Extraction and Characterization of Natural Higher Aliphatic Alcohol from Sugarcane Wax
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s i n S S al E n o u A i E s g R R s R e a C A of r H r G a U P n & f S o d I P R n n t R o i e O t g F O a IAPSIT i r a M c Y o t e T O s d s E T T I A I e l C O c a h n O N RESEARCH ARTICLE T n o i o S w o t w o a l a r o r n r d o g r s e m i t a o e n S T s I w s e u e o t e r r e a nd Prosp Estd. 2004 Sugar Tech 9(4) (2007) : 244-248 Extraction and Characterization of Natural Higher Aliphatic Alcohol from Sugarcane Wax GAN-LIN CHEN1* , HAI-RONG GUO2, XIAO-FEI WANG2 and YANG-RUI LI3 1Guangxi Sugarcane Research Institute, Nanning 530007, China 2College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China 3Guangxi Academy of Agricultural Sciences, Nanning 530007, China ABSTRACT Natural higher aliphatic alcohol (HAA), a bioactive substance, is widely found in waxy materials such as sugarcane wax, and can be extracted from filter mud or bagasse in sugar mill. This material is widely used in food, medicine and cosmetics industries as a kind of natural additive. In the present study, the purified HAA and its by-product of higher fatty acids (HFA) was extracted and purified through a series of processes that involve saponification and hydrolysis with alkali, calcification, Soxhlet extraction and crystallization by gradient cooling. The process of extracting the HAA from sugarcane wax was described and optimized using an orthogonal experimental design. From the analytic identification and quantification using GC-MS, the purified HAA obtained was found to be up to 90.7% in purity. The total content of the main bioactive components containing an even number of carbon atoms from C22 to C30 can reach up to 40.5%. Key words: higher aliphatic alcohol, higher fatty acid, extraction, characterization, by-products INTRODUCTION As the properties of sugarcane wax are similar to the rare Carnauba wax (Cai, 1994), sugarcane wax will be a likely Sugarcane wax, one of the higher vegetable waxes, is a substitute for Carnauba wax in the future. Sugarcane wax also kind of lipid found in sugarcane, which contains waxy lipids has a wide range of uses and contains other natural bioactive and fatty lipids, making up about 0.18~0.26% of the weight of substances such as phytosterol, octacosanol and other higher milling cane. Waxy lipids are a kind of compact crystalline aliphatic alcohols, which can be extracted and used as natural substance that is anchored on the cuticle of the stalk and the additive for functional foods, medicine and cosmetics (Chen node of sugarcane, comprising about 0.12% of the cane weight. et al., 2005). Fatty lipids are found inside of the sugarcane stalk and are the Higher aliphatic alcohol (HAA), which is also called higher major component of vegetable cell plasma. It's content is aliphatic alkanol or long-chain fatty alcohol, is a mixture of variable depending on the variety and the growth conditions higher saturated aliphatic primary alcohols containing 22-38 of sugarcane. In the milling process of sugarcane, about 60% carbons that has been widely found in waxy materials such as of the lipids is left in the bagasse. The rest is transferred into sugarcane wax, rice bran wax, beeswax, apple peel and wheat the mixed juice, of which 95% goes to the sediment of the filter germ (Kazuko et al., 1991). Its healthy benefits and biological mud when lime milk is added during the clarification of the activity include curing or healing hypercholesterolemia, mixed juice (Cai, 1994). Therefore, sugarcane wax is usually atherosclerotic complications as platelet hyperaggregabiulity, produced by extraction from filter mud or press mud waste ischemia and thrombosis, and preventing drug induced gastric and bagasse (Phukan et al., 1999; Chen et al., 2004). Most of ulcer and improving male sexual activity (Laguna et al., 1999). the sugarcane wax product used in the experiment was obtained from filter mud. The monocomponent of aliphatic alcohol has its own respective biological activity and health benefits. For instance, *Corresponding author e-mail: [email protected] ©2007 Society for Sugar Research and Promotion Gan-Lin Chen, Hai-Rong Guo, Xiao-Fei Wang and Yang-Rui Li docosanol (C22) can be used for restraining tumor of prostate, hours and then shaken at a constant temperature of 80oC for tetracosanol (C24) for strengtherning nerve system, 24-36 hours in order to obtain the mixture of higher fatty acids hexacosanol (C26) for lowering blood fat, octacosanol (C28) salts (K-HFA) and HAA. for lowering blood lipid or cholesterol (Arruzazabala et al., Calcification 1994), improving athletic performance (Rapport, 2000; Durden et al., 1993), offering cytoprotection role of trophic nerve The HFA components were separated from the HAA by (Carbajal et al., 1996), inhibiting blood platelet aggregation adding saturated calcium chloride (CaCl2) solution, equivalent (Carbajal et al., 1998), triacontanol (C30) for regulating and to the KOH used, in order to precipitate the water-insoluble controlling plant growth (Ries et al., 1977). calcium fatty acid salt (Ca-FA). The calcification reaction was maintained for 2-3 hours at 80oC and produced a precipitate of At present, there is a large gap between the demand of granules about 1 mm in diameter, which was washed with hot- sugarcane wax and higher aliphatic alcohol and the supply of water, dried at 60-65oC and crushed. the materials in the domestic and foreign markets, especially the mixture of HAA that is rich in the octacosanol (C28) Soxhlet Refluxing Extraction (SRE) ingredient. In order to optimize the operating conditions of the Guangxi Zhuang Autonomous Region, the biggest sugar- extraction solvent, the refluxing temperature and the extraction producing province in China, produced 7.18 million tons of time, the SRE experiments were performed in nine groups using sugar in 2006/2007 milling season, accounting for over 60% of 4 an orthogonal design of L9 (3 ) and the experiment results the total sugar output in the country. The sugar industry is were evaluated using the yield and production rate of the rich in raw materials of HAA, such as the sugarcane wax that is crude HAA and the purified HAA as measures. found in the filter mud and bagasse. This study deals with the extraction of HAA from sugarcane wax in filter mud. The The mixture of HAA and Ca-FA (5.00 g) was taken to operating processes were optimized to obtain the purified HAA extract the HAA mixture for 8-16 hours by Soxhlet extraction with high purity. The analyses of purified HAA and higher (SOTEX ANANTI) and all treatments were performed in the o fatty acids (HFA) products were carried out using GC-MS to range of 50-80 C in the medium containing 6-8 times (for the identify, quantify and characterize the monocomponents. ratio of weight of solid and volume of liquid, g/mL) of organic solvent such as acetone, anhydrous ether and mineral ether. At the given reaction time, samples of extractive liquor were MATERIALS AND METHODS taken from the Soxhlet extractor and dried and weighed. The sugarcane wax was derived from the filter mud of a Purification and crystallization sugar mill (Shangsi Sugar Mill, Guangxi, China) and refined by In the present study, the crude HAA obtained in each deliming, degreasing and decolouring. It was air-dried, crushed group was purified by crystallization using solvents such as and homogenized and stored until needed. Analyses of the n-hexane, methanol and chloroform. The crude HAA was air- refined sugarcane wax were carried out using methods reported dried and then recrystallized 3-4 times with n-hexane through by Chen (2006). The main physical and chemical properties of the crystallization of gradient cooling from 75oC to room the refined sugarcane wax are shown in Table 1. temperature. Samples of purified HAA were collected and Table 1: Main physico-chemical constants of sugarcane wax weighed at certain time. used in this study Recovery of HFA Physico-chemical constants Experimental data Color Brown The residues in each extraction thimble (that is, the Melting point (oC) 74～80 mixture of calcium fatty acids (Ca-FA)) were collected and Saponification value (mg KOH/g) 80.76 weighed. Samples of the Ca-FA mixture (2.0 g) were heated to Acid value (mg KOH/g) 25.24 80oC with 4 times of water (corresponding to the weight of Ca- Esters value (mg KOH/g) 55.52 FA) and adding the quantitative concentrated HCl to adjust Iodine value (g I2/100g) 18.23 pH to 1-1.5. After being boiled for 2-3 hours, the solid granule disappeared and the solid phase was left. At the end, the Saponification crude HFA was obtained through washing the acid solid phase The saponification (hydrolysis with alkali) of the to neutral products with hot water and drying at 50-55oC. sugarcane wax converts the component esters into the Analysis of HAA and HFA respective alcohols and acids. In this work, a 10.0 g sample of the sugarcane wax was added to 30 mL of triple-distilled water Thirty mL of acetylating agent was added to a 2.0 g containing a few drops of polyglycol as a surfactant, plus 20 sample of purified HAA and refluxed for 1 hour at 75 oC. The mL of 0.2 M KOH (Chen, 2006).