J Nat Med(2013)67:152-l58 D0110.1007/s11418-012-0666-7 ORIGINAL PAPER Rose oil (from Rosa x damascena Mill.)vapor attenuates depression-induced oxidative toxicity in rat brain Mustafa Naziroglu·Suileyman Kozlu· Emre Yorgancigil·Abduilhadi Cihangir Uguz· Kadir Karakus Received:31 January 2012/Accepted:14 March 2012/Published online:8 April 2012 The Japanese Society of Pharmacognosy and Springer 2012 Abstract Oxidative stress is a critical route of damage in oil vapor exposure.The vitamin A.vitamin E,vitamin C various physiological stress-induced disorders,including and B-carotene concentrations in the cerebral cortex were depression.Rose oil may be a useful treatment for lower in the CMS group than in the control group whereas depression because it contains flavonoids which include their concentrations were higher in the rose oil vapor plus free radical antioxidant compounds such as rutin and CMS group.The CMS-induced antioxidant vitamin chan- quercetin.We investigated the effects of absolute rose oil ges were not modulated by oral treatment.Glutathione (from Rosa x damascena Mill.)and experimental depres- peroxidase activity and reduced glutathione did not change sion on lipid peroxidation and antioxidant levels in the statistically in the four groups following CMS or either cerebral cortex of rats.Thirty-two male rats were randomly treatment.In conclusion,experimental depression is asso- divided into four groups.The first group was used as ciated with elevated oxidative stress while treatment with control,while depression was induced in the second group rose oil vapor induced protective effects on oxidative stress using chronic mild stress (CMS).Oral (1.5 ml/kg)and in depression. vapor(0.15 ml/kg)rose oil were given for 28 days to CMS depression-induced rats,constituting the third and fourth Keywords Rose oil·Depression·Glutathione groups,respectively.The sucrose preference test was used peroxidase.Oxidative stress.Antioxidant vitamins weekly to identify depression-like phenotypes during the experiment.At the end of the experiment,cerebral cortex Abbreviations samples were taken from all groups.The lipid peroxidation CMS Chronic mild stress levels in the cerebral cortex in the CMS group were higher GSH-Px Glutathione peroxidase than in control whereas their levels were decreased by rose GSH Glutathione MAO Monoamine oxidase PUFA Polyunsaturated fatty acid M.Naziroglu(☒，A.C.Uguz ROS Reactive oxygen species Department of Biophysics,Medical Faculty, SSRI Selective-serotonin reuptake inhibitor Suleyman Demirel University,32260 Isparta,Turkey e-mail:mnaziroglu@med.sdu.edu.tr M.Naziroglu Neuroscience Research Center, Introduction Suleyman Demirel University,Isparta,Turkey S.Kozlu.E.Yorgancigil Reactive oxygen species(ROS)including superoxide anion, Medical Faculty,Suleyman Demirel University, hydrogen peroxide and singlet oxygen act as subcellular Isparta,Turkey messengers in pathophysiological complex processes such K.Karakus as mitogenic signal transduction,gene expression,and reg- Department of Psychiatry,Medical Faculty, ulation of cell proliferation when they are generated in Suleyman Demirel University,Isparta,Turkey excess or when enzymatic and non-enzymatic defense Springer
ORIGINAL PAPER Rose oil (from Rosa 3 damascena Mill.) vapor attenuates depression-induced oxidative toxicity in rat brain Mustafa Nazırog˘lu • Su¨leyman Kozlu • Emre Yorgancıgil • Abdu¨lhadi Cihangir Ug˘uz • Kadir Karakus¸ Received: 31 January 2012 / Accepted: 14 March 2012 / Published online: 8 April 2012 The Japanese Society of Pharmacognosy and Springer 2012 Abstract Oxidative stress is a critical route of damage in various physiological stress-induced disorders, including depression. Rose oil may be a useful treatment for depression because it contains flavonoids which include free radical antioxidant compounds such as rutin and quercetin. We investigated the effects of absolute rose oil (from Rosa 9 damascena Mill.) and experimental depression on lipid peroxidation and antioxidant levels in the cerebral cortex of rats. Thirty-two male rats were randomly divided into four groups. The first group was used as control, while depression was induced in the second group using chronic mild stress (CMS). Oral (1.5 ml/kg) and vapor (0.15 ml/kg) rose oil were given for 28 days to CMS depression-induced rats, constituting the third and fourth groups, respectively. The sucrose preference test was used weekly to identify depression-like phenotypes during the experiment. At the end of the experiment, cerebral cortex samples were taken from all groups. The lipid peroxidation levels in the cerebral cortex in the CMS group were higher than in control whereas their levels were decreased by rose oil vapor exposure. The vitamin A, vitamin E, vitamin C and b-carotene concentrations in the cerebral cortex were lower in the CMS group than in the control group whereas their concentrations were higher in the rose oil vapor plus CMS group. The CMS-induced antioxidant vitamin changes were not modulated by oral treatment. Glutathione peroxidase activity and reduced glutathione did not change statistically in the four groups following CMS or either treatment. In conclusion, experimental depression is associated with elevated oxidative stress while treatment with rose oil vapor induced protective effects on oxidative stress in depression. Keywords Rose oil Depression Glutathione peroxidase Oxidative stress Antioxidant vitamins Abbreviations CMS Chronic mild stress GSH-Px Glutathione peroxidase GSH Glutathione MAO Monoamine oxidase PUFA Polyunsaturated fatty acid ROS Reactive oxygen species SSRI Selective-serotonin reuptake inhibitor Introduction Reactive oxygen species (ROS) including superoxide anion, hydrogen peroxide and singlet oxygen act as subcellular messengers in pathophysiological complex processes such as mitogenic signal transduction, gene expression, and regulation of cell proliferation when they are generated in excess or when enzymatic and non-enzymatic defense M. Nazırog˘lu (&) A. C. Ug˘uz Department of Biophysics, Medical Faculty, Suleyman Demirel University, 32260 Isparta, Turkey e-mail: mnaziroglu@med.sdu.edu.tr M. Nazırog˘lu Neuroscience Research Center, Suleyman Demirel University, Isparta, Turkey S. Kozlu E. Yorgancıgil Medical Faculty, Suleyman Demirel University, Isparta, Turkey K. Karakus¸ Department of Psychiatry, Medical Faculty, Suleyman Demirel University, Isparta, Turkey 123 J Nat Med (2013) 67:152–158 DOI 10.1007/s11418-012-0666-7
