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Potential of the aqueous extract of Terminalia chebula as an anticaries agent
Journal of Ethnopharmacology 68 (1999) 299 – 306 www.elsevier.com/locate/jethpharm
Short communication
Potential of the aqueous extract of Terminalia chebula as an anticaries agent A.G. Jagtap *, S.G. Karkera Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400098, India Received 6 January 1999; received in revised form 22 February 1999; accepted 20 April 1999
Abstract The aqueous extract from Terminalia chebula was tested for its ability to inhibit the growth and some physiological functions of Streptococcus mutans. The extract strongly inhibited the growth, sucrose induced adherence and glucan induced aggregation of S. mutans. Mouthrinsing with a 10% solution of the extract inhibited the salivary bacterial count and salivary glycolysis. Mouthrinsing with the extract significantly reduced total bacterial counts and the total streptococcal counts in the saliva samples obtained up to and including 3 h after rinsing, compared with the counts obtained prerinsing or after placebo rinsing. The extract successfully inhibited glycolysis of salivary bacteria for up to 90 min postrinsing. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Terminalia chebula; Streptococcus mutans; Anticaries agent
1. Introduction Dental caries, a localized, progressive decay of the teeth, results from colonization of the vulnerable surfaces of the teeth by a characteristic group of bacteria, the Streptococcus mutans (Scherp, 1971). S. mutans metabolizes sucrose in a peculiar way, producing an extracellular adhesive polysaccharide (dextran) from the glucose moiety and mainly lactic acid from the fructose moiety. The * Corresponding author. Tel.: +91-612-6284/9172; fax: + 91-614-0480. E-mail address: [email protected] (A.G. Jagtap)
synthesis of sticky, insoluble glucan promotes the firm adherence of the organism to the tooth surface that contributes to the formation of dental plaque. The accumulation of acids in the dental plaque subsequently leads to localized decalcification of the enamel surface (Ooshima et al., 1994). Despite several antiplaque agents being available in the market, the search for an effective agent still continues. Several undesirable side effects associated with these agents stimulated the search for alternate agents (Schee, 1989). Recently one focus is on plants or plant products used in folk dental practices or prescribed in Unani, homeopathic or Ayurvedic remedies (Memory, 1986).
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On examining the list of plants recommended for a particular dental therapeutic purpose, it is evident that the ripe fruit of Terminalia chebula is valuable in the prevention and treatment of several diseases of the mouth such as dental caries, spongy and bleeding gums, gingivitis and stomatitis (Date and Kulkarni, 1995). In India the role of T. chebula in decreasing the prevalence of caries has been common knowledge for many years (Chopra and Handa, 1958). This may be one of the reasons for unavailability of information regarding its mode of action in the oral cavity. Although many Ayurvedic practitioners realize that the finely powdered fruit of T. chebula can be used to strengthen the gums and as a remedy for carious teeth, they do not understand how it works. It was, therefore of interest to us to provide new insights into the mode of action of T. chebula in the oral cavity.
2. Materials and methods
2.1. Culture of the microorganism and preparation of the cell suspension The microorganism used for this study was S. mutans. The organism was originally isolated from the dental plaque of a healthy human volunteer. The strain from the human dental plaque was isolated onto the plates of Mitis sali6arius (MS) agar and incubated anaerobically at 37°C for 24 h. A cell suspension was prepared by removing the colonies of S. mutans into fluid thioglycolate medium and incubating at 37°C for 18 h.
2.2. Source of the plant material and preparation of the plant extract The dried ripe fruit of T. chebula was obtained from Yucca Enterprises, Mumbai, identified and authenticated by Dr K.S. Laddha, Department of Pharmacognosy, University Department of Chemical Technology, Mumbai. The ripe fruit (400 g) was suspended in 10 times its quantity of sterile distilled water in a round bottomed flask and kept at 4°C for 72 h. The aqueous extract was decanted, clarified by filtra-
tion through a muslin cloth and evaporated in a flat bottomed porcelain dish at 40°C. The dried extract was stored at 4°C prior to use. The extract was suspended in polyethylene glycol (PEG) 400 (20% v/v) and sterile distilled water to give a final concentration of 30% w/v. The concentrated extract was diluted with sterile distilled water to give concentrations of 2, 4, 8, 10, 15, 20, 25 and 30% w/v.
2.3. Antimicrobial acti6ity of the extract To study the effect of extract of T. chebula on the growth of S. mutans, the 18 h old culture of S. mutans was incorporated into brain heart infusion (BHI) agar and then poured into petriplates and allowed to set (0.1 ml of the culture fluid for every 10 ml agar). Sterile filter paper disks having a diameter of 6 mm were impregnated with known dilutions of the extract and placed on the agar. The zone of inhibition obtained with various concentrations of the extract was observed after 24 h incubation at 37°C. To determine the time required by the extract to exert the antimicrobial activity, 0.1 ml of the culture fluid of S. mutans was added to 5 ml of a known dilution of the extract. A loopful of this mixture was transferred to 5 ml of sterile BHI broth at time intervals of 2.5, 5, 10 and 15 min. The time was measured from the time of addition of the culture fluid of S. mutans to the extract. After incubation at 37°C for 24 h the tubes were observed for growth, i.e. the presence of turbidity.
2.4. Adherence inhibition To study the effects of various concentrations of the extract on the adherence of S. mutans to smooth glass surfaces, BHI broth containing 2% sucrose and 0.1 ml of various concentrations of the extract (from 2 to 10%) were inoculated with the overnight culture of S. mutans. Control consisted of cells grown in BHI broth containing sucrose and 0.1 ml PEG 400 (20% v/v). All tubes were inclined at 30° and incubated at 37°C for 24 h. The adherent and the nonadherent bacteria were quantitated spectrophotometrically as described by Segal et al. (1985).
A.G. Jagtap, S.G. Karkera / Journal of Ethnopharmacology 68 (1999) 299–306
To study the effects of the various concentrations of the extract on the adherence of S. mutans to the surface of the tooth, saliva coated sterile human teeth were kept in contact with 1 ml of the various concentrations of the extract (from 2 to 10%) for 1 min. Control consisted of teeth kept in contact with1 ml PEG 400 (20% v/v). The teeth were then added to 5 ml BHI broth containing 2% sucrose, inoculated with overnight cultures of S. mutans and incubated at 37°C for 24 h. The adherent bacteria (cells adhered to the surface of the glass and tooth) and the nonadherent bacteria were quantitated spectrophotometrically at 540 nm (Evans et al., 1977).
