Inhibitory effect of verbascoside on xanthine oxidase activity

International Journal of Biological Macromolecules 93 (2016) 609–614

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Inhibitory effect of verbascoside on xanthine oxidase activity Yin Wan a,b , Bin Zou a , Hailong Zeng a , Lunning Zhang c , Ming Chen d , Guiming Fu a,b,∗ a

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China School of Food Science and Technology, Nanchang University, Nanchang 330047, China c Institute for Food and Drug Control, Pingxiang 337055, China d The First Affiliated Hospital of Nanchang University, Nanchang 330006, China b

a r t i c l e

i n f o

Article history: Received 2 June 2016 Received in revised form 5 September 2016 Accepted 7 September 2016 Available online 9 September 2016 Keywords: Verbascoside Xanthine oxidase Inhibitory activity Fluorescence chromatographic Circular dichroism spectra Molecular simulation

a b s t r a c t In this study, we analyzed the inhibitory effect of verbascoside against xanthine oxidase (XOD) in vitro by using animal model and in vivo by direct inhibition assay. Results showed that verbascoside could reduce uric acid in rat serum and inhibit XOD activity in rat liver. The IC50 value of restraining XOD activity was 81.11 mg mL−1 . Fluorescence chromatographic analysis and circular dichroism spectroscopy indicated that the secondary structures of XOD were changed after incubation with verbascoside. The docking simulation showed that verbascoside could enter into the active site of XOD and form hydrogen bonding with amino acid residues (such as Lys-1045, Arg-880, Arg-912, Glu-1261 and Gln-1194). The results suggested that verbascoside, which is a naturally occurring water-soluble antioxidant, could be a potential low-toxicity XOD inhibitor for hyperuricemia treatment. © 2016 Published by Elsevier B.V.

1. Introduction Hyperuricemia, which is characterized by an elevated level of uric acid in human blood, is considered as an underlying cause of gout [1]. It can cause serious damages in cells and in human body. Xanthine oxidase (XOD), a key enzyme in purine metabolic pathway, is widely distributed in mammalian tissues, especially in liver and intestinal tract. The enzyme is responsible for catalyzing hypoxanthine oxidation to xanthine, and then to uric acid (UA) [2]. UA is the final metabolite compounds in humans, excreted into urine by the kidneys. Therefore, XOD inhibition and UA reduction production could provide therapeutic approaches against hyperuricemia and gout. XOD is a molecular mass homodimer of approximately 290 kDa, and each monomer can act independently in catalysis [3]. Each subunit contains one flavin adenine dinucleotide cofactor, two spectroscopically distinct (2Fe-2S) centers, and one molybdopterin cofactor (Mo-pt) [4]. Catalytic reaction occurs at molybdopterin cofactor. Electrons are introduced and then rapidly distributed to other centers by intramolecular electron transfer [5]. Purine

∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China. E-mail address: [email protected] (G. Fu). http://dx.doi.org/10.1016/j.ijbiomac.2016.09.022 0141-8130/© 2016 Published by Elsevier B.V.

analogs are among the first widely used synthetic inhibitors for clinical treatments of hyperuricemia and gout. However, allopurinol, the most effective purine derivative inhibitor, can lead to many serious side effects, such as impaired liver function, renal failure, and allergic reactions [6]. We pointed out natural antioxidants by screening of novel alternatives to allopurinol with high-inhibition effect, but with fewer side effects. In the last decades, experiments have reported a number of antioxidants extracted from plants as effective XOD inhibitors experimentally, such as flavonoids and phenolic compounds. Despite of high promising inhibitory effects and less toxicity, flavonoids components are limited to pharmaceutical field on account of their poor aqueous solubility and scarce absorption due to phenolic nature [7]. Hence, a search for more soluble XOD inhibitors is needed. Phenylethanoid glycosides are a kind of naturally occurring water-soluble compound with wide distribution in the plant kingdom. Verbascoside is a typical phenylethanoid glycoside that has numerous biological activities, including antioxidant [8], antiinflammatory [9], hepatoprotective [10], immunoregulatory [11], and neuroprotective activities [12]. More than 150 types of plants, such as Plantaginaceae, Herba cistanche, and Phoenix tree flower, were discovered to contain verbascoside [13]. Generally, verbascoside can be considered as a polyphenolic compound of four chemical moieties: caffeic acid (CA), hydroxytyrosol (HT;

