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Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 EFFECT OF MOUTH RINSING WITH SOLUTIONS CONTAINING DIFFERENT COMMERCIALLY AVAILABLE SUGAR SUBSTITUTES ON SALIVARY PH- AN IN VIVO RANDOMIZED CONTROLLED TRIAL. Tanushri MD, Puja CY. * Post graduate student, Department of Public Health Dentistry, Bapuji Dental Collage and Hospital, Davangere, India. Professor, Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere, India. Abstract Keywords: salivary pH, stevia rebaudiana, sucrose, mouth rinsing, sugar substitutes. Background: Nowadays sugar free food and drinks have become very popular because of increased concerns related to obesity and overweight; as it is well known that sucrose has a negative impact on both general as well as oral health. These artificial sweeteners also have some side-effects when consumed in excess. In recent decade, stevia rebaudiana, a plant derived sugar is considered as a natural and healthy alternative to sugar and artificial sweeteners. Literature pertaining to the salivary pH changes related to stevia is limited. Objective: to assess the effect of mouth rinsing with solutions containing different commercially available sugar substitutes on salivary pH. Method: the present study is a randomized controlled trial with concurrent parallel design. 85 female students aged 20-25 years were randomly allocated to four different groups. Salivary pH assessments were performed at baseline and after mouth rinsing with different sugar solutions containing Aspartame, Sucralose, Saccharin, Stevia and distilled water at 1, 20 and 60 minutes respectively. One way Analysis of variance and repeated measures Analysis of variance followed by Tukey’s post hoc tests were employed to analyze the data. Results: The results indicated that there was a statistical significant difference in the salivary pH values between group C (Stevia) and group E (Distilled water) after mouth rinsing at 20 minutes. In group C (Stevia) there was a significant rise in the salivary pH from baseline value to 1 min (p = 0.001) and 20 minutes (p = 0.005). Conclusions: Pattern of salivary pH changes after mouth rinsing with Stevia was similar to other artificial sweetener solutions which indicates that Stevia rebaudina can act as an excellent natural sugar substitute replacing the current artificial sweeteners and refined sugars and thereby help in tackling their side effects. Introduction The term intense sweeteners (IS) refers to various substances of plant origin or obtained by chemical synthesis, used in the food industry for their sweetening power and their low caloric value.1 Their sweetening power is a hundred to thousand times higher than that of sucrose. Sugars are recognized as the most important dietary factor in the development of dental caries. Furthermore, the role of acid fermented products of sugars in enamel dissolution by the action of bacteria, such as Streptococcus mutans and Lactobacillus casei, is clear. 2 The use and consumption of intense sweeteners have risen sharply over the last decades, probably due to the concerns linked to the doubling of prevalence of overweight and obesity. Numerous studies have shown that the form in which sugars are ingested and the frequency of their consumption are directly related to the prevalence of caries. 3,4,5 Nowadays sugar free food are © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [6] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 very much popular because of their less calorie content. The use of sucrose substitutes in sweets is believed to have contributed in part to the decline in the prevalence of dental caries in industrialized countries.6 These sugars tend to have desirable sweetness but are not metabolized in the human body and therefore do not provide calorie intake. A sweetener is a food additive, which mimics the effect of sugar on taste. Therefore, they are called as sugar substitutes.7 Research for alternatives to sucrose have resulted in the development of synthetic sweeteners, many of which are considered safe for teeth, such as Aspartame, Saccharin, Cyclamate, Xylitol, and Mannitol. However, in addition to their benefits, animal studies have shown that artificial sweeteners are associated with weight gain, brain