Optimization of Xanthine Oxidase Activity, Phytochemical Screening, Toxicity Assay, and Antigout Activity of Spatholobus littoralis Hassk. Extract

al. Optimization of Xanthine Oxidase Activity, Phytochemical Screening, Toxicity Assay, and Antigout Activity of Spatholobus littoralis Hassk. Extract


INTRODUCTION
The primary enzyme responsible for gout is a xanthine oxidase (XO). 1 Gout or the formation of uric acid crystals in the joints is led on by this enzyme activity. 2 The endogenous and exogenous nucleotides in the human body are metabolized into nucleosides and then to purine bases. Guanine from guanosine and hypoxanthine from inosine, generated from adenosine-by-adenosine deaminase, gives comparable breakdown products. Guanine deaminase converts the guanine to xanthine, which is ultimately converted to uric acid by XO. Hypoxanthine is converted by the XO into xanthine and then uric acid. 3 This activity also coincides with the production of reactive oxygen species (ROS). 4 The term "gout" itself is derived from the Latin word "gutta," which means "to drip," and in the thirteenth century, it was thought that poison drops that fell on the joints produced gout. 5 The disease primarily suffers people in Middle Ages men and their postmenopausal stages women. 6 If left untreated, this illness can intensify to chronic gout, even leading to kidney failure and a stroke. 7 The U.S. has reported that gout patients worldwide are rising annually. 8 Gout prevalence reaches 4.49% in Asian nations, for instance, Taiwan. 9 The in Indonesia, the prevalence of gout is in the range of 1.6-13.6 per 100.000 people. 10 The type of inhibition kinetics generated can describe how XO as a target is inhibited and how therapeutic candidate molecules bind to it, forming an affinity that may be either temporary (competitive and uncompetitive inhibition) or permanent (noncompetitive inhibition). 11,12 Due to the presence of secondary metabolites, substances with the ability to inhibit XO, some medicinal herbs can be mainly used to treat gout. 13 The five Indonesian medicinal herbs, Phaleria macrocarpa, Ruellia tuberosa, Sida rhombifolia, Cyperus rotundus, and Syzygium polyanthum, exhibit competitive inhibition kinetics against XO. 14 Hasskarl discovered bajakah tampala in 1842; its scientific name is Spatholobus littoralis Hassk. (S. littoralis Hassk.). Twenty-nine species in the genus Spatholobus are distributed throughout Southeast Asia. Consequently, it spreads from West India to South China, the Philippines, and West Malaysia. Spatholobus was expanded throughout in the Malay Peninsula, Borneo (Kalimantan Island), and southeast Sumatra. 15,16 Traditional medicines, including bajakah tampala, are frequently used to treat degenerative diseases. 17 According to Iskandar and Warsidah (2020), 18 the ethanol extract also apparently exhibits antioxidant activity, with an IC 50 of 8.25 g/mL. Ayuchecaria et al. (2020) 19 revealed that the bajakah tampala stem extract's total phenolic content is 12.32 mg GAE/g. In this current research work, we investigated the antigout activity of S. littoralis Hassk. extract. We applied the water extract and 70% ethanol extract of S. littoralis Hassk. stem. Response Surface Methodology (RSM) is a useful technique for optimizing XO activity. The experimental design chosen in the RSM is the Box-Behnken Design (BBD). BBD has the advantage of not containing any combination factors at the cube's vertices (high/low points), resulting in fewer tests. 20,21 Optimization of XO activity based on three combination factors: pH, temperature, and xanthine concentration. After obtaining these optimum conditions, we analyzed the inhibitory capacity of the water extract and 70% ethanol extract toward XO activity and their inhibition kinetics through Lineweaver-Burk plot analysis. We also evaluated the phytochemical screening and toxicity assay of both extracts.

Plant collection and sample preparation
The species S. littoralis Hassk. was collected in Muara Teweh, North Barito Regency, Central Kalimantan Province, and was identified by a botanist at the National Research and Innovation Agency at Cibinong, West Java, Indonesia. The Tropical Biopharmaca Research Center, IPB University, Indonesia, has deposited a voucher specimen of the sample (number BMK0490102021). The stem parts of the plant were cleaned, sundried, and ground to give 40 mesh-size powder to obtain the simplicia.

