Cerebroprotective effect of Ocimum gratissimum against focal ischemia and reperfusion-induced cerebral injury

Abstract

Context: Oxidative stress is believed to increase delayed neuronal death in the brain following ischemia. As a consequence, many attempts to reduce the damage resulting from cerebral ischemia under more highly oxidized conditions have focused on treatments aimed at maintaining the redox equilibrium of the local environment. Many antioxidants were shown to be neuroprotective in experimental models of cerebral ischemia and reperfusion.

Objective: The present study was designed to investigate the potential protective effects of ethanol extract of Ocimum gratissimum Linn. (Lamiaceae) (EEOg) against focal ischemia and reperfusion (I/R) insult in rat brain.

Materials and methods: The animal model of focal I/R was established by occluding the middle cerebral artery (MCA) of male Wistar rats for 2 h, followed by 24 h reperfusion. The thiobarbituric acid reactive substances concentration, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were determined by colorimetric assays. The characterization and quantitative analysis of phenolic content was determined using HPLC.

Results: MCA occlusion led to significant rise in cerebral infarct volume and lipid peroxidation, and depletion in SOD and GPx in brain. The neurological deficits were also significantly elevated by MCA occlusion. All the brain oxidative stress, damage and neurological deficits were significantly attenuated by pre-treatment with EEOg (150 or 300 mg/kg, p.o.).

Conclusion: The overall finding suggests the neuroprotective potential of O. gratissimum in cerebral ischemia, and is mediated through its antioxidant activity. Therefore, O. gratissimum should be investigated further as a possible strategy against cerebral stroke.

Keywords::

• Antioxidant
• middle cerebral artery occlusion
• neuroprotective
• Ocimum gratissimum
• phenolic contents

Introduction

Stroke is a major public health problem in the world. Stroke results in neurological deficits (E-Jian et al., 2009). It is well documented that oxidative stress is one of the primary factors that exacerbate damage by cerebral ischemia and reperfusion (Chan, 2001; Janardhan & Qureshi, 2004). Several components of reactive oxygen species (superoxide, hydroxyl radical, hydrogen peroxide, peroxynitrite radical, etc.) that are generated after ischemia and reperfusion injury play an important role in neuronal injury (Imaizumi et al., 1984; Oliver et al., 1990). Superoxide and hydroxyl radical cause destruction of the cell membrane by inducing lipid peroxidation (Bromont et al., 1989). Inducible nitric oxide synthase is up-regulated after ischemia and reperfusion injury. This results in excessive nitric oxide production. This excess nitric oxide reacts with superoxide to form peroxynitrite, a powerful radical that produces neuronal death after cerebral ischemia. The brain is particularly vulnerable to oxidative stress injury because of its high rate of oxidative metabolic activity, depletion of endogenous antioxidant systems, intense production of reactive oxygen species metabolites, high content of polyunsaturated fatty acids, relatively low antioxidant capacity, low repair mechanism activity and non-replicating nature of its neuronal cells (Evans, 1993; Gupta et al., 2003). Several natural and synthetic antioxidants have been shown to produce neuroprotective effect in ischemia and reperfusion-induced cerebral injury (Easton et al., 1998; Yousuf et al., 2009).

Ocimum gratissimum (Linn.) (Lamiaceae), an erect small shrub, is native from India to West Africa and has become naturalized in Europe and America (Offiah & Chikwendu, 1999). Though a lot of scientific work has been carried out on some members of this genus, sporadic information is available in the literature about the medicinal uses of O. gratissimum. Traditionally, the plant has been used as a stomachic, expectorant, anthelmintic, diaphoretic, laxative, febrifuge, for curing headache, angina and madness (Janssen et al., 1989; Ndounga & Ouamba, 1997), and in central nervous system diseases (Cristiana et al., 2006).

Phytochemically, O. gratissimum contains a high quantity of essential oil (3.2-4.1%), ocimol, gratissimin, β-sitosterol (List & Horhammer, 1977), flavonoids (Roberto et al., 2001), linolenic acid and polyphenolic compounds (Lukmanul et al., 2008).

