Effect of Anthocyanins Contained in a Blackberry Extract on the Circulatory Failure and Multiple Organ Dysfunction Caused by Endotoxin in the Rat

Abstract

Anthocyanins are a group of naturally occurring phenolic compounds related to the colouring of plants, flowers and fruits. These pigments are important as quality indicators, chemotaxonomic markers and for their antioxidant activities. Here we have investigated the therapeutic efficacy of anthocyanins contained in a blackberry extract on (i) circulatory failure, (ii) multiple organ dysfunction and (iii) activity of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclooxygenase (COX-2) in anaesthetised rats with endotoxic shock. In a model of endotoxic shock induced by lipopolysaccharide (LPS, E. coli, 10 mg/kg, i. v.) in the rat, pretreatment with anthocyanins present in the blackberry extract (5 mg/kg, i. v. 30 min before LPS) prevented the hypotension induced by LPS. Endotoxaemia also caused rises in the serum levels of (i) glutamyl oxaloacetic transaminase (GOT), glutamyl pyruvic transaminase (GPT), alkaline phosphates and bilirubin (hepatic dysfunction) (ii) creatinine (renal dysfunction), (iii) amylase and lipase (pancreatic injury), (iiii) NO_x and 6-keto-PGF_{1 α}. Anthocyanins attenuated the hepatic and pancreatic injury, the renal dysfunction and decreased NO_x and 6-keto-PGF_{1 α} levels. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX activity in rat lung, which was attenuated in rats pretreated with anthocyanins. Moreover, anthocyanins (0.02 - 0.32 mg/mL) inhibited in vitro iNOS and COX activity from lung of LPS-treated rats. Polymorphonuclear (PMN) infiltration (myeloperoxidase activity), lipid peroxidation (malondialdehyde levels) as well as tissue injury (histological examination) induced by LPS in rat lung and ileum was reduced by anthocyanins (5 mg/kg, i. v. 30 min before LPS). Furthermore, endotoxaemia induced the formation of nitrotyrosine and poly(ADP-ribose) synthetase (PARS) activation as determined by immunohistochemical analysis of lung and ileum tissues. The degree of staining was lowered by anthocyanin treatment. These results indicate that the anthocyanins contained in the blackberry extract exert multiple protective effects in endotoxic shock.

Abbreviations

NO_x:nitrite/nitrate

LPS:lipopolysaccharide

NO:nitric oxide

iNOS:inducible nitric oxide synthase

COX-2:cyclooxygenase-2

PMN:polymorphonuclear

PARS:poly(ADP-ribose) synthetase

PG:prostaglandin

ROS:reactive oxygen species

MODS:multiple organ dysfunction syndrome

MAP:mean arterial blood pressure

GOT:glutamyl oxaloacetic transaminase

GPT:glutamyl pyruvic transaminase

DEX:dexamethasone

MPO:myeloperoxidase

MDA:malondialdehyde

Key words

Blackberry - anthocyanins - endotoxic shock - iNOS - COX-2 - peroxynitrite - Vaccinium myrtillus - Gicaceae

References

• 1 Vane J R, Mitchell J A, Appelton I, Tomlinson A, Bishop-Bailey D, Croxtal J. et al . Inducible isoforms of cyclooxygenase and nitric oxide synthase in inflammation.  Proc Natl Acad Sci USA. 1994;  91 2046-50
• 2 Thiemermann C. The role of arginine: nitric oxide pathway in circulatory shock.  Adv Pharmacol. 1994;  28 45-79
• 3 Redl H, Gasser H, Schalg G, Marzi I. Involvement of oxygen radicals in shock related cell injury.  Br Med Bull. 1993;  49 556-65
• 4 Zingarelli B, Day B J, Crapo J D, Salzman A L, Szabo C. The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock.  Br J Pharmacol. 1997;  120 259-67
• 5 Szabo C, Zingarelli B, O’Connor M, Salzman A L. DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite.  P Proc Natl Acad Sci USA. 1996;  93 1753-8
• 6 Bagchi D, Bagchi M, Stohs S J, Das D K, Ray S D, Kuszynski C A. et al . Free radical and grape seed proanthocyanidin extract: importance in human health and disease prevention.  Toxicology. 2000;  148 187-97
• 7 Crespo M E, Galvez J, Cruz T, Ocete M A, Zarzuelo A. Anti-inflammatory activity of diosmin and hesperidin in rat colitis induced by TNBS.  Planta Medica. 1999;  65 651-3
• 8 Donovan JL; Meyer A S, Waterhose A L. Phenolic composition and antioxidant activity of prunes and prune juice (Prunus domestica).  J Agr Food Chem. 1998;  46 1247-52
• 9 Wang H, Cao G, Prior R L. Oxygen radical absorbing capacity of anthocyanins.  J Agr Food Chem. 1997;  45 304-9
• 10 Dugo P, Mondello L, Errante G, Zappia G, Dugo G. Identification of anthocyanins in berries by narrow-bore high performance liquid chromatography with electrospray ionization detection.  Journal of Agricultural and Food Chemistry. 2001;  49 3987-92
• 11 Caputi A P, Rossi F, Carney K, Brezenoff H E. Modulatory effect of brain acetylcholine on reflex-induced bradycardia and tachycardia in conscious rats.  J Pharmacol Exp Ther. 1980;  215 309-16
• 12 Leach M, Hamilton L C, Olbrich A, Wray G M, Thiemermann C. Effects of inhibitors of the activity of cyclooxygenase-2 on the hypotension and multiple organ dysfunction caused by endotoxin: a comparison with dexamethasone.  Br J Pharmacol. 1998;  124 586-92
• 13 Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Ciccolo A, Centorrino T. et al . Protective effects of N-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat.  The FASEB Journal. 2001;  15 1187-200
• 14 Rossi A, Longo R, Russo A, Borrelli F, Sautebin L. The role of the phenethyl ester of caffeic acid (CAPE) in the inhibition of rat lung cyclooxygenase activity by propolis.  Fitoterapia. 2002;  73 S30-7
• 15 Salter M, Knowles R G, Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases.  FEBS Lett. 1991;  291 145-9
• 16 Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976;  72 248-54
• 17 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.  Analytical Biochemistry. 1979;  95 351-8
• 18 Crow J P, Beckman J S. Reactions between nitric oxide, superoxide, and peroxynitrite: footprints of peroxynitrite in vivo .  Advances in Pharmacol. 1995;  34 17-43
• 19 Elliott S J. Peroxynitrite modulates receptor-activated Ca²⁺ signaling in vascular endothelial cells.  The Am J Physiol. 1996;  270 954-61
• 20 Serraino I, Dugo L, Dugo P, Mondello L, Mazzon E, Dugo G. et al . Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure.  Life Sciences. 2003;  73 1097-114