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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2019  |  Volume : 23  |  Issue : 1  |  Page : 73-77
 

Antiadherence and antimicrobial property of herbal extracts (Glycyrrhiza glabra and Terminalia chebula) on Streptococcus mutans: An in vitro experimental study


1 Department of Pedodontics and Preventive Dentistry, Index Institute of Dental Sciences, Indore, Madhya Pradesh, India
2 Department of Conservative Dentistry and Endodontics, Chhattisgarh Dental College and Research Institute, Rajnandgaon, Chhattisgarh, India
3 Department of Oral Pathology and Microbiology, Chhattisgarh Dental College and Research Institute, Rajnandgaon, Chhattisgarh, India
4 Department of Conservative Dentistry and Endodontics, VSPM Dental College and Research Centre, Nagpur, Maharashtra, India

Date of Submission01-May-2018
Date of Acceptance19-Dec-2018
Date of Web Publication17-Apr-2019

Correspondence Address:
Snehal S Sonarkar
Department of Conservative Dentistry and Endodontics, VSPM Dental College and Research Centre, Digdoh Hills, Hingna Road, Nagpur - 440 019, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.JOMFP_103_18

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   Abstract 


Background: Herbal agents are used for treating different forms of diseases since decades. In the current study, the antiadhesive property of herbal extracts has been evaluated using Glycyrrhiza glabra (GG) and Terminalia chebula (TC) herbal extracts on Streptococcus mutans.
Materials and Methods: The plant extracts (GG and TC) were powdered in mechanical grinder. Ten gram of each plant extract in powder form was placed in porous bag or thimble. The extract was placed in a round-bottom flask and was transferred into clean preweighed universal tubes. The yield strength of the extract was calculated. The antiadherence property of the herbal extract was evaluated using glass surface adherence test.
Statistical Analysis: The statistical analysis was done using one-way analysis of variance followed by post hoc Tukey's test.
Results: Both herbal extracts have significant antiadhesive and antimicrobial activity against S. mutans, however, high antiadherence property was seen with TC than GG.
Conclusion: Both the plant extracts exhibit inhibitory activity against S. mutans. However, TC had more clinically significant results than GG, but it was found statistically insignificant.


Keywords: Antiadherence activity, antimicrobial activity, Glycyrrhiza glabra, herbal agent, Terminalia chebula


How to cite this article:
Bhadoria N, Gunwal MK, Suryawanshi H, Sonarkar SS. Antiadherence and antimicrobial property of herbal extracts (Glycyrrhiza glabra and Terminalia chebula) on Streptococcus mutans: An in vitro experimental study. J Oral Maxillofac Pathol 2019;23:73-7

How to cite this URL:
Bhadoria N, Gunwal MK, Suryawanshi H, Sonarkar SS. Antiadherence and antimicrobial property of herbal extracts (Glycyrrhiza glabra and Terminalia chebula) on Streptococcus mutans: An in vitro experimental study. J Oral Maxillofac Pathol [serial online] 2019 [cited 2019 Jul 22];23:73-7. Available from: http://www.jomfp.in/text.asp?2019/23/1/73/256415





   Introduction Top


Dental caries is the most common oral disease that results in demineralization of tooth, pain and discomfort. The dental caries process starts with microbial adherence on tooth surfaces, after which dental plaque is formed which leads to localized demineralization of tooth enamel by acids of bacterial origin produced from the fermentation of dietary carbohydrates.[1] There are numerous bacteria which cause initiation and progression of dental caries; however, Streptococcus mutans and Lactobacillus species are considered as the principal microorganism for causing dental caries.[2] There are numerous agents which are used for the treatment and prevention of dental caries, of which herbal agents are considered as one of the options.

Herbal agents are used since ancient times for the treatment of various diseases. These herbal agents are used in the form of there extracts and essential oils, which have numerous biological properties that are confirmed by in vitro and in vivo studies. It has antibacterial, antiseptic, antifungal, antiviral, antiprotozoal and anthelminthic property. It was found to have anti-inflammatory, analgesic, anti-allergic, antitumor, antioxidative, anticonvulsant, antidepressant, contraceptive, antimutagenic and diuretic properties.[3],[4] Considering these properties, herbal agents have been used in the field of conservative dentistry and endodontics.[5]

Glycyrrhiza glabra (GG) also known as licorice or sweet root or mulethi. It belongs to the pea and bean family. It is extensively used in foods and is also used as traditional and herbal medicine. It is very sweet, moist and soothing herb. It is commonly used as a flavoring agent in Kampo medicines.[6] It is stated that the roots of GG contain high concentration of triterpene saponin glycyrrhizin (about 2.5%–9%). He compound glycyrrhizin is an diglucuronide of glycyrrhetinic acid, which is responsible for the positive properties,[1] and has anti-inflammatory (is because of glycyrrhetinic acid), antiviral and anticarcinogenic properties.[6]

