ASSESSING THE PROTECTIVE EFFECT OF PRIMULA HETEROCHROMA STAPF EXTRACTS AGAINST SODIUM FLUORIDE-INDUCED HEMOLYSIS IN RAT ERYTHROCYTES

Transcription

1 ASSESSING THE PROTECTIVE EFFECT OF PRIMULA HETEROCHROMA STAPF EXTRACTS AGAINST SODIUM FLUORIDE-INDUCED HEMOLYSIS IN RAT ERYTHROCYTES Heshmatollah Alinezhad, b Mahboobeh Zare, b Seyed Fazel Nabavi, a,c Alireza Naqinezhad, d Seyed Mohammad Nabavi a,c* Babolsar and Tehran, Iran SUMMARY: An investigation was conducted to determine the amount of protective effect of polyphenolic flavonoid extracts from the medicinal plant Primula heterochroma Stapf might have against hemolysis induced by sodium fluoride in rat erythrocytes. Blood samples from cardiac puncture of male rats sacrificed after anesthesia were collected for the preparation of erythrocyte suspensions in saline phosphate buffer. Hemolysis in the erythrocytes was induced by 24.7 ± 1.79% in 500 ppm NaF down to 10.2 ± 0.58% in 100 ppm NaF. All P. heterochroma extracts at 0.8 mg/ml showed moderate protective effects against NaF-induced hemolysis in the erythrocytes. Fractions extracted by ethyl acetate gave a 23.1 ± 1.16% protective effect in 100 ppm NaF, those by water 23.2 ± 0.97%, and those by n-hexane 22.5 ± 0.85%. Keywords: Hemolysis by NaF; Primula heterochroma extracts; Rat erythrocytes. INTRODUCTION It is well known that excessive levels of fluoride (F) in the diet can result in adverse health effects. 1 F intoxication in humans is manifested by dental and skeletal fluorosis, especially in regions with elevated exposure to F. 2 The ability of F anions to cross cell membranes and to enter soft tissues is also well documented. 3 Production of free radicals, lipid peroxidation, and change in antioxidant defense systems play an important role in F intoxication. 4,5 Various studies suggest that oxidative stress induced by F involves lipid peroxidation and disturbances in enzymatic antioxidant activities in vivo as well as in vitro. 6,7 Fortunately, cells have protective mechanisms to counter harmful effects of oxidative stress that include nucleotide damage and products of lipid peroxidation, or by pathways that directly diminish oxidative stress via enzymatic and nonenzymatic antioxidants. Certain natural products containing non-enzymatic antioxidants are also known to help protect against oxidative stress induced by free radicals and/or other toxic substances. In particular, polyphenolic compounds have been shown to reduce lipid peroxidation. 8,9 Primula heterochroma Stapf (Primulaceae) is one of five species of the genus Primula in Iran 10 that is indigenous to the shady habitats of Hyrcanian forests in northern Iran. This endemic plant is distributed across three northern provinces of Iran (Gilan, Mazandaran, and Golestan) as well as a record of its occurrence in the Talish part of the Republic of Azerbaijan. The Primula a,c* For correspondence: Seyed Mohammad Nabavi, Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran, PO Box ; Nabavisf@gmail.com; a National Elites Foundation of Iran, Tehran, Iran; b Department of Organic Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran; c Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran; d Department of Biology, Faculty of Sciences, University of Mazandaran, Babolsar, Iran.

2 genus has been cultivated as an ornamental plant, and used in traditional herbal medicine for many years. To the best of our knowledge, there has been no scientific report on the antihemolytic effect of this endemic medicinal plant. Accordingly, the aim of the present study was to investigate the extent of antihemolytic effects of extracts from P. heterochroma on oxidative stress induced by NaF in rat erythrocytes. MATERIALS AND METHODS Plant materials: Primula heterochroma Stapf was collected from Darabkola Forest in Mazandaran, Iran (located on the southeastern part of the town of Sari between to N and to E). The samples were identified by Dr Alireza Naqinezhad, Assistant Professor of Plant Systematics and Ecology, Department of Biology, University of Mazandaran. An identification voucher of the plant was deposited in the Herbarium of the University of Mazandaran under number Materials were transported to the laboratory and kept at <4ºC within 24 hr prior to sample preparation. The materials were oven dried at 38ºC, for 5 days. Extraction of flavonoids: After drying, the plant material was coarsely ground to a 2 3 mm mesh size before extraction. One hundred g of dried sample was defatted twice with 100 ml of chloroform to remove waxes, fatty materials, and chlorophyll, and then extracted twice with 100 ml of 60% aqueous acetone for 12 hr at room temperature. The extract was then separated from the sample residue by filtration through Whatman No.1 filter paper. The solvent in the combined filtrates was removed at room temperature with a rotary evaporator, leaving the crude acetone extract. After preparation of a 10% methanol slurry, the crude acetone extract was fractionated sequentially with 300 ml of n-hexane, ethyl acetate, and water. The n-hexane, ethyl acetate, and aqueous fractions and crude acetone extract were then used for determinations of bioactivity. 11 Preparation of erythrocytes: All the animal experiments were carried out with the approval of the institutional animal ethical committee. Male rats weighing between 180 and 220 g were housed in individual polypropylene cages and had free access to a standard rat diet and millipore deionized drinking water. After seven days of acclimatization the animals were sacrificed under anesthesia with ketamine (60 mg/kg) and xylazine (5 mg/kg), and then blood was collected by heart puncture in heparinized tubes. Erythrocytes were isolated and stored according to a recently described method. 12 Briefly, blood samples were centrifuged (1500 rpm, 10 min) at 4ºC, erythrocytes were separated from the plasma and buffy coat and were washed three times by centrifugation (1500 rpm, 5 min) in 10 volumes of 10 mm phosphate buffered saline (PBS, ph 7.4). The supernatant and buffy coats of white cells were carefully removed with each wash. Washed erythrocytes were stored at 4ºC and used within 6 hr for study. Estimation of antihemolytic activity: For the determination of antihemolytic activity, NaF at different concentrations of ppm NaF ( ppm F ion) was prepared in isotonic saline. One milliliter of 5% suspension of erythrocytes

