Of Calendula (Calendula Officinalis L.) and Cashew Tree (Anacardium Occidentale L.)

ISSN - 1519-0501 DOI: 10.4034/PBOCI.2014.141.08

Physical Properties and Antibacterial Activity of Herbal Tinctures of Calendula (Calendula officinalis L.) and Cashew Tree (Anacardium occidentale L.)

Julio Cesar Campos Ferreira Filho¹, Brenna Louise Cavalcanti Gondim², Diego Alves da Cunha², Christopher Cadete de Figueiredo¹, Ana Maria Gondim Valença³

¹ Undergraduate dental student, School of Dentistry, Federal University of Paraíba, Brazil. ² Dentist, Federal University of Paraiba, Brazil. ³ Associated Professor, Department of Clinical and Community Dentistry, School of Dentistry; Professor, Federal University of Paraiba, Brazil.

ABSTRACT

Objective: To evaluate in vitro the pH, Brix and antibacterial activity of herbal tinctures of calendula (Calendula officinalis L.) and cashew tree (Anacardium occidentale L.) against the following bacterial strains: Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 10557), Streptococcus salivarius (ATCC 9758), Enterococcus faecalis (ATCC 29212) and Eikenella corrodens (ATCC 23834). Material and Method: The strains were reactivated in agar Brain Heart Infusion (BHI) and seeded in blood agar with BHI. Susceptibility tests were performed and the strains were incubated in microaerophilia (37 °C) for 48 h. Chlorhexidine at 0.12% was used as a positive control and distilled water as a negative control. Dilution of the tinctures was carried out from 1:1 (pure form) to 1:64 in alcohol 70%, which is found in their composition, in order to evaluate a possible influence of alcohol on the antibacterial activity of the tinctures. oBx readings were made by refractometry using an Abbe refractometer and the pH was measured using a digital pH meter. Results: The calendula tincture had pH=5.43 and 18 °Brix and cashew tree tincture had pH=7.2 and 17 °Brix. The cashew tree tincture did not present antibacterial activity against S. oralis, but it inhibited the growth of all other bacteria up to the 1:64 dilution. The calendula tincture presented antibacterial activity against S. oralis up to 1:16 dilution (6.5 mm halo diameter); against S. mutans and S. salivarius up to 1:32 dilution (7 and 7.5 mm halo diameter, respectively); and against E. faecalis and E. corrodens up to 1:64 dilution. Alcohol 70% did not show any antibacterial activity against the bacterial strains. Conclusion: The tinctures of calendula and cashew tree have soluble solids, have different pH values and present a significant antibacterial activity against several bacterial strains, except for the cashew tree tincture against S. oralis, which was ineffective.

Key-words: Microbiology; Phytotherapy; Physical properties; Antimicrobials; Phytotherapic Drugs.

community structured three-dimensionally and INTRODUCTION composed by several microbial species that interact and communicate by signal transduction processes [1].

Streptococcus oralis is an acidogenic Dental biofilm is intimately associated with the microorganism present in the initial phase of dental onset, dissemination and progression of oral problems caries. It makes the environment more adequate for such as caries and periodontal diseases. The formation of plaque colonization by Streptococcus mutans, which is bacterial biofilm on teeth, restorations, implants or other considered as one of the most important pathogens surfaces exposed in the oral cavity is an omnipresent associated with the initiation and progress of dental phenomenon of microbial colonization at interfaces caries and has a key role in lesion formation [4]. between solid surfaces and biologic fluids [1]. It starts Streptococcus salivarius is one of the main components within a few minutes by the adsorption of salivary of biofilms formed on oral epithelium, tongue, dorsal proteins, glycoproteins and mucins, resulting in an epithelium [3] and corresponds to the most part of total “acquired enamel pellicle”. Shortly after, the first cultivable flora in the oral soft tissues [5]. Enterococcus bacteria adhere to start the colonization of this pellicle faecalis is a bacterium usually found in the human surface [2]. This process leads to the formation of the gastrointestinal tract, but it is associated with persistent dental plaque, a biofilm with multiple layers of bacteria periapical infections and failure of endodontic treatment resulting from the growth and co-adhesion of additional with calcium hydroxide [6], which suggests resistance to bacteria. Maturation of the extracellular matrix encasing the drug. Eikenella corrodens is an opportunistic bacillus the dental plaque is characterized by the formation of a and is found in the endogenous oral microflora (forming network of water-soluble and non-soluble glucans a subgingival plaque), the upper airways, and the synthesized by bacterial glycosyl transferases [3]. gastrointestinal and genitourinary tracts. This bacterium Therefore, oral biofilm is established as a may cause infections with slow progression, usually of