Terminalia chebula (TC) is an herbal plant having medicinal values and is known as Kadukka in Tamil Nadu, India. It contains hydrolysable tannins constituting gallic acid, chebulic acid, chebulagic acid and corilagin in a concentration of 13%. Because of these acids, it can be used for the treatment of oral diseases such as gingivitis and stomatitis and was found to have antibacterial activity.[7]

Considering the above properties, this experimental in vitro research has been undertaken to evaluate the effect of herbal extracts (GG and TC) against commonly found organism causing dental caries, i.e., S. mutans.


   Materials and Methods Top


Preparation of extract

The roots of GG were dried and powdered using mechanical grinder. Similarly, the dried ripen fruits of TC were taken and powdered using mechanical grinder. Soxhlet apparatus was used to prepare the extract. This apparatus has three sections, namely a percolator which circulates the solvent; a thimble made of thick filter paper, which retains the solid to be extracted and a siphon mechanism that empties the thimble.

Ten gram of each plant extract in powder form was placed in thimble. It was then fitted with appropriate size round-bottom flask with 100 ml water, and the upper part was fitted with condenser. The solvent was recycled by providing constant heat (50°–60°C) using Mantox heater. After complete extraction, the extract in round-bottom flask was transferred into clean and preweighed universal tubes.

Percentage of herbal extract

The percentage yield was calculated using the formula given below.



Where N indicates the final weight of extract.

Using the above formula, the yield obtained was about 30% and 36% for GG and TC, respectively.

Antiadhesive property

This property was determined by glass surface adherence test as described by Hamada et al.[8] The pure strains of S. mutans (ATCC 890) were obtained from Microbial Type Culture Collection, Chandigarh. These bacteria were grown for 24 h at 37°C at an angle of 30° in a glass tube with 10 ml of BHI broth (HiMedia Laboratories Pvt. Ltd., Mumbai, Maharashtra, India). The readings were obtained from spectrophotometer. The total free and adherent bacterial cells were determined.

Percentage of bacterial adherence



Antimicrobial activity

It was determined by agar well diffusion method. Whole experiment was repeated six times for each isolate, and the mean zone of inhibition was then calculated.

Statistical analysis was performed (IBM SPSS statistics software, version 18.0, IBM Corp., Armonk, NY, USA).


   Results Top


Antiadherence property

Antiadherence property was seen with both the herbal agents (GG and TC). However, TC showed higher antiadherence property of 59% at a concentration of 01:16, and lower percentage (37.46%) was seen at a concentration of 01:01. Similarly, higher antiadherence property of 49.90% at a concentration of 01:16 and lower percentage (40.00%) was seen at a concentration of 01:01 with GG. Hence, when compared in percentage, TC showed higher antiadherence property than GG [Graph 1].



Antimicrobial activity

The mean zone of inhibition was found to be maximum at a concentration of 01:02 (22.73) for GG; however, no zone of inhibition was seen at concentration of 01:16 (0.00). Similarly, for TC, maximum zone of inhibition was seen at a concentration of 01:01 (26.47) and minimum at a concentration of 01:16 (13.60) [Table 1].
Table 1: Descriptive statistics calculating the mean and standard deviation of inhibitory effect (zone of inhibition in mm) of Glycyrrhiza glabra and Terminalia chebula extract on Streptococcus mutans at different concentrations

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When GG and TC were compared using unpaired t-test, significantly higher inhibitory effect (zone of inhibition) of TC extract on S. mutans than GG extract was seen at a concentration of 01:01, 01:04, 01:08 and 01:16, respectively. At 01:02 concentration, inhibitory effect of GG was significantly higher than TC extract [Table 2].
Table 2: Comparison of inhibitory effect (zone of inhibition in mm) of Glycyrrhiza glabra and Terminalia chebula extract on Streptococcus mutans at different concentrations (using unpaired t-test)

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One-way analysis of variance showed a significant difference in inhibitory effect (zone of inhibition) of GG and TC extract on S. mutans at different concentrations [Table 3].
Table 3: Comparison of inhibitory effect of Glycyrrhiza glabra and Terminalia chebula extract at different concentrations using one-way analysis of variance