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visible minute clumps of cells in turbid fluid; 2+ , easily visible small clumps of cells in turbid fluid; 3 + , well defined clumps of cells in clear supernatant fluid; and 4+ , very large flocculent clumps of cells in clear supernatant fluid. For studying the sucrose-induced aggregation the cells were incubated with sucrose in the presence of the extract for 24 h. The degree of aggregation was observed after 24 h of incubation and scored on a zero to 4+ basis.
2.7. In 6i6o techniques
2.6. Inhibition of glucan-induced aggregation
2.7.1. Experimental plan Three adult subjects in good health and with 20 or more natural teeth free of dental caries were recruited into the study. The procedures, possible discomfort or risk were fully explained to the volunteers and their written consent obtained. The subjects continued their usual oral hygiene routines and no attempts were made to change or standardize diets or eating habits. The subjects gave a whole mouth saliva sample. After collection the sample were placed immediately on ice. This saliva sample served as the control. The subjects rested for 15 min and then rinsed the mouth with 10 ml of the assigned extract for 1 min. The mouthrinse was prepared by suspending the extract in PEG 400 (20% v/v) and water to give a concentration of 10%. None of the subjects rinsed with water afterwards. Further samples were collected after 10 min, 1 h and 3 h postrinsing. The subjects were not allowed to eat in between the sample collection. The placebo contained vehicle only with no active ingredient. The vehicle was a solution containing water and polyethylene glycol.
Aggregation of S. mutans in the presence of dextran T4133 (Sigma, St Louis, MO) was studied as described by Murchison et al. (1981). S. mutans cells were incubated with from 2 to 10% of the extract for 1 h at 37°C prior to the addition of the dextran. Control consisted of cells incubated with PEG 400 (20% v/v) for 1 h. The culture fluid was scored on a scale of 0 to 4+ for cell aggregation as follows: 0, no visible aggregation; 1+, slightly
2.7.2. Bacteriological examination of the sali6a Each saliva sample was immediately diluted 103 times with sterile saline and streaked on BHI agar plates and MS agar plates to determine the total bacterial and total streptococcal counts, respectively. The plates were incubated at 37°C for 24 h and the number of bacterial colonies was counted (Corner et al., 1990).
2.5. Inhibition of glycolysis The effect of the extracts on acid production was studied by two in vitro methods: 1. To 2 ml of fresh clarified human saliva was added 0.1 ml 5% glucose and 0.1 ml of the various concentrations of the extracts (from 2 to 10%). Control consisted of the saliva glucose mixes containing 0.1 ml PEG 400 (20% v/v). The pH of the saliva glucose mixes was recorded immediately and at 60 min intervals for the next 5 h (Corner et al., 1990). 2. To study the effects of the extract on acid production after 24 h incubation, 5 ml BHI broth containing 2% sucrose and 0.1 ml of various concentrations of the extracts (from 2 to 10%) were inoculated with the overnight cultures of S. mutans and incubated at 37°C for 24 h. The pH of the bacterial broth was recorded at the onset and after 24 h incubation (Ciardi et al., 1981).
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2.7.3. Inhibition of glycolysis Saliva samples were collected at both prerinsing and at 1, 15, 30, 45 and 90 min postrinsing. Each saliva sample (2 ml) was pipetted into test tubes containing 0.5 ml 5% glucose. All the test tubes were placed in a 37°C water bath for 1 – 2 min and the pH value of the saliva glucose mixes was determined immediately and at 60 min intervals thereafter for the next 5 h (Dolan et al., 1974). 2.8. Analysis of data Data were analyzed for inhibition of adherence, aggregation and acid production. The adherence index and the aggregation index were analyzed by the Wilcoxon’s matched-pairs signed ranks test. For in vitro adherence, data from the test and the control groups were compared using Student’s t-test. For in vitro glycolysis data from the test at various time intervals were compared with the data from the control at the same time interval using Wilcoxon’s matched-pairs signed ranks test. All values were considered significant when P B 0.05. Data were analyzed for the mouthrinse, for the inhibition of bacterial growth, by comparing the prerinsing values with the postrinsing values using a paired t-test. All values were considered significant when PB 0.05. Data were analyzed for the mouthrinses for the inhibition of salivary glycolysis by comparing the pH values of the postrinsing saliva samples, at various time intervals in the glycolytic reaction, with the pH values of the prerinsing saliva sample, at the same time interval in the glycolytic reaction, using Wilcoxon’s matched-pairs signed ranks test. All values were considered significant when P B0.05. 3. Results
Fig. 1. Inhibitory effects of T. chebula extract on the growth of S. mutans
Mouthrinsing with a 10% solution of the extract of T. chebula brought about a significant reduction in the total salivary bacterial count and also the total streptococcal count at 10 min, 1 h and 3 h postrinsing (PB0.05) (Table 2). Rinsing with the vehicle brought about no significant difference in the salivary bacterial count at any time interval (data not shown)
3.2. Effects of the extract on sucrose-induced in 6itro adherence Inhibition of in vitro adherence of S. mutans to glass was evident when the cells were grown in BHI broth containing sucrose and various concentrations of the extract (Table 3). At a concentration of 2% the extract showed more than 85% inhibition of adherence to the glass surface. However, the extract failed to inhibit adherence to the surface of the tooth even at a concentration as high as 10%. Table 1 Time required by the extract of T. chebula to kill S. mutans a Extract concentration (%)
3.1. Antimicrobial acti6ity against S. mutans When grown in agar containing from 6 to 30% of the extract, the aqueous extract of T. chebula was found to have antibacterial activity against S. mutans and had a MIC of 6% (Fig. 1). At a concentration of 10% of the extract, the organism was killed after a contact time of 15 min (Table 1)
Total time of contact of the extract with S. mutans (min) 2.5
5
10
15
6 8 10
+ + +
+ + +
+ + +
+ + –
Chlorhexidine
–
–
–
–
a
+, Growth observed; –, no growth observed; n =3.
