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Dept. of Botany, S.B. College, Changanacherry - 1, Kerala.
Dental caries is an infectious disease which damage the structures of teeth.Antibiogram studies were an alternative method for preventing enzymatic degradation of carbohydrates toacid for the prevention of bacterial growth and metabolism. Samples were collected fromtwenty five patients, severely affected by dental caries.They belonged to Villavancode Talukof Kanyakumari district,Tamil Nadu.Numerous bacterial isolates were obtained from thetwenty five samples analysed.Among this,antibiogram study of fifteen isolates were carriedout and five of them showed resistance and moderate sensitivity to different antibiotics(Ciprofloxacin, Ampicillin, Ceixime,Amoxycilin,Cephalexin and Erythromycin).The fiveisolates were then characterised based on their macroscopic, microscopic,biochemical andphysiological characteristics.An alternative to these drugs, antibacterial effect of leaves offive medicinal plants Piper betle, Areca catechu, Eucalyptus globules, Zingiber officinaleand Azadirachta indica were carried out in different solvent systems(acetone, ethanol,chloroform, methanol and water)by disc diffusion method. Among the five plants extracts ,three of them( Piper betle, Eucalyptus globules, Azadirachta indica) were more effective inproducing antibacterial property.These plants were then screened for the presence or absenceof certain phytochemicals such as saponins, steroids and terpenoids ,flavonoids ,carbohydratesand tannins.
Key words: Dental Caries, Antibiogram, Sensitivity, Phytochemicals.
Dental caries is a chronic endogenous infection caused by the normal oral commensal flora. The carious lesion is the result of demineralization of enamel and later of dentine byacids produced by plaque microorganisms as they metabolize dietary carbohydrates. However,the initial process of enamel demineralization is usually followed by remineralization, andcavitation occurs when the former process overtakes the latter. Once the surface layer ofenamel has been lost, the infection invariably progresses to dentine with the pulp becomingfirstly inflamed and then necrotic.
Oral flora comprises a diverse group of organisms and includes bacteria, fungi, mycoplasmas, protozoa and possibly a viral flora which may persist from time to time. Amongthis bacteria are the predominant group of organisms (Bowden,1990).
The bacteria primarily responsible for dental decay in man are Streptococcus mutans. Streptococci belong to four main species groups: mutan, salivarius, anginosus and mitis(Bowden, 1998). In addition to Streptococcis mutans, Lactobacillus acidophilus bacteriaprobably also pay a minor role in acid production in the plaque.
The stickiness of the plaque is caused by dextran, which is produced by the fermention of dietary sucrose by Streptococcus mutans. The plaque bacteria, particularly Streptococcusmutans, act on dietary fructose to produce lactic acid, which causes enamel decalcification(at below or above 5.5pH). The plaque and dietary carbohydrates are in the initiation ofenamel caries{Clark (1871, 1879), Tomes 1873 and Magitaol (1878)} The streptococcal organisms studied by Keyes and Fitzgerald were actually rodent- strains of Streptococcus mutans, a very powerful and efficient cariogenic microorganisms. Smutans is pandemic in its geographic distribution and is particularly prevalent in societieswhere sucrose consumption is high.
Certain cariogenic and highly acidogenic strains of streptococci, especially S. mutans have the ability to metabolize dietary sucrose and synthesize glucan by cell-surface andextracellular glucosyl transferase. This enzyme is considered to be of special importance inthe establishment of S. mutans in the dental plaque.
Dental caries affects persons of all races, countries and economic strata and can occur at any age and in either sex. A diet rich in fermentable carbohydrates promote caries (Malherbe,1944). Whereas a diet consisting of raw coarse food tends to reduce the incidence of caries.
The composition of teeth also has some bearing on incidence. Caries immune teeth havebeen shown to posses a higher fluoride content than caries susceptible teeth. The salivacontains an immunoglobulin called the secretary immunoglobulin A (IgA). Its main functionis to protect against viruses that invade the respiratory and intestinal tract.
Dental caries is an infectious disease which damage the structures of teeth. Tooth decay or cavities are consequences of caries. If left untreated, the disease can lead to pain toothloss, infection, and , in severe cases, death of the tooth [abc Dental cavities, 2006].
In a healthy mouth the pH is around 6.2 to 7.0. A pH of 7 is neutral. Thus a mouth with problem starts when the pH is less than 5.5. The tooth is now in an acid environment, andstarts to demineralise (lose calcium and minerals from the enamel). As the enamel loses it sminerals, it starts to break down. This is the start of a cavity (Freeth, 2007).Sticky foods aremore harmful than nonsticky foods because they remain on the surface of the teeth.
