Antimicrobial Effectiveness of an Herbal Mouthrinse Compared With an Essential Oil and a Chlorhexidine Mouthrinse
Anne D. Haffajee, BDS,
Tina Yaskell, BS and
Sigmund S. Socransky, DDS
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ABSTRACT
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Background. The authors investigated mouthrinses’ antimicrobial effectiveness against predominant oral bacteria, as determined by the minimum inhibitory concentration (MIC). Specifically, they evaluated an herbal mouthrinse, an essential oil rinse and a 0.12 percent chlorhexidine gluconate rinse.
Methods. The authors assessed the inhibitory effects of the three test agents against 40 oral bacteria at concentrations of 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 micrograms per millliter. They inoculated plates containing basal medium and the test agents with suspensions of the test species and incubated them anaerobically at 35°C. The authors interpreted the MIC as the lowest concentration of the agent that completely inhibited the growth of the test species.
Results. The herbal mouthrinse inhibited the growth of most of the 40 test species. Compared with the essential oil mouthrinse, the herbal mouthrinse exhibited significantly lower MICs for Actinomyces species, periodontal pathogens Eubacterium nodatum, Tannerella forsythia and Prevotella species, as well as the cariogenic pathogen Streptococcus mutans. The chlorhexidine gluconate rinse had the lowest MICs compared with the essential oil rinse and the herbal rinse for all test species examined.
Conclusions. Although less potent than the chlorhexidine gluconate rinse, the herbal rinse was more effective than the essential oil rinse in inhibiting the growth of oral bacteria in vitro.
Clinical Implications. The data suggest that the herbal mouthrinse may provide oral health benefits by inhibiting the growth of periodontal and cariogenic pathogens. In vivo clinical testing is essential to confirm in vitro results.
Key Words: Antimicrobial mouthrinse; oral bacteria; natural oral antimicrobial agentAbbreviations: MIC: Minimum inhibitory concentration.
There is growing interest throughout the oral health care profession in therapeutic agents that complement and enhance the mechanical removal of biofilms in the oral cavity. The gram-positive and gram-negative bacteria that compose oral biofilms produce various metabolites that induce gingival inflammation (that is, gingivitis). Gingivitis can lead to periodontitis, a condition in which gingival and bone tissues are destroyed.1
Periodontal diseases are among the most common infectious diseases affecting humankind and can lead to destruction of the periodontal ligament, cementum, gingiva and alveolar bone. Inflammation is key to the progression from gingivitis to periodontal disease, particularly when gingival inflammation transitions to an exaggerated inflammatory host immune response.2–4
The majority of the population may not perform mechanical plaque removal sufficiently. Thus, antimicrobial mouthrinses that augment daily home care may provide an effective means of removing or controlling bacterial plaque to limit gingivitis and periodontitis.5 In fact, it has been shown that chemotherapeutic mouthrinses are an effective adjunct to regular brushing and flossing for patients with gingivitis, providing a clinically significant and meaningful benefit in the reduction of plaque and gingivitis.6 Mouth-rinses have the ability to deliver therapeutic ingredients and benefits to all accessible surfaces in the mouth—including interproximal hard and soft tissues—and, depending on their composition, remain active for extended periods.2,7,8
Clinicians frequently recommend mouthrinses to their patients as useful in helping reduce dental plaque and control gingivitis. These recommendations are supported by documentation of the long-term plaque- and gingivitis-reducing characteristics of two specific antimicrobial mouthrinses: one based on essential oil (Listerine Cool Mint, Johnson & Johnson, New Brunswick, N.J.) and the other on chlorhexidine (Peridex, Zila Pharmaceuticals, Phoenix).9–11 Both have been shown to inhibit the development of plaque and gingivitis significantly.10,11
However, today’s dentists are practicing in an era of greater patient enlightenment relating to both their oral health and their overall medical well-being. Consumers in general take a more educated and whole-body approach to their health and care. In response, personal product manufacturers have begun to offer a variety of "natural" products designed to help consumers realize and maintain their whole-body health without "artificial" pharmaceuticals. Dental product manufacturers in the United States have been among those attentive to the need for natural oral care products for many years, particularly in their use of such plant extracts as grapefruit seed extract, which has been shown to inhibit a wide range of bacterial biotypes (that is, both gram-positive and gram-negative),12 as well as potent antioxidant and protective properties that have been beneficial in gastric health.13
In 2002, researchers reported that grapefruit seed extract demonstrates antimicrobial properties at concentrations that are safe.14 In 2004, others reported that the glyceric extract from grapefruit seeds exhibited better antioxidant properties when dissolved in aqueous solutions than when dissolved in alcoholic solutions.15
Given the growing evidence of the connection between oral health and whole-body health, dental practitioners may seek to respond to their patients’ oral hygiene needs by providing research-based products with "naturally occurring" active ingredients that achieve the desired antimicrobial and anti-inflammatory effects. Products that contain such natural ingredients as grapefruit seed extract may be appealing because they work without alcohol,15 artificial preservatives, or artificial flavors and colors.
