Study On Therapeutic Material Basis Of Compound Qianyu In Treatment Of Chronic Non-bacterial Prostatitis

Objective:Chinese medicine based on the theory of traditional Chinese medicine,using the principle of sovereign?minister?assistant and guide mixing variety herbal in appropriate doses.The therapeutic material base of the drug was complicated and various,which seriously hindered the process of modernization of traditional Chinese medicine.Compound Qianyu was the experience prescription of the third hospital of Wuhan in treatment of chronic non-bacterial prostatitis.Through the preliminary pharmacodynamic study,split experiment and long term clinical observation from different points of view,confirmed that it have satisfy efficacy.In this study,combined with the previous experiments,the prescription of the functions and indications,chemical composition,animal model preparation and pharmacological activities of the Chinese medicinal herbs were studied.Three classes of chemical components,that is,total saponins,total polysaccharides and total flavonoids,were separated from the water decoction,which is the formulation directly used in clinical practice,and further tested on CNP rats induced by carrageenan as the pharmacological models in this study.Thus clarity the effective substance basis of the compound Qianyu in treatment of chronic non-bacterial prostatitis,provide a theoretical and experimental basis in develop effective part of new drugs and new dosage forms provide a theoretical and experimental basis.Methods:This paper follows the traditional clinical dosage form of Chinese medicine that compound decoction,the chemical parts of compound Qianyu such as polysaccharides,total flavonids and total saponins were purified,using modern chromatography theory,chemical analysis,instrument analysis method to purification and content determination,so that it can achieve the requirements that the effective parts of traditional Chinese medicine can measure the total content of substances extracted from the total 50% in the new drug declaration.And then combined with pharmacological research models,the model of chronic non-bacterial prostatitis was prepared.By measured prostate index,HE staining pathological changes and associated cytokines,observed therapeutic material basis of composition in treatment chronic non-bacterial prostatitis.In the process of purification of total polysaccharides and total saponin,compound Qianyu boiled twice,combined liquid,crude polysaccharide with anhydrous ethanol,filtrate reserved the crude polysaccharide reconstitution with water,removal of protein,dialysis to get purified polysaccharide.The filtrate was dried,dissolved again,chloroform was extracted,the water layer was adjusted to the pH value,and then the purified the total flavonoids were purified by using the polyamide resin column.In the purification of total saponin,compound Qianyu water decoction was selected,with petroleum ether extraction,the water saturated n-butyl alcohol solution was extracted and using the macroporous resin column to extract it.In the purification and content determination of the total polysaccharides,using anhydrous glucose as the control,the purity of polysaccharides was determined by phenol sulfuric acid method.On the basis of the experiment of three single factors,the ratio of solid to liquid,the times of removing protein and the dialysis time,the best purification condition of the polysaccharide was optimized by orthogonal test.Weigh six prescription(prescription composition: radix astragali,herba epimedii,herba leonuri,cortex phellodendri and radix achyranthes,a total of 100g)common herbs 600 g,addition deionized water immersion 12 h,boiled twice,combined filtrate got water decoction,added ethanol to the decoction and alcohol precipitation overnight,filtered and dried to give crude polysaccharide,added water,setting on a magnetic stirrer 95? reconstituted,centrifugal,took the supernatant fluid,added 1/4 volume Sevag reagent,thoroughly shaking,centrifugation,put the upper layer in the treated dialysis bag dialysis overnight,remove,freeze dried to obtain the pure total polysaccharides.In the purification and content determination of total flavonids,the content of total flavonoids as index,used spectrophotometry and the method of static and dynamic combination.The effects of purified in the process of sample solution concentration,pH value,resin herbs ratio and elution solvent selection and dosage of process parameters in polyamide resin purification of total flavonoids was evaluated.The absolute ethanol was added in the water decoction,alcohol precipitation overnight,filtration,combined filtrate,filtrate was evaporated,dissolved in water to get the crude drug concentration,chloroform extraction,collected water layer,adjusted pH,through the polyamide resin,eluting with deionized water,ethanol boiled in turn,collected the elution liquid,dehydrated,got the pure total flavonoidsIn the purification and content determination of total saponins,used D101 macroporous resin enrichment,determination of content with vanillin sulfuric acid reaction.Took the water decoction of 1g raw medicinal,with petroleum ether extraction,collected water layer.Then used the water saturated n-butyl alcohol solution extracted,collected n-butyl alcohol to spin dry,added a few of D101 macroporous resin,shaking so that herbs completely adsorbed on the resin,continue to spin dry,removed the sample and first eluted with deionized water until the effluent was colorless,then eluted with 10% ethanol impurity,50% ethanol elute to get the total saponins,dried under reduced pressure to obtain the pure total saponins.Prepared the chronic non-bacterial prostatitis rat model,selected specific-pathogen-free(SPF)-bred Sprague-Dawley(SD)rats,aged eight weeks and weighed between 200 g and 220 g,feeded a week to adapt to the environment.The rats were randomized into 12 groups of 10 rats per group: the normal group,the sham group,the model group,the positive group and 8 test groups,that is,Qianyu decoction,polysaccharides,flavonoids,saponins,flavonoids-saponins,polysaccharides-saponins,polysaccharides-falconoid and polysaccharides-flavonoids-saponins combinations.The rats