Determination of moisture content 22
The porcelain cup was dried for 30 minutes in a 105 ℃ oven, then placed in a desiccator for 30 minutes of cooling before weighing. Next, the simplicia (3 g) was placed into the known-weight porcelain cup and dried in a 105 ºC oven for 3 hours. Finally, the porcelain cup was cooled in a desiccator for 30 minutes and weighed. The procedure was carried out repeatedly until a constant weight was obtained by a difference of less than 1 mg. Moisture content determination was carried out in triplicates. The moisture content was calculated using the following formula (1): (1) Abbreviations: W 1 = sample weight before drying (g); W 2 = sample weight after drying (g).

Extraction 23
The simplicia powder (100 g) was macerated with water, and 70% ethanol, at room temperature with a simplicia and solvent ratio of 1:10. The sample and solvent were stirred and allowed to stand for 24 hours at room temperature, then filtered. Next, the filtrate was separated, and the process was repeated three times. Finally, the filtrate was concentrated using a rotary evaporator at 40−60 ºC. The extracts were weighed, and the yield was calculated using the following formula (2): (2) Abbreviations: a = extract weight (g); b = sample weight (g); c = moisture content (%).

Phytochemical screening 24
A phytochemical assay was carried out to correlate the presence of secondary metabolites from the simplicia and the extracts with the inhibition of XO. This analysis used the Harborne method to identify alkaloids, triterpenoids, steroids, flavonoids, saponins, tannins, and quinones.

Toxicity assay 25
The toxicity assay was carried out using the BSLT (Brine Shrimp Lethality Test) method. Hatching of A. salina eggs was the first stage of the toxicity test. The eggs (50 mg) were weighed before being placed in an Erlenmeyer with sufficient filtered seawater. The Erlenmeyer was equipped with an aerator and incubated under light sources for 48 hours until all eggs hatched. Then, the toxicity test was performed on the hatched larvae. A 24-well plate containing seawater and extracts (water extract, 70% ethanol extract) was also added with 10 larvae per well. In a total volume of 2 mL with three replications, the concentration series were 1000, 500, 100, and 50 ppm. After 24 hours, the dead larvae were counted. Using the IBM SPSS Statistics version 21 application, the lethal concentration 50 (LC 50 ) was calculated by visualizing the mortality rate using probit analysis at a 95% confidence level.
Determination of the maximum wavelength (λ max ) of xanthine (Modification from Desma 2017) 26 Xanthine solution (1.0 mM) was prepared in potassium phosphate buffer (pH 7.5, 10 mL, 50 mM). The maximum wavelength was determined in the range of 200−400 nm. The determined wavelength was used as a reference in the XO inhibitory assay. Measurements were made using a U-2800 double-beam spectrophotometer (Hitachi, Tokyo, Japan).

Optimization of XO activity (Modification from Trivadila et al. 2020) 27
The experiment condition for XO activity determination was optimized at 20−30 °C temperature, 6.0−8.0 pH, and 0.70−1.40 mM xanthine concentration. RSM with BBD was used to optimize XO activity. This method was performed by entering a combination of factors of independent variables in the statistical software Minitab.v.14 English. Afterward, the experiment was carried out by determining three factors and 2-replicate. Thirty combinations generated the optimized condition. Then, the optimized results were verified by a one-sample t-test based on the value of the reacted xanthine concentration from the Minitab optimization results.
In Vitro xanthine oxidase inhibition assay and IC 50 determination (Modification from Iswantini et al. 2014) 11 The XO inhibitory was performed under previously determined optimum conditions. Various extract (1 mL) concentrations were added to the test tube depending on the toxicity value. At the optimum pH, 1.9 mL of potassium phosphate buffer (50 mM) was added. The optimum concentration of xanthine in 1 mL was then blended. The reaction was initiated by slowly adding 0.1 U/mL XO (0.1 mL), and it was then incubated for 45 minutes at the optimum temperature. The process was stopped by adding 0.58 M HCl (1 mL). In order to determine how much of the unreacted xanthine was still present in the test sample, the mixture's absorbance was then measured using a UV-Vis spectrophotometer at its maximum wavelength. The results were compared to a commercial antigout drug (allopurinol) to determine the inhibitory capacity of each extract. The formulas (3 and 4) were employed to express XO activity and inhibitory capacity (%): By entering 50 as the y value in the regression equation to obtain the x value as the inhibition concentration 50 (IC 50 ) of the sample, the inhibition concentration 50 (IC 50 ) was determined.

