br Morphological study br Untreated and treated cells with
2.7. Morphological study
Untreated and treated cells with 75 μg/mL of BHAE for 24 h were well washed with PBS (Gibco) then fixed for 30 min in the aqueous Bouin and colored with May Grunwald Giemsa (MGG) (Fluka; v/v). The observation was done and the using an inverted microscope (Zeiss).
2.8. Apoptotic cell death assay by FACS
Cell cycle analysis of Hep-2 cells was determined by flow cytometry using the propidium iodide (PI) (Sigma) staining as described previ-ously (Rocchi et al., 2004). In brief, Hep-2 cells were plated at the den-sity of 1.5 × 105 into 6 cm dishes. Cells were treated the day after as described above. After 24 h, the cells were trypsinized and collected then fixed on ice ethanol 70% overnight at −20 °C. The day of analysis, cells were incubated with RNase (Sigma) for 30 min then stained with PI. Cell cycle distribution was analyzed by measuring DNA content using a flow cytometer a LSRII SORP (Becton Dickinson). Rates of apo-ptosis analyzed using FlowJo software (Tree Star, Inc.). Each assay was performed in triplicate.
Fig. 2. The HPLC analysis reveals the presence of the berberine in the alkaloid extract of Berberis hispanica. (a) Calibration curve of berberine standard. (b) HPLC chromatograms of the commercial standard berberine. (c) Chromatogram of the BHAE and the times retention of the berberine present in the extract.
2.9. Determination of malondialdehyde (MDA)
The rate of lipid peroxidation was determined by estimating the MDA level in the intracellular compartments (ICC) of Hep-2 cancer cells (control and BHAE treated cells) following the method of (Heath and Packer, 1968). Briefly, an equal volume of sample and buffer (Na2HPO4/NaH2PO4) was added to a mixture of trichloracetic Prostaglandin J2 (TCA) and thiobarbituric acid (TBA) (Sigma) and boils at 100 °C. The complex formed was readable at 532 nm.
Colorimetric detection of cytochrome c, Akt, Erk1/2, p38MAPK and NF-kb in the ICC was performed in 96-well plates using Elisa kit (Invitrogen) according to the manufacturer's protocol. Absorbance was measured using a spectrophotometric micro plate reader at 450 nm (BioTek Instruments).
The Hep-2 cancer cells submitted to BHAE for 24 h and control were lysed in lysis buffer (1% v/v Triton X-100, 50 mM HEPES, 150 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM EGTA, 10 μM ZnCl2, 1 mM sodium orthovanadate) containing 4% v/v protease inhibitor cocktail (Roche). The lysate was centrifuged (30 min, 30,000 × rpm) and protein content was quantified fusing the BCA protein assay kit (Pierce). 30 μg of protein from each samples were mixed with Laemmli sample buffer and loaded on 10% of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane
(PVDF) (Milipore). The membrane was blocked overnight at 4 °C with 5% w/v non fat milk in Tris buffered saline (TBS).
For immunodetection, we used mouse anti- p53 monoclonal anti-body (Santa Cruz). Loading levels were normalized using rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam) poly-clonal antibody. Specific proteins were detected using an enhanced chemiluminescence (ECL) WB substrate (Pierce) and developed on Amersham Hyperfilm ECL films (GE Healthcare).
2.12. Statistical analysis
The data were expressed as the mean ± S.D. (Standard deviation). The results were calculated using Excel (Microsoft Office, Version 2007). Statistical analysis was performed with Student's t-test. Results with P ≤ .05 were considered statistically significant.
3.1. Extraction and HPLC analysis ofBerberis hispanica alkaloids extract
A calibration curve of berberine reference standard showing a linear-ity over the concentration range from to 100 μg/mL with a regression coefficient at r2 = 0.9999 (Fig. 2 a) was obtained. The HPLC profile of the BHAE and the standard berberine recorded at 350 nm was shown in Fig. 2 b and c.
HPLC analysis of BHAE showed that retention times (Rt) and berber-ine standard were respectively at 4.528 and 4.526 min. The comparison
Fig. 3. Effect of the Berberis hispanica alkaloids extract on proliferation of Hep-2 cancer cells. (a) The percent of cell inhibition was evaluated with MTT assay (n = 6). Data are expressed the percent of inhibition of the viability nozmalized to control cells and expressed as the mean ± S.D. Significant difference for each dose (75 and 90 μg/ml) versus control (**P ≤ .001, ****P ≤
.0001 respectively). (b)The cells were exposed 24 h to75 μg/ml of the BHAE, the evaluation of cells viability and proliferation was determined by cell counting using the trypan blue exclusion assay (n = 6). Data are expressed as the mean ± SD of control, DMSO and BHAE treated cells (**P ≤ .001). (c) Hep-2 cancer cells in culture were fixed and stained with May Grunwald-Giemsa (MGG). (c1) Control, (c2) Hep-2 treated cells with 75 μg/ ml of BHAE for 24 h. The observation was done with an inverted microscope (Zeiss) (GX400). Green arrow: cytosolic vacuolization, red arrow: condensed chromatin, blue arrow: dense cytoplasm and, black arrow: membrane blebbing.