Studies On Secondary Metabolites Of Halophytic Salicornia Bigelovii And Its Endophytic Fungi Salicorn-5

Studies have shown that plant endophytic fungi can produce compounds which are same or similar to constituents of the host plants. It has become a new way to the production of natural anti-cancer drugs and anti-oxidants. Due to the extreme living conditions, Halophile possess special hereditary and physiological character, and have the capability to produce special secondary metabolites with unique skeletons and bioactivities which have important applications in the field of biomedicine, functional foods, Chemistry and Chemical Engineering and so on. In this study, halophytic S.bigelovii as raw material, extracted with methanol extracts to obtain crude extract for isolation and structral identification of chemical constituents of S.bigelovii. And the other contents of this study include isolation and identification of the endophytic fungi from S. bigelovii, optimization of the fermentation conditions, isolation and identification of fermentation products, and it also conducted a preliminary evaluation on their antioxidant and anti-tumor activities. Experimental results are as follows:Nine pure compounds have been got from the methanol extract of Salicornia bigelovii by variety of separation and purification methods(column chromatography, sephadex LH-20, TLC, and so on). Then ID and 2D nuclear magnetic resonance(1H NMR,13C NMR, ’H-’H COSY, HSQC, HMBC, NOESY), infrared(IR), ultraviolet(UV), mass spectrometry (MS) and other modern spectroscopic techniques as well as related literature search were used to identify the chemical structure of the compounds. Two new compounds named Bigelovii C(3)å'ŒD(4) and seven known compounds, which were 8-hydroxy-9-16-enoate(1), 7-hydroxy-6-methoxycoumarin(2), Oleanic acid(5), gypsogenin(6), pheophorbide a(7), 15a-hydroxypheophorbide a(8), chlorin e6 di-Me ester(9) were separated from the methanol extract of Salicornia bigelovii.The associated one fungi, numbered Salicorn-5 was isolated and purified from Salicornia bigelovii by the PDA plate and the slant culture. It was identified as Cunninghamella sp. by molecular biology analysis combined with morphological observation. The fermentation conditions of Salicorn-5 were optimized by using the Plackett-Burman, the steepest hill-climbing experiment and the response surface method, to maximize its antioxidant activity. The optimum medium of Salicorn-5 was as follows:16 g·L-1 yeast extract,17 g·L-1 peptone,300 g·L-1 potato,30 g·L-1 maltose,30 g·L-1 Vannitol,5 g·L-11 glucose,10 g·L-1 monosodium glutamate and 10 g·L-1 sodium chloride, pH 7. Using the optimum culture medium, the maximum DPPH free radical scavenging rate of the fermentation products was 78.25%, which was significantly higher than before (66.55%).The strain Salicorn-5 have been fermented largely by the optimum medium, and the pure compounds have been got by the solvent extraction and variety of separation and purification methods, then the pure compounds’structures were determined by modern methods of spectroscopy.Nine compounds, including oleic aicd(10), linoleic acid(11), gamma-linolenic acid(12),3-monostearin(13), monostearin(14), P-sitosterol(15),1,3-oleic acid diglyceride(16),1,2-oleic acid diglyceride(17) and ergosterol peroxide(18) were isolated from the fermentation products of strain Salicorn-5.Total antioxidant (FRAP values) method and DPPH radical scavenging method were used to evaluate the antioxidant activity of the 18 compounds separating from fungi Salicorn-5 and the n-butanol extract of S. herbacea. Result shows that compound 2,3,4,7, 8 and 9 have stronger antioxidant activity, and 7-hydroxy-6-methoxycoumarin had the strongest antioxidant activity. In the conditions of 1 mM, the FRAP value and DPPH radical scavenging rate are as high as 255.08 mmol/100 g and 88.41% respectively,which are almost similar to Vc(positive control). Compound 9 had a moderate antioxidant level, its FRAP value and DPPH radical scavenging rate are 95.79 mmol/100 g and 75.33%. We concluded that the double bond, hydroxyl group in the structure exert antioxidant activity, they may the main active groups which can play antioxidant activity.The anti-tumor activities against several cancer cell lines of compounds with higer antioxidant activity were assayed by MTT method. Results show that compound 7 has higher anti-tumor activity against A549 and hepG2 cells, and their inhibition of cell proliferation rates and IC50 value were 88.68%,81.12% and 6.15,17.56μM respectively, which are higer than Etoposide. Compound 3 have a moderate anti-tumor activity against A549 cells, and its inhibition of cell proliferation rates were 48.49%.