The influence of chemical composition of potent inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants to their xanthine oxidase activities

Kheng Leong Ooi a, Rahimah Zakaria b, Mei Lan Tan a, Shaida Fariza Sulaiman a, *
aSchool of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, USM, Pulau Pinang, Malaysia
bSchool of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia
Xanthine oxidase inhibitory activity Myricetin
Quercetin Apigenin
Syzygium aromaticum Merr. & L.M.Perry Orthosiphon aristatus (Blume) Miq

Ethnopharmacological relevance: Anti-hyperuricemic plant parts that were selected for this study, are traditionally used to treat gout in Malaysia. Caffeic acid (a hydroxycinnamic acid), apigenin (a flavone), myricetin, quercetin and kaempferol (flavonols), were reported to act as potent xanthine oxidase inhibitors. These compounds can be found in some of the selected ethnomedicinal plants. However, there is still lack of published research works on the quantification of these inhibitors from these urate-lowering phytotherapies.
Aims of the study: The compounds were quantified from 21 hydrolyzed extracts of the phytotherapies for gout. The activity-content contributions of the compounds to the potent extracts were determined.
Materials and methods: The anti-hyperuricemic activities of the extracts and the compounds were determined using a xanthine oxidase inhibitory assay. Ultra-Performance Liquid Chromatography (UPLC) coupled with Photodiode Array Detector (PDA) was used to quantify the compounds in the extracts.
Results: The results revealed higher activity of the hydrolyzed extracts. The hydrolyzed extract of the flower bud of Syzygium aromaticum Merr. & L.M.Perry exhibited the highest activity (EC50 = 39.58 ± 0.10 μg/mL) due to the highest content of myricetin (42,297.55 ± 159.47 μg/g). The activity-content contribution of myricetin was 7.69%. Due to the highest activity of apigenin (EC50 = 3.27 ± 0.09 μg/mL), the highest contribution of this flavone (29.96%) to the hydrolyzed extract of Orthosiphon aristatus (Blume) Miq. was observed.
Conclusion: The results revealed different contents and activities of xanthine oxidase inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants can play a major role to influence the activity.

Gout is an incurable disease although gout symptoms can be treated successfully. It is characterized by the precipitation of monosodium urate monohydrate crystals within joints, resulting from hyperuricemia (Dalbeth and So, 2010). The metabolism of xanthine and hypoxanthine to uric acid is catalyzed by xanthine oxidase. Therefore, one of the therapeutic approaches to treat gout is by using xanthine oxidase in- hibitors. A review by Abu Bakar et al. (2018) on the antigout potential of Malaysian medicinal plants has suggested further research on the xanthine oxidase inhibitors from plants.
Many studies have discovered higher xanthine oxidase inhibitory activity of the aglycones than that of glycosides. This is due to the steric interaction and glycosylation reduce the inhibitory effect (Cos et al.1998). Therefore, to increase the effect of plant extracts, it is crucial to remove the glycosides from the extracts by hydrolysis process. Caffeic acid (a hydroxycinnamic acid), apigenin (a flavone), myricetin, quer- cetin and kaempferol (flavonols) are aglycones that were reported to inhibit xanthine oxidase (Cos et al., 1998; Chang et al., 2007; ¨Ozyürek et al., 2009). Based on the structure-activity relationship analysis, the catechol moiety on the benzene ring of caffeic acid is the main attribute to the activity (Chang et al., 2007). Flavones and flavonols shared some characteristics for high inhibition such as having hydroxyl moieties at C7 and C5, double bond between C3 and C2, and carbonyl moiety at C4 ( ¨Ozyürek et al., 2009).
Quantification of these xanthine oxidase inhibitors from the active plant extracts is needed to determine their individual contribution to the activity of each extract. Twenty-one plant samples that were selected for this study, are traditionally used to treat gout in Malaysia, as reported by verifiable references such as Hien and Widodo (1999), Kiew (1999), Chooi (2003, 2004, 2006, 2008, 2011), and Samy et al. (2005). Table 1 describes the ethnomedicinal use of different plant parts to treat gout by local communities. Methods of treatment such as consumption of a fresh juice, infusion or decoction, and external application as a poultice of a paste or a moist powder, thermal patch of a heated leaf, or topical treatment of a seed oil or a fresh juice of the selected plants, are believed to assist in minimizing the pain and/or inflammation and/or may inhibit xanthine oxidase.
The present study was undertaken with aims (i) to comparatively determine the xanthine oxidase inhibitory activity of the 21 extracts, (ii) to quantify the potent inhibitors in the hydrolyzed extracts, (iii) to determine the activity-content contribution of the potent inhibitors to the potent extracts.

