| Hyperuricemia is one of the most common metabolic disorder diseases in humans causing deposition of urate crystals in the joints and/or kidneys and increasing the incidence of gouty arthritis, uric acid nephrolithiasis, cardiovascular and renal diseases, especially hypertension. Xanthine oxidase is the key enzyme that is responsible for the catabolism of purines in humans and contributes to hyperuricemia through catalyzing the oxidation of hypoxanthine to xanthine and then to uric acid. So xanthine oxidase inhibitor is an useful treatment for hyperuricemia, but the most commonly-used xanthine oxidase inhibitor(allopurinol) may present adverse effects such as allergic reactions, skin rashes, fever,hepatitis and nephropathy. Thus the treatment of hyperuricemia needs more effective and safer inhibitors of xanthine oxidase. Active substances in natural plants are the potential inhibitors of xanthine oxidase, and help to curb the oxidative stress reaction. Walnut(Juglans regia L.) is widely distributed all over the world, the shell of which accounts for about 30% of total weight and is largely produced in walnut processing industry.This research plans to study the antihyperuricemia activities of walnut shell extract in vivo and carry out in vitro xanthine oxidase inhibitory activity-guided separation and purification in the hope of finding xanthine oxidase inhibitory substances with a new structure.The main results are summarized as follows:(1) The catalytic conditions for assessing xanthine oxidase inhibitory activity were optimized, and the spectrophotometric and HPLC methods for determining xanthine oxidase inhibitory activities were compared in this study. The optimal conditions were as follows:0.24 mM xanthine, 0.01 U/mL xanthine oxidase, pH 7.5, 25 oC, 30 min for catalytic reaction and 80 μL 1.0 M HCl for stopping reaction. Both of the methods in a corresponding concentration range showed a good linear relationship and high intra-day precision, but the limit of detection and limit of quantification by HPLC was one order of magnitude lower than the spectrophotometric method, thus the HPLC method was more sensitive. The polyphenols with a maximum absorption around 292 nm and inhibitors with a weak xanthine oxidase inhibitory activity had a higher inhibition ratio by the spectrophotometric method than the real inhibition ratio. In comparison, the current HPLC method eliminated interferences from substrate and inhibitors, and could be used for analyzing without temperature control for at least 21 hours. Thus the HPLC method could be employed as a high output method for screening xanthine oxidase inhibitors due to each run requiring only 8 min. The HPLCmethod had inter-day relative standard deviation values within 0.10-1.50%, which indicated a high level of inter-day precision.(2) In order to obtain suitable raw material form walnut fruit and by-products, the antioxidant activities and xanthine oxidase inhibitory effects of the different parts of walnut fruit, stem and leaf extracts were comparaed systematically. The extraction rate of walnut shell was low(extraction rate about 2.80%), however, walnut shell extract showed the highest polyphenol content(14.81 ± 0.50 gallic acid equivalent g / 100 g) and optimal antioxidant activities(ORAC 3423.44 ± 142.52 μmol TE/g, reducing force 115.86 ± 2.05 g / mL), and xanthine oxidase inhibitory activity only after the walnut green husk extract(the 50%inhibition concentrations of walnut shell and walnut green husk extracts were 988.36 ± 31.31μg / mL and 616.86 ± 17.84 μg / mL, respectively). These results confirmed that walnut shell extract had a potential application value in health food industry. There were certain differences between the spectra of different parts of walnut fruit(green husk, shell and defatted walnut kernel), stem and leaf extracts, but It could be speculated that they had the same characteristic peaks, which also indicated that the main composition of these samples were similar.(3) The antihyperuricemic activities of walnut shell extract in an animal model was studied, and xanthine oxidase inhibitory activity-guided purification was carried out in vitro.Stock diet was given at a dose of 166 mg/kg for 30 days, and walnut shell extract had no adverse effect on rat weights and the serum uric acid levels of the rats were obviously lower than those of the rats in model control group(walnut shell extract group vs model control group; 20 d, p < 0.05; 30 d, p < 0.05). Additionally, the levels of serum creatinine and urea nitrogen were significantly suppressed, suggesting that walnut shell extract possessed in vivo antihyperuricemia and nephroprotective effects(walnut shell extract group vs model control group; p < 0.05 and p < 0.01, respectively). The p-coumaric aldehyde, β-D-daucosterol,hydroxy benzaldehyde and juglone were obtained through the activity-guided separation and purification, and the p-coumaric aldehyde showed the best xanthine oxidase inhibitory activity with a mixed inhibition type. There was a synergy effect between p-coumaric aldehyde and allopurinol. The walnut shell extract and p-coumaric aldehyde could be used as active substances for the prevention and treatment for hyperuricemia and gout.(4) Structure-xanthine oxidase inhibitory activity was studied based on p-coumaric aldehyde and its analogues, and the inhibitory mechanism of p-coumaric aldehyde on xanthine oxidase was further discussed. In vitro xanthine oxidase inhibitory activitity and computational docking studies indicated that the aldehyde group, double bond and hydroxyl group at C-4 ofp-coumaric aldehyde were required for xanthine oxidase inhibitory activity. The major interactions included arene-arene interactions(between p-coumaric aldehyde and Phe-914,Phe-1009, respectively), two hydrogen bonds(between hydroxyl group and Glu-1261,Arg-880, respectively), two hydrogen bonds(between aldehyde group and Thr-1010,Val-1011, respectively). Structure-xanthine oxidase inhibitory activity of p-coumaric aldehyde was further explored by changing the position of groups on p-coumaric aldehyde and introducing new groups, meanwhile, and the results showed that development of stronger inhibitors based on p-coumaric aldehyde, introduction of a chemical group should aim to enhance the eletrophilicity of the aldehyde and keep the linear stuctrue. The importance of eletrophilicity of the aldehyde to xanthine oxidase inhibitory activity was further comfirmed by the inhibitory mechanism of p-coumaric aldehyde on xanthine oxidase... |
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