Protective efficacy of Psidium cattleianum and Myracrodruon urundeuva aqueous extracts against caries development in rats

Abstract

This study evaluated the influence of Psidium cattleianum Sabine (Myrtaceae) and Myracrodruon urundeuva Allemão (Anacardiaceae) aqueous extracts on S. mutans counts and dental enamel micro-hardness of rats submitted to a cariogenic challenge. Sixty Wistar rats were distributed in three groups and received water (control) or aqueous extracts of Psidium cattleianum or Myracrodruon urundeuva as hydration solution. Initially the animals had their sublingual and submandibular salivary glands surgically removed and the parotid ducts ligated. Then the rats were inoculated with 10⁶ CFU of Streptococcus mutans ATCC 35668 and were fed with a cariogenic diet. To detect and quantify the presence of S. mutans, oral biofilms were sampled and microbial DNA was extracted and submitted to amplification by means of real-time PCR (Polymerase Chain Reaction). After seven weeks the animals were sacrificed and enamel demineralization was analyzed by cross-sectional micro-hardness. Both extracts produced a significant reduction on S. mutans counts and decreased the enamel demineralization. It can be concluded that the extracts tested had a significant effect on S. mutans in oral biofilm of the rats, decreasing S. mutans accumulation and enamel demineralization.

Keywords:

• Antibacterial agents
• dental caries
• natural products
• Streptococcus mutans

Introduction

In spite of the significant reduction of dental caries in western countries observed in the past 20 years, some people still hold a large amount of caries lesions and are resistant to preventive methods (Narvai et al., 2006). Primary targets of dental caries prophylaxis involve control of microorganism accumulation, acid production and adherence to the tooth surface. In particular, inhibitory activity toward Streptococcus mutans has a close relationship with caries control due to their virulence factors, such as the ability to adhere to dental enamel surface and their acidogenic and aciduric properties (Thenisch et al., 2006).

New chemical agents derived from natural compounds such as propolis (Leitão et al., 2004), guajava (Razak et al., 2006), cacao (Matsumoto et al., 2004) and Camellia sinensis L. tea (Hamilton-Miller, 1995) have been successfully evaluated regarding their anticariogenic activity. These substances are largely used in traditional medicine or in the diet, especially in developing countries (Hebbar et al., 2004; Tayanin & Bratthall, 2006; Yu et al., 2006). Herbal medicines may have advantages over synthetic compounds, such as diversity, flexibility, accessibility, affordability and broad acceptance, and are used by people concerned about the adverse effects of synthetic drugs (WHO, 2002).

Psidium cattleianum (araçá) Sabine (Myrtaceae) and Myracrodruon urundeuva (aroeira) (Allemão) Enlg. (Anacardiaceae) are tropical trees that have been used by the local population as sources of anti-ulcer, analgesic and anti-inflammatory drugs (Jaiarj et al., 1999; Viana et al., 2003; Souza et al., 2007). Aqueous extracts of P. cattleianum and M. urundeuva are currently employed for prevention of oral infective diseases such as dental caries and periodontal diseases by people living near the margins of the Amazon forest and in the savanna of the Central, Northeastern, and Northern Brazil, where public and private medical care are scarce. However, the efficacy of these natural products has not been scientifically established.

Previous results of our research group revealed the antimicrobial properties of P. cattleianum leaf extract on S. mutans biofilms. This extract was able to reduce S. mutans survival, pH drop and the expression of proteins related to cell maintenance and carbohydrate metabolism (Brighenti et al., 2008).

Thus, studies to characterize the anticariogenic potential of these plant extracts may be valuable to improve oral health in populations with low income and non-compliant to conventional preventive procedures. This study aimed to evaluate the effect of Psidium cattleianum and Myracrodruon urundeuva aqueous extracts on S. mutans counts and on enamel demineralization of rats submitted to a cariogenic challenge.

Material and methods

This study was approved by the Ethical Committee in Animal Experimentation of the Araçatuba School of Dentistry, São Paulo State University-UNESP, Brazil (protocol number 92/05).

