research article i - sri lanka dental...

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Research Article I

An Invitro Study to Compare and Evaluate The Antimicrobial Efficacy of Two Natural Sugar Substitutes, Xylitol and Stevia Rebaudiana

on Streptococcus Mutans.

C. Yadav, P.V. Sanoop

Sri Lanka Dental Journal 2017; 47(03) 59-74

Yadav Chakravarthy

P.V. Sanoop

Vinyayaka Missions Dental College

Vinayaka Missions Dental College, Sankiri Main Road, Nh47, Ariyanoor 636308 (Pin) +919003378817 (Telephone Number) E-mail: [email protected]

AbstractObjective: Dental caries is one of the most common infectious diseases seen in atleast ninety percent of the world’s population. Over the years many preventive programs have been introduced to control dental caries, mainly focusing on dietary modification, the use of fluoride and pit and fissure sealants so as to increase the host resistance. It has been found that Streptococcus Mutans is the major causative bacteria that form dental caries.

Many clinical studies have been conducted to test the effect of suppression of Streptococcus Mutans. The use of sugar substitute therapy replacing harmful dietary habits seems to have positive effect on dental caries

Materials and Methods: The extracts of stevia rebaudiana and xylitol were prepared with dried powdered Stevia Rebaudaiana leaves and crystalline Xylitol powder in a soxhelet apparatus using ethanol and water as solvents.

The extracts were filtered using whatman’s filter paper and evaporated to dryness using a distillation apparatus. The crude sample obtained were labelled and stored in an eppendroff tube under 200°c until further analysis.

The antimicrobial activity was determined on Muller Hinton Agar by disc diffusion method

measuring the inhibition zone diameter ( IZD). Extracts were loaded onto a 10 mm sterile disc with concentration of 100 μl/disc using micropipette. The loaded discs were placed on the surface of medium and the extracts were allowed to diffuse for 5 minutes and the plates were kept for incubation at 37oC for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with measuring scale in millimetre. The data were recorded and analysed statistically.

Results: The values obtained for zone of inhibition in term of millimetre was tabulated and the mean values were obtained. Statistical analysis was done for evaluation, interpretation and presentation of the data obtained. The values were subjected to statistical analysis using the analysis of variance (ANOVA) and means ± standard deviation.

Conclusion: Within the limitation of the present, it can be concluded that stevia rebaudiana extracted with ethanol had higher antimicrobial property on streptococcus mutans followed by xylitol extracted with ethanol. There was no significant difference between extracts of stevia and xylitol with water.

Key words: Streptococcus Mutans, Xylitol, Stevia Rebaudiana, Ethanol, Disc Diffusion Method, ANOVA

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Introduction“An ounce of prevention is worth a pound of cure”

Henry de Bracton Dental caries is one of the most prevalent chronic diseases of people worldwide. The disease process may involve enamel, dentin and cement, causing decalcification of these tissues and disintegration of the organic substances14. Infection occurs due to demineralization of enamel and dentine (the hard tissues of the teeth) by organic acids formed by bacteria in dental plaque through the anaerobic metabolism of sugars derived from the diet5. Sugars or other fermentable carbohydrates when ingested, the resulting fall in dental plaque pH caused by organic acids increases the solubility of calcium hydroxyapatite in the dental hard tissues and demineralization occurs as calcium is lost from the tooth surface2.

Miller quoted that micro-organism in the oral cavity causes the breakdown of dietary carbohydrates due to the activity of enzymes they produce and this in turn leads to acid production and enamel demineralization. He considered that all bacteria in the mouth were potentially cariogenic, a concept now known as the ‘non-specific plaque hypothesis,4 It is believed that bacteria of the species Streptococcus mutans is the main factor that initiates caries,14 Dental caries can also be caused by other bacteria, including members of the Mitis, Anginosus and Salivarius groups of Streptococci, Enterococcus faecalis, Actinomyces naeslundii, A. viscosus, Rothia dentocariosa, Propionibacterium, Prevotella, Veillonella, Bifidobacterium and Scardovia14.