J Nat Med(2013)67:l52-l58 153 systems are impaired [1,2].Oxidative stress is a disparity vitamins A,C and E,and B-carotene values in experimental between the rates of ROS production and elimination depression in rats. through endogenous enzymatic mechanisms such as gluta- thione peroxidase(GSH-Px)and catalase as well as the low- molecular-weight reductants glutathione (GSH),vitamin A Materials and methods vitamin C.vitamin E and B-carotene.There are numerous studies indicating that ROS-induced neuronal damage has an Animals important role in the pathophysiology of depression,prob- ably via membrane omega-3 polyunsaturated fatty acids Thirty-two male Wistar albino rats weighing 200-250 g pathology [1],decreased activity of GSH-Px [3],and anti- (10-12 weeks)were used for the experimental procedures. oxidant vitamins [4,5],suggesting oxidative damage.GSH- Rats were allowed 1 week to acclimatize to their sur- Px,one of the major intracellular antioxidant enzymes, roundings before beginning any experimentation.Animals detoxifies hydrogen peroxide (H2O2)to water and also were housed in individual plastic cages with bedding. scavenges other peroxides.Although most of the oxygen Standard rat food and tap water were available ad libitum used in brain tissue is converted to CO2 and water,small for the duration of the experiments.Sucrose (1%)was amounts of oxygen form ROS.The existence of polyunsat- available ad libitum for I week preceding the experimental urated fatty acids(PUFAs)which are targets of the ROS in procedures to allow for adaptation to its taste.The tem- the brain make this organ more sensitive to oxidative damage perature was maintained at 22+2 C.A 12/12-h light/ [1].In depression-induced stress disorders,ROS induces dark cycle was maintained,with lights on at 06:00,unless several mechanisms of oxidative damage such as mito- otherwise noted.Study protocol (Protocol Number: chondrial dysfunction,dysregulation of calcium homeosta- 2010:07-08)was approved by the local ethical committee sis [6,7],disruption of energy pathways,damage to neuronal of Suleyman Demirel University.Animals were maintained precursor impairment of neurogenesis [8]and induction of and used in accordance with the Animal Welfare Act and signal events in apoptotic cell death [6].These ROS pro- the Guide for the Care and Use of Laboratory Animals duction events make a significant contribution towards the prepared by the Suleyman Demirel University. resultant disease pathophysiology,as evidenced by atrophy/ morphological changes in the brain characteristic of stress- Experimental design induced depression [9].Hence,psychological stress,which accompanies severe depression,may increase lipid peroxi- Animals were divided equally into four groups.Group I was dation [7,10]. control animals and they received placebo.GroupsⅡ，IⅢand Rose oil is produced from Damask rose (Rosa x da- IV rats had depression induced by chronic mild stress(CMS). mascena Mill.),which is grown in Middle East countries, Rose oil (1.5 ml/kg)was dissolved in ethyl alcohol and made especially Iran and Turkey.Rose oil is widely used in up to final volume (0.2 ml)with physiological saline (0.9 % perfumery and the cosmetic industry [11].One increasingly w/v),and was orally administered via gastric gavage to ani- popular type of alternative therapy is aromatherapy,but mals in the third group each day for 28 days [14].The fourth scientific validation in this field is still rare.Rose oil con- group was exposed to rose oil (0.15 ml/kg)vapor within an tains flavonoids such as geraniol and citronellol [11],which isolated cage for 15 min each day for 28 days [15].Control demonstrated a ROS scavenging activity in a model of animals were exposed to vapor cage stress and received autotoxication of rat cerebral membranes [12.In recent physiological saline gastric gavage,respectively. years,the antidepressant effects of rose oil have also been demonstrated although the molecular mechanisms of these Analysis of content of rose oil effects have not yet been clarified [13].Rose oil,with a potential antioxidant activity,may therefore be of value in The pure rose oil was bought from Gulbirlik Inc.(Isparta, psychiatric disorders including depression in which free Turkey)and its chemical content was analyzed by a gas radical generation is implicated,and this subject needs to chromatograph(GC)-mass spectrophotometer (MS)sys- be investigated. tem (Shimadzu,GC-MS QP 5050,Kyoto,Japan)[11]. Rose oil,as an antidepressant medication with a potential antioxidant activity,was therefore hypothesized Induction of depression to be a better alternative to other antidepressant flower oils like jasmine,chamomile and bergamot oils for patients Chronic mild stress (CMS) with depression exhibiting elevated oxidative stress levels. We therefore aimed at investigating the effects of rose oil The procedures for inducing CMS are summarized in on cerebral cortex lipid peroxidation,and GSH,GSH-Px, Table 1.The CMS procedure employed here has been ≌Springer
systems are impaired [1, 2]. Oxidative stress is a disparity between the rates of ROS production and elimination through endogenous enzymatic mechanisms such as glutathione peroxidase (GSH-Px) and catalase as well as the lowmolecular-weight reductants glutathione (GSH), vitamin A, vitamin C, vitamin E and b-carotene. There are numerous studies indicating that ROS-induced neuronal damage has an important role in the pathophysiology of depression, probably via membrane omega–3 polyunsaturated fatty acids pathology [1], decreased activity of GSH-Px [3], and antioxidant vitamins [4, 5], suggesting oxidative damage. GSHPx, one of the major intracellular antioxidant enzymes, detoxifies hydrogen peroxide (H2O2) to water and also scavenges other peroxides. Although most of the oxygen used in brain tissue is converted to CO2 and water, small amounts of oxygen form ROS. The existence of polyunsaturated fatty acids (PUFAs) which are targets of the ROS in the brain make this organ more sensitive to oxidative damage [1]. In depression-induced stress disorders, ROS induces several mechanisms of oxidative damage such as mitochondrial dysfunction, dysregulation of calcium homeostasis [6, 7], disruption of energy pathways, damage to neuronal precursor impairment of neurogenesis [8] and induction of signal events in apoptotic cell death [6]. These ROS production