World Journal of Dentistry
Volume 14 | Issue 9 | Year 2023

Embryonic Toxicology Evaluation of Dental Varnish Using Titanium Oxide Nanoparticles Synthesized Using Ginger and Rosemary

Twinkle Francis1, Jerry Joe Chokkattu2, Singamsetty Neeharika3, Mahesh Ramakrishnan4, Rajeshkumar Shanmugam5, Lakshmi Thangavelu6

1–3,6Department of Prosthodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

4Department of Pedodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

5Department of Pharmacology, Saveetha Institute of Medical and Technical Sciences, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

Corresponding Author: Jerry Joe Chokkattu, Department of Prosthodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India, Phone: +91 9841026569, e-mail: dr.jerryjoe@gmail.com

Received on: 03 August 2023; Accepted on: 04 September 2023; Published on: 13 October 2023


Aim: The aim of this study was to determine the embryonic toxicology of dental varnish using titanium oxide (TiO2) nanoparticles (NPs) synthesized using ginger and rosemary.

Materials and methods: The study was conducted in a span of 2 weeks, involving zebrafish embryos, NP synthesis, and dental varnish formulation. Ginger and rosemary extracts were prepared by shaking powdered forms in distilled water, followed by filtration. Random sampling ensures an unbiased dispersion of NPs. TiO2 NPs were synthesized by heating a solution of TiO2 and plant extract filtrate. Zebrafish embryonic toxicology studies adhered to Organization for Economic Co-operation and Development (OECD) guidelines. Embryos were exposed to dental varnish-containing NPs across various concentrations (1, 2, 4, 8, and 16 µL) and incubation durations (24–96 hours postfertilization) with developmental toxicity effects assessed via hatching rates, viability, and morphology. Data was statically analyzed using one-way analysis of variance (ANOVA) and post hoc Tukey’s test employing Statistical Package for the Social Sciences (SPSS) software.

Results: The results obtained showed the highest percentage of viability at 100% in 1 and 2 µL and the least viability at 80% in 16 µL concentration of the prepared extract. In contrast, the hatching rate exhibited a descending trend, with the most substantial rate (85%) manifesting at the 1 µL concentration, succeeded by 2 µL (75%), 4 µL (71%), 8 µL (68%), 16 µL (65%), and the control group at 100%.

Conclusion: Herbally mediated TiO2 NPs mouthwash can be researched in order to prepare higher nontoxic concentrations with respect to different organisms in order to broaden the spectrum of action.

Clinical significance: The investigation significantly impacts the progression of dental varnish formulations, distinguished by their integration of herbal ingredients and NP interventions. This pivotal research aims to cultivate safer and more efficacious formulations, thereby elevating patient care and safety in the domain of oral health treatments. In addition to modern NP approaches, this study also acknowledges the relevance of herbal formulations and traditional agents, emphasizing a comprehensive exploration of both innovative and time-honored avenues toward refining dental varnishes.

How to cite this article: Francis T, Chokkattu JJ, Neeharika S, et al. Embryonic Toxicology Evaluation of Dental Varnish Using Titanium Oxide Nanoparticles Synthesized Using Ginger and Rosemary. World J Dent 2023;14(9):791–796.

Source of support: The present study was supported by the following agency: Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University)

Conflict of interest: None

Keywords: Dental varnish, Embryonic toxicology, Titanium nanoparticles, Zebrafish


Dental varnish has the capability to halt the process of enamel demineralization, which primarily affects the outer layer of the tooth, known as enamel. The presence of lactic acid in the oral cavity, which can lead to the breakdown of crucial calcium in the enamel, is one of the main reasons for demineralization.1 Fluoride protection can stop and slow down this process. The proper quantity of fluoride intake can lower the prevalence of caries, but too much fluoride intake can result in fluorosis. Topical fluoride is recognized as a significant form of fluoride application for providing direct protection to the teeth.

The forms of topical fluorides include liquid, paste, gel, and varnish. The unique properties of dental varnish, including its strong attraction to the tooth surface and prolonged contact time, provide distinct advantages over other fluoride formulations. Unlike fluoride toothpaste and gel, dental varnish remains in close proximity to the tooth structure for an extended period, effectively reducing demineralization.2

The herb ginger (Zingiber officinale) has a number of active components that promote dental health and healing. Raffinose and gingerol stand out as the most noticeable. Plaque defense is made possible by raffinose, which also relieves pain, reduces swelling and infections, and prevents inflammatory conditions like periodontal disease. The bactericidal properties of gingerol are well established.3

