cosmetics

Article Antioxidant and Moisturizing Effect of Camellia assamica Seed Oil and Its Development into Microemulsion

Wantida Chaiyana 1,2,* ID , Pimporn Leelapornpisid 1,2, Jaroon Jakmunee 3,4 ID and Chawalit Korsamphan 5

1 Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] 2 Innovation Center for Holistic Health, Nutraceuticals, and Cosmeceuticals, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand 3 Department of Chemistry and Research Laboratory for Analytical Instrument and Electrochemistry Innovation, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] 4 Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 5 Agricultural Scientist, Highland Research and Training Center, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +66-84-616-5614

 Received: 4 June 2018; Accepted: 26 June 2018; Published: 1 July 2018 

Abstract: The present study aimed to investigate the fatty acid content, and antioxidant and moisturizing effect of Camellia assamica seed oil (CA). Additionally, microemulsions containing CA were also developed for topical use. The antioxidant activity of CA and two commercial Camellia oleifera seed oils were investigated by means of 1,1-diphenyl-2-picrylhydrazy radical (DPPH) assay and lipid peroxidation by ferric thiocyanate method. Moreover, the in vitro skin moisturizing effect was investigated on stillborn piglet skin by using a Corneometer®. CA microemulsions were developed and characterized by photon correlation spectroscopy, rheometer, and heating-cooling stability tests. The results revealed that the major fatty acid components of CA were cis-9-oleic acid, cis-9,12-linoleic acid, and palmitic acid. CA had a significantly higher lipid peroxidation inhibition and DPPH scavenging capacity compared to the commercial oils (p < 0.05). Lipid peroxidation inhibition of CA was 39.2% ± 0.6% at 37.5 mg/mL and the IC50 value of DPPH assay was 70.8 ± 27.1 mg/mL. The skin moisture content after applying CA, commercial oils, and tocopheryl acetate were significantly higher than untreated skin (p < 0.05) and the moisturizing efficacy increased with time. Interestingly, radical scavenging and antioxidant effect of CA microemulsions were significantly higher than the native oil even after the stability test (p < 0.05). In conclusion, incorporating CA into microemulsion increased its antioxidant activity indicating that it would be beneficial as a cosmeceutical application for anti-aging.

Keywords: Camellia assamica; antioxidant; moisturizing effect; microemulsion; seed oil

1. Introduction Tea originated in East and South Asia. was the first country to use tea as a beverage by infusing the dried leaves into hot water [1]. Since ancient times, tea has been widely used as a medicinal plant and popularly used in various traditional medicines such as Ayurveda, Unani, and Homoeopathy. Nowadays, tea beverages are widely distributed throughout the world and are the most consumed after water. Not only is the leaf part of tea utilized, but the seed can be used to produce

Cosmetics 2018, 5, 40; doi:10.3390/cosmetics5030040 www.mdpi.com/journal/cosmetics Cosmetics 2018, 5, 40 2 of 13 oil. is an edible oil widely consumed in China. It is estimated that more than 2.5 million tons of tea seed oil will be produced by the year 2020 [2]. Cultivated tea was originally classified as Thea sinensis and Thea bohea by Linnaeus in 1752. However, the classification was revised to (: small-leaves, resistant to cold) and Camellia assamica ( tea: large-leaves, less resistant to cold), both of which belong to the family Theacea [3]. Altogether there are more than 65 species in the genus Camellia distributed in China but only 18 species can produce seeds that can be used for oil production [4]. Tea seed oil is rich in vitamin A, B, and E while having no cholesterol, leading to be the “king of cooking oil” with high nutritional value and health benefits [2]. Moreover, tea seed oil has desirable nutritional properties (high oleic acid, medium linoleic acid, and low linolenic acid) [5] and contains many natural antioxidants and functional components which was not only good for cardiovascular disease, cirrhosis, hypertension, and hyperlipidemia, but also have the special function of cancer prevention [6]. The physical and chemical constants of tea seed oil are similar to those of olive oil, thus it is sometimes referred to “Eastern olive oil”. As it is high in antioxidant properties, it could be a promising oil to use in cosmetic formulations. Microemulsion (ME) is the isotropic colloidal system that forms spontaneously with the appropriate combinations of oil, water, surfactant, and co-surfactant [7]. ME is optically transparent since the internal phase droplet size ranges from 5 to 200 nm which is below the wavelength of visible light [8,9]. The advantages of ME over the conventional topical formulations such as creams, ointments and gels are easy production and thermodynamic stability which will lead to a good shelf-life [10]. Therefore, ME is of interest for pharmaceutical and cosmeceutical applications as a carrier system. The present study focused on tea seed oil extracted from C. assamica cultivated in the high-attitude area in the Northern part of Thailand. The cosmeceutical properties, including antioxidant and moisturizing effects, of C. assamica seed oil (CA) were investigated. ME from CA were developed and characterized for topical use as a carrier system in cosmetic formulations.

2. Materials and Methods

2.1. Materials CA was obtained from the Department of Chemistry and Research Laboratory for Analytical Instrument and Electrochemistry Innovation, Faculty of Science, Chiang Mai University. Two commercial Camellia oleifera seed oils (CO1 and CO2) were purchased from local market in Chiang Mai, Thailand. DPPH (1,1-diphenyl-2-picrylhydrazyl radical), linoleic acid, quercetin, Triton X-114, tocopheryl acetate, and propylene glycol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Polyethylene glycol sorbitan monolaurate (Tween 20), polyethylene glycol sorbitan monostearate (Tween 60), polyethylene glycol sorbitan monooleate (Tween 80), polyethylene glycol sorbitan trioleate (Tween 85), sorbitan monooleate (Span 80), and polyoxyethylene monooctylphenyl ether (Triton X-114) were purchased from Acros Organics (Morris Plains, NJ, USA). Glycerin, BP/USP was purchased from Malaysia. Polyethylene glycol 400, USP was purchased from Wilhelmshaven, Germany. Ammonium thiocyanate, and ferrous chloride were purchased from Fisher Chemicals (Loughborough, UK). Hydrochloric acid was analytical grade and purchased from Merck (Darmstadt, Germany). Ethanol, acetone, dimethyl sulfoxide (DMSO), and propan-2-ol were AR grade and purchased from Labscan (Dublin, Ireland).

