| Objective:(1)Taking the petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet as the research object,analyze the chemical composition in it.(2)Study the diuretic effect of petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet,and preliminarily explore the material basis of it.(3)Study the anti-inflammatory effect of petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet,and screen the effective components of the anti-inflammatory effect.(4)Preliminarily study the diuresis mechanism of petroleum ether extract of Abutilon indicum(L.)Sweet.(5)Establish a HPLC method for determination of vanillic acid in Abutilon indicum(L.)Sweet.Methods:(1)Use silica gel column chromatography and LC-MS to analyze the chemical composition of petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet.(2)Split the petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet into different polar fractions,adopt the metabolic cage method and the water-loaded model in mice,with hydrochlorothiazide as the positive control drug,lavage 8 days in a row,determine the total amount of urine in 4 hours after the treatment,screen the effective fraction and take further analysis.(3)Split the petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet into different polar fractions,induce the inflammatory model of mouse ear edema by using xylene,take dexamethasone acetate as he positive control drug,determine the ear swelling degree of the mice,screen and analyze the effective anti-inflammatory fraction.(4)Use the metabolic cage method to collect the total amount of urine in 4 hours after the treatment,and determine the potassium ion concentration in urine.Get the eyeball blood of the mice and snap their kidney,determine the concentration of Na-K-ATPase in serum and kidney and carbonic anhydrase in kidney.(5)Chromatographic column is Thermo BDS HYPERSIL C18 column(5 μm,4.6 mm×250 mm);Liquid phase: methanol-0.1% formic acid(21:79)for isocratic elution;Detection wavelength: 260 nm;Flow rate: 1ml/min;Column temperature: 30 ℃;The injection volume is 10 μL.Results :(1)The petroleum ether extract of Abutilon indicum(L.)Sweet was isolated by silica gel column chromatography and identified four compounds,and one compound was identified by LC-MS;A compound was isolated and identified by silica gel column chromatography from ethyl acetate extract,and nine compounds were identified by LC-MS.(2)The effective diuresis fraction in the petroleum ether part of Abutilon indicum(L.)Sweet is 50:1(petroleum ether: ethyl acetate)fraction;The effective diuretic fraction of ethyl acetate fraction was 50:1(chloroform: methanol)fraction and two compounds were isolated from it.One of the compounds was identified as an effective diuretic component by pharmacodynamic verification and identified as oleanolic acid by thin layer chromatography and infrared spectroscopy.(3)The anti-inflammatory active fraction of the petroleum ether fraction of Abutilon indicum(L.)Sweet is 10:1(petroleum ether: ethyl acetate)fraction,from which a compound is isolated,which is confirmed to be an anti-inflammatory active ingredient through pharmacodynamic verification and identified as β-sitosterol through thin layer chromatography and infrared spectroscopy;The anti-inflammatory active fraction of ethyl acetate fraction was 50:1(chloroform: methanol)fraction.Two compounds were isolated from the fraction and confirmed to be anti-inflammatory active ingredients through pharmacodynamic verification.They were identified as β-sitosterol and oleanolic acid through thin layer chromatography and infrared spectroscopy.(4)Compared with the blank group,hydrochlorothiazide significantly increased the potassium ion concentration in urine(P<0.05),and no significant difference was found in the 50:1 flow group.Compared with the blank group,hydrochlorothiazide significantly reduced the concentration of Na-K-ATPase in mice serum and kidney(P<0.05),and no significant difference was found in the 50:1 flow group.There was no significant effect on the concentration of carbonic anhydrase in mice kidney,and hydrochlorothiazide showed a tendency to decrease the concentration of carbonic anhydrase in the mice.(5)The content of vanillic acid in Abutilon indicum(L.)Sweet is 94.98 μg/g.Conclusion:(1)The chemical composition analysis of the petroleum ether extract and ethyl acetate extract of Abutilon indicum(L.)Sweet was carried out to provide a basis for the in-depth study of Abutilon indicum(L.)Sweet.(2)The main component of the diuretic action in the petroleum ether extract is 50:1(petroleum ether: ethyl acetate)flow group,and the main component of diuretic action in ethyl acetate extract is oleanolic acid.(3)The main component of the anti-inflammatory action in the petroleum ether extract is β-sitosterol,and the main component of anti-inflammatory action in ethyl acetate extract is β-sitosterol and oleanolic acid.(4)The petroleum ether extract of Abutilon indicum(L.)Sweet may not achieve diuretic effect by promoting potassium ion excretion,inhibiting the activity of Na-K-ATPase and carbonic anhydrase.(5)The method is simple,easy to operate,stable and reproducible,and can be used to determine the content of vanilla acid in Abutilon indicum(L.)Sweet. |
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