Journal of Herbmed Pharmacology J Herbmed Pharmacol. 2023; 12(1): 109-117. A phytochemical screening, antioxidant and antibacterial activity analysis in the leaves, stems and roots of Portulacaria afra Domonique Courtney Basson* ID , Thabiso Katlego Teffo ID , Ida Masana Risenga School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa *Corresponding author: Domonique Courtney Basson, Email: domoniquebasson@gmail.com Implication for health policy/practice/research/medical education: This study contributes new knowledge on the phytochemical and biological activities of P. afra. The high phytochemical presence, as well as antioxidant and antibacterial activities exhibited by the plant extracts in this study validate the use of P. afra among traditional healers. The plant displays the potential of enhancing human health. Please cite this paper as: Basson DC, Teffo TK, Risenga IM. A phytochemical screening, antioxidant and antibacterial activity analysis in the leaves, stems and roots of Portulacaria afra. J Herbmed Pharmacol. 2023;12(1):109-117. doi: 10.34172/jhp.2023.10. Introduction: Portulacaria afra is a medicinal plant commonly used among African traditional healers to treat skin conditions and dehydration. The aim of this study was to scientifically validate the use of P. afra among traditional healers. Methods: Standard phytochemical colour tests were used to determine the presence of ten phytochemicals, using four solvents of varying polarities (hexane, ethyl acetate, methanol, and water). The antioxidant activity was determined using 1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide scavenging assays. An agar-well diffusion assay was used to determine the antibacterial activities of the leaves, stems, and roots of P. afra against Staphylococcus aureus and Escherichia coli. Results: P. afra exhibited a high phytochemical presence in the methanolic extracts, with seven out of the 10 phytochemical groups present. Flavonoids and phlobatannins were absent in all of the plant’s extracts. The methanolic root extract exhibited the highest DPPH scavenging activity (IC50 = 0.39) whilst the hexane leaf extract (IC50= 14.83) was the only extract to exceed the acceptable upper limit. The scavenging activity of the plant was stronger against hydrogen peroxide than it was against DPPH. The methanolic and hot water stem extracts displayed the largest zone of inhibition (of 20 mm) against E. coli. The cold-water and room-temperature water extracts, of all three plant parts, showed no zone of inhibition against either bacterial strain. Conclusion: P. afra has the capacity to be used as a nutritional supplement for its antioxidant properties, while the antibacterial properties may provide relief against E. coli infections. A R T I C L E I N F O Keywords: African traditional medicine Medicinal plants Phytochemistry Plant extracts Secondary metabolites Article History: Received: 15 August 2022 Accepted: 8 October 2022 Article Type: Original Article A B S T R A C T Introduction Medicinal plants are defined as plants that have compounds within their structures, which can be extracted and used for medicinal purposes (1). These compounds are more commonly known as phytochemicals, which are synthesised by plants through primary or secondary metabolism. The combination of secondary metabolites in plants is generally responsible for the medicinal properties expressed by plants (2). Plants have the ability to produce an infinite number of these phytochemicals because they are synthesised in response to stressors such as herbivory or competition (3). Many chemicals have been extracted from plants and replicated to produce modern drugs, such as salicin (more commonly known as aspirin), which was extracted from the bark of a white willow tree (4). The use of traditional medicine is extensive and continues to increase. The World Health Organization (WHO) has estimated that 80% of the world population relies on traditional medicine as their primary source of health care (5). Thus, it is important to scientifically validate the use of medicinal plants for treatment. Despite African