Rosemary, known for its calcium content, has been explored as a potential remedy for various conditions. Studies have revealed its hepatoprotective, antihyperglycemic, antifungal, anticancer, and antiulcerogenic properties, attributed to its rich phytochemical composition, including carnosic acid, rosmarinic acid, and chlorogenic acid. The diverse phenolic components within organic rosemary extracts contribute to their antibacterial potency.4 Due to their high polyphenol content, which has a significant antioxidant effect and inhibits the growth of streptococci known to cause tooth decay, their use in the prevention of cavities is advised.5

As an antibacterial agent, titanium dioxide nanoparticles (TNPs) have recently been employed in dentistry. TNPs are more effective against germs than chlorhexidine, have a pleasant hue, and are highly biocompatible.6

Prior to their potential application in patients, these compounds, which are created through the green synthesis of ginger and rosemary and mediated by titanium NPs, need to be tested for their level of toxicity. Cell viability, which refers to the proportion of viable cells in a given sample, serves as a valuable metric for assessing the impact of toxicity on cellular systems.7 As stipulated by the researchers, fibroblasts emerge as the prevailing cellular models in the domain of toxicity assessments, owing to their pervasive utilization. Their predominance within the stromal composition, concomitant with their pivotal contribution to the biogenesis of oral mucosal tissue, substantiates their paramount significance. Various research has been conducted toward this formulation in combination with NPs, which has been used as a prototype for the current study.8-14

The primary objective of this preliminary research was to elucidate the potential toxicity of dental varnish containing ginger and rosemary-mediated titanium oxide (TiO2) NPs on embryonic development.


Study Setting

The study was held in the Nanomedicine Laboratory, Department of Pharmacology, Saveetha Dental College, after obtaining approval from the Scientific Review Board (SRB/SDC/PhD/PRSOTHO-2207/22/012). The experiment was initiated in March 2023 and was conducted within a time period of 2 weeks from acquiring the zebrafish embryos, synthesis of NPs, and formulation of dental varnish.

Preparation of Plant Extract

For this procedure, commercially available powdered forms of ginger and rosemary were utilized. Precisely 1 gm of ginger powder and 1 gm of rosemary powder were measured and mixed with 100 mL of distilled water in a beaker. The resulting mixture was subjected to overnight shaking in a shaker. Subsequently, the aqueous extracts were boiled at 50°C using a heating mantle and filtered using a Whatman filter paper. The filtered extracts were once again placed in the shaker overnight. In order to mitigate the potential for sampling bias, the methodology of random sampling was implemented, thereby guaranteeing the stochastic dispersion of NPs within the collected specimens.

Preparation of NPs

A total of 0.35 gm of TiO2 was mixed with 50 mL distilled water and was added to the plant extract filtrate previously prepared. The combined solution was then kept on a heating mantle with a magnetic stirrer at 80°C for the preparation of NPs. The procedure underwent rigorous validation by leading researchers and nanotechnology experts to ensure its accuracy and reliability.

The prepared solution was then assessed for a color change overnight and studied under ultraviolet–visible spectroscopy every 4 hours to assess NP formation, followed by embryonic toxicology studies to evaluate the cytotoxic properties of the prepared extract.

Zebrafish Embryonic Toxicology Studies

The experimental procedures followed the Organization for Economic Co-operation and Development (OECD) guideline 236 with slight modifications. Zebrafish (Danio rerio), comprising a cohort of 10 females and 15 males, were procured from suppliers based in Chennai, Tamil Nadu, India. Healthy pregnant embryos at similar developmental stages were selected for the study, and viable zebrafish embryos (approximately 6 hpf) were used. These specimens were individually housed within distinct tanks, maintained at a constant temperature of 28°C, and subjected to a light-dark cycle of 14 and 10 hours, respectively. The water pH was meticulously regulated within the range of 6.8–8.5. The piscine diet consisted of shrimp and dry flakes, administered twice daily. For the nocturnal separation of sexes, a transparent partition was employed, subsequently removed the following morning to facilitate reproduction. The reproductive process involved a single female zebrafish paired with two male counterparts, thereby generating viable fish embryos.

Recovered and viable zebrafish eggs were subjected to cleansing procedures utilizing an E3 medium. Fertilized eggs were incubated in 24-well U-shaped bottom culture plates and demarcated into experimental and control groups. Each well contained 3 mL of E3 medium alongside a standardized TiO2 contrast solution at a concentration of 100 mg/L. The control group comprised zebrafish embryos and E3 medium solely.

The experimental design encompassed five distinct experimental groups, each treated with dental varnish-containing NPs, alongside a control group comprising five embryos in an E3 medium. The concentrations of dental varnish utilized were 1, 2, 4, 8, and 16 µL. Incubation duration spanned from 24 hours postfertilization (hpf) to 96 hpf, coinciding with the zebrafish embryo’s natural hatching period, which ranges from 48 to 72 hpf (Fig. 1).

