INFLUENCE OF NATURAL POLYPHENOLIC FRACTIONS ON CELLULAR RESPIRATION
INFLUENCE OF NATURAL POLYPHENOLIC FRACTIONS
ON CELLULAR RESPIRATION
I. NEAC?U1*, P. ROTINBERG2, Daniela GHERGHEL2, C. MIHAI2,
V.V. COTEA1 , C.V. Z?NOAG?1, M. NICULAUA1
1Research Centre for Oenology – Ia?i Branch of the Romanian Academy
2Institute of Biological Research, Ia?i
ABSTRACT – We have studied the influence of two polyphenolic fractions – methanolic (PMF) and ethanolic (PEF) – extracted from the
leaves of the medicinal plant Asclepias syriaca upon cellular respiration.
Experiments were performed in vitro on the cells from liver, sartorius
striated muscle and stomach smooth muscle of frog (Rana ridibunda, Pall.),
determining the cellular oxygen consumption by the Warburg
micromanometric method. Polyphenol effects were different, depending on
the nature of used fraction and cellular type. Therefore, after 90 minutes of
experiencing on liver, the both polyphenolic fractions stimulated cellular
respiration, compared to the untreated control. The striated muscle PMF
has inhibited and PEF has stimulated respiratory processes. In stomach
muscle, reverse effects were noticed: PMF has stimulated and PEF has
slightly inhibited cellular respiration. The results pointed out the specific
action of these polyphenolic agents on cellular respiration and energetic
metabolism processes, also allowing the estimate of their useful
pharmacological properties.
Key words: polyphenolic fraction, cellular respiration, energetic metabolism
Rezumat – Influen?a unor produ?i polifenolici naturali asupra respira?iei celulare. S-a urm?rit influen?a a dou? frac?ii polifenolice asupra
respira?iei unor tipuri diferite de celule animale, studiindu-se o frac?ie
metanolic? (PMF) ?i alta etanolic? (PEF), extrase din frunzele plantei
medicinale Asclepias syriaca. Experimentele s-au realizat ” in vitro”,
determinandu-se, timp de 90 de minute, consumul respirator de oxigen al
celulelor de ficat, mu?chi striat sartorius ?i mu?chi neted stomacal de
broasc? (Rana ridibunda, Pall.), prin metoda micromanometric? Warburg. * E-mail: ineacsu@uaic.ro
Efectele respiratorii au fost diferite, ?n func?ie de natura frac?iei
polifenolice ?i tipul celulelor. Astfel, dup? 90 de minute, la ficat ambele
frac?ii au stimulat respira?ia celular?, comparativ cu lotul martor, netratat,
?ns?, la ?esutul muscular, efectele au fost mai diverse: la mu?chiul striat,
PMF a indus o inhibare, iar PEF o stimulare a respira?iei, ?n timp ce la
stomac, efectele au fost inversate – o stimulare sub ac?iunea PMF ?i o
u?oar? depresare a consumului de oxigen sub influen?a PEF. Rezultatele
eviden?iaz? efectele specifice ale acestor agen?i polifenolici asupra
proceselor respira?iei celulare ?i asupra metabolismului intermediar ?i
energetic celular ?i permit estimarea propriet??ilor lor farmacologice utile.
Cuvinte cheie: frac?ii polifenolice, respira?ie celular?, metabolism energetic
INTRODUCTION
Different investigations have signalled the presence of polyphenols in
different organs of some plants and, especially, in flowers, fruits and leaves (Bodea, 1965), as well as in juice, must and wine (Cotea, 1985; Ursini, Sevanian, 2002). There were shown some aspects regarding the bioactive characteristics of polyphenols from different plant extracts, such as the ones present at cell level,
expressed by membranotrope, bioelectric, and bioenergetic (Cr?ciun et al.,1995; Gonzales-Lebrero et al., 2003; Neac?u et al.,2004; Rotinberg et al., 2004; Rotinberg et al., 2005), metabolic (Rotinberg et al, 2005), antioxidant and liver protecting (Ursini, Sevanian, 2002), and anti-tumour actions (Rotinberg et al., 2004; Rotinberg et al., 2005; Rotinberg et al., 2004; Rotinberg et al., 2005) or by redox modulators (Karp, 1996; Ursini, Sevanian, 2002).
