INTRODUCTION
Periodontitis is a chronic destructive inflammatory disease involving Gram-negative bacteria, and the inflammatory response from the host; this chronic and inflammatory condition threats the integrity of the toot-supporting tissues, collectively known as the periodontium, they include the gingiva, the periodontal ligament and the alveolar bone (1). The severe form of this disease affects approximately 10-15% of adults worldwide, and if the disease is left untreated, periodontitis may not only cause tooth loss, but can also affect systemic health by increasing the patient´s risk for diabetes mellitus, pneumonia, heart disease and preterm birth (2,3).
It has been shown that there are specific associations (complexes) among bacteria in dental biofilms, these microbial complexes represent the sequential colonization and the composition of the most important biofilm formed in the oral cavity; the subgingival dental plaque. Some bacterial strains, mainly belonging to the genus Actinomyces (blue complex) and Streptococcus (yellow complex) have been identified as early colonizers of the dental surface. The second group of bacteria that functions as a bridge between the early and late colonizers are formed by species belonging to the green, purple, and orange complexes (i.e. genus Fusobacterium, Prevotella, Capnocytophaga sputigena and Eikenella corrodens). Finally, the third group of species that appear at late stages in biofilm development and that are considered actual periodontal pathogens are species of the red complex; Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia and Treponema denticola (T. denticola) (1,4).
From a clinical point of view, Actinomyces israelii (A. israelii) and Streptococcus (S. sanguinis) have been considered beneficial colonizers of the dental plaque. On the other hand, P. gingivalis has been associated with periodontal infections, and a higher proportion of this strain has been found in sites with gingival bleeding, inflammation, and suppuration on individuals with advanced periodontitis (4). Also, Prevotella intermedia (P. intermedia) is closely related to inflammatory conditions of the oral cavity, and the presence of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has been related to specific periodontal infections (1).
In the field of dentistry, homeopathy has been used as an adjuvant to conventional therapy, in different presentations, such as, liquids, drops, tablets, pills, granules, or creams. Even more, preparations derived from natural products of plants and mineral substances are currently employed for pathological conditions of the oral cavity, such as periodontitis and dental abscess.
However, there is insufficient information on the antibacterial properties of these products against pathogens responsible for dental infections. Also, the majority of published literature regarding homeopathy has been conducted in the context of toxicology and the immune response under allergic conditions (5).
Among homeopathic remedies available, Arsenicum album and Lycopodium clavatum, have been used to treat oral abscesses and gingivitis (6,7), but their activity against bacteria of clinical relevance in dentistry has not been fully explored. Additionally, reports regarding the antimicrobial properties of such remedies are scarce, although they are prescribed for representative symptoms of bacterial related diseases such as gingivitis and periodontitis (6,8).
On the other hand, drug-resistant bacterial strains have been creating serious treatment issues; therefore, the search for new antimicrobial substances effective against resistant pathogenic microorganisms to conventional antibiotic treatments is necessary. Natural resources have been exploited in the last years, and plant compounds could be an alternative source of new or at least coadjutant treatment (9).
The objective of this investigation was to determine the in vitro antimicrobial efficiency of Arsenicum album and Lycopodium clavatum, against five important periodontal bacteria stains associated with different periodontal health status.
MATERIALS AND METHODS
Bacterial strains
Five bacterial strains from the American Type Cell Culture Collection were used in this study and are listed in Table 1. All strains were grown on HK enriched agar plates, Brain Heart Infusion Agar (26 g/L; BBL, Becton-Dickinson and Co., Sparks, MD) Trypticase Soy Agar (20 g/L; BBL, Becton, Dickinson and Co., Sparks, MD), Yeast extract (10 g/L; BBL, Becton, Dickinson and Co., Sparks, MD), supplemented with 5 μg/mL of Hemin (Sigma-Aldrich, St. Louis, MO), 0.3 μg/mL of Menadione (Sigma-Aldrich, St. Louis, MO), and 5% defibrinated sheep blood (Microlab, Mexico City)], at 35ºC under anaerobic conditions (80% N, 10% CO2 and 10% N2). The bacterial strains were incubated for five days, and pure cultures of each strain were used in the experiments.
