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reperfusion-induced oxidative stress in rats
Journal of Ethnopharmacology 142 (2012) 35–40
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Neuroprotective activity of Cymbopogon martinii against cerebral ischemia/reperfusion-induced oxidative stress in rats Prakruti Buch, Vishal Patel, Vishavas Ranpariya, Navin Sheth, Sachin Parmar n Department of Pharmaceutical Sciences, Saurashtra University, Rajkot 360 005, Gujarat, India
a r t i c l e i n f o
abstract
Article history: Received 7 January 2012 Received in revised form 1 April 2012 Accepted 3 April 2012 Available online 17 April 2012
Ethnopharmacological relevance: Cymbopogon martinii (Roxb.) Watson (Family: Graminae), commonly known as Palmarosa, is traditionally prescribed for central nervous system (CNS) disorders such as neuralgia, epileptic fits and anorexia. Although the plant possesses diverse pharmacological actions, the neuroprotective action has got little attention. Aim of the study: The present study evaluated neuroprotective effect of essential oil of Cymbopogon martinii (EOCM) against global cerebral ischemia/reperfusion (I/R)-induced oxidative stress in rats. Materials and methods: Global ischemic brain damage was induced by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by 60 min reperfusion on Wistar albino rats. The biochemical levels of lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), total thiols and glutathione (GSH) were estimated and brain coronal sections and histopathological studies were performed. Results: BCCA occlusion, followed by reperfusion caused varied biochemical/enzymatic alterations viz. increase in LPO and decrease in SOD, CAT, total thiols and GSH. The prior treatment of EOCM (50 mg/kg and 100 mg/kg, p.o. for 10 days) markedly reversed these changes and restored to normal levels as compared to I/R groups. Moreover, brain coronal sections and histopathological studies revealed protection against ischemic brain damage in the EOCM-treated groups. Conclusion: This study, for the first time, shows potent neuroprotective effect of EOCM against global cerebral I/R-induced oxidative stress in rats, suggesting its therapeutic potential in cerebrovascular diseases (CVD) including stroke. & 2012 Elsevier Ireland Ltd All rights reserved.
Keywords: Cerebrovascular diseases Cymbopogon martinii Ischemia/reperfusion Neuroprotection Oxidative stress
1. Introduction Cerebrovascular disease (CVD) is a group of brain dysfunctions related to disease of the blood vessels supplying the brain that includes some of the most common devastating disorders such as ischemic stroke, hemorrhagic stroke and cerebrovascular anomalies. It is reported that CVD represents major cause of disability causing two lacks deaths each year (Wade et al., 2005). World Health Organization (WHO) data reflects 5.7 million deaths from CVDs out of 58 million global deaths in 2005. It is postulated that CVDs will be the second most frequent cause of projected deaths in the year 2020 (Anonymous, 1997; Huang and McNamara, 2004). Ischemic stroke has been shown as substantial public health problem that leads to long-term disability in major industrialized countries. Indeed, it is the third leading cause of death after heart
n
Corresponding author. Tel.: þ919898002327; fax: þ 912812585083. E-mail addresses: [email protected] (P. Buch), [email protected] (V. Patel), [email protected] (V. Ranpariya), [email protected] (N. Sheth), [email protected] (S. Parmar). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.04.007
disease and cancer (Stapf and Mohr, 2002). A number of drugs with potential neuroprotective activity have been used in the treatment of stroke. However, there is no clinically effective therapy for the management of acute stroke except tissue-plasminogen activator (t-PA) (Alavijeh et al., 2005). Recently, intense interest has been focused on the neuroprotective properties of a series of natural products. In particular, some natural products act as neuroprotecting agents by enhancing the survival of neurons while preventing their death and apoptosis (Zhu et al., 2004). It is well-established that excitotoxicity, oxidative stress, inflammation and apoptosis are the major pathobiological mechanisms of ischemia/reperfusion (I/R) injury (Ozbal et al., 2008; Yousuf et al., 2009). These changes are associated with mitochondrial dysfunction and rapid decrease in ATP, resulting in free radical generation and lipid peroxidation. It can be recalled that oxidative stress plays a central role in cerebral I/R injury (Gilgun-Sherki et al., 2002; Janardhan and Qureshi, 2004). Reactive oxygen species (ROS) have been found to be over produced during I/R of neural tissues (Gilgun-Sherki et al., 2002; Li and Jackson, 2002; Sugawara and Chan, 2003). The lethal process is accompanied by elevated free radicals, including superoxide
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anion (O2 ), hydroxyl radical (OH) and hydrogen peroxide (H2O2) as well as progressive depletion in endogenous antioxidant system including antioxidant enzymes such as superoxide dismutase and catalase or antioxidants glutathione, vitamin C and E (Sugawara and Chan, 2003). Cymbopogon martinii (CM), commonly known as Palmarosa in English and Rohisha in Sanskrit, is an essential oil yielding grass from the family Graminae. Traditionally, the essential oil obtained from Cymbopogon martinii (EOCM) is widely used for neuralgia, bronchitis, anorexia, cardiac debility, skin diseases, epileptic fits and fever (Kirtikar and Basu, 1999). The main chemical constituents reported from the plant are geraniol, geranyl acetate, linalool, geranyl octanoate, geranyl butyrate, monoterpenoid and cymbodiacetal (Anonymous, 2000). In the light of this background, the purpose of the present study was to evaluate the potent neuroprotective effect of EOCM against global cerebral I/R-induced oxidative stress in Wistar albino rats.
2. Materials and methods 2.1. Drugs and chemicals Trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 5-5dithiobis (2-nitrobenzoic acid), glutathione and ( 7)-epinephrine were purchased from Sigma-Aldrich Co., Spruce Street, St. Louis, MO, USA. 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Hi-Media, Mumbai. All other chemicals were of the highest purity commercially available. 2.2. Plant material and essential oil (EO) extraction Leaves of CM were collected from the botanical garden of Saurashtra University, Rajkot, Gujarat, India during the month of August. Herbarium was prepared and the specimen was authenticated by Dr. A.S. Reddy, Department of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India. A voucher specimen (SU/DPS/Herb/43) was deposited in the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India for future reference. The EO was obtained by hydro-distillation in Clevenger apparatus using 100 g of dried leaves. The oil obtained was dried over anhydrous sodium sulfate, producing yields of 0.625% (v/w). The oil was subjected to GC–MS analysis (Shimadzu, Switzerland, model QP2010) according to the following experimental conditions: silica capillary column: SGE BP X 5 (30 m 0.25 mm 0.25 mm), injector temperature: 230 1C, detector temperature: 250 1C, temperature program: the column was initially 50 1C, then raised to 200 1C at a rate of 4 1C/min and finally held at 200 1C for 10 min, electron impact: 70 eV, carrier gas: helium at 1 ml/min, scanning speed 0.84 scan s 1 from m/z 50 to 1000 Da. The identification of the separated volatile organic compounds was achieved through retention time and mass spectrometry by the GC–MS library. 2.3. Determination of LD50 The acute toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949) and LD50 was calculated accordingly. Briefly, the EOCM suspended in 1% tween 80 in saline at a dose of 125, 250, 500 and 1000 mg/kg was administered orally to different groups of mice (n ¼10). The animals were examined every 30 min up to a period of 3 h and then, occasionally, for further 4 h; finally, after 24 h mortality was recorded. All tests on rats were performed at two dose levels; 50 and 100 mg/kg of body weight corresponding to 10 and 20% of LD50 value (500 mg/kg) respectively.