traditional medicine being regarded as one of the oldest and most diverse uses of medicinal plants (6), it has not been well documented throughout history. An example of http://www.herbmedpharmacol.com doi: 10.34172/jhp.2023.10 https://orcid.org/0000-0003-4553-624X https://orcid.org/0000-0003-2871-6097 http://www.herbmedpharmacol.com https://doi.org/10.34172/jhp.2023.10 Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023 http://www.herbmedpharmacol.com110  Basson et al this is Portulacaria afra. Portulacaria afra is a succulent evergreen plant, indigenous to southern Africa (7). It is a large shrub or small tree and can grow up to a height of 5 m. The plant is characterised by its small flat leaves and a red stem. The economic importance of P. afra is heightened as a result of its wide-ranging uses, such as its carbon sequestration properties, which have been extensively studied. However, the studies on the medicinal properties of the plant are limited. Through previously conducted interviews, it was determined that traditional healers commonly use the leaves topically to treat skin conditions such as rashes and acne (8). Due to the limited phytochemical and pharmacological studies performed on the plant, this study aims to perform a phytochemical screening and determine the antioxidant and antibacterial activities in the stems, roots and leaves of P. afra. Four solvents, with varying polarities, were used to create a thorough examination of the plant parts, as the polarity of the solvent affects the yield of the extract (9). This study will contribute new knowledge on the phytochemical profile and biological activities of P. afra. Materials and Methods Plant material The whole plant of P. afra was collected from the Witkoppen Wildflower nursery (26°03’32.6″S 27°56’15.9″E) in December 2020 (South African summer). The plant was identified and authenticated by researcher Dr Ida Risenga and a voucher specimen was deposited in the institute’s herbarium centre (IRM 001). The plant was maintained in the Oppenheimer Life Sciences greenhouse at the University of Witwatersrand for a period of three months. The fresh leaves, stems, and roots of 20 P. afra were placed in a hot air dryer for four days at 40°C. The dried plant materials were ground into a fine crude powder. The powdered plant was then enclosed in foil and placed in an airtight container, which was stored in a room- temperature cupboard till tests commenced. Preparation of the extracts The crude leaf, stem, and root extracts were prepared using four solvents: 80% methanol, hexane, ethyl acetate, and 100% distilled water at three different temperatures (4°C, 25°C, and 40°C). All crude plant extracts were prepared by placing one gram of the powdered plant into separate bottles and adding 25 mL of a solvent. The methanol, hexane, ethyl acetate, and room-temperature water (25°C) extracts were placed in a mechanical shaker. The hot water extract was placed on a hot plate with a magnetic stirrer. The cold-water extract was placed in the fridge and physically shaken every few hours. All extracts were shaken or stirred for 48 hours. The water extract was centrifuged to decrease its viscosity. All extracts were then filtered through a filter paper into a resealable vial. The supernatants were collected and placed in a vial and stored in the refrigerator until the tests were conducted. Phytochemical screening A qualitative analysis was conducted in order to determine the presence or absence of 10 phytochemical groups in the leaves, stems, and roots of P. afra using standard colour test methods (10). Froth test for saponins In a test tube, 0.5 mL of the crude plant extract was mixed with 5 mL of distilled water. The test tube was then vigorously shaken. Three drops of olive oil were placed inside the test tube and shaken vigorously. The presence of a stable foam indicated the presence of saponins. FeCl3 test for phenolics In a test tube, 1 mL of the crude plant extract was mixed with 5 to 10 drops of 10% ferric chloride (FeCl3). The appearance of a green-blue or violet colour was an indication of the presence of phenolic