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

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Dr Mohan Z Mani

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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.
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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

Research Protocol
Year : 2026 | Month : September | Volume : 20 | Issue : 9 | Page : ZK01 - ZK04 Full Version

Enamel Remineralisation and Antimicrobial Efficacy of a Withania somnifera, Ficus religiosa, Salvia rosmarinus-based Polyherbal Gel: A Research Protocol


Published: September 1, 2026 | DOI: https://doi.org/10.7860/JCDR/2026/84981.24361
Rijuta Gujar, Punit Fulzele, Nilima Thosar, Anjori Raut

1. Postgraduate Student, Department of Paediatric and Preventive Dentistry, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. 2. Professor, Department of Paediatric and Preventive Dentistry, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. 3. Head, Department of Paediatric and Preventive Dentistry, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. 4. Postgraduate Student, Department of Paediatric and Preventive Dentistry, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India.

Correspondence Address :
Rijuta Gujar,
Postgraduate Student, Department of Paediatric and Preventive Dentistry, Sharad Pawar Dental College and Hospital, Wardha, Maharashtra, India.
E-mail: rijutasgujar@gmail.com

Abstract

Introduction: Dental caries is a multifactorial and chronic disease. Streptococcus mutans and Lactobacillus species are the primary microorganisms responsible for dental caries and enamel demineralisation. Caries can be prevented by fluoridated products like Casein Phosphopeptide- Amorphous Calcium Phosphate (CPP-ACP) or non fluoridated products. CPP-ACP can be used as a remineralising agent. Fluoride can also be used only as a remineralising agent. This article focuses on Withania somnifera, Ficus religiosa, and Salvia rosmarinus, which have all the three properties- antibiofilm, antimicrobial and remineralising.

Need of the study: Dental caries is a significant oral disease due to bacterial biofilms and enamel demineralisation. Traditional chemical medicaments such as fluoride have drawbacks of toxicity and resistance. Therefore, safer natural substitutes are desired. Withania somnifera, Ficus religiosa, and Salvia rosmarinus have antimicrobial, antioxidant, and remineralising activities. But they have to be studied in combination as a polyherbal gel for enamel. Therefore, the present study aims to assess the antimicrobial, antibiofilm, and remineralisation capability of this plant formulation, providing a biocompatible and effective means to prevent dental caries.

Aim: To evaluate the antimicrobial, antibiofilm and remineralisation efficacy of Withania somnifera, Ficus religiosa and Salvia rosmarinus-based polyherbal gel on enamel.

Materials and Methods: An in-vitro study will be conducted in the Department of Paediatric and Preventive Dentistry, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India, from December 2025 to December 2026. In the present study, teeth extracted for orthodontic purposes or teeth samples with non cavitated lesions suitable for remineralisation assessment will be selected. Total teeth will be 70, with two groups each containing 35 teeth, n=35. Group 1: Withania somnifera, Ficus religiosa, Salvia rosmarinus. Group 2: Control group CPP-ACP. Antimicrobial efficacy will be determined against oral pathogens with the agar diffusion and broth microdilution techniques. Antibiofilm activity will be evaluated using biofilm inhibition and disruption assays. The remineralisation ability will be examined by scanning electron microscopy. A paired t-test will be applied to compare biofilm reduction before and after treatment within the same group. An independent t-test will be used to compare outcomes between two independent groups (polyherbal gel and control-CPP-ACP). The p-value of ≤0.05 will be considered statistically significant.

Keywords

Biofilm, Dental caries, Phytotherapy, Plant extracts

Dental caries results from dietary sugar, the host’s oral environment, and the action of cariogenic bacteria, primarily Streptococcus mutans, which produce acids that demineralise tooth enamel. It involves dissolution of enamel apatite crystals with loss of calcium and phosphate; if untreated, it progresses to cavitation and weakens the tooth surface (2).

