The Antioxidative Substances in Cacao Liquor. Naomi OSAKABE,1,* Megumi YAMAGISHI,1 Chiaki SANBONGI,1 Midori NATSUME,1 Toshio TAKIZAWA1

Transcription

1 J Nutr Sci Vitaminol, 1998, 44, The Antioxidative Substances in Cacao Liquor Naomi OSAKABE,1,* Megumi YAMAGISHI,1 Chiaki SANBONGI,1 Midori NATSUME,1 Toshio TAKIZAWA1 and Toshihiko OSAWA2 1 Functional Foods Research and Development Laboratories, Meiji Seika Kaisha, Ltd., Sakado , Japan 2 Department of Applied Biological Sciences, Nagoya University, Chikusa-ku, Nagoya , Japan (Received July 10, 1997) Summary The antioxidative substances contained in cacao liquor, which is one of the major ingredients of chocolate, were separated by column chromatography and high-performance liquid chromatography. Three major compounds were purified and two of them were identified by 'H, 13C NMR and mass spectra as (-)-epicatechin (EC) and (+) -catechin (CA). Their antioxidative activity was measured by monitoring the peroxide value of linoleic acid and the thiobarbituric acid-reactive substance values of erythrocyte ghost membranes and microsomes. EC and CA had strong antioxidative effects in all three methods, but one unidentified peak was found to be less effective. Additionally, we ana lyzed the polyphenol concentration of cacao liquor extractions produced in several countries. The total polyphenol concentration was 7.0 to 13.0%, catechin concentration was 0.31 to 0.49%, and epicatechin con centration was 0.35 to 1.68% in the extractions. It is believed that cho colate is stable against oxidative deterioration on account of the presence of these polyphenolic compounds, and it is also expected to have a pro tective role against lipid peroxidation in living systems. Key Words cacao liquor, polyphenol, antioxidant, catechin, epicatechin It is well known that free radical oxidative stress is an important etiologic factor in many pathological processes, such as atherosclerosis (1, 2), cancer (3, 4) and diabetes mellitus (5). Defense against such prooxidant damage of biological molecules requires both intra and extra-antioxidative activity. Thus, much attention has been focused on the antioxidants in food with regard to their chemopreventive effects. * To whom correspondence should be addressed. Abbreviations: TFA, trifluoroacetic acid; TMS, tetramethylsilane; DW, distilled water; t-bhp, tent-butylhydroxyperoxide; SDS, sodium dodecyl sulfate. 313

2 314 N OSAKABE et al Among the various foods, chocolate is stable against oxidative deterioration. This is considered to be due to the fatty acid composition (6) and polyphenolics in cacao beans (7-9). According to a previous report, approximately 8% of the polyphenolics in unfermented beans is leukocyanidins and catechins (7). Furthermore, in the process of making cacao liquor, where fermentation, roasting and grinding of raw beans is conducted, it is assumed that these compounds undergo major change, however such changes have not been reported. In this study, we performed the isolation, structural elucidation, analysis of concentration and antioxidative evaluation of hydrophilic polyphenolics in cacao liquor as the major ingredients of chocolate. MATERIALS AND METHODS Cacao liquor. Fermented and dried cacao beans imported from Ghana Colombia, Ecuador, Ivory Coast and Brazil were roasted and grinded at Meiji, Seika Kaisha Ltd., Japan. Extraction of polyphenolics. Cacao liquor was defatted twice with 3 volumes of diethylether (Wako Pure Chemical Industries, Ltd.) in order to remove lipophilic antioxidants such as y-tocopherol. In general, it is contained at the level of 0.02% in cacao beans. The defatted sample was extracted with 5 volumes of boiling water for 30min. The extract was filtrated, concentrated in vacuo, charged on a Sephadex LH-20 column (Pharmacia Co., Ltd., 2.5cmƒÓ ~20cm) and eluted with distilled water (DW) with step-wise increases in the ratio of acetone (Wako Pure Chemical Industries, Ltd.). High-performance liquid chromatography (HPLC). Further purification of the crude polyphenolics was carried out by preparative HPLC using an ODS column (Toso Co., Ltd., 4.6 mmƒó ~150mm). The elution solvent was 25% methanol (Wako Pure Chemical Industries, Ltd.) containing 0.03% trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, Ltd.). Antioxidative activity. 1) Linoleic acid oxidation. Each sample was added at the concentration of 100ƒÊg/mL to linoleic acid (Wako Pure Chemical Industries, Ltd.). One gram of mixed solution in a conical flask was incubated at 30 Ž and the peroxide value was determined by iodometry. 2) Erythrocyte membrane ghost system. Erythrocyte ghosts were prepared from rat red blood cells (10). The induction of lipid peroxide with tent-butyl hydroxyperoxide (t-bhp, Sigma Chemical Co., Ltd.) in the erythrocyte ghost fraction was carried out by the method of Ames et al (11). Test chemicals dis solved with methanol were added to the erythrocyte ghost. After incubation, the thiobarbituric acid-reactive substance (TBARS) was measured according to the method of Ohkawa et al (12). The reaction mixture contained 0.1mL of sample 0.2mL of 8.1% sodium dodecyl sulfate (SDS), 1.5mL of 20% acetate buffer (ph, J Nutr Sci Vitaminol

