Antioxidant Profiling and Phytochemical Characterization of Salt‑Based Toothpastes
Shweta Manchanda, Yamini Sharma, Vishal Sareen, Jyoti Singh, Ketan Patil, Ranjan Mitra*
Department of Research and Development Centre (DRDC), Dabur India Ltd., 22, Site IV, Sahibabad,
Ghaziabad - 201010, Uttar Pradesh (UP), India.
*Corresponding Author E-mail: ranjan.mitra@dabur.com
ABSTRACT:
Salt‑based herbal dentifrices combine the abrasive cleansing action of salt with bioactive plant constituents. In this work, we profiled the antioxidant capacity and phytochemical composition of salt‑based herbal toothpaste, ayurvedic toothpaste and salt-based toothpaste, focusing on the flavor‑active markers like camphor, menthol, eugenol, and piperine. These were quantified by validated GC‑FID (Gas Chromatography with Flame Ionization Detector) and HPLC (High performance Liquid Chromatography) protocols. Free‑radical scavenging potential was determined via the DPPH assay (reported as Trolox Equivalent Antioxidant Capacity, TEAC) and salt content was measured titrimetrically. Across the formulations tested, salt‑based herbal toothpaste displayed the most robust antioxidant response and the most complex phytochemical signature, suggesting a contribution from both mineral and botanical components. These findings provide laboratory evidence in support of traditional herbal‑salt dentifrices and illustrate how analytical characterization can substantiate their functional claims.
KEYWORDS: Salt‑based herbal toothpaste, Ayurvedic toothpaste, Antioxidant activity, GC‑FID, HPLC.
INTRODUCTION:
Oral health is a critical determinant of overall wellbeing, influencing nutrition, speech, aesthetics, and systemic health. Dental problems such as plaque, caries, and periodontal diseases are primarily caused by microbial infections and remain among the most prevalent global health concerns1,2. Despite the widespread availability of commercial toothpastes, skepticism persists regarding their effectiveness, leading many individuals to rely on traditional practices such as chewing sticks, charcoal, and salt-based remedies3,4. These practices, deeply rooted in cultural heritage, often incorporate natural compounds with antimicrobial and antioxidant properties4,5.
Salt-based herbal toothpaste has gained attention as a promising alternative, combining the abrasive and antimicrobial properties of salt with the therapeutic benefits of phytochemicals5.
Salt contributes to mechanical plaque removal and microbial inhibition, while herbal constituents provide bioactive compounds that enhance oral health. Phytochemicals such as camphor, menthol, eugenol, and piperine are widely recognized for antimicrobial, analgesic, and their anti-inflammatory properties, making them valuable in dental applications3,4. Evaluating these compounds alongside antioxidant activity and salt content is essential to establish the scientific validity of salt-based herbal toothpaste5,6.
Oral health care has increasingly embraced herbal formulations due to their natural bioactive compounds and multifunctional benefits6,7. Camphor oil exhibits notable anti-inflammatory and analgesic properties, while its antiseptic activity makes it effective in relieving toothache and maintaining oral freshness8,9. Mint essential oil demonstrates significant antimicrobial and cytotoxic activities, making it valuable in oral care formulations for its refreshing effect and protective properties9. Eugenol, derived from clove, is well known for its analgesic and antibacterial activity, making it a valuable component in oral care formulations, and piperine enhances bioavailability of other phytochemicals while exhibiting antioxidant potential10. In addition to these phytochemicals, salt plays a traditional role in oral hygiene by aiding mechanical cleaning, reducing microbial load, and maintaining oral pH balance.
The growing popularity of natural oral care reflects a shift toward safer, evidence-backed alternatives. By investigating flavouring agents, antioxidant activity10, piperine content, and salt levels, this study provides a comprehensive scientific lens on salt-based herbal toothpaste. The goal is to bridge tradition and modern science, validating natural formulations as effective, sustainable solutions for oral health.
MATERIALS AND METHODS:
Samples:
Salt‑based herbal, ayurvedic, and salt‑only toothpaste samples were provided by the formulation team and stored at ambient conditions prior to testing.
