Identification of bioactive anti-angiogenic constitutes targeting tumor endothelial cells in Shenmai Injection using multidimensional pharmacokinetics

OBJECTIVE To identify the bioactive anti-angiogenic constitutes targeting tumor endothelial cells(TECs)in Shenmai Injection(SMI).METHEODS For pharmacokinetic(PK)studies,Balb/c mice harboring human colorectal cancer(LoVo)xenografts were treated with SMI 10 mL·kg^-1 daily for 1 or 8 d.Multidimensional PK profiles of ginsenosides in plasma,subcutaneous tumors,and TECs were investigated.For PD studies,the tumor-bearing mice Intravital multi-photon imaging and CD31 immunofluorescence staining were used to evaluate the number of microves⁃sels and braches.Double staining of CD31 and α-SMA was performed to evaluate pericytes coverage ratios around vessels.ELISA was performed to determine the concentrations of VEGF and FGF in tumor tissues.For synergistic anti-tumor study,the tumor-bearing mice were treated with SMI 10 mL·kg^-1 daily,Rd 5 mg·kg^-1 daily with or without 5-FU 15 mg·kg^-1 every 3 d for 20 d.HPLC-MS/MS was used to determine the concentrations of 5-FU in plasma and tumor tissues.RESULTS SMI decreased the number of microvessels(P<0.05)and vessel branches(P<0.05)and improved vascular pericytes coverage(P<0.05).PK studies showed that the concentrations of protopanaxadiol-type(PPD)ginsenosides(Rb1,Rb2/Rb3,Rc,and Rd)in both,plasma and tumors,were higher than those of protopanaxatriol-type(Rg1 and Re)and oleanane-type(Ro)ginsenosides.Among PPD ginsenosides,Rd exhibited the greatest concentrations in tumors and TECs after repeated injection.In fact,the proportion of Rd in the detectable components of SMI gradually increased in the following order:SMI formula(2.8%),plasma(16.0%),tumor tissues(34.3%),and TECs(40.3%).In vivo bioactivity results showed that Rd 5 mg·kg^-1 daily significantly decreased the number of microvessels(P<0.05)and vessel branches(P<0.05)and increased pericytes coverage(P<0.05)while Rd 0.5 mg·kg^-1 daily,Rb1 and Rg1 had no significant effect on them.Rd 5 mg·kg^-1 suppressed the expression of VEGF and FGF simultaneously.Rd 5 mg·kg^-1 enhanced the antitumor effect of 5-FU via increasing the distribution of 5-FU in tumor tissues(P<0.05)in xenograft mice.CONCLUSION Ginsenoside Rd may be the major bioactive anti-angiogenic constituent targeting TECs after SMI treatment.