Neuroprotective activity of Stereospermum suaveolens DC against 6-OHDA induced Parkinson's disease model.

OBJECTIVES: To evaluate the neuroprotective effect of Stereospermum suaveolens DC on 6-hydroxy dopamine induced Parkinson's disease model.
MATERIALS AND METHODS: The study was conducted on Sprague-Dawley rats where parkinson's disease was induced by producing the striatal 6-hydroxy dopamine lesions. The test animals received methanolic extract of Stereospermum suaveolens at dose of 125, 250 and 500 mg/kg for 42 days. Behavioral assessment, spontaneous locomotor activity and muscular coordination were studied. Antioxidant levels, striatal infraction area were assessed and histopathological studies were carried out.
RESULTS: The Stereospermum suaveolens DC methanolic extract showed significant dose dependent increase in behavioral activity, improved muscular coordination. Significant reduction of lipid peroxidation (LPO), increased antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and non-enzymatic activity of glutathione (GSH) and total thiol levels in extract treated groups was observed in test groups as compared to control group. Striatal infarction area was significantly reduced in extract treated groups as compared to control group.
CONCLUSION: The methanolic extract of Stereospermum suaveolens DC showed neuroprotective activity against 6-hydroxy dopamine induced Parkinson's disease in rats.

Introduction

Parkinson's disease (PD), the second most common neurodegenerative disorder worldwide is characterized by a marked loss of dopaminergic neurons of mainly the substantia nigra pars compacta (SNpc) leading to a reduction of dopamine (DA) in the striatum.[1] The dopaminergic deficit results in motor disabilities, such as rigidity, akinesia, tremor and postural abnormalities as well as cognitive and vegetative disturbances. The prevalence of PD is about 100 cases per 100,000 populations and the incidence is 20 cases per 100,000 people annually. An estimated 1 million Americans, or 1% of the population above the age of 65 years, have PD.[2] Numerous factors such as reactive oxygen species induced damage, excitotoxicity, mitochondrial dysfunction and inflammation-mediated cell injury have been implicated in the etiology of this disorder. Neuronal toxins such as 6-hydroxydopamine (6-OHDA)[3] 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) and rotenone[4] are commonly employed to induce parkinsonism in animals.

6-hydroxydopamine (6-OHDA) induced neurotoxicity is mediated by its ability to inhibit complex-I, leading to the mitochondrial adenosine triphosphate (ATP) depletion and generation of reactive oxygen species. 6-Hydroxydopamine also inhibits tyrosine hydroxylase, which is the rate-limiting enzyme for dopamine synthesis.[5] N-methyl-D-aspartate mediated toxicity and calcium accumulation are also involved in dopaminergic neurodegeneration following 6-hydroxydopamine.[6] 6-OHDA has been also reported to reduce the superoxide dismutase (SOD) activity and glutathione (GSH) content.[7] Various neurotoxic mechanisms are implicated in 6-OHDA induced neuronal impairment resulting in the development of a valid model for PD to test various drugs and formulations for their anti-parkinson activity.[8]

Stereospermum suaveolens DC (Bignoniaceae), commonly known as ‘patala’, is widely available in India. The plant contains lapachol, apigenin, dinatin, dianatin-7-glucuroniside1, β-sitosterol, saponins, palmitic, stearic and oleic acids.[9] It is widely used by traditional practitioners as an analgesic, anti-dyspeptic, astringent and liver stimulant and is known to have wound healing property. Flowers are used in semen debility. It is also used in brain disorders[10] and scientifically this plant is validated for hepatoprotective,[11] anti-hyperglycemic,[12] antioxidant, anti-inflammatory and anticancer activity.[10]

The aim of present study is to evaluate the neuroprotective effect of Stereospermum suaveolens DC. against 6-OHDA induced Parkinson's disease in Sprague-Dawley rats.

Materials and Methods

Drugs and Chemicals

6-hydroxydopamine (6-OHDA), Trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 5-5’-Dithiobis (2-nitrobenzoic acid), glutathione and (±)-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.

Plant Material

The fresh stem barks were collected in the month of January from Kolhapur district of Maharashtra. It was identified and authenticated by S. A. Kappali, Botanist, Department of Botany, Basaveshwar Science College, Bagalkot, Karnataka. The voucher specimen (BSc.Bot/15/08) of the plant was kept in Department of Botany. The barks were cleaned, air dried and then subjected to coarse powdering and passed through a sieve #44 to get uniform powder size. The collected powder was successively extracted with petroleum ether and then by methanol (60- 65°C) for 24 hours by using soxhlet apparatus. After the extraction, solvent was distilled, dried by lyophilization and stored in air tight container under refrigeration. The obtained methanolic extract of Stereospermum suveolens (MES) (yield of extract 0.34% per 50 g of powder) was used to assess neuroprotective activity. A preliminary phytochemical screening was done for the extract.

