Isolation And Identification Of Fungal Endophytes And Analysis Of Alkaloids In Sphaerophysa Salsula

1. Isolation and identification of fungal endophyte in Sphaerophysa salsulaIn order to investigate whether endophytes exist and to assess distribution of endophytes, fungal endophytes were isolated from Sphaerophysa salsula from the grassland of Alashan, Inner Mongolia. Sterilization of plant materials surface was taken to isolate the fungal endophytes, and species and genus of endophyte was identified by the traditional morphology and rDNA–ITS sequence analysis. In addition, in order to obtain a better result, this study determined the best time to different tissues in Sphaerophysa salsula, that were stems and seeds by using 75% ethanol and 2% NaClO for 90 s, 5min, 40 s, 2.5min, respectively. 70 strains were isolated from the stems, leaves, petioles, and seeds of Sphaerophysa salsula, which belonged to Alternaria sp., Fusarium sp., Cladosporium sp., Chloridium sp., Diplodia sp., Pleosporales sp., Didymella fabae, Laetisaria arvalis and Preussia australiis. There were three strains were identified to species, that were Didymella fabae, Laetisaria arvalis and Preussia australiis. The relative frequency of Alternaria sp.(61.43%) was highest which was dominant in all the fungal endophytes. The isolation rate(19.58%) of leaves was highest in all tissues, followed by seed(41.39%), which showed that seeds and leaves were the main parts of endophyte to infect and colonize.2. Pre-trial system of chemical composition of Sphaerophysa salsulaAccording to conventional methods of phytochemical system pretest, the chemical composition of Sphaerophysa salsula conducted a systematic pre-test. The results showed that: there were steroids, terpenoids, volatile oils, fats, sugars, organic acids, amino acids, peptides, proteins, alkaloids, flavonoids, coumarin, lactones, saponins, tannins, phenols, cardiac glycosides, anthraquinones in Sphaerophysa salsula.3. TLC analysis of alkaloids in Sphaerophysa salsulaTLC analysis, planar chromatography quantitative image analysis software and column chromatography separation techniques were used to analyses and separate the alkaloids of Sphaerophysa salsula. The results showed that 650.20 g total extract was extracted from 5.00 kg Sphaerophysa salsula by ethanol hot refluxmade. Through acidification and alkalization, the alkali liquor followed by acidic chloroform, chloroform alkaline, alkaline n-butanol, respectively, obtaining three-part extract 3.01 g, 0.53 g, 23.13 g, and the extraction rates were 1.51%, 0.27%, 11.57%, respectively. These results showed that alkaloid were focus on n-butanol parts, mainly was a large polar alkaloids based. TLC analysis to various parts showed that there were at least 5 alkaloids in alkaline chloroform and alkaline butanol extract alkaloids least. By comparing with standard samples,the resultshowed that swainsonine was the main alkaloid in all alkaloids of Sphaerophysa Salsula.