|Year : 2019 | Volume
| Issue : 5 | Page : 718-726
Antimicrobial activity and volatile constituent analysis of three commercial herbal toothpastes containing Aloe vera L. and Fragaria vesca L. extracts
FM Korkmaz1, MB Ozel2, T Tuzuner3, B Korkmaz4, N Yayli4
1 Department of Prosthodontics, Faculty of Pharmacy, Karadeniz Technical University, Trabzon, Turkey
2 Department of Orthodontics, Faculty of Pharmacy, Karadeniz Technical University, Trabzon, Turkey
3 Department of Paediatric Dentistry, Faculty of Pharmacy, Karadeniz Technical University, Trabzon, Turkey
4 Department of Pharmacognosy, Faculty of Pharmacy, Karadeniz Technical University, Trabzon, Turkey
|Date of Acceptance||04-Feb-2019|
|Date of Web Publication||15-May-2019|
Dr. F M Korkmaz
Karadeniz Technical University, Faculty of Dentistry, Kanuni Kampus, 61080 Trabzon
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: This work was designed to evaluate the antimicrobial activity of three different commercial herbal (Aloe vera L. and Fragaria vesca L. extracts) toothpastes [LR Aloe vera (HTP1), ESI Aloe fresh (HTP2) and ROCS Teens (HTP3)] against two microorganisms that cause tooth infections. Materials and Methods: An agar disk diffusion method was used to test the antimicrobial activity of three herbal gel toothpastes in the amount of 100 μL against Streptococcus mutans and Staphylococcus aureus. In the second part of the work, the volatile organic compounds of three different commercial herbal toothpastes (HTP1-3) were determined by solid-phase microextraction/gas chromatography-mass spectrometry-flame ionization detection (SPME/GC-MS-FID). Results: The sensitivity of the tested herbal toothpastes toward each microorganism was expressed as the mean of the clear zone within the range of 6–16 mm diameters. HTP1 and HTP2 were found to be more effective against both bacteria compared with HTP3. Oxygenated monoterpenes (99.34%, 91.44%, and 83.48%) were the most abundant groups in the SPME of HTP1-3, respectively. Menthol (25.41%, 35.82%, and 31.15%) and anethole (52.01%, 23.62%, and 38.79%) were the major compounds identified in the SPME analysis of HTP1-3, respectively. Carvone was found only in HTP3 (0.49%) in a small quantity. Conclusion: The commercial herbal toothpastes could have advantages in decreasing bacterial accumulation on teeth with protection of the oral cavity.
Keywords: Antimicrobial activity, herbal, gas chromatography-mass spectrometry, toothpaste
|How to cite this article:|
Korkmaz F M, Ozel M B, Tuzuner T, Korkmaz B, Yayli N. Antimicrobial activity and volatile constituent analysis of three commercial herbal toothpastes containing Aloe vera L. and Fragaria vesca L. extracts. Niger J Clin Pract 2019;22:718-26
|How to cite this URL:|
Korkmaz F M, Ozel M B, Tuzuner T, Korkmaz B, Yayli N. Antimicrobial activity and volatile constituent analysis of three commercial herbal toothpastes containing Aloe vera L. and Fragaria vesca L. extracts. Niger J Clin Pract [serial online] 2019 [cited 2019 May 23];22:718-26. Available from: http://www.njcponline.com/text.asp?2019/22/5/718/258280
| Introduction|| |
Despite great improvements in oral health, dental issues remain one of the most prominent diseases. In the mouth, numerous bacteria with great accumulation provide a biofilm-rich environment on the tooth surface., The plaque-related biofilm, which is known as the primary etiological factor of dental caries and periodontal disease, cannot be completely removed from dental tissues but can be decreased with routine oral care.,, Controlling supragingival plaque accumulation could be accomplished using a toothbrush and dentifrices. Thus, toothpastes can be considered as one of the main vehicles for chemical agents that have the potential to be used for therapeutic and preventive purposes. Natural herbs and related substances that act against cariogenic bacteria and their suitable usage as preventive and therapeutic products have been commonly analyzed in recent years. Compared with synthetic products, plant extracts in herbal toothpastes could exhibit much safer and better antimicrobial activity according to previous findings.,,,, Evaluating new herbal formulas regarding safety and efficiency is a means of understanding the mechanisms for clinical use., The essential oils of herbal ingredients that destroy the microorganisms on the enamel surfaces could hamper biofilm formation during the progressive process.
