Pedalium murex Linn leaves against LPS-induced oxidative stress, anxiety and depression behavioural alterations in rats
S. Gomathi1, R. Shanmuga Sundaram2, M. Vijayabaskaran1, C. Kannan3, R. Sambathkumar3
1Department of Pharmaceutical Chemistry, JKK Nattraja College of Pharmacy, Komarapalayam,
Namakkal (Dist), Tamil Nadu - 638183.
2Department of Pharmacology, JKK Nattraja College of Pharmacy, Komarapalayam,
Namakkal (Dist), Tamil Nadu - 638183.
3Department of Pharmaceutics, JKK Nattraja College of Pharmacy, Komarapalayam,
Namakkal (Dist), Tamil Nadu - 638183.
*Corresponding Author E-mail: gomathiswaminathan03@gmail.com
ABSTRACT:
Background: Studies of antioxidant substances in foods and natural products have gained increasing interest. The prevalence of neurodegenerative diseases are increasing globally. Effective treatment is necessary to minimize the neuronal damage and oxidative stress. Traditional medicines offer potent pharmacological activity with minimal side effects compared to synthetic drugs to treat such chronic disorders. Objective: The present study was aimed to evaluate the neuroprotective effect of ethanol extract of Pedalium murex Linn. (EEPM) leaves against lipopolysaccharide (LPS)-induced endotoxemia. Materials and Methods: Neurodegeneration was induced in rats with a single intraperitoneal injection of LPS (1 mg/kg). The neuroprotective activity was investigated following LPS-induced endotoxemia and then subjecting the animals to a battery of behavioural tests such as open field test (OFT), elevated plus-maze (EPM) and forced swim test (FST) to test anxiety and depression. At the end of the study, rats were sacrificed, brain hippocampal region was isolated and biochemical parameters were analysed. Results: These results highlight the neuroprotective potential of EEPM in LPS-induced anxiety and depressive illness model which may be partially due to inhibition of oxidative stress-neuroinflammatory cascade. Conclusion: The present study proved that EEPM has promising anti-anxiety, anti-depressant action and can be potential candidates for the management of LPS-induced neurodegeneration.
KEYWORDS: Anxiety, depression, lipopolysaccharide, oxidative stress, neuroprotection.
INTRODUCTION:
Pedalium murex Linn. (P. murex) is a small herb distributed in tropical Africa, Ceylon, India and Mexicol. A decoction of leaves is given in cases of gonorrhoea while that of roots is said to be antibilious2. The fruits are considered to be diuretic, antispasmodic and aphrodisiac3. In the earlier period several flavonoids have been isolated from the leaves and flowers of this plant, whereas the fruits and leaves are reported to yield a number of phenolic acids. Flavonoids of the leaves of P. murex such as pedalitin, diosmetin, dinatin, pedaliin, dinatin-glucuronide, diosmetin-7-glucuronide reported in the year 19724. In 1983 two new compounds were isolated from the fruits of P. murex characterized as heptatriacontan-4-one and tetratriacontanyl octacosanoate by spectral studies along with pentatriacontane, sitosterol, hexatriacontanoic acid, hentriacontanoic acid, ursolic acid and vanillin5. Another two new compounds were isolated and identified from the fruits of P. murex which were characterized as 2’,4’,5’-trihydroxy-5,7-dimethoxyflavone and tri acontanyldotriacontanoate with luteolin, rubusic acid, nonacosane, tritriacontane, triacontanoic acid, tritriacontanoic acid and sitosterol-b-D-glucoside in 19926. In Indian system of medicine, the plant P. murex is being used to treat many illnesses, like antiulcer, antimicrobial, antibacterial, antihepatotoxic, antioxidant7-9 and immune modulatory10, antidermatophytic, antipyretic, aphrodisiac, aldose reductase inhibitory11 antitussive, antihyperlipidemic12, insecticidal and antifeedant successfully for centuries. A numeral of studies has accredited the curative effect of P. murex due to its high content of flavonoids. But there is non-existence of studies related with protective effect of P. murex in neurodegenerative diseases in human and experimental animal models too.
