Introduction Camellia japonica is a native plant grown in China, Korea and Japan. Tsubaki (C. japonica) oil has a long history of cosmetic usage traditionally in the oriental region as a protectant, to keep health of skin and hair, and as a soothing agent. It has been reported that Tsubaki oil possesses anti-inflammatory activity (Kim et al., 2011), anti-ageing properties and a skin barrier function (Jung et al., 2007). It particularly shows the capacity to inhibit matrix metalloproteinase MMP-1 and to induce the synthesis of human type I procollagen. The present study was designed to fully describe the composition of Tsubaki oil (fatty acid profile and unsaponifi able matter) and highlight its moisturizing properties thanks to a double-bind placebo-control clinical trial. Due to its sensitivity to the oxidation, some Rancimat® experiments were performed to stabilize this oil. A new natural antioxidant solution, Olei-Protect™, was developed to protect Tsubaki oil in order to ensure its integrity and preserve its biological activities on skin. Materials & methods Fatty acid profile: Determination of fatty acid distribution is performed by a GC 3800 VARIAN equipped with CombiPal autosampler and a FID detector. Identification uses a Supelco® 37 Component FAME Mix (Sigma-Aldrich, ref. CMR47885) as referenced standards, after trans- esterification of the triglycerides, followed by conversion into the methyl ester. Phytosterol and tocopherol content: Analysis of tocopherols and sterols was performed with a VARIAN 4000 GC/MS equipped with a split injector and a ion trap mass detector. A 0.25-mm (i.d.) x 30-m bonded-phase 5% phenylmethyl capillary column with a 0.25-mm film thickness (VF- 5MS column) was used. The identification of sterols and tocopherols was confirmed using both a Mass Detector and the retention times of standards. Rancimat® experiments: A Metrohm Rancimat model 679 (Metrohm Instruments, Herisau, Switzerland) was employed for OSI determination and stabilisation experiments following AOCS Official Method Cd 12b-92 AOCS 1997. The tests were carried out with 5 g of oil at 110°C (for OSI determination) or 100°C (for stabilisation experiments) and with an air flow of 10 L.h-1. The volatile degradation products were trapped in distilled water, increasing the water conductivity. The oxidative stability index was the time necessary to reach the conductivity curve inflection point. Clinical study: • Double blind vs placebo under dermatological control. Volar forearms of 24 females subjects, with dry skin (age 50-60, corneometer value ≤ 40). Assessment of skin moisturizing property by measurement of Skin capacitance with a corneometer after application of 5% Tsubaki oil in neutral gel-cream. • Short term Clinical study under dermatological control. Volar forearms of 32 females subjects, with dry skin (age 40-60, corneometer value ≤ 40). Evaluation of product efficacy by self-assessment questionnaire after 2 weeks treatment, twice daily applied. Test product : 5% Tsubaki oil in neutral gel-cream Conclusion Altogether, these data highlight different benefits of Tsubaki oil on topical application as soothing, moisturizing and anti-wrinkle properties. However, due to its composition, this oil is paradoxically sensitive to oxidation and needs to be protected using antioxidant agents. In order to ensure the integrity of Tsubaki oil and preserve its biological activities, Naturex has developed new natural antioxidant solutions to stabilize fragile oils. As regards Tsubaki oil, the innovative Olei-protect™ process improves the resistance to oxidation up to 29 times. In a double-bind placebo-control clinical trial conducted by Naturex, Tsubaki oil at 5% in a gel-cream increases skin hydration by 14.4% vs. placebo. Alexis LAVAUD, Caroline SZEWEZYK, Marjolaine MEYER, Yohan ROLLAND, Marc ROLLER and Antoine BILY Naturex SA, Site d’Agroparc, 84911 Avignon Cedex 9, France a.lavaud@naturex.com Tsubaki ( Camellia japonica ) cold-pressed oil: composition, protection from oxidation and moisturizing properties References Kim S, Jung E, Shin S, Kim M, Kim YS, Lee J, Park D. Anti-inflammatory activity of Camellia japonica oil. BMB Rep. 2012,45(3):177-82. Jung E, Lee J, Baek J, Jung K, Lee J, Huh S, Kim S, Koh J, Park D. Effect of Camellia japonica oil on human type I procollagen production and skin barrier function. J Ethnopharmacol. 