{"id":55677,"date":"2024-03-20T11:32:17","date_gmt":"2024-03-20T11:32:17","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=55677"},"modified":"2024-04-01T19:10:58","modified_gmt":"2024-04-01T19:10:58","slug":"evaluation-of-anti-epileptic-effect-of-sinapis-alba-using-maximal-electroshock-seizure-model","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/evaluation-of-anti-epileptic-effect-of-sinapis-alba-using-maximal-electroshock-seizure-model\/","title":{"rendered":"Evaluation of Anti-epileptic Effect of Sinapis alba using Maximal Electroshock Seizure Model"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Mustard is one of the oldest widely-grown condiments,\ndating back to 3000 BC. The most commonly used mustard varieties include Sinapis\nalba<\/em> (yellow mustard), Brassica nigra <\/em>(black mustard), and B.\njuncea<\/em> (Indian mustard). Mustards belong to the Brassicaceae<\/em> family.\nSinapis alba,<\/em> also known as yellow or white mustard, is used both as a\nspice and for medicinal purposes in various conditions.1<\/sup> The seeds\ncontain a bland fixed oil of about 23-25%, sinalbin (a crystalline compound),\nsinapin-sulphocyanide, mucilage, lecithin, myrosin, and 4% ash, which consists\nof potassium, calcium, and magnesium phosphates. They also contain vitamin A,\nthiamine (B1), riboflavin, proteins, and water.2<\/sup> The phenolic groups\nfound abundantly in white mustard are sinapic acid and p-hydroxybenzoic acid,\nwhich contribute to its antioxidant activity.3<\/sup> S. alba has a\npungent, bitter, sharp, unctuous, and hot flavor. While white mustard contains\nglucosinolate-sinalbin, black mustard contains sinigrin. Their hydrolyzed\nproducts differ, which is responsible for the pungency and sharp taste. Among\nthese, black mustard is more pungent.4<\/sup><\/p>\n\n\n\n

According to literature on its use in traditional\nmedicine, S. alba<\/em> has been employed to treat cholera, dysmenorrhea,\namenorrhea, whooping cough, high fever, headaches, and swollen joints. It also\npossesses emetic and diuretic effects.2,5<\/sup> They are potent natural\nantioxidants.3<\/sup> Yellow mustard calms vata and kapha and is beneficial\nfor ailments related to the head and ear. It is applied in the treatment of\nskin diseases due to its antimicrobial and anti-inflammatory activities.6 <\/sup> The high levels of glucosinolates present make\nit valuable in anti-cancer therapy.7<\/sup> It aids in regulating irregular\nheartbeats, cholesterol, and blood sugar levels and provides relief from\nrespiratory congestion, largely due to the magnesium in it.3<\/sup><\/p>\n\n\n\n

Ayurvedic literature mentions the use of these seeds\nin the treatment of nerve disorders such as hysteria, delirium, and epilepsy,\nproving effective when taken internally.2<\/sup> There’s also evidence of\nits use for epilepsy in German Renaissance herbals.8<\/sup> While there\nhave been some studies on the anti-epileptic effect of B. nigra<\/em> showing\npositive results, there are none conducted with yellow mustard seeds to date.9,\n10  <\/sup>Hence, this research was\nembarked upon to evaluate the efficacy of Sinapis\nalba<\/em>\nin treating epilepsy. We\nhypothesize that the potential neuroprotective action of S. alba could be\nattributed to its potent antioxidant properties, which help in reducing\nneuronal oxidative damage during epileptogenesis. In this study, the efficacy\nof Sinapis alba<\/em> seed oil was examined using the MES model in Wistar\nrats.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

Sodium valproate (SV) (Encorate 300mg tablet) was\nindented from Radha Medicals, Manipal. Cold-pressed yellow mustard seed oil\n\u2013Brand Planton was obtained from Green Trade Company, New Delhi. <\/p>\n\n\n\n

Experimental animals<\/strong><\/p>\n\n\n\n

The study was conducted as per the guidelines set by\nthe Committee for Control and Supervision of Experiments on Animals (CCSEA). Thirty\nalbino Wistar rats of 200 -300 grams weight were sanctioned for the experiment\nand were housed at the Central Animal House, Manipal. Housing conditions were\nas follows: – 3 rats per cage with 12:12 hours of light: dark lighting\nconditions, the temperature maintained at 25\u02da\u00b13\u02daC and humidity was 50%. Normal\nrodent diet pellet (VRK Nutritionals, Maharashtra) and water ad libitum were\nprovided.<\/p>\n\n\n\n