Plant extract preparation

Myracrodruon urundeuva and Psidium cattleianum were grown at the São Paulo State University- UNESP, São Paulo, Brazil, in natural conditions, without addition of chemical compounds such as chemical fertilizers, pesticides and insecticides. Plants were collected in January 2005 and identified by the botanist Omar K. Khalil. The voucher specimen is deposited at the Herbarium of Pharmacognosy and Phytotherapy Research Laboratory, UNIRP, São Paulo, Brazil under the numbers of HLF 2006/56 (M. urundeuva) and HLF 2006/71 (P. cattleianum). Leaves were washed three times in deionized water and allowed to dry in a dark room (initially at room temperature for 5 days and then at 37°C for 15 days) and then ground.

The aqueous extracts were prepared by adding 25 g of plant powder to 125 mL of deionized water. After that, the mix was boiled at 100°C for 5 min, then at 55°C for 1 h and finally incubated at room temperature for 3 days to the extraction of water-soluble chemical compounds. The extracts were filter-sterilized using a 0.22 μm cellulose membrane (Millipore^™, Massachusetts, Billerica) and they were allowed to evaporate at 37°C to achieve final concentrations of 15 mg/mL (Iwaki et al., 2006). The extracts were prepared immediately before use to avoid oxidation or were stored at −40°C, for 10 days.

Bacterial strain, growth conditions and culture medium

Streptococcus mutans ATCC 35668 was used to inoculate the oral cavity of the rats. This microrganism was selected because it was able to produce persistent and significant colonization of the experimental animals and is currently employed in studies on antimicrobial or therapeutic activity of plant extracts on cariogenic bacteria (Nostro et al., 2004). This bacterium was provided by the Laboratory of Oral Microbiology of São José dos Campos Dental School and it was maintained at -196°C in skim milk (Gibson & Khoury, 1986). The microorganisms were inoculated in tryptic soy broth (Difco, Franklin Lakes, NJ) supplemented with yeast extract (0.5%) and incubated anaerobically (90% N₂/10% CO₂) at 37°C, for 24 h.

Experimental groups

Sixty specific pathogen-free male Wistar rats (Rattus norvegicus albinus), with an approximate weight of 140 g and 25 days old were used. The animals were provided by the Central Bioterium of Araçatuba Dental School - UNESP and maintained at the Department of Pathology and Oral Diagnostics laboratory. Initially, the presence of resident S. mutans in rats’ oral cavities was evaluated by culture (Gold et al., 1973) and real-time PCR (Yoshida et al., 2003).

The rats presenting resident S. mutans or S. sobrinus were substituted. After confirming the absence of resident S. mutans, the submandibular and sublingual glands of the rats were surgically removed and the parotid ducts were ligated as described by Bowen et al. (1988). All surgical procedures were performed under general anesthesia of the animals with sodium thiopental (50 mg/kg).

On the fourth post-surgical day, 10⁶ CFU of S. mutans ATCC 35668 were inoculated into the oral cavity of the rats, which were fed with a cariogenic diet (NIH 2000 diet, 56% sucrose) (Hamada et al., 1978) to stabilize the implantation of the microorganisms (Peres et al., 2002) and water ad libitum. Verification of implantation of Streptococcus mutans ATCC 35668 and colonization of the oral cavity were performed after seven days as previously described.

The animals were randomly assigned to three experimental groups (n = 20): I: P. cattleianum aqueous extract; II: M. urundeuva aqueous extract; and III: sterilized distilled water (control group). Just before the extracts were used, they were twice diluted with distilled drinking water, to achieve a final concentration of 7.5 mg/mL. Animals were weighed weekly and their physical appearances (weight, mobility, presence or absence of cutaneous lesions, amount of food ingested, volume of water drank and appearance of urinary excretions) were noted daily.

Quantification of Streptococcus mutans

Samples of biofilms from all animals were harvested weekly by scaling around gingival margins and by sterilized cotton balls on coronary surfaces. The enumeration of Streptococcus mutans was performed by real-time PCR. The samples were transferred to 300 μL of ultra pure Milli Q water and submitted to DNA extraction by commercial kits and amplification by real-time PCR using primers and probe specific for S. mutans, as previously described (Yoshida et al., 2003).