Medicinal plants were an important source of traditional remedies throughout history, and they have been used as popular folk medicines for hundreds of years. Certain medical plants serve as a source of therapeutic agents that have antimicrobial effects. Aloe vera L. and Fragaria vesca L. (wild strawberry) have been the most important medical plants that had been used., The invented herbal toothpaste has advantages in taste, protection of oral cavities, prevention of caries, and freshening of breath. Due to these factors, herbal toothpastes have been used for many years. It has been mentioned that a pleasant-tasting herbal toothpaste compound is effective against oral diseases and disorders including oral cancerous and precancerous lesions.
Aloe vera L. Burm is a species of the genus Aloe of the family Asphodelaceae. It grows wild in tropical climates around the world and has been cultivated for agricultural and medicinal uses. Aloe vera L. Burm has been added to many consumer products including toothpaste, cosmetics, skin lotion, beverages, and ointments for minor burns and sunburns. Phytochemical studies of A. vera showed many active natural compounds such as anthraquinones, saponins, vitamins, salicylic acid, amino acids, sugars, and lignin. Gas chromatography-mass spectrometry (GC-MS) analyses of A. vera revealed the presence of carvacrol, thymol, linalool, (Z)-9-octadecenoic acid, phenylmethyl ester, astaxanthin, normethadol, and fenretinide as the main constituents. Biological studies on A. vera demonstrated its antiviral, antibacterial, antifungal, anti-inflammatory, and antitumor activities. In a randomized controlled clinical study, it was reported that toothpaste containing A. vera demonstrated significant improvement in plaque and gingival status.
Fragaria vesca L. is a perennial herbaceous plant in the family of Rosaceae that grows naturally throughout the world and is known to be a wild strawberry, which produces edible fruits and is added to many commercial products such as toothpaste. Wild strawberry fruit has been used to make commercial jam, sauces, liqueurs, cosmetics, and alternative medicine in Turkey, and hundreds of tons of wild fruit are harvested annually. GC-MS analysis of the essential oil of F. vesca revealed that myrthenol, nonal, linalool and dibuthylphthalide, 2,5-dimethyl-4-methoxy-3-2H-furanone,2-penta decanone,2-pentadecanol,6-methyl-5-hepten-2-ol, decyl acetate, carvyl acetate, verbenone, Me nicotinate, citronellol, myrtenol, decyl butyrate, eugenol, Me anthranilate, Me N-formyl anthranilate, and vanillin are the main constituents., The essential oils of wild strawberry (F. vesca) have also shown antimicrobial activity. In the literature, wild strawberry species have been known to show a wide range of pharmacological activities. These species have been traditionally used for their antimicrobial, cardio protective, antiseptic, and antioxidant activities. Antimicrobial and antioxidant activities of the solvent extracts of the fruits, leaves, flowers, and roots of wild strawberry have been tested. This study mentioned that extract activity was reduced in blood serum and possessed wound-healing properties.
In the literature, comparative evaluation of the antimicrobial efficacy of A. vera tooth gel and toothpastes had similar antimicrobial effects against Candida albicans, Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Prevotella intermedia, and Peptostreptococcus anaerobius. In an in vitro study, it was shown that herbal toothpastes, including A. vera, demonstrated quite high antimicrobial effects against S. mutans. Antimicrobial activity of A. vera toothpaste inhibited the growth of Streptococcus sanguinis in a previous study. The antimicrobial activity of 10 different toothpastes, including herbal toothpastes with various concentrations against Porphyromonas gingivalis, Escherichia More Details coli, Staphylococcus aureus, and C. albicans, were examined and it was found that the toothpastes showed antimicrobial activity against these microorganisms in a wide spectrum. In the oral cavity, S. mutans is the key factor in the initiation of caries processes; however, S. aureus also has been implicated as another responsible bacterium., There still remains a need for the study of other commercial toothpastes with other microorganisms.