2.4. Animals The Wistar-albino rats of either sex (150–200 g) were used for study. The animals were housed in a five rats per cage under well controlled conditions of temperature (2571 1C), humidity (5575%) and 12 h/12 h light-dark cycle (light on 07.30– 19.30 h). Animals had access to standard pellet diet (Pranav Agro Industries Ltd., India) and water was given ad libitum. The protocol of the experiment was approved by the Institutional Animal Ethical Committee (Protocol approval no: SU/DPS/IAEC/ 2011/05) as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forest, Animal Welfare Division, Government of India, New Delhi, India. For the present study, animals were randomized into 6 groups of 8 animals each and allowed to acclimatize for 1 week before the experiments. 2.5. Experimental protocol for global cerebral ischemia/reperfusioninduced oxidative stress The Wistar-albino rats of either sex (150–200 g) were divided into 6 groups of eight rats each and fed with drug or vehicle for 10 days prior to the experiment and treated as follows (Shah et al., 2005): Group-I: 1% tween 80 in saline (10 ml/kg, orally), no ischemia. Group-II: 1% tween 80 in saline (10 ml/kg, orally), bilateral common carotid artery (BCCA) occlusion for 30 min. Group-III: 1% tween 80 in saline (10 ml/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-IV: Quercetin (25 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-V: EOCM, suspended in 1% tween 80 in saline (50 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-VI: EOCM, suspended in 1% tween 80 in saline (100 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min.
2.6. Induction of ischemia Animals of groups II–VI were subjected to BCCA occlusion under ketamine anesthesia (45 mg/kg, i.p.) (Farbiszewski et al., 1995). Animals were placed on the back; both carotid arteries were exposed and occluded by atraumatic clamps. Temperature was maintained around 37 70.5 1C throughout the surgical procedure and artificial ventilation (95% O2 and 5% CO2) provided with artificial respirator. 2.7. Preparation of post-mitochondrial supernatant Following decapitation, the brain was removed and washed in cooled 0.9% saline, kept on ice and subsequently blotted on filter paper, then weighed and homogenized as 10% (w/v) in cold phosphate buffer (0.05 M, pH 7.4). Homogenization procedure was performed as quickly as possible under completely standardized conditions. The homogenates were centrifuged at 10,000g for 10 min at 4 1C (MPW-350R, Korea) and post-mitochondrial supernatant (PMS) was kept on ice until assayed. 2.8. Biochemical estimation 2.8.1. Lipid peroxidation (LPO) Thiobarbituric acid reactive substances (TBARS) in the homogenate were estimated by using standard protocol (Prabhakar
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et al., 2006). Briefly, the 0.5 ml of 10% homogenate was incubated with 15% TCA, 0.375% TBA and 5 N HCl at 95 1C for 15 min, the mixture was cooled, centrifuged and absorbance of the supernatant measured at 512 nm against appropriate blank. The amount of LPO was determined by using e¼1.56 105 M 1 cm 1 and expressed as TBARS nM/mg of protein (Braughler et al., 1987). 2.8.2. Superoxide dismutase (SOD) SOD activity was determined based on the ability of SOD to inhibit the auto-oxidation of epinephrine to adrenochrome at alkaline pH (Misra and Fridovich, 1972). Briefly, 25 ml of the supernatant obtained from the centrifuged brain homogenate was added to a mixture of 0.1 mM epinephrine in carbonate buffer (pH 10.2) in a total volume of 1 ml and the formation of adrenochrome was measured at 295 nm. The SOD activity (U/mg of protein) was calculated by using the standard plot. 2.8.3. Catalase (CAT) CAT activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml homogenate (10%, w/v) in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of nM H2O2 consumed/min/mg protein. 2.8.4. Total thiols This assay is based on the principle of formation of relatively stable yellow color by sulfhydryl groups with DTNB (Moron et al., 1979). Briefly, 0.2 ml of brain homogenate was mixed with phosphate buffer (pH 8), 40 ml of 10 mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks. The total thiol content was calculated by using e¼13.6 1031 cm 1 M 1 (Sedlak and Lindsay, 1968). 2.8.5. Glutathione (GSH) GSH was estimated in various tissues by the method of Sedlak and Lindsay (1968). Briefly, 5% tissue homogenate was prepared in 20 mM EDTA, pH 4.7 and 100 ml of the homogenate or pure GSH was added to 0.2 M tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 ml of Ellman’s reagent (10 mM/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm. Samples were centrifuged before the absorbance of the supernatants was measured (Khynriam and Prasad, 2003).
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cerebral infarction area was observed and compared between various treatment groups and negative control group. 2.11. Histopathology The brains from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 mm thickness. The sections were stained with haemotoxylin and eosin dye for histopathological observation. 2.12. Statistical analysis All the data are presented as mean 7S.E.M. The significance of difference in means between control and treated animals for different parameters was determined by using One-way Analysis of Variance (ANOVA) followed by multiple comparisons Dunnett’s test. A value of po0.05 was considered statistically significant.
3. Results 3.1. LD50 value Acute toxicity study showed that LD50 of the EOCM in mice was 500 mg/kg by oral route. 3.2. GC–MS analysis GC–MS analysis confirmed the presence of geraniol (77.51%), geranyl acetate (7.97%), b-linalool (6.27%) and b-caryophyllene (1.68%) as the main compounds in EOCM (Table A1). 3.3. Biochemical estimation The results showed in Table A2 revealed neuroprotective activity of EOCM. The animals from BCCA-occluded ischemic group (NS þI) exhibited significant increase in LPO levels, whereas significant decrease in all the other enzymatic and non-enzymatic parameters (SOD, CAT, GSH and total thiols) was observed. These levels were further augmented in animals of I/R (NS þI/R) group. The animals from EOCM-treated groups had shown a significant protection by reducing the elevated levels of LPO (po0.001) and marked increase in SOD (po0.001), CAT (po0.01), GSH (p o0.001) and total thiol (po0.001) levels as compared to I/Rtreated group (NS þI/R group). 3.4. Cerebral infarction area
2.9. Total protein The total protein contents of 10% brain homogenates were determined by using the modified method of Lowry et al. (1951).
The cerebral infarction area revealed significant decrease in EOCM-treated groups as compared to negative control group especially in caudal and rostral side (Fig. B1).
2.10. Measurement of infarction area
3.5. Histopathology
The infarction area was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining method according to Bederson et al. (1986). Following ischemia or reperfusion after varied durations of ischemia, animals were decapitated and the brains were removed. After the brains were placed briefly in cold saline, four coronal brain slices (2 mm thick) were made. Then the slices were incubated in phosphate buffered saline (pH 7.4) containing 2% TTC at 37 1C for 10 min and then kept in neutral-buffered formalin overnight (Isayama et al., 1991). The images of the TTC-stained sections were acquired by scanning with a high resolution scanner (Hewlett-Packard Scanjet 6100C/T). Then the
As shown in Fig. B2, ischemia evoked marked congestion of blood vessels and neutrophil infiltration (Plate B) in group II (NS þI). These effects were further augmented in group III (NS þI/ R), i.e. lymphocytic proliferation and neuronal necrosis (Plate C). Significant protection from the brain damage was observed in the EOCM- and quercetin-treated groups (Plates E–F and Plate D, respectively). The protection offered by EOCM was well-comparable to quercetin group. The EOCM reversed the I/R-provoked neuronal damage which was similar to the normal rat brain as seen in the tween 80 in saline-treated rats (group I, no ischemia) (Plate A).
