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Year : 2016  |  Volume : 6  |  Issue : 2  |  Page : 71-76

Comparative evaluation of antimicrobial activity of herbal extracts with 5.25% sodium hypochlorite against multispecies dentinal biofilm

1 Department of Conservative Dentistry and Endodontics, Faculty of Dental Science, Dharamsinh Desai University, Nadiad, Gujarat, India
2 Department of Conservative Dentistry and Endodontics, Ahmedabad Dental College and Hospital, Ahmedabad, Gujarat, India
3 Department of Conservative Dentistry and Endodontics, Government Dental College and Hospital, Ahmedabad, Gujarat, India
4 Department of Pharmacology, Sardar Patel College of Pharmacy, Anand, Gujarat, India

Date of Web Publication18-Apr-2016

Correspondence Address:
Kunjal S Mistry
"Sharnam Dental Clinic," Office No. 101, Shailly Complex, Surdhara Circle, SAL Hosptial Road, Thaltej, Ahmedabad - 380 052, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1658-5984.180619

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Aims: To check the antimicrobial activity of Tinospora cordifolia (TC) (Giloy), Ocimum sanctum (Tulsi), and 5.25% sodium hypochlorite (NaOCl) against 21-day-old multispecies biofilm formed on tooth substrate of extracted human teeth. Settings and Design: In vitro dentin disinfection model used to check the antimicrobial efficacy of herbal extracts. Materials and Methods: The in vitro dentin disinfection model was used to check the antimicrobial activity of the methanolic extracts of medicinal plants along with NaOCl. The polymicrobial biofilm was allowed to grow on extracted teeth sections for a period of 21 days. Remaining microbial load in the form of CFU/ml after the antimicrobial treatment was tabulated and data were statistically analyzed using ANOVA and Bonferroni post-hoc tests. Statistical Analysis Used: SPSS version 17 one-way ANOVA, Bonferroni post-hoc test. Results: Both the plant extracts showed considerable antimicrobial efficacy as compared to negative control. About 5.25% NaOCl was the most effective antimicrobial agent having statistically significant difference against plant extracts and negative control (saline). Conclusions: The methanolic extract of TC (Giloy), O. sanctum (Tulsi), and 5.25% NaOCl has considerable antimicrobial activity against polymicrobial dentinal biofilm of Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, and Candida albicans.

Keywords: Antimicrobial activity, herbal extracts, multispecies dentinal biofilm

How to cite this article:
Mistry KS, Sanghvi Z, Parmar G, Shah S. Comparative evaluation of antimicrobial activity of herbal extracts with 5.25% sodium hypochlorite against multispecies dentinal biofilm. Saudi Endod J 2016;6:71-6

How to cite this URL:
Mistry KS, Sanghvi Z, Parmar G, Shah S. Comparative evaluation of antimicrobial activity of herbal extracts with 5.25% sodium hypochlorite against multispecies dentinal biofilm. Saudi Endod J [serial online] 2016 [cited 2023 Feb 1];6:71-6. Available from: https://www.saudiendodj.com/text.asp?2016/6/2/71/180619

  Introduction Top

A potential role of microorganisms in the initiation and perpetuation of endodontic infections has long been established. [1],[2] A combined chemo-mechanical approach is followed in an attempt to clear off the microorganisms from the pulp space. Microcomputed tomography images of the canal walls have brought into light the fact that mechanical instrumentation alone is not sufficient enough to touch all areas of the canal wall. [3],[4] This makes the use of irrigation solutions and intracanal medicament mandatory in conjunction with mechanical instrumentation to achieve disinfection and healing. Keeping into account the microbial cause of pulpal and periapical diseases, an ideal irrigation solution should have good antibacterial activity.

According to current literature, sodium hypochlorite (NaOCl) is the most commonly used irrigation solution in the concentration range of 0.5-6%. It has an excellent antimicrobial and tissue dissolution properties. However, it can elicit a severe soft tissue inflammation along with extreme pain and/or swelling, if expressed out of the confines of the pulp space. [5] In addition, it lacks the capacity to completely remove the smear layer from instrumented canal walls. [6],[7]

In the search for a novel irrigation solution with a good biocompatibility and antimicrobial activity, researchers have explored a number of potential agents of natural origin. A few examples of the plant products that have shown a potential to be used as irrigation solution include Morinda citrifolia, [8],[9] triphala, and green tea polyphenols, [10] Curcuma longa, [11] garlic, [12] and many more.