Sample Preparation:
For phytochemical analysis, toothpaste samples were weighed (1g to 3g, depending on formulation) and extracted with 25mL of methanol. The mixtures were vortexed at 2500rpm for 30minutes to facilitate extraction of bioactive compounds. Following vertexing, samples were centrifuged to remove insoluble material. The supernatant was filtered through polytetrafluoroethylene (PTFE) membrane filter to obtain clear extract suitable for chromatographic analyses.
Phytochemical Quantification:
GC‑FID Analysis of Camphor, Menthol, and Eugenol:
The quantification of camphor, menthol, and eugenol was carried out using GC‑FID (Agilent 8890). Separation of analytes was achieved on a DB‑Wax polyethylene glycol capillary column (30m × 0.25mm internal diameter, 0.25µm film thickness), selected for its high polarity and suitability for separating volatile essential‑oil constituents.
The oven temperature program was optimized to resolve structurally related monoterpenes and phenolic compounds. The temperature was held at 100°C for 5 min, followed by a rise of 10°C/min to 150°C (5min hold), and subsequently increased to 230°C (with 10°C/min), and a final hold for 20min to elute late‑retaining components.
The injector and detector (FID) temperatures were set at 230°C and 250°C, respectively. Detector parameters such as hydrogen and airflow were calibrated daily to ensure signal stability and adequate flame response.
Identification and Quantification of camphor, menthol, and eugenol was based on matching retention times with those of authenticated analytical standards processed under identical chromatographic conditions. Results were reported as % w/w.
HPLC Analysis of Piperine:
Piperine quantification was performed using a HPLC system (Shimadzu) equipped with UV detector set at 338nm, the wavelength corresponding to the absorption maximum of piperine. The chromatographic separation utilized an Inertsil reversed‑phase C18 column (250mm × 4.6mm, 5µm particle size), chosen for its strong retention and resolution of hydrophobic alkaloids.
The mobile phase consisted of acetonitrile and water in a 60:40 (v/v) ratio, delivering optimal elution strength for piperine while minimizing peak tailing. The mobile phase was filtered and degassed prior to use. Chromatographic runs were conducted at a flow rate of 1.0mL/min, under isothermal conditions at 30°C, ensuring consistent retention behaviour.
10µL aliquot of the extracted sample was injected into the system, having total run time as 15 minutes, sufficient for the complete elution of piperine with excellent peak resolution.
External calibration was performed using serial dilutions of pure piperine standard, allowing for quantification based on linear regression of peak area versus concentration. Peak purity index was found to be 0.999884. The method demonstrated high sensitivity, reflected by the reliable detection of piperine in trace‑level ppm concentrations.
All chromatographic analyses were carried out in duplicate, and the mean values were used for reporting piperine content in toothpaste samples.
Salt Determination:
Chloride was measured by Mohr’s titration11 using silver nitrate, and results were reported as salt content (% w/w).
Antioxidant Activity (DPPH):
Antioxidant activity10 of toothpaste formulations was quantified using 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH) as free radical scavenging assay. For this analysis, 2–6g of each toothpaste sample depending on the formulation’s texture and matrix was extracted with 25 mL of 80% methanol to obtain a clear test solution suitable for spectrophotometric measurement. A 100µL aliquot of sample extract was then transferred into the individual wells of microtitre plate. To each well, 100 µL of freshly prepared DPPH reagent in 80% methanol was added. The working concentration of DPPH was 390µM, prepared by dissolving 7.69mg in 50mL of the 80% methanol. DPPH solution was sonicated for 45–60 minutes, or until fully dissolved, avoiding heating to prevent degradation of the radical species.
The mixtures were incubated under dark conditions for about 30 minutes to allow the reaction. Trolox (6‑hydroxy‑2,4,7,8‑tetramethylchroman‑2‑carboxylic acid) used as reference standard. After incubation, absorbance values were recorded at 517 nm using a microplate reader. The decrease in absorbance was proportional to the ability of the samples to quench DPPH free radicals. Antioxidant activity was then expressed as Trolox Equivalent Antioxidant Capacity (TEAC). All experiments were carried out in duplicate, and the average values were used for interpretation.