For pointing out aspects typical of the action of some natural polyphenolic
agents at the level of animal cells, this paper has studied the specific effects on
cell respiration, determined by two types of plant polyphenolic extracts, at three
categories of animal cells – liver, striated and smooth muscular cells. We had in
view the correlation between the intensity of oxygen consumption and cellular
energy (Karp, 1996; Lehninger, 1987) and data enriching as concerns useful pharmacological properties of studied polyphenolic produces, as well as the explanation of aspects on the mechanism of their effects.
MATERIALS AND METHODS
We have studied the dynamics of the intensity of oxygen consumption at three categories of animal cells, as influenced by two types of polyphenolic preparations, extracted and purified from leaves of the medicinal plant Asclepias syriaca – methanolic (PMF) and ethanolic fraction (PEF), at rates of 1.5 mg DM/ mL normal Ringer solution (NR).
Experiments were conducted in vitro on three series of frog (Rana ridibunda, Pall.) tissue preparations, represented by liver fragments (L), sartorius striated muscle (SM) and
stomach smooth muscle (SSM), accurately weighed and incubated into the respiration vessel, in pure or with studied agent Ringer solution, at a constant temperature of 200C. Each experimental series was carried out on three groups of five preparations obtained from the same tissue: an untreated control group, kept in NR without agents, a group treated with PMF and another treated with PEF, at a concentration of 1.5 mg DM/mL NR. The oxygen consumption was determined by the Warburg micro-manometric method (Rotinberg et al., 2004), at a constant volume, for 90 minutes. Reading of the manometrical values and calculation of the oxygen consumption were carried out at intervals of 15-30 minutes. The results were expressed in mm3 O/g tissue/h and were calculated statistically according to the Student Test, by reporting to the quantity of studied fresh tissue (g) and to the control group, which was not treated with polyphenols.
For the estimate of the composition of studied polyphenolic fractions, their adsorption spectrum was registered in UV. Spectra were traced by the help of spectrometer UV-Vis Analytical Jena UV-Vis 200 PC with a slit of 5 nm and a scanning speed of 1 nm/s. We reported to water found in 2 mm quartz tanks. The dry mass was dissolved in water and, then, filtered through a sterilized nylon filter of 0.45 μm.
RESULTS AND DISCUSSION
The obtained data have shown a respiratory reactivity of the studied tissues to the action of polyphenolic produces, which differed according to the type of tissue, nature of polyphenolic fractions and periods of time at which the measurement of cell oxygen consumption was done.
At the liver tissue from the untreated control, a graduate diminution in the intensity of oxygen consumption was registered during 90 minutes, from 1.773 mm3 O/g/h (100%) until 0.919 mm3 O/g/h, and after 90 minutes (51.8%). It represented the normal dynamics of liver cellular respiration under experimental conditions (Table 1), the diminution in the intensity of respiration being caused by the decrease of in vitro cellular energetic stocks.