compounds. Hydrochloric acid test for flavonoids In a test tube, 1 mL of the crude plant extract was mixed with three drops of hydrochloric acid (HCl). After a yellow colour was observed, a few drops of dilute acid were then added. A colourless solution indicated the presence of flavonoids. Salkowski’s test for glycosides In a test tube, 0.5 mL of the crude plant extract was mixed with 2 mL of concentrated HCl. A reddish-brown colour indicated the presence of the steroidal aglycone part of the glycoside. Bromine water test for tannins In a test tube, 0.5 mL of the crude plant extract was mixed with 10 mL of bromine water. Discolouration of bromine water indicated the presence of tannins. Test for terpenoids In a test tube, 1 mL of crude plant extract was mixed with 0.5 mL of chloroform (CHCl3). A few drops of concentrated sulphuric acid (H2SO4) were added to the mixture. The formation of a reddish-brown precipitate indicated the presence of terpenoids. Test for steroids In a test tube, 2 mL of the crude plant extract was mixed with 2 mL of CHCl3 and 2 mL of concentrated HCl. A reddish-brown ring at the junction indicated the presence of steroids. Test for coumarins In a test tube, 1 mL of crude plant extract was reacted with 1 mL of 10% NaOH in a test tube. The formation of a yellow colour indicated the presence of coumarins. http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023http://www.herbmedpharmacol.com 111 Portulacaria afra: Scientific validation Test for phlobatannins In a test tube, 1 mL crude plant extract was mixed with a few drops of concentrated HCl. The appearance of red colour indicated the presence of phlobatannins Test for volatile oils In a test tube, 1 mL of the crude plant extract was added to 0.2 mL solution of 1% NaOH. The presence of a precipitate indicated the presence of volatile oils. Antioxidant scavenging activity Two antioxidant assays were conducted in this study to determine the antioxidant activities of the crude plant extracts. These assays included 1-diphenyl-2- picrylhydrazyl (DPPH) free radical scavenging activity (11) and a hydrogen peroxide test (12), which are described below. 1-Diphenyl-2-picrylhydrazyl scavenging assay The stable radical DPPH was used to determine the scavenging activity and hence, the antioxidant activity of the plant parts of P. afra. The crude plant extract was placed in capped test tubes at varying concentrations (10-50 µL) and mixed with 700 µL of the DPPH work solution. Methanol 80% was used to create a mixture with a volume of 1 mL. The test tubes were incubated in a dark cupboard, at room temperature for 45 minutes. Following the incubation period, the mixtures were placed into cuvettes, which were placed within a spectrophotometer (Genesys 10S UV-VIS). The absorbance was measured at a wavelength of 517 nm. The test was then triplicated while the percentage of inhibition was calculated using the following formula below. %Inhibition of DPPH radical= ([Abr-Aar]/Abr) × 100 Where Abr is the absorbance of the control and Aar is the absorbance of the sample. The concentrations were then plotted against percentage inhibition values, in order to calculate IC50. Hydrogen peroxide scavenging activity A 40mM solution of 30% aqueous hydrogen peroxide was prepared in a phosphate buffer (pH 7.4). The crude plant extracts were placed in capped test tubes at varying concentrations (10-50 µL) and mixed with 600 µL of the hydrogen peroxide solution. The test tubes were incubated in a dark cupboard, at room temperature for 10 minutes. The absorbance of the mixtures was determined using a spectrometer at a wavelength of 230 nm. The blank used was the phosphate buffer without the hydrogen peroxide solution. This test was then triplicated. The different absorbance values were recorded while the percentage of hydrogen peroxide scavenging activities was calculated using the following formula: % Scavenged [H2O2] = ([Abr-Aar]/Abr) x 100 Where Abr is the absorbance of the control and Aar is the absorbance of the sample. The concentrations were then plotted against percentage inhibition values to calculate the IC50. Determination