背景:杏叶(Sprengel)R.M.King和H.Robo.已被用作世界各地的传统土著医学,因为其多样化的治疗应用,如抗癌,镇痛药,退烧药,产热,防腐剂,抗菌和收敛。印度的各个民族使用植物部分来治疗伤口和伤口,有毒的昆虫叮咬,皮肤损伤,水泡,sc疮和其他皮肤刺激,胃炎和消化不良,咳嗽,胃痛和痢疾。葡萄牙人传统上从植物中提取汁液并将其用于癌症,糖尿病,肝脏疾病,胆囊和胃病.尼日利亚医生使用植物的不同部位来治疗糖尿病,发烧和炎症。
目的:本研究的目的是研究水醇叶提取物(AHL)对大肠癌(CRC)细胞系(HCT-116,HCT-15和HT-29)的细胞毒性潜力。与化疗药物5FU和顺铂以及活性氧(ROS)产生的协同潜力,根据从曼尼普尔的毛区收集的样本,印度。还通过HRLCMS进行AHL中生物活性植物化合物的鉴定。
方法:通过3-(4,5-二甲基噻唑-2-基)-2,5二苯基四唑(MTT)测定法评估AHL的细胞毒性和抗增殖活性,克隆和细胞迁移测定。总酚含量(TPC)和总类黄酮含量(TFC)分别通过Folin-ciocalteu和氯化铝测定法进行定量。使用胱天蛋白酶-3测定试剂盒评价胱天蛋白酶3活化。使用膜联蛋白V-FITC/PI凋亡检测试剂盒通过流式细胞术进行凋亡检测。凋亡细胞也通过Giemsa和4'可视化,6-Diamidino-2-苯基吲哚(DAPI)染色。还使用荧光探针2'评估了细胞内活性氧(ROS)的产生,流式细胞术中的7'-二氯二氢荧光素二乙酸酯(H2DCFDA)。还评估了AHL与化疗药物5FU和顺铂的联合作用。通过HR-LCMS分析了AHL的植物化学成分。
结果:AHL在HCT-116中显著诱导细胞毒活性,IC50为65.65±2.10μg/mL,但非癌细胞HeK-293细胞毒性最小。集落形成和细胞迁移以剂量和时间依赖性方式受到抑制。AHL处理后的细胞形态随着凋亡特征而显著改变。提取物中富含总酚(82.09±0.35mgGAE/g)和总黄酮(58.31±0.55mgQAE/g)含量。AnnexinV/PI检测到AHL诱导的细胞凋亡,通过激活caspase3和提高活性氧(ROS)的产生。AHL与5FU和顺铂组合起协同作用并增强提取物的治疗性质。倍半萜,通过HRLCMS在AHL中检测到具有抗癌特性的酚类和类黄酮衍生物,这些植物成分可能归因于AHL的抗癌特性。
结论:本研究评估了AHL对结直肠癌细胞系的有效性。AHL是细胞毒性的,并且通过半胱天冬酶3激活和可归因于倍半萜类的增加的ROS产生在HCT-116细胞中诱导凋亡。因此,植物A.adenophora具有治疗结直肠癌的潜力,可以进一步开发用于开发抗癌药物。
BACKGROUND: Ageratina adenophora (Sprengel) R.M.King & H.Rob. has been used as traditional indigenous medicine all across the globe for its diverse therapeutic applications such as anticancer, analgesic, antipyretic, thermogenic, antiseptic, antimicrobial as well as astringent. The various ethnic groups of India use plant parts to treat cuts and wounds, venomous insect bites, skin lesions, blisters, scabies and other skin irritations, gastritis and indigestion problems, cough, stomach ache and dysentery. The Portuguese traditionally extract the juice from the plant and use it for cancer, diabetes, liver disorder, gallbladder and stomach ailments. Nigerian healers use different parts of the plant to treat diabetes, fever and inflammation.
OBJECTIVE: The aim of this study is to investigate the cytotoxic potential of A. adenophora hydroalcoholic leaves extract (AHL) on Colorectal cancer (CRC) cell lines (HCT-116, HCT-15 and HT-29), synergistic potential with chemotherapeutic drugs 5FU and Cisplatin as well as reactive oxygen species (ROS) generation, based on the sample collected from Mao district of Manipur, India. Identification of bioactive phytocompounds in AHL was also performed by HRLCMS.
METHODS: The AHL was evaluated for its cytotoxic as well as antiproliferative activities by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, clonogenic and cell migration assays. The total phenolic content (TPC) and total flavonoid content (TFC) were quantified by Folin-ciocalteu and Aluminium chloride assays respectively. Caspase 3 activation was evaluated using Caspase-3 Assay Kit. Apoptosis detection by flow cytometry was carried out using annexin V-FITC/PI apoptosis detection kit. The apoptotic cells were also visualized by Giemsa and 4\',6-Diamidino-2-phenylindole (DAPI) staining. The intracellular Reactive oxygen species (ROS) generation was also evaluated using fluorescent probe 2\',7\'-dichlorodihydrofluorescein di-acetate (H2DCFDA) in flow cytometry. The combination effects of AHL with chemotherapeutic drugs 5FU and Cisplatin were also evaluated. The identification of phytochemical constituents of AHL were analysed by HR-LCMS.
RESULTS: The AHL induced cytotoxic activity significantly in HCT-116 with IC50 of 65.65 ± 2.10 μg/mL, but non-cancerous cell HeK-293 was least cytotoxic. Colony formation and cell migration were inhibited in a dose and time dependent manner. The cell morphology upon AHL treatment was significantly altered with apoptotic features. The extract was rich in total phenolic (82.09 ± 0.35mgGAE/g) and total flavonoid (58.31 ± 0.55 mgQAE/g) contents. AHL induced apoptosis as detected by AnnexinV/PI, via activation of caspase 3 and elevated production of Reactive oxygen species (ROS). AHL in combination with 5FU and Cisplatin acts synergistically and potentiates the therapeutic properties of the extract. Sesquiterpenes, phenolic as well as flavonoid derivatives with anticancer properties were detected in AHL by HRLCMS, and these phytoconstituents may be attributed for anticancer property of AHL.
CONCLUSIONS: The present study evaluates the effectiveness of AHL against Colorectal cancer cell lines. AHL is cytotoxic and induces apoptosis in HCT-116 cells by caspase 3 activation and increased ROS production that can be attributed to sesquiterpenoids. Thus, the plant A. adenophora has therapeutic potential for Colorectal cancer and can be further exploited for developing anticancer drug.