Chlorhexidine: The gold standard antiplaque agent

Abstract

Chlorhexidine is one of the most widely and commonly used antiplaque and antigingivitis agent. The properties and mechanism of action of chlorhexidine must be understood in order to be put into maximum use. Chlorhexidine was used as a broad spectrum antiseptic since the 1950's. Its antibacterial action is due to the disruption of the bacterial cell membrane by the chlorhexidine molecules, increasing the permeability and resulting in cell lysis. It can be either bacteriostatic or bactericidal depending on the dose. It is available in different formulations. However it does have some side effects like permanent staining of teeth and dysgeusia. This article discusses the various clinical applications, properties and adverse effects of chlorhexidine.

Full text

Chlorhexidine: The Gold Standard Antiplaque Agent
Shruti Balagopal1, Radhika Arjunkumar2
1Student,Saveetha Dental College, Chennai
2Senior Lecturer,Department of Periodontics
Saveetha Dental College, Chennai
Abstract
Chlorhexidine is one of the most widely and commonly used antiplaque and antigingivitis agent. The properties and mechanism of action of
chlorhexidine must be understood in order to be put into maximum use. Chlorhexidine was used as a broad spectrum antiseptic since the
1950’s. Its antibacterial action is due to the disruption of the bacterial cell membrane by the chlorhexidine molecules, increasing the
permeability and resulting in cell lysis. It can be either bacteriostatic or bactericidal depending on the dose. It is available in different
formulations. However it does have some side effects like permanent staining of teeth and dysgeusia. This article discusses the various
clinical applications, properties and adverse effects of chlorhexidine.
Keywords:
Chlorhexidine, mouthrinse, chemical plaque control
INTRODUCTION
Chlorhexidine is a gold standard against which other
antiplaque and antigingivitis agents are measured.
Understanding the properties and limitations of the molecule
can ensure that the efficacy of the agent is maximized and the
side effects are minimized allowing it to rightly remain the
gold standard.
Dental plaque
Dental plaque clinically is a structured resilient, grayish-
yellow substance that tenaciously adheres to the intraoral
hard surfaces including removable and fixed restorations [1].
Plaque control
It is the removal of microbial plaque and the prevention of its
accumulation on the tooth and adjacent gingival tissues to
prevent calculus formation. Plaque control can be of two
types
 Mechanical plaque control
 Chemical plaque control
Mechanical plaque control
Dental plaque is one of the most important etiological factors
in the onset of periodontal disease. Dental plaque mineralizes
to form dental calculus. Calculus formation is significantly
reduced by proper plaque control. Bacterial plaque can be
removed effectively by mechanical means. It is safe and
effective. The various methods include:
 Toothbrushes
 Interdental cleaning aids
 Dental floss
 Toothpick
 Interdental brush and swab
 Dentifrices
Chemical plaque control
Terminology
Antimicrobial agents: Chemicals that have a bacteriostatic or
bacteriocidal effect in vitro that alone cannot be extrapolated
to a proven efficacy in vivo against plaque.
Plaque reducing/inhibitory agents: Chemicals that have only
been shown to reduce the quantity and/or affect quality of
plaque which may or may not be sufficient to influence
gingivitis and/or caries.
Antiplaque agents: Chemicals that have an effect on plaque
sufficient to benefit gingivitis and/or caries.
Antigingivitis agents: Chemicals which reduce gingival
inflammation without necessarily influencing bacterial plaque
(includes anti-inflammatory agents).
Chemical plaque control agents [2]
The various chemical plaque control agents are listed in
Table 1.
Commercial mouthwashes can be classified into based on
their substantivity, range of antibacterial activity against
various plaque bacteria, possible anti inflammatory effect,
acceptable taste, ability to promote fresh mouth sensation.
They can also be classified as group A,B and C as follows,
 Group A agents- antiplaque
 Chlorhexidine, acidified sodium chlorate, salifluor and
delmopinol
 Group B agents-plaque inhibitory
 cetyl pyridinium chloride,
 essential oil and triclosan rinses
 used as adjuncts to mechanical cleaning
 Group C agents-Have a low to moderate activity and
are used for cosmetic purposes like breath freshening
 Sanguinarine , oxygenating agents, saturated pyrimidine,
hexetidine
Shruti Balagopal et al /J. Pharm. Sci. & Res. Vol.5(12), 2013, 270 - 274
270

Table 1- Chemical plaque control agents
Bisbiguanide antiseptics
Bisbiguanide compounds are a group of chemical plaque
control agents and comprises of the following agents
 Chlorhexidine
 Alexidine
 Octenidine
From a therapeutic point of view, the most obvious benefit of
using mouth rinses is the potential to reduce plaque and
gingivitis and particularly in youngsters where mechanical
plaque control is not optimal in maintaining gingival health.