2.2. Determination of Fatty Acid Composition of Tea Seed Oils Fatty acid composition of CA and commercial tea seed oils (CO1) were analyzed according to the method reported by Lucchetti et al. [11]. Briefly, fatty acids were converted to methyl esters before the analysis to reduce their polarity and increase their volatility. Then the methyl esters were quantified by gas chromatography (GC) equipped with a flame ionization detector (FID) (Perkin Elmer Autosystem, Cosmetics 2018, 5, 40 3 of 13

Waltham, MA, USA). The fatty acids were identified by their retention times as compared to those of standards purchased from Sigma–Aldrich (St. Louis, MO, USA).

2.3. Biological Activities Determination of Tea Seed Oils

2.3.1. Antioxidant Activity Determination

Inhibition of Lipid Peroxidation by the Ferric Thiocyanate Assay Antioxidant activity of CA and two commercial tea seed oils (CO1 and CO2) were tested for the inhibition of lipid peroxidation by the ferric thiocyanate method reported by Motamed and Naghibi with slight a modification [12]. Briefly, 100 µL of a test sample solution or tocopheryl acetate (TA) as standard solution in DMSO was mixed with 1 mL of 25 mM linoleic acid in acetone and 1 mL of 0.1 M phosphate buffer pH 7.0 in the test tube with cork lid stock. The reaction was carried out in the dark for 6 h at 60 ◦C. Then 50 µL of the mixture was mixed with 3 mL of 75% EtOH, 20 µL of 35% ammonium thiocyanate, and 20 µL of 20 mM ferrous chloride in 3.5% HCl. After mixing by vortex for 1 min, the absorbance was measured at 500 nm by using a UV-Visible spectrophotometer (Biochrom, Cambridge, UK). % Inhibition was calculated using the following equation;

% Inhibition = [(B − S)/B)] × 100, (1) where B is the absorbance of the mixture of 100 µL of acetone, 1 mL of 25 mM linoeic acid in acetone, and 1 mL of 0.1 M phosphate buffer pH 7.0 in the absence of test sample and S is the absorbance of 1 mL of 25 mM linoeic acid in acetone, and 1 mL of 0.1 M phosphate buffer pH 7.0 in the presence of 100 µL of test sample. The experiment was performed in triplicate.

Scavenging of 1,1-Diphenyl-2-picrylhydrazy Radical (DPPH Assay) Tea seed oils including CA, CO1, and CO2 were tested for radical scavenging activity against stable DPPH using the method reported by Blois [13] with a slight modification. Briefly, 20 µL of test sample solution or quercetin (Q) as a standard solution in DMSO was mixed with 180 µL of 167 µM DPPH• (1,1-diphenyl-2-picrylhydrazyl radical) solution. The reaction was carried out in the dark for 30 min at room temperature. Then the absorbance was measured at 520 nm using a DTX-880 Multimode Detector (Biochrom, Cambridge, UK). % Inhibition was calculated using the following equation;

% Inhibition = {[(PC − NC) − (S − B)]/(PC − NC)} × 100, (2) where PC is the absorbance of 20 µL of acetone and 180 µL of 167 µM DPPH mixture, NC is the absorbance of 200 µL of acetone, S is the absorbance of 20 µL of test sample and 180 µL of 167 µM DPPH mixture, and B is the absorbance of 20 µL of test sample and 180 µL of acetone mixture. The experiment was performed in triplicate.

2.3.2. In Vitro Skin Moisturizing Effect Determination The tea seed oils including CA, CO1, and CO2 were examined in comparison with tocopheryl acetate (TA). The tested skins were prepared from the flank area of stillborn piglets which were accidentally died before birth. The piglets were obtained fresh from a local farm. The fat layer was removed, and the skin was cut into 3 × 3 cm2. The skins were left at room temperature (25 ± 1 ◦C) with 50–60% relative humidity (RH) for at least 30 min before further testing. After preparation, 60 µL of each test sample was applied on the skin surface. The moisture content was measured before and after applying the sample for 15 and 30 min using a Corneometer® CM 825 (Courage and Khazaka, Köln, Germany). All the measurements were performed in triplicate. This method had been modified from O’Goshi et al. [14]. Cosmetics 2018, 5, 40 4 of 13

2.4. Development of Microemulsion Containing Tea Seed Oils

2.4.1. Pseudoternary Phase Diagram Construction Pseudoternary phase diagrams of CA were constructed using a slightly modified water titration method [15]. Three components used for the construction of pseudoternary phase diagrams were oil phase, water phase, and surfactant mixture (Smix). CA was used as the oil phase. Various non-ionic surfactants (Tween 20, Tween 60, Tween 80, Tween 85, Span 80, or Triton X-114) were mixed with various co-surfactants (ethanol, propan-2-ol, glycerine, PG, or PEG-400) at a weight ratio of 1:1 to obtain Smix. The effect of surfactant to co-surfactant weight ratio were also investigated by using the ratio of 6:1, 4:1, 2:1, 1:1, and 1:2. The oil phase and Smix were then mixed at various weight ratios (0:1, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0) and the resulting mixtures were subsequently titrated with water under moderate agitation at room temperature. The samples were classified as MEs when they appeared visually as clear liquids. The different formulations were made in triplicate. The pseudoternary phase diagrams were drawn by OriginPro 8 software. The ME regions were measured by ImageJ 1.47v software.

2.4.2. Microemulsion Development MEs were developed by mixing CA (oil phase) with Smix and water, when Smix were the combination of Tween 85 and ethanal (4:1) or Tween 85 and PG (2:1). Three ME formulations were developed and were classified as ME A, B, and C. Tween 85 and ethanal (4:1) were used as Smix in formulation A, where the CA:Smix:water ratio was 1:8:1. Tween 85 and propylene glycol (2:1) were used as Smix in formulation B and C, where the CA:Smix:water ratio were 1:8:1 and 1:7:2, respectively.

2.4.3. Characterization of Microemulsion

Photon Correlation Spectroscopy Particle size analysis was carried out using photon correlation spectroscopy (Zetasizer® version 5.00, Malvern Instruments Ltd., Malvern, UK). The sizing measurements were carried out at a fixed angle of 173◦. The reported results are mean ± standard deviation (S.D.) of at least ten measurements on the sample.

Rheology Study Viscosity of MEs was measured using a Brookfield DVIII rheometer (Brookfield Engineering Laboratories, Stroughton, MA, USA) fitted with a bob spindle. Brookfield Rheocalc operating software was used to control the measurement. A sample volume of 70 mL was used. The measurements were performed in triplicate at 25 ◦C.

