| Objective:Prunella vulgaris L.,a genus of perennial herbaceous plants in the Labiatae family,is a commonly used traditional Chinese medicines.The chemical components are complex,the pharmacological actions are broad.At present,the main researches on Prunella vulgaris L.focus on the pharmacological activities of small molecule substances,including flavonoids,phenolic acids,triterpenoids,etc,But few are about its macromolecular materials,such as Prunella vulgaris L.polysaccharide.In this paper,extraction,separation,purification and structure characterization of Prunella vulgaris L.polysaccharide were researched,using Prunella vulgaris L.as its raw material,and its antioxidant,anti-inflammatory activity,inhibitory activity of vascular smooth muscle cell proliferation and hypolipidemic effect are briefly discussed,which lay a foundation for the further study of Prunella vulgaris L.Polysaccharide and the development of polysaccharide-based health care products lay the foundation.Method:1.During the extracting process,six main factors including exacting time and temperature,cellulose enzyme concentration,solid-liquid ratio,enzyme digestion temperature and time were studied by using Plackett-Burman analysis method,Steepest Ascent Design and Central Composite Design,to optimize the best extraction process of Prunella vulgaris L.Polysaccharides.2.Crude polysaccharide（PVP）was gained through enzymes assisted ultrasonic extraction,alcohol precipitation,decolorization and lyophilization.Then,DEAE-cellulose and gel-filtration chromatography were used to isolate and purify the polysaccharides.Thus,purified components were gained.3.Using phenol-sulfuric acid method,m-hydroxydiphenyl method and coomassie brilliant blue method,Folin phenol reagent method and barium chloride-turbidimetric method determined the total sugar,the uronic acid,protein,the total polyphenols,The sulfate content,respectively.4.The purity and relative molecular weight of PVP and purified fractions were determined,and using infrared spectrometry,ultraviolet spectroscopy,HPLC method to study polysaccharide structure characterization.5.This paper studied the antioxidant ability of PVP in vitro by ABTS method,FRAP method,DPPH method.Inflammatory cell model was constructed by LPS acting RAW264.7 cell line,The concentration of IL-1β,NO and TNF-αwere assayed by using ELISA,To evaluate the anti-inflammatory activity.6.To study the effects of PVP on the proliferation of VSMC,cells proliferation model was set up by AngⅡstimulating VSMC,and cell proliferation was determined by MTT assay.7.Healthy SD 40 male rats were randomly divided into four groups:control group;model group;positive group;PVP group,Each group was given daily drink and food.The rats in control group were fed with basic diet,In addition to the normal group,high-fat diet was offered to other groups,which were made as the hyperlipidemia model.PVP group was fed with high-fat diet and PVP(1.6 g·kg-1),positive group was fed with high-fat diet and lovastatin(22.5 mg·kg-1),Food intake and body weight were regularly measured for 10 weeks.Five weeks later,the determination of fat volume was tested by Micro CT,to verify that hyperlipidemia model was successful.Ten weeks later,collecting their blood plasma by abdominal aortic method,calculating their liver weights and fixed the livers with4%paraformaldehyde solution,then those liver tissues were paraffin embed,routine HE stained and morphology observed by microscope.Serum biochemistry functions TC、TG、LDL and HDL were measured by routine methods by means of an auto-analyzer.ELISA method was used to test SOD,GSH-PX activity and MDA,TNF-a content in serum.The experimental data were analyzed by SPSS 21.0software.8.Based on metabonomics analysis by GC-MS method,To observe the metabolism characteristics of PVP on the lipid level of SD rat by high fat feed was observed.Serum were collected to do the metabolomics experiments.Then analyze the significant differences of the endogenous metabolites among the 3 groups by using principal component analysis and partial least square algorithm.Results:1.The optimal processing conditions were as follows:enzyme concentration5.00%,Enzymatic hydrolysis temperature 60℃,Enzymatic hydrolysis time 150 min,liquid to solid ratio 45 m L/g,extraction temperature 60℃and extraction time 120min.The experimental yield 5.48%under optimized conditions was closely agreed with the predicted yield 5.36%of the model.2.Crude polysaccharide（PVP）was gained through enzymes assisted ultrasonic