Xanthine oxidase inhibitory activity of a new isocoumarin obtained from Marantodes pumilum var. pumila leaves

Background In traditional Malay medicine, Marantodes pumilum (Blume) Kuntze (family Primulaceae) is commonly used by women to treat parturition, flatulence, dysentery, dysmenorrhea, gonorrhea, and bone diseases. Preliminary screening of some Primulaceae species showed that they possess xanthine oxidase inhibitory activity. Thus, this study aimed to investigate the xanthine oxidase inhibitory activity of three varieties of M. pumilum and their phytochemical compounds. Method Dichloromethane, methanol, and water extracts of the leaves and roots of M. pumilum var. alata, M. pumilum var. pumila, and M. pumilum var. lanceolata were tested using an in vitro xanthine oxidase inhibitory assay. Bioassay-guided fractionation and isolation were carried out on the most active extract using chromatographic techniques. The structures of the isolated compounds were determined using spectroscopic techniques. Results The most active dichloromethane extract of M. pumilum var. pumila leaves (IC50 = 161.6 μg/mL) yielded one new compound, 3,7-dihydroxy-5-methoxy-4,8-dimethyl-isocoumarin (1), and five known compounds, viz. ardisiaquinone A (2), maesanin (3), stigmasterol (4), tetracosane (5), and margaric acid (6). The new compound was found to be the most active xanthine oxidase inhibitor with an IC50 value of 0.66 ± 0.01 μg/mL, which was not significantly different (p > 0.05) from that of the positive control, allopurinol (IC50 = 0.24 ± 0.00 μg/mL). Conclusion This study suggests that the new compound 3,7-dihydroxy-5-methoxy-4,8-dimethyl-isocoumarin (1), which was isolated from the dichloromethane extract of M. pumilum var. pumila leaves, could be a potential xanthine oxidase inhibitor.


Background
Marantodes pumilum (Blume) Kuntze belongs to the Primulaceae family [1]. It was previously known as Labisia pumila (Blume) Fern.-Vill. from the Myrsinaceae family [2]. The taxonomic characteristics of eight varieties of M. pumilum have been described [3], and three of the varieties (var. alata Scheff., var. pumila, and var. lanceolata (Scheff.) Mez) are commonly used in Malaysia [2]. The close resemblance of var. alata and var. pumila leaves has made macromorphological identification quite difficult, as the leaf laminas of both varieties are either narrowly or broadly elliptic or ovate with 10-30 × 1.3-11 cm dimensions [3]. However, their petioles differ. The petiole of var. alata is 5-12 cm long and broadly winged (3-5 mm wide), whereas that of var. pumila is 4-15 cm long and slightly winged. Nonetheless, to differentiate them based on characteristic anatomical features and chemical profiling, a pharmacognostical study of these varieties was performed using microscopic, highperformance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC), and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) techniques [4].
Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid [24]. It plays a major role during the last step of purine nucleotide metabolism in humans, and serves as an important biological source of oxygen-derived free radicals. Free radicals can contribute to the oxidative damage to living tissues, which are involved in many pathological processes and various ischemic tissues, vascular injuries, and inflammation [25,26]. Xanthine oxidase is primarily distributed in the liver and intestine [27]. In humans, overproduction of xanthine oxidase elevates the blood stream uric acid concentration and leads to hyperuricemia [28]. Uric acid deposition begins when uric acid dissolves in the blood and forms urate monohydrate crystals in the joints and kidneys, leading to painful inflammation. Uric acid has been identified as a marker for gout and several metabolic and hemodynamic abnormalities [25,29,30]. Synthetic xanthine oxidase inhibitors such as allopurinol, febuxostat, and phenylpyrazol derivative Y-700, have been widely used to treat hyperuricemia and gout [27], but may have side effects. The extensively prescribed allopurinol has been reported to cause Stevens-Johnson syndrome, toxic epidermal necrolysis, hepatic disorders, and renal dysfunction [31]. Therefore, new alternatives such as medicinal plants, with fewer side effects, are desired [32,33].
Phytochemical constituents such as phenolics, flavonoids, coumarins, lignans, triterpenoids, and alkaloids have been reported to inhibit xanthine oxidase [27,[34][35][36]. Esculetin, a hydroxycoumarin derivative, displayed strong xanthine oxidase inhibitory activity [37] and was proposed as an appropriate bioactive quality control marker for a traditional Chinese medicine formula used in the treatment of hyperuricemia [38]. The extract of M. pumilum was reported to alleviate hyperuricemia in vivo [39]. Thus, in this study, potential xanthine oxidase inhibitors were determined by evaluating the xanthine oxidase inhibitory activity of M. pumilum varieties and isolated compounds using an in vitro assay. The compound could be used as an analytical marker for quality control purposes of M. pumilum-containing herbal products intended for hyperuricemia or gouty conditions.
For structural elucidation of the isolated compounds, ultraviolet (UV) spectra were recorded in ethanol using a Shimadzu UV1800 UV-Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan), and infra-red (IR) spectra were obtained using a Spectrum 100 FTIR spectrophotometer (PerkinElmer, Inc., Waltham, MA, USA) with an ATR technique. One-dimensional proton ( 1 H) and carbon ( 13 C) and two-dimensional nuclear magnetic resonance (NMR) spectra were determined using a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany), while high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and electron ionization mass spectrometry (EI-MS) spectra were obtained using an Ultimate 3000 system, MicrOTOF-Q II (Bruker Daltonics, Bremen, Germany).