3 Antioxidants in Cacao ), and 1.5mL of 0.8% TBA. The mixture was heated at 95 Ž for 60min. After cooling with tap water, 4.0mL of n-butanol was added, and the mixture was shaken vigorously. After centrifugation at 3,000rpm for 10min, the absorbance of the organic solvent layer was measured at 532nm. 3) Microsomal lipid peroxidation. Microsomes were prepared from rat liver. The induction of lipid peroxidation with ADP-Fe2+/NADPH was carried out according to Pederson et al (13). Test chemicals dissolved with methanol were added to the microsomal solution. After incubation, the TBARS was measured as described above. Instruments for structure elucidation. 1H and 13C NMR spectra were obtained using a JEOL JNM-JSX 400 spectrometer. Chemical shifts were recorded as a values using tetramethylsilane (TMS) as the internal standard. Mass spectra were obtained using a HITACHI M-80B. Analysis of polyphenol concentration. The total polyphenol concentration in the extracts was measured using the Prussian blue method according to Price and Butler (14). Epicatechin and catechin were analyzed by HPLC: column, ODS120T (4.6mmƒÓ ~150mm, Toso Co., Ltd.); solvent, DW:ethanol:acetic acid at 87:8:5; and detector, 280nm. RESULTS Isolation of antioxidants from cacao liquor The procedure adopted for the isolation and purification of polyphenols from cacao liquor is outlined in Fig, 1. Fig. 1. Isolation of polyphenolic substances from cacao liquor. Vol 44, No 2, 1998

4 316 N OSAKABE et al The antioxidative activity of each fraction measured with the linoleic acid system is shown in Fig. 2. The fraction eluted with 30% acetone (fraction 2) showed a prolonged induction period as compared with the control and the other fractions. Further purification of this fraction (fraction 2) was carried out by preparative HPLC. Three major peaks, compounds A, B and C detected at 280 nm (Fig. 3), were collected individually. Fig. 2. Effect of crude fraction from cacao liquor on linoleic acid autooxidation. Each fraction shown in Fig. 1 was added to linoleic acid at the concentration of 100ƒÊg/mL and incubated at 30 Ž., no addition;, fraction 1;, fraction 2; ƒ, fraction 3. Fig. 3. Typical HPLC chromatogram of fraction 2. conditions: column, ODS 120 T (4.6mmƒÓ ~150mm); solvent, 25% methanol containing 0.03% TFA; detector, 280nm. J Nutr Sci Vitaminol

5 Antioxidants in Cacao 317 Evaluation of antioxidative activity All experiments were done three times, and one of the typical results shown. 1) Effects on the linoleic acid oxidation. As shown in Fig. 4, compounds A and C had potent antioxidative activity equal to a-tocopherol. Compound B exhibited weak antioxidative activity. 2) Effects on the erythrocyte membrane ghost system. As shown in Fig. 5, compounds A and C showed marked antioxidative activity, but compound B showed Fig. 4. Effects of polyphenolic substances from cacao liquor on linoleic acid autooxidation. Each sample shown in Figs. 1 and 3 was added to linoleic acid at the concentration of 100ƒÊg/mL and incubated at 30 Ž., no addition;, fraction 2; œ, compound A; ƒ, compound B;, compound C;, ƒ -tocopherol. Fig. 5. Antioxidative activity of polyphenolic substances from cacao liquor on RBC ghost oxidation induced by t-bhp. Each sample was the same as in Fig. 4. Vol 44, No 2, 1998

