|Year : 2012 | Volume
| Issue : 2 | Page : 69-75
Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial
Gayathri Ramesh1, Ramesh Nagarajappa2, AS Madhusudan1, Nagarajappa Sandesh3, Mehak Batra2, Ashish Sharma1, Srikant Ashwin Patel1
1 Departments of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Airport Road, Debari, Udaipur, India
2 Public Health Dentisty, Pacific Dental College and Hospital, Airport Road, Debari, Udaipur, India
3 Department of Public Health Dentistry, Sri Aurobindo College of Dentistry, Indore Ujjain State Highway, Indore, India
|Date of Web Publication||20-Sep-2013|
Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Airport Road, Debari, Udaipur - 313 024, Rajasthan
Source of Support: None, Conflict of Interest: None
Objectives: The purpose of the study was to evaluate saliva and tongue coating pH and also to assess the degree of tongue coating in healthy subjects before and after chewing herbal leaves (tulsi, mint, and curry leaf).
Materials and Methods: A double-blind, randomized, concurrent, parallel-group study was conducted among 60 volunteer subjects, who were randomly assigned into three groups of 20 each (tulsi, mint, and curry leaf) and were asked to chew five to six fresh leaves of the respective plants twice daily for 7 days. Salivary and tongue coating pH were measured by a digital pH meter and color pH indicators. Data were analyzed statistically using repeated measure analysis of variance and Student's t-test.
Results: Mean salivary pH values showed an increase immediately and 30 min after chewing the herbal leaves. A significant difference (P < 0.01) was observed between mint and curry leaf groups immediately after chewing and between tulsi and curry leaf groups (P < 0.05) 30 min after chewing the leaves. Tongue coating pH showed an increase toward alkalinity in all the groups. The assessment of tongue coating showed an increase in scores among tulsi and curry leaf groups, but this difference was not statistically significant.
Conclusion: Chewing traditional medicinal plant leaves can be considered as safe, effective, and economical alternate options for maintaining good oral health.
Keywords: Medicinal, plants, saliva, tongue
|How to cite this article:|
Ramesh G, Nagarajappa R, Madhusudan A S, Sandesh N, Batra M, Sharma A, Patel SA. Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial. Ancient Sci Life 2012;32:69-75
|How to cite this URL:|
Ramesh G, Nagarajappa R, Madhusudan A S, Sandesh N, Batra M, Sharma A, Patel SA. Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial. Ancient Sci Life [serial online] 2012 [cited 2021 Sep 19];32:69-75. Available from: https://www.ancientscienceoflife.org/text.asp?2012/32/2/69/118531
| Introduction|| |
Saliva plays a critical role in maintaining oral homeostasis; it modulates the ecosystem through lubrication of the alimentary bolus, protection against microorganisms, buffer and repair of the oral mucosa, and helps in dental remineralization. Salivary pH is a fair indicator of health for extracellular fluids and their alkaline mineral reserves. Some of the most frequently occurring important pathological conditions of the teeth and oral cavity are strongly dependant on the pH changes.  In addition to the presence of certain types of bacteria, the type and amount of substrate, and oxygen and pH levels influence the occurrence and severity of tongue coating (a biofilm deposit over the tongue dorsum). ,
In developing countries, there is higher prevalence of dental and periodontal disease due to negligence and lack of awareness. Dental care services are often too costly, scarce, or totally absent, especially in the rural areas of these countries. There is a need for alternate prevention and treatment options that are safe, effective, and economical in view of increased resistance of bacteria to antibiotics, adverse effects of some antibacterial agents currently used in dentistry, and economic considerations. , Despite several agents being commercially available, these chemicals can alter oral microbiota and have undesirable side-effects such as allergic reactions, vomiting, diarrhea, and tooth staining.  Hence, the search for alternative products continues and natural phytochemicals isolated from plants used in traditional medicine are considered as good alternatives to synthetic chemicals.
