Summary translation

Physiological Function of Phytopharma-ceuticals and its Utilisation XIII: One of the functional materials recently getting much attention is green tea. Several effects of green tea on life-style related diseases have been reported, including antitumor activities (1), antioxidative activities (2), antiinflammatory activities (3). Extracts containing concentrated compounds like catechins and theanine are used in most studies on green tea in Japan. In Europe, green tea is regarded as beneficial material for phytopharmaceuticals and green tea extracts are distributed in the European market as drugs and as supplementary food. The 12th Food Development Fair 2001 was held on September 26-28, 2001. In the memorial seminar Mr. Marco Netsch from Emil Flachsmann AG hold a lecture titled “Aspects of Chemopreventive and Antiinflammatory Activities of Green Tea and its Preparations”. This article describes the pharmacological efficacy of EFLA®942 demonstrated in clinical trials and possible mechanisms evaluated in in vitro studies. Green tea had an official state of medicine in several European Pharmacopoeias, including Ph.Port. IV, Ph.Franç. VIII-X, BP Edition 1968 and Ph.Helv. V. Nowadays green tea is not listed in the Ph.Eur., which is the only legal pharmacopoeia for EC countries. Independently, in France green tea pharmaceuticals are approved as drugs on the basis of the Ph. Franç. and the French Gazette for Marketing Authorisation of Plant Medicines (1990). In contrast to the situation in France, no marketing authorisation for medicinal use is available in other EC countries, but there exist preparations on the base of green tea as supplementary food. The French Gazette for Marketing Authorisation of Plant Medicines (1990) listed some traditional indications of green tea: For oral use, 1) diuresis, 2) mild diarrhoea, 3) recovery from fatigue, and 4) dietary supplement for weight reduction and for external use, 1) calm for itching of skin ailment and 2) treatment of cracks, grazes, and insect bites, etc. (4). 2. Diuretic effect and anti-diarrhoea effect Regarding the diuretic effect of green tea there might exist two different mechanisms of action. On the one hand this effect could be exerted by the green tea component caffeine, which acts as a diuretic by increasing the plasma renin activity. On the other hand, diuresis could be influenced by the inhibition of the neutral endopeptidase (NEP). In an in vitro study EFLA®942 was shown to decrease NEP activity with an IC50 of 40 mg/ml (5). The membrane ectoenzyme NEP, which is abundant especially in kidney, lung, brain and neutrophils, cleaves endogenous peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and adrenomedullin (ADM). As ANP, BNP and ADM are hormones with a broad range of potent physiological effects including diuresis, it is suggested that one mechanism by which EFLA®942 promotes diuresis is the inhibition of NEP activity. In two clinical pilot studies the effect of EFLA®942 on drug-associated diarrhoea was investigated. Subjects were HIV-patients with protease inhibitor-associated diarrhoea, 50pathogenic diarrhoea or unspecific Consistency of stools
EFLA®942 for 8 days with a Fig. 1 : Consistency of stools with green tea extract in patients with drug-associated diarrhoea, without additional medication. tablet over a time period of 6 months. The first pilot study including 15 patients demonstrated a Consistency of stools
Fig. 2 : Stool frequency per day with green tea extract in patients with drug-associated diarrhoea, without To study the mechanism of the anti-diarrhoea effect observed in the pilot studies, necrosis suppression and anti-inflammatory effects using EFLA®942 were examined. In many cases diarrhoea is caused by necrosis. Therefore, in this study multicellular spheroids of human WiDr colon carcinoma cells that characteristically develop a central necrosis were used (Fig.3). In comparison to conventional monolayer cell models they resemble far more to the histogenic cells and the similarity is clear in many respects including structure, cellular functions of Fig.3 : metabolism, growth, differentiation and Complexity of cell death in multicellular spheroids. micromilieu of the cells (6). The treatment of multicellular spheroids with 100mg/ml EFLA®942 confirmed the effects of EFLA®942 described below: hypertrophy(enlargement of volume and diameter) of multicellular spheroids cm