Acute Toxicity Study

The acute toxicity study was performed and LD50 was calculated as per the method described by Litchfield and Wilcoxon.[13] MES in the dose range of 10-2000 mg/kg was administered orally to a different group of mice (n=10). The animals were examined at every 30 minutes up to a period of 3 hours and then for an additional period of 4 hours. Overnight mortality was recorded.

Animals

Sprague-Dawley rats (200-250g), of either gender, were obtained from the central animal house of H. S. K. College of Pharmacy and Research Centre, Bagalkot. The animals were housed at room temperature (22-28°C) with 55 ± 5% relative humidity for 12 hours dark/light cycle and given standard laboratory feed (Amruth, Sangli, Maharashtra) and water ad libitum. The study was approved and conducted as per the norms of the Institutional Animal Ethics Committee (HSKCP/IAEC, Clear/20010-11/1-8).

Experimental Protocol

The animals were divided into five groups of 9 rats each and treated as follows:

Group 1: Sham received 3μl of 0.9% normal saline containing 0.2% ascorbic acid by stereotaxic injection into striatum and distilled water orally for 42 days.

Group 2: Control received 3μl of 6-OHDA (5 μg/μl) in 0.9% normal saline containing 0.2% ascorbic acid by stereotaxic injection into striatum and distilled water orally for 42 days.

Group 3: Received 3μl of 6-OHDA (5 μg/μl) in 0.9% normal saline containing 0.2% ascorbic acid by stereotaxic injection into striatum and MES (125 mg/kg) orally for 42 days.

Group 4: Received 3μl of 6-OHDA (5 μg/μl) in 0.9% normal saline containing 0.2% ascorbic acid by stereotaxic injection into striatum and MES (250 mg/kg) orally for 42 days.

Group 5: Received 3μl of 6-OHDA (5 μg/μl) in 0.9% normal saline containing 0.2% ascorbic acid by stereotaxic injection into striatum and MES (500 mg/kg) orally for 42 days.

Striatal 6-OHDA lesions were performed as previously described[14]. Rats were anaesthetized with ketamine (150 mg / kg, i.p.) and fixed to a stereotaxic frame (BS4 72-4240, Harvard apparatus, USA). A tooth bar was set at 3.3 mm below the interaural line, and the skull was exposed by making a 2 cm incision. A burr hole was made with a dental drill according to coordinates as follows 0.5 mm anterior and 2.5 mm lateral to the bregma. A 28-gauge cannula was inserted vertically into the striatum to a depth of 6 mm from the dura. At a rate of 1μL/min, 3μL of 6-OHDA hydrobromide (5μg total weight/1.0μL in normal saline containing 0.2% ascorbic acid) was injected.

Behavioral assessment

Deficits in forepaw adjusting steps in this PD rat model provided a simple and consistent behavior, phenomenologically similar to akinesia in PD. After the 6-OHDA lesion, forepaw adjusting step deficits were measured as previously described.[15] The rats were held by the rear part of the torso with one forepaw bearing the weight and placed on the surface of treadmill moving at a rate of 90 cm/12 seconds in the direction opposite to the weight-bearing forepaw resulting in the outward lateral shifting of the torso. The number of forepaw adjusting steps, defined as the movement of the weight bearing forepaw toward the torso to compensate for the outward lateral movement of the body, was counted. Each stepping test consisted of five trials for each forepaw, alternating between forepaws lasting for 12 seconds. The average of the five trials for each forepaw was used for analysis. Adjusting steps were tested in each group of animals at beginning of experiment and 6, 12, 18, 24, 30, 36 and 42nd day after the lesion.

Spontaneous locomotor activity (SLA)

Rats were individually placed in a digital photoactometer (Dolphin, Bombay, India), acclimatized for 5 minutes and their locomotor activity score were recorded for 10 minutes. Interruption in the photo beams positioned in parallel inside the chamber resulted in activity count. The chamber was swabbed with 10% ethanol every time to avoid interference due to animal odor.[16] SLA was tested in each group at beginning of experiment and 6, 12, 18, 24, 30, 36 and 42nd day after the lesion.

