PHYTOCHEMICAL STUDIES ON CASUARINA EQUISETIFOLIA AND INVESTIGATION OF ITS EFFECT AGAINST PATHOGENIC ORAL FLORAHTML Full Text
PATHOGENIC ORAL FLORA
Shafi Thompson T.*1, Alen Paiva1, Greeshma G. Mohan1, Jincy Das 1, Arya Suresh 1, K.K. Dil Baseer Sabith 1, J. Densingh 2
Department of Biotechnology, Science Campus 1, Nalanchira P.O, Thiruvanathapuram Dist-695 015, Kerala, India
Department of Microbial Technology, Malankara Catholic College 2, Kanyakumari Dist- 629 153, Tamil Nadu, India
This research was targeted to reveal the phytochemical activity of Casuarina equisetifolia against susceptible pathogenic oral bacteria. Antibacterial effect of the plant was examined by well diffusion method. Different plant parts were screened for the phytochemicals using various polar solvents like ethanol, acetone, methanol, chloroform and water. Studies disclosed that Casuarina equisetifolia encompasses a wide range of phytochemicals with anticariogenic activity. During phytochemical analysis by thin layer chromatography four compounds were obtained in the acetone solvent system with highest Rƒ value of 0.911.
Phytochemicals, Casuarina, Dental caries, Oral flora
INTRODUCTION: Dental caries is an infection, predominantly bacterial in origin, which permanently affects the hard organic material of the tooth by acid hydrolysis and demineralization. Tooth ache associated with it is due to the exposure of the nerve of the tooth through the passages of the dentinal tubule. Dental caries can precede increased sensitivity (Fig. 1) of the tooth and even life- threatening complications like Ludwig’s angina 1.
Worldwide, most children and an estimated ninety per cent of adults have experienced caries. In 1940’s the prevalence of dental caries in India was 55.5%, while in 1960’s it was reported to be 68%. Recent studies show an incidence ranging from 44-73%. It is the primary pathological cause of tooth loss in children. Conditions in the oral cavity may be indicative of systemic diseases such as osteoporosis, diabetes, or cancer 2. Numerous studies have also revealed that gum disease is associated with an increased risk of diabetes, cardiac disorder, and preterm birth.
FIG. 1: PROGRESSION OF DENTAL CARIES
Opportunistic oral flora like Streptococcus mutans, Pseudomonas aeruginosa, Streptococcus salivarius, Streptococcus viridians, Bacillus megaterium, Neisseria catarrhalis, Lactobacillus acidophilus, Actinomyces viscosus and Nocardia spp.3 are most susceptible for dental caries. Bacteria accumulate around the teeth and gums in an adhesive, creamy-coloured mass called plaque, which serves as a biofilm. Grooves on the occlusal surfaces of molar and premolar teeth provide microscopic retention sites for plaque bacteria. Fermentation of carbohydrates by these bacteria reduces the pH and the demineralisation of the tooth enamel instigate at pH 5.5 4.
Lack of appropriate oral hygiene, presence of remnant fermentable carbohydrates, presence of pathogens, reduced production of saliva 5, certain medications and excessive use of tobacco 6 are some of the causes of dental caries.
Use of antibiotics is the most prevalent method of treatment of dental caries. Increased use of antibiotics can lead to the formation of the antibiotic resistant mutants. Phytomedicines can prove to be a reliable alterative in this context. Various plant species are ascertained to be effective phytomedicines against the dental caries causing pathogens. Casuarina equisetifolia is a dicot angiosperm with immense potential to combat dental caries 7.
Traditionally the decoction formulated from the tannin containing astringent bark of Casuarina used against tooth ache. Routine chewing of the root is also effective to maintain dental hygiene. Being a plant with anticariogenic properties, isolation of phytochemicals from Casuarina equisetifolia can prove to be an effective alternative for the side effects causing antibiotic drugs.
MATERIALS AND METHODS:
Selection of Bacterial Strains: Six different species (Pseudomonas aeruginosa, Streptococcus salivarius, Streptococcus viridans, Streptococcus mutans, Bacillus megaterium, and Neisseria catarrhalis) of bacterial strains which can enhance the formation of dental caries were collected from Microbial Technology Laboratory, Malankara Catholic College, Mariagiri, Kaliakkivilai, Tamil Nadu.
Collection of Medicinal Plants: The different plant parts such as root, stem, leaves and bark of Casuarina equisetifolia (Fig. 2) were collected from Maruthuvarmalai region of Western Ghats of Kanyakumari district to study its antibacterial efficiency against the selected strains of microbes and also to isolate secondary metabolites.
FIGURE 2: CASUARINA EQUISETIFOLIA
Preparation of Plant Extracts:
Powdered samples were prepared by shade drying and grinding the different plant parts. 10 grams of the samples were procured in screw cap bottles and mixed with solvent systems like acetone, ethanol, chloroform, methanol and water for the extraction of phytochemicals. These screw cap bottles were preserved at 20oC for fifteen days.
Examination of Antibacterial Activity of Plant Extracts: Well diffusion method was employed for analyzing the antibacterial effect of extracts from the medicinal plant.
Well Diffusion Method: The bacterial isolates were effectively swabbed on the prepared Mueller-Hinton agar plates. After allowing the inoculum to dry at room temperature, wells of six mm diameter were bored on the medium. The extract was introduced (50 μl of a 100mg/ml concentration) into the three duplicate wells. The plates were allowed to stand at room temperature for one hour for the extract to diffuse into the agar and further incubated at 37oC for 16-18 hours. After incubation the plates were observed for the results.