3 sample in PBS was diluted with 1 ml of isotonic saline. For the control comparison, two sets of spectrophotometric tubes were prepared: one set containing 1.0 ml of 5% erythrocytes suspension in PBS and the other containing 1.0 ml of isotonic saline. For the NaF treatment, different tubes containing 0.1 to 0.5 ml of the NaF solution were mixed with 1.0 ml of 5% erythrocytes suspension in PBS, and the final volume of the mixtures was made to 2.0 ml with isotonic saline. The concentrations of NaF in the 2.0 ml final volume ranged from 100 to 500 µg/ml in the reaction mixtures. To determine the effect of the P. heterochroma extract fractions on NaF intoxication of the rat erythrocytes, 0.1 ml of each extract fraction (0.8 mg/ml in isotonic saline) was mixed with 0.1 to 0.5 ml of NaF solution and 1.0 ml erythrocytes suspension. The final volume of each reaction mixture was made up to 2.0 ml with isotonic saline, providing NaF concentrations between 100 (lowest concentration) and 500 ppm (highest concentration). All tubes were incubated at room temperature for 4 hr, and absorbances were read spectrophotometrically at 540 nm. 13 Statistical analysis: The percentages of hemolysis calculated from the absorbances results are presented as means ± SEM. Differences between group means were estimated using a one-way analysis of variance followed by Duncan's multiple range tests. Results were considered statistically significant when p < Results are shown in the Figure. RESULTS AND DISCUSSION Figure. Dose-dependent in vitro increase by NaF in hemolysis (%) of rat erythrocyte cells and protective effect of Primula heterochroma Stapf extract fractions. (Mean ± SEM)

4 The control cells remained at the bottom of the tubes for 4 hr with clear supernatant, suggesting no hemolysis. Addition of NaF to the erythrocytes suspension incurred a significant increase in hemolysis of the erythrocytes compared with the control tubes. The cells stayed at the bottom of the tube, but the normal tube advanced a reddish color, suggesting hemolysis. The results demonstrated a significant increase in hemolysis in a dose-dependent manner. Incubation of each plant extract fraction with NaF present showed a decrease in F- induced hemolysis compared with the NaF-alone group. As seen in the Figure, NaF in the range of 100 to 500 ppm in the erythrocytes suspensions affected membrane permeability leading to influx of water into the cells thereby inducing hemolysis. This might also be caused by an increase in lipid peroxidation and oxidative injury. Furthermore, F anions can inhibit or activate different functions in living systems. Neutrophils influenced by F anions show increased uptake of oxygen and production of superoxide anions along with diminished phagocytic capacity. 14 Also, F anions interfere with erythrocyte membrane transport processes, e.g., by impeding prevent K + -Cl co-transport. 15 Change in cation pump activity induced by F anions occurs as a direct inhibition of Na + -K + -ATPase. 16 Furthermore, F anions in humans have been shown to decrease ATP levels in erythrocytes. 17 According to the present results, addition of the Primula heterochroma Staph extract fractions to the erythrocyte suspensions with NaF present decreased hemolysis compared to NaF alone. At 400 ppm of NaF, the n-hexane fraction showed better protection against hemolysis than the other extracts (15.7 ± 0.84 % hemolysis for n-hexane vs ± 0.96 % hemolysis in NaF alone). The extracts thus demonstrate in vitro scavenging activity under physiological and pharmacological conditions. Evidently they can protect membrane lipids, protein, and nuclear deoxyribonucleic acid from free radical induced oxidative stress This decrease in hemolysis by addition of the extracts can be ascribed to stimulating enzymatic antioxidant, hydroxyl radical scavenging, and enzymatic pro-oxidant inhibiting capacity. Also, there are indications that the extracts are effective in protecting against oxidative stress. According to the results of this study, the extract fractions demonstrate moderate protective effect against NaFinduced hemolysis in erythrocytes. The protective role of extracts may also be correlated with the presence of antioxidant constituents. In summary, this work has shown that flavonoid-rich extract fractions of Primula heterochroma Staph have moderate protective effect against NaF-induced induced hemolysis in rat erythrocytes and probably oxidative stress as well. After appropriate clinical studies, these results may be useful as a starting point for further applications of this plant or its flavonoids in pharmaceutical preparations. ACKNOWLEDGEMENT This work supported by research grant from the University of Mazandaran, Babolsar, Iran.