However, Mutans streptococci has been found to be the most cariogenic pathogen as they are highly acidogenic, producing short-chain acids which dissolve hard tissues of teeth14. The most important mutans streptococci isolated from tooth caries samples are S. mutans, which is able

to metabolise a number of sugars and glycosides such as glucose, fructose, sucrose, lactose, galactose, mannose, cellobiose, glucosides, trehalose, maltose and a previously unrecognised group of sugar-alcohols14. The primary habitats for S. mutans are mouth, pharynx, and intestine7.

Efforts to prevent dental caries have focused upon attempts to increase the resistance of teeth with various agents such as; fluorides, adhesive pit and fissure sealants,6

Plaque control by mechanical oral hygiene procedures,7 addition of antiplaque or antimicrobial agents to dental health care products to lower the number or reduce the cariogenic activity bacteria in contact with the teeth,6 and modify dietary practices by urging people to eat sweets, cookies and soft drinks less frequently,etc3.

The use of sugar substitute therapy, replacing harmful dietary habits seems to have positive effect on caries levels6. Various sugar substitutes have been introduced and are widely used in confections and beverages to avoid tooth decay from sugar and other fermentable carbohydrates8. Some of the natural sugar substitutes include raw honey, date sugar, coconut sugar, maple syrup, stevia, sugar alcohol such as isomalt, sorbitol, maltitol, manitol and xylitol., etc8,9.

The use of sugar substitutes may have contributed in a limited way to the decline in the prevalence of dental caries in industrialized countries2. In dentistry, the application of this principle to a caries control strategy involves replacing the ingestion of fermentable sugars primarily sucrose with the ingestion of nonfermentable sugar substitutes15.

Xylitol is a polyol- a pentatol that occurs widely in nature is used originally to sweeten a number of sugar-free products and is most frequently used as chewing gum6. Xylitol is a five-carbon sugar alcohol derived primarily from forest and


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agricultural materials10. Several recent studies have been published documenting the inhibitory effect of xylitol against dental caries6-8. Its anticariogenic potential was established in the landmark study of Turku, Scheinin and Makinen et al. (1975)9.

Xylitol sugar substitute exerts inhibitory caries effect by inhibiting the cariogenic bacteria to ferment it6. Other studies reported that it reduces the ability of Mutans streptococci to adhere, makes it more easily removed from plaque. Holgerson et al6 among school children, reported that chewing gum with xylitol can reduce the amount of dental plaque and acid production and interfere with the microbial composition6.

Oral microorganisms and specially S. mutans do not have enzymes to utilize Xylitol as a source of energy for acid production or for synthesis of extracellular polysaccharides.

Experimental studies in rats have demonstrated an extremely low caries rate in the presence of a Xylitol-containing diet. There are even indications that it can exert an anti-cariogenic effect. (Edgar WM et al. (1998)9.

There are three notable properties of xylitol that have made it an important sugar alcohol in the dental perspective:8

1) It is not readily fermented by oral bacteria, especially by streptococci.

2) It can induce the production of salivary enzymes which lead to the growth inhibition of bacteria in plaque.

3) It has been shown to reduce the numbers of S. mutans in the oral cavity by limiting the source of fermentable substrates for their survival.

Stevia Rebaudiana. is a plant of recent focus worldwide as sugar supplement. The plant

had its origin in South America (Paraguay and Brazil), belongs to the family Asteraceae and is a perennial, endemic, medicinal shrub. Stevia rebaudiana Bertoni, commonly known as sweet leaf, sugarleaf, or simply stevia, is widely grown for its sweet leaves9.

As a sugar substitute, stevia's taste has a slower onset and longer duration than that of sugar, although some of its extracts may have a bitter or licorice like aftertaste at high concentrations. With its extracts having up to 300 times the sweetness of sugar, stevia has garnered attention with the rise in demand for low-carbohydrate, low-sugar food alternatives9 Stevia contain several potential sweetening compounds, with S. rebaudiana Bertoni being the sweetest of all (Soejarto et al. 1982; Kinghorn et al. 1984) The use of S. rebaudiana as a sweetener can be found in many parts of Central and South America, where this species is indigenous (Melis 1992), as well as in Japan (Kinghorn et al. 1984)11,12.

