Potent suppressive activity of pheophytin a and b from the non-polyphenolic fraction of green tea (Camellia sinensis) against tumor promotion in mouse skin

doi:10.1016/S0304-3835(98)00113-X

Cancer Letters

Volume 129, Issue 2, 19 June 1998, Pages 223-228

https://doi.org/10.1016/S0304-3835(98)00113-X Get rights and content

Abstract

Chlorophyll-related compounds pheophytin a and b have been recently identified as antigenotoxic substances in the non-polyphenolic fraction of green tea (Camellia sinensis), which suppressed umu C gene expression in tester bacteria induced by various genotoxins (Okai and Higashi-Okai, Cancer Lett. 118 (1997) 117–123). In the present study, the authors analyzed in vivo and in vitro effects of pheophytin a and b from the non-polyphenolic fraction of green tea on tumor promotion in mouse skin as follows. (1) When pheophytin a and b from green tea were topically applied prior to each treatment with a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on BALB/c mouse skin initiated by 7,12 dimethylbenz[a]anthracene (DMBA), they caused suppression in a dose-dependent fashion against skin tumorigenesis. (2) Pheophytin a and b exhibited significant suppressions against TPA-induced inflammatory reaction, such as edema formation, in BALB/c mouse ear skin in a dose-dependent manner. (3) Pheophytin a and b from green tea showed inhibitory effects against early induction of ornithine decarboxylase (ODC) in BALB/c mouse skin fibroblasts caused by TPA. These results suggest that pheophytin a and b from the non-polyphenolic fraction have potent suppressive activities against tumor promotion in mouse skin.

Introduction

In Japan, various kinds of green tea have been traditionally consumed as a daily beverage and some epidemiological studies have indicated that the ubiquitous consumption of green tea might be a protective factor against certain types of cancer 1, 2. Furthermore, many experimental studies have shown that the hot water-soluble extract of green tea or tea-derived polyphenols have significant suppressive activities against carcinogenesis 3, 4. The major polyphenols derived from green tea have been known as catechins, such as (−)epicatechin, (−)epicatechin gallate, (−)epigallocatechin and (−)epigallocatechin gallate, and they are detected in the water-soluble fraction of green tea [5]. However, the anticarcinogenic activity in the water-insoluble fraction has been poorly analyzed. To study this problem, we analyzed the effects of the non-polyphenolic fraction of green tea on tumor initiation and promotion using in vitro and in vivo experiments and detected the suppressive activities of the non-polyphenolic fraction against umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) caused by genotoxins in a tester bacteria, tumor promoter-induced ornithine decarboxylase (ODC) induction of 3T3 fibroblasts and chemically-induced skin tumor initiation and promotion in BALB/c mice [6]. Furthermore, we have recently identified pheophytin a and b as antigenotoxic substances in the non-polyphenolic fraction of green tea [7].

In the present paper, we describe the effects of pheophytin a and b on tumor promotion in mouse skin using in vivo and in vitro experiments.

Section snippets

Chemicals

7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (St. Louis, MO).

Purification of pheophytin a and b in the non-polyphenolic fraction of green tea

The preparation of the non-polyphenolic fraction of green tea was carried out using our previous method [7]. Briefly, green tea leaves (Sansui-En, Kumamoto, Japan) were brewed with hot water (25 ml/g tea leaves) and tea extract was decanted. After the same treatment was repeated three times, the residual tea leaves were dried and minced extensively by an electric mill

Suppressive effects of pheophytin a and b from the non-polyphenolic fraction of green tea against tumor promotion in BALB/c mouse skin

We examined the effects of pheophytin a and b from the non-polyphenolic fraction of green tea on the promotion phase of chemically-induced mouse skin tumorigenesis. As shown in Table 1, as a positive control, the combined treatment with 50 nmol DMBA (initiator) and 2 nmol TPA (promoter) induced remarkable tumors. Namely, all tested mice developed skin tumors and a high incidence of tumors was observed (19.2±3.6 tumors per mouse). However, the application of 100 or 500 μg pheophytin a or b prior

