Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Mohan Z Mani

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Professor & Head,
Department of Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2024 | Month : February | Volume : 18 | Issue : 2 | Page : FC01 - FC05 Full Version

Antioxidant Effect of Levetiracetam and Brivaracetam Acting on Presynaptic SV2A Receptors: An In-vitro Study


Published: February 1, 2024 | DOI: https://doi.org/10.7860/JCDR/2024/66773.18996
Riya Kataria, Kranthi Karunai Kadal, Kavitha Ramasamy, Kruthi N Parikh

1. Intern, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India. 2. Assistant Professor, Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India. 3. Professor and Head, Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India. 4. Junior Resident, Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India.

Correspondence Address :
Kranthi Karunai Kadal,
Assistant Professor, Department of Pharmacology, SRIHER, No.1, Sri Ramachandra Nagar, Porur, Chennai-600116, Tamil Nadu, India.
E-mail: drkranthi10@gmail.com

Abstract

Introduction: Epilepsy is a serious psychological condition associated with social stigma, psychiatric co-morbidity, and a high economic burden. It is pertinent to develop novel treatment methods given the ever-evolving nature of the disease. Recently, various newer Antiepileptic Drugs (AEDs) have been under study, among which a newer targeted modality consists of SV2A receptors.

Aim: To compare the antioxidant properties or free radical scavenging activity of the newer heterogeneous AEDs acting on the SV2A receptors, namely Levetiracetam (LEV) and Brivaracetam (BRV).

Materials and Methods: This in-vitro study was conducted at Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India from July 2021 to August 2021. The study evaluated the antioxidant properties of LEV and BRV using various assays such as the Ferric Reducing Antioxidant Power (FRAP) test, DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, Nitric Oxide (NO) radical scavenging assay, Superoxide Dismutase activity (SOD), and catalase activity. The obtained results were expressed using specific formulas related to the absorbance of the respective chemicals, along with the percentage of inhibition, and have been tabulated in Microsoft excel Sheet Version 16.16.27. Half-maximal Inhibitory Concentration (IC50) values were calculated using scatter plot graphs.

Results: The FRAP values for LEV were reported to be significantly higher compared to BRV at concentrations of 20, 40, 60, 80 μg/mL, while at 100 μg/mL, BRV showed better ferric reducing activity. Although both drugs exhibited antioxidant activity, the results clearly identified LEV as a better NO radical scavenger, with a percentage of inhibition reaching up to 80.52%. During the SOD assay, the percentage inhibition of superoxide generation by LEV was found to be 99.02%, while that of BRV was 97.07% at a concentration of 100 μg/mL. LEV (0.573) also showed a higher degradation of H2O2 per minute than BRV (0.065) at a concentration of 100 μg/mL.

Conclusion: The results showed that both drugs, BRV and LEV, exhibited significant antioxidant capacity. However, LEV demonstrated increased antioxidant potency and efficacy compared to BRV.

Keywords

Antiepileptic drugs, Ferric reducing antioxidant power assay, Nitric oxide assay, Oxidative stress, Reactive oxygen species reduction

Epilepsy is a chronic non communicable brain disorder that affects people of all ages. A 2017 meta-analysis revealed that the overall incidence of epilepsy is 67.77 per 100,000 persons. Most drugs targeting epilepsy are ion channel blockers. It is estimated that 5 million people are diagnosed with epilepsy annually worldwide (1). Recurrent seizures stem from hyperexcitation of specific groups of neurons, leading to various transient clinical signs and laboratory findings (2).

Epileptic seizures can occur due to neuronal cell death or as a consequence thereof. Several contributing factors include genetic components, the extent of excitotoxicity due to glutamate, which ultimately leads to disruptions in intracellular electrolyte metabolism, increased cytokine concentration, and heightened oxidative stress (3). Oxidative stress occurs due to an imbalance between the body’s antioxidant defense system and free radical production. It remains an important mechanism that plays a role in the aetiology of seizure-induced neuronal death. The most perilous effect of free radicals is lipid peroxidation, leading to the disruption of cell membranes (4).

The human brain, utilising the maximum amount of oxygen compared to other bodily organs, is particularly susceptible to oxidative stress. It contains high concentrations of polyunsaturated fatty acids that are prone to lipid peroxidation and low levels of antioxidant enzymes. Additionally, owing to the high aerobic metabolic rate, the brain itself produces elevated amounts of Reactive Oxygen Species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide (5).