Tooth decay is caused by certain types of acid-producing bacteria (specifically Lactobacillus and Streptococcus mutans) which cause damage in the presence of fermentablecarbohydrates such as sucrose, fructose, and glucose.
Oral Streptococci, which are major members of oral flora, frequently cause bacteraemia and infective endocarditis (IE) (Douglas et al,1993). Streptococcus mutans, a major cause ofdental caries, has occasionally been isolated from the blood of patients with IE ( Vose et al.,1987; Ullman et al., 1988; Gauduchon et al., 2001).
The biochemical, serological and genetic characterization of four Streptococcus mutans strains identified from a total of 522 Streptococcal isolates from the blood of patients with variousinfectious diseases, including IE (141 isolates from 84 cases), sepsis (85 isolates from 54 cases)and bacteraemia following tooth extraction (15 isolates from 12 cases) (Fujiwara et al., 2001).
Johnson and Rozanis,( 1979) pointed out some of the problems in using drugs like nitrofuran,spiramycin, chlorhexidine, tetracycline which are used as anticariogenic agents inhumans. The possible induction of resistant strains of microorganisms, the possibility of allergicreactions, the occurrence of side-effects such as nausea and diarrhea, and the expense oflong-term use. They also characterized the ‘ideal’ antibiotic for use as an anticaries agent asbeing one which has a very narrow spectrum directed towards plaque forming microorganisms,is not in general use against systemic diseases, is neither toxic nor allergic and is retained inthe tissue in an active state for a prolonged period with a predilection for the oral cavity.
Many plants and plant-derived antimicrobial components are used in folklore therapeutics for the treatment of periodontal disorders and for the purposes of oral hygiene The screeningassay can target additional pathogens including other Streptococci (group A and B, andPneumococci) and periodontal pathogens such as Porphyromonas. The activity of someextracts varied against different oral bacteria. And the screening for antimicrobials from plantsis feasible approach to the identification of natural compounds with antimicrobial propertiesagainst pathogens, (Josef, 1998) Plants as dental drugs about 10 different oral/dental conditions treatable with plants are common in traditional health namely: toothache/decay, gingivitis, ulcerative gingivitis, angular
stomatitis, mouth ulcers, swollen tonsils ,oral thrush, tonsillitis and black tongue (Hollist,
2004). Most common plants in the field include Piper guineense, Xylopia aethiopica, Citrus
aurantifolia and Afamomum melegueta
. (El-Said et al, 1971).
Many plant drugs even in the crude form are well known in the international markets today and African countries are among the top world producers of such plants. Examples areRauwolfia vomitoria Afz, (Family: Apocynaceae) which is a major source of reserpine , amajor tranqulizer and an ntihypertensive; ginger (Zingiber officinale) ,(Family Zingiberaceae)which contains gingerol used as spice, carminative and important medicinal product.Capsicumannum (Family Solanaceae) produces caspsain as Capsacin, use as spice and medicine;Physostigma venenosum Balf. (Family Leguminoceae) which is the calabar bean, producesphysostigmine or eserine used in Ophthalmia, Syzigium aromaticum Linn (Family Myrtaceae)is a dental remedy and also Chrysanthemum cinerarifolium Vis (Family Asteraceae), calledpyrethrum flower, produces the natural pyrethrins, a class of insecticides (Wallis, 1967).
Work Design
Identification dental Caries patients
Collection of Tooth
Analysis of Tooth
Scum Samples
Scum Samples
Anti-biogram Studies of
Selection and
the Bacterial Isolates
Characterization of
Bacterial Isolates
Collection of Medicinal
Preparation of Plant
Checking the Antibacterial Effect of Medicinal Plant
Extract- Disc Diffusion / Well Diffusion Method
Phytochemical Screening of
Effective Plant Extracts
3.2. Identification and sample collection from dental caries patients
Twenty five people, severely affected by dental caries belonging to Villavancode Taluk of Kanyakumari District, Tamil Nadu, South India, were identified as potential samples (dentalcaries patients) for oral bacteriological studies.
The tooth scum samples from the twenty five selected dental caries patients were collected aseptically in screw cap bottles with saline using swabs. The collected samples were broughtto the laboratory in ice box and stored at 40c in refrigerator for further studies.
3.3. Bacteriological Analysis
The Sample collected were bacteriologically analysed as follows: Isolation and antibiogram study of Bacterial Flora
The tooth scum sample (in swabs impregnated in saline) was continuously streaked on nutrient agar plates aseptically and the plates were incubated at 370 c for 24 hours. After incubation tubes were observed and results were recorded. Gram staining of particular isolateswere also carried out. Then the pure bacterial isolate were prepared by repeated sub culturing.