Biological Activities Determination of Microemulsion Containing Tea Seed Oil Antioxidant activities of ME s containing tea seed oil were tested as mentioned in Section 2.3.1.

2.4.4. Stability Study MEs were kept in air-tight containers under the accelerated conditions, i.e., six heating-cooling cycles consisting of 4 ◦C for 24 h switching to 45 ◦C for 24 h. The ME were then characterized for the particle size by using photon correlation spectroscopy (Zetasizer® version 5.00, Malvern Instruments Ltd., Malvern, UK) and the viscosity by using a Brookfield DVIII rheometer (Brookfield Engineering Laboratories, Stroughton, MA, USA) fitted with a bob spindle. Cosmetics 2018, 5, 40 5 of 13

2.5. Statistical Analysis All data are presented as a mean ± standard deviation (S.D.). Individual differences were evaluated by t-test and One-Way ANOVA with post-hoc testing. Statistical difference was set at p < 0.05. Cosmetics 2018, 5, x FOR PEER REVIEW 5 of 13

3. Results3. Results

3.1. Fatty3 Acid.1. Fatty Composition Acid Composition of Tea of SeedTea Seed Oils Oils The fatty acid composition of CA and CO1 are shown in Table 1. The major components of CA The fatty acid composition of CA and CO1 are shown in Table1. The major components of CA were cis-9-oleic acid, cis-9,12-linoleic acid, and palmitic acid. CO1 had similar major components as were cis-9-oleicCA but with acid, higher cis-9,12-linoleic amount. acid, and palmitic acid. CO1 had similar major components as CA but with higher amount. Table 1. Fatty acid composition of tea seed oils (CA) and commercial tea seed oils (CO1).

Table 1. Fatty acid composition of tea seed oils (CA) and commercialAmount tea(%) seed oils (CO1). Fatty Acid Composition CA CO1 Myristic acid (C14:0) Amount0.09 (%) 0.04 Fatty Acid Composition Pentadecanoic acid (C15:0) CA0.02 COND1 MyristicPalmitic acid acid (C14:0) (C16:0) 0.0917.32 0.047.00 PentadecanoicHeptadecanoic acid acid (C15:0) (C17:0) 0.020.18 ND0.05 PalmiticStearic acid acid (C16:0) (C18:0) 17.323.7 7.003.02 Heptadecanoic acid (C17:0) 0.18 0.05 Arachidic acid (C20:0) 0.11 0.16 Stearic acid (C18:0) 3.7 3.02 ArachidicBehenic acid acid (C20:0) (C22:0) 0.110.05 0.160.39 TricosanoicBehenic acid acid (C22:0) (C23:0) 0.050.02 0.39ND TricosanoicLignoceric acid acid (C23:0) (C24:0) 0.020.07 ND0.11 Lignoceric acid (C24:0) 0.07 0.11 Palmitoleic acid (C16:1n7) 0.18 0.11 Palmitoleic acid (C16:1n7) 0.18 0.11 Trans-9-EladicTrans-9-Eladic acid acid (C18:1n9t) (C18:1n9t) NDND 0.260.26 cis-9-Oleiccis-9-Oleic acid acid (C18:1n9c) (C18:1n9c) 57.9757.97 79.9679.96 cis-11-Eicosenoiccis-11-Eicosenoic acid acid (C20:1n11) (C20:1n11) 0.850.85 0.440.44 ErucicErucic acid acid (C22:1n9) (C22:1n9) 0.060.06 0.040.04 Nervonic acid (C24:1n9) 0.09 0.04 cis-9,12-LinoleicNervonic acid acid ( (C18:2n6)C24:1n9) 18.570.09 8.320.04 alpha-Linoleniccis-9,12-Linoleic acid acid (C18:3n3) (C18:2n6) 0.6618.57 0.068.32 cis-11,14-Eicosadienoicalpha-Linolenic acid acid (C18 (C20:2):3n3) 0.040.66 0.020.06 cis-13,16-Docosadienoiccis-11,14-Eicosadienoic acid acid (C22:2) (C20:2) 0.010.04 ND0.02 cis-13,16-DocosadienoicTotal acid (C22:2) 99.990.01 100.02ND Total 99.99 100.02 3.2. Antioxidant Activity of Tea Seed Oils 3.2. Antioxidant Activity of Tea Seed Oils The antioxidant activity of tea seed oils, as shown in Figure1, indicated that CA possessed the The antioxidant activity of tea seed oils, as shown in Figure 1, indicated that CA possessed the significantlysignificantly highest highest antioxidant antioxidant activity activity in in bothboth lipid lipid peroxidation peroxidation and DPPH and DPPHassay. Interestingly, assay. Interestingly, CA showedCA ashowed significantly a significantly higher higher lipid lipid peroxidation peroxidation inhibition inhibition compared compared to tocopheryl to tocopheryl acetate (TA acetate) (TA) and significantlyand significantly higher higher scavenging scavenging effects effects against against DPPH DPPH.. Moreover, Moreover, CA alsoCA hadalso the hadhighest the DPPH highest DPPH inhibitory activity among the tea seed oils (Figure 1b) with the IC50 of 70.76 ± 27.14 mg/mL (Figure inhibitory activity among the tea seed oils (Figure1b) with the IC of 70.76 ± 27.14 mg/mL (Figure2). 2). 50

50 120 B A 100 A 40 A B

C 80 30

60 C 20

inhibition 40 D 10 20 % DPPH inhibition % DPPH % Lipid peroxidation % peroxidation Lipid 0 0 TA CA CO1 CO2 Q CA CO1 CO2

(a) (b)

Figure 1. (a) Lipid peroxidation inhibition; (b) DPPH inhibition of tocopheryl acetate (TA), quercetin (Q), CA, CO1, and CO2 at the concentration of 37.5 mg/mL (different letters (A, B, C, and D) denote significant statistically differences between groups; p < 0.05). Cosmetics 2018, 5, x FOR PEER REVIEW 6 of 13

Figure 1. (a) Lipid peroxidation inhibition; (b) DPPH inhibition of tocopheryl acetate (TA), quercetin (Q), CA, CO1, and CO2 at the concentration of 37.5 mg/mL (different letters (A, B, C, and D) denote significant statistically differences between groups; p < 0.05).

