Antitumor Activity of Methanolic Fractions Extracted From the Aerial Part of Algerian Hyoscyamus albus and apoptotic cell Aspect Visualization

 

Massinissa Yahia*^1,2, Mouloud Yahia¹, Afaf Benhouda¹

¹Biotechnology’s Laboratory of the Bioactive Molecules and the Cellular Physiopathology,

Department of Biology of Living Organisms, University of BATNA-2-, Algeria

² Departments of Pharmacy, University of Naples Federico II, 80131 Naples, Italy

³Department of Biochemistry, Biophysics and General Pathology,

Second University of Naples, 80138 Naples, Italy

 

ABSTRACT:

During the last years, the discovery of new anticancer drugs remains the mains concerns in oncology, The natural environment has always been a very important source of active Biological molecules and which 60% of the cancer drugs used currently are original from a natural products. H.albus is a plant which belongs to Solanaceae family, used generally in traditional medicine as a nervous sedative and para sympatholytic which is rich source of flavonoid, alkaloids and tropane. The present work is for anobjective to test the methanolic fractions extracted from the Aerial Parts of Hyoscyamus albus collected among aures region in Algeria, and evaluate their cytotoxic activity on different cancercells lines and the characterization of the microscopical morphology of apoptosis cell. The effects of four different selected methanolic fractions of H.albusextract( D, E, F, G)obtained by column chromatography over Sephadex LH-20 tested on DU-145, PC-3, U-87 MG and U-373 MG cancer cells lines were determined using  MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] assay and the cell were marked by orange acridine to visualize the apoptotic cells. Our results indicated that the fraction C has a cytotoxic activity against cells lines showed a marked anti-cancer activity with IC50 = 73µg/ml, 187 µg/ml, 96 µg/ml and 114 µg/ml for the DU-145, PC-3, U-87 MG and U-373 MG respectively, the morphotype apoptotic cell have showed the morphological change which attempt an apoptotic cell marked by a fluorescence. Therefore, the fraction D had just an activity against DU-145 with IC50=186 µg/ml, the fraction F has an IC50 =165 µg/ml and 198 µg/ml against DU-145 and PC-3 respectively. The fraction G showed the activity against DU-145, LNCaP and U-373 MG. The results demonstrated the anticancer properties of H.albusand reveal the therapeutic potential of this plant.

 

 

1. INTRODUCTION:

The cancer is known medically as a malignant tumor characterized by uncontrolled growth of abnormal cells. it is caused by endogenous and exogenous factors which lead to the accumulation of genetic alterations in organism[1].

H.albus is a Solanaceae family plants; which have been used in traditional medicine from a long time ago  as a nervous sedative, para- sympatholytic, mydriatic, ant cholinergic, antispasmodique and analgesic[2]and recently the anti diabetic activity has been demonstrated [6]. During time researchers have isolated some tropane alkaloids (scopolamine, hyoscyamine) and with spectral techniques they isolated 2,3–dimethyl nonacosane [3]. Recently, a new group of polyhydroxylated nortropane alkaloide named calystegines have been isolated from different species of solanaceae like in Atropa, Hyoscyamus and Datura [4,5]

 

The aim of this study is to screen the different fractions of H.albus for cytotoxic activity on different cells lines and apoptosis cell marking. The cytotoxic potential was studied by MTT assay. Moreover, the characterizations of apoptosis cell were used by biological colorant and fluorescent microscope.

 

2. EXPERIMENTAL:

2.1. Plant Material:

The aerial parts of H.albus were collected from Ighzer Naith Abdi, Batna, Algeria in Mai 2015. The plant was identified in Laboratory of Botanic, Department of Agronomy, University Batna 1, Algeria. Then were dried for forty days at an ambient temperature under shade, after that it were crushed to obtain a fine and homogeneous powder and kept in dry place.

 

2.2. Extraction:

The vegetal materials were powdered (1Kg) and extracted with ether of petrol, chloroform and methanol at room temperature. The solvents were removed in a rotary evaporator at 30°C for ether of petrol and chloroform and 40°C for methanol. The extracts were conserved and covered in refrigerator at 4°C until use[7].

