Antioxidant Propertity Of Citrus Essential Oil And Research On The Protective Effect On Skin Fibroblast Oxidative Damage

This study was to identify and analyze constituents differences of four citrus peel oils, namely Valencia orange, ponkan, lemon and mandarin. High-speed counter-current chromatography（HSCCC） was firstly adopted to separate citrus essential oil components. The antioxidant properties of obtained oils and their main components were tested using two antioxidant assays. Besides, we used the Hydrogen peroxide（H₂O₂）-induced oxidative damage in human dermal fibroblasts as a model to study the protective effects of citrus essential oil and high-active compounds, hoping to provide some support for their application in skin care products.1. Extraction and separation of peel oil and differences analysis of volatile constituents Hydrodistillation and pressing were used to extract oils from fruit peels. Oil constituents were identified using Gas chromatography-mass spectrometry（GC-MS） and the differences were analyzed.（1） Hydrodistillation yielded more oils than pressing with greater proportions of oxygenated compounds. A total of 48 compounds were identified in all oil samples with difference occurred based on species and extraction methods. α-pinene、β-pinene、limonene and farnesene existed in four citrus species and each variety possessed its unique marker compounds, which can be used to differentiate from others.（2） HSCCC was adopted to separate oil components of ponkan essential oil, using a two-phase solvent system consisting of petroleum ether-acetonitrile-acetone（5:3:2 v/v/v）. β-myrcene、limonene and γ-terpinene were isolated, with purity improved from 3.8% to 64.9%, from 74.9% to 87.1%, from 9.7% to 50.5%, respectively.2. Antioxidant properties of citrus essential oil and their main components Two in-vitro assays, 1,1-diphenyl-2-picrylhydrazyl（DPPH） free-radical scavenging assay and the thio-barbituric acid reactive species（TBARS） assay were applied to evaluate the antioxidant properties of citrus essential oils and their main components. On the whole, citrus essential oils exhibited concentration-dependent trend on DPPH free-radical scavenging and lipid peroxidation inhibition abilities, with ponkan oil behaving the best. The antioxidant activities of citrus essential oils were probably related to the ratio of hydrocarbon against oxygenated compounds. The EC50 values of oils in TBARS assay were much lower compared to DPPH assay. Two terpenes, namely γ-terpinene and terpinolene in the concentration of 100 μL/m L, scavenged DPPH free radicals for 75.8% and 64.2%, inhibited lipid peroxidation for 94.8% and 88.2%, respectively. Showing antioxidant effect close to BHT, these two constituents were considered as the highly active compounds in citrus essential oil.3. Research on the protective effect of citrus essential oil on skin cell oxidative damage（1） The results showed that oils in higher concentration（ > 0.3 μL/m L） dramatically declined cell survival rate and oils in lower concentration demonstrated varying degrees of proliferating effects. All tested authentic compounds showed cell proliferation effect on oxidative damaged fibroblasts except for α-pinene and 3-carene showing strong toxic effects. As a result, limonene and γ-terpinene were decided to continue with further study, in consideration of their considerate content in citrus oils and high antioxidant activity.（2） Exogenous H₂O₂ was used to establish the human dermal fibroblasts oxidative injury model. MTT assay was used to study the effect of H₂O₂ on cell viability, with result showing decreased cell viability when exposed to H₂O₂ on a concentration-dependent manner. The proper conditions for cell oxidative injury were confirmed with 0.65 m M/ 0.33 m M H₂O₂ treated for 6h.（3） Oils pre-treatment possessed a certain amount of protection against cell oxidative injury, with cell viability increased from 54.9%（H₂O₂ treated） to 58.8-71.9%. γ-terpinene（0.1 μL/m L） co-incubation with H₂O₂ exhibited the best protection effect with cell viability recovered to 83.1% from 24.5% of the H₂O₂-treated cell group. Ponkan oil and the active components inhibited lipid peroxidation in human dermal fibroblasts, with increased SOD, GSH-Px, CAT activities and lower MDA level. Our results implied the probable mechanism involved in antioxidant protection could be through directly scavenging reactive oxygen and enhancing activity of endogenous antioxidant enzymes in skin fibroblasts.