Antioxidant Effects of Tannins and Related Polyphenols - ACS

Chapter 7

Antioxidant Effects of Tannins and Related Polyphenols

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 3, 2016 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch007

Takuo Okuda, Takashi Yoshida, and Tsutomu Hatano Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan

Antioxidant effects were exhibited by polyphenolic compounds isolated from medicinal plants, in various experimental systems: autoxidation of ascorbic acid and methyl linoleate, lipid peroxidation in liver mitochondria and microsomes, lipoxygenase-dependent lipid peroxidation, arachidonic acid metabolism, lipid metabolic injury in rat (oral administration), cytotoxicity in cultured hepatocytes, oxidative damage model of ocular lens, superoxide anion radical generated in the hypoxanthine-xanthine oxidase system. Mechanistic study showed participation of radical-scavenging activity of polyphenols.

A large number of polyphenolic compounds of a variety of structures have been isolated from diverse plants used as food and medicine, and their biological and pharmacological activities have been investigated, as described in the chapter "Polyphenols from Asian Plants —Structural Diversity, and Their Antitumor and Antiviral Activities—" in this book. These polyphenolic compounds can be defined as tannins, based on the comparisons of their structures and properties with those of known polyphenolic compounds regarded as tannins, and also with those of a large number of new compounds isolated in recent years from "tannin-containing plants." Inhibition of various actions of active-oxygen species, which is one of the most important activities underlying the biological and pharmacological activities of these polyphenols (1,2), is reviewed in this chapter. Inhibition of Autoxidation of Ascorbic Acid and Methyl Linoleate Ascorbic acid, an antioxidant and a radical scavenger, is quickly decomposed via a free radical upon aerobic oxidation, particularly in the presence of metallic catalysts. However, this compound is more stable when it is in the infusion of green tea which is rich in "green-tea tannin." In the experiments of Cu(II)-catalyzed aerobic oxidation of ascorbic acid, geraniin and tannic acid (Figure 1) remarkably inhibited the oxidation, while polyphenols of small molecules such as gallic acid and (+)-catechin did not inhibit it appreciably. The inhibitory activity of geraniin was stronger than that of tannic acid (3).

0097-6156/92/0507-0087$06.00/0 © 1992 American Chemical Society

Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

This inhibition of oxidation may be attributable to the chelation of these phenolic compounds with the metallic ion. However, the complexation constant of Cu(II) with geraniin, determined by the method of Scatchard plots, was smaller than that with tannic acid. On the other hand, the electron spin resonance (ESR) signals of several tannins in alkaline dunethylsulfoxide (DMSO) showed that tannin radicals are stable. These results strongly suggested that the inhibition of the oxidation is due to the radical scavenging activity associated with the formation of stable tannin radicals, rather than the chelation of the tannins with Cu(II) (4). Stronger inhibitory effects on the autoxidation of ascorbic acid in the same experimental system were exhibited by ellagic acid and quercetin (4). Marked inhibitory effect of tannins and polyphenols of related structures was also observed in an experiment of autoxidation of methyl linoleate, initiated by photoirradiation of 2,2'-azobisisobutyronitrile (AIBN) in the solution, which is a model system of lipid peroxidation (5) (Figure 2). The mechanism of this inhibition will be discussed later. Inhibition of Lipid Peroxidation Inhibition of Lipid Peroxidation in Liver Mitochondria and Microsomes. Inhibition of lipid peroxidation, occurring both in food and in living tissues, has been attracting interests from the angle of health effects. Several tannins and related compounds, isolated from various medicinal plants, markedly inhibited lipid peroxidation in rat liver mitochondria and microsomes (6,7). In this experiment, the lipid peroxide produced by incubating a mitochondrial fraction from rat liver with adenosine 5'-diphosphate (ADP) and ascorbic acid, and that produced in a rat liver microsomal fraction incubated with A D P and nicotinamide adenine dinucleotide phosphate (NADPH), were remarkably lowered by some hydrolyzable tannins [i.e., pedunculagin (Figure 3), penta-O-galloyl-p-D-glucose and isoterchebin) (6). Dicaffeoylquinic acids (Figure 3), the main components in "tannin-rich" Artemisia species, also markedly inhibited lipid peroxidation in these systems. The inhibitory effects of dicaffeoylquinic acids were noticeably stronger than those of chlorogenic acid (monocaffeoylquinic acid) and caffeic acid (7). Mechanistic Study of Lipid Peroxidation. The inhibitory effects of polyphenols upon the autoxidation of methyl linoleate were studied by kinetic study and in situ ESR measurements using 25 compounds. The strength of this effect was dependent on the type of polyphenolic groups and their number in each molecule, and the effect of these polyphenols generally lasted longer than those of the antioxidants on the market. For instance, the duration of inhibition of autoxidation of methyl linoleate by geraniin was several times of that of ascorbic acid or α-tocopherol. The radical-scavenging effect of ellagitannins such as geraniin, having a hexahydroxydiphenoyl (HHDP) group in the molecule, was stronger than that of gallotannins composed of galloyl groups. In situ ESR detection of tannin radicals, under the experimental condition inhibiting autoxidation, showed transient ESR signals of tannin radicals (Figure 4), which were identical with the signals obtained in separate measurement of aerial oxidation of tannins (5). These results indicated that tannins are proton-donors to lipid free radicals in the peroxidation. Stable tannin radicals were formed upon this action, and stopped the chain-reaction of lipid autoxidation. Upon comparison of the intensity of radical-scavenging effect of these phenolic compounds with their scavenging effect on l,l-diphenyl-2-picrylhydrazyl (DPPH), which gives violet solution of its free radical, and is decolorized when its radical is scavenged, the orders of intensity of each compound in these two experiments were similar. The radical-scavenging activity of the polyphenols of comparatively large