extraction,alcohol precipitation,decolorization and lyophilization.The major purified polysaccharide fraction（PVP-1,PVP-2）from PVP was purified by DEAE-52 and Sephadex G-150 gel column.3.Using phenol-sulfuric acid method,m-hydroxydiphenyl method and coomassie brilliant blue method,Folin phenol reagent method and barium chloride-turbidimetric method determined the total sugar,the uronic acid,protein,the total polyphenols,The sulfate content,respectively.PVP,PVP-1 and PVP-2 contained high levels of total carbohydrates（59.48%,90.71%,88.58%）and uronic acid（20.82%,4.32%,39.38%）,low content of sulfuric radical（15.26%,1.73%,2.40%）,a small amount of total polyphenols（2.25%,0.45%,0.38%）;a small amount of protein（3.60%,0.00%,0.00%）.4.In this study,we determined the Mws of PVP-1 and PVP-2 and analyzed the monosaccharide composition of PVP,PVP-1 and PVP-2.The Mws of PVP-1,PVP-2were 9.12E+04 Da、3.16E+04 Da.PVP was composed of Man,Rib,Rha,Glc UA,Gal UA,Glc,Gal,Xyl+Ara,Fuc in the molar ratio of 14.19,18.88,4.23,1.18,0.46,6.26,22.68,28.49,3.63.PVP-1 was composed of Man,Rib,Rha,Glc UA,Gal UA,Glc,Gal,Xyl+Ara in the molar ratio of 21.87,18.93,1.35,2.27,1.67,7.76,22.74,23.41.PVP-2 was composed of Man,Rib,Rha,Glc UA,Glc,Gal,Xyl+Ara,Fuc in the molar ratio of 19.86:5.52:3.26:17.01:4.06:16.84:24.84:8.61.5.On the basis of antioxidant activities assay,PVP possessed strong hydroxyl radical scavenging activities and Fe2+chelating activity,and has a good concentration-response relationship to the mass concentration.Moreover,PVP had no cytotoxic effect for the growth of macrophages,PVP was revealed to show strong anti-inflammatory activities by inhibiting inflammatory-related mediator NO and cytokines TNF-αand IL-1βrelease compared to LPS treatment in RAW 264.7macrophage cell line.6.The cell proliferation was measured by MTT,and compared with the normal group,the model group could significantly promoting the proliferation of VSMC（P<0.05）;PVP showed anti-proliferative effect in vascular smooth muscle cells induced by angiotensionⅡ.The PVP intervention group showed significantly lower proliferation rate than the model group（P<0.05）.7.This experiment had lasted for 10 weeks,Compared with the normal group,the model group could significantly increase weight,TG,TC,LDL and Non-HDL-C.Compared with the model group,PVP group could significantly decrease weight,TC,LDL and Non-HDL-C.The MDA and TNF-a level of model group were significant higher than control group（P<0.01）and PVP group（P<0.05）.The GSH-PX and SOD activity of model group were significant lower than control group（P<0.01）,and PVP group were greatly increase GSH-PX activity（P<0.05）.Abdominal fat volume of model group were significantly larger than control group（P<0.01）,PVP group were significantly smaller than control group（P<0.05）.Liver tissue was examined for histology.The results indicates that PVP has certain repair effects in liver tissue morphological structure damage induced by high-fat diet,the cell boundaries visible,the lipid vacuoles within hepatocytes were decreased,the hepatic cord was orderly.8.Compared with the normal group,Serum endogenous metabolic in the model group rats have 9 significant increase differences,that is,Urea,threonine,alanine,valine,succinic acid,proline,inositol,trans-9-stearic acid,arachidonic acid.These metabolic markers are related to tricarboxylic acid cycle,sugar metabolism,amino acid metabolism and lipid metabolism.After PVP intervention,most of the metabolites returned to normal,These results suggest that PVP treatment can adjusts lipid metabolism disorder.Conclusion:In the present study,optimal extraction process of the ultrasound-assisted enzymatic extraction of PVP were scientifically selected by using statistical methods of Plackett-Burman,the steepest ascent method and central composite design（CCD）.Then PVP-1 and PVP-2 were obtained by separating and purifying the crude polysaccharide,and molecular weight distribution is relatively narrow;Structure and the physical and chemical properties were characterized by analysis.At last,the activities results show that PVP possessed strong hydroxyl radical scavenging activities and anti-inflammatory activities;PVP showed anti-proliferative effect in vascular smooth muscle cells induced by angiotensionⅡ;PVP may amend the metabolism of lipids,decreases the level of blood-lipid,and the mechanism may be related to adjust the sugar metabolism,amino acid metabolism and lipid metabolism,etc. |
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