Fit control
Data 100 µg EFLA®942/ml
Fit 100 µg EFLA®942/ml
Data 20 µg EFLA®942/ml
Fit 20 µg EFLA®942/ml
Days in culture
multicellular spheroids Fig.4 : Volume growth curves of multicellular WiDr spheroids. (Fig.6) Fig.6 : Frozen sections of multicellular WiDr Effect of EFLA®942 on cell diameter and thickness of the spheroids at day 16: a. Control, ∅ 1070.0 mm/ proliferative zone in multicellular WiDr spheroids (p<0.01). Data control
Fit control
Data 100 µg EFLA®942/ml
Fit 100 µ g EFLA®942/ml
contribute to the results of the pilot studies. Spheroid volume [10-5 cm3]
Fig.7 : Multiplicity of multicellular WiDr spheroids. In cancer research, a consequence frequently associated with free radicals is the development of tumors. Cell division is a critical factor in mutagenesis, where non-repaired DNA damages can be the origin of mutations in tumor suppressor genes or proto-oncogenes (7). The fact that oxidants are ranked to the class of cell division stimulating agents (8) underlines the important role of antioxidants. The antioxidative defence systems of the cell can be subdivided into enzymatic (e.g. superoxide dismutase) and non-enzymatic defence systems (e.g. glutathione (GSH), vitamin A, C and E). The tripeptide GSH is localised in liver, kidney, lung and digestive tract. In addition to the antioxidative activity GSH has a broad influence on the cellular metabolism including DNA synthesis and repair and metabolism of toxins and carcinogens. Since EFLA®942 suppressed necrosis, the influence of EFLA®942 on the cellular GSH concentration was examined. After treatment of human colon carcinoma spheroids with EFLA®942 a significant increase of the cellular GSH concentration by 15% was observed (Fig.8). It is not clear of this effect is due to the increase in GSH production or the stabilisation of reduced GSH by the antioxidative activity of green tea polyphenols. As green tea is generally known for its radical scavenging potential, it appears to exert a dual antioxidative effect including the increase of 100 µg EFLA®942/ml
GSH concentration. If necrosis is caused by oxidative Glutathione concentration of multicellular stress and excess of free radicals in multicellular spheroids, WiDr spheroids in the diameter range of EFLA®942 may enhance cellular viability in spheroids by 300-800 mm. delay of GSH exhaustion and scavenging free radicals. c) Antiinflammatory activity and elastase inhibition Inflammation is an important symptom of various diseases including rheumatoid arthritis (9), atherosclerosis (10), gastric mucosal injury (11) and tumor metastasis (12). Recently, green tea was demonstrated to suppress the effect of proinflammatory mediators (13). However, this article reports on the effect of EFLA®942 on the human leukocyte elastase (HLE = NE: neutrophil elastase), which is one of the most destructive proteases frequently involved in inflammatory events. HLE, a serine protease secreted by neutrophils from azurophilic granules, is an important mediator in the initiation of an inflammation. It is involved in inflammatory diseases such as gastric mucosal injury (11) and rheumatoid arthritis (14). EFLA®942 strongly inhibited HLE activity with an IC50 of 2.5 mg/ml. Compared to epigallocatechin gallate (EGCG) exhibited the strongest inhibition of HLE among single polyphenolic green tea compounds, but at 1/6 in comparison to EFLA®942. This indicates synergistic effects of multiple components including EGCG. As HLE is involved in stress-induced gastric mucosal injury by decreasing gastric production of prostacyclin (PGI2) (11), its inhibition by EFLA®942 might contribute to the effect of EFLA®942 on drug-associated diarrhoea. Recent research focused on EGCG as an active ingredient of green tea. Nevertheless, many results have demonstrated a superiority of the multicomponent system green tea extract to single compounds or combinations thereof. In addition to the inhibitory effect on HLE described above, some reports supporting this phenomenon are described below. a) EFLA®942 more efficiently reduced the relative metabolic cell activity in human WiDr colon carcinoma cells than single compounds or ] [% 120
combinations thereof (Fig.9).