Muscular coordination

Muscular coordination was evaluated by rota rod apparatus (Medicraft Electromedicals, Lucknow, India). It consists of a rotating rod, 75 mm in diameter and divided into four parts by compartmentalization, permitting four rats to be tested at a time. The speed was set at 15 rpm and cutoff time was 180 s. The animals were trained so that they could remain on it at for at least the cut-off time.[17] Motor coordination was tested in each group of animals at beginning of experiment and 6, 12, 18, 24, 30, 36 and 42nd day after the lesion.

Locomotor activity

The locomotor activity was carried out by open field in a sound attenuated room. The floor was white polyvinyl with a black grid divide the open field into 100 square (10 × 10). Illumination was provided with 60w bulb placed above centre of field, while rest of the room was darkened. The rat was initially placed at centre of the field and observed for 5 minutes in all parameters i.e. latency (sec), ambulatory movements (number), rearing (sec), grooming (number), rest (sec), rotations (clockwise and anti-clockwise). Every time the activity chamber was swabbed with 10% alcohol to avoid the interference due to animal odors.[18] Locomotor activity was tested in each group of animals at beginning of experiment and 6, 12, 18, 24, 30, 36 and 42nd day after the lesion.

Biochemical estimation

After 42 days, the animals were sacrificed by cervical decapitation. The brain was removed and washed in cooled 0.9% saline, kept on ice, blotted on filter paper, then weighed and homogenized in cold phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 10000 rpm for 10 min at 4°C (MPW-350 R, Korea) and post-mitochondrial supernatant (PMS) was used for the estimation of total protein and lipid peroxidation. The supernatant was again centrifuged at 15000 rpm for 1 hour at 4°C. The supernatant obtained was used for further estimation of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and total thiols.

Thiobarbituric acid reactive substances (TBARS) in the homogenate were estimated by the method of Ohkawa et al.[19] 0.5 ml of 10% homogenate was incubated with 15% TCA, 0.375% TBA and 5 N HCl at 95°C for 15 minutes, the mixture was cooled, centrifuged and absorbance of the supernatant measured at 512 nm against appropriate blank. The amount of lipid peroxidation was determined by using ε = 1.56 × 105M-1cm-1 and expressed as TBARS nmoles/mg of protein.

Superoxide dismutase activity was determined based on the ability of SOD to inhibit the auto-oxidation of epinephrine to adrenochrome at alkaline pH.[20] 25μl of the supernatant obtained from the centrifuged brain homogenate was added to a mixture of 0.1mM epinephrine in carbonate buffer (pH 10.2) and the formation of adrenochrome was measured at 295 nm. The SOD activity (U/mg of protein) was calculated by using the standard plot.

Catalase activity was assayed by the method of Caliborne.[21] 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. Catalase activity was calculated in terms of nM H2O2 consumed/min/mg protein.

GSH was estimated in various tissues by the standard method.[22] 5% tissue homogenate were prepared in 20mM EDTA, pH 4.7 and 100μl of the homogenate or pure GSH was added to 0.2 M Tris-EDTA buffer (1.0 ml, pH 8.2) and 20mM EDTA, pH 4.7 (0.9 ml) followed by 20μl of Ellman's reagent (10mmol/l DTNB in methanol). After 30 minutes of incubation at room temperature, samples were centrifuged and absorbance at 412 nm was recorded.

Total thiols were assayed on the principle of formation of relatively stable yellow color by sulfhydryl groups with DTNB.[22] Briefly, 0.2 ml of brain homogenate was mixed with phosphate buffer (pH 8), 40 μl of 10mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 minutes and the absorbance was measured at 412 nm against appropriate blank. The total thiol content was calculated by using ε = 13.6 × 1031cm-1M-1. Protein content in samples was determined by the method of Lowry et al.[23]

Measurement of infarction area

The infarction area was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining method according to Bederson et al.[24] The animals were decapitated and the brain was removed. After brains were placed briefly in cold saline and four coronal brain slices (2 mm thick) were made. Then the slices were incubated in phosphate buffered saline (pH 7.4) containing 2% of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for 10 minutes and then kept in neutral-buffered formalin overnight. The images of the TTC-stained sections were acquired by scanning by a high resolution scanner (Hewlett Packard Scanjet 6100 C/T) and the cerebral infarction area was observed.

Histopathological studies

The brains from control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 μm thickness. The sections were stained with haemotoxylin and eosin dye for histopathological observation.

Statistical Analysis

All the data are expressed in mean ± SEM. The significance of difference in means between control and treated animals was determined by One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Significance of difference between sham and control group were evaluated by un-paired student's t-test. P<0.05 was considered statistically significant.

