Evaluation of the effect of volatile oil extract of Nigella sativa seeds on maximal electroshock-induced seizures in albino rats.
An epileptic seizure has been defined as a paroxysmal discharge of cerebral neurons accompanied by clinical phenomena apparent to the patient or an observer. The phenomena can be motor, sensory, or autonomic, and there may also be impairment or complete loss of consciousness. Motor disturbances may include convulsions - Which are involuntary, violent, and spasmodic or prolonged contraction of skeletal muscles. 
Epilepsy is one of the most common serious neurological disorders, responsible for substantial morbidity and mortality due to the seizures and the available medications. The prevalence of epilepsy is around 0.5-1%, and its overall annual incidence ranges from 50 to 70 cases/100,000 in industrialized countries and up to 190/100,000 in developing countries. Around 80% of people with epilepsy reside in developing countries. The high incidence in developing countries is attributed to poor obstetric services and the greater risk of intracranial infections and head injuries. Furthermore, in these countries, 80-90% of epileptic patients have difficulties in accessing treatment. This treatment gap has been mainly ascribed to inefficient and unevenly distributed health-care systems, cost of treatment, cultural beliefs, and unavailability of antiepileptic drugs. 
In India, the prevalence rate is about 5-6/1000, which means approximately more than 45 lakhs Indians suffers from this disease. 
Phenytoin was the first antiepileptic drug discovered using an animal seizure model. Phenytoin was synthesized, in 1908, and was recognized as a first nonsedating antiepileptic drug after the pioneering studies of Merritt and Putnam using an electroshock-induced seizure model in cats. Trimethadione, the first treatment specifically for absence seizures was licensed in the1940s, following laboratory evaluation with the pentylenetetrazole (PTZ) animal seizure model by Richards and Everett, in 1944, and clinical evaluation by Lennox in 1945. 
Despite the introduction of several new therapeutic options in the 1990s, a significant fraction of the patients with epilepsy continue to live with uncontrolled seizure. 
Pharmacoresistant Epilepsy [2,4]
Pharmacoresistance may be defined as poor seizure control despite accurate diagnosis and carefully monitored pharmacologic treatment. Clinically available anticonvulsant drugs fail to control seizures in around 30% of epileptic patients. About 75% of patients diagnosed with mesial temporal lobe epilepsy have pharmacoresistant seizures and more than 50% of patients with Lennox-Gastaut syndrome are classified as pharmacoresistant. The condition is more complicated in certain brain abnormalities, for example, when hippocampal sclerosis is combined with focal dysplasia. Despite the introduction of new drugs, the problem of pharmacoresistance has not been solved, although most of the new drugs have better safety profiles than those of older drugs. Surgical treatment of epilepsy may be an alternative, but at present, surgery is possible in only a small proportion of pharmacoresistant patients, and after the surgery, most of the patients are still prescribed antiepileptic drugs for full seizure control. 
It is not well established, why and how epilepsy becomes drug resistant in some patients while others with seemingly identical seizure types and epilepsy syndromes can achieve seizure control with medication. Thus, there is a clear need to understand the pathological process involving epilepsy and for an ideal antiepileptic agent with properties such as broad spectrum activity, rapid onset of action, least side effects, good oral bioavailability, and low cost. [4,5] Three major mechanisms have been proposed to explain pharmacoresistance in around 30% of patients:
1. Disease-related: Two key hypotheses have been proposed as disease-related mechanisms. The target hypothesis proposes the alterations of pharmacological targets of antiepileptic drugs in the brains of pharmacoresistant patients that lead to the failure of antiepileptic drugs to block excitatory sodium or calcium currents or to enhance the gamma-aminobutyric acid-mediated inhibition, whereas the transporter hypothesis proposes that excessive expression of multidrug transporters could remove antiepileptic drugs from epileptogenic brain regions.
2. Genetics: Genetic alterations due to, for example, polymorphisms in drug efflux transporters may also lead to poor seizure control in these patients.
3. Drug-related mechanism: Finally, tolerance as a drug-related mechanism may be responsible for lower efficacy of antiepileptic drugs in these patients. 
Intensive research is being carried out based on these hypotheses. However, the detailed mechanisms leading to pharmacoresistance is still unknown.  The aim of treating with an antiepileptic drug is not only to abolish the occurrence of seizures but also to lead a self-sustained life. Hence, search should continue to develop newer more effective and safer neuroprotective agents for the treatment of epilepsy. 