2.Materials and methods
Allopurinol (≥99%), apigenin (≥97%), caffeic acid (≥98%), dimethyl sulfoxide (DMSO; ≥99.9%), kaempferol (≥97%), myricetin96%), quercetin (≥98%), xanthine (≥99%), and xanthine oxidase (≥
were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were of analytical grade.

2.2.Plant materials and extraction
As indicated in Table 1, different parts of plants were used. The plants were identified by the corresponding author using published plant descriptions. The voucher specimens were compiled into one file (USM:SFS:1–20), and were deposited to the mini-herbarium at School of Pharmaceutical Sciences, Universiti Sains Malaysia. The plant names were checked with The Plant List (http://www.theplantlist.org.) to include the latest accepted plant names with authorities.
The plant materials were dried in an oven at 50 ◦ C for 3 days. The dried samples were then pulverized using an electrical grinder. The extraction process was carried out by boiling the pulverized samples in methanol and water (8:2, v/v) for an hour. The extracts were filtered using filter paper (Whatman no 1, UK) and dried using a rotary evapo- rator. The dried extract was then weighed and an initial concentration of each sample at 5 mg/mL was prepared using DMSO. All extracts were hydrolyzed with 2 M hydrochloric acid (20 mL) in boiling water (100 ◦ C) for 40min. Ethyl acetate was used for extracting the hydrolyzed extract.

2.3.Xanthine oxidase inhibition assay
The xanthine oxidase inhibition assay was performed as described by
Nguyen et al. (2004) with some changes. Each well of a 96-well plate contained 25 μL of sample [extract (at final concentrations of 156.25 μg/mL, 312.5 μg/mL and 625 μg/mL) or allopurinol (at final concen- trations ranging from 9.77 μg/mL to 625 μg/mL) or compound (at final concentrations from 1.96 μg/mL to 125 μg/mL)], 50 μL of xanthine oxidase (0.3U/mL) and 75 μL of 0.1 M sodium phosphate buffer (pH 7.5). After incubation for 15min, at 25 ◦ C, 50 μL of 0.6 mM xanthine was added. The absorbance was determined at 290 nm using a Multiskan Spectrum (Thermo Scientific, Finland), after incubation for 30min, at 25 ◦ C. Allopurinol was used as positive control and DMSO was used as negative control. The inhibitory percentage was calculated as follows
= [(absorbance of negative control – absorbance of sample)/absorbance of negative control] x 100%. Only extracts with ≥50% activity at final concentration of 625 μg/mL were serially diluted (final concentrations ranging from 19.53 μg/mL to 625 μg/mL) to determine the EC50 (half maximal effective concentration).

2.4.Quantitative analysis of the compounds
The hydrolyzed extracts (2.5 mg/mL) were filtered using a syringe filter (0.2 μm). Ultra performance liquid chromatography (UPLC) [with a photo-diode array detector (PDA) and a reverse-phase Acquity BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.)] (Waters Acquity, Milford, MA) was used to quantify the compounds in the extracts. The fl ow rate was 0.2 mL/min and the injection volume was 5 μL. The mobile phase composed of solvent A (methanol/acetic acid/water; 18:1:1) and solvent B (2% acetic acid) with a gradient mode that was initially set at A/B ratio of 15:85 and then linearly increased to 35:65 at 1.5min, 70:30 at 3min, 95:5 at 4min and 15:85 at 5.5min until 7.0min for re-equilibration of the column. For the quantification of caffeic acid, the PDA detector was set at 320 nm, and for apigenin, myricetin, quercetin and kaempferol, it was set at 350 nm. The quantification was performed by comparison of their UV spectra (detected by the PDA detector) and retention times with those of standards. A standard calibration curve of different concen- trations of each standard was plotted. The concentration of the com- pound in each extract was calculated using the regression equation of its peak area to the peak area of a known concentration of standard from the calibration curve. The results were expressed as micrograms of each compound per 1 g of extract (μg/g extract).
The quantification method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ) precision and recovery (Juan et al., 2015). The calibration curves exhibited good linear trends with correlation coefficients (r2) higher than 0.999 for all compounds (Table 2(i)). The LOD and LOQ values for each compound were deter- mined in the range of 0.136–0.285 μg/mL and 0.413–0.864 μg/mL, respectively, by the analysis of each standard solution at known con- centrations of the analytes. Precision of the chromatographic procedure was measured by repeating analyses of the standard solution on same day and on three different days. The relative standard deviations (RSD) of intra- and inter-day precisions for the compounds were below 0.789% and 3.18%, respectively. The recoveries of these compounds were detected approximately 100%. The results strongly suggest the applied method is accurate and reproducible to quantify the compounds for this study. The activity-content contribution (%) of the compounds to potent extracts were also determined.an unsupervised multivariate method, was performed to process the acquired and normalized data using SIMCA-P+ software version 14.1 (Umetrics, Umea, Sweden).an unsu- pervised multivariate method, was performed to process the acquired and normalized data using SIMCA-P+ software version 14.1 (Umetrics, Umea, Sweden).