In recent years, the philosophy of minimally invasive dentistry has been increasingly accepted, focusing on healthy tooth structure and early diagnosis and treatment of caries. Non-invasive remineralisation is one of the most significant practices in this philosophy, and aims to arrest or reverse early enamel caries by replenishing lost minerals (3). Apart from conventional remineralising agents such as fluoride and calcium phosphates, there has been a growing emphasis on the use of herbal products for the preventive measures and treatment of caries. These products of animal, plant, or microbial origin have been checked for their excellent health benefits, antimicrobial, and anti-inflammatory properties. Natural products such as polyphenols, essential oils, and bioactive peptides are being checked for their ability to inhibit cariogenic bacteria and induce enamel repair. This aligns with a current trend in healthcare, the application of biocompatible treatment options, which is the future of natural products in caries management strategies (4).

Fluoride systems are used in dentistry to deliver fluoride to tooth surfaces. It is available in both topical and systemic forms. At-home use of low-level fluoride can be through toothpastes, gels, and mouth rinses (5). Routine use of low-concentration topical fluoride prevents dental caries by promoting remineralisation. Topically applied fluoride concentrates salivary fluoride and inhibits the solubility of enamel by increasing the formation of fluoroapatite. Furthermore, fluoride varnish can be shown to suppress bacterial metabolism and therefore prevent caries (5).

CPP-ACP came into the market as a remineralising toothpaste in 1998 as an attempt at escaping some of the flaws in fluoride. It contains milk protein CPP-ACP nanocomplexes. The combination of CPP and fluoride causes a synergistic action. Having a high saturation level with bulky minerals was believed to boost remineralisation of caries lesions as well as suppress cariogenic bacteria colonising dental surfaces (6).

CPP-ACP has been considered particularly useful since CPP stabilises phosphate and calcium ions against precipitation while ACP provides a reservoir for such remineralising ions (7). CPP-ACP is a milk-derived protein complex that stabilises calcium and phosphate ions as ACP. It binds to dental plaque, enamel, and oral soft tissues, delivering calcium and phosphate for remineralisation. CPP can also carry fluoride ions (CPP-ACPF), enhancing fluoride penetration into demineralised enamel and acting as a buffer to reduce acid damage while promoting subsurface enamel remineralisation (8).

Medicinal plants have been used for centuries in treating oral or systemic diseases with oral symptoms. Herbal mouthwashes and dentifrices are preferred since they contain no alcohol or colour and provide effective antimicrobial and anti-inflammatory action through phytochemicals. Withania somnifera, or the Queen of Ayurveda, is purported to have potent anticarcinogenic properties. Based on experimental laboratory findings, ashwagandha systematically possesses phytochemicals, trapino, withananine, alkaloids, and choline that possess anti-angiogenic properties that may inhibit tumour vascularisation (9). It has inhibitory action on S. Mutans. It consists primarily of mono and disaccharides in a methanolic extract, with a small quantity of alcohol sugars and some organic acid groups. These substances have both bacteriostatic and bactericidal actions at much higher concentrations. The extract possessed significant inhibitory activities on S. mutans at the optimal Minimum Inhibitory Concentration (MIC). This proves that, to a certain approximate tolerance level, this herbal extract may therefore inhibit the cariogenic activity against pathogenic bacteria (9).

The ethanol extract from the bark of F. religiosa was found to have modest antifungal efficacy against the opportunistic pathogenic yeast Candida albicans (10).

Salvia rosmarinus essential oil was found to inhibit the growth of E. Coli. It has an inhibitory action on S. mutans. Moreover, it possesses several beneficial properties, including anti-oxidant, antiviral, antibacterial, anti-inflammatory, and anti-mutagenic activities. Due to low toxicity and few side-effects, rosmarinic acid has potential as a therapeutic agent. In addition, rosmarinic acid has significant antiviral, antibacterial, and anti-inflammatory properties, which imply that it can be employed in treating infections and inflammatory conditions (11).

The present research aimed to evaluate a polyherbal gel with CPP-ACP as a control agent.

Primary Objectives
• To check the antimicrobial, antibiofilm and remineralisation efficacy of Withania somnifera, Ficus religiosa and Salvia rosmarinus-based polyherbal gel on enamel;
• To evaluate the remineralisation efficacy of CPP-ACP on enamel.