Real-time PCR amplification was performed as previously described (Yoshida et al., 2003). The TaqMan^® 5′nuclease assay PCR method was used. The probes were double labeled with a reporter dye (FAM: 6-carboxyfluorescein) covalently attached at the 5′ end, and a quencher dye (TAMRA: 6-carboxytetramethylrhodamine) covalently attached at the 3′ end. The sequences of the primers and probes are presented in Table 1.

Table 1.  Oligonucleotide primers and probes.

Microorganism	Sequence 5′→3′	Amplicon Size (bp)	Target
S. mutans	5′-GCC TAC AGC TCA GAG ATG CTA TTC T-3′	114	gtfB
	5′-GCC ATA CAC CAC TCA TGA ATT GA-3′
	5′-TGG AAA TGA CGG TCG CCG TTA TGA A-3′ (probe)
S. sobrinus	5′-TTC AAA GCC AAG ACC AAG CTA GT-3′	88	gtfT
	5′-CCA GCC TGA GAT TCA GCT TGT-3′
	5′-CCT GCT CCA GCG ACA AAG GCA GC-3′ (probe)

TaqMan^® reactions contained 12.5 μL 2× TaqMan^® Universal Master Mix (Applied Biosystems, Foster City, CA), 9 μL ultra-pure water, each primer pair at a concentration of 200 nM with probe concentration of 100 nM. The total volume of reactions was 25 μL and reactions were performed in duplicate. Taqman amplification and detection were performed with a Rotor Gene 6000 (Corbett Life Science, Mortlake, New South Wales, Australia). Assay conditions for all primer/probe sets consisted of an initial denaturation step at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. The correlation coefficients for all standard curves was >0.99. DNAs of Streptococcus mutans ATCC 35668 and Streptococcus sobrinus ATCC 27609 were used as positive controls.

Evaluation of enamel micro-hardness

After seven weeks, the animals were sacrificed. Their upper central incisors and molars were removed and fixed in formaldehyde 2% (pH 7) for 30 days. Next, the teeth were longitudinally sectioned through the center, embedded in acrylic resin and serially polished. Cross-sectional micro-hardness (CSMH) was measured using a Shimadzu HMV-2000 micro-hardness tester and a Knoop diamond under a 25 g load for 10 s. Three rows of four indentations were made at 10, 30, 50, and 70 μm from the outer enamel surface on the cervical area of incisors and on the bottom of fissures of molars. The mean values at the three measuring points at each distance from the surface were then averaged and the results were expressed in Knoop hardness number (KHN) (Shinoda, 1975).

Statistical analysis

The null hypothesis tested was that treatment with the extracts does not change S. mutans counts and cross-sectional micro-hardness in the animal model used. GMC software was used for the statistical analysis. Analysis of variance and Tukey test were carried out after confirmation of normality and homogeneity of the data (Fontana et al., 2000; Al-Qunaian, 2005). P < 0.05 values were considered statistically significant.

Results

All rats showed a gradual and statistically significant gain of weight along the seven weeks and no significant differences between treatment groups were observed (ANOVA, p >0.05) (Figure 1). Figure 2 shows that already from the third week it is possible to detect a significant reduction in S. mutans in the experimental groups treated with plant extracts when compared to water. There were no statistical differences on bacterial survival between the two extracts studied. In addition, no further decrease in bacterial survival was observed after the third week of experiment for the extracts groups (ANOVA, p <0.05).

Figure 1.  Weight variation of the animals during the experiment as a function of time (mean ± SD). Distinct letters show statistical difference between treatments within the same period or between the same treatment along the whole experiment (p <0.05).

Figure 2.  Streptococcus mutans counts from oral biofilm of rats from different experimental groups in time (mean ± SD, n = 10). Distinct letters show statistical difference between treatments within the same period or between the same treatment along the whole experiment (p <0.05).

