INTRODUCTION:

A total of 700 species of microorganisms have been identified as belonging to the oral microbiota¹.However the characteristics of the oral cavity does not provide for the colonization of all organisms. Only a set of specific microbes thrive within the oral cavity^2,3. Of these microorganisms only a select few have been identified as pathogens. Dental caries is the most common disease affecting the oral cavity^4,5. The Streptococcus mutans, Lactobacillus fermentum and S. sanguis have been identified as the most prevalent acidogenic bacterial species causing dental caries^6,7,8,9,10. Plaque/biofilm is considered the etiologic agent for the development of periodontal diseases.

A biofilm is defined as bacterial aggregates, usually existing as closely associated communities, which adhere to assorted natural or artificial surfaces, usually in aqueous environment that contains a sufficient concentration of nutrients to sustain the metabolic needs of the microbiota¹¹.

The biofilm is multibacterial however the presence of certain specific microorganisms such as Porphyromonas gingivalis, Actinomycetes and Aggregatibactor actinomycetemcomitans causes the alteration of the host immune system causing periodontal breakdown and destruction of alveolar housing¹².

Algae is a member of the Kingdom Protista. G.W. Prescott Classification 1969 includes: Chlorophyta(green algae), Euglenophyta (euglenoids), Chrysophyta(golden-brown algae), Pyrrophyta (fire algae), Rhodophyta (red algae), Paeophyta (brown algae), Cyanophyta, Cryptophyta and Chloromonadophyta¹³. The Paeophyta or brown algae are present in marine environments. They are about 1500 species thriving in cool waters. These photosynthetic plants have pigments such as chlorophylls a and c with the accessory pigments such as xanthophylls, carotenoids and fucoxanthin. Fucoxanthin is a brown-colored pigment which gives these algae their characteristic dark color. The antibacterial property of the marine algae has been proved in various studies^14,15. Thus the study employs everything in its potential to harness the best from the nature. The extraction from the seaweeds and the unconventional use of these algae are immaterial compared to the varied benefits that could be achieved.

OBJECTIVES:

· To extract antibacterial compounds from Turbinaria conoides.

· To determine the bactericidal property of Turbinaria conoides against Streptococcus mutans

· To determine the bactericidal property of Turbinaria conoides against Actinomycetes

· To determine the minimal inhibitory concentration of the extract

· To compare the antibacterial potential exhibited by Turbinaria conoides between oral microorganisms causing dental caries and periodontal pathogens.

METHODOLOGY:

Selection of test plant:

Of the brown seaweeds in the Tamil Nadu coastal region Turbinaria conoides is the one with the most phenolic content¹⁶. This property of the seaweed can be exploited for deriving its antibacterial potential. Turbinaria conoides has shown significant antibacterial action against common human pathogens such as Staphylococcus aureus, Salmonella typhi and Escherichia coli^17,18. Also this algae is present in abundance in the coastal areas in and around Rameshwaram. Classification of this species is as follows: Biota, Chromista (Kingdom), Harosa (Subkingdom), Heterokonta (Infrakingdom), Ochrophyta (Phylum), Phaeista (Subphylum), Limnista (Infraphylum), Fucistia (Superclass), Phaeophyceae (Class), Fucophycidae (Subclass), Fucales (Order), Sargassaceae (Family), Turbinaria (Genus), Turbinaria conoides (Species)¹⁹.

Collection of the test plant:

The brown seaweed Turbinaria conoides is collected from the intertidal regions of Mandapam (9°16'53.7"N 79°07'01.3"E) in the Gulf of Mannar, Ramanathapuram, Tamil Nadu, India in the month of December 2019. All dead and necrosed parts of the algae are removed and the seaweed is washed with water.

Preparation of the test sample:

The brown algae is washed thoroughly using clean water. The algae are laid over a clean newspaper over a woven basket and shade dried. There is a significant weight and volume loss appreciated after drying for over a few days. The algae are cut into smaller pieces and dried to fasten the process. The dried alga is inspected for presence of any dust or extraneous substances and is then blended in a blender into fine powder ready for extraction.

Extract preparation:

Petroleum ether solvent has been most efficient in producing antibacterial compounds from the T. conoides in certain preliminary studies.¹⁸ The test plant powder is extracted using petroleum ether using Soxhlet extractor²⁰. The extracted concentrates are titrated to various concentrations based on the phenol content.

