Comparative Study of Xanthine Oxidase Inhibitory Activity Of Methanolic Leaf Extracts Of CalophyllumBlancoi (Bitaog), Diospyrospilosanthera (Bolongeta) and SyzygiumCumini (Duhat)

Gout is characterized as an in lammation and warmth in the joints. It is associated with hyperuricemia wherein an upregulation of xanthine oxidase in purine degradation leads to increased levels of uric acid in the blood. Gout is not fatal. However, it affects one’s quality of life. Thus, this research primarily focuses in determining the inhibitory activity of xanthine oxidase in the methanolic leaf extract of bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera), and duhat (Syzygiumcumini) in gout. A quantitativeexperimental research method was used in the study and the data were obtained by measuring the percent inhibition of the samples using UV-Vis spectrophotometer at 290 nm. The results showed that the methanolic leaf extract of above stated plants exhibited exemplary inhibition in comparison with the standard drug, allopurinol. The IC50 value determines the ability of the inhibitor to decrease the biotransformation of a substrate. The principle behind IC50 is, the lower the value the higher the inhibition. The bitaog (Calophyllumblancoi) trials have the lowest IC50 valuewith an average of 124.3 after the standard drug, followed by bolongeta (Diospyrospilosanthera) have an average of 155.3 IC50 value. Then duhat (Syzygiumcumini) showed the highest IC50 an average of 208.8. The bitaog (Calophyllumblancoi), next to allopurinol, showed the highest inhibition among all the extracts followed by the bolongeta (Diospyrospilosanthera). The least inhibitory activity was observed in duhat (Syzygiumcumini). Hence, it can be concluded that bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera), and duhat (Syzygiumcumini) can inhibit xanthine oxidase using in vitro analysis.


INTRODUCTION
Gout is manifested by intense pain and in lammation of joints occurs when the soft tissue accumulate monosodium urate crystals (Roubenoff, 1990). It has been estimated that in 2016, it affects about 1.6 million of Filipinos as stated by Philippine Rheumatism Association President, Eric Amante (Crisostomo, 2015) and 4.3% in the US. Gouty arthritis can be caused by an increase of the urate serum levels, also known as uric acid in the blood.
Uric acid chemical is formed when there is a breakdown of substances called purines (Maiuolo et al., 2016). This uric-acid is created by the conversion of xanthine to hypoxanthine by the reactive oxygen species called xanthine oxidase. Xanthine oxidase (XO) is a signi icant biological source of free radicals that is oxygen-derived which contributes to the oxidative damages in tissues. It is widely distributed in liver and intestine of different species. It is the rate-limiting enzyme in the biosynthesis of uric acid and catalyzes the formation of uric acid. Thus, any up-regulation of the purine degradation pathway may result in the increase of uric acid known as hyperuricemia that eventually leads to gout (Duong et al., 2017).
Allopurinol is commonly used for the treatment of gout. However, it has induced severe cutaneous adverse reactions including Steven-Johnson's syndrome, renal toxicity and even the fatal liver necrosis (Stamp et al., 2016;Gupta et al., 2019;Klaewsongkram et al., 2016). In contrast, the use of natural therapy is being introduced to attain fewer side effects for an inexpensive price.
As stated by Jothy et al. (2011), major compounds that act as primary antioxidants is the plant's phenolic constituent. They are capable of scavenging reactive oxygen species, such as xanthine oxidase, due to its electron donating properties. Furthermore, according to Bakar et al. (2018), Momordicacharantia, Chrysanthemum indicum, Cinnamomum cassia, Kaempferiagalanga, Artemisia vulgaris, and Morindaelliptica had higher percentages of xanthine oxidase inhibitory activity (>85%) because of their diverse natural bioactive compounds, which include lavonoids, phenolics, tannin, coumarins, luteolin, and apigenin. Besides these plants, Alvionita et al. (2019) reported that a lavonoid compound found in Annona squamosal L., has higher binding af inity to the xanthine oxidase than allopurinol which indicated that this plant may be a promising herbal drug for gout treatment. In a review done by Kapoor et al. (2017) on over 130 medicinal plants, they concluded that these plants exhibit anti-gout effect by different mechanisms, such as XO inhibition, uricosuric activity, anti-in lammatory activity, and antioxidant activity. Moreover, plant extracts and isolated constituents which showed promising XO inhibition are also considered and most of the isolated elements are found to be phenolic glycosides and lavonoids.
The researchers found potential plants of interest that contain phenolic compounds and lavonoids to further study. Consequently, the researchers chose bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera) and duhat (Syzygiumcumini) as potential xanthine oxidase inhibitor in the study.
In addition, limited studies regarding xanthine oxidase inhibition of these plants have been carried out. This study was conducted to evaluate and determine the potential xanthine oxidase inhibitory activity of the three plants namely using in vitro assay.