CSMH of extract-treated groups was statistically higher than the control group in all depths (ANOVA, p <0.05), but no differences were found in CSMH between incisors and molars (Figure 3). It is important to note that even at higher depths (> 50 μm), when enamel demineralization is not expected to occur, the KHN of the groups treated with the extracts were still higher when compared to the rats treated with water.

Figure 3.  Cross-sectional micro-hardness (in KHN) from incisors (solid lines) or molars (dashed lines) as a function of depth after exposure to water or the extracts for 7 weeks (mean ± se, n = 10). Distinct letters show statistical difference between treatment groups and depth (ANOVA, p<0.05).

Discussion

Because of the importance of S. mutans in dental caries etiology, The control of mutans streptococci populations became the main objective of the preventive ecological procedures, which aim to selectively interfere on dental biofilm microbial populations and on the expression of their virulence factors (Featherstone, 2000). In addition, S. mutans counts in dental biofilm or saliva is an indicator of caries activity and has been used for caries risk evaluation (Carvalho et al, 2006). However, van Ruyven et al. (2000) showed a conflicting association between the presence of S. mutans and carious lesions development, which may be due to different methodologies to assess caries development. Despite the fact that clinical examination is the most common detection method used in dental caries animal models, several scoring systems and parameters are available. Moreover, it is limited because enamel demineralization starts before it can be visually detected. For this reason, we decided to carry out cross-sectional micro-hardness to evaluate caries development, which is considered a sensitive method to assess tissue demineralization (Featherstone et al., 1983).

Natural products are receiving increased attention due to their extensive range of biological properties, allowing the discovery of novel bioactive compounds, usually produced by plants as protective substances. P. cattleianum contains flavonoids (kaempferol, quercetin and cyanidin) and tannin (ellagic acid), which are recognized as having antibacterial activity (NGRP, 2005). In general, phenolic compounds are known to inhibit glucosyltransferase production in S. mutans (Matsumoto et al., 1999, Koo et al., 2006). Particularly, flavonoid activity is probably due to its ability to form complexes with extracellular and soluble proteins and with bacterial cell walls; tannins are able to inactivate proteins by forming irreversible complexes that may lead to loss of function of microbial adhesins, enzymes, cell envelope transport proteins (Cowan, 1999).

Despite the fact that the aqueous extract composition from M. urundeuva has not been established yet, data in the literature showed that this plant also presents phenols in its composition (Souza et al., 2007), suggesting a mechanism of action similar to that observed to P. cattleianum.

In the present study, the plant extracts led to a significant decrease in S. mutans counts and produced higher CSMH values when compared to the control group, and the effects of the P. cattleianum and M. urundeuva extracts were similar.

Phenolic agents have potent antimicrobial activity by enzyme inhibition (Badria & Zidan, 2004) and may interfere with microbial adhesion to host tissues by modifying microbial adhesins and host receptors involved in the process (Ooshima et al., 1993; Daglia et al., 2002; Yu et al., 2006). High contents of phenols might also lead to protein precipitation and inhibition of microbial growth (Matsumoto et al., 2004; Tayanin & Batthall, 2006). However, since the composition of the extracts is complex, their activity on microbial physiology probably represents a synergistic effect of the compounds.

Besides the inhibitory activity of the extracts, the reduction of microbial ability to ferment sugars may constitute an ecological disadvantage for S. mutans, allowing the stabilization of pH and reducing the mineral loss in the dental enamel, as observed in the present study. This ability of natural products to interfere with lactic acid and glucans production by cariogenic microorganisms was previously described by Leitão et al. (2004) and Nalina and Rahim (2007).

The lower demineralization shown by enamel micro-hardness and the reduction of S. mutans population during the experiment can be related to the inhibitory activity produced by the extracts, due to the interference in the microbial colonization or inhibition of essential proteins. Thus, the extracts of the present study may be an alternative for the prevention of dental caries, even in low concentrations, where collateral effects, if present, may be minimized.

Declaration of interest

This study was partially supported by grants of Fundação de Amparo à Pesquisa do Estado de São Paulo (proc. 2002/07571-0, 2003/12763-7 and 2007/54851-0). The authors declare that they do not have any commercial relationships or conflict of interests with any public or private foundations in the development or publication of the article.