Antibacterial activity of aqueous extracts of Indian chewing sticks on dental plaque: An in vitro study.

The anti-microbial efficacy of aqueous extracts of Indian chewing sticks against different kinds of plaque bacteria in vitro was investigated. Supra-gingival plaque is cultured and subjected to the antibacterial activity of the aqueous extracts of chewing sticks (Neem, Acacia, Pongamia glabra, Achyranthes aspera, Streblus asper) separately. The results of the study demonstrate that all the five chewing sticks under study possess inhibitory potential against bacteria present in dental plaque mainly on aerobes. The antibacterial efficacy of aqueous extracts has antibacterial effects and could be used as a therapeutic agent and therefore, it appears to be potent anti-microbial agents that could be considered as a medicinal plant. Results of this study showed chewing sticks contained antibacterial agents, but the concentration and composition of the active substances differed among the plants.

In many parts of the world, especially in Indian rural areas, the chewing sticks have been in use for centuries as tooth cleaning devices because they are readily available, affordable, and efficacious; though, the number of anti-microbial formulations has been evaluated to determine the effectiveness as antiplaque and or anti-gingivitis agents. The medicinal properties associated with gum healing, analgesia, hemostasis and astringence have been attributed to chewing sticks as well as consisting of anti-microbial and plaque inhibiting effects.[123]

There are indications that the presence of substances in Acacia gum which when compared with ordinary gum primarily inhibits the early deposition of plaque. Saliva extracted factors obtained by chewing the end of sticks produce an inhibitory effect on certain oral pathogens associated with development of gingivitis, periodontal diseases, and dental caries.[4] The boiled product of Acacia is used as a rinse for gingivitis in general and mouth ulcers in particular.[5] Neem extracts have been reported to possess antibacterial, antiviral properties. The stem and bark are reported to possess astringent effect. Tender twigs of neem are used to clean teeth particularly in pyorrhea.[6]

The World Health Organization has recommended and encouraged the use of chewing sticks as an effective tool for oral hygiene.[7] As chewing sticks have been in use traditionally for centuries and readily available, economical and claimed to be effective, we report here the in vitro susceptibility of oral microflora in general to aqueous extracts of five (5) different chewing sticks commonly used in India.

Aims and objectives

To assess the efficacy of five commonly used chewing sticks in the inhibition of oral microflora and thus dental plaque in vitro

To compare the efficacy of five different chewing sticks in the inhibition of oral microbial flora

To correlate the effect of chewing sticks with the existing common anti-bacterial agents.

Subjects and Methods

Chewing sticks

Five chewing sticks, which are commonly used in India, are selected for in vitro study [Figures 1–5 and Table 1].

Figure 1

Twig of Acacia arabica (babool)

Figure 5

Twig of Achyranthes aspera (Uttareni – tel)

Table 1

Botanical names and native names of various chewing sticks TEL

Twig of Azadirachta indica (Neem)

Twig of Pongamia glabra (Kanuga – tel)

Twig of Strebulus asper (Baraniki – tel)

Preparation of the aqueous extract

Aqueous extracts are all prepared in Centre for Cellular and Molecular Biology in Hyderabad.

First the stem portions of each plant were chopped into small portions as a whole including bark and pulp. Each part was weighed and stored separately in 100 mg amounts in clean wide mouthed 250 ml screw capped bottles. Extracts were made by grinding the chopped parts in a pestle and mortar adding 10 ml of distilled water to the resultant fibers. Each extract was centrifuged at 2000 g for 10 min. The supernate was passed slowly through 0.45-um membrane filter into screw-capped tubes. Each aqueous extract is stored in 10 ml portions at 0°C [Figure 6].

Figure 6

Aqueous extracts of five different chewing sticks

Selection of the clinical samples

The patients for this study were selected from the Department of Periodontia, Government Dental College and Hospital, Hyderabad.

Inclusion criteria

The age of the patients was between 20 and 40 years

Patients having more than 20 sound teeth in the dentition

Patients with moderate to advanced periodontal disease.

Exclusion criteria

History of any systemic diseases

Patients who had received any periodontal treatment within the 6 months prior to study

Patients with gross oral pathology

Subjects undergoing, antibiotic, anti-microbial and/or anti-inflammatory therapy or who had undergone such therapy within 6 months prior to the initiation of the study

Patients wearing partial dentures or orthodontic appliances were excluded.

Study design

A total of 20 patients were selected who satisfied the above criteria. The examination of the oral cavity of patients was done using mouth mirror and probe under artificial light. Any abnormalities or stains in the oral cavity were recorded. Clinical parameters were recorded including full mouth periodontal evaluation.

