| Kudzu root(Pueraria lobata)is a kind of traditional natural Chinese herbal medicine and mainly distributed in Southeast Asia,it has amount of starch,cellulose and flavonoids.Kudzu root(Pueraria lobata)has many physiological activities because of rich flavonoids,such as inhibition cerebrovascular disease,anti-inflammatory,antialcoholismic and antioxidant,therefore kudzu root(Pueraria lobata)was used to produce various products and development of its potential application value for human health.The amount of flavonoid of kudzu root(Pueraria lobata)exsit as glycosides flavonoids,so it has low physiologically activity,while,the diferent measures was used to conversion glycosides flavonoids to aglycone flavonoids,and then the major measures was microbial bioconversion which was the the best effective way.Eurotium cristatum HC-18 was isolated from Fuzhuan tea,which itself has a variety of metabolites,high physiological activity.The fungi of E.cristatum HC-18 could grew and metabolized on low water activity of substrate.Solid-state fermentation is a low cost and high yield way of production.The main results were as follow:1.The E.cristatum HC-18 isolated from Fuzhuan tea and was identified by the morphology and molecular biology,it was Eurotium cristatum and named Eurotium cristatum HC-18.The E.cristatum HC-18 solid-state fermented kudzu root(Pueraria lobata)could be obviously changed the taste of traditional Chinese medicine of kudzu root,and then endowed the unique "Golden Flower" fragrance of kudzu root.The ergosterol contents were 43.66±5.66 ?g/g DW at fermentation 14 day,and the ergosterol increased along with the prolonging of fermentation time.The signal factor test and response surface methodology were used to analyze the fermentation condition,the optimal condition were the inoculum amount of 3%,the fermentation time was 14,the water content of substrate was 20%,the flavonoid contents were 43.98±10.12 mg RE/g DW.2.The fermented kudzu root(FK)and non-fermented kudzu root(NFK)were extracted by 80%enthanol and water,the total flavonoid contents were 35.78±0.95 and 23.49±0.98 mg RE/g DW at fermentation 14 day and 16 day.The results expressed that the 80%ethanol extracted total flavonoid contents were the highest,as fermentation time prolonged the flavonoid contents were slowly decreased.Analyzed the change of enzymes activity during kudzu root solid-state fermented process with E.cristatum HC-18,the highest of cellulase,a-amylase and ?-glucosidase activities at fermentation 10 day,12 day and 14 day,it were 677.47,703.94 and 146.76 U/g DW,respectively.At these enzyme activities,?-amylase and(3-glucosidase expressed great correlation ship with the total flavoioid and phenolic contents,the R2 were greater than 0.8.3.The results were the glycosides flavonoids contents of NFK higher than FK,and the aglycone flavonoids contents of FK higher than NFK,which by RE-HPLC analyzed.The extracts of FK were great antioxidant activity than NFK at ABTS·+free radical scavenging activity,DPPH free radical scavenging activity,metal chealting,reducing power,and AgNP method and hydroxyl radical scavenging activity,and expressed the the great relationship with the total flavonoid contents,and then presented dose concentration dependence,principal component analysis showed strong correlation between total flavonoids and antioxidant activity.4.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay revealed that the enthanolic and water extracts of fermented kudzu root(Pueraria lobat a)at the dose of 1-15 mg/mL had no significant toxic effects on PC 12 cells,and exerted protective effects on PC 12 cells against 0.1 mM of H2O2-induced damage,furthermore,enthanolic and water extracts protected PC 12 cells from damage in a dose-dependent manner.Morphology of PC 12 cells observed by inverted phase contrast microscope showed that some PC 12 cells regained their inherent morphology after pretreated with enthanolic and water extracts,that was,a fibriform structure and a distinct boundary,morphology of PC 12 cells observed by fluorescence microscope indicated that enthanolic and water extracts could reduce the proportion of injuried cells.Enthanolic and water extracts could retained the integrity of cell membrane,thus markedly attenuated the release of lactate dehydrogenase(LDH)compared with the impaired cells.Moreover,enthanolic and water extracts could increase the activity of superoxide dismutase(SOD),catalase(CAT)and glutathione(GSH)contents of PC 12 cells in a dose-dependent manner.Further experiments indicated that enthanolic and water extracts could reduce reactive oxygen species(ROS)level and apoptosis in PC 12 cells.Cell cycle analysis indicated that enthanolic and water extracts could relieve S phase arrest induced by H2O2.Finally,the kit assay suggested the apoptosis induced by H2O2 could be improved by reducing the activity of Caspase-3.All the results showed that enthanolic and water extracts enriched of flavonoid and phenolic could protect PC 12 cells from H2O2-damaged by reducing the apotosis.This study identified the E.cristatum HC-18 and analyzed the changed of the enzyme activities of solid-state fermentation process,the optimum content of total flavonoids was obtained by optimizing the fermentation conditions.Antioxidant activity of the non-fermented and fermented kudzu root extracts was analyzed by using the in vitro antioxidant activity and protective effects of samples on PC 12 cells against H2O2-induced damage.It is helpful to understand the function of active substances of kudzu root(Pueraria lobata)and provide a theoretical basis for the study of the health effects of kudzu root(Pueraria lobata). |
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