Isolation And Identification Of Endophytes From Medicinal Plants(polygonatum Sibiricum And Astragalus)and Study On The Structure And Biological Activity Of Exopolysaccharide

It has been reported that polysaccharides isolated from a variety of traditional medicinal plants play a very significant role in disease treatment.Plant endophytes as a rich species,have a symbiotic relationship with the traditional medicinal plants and can produce metabolites similar to that of the host plants.This work mainly focus on the difference between the exopolysaccharide form plant endophytes and polysaccharide from Chinese herbal polysaccharides.Polysaccharide from polygonatum sibiricum and astragalus have unique biological characteristics.In this paper,polygonatum sibiricum and astragalus were used to isolate and identify plant endophytes.Their exopolysaccharides were separated and purifed.It was found that the exopolysaccharides form plant endophytes were different from their Chinese herbal polysaccharides on the structure or composition of monosaccharide.However,exopolysaccharides have many similar biological activity to their Chinese herbal polysaccharides.This paper is divided three parts.Part one is about screening and identification of plant endophytes(Chapter 2);Part two is about purification and characterization of exopolysaccharide B3(EPS-B3)from polygonatum sibiricum endophytes B3 and exopolysaccharide Z7(EPS-Z7)from astragalus endophytes Z7(Chapter 3).Part three is sbout studying on the biological activity of polysaccharides(Chapter 4).Part One:First,17 strains of plant endophytes were isolated from polygonatum and astragalus by tissue separation method,and the morphological characteristics of plant endophytes were identified.The 16S rDNA sequence showed that plant endophytes A2 and A3 were Enterobacteriaceae.Plant endophytes A1,B1,B2,B2,Z1,Z2,Z3,Z4,Z6,M1,M2 and M3 were Bacillus.Plant endophytes Z5 and M4 were Staphylococcus.Plant endophytes 7 was Arthrobacter..Most of the isolated plant endophytes were Bacillus.The antibacterial activity was determined by filter paper method.The results showed that the fermentation liquor of plant endophytes B3 had a little antibacterial activity on Bacillus subtilis,Staphylococcus aureus and Escherichia coli.Plant endophytes M1 and M2 have a little antibacterial effects on Escherichia coli.Z2,Z3,Z5,Z7 and M3 have a little antibacterial activity on Bacillus subtilis,while plant endophyte Ml had the strongest antibacterial activity.Plant endophytes Z6,M2 and M4 had a little antibacterial activity on Staphylococcus aureus.Based on the results,plant endophytes B3 and Z7 were selected to further research.Part Two:The multi angle laser light scattering instrument was used to determine the molecular weight of polysaccharide.The results show that,the molecular weight of EPS-B3 from polygonatum sibiricum endophytes B3 was 1.863×104 Da,the molecular weight of EPS-Z7 from astragalus endophytes Z7 was 5.001 ×104 Da,the molecular weight of polygonatum sibiricum polysaccharide from polygonatum sibiricum was 2.791 ×104 Da and the molecular weight of astragalus polysaccharide from astragalus is 6.004 x 105 Da.The monosaccharide components of polysaccharides were determined by ion chromatography.It was found that EPS-B3 was mainly composed of Gal,Glc and Man monosaccharides.EPS-Z7 was mainly composed of Gal,Glc and Man.However,polygonatum sibiricum polysaccharide mainly composed Fru,Rha,Gal,Glc,Man,Fru monosaccharides.Astragalus polysaccharide mainly composed Fru,Rha,Gal and Glc monosaccharide composition.These results show that the exopolysaccharide from plant endophytes were different from that Chinese herbal polysaccharide from traditional medicinal plants in the structure and composition of monosaccharide,Part Three:The antibacterial activity was determined.The results showed that neither Chinese herbal polysaccharides nor exopolysaccharides from plant endophytes have no obvious antibacterial activity.However,these were obvious antioxidant activity,the results showed that the scavenging capacity of DPPH free radical and Superoxide anion,EPS-B3 from polygonatum sibiricum endophytes and polygonatum sibiricum polysaccharide were close,EPS-Z7 from astragalus endophytes and astragalus polysaccharide were close.In reducing ability,the ability of exopolysaccharide from plant endophytes is higher than that of Chinese herbal polysaccharide.In the scavenging ability of hydroxyl radicals,the scavenging ability of polygonatum sibiricumpolysaccharides was higher than that of EPS-B3,and the scavenging ability of astragalus polysaccharide and EPS-Z7 were similar.We detected the effects of polysaccharides on proliferation of S180 cells in vitro.The results showed that the inhibition the proliferation of S180 cells,EPS-Z7 from astragalus endophytes was higher than astragalus polysaccharide and polygonatum sibiricum polysaccharide was higher than EPS-B3 from polygonatum sibiricum endophytes.Mice were subcutaneously implanted with S180 cells in the right axillary region.The therapeutic effects of polysaccharides were determined by analyzing tumor size.Compared with model group,polysaccharides group could inhibit S180 tumor growth,and production of IgG in the serum also increased.The results indicated that combined application of polysaccharides from traditional medicinal plants and exopolysaccharides from plant endophytes had the effects of anti-tumor activity.The exopolysaccharide has many similar biological activity to their host Chinese herbal polysaccharides.Therefore,they can be used as substitutes for their host Chinese herbal polysaccharides,being used in the treatment of human and animal diseases.