J. Cosmet. Sci., 69, 145–156 (March/April 2018) 145 Rapid Method for The Gas Chromatographic Quantitative Analysis to Determinate Safrole in Commercial Essential Oils SHENG-CHING CHAN, YOUK-MENG CHOONG, and SHUN-HSIANG WENG, Department of Nursing, Tajen University, Neipu, Pingtung 912, Taiwan (S.-C. C.), Department of Food & Technology, Chia Nan University of Pharmacy and Science, Rende District, Tainan 717, Taiwan (Y.-M. C), and Department of Food Nutrition, Meiho University, Yanpu Township, Pingtung, Taiwan (S.-H. W.) Accepted for publication March 28, 2018. Synopsis Safrole is a well-known carcinogenic agent that is present in camphor trees. In this study, a gas chromatographic method was established to quantitate the levels of safrole in essential oils using n-decyl alcohol as an internal standard. The method used a nonpolar column and was able to detect concentrations of safrole as low as 5 μg/ml in the samples. Following addition of 2–10 mg of safrole into 1 g of essential oil extracted from Stout Camphor wood (Cinnamomum kanehirai Hayata) or 1–10 mg of safrole into 1 g of essential oil extracted from Small-fl ower Camphor wood (Cinnamomum micranthum Hayat), the recovery rates of safrole were determined. With direct injection of samples into the gas chromatograph, the results showed that the recovery was more than 96.1%, with a coeffi cient of variation below 5.6%. We then analyzed 23 commercially available Stout Camphor and other essential oil samples and found that 21 of them contained safrole in the range of 37.65– 355.07 mg/g. In addition, in the heavier essential oil distilled from Small-fl ower Camphor wood, the safrole level was up to 642.98 mg/g. Our results demonstrated that most camphor essential oils on the market have a carcinogenic potential due to their high safrole levels. INTRODUCTION Safrole (4-allyl-1,2-methylene dioxybenzene) is a principal component of essential oils obtained from several herbs and spices, such as the piper betle fl ower, camphor, nutmeg, and sassafras (1–7). In Taiwan, the term “camphor tree” indicates several species of trees belonging to the Cinnamomum genus, which can be subclassifi ed into four groups based on the recovery rate of camphor (8): camphor tree, linalool tree (C. camphora CT linalool), C. longepaniculatum, and C. camphora (Linn.) Presl var. camphora. These four groups of “camphor trees” are very diffi cult to distinguish according to their morphology. Based on Address all correspondence to Shun-Hsiang Weng at ssweng@meiho.edu.tw.
JOURNAL OF COSMETIC SCIENCE 146 differences in the main component of essential oils obtained from their leaves, camphor trees can be subclassifi ed into fi ve subgroups: (a) camphor tree (C. camphora subsp. formosana var. oxidentalis), in which the main component is camphor (b) linalool tree (C. camphora CT linalool), in which the main component is linalool (c) cineole tree (C. camphora (Linn.) Presl var. camphora), in which the main component is cineole (d) sesquiterpene tree, in which the main component is nerolidol and (e) safrole tree, in which the main component is safrole. Although it is not easy to distinguish them according to their morphology, the main component in their leaf essential oil can help to classify these trees. A study of cam- phor tree essential oils (Fujita et al., [9]) showed that oils extracted from the leaves and fruits of C. camphora Sieb contain 85% and 30–43% camphor, respectively, and oils extracted from the fruits also contain 40–59% safrole. Safrole is a known carcinogenic substance that causes liver tumors (10–12). The metabolites of safrole, including 1-hydroxysafrole, isosafrole, and dihydrosafrole, are all carcinogens (11). A study has also shown that orally administered safrole in humans resulted in four major metabolites, of which safrole 2′,3′-oxide (SFO) is a reactive electrophilic metabolite and a high concentration of safrole (0.5%) increased the incidence of cancer in a mouse model (12). Exposure to SFO caused an increase in micronuclei in mouse red blood cells and signifi cantly induced DNA strand breaks, indicating that the toxicity of safrole is due to its metabolites having carcinogenic effects (13–15). At present, methods for the analysis of safrole include gas chromatographic methods (16–20) and HPLC methods (6,7,21,22). Among these methods, the Association of Offi cial Analytical Chemists (AOAC) method (19,23,24) uses steam to distill safrole, followed by chloroform extraction and subsequent analysis using gas chromatography. However, the protocol is tedious and time-consuming, and the yield of safrole is low because losses occur during the multiple steps of the process. In addition, chloroform is a toxic and controlled reagent. Therefore, the method is complicated and has many limitations. Currently, aro- matic therapy is becoming popular, and many essential oils are used in body sprays, evapo- rative aromatherapy, and even for oral administration. If essential oils have high safrole levels, serious negative health effects will result. Therefore, the development of a simple and rapid quantitative method is required for screening the safrole contents of essential oils on the market. This study aimed to develop a simple and reliable method to quantify safrole that only requires the addition of an internal standard (IS) to essential oil samples without pretreat- ment, and the sample can be directly analyzed using gas chromatography. METHODS AND MATERIALS MATERIALS Essential oil samples were purchased from the market, including 15 Stout Camphor essen- tial oil (prepared from C. kanehirai Hayata) samples from four different manufacturers in Taiwan (Joben Bio-Medical co., Pingtung I Chuan Bio-Tech Corp., Tainan Golden Dapu Biotech Crop., Chaiyi and Yu-jang Biotech Co., Taoyuan), one sample each of the upper layer and the lower layer of Small-fl ower camphor essential oil, and a cypress oil sample (from Joben Bio-Medical co.), all of which were included in the analyses in this study.
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