The present study was a triple blind, interventional randomized parallel clinical trial conducted at ACPM Dental College and Hospital, Dhule, Maharashtra, India, in the month of August 2014 over a period of four weeks. It was carried out on a total of 45 subjects who were enrolled for BDS course (2^nd and 3^rd BDS students in the age group of 19-21years). The ethical clearance for the study was obtained from the Institutional Ethical Review Board and informed consent was obtained from all the participants prior to the study.

Materials and equipments used were three herbal toothpastes (Patanjali Dant-Kanti Dental Cream, Himalaya Complete Care Toothpaste and Vicco Vajradanti Toothpaste), sterile plastic containers, insulated containers, calibrated test tubes, ALP reagent kit, ACP reagent kit and Erba Manheim Automatic Analyzer.

Inclusion Criteria: Subjects in the age group of 19-21years with good general and periodontal health following routine oral hygiene procedures and those who agreed to comply with the study visits were included in the study.

Exclusion Criteria: Subjects suffering from systemic diseases, currently using antibiotics, subjects undergoing orthodontic treatment, subjects using oral hygiene aids other than toothbrush & toothpaste, subjects having adverse habits like smoking, alcohol consumption were excluded from the study.

Study Design [Table/Fig-1]: Since this was a preliminary study, it was decided to select 15 subjects in each group (For such preliminary studies the recommendation is a sample size of 12 per Group) [9]. All the under graduate dental students studying in 2^nd and 3^rd year and residing in the hostel of dental college who fulfilled the inclusion and exclusion criteria were invited to participate in the study. Among them, 45 subjects were randomly selected employing random sampling. Investigator not involved in examinations generated the random numbers. Total of 45 subjects were randomly divided into three groups of 15 each (Group A using Patanjali Dant-Kanti Dental Cream, Group B using Himalaya Complete Care Toothpaste and Group C using Vicco Vajradanti Toothpaste) employing random sampling using lottery method.

[Table/Fig-1]:

Flow chart of study design.

The present study employed a triple blinding procedure. The investigator was blinded and was unaware of the toothpaste allocation to the subjects of that particular group. A male assistant who was unknown to the principle investigator randomly allocated the toothpastes to three groups. The subjects were also blinded with respect to the particular toothpaste they used throughout the study period. All the study toothpastes were covered with white sticker labels indicating group name and caps of all the toothpastes were replaced with white caps of same uniform size to achieve blinding. Statistician was also kept blind regarding the groups while analyzing the data.

Subjects were instructed to brush twice daily using the toothpaste which was given to them. Unstimulated saliva samples were collected before and after brushing and were transported to the laboratory where salivary ALP and salivary ACP levels were determined. The study was conducted over a period of 4 weeks on each subject and saliva samples were obtained at weekly intervals starting from day 1 i.e., on 1^st day, 7^th day, 14^th day, 21^st day and 28^th day.

Saliva collection and analysis: The required quantity of saliva sample was collected before and after tooth-brushing procedure. Subjects were asked to abstain from eating and drinking for 1 hour before sample collection. Tooth-brushing along with the allotted toothpaste for each male participant was monitored by the investigator himself; while for female participants, it was monitored by a female investigator assistant throughout the study period.

The unstimulated salivary samples were collected from the subjects in morning between 7am to 7.30am before and after brushing. Subjects were made to sit in an upright position and saliva was collected by spitting method before and after brushing [10]. For each participant at least 10 ml of saliva was collected in sterile plastic container. The plastic containers with saliva were stored in an ice container maintained at 2-4⁰C until analysis of salivary variables was performed. Each saliva sample was centrifuged at 3,000rpm for 10 min and clear supernatants were processed immediately for estimation of ALP and ACP. The enzyme activities in saliva were determined spectrophotometrically by the International Federation of Clinical Chemistry (IFCC) method on the Erba Manheim Automatic Analyzer using ALP reagent and ACP reagent as substrates [11].

Data was analyzed using SPSS (Statistical Package for Social Sciences) statistical software version 16. Chi square test was used to compare gender-wise distribution. Mean and standard deviation of the obtained pre (before brushing) and post (after brushing) mean levels of salivary ALP and ACP at 1^st, 7^th, 14^th, 21^st and 28^th day were compared within group A, group B and group C by paired ‘t’ test. Overall mean changes in the levels of Salivary ALP and ACP on 1^st and 28^th day within group A, group B and group C were compared by ANOVA test.

Chi square test showed no significant difference (p=0.913) in gender-wise distribution between Group A, B and C indicating a good match [Table/Fig-2].

All the three toothpastes showed reduction in salivary APL and ACP levels after brushing at each interval (1st, 7th, 14th, 21st and 28th day) and the difference in pre and post brushing levels was highly significant (p<0.001) in every group [Table/Fig-3,4 and 5].