Despite the existence of toothpaste or herbal toothpaste in the world, studies on the antimicrobial and the volatile organic compounds (VOCs) of different commercial toothpastes are limited. Our literature survey showed that no research has been performed on the VOCs with the solid-phase microextraction (SPME) method of herbal toothpastes. In light of this evidence, we aimed to evaluate the in vitro antimicrobial activity of commercial toothpastes (HTP1-3) against some microorganisms that cause tooth infections; we analyzed the volatile constituents of three HTPs with SPME obtained from a Turkish pharmacy. We hypothesized that three commercial herbal toothpastes containing A. vera L. and F. vesca L. extracts would show antibacterial activity against S. mutans and S. aureus.
| Materials and Methods|| |
Commercial herbal toothpastes (HTP1-3)
The commercial toothpastes [LR Aloe vera (HTP1) (LR Health & Beauty Systems GmbH, Ahlen, Germany), ESI Aloe fresh (HTP2) (ESI, Bra, Italy), and ROCS Teens (HTP3) (ROCS; Greinheim, Germany)] were purchased from local pharmacy in Trabzon, Turkey. The toothpastes evaluated in this study are shown in [Table 1].
|Table 1: Compositions and manufacturers of the toothpastes evaluated in this study|
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Agar disk diffusion test
All test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam (Ankara, Turkey) and were as follows: S. mutans RSKK07038 (S. mutans) and S. aureus ATCC 25923 (S. aureus). The amount of HTP1-3 was measured with micropipettes (100 μL each) and they were directly incubated. Antimicrobial activity results for HTP1-3 are shown in [Table 2]. The antimicrobial effects of the HTP1-3 were tested quantitatively in the respective broth media using double microdilution., The antibacterial assay was performed in Mueller-Hinton broth (Difco, Detroit, MI, USA) at pH 7.3 and buffered Yeast Nitrogen Base (Difco) at pH 7.0. The microdilution test plates were incubated for 18–24 h at 35°C. Brain Heart Infusion broth (Difco) was used for S. mutans and S. aureus and incubated for 48–72 h at 35°C. Antimicrobial activity was assessed by comparing the zone of inhibition generated by HTP1-3 against the test microorganisms with the zone of inhibition generated by standard drugs. Ampicillin (35 mg/mL) was used as the standard. The results were interpreted in terms of the diameter of the zone of inhibition and are given in [Table 2].
|Table 2: Screening for antimicrobial activity of the commercial herbal toothpastes (HTP1-3)|
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Solid-phase microextraction (SPME)/gas chromatography-mass spectrometry-flame ionization detection (GC-MS-FID) analysis
A manual SPME holder and one type of fiber (65-μm blue hub plain, polydimethylsiloxane/divinyl-benzene) and 10-mL vials from Supelco Inc. (Bellefonte, PA, USA) were used for the extraction of the volatile components. Commercial herbal toothpastes (HTP1-3) (100 μL) were placed in 10-mL vials that were sealed with a silicone rubber septum cap. The fiber coating was embedded in the head space to assess temperature, and time (incubation and extraction times) values were set according to the experiment. SPME was done at 50°C with an incubation time of 5 min and extraction time of 10 min. The fibers containing the extracted aroma compounds were then injected into the GC-MS injector (split mode).
GC analysis was carried out on the HTP1-3 using a Shimadzu 2010 Plus (Shimadzu Scientific Instruments, Columbia, MA, USA) gas chromatograph coupled with a Shimadzu QP2010 Ultra mass selective detector. The separation was analyzed by means of a Restek Rxi-5ms capillary column (30 m × 0.25 mm × 0.25 μm; Shimadzu, Japan). The fibers containing the extracted aroma compounds of HTP1-3 were then injected into the GC-MS injector in splitless mode at 230°C and analyzed with the column initially held at 60°C for 2 min and then increased to 240°C with a 3°C/min heating ramp. The oven program was as follows: the initial temperature was 60°C for 2 min, which was increased to 240°C at 3 min, and the final temperature of 250°C was held for 4 min. Helium (99.999%) was used as the carrier gas with a constant flow rate of 1 mL/min−1. Detection was implemented in electronic impact mode; the ionization voltage was fixed at 70 eV, and the scan mode (40–450 m/z) was used for mass acquisition. The VOCs of HTP1-3 were identified by a comparison of their retention indexes (RIs) (relative to C6–C30 alkane standards) and by comparison with the mass spectra of the two libraries (FFNSC1.2 and W9N11).,,,,, Each sample was analyzed and the mean value was reported.
