Indonesian Sidaguri (Sida rhombifolia L.) as Antigout and Inhibition Kinetics of Flavonoids Crude Extract on the Activity of Xanthine Oxidase
Sida rhombifolia L., or Sidaguri in Indonesian, is a traditional medicinal plant with potential as a remedy against gout. The earlier research reported that flavonoids crude extract from this plant could in vitro inhibit the activity of Xanthine Oxidase (XO) (xanthine: oxygen oxidoreductase EC 126.96.36.199) up to 55% and could be antigout. It is a need to investigate type of inhibition kinetic of Sida rhombifolia L.s extract and the effects of fractionation on the inhibitory effect by in vitro method. The results showed that the flavonoids crude extract yielded approximately 12% with LC50 of 501 mg L-1 and its inhibitory effect from 48 to 71% (100-800 mg L-1). The kinetic study resulted that the type of flavonoids crude extract inhibition was a competitive inhibition with inhibitor affinity (α) of 2.32 and p<0.01. The fractionation yielded 11 fractions and the fraction 4 has highest activity, i.e., 79%. The fractions did not increase the inhibition effect significantly. GC-MS analysis of 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 compound. By phytochemical assay, fraction 4 contain flavonoid.
Gout results from overproduction or underexcretion of uric acid and is associated
with nucleic acid rich diets and the catalysis of xanthine by Xanthine Oxidase
(XO) which produces uric acid. Allopurinol, an important treatment for gout
in modern medicine (Connor, 2009), acts as a substrate
for and competitive inhibitor of XO and as a noncompetitive inhibitor at higher
concentrations (Hardman and Limbird, 1975). XO inhibitors
have been found in a wide variety of plants used in traditional medicines for
the treatment of gout and rheumatism from South America (Theoduloz
et al., 1991; Hayashi et al., 1989;
González et al., 1995) and northeastern
North America (Owen and Johns, 1999). Sida rhombifolia
L., known as Sidaguri in Indonesian, is used in Indonesian herbal
medicine for treatment of gout with proven uric acid prohibitive action (Dharma,
1985) and anti-inflammatory action (Soedibyo, 1998).
Several natural compounds such as flavonoids have been reported as inhibitors
of XO and this class of compounds has promise for greater use in gout treatment.
Allopurinol, theaflavin, theaflavin-3-gallic theaflavin-3-3-digallic(-)-epigallocatechin-3-gallic
and gallic acid have been reported as inhibitors for XO through the competitive
inhibitor mechanism (ODriscoll et al., 1999;
Lin et al., 2000), while flavonol crisin, luteolin,
caempherol, kuercetin, myricetin and isorhamnetin, as well as (-)-epicatechin,
(-)-epigallocatechin and (-)-epicatechin gallic from tea are reported to inhibit
XO by the noncompetitive inhibitor mechanism (Aucamp et
al., 1997). The Materia Medica Indonesia (Departemen
Kesehatan Indonesia, 1995) records the presence of the alkaloid rhombiffolina
in Sida rhombifolia L. which could inhibit activity of the XO. The flavonoids
crude extract of Sida rhombifolia L. was shown to inhibit XO by up to
55% and to lower uric acid (Iswantini and Darusman, 2003;
Iswantini et al., 2005), however, there was no
experiment conducted to determine the inhibition kinetics of the extract or
the potential inhibitory effects of purified fractions of the extract. Therefore,
this research was conducted to determine the inhibition kinetics of the flavonoids
crude extract, the inhibition effects of the different fractions and to identify
the compounds of the active fraction.
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).
||Inhibitory effect of flavonoid crude extract of Sida rhombifolia
at various concentrations
||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 KM 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 KM value of the reaction containing
extract/inhibitor with the KM 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.
||Potent inhibitory effect of several purified fractions on
the activity of xanthine oxidase
|| 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
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.
||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).
||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.
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