2.5.Statistical analysis
All experiments were carried out in triplicate. Results were expressed as mean ± standard deviation. Statistical analysis was determined by one way analysis of variance (ANOVA) followed by Duncan’s test using SPSS version 16.0 software (SPSS Inc., Chicago, IL) and the differences were considered as significant with p < 0.05. 3.Results and discussion As depicted in Table 1, only six extracts were found to demonstrate inhibitions ≥50%. The extract of Syzygium aromaticum Merr. & L.M. Perry shows the highest xanthine oxidase inhibitory effect with the in- hibition percentage at 84.13 ± 0.47% (EC50 = 68.51 ± 0.85 μg/mL). After the extract was hydrolyzed, the EC50 was decreased to 39.58 ± 0.10 μg/mL. The antigout property of this plant is often associated with the anti-inflammatory and pain-relieving effects of its main constituent (eugenol). However, this compound was found to have weak xanthine oxidase inhibition (EC50 = 300 μM) (Jeng et al., 1994). As indicated in Table 2(ii&iii), the highest content of myricetin was quantified from S. aromaticum (42,297.55 ± 159.47 μg/g). Myricetin was found to be the second-most potent inhibitor with EC50 = 10.89 ± 0.12 μg/mL. Ac- cording to Cos et al. (1998), myricetin was categorized as xanthine ox- idase inhibitor with superoxide scavenging activity. Myricetin activity-content contribution for this extract was 7.69% (Table 2(iv)). This extract also has high contents of quercetin and kaempferol. The synergistic effect of these flavonols may contribute the xanthine oxidase inhibitory activity of this extract. Many studies had reported lower xanthine oxidase activity of fla- vonols than that of flavones. Although kaempferol shares a similar orientation as apigenin to the active site, the weak interaction may arise from the unstable effect of the substitution of hydroxyl moiety at C3 ( ¨Ozyürek et al., 2009). As indicated in Table 2(ii&iii), apigenin was found to be the most potent inhibitor with EC50 = 3.27 ± 0.09 μg/mL. It was quantified from Orthosiphon aristatus (Blume) Miq. extract (18,46030.24 μg/g). The highest activity-content contribution of apigenin±(29.96%) to the extract was observed (Table 2(iv)). Bioassay-guided isolation of antigout plants (such as Glechoma grandis (A.Gray) Kuprian) had identified apigenin as the most effective xanthine oxidase in- hibitor (Masuda et al., 2013). Apigenin was also considered as one of the key xanthine oxidase inhibitors in Chrysanthemum morifolium Ramat. (Peng et al., 2020).
Owing to the highest caffeic acid content (39,172.16 ± 303.68 μg/g) in O. aristatus and its EC50 = 12.97 ± 0.02 μg/mL, this compound activity-content contribution was 16.03% (Table 2(ii,iii&iv)). Many studies revealed rosmarinic acid with weak xanthine oxidase inhibition (EC50 > 200 μM) as a major constituent of O. aristatus (Masuda et al., 2013). The moderate activity (EC50 = 106.14 ± 0.61 μg/mL; Table 1) of this hydrolyzed extract could be due to the antagonist effect of its compounds. The root extract of Morinda citrifolia L. was ranked second in the caffeic acid content (16,785.29 ± 57.18 μg/g; Table 2(ii)) as well as the xanthine oxidase inhibition (EC50 = 61.44 ± 1.08 μg/mL; Table 1). It is known to be a rich source of anthraquinones such as purpurin. However, this anthraquinone has moderate xanthine oxidase inhibition
(EC50 = 105.13 μM) (Sheu and Chiang, 1997).
Low activities of the dried leaf extracts of Brassica oleracea varieties and Gnetum gnemon L. could be due to the application of their heated fresh leaves as thermal patch to treat gout (Chooi, 2011). The weak activities of the dried bulb of Allium sativum L. and dried tuber of Pachyrhizus erosus (L.) Urb. extracts could be due to the antigout values of the fresh preparation (Chooi, 2003, 2008). The plant extracts with no activity might alleviate gout through other mechanisms such as by triggering warm or cool effects to the inflamed joints for excreting dampness and ameliorating pain.
In conclusion, our results revealed different xanthine oxidase in- hibitions of different plant extracts. Further experiments to determine the antagonistic, synergistic and/or potentiation involvements of different contents of compounds in different extracts that act as potent xanthine oxidase inhibitors are greatly needed.

The authors would like to acknowledge the Universiti Sains Malaysia, Malaysia for providing the laboratory facilities and financing the project (research university grant: 1001/PBIOLOGI/811281).

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