Hepatoprotecting effects of some bioactive principles from red wines Ion Neacşu, Irina Anca Cotea, Marius Niculaua, Valeriu V. Cotea Romanian Academy - Iasi Branch – Research Centre for Oenology, Aleea M. Sadoveanu 9 700490 Iasi, Romania Tel/fax: +40-232-228228
[email protected] Key words: red wine and must, allylic alcohol, cell respiration, membrane potential, redox potential, hepatoprotecting effects Abstract We studied the in vitro action of some samples of red wine and concentrated must on the liver of frog (Rana ridibunda, Pall), following the intensity of cellular respiration, the resting bioelectrical membrane potential and the redox potential (rH). Both wine (1 mL/100mL phyysiological solution normal Ringer – NR) and must (2 mg dry matter/100mL NR) stimulate aerobe respiration (mm3 O2/g wet tissue), comparatively with the non-treated reference sample. On intoxicating the liver with allylic alcohol (3 mL/100mL NR), known as inducing effects similar to those of hepatopathies, or with ethanol (1 mL/100mL NR) an irreversible depolarization of the cellular membranes may be noticed, alongwith perturbation of their structure and permeability. Treatment with either red must or red wine induces, on a normal liver, a stimulation of cell respiration, and on the intoxicated liver – a slow recovery of the normal values of the membrane potential, similar with the action of the hepatoprotecting drugs Silimarine and Aspatofort, administered in usual doses.The red must and the red wine also determine recovery of the hepatic redox potential affected by alcoholic intoxication, their effect being similar to that of the reference drugs. The obtained results show certain hepatoprotecting and redox modulating properties of the bioactive principles from red must and wine. Introduction Various investigations have evidenced the pozitive biological effects of a moderate wine consumption, induced by some active compounds – such as the polyphenols – present mainly in the red wines [1, 2, 10, 12]. Such compounds show anti-oxidating, anti-inflamatory, membranotrope, vasodilatatory, anti-atherogenes, anti-tumoral properties, with acting as modulators of the cell redox and bioenergetic processes, usualy mentioned for explaining the so-called “French paradox” [10]. The hepatoprotecting properties of such wine compounds were less studied, although the hepatoprotecting effects of the ethanol present in wine, besides the principles inducing beneficial effects, have been intensely studied [3, 6, 9, 11, 14]. This situation challenged the authors of the present study to investigate the effects of some red wine and must samples upon cellular respiration, on the membrane bioelectrical potential and on the redox potential of the frog luiver cells, intoxicated with allylic alcohol and ethanol. Material and method The experiments were performed in vitro, on the liver of frog (Rana ridibunda, Pall) incubated into a normal Ringer physiological solution (NR) containing allylic alcohol
(3mL/100mL NR) or ethanol, (1mL/100mL NR) subsequently substituted by a NR containing red wine (1mL/100mL NR) or concentrated red must (2 mg dry matter/100 mL NR) the duration of each phase being of 1 hour. Cellular respiration has been studied by the Warburg micromanometric method [8], the membrane potential – by the glass intracellular microelectrodes technique and redox potential – by potentiometric method, the obtained values being expressed by parameter rH, calculated according the relation of Clark, correlated with the normal hydrogen electrode (factor f) [5, 13]:
rH =
Eh + 0.058 ⋅ pH + f 0.029
The data thus obtained have been processed statistically by Student test. Results and discussion Determination of the intensity of the oxygen respiratory consumption (mm3 O2/g wet tissue) on a normal liver, for 1 hour, at intervals of 15 minutes (Table 1) evidenced a progressive stimulation the aerobe cell respiration processes, in the presence of both wine and the concentrated must, comparatively with the control non-treated samples (100%). Generally, the stimulating effect of the must is more intense than that of the wine (Table 1), the respiratory consumption of oxygen recorded after 60 minutes being 76.79% higher in the presence of must, and only 43.11% higher, respectively, in the presence of wine, comparatively with the control batch (100%), which may be possibly explained by the reverse action of the ethanol of the wine [2, 4, 6, 10, 11].
