Antioxidative Polyphenolic Substances in Cacao Liquor - American

Chapter 11

Antioxidative Polyphenolic Substances in Cacao Liquor 1

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N. Osakabe , M. Yamagishi , M. Natsume , T. Takizawa , T. Nakamura , and T. Osawa 1

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Functional Food Research and Development Laboratory, Meiji Seika Kaisha 350-0289, Japan Laboratory of Food and Biodynamics, Nagoya University, Nagoya 464-0814, Japan

Downloaded by UNIV OF LEEDS on May 18, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch011

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We have found antioxidative polyphenolic substances in cacao liquor that is one of the ingredients of chocolate and cocoa. Epicatechin, catechin, clovamide, quercetine, and their glycosides were confirmed as major antioxidants by using several methods. Furthermore, the physiological effects of crude polyphenols derived from cacao liquor (CLP) were examined with experimental animal models. C L P showed 1) antiulceric activity induced by ethanol, 2) inhibition on oxidative stress in vitamin Ε deficient rats, 3) reduction on L D L oxidative susceptibility in hypercholesterolemic rabbit, 4) antimutagenic effect against heterocyclic amines, 5) inhibitory effect on tumor promotion in two-stage carcinogenesis in mouse skin.

It is well known that free radical induced oxidative stress is an important etiologic factor in many pathological processes. Much attention has been focused on the antioxidants in food. Among the various foods, chocolate and cocoa are stable against oxidative deterioration. This is believed to be because of the fatty acid composition (/) and the polyphenolic substances in cacao beans (2-4). However, in the process of making cacao liquor, where fermentation, roasting, and grinding of raw beans are conducted, it is assumed that these compounds undergo major changes. In this report, we performed the isolation and structural elucidation of polyphenols in cacao liquor which is a major ingredient of chocolate and cocoa. Furthermore, a crude polyphenol fraction derived from cacao liquor (CLP) was prepared, and its physiological effects were evaluated with in vitro system and experimental animal models.

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© 2000 American Chemical Society

Parliment et al.; Caffeinated Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Materials and Methods

Cacao Liquor Fermented and dried cacao beans imported from Ghana and roasted and cracked at Meiji Seika Kaisha Ltd.


Extraction of Polyphenols Cacao liquor was defatted twice with fivefold n-hexane and extracted twice with fivefold 80 % v/v ethanol for 16 hours. The extract was concentrated and charged on a Diaion HP2MG column (35mm#x310mm). To remove contaminants, including xanthine derivatives, the column was washed with 20% v/v ethanol, and eluted with 80% v/v ethanol. Eighty percent ethanol fraction was concentrated and freeze-dried. This was cacao liquor crude polyphenols (CLP). C L P contained approximately 50% of total polyphenols by the analysis method of Prussian blue (5), using epicatechin as the standard.

High Performance Liquid Chromatography Further purification of the crude polyphenols was carried out by the method described in the previous report (6,7), using preparative H P L C . The column was used an O D S column, and elution solvent was 0.1 % trifluoroacetic acid, containing 40 % v/v methanol /water.

Antioxidative Activity

Linoleic Acid Oxidation Each sample was added at the final concentration of 0.5 m M to 1 g of linoleic acid and incubated at 30°C; the degree of oxidation was measured iodimetory. Microsomal Lipid Peroxidation Microsomes were prepared from rat liver (8). The antioxidants were added to the microsome fraction, and incubated at 37°C with tert-butylhydroxy peroxide (t-BHP) as a radical generator. After incubation, thiobarbituric acid reactive substance ( T B A R S ) was measured (9).