When treating the liver preparations with polyphenolic fractions, a different dynamics of cellular respiration was registered, according to the nature of studied fraction (Table 1). Thus, the methanolic fraction (PMF) has induced a graduate increase in the intensity of oxygen consumption, while the ethanolic fraction (PEF) has resulted in a diminution in the oxygen consumption, which was maintained during the entire period of the experiment. By reporting the values of treated groups to those of the control, at each period of the measurements, considered as 100%, we found that both polyphenolic fractions have stimulated respiration, unlikely the untreated control (Table 1). The stimulation effect has been differentiated according to the nature of polyphenolic agents. PMF has induced an earlier (at minute 30) and stronger stimulation. At minute 90, respiration was more intense by 154% compared to the control, and in case of PEF, stimulation was later (minute 60) and weaker (by 60.2%), as we could also see in a previous work (Neac?u et al., 2004)
Table 1
Dynamics of the intensity of cell respiration (mm 3 O 2/g/h), as influenced by
polyphenolic agents in liver, striated muscle and smooth muscle
Duration (minutes) Tissue Agent Index 15 (±%C) 30 (±%C) 60 (±%C) 90 (±%C)
?X 1.773 (100%) 1.628 (100%) 1.061 (100%) 0.919 (100%) ±SE
0.613 0.458 0.283 0.144 Control
(C) VC% 37.374 32.908 39.268 34.997
?X
1.492 (?15.9) 1.996 (+2
2.6) 2.398* (+126.0) 2.335* (+154.1) ±SE
0.084 0.497 0.391 0.452 PMF VC% 13.820 30.992 39.396 43.317
?X 1.580 (?10.9) 1.435 (?11.9) 1.531* (+44.3) 1.472* (+60.2) ±SE
0.328 0.380 0.408 0.320 L e v e r (L )
PEF VC% 40.981 34.991 35.431 38.622
?X 1.912 (100%) 2.340 (100%) 1.839 (100%) 1.172 (100%) ±SE
0.046 0.231 0.144 0.271 C VC% 5.414 27.810 17.489 25.324
?X 2.190 (+14.7) 1.646 (?29.7) 1.376 (?25.2) 0.943 (?19.6) ±SE
0.369 0.488 0.382 0.293 PMF VC% 37.732 36.388 32.100 29.730
?X
1.776 (?7.2) 1.980 (?15.4) 1.780 (?3.2) 1.842* (+57.1) ±SE
0.445 0.338 0.109 0.131 M u s c l e s (S M ) PEF VC% 36.088 38.272 13.766 15.917
?X 0.738 (100%) 1.197 (100%) 1.101 (100%) 0.990 (100%) ±SE
0.150 0.099 0.023 0.038 C VC% 45.601 18.534 4.702 8.596
?X
1.570* (+11
2.7) 1.895* (+58.3) 1.690* (+5
3.5) 1.618* (+63.4) ±SE
0.110 0.161 0.166 0.145 PMF VC% 15.699 19.017 22.064 22.068
?X 0.967 (+31.0) 0.890 (?25.7) 0.835 (?24.2) 0.896 (?9.5) ±SE
0.236 0.199 0.125 0.146 S t o m a c h (S S M ) PEF VC% 34.781 43.347 33.634 36.521
?X = mean value; SE = standard error; VC = variability coefficient; C = control;
PMF= methanolic fraction of polyphenols; PEF = ethanolic fraction of polyphenols;
* = significant to C
At the striated muscle, the dynamics of respiration has shown some different aspects compared to that of the liver (Table 1). At the untreated control, respiration has registered an increase by 22.4% in minute 30, compared to minute 15 (1.912 mm3 O/g/h; 100%), then it diminished gradually, as in case of liver, until 1.172 mm3 O/g/h (by 38.4% compared to minute 15) at minute 90. The treatment of the striated muscle with PMF resulted in a decreasing dynamics of oxygen consumption values, from 2.190 mm3 O/g/h, at minute 15, to 0.943 mm3 O/g/h (by 56.9% to minute 15), 90 minutes after the experiment. In comparison with the values of the control group (100%), we found that PMF has induced a moderate inhibition of the respiratory processes by 29.7% at minute 30, and by 19.6% at minute 90, although, initially (at minute 15), a slight stimulation of respiration (by 14.7%) took place. The ethanolic fraction (PEF) had a reverse effect compared to the methanolic one (PMF), determining a diminution in the intensity of cell respiration in the first 60 minutes (by 3.2-15.4% compared to the control), followed by a stimulation (by 57.1%) after 90 minutes. We found that the reactivity of striated cells to the action of polyphenolic fractions was different from the one of liver cells, because of their structural-functional characteristics, the muscular cells being excitable and contractile, and the liver cells being of metabolic type (Karp, 1996).
The cells of stomach smooth muscles have shown a more diverse respiratory dynamics to the action of polyphenolic agents (Table 1), resembling both to striated muscular cells and to hepatocites, but being also different from these. Thus, at the control group, unlike liver and striated muscle, in vitro experimental conditions have required an intensification of cellular respiration during the entire period of measurements; after 90 minutes, the oxygen consumption was greater by 34.1% than after 15 minutes.