Activation of immune system in response to stress and infection or bacterial endotoxin lipopolysaccharide (LPS) produces profound neurophysiological, neuroendocrine and behavioural changes. Due to the prevalence, morbidity and mortality of the neurodegenerative diseases, they characterized significant medical and monetarist encumbrance in the society. The etiology of neurodegenerative diseases remains enigmatic; however its defects in energy metabolism, excitotoxicity and oxidative damage is increasingly compelling, it is likely that there is a complex interplay between these mechanisms. Despite the great number of ongoing investigations, neurodegenerative diseases remain incurable13. There are, indeed, a multitude of paradigms assessing various aspects of the behavioral performance and anxiety and depression abilities. Till now, some of the paradigms will be not used at all in the evaluation of P. murex leaf extract against behavioural consequences of rats in LPS-induced endotoxemia. Hence, a special attention is focused to understand the treatment of LPS-induced neurodegenerative diseases by ethanol extract from this plant.
MATERIALS AND METHODS:
Plant collection and authentication
P. murex leaves were collected from Komarapalayam and Amaravathinagar, Namakkal District, Tamilnadu, India. The Plant was authenticated by Dr. G.V.S. Murthy, Scientist F, Botanical survey of India, Coimbatore, Tamilnadu (No.BSI/SRC/5/23/2012-13/Tech/1934) and the specimen was preserved in Pharmacognosy Lab, JKKN College of Pharmacy, Komarapalayam.
Preparation of crude extracts
Dried leaves were crushed into coarse powder and passed through pharmaceutical sieve no 40 and 80. The material which passed sieve-40 and retained at sieve-80 was collected and used for the extraction process. About 2 kg of shaded and dried plant leaves of P. murex. was extracted in soxhlet successively with n-hexane, chloroform, ethyl acetate and 90% v/v ethanol. Each extract was evaporated by rotary vacuum evaporator. The dried crude extract of each solvent was weighed and percentage yield was calculated.
Preliminary phytochemical studies
All the four crude extracts were screened for the presence of phytoconstituents. The extracts were tested for flavonoids, alkaloids, carbohydrates, saponins, glycosides, tannins, steroids, proteins and amino acids, terpenoids and fixed oils with all respective testing procedures as described in the textbook by Harborne JB.14
In vitro antioxidant and radical scavenging studies
All the four crude extracts were subjected to DPPH (Blois, 1958)15 and Nitric oxide (Green 1982) 16 radical scavenging activities. The therapeutically active ethanol extract of P. murex was selected on the basis of in vitro free radical scavenging activities and phytochemical analysis to carry out the pharmacological studies in animals.
Experimental animals
Sprague Dawley rats (100-150 g) were used for the experiments. Animals were obtained from KMCH College of Pharmaceutical Sciences, Coimbatore, Tamilnadu, India and maintained at standard housing conditions. A standard commercially available diet was provided with water ad libitum during the experiment. The animals were kept in clean and dry polycarbonate cages and maintained in a well-ventilated animal house with 12h light/dark cycle. The institutional animal ethical committee ((IAEC) has approved the experimental protocol prior to the animal experimentation (Approval no. KMCRET/Ph.D./ 08/2015-16, dated 22.08.2015) for the purpose of control and supervision of experiments on animal (CPCSEA).
Acute toxicity study
Acute toxicity study was performed according to OECD-guidelines 423. Three animals of same sex were used in each group. Ethanol extract of Pedalium murex Linn. leaves (EEPM) was administered to each group at 5, 50, 300 and 2000 mg/kg body weight respectively. The animals were fasted over-night before the administration of extract. Animals were observed regularly for 14 days for any signs and symptoms of toxicity.
Experimental design
Animals were randomly allotted into six groups with 6 animals in each group. EEPM was administered for a period of 30 days (p.o). Then neurodegeneration was induced with administration of intraperitoneal LPS (1 mg/kg) in normal saline on day 31. Two hours after the administration of LPS, animals were subjected to behavioural tests and finally sacrificed and the brain was extracted for biochemical analysis.
Group I: Normal group. Animals received 0.1 ml of normal saline orally for 30 days.
