Abstract
Treatment of wounds using medicinal plants is a common practice in South Africa. Several plants from the South African flora are used in the treatment of a wide range of wounds such as septic wounds, sores, burns, ulcers, boils, cuts, circumcision wounds amongst others. However, the extent to which this flora is explored for efficacy as wound healing agents is not yet known. It is for this reason that the study collates an inventory of indigenous medicinal plants and plant combinations used to treat wounds in South Africa. A list of indigenous medicinal plants and plant combinations used in the traditional system of South Africa in the management and treatment of wounds was collated through extensive literature review. The investigated antimicrobial, antioxidant, anti-inflammatory, wound healing activity, toxicity, and phytochemical studies were also recorded.
A total of 519 indigenous plant species distributed amongst 110 families, and 317 genera were collated in the inventory, with family Asteraceae and genus Helichrysum identified as the top representatives. A closer analysis revealed that isolation and/ or chemical characterization studies (324 species; 62.43%) were the most prevalent followed by the antimicrobial activity
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(318 species; 61.27%), toxicity (287 species; 55.30%), antioxidant activity (230 species; 44.32%), anti-inflammatory activity (196 species; 37.76%), with the wound healing activity (31 species; 5.97%) being the least studied pharmacological aspect. Additionally, a total of 25 plant combinations were identified of which, except for the antimicrobial activity, none had been evaluated for their antioxidant, anti-inflammatory, wound healing activity, and toxicity.
Six scientifically unexplored indigenous medicinal plants species, Agapanthus inapertus P. Beauv. (root), Cheilanthes hirta Sw., Crassula capitella Thunb., Eriospermum flagelliforme (Baker) J. C. Manning, Euphorbia clavarioides Boiss and Pelargonium alchemilloides (L.) L'Hér., two plant combinations Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. + Dicoma anomala Sond., and Senecio asperulus DC. + Cotyledon orbiculata L., selected from the inventories were assessed for their anti-microbial, antioxidant, anti-inflammatory, wound healing activity, cytotoxicity, and chemical characterization in this present study.
The water, 70% aqueous ethanol (EtOH), 50% aqueous methanol (MeOH), dichloromethane (DCM), and petroleum ether (PE) extracts from the six species, the water and 50% aqueous methanol extracts from the two plant combinations were tested against bacteria and fungi implicated in wound infection. The microdilution assay which measures the minimum inhibitory concentration (MIC), with Neomycin and Amphotericin B as positive controls for the antibacterial, and antifungal activity, respectively was used. Most of the extracts displayed very weak activity (MIC>0.625 mg/mL). However, a few of the plants demonstrated significant growth inhibition (MIC <0.1 mg/mL) against some microbial strains. This includes C. hirta which demonstrated significant growth inhibition (MIC = 0.04 mg/mL) against Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus mirabilis. The C. capitella DCM extract significantly inhibited the growth of P. aeruginosa at MIC of 0.04 mg/mL. The P. alchemilloides water extract significantly inhibited the growth of
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Enterococcus faecalis and B. subtilis at MIC 0.04 mg/mL. Significant inhibition against Aspergillus niger demonstrated by the C. hirta 70% aqueous ethanol.
The different extracts were further investigated for their free radical scavenging and antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), ferric reducing antioxidant power (FRAP), and β-carotene-linoleic acid model system (β-CLAMS) assays. Polyphenols were also quantified using standard methods. The total phenolics, flavonoids and proanthocyanidin content quantified ranged from 3.57 ± 0.07 – 30.17 ± 0.18 mg GAE/g, 23.32 ± 0.50 – 96.12 ± 0.14 mg CE/g, and 8.22 ± 0.10 – 71.13 ± 2.17 mg CE/g. Most of the extracts displayed significant DPPH radical scavenging activity comparable to that of BHT (IC50 = 0.05 ± 0.10 mg/mL) and ascorbic acid (IC50 = 0.23 ± 0.12 mg/mL). A total of fourteen extracts displayed excellent nitric oxide radical scavenging effects better than that of BHT (IC50 = 0.65 ± 0.07 mg/mL), and ascorbic acid (IC50 0.41 ± 0.27 mg/mL). A total of ten extracts exhibited percentage ANT that is significantly comparable to that of BHT (%ANT = 82.35 ± 4.11, ORR value = 0.17 ± 0.04). A dose-dependent ferric reducing power that increased with an increase in concentration was displayed by all the tested extracts with the C. capitella water extract recording significant reducing power.