Procedure<\/strong><\/p>\n\n\n\n

Adult albino female Wistar rats weighing 200-300 grams\nwere utilized in this study. After acclimatization for 1 week, rats were divided\ninto five groups. Each group had 6 rats each (random grouping was done as shown\nin Table 1) and treated for 14 days as follows: <\/p>\n\n\n\n

Table 1: Different groups along with the treatment doses<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Treatment<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Group 1 Normal control<\/p>\n<\/td>\n

\n

Distilled water (10 mL\/kg, p.o.)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 2 Disease control<\/p>\n<\/td>\n

\n

Distilled water (10 mL\/kg, p.o.)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 3 Test drug<\/p>\n<\/td>\n

\n

Yellow mustard oil (200 mg\/kg) (p.o.)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 4 Standard drug<\/p>\n<\/td>\n

\n

Sodium valproate (300 mg.kg) (p.o.)<\/p>\n<\/td>\n<\/tr>\n

\n

Group 5 Test + standard<\/p>\n<\/td>\n

\n

Yellow mustard oil (200 mg\/kg) (p.o.) + sodium valproate (150 mg\/kg) (p.o.)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Dose of yellow mustard oil and sodium valproate was\nobtained from previous studies. 11,12,13<\/sup> Sodium valproate was\nfreshly prepared daily. A gap of half an hour was provided between the dosing\nof yellow mustard seed oil and valproate. After 14 days of treatment as shown\nin Table 1, generalized tonic-clonic seizures were induced. <\/p>\n\n\n\n

Seizure induction: Grand mal or generalized\ntonic-clonic seizures were induced using an electro-convulsive meter (Inco Company,\nAmbala City, India). Electric stimulation of intensity 150mA, 0.2 seconds\n(50-60 Hz frequency) via ear clip electrodes was used to induce seizures on the\n14th day of the experiment.14<\/sup> Induction was done after 1 hour of\ndosing with oil, valproate, and distilled water.<\/p>\n\n\n\n

The duration of all the following stages was noted\ndown: –<\/p>\n\n\n\n

Flexion<\/p>\n\n\n\n

Tonic hind limb extension (THLE)<\/p>\n\n\n\n

Clonic convulsions<\/p>\n\n\n\n

Stupor<\/p>\n\n\n\n

Death\/recovery after twenty-four hours of induction\nwas recorded.<\/p>\n\n\n\n

THLE was the endpoint (backward extension of hindlimb-\n> 90\u00b0 against the plane of body axis). Percentage inhibition of convulsions\nand percentage protection were calculated. On the 15th<\/sup>\nday, rats were euthanized with thiopentone sodium (120 mg\/kg i.p.). Rats were\ndissected, and the brain was removed; ice-cold saline (ICS) was used to clean.\nBrain samples were stored in phosphate buffer saline (PBS) for biochemical\nestimations and in 10% formalin for histopathological analysis.<\/p>\n\n\n\n

Parameters <\/strong><\/p>\n\n\n\n

The parameters assessed\nin the MES model were body weight, duration of seizures, latency to seizure\nonset, recovery or death, seizure score\/stage, and death after 24 hours.<\/p>\n\n\n\n

Assessment of biochemical parameters<\/strong><\/p>\n\n\n\n

 Oxidative\nstress markers<\/strong><\/p>\n\n\n\n

Brain homogenate was\nprepared, and the levels of superoxidase dismutase (SOD), malonaldehyde (MDA),<\/p>\n\n\n\n

Nitrite and glutathione\n(GSH) were estimated. <\/p>\n\n\n\n

Histopathological analysis<\/strong><\/p>\n\n\n\n

The brain tissue was stored in 10% formalin after\ndissection. Nissl staining technique was used to assess the extent of neuronal\ndegeneration. Serial coronal brain sections of 30-50\u00b5m thickness\nwere taken using a cryostat at frozen condition (\u221218\u2103). Sections were hydrated with xylene, 100%, 90%, and 70%\nalcohol. The sections were stained with Cresyl violet at 37-50 \u2103. This stain is\nhelpful since it stains neuronal cell bodies, and degenerating or pyknotic\nneurons can easily be identified under a light microscope. Dehydration of\nsections was carried out using 70 %, 95%, and 100% alcohol grades and later\nwashed with xylene. Mounting was done using mountant- DPX. Cresyl violet stained\nsections were then observed under a light microscope for the qualitative\nanalysis of pyramidal neurons of the hippocampal CA3 region.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

