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RESEARCH ARTICLE
Annals of Experimental Biology
2013, 1 (1):10-14
ISSN : 2348-1935
Do herbal toothpastes really work on food-borne bacteria resident
on teeth: An in vitro study
Neha Paul1, Chanda Patel1, Sulekha Yadav1, Shilpi Dadariya2, Monica Agrawal1 and
Manish Kumar Agrawal1*
1
Daksh Laboratories, 1370, Napier Town, Jabalpur, MP India
Hitkarini Dental College, Airport road, Jabalpur, MP India
2
_____________________________________________________________________________________________
ABSTRACT
Toothpastes which claim to be totally herbal use different preparations of herbs based either on ancient scientific
literature or folk medicines. The absence of strict quality control rule for such preparations has provoked us to
perform a real quality control test of the herbal toothpastes against five bacteria that were recovered from the
dental caries. Five food-borne bacteria namely Listeria spp., Salmonella spp., Alcaligenes spp., Micrococcus spp.
and Proteus spp. were isolated from the patients having dental caries. When ten known brands of herbal toothpastes
were screened against these bacteria, not only the overall sensitivity was different for the tested toothpastes, their
susceptibility towards individual bacteria differed a lot. The MIC differed 1000 times in most of the cases against
individual bacteria. Salmonella sp. was the most resistant bacteria against tested samples.
Key words: Herbal toothpastes, quality control, dental caries, food borne bacteria, MIC.
__________________________________________________________________________________________
INTRODUCTION
The oral cavity contains some of the most varied and vast flora in the entire human body. These bacteria are
responsible for some of the most common bacterial diseases in human i.e. germ disease and tooth decay (dental
caries). These bacteria are also blamed to cause dental plaque which is supposed to be the basis of tooth
deterioration. These bacteria digest the sugar and starches available in food and produce acids which dissolve tooth
enamel. Bacteria are also responsible for bad breath [1].
Several diseases that involve the gastrointestinal and respiratory systems manifest in the oral cavity. A variety of
organisms in the microenvironment of the oral cavity adhere to the teeth, the gingival sulcus, the tongue, and the
buccal mucosa. These specific conditions in the oral cavity may create foci of infection that in turn lead to sub acute
bacterial endocarditic and glomerulonephritis [2,3].
Most of the pathogenic bacteria come from the food and reside in the oral cavity primarily on teeth. Different food
products are known to harbor different kinds of bacteria, and in case of food contamination, one may expect some
more pathogenic forms of bacteria. Clostridium, E. coli, Salmonella, Lactobacillus, Listeria, Alcaligenes are few
food-borne notorious bacteria that harbor teeth primarily [4].
Regular cleaning of oral cavity using toothpaste is the most common activity that helps in maintaining a
bacteriostatic condition in the oral cavity. Though most of the cleaning is done by the action of toothbrush;
toothpaste helps in maintaining the clean status by providing antibacterial support. The chemical based toothpaste
consist hard acids, sorbitol and flocculants which kill these bacteria [5]. However, organic or herbal toothpastes do
not contain any chemical agent and the antimicrobial activity is supposed to come from the active pharmacological
ingredients. The plant based natural ingredients found in such toothpastes vary widely depending upon the choice of
herbs and their individual concentration [6]. Such information is mainly based on the folk or traditional medicine
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Manish K Agrawal et al
Ann. Exp. Bio., 2013, 1 (1):10-14
_____________________________________________________________________________
and/or one may prepare their own formula. This makes such preparation to skip any stringent quality control and one
has to believe the manufacturer’s claim for its antimicrobial properties. In this way, the authenticity of such herbal
preparations is always under suspicion.
Keeping the above facts in mind, we undertook a study using ten herbal toothpastes available in local market and
study their potency towards bacteria directly isolated from dental caries. The MIC value for toothpastes was
recorded as a potential of killing the hazardous mouth bacteria.
MATERIALS AND METHODS
(i)Collection of bacteria from dental caries
Dental samples were collected from the patients having dental caries who come for the dental treatment. The
samples were collected with the help of an excavator and immediately transferred to 1 ml thioglycollate medium
(M191, HiMedia, India) in sterile glass vials. Information of patient’s dental case history was also recorded along
with his/her consent. After inoculation, bottles were capped and sealed by Para film. The packed bottles were
brought to the laboratory immediately and placed in incubator at 37°C for 12 to 18 hours for bacterial enrichment.
Afterward, the bacteria were transferred to nutrient agar media (M001, HiMedia, India) for isolation and
identification processes.