The methanolic fraction has shown a respiratory effect quite similar to the one from liver, but different from the one of the striated muscle, requiring an increase in the intensity of cell respiration during the determinations. This increase was higher by 112.7%, at the first stage, after 15 minutes, compared to the control, and, then, moderate (by 63.4%) after 90 minutes.
The ethanolic fraction has caused a different effect, having a weak resemblance to the one from the striated muscle, and requiring a slight respiratory intensification after 15 minutes (by 31% compared to the control), followed by a total depression during the entire period of determinations. After 90 minutes, the diminution was by 9.5% compared to the control (Table 1). We remarked that stomach smooth muscular cells have shown a different reactivity to the action of polyphenolic fractions, in comparison with the striated ones, although they were excitable contractile cells, too. However, they presented some structural-functional differentiations to the striated ones, expressed by a bioelectric and contractile automatism. It determined a rhythmical contractile activity, also involving some metabolic specific features, different from other cellular types and
a different energetic metabolism. This required certain dynamism of cellular
respiratory processes.
At the same way, we could explain the respiratory response of liver cells,
which are the headquarters of multiple metabolic processes, involving oxygen
consumption within the processes of the Krebs cycle of aerobe cellular respiration
(Karp, 1996; Lehninger 1987).
By comparing the values of oxygen consumption for respiration at the
control groups, we found that the striated muscular tissue presented a higher
intensity of cellular respiration, which was correlated to the processes of oxidative phosphorilation, creating a superior energetic balance (Lehninger, 1987;Neac?u
et al., 1996).
The action of polyphenolic fractions on the three types of investigated cells
has interfered with the processes of their aerobe respiration, and implicitly, with
the energetic processes, correlated to the reactions of oxidoreduction and
oxidative phosphorilation within the Krebs cycle (Lehninger, 1987; Neac?u et al.,
1996). The effects were according to the characteristics of the cellular type and
the nature of studied polyphenols.
The characteristic effects of the two types of polyphenolic fractions have
been signalled in glucide, lipide and protide intermediary metabolism of tumour
cells, correlated with a stimulation of cellular respiration and their cytostatic characteristics (Cr?ciun, 1995; Karp, 1996; Neac?u et al., 2004; Neni?escu, 1968;
Rotinberg et al., 2005, Rotinberg et al., 2004; Rotinberg et al., 2005; Ursini,
Sevanian, 2002).
The differences between the effects of the two types of polyphenolic
fractions came from the different chemical composition, also shown by their UV
absorption spectra. From UV-Vis spectrum, the presence of a continuous
emission area could be noticed, which was due to a clearly marked fluorescence
in the entire UV field. The spectral nature of these alcoholic extracts, strongly
fluorescent (until four units of absorbance), has shown that they were not
compounds of anthocyanic type, but of polyphenol-quinol type.
A detailed analysis of similar fractions has pointed out the differences of
chemical composition between them, by the spectrophotometrical determination
of polyphenols, the total content being expressed in galic acid (0.76 g/L). Data are
presented in Table 2 (Karp, 1996).
Table 2
Composition of polyphenolic fractions
Compounds Methanolic fraction Ethanolic fraction
Total polyphenols 19% 20%
12–15% Flavones 10%
Catechol 2%
2.5%
10% Anthocians 9%
By thin layer chromatography, rutine, flavones, glucose, mannose, palmitic, stearic, limuli and arahidonic acids, as well as steroids (β-sistosterol, heterozide arahidonic) were found at different rates in the two fractions (Karp, 1996). This chemical composition has explained the multiple biological effects of polyphenolic agents and differences between studied fractions and justified the continuation of investigations, for establishing their pharmacological characteristics and specific action mechanism.
CONCLUSIONS
The polyphenolic fractions extracted from leaves of Asclepias syriaca have a great influence on the intensity of cellular respiration, and the type of studied cells (liver, striated muscular or smooth muscular cells).
The respiration of liver cells was stimulated by both types of polyphenolic fractions, but with different amplitude, according to the nature of fraction, the methanolic one having a stronger effect than the ethanolic one.
