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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). The mixture was boiled for 8-10 resulted liquid was extracted with three volumes of benzene

Sugar Tech 9(4), 2007 245 Extraction and Characterization of Natural Higher Aliphatic Alcohol from Sugarcane Wax

(50 mL) and distilled water (50mL). The benzene phases were were shown in Fig.1, where Ki (i=1, 2, 3) is the sum of all results combined and treated with anhydrous sodium sulfate (Na2SO4) at the same level, ki (i=1, 2, 3) is the average of Ki and R is the to remove the water and concentrated for 1-2 hours at 50-55oC range of the ki in a array. for analysis of HAA by GC-MS (QP5050A, SHIMADZU). Table 3: Range analysis of the PR of HAA crude & purified Thirty millilitres of carbomethoxy agent was added to a product 1.0 g sample of HFA and refluxed for 2 hours at 75oC. The resulted liquid was extracted with three times of n-hexane PR of crude HAA (%) PR of purified HAA (%) (50mL) and distilled water (50ml). The n-hexane phases were K A B C K A B C K1 127.6 153.4 150.6 K1 81.4 91.4 91.4 combined and dewatered using anhydrous Na2SO4 for analysis of HFA by GC-MS. K2 160.2 142.6 145.0 K2 93.2 86.8 88.6 K3 156.4 148.2 148.6 K3 92.6 89.0 87.2 The GC-MS analyses were carried out using temperature k1 42.53 51.13 50.20 k1 27.13 30.47 30.47 programming, a DB-1 column (30 m×0.25 mm× 0.25 µm) and a k2 53.40 47.53 48.33 k2 31.07 28.93 29.53 70 eV EI ion source. The operating temperature was as follows: k3 52.13 49.40 49.53 k3 30.87 29.67 29.07 injector, 280oC; detector, 30oC; initial oven temperature 150oC R 10.87 3.60 1.87 R 3.933 1.533 1.40 (held for 25min), followed by a ramp rate of 4oC /min to 250oC (held for 5 min), and finally a ramp rate of 8oC /min to 290oC. 70 The scanned range was 33~800 amu. Crude product ) 60 Purified product % (

A 50

RESULTS AND DISCUSSION A H

f 40 o

In this study, three organic solvents (acetone, anhydrous R 30 P ether and mineral ether) were investigated as SRE solvents. In 20 the experiment, the operating temperature for acetone was 70oC, o o o o o 10 r . r t e e h h h 75 C and 80 C and 50 C, 55 C and 60 C for both anhydrous 1 2 3 e p e n n 6 2 8 B B B h m h e i t m o t 1 1 v t l e e T e

e

ether and mineral ether. The refluxing time was 8 h, 12 h and 16 o l T c e a t S A r u l h, which could extract HAA products effectively. e o n i s Levels b M The yield and production rate (PR) (that is, per cent of A starting material) of the related HAA products and the HFA Fig.1 Graph of the PR of HAA crude & purified products in the products during the SRE are shown in Table 2. different factors In order to obtain the optimum extraction conditions for In order to explore the significant difference of extraction the crude HAA using SRE system, the range analyses of the conditions further, the variance analysis of the PR of crude PR of HAA crude product and purified product were HAA are worked out, and the results were shown in Table 4, conducted and the results were shown in Table 3, and the where SS is the quadratic sum of all results at the same factor, relationships between the extraction factors and the levels DF is the degree of freedom and MS is the mean squares.

Table 2: Experimental results on the yield of HAA crude & purified products and HFA (For 5.0 g sample) Soxhlet Refluxing Extraction of HAA (SRE) Purification of HAA Recovery of HFA Yield of Yield of PR of B C PR of No. A crude purified purified HFA Temp. Time crude Ca-FA (g) Extraction solvent HAA HAA HAA (g) (oC) (h) HAA (%) (g) (g) (%) 1 Acetone 1(80) 16 2.20 44.0 1.41 28.2 2.64 2.21 2 Acetone 2(70) 12 1.95 39.0 1.28 25.6 2.60 2.18 3 Acetone 3(75) 8 2.23 44.6 1.38 27.6 2.35 1.92 4 Anhydrous ether 1(60) 12 2.80 56.0 1.64 32.8 1.81 1.46 5 Anhydrous ether 2(50) 8 2.53 50.6 1.46 29.2 2.18 1.68 6 Anhydrous ether 3(55) 16 2.68 53.6 1.56 31.2 1.90 1.60 7 Mineral ether 1(60) 8 2.67 53.4 1.52 30.4 1.98 1.58 8 Mineral ether 2(50) 16 2.65 53.0 1.60 32.0 1.97 1.55 9 Mineral ether 3(55) 12 2.50 50.0 1.51 30.2 1.77 1.36