Pesq Bras Odontoped Clin Integr. 2014; 14(1):49-53 49 Ferreira Filho et al. – Physical Properties and Antibacterial Activity of Herbal Tinctures

polymicrobial nature and frequently associated with a Tropical Medicine Center (NUMETROP) of the Health putrid odor, simulating an anaerobic process. It is Sciences Center of the Federal University of Paraíba commonly associated with periodontal problems and (UFPB). The following bacterial strains were used: S. may be involved in bacterial endocarditis [7]. mutans (ATCC 25175), S. oralis (ATCC 0557), S. salivarius Calendula officinalis L. is an annual herbaceous (ATCC 9758), E. faecalis (ATCC 29212) and E. corrodens plant of European origin adapted to Brazil and belonging (ATCC 23834). Because of its scientifically confirmed to the Asteraceae family. It is widely employed in antibacterial action, 0.12% chlorhexidine gluconate was phytotherapy because of its antiinflammatory, healing used as a positive control, while distilled water as a and antibacterial properties [8]. Flavonoids are among negative control. the most widely researched secondary metabolites of The tinctures teted in this study were purchased this plant species, and are in great part responsible for its ready-to-use from a prescription pharmacy (Vita Flora biological activities, specially the rutin heterosidic Farmácia de Manipulação, João Pessoa, PB, Brazil). flavonoid [8]. Regarding the substances that produce the According to the technical report, both tinctures were therapeutic effects attributed to Calendula officinalis L., diluted 20% in alcohol 70%. In addition to this initial it has been confirmed the presence of flavonoids, dilution, a serial dilution of the tinctures was performed flavones/flavonols and xanthones, cathechins, starting from 1:1 (“pure” form) to 1:64 (6th dilution) in flavonones and alkaloids. There is also evidence of alcohol 70%, which is routinely used in the preparation of phenol in its constitution [9]. phytotherapic tinctures. Anacardium occidentale L. (cashew tree) The bacteria were reactivated in Brain Heart belongs within the Anacardiaceae family and is originally Infusion agar broth (BHI Agar; Difco®, São Paulo, SP, native to northeastern Brazil, in the area between the Brazil) and seeded on blood agar in Petri dishes using Atlantic rain forest and the Amazon rainforest. This disposable swabs and following the visual pattern of the evergreen tree has several edible pear-shaped “pseudo- McFarland scale. Bacterial aggregate inocula were fruits” or “false fruits” known as “cashew apples”, which immersed in test tubes containing saline (one tube per are actually floral peduncles, and small bean-shaped, bacterial strain) and left to grow until reaching a turbidity grey “true fruits” that are adhered to the lower end of corresponding to 0.5 on the McFarland scale, which the cashew apples and are known as “cashew nuts”. indicates the existence of approximately 1.5 x 108 colony These parts are used in the treatment of several forming units per milliliter (CFU/mL). inflammatory conditions (e.g.: arthritis) [10], fever, pain The susceptibility test was performed using the [11] and asthma [12]. Antimicrobial action against the agar dilution method to verify the presence of external rotavirus that causes diarrhea [13] and against Candida or internal contaminations (impure strains), and the albicans, Trichophyton rubrum and Cryptococcus strains were incubated at 37 °C in microaerophilia for 48 neoformans [14] has been investigated. Brazilian studies h. Hand calipers were used to measure the zones of have identified several chemical composites, such as bacterial growth inhibition formed around the strains. carotenoids (-carotene, -carotene, -cryptoxanthin, 9- The measurements were made in duplicate and the data cis- +13-cis-b-carotene, auroxanthin, 5,8-epoxy- were analyzed descriptively, considering as results the cryptoxanthin, 5,8-epoxy-lutein, z-carotene) and ascorbic arithmetic means of the diameter of the zones of acid [15]. bacterial growth inhibition in millimeters Research on the antibacterial action of these pH was measured with an pH meter (Analyser® two plants is still limited and there is need to widen the model 300; São Paulo, SP, Brazil) calibrated at room knowledge about their medicinal properties and possible temperature (25 C). Total soluble solids contents (oBrix) applications, looking forward to contributing to the were measured with a handheld refractometer (model development of new phytotherapic products in N1, Atago Co. Ltd, Honcho, Itabashi-ku, Tokyo; range: 0 Dentistry. The aim of this study was to evaluate in vitro to 32% Brix, precision: 0.2%). All measurements were the pH, Brix and antibacterial activity of herbal tinctures made in triplicate and averaged to obtain the arithmetic of calendula (Calendula officinalis L.) and cashew tree means. (Anacardium occidentale L.) against several bacterial strains. RESULT S