Statistics\nwere carried out using GraphPad Prism 8.0.1. One-way ANOVA followed by “Post\nHoc Tukey’s multiple comparison test” was performed for biochemical and\nbehavioural parameters. For body weight, two-way ANOVA was performed, after\nwhich post hoc Bonferroni’s test was carried out. All the data were expressed\nas mean \u00b1 standard error of the mean (SEM).<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

The results of the study are\nmentioned below:<\/p>\n\n\n\n

Gas chromatography-mass spectroscopy (GC-MS) analysis of yellow mustard (Sinapis alba<\/em>) seed oil<\/strong><\/p>\n\n\n\n

Analytical\nResearch & Metallurgical Laboratories Pvt Ltd, Bangalore, performed GC-MS\nof cold-pressed Sinapis\nalba<\/em>\nseed oil. The identified\ncompounds are depicted in Table 2. A total of 21 compounds were identified, of\nwhich major proportion was “9-Octadecenoic acid,\nmethyl ester (20.74%), 6,8-Difluoro-2,2,4,4,6,7,7,8,9,9-decamethyl-[1,3,5,2,4,6,7,8,9]trioxahexasilonane\n(14.87%), 9,12-Octadecadienoic acid (Z,Z)-, phenylmethyl ester(5.4%),\n9-Octadecenoic acid, 1,2,3-propanetriyl ester (4.83%), Hexadecanoic acid,\nmethyl ester (4.74%), 9,12,15-Octadecatrienoic acid,\n2-[(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester (3.61%).”\n<\/p>\n\n\n\n

Table 2: List of compounds identified from the yellow mustard (Sinapis alba<\/em>) oil sample along with its retention time and %area. <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Peak<\/strong><\/p>\n<\/td>\n

\n

Retention Time <\/strong><\/p>\n

(min)<\/strong><\/p>\n<\/td>\n

\n

Area<\/strong><\/p>\n<\/td>\n

\n

Area%<\/strong><\/p>\n<\/td>\n

\n

Name<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

3.157<\/p>\n<\/td>\n

\n

39756<\/p>\n<\/td>\n

\n

3.61<\/p>\n<\/td>\n

\n

9,12,15-Octadecatrienoic acid, 2-[(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

4.859<\/p>\n<\/td>\n

\n

112809<\/p>\n<\/td>\n

\n

1.04<\/p>\n<\/td>\n

\n

1-Butene, 4-isothiocyanato [1]\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

9.177<\/p>\n<\/td>\n

\n

55508<\/p>\n<\/td>\n

\n

0.51<\/p>\n<\/td>\n

\n

Tetradecane<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

14.736<\/p>\n<\/td>\n

\n

130209<\/p>\n<\/td>\n

\n

1.2<\/p>\n<\/td>\n

\n

N-Benzylisatoic anhydride<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

15.055<\/p>\n<\/td>\n

\n

56654<\/p>\n<\/td>\n

\n

0.52<\/p>\n<\/td>\n

\n

Phthalic acid, 3,5-dimethylphenyl 2-isopropylphenyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

16.033<\/p>\n<\/td>\n

\n

1608701<\/p>\n<\/td>\n

\n

14.87<\/p>\n<\/td>\n

\n

6,8-Difluoro-2,2,4,4,6,7,7,8,9,9-decamethyl-[1,3,5,2,4,6,7,8,9]trioxahexasilonane<\/p>\n<\/td>\n<\/tr>\n

