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Table of Contents   
ORIGINAL RESEARCH  
Year : 2015  |  Volume : 26  |  Issue : 5  |  Page : 533-536
In vitro antibacterial activity of ethanolic extract of Morus alba leaf against periodontal pathogens


1 Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum, Karnataka, India
2 Dr. Prabhakar Kore Basic Science Research Centre, KLE University, Belgaum, Karnataka, India

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Date of Submission18-Mar-2015
Date of Decision10-Oct-2015
Date of Acceptance16-Nov-2015
Date of Web Publication17-Dec-2015
 

   Abstract 

Context: Antibiotic resistance is a major problem with inadvertent usage. Thus, there is a need to search for new antimicrobial agents of herbal origin to combat antibiotic resistance. One such plant is Morus alba which has a long history of medicinal use in traditional Chinese medicine.
Aim: To compare the antibacterial activity of ethanolic extract of M. alba leaves with chlorhexidine gluconate against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia.
Settings and Design: Experimental in vitro study.
Methodology: Crude extract from the leaves of M. alba were prepared by Soxhlet extraction method by using ethanol as a solvent. Minimum inhibitory concentration (MIC) of the extract was assessed against A. actinomycetemcomitans, P. gingivalis and T. forsythia, and compared with that of chlorhexidine gluconate by broth dilution method.
Results: P. gingivalis was the most sensitive organism against the M. alba extract with an MIC value of 1.95 mg/ml; while T. forsythia and P. gingivalis both were most sensitive organisms against chlorhexidine gluconate with MIC values of 0.00781 mg/ml.
Conclusion: M. alba possess good antibacterial activity against A. actinomycetemcomitans, P. gingivalis and T. forsythia and thus would be beneficial for the prevention and treatment of periodontal disease. However, chlorhexidine gluconate was found to be more effective when compared to M. alba.

Keywords: Antibacterial activity, herbal extract, Morus alba, periodontal pathogens

How to cite this article:
Gunjal S, Ankola AV, Bhat K. In vitro antibacterial activity of ethanolic extract of Morus alba leaf against periodontal pathogens. Indian J Dent Res 2015;26:533-6

How to cite this URL:
Gunjal S, Ankola AV, Bhat K. In vitro antibacterial activity of ethanolic extract of Morus alba leaf against periodontal pathogens. Indian J Dent Res [serial online] 2015 [cited 2020 Feb 24];26:533-6. Available from: http://www.ijdr.in/text.asp?2015/26/5/533/172082
Periodontal disease is one of the two major dental diseases that affect human populations worldwide at high prevalence rates. [1] Periodontitis is a chronic inflammatory disease of the tooth and supporting tissues with clinical signs of bone and connective tissue loss and is mediated by a combination of periodontal pathogens and host defense systems. [2] More than 700 different bacterial species colonize the oral cavity, but only a few of these are thought to be potential periodontal pathogens. [3] Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia are known as the main pathogens of periodontal disease and treatment of periodontal disease is associated with success in removing and reducing these microorganisms. [4],[5],[6] The most common treatment for periodontal disease is scaling and root planing (SRP) which has been confirmed as a gold standard treatment of periodontitis. [7] However, the limitations of SRP are the inability to access deeper areas of gingival sulcus which leads to early microbial re-colonization and recurrence of periodontitis. [8],[9] This has led researchers to investigate regarding local delivery of drugs which can reach the inaccessible areas of the gingival sulcus. One such antimicrobial agent is chlorhexidine.

Chlorhexidine is the most effective antiplaque agent until date with its mechanism of action being; decreasing pellicle formation, alteration of bacterial adhesion to the tooth surface and alteration of bacterial cell wall, ultimately leading to cell death. [10] The major advantage of chlorhexidine over other compounds lies in its substantivity, as it binds to soft and hard tissues in the mouth, enabling it to act over an extended period after application of a formulation. It is safe and has an inherent advantage over antibiotics by not producing resistant microorganisms. In the present study, chlorhexidine has been used as a positive control as it is considered the gold standard in dentistry and its disadvantages on long-term usage are staining of teeth and altered taste sensation. In rare cases, serious adverse effects like oral mucosal erosion, parotid swelling, enhanced supragingival calculus has been reported. [11]