M ATERIAL AND METHODS Data from the antibacterial activity of the calendula and cashew tree tinctures against the five bacterial species are presented in Tables 1 and 2, An inductive approach with descriptive statistics respectively. Table 3 presents the BRIX and pH results. and direct laboratory documentation technique was adopted. The bacterial strains used in this study were obtained from Laboratory of Reference Materials of the DISCUSSION National Institute for Health Quality Control - Oswaldo

Cruz Foundation (FIOCRUZ, Rio de Janeiro, RJ, Brazil) and provided by the Laboratory of Oral Microbiology of the The antibacterial agents most frequently used

50 Pesq Bras Odontoped Clin Integr. 2014; 14(1):49-53 Ferreira Filho et al. – Physical Properties and Antibacterial Activity of Herbal Tinctures

Table 1. Antibacterial action according to the bacterial growth inhibition zones (mm) produced by the Calendula officinalis L. tincture against the bacterial strains. Bacterial growth inhibition zones (mm) 1:1 Distilled Alcohol 1:2 1:4 1:8 1:16 1:32 1:64 Chlorhexidine** (pure water* 70% dilution dilution dilution dilution dilution dilution form) S. mutans 0 0 14 10.5 10 8 8 8 7 0 S. oralis 0 0 13 8 8 8 7 6.5 0 0 S. salivarius 0 0 12 10 10 9 9 7.5 7.5 0 E. faecalis 0 0 12 9 9 9 7 7 7 6 E. corrodens 0 0 14 12.5 11.5 11.5 9 8.5 8 6.5 *: Negative control. ** Positive control.

Table 2. Antibacterial action according to the bacterial growth inhibition zones (mm) produced by the Anacardium occidentale L. tincture against the bacterial strains. Bacterial growth inhibition zones (mm) 1:1 Distilled Alcohol Chlorhexid.* 1:2 1:4 1:8 1:16 1:32 1:64 (pure water* 70% * dilution dilution dilution dilution dilution dilution form) S. mutans 0 0 17 12 11 10 9.5 9 8 7 S. oralis 0 0 13 0 0 0 0 0 0 0 S. salivarius 0 0 15 13 9 9 8.5 8 7.5 7,5 E. faecalis 0 0 17 11 9 9 8 8 7 7 E. corrodens 0 0 19 15 9 9 9 9 7.5 7 *: Negative control. ** Positive control.