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4. Discussion The present study revealed neuroprotective potential of EOCM at 50 mg/kg and 100 mg/kg. We estimated LPO, SOD, CAT, GSH and total thiol levels in the brain tissue as an index to assess the severity of oxidative damage. Traditionally, the dose up to 10 g is suggested to treat epilepsy and other CNS related conditions suggesting the plant is considerably safe in human (Anonymous, 2006). In the present study also, the selected dose levels 50 and 100 mg/kg were devoid of any sign of toxicity in animals. Oxygen is essential for aerobic life, but it is also precursor to the formation of harmful reactive oxygen species (ROS) (Albert and Yong-Mei, 1998). Free radicals in the living organism are generated both enzymatically and non-enzymatically, leading to the formation of ROS. It has been reported that hydroxyl and peroxynitrite are the most potent ROS that can damage proteins, lipids and nucleic acids, resulting in the inactivation of some enzyme activities, disruption of ion homeostasis and modification of genetic apparatus and apoptotic death (Albert and Yong-Mei, 1998). An interesting finding of the present study was amelioration of LPO by EOCM, an effect that could be attributed to its capacity to scavenge free radicals, as shown by the observed restoration of the antioxidant enzyme activities. We may recall that ROS produces malondialdehyde (MDA), an end product of LPO, a process that leads to dysfunction of membrane bound receptors and enzymes (Love, 1999; Fisher, 2001). Moreover, recent studies showed that global cerebral ischemia (Gupta and Sharma, 2006) and reperfusion (Irmak et al., 2003) significantly augmented MDA levels in animals. In consistent with this, we found that the ischemia and reperfusion resulted into augmentation of LPO process and thereby elevated the MDA levels in the brains of animals. There is strong indirect evidence that free radical production appears to be an important mechanism of brain injury after exposure to I/R (Traystman et al., 1991). To further characterize the neuroprotective actions of EOCM, we evaluated effect of EOCM on endogenous enzymatic and nonenzymatic antioxidant levels in the brain homogenates. It is well established that antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) play central role in the defense mechanisms against free radicalinduced oxidative stress (Islekel et al., 1999; Nita et al., 2001). Similarly, non-enzymatic antioxidants such as ascorbic acid (vitamin C), a-tocopherol (vitamin E), glutathione (GSH), and carotenoids have tonic inhibitory role in the oxidative damage induced by free radicals (Dringen, 2000; Aabdallah and Eid, 2004; Ege et al., 2004). In the present study, prior treatment of EOCM significantly increased GSH levels. It can be recalled that GSH acts directly by detoxifying ROS and indirectly as a substrate for various peroxidases thus, protecting the cells against oxidative damage (Dringen, 2000). Dysfunction of the GSH system has been implicated in a number of neurodegenerative diseases such as Alzheimer and Parkinson’s diseases (Schulz et al., 2000) and is a potential contributor to oxidative damage following temporary ischemia in rodents (Chen et al., 2000). In the current study, GSH levels were moderately reduced due to I/R insult which is in consistence with other studies (Islekel et al., 1999; Nita et al., 2001; Ege et al., 2004; Shah et al., 2005). Furthermore, administration of EOCM showed significant increase in SOD levels in brain. It has been shown that the SOD, a major antioxidant enzyme located in cytoplasm and mitochondria of cells, forms the principal defense system against excess O2 production by direct scavenging of O2 during reperfusion (Nita et al., 2001). We also evaluated effect of EOCM on the CAT levels in the rat brain. As compared to the control animals, EOCM-treated group showed significant increase in the CAT levels. The reports about the effect
of I/R on CAT activity are controversial in the literature. Some studies reported that I/R in rats lowered CAT activity (Aabdallah and Eid, 2004; Shah et al., 2005). On the other hand, Islekel et al. (1999) showed significant increase in the CAT activity after ischemia. However, in our experiments, the mean CAT activity was significantly decreased in I/R group. Furthermore, the histopathological studies revealed significant decrease in neutrophil infiltration and brain damage by the EOCM as compared to control group. Additionally, the cerebral infraction area was very less in EOCM-treated groups as compared to control groups indicating protection from I/R damage in the brain tissue. These results indicated neuroprotection by EOCM against BCCA occlusion-induced I/R brain damage. GC–MS study showed that EOCM is rich in geraniol (77.51%). The earlier study reported marked in vitro free-radical scavenging activity of geraniol against the DPPH model (Choi et al., 2000). Tiwari and Kakkar (2009) reported the antioxidant potential of geraniol using tsigniertiary-butyl hydroperoxide stressed rat alveolar macrophages and concluded that geraniol significantly increased the cell viability, SOD activity, GSH content and restored the mitochondrial membrane potential. Further, it was suggested that geraniol significantly decreased LPO, inhibited NO release and generated ROS in the pre-treated cells as compared to stressed cells. The protection offered by EOCM against cerebral ischemia could, at least in part, be attributed to the free-radical scavenging activity of geraniol. In conclusion, these findings suggest a potential protective role of EOCM in the global model of ischemia, a standard model for stroke in rats and gain importance in view of the fact that stroke is at present the third leading cause of death worldwide and CVDs constitute second most frequent cause of projected death in the years to follow. We suggest that the mechanism with which EOCM has normalized the damage is probably, by the antioxidant property of the geraniol, is a major constituent of EOCM. As evident by the results of the present study, it seems that EOCM has enhanced the defense mechanism, thereby reducing the damaged produced by global ischemia. Further studies are required to pursue the interesting leads emerging from the present results to exploit the full therapeutic potential of EOCM for neuroprotection.
Acknowledgments The authors are grateful to Dr. A.S. Reddy, Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, for identification of medicinal herb and Dr. P.S. Parsania, Department of Chemistry, Saurashtra University, Rajkot, for GC–MS analysis.
Table A1 Composition of essential oil. Rta time
Area
% area
Molecular formula
Compound name
8.558 9.442 10.292 12.525 12.758 14.033 14.933 16.333 17.167 18.400 18.675 20.858
145267 1188499 4660721 57656438 910842 5930217 1252613 205023 349126 776018 1091639 222139
0.20 1.60 6.27 77.51 1.22 7.97 1.68 0.28 0.47 1.04 1.47 0.30
C10H16 C10H16 C10H18O C10H18O C10H16O C12H20O2 C15H24 C12H20O2 C15H24O C15H26O C12H20O2 C12H20O2
b-pinene b-cis-Ocimene b-Linalool
a
Rt—Retention time.
Geraniol Citral Geranyl acetate b-Caryophyllene Nerol acetate Caryophyllene oxide Farnesol Neryl acetate Bay pine (oyster) oil
P. Buch et al. / Journal of Ethnopharmacology 142 (2012) 35–40
Appendix A
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Appendix B
See Tables A1 and A2 here.
See Figs. B1 and B2 here.
Table A2 Effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion-induced oxidative stress in rats. Group no.
Treatment
Lipid peroxidation (nM/mg of protein)
Superoxide dismutase (U/mg of protein)
Catalase (U/mg of protein)
Glutathione (nM/mg of protein)
Total thiols (lM/mg of protein)
I II III IV V VI
Normal NSþ Ischemia NSþ I/R Quercetin þI/R 50 mg/kg EOCM þI/R 100 mg/kg EOCMþ I/R
10.34 70.74 23.45 71.98nnn 51.73 71.68nnn 4.238 70.48b 8.625 70.54b 4.713 70.43b
151.9 74.99 43.28 70.54nnn 39.70 70.82nnn 187.8 73.58b 109.8 72.52b 132.2 75.02b
0.01886 7 0.0015 0.01222 7 0.0020n 0.008967 7 0.0017nn 0.02358 7 0.0010b 0.01445 7 0.0016a 0.01866 7 0.0008b
7.012 70.30 6.344 70.09n 5.964 70.13nn 8.120 70.12b 6.823 70.15b 7.475 70.14b
18.83 70.36 13.97 70.26nnn 10.61 70.25nnn 17.97 70.25b 16.45 70.30b 16.94 70.33b
Values are mean 7S.E.M., n¼ 8, One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test, *p o 0.05, **p o0.01 and ***p o 0.001 vs. normal and a p o0.05 and bp o 0.001 vs. NS þI/R. NS—Normal Saline, I—Ischemia, I/R—Ischemia/Reperfusion, EOCM—Essential oil of Cymbopogon martinii.
Fig. B1. Neuroprotective effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion damage in rats evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain coronal sections were prepared (2 mm thickness) and then each section was stained with TTC. A: normal; B: normal salineþ ischemia for 30 min; C: normal salineþ ischemia for 30 min followed by 60 min reperfusion (I/R); D: quercetin 25 mg/kgþ I/R; E and F: 50 mg/kgþI/R and 100 mg/kgþI/R of EOCM respectively. A large infarction area observed mainly in the caudal and rostral side of hippocampus in the damaged brain of I/R-treated rats (C) whereas the infarction was markedly reduced in the rat brains treated with 50 mg/kg (E) and 100 mg/kg (F) of EOCM and also in quercetin-treated animals (D). n¼3.