of antibacterial properties To determine whether P. afra displayed anti-microbial properties, the agar-well diffusion assay was performed (13). The plant extracts were tested against gram-positive Staphylococcus aureus (ATCC 25923) and gram-negative Escherichia coli (ATCC 25922). Agar-well diffusion assay Petri dishes were sterilised and autoclaved before the introduction of the respective agar medium. This experiment used the recommended Mueller-Hinton and Baird Parker agar. After solidification of the media, sterile cotton swabs were used to evenly inoculate the entire surface of the agar medium with the respective bacteria strain. S. aureus was inoculated on the Baird Parker agar plates and E. coli was inoculated onto the Mueller-Hinton agar plates. Six wells were then bored in the media using a 6 mm sterile borer. The various plant extracts (100 µL) were then impregnated into five of the wells, whereas the sixth well was impregnated with the negative control, dimethyl sulfoxide (DMSO). The plates were then sealed and placed in the fridge for 30 minutes to allow for efficient diffusion. The plates were then transferred to the incubator set at 37℃ for 24 hours. Following the incubation period, the zones of inhibition were then measured in millimetres using a ruler. The presence of an inhibitory zone surrounding the well indicated a positive antibacterial activity result. Results Phytochemical screening A qualitative study was conducted and made use of visual cues and colour changes to detect whether phytochemical groups were present or not in the crude plant extract. The intensity of the visual difference was then used to determine the expected amount of the phytochemical groups (absent, present, moderate presence, or high presence) as shown in Table 1. The methanolic extracts of all three plant parts were able to extract the most phytochemical groups; seven out of the 10 phytochemical groups were detected in each plant part, namely: saponins, glycosides, phenolics, terpenoids, steroids, coumarins, and volatile oils. However, methanolic extracts were unable to extract tannins. The weakest extraction was exhibited by the room-temperature water and hexane extracts. There was a higher number of phytochemical groups extracted by the hot water in comparison to the cold water and room temperature water. The presence of coumarins was high in the leaves and stem, whilst only the methanolic extract was able to detect http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023 http://www.herbmedpharmacol.com112  Basson et al coumarins in the roots. Ethyl acetate extracts of the leaves and roots showed a high amount of phenolics. The plant showed no presence of flavonoids or phlobatannins in either plant part. The leaves had the highest number of phytochemical groups present in all solvents among the plant parts. Antioxidant activity In this study, the radicals DPPH and H2O2 were used to determine the scavenging activities of various crude plant extracts of P. afra. IC50 value was calculated for each extract and plotted in the following graphs. One-way ANOVA tests were used to determine the statistical significance between the different extracts for each plant part. Tukey’s test was used as the post-hoc test. In the DPPH assay, the hexane leaf extract (IC50= 14.83 mg/mL) was the only extract to exceed the acceptable upper limit of 10 (Figure 1A). Ethyl acetate experienced the lowest IC50 value (2,65 mg/mL). The stems displayed the lowest IC50 value against DPPH in the ethyl acetate extracts (Figure 1B). The methanol, cold water, and room temperature extracts exhibited IC50 values in the acceptable upper range. The methanolic root and the ethyl acetate root extracts displayed the highest antioxidant activity with IC50s of 0.39 mg/mL and 0.31 mg/mL, respectively. The root extracts outperformed the stems and the leaves counterparts in five out of the six extracts – with the exception of the hot Table 1. Colour screening results for the methanol, cold water, room-temperature water, hot water, ethyl acetate, and hexane extracts of the stems, leave, and roots of Portulacaria