Anticaries Efficacy of Liquorice Lollipop: an Ex Vivo Study

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Anticaries10.5005/jp-journals-10024-2361 Efficacy of Liquorice Lollipop Original Research

Anticaries Efficacy of Liquorice Lollipop: Anex vivo Study 1Gauri Krishnakumar, 2Dhananjaya Gaviappa, 3Shwetha Guruswamy

ABSTRACT Source of support: Nil Aims: To investigate the anti-bacterial efficacy of liquorice Conflict of interest: None lollipop and also to investigate the remineralizing potential of the extract. INTRODUCTION Materials and methods: An interventional study was carried out on 20 volunteers aged between 18 and 21 years. Antibacterial Dental caries still remains the most common dental efficacy was investigated by evaluating the bacterial counts problem to affect individuals across the globe. With the of salivary Streptococcus mutans and Lactobacilli before and gradual change in lifestyle trends and food habits, adults after the consumption of liquorice lollipops for 10 days while the and especially children are becoming more susceptible remineralization potential was studied with scanning electron to caries. Thus, there is a pressing need to develop new microscopy (SEM) analysis of the enamel block embedded on a Hawley’s appliance worn by volunteers for 14 days after natural and innovative antimicrobial agents for the consumption of the liquorice lollipop. treatment of dental caries.1,2 Herbal extracts in the form of leaves, fruits, flowers, seeds, roots, and barks have Results: A reduction in the bacterial counts of S. mutans which was statistically significant was observed. However, been studied by researchers and have shown to possess Lactobacillus growth was not observed before or after use of anti-inflammatory, antibiotic, analgesic, and antinocicep- liquorice lollipop. No structural or morphological changes were tive properties. Validation of the uses of these different observed on the enamel surface through SEM imaging. therapeutic modalities is particularly important to help Conclusion: Thus, liquorice lollipop was effective in reducing serve as a potent, safer, and cost-effective solution to S. mutans but did not show to possess a remineralizing potential. dental diseases.3 Clinical significance: The unique approach of a lollipop delivery Root extracts like ginger, garlic, turmeric, and system to reduce S. mutans count in children is also observed liquorice (mulethi) are traditionally used in India and have to be palatable for children. This raises hopes of a simple and been known to have an antibacterial potential. The anti- effective way to deliver a targeted intervention to young children bacterial efficacy of liquorice has been studied by various who are at risk for dental caries. researchers, but the effective mode of administration of Keywords: Adolescent, Antibacterial, Anticarious, Herbal, the extract by which the optimal antibacterial potential Liquorice, Remineralizing, Scanning electron microscope can be attained has not been established.4 Literature analysis. reveals that liquorice has an inhibitory effect on S. mutans How to cite this article: Krishnakumar G, Gaviappa D, and the modes of administration reported are either as Guruswamy S. Anticaries Efficacy of Liquorice Lollipop: An ex mouthwashes or through a lollipop delivery system.5,6 vivo Study. J Contemp Dent Pract 2018;19(8):937-942. The novel approach of incorporation of the liquorice extract with active compound Glycyrrhizol A into a lollipop delivery system had been devised by Hu et al,7 and 1-3 Department of Pedodontics and Preventive Dentistry, Faculty a reduction in salivary S. mutans levels postconsumption of Dental Sciences, M. S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India of the liquorice lollipops has been observed and it has also been reported that the mode of delivery was well Corresponding Author: Gauri Krishnakumar, Department of Pedodontics and Preventive Dentistry, Faculty of Dental accepted by children. Sciences, M. S. Ramaiah University of Applied Sciences The potential of liquorice lollipop has been proven Bengaluru, Karnataka, India, Phone: +919650232720, e-mail: but has not yet been investigated in the Indian scenario. [email protected] Further, the remineralizing potential of liquorice, if