tumors, bladder cancers and many other health hazards. Advancing research in this area has oriented towards discovery and evaluation of natural non-cariogenic sweeteners. Stevioside is one such natural non-cariogenic sweetener, it is a steviol glycoside extracted from stevia rebaudina. The literature search revealed very few studies which have assessed and compared the salivay pH changes after consumption of different commercially available natural and artificial sugar substitutes such as Stevia, Aspartame, Sucralose, and Saccharin. So, study is planned to evaluate and compare the effect of mouth rinsing with solutions containing different commercially available natural and artificial sugar substitutes on salivary pH. The present study wants to evaluate whether there is a difference in salivary pH changes after mouth rinsing with solutions containing commercially available sugar substitutes Sugar free Gold (Aspartame), Sugar Free Natura (Sucralose), Apollo Pharmacy Stevia (Stevia) and Saccharin Helios Pharmaceuticals (Saccharin). Null hypothesis states that there is no difference in salivary pH changes after mouth rinsing with solutions containing commercially available sugar substitutes Sugar free Gold (Aspartame), sugar free Natura (Sucralose), Apollo Pharmacy Stevia (Stevia) and Saccharin Helios Pharmaceuticals (Saccharin). Materials and methods An Experimental, in vivo, concurrent parallel study was planned. Sample size was determined using sample size and power calculator software with R code application on www.http://powerandsamplesize.com/Calculators/Compare-2Proportions/2-Sample-Equality website. (powerandsamplesize.com)8 sample size was calculated using the formula9 2 n= 2 z1−α/(2τ)+z1−β σ μA−μB based on values from the study by Steinberg L 10, where µA is group A mean = 6.04 µB is group B mean = 5.35 Standard deviation σ = 1.13 Power of the study (1- β) =0.8 Type 1 error rate (α ) =0.05 Overall, sample size was estimated to be 83 which was rounded off to 85 (17 in each group.) Sampling frame comprised of female undergraduate students aged 20-25 years of Bapuji Dental College and Hospital, Davangere. A sample of 85 undergraduate female students who fulfilled the eligibility criteria were randomly selected. Subjects who were on medications systemic diseases that affected their salivary flow rate and who were unable to comply with the study appointment schedules were excluded and the study was conducted at the clinical premises of Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere. Ethical clearance was obtained from the Institutional Review Board of Bapuji Dental College and Hospital, Davangere. Voluntary informed consent was obtained from the study participants prior to the start of the study after informing about the research details through a participant information form. Investigator and participants were blinded. Interventional groups: 1. Group A -20ml of distilled water with one tablet of Sugar free Gold (Aspartame) 2. Group B -20ml of distilled water with one tablet of Sugar free Natura (Sucralose) 3. Group C -20ml of distilled water with one tablet of Apollo Pharmacy Stevia (Stevia) 4. Group D-20ml of distilled water with one tablet of Saccharin Helios pharmaceuticals (Saccharin) 5. Group E -20ml of distilled water Random allocation: Concealed randomization method was followed © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [7] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 Random assignment of the participants to five interventional groups was done by a separate person not involved in the study using computer assisted software for generating random numbers.(random no table)11 Preparation of the test solutions:The test solutions were prepared by using commercially available sugar substitutes which are Sugar free Gold containing Aspartame, Sugar free Natura containing Sucralose, Apollo Pharmacy Stevia containing Stevia and Saccharin in form of tablets. One tablet of each commercial sugar substitute was added separately to 20 ml of distilled water and stirred (10 seconds) until it completely dissolved in it. The test solutions were then given to each subject for mouth rinsing. Intervention details: After selection of the students, they will be randomly allocated to different groups and their unstimulated