The oxygen consumption by striated muscular cells was slightly diminished by both polyphenolic fractions, but, at the end of the experimental period, the ethanolic fraction has resulted in a slow effect of stimulating the cellular respiration.
The reactivity of stomach smooth muscular cells to the action of polyphenols was different, both to the action of liver cells and to the one of striated muscular cells. The methanolic function has determined stimulation and the ethanolic fraction – slight inhibition of cellular respiration.
The results indicate the interference of investigated polyphenolic agents to the processes of aerobe respiration and cellular energetic and intermediary metabolism and allow the assessment of useful pharmacological characteristics of these compounds.
REFERENCES
Bodea C. (coordinator), 1965 – Treatise of plant biochemistry, vol II, Academia Roman?, Bucharest, 768, 784
Cotea V.D., 1985- Treatise of oenology, vol. I, Ceres, Bucharest, 224-250
Cr?ciun V., Cr?ciun M., Agrigoroaei ?., Neac?u I., Rotinberg P., Kelemen S., 1995 - The specific action of some polyols and polyphenolic fractions on the Na+?K+?ATP-
ase activity, Journal Roum. Biol.-Biol. Anim., 40, 2, 141-144
Gonzales-Lebrero R.M., Kaufman S.B., Garrahan P.J., Rossi R.C., 2003 - The sidedness of the direct route of occlusion of K+ in the Na+/K+-ATPase, Ann. N.Y.
Acad. Sci., 986, 301-303
Karp G., 1996, Cell and Molecular Biology, John Wiley and Sohns Inc., New York, Chichester, Brisbane, Toronto, Singapore, 177-205, 675
Lehninger A.L., 1987 - Biochemistry, vol. II, Tehnica, Bucharest, 473-503
Neac?u I., Agrigoroaei ?., Cr?ciun V., Cr?ciun M., Rotinberg P., Kelemen S., Nu?? V., Oi?? N., 1996 - Bioelectrical effects of some polyolic and polyphenolic compounds in normal environment conditions, Journal Roum. Biol.-Biol. Anim., 41, 2, 165-169 Neac?u I., Rotinberg P., Gherghel D., Mihai C., Giread? O., 2004 – Influence of some polyphenolic products on cellular respiration in frog liver. Bul. Soc. Nat. Biol. Cel., 32, 190
Neni?escu C.D., 1968 – Organic chemistry, sixth ed., vol. II, Didactic??i Pedagogic?Publishing House, Bucharest, 24
Nu?? G., Bu?neag C., 1977 – Biochemical investigations, Didactic??i Pedagogic?Publishing House, Bucharest, 283-286
Rotinberg P., Gherghel D., Neac?u I., Rotinberg H., Mihai C., 2004 - The interference of some active cytostatic autochtonous polyphenolic biopreparations with membranary Na+?K+?ATP-ase activity of the tumoral cells, Scientific Annals of “Al.
I. Cuza” University of Ia?i, Genetics and Molecular Biology, V, 44-50
Rotinberg P., Neac?u I., Gherghel D., Gr?mescu M., Mihai C., 2005 - Reactivity of cellular respiration at tumour cells, under the action of new cytostatic agents of polyphenolic nature,Bul. Soc. Nat. Biol. Cel., 33, 197
Rotinberg P., Gherghel D., Neac?u I., Rotinberg H., Mihai C., 2004 - Effect of some active cytostatic vegetable extracts of polyphenolic nature upon membrane bioelectric potential of tumour cells, Scientific Annals of “Al. I. Cuza” University of Ia?i, Genetics and Molecular Biology V, 51-56
Rotinberg P., Gherghel D., Gr?mescu M., Mihai C., Neac?u I., Hefco V., Rotinberg H., 2005 - The reactivity of the metabolic processes of the Hep-2p tumoral cells to the action of some active cytostatic biopreparations of polyphenolic nature, Scientific Annals of “Al. I. Cuza” University of Ia?i, Genetics and Molecular Biology VI, 75-82 Ursini F., Sevanian A., 2002 - Wine polyphenols and optimal nutrition, Annals N.Y. Acad.
Sci., 957, 200-209