100

75

50

25 % DPPH inhibition 0 0.75 1.00 1.25 1.50 1.75 2.00 log concentration (mg/mL)

Figure 2. Dose response curve of DPPH inhibition versus log concentration of CA (R2 = 0.998 ± 0.002). Cosmetics 2018, 5, x FOR PEER REVIEW 6 of 13 Cosmetics 2018, 5, x FOR PEER REVIEW 6 of 13

FigureFigure 1. (a )1 .Lipid (a) Lipid peroxidation peroxidation inhibition; inhibition; ((b) DPPH inhibition inhibition of oftocopheryl tocopheryl acetate acetate (TA) ,( quercetinTA), quercetin Cosmetics 2018(Q),, 5CA(,Q 40),, COCA,1, CO and1, and CO CO2 at2 atthe the concentration concentration of 37..55 mg mg/mL/mL (different (different letters letters (A, B,(A, C, B, and C, Dand) denote D) denote 6 of 13 In Vitro Skin Moisturizing Effect of Teasignificant significantSeed statistically statistically Oils differences differences between between groups;groups; p p < < 0 .005.05). ).

The skin moisturizing effect of tea seed oils was100100 investigated using a Corneometer. The results 75 are shown in Figure 3. The so called short-term moisturizing75 effect of all tested substances presented 50 significantly different from the controls (untreated area50 ) at p < 0.05 with increasing efficacy over time. These may be due to the occlusive property of the oils25. CO1 had the highest skin moisturizing effect, % DPPH inhibition DPPH % 25 0 followed by CA (which was equal to TA) and then inhibition DPPH % CO0.752, respectively1.00 1.25 1.50 1.75. 2.00 0 log concentration (mg/mL) 0.75 1.00 1.25 1.50 1.75 2.00 Figure 2. Dose response curve of DPPH inhibition versus log concentration of CA (R2 = 0.998 ± 0.002). log concentration (mg/mL) 2 60 Figure 2. Dose response curve of DPPH inhibition versus log concentration of CA (R = 0.998 ± 0.002). InFigure Vitro Skin2. Dose Moisturizing response curve Effect of ofDPPH Tea Seed inhibition Oils versus log concentration of CA (R2 = 0.998 ± 0.002). In Vitro SkinThe Moisturizing skin moisturizing Effect effect of Teaof tea Seed seed Oilsoils was investigated using a Corneometer. The results In Vitroare shownSkin Moisturizing in Figure 3. The Effect so called of Tea short Seed-term Oils moisturizing effect of all tested substances presented 50 The skin moisturizing effect of tea seed oils was investigated using a Corneometer. The results Thesignificantly skin moisturizing different from effect the controls of tea seed (untreated oils was area investigated) at p < 0.05 with using increasing a Corneometer efficacy over. The time results. are shown in Figure3 . The so called short-term moisturizing effect of all tested substances presented are shownThese mayin Figure be due 3 .to The the soocclusive called propertyshort-term of the moisturizing oils. CO1 had effect the highest of all testedskin moisturizing substances effect, presented followed by CA (which was equal to TA) and then CO2, respectivelyp . 40 significantly*significantly different different from from the the controls controls (untreated (untreated area area)) at at p < 0<.05 0.05 with with increasing increasing efficacy efficacy over time over. time. TheseThese may may be duebe due to theto the occlusive occlusive property property ofof the oils oils.. COCO1 1had had the the highest highest skin skin moisturizing moisturizing effect, effect, 60 followedfollowed by CA by CA(which (which was was equal equal to toTA TA)) and and thenthen CO2,2, respectively respectively.. 30 * 50 6040 *

20 5030 * 4020 * Moisture content (a.u.) Moisture content Moisture content (a.u.) 10 10 30 * 0 20 Control TA CA CO1 CO2

Moisture content (a.u.) Moisture content 0 Figure 3. Skin moisture10 content before application (■), 15 min after application (■), and 30 min after application (□) of TA, CA, CO1, and CO2. (Asterisks denote significant statistically differences from Controlbefore application; TA p <0 0.05). CA CO1 CO2 Control TA CA CO1 CO2

3.3. Development of Microemulsion Containing Tea Seed Oils Figure 3. Skin moisture content before application (■), 15 min after application (■), and 30 min after Figure 3. Skin moisture content before application (), 15 min after application ( ), and 30 min after Figure 3. Skin moisture content beforeapplication3.3.1 . applicationPseudoternary (□) of TA ,Phase CA, CO Diagram(■)1, and, 15 ConstructionCO 2min. (Asterisks after denote application significant statistically ( ■differences), and from 30 min after application ( ) of TA, CA, CO1, and CO2. (Asterisks denote significant statistically differences from before application; p < 0.05). application (□) of TA, CA, CO1, andbefore CO application;Pseudoternary2. (Asterisksp < 0.05). phase diagram denote is an important significant tool for the statistically screening of self-dispersible differences from formulation components and evaluating the effect of different components of the ME [16]. The effect 3.3. Development of Microemulsion Containing Tea Seed Oils before application; p < 0.05). of surfactant type on ME development was investigated. Six surfactants (Tween 20, Tween 60, Tween 3.3. Development80, Tween of 85, Microemulsion Span 80, and Triton Containing X-114) were Tea Seedused in Oils this study. Only Tween 85 produced a ME 3.3.1.region Pseudoternary in the phase Phase diagram Diagram (Figure Construction 4a). 3.3.1. Pseudoternary Phase Diagram Construction PseudoternaryThe effect of phaseco-surfactant diagram type iswas an also important investigated tool when for the the surfactant screening was Tween of self 85-dispersible. The five co-surfactants were ethanol, isopropanol, propylene glycol, glycerin, and polyethylene glycol- 3.3. Development of Microemulsion ContainingformulationPseudoternary components phaseTea diagram Seedand evaluating isOils an important the effect toolof different for the components screening of of self-dispersible the ME [16]. The formulation effect 400. The results (Figure 4) indicated that ethanol, isopropanol, and propylene glycol produced MEs componentsof surfactant and type evaluating on ME development the effect of was different investigated components. Six surfactants of the ME (Tween [16]. 20, The Tween effect 60, of Tween surfactant

