|Year : 2022 | Volume
| Issue : 3 | Page : 134-141
Potential Efficacy of Ocimum sanctum Hydro-Alcoholic Leaf Extract as an Adjuvant Role with Phenobarbital: Acute Models of Epilepsy on Mice
Aman Shrivastava1, Jeetendra Kumar Gupta1, Manoj Kumar Goyal2
1 Department of Pharmacology, Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh, India
2 Jai Institute of Pharmaceutical Sciences and Research, Gwalior, Madhya Pradesh, India
|Date of Submission||23-Mar-2022|
|Date of Decision||16-Apr-2022|
|Date of Acceptance||18-Apr-2022|
|Date of Web Publication||08-Jul-2022|
Research Scholar, Department of Pharmacology, Institute of Pharmaceutical Research, GLA University, Mathura (UP), 17km Stone, NH-2, Chaumuhan, India - 281406
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: In Ayurveda, various Ocimum species have therapeutic potential to enhance the therapeutic efficacy of some antiepileptic drugs in epilepsy. O. sanctum has two flavonoid compounds that are orientin and vicenin, and both are responsible for their anti-seizure properties in epilepsy. Material and Methods: The ultraviolet spectroscopy instrument was used to detect the absorbance of light by the active constituent present in the herbal extract at various concentrations. A turbidity meter was used to detect the amount of turbidity in the sample. Phenobarbital (PB 40 mg/kg, per oral [p.o.]) and O. sanctum hydroalcoholic leaf extract (OSHE 1000 mg/kg, p.o.) were administered every other day for 2 weeks in which two acute models, maximal electroshock and pentylenetetrazole (PTZ), models of epilepsy were used. Result: The outcome result data were statistically compared and showed Tukey test P < 0.05 of significant anticonvulsive activity as compared to control and standard (phenobarbitone). Conclusion: We have investigated the anti-seizure activity of PB with hydroalcoholic leaves extract of O. sanctum L. by using the electrically maximal electroshock seizure and chemically (PTZ) induced convulsion acute models of epilepsy on mice. As per the histopathological study, photomicrographs (×40) of mice brain tissue showed no neuronal degenerations or focal microglia sensitivities in OSHE + PB treated group. We concluded that the standard drug exhibits a synergistic effect with OSHE for the treatment of acute seizures in mice.
Keywords: Adjuvant role, anticonvulsant activity, antiepileptic drugs, models of epilepsy, Ocimum sanctum L., phenobarbital
|How to cite this article:|
Shrivastava A, Kumar Gupta J, Kumar Goyal M. Potential Efficacy of Ocimum sanctum Hydro-Alcoholic Leaf Extract as an Adjuvant Role with Phenobarbital: Acute Models of Epilepsy on Mice. Int J Nutr Pharmacol Neurol Dis 2022;12:134-41
|How to cite this URL:|
Shrivastava A, Kumar Gupta J, Kumar Goyal M. Potential Efficacy of Ocimum sanctum Hydro-Alcoholic Leaf Extract as an Adjuvant Role with Phenobarbital: Acute Models of Epilepsy on Mice. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 Nov 29];12:134-41. Available from: https://www.ijnpnd.com/text.asp?2022/12/3/134/357251
| Introduction|| |
Epilepsy is a fourth common neurological condition characterized by repetitive uncontrolled seizures that affect 1% to 2% of the population around the world. The most effective treatment for epilepsy is the long-term use of anticonvulsant medicines. The potency of an anticonvulsant drug depends on different kinds of epileptic seizures as well as also includes the effect of its tolerability and stability. Both brain hemispheres might be affected during generalized epileptic seizures. Only one region of the brain is affected by a simple partial seizure. Partial (focal) seizures are more common and occur in the temporal part of the brain. The type of epilepsy or epileptic syndrome can be categorized by patient-related physical characteristics such as seizure type, epidemiology, time of onset, and age [Figure 1]. Epilepsy affects more than 1% of the total population. Approximately 30% of patients around the world do not give a response to traditional drug therapies but the use of some traditional drugs has better therapeutic efficacy when taken with suitable antiepileptic drugs (AEDs). Many theoretical epileptic models are commonly used for simulating epileptic activity and exploring various medication effects that have been utilized to evaluate the neurochemical, neurological, biological, and cellular pathways that regulate epileptic convulsions. Furthermore, studies on all of these models have resulted in the analysis of pathways that are common in human epilepsy. Experimental models of epilepsy are primarily induced by chemical convulsing substances and can be classified into several types. The traditional system is considered to be a significant component of natural products with therapeutic potential. Herbs can also have anticonvulsant properties in a variety of ways. For years, epileptic seizures have been treated using a variety of AEDs. AEDs such as carbamazepine, phenytoin, and valproic acid, as well as recent AEDs such as levetiracetam, tiagabine, vigabatrin, and phenobarbital (PB), are safer than other drugs. Previous research studies in India reported that 9.8% to 59.9% of patients with epilepsy were using PB. Recently, various AEDs drugs are used as a monotherapy or as an adjuvant role with other traditional drugs.