Fig. B2. Neuroprotective effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion damage in rats. Photographs of brain sections from different treatment groups stained with Haemotoxylin & Eosin, 10 . Plates; A: normal; B: normalþ ischemia for 30 min; C: normal saline þischemia for 30 min followed by 60 min reperfusion (I/R); D: quercetin 25 mg/kgþ I/R; E and F: 50 mg/kgþ I/R and 100 mg/kgþ I/R of EOCM respectively. Ischemia (B) caused marked congestion of blood vessels and neutrophil infiltration. These effects were further augmented by reperfusion i.e. lymphocytic proliferation and neuronal necrosis (C). There is significant reversal of damage observed in EOCM-treated groups (E and F) and also in quercetin-treated group (D). The reversal was marked as the values were mostly comparable with saline-treated rats without ischemia (A).
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References Aabdallah, D.M., Eid, N.I., 2004. Possible neuroprotective effects of lecithin and alpha tocopherol alone or in combination against ischemia/reperfusion insult in rat brain. Journal of Biochemical and Molecular Toxicology 18, 273–278. Alavijeh, M.S., Chishty, M., Qaiser, M.Z., Palmer, A.M., 2005. Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug delivery. The Journal of the American Society for Experimental NeuroTherapeutics 2, 554–571. Albert, Y.S., Yong-Mei, C., 1998. Oxidative stress and neurodegenerative disorders. Journal of Biomedical Science 5, 401–414. Anonymous, 1997. From what will we die in 2020? Lancet 349, 1263–1264. Anonymous, 2000. The Wealth of India. Council of Scientific & Industrial Research, New Delhi (pp. 324–325). Anonymous, 2006. The Ayurvedic Pharmacopoeia of India, vol. 5. Department of Ayush, Government of India, New Delhi (pp. 187–189). Bederson, J.B., Pitts, L.H., Germano, S.M., Nishimura, M.C., Davis, R.L., Bartkowski, H.M., 1986. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17, 1304–1308. Braughler, J.M., Chase, R.L., Pregenzer, J.F., 1987. Oxidation of ferrous iron during peroxidation of various lipid substrates. Biochimica et Biophysica Acta 921, 457–464. Chen, Y.H., Du, G.H., Zhang, J.T., 2000. Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats. Acta Pharmacologica Sinica 21, 463–466. Choi, H.S., Song, H.S., Ukeda, H., Sawamura, M., 2000. Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2picrylhydrazyl. Journal of Agricultural and Food Chemistry 48, 4156–4161. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), Hand Book of Methods for Oxygen Radical Research. CRC Press, Boca Raton, Florida, pp. 283–284. Dringen, R., 2000. Metabolism and functions of glutathione in brain. Progress in Neurobiology 62, 649–671. Ege, E., Ilhan, A., Gurel, A., Akyol, O., Ozen, S., 2004. Erdosteine ameliorates neurological outcome and oxidative stress due to ischemia/reperfusion injury in rabbit spinal cord. European Journal of Vascular and Endovascular Surgery 28, 379–386. Farbiszewski, R., Bielawski, K., Bielawska, A., Sobaniec, W., 1995. Spermine protects in vivo antioxidant enzymes in transiently hypoperfused rat brain. Acta Neurobiologiae Experimentalis 55, 253–258. Fisher, M., 2001. Stroke Therapy, second ed. Butterworth-Heinemann, Boston (pp. 25–50). Gilgun-Sherki, Y., Rosenbaum, Z., Melamed, E., Offen, D., 2002. Antioxidant therapy in acute central nervous system injury: current state. Pharmacological Reviews 54, 271–284. Gupta, S., Sharma, S.S., 2006. Neuroprotective effects of trolox in global cerebral ischemia in gerbils. Biological and Pharmaceutical Bulletin 29, 957–961. Huang, Y., McNamara, J.O., 2004. Ischemic stroke: ‘‘acidotoxicity’’ is a perpetrator. Cell 118, 665–666. Irmak, M.K., Fadillioglu, E., Sogut, S., Erdogan, H., Gulec, M., Ozer, M., Yagmurca, M., Gozukara, M.E., 2003. Effects of caffeic acid phenethyl ester and alphatocopherol on reperfusion injury in rat brain. Cell Biochemistry and Function 21, 283–289. Isayama, K., Pitts, L.H., Nishimura, M.C., 1991. Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke 22, 1394–1398. Islekel, S., Islekel, H., Guner, G., Ozdamar, N., 1999. Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion. Research in Experimental Medicine 199, 167–176. Janardhan, V., Qureshi, A.I., 2004. Mechanisms of ischemic brain injury. Current Cardiology Reports 6, 117–123.
Khynriam, D., Prasad, S.B., 2003. Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin. Brazilian Journal of Medical and Biological Research 36, 53–63. Kirtikar, K.R., Basu, B.D., 1999. Indian Medicinal Plants, sixth ed. International Book Distributors, Dehradun, India (pp. 2677–2681). Li, C., Jackson, R.M., 2002. Reactive species mechanisms of cellular hypoxiareoxygenation injury. American Journal of Physiology. Cell Physiology 282, C227–C241. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. The Journal of Pharmacology and Experimental Therapeutics 96, 99–113. Love, S., 1999. Oxidative stress in brain ischemia. Brain Pathology 9, 119–131. Lowry, O.H., Rosebrough, N.J., Fair, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193, 265–275. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. The Journal of Biological Chemistry 2479, 3170–3175. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 582, 67–78. Nita, D.A., Nita, V., Spulber, S., Moldovan, M., Popa, D.P., Zagrean, A.M., Zagrean, L., 2001. Oxidative damage following cerebral ischemia depends on reperfusion—a biochemical study in rat. Journal of Cellular and Molecular Medicine 5, 163–170. Ozbal, S., Erbil, G., Kocdor, H., Tu˘gyan, K., Pekcetin, C., Ozo˘gul, C., 2008. The effects of selenium against cerebral ischemia-reperfusion injury in rats. Neuroscience Letters 438, 265–269. Prabhakar, K.R., Veerapur, V.P., Parihar, K.V., Priyadarsini, K.I., Rao, B.S., Unnikrishnan, M.K., 2006. Evaluation and optimization of radioprotective activity of Coronopus didymus Linn. in g-irradiated mice. International Journal of Radiation Biology 82, 525–536. Schulz, J.B., Lindenau, J., Seyfried, J., Dichgans, J., 2000. Glutathione, oxidative stress and neurodegeneration. European Journal of Biochemistry 267, 4904–4911. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25, 192–205. Shah, Z.A., Gilani, R.A., Sharma, P., Vohora, S.B., 2005. Cerebroprotective effect of Korean ginseng tea against global and focal models of ischemia in rats. Journal of Ethnopharmacology 101, 299–307. Stapf, C., Mohr, J.P., 2002. Ischemic stroke therapy. Annual Review of Medicine 53, 453–475. Sugawara, T., Chan, P.H., 2003. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia. Antioxidants and Redox Signaling 5, 597–607. Tiwari, M., Kakkar, P., 2009. Plant derived antioxidants-geraniol and camphene protect rat alveolar macrophages against t-BHP induced oxidative stress. Toxicology In Vitro 23, 295–301. Traystman, R.J., Kirsch, J.R., Koehler, R.C., 1991. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. Journal of Applied Physiology 71, 1185–1195. Wade, S.S., Johnson, S.C., Easton, J.D., 2005. Cerebrovascular Disease, Principles of Internal Medicine. McGraw-Hill, Noida, Uttar Pradesh, India (pp. 2372). Yousuf, S., Atif, F., Ahmad, M., Hoda, N., Ishrat, T., Khan, B., Islam, F., 2009. Resveratrol exerts its neuroprotective effect by modulating mitochondrial dysfunctions and associated cell death during cerebral ischemia. Brain Research 1250, 242–253. Zhu, Y.Z., Huang, S.H., Tan, B.K., Sun, J., Whiteman, M., Zhu, Y.C., 2004. Antioxidants in Chinese herbal medicines: a biochemical perspective. Natural Product Reports 21, 478–489.
