| Hyperuricemia(HUA)is a chronic metabolic disease caused by metabolic disorder of purine nucleotide or uric acid excretion dysfunction in the body,with the incidence rising in many countries.At present,there is not effective treatments in clinical practice,and anti-gout drugs such as allopurinol,febuxostat,benzbromarone are mostly used.However,these drugs can not only regulate uric acid,but also lead to the adverse effects of metabolic disease complications.And the continuous use of these drugs can also lead to liver and kidney damage,which greatly limits their clinical application.Traditional Chinese medicines have the following characteristics:multiple ingredients,multiple targets and multiple ways to be effective.From the perspective of the whole,it has many advantages for the treatment of HUA,such as treatment according to syndrome differentiation,treating both manifestation and root cause of disease,and rarely induced side effects.In this study,Paecilomyces Cicadidae and Astragalus membranaceus were selected as materials for bi-direction fermentation.The Paecilomyces Cicadidae has the effect of warming kidney and enhancing yang and the Astragalus membranaceu could replenish qi to invigorate the spleen.Therefore,using Astragalus membranaceus as the medicinal substrate and Paecilomyces cicadae as the fermentation strain to prepare Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance.The single-factor test and orthogonal experiments were used to optimize the fermentation substrate enzymatic hydrolysis conditions and the bidirectional fermentation technology.At the same time,the chemical composition and anti-hyperuricemia activity of the fermentation fungal substance were preliminary studied.Besides,the metabonomics technology based on ultra high performance liquid chromatography-mass spectrometry technology was used to further study the interference effect of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance on HUA rats.It provides a theoretical basis and reference for the development of fermentation related product.The main contents and results were described as follows:1.Study on the application rationality of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance.The rationality of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance in the treatment of HUA was elaborated from three aspects:network pharmacology,clinical principles and bidirectional fermentation theory of traditional Chinese medicine.The results showed that Astragalus membranaceus can regulate HIF-1,MAPK,PI3K/Akt,VEGF,AMPK/p53 and insulin pathways,which are related to inflammation and oxidative stress,to play an anti-hyperuricemia role through the joint action of astragalosides,luteolin,genistein,genistein and genistein.Paecilomyces cicadae/Astragalus membranaceus has the effect on reinforcing kidney and invigorate spleen.The combination of them can benefit Qi,warm the kidney,ascending lucidity,descending turbidity,promoting diuresis and clearing the arthralgia,which is in accordance with the therapeutic principle of HUA in the traditional Chinese medical principles.Besides,the fermentation treatment of Astragalus membranaceus with Paecilomyces Cicadidae can improve the efficacy and utilization rate of Astragalus membranaceus,and provide a strongly scientific foundation of the sequential studies.2.Optimization of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance processing.In this part,taking the content of astragaloside Ⅳ in the fermentation product as an index,using Astragalus membranaceus as the medicinal substrate and Paecilomyces cicadae as the fermentation strain to evaluate Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance.The single factor test and orthogonal test optimization methods were used to optimize enzymolysis technology and fermentation technology.Firstly,investigating the effects of the amount of hydrolytic enzyme,enzymolysis time and enzymolysis temperature on the results of Astragalus membranaceus.The optimum conditions were determined as follows:0.3%of hydrolytic enzyme,50℃ of enzymolysis temperature and 120 min of enzymolysis time.Then,the amount of Astragalus membranaceus,the inoculation amount of Paecilomyces cicadae,the initial pH and the fermentation time on the fungal substance were investigated.The optimal fermentation process were determined as follows:the amount of Astragalus membranaceu addition 20%,the inoculation amount of Paecilomyces cicada 5 mL·100 mL-1,the initial pH was 6.5 and the fermentation time was 7 days.3.Study on change of chemical component in Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance.In this part,the contents of polysaccharide and total flavonoids were determined by sulfuric acid anthrone colorimetry and NaNO2-Al(NO3)3 colorimetry.The