In the recent years, the extract of Stevia leaf has been subjected to various pharmacological, clinical and toxicological investigations and results revealed interesting therapeutic applications13. It also has been used in developing broiler embryos13. In addition there has been an immense interest in utilization of natural plant extracts as antimicrobial activity due to the increase in outbreak of food borne diseases and to minimize the health

causing diseases over synthetic drugs. Few literatures have described antimicrobial activity of Stevia Rebaudiana leaf extracts using different methods13.

Thus, the purpose of this invitro study was to compare and analyze the antimicrobial efficacy of two natural sugar substitutes; Stevia Rebaudiana and Xylitol against Streptococcus Mutans, the main causative microorganism of dental caries.

An Invitro Study to Compare and Evaluate The Antimicrobial Efficacy of Two Natural Sugar Substitutes, Xylitol and Stevia Rebaudiana on Streptococcus Mutans.

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Materials and MethodsMaterials Used: 1. Dried Stevia rebaudiana leaves

(NUTRIVALUE) 2. Xylitol crystalline powder (HI MEDI-RM

7625-100g) 3. Weighing balance 4. Distilled water 5. Ethanol 99.9% 6. Soxhelet apparatus 7. Whatman no: 1 filter paper 8. Distillation apparatus 9. Eppendroff tube 10. Streptococcus mutans (MTCC- 497) 11. Nutrient agar (HI MEDIA) 12. Mueller Hinton agar (HI MEDIA-M173-

500G ) 13. Petridish 14. Laminar air flow 15. Sterile discs (HIMEDIA-DD036) 16. Micropipette 17. Incubator 18. Measuring Scale

Bacterial strain and medium: The Streptococcus mutans (MTCC-497) strains were obtained from Microbial Type Culture Collection and Gene Bank; Chandigarh. Pure culture was maintained by routine sub-culturing in nutrient agar.

Preparation of Extracts: Stevia Rebaudiana Extract: Dried Stevia rebaudiana leaves were grounded into powder weighing 20 g and were extracted separately with 200 ml of water and ethanol (99.9%) as individual solvents in the flask of soxhelet apparatus at 20o C for 3-4 hours. The extract was filtered using whatman’s no: 1 filter paper and the filtrates were evaporated in a distillation unit to obtain the crude sample. The residue were labeled and stored in an eppendroff tube under 200c until further analysis.

Xylitol Extract: Xylitol extract were prepared from pure crystalline xylitol powder (HI MEDIA RM7625) weighing 20g in the flask of soxhelet apparatus with water and ethanol (99.9%) as individual solvenst at 20o C for 3-4 hours.

The extract was filtered using whatman’s no: 1 filter paper and the filtrates were evaporated in a distillation apparatus to obtain the crude sample. The residue were labeled and stored in an eppendroff tube under 200c until further analysis.

Antimicrobial Procedure: Bacteria tested Bacterial strains used throughout investigation were Streptococcus mutans. (MTCC 497). The bacterial cultures were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. The young bacterial broth cultures were prepared before the screening procedure.

Preparation of inoculums Stock cultures were maintained at 4oC on slopes of nutrient agar. Active cultures of experiment were prepared by transferring a loopful of cells from the stock cultures to test tube of Muller-Hinton broth (MHB) for bacteria that were incubated without agitation for 24 hrs at 37oC and 25oC respectively. The cultures were diluted with fresh Muller-Hinton broth to achieve optical densities corresponding to 2.0. 106 colony forming units (CFU/ml) for bacteria.

Antimicrobial susceptibility test The disc diffusion method (Bauer et al., 1966) was used to screen the antimicrobial activity. In-vitro antimicrobial activity was screened by using Muller Hinton Agar (MHA) obtained from Hi-media (Mumbai). The MHA plates were prepared by pouring 15 ml of molten media into sterile petriplates. The plates were allowed to solidify for 5 - 10 minutes and inoculums suspension was swabbed uniformly and was


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allowed to dry for 5 minutes. The concentration of extracts is 100 μl/disc was loaded on 10 mm sterile disc using micropipette. The loaded disc was placed on the surface of medium and the extract was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37oC for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with measuring scale in millimeter.

