Study On Chemical Constituents Of Shenfu Injection

Shenfu injection derived from "shenfu tang" which is ancient formula of China. It iscomposed of extracts of Ginseng Radix Et Rhizoma Rubra, and Aconiti Lateralis RadixPraeparata. The injection has the function of protecting against myocardial ischemia-reperfusion injury and improving the systolic function, anti-heart failure andantiarrhythmic.In order to improve the quality standards, ensure the safety and prove thepharmacodynamic material basis of Shenfu injection, we studied the chemical compositionand the the activity of Shenfu injection in depth.Firstly, the extracts of Ginseng Radix Et Rhizoma Rubra and Aconiti Lateralis RadixPraeparata were preparated according to the preparation process of the Shenfu injection,and then, the chemical constituents of the two extracts were isolated. Twenty compoundsincluding12kinds major ginsenosides and10kinds rare ginsenosides were separated fromthe extracts of Ginseng Radix Et Rhizoma Rubra and identified as ginsenoside Rg₁,ginsenoside Re, ginsenoside Rf, ginsenoside Rg₂, ginsenoside Rb₁, ginsenoside Rc,ginsenoside Rb₂, ginsenoside Rb₃, ginsenoside Rd, ginsenoside F₁, ginsenoside F₃andginsenoside F₅, ginsenoside Rk₁, ginsenoside Rg₅, ginsenoside Rk₃, ginsenoside Rh4,ginsenoside Rg₆, ginsenoside F₄, ginsenoside Rg₃, ginsenoside20(R)-Rg₃, ginsenoside Rh₁and ginsenoside20(R)-Rh₁. Six compounds were separated and identified from the extractsof Aconiti Lateralis Radix Praeparata, they are benzoylhypaconitine, benzoyldeoxy-aconitine, benzoylmesaconitine, aconine and hypaconine.In this paper,4kinds of alkaloids and20kinds of ginsenosides in Shenfu injectionwere determined by different approaches. A method for the determination ofbenzoylaconine (BA), benzoylmesaconine (BM), benzoylhypaconine (BH) and benzo-yldeoxyaconine (BD) in Shenfu injection based on homogeneous ionic liquidmicroextraction coupled with high performance liquid chromatographic (HPLC) separationwere developed. The experimental parameters including volume of ionic liquid, pH,extraction time and salt concentration were optimizated when0.058g [C₆Mim][BF₄] and 0.26g NH4PF6were used as extraction solvent,the target compounds can be extracted from5mL of injection, the extraction time is1min, and the concentration of NaCl is50g·L^-1.The detection limits for BM, BA, BH and BD were45.5,19.8,26.3and12.2μg·L^-1respectively. The test of samples purchased from Yaan San-Jiu Pharmaceutical showed thatthe content ranges are0.2^-1.2(BM),0.2-0.3(BA),0.1-0.7(BH) and0.04-0.1(BD) μg·mL^-1,respectively, and the values are quite different between samples with different batchnumber. This method was proved to be fast, easy and have good enrichment effect which issuitable for the preconcentration and determination of trace components in traditionalChinese medicine injection.Twenty kinds of ginsenosides including10kinds major ginsenosides Rg₁, Re, Rb₁,Rb₂, Rb₃, Rc, Rd, Rf, Rg₂, F₁and10kinds rare ginsenosides Rh₁,20(R)-Rh₁, Rk₃, Rh4,Rg6, F₄, Rg₃,20(R)-Rg₃, Rk₁and Rg₅were detected in Shenfu injection by SPE and HPLC.The results showed that the contents of the major ginsenosides are larger than the rare ones.It also found that the amounts of the ginsenosides are different in the samples, the contentschanged in the ranges as follow:85^-119(Rg₁),58-89(Re),28-31(Rf),13^-15(Rg₂),198-227(Rb₁),97^-114(Rc),50-64(Rb₂),8^-14(Rb₃),3-6(F1),36-49(Rd),27-42(Rh₁),14-25(20(R)-Rh₁),3-6(Rg6),4-8(F₄),8^-11(Rk₃),6^-11(Rh4),21-36(Rg₃),16-30(20(R)-Rg₃),8^-15(Rk₁), and12-20(Rg₅) μg·mL^-1.In this article, we also investigated the contents of20ginsenosides in the other twocommonly used injections which are also composed of Ginseng Radix Et Rhizoma Rubra,Shenmai injection and Shengmai injection. The results showed that the contents of20ginsenosides are with the same trend in those three kinds of injections but for specificcontent difference.Shenfu injection was proved to be effective to protect against myocardial ischemia-reperfusion injury clinically. In order to explore the material basis and the mechanism ofthis effect, we studied the protective effects of Shenfu injection, the mixture ofginsenosides and alkaloids, the mixture of ginsenosides, and the mixture of alkaloids onhuman erythrocytes against AAPH-induced hemolysis. The results showed that Shenfuinjection has the most obvious anti-hemolysis effect, which proved that the protective effectof Shenfu injection related with not only the ginsenosides and alkaloids but also other ingredient in it, and the protective effect of Shenfu injection against myocardial ischemia-reperfusion injury may be related to the free radical scavenging effect. The results alsoshowed that the activity of Shenfu injection is closed linked with its concentration, theanti-hemolytic activities is strongest when the concentration is10%(v/v), and weakenedgradually with the increase or decrease of concentration. Shenfu injection showedpro-hemolytic activities until the concentration is higher than50%.Finally, the effects of monomers of20ginsenosides on human erythrocytes duringAAPH-induced hemolysis were studied. The results showed that the activities ofginsenosides are in relation with their structures.10major ginsenosides and3rareginsenosides can performance anti-hemolytic activity with the order as Rf>20(R)-Rh₁>Rg₁>Rb₁>Rd>Rc>Re>Rh1>Rg₂^Rb₃^F1²0(R)-Rg₃>Rb₂, and other7rare ginsenosides gavehemolytic activity in the order of Rg₃^Rk1^Rg₅>Rg6^F4>Rk3>Rh4.