LEV and BRV belong to a group of pyrrolidone compounds derived from piracetam, binding at the presynaptic SV2A receptor site, representing a group of AEDs with a distinctive mode of action (6),(7). In the current treatment method, drugs having combined antiepileptic and antioxidant actions are limited (2),(8). Older generation antiepileptic drugs have been shown to increase the oxidant load, leading to an elevated damage rate; however, a few clinical studies have hypothesised probable neuroprotective effects of LEV in neurodegenerative diseases (9),(10),(11). The present study is the first to evaluate the antioxidant effects of the above-mentioned AEDs using an in-vitro model, which should be further validated in an in-vivo model.

The current study aimed to evaluate the antioxidant properties of two newer generation AEDs, LEV and BRV, with the help of an in-vitro model. The experiment was designed to assess the antioxidant properties of these drugs by comparing their antioxidant efficacy not only with a standard but also by analysing and establishing the superior antioxidant agent among the two novel AEDs.

Material and Methods

It is an in-vitro study model using standard assays under Good Laboratory Practice (GLP). The study was carried out at Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India for a period of two months-July 2021 to August 2021. The study was conducted after obtaining Institutional Ethics Committee approval (REF:CSP/21/JAN/89/54).

Study Procedure

Drugs and chemicals: LEV and BRV-4 tablets of 250 mg each were obtained from Intas. These tablets were dissolved in buffered saline and sonicated for 10 minutes. This was followed by centrifugation and filtration, and then serial dilution. Finally, these were tested in various concentrations of -20, 40, 60, 80, 100 μg/mL.

All the reagents and other chemicals used in the present study were of analytical grade. The chemicals DPPH (1,1-diphenyl-2-picrylhydrazyl), TPTZ (2,4,6-tripyridyl-S-triazine), 2-thiobarbituric acid, ascorbic acid, and Folin-Ciocalteu reagent were obtained from Sigma Chemical Co., 2,2’-Azobis (2-amidinopropane) dihydrochloride (AAPH) and Trolox (6-hydroxy-2,5,7,8-tetramethyl chromane 2-carboxylic acid) were obtained from Sigma Aldrich Chemicals Co., Mumbai, India.

1. FRAP Assay: Ferric Reducing Antioxidant Power (FRAP): The method is based on the principle of electron donating antioxidants that lead to the reduction of Fe3+ TPTZ complex (colourless complex) to Fe2+-tripyridyltriazine (a blue-coloured complex) at a low pH (12). The next step is to observe and measure the change in absorbance at 593 nm. The FRAP reagent was prepared by mixing 300 mM acetate buffer, 10 mL TPTZ in 40 mM HCl, and 20 mM FeCl3.6H2O in the proportion of 10:1:1 at 37°. It was then pipetted using a 1-5 mL variable micropipette (3.995 mL) and mixed thoroughly with various concentrations (20 mg/mL, 40 mg/mL, 60 mg/mL, 80 mg/mL, 100 mg/mL) of test samples. The ferric tripyridyl triazine (Fe3+ TPTZ) complex was reduced to the ferrous (Fe2+) form, and a profound blue-coloured complex was observed. The absorbance at 593 nm was further recorded against a reagent blank (3.995 mL FRAP reagent + 5 μL distilled water) spectrophotometrically following the procedure of Benzie and Strain after 30 min incubation at 37° (10). All the determinations were performed in triplicates. After plotting the absorbance at 593 nm versus different concentrations of FeSO4, a calibration curve was created. The concentrations of FeSO4 were also compared against various concentrations of the standard

FRAP Value (μmol/L)= 4´ Absorbance at 590 nm of test sample reaction mixture/ 4´ Absorbance at 590 nm of Fe2+ STANDARD reaction mixture ×Fe2+ Standard concentration

antioxidant Ascorbic acid. The FRAP values were finally obtained after contrasting the absorbance change in the test mixture against those observed from increasing concentrations of Fe3+. These values are expressed here as mg of Ascorbic acid equivalent per gram of the sample. The FRAP values of test samples were calculated from their absorbance readings in relation to the absorbance reading of the standard according to the following formula (13).