Figs 1A to C: Microscopic observation of embryonic development of zebrafish showing stages of growth on (A) Day 1; (B) Day 2; and (C) Day 3

At 24-hour intervals, the hatching rate and viability of embryos were meticulously documented based on the following calculation:

  • Hatching rate: number of embryos hatched among the five embryos that were incubated in each well.

  • Viability rate: number of hatched embryos that were alive among the five embryos that were incubated in each well.

  • Malformations were evaluated in comparison with a healthy hatched embryo under the observation of stereomicroscope.

Concurrently, a control group of five embryos was maintained, and mortalities were duly recorded and eliminated. Throughout the exposure period to dental varnish, embryonic development was scrutinized every 2 hours using a stereomicroscope (Lawrence and Mayo India Pvt Ltd, Maharashtra, India) at an optical zoom of 80×. The overarching aim of the study was to assess developmental toxicity markers, encompassing mortality rates, embryo hatching rates, and larval viability. Photographic documentation of the developing embryos was facilitated using a stereomicroscope. The factors considered to evaluate the embryos’ hatching capacity, survival duration, mortalities (after 24, 48, and 72 hours postfertilization) and the malformations were verified constantly through periodic photographs taken through stereomicroscopic observations on comparison with the previously incubated embryos.

The collected data were transcribed into an Excel spreadsheet, followed by one-way analysis of variance (ANOVA) and post hoc Tukey’s statistical analysis was performed employing Statistical Package for the Social Sciences (SPSS) software.


During the investigation, zebrafish embryos demonstrated successful hatching and viability without any anomalies or malformations (edema, bent tail, and bent spinal cord) during the period of observation (after 24, 48, and 72 hours postfertilization) on subjecting to various concentrations of dental varnish. Figures 2 and 3 show the hatching rate and viability rate of zebrafish embryos against the newly prepared dental varnish. The hatching rate exhibited a descending trend as the concentration increased, with the most substantial rate (85%) manifesting at the 1 µL concentration, succeeded by 2 µL (75%), 4 µL (71%), 8 µL (68%), 16 µL (65%), and the control group at 100%. Parallelly, the viability percentage mirrored the hatching pattern, demonstrating the highest viability rate of 100% at the lower concentrations—1 µL (100%), 2 µL (100%), and 4 µL (100%), indicating that the embryos remained healthy and viable under lower concentrations. As the concentration increased beyond 4 µL, the viability percentage started to decrease. At 8 µL, the viability remained relatively high at 91%, but a more noticeable decrease was observed at 16 µL, where viability dropped to 80%.

Figs 1A to C: Fig represents the percentage of zebrafish hatching rate where the x-axis represents the concentration of extract in microliters (μL/mL), and the Y-axis represents hatching rate (%)

Fig. 3: Fig represents the percentage viability rate of zebrafish where the X-axis represents the concentration of extract in microliters (μL/mL), and the Y-axis represents viability rate (%)

One-way ANOVA analysis revealed a significant (p = 0.00) difference in the hatching rate and mortality rate between the groups at various concentrations (Tables 1 and 2). On intergroup comparisons of different concentrations using post hoc Tukey’s test (Tables 3 and 4), the hatching rate and mortality of the zebrafish embryos showed significant differences (p = 0.00) in most of the concentrations that were compared, and few didn’t.

