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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 52-56

Differences in antimicrobial activity of 0.2% Chlorhexidine mouthwash and a Herbal mouth rinse formulation—A salivary bacterial counts study


1 Department of Periodontology, P.M. Nadagouda Memorial Dental College and Hospital, Bagalkot, Karnataka, India
2 Department of Periodontology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
3 Consultant Periodontist, Bangalore, Karnataka, India
4 Consultant Periodontist, Pune, Maharashtra, India
5 Department of Periodontology, MNR Dental College, Sangareddy, Telangana, India
6 Department of Oral pathology, Subbaiah Institute of Dental Sciences, Shimoga, Karnataka, India

Date of Submission05-Jun-2019
Date of Acceptance13-Jan-2020
Date of Web Publication14-May-2020

Correspondence Address:
Dr. C Naresh Kumar
Department of Periodontology, Vishnu Dental College, Bhimavaram
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_66_19

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  Abstract 


Background: Chlorhexidine gluconate (CHX) mouthwash has earned eponym of gold standard to treat or prevent periodontal disease. However, it has been reported to have local side-effects on long-term use. To explore a herbal alternative, the present study was carried out to evaluate the clinical antimicrobial activity of two mouth rinses. One containing 0.2% CHX and other containing herbal mouth rinse (HM).
Methods: This randomized, crossover salivary bacterial counts, was carried out on a sample of 10 healthy volunteers rinsed for 1 minute with 0.2% CHX and for 1 minute with HM. Saliva samples were obtained before rinsing, and after 5 min, and 1, 3, 5 and 7 h. These samples were cultured both aerobically and anaerobically. Percentages of survival, in regard to baseline, were calculated for each time point.
Results: Results were available both for aerobic and anaerobic salivary bacteria. Statistically significant difference is observed between Herbal and 0.2% CHX mouth wash at 1 hr (P < 0.05), 3 hrs (P < 0.01), 5 hrs (P < 0.01) as well as at 7 hrs (P < 0.001) and HM and 0.2% CHX mouth wash at 5 minutes after using mouth wash (P < 0.05), 1 hr (P < 0.05), 3 hrs (P < 0.01), 5 hrs (P < 0.01) as well as at 7 hrs (P < 0.001).
Conclusion: Based on the results, this clinical study demonstrated that significant reduction of microbial activity was observed in 0.2% CHX when compared with HM containing bibhitaka, nagavali and salvodora persica as main ingredients.

Keywords: 0.2% Chlorhexidine, antimicrobial activity, colony forming unit, herbal mouth rinse


How to cite this article:
Rao SM, Kumar C N, Jumani PN, Patil AS, Kumar RS, Marullappa R. Differences in antimicrobial activity of 0.2% Chlorhexidine mouthwash and a Herbal mouth rinse formulation—A salivary bacterial counts study. J NTR Univ Health Sci 2020;9:52-6

How to cite this URL:
Rao SM, Kumar C N, Jumani PN, Patil AS, Kumar RS, Marullappa R. Differences in antimicrobial activity of 0.2% Chlorhexidine mouthwash and a Herbal mouth rinse formulation—A salivary bacterial counts study. J NTR Univ Health Sci [serial online] 2020 [cited 2020 Sep 26];9:52-6. Available from: http://www.jdrntruhs.org/text.asp?2020/9/1/52/284318




  Introduction Top


Chlorhexidine is considered at present to be the most effective antiplaque agent[1],[2] and has been recommended for a variety of uses in clinical dentistry.[3],[4] Commercially produced mouthwashes are perphaps most commonlyprescribed and a number of products are available. The products vary not only in chlorhexidine concentration and formulation but also regimens of use.[5]