A.G. Jagtap, S.G. Karkera / Journal of Ethnopharmacology 68 (1999) 299–306 Table 2 Temporal Effects of Mouthrinsing with a 10% solution of the extract of T. chebula on total salivary bacterial and total salivary streptococcal countsa Time (min)
Reduction of the to- Reduction of the tal salivary bacterial total salivary strepcount (%) tococcal count (%)
10 60 180
62.1 95.17* 54.6 98.08* 49.8 99.34*
64.3 9 10.9* 58.6 94.85* 56.89 3.88*
a Values are expressed as the mean 9S.E.M. of the readings obtained with three subjects. * Significant difference from the control at PB0.05.
3.3. Effect of the extract on acid production by S. mutans The results of the in vitro salivary glycolytic assay revealed that at concentrations of 2 and 4% the extract failed to inhibit acid production. At higher concentrations of 8 and 10% the pH values did not change significantly over a period of 5 h into the glycolytic reaction. The significantly lower pH values of all the test samples as compared with the control values is due to the low pH of the extract itself (Table 4) The extract even failed to inhibit acid production in the bacterial broth containing glucose and the extract. With all the concentrations tested the pH values of the 24 h old bacterial broth were significantly lower than the control pH values Table 3 Inhibitory effects of T. chebula extract on sucrose-induced adherence of S. mutans a Extract concentration (%)
Inhibition of adherence to glass surface (%)
Inhibition of adherence to tooth and glass surfaces (%)
2 4 8 10
89.6 92.2* 84.62 91.67* 76.61 94.28* 83.46 93.62*
15.3 9 8.53b 35.069 13.7b 13.2 9 6.46b 8.24 9 4.93b
Chlorhexidine
99.8 90.02*
77.6 9 1.56*
Values are expressed as the mean 9 S.E.M. of six readings. Non-significant difference between the control and treated groups. * Significant difference from the control at PB0.05. a
b
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(Table 5). The pH of the saliva samples collected at 1, 15, 30, 45 and 90 min after rinsing with the extract were not significantly different from the pH of the prerinsing saliva sample. However at the 2nd, 3rd, 4th and 5th h into the glycolytic reaction the pH of the saliva samples collected at the various time intervals after rinsing with the extract were significantly higher than the pH of the saliva sample collected prerinsing (Table 6)
3.4. Effects of the extract on glucan-induced aggregation The addition of dextran to the cell suspension of S. mutans resulted in a rapid, dramatic cellular aggregation. Aggregation resulted from dextraninduced cross linking of cells and occurred within 1 h. Sucrose-induced aggregation depended upon the synthesis of dextran by dextransucrase with the subsequent cellular binding of the product and was, therefore, observed after 24 h of incubation of the bacterial broth containing sucrose and the extract. When the cells of S. mutans were incubated with the extract for 1 h prior to the incorporation of dextran, the subsequent addition of dextran resulted in rapid flocculation followed by sedimentation of the cells and dextran particles. Hence a score of 4+ was given for all the concentrations, i.e. 2, 4, 8 and 10%. When this flocculated suspension was examined microscopically it was seen that the particles of dextran were joined by individual bacterial cells or single chains of cells and that massive complexes of cells, characteristic of cell aggregation as seen in the control, were not present. Similar results were observed with sucrose-induced cellular aggregation.
4. Discussion and conclusions Tannic acid represents the major constituent of the ripe fruit of T. chebula and is present in a concentration of 20–40% (Chopra and Handa, 1958). Studies carried out at our laboratory indicate that the amount of tannic acid in the aqueous extract of T. chebula is 13% (Ramamurti, 1979).
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Table 4 Temporal effects of the aqueous extract of T. chebula on in vitro salivary glycolysis of S. mutans a Incubation time (h)
pH values of the saliva glucose mixes at different time intervals (control)
7 90.02 6.9 90.13 6.57 9 0.1 6.37 9 0.2 5.6 90.2 5.2 9 0.1
0 1 2 3 4 5
pH values of the saliva glucose mixes containing various concentrations of the extract at different time intervals 2%
4%
8%
10%
6.3 9 0.6* 6.19 9 0.08* 5.6890.09* 5.879 0.1* 5.58 90.1 4.959 0.05*
6.05 90.1* 5.9 90.07* 5.36 90.09* 5.57 90.12* 5.24 9 0.09* 4.95 90.05*
5.1 9 0.06* 5.1 9 0.04* 5.2 9 0.06* 5.4 9 0.01* 5.07 9 0.01* 4.99 9 0.01*
5 9 0.02* 4.99 90.03* 5 9 0.04* 5.24 90.04* 4.9 90.02* 4.9 90.05*
Values are expressed as the mean 9S.E.M. of six readings. * Significant difference from the control at PB0.05.