2. Reduction of DPPH Assay: DPPH (1,1-diphenyl-2-picrylhydrazyl) Assay: The 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay method is based on the reduction of a stable free radical, DPPH. It is a rapid, straightforward, inexpensive method to assess the ability of drugs and compounds to act as free radical scavengers or hydrogen donors while evaluating their antioxidant activity. A maximum absorption at 517 nm (purple colour) is recorded with the free radical DPPH, which contains an odd electron. The absorbance is decreased from DPPH when it reacts with antioxidants and is reduced to DPPH-H. The radical to the DPPH-H form then results in the decolourisation of the assay to yellow. An increase in the decolourisation signifies intense reducing ability.

The DPPH solution was prepared by dissolving 4 mg of DPPH in 50 mL of methanol. The standard, Butylated Hydroxytoluene (BHT) solution, was prepared by mixing 10 mg of BHT in 10 mL of methanol.
The test drugs, LEV and BRV, were tested in concentrations of 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL. An aliquot of 3.7 mL of absolute methanol was taken in all test tubes, and 3.8 mL of absolute methanol was added to the blank, as described by Molyneux P (14). The scavenging effect of LEV and BRV on the DPPH radical was calculated using the following formula (15).

Calculation:

% Antioxidant activity= (Absorbance at blank)-(Absorbance at test)/ (Absorbance at blank) ×100

3. Nitric Oxide (NO) Radical Scavenging Assay: The activity of this assay was prepared as per Kumar S et al., who described the preparation of the reagent to measure the NO radical scavenging activity of methanolic fruit extracts, and a similar procedure was followed in the present study (16). The NO radical scavenging activity of LEV and BRV on the DPPH radical was calculated using the following formula (17).

Formula:

% scavanging= OD of control-OD of Test/ OD of control ×100

4. Assay of Superoxide Dismutase (SOD): SOD is an important antioxidant enzyme that plays a critical role in protecting cells from oxidative stress. An assay for SOD activity can be used to measure the enzyme’s ability to convert superoxide anions into Hydrogen Peroxide (H2O2) and Oxygen (O2). One commonly used assay for SOD activity is the Nitro Blue Tetrazolium (NBT) reduction assay. In this assay, SOD activity is measured by monitoring the reduction of NBT by superoxide anions. The assay mixture contained 1.8 mL of Sodium pyrophosphate buffer, 0.1 mL of PMS, 0.3 mL of NBT, 0.6 mL of the enzyme preparation, and water in a total volume of 2.8 mL mixed with the sample according to the modified spectrophotometric assay model described by Kakkar P et al., (18). The sample is exposed to light, which then generates superoxide anions. The superoxide anions react with NBT, reducing the blue formazan dye. SOD converts superoxide anions to H2O2, which does not react with NBT, resulting in less blue formazan dye being formed. The amount of blue formazan dye formed is measured spectrophotometrically at 560 nm, and the decrease in absorbance due to SOD activity is used to calculate the compound’s activity (19).

Calculation:

% INHIBITION=(A560 nm of blank-A560 nm of test)/A560 nm of blank×100

5. Assay of Catalase: The activity of Catalase was determined as described by Sinha AK (20). Catalase is a ubiquitous antioxidant enzyme that degrades hydrogen peroxide into water and oxygen, eventually protecting cells from oxidative damage. The catalase assay is a laboratory technique used to measure the activity of the enzyme in a sample. The steps involve first preparing the sample, and then the reaction mixture is prepared by mixing the sample with a substrate solution containing hydrogen peroxide. The mixture is then incubated for a specific period of time, during which catalase in the sample reacts with the hydrogen peroxide and produces oxygen and water. The amount of oxygen produced in the reaction is measured calorimetrically using a suitable reagent. The catalase activity is calculated based on the change in absorbance of the reaction mixture over time. An overview of the study flowchart is given in (Table/Fig 1).

Statistical Analysis

Spectrophotometry using UV probe software is used to gather the data. The obtained results are expressed using specific formulas regarding the absorbance of the concerned chemical, along with the percentage of inhibition, and have been tabulated in Microsoft excel sheet version 16.16.27. Half-maximal Inhibitory Concentration (IC50) values are calculated using a scatterplot graph.

Results

1. FRAP Assay: The reducing ability of the drugs was found to be in the range of 1298 μmol/L to 801.11 μmol/L. The FRAP values for LEV were recorded at an elevated level compared to BRV at concentrations of 20, 40, 60, 80 μg/mL. At these concentrations, both BRV and LEV showed superior ferric reducing potential than standard ascorbic acid (Table/Fig 2). The results suggested that LEV has a consequential ability to react with free radicals, altering them into more stable non reactive species and finally terminating the radical chain reaction. The IC50 values of BRV and LEV were noted as 251.13 μg/mL and 117.92 μg/mL, respectively.

