Study On The Diuretic Activity,Mechanism And Chemical Constituents Of Astragalus Rigidulus

Astragalus rigidulus Benth.ex Bunge is a perennial herb of the genus Astragalus.According to the records of Flora of my country,Astragalus rigidulus is produced in eastern and southern Tibet（Jiangda,Nagqu,Basu,Lhasa,Gyantse,Zanda,Yadong）.Born in the hillside grassland or river beach gravel at an altitude of 3800-5200 meters.Astragalus rigidulus is used as the base of Tibetan medicine"Sagar"in Tibetan areas of my country.The whole herb is used as medicine to treat ascites,relieve intestinal pain,and cure chronic nephritis,edema,carbuncle,swollen,sore and furuncle.Although domestic and foreign scholars have reported many studies on the chemical constituents and pharmacology of Astragalus plants,there is no relevant research report on Astragalus rigidulus.In order to verify the traditional efficacy of Astragalus as the base of Tibetan medicine"Sagar",and to explore its related mechanism of action and pharmacodynamic material basis,this paper evaluated the diuretic effect of Astragalus rigidulus for the first time.A more systematic separation of chemical components was carried out in the n-butanol extraction part.The specific research contents are as follows:1.Evaluation and mechanism of diuretic efficacy of Astragalus rigidulus.In this experiment,a rat model of normal saline was used to study the diuretic effect of Astragalus rigidulus and its mechanism.Rats qualified for urination were screened and randomly divided into normal control group（normal saline group）,model group（water load model group）,furosemide positive drug group,low-dose ethanol group,high and low-dose petroleum ether group,dichloromethane High and low dose group,high and low dose group of ethyl acetate,high and low dose group of n-butanol,high and low dose group of water site.After the first administration,the urine volume of the rats in the first 6 hours was collected and recorded,the concentration and p H of Na⁺,K⁺,Cl^-,Ca²⁺in the urine were measured,and the diuretic effect was evaluated.After continuous administration for seven days,blood was collected,and serum renin,angiotensin II,aldosterone,atrial natriuretic peptide and antidiuretic hormone were determined by enzyme-linked reaction adsorption method（ELISA）,and AQP1、AQP2 and AQP3 protein content in kidney were determined by western blot method.The results showed that the site of petroleum ether,ethyl acetate and n-butanol could significantly increase the urine output of rats（P<0.05,P<0.01）,and the diuretic effect of petroleum ether,ethyl acetate and n-butanol in high-dose groups was slightly stronger than that of positive drug group;low dose of total extract,low dose of petroleum ether,high dose of ethyl acetate,low dose of n-butanol and low dose of water can significantly increase the p H value of urine output in rats（P<0.05,P<0.01）;The high and low dose of n-butanol significantly increased the concentration of K⁺in the urine of rats（P<0.05）,high dose of total extract,high and low dose of petroleum ether,high dose of ethyl acetate,and high dose of n-butanol can significantly increase the Cl^-concentration in rat urine（P<0.05,P<0.01）.The concentration of Ca²⁺in the urine of rats with high and low dose of total extract and high dose of petroleum ether increased significantly（P<0.05,P<0.01）;Compared with the model group,the high-dose n-butanol group can significantly reduce the renin content（P<0.05）,and the high-dose ethyl acetate,and low-dose n-butanol can significantly reduce the content of aldosterone in serum of rats（P<0.05,P<0.01）,and high-dose ethyl acetate and high-dose n-butanol groups can significantly reduce the level of angiotensin II（P<0.05）,and high-dose ethyl acetate group can significantly reduce antidiuretic hormone level（P<0.05）;compared with model group,petroleum ether site significantly decreased AQP3protein expression（P<0.05）,ethyl acetate site significantly decreased AQP2 protein expression（P<0.05）,n-butanol The expression of AQP1 protein was significantly decreased in the site（P<0.05）.2.Study on the chemical constituents of Astragalus rigidulus.The n-butanol fractions were systematically separated by macroporous resin,gel,reverse silica gel,preparative HPLC and other chromatographic techniques,and the structures of the separated monomers were identified by 1D/2D-NMR,MS,UV,IR and other spectra..Twenty flavonoids and one fatty glycoside were isolated and identified from the ethanol extract of Astragalus rigidulus,including eight flavonols and their glycosides kaempferol（AR1）,kaempferol-3-O-β-D-glucopyranoside（AR8）,quercetin（AR30）,kaempferol-3-O-β-D-6′-acetylglucopyranoside（AR34）,kaempferol-3-O-α-L-rhamnopyranosyl-（1→6）-β-D-glucopyranoside（AR58）,5,7,4′-trihydroxy-3′-methoxyflavonol-3-O-rutinoside（AR66）,kaempferol-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranoside（AR68）,quercetin-3-O-β-D-glucoside（AR70）,8 isoflavones and their glycosides 7-O-methylorobol-4′-O-β-D-glucopyranoside（AR5）,5,7,4′-trihydroxyisoflavone（AR6）,Mildiside A（AR10）,calycosin（AR21）,purine 4′-O-β-D-Glucoside（AR28）,orobot（AR32）,5,7-dihydroxy-4′-methoxyisoflavone-2′-O-β-D-glucopyranosid（AR38）,pratensein-7-O-β-D-glucoside（AR40）,3 isoflavanes and their glycosides（R,S）-（+）-mucronulatol（AR13）,astriginosideA（AR14）,2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-D-glucose（AR45）,a dihydroflavonoid naringenin（AR23）and a fatty glycoside amarantholidoside IV（AR63）.Among them,AR14 is a new compound,and 9 compounds,such as AR5,AR10,AR23,AR28,AR32,AR34,AR38,AR63,AR68,are found in Astragalus for the first time,and the rest of the compounds are isolated from Astragalus rigidulus for the first time..In conclusion,animal models were used to verify the diuretic activity of the Tibetan medicine Astragalus rigidulus,and its active sites were petroleum ether,n-butanol and ethyl acetate.Further research shows that each part may exert diuretic effect through different mechanisms:petroleum ether part plays a diuretic effect by regulating chloride,calcium ion and AQP3 protein content,ethyl acetate part plays a diuretic effect mainly by chloride,antidiuretic hormone,regulating angiotensin II,aldosterone hormone and AQP2 protein content,The n-butanol site has a diuretic effect by regulating the content of potassium ion,chloride,renin,angiotensin II,aldosterone hormone and AQP1 protein.The research on the chemical composition of the n-butanol part of the active site shows that the chemical composition of this part is isoflavone and flavonol derivatives.The results of this study laid the foundational data for the further development and utilization of the Tibetan medicine Astragalus hard（Sagar）resources.