\n

7<\/p>\n<\/td>\n

\n

16.506<\/p>\n<\/td>\n

\n

525131<\/p>\n<\/td>\n

\n

4.85<\/p>\n<\/td>\n

\n

Hexadecanoic acid, methyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

8<\/p>\n<\/td>\n

\n

18.215<\/p>\n<\/td>\n

\n

2243696<\/p>\n<\/td>\n

\n

20.74<\/p>\n<\/td>\n

\n

 9-Octadecenoic acid, methyl ester, (E)<\/p>\n<\/td>\n<\/tr>\n

\n

9<\/p>\n<\/td>\n

\n

18.571<\/p>\n<\/td>\n

\n

52857<\/p>\n<\/td>\n

\n

0.49<\/p>\n<\/td>\n

\n

Oleyl Alcohol<\/p>\n<\/td>\n<\/tr>\n

\n

10<\/p>\n<\/td>\n

\n

19.992<\/p>\n<\/td>\n

\n

512335<\/p>\n<\/td>\n

\n

4.74<\/p>\n<\/td>\n

\n

 9-Hexadecenoic acid, methyl ester, (Z)<\/p>\n<\/td>\n<\/tr>\n

\n

11<\/p>\n<\/td>\n

\n

20.182<\/p>\n<\/td>\n

\n

91401<\/p>\n<\/td>\n

\n

0.84<\/p>\n<\/td>\n

\n

Eicosanoic acid, methyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

12<\/p>\n<\/td>\n

\n

21.448<\/p>\n<\/td>\n

\n

583762<\/p>\n<\/td>\n

\n

5.4<\/p>\n<\/td>\n

\n

 9,12-Octadecadienoic acid (Z,Z)-, phenylmethyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

13<\/p>\n<\/td>\n

\n

21.633<\/p>\n<\/td>\n

\n

2206619<\/p>\n<\/td>\n

\n

20.4<\/p>\n<\/td>\n

\n

 9-Octadecenoic acid, methyl ester, (E)<\/p>\n<\/td>\n<\/tr>\n

\n

14<\/p>\n<\/td>\n

\n

22.18<\/p>\n<\/td>\n

\n

143823<\/p>\n<\/td>\n

\n

1.33<\/p>\n<\/td>\n

\n

Phosphine imide, P,P,P-triphenyl[1]\n<\/td>\n<\/tr>\n

\n

15<\/p>\n<\/td>\n

\n

23.85<\/p>\n<\/td>\n

\n

64822<\/p>\n<\/td>\n

\n

0.6<\/p>\n<\/td>\n

\n

1,5-Di-p-tolyl-anthraquinone<\/p>\n<\/td>\n<\/tr>\n

\n

16<\/p>\n<\/td>\n

\n

24.492<\/p>\n<\/td>\n

\n

522751<\/p>\n<\/td>\n

\n

4.83<\/p>\n<\/td>\n

\n

 9-Octadecenoic acid, 1,2,3-propanetriyl ester<\/p>\n<\/td>\n<\/tr>\n

\n

17<\/p>\n<\/td>\n

\n

27.486<\/p>\n<\/td>\n

\n

97870<\/p>\n<\/td>\n

\n

0.9<\/p>\n<\/td>\n

\n

Ergosta-8(14),15,22-trien-3-ol, (3.beta.,5.alpha.,22E)<\/p>\n<\/td>\n<\/tr>\n

\n

18<\/p>\n<\/td>\n

\n

28.25<\/p>\n<\/td>\n

\n

206902<\/p>\n<\/td>\n

\n

1.91<\/p>\n<\/td>\n

\n

beta.-Sitosterol<\/p>\n<\/td>\n<\/tr>\n

\n

19<\/p>\n<\/td>\n

\n

28.536<\/p>\n<\/td>\n

\n

58037<\/p>\n<\/td>\n

\n

0.54<\/p>\n<\/td>\n

\n

5-(p-Aminophenyl)-4-(p-tolyl)-2-thiazolamine<\/p>\n<\/td>\n<\/tr>\n

\n

20<\/p>\n<\/td>\n

\n

29.447<\/p>\n<\/td>\n

\n

496285<\/p>\n<\/td>\n

\n

4.59<\/p>\n<\/td>\n

\n

gamma. \u2013Sitosterol<\/p>\n<\/td>\n<\/tr>\n

\n

21<\/p>\n<\/td>\n

\n

30.125<\/p>\n<\/td>\n

\n

656236<\/p>\n<\/td>\n

\n

6.07<\/p>\n<\/td>\n

\n

2,4-Di-tert-butyl thiophenol<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Weight of rats<\/strong><\/p>\n\n\n\n

The total body weights of animals on Day 1, day 14, and day 15 is shown in Table no. 3. On the first day, the mean weights of different group were not statistically different. In 2 weeks after treatment, there was an increase in the weight of rats of group 3 (oil 200 mg\/kg) and group 5 (oil 200 mg\/kg + SV 150 mg\/kg), which was significant (p-value <0.05 and p<0.01 respectively) as compared to control.<\/p>\n\n\n\n

On the 15th<\/sup> day\n(i.e., 24 hours after MES induction), we could observe a significant difference\n(p-value <0.05) in weight of rats administered with oil 200 mg\/kg (group 3)\nand oil 200 mg\/kg + SV 150 mg\/kg (group 5) when compared to disease control\n(where the weight reduced post-induction).<\/p>\n\n\n\n

Table 3: Body weight of different groups measured at day 1, 14 and 15.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Sl. No.<\/strong><\/p>\n<\/td>\n