The pure cultures were stored at 40 c in nutrient agar slants for further studies.
Antibiogram study of the predominant bacterial isolates against commercial antibiotics (amoxicillin, ampicillin, ciprofloxacin, cephalexin, cefixime, erythromycin) commonly usedfor dental caries therapy was made by Kirby-Bauer disc diffusion method (Cappuccino 1999,Dey 1991). This is to detect the presence of any drug resistant oral bacteria in the selectedsamples.
3.4. Characterization of Bacterial Isolates
The bacterial isolates with drug resistance and moderate drug resistance were characterized by microscopic examination includes gram staining and motilitytest,macroscopic examination by plating and also by biochemical and physiologicalcharacteristics of bacterial isolates 3.5 Collection and preparation of Medicinal Plant extracts
1) The medicinal plant samples were collected from Villavancode Taluk of Kanyakumari
district, Tamil Nadu, South India. The leaves of five plants Piper betle (Betel), Betel Nut(Areca catechu), Eucalyptus globules, Zingiber officinale and Azadirachta indica (Neem)were selected for testing its antibacterial effects and characterization of secondary metabolitesof effective ones. For that plant samples were shade dried and ground well. 10 gram ofpowdered sample was taken in screw cap bottles with 10 ml of different solvent systems(acetone, ethanol, chloroform, methanol and water).
2) 3.6.) Checking the Antibacterial Effect of Medicinal Plant Extracts and their
phytochemcal screenig
Antibacterial effect of medicinal plant extracts were checked by Well- diffusion method.
Then plants with effective antibacterial activity were taken to screen for the presence offollowing phyto chemicals Liebermann-Burchard Test for Steroids/ Terpenoids
0.1 ml of the different solvent extracts were dissolved in chloroform in a dry test tube.
Add one ml of acetic anhydride followed by concentrated H SO along the sides of the test tube. The tubes were observed and the results were recorded.
b) Shinoda’s Test for Flavonoids
0.1ml of the extracts were dissolved in methanol. A few drops of magnesium turnings were added followed by few drops of concentrated HCl. It was observed and the result wasrecorded.
c) Molisch’s Test for Carbohydrates
5ml of the extracts were dissolved in alcohol or water and few drops of 10% alcoholic solution of â-naphthol was added followed by the addition of one ml of concentrated Sulphuricacid along the side of the test tube. It was observed for result.
Test for Saponins
The extracts were dissolved in few milliliter of water in a test tube and shaked well and e)
Test for Tannins
A few drops of 0.1%ferric chloride were added to 0.5 ml of extract and observed for f) Test for Phenolic Compounds
The extract was diluted to 5 ml with the distilled water. To this a few drops neutral 5%ferric chloride was added .A dark colour indicates the presence of phenolic compounds.
4.1. Bacteriological analysis of Dental caries samples
4.1. a) Isolation and Selection of Dental Caries Bacteria
Numerous bacterial isolates were obtained on Nutrient agar plates out of which fifteen were selected based on their morphological characterization 4.1.b) Gram’s Staining
Out of the twenty five samples analyzed eleven were with only Gram positive bacterial cells, and four samples showed the presence of Gram negative cells alone. Whereas ten samplewere with both Gram positive and negative cells .
4.2. Antibiogram Studies
Antibiogram of fifteen isolates were made and their response was tabulated .Among this five bacterial Isolates showed resistance and moderate sensitivity towards five antibioticssuch as ampicillin, ciprofloxacin, cefixime, amoxicillin and erythromycin. In other cases theorganisms analysed were sensitive towards the selected antibiotics.
4.3. Characterization of Bacterial Isolates
The five bacterial isolates with resistance and moderate sensitivity to selected antibiotics were characterized as Pseudomonas aeruginosa, Streptococcus salivarius, Streptococcusviridans, Streptococcus mutans, and Bacillus megaterium based on their macroscopic,microscopic, biochemical and physiological characters 4.4. Antimicrobial Effect of Medicinal Plant Extracts
The antimicrobial activity of selected medicinal plant’s (Piper betle, Areca catechu, Eucalyptus globules, Zingiber officinale, Azadirachta indica) in different solvent extractsagainst bacterial isolates (Pseudomonas aeruginosa, Streptococcus salivarius, Strptococcusviridans, Strptococcus mutants and Bacillus megaterium) were tabulated (Table No. 5.5.a,5.5.b, 5.5.c) The crude acetone extract of Piper betle showed high inhibitory activity against Bacillus megaterium and Pseudomonas aeruoginosa with a zone of 34mm, 38mm respectively.
