| ObjectiveComparing the flavonoids and polysaccharides of the stem and leaves of four different dendrobium species firstly,the antioxidant sites of Dendrobium denneanum with the most abundant flavonoids were selected,in order to find the material basis of the antioxidant activity of Dendrobium denneanum,as well as provide a basis for the research and development of natural antioxidants and lay a scientific foundation for the effective integration and utilization of resources.Aiming at the phenomenon that researches on the components of different dendrobium were mostly based on dried products,a comparative study was conducted on the fresh and dried Dendrobium officinale with highpolysaccharide content,which wound provide a theoretical basis for the further development and clinical application of dendrobium.Methods:(1)The method of UPLC-PDA and phenol sulfuric acid were used to determined the content of flavonoids and total polysaccharides in stems and leaves of four different dendrobium,including Dendrobium officinale,Dendrobium nobile,Dendrobium chrysanthum and Dendrobium denneanum.(2)The quality of flavonoids in Dendrobium denneanum was analyzed by the method UPLC-PDA and the whole component analysis and similarity evaluation of fingerprints of 10 batches leaves of Dendrobium denneanum were carried out.A method for simultaneous determination of aperitin-6,8-2-c-glucoside,isafutaglycoside,rutin and determination of total flavonoids was also established.Then,the extraction process of Dendrobium denneanum was selected with antioxidant activity index,and the extract was extracted with different polar solvents,which were the petroleum ether layer,dichloromethane layer,ethyl acetate layer,n-butanol layer and water layer.Moreover,the components of each part were analyzed with the correlation analyzed by grey correlation degree.(3)According to the pharmacopoeia aimed at dried dendrobium,section drying method was used to determine the water content of fresh dendrobium compared with the pharmacopoeia method.In addition,antioxidant capacity of polysaccharides of fresh and dried Dendrobium denneanum and Dendrobiumofficinale was determined by different antioxidant methods,including hydroxyl radical scavenging capacity,total antioxidant capacity and reducing capacity.(4)The composition,microstructure and rheological properties of fresh and dried polysaccharides of Dendrobium officinale and Dendrobium denneanum were analyzed by liquid chromatography derivatization,sem and rheometer.(5)The MTT method was used to determine the cell viability of RAW264.7 macrophage induced by polysaccharides of Dendrobium officinale and Dendrobium denneanum to RAW264.7 macrophage and lps-induced RAW264.7 macrophage.Besides,the phagocytic capacity of cells was determined by neutral red method.Results:(1)The content of flavonoids in leaves of four dendrobium species was significantly higher than that of stems,among which,the content of flavonoids in Dendrobium denneanum was the highest,reaching more than 2%.The content of polysaccharides in the stems of the four dendrobium species was higher than that in the leaves,and the polysaccharide content of Dendrobium denneanum was the second,up to 10%.(2)The contents of aperitin-6,8-2-c-glucoside,isafutaglycoside,rutin and total flavonoids in leaves of Dendrobium denneanum were determined simultaneously by UPLC-PDA.A total of 29 peaks were identified by thefingerprint,including 25 peaks of flavonoids and 23 common peaks and the similarity of samples from the same origin was 0.968?0.995.Compared with the leaves of Dendrobium denneanum in Yunnan province,the content of flavonoids in Sichuan province was more,and the content of apigenin-6,8-2-c-glucoside was lower,but the content of rutin and total flavonoids was higher.After the screening of antioxidant active sites,it was found that the total amount of flavonoids and polysaccharides in n-butanol layer and water layer were high,which was the main active site of anti-oxidation,in the consequence of the synergistic action of flavonoids and polysaccharides.(3)In the determination of water content of fresh dendrobium,the relative deviation of the determined results of two methods was less than 5%,between which the section processing drying method was easier and faster,while the pharmacopoeia method was only used for dried dendrobium.In the determination of antioxidant index of polysaccharides,the polysaccharides of two types of fresh and dried dendrobium had a certain degree of antioxidant capacity,among which the polysaccharide activity of fresh Dendrobium denneanum was better in terms of the scavenging ability of hydroxyl radical(0.8?4.0 mg/mL)and the reducing power(0.1?0.9 g/mL),when in terms of total antioxidant capacity(3.0?7.0 mg/mL),polysaccharides of fresh dendrobium were more active than dried polysaccharides.(4)The detected results of liquid chromatography-derivatization method were that four monosaccharides including mannose,glucose,galactose,andarabinose were detected.The content of mannose in polysaccharides of Dendrobium officinale was higher than that of Dendrobium denneanum.Mannose content and polysaccharide purity of fresh polysaccharides was also slightly higher than dried.The results scanned by scanning electron microscope(sem)were that compared with dried polysaccharides,fresh polysaccharides have smoother surface and looser structure.The results detected by rheometer were that the polysaccharide solution was a shear dilute pseudoplastic fluid,and the viscosity of the solution was concentration dependent.Under acidic conditions,the polysaccharide structure of fresh Dendrobium officinale was the most stable,when under extremely alkaline conditions(pH=13),the polysaccharide structure of fresh and dried dendrobium was destroyed.(5)Within the concentration range of 313?1000 ?g/mL,polysaccharides of fresh Dendrobium officinale and Dendrobium denneanum had no inhibitory effect on RAW264.7 macrophages.Within the concentration range of6.25?125 ?g/mL,polysaccharide of fresh dendrobium can reduce the inhibition of LPS on cells and enhance the phagocytosis of cells to a certain extent.Conclusion:(1)The UPLC-PDA and "point-line-surface" quality analysis method established had strong specificity and high accuracy,which was more beneficial to the quality control of dendrobium as a method for thedetermination of flavonoids constituent.The content of flavonoids was highest in leaves of Dendrobium denneanum,and the content of polysaccharides was higher in Dendrobium officinale and Dendrobium denneanum.(2)The results showed that Dendrobium denneanum could be used as a new type of plant antioxidant in food and treatment,and the extraction layer with strong antioxidant activity could be screened,and the compounds with strong antioxidant activity could be further extracted,which laid a solid foundation for the comprehensive development and utilization of Dendrobium denneanum.(3)The section drying method was determined as a method for water determination of fresh dendrobium.Among the different antioxidant activity indexes,the polysaccharide activity of two varieties of dendrobium was better at scavenging hydroxyl radicals.Besides,the polysaccharides of fresh Dendrobium denneanum had relatively strong antioxidant ability,which could provide certain guidance for the development of natural antioxidant products with the fresh polysaccharides of Dendrobium denneanum as the effective component.(4)The rheological behavior of shear thinning of polysaccharides of fresh and dried Dendrobium officinale and Dendrobium denneanum could provide reference for the research and development of polysaccharides of dendrobium officinale in the field of food.In addition,the polysaccharide of fresh Dendrobium officinale was stable in acidic condition,and its viscositywas conducive to the formation of gastric protective film.Besides,as dendrobium itself was beneficial to the production of gastric fluid,the curative effect of polysaccharide of fresh dendrobium on the stomach wound be further explored to provide guidance for the research and development of auxiliary drugs for gastric diseases in the future.(5)The polysaccharides of fresh Dendrobium officinale and Dendrobium denneanum had an immune regulatory effect on RAW264.7 macrophages and a protective effect on lps-induced macrophage damage,which could promote the phagocytosis of cells.The difference of pharmacological activity among polysaccharides of different Dendrobium officinale might be the result of the difference of polysaccharides composition and microstructure,and would be come under review. |
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