Université de Toulouse; UPS; UMR 152 Pharma-DEV; Université Toulouse 3; Faculté, des Sciences Pharmaceutiques; 35 Chemin des Maraîchers, F-31062 Toulouse cedex 9, France; Institut de Recherche pour le Développement; IRD; UMR 152 Pharma-DEV, F-31062 Toulouse cedex 9, France.
Université de Toulouse; UPS; UMR 152 Pharma-DEV; Université Toulouse 3; Faculté, des Sciences Pharmaceutiques; 35 Chemin des Maraîchers, F-31062 Toulouse cedex 9, France.
J Ethnopharmacol. 2015 May 26;166:185-99. doi: 10.1016/j.jep.2015.02.028. Epub 2015 Feb 19.
The high incidence of human hepatocellular carcinoma (HCC) in Peru and the wide use of medicinal plants in this country led us to study the activity against HCC cells in vitro of somes species used locally against liver and digestive disorders.
Ethnopharmacological survey: Medicinal plant species with a strong convergence of use for liver and digestive diseases were collected fresh in the wild or on markets, in two places of Peru: Chiclayo (Lambayeque department, Chiclayo province) and Huaraz (Ancash department, Huaraz province). Altogether 51 species were collected and 61 ethanol extracts were prepared to be tested. Biological assessment: All extracts were first assessed against the HCC cell line Hep3B according a 3-step multi-parametric phenotypic assay. It included 1) the evaluation of phenotypic changes on cells by light microscopy, 2) the measurement of the antiproliferative activity and 3) the analysis of the cytoskeleton and mitosis by immunofluorescence. Best extracts were further assessed against other HCC cell lines HepG2, PLC/PRF/5 and SNU-182 and their toxicity measured in vitro on primary human hepatocytes.
Ethnopharmacological survey: Some of the species collected had a high reputation spreading over the surveyed locations for treating liver problems, i.e. Baccharis genistelloides, Bejaria aestuans, Centaurium pulchellum, Desmodium molliculum, Dipsacus fullonum, Equisetum bogotense, Gentianella spp., Krameria lapacea, Otholobium spp., Schkuhria pinnata, Taraxacum officinale. Hep3B evaluation: Fourteen extracts from 13 species (Achyrocline alata, Ambrosia arborescens, Baccharis latifolia, Hypericum laricifolium, Krameria lappacea, Niphidium crassifolium, Ophryosporus chilca, Orthrosanthus chimboracensis, Otholobium pubescens, Passiflora ligularis, Perezia coerulescens, Perezia multiflora and Schkuhria pinnata) showed a significant antiproliferative activity against Hep3B cells (IC50≤ 50µg/mL). This was associated with a lack of toxicity on primary human hepatocytes in vitro. Immunofluorescence experiments on Hep3B cells showed that crude extracts of Schkuhria pinnata and Orthrosanthus chimboracensis could block Hep3B cells in mitosis with an original phenotype. Crude extracts of Perezia coerulescens, Perezia multiflora, Achyrocline alata, Ophryosporus chilca, Otholobium pubescens and Hypericum laricifolium could modify the overall microtubule cytoskeletal dynamics of Hep3B cells in interphase by an original mechanism.
Our method allowed us to select 9 extracts which displayed antiproliferative activities associated with original cellular phenotypes on Hep3B cells, regarding known microtubule-targeting drugs. Both chemical and cellular studies are ongoing in order to elucidate natural compounds and cellular mechanisms responsible of the activities described.
秘鲁肝癌（HCC）的高发率和该国对药用植物的广泛使用，促使我们研究了当地用于治疗肝脏和消化系统疾病的一些物种对 HCC 细胞的体外活性。
民族药理学调查：在秘鲁的两个地方，奇克拉约（兰巴耶克省，奇克拉约地区）和瓦拉斯（安卡什省，瓦拉斯地区），我们采集了当地用于治疗肝脏和消化系统疾病的药用植物的新鲜样本进行研究。共采集了 51 种植物，制备了 61 种乙醇提取物进行测试。生物评估：所有提取物首先根据三步多参数表型测定法对 Hep3B 肝癌细胞系进行评估。它包括 1）通过光学显微镜评估细胞表型变化，2）评估抗增殖活性，3）通过免疫荧光分析细胞骨架和有丝分裂。对最佳提取物进行进一步评估，以检测其他 HCC 细胞系 HepG2、PLC/PRF/5 和 SNU-182，并在原代人肝细胞上检测其体外毒性。
民族药理学调查：在所调查的地点，一些被采集的物种因其治疗肝脏问题的高声誉而被广泛使用，例如 Baccharis genistelloides、Bejaria aestuans、Centaurium pulchellum、Desmodium molliculum、Dipsacus fullonum、Equisetum bogotense、Gentianella spp.、Krameria lapacea、Otholobium spp.、Schkuhria pinnata、Taraxacum officinale。Hep3B 评估：来自 13 种植物的 14 种提取物（Achyrocline alata、Ambrosia arborescens、Baccharis latifolia、Hypericum laricifolium、Krameria lappacea、Niphidium crassifolium、Ophryosporus chilca、Orthrosanthus chimboracensis、Otholobium pubescens、Passiflora ligularis、Perezia coerulescens、Perezia multiflora 和 Schkuhria pinnata）对 Hep3B 细胞表现出显著的抗增殖活性（IC50≤50µg/mL）。这与体外原代人肝细胞无毒性相关。对 Hep3B 细胞进行免疫荧光实验表明，Schkuhria pinnata 和 Orthrosanthus chimboracensis 的粗提取物可使 Hep3B 细胞在有丝分裂中停滞，表现出原始的表型。Perezia coerulescens、Perezia multiflora、Achyrocline alata、Ophryosporus chilca、Otholobium pubescens 和 Hypericum laricifolium 的粗提取物可通过一种原始机制改变 Hep3B 细胞在有丝分裂间期的整体微管细胞骨架动力学。
我们的方法允许我们选择 9 种提取物，这些提取物在 Hep3B 细胞上显示出与已知微管靶向药物相关的抗增殖活性和原始细胞表型。正在进行化学和细胞研究，以阐明负责所描述活性的天然化合物和细胞机制。