RESULTS AND DISCUSSION:
Phytochemical and Salt Profile:
Camphor was detected highest in salt‑based herbal (0.51–0.58 %w/w) and ayurvedic (0.50–0.52 %w/w) toothpaste samples but was absent in salt‑only toothpaste samples, confirming its origin in herbal inputs. Menthol was present across all products, salt-based herbal variant (0.48–0.54 %w/w), and salt‑only (0.52–0.58 %w/w). Eugenol was highest in salt‑based herbal (~0.58–0.62 %w/w), moderate in ayurvedic (~0.48–0.51 %w/w), and minimal in salt‑only (~0.10–0.12 %w/w). Piperine was consistently present in salt‑based herbal (2.0–2.5ppm), variable in ayurvedic (0–1.9ppm), and absent in salt‑only. Salt was highest in salt‑based herbal (2.86–3.33 %w/w), moderate in salt‑only (0.55–0.66 %w/w), and absent in ayurvedic (refer Table 1).
Table 1. Phytochemical Composition of Toothpaste Samples
|
Sample Type |
Sample |
Camphor (% w/w) |
Menthol (% w/w) |
Eugenol (% w/w) |
Piperine (ppm) |
Salt (%w/w) |
|
Salt based herbal toothpaste |
1 |
0.58 |
0.54 |
0.62 |
2.3 |
2.86 |
|
2 |
0.54 |
0.51 |
0.60 |
2.4 |
2.88 |
|
|
3 |
0.51 |
0.48 |
0.58 |
2.0 |
3.24 |
|
|
Ayurvedic toothpaste |
1 |
0.50 |
0.42 |
0.49 |
ND† |
ND€ |
|
2 |
0.52 |
0.44 |
0.51 |
0.5 |
ND€ |
|
|
3 |
0.52 |
0.43 |
0.48 |
1.9 |
ND€ |
|
|
Salt based toothpaste |
1 |
ND* |
0.55 |
0.11 |
ND† |
0.58 |
|
2 |
ND* |
0.58 |
0.11 |
ND† |
0.66 |
|
|
3 |
ND* |
0.54 |
0.10 |
ND† |
0.62 |
*ND: Not detected (camphor LOD 3 ppm)
†ND: Not detected (piperine LOD 0.2 ppm)
€ ND: Not detected (salt LOD 0.1 %w/w).
LOD: Limit of detection
Antioxidant Capacity (TEAC, DPPH Assay):
The salt‑based herbal toothpaste showed the strongest radical‑scavenging activity (TEAC range 28.1–28.8 µM/g; mean ≈ 28.6 µM/g). The ayurvedic toothpaste displayed similarly high activity (25.90–29.0µM/g; mean ≈ 26.4µM/g). In contrast, the salt‑only toothpastes exhibited markedly lower TEAC values (2.4–2.7µM/g; mean ≈ 2.6µM/g). These patterns indicate that herbal load and composition are major determinants of antioxidant potential, whereas salt alone does not elevate TEAC (refer Table 2 and figure 1).