The Journal of Contemporary Dental Practice, August 2018;19(8):937-942 937 Gauri Krishnakumar et al any, has also not been investigated. Thus, the scope of Assessment of Remineralizing Potential this study is to investigate the antibacterial efficacy of Twenty premolars extracted for orthodontic purpose were liquorice lollipops among volunteers and to investigate obtained from the Oral Surgery Department of the same if the extract has a remineralizing potential. The hypoth- institution. The radicular part of each tooth was removed esis is that there is no difference in the bacterial counts and the coronal part was then sectioned along the long of salivary S. mutans and Lactobacilli before and after the axis of the tooth into two portions of buccal and lingual consumption of liquorice, and there is no remineralizing using a high-speed diamond-tipped disk. Further, the tooth effect of liquorice on the enamel surface. was sectioned into three sections of occlusal, middle, and cervical third using the high-speed diamond-tipped disk. MATERIALS AND METHODS Four enamel specimens were prepared from each tooth and The study was carried out in the Department of only the buccal sections were selected for the study (Fig. 1). Pedodontics and Preventive Dentistry, M. S. Ramaiah Each enamel block was standardized to 3 × 2 mm in size. University of Applied Sciences, Bengaluru, Karnataka, The prepared enamel blocks were further subjected India, from March 2017 to September 2017. The volunteers to demineralization by suspending each section of the were selected from the undergraduate students of the enamel block into a glass bottle containing 20 mL of institution and were in the age group of 18 to 21 years. demineralization solution for 72 hours in an incubator at The exclusion criteria included adolescents who were a temperature of 35°C.8 The composition of the deminer- on any antibiotic regimen for the past 3 months, under- alizing solution used was CaCl2 = 2.2 mM, NaH2PO4 = going any orthodontic treatment, and if suffering from 2.2 mM, lactic acid = 0.05 M, fluoride = 0.2 ppm. Solution any systemic disease or condition. All volunteers of the was adjusted with 50% NaOH to a pH of 4.5.9 The pH was study had signed consent for the same. The sample size recorded each time with litmus paper before subjecting determination was done in accordance with a previous the enamel blocks to demineralization. study by Hu et al,7 power of 80%, error of 5%, dropout Maxillary and mandibular alginate impressions were of 10%, and confidence level of 5%. recorded in order to fabricate a Hawley’s appliance for each The liquorice lollipops were procured from the subject. Once the Hawley’s appliances were fabricated, a LOLOZTM Company who had fabricated the lollipops to window measuring 5 × 5 mm was prepared in the palatal contain 7 to 15 mg of the active compound, Glycyrrhizol A. aspect of the acrylic. The demineralized enamel blocks The genotoxicity and cytotoxicity of this liquorice lollipop were incorporated into this prepared window with cold had been earlier studied7 and is reported to be safe for cure acrylic (Fig. 2). The incorporated appliances were ster- children and adults. The recommended regimen is twice ilized by 3% ethylene oxide gas sterilizer. The subjects were daily for 10 days for an antibacterial effect. instructed to wear the appliance while consumption and 20 minutes postconsumption of the lollipop. When not Assessment of Antibacterial Efficacy worn, the Hawley’s appliance was stored in a plastic con- tainer. The liquorice lollipops were consumed for 14 days. Unstimulated saliva (2–2.5 mL) was collected for the Post the treatment regimen, the enamel blocks were sub- purpose of evaluating the antibacterial efficacy of jected to SEM analysis for evaluation of remineralization. liquorice extract. The samples were collected between 8 and 10 am in accordance with the circadian rhythm, and at least 1 hour after the consumption of any food or drink. The collected samples were transported to the laboratory within 3 to 4 hours for processing and estimation of the bacterial counts of S. mutans and Lactobacillus. Saliva samples were inoculated into mitis salivarius bacitracin (MSB agar) and Rogosa agar for specific bacterial culturing and identification of colony characteristics, and counting was done by digital colony counter. Saliva collection was performed twice during the study period. Saliva was first recorded at baseline before start of the liquorice regimen to evaluate the salivary bacterial status of the subject. The liquorice regimen was then followed for a period of 10 days. Similar procedure was performed again to evaluate the salivary bacterial status posttreatment. Fig. 1: Enamel block preparation 938 JCDP

Anticaries Efficacy of Liquorice Lollipop

Fig. 2: LOLOZ liquorice lollipops used in the study Fig. 3: Hawley’s appliance with the incorporated enamel blocks

ETHICAL AND HUMANE CONSIDERATIONS The study was approved by the ethical review board University Ethics Committee for Human Trials of the university and is in accordance with the Declaration of Helsinki. The commercially produced liquorice lollipop was procured from LOLOZTM 601 SW 2nd Ave, Portland, OR (Fig. 3). Data were subjected to statistical analysis performed using Statistical Package for the Social Sciences software version 20.0. Paired Student’s t-test was performed as analytical statistics to measure the S. mutans count before and after consumption of liquorice.

RESULTS Graph 1: Difference in CFU counts before and after treatment A considerable reduction in the S. mutans count was obser ved from 91.70 ± 63.814 to 11.20 ± 11.448 colony-forming units (CFU)/100 mL, which was statistically significant observed at 5,000× and 10,000× magnifications remained with p-value <0.05 (Table 1), suggesting that consumption the same with no presence of any mineral deposition or of liquorice lollipops showed antibacterial potential against collagen formation, which is suggestive of the fact that S. mutans. However, Lactobacillus growth was not observed liquorice root extract did not show any remineralizing before or after use of liquorice lollipop (Graph 1). potential following the study period of 14 days. Figures 4A and 5A show the initial enamel lesion created on the surface with significant porosity. The DISCUSSION porosity observed was assumed to help in penetration Several researchers have investigated the chemical constit- of solution ion constituents and allows a greater area for uents and biological activities of liquorice roots (Glycyrrhiza reaction of enamel mineral. The morphological evaluation glabra) and many phenolic compounds have been identi- of the surface enamel subjected to the action of liquorice fied that are shown to have an inhibitory effect against root extract was observed to have no change worthy of bacterial growth. Literature reveals the antibacterial role observations (Figs 4B and 5B). The structural features of polyphenols on cariogenic streptococci, suggesting the mechanism of action as a direct effect against bacteria or an Table 1: Difference in S. mutans count (CFU/100 mL) among interaction with microbial membrane proteins, thus inhib- subjects before and after the use of liquorice lollipops iting the adherence of bacterial cells to the tooth surface Mean ± SD t-value p-value or also by inhibiting glucosyl transferase and amylase Before 91.70 ± 63.814 4.427 0.002* enzymes that disrupt the glucose mechanism. After 11.20 ± 11.448 A sugar-free orange-flavored lollipop has been devel- SD: Standard deviation; *< 0.05 oped containing the novel compound Glycyrrhizol A against The Journal of Contemporary Dental Practice, August 2018;19(8):937-942 939 Gauri Krishnakumar et al

