Faculty of Applied and Computer Science

Permanent URI for this community

https://hdl.handle.net/10352/31

Browse

Browsing Faculty of Applied and Computer Science by Subject "2³ factorial design"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    The effect of Carpobrotus edulis and Cotyledon orbiculata on the migration, proliferation and differentiation of keratinocytes in vitro
    (Vaal University of Technology, 2020) Terblanche, Unisa; Ssemakalu, Dr. C.C.; Pillay, Prof. M.
    Cutaneous wounds are an epidemic that affects millions of people worldwide. Healing and regeneration of wounds is a complex process that involves several molecular and cellular pathways. Traditional healers have used plants for the healing and regeneration of cutaneous wounds for centuries. In South Africa, two species, Carpobrotus edulis and Cotyledon orbiculata, have been used in traditional medicine for the treatment of various skin conditions and wounds. There is limited empirical evidence on the effectiveness of some of these commonly used plants on wound healing. The aim of this study was to use a factorial design to develop a protocol for optimal extraction of bioactive compounds from Carpobrotus edulis and Cotyledon orbiculata using water and methanol as extractants and evaluate the effectiveness of these extracts on the migration, proliferation and differentiation of keratinocytes in vitro. To achieve this the study followed a 2 step integrated approach. In the first part of this study, 23 full factorial designs were employed to investigate the effect of different extraction parameters on the yield of crude extracts obtained from the leaves of Carpobrotus edulis and Cotyledon orbiculata, respectively. The impact of these parameters on the phytochemical constituents were assessed using standard colorimetric tests, whereas the Folin-Ciocalteau and Aluminium Chloride colorimetric assays were used to determine the impact on the total phenolic (TPC) and total flavonoid (TFC) contents, respectively. This was followed by the utilisation of two cell viability assays, CellTiter® Blue (CTB) and Crystal violet (CVS) staining, to assess the effect of these rude extracts on an immortalised human skin keratinocyte (HaCaT) cell line, in vitro. The response values from these assays were used in a 25 factorial design to develop the best-fit regression model to predict which aqueous and methanolic extract (for each plant) expressed the highest proliferative potential. A pH of 9 for 72 h at 40°C proved to be the best extraction conditions for the aqueous extraction of Carpobrotus edulis, resulting in a yield of 31.03% whereas the methanolic extract produced a maximum yield of 64.21% at a pH of 9 for 168 h at 40°C. For Cotyledon orbiculata, the highest yield of 44.13% was achieved for the aqueous extract at a pH of 9 for 4 h at 60°C whereas 50% methanol for 168 h at 25°C resulted in a maximum yield of 33.64% for the methanolic extraction. Preliminary tests of the plant leaf extracts indicated the presence of reducing sugars, phenols, tannins, flavonoids, cardiac glycosides, diterpenes, phytosteriods and triterpenoids in both species. The highest TPC and TFC for the aqueous extracts of Carpobrotus edulis were 16.43 ± 0.07 mg GAE/g and 7.42 ± 0.53 mg QE/g respectively, whereas values of 18.92 ± 0.23 mg GAE/g and 6.88 ± 0.44 mg QE/g were obtained for the methanolic extracts. The highest TPC and TFC for the aqueous extract of Cotyledon orbiculata were 38.79 ± 1.07 mg GAE/g and 2.60 ± 0.04 mg QE/g, respectively. For the methanolic extract, maximum values for TCP and TFC were 56.49 ± 1.79 mg GAE/g and 12.35 ± 0.21 mg QE/g, respectively. The aqueous extract of Carpobrotus edulis increased the viability of the HaCaT cells by approximately 67% ± 3.93%, whereas a similar extract of Cotyledon orbiculata only increased cell viability of HaCaT by 29% ± 0.99%. Based on the experimental observations and developed regression models, the extraction parameters that predicted the highest proliferative potential across both assays were a pH of 5 for 168 h at 40°C for the aqueous extract of Carpobrotus edulis, and a pH of 5 for 168 h at 