Antibacterial activities, proposed mode of action and cytotoxicity of leaf extracts from Triumfetta welwitschii against Pseudomonas aeruginosa

The cell membrane damage potential of the DCM/methanol, acetone and ethanol extracts from leaves of T. welwitschii against the ATCC strain of P. aeruginosa was determined using propidium iodide as described by Moyo and Mukanganyama [9], with modifications. Propidium iodide is a dye capable of binding to nucleic acids of non-viable cells with damaged membranes only [18]. The dye is unable to enter viable cells, thus, it is useful for determining the effects of plant extracts on bacterial membranes. P. aeruginosa cells were grown by pipetting 200 μl of overnight inoculum into 200 ml TSB and incubating overnight at 37 °C with shaking in an incubator. The optical density of the cells was adjusted to an OD₆₀₀ = 1.5 equivalent to 2 × 10⁹ c.f.u/ml using PBS. Cell suspensions were exposed to different concentrations of the extracts of a final concentration of 50 μg/ml, 100 μg/ml and 200 μg/ml for 30 min at 37 °C with shaking in an incubator. The negative control contained cells with no extract added. All test samples were prepared in triplicate. After incubation, 1 ml of each test sample was centrifuged at 11000 rpm. The pellet was washed with saline solution, resuspended in PBS and propidium iodide of a final concentration of 10 μg/ml added to the suspension. The mixture was kept in the dark for 10 min after which 200 μl of test samples were transferred to a 96-well plate. Fluorescence was measured at 544 nm Excitation and 612 Emission using an f_max spectrofluorometer (Molecular Devices, Sunnyvale, USA).

The transport of R6G dye out of cells as described by Chitemerere and Mukanganyam [19] was used to evaluate the effects of the acetone, ethanol and DCM/methanol leaf extracts as potential efflux pump inhibitors. Duplicate standards of R6G (0 μM to 3 μM) were prepared in PBS and their absorbance values determined at 527 nm using a Shimadzu UV/VIS UV-1601spectrophotometer (Shimadzu, Kyoto, Japan). A calibration curve was generated from values of absorbance obtained as a function of concentration using Graphpad™ version 5 for Windows (Graphpad™ Software Inc., San Diego, California, USA).

All samples were incubated at 37 °C for 1 h. Cells were collected by centrifugation at 4000 r.p.m. for 15 min and the supernatant was used for R6G efflux quantification. Optical density values of the R6G pumped out of the cells was determined using a Shimadzu UV/VIS UV1601 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 527 nm. The calibration curve was used to interpolate concentrations of R6G in samples in the efflux assay based on their absorbance values.

The cytotoxicity effects of the DCM/methanol, acetone and ethanol extracts from leaves of T. welwitschii against erythrocytes from sheep was determined using the haemolysis assay as described by Malagoli [20], with modifications. A volume of 50 ml sheep blood was collected and added to an equal volume of Alsever solution. Blood was centrifuged at 3000 r.p.m. for 10 min and the supernatant was discarded. The residue was washed three times with a 1:5 volume of PBS. The resulting cells were diluted four-fold using PBS to give an erythrocyte suspension. Extracts were prepared in PBS and final concentrations of 50 μg/ml (¹/₂MIC), 100 μg/ml (MIC) and 200 μg/ml (2MIC) were used in the assay. The erythrocyte suspension (500 μl) was mixed with 500 μl test sample extract and incubated for 90 min at 37 °C. All test samples were prepared in triplicate. After incubation, the tubes were spurn at 3000 r.p.m. for 1 min in a microcentrifuge (Geratebau Eppendorf GmbH, Engelsdorf, Germany). The positive control with 100% haemolysis was obtained by mixing 200 μl erythrocyte suspension with 1.5 ml Drabkin’s reagent; the negative control was a mixture of 500 μl erythrocyte suspension and 500 μl PBS. Aliquots of 200 μl of supernatant were transferred into 96-well plates. The absorbance (Abs) of haemoglobin released was measured at 590 nm using a Tecan Genios microplate reader (Grödig, Austria). The percentage haemolysis for each sample was calculated using the equation [21]:

This work on animals was conducted in accordance with the internationally accepted principles for the protection of animals used for scientific purposes [22]. Six weeks old male laboratory-bred strain of the house mice (BALB/c) of 20–25 g weight were collected from the Animal House at the University of Zimbabwe (Harare, Zimbabwe) and used. The research was carried out according to the rules governing the use of laboratory animals and the experimental protocol was approved by the Faculty of Higher Degrees Committee, Harare, Zimbabwe (HD/71/16). To increase the number of peritoneal cells within the mice, 20% sterile starch solution was intraperitoneally introduced into the mice using a syringe with a 27 g needle. The mice were left for 48 h in plastic cages with unlimited access to food and water in order to allow peritoneal cell yield increase. Total peritoneal cells were isolated as described by Ray and Dittle [23]. Each mouse was euthanized by cervical dislocation. Then sprayed with 70% ethanol and mounted on a styrofoam block on its back. Scissors and forceps were used to cut the outer skin of the peritoneum to expose the inner skin lining the peritoneal cavity. A volume of 5 ml of ice cold PBS with 3% FCS was introduced into the peritoneal cavity using a 27 g needle. Due care was taken to avoid puncturing of organs. After injection, the peritoneum was gently massaged to remove any attached cells into the PBS solution. A 25 g needle, attached to a 5 ml syringe was used to collect the fluid from the peritoneum into tubes kept on ice after removing the needle from the syringe. The cell suspension collected was spurn at 1500 r.p.m. for 10 min in a Rotofix 32A centrifuge. The supernatant was discarded and cells resuspended the cells in RPMI. Cells were cultured in RPMI medium supplemented with 10% Fetal bovine serum (FBS) and 1% PNS (penicillin, neomycin and streptomycin) and incubated in a Shellab incubator (CO₂ series Sheldon Mfg. Inc., Cornelius, USA) at 37 °C in a controlled atmosphere with 5% CO₂ for 24 h. Cells were stained with 0.4% trypan blue and viable cells counted using a haemocytometer counting chamber under a Celestron digital light microscope (Celestron, Los-Angeles, USA) using the × 10 objective lens. Toxicity was determined using the MTT assay as described by Mapfunde et al., [24]. Extracts were dissolved in DMSO. Each of the three extracts was double diluted to give concentrations of 12.5, 25, 50 and 100 μg/ml. The final concentration of DMSO in each well was 1%. A typical plate set up is as shown in Fig. 1. The cells were incubated in 96-well plates in the presence of extracts for 24 h at 37 °C in a 5% CO₂ Shel lab incubator. Each well contained 100 μl of the test substance and 100 μl of 0.5 × 10⁵ cells/ml in RPMI. Cells exposed to the standard anticancer drug daunorubicin (10 μg/ml) were used as the positive control. Cells in RPMI were used as the negative control. After the 24 h incubation, 25 μl of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was added to each well and plates were incubated for 4 h. A volume of 50 μl of DMSO was added and the absorbance of the contents in wells was measured at 590 nm using a Tecan Genios-Pro microplate reader (Tecan Group Ltd. Mӓnnedorf, Switzerland).