Wang Baolin, Wang Qinqin, Yuan Renyikun, Yang Shilin, Lu Meilin, Yuan Fuhong, Dong Zhidan, Mo Menghuan, Pan Qiming, Gao Hongwei
College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530200, China.
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China.
Chin Med. 2023 Nov 23;18(1):153. doi: 10.1186/s13020-023-00860-3.
The successful launch of icaritin, a therapeutic drug for liver cancer derived from Epimedium brevicornu, has provided new impetus for the development of prenylated flavonoids in the field of oncology. Flemingia macrophylla is reported to contain characteristic prenylated flavonoids which can regulate the p53 protein. We aimed to isolate these constituents and conduct activity evaluation, structure-activity relationship, and mechanism studies to provide candidate compounds for antitumor drug development.
In this study, chromatographic techniques combined with spectroscopic methods were used to separate, purify, and identify the constituents of Flemingia macrophylla methanol extract. The cytotoxic activity of the constituents was evaluated using an MTT assay with A549 and H1975 cells as the model. The binding mechanism between the compounds and the p53 protein was investigated with molecular docking and validated with cellular thermal shift assay (CETSA). Western blotting (WB) was employed to detect the expression of p53 protein and apoptosis-related proteins in cells.
Chiral HPLC separation of racemates 1 and 7 provided two pairs of undescribed enantiomers (1a/1b and 7a/7b), along with eight known compounds (2 - 9) isolated from Flemingia macrophylla roots. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of the enantiomers were determined from experimental and calculated electronic circular dichroism data. Compounds 1 - 7, and the non-prenyl analogues 10 - 13, were evaluated for cytotoxic activity against the human lung cancer A549 and H1975 cell line. Compounds 5 - 7 displayed better cytotoxicity than the positive control icaritin in A549 and H1975, with IC values ranging from 4.50 to 19.83 μmol·L and < 5 μmol·L, respectively. The structure-activity relationships of the chromone or flavonoid analogues against A549 cells were discussed. Molecular docking results demonstrated that compound 7a has strong interaction with p53 and WB indicated that 7a induced apoptosis by increasing the p53 protein, decreasing the anti-apoptotic protein Bcl-2, and activating the caspase family in A549 cells. These results suggest that prenylated flavonoids are potential p53 protein activators.
This study demonstrates that Flemingia macrophylla is rich in prenylated flavonoid constituents, among which compounds 5 and 7 exhibited significant cytotoxic activity against A549 cells and served as reference candidates for the design and development of prenylated compounds as antitumor therapeutic drugs.
淫羊藿素（一种从淫羊藿中提取的肝癌治疗药物）的成功上市为肿瘤学领域中异戊烯基黄酮类化合物的发展提供了新的动力。据报道，大叶千斤拔含有能够调节p53蛋白的特征性异戊烯基黄酮类化合物。我们旨在分离这些成分并进行活性评估、构效关系及作用机制研究，为抗肿瘤药物开发提供候选化合物。
本研究采用色谱技术结合光谱方法对大叶千斤拔甲醇提取物的成分进行分离、纯化和鉴定。以A549和H1975细胞为模型，采用MTT法评估各成分的细胞毒性活性。通过分子对接研究化合物与p53蛋白之间的结合机制，并采用细胞热位移分析（CETSA）进行验证。利用蛋白质免疫印迹法（WB）检测细胞中p53蛋白及凋亡相关蛋白的表达。
通过手性HPLC拆分外消旋体1和7，得到两对未报道的对映体（1a/1b和7a/7b），同时从大叶千斤拔根部分离得到8个已知化合物（2 - 9）。通过光谱分析确定了它们的结构，并根据实验和计算的电子圆二色光谱数据确定了对映体的绝对构型。评估了化合物1 - 7以及非异戊烯基类似物10 - 13对人肺癌A549和H1975细胞系的细胞毒性活性。在A549和H1975细胞中，化合物5 - 7表现出比阳性对照淫羊藿素更好的细胞毒性，IC值分别为4.50至19.83 μmol·L和<5 μmol·L。讨论了色酮或黄酮类类似物对A549细胞的构效关系。分子对接结果表明化合物7a与p53有强烈相互作用，WB结果表明7a通过增加p53蛋白、降低抗凋亡蛋白Bcl-2并激活A549细胞中的半胱天冬酶家族来诱导凋亡。这些结果表明异戊烯基黄酮类化合物是潜在的p53蛋白激活剂。
本研究表明大叶千斤拔富含异戊烯基黄酮类成分，其中化合物5和7对A549细胞表现出显著的细胞毒性活性，可作为设计和开发异戊烯基化合物作为抗肿瘤治疗药物的参考候选物。
