Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Xianlin Road 138#, Nanjing 210023, China.
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Xianlin Road 138#, Nanjing 210023, China.
J Ethnopharmacol. 2018 Jun 12;219:152-160. doi: 10.1016/j.jep.2017.11.005. Epub 2017 Nov 7.
Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated.
To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents.
The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR.
A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P < 0.01, P < 0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P < 0.01, P < 0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P < 0.05, P < 0.01, P < 0.001).
A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.
茜草是一种常见的中药，具有活血化瘀的功效，临床上已用于治疗与血瘀证相关的疾病多年。先前的研究表明，抗血栓形成和促进血管生成可以改善血瘀证。然而，茜草的抗血栓形成和促血管生成作用尚未得到很好的研究。
确定茜草的潜在抗血栓形成和促血管生成活性，并阐明其潜在机制。此外，通过 UPLC-Q-TOF/MS 对茜草提取物（QC）的主要化学成分进行定性分析，以探讨药理活性与化学成分之间的关系。
QC 样品由 95%乙醇提取物和水提物组成，采用 95%乙醇提取。使用 UPLC-Q-TOF/MS 分析 QC 的主要化学成分。对于 QC 的抗血栓形成实验，采用苯肼（PHZ）诱导的 AB 品系斑马鱼血栓形成模型。用邻苯二胺对斑马鱼幼虫进行染色，用荧光显微镜观察和拍摄斑马鱼心脏和尾静脉的图像。观察和分析各组心脏中红细胞的染色强度（SI）和尾静脉中的血栓形态，以评估 QC 的抗血栓形成作用。对于 QC 的促血管生成实验，采用 VEGF 受体酪氨酸激酶抑制剂 II（VRI）诱导的 Tg（fli-1：EGFP）y1 转基因斑马鱼（Flik 斑马鱼）的节间血管（ISV）不足模型。观察和分析完整 ISV 和缺陷 ISV 的形态，以评估 QC 的促血管生成活性。采用实时 PCR 研究促进血管生成的机制。
根据标准化合物和参考文献，从 QC 中鉴定出 12 种成分，包括 9 种蒽醌和 3 种萘醌。用 QC 处理后，PHZ 诱导的 AB 品系斑马鱼幼虫的血栓形成在一定程度上减少，我们认为这与 QC 的剂量有关，50-200μg/ml QC 处理组的治疗效果尤为明显（P<0.01，P<0.001），明显优于 PHZ 模型组。同样，QC 也恢复了 VRI 诱导的 Flik 斑马鱼幼虫中缺失的 ISV，这与 QC 的一定剂量效应有关。QC 的促血管生成活性也很明显（P<0.01，P<0.001），明显优于 VRI 模型组。实时 PCR 检测结果表明，QC 显著恢复了 VRI 诱导的 Flik 斑马鱼中 vWF、VEGF-A、kdrl 和 flt-1 的下调（P<0.05，P<0.01，P<0.001）。
通过 UPLC-Q-TOF/MS 对 QC 中的 12 种化合物进行了分析。药理学实验数据表明，QC 在斑马鱼中具有抗血栓形成和促血管生成活性，主要活性成分可能是蒽醌和萘醌。因此，本研究为茜草作为一种活血化瘀的中药在临床上的应用提供了理论依据。