The Natural Dentist Healthy Gums Daily Oral Rinse (The Natural Dentist, Medford, Mass.) (formerly named Herbal Mouth and Gum Therapy) has been shown to have beneficial effects in the oral environment—such as reducing gingival bleeding and gingivitis16 and inhibiting the growth of aerobic, microaerophilic and anaerobic bacteria.17 It also has been shown to have in vitro efficacy against three specific strains of oral bacteria: Streptococcus mutans, Streptococcus sanguinis and Actinomyces viscosus.18 The mouth-rinse contains several naturally occurring anti-inflammatory agents, such as aloe vera and calendula, and antimicrobial agents such as goldenseal and grapefruit seed (Table 1
).14,19–25
When applied specifically against the broad spectrum of oral bacteria responsible for gingivitis, plaque and periodontal disease, this herbal mouthrinse may prove beneficial for maintaining oral health. Thus, we undertook a study to test the in vitro efficacy of an herbal mouth-rinse (The Natural Dentist Healthy Gums Oral Rinse), an essential oil mouthrinse (Listerine Cool Mint) and a 0.12 percent chlorhexidine gluconate mouthrinse (Peridex) against predominant oral bacteria, as determined by the minimum inhibitory concentration (MIC).
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MATERIALS AND METHODS
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We conducted in vitro testing to determine the efficacy of a natural oral rinse compared with those of two other oral mouthrinses (Listerine Cool Mint and Peridex [0.12 percent chlorhexidine gluconate]), as determined by the MIC, against reference strains of 40 predominant oral bacterial species. We performed all MICs in duplicate, and we used an agar dilution method to assess the inhibitory effect of the three test agents.
Strains employed.
Table 2 (page 609) summarizes the strains we used. The numbers next to each species indicate the American Type Culture Collection (Manassas, Va.) strain designation.
Basal medium.
One of the authors (T.Y.), a laboratory technician, prepared the basal medium, which consisted of trypticase soy agar (40 grams per liter), hemin (5 milligrams/L), N-acetylmuramic acid (10 mg/L), menadione (0.5 mg/L) and sheep blood (50 milliliters/L). She added the last three ingredients aseptically after autoclaving. She sterilized the basal medium in individual tubes (one per agar plate) by autoclaving the tubes at 121°C for 15 minutes and then placing them in a 50°C water bath. She adjusted the volume in each tube so that the final volume totaled 20 mL when the filter-sterilized test agents were added.
Preparation of test media.
The laboratory technician filter-sterilized the test herbal mouthrinse (which was supplied by The Natural Dentist) and Listerine Cool Mint and Peridex (which were supplied by The Forsyth Institute) by passing them through a 0.22-micrometer Nalgene (Nalge Nunc International, Rochester, N.Y.) filter. She prepared dilutions of each agent to provide final concentrations of 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 micrograms/mL. She dispensed the media aseptically in a total of 20 mL in 100-millimeter plastic Petri plates, allowed them to set and used them the next day.
Preparation of inoculum.
The laboratory technician grew the organisms on blood agar plates under an atmosphere of 80 percent nitrogen, 10 percent hydrogen and 10 percent carbon dioxide at 35°C for three days. She harvested growth from the plate aseptically and suspended it in 1 mL of sterile mycoplasma broth. She adjusted each suspension to an optical density approximating a McFarland 0.5 standard.
Inoculation of test media.
For each strain suspension, the laboratory technician placed 100 microliters in the wells of a sterile microtiter plate. She used an MIC 2000 inoculator (Dynatech, Boonville, Ind.) to transfer 1.5 µL of inoculum to the surface of the prepared plates. She inoculated control plates without the test agents at the beginning and end of the series of inoculations. She used these plates to indicate that the inoculum was present and viable throughout the series. The laboratory technician performed all tests in duplicate.
Reading of plates and interpretation of data.
The laboratory technician read the plates, and the other two authors (A.D.H. and S.S.S.) interpreted her findings. The laboratory technician incubated the plates under an atmosphere of 80 percent nitrogen, 10 percent hydrogen and 10 percent carbon dioxide at 35°C and evaluated daily. We interpreted the MIC as the lowest concentration of the agent that completely inhibited the growth of the test species.