Chemicals and reagents
Analytical grade organic solvents, including dichloromethane (DCM), methanol (MeOH), chloroform (CHCl 3 ), dimethyl sulfoxide (DMSO), hexane, ethyl acetate (EtOAc), toluene, acetone, and ethanol (EtOH), were purchased from Merck (Darmstadt, Germany). For the bioassay, allopurinol, xanthine, and xanthine oxidase (cow's milk) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA), while dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), sodium hydroxide (NaOH), and potassium dihydrogen phosphate (KH 2 PO 4 ) were purchased from Merck (Darmstadt, Germany). Leaves and roots (consisting of both stems and roots) of the fresh plants were separated and air-dried under shade. Following this, they were coarsely ground to obtain six powdered plant materials: var. alata leaves (0.2 kg) and roots (0.8 kg), var. pumila leaves (0.8 kg) and roots (2.0 kg), and var. lanceolata leaves (0.2 kg) and roots (0.5 kg). Within 1 week, each plant powder was successively macerated with dichloromethane in a powder-to-solvent ratio of 1:5, followed by methanol (ratio of 1:5). The methanol residue was refluxed with distilled water in a residue-to-solvent ratio of 1:13 for the leaves and 1:10 for the roots. The dichloromethane and methanol fluid extracts were vacuum-dried, and the water extracts were freeze-dried. This process resulted in eighteen dried extracts, which were stored in a refrigerator at 4°C until further analyses.