a
Some studies have reported that tannic acid is bacteriostatic or bactericidal to some Gram ( + )ve and Gram (−)ve pathogens (Kau, 1980). The result of our study has also demonstrated the antibacterial activity of the extract of T. chebula against S. mutans. At higher concentrations the extract even possessed the property of contact inhibition. As the bacteriological in vitro tests at their best served only as very simplified models of the in vivo conditions in the mouth, we also tested for the ability of the extract to reduce the bacterial counts in saliva samples after mouthrinsing. At concentrations of 10%, the extract had an immediate effect on the salivary bacteria, and this effect was retained for 3 h. For an agent to work successfully in the oral cavity, it should have an immediate effect that is sustained over time. The prolonged release of the tannic acid is essential as high antibacterial activity per se is not sufficient to obtain inhibition of plaque formation. Thus a temporary intra oral depot of rather firmly bound tannic acid seems to have formed during the rinsing. The large number of phenolic groups in tannins provides them with unique binding properties. The tannin must have adsorbed to the hydroxyapatite of the teeth or to the salivary mucins or, alternately, to the bacterial surface or to the mucosal cells in order to ensure its retention. The inhibition of acid production in the saliva samples after mouthrinsing with the 10% extract may be related to the antibacterial effect of the
extract on the salivary bacteria in situ at that concentration. This could be concluded from the inability of the extract to inhibit acid production in the in vitro salivary glycolytic assay at concentrations of 2 and 4%. At higher concentrations of the extract (8 and 10%) the pH values did not change significantly throughout the glycolytic reaction. The mechanism of the observed long term effect upon the acidogenicity of the saliva on mouthrinsing with the extract could therefore be related to the extracts bactericidal or bacteriostatic effect. Initially it was conceived that the inhibition of the adherence of S. mutans to the glass surface could be related to the antibacterial activity of the extract. However, previous studies on tannins have reported that the polyphenolic compounds have a non competitive inhibitory effect on the Table 5 Effect of the extract of T. chebula on acid production by S. mutans after 24 h incubationa Extract concentration (%)
pH at the onset pH after 24 h of incubation
Control 2 4 8 10
7.08 90.06 7 90.01 6.96 90.007 6.9 90.02 6.88 90.02
Chlorhexidine
7.15 90.02
4.45 90.02 4.4790.01 4.3 90.02* 4.27 90.023* 4.33 90.02* 7 90.04*
Values are expressed as the mean 9S.E.M. of six readings. * Significant difference from the control at PB0.05. a
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Table 6 Temporal effects of mouthrinsing with a 10% solution of the extract of T. chebula on in vitro salivary glycolysisa Incubation time (h)
pH of the prerinse saliva sample
7.1 90.07 7.129 0.2 6.5 90.05 6.1 90.02 5.8 90.04 5.3 90.01
0 1 2 3 4 5
pH of the postrinse saliva samples collected at various time intervals 1 Min
15 Min
30 Min
45 Min
90 Min
7.1 90.2 7.59 0.7* 7.19 0.1* 79 0.6* 6.89 0.8* 6.69 0.1*
7.1 90.8 7.3 9 0.8* 7.4 9 0.1* 7.5 9 0.2* 7.3 9 0.2* 7.2 9 0.2*
7.1 9 0.1 7.2 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.4 9 0.3* 7.2 9 0.2*
7.1 9 0.1 7.2 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.2 9 0.1* 7.1 9 0.2*
7.1 9 0.9 7.3 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.3 9 0.2* 7.3 9 0.2*
Values are expressed as the mean 9S.E.M. of the readings obtained with three subjects. * Significant difference from the control at PB0.05.
a
activity of the enzyme glucosyltransferase (GTF) (Schee, 1989). This enzyme is responsible for the conversion of sucrose to sticky insoluble glucan, which promotes the firm adherence of S. mutans to the surface of the tooth. Thus plaque prevention must have been achieved by the inhibition of the GTF. Once S. mutans adhere to the surface of the tooth, they aggregate by virtue of their glucan binding protein, leading to the formation of a confluent microbial layer. Dextran-induced aggregation is enhanced in the presence of the cell bound GTF, as it serves as a second type of glucan receptor. As aggregation was significantly inhibited in the presence of the extract, it may be suggested that the tannins must have inhibited the enzyme by virtue of some tannin – protein interaction. In the absence of the cell bound GTF, aggregation occurred, albeit weakly, by the dextran molecule that bound to the receptors on the cell surface. Thus, the extract could successfully prevent plaque formation on the surface of the tooth, as it inhibited the sucrose-induced adherence and the glucan-induced aggregation, the two processes which foster the colonization of the organism on the surface of the tooth. The in vitro plaque assay revealed that as the concentration of the extract increased, the inhibition of adherence to the saliva coated tooth decreased. For the extract to inhibit plaque formation on the surface of the tooth, tannic acid should adsorb well to the hydroxyap-
atite of the tooth, or to the salivary mucins during the 1 min exposure period. To ensure its retention, the tannic acid will bind strongly to the carboxyl groups of the salivary glycoproteins present on the pellicle. The ionization of these carboxyl groups will be greatly reduced when the pH of the surrounding medium is lowered. As the extract itself had a low pH, the smaller retention of tannic acid at higher concentration coincided with the reduction in the number of ionized carboxyl groups of some of the glycoproteins in the salivary pellicle. However the sustained antibacterial and antiglycolytic effect observed in the oral cavity on mouthrinsing with the extract suggested that the extract had a good substantivity in the oral cavity. It can thus be conceived that in the oral cavity the tannic acid must be binding to salivary proteins other than those on the pellicle and which also remained ionized at the pH of the extract. Alternately, they may also have bound to the anionic groups on the surface of the bacterial cell or to the mucosal cells to ensure its retention. Some studies have reported that the denaturing of proteins at low pH alters the amount and the nature of the binding sites for cationic groups (Bonesvoll et al., 1974). Denatured proteins have a higher affinity for cationic groups than the native proteins. An increased denaturing of proteins at low pH could thus counterbalance the reduced binding of tannic acid to the proteins of the tooth pellicle. In conclusion we can say that the extract of T. chebula may be an effective agent
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in the treatment of carious teeth, owing to its ability to inhibit the growth and accumulation of S. mutans on the surface of the tooth. This would prevent the accumulation of acids on the surface of the tooth, and thus the further demineralization and the breakdown of the tooth enamel.
Acknowledgements We are grateful to the University Grant Commission (UGC) for providing us with financial aid.
References Bonesvoll, P., Lokker, P., Rolla, G., Paus, P.N., 1974. Retention of chlorhexidine in the human oral cavity after mouthrinses. Arch. Oral. Biol. 19, 209–212. Chopra, R.N., Handa, K.L., 1958. Chopra’s Indigenous Drugs in India, 2nd edn. U.N. Dhurs and Sons, Calcutta, pp. 658 – 659. Ciardi, J.E., Rosenthal, A.B., Bowen, W.H., 1981. Rapid quantitative determination of the effect of anti-plaque agents and antisera on the growth, acid production and adherence of S. mutans. J. Dent. Res. 60, 756–762. Corner, A.M., Brightman, V.J., Cooper, S., Yankell, S.C., Mahamud, D., 1990. Clinical study of C31G containing mouthrinses: effect on salivary microorganism. J. Clin. Dent. 2, 34 – 38.