The ADA council for scientific affairs has proposed a
program for acceptance of plaque control agents. These
include that the patients be evaluated in placebo control trials
of 6 months or longer and demonstrate significantly improved
gingival health compared with controls [3].
To date the ADA has accepted 2 agents for treatment of
gingivitis which include prescription solution of
chlorhexidine digluconate oral rinse and non prescription
essential oil rinse.
CHLORHEXIDINE
History
Chlorhexidine was developed by Imperial Chemical
Industries in England during 1940’s.It was marketed as a
general antiseptic in the year 1950. In 1957 chlorhexidine
was introduced for human use in Britain as an antiseptic for
skin. Later it was widely used in medicine and surgery.
Plaque inhibition first investigated by Schroeder in 1969[4]. A
definitive study for caries inhibition by inhibition of dental
plaque was done by Loe and Schiott 1972[5].
Forms
Chlorhexidine is available in various forms such as
digluconate, acetate and hydrochloride salts which are
sparingly soluble in water.
Structure
Chlorhexidine is a symmetrical molecule.It has four
chlorophenyl rings and two biguanide groups connected by a
central hexamethylene bridge. (figure 1)
Figure 1- Structure of chlorhexidine
Characteristics
Chlorhexidine is an antimicrobial agent. It acts on the inner
cytoplasmic membrane hence it is a membrane active type of
substance. It is dicationic at pH levels above 3.5. It prevents
plaque accumulation, hence it is a antiplaque and
antigingivitis agent[6] and reduces the adherence of
Porphyromonas gingivalis to epithelial cells[7]. It can be
bacteriostatic or bactericidal depending on the dose. It acts
against a wide array of bacteria including Gram positive and
Gram negative bacteria, dermatophytes and lipolytic viruses.
It also acts against fungi, yeasts and some viruses including
Hepatitis B virus and Human Immunodeficiency Virus. It
acts against Streptococcus mutants making it anticariogenic
in nature. Studies have also shown that chlorhexidine has the
ability to neutralize pathogenic agents such as Streptococcus
aureus, Porphyromans gingivalis and Prevotella intermedia.[8]
Another most important unique property of chlorhexidine is
its substantivity. Substantivity refers to the oral retentiveness.
It depends upon various factors such as concentration, pH,
temperature and time of contact of the solution with oral
structures.[9]
Mechanism of action of chlorhexidine:
The mechanism of action of chlorhexidine is outlined in
table 2.
COMPOUNDS AGENTS
Enzymes Protease, Lipase, Nuclease, Dextranase, Mutanase, Glucoseoxidase, Amyloglucosidase
Bisbiguanides Chlorhexidine, Alexidene, Octenidine
Quaternary Ammonium
Compounds Cetyl pyridinium chloride, Benzalconium Chloride
Phenolic compounds Thymol, 4-Hexylresorcinol, 2-Phenylphenol Eucalyptol, Listerene
Fluorides Sodium fluoride, Sodium monofluorophosphate Stannous fluoride, Amine fluoride
Metal ions Copper, Zinc, Tin
Oxygenating agents Peroxides
Other Antiseptics Iodine, Povidone iodine, Chloramine-T Sodium hypochlorite, Hexetidine, Triclosan
Salifluor, Delmopinol
Shruti Balagopal et al /J. Pharm. Sci. & Res. Vol.5(12), 2013, 270 - 274
271

Table 2- Mechanism of action of chlorhexidine
CHLORHEXIDINE FORMULATIONS
Mouthrinses
Chlorhexidine mouth rinses are available in the form of 0.2%
and 0.12%.There is equal efficacy for 0.2%and 0.12% rinses
when used at appropriate similar doses [10]. The time of
rinsing is 30 or 60 seconds depending on the adsorption rate
of antiseptics to the oral surfaces (50% of chlorhexidine binds
to receptors within 15 seconds) but this does vary from
individual to individual. The plaque inhibiting effect of a
0.2%chlorhexidine with rinsing times of 15, 30 and 60
seconds following a 72 hour non brushing period showed no
difference [11]. The ideal regimen is twice daily (morning and
night) which will have a substantivity for 12 hours.