A B Figs 4A and B: Scanning electron microscopy analysis of specimen—pre- and posttreatment

A B Figs 5A and B: Scanning electron microscopy analysis of specimen—pre- and posttreatment

S. mutans in children who are at risk for caries. A study by also salivary and dietary parameters were not taken into Peters et al5 to determine the antibacterial effect of consump- consideration which could be confounding factors. tion of the liquorice lollipop twice daily for 3 weeks reported Regarding the Lactobacillus species not showing any a significant decrease in S. mutans count and also observed growth in the culture media in this study, it could be a steeper decrease in children who were at a higher risk for inferred that, Lactobacilli usually comprise less than 1% caries. A similar study was carried out by Hu et al,7 in which of the total cultivable microbiota and a strong correlation two pilot human studies were conducted to observe the has been established between the Lactobacillus count and effect of the developed liquorice lollipop against S. mutans. caries. The higher the decayed, missing, and filled teeth They concluded that a brief twice-daily application for (DMFT) index, the higher would be the number of sub- 10 days led to a marked reduction of cariogenic bacteria in jects harboring a high Lactobacillus count.11,12 Thus, the the oral cavity among the human subjects tested. growth is dependent on the DMFT status of the subject Among the Indian population also, the antibacte- as well as the sensitivity of the culture method used. rial effect of liquorice has been studied, and a similar Pre- and posttreatment, the enamel blocks were inhibitory effect on S. mutans by the ethanolic extract of analyzed by SEM. Scanning electron microscope allows liquorice has been reported.6,10 assessing qualitatively the process of demineralization In our study, we followed regimen of twice daily for through the observation of specific morphological and 10 days and observed a significant reduction in theS. mutans structural features, such as opening and size of the count, which was in accordance with previous studies. But, tubules, the presence/absence of peritubular mineral, the present study had certain limitations that, the age group and the evolution of the organic network of collagen.13 comprised of adolescents, which makes it not generalizable The remineralization potential of liquorice has not to pediatric patients. The sampling was convenient and been studied much. But, flavanoid compounds, such as 940 JCDP

Anticaries Efficacy of Liquorice Lollipop

1-methoxyficifolinol, Licorisoflavan A, and 6, 8-diprenyl- root extract did not show any remineralizing potential. genistein have been isolated from liquorice root extracts It is a known fact that children love sweets and confec- (G. glabra).14 Flavanoids have been reported to increase tionary. Overindulgence in high sugar-containing food the availability of calcium and phosphate ions, increasing items, along with improper oral hygiene methods, leads the possibility of remineralization. to children being at a higher risk for caries. Liquorice So, in our study, we made an attempt to investigate shows to have an antibacterial efficacy and at the same the remineralizing potential of liquorice extract through time is palatable for children. SEM analysis, and the results show that liquorice root extracts do not show any collagen formation/reminer- Clinical Significance alizing potential. In the literature, studies regarding the The unique approach of a lollipop delivery system coun- remineralization potential of any material reveal that the teracts this problem as it is in the form of a candy that is material should be studied for a period of 7, 14, 21, and sugar-free and is shown to have an antibacterial efficacy 28 days to observe structural and morphological changes while at the same time being esthetic and palatable for on the enamel surface. But we studied the material only children. This raises hope of a simple and effective way for a period of 14 days which could be one of the reasons to deliver a targeted intervention to young children who for the material not showing any remineralizing ability.15 are at risk for dental caries. Thus, we suggest that a study for more than 14 days must be carried out before concluding that liquorice root extract has no remineralizing potential. ACKNOWLEDGMENTS The observed adverse effect associated with exces- The authors would like to acknowledge the Department sive intake of glycyrrhizin has been termed as liquorice- of Molecular Biology, Indian Institute of Sciences, induced pseudoaldosteronism. It consists of a triad of Bengaluru, and the Department of Pharmaceutics, Faculty increased blood pressure, hypernatremia, and hypoka- of Pharmacy, M. S. Ramaiah University of Applied lemia. Glycyrrhizin blocks the activity of the enzyme Sciences, Bengaluru, Karnataka, India for their contribu- 11β-hydroxysteroid dehydrogenase type II, which converts tions in the study. cortisol to inactive cortisone. Cortisol, in turn, binds to mineralocorticoid receptors, promoting sodium reabsorp- REFERENCES tion, potassium excretion, and hypertension. Nowadays, 1. Messier C, Epifano F, Genovese S, Grenier D. 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