Studies of the in vitro anticancer, antimicrobial and antioxidant potentials of selected Yemeni medicinal plants from the island Soqotra

The following microorganisms were used as test organisms in the screening: 3 Gram-positive strains namely, Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6059), Micrococuss flavus (SBUG 16), 2 Gram-negative strains namely, Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 27853) and one fungal strain Candida maltosa (SBUG 700). In addition, three multiresistant Staphylococcus strains namely, Staphylococcus epidermidis 847, Staphylococcus haemolyticus 535, and Staphylococcus aureus North German Epidemic Strain (supply from the Institute of Hygiene of Mecklenburg-Vorpommern, Greifswald, Germany) were also employed as test organisms. Stock cultures were maintained at 4°C on slopes of nutrient agar. The cultures were diluted to achieve optical densities corresponding to 2.0 × 10⁶ colony forming units (CFU/ml) for bacterial and 2.0 × 10⁵ spore/ml for fungal strains.

The disc-diffusion assay [7] was used to determine the antimicrobial potential of the investigated extracts. Nutrient agar (OXOID LTD, Basingstoke, Hampshire, England) was prepared by dissolving of 27 g/l in water. The sterile nutrient agar was inoculated with microbial cells (200 μl of microbial cell suspension in 20 ml agar medium) and poured into sterile petri dishes. Sterile filter paper discs of 6 mm diameter (Schleicher and Schuell, ref. No. 10321260, lot. DG0274-1) were impregnated with 20 μl of the extract solution (equivalent to 4 mg of the dried extract). The paper discs were dried and placed on the surface of the inoculated agar plates. Plates were kept for 2 hours in refrigerator to enable prediffusion of the extracts into the agar. Then the plates were incubated overnight (18 hours) at 37°C. In contrast, M. flavus was incubated at room temperature for 48 h and C. maltosa was incubated at 28°C for 48 h. Ampicillin, gentamicin and amphotericin B were used as positive control. Negative controls were performed with paper discs loaded with 20 μl of organic solvents (methanol and 5% ethanol) and dried. At the end of the incubation period the antimicobiral activity was evaluated by measuring the inhibition zones (diameter of inhibition zone plus diameter of the disc). An inhibition zone of 15 mm or more was considered as high antibacterial activity.

The broth micro-dilution method described by [8] with modifications was used to determine the MIC of extracts against the three standard Gram-positive strains. With sterile round-bottom 96-well plates, duplicate two-fold serial dilutions of extract (100 μl/well) were prepared in the appropriate broth containing 5% (v/v) DMSO to produce a concentration range of 2000 to 15.6 μg of extract/ml. Two-fold dilutions of ampicillin were used as a positive control. A bacterial cell suspension (prepared in the appropriate broth) of 100 μl, corresponding to 1 × 10⁶ CFU/ml, was added in all wells except those in columns 10, 11 and 12, which served as saline, extract and media sterility controls, respectively. Controls for bacterial growth without plant extract were also included on each plate. The final concentration of bacteria in the assay was 5 × 10⁵ CFU/ml. The final concentration of extracts ranged between 1000 to 7.8 μg/ml. Plates were then incubated at 37°C for 18 h overnight. After incubation, the MIC of each extract was determined as the lowest concentration at which no growth was observed in the duplicate wells. Twenty microliters of a p-iodonitro-tetrazolium violet solution (0.04%, w/v) (Sigma, USA) was then added to each well. The plates were incubated for a further 30 min, and estimated visually for any change in color from yellow to pink indicating reduction of the dye due to bacterial growth. The highest dilution (lowest concentration) that remained yellow corresponded to the MIC. Experiments were performed in duplicate.

For the estimation of the in vitro cytotoxic potency of the investigated extracts, an established microtiter plate assay [9] was used with three human cancer cell lines: one lung cancer (A-427), one urinary bladder cancer (5637) and one breast cancer (MCF-7) line. Cell lines were obtained from the DMSZ, Braunschweig, Germany, and culture in RPMI 1640 medium with 10% FCS. Cytotoxicity determinations are based on cellular staining with crystal violet and were performed as previously described in detail. Briefly, a volume of 100 μl of a cell suspension was seeded into 96-well microliter plates at a density of 1000 cell/well. Twenty-four hours later, cells were treated with the plant extracts at five dilutions and exposed continuously to the extracts for the next 96 h. Etoposide was used as a positive control. At the end of the exposure time, the medium was removed and the cells were fixed with a glutaraldehyde solution. The cells were then stained with crystal violet and the optical density (OD) was measured at λ = 570 nm with a plate reader. The percent growth values were calculated by the following equation:

Growth (%) = OD_T - OD_{c, 0}/OD_c - OD_{c, 0} × 100

Where OD_T is the mean absorbance of the treated cells, OD_c is the mean absorbance of the controls, OD_{c, 0} is the mean absorbance at the time the extract was added. The IC₅₀ values were estimated by a linear least-squares regression of the growth values versus the logarithm of the extract concentration; only concentrations that yielded growth values between 10% and 90% were used in the calculation.

The DPPH free radical scavenging assay was carried out for the evaluation of the antioxidant activity. This assay measures the free radical scavenging capacity of the investigated extracts. DPPH is a molecule containing a stable free radical. In the presence of an antioxidant, which can donate an electron to DPPH, the purple colour typical for free DPPH radical decays, and the absorbance change at λ = 517 nm is measured. This test provides information on the ability of a compound to donate a hydrogen atom, on the number of electrons a given molecule can donate, and on the mechanism of antioxidant action. The method was carried out as previously described by [10]. The methanolic and aqueous extracts were redissolved in methanol and 5% ethanol, respectively, and various concentrations (10, 50, 100, 500 and 1000 μg/ml) of each extract were used. Similar concentrations of ascorbic acid were used as positive control. The assay mixture contained in a total volume of 1 ml, 500 μl of the extract, 125 μl prepared DPPH (1 mM in methanol) and 375 μl solvent (methanol or 5% ethanol). After 30 min incubation at 25°C, the decrease in absorbance was measured at λ = 517 nm. The radical scavenging activity was calculated from the equation:

% of radical scavenging activity = Abs_control - Abs_sample/Abs_control × 100

The screening of chemical constituents was carried out with the methanol extracts by using chemical methods and thin-layer chromatography (TLC) using different mixtures of organic solvents as mobile phases. Several chemical reagents e.g. Dragendorf's reagent for alkaloids, borntraeger reagent for anthraquinons and etc. were used in the detection according to previously published methodology [11].