 

2.3. Purification on Sephadex Gel:

The methanolic extract of H. albus‘s (HAMeOH) was submitted to column chromatography over Sephadex LH-20 (D. Farmacia. Italy), using methanol as eluent. The obtained fractions (A,B,C,D,E,F)  were analyzed by Thin layer chromatography (TLC) on Selica gel 60 F ₂₅₇ plate (Merck)recoated aluminum plates (thickness = 200 μm) using butanol - glacial acetic acid-water system and anisaldehyde sulfuric and FeCl₃ reagents as a spray reagent, finally the similar profiles were combined.

 

All the reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and Merck KGaA (Darmstadt, Germany).

 

We obtained seven fractions FA (0.147 g), FB (1.35 g) ,FC (4.08 g) ,FD (4.22 g) ,FE (0.71 g) ,FF (0.65 g) ,FG (1.76 g)[8].

 

2.4. Cytotoxic Activity of Fractions:

The antitumoral activity of the four selected fractions F , C , D ,G of HAMeOH was evaluated with MTT [3-(4,5-dimethylthiazole-2-yl) -2,5diphenyltetrazolium bromide; Sisco, Italy] with method of Mosmann [9,2] with some modifications. DU-145 (human prostate cancer cell lines), PC-3(human prostate cancer cell lines), LNCaP, LN-229(cells are androgen-sensitive human prostate adenocarcinoma), U-87 MG (human primary glioblastoma) and U-87, U-373 MG (human glioblastoma line cells) were kindly provided by the United States National Cancer Institute (NCI).

 

The cells were grown in the Dulbecc modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), Penicillin G (100 U/mL) and streptomycin sulfate (100 μg/mL) at 38°C and 4.7% of CO₂, Our fractions were solubilized in 10 % of Dimethyl sulfoxide DMSO (1µg/ml, 10 µg/ml, 100 µg/ml and 1000 µg/ml) which is prepared in DMEM concentrations (10, 20, 30, 40 and 50 µg /ml) and incubated 72 hours. DMEM and the DMSO were used as controls and final DMSO concentration did not affect cell viability.

 

After 72 h of incubation, 25 µl de MTT were added in each well and after  3 hours of incubation we added  also  100 µl of Lysis buffer of MTT and the absorbance was measured in spectrophotometric quantification  (Mutiskan Ex)  at 620 nm. Experiment was conducted in triplicate.

 

The cellular viability and mortality was calculated as described by:

% Viability = (Abs test /Abs control) x 100

% Mortality = 100- % Viability.

IC50 values were calculated as the concentrations that show 50% inhibition of proliferation on any tested cell line.

 

2.5. Apoptosis Cells Marking:

Acridine 5C₃₄H₄₀Cl₄N₆Zn) is a vital biological colorant which land on nucleic acid , we have incubate 25 ml of cell suspension mixture with 1 ml of acridine solution (Thermo Fisher A1301) for 10 to 20 minutes , the samples were mixed and visualized just after the incubation[11]

 

We have placed 10 ml of cell suspension of microscope flake covered by a glass and to be cheeked with fluorescent microscope with fluorescein filtered lens X60.

 

3. STATISTICAL ANALYSIS:

The results of activity and cell proliferation inhibition was calculated by microplate reader Brand BioTek ELISA

 

4. RESULTS AND DISCUSSION:

4.1. Cytotoxic activity of Fraction C,D,F,G:

The graphson figure 1 present the percentage of viability of different cells DU-145, PC-3, LNCaP, U-87 MG and U-373 MG after treatment with different concentrations of fractions C,D,F,G ( Fig1).

 

Fig. 1.Percentage of viability of lines cells after treatment with fractions of HAMeOH C, D, F and G.

 

The MTT is a colorimetric assay which measures the enzymatic activity and depends to the reduction of MTT to formazan. The results IC 50 values of 4 fractions of methanolic extract of H.albus  are summarized in Table 1.

 

Table 1: IC 50 values of four selected fractions of methanolic extract of H.albus .