Group II: Disease control group. Single dose of LPS (1mg/kg)
Group III: Pre-treatment group. EEPM 100 mg/kg for 30 days + LPS (1mg/kg)
Group IV: Pre-treatment group. EEPM 200 mg/kg for 30 days + LPS (1mg/kg)
Group V: Pre-treatment group. EEPM 400 mg/kg for 30 days + LPS (1mg/kg)
Group VI: Standard pre-treatment group. Dexamethasone (0.5 mg/kg) for 30 days + LPS (1mg/kg)
Behavioural Test for Anxiety and Depression
The anxiolytic activity was examined by using open field test, elevated plus-maze test and forced swim test.
Open field test (OFT) 17
The rats were observed in a square open field arena (68×68×45 cm) equipped with 2 rows of 8 photocells, sensitive to infrared light, placed 40 and 125 mm above the floor respectively. The photocells were spaced 90 mm apart and the last photocell in a row was spaced 25 mm from the wall. Measurements were made in the dark in a ventilated, sound-attenuating box. Interruptions of photocell beams were collected by a microcomputer and the following variables were evaluated.
Elevated plus maze (EPM) 18
The apparatus comprised of two open arms (35x5 cm) and two closed arms (30x5x15 cm) that extended from a common central platform (5x5 cm). The floor and walls of the closed arms were made of wood and painted black. The entire maze was elevated to a height of 50 cm above the ground level. Rats were housed in a pair of 10 days prior to the test in the apparatus. During this time the rats were handled by the investigator on alternate days to reduce stress. 30 min and 60 min after oral administration of the drug treatment, each rats were placed at the centre of the maze facing one of the enclosed arm. During five min session, number of entries into open arm and closed arm was noted. The procedure was conducted preferably in a sound attenuated environment.
Forced swim test (FST) 19
Rats were individually forced to swim inside a vertical Plexiglas cylinder containing 15 cm of water maintained at 25°C. Rats placed in the cylinders for the first time were initially highly active vigorously swimming in circles, trying to climb the wall or diving to the bottom. After 2-3 min of activity they began to subside and to be interspersed with phases of immobility or floating of increasing length. After 5-6 min immobility reached a plateau where the rats remained immobile for approximately 80% of the time. After 15 min in the water the rats were removed and allowed to dry in a heated enclosure (32°C) before being returned to their home cages. They were again placed in the cylinder 24 hrs later and the total duration of immobility was measured during a 5 min test. Floating behaviour during this 5 min period had been found to be reproducible in different groups of rats.
Biochemical Analysis
At the end of study, animals were sacrificed and brain hippocampal region was carefully isolated, homogenized in a Potter–Elvehjem homogenizer with 0.1M phosphate buffer (pH 8) at temperature of 0◦C. The homogenate was then centrifuged at 10,000×g for 5 min at 4◦C and used for biochemical estimations like Lipid Peroxidation ( LPO)20, Superoxide Dismutase (SOD)21, Catalase (CAT)22 and Glutathione Peroxidase (GPx)23.
Statistical Analysis
The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by dunnet’s test. The results were expressed as mean ± SEM of six animals from each group. P values < 0.05 were considered significant.
RESULTS:
Preliminary phytochemical studies
Ethanol extract showed high extractive yield of 8.34 %w/w when compared to other extracts of P. murex respectively. Preliminary phytochemical screening revealed, n-hexane extract contains the compounds includes fixed oils and fats, steroids, gum and mucilage. Chloroform extract contains glycosides, tannins, phenolic compounds. Ethyl acetate extract contains alkaloids, carbohydrates, proteins, saponins. Ethanol extract contains flavonoids, glycosides, tannins, carbohydrates, phenolic compounds, triterpenoids and saponins.
In vitro antioxidant and radical scavenging studies
DPPH radical scavenging activity
The DPPH radical scavenging activity of P. murex leaf extracts were evaluated using ascorbic acid (positive control). DPPH reduction is directly proportional to the antioxidant content in the extract. Higher the antioxidant contents produced higher DPPH reduction. Among the solvents used in the study, ethanol extract of P. murex leaves exhibited a significant dose dependent DPPH radical scavenging activity. Percentage of scavenging activity or percentage inhibition was calculated by linear regression method. The IC50 values of ascorbic acid and ethanol extract was found to be <12.5 μg/ml and 21.91 μg/ml respectively. The DPPH radical is considered to be a model for a lipophilic radical chain reaction initiated by the lipid auto oxidation. The DPPH radical scavenging activity of various solvent extracts P. murex leaves are presented in figure 1.