Secondary Objectives
To compare the efficacy of polyherbal gel and CPP-ACP on enamel.

Review of Literature

Interest in drugs derived from medicinal plants has significantly increased over the last decade. As there are some drawbacks to conventional products used in dentistry for a long time, including immune suppression, hypersensitivity, allergic reactions, and resistance, some of which are mutagenic and cytotoxic, a shift to herbal products is necessary (12). Therefore, the need to convert to herbal products led many researchers to experiment on different medicinal and herbal plants. The current research aimed to provide a natural, plant-derived antimicrobial agent for preventing oral diseases against the backdrop of the global rise in antimicrobial resistance.

Gunther M et al., demonstrated the use of Salvia rosmarinus extracts as an herbal adjuvant, offering an alternative to synthetic chemicals for caries and periodontitis. The extract showed a marked effect on both aerobic and anaerobic bacteria, exhibiting bactericidal activity, with Colony-Forming Units (CFU) significantly reduced after treatment compared to pre-treatment levels (11).

Sienkiewicz M et al., found Salvia rosmarinus essential oil affects the growth of E. coli. A series of studies also testified to the efficacy of Salvia rosmarinus against S. aureus (13).

Hickl J et al., utilised in-vitro models for the first time to determine the action of different plant extracts, including Salvia rosmarinus, on oral microbes. The authors concluded that the growth of all the tested bacteria was severely suppressed by rosemary extract. Apart from reducing total bacterial numbers and viable counts, the antimicrobial activity of Salvia rosmarinus extract was also demonstrated by the total number of bacterial species present, which substantially reduced after treatment with the extract (14).

Sharma H et al. reported in an in vitro study that the 67% ethanolic stem extract of Ficus religiosa exhibited antibacterial activity against primary plaque colonisers only after 48 hours, producing a mean Zone of Inhibition (ZOI) of 2.6±0.54 mm. There was no significant activity in the extract at 24 h or 72 h, indicating that the antibacterial action was only transient. Findings revealed that although F. religiosa exhibited some antimicrobial properties, the effectiveness was low and short-lived, attributed to the fact that unrefined ethanol extraction may fail to provide all the effective phytochemicals. The researchers suggested conducting studies on methods of extraction, concentration, and human testing to understand antimicrobial purposes (15).

Dausage P et al., found that, in comparison to normal saline, ashwagandha preparations were superior for ion diffusion across the dentinal tubules over an extended period (up to 168 hours). For intracanal application, it can be used as an ideal vehicle for calcium hydroxide (16).

Abd El Aziz PM et al., illustrated that the richness of ashwagandha, in terms of biologically active compounds and minerals, has been well documented; elemental analysis has supported this with a high calcium and phosphorus content. Alkaloids and steroidal compounds in ashwagandha possess intense antibacterial, anti-inflammatory, and antioxidant activity. These molecules can bind and precipitate macromolecules, such as bacterial enzymes involved in bacterial metabolism. 0.5% ginger, ashwagandha, and maca are potent remineralising agents for early enamel carious lesions and possess antibacterial properties against Streptococcus mutans. They are a cheap and safe substitute for mouthwash and fluoride. They serve as essential minerals and phenolics. He also used laser fluorescence to confirm the remineralisation of early enamel carious lesions (17).

The antibacterial activity of the ancient Indian medicinal plants is tremendous. F. religiosa aqueous extracts exhibited marked antibacterial activity against isolated bacteria, which are primarily associated with dental caries (18). Ficus religiosa helps in wound healing and acts as a disinfectant. It can also be used as a dye and applied as a varnish. Salvia rosmarinus has an antifungal effect; it acts against Candida albicans and can also be used with titanium dioxide and zinc oxide nanoparticles. It also has better dentin adhesive properties. The main property of Withania somnifera is its antibacterial, mainly against S. aureus, E. faecalis, E. coli, and P. aeruginosa. Also, Ashwagandha is water-soluble, and Rosemary can be both water and oil-soluble, so the combination of these three products forms an hydrogel. Due to their capacity to treat oral diseases, these medicinal plants should be utilised. Therefore, this study is different from other studies. Thus, the study aims to evaluate the antimicrobial, antibiofilm and remineralisation efficacy of Withania somnifera, Ficus religiosa and Salvia rosmarinus-based polyherbal gel on enamel.