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Date, B.B., Kulkarni, P.H., 1995. Assessment of Rasadanti in various oral disorders. Ayurveda Res. Pap. 2, 167 – 175. Dolan, M.M., Harding, E.T., Yanktee, S.E., 1974. Salivary glycolysis after mouthrinses. Odont. Acta/Suppl. 8, 54 – 56. Evans, R.T., Baker, P.J., Coburn, R.A., Genco, R.J., 1977. Comparison of antiplaque agents using an in vitro assay reflecting oral conditions. J. Dent. Res. 56, 559 – 567. Kau, P.C., 1980. In: Wu, B. (Ed.), New Chinese Medicine Handbooks, vol. 1. Shing Wen Fang Publishing, China, pp. 288 – 291. Memory, F.E., 1986. In: Saxena, R.C., Gupta, T.K. (Eds.), Proceedings of the International Seminar on Clinical Pharmacology in Developing Countries, pp. 1 – 9. Murchison, M., Larrimore, S., Curtis, R., 1981. Isolation and characterization of S. mutans mutants defective in adherence and aggregation. Infec. Immun. 34, 1044 – 1055. Ooshima, T., Minami, T., Aono, W., Tamura, Y., Hamada, S., 1994. Reduction of dental plaque formation in humans by Oolong tea extracts. Caries Res. 28, 146 – 149. Ramamurti, R.C., 1979. Pilot scale extraction of tannic acid from indigenous natural source and development and evaluation of its formulation for burns. Thesis submitted to the University of Bombay for the Degree of Masters of Pharmacy, The Bombay College of Pharmacy, Kalina, Bombay 400 098, pp. 22. Schee, A., 1989. Modes of action of currently known chemical antiplaque agents other than chlorhexidine. J. Dent. Res. 68, 1609 – 1616. Scherp, H.W., 1971. Dental caries: prospects for prevention. Science 173, 1199 – 1205. Segal, R., Pisanty, S., Wormser, R., Azaz, E., Sela, M.N., 1985. Anticariogenic activity of Licorice and glycyrrhizine. I: Inhibition of in vitro plaque formation by S. mutans. J. Pharm. Sci. 74, 79 – 81.
Short communication
Potential of the aqueous extract of Terminalia chebula as an anticaries agent A.G. Jagtap *, S.G. Karkera Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400098, India Received 6 January 1999; received in revised form 22 February 1999; accepted 20 April 1999
Abstract The aqueous extract from Terminalia chebula was tested for its ability to inhibit the growth and some physiological functions of Streptococcus mutans. The extract strongly inhibited the growth, sucrose induced adherence and glucan induced aggregation of S. mutans. Mouthrinsing with a 10% solution of the extract inhibited the salivary bacterial count and salivary glycolysis. Mouthrinsing with the extract significantly reduced total bacterial counts and the total streptococcal counts in the saliva samples obtained up to and including 3 h after rinsing, compared with the counts obtained prerinsing or after placebo rinsing. The extract successfully inhibited glycolysis of salivary bacteria for up to 90 min postrinsing. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Terminalia chebula; Streptococcus mutans; Anticaries agent
1. Introduction Dental caries, a localized, progressive decay of the teeth, results from colonization of the vulnerable surfaces of the teeth by a characteristic group of bacteria, the Streptococcus mutans (Scherp, 1971). S. mutans metabolizes sucrose in a peculiar way, producing an extracellular adhesive polysaccharide (dextran) from the glucose moiety and mainly lactic acid from the fructose moiety. The * Corresponding author. Tel.: +91-612-6284/9172; fax: + 91-614-0480. E-mail address: [email protected] (A.G. Jagtap)
synthesis of sticky, insoluble glucan promotes the firm adherence of the organism to the tooth surface that contributes to the formation of dental plaque. The accumulation of acids in the dental plaque subsequently leads to localized decalcification of the enamel surface (Ooshima et al., 1994). Despite several antiplaque agents being available in the market, the search for an effective agent still continues. Several undesirable side effects associated with these agents stimulated the search for alternate agents (Schee, 1989). Recently one focus is on plants or plant products used in folk dental practices or prescribed in Unani, homeopathic or Ayurvedic remedies (Memory, 1986).
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 5 8 - 6
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A.G. Jagtap, S.G. Karkera / Journal of Ethnopharmacology 68 (1999) 299–306
On examining the list of plants recommended for a particular dental therapeutic purpose, it is evident that the ripe fruit of Terminalia chebula is valuable in the prevention and treatment of several diseases of the mouth such as dental caries, spongy and bleeding gums, gingivitis and stomatitis (Date and Kulkarni, 1995). In India the role of T. chebula in decreasing the prevalence of caries has been common knowledge for many years (Chopra and Handa, 1958). This may be one of the reasons for unavailability of information regarding its mode of action in the oral cavity. Although many Ayurvedic practitioners realize that the finely powdered fruit of T. chebula can be used to strengthen the gums and as a remedy for carious teeth, they do not understand how it works. It was, therefore of interest to us to provide new insights into the mode of action of T. chebula in the oral cavity.
2. Materials and methods
2.1. Culture of the microorganism and preparation of the cell suspension The microorganism used for this study was S. mutans. The organism was originally isolated from the dental plaque of a healthy human volunteer. The strain from the human dental plaque was isolated onto the plates of Mitis sali6arius (MS) agar and incubated anaerobically at 37°C for 24 h. A cell suspension was prepared by removing the colonies of S. mutans into fluid thioglycolate medium and incubating at 37°C for 18 h.
2.2. Source of the plant material and preparation of the plant extract The dried ripe fruit of T. chebula was obtained from Yucca Enterprises, Mumbai, identified and authenticated by Dr K.S. Laddha, Department of Pharmacognosy, University Department of Chemical Technology, Mumbai. The ripe fruit (400 g) was suspended in 10 times its quantity of sterile distilled water in a round bottomed flask and kept at 4°C for 72 h. The aqueous extract was decanted, clarified by filtra-
tion through a muslin cloth and evaporated in a flat bottomed porcelain dish at 40°C. The dried extract was stored at 4°C prior to use. The extract was suspended in polyethylene glycol (PEG) 400 (20% v/v) and sterile distilled water to give a final concentration of 30% w/v. The concentrated extract was diluted with sterile distilled water to give concentrations of 2, 4, 8, 10, 15, 20, 25 and 30% w/v.
