F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.142449.1 Study Protocol Articles Comparative evaluation of dose dependent efficacy of vitamin C, in relation to oxidative stress level in subjects  with Oral Submucus Fibrosis

[version 1; peer review: 1 approved, 1 approved with reservations]

 Patel Shraddha Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-1925-0590 a 1 Patel Aditya Supervision Writing – Review & Editing 2 Lohe Vidya Supervision Writing – Review & Editing 1 Mohod Swapnil Writing – Review & Editing 1 Urkude Minakshi Resources https://orcid.org/0000-0001-7376-3584 3 Patel Satyawansingh Conceptualization Formal Analysis Project Administration Supervision Validation 4 Oral Medicine and radiology, DMIHER, Wardha, Maharashtra, 442001, India Conservative Dentistry and endodontics, DMIHER, wardha, maharashtra, 442001, India Agadtantra, DMIHER, Wardha, MAHARASHTRA, 442001, India Pharmacology, DMIHER, wardha, maharshtra, 442001, India shraddhaadityapatel@gmail.com

No competing interests were disclosed.

 10 11 2023 2023 12 1455 1 11 2023 Copyright: © 2023 Patel S et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Oral submucus fibrosis (OSMF) has a 0.2-0.5 prevalence rate in India. The malignancy rate of OSMF was found to be 7.6%. The focus of pathogenesis is currently on reactive oxygen species levels which can rise to extremely high levels, exceeding the human body’s antioxidant defense system and causing oxidative stress that goes beyond physiological limitations. Since oxidative stress plays such a significant part in carcinogenesis and other degenerative diseases, it is fair to believe that antioxidants will help to reduce or even stop these processes. Vitamin C is known for its antioxidant properties which act as a lipid-soluble free radical scavenger in cell membranes. But in some of studies a paradoxical effect of antioxidant doses was found. Thus, the present study will be conducted to evaluate the dose dependent efficacy of Vitamin C level in relation to oxidative stress level in subjects of OSMF.

 Methods

After informed consent, the patient’s examination will be conducted. For each individual, 5ml of the blood sample will be withdrawn under all the aseptic precautions and oxidative stress level will be assessed depending on MDA & SOD level in a central research laboratory. Depending on oxidative stress level patients will be divided into two groups.

Group I - Moderate oxidative stress in OSMF SI less than 240 but more than 192

Group II - High Oxidative Stress in OSMF SI more than 240

Single and double doses of Vitamin C will be given to patients accordingly and the MDA & SOD levels will be calculated on day 30,90 days and on 180 th day

 Conclusions

Positive results of the study will give direction for choosing the correct dose of the antioxidant Vitamin C for OSMF depending on oxidative stress level.

 oral submucous fibrosis reactive oxygen species oxidative stress vitamin C Intramural Funding DMIHER This study received Intramural Funding from DMIHER. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Oral submucous fibrosis (OSMF) is a common oral precancerous lesion in Asian countries, particularly in regions where betel nuts are chewed. OSMF is caused by abnormal collagen deposition in connective tissues and has an impact on mouth functions. OSMF impairs a patient’s quality of life due to discomforting symptoms such as ulceration, xerostomia, a burning sensation, and mouth-opening opening limitation. 1

The pathogenesis is still believed to be multifactorial in origin. The focus of pathogenesis is currently on reactive oxygen species. During normal metabolism, reactive oxygen and nitrogen species (ROS and RNS) are produced. ROS causes an increase in the production of enzymes such as Super Oxide Dismutase (SOD), which neutralizes reactive oxygen species at low and moderate quantities. 2 So, in everyday life, oxidation and reduction processes are linked with each other. However, in stressful situations, ROS levels can rise to extremely high levels, exceeding the human body’s antioxidant defense system and causing oxidative stress that goes beyond physiological limitations. At this point, ROS begins to harm cells in biological systems, resulting in a vicious cycle. 3 In addition, this may also encourage unmanageable lipid peroxidation, which may cause cell injuries through DNA damage and directly inhibit proteins. 4 , 5

