect greater than 50%. Lineweaver-Burk plot analysis was performed to determine the mode of inhibition by the active compounds. This kinetics study was carried out in the absence and presence of active compounds with varying concentrations of xanthine as the substrate.

Fractionation of flavonoid crude extract: Fractionations were performed using column chromatography with silica gel column G-60 as solid adsorbent and the best eluent (CHCl₃:MeOH), in a gradient of polarity level. Flavonoids crude extract (4.609 g) were fractionated twice, consisted of 1.000 g on 2.4x33 cm column (first column) and 3.608 g on 3.0x76.5 cm (second column) with a constant flow rate of 1-1.54 mL min^-1. Eluates from fractionations were collected for every 5 mL and the eluates with similar Rf and chromatogram pattern were combined and viewed using analytical TLC (Thin Layer Chromatography). The fractions were freeze-dried, the yields were measured and the cytotoxicity was tested on Artemia salina. The cytotoxicity values were subsequently used to determine the maximum concentration limited tolerance in the inhibitory effect assay on XO.

Compound identification of active fraction: Identification was performed on the fraction showing the highest inhibition power. All fractions were identified using a Fourier transformed infrared (FTIR) spectrometer instrument. To identify the purity of the fraction of crude extract including flavonoid and component fragmentation pattern, we conducted GC-MS analysis using an Agilent Technologies 6890 Gas Chromatograph with Auto Sampler and 5973 Mass Selective Detector and Chemstation data system with 17 mx0.25 mm i.d. HP Ultra 2 with 0.25 μm film thickness. Initial oven temperature was held at 70°C for 1 min, then increased at 5°C min^-1 to 150°C, held for 4 min, then increased at 10°C min^-1 to 260°C and held for 35 min, with helium carrier gas and constant column flow (0.9 μL min^-1), injection volume 5 μL and split 10:1.

RESULTS AND DISCUSSION

The potency of flavonoids crude extract against gout could be determined by examining the inhibition effect of the extract on the XO. The dried sample yielded 11.85% crude extract and LC₅₀ of 501.23 mg L^-1 using probit analysis. Furthermore, assay of inhibitory effect of the crude extract have been performed with concentration less than its LC₅₀ value.

Table 1 shows the inhibitory effect increasing with increasing extract concentration, except for a decrease at 300 mg L^-1. The decreasing of inhibitory effect at that concentration may be caused by various components in the sample extracted by methanol-water. Compounds extractable by methanol-water mixtures are carbohydrates, terpenoids and to a small extent alkaloids (Harborne, 1987), including NADH which acts as a XO activator (Millar et al., 2002).

Table 1:	Inhibitory effect of flavonoid crude extract of Sida rhombifolia at various concentrations

Fig. 1:	Lineweaver-Burk plot of XO inhibition with various concentrations of xanthine, addition of 200 ppm of flavonoids extract of Sida rhombifolia L., no extract and Linewaver-Burk transformed data were plotted and followed by linear regression of the points

Noro et al. (1983) reported that an extract can be considered as useful for gout treatment if its inhibitory effect on XO is more than 50%. Present findings indicate that concentrations above 200 ppm have inhibition of more than 50%, suggesting that the flavonoids crude extract has potential as a treatment against gout.

The inhibition kinetic assay was conducted only for 200 mg L^-1 of the flavonoid crude extract. At higher concentrations, various compounds in the extract may interfere with the enzymatic reaction. To understand the mode of the enzyme inhibition, the Lineweaver-Burk plot was established (Fig. 1).

The extract caused a change of kinetic parameters. Michaelis-Menten constant of K_M increased from 0.87 to 2.02 mM, Vmax decreased from 0.01892 mM min^-1 to 0.01893 mM min^-1. ANOVA analysis on Vmax values showed a non-significant value (p<0.01). Based on this data, the inhibition of XO was in a competitive mode. The inhibitor affinity value (α) on XO was calculated by ratio of the K_M value of the reaction containing extract/inhibitor with the K_M value of the reaction without extract/inhibitor. The resulting α was 2.32, which is better than pyrazolopirimidin (α = 2.10; Tamta et al., 2005), but less than quercetin from Allium cepa (α = 2.92; Hanaee et al., 2004). This data showed that the competitive inhibition is strong (α>2). The competitive inhibition kinetic pattern from this research supports former research (Lin et al., 2000, 2002), suggesting that flavonoids may be competitive inhibitors of XO.