The use of indigenous plant medicines in developing countries became the World Health Organization policy since 1970. Of the 520 new drugs approved in the period 1983-1994 by either the US Food and Drug Administration or comparable entities in other countries, 30 drugs came directly from natural product sources, 173 were either semi-synthetics or synthetics originally modeled on a natural parent product. 
Nigella sativa is an annual herb of the Ranunculaceae family, which grows in countries bordering the Mediterranean Sea, Pakistan, and India. For thousands of years, this plant has been used in many Asian, Middle Eastern, and Far Eastern Countries as a spice and food preservative as well as a protective and health remedy in traditional folk medicine for the treatment of numerous disorders. 
The seed of this plant is commonly known as black seed and is referred by the Prophet Muhammad as having healing powers. The Prophet Muhammad (Peace be upon him) once stated that the black seed can heal every disease except death. It is also included in the list of natural drugs of "Tibb-e-Nabavi" or "Medicine of the Prophet Muhammad (Peace be upon him)" according to the tradition "hold onto the use of black seeds for healing all diseases." 
Other names for the seed include black caraway seed: Habbat Al Sawda and Habat Al Baraka, and the blessed seed:  Ajemuz, neguilla,  kalaunji, upakunchika, ajaji, kalvanjika, kalika, and kunchika. 
N. sativa seeds contain two types of oils, i.e., fixed oil (30-36% w/w) and volatile oil (0.43-0.72% w/w). 
The fixed, or fatty, oil is rich in unsaturated fatty acids, mainly linoleic acid (44.7-56%), oleic acid (20.7-24.6%), and eicosadienoic acid (3%). Saturated fatty acids (palmitic, stearic acid) amount to about 30% or less. 
Volatile oil of N. sativa seeds is composed mainly of thymoquinone (2-isopropyl-5- methyl- 1,4- benzoquinone) and monoterpenes. Thymoquinone contents range from 18.4% to 24% w/w of the volatile oil. The monoterpenes in the volatile oil amount to 46% w/w. The major components of these monoterpenes are p-cymene (isopropyl toluene), which comprises 31.7% of the volatile oil, and alpha-pinene, which comprises 9.3% of the volatile oil. Other components include phenols (1.7%), esters (16%), thymol, dithymoquinone, and thymohydroquinone. 
Black seed's constituents, in particular, its major constituent thymoquinone, have recently shown antiepileptic effects in mice. Literature has indicated that the whole oil from black seeds is effective against PTZ-induced kindling in mice. Some other studies have pointed out that the treatment of mice with thymoquinone reduced the duration of myoclonic seizures and effectively protected the mice from mortality. 
Here, we have evaluated the antiepileptic activity of volatile oil extract of N. sativa seeds with the standard sodium valproate in electrically induced seizures. We have also evaluated the influence of N. sativa volatile oil on sodium valproate.
Aims and Objectives
i. To evaluate the anticonvulsant activity of volatile oil extract of N. sativa seeds in albino rats by electrically induced seizure model
ii. To evaluate the influence of volatile oil extract of N. sativa seeds on the anticonvulsant activity of sodium valproate in albino rats by electrically induced seizure model.
MATERIALS AND METHODS
The study was conducted after the Institutional Animal Ethical Committee clearance.
Electroconvulsiometer with accessories, 1 ml tuberculin syringe, electronic weighing balance, animal weighing balance.
* Sodium valproate was obtained from Sun Pharmaceuticals. Dose used was 300 mg/kg  and was given intraperitoneally. Vehicle used was 5% gum acacia
* Volatile oil of N. sativa (200, 400, and 600 mg/kg body weight) 
* Gum acacia: 5%
* Distilled water.
Collection of seeds
The seeds of N. sativa were obtained from a local market in Mysore, Karnataka state and authenticated by the Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore. The seeds were crushed and grounded into a fine powder. The powdered sample was stored in clean and dry container until used.