Discussion

Previous experimental studies have indicated that green tea extract or tea-derived polyphenols known as catechins have antitumor promoting activities 3, 4. However, the effect of the non-polyphenolic fraction of green tea against tumor promotion has been poorly analyzed. Therefore, the authors analyzed the effect of the non-polyphenolic fraction of green tea against tumor promotion using in vitro and in vivo experiments [6]. On the other hand, the authors have recently identified

Acknowledgements

The authors would like to thank Dr Y. Nakamura (Faculty of Agriculture, Kyoto University) for the useful information on tumor promotion in mouse skin and Dr Y. Yano (Department of Second Biochemistry, Osaka City University, Medical School) for his kind suggestion for the ODC assay.

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Aqueous and hydroalcoholic extracts of Tagetes minuta displayed anti-inflammatory activity in vitro, the hydroalcoholic extract being the most active (IC₅₀ between 59.72 and 66.42 μg/mL) in all cell lines. Bio-guided hydroalcoholic extract fractionation led to the isolation and characterisation of two pheophytins, pheophytin a (1) and 13²-hydroxy pheophytin a (2). Both compounds inhibited the production of NF-κB with IC₅₀ values in the low micromolar range, with an IC₅₀ between 12.32 and 16.01 μM for compound 1 and 7.91–9.87 μM for compound 2.
The two pheophytins isolated in this study inhibit the production of NF-κB, thus showing that the traditional anti-inflammatory use of Tagetes minuta can be proved through pharmacological assays. This contributes to understanding the anti-inflammatory activity of the Huacatay extracts and their use in the treatment of stomach and intestinal discomfort.
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Chung et al [70] found that orally administered SCC inhibited both tumor promotion and progression of papillomagenesis in 7,12-dimethylbenz[a]anthracene– and 12-O-tetradecanoyl-phorbol-13-acetate–induced mouse skin carcinogenesis. Higashi-Okai et al [85] further demonstrated that magnesium-free chlorophyll derivatives pheophytin a and b significantly suppressed tumor promotion and progression of skin tumorigenesis in a BALB/c mouse skin model. Recently, epidemiological evidence has linked diets high in chlorophyll with a reduced risk of colon cancer in humans [4].
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Cancer Letters

Abstract

Introduction

Section snippets

Chemicals

Purification of pheophytin a and b in the non-polyphenolic fraction of green tea

Suppressive effects of pheophytin a and b from the non-polyphenolic fraction of green tea against tumor promotion in BALB/c mouse skin

Discussion

Acknowledgements

References (14)

Green tea and skin-anticarcinogenic effects

J. Inv. Dermatol.

Green tea composition and polyphenol chemistry

Prev. Med.

Suppressive effects of the extracts of Japanese edible seaweeds on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells

Cancer Lett.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing protein-dye binding

Anal. Chem.

Suppressive effects of chlorophyllin on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells

Mutat. Res.

Inhibitory effects of pheophorbide a, a chlorophyll-related compound on skin tumor promotion in ICR mouse

Cancer Lett.

A case-control study of gastric cancer and diet in Northern Kyushu

Jpn. J. Cancer Res.

Cited by (26)

Hydroalcoholic extract of Tagetes minuta L. inhibits inflammatory bowel disease through the activity of pheophytins on the NF-κB signalling pathway

Digestion, absorption, and cancer preventative activity of dietary chlorophyll derivatives

Effect of supplemental ultraviolet radiation on the carotenoid and chlorophyll composition of green house-grown leaf lettuce (Lactuca sativa L.) cultivars

Chlorophylls as food additives

A comprehensive insight on the health benefits and phytoconstituents of camellia sinensis and recent approaches for its quality control

Lipopolysaccharide-induced Nitric oxide, prostaglandin E2, and cytokine production of mouse and human macrophages are suppressed by pheophytin-b