afra Plant Part Phytochemical Solvent Methanol Cold water Room temperature water Hot water Ethyl acetate Hexane Stems Saponins ++ + - ++ + ++ Flavonoids - - - - - - Glycosides ++ - - + - - Phenolics + - - - - - Tannins - + + + ++ - Terpenoids + - - - - - Steroids + - - - - - Coumarins +++ + + + ++ - Phlobatannins - - - - - - Volatile oils ++ - - + + + Leaves Saponins + - - - + ++ Flavonoids - - - - - - Glycosides ++ + - + - - Phenolics + + + + +++ - Tannins - + + + +++ + Terpenoids + + - - - + Steroids ++ - - + - - Coumarins ++ +++ ++ +++ - - Phlobatannins - - - - - - Volatile oils + + - ++ + + Roots Saponins + ++ ++ ++ - - Flavonoids - - - - - - Glycosides ++ +++ + ++ - - Phenolics + - - - +++ - Tannins - - - - - + Terpenoids +++ - - - + - Steroids + - - - - + Coumarins + - - - - - Phlobatannins - - - - - - Volatile oils + - ++ + - ++ (-): Absent; (+): Present; (++): Moderate presence; (+++): High presence. http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023http://www.herbmedpharmacol.com 113 Portulacaria afra: Scientific validation water extracts. In the H2O2 assay, the highest scavenging activity was exhibited by the hot water leaf extract (0.217 mg/mL). The extracts showed high antioxidant activity in the leaves (Figure 2A). All of the leaf extracts exhibited IC50 values below 10. The results between the different extract types were calculated as being statistically significant. The cold-water stem extract exceeded the IC50 threshold at 11.01 mg/mL. The hot water stem extract exhibited the lowest IC50 value of 0,46 mg/mL (Figure 2B). The IC50 values for the root extracts were consistently low. The statistical analysis determined that there was no statistical significance between the root extract types (Figure 2C). Four out of the six extract types showed that the leaves outperformed the stems and roots counterparts. The cold-water extracts had the highest IC50 value among all three plant parts. All IC50 values for the leaves, stem, Figure 1. IC50 values of the leaves (A), stems (B) and roots (C) extracts of Portulacaria afra using methanol, cold water, room temperature water, hot water, hexane, and ethyl acetate against 1-diphenyl-2-picrylhydrazyl (DPPH). Different small letters (a, b, c, and d) between the different groups indicate a statistical difference between extracts type (P < 0.05). (A) (B) (B) 4.76 5.09 7.41 3.71 14.83 2.65 0 2 4 6 8 10 12 14 16 Methanol Cold water Room temperature water Hot water Hexane Ethyl acetate IC 50 (m g/ m L) Extract type Antioxidant activity of leaves extracts against DPPH a a a a a b 7.23 8.54 6.85 2.98 2.48 1.5 0 2 4 6 8 10 12 14 16 Methanol Cold water Room temperature water Hot water Hexane Ethyl acetate IC 50 (m g/ m L) Extract type Antioxidant activity of the stems extracts against DPPH ac ac ac bd bc bc 0.39 1.443 4.12 5.59 1.96 0.31 0 2 4 6 8 10 12 14 16 Methanol Cold water Room temperature water Hot water Hexane Ethyl Acetate IC 50 (m g/ m L) Extract type Antioxidant activity of the roots extracts against DPPH a ab b b ab a http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023 http://www.herbmedpharmacol.com114  Basson et al and roots were below 5mg/mL, except for the hexane stem extract (6.375 mg/mL); the cold-water stem extract (11.002 mg/mL) was the only extract above 10. Antibacterial activity The antibacterial properties of P. afra were measured against two different bacterial strains, E. coli and S. aureus. The zones of inhibition were measured with a ruler and compared to the standard zone of inhibition ranges. The methanolic stem and hot water stem extracts displayed the largest zone of inhibition of 20 mm against E. coli. The cold-water and room-temperature water extracts of all three plant parts showed no zone of inhibition against either bacterial strain (Table 2). In all three plant parts, the methanolic and hot water extract zones of inhibition were identical against E. coli. The largest zones of inhibition were against E. coli, with the exception of the hexane extracts, which exhibited a greater or equal activity against S. aureus. Discussion The phytochemical constituents of a plant