Since oxidative stress plays such a significant part in carcinogenesis and other degenerative diseases, it is fair to believe that antioxidants will help to reduce or even stop these processes. In some situations, by preventing free radical generation, detecting free radicals, aiding repair, and creating a favorable environment. Vitamin C is known for its antioxidant properties which act as a free radical scavenger in cell membranes. 6 , 7 It also prevents the carcinogenic toxin nitrosamine from developing from nitrites in specific meals, as well as improves immune system activity. 8

Rationale

In 2014 Potter et al., published their review article in which they presented the major problems with chemoprevention remarking that some substances like beta carotin fail to provide evidence of benefit and one trial suggested excess of large colorectal polyp. 5 The author further remarked that supplemental high dose beta carotene not only reduces cancer incidence, it clearly increases risk in high risk groups. The author proposed that ROS might bring out beneficial effect through elimination of cancer cell via apoptosis and an active antioxidant may abrogate the beneficial effect of ROS.

One of the reason of these controversial observation could be paucity of knowledge regarding optimal doses of antioxidants to be used in relation to the level of oxidative stress.

Dose Related Paradoxical Effect

In 1997 Carney, in 2007 Chemilos G and in 2011 Borkar et al. have studied the efficacy of low and high dose of antioxidant in patients and found that low doses of antioxidants were found to be more efficacious whereas with the higher dose statistically significant reduction in oxidative stress was not observed. 8 – 10

From the literature search, we gathered some observations as follows

Benefits of antioxidants in case of OSMF are still not clear. Observations and conclusions of different studies differ.

For dose dependent actions of antioxidants correlating to oxidative stress levels, literature search through leading search engine the scholar could not find any study regarding efficacy of antioxidant of low and high dose of Vit. C is related to oxidative stress level in subjects of OSMF.

In view of above point it is worthwhile to conduct a clinical study to answer the to Identify knowledge gaps about the importance of a dose related efficacy of antioxidant vitamin C is related to lower and higher oxidative stress levels in subjects of Oral submucous Fibrosis?

Aim

To determine oxidative stress level and compare the dose dependent efficacy of Vitamin C in relation to oxidative stress level in subjects with Oral Submucous Fibrosis.

 Objective

To assess the efficacy of low dose Vit. C on mean SOD & MDA level in subject with low oxidative stress.

To assess the efficacy of high dose Vit. C on mean SOD & MDA level in subject with low oxidative stress.

To assess the efficacy of low dose Vit. C on mean SOD & MDA level in subject with high oxidative stress.

To assess the efficacy of high dose Vit. C on mean SOD & MDA level in subject with high oxidative stress.

To compare the efficacy of low & high dose Vit. C in low & high oxidative stress level.

To correlate the effect of low & high dose Vit. C with low & high oxidative stress level.

Methods Study design

The study will be a randomized single blind four arm Interventional Comparative Clinical Study. See Figure 1 for a plan of the study.

Plan of study.

The study will be carried out in Sharad Pawar Dental College, Sawangi MegheWardha. All the biochemical procedures will be carried out in Central Research Lab, DMIMS, Sawangi(M) Wardha.

Patients attending OPD of Oral Medicine & Radiology department of Sharad Pawar Dental College –Sawangi Meghe, Wardha will be recruited into the study.

Random allocation of participants through computer generated random number.

The study will be conducted in two phases:

Phase 1 Pilot study

A pilot study will be carried out for the purpose of calculating the sample size.

Phase II Conduction of detailed study

Patients attending OPD shall be assessed for eligibility criteria.

 IEC clearance

The Institutional Ethics Committee at Datta Meghe Institute of Higher Education and Research (Deemed to be University), Nagpur, approved the study. Written informed consent will be requested from all patients with OSMF. All patients will be subjected to a thorough history check regarding dietary habits and addiction. They will be clinically examined to determine various stages of oral submucous fibrosis and shall be assessed for eligibility criteria.