Extraction of the volatile oil 
The steam distillation method was employed in the extraction process. 250 ml of distilled water was added to 50 g of finely powdered sample placed in a distillation flask connected to a steam distiller, condenser, and a receiver and was hydro distilled for 4 h. The distillate was collected in coated glass bottles. Diethyl ether was added to separate the water phase. Anhydrous sodium sulfate was added to the supernatant fraction with simple agitation to dry volatile oil and then filtered. The filtrate was concentrated by distilling off the solvent in a rotary evaporator. The final residue of volatile oil was stored at 4[degrees]C in coated glass bottles.
Animals: Healthy male swiss albino rats of 150-200 g
A total of 36 adult healthy male albino rats of Wistar strain of similar characteristics weighing 150-200 g were selected from the central animal facility, Mysore Medical College and Research Institute, Mysore, and divided into six groups of six each.
All the test animals were allowed food and water ad libitum both being withdrawn just before experimentation. The test animals were subjected to further study after a gap of 24 h to avoid any possible "kindling" effect. The drug preparations were administered intraperitoneally as shown in Table 1. To evaluate the influence of volatile oil extract of N. sativa seeds on sodium valproate, a combination of subanticonvulsive dose of volatile oil of N. sativa seeds and sodium valporate were studied, and results obtained were compared with anticonvulsive dose of sodium valproate alone. After an interval of 30 min, animals were subjected to electroshock of 150 mA intensity for 0.2 s, through auricular electrodes (covered in cotton wool and saline moistened). The duration of different parameters namely tonic flexion of fore and hind limbs with tail erection, tonic extension of both fore and hind limbs, clonus, stupor followed by postictal depression and recovery were noted.
Maximal electroshock (MES) seizure model
The most commonly used simple model for evaluation of drugs useful in generalized seizures is the electroshock model, which has been validated both clinically and electroencephalographically. The credit for standardizing this model goes to Woodbury and Davenport. 
Electroshock seizures are either threshold or maximal. It is the most specifically predictive test among the available anticonvulsant screening models.
MES model evaluates the ability of drugs to prevent electrically induced tonic hind limb extension (THLE) in mice or rats. Efficacy of drugs in this model has been shown to correlate with their ability to prevent partial and generalized tonic-clonic seizures in man and is said to evaluate the capacity of a drug to prevent the spread of seizures. Drugs that are active in the MES test often have a phenytoin like effect on voltage-dependent Na+ channels, viz., phenytoin, carbamazepine, phenobarbital, and valproate.
All animals are maintained on an adequate diet and allowed free access to food and water, except during testing as pre-test starvation modifies the MES-induced seizure pattern (shortens tonic flexion and prolongs tonic extension). Stimulation can be carried out through directly applied corneal electrodes. However, as this method causes pain and bleeding, hence transauricular electrodes (applied to the pinna with small crocodile clips covered with cotton wool and saline-moistened) are used. Pre-test saline moistening is mandatory to ensure better contact and to reduce fatalities resulting from MES-induced seizures. Maximal seizures are evoked by supramaximal electroshock stimulation of150 mA, 50 HZ, for 0.2 s using conventional electroconvulsiometer.
A stopwatch can be used for timing the various events of each phase.
The parameters studied were:
1. Tonic hind limb flexion
4. Stupor ([Unconsciousness] from the end of clonus to regain consciousness)
5. Postictal depression (from the regain of consciousness till the animals starts walking) duration of each parameter was recorded in seconds.
The abolition of the hind limb tonic extension is taken as an index of anticonvulsant activity.
Analysis of normal distribution and equivalence of variances between different phases of convulsions of MES model was confirmed using Shapiro-Wilks normality test and Barlett's Chi-square with Levene tests, respectively (Tables 2 and 4). As the phases of convulsions are naturally dependent on each other, Karl Pearson correlation analysis was used, supported by Barlett's Chi-square and Scatter plot matrix. (Table 5 and Graph 1). Multivariate analysis was used for testing the equality of sequences of vector means of different phases of convulsion across treatment groups simultaneously, which indicated that vector means are significant (Table 7). As multivariate test provide the significance in the vector sequences, it was customary to find out which of the convulsion phases contribute for the overall all significance. As a result, one-way ANOVA (Table 8 a) was used for multiple comparisons since the normality assumptions were met by the variables, followed by post-hoc test for comparison between groups.
In this study, the anticonvulsant activity of volatile oil extract of N. sativa seeds was evaluated against MES-induced convulsions. The present study demonstrates abolition of THLE, suggesting that the drug possesses anticonvulsant property.