246 Sugar Tech 9(4), 2007 Gan-Lin Chen, Hai-Rong Guo, Xiao-Fei Wang and Yang-Rui Li

Table 4: Variance analysis of the PR of crude HAA The mass spectrogram of the purified HAA was shown in Fig.2 and of the mixture of HFA was shown in Fig.3. Variance Nota- SS DF MS F-value Fα source bility A Extraction F 211.8489 2 105.9245 13.5491 0.05 * solvent (2,2)=19 o B Temp. ( C) 19.4489 2 9.7245 1.2439 F0.1 (2,2)=9 C Time (h) 5.3689 2 2.6845 <1 Error 15.6356 2 7.8178 Fig. 2 The mass spectogram of the purified HAA The results of range and variance analysis of the PR of crude HAA in Table 3 and Table 4 showed that extraction solvent had a greater impact on the extraction of HAA than the temperature or the length of time for SRE. It is observed that the PR of crude HAA reached a maximum value of 56.0% when performed at 60oC and 12 h using anhydrous ether and reached a minimum value of 39% when performed at 70oC and 12 h using acetone. However, the samples of crude HAA extracted using acetone had a better Fig. 3 The mass spectogram of the mixture of HFA color (light to dull yellow) and a better luster than the samples Based on reference of mass spectrograms (Pretsch, 2000) extracted using anhydrous ether and mineral ether, which were and eliminating the interference spectra and mixed spectra, generally grey or brownish-yellow and had a poorer luster, the components and contents of purified HAA and mixture of suggesting the presence of significantly more impurities. HFA were identified and quantified as shown in Tables 5 and Therefore, it is conclude that the preferred extraction solvent 6. is acetone (with a PR value of 42.53%), because the purity of the crude HAA is the preferred technical parameter for further Table 5: Components and contents of the purified HAA processing of HAA. HAA Carbon number Peak Content (%) It can be remarked that the extraction efficiency increases Eicosanol C20 2 0.68 with refluxing temperature, because the solvent is volatilized Heneicosanol C21 3 2.2 and condensed more quickly as the temperature increases, Docosanol C22 4 4.59 which results in the material in the extraction thimble being Tricosanol C23 5 6.55 exposed to a greater quantity of extracting liquid in a given amount of time. Tetracosanol C24 6 8.59 Pentacosanol C25 8 9.07 The data also showed that the extraction time had Hexacosanol C26 11 10.23 relatively small effect on the PR of crude HAA. For example, Heptacosanol C27 14 9.81 the relative higher PR value reached 53.60% at 16 h of extraction Octacosanol C28 17 9.98 at 55oC, whereas the PR value of 53.4% was at 8 h of extraction that was nearly in accordance with the PR value at 12 h of Nonacosanol C29 21 8.86 extraction. Therefore, the extraction time of 8 h was the preferred, Triacontanol C30 26 7.11 and the PR value of crude HAA was 40%-50%. Hentriacontane C31 29 8.37 Dotriacontanol C32 31 2.95 The data presented in Table 3 showed that, in general, Psyllic alcohol C33 34 1.71 the PR of purified HAA was proportional to the PR of crude HAA, indicating that the impurities in the various trials were Others 9.3 similar and were produced at a similar rate. The highest PR The HAA monocomponents constituted more than 90% value of purified HAA reached 32.8%. More generally, PR of the purified HAA mixture (Table 5). Furthermore, the alcohols values were in the range of 25%~30%. with even numbers of carbons from 20 to 33 (which had greater Fatty acid calcium (Ca-FA) was the main component of biological activity) constituted more than 40% of the HAA the residue in the thimble after extraction of the crude HAA. mixture. These data showed that the SRE method of obtaining The yield value from the 5.0 g sample of sugarcane wax of Ca- HAA had resulted in a mixture that was rich in components FA ranged from 1.81 g to 2.64 g with the PR range of 30%-40% (those with an even number of carbon atoms) that would be in average. particularly useful in commercial applications.

Sugar Tech 9(4), 2007 247 Extraction and Characterization of Natural Higher Aliphatic Alcohol from Sugarcane Wax