Cell lines	Fraction C µg/mL IC50	Fraction D µg/mL IC50	Fraction F µg/mL IC50	Fraction  G µg/mL IC50
DU-145	73	186	165	110
PC-3	187	……	198	……
LNCaP	…….	…….	……...	142
U-87 MG	96	……	……	…...
U-373 MG	114	…….	……..	133

 

4.2. Apoptosis Cell Coloring:

We reorganize that for the presence of many yellow dot’s on the base of nuclei which show a chromatin condensation on the nuclei and also it fragmentation we also reorganize the budding of cytoplasm which define and confirm  the biological mechanism of apoptosis  the yellow dots  have been shown on (Fig.2)  by   arrows.

 

Fig. 2.  Visualization of apoptosis cell marked by acradine orange . Comparison between control cells treated by Fraction C on cell line PC-3 (a, b) and DU-145 cells (c, d).

 

The results indicated that the fraction C of HAMeOH possessed a strong activity against cells lines showed marked anti-cancer activity, with IC50 = 73µg/ml, 187 µg/ml, 96 µg/ml and 114 µg/ml for the DU-145, PC-3, U-87 MG and U-373 MG respectively.

 

 Therefore, the fraction D had just an activity against DU-145 with IC50=186 µg/ml, the fraction F has an IC50 =165 µg/ml and 198 µg/ml against DU-145 and PC-3respectively. The fraction G showed the activity against DU-145, LNCaP and U-373 MG.

 

About the Fraction Cwe estimate the growth inhibition of the human prostate cancer cell lines, DU-145 and PC-3, and the human glioblastoma cell lines, LN-229 and U-373 MG, after 72 hours of treatment.

The observed content of methanolic extracts during their phytochemical screening of flavonoids, terpen and alcaloidsmust explain their anticancer activity of h.Albus.

They found that the flavonoids are the best candidates with protective effects against the different kinds of cancer[12].A previous study evaluating the cytotoxicity of more than 100 low molecular weight polyphenols on normal and tumor cell lines, it appears that the compounds are more active on cancer strains than the healthy ones[13,14].

The plant rich of flavonoids act in different levels of the carcinogenesis process: reducing the activation of procarcinogens to carcinogens by interacting with cytochromes P450, or by inducing the synthesis of certain cytochromes (CYP1A1 and CYP1A2, CYP1B1), Either by being metabolized by certain cytochromes, or by modulating the enzymatic activities of certain [15]. Cytochromes CYP1A1 and CYP1B1 are over expressed in tumor tissues and metabolize procarcinogens to carcinogens.

The anticancer activity of our extracts can be attributedalso at abundance of terpenic compounds, Indeed, studies have demonstrated the anti-cancer activity of terpenes [17] According to the same author, monoterpenes prevent the process of carcinogenesis during initiation and the stages of promotion / progression. Monoterpene pirillyl alcohol has been described to have anti-proliferative activity on glioblastoma by the inhibition of the Na / K-ATPase pump [17].Other monoterpenes, such limonene, have been shown to prevent mammary, liver, lung, and other cancers.  The activity of these constituents is related to the activation of cell death induced by the caspases proteins in cancer cells[18].

Some plant alkaloids have already been cited to inhibit proliferation of breast cancer cells by inhibiting anoïkis resistance, or detachment-induced apoptosis, may prevent cancer progression and metastasis by blocking signals necessary for survival of localized cancer cells[19,20,21].

In our study, we found that the fraction Cinduce apoptosis while cell proliferation inhibition and we reorganize that for the presence of many dot’s on the base of nuclei which show a chromatin condensation on the nuclei and also it fragmentation we also reorganize the budding of cytoplasm which define and confirm the biological mechanism of apoptosis [22].

 

5. CONCLUSION:

In conclusion, the present investigation demonstrated that H. albus have the anticancer activity against different cells line which gives more importance to medicinal plants  used specially by the fraction C and fraction F and induce apoptosis while proliferation inhibition , for this reason we open the gate for future research to identify the unknown molecules on fractions and which could be the first responsible of the obtained activity and investigate the molecular pathway induced on the anticancer activity.

 

6. CONFLICTS OF INTERESTS:

All authors have none to declare

 

7. ACKNOWLEDGMENTS:

This work was supported by the department of Pharmacy University Federico II- Naples Italy, we also thank the Department of Biochemistry, Biophysics and General Pathology, Second University of Naples,80138 Naples, Italy.  LBMBPC University of Batna 2, Algerie .

 

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Accepted on 28.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(11): 3676-3680.