The results of the experiment are tabulated in Tables 2-15, Bar Diagrams 1-5, and Line Diagrams 1-5. The results were analyzed statistically, and tests of significance were found out.
Table 2 provides the SW value and P value for different phases of convulsion across different treatment groups. Only 2 significant value in extension for TG2 and TG3. The rest follow normal distribution.
Table 3 provides the information on mean, variance, and median of different phases of convulsion in seconds across different treatment groups.
Table 4 gives the information on the equality of variance across treatment groups for different phases of convulsion.
Table 5 provides the correlation among several phases of convulsion. It is very clear that positive correlations exist and most are >80% indicating strong relationship among phases of convulsion. The same is supported by the scatter plot matrix (Graph 1).
Bartlett Chi-square statistic: 195.734, df = 10 P < 0.0005.
Bartlett Chi-square test for equality of several correlations shows that there is a significant difference in the equality of correlations indicating that there may be some real correlations among the variables.
Table 6 shows cell-wise correlation test, given by Bonferroni-adjusted correlation test at 5% level of significance. Indicating significance.
Table 7 is a multivariate statistical test, for testing the equality of mean vectors of different phases across treatment groups. It shows that vector means are significant.
One-way ANOVA is provided in the Table 8a. Furthermore, cross checking is been done with the nonparametric test which also supports the same and is given in Table 8b. P value in the last column indicates the significance.
Table 9 provides the complete detail of comparison of THLE duration in seconds among various groups in MES seizure model.
Tables 10-14 represents the post-hoc Fisher's test and P values for tonic hind limb flexion, THLE, clonus, stupor, and postictal depression, respectively, and are tabulated in the respective cells.
Table 10 (flexion) shows test Group 1 (TG1)is statistically insignificant to control group, but TG2 is significant to the control group. It is also observed from the table that TG3 and TG4 are significant with respect to control group, however, are insignificant with standard group, so comparable to standard group. Also that TG3 and TG4 are insignificant, thus comparable to each other.
Table 11 (extension) represents that TG1 is statistically insignificant to control group. It is observed from the table that TG2, TG3, and TG4 are significant with respect to control group.
Table 12 (clonus) represents that TG1 is statistically insignificant to control group, but TG2 is statistically significant to control group. It is also observed that TG3 and TG4 are significant with respect to the control group, however, are insignificant with standard group, so comparable to standard group. Furthermore, that TG3 compared to TG4 is insignificant, thus comparable to each other.
Table 13 (stupor) shows that TG1 is statistically insignificant to control group, but TG2 is statistically significant to control group. It is observed from the table that TG3 and TG4 are significant with respect to control group, however, are insignificant with Standard group, so comparable to standard group. Also that TG3 compared to TG4 is insignificant, so comparable to each other.
Table 14 (postictal depression) represents that TG1 is statistically insignificant to control group. TG2 and TG3 are statistically significant to control group, but not comparable to standard. TG4 is significant with respect to control group, however, is insignificant with standard group, so comparable to standard group.
Table 15 shows percentage protection in THLE among various treatment groups in MES seizure model.
Almost 30% of epileptic patients suffer from pharmacoresistance. The treatment of pharmacoresistant patients usually requires polytherapy. The long-term use of antiepileptic drugs is limited due to their adverse effects, withdrawal symptoms, deleterious interactions with other drugs and economic burden, especially in developing countries. 
There is still a need for an ideal antiepileptic agent with properties such as broad spectrum activity, rapid onset of action, least side effects, good oral bioavailability, and low cost.  Natural products from folk remedies have contributed significantly in the discovery of modern drugs and can be an alternative source for the discovery of antiepileptic drugs with novel structures and better safety and efficacy profiles. Hence, the search should continue to develop newer more effective and safer neuroprotective agents for the treatment of epilepsy. 
In this study, the anticonvulsant activity of volatile oil extract of N. sativa seeds was evaluated against MES-induced convulsions. The present study demonstrates the abolition of THLE showing that drug possesses anticonvulsant activity.
Dose 1 - 200 mg/kg Body Weight
Analysis of results of TG1 animals in MES model (Tables 10-14 and Line Diagrams 1-5) that received 200 mg/kg of test compound showed a reduction in all phases, namely, tonic hind limb flexion, THLE, clonus, stupor, and postictal depression. When compared to control group, reduction in the duration of clonus was statistically significant, whereas remaining phases were not statistically significant.
