|Year : 2013 | Volume
| Issue : 3 | Page : 146-152
Liquorice root extracts as potent cariostatic agents in pediatric practice
Eesha Jain, Ramesh K Pandey, Richa Khanna
Department of Pedodontics, Faculty of Dental Sciences, King George’s Medical University, Lucknow, Uttar Pradesh, India
|Date of Web Publication||11-Sep-2013|
Ramesh K Pandey
Department of Pedodontics, Faculty of Dental Sciences, King George’s Medical University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The present study was designed to evaluate the in vitro as well as in vivo cariostatic efficacy of aqueous and ethanolic extracts of liquorice and assess their acceptability among child patients. Materials and Methods: Minimum bactericidal concentrations of aqueous and ethanolic extracts of liquorice against mutans streptococci were evaluated and their toxicity profiles were tested using the model organism Caenorhabditis elegans. The clinical trial was conducted as a double-blind pilot study where pediatric patients (N = 60), aged 7-14 years, were equally divided by randomization into three groups, namely, Group 1 using aqueous liquorice mouthwash (15%), Group 2 using ethanolic liquorice mouthwash (3.75%), and Group 3 using chlorhexidine gluconate (0.156%) as positive control. A baseline pre-rinse and three post-rinse saliva samples were evaluated for the changes in pH and mutans streptococci colony counts. Palatability of liquorice extracts was assessed through a self-designed questionnaire having structured categorical responses. Statistical Analysis: Parametric evaluations were done using Analysis of Variance (ANOVA) and Dunnett's "t" test. Results: The mean mutans streptococci colony counts in all three groups decreased significantly (P < 0.001) immediately after the oral rinsing. The reduction in colony counts was significant in ethanolic liquorice group as compared to the control (P < 0.01). Liquorice extracts also led to an immediate rise in salivary pH. The results showed an immediate antimicrobial action of liquorice extracts, with limited retentivity. Conclusion: The study affirms that both aqueous and ethanolic liquorice extracts are potent cariostatic agents and are found to be palatable by child patients.
Keywords: Chlorhexidine, dental caries, liquorice root extracts, mutans streptococci
|How to cite this article:|
Jain E, Pandey RK, Khanna R. Liquorice root extracts as potent cariostatic agents in pediatric practice. J Indian Soc Pedod Prev Dent 2013;31:146-52
|How to cite this URL:|
Jain E, Pandey RK, Khanna R. Liquorice root extracts as potent cariostatic agents in pediatric practice. J Indian Soc Pedod Prev Dent [serial online] 2013 [cited 2020 Mar 30];31:146-52. Available from: http://www.jisppd.com/text.asp?2013/31/3/146/117964
| Introduction|| |
Dental caries is a dynamic process, promoted by a vicious multifactorial discrepancy between demineralization and remineralizing factors in the oral environment. Any attempt which can impede or even halt one or more causative factors will contribute in intercepting the disease process. Moreover, reversal of the early carious lesion is desirable rather than surgical intervention at later stages, as the preventive approach is more time effective and patient friendly.
The treatment of dental caries also weighs down heavily on public health economy as a whole, more so in developing countries. This financial burden is attributed to the management of infrastructure and the cost involved in procedures. Thus, prevention at primary level has always been accepted as the best management protocol for dental caries. The upsurge of preventive dentistry is accompanied by a need to delineate novel therapeutic protocols which can minimize the cariogenic microbial load and provide conducive pH in the oral cavity, thus exhibiting a cariostatic action.
Oral rinses are a common tool employed to deliver such therapeutic ingredients to all accessible surfaces of the mouth, including interproximal hard surfaces. Mouthrinses forgo the need for much manual dexterity and the duration of the procedure can also be regulated. However, several adverse effects have been attributed to the use of mouthwashes currently available in the market, such as unpleasant taste, increased risk of caries due to fermentation and alcohol content, and discoloration of teeth.  Chlorhexidine, considered to be the "gold standard" of oral preventive therapy due to its prolonged broad-spectrum antimicrobial property, is accompanied by altered taste perception, metallic taste, and staining of teeth.  Such lacunae of conventional preventive measures have led to a renewed interest in herbal medicine.
Medicinal plants are a well-recognized source of potential bioactive compounds.  They offer advantages such as less cost, ease of availability, and relatively safe toxicity profile. Glycyrrhiza glabra Linn., commonly known as liquorice (mulethi), is one such medicinal plant used by various cultures for thousands of years to relieve coughs, sore throats, and gastric inflammation. The Food and Drug Administration (FDA) also lists liquorice as GRAS (generally regarded as safe) when used as food flavoring and sweetening agent.  Recent research suggests that attributing to its anti-adherence, antimicrobial, and anti-inflammatory properties, liquorice and its various bioactive ingredients have potential benefits in oral diseases, including dental caries. ,
The present study was undertaken to determine the cariostatic potential and antimicrobial efficacy of aqueous and ethanolic extracts of liquorice roots against salivary mutans streptococci. The present study also aimed to evaluate acceptability of the extracts among pediatric patients, when delivered for oral rinsing.
| Materials and Methods|| |
The present study was conducted as a subject- and observer-blind, randomized, controlled trial in the Department of Pedodontics, Faculty of Dental Sciences, King George's Medical University, Lucknow in collaboration with Herbal Medicinal Product Department, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow and Department of Microbiology, King George's Medical University, Lucknow. The present study was conducted from June 2010 to November 2011, including plant material procurement, preparation of extracts, in vitro assay, and the clinical study, encompassing patient enrollment, data collection, and interpretation. Ethical clearance for the trial was obtained from the Institutional Ethics Committee of King George's Medical University.
Preparation of liquorice extracts
Liquorice roots were obtained from Yamuna Nagar district in Haryana where it is widely cultivated. The taxonomical verification of the plant material was done at the Herbarium, Department of Botany, Forest Research Institute, Dehradun where the voucher specimen was deposited in duplicate and accorded accession number 163917.
Liquorice roots were dried in shade and coarse ground in an electric blender. Two samples of the powder were soaked in distilled water and ethyl alcohol, respectively, for 24 h with intermittent shaking. The active ingredients leached out in the solvent were subsequently filtered using several folds of muslin cloth and Whatman No. 1 filter paper. The filtrate for each extract was concentrated using a rotavapor and freeze dried using lyophilization, following which the residues were finely ground, weighed, and stored at 4°C for further experiments.
In vitro evaluation of antimicrobial efficacy and toxicity of liquorice extracts
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the aqueous and ethanolic extracts against mutans streptococci were determined by broth tube dilution method (macrodilution) in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) guidelines.  Mutans streptococci strain was isolated from the saliva samples of child patients with high caries activity by culturing using selective medium mitis salivarius-bacitracin agar.  Colonies with a typical pale blue, granular, frosted glass appearance were identified and subcultured using blood agar plates. 
A stock solution (30% concentration of the extract in normal saline) was taken and 10 subsequent doubling dilutions of each extract were made to obtain concentrations of 15%, 7.5%, 3.75%, 1.88%, 0.94%, 0.47%, 0.23%, 0.12%, 0.06%, and 0.03%, respectively. To each of the 10 test tubes, brain heart infusion (BHI) broth and an equal volume of mutans streptococci adjusted to 0.5 McFarland were added. After incubation, MIC was detected by visual inspection. The lowest concentration of the test agent with clear supernatant is considered as the MIC. Small aliquots were taken from all the tubes in which no visible bacterial growth was observed, and seeded into Mueller-Hinton Agar (MHA) and incubated overnight at 37°C. The concentration at which no colonies of mutans streptococci appeared was inferred to be the MBC.
Toxicity of the extracts was evaluated using the nematode Caenorhabditis elegans, which simulates the human model. An acute toxicity test was performed in CIMAP according to the procedure described by Donkins and Williams.  The lyophilized plant extracts were analyzed for their major constituents using standard qualitative methods. ,
Selection of subjects for clinical trial
The clinical study was conducted as a subject- and observer-blind, randomized, chlorhexidine-controlled trial. Male and female patients, aged between 7 and 14 years, voluntarily willing to participate in the trial, with at least five active decayed tooth surfaces (caries status determined by DMFS and dmfs scores)  and with poor oral hygiene [determined by Oral Hygiene Index-Simplified (OHIS)]  were included in the present study. A written informed consent was obtained from the parents/guardians of the participants. The exclusion criteria included patients undergoing dental treatment, who had received antibiotic therapy within past 1 month, using any mouthwash for maintaining oral hygiene, having any painful, debilitating oral condition, and patients who failed to comprehend the task. Demographic and other categorical data regarding patients' response to the rinses, their taste, and post-rinse effects were collected from the participants through a self-designed questionnaire with structured categorical responses.
Clinical trial: Rinsing procedure and saliva collection
All the children participating in the present study were instructed not to brush their teeth on the day of sampling and to follow their daily diet intake. Unstimulated saliva samples were collected at least 2 h after meals. The children were randomly divided into the groups and the pre-weighed dose of the allocated drug material was delivered through color-coded vials by an additional blinded observer. The concentrations of the three drugs were based on their respective MBC values against mutans streptococci, evaluated in the in vitro experiment. The quantities of oral rinses given in each group are as follows:
Child patients were instructed to rinse their mouth with 10 ml of the prepared suspension in their respective groups for a period of 1 min, similar to a study conducted by Axelsson and Lindhe.  The subjects were then asked to let saliva pool in their mouth for 2 min and collect the accumulated saliva into sterile, labeled saliva collecting cups. Thus, for each patient, four saliva samples were collected, i.e. pre-rinse sample that was collected before the child performed oral rinsing, and three post-rinse samples collected immediately, 15 min, and 30 min after the intervention. The pH of the unstimulated whole saliva collected at each interval was analyzed using a chairside kit (GC Saliva Check). The pH test paper was dipped in the sample for at least 10 sec and the color changes were compared with the chart provided by the manufacturer.
- Group 1: Aqueous liquorice extract-1.5 g/10 ml saline
- Group 2: Ethanolic liquorice extract-375 mg/10 ml
- Group 3: Chlorhexidine mouthwash was used in a concentration of 0.0156%, such that 10 ml was dispensed at one time.
Mutans streptococci colony counting
The saliva collecting cups were stored at 4°C till all the required samples for each individual were collected, and then transported to microbiological laboratory within 1 h in a refrigerated container. Mutans streptococci colony counts were estimated by a semi-quantitative method, i.e. standard loop method as described by Collee et al.  Saliva samples were vortexed and diluted in a 1:50 ratio with normal saline. A nichrome SWG 28 wire loop was used to take a loopful (0.004 ml) aliquot of mixed, uncentrifuged, diluted saliva which was spread over a plate of selective media for mutans streptococci, i.e. mitis salivarius-bacitracin agar. All plates were incubated at 37°C in a micro-aerophilic environment at 5% CO 2 for 48 h. The colonies were identified on the basis of their morphology and counted using a digital colony counter. Confirmation of mutans streptococci was performed under light microscope after staining a heat-fixed smear slide. Microbial counts were expressed as colony forming units (CFUs) per milliliter of saliva. The values of pH and colony counts were recorded and intergroup comparisons were made at baseline and after the intervention.
Parametric evaluations were done using Analysis of Variance (ANOVA) and Kruskall Wallis test. Differences from control were evaluated using Dunnett's "t"-test. Pairwise comparisons at different time intervals were done using paired "t"-test. Discrete (categorical) data were compared by chi-square (χ2 ) test. All analyses were performed using Statistical Package for Social Sciences (SPSS) Version 15.0.
| Results|| |
In the present study, the MBCs of aqueous and ethanolic extracts of liquorice and Chlorhexidine as control were 150 mg/ml, 37.5 mg/ml, and 0.0156 mg/ml, respectively. The concentrations of the testing extract for assay were 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, and 10 mg/ml for toxicity testing on C. elegans. In the assay, effects were observed after 30 min, 1 h, 3 h, and 24 h in treated worms. No toxicity was observed at all the concentrations up to 24 h.
The demographic characteristics of the three groups at admission are summarized in [Table 1]. The demographic factors, viz. sex, mean age, mean OHIS, Caries Index Score, frequency of daily tooth brushing, and mean time elapsed since last eatable consumed did not influence the study outcome measures. Among the children participating in the present study, 35% children expressed dislike for the chlorhexidine mouthwash. In contrast, 45% children preferred the ethanolic liquorice extract oral rinse, while 40% reported of liking for the aqueous liquorice oral rinse. Moreover, none of the herbal rinses had any adverse residual effects, either local (irritation, burning) or systemic (nausea, vomiting, headache). The frequency of residual sweetness was observed to be the maximum in aqueous liquorice group (90%) followed by ethanolic liquorice (60%) group, while no residual sweetness was reported in the control group [Table 1].
|Table 1: Baseline characteristics of the patients in three groups at the time of enrollment|
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The mean mutans streptococci colony counts at baseline (pre-treatment) did not show significant difference between the three groups. The mean colony counts in all three groups decreased significantly (P < 0.001) immediately after rinsing. The reduction in colony count was significant in ethanolic liquorice group as compared to the control group. In the control group, mutans streptococci colony counts showed a further decreasing trend from immediately after rinsing till 30 min post-rinse [Table 2]. Both test groups did not show any decline in the colony count at intervals of 15 min and 30 min. At the final evaluation, the colony counts in all three groups were found to be equivalent [Figure 1].
|Table 2: Pre- and post-rinse mutans streptococci colony counts (CFU per ml of saliva) in three groups (values are Mean±SD of log10)|
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|Figure 1: Mean colony counts of mutans streptococci in the three groups at different time intervals|
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The mean pH values in the three groups at the different time intervals are plotted as shown in [Figure 2]. In the control group, there was a drop in pH at the immediate post-rinse interval following which the pH continued rising till the end point. In both the test groups, immediate post-rinse samples showed a rise in pH, which decreased subsequently, with the end point pH well above the baseline pH in both the groups.
The preliminary qualitative phytochemical screening revealed the presence of tannins, saponins, and glycosides in both the extracts, while alkaloids were found to be absent. Additionally, the aqueous extract also tested positive for phenols while the ethanolic extract showed the presence of flavanoids.
| Discussion|| |
Liquorice, commonly known as "mulethi" in the Indian subcontinent, has been used extensively over the centuries for sore throat and cough. The present study was designed to evaluate the cariostatic properties of liquorice to ascertain if it could be developed into a caries-preventive regimen basically targeted for use in the pediatric population. The findings of the present study indicate that liquorice is safe, well accepted among child patients, has shown fall in mutans streptococci colony counts and a rise in pH after a rinse with both aqueous and ethanolic extracts. The efficacy of liquorice extracts was evaluated in vitro as well as after a single topical application in the oral cavity, using chlorhexidine as a positive control.
The antimicrobial activity of the extracts was assessed by estimating their inhibitory and cidal action against mutans streptococci and determining their MIC/MBC. Results of the in vitro experiment revealed that ethanolic extract of liquorice had better antimicrobial activity than the aqueous extracts. These findings are in agreement with the observations of Ahmad et al., who concluded that alcohol is a better solvent than water.  This might be attributed to the polar nature of the solvent, i.e. ethanol, which resulted in leaching of more active ingredients during extraction. Variation of susceptibility of the pathogens to aqueous and ethanolic extracts indicates the involvement of more than one active principle of biological significance. In case of ethanolic extracts, the solvent did not contribute to the antimicrobial activity since it had been completely removed by lyophilization and freeze-dried extracts were delivered to the patients in saline.
Toxicity testing of the plant extracts was performed in an attempt to assess their biocompatibility, using the nematode C. elegans, a model organism introduced by Brenner.  Model system C. elegans attains significance because of its 60-80% homology of gene sequences with those of human beings, along with several disease genes and disease pathways, and 12 out of 17 known signal transduction pathways conserved between humans and C. elegans, as confirmed by Kaletta and Hengartner.  In the present study, the toxicity of the used plant extracts was not noticed.
The subjects in the present study were selected in the age group of 7-14 years. Piaget has advocated that at age of 7 years, a child largely corresponds to an increase in cognitive development and intellectual ability.  Hence, in this age group, a positive compliance could be expected from the child and that the child would not ingest the suspension of the extract administered to them.
In the present study, dose of each extract was predetermined by the MBC values ascertained in vitro. Thus, only a limited fraction of delivery dosages could be divided from the final yield of the extracts obtained. The sample size was also limited by the number of patients in the dental outpatient department, who fulfilled the selection criteria, during the study period. Randomization of the children in each group was done to eliminate selection bias. All the in vitro experiments were performed in triplicates to mitigate procedural errors. The subjects as well the observer who administered the rinse to the children, both were kept blind to further abolish any observer bias. The baseline characteristics were thus similar in all the groups and would not have influenced the outcome.
In the present study, chlorhexidine was used in a concentration of 0.0156% in accordance with the MBC assessed for the study. Segreto et al. also concluded that 0.1% twice daily administration offers the same clinical benefits as a 0.2% chlorhexidine solution.  Moreover, the reduced concentration of chlorhexidine makes it more acceptable to child patients for oral rinsing.
The samples for the trial were collected at four periods of time allowing observing the immediate as well as the sustained action of the extracts. The samples were prepared to obtain a 50-fold dilution and subjected to automated colony counting. The colony counts were best done on samples with 20-200 colonies per plate, with a reported error of less than 4% in reproducibility. 
The results showed an immediate antimicrobial action of liquorice, which was significant compared to the control group. The antimicrobial action of liquorice has been reported by several authors. , Chu Hong Hu et al. discovered a novel compound, glycyrrhizol A, from the extract of liquorice roots, which exerted strong antimicrobial action against cariogenic bacteria.  It has been proposed that one of its main ingredients, glycyrrhizin, dose-dependently inhibits the glucosyltransferase activity of mutans streptococci, which is involved in the formation of insoluble glucans required in biofilm formation.  Gedalia et al. reported that glycyrrhizin, when added to an acidulated phosphate-fluoride solution, increases fluoride uptake and reduces enamel solubility, most likely due to a surface-coating effect and its deposition in the porous structure of demineralized enamel.  On the other hand, chlorhexidine reacts with the microbial cell surface, destroys the integrity of the cell membrane, penetrates the cell, and precipitates the cytoplasm leading to cell death.  Chlorhexidine also possesses the unique property of substantivity, pertaining to which it has slow, sustained release in the oral cavity and, hence, prolonged action.
Estimation of pH of the salivary samples also indicates that a single exposure to liquorice aqueous as well as ethanolic extracts resulted in a rise in the pH of saliva, whereas chlorhexidine, which is established to have a neutral pH, led to a decrease in pH of immediate post-rinse salivary samples. Söderling et al . reported that in an in vivo acid production test, liquorice-containing gel was shown to inhibit acid production.  Liquorice is also known to be an alkaline food and has a protective effect in Gastro esophageal reflux disease (GERD) and peptic ulcers. Moreover, the organoleptic properties of liquorice further stimulate salivary flow. Stimulated saliva contains greater concentration of bicarbonate ions and, thus, has increased buffering capacity. Added to its ability to clear acids and substrates from plaque and its improved pH, stimulated saliva increases the resistance to caries.
The presence of active principles in the phytochemical analysis also validates the clinical findings. The antimicrobial activity may be mostly due to tannins, triterpenoid saponins, and flavonoids. The presence of saponins also confirmed the extracts to be positive for glycyrrhizin, the active principle known to reduce bacterial growth and acid production. However, when delivered as a rinse, this duration was found to be too short to provide optimum contact of the herbal extracts with the oral cavity. Moreover, the sweet taste promoted early swallowing of saliva and consequent washout of the residual drug from the oral cavity. Therefore, the property of substantivity was not evident in liquorice extracts.
The mode in which liquorice is delivered to the subjects is a major criterion to effectuate prolonged duration of action. Liquorice, delivered as a hard candy or chewing gum, would have an extended release in the mouth, which would significantly enhance its sustained action.  It can be used as a viscous suspension or an oral gel to increase its action. Liquorice roots have been conventionally used as chew twigs to clean teeth and also as teething sticks which exert a numbing and cooling effect on infants' gums. Liquorice powder, ammoniated glycyrrhizin, and monoammonium glycyrrhetic acid competitively reduced the metabolism of sucrose, fructose, and glucose, but were themselves minimally fermentable. For infants, liquorice can be incorporated in infant swipes and pacifiers, which would serve as a non-cariogenic sweet-tasting alternative to the commonly used honey-dipped pacifiers. Further optimizing of the drug delivery can be done by delivering using controlled-release devices, mucosal adherent patches, and even as liquorice-containing resorbable fibers which can be inserted into diseased periodontal site and healing sockets.
The present study was limited by some factors such as the efficacy of the extracts was observed for a short period of 30 min. Besides, quantitative analysis of the extracts along with their qualitative screening would further assist to elucidate the active principles responsible for the antimicrobial activity. Further studies can be undertaken to explore the efficacy of liquorice in other oral ailments. Thus, liquorice can be a promising natural alternative agent for maintaining oral health in pediatric population.
| Conclusion|| |
The present study leads to the conclusion that liquorice extracts used in child patients showed antimicrobial efficacy and led to a rise in the pH of saliva. The present study confirms the antimicrobial and cariostatic efficacy of liquorice extracts and recommends that liquorice can be used as a preventive regimen in pediatric practice.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]