Test procedure:

Agar disc diffusion method:

Antibacterial effect of the extracts was determined by disc diffusion method on Muller Hinton agar (MHA) medium. Muller Hinton Agar (MHA) medium is solidified on petri plates, the inoculums are spread on the solid plates with sterile swab moistened with the bacterial suspension. The disc were placed in MHA plates and 20µl of sample (Concentration: 1000µg, 750µg and 500µg) were placed in the disc. The plates were incubated at 37ºC for 24 hrs. Then the antimicrobial activity was determined by measuring the diameter of zone of inhibition^21,22.

Determination of minimum inhibitory concentration:

Preparation of inoculum:

1. A direct broth suspension of isolated colonies is made from an 18- to 24-hour agar plate.

2. The suspension is kept at a turbidity equivalent to a 0.5 McFarland turbidity standard.

3. Every tube contains approximately 5 x 10⁵ CFU/mL.

4. The 0.5 McFarland suspension is diluted to 1:150, resulting in a tube containing approximately 1 x 10⁶ CFU/mL.

The determination of Minimum Inhibitory concentration (MIC) for the sample through the method of successive dilution.

1. 1ml of sample (1mg/ml) is added to the LB broth tube.

2. Serial dilution was carried out until tube 6.

3. 100μl of bacterial culture gets incorporated in each tube and incubated at 37°C for 24 hrs.

4. MIC is the concentration at which the highest dilution shows no bacterial growth

5. At 450nm the optical absorbance is tested using a spectrometer.

RESULTS:

The agar disc diffusion after 24 hours reveals an inhibitory zone surrounding the disc which is identified as a zone of no bacterial growth. Each disc was laden with 20µl of sample but having a varying concentration of 1000µg, 750µg, 500µg. The antibiotic (ampicillin) is also placed as a positive control²³.

The sample with a concentration of 500µg reveals an inhibitory zone of 6mm while that with 750 and 1000µg reveals an inhibitory zone of 9mm. While the positive control ampicillin reveals a zone of 28mm on the Streptococcus mutans agar plate. (Table 1) (Figure 1)

On the Actinomyces sp. the sample reveals an inhibitory zone of 12mm at 1000µg, 10mm at 750µg and 9mm at 500µg. The control ampicillin reveals an inhibitory zone of 29mm. (Table 1) (Figure 2)

Table 1: Zone of Inhibition against Streptococcus and Actinomycetes

Organisms	Zone of Inhibition (mm)			Antibiotic (1mg/ml)
	Sample (1mg/ml)
	1000	750	500
Streptococcus sp	9	9	6	28
Actinomycetes sp	12	10	9	29

Figure 1: Zone of inhibition against Streptococcus mutans

Figure 2: Zone of inhibition against Actinomycetes

The minimum inhibitory concentration was identified using the serial dilution method. The extract proved to possess inhibitory effect at a dilution of 1000µg showing the least optical absorbance as 0.116 for Streptococcus and 0.123 for Actinomycetes (Table 2)

Table 2: MIC values for Streptococcus mutans and Actinomycetes

S. No	Dilutions (µg/ml)	Absorbance (O.D)
S. No	Dilutions (µg/ml)	*Streptococcus mutans*	*Actinomycetes*
1	1000	0.116	0.123
2	500	0.693	0.916
3	250	0.863	1.252
4	125	1.156	1.384
5	62.5	1.471	1.485
6	31.2	1.781	2.432

DISCUSSION:

It is no hidden fact the distress the oral diseases like dental caries and gingivitis pose to the society. The microorganisms that cause these dental issues could lay the seeds for more serious medical conditions and life-threatening ailments. The use of antibiotics has given power to dentist to alleviate patients from sufferings. However many of these microbes have developed antibiotic resistance of various degree. Dental professionals use antibiotics for both prophylactic and therapeutic purposes. Literature often blames the dental surgeons of inappropriate use of antimicrobial drugs leading to resistance. Antibiotic resistance puts the society in grave danger. Hence care should be taken to develop more natural remedies.^10,24,25,26

Marine algae are known to possess various unscathed molecular compounds and chemicals which would be of much importance in the pharmaceutical field. The correlation between the antibacterial activity of the crude algal extracts and the presence of phenolic compounds has been established in various literature.^{14,15,25,26,27} The effect of phenols against the microbes is in way of inhibiting their metabolism and growth. On the basis of concentration the microorganisms can be inhibited from their growth and reproduction. The crude extracts from marine algae have shown to possess these phenolic compounds.^28,29