Dose 2 - 400 mg/kg Body Weight
Analysis of results of TG2 animals in MES model (Tables 9-14 and Line Diagrams 1-5), which received 400 mg/kg of the test compound, showed a reduction in all phases, namely, tonic hind limb flexion, THLE, clonus, stupor, and postictal depression which were statistically significant when compared to control group. Reduction in all phases of convulsion was statistically not comparable to standard; that means the reduction in the duration of phases of convulsion was less when compared to standard. There was complete abolition of THLE in 3 animals out of 6 animals. 50% abolition in THLE at this dose was observed.
Dose 3 - 600 mg/kg Body Weight
Analysis of results of TG3 animals in MES model (Tables 10-14 and Line Diagrams 1-5) that received 600 mg/kg of test compound showed a reduction in all phases, namely, tonic hind limb flexion, THLE, clonus, stupor, and postictal depression. Reduction in duration of tonic hind limb flexion, extension, clonus, and stupor was statistically significant when compared to control group and comparable to standard group. However, the reduction duration of postictal depression was statistically significant when compared to control but not comparable to the standard. There was the complete abolition of THLE in four animals out of six animals. 66.66% abolition in THLE at this dose was observed.
Dose 4--Volatile Oil of N. sativa 200 mg/kg + Sodium Valproate 150 mg/kg Body Weight
Analysis ofresults of TG4 animals in MES model (Table 10-14 and Line Diagrams 1-5) that received volatile oil of N. sativa 200 mg/kg body weight and sodium valproate 150 mg/kg body weight (sub anticonvulsant dose) showed a reduction in all phases, namely, tonic hind limb flexion, THLE, clonus, stupor, and postictal depression. Reduction in duration of tonic hind limb flexion, clonus, stupor, and postictal depression was statistically significant when compared to control group and comparable of standard group. There was complete abolition of THLE in all six animals. 100% abolition in THLE at this dose was observed. Furthermore, reduction in duration of tonic hind limb flexion, clonus, and stupor but not postictal depression were statistically insignificant and comparable to TG3.
Swinyard et al. have considered abolition of hind limb tonic extension as the protective end point against MES-induced seizures. 
According to this study, sodium valproate showed 100% protection against hind limb extension. At doses of 200 mg/kg of volatile oil of N. sativa, there was no significant anticonvulsant activity. At the dose of 400 and 600 mg/kg, THLE was abolished in 50% and 66.66% of animals, respectively, when compared to standard. This indicates that the anticonvulsant activity of volatile oil of N. sativa is less when compared to the standard drug, sodium valproate. The combination of volatile oil of N. sativa 200 mg/kg body weight with sodium valproate (150 mg/kg) showed statistically significant reduction in all phases of convulsion when compared to control group and were comparable of standard group. The combination of volatile oil of N. sativa 200 mg/kg body weight with sodium valproate (150 mg/kg) showed complete abolition of THLE in all 6 animals, thus showed 100% protection against THLE.
Bar diagram 1: Comparison of mean duration of tonic hind limb flexion across different treatment groups Different treatment groups CT 7.82 STD 4.16 TGRP1 7.53 TGRP2 5.95 TGRP3 4.48 TGRP4 4.12 Note: Table made from bar graph. Bar diagram 2: Comparison of mean duration of tonic hind limb extension across different treatment groups Different treatment groups CT 11.68 TGRP1 11.29 TGRP2 4.38 TGKP3 4.38 Note: Table made from bar graph. Bar diagram 3: Comparison of mean duration of clonus across different treatment groups Different treatment groups CT 19.54 STD 9.12 TGRP1 14.55 TGRP2 11.42 TGRP3 9.40 TGRP4 8.99 Note: Table made from bar graph. Bar diagram 4: Comparison of mean duration of stupor across different treatment groups Different treatment groups CT 114.5 STD 60.83 TGRP1 105.33 TGRP2 70.83 TGRP3 63.83 TGRP4 62.83 Note: Table made from bar graph. Bar diagram 5: Comparison of mean duration of postictal depression across different treatment groups Different treatment groups CT 131.16 STD 53.16 TGRP1 127.66 TGRP2 108.83 TGRP3 77 TGRP4 54.16 Note: Table made from bar graph.