Chlorhexidine (CHX) is, beyond a doubt, the most studied and efficacious antimicrobial agent in the chemical control of dental plaque, being considered as the positive control (goldstandard), to which all other antiplaque agents should be compared.[6] It is a cationic bis-biguanide, with a wide antibacterial activity, low toxicity on mammalian cells, and a high affinity to attach to skin and mucous membranes. Its mechanism of action includes direct damage to the internal cytoplasmatic membrane, being bacteriostatic at low dosages and bactericidal at high concentrations. Its benefits doesn't seem to be solely supported its antimicrobial properties however addtionalon its affinity to connect to a large sort of substrates. This property, called substantivity, permits this compound to achive efficacious antibacterial drug levels, employing a cheap dose (twice a day), and hence permitting patients to fits itsuse.[7]

Because of this demonstrated efficacy, chlorhexidine has been included in different vehicles for intraoral use, such as mouthwashes, gels, dentifrices, sprays, chewing gums, etc.; at different concentrations; and in different formulations, either associated with other active products, or with the goal of obtaining a more stable formulation, or reducing the secondary effects.[8]

Local side effects, notably toothstaining, ever limit the long term use of this antiseptic and regimens of use are typically short to meditim term, particularly when mechanical cleaning is difficult or impossible.

Research in chemical plaquecontrol continues, but, with the exception of chlorhexidine, success has been limited and this antiseptic remains the positive control by which many agents are compared.[9]

In recent years, there is a growing interest in the herbal products as mouthwash or oral care products. Hence, the quest for a long-term, ideal and safe antiplaque and anti-gingivitis agent continues.

In this context, synthetic antimicrobials have been analyzed, but the increasing problems of resistance have encouraged the search for alternative agents based on herbal extracts.

The herbal mouthrinse (HM) that contains bibhitaki, nagavalli, pilu, peppermint satva, yavani satva, gandhapura taila, and ela. Research has shown that pilu (Salvadora persica), one of the primary constituent, possess antiplaque activity that is comparable to that of chlorhexidine.[10] Nagavalli (Piper betle) has also been proven to have plaque inhibitory activity in in-vitro studies.[11],[12] Other constituents (bhibhitika, peppermint satva, gandhapura taila) have encouraging antimicrobial activity that may be helpful in providing better oral care.[13],[14]

A review of the literature has shown that no previous investigation has assessed the comparative antimicrobial activity of 0.2% Chlorhexidine and Herbal mouthrinse.

So, the aim of this study was to assess the clinical antimicrobial activity of two mouth rinses. One containing 0.2% Chlorhexidine and other containing Herbal mouthrinse.


  Materials and Methods Top


The study was carried out on 10 healthy volunteer subjects of aged between 25 and 40 years (8 men and 2 women) the post graduate students in the AECS Maaruti College of Dental Sciences and Research Centre, Bangalore. Institutional Ethical Committee clearance (No. MDS/11012013/05) and informed consent was taken was taken from all the participants before commencement of the study and approved on 11th Jan 2013. The objectives of the study, was done to evaluate the clinical antimicrobial activity of two mouth rinses, one containing 0.2% Chlorhexidine and other containing Herbal mouth rinse were described to each subject. This sample was selected because it comprised a reasonably homogeneous population among whom the knowledge of oral hygiene was similar. None had any underlying systemic illness. They did not take any drugs (including antibiotics), no antiseptic mouthwash use in the week before the study and during thestudy period. They agreed to avoid any oralhygiene measures in the morningsduring the investigation period, they were nonsmokers, and they had no fixed or removable prostheses or fixed or removable orthodontic appliances.

The mouthwashes used in the study were as follows:

  1. 0.2% Chlorhexidine. (Rexidin by Indoco health products ltd, India)
  2. Herbal formulation: Bibhitaka, 10.0 mg; Nagavali, 10.0 mg; Salvodora Persica, 5.0 mg. (Hiora Regular by Himalaya Herbal Healthcare, India).


