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Biol. Pharm. Bull. 25(9) 1133—1136 (2002)
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Beneficial Effects of Hippophae rhamnoides L. on Nicotine Induced Oxidative Stress in Rat Blood Compared with Vitamin E Halis SULEYMAN,a Kenan GUMUSTEKIN,b Seyithan TAYSI,c Sait KELES,c Nuray OZTASAN,b Omer AKTAS,b Konca ALTINKAYNAK,c Handan TIMUR,b Fatih AKCAY,c Sedat AKAR,b Senol DANE,b and Mustafa GUL*,b a
Department of Pharmacology, Faculty of Medicine, Ataturk University; b Department of Physiology, Faculty of Medicine, Ataturk University; and c Department of Biochemistry, Faculty of Medicine, Ataturk University; 25240, Erzurum, Turkey. Received April 1, 2002; accepted May 13, 2002 The aim of this study was to determine the effects of Hippophae rhamnoides L. extract (HRe-1) and also vitamin E as a positive control on nicotine-induced oxidative stress in rat blood, specifically alterations in erythrocyte malondialdehyde (MDA) level, activities of some erythrocyte antioxidant enzymes, and plasma vitamin E and A levels. The groups were: nicotine (0.5 mg/kg/d, intraperitoneal, i.p.); nicotine1vitamin E (75 mg/kg/d, intragastric, i.g.); nicotine1HRe-1 (1 ml/kg/d, i.g.); and control group (receiving only vehicles). There were 8 rats per group and the supplementation period was 3 weeks. Nicotine-induced increase in erythrocyte MDA level was prevented by both HRe-1 and vitamin E. Nicotine-induced decrease in erythrocyte superoxide dismutase (SOD) activity was prevented by HRe-1, but not vitamin E. HRe-1 increased the erythrocyte glutathione peroxidase (GSH-Px) activity compared with nicotine and the vitamin E groups. Catalase activity was not affected. Vitamin E supplementation increased plasma vitamin E level. Plasma vitamin A level was higher in both vitamin E and HRe-1 supplemented groups compared with nicotine and control groups. The results suggest that HRe-1 extract can be used as a dietary supplement, especially by people who smoke, in order to prevent nicotine-induced oxidative stress. Key words Hippophae rhamnoides L.; nicotine; oxidative stress; vitamin E; antioxidant enzyme; malondialdehyde
It is known that nicotine, a major toxic component of cigarette smoke, induces oxidative stress both in vitro and in vivo. Increased free radical production and cooperative lipid peroxidation levels in pancreatic tissue of rats incubated with nicotine,1) and increased lipid peroxidation in Chinese hamster ovary cells incubated with nicotine and smokeless tobacco extract2) have been reported. In addition, elevated oxidative DNA damage in various tissues of mice exposed to side-stream cigarette smoke,3) and increased lipid peroxidation levels in tissues of intraperitoneal nicotine administered rats4) have been found. Furthermore, increased lipid peroxidation in blood of smokers5) has also been reported. It seems that people who smoke and also who are exposed to cigarette smoke indirectly by breathing the air in the same environment are exposed to nicotine-induced oxidative stress. On the other hand, Beser et al. have reported that smokers consume fewer green vegetables and fruits, which are rich in antioxidants, than non-smokers of both sexes.6) Differences in dietary habits may also exacerbate the nicotine-induced oxidative stress in smokers. Oxidative stress is considered to take part in the pathogenesis of various diseases, including cancer, diabetes, and cardiovascular diseases.7—9) Thus, nicotine-induced oxidative stress may play an important role in the development of cardiovascular disease and lung cancer in smokers. Hippohae rhamnoides L., a member of the Elaeagnaceae family, is a perennial plant native to Europe and Asia10) and also widely distributed in the fields of north and east Anatolia.11) Its fruits are orange colored, sour to the taste, singleseeded and 3—7 mm in diameter12) and contain carotenes (a , b , d ) vitamins C, E, riboflavin, folic acid, tannins, sugar, glycerides of palmitic, stearic and oleic acids, polyphenols and some essential amino acids.13—15) Fruits of Hippophae ∗ To whom correspondence should be addressed.