7. OKUDA ET AL.


Figure 1. Chemical structures of geraniin and tannic acid.

NC - C (CHafe - Ν = Ν - C (CH ) - CN 3

2

Inert Product

Figure 2. Schemes of autoxidation of methyl linoleate. L H means methyl linoleate.


89


90


Caf= -CO OH

\

oc

OH

oc

HO-^^—^-OH HO HO OH OH

3,5-Di-O-caffeoylquinic acid: R = R = caf, R = H 3.4- D i - O - c a f f e o y l quinic acid: R = H, R = R = caf 4.5- D i - O - c a f f e o y l quinic acid: R = R = Caf, R = H 1

1

1

Pedunculagin

3

2

2

2

3

3

Figure 3. Chemical structures of pedunculagin and dicaffeoylquinic acids.

a = 0.8 G Figure 4. In situ ESR signal of geraniin. This signal was observed upon the photo-irradiation of a mixture of methyl linoleate (0.4 M), A I B N (25 mM) and geraniin (10 mM) in DMSO-water (9:1) in an ESR cell.


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molecular weight (~1000) was generally strong, although some small polyphenols of certain structures, such as (-)-epicatechin gallate, also showed strong activity (8). Upon the reaction of the DPPH radical with various alkyl gallates, dialkyl hexahydroxydiphenates were produced and were isolated in high yields (Figure 5). These products, most probably from mutual coupling of C-centered galloyl radicals, and the ESR spectra of alkyl gallates in alkaline DMSO, verified that the elimination of the DPPH color is due to the scavenging activity of these polyphenols which formed stable free radicals (8). Inhibition of Lipoxygenase-dependent L i p i d Peroxidation. Tannins and related polyphenols also inhibited the autoxidation of linoleic acid, which was initiated by abstraction of its hydrogen with soybean lipoxygenase (9). Among these compounds, vescalagin and casuarinin (Figure 6), having two HHDP moieties in each molecule, exhibited stronger inhibition of the autoxidation than the other compounds such as geraniin having an HHDP group (9). This observation is in accord with the stronger radical-scavenging effect of ellagitannins than that of the other types of polyphenols (5). Several caffeic acid derivatives remarkably inhibited the peroxidation of linoleic acid. Among them, 3,5-di-O-caffeoylquinic acid, isolated from Artemisia species, and rosmarinic acid, so-called labiataetannin which is widely distributed in plants of Labiatae family, having two caffeic acid (or equivalent) moieties, showed stronger inhibition than chlorogenic acid and caffeic acid, and the effect of ferulic acid was the lowest. This order of the inhibitory effect among these compounds on the peroxidation was the same as that of the scavenging effect on the DPPH radical. Ferulic acid showed no ESR signal in alkaline D M S O , while all other four compounds (caffeic acid and its esters) showed the signals of their stable radicals under the same condition. These results show that the inhibition of the autoxidation of linoleic acid by these polyphenols is due to their activity scavenging lipid peroxide radicals (9). Analogous mechanism can be assumed for the inhibition in the other systems of enzyme-dependent lipid-peroxidation, including that caused by A D P and N A D P H in rat liver. Effects on Arachidonic Acid Metabolism. Inhibition of the peroxidation catalyzed by a lipoxygenase was also observed in the products from arachidonic acid metabolism. Several tannins and related polyphenols affected the enzyme-dependent peroxidation in arachidonic acid metabolism in polymorphonuclear leukocytes (10,11). 