b) Regarding the inhibitory effect on the expression of a cytochrome responsible for the metabolism of many carcinogens, green tea extract was more EFLA®9421 EGCG2
Theanin Caffeine
c) Addition of epicatechin significantly increased the +Theanine
+Caffeine3
induction of apoptosis, the tumor growth reduction and the inhibition of tumor necrosis factor a release Fig.9 : by other single polyphenolic green tea compounds in Relative metabolic cell activity of human WiDr human lung tumor cells (16). colon carcinoma cells treated with 100 mg d) Oral administration of green tea was markedly more EFLA®942/ml or green tea compounds or effective than decaffeinated green tea at inhibiting combinations thereof corresponding to 100 mg the tumorigenic effect of UVB light in SKH-1 mice green tea extract/ml. (17, 18). The various kinds of efficacy demonstrated for green tea appears to result from synergistic effects of a variety of compounds and from complex mechanisms. The green tea extract EFLA®942 described in this article is developed on the basis of the multicomponent system. EFLA®942 is manufactured in accordance to the Good Manufacturing Practice (GMP) and has a standardised reproducible composition with regard to the content of polyphenols, xanthines, and theanine. It is the extract that addresses the most importance on phytoequivalence with the “green tea beverage” proved for the safety and efficacy through the long history and food culture. The development of EFLA®942 has made feasible the research to prove scientifically many benefits known for green tea beverages. Presently, research on the efficacy of green tea is conducted with EFLA®942 in Europe, Japan and the USA. 1) Fujiki H (1999) Two stages of cancer prevention with green tea. J Cancer Res Clin Oncol 125(11):589-97 2) Benzie IF, et al. (1999) Consumption of treen tea causes rapid increase in plasma antioxidant power in 3) Haqqi TM, et al. (2000) Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green 4) Ministère des affaires sociales et de la solidarité (1990). Avis aux fabricants concernant les demandes d’autorisation de mise sur le marché des médicaments a base de plantes. Fascicule spécial no 90/22 bis 5) Major H, Kreuter MH, Rennert B, Netsch M, Melzig MF (2001) Effects of green tea and its polyphenols on various peptidases. Poster at the 49th Annual Meeting of the Society for Medicinal Plant Research, Erlangen, Germany 6) Müller-Klieser W (1997) Three-dimensional cell cultures: from molecular mechanisms to clinical applications. 7) Ames BN, Gold LS (1990) Chemical carcinogenesis: Too many rodent carcinogens. Proc Natl Acad Sci 8) Cerutti PA (1991) Oxidant stress and carcinogenesis. Eur J Clin Invest 21(1):1-5 9) Feldmann M, et al. (1996) Rheumatoid arthritis. Cell 85:307-10 10) Lusis AJ (2000) Atherosclerosis. Nature 407:233-41 11) Harada N, et al. (2000) Activated neutrophils impair gastric cytoprotection role of neutrophil elastase. Dig Dis 12) Dong G, et al. (2001) Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-kappaB signal pathway. Cancer Res 61(12):4797-4808 13) Ahmad N, et al. (2000) Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappa B in cancer cells versus normal cells. Arch Biochem Biophys 376(2):338-46 14) Momohara S, et al. (1997) Elastase from polymorphonuclear leukocyte in articular cartilage and synovial fluids of patients with rheumatoid arthritis. Clin Rheum 16(2):133-40 15) Williams SN, et al. (2000) Comparative studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression. Chem Biol Interact 128:211-29 16) Suganuma M, et al. (1999) Synergistic effects of (-)-epigallocatechin gallate with (-)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Res 59:44-7 17) Lou YR, et al. (1999) Effects of oral administration of tea, decaffeinated tea, and caffeine on the formation and growth of tumors in high-risk SKH-1 mice previously treated with ultraviolet B light. Nutr Cancer 33(2):146-53 18) Huang MT, et al. (1999) Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogenesis in SKH-1 mice: demonstration of caffeine as a biologically important constituent of tea. Cancer Res 57:2633-29

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