In vitro xanthine oxidase assay
The xanthine oxidase inhibitory assay was carried out using a previously reported method [40] with slight modifications. Initially, allopurinol (the positive control) and the dichloromethane and methanol extracts were dissolved in dimethyl sulfoxide (DMSO), and the water extracts were dissolved in distilled water. This was followed by dilution with potassium phosphate buffer (0.05 M, pH 7.5) to achieve the desired concentrations. Each test solution contained 0.5% DMSO. The assay was performed in triplicates in a 96-well microplate. The assay reaction mixture, which consisted of 130 μL of buffer, 10 μL of either test solution (400 μg/mL for extracts and 100 μg/mL for isolated compounds) or allopurinol (100 μg/mL), and 10 μL of xanthine oxidase (0.2 U/well), was incubated at 25°C for 15 min. Then, 100 μL of substrate solution, xanthine (0.15 mM, pH 7.5), was added before further incubating at 25°C for 10 min. The final assay mixture was spectrophotometrically measured at 295 nm. Xanthine oxidase inhibitory activity was expressed as the percentage of xanthine oxidase inhibition and calculated using the following formula: Where A is the optical density without the test solution or allopurinol, B is the optical density of blank solution containing only potassium phosphate buffer (0.05 M, pH 7.5), C is the optical density of the test solution or allopurinol with the presence of xanthine oxidase, and D is the optical density of the test solution or allopurinol without xanthine oxidase. Test solutions with more than 50% xanthine oxidase inhibition were reassayed at concentrations of 25, 50, 100, 200, and 400 μg/ mL for extracts, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for compound 1, and 6.25, 12.5, 25, 50, and 100 μg/mL for compound 2. Their half-maximal inhibitory concentration (IC 50 ) values were determined from percentages of xanthine oxidase inhibition of the respective concentration range using GraphPad Prism 5 software (La Jolla, CA, USA) and compared with that of allopurinol (0.0064, 0.032, 0.16, 0.8, 4, 20, and 100 μg/ mL).

Isolation and structural elucidation of compounds from M. pumilum var. pumila
The screening assay revealed that the dichloromethane extract of M. pumilum var. pumila leaves was most active. The extract (20.0 g) was fractionated by vacuum liquid chromatography using silica gel and gradient elution with increasing polarity mobile phase, that is, 3 L of hexane-ethyl acetate (9:1, 8:2, 7:3, 6:4, 5:5, 3:7, 2:8, and 1:9) followed by 2 L of 100% ethyl acetate and 2 L of 100% methanol. Eluents (250 mL each) were collected and combined based on the similarity of TLC profiles to obtain 16 fractions (CC1: F 1-16 ) (Fig. 1). The fractions were further fractionated using various chromatographic techniques with different solvent compositions to obtain six pure compounds.

Ardisiaquinone A (2)
The fraction CC1-F 15 (0.69 g) was fractionated using Sephadex LH-20 column chromatography with 1% methanol in chloroform to yield eight fractions. The third fraction was then eluted using silica gel column chromatography with chloroform-methanol (9:1) to obtain five fractions. The fourth fraction was triturated with hexane-methanol (1:1) to give compound 2. Compound 2 was obtained as a yellow powder (5.0 mg), and the data for its structural elucidation were as follows: UV (EtOH) λ max nm (log  Maesanin (3) The fraction CC1-F 3 (0.62 g) was fractionated using Sephadex LH-20 column chromatography with 1% methanol in chloroform to yield eight fractions. The fourth fraction was then eluted using silica gel column chromatography with chloroform-ethyl acetate (9:1), followed by trituration with hexane-methanol (1:1) to obtain compound 3.

Stigmasterol (4)
The fraction CC1-F 7 (2.0 g) was fractionated using Sephadex LH-20 column chromatography with 1% methanol in chloroform to yield ten fractions. The fifth fraction was purified by re-crystallization in methanol to yield compound 4. Tetracosane (5) The fraction CC1-F 1 (0.12 g) was precipitated to obtain compound 5. Compound 5 was obtained as a white

Margaric acid, (6)
The fraction CC1-F 10 (1.84 g) was fractionated using Sephadex LH-20 column chromatography with 1% methanol in chloroform to yield ten fractions. The fourth fraction was then eluted using silica gel column chromatography with chloroform-ethyl acetate (4:1) to obtain ten more fractions. The fourth fraction was further purified using silica gel column chromatography

Statistical analysis
Assay data obtained were subjected to one-way ANOVA with post-hoc Tukey's multiple comparisons test using GraphPad Prism 5 software (La Jolla, CA, USA). The data are expressed as mean ± standard error of the mean (S.E.M.) with triplicate measurements (n = 3). The difference between means was determined at 95% confidence intervals, with p value < 0.05 considered as significantly different.