Turbinaria conoides has shown high phenolic activity as compared to other marine brown algae¹⁶. The results show that the brown algae show a clear-cut antibacterial activity against the oral pathogens. The crude extract from petroleum ether has shown inhibition of the bacterial growth. The inhibition zones procured by the extract seems to increase in size with an increase in concentration. This implies an increase in the phenolic content in the test extract. Also with a 12mm inhibition zone when used against Actinomycetes reveals a greater efficiency against the anaerobes which are the usual causative organisms of root caries and the periodontal destruction.^30,31

CONCLUSION:

The antibacterial nature of the Turbinaria conoides has been explored in the study. The marine brown algae shows inhibition of the microbial growth of both aerobes and anaerobic organisms. The antibacterial effect however is more pronounced among the Actinomycetes species showing a 12mm zone of inhibition as compared to Streptcoccus mutans which shows a 9mm zone of inhibition. The results prove that the extracts from Turbinaria conoides shows promising results for use against the oral pathogens causing gingivitis and root caries. The study reflects the need for newer antimicrobials due to the deafening rise of drug resistant microbes. The study is contemplated to provide the society a revival into the healing from nature. The seaweeds are present in abundance and provide a cost-effective source for extraction of antibacterial compounds. With further research on the algae the full potential of the plant can be harnessed.

REFERENCE:

1. Faran Ali SM. Tanwir F. Oral microbial habitat a dynamic entity. Journal of Oral Biology and Craniofacial Research. 2012; 2(3):181-187. doi.10.1016/j.jobcr.2012.07.001.

2. Marsh PD. Martin MV. Mouth as a microbial habitat. In: Lewis M.A., editor. Oral Microbiology Textbook. Churchill Livingstone Elsevier; Edinburgh, London, New York, Oxford: 2009. p. 8–23.

3. Jyothi S. Subha M. Oral Health Awareness among the General Population in Chennai. Research Journal of Pharmacy and Technology. 2017; 10(11):3873-3876. doi.10.5958/0974-360X.2017.00703.X

4. Nilima T. Madhura T. Pranali S. Review on Role of Herbs in Management of Oral diseases. Asian Journal of Pharmaceutical Research. 2020; 10(4):321-326. doi.10.5958/2231-5691.2020.00055.6

5. Suganya M. Soondron Y. Rao MUS. Oral and Dietary Habits, and Immunological and Clinical Impacts on the Incidence of Dental Caries – A Review. Research Journal of Pharmacy and Technology. 2014; 7(11):1348-1353.

6. Strużycka I. The Oral Microbiome in Dental Caries. Polish Journal of Microbiology. 2014; 63(2):127-135.

7. Caufield P. Schön C. Saraithong P. Li Y. Argimón S Oral Lactobacilli and Dental Caries. Journal of Dental Research. 2015; 94(9_suppl):110S-118S. doi.10.1177/0022034515576052.

8. Kuzhalvaimozhi P. Geetha RV. Antibacterial Property of Different types of Tea Extracts against Cariogenic Bacteria, Streptococcus mutans. Research Journal of Pharmacy and Technology. 2016; 9(10):1732-1733. doi.10.5958/0974-360X.2016.00348.6

9. Christy S. Nivedhitha MS. Antimicrobial Efficacy of Azadirachta indica against Streptococcus mutans– An In vitro Study. Asian Journal of Pharmacy and Technology. 2019; 9(3):149-153. doi.10.5958/2231-5713.2019.00025.4

10. Murthykumar K. The Impact of mushroom, chicory extracts, fresh fruits, xylitol containing chewing gums and milk products on dental caries-A Review. Research Journal of Pharmacy and Technology. 7(2): Feb. 2014; p.266-8.

11. Egert M. Simmering R. The Microbiota of the Human Skin. Advances in Experimental Medicine and Biology. 2016; 902:61-81. doi.10.1007/978-3-319-31248-4_5.

