Policosanol Contents and Compositions in Wax-Like Materials Extracted from Selected Cereals of Korean Origin

Cereal Chern. 82(3):242-245 Policosanols. long-chained alcohols. have been reported to have beneficial physiological activities. Content and composition of policosanols in wax-like materials extracted from selected cereals of Korean origin were determined. Wax-like materials were extracted using hot hexane. Yields of wax-like materials from unpolished grain sorghum, polished grain sorghum. brown rice. purple rice, wheat, and maize were 223, 37, 33, 61, Policosanols are a mixture of primary long-chained alcohols and are available commercially as nutritional supplements containing mostly octacosanol (28:0), triacontanol (30:0), hex acosanol (26:0), and dotriacontanol (32:0). They have been produced from sugar cane, beeswax, and kernels of cereal grains. Policosanols have beneficial physiological activities such as improving blood lipid levels (Aruzazabala et al 1994; Kato et al 1995; Gouni-Berthold and Berthold 2002), reducing platelet aggregation (Aruzazabala et al 1996), improving exercise performance of coronary heart disease patients (Sttisser et al 1998), and increasing muscle endurance (Kabir and Kimura 1995). However, Wang et al (2003) reported that policosanols have no significantly favorable effect in changing blood lipid levels of hamsters. Recently, nutritional significance and metabolism of policosanols were reviewed by Hargrove et al (2004). Surfaces of kernels of cereal grains are covered with epicuticular wax, which appears to regulate the water balance of the parent plant by reducing evaporation to minimize mechanical damage to cells and to inhibit fungal and insect attacks. Bianchi et al (1979) and Avato et al (1990) extracted wax-like materials (WLM) from grain sorghum grown in Italy and reported that the WLM consisted of alcohols (32 and 34%, w/w, db, respectively), otherwise known as policosanols, acids (24 and 27%, respectively), and aldehydes (21 and 32%, respectively), fractionated by column chromatography. Recently, Hwang et al (2002a,b; 2004) confirmed that aldehydes, alcohols (policosanols), and acids were the major components of the WLM extracted from grain sorghum grown in the United States in Nebraska, comprising 46-55% (w/w, db), 37-41 %, and 4-7%, respectively, as determined by HPLC. Other cereal grains have not been studied extensively regarding their policosanol contents. Bianchi et a1 (1984) extracted wax from maize kernels using chloroform, reporting a wax mass of 2.7 mg recovered from 20 hand-harvested and hand-shelled ears. They reported esters comprised 76% (w/w, db) of the wax and alcohols comprised 2 %. According to the study by Kawanishi et al (1991), octacosanol contents in germs of rice, wheat, and maize were 0.46, 0.22, and 0.82 mgll 00 g, respectively. They reported petroleum ether-extractable materials (including waxes and oils) in the germs, but did not report levels of WLM. I Department of Food Science and Human Nutrition. and Center for Healthcare Technology Development. Chonbuk National University, Jeonju. leonbuk. 561756. Korea. 2 Corresponding author. Phone: +82-63-270-3857. Fax: +82-63-270-3854. E-mail: [email protected] 3 Department of Biological Systems Engineering. University of Nebraska. Lincoln. NE 68583. 001: 10.1 094/CC-82-0242 © 2005 AACC International, Inc. 242 CEREAL CHEMISTRY 10, and 10 mg/lOO g of dry kernels, respectively. Policosanol contents, as determined using HPLC, in the wax-like materials from the cereals were 33,29,6.0, and 2% (w/w, db), respectively. Major alcohols in the policosanols from grain sorghum were octacosanol and triacontanol. Docosanol was the major alcohol in the policosanols from brown rice. purple rice, wheat. and maize. The objectives of this study were to determine and compare yield and composition of WLM (recovered using hot hexane and precipitated in a cold environment) extracted from grain sorghum, brown rice, purple rice, wheat, and maize, all of Korean origin, and to determine the content and composition of policosanols in theWLM. MATERIALS AND METHODS Raw Materials Intact kernels of unpolished grain sorghum (purple), abrasively polished grain sorghum (purple color partially retained), brown rice (round japonica type), purple rice (unpolished long grain), wheat (soft, winter, red), and maize (flint, yellow) were obtained from a local market in Jeonju, Korea. The cereal kernels used in the study were grown in Korea. The parent grain sorghum plants were tall and their heads drooped upon ripening. The color of the pericarp of the grain sorghum kernels was purplish, whereas the endosperm was creamy white. An abrasive type of polishing machine (IDP-3000, Ideal System Co., Daegu, Korea) was used for polishing of grain sorghum. Yield of polished sorghum from whole grains was 70-75%. Polished sorghum kernels retained a portion of the purple pericarp on their surfaces. The polished sorghum kernels are a common food material in Korea. Extraction of Wax-Like Materials from Cereals Extraction of WLM from whole cereal kernels followed the same method as in Hwang et al (2004). Cereal kernels were washed with tap water and dried at 45°C over 24 hr. Kernels (800 g at ~ 1 0% [wb] moisture) with 800 mL of hexane were refluxed for 30 min. The mixture was filtered through a coffee filter paper lying on top of a Whatman