were drugged by urethane,fixed,sterilized the surgical site,opened in the lower right part of the abdomen to expose the prostate tissue,Briefly,the prostate of each rat was injected with 0.1 ml filter sterilized 0.1% solution of carrageenan in water.For the sham group,carrageenan solution was replaced by the same volume of saline during injection.Seven days later,rats in drug administration and positive groups were kept for oral administration of corresponding drug for three weeks,the sham and model groups were given saline at the same time.The general physical condition of each rat was observed throughout the test period.Food consumption and body weight were measured once per week.After final administration,the SD rats were deprived of food,then weighed and blood samples were collected.Finally,the rats were sacrificed and the prostates were excised and weighted.The prostates were done ELISA analysis and tissue biopsy respectively.Observed the pathological changes of each rat prostate tissue and the changes of some immune index,evaluation and select the polysaccharides,flavonoids,saponins chemical parts of parts and their combination of the compound Qianyu in the treatment of chronic non-bacterial prostatitis effective,exploring the material basis of its effectiveness.Results:With the aim to elucidate the effective substance of Qianyu,in this experiment,modern analysis and test means were used to isolate the total polysaccharides,total flavonoids and total saponins chemical parts from the compound Qianyu,combined with animal disease model to study the biological activity.In the purification and content determination of total polysaccharides,investigated the key technologies in purification process of polysaccharides,such as liquid ratio,the number of protein and dialysis hours,single factor test and orthogonal test was used to optimize the optimum purification conditions of the total polysaccharides.The best purification technological conditions of polysaccharide were: Liquid-solid ratio was 1:40(g/ml,w/v),protein removal 10 times,dialysis 18 hours,the content of polysaccharides can reach 69.04% under this condition,the transfer rate was 51.84%.The optimized purification process was simple and accurate,and can be used for polysaccharides purification in compound Qianyu.Study on purification technology and determination of total flavonoids.The purity of total flavonoids as index,polyamide resin was used to study the concentration of sample solution,pH,ratio of resin and herbs,elution volume and other factors.The best purification technology of total flavonoids involved 1.0 g·ml-1 crude drug solution,pH 3.0,5 BV water was applied to remove the impurities and 4 BV 50% ethanol was to elute.Under these conditions,the purity of total flavonoids was up to 63.67% from 8.89%.Purification effect was better,can significantly improve the purity of flavonoids and the process conditions were stable and feasible.In the purification and determination of the total saponins,macroporous resin was used,wet packed column dry sample,eluting with deionized water,10%,30%,50%,70%,90% ethanol in turn,collected the elution liquid,and determine the content of total saponin.The best purification technology was deionized water,and 30% ethanol impurity,70% ethanol eluted and dried to obtain high purity total saponins,with a purity of 56.93%.In this study CNP rats was induced by carrageenan as the pharmacological models.Prostate index in the model group was significantly increased,observed tissue swelling and adhesion with the surrounded tissues.In histopathology,the model group rat prostate tissue severe diffuse chronic inflammatory characteristics,such as prostatic intraepithelial increase,foliate papillae deep glandular,lateral lobe prostate gland luminal diameter becomes smaller,lymphocytes and other inflammatory cell infiltration.In the experiment,the CNP animal model was successfully prepared,and the effect of physiological saline on the inflammation was excluded.After administration,compared with model group,the positive drug group,Qianyu decoction,chemical parts and the combination group of prostate inflammation of the situation has improved,among them,the positive group,Qianyu decoction group and the polysaccharides-flavonoids-saponins combinations group significant improved,gland epithelial cells arranged in order,less inflammatory cells infiltration.The test results showed that the total polysaccharides,total flavonoids,total saponins of three chemical components have inhibitory effect in the model group of the abnormal cytokines.The effect was not significant of the single chemical composition,although they have reduced effect on some cytokines,such as,the polysaccharides can reduce the amount of COX-2,the saponins can be reduced iNOS activity and NO content to a certain extent,the flavonoids can reduce the abnormal expression of PGE2.In the compatibilities of two chemical components,effect of TGF-?1,TNF-?,IL-1?,NO main content of total flavonoids and total saponins,about PGE2,iNOS inhibition mainly from polysaccharides and total saponins,the impact of COX-2 content mainly from the total polysaccharides and total falconoid,three chemical components were have reduced effect on cytokines,such as,TNF-??PGE2?IL-1??TGF-?1?COX-2?iNOS and NO.Conclusion:This experiment studies the purification process of different chemical components.In the total polysaccharides purification process used water extraction and alcohol precipitation,removed protein,dialysis to get higher pure total polysaccharides,using polyamide resin to purify the total flavonoids.Used the macroporous resin packed column to get the high purity total saponins.Then the rat model of chronic non-bacterial prostatitis was prepared by the method of chemical stimulation.The model of the disease was used to screen the therapeutic material basis of the compound Qianyu in treatment of chronic non-bacterial prostatitis.Three chemical components have different effects on the disease states of rats.The combination treatment effect of three chemical components was greater than single chemical component and other chemical components group,so the three chemical components were the therapeutic material basis.