Antimicrobial Activity Index: The values obtained for zone of inhibition in term of millimetre was tabulated and the mean values were obtained. Statistical analysis was done for evaluation, interpretation and presentation of the data obtained. The values were subjected to statistical analysis using the analysis of variance (ANOVA) and means ± standard deviation.

Statistical Value:

SteviaHours Xylitol

Ethanol Aqueous34 ± 0.40 18 ± 0.40

34 ± 0.40 18 ± 0.81

18

24

Ethanol Aqueous32 ± 0.81 18 ± 0.0

31 ± 0.70 18 ± 0.0

Statistical Value in 18 hours

Statistical Value in 24 hours


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Result obtained from this study by measuring the inhibition zone diameter and evaluating the means value, it was observed that stevia rebaudiana extracted with ethanol had higher antimicrobial property on streptococcus mutans followed by xylitol extracted with ethanol. There

Table 1: Anti-Microbial Activity in 18 hours

Graph 1: Anti-Microbial Activity in 18 hours

Colour Plate 22: Anti-Microbial Activity in 18 hours Anti-Microbial Activity in 24 hours.

Concentration with 100 µl.

was no significant difference between extracts of stevia and xylitol with water.

ResultsAnti-Microbial Activity in 18 hours:Concentration with 100 µl.

S.no. Name of Stevia Xylitol Organism Ethanol Aqueous Ethanol Aqueous1. S.mutans 34 35 34 17 18 18 30 32 32 18 18 18


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Table 2: Anti-Microbial Activity in 24 hours

Graph 2: Anti-Microbial Activity in 24 hours

Colour Plate 22: Anti-Microbial Activity in 18 hours Anti-Microbial Activity in 24 hours.

Concentration with 100 µl.

S.no. Name of Stevia Xylitol Organism Ethanol Aqueous Ethanol Aqueous

1. S.mutans 34 35 34 16 18 18 30 32 31 18 18 18


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DiscussionDental caries is an infectious microbiological disease that results in the localized dissolution and destruction of the calcified tissues of the teeth. It is the second most common cause of tooth loss and is found universally irrespective of age, sex, caste, creed or geographic location. It is considered to be a disease of the civilized society, related to lifestyle factors but heredity also plays a role. However, it is preventable to a certain extent. The prevalence of dental caries in India is 50%–60%24.

Dental caries is a chronic infectious disease20 in which organic acid metabolites produced by the metabolism of oral microorganisms lead to gradual demineralization of tooth enamel, followed by rapid proteolytic destruction of the tooth structure30. It is a multifactorial disease that is caused by interplay of three major factors, i.e., teeth, cariogenic bacteria, and fermentable sugars. Epidemiological studies have revealed the relationship between caries prevalence and sugar consumption20.

Bacteria are an essential part of the tooth decay process. Several microorganisms are capable of fermenting dietary carbohydrates, of which Streptococcus mutans is the most prevalent followed by Lactobacillus casein and Streptococcus sanguis30.

Dental caries is caused by streptococcus mutans. The mutans streptococci comprise a group of seven species of which Streptococcus mutans and Streptococcus sobrinus are the predominant species isolated from human saliva and dental plaque(Loesche, 1986)23

Streptococcus mutans is the most important oral bacteria which play a major role in dental caries, they are Gram-positive, non-motile, non-spore forming, catalase negative, facultative anaerobic cocci bacterium commonly found in the human oral cavity23. S. Mutans are the most cariogenic pathogens, as they are highly

acidogenic and produce short-chain acids which dissolve hard tissues of teeth. They metabolize sucrose to synthesize insoluble extracellular polysaccharides which enhance their adherence to the tooth surface and encourage biofilm formation14.

The most important virulence factor of streptococcus mutans is glucosyltransferase which synthesize adhesive, water-insoluble glucans from sucrose20. S. Mutans produce three glucosyltransferases, whose cooperative actions is essential for adhesive glucan synthesis. Adhesive glucans mediate attachment of bacteria to the tooth surface as well as to each other20.