(ii) Growth of bacteria from Dental samples
After enrichment; a loop full culture of bacterial growth in thioglycollate medium, was streaked onto nutrient agar
plate. The plates were incubated at 37°C for 24 to 48h.
Each distinct colony on the plate was picked up with the use of an inoculating loop. Each colony was transferred to
the slants and again grown for 24-48 h. The pure cultures were transferred periodically into a fresh nutrient broth
medium (sub-culturing) to allow continuous growth and viability of isolated microorganism [7].
(iii) Identification of bacteria
The bacteria were identified up to genera level by different procedures involving morphological characters of the
colony, staining by Gram’s stain and biochemical tests [8,9]. Different biochemical test i.e. indole production,
methyl red test, Voges Proskaur test, citrate utilization, urease production and triple sugar iron agar test were used
for the identification of bacteria up to genera level [8,10].
(iv) Collection of toothpaste samples
All leading herbal toothpaste and toothpowder brands were purchased from local market. The care was taken to
procure the completely herbal (organic) toothpastes only. Ten samples namely Babool, Dabur red, Babool mint,
Miswak, Himalaya, Dant-kranti, Colgate Herbal, Amar, Agro herbal powder and Vicco were collected.
(v) Preparation of dilutions of toothpastes
500 mg of toothpaste or toothpowder was dissolved in 500 µl of distilled water, shaken and kept as a stock solution.
Five dilutions ranging from 10-1 to 10-5 were prepared for all the toothpaste solutions using sterile water.
(vi) Preparation of toothpastes disc for testing antibacterial activity
The discs was prepared by cutting the sterilized Whitman’s No. 1 filter paper through the paper-punch machine in
order to get the diameter of 6 mm and autoclaved. The separate discs were loaded with 5 µl of each toothpaste
solution of each dilution. A control disc was also prepared with 5 µl autoclaved distilled water. The loaded discs
were allowed to dry.
(vii) Antimicrobial susceptibility tests
For antibacterial activity in vitro, the test bacterium was streaked onto Muller Hinton agar (M173, HiMedia
Laboratories, India) plates. After solidification, bacterial suspension was streaked. The turbidity of the bacterial
suspension was adjusted to the absorbance of 0.8 to 1.0 at 620 nm to get 105 to 106 cells per ml [11]. Streaking was
done with the help of sterilized cotton swabs (Johnson & Johnson, USA). The inoculum was allowed to dry before
inserting the test discs. Discs were placed in equal distance on media with the help of sterile forceps. At the centre, a
control disc was placed. Plates were allowed to grow at 37°C for 24h in a bacteriological incubator. After incubation
diameter of the clear zone was measured using antibiotic zone measurement scale (HiMedia, Labs India). The
minimum inhibitory concentration was calculated as described by manual of clinical microbiology [12,13].
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Manish K Agrawal et al
Ann. Exp. Bio., 2013, 1 (1):10-14
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RESULTS
During the course of study dental samples were collected from dental caries from around 25 patients using standard
dental procedures. These samples were plated on to nutrient agar media and the isolated colonies were picked. Total
five bacterium Listeria spp., Proteus spp., Micrococcus spp., Salmonella spp. and Alcaligenes spp. were isolated
from dental caries. The microbial cultures were identified by Gram staining and biochemical test up to the genera
level. Figure 1 shows gram staining of bacteria isolated from dental caries and Table 1 shows biochemical
characteristics of isolated bacteria.
Table 1: Microbiological and biochemical characteristics of bacteria isolated from dental caries
No. of colonies
9
6
5
6
4
Color
Culture
characteristics
Elev.
Margin
Shape
Microscopic characteristics
Biochemical
tests
CIT URE TSI
Gram
Shape
I MR VP
Stain
Cream
Flat
Entire
Circular
Rod
+
+
+
+
Yellow Convex
Entire
Circular
Rod
White
Convex
Entire
Circular
Cocci
+
+
+
White
Flat
Entire
Circular
Rod
+
+
+
Cream
Flat
Entire
Circular
Rod
+
+
I – Indole, MR – Methyl Red, VP – Voges Proskaur, CIT – Citrate, URE – Urease, TSI – Triple Sugar Iron Agar.
Result
Proteus sp.
Alcaligenes sp.
Micrococcus sp.
Listeria sp.
Salmonella sp.
+
+
+
+
Table 2: The minimum inhibitory concentration (MIC) of herbal toothpaste against bacteria isolated from
dental caries.