Chloroform extract revealed activity against Streptococcus viridans with a clear zone of 22mm.
Whereas ethanol and methanol extracts showed inhibitory activity against Bacillus megateriumwith zone of 23mm. It was observed that water extract don’t have any inhibitory effect on fiveof the test organisms (Table 5.5.a).
The acetone extract of Eucalyptus globulus showed high inhibitory activity against Streptococcus mutans, Streptococcus viridans and Bacillus megaterium with 34mm, 36mmand 34mm respectively. Whereas the ethanol extract revealed activity against Bacillusmegaterium (24mm) and Streptococcus salivarius (28mm). It was observed that water extractinhibited the growth of Streptococcus viridans with a zone of 29mm (Table 5.5.c).
Acetone extract of Azardirachta indica showed maximum inhibitory activity against Bacillus megaterium and Streptococcus mutans with 25mm and 22mm respectively. Whereasthe chloroform, ethanol and methanol extracts were with moderate inhibitory effect on alltested organisms.
4.5 Phytochemical Screening
Phytochemical contents of solvent extracts of three plants, (Piper betle, Eucalyptus globules, Azadirachta indica)) with effective antibacterial property were studied .All the fivephytochemicals such as saponins,steroids and terpenoids,flavonoids,carbohydrates and tanninswere present in different solvent extract of piper betle and Azadirachta indica.In the case ofsolvent extracts of Eucalyptus globules,phytochemicals such as steroids and terpenoids,flavonoids and saponins were shown positive result.The phytochemicals showed differencein their presence or absence in different solvent extract.That is,presence of saponins wasdetected in the chloroform, methanol and water extract of Piper betle. The test for steroidsand terpenoids were positive in acetone, ethanol and methanol extracts. The test for flavonoidsgave positive result only in ethanol extract. The test for phenolic compounds was positive inacetone and ethanol extracts. Whereas the test for tannins were positive only in acetone andmethanol extracts. Carbohydrates present in ethanol, methanol and water extract.
In Eucalyptus globules the test for steroids and terpenoids give positive result in chloroform, ethanol, methanol and water extracts and negative in acetone. The tests forflavonoids were positive in acetone, ethanol and methanol extracts. Test for carbohydrates,phenolic compounds and tannins showed negative response in all the extracts. Whereaschloroform and water extracts showed the presence of saponins .
In Azadirachta indica test for steroids terpenoids and flavonoids were positive in acetone and ethanol extracts. Except the chloroform extract others were positive to carbohydrateswhere as Phenolic compounds were present in acetone alone. Test for saponins gave negativeresult in ethanol extract and others gave positive result. It is observed that tannins were presentin acetone and methanol extracts.
Table.5.5.a. Zone of Inhibition of Solvent Extracts of Piper betle
Table.5.5.b. Zone of Inhibition of Solvent Extracts of Eucalyptus globulus.
Table.5.5.c. Zone of Inhibition of Solvent Extracts of Azadirachta indica.
In the present oral bacteriological study, potential samples were collected from twenty five patients, severely affected by dental caries belonging to Villavancode Taluk ofKanyakumari District, Tamil Nadu, South India. Among the twenty five samples analysednumerous bacterial isolates were obtained. The majority of these isolates were gram positive than Gram negative bacteria. Both gram positive and Gram Negative bacteria were also isolatedfrom some patients. (Bowden. 1998). In the early stages of the disease, there was an alterationin abundance of certain forms of microorganisms in the oral cavity. Even though there was apossibility exists that one or more organisms are responsible for the initiation of dental caries.
Antibiogram studies were an alternative method for preventing enzymatic degradation of carbohydrates to acid for the prevention of bacterial growth and metabolism. In thisantibiogram study, Kirby-Bauer disc diffusion method were used for the fifteen isolates. Sixantibiotics were selected for the study are Ampicillin Cefixime, Ciprofloxacin, Amoxicillin,Cephalexin and Erythromycin. Each of the fifteen isolates showed variations in their responseto the selected antibiotics.
It was observed that among the fifteen bacterial isolates, five of them Pseudomonas aeruginosa, Streptococus Salivarius, Streptococcus Viridans, Streptococcus mutans andBacillus megaterium showed resistance and moderate sensitivity to the selected antibiotics.
These five isolates were characterised based on their macroscopic. Macroscopic, biochemicaland physiological characters.
