|Year : 2020 | Volume
| Issue : 2 | Page : 96-100
Antibacterial Efficacy of the Extract of Sargassum Wightii Against Oral Pathogen − An In Vitro Study
KT Magesh M.D.S. , R Aravindhan, M Sathya Kumar, A Sivachandran
Department of Oral pathology and Microbiology, SRM Kattankulathur Dental College and Hospital, SRM Institute of Science and Technology, Kattankulathur, Kanchipuram, Tamil Nadu, India
|Date of Submission||04-May-2020|
|Date of Acceptance||18-Jul-2020|
|Date of Web Publication||16-Feb-2021|
Dr. K T Magesh
Department of Oral pathology and Microbiology, SRM Kattankulathur Dental College, Potheri 603203, Kattankulathur, Kanchipuram, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Introduction: Oral cavity contains a plethora of microorganisms. These microorganisms have shown both beneficence and maleficence. The most common disease affecting the oral cavity not only to the human being but also for the animals was dental caries that are proved to have multi-bacterial origin. Bacterial species such as Streptococcus mutans (S.mutans), S.sanguis and Lactobacillus fermentum plays a major role in the pathogenesis of this disease. In this study, the crude extract of sargassum wightii is tested for its antibacterial efficacy against the S.mutants. Materials and Methods: The brown seaweed S. wightii was collected from the Gulf of Mannar. The samples were cut into small pieces and shade dried for five days. After drying, the samples were grounded and stored. Stock solution was prepared using soxhlet apparatus. The organic extract of the seaweed S. wightii was assayed for the antimicrobial activity against S. mutans using agar diffusion method and the result was compared with the positive control sample (Ampicillin). Results: Stock solution of S.wightii used in this study was prepared in the following grades 500, 750, and 1000 μg. It was found that zone of inhibition increased steadily as the concentration of the extract increased. Highest inhibition zone of about 23 mm was found out at the concentration of 1000 μg, whereas the control showed 38 mm. Result showed the definite antibacterial activity of S.wightii even with the crude extract. Conclusion: Majority of the treatment and preventive approaches for dental caries are based on the use of synthetic antibiotics and chemicals. Their excessive use leads to alterations in the oral and intestinal microbiome, development of resistance and other undesirable side effects. The use of natural resources or products has proven to be copious source of biologically active compounds.
Keywords: Antibacterial, algae, dental caries, seaweed, S. wightii, tooth decay
|How to cite this article:|
Magesh K T, Aravindhan R, Kumar M S, Sivachandran A. Antibacterial Efficacy of the Extract of Sargassum Wightii Against Oral Pathogen − An In Vitro Study. J Orofac Sci 2020;12:96-100
|How to cite this URL:|
Magesh K T, Aravindhan R, Kumar M S, Sivachandran A. Antibacterial Efficacy of the Extract of Sargassum Wightii Against Oral Pathogen − An In Vitro Study. J Orofac Sci [serial online] 2020 [cited 2021 Mar 7];12:96-100. Available from: https://www.jofs.in/text.asp?2020/12/2/96/309591
| Introduction|| |
Oral health is the gateway for the general well being and relates to the overall health of an individual. Oral diseases continue to be a major health problem worldwide. Dental caries and diseases of periodontium are the most common of all the oral diseases since ancient times. The incidence of dental caries remains high among all the age groups in today’s era despite the fact of the vast knowledge of pathogenesis of this disease and technologies used in their detection and treatment in the dental practice.
Oral cavity is unique in its structure as it is made up of tissues of varying nature like oral mucosa, teeth, gums and the tongue, which are always kept moist by means of saliva. This composite nature of the tissue can be suitable habitats for microbial growth. Oral cavity shows extreme microbial diversity that consists of several hundreds of different species of microorganisms. Normal resident microbial flora is involved in the disease process also by means of multitude of factors. Virulent bacterial strains attached to the surface of the tooth and form bioflims subsequently matures to form dental plaque will create their own habitat, and microbial community interaction are considered to be the prime biological factors associated with the dental caries. More than 700 different microbial species contributes this complex and dynamic processes.
Of all the microbes, the streptococcus species are considered to be highly cariogenic and are significant for the initiation and the growth of the pathogenic plaque., Streptococcus genera shows extreme diversity that consists of 79 different species belonging to the order of lactobacillus and the family of streptococcaceae. Streptococci possess variety of virulence factors which is responsible for an array of diseases that ranges from non-life threatening conditions like dental caries to life-threatening diseases like necrotizing fasciitis and meningitis. Among all the types, Streptococcus mutans ns) is considered to be the etiological agent of dental caries.