Table 6: Components and contents of HFA mixture product Guangxi University from US Environmental Protection Agency, for his warmhearted assistance in the revision and review of HFA Carbon number Peak Content (%) this paper. Cydonic acid C17 3 9.9 Nondecylic acid C19 4 1.75 REFERENCES Heneicosanic acid C21 7 0.44 Docosanoic acid C22 8 3.24 Laguna Granja, A., Magraner Hernandez, J., Carbajal Tricosanic acid C23 9 5.04 Ouintana, D., Arruzazabala Valmana, L., Mas Ferreiro, R. Tetracosanic acid C24 11 6.43 and Garcia Mesa, M. (1999). Mixture of higher primary aliphatic Pentacosanoic acid C25 13 8.44 alcohols, its obtention from sugar cane wax and its pharmaceutical Hexacosanoic acid C26 15 9.23 uses. United States Patent, 5856316 Carboceric acid C27 18 10.08 Phukan, A.C. and Boruah, R.K. (1999). Extraction and evaluation Octocosoic acid C28 21 8.95 of microcrystalline wax from press mud waste of the sugar Montanic acid C29 24 8.33 industry. Separation and Purification Technology, 17: 189-194 Triacontanoic acid C30 27 5.74 Arruzazabala, M. L., Carbajal, D., Mas, R., Molina, V., Valdes, Myricinic acid C31 28 3.47 S. and Laguna, A. (1994). Cholesterol-lowering effects of Lacceroic acid C32 29 1.71 policosanol in rabbits. Biological Research, 27: 205-208 Others 17.25 Cai, D.W. (1994). Production of sugarcane wax from filter mud. Fujian Cane Sugar, 2: 46-62 Similarly, the data in Table 6 showed that the HFA mixture consisted more than 80% of aliphatic fatty acids with chains Carbajal, D., Arruzazabala, M. L. Valdes, S. and Mas, R. (1998). Effect of policosanol on platelet aggregation and serum levels of ranging from 17 to 32 carbon atoms. The fatty acids with an arachidonic acid metabolites in healthy volunteers. even number of carbon atoms constituted more than 40% of Prostaglandins, Leukotrienes and Essential Fatty Acids, 58(1): the total, while the fatty acids with an odd number of carbon 61-64 atoms constituted more than 50% of the total. Carbajal, D., Molina, V., Valdes, S., Arruzazabala, L., Rodeiro, I., Mas, R. and Magraner, J. (1996). Possible cytoprotective CONCLUSION mechanism in rats of D-002, an antiulcerogenic product isolated from beeswax. Journal of Pharmacy and Pharmacology, 48: 858- The present study showed that the sugarcane wax from 860 filter mud of sugar mill was good raw material to extract HAA Chen, F., Cai, T.Y., Zhao, G.H., Liao, X.J., Guo, L.Y. and Hu, product. The yield of crude HAA reached up to 40%-50% by X.S. (2005). Optimizing conditions for the purification of crude the processes of saponification, hydrolysis, calcification, octacosanol extract from rice bran wax by molecular distillation dewatering, and Soxhlet refluxing extraction using different analyzed using response surface methodology. Journal of Food Engineering, 70:47-53 extraction solvents, extraction temperatures and extraction times. The yield of purified HAA ranged from 25% to 30%. Chen, G.L. (2006). Studies on extraction of higher aliphatic alkanol from filter mud in sugarcane factory [D]. Master Degree Thesis The identification and quantification of the extracts of Guangxi University, 37-44 indicated that the HAA product and HFA product obtained Chen, G.L., Guo, H.R. and Zhang, S.Y. (2004). Study on extraction had many components, most of them were aliphatic of sugarcane wax and lipid from bagasse. Guangxi Sugarcane, compounds with the number of atoms between 20 and 34. The Canesugar, 4: 26-30 recoveries of the HAA product reached over 90% and the Durden Beltz, S. and Doering, P.L. (1993). Efficacy of nutritional components with significant bioactivity accounted for more supplements used by athletes. Clin Pharm, 12: 900 than 40%. E. Pretsch, Bûhlmann, P. and Affolter, C. (2000). Structure determination of organic compounds table of spectral date. (in The purified HAA produced by this SRE method has US). Translated by Rong G.B. (2002), Shanghai: East China good color (light yellow) and luster, and can be used as a kind University of Science and Technology Press, pp.245-312 natural supplement in industrial applications such as food, Kazuko, K., Kumlko, A. and Yohel, H. (1991). Free primary medicine and cosmetics. The extraction method developed in alcohols in oils and waxes from germs kernels and other the present study can be applied to commercial production. components of nuts, seeds, fruits and cereals. Journal of the However, additional work would be needed to further improve American Oil Chemists’ Society, 68(11): 869-872 the yield, purity and separation of HAA components. Rapport, L. J. (2000). Nutraceuticals (3): octacosanol. Pharmaceutical Journal, 265: 170-171 ACKNOWLEDGEMENT Ries, S. K., Wert V., Sweeley, C. C. and Leavitt, R. A. (1977). Triacontanol: a new naturally occurring plant growth regulator This study was supported by Guangxi Science [J]. Science, 195(4284):1339-1341 Foundation (Gui Ke Ji 0663023), China. The authors would like Received : August 2, 2007; Revised : October 7, 2007; to thank Donald G. Barnes, a Visiting Professor of chemistry at Accepted : November 30, 2007

248 Sugar Tech 9(4), 2007