Assessment of the antimicrobial activity
For the antimicrobial analysis, HK enriched agar plates were prepared by adding dilutions of the mother tinctures of Arsenicum album or Lycopodium clavatum, to obtain decimal (1dH) and centesimal (1cH) dilutions of each tincture in the agar plates.
The growth from 5 day-cultures of each strain was harvested and placed in individual sterile Eppendorf tubes, containing 900 μL of enriched broth (EB) prepared with Brain heart broth (26 g/L), Trypticase soy broth (20 g/L), Yeast extract (10 g/L), supplemented with Hemin (5 μg/mL), and Menadione (0.3 μg/mL). Each tube was adjusted to have an optical density of 1 at 600 nm in a spectrophotometer. Five serial dilutions of each strain were done, and 100 μL of each dilution were plated on the HK enriched agar plates with or without the homeopathic solutions.
The homeopathic medications of Arsenicum album and Lycopodium clavatum from Similia laboratories (Similia, México, D.F.) were used. The 1cH and 1dH dilutions were prepared according to Mexican Pharmacopoeia (10, 11). Positive control plates were prepared by adding 32 μg/mL of tetracycline, and negative control plates were the HK agar plates only. Another control was included by making HK enriched agar plates added with 1cH and 1dH dilutions of the same alcohol (87°) used to prepare the mother tinctures.
Plates were incubated at 35ºC under anaerobic conditions for seven days. After incubation, the number of Colony Forming Units (CFUs) was counted using the colony counter aCOlyte 3 HD (Synbiosis, USA), accordingly to the manufacture´s instruction. All experiments were performed in triplicate, with three replicates each time.
The antibacterial effectiveness of the homeopathic tinctures was expressed as the inhibition ratio according to the following equation:
Inhibition Radio (%) - (A1-A2/A1) x 100
Where,
A1=total number of CFUs on the plates without the homeopathic remedies (negative control).
A2=total number of CFUs in the presence of a bacterial growth inhibitor; diluted tincture, antibiotic (positive control), or the diluted alcohol containing plates.
DATA ANALYSIS
Data are presented as the mean ± standard error of the mean (SEM), and as the percentage of inhibition. Significant differences in the number of CFU´s in each type of media were calculated using the Student's t-test, and significant differences between groups were determined using Bonferroni´s Modification of Student t-test.
RESULTS
Antimicrobial effect on the total number of colony forming units (CFU´s)
The results revealed that the tinctures from Arsenicum album and Lycopodium clavatum are potent antimicrobials against five bacterial strains from the microbiota of the oral cavity associated with different periodontal health status. The total number of CFU´s and the percentage of inhibition or periodontal bacteria using two different dilutions (1dH and 1cH) of the tinctures are presented in Tables 2 and 3.
The plates contained the Arsenicum album and Lycopodium clavatum tinctures showed significant activity against the bacterial growth at 1dH, and 1cH dilution in comparison with the plates contained the alcohol at 87º alone (p <0.05). The Arsenicum album tincture, exhibited a strong antimicrobial effect (100% of growth inhibition) similar to tetracycline; a common antibiotic used for the treatment of periodontitis (p <0.05; Table 3). Based on the results of the present study the antibacterial action of the tincture from Arsenicum album was more pronounced in all the bacterial strains used, compared with the tincture from Lycopodium clavatum (Table 2).
According to the manufacturer, the tinctures are ethanol extracts prepare with 87º alcohol. With the purpose of comparison, in the present study, the 87º alcohol alone was also tested on the agar plates. The 87º alcohol alone showed more than 90% inhibition of the CFU´s at 1dH for the Gram-negative bacteria, A. actinomycetemcomitans serotype b (96.99%), P. gingivalis (91.54%), and P. intermedia (95.54%). It also showed good inhibition of CFU´s for the Gram-positive, S. sanguinis (84.96%), and A. israelii (80.36%) (Table 2 and 3). However, for the 1cH dilution, less than the 9% of the growth inhibition was observed in all the strains tested, confirming the antibacterial properties of the homeopathic tinctures used in the present study.
Arsenicum album tincture
This tincture showed a 100% of inhibition of the CFU´s of all the Gram-negative and Gram-positive periodontal bacteria tested, regardless the dilution used, and its activity was identical to the tetracycline used as positive control. It appears that the antibacterial action of the mineral origin extracts, i.e. Arsenicum album, could be much more efficient than plant origin extracts like Lycopodium clavatum.