6 318 N OSAKABE et al Fig. 6. Antioxidative activity of polyphenolic substances from cacao liquor on ADP-Fe2+/NADPH dependent oxidation of rat microsomes. Each sample was the same as in Fig. 4., fraction 2; œ, compound A; ƒ, compound B;, compound C;, ƒ -tocopherol. only weak activity. These results are roughly the same as in the case of the linoleic acid system. 3) Effects on the microsomal system. All three components used in this study were more effective than a-tocopherol in this method, as shown in Fig. 6. Structural elucidation Compound A was identified as (-)-epicatechin from the mass and NMR data: El-MS (m/z) 290 [M+]; 1 H-NMR (in DM SO d6) ƒâ 2.47 (1H, dd, H4), 2.68 (1H, dd, H4), 4.00 (1 H, m, H3), 4.66 (1H, d, OH3), 4.73 (1H, s, H2), 5.71 (1 H, d, H8), 5.89 (1H, d, H6), 6.65 (1H, d, H5'), 6.66 (1H, d, H6 L), 6.89 (1 H, s, H2 L);13C-NMR (in DMSO d6) ( (CS), (C7), (C9), (C4 L), (C3 L), (C 1 L), (C6 L), (CS L), (C2 L), 99.0 (C 10), 95.1 (C6), 93.8 (C8), 81.0 (C2), 66.3 (C3), 27.8 (C4) (15). Compound C was identified as (+)-catechin from the mass and NMR data: El-MS (m/z) 290 [M+]; 1 H-NMR (in DMSO d6) (52.34 (1 H, dd, H4), 2.65 (1H, dd, H4), 3.81(1 H, m, H3), 4.47 (1H, d, H2), 4.87 (1H, d, 0H3), 5.68 (1H, d, H8), 5.89 (1H, d, H6), 6.59 (1H, dd, H6 L), 6.68 (1H, d, H5 L), 6.72 (1H, d, H2 L); 13C-NMR (in DMSO d6) b (C7), (C5), (C9), (C4 L), (C3 L), (C 1 L), (C6 L), (C2 L), (C5 L), 98.5 (C 10), 95.0 (C6), 94.0 (C8), 78.0 (C2), 64.9 (C3), 28.2 (C4) (15). The chemical structures of these compounds are shown in Fig. 7. The retention times of compounds A (9.5min) and C (4.2min) in the HPLC analysis were identical with those of respective commercial HPLC-grade chemicals (Funakoshi, Japan). J Nutr Sci Vitaminol

7 Antioxidants in Cacao 319 Fig. 7. The chemical structures of (-)-epicatechin and (+)-catechin. Table 1. Concentration of total polyphenols, catechin and epicatechin in extracts of cacao liquor made in several countries. Concentration of polyphenols in extractions The total polyphenol concentration in the extracts derived from cacao liquor was 6.7 to 13.0% as shown in Table 1. These results show that the polyphenol content in cacao liquor differs with the place of production. The concentration of catechin was 0.31 to 0.49%, and that of epicatechin was 0.35 to 1.68%. These contents correlate with the total polyphenols of each sample. On the contrary, the extract derived from raw beans imported from Ghana showed a polyphenol content of about twofold before processing when compared with that of the cacao liquor made from the same beans. Furthermore, the concentrations of epicatechin and catechin in the extract from raw beans were about seven and tenfold when compared with that in the extract from cacao liquor. DISCUSSION Epicatechin and catechin are widely distributed in plants and have potent antioxidative activity (16, 17). We confirmed their effectiveness using three model experiments, and their antioxidative activities were similar to previous reports (17). In cacao liquor, their concentrations are low as shown in Table 1. However, it is Vol 44, No 2, 1998

