Twenty southern African plants were selected based on traditional use. The ethanol extracts were tested for their antimicrobial activity against P. acnes (ATCC 11827) [Propionibacterium acnes (Gilchrist) Douglas and Gunter deposited as Corynebacterium acnes (Gilchrist) Eberson]. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of the extracts were also determined.
Cytotoxicity on human macrophage cells (U937) was performed using the 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) reduction assay. The cytotoxicity was performed to ensure that the extracts are not toxic to human macrophage cells (U937) and to obtain a non-lethal range of concentrations to be tested in the anti-inflammatory assay. The anti-inflammatory potential was tested using IL-8 as a marker cytokine. This is a pro-inflammatory cytokine secreted when cells are stimulated with heat-killed P. acnes cultures.
During the investigation of the antimicrobial activity, four plant extracts were found to have significant inhibitory activity against P. acnes. Helichrysum odoratissimum showed the best activity with a minimum inhibitory concentration (MIC) at 7.81μg/ml. Clausena anisata, Rapanea melanophloeos and Helichrysum kraussii were also active with MICs at 31.25μg/ml, 15.63μg/ml and 125μg/ml, respectively. All MICs were selected based on PrestoBlue as the growth indicator. Helichrysum odoratissimum showed also showed the best antioxidant activity with an IC50 of 3.86 ± 0.24μg/ml. It was also reported to have the best selectivity index (SI) of 2.76 on U937 cells. Clausena anisata exhibited good antimicrobial activity and low toxicity on U937 cells with an IC50 at 74.46μg/ml and an SI of 2.38.
During the investigation of the synergistic activity of the extracts, the combination of 3.13μg/ml of Helichrysum odoratissimum and Helichrysum kraussii at 0.78μg/ml showed better antimicrobial activity of either of the plant extracts acting alone against P. acnes with a fractional inhibitory index (ΣFIC) of 0.42.
Clausena anisata was selected as the extract for the Interleukin-8 (IL-8) inhibition assay as it was shown to be traditionally used for treatment of many inflammatory disorders or symptoms. The inhibition of IL-8 by C. anisata in vitro when plant extract was added to stimulated U937 cells was low but there was some inhibition. The IL-8 protein concentration produced by U937 cells treated with 100μg/ml of heat-killed P. acnes was 840.52pg/ml. Low levels of IL-8 inhibition were observed when cells stimulated with P. acnes were treated with non-lethal concentrations of C. anisata extract. Treatment with 50μg/ml, 25μg/ml, 12.5μg/ml and 6.25μg/ml showed a decrease in IL-8 to 322.48 ± 0.07pg/ml, 365.98 ± 0.24pg/ml, 383.62 ± 0.08pg/ml and 409.52 ± 0.13pg/ml, respectively. The untreated cell control however, seemed to show spontaneous production of IL-8 with quantified as 488.76 ± 0.06pg/ml, making it difficult to analyse effects of other cell stimulants. This spontaneous release was also inhibited with the addition of C. anisata extract at 50μg/ml, 25μg/ml, 12.5μg/ml and 6.25μg/ml which showed IL-8 levels at 299.24 ± 0.13pg/ml, 357.82 ± 0.07pg/ml, 387.14 ± 0.12pg/ml and 388.74 ± 0.19pg/ml, respectively
The use of many polyherbal formulations is becoming popular practice. Due to the variety of symptoms observed with acne vulgaris it would be beneficial to investigate mixtures of plants showing good antimicrobial, antioxidant, anti-inflammatory and anti-pathogenic activity as potential treatments for acne vulgaris.
This is the first report of the synergistic activity of Helichrysum odoratissimum and Helichrysum kraussii crude ethanol extracts used in a synergistic combination. Also the production of hyaluronidase by the tester strain P. acnes (ATCC 11827). The combination and some other active plants shown in the study should be further investigated as possible novel medicinal agents against acne vulgaris.