The above analysis gives us the result that:
* Volatile oil of N.sativa by itself has significant anticonvulsant activity in the dose of 400 and 600 mg/kg body weight. However, anticonvulsant activity produced by it is less when compared to the standard drug sodium valproate in the dose of 300 mg/kg
* The combination of volatile oil of N. sativa with sodium valproate at their sub anticonvulsant doses showed significant anticonvulsant activity. This suggests that volatile oil of N. sativa has potentiated the effect of sodium valproate.
Hence, the test compound may be useful in generalized tonic-clonic seizures (grand mal) and partial epilepsy.
The anticonvulsant activity of volatile oil of N. sativa in different dosage profiles - 200, 400, and 600 mg/kg body weight and combination of volatile oil of N. sativa 200 mg/kg with sodium valproate 150 mg/kg were evaluated by MES model, and the results were compared with that of respective control groups and respective standard groups. The volatile oil of N. sativa has shown the significant anticonvulsant activity at the dose of 400 and 600 mg/kg body weight. Further, the combination of volatile oil of N. sativa 200 mg/kg with sodium valproate 150 mg/kg has shown significant anticonvulsant activity. The anticonvulsant activity of volatile oil of N. sativa was less when compared to sodium valproate. The anticonvulsant activity of combination, volatile oil of N. sativa 200 mg/kg with sodium valproate 150 mg/kg (in their sub anticonvulsive dose) was comparable to standard drug. From this, it may be predicted that volatile oil of N. sativa may be useful either alone or in combination with sodium valproate in the treatment of generalized tonic-clonic seizures (grand mal) epilepsy and partial seizures. The combination of subtherapeutic doses of valproate volatile oil of N. sativa resulted in the potentiation of sodium valproate. Thus, the reduction in the valproate dose required for anticonvulsant activity may be valuable in suppressing its unwanted effects such as hepatotoxicity and teratogenic implications. However, further studies are required to confirm the same.
We wish to thank Dr. Hema N G, Professor and Dr. Basavanna P L, Professor, Department of Pharmacology, Mysore Medical College and Research Institute, Mysore, for their precious and timely suggestions and advice. Our thanks to Dr. Vadiraja N, Assistant Professor, Department of Community Medicine, Mysore Medical College and Research Institute, Mysore, for helping to carry out the statistical analysis of the study.
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Asmatanzeem Bepari (1), Parashivamurthy B M (2), Shaik Kalimulla Niazi (3)
(1) Department of Pharmacology, Vijayanagara Institute of Medical Sciences, Ballari, Karnataka, India, (2) Department of Pharmacology, Mysore Medical College and Research Institute, Mysore, Karnataka, India, (3) Department of Biochemistry, Riyadh Colleges of Dentistry and Pharmacy, Riyadh, Kingdom of Saudi Arabia
Correspondence to: Asmatanzeem Bepari, E-mail: email@example.com
Received: October 01, 2016; Accepted: October 13, 2016