The addition of fluoride to chlorhexidine is considered
questionable [12]. The concentration of 0.06% and sodium
fluoride 0.2% and 0.055% of stannous fluoride was
considered compatible with fluoride [13]. The chlorhexidine
monofluorophosphate complexes was considered
incompatible without fluorides [14].
Gel
The different available concentrations of chlorhexidine gel
are 1%, 0.2%, 0.12%. They are delivered in trays and
toothbrushes. Chlorhexidine gel, that is applied once a day
has therapeutic effects, like reducing oral malodour and
also reduces chlorhexidine staining [15].
Toothpastes
0.12% of chlorhexidine with 1 parts per million of fluoride
has antiplaque effects similar to chlorhexidine mouthwash.
However there were difficulties in incorporating
chlorhexidine into gels and toothpastes.1% chlorhexidine
used as slurries and rinsed twice per day for one minute
causes significant reduction in the plaque and gingival scores
but also causes stains. Chlorhexidine in dentifrices gained
little attention due to its possible interaction with anionic
ingredients contained in toothpaste and competition for oral
retention sites [16].
Sprays
0.1% and0.2% sprays have similar plaque inhibition
properties of 0.2% mouthwash. It is well received by
physically and mentally handicapped patients [17].
Varnishes
Chlorhexidine varnishes are used for prophylaxis against root
caries [18].
Sugar free chewing gum
Chlorhexidine remains unbound in this form. It contains
20mg of chlorhexidine diacetate. It is advised to chew 2
The bacterial cell wall is negatively charged and contains sulphates and phosphates
Dicationic positively charged chlorhexidine is attracted to the negatively charged bacterial cell wall with specific and strong
adsorption to phosphate containing compounds
Alters the integrity of the bacterial cell membrane and chlorhexidine is attracted to the inner cell membrane
Chlorhexidine binds to the phospholipids in the inner membrane and there is leakage of low molecular weight compounds
like potassium ions
By increasing the concentration of chlorhexidine there is progressive damage to the membrane
There is coagulation and precipitation of the cytoplasm by the formation of phosphate complexes which include adenosine
triphosphate and nucleic acids
Cytoplasm of the cells are chemically precipitated
Bactericidal stage which is irreversible
Shruti Balagopal et al /J. Pharm. Sci. & Res. Vol.5(12), 2013, 270 - 274
272

pieces twice per day for 10 minutes. This procedure is said to
cause less stains. It is a good method of using chlorhexidine
for a long period of time [19].
CLINICAL APPLICATIONS OF CHLORHEXIDINE
It is used as an adjunct to oral hygiene and professional
prophylaxis. It is used post oral surgery in periodontal
surgery or root planing. Studies have shown that the daily use
of mouthrinse combined with toothbrushing resulted in
reduced interproximal plaque when compared with
toothbrushing and daily flossing [20]. Chlorhexidine is of
importance in the maintainance protocol in immediate
function implants as there is a correlation between plaque and
bleeding index revealed a good result for 0.2% chlorhexidine
gel for daily implant self care at 6 months [21]. It is used in
patients with intermaxillary fixation and in patients who are
under high risk of caries. For those patients who are
physically and mentally handicapped chlorhexidine sprays
can be used [22]. It is used in medically compromised patients
who are predisposed to oral candidiasis. Chlorhexidine is
used to limit the bacteremia and operatory contamination by
oral bacteria and as an adjunct to antibiotic prophylaxis.
Other uses of chlorhexidine include sub gingival irrigation,
management of denture stomatitis, hypersensitivity and for
oral malodour. Full mouth disinfection has been introduced
with scaling and root planing and the application of
chlorhexidine into periodontal pockets with daily use of
chlorhexidine rinses at home for 2 months [23]. Chlorhexidine
formulations have well proven short to medium term
application as adjuncts and even replacements for mechanical
cleaning but it is still controversial [24]. Wound healing is
enhanced when chlorhexidine rinses are used before
extractions and after scaling and root planing or periodontal
surgery [25]. Chlorhexidine is shown to induce changes to
human gingival fibroblast collagen production and non
collagen protein production [26]. There is 65% reduction in
collagen production and a 75% reduction in non collagen
protein production.