Research Design
The study used quantitative-experimental method of research in the determination of the inhibition activity of xanthine oxidase of the leaf methanolic tract from the leaves of (a) bitaog (Calophyllumblancoi), (b) bolongeta (Diospyrospilosanthera), and (c) duhat (Syzygiumcumini) which permits the measurement of variables within the desired collection resulting in numerical data which then subjected to statistical analysis. Besides, it con irms the presence phenolic and lavonoid. The methanolic extract of the plants and the standard drug, allopurinol, were manipulated to determine the effect of the material under the study. The dependent variable of the study was of xanthine oxidase inhibition of the methanolic extract of each plant. The analysis is between the positive control of the standard drug allopurinol, and sample test group composed of (a) bitaog (Calophyllumblancoi), (b) bolongeta (Diospyrospilosanthera), and (c) duhat (Syzygiumcumini) methanolic leaf extracts.

Collection and Preparation of the Plant and Plant Extract
The leaves of bitaog (Calophyllumblancoi) and bolong-eta (Diospyrospilosanthera) were collected at Dasmariñas City, Cavite and duhat (Syzygiumcumini) was collected from Indang, Cavite, Philippines in the month of January 2018.
The dried leaf samples were crushed using blender until it turned into powdered form. Every leaf samples were weighed for three times having 10 g each. Each sample was placed in of 90% methanol for 72 hours and stored in a tightly closed container wrapped with aluminum foil.

Total Phenolic Content
The phenolic content was determined using the Folim-Ciocalteu assay. An exactly 100 µL of each methanolic extract sample was diluted with 0.50 mL with distilled water in a test tube. Folin-Ciocalteu Phenol reagent (0.25 mL, 10x diluted) was added to each sample and were incubated. Afterwards, 1.50mL of 7% sodium carbonate (Na 2 CO 3 ) After the incubation, each sample was subjected to vortex mixer to mix thoroughly and some amounts were transferred to cuvettes. The absorbance of each sample was read using UV-Vis spectrophotometer. Dilutions were carried out as needed. Gallic acid was used as standard in different concentrations (0.5, 1.0, 2.5, and 5 µg/mL). The total phenolic content (TPC) was expressed as µg of gallic acid equivalent per gram of sample (µg GAE/g).

Total Flavonoid Content
The total lavonoids content determination of the methanolic extract was done using the aluminum chloride colorimetric method following procedures outlined by Baba and Malik (2015). A 50 µL of methanolic extract (1mg/mL ethanol) was diluted with 1mL with methanol, mixed with 4 mL of distilled water and then 0.3 mL of 5% sodium nitrite (NaNO 2 ) solution; 0.3 mL of 10% aluminum chloride (AlCl 3 ) solution was added after ive minutes of incubation at dark at room temperature, and the mixture was allowed to stand for six minutes. The mixtures were mixed thoroughly using vortex mixer and transferred to the cuvettes. Then, absorbance was read immediately at 510 nm versus blank sample (methanol) using UV-Vis spectrophotometer. Dilutions were carried out as necessary. Calibration curve was constructed using quercetin as standard with varying concentrations (0.10, 0.20, 0.50, 1 and 2 µg/mL). The total lavonoids content was expressed as µg of quercetin equivalent per gram of samples (µg of QE/g).

Thin Layer Chromatography
Each solvent extract was subjected to thin layer chromatography (TLC) as per conventional onedimensional ascending method using CAMAG TLC Silica Gel 60 F254 Aluminum sheet. The starting point to inish line measures about 7 centimeters. The solvent system used was (1:4) acetonechloroform. The TLC was set up by placing the sheet in a slanting position inside of 500 mL beaker with a #1 Whatman ilter paper and 50 mL of the solvent system and cover by aluminum foil. Then, retention factor (R f ) was measured where retention factor is distance travelled by the solute divided by distance travelled by the solvent as shown in Equation (1).  (Sarawek, 2007).

Xanthine Oxidase (Enzyme)
The xanthine oxidase solution was prepared by dissolving 0.003 g of xanthine oxidase from bovine's milk in 5 mL of buffer pH 7.4 cold 0.2 M phosphate (Sarawek, 2007).
The irst control sample was made by adding a 25 µL of xanthine oxidase solution to 0.2 M phosphate buffer (1 mL) and the mixture was incubated. Then, 0.3 mL xanthine oxidase buffer solution was added to the mixture. The second control sample was prepared by diluting 1.025 mL of 0.2 M phosphate buffer (pH 7.4) to 0.3 mL of xanthine oxidase buffer solution and each control sample was made for four replicates. The blank sample contains only 0.2 M phosphate buffer solution (Sarawek, 2007).

Xanthine Oxidase Inhibition Assay
The modi ied method of Sivashanmugam and Chatterjee (2012) was used for the determination of xanthine oxidase inhibitory activity of plant extracts. Exactly 0.3 mL of test solution (25, 50, 100 and 300 µg extracts/mL phosphate buffer, 0.8 mL of 0.2 M phosphate buffer (pH 7.4) and 25 µL of xanthine oxidase were combined. The mixtures were incubated in dark at room temperature for 15 minutes and the reaction was initiated by addition of 0.3 mL of xanthine oxidase buffer. Each sample was transferred to quartz cuvette and the absorbance of each was mea-sured using JASCO V-530 UV-Vis spectrophotometer at 290 nm. The percentage of inhibition was calculated using Equation (2).

