Study On The Extraction,Separation And Purification Of Polygonatum Polysaccharide And Its Anti-Inflammatory And Immunological And Activities

Objective:Rhizoma polygonati is a traditional Chinese medicine,which was originally recorded in the Shennong Herbal Classic.It is an important medicinal material of the same origin in medicine and food.It has the functions of replenishing qi and nourishing yin,strengthening spleen and lungs,benefiting kidney,etc.In the 2020 edition of the Chinese Pharmacopoeia,the indigenous polygonal plant Polygonatum kingianum Coll.et Hemsl、 Polygonatum sibiricum Red.、Polygonatum cyrtonema Hua.The three kinds of original polygonatum are similar in shape and difficult to identify.They are usually identified by "Yunnan polygonatum looks like cow’s head,polygonatum looks like chicken’s head,polygonatum looks like ginger" and other forms.There has been a longterm mixed use on the market,which has seriously affected the quality control of polygonatum.In recent years,studies have shown that the chemical components of Rhizoma Polygonatum mainly include polysaccharides,steroidal saponins and flavonoids,among which the content of polysaccharides in Rhizoma Polygonatum is the highest,which is its quality marker(Q-marker)and the basis of its main pharmacological substances,and is listed as the quality control index component in the Chinese Pharmacopoeia(2020).Polygonatum polysaccharide has shown potential medicinal value in regulating immunity,anti-aging,anti-tumor,antibacterial and other aspects.As one of the prominent pharmacological activities of polygonatum polysaccharide,anti-inflammatory immunity has become a hotspot and difficulty in recent years.The biological activities of polysaccharides are closely related to their intrinsic structural characteristics.Only polysaccharides with a certain characteristic molecular structure will have specific physiological activities.The relationship between the molecular structure of this polysaccharide and its biological activity is called the structure-activity relationship of polysaccharide.According to the searches performed,studies on the three types of polysaccharides currently included in the pharmacopoeia mainly focus on the extraction,separation and purification of one polysaccharide and on the structural characterisation,but there are relatively few studies comparing the material basis and structure-activity relationship of the anti-inflammatory and anti-immune activity of the three types of polysaccharides.Therefore,this study took three kinds of rhizoma polygonatum from different places of origin as the research object,and collected a total of 12 batches of three kinds of rhizoma polygonatum from different places of origin in the country.The quality of rhizoma polygonatum polysaccharides from different places of origin was evaluated by using near-infrared spectroscopy combined with cluster analysis,principal component analysis and other statistical analysis methods,providing scientific reference for the identification of rhizoma polygonatum counterfeits.In addition,the extraction and purification methods of ethanol fractionation and DEAE-52 anion exchange column chromatography,combined with the DPPH antioxidant activity test,were used to screen the three components of polygonatum polysaccharide with the strongest activity,and the physical and chemical properties,structural characterisation,anti-inflammatory and antiimmune activities of the three polysaccharide components of the polyconate were compared by chemical identification and spectroscopic methods.Methods:1.12 batches of Rhizoma Polygonatum with different origins were collected from all over the country,and 12 batches of crude polysaccharide of Rhizoma Polygonatum were obtained by water extraction and alcohol precipitation.The infrared spectral data were collected by Fourier transform infrared spectroscopy,and the 4000-400 cm-1 section was selected as the analysis object.The original spectral data were preprocessed by automatic smoothing,baseline correction,coordinate normalization,and second-order derivative.The quality control and identification of 12 batches of crude polysaccharides from polygonatum officinalis from 3 kinds of raw materials were realized by using chemometrics methods such as principal component analysis(PCA),cluster analysis(HCA),and partial least squares discriminant analysis(PLS-DA).2.The extraction and separation methods of ethanol