Suppression of the Formation of Advanced Glycosylation Products by

Chapter 7

Suppression of the Formation of Advanced Glycosylation Products by Tea Extracts

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N. Kinae, K. Shimoi, S. Masumori, M . Harusawa, and M . Furugori Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422, Japan

Three kinds of tea extracts were prepared from green tea (Camellia sinensis, unfermented), polei tea (Camellia assamica, fermented with yeast) and rooibos tea (Aspalathus linearis, irradiated with sunlight). Each tea extract was incubated with a mixture of D-glucose and human serum albumin under physiological conditions (pH 7.4, 37°C). A l l tea extracts showed suppression effects on the formation of glycated albumin including fluorescent advanced glycosylation endproducts (AGEs). The determination of ESR spectra of reaction mixture and tea extract suggested that the suppression activity is correlated to the radical scavenging potency of tea extracts. Several tea components such as catechins and flavonoids may play important roles in the disappearance of free radicals containing superoxide formed in the early stage of the Maillard reaction.

An amino-carbonyl reaction, the so-called Maillard reaction, is well known as one of the major phenomena observed in the process of cooking, manufacturing and storage of foodstuffs (1). Recent studies show that the reaction also occurs in our body tissues, especially in hemoglobin (2), lens crystallins (3) and collagens (4). The glycated Cu-Zn-superoxide dismutase was isolated from human erythrocytes and the content increased as a function of aging and diabetes development (5). In the early stage of the reaction, Schiff s base and Amadori product are formed and then converted to deoxyglucosones, which are key substances in the intermediate stage. In the last stage, AGEs are formed in vivo and in vitro as yellow fluorescent condensates (6). The AGEs content in the body tissues of old people and diabetic patients is significantly higher than those of young and healthy individuals (7,8). Tea extract is one of the most ancient and popular beverages in the world. Green tea is an attractive beverage because of its antioxidative, antimicrobial, antimutagenic and anticarcinogenic activities (9-11). In this paper, we report on the inhibitory effects of three kinds of tea extracts on the formation of AGEs and discuss the inhibitory constituents and the mechanism of inhibition.

0097-6156/94/0547-0068$06.00/0 © 1994 American Chemical Society

Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

7. KINAE ET AL.

Suppression of the Formation of Glycosylation Products

Preparation of Tea Extracts Green tea was supplied by Shizuoka Tea Experiment Station (Shizuoka, Japan). Polei tea (produced in China) and rooibos tea (produced in South Africa) were supplied by JACOS Co. (Tokyo, Japan) and Rooibos Tea Japan Co. (Osaka, Japan), respectively. Ten grams of tea leaves were added to 300 ml boiling distilled water and kept at 100°C for an appropriate time (green tea: 0.1 min; polei tea: 15 min; rooibos tea: 15 min). Each tea infusion was filtered through gauze and the filtrate was lyophilized. The lyophilizate was referred to as tea extract. Inhibitory Effects of Tea Extracts on AGEs Formation Tea extract (10-50 mg) was added to 50 ml of phosphate buffered saline (PBS) containing 500 mg HSA and 5 g D-glucose then incubated at 37°C for 47 days. Aliquots of the reaction solution were taken out periodically and dialyzed against PBS. The fluorescence intensity of the lyophilizate (AGEs) was measured at the wavelength of 440 nm (excitation at 370 nm). Time course of the relative fluorescence intensity of each reaction solution is shown in Figure 1. A l l tea extracts (10 mg) suppressed the formation of AGEs to 52-80% of the control. The order of decreasing inhibitory strength after 47 days was green tea, polei tea and rooibos tea.

0

10

20 30 Incubation time (days)

40

50

Figure 1. Inhibitory effect of tea extracts on the formation of fluorescent compounds.

Chemical Analysis of Tea Extract In previous papers (12,13), we demonstrated that several catechins containing (-)-epigallocatechin gallate (EGCg) were isolated from the ethyl acetate-soluble fraction of green tea extract and possessed high inhibitory activity against A G E formation. The other two kinds of tea extracts were submitted to H P L C analysis to determine catechin contents using the following conditions; column: T S K gel ODS-80 T M (φ4.6 χ 150 mm), eluant: CH CN:50 m M K H P 0 (l:9)->(4:6), flow 3