The study was a randomized, crossover design. The washout period, between evaluations, was at least 1 week. Each subject used all test products and the negative control, in a randomized order, according to a computergenerated list. All the products were codified by an external agent, in identical bottles with measuring cups. During the study days, saliva samples were obtained in the morning (at approximately 9.05 am). Then subjects were asked to rinse with their assigned product as per manufacturer instructions 10 ml for 1 min with 0.2% chlorhexidine and 15 ml for 30 seconds with herbal mouth rinse. Additional saliva samples were collected after 5 min and 1, 3, 5, and 7 h.

Saliva sampling

Unstimulated saliva samples were obtained by asking the patient to spit approximately 1 ml of saliva in a graduated tube. Samples were processed in the laboratory within 30 min, following the usual bacteriological procedures of dispersion (vortexing, 30 sec), serial dilution in PBS, and inoculation on two series of nonselective 5% horse blood agar plates (BD™), supplemented with haemin (5 mg/l) and menadione (1 mg/l). One series was incubated in air at 37°C for 24 h, and the other in anaerobic atmosphere for 48 h. After the incubation time, counting was performed on the most suitable plates (those with 30–300 colonies).

Statistical analyses

Total bacterial counts for each sampling time and product were available both for aerobic and anaerobic counts, from 10 subjects. Log-transformed CFU were calculated in order to obtain a normal distribution. The percentage of survival was calculated by dividing the CFU in each sampling time by the CFU at baseline.

Mann-Whitney test was performed at each sampling time for determining if significant differences existed between the percentages of survival for the different tested products. The Statistical Package for Social Sciences software (SPSS Inc 21.0, Chicago, II, USA) was used for data processing and data analysis.


  Results Top


Results were available both for aerobic and anaerobic salivary bacteria. Statistically significant difference was observed between Herbal and 0.2% Chlorhexidine mouth wash at 1 hr (P < 0.05), 3 hrs (P < 0.01), 5 hrs (P < 0.01), as well as at 7 hrs (P < 0.001). No significant difference was observed before using mouth wash and at 5 mins after using mouth wash (P > 0.05) [Table 1].
TABLE 1: On the day of trial

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Statistically significant difference was observed between Herbal and 0.2% Chlorhexidine mouth wash at 5 mins after using mouth wash (P < 0.05), 1 hr (P < 0.05), 3 hrs (P < 0.01), 5 hrs (P < 0.01), as well as at 7 hrs (P < 0.001). No significant difference was observed before using mouth wash (P > 0.05) [Table 2].
TABLE 2: 2ND week trial

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No significant difference was observed between aerobic and anaerobic groups at any of the time interval (P > 0.05) in Herbal group [Table 3]. No significant difference was observed between aerobic and anaerobic groups at any of the time interval (P > 0.05) in 0.2% Chlorhexidine group [Table 4].
TABLE 3: Aerobic and anaerobic differences within herbal group

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TABLE 4: Aerobic and anaerobic differences within 0.2% chlorhexidine group

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  Discussion Top


This study design allows comparisons to be made of the magnitude of the antimicrobial activity in the saliva of any mouth rinse formulation. Therefore, it was considered a useful test to assess substantivity and plaque inhibition properties.

Especially Addy et al.[7] have used this study design extensively in the literature, although slight modifications canbe found in different publications. The first postrinsing sample is usually taken after 30 min;[15],[16],[17],[18] however, in this study we chose 5 min to evaluate the immediate effect of the rinsing. The bacteriologic methodology may additionally disagree and this may additionally influence the results. Most of the studies have only assessed anaerobic bacteria;[15],[16],[17],[18] however, the addition of aerobic bacteria can provide valuable information to the results, as shown in the present and other studies.[19] The time of incubation also shows some variability. One study[7] employed an anaerobic incubation period of 12 h, while others extended that period up to 72 h.[16],[18] The most frequent incubation period, also used in the present study, is 48 h.[16],[17],[18] For aerobic incubation, 24 h were used.[19] Singh et al.[20] concluded that herbal mouthrinse had a promising plaque inhibitory potential but it not as efficacious as chlorhexidine in preventing plaque regrowth, which also agreed with our present study. In another invitro study Bhat et al.[21] compared antiplaque effects of herbal and chlorhexidine rinse and found that both mouthwashes were equally effective in invitro and therefore suggested that the herbal mouthwash may be used therapeutically in future to inhibit oral microbial growth.