Fig. 2:	Potent inhibitory effect of several purified fractions on the activity of xanthine oxidase

Table 2:	Profile of chromatography column and yield of fractions

An active compound from wheat leaf (4-aminopyrazolo[3,4-d]pyrimidine) also exhibited the competitive inhibition of XO (Hsieh et al., 2007).

Fractionation of flavonoids crude extract was done in gradient mixture of eluents to obtain good separation pattern. The best eluent was CHCl₃:MeOH 9:1. Fractionation of 4.6086 g crude extract gave the highest yield on the 11th fraction (30.18%), whereas the fraction with the lowest yield was the 4th fraction (0.41%) (Table 2).

Each fraction from the purification step has a different inhibitory effect (Fig. 2). Among the 11 fractions, fraction 4 showed the strongest inhibitory effect at concentration assay 50 mg L^-1. And the inhibitory effect of fraction 4 at 50 mg L^-1 was stronger than the highest tested concentrations of the six other fractions that were tested at concentrations higher than 50 mg L^-1. Based on this, we assume that the compounds in fraction 4 have potential as gout medicine. If we compare with allopurinol at 300 mg L^-1 (inhibitory effect 68.07%, data not shown), fraction 4 has better inhibitory effect. However, compared with the crude extract in the same concentration, allopurinol inhibitory effect is 1.23 times stronger (Table 1). Based on these facts, fraction 4 and the crude extract are potential as antigout/gout medicine.

Fig. 3:	The inhibitory effect of crude extract and its fractions on the activity of xanthine oxidase. CE: Crude extract; F: Fraction

The increase of concentration did not always show with a linear increase in the inhibitory effect. This is indicated by fraction 5. When the concentration reached 450 mg L^-1, the inhibitory effect increased to 71.7% but when the concentration was increased to 2500 mg L^-1 the inhibitory effect was only 69.2%.

Figure 3 shows the potent inhibitory effect of flavonoid crude extract compared with fractions at the concentrations 50 and 100 mg L^-1. It shows that fractionation generally had little effect on increasing the inhibition power. The purified fraction 3 at the concentration 100 mg L^-1only showed a slight increase of 8.11% in the inhibitory effect (51.58%) as compared with the crude extract at the same concentration (47.71%). Generally, the potent inhibitory effect of the purified fraction at 50 mg L^-1 was similar or much lower than the potent inhibitory effect of crude extract of 100 mg L^-1 (Fig. 3). Fraction 4 was the exception, showing a stronger potent inhibitory effect (79.1%) compared to that of the flavonoids crude extract (47.7%).

Since, fraction 4 was the most active fraction in term of inhibitory effect we conducted further analysis to characterize its compounds. We used FTIR to reveal the functional groups contained in fraction 4. To estimate the degree of purification of this fraction, we analyzed this fraction with GC-MS.

The FTIR analysis showed some absorbencies with peaks at 3437.50, 2928.28, 2361.30, 1654.82, 1461.79, 1377.42, 1172.50 and 722.19 cm^-1 (Table 3). Wide peak at 3437.50 cm^-1 showed an absorbance caused by -OH stretching from bounded phenol.

GC-MS analysis from fraction 4 showed that there were 39 organic compounds and flavonoid fragments with retention times of 4.14, 6.53 and 6.74 that have resemble fragmentation of benzoic acid compounds based on instrument database (chromatograms not shown).

Table 3:	The IR absorbance patterns and functional group estimation of fraction 4
*Pavia et al. (1996)

These results indicated that fraction 4 was not completely pure. The phytochemical assay was determined on fraction 4. Result indicated that flavonoids are the class of compound identified in this particular fraction. This result is also supported by earlier studies that several flavonoids such as apigenin and isovitexin, quercetin, myricetin and genistein can inhibit XO and exhibit competitive inhibition (Lin et al., 2002). Other research also reported that the size and geometry of the flavonoids are important factors in the positioning of the molecule in order to block the access of the physiologic substrate (hypoxanthine) to the catalytic center of XO. Flavonoids will be good as XO inhibitor when the size is small and the hydrophobic character high (Silva et al., 1996). We did not find research related to identification of flavonoids from S. rhombifolia or the genus Sida. But some flavonoids have been found in some plants belong to the family of Malvaceae, such as hibiscin, hibiscitrin, hibiscetin, gossypitrin, sabdaritrin, gossypetin and quercetin from Hibiscus sabdariffa, luteolin and quercetin from Kitaibelia vitifolia, herbacetin, gossypetin and isoscutellarein from Malope trifida. These or other flavonoids may also be found in S. rhombifolia.