Comparison Research Of Biotransformation And Pharmacokinetics Of Antitumor Effective Fraction EVn50 And Its Active Constituents VBE-1, VBE-2 From Fruits Of Vitex Negundo

Fructus Viticis Negundo is a famous tranditional Chinese medicine widely used in South of China.The previous study of antineoplastic activity indicated that a great deal of malignant tumour cells were inhibited by the fruits extracts(EVn50).VBE-1 and VBE-2,respectively chemical name described as 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl) -3-hydroxymethyl-7-methoxy-3,4-2H(3R,4S)-2-aldehydenaphthalene and 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-5-methoxy-3,4-2H(3R,4S)-2-aldehydenaphthalene,were two main lignan compounds isolated from EVn50.VBE-1 was the first inhibitor of PI3K/Akt/mTOR signal transduction pathway originated from natural lignans,suggesting VBE-1 has the potential to be an antitumor drug in the future.It was demonstrated that the effects of VBE-1 on the ovarian cancer cell line COC1 growth inhibition in vitro were potent in several antineoplastic activity tests,and the IC₅₀ value reached to 1.40μg·mL^-1.In contrast,the effects of VBE-1 on the tumor of nude mice model of human ovarian carcinoma growth inhibition were inconspicuous.Comparison of EVn50 and VBE-1 of inhibition of ovarian cancer cell lines indicated that EVn50 both showed powerful inhibition of ovarian cancer cell lines in vitro and in vivo;VBE-1 was potent in vitro but had no significant inhibition in vivo.In addition,the three metabolites isolated from the excreta after rats oral adminstration of VBE-1 suggested that the metabolites were weak inhibitor of tumor cells,and the metabolic process was inactive.In this paper,metabolism of VBE-2 and EVn50 in rats were studied.There were three metabolites identified from rats excreta after oral adminstration of VBE-2,and two metabolites were isolated by chromatographic means.The two metabolites were identified as 6-hydroxy-4-(4-GlcA-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-2H(3R,4S)-2-aldehydenaphthalene and 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-2,3-dihydroxymethyl-7-methoxy-3,4-2H(3R,4S)-napht halene.The metabolic sites of VBE-2 were glucuronidation of 4' phenolic hydroxyl and reduction of aldehyde group,which were similar to those of VBE-1.The main constituents of EVn50 were detected in the urine of rats after oral adminstration of EVn50,as well as small amount of metabolites.The HPLC-MS-MS analytical method of VBE-1 in rats was developed and validated.The result of method validation indicated that the method was highly sensitive and stable,which was suitable for study of VBE-1 in pharmacokinetics of rats.Three groups of rats,including oral administration of VBE-1,oral administration of VBE-1 and VBE-2 and oral administration of EVn50,were studied in order to demonstrate the differences of plasma concentration and parameters of pharmacokinetics of VBE-1 and its two metabolites,M-1 and M-3.The one-compartmental model was all fit to three groups of kinetics process of VBE-1 in rats.The pharmacokinetic parameters of VBE-1 was as follows,AUC_0-4 was 1.12±0.50 mg·h·L^-1,5.12±2.61mg·h·L^-1 and 5.38±1.50 mg·h·L^-1,respectively; AUC_0-∞ was 1.26±0.50 mg·h·L^-1,5.79±3.14 mg·h·L^-1 and 6.59±1.98 mg·h·L^-1,respectively;t_1/2 was 0.90±0.18 h,1.23±0.40 h and 1.83±0.34 h,respectively;C_max was 0.97±0.57 mg·L^-1,4.68±2.81 mg·L^-1 and 4.69±2.52 mg·L^-1,respectively;T_max was 0.30±0.23 h,0.23±0.05 h and 0.31±0.17 h,respectively.It was concluded that in the same dosage of VBE-1,there were significant differences in pharmacokinetic parameters of VBE-1 monomer administration group and the mixture administration groups(P＜0.05).The plasma concentration,AUC and C_max of VBE-1 increased significantly in complex administration groups,which demonstrated that the low plasma concentration and poor pharmaco -kinetic parameters were probably the reason why VBE-1 activited low in vivo.There were no significant differences in AUC and C_max of VBE-1 in two mixture administation groups(P＞0.05),which showed that VBE-2 in EVn50 administration group have synergistic action to VBE-1,increasing the effective plasma concentration of VBE-1.The variation of plasma concentration of two metabolites of VBE-1 were similar to that of VBE-1 in three groups,which suggested that metabolites of VBE-1 was positive correlation to parent drug. Based on the research of biotransformation and pharmacokinetics of VBE-1,VBE-2 and EVn50,the probable reason of different activities of VBE-1 in vitro and in vivo was the low absorption amount of VBE-1 in VBE-1 monomer adminstration.VBE-2 in EVn50 increased the plasma concentration of VBE-1 by promoting absorption or competitive metabolism.More studies on mechanism of drug absorption are required in the future.