Comparison of antibacterial efficacy of herbal extracts of aloe vera leaves and mushroom against streptococcus mutansand lactobacillus - an in vitro study

INTRODUCTION

Dental caries is one of the common infectious diseases of oral cavity.1 It is an irreversible process which causes demineralization of inorganic and destruction of the organic substance of the tooth leading to cavitation. It has a multifactorial aetiology that depends on the presence of bacterial plaque, sugars, quantity and quality of saliva.2 Streptococcus mutans and lactobacillus are the main micro-organisms in the aetiology of dental caries.

Literature suggests that mutans streptococci, a gram-positive facultative anaerobe as the major pathogen and the initiator of dental caries (induce caries formation in animals fed with a sucroserich diet).3 MS has been isolated from cavitated carious lesions. They are highly acidogenic and aciduric, able to produce surface antigens I/ II and water-insoluble glucan, which promote bacterial adhesion to both tooth surface and other bacteria.4 Lactobacilli a gram positive, nonspore forming rods that grow best under microaerophilic conditions, are also acidogenic, aciduric and multiply in low pH. They are found in pit and fissure and deep dentinal caries, which favour their retention and thus help in progression of caries.2

MS metabolizes dietary sugar to produce a sticky, extracellular, dextran-based polysaccharide thus forming dental plaque. This is a biological film where various bacterial species interact and forms a protective barrier against anti-microbial agents used in clinical dentistry.4 Controlling the levels of these microorganisms in the dental plaque will aid in the prevention of caries. Hence, a caries prevention program primarily should be aimed at reducing the cariogenic bacterial plaque.5

Many attempts have been made to eliminate MS and lactobacillus from the oral cavity. Antimicrobial mouthwashes are one of the commonly used methods to inhibit plaque formation, reduce gingival inflammation and prevent dental caries.

Chlorhexidine (CHX) is the most commonly used mouth rinse and is a gold standard against which other antimicrobial agents are compared.6 CHX is a bisbiguanide that has bactericidal activity against both gram- positive and gram-negative bacteria. It is positively charged and hence binds to various surfaces including enamel pellicle, hydroxyapatite and mucous membranes that are negatively charged. It has been studied extensively and is currently the most potent chemotherapeutic agent against MS and lactobacillus.7

However, its excessive use can result in alterations of the oral flora and development of bacterial tolerance along with certain undesirable side effects like unpleasant taste, staining of teeth, tongue and restorations, desquamation and soreness of the oral mucosa.8 These problems necessitate further research for alternate antimicrobial agents that are safe and potent against specific oral pathogens.

Herb extracts contain wide variety of active components, which has antimicrobial, antiinflammatory, anti-oxidant and biocompatible properties. The advantage of herbal medicine is that it is less likely to form allergies and side effects.9

Mushroom extracts have wide range of biomolecules with nutritional and medicinal substance with anti-inflammatory and antimicrobial properties. Data available from the literature indicate a higher antimicrobial activity of mushroom extracts against gram-positive bacteria.10

Aloe barbadens miller has a long history of use as a therapeutic agent with many reported medicinal properties. Amongst its therapeutic properties, it has been shown to have anti-inflammatory, immune stimulatory, cell growth stimulatory, antibacterial, anti-fungal and anti-viral property.11

Hence in search of novel antimicrobial agent, this study was done to evaluate and compare the antimicrobial efficacy and minimal inhibitory concentration (MIC) of chlorhexidine 0.12% and extracts of two different herbs mushroom and aloe vera on Streptococcus mutans and lactobacillus.

MATERIALS AND METHODOLOGY

50 grams of dried herbs was boiled in 500ml of deionized distilled water to a final volume of 10- 20 ml. The concentrated mixture was filtered and stored in refrigerator for antimicrobial studies.