events make a significant contribution towards the resultant disease pathophysiology, as evidenced by atrophy/ morphological changes in the brain characteristic of stressinduced depression [9]. Hence, psychological stress, which accompanies severe depression, may increase lipid peroxidation [7, 10]. Rose oil is produced from Damask rose (Rosa 9 damascena Mill.), which is grown in Middle East countries, especially Iran and Turkey. Rose oil is widely used in perfumery and the cosmetic industry [11]. One increasingly popular type of alternative therapy is aromatherapy, but scientific validation in this field is still rare. Rose oil contains flavonoids such as geraniol and citronellol [11], which demonstrated a ROS scavenging activity in a model of autotoxication of rat cerebral membranes [12]. In recent years, the antidepressant effects of rose oil have also been demonstrated although the molecular mechanisms of these effects have not yet been clarified [13]. Rose oil, with a potential antioxidant activity, may therefore be of value in psychiatric disorders including depression in which free radical generation is implicated, and this subject needs to be investigated. Rose oil, as an antidepressant medication with a potential antioxidant activity, was therefore hypothesized to be a better alternative to other antidepressant flower oils like jasmine, chamomile and bergamot oils for patients with depression exhibiting elevated oxidative stress levels. We therefore aimed at investigating the effects of rose oil on cerebral cortex lipid peroxidation, and GSH, GSH-Px, vitamins A, C and E, and b-carotene values in experimental depression in rats. Materials and methods Animals Thirty-two male Wistar albino rats weighing 200–250 g (10–12 weeks) were used for the experimental procedures. Rats were allowed 1 week to acclimatize to their surroundings before beginning any experimentation. Animals were housed in individual plastic cages with bedding. Standard rat food and tap water were available ad libitum for the duration of the experiments. Sucrose (1 %) was available ad libitum for 1 week preceding the experimental procedures to allow for adaptation to its taste. The temperature was maintained at 22 ± 2 C. A 12/12-h light/ dark cycle was maintained, with lights on at 06:00, unless otherwise noted. Study protocol (Protocol Number: 2010:07–08) was approved by the local ethical committee of Suleyman Demirel University. Animals were maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Suleyman Demirel University. Experimental design Animals were divided equally into four groups. Group I was control animals and they received placebo. Groups II, III and IV rats had depression induced by chronic mild stress (CMS). Rose oil (1.5 ml/kg) was dissolved in ethyl alcohol and made up to final volume (0.2 ml) with physiological saline (0.9 %, w/v), and was orally administered via gastric gavage to animals in the third group each day for 28 days [14]. The fourth group was exposed to rose oil (0.15 ml/kg) vapor within an isolated cage for 15 min each day for 28 days [15]. Control animals were exposed to vapor cage stress and received physiological saline gastric gavage, respectively. Analysis of content of rose oil The pure rose oil was bought from Gulbirlik Inc. (Isparta, Turkey) and its chemical content was analyzed by a gas chromatograph (GC)–mass spectrophotometer (MS) system (Shimadzu, GC-MS QP 5050, Kyoto, Japan) [11]. Induction of depression Chronic mild stress (CMS) The procedures for inducing CMS are summarized in Table 1. The CMS procedure employed here has been J Nat Med (2013) 67:152–158 153 123
154 J Nat Med(2013)67:l52-158 Table 1 Procedures for inducing chronic mild stress in rats Sunday Monday Tuesday Wednesday Thursday Friday Saturday Taking water bottles(h) 16:00→ 08:00 Adding empty water bottles (h) 08:00-09:00 Continuous illumination (h) 16:00→ 08:00 17:00→10：00 40°cage tilt 11:00-17:00 Paired housing →→→ 08:00 18:00→ 14:00 10:00 Damp bedding (300 ml) 17:00→ 10:00h) White noise(90 dB) 10:00-13:00h) Stroboscopic illumination 11:00-16:00h) 13:00-15:00 (300 flashes/min) described previously [4,5]and was designed to maximize dissected out of the head of the animals and split in the the unpredictable nature of the stressors.The CMS group mid-sagittal plane.The cerebral cortex of the brain was was exposed to the following stressors in random order: then dissected from the whole brain. continuous overnight illumination,40 cage tilt,paired The cortex of the brain was washed twice with cold housing,damp bedding (300 ml water spilled into bed- saline solution,placed into glass bottles,labeled and stored ding),exposure to an empty water bottle immediately in a deep freeze (-33 C)until processing (maximum following a period of acute water deprivation,stroboscopic 10 h).After weighing,the cortex was placed on ice,cut illumination (300 fashes/min),and white noise (approx. into small pieces using scissors,and homogenized(2 min 90 dB).The stressors were presented in the order shown at 5000 rpm)in five volumes (1:5,w/v)of ice-cold Tris- during the first week and repeated during each of the fol- HCI buffer (50 mM,pH 7.4)using a glass ultrasonic lowing weeks for a total of 4 weeks.Control animals were homogenizer.All preparation procedures were performed left undisturbed in the home cages with the exception of at 4 C general handling (i.e.,regular cage cleaning and measuring body weight),which was comparable to the activities of the Lipid peroxidation level determination CMS group. Malondialdehyde is a secondary product of lipid peroxi- Sucrose preference tests dation and is used as an index of lipid peroxidation.Lipid peroxidation levels as malondialdehyde in the brain cortex Sucrose preference tests as employed previously [16]were homogenate were measured by the method of Placer et al. used to operationally define anhedonia.Specifically, [17]as described in previous studies [4,5].The samples anhedonia was defined as a reduction in sucrose intake and were incubated at 100C for 30 min in acid medium sucrose preference relative to the intake and preference of containing 0.45 sodium dodecyl sulfate and 0.67% the control group.The sucrose preference test consisted of thiobarbituric acid.Quantification of thiobarbituric acid first removing the food and water from each rat's cage reactive substances was done by comparing the absorption (both CMS and control groups)for a period