Supra-gingival plague collection and inoculation

In this study, a sterile 11/12 curette was selected to collect the supra-gingival plaque which was drawn coronally from the gingival margin. If the removed plaque was not sufficient to visibly cover the curette tip the process was repeated. Chair side samples were collected and inoculated into Blood agar and MacConkey agar culture media plates by streaking with a sterile loop. These culture media plates were immediately transported to Microbiology Department for incubation.

Direct smear examination

The direct smears were made on a clean glass slides by chair side. The smear is heat fixed by passing the slide over the flame that is then stained by Jansen's modification of Gram's Method. The various organisms were observed and noted.

Aerobic culture

Chair side inoculation of the sample into blood agar and MacConkey agar culture media with sterile wire loop of 3.26 mm internal diameter was done. The inoculated blood agar and MacConkey agar plates were incubated aerobically at 37°C for 24 h. After overnight incubation, the blood agar and MacConkey agar plates were examined for evidence of growth. If there was growth, the colony characters were studied.

Smears were made from different colonies and examined under the ×100 objective after Gram's stain.

Gram-positive cocci were sub-cultured into Hartley's broth for further study and Gram-negative Bacilli were sub-cultured into peptone water for study of bio-chemical properties and special tests.

The bacterial species isolated from the primary culture were identified by their morphology, cultural characteristics and bio-chemical reactions according to the standard techniques.

Streptococci were identified by colony characteristics, type of hemolysis on blood agar and smear examination. Viridans streptococci produced alpha hemolysis on blood agar and were processed further by bio-chemical reactions. Beta-hemolytic streptococci were identified by beta hemolysis (complete lysis of red blood cells) and bacitracin sensitivity).

Staphylococci were identified by colony pigmentation, mannitol fermentation and coagulase tests. The slide coagulase test was done as per William and Harper 1946 method and the tube coagulase test was done as per Gillespie 1945 method. By these tests, staphylococci were grouped into two categories, namely coagulase positive staphylococcus and coagulase negative staphylococcus. The Gram-negative Bacilli were tested for motility by doing a hanging drop preparation and they were subjected to other bio-chemical and sugar fermentation tests. The tests were read after incubation at 37°C at the end of 24 h and 48 h.

Gram-negative lactose-fermenting Bacilli were classified on the basis of their motility, fermentation of sugars, indole production, methyl red reaction, Voges-Proskauer test, and utilization of citrate and nitrate reduction tests into Escherichia coli, Klebsiella etc.

Gram-negative nonlactose fermenting Bacilli were identified by motility, Oxidase, Catalase tests, fermentation of sugars, IMViC reaction, and urease tests. These species were categorized into miscellaneous groups.

Susceptibility to aqueous extracts in vitro

Muller and Hinton agar culture media plates were prepared containing each aqueous extract in doubling dilutions as 2%, 4%, 8%, and 16% code number of aqueous extract and dilution was noted, underneath the media plate.

Inoculating procedure

Eight aerobic sub-culture tubes were taken which contains isolate strains

Staphylococci

Klebsiella

Enterococci

Streptococci

E. coli

Coagulase positive Staphylococci

Coagulase negative Staphylococci.

These plates were divided into seven parts on the glass plate with marking pencil and code number was given. Each divided part of that plate was given the microorganism code also.

Sterile wire loop of 3.26 mm internal diameter inoculating loop was taken. The loop was inserted vertically into the sub-cultured sample to allow the sample to adhere to the loop. The loopful of the sample was dropped over the divided part of the culture media.

These inoculated Muller and Hinton agar culture media plates were incubated at 37°C for 24 h. After overnight incubation of the agar plates at 37°C these culture plates were taken out and examined for growth and inhibition. The dilutions at which the strains were inhibited if any were noted.

Results

This is an in vitro study and was conducted at microbiological laboratory. The study included 20 patients of both sexes belonging to the age group of 20–40 years. Plaque was collected from each patient and subjected to the culture and sensitivity to the aqueous extracts of five chewing sticks in the microbiology laboratory. Aerobic organisms were sub-cultured and sensitivity test was done against the isolated strains.

The results are presented in the following tabular form [Table 2].

Table 2

Results - susceptibility of plaque bacteria to extracts of various chewing sticks

Discussion

In this study of 20 patients, supra-gingival plaque is cultured and subjected to the antibacterial activity of the aqueous extracts of chewing sticks (Neem, Acacia, Pongamia glabra, Achyranthes aspera, Streblus asper) separately. The results of the study demonstrate that all five chewing sticks understudy possess inhibitory potential against bacteria present in dental plaque mainly on aerobes. This is in agreement with early report demonstrating the anti-microbial properties of some of the sticks.[1] There were however no marked differences in the susceptibility of various bacterial types to any aqueous extract of the chewing sticks mentioned. Furthermore, there are no marked differences in the susceptibility of bacteria from one chewing stick to the other.

