br of these times confirmed
of these times confirmed that the peak in the sample (BHAE) is corre-sponding to the berberine. The content of berberine in the BHAE was de-termined from the area of the chromatogram, to be 3.30 mg in 100 mg of total alkaloids (i.e., 3.30% weight by weight of alkaloids) with devia-tion less than 1% in duplicated analysis.
Berberine is a potent alkaloid widely embraced in herbal medicines andpresent in several plant species such as Copitidis rhizoma (Iizuka, 2000) or Berberis vulgaris (Imanshahidi and Hosseinzadeh, 2008). This molecule is well known for its ability to induce cell death and apoptosis in numerous cancers as breast cancer 6diazo5oxo-L-nor-Leucine MCF-7 (Patil et al., 2010) or head and neck squamous carcinoma (Seo et al., 2015).
3.2. BHAE induced cell death and morphologicals changes in Hep-2 cancer cells
In order to show the effect of the BHAE, the Hep-2 cells, were treated with 75 μg/mL of BHAE for 24 h. The evaluation of cytotoxicity of the BHAE with the MTT assay (Fig. 3 a) showed a significant decrease of the proliferation in BHAE submitted cells at 75 μg/mL compared to the control cells (decrease of 45%;***P ≤ .0001) and decrease remarkably at the dose of 90 μg/mL (****P ≤ .0001). These data were confirmed by the cell counting using the trypan blue exclusion assay (Fig. 3 b) and showed that the proliferation decreased significantly in treated cells compared to the control and was 1.21 × 106 and 2.19 × 106cells respec-tively. We also observed that the use of DMSO at 0.05% as vehicle control did not affect significantly the proliferation of Hep-2 cells.
Furthermore, morphological analysis as shown in Fig. 3 c showed numerous differences between the controls cells (Fig. 3 c1) and cells
treated with BHAE (Fig. 3 c2). The cellular density of treated cells de-creased remarkably, the cytoplasm appears dense with cytoplasm vacuolization, blebbing of the membrane and condensation of the chromatin.
These results are in accordance with those of Sichaemet al. (2010) which observed cytotoxic and antiproliferative effects after treating HeLa and KB cells with alkaloids extracted from the roots of Nauclea orientalis.
Fig. 4. BHAE induced apoptosis in Hep-2 cancer cells. Control and treated cells with 75 μg/ mL of BHAE or 0.05% of DMSO for 24 h were analyzed by flow cytometry. The experiment was repeated in triplicate. Data show the percentage of cells undergoing apoptosis (cells in Sub G0). Values are mean ± S.D. Significant difference versus control and vehicle control (**P ≤ .01, **P ≤ .01 respectively) by Student's t test.
Fig. 5. Evaluation in the ICC of (a) molondialdehyd (nmol/106cells) and (b) cytosolic cytochrome c (ngr/mL/106) in control and cells treated with 75 μg/mL of BHAE during 24 h. Data are expressed mean ± SD. The asterix indicate significant difference between Hep-2 cells treated with BHAE and control cells for the MDA and cytochrome c respectively (****P ≤ .0001, **P ≤ .0001).
Previously, it was also demonstrated that berberine induced cell death in breast cancer cells MCF-7 and head and neck squamous carci-noma (Patil et al., 2010; Seo et al., 2015). Further, Kim et al. (2015), also highlighted the antiproliferative effect of berberine in KB oral can-cer cells and its ability to induce apoptosis, morphological changes and DNA fragmentation
3.3. BHAE induced apoptosis by increasing the SubG0 fractions
In this study, the effect of BHAE on cell cycle distribution was also an-alyzed as indicated in the Fig. 4. The data showed that the BHAE caused an increase of Sub G0 fractions (**P ≤ .01), the indicator of apoptotic
population in BHAE treated cells, but no significant changes in the G1 and G2/M phases (data not shown). Thus, the Sub G0 population in-creased from 4.74% in the control cells to 15.4% in BHAE submitted cells, resulting in three-fold increase over the control.
These data suggest that BHAE acted upstream of cell division by preventing the transition to the G1/S phase (Kim et al., 2002; Kishi et al., 2001). This is consistent with previous studywhich demonstrated the ability of pancratistatin to stop the progression of the cell cycle at G0/G1 phase and induction of apoptosis in HL-60 leukemic cells (Mutsuga et al., 2002). Recently Du (2017) demonstrated the synergis-tic effect of berberine and evodiamine through inductionof apoptosis and cell cycle arrest in G0/G1 phase in breast cancer cells MCF-7.
Stress markers Control BHAE 75 μg/mL
3.4. BHAE induced lipid peroxidation in the Hep-2 cancer cells and causeds cytochrome c release
To determine whether BHAE induced cell death and apoptosis, the rate of MDA and cytosolic cytochromec were quantified. Our results in-dicated that after 24 h, the rate of MDA in BHAE submitted cells com-pared to their control increase significantly in the ICC (p = 0,00017) (Fig. 5).