Plant material
Plant material was collected during the period of March-July 2014 from Shandi, River Nile state, Sudan. The taxonomic identification of this plant was carried out at the Medicinal and Aromatic Plants Research Institute (MAPRI), National Center for Research-Sudan by Dr. W.E.A/Alla, and the voucher specimen (Ref. No. 30593/2014) was deposited at the herbarium of the institute.
Preparation of extracts
The dried stem bark was ground to powder; the plant material was extracted successively with n-hexane, chloroform, methanol and water using maceration method as described previously [17, 18]. All the extracts were prepared by 250 ml of the solvents using maceration method (40 °C) with shaking. The extracts were filtered; evaporated and stock solutions of the extracts were prepared at 10 mg/ml in dimethyl sulfoxide (DMSO). The stock solutions as well as DMSO (vehicle) were diluted with cell culture medium, so the highest DMSO concentration exposed to the cells or implanted tissue was 1 % v/v.
Determination of plant extract yield
The yields of the stem bark extracts of B. aegyptiaca based on dry weight basis were calculated from the following equation:
$$ \mathrm{Yield}\kern0.5em \left(\mathrm{g}/20\;\mathrm{g}\kern0.5em \mathrm{of}\kern0.5em \mathrm{dry}\kern0.5em \mathrm{plant}\kern0.5em \mathrm{material}\right)=\mathrm{W}1/\mathrm{W}2\times 100 $$
Where, W1 and W2 were the weight of the extract after the solvent evaporation and the weight of the dry plant material, respectively.
Chemicals and reagents
Endothelial Cell Medium (ECM) supplied with endothelial cell growth supplements (ECGS) was purchased from ScienCell, USA. Cell culture RPMI 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from GIBCO; Trypsine and heat inactivated foetal bovine serum (HIFBS) were obtained from GIBCO, UK. MTT reagents, suramin, aprotinin, 6-aminocarpoic acid, L-glutamine, thrombin, gentamicin were purchased from Sigma-Aldrich, USA.
Cell lines and culture conditions
Human umbilical vein endothelial cells (HUVECs), human colorectal carcinoma cell line HCT-116, human hormone sensitive and invasive breast cancer cell line MCF-7, human leukemia K562 and U937 were purchased from ScienCell, USA. HUVECs were maintained in ECM medium supplemented with 5 % HIFBS, 1 % PS and 1 % ECGS, whereas HCT-116, K562 and U937 were maintained in RPMI; MCF-7 cell line was maintained in DMEM. Cells were cultured in a humidified incubator at 37 °C supplied by 5 % CO2.
Experimental animals
The 12–14 weeks old Sprague Dawley male rats were obtained from animal house facility of USM. The rats were kept in well ventilated cages at 12 h light with food and water in animal transient house (School of Pharmaceutical Sciences, USM) for one week prior to the experiment. Athymic NCR nu/nu nude mice were obtained from Taconic Farms Inc., USA. The mice of the same gender were housed in specific pathogen-free (SPF) cages supplied with high efficiency particulate air (HEPA) filters. Free access to autoclaved food and water was provided and the autoclaved bedding was changed twice weekly. The procedures were approved by the USM Animal Ethics Committee with a reference number USM/Animal Ethic approval/2012/ (81) (475).
Ex vivo rat aortic ring assay
The Rat aortic ring assay was used according to techniques established by [19] with slight modification. In brief, aortic rings (1 mm thickness) taken from thoracic aortas of 10–12 weeks old male Sprague Dawley rats were seeded individually in 48-wells plate in 300 μL serum free M199 media containing 3 mg/mL−1fibrinogen and 5 mg ml−1aprotinin. 50 NIHUmL−1thrombin in 0.15 M NaCl were added in each well. After 90 min incubation at 37 °C, various concentrations of each extract were dissolved in 0.3 ml M 199 medium supplemented with 20 % HIFBS, 0.1 % έ-aminocaproic acid, 1 % L-Glutamine, 2.5 μg/mL amphotericin B, and 60 μg/mL gentamicin were added to each well. Suramin used as positive and DSMO as a negative controls. On the fourth day, the medium was replaced with a fresh one containing the extracts. On fifth day, aortic rings were photographed at 4x magnification using an inverted light microscope (EVOS). The angiogenic response was determined by measuring the distance of blood vessels outgrowth from the primary tissue ex-plants using the same instrument with the aid of Leica Quin software package [20]. Results are presented as mean percent inhibition to the negative control ± SD, (n = 3).
