Evaluation of anti-ageing properties of Moringa oliefera Lam., Centella asiatica (L.) Urban, Clitoria ternatea L. and Cosmos caudatus Kunth. for potential application as cosmeceuticals.

Background.
In 2010, the launch of the Malaysian Economic Transformation Programme (ETP) changed the agriculture sector in Malaysia from “traditional” agriculture into an agribusiness sector. The National Key Research Area (NKRA) in the agriculture sector focuses on raising the status of local herbs for potential commercialisation. Application of local herbs in the cosmetics industry seems to be a promising investment with the current high demand for natural and organic cosmetics worldwide. Given this scenario, this study focused on investigating the anti-ageing properties of selected medicinal plants which are considered as underutilised in Malaysia, as scientific evidence to support their traditional and cosmetic usages is lacking.

Aim.
This project aims to add agricultural and commercial value to the selected medicinal plants; Moringa oliefera Lam., Centella asiatica (L.) Urban, Clitoria ternatea L. and Cosmos caudatus Kunth. which were investigated for their anti-ageing properties (antioxidant, anti-collagenase, anti-elastase and anti-tyrosinase activities) and their protective effects against oxidative-stress induced by hydrogen-peroxide and UV radiation in vitro.

Methods:
Two extracts per plant species were prepared using water and 70% ethanol to mimic either the traditional methods or industrial extract preparations. The total phenolic content (TPC) was determined by the Folic Ciacalteau method. High-performance liquid chromatography-mass spectrophotometry was performed to characterise the individual components of the extracts and to chemically authenticate the plant samples. Antioxidant activities were determined using diphenyl-picryl hydrazine (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS). The results are expressed as Trolox equivalent (mg/g dried extract) and IC50 (μg/mL). The anti-collagenase and anti-elastase activities were measured by fluorescence assays and anti-tyrosinase activity using a colourimetric assay. The results are expressed as percentage inhibition (%) compared to control (untreated enzymes). HaCaT cells (human keratinocytes) were used to investigate the protective effect against H2O2-induced cytotoxicity by measuring the percentage cell viability of HaCaT treated with plant extracts compared with the control (untreated HaCaT). The protective effect against UV radiation was evaluated using polymerase chain reaction (PCR) targeting mitochondrial DNA (mtDNA) damage as a biomarker.

Results:
In the antioxidant assays. M. oliefera ethanol extract (MOE) showed the highest antioxidant compared with other extracts, with 941 μM/mg, 135 μM/mg, 312 μM/mg of Trolox equivalent (TE)/mg dried extract in the ABTS, DPPH and FRAP assays respectively. Other plant extracts also demonstrated antioxidant activities, but at different levels between assays. The correlation analysis of TPC vs. antioxidant activities of all plant extracts showed a strong positive correlation (R=0.74), which suggests that phenolics might be responsible for the observed activities.

The collagenase, elastase and tyrosinase assays showed that C. caudatus and C. ternatea extracts were able to inhibit the enzymes in a dose-dependent manner. At the highest concentration tested (1 mg/mL), C. caudatus water extract (CCW) inhibited collagenase, elastase and tyrosinase activities by 48.7%, 64% and 72.6% respectively, while its ethanolic extract (CCE) inhibited collagenase and elastase by 46.7% and 26% respectively. C. ternatea extracts (CTW and CTE) inhibited collagenase, whereas CTE showed higher inhibition compared with CTW (70.1% vs. 82.1% respectively). Both extracts also inhibited elastase with 28% and 24 % inhibition for CTW and CTE respectively. C. asiatica extract showed collagenase inhibition, where its ethanol extract (CAE) inhibited collagenase at 77.4%. Meanwhile, M. oliefera extracts exhibited very low (6-22%) or no activity against the enzymes.

In the H2O2-induced cytotoxicity assay, only the water extracts of M. oliefera (MOW), C. asiatica (CAW) and C. ternatea (CTW) demonstrated significant protective effects (p<0.05). Such activity was absent in the ethanol extracts of the plant species, mainly due to the lower concentrations that had to be used since the ethanol extracts were more toxic to HaCaT compared with the water extracts. Similar results were observed in the UV-induced mtDNA damage assay where the water extracts showed significant protective effects compared to the ethanol extracts. In the UV filter experiment, the water extracts of C. asiatica, C. ternatea, C. caudatus (CAW, CTW, CCW) and MOE showed significant protective effects against UV-induced mtDNA damage (p <0.05). The UV spectra of each plant extract demonstrated UV absorbing properties, which may explain the protective effect observed. Other than that, the antioxidant properties of the extracts could also play a role by lowering UV-induced ROS level in the cells. This is further supported by the comparison of potency, which showed that MOE exerted protective effects in both UV filter and pre-incubation experiments, where MOE was shown to have the highest antioxidant activities in comparison with the other extracts in the DPPH, ABTS and FRAP assays.

Conclusion.
The findings in this study demonstrate that the prepared extracts of the selected medicinal plants have high potential to be developed into antioxidant, anti-wrinkle, hypopigmentation agents and UV-filters. Significant protective effects against the targets of interest were observed that may provide a scientific explanation for the putative traditional and commercial use of the plants. These findings should therefore increase the value of these underutilised medicinal plants.