of 20 h.Water to the standard curve of malondialdehyde equivalents and 1 sucrose were then placed on the cages in pre- generated by acid catalyzed hydrolysis of 1,1,3,3-tetra- weighed glass bottles,and animals were allowed to con- methoxypropane.The values of lipid peroxidation in the sume the fluids freely for a period of I h.Two baseline cerebral cortex were expressed as umol/g protein preference tests were performed,separated by at least 5 days,and the results were averaged.A preference test Reduced glutathione (GSH),glutathione peroxidase was also conducted following the 4-week CMS period. (GSH-Px)and protein assay Anesthesia and tissue and blood sampling Cerebral cortex GSH levels were measured as nonprotein thiols based on the protocol developed by Sedlak and Animals were rested and fasted for 12 h after the last Lindsay [18].Cerebral cortex homogenates were precipi- supplementation before killing.Rats were anesthetized tated in cooled trichloroacetic acid 10%and centrifuged at with a cocktail of ketamine hydrochloride (50 mg/kg; 15,000g for 2 min,and the supernatant was incubated with Ketalar,Eczacibasi Inc.,Istanbul,Turkey)and xylazine DTNB in a 1 M phosphate buffer,pH 7.0.Absorbances (5 mg/kg;Rompun,Eczacibasi)administered intraperito- were measured at 412 nm.A standard curve of reduced neally (i.p.)before killing,and the whole brain was glutathione was used to calculate GSH levels. Springer
described previously [4, 5] and was designed to maximize the unpredictable nature of the stressors. The CMS group was exposed to the following stressors in random order: continuous overnight illumination, 408 cage tilt, paired housing, damp bedding (300 ml water spilled into bedding), exposure to an empty water bottle immediately following a period of acute water deprivation, stroboscopic illumination (300 flashes/min), and white noise (approx. 90 dB). The stressors were presented in the order shown during the first week and repeated during each of the following weeks for a total of 4 weeks. Control animals were left undisturbed in the home cages with the exception of general handling (i.e., regular cage cleaning and measuring body weight), which was comparable to the activities of the CMS group. Sucrose preference tests Sucrose preference tests as employed previously [16] were used to operationally define anhedonia. Specifically, anhedonia was defined as a reduction in sucrose intake and sucrose preference relative to the intake and preference of the control group. The sucrose preference test consisted of first removing the food and water from each rat’s cage (both CMS and control groups) for a period of 20 h. Water and 1 % sucrose were then placed on the cages in preweighed glass bottles, and animals were allowed to consume the fluids freely for a period of 1 h. Two baseline preference tests were performed, separated by at least 5 days, and the results were averaged. A preference test was also conducted following the 4-week CMS period. Anesthesia and tissue and blood sampling Animals were rested and fasted for 12 h after the last supplementation before killing. Rats were anesthetized with a cocktail of ketamine hydrochloride (50 mg/kg; Ketalar, Eczacibasi Inc., Istanbul, Turkey) and xylazine (5 mg/kg; Rompun, Eczacibasi) administered intraperitoneally (i.p.) before killing, and the whole brain was dissected out of the head of the animals and split in the mid-sagittal plane. The cerebral cortex of the brain was then dissected from the whole brain. The cortex of the brain was washed twice with cold saline solution, placed into glass bottles, labeled and stored in a deep freeze (-33 C) until processing (maximum 10 h). After weighing, the cortex was placed on ice, cut into small pieces using scissors, and homogenized (2 min at 5000 rpm) in five volumes (1:5, w/v) of ice-cold Tris– HCl buffer (50 mM, pH 7.4) using a glass ultrasonic homogenizer. All preparation procedures were performed at 4 C. Lipid peroxidation level determination Malondialdehyde is a secondary product of lipid peroxidation and is used as an index of lipid peroxidation. Lipid peroxidation levels as malondialdehyde in the brain cortex homogenate were measured by the method of Placer et al. [17] as described in previous studies [4, 5]. The samples were incubated at 100 C for 30 min in acid medium containing 0.45 % sodium dodecyl sulfate and 0.67 % thiobarbituric acid. Quantification of thiobarbituric acid reactive substances was done by comparing the absorption to the standard curve of malondialdehyde equivalents generated by acid catalyzed hydrolysis of 1,1,3,3-tetramethoxypropane. The values of lipid peroxidation in the cerebral cortex were expressed as lmol/g protein. Reduced glutathione (GSH), glutathione peroxidase (GSH-Px) and protein assay Cerebral cortex GSH levels were measured as nonprotein thiols based on the protocol developed by Sedlak and Lindsay [18]. Cerebral cortex homogenates were precipitated in cooled trichloroacetic acid 10 % and centrifuged at 15,000g for 2 min, and the supernatant was incubated with DTNB in a 1 M phosphate buffer, pH 7.0. Absorbances were measured at 412 nm. A standard curve of reduced glutathione was used to calculate GSH levels. Table 1 Procedures for inducing chronic mild stress in rats Sunday Monday Tuesday Wednesday Thursday Friday Saturday Taking water bottles (h) 16:00 ? 08:00 Adding empty water bottles (h) 08:00–09:00 Continuous illumination (h) 16:00 ? 08:00 17:00 ? 10:00 408 cage tilt 11:00–17:00 Paired housing ??? 08:00 18:00 ? 14:00 10:00 ??? Damp bedding (300 ml) 17:00 ? 10:00 (h) White noise (90 dB) 10:00–13:00 (h) Stroboscopic illumination (300 flashes/min) 11:00–16:00 (h) 13:00–15:00 154 J Nat Med (2013) 67:152–158 123