12. Sochalska M. Potempa J. Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis. Frontiers in Cellular and Infection Microbiology. 2017; 7:197. doi.10.3389/fcimb.2017.00197

13. Baweja P. Sahoo D. Classification of Algae. The Algae World. 2015:31-55. doi.10.1007/978-94-017-7321-8.

14. Magesh KT. Aravindhan R. Kumar M S. Sivachandran A. Antibacterial Efficacy of the Extract of Sargassum Wightii Against Oral Pathogen − An In Vitro Study. Journal of Orofacial Sciences. 2020; 12:96-100. doi.10.4103/jofs.jofs_80_20

15. Sangeetha J. Gayathri S. Rajeshkumar S. Antimicrobial assessment of marine brown algae Sargassum whitti against UTI pathogens and its phytochemical analysis. Research Journal of Pharmacy and Technology. 2017; 10(6):1905-1910. doi.10.5958/0974-360X.2017.00334.1

16. Chakraborty K. Praveen N. Vijayan K. Rao G. Evaluation of phenolic contents and antioxidant activities of brown seaweeds belonging to Turbinaria spp. (Phaeophyta, Sargassaceae) collected from Gulf of Mannar. Asian Pacific Journal of Tropical Biomedicine. 2013; 3(1):8-16. doi.10.1016/S2221-1691(13)60016-7.

17. Ponnan A. Ramu K. Marudhamuthu M. Marimuthu R. Siva K. Kadarkarai M. Antibacterial, antioxidant and anticancer properties of Turbinaria conoides (J. Agardh) Kuetz. Clinical Phytoscience. 2017; 3(1). doi.org/10.1186/s40816-017-0042-y.

18. Sridharan MC. Dhamodharan R. Antibacterial activity of marine brown alga Turbinaria conoides. Journal of Chemical and Pharmaceutical Research. 2012. 4(4):2292-2294.

19. Rohfritsch. Audrey P. Claude S. Valérie B. Francois. Molecular and morphological relationships between two closely related species, Turbinaria ornata and T. conoides (Sargassaceae, Phaeophyceae). Biochemical Systematics and Ecology. 2007; 3(5):91-98. doi.10.1016/j.bse.2006.09.002.

20. Vijayabaskar P. Shiyamala V. Antibacterial Activities of Brown Marine Algae (Sargassum wightii and Turbinaria ornata) from the Gulf of Mannar Biosphere Reserve. Advances in Biological Research. 2011; 5(2):99-102.

21. Bauer AW. Kirby WM. Sherris JC. Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 1966; 45(4):493-496.

22. Syed J. Dileep N. Rakesh KN. Prashith KTR. Anticaries Activity of Selected Plants against Clinical Isolates of Streptococcus mutans. Asian Journal of Pharmacy and Technology. 2013; 3(3):107-108.

23. Chandrasekaran S. Geetha RV. Antibacterial Activity of the Three Essential Oils on Oral Pathogens- An In-vitro Study. Research Journal of Pharmacy and Technology. 2014; 7(10):1128-1129.

24. Haque M. Sartelli M. Haque SZ. Dental Infection and Resistance-Global Health Consequences. Dentistry Journal (Basel). 2019;7(1):22. doi.10.3390/dj7010022.

25. Marra F. George D. Chong M. Sutherland S. Patrick DM. Antibiotic prescribing by dentists has increased: Why? Journal of the American Dental Association. 2016; 147(5):320–327. doi.10.1016/j.adaj.2015.12.014

26. Vaishali M. Geetha RV. Antibacterial activity of Orange peel oil on Streptococcus mutans and Enterococcus - An In -vitro study. Research Journal of Pharmacy and Technology. 2018; 11(2):513-514. doi.10.5958/0974-360X.2018.00094.X

27. Kavya R. Glycyrrhiza glabra- An Ayurvedic Medicine in Dentistry. Research Journal of Pharmacy and Technology. 2014;7(7):821-822.

28. Pandian V. Shiyamala V. Antibacterial Activities of Brown Marine Algae (Sargassum wightii and Turbinaria ornata) from the Gulf of Mannar Biosphere Reserve. Advances in Biological Research. 2011; 5(2):99-102.

29. Heipieper HJ. Keweloh H. Rehm HJ. Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli. Applied and Environmental Microbiology. 1991; 57(4):1213–1217. doi.10.1128/aem.57.4.1213-1217.1991.

30. Loesche WJ. Grossman NS. Periodontal Disease as a Specific, albeit Chronic, Infection: Diagnosis and Treatment. Clinical Microbiology Reviews. 2001;14(4):727–752. doi.10.1128/CMR.14.4.727-752.2001

31. Dame-Teixeira N. Parolo CC. Maltz M. Tugnait A. Devine D. Do T. Actinomyces spp. gene expression in root caries lesions. Journal of Oral Microbiology. 2016; 8:32383. doi.10.3402/jom.v8.32383.

Received on 04.03.2021 Modified on 18.05.2021

Research J. Pharm. and Tech. 2022; 15(5):2236-2239.

DOI: 10.52711/0974-360X.2022.00371