In Vitro inhibition kinetics of xanthine oxidase assay (Modification from Iswantini et al. 2014) 11
The extract concentrations were determined based on the IC 50 and LC 50 values to evaluate the XO inhibition kinetics. The xanthine concentration ranged from 0.10 to 1.60 mM. (0.10 mM interval). The data were interpreted into the Lineweaver-Burk plot analysis to obtain the maximum reaction rate (V max ), Michaelis-Menten constant (K m ), and constant of inhibition (K I ) following the formulas (5 and 6):

Statistical analysis
Data were presented as the mean of standard deviation (SD) (n = 3), and one-way analysis of variance (ANOVA) was used to examine them well before the Tukey test at a 95% confidence level. The P-value<0.05 indicates that the differences between samples are significant.

Moisture content
In this study, the cleaned, dried (Figure 1), ground to 40-mesh powder of stem pieces of bajakah tampala was used ( Figure 2). The simplicia powder was brown and had a distinctive aroma. Simplicia drying was intended to avoid microbial attack. In addition, the grinding of samples was intended to facilitate solvent diffusion into the plant cell walls to optimize extraction. Meanwhile, sieving aims to homogenize the size of the simplicia powder.
The moisture content of the simplicia was determined by the gravimetric method. Moisture content determination is useful for determining how to store the simplicia and avoid damage due to microbial activity (fungi and bacteria). In addition, the moisture content also acts as a correction factor for calculating the extract yield. The moisture content in this study was 5.81% ± 0.0827. The simplicia can be stored relatively long, depending on the findings for further use at the extraction stage, following the requirements of the Indonesian Food and Drug Authority (2014), 28 where the simplicia moisture content should be less than 10%. Microbial development may be slowed down in this optimum condition.

Extract yields
Maceration was used to extract secondary metabolites such as antigout. The mechanism of maceration is the process of solvent diffusion into the plant cell wall to extract the plant's compounds. The advantage of maceration is that the equipment used is simple, has low energy consumption, and is relatively inexpensive. The maceration method, on the other hand, requires a longer extraction time. 29,30   The use of water and ethanol solvents in this study refers to the regulation of the Indonesian Food and Drug Authority (2012) 23 regarding the reference to Herbal Preparations (Acuan Sediaan Herbal). People use water to prepare traditional drink ingredients. Meanwhile, ethanol is generally considered safe for human consumption and a solvent for natural food and medicine ingredients. 31 Water is a polar solvent with the highest polarity (1.000). On the other hand, ethanol is a semipolar solvent with a polarity of 0.654. 32 The yield of 70% ethanol extract was higher (11.24%) than the water extract (7.29%) ( Figure 3). It indicates semipolar compounds can be extracted more easily in 70% ethanol than in water extract. The "like-dissolvelike" rule states that chemical components will separate into two phases based on their level of polarity. 33 Meanwhile, semipolar compounds are flavonoids, phenolic compounds (tannins, quinones), and saponins. 24

Phytochemical composition
The phytochemical assay is a preliminary test to determine the secondary metabolite content that can inhibit XO in simplicia and extracts. Table 1 showed that the simplicia and extracts (water and 70% ethanol) were positive for flavonoids, tannins, saponins, and steroids. The flavonoids in the 70% ethanol extract were higher than in the water extract. Saputera et al. (2019) 34 showed a negative result for alkaloids in 70% ethanol extract of the same species collected in Pulang Pisau Regency, Central Kalimantan and this result is the same as this research. In addition, the 96% ethanol extract from Lamandau Regency, Central Kalimantan, also displayed negative results for alkaloids. 35 However, in the Ayuchecaria et al. (2020) 19 study, the ethanol extract showed a negative result for saponins, which is different from our findings. Meanwhile, Iskandar and Warsidah (2020) 18 stated that the ethanol extract of the same species from Palangkaraya, Central Kalimantan was positive for alkaloids. This difference may occur due to internal factors, such as gene regulation and external environmental factors (the growing site, the presence of light, temperature, moisture content, salt content, and soil type). 36 The quantity of secondary metabolites in plants is also affected by the harvest time and the post-harvest handlings. 37