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Neuroprotective activity of Cymbopogon martinii against cerebral ischemia/reperfusion-induced oxidative stress in rats Prakruti Buch, Vishal Patel, Vishavas Ranpariya, Navin Sheth, Sachin Parmar n Department of Pharmaceutical Sciences, Saurashtra University, Rajkot 360 005, Gujarat, India
a r t i c l e i n f o
abstract
Article history: Received 7 January 2012 Received in revised form 1 April 2012 Accepted 3 April 2012 Available online 17 April 2012
Ethnopharmacological relevance: Cymbopogon martinii (Roxb.) Watson (Family: Graminae), commonly known as Palmarosa, is traditionally prescribed for central nervous system (CNS) disorders such as neuralgia, epileptic fits and anorexia. Although the plant possesses diverse pharmacological actions, the neuroprotective action has got little attention. Aim of the study: The present study evaluated neuroprotective effect of essential oil of Cymbopogon martinii (EOCM) against global cerebral ischemia/reperfusion (I/R)-induced oxidative stress in rats. Materials and methods: Global ischemic brain damage was induced by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by 60 min reperfusion on Wistar albino rats. The biochemical levels of lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), total thiols and glutathione (GSH) were estimated and brain coronal sections and histopathological studies were performed. Results: BCCA occlusion, followed by reperfusion caused varied biochemical/enzymatic alterations viz. increase in LPO and decrease in SOD, CAT, total thiols and GSH. The prior treatment of EOCM (50 mg/kg and 100 mg/kg, p.o. for 10 days) markedly reversed these changes and restored to normal levels as compared to I/R groups. Moreover, brain coronal sections and histopathological studies revealed protection against ischemic brain damage in the EOCM-treated groups. Conclusion: This study, for the first time, shows potent neuroprotective effect of EOCM against global cerebral I/R-induced oxidative stress in rats, suggesting its therapeutic potential in cerebrovascular diseases (CVD) including stroke. & 2012 Elsevier Ireland Ltd All rights reserved.
Keywords: Cerebrovascular diseases Cymbopogon martinii Ischemia/reperfusion Neuroprotection Oxidative stress
1. Introduction Cerebrovascular disease (CVD) is a group of brain dysfunctions related to disease of the blood vessels supplying the brain that includes some of the most common devastating disorders such as ischemic stroke, hemorrhagic stroke and cerebrovascular anomalies. It is reported that CVD represents major cause of disability causing two lacks deaths each year (Wade et al., 2005). World Health Organization (WHO) data reflects 5.7 million deaths from CVDs out of 58 million global deaths in 2005. It is postulated that CVDs will be the second most frequent cause of projected deaths in the year 2020 (Anonymous, 1997; Huang and McNamara, 2004). Ischemic stroke has been shown as substantial public health problem that leads to long-term disability in major industrialized countries. Indeed, it is the third leading cause of death after heart
n
Corresponding author. Tel.: þ919898002327; fax: þ 912812585083. E-mail addresses: [email protected] (P. Buch), [email protected] (V. Patel), [email protected] (V. Ranpariya), [email protected] (N. Sheth), [email protected] (S. Parmar). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.04.007
disease and cancer (Stapf and Mohr, 2002). A number of drugs with potential neuroprotective activity have been used in the treatment of stroke. However, there is no clinically effective therapy for the management of acute stroke except tissue-plasminogen activator (t-PA) (Alavijeh et al., 2005). Recently, intense interest has been focused on the neuroprotective properties of a series of natural products. In particular, some natural products act as neuroprotecting agents by enhancing the survival of neurons while preventing their death and apoptosis (Zhu et al., 2004). It is well-established that excitotoxicity, oxidative stress, inflammation and apoptosis are the major pathobiological mechanisms of ischemia/reperfusion (I/R) injury (Ozbal et al., 2008; Yousuf et al., 2009). These changes are associated with mitochondrial dysfunction and rapid decrease in ATP, resulting in free radical generation and lipid peroxidation. It can be recalled that oxidative stress plays a central role in cerebral I/R injury (Gilgun-Sherki et al., 2002; Janardhan and Qureshi, 2004). Reactive oxygen species (ROS) have been found to be over produced during I/R of neural tissues (Gilgun-Sherki et al., 2002; Li and Jackson, 2002; Sugawara and Chan, 2003). The lethal process is accompanied by elevated free radicals, including superoxide
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anion (O2 ), hydroxyl radical (OH) and hydrogen peroxide (H2O2) as well as progressive depletion in endogenous antioxidant system including antioxidant enzymes such as superoxide dismutase and catalase or antioxidants glutathione, vitamin C and E (Sugawara and Chan, 2003). Cymbopogon martinii (CM), commonly known as Palmarosa in English and Rohisha in Sanskrit, is an essential oil yielding grass from the family Graminae. Traditionally, the essential oil obtained from Cymbopogon martinii (EOCM) is widely used for neuralgia, bronchitis, anorexia, cardiac debility, skin diseases, epileptic fits and fever (Kirtikar and Basu, 1999). The main chemical constituents reported from the plant are geraniol, geranyl acetate, linalool, geranyl octanoate, geranyl butyrate, monoterpenoid and cymbodiacetal (Anonymous, 2000). In the light of this background, the purpose of the present study was to evaluate the potent neuroprotective effect of EOCM against global cerebral I/R-induced oxidative stress in Wistar albino rats.
2. Materials and methods 2.1. Drugs and chemicals Trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 5-5dithiobis (2-nitrobenzoic acid), glutathione and ( 7)-epinephrine were purchased from Sigma-Aldrich Co., Spruce Street, St. Louis, MO, USA. 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Hi-Media, Mumbai. All other chemicals were of the highest purity commercially available. 2.2. Plant material and essential oil (EO) extraction Leaves of CM were collected from the botanical garden of Saurashtra University, Rajkot, Gujarat, India during the month of August. Herbarium was prepared and the specimen was authenticated by Dr. A.S. Reddy, Department of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India. A voucher specimen (SU/DPS/Herb/43) was deposited in the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India for future reference. The EO was obtained by hydro-distillation in Clevenger apparatus using 100 g of dried leaves. The oil obtained was dried over anhydrous sodium sulfate, producing yields of 0.625% (v/w). The oil was subjected to GC–MS analysis (Shimadzu, Switzerland, model QP2010) according to the following experimental conditions: silica capillary column: SGE BP X 5 (30 m 0.25 mm 0.25 mm), injector temperature: 230 1C, detector temperature: 250 1C, temperature program: the column was initially 50 1C, then raised to 200 1C at a rate of 4 1C/min and finally held at 200 1C for 10 min, electron impact: 70 eV, carrier gas: helium at 1 ml/min, scanning speed 0.84 scan s 1 from m/z 50 to 1000 Da. The identification of the separated volatile organic compounds was achieved through retention time and mass spectrometry by the GC–MS library. 2.3. Determination of LD50 The acute toxicity study was performed as per the method described by Litchfield and Wilcoxon (1949) and LD50 was calculated accordingly. Briefly, the EOCM suspended in 1% tween 80 in saline at a dose of 125, 250, 500 and 1000 mg/kg was administered orally to different groups of mice (n ¼10). The animals were examined every 30 min up to a period of 3 h and then, occasionally, for further 4 h; finally, after 24 h mortality was recorded. All tests on rats were performed at two dose levels; 50 and 100 mg/kg of body weight corresponding to 10 and 20% of LD50 value (500 mg/kg) respectively.