|Figure 2 Graphical representation of studies on acute models of epilepsy|
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Traditional health care professionals use medicinal plants frequently in their everyday practice to treat various diseases and disorders. In different regions of the world, various Ocimum species are used for the treatment of severe disorders. The specification of plant (Botanical name: Ocimum tenuiflorum L.; Family: Lamiaceae; Synonym: O. sanctum l.; Traditional name: Tulsi) that are extensively used to treat central nervous system disorders and its therapeutic psychotic effects have been already reported. When the leaves of Ocimum species are squashed between the palms, they release a pleasant odor that can also be used as a culinary condiment or for pest control. Therefore, O. sanctum L. species herbs can be used for the treatment of epilepsy as well as also reducing the epileptogenic effect of some drugs. For a variety of reasons, experimental models have been used in various epilepsy studies. They have also been used for evaluating more effective antiepileptic medications. Animal models are frequently used to optimize the compound’s efficacy and safety in various forms of epilepsy. Epilepsy generally requires long-term treatment to minimize the frequency of seizures. PB is mainly used to treat cognitive impairment medications (pharmacokinetics or pharmacodynamic parameters) with a broad range of other drugs and vitamin supplements. Various neuroprotective strategies have been investigated in various animal models. It is also used in Ayurveda for the treatment of cardiovascular and nervous disorders. Phytoconstituents also improve the bioavailability of the O. sanctum and enhance therapeutic value. According to Ayurveda studies, plants and phytoconstituents have been used to cure diseases since ancient times. O. sanctum L. has been used to heal epileptic disorders. The occurrence of oleanolic acid and ursolic acid as the main ingredients of O. sanctum extract was detected through phytochemical analysis. In the study, we stated the adjuvant value of O. sanctum L. with PB by pharmacological evaluation of their antiepileptic activity on different experimental models on mice. Previous in vivo research and folkloric uses based on the anticonvulsant efficacy of Ocimum leaves extracts were limited to acute seizure models such as pentylenetetrazole (PTZ) and maximal electroshock seizure (MES) model. We have evaluated both acute seizure models on animals. These studies indicate that PB may react pharmacodynamically with several other neuro-modulatory compounds and herbal supplements. For acute seizures, the MES model is a very standard common technique for identifying drug molecules with anticonvulsant efficacy in various models. However, the chronic seizure model is more important for understanding epileptogenesis and discovering new AEDs. The pathophysiology of epileptic seizures was investigated using laboratory epilepsy models. A well-coordinated research methodology is essential for understanding epileptic pathways as well as for innovating effective forms. Pharmacokinetic herb-drug interactions may also affect the drug absorption, distribution, metabolism, or excretion of a compound.
| Material and Methods|| |
Healthy Swiss albino mice were selected and weighed between 18–25 g of either sex chosen for In-vivo study. All experimental animals were procured and placed under (12:12 h light-dark schedule) in the animal house facility of PBRI (Bhopal, MP) to maintain a standard pelleted diet and ad libitum water with suitable ventilation and hygienic conditions. The animals were maintained in polypropylene cages with stainless steel wire meshes over a paddy husk bed. After receiving prior permission from Institute Animal Ethics Committee (IAEC), protocol approval no. PBRI/IAEC/2021/008, all animal studies were accomplished as per procedures of the Committee for the Purpose of Control and Supervision of Experiments on Animals.
