Annals of RSCB Vol. XVI, Issue 1

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1 ANTIOXIDANT EFFECT OF ARONIA VERSUS SAMBUCUS ON MURINE MODEL WITH OR WITHOUT ARTERIAL HYPERTENSION Anca Iuliana Moroşanu 1,2, Manuela Ciocoiu 2, L. Bădescu 3, Magda Bădescu 2 UNIVERSITY OF MEDICINE AND PHARMACY GR. T. POPA IASI, ROMANIA 1 DEP. OF GERIATRICS AND GERONTHOLOGY C. I. PARHON HOSPITAL; 2 DEPARTMENT OF PATHOPHYSIOLOGY; 3 DEPARTMENT OF CELLULAR AND MOLECULAR BIOLOGY Summary The data provided in literature reveal the complex biologically-active properties of polyphenols: antioxidant, anti-inflammatory, platelet antiaggregant, hypocholesterolemic, vasorelaxant. Due to their anthocyanins content, both Aronia melanocarpa and Sambucus nigra may have beneficial effects on the evolution and complications of arterial hypertenison. The experiment was conducted on five groups of white Wistar rats that enjoyed Aronia and Sambucus extract protection without suffering from drug-induced high blood pressure versus those that suffered from L-NAME induced high blood pressure. Their blood pressure as well as their SOD and GSH-Px expressed antioxidant status were measured on a weekly basis throughout the experiment, and their kidneys were sampled for a histological examination performed at the end of the experiment. The antioxidant capacity was either highly statistically significant for Aronia versus the purely high blood pressure group, or just statistically significant for Sambucus; moreover, the high blood pressure group treated with Aronia proved more protected (p<0.001) than the unprotected group and more highly statistically significant than the group that received Sambucus. The anatomicpathological examination of the kidney sample revealed alterations such as chronic lymphocytic inflammatory infiltrate, hydropic degeneration injuries, perivascular fibrosis. These alterations were considerable in the high blood pressure group, whereas in the polyphenol protected groups they were much diminished. The considerable oxidative stress revealed in the high blood pressure rats was favorably modulated by vegetable polyphenols delivery. The histological examination conducted on the kidney samples supported the vessel protection effect of the two extracts. Key words: Aronia melanocarpa, sambucus nigra, oxidative stress, arterial hypertension. magda.badescu@gmail.com Introduction It is known that natural polyphenols posses complex biological properties such as: antioxidant, antiinflammatory, cardioprotective, antiplatelet activities. The Aronia genus include Aronia melanocarpa specie, are one of the richest plant sources of phenolic substances, mainly anthocyanins. Most of the effects of Aronia melanocarpa anthocyanins are due to their high antioxidative activity. Numerous health-promoting activities such as antioxidative, cardioprotective, 222 antidiabetic, antiinflammatory, antibacterian, antiviral, immunomodulatory have been demonstrated for black chokeberry (Aronia melanocarpa) extracts by both in vitro and in vivo studies (Kokotkiewicz et al., 2010). Also, the fruit of Sambucus nigra (elderberries) contain several anthocyanin flavonoids known to possess significant antioxidant properties. As a cardiovascular risk factor, hypertension is associated to a endothelial dysfunction and oxidative stress. It has