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phenylethanoid aglycone), glucose (central saccharide), and rhamnose group [14]. In our laboratory, phenylethanoid glycosides extract from seeds of Plantago asiatica L., a kind of traditional Chinese medicine herbs, was found to reduce blood uric acid level and inhibit liver XOD activity of hyperuricemic model rat. As one kind of phenylethanoid glycosides, verbascoside was separated and identified the structure from Plantago extracts [15]. Phenolic compound and CA have been reported to have an inhibitory effect on XOD activity [16]. Since these previous studies, we put forward a scientific hypothesis that verbascoside could inhibit XOD activity. In this study, verbascoside extracted from seeds of P. asiatica L. was reported for the first time to have XOD inhibition activity by in vivo and in vitro tests. Furthermore, inhibition mechanism was proposed after the interaction between verbascoside and enzyme was investigated by fluorescence titration and circular dichroism (CD) analysis. 2. Materials and methods

length with xanthine as the substrate, according to the report [17] with slight modification. In brief, assay solution series (0.2 mol L−1 PBS, pH 7.5) containing various amounts of verbascoside and fixed XOD concentration (0.1 U mL−1 ) were incubated for 15 min at 37 ◦ C. The assay was initiated by adding substrate xanthine (2.0 mmol L−1 final concentration) and NBT solution (0.02 m mol L−1 final concentration) to the verbascoside–XOD complex solution. The final volume of the reaction system was 5.0 mL, which was incubated for 30 min at 37 ◦ C. The absorbance at 560 nm wavelength (B) was collected by using a UV–vis spectrometer (UV-2450 Spectrophotometer, Shimadzu, Japan). PBS was used to replace verbascoside in blank group to obtain absorbance (A) through the same procedure. All assays were performed in triplicates. Inhibition rate to XOD activity was calculated by using the following equation: Inhibition rate% = (1 −

B ) × 100% A

(1)

The inhibition rates were presented as means ± standard deviation (n = 3). Furthermore, IC50 of verbascoside-inhibiting XOD activity was determined by using SPSS 16.0 software.

2.1. Reagents 2.5. Interaction of verbascoside and XOD in vitro Verbascoside was isolated from P. asiatica L. seeds in our laboratory (HPLC purity >98%) [15], and was dissolved in distilled water. Methanol (HPLC grade) was purchased from Honeywell International Inc. (Morris Plains, NJ, USA). Nitroblue tetrazolium chloride (NBT), xanthine, and allopurinol were purchased from Aladdin Industrial Corporation (Shanghai, China) and were dissolved in 0.2 mol L−1 sodium phosphate buffer (PBS; pH 7.5), respectively. XOD from bovine milk and oxygen oxazine acid potassium were purchased from Sigma-Aldrich (St. Louis, MO, United States), and XOD was dissolved in 0.2 mol L−1 PBS (pH 7.5). XOD and UA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents used in the study were of analytical grade. Only ultrapure water was used throughout the experiment. 2.2. Animals Male Sprague-Dawley (SD) rats, weighing 180–220 g, were obtained from Beijing Vital River Company (SCXK2012-0001). 2.3. Verbascoside effects on serum UA value and liver XOD activity in hyperuricemic rats Sixty SD rats were randomly divided into five groups, namely, normal, model, low-dose verbascoside (18 mg kg−1 wt.), high-dose verbascoside (54 mg kg−1 wt.), and allopurinol (positive control, 10 mg kg−1 wt.). Animals were fed with normal food, supplied with water, and acclimatized to the facilities for 3 days prior to the experiments. Normal rat group was fed with distilled water in the breeding process, while other rats were fed a dose of verbascoside or allopurinol, dissolved in distilled water by gavage, for 7 days. Hyperuricemia animal model was established by intraperitoneal administration of oxygen oxazine acid potassium before the last gavage. Rats were killed after 1 h, and their serum and livers were obtained. XOD activity in liver and UA value in serum of each group were determined with XOD and UA assay kits, respectively. Data were expressed as means ± standard deviation. One-way analysis of variance was performed by using Origin 8.0 to judge the significant difference at p < 0.05. 2.4. Effects of verbascoside on XOD activity in vitro XOD activity in vitro was measured spectrophotometrically by continuously measuring formazan formation at 560 nm wave-