India has a rich source of medicinal plants that are widely distributed throughout the country. Known in English as holy basil and botanically called Ocimum sanctum, Tulsi belongs to genus Ocimum and family Labiatae. Tulsi is an herbaceous sacred plant of Hindus and is worshiped in both homes and temples. It has made an important contribution to the field of science from ancient times as also to modern research due to its large number of medicinal properties. Almost every part of the plant such as stem, flower, seed, leaves, root, etc., have been shown to possess important medicinal properties including anti-inflammatory, [13] analgesic, [14],[15] and antibacterial activity. [16],[17]

Giloy or Guduchi or Amrita, botanically known as Tinospora cordifolia (TC) is a well-known rasayana plant and its rejuvenating property is well reported in ancient literature. TC is a large extensively spreading glabrous shrub belonging to the family Menispermaceae. The leaves, stem bark, and roots of TC have been proven effective against various ailments such as fever, jaundice, diarrhea, dysentery, general debility, cough, asthma, and skin diseases. [18] The plant has proven antibacterial activity and antifungal activity against various microorganisms. [19] To our knowledge, there is no study reported till date comparing the antimicrobial efficacy of the above mentioned herbal extracts with NaOCl, therefore, the purpose of this in vitro study was to compare the antimicrobial efficacy of Giloy and Tulsi and 5.25% NaOCl against 21-day-old multispecies biofilm formed on tooth substrate of extracted human teeth.

  Materials and methods Top

The microbial strains investigated in the study were obtained from Imtech-Chandigarh. The strains used are Enterococcus faecalis (MTCC 439), Streptococcus mutans (MTCC 497), Staphylococcus aureus (MTCC 737), and Candida albicans (MTCC 227).

Preparation of test solutions

The stem of Giloy and leaves of Tulsi were collected from the courtyard. Plant materials were washed with distilled water and dried under shade for 10-12 days. All the material was ground in an electric grinder to produce a powder. The powdered material was again dried in an oven at 40°C for 4 h and used for extraction. Accurately, weighed 50 g of powdered leaf sample was extracted with 500 ml methanol. This process was repeated till the marc was obtained and finally extracts were pooled and evaporated in rota-evaporator. The extracts were concentrated under partial vacuum at 80°C to dryness, leaving behind thick semi-solid residue. This extract was dissolved in dimethyl sulfoxide (DMSO) to get six different concentrations to be tested.

Agar diffusion test was carried out as a preliminary screening test, to check the antimicrobial efficacy of the test solutions on all the micro-organisms in their planktonic form. The micro-organisms were subcultured on specific media procured by Himedia Laboratory Pvt. Ltd., Mumbai, India and incubated aerobically at 37°C for 24 h. A total of six wells were made into a nutrient agar plate using sterile cork (6 mm in diameter) and inoculums containing 1 × 10 5 CFU/ml of bacteria were spread on the solid plate with the bacterial suspension. One hundred microliters of the working solution of different medicinal plant extract carrying different concentration of the medicine were filled in the wells with the help of micropipette. The plates were then incubated at 37°C for 24 h in an aerobic environment. After overnight incubation, the plates were observed for the zone of inhibition and the diameter of the inhibition zone in millimeters was measured using a scale. Each extract was tested three times and mean values were recorded. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined using micro-broth dilution method.

Preparation of tooth samples

A total of 31 single rooted, intact, noncarious human anterior teeth with fully formed apices were selected for the study. The teeth were cleaned to remove superficial debris, calculus and tissue tags, and were stored in normal saline to prevent dehydration before use. The access cavity was opened with the help of a round bur in an air rotor. The working length was determined as the negotiating file was just visible at the apical foramen. The root canals were instrumented using the crown-down technique with rotary instruments and canals were prepared to an apical size 30 by using ProTaper F3 (ProTaper, Dentsply Maillefer, Ballaigues, Switzerland). During the preparation 2 ml of 3% NaOCl (Vishal Dento care, Ahmedabad) was used as an irrigant between each instrument. The teeth were decoronated at cemento-enamel junction as well as splited vertically along the mid-sagittal plane into two halves.

The teeth sections were autoclaved at 121°C for a period of 20 min. The sterilized sections were stored in 2 ml Ependroff tubes. The vials containing tooth sections were inoculated with 2 ml of bacterial suspension of all four micro-organisms and incubated at 37°C for a period of 21 days to allow biofilm formation. The culture medium was replaced every alternate day to avoid nutrition depletion and accumulation of toxic end products. During every change of the culture medium, the samples were taken from each well with a sterile paper point, inoculated onto Mueller-Hinton agar plates, and incubated at 37°C for a period of 24 h to check for cell viability and purity of culture.