Results
In vitro xanthine oxidase inhibitory activity of M. pumilum varieties Among the eighteen extracts assayed, five exhibited more than 50% xanthine oxidase inhibition, and their inhibitions were less than that of the positive control, allopurinol (99.82 ± 0.00%, IC 50 = 0.24 ± 0.00 μg/mL).  1 and 2). The dichloromethane extract of var. pumila leaves was considered to be more active than the other extracts because it had the highest percentage of xanthine oxidase inhibition and the lowest IC 50 value. Thus, the extract was subjected to further fractionation processes that led to the isolation of six pure compounds.

Discussion
The extract of M. pumilum var. pumila leaves inhibited xanthine oxidase in vitro. The findings of this study support the previous report [39] in which ethanol (80%) M. pumilum var. pumila leaf extract showed antihyperuricemic effect by inhibiting hepatic xanthine oxidase and reducing serum uric acid levels in hyperuricemic-induced male Sprague-Dawley rats 14 days after treatment with 200 mg/kg extract.
In this study, a new compound (3,7-dihydroxy-5-methoxy-4,8-dimethyl-isocoumarin) was isolated from the dichloromethane extract of M. pumilum var. pumila leaves, which was found to be the most active extract (IC 50 = 161.6 ± 7.35 μg/mL). The compound had an IC 50 value (0.66 ± 0.01 μg/mL) that was comparable to that of allopurinol (IC 50 = 0.24 ± 0.00 μg/mL) and could be a potential xanthine oxidase inhibitor. A study by Lin et al. [46] demonstrated competitive inhibition of selected coumarins (e.g., coumarin, 4-hydroxycoumarin, 7-hydroxycoumarin, esculetin, scopoletin, dihydrocoumarin, and 7-hydroxy-4-methylesculetin) against xanthine oxidase. Esculetin was found to be the most potent inhibitor through substrate binding blockade. It was suggested that the two hydroxyl moieties on its benzene ring contributed to its activity by forming hydrogen bonds with the active site of xanthine oxidase. Therefore, the presence of two hydroxyl groups in the structure of 3,7-dihydroxy-5methoxy-4,8-dimethyl-isocoumarin (1) could explain the basis of its xanthine oxidase inhibitory activity. Another study also reported that the xanthine oxidase inhibitory activity of 5,7-dihydroxy-3-(3-hydroxyphenyl) coumarin was 7fold better than that of allopurinol [47]. The low activity of ardisiaquinone A (2) and lack of activity of the other isolated compounds (3)(4)(5)(6) obtained in this study could be explained by the difference in molecular structure that influences the stability of hydrophilic and hydrophobic characteristics on the xanthine oxidase active binding site [48].
There are several reviews on the anti-hyperuricemic effects of foods [49], Chinese herbs [50], and natural products [51]. Hyperuricemia has been linked with cardiovascular disease, hypertension, diabetes, obesity, chronic kidney disease, and many other diseases [52,53]. Its prevalence in the female population and postmenopausal women has been reported [54][55][56]. The data from the Third National Health and Nutrition Examination Survey showed that menopause was associated with higher serum uric acid levels and postmenopausal hormone replacement was associated with lower serum uric acid levels, suggesting that estrogen plays a key role in protecting women from hyperuricemia and gout [57]. Several publications have reported on the potential use of M. pumilum extract to alleviate postmenopausal conditions due to estrogenic properties [58][59][60], hypercholesterolemia [61], and hypertension [62]. Thus, the extract of M. pumilum var. pumilum could be beneficial in preventing or treating hyperuricemic-related diseases, while 3,7-dihydroxy-5-methoxy-4,8-dimethylisocoumarin (1) could be used as an analytical marker to standardize the extract and formulated herbal products. Standardization by simultaneous quantification of xanthine oxidase inhibitors from Zanthoxylum armatum fruits using high-performance liquid