Table 1. Red wine and must influence upon hepatic cellular respiration (mm3 O2/g wet tissue of frog). 15 min 30 min 45 min 60 min Batch 3 3 3 3 mm mm mm mm (treatment) % % % % O2/g O2/g O2/g O2/g Control 0.4483 100 1.176 100 1.614 100 1.9727 100 Must 0.7654 170.73 2.031 172.7 2.8831 178.63 3.4876 176.79 Wine 0.9704 216.46 1.9409 165.04 2.6466 163.98 2.8231 143.11 Similar results have been attained in the determination of the resting bioelectrical membrane potential of the hepatic cells intoxicated through incubation, for 60 min, in a NR physiological solution containing allylic alcohol (3 mL/100 mL NR), subsequently treated with either red concentrated must (Table 2), or red wine (Table 3), for 60 min. The allyylic alcohol, frequently utiliyed in such experiments, induces, by its toxicity, major morpho/functional disorders of the liver cells, similar to the pathological ones [6, 7, 9, 11], expressed, in the present experiments, byy an irreversible decrease of the membrane potential, on the average with 13% of the normal value recorded initially in NR (Tables 2 and 3), as associated with severe modifications of the molecular structure and of the permeability of hepatocytes’ membranes [3, 6, 7, 9, 11]. The treatment with red must or wine of the hepatic cells depolarized by allylic alcohol results in a slow recovery (within about 60 min) of the normal values of the membranary biopotential, up to the manifestation, in the end, of a slight hyperpolarization tendency (of +0.36%, and, respectively, +0.31%, versus the initial normal value in NR). Such positive bioelectrical effects of the red must and wine are similar to those of the reference
hepatoprotecting drugs, Silimarine and Aspatofort (Table 3), administered in normal clinical doses (1.4 mg/100mL NR and, respectively, 0.5 mL/100mL NR), which indicates, once more, the manifestation of the hepatoprotecting properties of both red wine and must, most probably induced by the polyphenols they contain, possessing membranotropic, stabilizing and antioxidating properties [1, 4, 10]. Mention should be also made of the fact that the ethanol manifests hepato-toxic effects similar to those of the allylic alcohol, thus inducing an irreversible membranary depolarization of hepatocytes (Table 2), which may be anulated, however, by the action of the polyphenolic compounds present in wine and must [4, 10, 12, 14]. Table 2. Influence of treatment with ethanol, allylic alcohol and must upon membrane potential (MP) of frog hepatic cells (NR – normal Ringer physiological solution). Effect MP ±ES Exp. No. Values Minutes n (mV) (mV) ±mV ±% NR 0 29.87 0.45 18 0.00 100.00 10 28.62 0.35 17 Ethanol 30 27.73 0.41 17 1% v/V 1 60 26.75 0.43 18 –3.12 –10.44 10 27.21 0.54 16 NR 30 27.50 0.37 17 60 27.74 0.33 18 –2.13 –7.13 NR 0 30.55 0.43 17 0.00 100.00 10 27.44 0.55 18 Allyl-alcohol 30 27.01 0.48 16 3% v/V 2 60 26.30 0.37 18 –4.25 –13.91 10 27.07 0.47 17 NR 30 27.11 0.61 18 60 27.20 0.45 16 –3.35 –10.96 NR 0 30.27 0.46 16 0.00 100.00 10 28.73 0.51 18 Allyl-alcohol 30 27.21 0.37 17 3% v/V 3 60 26.67 0.41 18 –3.60 –11.89 10 27.32 0.53 17 Must 30 29.27 0.37 16 2 mg/100 mL 60 30.38 0.42 18 +0.11 +0.36 The results obtained permit drawing of another conclusion, namely that the red wine also causes normalization in the redox potential of the liver intoxicated with allylic alcohol, which provokes an abnormal increase of the rH values, both in the solutions containing the agents employed in the experiments and in the hepatic tissue (Table 4). The effects of the red must and wine of normalization of the redox potential are similar to those of the reference drugs Silimarine and Aspatofort, which evidences the multiple cellular influences of the bioactive principles present in must and wine.