Sample name
Babool Herbal
(Dabur India Limited)
Dabur Red Herbal
(Dabur India Limited)
Babool Mint Herbal
(Dabur India Limited)
Dantkranti Herbal
(Patanjali Ayurved Ltd.)
Colgate Herbal
(Palmolive India Limited)
Vicco Herbal
(Vicco Laboratory)
Agro Herbal Powder
(Shree Balaji Agro Pharma)
Amar Herbal
(Amar Remedies Ltd)
Miswak Herbal
(Dabur India Limited)
Himalaya Herbal
(The Himalaya Drug Company)
Alcaligenes sp.
50µg
Minimum inhibitory concentration (MIC) of toothpastes
Listeria sp.
Proteus sp.
Salmonella sp.
5µg
50µg
-
Micrococcus sp.
50µg
500ng
500ng
500µg
5mg
50µg
-
50µg
500ng
-
500µg
50ng
5mg
500µg
500 µg
50µg
50µg
500µg
500µg
-
50µg
5mg
5mg
500µg
5mg
5mg
50µg
50µg
500µg
-
500µg
-
5µg
50µg
-
-
500µg
5mg
500µg
-
5mg
500µg
500µg
500µg
-
50µg
When 10 herbal toothpastes, collected from local shops were screened against these isolated bacteria, the overall
sensitivity was different for all the tested toothpastes. All the toothpastes were unable to kill all tested bacteria and
their minimum inhibitory concentration was also different against all bacteria. In over all the study, Salmonella spp.
was the most resistant bacteria against tested toothpaste.
In our study Dant-kranti was found most effective. Agro herbal powder showed least inhibition towards test
bacteria. Dabur red herbal toothpaste was found effective with lower dilutions. Other failed to inhibit bacteria when
used in the dilutions less than 10-2 and 10-3. Table 2 shows the minimum inhibitory concentration of all tested
bacteria against herbal toothpaste. Salmonella sp. has shown great degree of resistance towards tested toothpaste.
Vicco produced a very high MIC of 5 mg against Salmonella while other failed to inhibit the Salmonella growth. In
most of the cases the MIC produced by the toothpaste against any specific bacterium was same; the difference in
MIC specifies the difference in sensitivity against bacterium.
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Ann. Exp. Bio., 2013,
201 1 (1):10-14
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Proteus spp.
Salmonella
monella spp.
Micrococcus spp.
Alcaligenes spp.
Listeria spp.
Fig 1: Gram staining of food born bacteria isolated from dental caries.
caries
astes against bacteria.
Fig 2: Minimum inhibitory concentration of selected herbal toothpastes
Dant-kranti herbal toothpaste vs. A. Alcaligenes spp., B. Listeria spp., C. Proteus spp., D. Salmonella spp.,
spp. E. Micrococcus spp.
Agro herbal powder vs. a. Alcaligenes spp., b. Listeria spp., c. Proteus spp., d. Salmonella spp.,, e. Micrococcus spp.
DISCUSSION
The herbal drugs, cosmetics and life style product are based on the formulation of specified in Ayurveda in India.
Due to large herbal recipes and mixture combination, there is no single formula to produce any product. Hence
different companies use different
nt formulas either fully based on Ayurveda or related sources. This makes the quality
control of such preparation rather difficult and because of this there is no strict quality control system for such
preparation.
Previous research indicates that most of
of the herbs used in toothpastes vary considerably [14]. The role of the
toothpaste is to kill pathogenic bacteria of mouth which is responsible for dental diseases [15]. The primary
etiological factor for dental diseases is dental plaque. The formation of dental plaque on the tooth surface is
characterized by the progression form a limited number of microbial species to the complex flora of mature dental
plaque. This involves initial adherences of bacteria to the tooth surface. This tooth surface coated with
wit dense,
complex micro community ends up in the destruction of hard enamel tissue [16] and these are initiation of various
dental disorders [17].
We investigated the action of herbal toothpaste against pathogenic microorganism developed in oral cavity. The
Th
manufacturer’s instructions indicate that these toothpastes do have antibacterial and plaque inhibitory activities.
Earlier it has been shown that such toothpastes prevent plaque formation by interfering with bacterial adherence to
the tooth surface [18,19,20].
19,20]. When we screened these herbal toothpastes against five major food-born
food
bacteria, the
result obtained show that only some tooth pastes had good antibacterial activity. Other toothpastes failed to inhibit
bacterial growth whenever diluted up to a concentration
con
of 10-2 to 10-3. Previous research has indicated that some the
tested bacteria may cause serious dental and other problem in human beings i.e. Listeriosis caused by Listeria spp.