5-Lipoxygenase in leukocytes catalyzes peroxidation of arachidonic acid, producing 5-hydroperoxy-6,8,ll,14-eicosatetraenoic acid (5-HPETE), and this product is further converted into 5-hydroxy-6,8,ll,14-eicosatetraenoic acid (5HETE) and leukotrienes. On the other hand, cyclooxygenase catalyzes formation of prostagrandin G 2 ( P G G 2 ) from arachidonic acid, and subsequent reactions in leukocytes give various prostagrandins and related products, including 6ketoprostagrandin F i a (6KF), thromboxane B ( T X B ) and 12-hydroxy-5,8,10heptadecatrienoic acid (HHT). Geraniin and corilagin appreciably inhibited the formation of 5-HETE in a dose-dependent manner, while they inhibited the formation of 6KF, T X B 2 and HHT only at much higher concentrations (10). Caffeic acid and its esters also inhibited the peroxidation catalyzed by 5lipoxygenase, but they stimulated the formation of prostagrandin E (PGE ). Since some radical scavengers stimulate the formation of P G E , these polyphenols may be radical scavengers in the stimulation processes (77). 2

2

2

2

2




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Inhibition of Oxidative Damages Associated with Lipid Peroxidation Inhibition of Lipid Metabolic Injury by Oral Administration of Polyphenols. Lipid metabolic injury in rat was also inhibited by oral administration of tannins or tannin-rich plant extracts (12-14). The elevation of lipid peroxide levels in serum and in liver, and that of the serum transaminases [glutamicoxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT)] levels, induced by feeding peroxidized corn oil to rats, were inhibited by oral administration (300 mg/kg/day) of the extracts of Geranium thunbergii [containing geraniin (11.7 or 18.4 %) or corilagin (3.0-7.6 %)]. The levels of total cholesterol and free fatty acids in serum, elevated by peroxidized corn oil, were lowered, too (12). Oral administration of geraniin showed similar effects even in a dose of 50 mg/kg/day (12). Analogous effects were also observed upon administration of extracts of Artemisia species containing 3,5-, 3,4- and 4,5-di-O-caffeoylquinic acids (9.4-21.6 % in total) and chlorogenic acid (3.4-3.9 %), and upon administration of caffeic acid, and of chlorogenic acid (73). Extracts of fermented and non-fermented tea leaves also showed some improving effects against lipid metabolic injury (74). Inhibition of Cytotoxicity in Primary Cultured Hepatocytes. Primary cultured rat hepatocytes have been used for searching compounds with liverprotecting activity. Addition of carbon tetrachloride to the culture medium of the hepatocytes causes increase of the GPT activity in the medium, due to its cytotoxicity (75). This cytotoxic effect was inhibited by several hydrolyzable tannins [such as corilagin, pedunculagin, granatin A and gemin A (Figure 7)], galloylated condensed tannins (procyanidin B2 3'-0-gallate, procyanidin B2 3,3 -di-0-gallate) and related polyphenols with low molecular weight (pyrocatechol, gallic acid, epicatechin gallate and epigallocatechin gallate) (76). Since the hepatotoxicity of carbon tetrachloride is attributable to the lipid peroxidation caused by •CCb radical (75), tannins and related polyphenols may act as radical scavengers against this lipid peroxidation. Galactosamine-induced cytotoxicity in cultured rat hepatocytes were also inhibited by several tannins and related polyphenols (76). f

