Hermannia depressa roots were collected in Thaba ‘Nchu, Free state, South Africa. Standard methods were used in plant material collection, drying, mounting, preparation, and preservation. The plant was identified first by its vernacular names and later validated by the South African National Biodiversity Institute (SANBI) personnel. Voucher specimen (B007) was deposited at the Central University of Technology, Centre for Applied Food Sustainability and Biotechnology. The roots were collected, washed, oven-dried at 40 °C for 72 h, and ground to powder. The dry, powdered material (50 g each) of the plants was weighed, pulverised, and soaked in 100% methanol, acetone, or water for 48 h on a rotary shaker. After filtering the extracts, further extraction with a new solvent was carried out until the solvent was clear. The solvents of extraction were evaporated at 40 °C, under a vacuum, using a Buchi Rotavapor, while the aqueous extracts were dried through lyophilisation. All extracts were stored in the fridge at 4 °C until required. All aqueous, methanol and acetone extracts were used throughout the experiments. The leaves, stems, and roots of Hermannia depressa can be seen in Figure 1.

The total phenolic content of the plant extracts was performed using the Folin-Ciocalteu method by Singleton, Orthofer and Lamuela-Raventós (1990) with modifications from Tahrani et al. (2019). A 100 μL of each extract solution at a concentration of 1 mg/mL, 100 μL of 50% Folin–Ciocalteu reagent and 2 mL of 7.5% sodium carbonate was prepared in triplicates. The mixture was incubated for 30 min at room temperature and the absorbance was measured at a wavelength of 720 nm. Gallic acid was used to formulate a standard curve (0–350 μg/mL; y = 0.0029x – 0.00121; R² = 0.995), and the total phenolic content of gallic acid equivalent (GAE) extracts was calculated using the following formula: C = c.V/m, where C is the total amount of phenolic compounds in mg/g of plant extract in GAE, c is the concentration of gallic acid from the calibration curve (mg/mL), V is the extract volume (mL), and m refers to the weight of pure organic plant extract (g).

The total flavonoid content was determined using aluminium the colorimetric method by Pothitirat et al. (2009) with modifications from Vongsak et al. (2013). A 50 μL of crude extracts (1 mg/mL ethanol) was made up to 1 mL with methanol, mixed with 4 mL of distilled water and then 0.3 mL of 5% NaNO₂ solution; 0.3 mL of 10% AlCl₃ solution was added after 5 min of incubation, and the mixture was incubated for 10 min. Then, 2 mL of 1 mol/L NaOH solution was added and the final volume of the mixture was brought to 10 mL with double-distilled water. The absorbance of the mixture was measured at 415 nm against a blank sample without aluminium chloride using UV–VIS spectrophotometer. The content of total flavonoids was calculated as mean ± SD (n = 3) and expressed as grams of quercetin equivalents (IQE) in 100 g of the extract and dried powder.

Liquid Chromatography with tandem mass spectrometry (LC-MC/MS) analysis for the identification of active compounds from H. depressa crude extracts was carried out using the Waters Synapt G2, Electrospray Ionisation (ESI) positive or negative Cone Voltage 15 V, lock mass: leucine encephalin instrument. This is an Agilent 1100 LC system consisting of degasser, binary pump, autosampler and column heater. The column outlet was coupled to an Agilent MSD Ion Trap XCT mass spectrometer equipped with an ESI ion source. Data acquisition and mass spectrometric evaluation were carried out on a computer with an Acquity binary solvent manager instrument system. For the chromatographic separation, Waters BEH C18, 2.1 × 100 mm column was used. The column was held at 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1% formic acid in acetonitrile) for 1 min, followed by an 11 min step gradient from 5% B to 100% B; then it was kept for 4 min with 100% B; finally, the elution was achieved with a linear gradient from 100% B to 5% B for 2 min. The flow rate was 0.4 mL/min and the injection volume was 0.01 mL. The following parameters were used throughout all mass spectrometry (MS) experiments: for electrospray ionisation with negative ion polarity, the capillary voltage was set to 3 kV, the drying temperature to 350 °C and cone voltage of 15 V, the maximum nebulizer pressure to 15 000 psi and the seal wash was 5 min. The total run time was 15 min, the scan speed was 26 000m/z/s (ultra-scan mode) and the lock mass was Leucine enkephalin. The phenolics were identified using a combination of High-Performance Liquid Chromatography (HPLC) with diode array detection and liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (ESI-LC/MS/MS) based on their ultraviolet spectra, mass spectra and by comparison of the spectra with those of available authentic standards.

