基于质谱分子网络从近缘亚属发现重楼药材可替代资源的评价研究

Paridis Rhizoma (Chonglou) is widely used in traditonal Chinese medicine as hemostatic, antitumor, and anti-inflammatory effects, and is the main raw material of more than 70 Chinese patent medicines, such as ‘Yunnan Baiyao’. Chinese Pharmacopoeia (2015) rules the original plant of traditional Chinese drug “Chonglou” is the rhizome of Paris polyphylla var. yunnanensis or Paris polyphylla var. chinensis, which belong to the variations of Paris polyphylla. Due to the long-term dependence on wild resources, slow-growing (more than 5 years) and the domestic demand of more than 1000 tons every year, we face an increasing problem with the resource shortage of the two plants, which have been listed as near threatened (NT) species. However, there aren’t the related research reports on the most species of the allied subgenus Paris of this herb, which have a certain amount of resources and have been planted in the southwest of China. Our group found that the extracts of four species of subgenus Paris showed comparable to that of the extract of P. polyphylla var. yunnanensis or stronger on hemostatic and antitumor activities. The results of HPLC analysis showed that the contents of the total steroidal saponins of the three speices were higher than that of P. polyphylla var. yunnanensis. Furthermore, there are some similarities as well as differences in their chemical constituents between the five species by MS/MS molecular networking. Our group are investigating the chemical components of P. axialis and obtained a series of highly oxygenated steroidal saponins with multiple sugar chains by the molecular networking deduplication technology and the directional separation technology. The project is firstly planned to quickly investigate the chemical constituents with hemostatic and antitumor effects of the five species of the subgenus Paris based on the MS/MS molecular networking dereplication technology and illuminate their structure-activity relationships. Furthermore, we will clarify the differences of the active compounds between the five species and P. polyphylla var. yunnanensis by the qualitative and quantitative analysis. On the basis of the research results of the bioactivity, the therapeutic material basis, and the active ingredient content, we will clarify what species of the subgenus Paris can be used as Paridis Rhizoma. This study will be significant to alleviate the resource shortage of Paridis Rhizoma.

重楼具有止血、抗肿瘤、消炎等功效,为云南白药等70余种中成药的原料。《中国药典》规定重楼药材的基原为多叶重楼的两个变种云南重楼或七叶一枝花的根茎。由于长期依赖野生资源,5-7年才能入药,年需求量超过千吨,导致其资源短缺和物种近危。其近缘中轴亚属在西南地区有一定资源量和种植,但大部分种无研究报道。申请人前期研究发现中轴亚属4种重楼的提取物在止血、抗肿瘤活性上与云南重楼相当或更强,HPLC分析发现其中3个种的总皂苷高于云南重楼,质谱分子网络技术发现它们在化学成分上有一定相似性和差异性。通过该技术去重复和定向分离,已从五指莲中发现了一些高氧化度多糖链的皂苷。本项目拟在前期研究基础上,借助分子网络技术,快速识别分离未知皂苷,阐明中轴亚属5种可用重楼的止血抗肿瘤物质基础和构效关系;进而从生物活性、物质基础、活性成分含量三个层次阐明这5个种哪些可以作为重楼药材使用,对缓解重楼资源短缺具有重要意义。

重楼具有止血、抗肿瘤、消炎等功效，为云南白药等70余种中成药的原料。《中国药典》规定重楼药材的基原为滇重楼或七叶一枝花的根茎，其近缘中轴亚属植物在西南地区有一定资源量和种植，常用做重楼使用，但大部分种无化学成分及生物活性研究报道。为了评价中轴亚属植物是否可以用做重楼药材，本项目从有效成分含量、化学成分差异性、物质基础和生物活性四个方面展开了研究。.本项目按照《项目任务书》执行，利用HPLC分析及质谱分子网络技术等对重楼中轴亚属6种植物五指莲、平伐重楼、长柱重楼、独龙重楼、皱叶重楼和腾冲重楼开展了有效成分含量、化学成分差异性、物质基础和生物活性的系统研究。研究结果表明：（1）按照2015版《中国药典》的规定，除五指莲外，其它5种重楼的总皂苷均达到药典的标准，平伐重楼、长柱重楼、独龙重楼、皱叶重楼和腾冲重楼总皂苷的含量分别为1.124%、0.847%、0.680%、1.496%和1.322%；（2）发现五指莲富含17-羟基纤细薯芋皂苷，最高含量可以达到3.80%；4个种富含重楼皂苷I，含量均超过1%，其中腾冲重楼含量高达3.06%；（3）皱叶重楼、长柱重楼、独龙重楼和五指莲提取物诱导兔子血小板聚集活性与滇重楼提取物相当，其中五指莲和皱叶重楼的活性强于滇重楼；（4）根据分子网络的分析，发现皱叶重楼、平伐重楼、腾冲重楼的化学成分与滇重楼的相差较大，进而从这3种重楼中分离鉴定了63个化合物，其中新化合物21个；（5）生物活性研究发现，皂苷parisyunnanoside D和dichotomin对四株肿瘤细胞株的抑制活性均强于阳性对照药顺铂，其中dichotomin对A-549和SMMC-7721细胞株的IC50值为0.50 μM；首次发现部分薯蓣皂苷具有显著的止血活性。综上所述，从物质基础角度，这6种中轴亚属植物均可用作重楼药材；由于其微量成分有差异，因此其安全性和药效学仍需进行评价和深入研究。此外，根据其所含皂苷的类型及含量不同，可以根据药品生成和药材配伍需要来进一步开发利用这些重楼中轴亚属植物资源，或者从高含量的种中获得活性单体，用于新药研发。