type80, on Tween ME development 85, Span 80, wasand investigated.Triton X-114) were Six surfactants used in this (Tween study. Only 20, Tween Tween 60, 85 Tweenproduced 80, a Tween ME 85, 3.3.1. Pseudoternary Phase DiagramSpanregion 80, Construction and in Tritonthe phase X-114) diagram were (Figure used in4a). this study. Only Tween 85 produced a ME region in the phase The effect of co-surfactant type was also investigated when the surfactant was Tween 85. The diagram (Figure4a). five co-surfactants were ethanol, isopropanol, propylene glycol, glycerin, and polyethylene glycol- The effect of co-surfactant type was also investigated when the surfactant was Tween 85. The five Pseudoternary phase diagram400 . Theis resultsan (Figureimpo 4) indicatedrtant that tool ethanol, isopropanol,for the and screeningpropylene glycol produced of self MEs -dispersible co-surfactants were ethanol, isopropanol, propylene glycol, glycerin, and polyethylene glycol-400. formulation components and evaluatingThe results (Figure the4) effect indicated thatof different ethanol, isopropanol, components and propylene glycolof the produced ME MEs[16 with]. The effect a ME region of 6.17%, 3.08%, and 5.32%, respectively, whereas, glycerin and polyethylene glycol-400 of surfactant type on ME developmentdid not produce was any investigated MEs (Figure not shown).. Six surfactants (Tween 20, Tween 60, Tween 80, Tween 85, Span 80, and Triton X-114) were used in this study. Only Tween 85 produced a ME region in the phase diagram (Figure 4a). The effect of co-surfactant type was also investigated when the surfactant was Tween 85. The five co-surfactants were ethanol, isopropanol, propylene glycol, glycerin, and polyethylene glycol- 400. The results (Figure 4) indicated that ethanol, isopropanol, and propylene glycol produced MEs

Cosmetics 2018, 5, x FOR PEER REVIEW 7 of 13

withCosmetics a ME2018 ,region 5, x FOR ofPEER 6.17 REVIEW%, 3.08 %, and 5.32%, respectively, whereas, glycerin and polyethylene 7 of 13 Cosmetics 2018, 5, x FOR PEER REVIEW 7 of 13 Cosmeticsglycol-4002018 did, 5, 40 not produce any MEs (Figure not shown). 7 of 13 with a ME region of 6.17%, 3.08%, and 5.32%, respectively, whereas, glycerin and polyethylene with a ME region of 6.17%, 3.08%, and 5.32%, respectively, whereas, glycerin and polyethylene glycol-400 did not produce any MEs (Figure not shown). glycol-400 did not produce any MEs (Figure not shown).

(a) (b) (c)

Figure 4. Pseudoternary phase diagram of CA/Tween 85 and co-surfactant (1:1)/water, when the co- Figure 4. Pseudoternary(a) phase diagram of CA/Tween(b) 85 and co-surfactant (1:1)/water,(c) when the surfactant was(a) (a) ethanol; (b) isopropanol; (c()b propylene) glycol. The dark area represents(c) the co-surfactant was (a) ethanol; (b) isopropanol; (c) propylene glycol. The dark area represents the Figure 4. Pseudoternary. phase diagram of CA/Tween 85 and co-surfactant (1:1)/water, when the co- microemulsionFigure 4. Pseudoternary region phase diagram of CA/Tween 85 and co-surfactant (1:1)/water, when the co- microemulsionsurfactant was region.(a) ethanol; (b) isopropanol; (c) propylene glycol. The dark area represents the surfactant was (a) ethanol; (b) isopropanol; (c) propylene glycol. The dark area represents the microemulsion region. Sincemicroemulsion ethanol andregion propylene. glycol produced a promising ME region, the various ratios of Smix (6:1, Since4:1, 2 ethanol:1, 1:1, and and 1 propylene:2) were investigated glycol produced using athese promising two co ME-surfactants region, the. The various results ratios (Figure of Smix 5) (6:1,indicated 4:1,Since 2:1, that ethanol 1:1, the andoptimum and 1:2) propylene were ratio investigatedof glycol Tween produced 85 to using ethanol a thesepromising was two 4:1 co-surfactants.MEas it region, produced the variousthe The largest results ratios ME( Figureof region Smix5 ) (6:1, Since4:1, 2 ethanol:1, 1:1, and and 1propylene:2) were investigated glycol produced using a promisingthese two coME-surfactants region, the. Thevarious results ratios (Figure of Smix 5) indicatedin(6 :the1, 4 phase:1, 2 that:1, diagram 1 the:1, optimumand. On1:2 )the were ratio other investigated of hand, Tween the 85 optimum using to ethanol these ratio was two of 4:1 Tweenco as-surfactants it produced85 to propylene. The the results largest glycol (Figure ME was region 2 5:1) indicated that the optimum ratio of Tween 85 to ethanol was 4:1 as it produced the largest ME region in(indicatedFigure the phase 6). that diagram. the optimum On the ratio other of Tween hand, the 85 to optimum ethanol ratiowas 4 of:1 Tweenas it produced 85 to propylene the largest glycol ME region was 2:1 in the phase diagram. On the other hand, the optimum ratio of Tween 85 to propylene glycol was 2:1 (Figurein the phase6). diagram. On the other hand, the optimum ratio of Tween 85 to propylene glycol was 2:1 (Figure 6). (Figure 6).

(a) (b) (c) (d) (e)

Figure(a) 5 . Pseudoternary phase(b) diagram of CA/Tween(c) 85 and ethanol/water,(d) when the Smix ratio(e) was (a) 6:(1;a) ( b) 4:1; (c) 2:1; (d) 1(:1;b) ( e) 1:2. The dark area(c )represents the microemulsion(d) region. (e) Figure 5. Pseudoternary phase diagram of CA/Tween 85 and ethanol/water, when the Smix ratio was FigureFigure 5.5. Pseudoternary phase diagram of CA//TweenTween 85 85 and and ethanol ethanol/water,/water, when when the the Smix Smix ratio ratio was was (a) 6:1; (b) 4:1; (c) 2:1; (d) 1:1; (e) 1:2. The dark area represents the microemulsion region. ((aa)) 6:1;6:1; ((bb)) 4:1;4:1; ((cc)) 2:1;2:1; ((dd)) 1:1;1:1; ((ee)) 1:2.1:2. TheThe darkdark areaarea representsrepresents the microemulsion region region..