Pharmacological reports revealed that O. gratissimum possesses antioxidant, anti-inflammatory (Lukmanul et al., 2008), antimicrobial, antifungal, antimutagenic, immunomodulating, smooth muscle contraction effects of lipid-soluble principles, hepatoprotective (Lin et al., 1995; Veronica & Unoma, 1999), antimycobacterial and antidiarrheal effects in experimental animals (Offiah & Chikwendu, 1999). The ethanol extract of leaves of O. gratissimum has been reported to exhibit significant hypoglycemic effect in normal and alloxan-induced diabetes in rats (Aguiyi et al., 2000).

Recently, it has been reported that O. gratissimum possesses antioxidant and anti-inflammatory properties, (Lukmanul et al., 2008) but so far no work has been carried out to evaluate neuroprotective effect of O. gratissimum on ischemia and reperfusion-induced cerebral injury. Therefore, the present study has been designed to investigate the effect of O. gratissimum on focal cerebral ischemia and reperfusion-induced cerebral injury in rats.

Materials and methods

Drugs and chemicals

Thiobarbituric acid (TBA), triphenyltetrazolium chloride (TTC), trichloroacetic acid (TCA), nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), xanthine oxidase, gallic acid (GA), glutathione and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma, St. Louis, MO. Chloral hydrate was obtained from Reidel-de Haen, Seelze, Germany. All chemicals were of the highest purity commercially available. All drug solutions were freshly prepared before use.

Plant materials and preparation of the extract

Aerial parts of the plant O. gratissimum were obtained in August 2009 from the medicinal garden, Panjab University, Chandigarh, India. Identity of the plant material was authenticated by S. Negi, Director, Forest Research Institute, Dehradoon, India. Voucher specimen of the plant was deposited in Punjabi University Herbarium. Dried plant material was pulverized using a mechanical grinder. Powdered material (100 g) was extracted with ethanol (95%) continuously for 15 h using a Soxhlet apparatus. Thereafter, the resulting ethanol extract was dried under reduced pressure using a Buchi 461 rotary vacuum evaporator (Postfach, Switzerland) and was preserved in a vacuum desiccator containing anhydrous silica gel blue. Phytochemical screening (Farnsworth, 1966; Evans, 2002) of the extract revealed the presence of polyphenolic compounds, terpenes, flavonoids and carbohydrates.

Standardization of extract

Determination of total phenolic content

Total phenol content was estimated as gallic acid equivalents (Singleton et al., 1999). Briefly, a 100 μL aliquot of dissolved extract was transferred to a 10 mL flask, containing 6 mL double distilled water, to which was subsequently added 500 μL undiluted Folin-Ciocalteu reagent. After 1 min, 1.5 mL Na₂CO₃ (20%, w/v) was added and the volume was made up to 10 mL with ultra-pure water. After 30 min incubation at 25°C, the absorbance was measured spectrophotometrically (Beckman DU 640B, Nyon, Switzerland) at 760 nm.

High-performance liquid chromatography - Phenolic compounds analysis

A high-performance liquid chromatography (HPLC) system comprising a vacuum degasser, quaternary pump, autosampler, thermostat column compartment, and photodiode array detector (Analytical Technologies, Vadodara, India) was used for the quantification of individual phenolic compounds. The column, Phenomenex C18 5 μm (250 × 4.6 mm), was maintained at 26°C. Different proportions of solvents such as acetonitrile/water/acetic acid (15:84:0.85) as eluant B and methanol as eluant A were used for the separation. The multiple gradient used for chromatographic separation consisted of different proportions of eluant A/B (50:50 for 5 min, 40:60 for 5–10 min, 30:70 for 10–15 min, 15:85 for 15–20 min). The flow rate was 1 mL/min, the sample injection volume was 50 μL and the chromatogram monitored at 330 nm. The peak purity of the tested sample was determined by comparing its ultraviolet spectra to that of the reference standards. Quantification was made on the basis of the corresponding peak area recorded by chromatopac c-R6A. Reference standards were used for the preparation of standard curves.