salivary sample will be collected at the baseline and pH will be determined with the help of salivary pH indicator strips. After determination of baseline pH, the subjects will be instructed to mouth rinse with test solutions for 30 seconds12 by swishing the entire content in the mouth at once and expectorate after which the unstimulated salivary pH will be again assessed at 1, 20 and 60 min respectively.13 Method of saliva collection: All subjects were given clear instructions to refrain from eating for one hour before collection of saliva. Passive drool method of saliva collection was followed. The subjects were instructed to let saliva pool in the floor of the mouth for at least 1 minute and then expectorate into a sterile disposable cup.14 Salivary pH estimation: Salivary pH was checked using dental salivary pH indicator strips (GC company). The pH strip was dipped into the collected saliva and taken out immediately and observed for 10 seconds for the colour change. The change in colour was compared with the reference given by the manufacturer and readings were entered.15 Salivary pH was recorded at baseline and then after mouth rinsing with the sugar substitute containing solutions at one minute, 20 minutes and 60 minutes respectively. Statistical Analysis: The data obtained during the course of the study was systematically entered in Microsoft Excel sheet. Data analysis was performed using SPSS software version 20. Data was normally distributed, so parametric tests have been employed. Repeated measures analysis of variance was employed to compare the means of salivary pH within the group at different time intervals. One way analysis of variance was employed to compare the means of salivary pH between the groups. Post hoc Tukey’s test was performed as significant difference was found between the groups and within the groups at different time intervals. Results There was a significant difference in mean salivary pH between groups after mouth rinsing with solutions containing Aspartame (group A), Sucralose (group B), Stevia (group C), Saccharin (group D) and distilled water (group E) at 20 minutes. Table 1 (p = 0.040). At baseline, there was no statistical difference in mean salivary pH values between the interventional groups allowing for valid comparisons between groups post intervention (table 1). At 20 minutes post mouth rinsing, there was a statistically significant difference (p = 0.040) between the mean salivary pH values of Stevia (group C) and Distilled water (group E). No significant difference in mean salivary pH values was observed between interventional groups at different time intervals (Tables 1, 2). Tukey’s Post-hoc comparisons of ANOVA test between the groups at different time intervals showed a statistically significant difference between Stevia (group C) and Distilled water (group E) at 1 minute (p=0.046) and 20 minutes time interval (p= 0.037), (Table 2).There was an initial rise in the salivary pH after mouth rinsing with all five groups at one minute which was not statistically significant followed by a gradual drop from 1 minute to 20 minutes in all the groups among which the mean salivary pH drop was less in group C (table 1, graph 1). From 20 minutes to 60 minutes, there was a gradual drop in the mean salivary pH values in all the groups and it remained above the baseline value at the end of 60 minutes after mouth rinsing with respective solutions. In group C (Stevia) there was a significant rise in the salivary pH from baseline value to 1 minute and 20 minutes (p = 0.005), (tables 3, 4, graph 1). At the end of 60 minutes, the mean salivary pH remained above normal in all the five interventional groups (table 4, graph 1). There were no adverse outcomes or unintended effects reported during and after the study. © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [8] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 Schematic representation of random allocation: Fig (1) Study plan and hypothesis formulation Ethical clearance obtained from Institutional Review Board of Bapuji Dental College and Hospital, Davangere Female students aged 20 – 25 years of Bapuji Dental College and Hospital, Davangere city were invited to participate in the study Excluded - not meeting inclusion criteria, declined to participate 