Chapter 12
Inhibitory Effect of Rooibos Tea (Aspalathus linearis) on the Induction of Chromosome Aberrations In Vivo and In Vitro 1
1
2
2
3
K. Shimoi , Y. Hokabe , Y. F. Sasaki , H. Yamada , K. Kator , and N. Kinae 1
1
Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422, Japan Biological Laboratory, School of Science, Kwansei Gakuin University, Nishinomiya, Hyogo 662, Japan Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan
Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch012
2
3
The treatment of Chinese hamster ovary cells with Rooibos tea extract (RT, lyophilizate of Rooibos tea infusion, 100-1000 μg/ml) simultaneously with or subsequent to mitomycin C or benzo[a]pyrene (BP) significantly suppressed chromosome aberrations induced in the presence or absence of a metabolic activation system. Further more, gastric intubation of RT (0.05-0.1%) to ICR male mice at 1 ml per mouse per day for 28 days before intraperitoneal mitomycin C or B P treatment reduced micronucleus induction in peripheral blood reticulocytes. When mice received oral administration of RT (0.05%) in the drinking water ad libitum for 28 days, the frequency of micronuclei induced by mitomycin C (i.p.) or gamma rays was also decreased. As the dose of tea extract used in the experiments was almost the same as that of typical human daily consumption, RT intake might be a useful tool for chemoprevention of human cancer. Catechins in green tea leaves have been known to possess antimutagenic and anticarcinogenic potency (1-6). In 1992, Wang et al. (7) reported that green tea extract as the sole source of drinking water inhibited UV-induced skin tumori genesis. Antimutagenic and anticarcinogenic effects of Rooibos tea, an herb tea from South Africa, however, have not been reported so far. In this study, we investigated the suppressing effect of a crude aqueous extract of Rooibos tea, at a concentration equivalent to typical human daily intake, on the induction of chromo some aberrations in vivo (ICR male mice) and in vitro [Chinese hamster ovary (CHO) cells] in order to evaluate its chemopreventive activity. Rooibos Tea (Aspalathus linearis) Rooibos tea is one of the major crops cultivated in the western districts of the Cape Province of South Africa and is consumed as an herb beverage in Africa and 0097-6156/94/0547-0105$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.
FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS
106
Europe. This tea contains several polyphenols and flavonoids like quercetin, but no caffeine or theaflavins (8-9). As shown in Figure 1, we demonstrated from H P L C analysis that Rooibos tea contains little catechins which were characteristic to green tea. Preparation of Tea Extract
Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch012
Rooibos tea leaves (50 g) were boiled in 1.5 1 distilled water for 15 min. The infusion was cooled, filtered with a glass fiber filter and then freeze dried. About 7 g of solids was obtained. This solid extract, called RT, was dissolved in distilled water for CHO cells or tap water for mice just before use. Suppressing Effect of RT on Chromosome Aberration in Cultured CHO Cells C H O cells originally obtained from American Type Cell Collection were used. Cells were grown in Ham's F12 medium (Nissui Pharmaceuticals Inc., Tokyo) supplemented with 10% fetal bovine serum (HyClone Laboratories Inc., USA) and several antibiotics. In the simultaneous treatment study, the cultured C H O cells were exposed to 100 μΜ benzo[a]pyrene (BP) for 3 h or to 1 μΜ mitomycin C for 1 h with or without S9 mix and RT (50-1000 μg/ml). Subsequent treatment with RT was performed for 20 h in the absence of S9 mix and for 3 h in the presence of S9 mix after washing the cells treated with mutagens. Chromosome preparations were made according to the air drying method and then were stained with Giemsa. One hundred metaphases per culture were analyzed for chromosome aberrations. The numbers of aberrant cells were statistically analyzed using the χ test. As shown in Figures 2 and 3, simultaneous or subsequent treatment of RT with or without S9 mix suppressed the induction of chromosome aberrations by B P or mitomycin C. The suppressing effects of RT were stronger in simultaneous treatment than in subsequent treatment. 2
Suppressing Effect on Micronucleus Induction in Mice Male ICR mice (8 weeks old at the beginning of the study, Japan SLC Inc., Hamamatsu, Japan) were used. They were housed in an air conditioned room, where ambient lightning was controlled to provide 12 h light/dark cycles, and were given CE-2 commercial food pellets (Créa Japan Co., Tokyo). Tap water or RT dissolved in tap water was administered ad libitum or by gastric intubation. Five mice were assigned to each experimental group. The effects of preadministration of RT on the induction of micronuclei in mouse peripheral blood reticulocytes by B P (125 mg/kg, i.p.), mitomycin C (2 mg/kg, i.p.) and γ-rays (1.5 Gy) were examined according to the acridine orange-coated slide method of Hayashi et al. (10). One thousand peripheral reticulocytes per mouse were scored and the number of micronucleated peripheral reticulocytes (MNRETs) was statistically analyzed using Student's i-test. Gastric intubation of RT (0.05-0.1%) at 1 ml/mouse/day for 28 days reduced the frequency of MNRETs induced by B P and mitomycin C, but no effect was observed on γ-ray-induced MNRETs (Table I). On the other hand, oral administration of RT (0.05%) ad libitum for 28 days decreased the frequency of mitomycin C and γ-ray-induced MNRETs (Table II). As shown in these Tables, γ-ray treatment exhibited different results between gastric intubation and oral
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
Inhibitory Effect of Rooibos Tea (Aspalathus linearis)
Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch012
12. SHIMOI ET AL.
Figure 1. HPLC elution pattern of AcOEt fraction of RT. G A : gallic acid, EC: (-)-epicatechin, ECg: (-)-epicatechin gallate, EGC: (-)-epigallocatechin, EGCg: (-)-epigallocatechin gallate. Conditions: column, TSK gel ODS-80TM (04.6 χ 150 mm); eluant, CH CN-50 mM K H P 0 (l:9->4:6), 0.96 ml/min; detection, U V 280 nm. 3
2
4
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
107
Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch012
108
FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS
Post treatment
Simultaneous treatment
+ S9 mix
+ S9 mix
MMC 1 μΜ
MMC 1 μΜ
125 5001000 RT (u,g/ml) - S9 mix
125 500 1000 RT ^g/ml) - S9 mix
50 40 30 20 10
MMC 1 μΜ
125 500 1000 RT (μ /ηι1) β
MMC 1 μΜ
125 500 1000 RT^g/ml)
Figure 2. Effects of simultaneous or subsequent treatment of RT on mitomycin C-induced chromosome aberrations in CHO cells. * Significantly different from control (p