The susceptibility of staphylococci, streptococci, and E. coli, to the 2% extract shows antibacterial activity of neem. It is in agreement with.[6] Neem extract shows that there is no difference of the spectrum of antibacterial activity from 2% to 8%, respectively. It shows that the minimum inhibitory concentration (MIC) is 2% whereas the susceptibility of Klebsiella in addition to Staphylococci. Streptococci and E. coli show the varying susceptibility of each species, which may be a function of available binding sites on the bacterial cell walls; these are probably bacterial surface proteins. Tannins have been shown to form irreversible complexes with proline rich proteins which would lead to inhibition of cell wall protein synthesis, a property that may explain the mode of action of these chewing stick extracts.[8]

The susceptibility of Staphylococci, Streptococci and E. coli to 2% Acacia extract indicates its antibacterial activity. This is in agreement with the earlier studies of Acacia gum compared with ordinary gum primarily inhibited the early deposition of plaque. The susceptibility of Staphylococci, Streptococci and E. coli only at 4%, 8% and 16% show no difference of spectrum. The concentrations from 2% to 16% show the MIC in this study is 2%. It is in the range of 0.04-10% W/V reported for various tooth paste preparations against different bacteria using a similar agar dilution method (Moran et al. 1988).[9]

However Acacia chewing stick users often chew for a long time before use to make the ends soft. Thus, acacia may prove relatively more effective in vivo than the MIC figures alone suggest.[10]

The susceptibility of Staphylococci, Streptococci and E. coli to 2% Pongamia, S. asper, A. aspera extracts shows their antibacterial activity. The susceptibility of the same strain to 4%, 8% and 16% Pongamia shows no difference of its antibacterial spectrum with increasing concentrations. Thus, MIC for Pongamia in this study is 2%. Whereas the inhibition of Enterococci at 8% concentration and 16% concentration of S. asper extract in addition to Staphylococci, Streptococci and E. coli shows its wide spectrum at increasing concentrations. Thus, the MIC of S. asper to Enterococci is 8%, whereas 2% is the MIC for Staphylococci, Streptococci, and E. coli.

The zones of inhibition obtained on the primary impregnated with aqueous extracts shows the sensitivity of the anaerobes to the chewing sticks. This is in coordination with earlier studies.[10] However, the different anaerobes were not isolated and tested; thus further detailed study is required against specific anaerobic organism to prove the efficacy of chewing sticks.

All the isolated aerobic strains are inhibited at 2% concentrations of all aqueous extracts except the Enterococci which is inhibited to 8% concentration of S. asper and Klebsiella to 16% of neem extract. This shows that there is no marked difference between the antibacterial spectrums of all the chewing sticks at 2%. The inhibitory action of chewing sticks against these organisms is thus of possible clinical significance.

It could be argued that the effectiveness of the chewing sticks could be limited by the inhibitory action of the components to gain access to deep periodontal pockets containing mainly anaerobic organisms. However, small number of these pathogens is present in the deep layer of mature, supra-gingival plaque and in accordance with their traditional use as an oral hygiene aid; they might assist in the prevention of periodontal disease by restricting their growth. This preventive action might be explained by the incorporation of these extracts in toothpaste preparations. They can also be used in the treatment of periodontal pockets if it were applied subgingivally, either mixed with gel or absorbed into a solid slow releasing material. Such devices have also been used to deliver conventional antibiotics such as tetracycline.[11] They may have an advantage over such agents in that they are natural products, which have been used for many centuries without any reported side-effects. In addition, these sticks have been in use traditionally for centuries and easily available and cost-effective.

Conclusion

Our studies described here confirm the presence of material possessing antibacterial activity in extracts of chewing sticks and tannin like substances reported earlier to be present in African chewing sticks may be responsible for this activity.

Apart from the ready availability, cost-effectiveness, natural products like chewing sticks with medicinal properties are effective in gum healing, analgesia, hemostasis, and antibacterial properties. In search of an alternative, safe and effective anti-plaque agent, an in vitro study on the antibacterial activity of aqueous extracts of five commonly used chewing sticks was done on dental plaque collected over 20 patients.

It can be concluded from this in vitro study that Indian chewing sticks appeared to have an inhibitory effect on the plaque microbiota. The nature of this inhibition however is not known. Different anaerobes were not isolated, so, further studies are suggested to identify its active ingredients against different strains for future trials in toothpastes and mouth rinse formulae.