Plant material and preparation of the extract
In the present study, we examined two extracts of Acalypha indica: aerial parts and root extracts. Acalypha indica was planted at the School of Biomedical Engineering and Health Sciences at the corresponding university. The whole fresh plants were washed then dried at room temperature for seven days. Dry plants were then separated into aerial parts and roots. Each of the two partitions was chopped into small pieces and extracted for 24 h with 30% ethanol using a Soxhlet extractor. The solvent containing the extracted compounds was then concentrated using a rotary evaporator before freeze-drying the extracts for 48 h.
Chemicals
Ethanol (96%), Methanol, DMSO (Dimethyl Sulfoxide) (Merck). DPPH (2,2-diphenyl-1-picrylhydrazyl), L-ascorbic acid, Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Trypan blue, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), PBS (Phosphate Buffered Saline) (Sigma-Aldrich). ABS (Bovine Serum Albumin) (Vivantis) Pen Strep (Penicillin and streptomycin) (Gibco). Animal tissue culture lab in the Faculty of Sciences, Department of Bioscience in the corresponding university provided the Human Skin Fibroblast cell line (HSF 1189).
Antioxidant DPPH free radical-scavenging activity
We evaluated the antioxidant activity based on the scavenging activity of the aerial parts and root ethanolic extracts on DPPH [20, 21]. Using methanol as solvent, we prepared 0.5 mg/mL ascorbic acid as standard and (0.04% w/v) DPPH as a stock solution. DPPH scavenging activity was studied on concentration gradient of the extracts (500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.63 µg/mL and 7.81 µg/mL). DPPH solution in methanol was used as a control, and pure methanol was used as blank. After incubation for 30 min at room temperature in the dark, the absorbance was taken at 517 nm, and the antioxidant activity was expressed as IC50 (µg/mL). The ability to scavenge the DPPH radicals was calculated using the following equation:
$$\%{\text{Radical}}\;{\text{scavenging}} = { }\left\{ {\frac{{\left[ {{\text{Absorbance}}\;{\text{of}}\;{\text{control}} - \left( {{\text{Absorbance}}\;{\text{of}}\;{\text{sample}} - {\text{Absorbance}}\;{\text{blank}}} \right)} \right]}}{{{\text{Absorbance}}\;{\text{of}}\;{\text{control}}}}} \right\} \times 100$$
The assay was performed in triplicates, and the results were averaged. A dose–response curve was plotted with the percentage of inhibition against the concentration of crude extracts on a log scale. The IC50 value was then obtained from the graph.
Cell viability (MTT assay)
The cell viability effect of A. indica aerial parts and root ethanolic extracts on fibroblasts was carried out using MTT assay [22, 23]. Human skin fibroblast cells (HSF 1189) were seeded in a 96-well plate at a density of 2 X 105 cells/well and supplemented with DMEM (containing 10% FBS and 1% pen strep). After incubation for 24 h at 37 ºC in a humidified 5% CO2 atmosphere, we treated–80% confluence–cells with concentration gradient of the extracts (1000 µg/mL, 100 µg/mL, 10 µg/mL, 1 µg/mL, and 0.1 µg/mL) dissolved in DMSO and left one well untreated: DMEM only. After treating the cells, we incubated them again for 24 h. Then, we added MTT 10% to each well, and the cells were further incubated for 4 h. After that, we removed the media and added DMSO to dissolve the formed formazan. The absorbance was measured at 575 nm and 655 nm. The absorbance of formazan in untreated cells was considered as 100% proliferation or viability. Cell viability percentage of the samples was calculated according to the equation:
$$\%{\text{Cell viability}} = { }\left\{ {\frac{{\text{Absorbance of Treated cells}}}{{\text{Absorbance of Untreated cells}}}} \right\} \times 100$$
The assay was performed in nine replicates, and the results were averaged. A dose–response curve was plotted with the viability percentage against the concentration of crude extracts on a log scale.
Wound scratch assay
In vitro wound healing activity of A. indica aerial parts and root, ethanolic extracts were determined by using scratch assay [22, 24, 25]. Fibroblasts were seeded in a 6-well plate at a density of 3 X 105 cells/well and incubated for 24 h to get 80% of cell confluence. We then made an artificial wound using 200 µL pipette tips. The treatment was then done by applying five different concentrations (1000 µg/mL, 100 µg/mL, 10 µg/mL, 1 µg/mL and 0.1 µg/mL) and one untreated well, which only contain growth medium (DMEM). The wound area was monitored under an inverted microscope for up to 48 h to observe fibroblasts migration. Images of the wound area were captured using an optical microscope (at 4 × magnification) to determine the percentage of the wound area. Duplicate wells were used per condition, and two fields per well were captured at each time point. ImageJ 1.48v software was used to analyse the images and calculate the wound area.
$$\%{\text{Wound closure}} = \left\{ {\frac{{{\text{Average scratch area }}\left( {0{\text{ h}}} \right) - {\text{Average scratch area }}\left( {48{\text{ h}}} \right)}}{{{\text{Average scratch area }}\left( {0{\text{ h}}} \right)}}} \right\} \times 100$$
Statistical analysis
The statistical evaluation was studied by using Microsoft Excel 2010 and IBM SPSS Statistics version 20. The normality test was completed at a 0.95 confidence level, and the data were considered normally distributed when p > 0.05. One-way analysis of variance (ANOVA) and Kruskal–Wallis tests for parametric and non-parametric data were respectively used to determine the mean differences between the variables. The differences were considered statistically significant when p < 0.05. For the Post-Hoc test of ANOVA, Dunnett t was used for multiple data comparisons with homogeneous variances, whereas Games-Howell was used for data with non-homogeneous variances. Mann–Whitney test was used to study the differences in non-parametric data.