2.3. Antimicrobial acti6ity of the extract To study the effect of extract of T. chebula on the growth of S. mutans, the 18 h old culture of S. mutans was incorporated into brain heart infusion (BHI) agar and then poured into petriplates and allowed to set (0.1 ml of the culture fluid for every 10 ml agar). Sterile filter paper disks having a diameter of 6 mm were impregnated with known dilutions of the extract and placed on the agar. The zone of inhibition obtained with various concentrations of the extract was observed after 24 h incubation at 37°C. To determine the time required by the extract to exert the antimicrobial activity, 0.1 ml of the culture fluid of S. mutans was added to 5 ml of a known dilution of the extract. A loopful of this mixture was transferred to 5 ml of sterile BHI broth at time intervals of 2.5, 5, 10 and 15 min. The time was measured from the time of addition of the culture fluid of S. mutans to the extract. After incubation at 37°C for 24 h the tubes were observed for growth, i.e. the presence of turbidity.
2.4. Adherence inhibition To study the effects of various concentrations of the extract on the adherence of S. mutans to smooth glass surfaces, BHI broth containing 2% sucrose and 0.1 ml of various concentrations of the extract (from 2 to 10%) were inoculated with the overnight culture of S. mutans. Control consisted of cells grown in BHI broth containing sucrose and 0.1 ml PEG 400 (20% v/v). All tubes were inclined at 30° and incubated at 37°C for 24 h. The adherent and the nonadherent bacteria were quantitated spectrophotometrically as described by Segal et al. (1985).
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To study the effects of the various concentrations of the extract on the adherence of S. mutans to the surface of the tooth, saliva coated sterile human teeth were kept in contact with 1 ml of the various concentrations of the extract (from 2 to 10%) for 1 min. Control consisted of teeth kept in contact with1 ml PEG 400 (20% v/v). The teeth were then added to 5 ml BHI broth containing 2% sucrose, inoculated with overnight cultures of S. mutans and incubated at 37°C for 24 h. The adherent bacteria (cells adhered to the surface of the glass and tooth) and the nonadherent bacteria were quantitated spectrophotometrically at 540 nm (Evans et al., 1977).
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visible minute clumps of cells in turbid fluid; 2+ , easily visible small clumps of cells in turbid fluid; 3 + , well defined clumps of cells in clear supernatant fluid; and 4+ , very large flocculent clumps of cells in clear supernatant fluid. For studying the sucrose-induced aggregation the cells were incubated with sucrose in the presence of the extract for 24 h. The degree of aggregation was observed after 24 h of incubation and scored on a zero to 4+ basis.
2.7. In 6i6o techniques
2.6. Inhibition of glucan-induced aggregation
2.7.1. Experimental plan Three adult subjects in good health and with 20 or more natural teeth free of dental caries were recruited into the study. The procedures, possible discomfort or risk were fully explained to the volunteers and their written consent obtained. The subjects continued their usual oral hygiene routines and no attempts were made to change or standardize diets or eating habits. The subjects gave a whole mouth saliva sample. After collection the sample were placed immediately on ice. This saliva sample served as the control. The subjects rested for 15 min and then rinsed the mouth with 10 ml of the assigned extract for 1 min. The mouthrinse was prepared by suspending the extract in PEG 400 (20% v/v) and water to give a concentration of 10%. None of the subjects rinsed with water afterwards. Further samples were collected after 10 min, 1 h and 3 h postrinsing. The subjects were not allowed to eat in between the sample collection. The placebo contained vehicle only with no active ingredient. The vehicle was a solution containing water and polyethylene glycol.
Aggregation of S. mutans in the presence of dextran T4133 (Sigma, St Louis, MO) was studied as described by Murchison et al. (1981). S. mutans cells were incubated with from 2 to 10% of the extract for 1 h at 37°C prior to the addition of the dextran. Control consisted of cells incubated with PEG 400 (20% v/v) for 1 h. The culture fluid was scored on a scale of 0 to 4+ for cell aggregation as follows: 0, no visible aggregation; 1+, slightly
2.7.2. Bacteriological examination of the sali6a Each saliva sample was immediately diluted 103 times with sterile saline and streaked on BHI agar plates and MS agar plates to determine the total bacterial and total streptococcal counts, respectively. The plates were incubated at 37°C for 24 h and the number of bacterial colonies was counted (Corner et al., 1990).
2.5. Inhibition of glycolysis The effect of the extracts on acid production was studied by two in vitro methods: 1. To 2 ml of fresh clarified human saliva was added 0.1 ml 5% glucose and 0.1 ml of the various concentrations of the extracts (from 2 to 10%). Control consisted of the saliva glucose mixes containing 0.1 ml PEG 400 (20% v/v). The pH of the saliva glucose mixes was recorded immediately and at 60 min intervals for the next 5 h (Corner et al., 1990). 2. To study the effects of the extract on acid production after 24 h incubation, 5 ml BHI broth containing 2% sucrose and 0.1 ml of various concentrations of the extracts (from 2 to 10%) were inoculated with the overnight cultures of S. mutans and incubated at 37°C for 24 h. The pH of the bacterial broth was recorded at the onset and after 24 h incubation (Ciardi et al., 1981).