5 REFERENCES 1 Cicek E, Aydin G, Akdogan M, Okutan H. Effects of chronic ingestion of sodium fluoride on myocardium in a second generation of rats. Hum Exp Toxicol 2005;24: Shivarajashankara YM, Shivashankara AR, Bhat PG, Rao SH. Effect of fluoride intoxication on lipid peroxidation and antioxidant systems in rats. Fluoride 2001; 34: Jacyszyn K, Marut A. Fluoride in blood and urine in humans administered fluoride and exposed to fluoride-polluted air. Fluoride 1986;19: Sharma A, Chinoy NJ. Role of free radicals in fluoride-induced toxicity in liver and kidney of mice and its reversal [abstract]. Fluoride 1998;31(3):S26. 5 Shan KR, Qi XL, Long YG, Nordberg A, Guan ZZ. Decreased nicotinic receptors in PC12 cells and rat brains influenced by fluoride toxicity a mechanism relating to a damage at the level in post-transcription of the receptor genes. Toxicology 2004;200: Shanthakumari D, Srinivasalu S, Subramanian S. Effect of fluoride intoxication on lipid peroxidation and antioxidant status in experimental rats. Toxicology 2004;204: Rzeuski R, Chlubek D, Machoy Z. Interactions between fluoride and biological free radical reactions. Fluoride 1998;31: Altuntas I, Delibas N, Sutcu R. The effects of organophosphate insecticide methidathion on lipid peroxidation and anti-oxidant enzymes in rat erythrocytes: role of vitamins E and C. Hum Exp Toxicol 2002;21: Gultekin F, Delibas N, Yasar S, Kilinc I. In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats. Arch Toxicol 2001;75: Wendelbo P. Primulaceae. In: KH Rechinger, editor. Flora Iranica vol 9. Graz, Austria: Akademische Druck- U. Verlagsanstalt; Chung SK, Chen CYO, Blumberg JB. Flavonoid-Rich fraction from Sageretia theezans leaves scavenges reactive oxygen radical species and increases the resistance of lowdensity lipoprotein to oxidation. J Med Food 2009;12: Nabavi SF, Nabavi SM, Abolhasani F, Moghaddam AH, Eslami SH. Cytoprotective effects of curcumin on Sodium Fluoride-Induced Intoxication in rat erythrocytes. Bull Environ Contam Toxicol 2011; DOI: /s Rao MV, Vyas DD, Meda RB, Chawla SL. In vitro protective role of melatonin against hemolysis induced by sodium fluoride in human red blood cells. Fluoride 2011;44: Bober J, Kucharska E, Zawierta J, Machoy Z, Chlubek D, Ciechanowski K. The influence of fluoride ions on the viability, reduction of NBT, cytolysis, degranulation, and phagocytosis of human and rabbit neutrophils. Fluoride 2000;33: O Neill WC. Swelling-activated K-Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride. Am J Physiol 1991;260:C Grabowska M, Gumińska M. Effect of sodium fluoride on magnesium-activated ATPase from human erythrocyte membranes. Folia Med Cracov 1985;26:29-33 [in Polish]. 17 Gumińska M, Kedryna T, Marchut E, Stachurska MB. ATP, glucose and lactate levels in the blood of persons exposed to fluorides before and after preventive administration of magnesium salts. Folia Med Cracov 1985;26:93-7 [in Polish]. 18 Reiter RJ, Melchiorri D, Sewerynek E, Poeggeler B, Barlow-Walden L, Chuang J, et al. A review of the evidence supporting melatonin s role as an antioxidant [review]. J Pineal Res 1995;18: Romero MP, Osuna C, García-Perganeda A, Carrillo-Vico A, Guerrero JM. The pineal secretory product melatonin reduces hydrogen peroxide-induced DNA damage in U-937 cells. J Pineal Res 1999;26: Wakatsuki A, Okatani Y, Ikenoue N, Shinohara K, Watanabe K, Fukaya T. Melatonin protects against oxidised low-density lipoprotein-induced inhibition of nitric oxide production in human umbilical artery. J Pineal Res 2001;31: Vijayalaxmi, Reiter RJ, Tan DX, Herman TS, Thomas CR. Melatonin as a radio protective agent: a review. Int J Radiat Oncol Biol Phys 2004;59(3): Copyright 2011 The International Society for Fluoride Research Inc Editorial Office: 727 Brighton Road, Ocean View, Dunedin 9035, New Zealand.