Table 1: Drug preparations administered Group Drugs administered I (control group) 5% gum acacia 0.25 ml/100 g II (standard group) Sodium valproate 300 mg/kg III (TG1) Volatile oil extract of N. sativa seeds 200 mg/kg IV (TG2) Volatile oil extract of N. sativa seeds 400 mg/kg V (TG3) Volatile oil extract of N. sativa seeds 600 mg/kg VI (TG4) Sodium valproate 150 mg/kg+volatile oil extract of N. sativa seeds 200 mg/kg N. sativa: Nigella sativa, TG: Test group Table 2: Subgroup normality assumption check SW normality tests results Drugs Statistic Flexion Extension Clonus Stupor Postictal test depression Control SW 0.954 0.941 0.987 0.902 0.981 value SW P 0.769 0.667 0.981 0.386 0.959 value Standard SW 0.926 0.940 0.992 0.928 value SW P 0.552 0.662 0.993 0.566 value TG1 SW 0.960 0.905 0.949 0.940 0.924 statistic SW P 0.820 0.406 0.729 0.660 0.532 value TG2 SW 0.943 0.729 0.958 0.818 0.878 statistic SW P 0.682 0.012 (*) 0.808 0.085 0.258 value TG3 SW 0.992 0.665 0.946 0.888 0.854 statistic SW P 0.993 0.003 (*) 0.708 0.307 0.170 value TG4 SW 0.913 0.875 0.915 0.965 statistic SW P 0.458 0.247 0.473 0.854 value (*) Indicates significant. SW: Shapiro-Wilks Table 3: Descriptive statistics of phases of convulsion across different treatment groups Phases of Groups Mean Variance Median convulsion Flexion Control 7.820 1.148 7.865 Standard 4.167 0.638 3.990 TG1 7.535 0.713 7.490 TG2 5.957 0.468 6.010 TG3 4.487 0.248 4.475 TG4 4.120 0.423 3.895 Clonus Control 19.545 1.627 19.605 Standard 9.122 1.580 9.105 TG1 14.555 3.762 14.740 TG2 11.420 0.784 11.445 TG3 9.407 1.160 9.385 TG4 8.997 0.981 8.985 Stupor Control 114.500 103.900 115.000 Standard 60.833 71.767 60.500 TG1 105.333 31.467 105.00 TG2 70.833 130.967 76.500 TG3 63.833 40.167 62.000 TG4 62.833 24.567 64.000 Postictal Control 131.167 180.167 133.000 depression Standard 53.167 52.167 52.500 TG1 127.667 154.267 131.000 TG2 108.833 44.167 106.500 TG3 77.000 164.400 79.500 TG4 54.167 19.367 53.500 TG: Test group Table 4: Barlett's Chi-square test and Levene's test for equality of several variances Phases of Bartlett's P value Levene's P value convulsions Chi-square of test of Levene's test Chi-square test test Flexion 3.065 0.690 0.640 0.671 Clonus 3.925 0.560 1.575 0.1971 Stupor 5.128 0.400 0.919 0.482 Depression 8.043 0.154 0.815 0.549 Table 5: Correlation analysis: Karl Pearson correlation matrix Flexion Extension Clonus Stupor Depression Flexion 1.000 Extension 0.783 1.000 Clonus 0.810 0.811 1.000 Stupor 0.695 0.859 0.809 1.000 Depression 0.782 0.852 0.816 0.798 1.000 Table 6: Matrix of Bonferroni probabilities Flexion Extension Clonus Flexion <0.0005 (*) Extension <0.0005 (*) <0.0005 (*) Clonus <0.0005 (*) <0.0005 (*) <0.0005 (*) Stupor <0.0005 (*) <0.0005 (*) <0.0005 (*) Postictal <0.0005 (*) <0.0005 (*) <0.0005 (*) depression Stupor Postictal depression Flexion Extension Clonus Stupor <0.0005 (*) Postictal <0.0005 (*) <0.0005 (*) depression (*) Indicates the statistical significance Table 7: Multivariate test statistics Statistic Value F-statistic df P value Wilks's 0.006 11.803 25.98 <0.0005 (*) Lamda Pillai 2.068 4.233 25.150 <0.0005 (*) trace Hotelling-Lawley 35.595 34.741 25.122 <0.0005 (*) trace (*) Indicates the statistical significance Table 8a: Univariate ANOVA table Phases of SS Df MS F P value convulsion Flexion Between 85.47 5 17.094 0.902 <0.0005 (*) groups Error 18.19 30 0.606 0.386 Extension Between 842.018 5 168.404 0.992 <0.0005 (*) groups Error 202.580 30 6.753 0.993 Clonus Between 525.838 5 105.168 0.940 <0.0005 (*) groups Error 49.470 30 1.649 0.660 Stupor Between 17,033.47 5 3406.694 0.818 <0.0005 (*) groups Error 2014.167 30 67.139 0.085 Postictal depression Between 37,523.33 5 7504.667 0.888 <0.0005 (*) groups Error 3072.667 30 102.422 0.307 SS: Sum of squares, Df: Degrees of freedom, MS: Mean