Table 3. Mean ºBrix (percentage) and pH values of predilection. The idea was to provide an overview of the Calendula officinalis L. and Anacardium occidentale L. antimicrobial action of the two herbal tinctures against tinctures. important pathogens of the oral microbiota. The agar Herbal tincture ºBrix(%) pH diffusion method was used because it is usually Calendula officinalis L. 17 5.43 employed in studies assessing the antimicrobial action of Anacardium occidentale L. 18 7.20 artificial or natural products [18-20]. The 0.12% chlorhexidine digluconate was used as a positive control because it has a recognized efficacy for prevention and treatment of oral diseases, including against a wide range of microorganisms and has long cetylpyridinium chloride, chlorhexidine, amine fluorides been considered as a gold standard antimicrobial agent. or products containing these agents, are reported to Its cationic nature provides connection with ammonium- present toxicity and cause pigmentation of teeth and based compounds of the bacterial cell surface (teichoic microbial resistance as a result of prolonged daily use acid phosphate groups in Gram-positive bacteria and [4,16]. Traditional plants used for health care are an lipopolysaccharides in Gram-negative bacteria) and able important source of new compounds to serve as the base to alter the bacterial integrity. Alteration of the of modern pharmacology, since a great part of synthetic permeability of the cytoplasmic membrane produces drugs is directly or indirectly derived from medicinal precipitation of cytoplasmic proteins, alters the cell plants [17]. osmotic balance, affects cell metabolism, growth and In this way, studies using phyto-pharmaceutical division, inhibits cell membrane ATP-ase and the drugs or medicinal products derived from plant parts anaerobic processes [21], acting as a scientifically proven should receive special emphasis in both social and antimicrobial agent. scientific contexts, being an alternative for the treatment It is difficult to confront our results of of different diseases and conditions in several health antimicrobial activity with the literature in view of the areas. Studies assessing the antimicrobial activity of lack of studies evaluating the same tinctures against the phytotherapic products are important predecessors of same bacteria and using the same methodology. This fact more specific and accurate evaluations. emphasizes the importance of this study for future In this study, the antimicrobial action of herbal research that supports the clinical use of the calendula tinctures of calendula (Calendula officinalis L.) and (Calendula officinalis L.) and cashew tree (Anacardium cashew tree (Anacardium occidentale L.) was evaluated occidentale L.) tinctures. It is important to highlight that against reference strains of S. mutans (ATCC 25175), S. the diameter of the microbial growth inhibition zones oralis (ATCC 0557), S. salivarius (ATCC 9758), E. faecalis produced by the tinctures were relatively close to those (ATCC 29212) and E. corrodens (ATCC 23834) because produced by the positive control (0.12% chlorhexidine), these microorganisms have an important participation in especially the cashew tree tincture, which inhibited the oral microflora, have specific behaviors, and bacterial growth up to the 1:64 dilution, except for S. represent a diverse group of bacteria with different site oralis.

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The initial formulation of hydroalcoholic extracts prescription pharmacy, which hampers obtaining is made with alcohol 70% and for this reason it was used information on the plants. Additionally, dilution in for dilution of the samples [19]. Additionally, its alcohol in the “pure” form at 20% might have influenced interaction with bacteria was evaluated, as reported the results by underestimating the diameters of the elsewhere [19], in order to verify whether alcohol had bacterial growth inhibition zones. Consequently, some of any action against the bacterial strains, reducing the these factors may have acted to alter the antimicrobial chances of research bias. The results ruled out this properties of the tinctures, but might as well explain the hypothesis, demonstrating bacterial resistance to alcohol different pH found in a previous study, even though they and indicating that the antimicrobial effect of the are within the normal range of pH values [9]. tinctures seems to be not influenced by this substance There are important limitations in the in vitro [19]. If this hypothesis were not rejected, it would be experimental models with planktonic cells, but their impossible to attribute the antibacterial action to the results cannot be overlooked because of their phytotherapic compound, since it could be caused by the importance for the preliminary evaluations of alcohol 70% present in the tincture formulation or by the antimicrobial agents against some specific bacterial association of tincture with alcohol 70%. species [27]. Refractometry or oBrix is a physical method for Considering that there is a growing interest for measuring the quantity of soluble substances (sugar natural therapies like phytotherapy and an emphasis to salts, proteins, acids and other) in a water solution [22]. use phytotherapic agents in public health treatments, the A high level of sugars in the formulations is directly promising results of this study suggest further research related to their cariogenic potential. In the present study, with the calendula and cashew tree tinctures for a better the calendula and cashew tree tinctures presented 17 understanding of their antimicrobial activity and their and 18 oBrix respectively, which corresponds to 17 and potential clinical use in Dentistry.