When overall mean reduction for all the intervals was compared, group A toothpaste showed significantly more reduction (p<0.05) of ACP and ALP levels when compared to group B toothpaste. Similar result was seen when group B and group C toothpaste were compared where group C toothpaste was found to be more effective than group B toothpaste (p<0.05). However, no significant difference was seen in the efficacy of group A and group C toothpaste in reducing levels of ACP and ALP (p>0.05) [Table/Fig-6].

In the present study, overall reduction in baseline (pre-brushing) values of salivary ACP on 1^st day and 28^th (last) day was 0.04 for group A, 0.10 for group B and 0.27 for group C. ANOVA test showed very highly statistically significant difference (p<0.001) in all the three groups [Table/Fig-7].

Overall reduction in baseline (pre-brushing) values of salivary ALP on 1^st day and 28^th (last) day was 0.18 for group A, 0.12 for group B and 0.14 for group C. ANOVA test showed statistically significant difference (p<0.050) in all the three groups [Table/Fig-7].

The present study was an in-vivo, three parallel groups and randomized clinical trial that evaluated the comparative efficacy of three different herbal toothpastes on salivary parameters i.e., ALP and ACP. From the results, it can be seen that there was reduction in salivary ALP and ACP levels after using each toothpaste. Vicco Vajradanti Toothpaste and Dant Kanti Dental Cream performed better than Himalaya Complete Care toothpaste. No clinically significant adverse reactions of the tested herbal toothpastes, were reported or observed, during the entire study period.

The present study employed only second and third year BDS students based on the availability during the actual study period and keeping in mind the future study compliance. The present study incorporated only herbal toothpaste because of increased public interest in the usage of alternative health care, including use of natural or herbal health care products. According to the World Health Organization (WHO), up to 80% of the population in developing countries uses plants and its products as traditional medicine for primary health care. In the developed countries, 25% of the medical drugs are based on herbs and their derivatives [12]. Ease of availability, least side effects compared to conventional chemical toothpastes and low cost make the herbal preparations as the key player of all available therapies, especially in rural areas [13]. Available scientific literature shows that herbal toothpastes are as effective as conventional toothpastes [14,15]; but there is a dearth of scientific literature showing comparative efficacy of one herbal toothpaste over the other. Hence, only herbal toothpastes have been selected for the present study to assess the efficacy of one over another.

Composition of the three herbal toothpastes employed in the present study was:

As seen from the composition, there is variation in the contents of the toothpastes which might have influenced the study results.

Unstimulated saliva was preferred over stimulated saliva in the present study, as unstimulated saliva is secreted by the salivary glands in normal physiological conditions. The unstimulated whole saliva is often preferred in the diagnosis of biomarkers, as stimulated saliva contains a diluted concentration of ingredients which are intended to be detected. The dilution of salivary sample may pose difficulties in detection of these ingredients and may result in inferior levels of these ingredients in the processed salivary sample [16].

ALP and ACP can act as good biomarkers for assessing periodontal disease. ALP aggravates the bone loss resulting in rapid progression of periodontitis and ACP is closely associated with salivary calculus formation. Also, there is lack of scientific literature regarding the effect of herbal toothpaste on these salivary parameters. Hence, these parameters were selected for the present study.

Salivary ALP and ACP showed reduction in all the three groups after brushing. Levels of ALP and ACP activities in saliva and GCF directly correlate with the intensity of inflammatory process of periodontal tissue and act as inflammatory markers in periodontal diseases. As the inflammation subsides, level of ALP and ACP in saliva also decreases [17,18]. In the present study, gingival inflammation present in some individuals might have come down due to various anti-inflammatory substances present in toothpastes such as pilu, akarkara, vidang, chamomile, babool, jamun, bor, liquorice, harda, neem etc [19–21] and thus, helped in reducing the levels of ALP and ACP in saliva.

Another reason for reduction in these salivary enzyme levels following use of herbal toothpaste may be inhibition of plaque bacteria. Plaque bacteria secrete ALP and ACP [18,22]. Activity of anti-bacterial ingredients present in herbal toothpaste resulted in inhibition of plaque bacteria and ultimately reduction in levels of acid and alkaline phosphatase in saliva.

Patanjali Dant Kanti and Vicco Vajradanti performed better than Himalaya Complete care toothpaste in terms of reduction of salivary ALP and ACP levels as these toothpastes contain more number of anti-inflammatory and anti-bacterial ingredients than Himalaya Complete Care toothpaste. Valid and accurate comparison of results for salivary ALP and ACP in the present study could not be done with other scientific studies, as no previous research work has conducted to assess the efficacy of herbal toothpastes on these salivary parameters.

Long term clinical trials taking larger samples with wider geographical representation need to be conducted to quantify the efficacy of herbal toothpaste. Studies should also be carried out to assess the combine effect of herbal toothpastes and herbal mouthwashes on saliva.

The herbal toothpastes possess significant ALP and ACP inhibitory activity and can prove to be an alternative to conventional toothpastes. Among the three tested toothpaste, Dant Kanti and Vicco Vajradanti were found to be more effective than Himalaya Complete Care toothpaste.

Conflict of Interest: None of the authors is associated with the herbal toothpaste companies used in the present trial.