Halita is the name of a mouth rinse containing 0.05% of
chlorhexidine, 0.05% cetyl pyridinium chloride and 0.14% of
zinc lactate. It is used in the management of halitosis [27, 28].
Zinc is added as it has the ability to convert volatile sulfur
compounds and it also acts synergistically with
chlorhexidine. Other clinical benefits include its use as a root
canal irrigant [29] and in atraumatic restorative treatment
where chlorhexidine containing glass ionomer cement [30].
Chlorhexidine is also used for surgical skin preparation for
the patient and the surgeon. Chlorhexidine is used as a local
drug delivery system in the form of a bio-degradable chip to
be used in the subgingival environment [31]. There is
controlled delivery of chlorhexidine to the periodontal
pocket. A slow sub-gingival release of 2.5mg of
chlorhexidine is found to have an average drug concentration
greater than 125 microgram per milliliter for 7 to 10 days.
The concentration of the drug remains above the minimum
inhibitory concentration for more than 99% of the
subgingival micro-organisms from the periodontal pockets.
The results of several clinical trials have shown that the use
of the chlorhexidine chip in conjunction with scaling and root
planing is effective in reducing periodontitis, clinical
attachment loss and bleeding on probing over a period of 6 to
9 months. The use of the controlled release of chlorhexidine
delivery system during maintenance therapy allows greater
improvement in clinical signs of periodontitis. In subgingival
exudates, the serum proteins may effectively compete with
the bacteria for the chlorhexidine, thus reducing the
availability of the drug. This might account for the occasional
lack of clinical effectiveness when the agent is administered
subgingivally [32].
Toxicology and side effects [33]
The side effects of chlorhexidine include brown
discolouration of the teeth, restorative materials and dorsum
of tongue. There is taste perturbation. There can be oral
mucosal erosion which is an idiosyncratic reaction and is
dose dependent. The bitter taste is difficult to mask.
Chlorhexidine staining
There is degradation of the chlorhexidine molecule to release
parachloroaniline. Non enzymatic browning reactions take
place called catalysis of Maillard. Protein denaturation and
metal sulfide formation occurs and there is precipitation of
anionic dietry chromogens [34].
Metabolism of chlorhexidine
The chlorhexidine that is swallowed undergoes minimal
metabolic changes. It has a half life of 4 days and it is
excreted in faeces.
Safety of chlorhexidine
Chlorhexidine is poorly absorbed in the gut and displays very
low toxicity. It does not cause any teratogenic alterations.
There is no evidence of formation of carcinogenic substances.
Precautions
After the use of chlorhexidine mouthwash the intake of tea,
coffee and red wine must be avoided. The usage is restricted
in cases of anterior composite restorations and glass ionomer
restorations. There should be a 30 minute lapse between the
usage of a dentifrice and chlorhexidine mouth wash [35]. It is
so advised because the toothpastes contain detergents which
are predominantly anionic agents. Chlorhexidine molecule
being dicationic tends to bind with the anionic agents leading
to a reduction in the substantivity of chlorhexidine
mouthrinse.
Comparative studies
Increasing the rinsing frequency of cetyl pyridinium chloride
to four times a day has been suggested to produce efficacy
equivalent to that of chlorhexidine [8]. Listerine has a
moderate plaque inhibitory effect poor oral retention and
some antigingivitis effects [36]. Substantivity of hexitidine
is one to three hours and has some plaque inhibitory effects
[37]. The effect of three commercial mouth rinses on cultured
human gingival fibroblast, an in vitro study revealed that
chlorhexidine, listerine and povidone iodine are capable of
inducing a dose dependant reduction in cellular proliferation
of fibroblasts [38].
Shruti Balagopal et al /J. Pharm. Sci. & Res. Vol.5(12), 2013, 270 - 274
273

CONCLUSIONS
Chronic periodontitis is always preceded by chronic
gingivitis; chemicals that inhibit plaque may be expected to
be of value in both the prevention and management of
periodontal disease in some individuals. Thus, the use of a
chemical plaque-inhibitory mouthwash as an adjunct to tooth
brushing may have a major effect on improving the oral
health of the individual. Chlorhexidine is one chemical
plaque control agent which has various clinical applications
in dentistry especially in Periodontics . Chlorhexidine in its
various formulations has come to stay and it is appropriate to
call it the gold standard chemical plaque control agent.