2

4


FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

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rate: 0.96 ml/min, detection: U V (280 nm). The elution profile of the ethyl acetatesoluble fraction of polei tea extract is shown in Figure 2. The catechin content of polei tea and rooibos tea was extremely low compared to green tea (Table I). Other chemical analysis was performed according to the Standard Method of Analysis for Hygienic Chemist (1990) in Japan. Determination of Glycated Albumin by High Performance Chromatography

Affinity

The content of glycated albumin, which contains mainly Amadori product, was determined by high performance affinity chromatography using T S K gel borate column (φ4.6 χ 150 mm) (14). First, unreacted materials were removed with a solvent (0.25 M CH COONH /0.05 M M g C l , pH 8.5) and then glycated albumin was eluted with an eluant (0.2 M sorbitol/0.1 M Tris, pH 8.5). The elution profile and time course of glycated albumin content are shown in Figure 3. The formation of glycated albumin was inhibited in order of decreasing strength by rooibos tea, polei tea and green tea. 3

4

2

Table I. Chemical Analysis of Tea Extracts (%)

Total nitrogen Crude protein Reducing sugars Crude fat Ash Tannins Gallic acid EC ECg EGC EGCg b

a b

3

Green tea

Polei tea

Rooibos tea

4.01 25.10 16.30 1.06 6.59 30.20 0 4.19 4.24 8.79 13.20

6.09 38.10 11.40 1.10 14.50 1.14 0.08 0.28 0.39 0 0.39

0.38 2.38 17.30 0.79 6.88

trace trace

Equivalent to D-glucose Equivalent to tannic acid

Determination of Radical Scavenging Activity of Tea Extract l,l-Diphenyl-2-picrylhydrazil (DPPH) Method. DPPH contains a free radical in the molecule and combines with other free radicals to form a stable complex. The radical scavenging activity of each tea extract was evaluated by determination of the 50% inhibitory dose (ED ) at OD o nm of DPPH-methanol solution according to the method of Fugita et al. (15). Compared with the E D of EGCg (2.58 μg), the relative activities of green tea, polei tea and rooibos tea were 0.24, 0.15, 0.16, respectively (Table II). 50

52

5 0


7. KINAE ET AL.


*

*

2

£

a

a

g

Retention time (min)

Figure 2. HPLC elution profile of the ethyl acetate fraction of polei tea extract.


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>1

0

1

1

,

2

1

,

1

3 4 5 6 Reaction time (days)

«

1

7

—>

8

Figure 3. Determination of glycated albumin. Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

7. KINAE ET AL.


Table II. Radical Scavenging Activity of Tea Extracts by the DPPH Method Procedure 4

DPPH (2.0 χ 10' M) 4 ml + _3

Sample (0-1.4 χ 10 %) 1 ml I Mix and shake for 5 min. Let stand for 30 min. I Mixture Measure O D ED

5 2 0 n m

5 0

S a m

P

l e

E D

50^g)

10.60 Green tea 16.98 Polei tea Rooibos tea 16.03 2.58 EGCg

Relative activity 0.24 0.15 0.16 1.00

ESR Spin-trapping Method. Superoxide radical (0 ") supplied from a hypoxanthine-xanthine oxidase system (16) was trapped with 5,5-dimethyl-l-pyrrolineN-oxide (DMPO) to produce the spin adduct (DMPO-0 "). When each tea extract was added to the system, a decrease in ESR signal intensity was observed in the superoxide formation system. The inhibitory activity of green tea was the highest among the 3 kinds of tea extract (Figure 4). 2

2

Procedure Hypoxanthine (2.0 mM) 20 μΐ D E T A P A C (5.5 mM) 14 μΐ Sample (dissolved in 0.1 M Na phosphate buffer, pH 7.4) 20 μΐ DMPO 6 μΐ Xanthine oxidase (0.33 unit/ml) 20 μΐ

Sample (concentration) Activity (%) None SOD (1.38 units/ml) Green tea (0.006 mg/ml) Polei tea (0.006 mg/ml) Rooibos tea (0.006 mg/ml) EGCg (0.003 mg/ml)

0.0 12.9 31.9 19.7 4.6 39.5

Figure 4. Superoxide scavenging activity of tea extracts by ESR spin-trapping method.