2.5.1. Fluorescence titration Fluorescence titration assay was further used to analyze the interaction between verbascoside and XOD. Briefly, XOD mixture (0.1 U mL−1 ) and verbascoside with different concentrations (0–20 mg L−1 ) was incubated for 30 min. Afterwards, fluorescence emission spectra of each mixture were collected using Hitachi spectrofluorometer Model F-7000 (Hitachi Ltd., Chiyoda, Japan) in 290–500 nm wavelength range, under 280 nm excitation wavelengths. The widths of both excitation and emission slits were set at 5 nm. Background fluorescence of the buffer (0.2 mol L−1 PBS solution, pH 7.5) was deducted from verbascoside–XOD clathrate. Fluorescence-quenching data were analyzed by Stern–Volmer equation [18], which is as follows: F0 = 1+KSV [Q ] = 1+Kq ␶0 [Q ] F

(2)

F0 and F are steady-state fluorescence intensities of fluorophore in the absence and presence of quencher, respectively. KSV and [Q] are Stern–Volmer dynamic quenching constant and quencher concentration, respectively. Kq is quenching rate constant of biomolecule.  0 is average biomolecule lifetime (Kq = Ksv / 0 ), and its value is 10−8 s [19]. KSV can be obtained from linear regression plot slope of F0 /F versus [Q]. 2.5.2. CD spectrum analysis CD measurements of XOD were conducted by using BioLogic MOS 450 CD spectrometer (Bio-Logic Inc, Claix, France) in 180–260 nm wavelength range, under constant nitrogen flush. XOD concentration was fixed at 0.1 U mL−1 , while verbascoside concentrations were verified from 0, 20, 50, to 200 ␮g mL−1 . All observed CD spectra were corrected for buffer signal (0.2 mol L−1 PBS solution, pH 7.5), and results were expressed as ellipticity in millidegrees. Percentages of different secondary XOD structures were analyzed from CD spectroscopic data by an online server, Dichroweb (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml). 2.5.3. Molecular simulation AutoDock (version 4.2) was used to explore the probable binding site between XOD and verbascoside. The X-ray crystal structure of XOD from bovine milk (PDB ID code 3ETR, Protein Data Bank: http:// www.rcsb.org/pdb) was used for the docking studies [20]. Before the docking program running, the water molecules in XOD were removed, and then the polar hydrogen atoms and Gasteiger charges

Y. Wan et al. / International Journal of Biological Macromolecules 93 (2016) 609–614 Table 1 Effects of verbascoside on serum uric acid (UA) in hyperuricemic rats (n = 12).

611

Table 3 Results of inhibitory rates of different samples on XOD.

Group

UA(mg L−1 )

Group (Concentration)

Relative inhibition rate%

Normal Model Verbascoside (low dose) Verbascoside (high dose) Allopurinol (positive control)

30.2 ± 1.52 53.1 ± 2.86b 40.6 ± 2.02a , b 32.7 ± 3.54a 16.8 ± 1.14a , b

Blank Allopurinol (6.25 ␮g mL−1 ) Verbascoside (6.25 ␮g mL−1 ) Verbascoside (15.625 ␮g mL−1 ) Verbascoside (31.25 ␮g mL−1 ) Verbascoside (62.5 ␮g mL−1 ) Verbascoside (93.75 ␮g mL−1 ) Verbascoside (156.25 ␮g mL−1 )

0 87.13 ± 1.13 18.85 ± 1.58 32.39 ± 1.13 39.50 ± 0.34 48.31 ± 0.90 52.26 ± 0.79 55.42 ± 0.56

a b

Significant difference compared with the model group (p < 0.05). Significant difference compared with the normal group (p < 0.05).

Table 2 Effects of verbascoside on XOD in hyperuricemic rats (n = 12). Group

XOD (U gprot−1 )

Normal Model Verbascoside (low dose) Verbascoside (high dose) Allopurinol

25.2 ± 0.17 25.4 ± 0.23 23.8 ± 1.18 22.8 ± 0.69a , b 19.5 ± 1.13a , b

a b

Significant difference compared with model group (p < 0.05). Significant difference compared with normal group (p < 0.05).