8 320 N OSAKABE et al considered that these catechins contribute the most antioxidative effects in this extract. In the study, using microsomes, 80% of the antioxidative activity of the extracts was explained as epicatechin and catechin contribution (Fig. 6). In this study, we detected one unidentified peak in the HPLC chromatograph which might be a phenolic compound since a black sediment was produced after incubation with iron(iii) chloride. This component showed antioxidative activity only on the microsomel system. It is believed that this component might produce a chelate compound with iron, which was added in order to induce oxidation. In this study, the total polyphenols recovered from cacao liquor produced by fermentation and roasting of raw cacao beans was twofold less as compared to the extract derived from raw beans. Catechin contents were also 7 to 10 times less. We did not detect other reported phenolic substances such as leukocyanidine (7). These results suggest two possibilities. The first one is concerned with the source of the extract. In the process of making cacao liquor, cacao beans are fermented and roasted. These steps might influence the contents and chemical structures of the polyphenolic substances. The recovery of total polyphenols and catechins was not correlated, and this might lead to disparity. The second possibility is the method of extraction. In the case of green tea (16), low molecular phenols such as catechins are extracted sufficiently by hot water. In the case of cacao liquor, however, the polyphenols might not have been extracted in sufficient quantities. According to previous reports, catechins are polymerized during fermentation. For instance, theaflavins were produced in black tea (18) and procyanidins were increased in red wine (19). It is believed that these compounds were produced with the catechins during fermentation. A similar process as that for black tea and red wine was performed to make cacao liquor. The polymerization of catechins was expected, but we did not detect such a change. According to these findings, further experiments are required to explain the antioxidative activities of cacao liquor. REFERENCES 1) Gey KF Ten-year retrospective on the antioxidant hypothesis of arteriosclero sis: Threshold plasma level of antioxidant micronutrients related to minimum cardiovascular risk. J Nutr Biochem 6: ) Reavan PD, Ferguson E, Navab M, Powell FL Susceptibility of human LDL to oxidative modification. Effects of variations in /3-carotene concentration and oxygen tension. Arterioscler Tromb 14: ) Flagg EW, Coats RJ, Greenberg RS Epidemiologic studies of antioxidants and cancer in humans. J Am College Nutr 14: ) Weitsman SA, Gorton LI Inflammation and cancer: Role of generated oxidants in carcinogenesis. Blood 76: ) Thompson KH, Godin DV Micronutrients and antioxidants in the progression of diabetes. Nutr Res 15: ) Kattenberg HR The quality of cacao butter. Munufact Confect 76: J Nutr Sci Vitaminol

9 Antioxidants in Cacao 321 7) Forsyth WGC Cacao polyphenolic substances 3. Separation and estimation on paper chromatograms. Biochem J 60: ) Forsyth WGC Cacao polyphenolic substances 5. The structure of cacao leucocyanidin. Biochem J 74: ) Kim H, Keeney PG (-)-Epicatechin content in fermented and unfermented cocoa beans. J Food Sci 49: ) Osawa T, Ide A, Su J, Namiki M Inhibition of lipid peroxidation by ellagic acid. J Agric Food Chem 35: ) Ames BN, Cathcart R, Schwiers E, Hochstein P Uric acid provides an antioxidant defense in humans against oxidant and radical caused cancer: A hypothesis. Proc Natl Acad Sci USA 78: ) Ohkawa H, Ohishi N, Yagi K Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: ) Pederson TC, Buege JA, Aust SD Microsomal electron transport. The role of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in liver microsomal lipid peroxidation. J Biol Chem 248: ) Price MP, Butler LG Rapid visual estimation and spectrophotometric de termination of tannin content of sorghum grain. J Agric Food Chem 25: ) Chien-Chang S, Yuan-Shiun C, Li-Kang H Nuclear magnetic resonance studies of 5,7-dehydroxyflavonoids. Phytochemistry 34: ) Matsuzaki T, Hara Y Antioxidant activity of tea leaf catechin. Nippon Nogeikagaku Kaishi 59: ) Namiki M, Osawa T Antioxidants/antimutagens in foods. Basic Life Sci 39: ) Collier PD, Bryce T, Mallows R, Thomas PE The theaflavins of black tea. Tetrahedron 29: ) Auw JM, Blanco V, O'keefe SF, Sims CA Effect of processing on the phenolics and color of Cabernet Sauvignon, Chambourcin and Noble wines and juices. Am J Enol Vltic 47: Vol 44, No 2, 1998