Evaluation Of Uric Acid-lowering Effect Of Mulberry Flavonoids And The Development Of Uric Acid-lowering Tea Substitute

Uric acid（UA）in the body is mainly produced through endogenous purine metabolism and food intake of nucleotides.When there is an imbalance between the production and excretion of uric acid,the accumulation of uric acid in the body causes the blood uric acid level to rise above a certain level,which is called hyperuricemia（HUA）.Long-term HUA can easily lead to chronic kidney disease,cardiovascular disease,type II diabetes,and a series of other diseases.The current clinical uric acid-lowering drugs not only require long-term administration but also all have strong toxic side effects.Therefore,it is of great importance to search low toxic and efficient natural active ingredients to intervene in hyperuricemia.As a medicinal fungus,mulberry is rich in flavonoids and other bioactive components.Numerous studies have shown that flavonoids can effectively inhibit the activity of Xanthine Oxidase（XO）,reduce the production of uric acid and have potential nephroprotective effects.However,there are differences in the physiological activity of mulberry due to different varieties,growth years,cultivation methods and growing regions.At present,few literatures have reported on mulberry to reduce uric acid,and they mainly focused on the inhibitory effect of a single mulberry species on XO.There are few reports on the comparison of the effect of different mulberry and different extracts of the same mulberry to reduce uric acid.Coupled with the fact that there are few functional mulberry products available in the market for people with high uric acid,which provides the possibility of developing mulberry products to reduce uric acid.Based on the above research background,this study collected nine mulberry seeds of different origins,varieties,cultivation patterns and growth years,and systematically determined the differences in the contents of four bioactive components,namely,total flavonoids,total phenols,triterpene acids and polysaccharides.The differences in the moisture content of the samples and the differences in the in vitro uric acid-lowering effects of the extracts among the nine samples were also measured.On this basis,screening was conducted to further investigate the effects of different mulberry extracts on XO,we investigated the potential mechanisms of in vivo uric acid lowering through animal models of hyperuricemia in order to screen out the best varieties and develop tea substitutes that can reduce uric acid levels.The main research contents and conclusions are as follows:（1）By comparing a variety of flavonoid extraction methods,and combining the laboratory conditions and extraction efficiency,we finally decided to choose the ultrasonic-assisted ethanol extraction to gain mulberry alcohol extracts and ultrasonic-assisted hot water to gain mulberry water extracts.The results showed that the yield of the alcoholic extract of mulberry was generally higher than that of the water extract,which may be due to the fact that the flavonoids are more soluble in ethanol.By calculating the yield of crude extracts,it was found that No.2,No.4,No.5,No.7 and No.9 mulberry alcohol extracts had a higher yield,and No.2,No.7 and No.9mulberry water extracts had a higher yield.This result may provide a basis for subsequent comparison of the flavonoid content of mulberry.（2）The total flavonoid content,total phenolic content,triterpene acid content and polysaccharide content of the nine mulberry alcoholic extracts and water extracts were determined by chemical methods.The results showed that there were significant differences in the total flavonoid contents of the nine species of mulberries,which might be related to many factors such as the variety of mulberries,growing age,cultivation method and growing area.The total flavonoid content was the highest in the No.5 mulberry,while the content of the remaining eight varieties of mulberries was relatively small and did not differ significantly.The total phenolic content was the highest in No.5 and No.9 mulberries,at 18.74±2.87 mg/g and 18.55±2.15 mg/g,while the differences in the content of the remaining seven mulberries were small.The triterpene acids and polysaccharides were measured to be low,which was greatly related to the extraction conditions of the crude extracts.The contents of all the above four bioactive components were not much different from those measured by other researchers and have a reference value.（3）The inhibition effect of the alcoholic extracts and water extracts of mulberries on XO was compared by in vitro enzyme activity inhibition experiments,and the correlation analysis was done between the XO inhibition rate and the content of bioactive components.The results showed that both alcoholic and water extracts had certain inhibitory effects on XO activity and showed a dose-effect relationship.The inhibition effects of alcoholic and water extracts were significantly different,and the inhibition effects of the nine alcoholic extracts were generally higher than those of the water extracts.Among the alcoholic extracts,No.5 and No.9 mulberry varieties had the highest inhibition rate on XO,and the half inhibition(IC₅₀)were 0.03±0.01mg/m L and 0.02±0.01 mg/m L.No.9 mulberry have shown the best inhibiting ability among the water extract of mulberry,and the IC₅₀of No.9 mulberry was 0.1±0.01mg/m L.The correlation analysis results showed that the inhibition effects of each mulberry extract on XO were highly significant and negatively correlated with its total flavonoid and total phenolic contents（p<0.01）.Therefore,it is assumed that in addition to flavonoids,phenolic substances may also inhibit XO activity.（4）Through the above experimental screening,it is concluded that No.5 and No.9 mulberry are the most effective in lowering uric acid,and the reversibility of enzyme activity inhibition and the type of inhibition are conducted.The results of the inhibition reversibility experiments showed that all three lines in the inhibition images of the different extracts intersected at the origin,indicating that the inhibition of XO by the measured samples was reversible.The results of the inhibition type experiments showed that all the different extracts inhibited XO in a mixed manner.After that,by comparing K_iand K_is,the No.5 mulberry alcohol extract,No.5 mulberry water extract and No.9 mulberry water extract were more likely to bind to the free enzyme to reduce the catalytic reaction rate.And the No.9 mulberry alcohol extract was more likely to bind to the enzyme-substrate complex to reduce the catalytic reaction rate.Considering other factors such as price and yield,No.5 mulberry was finally selected for the subsequent animal experiments on uric acid reduction.（5）The animal results showed that both the alcoholic extract and the water extract of No.5 mulberry had the effect of reducing uric acid level in mice.Compared with the alcoholic extract,the water extract had a better effect of reducing uric acid level,their uric acid values were reduced by about 40.61%and 49.17%compared to the model group,but there was no significant difference between them.The effect of XO inhibition in in vivo animal experiments is slightly different from that in vitro,which is probably due to the difference in absorption and metabolism of the active ingredient in vivo and the change in the target site of action.In addition,the alcoholic extract and the water extract of No.5 mulberry can effectively inhibit the activity of key enzymes of uric acid production pathway,which can inhibit the production of uric acid and its precursors in mice with hyperuricemia from the source and achieve the effect of reducing uric acid level.（6）The experiment was determined to add mulberry,goldenrod,cornus,poria,and gardenia as raw materials to develop mulberry uric acid lowering substitute tea.The best formulation of the tea substitute was determined by response surface experiment as follows:mulberry 2.01 g,goldenrod 1.23 g,corn mullein 1.6 g,poria0.5 g and gardenia 0.5 g.The best brewing condition was optimized by orthogonal test as follows:brewing at 80°C with 250 m L of water for 20 minutes.Through sensory evaluation and determination of the corresponding component content,it was determined that the best value of the tea substitute could be brought into play when the number of brewings was 4 times,and on this basis,the product standard of mulberry uric acid reduction tea substitute was established.In conclusion,mulberry extracts exert good in vitro XO inhibitory effect and in vivo uric acid lowering effect,while it can significantly reduce the urea nitrogen and creatinine content in mice,alleviate hyperuricemia-induced renal enlargement and effectively improve kidney injury.On this basis,mulberry extract was compounded with goldenrod,cornus,poria and gardenia to develop a substitute tea.This research can provide a reference for mulberry functional food to lower uric acid,and also to provide a scientific basis for mulberry flavonoids to lower uric acid.