|Year : 2023 | Volume
| Issue : 2 | Page : 150-159
|Six-month follow-up of salivary antioxidant defense outcomes of individuals using medicated toothpaste
Mithra N Hegde1, N Suchetha Kumari2, Nireeksha Shetty1, V Chaithra Lakshmi1, Nidarsh Divakar Hegde3, Nishmitha N Hegde3, Jyoti Singh4, Amit Sirdesai4, Prasun Bandyopadhyay5
1 Department of Conservative Dentistry and Endodontics, A. B. Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangalore, Karnataka, India
2 Department of Biochemistry, Central Research Laboratory, KSHEMA, Mangalore, Karnataka, India
3 Department of Conservative Dentistry and Endodontics, A. B. Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
4 Research Scientist, R & D, Dabur India Ltd, Sahibabad, Ghaziabad, Uttar Pradesh, India
5 Global VP Home & Personal Care, Dabur India Ltd, Sahibabad, Ghaziabad, Uttar Pradesh, India
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|Date of Submission||01-Oct-2022|
|Date of Decision||07-Nov-2022|
|Date of Acceptance||28-Nov-2022|
|Date of Web Publication||16-Mar-2023|
| Abstract|| |
Objective: The objective of this study was to study the effect of medicated toothpaste on oral health, a 6-month follow-up.
Methods: Four hundred and twenty-seven participants were screened and followed up for 6 months. The intraoral examination was performed to record caries, gingival bleeding, and plaque index. Saliva collected was evaluated for pH, total antioxidant capacity (TAC), malondialdehyde (MDA), and Vitamin C level for 6 months, and data were analyzed.
Results: On the usage of medicated toothpaste with herbal extract for 6 months, the salivary pH levels were increased, the interquartile range for plaque, and the gingival bleeding index decreased. The percentage change in salivary TAC, MDA, and Vitamin C levels in the caries-free group of subgroup I was 174.8, 58.06, and 59.98, respectively, in subgroup II was 133.3, 52.08, and 58.51, and in subgroup III was 63.77, 45.11, and 47.77. The percentage change in salivary TAC, MDA, and Vitamin C levels in the caries-active group of subgroup I was 136.62, 57.27, and 72.83, subgroup II was 108.59, 37.50, and 61.55, and in subgroup III was 35.62, 30.82, and 54.10, respectively.
Conclusion: The salivary pH levels increased on the usage of medicated toothpaste with herbal extract; plaque and the gingival bleeding index scores were decreased. The salivary antioxidant defense was increased in individuals using medicated toothpaste with herbal extracts which signifies an improvement in overall oral health in the 6-month follow-up.
Keywords: Dental caries; dental plaque; free radical scavengers; gingivitis; oral health; oxidative stress
|How to cite this article:|
Hegde MN, Kumari N S, Shetty N, Lakshmi V C, Hegde ND, Hegde NN, Singh J, Sirdesai A, Bandyopadhyay P. Six-month follow-up of salivary antioxidant defense outcomes of individuals using medicated toothpaste. J Conserv Dent 2023;26:150-9
|How to cite this URL:|
Hegde MN, Kumari N S, Shetty N, Lakshmi V C, Hegde ND, Hegde NN, Singh J, Sirdesai A, Bandyopadhyay P. Six-month follow-up of salivary antioxidant defense outcomes of individuals using medicated toothpaste. J Conserv Dent [serial online] 2023 [cited 2023 May 28];26:150-9. Available from: https://www.jcd.org.in/text.asp?2023/26/2/150/371794
| Introduction|| |
Oral disease is a major global public health problem; dentistry has been unable to tackle this problem completely. Toothbrushing plays a major role in oral health maintenance, stimulation of the gingiva, and also physically dislodging food particles. The clinical significance of toothpaste can be studied based on many factors: its composition, its activity to prevent dental caries and gingivitis, its sustainability, and the effects of components within the oral cavity.
Salivary compounds can drastically change their concentration in a consequence of a great variety of physiological states, stimuli, and stress states, so their identification and quantification can be useful for their early diagnosis. Wagle, et al. showed that saliva contains various antioxidants; thus, it serves as the first line of defense against oxidative stress. The total antioxidant level is evaluated as total antioxidant capacity (TAC), a biomarker for a number of oral and systematic diseases. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in the cells. An increase in free radicals causes the overproduction of MDA. Thus, MDA level is commonly known as a marker of oxidative stress.
