Fermented Production Technology Of Cajaninstibene Acid(CSA)in Fungal Endophytes From Pigeon Pea[Cajanus Cajan(L.)Millsp.]and Antioxidant Mechanism Study

Pigeon pea[Cajanus cajan(L.)Millsp.],is mainly distributed in tropical and subtropical countries,has relieving heat,detoxify,antibacterial,anti-inflammory activities.Cajaninstilbene acid(CSA),3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid,is the main active constituent in pigeon pea,can be used to treat diabetes,measles,hepatitis,dysentery and many other illness.However,pigeon pea as a plant resource has some defects such as long growth cycle,low resource utilization rate,large environment effect and need a substitute resource.Endophytes may produce the same or similar metabolism substance,isolation the endophytes from pigeon pea will expand source of active pigeon pea metabolism substance,provide a new pathway for pigeon pea protection and utilization.After characterization of the five fungal endophytes by morphology and molecular methods,we choose K4 isolate with high CSA amount to make an optimization of fermented conditions.Furthermore,separation and purifying CSA from fungal endophyte K4,evaluation the antioxidant activities and preliminary study the antioxidant mechanism pathway of fungal CSA were carried out.Conclusions were summarized below:1.Five fungal endophytes producing CSA were isolated from pigeonpea and systematic identified;Isolation,screening and identification of fungal endophytes from pigeon pea roots,stems,leaves,flowers,bean-pod and seeds,112 strains of fungal endophytes were obtained totally.L45?L53?R64?R66?L77?L89?L92?K4?K5?K6?K9?K14 strains with good antioxidant activities was screened by preliminary study and five fungal endophytes(K4,K5,K6,K9,K14)producing CSA were obtained by LC-MS/MS method.The five fungal endophytes were characteristic by morphology and molecular methods.K4,K5,K9 and K14 are most closely related to Fusarium oxysporum.K6 is more closely related to Neonectria macrodidyma.2.The best fermented technology conditions of fungal endophyte K4 were obtained byoptimization of the fermented medium and culture conditions;K4 was choosed to optimize the fermentation conditions for CSA production effected by different factors.It is mainly refers to kinds of fermented medium,carbon source,nitrogen source,inorganic salt,pH value,temperature,time and so on.Three main factors were selected to central composite design and response surface optimization.The best fermented techonology conditions were:PDB medium,carbon source:glucose,pH value:7,temperature:300C;culture time:5 days.The maximum production of CSA from K4 was 1.454 mg/L.3.The purification technology of fermented extracts CSA were obtained by normal-phase medium pressure silica gel column chromatography and reverse-phase medium pressure ODS column chromatography;Fungal endophyte K4 fermented extracts were separated and purified for CSA by normal-phase medium pressure silica gel column chromatography and reverse-phase medium pressure ODS column chromatography.Purification technology conditions:Normal-phase conditions:The quantity ratio of sample/silca gel was 1:5;an gradient elution was performed with CHC 13-Petroleum Ether(10-15/1,v/v)on the column,the flow rate was 15 BV/h.Reverse-phase conditions:The quantity ratio of sample/ODS silica gel was 1:50;the flow phase was MeOH-H2O-CH2O2(76.5:23.2:0.3,v/v/v),the flow rate was 5 BV/h.Fungal endophytes EtoAc extracts was puried to 96.5%of CSA by normal-phase medium pressure silica gel column chromatography and reverse-phase medium pressure ODS column chromatography.4.Determination antioxidant activity of fungal endophyte fermented products CSA by chemistry and cellular levels;DPPH radical scavenging,?-carotene-linoleic acid bleaching,reducing power assay,lipid peroxidation assay and DNA damage protection assay were used to evaluated the antioxidant properties of fungal CSA and plant CSA.The IC50 value of fungal CSA were 0.41 ±0.04,6.11±0.77,0.88±0.05,5.77±0.78(?g/mL)by DPPH radical scavenging,??carotene-linoleic acid bleaching,reducing power assay,lipid peroxidation assay,respectively.Furthermore,XOD,SOD and GR activities was tested in HepG2 cell to evaluate effects of enzyme system.The results showed CSA could inhibit XOD activity,the inhibition was 62.57%when CSA concentration was 100 ?g/mL.The SOD and GR activities in HepG2 cell was increased with CSA concentration dependment and enhanced 2.77 mg/prot and 0.49 g/prot,respectively.Furthermore CSA showed protection ability for DNA damage from hydroxyl radicals.5.Nrf2 antioxidant mechanism of CSA was revealed by determination of Nrf2,AKT,ERK,JNK and HO-1;Determination expression of Nrf2 AKT,ERK,JNK,HO-1 and NQO1 proteins of CSA induced HepG2 cell by western blotting method.The results revealed that CSA treatment enhanced the phosphorylation-dependent activation of signaling components,such as PI3K/AKT,ERK,and JNK,and activated PI3K/AKT,ERK and JNK signaling pathways.Expression of Nrf2 was incrseaed in nucleus and decreased in cytoplasm and revealed Nrf2 nuclear translocation.Activation Nrf2 pathway induced by CSA was identified.Five fungal endophytes producing CSA were isolated from pigeonpea and identified systematic;The best fermented technology conditions of fungal endophyte K4 with high level CSA were obtained by optimizing the fermented medium and culture conditions;the purification technology of fermented extracts CSA were obtained.Addtionally,antioxidant activity of fungal endophyte fermented products CSA was determined and Nrf2 antioxidant mechanism of CSA was revealed.The study provides a new pathway and method for CSA resource.The good pharmacological activity of CSA provide new scientific basis for natural antioxidant in medicine,food,health care products application and development.