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Antibiotic Effects of a Natural Extracts Mouthwash
Int J Clin Prev Dent 2021;17(1):6-11
Published online March 31, 2021;  https://doi.org/10.15236/ijcpd.2021.17.1.6
© 2021 International Journal of Clinical Preventive Dentistry.

Se-Hyun Hwang1, Inchul Hwang2, Ji-Young Lee1

1Department of Dental Hygiene, Dongju College, 2Department of Health Sciences, Kosin University, Busan, Korea
Correspondence to: Inchul Hwang
E-mail: hwang@kosin.ac.kr
https://orcid.org/0000-0002-1664-9854
Received March 9, 2021; Revised March 17, 2021; Accepted March 20, 2021.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Objective: Oral mouthwash is widely used in effective management of periodontal disease prevention, plaque removal, and bad breath removal. Commercially available oral mouthwash is a chemical substance that is likely to cause side effects, and significant research on natural substances is being conducted. In this study, a safe natural extracts mouthwash was developed and compared with Listerine, which has the largest market share, to confirm antibacterial activity.
Methods: In order to evaluate the antibacterial effects of natural mouthwash Natura-A, a natural extracts extracted from 30 kinds of plants and aquatic products, with the positive control Listerine, oral samples of 10 college students in their 20s for each group were analyzed using the polymerase chain reaction (PCR) technique for strain and concentration of 18 species of oral bacteria.
Results: The antibacterial effects of Listerine and Natura-A against Agregatibacter actinomytans, Tannerella forsythia, Treponema denticola, Parvimonas micra, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens, Streptococcus mutans., Streptoccuc sobrinus, Lactobacillus casei, Staphylococcus aureus, Enteroccus faecalis, and Actinomyces viscosus apeared to be excellent. For Prevotella intermedia, Prevotella nigrescens, Streptococcus mitis, Fusobacterium nucletum, and Porphyromonas gingivals showed good antibacterial action.
Conclusion: We suggest that Natura-A has an excellent inhibitory effect on oral microbes compared to Listerine and then that this natural extracts mouthwash can be used effectively to prevent oral diseases including dental caries and periodontal diseases safely in the long term without side effects.
Keywords : mouthwash, antibiotic, Listerine, anti-bacterial, extract
Introduction

The humidity and temperature of the oral cavity are always suitable for bacteria to reside and increase. In the mouth, more than 700 species of bacteria are involved in the health and pathological conditions of the oral cavity [1] and settle in the teeth, palate, gingival, tongue, and buccal mucosa, which are suitable for the characteristics of the bacteria [2]. Dental plaque or dental biofilm is a combination of numerous bacteria in the oral cavity and contains more than 100 billion bacteria per 1 mg [3], which form mainly on the tooth surface [4]. Normally, it is present as a normal flora, however, if the number of specific bacteria in the flora increases or decreases for some reason, infection or inflammation of the periodontal tissue may occur [5]. In other words, the bacterial flora is stable in a healthy state, but the pathological flora forms a bacterial membrane in the oral cavity of a host, causing bad breath, dental caries, gingivitis, and periodontitis [6]. Therefore, interest in oral hygiene management for removal of dental biofilm is increasing in order to prevent systemic diseases related to oral bacteria as well as oral diseases.

The salivary microflora of Streptococcus salivarius, Strep-tococcus mitis, and Streptococcus sanguis, tongue microflora of S. salivarius, buccal microbiota of S. mitis, dental microflora of S. sanguis, S. salivarius, Prevotella nigrescens, and Porphyromonas gingivalis, etc. are distributed [7], Actino-bacillus actinomycetemcomitans is a bacterium associated with local aggressive periodontitis [8], and P. gingivalis, Tan-nerella forsythia, and Treponema denticola are the causative bacteria that play an important role in adult periodontal disease [9,10]. Acute progressive periodontitis, of which the main causative bacterium is P. gingivalis, occurs in most teeth without any destructive pattern, and is a periodontal disease that occurs from puberty to around 30 years of age. P. gingivalis, a gram-negative anaerobic organism, has been identified as a major causative agent of periodontitis in studies related to etiology and toxicity factors [11,12]. Periodontal disease is a chronic inflammatory disease of the tissues that support the teeth, and can lead to tooth loss due to the destruction of the alveolar bone [13]. In addition, periodontitis has been reported to show an association with a variety of systemic diseases including cardiovascular disease, diabetes, and rheumatoid arthritis [14]. Tooth brushing is the most basic and most effective for oral hygiene management to improve general and oral health through proper plaque control. However, there is a limitation in oral hygiene management using only mechanical or physical methods, therefore oral gargling solutions are widely used as a chemical method to increase the effect of removing plaque and oral bacteria [15]. According to the 2012 National Oral Health Survey, among various oral supplements other than toothbrushes and toothpastes, the use of mouthwash was the highest at 17.32% [16].