Evans and Omaye proved that there is a potential relationship between varying free radicals and antioxidant concentrations related to cell life and longevity. Carr and Maggini observed that Vitamin C enhances the differentiation and proliferation of B and T-lymphocytes, thereby enhancing the antibody levels, which in turn boosts immunity.
The composition of medicated toothpaste: maricha, pippali, and tomar have an active ingredient Piperine which has antioxidant, antimicrobial, anti-inflammatory, and mucosa protective activities. Gingerol, shogaol, and zingerone are the antioxidant compounds present in the component ginger or Shunti acts as free radical scavengers. Dadim has a significant antibacterial effect on common oral bacterial pathogens with maximum effect on Streptococcus mutans, which is the main microorganism responsible for dental caries. Cinnamon has antifungal activity against Candida albicans. Clove inhibits S. mutans as it contains eugenol, which is an antimicrobial agent. The use of Meswak proved to lower the gingival and plaque indices.
The lack of clinical evidence on long-term follow-up and oral health outcomes of using medicated toothpaste was the need for the study.
A 6-month follow-up, an active-controlled trial was done to analyze various parameters of oral health: caries, gingivitis, plaque, and salivary pH over 6 months. At five intervals, the salivary analysis of TAC, malondialdehyde (MDA) level, and Vitamin C, the nonenzymatic salivary antioxidant markers were analyzed.
| Methods|| |
The accrual of participants commenced from July 2020, and the study was completed in May 2021. In this active-controlled trial, participants aged between 25 to 50 years, who fulfilled the inclusion criteria, were enrolled. The study protocol and procedures were accepted by the University Ethics Committee (NU/CEC/2020/0302) and Clinical trial registered (CTRI/2020/06/026082). The reporting guidelines are followed as per the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) Statement.
A total of 427 participants were screened, subjects undergoing orthodontic treatment, edentulous, history of smoking, alcohol consumption, pregnancy, lactating, or nursing, under continuous nutritional supplementation, undergoing radiotherapy, chemotherapy, under long-term medication, underlying uncontrolled medical illness, including hypertension, diabetes mellitus, and hepatitis, were excluded from this study (126 participants). Three hundred one healthy subjects free from systemic diseases affecting salivary flow patterns and available for follow-up of 6 months, with and without caries, gingivitis, and periodontal diseases were included and grouped as caries-free (n = 140) and caries-active (n = 161). They were further subgrouped into four depending on the toothpaste given to them, three subgroups using marketed medicated toothpaste (Dabur Red Paste, Dabur Dant Rakshak Paste, and Dabur Meswak Toothpaste) and one control (marketed nonherbal toothpaste). Twenty-one participants missed follow-up during the course of the trial, and thus, a total of 280 participants were followed up and included in the 6-month analysis. Voluntary written consent was obtained from patients. The WHO Oral Survey Form for adults, 2013 annex 1, was used to document the case history and intraoral examination details [Figure 1].
Dabur Red Paste contains herbal extracts derived from Maricha, Pippali, Shunti, Tomar, Lavang, Karpura, Pudina, and garlic powder. Dabur Dant Rakshak Paste contains herbal extracts derived from Mulethi, ajwain, Pudina Satva, Pudina oil, Babul, Manjistha, Dalchini, Khair, Patang, Kababchini, Jambhul, Majuphal, Ber, Vajradanti, Tejbal, Lavang oil, Anantmool, Bakul, Beheda, Dadim, Peelu, Nilgiri, Sonf, Akarkara, Neem, Maricha, Pippali, Shunti, Badam, Elaichi, and garlic powder. Dabur Meswak contains calcium carbonate, sorbitol, water, silica, sodium lauryl sulfate, flavor, Meswak extract, cellulose, gum, carrageenan, sodium silicate, sodium silicate, PVM/MA copolymer, sodium saccharin, zinc gluconate, sodium benzoate, Cl 77891, benzyl alcohol, and p-Thymol. Control toothpaste consists of calcium carbonate, sorbitol, sodium lauryl sulfate, arginine, silica, titanium dioxide, sodium silicate, flavor, carrageenan, sodium monofluorophosphate, sodium bicarbonate, potassium nitrate, benzyl alcohol, sodium saccharin, limonene, and in aqueous base.