Dental caries was proved to be a multifactorial disease and the factors associated with demineralization of enamel are complex. So due to this reason new agents were needed forthe treatment of periodontal disorders and for the purposes of oral hygiene. So in the presentstudy, leaves of the five medicinal plants such as piper betle, Areca catechu, Eucalyptusglobulus,Zingiber officinale and Azadirachta indica were selected from Villavancode Talukof Kanyakumari District, Tamil Nadu, South India.
These selected medicinal plants were then observed for their antibacterial activity by dissolving the powdered plant extract in to different solvent systems (acetone, ethanol,chloroform, methanol and water). Then each of these selected plant extracts were studied fortheir antibacterial activity against the selected five bacterial isolates by using well diffusionmethod.
Each of the plant solvent extract showed diversity in their activity against five isolates.
But the water extract of piper betle. Areca catechu and Zingiber officinale do not have anyinhibitory effect on the five bacterial isolates. Where as water extract of Eucalyptus globulusshowed growth inhibition on streptococcus viridans with a zone of 29mm. Other than thatwater extract of Piper betle and Eucalyptus globulus only were able to produce a zone ofinhibition against Streptococcus Salivarius,streptococcus viridans and Bacillus megaterium.Itwas noted that the zone of the inhibition of solvent extracts of Areca catechu was very lowamong the five plant except in the case of Acetone extract.] Thus the study about antibacterial property among the five plant extracts were showed that the solvent extracts of three plants: Piper betle, Eucalyptus globulus and Azadirachtaindica were more effective in producing antibacterial properties.
So the plants with effective antibacterial activity were taken for screening for the presence of certain phytochemicals such as steroids, terpenoides, flavanoids, carbohydrates, Saponinsand phenolic compounds. The different plant solvent extracts showed difference in the presenceor absence of phytochemical contents.
The present study were carried out based on the bacteriological analysis of dental caries samples. The antibiogram study showed that among the fifteen bacterial isolates five of themsuch as pseudomonas aeruginosa,streptococcus salivarius,streptococcus viridans,streptococcus mutans and bacillus megaterium were resistant or moderately sensitive to certainselected antibiotics (Ciprofloxacin,Ampicillin,Ceixime,Amoxycilin,Cephalexin andErythromycin). This isolates were characterized based on their macroscopic, microscopic,biochemical and physiological characters.
So the antibacterial effect of five medicinal plants against these five bacterial isolates were carried out. The extracts of these plants were made in five different solvents such asethanol, acetone,chloroform,methanol and water extracts and were checked against thebacterial isolates.
The plant extracts in solvents showed differences in their antibacterial activity against the five bacterial isolates.Which were then screened for the presence of phytochemicalcontents.The study concluded that some of the oral bacterial isolates showed resistance tocertain antibiotics and at the same time some medicinal plant extracts were able to inhibit thegrowth of this particular isolates.The solvent extracts of this plants differed in their efficiencyin producing antibacterial activity against the particular oral bacterial isolates. Among fiveplant extracts three of them were more resistant to the particular bacterial isolates.
Bowden, GHW 1990. Microbiology of root Surface caries. Journl of Dental Reserch., 69 :1205 - 1210 Bowden, GHW and I.R. Hamilton 1998. Survival of Oral bacteria Cretical Reviews in oralBiology and Medicine., 9 : 54 - 54.
Clark, F.Y 1879. Bacteremia. Johnstons Dental Miscellany., 6 : 447.
El-Said, F. et al 1971. Nature cures in Nigeria. Part II : The antimicrobial properties of thebuffer extracts of chewing sticks. Lloydia., 34(1) : 172.
Fujiwara, T., nakano, Kawaguchi, M., Ooshima, T., Sobue, S., Kawaabata, S., Nakagawa, l.
& Hamada, S. 2001. Biochemical and genetic characterization of serlogically untypableStreptococcus mutans strains isolated from patients with bacteremia. Eur J Oral Sci., 109 :330-334.
Josef Tichy, Jan Novak 1998. The Journal of Alternative and complementary Medicine., 39- 45.
Magitot. E 1878. Treatise on Dental caries; Experimental and Therapeutic InvestigationsTranslated by T.H. Chandler.Boston, Houston, Osgood and company.
Ullman, R. F., Miller, S. J., Strampfer, M. J. & Cunha, B.A 1988. Streptococcus mutansendocarditis: report of three cases and review of the literature. Herat Lung., 17 : 209-212.
Vose, J. M., Smith, P. W., Henry, M. & Colan, B. 1987. Recurrent Streptococcus mutansendocarditis. Am J. Med., 82 : 630-632.



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