Xanthine Oxidase Inhibitory Activity and Chemical Composition of Pistacia chinensis Leaf Essential Oil

Gout is a common metabolic disease caused by abnormal purine metabolism that promotes the formation and deposition of monosodium urate crystals within joints that causes acute arthritis and can seriously affect the daily life of patients. Pistacia chinensis is one of the traditional medicinal plants of the Anacardiaceae family, and there have been many studies on its biological activity, including anti-inflammatory, antidepressant, antibacterial, antioxidant, and hypoglycemic activities. The aim of this study was to evaluate the antigout effect of P. chinensis leaf essential oil and its constituents through xanthine oxidase inhibition. Leaf essential oil showed good xanthine oxidase inhibitory activity for both substrates, hypoxanthine and xanthine. Six fractions were obtained from open column chromatography, and fraction E1 exhibited the best activity. The constituents of leaf essential oil and fraction E1 were analyzed by GC-MS. The main constituents of both leaf essential oil and fraction E1 were limonene and 3-carene; limonene showed a higher inhibitory effect on xanthine oxidase. Based on the enzyme kinetic investigation, limonene was the mixed-type inhibitor against xanthine oxidase. The results revealed that Pistacia chinensis leaf essential oil and limonene have the potential to act as natural remedies for the treatment of gout.


Introduction
Gout is a disorder of purine metabolism resulting from the high uric acid level in serum (hyperuricaemia), which causes urate or uric acid crystal deposition within the joints. The symptoms of acute gout include severe pain, swelling, and redness in the joints, and the disease can seriously affect the daily life and diet of patients. Purine catabolic enzymes convert dietary and endogenous purines to hypoxanthine and xanthine by complex enzyme systems. Xanthine oxidase is a key enzyme that catalyzes the oxidation of hypoxanthine to xanthine, and further catalyzes xanthine to uric acid [1][2][3][4]. Allopurinol, a xanthine oxidase inhibitor, has been used in treatments for hyperuricemia and gout; febuxostat, topiroxostat, uricosurics, probenecid, etc., are relatively new drugs. Some therapeutic agents may cause adverse effects; for example, febuxostat can cause side effects such as diarrhea, nausea, and elevation of liver enzymes [5][6][7].
The genus Pistacia (Anacardiaceae) is widely distributed in North and Central America, Africa, southern Europe, and Asia. P. vera is the species in this genus famous for the production of edible pistachio nuts [17,18]. The bioactivities of folk medicinal plants in 2.2. Column Chromatography and Thin Layer Chromatography P. chinensis leaf essential oil (50 g) was further subjected to classical preparative silica gel column chromatography (CC) with a gradient elution of n-hexane and ethyl acetate of increasing polarity, and then separated into six fractions (E1-E6) using thin-layer chromatography (TLC) to analyze the elution profiles at both 254 nm and 365 nm UV light [41][42][43].

GC-MS Analysis of Leaf Essential Oil and Fraction E1
Chemical constituents in leaf essential oil and fraction E1 were analyzed using a Thermo Trace GC Ultra gas chromatograph equipped with a Polaris Q MSD mass spectrometer (Thermo Fisher Scientific, Austin, TX, USA). Analyte (1 µL) was injected into the capillary column (DB-5MS, Crossbond 5% phenyl methyl polysiloxane, 30 m length × 0.25 mm i.d. × 0.25 µm film thickness). The GC column temperature program was set as follows: initial temperature of 60 • C for 3 min; 2 • C/min up to 120 • C with a 3 min hold; 3 • C/min up to 180 • C; 10 • C/min up to 250 • C with a 5 min hold. The flow rate of the carrier gas, helium, was 1 mL/min and the split ratio was 1:10. The compound was characterized by comparing the mass spectra (m/z 50-650 amu) with library databases, including National Institute of Standards and Technology (NIST) and Wiley, and Arithmetic index (AI) [44]. The quantification of constituents was analyzed by integrating the peak area of the chromatogram [45,46].