Subjects who fulfilled the enrolment criteria were provided with a commercially available nonherbal toothpaste (Colgate Cibaca) and an adult Colgate Sensitive UltraSoft Toothbrush to use for 1 week. Subjects were instructed to use the toothpaste and toothbrush for 2 min twice a day as per the direction of use mentioned on the toothpaste pack in place of their customarily used toothpaste and toothbrush. This allows for an attenuation of the residual effects of the subject's previously used toothpaste and enables every subject on the same toothpaste background.
Active control trial
The 280 participants were assigned to subgroups by the researcher depending on the time of recruitment.
A baseline examination was performed to assess caries, plaque, and gingival bleeding index. Participants were given the allocated toothpaste and toothbrush for home use and advised to brush twice daily for 2 min as per the direction of use mentioned on the toothpaste pack for 24 weeks.
World Health Organization; Oral Health Assessment Form for Adults, 2013 Annex 1 was used. The number of carious teeth, plaque index, and gingival bleeding index were calculated. These parameters were recorded at two intervals, i.e., 1st visit (preusage of toothpaste) and 5th visit (24 weeks).
Collection of saliva
The saliva collection was done 2 h postbrushing teeth and eating/drinking. 5 mL of unstimulated saliva was collected in Tarsons centrifuge tube (15 mL), centrifuged, and the supernatant obtained was stored at −80°C for subsequent analysis. The saliva samples were collected at five intervals, i.e., 1st visit (preusage of toothpaste), 2nd visit (1 week), 3rd visit (4 weeks), 4th visit (12 weeks), and 5th visit (24 weeks).
The salivary pH was determined at two intervals, i.e., at 1st visit and 5th visit. The pH paper was dipped in the saliva present in the vial, and color change was observed. The interpretation was made based on the color guide present in the GC Salivary Check kit (S642-090).
Salivary biochemical analysis of TAC, MDA, and Vitamin C was done for the saliva collected at 5 intervals. Before analysis, bench cooling of the salivary samples was done to liquefy the saliva samples stored at −80° C.
Estimation of salivary total antioxidant capacity
The TAC of the saliva sample was evaluated by the phosphomolybdenum method. 0.1 ml of sample solution containing a reducing species (in water, methanol, ethanol, dimethyl sulfoxide, or hexane) was combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were capped and incubated in a thermal block at 95°C for 1 h 30 min. After the incubation, the samples were cooled to 25°C, and the absorbance of the solution was measured using a double-beam spectrophotometer (Systronics, India, Model 2202) at 695 nm against a blank.
Estimation of salivary malondialdehyde level
The salivary MDA level was estimated by the thiobarbituric acid-trichloroacetic acid (TBA-TCA) method, and the samples were heated with TBA-TCA reagent (20% TCA, 0.5% TBA, and 2.5N HCl) in a water bath at 100°C for 20 min. After cooling to room temperature, the precipitate was removed by centrifuging the solution at 2000 rpm for 10 min. The supernatant's absorbance was determined at 532 nm against blank.
Estimation of salivary Vitamin C
The dinitrophenylhydrazine (DNPH) technique was used to determine the salivary Vitamin C levels in the samples. Vitamin C, also known as ascorbic acid, is a good reducing agent that undergoes reversible conversion to its oxidized form, dehydroascorbic acid; both of these forms a pair with 2,4-dinitrophenyl hydrazine to make an osazone, which produces a yellow color when combined with 4.5M sulfuric acid. Copper functions as a catalyst in the DNPH reagent, and the color intensity was measured at 520 nm.
The data collected were entered into a Microsoft Excel spreadsheet and analyzed using IBM SPSS Statistics, Version 22 (Armonk, NY, USA: IBM Corp). Descriptive data were presented in mean and standard deviation. The P < 0.05 was considered statistically significant.
| Results|| |
Three hundred one healthy subjects with and without caries, gingivitis, and periodontal diseases were included in this study, lost a follow-up of 21 participants during the course of the study, 280 participants were followed up and included in the analysis.