Cell proliferation assay
Cytotoxicity of extracts was evaluated by MTT assay. Cells were treated for 48 h with different extracts of B. aegyptiaca or 1 % DMSO as a negative control. Viability of cells was determined by MTT test as described previously [21]. Assay plates were read using a microplate reader (Tecan infinite bro 2000) at absorbance 570. The results are presented as percentage inhibition to the negative control (n = 3).
Migration assay
The assay was carried out as described previously [22]. In brief, HUVECs were seeded in 6 well plates till the formation of a confluent monolayer after which a wound was created using 200 μl micropipette tip. The detached cells were removed by washing with PBS and the plates were treated with (MBA). The wounds were photo-graphed after 12 and 18 h, and the width of the cell-free wounds was measured using an inverted light microscope supplied with Leica Quin computerized imaging system. Ten fields per well were photographed and minimum of 30 readings per field were taken. The results are presented as mean percentage of migration inhibition compared to control ± SD, (n = 3).
Determination of VEGF levels
Concentration of human VEGF in HUVEC cells lysates was determined by human VEGF ELISA kit (China) according to manufacturer’s instructions. The kit consists of anti-human VEGF-1 mouse IgG monoclonal antibody and a horseradish peroxidase conjugated secondary antibody and recombinant human VEGF 165 as a standard. HUVEC cells were seeded in 6-well plates at 1 × 106 in 3 mL of ECM medium. After overnight attachment, the cells were treated for 6 h with (MBA) at 50 and 100 μg/ml. Calibration curve of VEGF standard was prepared simultaneously with the samples, and concentration of VEGF-1 in cell lysates was determined by applying the linear regression equation, (y = 0.004x + 0.3114, R2 = 0.982).
Tube formation assay
The ability of HUVECs to form tube-like structures was assessed on a matrigel matrix. Briefly, the matrigel matrix was allowed to polymerize for 45 min at 37 °C and 5 % CO2. HUVECs were trypsinized and seeded (3 × 104 cells per well) in 100 μl of ECM containing various concentrations of (MBA) in triplicates. After 6 h tubular structures were imaged under an inverted light microscope at 4X magnification. The quantitative assessment of tube formation inhibition was achieved by measuring the area occupied by tubular structures using the Scion Image analysis program [23]. The results are presented as a mean percentage of inhibition ± SD, (n = 3).
Determination of total phenolic content
Total phenolic contents in the four extracts of stem bark were determined using Folin-Ciocalteu reagent according to previously described method [24]. Twenty μl of extract and gallic acid solutions were pipetted into separate test tubes followed. Then, 1.58 ml of distilled water and 100 μL of 2 N Folin-Ciocalteu reagent were added and mixed thoroughly. After that, 300 μl of 20 % sodium carbonate solution were added and the mixture was incubated for 2 h at room temperature (22–25.6 °C). The absorbance was measured at 765 nm using a Hitachi U-2000 spectrophotometer (Hitachi, Japan). The phenolic content was expressed as mg of gallic acid equivalent/g extract.
Determination of total flavonoids
The total flavonoids contents in the four extracts of stem bark were determined using aluminum chloride colorimetric method with quercetin as a standard [25]. A solution of 6 mg/mL of B. aegyptiaca extracts in 80 % methanol and different concentrations of quercetin (0.007, 0.015, 0.0313, 0.0625, 0.125, 0.25, 0.5, and 1 mg/ml in 80 % methanol) were prepared. 500 μl of plant extracts and each concentration of quercetin (Sigma Aldrich, Germany) were pipetted into respective test tubes followed by 0.1 ml of 10 % (w/v) aluminum chloride (R & M Chemicals, UK), 0.1 ml of 1 M potassium acetate (Merck, Germany), 1.5 ml of methanol and 2.8 ml of distilled water. The test tubes were thoroughly mixed and after incubating at room temperature (24 to 26 °C) for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Tecan infinite pro-2000 spectrophotometer against blank. The amount of 10 % (w/v) aluminum chloride was substituted by the same amount of distilled water in a blank. The concentration of total flavonoids contents of the extracts were determined using a standard curve and quercetin was used as a standard. The data were presented as mean ± SD (n = 3).
DPPH free radical scavenging activity
The DPPH radical scavenging activity of the samples was calculated as described by Molyneux 2004 [26] with some modification, in brief, 100 μl of each extract or scorbic acid (prepared in methanol) were added to 100 μl of DPPH radical dissolved in methanol (200 μM), and the reaction mixture was incubated for 30 min. Then, the absorbance of the mixture was measured at 517 nm, which shows the amount of DPPH radical remaining in the solution. The scavenging activity expressed as the IC50 (represents the sample concentration required to scavenge 50 % of DPPH free radicals) and calculated by using the following formula:
$$ \%\kern0.5em \mathrm{inhibition}=\left[\left(\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s}\right)/\mathrm{A}\mathrm{c}\right]\times 100 $$
Where Ac is the absorbance of the control, and As is the absorbance of sample.