As far back as the 1970s, it was suggested that the regular use of the African chewing stick, acting as an antiseptic, may control the formation and activity of dental plaque and, therefore, reduce the incidence of gingivitis and possibly dental caries.  Studies from different parts of the world appear to support the view that chewing sticks may contain substances that promote dental health or substances that have antibacterial activity against plaque-forming bacteria. ,
In West Africa, antibacterial activity has been reported from the aqueous extract of Citrus sinensis chewing sticks.  Work done on the aqueous extract from twigs of Mangifera indica revealed antibacterial activity against several oral bacteria, namely, Streptococcus mutans, Prevotella intermedia, and Porphyromonas gingivalis. , The compound "mangiferin" was found to be responsible for this activity. In India, both branches  and leaves  of M. indica (mango) are used in oral hygiene and care.
Singh et al. (2005)  in their study suggested that higher content of linoleic acid in Ocimum sanctum L. (Tulsi) fixed oil could contribute toward its antibacterial activity. The oil showed good antibacterial activity against Staphylococcus aureus, Bacillus pumius, and Pseudomonas aeruginosa, where Sta. aureus was the most sensitive organism. Geeta et al. (2001)  reported that in comparison to the alcoholic extract, the aqueous extract of O. sanctum L. (60 mg/kg) showed wide zones of inhibition against Klebsiella, Escherichia More Details coli, Proteus, Sta. aureus, and Candida albicans, when studied by agar diffusion method.
A need to explore, develop, and promote the use of locally available and accessible methods of dental disease prevention gave us an opportunity to evaluate the salivary and tongue coating pH and also assess the degree of tongue coating during various appointments of chewing three traditional medicinal plant leaves (Tulsi, mint, and curry leaf).
Null hypothesis - Household herbal leaves do not have any beneficial effect on salivary and tongue coating pH and also the degree of tongue coating.
Properties of the household herbal leaves used in our study
Tulsi (O. sanctum) is an excellent mouth freshener and oral disinfectant and its freshness lasts longer in the mouth. Tulsi destroys more than 99% of germs and bacteria in the mouth that are responsible for dental cavities, plaque, tartar, bad breath, etc., and protects the teeth. It also has astringent properties that make the gums hold the teeth tighter, thereby protecting them from falling. It cures ulcer in the mouth and is known to help inhibit growth of oral cancer caused due to chewing tobacco, etc.  Some of the main chemical constituents of Tulsi are: Oleanolic acid, ursolic acid, rosmarinic acid, eugenol, carvacrol, linalool, β-caryophyllene (about 8%), β-elemene (c. 11.0%), and germacrene D (about 2%). 
Mint, scientifically known as menthe (Mentha spicata), with over two dozens of species and hundreds of varieties, is a herb with remarkable medicinal properties. Being a germicidal agent and breath freshener, it takes care of oral health by inhibiting harmful bacterial growth inside the mouth and by cleaning the tongue and teeth. Besides its wide industrial use in foodstuffs such as ice creams, chocolates, alcoholic and non-alcoholic beverages, cosmetics, medicines, inhalers, and mouth and breath fresheners etc. It is also used as a condiment and decorative item for culinary purposes worldwide.  Analysis of essential oil of M. spicata mainly revealed the presence of oxygenated monoterpenes (65.8%), followed by sesquiterpene hydrocarbons (12.4%), monoterpene hydrocarbons (9.7%), oxygenated sesquiterpenes (2.8%), and oxygenated diterpenes (1.2%). 
The curry leaf (Murraya koenigii; Rutaceae), a green leafy vegetable, is grown all over India and other countries and is used as a spice everyday in small quantities for its distinct aroma, which makes it an ingredient in Indian cuisine. It is also used for its preservative properties. Fresh curry leaves contain 2.6% volatile essential oils (containing sesquiterpenes and monoterpenes), and the essential oils in the curry leaves are sufficiently soluble in water. The cold extract of curry leaves (10 g of cut fresh curry leaves in 200 ml of distilled water) has a pH of 6.3-6.4 (unpublished personal observations). Chlorophyll has been proposed as an anticariogenic agent and it also helps to reduce halitosis, and the terpenes have been found to reduce airborne chemicals and bacteria. 