S. mutans is able to metabolise a number of sugars and glycosides such as glucose, fructose, sucrose, lactose, galactose, mannose, cellobiose, glucosides, trehalose, maltose and a previously unrecognised group of sugar-alcohols. In the presence of extracellular glucose and sucrose, S. mutans synthesizes intracellular glycogen-like polysaccharides. S. mutans produces also mutacins (bacteriocins), what is considered to be an important factor in the colonization and establishment of S. mutans in the dental biofilm14.

Prevention is the most cost-effective means of controlling oral diseases and improving oral health. Dentistry has increasingly incorporated the medical mode of treatment, turning to pharmacological agents to arrest and reverse the disease process, instead of relying solely on surgical interventions (as in the past) to repair dentition after the disease has taken its toll. Presently, several methods are employed for the prevention and remineralization of caries,34 such as;

1) Increase the resistance of the teeth.24 a) Systemic use of fluoride which include

fluoridation of water, milk and salt; fluoride supplementation in the form of tablets and lozenges; and consuming a fluoride-rich diet such as tea, fish, etc.


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b) Topical: Use of fluoridated toothpaste and mouth wash; use of fluoride varnishes (in-office application, longer duration of action, high fluoride content); use of casein phosphopeptide–amorphous calcium phosphate (CPP–ACP), helps to remineralize the soft initial carious, demineralized areas of the teeth.

2) Combat the microbial plaque by physical and chemical methods24.

(i) Physical methods. a) The correct method and frequency of

brushing should be followed. b) The use of various interdental cleaning

aids such as dental floss, interdental brush, etc.

c) Use of an electronic toothbrush in children and persons with decreased manual dexterity is recommended.

(ii) Chemical methods. a) Use of fluoride-containing toothpaste. b) Mouth rinses and 0.2% chlorhexidine

and povidine–iodine mouthwash.

3) Preventive interventions24. a) The use of pit and fissure sealants. b) Application of fluoride varnish. c) Atraumatic restorative treatment (ART).

4) Modify the diet.24 a) Reduce the intake and frequency of

refined carbohydrates. b) Avoid sticky foods and replace refined

with unrefined natural food. c) Increase the intake of fibrous food

to stimulate salivary flow which is protective against caries.

d) Consume caries-protective foods such as cheese, nuts, raw vegetables, fruits, etc.

e) Use of natural sugar substitutes such as raw honey, date sugar, coconut sugar, maple syrup, stevia, sugar alcohol such as isomalt, sorbitol, maltitol, manitol and xylitol., etc8,9.

Oral health is related to diet in many ways, for example, nutritional influences on craniofacial development, oral cancer and oral infectious diseases2. Sugars and other fermentable dietary carbohydrates are substrates to microorganisms that ferment carbohydrates and generate acids. The most significant effect of nutrition on teeth is the local action of diet in the mouth on the development of dental caries and enamel erosion. Dental erosion is increasing and is associated with dietary acids2, these acidity causes demineralisation of the tooth enamel which is the initial step in dental caries lesions. Hence, blocking any of the aetiological factors will decrease caries activity. These include suppressing acidogenic bacteria in the mouth by maintaining good oral hygiene and limiting consumption of fermentable carbohydrates. In addition, use of fluoride, which reduces enamel liability to acid dissolution, leads to less dental caries6.

Sugar substitutes have been introduced and widely investigated in limiting the dietary source of caries hazards2. Various sugar substitutes have been introduced to avoid tooth decay from sugar and other fermentable carbohydrates6. Some of the natural sugar substitutes include raw honey, date sugar, coconut sugar, maple syrup, stevia, sugar alcohol such as isomalt, sorbitol, maltitol, manitol and xylitol., etc8,9.

Among the sugar alcohols, the most common is a five carbon sugar alcohol named xylitol claimed to have a significant correlation with reduction of caries incidence by different mechanisms. It is a member of the group of compounds known as sugar alcohols or polyols28. Polyols offer many benefits, i.e. they taste like sugar but have fewer calories than sugar25.

