Study On The Processing Chemistry In Polygonatum Cyrtonema Hua And The Enhancement Mechanism Of Processing

Polygonati Rhizoma(PR)is a traditional tonic herbal medicine of China.Polygonatum kingianum Coll.et Hemsl.,Polygonatum sibiricum Red.andP olygonatum cyrtonema Hua are three medicinal resources of PR recorded in the Chinese Pharmacopoeia 2020 edition.According to the theory of traditional Chinese medicine(TCM),its efficacy is derived by supplementing qi,nourishing yin,replenishing the kidney system,and improving the strength of the spleen and lungs.Processed Polygonatum cyrtonema Rhizoma(PCR)is usually used as tonic.The main purpose of processing is to eliminate irritation and enhance the effect.There are some shortcomings in the present research of modern mechanism of PCR processing.The main effective and tonic substances in PCR are not clear.At present,less attention is paid to the changes of chemical structure and pharmacological activity of Polygonatum cyrtonema polysaccharides(PCP),the only quality control component of PCR in Pharmacopoeia,before and after processing.Based on the scientific problems and deficiencies of PCR processing,the main research contents of this thesis were listed as follows:1.Comparison on pharmacological effects between R-PCR and P-PCRThe spleen and kidney deficiency model of wistar mouse was selected to compare the tonic effects of raw Polygonatum cyrtonema Rhizoma(R-PCR)and processed Polygonatum cyrtonema Rhizoma(R-PCR).In this study,the serum biochemical indexes in experimental rats about hypothalamus-pituitary-adrenal(HPA)axis,hypothalamic-pituitary-gonadal(HPG)axis,hypothalamus-pituitary-testis(HPT)axis,immune system,digestive system and oxidative stress were detected.The visceral index and pathological section of each experimental group were compared.The results showed that the pharmacological activity of P-PCR on regulating the levels of GAS,MTL,IL-6,TNF-α,SOD,GSH-Px and MDA in spleen and kidney deficiency rats was stronger than R-PCR.The P-PCR is more conducive on the recovery of damaged spleen and testis in spleen and kidney deficiency rats.The pathological tissue of P-PCR group rats was more access to normal group rats.2.Research on processing-induced chemical variations in PCR by integrating metabolomics and glycomicsIn the present study,the overall chemical variations in PCR during processing were elucidated by integrating metabolomics and glycomics.The study was designed as follows.First,PCR materials steamed for 0,1,2,3,4,6,8,10,12,14,16,and 18 h were prepared as research objects.According to the data of electronic eye and polysaccharide content,the time period of processing chemistry research was judged.Second,the non-saccharide micromolecules,monosaccharides,and oligosaccharides in PCR materials of different processing times were determined by UPLC-QTOF-MS/MS,whereas the qualitative and quantitative information of PCP was investigated by multiple chromatographic techniques.A precolumn derivatization method for high performance liquid chromatography-photo diode array(HPLC-PDA)was applied to characterize variations in monosaccharide composition.A high-performance gel permeation chromatography-refractive index detector(HPGPC-RID)was used to determine the molecular weight of PCP.An ultraviolet(UV)detector was utilized to measure the polysaccharide,uronic acid,and glycoprotein contents of PCP during processing.Third,in order to distinguish PCR in different processing stages,multivariate statistical analysis was conducted to screen differential markers from nonsaccharide micromolecules,monosaccharides,and oligosaccharides during processing,and multiple chemical parameters of polysaccharides were obtained and analyzed.In this research,83 chemical components including non-saccharide micromolecules,monosaccharides,and oligosaccharides in PCR of varying processing times were identified or tentatively assigned by UPLC-QTOF-MS/MS.The results of principal component analysis(PC A)demonstrated that the processing of PCR could be divided into three stages,namely the raw stage(0 h),the middle stage(1-6 h),and the late stage(8-18 h).The partial least-squares discriminant analysis(PLS-DA)model further screened 20 differential markers from three different processing stages of PCR samples to distinguish PCR of different processing stages.The chemical transformation stages of PCP were consistent with secondary metabolites,oligosaccharides,and monosaccharides.The physicochemical properties of PCP changed significantly during processing.The polysaccharide purity gradually decreased,and the content of uronic acid and glycoprotein gradually increased.From the perspective of monosaccharide composition,the polysaccharides of different processing time were mainly composed of Man,Rib,GalA,Glc,Gal and Xyl.However,the PCP monosaccharide proportion of different processing time is quite