REFERENCES:
1. Beck JD, Koch GG, Rosier RG, Tudor GE; Prevalence and risk
indicators for periodontal attachment loss in population of older
community-dwelling blacks and whites, J Periodontol 1971;61:521
2. Addy et al Chlorhexidine digluconate–an agent for chemical plaque
control and prevention of gingival inflammation: J Periodont res 1986;
21:16 74-89.
3. Rethman MP, Beltrán-Aguilar ED, Billings RJ, Hujoel PP, Katz BP,
Milgrom P, Sohn W, Stamm JW, Watson G, Wolff M, Wright JT, Zero
D, Aravamudhan K, Frantsve-Hawley J, Meyer DM; American Dental
Association Council on Scientific Affairs Expert Panel on Nonfluoride
Caries-Preventive Agents. Nonfluoride caries-preventive agents:
executive summary of evidence-based clinical recommendations. J Am
Dent Assoc. 2011; 142(9):1065-1071.
4. Schroeder H E. Formation and Inhibition of Dental Calculus. Hans
Huber, Berlin 1969; 145-172.
5. Löe H, Von der Fehr FR, Schiött CR. Inhibition of experimental caries
by plaque prevention. The effect of chlorhexidine mouthrinses Scand J
Dent Res. 1972;80(1):1-9
6. Corbet EF, Tam JO, Zee KY, Wong MC, Lo EC, Mombelli AW.
Therapeutic effects of supervised Chlorhexidine mouthrinses on
untreated gingivitis. Oral Dis. 1997; 3:9–18.
7. Grenier D, Effect of chlorhexidine on the adherence properties of
Porphyromonas gingivalis, J Clin Periodontol. 1996 Feb; 23(2):140-2.
8. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-
Filho FJ. In vitro evaluation of the antimicrobial activity of
chlorhexidine and sodium hypochlorite. Oral Surg, Oral Med, Oral
Pathol, Oral Radiol Endod. Jan 97(1), 79–84.
9. Bonesvoll P,Gjermo P. A comparison between chlorhexidine and some
quaternary ammonium compounds with regard to retention,salivary
concentration and plaque inhibiting effect in human mouth after mouth
rinses. Arch Oral biol 1978, 23; 289-294.
10. Hoffmann, T., Bruhn, G., Richter, S., Netuschil,L. & Brecx, M. clinical
controlledstudy on plaque and gingivitis reduction under long term use
of low dose chlorhexidine solutions in a population exhibiting good
oral hygiene. Clin Oral Investig 2001, 5: 89–95.
11. Van de weijden GA, Timmerman MF, Novtny GA, Rosema N, Verkerk
A, Three different rinsing times and inhibition of plaque accumulation
with chlorhexidine, J Clin Periodontol 2005, 32(1):89-92.
12. Regolati, B.; Schmid, R.; and Muhlemann, H.R.: Combination of
Chlorhexidine and Fluoride in Caries Prevention. An Animal
Experiment, Helv Odont Acta 1974,18: 12-16.
13. Lorenz K, BruhnG, Heumann C, NetuschilL, Brecx M, Hoffmann;
Effect of two new chlorhexidine mouthrinses on the development of
dental plaque, gingivitis, and discolouration. A randomized,
investigator-blind, placebo-controlled, 3-week experimental gingivitis
study, J Clin Periodontol, Aug 2006; 33(8):561-7
14. Barkvoll, Marinho, Home use oral hygiene products: mouthrinses, J
Clin Periodontol, Oct 2008,48 (1):42-53,
15. Addy M,Moran JM, Clinical indications for the use of chemical
adjuncts to plaque control, Periodontol 2000, 1997 Oct;15:52-54.
16. Dolles, Gjermo, The effects of a chlorhexidine toothpaste on the
development of plaque,gingivitis and tooth staining, J Clin
Periodontol, January 1993, 20,59-62.
17. Kalaga A, Addy M, Hunter B: The use of a 0.2% chlorhexidine spray
as an adjunct to oral hygiene and gingival health in physically and
mentally handicapped adults. J Periodontol 1989, 60: 381–385.
18. Matthijs S, Adriaens P A, Chlorhexidine varnishes: a review, J
Periodontol, 2002, 29:1-8.
19. Ainamo J, Etemadzadeh H, Prevention of plaque growth with chewing
gum containing chlorhexidine acetate, J Clin Periodontol
1987,14(9):524-7
20. Zimmer, S., Kolbe, C., Kaiser, G., Krage, T., Ommerborn, M. &
Barthel, C. Clinical efficiency of flossing versus use of antimicrobial
rinses. J Periodontal 2006 Aug; 77(8):1380-5.