The acetone and water extracts from the six plants and two plant combinations were evaluated for their anti-inflammatory activity against the xanthine oxidase (XO), 15- Lipoxygenase (15-LOX), cyclooxygenase-1 (COX-1), and cyclooxygenase -2 (COX-2) enzymes. Several extracts displayed significantly high anti-xanthine activity, outperforming the positive control, quercetin, 101.65 ± 0.57 μg/mL. Out of all the tested extracts, only the S. asperulus + C. orbiculata water polyherbal extract significantly inhibited the 15-LOX enzyme with IC50 value of 1.04 ± 0.09 μg/mL which was comparable to that of the standard NDGA (0.45 ± 0.01 μg/mL). Majority of the tested extracts had some degree of protection of the COX-1 enzyme activity. The acetone extracts from C. capitella (3.16 ± 0.06 μg/mL), E. flagelliforme (1.13 ±
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0.09 μg/mL), E. clavarioides (1.84 ± 0.18 μg/mL), P. alchemilloides (1.13 ± 0.14 μg/mL), and P. prunelloides + D. anomala polyherbal (1.19 ± 0.06 μg/mL), and the E. clavarioides water extract (1.18 ± 0.11 μg/mL) displayed COX-2 activity comparable to that of standard aspirin, 1.11 ± 0.03 μg/mL. The E. flagelliforme acetone, P. alchemilloides acetone and the E. clavarioides water extracts displayed potential as dual COX-2/15-LOX inhibitors.
The extracts with excellent anti-inflammatory activity were further investigated for cell viability, cytotoxic as well as wound healing potential on WS1 human skin fibroblast cells. The effect of the extracts on cell viability was evaluated using the Adenosine triphosphate (ATP) luminescence cell viability assay, whilst the potential extracts cytotoxic was determined using the Lactate Dehydrogenase (LDH) cytotoxicity assay. All cells treated with the tested extracts displayed good cell viability. The extracts did not display any signs of toxicity after 48 h except for the C. orbiculata + S. asperulus water extract. Non-toxic extracts at concentration 50 μg/mL were further evaluated for their ability to promote cell migration in vitro through measuring the cells’ migration rate, percentage wound closure and assessing the time-lapse micrographs at 0, 24 and 48 h using the in vitro wound healing scratch assay. All test extracts displayed a significant degree in promoting cell migration with the wounded cells treated with P. prunelloides + D. anomala acetone extract displaying complete wound closure at 48 h. All the tested extracts had no toxicity on WS1 human skin fibroblast cells at 24 and 48 h post-wounding.
Overall, the study revealed that there is still a huge gap for scientific validation of medicinal plants and plant combinations used in South Africa to treat wounds in respect to their wound healing properties. The study also suggest that several plants are still consumed without their toxicity knowledge. The wound healing activity exerted by the investigated plants could be attributed to the quantified polyphenols which possess antioxidant and anti-inflammatory effects. Findings from the in vitro wound healing study lends credence to the folkloric use of
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the investigated plants and plant combinations as wound healing agents. However, further studies elucidating the extracts’ molecular mechanism of wound healing are of significance.
Keywords: Indigenous medicinal plants, Antimicrobial, Antioxidant, Xanthine oxidase, Cyclooxygenase, Lipoxygenase, Fibroblasts, Cell viability, Cytotoxicity, Adenosine triphosphate, Lactate dehydrogenase, Wound healing, Cell migration, Scratch assay, South Africa.