\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Body weight in grams<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Day 1<\/strong><\/p>\n<\/td>\n

\n

Day 14<\/strong><\/p>\n<\/td>\n

\n

Day 15<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Normal control<\/p>\n<\/td>\n

\n

271.66\u00b16.78<\/p>\n<\/td>\n

\n

279.16\u00b17.28<\/p>\n<\/td>\n

\n

281\u00b17.21<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Disease control<\/p>\n<\/td>\n

\n

258\u00b14.78<\/p>\n<\/td>\n

\n

293.5\u00b15.32<\/p>\n<\/td>\n

\n

275.33\u00b16.46<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Oil 200mg\/kg<\/p>\n<\/td>\n

\n

277.5\u00b112.48<\/p>\n<\/td>\n

\n

324.33\u00b111.15*<\/sup><\/p>\n<\/td>\n

\n

317.5\u00b113.22a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

SV 300mg\/kg<\/p>\n<\/td>\n

\n

284.5\u00b111.17<\/p>\n<\/td>\n

\n

306.83\u00b112.10<\/p>\n<\/td>\n

\n

309.83\u00b111.53<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

Oil 200mg\/kg + SV 150mg\/kg<\/p>\n<\/td>\n

\n

267.83\u00b19.69<\/p>\n<\/td>\n

\n

327.83\u00b19.86a<\/sup><\/p>\n<\/td>\n

\n

324.5\u00b111.53*, a<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Two-way ANOVA. Post hoc – Bonferroni’s multiple comparisons test. Data expressed as\nMean\u00b1SEM. <\/p>\n\n\n\n

*<\/sup>p <0.05 vs normal control (group 1), a <\/sup>p-value\n<0.05 vs disease control (group 2)<\/p>\n\n\n\n

Anti-epileptic effect of Sinapis alba<\/em> in Maximal electroshock model<\/strong><\/p>\n\n\n\n

The average duration of flexion,\nTHLE, clonic-convulsions and stupor are presented in Table 4, along with the percentage\ninhibition of THLE in comparison to MES disease control. There was a significant\nreduction in the duration of flexion, clonic convulsion and stupor period in\ntest, standard and combination groups compared to disease control. The duration\nof THLE in group 3, group 4 and group 5 was significantly low (p<0.05). THLE\nwas completely inhibited in sodium valproate 300mg\/kg. SV 300mg\/kg showed 100%\nprotection from THLE, while the mustard oil 200mg\/kg and combination of mustard\noil 200mg\/kg + SV 150 mg\/kg showed 33.33% and 66.6% protection respectively.\nThere was no mortality recorded after 24 hours of induction in any of the groups.<\/p>\n\n\n\n

Table 4: Effect of yellow mustard seed oil (Sinapis alba<\/em>) in MES-induced epilepsy in rats<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Sl. No.<\/strong><\/p>\n<\/td>\n

\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Flexion
<\/strong>(Duration in seconds)<\/strong><\/p>\n<\/td>\n

\n

Tonic hind limb extension (THLE)<\/strong>(Duration in seconds)<\/strong><\/p>\n<\/td>\n

\n

Clonic convulsions
<\/strong>(Duration in seconds)<\/strong><\/p>\n<\/td>\n

\n

Stupor
<\/strong>(Duration in seconds)<\/strong> <\/strong><\/p>\n<\/td>\n

\n

% Inhibition of convulsions
<\/strong>(THLE) <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Disease<\/p>\n<\/td>\n

\n

10.02\u00b11.48<\/p>\n<\/td>\n

\n

9.97\u00b10.69<\/p>\n<\/td>\n

\n

33.71\u00b14.07<\/p>\n<\/td>\n

\n

130.1\u00b110.35<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Oil 200mg\/kg<\/p>\n<\/td>\n