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2.7.3. Inhibition of glycolysis Saliva samples were collected at both prerinsing and at 1, 15, 30, 45 and 90 min postrinsing. Each saliva sample (2 ml) was pipetted into test tubes containing 0.5 ml 5% glucose. All the test tubes were placed in a 37°C water bath for 1 – 2 min and the pH value of the saliva glucose mixes was determined immediately and at 60 min intervals thereafter for the next 5 h (Dolan et al., 1974). 2.8. Analysis of data Data were analyzed for inhibition of adherence, aggregation and acid production. The adherence index and the aggregation index were analyzed by the Wilcoxon’s matched-pairs signed ranks test. For in vitro adherence, data from the test and the control groups were compared using Student’s t-test. For in vitro glycolysis data from the test at various time intervals were compared with the data from the control at the same time interval using Wilcoxon’s matched-pairs signed ranks test. All values were considered significant when P B 0.05. Data were analyzed for the mouthrinse, for the inhibition of bacterial growth, by comparing the prerinsing values with the postrinsing values using a paired t-test. All values were considered significant when PB 0.05. Data were analyzed for the mouthrinses for the inhibition of salivary glycolysis by comparing the pH values of the postrinsing saliva samples, at various time intervals in the glycolytic reaction, with the pH values of the prerinsing saliva sample, at the same time interval in the glycolytic reaction, using Wilcoxon’s matched-pairs signed ranks test. All values were considered significant when P B0.05. 3. Results
Fig. 1. Inhibitory effects of T. chebula extract on the growth of S. mutans
Mouthrinsing with a 10% solution of the extract of T. chebula brought about a significant reduction in the total salivary bacterial count and also the total streptococcal count at 10 min, 1 h and 3 h postrinsing (PB0.05) (Table 2). Rinsing with the vehicle brought about no significant difference in the salivary bacterial count at any time interval (data not shown)
3.2. Effects of the extract on sucrose-induced in 6itro adherence Inhibition of in vitro adherence of S. mutans to glass was evident when the cells were grown in BHI broth containing sucrose and various concentrations of the extract (Table 3). At a concentration of 2% the extract showed more than 85% inhibition of adherence to the glass surface. However, the extract failed to inhibit adherence to the surface of the tooth even at a concentration as high as 10%. Table 1 Time required by the extract of T. chebula to kill S. mutans a Extract concentration (%)
3.1. Antimicrobial acti6ity against S. mutans When grown in agar containing from 6 to 30% of the extract, the aqueous extract of T. chebula was found to have antibacterial activity against S. mutans and had a MIC of 6% (Fig. 1). At a concentration of 10% of the extract, the organism was killed after a contact time of 15 min (Table 1)
Total time of contact of the extract with S. mutans (min) 2.5
5
10
15
6 8 10
+ + +
+ + +
+ + +
+ + –
Chlorhexidine
–
–
–
–
a
+, Growth observed; –, no growth observed; n =3.
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Reduction of the to- Reduction of the tal salivary bacterial total salivary strepcount (%) tococcal count (%)
10 60 180
62.1 95.17* 54.6 98.08* 49.8 99.34*
64.3 9 10.9* 58.6 94.85* 56.89 3.88*
a Values are expressed as the mean 9S.E.M. of the readings obtained with three subjects. * Significant difference from the control at PB0.05.
3.3. Effect of the extract on acid production by S. mutans The results of the in vitro salivary glycolytic assay revealed that at concentrations of 2 and 4% the extract failed to inhibit acid production. At higher concentrations of 8 and 10% the pH values did not change significantly over a period of 5 h into the glycolytic reaction. The significantly lower pH values of all the test samples as compared with the control values is due to the low pH of the extract itself (Table 4) The extract even failed to inhibit acid production in the bacterial broth containing glucose and the extract. With all the concentrations tested the pH values of the 24 h old bacterial broth were significantly lower than the control pH values Table 3 Inhibitory effects of T. chebula extract on sucrose-induced adherence of S. mutans a Extract concentration (%)
Inhibition of adherence to glass surface (%)
Inhibition of adherence to tooth and glass surfaces (%)
2 4 8 10
89.6 92.2* 84.62 91.67* 76.61 94.28* 83.46 93.62*
15.3 9 8.53b 35.069 13.7b 13.2 9 6.46b 8.24 9 4.93b
Chlorhexidine
99.8 90.02*
77.6 9 1.56*
Values are expressed as the mean 9 S.E.M. of six readings. Non-significant difference between the control and treated groups. * Significant difference from the control at PB0.05. a
b
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(Table 5). The pH of the saliva samples collected at 1, 15, 30, 45 and 90 min after rinsing with the extract were not significantly different from the pH of the prerinsing saliva sample. However at the 2nd, 3rd, 4th and 5th h into the glycolytic reaction the pH of the saliva samples collected at the various time intervals after rinsing with the extract were significantly higher than the pH of the saliva sample collected prerinsing (Table 6)
3.4. Effects of the extract on glucan-induced aggregation The addition of dextran to the cell suspension of S. mutans resulted in a rapid, dramatic cellular aggregation. Aggregation resulted from dextraninduced cross linking of cells and occurred within 1 h. Sucrose-induced aggregation depended upon the synthesis of dextran by dextransucrase with the subsequent cellular binding of the product and was, therefore, observed after 24 h of incubation of the bacterial broth containing sucrose and the extract. When the cells of S. mutans were incubated with the extract for 1 h prior to the incorporation of dextran, the subsequent addition of dextran resulted in rapid flocculation followed by sedimentation of the cells and dextran particles. Hence a score of 4+ was given for all the concentrations, i.e. 2, 4, 8 and 10%. When this flocculated suspension was examined microscopically it was seen that the particles of dextran were joined by individual bacterial cells or single chains of cells and that massive complexes of cells, characteristic of cell aggregation as seen in the control, were not present. Similar results were observed with sucrose-induced cellular aggregation.
4. Discussion and conclusions Tannic acid represents the major constituent of the ripe fruit of T. chebula and is present in a concentration of 20–40% (Chopra and Handa, 1958). Studies carried out at our laboratory indicate that the amount of tannic acid in the aqueous extract of T. chebula is 13% (Ramamurti, 1979).
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Table 4 Temporal effects of the aqueous extract of T. chebula on in vitro salivary glycolysis of S. mutans a Incubation time (h)
pH values of the saliva glucose mixes at different time intervals (control)
7 90.02 6.9 90.13 6.57 9 0.1 6.37 9 0.2 5.6 90.2 5.2 9 0.1
0 1 2 3 4 5
pH values of the saliva glucose mixes containing various concentrations of the extract at different time intervals 2%
4%
8%
10%
6.3 9 0.6* 6.19 9 0.08* 5.6890.09* 5.879 0.1* 5.58 90.1 4.959 0.05*
6.05 90.1* 5.9 90.07* 5.36 90.09* 5.57 90.12* 5.24 9 0.09* 4.95 90.05*
5.1 9 0.06* 5.1 9 0.04* 5.2 9 0.06* 5.4 9 0.01* 5.07 9 0.01* 4.99 9 0.01*
5 9 0.02* 4.99 90.03* 5 9 0.04* 5.24 90.04* 4.9 90.02* 4.9 90.05*
Values are expressed as the mean 9S.E.M. of six readings. * Significant difference from the control at PB0.05.