Results

Phytochemical analysis showed that methanol stem bark extract of Stereospermum suaveolens DC contain alkaloids, phenols, saponins, flavonoids and tannins.[17]

Neurobehavioral Studies

The results of this study revealed the potential neuroprotective activity of Stereospermum suaveolens DC. methanol extract. In behavioral assessment the number of contralateral forepaw adjusting steps decreased significantly (P<0.001) in control group and treatment with methanol extract of Stereospermum suaveolens significant (P<0.001) reduced the contralateral adjusting step deficits [Tables 1 and 2]. A significant decrease (P<0.001) in SLA was observed in control group as compared with sham group and in contrast there is a significant (P<0.001) increase in methanol extract of Stereospermum suaveolens treated groups (125, 250 and 500 mg/kg) as compared to control [Table 3]. The muscular coordination was significantly (P<0.001) reduced in control group as compared with sham group and in methanol extract of Stereospermum suaveolens treated groups (125, 250 and 500 mg/kg) it was increased significantly (P<0.001) and dose dependent improvement was observed [Table 4].

Table 1

Effect of methanol extract of Stereospermum suaveolenson behavioral assessment (ipsilateral forepaw adjusting steps) in 6-OHDA induced Parkinson's disease model

Table 2

Effect of methanol extract of Stereospermum suaveolens on behavioral assessment (contralateral forepaw adjusting steps) in 6-OHDA induced Parkinson's disease model

Table 3

Effect of methanol extract of Stereospermum suaveolens on spontaneous locomotor activity in 6-OHDA induced Parkinson's disease model

Table 4

Effect of methanol extract of Stereospermum suaveolens on muscular coordination in 6-OHDA induced Parkinson's disease model

In locomotor activity, a significant (P<0.001) increase in latency, clockwise rotation (ipsilateral) and rest was observed in control group as compared with sham group and a significant (P<0.001) recovery of latency [Table 5], clockwise rotation (results not shown) and rest [Table 6] period was observed in MES treated groups (125, 250 and 500 mg/kg). A significant (P<0.001) decrease in ambulatory movement, rearing and grooming was seen in control group as compared with sham group and MES treatment increased it significantly (P<0.001) [Table 7]. Results for rearing and grooming are not shown. There was no significant effect on anticlockwise rotation (contralateral) in control and even with other treatment groups.

Table 5

Effect of methanol extract of Stereospermum suaveolens on latency in 6-OHDA induced Parkinson's disease model

Table 6

Effect of methanol extract of Stereospermum suaveolens on rest in 6-OHDA induced Parkinson's disease model

Table 7

Effect of methanol extract of Stereospermum suaveolens on ambulatory movements in 6-OHDA induced Parkinson's disease model

Biochemical Estimation

The enzymatic and non enzymatic antioxidant estimations were done (results are not shown). The brain homogenate showed significantly reduced activities of SOD, CAT, GSH and total thiols and increase in LPO in control group as compared to sham group. The methanol extract of Stereospermum suaveolens showed a significant protection by reducing the elevated levels of LPO (P<0.001) and increasing the SOD (P<0.01), CAT (P<0.01), GSH (P<0.001) and total thiols (P<0.001) levels as compared to the control group.

Measurement of Infarction Area

A significant decrease in infarction area was observed in MES treated groups as compared with control groups especially in basal ganglia reason of brain [Figure 1] This decrease in infarction was comparable with sham group.

Figure 1

Effect of methanol extract of Stereospermum suaveolens in 6-OHDA induced Parkinson's disease can be evaluated by 2,3,5- triphenyltetrazolium chloride (TTC) staining. A: Sham group, B: Control, C: MES 125 mg/kg, D: MES 250 mg/kg, E: MES 500 mg/kg. In plate B a large infarction area was observed in basal ganglia reason, whereas the infarction area was markedly reduced in rats brains treated with MES

Histopathological Studies

The histopathological study confirmed the neuroprotective activity of MES as a significant recovery of neuronal damage and decreased necrosis was evident [Figure 2].

Figure 2

Photography shows, plates; A1 and A2: Sham group, B1 and B2: Control group, C1 and C2: MES 125 mg/kg, D1 and D2: MES 250 mg/kg, E1 and E2: MES 500 mg/kg; In B1 and B2 there was marked infiltration of neutrophils, intracellular space increased and more vacuoles seen, density of cells decreased, architecture completely altered, also hemorrhage and neuronal cell death was observed in striatum. There is significant reversal of damage observed in MES treated groups (C1-E2), reversal observed in treated groups

Discussion

Oxidative stress to dopamergic neurons of SNpc is believed to be one of the leading causes of neurodegeneration in PD. Antioxidants may play an important role in the prevention of PD and combat against oxidative stress induced progressive neurodegeneration by reactive oxygen species. However, medicinal plants like Gingko biloba,[25] Camellia sinensis[15] and Withania somnifera[16] have shown neuroprotective activity in 6-OHDA induced PD due to their antioxidant property. Extract of Stereospermum suaveolens has proven free radical scavenging activity in in-vitro evaluation[12] and the methanolic extract has been found to contain alkaloids, phenols, saponins, flavonoids and tannins,[11] responsible for its antioxidant property.