Table 2. Trolox Equivalent Antioxidant Capacity (TEAC) of Toothpaste Samples (µM/g)
|
Sample Type |
Sample |
TEAC (µM/g) |
|
Salt based herbal toothpaste |
1 |
28.79 |
|
2 |
28.80 |
|
|
3 |
28.13 |
|
|
Ayurvedic toothpaste |
1 |
25.11 |
|
2 |
25.05 |
|
|
3 |
29.03 |
|
|
Salt based toothpaste |
1 |
2.74 |
|
2 |
2.64 |
|
|
3 |
2.44 |
Figure 1. Graphical Summary of Trolox Equivalent Antioxidant Capacity (TEAC)
Measurement Principle of Antioxidant Activity:
DPPH has deep violet color due to its radical nature. Antioxidants in the toothpaste extracts reduce DPPH, causing a change in color from violet to pale yellow. The decrease in the absorbance value at 517nm was recorded, reflecting radical‑scavenging activity of each formulation. The choice of 390µM was deliberate to ensure a stable and reproducible radical concentration. At lower concentrations (e.g., 300µM or 250µM), we observed that the calculated Trolox Equivalent Antioxidant Capacity (TEAC) values were substantially reduced, especially in samples rich in antioxidants. This happens because when the DPPH pool is too small, strong antioxidants in the sample can rapidly quench most of the radicals, leading to underestimation of antioxidant capacity. Using a sufficiently high concentration (390µM) ensures that the reaction proceeds in a linear, measurable range. Additionally, the antioxidant activity is not underestimated in potent samples. Results are comparable across formulations and aligned with standard DPPH assay protocols.
Integrated Interpretation:
The salt‑based herbal toothpaste combines higher camphor/eugenol/piperine with elevated salt content and achieves the top antioxidant performance. The salt‑only product shows minimal phytochemicals and low TEAC, underscoring the functional role of botanicals.
CONCLUSION:
Salt‑based herbal toothpaste exhibits a chemically richer profile and superior antioxidant capacity relative to salt‑only products. The composition function link is most evident in the salt‑based herbal variant, which combines higher levels of camphor, eugenol, and piperine with salt and achieves the greatest TEAC values. These data substantiate the functional advantages of herbal‑salt formulations and support their use as natural dentifrices.
CONFLICT OF INTEREST:
All authors declare no conflicts of interest.
REFERENCES:
1. Janakiram C et al. Effectiveness of herbal oral care products in reducing dental plaque and gingivitis: a systematic review and meta-analysis. BMC Complement Med Ther. 2020; 20: 43. doi:10.1186/s12906-020-2812-1.
2. Nagansurkar SB et al. Preparation and evaluation of herbal toothpaste. Int J Adv Res Sci Commun Technol. pp. 222–233. doi:10.48175/ijarsct-8094.
3. Bangar PK, Shelke DS. Formulation and evaluation of herbal toothpaste. Int J Adv Res Sci Commun Technol. 2022; 2(2). doi:10.48175/568.
4. Mangilal T, Ravikumar M. Preparation and evaluation of herbal toothpaste and comparison with commercial herbal toothpaste: an in vitro study. Int J Ayurvedic Herb Med. 2016; 6(3): 2266–2273.
5. Girija ASS et al. Investigation on the antioxidant, antibacterial and antibiofilm properties of three distinct herbal tooth powders. BOHR Int J Curr Res Dent. 2023; 2(1): 22–28. doi:10.54646/bijcrid.2023.14.
6. Deshmukh P, Shende V, Gunde M. Formulation and evaluation of herbal toothpaste. Int J Pharm Drug Anal. 2017.
7. Davies R, Scully C, Preston AJ. Dentifrices: an update. Med Oral Patol Oral Cir Bucal. 2010; 15: 976–982.
8. KCC College. JIGYAASA Vol V Issue II [Internet]. 2024. Available from: https://kccollege.edu.in/wp-content/uploads/2024/05/JIGYAASA-VOL-V-Issue-II.pdf
9. Rajale PA, Gaikwad VA. A review on herbal toothpaste. J Emerg Technol Innov Res. 2024; 11(6).
10. Baliyan S et al. Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of Ficus religiosa. Molecules. 2022; 27: 1326.
11. Food Safety and Standards Authority of India. Manual of methods of analysis of foods: fruits and vegetables [Internet]. 2016. Available from: https://fssai.gov.in/upload/uploadfiles/files/Manual_Fruits_Veg_25_05_2016(1).pdf
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Received on 07.11.2025 Revised on 09.02.2026 Accepted on 01.05.2026 Published on 05.06.2026 Available online from June 06, 2026 Research J. Pharmacy and Technology. 2026;19(6):2531-2534. DOI: 10.52711/0974-360X.2026.00362 © RJPT All right reserved
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