90 Effect of C L P on Gastric Mucosal Lesion Induced by Ethanol


Experimental Ulcers Male 9-week old Sprague-Dawley rats were used. Twenty-four hours before the experiment, the rats were deprived of food. The samples were dissolved or suspended in 0.1% w/v carboxylmethyl cellulose (CMC). Five-mL/kg body weight of test solution was intragastrically given to the animals 30 min before the administration of 5 mL/kg ethanol. After 60 min, the animals were sacrificed under anesthesia and their stomachs removed, and opened along the curvature. The degree of mucosal damage was evaluated by a computerized video-scanning system. Measurement of Lipid Peroxide The gastric mucosa was homogenized with 1.15 % K G solution, and the protein concentration of homogenates was measured by the Lowly method (10). The level of T B A R S in gastric mucosa was determined (9). Measurement of Xanthine Oxidase (XOD) Gastric mucosa homogenized was centrifuged at 4000 g for 10 min at 4°C, and supernatant was collected. The reaction mixture was 0.6 m M xanthine, and 1 mg protein of the sample containing 30 m M phosphate boric acid buffer (pH 8.2). The level of uric acid in the reaction mixture was measured by a Wako uric acid test kit after incubation for 3 hours at 37°C (//). Measurement of Myeloperoxidase (MPO) The reaction mixture was 0.4 m M tetramethyl benzidine, 0.3 % H2O2, and 1 mg protein of the sample. Absorbance at 655 nm of mixture was immediately recorded for 5 min. Activity was calculated from optical density per minute (12).

Effect of C L P on Oxidative Stress of Vitamin Ε Deficient Rats.

Experimental Procedure Male 3-week old Sprague-Dawley rats were fed a normal diet that was reformed AIN-76 diet, a vitamin Ε ( V E ) deficient diet that contained less than 6.0 μg/kg octocopherol, or they fed 0.25, 0.5, 1.0 % C L P containing VE-deficient diets, for 7 weeks. After the experimental period, animals were sacrificed under anesthesia, and blood and several tissues were removed. Analysis α-Tocopherol concentrations in plasma and tissues were analyzed by the H P L C method (13). T B A R S in plasma and tissues were determined as described above (9).


91 Effect of C L P on L D L Oxidative Susceptibility in Hypercholesterolemic Rabbits


Experimental Procedure Male 13-week old Japanese white rabbits were fed a high cholesterol diet, which contained 1.0 % cholesterol. After 4 weeks of the feeding, the plasma total cholesterol was raised 12 times, compared with before experiment (1,639±397 mg/dL), and initial L D L oxidation was measured. One percent CLP-containing diet was fed to the animals after 10 days. L D L oxidation was measured 4, 7, and 10 days after administration. Analysis L D L was prepared by the sequential ultracentrifugation method of Havel et al. (14). Thirty-five μg protein/mL of L D L fraction and 200 μΜ 2-2'azobis (4methoxy-2,4-dimethylvaleronitrile) (V-70) as radical generators were incubated at 37°C (15). The kinetics of L D L oxidation was determined by monitoring the conjugated-diene formation at 234-nm absorbance. T B A R S in L D L fraction induced by V-70 was also determined as described above (9). Plasma lipids were measured by using commercially available kits.

Antimutagenic Activity Against Heterocyclic Amine of C L P

Ames Test of Heterocyclic Amines With S-9 Mix The mutation test was carried out by the preincubation method (16), which was a slightly modified method of Ames et al. (17). Test samples, 3-amino-1 methyl-5Hpyrido(4,3-b)indole (Trp-P-2) or 2-amino-3,4-dimethyl-3H-imidaz(4,5-f)quinoline (MelQ), S-9 mix, and bacterial suspension were mixed and preincubated at 37°C for 20 min. Soft agar was added to the bacterial mixture and poured onto the modified Vogel-Bonner medium agar plate. These plates were incubated at 37°C for 2 days, and the revertant colonies (his ) were counted. +

Ames Test of Activated Heterocyclic Amine Trp-P-2 and M e l Q were incubated with S-9 mix at 37°C for 20 min, after incubation cold-acetone was added to the mixture and centrifugated (18). The supernatants were concentrated and used. The mutation test was carried out as described above, except that phosphate buffer was added to the bacterial mixture instead of S-9 mix. The concentrations of activated Trp-P-2 and M e l Q in this study were 20 nmol/plate and 0.1 nmol/plate.