Nitric oxide (NO) production and viability of lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were used to determine the anti-inflammatory potential of the selected plants. RAW 264.7 cell lines, seeded into 96-well plates at a density of 1 × 10⁵ cells per well and allowed to attach overnight. The extracts were dissolved in Dulbecco’s Modified Eagle’s Medium (DMEN) complete medium to give final concentrations of 50, 100 and 200 μg/mL, respectively, and stored in room temperature until needed. To assess the anti-inflammatory activity, 50 μL of LPS (final concentration of 500 μg/mL) containing medium was added to the corresponding wells. Aminoguanidine (AG) was used as the positive control for this experiment. Cells were incubated for a further 18 h. To quantify NO production, 50 μL of supernatant from each well of cell culture plates was transferred to a new 96-well plate and 50 μL Griess reagent was added. Absorbance was measured for 10 min at 540 nm, and the results were expressed relative to the appropriate untreated control. A standard curve using sodium nitrite dissolved in a culture medium was used to determine the concentration of NO in each sample. Percentage inhibition was calculated based on the ability of extracts to inhibit nitric oxide formation by cells compared with the control (cells in media without extracts containing triggering agents and dimethyl sulfoxide [DMSO]), which was considered as 0% inhibition (Dzoyem & Eloff 2015).

The DPPH method was used to test the radical scavenging activity of the aqueous, methanol, and acetone extracts of H. depressa. The DPPH was dissolved in methanol to give 0.1 mM solution; 10 μL of extract samples in methanol (or methanol itself as blank control) was added to 175 μL of the methanol DPPH solution. For each extract, different concentrations were tested (200, 100, 50, 25, 12.5, 6.25, 0 μg/mL). The decrease in absorbance was measured at 520 nm after 20 min. The decrease in absorption induced by the test sample was calculated by subtracting that of the control. The antioxidant activity of each test sample was expressed as an IC₅₀ value, that is, the concentration in μg/mL that inhibits DPPH absorption by 50% and was calculated from the concentration-effect linear regression curve. The percentage DPPH inhibition was calculated using the formula: (A0-A1)/A0*100 where A0 is the absorbance of the control and A1 is the absorbance of the test sample. Trolox was used for positive control. Trolox equivalents were calculated as the IC₅₀ of the test sample relative to that of Trolox expressed as a dry mass.

The cytotoxicity of plant extracts was carried out using the Hoechst 33342 nuclear dye and propidium iodide exclusion method. African green monkey kidney cell line was maintained at 37 °C in a humidified incubator with 5% CO₂ in 10 cm culture dishes. The complete growth medium consisted of 10% foetal bovine serum (FBS) supplemented by Dulbecco’s Modified Eagle’s Medium (DMEM). In each well, cells were seeded into 96 well microtiter plates with a density of 4000 cells/well using a volume of 100 μL. The microtiter plates were incubated for 24 h at 37 °C, 5% CO₂ and 100% relative humidity before adding test compounds to allow cell attachment. Cells were treated with 100, 200 and 400 μg/mL of 100 μL of each extract diluted in culture medium. The cells were also treated with the positive control called melphalan at 100 μg/mL. Cells were further incubated for 48 h. The treatment medium was aspirated from all wells and replaced with 100 μL of Hoechst 33342 nuclear dye (5 μg/mL) and incubated for 10 min at room temperature. Thereafter, cells were stained with propidium iodide (PI) at 100 μg/mL to enumerate the proportion of dead cells within the population. Cells were imaged immediately after the addition of PI using the ImageXpress Micro XLS Widefield Microscope (Molecular Devices) with a 10× Plan Fluor objective, DAPI and Texas Red filter cubes. The dye exclusion technique assumes that non-viable cells will absorb the dye, whereas viable ones would not. After 24 h of treatment with plant extracts, the total cell count was determined using a haemocytometer. Extracts with cytotoxic potential are identified by a 50% or greater reduction in viable cell count compared to untreated control cell cultures. The percentage growth inhibition was calculated using the following formula: % Inhibition = (Total cells-dead cells/Total cells) × 100.

