Microbiol. Immunol. Vol. 36 (9), 999-1001, 1992

The

Protective

Activity

Experimental

of Tea

Infection

by

Catechins

against

Vibrio cholerae O1

Masako TODA,*,1Sachie OKUBO,1Hajime IKIGAI,1 Tatsuo SUZUKI,2 Yurniko SUZUKI,2Yukihiko HARA,3 and Tadakatsu SHIMAMURA1 1

Departmentof Microbiologyand Immunology,Showa UniversitySchoolof Medicine, Shinagawa-ku, Tokyo 142, Japan, 2Department of ResearchPlanning, Kitasato Institute Hospital, Minato-ku, Tokyo108, Japan, and 3 Food ResearchLaboratories, Mitsui Norin Co., Fujieda, Shizuoka 426, Japan (Accepted for publicaTion, May 20, 1992)

Abstract toxin

Tea in sealed

catechins adult

Vibrio cholerae Olin tea catechins

may

inhibited mice.

ligated possess

The intestinal

protective

the

fluid

catechins

accumulation also

reduced

loops

of rabbits.

activity

against

induced fluid

These

by

cholera

accumulation

findings

suggest

by that

V. cholerae O1.

Tea possesses bactericidal activity against various bacteria that cause diarrheal diseases (7, 8), and inhibits the activity of their exotoxins in vitro (5). We recently found that tea protects against Vibrio cholerae O1 in animal models (10), and that catechins obtained from green tea leaf have both bactericidal and toxin-inhibitory activities (4, 9). We therefore investigated the inhibitory activity of tea catechins against V. cholerae O1 in vivo. A catechin mixture was prepared from Japanese green tea as described previously (3). The purity of catechins was 91.2%. The mixture contained (+)gallocatechin (1.6 w/w%), ( -)epicatechin (6.4%), ( -)epigallocatechin (19.2%), ( -)epicatechin gallate (13.7%) and (-)epigallocatechin gallate (59.1%). The catechins were dissolved in phosphate-buffered saline (PBS) at appropriate concentrations just before use. V. cholerae O1 Classical Inaba strain 569B was used. The origin of this strain has been previously described (2). Pure cholera toxin (Choletox) was purchased from Seikagaku Kogyo Co., Ltd. (Tokyo). Male C57BL/6 mice were purchased from Charles River Japan (Atsugi, Japan). Mice were used at 6 weeks of age. Male New Zealand White rabbits weighing 2.5 kg were purchased from Nippon Bio-supp Center (Tokyo). A modification of sealed adult mouse test (6) was used. Mice were administered 0.1 ml of each test material per os and then lightly anesthetized with pentobarbital sodium (somnopentyl, Pitman-Moore, New Jersey, U.S.A.). Their anuses were sealed with Aron Alpha (Toagosei Chemical Co., Industry Ltd., Tokyo). After 6 hr, their body weight (gram) and gut weight (gram) were measured and FA values were 99 9

1000

M.

calculated

FA=

with

the

following

formula

gut weight body

weight-gut

TODA

ET AL

:

×1,000.

weight

A modification of the rabbit intestinal loop test (1) was used. Ligated loops of intestine (approximately 10 cm long) made in vivo in adult rabbits were each injected with 0.5 ml of test material. The ligated loops were removed (18 hr after injection) to be measured and have the volume (ml) /length (cm) ratios determined. Inhibitory activity of tea catechins against cholera toxin was first determined using sealed adult mice. Cholera toxin was administered to mice 5 min before administration of tea catechins. The catechins significantly inhibited fluid accumulation induced by cholera toxin in sealed adult mice (Table 1). We next determined whether tea catechins prevent cholera infection in a rabbit model. Cholera vibrios were injected to the rabbit intestinal loops 5 min before injection of tea catechins. The catechins completely inhibited fluid accumulation induced by strain 569B in the rabbit intestinal loops (Table 2). Since each Table

Table

2.

1.

Inhibition

Protective

of fluid accumulation in sealed adult mice

activity in rabbit

of tea

catechins

intestinal

loops

by tea

against

catechins

cholera

infection

NOTES

10 0 1

original intestinal loop was empty and the amounts of test materials administered were relatively very slight, the amount of liquid that could be recovered (Table 2) was negligible in the catechin-treated loops. Hence the volume/length ratio was practically zero. Our results indicated that tea catechins inhibit the activity of cholera toxin, and protect against cholera infection in an animal model. We recently reported that tea extract protects against experimental infection by V. cholerae O1 (10). The present results further demonstrated that catechin was one of the components responsible for the protective activity of tea against cholera infection. The doses of the catechins used in mice and rabbits were not cytotoxic to enterocytes, since no signs or symptoms were observed. A cup of tea beverage (about 150 ml) contains 250 to 300 mg of catechins. Effectiveness of the catechins could be expected to be achieved in the human small intestine. Oral rehydration therapy (11) without antibiotics has been used for the treatment of cholera patients in epidemic areas. There is a possibility that tea catechins added to rehydration solutions may show therapeutic activity against V. cholerae O1, since the catechins are vibriocidal (9) and inhibit cholera toxin and cholera hemolysin (4, 9) in addition to their protective activity. This possibility remains to be examined on humans in the future. We thank Dr. G. M. Fukui and Dr. A. Simpson for reviewing this manuscript. This work was supported by grants from the Japanese Cholera Panel of the United States-Japan Cooperative Medical Science Program and from the Waksman Foundation of Japan Inc. REFERENCES

1) 2) 3) 4)

5) 6) 7) 8) 9) 10) 11)

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