2. Reduction of DPPH assay: There was a dose-dependent increase in antioxidant potential of both drugs, similar to that of standard BHT (Table/Fig 3). IC50 values of BRV and LEV were recorded as 24.475 μg/mL and 23.719 μg/mL, respectively (Table/Fig 4). These results indicate that BRV and LEV have a marked effect on scavenging free radicals, especially at higher doses.

3. Nitric Oxide (NO) radical scavenging assay: Though BRV showed potent antioxidant capacity, the IC50 value remained at 9.076 μg/mL, with its inhibition percentage reaching up to a maximum of 79.20% (Table/Fig 5). The results distinctly identified LEV as a better NO radical scavenger, where the percentage of inhibition reached up to 80.52% with an IC50 value of 8.432 μg/mL.

4. Assay of Superoxide Dismutase (SOD): The superoxide scavenging activity of both drugs was noted to be significant in contrast to ascorbic acid used as a standard (Table/Fig 6). The percentage inhibition of superoxide generation by LEV was found to be 99.02%, while that of BRV was at 97.07% at a concentration of 100 μg/mL (Table/Fig 7). These results indicate that LEV has a more potent effect of scavenging the free radicals, with an IC50 value of 11.66 μg/mL, while that of BRV was noted as 109.19 μg/mL.

5. A Assay of Catalase: LEV (0.573) showed higher degradation of H2O2/min than BRV (0.065) at a concentration of 100 μg/mL. Catalase activity was expressed as mol of H2O2 consumed/min/mg of protein (Table/Fig 8). The results demonstrated better hydrogen peroxide scavenging activity of LEV compared to BRV. Considering all the assays, both the drugs BRV and LEV, compared to the standard, exhibited significant antioxidant capacity. However, LEV showed increased antioxidant potency and efficacy than BRV.

Discussion

The role of oxidative stress in neuronal death is receiving significant interest in epilepsy research. This interest stems from the brain’s relatively weak antioxidant defense mechanisms, making it highly vulnerable to oxidative stress due to its high oxygen consumption, accounting for about 20% of the body’s total oxygen consumption. Furthermore, the neuronal membrane contains numerous polyunsaturated fatty acids, making it susceptible to lipid peroxidation, a crucial component of oxidative stress (21). Additionally, the brain harbours substantial amounts of iron and copper, which facilitate the synthesis of hydroxyl radicals (22). Neuronal cells in the brain are particularly susceptible to oxidative injury, and under pathological conditions, excessive Reactive Oxygen Species (ROS) brought on by altered redox equilibrium lead to oxidative stress, resulting in neuronal death through various mechanisms, including apoptosis, autophagy, necroptosis, pyroptosis, and ferroptosis. Prolonged convulsions induce oxidative stress, contributing to neuronal death. This supports the concept that oxidative stress plays a significant role in seizure-induced neuronal death and/or the onset of epilepsy (23),(24).

In 2004, Liang LP and Patel M demonstrated that superoxide radicals initiate chronic mitochondrial oxidative stress, which is sufficient to increase seizure susceptibility due to aging, environmental stimulation, or excitotoxin administration (25). Another study conducted by Sashindranath M et al., examined early seizure-induced oxidative stress in wild type and SOD2 (-/+) mice using the Rapid Electrical Amygdala Kindling (REAK) model, which does not induce cell death (26). Additionally, LEV has previously been shown to protect against neurotoxicity induced by oxidative stress in experimental animal seizure models (27),(28). The role of free radicals in seizures has also been supported by the successful use of exogenously administered antioxidants such as vitamin C and vitamin E to protect the brain against seizure-induced damage (29),(30).

Pharmacological studies of exogenous substances or therapeutic treatments have, for a number of years, focused on potential interactions with ROS to assess their ability to prevent or limit free radical damage to biological targets (26),(31),(32). The second-generation AEDs, BRV and LEV, are novel molecules clearly distinguished from traditional AEDs by their pharmacological properties and mechanism of action. Although the literature suggests that the anticonvulsant effects of AEDs could be directly related to their antioxidant properties, the basis of these properties remains unclear (33). The results of the present study clearly demonstrate a significant antioxidant effect of both drugs, which increased with dosage when compared to ascorbic acid. Previously reported data and studies have also demonstrated the well-recognised antioxidant effects of vitamin C. This fact suggests that vitamin C acts as a neuroprotective agent capable of reducing behavioural and pro-oxidative changes associated with the development of seizures (30).

