br Alkaloids are secondary metabolites Kutchan known
Alkaloids, are secondary metabolites (Kutchan, 1995), known to possess several biological activities including antiviral, cholesterol low-ering or antifungal activities (Chen et al., 2012; Wang et al., 2017; Yokozawa et al., 2003; Zhang et al., 2009). Alkaloids also exhibit anti-cancer activity (Havelek et al., 2014; Pierpaoli et al., 2015). Indeed, anticancer effects of alkaloids and their ability to induce apoptosis have been demonstrated on numerous cell lines with differ-ent alkaloids such as the Vinca alkaloid on lung adenocarcinoma cells
by the induction of DNA damage and apoptosis through aberrant ROS-generation (Chiu et al., 2012), the Vinblastine on human KB-3 carci-noma cells by inducing cell deaththrough activation of Jnk pathway (Stone and Chambers, 2000), or berberine in prostate cancer cells which induced mitochondrial pathway of apoptosis (Meeran et al., 2008).
Berberine is an isoquinoline alkaloids (Fig. 1) found in roots and rhi-zome of several medicinal plants species as Coptidis Rhizoma and Ber-beris vulgaris (Iizuka, 2000; Ivanovska and Philipov, 1996). This alkaloid possesses numerous properties as antifungal, antimicrobial and anticancer activities (Dhamgaye et al., 2014; Peng et al., 2015; Pierpaoli et al., 2015).
In this study, we evaluated anticancer potential of total alkaloids ex-tracted from Algerian Berberis hispanica, a plant living in the hills of the Algerian mountains. In this purpose, we investigated the presence of the berberine in the alkaloids extract and using human laryngeal carcinoma cells Hep-2, we studied the in vitro effects of the extract as well as their possible involvement in the induction of cell cycle arrest and apoptosis.
2. Material and methods
2.1. Collection of plant specimen
Berberis hispanica Boiss. & Reut was collected from the Mountain of Djurdjura city of Bouira, Algeria in October, 2011. The plant was identi-fied by Professor SMATI in the Laboratory of botanic of Algiers University and authenticated in laboratory of botanic of Higher National Agronomic School (HNAS) according to the classification of Quezel and Santa (1962). The reference number in the herbarium is in progress of digitalization.
2.2. Extraction of alkaloids from Berberis hispanica
Roots Suramin was dried and ground before extraction as described by (Bruneton, 1999). Tengram of dried powder was delipidated in petro-leum ether during 3 h and alkalized with ammonia (0.5 N) during 8 h then extracted four times with dichloromethane on Soxhlet extractor then the extract was purified 3X with sulfuric acid (0.5 N) and 3X with chloroform and finally dried with anhydrous sulfate sodium before evaporation to dryness by vacuum at 60 °C. The dry extract obtained was the Berberis hispanica alkaloids extract (BHAE) and was used in the following experiments.
By applying high-performance liquid chromatography (HPLC) with gradient elution, determination of presence of berberine in the extract was carried out on an Agilent HPLC system model 1100, having four pumps and Shimadzu SPD-20 AV- UV/Vis detector with 20 μl-loop
Fig. 1. Chemical structure and molecular weight of Berberine chloride (C₂₀H₁₈NO₄ + Cl-)
Human laryngeal epidermoid carcinoma (Hep-2) cells were ob-tained from the Algerian Pasteur Institute; (Algiers, Algeria). The cells were maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1.2% L-glutamine and 100 UI/mL penicillin–streptomycin (both from Sigma Aldrich) at 75 cm2 cell cul-ture flasks (Nunc) and incubation was carried out at 37 °C in a 5% CO2 humidified atmosphere.
2.5. Viability assay
In order to evaluate the cytotoxic activity, the alkaloids were essayed on Hep-2 cells lines. The cell viability was determined by 3-(4,5-di-methylthiazol-2-yl)-2,5- diphenyl tetrazolium (MTT) assay (Mosmann, 1983). In brief, Hep-2 cells were seeded in 96-well plates (0.75 × 104 cells/well), after 24 h of incubation media was removed and cells were incubated or no with BHAE dissolved in dimethylsulfoxide (DMSO) (b0.05%) and passed through 0.45 μm filter at 75 and 90 μg/mL for 24 h. After the indicated time, the cells were in-cubated with 20 μl of MTT (5 mg/mL in PBS) for 3 h 30 at 37 °C. The su-pernatant was then removed and acidified Isopropanol with 0.1% of NP40 was added to dissolve the formazon crystals. The optical density was read at 570 nm using microplate reader (BioTek Instruments). All experiments were repeated at least three times. Data are expressed as the percentage of viability of Hep-2 cells treated with BHAE compared to the control.
2.6. Proliferation essay
Cells were seeded at density of 0.8 × 106 cells on 6-well plate dishes, at 70% of confluence, 75 μg/mL of BHAE dissolved in DMSO (b0.05%) and filtrated extract was added. Cells were also treated with 0.05% of DMSO only as control vehicle. After 24 h of incubation, viability was deter-mined by counting using the trypan blue staining (0.4%) using an inverted microscope (Zeiss).