Animals

Male Wistar rats (250–300g) were maintained on standard environmental conditions and fed with standard rodent diet and tap water ad libitum. They were housed in the institutional animal house and were exposed to natural photoperiod. The experimental protocol was approved by Punjabi University Animal Ethics Committee and care of the animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India (Reg. no. 107/1999/CPCSEA).

Experimental protocol

A total of four groups of fourteen rats each were employed in the present study. Half the animals from each group were subjected to the elevated plus maze test, inclined beam test and rota-rod test before focal cerebral ischemia, and after 24 h reperfusion. The remaining half of the animals from each group were used to estimate infarct volume, lipid peroxidation, superoxide dismutase, and glutathione peroxidase activity.

The first group (n = 7) were sham operated (animals were subjected to surgical procedure, but did not suffer middle cerebral artery occlusion (MCAO), except for exposure of the right internal carotid artery (ICA) and right external carotid artery (ECA)). A second group (n = 7) served as control [the rats were orally administered vehicle (distilled water + 5% Tween 80, 10 mL/kg) 60 min before subjecting to 2 h focal ischemia by MCAO followed by 24 h reperfusion]. The third and fourth groups of rats (n = 7 each) received 100 mg kg⁻¹ and 200 mg kg⁻¹ doses of ethanol extract of O. gratissimum, respectively. The vehicle and drugs were administered orally, 60 min before subjecting them to focal cerebral ischemia. A pilot study using infarct volume as the end point was performed to select the doses.

Middle cerebral artery occlusion

The rats were subjected to 2 h right MCAO using the intraluminal filament technique (Longa et al., 1989). The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.). The right common carotid artery was exposed at the level of the ECA and ICA bifurcation. A 4-0 monofilament nylon suture was inserted into the ECA and advanced into the ICA for 17–20 mm until a slight resistance was felt, to block the origin of the middle cerebral artery. Two hours after MCAO, the suture was slowly withdrawn. The animals were then returned to their cages for 24 h.

Cerebral infarct volume assessment

After 24 h reperfusion, the animals were sacrificed by spinal dislocation and the brains removed. The brains were kept overnight at −4°C. Frozen brain was sliced into uniform sections of about 1 mm thickness. The slices were incubated in 1% triphenyl tetrazolium chloride (TTC) at 37°C in 0.2 M tris buffer (pH 7.4) for 20 min (Bochelen et al., 1999). TTC is converted to red formazone pigment by NAD and dehydrogenase present in living cells. Hence viable cells were stained deep red. The infarcted cells had lost the enzyme and thus remained unstained dull yellow. The area of infarction was measured in coronal brain sections using image analysis software Leica Qwin (Leica Microsystems, Cambridge, UK.). Infarct areas of all sections were cumulated to give the total infarct area which was multiplied by the thickness of the brain sections to get the volume of infarction.

Estimation of mitochondrial thiobarbituric acid reactive substances (TBARS)

The brain was weighed, minced and suspended in a buffer containing 30 mM Tris-HCl and 2.5 mM CaCl₂ (pH 7.6). The above mixture was homogenized and the homogenate was centrifuged at 750 rpm to separate cellular debris. The supernatant was accurately divided into two parts. Both portions were centrifuged at 8200 rpm to obtain the mitochondrial fraction. One was utilized for determination of TBARS (Yagi, 1982), and the other one was employed for protein estimation (Lowry et al., 1951).

Superoxide dismutase

Superoxide dismutase (SOD) activity was measured by the method explained by Beauchamp and Fridovich (1971). The reaction mixture of total volume 1 mL consisted of 0.6 mL phosphate buffer (0.5 M, pH 7.4), 0.1 mL PMS (10%, w/v), 0.1 mL of xanthine (1 mM), 0.1 mL of NBT (57 µM) was incubated for 15 min at room temperature and reaction was initiated by the addition of xanthine oxidase (50 mU). The rate of reaction was measured by recording change in the absorbance at 550 nm due to formation of formazan, a reduction product of NBT.

