硫磺熏制白芍的安全性评价初步研究

黄山君; 王瑞; 石燕红; 杨莉; 王再勇; 王峥涛

摘要:

建立硫磺熏制白芍中芍药苷及其衍生物芍药苷亚硫酸酯、芍药内酯苷的高效液相色谱含量测定方法, 并通过对芍药苷亚硫酸酯的细胞毒性以及白芍药材的小鼠急性毒性评价, 对硫磺熏制前后的白芍进行安全性评价研究。含量测定采用Shiseido Capcell PAK C₁₈ (250 mm × 4.6 mm ID, 5 μm) 色谱柱, 流动相乙腈−0.02%磷酸水溶液 (15∶85), 检测波长230 nm, 流速1.0 mL·min⁻¹, 柱温30 ℃; 采用MTT法考察芍药苷亚硫酸酯对小鼠原代肝细胞和人原代肝细胞的毒性; 采用小鼠灌胃最大给药量法考察芍药苷亚硫酸酯和白芍的急性毒性。芍药苷亚硫酸酯、芍药苷和芍药内酯苷分别在0.041 8～1.045 0 mg·mL⁻¹、0.023 5～0.587 5 mg·mL⁻¹和0.039 8～0.995 0 mg·mL⁻¹内呈良好线性, r > 0.999 8, 平均回收率99.11%～101.71%, RSD < 2%; 芍药苷亚硫酸酯的浓度≤300 μmol·L⁻¹, 各检测浓度均无明显细胞毒性; 芍药苷亚硫酸酯水溶液和白芍水提物分别进行小鼠灌胃给药, 最大耐受量分别为 5 g·kg⁻¹和80 g·kg⁻¹。建立的白芍中3个成分含量测定方法简单快速、准确可靠、重复性好; 硫磺熏制后白芍中芍药苷的含量显著降低, 且芍药苷亚硫酸酯的含量与芍药苷的含量呈负相关; 体外肝细胞和体内小鼠急性毒性实验未发现芍药苷亚硫酸酯的毒性, 硫磺熏制白芍及白芍水提物比较无显著的毒性。

Abstract:

The paper is to report the development of a method of quantitative analysis of multi-components by high performance liquid chromatography (HPLC) for simultaneously determining paeoniflorin sulfonate (PS), paeoniflorin (PF) and albiflorin (AF) in sulfated Paeoniae Radix Alba. Moreover, the cytotoxicity of paeoniflorin sulfonate by MTT-assay and the acute toxicity of mice by administration of paeoniflorin sulfonate were evaluated. Chromatographic separation of paeoniflorin sulfonate, PF and AF were performed on a SHISEIDO CAPCELL PAK C18 column (250 mm × 4.6mm, 5 μm) for HPLC and a mixture of acetonitrile and 0.02% phosphoric acid solution (15∶85) as the mobile phase. As detector a spectrophotometer set at 230 nm; column temperature 30 ℃; flow rate 1.0 mL?min⁻¹. The toxicity of paeoniflorin sulfonate was evaluated by in vitro cytotoxicity carried out on mouse and human primary hepatocytes, and by acute oral toxicity test carried out on mice. The calibration curve of paeoniflorin sulfonate, PF and AF revealed linearity in the range of 0.041 8 − 1.045 0, 0.023 5 − 0.587 5, and 0.039 8 − 0.995 0 mg·mL⁻¹, respectively (r > 0.999 8). The average recovery was ranged from 99.11% to 101.71%, RSD < 2%. Paeoniflorin sulfonate does not have any cytotoxicity to cells at all the tested concentrations (≤300 μmol·L⁻¹) in the in vitro cytotoxicity assay. The maximum tolerance dose of paeoniflorin sulfonate solution and extraction of Paeoniae Radix Alba to mouse is 5 g·kg⁻¹ and 80 g·kg⁻¹ respectively. The contents of these three components in the samples were determined with the developed method. It is a rapid, convenient and accurate method to determine multi-components. The content of PF in sulfated Paeoniae Radix Alba is significantly lower, and there is negative correlationship between the content of paeoniflorin sulfonate and PF. The in vitro cytotoxicity assay and in vivo mouse acute toxicity test showed that there is no obvious toxicity of paeoniflorin sulfonate and water-soluble extract of sulfated Paeoniae Radix Alba.

硫磺熏制白芍的安全性评价初步研究

Primary safety evaluation of sulfated Paeoniae Radix Alba