In 6-month follow-up, there was no new caries occurrence, i. e., the number of caries remained the same throughout the follow-up. The P > 0.05 indicating that results were not statistically significant in all groups.
The gingival bleeding index and plaque index were decreased in all groups using medicated toothpaste over a period of 6 months and the results were statistically significant [Table 1].
|Table 1: Effect of toothpaste on gingival bleeding and plaque index (baseline to 24 weeks)|
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The salivary pH was significantly increased after using medicated toothpaste with herbal extract over a period of 6 months compared to the control group [Table 2].
|Table 2: Salivary biochemical parameters and pH at 5 intervals over a period of 6 months|
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The mean salivary TAC and Vitamin C levels have been increased in both caries-free and caries-active groups using medicated toothpaste with herbal extract, whereas the mean salivary TAC and Vitamin C levels were decreased from preusage to 24 weeks in individuals using control toothpaste. The mean salivary MDA levels have been decreased in all groups using medicated toothpaste with herbal extract, whereas the mean salivary MDA levels have been increased in groups using control toothpaste [Table 2].
The post hoc analysis of biochemical parameters at five intervals shows that the variation in salivary MDA, TAC, and Vitamin C levels in four subgroups at five intervals was statistically significant (P < 0.001), indicating that the play of chance is very less. The mean difference of salivary MDA levels in three subgroups using medicated toothpaste with herbal extract was positive, indicating a decrease in salivary MDA levels over a period of 6 months. The mean difference of salivary TAC and Vitamin C levels in groups using medicated toothpaste with herbal extract showed negative values that denote an increase in their levels over 6 months, whereas the mean difference of salivary TAC and Vitamin C levels in individuals using control toothpaste showed positive values indicating a decrease in their levels over a period of 6 months [Table 3].
| Discussion|| |
The active-controlled trial estimated oral health parameters like; number of dental caries lesions, salivary pH, plaque index, and gingival bleeding index at pre and post usage of toothpaste; also, the salivary total antioxidant capacity, malondialdehyde, and vitamin C levels were estimated at 5 intervals over a period of 6-months in 280 participants.
The study showed no new caries lesions throughout the follow-up in both caries-active and caries-free groups; this could be due to the oral hygiene method and awareness among the participants during the preusage and follow-up period. As per de Oliveira Carvalho et al., the anti-cariogenic and anti-biofilm of toothpaste formulated with essential oils proved to have an inhibitory effect against bacteria associated with cavities and periodontitis.
The salivary pH depends on the H + ion concentration, which in turn is affected by food, beverages, vigorous exercise, and enzymes in the oral cavity. Hence, the pH was evaluated 2 h before or after food intake and brushing. The salivary pH did not fall below 5.5 (critical pH) in any of the groups.
At 6-month follow-up, there was a significant rise in salivary pH on the usage of medicated toothpaste with herbal extracts which could be attributed by the ingredients: Peelu, Neem, and Dalchini in the toothpaste. A study by Khairnar et al. on the efficacy of herbal toothpaste on salivary pH showed that the ingredients; Pilu, Neem, Liquorice, and Cinnamon raise the salivary pH. Khairnar et al. proved that the salivary pH influences the caries process and the demineralization, remineralization processes of the tooth are dependent on the pH of the saliva.
Food particles which are accumulated on the tooth surface become the source of nutrition, and the plaque becomes the host for the cariogenic bacteria (Streptococcus mutans and Lactobacillus acidophilus) in the oral cavity leading to the fermentation of food particles, thereby producing free radicals, which leads to various pathologies: such as decrease in pH leading to cavitation. The use of medicated toothpaste with herbal extract has shown to have a higher antioxidant capacity to scavenge these free radicals, thus helping in the regulation of pH and control of caries [Figure 2].