Figure 1
Nitric oxide radical scavenging activity
Nitric oxide is a free radical produced from mammalian cells and it involved in various physiological processes. The excess production of nitric oxide is directly toxic to the cells and associated to septic shock, vascular collapse and arthritis. Our study results showed crude extracts of P. murex leaves scavenge the free nitric oxides in the medium and prevent the formation of nitrite. The IC50 values of ethanol extract was found to be 156.13 μg/ml; ethanol extract exhibited higher nitric oxide scavenging activity Nitric oxide radical scavenging activity of various solvent extracts P. murex leaves are presented in figure 2.
Figure 2
The ethanol extract of Pedalium murex Linn (EEPM) showed the dose dependent free radical scavenging activity in in vitro assay models and it was selected for the pharmacological screening based on the preliminary phytochemical studies and in vitro free radical scavenging activities.
Anti-Anxiety and Depression Studies
Open field test
When tested on an open field apparatus, LPS-treated group had significantly decreased in movement levels when compared to control animals. A significant improvement in movement activity was seen in LPS-treated animals administered with EEPM at different doses (100 mg/kg: 14.50±0.42; 200 mg/kg: 16.83±0.47; 400 mg/kg: 17.17±0.70). The movement activity of 400 mg/kg of EEPM showed highly significant activity which was comparable with control animals (19.50± 0.42). The results are shown in table 1.
Table 1
Groups |
Treatment |
No of movements in 5 mins |
I |
Control |
19.50± 0.42 |
II |
LPS (1mg/kg) |
07.80±0.30*** |
III |
EEPM 100mg/kg |
14.50±0.42* |
IV |
EEPM 200mg/kg |
16.83±0.47** |
V |
EEPM 400mg/kg |
17.17±0.70*** |
VI |
Dexamethasone(0.5mg/kg) |
17.50±0.99*** |
(Values are expressed as mean± SEM observations from six animals in each group) *P<0.05; **P<0.01; ***P<0.001
Elevated plus-maze test
An elevated plus-maze, LPS-treated group had significantly increased in anxiety levels (time spent in open arms [OP]: 7.33±1.25; time spent in closed arms [CL]: 234.2±6.10), when compared with control animals (OP: 42.83±2.72; CL: 234.2±6.10). A significant reduction in anxiety levels were observed in EEPM treated rats at different doses (100 mg/kg: OP: 22.83±2.00 and CL: 192.3±6.79; 200 mg/kg: OP: 32.17±2.79 and CL: 181.8±3.68; 400 mg/kg: OP: 40.17±2.08and CL: 115.2±4.01). The behavioural effect of 400mg/kg of EEPM showed excellent activity which was comparable with control animals and the results are shown in table 2.
Table 2
Groups |
Treatment |
Time spent in Arms ( Secs) |
|
Open Entry ( Day 30) |
Closed Entry ( Day 30) |
||
I |
Control |
42.83±2.72 |
172.2±5.00 |
II |
LPS (1mg/kg) |
07.33±1.25*** |
234.2±6.10*** |
III |
EEPM 100mg/kg |
22.83±2.00* |
192.3±6.79* |
IV |
EEPM 200mg/kg |
32.17±2.79** |
181.8±3.68** |
V |
EEPM 400mg/kg |
40.17±2.08*** |
142.2±4.01*** |
VI |
Dexamethasone (0.5mg/kg) |
30.17±0.70*** |
136.7±3.43*** |
(Values are expressed as mean± SEM observations from six animals in each group) *P<0.05; **P<0.01; ***P<0.001
Forced swim test
A significant increase immobility time was observed in LPS-treated rats (43.54±1.07), when compared with control animals (19.72±1.06) and decrease in immobility time was seen in EEPM-treated animals at different doses (100 mg/kg: 32.08±0.61; 200 mg/kg: 27.00±0.78; 400 mg/kg: 22.52±0.63) in forced swim test. The results are shown in table 3.