Null Hypothesis
The polyherbal gel does not exhibit any significant antimicrobial, antibiofilm, or remineralisation effects on enamel when compared with control groups.

Alternate Hypothesis
The polyherbal gel exhibits significant antimicrobial, antibiofilm, and remineralisation effects on enamel when compared with control groups.

Material and Methods

An in-vitro study will be conducted at Datta Meghe Institute of Higher Education and Research, Wardha, from December 2025- December 2026. Ethical clearance was obtained with the ethical clearance no.- DMIHER(DU)/IEC/2025/552. Informed consent was obtained before commencing the study.

Inclusion criteria:
1. Teeth extracted for orthodontic purposes;
2. Teeth samples with non cavitated lesions;
3. Teeth of children aged 10-14 years.

Exclusion criteria:
1) Teeth samples with fractures, restorations, or other defects unrelated to caries;
2) Teeth with fluorosis or eroded teeth;
3) Teeth with root caries or teeth exposed to fluoride varnish or bleaching agents.

Sample size calculation:

n≥{Z_(1-α/2)+Z_(1-β)}2×(σ1222/r)/(μ12) 2
Remineralisation potential (Mean) in Group 1 (μ1): 3.10 (17)
Remineralisation potential (Mean) Mean Group 2 (μ2): 2.40 (17)
Standard deviation in Group 1 (σ1): 0.88 (17)
Standard deviation in Group 2 (σ2): 1.17 (17)
Ratio (r, Group 2/Group 1): 1
σ12 + (σ22/r)=0.7744+1.3689=2.1433
Zα/2=1.96 (for 95% confidence level)
Zβ=0.84 (for 80% power)
(1.96+0.84) 2=(2.8) 2=7.84
n=7.84×2.1433 (0.70) 2
(0.70) 2=0.49
7.84×2.1433=16.80
n=16.80 0.49
n=34.29
Sample size per group: 35

A total of 70 teeth will be required. 35 teeth will be in Group 1: Withania somnifera, Ficus religiosa, Salvia rosmarinus, and the remaining 35 teeth will be in Group 2: Control group CPP-ACP.

Study Procedure

Source of plant material: Plants of Ashwagandha, Peepal and Rosemary will be taken from the Ayurvedic college of Datta Meghe Institute of Higher Education and Research, Wardha.

Exact composition of polyherbal gel:
1. Withania somnifera extract- Antimicrobial, anti-inflammatory- 2% w/w
2. Ficus religiosa leaf extract- Antibacterial, antioxidant- 2% w/w
3. Salvia rosmarinus (Rosemary) extract- Antimicrobial, antibiofilm- 2% w/w
4. Propylene glycol- Humectant- 5% w/w
5. Methyl paraben- Preservative- 0.1% w/w
6. Distilled water- Vehicle
7. Carbopol 940- Gelling agent- 1% w/w
8. Triethanolamine- pH adjustment- 0.5–1%

Preparation of polyherbal gel:
First leaves of Ashwagandha, Peepal and Rosemary will be collected. The gel formulation is prepared by first dispersing Carbopol 940 in 50 mL of distilled water to form the base. Each herbal extract is then dissolved in propylene glycol to create the herbal phase. This mixture is gradually incorporated into the hydrated Carbopol gel with continuous stirring to ensure uniformity. Methyl paraben, dissolved separately, is added as a preservative. The pH of the formulation is adjusted to 6-7 by the dropwise addition of triethanolamine, which neutralises the Carbopol and yields a clear gel. Distilled water is then added to bring the final weight to 100 g. The formulation is homogenised using a magnetic stirrer or homogeniser until a smooth, uniform gel is obtained. Finally, the gel is transferred into sterile airtight containers and stored at room temperature for subsequent use.