different.From the perspective of molecular weight,polysaccharides of P-PCR produced a part of high molecular weight polysaccharide and a part of low molecular weight polysaccharide.Multiple chemical mechanisms,including hydrolysis,oxidative decomposition,dehydration,Maillard reaction,and polymerization,were involved in PCR processing and caused qualitative and quantitative changes in chemical ingredients.3.The tonic chemical fraction of PCR was screened based on the spleen and kidney deficiency modelIn this study,the tonic chemical fraction of PCR was screened based on the spleen and kidney deficiency model.The regulating effects of non-saccharide micro molecules,oligosaccharides and polysaccharides in PCR on serum biochemical indexes and visceral indexes of spleen and kidney deficiency rats were compared.The pathological sections of each experimental group rats were observed and compared.The results showed that the tonic activity of saccharide fraction was better than non-saccharide micro molecules,polysaccharides>oligosaccharides>micromolecules.Therefore,the following study should focus on polysaccharides.The PCR regulatory effects on digestive function,immune function,antioxidative function of spleen and kidney deficiency rats were significantly better than hypothalamus-pituitary-testis(HPT)axis,hypothalamus-pituitary-adrenal(HPA)axis and hypothalamic-pituitary-gonadal(HPG)axis.Therefore,the following research on polysaccharide activity could be carried out from the aspects of enhancing immunity and antioxidation.4.Separation,purification and chemical difference research of homogeneous molecular weight polysaccharides in R-PCR and P-PCRIn this study,five homogeneous molecular weight polysaccharides were separated by negative ion-exchange chromatography and gel chromatography.The molecular weight changes of five homogeneous polysaccharides in R-PCR and P-PCR were characterized by HPGPC-RID.The monosaccharide composition of five homogeneous molecular weight polysaccharides was further detected by ion chromatography.The results showed that the molecular weight of PCP-1 and PCP-2 ranged from 2000 to 3000 Da,and the molecular weight of PCP-3,PCP-4 and PCP-5 ranged from 150 to 250 kDa.PCP-1 is mainly composed of Fru and Glu,and the proportion of Fru and Glu can reach 93.8%.The monosaccharide composition of PCP-2 is more complex,including Gal,Glc,Rib,Fru,Ara and Man.Gal has the highest proportion of PCP-2,and Gal can reach 57.8%.The high molecular weight acidic polysaccharides PCP-3,PCP-4 and PCP-5 are mainly composed of GalA,Gal and Ara.The above results revealed that processing caused great changes in physical and chemical properties of PCP.5.The immunological regulation activities of PCP on RAW 264.7 macrophagesIn this study,RAW 264.7 macrophages were selected to study the immunological regulation activities of PCP in vitro.The pharmacological activities of crude polysaccharides in different processing stages,homogeneous molecular weight polysaccharides in R-PCR and P-PCR,and oligosaccharides were compared by detecting the proliferation ability,phagocytosis activity,and secretion activities of NO,IL-1β and TNF-α of each cell group.The results showed that the high molecular weight acidic polysaccharides PCP-3,PCP-4 and PCP-5 are more active in promoting cellular NO secretion than the low molecular weight neutral polysaccharides PCP-1 and PCP2.It is hypothesized that the immunostimulatory capacity of high molecular weight polysaccharides was stronger than low molecular weight polysaccharides,prompting a stronger immune response of RAW264.7 cells.The polysaccharides are more potent than oligosaccharides in promoting cell proliferation and NO secretion.It is speculated that the activity differences of polysaccharides may be related to two reasons.On the one hand,the molecular weight parameters of PCP are different.On the other hand,the activities of neutral polysaccharides and acidic polysaccharides in stimulating cell immune responses are different.In summary,this study confirmed that P-PCR had better preventive and therapeutic effect on spleen and kidney deficiency rats than R-PCR.The non-saccharide micromolecules and saccharides were taken into account for holistic chemical composition characterization by integrating metabolomics and glycomics.The study compared the chemical changes of PCR in different processing stages,and the chemical mechanisms involved in PCR processing were investigated.Then,the study confirmed that the main tonic chemical fraction of PCR was polysaccharide.Five homogeneous molecular weight polysaccharides were separated from R-PCR and P-PCR and their chemical properties were compared.Furthermore,the immunomodulatory activities on RAW 264.7 macrophages of different polysaccharides were compared.This paper provided data support and scientific basis for the study of PCR processing mechanism.