THAI AROMATIC PLANT EXTRACTS IN ANTIWRINKLE BODY CREAMS 221 purchased from Sigma-Aldrich Inc (St. Louis, MO), Cholesterol from lanolin (C27H46O) was purchased from Flukachemie GmbH., (Tokyo, Japan). Phosphatidylcholine (Epikuron 200) was purchased from Degussa, (Frankfurt, Germany). The 2,2´-azobis(2-amidino- propane) dihydrochloride (AAPH) was purchased from Wako Pure Chemical Industries, (Osaka, Japan). Other chemicals and solvents were of analytical grade. All the plants used were cultivated and collected from Chiang Mai Province, Thailand. PREPARATION OF THE EXTRACTS The seven kinds of rhizomes of Zingiberaceous plants [Zingiber cassumunar Roxb., Curcuma domestica Val., C. mangga Val. & Zijp., Z. offi cinale L., Alpinia galanga (L.) Swartz, Z. ottensii Val. and Curcuma sp.], leaves petiole of Cymbopogon citratus, and leaves of Ocimum sanctum collected in northern provinces of Thailand were hydrodistilled for 5 h using a Clevenger- type apparatus. The obtained volatile oils were dried over anhydrous sodium sulfate. The petals or whole fl owers of 10 plants [Mammea siamensis Kosterm., Michelia alba, Plumeria alba, C. odorata (Lam.) Hook. f. & Thomson var. fruticosa (Craib) J. Sinclair, Millingtonia hortensis L., Alangium salviifolium subsp. hexapetalum Wang., Alstonia scholaris (L.) R. Br., Gardenia augusta, Saraca thaipingensis Cantley ex King, and Quisqualis indica L.] collected in northern provinces of Thailand were extracted with petroleum ether for 24 h. After fi ltering and evaporating to dryness in a vacuum, the concretes obtained were treated with ethanol (EtOH) to produce absolutes. All of the volatile oils and absolutes were stored in the dark at 4°C until tested. DETERMINATION OF ANTIOXIDANT ACTIVITIES Antioxidant activities of all extracts were determined by two different methods, scaveng- ing effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and lipid peroxidation [thiobarbituric acid reactive substance (TBARs)]. Determination of antioxidant activity with DPPH radical scavenging method. In this assay, the stable free radical DPPH, which has a strong absorption at 520 nm, reacts with antioxidants and produces colorless 2,2-diphenyl-1-picrylhydrazine independently of enzymatic activi- ties (7). Dilution series of test compounds, dissolved in EtOH, were performed in sterile disposable microplates, using freshly prepared 167 μM DPPH•/EtOH solutions, 180 μl. Trolox, quercetin, and thyme oil served as reference antioxidants. The samples were tested in dilutions ranging from 0.5 to 500 mg/ml with a fi nal volume of 200 μl for all of the as- says. The results were determined after 30 min of reaction time to analyze antiradical ac- tivities. The disappearance of the free radical DPPH• was measured spectrophotometrically at 520 nm with a microplate reader. The percentage inhibition was calculated by the equa- tion: % inhibition = (Acontrol − Asample/Acontrol) × 100 where Acontrol is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absor- bance of test compound. Extract concentration providing 50% inhibition was calculated from the graph plotted inhibition percentage against extract concentration. Determination of antioxidant activity with TBARs method. A modifi ed TBARS assay was used to measure the antioxidant activity of the extracts in terms of inhibition on lipid peroxidation (8–10). A liposome suspension, consisting of cholesterol (0.25 g),

JOURNAL OF COSMETIC SCIENCE 222 phosphatidylcholine (0.03 g), and 20 ml of 0.2 M potassium phosphate buffer (pH 7.2), was prepared in a sonicator. The essential oil or absolute in EtOH (100 μl), was mixed with a mixture of the sonicated solution (600 μg/ml) and AAPH (0.07 M, 60 μl). The resulting mixture was incubated at 50°C. After incubation, the solution (80 μl) was mixed with 0.2% BHT (24 μl), 3% Triton-X (100 μl), 20% acetic acid (500 μl), and 0.6% TBA (250 μl). Then the mixture was heated for 30 min, cooled to room tempera- ture and absorbance was measured at 540 nm with a microplate reader. The percentage inhibition was calculated same as in DPPH assay. Extract concentration providing 50% inhibition was calculated from the plot of inhibition percentage against its concentration. Trolox, quercetin, and thyme oil that are known antioxidants served as positive controls. GAS CHROMATOGRAPHY ANALYSIS OF THE ESSENTIAL OILS AND FLOWER ABSOLUTES Essential oils 0.5 % v/w in ethanol and fl ower absolutes were characterized by gas chromatography–mass spectrometry (GC-MS) using a Shimadzu model GCMS-QP2010 Plus (Kyoto, Japan) with mass-selective detector with electron impact ionization. The samples were separated using a DB-5 MS capillary column (5% phenylmethylpolysiloxane, 30 m × 0.25 mm, 0.25 μm fi lm thickness) Helium was used as carrier gas. Injector temperature 200°C, split ratio 1:50. Column temperature was 90°C, ramp to 130°C with the rate of 5°C/min, ramp to 133°C with the rate of 1°C/min for 3 min, ramp to 134°C with the rate of 0.5°C/min for 3 min, ramp to 136°C with the rate of 0.5°C/min for 8 min, and then ramp to 230°C/min with the rate 15°C/min for 5 min. Chemical constituents were characterized by matching mass spectra with reference com- pounds mass spectra in WILLEY 7 and NIST 2005 Library. PREPARING OF ESSENTIAL OIL BLEND Essential oils and absolutes with good antioxidant activity were selected for odor evalua- tion via fragrance by 21 volunteers. Each volunteer smelled the samples and scored the odor from 1 (least like) to 5 (most like). The essential oils and absolutes that presented high odor satisfaction were blended into the essential oil blend (EOB). FORMULATION AND STABILITY TEST OF CREAM BASE AND EOB BODY CREAM A cream base containing 45.6% of oil phase (jojoba oil, avocado oil, vitamin E acetate, cyclomethicone, glyceryl stearate, stearic acid, stearyl alcohol, isopropyl myristate, BHT, and ceteareth-25) and 55.4% of water phase (propylene glycol, triethanolamine, purifi ed water, sodium metabisulfi te, and preservative) was formulated. Texture smoothness, color, odor, spreadability, feel on skin, and pH were observed at the starting point and after stability testing under storage at eight cycles of heating and cooling (alternation of storage conditions from 50°C for 48 h to 4°C for 48 h as one cycle). Creaming and crack- ing were also observed. The EOB (1.5% w/w) was then incorporated into the stable cream base and its stability was also determined as described above.