J Nat Med(2013)67:l52-l58 155 GSH-Px activities of cerebral cortex were measured p-Values of less than 0.05 were regarded as significant. spectrophotometrically(UV-1800,Shimadzu,Kyoto,Japan) Significant values were assessed with the Mann-Whitney at 37 C and 412 nm according to the method of Lawrence U test.Data were analyzed using the SPSS statistical pro- and Burk [19].GSH-Px uses GSH to reduce tert-butyl gram (version 9.05,SPSS Inc.Chicago,IL,USA). hydroperoxide,producing oxidized glutathione,which is readily reduced to GSH by GSH reductase using NADPH as a reducing equivalent donor.The protein content in the Results cerebral cortex was measured by the method of Lowry et al. 20]with bovine serum albumin as the standard. Among the 15 chemical constituents identified by GC-MS analysis of rose essential oil,citronellol was found to be the Determination of vitamins A.E and C major compound (33.74%),followed by geraniol and B-carotene concentrations (24.85 %)nerol (10.77 %and nonadecene (9.30 %) Trace amounts of other chemical compounds were also Vitamins A(retinol)and E(a-tocopherol)were determined identified.These results are in agreement with previous in the cerebral cortex samples by a modification of the studies [11](Table 2). method described by Desai [21]and Suzuki and Katoh [22].Cerebral cortex samples (250 mg)were saponified by Table 2 Major components of rose oil the addition of 0.3 ml 60 %(w/v in water)KOH,followed by heating at 70 C for 30 min.After cooling the samples Compounds 名 on ice,2 ml of water and 1 ml of n-hexane were added and Ethanol 0.65 mixed with the samples and then rested for 10 min to allow a-Pinene 0.81 phase separation.An aliquot of 0.5 ml of n-hexane extract B-Pinene 0.21 was taken and vitamin A concentrations were measured at Myrcene 0.47 325 nm.Reactants were then added and the absorbance PEA 1.12 value of hexane was measured in a spectrophotometer at Linalool 0.85 535 nm.Calibration was performed using standard solu- Terpinen-4-ol 0.39 tions of all-trans-retinol and a-tocopherol in hexane. Nerol 10.77 The concentrations of B-carotene in the brain samples Citronellol 33.74 were determined according to the method of Suzuki and Geraniol 24.85 Katoh [22].Two ml of hexane were mixed with 250 mg Eugenol 0.89 cerebral cortex sample.The concentration of B-carotene in Citronellyl acetate 0.82 hexane was measured at 453 nm in a spectrophotometer. Geranyl acetate 3.18 Quantification of ascorbic acid in the brain samples was Methyl eugenol 2.04 performed using the method of Jagota and Dani [231.The Germacrane-D absorbance of the samples was measured spectrophoto- 0.39 metrically at 760 nm. Farnesol 0.56 Heptadecane 1.25 Statistical analyses 9-Nonadecene 2.18 Nonadecene 9.30 All results are expressed as means+SD.To determine the Eicosane 0.62 effect of treatment,data were analyzed using the LSD test. Heneicosane 3.63 Table 3 Sucrose (1 %w/v)test results in the control,chronic mild stress (CMS)depression,depression plus oral and vapor rose oil supple- mented groups(n 8,mean SD) Weeks Control (ml/kg) CMS (ml/kg) CMS oral rose CMS vapor rose oil (ml/kg) oil (ml/kg) Basal 25.18±4.05 28.97±8.78 28.65±10.26 25.66±10.11 Ist 25.31±6.47 25.30±6.47 25.00±10.20 21.70士10.70 2nd 24.56±8.07 29.60±5.16 27.80±6.57 30.70±13.50 3rd 23.93±9.45 19.56±7.53 26.56±8.53 33.00±9.94 4th 21.43±9.69 18.94±8.37 22.80±9.76 35.40±8.63 ap<.05 versus basal values ≌Springer
GSH-Px activities of cerebral cortex were measured spectrophotometrically (UV-1800, Shimadzu, Kyoto, Japan) at 37 C and 412 nm according to the method of Lawrence and Burk [19]. GSH-Px uses GSH to reduce tert-butyl hydroperoxide, producing oxidized glutathione, which is readily reduced to GSH by GSH reductase using NADPH as a reducing equivalent donor. The protein content in the cerebral cortex was measured by the method of Lowry et al. [20] with bovine serum albumin as the standard. Determination of vitamins A, E and C and b-carotene concentrations Vitamins A (retinol) and E (a-tocopherol) were determined in the cerebral cortex samples by a modification of the method described by Desai [21] and Suzuki and Katoh [22]. Cerebral cortex samples (250 mg) were saponified by the addition of 0.3 ml 60 % (w/v in water) KOH, followed by heating at 70 C for 30 min. After cooling the samples on ice, 2 ml of water and 1 ml of n-hexane were added and mixed with the samples and then rested for 10 min to allow phase separation. An aliquot of 0.5 ml of n-hexane extract was taken and vitamin A concentrations were measured at 325 nm. Reactants were then added and the absorbance value of hexane was measured in a spectrophotometer at 535 nm. Calibration was performed using standard solutions of all-trans-retinol and a-tocopherol in hexane. The concentrations of b-carotene in the brain samples were determined according to the method of Suzuki and Katoh [22]. Two ml of hexane were mixed with 250 mg cerebral cortex sample. The concentration of b-carotene in hexane was measured at 453 nm in a spectrophotometer. Quantification of ascorbic acid in the brain samples was performed using the method of Jagota and Dani [23]. The absorbance of the samples was measured spectrophotometrically at 760 nm. Statistical analyses All results are expressed as means ± SD. To determine the effect of treatment, data were analyzed using the LSD test. p-Values of less than 0.05 were regarded as significant. Significant values were assessed with the Mann–Whitney U test. Data were analyzed using the SPSS statistical program (version 9.05, SPSS Inc. Chicago, IL, USA). Results Among the 15 chemical constituents identified by GC-MS analysis of rose essential oil, citronellol was found to be the major compound (33.74 %), followed by geraniol (24.85 %), nerol (10.77 %) and nonadecene (9.30 %). Trace amounts of other chemical compounds were also identified. These results are in agreement with previous studies [11] (Table 2). Table 2 Major components of rose oil Compounds % Ethanol 0.65 a-Pinene 0.81 b-Pinene 0.21 Myrcene 0.47 PEA 1.12 Linalool 0.85 Terpinen-4-ol 0.39 Nerol 10.77 Citronellol 33.74 Geraniol 24.85 Eugenol 0.89 Citronellyl acetate 0.82 Geranyl acetate 3.18 Methyl eugenol 2.04 Germacrane-D 0.39 Farnesol 0.56 Heptadecane 1.25 9-Nonadecene 2.18 Nonadecene 9.30 Eicosane 0.62 Heneicosane 3.63 Table 3 Sucrose (1 %, w/v) test results in the control, chronic mild stress (CMS) depression, depression plus oral and vapor rose oil supplemented groups (n = 8, mean ± SD) Weeks Control (ml/kg) CMS (ml/kg) CMS ? oral rose oil (ml/kg) CMS ? vapor rose oil (ml/kg) Basal 25.18 ± 4.05 28.97 ± 8.78 28.65 ± 10.26 25.66 ± 10.11 1st 25.31 ± 6.47 25.30 ± 6.47 25.00 ± 10.20 21.70 ± 10.70 2nd 24.56 ± 8.07 29.60 ± 5.16 27.80 ± 6.57 30.70 ± 13.50 3rd 23.93 ± 9.45 19.56 ± 7.53a 26.56 ± 8.53 33.00 ± 9.94a 4th 21.43 ± 9.69 18.94 ± 8.37a 22.80 ± 9.76 35.40 ± 8.63a a p\0.05 versus basal values J Nat Med (2013) 67:152–158 155 123