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For GG, when post hoc Tukey's test was applied for pairwise comparison, it showed that at 01:01 and 01:02 concentrations, inhibitory effect was significantly higher than other concentrations, whereas for TC, 01:01 concentration showed highest inhibitory effect. However, minimum zone of inhibition was seen at 01:16 concentration for both GG and TC. Further, inhibitory effect was not significant between 01:01 and 01:02 concentrations and between 01:02 and 01:04 concentration for GG and TC, respectively [Table 4].
Table 4: Tukey's post hoc test for pairwise comparison of inhibitory effect of Glycyrrhiza glabra and Terminalia chebula extract on  Streptococcus mutans Scientific Name Search rent concentrations

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   Discussion Top


Dental caries is considered to be one of the major oral health problems and has resulted in increased use of mouthwash. To overcome the side effects of major component of mouthwashes i.e. antiseptics such as chlorhexidine, various studies have investigated anticariogenic and antiperiodontitis properties using herbal agents extracted from teas, cranberries or propolis.[9] The major strength of these natural herbs is that their use has not been reported with any side effects till date. Apart from this, all herbal mouth rinses do not contain alcohol and/or sugar, two of the most common ingredients found in products. The problem is that the microorganisms that cause bad breath and halitosis love to feed on these ingredients.[10] In this study, the biological potential of two herbal extracts was investigated, which are already used as nutritional complements because of their polyphenolic components.

Root of licorice plant has several useful pharmacological properties such as anti-inflammatory, antiviral, antimicrobial and anticancer activities in addition to immunomodulatory, hepatoprotective and cardioprotective effects.[11] Sedighinia et al. stated that GG can be used for the treatment of various oral diseases (periodontal, endodontic and dental caries infection).[12] In the current study, minimal inhibitory concentration (MIC) of GG was 15% at 1:2 dilution, which was in consonance with other study. Wittschier et al. found that licorice has strong antiadhesive effects against Porphyromonas gingivalis,[13] prevents production of matrix metalloproteinase by host cells and is as useful as antibiotic like doxycycline.[14] Ahn et al., in their study, found that when glucose or sucrose concentration of 4 μg/ml and 16 μg/ml, respectively, was used, then the antibacterial activity (S. mutans) was strongest with licorice root extract.[15] The results of the present study were in accordance with other study where they found that licorice extracts inhibit the growth of cariogenic pathogens[16] and have good antibacterial activity.[12]

Parts of TC have been used for the prevention and cure of diseases. Tannic acid, gallic acid and chebulic acid are the major constituents of the ripe fruit of TC. The amount of tannic acid in the aqueous extract of TC is 13%. Tannic acid is bacteriostatic or bactericidal to some Gram-positive and Gram-negative pathogens.[17] The extract of TC prevents plaque formation on the surface of tooth by inhibiting sucrose-induced adherence and glucan-induced aggregation, two processes which foster the colonization of S. mutans on the surface of tooth. Tannic acid can be well adsorbed to the hydroxyapatite of the tooth or to the salivary mucins; alternatively, it can bound to the anionic groups on the surface of bacterial cells, which resulted in protein denaturation and ultimately bacterial cell death.[10]

In this study, both the extracts inhibited the adherence of bacteria on smooth glass surface. It was also found that as the concentration of extract increased, the adherence of bacteria on glass surface decreased. The antiadherence property of both the extracts can be attributed to the presence of phytochemicals in plants such as alkaloids, tannins, essential oils and flavonoids. It also exhibits anticariogenic property through various modes of action, involving bactericidal effects on oral bacteria, prevention of adherence of bacteria to the tooth surfaces, inhibition of glucan production and inhibition of amylases.[18] Glabridine, one of the important substances in GG, is active against Gram-positive strains then Gram-negative strains. In addition, the presence of glycyrrhizin, glycyrrhetinic acid, flavonoids, asparagine, isoflavonoids and chalcones in GG further improves its antiadhesive and antimicrobial properties.[19]

According to the NCCLS 2000 standards, the antimicrobial efficacy of any agent can be evaluated by broth dilution method, agar dilution method, disc diffusion method, agar well diffusion method and ditch plate method. The present study employed agar well diffusion method because of its reliability and acceptability. The ethanolic extracts were used because the antimicrobial efficacy can be enhanced if the phytoconstituents of these plant extracts are purified using different solvents such as ethanol, methanol and acetone. In the present study, MIC of TC came about 9% at 1:4 dilution which correlated with a study by Carounanidy et al. in which 10% aqueous extracts of TC were used.[20] In an in vivo study by Jagtap and Karkera, found that TC extract successfully inhibits glycolysis of salivary bacteria for up to 90 min after rinsing.[21]

S. mutans has the ability to adhere to a smooth surface, providing suitable conditions for it and for other bacteria to aggregate at the tooth surface. The present results showed that both the extracts strongly inhibited the adherence ability of S. mutans on a smooth glass surface. However, few studies are documented on the adherence inhibition of S. mutans by GG. The mechanism of adherence inhibition should be studied further. Hence, this plant should be studied further for their active compounds and further developed into products for the prevention of dental caries.