Ferric reducing antioxidant power (FRAP) assay
The reducing power of the extracts was measured using a modification of the FRAP assay conducted by Benzie and Strain in 1999 [27]. The FRAP reagent was prepared by mixing 2.5 mL of a 10 mmol/L TPTZ solution in 40 mmol/L HCl with 2.5 ml of 20 mmol/L FeCl3 · 6H2O and 25 mL of 0.3 mmol/L acetate buffer (PH 3.6). 20 μl of the plant extract or standard was mixed with 150 μL of FRAP reagent prepared freshly and the reaction mixture was incubated at 37 °C for 4 min. Absorbance at 593 nm was read against the blank (methanol). Calibration curve was determined by using different concentrations in the range of 0.003–0.12 mg/ml (FeSO4 · 7H2O) and by applying a standard curve equation (Y = 4.2669x + 0.1814) (R2 = 0.992).
The data were represented as ferrous sulfate [Iron (II) sulfate] equivalent μmol/ml. Sample determinations were all performed in triplicate.
ABTS radical scavenging assay
ABTS radical scavenging activity of extracts was determined as previously described [28]. Initially, ABTS stock solution containing 5 ml of 2.45 mM potassium persulfate was mixed with 5 ml of 7 mM ABTS+ solution to produce ABTS radical cation (ABTS+). Then, the mixture was allowed to stand in the dark at room temperature for 12–16 h prior to use. Methanol was used to dilute the ABTS+ solution to a final concentration that would give an absorbance of 0.70 ± 0.02 at 734 nm. About 100 μl of extracts was added to 900 μl of ABTS solution. This solution was vortexed for 15 s and the absorbance was measured at 734 nm after 6 min using a UV visible spectrophotometer (Hitachi U-2000; Hitachi, Tokyo, Japan). Vitamin C was used as reference standard at final concentrations in range of 0.025 to 1 mg/ ml. The scavenging activity of test sample was calculated and expressed as the IC50 using the following formula:
$$ \%\kern0.5em \mathrm{inhibition}=\left[\left(\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s}\right)/\mathrm{A}\mathrm{c}\right]\times 100 $$
Where Ac is the absorbance of the control, and As is the absorbance of sample.
In vivo antitumor study
HCT-116 human colorectal carcinoma cells was selected as a model of colon cancer [29]. The cells were propagated in RPMI 1640 medium with 10 % FBS and 1 % PS solution. Eighty percent confluent HCT-116 cells cultures in T75 flasks were trypsinized and re-suspended in 10 ml fresh medium, the cells were collected by centrifugation at 1000 rpm for 5 min and washed with a sterile PBS. The nude mice with aged 5–7 weeks were injected subcutaneously in the right flank with 5 × 106 cells in 200 μl culture medium using 1 ml insulin syringe (27 G needle).
After one week of tumor initiation, animals were divided randomly into four groups of 6 animals each. Group 1 received 0.2 ml distilled water (control), and Groups 2, 3 and 4 received oral treatments with 400, 200 and 100 mg/kg bodyweight of (MBA), respectively. The tumor size and body weight were recorded before starting the treatment and at weekly basis. Treatment of animals was performed orally by oral gavages (wt/wt) once a day for a period of 5 weeks. The tumor dimensions were measured by a caliber in 2 angles, length and width as well as depth [30]. The tumor size was calculated by applying the formula: Tumor volume (mm3) = (W + L) / 2) ^ 3) × 2 Where W is the width and L is the length. Also following value was calculated :% ∆T/∆C, where, ∆T = T − ∆0 and ∆C = C − ∆0 (∆0 is the average tumor volume at the beginning of the treatment, T and C are the tumor volumes at a specified day for treated and control groups, respectively). Generally, the ∆T/∆C value in percent is used as an indication of antitumor effectiveness, and a value of ∆T/∆C ≤42 % is considered as significant antitumor activity by the Division of Cancer Treatment, NCI, NIH [31].
Statistical analysis
Results were presented as means ± SD and differences between groups were compared by the one way ANOVA and considered significant at P < 0.05, 0.01 or 0.001. The statistical analysis was carried out by using SSPS edition 20.