156 J Nat Med(2013)67:l52-158 Sucrose (1 %w/v)test results are shown in Table 3: oil vapor group were significantly (p<0.001)higher than consumption of water with sucrose at 3rd and 4th weeks in in the CMS group. ml/kg body weight was significantly (p<0.05)lower in The mean GSH and GSH-Px values in the cortex of the the depression group than basal values.In addition,con- brain in the four groups are also shown in Table 4.There sumption of water with sucrose at 3rd and 4th weeks in ml/kg were no statistically significance differences in GSH and body weight was significantly (p<0.05)higher in the rose GSH-Px values between the groups vapor supplemented groups than in the basal group. The mean lipid peroxidation values in the cerebral cortex of the four groups are shown in Fig.1.The results Discussion showed that the lipid peroxidation levels in CMS and CMS plus oral rose oil groups were significantly (p<0.05) We observed that lipid peroxidation levels in cerebral higher than in control.Exposure to rose oil vapor in cortex were increased by CMS-induced experimental depressed rats caused a decrease in the lipid peroxidation depression and oral rose oil supplementation,while the levels(p<0.001)compared to the depression-only group. antioxidant vitamins A,C and E and B-carotene levels The mean vitamin A.vitamin C.vitamin E and B-car- decreased.Therefore,CMS-induced depression in the otene concentrations in the cerebral cortex of the four animals is characterized by decreased antioxidants and groups are shown in Table 4.The results showed that increased lipid peroxidation levels.As another novel result vitamin A,vitamin C,vitamin E and B-carotene concen- of the current study,28 days'rose oil vapor supplemen- trations in the cerebral cortex of the depression and oral tation caused a decrease in lipid peroxidation levels,and rose oil groups were significantly (p<0.001)lower than in the antioxidant values increased. control.The vitamin A,vitamin C,vitamin E and B-caro- Numerous attempts have been made to set up an animal tene concentrations in the cerebral cortex in CMS plus rose model for depression or at least for some disease aspects. The weekly sucrose preference tests revealed that control animals typically exhibited a high preference for palatable 250 sucrose solutions [16],while this preference was markedly reduced following exposure to uncontrollable restraint 200 stress,in agreement with previous studies [4,5,10].This (3/oum) progressive decline in the sensitivity to a rewarding stim- ulus (sucrose)observed due to chronic stress exposure is thought to represent anhedonia (the loss of interest or pleasure)in animals,one of the two core symptoms required for diagnosis of a major depressive episode in human and rodents [16]. 50 Control Depression Depression+oral Depression+vapor Exposure to stress can induce psychiatric conditions, a p<0.05 versus control. including depression.Alterations in oxidative stress are bp<0.001 versus depression group. increasingly being recognized as a critical route of damage Fig.1 The effects of oral and vapor rose oil administrations on lipid towards the pathology of stress-induced psychiatric disor- peroxidation levels in brain of control and rats with depression ders [24].Significant correlations were found between the induced by chronic mild stress (CMS)(mean SD.n=8) severity of depression,as well as length of index episode Table 4 The effects of oral and vapor rose oil supplementation on glutathione peroxidase(GSH-Px),reduced glutathione(GSH)and antioxidant vitamin values in cerebral cortex of rats with chronic mild stress (CMS)-induced depression (mean+SD) Parameters Control (n 8) Depression(n =8) CMS oral (n =8) CMS vapor GSH(umol/g protein) 9.78±0.95 8.69±0.49 9.60±0.66 9.36±0.36 GSH-Px (IU/g protein) 64.84±6.63 62.31±2.99 64.98±3.41 66.78±4.17 Vitamin A (umol/g tissue) 2.42±0.21 0.73±0.13 0.72士0.114 2.27±0.36h Vitamin C(umol/g tissue) 83.04±16.33 41.16±5.87 33.36±6.39 89.14±18.74 Vitamin E(umol/g tissue) 20.10±1.74 7.63±1.04 7.57±0.98 20.4±1.68b B-Carotene (umol/g tissue) 1.21±0.08 0.65±0.10 0.59±0.08 1.10±0.10 ap<0.001 versus control p01 versus depression group Springer
Sucrose (1 %, w/v) test results are shown in Table 3; consumption of water with sucrose at 3rd and 4th weeks in ml/kg body weight was significantly (p\0.05) lower in the depression group than basal values. In addition, consumption of water with sucrose at 3rd and 4th weeks in ml/kg body weight was significantly (p\0.05) higher in the rose vapor supplemented groups than in the basal group. The mean lipid peroxidation values in the cerebral cortex of the four groups are shown in Fig. 1. The results showed that the lipid peroxidation levels in CMS and CMS plus oral rose oil groups were significantly (p\0.05) higher than in control. Exposure to rose oil vapor in depressed rats caused a decrease in the lipid peroxidation levels (p\0.001) compared to the depression-only group. The mean vitamin A, vitamin C, vitamin E and b-carotene concentrations in the cerebral cortex of the four groups are shown in Table 4. The results showed that vitamin A, vitamin C, vitamin E and b-carotene concentrations in the cerebral cortex of the depression and oral rose oil groups were significantly (p\0.001) lower than in control. The vitamin A, vitamin C, vitamin E and b-carotene concentrations in the cerebral cortex in CMS plus rose oil vapor group were significantly (p\0.001) higher than in the CMS group. The mean GSH and GSH-Px values in the cortex of the brain in the four groups are also shown in Table 4. There were no statistically significance differences in GSH and GSH-Px values between the groups. Discussion We observed that lipid peroxidation levels in cerebral cortex were increased by CMS-induced experimental depression and oral rose oil supplementation, while the antioxidant vitamins A, C and E and b-carotene levels decreased. Therefore, CMS-induced depression in the animals is characterized by decreased antioxidants and increased lipid peroxidation levels. As another novel result of the current study, 28 days’ rose oil vapor supplementation caused a decrease in lipid peroxidation levels, and the antioxidant values increased. Numerous attempts have been made to set up an animal model for depression or at least for some disease aspects. The weekly sucrose preference tests revealed that control animals typically exhibited a high preference for palatable sucrose solutions [16], while this preference was markedly reduced following exposure to uncontrollable restraint stress, in agreement with previous studies [4, 5, 10]. This progressive decline in the sensitivity to a rewarding stimulus (sucrose) observed due to