S. mutans are proved to be the chief architect behind the initiation and progression of dental caries by the following mechanisms. It synthesizes an enzyme called as glucosyltransferases that assist in the adhesion of the bacteria to the tissue surface by the production of insoluble glucan from the sucrose. Attachment is followed by proliferation of primary and other microbial colonies, stabilization of the architecture by exopolysaccharides production and subsequently results in the maturation of the bio-film. Also it was proved that these bio-films have the capability to reduce the action of antimicrobial agents thereby resulting in the development of more resistant strains and leads to persistent disease process. S. mutans utilize the glycolytic pathway and result in the metabolism of glucose into lactic acid and causes a drop in the pH of the dental plaque. Also it develops a tolerance mechanism to protect itself from the acidic environment. Acidic nature of the plaque matrix leads to the demineralization of the inorganic component and result in the initiation of the dental caries. Many streptococci use cell to cell communication, often referred as “Quorum sensing” to regulate unnecessary over population of bacterial colonies and nutrient competition.
Meticulous personal oral hygiene can control the oral pathogen but cannot be eliminated completely. Many of the oral health care products are formulated to keep the dental plaque under control. Most of these anticariogenic products including the antimicrobials are synthetic and results in adverse side effects like vomiting, diarrhea, alteration in the oral and intestinal microflora, staining of the teeth and even linked to the oral cancer (ethanol in mouthwashes).
Keeping the side effects of synthetic products in mind, there arises global needs for the search of alternative treatment and prevention strategies. One such approach would be to verify the value of medicinal plants. Literature in the past showed promising results over the use of traditional plants and their products for the treatment of oral diseases., In a country like India, higher percentage of population use remedies of natural origin as a practice for curing illness since ancient times.
As more than 70% of the world’s surface is covered by oceans, marine organisms offer a wider range of natural products. Marine organism lives in a challenging environment and produces structurally novel and biologically active metabolites. Many of these marine products exhibit structural features that are found to be beneficial. Among marine organisms, marine algae offer structurally diverse bioactive compounds with various biological activities.
Being a plant, marine algae or seaweed can synthesize the three basic classes of pigments as a result of photosynthesis namely chlorophylls, carotenoids and phycobiliproteins, and thereby allowing classification of seaweed into Chlorophyceae (green algae), Phaeophyceae (brown algae) and Rhodophyceae (red algae). Different seaweed species have been extensively studied due to their varied biological activities like antimicrobial, anti-inflammatory, neuroprotective, anti tumor, antilipidic, antioxidant, and anticomplementary properties, antithrombotic activity, antacids and therapeutic potential agents in surgery.
Sargassum is large brown seaweed comprises of 400 species abundantly seen in warm temperate waters. Around 16 species are seen in Indian coastal waters. Sargassum wightii (S.wightii) found commonly in South Indian coastal belt as an inhabitant of shallow water and coral reefs. In this background the present study was conducted to evaluate the antibacterial capability of Sargassum wightii against oral bacterial pathogen, S. mutans.
| Materials and Methods|| |
Collection of seaweed
The brown seaweed Sargassum wightii was collected from the Gulf of Mannar, Tamilnadu, India by handpicking method at a depth of 10 m, and its identification was confirmed by a qualified botanist [Figure 1] and [Figure 2]. Surface of the seaweed was washed with tap water and then with distilled water and sent to the laboratory. The samples were cut into small pieces and shade dried for five days. After drying, the samples were grounded and stored for further analysis. Ethical approval for this study (Ethical clearance number 13/ISC/2019 dated 09.12.2019) was provided by the Ethical Committee SRM University, Potheri, on 09 December 2019.
|Figure 1 Macroscopic appearance of S. wightii upon collection by hand picking method.|
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|Figure 2 Zone of inhibition was determined by disc diffusion method on Muller Hinton agar (MHA) medium.|
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Preparation of crude extract
Using soxhlet apparatus, twenty-five grams of the sample was extracted with petroleum ether for ten hours. The extracts were condensed using rotary evaporator at 50°C under reduced pressure. After condensation the samples were reconstituted in their respective solvents to obtain a stock of 100 mg/ml and were stored in a refrigerator. The extracts were used for further analysis.
Preparation of inoculum
Pure bacterial cultures of S. mutans were purchased from the microbial type culture collection (MTCC), Chandigarh. Stock cultures were maintained at 4°C on Nutrient agar Slant. Active cultures for experiments were prepared by transferring a loop full of culture from the stock cultures into the test tubes containing nutrient broth, that was incubated at 24 hrs at 37°C.
Antibacterial activity assay
Antibacterial activity of extracts was determined by disc diffusion method on Muller Hinton agar (MHA) medium. Muller Hinton Agar (MHA) medium is poured into the petriplate. After the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the bacterial suspension. Ampicillin, a broad spectrum antibiotic was used as a control in this study as it is effective against both Gram-positive and Gram-negative bacteria.