Quality Control

Quality control tests will be carried out for macroscopic evaluation- colour, odour and texture; microscopic evaluation- identification of diagnostic plant structures.

Quality Evaluation of the Polyherbal Gel

The prepared gel will be evaluated for pH, viscosity, spreadability, homogeneity, drug content uniformity, and stability testing (25°C and 40°C).

OUTCOMES

1. For antimicrobial testing, the pathogens will be cultured on agar plates. Antimicrobial efficacy of the polyherbal gel against cariogenic microorganisms assessed by ZOI. The agar well diffusion method will be used, diameter will be recorded in mm using a vernier calliper. MIC protocol will be- dilutions of gel extract prepared in broth, both positive and negative controls will be taken, with the positive control being a bacterial suspension without extract, and the negative control will be broth. Incubated for 24 hours at 37°C.
2. Antifungal testing will be done by culturing Candida albicans on culture plates and then by the agar diffusion method.
3. For antibiofilm testing, the treated biofilm will be examined using a Scanning Electron Microscope (SEM). Antibiofilm efficacy will be assessed by biofilm formation reduction using the crystal violet assay. Reduction percentage will be calculated by using the formula- {OD (control) - OD (test)/OD (control)}×100 (19).
4. Remineralisation efficacy assessed by change in enamel surface microhardness by using the Vickers Microhardness test. Measurement intervals will be at baseline, Post-demineralisation and post treatment- 7 and 14 days. It will be calibrated using a reference block before testing.
5. SEM analysis- Imaging parameters will be magnification 1000, 3000 and 5000, and resolution will be high resolution mode.
6. SEM-EDX- lesion depth will be standardised by immersing all the samples in the same demineralising solution for the same duration. Elemental analysis of Ca and P will be evaluated, and Ca/P ratio will be calculated for each sample.
7. For stability and handling properties, pH, viscosity, and shelf-life will be evaluated.
8. Handling properties like spreadability will be evaluated by the two glass slides method, and acceptability will be evaluated by a 5-point Likert scale.

STATISTICAL ANALYSIS

Statistical analysis will be performed using SPSS software (Version 30). A paired t-test will be applied to compare biofilm reduction before and after treatment within the same group. An independent t-test will be used to compare outcomes between two independent groups (polyherbal gel and control-CPP-ACP). A p-value ≤0.05 will be considered statistically significant.