21. De Araujo Nobre M, Cintra N, Malo P, Peri implant maintainance of
immediate function implants:a pilot study comparing hyaluronic acid
and chlorhexidine, Int J Dent Hyg. 2007 May;5(2):87-94.
22. Roncati M, Polizzi E, Cingano L, Lucchese A, An oral health aid for
disabled patients, Dent Cadmos 2013; 81(7):447-452
23. Quirynen M, Mongradi, van steenberghe D, The effect of a one stage
full mouth disinfectionon oral malodour and microbial colonization of
tongue in periodontitis, J Periodontol 1998,69(3): 374-82.
24. Greenstein G, Berman C, Jaffin R, Chlorhexidine. An adjunct to
periodontal therapy. J Periodontol 1986; 57 (6): 370-6.
25. Sridhar V, Wali GG, Shyla HN, Evaluation of the Perioperative Use of
0.2% Chlorhexidine Gluconate for the Prevention of Alveolar Osteitis
After the Extraction of Impacted Mandibular Third Molars: A Clinical
Study. J Maxillofac Oral Surg. 2011 Jun;10(2):101-11
26. Angelo J. Mariotti, Chlorhexidine-Induced Changes to Human
Gingival Fibroblast Collagen and Non-Collagen Protein Production, J
Periodontol, Dec 1999; 70(12):1443-1448
27. Winkel EG, Roldán S, Van Winkelhoff AJ, Herrera D, Sanz M,
Clinical effects of a new mouthrinse containing chlorhexidine,
cetylpyridinium chloride and zinc-lactate on oral halitosis. A dual-
center, double-blind placebo-controlled study, J Clin Periodontol. 2003
Apr;30(4):300-6
28. van Steenberghe D, Avontroodt P, Peeters W, Pauwels M, Coucke
W, Lijnen A, Quirynen M, Effect of different mouthrinses on morning
breath, J Periodontol, 2001; 72(9):1183-1191.
29. Ferguson JW, Hatton JF, Gillespie MJ, Effectiveness of intracanal
irrigants and medications against the yeast Candida albicans. J Endod
2002; 28(2): 68–71.
30. Takahashi Y, Turkun M, Ertugul,Ates M,Brugger S, Long term
antibacterial effects and physical properties of chlorhexidine containing
glass ionomer cement, J Esthet Restor Dent, 2008, 20(11):29-45.
31. Soskolne WA, Heasma PA, Stabholz A, Smart GJ, Palmer M, Flashner
M, Newman HN, Sustained local drug delivery of chlorhexidine in the
treatment of periodontitis. J Periodontol. 1997 Jan;68(1):32-8
32. Jan Lindhe. Clinical Periodontology and Implant Dentistry, Fifth
edition. Blackwell Munksgard Publications, 2007.
33. Flotra L, Gjermo P, Rolla G, Waerhaug J: Side effects of chlorhexidine
mouthwashes. Scand J Dent Res, 1971; 79: 119–125.
34. EriksonH M, Nordbo H, Kantanen H, Ellingsen J E: Chemical plaque
control and extrinsic tooth discolouration. A review of possible
mechanisms. J Clin Periodontol, 1985; 12: 345–350.
35. Kolahi J, Soolari A, Rinsing with chlorhexidine gluconate solution
after brushing and flossing teeth: a systematic review of effectiveness.
Quintessence Int. 2006 Sep;37(8):605-12
36. Gordon J M, Lamster I B, Sieger M C. Efficacy of Listerine antiseptic
in inhibiting the development of plaque and gingivitis. J Clin
Periodontol 1985; 12: 697-704.
37. Harper P R, Milsom S, Wade W, Addy M, Moran J, Newcombe R G.
An approach to efficacy screening of mouthrinses: studies on a group
of French products (II) Inhibition of salivary bacteria and plaque in
vivo. J Clin Periodontol 1995; 22: 723-727
38. Flemingson , Emmadi P, Ambalavanan N,Ramakrishnan T
Vijayalakshmi R, Effect of three commercial mouth rinses on cultured
human gingival fibroblast: an in vitro study, Indian J Dent Res 2008
Jan-Mar; 19(1) :29-35.
Shruti Balagopal et al /J. Pharm. Sci. & Res. Vol.5(12), 2013, 270 - 274
274