It has also been reported that curry leaves have medicinal value and is traditionally used in eastern Asia. It has been shown to have antioxidant, anti-diabetic, anticarcinogenic, antidysenteric, stimulant, hypoglycaemic, and antimicrobial activities. Reports of its antioxidant potency have stimulated interest in using curry leaves and this activity is attributed to the presence of mahanimbine, murrayanol, and mahanine.  The major constituents identified were α-pinene (51.7%), sabinene (10.5%), β-pinene (9.8%), β-caryophyllene (5.5%), limonene (5.4%), bornyl acetate (1.8%), terpinen-4-ol (1.3%), γ-terpinene (1.2%), and α-humulene (1.2%). 
| Materials and Methods|| |
A double-blind, randomized, concurrent, parallel-group study was carried out among the dental students of Pacific Dental College and Hospital, Udaipur to assess the effect of chewing household herbal leaves on salivary and tongue coating pH and also the degree of tongue coating. The study protocol was reviewed and approved by the Institutional Ethical Committee. Sixty orally and systemically healthy dental students, who satisfied the eligibility criteria, aged over 18 years volunteered to participate in this study.
The exclusion criteria were subjects with medical disorders such as diabetes mellitus, renal disease, gastrointestinal disorders, respiratory diseases, evidence of recent bronchitis, sinusitis, or tonsillitis, patients undergoing antibiotic or other antimicrobial therapy, smokers, those who, on pre-study clinical screening, presented a probing depth ≥4 mm, subjects with cavitated caries lesion or naso-pharyngeal alterations, mouth breathers, and patients with prostheses, orthodontic or dental appliances. In addition, the participants in the study were required to have a normal unstimulated (resting) whole saliva flow rate (0.5 ml/min).
The nature of the study was explained and a written informed consent obtained from all the subjects. Oral prophylaxis that includes scaling and polishing with prophylactic paste applied with a rubber cup was performed for all the subjects by one of the investigators. The absence of plaque was confirmed by careful examination of the teeth using a mouth mirror and probe. The baseline data were collected on 4 October 2011 and the outcome data were collected a week later.
All participants attended three consultations (1 st , 2 nd , and 7 th days) which was conducted by the same examiner. In the first consultation, all subjects were seen in the morning at around 7 a.m., fasting for at least 8 h and without having performed any oral hygiene procedures on the day of consultation, but having performed oral hygiene as usual the previous night at bedtime. The participants followed these guidelines to improve standardization of data collection.
On the first day, baseline tongue coating pH, degree of tongue coating, and collection of baseline saliva was performed. Following this procedure, using the random number table, the participants (N = 60) were randomly allocated to three groups (Tulsi, mint, and curry leaf) consisting of 20 subjects in each group by another investigator [Figure 1]. They were asked to chew five to six fresh (Tulsi, mint, or curry) leaves with 5 ml of water in the mouth, rinse for 5-7 min, and expectorate. Subsequent to baseline estimation, each participant underwent two collections of saliva samples along with estimation of tongue coating pH in two phases: Immediately after chewing and 30 min after chewing leaves of a specific herb. None were allowed to eat or drink during the phases.
Later, the participants were asked to chew the respective herbal leaves distributed to them at night after they performed their regular oral hygiene practice. In the second consultation, the next morning before brushing, the degree of tongue coating along with the pH of saliva and tongue coating was estimated to check if any difference was found.
Chewing of herbal leaves continued on subsequent days, twice daily (once in the morning and once at night). Third consultation was on the morning of the 7 th day, where the procedure of estimating all the components was repeated.
Before saliva collection, patients were kept seated for 5 min, relaxed and silent. Following an initial swallow, unstimulated saliva was collected in a sterilized airtight container by allowing the saliva to flow over a period of 5 min into the containers by tilting their heads forward.
