Research On The Antidiabetic Activity Of The Constituent Of Siraitia Grosvenorii As Well As Their Endophytic Fungi

Siraitia grosvenorii,as a special plant in Guangxi,has more than 300 years of medicinal history.Its ripe fruits and extracts are not only high in sweet,low in calories,but also have obvious hypoglycemic effect,which could be a substitute for sugar by diabetes patients and obese patients.In the process of growing,there were a large number of fruits which could not turn into ripe naturally due to the influence of low temperature in the later growth stage.Up to now,the biological activities of these fruits were rarely studied.In addition,based on the symbiosis theory,endophytic fungi may have the same or similar biological activities as their hosts,and endophytic fungi,as a special kind of microbial resources,can produce active substances through fermentation with the advantages of short regeneration cycle,easy access to resources,no seasonal restrictions,low cost and no pollution.Hence,the hypoglycemic and anti-inflammatory activities of extracts and monomers from ripe and unripe fruits of S.grosvenorii,as well as the extracts of endophytic fungi isolated from S.grosvenorii,were studied,aiming to provide a theoretical basis for the application of unripe fruits and the endophytic fungi of S.grosvenorii,also promoting the industry development of S.grosvenorii.The main results are as follows.1.The extracts of S.grosvenorii were preliminary separated using macroporous adsorption resin.Then the new technology high speed countercurrent chromatography（HSCCC）was used to prepare mogrosideⅤwith the purity as high as 94.72%by ternary cycle.The separation conditions were as follows:the two-phase solvent system was composed of ethyl acetate-n-butanol-water-ethanol（1:3:4:0.5,v/v/v/v）.The upper phase was the stationary phase,and the lower phase was the mobile phase.The rotational speed was 860 r/min,with the flow rate of 2.5 mL/min.The detection wavelength was 210 nm.2.A total of 150 endophytic fungi were obtained by a tissue culture method,including96 and 54 fungal isolates respectively isolated from pistillate plant and staminiferous plant of S.grosvenorii.122 isolates were classified into 9 genera based on the morphology identification and sequencing analysis of ITS gene,which belong to phylum Ascomycota,including Dothideomycetes and Sordariomycetes.Dothideomycetes contains 3 families and3 genera,and Sordariomycetes contains 6 families and 6 genera.The most frequent genera were Colletotrichum and Fusarium.There were different colonization rate and isolation rate between pistillate plant and staminiferous plant of S.grosvenorii,i.e.,in the pistillate plant,the root possessed the highest colonization rate and isolation rate,while the leaves possessed the lowest.The opposite happens in staminiferous plant.Additionally,the distribution of endophytic fungi was different in different tissues of both pistillate plant and staminiferous plant of S.grosvenorii.Combined with the similarity indices of community composition of endophytic fungi in different tissues,the result indicated that endophytic fungi have the hosts and tissues preferences.The diversity indices of endophytic fungi in different tissues ranged from 0.11 to 0.69,among which the root of pistillate plant had the highest diversity indices.This is the first systematic isolation and identification the endophytic fungi that isolated from different tissues of Siraitia grosvenorii of pistillate staminiferous plant.3.Extracts from ripe and unripe fruits of S.grosvenorii,both showed good inhibitory activity againstαα-glucosidase.The inhibitory activity increased with the increase of the concentration of extracts,and the IC₅₀ values were 12.88 mg/mL and 5.57 mg/mL,respectively.The results suggested that of the extract of the unripe fruit toα-glycosidase inhibition activity was higher than that of ripe.4.The inhibitory activity of extracts of endophytic fungi onα-amylase were generally much higher,with the IC₅₀ in the range of 2.08-16.27 mg/mL.The IC₅₀ values of the extracts of ripe and unripe fruits againstα-amylase were 10.95 mg/mL and 19.45 mg/mL,respectively.Two monomers,V andⅡE,had poor inhibitory effects onα-amylase,with IC₅₀of 62.02 mg/mL and 378.59 mg/mL,respectively.Results showed that extracts of endophytic fungi had best inhibitory activities againstα-amylase,followed by of ripe and unripe fruits,and the inhibitory activity of two monomers,V andⅡE,againstα-amylase were lowest.5.Toxicity of extracts of fruits and mogrosides to the RAW 246.7 cells increased with the increase of the treated concentration.When treated with the concentration,extracts of ripe fruit displayed more toxicity than the extracts of unripe fruit,mogrosideⅤmore toxicity than mogrosideⅡE.When treated with extracts of fruits and mogrosides,the contents of NO released by macrophages（RAW 246.7）induced by LPS were in the range from 23.45 to64.81μmol/L,and the concentration of NO was the lowest（23.45μmol/L）,treated with the concentration of 0.63 g/mL of extracts of unripe fruit.Combined with the above cytotoxicity test results,it could be concluded that extracts of unripe fruit had both low cytotoxicity and good anti-inflammatory activity.6.The extracts of unripe fruit with low dose and high dose of shown good hypoglycemic effect onⅠdiabetes mice for a long time.Continuous administration to the 29th day,blood glucose ofⅡdiabetes mice were dropped to 8.26±4.76 mmol/L and 7.41±2.63 mmol/L,respectively,nearly equating with the blood glucose of normal groups（8.30±4.66 mmol/L）.Moreover,it had no significant effect on body weight,specific gravity of liver and kidney.This study laid a foundation for the application of unripe fruit and the endophytic fungi isolated from S.grosvenorii in hypoglycemic aspects,and also provided a new resource for the discovery of anti-diabetes drugs.This study provides a new approach for the comprehensive utilization of unripe fruits and endophytic fungi of Siraitia grosvenorii,as well as their application in hypoglycemia.