RIs of the components were determined by Kovats method using n-alkanes (C6–C32) as standards. The volatile compounds were identified by comparison of their RIs and mass spectra with those of the available analytical standards and by matching mass spectra held in the libraries; FFNSC1.2, W9N11, and NIST confirmed by comparing the results with the literature.,,
| Results|| |
This study analyzed the uses of plants and describes an in vitro investigation that compared the antimicrobial effectiveness and VOCs of A. vera and wild strawberry (F. vesca) toothpastes, which are known as commercially available dentifrices [LR, Aloe vera (HTP1), ESI Aloe fresh (HTP2), and ROCS Teens (HTP3)]. Two of those commercial herbal toothpastes had an A. vera extract (HTP1-2) and one of toothpaste had a wild strawberry (F. vesca) extract (HTP3).
The antimicrobial activity of the HTP1-3 was tested in vitro using the agar disk diffusion method against the microorganisms listed in [Table 2]. The results obtained are expressed as diameters of inhibition. HTP1-3 were active in all tested microorganisms (S. mutans and S. aureus). All the HTPs were active against S. mutans in the range of 6–16 mm zones of inhibition. The toothpastes containing A. vera L. extract (HTP 1 and 2) were able to inhibit the growth of S. mutans and S. aureus, revealing an antimicrobial activity higher than that of herbal toothpaste containing F. vesca L. (HTP 3) extract.
Chemical compositions of VOCs of HTP1-3 were identified by SPME GC-MS flame ionization detection (FID). GC-MS chromatograms of HTP1-3 are shown in [Figure 1], [Figure 2], [Figure 3], respectively. The identity, retention time, and the percentage of the composition of VOCs from the commercial herbal toothpastes are given in [Table 3]. Identifications were made on the basis of comparisons of GC Kovats RIs with reference to a homologous series of n-alkanes. The percentage compositions of VOCs are presented as relative peak area. A total of 5, 24, and 26 compounds from the three HTPs were identified and quantified, accounting for ratios of 99.23%–99.89%. Among the herbal toothpastes studied, the highest numbers of compound diversity were observed in HTP3 and HTP2 with 26 and 24 compounds, respectively [Table 3]. According to the VOC results, the major compounds identified in the SPME of the three herbal toothpastes were anethole (52.01%), menthol (25.41%), and eucalyptol (21.83%) in HTP1, menthol (35.82%), anethole (23.62%), and menthone (19.37%) in HTP2, and anethole (38.79%), menthol (31.15%), and menthone (10.53%) in HTP3, which were the most abundant components in the investigated HTPs [Table 3]. Major volatile components (anethole and menthol) varied in all HTPs in the SPME GC-MS spectra. The chemical class distribution of the VOCs for the three HTPs is listed in [Table 4], and they were classified into seven classes, namely, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, alcohols, esters, aliphatic hydrocarbons, and others. Comparative chemical class evaluation in the HTP1-3 resulted that oxygenated monoterpenes (99.34%, 91.44%, and 83.48%, respectively) were the main class of organic compounds in all samples that were studied.
