According to the World Health Organization (WHO), oral health is defined as the absence of chronic orofacial pain, mouth or throat cancer, oral ulcers, congenital defects such as cleft palate, periodontal disease, dental caries, tooth loss and other diseases. The most frequent oral problems in Mexican population are dental caries and periodontal disease. Both have an impact on quality of life and can be prevented and controlled with simple and economical measures as well as with the help of dental professionals working together with the community1.
Periodontal disease is characterized by inflammation and loss of supporting tissues, in final stages it causes mobility and loss of teeth2. In Mexico, periodontal disease is found in 67.2% of adults between 40 and 49 years of age and in 90% of individuals between 60 and 90 years of age3.
Chronic periodontitis starts and is sustained by gingival biofilm. This biofilm modifies the pathogenesis of the disease according to age or genetic factor. Periodontitis is polymicrobial and progresses slowly. The microorganisms most frequently related with this disease are Porphyromonas gingivalis, Tannerella fosythensis, Prevotella intermedia, Aggregatibacter actinoinomycentencomitans, Fusobacterium nucleatum, Campylobacter spp., Eikenella corrodens and others4.
Plants are a valuable resource in health systems in developing countries. Although there are no precise data to evaluate the use of medicinal plants, the WHO has estimated that more than 80% of the world population routinely uses traditional medicine to satisfy their primary care health needs and a large part of these traditional treatments involve the use of plant extracts or their active substances5.
Psacalium decompositum (Asteraceae) grows throughout Mexico from Chihuahua to Chiapas and has several popular names: Matarique, Matari, Materi, Matariki, Maturín, Maturi, Mataril and Pitacawi. The root is traditionally prepared as tea and used for rheumatism, jaundice, colicky pain in infants, diabetes, renal disease, malaria, fever, arthritis, muscle pain, urinary tract diseases as a diuretic, tonic, aseptic and for wound healing. The root is also applied as a cataplasm for wounds. A portion of the root is placed in dental caries to relieve pain6.
Hypoglycemic sesquiterpenes such as cacalol and a mixture of 3-hydroxycacalolide and epi-3-hydroxycacalolide have been identified; however, their mechanism of action is unknown. The plant also contains alkaloids, essential oils, resins, tannins and glycosides7.
Different chemical substances are currently used for the treatment of periodontal disease, such as ammonium derivatives, fluorides, phenolic compounds and chlorhexidine gluconate. They are used as oral rinses to eliminate dental plaque but they possess side effects such as pigmenting teeth, irritating the mucosa and altering the sense of taste8.
The objective of this study was to determine the antimicrobial a ctivity of the methanolic extract of P. decompositum using the plate diffusion method on the ATCC bacteria P. gingivalis, P. intermedia and S. mutans in vitro and determine its Minimum Inhibitory Concentration (MIC).
MATERIALS AND METHODS
Plant material: Psacalium decompositum was collected in Real de Catorce, Mexico and identified in the Botany Department of the School of Biological Sciences of the UANL. The root was separated, washed with water, cut into slices and dried in the shade for 7 days, afterwards, it was ground and stored in refrigeration until its use.
Preparation of methanolic extract: Two hundred grams sample of pretreated P. decompositum was placed in a 500 mL Erlenmeyer flask. Briefly, 120 mL of 99.9% methanol was added and the flask was hermetically sealed and shaken constantly for 7 days using a dual action Lab-line shaker (Thermo Fisher Scientific Inc., Waltham, MA). The solvent was separated from the rest of the plant material and filtered with Whatman No. 1 filter paper; afterwards, the same plant material was extracted two more times. The methanolic extract was concentrated using a Büchi rotavapor at a temperature below 60°C, the extract was then totally dried at room temperature and stored in amber colored vials at 4°C until use.
The yield of the extract was obtained with the following formula:
where, weight obtained refers to the weight obtained by extraction and initial weight is the weight of the plant material to be extracted.
Phytochemical screening: The variety of compounds with a different chemical nature, found in the plants was named phytocomplex8. This contained secondary metabolites with biologic activity in the oral cavity (antimicrobial, anti-inflammatory and astringent among others)9.
Phytochemical tests were performed to determine the presence of unsaturated compounds (tetranitromethane), tannins (phenolic oxyhydriles), sterols and triterpenes (Liebermann-Burchard), saponins, carbohydrates, quinines, coumarins, alkaloids (Dragendorff), sesquiterpen lactones (Baljet) and flavonoids (Shinoda)10.