results showed that the content of total polysaccharide increased by 1.37 times as much as that of Astragalus group,and the content of total flavonoids decreased by 1/3 as much as that of Astragalus group.In addition,based on UHPLC-LTQ-Orbitrap MS technology,the chemical components of Astragalus membranaceus and fermentation fungal substance were screened and identified.Combined with chromatographic retention behavior,accurate molecular weight,diagnostic fragment ions and related literature reports,107 chemical components were analyzed and identified,including 42 saponins and 65 flavonoids.Specifically,the number of saponins decreased from 42 to 29 after fermentation and the content of long-chain saponins saponins decreased significantly.The content of secondary glycosides or aglycones increased significantly.The content of flavonoids increased from 47 to 52,which was consistent with the determination results of total flavonoids.4.Anti-hyperuricemia activity of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance.The male SD rats were randomly divided into a blank group,HUA model group,fermentation fungal substance high-dose and low-dose group,and Astragalus high-dose and low-dose group.The blank group was orally administered with distilled water;the other groups were orally administered with 300 mg·kg-1 potassium oxazinate for modeling.One hour after the establishment of the model,20 mg·kg-1 benzbromarone was given to the positive drug group,150 mg·kg-1,300 mg·kg-1 Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance was given to fungal group and 150 mg·kg-1,300 mg·kg-1 Astragalus was given to Astragalus group,respectively.Rats serum were collected after 14 days of administration.Using Automatic Biochemistry Analyzer to detect uric acid(UA),total cholesterol(TC),triglyceride(TG),creatinine(CRE),urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Compared with the blank group,the level of UA,BUN,TC,TG,ALT,and AST in the serum of the HUA model group were significantly increased(p<0.05).Compared with the HUA model group,the levels of UA,BUN,TC,TG,ALT and AST were significantly reduced in the serum of the Astragalus group and the fermentation fungal group(p<0.05 or p<0.01),and the effect of the fermentation fungal group was better than that of the Astragalus group.5.Metabolomics study of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance based on UHPLC-HRMS technology for intervention of hyperuricemia.UHPLC-Q-Exactive was used to determine the profile of rat plasma metabolites.Orthogonal Partial Least-Squares Discriminant Analysis(OPLS-DA)was used to study the phenotype changes of plasma metabolism in rats of HUA group and drugs groups.Using variable importance in the projection(VIP),t-test,and fold change to screen for potential biomarkers associated with hyperuricemia.The results showed that a total of 16 potential biomarkers related to hyperuricemia were selected.Among them,9 potential biomarkers were significantly recalled by fermentation fungal,11 potential biomarkers were significantly recalled by Astragalus.It mainly involves sphingolipid metabolism,pyrimidine metabolism,tryptophan metabolism,pantothenic acid and coA biosynthesis,glycine,serine and threonine metabolism and so on.The main pathway is sphingolipid metabolism,and the recovery ability of bacterial group is better than that of Astragalus group.Mainly involved in sphingolipid metabolism,pyrimidine metabolism,tryptophan metabolism,pantothenic acid and coenzyme A biosynthesis,glycine,serine and threonine metabolism and other pathways.The main pathway is sphingolipid metabolism,and the recovery ability of fermentation fungal group is better than that of Astragalus group.In conclusion,this study used bidirectional luquid state fermentation technology to optimize the process of Paecilomyces cicadae/Astragalus membranaceu bidirectional fermentation fungal substance with anti-hyperuricemia activity,and elucidated the effect of bidirectional fermentation on the metabolism of Paecilomyces cicadae/Astragalus membranaceu in rats of hyperuricemia model rats by plasma metabolomics.The results showed that saponins,flavonoid glycosides and polysaccharides contained in Astragalus membranaceus could be pre-converted into small molecules such as secondary glycosides,aglycones and oligosaccharides after bidirectional fermentation,which will not only improve the absorption and utilization of active ingredients,but also adjust the metabolic network,intervene sphingolipid metabolism and pyrimidine pathways.And it could effectively treat renal damage of rats caused by potassium oxyzinate,thus exert uric acid-lowering effects based on multi-component,multi-target and multi-pathway. |
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