No.2 filter paper (Whatman, Maidstone, Kent, UK). The filtrate was placed in a freezer at -18°C for at least 8 hr. Filtering the cold miscella on to Whatman No. 42 filter paper, which was then desolvented under vacuum, collected the precipitate of the WLM. TLC of Wax-Like Materials Extracted from Cereals Thin-layer chromatography (TLC) separation of WLM extracted from cereals was completed as in the previous report (Hwang et al 2002a). The WLM dissolved in hexane (~100 /-lgllO /-lL) were spotted on a TLC plate (general purpose, 20 x 20 em, particle size 250 /-lm) (Aldrich Chemical Co., Milwaukee, WI). The developing solvent was hexane, diethyl ether, and acetic acid (85:15:2, v/v). Developed bands were visualized by dipping the plate in a solution of 10 g of cupric sulfate dissolved in 100 mL of 8% phosphoric acid for 5 sec, letting it dry for 5 min, and heating it in an oven at ~150°C until the developed bands were charred. Compositional Analysis of Wax-Like Materials Using HPLC Policosanol content along with contents of major components in the WLM extracted from cereal kernels were determined using an HPLC system as in Hwang et al (2002b; 2004). Two HPLC pumps (SI 0, Waters Corp. , Milford, MA) were operated in gradient modes. Flow rate of mobile phase was 1 mL/min. Column was a Luna S J.l si lica column (2S0 mm length x 4.6 mm i.d.) (Phenomenex, Torrance, CA). The column was heated at 40°C using a Waters column heater module. Exposed lines between injection loop and detector connection were maintained at ::d8-40°C by wrapping in heating tape. Detector was a Varex ELSD II (Rockville, MD) operated at SO°C with nitrogen pressure of 930 kPa. Samples were dissolved in hexane at 20 J.lg/l 00 J.lL, and 100 J.lL of each was injected. Compositional Analysis of Policosanols Fractionated from Wax-Like Materials of Cereals Using GC Policosanol composition in WLM from whole cereal followed the same method as Hwang et al (2004). Policosanol fraction (2 mL) was collected from 20 !-lg of WLM using HPLC. Policosanol composition was determined using Gc. To desolventized policosanol fraction, 0.2 mL of chloroform and O.OS mL of N-methyl-N(trimethylsilyl)trifluoroacetamide (Sigma Chemical, St. Louis, MO) were added. The content was heated at 60°C for 10 min to make trimethylsilyl (TMS) ether derivatives. A standard mixture of palmityl alcohol, stearyl alcohol, eicosanol, behenyl alcohol, tricosanol , lignoceryl alcohol (Nu-Chek Prep, Inc. , Elysian, MN), hexacosanol , heptacosanol, octacosanol , and triacontanol (Sigma Chemical) containing 1-8 )lg of each in 0.2 mL of chloroform was prepared and derivatized in the same manner as above for identifying the retention times of the alcohols and for calculating their response factors. The TMS ether derivative solution (2 )lL) was injected into a gas chromatography system (GC) (HP6890, Hewlett-Packard Co. , Wilmington, DE) equipped with a DB-S column (30 m, 0.2S mm i.d. , and 0.2S !-lm film thickness) (J&W Scientific, Folsom, CA) and using a carrier gas of helium. Detector was a flame-ionization detector. Injector and detector temperatures were both set at 31S°C. Oven was programmed to start and hold at IS0°C for 1 min before increasing to 210°C at 20°C/min, increasing to 310°C at 4°C/min, holding at 310°C for 1 min, increasing to 31SoC at 2SoC/min, and finally holding for S min. Content, yield , and composition values are means of three determinations except for composition of the WLM and policosanol composition of maize. RESULTS AND DISCUSSION Moisture and crude lipid contents of cereal grains observed in the study are shown in Table 1. Crude lipids in unpoli shed and polished grain sorghum were 4.8 and 2.6% (w/w, wb), respectively, indicating lipids still remain after the poli shing of grain sorghum kernel s. Purple rice was a little higher in lipid content than brown rice . Whole wheat contained < ] % lipids. Yield of WLM extracted from grain sorghum was 223 mg/] 00 g of dry kernels (Table I), similar to the values from various grain sorghum types produced in Nebraska (200-300 mg/IOO g of dry kernels (Hwang et al 2004). A fairly large amount of WLM (37 mg/lOO g of dry kernels) still remained on the polished grain sorghum. WLM leve ls from brown rice, purple lice, wheat, and maize were fairly low compared with those of grain sorghum. TLC of WLM extracted from whole grain sorghum resulted in two dark spots representing aldehydes and poJicosanols and some light spots representing hydrocarbons, wax esters and steryl esters (WE/SE), and acids (Fig. 1). Quantitative composition of the WLM of whole grain sorghum, analyzed by HPLC, was S7 % (w/w ) aldehydes, 33% policosanols, 4% acids, 2% hydrocarbons, and 1% triacylglycerols (Fig. 2, Table I). WEISE did not appear on the HPLC results. TLC of WLM from polished grain sorghum showed darker spots for WEISE, triacy lglycerols, and acids compared with TLC of unpoli shed grain sorghum (Fig. 1). It is consistent with the HPLC results (Fig. 2, Table I). Aldehydes (34%) and policosanol s (29%) were still major components of the WLM extracted from polished grain sorghum. While WEISE were not detected in the WLM from whole grain sorghum, a fairly large quantity of WEISE ( l3 o/c) was detected in the WLM from the polished grain sorghum. It implies that WEISE may exist in the inner parts of grain sorghum kernels rather than on the surfaces.