Pure strains of MS (ATCC 25175) and lactobacillus (ATCC 11975) were obtained from standard microbiology lab and maintained at 4°C on slopes of nutrient agar medium. Active cultures were freshly prepared before every antibacterial assay. 0.5 McFarland standard was used for visual comparison to adjust the bacterial suspension to a density approximately equivalent to 1.5 × 108CFU/ml. Antimicrobial assay was performed against pathogenic strain by agar disc diffusion method. The groups were divided as follows;

Streptococcus mutans groups:

Group I: Positive control - 0.12% Chlorhexidine solution

Group II: Aqueous extract of aloe vera

Group III: Aqueous extract of mushroom

Lactobacillus groups:

Group I: Positive control - 0.12% Chlorhexidine solution

Group II: Aqueous extract of aloe vera

Group III: Aqueous extract of mushroom 0.1 ml (0.5 McFarland standard) of microbial suspensions was spread evenly onto the surface of the sterilized MHA agar plates using sterile glass spreader. The plates were dried for 3 to 5min. Sterile Whitman No. The filter paper disks of 6 mm diameter were saturated with 50 μl of each extract. These were air dried to remove any residual solvent. Three MHA plates inoculated with test bacteria were used for every group. Four disks of each extract were then placed on the surface of each MHA plate (n=12) and incubated at 37°C. After 24 hours of incubation, each plate was examined for inhibition zones that were measured with Vernier calliper. The bigger the diameter of inhibition zone, the more susceptible is the microorganism to antimicrobial agent. Chlorhexidine was used as positive control against MS and lactobacillus. The minimal inhibitory concentration was determined for two plant extracts against Streptococcus mutans and lactobacillus using ELISA micro dilution method.

RESULTS

Statistical analysis was done using Analysis of Variance (ANOVA) technique and multiple comparisons were done using (post-hoc test) Bonferroni test.

Minimum inhibitory concentration for MS was 80mg/ml of aloevera extract,

80mg/ml of mushroom extractand 60mg/ml of chlorhexidine. Minimum inhibitory concentration for Lactobacillus was 60mg/ml aloevera extract, 60mg/ml of mushroom extract and 60 mg/ml of chlorhexidine.

DISCUSSION

Dental plaque is a biofilm, i.e. a group of microorganisms embedded in a polysaccharide matrix attached to the tooth surface.4 Epidemiological studies have shown that tooth decay is the most common sequelae of dental plaque formation.5

MS was chosen as the test organism because it is one of the predominant inhabitants of dental plaque and has been implicated in the formation of dental caries because of its acidogenic and aciduric property. MSadhere by hydrophobic bonds to the enamel surface and ferment dietary carbohydrates, notably sucrose. Acid production resulting from carbohydrate metabolism by these bacteria subsequently decrease the environmental pH and are responsible for the demineralization of tooth surfaces.12 MS play a critical role in destabilizing the homeostasis of the plaque by facilitating a shift of the demineralization/ remineralization balance from ‘net mineral gain’ to ‘net mineral loss’ (acidogenic stage). Once the acidic environment has been established, MS and other aciduric bacteria may increase and accumulation of acids in the dental plaque initiate dissolution of the tooth enamel subsequently leading to localized decalcification, cavitation and breakdown of calcified dental by sustaining an environment characterized by ‘net mineral loss’ (aciduric stage).13

Lactobacillus acidophilushas been isolated from carious teeth and its number in plaque has been shown to increase only after caries has developed. Lactobacilliare relevant secondary pathogen involved in the progression of caries.14

The removal of bacterial biofilms is a decisive component in the prevention and treatment of dental caries. The levels of microorganisms in the biofilm should be reduced for controlling plaque. Thus several methods to reduce the amount of dental plaque include mechanical and chemical methods. The use of mechanical agents is a simple cost effective method. The effectiveness of this method however is influenced by the individual’s dexterity and motivation. Mouthwashes are the commonly advocated chemical method for plaque elimination.Mouth rinsing is favored because of its ease of use and instant breath freshening effect. Among all available mouthwashes, chlorhexidine has good antimicrobial property.15 prolonged use of these has certain adverse effects such as alterations of normal flora, unpleasant taste, staining of teeth and restorations, soreness in the oral mucosa etc. So researches are trying to pay more attention to herbal drugs, which are a good alternative to synthetic chemical substances for caries prevention.Herbs have shown to possess antibacterial, antiviral, insecticidal and antioxidant properties due to the presence of wide variety of active phytochemicals, including flavonoids, terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols, curcumins, and phthalides.Their antimicrobial action is basically on the cell membrane, which disrupts its structure, blocks membrane synthesis, inhibits cellular respiration thereby causing cell leakage and death.16