P ercentage of inhibition = (
where A is equivalent to the change in absorbance of the assay without test samples, and B is equal to the change in absorbance of the test assay with test sample. The IC 50 was determined by linear regression and compared to the value of the standard drug allopurinol.

Statistical Analysis
All data were statistically treated by one-way analysis of variance (ANOVA), T-Test, and F-Test followed by post-hoc studies using Tukey's Honestly Significant Difference to assess signi icant differences of mean between groups. The calculations and all values were expressed as mean ± S.E.M (standard error of mean) at 5% signi icant level. Values with P<0.05 is considered statistically signi icant.

Total Phenolic Content
The Folin-Ciocalteu assay con irms the presence of the phenolic compounds of the extracts by having an absorbance ≤1. The TPC test used Gallic acid as the reference standard. Figure 1 demonstrates the calibration curve obtained from the absorbance of Gallic acid under 750 nm.

Figure 1: Calibration curve obtained from the absorbance of Gallic acid
On the other hand, Table 1 collates result of the absorbance of the samples from bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera), and duhat (Syzygiumcumini) methanolic leaf extract. The results displayed above showed that the phenolic content primarily contributes to the antioxidant activities of the above-mentioned plants, therefore The results shows that the Bitaog (Calophyllumblancoi) has the greatest amount of phenolic contents with an average of 944.8 µg GAE/ g sample, followed by bolongeta (Diospyrospilosanthera) having a 851.1 µg GAE/ g sample. The least amount of phenolic content was found in duhat (Syzygiumcumini) with 647.5 µg GAE/ g sample.

Total Flavonoid Content
Aluminum chloride colorimetric method af irms the presence of lavonoids compounds of the extracts by having an absorbance of ≤1. Quercetin was used as the reference standard for the test. Figure 2 draws the calibration curve quercetin after subjected to the     Table 2 shows the calculated total lavonoid content of bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera), and duhat (Syzygiumcumini). As seen in Table 2, the total lavonoid content of the plants has an average that ranges from 345.9 to 593.8 µg QE/ g sample. Bolongeta (Diospyrospilosanthera) showed highest the lavonoid content (593.8 µg QE/ g sample), followed by bitaog (Calophyllumblancoi) (389.1 µg QE/ g sample), whereas duhat (Syzygiumcumini) has the lowest phenolic content (345.9 µg QE/ g sample). This result supported the literature that the lavonoid content predominantly contributes to the antioxidant effect of the said plants, and therefore, the lavonoid con-tent of the said plants could play a signi icant role in blocking xanthine oxidase.
As shown in Figures 3 and 4, the functional group that has more af inity to the stationary phase adhered and travelled slower than others. The pinkish color, as seen in Figure 4, represents the phenolic groups that include lavonoids. Figures also shows that the plate is best viewed under the UV light long and short wave.

Xanthine Oxidase Inhibition Assay
The xanthine oxidase inhibition assay used allopurinol as the positive control. Figures 5, 6, 7 and 8 Figures 5, 6, 7, and 8 illustrate the xanthine oxidase inhibition of allopurinol, bitaog (Calophyllumblancoi), Bolongeta (Diospyrospilosanthera) and duhat (Syzygiumcumini) respectively. Table 3 summarizes the inhibitory concentration (IC 50 ) value of the samples in comparison with the positive control, allopurinol. The IC 50 value determines the ability of the inhibitor to decrease the biotransformation of a substrate. The principle behind IC 50 is, the lower the value the higher the inhibition. The bitaog (Calophyllumblancoi) trials have the lowest IC 50 value with an average of 124.3 after the standard drug, followed by bolongeta (Diospyrospilosanthera) have an average of 155.3 IC 50 value and duhat (Syzygiumcumini) showed the highest IC 50 an average of 208.8.
As per the interpretation in Table 4, the methanolic leaf extract of bitaog (Calophyllumblancoi), bolongeta (Diospyrospilosanthera) and duhat (Syzygiumcumini) has a signi icant inhibitory effect in xanthine oxidase. The table also lead to the conclu-sion that the bitaog (Calophyllumblancoi) have the highest inhibitory activity after the standard drug, allopurinol, followed by duhat (Syzygiumcumini), whereas the bolongeta (Diospyrospilosanthera) have the lowest xanthine oxidase inhibition.

CONCLUSION
In conclusion, based on the study done on xanthine oxidase inhibitory activity of methanolic leaf extracts of the three plants, it was proven that these plants exhibited exemplary inhibition in comparison with the standard drug, allopurinol. Among the three plants, bitaog (Calophyllumblancoi) had the highest inhibition, followed by bolongeta (Diospyrospilosanthera), and lastly duhat (Syzygiumcumini).