Characterization Of Endophytic Fungi Producing Vitexin Isolated From Pigeon Pea And Its Effect On The Proliferation Of MC3T3-E1 Cells

Endophytic fungi are important components of plant micro ecosystem, which are widely existed in healthy plants, could produce the same or similar compounds with host plants, many kinds of secondary metabolites and different biological activities in them, in agriculture, medicine, industry and other fields show a good prospect of application.Pigeonpea belongs to legumes pigeonpea genus, has many pharmacological activities, the main active ingredients of pigeonpea are flavonoid and stilbeness, the active ingredients separation from Pigeonpea has very important role in anti-tumor, anti-inflammatory, antiviral, antioxidant and antibacterial.Vitexin, is an important class of flavonoid in pigeon pea. Clinically used for the treatment of cardiovascular and cerebrovascular diseases, has a good protective effect on ischemic myocardial injury. Its mechanism is the increase of coronary blood flow, reduce the ejection resistance and plasma viscosity, improve red blood cell deformability, inhibit the formation of thrombus. The subject are to isolation and identification of vitexin produced from endophytic fungi from pigeonpea and research the promote proliferation on osteoblast MC3T3-E1. Results are as follows:1. The root, stem and leaf as materials, the sterilized plant materials were inoculated to PDA for isolation and culture, subculture and purification. Finally, a total of 154 strains were purified, among them,76 strains were obtained from the roots,15 strains were obtained from the stem, and 63 strains were obtained from the leaves, on PDA slant tube, preservation at 4 ℃.2. Reference< fungal identification manual> and identification of molecule biological methods, according to the different forms of pigeonpea endophytic fungi, these fungies are classified into 6 types:Fusarium sp., Chaetomium sp., Epicoccum sp., Arthrinium sp., Actinomucor sp., Alternaria sp., respectively.3. Using HPLC-UV and LC-MS/MS method, determination the detection conditions of pigeonpea producing endophytic fungi of vitexin as follows:Chromatographic column HiQ sil C18W (4.6 mmψ×250 mm,5 μm); mobile phase:(A)acetonitrile-(B)water(v/v) was 20%-80%; detection wavelength:332 nm; flow rate:1 mL/min; injection volume:15 μL. Founding that two strains of endophytic fungi production vitexin, belong to Fusarium sp. and Chaetomium sp. respectively.4. Using single factor, the response surface central composite design for fermentation culture medium type, medium composition and liquid culture conditions were optimized. G6 strain as the experimental object, the influence of different factors on the yield of vitexin were investigated, including different fermentation culture medium and culture medium carbon source, nitrogen source culture medium, culture time, temperature and medium initial pH value. Determinedthe best fermentation of endophytic fungi of G6 culture conditions as follows:PDB liquid medium; 20 g/L glucose as carbon source; 10 g/L ammonium sulfate asnitrogen source; medium initial pH of 5, for 6 days of incubation time, incubation temperature for 28℃ , train G6 obtained under the condition of the vitexin amount reached 18.51 mg/L.5. The proliferation promoting effect to osteoblast MC3T3-E1 for endophytic fungi producing vitexin from pigeonpea. Take the vitexin standard as a positive control, vitexin extract as the sample. Results are as follows:endophytic fungi producing vitexin have varying degrees of impact to osteoblast MC3T3-E1.In this paper, the health pigeonpea plant used as the host plants, the endophytic funguses were isolated from the roots, stems and leaves. Identificated the species of endophytic fungi from morphology and molecular biology respectively; used the HPLC, LC-MS and other methods to screen out the endophytic fungus producing Vitexin and its yield was determined. Using single factor-the response surface central composite design for fermentation culture medium type, medium composition and liquid culture conditions were optimized. The osteoblast MC3T3-E1 as vitro model, study on the influence of vitexin endophytic fungi on proliferation of MC3T3-E1 cells.