Although chemical oral mouthwash is very effective in suppressing oral microbes, there is a tendency to be reluctant to use it due to side effects. Therefore, for refreshing long-term healthy oral hygiene management, there has been a demand for a natural oral mouthwash that has no side effects and can be used safely for a long period of time. Interest in natural products for plaque management and periodontal disease prevention has resulted in conduct of significant researches on natural products that have the effect of inhibiting oral microbes [17]. This study was conducted to identify the appropriate antibacterial effect as an oral mouthwash along with the results of confirming that a toothpaste produced using a patented natural extract (Patent Registration No.: 10-1605623) appeared effective in reducing bad breath [18] and plaque [19]. To this end, 18 species of oral microbes were examined using real-time PCR (polymerase chain reaction) to compare this natural extracts mouthwash with the antibacterial effect of Listerine, which has a high market share in Korea.

Materials and Methods

1. Subjects

This study was conducted in June 2016 on 20 female students in their 20s at universities located in the Gyeongsang province. All subjects agreed to participate after comprehending the purpose of this study with use of the currently marketed Listerine and edible natural extracts mouthwash. The safety of the natural extracts mouthwash was confirmed by observing the allergic reaction with a cotton swab moistened with the natural extracts mouthwash on the subject’s lip. Among the subjects without allergic hypersensitivity reactions, this experiment included those who did not have systemic disease and did not have specifics in the oral cavity. This study was conducted after obtaining approval from the Institutional Review Board (IRB) Committee of Kosin University (IRB no. KU IRB 016-54).

2. Preparation of a natural extracts mouthwash, Natura-A

Natura-A, a natural extracts mouthwash used in this study, was prepared by mixing natural ingredients as indicated in the patent invention as shown in Table 1.

Table 1 . The composition of a natural extracts mouthwash (Natura-A)

ItemNatural ingredientComposition
Mixture 1Campsis grandiflora2-6 (v/v)%
Flower leaf of Camellia japonica2-6 (v/v)%
Flower leaf of Albizia julibrissin Durazz14-18 (v/v)%
Allium cepa6-10 (v/v)%
Portulaca oleracea L.10-14 (v/v)%
Aloe ssp14-18 (v/v)%
Bone of Squid (Todarodes paciftcus)14-18 (v/v)%
Mixture 2Magnolia kobus4-10 (v/v)%
Allium tuberosum20-26 (v/v)%
Petasites japonicus4-10 (v/v)%
Lactuca sativa27-33 (v/v)%
Raphanus sativus27-33 (v/v)%
Mixture 3Forsythia koreana7-11 (v/v)%
Zingiber officinale2-6 (v/v)%
Platycodon grandiflorus2-6 (v/v)%
Saccharina japonica11-15 (v/v)%
Engraulis japonicus29-33 (v/v)%
Solanum tuberosum16-20 (v/v)%
Vigna radiata2-6 (v/v)%
Mixture 4Hyssopus officinalis60-66 (v/v)%
Camellia sinensis15-21 (v/v)%
Allium sativum15-21 (v/v)%
Mixture 5Leaf of Morus alba31-35 (v/v)%
Toxicodendron vernicifluum5-9 (v/v)%
Zanthoxylum piperitum1-5 (v/v)%
Undaria pinnatifida20-24 (v/v)%
Brassica oleracea var. capitata20-24 (v/v)%
Glycine max24-28 (v/v)%


3. Experimental method

The subjects were the positive control group using Listerine and the experimental group using Natura-A; each group was randomly selected and included 10 females. All examiners ate the same type of Korean food at the same time, then put 20 ml of their own mouthwash in the oral cavity for 20 seconds, then put it in a container for testing and sealed it. The collected samples were quantitatively analyzed using multiplex real- time PCR for 18 species of oral microbes.