\n

5.987\u00b11.19a<\/sup><\/p>\n<\/td>\n

\n

5.48\u00b11.13a,b<\/sup><\/p>\n<\/td>\n

\n

21.25\u00b15.82<\/p>\n<\/td>\n

\n

72.33\u00b19.39a<\/sup><\/p>\n<\/td>\n

\n

33.33<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

SV 300 mg\/kg<\/p>\n<\/td>\n

\n

4.63\u00b10.41a<\/sup><\/p>\n<\/td>\n

\n

 0a<\/sup><\/p>\n<\/td>\n

\n

14.37\u00b11.83a<\/sup><\/p>\n<\/td>\n

\n

58.33\u00b18.71a<\/sup><\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

Oil 200mg\/kg+ SV 150 mg\/kg<\/p>\n<\/td>\n

\n

5.67\u00b10.45a<\/sup><\/p>\n<\/td>\n

\n

3.84\u00b11.31a,b<\/sup><\/p>\n<\/td>\n

\n

16.02\u00b12.21a<\/sup><\/p>\n<\/td>\n

\n

59.83\u00b110.96a<\/sup><\/p>\n<\/td>\n

\n

66.66<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/p>\n\n\n

All values are expressed as Mean\u00b1SEM. One-way ANOVA, post hoc – Tukey’s multiple comparison test was performed. All the values were compared with disease control (group 2).<\/p>\n\n\n\n

a<\/sup>p <0.05 vs disease control<\/p>\n\n\n\n

b<\/sup>p <0.05 vs SV 300mg\/kg<\/p>\n\n\n\n

Effect of Sinapis alba<\/em> seed oil on brain oxidative stress <\/strong><\/p>\n\n\n\n

The levels of MDA were expressed as nmol\/gram protein.\nThere was an elevation in MDA levels in MES control compared to the normal\ncontrol (p<0.05). There is a significant reduction (p<0.05) in the MDA\nlevels in oil 200mg\/kg, SV 300mg\/kg, and oil 200mg\/kg + SV 150mg\/mg compared to\ndisease control. MDA levels of treatment groups 4 and 5 were comparable with\nnormal (p>0.05).<\/p>\n\n\n\n

Glutathione levels decreased significantly in disease\ncontrol (p<0.05) compared to normal control. In comparison with disease\ncontrol, there was a significant difference in GSH (reduced form) in those\ntreated with either oil 200mg\/kg, SV 300 mg\/kg or oil 200mg\/kg + SV 150 mg\/kg\n(p<0.05). GSH levels of group 4,5 were comparable with normal control\n(p>0.05). <\/p>\n\n\n\n

Nitrite levels in disease control increased\nsignificantly when compared to the normal group (p<0.05). There was a\ndecrease in group 3,4,5 nitrite levels when compared to group 2(disease\ncontrol). Group 4 (SV 300mg\/kg) nitrite levels reduced significantly (p<0.05).\nNitrite levels in brain of rats treated with Oil 200mg\/kg (group 3) alone and\nin combination with sodium valproate 150 mg\/kg (group 5) were comparable to\nsodium valproate 300mg\/kg (group 4). <\/p>\n\n\n\n

Superoxide dismutase\n(SOD) levels reduced significantly in disease control when compared to normal\n(p<0.05). In SV 300mg\/kg (group 4), the SOD levels increased significantly\nwith respect to disease control (p<0.05) and almost matched normal levels.\nThe levels of group 3 and group 5 increased significantly (p<0.01) compared\nto MES control and were comparable to group 4 (Table 5). <\/p>\n\n\n\n

Table 5: Effect of Sinapis alba<\/em> seed oil on brain oxidative stress status (MES model)<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

MDA (nM\/g)<\/strong><\/p>\n<\/td>\n

\n

GSH (\u00b5M\/mg)<\/strong><\/p>\n<\/td>\n

\n

Nitrite (mM\/L)<\/strong><\/p>\n<\/td>\n

\n

SOD (U\/mg)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Normal<\/p>\n<\/td>\n

\n

45.35\u00b15.57<\/p>\n<\/td>\n

\n

10.68\u00b10.09<\/p>\n<\/td>\n

\n

31.99\u00b13.41<\/p>\n<\/td>\n

\n

52.14\u00b11.24<\/p>\n<\/td>\n<\/tr>\n

\n

Disease<\/p>\n<\/td>\n

\n

101.9\u00b15.4*<\/p>\n<\/td>\n

\n

8.7\u00b10.19*<\/p>\n<\/td>\n

\n

66.08\u00b14.12*<\/p>\n<\/td>\n

\n

36.54\u00b12.05*<\/p>\n<\/td>\n<\/tr>\n

\n

Oil 200mg\/kg<\/p>\n<\/td>\n

\n

71.04\u00b18.19*,a<\/sup><\/p>\n<\/td>\n

\n

9.71\u00b10.06*, a,b<\/sup><\/p>\n<\/td>\n

\n

47.20\u00b12.19*,a<\/sup><\/p>\n<\/td>\n

\n

46.7\u00b12.54 a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

SV 300 mg\/kg<\/p>\n<\/td>\n

\n

50.72\u00b11.79a<\/sup><\/p>\n<\/td>\n

\n

10.32\u00b10.22a<\/sup><\/p>\n<\/td>\n

\n

36.75\u00b13.58a<\/sup><\/p>\n<\/td>\n

\n

51.81\u00b11.25a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Oil 200mg\/kg + SV 150 mg\/kg<\/p>\n<\/td>\n