18 g of solids dissolved in 100 g of solution. Speaking generally, the higher this value, the greater the possibility of the tincture to present a sweet nature. It CONCLUSION should be pointed out that such values are lower that those found in mouthwashes available on the Brazilian market, such as Clinerize (23.7%), Listerine Cool Citrus The calendula (Calendula officinalis L.) and (22.7%), Prevident 220 (20.9%) and Equate (20%) [23]. cashew tree (Anacardium occidentale L.) tinctures have A pH value equal or lower than 5.5 is considered different pH values, possess soluble solids in their critical for dissolution of dental enamel, but the mineral composition and present a significant antimicrobial loss may start with a higher pH value [24]. Therefore, activity against several oral bacterial strains. prolonged use of mouthwashes with pH below this value Antimicrobial activity was not verified only for the may be potentially harmful to the dental tissues. This is cashew tree tincture against S. oralis. In view of the important when using calendula tinctures, which have above findings, further studies with different low or very close pH values. In a previous study [9], a methodologies are recommended in order to Calendula officinalis L. tincture presented a pH value of corroborate the promising results of this experiment. 4.79, which is lower than the one obtained in the present study (5.43). On the other hand, the cashew tree tincture had a neutral pH (7.20), which is not aggressive to REFERENCES enamel (low pH values). Increased acidity of the medium reduces the antibacterial action of basic substances such as 1. Rupf S, Balkenhol M, Sahrhage TO, Baum A, Chromik JN, streptomycin and, on the other hand, creates favorable Ruppert K, Wissenbach DK, Maurer HH, Hannig M. Biofilm conditions for a more intense activity of acid substances inhibition by an experimental dental resin composite containing like penicillin [25]. This could have a negative influence octenidine dihydrochloride. Dent Mater 2012; 28(9):974-84. 2. Hannig C, Hannig M. The oral cavity – a key system to on the antimicrobial activity of the calendula tincture. understand substratum-dependent bioadhesion on solid The physicochemical properties of the products surfaces in man. Clin Oral Investig 2009; 13(2):123-39. may influence on their diffusion in culture media and 3. Bowen WH, Koo H. Biology of Streptococcus mutans – interfere in the results of antimicrobial evaluation [20]. derived lucosytransferases: role in extracellular matrix Other factors that can affect the pharmacological formation of cariogenic biofilms. Caries Res 2011; 45(1):69-86. properties of phytotherapic drugs must also be 4. Knoll-Köhler E, Stiebel J. Amine fluoride gel affects the considered, such as the age of the plant, site and season viability and the generation of superoxide anions in human of plant collection, quality of the used solvent, type of polymorphonuclear leukocytes: an in vitro study. Eur J Oral Sci packaging (hermetically sealed or not), as well as product 2002; 110(4):296-301. 5. Tamura S, Yonezawa H, Motegi M, Nakao R, Yoneda S, evaporation during storage possibility increasing the Watanabe H, Yamazaki T, Senpuku H. Inhibiting effects of concentration of the individual components. Streptococcus salivarius on competence-stimulating peptide- Additionally, tannin contents may vary significantly with dependent biofilm formation by Streptococcus mutans. Oral the used extraction method [26]. This is a limitation to Microbiol Immunol 2009; 24(2):152-61. the study because the tinctures were purchased from a 6. Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J.

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