The possible role of natural products in the ...

Acta Alimentaria DOI: 10.1556/AAlim.2007.0009

THE POSSIBLE ROLE OF NATURAL PRODUCTS IN THE DIETOTHERAPY OF CANCER-RELATED WEIGHT LOSS: AN ANIMAL MODEL V. VIRÁGa, T. VARJASa, Z. GYÖNGYIa, G. SOMLYAIb, I. EMBERa and E. NÁDASIa* a Department

of Public Health and Preventive Medicine, Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti út 12. Hungary b HYD Ltd for Research and Development, H-1113 Budapest, Ábel Jenő u. 12. Hungary (Received: 9 August 2006; accepted: 29 November 2006)

Despite many different trials, no effective dietotherapy exists for curing enormous weight loss caused by malignant diseases yet. The present study was aimed at determining in an animal model, weather some natural products might be included in the dietotherapy of cancerous patients with cachexia. Tumour was transplanted into Fischer 344 rats drinking either seabuckthorn extract, green tea, deuterium depleted water, trace elements preparation, fruits’ extract or multivitamin solution. Weight loss, tumour growth and expression of Ha-ras gene were determined. All the investigated natural products have significantly decreased tumour growth, and trace elements preparation has significantly decreased weight loss. Green tea, seabuckthorn extract and deuterium depleted water have notably diminished Ha-ras gene expression. Our results suggest that these natural products may be useful in inhibiting tumour growth, and some of them may be applied in the dietotherapy of cancer-related weight loss. Keywords: cancer-related weight loss, tumour growth, natural products, dietotherapy, animal model

At the time of presentation, as many as 40% of cancer patients are already malnourished before the onset of any medical or surgical treatment (SHIKE, 1996). Several years ago, in a large series of cancer patients, it was demonstrated that more than 50% of them had lost some weight and 15% had lost more than 10% of their pre-illness weight (DEWYS et al., 1980). Weight loss in cancer patients is associated with shortened survival (DEWYS et al., 1980; NIXON et al., 1981), poor quality of life (OVESEN et al., 1993b), and may itself be a cause of mortality in a small but significant proportion of cancer patients (INAGAKI et al., 1974). Cancer patients with a known involuntary 5% weight * To whom correspondence should be addressed. Phone: 36/72/536-394; fax: 36/72/536-395; e-mail: [email protected] © 2007 Akadémiai Kiadó, Budapest

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VIRÁG et al.: NATURAL PRODUCTS IN DIETOTHERAPY

loss have a shorter median survival rate than patients with stable weight (DEWYS et al., 1980). Lean body weight loss leads to a failed performance and immune status (CONNEY et al., 2002), which together with cancer-induced weight loss shown to be an independent adverse prognostic factor (BODY, 1999), may cause patients with weight loss respond poorly to chemotherapy and experience increased toxicity (ROSENBAUM et al., 2000). The best way to treat cancer-induced weight loss is to cure the cancer, but unfortunately, this remains an infrequent achievement among adults with advanced solid tumours (BARBER et al., 1999). Thus, a number of approaches have been taken to improve nutritional status in such patients. Attempts to raise energy and protein intake by counselling have been successful, but despite improvements over a 3-month-period, no improvement in weight, anthropometric measures, response rate, survival, or quality of life have been demonstrated (EVANS et al., 1987; OVESEN et al., 1993a). Since several natural products such as green tea have been demonstrated to inhibit carcinogenesis, the aim of our present study was to determine that natural products sold commercially and recommended for cancer patients have an effect both on tumour growth and cancer-related weight loss. 1. Materials and methods 1.1. Tumour transplantation Two mg/animal Ne/De tumour mass (mesenchymal renal tumours induced by a single dose of dimethylnitrosamine in Fisher 344 newborn rats, courtesy of Prof. Pál Kertai, University of Debrecen, Debrecen, Hungary, described in reference DEZSŐ and coworkers (1990)) was transplanted underneath the renal capsule of Fischer 344 rats (10 animals per group, 5 females and 5 males. 1.2. Treatments Each group of animals was treated per os for 14 days with seabuckthorn extract (HT; HéBé Ltd, Tata, Hungary), green tea (GT) (1 l tea prepared with 5 g dried tea leaves in tap water), deuterium depleted water (ddW; HYD Ltd, Budapest, Hungary), trace elements preparation (Béres drops/B; University of Pécs, Pharmacy, Pécs, Hungary) completed with Béres Vitamin C (University of Pécs, Pharmacy, Pécs, Hungary), Flavin7 fruits’ extract (F7; Crystal Institute Kft, Eger, Hungary), or multivitamin and mineral tablet (Centrum/C; University of Pécs, Pharmacy, Pécs, Hungary), while the untreated group (K) received tap water as drinking fluid. Details of treatments are given in Table 1. Tablets and extracts were dissolved in tap water and solutions were freshly prepared each day. Doses of the natural products were the body mass equivalent of human doses recommended by the manufacturers. Body weight of the animals was measured daily by using a precision scale.