were added to the macromolecule file. The 3D structures of verbascoside were obtained from National Center for Biotechnology Information (http://pubchem.ncbi.nlm.nih.gov). The dimension of docking center was set as 20 × 20 × 20 points. Pymol was used to obtain the visible combination models. 3. Results and discussion 3.1. Verbascoside effect on serum UA value and liver XOD activity in vivo 3.1.1. Verbascoside effects on serum UA value To examine potential verbascoside inhibition against XOD activity, verbascoside effect on serum UA production was analyzed with UA assay kit by in vivo lavage experiment. As results in Table 1 show, serum UA value in model group was remarkably higher than that in the normal group, which indicates that hyperuricemic animal model was established successfully. Serum UA concentrations in low-dose and high-dose verbascoside rat group, and allopurinol rat group (positive control) were significantly lower (p < 0.05) than that in model rat group. These results indicate that both verbascoside and allopurinol could significantly restrain serum UA value. In addition, serum UA concentration in high-dose verbascoside rat group was found significantly lower than that in low-dose rat group. Results indicate that verbascoside dose might be inversely correlated to serum UA value. As allopurinol is known to alleviate UA concentration by inhibiting XOD activity [6], it is possible that verbascoside might play a similar role, which results in decrease of serum UA value. Moreover, the serum UA concentration in high-dose verbascoside (54 mg kg−1 wt.) rat group was much closer to that in normal rat group. 3.1.2. Verbascoside effects on XOD activity in hyperuricemic rats XOD activity in rat liver extracts from each rat group was further examined. Results are shown in Table 2. XOD activity of normal and model groups were 25.2 ± 0.17 U gprot−1 and 25.4 ± 0.23 U gprot−1 , respectively, which meant that difference between them was not significant. However, XOD activity in high-dose verbascoside and allopurinol rat groups significantly decreased. Additionally, XOD activity decreased with the increase of verbascoside concentration by lavage. As expected, results from XOD activity measurement are

consistent with those from serum UA assay reported in Table 1. Therefore, high dose of verbascoside might have a certain inhibitory ability against XOD through in vivo analysis. 3.2. Effects of different verbascoside concentrations on XOD by in vitro analysis The effect of verbascoside toward pure XOD was then evaluated. The inhibitory effects of allopurinol and verbascoside with various concentrations on XOD activity are shown in Table 3. Inhibition rate increased significantly with the increase of verbascoside, and reached 55% when verbascoside concentration was 156.25 ␮g mL−1 . When inhibitor concentration was 6.25 ␮g mL−1 , inhibition rates of verbascoside and allopurinol on XOD were 18.85 ± 1.58% and 87.13 ± 1.13%, respectively. Using the formula by SPSS 16.0 software shown in Fig. 1, verbascoside concentration, which led to 50% activity loss (IC50 ), was estimated to be 81.11 ␮g mL−1 . Results indicate that XOD inhibitory activity of verbascoside was weaker than that of allopurinol. 3.3. Interaction of verbascoside with XOD in vitro 3.3.1. Fluorescence chromatographic analysis Fluorescence chromatographic analysis was further used to investigate the binding mechanism of verbascosides to XOD. XOD fluorescence spectra, in the absence and presence of verbascoside under 280 nm excitation wavelength, are shown in Fig. 2. XOD had two strong fluorescence emissions at 341 nm and 405 nm wavelengths [21]. For XOD, three intrinsic fluorophores exist, namely, tryptophan, tyrosine, and phenylalanine [21]. As phenylalanine has low quantum yield, and tyrosine fluorescence is almost completely quenched when it is near an amino group, a carboxyl group, or a tryptophan, change in intrinsic fluorescence intensity of XOD will be mostly due to tryptophan residues and slightly attributed to tyrosine residues [22]. Under the same experimental conditions, verbascoside has no intrinsic fluorescence. With increase of verbascoside concentrations, XOD emission intensities both at 341 nm and 405 nm decreased without any significant peak shift. The phenomenon indicates that verbascoside could directly interact with XOD and quench its intrinsic fluorescence. Furthermore, Ksv and Kq values with maximal emission at 340 nm were calculated to be 37,510 L mol−1 and 3.75 × 1012 L mol−1 s−1 , respectively. Kq value was far above the threshold of scattering collisional quenching constant (2.0 × 10−10 s) in water [23], which implies that fluorescence quenching mechanism of XOD by verbascoside is static quenching rather than dynamic quenching, i.e., fluorescent compounds were converted into non-fluorescent compounds [24]. 3.3.2. CD spectrum detection Following the fluorescent experiment, we further analyzed CD spectra of XOD, in the absence and presence of verbascoside, to look into the possible mechanism of interaction between verbascoside and XOD. As shown in Fig. 3, CD spectra of XOD were characterized mainly by two negative bands at 208 nm and 222 nm, which

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Fig. 1. Effects of different relative concentrations of verbascoside on XOD.

Relative fluorescence intensity

1400

XOD chain and destroyed hydrogen-bonding networks [27]. Meanwhile, ␤-sheet content increased notably. Results indicate that verbascoside caused rearrangement and conformational changes of XOD molecular structure.

a

1200 1000 800

k

600 400 200 0 250

300

350

400

450

500

Wavelenth(nm) Fig. 2. Fluorescence spectra of XOD in the presence of verbascoside at different concentrations. c (XOD) = 0.1 U mL−1 ; and c(verbascoside) = 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 ␮g mL−1 for curves a → k, respectively.

