Yunnan University of Traditional Chinese Medicine, 1076 Yuhua Road, Chenggong District, Kunming, Yunnan Province, China.
J Ethnopharmacol. 2011 Oct 11;137(3):1291-9. doi: 10.1016/j.jep.2011.07.055. Epub 2011 Aug 4.
Radix Polygoni Multiflori (RPM) and Radix Polygoni Multiflori Praeparata (RPMP) were traditionally widely used as Chinese herbal medicine. However, liver adverse reactions caused by RPM or RPMP were frequently reported all around the world recent years. The aim of this study was to study the cytotoxicities of RPM, RPMP and their major constituents on human liver cell L-02 simultaneously.
Multi-assays, including MTT assay, neutral red uptake (NRU) assay, LDH leakage percentage and liver enzyme secretion (AST, ALT and ALP) were used. Cytotoxicities of major chemical constituents of RPM, 2, 3, 5, 4'-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG), physcion and emodin, were tested. The cytotoxicities of water, 50% ethanol and 95% ethanol extractions of RPM and RPMP were tested. HPLC-DAD analysis was carried to reveal the content change of TSG, physcion and emodin after the processing procedure.
The TD(50) of TSG, physcion and emodin in MTT assay were >10,000 μM, 2853.61 μM and 520.37 μM. In the NRU assay, the TD(50) of TSG, physcion and emodin were much smaller (1401.53 μM, 1140.00 μM, and 3.80 μM). Emodin induced much severe liver enzyme secretion than TSG and physcion. Cell proliferation and LDH leakage rate showed no difference between RPM and RPMP extractions, but ALP, AST and ALT secretions in RPMP extractions were significant lower than that of PMR groups. Water extractions of RPM and RPMP were less toxic than any other solvent in most of the assays. Positive correlation was found between the TSG/emodin ratio and MTT survival rate. The emodin/physcion ratio also showed positive correlation with the LDH leakage percentage.
In conclusion, Radix Polygonum multiflorum and Radix Polygonum multiflorum Praeparata were not liver injure inducing in our in vitro assays. However, the processing produce of RPM could reduce its effect on both cell proliferation and enzyme secretion of liver cell. Judging from cell proliferation, integrity of cell membrane and enzyme secretion, three major chemical constituents of RPM: TSG, physcion and emodin showed no, moderate and severe cytotoxicity against human liver cell line L-02 respectively. Chemical constituents-cytotoxicity relationship investigation revealed that TSG and physcion probably had attenuating effect to emodin. The attenuating mechanisms were still under investigation.
ETHNOPHARMACOLOGICAL 相关性：何首乌（RPM）和制何首乌（RPMP）传统上被广泛用作中草药。然而，近年来世界各地经常报告 RPM 或 RPMP 引起的肝不良反应。本研究旨在同时研究 RPM、RPMP 及其主要成分对人肝细胞 L-02 的细胞毒性。
采用 MTT 法、中性红摄取（NRU）法、LDH 漏出率和肝酶分泌（AST、ALT 和 ALP）等多项检测方法。测试了 RPM 的主要化学成分 2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（TSG）、大黄素和大黄素的细胞毒性。测试了 RPM 和 RPMP 的水、50%乙醇和 95%乙醇提取物的细胞毒性。采用 HPLC-DAD 分析揭示了加工过程后 TSG、大黄素和大黄素含量的变化。
TSG、大黄素和大黄素在 MTT 测定中的 TD（50）均大于 10,000μM、2853.61μM 和 520.37μM。在 NRU 测定中，TSG、大黄素和大黄素的 TD（50）要小得多（1401.53μM、1140.00μM 和 3.80μM）。大黄素诱导的肝酶分泌比 TSG 和大黄素更严重。RPM 和 RPMP 提取物的细胞增殖和 LDH 漏出率无差异，但 RPMP 提取物的 ALP、AST 和 ALT 分泌明显低于 RPM 组。RPM 和 RPMP 的水提取物在大多数测定中毒性低于其他任何溶剂。TSG/大黄素比值与 MTT 存活率呈正相关。大黄素/大黄素比值与 LDH 漏出率也呈正相关。
总之，在我们的体外试验中，何首乌和制何首乌不会引起肝损伤。然而，RPM 的炮制产物可能会降低其对肝细胞增殖和酶分泌的影响。从细胞增殖、细胞膜完整性和酶分泌来看，RPM 的三种主要化学成分 TSG、大黄素和大黄素对人肝细胞系 L-02 的细胞毒性分别为无、中度和重度。化学物质-细胞毒性关系的研究表明，TSG 和大黄素可能对大黄素有减弱作用。减弱机制仍在研究中。