represent a good indication of the medicinal value of the plant (14). Of the 10 phytochemicals tested in this study, eight of them were present in all plant parts. Methanol is a commonly suggested solvent in literature for phytochemical a 0.385 b 4.55 a 0.89 a 0.22 b 3.9 a 0.85 0 2 4 6 8 10 12 Methanol Cold water Room temperature water Hot water Hexane Ethyl acetate IC 50 (m g/ m L) Extract type Antioxidant activity of the leaves extracts against H2O2 acd 1.08 bcd 11.01 acd 2.48 ac 0.46 ad 6.38 acd 2.67 0 2 4 6 8 10 12 Methanol Cold water Room temperature water Hot water Hexane Ethyl acetate IC 50 (m g/ m L) Extract type Antioxidant activity of the stems extracts against H2O2 a 0.561 a 2.22 a 1.65 a 1.22 a 0.41 a 0.33 0 2 4 6 8 10 12 Methanol Cold water Room temperature water Hot water Hexane Ethyl acetate IC 50 (m g/ m L) Extract type Antioxidant activity of the roots extracts against H2O2 (A) (B) (C) Figure 2. IC50 values of the leaves (A), stems (B) and roots (C) extracts of Portulacaria afra using methanol, cold water, room temperature water, hot water, hexane, and ethyl acetate against H2O2. Different small letters (a, b, c, and d) between the different groups indicate a statistical significance between the extract type (P<0.05). http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023http://www.herbmedpharmacol.com 115 Portulacaria afra: Scientific validation extraction due to its high polarity of 0.763 (15). This study conforms with the current literature review, as methanolic solvents detected the highest number of phytochemicals in all three plant parts. The hot water extracts showed the presence of five phytochemical groups in all three plant parts and indicated that hot water was able to extract a larger number of phytochemicals than both cold water and room-temperature water. This validates the use of hot water extractions performed traditionally. Hexane has the lowest polarity among all solvents used in this study (15) and the hexane extracts had the lowest presence of phytochemical groups in the leaves, stems, and roots. Coumarins are a phytochemical group, which displayed a high presence in the stems and leaves of the plant. This suggests a large biological potential of these plant parts because coumarins have shown to possess high antioxidant and antibacterial activities and have recently been associated with anti-cancer, anti-inflammatory, and analgesic properties (16). In addition, saponins, phenolics, and glycosides are phytochemical groups which appear frequently in the phytochemical screening. The high frequency of phytochemicals detected in P. afra coincides with the study performed by Tabassum et al (17). The leaves and stems showed a higher overall presence of Table 2. The zone of inhibition (mm) of the different plant extracts of Portulacaria afra against Escherichia coli and Staphylococcus aureus Plant part Extract Zone of inhibition (mm) E. coli S. aureus Stem Methanol 20 16 Cold water - - Room temperature water - - Hot water 20 15 Ethyl acetate - - Hexane 13 13 Leaves Methanol 19 16 Cold water - - Room temperature water - - Hot water 19 15 Ethyl acetate - 14 Hexane 15 18 Roots Methanol 12 15 Cold water - - Room temperature water - - Hot water 12 16 Ethyl acetate - - Hexane 15 17 photochemical groups. Phytochemicals are the bioactive compounds within plants, which produce medicinal properties (18). As such, the leaves may exhibit a larger range of health benefits than the stems and roots. Antioxidants are important in overall health because they stop the effects of free radicals, which attack the human body, and have been associated with the risk of chronic human diseases (19). The IC50 value represents the extracts’ ability to reduce oxidation activity by half. The lower the IC50 value, the greater the scavenging activity of the extracts and thus the greater the antioxidant activity (20). The DPPH scavenging assay is the commonly used antioxidant test because it is rapid and inexpensive; it also provides a general idea of the extracts’ ability to donate an electron, neutralising oxidative molecules (11). All IC50 values were below 10, with