2 been established that endothelial cell dysfunction results in changes in the redox status of cell (Kehrer, 1993). Based on this presmise and on elderberry s antioxidant potential, Youdin et al. demonstrated elderbery anthocyanin Material and methods Elder fruits (Aronia melanocarpa and Sambucus nigra) were washed and shade dried. Dried and powdered fruits (50 g) were extracted with 2 x 250 ml acidulated methanol (0.5% HCl) using a magnetic stirrer, each time for one hour. The total phenolic content in elder fruit extract was determined by the Singleton & Rossi method. The amount of total phenolic content was expressed as g gallic acid equivalents (GAE)/100 g extract. The absorbencies of all the solutions were determined by using a V- 550 Able Jasco UV-VIS spectrophotometer. The result is the mean of triplicates ± standard deviation. The research was performed on Wistar white rats, with an average weight of g, wich were divided into five groups of ten, namely: - Group A animals wich were administered the extract obtained from the Aronia melanocarpa fruit, with a dosage of (?) p.o. (by tube feeding) at every two days for eight weeks; - Group S animals wich were administered the extract obtained from the Sambucus nigra fruit, with a dosage of (?) p.o. (by tube feeding) at every two days for eight weeks; - Group animals wich were administered L-NAME 40 mg/kg body/day i.p. at every two days for eight weeks; - Group + A animals wich were administered the Aronia melanocarpa extract p.o. at every two days concomitantly with L-NAME for eight weeks; - Group + S animals wich were administered animals wich were administered the Sambucus nigra extract p.o. at every two days concomitantly with L-NAME for eight weeks. incorporation against oxidative stress. Elderberry s ability to incorporate into endothelial cells and potentially improve endothelial function may also indicate a role in prevention in vascular disease such as hypertension. The experimental study fulfils all the requirements of the guide regarding the use of laboratory animals and biological preparations issued by the International Society of Pain Study (IASP) and the European Council Committee (86/609/EEC). Glutathion peroxidase (H2O2: GSH oxidoreductase) (GSH-Px) was determined by the Gross and Beutler method (Beutler et al., 1990); the GSH- Px activity was expressed in µm oxidized GSH per minute/ g Hb or mg protein. Superoxide dismutation (SOD) determination was achieved by the Minami method (Minami and Yoshokawa, 1979) in which the pyrogalol is used as O - 2 anion generator. Statistical data interpretation. All the data are shown as mean value ± standard error of the mean (SEM). Statistical analysis was performed with the paired or unpaired t-test. Statistical data interpretation considered the corresponding differences for a given significance threshold: p>0.05 statistically insignificant; p<0.05 statistically significant; p<0.01 strong statistical significance; p<0.001 very strong statistical significance. Results and discussion Serum superoxide-dismutases (SOD) and GSH-Pxs are two major antioxidant enzyme families that may act co-operatively in protecting cells against oxidative stress (Hu et al., 1995). GSH-Px is an enzyme localized in the cytoplasm or in the mitochondrial matrix and catalyzes the reduction of H2O2 and of organic hydroperoxides. The activity of GSH-Px is tightly linked to the glycolysis dependent metabolic processes 223

3 and is correlated with the oxidation of LDL (Ursini et al., 1995). A strong statistic significance reveals bitween the hypertension group that enjoyed protection and those that did not (p<0,01). The experiment shows no difference between the two types of protection Aronia versus Sambucus (Table I). Table I. Blood pressure in white Wistar rats TAS (mmol/l) Lot A Lot S Lot Lot +S Mean value ± standard deviation 1,5812 ± 0, ,8221 ± 0, ,3577 ± 0, ,7979 ± 0, ,9060 ± 0,22624 Variation coefficient, % 0,101 0,262 0,027 0,031 0,051 Statistical significance Lot A/Lot S p=0,415 Lot +S / Lot +S p=0,229 The serum activity of SOD in the rats belonging to group A versus group S has know statistic significance, but reveals highly significant values () in group A when compared with group. Also, a strong statistical significance appears (p<0,01) for group with Aronia protection versus group, but not the same thing happens with Sambucus protection (Table II). Table II. Serum SOD activity in white Wistar rats SOD (U/mL) Lot A Lot S Lot Lot +S Mean value ± standard deviation Variation coefficient, % 9,7833 ± 0, ,1583 ± 0, ,5250 ± 0, ,3600 ± 0, ,4250 ± 0, ,442 0,959 0,498 0,761 0,662 Statistical significance Lot A/Lot S Lot A/ Lot Lot S/ Lot Lot +S Lot +S / p=0,282 p=0,001 p=0,463 p=0,002 (p<0,01) p=0,012 (p<0,05) The activity of GSH-Px in the group with Aronia reveals highly significant values () versus group and the same thing appears () if the group is and has Aronia protection. On the other hand, in the group with Sambucus, the activity of GSH-Px has statistical significance values (p<0.05), but not very strong significance as Aronia (Table III). 224