85 female students who fulfilled the eligibility criteria were the study sample Random allocation Sugar free Gold (Aspartame) with distilled water n n= 17 n- Group A Sugar free Natura (Sucralose) with distilled water Apollo Stevia (Stevia) with distilled water n= 17 n= 17 Group B Group C Saccharin Helios Pharmaceuticlas (Saccharin) with distilled water n= 17 Group D Distilled water n= 17 Group E Salivary pH was assessed at baseline, 1 min, 20 min, 60 min Compilation, analysis and reporting of results © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [9] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 Tables: Table 1: Comparison of salivary pH at baseline and different time intervals between interventional groups Interventional groups Mean salivary pH and Standard Deviation at baseline 6.82±0.39 Mean salivary pH and Standard Deviation at 1 minute 7.05±0.44 Distilled Water With Aspartame (Group A) 7.08±0.42 Distilled Water 6.84±0.39 With Sucralose (Group B) 7.17±0.44 Distilled Water 6.72±0.19 With Stevia (Group C) 7.04±0.36 Distilled Water 6.83±0.37 With Saccharin (Group D) 6.78±0.30 Distilled Water 6.74±0.19 (Group E) 0.498 2.215 F Value (One Way Anova) 0.737 0.075 Probability (P Value) ‘A’ denotes between groups statistically significant at p < 0.01 Mean salivary pH and Standard Deviation at 20 minutes 6.81±0.14 Mean salivary pH and Standard Deviation at 60 minutes 6.88±0.17 7.01±0.34 7.04±0.43 7.12±0.41A 6.98±0.19 6.94±0.29 6.83±0.26 6.82±0.26A 6.85±0.20 2.633 1.900 0.040 0.119 Table 2: Tukey’s post hoc comparison of ANOVA to find significant difference in mean salivary pH values in the interventional groups at different time intervals Interventional groups Aspartame vs Sucralose (A vs B) Aspartame vs stevia (A vs C) Aspartame vs Saccharin (A vs D) Aspartame vs Distilled water ( A vs E) Sucralose vs stevia (B vs C) Sucralose vs Saccharin (B vs D) Baseline (p value) 1 minute (p value) 1.00 1.00 0.666 0.401 0.915 0.912 0.110 0.788 1.00 1.00 0.962 0.987 0.66 0.291 0.992 0.999 0.828 0.959 0.796 0.970 1.000 0.999 © Indian Journal of Medical Research and Pharmaceutical Sciences 20 minutes (p value) 0.962 60 minutes (p value) 0.166 http://www.ijmprs.com/ [10] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 Sucralose vs 0.876 Distilled water (B vs E) Stevia vs Saccharin 0.876 (C vs D) Stevia vs Distilled 1.000 water (C vs E) Saccharin vs 0.915 Distilled water (D vs E) p value –probability value * Statistically significant at p =0.01 (2-tailed). 0.214 0.387 0.268 0.880 0.387 0.478 0.046* 0.037* 0.638 0.035* 0.796 0.999 Table 3: Comparison of mean salivary pH at different time intervals within the groups. Interventional groups Mean salivary pH and Standard Deviation at baseline 6.82±0.39 Mean salivary pH and Standard Deviation at 1 minute 7.05±0.44 Mean salivary pH and Standard Deviation at 20 minutes 6.81±0.14 Distilled Water With Aspartame (Group A) 6.84±0.39 7.08±0.42 7.01±0.34 Distilled Water With Sucralose (Group B) 6.72±0.19 7.17±0.44 7.12±0.41A Distilled Water With Stevia (Group C) 6.83±0.37 7.04±0.36 6.94±0.29 Distilled Water With Saccharin (Group D) 6.74±0.19 6.78±0.30 6.82±0.26A Distilled Water (Group E) ** Statistically highest significant at p < 0.01 (2-tailed). Mean salivary pH and Standard Deviation at 60 minutes 6.88±0.17 F Value (Repeated Measures ANOVA) Probability (P Value) 2.021 0.120 7.04±0.43 1.402 0.250 6.98±0.19 5.690 0.002** 6.83±0.26 1.542 0.212 6.85±0.20 1.604 0.370 Table 4: Tukey’s post hoc comparisons of repeated measures ANOVA to find significant difference in mean salivary pH between different time intervals of different interventional groups Time intervals Baseline vs 1 minute Baseline vs 20 minutes Baseline vs one hour Distilled Water With Aspartame (Group A) 0.104 Distilled Water With Sucralose (Group B) 0.286 Distilled Water With Stevia (Group C) 0.002* Distilled Water With Saccharin (Group D) 0.192 Distilled Water (Group E) 0.551 0.970 0.522 0.006** 0.529 0.339 0.909 0.328 0.120 0.930 0.702 © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [11] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 1 minute vs 20 minutes 1 minute vs 1 hour 20 minutes vs 1 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 0.262 0.976 0.990 0.915 0.984 0.370 1.000 0.483 0.509 0.995 0.996 0.987 0.674 0.880 0.993 hour * Statistically significant at p < 0.05 (2-tailed). ** Statistically highly significant at p < 0.01 (2-tailed) Line graph 1: Depicting the Salivary pH changes at different time intervals in five Interventional groups 7.30 7.20 aspartame 7.10 7.00 sucralose 6.90 stevia 6.80 6.70 saccharin 6.60 distilled water 6.50 6.40 0 10 20 30 40 50 60 Discussion The present study assessed and compared the baseline salivary pH changes after mouth rinsing with commercially available sugar substitutes (Aspartame, Sucralose, Stevia and Saccharin) at one minute, twenty minutes and one hour. To the author’s best knowledge, this is the first study of its kind to compare salivary pH changes between commercially available natural and artificial sugar substitutes. The results indicated that there was a statistically significant rise in the salivary pH values in Stevia group (group C) at 20 minutes after mouth rinsing with the respective solutions. This result is in accordance with the study result of Goodson J et al.16 where statistically significant rise in plaque pH was found in stevia oral rinse group when compared with sucrose oral rinse group. In the present study, there was a rise in the salivary pH in all the groups post mouth rinsing with commercially available sugar substitutes from baseline to 1 minute. Between one to twenty minutes there was a gradual decline in the salivary pH in all the 5 groups out of which the salivary pH drop in group C (Stevia) was less than other groups and it was statistically significant. From 20 minutes to 60 minutes; the salivary pH values further decreased for aspartame and stevia group as compared to sucralose, saccharin and distilled water but the pH remained above the baseline for all groups and the pH change was not statistically significant. Our study findings are concurrent with those of Stephan and Kumar et al 17 in which the study participants were given 10 ml (20%) of sucrose solution and the salivary pH reduced gradually to a point and then increased gradually toward the baseline level, which was not reached even 60 minutes after sucrose exposure. They suggested that although organisms in the saliva may be © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [12] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 ISSN: ISSN: 2349-5340 Impact Factor: 3.052 producing some acid, the acids arising in the dental plaque or from bacteria colonizing on the tongue and other soft tissues might appear in the saliva, giving rise to a 'Salivary Stephan Curve'. In the present study, in stevia group, there was drop in mean salivary pH from 20 minutes to 60 minutes but didn’t reach the baseline mean salivary pH value even at the end of one hour. The pattern of salivary pH changes in the stevia group was similar to all other groups which shows that it can replace other artificially available commercial sugar substitutes. Results could not be compared with other studies as literature search revealed no studies in this regard. The drop in salivary pH in stevia group is similar to the study findings of Giongo et al.18 and Goodson J et al 16 In the study conducted by Goodson J et al16, rinses with stevia demonstrated a minimum pH of 6.92 ± 0.08, (mean ± SE) that was significantly higher (p <0.0001) than that of sucrose rinse (5.62 ± 0.13). Certain in vivo studies have shown pH rise. In vitro and in vivo studies have shown that stevia extracts have anti-bacterial activity on Streptococcus mutans, Streptococcus sobrinus and Lactobacillus acidophilus organisms that are closely related to the production and development of tooth decay.19 Hence it can be a potential substitute to sugar. Stephan’s curve describes the changes in dental plaque pH in response to a carbohydrate challenge over a period of time. Characteristically, the curve reveals a rapid drop in plaque pH that is attained after consumption of sugar. It normally takes at least twenty minutes for the plaque pH to reach its resting value.13 In some studies, the salivary pH did not return to its original baseline even at the end of one hour and remained below baseline mean salivary pH.19 So, it was decided to check salivary pH changes at one, twenty and sixty minutes time interval. A study done by Motamayell FA 20 showed that the pH of saliva was lower in males as