Lycopodium clavatum tincture
In the case of the Lycopodium clavatum tincture, the 1dH dilution showed strong activity (100% of inhibition) on the Gram-negative strains; A. actinomycetemcomitans serotype b, P. gingivalis, and P. intermedia. Additionally, an excellent inhibitory effect was observed against the Gram-positive S. sanguinis and A. israelii (100 and 99.17% of inhibition; Table 3).
DISCUSSION
Studies with a similar approach have focused on the antimicrobial effects of plant extracts on periodontal bacteria. For example, Iauk et al.(12) evaluated the antimicrobial effect of Althaea officinalis, Arnica montana, Calendula officinalis, Hamamelis virginiana, Illicium verum and Melissa officinalis against 18 bacterial strains isolated from the periodontal pocket´s gingival crevicular fluid of periodontal patients. Their results included five strains of P. gingivalis and three strains of Actinomyces odontolyticus, and showed a good antimicrobial activity of the extracts of Arnica montana, and Hamamelis virginiana, which have been already studied for their use in dentistry (12,13). In the current study, we observed a strong in vitro antimicrobial activity for the tinctures of Arsenicum album and Lycopodium clavatum, and the results suggested that the compounds could be a potential target for drug development.
Arsenicum album tincture is obtained from dilutions of Arsenic trioxide (AS2O3) in a potentization process to the point in which it is supposed to lose its toxic properties. Many skeptics of homeopathy dismiss its credibility, mostly because the homeopathic remedies are made by repeated dilutions that lead them to contained extremely low or undetectable amounts of the active substance, therefore, at such small concentrations of the compounds, their effects must be small and weak (14).
However, the effect that homeopathic preparations of Arsenicum album showed against intoxication has been well documented (15). On this regard, various studies have suggested that low molecular dose effects appear to be opposite to those caused by high molecular doses (16).
As for the results presented in this study, they showed that dilution of the Arsenicum album tincture does not cause a significant change in its antimicrobial activity in vitro. Recent physicochemical studies of Chikramane et al. detected nanoparticles of the starting raw material (metals) by transmission electron microscopy in extremely high dilutions prepared with traditional homeopathic manufacturing (17), and our results appear to be consistent with their observations.
It is worth to mention that in the case of Lycopodium clavatum tincture, although the inhibition ratio was lower compared to those of the antibiotic tetracycline, the inhibition of the CFU´s for the tincture confirmed its antibacterial activity. These results are of interest since they have been obtained with a dilution of the tincture and such dilutions could be considered to have a good potency level.
The Lycopodium clavatum is deemed to be a montane plant in the Neotropics, with high alkaloid content, used in folk medicine for the treatment of conditions related to the central nervous system and for motor disorders. Their properties have been extensively reviewed, and it is well accepted that the alkaloids of the plant are responsible for the anticholinesterase and antioxidant activities in diseases such as Alzheimer (18). However, the results of the present study regarding the antimicrobial effects against the periodontal bacteria, suggest that more studies to evaluate the antimicrobial properties of the plant should be carried out.
CONCLUSIONS
In the present study, we found that both of the compounds used could be effective inhibitors of periodontal pathogens like P. gingivalis. However, the mineral extract tincture Arsenicum album was more active in the bacterial growth inhibition than the plant extract tincture Lycopodium clavatum. Additionally, our results indicate that high potencies were less effective than low potencies, at least with the conditions tested.
The results of the present study showed that is worthy to investigate the potential of other therapies or new compounds that could effectively treat infectious diseases like periodontal infections, and such studies represent a contribution to reduce antibiotic resistance.
Funding
Research reported in this manuscript was supported by the National Autonomous University of Mexico, grant number UNAMDGAPA-PAPIIT-IN220416.
Conflict of interests
The authors declare no conflicts of interest.
Compliances with Ethics Guidelines
This article does not contain any studies with human or animal subjects performed by any of the authors.
Acknowledgments
We specially thank Julia Carrasco for the homeopathic preparations of Lycopodium clavatum and Arsenicum album, and for her technical assistance in performing the experiments.
Disclaimer
The content of the manuscript is solely responsibility of the authors and does not necessarily represents the official views of the National Autonomous University of Mexico.
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