4. Statistics

The mean and standard deviation of all data were calculated using the IBM SPSS Statistics ver. 21.0 program (IBM Co., Armonk, NY, USA), and the difference test between the control group, the positive control group, and the experimental group and immediately after the use of mouthwash was analyzed using the Kruskal–Wallis test. The post hoc test was performed using the Tukey HSD (honestly significant difference) by making a ranking variable with the Savage value. The statistical significance level was set to 0.05.

Results

Of 18 oral microbiological species examined immediately after the use of Listerine and natural extracts mouthwash, Natura-A, 13 species of Agregatibacter actinomytans, Tan-nerella forsythia, Treponema denticola, Parvimonas micra, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens, Streptococcus mutans., Streptoccuc sobrinus, Lacto-bacillus casei, Staphylococcus aureus, Enteroccus faecalis, and Actinomyces viscosus were not detected in all groups.

As shown in Table 2, in the positive control group using Listerine, 4 types of oral microorganisms were found. Pre-votella intermedia and Prevotella nigrescens were detected in 1 out of 10, showing an appearance rate of 10.0%, and Streptococcus mitis in 4 out of 10, resulting in a 40.0% appearance. Fusobacterium nucletum was detected in 9 out of 10, and the appearance rate was 90.0%. In the experimental group using mouthwash Natura-A, 3 types of oral microorganisms were found. Porphyromonas gingivals was detected in 1 out of 10, showing an appearance rate of 10.0%, and F. nucletum was detected in 7 out of 10, showing a 70.0% prevalence. S. mitis was detected in 9 out of 10 patients, showing 90.0%.

Table 2 . Prevalence of oral microbes immediately after using Listerine and a natural extracts mouthwash (Natura-A) (unit: ×105copies/ml)

BacteriaGroupFrequencyPrevalence (%)Mean±SD MinimumMaximum
PgControla00.0NDNDND
ExperimentalbND10.00.011±ND0.0110.011
FnControl990.07.476±7.4400.00020.000
Experimental770.03.322±6.2610.00117.000
PiControl110.017.000±ND17.00017.000
Experimental00.0NDNDND
PnControl110.00.028±ND0.0280.028
Experimental00.0NDNDND
SmControl440.00.029±0.0370.0000.083
Experimental990.00.082±0.1020.0000.270

Pg: Porphyromonas gingivalis, Fn: Fusobacterium nucletum, Pi: Prevotella intermedia, Pn: Prevotella nigrescens, Sm: Streptococcus mitis, SD: standard deviation, ND: not detected. aPositive control group using Listerine. bExperimental group using a natural extracts mouthwash (Natura-A).



Immediately after gargling, F. nucletum showed lower in Listerin than in the control group (p=0.074), and in Natura-A was lower than in Listerine, however no statistically significant difference between Natura-A and Listerine was observed (p=0.655) (Figure 1). S. mitis was lower than that of the control group in Listerine and Natura-A (p<0.001), and no statistical difference was observed between the two groups. (Figure 2).

Figure 1. Comparison of Fusobacterium nucletu between Lis-terine and a natural extracts mouthwash (Natura-A) immediately after gargling. By Kruskal–Wallis test, p=0.015. By Tukey honestly significant difference; Control vs Listerine (p=0.074), Control vs Natura-A (p=0.016), Listerine vs Natura-A (p=0.655).

Figure 2. Comparison of Streptococcus mitis between Listerine and a natural extracts mouthwash (Natura-A) immediately after gargling. By Kruskal–Wallis test, p=0.001. By Tukey honestly significant difference; Control vs Listerine (p=0.000), Control vs Natura-A (p=0.000), Listerine vs Natura-A (p=0.945).
Discussion

In the research of Moon et al. [20], the oral microbial prevalence in adults was 100% for F. nucleatum, 85.5% for P. micra, 76.1% for P. intermedia, 72.6% for T. forsythia, 17.1% for A. actinomytans, 53.0% for P. gingivals, 64.1% for T. denticola, 70.1% for P. nigrescens, 43.6% for C. rectus, 53.8% for E. nodatum, and 67.5% for E. corrodens. Jeon et al. [21] reported that the prevalence of specific intra-oral microbes in patients in their 20s with early gingivitis were A. actinomycetem-comitans 7.9%, P. gingivalis 11.8%, T. forsythia 26.0%, T. denticola 28.7%, P. intermedia 47.6%, F. nucleatum 100%, P. micra 82.7%, C. rectus 9.8%, E. corrodens 90.6%, S. mutans 16.5%, and S. sobrinus 1.2%. Both Listerine and natural extracts mouthwash have immediate antibacterial effects for 13 species of A. actinomytans, T. forsythia, T. denticola, P. micra, C. rectus, E. nodatum, E. corrodens, S. mutans, S. sobrinus, L. casei, S. aureus, E. faecalis, and A. viscosus were not detected in all groups of 18 oral microbiological species.