Salivary pH was measured by a digital pH meter (Sentron Model 1001 pH System; Sentron Incorporated, Fort Lauderdale, USA), calibrated with standard solutions of pH 4.0 and 7.0. The electrode was washed with distilled water and dried with absorbent paper after each analysis.
In the same consultation, tongue coating pH was measured using pH indicator strips (pH 0-14; Merck, Darmstadt, Germany). One strip was placed on posterior tongue region, with the participants' mouth kept opened for 1 min. The color change in the strip indicated tongue coating pH.
The degree of tongue coating was assessed based on the conventional criteria, with a simple modification (0, no tongue coating; 1, thin tongue coating covering less than one-third of the tongue dorsum; 2, thick tongue coating covering approximately one-third of the tongue dorsum or thin tongue coating covering one-third to two-thirds of the tongue dorsum; 3, thick tongue coating covering one-third to two-thirds of the tongue dorsum or thin tongue coating covering more than two-thirds of the tongue dorsum; and 4, thick tongue coating covering more than two-thirds of the tongue dorsum). 
Neither local nor systemic side effects were noted in any of the groups. To safeguard against bias, the investigator who obtained outcome measurements was not informed of the group assignment, which ensured strict blinding.
The data obtained were analyzed using Statistical Package for Social Sciences software version 15.0 (SPSS Inc., Chicago, IL, USA). Repeated measures analysis of variance (ANOVA) and Student's t-test were used for phase and group comparisons, respectively. The level of significance was set at P ≤ 0.05.
| Results|| |
Sixty subjects complied with the protocol and completed the study. Throughout the study, there were no adverse effects on the soft and hard tissues of the oral cavity observed by the examiner, or reported by the subjects when questioned. The baseline values were not significant between the groups.
Mean salivary pH values showed an increase immediately and 30 min after chewing the herbal leaves. There was a drop in the subsequent estimation on the 2 nd day, but again rose to a slightly higher level on the 7 th day [Figure 2]. A significant difference (P < 0.01) was observed between the mint and curry leaf groups immediately after chewing and between Tulsi and curry leaf groups 30 min after chewing the herbal leaves (P < 0.05). There was no statistically significant difference (P > 0.05) between the Tulsi and other groups, and also among all groups in the 2 nd and 3 rd consultation phases [Table 1].
|Figure 2: Mean salivary pH values of the herbal leaves at different phases of consultations|
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Similarly, the mean tongue coating pH values increased immediately and 30 min after chewing. During the second consultation, the values consolidated around the values obtained immediately after chewing and again showed an increase on the final day [Figure 3]. A significant difference was found between the Tulsi and curry leaf groups in the third consultation [Table 2].
|Figure 3: Mean tongue coating pH values of the herbal leaves at different phases of consultations|
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The assessment of tongue coating showed an increase in the scores among Tulsi and curry leaf groups, but the difference observed was not statistically significant. Interestingly, the tongue coating decreased among mint chewers in the final estimation [Figure 4]. However, no significance was observed between any of the groups or consultations [Table 3].
|Figure 4: Mean tongue coating scores of the herbal leaves at different consultations|
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|Table 3: Mean difference in scores in the degree of tongue coatings between groups|
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| Discussion|| |
The normal pH of saliva is considered to be around 6.8. If the pH becomes higher, it indicates that our body can respond easily to strong stimuli. Saliva of individuals with "dry mouth" (common situation after a night of sleep) often shows acidic pH. However, there is a tendency for salivary pH to become more alkaline during the day because of talking or chewing. Salivary pH is slightly acidic before its secretion in the oral cavity. It becomes alkaline at the time of gland's secretion due to loss of carbon dioxide (CO 2 ) and increase in saliva's bicarbonate concentration when the salivary flow increases.  When salivary pH decreases, and stays low in time, it results in the symptoms such as cavities, gingival recession, and demineralization in the neck of the tooth, and white spots in the enamel.
According to the present study, the most notable changes in salivary pH were observed immediately and 30 min after chewing the herbal leaves. Between the mint and curry leaf groups, there was a significant increase in the pH levels immediately after chewing, and between Tulsi and curry leaf groups, the increase was observed 30 min after their use; however, there is no information in literature about the influence of herbal leaves on pH. This can be explained by the fact that chewing herbal leaves stimulates salivation which increases the saliva's bicarbonate concentration and thus increases salivary pH. 
An increased salivary flow actively reduces demineralization and improves the rate of remineralization. Stimulated saliva contains more calcium and bicarbonate and has a higher pH than unstimulated saliva. This results from a higher degree of saturation, making it even more effective at remineralizing the enamel crystals damaged by an acid attack and is therefore better at fighting tooth decay. Willershausen et al.  in their study have observed a shift in the salivary pH values to the alkaline range in the herbal extract group, which demonstrates the usefulness of herbal extracts as an adjunct in the treatment of periodontal disease and in a routine preventive regimen.
Chewing the herbal leaves in all groups increased the pH immediately and 30 min after their use; however, the mechanism of action is not clearly described in the literature. In our research, the analysis of tongue coating pH showed a statistically significant difference between the Tulsi and curry leaf groups, only during the final consultation phase. pH is the major regulating factor in the formation of bad breath, and it is clearly established that acidity inhibits the production of odors while neutrality and alkalinity favor it.  The same context is applied to tongue coating pH, which has alkaline pH due to the production of odorivetores during proteolysis. Among the final products of proteolysis are amines, ammonia, and urea, which have alkaline pH, characteristic of physiological or pathological halitosis.  It is questioned whether the use of herbal leaves could change these pH values and influence in the reduction of saliva and tongue coating pH.
Results and discussion
Mikkonen et al. studied the relationship between the different morphological forms of the tongue surface and the salivary pH and concluded that retention of food debris including carbohydrates resulted in the formation of acids by the bacteria on different morphological forms of the tongue surface and also showed that there was no alteration in the buffering capacity.
Saliva is one of the main sources of bad breath because volatile sulphur compounds (VSC) are produced from proteins and sulfur-containing amino acids present in the saliva. The reduction in salivary flow during sleep and consequent increase in the number of epithelial cells scaled from oral mucosa lead to tongue coating formation, even in healthy patients.  As these cells, in particular leukocytes, are found to be in higher numbers in saliva from patients with periodontal disease and accumulate on the tongue surface, tongue coating would be expected to increase in patients with periodontal disease.
The tongue coating score could be an indicator of whether a person has an unpleasant mouth odor or not. Therefore, it is very important to study the accumulation process or to investigate the influence of factors against tongue coating. Regular (twice daily) brushing reduces the thickness of plaque. Plaque that is kept thin (with the biofilm brushed away regularly) does not have low oxygen micro-environments within it. Conversely, the oxygen tension in plaque falls if there is no brushing. As plaque thickens, the deeper layers with lower oxygen tension favor the growth of facultative anaerobes, including mutans streptococci (which are associated with caries), and Gram-negative rods (associated with periodontitis). 
The safety and possible side-effects in relation to natural products used in dentistry have recently been reviewed by Groppo et al. Given the possibility of adverse interactions between herbal formulations and conventional drugs, caution should be exercised when using herbal medicines. In this context, the need for more clinical studies is also emphasized.  The antibacterial principle in the leaves studied may probably be attributed to one or more of the following phytochemicals previously identified: Essential oils, flavonoids, terpenes, and terpenoids. From our study, it is not possible to attribute the antibacterial activity observed here to any particular phytochemical group. However, the plant extracts have exhibited a strong inhibition of the growth, acid production, sucrose-induced adherence, and glucan-induced aggregation of Str. mutans. 
The findings of this study also confirm that chewing household herbal leaves has a beneficial effect on salivary and tongue coating pH and therefore justify the use of above leaves in traditional medicine. Antimicrobial properties of these herbs, which are possible sources of cheap dental health care for the rural poor, should be effectively utilized to fight against common and prevalent oral health problems.
| Acknowledgment|| |
The authors would like to thank the study participants for their participation and kind cooperation throughout the study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]