Drug instruments and treatment schedule
O. sanctum hydroalcoholic extract (OSHE) 1000 mg/kg, p.o., pentobarbitone sodium (40 mg/kg PB, Abbott India Ltd.), PTZ (30 mg/kg i.p.; Sigma Chemical Co., CAS Number:54-95-5) were used in this study. O. sanctum leaves extract, PB, normal saline (NS), PTZ, ethyl alcohol, distilled water, and the instruments Soxhlet extraction assembly, UV spectrophotometer V-630 Jasco, turbidity meter, and heating mantle were used in the current study. The following groups were included in the research protocol (n = 6):
- Group 1 (PTZ): PTZ (30 mg/kg, i.p.) every other day for period of 14 days
- Group 2 (PTZ + PB): PTZ (30 mg/kg, i.p.) + PB (40 mg/kg, p.o.) every other day for period of 14 days
- Group 3 (PTZ + OSHE): PTZ (30 mg/kg, i.p.) + OSHE (1000 mg/kg, p.o.) every other day for period of 14 days
- Group 4 (PTZ + PB + OSHE): PTZ (30 mg/kg, i.p.) + PB (40 mg/kg, p.o.) + OSHE (1000 mg/kg, p.o.) every other day for period of 14 days
Preparation of plant extract
The OSHE was used in the current study. O. sanctum L. fresh leaves were collected from the medicinal garden of the Institute of Professional Studies, College of Pharmacy, Gwalior and then washed under running water. The leaves were dried at room temperature in the shade and 1 kg of dried leaf coarse powder was collected in a beaker. The powder material was soaked in water overnight and after that the solution was filtered with Whatman no. 1 filter paper and then Soxhlet apparatus was used for extraction of active compound from O. sanctum L. (Tulsi) leaves.
Preliminary phytochemical study
Phytochemical screening was performed to detect the existence and absence of individual phytoconstituents such as alkaloid, flavonoid, and carbohydrates in O. sanctum leaves extract [In [Table 1].
a. Test for alkaloids: The isolated extract of the drug was prepared with ammonia and mixed with chloroform addition. Then acidified and well shaken with hydrochloric acid diluted and now filter this medium. This extract of filtrate was used to test for alkaloids.
b. Hager test: Hager reagent was processed with the filtrate or added with a few drops of Hager reagent. The formation of yellow-precipitated particles indicated the presence of alkaloids.
c. Wagner test: With a few drops of Wagner reagent, an acid layer was formed then precipitated reddish-brown formation showed the existence of alkaloids.
d. Flavonoid test: A few drops of 1% liquor nitrate were put into the test tube and then test samples were taken on the test tube. If a yellow color forms after adding the sample, this indicates the presence of flavonoids in the sample.
e. Test for terpenoids: Take 2 mL of chloroform, add 3 mL of concentrated sulfuric acid, and then add 5 mL of the sample. A reddish-brown color formation means terpenoids are present in the sample.
f. Cardiac glycosides: Add 5 mL of the sample with 2 mL of glacial acetic acid or add two drops of ferric chloride. The shape of the brown ring indicates a valid experiment outcome.
g. Test for tannins: Add 5 mL of the sample with a few drops of 0.1% ferric chloride and pour to the beaker. The black color indicated that the drug contains tannins.
h. Test for saponins: Take 5 mL of the drug sample with 3 mL of distilled water and shake well and then add a few drops of the drug sample. The solution is converted into foam, indicating the presence of saponins.
UV spectrophotometers are mainly used in the study to determine the visible regions of UV light and can provide valuable information about the concentration of active ingredients present in herbal extracts as well as used to detect any impurities in the sample. A UV/Vis spectrophotometer (Jasco) was used to detect the absorbance of light from a sample. The different concentrations of plant extract (100, 500, and 1000 μg/mL) were used for the determination of the absorbance value at 270. This quantitative method was used in the study to detect the impurities and active constituents in the sample drug (O. sanctum L.) that are responsible for the therapeutic effect in epilepsy.
Acute toxicity study
The study was followed as per Organization for Economic Co-operation and Development (OECD) 423 guidelines. An acute toxicity study was conducted to identify the safe dose range by using acute oral toxicity.
Acute epilepsy models on experimental animals
Maximal electroshock induced convulsion in mice
The Swiss albino mice (18–25 g) of either gender were divided into four groups with six animals in each group. Mice were divided into four groups (control [NS], PB, OSHE, PB + OSHE). Group I received NS; Group II was given as a standard and received PB (40 mg/kg, p.o.); Group III was used as a test group and was administered with OSHE (1000 mg/kg, p.o.); and Group IV was served as test group and received the combination of PB (40 mg/kg, p.o.) and OSHE (1000 mg/kg, p.o.). This investigation was conducted on the 14th day of administration of OSHE and PB [Table 4], different stages of tremor and latency of convulsion were recorded after 60 min, 60 mA voltage current was administered trans auricularly for 0.2 second via minor alligator clips anchored to the cornea using an extracellular matrix [Figure 5].