 Inclusion criteria

Patients who are above the age of 18 years and are clinically diagnosed with Oral Submucous Fibrosis, diagnosed on the basis of the following clinical parameters. Stomatitis, burning sensation, inter incisal distance, tongue protrusion.

 Exclusion criteria

Subjects with previous radio/chemotherapy or any other treatment modalities for oral submucous fibrosis.

Subjects with systemic disorders, temporomandibular disorder, or pericoronitis.

Pregnant or lactating mothers.

Protocol

The patients will be instructed to discontinue consuming betel nut, tobacco, and pan masala. They will need to quit smoking, and practice good dental hygiene. The study’s goal and purpose will be clearly described to each patient, and a formal agreement will be taken. The patient’s examination will be conducted by using the diagnostic instrument and natural light. For each individual, 5 ml of the blood sample will be withdrawn under all the aseptic precautions and oxidative stress level will be assessed depending on MDA & SOD level in central research laboratory DMIMS (DU). Method for calculating MDA & SOD The median cubital vein will be used to collect five millilitres of venous blood: For 1 to 2 hours, the blood will be allowed to coagulate at room temperature. To obtain a clear serum sample, the serum will be separated using a centrifuge machine at 3000 rpm for 10 minutes. SOD level will be assessed by Marklund S & Marklund:1974 11 method MDA level will be carried out by the Thiobarbituric acid method. From the collected blood sample using spectrophotometer on day 1, day 30 day 90 days, and on 180th day MDA & SOD will be measured.

 Statistical analysis

It will be done by using descriptive & inferential statistics using one way ANOVA, Dunnet D test, Multiple comparison Tukeytest. Software to be used in the analysis will be SPSS latest version.

 Implication

Positive results of study will give direction for choosing best suitable dose of antioxidant Vitamin C for OSMF depending on oxidative stress level.

Dissemination: The study will be published in an indexed journal.

Study status: Study is yet to commence.

 Limitation

The research might be subjected to response bias as we have taken only two doses of antioxidants which could be considered a crucial factor of limitation.

 Discussion

Antioxidants are supposed to maintain the health of cells. This presumption encouraged people worldwide to consume a lot of phytochemical supplements bearing antioxidant properties. In few of articles on antioxidant therapeutics it was observed that antioxidants can work as a double-edged sword if inappropriately used. It has been opined recently that excessive antioxidant therapy can act as a potential immunosuppressant and increase the likelihood of developing malignancies by compromising the natural immune protection system and direct cell and DNA damage. Hence, the best suitable dose of antioxidant must be dependent upon oxidative stress level. Further great care should be taken in selection of antioxidant agents, selection of dosage of antioxidants not to produce harmful effects.

 Data availability

No data are associated with this article.

 References World Health Organization: IARC Working Group on the Evaluation of carcinogenic risk to humans, International agency for research on cancer, Betel quid & areca nut chewing & some areca nut derived nitrosamines. IARC;2004. Murti PR Bhonsle RB Pindborg JJ : Malignant transformation rate in oral submucous fibrosis over a 17-year period. Community Dent. Oral Epidemiol. 1985 Dec;13(6):340–341. 10.1111/j.1600-0528.1985.tb00468.x Naik V Prakash S Thomas LA : Role of free radicals and effectiveness of antioxidants in patients with OSMF & OLP- A Systematic Review. Int. J. Life Sci. Pharma. Res. JANUARY 2018;8(1). Potter JD : The failure of cancer chemoprevention. Carcinogenesis. 2014 May;35(5):974–982. 24618374 10.1093/carcin/bgu063 Cai L Scott E Kholgi : Cancer Chemoprevention: Evidence of a non linear dose response for the protective effectsof resveratrol in humans & mice. Transl. Med. 2015 July;7(298):298 ra 117. 10.1126/scitranslmed.aaa7619 Carney SL : Calcitonin& human renal calcium & electrolyte transport. Miner. Electrolyte Metab. 1997 Jan;23(1):43–47. 9058369 Chemilos G Lewis R Albert N : Paradoxical effect of Echinocandins across Candida Species in vitro: Evidence for Echinocandins-Specific & Candida species related differences. Antimicrob. Agents Chemother. 2007 Jun;51(6):2257–2259. 17438060 10.1128/AAC.00095-07 PMC1891358 Borkar MA Patel SS : Clinical Evaluation of HemidesmusIndicus, Vit. E and Vit. C Co-administration in Patients Suffering from Tuberculosis. JDMIMSU. 2011;6(3). Kaugars GE Silverman S Jr Lovas JGL : A clinical trial of antioxidant supplements in the treatment of oral leukoplakia. Oral Surg. Oral Med. Oral Pathol. 1994 Oct;78(4):462–468. 7800378 10.1016/0030-4220(94)90039-6 Cathcart RF 3rd. : Vitamin C: the nontoxic, nonrate-limited, antioxidant free radical scavenger. Med. Hypotheses. 1985 Sep;18(1):61–77. 4069036 10.1016/0306-9877(85)90121-5 Marklund S Marklund G : Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 1974 Sep 16;47(3):469–474. 4215654 10.1111/j.1432-1033.1974.tb03714.x 10.5256/f1000research.156003.r401186 Reviewer response for version 1 Mahdani Fatma Yasmin 1 Referee https://orcid.org/0000-0001-6919-418X Universitas Airlangga, Surabaya, Indonesia

