| Objective:To establish the HPLC fingerprint chromatography,mouse’s serum HPLC fingerprint chromatography and a method for the determination of total alkaloids in SZS.To observe the effect of total alkaloids from SZS on mice’s behaviors and cerebral monoamine oxidase(MAO)with chronic unpredictable mild stress model.Methods:1.With acid water extraction method then being purified by macroporous resin,total alkaloids was prepared from SZS.Then the compound was isolated by alumina column chromatography followed preparative liquid chromatography,its structure was identified on the basis of spectral analysis.The HPLC fingerprint chromatography of total alkaloids in SZS was established with HPLC-DAD method and the content of zizyphusine in total alkaloids was determined at the same time.The method of determine the content of total alkaloids in SZS was established with UV method,providing a basis for quality control of total alkaloids from SZS.2.The composition of total alkaloids in SZS was analyzed with HPLC-ESI-MS method and its constituents in mice’s blood were comfirmed.3.With chronic unpredictable mild stress(CUMS)method,The forced swimming test(FST),tail suspension test(TST),independent activities experiment and sugar water consumption test were carried out to evaluate the antidepressant effect of SZS total alkaloid fraction.CUMS mice’s MAO-A and MAO-B was determined with UV method,providing a basis for elucidating the mechanism of the antidepressant effect of SZS total alkaloid fraction.Results:1.The compound isolated from total alkaloids in SZS was identified with 1H-NMR and 13C-NMR as zizyphusine and could be used as a reference substance for the quality control of SZS total alkaloids.A HPLC Fingerprint Chromatography of total alkaloids from SZS was established with HPLC-DAD.Chromatogram condition:Agilent TC-C18 chromatographic column(4.6 mm×250 mm,5 μm)and Agilent TC-C18 protection column was selected,with a mixture of A-acetonitrile and B-water as mobile phase,gradient elution at 0 1 Omin,0～15%A;10～20min,15～20%A;20～30min,20～30%A;30～40min,30～40%A;40～50min,40～60%A;50～60min,60～95%A,flow rate at 1 mL/min,column temperature at 25 ℃,the injection volume was 20μL,detection wavelength was 282 nm.All chromatographic peaks were completely separated from one another.The content of zizyphusine in SZS total alkaloids was determined with HPLC method;the result shows that the concentration of zizyphusine was in linear relationship with absorbance in the range of 10.0-20.0μg/mL.The equation of linear regression was Y=3593844X-2x107(r=0.9994).The average recovery was 100.2%and RSD was 0.63%.The content of total alkaloids in SZS alkaloids fraction was determined by UV and with berberine as its reference substance,the result shows the concentration of berberine was in linear relationship with absorbance in the range of 10.0-50.0ug/mL.The equation of linear regression was Y = 0.0504X-0.009(r =0.9998).The average recovery was 100.3%and RSD was 0.69%.This method is simple,accurate and reproducible,its detection period is short therefore suitable for quality control of SZS,providing a reliable basis for quality control of total alkaloids from SZS.2.Two ingredients of SZS total alkaloids were assigned with HPLC-ESI-MS:zizyphusine(342.4)and sanjoinine D(566.7).It was primary confirmed that zizyphusine could be absorbed orally by mouse and then entering the serum.3.With CUMS model,the forced swimming test(FST),tail suspension test(TST),independent activities experiment and sugar water consumption test were carried out,the result shows the weight,the sugar water intake and independent activities times of each administration group increased significantly compared to control group;yet the immobility time of FST and TST significantly decreased,shows the antidepressant effect of SZS total alkaloid fraction is quite remarkable.The results of the determination of CUMS mice’s MAO-A and MAO-B indicate that SZS total alkaloids has MAO inhibit effect which would be related with the antidepressant effect of SZS total alkaloid fraction.Conclusion:This HPLC fingerprint chromatography of SZS total alkaloids was established with high resolution,both accurate and reproducible,is suitable for qualitative identification of index components.The content of zizyphusine in SZS total alkaloids was determined with HPLC-DAD;this method is simple,accurate and reproducible,with short detection period.The content of total alkaloids in SZS alkaloids fraction was determined by UV and with berberine as its reference substance,the method is both simple and accurate,could be used for quality control of total alkaloids from SZS.The pharmacodynamic result shows alkaloids.from SZS could improve behaviors of CUMS mice.In addition,the result has a relationship of time-effect and dose-effect.The determination of MAO shows that the antidepressant mechanism of alkaloid fractions in SZS was related with the decreasing of MAO activity and its synergy with increasing 5-HT&NE in hippocampus and cortex,and several other mechanisms. |
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