Natural flora and fauna are known to play an important role in human life. India, as a tropical country is rich in medicinal plants, herbs and is known for its ancient traditional medicine used in the treatment of various ailments. The different parts of the plants such as roots, leaves, bark, and fruits have been used as an important source of traditional medicine since ages. In the recent years, most of the researchers' interest has shifted in evaluating the antimicrobial properties of different parts of the plants. A lot of research has been carried out on the utilization of medicinal plants in the treatment of a variety of ailments, especially in the last two to three decades. [12],[13],[14],[15],[16] As a result, the commercial use and exploitation of these herbal medicines has increased drastically. [17]

Morus alba is one such invaluable member of the plant kingdom belonging to the family Moraceae and the genus Morus. M. alba also known as the mulberry is a short-lived, fast-growing, small to medium-sized tree, native to northern china, and is widely cultivated elsewhere. It is extensively cultivated for leaf yield in sericulture. [18] Traditionally, mulberry fruit has been used as a medicinal agent to nourish blood, benefit the kidneys and treat weakness, fatigue, anemia and premature graying of hair. [19] Even the root bark has been traditionally used in Asian countries for medicinal purposes due to its anti-inflammatory, hypoglycemic, antibacterial activities, etc. [20] On exploration of literature, no study has been reported on the antibacterial activity against potential periodontal pathogens. Therefore, an attempt has been made to compare the antibacterial activity of ethanolic extract of M. alba leaf with chlorhexidine against A. actinomycetemcomitans, P. gingivalis and T. forsythia.


   Methodology Top


Prior to the start of the study, ethical approval was obtained from recognized ethical board. This study is a part of the ongoing clinical trial.

Materials

Chlorhexidine gluconate solution BP grade (Unilab Chemicals and Pharmaceuticals Pvt. Ltd., Mumbai), dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific India Pvt. Ltd., Mumbai), brain heart infusion (BHI) broth, horse serum, ethanol (Hi Media Laboratories Pvt. Ltd., Mumbai) were procured for the present study.

Collection and identification of plant material

The leaves of M. alba were collected during May (2014) from Belgaum district, Karnataka state, India. The plant was authenticated from the taxonomist in Regional Medical Research Centre, ICMR, Belgaum (Voucher specimen is RMRC-988).

Preparation and extraction of plant material

The fresh leaves were cut from the M. alba plant, washed thoroughly three times in water, shade dried at room temperature. After complete drying, the leaves were subjected to size reduction in a grinder and then sieved through sieve number 40 to get a coarse powder and stored in airtight glass jar. Coarse powder (70 g) was extracted with solvent (1000 ml of 99.9% ethanol) at room temperature using Soxhlet method of extraction. After the completion of Soxhlet process, the ethanol was evaporated using IKA Rotavapour evaporator at 40°C, leaving a small yield of extracted plant material (about 2-3 ml) in the glass bottom flask. The extract was further dried at room temperature until the remaining ethanol was completely evaporated. The yield obtained was 11.4 g. The extract was stored at 4°C until further microbial tests are carried out. [21]

Source of microorganisms

The standard strains of A. actinomycetemcomitans (ATCC 29523), P. gingivalis (ATCC 33277) and T. forsythia (ATCC 43037) were procured from Promochem, Bangalore.

Antibacterial activity

The lowest concentration of the extract/drug that will inhibit the growth of test microorganisms is the Minimum inhibitory concentration (MIC). For the bacteriological suspension, 1.5 × 10 8 CFU/ml was prepared by comparing the growth turbidity with 0.5 McFarland standards. BHI broth supplemented with horse serum was used as media for all the 3 microorganisms. For T. forsythia n-acetyl, muramic acid was added to the BHI broth supplemented with horse serum. The stock solution of the M. alba crude extract used for MIC was 500 mg in 1 ml of DMSO 50%. Nine dilutions (50%, 25%, 12.5%, 6.25%, 3.125, 1.562%, 0.781%, 0.390%, and 0.195%) were tested against selected organisms by broth dilution method as per Clinical and Laboratory Standards Institute guidelines. [22] Chlorhexidine being the standard in the periodontal regimen, MIC of chlorhexidine gluconate 2% was also performed to compare with that of M. alba crude extract. Nine dilutions of chlorhexidine gluconate were 2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.0312%, 0.0156%, and 0.00781%. Then the tubes were kept for incubation for 48 h at 37°C in the bacteriological incubator and observed for turbidity (P. gingivalis and T. forsythia being an obligate anaerobe was incubated in an anaerobic jar while A. actinomycetemcomitans being a facultative anaerobe was incubated in CO 2 dessicator). Turbidity was taken as an indication of growth, and the lowest concentration which remained clear was recorded as the relative MIC. [23] All the tests were performed in triplicate, and the mean value was taken.