Phytochemical Screening: Screening for secondary metabolites like steroids or terpenoids (Liebermann-Burchard Test), flavonoids (Shinoda’s Test), Carbohydrates (Molisch’s Test), saponins, tannins and phenolic compounds 8 was conducted to reveal the phytochemical properties of the plant.
Phytochemical Analysis (TLC): Silica gel G slurry 1: 2 (W/V) with thickness of 0.25 mm was prepared on a head glass plate. It was air dried for 15 to 30 min followed by hot treatment in an oven at 100oC for one to two hours. The samples were applied at one end (2.5 cm away from ends) of the gel plate with equal distance between them. The plates were dipped in solvent tanks to a depth of 1.5 cm from bottom and allowed to cover the solvent over the top. After that the plates were removed dried and processed for the identification of separated compounds (as colored spots) and the Rƒ values were calculated using the formula;
Distance (cm) moved by the solute (extract) from the origin
Distance (cm) moved by the solvent from the origin
* Rf - Retention Factor
RESULTS & DISCUSSION:
Antimicrobial effect of Casuarina equisetifolia extracts: Acetone extract of Casuarina equisetifolia showed inhibitory activity against Streptococcus mutans and Pseudomonas aeruginosa with 29mm and 26mm respectively. Ethanol extract showed activity against Streptococcus viridians (Fig:3) and Neisseria catarrhalis with a zone of inhibition of 16mm. Whereas the methanol extract showed activity against Bacillus megaterium with a zone of 16mm.Water extract gave activity only against Streptococcus salivarius with a zone of 9mm (Table 1).
FIG. 3: ZONE OF INHIBITION (WELL DIFFUSION METHOD)
TABLE 1: DIAMETER OF ZONE OF INHIBITION OF DIFFERENT EXTRACTS OF CASUARINA EQUISETIFOLIA AGAINST BACTERIA
|Bacteria||Acetone (mm)||Chloroform (mm)||Ethanol (mm)||Methanol (mm)||Water (mm)|
Phytochemical Screening: The chloroform, methanol and water extract of Casuarina equiestifolia revealed the presence of saponins. The test for steroids and terpenoids were positive in acetone, ethanol and methanol extracts. The test for flavonoids gave positive result only in ethanol extract. The test for phenolic compounds was positive in acetone and ethanol extracts. Whereas the test for tannins were positive in acetone and methanol extracts (Table 2).
ABLE 2: RESULT OF PHYTOCHEMICAL SCREENING OF CASUARINA EQUISETIFOLIA
|Experiment||Acetone Extract||Chloroform Extract||Ethanol Extract||Methanol Extract||Water Extract|
|Liebermann –Buchard test for steroids and terpenoids||Present||Absent||Present||Present||Absent|
|Shinodas test for flavanoids||Absent||Absent||Present||Absent||Absent|
|Molisch’s test for carbohydrates||Absent||Absent||Present||Present||Present|
|Test for Phenolic compounds||Present||Absent||Present||Absent||Absent|
|Test for saponins||Absent||Present||Absent||Present||Present|
|Test for tannins||Present||Absent||Absent||Present||Absent|
Phytochemical Analysis by Thin Layer Chromatography (TLC): Casuarina equisetifolia produced four compounds in acetone solvent system. Compound 4 showed highest Rƒ value of 0.911 and the compound 1 showed the least Rƒ value of 0.333. In water solvent system three compounds were obtained with highest Rƒ value of 0.900. Ethanol solvent system showed two spots with highest Rƒ value of 0.612. Chloroform solvent system produced two spots with highest Rƒ value of 0.691. Methanol solvent system gave two compounds with highest Rƒ value 0.780 (Table 3).
The ill effect of antibiotics includes the formation of multiple drug resistant mutants, emphasizing the necessity of alternative therapeutics. The use of plant compounds to treat infections is an age-old practice in a large part of the world, especially in developing countries, where there is dependence on traditional medicine for a variety of diseases. Interest in plants with antimicrobial properties has revived as a result of current problems associated with the use of antibiotics.
TABLE 3: RESULTS OF TLC DONE ON CASUARINA EQUISETIFOLIA
|Solvent system||No. of spots obtained||Rf value|
CONCLUSION: The research accomplishes that the plant extract from Casuarina equisetifolia has antibacterial action against microbes accountable for the tooth caries formation and thus helps avert the caries formation. The treatment involving the phytomedicines obtained from this plant can be employed as an alternative for the antibiotics that generates various side effects.
ACKNOWLEDGEMENTS: The authors are thankful to the authorities of Malankara Catholic College, Mariagiri, Tamilnadu and Science Campus, Nalanchira, Trivandrum for providing facilities and support.
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How to cite this article:
Thompson ST, Paiva A, Mohan GG, Das J, Suresh A, Sabith KKDB, Densingh J: Phytochemical studies on Casuarina equisetifolia and investigation of its effect against pathogenic oral flora. Int J Pharm Sci Res. 3(12); 4807-4810.
Shafi Thompson T.*, Alen Paiva, Greeshma G. Mohan, Jincy Das , Arya Suresh , K.K. Dil Baseer Sabith , J. Densingh
Chief Scientific Officer, Science Campus, Nalanchira, Thiruvananthapuram Dist-695 015, Kerala, India
18 August, 2012
07 November, 2012
24 November, 2012
01 December, 2012