Table 1: One-way ANOVA performed at various concentrations of test solution in comparison with hatchability of zebrafish embryos
Hatching rate
Sum of squares Degree of freedom (df) Mean square F Significance
Between groups 3233.333 5 646.667 215.556 0.000
Within groups 54.000 18 3.000
Total 3287.333 23
Table 2: One-way ANOVA performed at various concentrations of dental varnish in comparison with viability of zebrafish embryos
Viability percentage
Sum of squares df Mean square F Significance
Between groups 1283.375 5 256.675 117.711 0.000
Within groups 39.250 18 2.181
Total 1322.625 23
Table 3: Post hoc Tukey’s tests performed between various concentrations toward their hatchability
Multiple comparisons
Dependent variable: hatching rate
Tukey honestly significant difference (HSD)
(I) Concentration (J) Concentration Mean difference (I − J) Standard error Significance 95% confidence interval
Lower bound Upper bound
1 µL 2 µL 10.500* 1.225 0.000 6.61 14.39
4 µL 13.500* 1.225 0.000 9.61 17.39
8 µL 16.500* 1.225 0.000 12.61 20.39
16 µL 20.750* 1.225 0.000 16.86 24.64
Control −13.750* 1.225 0.000 −17.64 −9.86
2 µL 1 µL −10.500* 1.225 0.000 −14.39 −6.61
4 µL 3.000 1.225 0.191 −0.89 6.89
8 µL 6.000* 1.225 0.001 2.11 9.89
16 µL 10.250* 1.225 0.000 6.36 14.14
Control −24.250* 1.225 0.000 −28.14 −20.36
4 µL 1 µL −13.500* 1.225 0.000 −17.39 −9.61
2 µL −3.000 1.225 0.191 −6.89 0.89
8 µL 3.000 1.225 0.191 −0.89 6.89
16 µL 7.250* 1.225 0.000 3.36 11.14
Control −27.250* 1.225 0.000 −31.14 −23.36
8 µL 1 µL −16.500* 1.225 0.000 −20.39 −12.61
2 µL −6.000* 1.225 0.001 −9.89 −2.11
4 µL −3.000 1.225 0.191 −6.89 0.89
16 µL 4.250* 1.225 0.028 0.36 8.14
Control −30.250* 1.225 0.000 −34.14 −26.36
16 µL 1 µL −20.750* 1.225 0.000 −24.64 −16.86
2 µL −10.250* 1.225 0.000 −14.14 −6.36
4 µL −7.250* 1.225 0.000 −11.14 −3.36
8 µL −4.250* 1.225 0.028 −8.14 −0.36
Control −34.500* 1.225 0.000 −38.39 −30.61
Control 1 µL 13.750* 1.225 0.000 9.86 17.64
2 µL 24.250* 1.225 0.000 20.36 28.14
4 µL 27.250* 1.225 0.000 23.36 31.14
8 µL 30.250* 1.225 0.000 26.36 34.14
16 µL 34.500* 1.225 0.000 30.61 38.39

*The mean difference is significant at the 0.05 level

Table 4: One-way ANOVA performed at various concentrations of dental varnish in comparison with viability or mortality of zebrafish embryos
Multiple comparisons
Dependent variable: viability percentage
Tukey HSD
(I) Concentration 2 (J) Concentration 2 Mean difference (I − J) Standard error Significance 95% confidence interval
Lower bound Upper bound
1 µL 2 µL 0.250 1.044 1.000 −3.07 3.57
4 µL 1.000 1.044 0.925 −2.32 4.32
8 µL 8.500* 1.044 0.000 5.18 11.82
16 µL 19.750* 1.044 0.000 16.43 23.07
Control −0.250 1.044 1.000 −3.57 3.07
2 µL 1 µL −0.250 1.044 1.000 −3.57 3.07
4 µL 0.750 1.044 0.977 −2.57 4.07
8 µL 8.250* 1.044 0.000 4.93 11.57
16 µL 19.500* 1.044 0.000 16.18 22.82
Control −0.500 1.044 0.996 −3.82 2.82
4 µL 1 µL −1.000 1.044 0.925 −4.32 2.32
2 µL −0.750 1.044 0.977 −4.07 2.57
8 µL 7.500* 1.044 0.000 4.18 10.82
16 µL 18.750* 1.044 0.000 15.43 22.07
Control −1.250 1.044 0.833 −4.57 2.07
8 µL 1 µL −8.500* 1.044 0.000 −11.82 −5.18
2 µL −8.250* 1.044 0.000 −11.57 −4.93
4 µL −7.500* 1.044 0.000 −10.82 −4.18
16 µL 11.250* 1.044 0.000 7.93 14.57
Control −8.750* 1.044 0.000 −12.07 −5.43
16 µL 1 µL −19.750* 1.044 0.000 −23.07 −16.43
2 µL −19.500* 1.044 0.000 −22.82 −16.18
4 µL −18.750* 1.044 0.000 −22.07 −15.43
8 µL −11.250* 1.044 0.000 −14.57 −7.93
Control −20.000* 1.044 0.000 −23.32 −16.68
Control 1 µL 0.250 1.044 1.000 −3.07 3.57
2 µL 0.500 1.044 0.996 −2.82 3.82
4 µL 1.250 1.044 0.833 −2.07 4.57
8 µL 8.750* 1.044 0.000 5.43 12.07
16 µL 20.000* 1.044 0.000 16.68 23.32

*The mean difference is significant at the 0.05 level

The results may suggest a potential threshold effect, where lower concentrations of TiO2-mediated dental varnish are not significantly detrimental to hatching and viability. However, as the concentration exceeds a certain point (16 µL in this case), the viability starts to decline noticeably.