The percentage inhibition versus concentration was plotted and the CTC50 (concentration needed to decrease viability by 50%) value was determined for the cell line. All experiments were carried out in triplicates. The results were calculated as mean ± standard deviation (SD) values.

The values were presented as the mean ± standard deviation of three separate measurements. Correlation and regression analyses were performed using the EXCEL software from Microsoft Corporation, USA. The results were further analysed using one-way analysis of variance (ANOVA) and Tukey-Kramer post hoc test and statistical significance was determined by considering p-values less than 0.05 when comparing the different groups using EXCEL software from Microsoft Corporation, USA.

The study does not require ethical clearance since there was no direct contact with either human or animal subjects.

Total phenolic content (TPC) was conducted to estimate the total amount of phenols present in each concentration of extracts of H. depressa shown in Table 1. The total phenol content was extrapolated from the gallic acid standard/calibration graph equation; y = 0.1942x – 0.0174, R2 = 0.9508 and was expressed as mg gallic acid equivalent (GAE)/g from the equation CV/m. The total phenolic content of H. depressa extracts exhibited a range of 2.93 to 8.45 mg GAE/g when assessed at a concentration of 1mg/mL for the extracts. The highest concentration of phenolics was found in acetone (8.45 ± 0.006 mg of GAE/g dried extract), followed by methanol and aqueous extracts of H. depressa (Table 1). The total flavonoid content (TFC) was calculated to estimate the total amount of flavonoids present in each concentration, as shown in Table 1. The TFC was calculated using the equation calibration curve of quercetin standard (y = 0.6961 x 0.0046, R² = 0.997) and was expressed as mg quercetin equivalent (QE)/g from the equation C = c.V/m. The TFC of all the extracts ranged from 0.97 to 0.53 mg QE/g of extract in 1mg/mL of extract. Hermannia depressa aqueous extracts had the highest amount of flavonoids, while acetone was the second highest followed by methanol extracts.

The LC-MS/MS results showed spectral data of major bioactive compounds with important pharmacological properties identified in H. depressa methanolic, acetone and aqueous crude extracts detected with mass spectrometry (MS) in negative and positive modes (Table 2). The spectral data obtained from the peaks in the aqueous extracts matched the spectra of several compounds of various classes alkaloids, flavones, and flavonoids. Meanwhile in methanol extracts, compounds classified as steroids, flavonoids, and fatty acids were detected. Acetone extracts contained compounds that are classified as polyphenolics. The identification of the compounds was based on LC-MS/MS results and in comparison with literature.

Table 3 displays the results of antimicrobial activity of H. depressa extracts. The aqueous extracts of H. depressa showed no antimicrobial activity against all the microbial organisms except against C. krusei, where activity was at MIC of 1.25 mg/mL. Hermannia depressa methanolic extracts demonstrated significant antimicrobial activity against all the microorganisms tested, at MICs ranging from 0.3 to 1.25 mg/mL with B. cereus and C. albicans being the most affected. Hermannia depressa acetone extracts also demonstrated antimicrobial activity against 10 of 13 microbial species tested against at MICs ranging from 0.1 to 1.25 mg/mL with C. albicans being the most affected.