2.4. Animals The Wistar-albino rats of either sex (150–200 g) were used for study. The animals were housed in a five rats per cage under well controlled conditions of temperature (2571 1C), humidity (5575%) and 12 h/12 h light-dark cycle (light on 07.30– 19.30 h). Animals had access to standard pellet diet (Pranav Agro Industries Ltd., India) and water was given ad libitum. The protocol of the experiment was approved by the Institutional Animal Ethical Committee (Protocol approval no: SU/DPS/IAEC/ 2011/05) as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forest, Animal Welfare Division, Government of India, New Delhi, India. For the present study, animals were randomized into 6 groups of 8 animals each and allowed to acclimatize for 1 week before the experiments. 2.5. Experimental protocol for global cerebral ischemia/reperfusioninduced oxidative stress The Wistar-albino rats of either sex (150–200 g) were divided into 6 groups of eight rats each and fed with drug or vehicle for 10 days prior to the experiment and treated as follows (Shah et al., 2005): Group-I: 1% tween 80 in saline (10 ml/kg, orally), no ischemia. Group-II: 1% tween 80 in saline (10 ml/kg, orally), bilateral common carotid artery (BCCA) occlusion for 30 min. Group-III: 1% tween 80 in saline (10 ml/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-IV: Quercetin (25 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-V: EOCM, suspended in 1% tween 80 in saline (50 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min. Group-VI: EOCM, suspended in 1% tween 80 in saline (100 mg/kg, orally), BCCA occlusion for 30 min, followed by reperfusion for 60 min.
2.6. Induction of ischemia Animals of groups II–VI were subjected to BCCA occlusion under ketamine anesthesia (45 mg/kg, i.p.) (Farbiszewski et al., 1995). Animals were placed on the back; both carotid arteries were exposed and occluded by atraumatic clamps. Temperature was maintained around 37 70.5 1C throughout the surgical procedure and artificial ventilation (95% O2 and 5% CO2) provided with artificial respirator. 2.7. Preparation of post-mitochondrial supernatant Following decapitation, the brain was removed and washed in cooled 0.9% saline, kept on ice and subsequently blotted on filter paper, then weighed and homogenized as 10% (w/v) in cold phosphate buffer (0.05 M, pH 7.4). Homogenization procedure was performed as quickly as possible under completely standardized conditions. The homogenates were centrifuged at 10,000g for 10 min at 4 1C (MPW-350R, Korea) and post-mitochondrial supernatant (PMS) was kept on ice until assayed. 2.8. Biochemical estimation 2.8.1. Lipid peroxidation (LPO) Thiobarbituric acid reactive substances (TBARS) in the homogenate were estimated by using standard protocol (Prabhakar
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et al., 2006). Briefly, the 0.5 ml of 10% homogenate was incubated with 15% TCA, 0.375% TBA and 5 N HCl at 95 1C for 15 min, the mixture was cooled, centrifuged and absorbance of the supernatant measured at 512 nm against appropriate blank. The amount of LPO was determined by using e¼1.56 105 M 1 cm 1 and expressed as TBARS nM/mg of protein (Braughler et al., 1987). 2.8.2. Superoxide dismutase (SOD) SOD activity was determined based on the ability of SOD to inhibit the auto-oxidation of epinephrine to adrenochrome at alkaline pH (Misra and Fridovich, 1972). Briefly, 25 ml of the supernatant obtained from the centrifuged brain homogenate was added to a mixture of 0.1 mM epinephrine in carbonate buffer (pH 10.2) in a total volume of 1 ml and the formation of adrenochrome was measured at 295 nm. The SOD activity (U/mg of protein) was calculated by using the standard plot. 2.8.3. Catalase (CAT) CAT activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml homogenate (10%, w/v) in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of nM H2O2 consumed/min/mg protein. 2.8.4. Total thiols This assay is based on the principle of formation of relatively stable yellow color by sulfhydryl groups with DTNB (Moron et al., 1979). Briefly, 0.2 ml of brain homogenate was mixed with phosphate buffer (pH 8), 40 ml of 10 mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks. The total thiol content was calculated by using e¼13.6 1031 cm 1 M 1 (Sedlak and Lindsay, 1968). 2.8.5. Glutathione (GSH) GSH was estimated in various tissues by the method of Sedlak and Lindsay (1968). Briefly, 5% tissue homogenate was prepared in 20 mM EDTA, pH 4.7 and 100 ml of the homogenate or pure GSH was added to 0.2 M tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 ml of Ellman’s reagent (10 mM/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm. Samples were centrifuged before the absorbance of the supernatants was measured (Khynriam and Prasad, 2003).
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cerebral infarction area was observed and compared between various treatment groups and negative control group. 2.11. Histopathology The brains from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 mm thickness. The sections were stained with haemotoxylin and eosin dye for histopathological observation. 2.12. Statistical analysis All the data are presented as mean 7S.E.M. The significance of difference in means between control and treated animals for different parameters was determined by using One-way Analysis of Variance (ANOVA) followed by multiple comparisons Dunnett’s test. A value of po0.05 was considered statistically significant.
3. Results 3.1. LD50 value Acute toxicity study showed that LD50 of the EOCM in mice was 500 mg/kg by oral route. 3.2. GC–MS analysis GC–MS analysis confirmed the presence of geraniol (77.51%), geranyl acetate (7.97%), b-linalool (6.27%) and b-caryophyllene (1.68%) as the main compounds in EOCM (Table A1). 3.3. Biochemical estimation The results showed in Table A2 revealed neuroprotective activity of EOCM. The animals from BCCA-occluded ischemic group (NS þI) exhibited significant increase in LPO levels, whereas significant decrease in all the other enzymatic and non-enzymatic parameters (SOD, CAT, GSH and total thiols) was observed. These levels were further augmented in animals of I/R (NS þI/R) group. The animals from EOCM-treated groups had shown a significant protection by reducing the elevated levels of LPO (po0.001) and marked increase in SOD (po0.001), CAT (po0.01), GSH (p o0.001) and total thiol (po0.001) levels as compared to I/Rtreated group (NS þI/R group). 3.4. Cerebral infarction area
2.9. Total protein The total protein contents of 10% brain homogenates were determined by using the modified method of Lowry et al. (1951).
The cerebral infarction area revealed significant decrease in EOCM-treated groups as compared to negative control group especially in caudal and rostral side (Fig. B1).
2.10. Measurement of infarction area
3.5. Histopathology
The infarction area was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining method according to Bederson et al. (1986). Following ischemia or reperfusion after varied durations of ischemia, animals were decapitated and the brains were removed. After the brains were placed briefly in cold saline, four coronal brain slices (2 mm thick) were made. Then the slices were incubated in phosphate buffered saline (pH 7.4) containing 2% TTC at 37 1C for 10 min and then kept in neutral-buffered formalin overnight (Isayama et al., 1991). The images of the TTC-stained sections were acquired by scanning with a high resolution scanner (Hewlett-Packard Scanjet 6100C/T). Then the
As shown in Fig. B2, ischemia evoked marked congestion of blood vessels and neutrophil infiltration (Plate B) in group II (NS þI). These effects were further augmented in group III (NS þI/ R), i.e. lymphocytic proliferation and neuronal necrosis (Plate C). Significant protection from the brain damage was observed in the EOCM- and quercetin-treated groups (Plates E–F and Plate D, respectively). The protection offered by EOCM was well-comparable to quercetin group. The EOCM reversed the I/R-provoked neuronal damage which was similar to the normal rat brain as seen in the tween 80 in saline-treated rats (group I, no ischemia) (Plate A).