Toxicity
The BSLT method was used to determine the toxicity of S. littoralis Hassk. extracts. This method is a simple and direct bioassay for measuring the toxicity of natural products. The percentage of A. salina larvae mortality was used to determine the probit value. The obtained probit value is then plotted against the concentration log in a graph to determine the LC 50 value. According to Meyer's criteria, an extract is considered very toxic if its LC 50 value is less than 30 mg/L, toxic if its LC 50 value is between 30 and 1,000 mg/L, and not toxic if its LC 50 value is greater than 1,000 mg/L. 25 Figure 4 displays the LC 50 of each extract and the negative control (without extract). The LC 50 for the 70% ethanol extract (662.25 mg/L) has a lower value than the water extract (774.68 mg/L), indicating that the 70% ethanol extract is more toxic than the water extract. contains more secondary metabolites and is more toxic to A. salina L. The high toxicity of 70% ethanol extract suggests that it contains a higher concentration of secondary metabolite components. 38 These results are consistent with phytochemical screening that 70% ethanol extract has a higher content of flavonoids. After the secondary metabolite components are absorbed, they are distributed throughout the larvae body, going to cause metabolic disorders. The mortality rate of larvae was closely related to the concentration and synergistic effect of several compounds in the extract. 39 The LC 50 of each extract determines the concentration in the XO inhibition assay because it is safe and secure in the drug formula when employed below its LC 50 . 40     The maximum wavelength (λ max ) of xanthine Determination of the maximum wavelength was carried out before the xanthine oxidase inhibition assay. Measurement of the absorption of a solution at the maximum wavelength can reduce errors or have high accuracy in determining a compound by spectrophotometry. This is because measurements at the wavelength (λ) with maximum absorption will increase the sensitivity of the analysis. Ultraviolet (UV) measurements were carried out in the wavelength range of 190-400 nm because the compounds to be measured are colorless. 41,42 The maximum wavelength (λ max ) of xanthine was 270.0 nm ( Figure 5). A similar result was reported by Kim et al. (2020) 43 and Desma (2017). 26 Optimum conditions for XO activity  Table 2 describes the results of the optimization conditions for determining XO activity with reacted xanthine as a response.
Analysis of Variance (ANOVA) is a method used to determine the optimized XO activity from each response, such as pH, temperature, and xanthine concentration. 47 Based on the ANOVA (Table 3) of various pHs, temperatures, and xanthine concentrations in the linear regression model, the P-value was 0.000. It indicates that the three variables have a significant effect (P<0.05) on the response (concentration of the reacted xanthine). Meanwhile, in the squared regression model, temperature and temperature had no significant effect, with a P-value of 0.832. In addition, for 2-way interaction, the interaction between pH and temperature; the interaction of pH and xanthine concentration had a significant effect with P-values of 0.040 and 0.001 respectively; while the interaction of temperature and xanthine concentration had no significant effect with a P-value of 0.904. Data on lack of fit obtained a P-value of 0.000, which means that the model used is suitable to be applied in determining optimum conditions. It is also supported by the R-sq value of 95.22% which states that the variables analyzed affect the resulting response.
The rate of a chemical reaction increases, as the temperature increases, increasing the kinetic energy of the system. However, if the temperature of the system exceeds its optimum conditions, the enzymes will be denatured.   The contour plot and surface plot analysis in Figure 6 shows the relationship between the 3 variables and the response (reacted xanthine concentration). Both of these plots produce XO activity at optimum conditions. The darkest green color indicates the maximum response, while the darkest blue color shows the minimum response. Figure 6 shows that the maximum response will be obtained in the pH range of 7.5−8.0; the temperature of 20 ℃; and the xanthine concentration of 1.40 mM. These results are also following the optimization produced by the Minitab software through response optimizer (RO).
The RO analysis explained that the optimum activity conditions of XO were reached at pH 8.0, the temperature of 20 ℃, and the xanthine concentration of 1.40 mM (Figure 7). The desirability value of the RO shows that it reaches 1.0. The higher the desirability value (up to 1.0), the higher the suitability of the variable combinations to achieve the response. 49 The expected value of the Minitab reacted xanthine (optimized result) was 1.1146 (Table 4). When tested based on the optimum conditions, the average value of xanthine concentration was reached at 1.1067. When H 0 ; μ= 1.1067; H 1 : μ ≠1.1067; then the P-value of the one-sample t-test is 0.423 or greater than 0.05. It means that H 0 is accepted (not significantly different), and the verification follows the expected value (Table 5).
The research data for optimizing XO activity in the study also did not differ significantly, as shown in Table 6. The difference in the optimum conditions for XO activity was caused by measurement time, environmental conditions, and differences in laboratory equipment. 11 Furthermore, differences in the enzyme type used will impact XO, typically isolated from mammalian milk, which is influenced by the animal's sex, season, and diet. 50 Jadhao et al. (2018) 51 reported that the optimum temperature and pH obtained from cow's milk were 25 ℃ and 7.4. Meanwhile, the optimum pH of XO obtained from buffalo milk is 8.0. 51