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Table 1. Protocol of per os treatments of tumour-bearing F344 rats Animal group Untreated Seabuckthorn extract Green tea Deuterium depleted water Flavin7 Centrum multivitamin tablets Béres drops

Treatment Tap water (no treatment) 0.25 ml/day dissolved in tap water Ad libitum (5 g dried green tea leaves in1 l prepared tea) Ad libitum 0.25 ml/day, dissolved in tap water 1/5 tablet/day multivitamin and mineral tablet, dissolved in tap water 2 drops/day, completed with 0.25 tablet of Béres Vitamin C 50 mg

1.3. Composition of natural compounds Composition of used naturals products were as follows. Seabuckthorn extract: vitamin A 1310 mg, carotenoids 225 mg, vitamin E 432 mg in 100 ml extract. Deuterium depleted water: 21 ppm±3 ppm deuterium (tap water: 150 ppm±3 ppm deuterium). Béres drops: 2.00 mg iron, 1.14 mg zinc, 0.40 mg magnesium, 0.31 mg manganese, 0.25 mg copper, 0.19 mg molybdenum, 0.12 mg vanadium, 0.11 mg nickel, 0.10 mg boron, 0.09 mg fluor, 0.025 mg cobalt in 1 ml purified water. Flavin7: total energy content 9.7 kJ, protein 0.07 g, fat 0.02 g, carbohydrates 0.16 g, total polyphenols 85 mg (flavonoid 75 mg, resveratrol 0.16 mg) 8 v/v% alcohol in 10 ml solution. Centrum multivitamin and mineral tablet: vitamin A 4000 IU, vitamin B1 1.5 mg, vitamin B2 1.7 mg, vitamin B3 20 mg, vitamin B6 2 mg, vitamin B12 6 µg, vitamin C 60.0 mg, vitamin D 400 IU, vitamin E 30 IU, vitamin H 30 µg, vitamin K 29 µg, folic acid 400 µg, boron 150 µg, calcium 162 mg, chlorine 72 mg, chromium 130 µg, copper 2 mg, iodine 150 µg, iron 18 mg, magnesium 100 mg, manganese 2 mg, molybdenum 80 µg, nickel 5 µg, phosphorus 109 mg, potassium 80 mg, selenium 24 µg, silicon 2 mg, stannic 11 µg, vanadium 10 µg, zinc 15 mg per tablet. Tap water contains the following minerals: sodium 18 mg l–l, potassium 1.5 mg l–l, calcium 89 mg l–l, magnesium 41 mg l–l, chlorine 25 mg l–l, fluor 0.2 mg l–l, sulphate 56 mg l–l, hidrogencarbonate 427 mg l–l, nitrate 7 mg l–l). 1.4. Tumour growth, body weight loss, RNA isolation Rats were autopsied after 2 weeks of survival. Tumours were removed and weight was determined by precision scale. Tumour weight was subtracted from the body weight measured the last day to determine the absolute body weight loss. One hundred mg tissue samples of liver, spleen, kidney, lung and thymus were removed from each animal and total cellular RNA was isolated by using TRIZOL Reagent (GIBCO, Grand Island, NY, USA). After RNA concentration and quality check at 260/280 nm, 5 µg of RNA was dot blotted onto Hybond N+ nitrocellulose membranes and hybridised with chemiluminescently-labelled (ECL Kit, Amersham, Little Chalfont, UK) Ha-ras (Prof. J. Szeberényi, University of Pécs, Hungary) DNA probe. Signals were detected on X-ray films and quantified by Quantiscan software (Biosoft, Cambridge, UK). Gene expressions were evaluated as percentages of β-actin. The arbitrary unit in Fig. 1 is equivalent to 100% expression of β-actin.

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1.5. Statistical analysis Influence of the applied treatments on tumour growth, body weight loss and expression of Ha-ras gene was determined by Student’s t-test. 2. Results and discussion 2.1. Tumour growth and body weight loss Two Fischer 344 rats from the HT group and 1 from the GT group had to be excluded from the evaluation because of caducity prior to the end of the experiment. Tumour growth in different groups is shown in Table 2. All the investigated natural products have significantly reduced the tumour growth in the 2-week-long experiment (P