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4. Discussion The present study revealed neuroprotective potential of EOCM at 50 mg/kg and 100 mg/kg. We estimated LPO, SOD, CAT, GSH and total thiol levels in the brain tissue as an index to assess the severity of oxidative damage. Traditionally, the dose up to 10 g is suggested to treat epilepsy and other CNS related conditions suggesting the plant is considerably safe in human (Anonymous, 2006). In the present study also, the selected dose levels 50 and 100 mg/kg were devoid of any sign of toxicity in animals. Oxygen is essential for aerobic life, but it is also precursor to the formation of harmful reactive oxygen species (ROS) (Albert and Yong-Mei, 1998). Free radicals in the living organism are generated both enzymatically and non-enzymatically, leading to the formation of ROS. It has been reported that hydroxyl and peroxynitrite are the most potent ROS that can damage proteins, lipids and nucleic acids, resulting in the inactivation of some enzyme activities, disruption of ion homeostasis and modification of genetic apparatus and apoptotic death (Albert and Yong-Mei, 1998). An interesting finding of the present study was amelioration of LPO by EOCM, an effect that could be attributed to its capacity to scavenge free radicals, as shown by the observed restoration of the antioxidant enzyme activities. We may recall that ROS produces malondialdehyde (MDA), an end product of LPO, a process that leads to dysfunction of membrane bound receptors and enzymes (Love, 1999; Fisher, 2001). Moreover, recent studies showed that global cerebral ischemia (Gupta and Sharma, 2006) and reperfusion (Irmak et al., 2003) significantly augmented MDA levels in animals. In consistent with this, we found that the ischemia and reperfusion resulted into augmentation of LPO process and thereby elevated the MDA levels in the brains of animals. There is strong indirect evidence that free radical production appears to be an important mechanism of brain injury after exposure to I/R (Traystman et al., 1991). To further characterize the neuroprotective actions of EOCM, we evaluated effect of EOCM on endogenous enzymatic and nonenzymatic antioxidant levels in the brain homogenates. It is well established that antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) play central role in the defense mechanisms against free radicalinduced oxidative stress (Islekel et al., 1999; Nita et al., 2001). Similarly, non-enzymatic antioxidants such as ascorbic acid (vitamin C), a-tocopherol (vitamin E), glutathione (GSH), and carotenoids have tonic inhibitory role in the oxidative damage induced by free radicals (Dringen, 2000; Aabdallah and Eid, 2004; Ege et al., 2004). In the present study, prior treatment of EOCM significantly increased GSH levels. It can be recalled that GSH acts directly by detoxifying ROS and indirectly as a substrate for various peroxidases thus, protecting the cells against oxidative damage (Dringen, 2000). Dysfunction of the GSH system has been implicated in a number of neurodegenerative diseases such as Alzheimer and Parkinson’s diseases (Schulz et al., 2000) and is a potential contributor to oxidative damage following temporary ischemia in rodents (Chen et al., 2000). In the current study, GSH levels were moderately reduced due to I/R insult which is in consistence with other studies (Islekel et al., 1999; Nita et al., 2001; Ege et al., 2004; Shah et al., 2005). Furthermore, administration of EOCM showed significant increase in SOD levels in brain. It has been shown that the SOD, a major antioxidant enzyme located in cytoplasm and mitochondria of cells, forms the principal defense system against excess O2 production by direct scavenging of O2 during reperfusion (Nita et al., 2001). We also evaluated effect of EOCM on the CAT levels in the rat brain. As compared to the control animals, EOCM-treated group showed significant increase in the CAT levels. The reports about the effect
of I/R on CAT activity are controversial in the literature. Some studies reported that I/R in rats lowered CAT activity (Aabdallah and Eid, 2004; Shah et al., 2005). On the other hand, Islekel et al. (1999) showed significant increase in the CAT activity after ischemia. However, in our experiments, the mean CAT activity was significantly decreased in I/R group. Furthermore, the histopathological studies revealed significant decrease in neutrophil infiltration and brain damage by the EOCM as compared to control group. Additionally, the cerebral infraction area was very less in EOCM-treated groups as compared to control groups indicating protection from I/R damage in the brain tissue. These results indicated neuroprotection by EOCM against BCCA occlusion-induced I/R brain damage. GC–MS study showed that EOCM is rich in geraniol (77.51%). The earlier study reported marked in vitro free-radical scavenging activity of geraniol against the DPPH model (Choi et al., 2000). Tiwari and Kakkar (2009) reported the antioxidant potential of geraniol using tsigniertiary-butyl hydroperoxide stressed rat alveolar macrophages and concluded that geraniol significantly increased the cell viability, SOD activity, GSH content and restored the mitochondrial membrane potential. Further, it was suggested that geraniol significantly decreased LPO, inhibited NO release and generated ROS in the pre-treated cells as compared to stressed cells. The protection offered by EOCM against cerebral ischemia could, at least in part, be attributed to the free-radical scavenging activity of geraniol. In conclusion, these findings suggest a potential protective role of EOCM in the global model of ischemia, a standard model for stroke in rats and gain importance in view of the fact that stroke is at present the third leading cause of death worldwide and CVDs constitute second most frequent cause of projected death in the years to follow. We suggest that the mechanism with which EOCM has normalized the damage is probably, by the antioxidant property of the geraniol, is a major constituent of EOCM. As evident by the results of the present study, it seems that EOCM has enhanced the defense mechanism, thereby reducing the damaged produced by global ischemia. Further studies are required to pursue the interesting leads emerging from the present results to exploit the full therapeutic potential of EOCM for neuroprotection.
Acknowledgments The authors are grateful to Dr. A.S. Reddy, Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, for identification of medicinal herb and Dr. P.S. Parsania, Department of Chemistry, Saurashtra University, Rajkot, for GC–MS analysis.
Table A1 Composition of essential oil. Rta time
Area
% area
Molecular formula
Compound name
8.558 9.442 10.292 12.525 12.758 14.033 14.933 16.333 17.167 18.400 18.675 20.858
145267 1188499 4660721 57656438 910842 5930217 1252613 205023 349126 776018 1091639 222139
0.20 1.60 6.27 77.51 1.22 7.97 1.68 0.28 0.47 1.04 1.47 0.30
C10H16 C10H16 C10H18O C10H18O C10H16O C12H20O2 C15H24 C12H20O2 C15H24O C15H26O C12H20O2 C12H20O2
b-pinene b-cis-Ocimene b-Linalool
a
Rt—Retention time.
Geraniol Citral Geranyl acetate b-Caryophyllene Nerol acetate Caryophyllene oxide Farnesol Neryl acetate Bay pine (oyster) oil
P. Buch et al. / Journal of Ethnopharmacology 142 (2012) 35–40
Appendix A
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Appendix B
See Tables A1 and A2 here.
See Figs. B1 and B2 here.
Table A2 Effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion-induced oxidative stress in rats. Group no.
Treatment
Lipid peroxidation (nM/mg of protein)
Superoxide dismutase (U/mg of protein)
Catalase (U/mg of protein)
Glutathione (nM/mg of protein)
Total thiols (lM/mg of protein)
I II III IV V VI
Normal NSþ Ischemia NSþ I/R Quercetin þI/R 50 mg/kg EOCM þI/R 100 mg/kg EOCMþ I/R
10.34 70.74 23.45 71.98nnn 51.73 71.68nnn 4.238 70.48b 8.625 70.54b 4.713 70.43b
151.9 74.99 43.28 70.54nnn 39.70 70.82nnn 187.8 73.58b 109.8 72.52b 132.2 75.02b
0.01886 7 0.0015 0.01222 7 0.0020n 0.008967 7 0.0017nn 0.02358 7 0.0010b 0.01445 7 0.0016a 0.01866 7 0.0008b
7.012 70.30 6.344 70.09n 5.964 70.13nn 8.120 70.12b 6.823 70.15b 7.475 70.14b
18.83 70.36 13.97 70.26nnn 10.61 70.25nnn 17.97 70.25b 16.45 70.30b 16.94 70.33b
Values are mean 7S.E.M., n¼ 8, One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test, *p o 0.05, **p o0.01 and ***p o 0.001 vs. normal and a p o0.05 and bp o 0.001 vs. NS þI/R. NS—Normal Saline, I—Ischemia, I/R—Ischemia/Reperfusion, EOCM—Essential oil of Cymbopogon martinii.
Fig. B1. Neuroprotective effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion damage in rats evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain coronal sections were prepared (2 mm thickness) and then each section was stained with TTC. A: normal; B: normal salineþ ischemia for 30 min; C: normal salineþ ischemia for 30 min followed by 60 min reperfusion (I/R); D: quercetin 25 mg/kgþ I/R; E and F: 50 mg/kgþI/R and 100 mg/kgþI/R of EOCM respectively. A large infarction area observed mainly in the caudal and rostral side of hippocampus in the damaged brain of I/R-treated rats (C) whereas the infarction was markedly reduced in the rat brains treated with 50 mg/kg (E) and 100 mg/kg (F) of EOCM and also in quercetin-treated animals (D). n¼3.