References

1.
Selwitz RH, Ismail AI, Pitts NB. Dental caries. The Lancet. 2007;369(9555):51- 59. [crossref] [PubMed]
2.
Takahashi N, Nyvad B. The role of bacteria in the caries process: Ecological perspectives. Journal of Dental Research. 2011;90(3):294-303. [crossref] [PubMed]
3.
Prabhakar AR, Karuna YM, Yavagal C, Deepak BM. Cavity disinfection in minimally invasive dentistry-comparative evaluation of Aloe vera and propolis: A randomized clinical trial. Contemporary Clinical Dentistry. 2015;6(Suppl 1):S24- S31. [crossref] [PubMed]
4.
Kumar G, Jalaluddin MD, Rout P, Mohanty R, Dileep CL. Emerging trends of herbal care in dentistry. Journal of clinical and diagnostic research: JCDR. 2013;7(8):1827.
5.
Munteanu A, Holban AM, Pa? una MR, Imre M, Farcas¸ iu AT, Farcas¸ iu C. Review of professionally applied fluorides for preventing dental caries in children and adolescents. Applied Sciences. 2022;12(3):1054. [crossref]
6.
Bhargavi H, Naik SB, Kumar NK, Merwade S, Brigit B, Bhumralkar SS, et al. Comparative evaluation between self-assembling peptide P11-4 and ACP CPP in enamel remineralisation post Er: YAG laser irradiation-a confocal laser scanning microscopic study. Contemporary Clinical Dentistry. 2025;16(1):03-09. [crossref] [PubMed]
7.
Abufarwa M, Noureldin A, Dziak R, Covell Jr D. Efficacy of CPP-ACP fluoride varnish applied with and without acid etching in preventing enamel demineralisation compared to light-curable fluoride varnish. The Angle Orthodontist. 2022;92(2):213-19. [crossref] [PubMed]
8.
Reynolds E. Casein phosphopeptide-amorphous calcium phosphate: The scientific evidence. Advances in Dental Research. 2009;21(1):25-29. [crossref] [PubMed]
9.
Thribhuvanan L, Saravanakumar MS, Hans Y, Thankachan C, Singh M, Thribhuvanan L. Clinical uses and application of withania somnifera (ashwagandha): In dentistry: A narrative review. Chelonian Conservation and Biology. 2023;18(2):1016-25. Doi: doi.org/10.18011/2023.11(2).1016.1025.
10.
Murugesu S, Selamat J, Perumal V. Phytochemistry, pharmacological properties, and recent applications of Ficus benghalensis and Ficus religiosa. Plants. 2021;10(12):2749. [crossref] [PubMed]
11.
Günther M, Karygianni L, Argyropoulou A, Anderson AC, Hellwig E, Skaltsounis AL, et al. The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo. Clinical Oral Investigations. 2022;26(6):4369-80. [crossref] [PubMed]
12.
Sinha DJ, Sinha AA. Natural medicaments in dentistry. AYU (An international quarterly journal of research in Ayurveda). 2014;35(2):113-18. [crossref] [PubMed]
13.
Sienkiewicz M, Lysakowska M, Pastuszka M, Bienias W, Kowalczyk E. The potential of use basil and rosemary essential oils as effective antibacterial agents. Molecules. 2013;18(8):9334-51. [crossref] [PubMed]
14.
Hickl J, Argyropoulou A, Sakavitsi ME, Halabalaki M, Al-Ahmad A, Hellwig E, et al. Mediterranean herb extracts inhibit microbial growth of representative oral microorganisms and biofilm formation of Streptococcus mutans. PLoS One. 2018;13(12):e0207574. [crossref] [PubMed]
15.
Sharma H, Yunus GY, Mohapatra AK, Kulshrestha R, Agrawal R, Kalra M. Antimicrobial efficacy of three medicinal plants, Glycyrrhiza glabra, Ficus religiosa, and Plantago major on inhibiting primary plaque colonizers and periodontal pathogens: An: in-vitro study. Indian Journal of Dental Research. 2016;27(2):200-04. [crossref] [PubMed]
16.
Dausage P, Dhirawani RB, Marya J, Dhirawani V, Kumar V. A comparative study of ion diffusion from calcium hydroxide with various herbal pastes through dentin. Int J Clin Pediatr Dent. 2017;10(1):41-44. Doi: 10.5005/jp-journals-10005- 1405. [crossref] [PubMed]
17.
Abd El Aziz PM, Kamh R, El Gezawy EA. Remineralisation and Antibacterial efficacy of Ashwagandha, Maca and Ginger-based mouthwashes versus Fluoride-based mouthwash on initial enamel caries: An in-vitro study. Egyptian Dental Journal. 2024;70(2):1997-2009. [crossref]
18.
Ambulkar S, Tale V, Khilari S. Evaluation of the antibacterial potential of traditional medicinal plants against bacteria isolated from dental caries. J Pure Appl Microbiol. 2021;15(3):1204-11. [crossref]
19.
Bazaid AS, Alsolami A, Patel M, Khateb AM, Aldarhami A, Snoussi M, et al. Antibiofilm, antimicrobial, anti-quorum sensing, and antioxidant activities of Saudi Sidr honey: In-vitro and molecular docking studies. Pharmaceutics. 2023;15:2177. Doi: 10.3390/pharmaceutics15092177 [crossref]. [PubMed]

DOI and Others

DOI: 10.7860/JCDR/2026/84981.24361

Date of Submission: Oct 18, 2025
Date of Peer Review: Dec 18, 2025
Date of Acceptance: May 30, 2026
Date of Publishing: Sep 01 , 2026

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Dec 04, 2025
• Manual Googling: May 26, 2026
• iThenticate Software: May 28, 2026 (1%)

ETYMOLOGY: Author Origin

EMENDATIONS: 9

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