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rhamnoides L. have been used extensively in traditional medicine in Turkey as well as China and former Soviet Republics to treat constipation, gastric ulcer, skin wounds and influenza infections.12) Beneficial affects of HRe-1 have been shown in experimental gastric ulcer models.16,17) Antioxidant activity of Hippophae rhamnoides L. has been shown in in vitro, cell culture and animal studies. Different fractions of sea buckthorn (Hippophae rhamnoides L.) fruits inhibit 2,2-azobis (2,4-dimethylvaleronitrile) and ascorbateiron induced lipid peroxidations in vitro.18) Hippophae rhamnoides, as well as vitamin E, decreases the malondialdehyde (MDA) content in hyperlipidemic rabbit serum cultured smooth muscle cells.19) Seed oil of Hippophae rhamnoides L. inhibits MDA formation of liver induced by CCl4, acetaminophen and ethyl alcohol and also prevents the acetaminophen-induced glutathione depletion in liver.20) The prevention of glutathione depletion by HRe-1 is also reported in gastric tissue in ethanol administered rats.16) However, no study has reported the effects of HRe-1 on nicotine-induced lipid peroxidation. Vitamin E, the term for a group of tocopherols and tocotrienols, is well accepted as nature’s most effective lipidsoluble, chain-breaking antioxidant, protecting cell membranes from peroxidative damage.21) Studies show that vitamin E also counteracts against nicotine-induced lipid peroxidation in animals4) and also humans.22) Hence, the aim of this study was to determine the effects of HRe-1 and vitamin E supplementation as a positive control on nicotine-induced oxidative stress in rat blood, specifically alterations in erythrocyte MDA level, activities of some erythrocyte antioxidant enzymes, and plasma vitamin E and A levels. The results of the study may have clinical implications since HRe-1 is a non-toxic plant extract that can easily © 2002 Pharmaceutical Society of Japan
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be used as a dietary supplement. MATERIALS AND METHODS Animals Thirty-two rats (Sprague-Dawley strain with a body weight of 225628 g), fed with standard laboratory chow and water, were used in the study. They were randomly divided into 4 groups (8 rats per group) and placed in separate cages during the study. The groups were as follows: Group I: nicotine (0.5 mg/kg/day, i.p.) Group II: nicotine (0.5 mg/kg/day, i.p.)1vitamin E (75 mg/kg/day, i.g.) Group III: nicotine (0.5 mg/kg/day, i.p.)1HRe-1 (1 ml/kg/ day, i.g.) Group IV: control group (received only the same amounts of vehicles, 0.9% NaCl solution, i.p., and corn oil, i.g.). Supplementation period was 3 weeks. Animal experimentations were carried out in an ethically proper way by following guidelines as set by the Ethical Committee of the Ataturk University. Preparation and Administration of Hippophae rhamnoides L. Extract The ripe fresh fruit of Hippophae rhamnoides L. were collected from the Tortum area (altitude of 1600 m), a town in Erzurum, Turkey. The plant was identified by Dr. Ali Aslan from the Department of Pharmaceutic Botany, Faculty of Pharmacy, Ataturk University, Turkey. Fruits of Hippophae rhamnoides L. were removed from the branches, washed with tap water and dried, then crushed in a mortar and mixed. Fruit mash was placed in a glass jar and hexane was added in equal volume. Forty-eight hours later, juice was obtained from the mixture by squeezing and centrifuging at 10003g for 15 min; clear supernatant was removed by a drip. Hexane was evaporated from the liquid by an evaporator (Büchi, Rotavapor, R110 , Switzerland). Hippophae rhamnoides L. extract (HRe-1) was also mixed with corn oil (1/1, v/v), and administered orally by a stomach tube to group 3 for 3 weeks at 1 ml/kg/d. Preparation and Administration of Nicotine Hydrogen tartrate salt of nicotine (Sigma N-5260) was dissolved in 0.9% NaCl solution to get a 0.15 mg/ml concentration of nicotine. Then, pH of the nicotine solution was adjusted to 7.4 by 0.1 N NaOH. Nicotine (0.5 mg/kg/d) was administered by intraperitoneal injection to groups 1, 2 and 3 for 3 weeks. Preparation and Administration of Vitamin E Vitamin E (Ephynal 300 capsule, Roche, France) was