|Table 3: Volatile constituent of the three commercial herbal toothpastes (HTP1-3)|
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|Table 4: The chemical class distribution of the volatile components of the three commercial herbal toothpastes (HTP1-3)|
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| Discussion|| |
Under the conditions described, this study showed that toothpastes that include Aloe vera and wild strawberry extracts exhibited antimicrobial activity against two important cariogenic bacteria (S. mutans and S. aureus). Therefore, the hypothesis of this study was accepted. The antimicrobial activity of A. vera–containing toothpastes was significantly higher than that of wild strawberry–containing toothpastes. HTP1 generated higher antimicrobial activity against S. mutans comparing to S. aureus, and HTP2 showed almost similar effects for both bacteria. When considering HTP3, the comparison was found to be equal for S. mutans and S. aureus. According to this study, the VOCs that contained oxygenated monoterpenes (99.34%, 91.44%, and 83.48%) were the most abundant groups of HTP1-3, respectively. Moreover, menthol (25.41%, 35.82%, and 31.15%) and anethole (52.01%, 23.62%, and 38.79%) were found to be the prominent compounds identified in the SPME analysis of HTP1-3, respectively. Carvone was also found only in HT-3 (0.49%); however, this compound was found only in a small quantity.
The agar diffusion test was used to evaluate the antimicrobial activity of the herbal toothpastes in this study. While the agar diffusion test method can be used for fluid materials, its potential benefit on the evaluation of semisolid materials such as toothpaste should not be overlooked., Although the agar diffusion test method is accepted as a way of screening the antimicrobial activity of toothpastes before in vivo testing, it has limitations such as analyzing the antimicrobial effects on the ability of toothpastes to penetrate a biofilm matrix, which could be problematic when mimicking in vivo conditions. Nevertheless, it is still an easily performed test commonly used to detect the antimicrobial activity of natural products.
Even though human behavior cannot be controlled easily, brushing the teeth regularly could provide lower biofilm accumulation and bacterial colonization. Previous reports indicated prominent antibacterial activities,,, and antierosive effects on teeth of herbal substances against caries-associated bacteria. Thus, we have selected various toothpastes that included A. vera and wild strawberry. S. mutans has previously been indicated as the bacterium responsible for the initiation and progression of caries, while the facultative pathogen S. aureus was also considered as another factor. Thus, two types of bacteria with great importance for the caries processes were chosen for this study design. HTP-1 showed better antibacterial effects against S. mutans compared with HTP2 and HTP3. In addition, HTP2 also showed higher antimicrobial activity compared with HTP1 and HTP3. These may be attributed to the higher oxygenated monoterpenes that were found in the structures of HTP1 and HTP2.
In recent years, a number of toothpaste preparations containing herbal ingredients have been found useful for oral and dental health. Previous studies have reported large variations in the antimicrobial effects of herbal dentifrices, which is consistent with the current results.,,,,,,, This activity can be based on the low solubility of their contents such as essential oils.
In the literature, three different solvent extracts (aqueous, ethanol, and acetone) were obtained from the leaves of A. vera to screen the antibacterial activity against selected human pathogens by the agar diffusion method; the acetone extract had maximum antibacterial activity compared with the aqueous and ethanol extracts. It has been mentioned that due to its phytochemical compounds, an A. vera leaf gel may show promise in alleviating symptoms associated with/or prevention of cardiovascular diseases, neurodegeneration, diabetes, and cancer. Chemical constituents of solvent extracts for the dry and fresh flowers of A. vera were also mentioned. Ten and twenty-four compounds had been identified from dry and fresh flowers by GC-MS, respectively. In a previous study, the hydromethanolic extracts of wild F. vesca L. showed high antibacterial activities and were able to inhibit the formation of bacterial biofilms. The antiviral activity of total methanol extract as well as that of the anthocyanins and the non-anthocyanins from wild strawberry (F. vesca) picked in Bulgaria was reported. The antiviral effect has been tested against viruses that are important human pathogens and for which chemotherapy and/or chemoprophylaxis was indicated, namely, as poliovirus type 1 (PV-1) and coxsackievirus B1 (CV-B1), human respiratory syncytial virus A2 (HRSV-A2), and influenza virus A/H3N2. Investigations on the solvent extracts and essential oils of other herbal toothpastes have shown carvacrol, thymol, linalool, (Z)-9-octadecenoic acid, astaxanthin, normethadol, fenretinide, myrthenol, nonal, dibuthylphthalide, 2,5-dimethyl-4-methoxy-3-2H-furanone,2-pentad ecanone,2-pentadecanol,6-methyl-5-hepten-2-ol, decyl acetate, carvyl acetate, verbenone, Me nicotinate, citronellol, myrtenol, decyl butyrate, eugenol, Me anthranilate, Me N-formyl anthranilate, and vanillin as the main components., The antimicrobial activity of the extracts and essential oils was generally attributed to their main compounds. Antimicrobial activity of A. vera showed activity against human pathogens, and phytochemical analysis by GC-MS revealed squalene, oleic acid, and dodecanoic acid as the major components. The ethanolic extract of lyophilized A. vera gel was analyzed by GC-MS, and hexadecanoic acid (22.22%), sitosterol (2.89%), and stigmasterol (2.1%) were reported as major compounds in the extracts of A. vera.