4 Table III. Serum GSH-Px activity in white Wistar rats GSH-Px (U/L) Lot A Lot S Lot Lot +S Mean value ± standard deviation Variation coefficient, % 1562,2832 ± 85, ,4584 ± 242, ,9640 ± 96, ,1224 ± 118, ,4543 ± 252, , , , , ,744 Statistical significance Lot A/Lot S Lot A/ Lot Lot S/ Lot Lot +S / Lot +S p=0,945 p=0,020 (p<0,05) 0,005 (p<0,01) 0,539 For histopatologycal lesions, the study uses usual (hematoxylin eozyne) and special (Van Gieson with elastine) stains. The sections were examined at the optical microscope with a x 100 lens. The histologycal examination of the kidney sample releaves important congestia in the interstice of medular area in hypertension group, not so evident in hypertension group treated with Aronia (Fig. 1). But in this last group, is observed chronic lymphocytic inflammatory infiltrate and diffuse microhemorrhage in the interstice (Fig. 2). On the other hand, the chronic lymphocytic inflammatory infiltrate is more evident in hypertension group treated with Sambucus (Fig. 3). Also appears in the cortical area TCP lines with cells on with hydropic degeneration injuries or areas with flat cells. Fibrosis (peritubular or nodular forms) like the expression of hypertension impact on kidney appears in specials stain (Van Gieson with elastine) on pure hypertension group (Fig. 4). Fig. 1 Cross section through the kidney, Group (HE, x100) Fig. 2 Cross section through the kidney, + A Group (HE, x100) 225

5 Fig. 3 Cross section through the kidney, + S Group (HE, x100) Fig. 4 Cross section through the kidney, Group (VG, x100) Conclusions The considerable oxidative stress revealed in the high blood pressure rats was favorably modulated by vegetable polyphenols delivery. The histological examination conducted on the kidney samples supported the vessel protection effect of the two extracts. Antioxidant suplementation with Aronia melanocarpa or Sambucus nigra extract can reduse endothelial dysfunction and has cardiovascular and renal protection effects, but additional studies are needed. Acknowledgements. The work was supported by the Romanian Ministry of Education and Research, CNCSIS, plan PN2, program IDEI, section PCE, research grant ID-2519/2008. References Abuja P.M, Murkovic M., Pfannhauser W. Antioxidant and prooxidant activities of elderberry (Sambucus nigra) extract in lowdensity lipoprotein oxidation. J. Agir.g Food Chem. 1998; 46: Bell, D.R; Gochenaur, K.: Direct vasoactive and vasoprotective properties of anthocyanin rich extracts. J. Appl. Physiol., 100, , Beutler, E; Durion, O; Kelly, B.J.: Diabetic heart and kidney exhibit increased resistance to lipid peroxidation. Biochem. Biophys. Acta, 1047, 63-69, Bitsch I., janssen M., Netzel M. Bioavailability of anthocyanidin-3-glycosides following consumption of elderberry extract and blackcurrand juice. Int. J. Clin. Pharmacol. Ther. 2004; 42: Groot, H.; Rauen, U.: Tissue injury by reactive oxygen species and the protective effects of flavonoids. Fundam. Clin. Pharmacol., 12, , Hu, J.P.; Calomme, M.; Lasure, A.; De Bruyne, T.; Pieters, L.; Vlietinck, A.: Structure activity relationship of flavonoids with superoxide scavenging activity. Biol. Trace. Elem. Res., 47, , Kehrer J.P. Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 1993;23: Kokotkiewicz A., Jaremicz Z., Luczkiewicz M., Aronia plants: a review of traditional use biological activites, and perspectives for modern medicine. Med. Food 2010;13(2): Masella, R.; Di Benedetto, R.; Vari, R.; Filesi, C.; Giovannini, C.: Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathionerelated enzymes. J. Nutr. Biochem., 16, 10, , Milbury P.E., Cao G., Prior R.L., Blumberg J. Bioavailability of elderberry anthocyanins. Mech. Aging Dev. 2002;123: Minami, M.; Yoshokawa, H.: A simplified assay method of superoxid dismutase activity for clinical use. Clinica Chimica Acta, 92, , Mulleder U., Murkovic M., Pfannhauser W. Urinary excrection of cyanidin glycosides. J. Biochem. Biophys. Methods 2002;53:

6 Sies, H.: Oxydative stress: oxidants and antioxidants. Experim. Physiolog., 82, , Ursini, F., Maiorino, M.; Brigelius-Flohe, R.; Aumann, K.D.; Roveri, A.; Schomburg, D.:The diversity of glutathione peroxidases. Methods Enzymol., 252B, 38 53, Ursini, F.; Tubaro,.F.; Rong, J.; Sevanian, A.: Optimization of nutrition: polyphenols and vascular protection. Nutr. Rev., 57, , Yang, D.; Feletou, M.; Boulanger, C.M.; Wu, H.F.; Levens, N.; Zhang, J.N.; Vanhoutte, P.M.: Oxygen derived free radicals mediate endothelium-dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats. Br. J. Pharmacol., 136, , Youdin K.A., Martin A., Joseph J.A. Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Radic. Biol. Med. 2000;29: