Effect of Liquorice Candies on Remineralization of Initial Enamel Carious Lesion

ABSTRACT

Background

Glycyrrhiza glabra (G. glabra), known as liquorice root is one such herbal remedy that has been shown to have immense potential in the treatment of orofacial diseases.

Aim

To evaluate the remineralizing potential of liquorice extract on initial enamel carious lesions.

Materials and methods

An in vitro and in vivo study was conducted, wherein the sound-extracted human premolars were sectioned longitudinally and into enamel blocks of size 4 × 4 mm. Enamel blocks were demineralized to produce artificial carious lesions. In vitro study, the demineralized enamel block was exposed to artificial saliva for 5 minutes and artificial saliva containing liquorice for 4 minutes 14 times in a sequential manner. An in vivo study was carried out on six volunteers aged between 6 and 12 years. The demineralized enamel block was inserted into the 5 × 5 mm window created on the full palatal coverage appliance. Patients were given commercially available liquorice candies (Ricola) to consume for 7 days twice daily. The remineralization potential of liquorice on demineralized enamel block was assessed using confocal microscopy analysis.

Results

Liquorice extract containing candies showed visual remineralization of the demineralized area on the enamel surface through confocal images.

Conclusion

Liquorice extract possesses remineralizing potential with its other medicinal properties.  

How to cite this article

Pooja HR, Nagar P, Mascarenhas AN, et al. Effect of Liquorice Candies on Remineralization of Initial Enamel Carious Lesion. Int J Clin Pediatr Dent 2023;16(S-1):S33–S38.

Introduction

Liquorice [Glycyrrhiza glabra (G. glabra)], well-known as Jeshthamadh, Madhuca, and Yashtimadhu in India, belongs to the genus Glycyrrhiza and has been widely used for enormous purposes for 4000 years.¹

Liquorice root or radix Glycyrrhizae has prevailed in certain regions of Asia, Mediterranean countries, and the Southern part of Russia. The word “Glycyrrhiza” is derived from the ancient Greek word “glycos” which defines “sweet” and “rhiza” which defines “root.”²

Liquorice contains important rich specialized metabolites, hence it has antiinflammatory, antiviral, antioxidant, anticarcinogenic, antiulcerative, and many more properties.

Hence, various diseases such as gastric ulcers, atherosclerosis, cancer, hepatitis, tuberculosis, immunodeficiency, and bacterial infections can be treated with liquorice and also has proven to be one of the medicines in treating orofacial diseases.¹

Materials and Methods

In the present study, six patients were selected, who were indicted for any sound tooth extraction (preshedding mobility, mesiodens, serial extraction, etc.) and were undergoing or required removable orthodontic treatment. The extracted tooth was stored in 10% formalin.³ Then the root part was sectioned and removed. The crown portion was sectioned into two portions along the long axis of the tooth, that is, the lingual and buccal portion with the help of a high-speed diamond disk (Fig. 1).

Fig. 1.

Armamentarium

Two enamel blocks were prepared from each tooth using a high-speed diamond-tipped disk (Fig. 2). To systematize the procedure, each enamel block was sectioned to 4 × 4 mm in size (Fig. 3).⁴

Fig. 2.

Enamel blocks were prepared using a high-speed diamondtipped disk

Fig. 3.

Enamel block was standardized to 4 × 4 mm in size

From each tooth, two enamel blocks prepared were used in vitro and in vivo on the same patient in their removable orthodontic appliance.

Demineralization was carried out by suspending each enamel block into 20 mL of demineralization solution for 72 hours in an incubator at a temperature of 35°C.⁵ The demineralizing solution consists of calcium chloride (CaCl2) = 0.2 mm, monosodium phosphate = 0.2 mm, and acetic acid = 0.05 mm which was indigenously prepared in a Biochemistry laboratory, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India (Fig. 4). Around 40% sodium hydroxide (NaOH) solution was used to adjust the pH of 4.5 (Fig. 5).⁶ Before subjecting the enamel blocks to the demineralizing solution, the pH was adjusted and recorded with the help of litmus paper.

Fig. 4.

Demineralizing solution

Fig. 5.

Solution was adjusted with 40% NaOH to a pH of 4.5

Then the enamel block was assessed for demineralization using a confocal microscope (Fig. 6).

Fig. 6.

Confocal microscope (model OLS 4000)

In Vitro study

In the present in vitro study, the composition of the artificial saliva was sorbitol 30.0 gm, carboxymethylcellulose sodium 11.7 gm, dipotassium monohydrogen phosphate 804 mg, potassium chloride 625 mg, potassium dihydrogen phosphate 366 mg, calcium chloride 166 mg, magnesium chloride 59 mg, and purified water ad 1000 mL,⁷ which was indigenously prepared in Biochemistry laboratory, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India (Fig. 7).

Fig. 7.

Artificial saliva

The remineralizing solution was prepared in the Biochemistry laboratory, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India, by grinding the liquorice lozenges using mortar and pestle into powder. The powdered liquorice is dissolved in the artificial saliva (Fig. 8).

Fig. 8.

Remineralizing solution (artificial saliva containing liquorice)

The demineralized enamel block was immersed in artificial saliva for 5 minutes and remineralizing solution for at least 4 minutes, in a sequential manner 14 times. Following, remineralization of the enamel blocks was assessed.

A confocal microscope was used to assess the potential of liquorice in the remineralization of demineralized enamel blocks.

In Vivo Study

An in vivo study was carried out in the Department of Pediatrics and Preventive Dentistry, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India. Six patients who were undergoing extraction of the sound tooth and undergoing or required removable orthodontic treatment, of age-group 6–12 years were selected. Child assent form and informed consent were taken from the patient and their parents respectively after providing them with the participant information sheet.

The enamel block was prepared as the earlier mentioned procedure using the extracted tooth of each respective patient to prevent cross-contamination. The enamel blocks were demineralized in 20 mL of demineralization solution for 72 hours at a temperature of 35°C, using an incubator. Then, enamel blocks that are demineralized are embedded in an acrylic on the full palatal coverage appliance using a micromotor (Fig. 9). The incorporated appliances were sterilized in an autoclave with 210° C for 30 minutes and delivered. Patients were asked to consume liquorice lozenges (Ricola) for 7 days twice daily (Fig. 10). After the treatment regimen, the enamel blocks were removed from the appliance, and the appliance was rebased for patient comfort. The samples were analyzed under a confocal microscope and remineralization was evaluated.

Fig. 9.

Appliance with the incorporated enamel blocks

Fig. 10.

Liquorice lozenges (Ricola)

Results

The compounds present in the dried root of the liquorice plant has an advantageous to maintain oral health and also mainly prevent tooth decay.

Licoricidin and licorisoflavan A are the principal components present in liquorice, which inhibits the growth of cariogenic bacteria.²

In our present study, the enamel blocks immersed in the demineralizing solution showed the visual demineralized layer in the confocal images (Fig. 11) obtained from the confocal microscope. All the samples showed a good amount of visual remineralization after analyzing through confocal images (Fig. 12).

Fig. 11.

Demineralized layer seen on the confocal images of enamel blocks (in vitro study)

Fig. 12.

Remineralized layer seen on the confocal images of enamel blocks (in vitro study)

In the present in vivo study, the enamel blocks of the respective patient were demineralized in the same manner as mentioned above in the in vitro study, and the confocal images were analyzed. The enamel blocks showed visual remineralization through the formation of a demineralized layer (Fig. 13).

Fig. 13.

Demineralized layer seen on the confocal images of enamel blocks (in vivo study)

Among all the patients, one child discontinued the consumption of liquorice lozenges, so that particular sample could not be assessed. Confocal images showed the demarcated area on the enamel block, indicating remineralization (Fig. 14).

Fig. 14.

Remineralized layer seen on the confocal images of enamel blocks (in vivo study)

Discussion

Dental caries, a chronic disease is one of the most commonly seen and important global oral health problems in the world. Gradual changes in diet and increase in sugar uptake, especially in children become the culprit and more susceptible to caries. Thus, it necessitates developing natural, novel antimicrobial agents not only to treat dental caries but also to prevent it.²

Various parts of herbal plants like leaves, buds, fruits, flowers, seeds, roots, and barks have been used and studied by researchers for ages. It was shown to have immense potential for antiinflammatory, antibiotic, analgesic, and antinociceptive properties.⁴

Liquorice contains important metabolites like more than triterpenoids and flavonoids like glycyrrhizin (GL), liquiritigenin, 18-β-glycyrrhetinic, licochalcone A, licochalcone E, licorisoflavan A, and glabridin having both antimicrobial and antiviral activities.⁸ The studies on the remineralization potential of liquorice are minimal. But, flavonoid compounds, such as 1-methoxyficifolinol, licorisoflavan A, and 6, 8-diprenylgenistein have been isolated from liquorice root extracts (G. glabra).

Studies on flavanoids have found that it has the potential of remineralization by increasing the availability of calcium and phosphate ions.⁴

In the present in vitro study, all the samples showed a good amount of visual remineralization after analyzing through confocal images.

Sterilization of the enamel block was done using humid steam vapor in an autoclave. According to Barcelos et al., the method to sterilize the extracted teeth has not been well defined.⁹ The most commonly used method of sterilizing the biological restoration is humid steam vapor and is one of the most recommended techniques. Microbiological culturing and scanned electron microscope (SEM) analysis was has verified that humid -steam vapor has the potential to eliminate microorganisms.¹⁰ In the present study, the enamel blocks incorporated appliances were sterilized in an autoclave at 210° C for 30 minutes and delivered.

The enamel block was placed in the center of the full palatal coverage appliances just below the rugae area and not close to the palatal surfaces of the maxillary teeth since the pooling of the saliva is more toward it and to eliminate the bias. Saliva has important protective properties including antimicrobial property, cleansing action, buffering capacity, and remineralization activity (Dowd, 1999). Directly or indirectly liquorice simulates the gustatory function, followed by an increase in salivary flow, thereby aids in multiaction of saliva helping to minimize the occurrence of dental caries.¹¹

Gedalia et al. conducted a study and found that when GL was added to Acidulated phosphate fluoride (APF) solution, had immense potential in increasing the uptake of fluoride and reduced enamel solubility. Also found a special property of surface-coating effect.

Another study by Deutchman et al. found that the effect of GL with respect to mineral loss in an artificial carious lesion was not significant. Also suggested that it might be related to insufficient GL concentration or time of exposure. He found that glycyrrhizic acid is the one to minimize the dissolution of enamel by hindering the production of acid by dental plaque.¹²

Thus, a study was conducted both in vivo and in vitro to evaluate the remineralizing potential of liquorice and the effective means of delivery of liquorice with its therapeutic potential to children, which is difficult and challenging.

A very simple and effective means of delivery of mineralizing agent is presented here, which increases the time of exposure of liquorice to the tooth surfaces. The intake of sugar was reduced tremendously and children accepted the taste of lozenges with therapeutic agents. Modification of Behavior for children was also not required for consuming lozenges. Liquorice is approved by Food and Drug Administration, similar to other agents, with no or minimal adverse effects if overused.¹³ In the present study, a limited amount of product was given to the patient, and was strictly instructed to use only the required amount at assigned time slots only.

Nowadays, the use of liquorice has increased as a flavorants. However, a maximum recommended daily dose of liquorice containing GL was found to be 100 mg/kg by WHO.⁴

Summary

Various parts of Yashtimadhu like roots, dried peeled or unpeeled liquorice, underground stem, and oil constitute the drug, it is one of the principal constituents used in cough and catarrh syrups, throat lozenges, etc. for >4000 years. It also, helps in the healing of ulcers and quenching of thirst, as found by Hippocrates (400 BC).

Routinely, glycyrrhizic acid, a chief component in liquorice has been utilized in various parts of Japan mainly to prevent and treat chronic viral hepatitis.¹⁴ Liquorice roots contain numerous compounds, the glucoside, and GL being the most important, which on hydrolysis gives glycyrrhetinic acid. GL has a great many medicinal properties like antiinflammatory, expectorant, antidiuretic, antihistaminic, and antiulcerous.

As there is enormous usage of liquorice in the medicinal field but shows only minimal study conducted to evaluate its efficacy in dentistry. Therefore, this study aimed to understand and examine its remineralizing potential on tooth surfaces with initial enamel carious lesions.¹³ And it was observed that there is a visible effect of remineralization in the in vitro as well as in vivo studies.

Conclusion

The distinctive approach of delivery of lozenges forestalls the problem of consumption by children and if found to have an antibacterial potential, at the same time being candy-like and appetizing for children. This build-up hopes in compliance of young children in regular consumption of lozenges with caries high-risk patients.

Orcid

Pooja HR https://orcid.org/0000-0003-3349-6711

Andrea N Mascarenhas https://orcid.org/0000-0002-5592-0969

Chandana KCH https://orcid.org/0000-0001-5508-7818