Evaluation of the antibacterial activity of the methanolic extract of Psacalium decompositum: This was determined by the plate diffusion method. Plates with sheep blood agar were inoculated with 100 μL of the bacterial supernatant. Plates were divided into five zones; in three, sterile Whatman No. 1 filter paper was placed with 10 μL of the extract to be tested, in zone 4, the negative control, 99.9% ethanol was placed depending on the extract and in zone 5, 0.12% chlorhexidine was used as a positive control. The procedure was carried out in triplicate. Plates were incubated for 24 h at 37°C in anaerobiosis and inhibition halos were measured to determine activity.
Determination of the minimum inhibitory concentration of the methanolic extract P. decompositum: The MIC was determined with the microdilution technique in 96-well plates11. The concentrations evaluated were: 1000, 900, 700, 500, 400, 300, 200, 100 and 50 μg mL1, each concentration was tested 4 times in triplicate. Target samples with the extract in tripticase in soy culture medium were included with the positive control 0.2% chlorhexidine (final concentration in the medium). With this, different concentrations of 1200, 1000, 900, 700, 500 and 400 μg mL1 were evaluated using 5% ethanol as a negative control and microorganisms as a growth control, each of these were performed 4 times in triplicate.
The strains S. mutans ATCC® 700610TM UA159 [UAB577], P. gingivalis ATCC® BAA-308TM W83 and P. intermedia ATCC® 25611TM were activated. An aliquot of 100 μL of each strain was collected and inoculated in 5 mL of trypticase soy broth and incubated at 37°C for 12 h in reduced oxygen conditions necessary for each bacterial strain. An inoculum was prepared adjusting it to 0.5 McFarland (1.5×108 CFU). Mother solution was prepared from the extract. About 50 mg portion of the extract was dissolved in 1 mL of solvent (100% ethanol) and a 1/20 dilution was made, 50 μL of the initial extract solution and 950 μL of a fluid medium were mixed to a final volume of 1 mL (2500 μg mL1). From this, the different concentrations mentioned were prepared. Stock solution of the positive control (chlorhexidine) was prepared at a concentration of 2000 μg mL1. With this, the previously mentioned concentrations were prepared. A stock solution of 100% ethanol and liquid culture medium were prepared both in the same proportion as the extract as a negative control and the different concentrations to be evaluated were made.
A volume of 200 μL per well was deposited in each microplaque with a different volume of mother solution for each concentration of the extract, plus 100 μL of the inoculum of each bacterial strain, completing the final volume of each well with a fluid medium. This was run in quadruplicate: for the target of the extract a different volume of mother solution, for the chlorhexidine control a different volume of the stock solution was added for each concentration of the positive control, for the negative control a different volume of stock solution for each concentration and for growth control a 100 μL inoculum of each bacterial strain and 100 μL of liquid medium. The plaque was incubated at 37°C for 24 h in appropriate redox conditions for each bacterial strain; after this optical density was measured at 595 nm on a BIO-RAD® iMark Microplate Absorbance Reader (Bio-rad Laboratories, Inc., Hercules, CA). The lowest absorbance between the tested concentrations after subtracting the turbidity of the extract was considered the minimum inhibitory concentration.
Statistical analysis: A statistical analysis Leven test was performed to assess whether there are significant differences in the variances of the treatments. The nonparametric Kruskal-Wallis statistical test was used to compare three treatments and the Mann-Whitney test for comparing each pair of treatments to see if there is a significant difference.
The methanolic extract of P. decompositum, presented a significant antimicrobial activity against the three bacteria studied (Table 1). The inhibition halos in the antibiograms of the extract against the microorganisms of the study are shown in Fig. 1 with the against P. gingivalis being noteworthy. Minimum Inhibitory Concentration (MIC) of the methanolic root extract of P. decompositum was higher for the cariogenic bacterium S. mutans at 700 μg mL1 and lower for the periodontal pathogenic bacteria P. gingivalis and P. intermedia at 500 μg mL1 for both and 900 μg mL1 for chlorhexidine (positive control) for the three bacteria tested (Fig. 2). Levenes test showed variances that differed significantly, p<0.001, therefore, the Kruskal-Wallis test was used to compare the three treatments with their absorbances with a significance of p<0.002, demonstrating that there was a significant difference between the absorbances of the three treatments. Based on the range table, the Mann-Whitney test was applied for each treatment pair of the methanolic extract P. decompositum and the bacteria P. gingivalis, P. intermedia and S. mutans.
Measurement of the inhibition halo of the methanolic extract
of P. decompositum
activity against (a) P. gingivalis
BAA-308TM, (b) P. intermedia
25611TM and (c) S. mutans
|Fig. 2:||Minimum inhibitory concentration of the methanolic extract of P. decompositum and the positive and negative controls against S. mutans, P. gingivalis and P. intermedia
|Table 1:||Antimicrobial activity of the methanolic extract of P. decompositum on periodontopathogenic and cariogenic bacteria by the plate diffusion method
|Positive control: 0.12% clorhexidine and Negative control: 100% ethanol|
It was found a significant difference in the first group of p<0.040 for the second group the p-value was p<0.001 and for the third group there was no significant difference (p<0.906).
