Elastase inhibitory, anti-inflammatory, and antioxidant potential of South African plants in a multi-faceted anti-ageing approach
Ageing is a complex biological process constituting states of oxidative stress and chronic inflammation that result in the abnormal degradation of the structural components within the dermis. These components, namely elastin and collagen are vital in conferring integrity to the structure of the skin. Oxidative stress due to hydrogen peroxide and superoxide, and chronic inflammation due to the action of cyclooxygenase – II and 5-lipoxygenase result in the release of proteases such as elastase which hydrolyze the structural components if the dermis undesirably. This results in the formation of wrinkles. In the present study eleven terrestrial and wetland plants (Aloe ferox, Aloe spicata, Annona senegalensis, Barleria albostellata, Bulbine frutescens, Bulbine latifolia, Commelina benghalensis, Elephantorrhiza elephantina, Equisetum ramosissimum, Persicaria senegalensis and Plantago longissima) were investigated based on traditional reports of their use in skin care for the treatment of eczema, psoriasis and wound healing, in which elastase has been implicated. Water and ethanolic extracts were prepared for each plant based on their relevance to traditional means of preparation and industry standards. The ability of Streptococcus mutans (ATCC 25175) to enhance the elastase inhibitory potential of these extracts through biotransformation was investigated using liquidstate fermentation. Bioautography revealed minor zones of inhibition for P. senegalensis (water), B. latifolia (water and ethanol), A. senegalensis (bark and twigs, ethanol), A. ferox (ethanol), C. benghalensis (ethanol), B. albostellata (ethanol and water), B. latifolia and B. frutescens. Despite this, the fermentation was considered successful as all extracts exhibited visual differences in the chemical profile of fermented and non-fermented counterparts when observed using thin layer chromatography. The elastase inhibitory potential of the extracts was investigated by measuring the binding affinity of porcine pancreatic elastase to N-succinyl-(Ala)3-p-nitroanilide in the presence of the respective extracts. A one-way analysis of variance (ANOVA) indicated that the half maximal inhibitory concentrations (IC50) ` iv were significantly different (P < 0.001) when comparing the non-fermented and fermented counterparts, confirming the success of the fermentation. The fermented ethanolic extract of P. senegalensis exhibited the lowest IC50 (16.89 ± 4.15µg/mL), however, a one-way ANOVA found this value to be not significantly different from the P. senegalensis ethanolic extract. The most noteworthy activity was observed for the non-fermented ethanolic extracts of A. senegalensis (leaves), A. senegalensis (bark and twigs) and P. senegalensis (leaves) respectively (79.66 ±2.58 µg/ mL, 85.30 ± 1.95 µg/ mL and 56.07 ± 0.85 µg/ mL) compared to the positive drug control, Ursolic acid (5.03±0.47 µg/mL). These lead extracts were selected for further investigation to determine their in vitro cytotoxicity, anti-inflammatory potential using cyclooxygenase-I, cyclooxygenase-II and 5-lipoxygenase, and their propensity to scavenge hydrogen peroxide and superoxide. In vitro cytotoxicity was conducted on the human keratinocyte cell line using the XTT viability reagent and found the extracts of A. senegalensis (leaves) and P. senegalensis (leaves) to exhibit no toxicity at the highest concentration tested (>400 µg/mL) while the bark and twigs of A. senegalensis exhibited moderate toxicity with an IC50 of 139.7±2.97mL. The cyclooxygenase-I and -II inhibitory potential was investigated through measuring the formation of prostaglandin E2 as a product of the inflammatory reaction in the presence of the test sample. Cyclooxygenase-I was included due to the crucial role it fulfils in many tissues of the body where it is constitutively expressed. Selective inhibitors of cyclooxygenase-II are thus desirable. P. senegalensis exhibited the most significant potential as an anti-inflammatory agent as no cyclooxygenase-I inhibition was observed, while 50% inhibition towards cyclooxygenase-II was observed at 2.27±2.04 µg/mL. The bark/twig extract of A. senegalensis exhibited 50% inhibition of cyclooxygenase-I at 1.90 x 10- 2±8.90 x 10-3 µg/mL, comparable to the positive control, Ibuprofen (6.3 x 10-2±8.79 x 10-4 µg/mL), and cyclooxygenase-II (5.28±0.62 µg/mL). The leaf extract of A. senegalensis exhibited 50% inhibition of COX-I at 1.19 ±0.21 µg/mL, significantly higher than the bark/twig extract, and 2.41±0.37 µg/mL towards cyclooxygenase-II, significantly lower than the bark/twig extract. ` v The ability of these extracts to inhibit 5-lipoxygenase was determined by measuring the ferrous oxidation of xylenol orange in the presence of the extracts using linoleic acid as a substrate. The most noteworthy inhibition of 5-lipoxygenase was observed for the leaf extract of A. senegalensis (3.02±9.75 µg/mL), highly comparable to Caffeic acid, the positive drug control (3.31±4.47 ± µg/mL). The bark/twig extract exhibited the second lowest IC50 5.19±5.35 µg/mL, followed by P. senegalensis (13.30±7.14µg/mL). Superoxide radicals were generated using alkaline DMSO and the propensity of the extracts to scavenge this radical spectrophotometrically measured using nitro blue tetrazolium chloride as an indicator. The propensity of the extracts to scavenge hydrogen peroxide was determined by measuring the ferrous oxidation of xylenol orange in the presence of the radical and extracts. P. senegalensis exhibited the highest propensity to scavenge the superoxide radical with an IC50 of 27.22±4.3 µg/L, followed by the bark and twig extract of A. senegalensis (43.29±8.89 µg/mL) and the leaf extract 70.38±5.90 µg/mL. The results obtained for P. senegalensis were highly comparable to the positive control, L-ascorbic acid (12.69±3.90 µg/mL). The best hydrogen peroxide scavenging activity was observed for the leaf extract of A. senegalensis (3.02±9.75 µg/mL) followed by the bark and twig extract of A. senegalensis (5.19±5.35 µg/mL) which were both significantly better than P. senegalensis (71.66±9.86 µg/mL) and Quercetin, the positive drug control (47.37±2.72 µg/mL). Two-way ANOVA analysis revealed significant differences in the values obtained for A. senegalensis leaf and bark/twig extracts in the current study, confirming the contingency of biological activity on plant part substitution. The findings from this investigation identify P. senegalensis as the lead extract to be further investigated in future studies as it exhibited the best inhibitory action towards elastase and cyclooxygenase-II, in addition to acting as a selective inhibitor. It also exhibited the highest propensity to scavenge superoxide radicals, thus acting on multiple targets.
Elastase, Cytotoxicity, Free radicals, Fermentation, Inflammation
Networked Digital Library of Theses and Dissertations