Fig. B2. Neuroprotective effect of essential oil of Cymbopogon martinii against global cerebral ischemia/reperfusion damage in rats. Photographs of brain sections from different treatment groups stained with Haemotoxylin & Eosin, 10 . Plates; A: normal; B: normalþ ischemia for 30 min; C: normal saline þischemia for 30 min followed by 60 min reperfusion (I/R); D: quercetin 25 mg/kgþ I/R; E and F: 50 mg/kgþ I/R and 100 mg/kgþ I/R of EOCM respectively. Ischemia (B) caused marked congestion of blood vessels and neutrophil infiltration. These effects were further augmented by reperfusion i.e. lymphocytic proliferation and neuronal necrosis (C). There is significant reversal of damage observed in EOCM-treated groups (E and F) and also in quercetin-treated group (D). The reversal was marked as the values were mostly comparable with saline-treated rats without ischemia (A).
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References Aabdallah, D.M., Eid, N.I., 2004. Possible neuroprotective effects of lecithin and alpha tocopherol alone or in combination against ischemia/reperfusion insult in rat brain. Journal of Biochemical and Molecular Toxicology 18, 273–278. Alavijeh, M.S., Chishty, M., Qaiser, M.Z., Palmer, A.M., 2005. Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug delivery. The Journal of the American Society for Experimental NeuroTherapeutics 2, 554–571. Albert, Y.S., Yong-Mei, C., 1998. Oxidative stress and neurodegenerative disorders. Journal of Biomedical Science 5, 401–414. Anonymous, 1997. From what will we die in 2020? Lancet 349, 1263–1264. Anonymous, 2000. The Wealth of India. Council of Scientific & Industrial Research, New Delhi (pp. 324–325). Anonymous, 2006. The Ayurvedic Pharmacopoeia of India, vol. 5. Department of Ayush, Government of India, New Delhi (pp. 187–189). Bederson, J.B., Pitts, L.H., Germano, S.M., Nishimura, M.C., Davis, R.L., Bartkowski, H.M., 1986. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17, 1304–1308. Braughler, J.M., Chase, R.L., Pregenzer, J.F., 1987. Oxidation of ferrous iron during peroxidation of various lipid substrates. Biochimica et Biophysica Acta 921, 457–464. Chen, Y.H., Du, G.H., Zhang, J.T., 2000. Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats. Acta Pharmacologica Sinica 21, 463–466. Choi, H.S., Song, H.S., Ukeda, H., Sawamura, M., 2000. Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2picrylhydrazyl. Journal of Agricultural and Food Chemistry 48, 4156–4161. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), Hand Book of Methods for Oxygen Radical Research. CRC Press, Boca Raton, Florida, pp. 283–284. Dringen, R., 2000. Metabolism and functions of glutathione in brain. Progress in Neurobiology 62, 649–671. Ege, E., Ilhan, A., Gurel, A., Akyol, O., Ozen, S., 2004. Erdosteine ameliorates neurological outcome and oxidative stress due to ischemia/reperfusion injury in rabbit spinal cord. European Journal of Vascular and Endovascular Surgery 28, 379–386. Farbiszewski, R., Bielawski, K., Bielawska, A., Sobaniec, W., 1995. Spermine protects in vivo antioxidant enzymes in transiently hypoperfused rat brain. Acta Neurobiologiae Experimentalis 55, 253–258. Fisher, M., 2001. Stroke Therapy, second ed. Butterworth-Heinemann, Boston (pp. 25–50). Gilgun-Sherki, Y., Rosenbaum, Z., Melamed, E., Offen, D., 2002. Antioxidant therapy in acute central nervous system injury: current state. Pharmacological Reviews 54, 271–284. Gupta, S., Sharma, S.S., 2006. Neuroprotective effects of trolox in global cerebral ischemia in gerbils. Biological and Pharmaceutical Bulletin 29, 957–961. Huang, Y., McNamara, J.O., 2004. Ischemic stroke: ‘‘acidotoxicity’’ is a perpetrator. Cell 118, 665–666. Irmak, M.K., Fadillioglu, E., Sogut, S., Erdogan, H., Gulec, M., Ozer, M., Yagmurca, M., Gozukara, M.E., 2003. Effects of caffeic acid phenethyl ester and alphatocopherol on reperfusion injury in rat brain. Cell Biochemistry and Function 21, 283–289. Isayama, K., Pitts, L.H., Nishimura, M.C., 1991. Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke 22, 1394–1398. Islekel, S., Islekel, H., Guner, G., Ozdamar, N., 1999. Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion. Research in Experimental Medicine 199, 167–176. Janardhan, V., Qureshi, A.I., 2004. Mechanisms of ischemic brain injury. Current Cardiology Reports 6, 117–123.
Khynriam, D., Prasad, S.B., 2003. Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin. Brazilian Journal of Medical and Biological Research 36, 53–63. Kirtikar, K.R., Basu, B.D., 1999. Indian Medicinal Plants, sixth ed. International Book Distributors, Dehradun, India (pp. 2677–2681). Li, C., Jackson, R.M., 2002. Reactive species mechanisms of cellular hypoxiareoxygenation injury. American Journal of Physiology. Cell Physiology 282, C227–C241. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. The Journal of Pharmacology and Experimental Therapeutics 96, 99–113. Love, S., 1999. Oxidative stress in brain ischemia. Brain Pathology 9, 119–131. Lowry, O.H., Rosebrough, N.J., Fair, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193, 265–275. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. The Journal of Biological Chemistry 2479, 3170–3175. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 582, 67–78. Nita, D.A., Nita, V., Spulber, S., Moldovan, M., Popa, D.P., Zagrean, A.M., Zagrean, L., 2001. Oxidative damage following cerebral ischemia depends on reperfusion—a biochemical study in rat. Journal of Cellular and Molecular Medicine 5, 163–170. Ozbal, S., Erbil, G., Kocdor, H., Tu˘gyan, K., Pekcetin, C., Ozo˘gul, C., 2008. The effects of selenium against cerebral ischemia-reperfusion injury in rats. Neuroscience Letters 438, 265–269. Prabhakar, K.R., Veerapur, V.P., Parihar, K.V., Priyadarsini, K.I., Rao, B.S., Unnikrishnan, M.K., 2006. Evaluation and optimization of radioprotective activity of Coronopus didymus Linn. in g-irradiated mice. International Journal of Radiation Biology 82, 525–536. Schulz, J.B., Lindenau, J., Seyfried, J., Dichgans, J., 2000. Glutathione, oxidative stress and neurodegeneration. European Journal of Biochemistry 267, 4904–4911. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25, 192–205. Shah, Z.A., Gilani, R.A., Sharma, P., Vohora, S.B., 2005. Cerebroprotective effect of Korean ginseng tea against global and focal models of ischemia in rats. Journal of Ethnopharmacology 101, 299–307. Stapf, C., Mohr, J.P., 2002. Ischemic stroke therapy. Annual Review of Medicine 53, 453–475. Sugawara, T., Chan, P.H., 2003. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia. Antioxidants and Redox Signaling 5, 597–607. Tiwari, M., Kakkar, P., 2009. Plant derived antioxidants-geraniol and camphene protect rat alveolar macrophages against t-BHP induced oxidative stress. Toxicology In Vitro 23, 295–301. Traystman, R.J., Kirsch, J.R., Koehler, R.C., 1991. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. Journal of Applied Physiology 71, 1185–1195. Wade, S.S., Johnson, S.C., Easton, J.D., 2005. Cerebrovascular Disease, Principles of Internal Medicine. McGraw-Hill, Noida, Uttar Pradesh, India (pp. 2372). Yousuf, S., Atif, F., Ahmad, M., Hoda, N., Ishrat, T., Khan, B., Islam, F., 2009. Resveratrol exerts its neuroprotective effect by modulating mitochondrial dysfunctions and associated cell death during cerebral ischemia. Brain Research 1250, 242–253. Zhu, Y.Z., Huang, S.H., Tan, B.K., Sun, J., Whiteman, M., Zhu, Y.C., 2004. Antioxidants in Chinese herbal medicines: a biochemical perspective. Natural Product Reports 21, 478–489.