In the current study, we tested two medicinal plants aloe vera and mushroom for their antimicrobial effectiveness against MS and lactobacilli. For antibacterial susceptibility testing, disc diffusion and broth microdilution methods were followed as per the NCCLS guidelines.17

Disk diffusion is a qualitative test. Diameter of the zone of growth inhibition around an antibiotic disk predicts the effectiveness of the antimicrobial agent. Broth microdilution is a quantitative method used for determining the MIC of antimicrobial agents and is considered to be ‘gold standard’ reference testing methodology against which other methods, such as disc diffusion, are calibrated.

In present study CHX showed better zones of inhibition than other groups. Chlorhexidine gluconate, is charged positively, shows high affinity for negative ions found in cell membranes of microorganisms. It indirectly affects the enzymatic function of dehydrogenase and adenosine triphosphatase present in the cell wall of bacteria resulting in the disruption of cell membrane. It is evident in this study that the chlorhexidine showed a definite reduction in the microbial activity which has marked anticariogenic effect. However the side effect of chlorhexidine may limit the long term use of chlorhexidine.18,19

In this study, aqueous extract of aloe vera and mushroom was chosen, based on the results of pilot study, which was conducted using different solvents like methanol, water, and chloroform.

Higher mean MS and lactobacillus was recorded in Group 1 followed by Group 3 and Group 2 respectively. The difference in mean MS among the groups was found to be statistically significant (P<0.001).

Bonferroni method was used for multiple comparisons. Mushroom extract showed higher antibacterial activity against MS and lactobacillus. Mushroom extracts have some active, low molecular weight compounds (plectasin, confuentin, grifolin, neogrfolin) that show antibacterial action.20,21,22 A study done by Signoretto et al showed that low molecular weight mass fraction of an aqueous mushroom extract had antimicrobial activity against potential oral pathogens.23 The literature shows that mushroom has highest activity against gram-positive bacteria and gel form of extract increases the contact time, which enhances the performance.20

Aloe vera contains active ingredients like anthraquinones, tannins, myristicacid, curcumin and nimbidin that contribute to its antimicrobial action.24,25 In this study, aloe vera showed less antibacterial activity than mushroom. Molecular weight (1000kDa) of the aloe vera is more than that of mushroom (12kDa), that can result in lesser penetration of aloe vera through the agar medium and hence lesser action.26,27

On reviewing the literature of various studies done previously and on analyzing the results of the present study it can be inferred that herbal extracts can be a promising substitute for the artificial antimicrobial agents, thereby limiting their side effects. For any mouth wash to be used clinically, there is a need to investigate its substantivity action on bacterial tolerance and discoloration on long term usage. The results of laboratory studies may not reflect the real effect of a material when applied in an in vivo condition but in vitro research gives support to clinical trials. Further long-term in vivo studies need to be conducted before it can be recommended for routine clinical usage.

CONCLUSION

Within the limitations of this in- vitro study, it can be concluded that

1. Both the herbal extracts tested in this study demonstrated antibacterial activity against MS and lactobacillus.

2. Chlorhexidine showed maximum antibacterial action against Streptococcusmutans and lactobacillus followed by mushroom extract and aloe vera extract with the zone of inhibition of (20.3mm/ 24.13), (17.03/ 16.05),(14.09/14.26) respectively.

3. Tested extracts demonstrated higher antimicrobial activity against lactobacillus than Streptococcus mutans.

Herbal products have potent antimicrobial activity that can be looked at as an alternative to chlorhexidine. However, further in vitro and longterm in vivo studies are recommended to confirm and correlate the findings of this study to a clinical situation.