(a) (b) (c) (d) (e)

Figure(a) 6 . Pseudoternary phase(b) diagram of CA/Tween(c) 85 and propylene( dglycol) /water, when the(e Smix) ratio( wasa) (a) 6:1; (b) 4:1; (c)( b2:)1; (d) 1:1; (e) 1:2. The(c )dark area represents( dthe) microemulsion region(e) . Figure 6. Pseudoternary phase diagram of CA/Tween 85 and propylene glycol/water, when the Smix Figure 6. Pseudoternary phase diagram of CA/Tween 85 and propylene glycol/water, when the Smix 3.3.2Figure.ratio Microemulsion was 6. Pseudoternary (a) 6:1; (b Formulations) 4:1; ( phasec) 2:1; diagram(d ) 1:1; (e of) 1CA:2./Tween The dark 85 area and represents propylene the glycol/water, microemulsion when region the Smix. ratio was (a) 6:1; (b) 4:1; (c) 2:1; (d) 1:1; (e) 1:2. The dark area represents the microemulsion region. ratio was (a) 6:1; (b) 4:1; (c) 2:1; (d) 1:1; (e) 1:2. The dark area represents the microemulsion region. 3.3.2Three. Microemulsion formulations Formulations of ME (A, B , and C) which contained 10% of oil phase were formulated and characterized3.3.2. Microemulsion. The compositions Formulations of each formulation are shown in pseudoternary phase diagrams in 3.3.2.Three Microemulsion formulations Formulations of ME (A, B, and C) which contained 10% of oil phase were formulated and FigureThree 7. The formulations MEs were isotropicof ME (A systems, B, and Cwith) which a yellow contained color .10 % of oil phase were formulated and characterized. The compositions of each formulation are shown in pseudoternary phase diagrams in characterizedThree formulations. The compositions of ME (A of, Beach, and formulationC) which contained are shown 10% in pseudoternary of oil phase were phase formulated diagrams andin Figure 7. The MEs were isotropic systems with a yellow color. characterized.Figure 7. The MEs The were compositions isotropic ofsystems each formulation with a yellow are color shown. in pseudoternary phase diagrams in Figure7. The MEs were isotropic systems with a yellow color.

Cosmetics 2018,, 5,, 40x FOR PEER REVIEW 8 of 13 Cosmetics 2018, 5, x FOR PEER REVIEW 8 of 13

(a) (b) (a) (b) Figure 7. (a) Formulation A composing of CA/Tween 85 and ethanol/water, when the Smix ratio was Figure 7. (a) Formulation A composing of CA/Tween 85 and ethanol/water, when the Smix ratio was 4:1;Figure (b) Formulation 7. (a) Formulation B and C A composing composing of of CA CA//TweenTween 8585 andand propyleneethanol/water, glycol when/water, the whenSmix ratiothe Smix was 4:1;4:1; (b ()b Formulation) Formulation B B and and C C composing composing of ofCA CA/Tween/Tween 85 85 and and propylene propylene glycol/water,glycol/water, when the Smix ratio was 2:1. ratioratio was was 2:1. 2:1. 3.4. Characterization of Microemulsion 3.4.3.4 Characterization. Characterization of of Microemulsion Microemulsion The internal internal droplet droplet size size and and polydispersity polydispersity index index of formulation of formulation A, B, Aand, B ,C and are shownC areshown in Figure in 8a. TheThe formulation internal droplet containing size and 10% polydispersity of oil phase (indexA and of B formulation) had significantly A, B, and smaller C are showninternal in droplet Figure Figure8a. The8a. formulation The formulation containing containing 10% of 10% oil phase of oil phase(A and ( BA) andhad Bsignificantly) had significantly smaller smallerinternal internaldroplet size than that containing 20% (C) (p < 0.05). Therefore, the oil phase content had a strong effect on the dropletsize than size that than containing that containing 20% (C 20%) (p < (C 0)(.05p).< Therefore, 0.05). Therefore, the oil thephase oil content phase content had a strong had a strongeffect on effect the internal droplet size, whereas, the type of co-surfactants had no effect. The internal droplet size of A oninternal the internal droplet droplet size, size,whereas, whereas, the type the typeof co- ofsurfactants co-surfactants had no had effect no effect.. The internal The internal droplet droplet size of size A and B was almost the same although PG has a larger molecular size compared to ethanol. ofandA and B wasB was almost almost the thesame same although although PG PGhas hasa larger a larger molecular molecular size sizecompared compared to ethanol to ethanol.. All formulations developed in this study showed a Newtonian flow behavior which is a AllAll formulations formulations developed developed in in this this study study showed showed a a Newtonian Newtonian flow flow behavior behavior which is a characteristic of ME. The formulations using PG as a co-surfactant (B and C) had a higher viscosity characteristiccharacteristic of of ME. ME The. The formulations formulations using using PG PG as aas co-surfactant a co-surfactant (B and (B andC) had C) ahad higher a higher viscosity viscosity than than that using ethanol (A) (Figure 8b). The explanation might be that the viscosities of the thatthan using that ethanol using (A ethanol) (Figure (A8)b). (Figure The explanation 8b). The might explanation be that might the viscosities be that of the the viscosities formulations of are the formulations are derived from the native viscosity of each co-surfactant. derivedformulations from the are native derived viscosity from the of native each co-surfactant. viscosity of each co-surfactant.

(a()a ) ((bb))

FigureFigure 8. 8Internal. Internal droplet droplet size size and and polydispersity polydispersity index index ( a(a) )and and viscosity viscosity ((bb)) ofof microemulsionmicroemulsion A, B, Figure 8. Internal droplet size and polydispersity index (a) and viscosity (b) of microemulsion A, B, and andand C C (different (different letters letters (* (* and and # )# )denote denote significant significant statistically statistically differentdifferent internalinternal dropletdroplet size and C (different letters (* and #) denote significant statistically different internal droplet size and viscosity viscosityviscosity between between groups; groups; p p< <0 .005,.05, whereas, whereas, different different symbols symbols ( α(α and and β β)) denote denote significantsignificant statistically between groups; p < 0.05, whereas, different symbols (α and β) denote significant statistically different differentdifferent polydispersity polydispersity index; index; p p< <0 .005.05).). polydispersity index; p < 0.05). 3.53.. 5Antioxidant. Antioxidant Activity Activity of of Microemulsion Microemulsion Containing Containing Tea Tea Seed Seed Oils Oils 3.5. Antioxidant Activity of Microemulsion Containing Tea Seed Oils TheThe antioxidant antioxidant activity activity of of 10 10%% CA CA and and the the ME ME containing containing 1010%% CACA werewere determineddetermined by lipid The antioxidant activity of 10% CA and the ME containing 10% CA were determined by lipid peroxidationperoxidation and and DPPH DPPH assay assay. .The The results results from from both both methodsmethods werewere comparablecomparable to each other as peroxidation and DPPH assay. The results from both methods were comparable to each other as shown shownshown in in Figure Figure 9 .9 The. The MEs MEs had had a asignificantly significantly higher higher antioxidant antioxidant effecteffect comparedcompared to the native CA in Figure9. The MEs had a significantly higher antioxidant effect compared to the native CA oil. oiloil. .