   Results Top


In the present study, the antibacterial activity of ethanolic extract of M. alba leaves and chlorhexidine gluconate was determined against three most potential periodontal pathogens. The results are summarized in [Table 1]. Among the tested microorganisms, P. gingivalis was the most sensitive organism against the M. alba extract with an MIC value of 1.95 mg/ml; while T. forsythia and P. gingivalis were the most sensitive organisms against chlorhexidine gluconate with an MIC value of (0.00781 mg/ml). T. forsythia was least sensitive against M. alba extract (15.63 mg/ml), while for chlorhexidine gluconate, A. actinomycetemcomitans was least sensitive (0.031 mg/ml).
Table 1: MIC values of M. alba extract and chlorhexidine gluconate against three periodontal pathogens


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   Discussion Top


M. alba has been used in traditional oriental medicine, and almost all the parts of this plant like root, bark, stem, leaves and fruit have been used as medicine. Traditionally, mulberry is chewed in a toothache to avoid further destruction and cavitation of the tooth. M. alba has gained great attention in the recent years for its antioxidative and antidiabetic effects and is an important ingredient of herbal tea. [24] Park et al., flavanoid isolated from the root bark of M. alba has shown antibacterial activity against Streptococcus mutans, [25] 1-deoxynojirimycin, isolated from leaves of M. alba also showed anti-adherence activity against S. mutans. [26] The above evidence suggests the antibacterial activity of M. alba against dental caries. The root bark of M. alba has also been tested for antibacterial activity against periodontal microorganisms like P. gingivalis (W50) and A. actinomycetemcomitans (ATCC 33384) showing MIC value of 8 μg/ml and 10,008 μg/ml, respectively. [25] However, the leaves of M. alba has not been tested against the potential periodontal pathogens. To the best of our knowledge and thorough review of literature, this is the first report to provide evidence that the leaf extract of

M. alba has antibacterial activity against potential periodontal microorganisms.

M. alba plant is reported to contain phytoconstituents such as tannins, phytosterols, sitosterols, saponins, triterpenes, flavonoids, benzofuran derivatives, morusimic acid, anthocyanins, anthraquinones, glycosides, and oleic acid as the main active principle. [27],[28],[29],[30] While leaves contain tannins, triterpenes, sterols, bioflavonoids, coumarins, volatile oil, alkaloids, organic acids and amino acids, glycosides, and saponins. [31] Plants rich in tannin and phenolic compounds have shown to possess antimicrobial activity against a number of microorganisms. [32] It has also been reported that plants consisting of triterpene compounds effectively inhibits the growth of periodontopathic bacteria. [33] As the exact mechanism of action and chemical compounds responsible for the antibacterial activity are not known, the antibacterial activity of M. alba can be attributed majorly to the triterpenes, and also to the tannins present in the leaves. The results of the present study could not be compared with that of other studies as this is the first of its kind.

In the present study, the antimicrobial activity of leaves of M. alba was assessed against ATCC standard strains of potential periodontal pathogens such as A. actinomycetemcomitans, P. gingivalis, and T. forsythia, as these three bacteria were officially designated as etiological agents of periodontitis in 1996. [34] The presence of these bacteria have been shown to be a useful indicator of active disease and of increased risk of gingival attachment loss. [35]


   Conclusion Top


Periodontal disease is a multifactorial disease, caused by the interaction of host, agent, and environmental factors. It is important to remember that in vitro tests do not reflect the real condition found in periodontal pockets. They do not consider the above factors into consideration. From the present study, it can be concluded that M. alba possess antibacterial activity against periodontal pathogens, but chlorhexidine gluconate was found to be more effective. As the antibacterial activity is assessed against single microorganism, the exact interaction cannot be assessed by in vitro studies. Further in vivo studies should be conducted to prove its antibacterial activity against periodontal microorganisms without causing major local or systemic adverse effects. Indeed, further work should be done to elucidate main chemical compounds and their exact mechanism of action responsible for the antibacterial activity.

Acknowledgements

We would like to thank Dr. Kiran Kumar Hullatti, Associate Professor, Department of Pharmacognosy, KLE College of Pharmacy for his extended help in the Soxhlet extraction procedure.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Shilpa Gunjal
Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.172082

Clinical trial registration REF/2014/07/007209

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    Tables

  [Table 1]

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