Xylitol with sweetness equal to that of table sugar (sucrose) but with 40% fewer calories39 are produced commercially from birch trees and other hardwoods containing xylan (polymeric pentosans of xylose which are the binding


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material of the fibres of cellulose in plants)35. It can also be found in small quantities in fruits and vegetables. Xylitol contains 40 percent fewer calories than sucrose. Because xylitol is absorbed slowly by the human gastrointestinal tract, the main side effect associated with its consumption is osmotic diarrhea. This usually occurs only when xylitol is consumed in large quantities, four to five times those needed for the prevention of dental caries. This side effect is common to all sugar alcohols28.

Xylitol, a naturally occurring sugar substitute with anticariogenic properties27 and sweetness equal to that of table sugar25 has been a focus of scientific inquiry for several decades28. Numerous field studies and clinical trials have demonstrated that the regular use of xylitol in the diet can reduce the occurrence of tooth decay (dental caries) by as much as 100%27.

The inhibitory effects of xylitol against tooth decay were first demonstrated during the Turku Sugar Studies, at the beginning of the 1970’s27. Xylitol has been shown to actively inhibit the growth of cariogenic bacteria, and over time, to reduce their overall numbers in the plaque. It also causes a shift in the Mutans Streptococci population; to one that is less virulent, having a reduced ability to ferment sugars to organic acids and exhibiting reduced adhesion characteristics (particularly the ability to adhere to tooth enamel)27. Xylitol decreases the level of S.mutans which is in saliva and plaque; this is done by interrupting the bacteria’s energy production so that they will die and it reduces the inherence of the bacteria to the teeth and reduces acid release29.

Microorganisms do not readily metabolize xylitol and its consumption has minimal effect on plaque pH. However, xylitol does accumulate intracellularly in S. mutans. This accumulation inhibits the bacteria’s growth. This has been demonstrated in-vitro and may contribute to a reduction of S. mutans levels in the plaque and

saliva of those consuming xylitol28.

In addition, xylitol has a number of effects on S. mutans that may account for some of its clinical effects in caries reduction28. Xylitol disrupts the energy production processes leading to a futile energy consumption cycle and finally cell death. Further, consumers of clinically effective levels of xylitol have shown streptococcus mutans strains with reduced adhesion to the teeth and other reduced virulence properties such as less acid production36.

Short-term consumption of xylitol is associated with decreased S. mutans levels in both saliva and plaque28. Long-term habitual consumption of xylitol appears to have a selective effect on S. mutans, resulting in selection for populations less adherent to tooth surfaces. These colonies, therefore, are shed more easily from plaque into saliva. This effect may not only be important to the individual’s decay experience, but may also influence the transmission of S. mutans from mothers who consume xylitol to their children28.

Xylitol having anticariogenic property has also got its own drawback as well. Xylitol is safe for children when consumed in therapeutic doses for dental caries prevention. Common side effects that may occur with the use of xylitol are gas and osmotic diarrhea. Hence these symptoms usually occur at higher dosages and will subside once xylitol consumption is stopped. To minimize gas and diarrhea, xylitol should be introduced slowly, over a week or more, to acclimate the body to the polyol, especially in young children36.

Stevia Rebaudiana is another natural sugar substitute with high nutritional value beneficial in the battle against dental caries30. its scientific name is Stevia Rebaudiana Bertoni, commonly is known as sweet grass or paraguayan sweet grass31 is an herbaceous perennial shrub indigenous to Paraguay and Brazil. Stevioside, the main sweet component in the leaves of


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this plant, is approximately 300 times sweeter tasting than sucrose32. it is a natural non-caloric sweetener, with more sweetness than sucrose, without adverse effects, which has demonstrated to have multiples benefits to the systemic health and recently to the oral health30. The ancient Ayurvedic system of medicine has a long history regarding the use of S. rebaudiana (Megeji et al., 2005)30. it is traditionaly known as “Sweet Leaf/Meethi Patti”, “Sweet Herbs” and “Honey Leaf”,37