* Cosmetics 2018, 5, 40 9 of 13 Cosmetics 2018, 5, x FOR PEER REVIEW 9 of 13

30 20 * * * * * 25 * 15 20 15 10 10 inhibition 5 5 % DPPH inhibition DPPH % % Lipid peroxidation peroxidation % Lipid 0 0 CA A B C CA A B C (a) (b)

Figure 9. (a) Lipid peroxidation inhibition; (b) DPPH inhibition of microemulsion A, B, and C Figure 9. (a) Lipid peroxidation inhibition; (b) DPPH inhibition of microemulsion A, B, and C compared compared to 10% CA (Asterisks (*) denote significant statistically differences from 10% CA; p < 0.05). to 10% CA (Asterisks (*) denote significant statistically differences from 10% CA; p < 0.05).

3.6. Stability of Microemulsion Containing Tea Seed Oils 3.6. Stability of Microemulsion Containing Tea Seed Oils Viscosity and internal droplet size of each ME is shown in Table 2. After the heating-cooling Viscosity and internal droplet size of each ME is shown in Table2. After the heating-cooling stability test, the ME A, in which ethanol was used as a co-surfactant, significantly increased in stability test, the ME A, in which ethanol was used as a co-surfactant, significantly increased in viscosity viscosity (p < 0.05). The increment of viscosity may be due to the evaporation of ethanol during the (p < 0.05). The increment of viscosity may be due to the evaporation of ethanol during the storage time. storage time. Since PG was used in ME B and C, the evaporation did not occur and no alteration in Since PG was used in ME B and C, the evaporation did not occur and no alteration in the viscosity the viscosity occurred after the stability test. occurred after the stability test.

Table 2. Viscosity and internal droplet size of microemulsions before and after the heating-cooling Table 2. Viscosity and internal droplet size of microemulsions before and after the heating-cooling stability test. stability test. Viscosity (Pas) Internal Droplet Size (nm) Viscosity (Pas) Internal Droplet Size (nm) MicroemulsionMicroemulsion Before After Before After Before Stability Test After Stability Test Before Stability Test After Stability Test A 0.38Stability± 0.03 Test Stability 0.52 ± 0.03 Test * Stability 426.07 Test± 56.96 Stability 351.43 Test± 47.14 B A 0.950.±380.07 ± 0.03 0.52 1.24 ± ±0.0.1203 * 426.07 374.67 ± 56.±9643.05 351.43 1126.00 ± 47.14± 125.04 * C 1.01 ± 0.03 0.95 ± 0.01 801.47 ± 40.13 1013.93 ± 140.90 A: 10% CA, 64%B Tween 85, 16%0.95 ethanol, ± 0.07 10% water;1.24B: ± 10% 0.12CA , 53.3374 Tween.67 ± 85, 43 26.7%.05 propylene1126.00 glycol,± 125. 10%04 * water; C: 10% CA, 46.7C Tween 85, 23.4%1.01 propylene± 0.03 glycol,0.95 20% ± water;0.01 Asterisks801.47 (*) ± denote 40.13 significantly 1013.93 different ± 140.90 between the results before and after stability test, p < 0.05. A: 10% CA, 64% Tween 85, 16% ethanol, 10% water; B: 10% CA, 53.3 Tween 85, 26.7% propylene glycol, 10% water; C: 10% CA, 46.7 Tween 85, 23.4% propylene glycol, 20% water; Asterisks (*) denote Thesignificantly antioxidant different activity between of the MEs results containing before and after tea stability seed oil test, was p < 0 also.05. investigated after the heating-cooling cycles (Table3). The DPPH inhibition of all MEs was not altered by the storage conditions,The antioxidant whereas, lipidactivity peroxidation of MEs containing inhibition tea was seed significantly oil was also decreased investigated (p < after 0.05). the heating- cooling cycles (Table 3). The DPPH inhibition of all MEs was not altered by the storage conditions, Table 3. Lipid peroxidation and DPPH inhibition of microemulsions before and after the heating-cooling whereas, lipid peroxidation inhibition was significantly decreased (p < 0.05). stability test.

Lipid Peroxidation Inhibition (%) DPPH Inhibition (%) Microemulsion Before Stability Test After Stability Test Before Stability Test After Stability Test A 22.07 ± 2.89 15.08 ± 0.93 * 15.39 ± 0.82 15.08 ± 0.67 B 24.47 ± 2.50 15.80 ± 1.74 * 16.72 ± 1.75 16.25 ± 2.89 C 24.05 ± 1.88 15.58 ± 1.02 * 16.52 ± 1.35 15.16 ± 0.86 A: 10% CA, 64% Tween 85, 16% ethanol, 10% water; B: 10% CA, 53.3 Tween 85, 26.7% propylene glycol, 10% water; C: 10% CA, 46.7 Tween 85, 23.4% propylene glycol, 20% water; Asterisks (*) denote significantly different between the results before and after stability test, p < 0.05.