Chemical induced seizures pentylenetetrazole
Swiss albino mice (18–25 g) were divided into four groups (Negative control [PTZ], PTZ + PB, PTZ + OSHE, PTZ + PB + OSHE) with six animals in each group. Group I was used as a negative control and was administered PTZ (30 mg/kg i.p.) 60 min after the administration of NS; Group II served as a standard group and received PB (40 mg/kg, p.o.) and after 60 minutes they were treated with PTZ (30 mg/kg); Group III served as a test group and was treated with PTZ (30 mg/kg) 30 minute after administration of OSHE (1000 mg/kg, p.o.); and Group IV was also taken as a test group and received the combination of PB (40 mg/kg, p.o.) and OSHE (1000 mg/kg, p.o.) and after 60 min they were treated with PTZ. This investigation was conducted on the 14th day of administration of OSHE and PB; different phases of convulsion of every group, after administration of drugs, the time of latency of convulsion (sec), and neurobehavioral parameters were recorded [Figure 6]. After injecting PTZ through the intraperitoneal route, the onset of action (indicated by Straub tail jerky movement of whole body and convulsion) as well as the duration of latency of convulsion was observed in seconds [Table 5].
Histopathological study of mice hippocampus tissue
Half of the mice’s brain cerebral cortex was removed to isolate hippocampus and preserved in 10% formalin solution for histopathology investigation. Dehydrated tissues were implanted in a Petri dish. A blinded investigator sliced a 5 μm paraffin segment, colored it with hematoxylin dye, mounted it on a microscopic slide, and viewed it under a biological scientific microscope (Quasmo digital microscope, Pvt., Lmt., India). The remaining cells were identified as round-shaped nuclei with complete cytoplasmic membranes and no nuclear shrinkage or deformed appearance. The acidophilus cell, which was characterized by cytoplasmic eosinophilia, chromatin scatter, and loss of nuclear membrane permeability, was considered to be a biological marker for incurable neurodegeneration. The Golgi type II cells showed larger than the nuclei of granule cells, medulla of white matter, Purkinje cell layer.
All experimental values are presented as the mean ± standard error of the mean. All the graphs and charts were prepared by MS Excel 2016 Version. The results were determined statistically by using an analysis of variance followed by Tukey test; P < 0.05 was considered to be as significant.
| Result|| |
Phytochemical screening of O. sanctum L.
Determination of absorption maxima
The leaf extract of Tulsi (O. sanctum) was taken and diluted by 100, 5, and 1000 ug/ml.Three samples were scanned by UV and the absorption maximum was found at 270 for the leaf extract of O. sanctum [Figure 3].
|Figure 3 UV spectrophotometric analysis of an extract of O. sanctum L. absorption in different concentrations|
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Turbidity estimates by nucleation assay
The formula for determining the turbidity is as per the Lamber-beer method [Table 2] and [Figure 4].
|Figure 4 UV spectrophotometric analysis of turbidity in an O. sanctum L. extract at various concentrations|
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|Figure 5 Maximal electroshock induced convulsion: Group I: Control group showed 100% convulsion. Group II: PB showed 100% protection against convulsion. It does not cause major convulsion. Group III: Test I treated with OSHE showed 50% protection and delays onset of the time of convulsion. Group IV: Test II treated with PB+OSHE showed 83.33% protection and delays the onset of the time of convulsion|
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|Figure 6 PTZ-induced convulsion: Group I: Negative control group (showed 100% convulsion). Group II: PB (showed 100% protection against convulsion). It does not cause major convulsion. Group III: Test I treated with OSHE showed 66.66% protection and delays the onset of the time of convulsion. Group IV: Test II treated with PB+OSHE showed delays in the onset of the time of convulsion and 83.33% protection in convulsion|
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Turbidity = (2.2 absorbance)/L
Acute oral toxicity study
According to OECD 425 guidelines, a study of OSHE acute oral toxicity was carried out to attain the optimum tolerable dose of the test preparation under study. There was no mortality observed with the doses 500, 1000, and 2000 mg/kg of body weight [Table 3].