2007,112(1):127-31. Palmitic acid C16:0 Stearic acid C18:0 Oleic acid (ɷ9) C18:1 Linoleic acid (ɷ6) C18:2 Linoleic acid (ɷ3) C18:3 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Olive Palm Soybean Sunflower Tsubaki 40 30 20 10 C16:0 palmitic C18:0 stearic C18:1n9c oleic C18:2n6c linoleic C20:1 gadoleic 40 mVolts 30 20 10 0 -4 Figure 1: Tsubaki Oil - From growing to oil processing industry Figure 2A: Comparison of the fatty acid profile between conventional vegetable oils and Tsubaki oil. The main fatty acids are oleic acid C18:1 (>80%), linoleic acid C18:2 (1-5%) and palmitic acid C16:0 (4-12%). Figure 2B: GC Analysis revealed a relative low content in tocopherols (190 mg/kg) and sterols content (470 mg/kg). Interestingly, Tsubaki oil is highly concentrated in oleic acid (ɷ9) and is composed by uncommon phytosterols, a-spinasterol and 24-methylcholest-7-enol. The analysis of the unsaponifi able matter allow ensuring the botanical origin of Naturex Tsubaki oil. Vitamin E content Value α-tocopherol 191 mg / kg γ-tocopherol 3 mg / kg Phytosterols composition Value Cholesterol 3.0 % Brassicasterol 3.0 % Campesterol 0.9 % 24-methylcholest-7-enol 2.9 % α-spinasterol 38.3 % Sitostanol 0.8 % δ-5-avenasterol 3.3 % δ-5,24-stigmastadienol 1.8 % δ-7-stigmasterol 37.7 % δ-7-avenasterol 8.2 % Total content 470 mg / kg Flower Fruit Mature fruit and seeds Dry seeds Oil Figure 4A: Double blind vs placebo under dermatological control. Volar forearms of 24 females subjects, with dry skin (age 50-60, corneometer value ≤ 40). Assessment of skin moisturizing property by measurement of Skin capacitance with a corneometer after application of 5% Tsubaki oil in neutral gel-cream. ***p < 0.001 versus placebo group. Figure 4B: Short term Clinical study Under dermatological control. Volar forearms of 32 females subjects, with dry skin (age 40-60, corneometer value ≤ 40). Evaluation of product efficacy by self-assessment questionnaire. Test product: 5% Tsubaki oil in neutral gel-cream. Placebo Tsubaki oil a.u. Skin capacitance + 14,4 % 0h 1h 2h 0 5 10 15 20 25 30 35 40 94% of volunteers agree that “the test product moisturizes the skin” 81% of volunteers declare that “the test product has good caring properties” A A B B Figure 3A: The Rancimat® method is a test used to measure the stability of oil and fat during an accelerated oxidation test. Fat oxidation is decomposed in 2 steps: the initiation (the oxidation is slow and does not influence the quality product) and the propagation (the oxidation is fast and the product deteriorates). The longer the initiation step is, the more stable the product is. Figure 3B: Tsubaki oil is sensitive to oxidation, which was confirmed by Rancimat® experiments (Induction time = 3.6h ± 0.2h – T°C = 110°C, Q = 10L/h). The use of Olei-Protect™ solution allow increasing the protection factor up to 29 times. All experiments were done in triplicate (n=3). Figure 3C: Two main Olei-Protect™ solutions were developed using natural vitamin C. Olei-Protect™EC is a 100% clear, oil soluble Vitamin C solution, using a natural emulsifier. Olei-Protect™ C, micro-dispersible Vitamin C solution, using a physical process developed by Naturex. Olei-Protect™ EC is a 100% clear, oil soluble Vitamin C solution Olei-Protect™ C, micro-dispersible Vitamin C solution Initiation Propagation Time Conductivity Induction time Control, no oxydant 0.1% natural tocopherol 90% 0.05% Olei-Protect C 1% Olei-Protect EC Protection factor 0 5 10 15 20 25 30 HO HO HO OH O O H A B C lipophilic tail hydrophilic head posterNAToleis_Tsubaki_841x1189.indd 1 03/12/13 09:39 View publication stats View publication stats