Competing interests: No competing interests were disclosed.

 28 8 2025 Copyright: © 2025 Mahdani FY 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Background and Objectives

The background and objectives are clearly presented; however, the rationale could be strengthened by more explicitly highlighting the research gap and the significance of the study. In the abstract, the statement  “Vitamin C is known for its antioxidant properties which act as a lipid-soluble free radical scavenger in cell membranes” appears questionable. Vitamin C is a water-soluble antioxidant, though it can indirectly protect lipid membranes by regenerating lipid-soluble antioxidants such as Vitamin E. Clarifying this distinction in the background would enhance accuracy and scientific rigor. Furthermore, explicitly explaining the choice to focus on Vitamin C—rather than lipid-soluble Vitamin E—could serve as a key justification for the study’s focus and strengthen the reader’s understanding of the study’s novelty.

Study Design

The chosen design is appropriate for addressing the research question. Nevertheless, it would be beneficial to provide a brief justification for selecting this design over possible alternatives. In addition, please indicate the duration of the study, specifying when recruitment and data collection began for all patients, and include the total study period.

Methods

The methodology is generally described; however, the inclusion criteria, data collection procedures, and detailed statistical analysis steps should be elaborated to enable full replication. Furthermore, please clarify whether a grading or staging system for OSMF was applied, and specify which classification was used. Additionally, please provide the rationale for selecting blood-based measurements over alternative matrices such as saliva, as this methodological choice may influence both the interpretation and applicability of the findings.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

Oral medicine, with a particular focus on salivary gland diseases, oral mucosal pathology, and the role of antioxidants in oral potentially malignant disorders such as Oral Submucous Fibrosis (OSMF).

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.156003.r401182 Reviewer response for version 1 Manmuan Suwisit 1 Referee Burapha University, Mueang Chonburi District, Chon Buri, Thailand

Competing interests: No competing interests were disclosed.

 27 8 2025 Copyright: © 2025 Manmuan S 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

- The introduction describes in detail the role of reactive oxygen species (ROS) in OSMF pathophysiology, as well as the potential advantages and hazards of antioxidant supplements.

- The authors accurately identify a gap in the literature about the association between oxidative stress levels and adequate vitamin C intake.The terms low dose and high dose of vitamin C are used, but the specific amounts (mg/day) are not provided. Without identifying the dose, repeatability and clinical translation are limited.

- The trial does not include a placebo or standard care control group, making it impossible to separate treatment effects from natural illness progression or placebo effects.

- MDA and SOD levels are clearly measured on Days 1, 30, 90, and 180, but clinical endpoints (e.g., mouth opening, burning sensation) are not reported, despite their importance for patient outcomes.

- Adjustments for baseline oxidative stress differences between groups should be considered.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

-

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.