Glutathione peroxidase (GPx)

Glutathione peroxidase (GPx) activity was measured according to the procedure described by Mohandas et al. (1984). The reaction mixture consisted of 1.44 mL phosphate buffer (0.05 M, pH 7.0), 0.1 mL EDTA (1 mM), 0.1 mL sodium azide (1 mM), 0.05 mL of glutathione reductase (1 EU/mL), 0.1 mL of glutathione (1 mM), 0.1 mL NADPH (0.2 mM), 0.01 mL hydrogen peroxide (0.25 mM) and 0.1 mL PMS (10%, w/v) in a final volume of 2 mL. The disappearance of NADPH at 340 nm was recorded at room temperature. The enzyme activity was calculated as nM NADPH oxidized/min/mg protein.

Short-term memory evaluation

The elevated plus maze was employed to evaluate the short-term memory, which consisted of two enclosed (500 × 100 × 400 mm) and two open (500 × 100 mm) arms. It was elevated to a height of 500 mm above the floor (Kulkarni, 2007). All the animals were given a single trial on the plus maze. Individually, each rat was placed at the end of the open arm facing away from central platform of the maze. The time taken by the rat to enter from the open arm with all four legs within the enclosed arm was taken as transfer latency time (TLT). If the animal did not enter the enclosed arm within 90 s, it was gently pushed into the enclosed arm and a TLT of 90 s was assigned to it. The animal was allowed to explore the maze for an additional 10 s after the measurement of TLT (Itoh et al., 1990). With an aim to attain a low level of TLT the exposure to the elevated plus maze was repeated on days 2 and 3 (Zdenek & Ivan, 1998). After the third training trial the animals were subjected to focal cerebral ischemia for 90 min followed by reperfusion for 24 h. Then they were put on the elevated plus maze. TLT measured on the plus maze on the third training trial served as an index of learning or acquisition, whereas TLT recorded 24 h after reperfusion (day 4) served as an index of retrieval or memory.

Inclined beam walking test

To evaluate fore and hind limb motor coordination, the inclined beam walking test was employed (Feeney et al., 1981). The inclined beam walking test was performed before ischemia and 24 h after reperfusion.

Rota-rod test

The Dunham and Miya (1957) method was used to evaluate the rota-rod performance. It is used to evaluate motor coordination by testing the ability of rats to remain on a revolving rod. The apparatus consisted of a horizontal rough metal rod of 30 mm diameter attached to a motor with a variable speed. The rod was 700 mm in length and was divided into four sections by wooden partitions, thereby allowing the simultaneous testing of four rats. The rod was at a height of about 500 mm above the table top in order to discourage the animals from jumping off the roller. The rate of rotation of the rod was adjusted so that the normal rat was able to stay on the rotating rod for a period of 5 min. The difference in the fall-off time from the rotating rod between the control and treated animals is taken as an index of motor uncoordination. Each rat was given four or five trials before the actual reading was taken. The rats that were able to stay on the rotating rod for a period of 5 min before focal cerebral ischemia were selected, and the test was again performed 24 h after focal cerebral ischemia and reperfusion.

Statistical analyses

All the data are presented as mean ± SD. The data of infarct volume and biochemical parameters were statistically analyzed using one-way ANOVA followed by Tukey’s post hoc multiple range test for multiple comparisons among various groups. However, the data of percentage change in TLT was statistically analyzed using repeated measure ANOVA followed by Tukey’s post hoc multiple range test. The inclined beam walking test and Rota-rod test were statistically analyzed using one-way ANOVA followed by post hoc Kruskal-Wallis test and Bonferroni’s correction test respectively. A p value of <0.05 was considered statistically significant.

Results

Extract yield, total phenolic content and HPLC analysis

The yield of extract was 8.45% (w/w). The total phenolic content was estimated by the Folin-Ciocalteu reagent method. The total phenolic content was estimated to be 166.2 ± 4.1 mg GA equivalents/g dry extract of O. gratissimum from triplicate measurements.