All experiments were conducted in triplicates.

The anti-inflammatory activity was expressed by the ability of plant extracts to inhibit the release of NO from live cells. A treatment plant is considered anti-inflammatory potent when NO production inhibition from the cells is greater than 60% (Figure 2). Nitric oxide production inhibitory activity of H. depressa methanolic, acetone and aqueous plant extracts were tested using the liposaccharide (LPS)-activated malignant macrophage cell line RAW264.7. The acetone extracts of H. depressa demonstrated the highest level of potency in inhibiting NO, with a remarkable inhibition rate of 77.5% at the highest concentration. Similarly, the methanol extracts displayed significant inhibition of NO, reaching 62.6% at the highest concentration. Hermannia depressa aqueous extracts demonstrated less potency in inhibiting NO, thus no anti-inflammatory activity was displayed. An analysis of variance (one-way ANOVA) yielded significant variation among anti-inflammatory activity with different concentrations of the H. depressa, F = 22.85, p = 1.22 × 10⁻⁸< 0.05. According to Tukey’s test nitric oxide percentage inhibition data were divided into 10 groups and the results are presented in supplementary data folder. The results suggest that H. depressa has anti-inflammatory activity by inhibiting NO production in a malignant macrophage cell line activated with LPS.

Free radical scavenging activities of methanolic, acetone and aqueous extracts of H. depressa were measured by the DPPH assay. Table 4 shows the IC₅₀ and DPPH percentage inhibition of H. depressa extracts for antioxidant activity. The DPPH percentage inhibition of the extracts was tested at concentrations ranging from 6.25 to 200 μg/mL. Hermannia depressa methanolic, acetone, and aqueous extracts inhibited DPPH by more than 50% at the highest concentrations (100 μg/mL – 200 μg/mL), indicating strong free radical scavenging activity. When compared with synthetic antioxidant agents such as Trolox and ascorbic acid, the H. depressa acetone and methanol extracts demonstrated significant antioxidant activity, at IC₅₀ values of 3.57 and 5.197 μg/mL, respectively. One-way ANOVA showed significant variation between antioxidant activity and different concentrations, F = 480.6, p = 2.82 × 10⁻³³<0.05. Tukey’s test DPPH data were divided into 16 groups to determine the variance in activity between the extract groups and the positive control (Online Appendix 1).

A low correlation (R² = 0.744) was observed between the antioxidant activity data obtained through DPPH assays (IC₅₀) and the total phenolic contents of H. depressa extracts examined in the study (Figure 3). A significant correlation (R² = 0.744) was witnessed between antioxidant activity and total flavonoid content for the same extracts (Figure 4). This suggests that the flavonoid constituent of the extracts had an influence on the antioxidant properties of H. depressa extracts.

The cytotoxicity results are presented as the percentage of viable cells following exposure to the extracts at a specified range of concentrations (Figure 5). If the cell viability drops below 50% after being exposed to the highest concentration of the extract, the extract is considered toxic. The treatment of Vero cells with H. depressa acetone extracts at the highest concentration reduced the percentage cell viability to 24.52%, which is also an indication of the extract’s toxicity towards the cells. When the Vero cells were exposed to H. depressa acetone and methanol extracts at the highest concentration, the percentage of viable cells decreased to 24.52% and 37.49%, respectively. Aqueous extracts showed no toxicity against Vero cells because the cell viability was increased above 50% after treatment with the extracts at the highest concentration. This decrease in cell viability at the highest concentration further confirms the toxic effects on the cells. An analysis of variance using one-way ANOVA showed significant variability among cell viability values with different concentrations of extracts, F = 677.70, p = 3.98 × 10⁻³². According to Tukey’s test, cell viability data were divided into 10 groups to determine the variance in activity between the extract groups and the positive control (Online Appendix 1)