At the end of 21 days, two tooth sections were selected randomly and sent for scanning electron microscopic (Leo's 440i) evaluation for confirmation of polymicrobial biofilm growth [Figure 1]a and b. Remaining 60 sections were divided into four groups according to the medicaments to be tested. Normal saline was served as negative control. The four groups (15 each) were divided as follows:
Figure 1: (a, b) The presence of microorganisms on root canal wall after 21 days of growth

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  • Group 1: 5.25% NaOCl
  • Group 2: O. sanctum (75 mg/ml in 10% DMSO)
  • Group 3: TC (75 mg/ml in 10% DMSO)
  • Group 4: Normal saline.
All the samples were immersed in the 2 ml of test solutions according to their groups for 10 min. Then, the biofilm on the root canal portion was scraped off with the help of a scalpel to collect well attached, deeply seated as well as loosely attached superficial bacteria from the formed biofilm. The scrapings were inoculated on Muller-Hinton agar plates and incubated for 24 h at 37°C for qualitative analysis where n = 2 for each group. The quantitative analysis was performed by vortexing the scraped biofilm material of the treated tooth samples with sterile saline for a few minutes followed by serial dilution method and CFU counts, where n = 13 for each group. The mean and standard deviations were calculated.

The collected data was tabulated and analyzed using SPSS software version 17 (Inc., Chicago, USA) for ANOVA. Multiple comparisons were carried out using Bonferroni Post-hoc test to check the difference within the groups.

  Results Top

[Table 1] shows the number of bacteria remaining after the antimicrobial treatment of the tooth samples with different test solutions. 5% NaOCl proved to be the best as shown complete eradication of microorganisms in both the analysis methods. Plant extracts also showed inhibition of microbial growth and their effect was statistically more significant than saline group. However, they were less effective than NaOCl.
Table 1: Quantitative analysis of 3-week polymicrobial biofilm formed on tooth substrate for different groups

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  Discussion Top

Herbal products have been used since ancient times in folk medicine involving both eastern and western medical tradition. In dentistry phytomedicine has been used as anti-inflammatory, antibiotic, analgesic, sedative agents, and also as an endodontic irrigant. [20] The beneficial medicinal properties exerted by the plants are because of the presence of secondary metabolites which are responsible for antimicrobial effect also. Antimicrobial agents of plant origin have enormous therapeutic potential. They have been explored for their properties against a variety of infectious diseases and in a way they help to overcome the side effects and ineffectiveness associated with synthetic antimicrobials. [21] In recent years, there has been a tremendous increase in the studies related to the use of natural plant extract as an antimicrobial agent against microorganisms responsible for endodontic infections. All of these herbal extracts have shown considerable antimicrobial efficacy against selected endodontic pathogens.

In the present study, we have used methanolic extracts of O. sanctum (Tulsi), and TC (Giloy). These plants have been tested for their medicinal properties for the treatment of various human ailments. However, the studies proving their effectiveness as an endodontic irrigants are very rare. Hence, the purpose of this study was to compare these plant extracts for their antimicrobial effectiveness with NaOCl against common endodontic pathogens in the form of polymicrobial biofilm growth in an ex-vivo condition.

By definition, biofilms are matrix-enclosed microbial communities in which cells adhere to each other and/or to surfaces or interfaces. [22],[23] The concept of biofilm in endodontics was first introduced by Nair in 1987. [24] Later on in 1995, the scanning electron microscope examination of the root canal wall of extracted human maxillary and mandibular molars demonstrated the presence of cocci, rods, and yeast into the dentinal tubules. Penetration of bacteria into the dentinal tubules ranged from 10 to 150 μm. [25] The relation between biofilms and endodontic infections has recently been revised by Svens€ater and Bergenholtz. [26] After the introduction of the concept of biofilm in endodontic infections, it is more prudent to check the efficacy of antimicrobial agents against the bacteria in their biofilm mode of growth rather than in their planktonic counterpart. Numerous biofilm models have been proposed from time to time by various researchers to study the antimicrobial strategies of endodontic irrigants and intracanal medicaments. Most of the studies performed to evaluate the antimicrobial efficacy of various medicaments have been done either on planktonic cultures or monoculture biofilm of E. faecalis.[27],[28] However, it has been well established that endodontic infections are polymicrobial in nature and E. faecalis has been reported to coexist with other several taxa in root canal treated teeth. [29],[30] Therefore, in the current study, we have used a polymicrobial biofilm model to investigate the antimicrobial activity of plant extracts along with 5% NaOCl.