fractionation and DEAE-52 anion exchange column chromatography,combined with the DPPH antioxidant activity experiment,were used to select the three most active components of refined polysaccharide from Rhizoma Polygonati,PKP 4-1,PSP 1-2,PCP 3-2.The infrared spectrometer(FT-IR),high-performance gel permeation chromatography(HPGPC)Its physical and chemical properties and structure were characterized and discussed by gas chromatography-mass spectrometry(GC-MS)and other modern biological analysis methods.Use TNF-α The induced MH7 A inflammatory cell model was used to investigate the material basis of the anti-inflammatory and immune effects of the three different glycogen polygonatum polysaccharides and their structure-activity relationship with polysaccharides.Results:1.The second derivative infrared spectrum is different in the range of 2000-400(cm-1)wave number.The three kinds of crude polysaccharides of Rhizoma Polygonatum can be identified by the peak shape and peak strength of the absorption peaks in the I,II and III regions.PCA,HCA and PLS-DA analysis showed that 12 batches of three original polygonatum polysaccharides can be roughly divided into three categories: S1-S4 is polygonatum polysaccharides,S5-S8 is polygonatum polysaccharides,and S9-S12 is polygonatum polysaccharides.In the model,R2 X,R2Y and Q2 are greater than 0 5.It shows that the model has good stability and predictability.The results of the principal component comprehensive score of 12 batches of polygonatum polysaccharide samples showed that the principal component comprehensive score of polygonatum polysaccharide from Yunnan was the highest,followed by polygonatum,and polygonatum multiflorum ranked the lowest.S11(Yunnan Pu’er)in four batches of Yunnan Polygonatum has the highest score,S2(Shaanxi Danfeng)in four batches of Polygonatum has the highest score,and S6(Sichuan Nanchong)in four batches of Polygonatum multiflora has the highest score.2.using graded alcohol precipitation and DEAE-52 cellulose column chromatography,combined with DPPH antioxidant activity experiment,three batches of the most active homogeneous polysaccharide components PKP 4-1,PSP 1-2,and PCP 3-2 were selected from 12 batches of Polygonatum polysaccharides.GC-MS,FT-IR,HPGPC and other analytical methods were used to analyze and characterize its physicochemical properties and chemical structure.The results showed that PKP4-1,PCP3-2 and PSP1-2 were heteropolysaccharides,mainly composed of glucose,mannose,galactose and glucuronic acid.Methylation analysis shows that the components of PKP4-1 and PCP3-2 are mainly composed of 3-Glcp,while the components of PSP1-2 are mainly composed of 3,5-Manp.The relative molecular weights of PKP 4-1,PCP 3-2,and PSP 1-2 are 22.4kd,35.5kd,and 36.2kd,respectively.The results of in vitro cell experiments indicate that PKP 4-1,PCP 3-2,and PSP 1-2 have an effect on TNF-α The induced proliferation of MH7 A cells has a concentration dependent inhibitory effect.As the drug concentration increases,cell viability gradually decreases.Scratch and apoptosis experiments show that PKP 4-1,PCP3-2,and PSP 1-2 can inhibit the migration of MH7 A cells,promote their apoptosis,and have a concentration dependent effect.Conclusion:1.The infrared spectrum fingerprints of crude polysaccharides of polygonatum from three different origins were established by using infrared spectrum technology,and their common characteristic peaks were characterized and analyzed.Combined with the chemometrics analysis methods such as cluster analysis and principal component analysis,the identification of three kinds of polygonatum polysaccharides was successfully realized.This method can effectively solve the identification problems of polygonatum from different origins and different origins,It opens up a new way for the authenticity identification and quality evaluation of Rhizoma Polygonatum.2.In this paper,three active polysaccharides,PKP 4-1,PCP 3-2 and PSP 1-2,were selected and purified from 12 batches of polygonatum polysaccharides from three glycogen sources.Their physicochemical properties and structural composition were investigated by chemical and spectroscopic methods.The anti-inflammatory and immune activities of polygonatum polysaccharide were studied,and the structure-activity relationship of polysaccharide was further explored on this basis,which provided a reference for the development and utilization of polygonatum polysaccharide.