|Figure 2: Schematic representation of production and regulation of free radiacls in the oral cavity. (a) Food particles which is accumulated on tooth surface becomes the source of nutrition and the plaque becomes the host for the cariogenic bacteria (Streptococcus mutans and Lactobacillus acidophilus) in the oral cavity leading to fermentation of food particles thereby by producing free radicals, which leads to various pathologies: such as decrease in pH leading to cavitation. (b) The use of medicated toothpaste with herbal extract shown to have higher antioxidant capacity scavenge these free radicals thus help in regulation of pH and control of caries|
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The postusage of medicated toothpaste with herbal extract showed a reduction in plaque and gingival bleeding index. The control group also showed plaque index (Silness and Löe, 1964) and gingival bleeding index (Löe and Silness, 1963) scores between 0.1 and 1.9, i. e., in good and fair criteria, none of the groups showed poor criteria. As the difference is minimal, the results were expressed in the Interquartile range. The resolution of GBI and PI could be associated with an increase in salivary TAC over a period of 6 months. Thus, the medicated toothpaste with herbal extract provided the environment in improving gingival health as its usage significantly reduced GBI and PI scores. Similar results were observed by Hosadurga, et al.
The 6-month follow-up also showed that the usage of medicated toothpaste with herbal extract increased salivary TAC and Vitamin C levels. The results are in accordance with the in vitro study on the redox potential of different herbal toothpaste. The baseline data of the caries-active subgroup using medicated toothpaste showed higher TAC and MDA levels than the caries-free group this is due to the increase in the free radical activity., After using medicated toothpaste with herbal extracts for 6 months, the MDA levels decreased and TAC levels increased which was an important finding in all experimental groups. The herbal extracts found in the medicated toothpaste have proved to be having medicinal and therapeutical properties which have caused this change in the TAC and MDA levels.
Natural phytochemicals provide an alternate strategy for the treatment and prevention of oral diseases. The isoprenoid group of neem has anti-inflammatory, anti-bacterial, anti-fungal, and immunomodulatory properties. Eugenol, which makes up 80 to 90% of clove oil, stimulates the phospholipid bilayer in the microorganism's cell membranes, increasing penetrability. This disturbs the bacterial cell's membrane, which eventually causes cellular contents to leak out, resulting in the death of the bacterial cell.
The oral mucosal cell membrane is made up of lipid bilayers (mainly unsaturated fatty acids) which help in maintaining the fluidity of the membrane. Increased levels of free radicals/ROS convert unsaturated fatty acids into saturated fatty acids leading to a decrease in the fluidity of the membrane and increased MDA production, which in turn increases the toxic stress in cells leading to impairment of cellular functions [Figure 2].
The postusage of medicated toothpaste with herbal extract increased salivary TAC levels and led to a decrease in MDA levels. Decreased MDA levels and increased TAC could be directly related to the increase in pH and improved gingival health in patients using medicated toothpaste with the herbal extract.
Vitamin C, a nonenzymatic salivary antioxidant level increased in the 6-month follow-up of subgroups using medicated toothpaste. The salivary Vitamin C level is directly related to serum Vitamin C levels. These levels are correlated with increased antioxidant and decreased MDA levels. Increased free radicals are responsible for various oral pathologies; these free radicals are neutralized by antioxidants, thus resulting in less utilization of Vitamin C in the oral cavity and could have increased salivary Vitamin C levels. A study by Tada A, and Miura H., showed salivary Vitamin C reduces gingival scores of inflammations and thus helps in the reduction of gingival bleeding in patients affected by chronic periodontitis. Study by Shetty et al. has shown that Vitamin C is an essential nutrient that maintains tooth integrity, and prolonged deficiency of Vitamin C results in tooth loss.
Subgroup 1 showed the best clinical results with a decrease in the plaque index, gingival bleeding index, and salivary MDA levels and an increase in salivary pH, TAC, and Vitamin C over a period of 6 months. The possible limitation of the study could be that this study was carried out for 6 months, a long-term study is required to check the effect of toothpaste on caries progression.
| Conclusion|| |
After 6 months of trial, it can be concluded that both the experimental and control group showed effective control of caries. No adverse reactions were observed during the trial. Medicated toothpaste with herbal and Ayurvedic extracts (Dabur Red Paste, Dabur Dant Rakshak Paste, and Dabur Meswak Toothpaste) enhanced overall oral health by control of plaque, gingivitis, and maintained high salivary pH, which was statistically significant. Medicated toothpaste with herbal extracts also increased the salivary TAC and Vitamin C levels, and decreased MDA levels which were statistically significant.
Financial support and sponsorship
Dabur India Ltd.
Conflicts of interest
There are no conflicts of interest.
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Prof. Mithra N Hegde
A. B. Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangalore - 575 018, Karnataka
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]