dissolved in corn oil (30 mg/ml) and administered orally by a stomach tube (approximately 75 mg/kg/d) to group 2 for 3 weeks. Determination of Erythrocyte MDA Level, and the Activities of SOD, Catalase and Glutathione Peroxidase (GSH-Px) At the end of the experiment, the animals were anesthetized with ketamine-HCl (Ketalar, 20 mg/kg, i.p.), and the blood was collected by cardiac puncture after thoracotomy. Blood samples were collected in vacutainer tubes with K3-EDTA as anticoagulant (1/10, v/v). They were centrifuged at 30003g for 15 min and plasma was removed by a Pasteur pipette. Then, erythrocytes were washed with 0.9 % NaCl solution three times, and washed erythrocytes were hemolysed by the addition of Tris–HCl buffer, pH 7.2, 0.015 M. Hemoglobin (Hb) values of the samples were measured by a GEN-S counter hematology analyser. The hemolysate and plasma of the subjects were kept in 280 °C until biochemical
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determinations. Erythrocyte MDA level was measured by the spectrophotometric method of Beuge and Aust.23) Briefly, the sample was mixed with two volumes of a stock solution of 15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid and 0.25 N hydrochloric acid. The combination of sample and stock solution was heated for 30 min in a boiling water bath. After cooling, the precipitate was removed by centrifugation and the absorbance of the supernatant was determined at 535 nm. CuZn-superoxide dismutase (SOD) activity was determined by inhibition of the reduction of nitroblue tetrazolium by superoxide anion radicals, which are produced by the xanthine–xanthine oxidase system.24) GSH-Px activity was measured by coupled spectrophotometric assay at 340 nm from the oxidation of NADPH in the presence of H2O2 used as substrate.25) Catalase activity was determined by measuring the rate of decay of H2O2 absorbance at 240 nm.26) Determination of Plasma Vitamin E and A Levels with HPLC Plasma vitamin E and A levels were determined by HP 1100 HPLC system using a Chromosystems analytical column and mobile phase and other reagents and standards provided by the same company (Chromosystems instruments and Chemicals GmbH, Munich, Germany).27) The instrument parameters were: injection volume 50 m l, analytical run time 15 to 17 min, sample was light protected, flow rate 1.5 ml/min, column temperature 20 °C, and the UV detector l was initially set at 325 nm and switched to 295 nm after 3 min. Statistical Analysis One-way analysis of variance (ANOVA) with post-hoc LSD test was used to compare the group means and p,0.05 was considered statistically significant. SPSS-for windows (version 9.0.0) was used for statistical analyses. RESULTS Nicotine increased the erythrocyte MDA level compared with the control group. This nicotine-induced increase was prevented in both vitamin E and HRe-1 supplemented nicotine-administered groups (Fig. 1). Nicotine decreased the activity of erythrocyte SOD compared with the control group (Fig. 2A). This nicotine-induced decrease in activity was prevented by HRe-1, but not vitamin E (Fig. 2A). Nicotine plus HRe-1 increased the erythrocyte GSH-Px activity compared with the nicotine and the nicotine plus vitamin E groups, however, neither nicotine
Fig. 1.
Erythrocyte Malondialdehyde (MDA) Levels
∗: nicotine vs. control (p50.004), nicotine1vitamin E (p50.035), nicotine1HRe-1 (p50.015), one-way ANOVA with post-hoc LSD test. HRe-1: Hippophae rhamnoides L. extract.
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Fig. 2.
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Erythrocyte Superoxide Dismutase (SOD, A) and Glutathione Peroxidase (GSH-Px, B) Activities
∗: control vs. nicotine (p50.04), nicotine1vitamin E (p50.011); Y : nicotine1HRe-1 vs. nicotine (p50.017), nicotine1vitamin E (p50.042), one-way ANOVA with post-hoc LSD test. HRe-1: Hippophae rhamnoides L. extract.
Fig. 3.
Plasma Vitamin E (A) and A (B) Levels
∗: nicotine1vitamin E vs. control (p50.021), nicotine (p50.041); Y : nicotine1vitamin E vs. control (p50.001), nicotine (p50.001); #: nicotine1HRe-1 vs. control (p50.001), nicotine (p50.001), one-way ANOVA with post-hoc LSD test. HRe-1: Hippophae rhamnoides L. extract.