compared to females. Hence, for the purpose of standardisation, only female students were included in the present study. A study done by Paraskevas et al.12 showed that, rinsing for 30 seconds appeared to be sufficient for all plaque-covered surfaces of the dentition to come into contact with the mouthwash. Hence, the time duration of mouth rinsing was set at 30 seconds for all the groups. Unstimulated whole saliva reflects basal salivary flow rate, is present for about fourteen hours a day, and is the secretion that provides protection to oral tissues.21 Stimulated saliva cause alterations in salivary pH hence unstimulated saliva collection was preferred. The study was done on a small sample. So, further studies should be done involving larger sample size. Though plaque pH changes are concurrent with salivary pH changes, it would have been better if plaque pH was assessed as the process of dental caries starts at plaque tooth interface. Conclusion There was a significant rise in salivary pH after mouth rinsing with Stevia solution. Pattern of salivary pH changes after mouth rinsing with Stevia was similar to other artificial sweetener solutions. From baseline to one minute after mouth rinsing with the respective solutions there was a rise in the salivary pH in all the 5 groups but Stevia group showed more rise as compared to other groups. There was a gradual fall in the salivary pH values from one minute to 20 minutes in all the groups. From 20 minutes to 60 minutes, the salivary pH remained above the baseline values in all the groups. The ability of stevia to avoid high salivary pH drop when consumed along with its ability to reduce the bacterial load and formation of biofilm together with its anti-inflammatory properties point out the potential of research towards anti-cariogenic properties of stevia. If proved effective, stevia can serve as an excellent sugar substitute replacing the current artificial sweeteners and refined sugars thereby help in tackling the lifestyle associated diseases like dental caries, obesity etc. References 1. 2. 3. 4. 5. Olivier B, Serge AH, Catherine A, Jacques B, Murielle B, Marie-Chantal CL et al. Review of the nutritional benefits and risks related to intense sweeteners. Arch Public Health 2015 Oct 1; 73:41-6. Sheiham A, James W.P.T. A reappraisal of the quantitative relationship between sugar intake and dental caries: The need for new criteria for developing goals for sugar intake. BMC Public Health 2014 Sep 16; 14: 863-71. Burt BA, Eklund SA, Morgan KJ, Larkin FE, Guire KE, Brown LO et al. The effects of sugars intake and frequency of ingestion on dental caries increment in a three-year longitudinal study. J Dent Res 1988 Nov; 67(11):1422-429. Newbrun E. Frequent sugar intake-then and now: interpretation of the main results. Scand J Dent Res. 1989 Apr; 97(2):103-9. Gupta P, Gupta N, Pawar AP, Birajdar SS, Natt AS, Singh HP et al. Role of sugar and sugar substitutes in dental caries: A review. ISRN Dent 2013; 2013:519421. © Indian Journal of Medical Research and Pharmaceutical Sciences http://www.ijmprs.com/ [13] Open Access Journal Indian Journal of Medical Research and Pharmaceutical Sciences May 2017; 4(5) DOI: 10.5281/zenodo.572580 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 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Abstract presented at the International Association for Dental Research General Sessions, Barcelona; 2010 Jul :14-17 Kumar A, Hedge R, Dixit U. Role of plaque in the clearance of salivary sucrose and its influence on salivary pH. J Indian Soc Pedod Prev Dent 2011; 310-314. Giongo FC, Mua B, Parolo CC, Carlén A, Maltz M. Effects of lactose-containing stevioside sweeteners on dental biofilm acidogenicity. Braz Oral Res. 2014;28. Contreras S. Anticariogenic properties and effects on periodontal structures of Stevia rebaudiana Bertoni. Narrative review. J Oral Res, (2013); 2(3), 158-166. Motamayell FA, Goodarzi MT, Hendi SS, Abdolsamadil H, Rafieian N. Evaluation of salivary flow rate, pH, buffering capacity, calcium and total protein levels in caries free and caries active adolescence. J Dent Oral Hyg. 2013; 5(4):35-9 Fenoll-Palomares C, Muñoz Montagud JV, Sanchiz V, Herreros B, Hernández V, Mínguez M, Benages A. 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