Abstract:Objective To investigate the effects of different doses of active ingredients in Salvia miltiorrhiza and Rhizoma Ligustici Chuanxiong on rat brain microvascular endothelial cells (rBMECs) during hypoxic injury and to determine the preferred dose combinations. Methods rBMECs were cultured in vitro and were identified with rabbit-anti-rat factor Ⅷ antibody. The rBMECs were divided into normal group, model group, and test groups. An in vitro cell model of hypoxia was induced in the model group and test groups. The test groups were treated with different doses of tanshinol (A), tanshinone ⅡA (B), ligustrazine (C), ligustilide (D), and ferulic acid (E). An orthogonal test was performed to investigate the effects of these active ingredients in Salvia miltiorrhiza and Rhizoma Ligustici Chuanxiong (each at three dose levels) on superoxide dismutase (SOD) activity in cells, lactate dehydrogenase (LDH) content in cell supernatant, cell apoptosis rate, and cell cycle. Orthogonal analysis of variance and direct comprehensive analysis were used to optimize the dose combination for these indices. Results Compared with the model group, the test groups had increased SOD activity, decreased LDH content, reduced early apoptosis, and inhibited G1/S arrest induced by hypoxia. The orthogonal analysis of variance showed that A2B1C3D2E1 was the preferred dose combination for SOD, A3B1C1D2E3 was the preferred dose combination for LDH, A2B1C2D3E2 was the preferred dose combination for early apoptosis, A2B3C3D3E2 was the preferred dose combination for G1 phase, and A2B3C3D2E2 was the preferred dose combination for S phase. The comprehensive analysis showed that A2B1C1D3E2 was the preferred dose for multiple indices. Conclusion Different doses of active ingredients in Salvia miltiorrhiza and Rhizoma Ligustici Chuanxiong can reduce the hypoxic injury of rBMECs, and their action mechanism varies in different dose combinations.