Inhibitory capacity and IC 50 of extracts toward XO activity
Allopurinol from Sigma and commercial allopurinol were used as positive controls. Figure 8 represents the effect of increasing the concentration of allopurinol on XO. The analysis results explained that allopurinol exhibited a much stronger inhibitory power than water extracts and 70% ethanol. The IC 50  The IC 50 of 70% ethanol extract is 224.14±8.62 mg/L. This value is lower than the water extract (348.83±4.85 mg/L) and this condition indicates that 70% ethanol extract is more active than water extract. The inhibitory activity is increasing with the increasing polarity of the fractions ( Figure 5). This result is in line with the LC 50 result obtained previously. Semipolar compounds are assumed to be present in the 70% ethanol extract. XO inhibitors are phenolic compounds such as phenolic acids and flavonoids. 55 The total phenolic content of the ethanol extract of bajakah tampala stem reported by Ayuchecaria et al. 2020 19 is 12.33 mgGAE/g. Meanwhile, according to Kurnianto et al. (2020), 56 the total phenolic content is 14.952 mg/100g GAE and the flavonoid content of the ethanol extract of the stem is 79.739 mg/100g QE. The pharmacological effects of these compounds not only reduce uric acid concentrations but also as anti-inflammatory and suppress the formation of free radicals such as superoxide anion and hydrogen peroxide. 57,58         affinity of the enzyme for the substrate. The ratio of the value of Km in the condition of adding the inhibitor and the Km in the condition without the inhibitor will reach the value of α. This value is also the affinity of the inhibitor for XO. The greater the value of α, the higher the competitive inhibition that occurs. 45,59 The concentrations of inhibition kinetics on the water and 70% ethanol extracts were 400 and 300 mg/L, respectively. During the inhibition kinetics assay, the activity rate of XO was measured both in the absence and presence of the inhibitor expressed in mM/min at various substrate (xanthine) concentrations ranging from 0.10 to 1.60 mM. In addition, a Lineweaver-Burk plot investigation was carried out to establish the type of inhibition kinetics from extracts toward XO activity ( Figure 6).
The V max and K m values without inhibitors were 0.0144 mM/min and 0.7320 mM. This condition means that XO will reach half of its maximum rate at a substrate concentration of 0.7320 mM with a maximum rate of 0.0144 mM/min. Table 6 shows that V max values for extracts remained relatively unchanged, while K m values increased. The highest α value was obtained in the 70% ethanol extract (2.36), while the lowest was in the water extract (2.06). The value of α which is greater than 1 indicates a strong inhibitory power. The value of the inhibition constant (K I ) for the 70% ethanol extract was obtained as the smallest (272.68 mg/L), which means that the inhibition is stronger and the binding affinity of the inhibitor-enzyme exceeds the binding affinity for the enzyme-substrate. 45 The type of inhibition kinetics for the extracts obtained is competitive inhibition kinetics. This is based on an increase in the value of K m but a relatively constant V max . 60 The Lineweaver-Burk plot ( Figure 9) shows a decreasing x-intercept due to the increasing K m value. However, in the competitive inhibition mechanism, the value of V max tends to remain constant, resulting in no change in the y-intercept in the Lineweaver-Burk plot.
In the mechanism of competitive inhibition, the structure of the inhibitor compound resembles that of the substrate so that it competes with the substrate in binding to the active site of the enzyme. Competitive inhibitors and substrates have the same affinity for occupying the active site of the enzyme so that when the concentration of the inhibitor exceeds the concentration of the substrate, it will form an enzyme-inhibitor complex (EI), and no product is formed. 45,59,61 In the world of pharmacokinetics, the selected drug candidates are competitive inhibitor types because they are easily absorbed in the body's metabolism (bioavailability) and reduce the effects of toxicity in the body. 62 In general, flavonoids can be absorbed by the small intestine around 0-60% of the dose and the elimination half-life ranges from 2-28 hours. 63 Flavonoids that lead to competitive inhibitors of XO are isoflavones (genistein), flavonols (kaempferol), flavones (luteolin, apigenin), flavanones (eriodictyol), and flavanols (catechins). 14 These flavonoid compounds are thought to be contained in the water and 70% ethanol extracts from the stem of S. littoralis Hassk.

CONCLUSIONS
The Box-Behnken Design was successfully employed to optimize XO activity. Water and 70% ethanol extracts from the stem of S. littoralis Hassk. competitively inhibit xanthine oxidase activity. The yield, phytochemical screening, and toxicity correspond with these findings. However, further fractionation of the extracts is required to increase their inhibitory activity.