4. Discussion Tea seed oil is rich in vitamin A, B, and E with no cholesterol [2,17]. The major fatty acids of CA were cis-9-oleic acid (57.97%), cis-9,12-linoleic acid (18.57%), and palmitic acid (17.32%). The fatty acid

Cosmetics 2018, 5, 40 10 of 13 contents of CO1 were slightly different from CA, as cis-9-oleic acid, the major component in both oils, was more pronounced in CO1 (79.96%) rather than that in CA (57.97%). Furthermore, the amount of cis-9,12-linoleic acid and palmitic acid in CO1 was about half of the amount that detected in CA. However, the results in this study were in an accordance with the literature which noted that tea seed oil contained high amount of oleic acid, medium amount of linoleic acid, and low amount of linolenic acid [5]. The antioxidant activities of tea seed oil from Camellia oleifera Abel. and Camellia sinensis L. have been extensively investigated and their prophylactic properties against free radical related conditions have been reported [18,19]. However, the antioxidant activity of CA is first described in the present study. The antioxidant activity of the oil was determined in a comparison with two commercial oils (CO1 and CO2) by using the inhibition of lipid peroxidation and DPPH assays. These two methods were used as the previous studies emphasized that the antioxidant activity was depended on the method used and it recommended the use of at least two different test methods [20]. DPPH assay is the test system using a stable free radical to give information on radical scavenging or antiradical activity, whereas, the lipid peroxidation assay is the most studied biologically relevant free radical chain reaction that gives information on the antioxidant activity. Both assays revealed that CA possessed higher antioxidant activity compared to the commercial oils. Therefore, tea seed oil is rich in antioxidant activity and can be used as alternative source of oil phase in cosmetics production. Additionally, CA possessed moisturizing efficacy on a pig skin model which has structural similarities to human skin [21]. Therefore, it would be an attractive component for ME development for topical use purposes. ME is one of the delivery systems that is suitable for topical use. The construction of pseudoternary phase diagrams greatly helps to elucidate the ME region that exists in the diagram depending upon the composition ratios since the variety of structure of ME is a function of the formulation’s composition [22]. The previous study indicated that among various surfactants, Tween 85 can produce ME, when the oil phase was isopropyl myristate, the co-surfactant was ethanol, and Smix ratio was 1:1 [17,23]. In some cases, Tween 85 produces smaller ME regions compared to the others [17]. Therefore, there was no universal surfactant that suitable for all types of oil in ME development. For tea seed oil, Tween 85 was suggested. In the present study, co-surfactants (ethanol and PG) and the ratios of Smix affected the ME region in the phase diagram. The results agree with the previous study where different types of co-surfactant brought about the various ME formations and ME regions in the phase diagram [24]. Additionally, the higher proportion of surfactant in Smix, the larger area of ME region. Gao et al. reported that ME region respectively increased when the ratio of Cremophor EL to Transcutol increased from 0.5:1 to 4:1 [25]. Similarly, Guan et al. reported that ME region increased when the ratio of Tween 20 to PEG-400 increased from 1:2 to 4:1 [17]. However, in the present study, there was an optimum ratio of the Smix. The ME region decreased at a higher proportion of surfactant and the lower proportion of co-surfactant because there was not enough co-surfactant to ensure flexibility of interfacial layer and reduce the interfacial tension. In the present study, the tea seed oil content affected the ME formation. This agrees with the previous study that droplet size of oil-in-water MEs were influenced by the total content of the oil phase [26]. In additions, the surfactant to co-surfactant ratio was an important parameter in determining the size of the internal phase of the ME systems [27]. ME A and C were isotropic systems with a yellow color which had the same appearance as the original after stability testing. On the other hand, ME B separated into two layers made of a ME (top) and water-rich region (bottom) which was the characteristic of a Winsor type II system. The internal droplet size of ME B significantly increased after the heating-cooling cycles (p < 0.05) which related well with a change of external appearance. Normally, ME containing a non-ionic surfactant is a Winsor type I system (oil in water ME with excess oil) at low temperature and turns to a Winsor type II system (water in oil ME with excess water) when the temperature increases. Increasing temperature decreases the degree of hydration of the head group of non-ionic surfactants, thereby decreasing its effective Cosmetics 2018, 5, 40 11 of 13 area at the interface [28,29]. ME B turned into a Winsor type II system during the storage at high temperature (45 ◦C) which led to the phase separation. After the stability test, the DPPH inhibition of all MEs still remained the same while lipid peroxidation inhibition was significantly decreased (p < 0.05). These two different methodologies revealed distinctly different results. Lipid peroxidation is a complex process involving various mechanisms, including lipid radical formation, oxygen uptake, and rearrangement of the double bonds in unsaturated lipids [30], whereas, DPPH assay was only related to the scavenging ability. In this study, the scavenging ability of MEs containing CA did not decrease over the time but the ability to inhibit the rearrangement of the double bonds in unsaturated lipids could be reduced. In conclusion, the ME retained the radical scavenging activity; however, the antioxidant activity decreased during the storage time.

5. Conclusions The antioxidant and moisturizing properties of CA was firstly described in the present study. CA had a significantly higher lipid peroxidation and DPPH inhibition compared to two commercial tea seed oils (CO1 and CO2). The lipid peroxidation inhibition of CA was 39.20% ± 0.63% at a concentration of 37.5 mg/mL and the IC50 against DPPH was 70.76 ± 27.14 mg/mL. The moisturizing effect of CA, TA, CO1, and CO2 were not significantly different. However, the short-term moisturizing effect of CA, TA, CO1, and CO2 was significantly higher than that of controls (untreated area) and the increasing moisturizing efficacy with time of the oils was also observed. The ME of CA was then developed and the factors affecting ME formation were investigated. The surfactant types, co-surfactant types, and Smix ratio influenced ME region based on observation of the pseudoternary phase diagrams. Tween 85 was found to be the only surfactant suitable for the ME formation. The ME containing CA were prepared by mixing CA with Smix and water. Smix was the combination of Tween 85 and ethanal (4:1) or Tween 85 and propylene glycol (2:1). The oil phase content had a strong effect, whereas, the type of co-surfactant had no effect on the internal droplet size. The radical scavenging activity and antioxidant effect of MEs were significantly higher compared to the native CA oil. Furthermore, the ME retained the radical scavenging activity after 6 cycles of heating-cooling stability test, but the antioxidant activity decreased during a storage time. It concluded that the ME of CA was suitable for topical use as carrier system in cosmetic formulations.

Author Contributions: Conceptualization, W.C., P.L.; Methodology, W.C., J.J., C.K.; Investigation, W.C.; Writing-Original Draft Preparation, W.C.; Supervision, P.L.; Funding Acquisition, P.L. Funding: This research was funded by the Research Administration Center, Chiang Mai University, Thailand. Acknowledgments: We thank Karl Bailey, Scientific Officer, Department of Human Nutrition, University of Otago, New Zealand and Pummy Krittaphol, assistant research fellow, School of Pharmacy University of Otago, New Zealand for improving the use of English in the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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