alone nor nicotine plus vitamin E affected the erythrocyte GSH-Px activity (Fig. 2B). Of the erythrocyte antioxidant enzymes studied, catalase activity was not affected by any of the treatments (data not shown). Vitamin E supplementation increased plasma vitamin E level compared with control and nicotine-administered groups (Fig. 3A). Plasma vitamin A levels in both vitamin E and HRe-1 supplemented groups were higher than control and nicotine administered groups (Fig. 3B). DISCUSSION To our knowledge, prevention of nicotine-induced oxidative stress by HRe-1 in erythrocytes of nicotine-administered rats is reported here for the first time. Our result, increased oxidative stress determined as a higher erythrocyte MDA level in nicotine-administered rats, agrees with studies showing increased MDA levels in blood and serum of smokers,5) elevated 8-hydroxy-29-deoxyguanosine levels showing oxidative DNA damage in heart, lung, and liver tissues of mice exposed to side-stream cigarette smoke,3) increased TBARS, conjugated diene and hydroperoxide levels in heart, liver and lung tissues of intraperitoneally nicotine-administered rats.4) There are also in vitro studies demonstrating higher chemiluminescence and lipid peroxidation levels in the homogenate of pancreatic tissue of rat incubated with nicotine,1) and increased MDA levels in Chinese hamster ovary cells incubated with nicotine and smokeless tobacco extract.2) Nicotine-induced increase in erythrocyte MDA level was
prevented by vitamin E in our study. This finding is in line with the studies reporting decreased susceptibility of erythrocytes to hydrogen peroxide-induced lipid peroxidation in vitro in both smokers and non-smokers,22) and prevention of the increase in lipid peroxidation in liver, lungs and heart tissues of intraperitoneally nicotine-injected rats4) by vitamin E supplementation. In our study, nicotine decreased erythrocyte SOD activity but did not alter erythrocyte catalase or GSH-Px activities. Proposed mechanisms by which nicotine produces oxidative stress include disruption of the mitochondrial respiratory chain leading to leakage from the electron transport in cardiomyocytes of rabbits,28) decrease in glutathione level in Chinese hamster ovary cells,2) and decreased activities of catalase and SOD in various tissues of rat.4) It is also shown that addition of free radical scavenging enzymes, SOD and catalase, prevent nicotine-induced increase in lipid peroxidation in pancreatic tissue of rat1) and decrease in cellular glutathione level in Chinese hamster ovary cells.2) The results of these studies suggest that superoxide anions and hydrogen peroxide are the main source of nicotine-induced free radical production depleting the cellular glutathione level. In agreement with the decreased SOD activity found in various rat tissues,4) decreased erythrocyte SOD activity would have taken part in the nicotine-induced oxidative stress in the present study. In contrast to the alterations in activity levels of antioxidant enzymes due to nicotine and vitamin E supplementation in our study, increased erythrocyte cytosolic antioxidant enzyme activities in smokers compared with non-smokers, and
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further increase in erythrocyte catalase activity but a decrease in erythrocyte SOD activity due to a 10 week vitamin E supplementation in smokers have also been reported22). HRe-1 supplementation prevented the nicotine-induced decrease in erythrocyte SOD activity and also remarkably increased erythrocyte GSH-Px activity in nicotine-treated rats, which would have contributed to the prevention of nicotineinduced increase in erythrocyte MDA level. It seems that alterations of antioxidant enzyme activities in response to nicotine-induced lipid peroxidation and also antioxidant supplements such as vitamin E and Hippophea rhamnoides L. are complicated. In our study, nicotine-induced increase in erythrocyte MDA level was prevented by vitamin E as well as HRe-1 supplementation. Erythrocyte GSH-Px activity was remarkably increased by HRe-1 compared to the nicotine and the nicotine plus vitamin E groups. Nicotine-induced decrease in erythrocyte SOD activity was prevented by HRe-1, but not by vitamin E. Remarkable increase in erythrocyte GSH-Px activity, and the increase in plasma vitamin A level in HRe1-supplemented group may have played a role in the prevention of nicotine-induced oxidative stress in our study. GSHPx takes part in detoxification of hydrogen peroxide by converting it to water and molecular oxygen using glutathione as substrate.7) Increased plasma vitamin E and A levels may be responsible for the prevention of nicotine-induced lipid peroxidation in vitamin E-supplemented group. Vitamin E supplementation can explain the increase in plasma vitamin E level. Parallel to our finding, a higher level of vitamin A in liver tissue of vitamin E-supplemented rats has also been reported by Helen et al.4) It is possible that vitamin E supplementation might have increased the absorption of vitamin A from the intestine. The increase in plasma vitamin A level in the HRe1-supplemented group may be explained by carotenes present in the HRe-1 extract.13—15) In conclusion, HRe-1, as well as vitamin E, prevented the increase in oxidative stress in erythrocytes of nicotine-administered rats. Remarkable increase in erythrocyte GSH-Px activity, and the increase in plasma vitamin A level in HRe-1 supplemented group might have contributed to the prevention of nicotine-induced oxidative stress. The results of this study suggest that HRe-1 extract can be used as a dietary supplement, especially by people who smoke, in order to prevent nicotine-induced oxidative stress. Acknowledgements This study was partly supported by
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The Research Foundation of Ataturk University, Erzurum, Turkey (project no: 2001/53). The authors thank Delali Camgoz, who has taken care of the animals. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15)
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