In the present study, treatment with BRV and LEV (at all doses tested) revealed increased ferric reducing potential compared to the standard at lower concentrations, which reduced as the concentration of the drugs increased. Regarding the scavenging activity of the DPPH radical, both AEDs demonstrated increased activity at higher concentrations, such as 100 μg/mL. In terms of nitrite-nitrate concentrations, LEV and BRV showed the capacity to reduce the changes induced by heating with an increased inhibition percentage. Subsequently, the SOD activity was recorded at 99.02% at 100 μg/mL for LEV, with a slight difference from BRV at the same concentration. Finally, catalase activity of LEV was significantly higher than BRV at a concentration of 100 μg/mL, with a degradation rate of hydrogen peroxide of 0.573/min.

The existing literature reports that numerous AEDs, especially from the older generation including phenobarbitone, carbamazepine, and valproic acid, produce reactive metabolites. These metabolites can covalently bind to different endogenous macromolecules, increasing the formation of ROS and inducing oxidative damage and toxicity (31),(34). Martinc B et al., evaluated the influence of older AEDs using a meta-analysis, showing increased or inconclusive Malondialdehyde (MDA) levels in the group treated with older generation AEDs (2). Similarly, multiple studies have confirmed the theory of increased lipid peroxidation in conventional AEDs like phenobarbitone, phenytoin, and carbamazepine (35),(36). However, in the present study, results demonstrated significant antioxidant and free radical scavenging activity of LEV and BRV at certain concentrations. Additionally, when comparing the two drugs, LEV showed higher potency and efficacy when tested in-vitro using various assays.

Previously, a few studies have revealed a novel spectrum of activity in experimental seizure models (37),(38),(39). To date, the mechanisms underlying the neuroprotective effect are not well understood. Some AEDs, such as phenytoin, have been observed to reduce oxidative stress by increasing Glutathione (GSH) reductase activity (8). Considering the findings given by Marini H et al., of blunting brain MDA both in cortex and diencephalon in a kainic acid-induced seizures model due to LEV pretreatment, it is possible to speculate an activity against oxidative stress (40). Apart from epilepsy, LEV has also shown antioxidant and neuroprotective effects in Alzheimer’s disease in a number of animal studies (9). Similarly, the present findings also support this notion, as the drugs showed the ability to reduce lipid peroxidation and increase NO radical scavenging activity after inducing exogenous oxidative stress.

The conversion of H2O2 to H2O can be achieved by catalase and GSH. As stated earlier, epileptogenesis and episodes of status epilepticus lead to increased production of free radicals while suppressing the brain’s ability to activate free radical scavenging mechanisms, leading to cell death (41),(42). However, the present data shows enhanced catalase activity of BRV that is directly proportional to the dosage. Thus, the present study suggests the ferric reducing potential, free radical scavenging, increased catalase activity, and SOD potential of newer AEDs-LEV and BRV. Although a few models have been designed to study the clinical aspect of BRV and LEV, in-vitro results have not been demonstrated previously for BRV, to the best of the present knowledge (11),(22),(37).

Limitation(s)

In-vitro studies cannot replicate the human internal milieu because various physiological interactions and biochemical reactions are not accounted for. In-vitro studies lack the ability to incorporate xenobiotic metabolism, and there are practical difficulties in translating correct in-vivo doses from in-vitro concentrations. Furthermore, the long-term consequences with the help of in-vivo studies cannot be ascertained.

Conclusion

In the present study, both drugs exhibited antioxidant activity, with LEV showing increased antioxidant potency and efficacy compared to BRV, which may suggest a neuroprotective action in addition to their already known mechanisms of action. The authors propose further extended clinical research and in-vitro model designs to gather enough evidence to advocate their use in other neurodegenerative diseases.

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DOI and Others

DOI: 10.7860/JCDR/2024/66773.18996

Date of Submission: Jul 30, 2023
Date of Peer Review: Aug 11, 2023
Date of Acceptance: Dec 19, 2023
Date of Publishing: Feb 01, 2024

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Jul 30, 2023
• Manual Googling: Sep 19, 2023
• iThenticate Software: Dec 16, 2023 (17%)

ETYMOLOGY: Author Origin

EMENDATIONS: 9

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