In present study we employed hemi-parkinsonian rat model by unilateral lesioning of striatum with 6-OHDA. Partial dopamine depletion by local injection of 6-OHDA in the striatum has been shown to be a good model for examining the effects of neuroprotective therapies[14] in the early and moderate stages of PD. Behavioral tests in PD models can be used to characterize the extent of lesion and/or to detect therapeutic effects. Forepaw adjusting step deficits after 6-OHDA lesion in rats was suggested as a model for akinesia of Parkinson's disease.[26] In our study we found reduction in akinesia in animals treated with methanolic extract of Stereospermum suaveolens, where significant decrease in forepaw adjusting step deficit was observed as compared to control animals. Spontaneous locomotor activity, as tested using a photoactometer, showed significant improvement in treated groups as compared to control group animals. The muscular coordination tested using a rota-rod, is an established method used for the assessment of neurological deficits in rodents.[27] Significantly enhanced muscular coordination was seen in methanol extract of Stereospermum suaveolens treated groups, as compared to control group. Hypokinesia, a main symptom of PD was studied by open field test by monitoring the locomotor activities of animals. Significant improvement of locomotor activity was observed by increased ambulatory movements, rearing, grooming and decreased latency period, rest, ipsilateral rotations in methanol extract of Stereospermum suaveolens treated animals. These behavioral parameters reveal an enhanced motor function, which is usually disturbed in PD.

6-OHDA has been reported to cause its dopaminergic toxicity by enhancing lipid peroxidation and oxidant stress[28] and many reports have implicated antioxidant demonstrating protection against 6-OHDA toxicity. In the present study, a marked increase in LPO (TBARS) and depletion of GSH, SOD, CAT and total thiols in 6-OHDA lesioned group (control) was observed, which is similar as per the earlier reports.[29] The methanolic extract of Stereospermum suaveolens significantly reversed these toxic effects of 6-OHDA, revealing the antioxidant activity of the extract, which is supported by previous reports of in-vitro antioxidant activity.[12] Oxidative stress induced injury is believed to result from an imbalance between the production of reactive cellular oxidants and the ability of a cell's endogenous antioxidant defenses to control them. Cellular oxidants are generated normally throughout the life of a cell by standard metabolic processes with a major source of reactive oxygen species (ROS). Additional oxidants can be generated by non-metabolic and aberrant processes within the cell, through exposure to cytotoxic chemicals, ionizing radiation or certain drugs.[30] Once generated, these reactive oxidants can inflict damage on proteins, DNA and lipids because of their ability to oxidize these important cellular components and alter them both structurally and functionally, unless neutralized. Indeed, markers of oxidative stress like lipid peroxidation and protein nitration are found in postmortem examination of brain tissues from patients with neurodegenerative disorders. Under normal circumstances, an array of endogenous cellular defense system exist to counterbalance ROS,[31] which include enzymatic and non-enzymatic antioxidants such as thiols, glutathione and its related enzyme system. Catalase removes most H2O2 in the brain and GSH is the primary low molecular weight thiol in the cytoplasm and a major reserve for cystein. GSH in conjunction with the reductant NADPH can reduce lipid peroxides, free radicals and H2O2. Similarly, in our study the antioxidant enzymes SOD, catalase, non enzymatic GSH and total thiol levels were elevated as compared to control group which indicates the potential antioxidant property of the extract.

Histopathological findings showed that methanol extract of Stereospermum suaveolens DC. treated animals had decreased infiltration of neutrophils, reduced intracellular space, and increased density of cells, regained normal architecture and moderate necrosis in striatum region of brain. The decreased infarction area at striatum and basal ganglia in extract treated groups, as compared to control group, further substantiates the neuroprotective activity against 6-OHDA induced Parkinson's disease model.

In conclusion, the present study suggests a potential role of methanolic extract of Stereospermum suaveolens DC. against 6-OHDA induced Parkinson's disease model. Further studies are required for understand the basic mechanism and characterization of active constituents responsible for neuroprotective effect.