In the HPLC analysis, phenolic compounds of O. gratissimum were quantified at 330 nm as phenolic acids, flavonoids and hydroxycinnamates. The components rosmarinic acid, p-coumaric acid, hydroxybenzoic acid, ferulic acid, cinnamic acid and lithospermic acid (Table 1) were identified by comparing their retention time with those of standards, and quantitatively determined by their respective peak areas.

Table 1.  Quantitative analyses of phenolic content of O. gratissimum ethanol extract (EEOg).

RA (% w/w)	pCA (% w/w)	HBA (% w/w)	FA (% w/w)	CA (% w/w)	LA (% w/w)
0.025 ± 0.01	0.019 ± 0.03	0.012 ± 0.04	0.015 ± 0.01	0.006 ± 0.03	0.010 ± 0.02

RA, rosmarinic acid; pCA, p-Coumaric acid; HBA, Hydroxy benzoic acid; FA, Ferulic acid; CA, Cinnamic acid; LA, Lithospermic acid.

Protective effect of O. gratissimum

Focal cerebral ischemia of 2 h followed by reperfusion for 24 h produced significant increase in cerebral infarct volume (37.13%) (Figure 1A). Administrations of ethanol extract of O. gratissimum markedly attenuated (about 51%, p < 0.05) ischemia and reperfusion-induced increase in cerebral infarct volume.

Figure 1.  A) Neuroprotective effect of ethanol extract of O. gratissimum (EEOg) on brain infarction in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Tukey’s post hoc multiple range test; *p <0.05 versus control; #p < 0.05 versus sham; ^ap <0.05 versus 150 mg/kg dose of EEOg. B) Effect of EEOg on impairment of short-term memory (plus maze test) in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Tukey’s post hoc multiple range test; *p <0.05 versus control; #p < 0.05 versus sham; ^ap <0.05 versus 150 mg/kg dose of EEOg.

Biochemical observations

TBARS concentration in brain mitochondrial and supernatant fractions was significantly increased in rat ischemic brain due to ischemia and reperfusion injury (Figure 2). Administrations of ethanol extract of O. gratissimum markedly decreased ischemia and reperfusion-induced increase in TBARS concentration in brain mitochondrial and supernatant fractions.

Figure 2.  Effect of ethanol extract of O. gratissimum (EEOg) on thiobarbituric acid reactive substances (TBARS) formation in brain of rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Tukey’s post hoc multiple range test; #p <0.05 versus sham; *p <0.05 versus control; ^ap <0.05 versus 150 mg/kg dose of EEOg.

SOD and GPx showed a significant decrease in the MCAO induced control group (Figures 3A and 3B). Pre-treatment with ethanol extract of O. gratissimum significantly reversed the alterations in biochemical parameters brought by ischemia and reperfusion. SOD and GPx were significantly elevated.

Figure 3.  A) Effect of ethanol extract of O. gratissimum (EEOg) on superoxide dismutase activity in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Tukey’s post hoc multiple range test; *p <0.05 versus control; #p <0.05 versus sham; ^ap <0.05 versus 150 mg/kg dose of EEOg. B) Effect of EEOg on glutathione peroxidase (GPx) activity in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Tukey’s post hoc multiple range test; *p <0.05 versus control; #p <0.05 versus sham; ^ap <0.05 versus 150 mg/kg dose of EEOg.

Behavioral observations

Focal cerebral ischemia and reperfusion produced a marked impairment of short-term memory by decreasing the TLT using the elevated plus maze. Pre-treatment with ethanol extract of O. gratissimum significantly prevented ischemia and reperfusion-induced increase in the percentage change in TLT (Figure 1B). In the same way, focal cerebral ischemia and reperfusion produced a marked impairment of motor performance. Administration of ethanol extract of O. gratissimum significantly prevented ischemia- and reperfusion-induced impairment of motor performance (Figure 4A and 4B).

Figure 4.  A) Effect of ethanol extract of O. gratissimum (EEOg) on impairment of motor performance (inclined beam test) in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± S.D.; n = 7; ANOVA followed by post hoc Kruskal-Wallis test; #p <0.05 versus sham; *p <0.05 versus control; ^ap <0.05 versus 150 mg/kg dose of EEOg. B) Effect of EEOg on impairment of motor performance (rota-rod test) in rats subjected to focal cerebral ischemia and 24 h reperfusion injury. The data are expressed as mean ± SD; n = 7; ANOVA followed by Bonferroni’s post hoc correction test; *p <0.05 versus control; #p <0.05 versus sham.