square, SE: Standard error, ANOVA: Analysis of variance (*) Indicates the statistical significance. Table 8b: Kruskal-Wallis test Phases of Kruskal-Wallis P value Convulsion H test Flexion 28.135 <0.0005 (*) Extension 29.207 <0.0005 (*) Clonus 29.390 <0.0005 (*) Stupor 25.164 <0.0005 (*) Postictal depression 30.536 <0.0005 (*) (*) Indicates the statistical significance Table 9: Comparison of THLE duration in seconds among various groups in MES seizure model Groups Number of rats 1 2 3 4 5 6 Control 11.32 13.17 12.23 11.56 10.67 11.13 group Standard 0 0 0 0 0 0 group TG1 10.87 11.44 11.68 11.37 10.63 11.75 TG2 0 9.34 0 8.13 0 8.86 TG3 0 0 8.23 0 7.35 0 TG4 0 0 0 0 0 0 Groups Mean[+ or -]SE Control 11.68[+ or -]0.365 group Standard 0[+ or -]0 group TG1 11.29[+ or -]0.183 TG2 4.38[+ or -]1.969 TG3 2.59[+ or -]1.646 TG4 0[+ or -]0 THLE: Tonic hind limb extension, MES: Maximal electroshock, SE: Standard error, TG: Test group Table 10: Flexion matrix of pair-wise comparison probabilities Control Standard TG1 Control 1.000 Standard <0.0005 (*) 1.000 TG1 0.531 <0.0005 (*) 1.000 TG2 <0.0005 (*) <0.0005 (*) 0.001 (*) TG3 <0.0005 (*) 0.482 <0.0005 (*) TG4 <0.0005 (*) 0.918 <0.0005 (*) TG2 TG3 TG4 Control Standard TG1 TG2 1.000 TG3 0.003 (*) 1.000 TG4 <0.0005 (*) 0.421 1.000 (*) Indicates the statistical significance. TG: Test group Table 11: Extension matrix of pair-wise comparison probabilities Control Standard TG1 Control 1.000 Standard <0.0005 (*) 1.000 TG1 0.797 <0.0005 (*) 1.000 TG2 <0.0005 (*) 0.007 (*) <0.0005 (*) TG3 <0.0005 (*) 0.094 <0.0005 (*) TG4 <0.0005 (*) 1.000 <0.0005 (*) TG2 TG3 TG4 Control Standard TG1 TG2 1.000 TG3 0.242 1.000 TG4 0.007 (*) 0.094 1.000 (*) Indicates the statistical significance. TG: Test group Table 12: Clonus matrix of pair-wise comparison probabilities Control Standard TG1 TG2 Control 1.000 Standard <0.0005 (*) 1.000 TG1 <0.0005 (*) <0.0005 (*) 1.000 TG2 <0.0005 (*) 0.004 (*) <0.0005 (*) 1.000 TG3 <0.0005 (*) 0.703 <0.0005 (*) 0.011 (*) TG4 <0.0005 (*) 0.867 <0.0005 (*) 0.003 (*) TG3 TG4 Control Standard TG1 TG2 TG3 1.000 TG4 0.584 1.000 (*) Indicates the statistical significance. TG: Test group Table 13: Stupor matrix of pair-wise comparison probabilities Control Standard TG1 TG2 Control 1.000 Standard <0.0005 (*) 1.000 TG1 0.062 <0.0005 (*) 1.000 TG2 <0.0005 (*) 0.043 (*) <0.0005 (*) 1.000 TG3 <0.0005 (*) 0.531 <0.0005 (*) 0.149 TG4 <0.0005 (*) 0.675 <0.0005 (*) 0.101 TG3 TG4 Control Standard TG1 TG2 TG3 1.000 TG4 0.834 1.000 (*) Indicates the statistical significance. TG: Test group Table 14: Postictal depression matrix of pair-wise comparison probabilities Control Standard TG1 TG2 Control 1.000 Standard <0.0005 (*) 1.000 TG1 0.554 <0.0005 (*) 1.000 TG2 0.001 (*) <0.0005 (*) 0.003 (*) 1.000 TG3 <0.0005 (*) <0.0005 (*) <0.0005 (*) <0.0005 (*) TG4 <0.0005 (*) 0.865 <0.0005 (*) <0.0005 (*) TG3 TG4 Control Standard TG1 TG2 TG3 1.000 TG4 <0.0005 (*) 1.000 TG: Test group, (*) Indicates the statistical significance. Table 15: Percentage protection in THLE among various treatment groups in MES seizure model Treatment groups Percentage protection Standard group 100 TG1 Nil TG2 50 TG3 66.66 TG4 100 THLE: Tonic hind limb extension, MES: Maximal electroshock
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Bepari, Asmatanzeem; Parashivamurthy B.M.; Niazi, Shaik Kalimulla|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Mar 1, 2017|
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