P. gingivalis was not detected in Listerine, while it was rarely found at very low concentrations in a natural extracts mouthwash, Natura-A. We would suggest that as both Listerine and Natura-A had inhibitory effect on P. gingivalis which causes pulp and apical diseases, periodontitis, and periodontal abscess [14,22,23], these mouthwashes may prevent periodontitis and oral microbial diseases. P. intermedia and P. nigrescens were found to be low in Listerine immediately after gargling, but not in Natura-A, therefore this natural mouthwash was evaluated as excellent in inhibiting these bacteria.

Natura-A showed better antimicrobial effect against F. nucleatum than Listerine, but no statistical difference. F. nucleatum is the most common bacterium found in gingival, swallowing, and plaque [16,17], Natura-A effectively controls this fungus, thus reducing plaque formation and preventing gingivitis.

S. mitis is a bacterium known to be involved in acute pulpitis and root canal and dental caries of acute apical abscess [24], which was effectively inhibited by both Natura-A and Listerine. S. mutans proliferates on the tooth surface and creates plaque, and it is a representative bacterium that causes dental caries by participating in tooth enamel decay together with other bacteria in the oral cavity [22]. Listerine and Natura-A show an inhibitory effect against S. mutans.

Yun JH et al. [25] suggested that T. forsythensis, C. rectus, P. gingivalis, and F. alocis in Korean chronic periodontitis patients may be putative periodontal pathogens. Natural extracts Natura-A, which has an excellent antibacterial effect, is expected to show excellent results in preventing periodontitis.

Commercially available oral mouthwashes are effective in killing or inhibiting oral microbes, and they greatly contribute to periodontal disease prevention, dental caries prevention, plaque removal, and bad breath removal [13,26], however side effects may occur due to their chemical components. Listerine, which occupies the highest share in the market, claims to kill 99% of germs that cause bad breath, plaque and the gum disease gingivitis. It contains many harmful chemicals such as ethanol (21.6%) which can cause xerostomia (dry mouth), eucalyptol (0.092%), insecticidal Thymol (0.064%), allergic methyl salicylate (0.060%), menthol (0.042%), potentially carcinogenic Poloxamer 407, benzoic acid, sodium benzoate, and tar pigment Green 3.

Many studies on the antibacterial activities of Dalbergiae Odoriferae Lignum extracts against S. mutans [27], of Prunus mume extract against oral microbes [28] of propolis against oral candidiasis [29] and of edible plant extracts against oral bacteria [30] have been conducted. Research will continue, and it may be regarded that the successful implementation of this study has resulted in great progress in these similar studies.

Conclusion

To evaluate whether the natural extracts Natura-A is suitable as an oral mouthwash, 18 oral microbes known to be involved in inducing periodontal disease and dental caries were quantitatively analyzed using a PCR method. Listerine was applied to 10 subjects as a positive control group, while Natura-A was applied to 10 subjects in the experimental group. The results were as follows.

1. Of 18 oral microbiological species examined immediately after the use of Listerine and Natura-A in the study subjects, 13 species of A. actinomytans, T. forsythia, T. denticola, P. micra, C. rectus, E. nodatum, E. corrodens, S. mutans, S. sobrinus, L. casei, S. aureus, E. faecalis, and A. viscosus were not detected in both groups.

2. F. nucleatum, P. intermedia, P. nigrescens, and S. mitis were detected in the Listerine group immediately after gargling, while P. gingivalis, F. nucleatum, and S. mitis were detected in Natura-A. However, they were lower than those of the control group. The antibacterial effect of Listerine and Natura-A against these strains was demonstrated.

3. The antibacterial effect of the natural extract was found to be better than that of Listerine, but there was no statistical significance.

Judging from these results, natural extracts mouthwash, Natura-A has an excellent inhibitory effect on oral microbes. Therefore, we suggest that Natura-A can be used effectively for the prevention of oral diseases including dental caries and periodontal disease long-term without side effects.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

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March 2021, 17 (1)