The yield of the extract was 8.1% using the previously mentioned formula. Phytochemical screening of the methanolic extract demonstrated the following metabolites or functional groups: Unsaturated compounds, carbonyl group, phenolic oxyhydril-tannins and sterols.
The methanolic extract of Psacalium decompositum was active against the periodontopathogenic bacteria P. gingivalis and P. intermedia with an MIC of 500 μg mL1 and the cariogenic bacteria S. mutans with an MIC of 700 μg mL1. In dental practice substances currently available for treating periodontal problems are used in higher concentrations. The main product used as a mouthwash is clorhexidine 0.12 or 0.2% (this corresponds to 1200-2000 μg mL1).
With these results we can conclude that the P. decompositum extract represents a natural alternative for the control of the microorganisms that cause oral diseases12. In addition, the concentrations used in this study can be achieved in clinical solutions. These could be at approved concentrations used commercially or at the concentration reported in this study because the extract is active at a lower concentration than commercial products. It is also important to mention that since periodontal disease is chronic and clorhexidine causes teeth discoloration with prolonged use, the use of this natural product could be an advantage.
Marsh and Martin13, Van Winkelhoff et al.14 and Urena15 described P. gingivalis as the most pathogenic Gram-negative anaerobe bacilli emphasizing its ability to colonize and destroy periodontal tissue and evade host defenses.
Prevotella intermedia are periodontopathogenic microorganisms that play an important role in the establishment and development of polymicrobial periodontal diseases. It is characterized by its ability to adhere and invade gingival epithelial cells, fibroblasts and endothelial cells, although the role that gram-negative enteric bacilli play in the pathogenesis of periodontal disease is unknown. Researchers agree that they have an impact on the progress and treatment of the disease in addition to being invasive and toxigenic bacteria16. The S. mutans is a lactic acid producing Gram-positive immobile coccus that is capable of changing alkaline pH to acid pH in 24 h. It is a microorganism that is closely related to the most prevalent infectious disease in humans, dental caries, which is characterized by demineralization and remineralization17.
Caries and periodontitis are caused by an imbalance of bacterial populations that naturally form biofilms. The bacteria in these biofilms are saprophytes that help maintain normal conditions in the oral cavity but when changes occur in host defenses or in the oral environment, the virulence of these bacteria increases.
Almanza-Perez 18 and Alarcon-Aguilar et al.19 reported the presence of alkaloids in rhizomes and roots of P. peltatum, in this study of P. decompositum, these compounds were not found. In a review performed to study for secondary metabolites present in the family Asterecea. De Vivar et al.20 reported the presence of pyrrolizidine alkaloids in all species of the genus Psacalium. Secondary metabolites such as coumarins, which have antimicrobial activity21 and sesquiterpene lactones, that also have antimicrobial activity, were found in the methanolic extract of P. decompositum22,23. This study with P. gingivalis, P. intermedia and S. mutans coincides with that of Jimenez et al.24, in that some compounds from the organic extracts of roots of P. decompositum, such as cacalol and some of its derivatives have antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and certain fungi such as Candida albicans and Cryptococcus neoformans25,26.
In this study, it was found that P. decompositum has a broader antimicrobial effect since it inhibited the growth of the three bacteria studied in comparison with the results obtained by Almanza-Perez18 who used hexanic and dichloromethane extracts from a plant of the same genus, P. pelatum, which inhibited the growth of only one bacteria, Bacillus subtilis of six that were evaluated at a concentration of 10 and 100 μg mL1 for both extracts.
These results show that plants from different species have different properties despite belonging to the same genus. Antimicrobial activity has been reported with diverse plants belonging to the same family Asteraceae, such as the study by Portalatino and Medina27 with hydroalcoholic extracts of Baccharis genistelloides (callua), Jungia paniculata (matico) and Perezia multiflora (escorzonera) which inhibited Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 11774.
Anti-inflammatory and hypoglycemic properties have been reported for P. decompositum; however, there are few studies on its antimicrobial activity, making this an area of opportunity for new study.
The methanol extract Psacalium decompositum was active against the peridontopathogenic bacteria P. gingivalis and P. intermedia and the cariogenic bacteria, S. mutans and P. decompositum represent a natural alternative for the control of these microorganisms which produce oral diseases. It is important in future studies to fractionate this methanolic extract to identify the specific molecule that produces this antimicrobial activity. Also, possible toxicity of the extract should be considered. For this reason, toxicity testing and the Ames test are currently being carried out.
We thank Sergio Lozano-Rodriguez, M.D. for his help in transalting the manuscript. García Palencia was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico) No 557089.