Discussion

Experimental models of stroke have been developed in animals in an attempt to mimic the events of human cerebral ischemia. The focal ischemic animal model involves the transient or permanent occlusion of the middle cerebral artery to be used as a model of cerebral ischemia (Crack et al., 2001). In the present study we used the temporary MCAO rat model to determine the actions of in vivo administration of O. gratissimum ethanol extract. The extract was standardized on the basis of total phenolic and flavonoid content. The results demonstrated that ethanol extract of O. gratissimum exhibited neuroprotection as was evident from the significant reduction in neuronal cell death (decreased infarct volume).

It is well documented that free radicals are involved in ischemia- and reperfusion-induced cerebral injury, among which, superoxide and hydroxyl radicals are potent inducers of lipid peroxidation (Bromont et al., 1989). Reactive oxygen species (ROS) produce malondialdehyde (MDA), an end product of lipid peroxidation. MDA reacts with TBA and is, thus, estimated as TBARS (Dib et al., 2002). Therefore, in the present study, MDA was estimated using TBARS assay to estimate extent of ROS. Administration of ethanol extract of O. gratissimum significantly decreased the extent of lipid peroxidation (decreased level of TBARS).

Mitochondria produce superoxide anion radicals and hydrogen peroxide under normal physiological conditions (Taku et al., 2004). These constantly produced ROS are scavenged by SOD and GPx. SOD specifically processes superoxide anion radicals and produces hydrogen peroxide, which is then detoxified by GPx, and finally changed to water and superoxide. In MCAO groups, SOD and GPx activity were significantly reduced. Pre-treatment with ethanol extract of O. gratissimum significantly prevented MCAO-induced decline in SOD and GPx activity.

It is well documented that hippocampal neurons are highly susceptible to ischemia- and reperfusion-induced injury (Jenkin et al., 1981). Therefore, in the present study, the elevated plus maze model has been employed to evaluate impairment of short-term memory as a result of MCAO. Moreover, neuronal ischemia is reported to impair sensory motor ability. The inclined beam and rota-rod test have been employed to investigate the effect of cerebral ischemia and reperfusion on motor performance. Oral administration of ethanol extract of O. gratissimum, before focal cerebral ischemia has prevented ischemia- and reperfusion-induced impairment of short-term memory and motor uncoordination.

Ocimum species have been extensively reported to contain essential oil; however, the antioxidant capacity of the plant extracts is mainly dependent on phenolic compounds. Phenolic compounds are an important group of secondary metabolites, which are synthesized by plants due to plant adaptation in response to biotic and abiotic stresses (infection, water stress, cold stress, high visible light) (Pitchersky & Gang, 2000). In the present study, we quantified our extract for total phenolic content. The phenolic components rosmarinic acid, p-coumaric acid, hydroxybenzoic acid, ferulic acid, lithospermic acid and cinnamic acid were identified and quantified at 330 nm. In our study, rosmarinic acid was the most abundant component identified in O. gratissimum extract (0.025% w/w) as compared to other compounds.

In conclusion, O. gratissimum exhibited neuroprotection by reducing brain infarction, restoring endogenous antioxidant system, and attenuating impairment in short-term memory and motor uncoordination. The protection may be due to the reduction of oxidative stress by phenolic compounds which are identified and quantified in the present study. These observations suggest that O. gratissimum may be a clinically viable protective agent against a variety of conditions where cellular damage is a consequence of oxidative stress. In addition, O. gratissimum may have the potential to be used in the prevention of neurodegenerative diseases such as cerebral stroke.

Declaration of interest

Authors are grateful to the L.L.R. Educational Trust, which runs the L.R. Institute of Pharmacy, Solan, Himachal Pradesh, India, for providing funds to carry out the present investigation. The authors alone are responsible for the content and writing of the paper.