In this study comparing Herbal formulations with 0.2% CHX assessing their antibacterial efficacy using the study of reductions of salivary counts, demonstrated effect at 5 mins, 1 h, 3 h, 5, and as well as at 7 h, therefore CHX showed significant reduction of microbial activity then herbal mouth rinse.


  Conclusion Top


Based on the results, this clinical study demonstrated that significant reduction of microbial activity was observed in 0.2% Chlorhexidine when compared with Herbal mouth rinse containing bibhitaka, nagavali and salvodora persica as main ingredients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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2.
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Addy M, Greenman J, Renton-Harper P, Newcombe R, Doherty FM. Studies on stannous fluoride toothpaste and gel. (2). Effects on salivary bacterial counts and plaque regrowth in vivo. J Clin Periodontol 1997;24:86-91.  Back to cited text no. 7
    
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Yates R, Moran J, Addy M, Mullan PJ, Wade WG, Newcomhe R. The comparative effect of acidified sodium chlorite and chlorhexidinemouthrinses on plaque regrowth and salivary bacterial counts. J Clin Periodontol 1997;24:603-9.  Back to cited text no. 9
    
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Rahmani ME, Radvar M. The anti-plaque activity of Salvodorapersica and padina essential oil solution in comparision to chlorhexidine in human gingival disease: A randomized placebo-controlled clinical trial. Int J Pharmacol 2005;1:311-5.  Back to cited text no. 10
    
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Razak AF, Othman RY, Rahim ZHA. The effect of Piper betle and Psidiumguajava extracts on the cell surface hydrophobicity of selected early settlers of dental plaque. J Oral Sci 2006;48:71-5.  Back to cited text no. 12
    
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Sreenivasan P, Jayakumar M, Ignacimuthu S. In vitro antibacterial activity of some plant essential oils. BMC Complement Altern Med. 2006;6:39.  Back to cited text no. 14
    
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Jenkins S, Addy M, Newcombe R. Triclosan and sodium lauryl sulphate mouthwashes (I). Effects on salivarybacterial counts. J Clin Periodontol 1991;18:140-4.  Back to cited text no. 15
    
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Harper PR, Milsom S, Wade W, Addy M, Moran J, Newcombe RG. 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;22:723-7.  Back to cited text no. 16
    
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Moran J, Addy M, Wade W, Milson S, McAndrew R, Newcombe RG. The effect of oxidising mouthrinses compared with chlorhexidine on salivary bacterial counts and plaque regrowth. J Clin Periodontol 1995;22:750-5.  Back to cited text no. 17
    
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Elworthy A, Greenman J, Doherty FM, Newcombe RG, Addy M. The substantivity of a number of oral hygiene products determined by the duration of effects on salivary bacteria. J Periodontol 1996;67:572-6.  Back to cited text no. 18
    
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Moran J, Addy M, Newcombe R. The antibacterial effect of toothpastes on the salivary flora. J Clin Periodontol 1988;15:193-9.  Back to cited text no. 19
    
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Singh A, Dain A, Dixit J. The effect of herbal, essential oil and chlorhexidinemouthrinse on de novo plaque formation. Int J Dent Hygiene 2013;11:48-52.  Back to cited text no. 20
    
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Bhat N, Mitra R, Reddy JJ, Oza S, Vinayak. Evaluation of efficacy of Chlorhexidine and a Herbal mouthwash on dental plaque: An invitro comparative study. Int J Pharm Bio Sci 2013;4:625-32.  Back to cited text no. 21
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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