Leaves of S. rebaudiana has been recommended as a treatment against various chronic and non-chronic diseases like diabetes, cardiovascular disease, cancer, renal disease, obesity, inflammatory bowel disease and dental caries30. In addition to its natural, noncaloric sweetening properties, extracts of the leaf of stevia have produced beneficial antihypertensive,antihyperglycemic, antioxidant, chemoprotective,anti-inflammatory, nomodulatory, and antiviral effects on human health32

Stevia is thought to be able to inhibit the growth of certain bacteria and other infectious organisms33. Extract of Stevia leaves and its major secondary metabolites, steviol, isosteviol, stevioside and rebaudioside A, B, C and E are noncariogenic and have been found to inhibit glucan induced aggregation of cariogenic organism, Thus Stevia have potential of providing oral health benefits (Wu et al., 1998)30. In vitro researches have shown that Stevia extracts have anti-bacterial activity on Streptococcus mutans31.

The application of Stevia in the dental treatment is a barely explored field. In order to materialize their contribution to this area, further studies are needed on the isolation, characterization and identification of substances present in the extracts. It has to be found the solvent that achieve the best use of the active components of this plant and make it biocompatible; the concentration has to be selected to suits the standards of acceptable daily intake and make it

effective at the same time, and has to be found a mean of administration considering the time spent at the site of action so that the active compound will achieve the desired effect31.

Hence, this study was performed to compare and evaluate the antimicrobial efficacy of Stevia Rebaudiana and Xylitol against streptococcus mutans, the main causative agent of dental caries.

Powdered Stevia rebaudiana leaves (nutrivalue stevia 50gm, GrassRoot Nutrition) and pure crystalline xylitol powder (HI MEDIA RM 7625-100g) weighing 20 gms were used to prepare the extract in the flask of soxhelet apparatus using ethanol and water as solvent for 3-4 hours. The extracts were filtered using whatman no: 1 filter paper to remove any leaf residue. The filtrated were then evaporated to dryness in a distillation apparatus. The residual yield of water and ethanol extracts for stevia rebaudiana and xylitol were 4.039 g, 2.761g, 5.828g and 4.611 g respectively.

Disc diffusion technique was used to determine the inhibition zone diameter by using Muller Hinton Agar (MHA) obtained from Hi-media (Mumbai). 0.1% inoculums were swabbed uniformly and the inoculums were allowed to dry for 5 minutes. 10 mm sterile discs were loaded onto the plate with 100 mg/disc concentration of the extract. The extracts were then allowed to diffuse for 5 minutes and were kept for incubation at 370c for 24 hours. After the incubation, the presence or absence of inhibition zones was determined. Each assay was performed in triplicate and the average reported in mm.

The results obtained from this study was done measuring the inhibition zone diameter (IZD) and evaluating the means value, it was observed that stevia rebaudiana extracted with ethanol had higher antimicrobial property on streptococcus mutans followed by xylitol extracted with


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ethanol. The statistical value obtained [ Table 3] shows that extracts of stevia in ethanol performed better after 18 hours [34 ± 0.40] and 24 hours [34 ± 0.40] when compared to xylitol in ethanol after 18 hours [32 ± 0.81] and after 24 hours [18 ± 0.0].

However the statistical value for extracts of stevia rebaudiana and xylitol in the aqueous medium after 18 and 24 hours show no significant difference [Table 3].

Xylitol sugar substitutes exhibits inhibitory properties against dental caries by inhibiting the cariogenic bacteria to ferment it and by reducing the levels of Streptococcus mutans in plaque and saliva and thereby reducing the level of lactic acid produced by these bacteria32. Studies have reported that xylitol reduces the ability of streptococcus mutans to adhere, making it more easily removable from plaque. Holgerson et al; reported that xylitol can reduce the amount of dental plaque and acid production and interface with the microbial composition6.

However, in this study stevia rebaudiana performed better when compared to xylitol [Table 3], the probable reason for the superior performance could be associated due to the presence of phytochemicals in stevia rebaudiana.

Phytochemicals are non-nutrient bioactive plant origin compounds naturally present in fruits, vegetables and whole grains43. Stevia rebaudiana, is found to contain over 100 phytochemicals,41 such as flavonoids, phenolic acids, fatty acids, proteins and vitamins etc40. Due to the high content of various phytoconstituents, stevia extracts have showed significant antimicrobial property42. The full chemical composition of the Stevia rebaudiana is not available yet31.