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RESULTS
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The herbal mouthrinse formulation effectively inhibited the growth of the majority of the 40 test species (Table 2
). Compared with Listerine Cool Mint, The Natural Dentist Healthy Gums Oral Rinse exhibited significantly lower MICs (that is, a greater than twofold difference in MICs) for Actinomyces species; the periodontal pathogens Eubacterium nodatum, Tannerella forsythia and Prevotella species; and the cariogenic pathogen S. mutans (Table 2; Figure). For all test species examined, Peridex had the lowest MICs compared with Listerine Cool Mint and The Natural Dentist Healthy Gums Oral Rinse.
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Figure. Minimum inhibitory concentration (MIC) values for the 40 test species for the three mouthrinses tested. The species appear in order from those with the lowest MIC scores to those with the greatest MIC scores. Peridex is manufactured by Zila Pharmaceuticals, Phoenix. The Natural Dentist Healthy Gums Oral Rinse is manufactured by The Natural Dentist, Medford, Mass. Listerine Cool Mint is manufactured by Johnson & Johnson, New Brunswick, N.J.
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DISCUSSION
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The results of this in vitro study indicated that the mouthrinses we evaluated demonstrated a range of inhibitory effects on the test bacteria. Not surprisingly, the chlorhexidine gluconate rinse had the greatest effect. However, the herbal mouthrinse compared favorably, particularly in inhibiting the growth of Actinomyces species; the periodontal pathogens E. nodatum, Prevotella intermedia, Prevotella melaninogenica, Prevotella nigrescens and T. forsythia; and the caries pathogen S. mutans.
The effectiveness of the herbal mouthrinse likely was due to the antimicrobial effect of several of the ingredients, as listed in Table 1. Goldenseal has been shown to have antimicrobial properties against such oral pathogens as S. mutans and Fusobacterium nucleatum.19 Grapefruit seed extract also is a proven antimicrobial agent, having shown its effectiveness against various bacteria, yeasts and viral strains—including a wide range of gram-positive and gram-negative organisms.14,20 Furthermore, the antibacterial mechanisms of grapefruit seed extract, even at more dilute concentrations, disrupt bacterial membranes, releasing the cytoplasmic contents within 15 minutes after contact.14
In vitro evaluation of the antimicrobial activity of test agents or mixtures of test agents is a common first step in determining potential therapeutic efficacy. To permit comparisons of efficacy among the test mouthrinses, the data routinely are expressed as MICs of the test agent(s) against a range of microbial species of interest—in this case, 40 species. The MIC usually is performed using broth or agar dilution techniques; serial dilutions of the agent(s) are incorporated in agar (or broth) media, and the lowest concentration of the agent that completely inhibits growth of a given strain is recorded as the MIC. While the methods are routine, occasionally one encounters pitfalls for some agents or combinations of agents. These include insolubility of the agent(s) in the test medium; unforeseen interaction of a medium constituent with one or more of the test agents (for example, precipitation); or volatility of an important ingredient of the test mixture, such as alcohol.
Another important consideration is the range of expected MICs. For antibiotics, this typically would be in the microgram- or submicrogram-per-milliliter range; for other antibacterial agents, the range might be much higher, leading to difficulty in dissolving the test product.
Nonetheless, if possible to perform on a given formulation, the MIC is the most reliable and easily interpreted method of comparison of formulations in use today. In this evaluation, we found the ingredients in the natural herbal mouthrinse to inhibit the growth of predominant oral bacteria and cariogenic pathogens.
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CONCLUSION
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In this study, we found that the ingredients in the herbal oral rinse were effective in inhibiting oral bacteria, thereby demonstrating the agent’s antimicrobial efficacy and its potential usefulness in controlling plaque and gingivitis. Although less potent than chlorhexidine, the herbal mouthrinse was a more effective antimicrobial agent than was the essential oil mouthrinse in inhibiting the growth of oral bacteria in vitro. Furthermore, it may serve as a natural antimicrobial mouthrinse alternative for patients who wish to avoid alcohol, artificial preservatives, and artificial flavors and colors. Our research group is conducting clinical studies in humans to further clarify and broaden our understanding of the role of this herbal mouthrinse in the management of periodontal disease.
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FOOTNOTES
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Dr. Haffajee is a senior member of the staff and the head, Department of Periodontology, The Forsyth Institute, 140 The Fenway, Boston, Mass. 02115-3799, e-mail "ahaffajee{at}forsyth.org". Address reprint requests to Dr. Haffajee.
Ms. Yaskell is a staff associate, The Forsyth Institute, Boston.
Dr. Socransky is a senior member of the staff, The Forsyth Institute, Boston.
Disclosure. The research described in this article was supported by an educational grant to The Forsyth Institute under the direction of Dr. Haffajee from The Natural Dentist, Medford, Mass.
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ABSTRACT
MATERIALS AND METHODS