From the current study, it can be deduced that no anomalies, such as a bent spinal cord, a bent tail, or edema, were seen. At greater quantities, such as 8 and 16 gm/mL, delayed hatching was noted. This demonstrates that lower quantities of the extract are favored during the embryonic phases of development in order to encourage the growth of the embryo. According to the present research, exposure to titanium dioxide resulted in decreased developmental toxicity and a normal and healthy effect on the hatching of immature zebrafish embryos, which also correlates with the studies conducted earlier on a dental varnish formulation using ginger and clove as the herbal combination mediated with TiO2 NPs.13

Because of its optical characteristics, high stability, and nontoxicity, titanium dioxide has been widely employed as an eco-friendly photocatalyst. To shield the skin from UV rays and whitening, TiO2 NPs have been utilized in skincare products, medicines, and cosmetics.15 Additionally, it is utilized in toothpaste, inks, paints, plastics, papers, and food coloring. According to Riad and Ibrahim, nanoparticulate TiO2, which is employed in antibacterial coatings, produces reactive oxygen species when exposed to UV radiation.16

Zebrafish embryo viability exhibited consistent preservation within the range of 1–4 gm/mL, while a discernible decline occurred at 16 gm/mL concentration. Over recent decades, the zebrafish has emerged as a pivotal model organism for scrutinizing human ailments. Although zebrafish, being nonmammalian, exhibit a comparatively distant evolutionary relation to humans compared to rodents, their pragmatic attributes such as facile management, cost-effectiveness, rapid developmental kinetics, prolificacy, substantial genetic resemblance to humans, and optically translucent bodies have propelled their prominence in disease research.17

Because of their great stability, anticorrosive, and photocatalytic qualities, TiO2 NPs are currently produced in large quantities and employed extensively. It has been suggested that TiO2 NPs’ high surface area and predominant anatase rather than rutile composition are responsible for their higher catalytic activity.18 TiO2 NPs can be employed in catalytic reactions, such as semiconductor photocatalysis, in the treatment of water contaminated with toxic industrial by-products, and in nanocrystalline solar cells as a photoactive material. TiO2 NPs’ photocatalytic effect has been used industrially for a variety of purposes, including for self-cleaning and antifogging materials like self-cleaning tiles, windows, textiles, and antifogging automobile mirrors.19

Furthermore, ginger and rosemary have served as both culinary spices and medicinal plants for numerous centuries. These botanicals are derived from natural sources and have demonstrated a lack of toxicity. They have also received the ”generally recognized as safe” designation from the United States Food and Drug Administration.20 The active phytonutrient components of ginger are phenols, hydrocarbons and oleoresins, which show well-documented therapeutic properties like anti-inflammatory, antioxidant, anticancer, antidiabetic and antimicrobial.21 In comparison with ginger, rosemary has >12 phytocompounds (rosmarinic acid, carnosic acid, carnosol, rosmanol, etc.) that demonstrate similar pharmacological properties.22 Numerous research endeavors have underscored the capacity of these substances to effectively counteract the proliferation of fungi and microorganisms within the oral cavity. These inhibitory effects have been observed across a spectrum of concentrations, exhibiting a compelling alignment with the outcomes of the ongoing in situ analysis.20 This congruence substantially bolsters the effectiveness of the dental varnish, which ingeniously amalgamates herbal elements and nanomaterials.


This investigation is subject to certain limitations. Its preliminary character underscores the potential requirement for more extensive inquiries to ensure comprehensive validation. The study’s specific emphasis on zebrafish embryos could restrict the extrapolation of findings to a broader range of organisms. The variability in outcomes observed across various species or conditions might impact the broader applicability of the results. Despite these constraints, the study provides valuable insights into the potential toxicity of dental varnish containing ginger and rosemary-mediated TiO2 NPs on embryonic development.

Areas of Future Research

The characterization of active constituents and intricate molecular interactions within herbal formulations poses a significant challenge, impeding the process of product standardization. While the available data remains insufficient to establish the efficacy and appropriate dosing regimens for addressing oral diseases, the potential for extensive large-scale clinical trials offers a promising avenue for investigation. These trials hold the capacity to unveil the efficacy and safety profiles of herbal formulations within the realms of medicine and dentistry.


Titanium dioxide NPs (TNPs) are being researched as potential instruments for enhanced imaging and nanotherapeutics in the field of nanomedicine. Additionally, research can be done to develop higher nontoxic concentrations of rosemary and ginger-mediated TiO2 NP dental varnishes that are nontoxic to other organisms in order to widen the range of action. However, following further processing of mouthwash with chemicals and preservatives, our study has shown a safe concentration that can be utilized for trials.


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