25°C for the methanolic extract. For the aqueous extract of Cotyledon orbiculata, a pH of 7 for 4 h at 30°C predicted the highest proliferative potential whereas best results for the methanolic extract was predicted to be achieved with full strength methanol, for 168 h at 25°C. In the second part of this study, these optimised extraction models were used to assess the in vitro wound healing ability of Carpobrotus edulis and Cotyledon orbiculata as a measure of its ability to promote re-epithelialisation. Cytotoxicity of the extracts that was assessed morphologically and with the Lactate Dehydrogenase (LDH) Cytotoxicity assay showed that the aqueous extract of Carpobrotus edulis showed little to no morphological changes at concentrations ranging from 100 to 1000 μg/mL which correlated with the non-significant (p > 0.05) LDH activity whereas the methanolic extract exhibited morphological changes at all tested concentrations. On the contrary, both the aqueous and methanolic extracts of Cotyledon orbiculata induced morphological changes of the HaCaT cells at most concentrations. The significant (p < 0.05) increase in LDH activity for the aqueous (8.4% ± 2.54% to 26.9% ± 2.38%) and methanolic extract (5.3% ± 1.14% to 10.6% ± 1.64%) verified this result. The proliferation of HaCaT cells after treatment with the crude extracts, measured with the sulforhodamine B (SRB) assay and the xCELLigence Real-Time Cell Analysis (RTCA) system, showed a significant reduction in doubling times of the cells when they were treated with the aqueous extract of Carpobrotus edulis. The methanolic extracts of the species showed some increase in proliferation rates with 500 to 1000 μg/mL after 24 h of treatment, but this was not supported with the RTCA system. For the Cotyledon orbiculata, the SRB assay showed that the aqueous extract at concentrations of 1, 10 and 25 μg/mL significantly increased the proliferation of HaCaT cells after 48 h of treatment; however, the RTCA system did not support this proliferation rates or doubling times. For the methanolic extract, an antiproliferative effect was observed for all treatments 24 h after exposure. This was supported by the absence of any evidence of a proliferative effect or decreased doubling time using the RTCA system. The migration potential of HaCaT cells, evaluated using an in vitro scratch assay and the RTCA system, showed that the aqueous extract of Carpobrotus edulis increased the wound closure by 31% compared to the untreated cells between concentrations of 1000 to 2000 μg/mL. The methanolic extract increased the migration rate by 17% only at 10 μg/mL. On the contrary, the aqueous extracts of Cotyledon orbiculata decreased the rate of migration by 10% and 30% for both the 10 μg/mL and 50 μg/mL concentrations, respectively. The methanolic extract also decreased migration rates for the 10 μg/mL and 50 μg/mL by 38% and 37%, respectively. The expression of selected cytokines by migrating HaCaT cells were assessed using a Luminex MAP® cytokine kit. Cytokine expression for Carpobrotus edulis showed that both solvent extracts did not significantly influence the expression of TNF-α, IL-6, and IL-8. It was, however, found that the expression of TGF-α and VEGF were reduced for both solvent media. Cytokine expression was not investigated for Cotyledon orbiculata as the plant showed an inhibitory effect on migration for both aqueous and methanolic extracts. The evaluation of the effect of the extracts on keratinocyte differentiation, measured by the expression of differentiation markers K1,10 and involucrin showed either no change, or a reduction in K1,10 expression for both aqueous and methanolic extracts for both species. Involucrin expression was inconclusive for both plants. The results of this study showed that within a concentration range of 1000 and 2000 μg/mL, the aqueous extract of Carpobrotus edulis promoted keratinocyte proliferation and migration. This supports the ethnomedicinal use of the plant for the treatment of cutaneous wounds. While Cotyledon orbiculata is also used as folk medicine for wound healing, this study found no evidence to support such a claim.