a
Some studies have reported that tannic acid is bacteriostatic or bactericidal to some Gram ( + )ve and Gram (−)ve pathogens (Kau, 1980). The result of our study has also demonstrated the antibacterial activity of the extract of T. chebula against S. mutans. At higher concentrations the extract even possessed the property of contact inhibition. As the bacteriological in vitro tests at their best served only as very simplified models of the in vivo conditions in the mouth, we also tested for the ability of the extract to reduce the bacterial counts in saliva samples after mouthrinsing. At concentrations of 10%, the extract had an immediate effect on the salivary bacteria, and this effect was retained for 3 h. For an agent to work successfully in the oral cavity, it should have an immediate effect that is sustained over time. The prolonged release of the tannic acid is essential as high antibacterial activity per se is not sufficient to obtain inhibition of plaque formation. Thus a temporary intra oral depot of rather firmly bound tannic acid seems to have formed during the rinsing. The large number of phenolic groups in tannins provides them with unique binding properties. The tannin must have adsorbed to the hydroxyapatite of the teeth or to the salivary mucins or, alternately, to the bacterial surface or to the mucosal cells in order to ensure its retention. The inhibition of acid production in the saliva samples after mouthrinsing with the 10% extract may be related to the antibacterial effect of the
extract on the salivary bacteria in situ at that concentration. This could be concluded from the inability of the extract to inhibit acid production in the in vitro salivary glycolytic assay at concentrations of 2 and 4%. At higher concentrations of the extract (8 and 10%) the pH values did not change significantly throughout the glycolytic reaction. The mechanism of the observed long term effect upon the acidogenicity of the saliva on mouthrinsing with the extract could therefore be related to the extracts bactericidal or bacteriostatic effect. Initially it was conceived that the inhibition of the adherence of S. mutans to the glass surface could be related to the antibacterial activity of the extract. However, previous studies on tannins have reported that the polyphenolic compounds have a non competitive inhibitory effect on the Table 5 Effect of the extract of T. chebula on acid production by S. mutans after 24 h incubationa Extract concentration (%)
pH at the onset pH after 24 h of incubation
Control 2 4 8 10
7.08 90.06 7 90.01 6.96 90.007 6.9 90.02 6.88 90.02
Chlorhexidine
7.15 90.02
4.45 90.02 4.4790.01 4.3 90.02* 4.27 90.023* 4.33 90.02* 7 90.04*
Values are expressed as the mean 9S.E.M. of six readings. * Significant difference from the control at PB0.05. a
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Table 6 Temporal effects of mouthrinsing with a 10% solution of the extract of T. chebula on in vitro salivary glycolysisa Incubation time (h)
pH of the prerinse saliva sample
7.1 90.07 7.129 0.2 6.5 90.05 6.1 90.02 5.8 90.04 5.3 90.01
0 1 2 3 4 5
pH of the postrinse saliva samples collected at various time intervals 1 Min
15 Min
30 Min
45 Min
90 Min
7.1 90.2 7.59 0.7* 7.19 0.1* 79 0.6* 6.89 0.8* 6.69 0.1*
7.1 90.8 7.3 9 0.8* 7.4 9 0.1* 7.5 9 0.2* 7.3 9 0.2* 7.2 9 0.2*
7.1 9 0.1 7.2 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.4 9 0.3* 7.2 9 0.2*
7.1 9 0.1 7.2 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.2 9 0.1* 7.1 9 0.2*
7.1 9 0.9 7.3 9 0.1* 7.3 9 0.1* 7.3 9 0.1* 7.3 9 0.2* 7.3 9 0.2*
Values are expressed as the mean 9S.E.M. of the readings obtained with three subjects. * Significant difference from the control at PB0.05.
a
activity of the enzyme glucosyltransferase (GTF) (Schee, 1989). This enzyme is responsible for the conversion of sucrose to sticky insoluble glucan, which promotes the firm adherence of S. mutans to the surface of the tooth. Thus plaque prevention must have been achieved by the inhibition of the GTF. Once S. mutans adhere to the surface of the tooth, they aggregate by virtue of their glucan binding protein, leading to the formation of a confluent microbial layer. Dextran-induced aggregation is enhanced in the presence of the cell bound GTF, as it serves as a second type of glucan receptor. As aggregation was significantly inhibited in the presence of the extract, it may be suggested that the tannins must have inhibited the enzyme by virtue of some tannin – protein interaction. In the absence of the cell bound GTF, aggregation occurred, albeit weakly, by the dextran molecule that bound to the receptors on the cell surface. Thus, the extract could successfully prevent plaque formation on the surface of the tooth, as it inhibited the sucrose-induced adherence and the glucan-induced aggregation, the two processes which foster the colonization of the organism on the surface of the tooth. The in vitro plaque assay revealed that as the concentration of the extract increased, the inhibition of adherence to the saliva coated tooth decreased. For the extract to inhibit plaque formation on the surface of the tooth, tannic acid should adsorb well to the hydroxyap-
atite of the tooth, or to the salivary mucins during the 1 min exposure period. To ensure its retention, the tannic acid will bind strongly to the carboxyl groups of the salivary glycoproteins present on the pellicle. The ionization of these carboxyl groups will be greatly reduced when the pH of the surrounding medium is lowered. As the extract itself had a low pH, the smaller retention of tannic acid at higher concentration coincided with the reduction in the number of ionized carboxyl groups of some of the glycoproteins in the salivary pellicle. However the sustained antibacterial and antiglycolytic effect observed in the oral cavity on mouthrinsing with the extract suggested that the extract had a good substantivity in the oral cavity. It can thus be conceived that in the oral cavity the tannic acid must be binding to salivary proteins other than those on the pellicle and which also remained ionized at the pH of the extract. Alternately, they may also have bound to the anionic groups on the surface of the bacterial cell or to the mucosal cells to ensure its retention. Some studies have reported that the denaturing of proteins at low pH alters the amount and the nature of the binding sites for cationic groups (Bonesvoll et al., 1974). Denatured proteins have a higher affinity for cationic groups than the native proteins. An increased denaturing of proteins at low pH could thus counterbalance the reduced binding of tannic acid to the proteins of the tooth pellicle. In conclusion we can say that the extract of T. chebula may be an effective agent
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in the treatment of carious teeth, owing to its ability to inhibit the growth and accumulation of S. mutans on the surface of the tooth. This would prevent the accumulation of acids on the surface of the tooth, and thus the further demineralization and the breakdown of the tooth enamel.
Acknowledgements We are grateful to the University Grant Commission (UGC) for providing us with financial aid.
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