The disc was placed in MHA plates and 20 μl of sample (Concentration: 1000μg, 750 μg and 500 μg) and control (Ampicillin: 1mg/ml) were placed in the disc. The plates were incubated at 37°C for 24 hrs. Then the antimicrobial activity was determined by measuring the diameter of zone of inhibition.,
| Results|| |
The organic extract of the seaweed S. wightii was assayed for the antimicrobial activity against S.mutans using agar diffusion method and the result were compared with the positive control sample (Ampicillin). Zone of inhibition indicates the antibacterial activity. Among the concentration used in our study (500, 750, and 1000 μg), it was found that zone of inhibition increased steadily as the concentration of the extract increased. Highest inhibition zone of about 23 mm was found out at the concentration of 1000μg, whereas the control showed 38 mm. Result showed the definite antibacterial activity of S.wightii even with the crude extract [Table 1] and [Figure 1].
|Table 1 Inhibition halo diameter of S. wightii extract at various concentration and control against S. mutans|
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| Discussion|| |
Marine algae or seaweeds are group of organisms with little tissue differentiation that grow along the seashores. Seaweeds are available in different forms and sizes, and ranges from unicellular microscopic organisms (microalgae) to multi cellular of great size (macro algae). Humans are closely associated with these algae in numerous ways as a source of food, feed, fertilizer, medicine and as phycocolloids. Literature shows that these seaweeds contain higher concentrations of more than 60 trace elements when compared with terrestrial plants.
Crude extracts of algae and their components exhibit medicinal values like antimicrobial, anticoagulant, antiviral, antineoplastic, antioxidant, and anti-inflammatory properties. Sargassum wightii, a brown marine algae that grows up to 20-30 cm in length, dark-brown in color and grows in intertidal and subtidal habitats.
The efficacy of S.wightii on oral pathogen which is responsible for dental caries has not been explored so far. The study is first of its kind to test the antimicrobial activity of crude extracts of S. wightii against S.mutans. In this pilot study, using the crude petroleum ether extracts of S. wightii showed significant antibacterial effect over S. mutans and had maximum inhibition zone at the concentration of 1000 μg.
In a study conducted by Shanmugapriya et al., the extracts of different species belonging to green, red and brown algae that includes S. wightii showed antimicrobial activities against multi-resistant strains of bacteria, with significant effects over the gram-negative bacteria than gram-positive bacteria. Also in the study the author has stated that the factors like extraction methods, solvent used, seasonal variations and drying of the seaweeds can alter the results by the elimination of certain active ingredients.
In a study by Choi et al., the extract of a seaweed called Enteromorpha Linza was tested against Gram-negative pathogens, such as Prevotella intermedia nd Porphyromonas gingivalis, the bacteria responsible for periodontitis. The study displayed antimicrobial activity against P. intermedia and P. gingivalis without any undesirable effects at a moderate dose and in a mouth rinse, the efficacy is similar to commercially available Listerine® against gingivitis.In another study, Kim et al. proved the antimicrobial activity of extracts of L. japonica against oral microbes. Venugopal et al. showed the antimicrobial property of S. wightii against B.cereus using four different solvents, and among those petroleum ether extract showed highest efficacy. Marudhupandi et al. showed that a substance called fucoidan, a polysaccharide seen in the cell wall of S. wightii showed the antimicrobial activity against selected human pathogens. Bibiana et al. showed the antibacterial and antifungal efficacy of S.wightii using four different solvents and the biochemical analysis showed significant amount of carbohydrates in it.
In our study, the concentration used were (500, 750, and 1000 μg), which showed that zone of inhibition increased steadily as the concentration of the extract increased. Highest inhibition zone of about 23 mm was found out at the concentration of 1000 μg, which shows the definite antibacterial activity of S. wightii even with the crude extract. Therefore, it is evident that seaweed could provide promising bioactive metabolites that can be used in the treatment of various oral diseases including the dental caries.
Limitations of the Present Study
Even though S.mutans proved to be the chief organism for intiation and progression of dental caries, efficacy of S.wightii on other oral microrganisms should also be evaluated and also the antimicrobial property of this seaweed should be evaluated and subsequently should be compared with different extracting agents.
| Conclusion|| |
The use of the medicinal plants for curing various diseases has been well documented in history of all civilizations. Before onset of synthetic antibiotics era, man was completely dependent on natural resources for prevention and treatment of diseases. Upon understanding the knowledge of its nature and their function of active components, plants extracts and its purified phytochemicals can be developed into agents which can be used as a preventive or treatment therapies for oral diseases such as dental caries. There has been constant effort and researches on interception and correction of the dental caries over a century now and today our horizon has broadened and our approach should be toward preventing the disease process rather than to correct it. The waters of the Gulf of Mannar of the Indian subcontinent have abundant resources of brown seaweed belonging to S. wightii, which have proven to produce effective antimicrobial properties due to their adverse habitat. This is a pilot study where an attempt was made to check the efficacy of S. wightii over S.mutants. Further studies on this seaweed with large number of extraction techniques and other oral microorganisms should be performed to validate the hypothesis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]