chronic stress exposure is thought to represent anhedonia (the loss of interest or pleasure) in animals, one of the two core symptoms required for diagnosis of a major depressive episode in human and rodents [16]. Exposure to stress can induce psychiatric conditions, including depression. Alterations in oxidative stress are increasingly being recognized as a critical route of damage towards the pathology of stress-induced psychiatric disorders [24]. Significant correlations were found between the severity of depression, as well as length of index episode 50 75 100 125 150 175 200 225 250 Control Depression Depression+oral Depression+vapor (µmol/g protein) a b a a p<0.05 versus control. bp<0.001 versus depression group. Fig. 1 The effects of oral and vapor rose oil administrations on lipid peroxidation levels in brain of control and rats with depression induced by chronic mild stress (CMS) (mean ± SD, n = 8) Table 4 The effects of oral and vapor rose oil supplementation on glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and antioxidant vitamin values in cerebral cortex of rats with chronic mild stress (CMS)-induced depression (mean ± SD) Parameters Control (n = 8) Depression (n = 8) CMS ? oral (n = 8) CMS ? vapor GSH (lmol/g protein) 9.78 ± 0.95 8.69 ± 0.49 9.60 ± 0.66 9.36 ± 0.36 GSH-Px (IU/g protein) 64.84 ± 6.63 62.31 ± 2.99 64.98 ± 3.41 66.78 ± 4.17 Vitamin A (lmol/g tissue) 2.42 ± 0.21 0.73 ± 0.13a 0.72 ± 0.11a 2.27 ± 0.36b Vitamin C (lmol/g tissue) 83.04 ± 16.33 41.16 ± 5.87a 33.36 ± 6.39a 89.14 ± 18.74b Vitamin E (lmol/g tissue) 20.10 ± 1.74 7.63 ± 1.04a 7.57 ± 0.98a 20.4 ± 1.68b b-Carotene (lmol/g tissue) 1.21 ± 0.08 0.65 ± 0.10a 0.59 ± 0.08a 1.10 ± 0.10b a p\0.001 versus control b p\0.001 versus depression group 156 J Nat Med (2013) 67:152–158 123
J Nat Med(2013)67:152-l58 157 and duration of illness,and alterations in superoxide dis- known that antioxidant flavonoids induce modulator effects mutase and lipid peroxidation levels [3].Increased oxida- in SSRIs [10.301.Rose oil contains flavonoids such as tive stress occurs in depression,as evidenced by defective geraniol and citronellol which demonstrated ROS scav- cerebral cortex antioxidant defenses in conjunction with enging activity in a model of auto-oxidation of rat cerebral enhanced lipid peroxidation in patients [3]and experi- membranes [12].Therefore,rose oil,with potential anti- mental animals [4,5,24].In stress disorders,several routes oxidant activity and regulator effects on SSRIs,may be of of damage are triggered,such as mitochondrial dysfunction value in depression.Furthermore,membrane lipid peroxi- [1,6]and abnormalities of Ca2+influx [7]and the dation also modifies neurotransmitter release and uptake, phagocyte immune system [6].These events make a sig- ion-channel activity,the function of ATPases and glucose nificant contribution towards the resultant pathophysiology transporters,and the coupling of cell surface receptors to in brain characteristics in stress-induced depression. GTP-binding proteins,to impair mitochondrial function Depression is also characterized by oxidative stress and promote a cascade of events that culminates in apop- production pathways such as mitochondrial dysfunction, totic cell death [31,32].Prevention of these potentially dysregulation of Ca2+homeostasis [6,7].disruption of damaging factors during CMS-induced lipid peroxidation energy pathways,damage to neuronal precursor impair- may possibly be a target of the antioxidant action of rose ment neurogenesis [8]and induction of signal events in oil,relevant to its therapeutic benefits. apoptotic cell death [6].Lipid peroxidation levels such as This reported antidepressant-like effect of rose oil vapor malondialdehyde is a major oxidative degradation product may be also dependent on different properties of this flower of membrane unsaturated fatty acid and has been shown to oil,such as its neuromodulatory and antioxidant actions be biologically active with ROS properties [1,2].In the Studies report that in the central nervous system(particu- current study,CMS-induced depression enhanced cerebral larly in neurons)flavonoid is maintained at elevated con- cortex lipid peroxidation levels in the animal system, centrations and may act as a neuromodulator,facilitating although fat-soluble antioxidants(vitamin A,vitamin E and the release of some neurotransmitters and inhibiting neu- B-carotene)concentrations decreased.The brain has a high rotransmitter binding to receptors,including responses content of oxidizable polyunsaturated fatty acids and pre- mediated by the glutamatergic system [12.331.which is vention of lipid peroxidation by antioxidants serves to proposed to play a key role in the pathophysiology of maintain membrane integrity by protecting membrane depression [13]. phospholipids from damage,which is the result of a com- In conclusion,oxidative stress plays a role in the path- plex cascade involving impairment of membrane-transport ogenesis of CMS-induced depression in rat brain.The protein function and cation channels that,in turn,mediates beneficial effect of rose oil vapor on antioxidant vitamin membrane lipid peroxidation-induced disruption of neuro- systems in CMS-induced depression was shown by nal ion homeostasis [24.251. upregulation of vitamins A,vitamin C,vitamin E and Treatment of the CMS-induced depressed rats with rose B-carotene concentrations in the cerebral cortex.The oil vapor effectively protected the rats against depression- results may be helpful to physicians and for the treatment induced brain damage,as shown by increased antioxidant of depression with rose oil vapor,as well as to scientists for concentrations and decreased lipid peroxidation levels clarification of the etiology of depression. in the cerebral cortex.Rose oil showed antioxidant and antinociceptive properties in the treatment of recurrent Acknowledgments The study was partially supported as a graduate aphthous stomatitis [26].The destruction of membrane student project by TUBITAK,Ankara,Turkey. phospholipids alters membrane viscosity and it is supposed Conflict of interest All authors reported that they have no conflicts to influence several stages in biogenic amine function,such of interest. as receptor density or function of serotonergic/catechola- minergic receptors [3].The oxidation of catecholamines such as dopamine and serotonin by monoamine oxidase References (MAO)may result in an increased radical burden.The inhibitory effects of serotonin on depression are very well 1.Halliwell B (2006)Oxidative stress and neurodegeneration: documented [271:this effect has been attributed to a where are we now?J Neurochem 97:1634-1658 2.Kovacic P,Somanathan R (2008)Unifying mechanism for eye serotonin-induced decrease in dopamine in the central toxicity:electron transfer,reactive oxygen species,antioxidant nervous system [28].Selective serotonin reuptake inhibi- benefits,cell signaling and cell membranes.Cell Membr Free tors (SSRIs)are effective in the treatment of depression: Radic Res 2:56-69 the probable mechanism of these drugs is the enhancement 3.Bilici M,Efe H,Koroglu MA,Uydu HA,Bekaroglu M,Deger O (2001)Antioxidative enzyme activities and lipid peroxidation in of net serotonergic transmission by blocking presynaptic major depression:alterations by antidepressant treatments. 