the exception of the hexane leaf extract. The high IC50 coincides with the low phytochemical presence in the hexane extracts. Hydrogen peroxide is not a very reactive molecule; however, it gives rise to a hydroxyl radical, which is highly reactive (12). Hydroxyl radicals have a highly oxidising nature, as such, are toxic to cells. Thus, a strong H2O2 scavenging activity is important in reducing oxidative substrates in the body. All IC50 values for the leaves, stem, and roots were below 5 mg/mL, except for the hexane stem extract (6.375 mg/mL) and cold-water stem extract which was above 10 (11.002 mg/mL). The strongest activity against H2O2 was displayed by the hot water leaves extracts (0.217 mg/mL). In the study performed by Tabassum et al (17), it was also determined that the antioxidant activity against DPPH was considered moderate whilst the plant showed strong antioxidant activity against other methods of antioxidant testing. The overall low IC50 values obtained in this study suggest the use of the plant as an antioxidant to improve overall health. The antibacterial activity was tested against E. coli, a prominent bacterial strain in South Africa (21) and S. aureus, a bacterial strain which is a common cause of skin diseases (22). The methanolic stem and hot water stem extracts displayed the largest zone of inhibition of 20 mm against E. coli. Thus, E. coli is susceptible to these extracts. The rest of the extracts, which displayed inhibition zones, varied between 12 and 19 mm and thus fall within the intermediate category. The cold water and room- temperature water extracts showed no zones of inhibition against either bacterial strain, indicating no bacterial activity. The antibacterial activity against S. aureus was considered intermediate with the zones of inhibition ranging from 13 to 18 mm. Due to S. aureus being one of the most common skin pathogens, the intermediate bacterial activity of P. afra against S. aureus may provide relief against diseases caused by S. aureus, such as skin and soft skin diseases (22). http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023 http://www.herbmedpharmacol.com116  Basson et al This study provides a good general assessment of the potential medicinal properties of the different plant parts of P. afra. The use of different solvents of varying polarities allowed for a more comprehensive analysis of the phytochemicals present. Future studies on P. afra should include antibacterial activity analyses against other common skin pathogens in order to further validate the use of P. afra to treat skin conditions. Conclusion In conclusion, P. afra possesses phytochemical groups, which contribute to the medicinal properties exhibited by the plant. The plant is rich in coumarins, making the plant a potential species of interest in anticancer studies. The plant extracts displayed high antioxidant properties against DPPH and H2O2, thus confirming the nutritional properties of the plant. The antibacterial properties indicated that the stem and leaf extracts might be used as antibacterial agents against E. coli and might provide relief against S. aureus infections. P. afra is a medicinal plant species with a high phytochemical presence and strong biological activities. Authors’ contributions DCB: Data collection, contribution to methodology, data analysis and interpretation, manuscript writing. TKT: Contribution to methodology, study supervision, manuscript edit and review. IMR: Conceptualisation, contribution to methodology, study supervision, manuscript edit and review. Conflict of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical considerations Ethical issues regarding authorship, data acquisition, review, and analysis have been carefully observed by authors. Funding/Support National Research Foundation (Reference number: TTK190401426371). References 1. Sofowora A, Ogunbodede E, Onayade A. The role and place of medicinal plants in the strategies for disease prevention. Afr J Tradit Complement Altern Med. 2013;10(5):210-29. doi: 10.4314/ajtcam.v10i5.2. 