Liquid–liquid extraction is a conventional method that is used to obtain the natural volatile component. However, it is a time-consuming, unhealthy and expensive method. Instead of this method, a less expensive and healthful SPME method was used to extract volatile components from natural sources. SPME is a reliable all-in-one sample preparation technique that has been used for the analysis of essential compounds. SPME is economical and fast, and it eliminates solvent hazards. As far as we know, this method has not been used for the analysis of herbal toothpaste, which we used in this work.
In this study, anethole, menthol, eucalyptol, isomenthone, and menthone were found to be abundant compounds in HTP1-2 due to SPME techniques that were used for the analysis of VOCs [Table 3]. Since the significant antimicrobial activity of the oxygenated monoterpenes including menthol against S. mutans has been revealed previously, menthol could be responsible for the antimicrobial activity that we observed in this work. The chemical class distribution of the VOCs from the toothpastes that had A. vera extracts revealed that the main class of organic compounds were monoterpene hydrocarbons and oxygenated monoterpenes. The A. vera extracts commonly show antimicrobial activity because of monoterpenoids. The activity of monoterpenoids is explained by their lipophilic characteristics, which cause cell membrane damage and deterioration, increased fluidity, and suppression of membrane-bound enzymes. The findings of this in vitro study are in agreement with those of Villalobos et al., de Oliveira et al., and Pradeep et al. who observed significant reduction in plaque accumulation after use of mouth rinses and toothbrushes containing A. vera. Previous reports indicated that the A. vera components in toothpastes showed significant antibacterial effects., Both A. vera toothpastes tested (HTP 1 and HTP 2) showed satisfactory antimicrobial activity compared with HTP 3 (F. vesca) against both bacteria tested. The differences that were found between the toothpastes might be attributed to the volatile chemical profiles of the three HTPs, which proved that they differed markedly in their volatile constituents and in their antimicrobial activities. Moreover, our results can be defined as consistent with the previous articles,,, and the tested toothpastes might indicate that the commercial production of herbal toothpastes plays a decisive role in the amount and number of bioactive compounds.
Even though all the herbal dentifrices showed antimicrobial activity, the study had certain limitations. Pathogenicity of bacteria is a multifactorial process with confounding factors, such as virulence and host responses, genetic and environmental factors, saliva buffering, and diet. Thus, dentists must recommend toothpaste options for patients' specific expectations.
Moreover, it must be noted that there is a necessity for carrying out comprehensive high-quality research, and evidence-based studies are still necessary for herbal oral healthcare products because of the lack of prominent conclusions regarding chemical testing, inadequate clinical trials with poor study designs, lack of multidisciplinary work, and partner-based relations with the industry.
In conclusion, VOC studies in HTPs showed a large variety of oxygenated monoterpenes as bioactive compounds. The observed antimicrobial activity, specifically for A. vera–based activity, indicates that commercial herbal toothpastes could have advantages in decreasing bacterial accumulation on teeth with protection of the oral cavity and decreasing caries risk. Therefore, it can be deduced that herbal toothpastes that have A. vera and wild strawberry extracts can be suggested similar to the conventional contents. The concentration of the active volatile components in toothpaste deserves further study. This is the first report on the SPME GC-MS analysis of the above-mentioned commercial herbal toothpastes.
The authors are thankful to Professor Şengül Alpay Karaoğlu for the help of the antimicrobial activity determination.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]