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74 Discussion

Recent reports have shown that glycated proteins are significantly accumulated in the several body tissues of aged individuals and diabetes patients (5). Aminoguanidine, a nucleophilic hydrazine compound, is noteworthy as an inhibitor of AGE formation in vivo and in vitro (17). Aspirin (78) and Diclofenac (79) have also been reported as glycation blockers. Catechin-aluminum complex decreased the blood glucose level of dia betic mice (20). There is one report which showed a positive correlation between green tea production and average human life span (21). In this study, we demonstrated that glycation of human serum albumin was inhibited by tea extract and polyphenols including catechins. In consideration of the generation of superoxides in the early stage of the reaction (22), these superoxides might be trapped into tea extract and therefore, AGE formation will be suppressed. As the catechin content in polei tea and rooibos tea is very low, different effective components from catechins may play roles as radical scavengers. Acknowledgements We thank Dr. Y. Hara (Mitsui Norin Co., Shizuoka, Japan) for providing standard tea catechins and Dr. I. Oguni (Hamamatsu College, University of Shizuoka) for helpful discussion of this work. This research was supported by a grant from the Ministry of Education, Science and Culture of Japan. Literature Cited 1. Hodge, J. E. J. Agric. Food Chem. 1953, 1, 928-943. 2. Bunn, H. F.; Gabbay, K. H.; Gallog, P. M. Science 1978, 200, 21-27. 3. Monnier, V. M.; Cerami, A. Science 1981, 211, 491-493. 4. Monnier, V. M. Dev.Food Sci. 1986, 13, 459-474. 5. Arai, K.; Maguchi, S.; Fujii, H.; Ishibashi, K.; Taniguchi, N. J. Biol. Chem. 1987, 262, 16969-16972. 6. Monnier, V. M.; Kohn, R. R.; Cerami, A. Proc. Natl. Acad. Sci. USA 1984, 81, 583-587. 7. Hayase, F.; Nagaraj, R.H.; Miyata, S.; Njoroge, F. G.; Monnier, V. M. J. Biol. Chem. 1989, 264, 3758-3764. 8. Monnier, V. M.; Sell, D. R.; Miyata, S.; Nagaraj, R. H. In Maillard Reaction; Finot, P. A.et al. Eds; Adv. in Life Sci.; Birkhauser Verlag: Basel, 1990; pp 393-414. 9. Matsuzaki, T.; Hara, Y. Nippon Nogeikagaku Kaishi (in Japanese) 1985, 59, 129-134. 10. Stich, H. F.; Rosin, M. P. Adv. Exp. Med. Biol. 1984, 177, 1-29. 11. Wang, Z. Y.; Khan, W. Α.; Bicher, D. R.; Mukhtar, H. Carcinogenesis 1989, 10, 411-415. 12. Kinae, N.; Yamashita, M.; Esaki, S.; Kamiya, S. In Maillard Reaction; Finot, P. A. et al. Eds; Adv. in Life Sci.; Birkhauser Verlag: Basel, 1990; pp 221-226. 13. Kinae, N.; Masumori, S.; Nakada, J. Saito, T.; Furugori, M.; Esaki, S.; Kamiya, S.; Owada, K.; Masui, T. Proc. Int. Symp. on Tea Science (Shizuoka, Japan) 1991,14, 649-661. 14. Mallia, A. K.; Hermanson, G. T.; Krohn, R. I.; Fujimoto, Ε. K.; Smith, P. K. Anal. Letters 1981, 14, 649-661. Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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15. Fujita, Y.; Uehara, I.; Morimoto, Y.; Nakajima, M.; Hatano, C.; Okuda, T. Yakugaku Zassi (in Japanese) 1988, 108, 129-135. 16. Miyagawa, H.; Yoshikawa, T.; Tanigawa, T.; Yoshida, N.; Sugino, S.; Kondo, M.; Nishikawa, H.; Kohno, M. J. Clinic. Biochem. Nutr. 1988, 5, 1-7. 17. Brownlee, M.; Vlassara, H.; Kooney, Α.; Ulrich, P.; Cerami, A. Science 1986, 232, 1629-1632. 18. Ajiboye, R.; Harding, J. J. Exp. Eye Res. 1989, 49, 31-41. 19. van Boekel, M . A. M.; van den Bergh, P. J. P. C.; Hoenders, H. J. Biochem. Biophys. Acta. 1992, 1120, 201-204. 20. Asai, H.; Kunou, Y.; Ogawa, H.; Hara, Y.; Nakamura, K. Kiso to Rinsho (in Japanese) 1987, 21, 4601-4604. 21. Oguni, I. (personal communication) 22. Sakurai, T.; Tsuchiya, S. FEBS Letters 1988, 236, 406-410. R E C E I V E D May 4, 1 9 9 3


Suppression of the Formation of Advanced Glycosylation Products by

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