Xanthine Oxidase Assay
Xanthine oxidase inhibitory activity of specimens was evaluated by using in vitro spectrophotometric analysis [11,47]. Both hypoxanthine and xanthine were used as the substrate in the assay, respectively. We added 117 µL of potassium phosphate buffer (50 mM, pH 7.8), 3 µL of specimen solution, and 60 µL of 0.025 unit/mL xanthine oxidase (EC 1. 1.3.22) and mixed well in a 96-well microplate, and the mixture was incubated for 10 min at room temperature (25 • C). We added 100 µL of 0.15 mM substrate (hypoxanthine/xanthine) into the well, and the solution was incubated in the dark for 30 min at 37 • C. Then, 20 µL of 1 N HCl was added to stop the reaction. Absorbance at 290 nm of each well was measured using an ELISA (enzyme-linked immunosorbent assay) microplate reader (SPECTROstar Nano, BMG LABTECH, Offenburg, Germany) after incubation. Allopurinol, a therapeutic agent for gout, was used as the positive control. All the experiments were performed in triplicate. Inhibition of the xanthine oxidase inhibitory activity was determined by the following formula: 50 , half maximal inhibitory concentration, was calculated from the concentration-response curve of the specimen.

Enzyme Kinetic Study
The Lineweaver-Burk reciprocal plot of the reaction rate and concentration of the substrate was used to determine kinetic parameters for enzyme kinetic study, and to evaluate the interaction of the compound on the affinity of the substrate and enzyme. The concentration of xanthine oxidase was constantly kept at 0.025 unit/mL, and the concentration of substrate (hypoxanthine/xanthine) was varied at the range of 0.0125-0.20 mM. The reaction was similar to the xanthine oxidase assay as described above; 117 µL of potassium phosphate buffer (50 mM, pH 7.8), 3 µL of specimen solution, 60 µL of 0.025 unit/mL xanthine oxidase, and 100 µL of the substrate (hypoxanthine/xanthine) was added into the 96-well microplate and mixed well, and kinetic measurements of the solution were immediately taken for a period of 3 min at 290 nm at 37 • C. Kinetic parameters, including Michaelis-Menten constant (K m ) and maximum velocity (V max ), were measured from the Lineweaver-Burk linear equation [48,49]. Main types of inhibition of enzyme inhibitors included competitive inhibition, noncompetitive inhibition, uncompetitive inhibition, and mixed inhibition.

Statistical Analysis
Statistical analysis of the data was performed by SPSS (Statistical Product and Service Solutions) (Chicago, IL, USA) Version 16 using Scheffe's multiple comparison test (a post hoc multiple comparison method). The confidence interval was computed at the confidence level of 95%.