Table 3
Groups |
Treatment |
Immobility Time |
I |
Control |
19.72 ± 1.06 |
II |
LPS (1mg/kg) |
43.54 ± 1.07*** |
III |
EEPM 100mg/kg |
32.08 ± 0.61 |
IV |
EEPM 200mg/kg |
27.00 ± 0.78* |
V |
EEPM 400mg/kg |
22.52 ± 0.63** |
VI |
Dexamethasone (0.5mg/kg) |
19 .89 ± 0.92*** |
(Values are expressed as mean±SEM observations from six animals in each group) *P<0.05; **P<0.01; ***P<0.001
Biochemical Analysis
LPO levels were found to be elevated and the SOD, CAT, GPx levels were decreased in LPS-treated rats when compared to control rats, due to neuroinflammation and oxidative stress. The perturbations in the levels of LPO, SOD, CAT, GPx were found to be almost normalized following treatment with different doses of EEPM when compared with control group.
DISCUSSION:
The open-field test has been used widely to assess emotionality and locomotor performance.24, 25 OFT was used to exclude these false effects that could be associated with hyperkinesia. The main difference between antidepressants and psycho-stimulants is that antidepressants do not increase general motor activity.26 In our study, repeated administration of EEPM increase locomotor activity at doses that produced an antidepressant-like effect, indicating that the specific actions of this extract on the behavioural model are predictive of anti-depressant activity. In addition, the antidepressant effect of EEPM was influenced by changes in locomotor activity.
The elevated plus maze is considered to be an etiologically valid animal model of anxiety because it uses natural stimuli viz. fear of a novel open space and fear of balancing on a relatively narrow, raised platform that can induce anxiety in humans.27 The ratio of open/closed area entries and the time spent reflect a specific effect on anxiety. In the present study, oral administration of EEPM (100, 200 and 400 mg/kg) exhibited an anxiolytic-like effect in rats, since it increased the number of entries and the time spent on open arms and decreased the time spent in closed arms in the EPM test. In agreement with previously published reports, diazepam increased the percentage of time spent on open arms and the number of entries into the open arms.28
The present result confirmed that administration of EEPM (100, 200 and 400 mg/kg) had a specific anti-depressant-like effect in FST in rats by significantly reducing the immobility time as compared to LPS-treated group. Moreover, the anti-immobility effect produced by EEPM shared some pharmacological mechanisms with established anti-depressant drugs in this investigation and showed dose-dependent anti-depressant effect. Again, FST increase the cortisol level of mice by altering hyperactivity.29 EEPM showed anti-depressant effect which was probably due to reduction of the corticosterone concentration in rats exposed to FST. In fact, hyperkinesia also causes false positive effect in FST by shortening the immobility time in tests.
EEPM alleviates anxiety in open field test, elevated plus-maze and forced swim tests, the studied antioxidant pre-treatments were effective in reducing LPS-induced brain oxidative stress as evidenced by quenching of free radicals and elevation of the antioxidant GSH. The behavioural effects were significantly attenuated by EEPM in a dose dependent manner. All treatments elicited significant protection but EEPM 400 mg/kg produced the best results.
CONCLUSION:
In the field of neuroscientific research, neurodegenerative diseases is one of the most active in respect of both medical and associated social issues. Recent advances in the basic knowledge of such diseases have led to a re-evaluation of new therapeutic approaches from nature. In our findings, EEPM showed significant results in preliminary phytochemical studies and in vitro free radical scavenging activity prompted us to select the ethanol extract for pharmacological screening. In this study, results of behavioural tests for anxiety and depression, agitated levels of in vivo non- enzymatic and enzymatic anti-oxidant parameters indicated that the endotoxin, LPS, lead to anxiety, depressive like behaviour in rats, which were found to be reversed by EEPM when compared to control treated groups. These results indicated that EEPM may be a potential candidate for LPS-induced brain damage which may be attributed to the presence of potent antioxidants in EEPM. Such new findings may be included as strategies for more effective neuroprotection in addition to current therapies.
ACKNOWLEDGEMENT:
The authors acknowledge the Principal, KMCH College of Pharmacy, Coimbatore, India for all supports during the study and permission granted to carry out the pharmacological work at their premises.
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Received on 29.01.2017 Modified on 31.03.2017
Accepted on 04.04.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(5): 1333-1338.
DOI: 10.5958/0974-360X.2017.00236.0