Study On Activities Of The Anti-inflammatory Ingredients From Stephania Hainanensis

Stephania hainanensis,a species of genus Stephania(Menispermacae),is representative southern herb and Li medical plants.It has been showed that the main substances with biological activity in S.hainanensis are isoquinoline alkaloids.In order to further explore the chemical composition,19 monomer compounds were obtained with alcohol extraction and various separation methods from S.hainanensis.In this paper,in order to explore the activity compounds,we used the classical inflammatory model to investigate the anti-inflammatory effect of 12 compounds that were popular in the present study.The classical inflammatory model was built by stimulating RAW 264.7 macrophages by lipopolysaccharide(LPS)for 24h.Then the levels of NO were measured by NO detection kit in the supernatant of RAW 264.7 which were cultured by different concentrations of the compound which was selected in the inflammatory experiment.The mRNA expression of TNF-? and COX-2 were observed by qPCR.At the same time,the ability of scavenging free radical were detected in vitro at different concentrations of this compound.Finally,to establish a UPLC-MS/MS analysis method to determine tetrahydroberberine,cepharanthine,tetrandrine,fangchinoline,magnoflorine,and dauricine of S.hainanensis,we used Waters Acquity UPLC HSS T3 chromatographic column(2.1mm×100 mm,1.7 m),0.1%formic acid(A)-acetonitrile(B)with a mobile phase gradient elution(0-1 min,18%B;1-7 min,18%-28%B;7-9 min,28%-59%B;9-18 min,59%-100%B)at a flow rate of 0.3 mL/min with 1 ?L sample.Mass spectrometry conditions were monitored by MRM and triple quadrupole mass spectrometer parameters and mass spectral parameters of 6 index components were optimized.All datas were processed using independent sample t-test.Through the physicochemical properties and spectral analysis,the 19 compounds were identified respectively as:dicentrine(1),crebanine(2),demethydicentrine(3),demethycrebanine(4),oxocrebanine(5),4-Oxodicentrine(6),asimilobine(7),p-hydroxyphenethylalcohol(8),tetrahydropalerine(9),cepharanthine(10),Palmatine(11),tetrahydropalmatine(12),tetrandrine(13),fangchinoline(14),chelerythrine(15),magnoflorine(16),dauricine(17),berbamine hydrochloride(1 8),sinomenine hydrochloride(19).The results showed THB had better anti-inflammatory effect,so THB was selected for the following study.THB(5 ?g/mL,10 ?g/mL,20 ?g/mL)could significantly inhibit the production of NO and the mRNA expression of TNF-? and COX-2 in RAW 264.7 cells induced by LPS.At the same time,THB showed a certain effect of scavenging free radical in vitro.Therefore,the anti-inflammatory effect of THB in RAW264.7 macrophages induced by LPS were determined,and the mechanism may be related to the inhibition effect of TNF-? and COX-2.The results showed that the linear relationship between the six components was good within a certain concentration range(R2>0.999).The precision test RSD was 0.41%-2.13%.The duplicate test RSD was 0.77%-1.40%,and stability test RSD was 0.89%-1.99%.The average additive recovery rate was 99.61%-104.55%,RSD ?2.07%.Through this method,the contents of tetrahydropalerine,gepharanthine,fangchinoline,tetrandrine,magnoflorine and dauricine in S.hainanesnsis were 1.647?0.05815?7.913?0.4043?424.5?0.4993 ?g/g.Therefore,the method was highly sensitive,stable and reproducible,and provided a theoretical basis for determination of the alkaloid composition content of S.hainanensis.