5-hydroxytryptamine (5-HT)uptake sites [29].It is well J Affect Disord 64:43-51 ≌Springer
and duration of illness, and alterations in superoxide dismutase and lipid peroxidation levels [3]. Increased oxidative stress occurs in depression, as evidenced by defective cerebral cortex antioxidant defenses in conjunction with enhanced lipid peroxidation in patients [3] and experimental animals [4, 5, 24]. In stress disorders, several routes of damage are triggered, such as mitochondrial dysfunction [1, 6] and abnormalities of Ca2? influx [7] and the phagocyte immune system [6]. These events make a significant contribution towards the resultant pathophysiology in brain characteristics in stress-induced depression. Depression is also characterized by oxidative stress production pathways such as mitochondrial dysfunction, dysregulation of Ca2? homeostasis [6, 7], disruption of energy pathways, damage to neuronal precursor impairment neurogenesis [8] and induction of signal events in apoptotic cell death [6]. Lipid peroxidation levels such as malondialdehyde is a major oxidative degradation product of membrane unsaturated fatty acid and has been shown to be biologically active with ROS properties [1, 2]. In the current study, CMS-induced depression enhanced cerebral cortex lipid peroxidation levels in the animal system, although fat-soluble antioxidants (vitamin A, vitamin E and b-carotene) concentrations decreased. The brain has a high content of oxidizable polyunsaturated fatty acids and prevention of lipid peroxidation by antioxidants serves to maintain membrane integrity by protecting membrane phospholipids from damage, which is the result of a complex cascade involving impairment of membrane-transport protein function and cation channels that, in turn, mediates membrane lipid peroxidation-induced disruption of neuronal ion homeostasis [24, 25]. Treatment of the CMS-induced depressed rats with rose oil vapor effectively protected the rats against depressioninduced brain damage, as shown by increased antioxidant concentrations and decreased lipid peroxidation levels in the cerebral cortex. Rose oil showed antioxidant and antinociceptive properties in the treatment of recurrent aphthous stomatitis [26]. The destruction of membrane phospholipids alters membrane viscosity and it is supposed to influence several stages in biogenic amine function, such as receptor density or function of serotonergic/catecholaminergic receptors [3]. The oxidation of catecholamines such as dopamine and serotonin by monoamine oxidase (MAO) may result in an increased radical burden. The inhibitory effects of serotonin on depression are very well documented [27]; this effect has been attributed to a serotonin-induced decrease in dopamine in the central nervous system [28]. Selective serotonin reuptake inhibitors (SSRIs) are effective in the treatment of depression; the probable mechanism of these drugs is the enhancement of net serotonergic transmission by blocking presynaptic 5-hydroxytryptamine (5-HT) uptake sites [29]. It is well known that antioxidant flavonoids induce modulator effects in SSRIs [10, 30]. Rose oil contains flavonoids such as geraniol and citronellol which demonstrated ROS scavenging activity in a model of auto-oxidation of rat cerebral membranes [12]. Therefore, rose oil, with potential antioxidant activity and regulator effects on SSRIs, may be of value in depression. Furthermore, membrane lipid peroxidation also modifies neurotransmitter release and uptake, ion-channel activity, the function of ATPases and glucose transporters, and the coupling of cell surface receptors to GTP-binding proteins, to impair mitochondrial function and promote a cascade of events that culminates in apoptotic cell death [31, 32]. Prevention of these potentially damaging factors during CMS-induced lipid peroxidation may possibly be a target of the antioxidant action of rose oil, relevant to its therapeutic benefits. This reported antidepressant-like effect of rose oil vapor may be also dependent on different properties of this flower oil, such as its neuromodulatory and antioxidant actions. Studies report that in the central nervous system (particularly in neurons) flavonoid is maintained at elevated concentrations and may act as a neuromodulator, facilitating the release of some neurotransmitters and inhibiting neurotransmitter binding to receptors, including responses mediated by the glutamatergic system [12, 33], which is proposed to play a key role in the pathophysiology of depression [13]. In conclusion, oxidative stress plays a role in the pathogenesis of CMS-induced depression in rat brain. The beneficial effect of rose oil vapor on antioxidant vitamin systems in CMS-induced depression was shown by upregulation of vitamins A, vitamin C, vitamin E and b-carotene concentrations in the cerebral cortex. The results may be helpful to physicians and for the treatment of depression with rose oil vapor, as well as to scientists for clarification of the etiology of depression. Acknowledgments The study was partially supported as a graduate student project by TUBITAK, Ankara, Turkey. Conflict of interest All authors reported that they have no conflicts of interest. References 1. Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J Neurochem 97:1634–1658 2. Kovacic P, Somanathan R (2008) Unifying mechanism for eye toxicity: electron transfer, reactive oxygen species, antioxidant benefits, cell signaling and cell membranes. Cell Membr Free Radic Res 2:56–69 3. 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