Xanthine Oxidase Inhibition Activity and Enzyme Kinetic Study of Main Constituents of Fraction E1
The xanthine oxidase inhibitory activity of main constituents in the active fraction E1 is represented in Table 4. The IC 50 values of limonene and 3-carene were 37.69 µg/mL (0.28 mM) and 110.34 µg/mL (0.81 mM), respectively, using hypoxanthine as the substrate. When xanthine was the substrate, limonene was still effective, with an IC 50 value of 48.04 µg/mL (0.35 mM); 3-carene did not display activity against xanthine oxidase. Limonene showed inhibitory effects of reducing the formation of uric acid in both substrates.  Allopurinol 0.15 ± 0.02 c (1.10 ± 0.15) *** 0.12 ± 0.02 B (0.88 ± 0.15) *** *: µg/mL; **: mM; ***: µM; Allopurinol: positive control; different letters in the table represent significantly different IC 50 values between specimens in the same substrate at the level of p < 0.05 according to Scheffe's test.
Two flavonoids, apigenin and rutin, were reported to exhibit xanthine oxidase inhibition activity with IC 50 values of 35 and 61 µg/mL, respectively, using hypoxanthine as the substrate [47]. Priyatno et al. analyzed the xanthine oxidase inhibition activity of ethyl acetate extract from snake fruit (Salacca edulis), and found the active compound, 2-metyl ester-1H-pyrrole-4-carboxilyc acid, showed the best efficacy with an IC 50 value of 48.86 µg/mL, using xanthine as the substrate [51].
The main modes of enzyme inhibition include competitive, uncompetitive, noncompetitive, and mixed types [16,52]. The inhibition mechanisms of allopurinol and limonene were elucidated by an enzyme kinetic study. Figure 3 shows the Lineweaver-Burk plots of the positive control, allopurinol, with both substrates, hypoxanthine and xanthine. The linear regression lines of allopurinol under different dosage levels had the same intercept on the y-axis and increasing slopes. Table 5 shows the kinetic parameters of allopurinol Pharmaceutics 2022, 14,1982 7 of 10 against xanthine oxidase; an increase in K m and a constant in V max were observed. The results indicated the inhibition type of allopurinol on xanthine oxidase was a competitive model to suppress the production of uric acid. Chen et al. reported that allopurinol exhibited competitive-type inhibition, which is consistent with our results [52]. Competitive inhibition demonstrated that allopurinol would bind to free xanthine oxidase with strong affinity, and prevent substrate, hypoxanthine/xanthine, binding to xanthine oxidase.
The main modes of enzyme inhibition include competitive, uncompetitive, noncompetitive, and mixed types [16,52]. The inhibition mechanisms of allopurinol and limonene were elucidated by an enzyme kinetic study. Figure 3 shows the Lineweaver-Burk plots of the positive control, allopurinol, with both substrates, hypoxanthine and xanthine. The linear regression lines of allopurinol under different dosage levels had the same intercept on the y-axis and increasing slopes. Table 5 shows the kinetic parameters of allopurinol against xanthine oxidase; an increase in Km and a constant in Vmax were observed. The results indicated the inhibition type of allopurinol on xanthine oxidase was a competitive model to suppress the production of uric acid. Chen et al. reported that allopurinol exhibited competitive-type inhibition, which is consistent with our results [52]. Competitive inhibition demonstrated that allopurinol would bind to free xanthine oxidase with strong affinity, and prevent substrate, hypoxanthine/xanthine, binding to xanthine oxidase.   The Lineweaver-Burk plots and kinetic parameters of limonene are shown in Figure 4 and Table 6. In the presence of limonene, an increase in Km and a decrease in V max were observed. It revealed limonene was a mixed-type inhibitor to reduce the formation of uric acid. The inhibition type of quercetin, a versatile flavonoid, against that of xanthine oxidase was also a mixed type, containing competitive and noncompetitive types, as demonstrated in previous studies [48,49].  The Lineweaver-Burk plots and kinetic parameters of limonene are shown in Figure  4 and Table 6. In the presence of limonene, an increase in Km and a decrease in Vmax were observed. It revealed limonene was a mixed-type inhibitor to reduce the formation of uric acid. The inhibition type of quercetin, a versatile flavonoid, against that of xanthine oxidase was also a mixed type, containing competitive and noncompetitive types, as demonstrated in previous studies [48,49].

Conclusions
Gout is a metabolic disease caused by abnormal purine metabolism, which promotes the formation of uric acid. A daily diet with purine-rich foods might easily cause gout, and it is imperative to develop medicines or remedies with low side effects for gout therapy. The xanthine oxidase inhibitory effects of P. chinensis leaf essential oil and its constituents were evaluated in this study. The IC 50 values of leaf essential oil against xanthine oxidase were 43.52 and 55.40 µg/mL when using hypoxanthine and xanthine as the substrate, respectively. Among the examined fractions separated from leaf essential oil, fraction E1 had the best xanthine oxidase inhibition activity to reduce the formation of uric acid with IC 50 values of 40.55 and 51.84 µg/mL when using hypoxanthine and xanthine as the substrate, respectively. Limonene, a major constituent of fraction E1, showed an inhibitory effect on xanthine oxidase with IC 50 values of 37.69 and 48.04 µg/mL using hypoxanthine and xanthine as the substrate, respectively. Through an enzyme kinetic study, we found that limonene was a mixed type inhibitor of xanthine oxidase for both substrates. The results indicated P. chinensis leaf essential oil and limonene have potential as natural xanthine oxidase inhibitors for the treatment of gout. Further investigation is required for potential clinical application.