Inhibition of Oxidative Damage of Ocular Lens. Opacification of ocular lens in human senile cataract and diabetic cataract, and several experimental models for cataracts, has been correlated with lipid peroxidation in lens. Geraniin, penta- Ogalloyl-p-D-glucose, (-)-epigallocatechin gallate and several polyphenols, inhibited lipid peroxidation in lens. The lipid peroxidation in intact lens, which was induced by incubating the lens in a medium containing xanthine, xanthine oxidase, A D P and FeCb, was inhibited by further incubation of the lens in a medium containing each polyphenol, to various extent depending on the structure of each polyphenol (77). The increase of the N a / K ratio, and the decreases of the glutathione level and of the activities of glutathione reductase and Na,K-ATPase, which are accompanied by this lipid peroxidation, were also restored by the incubation of the lens with these polyphenols. The action site of the polyphenols was the plasma membrane of the lens (77). Inhibition of the generation of superoxide anion radical in the hypoxanthineX O D system was exhibited by tannins and related compounds (18). The generated superoxide radical in this system was detected by ESR spectrometry in the presence of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO). The ESR signal due to the D M P O adduct of superoxide radical decreased its amplitude upon addition of polyphenolic compound to the reaction mixture, in a dose-dependent manner (Figure 8). The signals of two new radical species assignable to the D M P O adducts of hydrogen (DMPO-H) and of a C-centered radical, then appeared. The latter signal shows production of phenoxy radical from polyphenol upon scavenging the superoxide +

+


94



HO HO

OH

Casuarinin

OH

HO HO

OH

OH

Vescalagin

Figure 6. Chemical structures of casuarinin, vescalagin and rosmarinic acid.

HO

HO

OH

OH

Gemin A Figure 7. Chemical structures of corilagin, granatin A , procyanidin (9-gallates and gemin A .



7. OKUDA ET AL.


Figure 8. Effect of penta-O-galloyl-p-D-glucose on the ESR signal of the D M P O adduct of superoxide anion radical. The spectra were recorded in the absence [(a)] and in the presence [(b) 1.0 χ ΙΟ" M, (c) 2.5 χ 10' M, (d) 5.1 χ Ι Ο M, (e) 1.0 χ 10" M, (f) 2.5 χ 10" M, (g) 1.0 χ ΙΟ" M of penta-O-gaUoyl/9-D-glucose. The solid (A) and broken (B) lines below the spectrum (g) indicate the assignments of the hyperfine splitting patterns for the DMPO adduct of a C-centered radical and the hydrogen adduct of DMPO, respectively. 6

6

5

5

6

5


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II OH


OH

Cornusiin A

Coriariin A Figure 9. Chemical structures of cornusiin A and coriariin A .

radical. Strong scavenging activity has been found for the polyphenolic compounds with ortho-tnhyaroxy (pyrogallol) structure (18). The compounds which strongly inhibited generation of superoxide radical in the hypoxanthine-XOD system [such as geraniin, cornusiin A , coriariin A (Figure 9), epigallocatechin and epigallocatechin gallate], however, showed only weak inhibition of the X O D activity catalyzing the formation of uric acid. This result substantiated that their inhibitory effect on the generation of the superoxide radical is ascribable to direct scavenging of the radical (79). Conclusion The recent findings concerning the antioxidant effects of polyphenolic compounds have revealed the possibility that the effects of these compounds, considerable amount of which is taken in by the people in the world, are underlying various favorite health effects of foods and medicinal plants. The recent investigations have also shown marked difference of these effects due to the difference of the structure of each polyphenolic compound and of the oxidation system. Further detailed investigations of their effects, particularly those occurring after they are taken as food and medicine, must be carried out. Acknowledgments The authors thank Prof. Y . Fujita and Prof. A . Mori (Okayama University), the late Prof. S. Arichi and Dr. Y . Kimura (Kinki University), Prof. H . Okuda (Ehime University), the late Prof. H . Hikino (Tohoku University), the late Prof. S. Iwata (Meijo University), Prof. T. Noro (University of Shizuoka) and their co-workers for their collaboration.