In dentinal biofilm model, we have done qualitative and quantitative analysis of the teeth samples treated with the different antimicrobial agents. The results show that 5% NaOCl was the most effective of all the tested agents in both the analysis. The qualitative analysis showed complete eradication of micro-organisms, whereas quantitative analysis did not show any growth of micro-organism after 24 h of incubation period. This is in accordance with the studies where NaOCl has been shown to be the most effective antibacterial agent against selected micro-organism in their planktonic form as well as in the form of mono species and polymicrobial biofilm. [31],[32],[33],[34]

In this study, the teeth samples were treated with the solution of plant extracts in the concentration of 75 mg/ml. The qualitative analysis has shown the growth of microorganisms after the treatment with both the herbal agents. Quantitative analysis has shown an average of 17.76 × 10 7 , 20.23 × 10 6 CFU/ml against Tulsi and Giloy, respectively. Giloy was the most promising among both the extracts as it has statistically significant difference in antimicrobial efficacy as compared to Tulsi and saline. The results of plant extracts on biofilm are promising as compared to negative control (saline) which has shown the growth of 36.38 ± 4.682 × 10 7 CFU/ml of micro-organisms.

A comparatively less pronounced effect of plant extracts can be explained by certain factors. The higher concentration of NaOCl (5%) may account for the better performance of this synthetic agent, as the antimicrobial potential of this agent is concentration dependent, and, therefore, the use of lower concentration of NaOCl and increasing the concentration of plant extracts may show comparable or even better performance of herbal extracts.

The microorganisms undergo certain genetic and phenotypic variation in biofilm growth as compared to their planktonic counterpart. These changes result in increased resistance of the microorganisms to topical antimicrobials. In previous study, [35] both the plant extracts have shown considerable antimicrobial effect against individual microorganisms in agar diffusion test, whereas in this study where the microorganisms have been grown in the form of polymicrobial biofilm the effectiveness of plant extracts has significantly reduced. This may be because of synergistic effect that exists among microorganisms in the biofilm mode of growth that increases the resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms. [36] Furthermore, the biofilm cells can be 100-1,000 fold more resistant to antimicrobial agents than their planktonic counterpart. [37]

The age of Biofilm also plays a vital role in the resistance of micro-organisms to antimicrobials. [38] In this study, we have allowed the growth of biofilm for a period of 21 days, which more closely simulates the in vivo condition. Mature biofilms may develop their own localized environments that dictate the metabolic activities of cells and better protect them against changes in the environment. Shen et al. [39] demonstrated that if young, nonmatured biofilms are used to assess the antibacterial efficacy of disinfecting agents, the results give a far too optimistic picture of their effect. It is, therefore, important to use mature biofilms when evaluating the antibacterial efficacy of endodontic irrigants. In addition to this, the time period this extracts are kept in contact with the biofilm may be a significant factor to be considered. In a similar kind of study, Gupta et al. [40] evaluated the antimicrobial efficacy of ethanolic extracts of three different plants including O. Sanctum and NaOCl on 3-week-old E. faecalis biofilm formed on cellulose nitrate membrane models as well as tooth model. Cinnamomum zeylanicum, Syzygium aromaticum, O. sanctum, and NaOCl showed complete bacterial inhibition in planktonic form after exposure of 30, 15, 35, and 1 min, respectively. In biofilm susceptibility assay on cellulose nitrate membrane, NaOCl was associated with complete bacterial inhibition after contact of 2 min, while 10% C. zeylanicum, 10% S. aromaticum, and 40% O. sanctum showed cessation of growth after 12, 12, and 24 h, respectively. Their results clearly show that the plant extracts considerably take a longer time to kill microorganisms than NaOCl. In our study, we have used only one-time interval of 10 min for all the test agents. If the plant extracts are allowed to act for a longer time on the biofilm microorganisms, they could perform better.

  Conclusion Top

Within the limitations of this study, it can be concluded that 5% NaOCl exhibits maximum antimicrobial efficacy against 21-day-old polymicrobial biofilm formed on tooth substrate. Methanolic extracts of O. sanctum (Tulsi) and TC (Giloy) also showed considerable effectiveness as an antimicrobial agent, however, they need to be tested against a wide number of endodontic pathogens before recommending as an endodontic irrigant.

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  [Figure 1]

  [Table 1]

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