| Polygonatum sibiricum Red.（P.sibiricum Red.）is a rhizome of natural plant in the Polygonatum of Liliaceae family in Chinese Pharmacopoeia（2020 edition）.P.sibiricum contains polysaccharides,saponins,alkaloids and other active ingredients.As an important functional component of Fructus flavesculi,P.sibiricum polysaccharides have a variety of biological activities,such as antioxidant,hypoglycemic,anti-tumor,immune regulation and so on.Polysaccharide is not a single pure chemical substance,but a mixture composed of sugar chains with different degrees of polymerization.Therefore,the chemical composition,structural characteristics and biological activity of polysaccharide obtained by different extraction methods may have potential differences.Unfortunately,the current research on P.sibiricum polysaccharide mainly focuses on single extraction method and subsequent biological activity evaluation,mechanism of action and product development based on this method,and lacks systematic research on the influence of different extraction methods on the chemical composition,structural characteristics and biological activity of P.sibiricum polysaccharide.In this study,P.sibiricum was selected as the research object,and different extraction methods were used to obtain the crude extract of P.sibiricum polysaccharide,and then different P.sibiricum polysaccharide components were prepared through separation and purification.The chemical constituents of total polysaccharide,protein and uronic acid were analyzed by phenol-sulfuric acid method,Coomasil bright blue method and sulfuric acid-carbazole method.The common and different structural characteristics of P.sibiricum polysaccharides were characterized by high performance liquid chromatography（HPLC）,infrared chromatography（IR）,nuclear magnetic resonance（NMR）and Congo red triple helix determination.At the same time,the biological activity difference of different polygonatum polysaccharides was studied.Finally,the pharmacokinetic characteristics of PSP-5 inhibiting acetylcholinesterase（ACh E）and its influence on the toxicity and side effects of first-line ACh E inhibitors in the market were investigated.This study aims to clarify the effects of different extraction methods on the chemical composition,structural characteristics and biological activities of P.polygonum polysaccharide,and provide a theoretical basis for the development and utilization of P.polygonum polygonum polysaccharide.The main research contents and results of this paper are as follows:（1）Preparation of P.polygonatum polysaccharide based on different extraction methods.PSP-1～PSP-3 polysaccharides were extracted from P.polygonatum at 24℃,boiling water and 80℃,respectively.At the same time,at 24℃,50%ethanol,25%ethanol and water were used to extract PSP-4～PSP-6,respectively.PSP-1～PSP-6 was obtained by 3%trichloroacetic acid deproteinization,dialysis bag dialysis and 80%ethyl alcohol precipitation,and the yield rates were 6.64±0.33%,1.89±0.23%,8.11±0.50%,5.55±0.41%,5.04±0.38%,1.44±0.28%,respectively.The yield of PSP-3was significantly higher than that of the other 5 PSPs(**p<0.01).（2）Analysis of chemical composition and sructural characteristics of P.polygonatum polysaccharide.The contents of total sugar,protein and uronic acid were determined by phenol-sulfuric acid method,Coomassie bright blue method and sulfuric acid-carbazole method,respectively.At the same time,the structure characteristics were analyzed by high performance liquid chromatography（HPLC）,infrared spectroscopy（IR）,nuclear magnetic resonance（NMR）and Congo red triple helix structure.The results showed that the total sugar content of PSP-1～PSP-6 was more than 70%,but the total sugar content of PSP-3 could reach 92.77±1.34%.The six PSPs all contained a small amount of protein.Compared with other PSPs,the uronic acid content of PSP-3 was the highest（7.49±0.62%）（*p<0.05）,which was1.1～2.0 times of that of other five PSPs.PSP-1～PSP-6 contained the same monosaccharides,mainly mannose,glucose and galactose,but their proportions were significantly different.Infrared spectrum analysis showed that PSP-1～PSP-6 had characteristic absorption peaks of glucoside bond,and there wereαandβ-types of glucoside bond.1H and 13C spectra confirmed that the glycosidic bond types of PSP-1～PSP-6 wereαandβ-types.Congo red structure analysis showed that PSP-1～PSP-6 had triple helix structure.（3）Evaluation of biological activity of PSP-1～PSP-6.The in vitro activity evaluation models of ACh E,DPPH,ABTS,OH,α-glucosidase andα-amylase were constructed,and PSP-1～PSP-6 were evaluated in vitro.The results showed that PSP-5was 1.2～1.5 times more active in inhibiting ACh E than other PSPs,and the activity of PSP-5 was comparable to that of the control group.In terms of antioxidant activity,there was no significant difference between PSP-2 and PSP-3（*p>0.05）,but there was significant difference between PSP-2 and the other four PSPs（*p<0.05）.In terms of hypoglycemic activity,there was no significant difference between PSP-2and PSP-3（*p>0.05）,but there was significant difference between PSP-3 and the other four PSPs in the concentration range of 1.0 to 10.0 mg/m L（*p<0.05）.In conclusion,different PSPs showed different pharmacological activities in vitro,and it is worth mentioning that PSP-5 showed excellent ACh E inhibitory activity,which is worthy of further study.（4）Study on the pharmacokinetic characteristics of PSP-5 inhibiting ACh E and its effects on the side effects of first-line ACh E inhibitors.The pharmacokinetic characteristics of PSP-5 in inhibiting ACh E were studied by double reciprocal method of enzymatic kinetics method,and the activities of PSP-5 in inhibiting ACHE were compared with those of tacholine,donepezil,galantamine,carbalatine and other side effects.The results showed that the optimal conditions for PSP-5 to inhibit ACh E were p H=7.4 and reaction time 25 min.The pharmacokinetic inhibition type was mixed type I competition reversible inhibition.In terms of ACh E inhibition activity,PSP-5 was similar to the four selected positive drugs.However,PSP-5 could significantly improve the dyspepsia caused by tarcrine and donepezil,significantly reduce the allergic reaction caused by tarcrine and galantamine,and the myocardial injury caused by tarcrine and galantamine.In conclusion,this study can provide scientific basis for the precise development and utilization of P.polygonum polysaccharide,especially for the development of novel therapeutic agents for Alzheimer’s disease with P.polygonum as the core raw material. |
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