Assessment of safety and health-benefits of Citrus hystrix DC. peel essential oil, with regard to its bioactive constituents in an in vitro model of physiological and pathological skin conditions.

Citrus hystrix DC. peel essential oil (ChEO) has been reported to have many biological activities and is promoted for topical application. However, its effect on skin functioning has not yet been studied. This study aimed to evaluate its safety for normal skin cells as well as its potential activity against human melanoma. In addition, pro-inflammatory and anti-inflammatory activity was assessed, as well as inhibitory effects on important skin enzymes: tyrosinase and hyaluronidase. To better understand the complexity of the action of ChEO and the role of individual components, the study also included an evaluation of the activity of its main constituents: limonene, β-pinene, and terpinen-4-ol as well as two mixtures of these compounds, specially designed for this purpose: M1 in equal proportions (1:1:1) and M2 in proportions mimicking those found in the ChEO (2.6:1.7:1). The results showed that the essential oil of the C. hystrix peel, as well as its major components, was not cytotoxic to normal human skin cells representing various skin layers, namely keratinocytes (HaCaT), melanocytes (HEM), and fibroblasts (HDF), even after prolonged exposure of 72 h. The pro-inflammatory effect of ChEO, tested by caspase-1 activation in HaCaT cells, was less pronounced compared to limonene, β-pinene and terpinen-4-ol, and generally very low. On the other hand, its anti-inflammatory effect was noticeable and was half the potency of diclofenac sodium used as the reference drug. Although the anti-hyaluronidase activity of C. hystrix peel essential oil was lower compared to β-pinene and terpinen-4-ol, ChEO revealed fairly high anti-tyrosinase activity, with an enzyme inhibition level of over 80% at a concentration of 150-220 μg/ml. Studies on the potential anti-melanoma effect were performed using the LDH assay on three human cell lines of varying degrees of malignancy, namely WM793, A375, and HTB140. ChEO was more active than the tested single compounds or their mixtures. WM793 cells were found to be most susceptible, while HTB140 and A375 cells were slightly more resistant (IC 59, 88 and 70 μg/ml, respectively). Our data indicate that ChEO is safe for the skin and has a perspective as an anti-melanoma agent.