In different treated groups, histopathology study demonstrated neuropathological changes in tissue such as Golgi type II cells, the white matter medulla, the Purkinje cell layer, dendrites of mice brain tissue [Figure 7]. The histological findings of seizure induced by PTZ, OSHE + PB treated group showed no neuronal degenerations or focal microglia sensitivities. In the PB-treated group, there was substantial neuronal degeneration, Microglia play the vital role in initiation of neuronal disruption. Moderate neuronal degenerations with regional microglial reactions were reported in the OSHE-treated group.
|Figure 7 Photomicrographs (×40) of mice hippocampus brain tissue: In different treated groups, histopathology study demonstrated neuropathological changes in tissue such as Golgi type II cells, the white matter medulla, and the Purkinje cell layer, dendrites of mice brain tissue. The histological findings of seizure-induced by PTZ, OSHE + PB treated group was showed no neuronal degenerations or focal microglia sensitivities. In the PB-treated group, there was substantial neuronal degeneration, no neuronal disruption, and microglia. Moderate neuronal degenerations with regional microglial reactions were reported in the OSHE-treated group|
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| Discussion|| |
In our study, we discovered the synergistic effect of O. sanctum with PB drug on the MES and PTZ model of epilepsy. PTZ has been shown to activate membrane phospholipases, proteolytic enzymes, and nucleases, leading to the destruction of lipid membranes, proteolysis of microtubule proteins, and protein oxidative phosphorylation in the cell. The latency period of convulsion data has been recorded for all models, which is consistent with previous findings. We have also found out the percentage of protection through different models. The current study demonstrated the antiepileptic effect of PB with OSHE. We have investigated the anti-seizure activity of PB with hydroalcoholic leaves extract of O. sanctum L. by using the electrically (MES) and chemically (PTZ) induced convulsion acute models of epilepsy on mice [Figure 2]. According to previous research, PB is another AEDs that has minimal or no interaction with other drugs. It has been found that numerous plant-derived bioflavonoids exhibit their synergistic action in animal models of epilepsy. The current study found that PB has no harmful effect when it is taken with O. sanctum L. (Tulsi). This result indicated that Ocimum leaves extract has a synergistic effect with anticonvulsant drugs and can be used to minimize the frequency of epileptic seizures. About one-third of the population probably suffers from unpreventable brain abnormalities caused by neurological complications, as well as some usually related adverse reactions and neurotoxicity. It was concluded that frequent use of GABA-enhancing substances can result in reduced GABAergic mechanisms, major changes in GABAA receptors, and also sensitivity and dependence, particularly to sedatives and the effects of anticonvulsants. Ocimum extract has the potential to act as an adjuvant with low doses of standard AEDs, as a reduction in antiepileptic medication dose levels can reduce the undesirable effect of pharmaco-resistance and respond through a multi-targeted strategy.
| Conclusion|| |
Based on the above discussion, it is clear that Ocimum enhanced the therapeutic potential and may increase the efficacy of PB in epilepsy. Neurodegeneration is primarily caused by seizures. In addition, group of neuronal networks fluctuates by impulse conductance occurs in the hippocampus region in the brain. Some epileptogenic drugs are also responsible for decreasing the effect of PB. There is little evidence of O. sanctum efficacy in acute models of epilepsy. This seems to be the first study of its kind to demonstrate the neuroprotective and anticonvulsant properties of Ocimum leave extract in acute seizure models. Similarly, this is a preliminary attempt to investigate the pharmacodynamics interaction study of O. sanctum L. with AED (i.e., PB). A spectrophotometer UV/Vis was used to measure the absorbance of the sample and a turbidity device was used to distinguish active compounds by the quantity of turbidity in the sample. Both the assays showed positive results as per the objective of the study. The histological findings of seizure induced by PTZ, OSHE + PB treated group showed no neuronal degenerations or focal microglia sensitivities. In the PB-treated group, there was substantial neuronal degeneration, Microglia play the vital role in initiation of neuronal disruption.. Moderate neuronal degenerations with regional microglial reactions were reported in the OSHE-treated group. Further investigations are needed to figure out the exact bioflavonoids responsible for the mechanism behind O. sanctum L. for their anticonvulsant potency in combination with PB as well as molecular/receptor-based investigations are required.
The authors are thankful to G.L.A University, Mathura, (UP) and the Institute of Professional Studies College of Pharmacy, Gwalior for their support. Special thanks to Dr Jeetendra Kumar Gupta, Assistant Professor of G.L.A University, for his guidance and support.
IAEC protocol approval no. PBRI/IAEC/2021/008
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]