92 Effects of C L P on Tumor Promotion in Two-Stage Carcinogenesis in Mouse Skin Four-week old female ICR mice were shaved and treated with 200 nmol 7,12dimethylbenz[a]anthrathene in 200 μL· acetone. After 1 week, the animals were treated with 200 μ ι acetone, or 5 nmol 12-0-tetradecanoylphorbol-13-acetate (TPA), or 5 nmol with 5 or 10 mg C L P in acetone twice weekly for 20 weeks. Skin tumors more than 1 mm diameter were counted every week.


Statistical Analysis Results were expressed as the mean and standard deviations. A l l analyses were done by using SPSS statistical software. Mean values were calculated by A N O V A and multiple-range comparisons, Student's t-test, or paired t-test. Values of p epicatechin > catechin > quercetine 3-glucoside, quercetine-3arabinoside, dideoxyclovamide (Figure 2) (7). Microsomal Lipid Peroxidation In this experiment, flavans such as epicatechin and catechin had a potent antioxidative activity, as shown in Figure 3 (7). Effect of C L P on Gastric Mucosal Lesion Induced by Ethanol Five mL/kg of ethanol given intragastrically consistently caused lesions in the mucosa in glandular stomach in the control group. As shown in Figure 4, the groups



Figure I. Chemical structure of antioxidative polyphenols in cacao liquor


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addition

250.0

Dooxy-CLV

Quercotln

Catechin

Epicatechin


Clovamide

0.0

20.0

40.0 Hours

Figure 2. Effects of polyphenolic substances from cacao liquor on linoleic acid autooxidation

1000.0

- d — Clovamide .«.«B Dideoxy-clovamide Quercetin-3-G , •Ά Querceiin-3-A , - - > - - Quercetin » ~-Ο·-Epicatchln , —# t

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Catechin

Figure 3. Effects of polyphenolic substances from cacao liquor on t-BHT-induced oxidation of rat microsomes



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Ethanol

α-tocopherol

CUP

Cimetidlne

Sucralfate

Figure 4, Effects of α-tocopherol, CLP, cimetidine and sucralfate on gastric mucosal Injury induced by ethanol administration


96 treated C L P , cimetidine, and sucralfate exhibited a marked reduction of these lesion (19). α-Tocopherol, a typical antioxidant, slightly reduced lesion formation. The level of T B A R S in gastric mucosa, increased 60 min after the administration of ethanol shown in Table 1(19). This change was significantly inhibited by the C L P treatment. X O D activity that has been suggested as a major source of oxygen radicals was not increased by ethanol treatment (Table I) (19). The level of T B A R S did not correlate with the X O D activity (r =0.232). M P O , is the marker enzyme of leukocytes, was raised by ethanol treatment, and this change was significantly inhibited by C L P (Table I) (19). The correlation between M P O activity and T B A R S is positive (r =0.634). C L P showed inhibition of leukocytes infiltration in gastric mucosa. In conclusion, the results of this study indicate that the mechanism of antiulcer effect of C L P is reduction of the migration of activated leukocytes to the inflammation area, and so the following attack of oxygen radical generation by these cells. 2


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Table I. Effect of C L P on TBARs Production, XOD and MPO Activity in Gastric Mucosa Lesions Induced by Ethanol Administration

No treatment Ethanol CLP

TBARS (nmol/mg protein 4.26 ± 0.76 7 . 6 4 ± 1.13 + 3.8410.64*

XOD (10-3U/protein) 1.27 ± 0 . 4 9 1.52 ± 0 . 3 4 0.38 ± 0 . 1 3 **

MPO (U/mg protein) 8.21 ± 1 . 2 9 19.62 ±4.11 + 2.67 ± 2 . 6 7 **

Significant difference from no treatment : +, pO.01 ; Significant difference from ethanol : *, p

Antioxidative Polyphenolic Substances in Cacao Liquor - American

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