\n

59.19\u00b12.54a<\/sup><\/p>\n<\/td>\n

\n

10.26\u00b10.21a<\/sup><\/p>\n<\/td>\n

\n

40.81\u00b11.26a<\/sup><\/p>\n<\/td>\n

\n

48.41\u00b11.24 a<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

<\/p>\n\n\n<\/p>\n

One way ANOVA and then post hoc Tukey’s multiple comparison test. Values expressed as Mean\u00b1SEM.<\/p>\n

\n\n\n\n<\/p>\n

*<\/sup>p <0.05 vs normal control<\/p>\n

\n\n\n\n<\/p>\n

a<\/sup>p <0.05 vs disease control<\/p>\n

\n\n\n\n<\/p>\n

b<\/sup>p <0.05 vs sodium valproate 300mg\/kg\n\n\n

<\/p>\n\n\n

Qualitative analysis of Cresyl violet stained pyramidal neurons of hippocampal CA3 region<\/strong><\/p>\n\n\n\n

Cresyl violet stained\npyramidal neuronal cell bodies of the hippocampal CA3 region of rats of group 1\n(normal control) showed normal healthy neurons (indicated by the yellow arrow\nin figure 1) with healthy-looking cell membrane, clear cytoplasm, and prominent\nnucleus. Cresyl violet stained hippocampal CA3 region of disease group 2\n(disease control group) also showed predominantly healthy neurons and very few\ndegenerating, flame-shaped, pyknotic cell bodies of pyramidal neurons (indicated\nby the red arrow in Figure 1). The flame-shaped cells that look deeply\nbasophilic indicate karyopyknosis of the hippocampus’s neurons. Based on this\nobservation, it can be stated that the acute model of epilepsy is unable to\nelicit structural changes in the hippocampal CA3 region. Further, it can be\nnoted that hippocampal CA3 neurons of group 3 (treated with 200 mg\/kg yellow\nmustard oil), group 4 (treated with 300mg\/kg Sodium valproate), and group 5\n(treated with 150mg\/kg sodium valproate + 200mg\/kg yellow mustard oil) looked\nhealthy and normal (indicated by the yellow arrow in figure 1). It can be noted\nthat all the groups exhibited normal hippocampal structures with predominantly\nhealthy neurons (indicated by the yellow arrow). However, Group 2 showed very\nfew degenerating pyknotic neurons (indicated by the red arrow). <\/p>\n\n\n\n

It can be noted that the neurons that\nlook characteristically flame-shaped are relatively more in group 2 when\ncompared to those of other groups. Groups 3, 4, and 5 showed predominantly\nhealthy neurons (indicated by yellow arrow) when compared to group 2. The\nneurons of groups 4 and 5 looked almost at par with the normal control group. <\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Representative photomicrographs of Cresyl violet stained hippocampal CA3 region of group 1 (normal control) \u2013 (a), group 2 (Disease control) \u2013 (b), group 3 (Treated with 200 mg\/kg yellow mustard oil) \u2013 (c), group 4 (Treated with 300mg\/kg Sodium valproate) \u2013 (d)<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Sinapis\nalba<\/em> (yellow\/white\nmustard) is a widely used spice in India and has valuable medicinal properties.\nYellow mustard has anti-microbial, anti-inflammatory, anti-proliferative,\nantioxidant, analgesic and immunomodulatory properties.6,7,15,16<\/sup> It\nis also known for its stimulant, expectorant, rubefacient, appetizing,\ndiuretic, emetic, carminative, digestive, and diaphoretic properties.3<\/sup>\nAyurvedic literature mentions the use of S.\nalba<\/em> in the treatment of diseases involving the nervous system, such as\nhysteria and epilepsy.2,17 <\/sup>Even it is present in 16-17th<\/sup>\ncentury Renaissance period literature. It is mentioned in the list of the\nherbal plants used in epilepsy therapy along with other species belonging to the\nfamily i.e., Brassica nigra<\/em> during\nthe 16 \u2013 17th<\/sup> century Renaissance period.8 <\/sup>There are two\nstudies already done on B. nigra<\/em> (black\nmustard) which belongs to same family, conducted using PTZ model in mice and\npenicillin model in rabbits. And it has been proven to have anti-convulsant\nproperty.9,10<\/sup> Though S. alba<\/em>\nis extensively used in alternative medicine, there are no published scientific\nreports regarding the same. So, in this study we evaluated the effect of Sinapis alba<\/em> seed oil (yellow mustard)\nin maximal electroshock-induced epileptic rat models. Several studies have been\ncarried to check the efficacy of anti-epileptic drug sub-therapeutic dose\ncombinations with products of plants with antioxidant properties in different\nepileptic models.13, 18  <\/sup>Therefore,\nwe wanted to check if the combination of S.\nalba<\/em> oil with sub-therapeutic dose of sodium valproate had some synergistic\neffect.<\/p>\n\n\n\n

