Study On The Extraction And Isolation Of Polysacchrides And Cordycepin From Cordyceps Militaris And Their Activities

Cordyceps militaris?L.ex Fr?Link is a traditional edible medicinal fungus that is comparable to Cordyceps sinensis.It has antioxidant,immune-regulating,antibacterial and anti-cancer effects.Cordyceps militaris polysaccharides and cordycepin are the main active substances in Cordyceps militaris.Based on the optimization of the extraction and purification process of polysaccharides from Cordyceps militaris,the chemical composition?structural characteristics and antioxidant activity of Cordyceps militaris polysaccharides were investigated preliminarily.Meanwhile,this research also extracted and purified the cordycepin from Cordyceps militaris,studied its antibacterial effect,and explored the mode of antibacterial mechanism for the first time.The in-depth development on functional components provides a theoretical basis to Cordyceps militaris.The main results are as follows:1.Cordyceps militaris polysaccharides?CMP?extraction.PEG-based microwave extraction method was employed to extract CMP.The optimal extraction conditions by response surface methodology were as follows:21%concentration of PEG200,360 W and 8 min for microwave power and extraction time,respectively.The CMP extraction rate was 93.2%,which was 21.9%higher than that of water extraction.Scanning electron microscopy?SEM?observations showed a clear rupture of tissue surface in Cordyceps militaris.UV-visible and FT-IR spectra analysis showed that both polysaccharides extracted by the two methods had homogenous characteristic absorption signals.Thermal analysis confirmed that both polysaccharides had high thermal stability.2.Cordyceps militaris polysaccharides purification.A neutral Cordyceps militaris polysaccharides?CMP-1?was obtained after water washing with DEAE-52 column.The UV-visible spectrum showed that CMP-1 had no significant absorption peaks at 260 nm and280 nm,and the purity was 76.6%,which was 70%higher than that of crude polysaccharide?45.0%?.Fourier transforming-infrared spectrum?FT-IR?revealed that CMP-1 was?-type polysaccharide.The monosaccharide composition of CMP-1 was determined by Gas chromatography-mass spectrometry?GC-MS?,and it was composed by mannose,galactose and glucose,while the molar ratio was 1.0:1.51:3.9.Additionally,the molecular weight?Mw?of CMP-1 was 2.432×10⁷ g/mol and root mean square rotation radius?Rg?was 31 nm which were analysed by multi-angle laser light scattering and gel permeation chromatography with reflective index detector?GPC-MALLS-RI?.CMP-1 exhibited a highly branched structure in solution.Meanwhile,antioxidant experiment indicated that CMP-1 held up fairly well antioxidant activity,the IC₅₀ of DPPH and ABTS free radical scavenging capacity of CMP-1 were 0.8mg/m L and 1.4 mg/m L,respectively.3.Cordycepin purification.The content of cordycepin in the fruit body of Cordyceps militaris was 0.56%determining by high performance liquid chromatography.After cordycepin was isolated and purified by NKA-II macroporous resin and Sephadex G-10column,the purity of cordycepin was improved to 98.8%.The purified substance was characterized by UPLC-TOF-MS,the parent ion m/z 252[M+H]⁺and the secondary fragment ion m/z 136[M+H]⁺were identical to the standard product,so the purified substance was cordycepin.4.Antibacterial effect and mechanism of cordycepin.The minimal inhibitory concentration?MIC?antibacterial test proved that cordycepin can effectively inhibit the growth of Gram-positive bacteria and negative bacteria,especially on Escherichia coli and Bacillus subtilis,the MIC were 2.50 mg/mL and 1.25 mg/m L,respectively.Scanning electron microscopy?SEM?and transmission electron microscopy?TEM?showed that cordycepin disrupted the bacterial cell membrane and cell wall;The nucleic acid leakage examination showed that the cell membrane permeability of the bacteria was increased,and cytoplasmic contents was leaked out.Propidium iodide?PI?and carboxyfluorescein diacetate?cFDA?double staining approach coupled with flow cytometry?FCM?analysis found that the integrity of cell membrane was severely damaged in 30 minutes,while the intracellular enzyme system still possessed activity.It clearly suggested that membrane damage was one of the main reasons for killing bacteria.Additionally,the results from UV,fluorescence and circular dichroism analysis demonstrated that there were two interaction modes between cordycepin and bacteria genome DNA,the intercalation binding mode with the hydrophobic bases and electrostatic binding mode with the phosphate groups of DNA.Thus it affected the copy?transcription and expression of DNA.On the basis of above results,the mode of bactericidal action of cordycepin against E.coli and B.subtilis was found to be a dual mechanism:disrupting bacterial cell membrane and changing bacterial genomic DNA structure,ultimately leading to cell death.