This study showed promising results of both the extracts against S. mutans. However, the evaluation of these extracts on secondary and tertiary plaque colonizers should also be evaluated such that it inhibits both caries and plaque microorganisms.


   Conclusion Top


The development of new therapies for the treatment of oral diseases is of great importance. From this study, we found that both GG and TC showed significant inhibitory activity against S. mutans at different concentrations. Thus, from this in vitro study, we can propose that both GG and TC can help in the reduction of dental caries. However, further studies can be done by incorporation of GG and TC in mouth rinse and their effect on S. mutans and other caries causing microorganisms.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Ajagannanavar SL, Battur H, Shamarao S, Sivakumar V, Patil PU, Shanavas P, et al. Effect of aqueous and alcoholic licorice (Glycyrrhiza glabra) root extract against Streptococcus mutans and Lactobacillus acidophilus in comparison to chlorhexidine: An in vitro study. J Int Oral Health 2014;6:29-34.  Back to cited text no. 1
    
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Haidari M, Ali M, Ward Casscells S 3rd, Madjid M. Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir. Phytomedicine 2009;16:1127-36.  Back to cited text no. 4
    
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Aluckal E, Ismail A, Paulose A, Lakshmanan S, Balakrishnan MS, Mathew B, et al. Assessment of total antioxidant capacity and antimicrobial activity of Glycyrrhiza glabra in saliva of HIV-infected patients. J Pharm Bioallied Sci 2017;9:S237-40.  Back to cited text no. 5
    
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Anand S, Rajan M, Venkateshbabu N, Kandaswamy D, Shravya Y, Rajeswari K. Evaluation of the antibacterial efficacy of Azadirachta indica, Commiphora myrrha, Glycyrrhiza glabra against Enterococcus faecalis using real time PCR. Open Dent J 2016;10:160-5.  Back to cited text no. 6
    
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Hamada S, Torii M, Kotani S, Tsuchitani Y. Adherence of Streptococcus sanguis clinical isolates to smooth surfaces and interactions of the isolates with Streptococcus mutans glucosyltransferase. Infect Immun 1981;32:364-72.  Back to cited text no. 8
    
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Malhotra R, Grover V, Kapoor A, Saxena D. Comparison of the effectiveness of a commercially available herbal mouthrinse with chlorhexidine gluconate at the clinical and patient level. J Indian Soc Periodontol 2011;15:349-52.  Back to cited text no. 10
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Sedighinia F, Safipour Afshar A, Soleimanpour S, Zarif R, Asili J, Ghazvini K, et al. Antibacterial activity of Glycyrrhiza glabra against oral pathogens: An in vitro study. Avicenna J Phytomed 2012;2:118-24.  Back to cited text no. 12
    
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Farhad SZ, Aminzadeh A, Mafi M, Barekatain M, Naghney M, Ghafari MR, et al. The effect of adjunctive low-dose doxycycline and licorice therapy on gingival crevicular fluid matrix metalloproteinase-8 levels in chronic periodontitis. Dent Res J (Isfahan) 2013;10:624-9.  Back to cited text no. 14
    
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Ahn SJ, Cho EJ, Kim HJ, Park SN, Lim YK, Kook JK, et al. The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures. Anaerobe 2012;18:590-6.  Back to cited text no. 15
    
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Kumar D, Sidhu P. The antimicrobial activity of Azardirachta indica, Glycyrrhiza glabra, Cinnamomum zeylanicum, Syzygium aromaticum, Acacia nilotica on Streptococcus mutans and Enterococcus faecalis – An in vitro study. Endodontology 2011;23:16-23.  Back to cited text no. 16
    
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Nayak SS, Ankola AV, Metgud SC, Bolmal U. Effectiveness of mouthrinse formulated from ethanol extract of Terminalia chebula fruit on salivary Streptococcus mutans among 12 to 15 year old school children of Belgaum city: A randomized field trial. J Indian Soc Pedod Prev Dent 2012;30:231-6.  Back to cited text no. 17
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Steinberg D, Sgan-Cohen HD, Stabholz A, Pizanty S, Segal R, Sela MN. The anticariogenic activity of glycyrrhizin: Preliminary clinical trials. Isr J Dent Sci 1989;2:153-7.  Back to cited text no. 19
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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