基于人参皂苷抗炎活性叠加作用的人参质量评价新体系构建

To evaluate the quality of traditional Chinese medicines by the overall view of traditional Chinese medicine scientifically is an important affair now. Ginseng has anti-inflammatory effect, which are associated with immune, cardiovascular, neurological function. Moreover, it is also highly related to the traditional efficacy of invigorating renal qi, as well as spleen and lung benefiting. Ginsenosides are the main effective constituents and 18 kinds of ginsenosides are known to have anti-inflammatory activity. Among them, Rg1, Rb1 and Re are the ginseng quality control markers prescribed by the chinese pharmacopoeia. However, there are still many trace-content of ginsenosides which possess the same structural skeleton in the ginseng (334 constituents reported). And they may have the same pharmacological activity and target of action. Thus, it is worthy to pay attention to their potential pharmacological action. In our perious work, we found that ginsenosides Rh2 etc. had significant additive effect on anti-inflammation. This indicates that a large number of trace-content ginsenosides may have combination and produce a greater additive effect to exert ginseng efficacy. Hence, this project intend to carry out experiments at target, cell and animal levels to certify that over 10 kinds of ginsenosides can exert additive effect of anti-inflammatory activity through glucocorticoid receptor and estrogen receptor. We plan to use a new strategy for screening active constituents (utilize the correlation between drug efficacy and plasma concentration difference caused by animal individual differences), based on the inflammatory model, to find more potential anti-inflammatory active constituents and verify their activity.Our final goal of the present project is to establish novel system and standard for quality evalution of ginseng with the overall view of traditional chinese medicine based on anti-inflammation related additive effect of Ginsenosides.

结合中医药整体观科学评价中药质量是当前要务。人参抗炎活性与其心血管、神经保护作用乃至传统功效大补元气、补脾益肺等密切相关。人参皂苷为其主要活性成分，已知18种人参皂苷具有抗炎活性，其中Rg1、Rb1、Re为药典规定的人参质控指标。但人参中尚存在众多结构母核相同的微量皂苷类成分（报道334种），它们很可能具有相同药理活性及作用靶点，其药理作用值得关注。我们前期发现Rh2等化合物具有显著抗炎叠加作用，提示种类众多的微量人参皂苷很可能加和产生显著叠加作用。故本项目拟开展靶点、细胞、动物水平实验，证明10种以上人参皂苷能通过糖皮质激素受体、雌激素受体产生抗炎活性叠加作用。拟采用基于炎症模型的活性化合物筛选新策略（利用动物个体差异产生的血药浓度差异与药效强度相关性），发现更多潜在抗炎活性化合物并验证其活性。最终基于具有抗炎活性叠加作用的皂苷类化合物，创立符合中医药整体观的人参质量评价新体系与新标准。

中药成分种类复杂，少数几种成分的含量测定不能满足中药质量评价的需求，是限制中药质量评价及质量控制发展的关键问题。我们提出的中药药效物质“叠加作用”理论为中药质量评价提供了新思路：中药成分是否能在相同靶点上产生叠加作用，进而产生药效叠加作用？能否基于具有叠加作用的众多成分，构建符合中医药整体观的质量评价新模式? 为此，我们以中药人参及其主要活性成分人参皂苷类化合物（GNs）为研究对象，以糖皮质激素受体（GR）为靶点，采用分子对接技术从69种GNs中筛选出10种可能与GR结合的GNs，用细胞实验验证其中10种GNs对LPS诱导的RAW264.7细胞NO释放有抑制作用，并欣喜地意外地发现其中5种GNs（PPT、PPD、Rh2、F1、Rd）可以增强地塞米松（Dex）的抗炎作用，是GNs增强Dex活性的“显效形式”。此外，当这5种GNs在低浓度下以一定比例组合时，在增强Dex抗炎活性方面具有浓度上的“叠加作用”， 这是一个重要成果，表明具有相同作用靶点的化合物确实可以产生药效“叠加作用”。进一步以我们提出的口服给药大鼠个体差异造成的中药入血成分血药浓度差异与药理作用强度差异之间相关性寻找体内活性化合物新策略，利用角叉菜胶诱导的大鼠抗炎模型，筛选出人参总皂苷的与抗炎活性呈强正相关性的25种和呈中等正相关性的18种化合物，其中19种化合物为已有文献报道的抗炎活性化合物，表明该筛选活性化合物的新策略的可靠性很高；其中24种为潜在抗炎活性化合物，经对24种中的2种化合物（人参皂苷Mb和人参皂苷Rs2）的细胞实验，成功验证它们确实具有抗炎活性。最后，测定3种不同来源人参样品中GNs的种类和含量，基于人参皂苷类化合物的转化关系以及化合物之间的叠加作用，我们提出了更贴近于符合中医药整体观的人参质量评价新模式：一是控制人参中14种GNs（人参皂苷Rg1、Re、Rf、Rh1、F1、Rg2、Rb1、Rd、Rb2、Rb3、Rg3、Rc、F2和三七皂苷R1）的含量；二是控制人参中包含上述14种和其他29种化合物在内的人参总皂苷的含量，进行人参的质量评价。