Table 3. Influence of treatment with red wine, Silimarine and Aspatofort upon membrane potential (MP) of frog hepatic cells intoxicated with allylic alcohol (NR – normal Ringer physiological solution). Effect MP ±ES Exp. No. Values Minutes n (mV) (mV) ±mV ±% NR 0 31.47 0.56 17 0.00 100.000 10 30.10 0.43 18 Allyl-alcohol 30 28.82 0.39 18 3% v/V 4 60 27.26 0.40 17 –4.21 –13.38 10 30.03 0.45 16 Wine 30 30.75 0.37 18 1% v/V 60 31.57 0.66 16 +0.10 +0.31 NR 0 32.27 0.38 15 0.00 100.00 10 29.78 0.47 17 Allyl-alcohol 30 28.35 0.52 16 3% v/V 5 60 28.07 0.39 18 –4.20 –13.01 10 29.75 0.47 17 Silimarine 30 31.15 0.58 16 1.4 mg/100 mL 60 32.50 0.41 16 +0.23 + 0.71 NR 0 34.10 0.48 16 0.00 100.00 10 32.81 0.35 18 Allyl-alcohol 30 29.94 0.43 16 3% v/V 6 60 29.70 0.52 17 –4.40 –12.90 10 30.37 0.40 17 Aspatofort 30 32.94 0.43 16 0.5% v/V 60 34.06 0.38 18 –0.04 –0.11 Table 4. Influence of alcoholic and pharmacological agents upon rH values (V) of solutions and hepatic tissue of frog (NR – normal Ringer physiological solution). NR + agent Hepatic tissue Agent rH Effect rH Effect % % (V) (± %) (V) (±%) NR (control) 28.27 100 0 22.33 100 0 NR + Ethanol 28.31 100.14 +0.14 23.24 104.07 +4.07 NR + Allyl-alcohol 32.62 115.39 +15.39 23.89 106.99 +6.99 NR + Must 26.96 95.37 –4.63 22.4 100.31 +0.31 NR + Wine 27.17 96.11 –3.89 22.15 99.19 –0.81 NR + Silimarine 27.05 95.68 –4.32 22.11 99.01 –0.99 NR + Aspatofort 27.41 96.96 - 3.04 22.22 99.95 - 0.05
Conclusions The results of the study indicate the positive effects of the active principles present in vine and must – the red ones, especially – expressed by the normalization of the membrane potential and the redox potential of the hepatocytes intoxicated with alcohol, as well as by the intensigfication of the processes of aerobe cellular respiration. In this way, some hepatoprotecting and redox modulation properties of these active principles are put into evidence, which support the idea of some benefic effects of a moderate wine consumption, depending, however, on the individual physiological particularities. References 1. Burda S.,Oleszek W., 2001 – Anntioxidant and antiradical acticities of flavonoids, J. Agric. Food. Chem. 49, 2774-2779 2. Cotea V.D.,1985 – Tratat de Oenologie, vol.I, Ed. Ceres, Bucureşti, 224-250 3. Esterbauer H., 1996 – Estimation of peroxide damage, Path. Biol., 44, 25-28 4. Hollman P.C.H., Hertog M.G.L., Kata M.B., 1996. – Analysis of health effects of flavonoids, Food. Chem., 57, 43-46 5. Ingold W. 1982, – Redox measurements principles and problems, INGOLD, Urdorf, Switzerland 6. Naum E., Maftei A., Neacsu I., Maniu C.,2002 – Disturbance of redox processes determined by the action of ethanol at the cellular level, Sci. Anal. Univ. Agric. Sci. Vet. Med. Ion Ionescu de la Brad Iaşi, Horticulture, XLV, 97-102 7. Neacşu I., Zănoagă C.V., Niculaua M. 2006 – Hepatoprotective effects of some anthocyanic vegetal extracts,St. Cercet. Biol. Univ. Bacău (Romania), 11, 141-145 8. Nuţă G., Buşneag C. 1977 – Investigaţii biochimice, Ed. Didactică şi pedagogică, Bucureşti,283-286 9. Reid W.D., 1972 – Mechanism of allyl alcohol-induced hepatic necrosis, Experientia, 28, 1058-1061 10. Renaud S., Lorgeril M., 1992 – Wine, alcohol, platelet and “French paradox” for coronary disease, Lancet, 339, 1523-1526 11. Strubelt O., Deters M., Pentz R., Siegers C.-P., Younes M., 1999 – The toxic and metabolic effects of 23 aliphatic alcohols in the isolated perfused rat liver, Toxicological Sciences, 49, 133-142 12. Ursini F., Sevanian A., 2002 – Wine polyphenols and optimal nutrition, Annals N.Y. Acad. Sci., 957, 202-209 13. Zănoagă C.V., Neacşu I., Zănoagă M., 1988 – Consideraţii asupra tehnicii de determinare a rH-ului unor probe biologice, St. Cerc. Biochim., 31, 1, 53-58 14. Watkins T.R., 1977 – Wine-nutritional and therapeutic benefits, ACS Symposium Series 661, American Chemical Society, Washington D.C.