Salmonella spp. cause typhoid fever, Micrococcus spp. is an opportunistic pathogen, particularly in hosts with
compromised immune system.
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Manish K Agrawal et al
Ann. Exp. Bio., 2013, 1 (1):10-14
_____________________________________________________________________________
Our result clearly showed that effectiveness towards Salmonella spp. was least with all tested tooth pastes though
they were effective against Proteus spp. The MIC varied greatly suggestive poor quality of such toothpastes. Since
herbal toothpaste do not disclose the active antimicrobial ingredients in their formulas, it is almost impossible to
judge their effectiveness without proper microbiological quality control. This study was conducted to measure
minimum inhibitory concentration of herbal toothpastes. It was clear from the result that herbal toothpastes were not
effective against many of the food born bacteria.
CONCLUSION
Organic toothpastes are herbal prepared formulas. However, the quality control system of these pastes is based on
their ability to kill the pathogenic forms of the bacteria resident on tooth. Hence, the quality control of such chemical
compounds is entirely microbiological. Our study advocates the testing of herbal toothpastes against some more
bacterial forms especially derived from food i.e. Proteus, Alcaligenes Micrococcus, Listeria and Salmonella spp. We
strongly advocate the need of a stringent quality control test mandatory to all the manufacturers with elaborated
quality control parameters. This will not only spread the awareness amongst users, but will also help in selecting
most effective one.
Abbreviation
MIC - Minimum Inhibitory Concentration.
CLSI - Clinical and Laboratory Standards Institutes.
ADA - American Dental Association.
I
- Indole.
MR - Methyl Red.
VP - Voges proskaur.
CIT
- Citrate.
URE - Urease.
TSI
- Triple sugar iron agar.
REFERENCES
[1] W.J. Loesche. Microbiol. Rev., 1986, 50, 353-380.
[2] R.J. Gibbons, J.V. Van Houte. Annu. Rev. Med., 1975, 29, 19-44.
[3] H.J. Sanders. Chem. Eng. News., 1980, 58, 30-42.
[4] P.D. Marsh., Horizon Scientific Press., 2000, 32, 11-65.
[5] American Dental Association. 2010, 12, 04-15.
[6] L.A. Mitcher, Y.H. Park, D. Clark, J.L. Beal. J. Nat. Prod. Res., 1980, 43,259-69.
[7] R.P. Singh. Textbook of Microbiology, 2nd ed., Kalyani publishers, New Delhi 2009, 811- 812.
[8] K.R. Aneja. In Experiments in Microbiology, plant pathology, tissue culture and mushroom production
technology. New Age International (P) Limited, New Delhi, 2000.
[9] B. Potter. Biochemical Tests, Microbiology, Penn State Erie, The Behrend College, 2008, 202.
[10] M.O. Fawole, B.A. Oso. Characterization of bacteria in laboratory. In Manual of Microbiology, Spectrum
Books Limited, Nigeria. 2004, 22-33.
[11] Performance standards of antimicrobial disc susceptibility tests. CLSI, 2010, 30, 1.
[12] J.M. Andrews. J. Antimicrob. Chemother., 2001, 48, 5-16.
[13] J.D. Turnidge, M.J. Ferraro, J.H. Jorgensen. Susceptibility Test Methods: General Considerations, Manual of
Clinical Microbiology. 8th Ed. Washington., 2003, 1103.
[14] S. Anil. J. Contemp. Dent. Practice, 2007, 8, 60-66.
[15] J. Moran, M. Addy, R.G. Newcombe, I. Marlow. J. Clin. Periodontol., 1988, 27, 806-809.
[16] F. Gamboa, M. Estupinan, A. Galindo. Dent. Res. J., 2004, 9, 23-27.
[17] S.S. Lee, W. Zhang, Y. Li. J. Am. Dent. Assoc., 2004, 135, 1133-1141.
[18] S. Jenkins, M. Addy, R. Newcombe. J. Clin. Periodontol., 2000, 17, 85-89.
[19] D.H. Fine, D. Furgang, K. Markowitz, P.K. Sreenivasan, K. Klimpel, W. Vizio. J. Am. Dent. Assoc., 2006, 135,
1123-1128.
[20] D. Herrera, S. Roldan, I. Santacruz, S. Santos, M. Masdevall, M. Sanz. J. Clin. Periodontol, 2003, 30, 315-318.
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