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Literature Cited 1. Okuda, T.; Yoshida, T.; Hatano, T.; Fujita, Y. In Free Radical and Cino-Japanese Medicine; Okuda, T.; Yoshikawa, T., Eds.; Kokusai-ishoshuppan: Tokyo, 1990; pp 42-70. 2. Okuda, T.; Fujita, Y.; Yoshida, T.; Hatano, T. In Free Radicals in Clinical Medicine; Kondo, M.; Oyanagi, Y.; Yoshikawa, T., Eds.; Nihon-igakukan: Tokyo, 1990, Vol. 4; pp 19-30. 3. Yoshida, T.; Koyama, S.; Okuda, T. Yakugaku Zasshi 1981, 101, 695. 4. Fujita, Y.; Komagoe, K.; Sasaki, Y.; Uehara, I.; Okuda, T.; Yoshida, T. Yakugaku Zasshi 1987, 107, 17. 5. Fujita, Y.; Komagoe, K.; Uehara, I.; Okuda, T.; Yoshida, T. Yakugaku Zasshi 1988, 108, 528. 6. Okuda, T.; Kimura, Y.; Yoshida, T.; Hatano, T.; Okuda, H.; Arichi, S. Chem. Pharm. Bull. 1983, 31, 1625. 7. Kimura, Y.; Okuda, H.; Okuda, T.; Hatano, T.; Agata, I.; Arichi, S. Planta Med. 1984, 50, 473. 8. Yoshida, T.; Hatano, T.; Okumura, T.; Uehara, I.; Komagoe, K.; Fujita, Y.; Okuda, T. Chem. Pharm. Bull. 1989, 37, 1919. 9. Fujita, Y.; Uehara, I.; Morimoto, Y.; Nakashima, M.; Hatano, T.; Okuda, T. Yakugaku Zasshi 1988, 108, 129. 10. Kimura, Y.; Okuda, H.; Okuda, T.; Arichi, S. Planta Med. 1986, 52, 337. 11. Kimura, Y.; Okuda, H.; Okuda, T.; Hatano, T.; Arichi, S. J. Nat. Prod. 1987, 50, 392. 12. Kimura, Y.; Okuda, H.; Mori, K.; Okuda, T.; Arichi, S. Chem. Pharm. Bull. 1983, 31, 2501. 13. Kimura, Y.; Okuda, H.; Okuda, T.; Hatano, T.; Agata, I.; Arichi, S. Chem. Pharm. Bull. 1985, 33, 2028. 14. Kimura, Y.; Okuda, H.; Mori, K; Okuda, T.; Arichi, S. Nippon Eiyo Shokuryo Gakkai Shi 1984, 37, 223. 15. Kiso, Y.; Tonkin, M.; Hikino, H. Planta Med. 1983, 49, 222. 16. Hikino, H.; Kiso, Y.; Hatano, T.; Yoshida, T.; Okuda, T. J. Ethnopharmacology, 1985,14,19. 17. Iwata, S.; Fukaya, Y.; Nakazawa, K.; Okuda, T. J. Ocular Pharmacol. 1987, 3, 227. 18. Hatano, T.; Edamatsu, R.; Hiramatsu, M.; Mori, Α.; Fujita, Y.; Yasuhara, T.; Yoshida, T.; Okuda, T. Chem. Pharm. Bull. 1989, 37, 2016. 19. Hatano, T.; Yasuhara, T.; Yoshihara, R.; Agata,I.;Noro, T.; Okuda, T. Chem. Pharm. Bull. 1990, 38, 1224. RECEIVED

December 17, 1991


Antioxidant Effects of Tannins and Related Polyphenols - ACS

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