In the MES model, the standard sodium valproate had\n100% protection, while yellow mustard seed oil showed 33.33% protection and a\ncombination of half-dose sodium valproate (150mg\/kg) and yellow mustard oil 200\nmg\/kg showed 66.66% protection. Prevention of THLE correlates with reduced\nseizure spread to the brain and hence inhibits neuronal death.14<\/sup>\nHistopathological results showed no significant difference since this was an\nacute model; there was not much of structural changes in the CA3 region of the\nhippocampus. However, when compared, the MES-disease control showed some flamed\npyknotic cells, while the test, standard and combination group was at par with\nthe normal control. This suggests that mustard oil and its combination was able\nto prevent neuronal loss.<\/p>\n\n\n\n

The biochemical tests conducted showed raised MDA and\nnitrite levels, levels of SOD and GSH (reduced form) declined in the MES\ncontrol, this is in agreement with previous studies conducted.19<\/sup> Our\nstudy evidently reduced MDA and nitrite levels and enhanced SOD, GSH levels\nsuggesting the anti-oxidative action of S.\nalba<\/em> seed oil. The oxidative enzyme levels almost matched the normal levels\nin animals administered with standard sodium valproate and combination of\nhalf-dose SV and yellow mustard oil. The overall result obtained conveys that\ncombination of yellow mustard oil (200 mg\/kg) and sub-therapeutic dose\nvalproate (150mg\/kg) has some additive effect in protection against THLE and\ncontrolling GTCS, since the data was comparable with the standard. <\/p>\n\n\n\n

Antioxidants are ideal in amelioration of the diseases\nlike Alzheimer’s, epilepsy, stroke etc.20<\/sup> Metabolic oxidative stress\ncreates imbalance between levels of pro-oxidants and antioxidant species. ROS, reactive\nnitrogen species and lipid peroxides form the pro-oxidative agents, while\ncatalase, SOD, GSH helps in neutralizing these. Brain consumes higher amount of\noxygen to cope up with the extensive metabolic processes. Hence, it undergoes a\nlot of oxidative damage.21<\/sup> Oxidative stress with marked increase in\ngeneration of free radicals is observed after seizure insult, which further suppresses\nthe neuronal system and attributes to epileptogenesis, followed by\nprecipitation of spontaneous recurrent seizures after a few months to years.22<\/sup>\nFrantseva et al., have shown that free radicals are generated upon incidence of\nseizure, and they are linked to seizure-induced neuronal cell death mainly in\nthe hippocampal region.23<\/sup> However, it is also observed that\nantioxidants can inhibit the lipid peroxidation reactions and reduce cell loss\nin the hippocampus.23<\/sup> In vivo study conducted earlier showed that mustard\nseed can enhance the levels of SOD, GSH, catalase and reduced the levels of\nlipid peroxide marker-MDA.15,24-26<\/sup><\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The current study shows that Sinapis alba<\/em> oil has demonstrated anti-epileptic effect in MES\nmodel and it can be considered as an add-on drug to the current anti-epileptic\nregimens. Hence, this study adds to the scientific literature and provides a\nplatform for detailed molecular studies on Sinapis alba extract in different\nforms of epilepsy. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

None<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is are no conflict of interest.<\/p>\n\n\n\n

Ethical clearance<\/strong><\/p>\n\n\n\n

The protocol was approved by (IAEC\/KMC\/65\/2021) by the Institutional Animal Ethics Committee, KMC, MAHE, Manipal. The study was conducted as per Committee for Control and Supervision of Experiments on Animals (CCSEA) guidelines.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The study is funded by postgraduate thesis grant, Manipal Academy of Higher Education, Manipal, Thesis Research Grant number: PGR600<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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