Plant collection and preparation of the extract
S. heyneanus plants were collected from Palchuram, Kottiyoor Road, Wayanad, Kerala, India. The specimen was deposited at the Centre for Repository of Medicinal Resources (C-RMR), Bangalore (Voucher No. FRLH 120018). The roots were separated, washed in distilled water, and dried for 2 days. The roots were powdered by using the mechanical blender and immersed in methanol for 3 days at room temperature. The clear supernatant above the plant powder residue was drained out through the filter paper and condensed by the rotary evaporator at 50 °C, which yields blood-red colour viscous extract [5, 6].
Phytochemical investigation
The root extract of S. heyneanus was tested for phytochemical analysis for different categories of secondary metabolites of phytochemicals using specific chemical reagents [7, 8].
Chemicals and reagents
DPPH, ABTS, and Folin–Ciocalteu phenol reagents were procured from SRL India. Quercetin, sodium carbonate (Na2CO3), absolute ethanol, Mueller-Hinton (MH) agar, and gallic acid were obtained from Himedia, India. The other analytical grade common chemicals were purchased from the local vendors (Chennai, India).
Total phenol estimation
The total phenolic content was determined by the Folin-Ciocalteu reagent method with small modifications [9]. One hundred microlitres of the root extract (1 mg/mL) of S. heyneanus was mixed with 900 μL of methanol and 1 mL of 1:10 diluted, Folin-Ciocalteu reagent. Next, 1 mL of 20% (w/v) Na2CO3 solution was added and shaken well. The reaction mixture was incubated in dark for 30 min at room temperature. At 765 nm, the absorbance was measured and the total phenolic content was calculated as gallic acid equivalent (μg/mg of extract).
Total flavonoid estimation
The total flavonoid content of the root extract of S. heyneanus was estimated by the aluminium chloride reagent method with small modifications [10]. Five hundred microlitres of extract (1 mg/mL) was mixed with 500 μL of methanol, and to this, 0.5 mL of 5% (w/v) sodium nitrite solution was added. Next, 500 μL of aluminium chloride solution (10%, w/v) was added, followed by 50 μL of 1 M NaOH solution and shaken well. The reaction mixture was incubated for 30 min at room temperature, and the absorbance was measured at 510 nm. The flavonoid content was calculated as quercetin equivalent (μg/mg of extract).
DPPH● radical scavenging activity
The antioxidant activity of the root extract of S. heyneanus was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay method [11]. One millilitre of the root extract with different concentrations (20–120 μg/mL) was mixed with 1 mL of 0.1 mM DPPH solution in methanol. One millilitre of DPPH solution mixed with 1 mL of methanol was used as the control. Then, the mixture was incubated in dark for 30 min, and the absorbance was measured at 517 nm. Ascorbic acid was used as the standard. The percentage of inhibition was calculated as:
$$ \%{\mathrm{of}\ \mathrm{DPPH}}^{\bullet}\kern0.5em \mathrm{radical}\ \mathrm{inhibition}\kern0.5em =\kern0.5em \frac{\mathrm{Control}-\mathrm{Sample}}{\mathrm{Control}}\times 100 $$
Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity was analysed by the salicylic acid method with some minor modifications [12]. To 1 mL of various concentrations of the extract, 300 μL of FeSO4 (8 mM) solution and 250 μL of H2O2 (20 mM) were added and shaken well. To start the reaction, 250 μL of salicylic acid in methanol (3 mM) was added, and then, the reaction mixture was incubated at 37 °C for 30 min. Then, 200 μL of distilled water was added, and the mixture was centrifuged at 5000 rpm for 5 min. At 510 nm, the absorbance of the supernatant was measured and the percentage of OH● radical scavenging activity was calculated as:
$$ \%\mathrm{of}\ {\mathrm{OH}}^{\bullet}\kern0.5em \mathrm{radical}\ \mathrm{inhibition}\kern0.5em =\kern0.5em \frac{\mathrm{Control}-\mathrm{Sample}}{\mathrm{Control}}\times 100 $$
ABTS●+ radical cation scavenging activity
The antioxidant ability of the root extract of S. heyneanus was evaluated by the ABTS●+ radical cation scavenging assay method with slight modifications [13]. ABTS●+ radical cation was obtained by 7 mM of ABTS reacted with 2.45 mM of potassium persulfate in 200 mM of PBS (pH 7.4) and incubated for 12–16 h in the dark before use. The ABTS●+ radical cation solution was diluted with PBS and set an absorbance of 0.70 ± 0.02 at 734 nm. Diluted ABTS●+ radical cation solution was prepared freshly every day for this assay. Different concentrations (5–30 μg/mL) of the root extract was mixed with 500 μL of diluted ABTS●+ radical cation solution, and after 5 min, the absorbance was measured at 734 nm. Ascorbic acid was used as the standard reference. The ABTS●+ radical cation scavenging activity was expressed as:
$$ \%{\mathrm{of}\ \mathrm{ABTS}}^{\bullet +}\kern0.5em \mathrm{radical}\ \mathrm{cation}\ \mathrm{inhibition}\kern0.5em =\kern0.5em \frac{\mathrm{Control}-\mathrm{Sample}}{\mathrm{Control}}\times 100 $$
Phosphomolybdenum reduction assay
The antioxidant capacity of the root extract of S. heyneanus was assessed by phosphomolybdenum reduction assay method [14]. One millilitre of various concentrations of the root extract (20–120 μg/mL) was mixed with 1 mL of reagent solution including ammonium molybdate (4 mM), sodium phosphate (28 mM), and sulphuric acid (600 mM). The reaction mixture was incubated in a water bath at 95 °C for 90 min, and the absorbance of the coloured complex was measured at 695 nm. Ascorbic acid was used as the standard. The percentage of Mo6+ reduction was calculated as:
$$ \%\mathrm{of}\ {\mathrm{Mo}}^{6+}\kern0.5em \mathrm{reduction}\kern0.5em =\kern0.5em \frac{\mathrm{Sample}-\mathrm{Control}}{\mathrm{Sample}}\times 100 $$
Ferric (Fe 3+) reducing power assay
The assay for estimating the reducing power of the root extract of S. heyneanus was the potassium ferricyanide method with minor modifications [15]. One millilitre of the root extract of different concentrations (20–120 μg/mL) was mixed with 1 mL of potassium ferricyanide [K3Fe(CN)6] (1% w/v) solution and 1 mL of phosphate buffer (0.2 M, pH 6.6) solution. Then, the mixture was incubated at 50 °C in a water bath for 20 min. Five hundred microlitres of trichloroacetic acid (10% w/v) was added to each mixture followed by 100 μL of freshly prepared FeCl3 (0.1%, w/v) solution which was added and shaken well. The absorbance was measured at 700 nm. Ascorbic acid was used as the standard. The percentage of Fe 3+ reduction was calculated as:
$$ \%\mathrm{of}\ {\mathrm{Fe}}^{3+}\kern0.5em \mathrm{reduction}\kern0.5em =\kern0.5em \frac{\mathrm{Sample}-\mathrm{Control}}{\mathrm{Sample}}\times 100 $$
Antibacterial activity
Organisms
The Gram-positive organisms such as Bacillus subtilis (MTCC 441) and Staphylococcus aureus (MTCC 98), as well as the Gram-negative organisms such as Salmonella typhi (MTCC 734) and Klebsiella pneumonia (MTCC 109), were chosen for the antibacterial activity study.
Standard
Tetracycline was used as the standard compound for all the bacterial cultures.
Mueller-Hinton agar
MH agar was procured from HiMedia (HIMEDIA-M173-500 G) to make up the medium for bacteria. The media was calculated (38.0 g/L) depending upon the availability of strains and suspended in 150 mL of distilled water in a conical flask, stirred, boiled to dissolve, and then autoclaved at 15 lbs and 121 °C for 15 min. The pH range was between 7.0 and 7.5 [16]. The hot medium was dispensed in sterile petri plates which were kept in sterile laminar airflow chamber and allowed to solidify for 15 min.
Disc diffusion method
Antibacterial activity of root extract of S. heyneanus was carried out using the agar disc diffusion method. The MHA in the petri plates was inoculated by dispensing the inoculum using sterilised cotton swabs which are previously immersed in the inoculum containing test tube and spread evenly onto the solidified agar medium. Five sterile discs were taken, and the root extract was loaded onto the discs with different concentrations of 250 μg/mL, 375 μg/mL, and 500 μg/mL concentrations. The discs loaded with extract were placed onto an organism swabbed medium in the petri plates which were incubated for 24 h at 37 °C. The antibacterial activity was expressed by measuring the diameter zone of inhibition formed around the disc [17, 18]. Tetracycline (25 μg) was used as a positive control (standard).
Minimum inhibitory concentration
The antimicrobial activity of the root extract was determined by resazurin microtiter assay method [19]. The inoculum, equivalent to a 0.5 McFarland turbidity standard, was prepared from each bacterial isolate in MH broth. Sterile PBS (50 μL) was dispensed in each well of a sterile 96-well flat bottom plate. The root extract of various concentrations from 1000 μg/mL to 0.48 μg/mL was added by twofold serial dilutions. Tetracycline was used as the positive control in separate wells. Separate wells with negative control (culture with no plant extract) were also performed. To each well, 10 μL of resazurin indicator solution was added, followed by the addition of 30 μL of threefold strength MH broth. The plates were incubated at 37 °C in an incubator for 12–24 h for further growth if any was detected. The change in colour of the dye added to the wells from blue to pink indicated the growth of bacteria. The MIC was defined as the lowest concentration of each test compound or drug that prevented any colour change observed visually, i.e., the concentration that inhibited the microbial growth.
Cytotoxicity
MTT assay
Cell viability was measured with the conventional MTT reduction assay, as described previously with slight modification. Briefly, human dermal fibroblast (HDF) cells were seeded at a density of 5 × 103 cells/well in 96-well plates for 24 h, in 200 μL of RPMI with 10% FBS. Then, the culture supernatant was removed and RPMI containing root extract of S. heyneanus of various concentrations of 10–500 μg/mL was added and incubated for 48 h. After treatment, the cells were incubated with MTT (10 μL, 5 mg/mL) at 37 °C for 4 h and then with DMSO at room temperature for 1 h. The plates were read at 595 nm on a scanning multi-well spectrophotometer [20]. Data are represented as the mean values for three independent experiments:
$$ \mathrm{Cell}\ \mathrm{viability}\ \left(\%\right)\kern0.5em =\kern0.5em \left(\frac{\mathrm{Mean}\ \mathrm{OD}}{\mathrm{Control}\ \mathrm{OD}}\right)\times 100 $$
Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) using Graph Pad Prism 8.0.1. Differences in the mean between observations were considered significant at level p < 0.05. All values acquired are cumulative of three replicates, and it was represented as a mean ± standard deviation.