Table 4 Effects of verbascoside on secondary structure of xanthine oxidase. Verbascoside (mg L−1 )

␣-helix (%)

␤-sheet (%)

␤-turn (%)

random coil (%)

0 20 50 200

40.16 24.12 18.98 2.01

0.90 31.26 40.92 43.44

22.58 20.40 20.31 21.62

36.26 24.32 19.79 33.13

3.3.3. Computational docking of the verbascoside–XOD complex Molecular docking was used to improve the understanding of the interaction between verbascoside and XOD. As shown in Fig. 4(a), the docking simulation proposed that verbascoside could be located well in the hydrophobic pocket of XOD (molybdopterin domain) with the lowest binding energy of −6.0 kcal mol−1 . As shown in Fig. 4(b), one hydrogen bond was formed between the oxygen atom from CA group and the hydrogen atom of Lys-1045 of XOD. Two hydrogen bonds were formed between the hydrogen atom from HT group and the oxygen atom of Glu-1261 of XOD, as well as between the oxygen atom from HT group and the hydrogen atom of Arg-880 of XOD, respectively. All of these suggest that weak hydrogen bond interaction existed between XOD and verbascoside. The interaction was similar with the previous moleculardocking analysis on the interaction between CA and XOD [28]. Ortho-position phenolic hydroxyl group of CA and HT could combine XOD molybdopterin domain with hydrogen bonds. Two aromatic rings from its two-phenethyl ester groups also come close to hydrophobic XOD residues to establish hydrophobic stabilizing forces, which eventually contribute stronger XOD-inhibiting activity. Therefore, verbascoside, as glycoside derivative of CA and HT, which has two phenyl rings with ortho-position phenolic hydroxyl group, inhibit XOD in the same mode, i.e., by altering hydrogenbond network in XOD, resulted in conformation changes. 4. Conclusions

were caused by a negative Cotton effect characteristic of ␣-helical structure [25]. These negative bands were rationalized by n → * transition in ␣-helical peptide bond [26]. With the increase of verbascoside concentrations, CD intensity in both peaks decreased without any significant peak shift, indicating the decrease of ␣helical content in protein. Contents of different secondary XOD structures were calculated by Dichroweb, and results are listed in Table 4. After verbascoside was added, proportion of different secondary XOD structures changed significantly. With the increase of verbascoside concentrations, ␣-helix content declined remarkably. Verbascoside was indicated to be bound with amino acid residues of main polypeptide

In summary, XOD can be inhibited by verbascoside, which is a naturally occurring water-soluble antioxidant extracted from P. asiatica L. seeds. In vivo results showed that verbascoside could significantly reduce uric acid concentration in hyperuricemic animal model and inhibit XOD activity. Also, 54 mg kg−1 verbascoside weight dose could reduce serum UA value to normal concentration in hyperuricemic rats. Meanwhile, IC50 value of restraining XOD activity was found as 81.15 ␮g mL−1 by in vitro experiment. XOD activity inhibition was demonstrated by fluorescence titration assay and CD analysis. Mechanism inhibition of XOD activity with verbascoside was suggested to be attributed to phenyl rings of verbascoside complete with Trp to combine with molybdopterin

Y. Wan et al. / International Journal of Biological Macromolecules 93 (2016) 609–614

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Fig. 3. CD spectra of XOD in the absence and presence of increasing amounts of verbascoside. c (XOD) = 0.1 U mL−1 ; and c (verbascoside) = 0, 20, 50, and 200 ␮g mL−1 for curves 1 → 4, respectively.

Dr. Longyan Chen (Department of Mechanical Engineering, McGill University), who polished this paper.

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Fig. 4. The docking models of xanthine oxidase inhibition by verbascoside: (a) predicted binding mode of verbascoside docked into xanthine oxidase’s active site; (b) verbascoside interacted with the amino acid residues within the active site of xanthine oxidase.

domain in XOD, which subsequently destroyed hydrogen-bond network of XOD and changed its conformation. Our results indicate the considerable potentials of verbascoside from Plantago, a food based ingredient, to be a dietary supplement and an assisted additive to allopurinol for treatments of gout and other hyperuricemic conditions. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 31160316) and Nanchang University Testing Fund (No. 2008042). The authors also appreciate the work of

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