Regular consumption of nutritive sweeteners also known as caloric sweeteners or sugars provide energy in the form of carbohydrate, causes cavities which encourage the growth

of harmful bacteria in the mouth contributing to plaque formation and gingivitis. There is a requirement to substitute sucrose with natural sweetener which should be nutritionally appropriate and not being detrimental to the overall general health of the individual30.

Unlike the metabolism of sucrose, which produces energy and promotes bacterial growth, S mutans expends energy to break down the accumulated xylitol without yielding energy in return. Furthermore, the energy-producing intermediates are consumed and not reproduced by xylitol metabolism. This has been demonstrated in vitro and may contribute to a reduction of S mutans levels in plaque and saliva and a reduction in acid production among those consuming xylitol39.

Stevia, as a non nutritive sweetener are zero- or low-calorie alternatives to nutritive sweeteners, such as table sugar possess bacteriostatic and bacteriocidal properties benefit oral health by eliminating the cause of dental decay and gingivitis. Stevia is a natural sucrose substitute with high nutritional value beneficial in the battle against dental caries. Extract of Stevia leaves and its major secondary metabolites, steviol, isosteviol, stevioside and rebaudioside A, B, C and E are noncariogenic and have been found to inhibit glucan induced aggregation of cariogenic organism, Thus Stevia have potential of providing oral health benefits (Wu et al., 1998).

A study conducted by Grenby, 1991 shows that the major cariogenic organism, S. mutans, experiences growth suppression and secretes less acid when grown on media containing stevioside than when grown on sucrose, glucose or fructose media30.

Buitrago et al. evaluated the antimicrobial effect of Stevia extracts in methanol and concluded that concentrations starting from 200 mg/ml lead to an inhibitory effect on Streptococcus


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mutans. It was found that Streptococcus mutans experiments higher growth suppression when it is grown in mediums that contain stevioside, than when it’s grown in mediums with sucrose, glucose or fructose31.

Das et al. 1992 conducted a study to test whether stevioside and rebaudioside A may have the potential of causing dental caries from prolonged use. Rats were fed a diet containing 0.5% stevioside or 0.5% rebaudioside A for 5 weeks. Neither compound showed a potential of increasing the risk of developing dental caries38. He concluded that development of dental caries in rat are triggered in presence of sucrose solution while it is not with stevioside30.

In order to maintain oral health, natural sweeteners are the best substitute to sugar. It has been suggested that a sugar substitute should be non-toxic and non-carcinogenic. Controlled

human and other animal studies have concluded that sugar substitutes play a significant role in preventing dental caries. Animal caries experiments have observed a significant difference in the sulcal caries scores caused by a sucrose group and a Stevia sweeteners group (Matsukubo and Takazoe, 2006). It has been reported that higher amount of stevioside and Stevia extract has the ability to reduce the bacterial growth. Interestingly, the required concentration of stevioside as sweetener is rather low compared to sugar. Thus, stevioside can be a substitute for a certain cariogenic compound present in sucrose (Geuns, 2003). Various studies have concluded that stevioside as well as rebaudioside A are non-cariogenic sweeteners (Matsukubo and Takazoe, 2006)38.

Stevia presents properties that potentially are anti-caries and anti-periodontal diseases. However, further studies are necessary to

Figure 1. Xylitol in powder form

Figure 3. Stevia

Figure 2. Ethanol

Figure 4. Petri dish used for culture of microorganisum


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confirm these assumptions and provide a greater understanding of the mechanisms of action of this plant and the components involved.

If deeper studies are done regarding this subject, Stevia Rebaudiana Bertoni could become a complement to oral care used in the form of mouthwashes, toothpastes, chewing gum, artificial saliva, chewable tablets, among others, being especially beneficial in patients with obesity, diabetes and hypertension31.

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Evidence on Interventions to Prevent Dental Caries, Oral and Pharyngeal Cancers, and Sports-Related Craniofacial Injuries. American journal of Preventive Medicine, Volume 23.

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