2. Jain C, Khatana S, Vijayvergia R. Bioactivity of secondary metabolites of various plants: a review. Int J Pharm Sci Res. 2019;10(2):494-504. doi: 10.13040/ ijpsr.0975-8232.10(2).494-04. 3. Ahn K. The worldwide trend of using botanical drugs and strategies for developing global drugs. BMB Rep. 2017;50(3):111-6. doi: 10.5483/bmbrep.2017.50.3.221. 4. Shara M, Stohs SJ. Efficacy and safety of white willow bark (Salix alba) extracts. Phytother Res. 2015;29(8):1112-6. doi: 10.1002/ptr.5377. 5. Mahomoodally MF. Traditional medicines in Africa: an appraisal of ten potent African medicinal plants. Evid Based Complement Alternat Med. 2013;2013:617459. doi: 10.1155/2013/617459. 6. van Wyk B, Wink M. Medicinal Plants of The World. Wallingford, Oxfordshire, UK: CABI; 2018. p. 12. 7. Van Jaarsveld E, Le Roux A. Portulacaria afra (L.) Jacq.: variability and distribution. Bradleya. 2021(39):138-52. doi: 10.25223/brad.n39.2021.a13. 8. De Wet H, Nciki S, van Vuuren SF. Medicinal plants used for the treatment of various skin disorders by a rural community in northern Maputaland, South Africa. J Ethnobiol Ethnomed. 2013;9:51. doi: 10.1186/1746-4269- 9-51. 9. Nawaz H, Shad MA, Rehman N, Andaleeb H, Ullah N. Effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals from bean (Phaseolus vulgaris) seeds. Braz J Pharm Sci. 2020;56:e17129. doi: 10.1590/s2175-97902019000417129. 10. Pandey A, Tripathi S. Concept of standardization, extraction and pre phytochemical screening strategies for herbal drug. J Pharmacogn Phytochem. 2014;2(5):115-9. 11. de Torre MP, Cavero RY, Calvo MI, Vizmanos JL. A simple and a reliable method to quantify antioxidant activity in vivo. Antioxidants (Basel). 2019;8(5):142. doi: 10.3390/ antiox8050142. 12. Keser S, Celik S, Turkoglu S, Yilmaz O, Turkoglu I. Hydrogen peroxide radical scavenging and total antioxidant activity of hawthorn. Chem J. 2012;2(1):9-12. 13. Jain I, Jain P, Bisht D, Sharma A, Srivastava B, Gupta N. Comparative evaluation of antibacterial efficacy of six Indian plant extracts against Streptococcus mutans. J Clin Diagn Res. 2015;9(2):ZC50-3. doi: 10.7860/ jcdr/2015/11526.5599. 14. Aborisade AB, Adetutu A, Owoade AO. Phytochemical and proximate analysis of some medicinal leaves. Clin Med Res. 2017;6(6):209-14. doi: 10.11648/j.cmr.20170606.16. 15. Abubakar AR, Haque M. Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes. J Pharm Bioallied Sci. 2020;12(1):1-10. doi: 10.4103/jpbs.JPBS_175_19. 16. Wu L, Wang X, Xu W, Farzaneh F, Xu R. The structure and pharmacological functions of coumarins and their derivatives. Curr Med Chem. 2009;16(32):4236-60. doi: 10.2174/092986709789578187. 17. Tabassum S, Ahmad S, Rehman Khan KU, Tabassum F, Khursheed A, Zaman QU, et al. Phytochemical profiling, antioxidant, anti-inflammatory, thrombolytic, hemolytic activity in vitro and in silico potential of Portulacaria afra. Molecules. 2022;27(8):2377. doi: 10.3390/ molecules27082377. 18. Bachar SC, Bachar R, Jannat K, Jahan R, Rahmatullah M. Chapter Seven - Hepatoprotective natural products. In: Sarker SD, Nahar L, eds. Annual Reports in Medicinal Chemistry. Vol 55. Academic Press; 2020. p. 207-49. doi: 10.1016/bs.armc.2020.06.003. http://www.herbmedpharmacol.com Journal of Herbmed Pharmacology, Volume 12, Number 1, January 2023http://www.herbmedpharmacol.com 117 Portulacaria afra: Scientific validation 19. Kim KH, Tsao R, Yang R, Cui SW. Phenolic acid profiles and antioxidant activities of wheat bran extracts and the effect of hydrolysis conditions. Food Chem. 2006;95(3):466-73. doi: 10.1016/j.foodchem.2005.01.032. 20. Olugbami JO, Gbadegesin MA, Odunola OA. In vitro evaluation of the antioxidant potential, phenolic and flavonoid contents of the stem bark ethanol extract of Anogeissus leiocarpus. Afr J Med Med Sci. 2014;43(Suppl 1):101-9. 21. Edokpayi JN, Odiyo JO, Durowoju OS. Impact of wastewater on surface water quality in developing countries: a case study of South Africa. Water Qual. 2017;10:401-16. 22. McCaig LF, McDonald LC, Mandal S, Jernigan DB. Staphylococcus aureus-associated skin and soft tissue infections in ambulatory care. Emerg Infect Dis. 2006;12(11):1715-23. doi: 10.3201/eid1211.060190. http://www.herbmedpharmacol.com