JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2014, 1–5 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0181
FULL COMMUNICATION
Evaluation of Melia azedarach Extracts Against Streptococcus mutans Alvaro Della Bona1 and Fernanda Nedel 2 1
Post-Graduate Program in Dentistry, Dental School, University of Passo Fundo, Passo Fundo, Brazil. 2 Post-Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, Brazil.
ABSTRACT Although the incidence of caries worldwide has declined in recent years, it is necessary to search for new means to overcome this disease and its microbiological agents. Phytochemistry can become an effective alternative to antibiotics, offering a promising strategy in the prevention and therapy of dental caries. This study aimed to evaluate in vitro the bactericide activity of a bioactive phytocomponent from Melia azedarach against Streptococcus mutans. The crude extract (CEx) from leaves and stem barks of M. azedarach in chloroform, petroleum ether, acetate ethyl, butanol, and aqueous fractions was evaluated using seven different concentrations. Disk diffusion and minimum inhibitory concentration assays were used to evaluate the antibacterial activity. 0.12% chlorhexidine was used as a positive control. The CEx and the petroleum ether fraction from M. azedarach showed significant antibacterial activity against S. mutans, confirming its antibiotic potential.
KEY WORDS: antibacterial activity bioactive phytocomponents caries M. azedarach S. mutans
tive to traditional antibiotics and represent a promising practice in the prevention and treatment strategy for various diseases, including dental caries.4 In a recent review on the potetial of plant extract and natural products to overcome oral bacteria, authors stated that natural products derived from medicinal plants have proven to be a rich source of biologically active compounds, many of which have been the basis for the development of new chemicals in the pharmaceutical industry. In addition, the authors emphasized that there are about 500,000 plant species worldwide and only 1% had their bioactive phytocomponents investigated.5 The antibacterial activity of mauve dyes, sage, cocoa, chamomile, thyme, and propolis has been studied against S. mutans and Streptococcus sobrinus. Only thyme, cocoa, and propolis showed antibacterial activity.6 Riihinen et al.7 suggested that the polyphenol constituent in the high molecular size fraction of bilberry, blackcurrant, lingonberry, and cranberry juice can affect the aggregation of oral bacteria.7 Vasconcelos et al.8 evaluated a herbal gel of Punica granatum Linn (pomegranate) against S. mutans, Streptococcus mitis, and Candida albicans, using the miconalzole (Daktarin oral gel) as a positive control. The pomegranate gel showed a higher efficacy than miconalzole in experiments with three and four associated microorganisms.8 Together, these results suggest the possibility of using natural substances as a controlling agent of plaque microorganism. Melia azedarach L., popularly known as Persian lilac or chinaberry, is native to tropical Asia. However, this plant has been widely spread, it was introduced and naturalized in Philippines, United State of America, Brazil, Argentine, and
INTRODUCTION
D
ental caries is considered a public health problem where Streptococcus mutans plays a key role in its etiology. In addition to local and systemic changes resulting from the cariogenic process, psychosocial changes are also a consequence of inadequate dentition. Dental caries development is linked to the accumulation of microorganisms in the biofilm formed on the surface of the teeth (dental plaque) associated with the frequent intake of sugar. Thus, the primary objective in terms of oral health is to maintain dental plaque control, by mechanical means, and regulate the intake of sugar. However, one should consider the difficulties for patients to maintain adequate plaque control and a balanced diet.1 Indeed, studies have demonstrated that after 3 months of complete plaque removal by professional prophylaxis and oral hygiene orientation, the plaque index reached 60–80% from the initial state. In conclusion, as a function of time, people feel discouraged regarding proper oral hygiene. These observations encouraged the use of substances as an attempt to make up for the lack of motivation and maintain a reduced plaque index.2,3 Although caries prevalence in Brazil has decreased in recent years, mainly due to the addition of fluoride to water and tooth pastes, it is necessary to search for new ways to overcome this disease and its microbiological agents. Bioactive phytocomponents can offer an effective alternaManuscript received 10 December 2013. Revision accepted 18 June 2014. Address correspondence to: Alvaro Della Bona, DDS, MS, PhD, Post-Graduate Program in Dentistry, Dental School, University of Passo Fundo, Campus I, BR285, km 171, Passo Fundo, RS 99001-970, Brazil, E-mail: [email protected]
1
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DELLA BONA AND NEDEL
many African and Arab countries.9,10 Traditional medicine has used M. azedarach leaves, roots, fruits, flowers, and seeds for the treatment of a large spectrum of diseases. Leaves have been used in the treatment of leprosy, vitiligo, anthelmintic, as an antidote against toxins, diuretic, chronic intestinal obstruction, and purulent sores. Roots are effectively used as a resolvent and deobstruent. Fruits have been used in the treatment of leprosy, vitiligo, head wounds, phlegmatic fevers, and as cough relievers. The flower is used for brain obstructions, as a temperament normalizer, headaches, and as other head pain relievers. Seed oil is the most active medicinal product of the plant and used as an antiseptic for sores and ulcers that show no tendency to heal. It is also used for rheumatism and skin disease such as ringworm and scabies. Internally, the oil is useful in malaria fever and leprosy. Fresh leaf extract is applied externally for burns and as a mouthwash constituent for gingivitis.9,11 Bioactive phytocomponent investigations of M. azedarach have identified antiviral activity against several RNA and DNA viruses,12 antimalarial, antifungal,13 antifeedant activity on a variety of insects,14 rodenticidal,15 insecticide,16 nematotoxic,17 ovicidal and larvicidal of anthelmintic,18 antitumoral,19,20 and antibacterial activity.9 However, the literature has poorly explored the antibacterial activity of M. azedarach related to dental caries. Therefore, this study aimed to evaluate in vitro the bactericide activity of M. azedarach against S. mutans. Additionally, we compared the bactericide activity of M. azedarach to chlorhexidine, a broad-spectrum antibacterial agent commonly used in dental practice, testing the hypothesis that the extract of M. azedarach has a bactericidal action similar to 0.12% chlorhexidine. MATERIALS AND METHODS Plant material and extract preparation Leaves and stem barks of Melia azedarach L. (Meliaceae) were collected at S 28 130 35.700 ; W 52 230 14.500 .
They were dried and powdered. Samples were taken to the herbarium of the University of Passo Fundo for botanical identification and storage. The plant powder was refluxed with ethanol in the ratio 1:10 (w/v) for 30 min. The crude ethanol extract was cooled and filtered. A portion of it was reserved as crude extract (CEx) and the remaining was used to carry out liquid–liquid separation. The solvent was revaporated under reduced pressure in a rotary evaporator (BUCHI Rotavapor-R; Buch Laboratories, Flawil, Switzerland). After separation by a polarity gradient, fractions of petroleum ether (PE), chloroform (CF), ethyl acetate (EA), butanol (BT), CEx, and aqueous (Aq), residue were collected. A standard concentration of 0.1 g/mL was used for disk diffusion and minimum inhibitory concentration (MIC) assays. The solvent used was dimethylsulfoxide (DMSO; MERCK KGaA, Darmstadt, Germany). Disk diffusion test S. mutans strain (ATCC 25175) was obtained from the State University of Campinas. The assays to evaluate the antibacterial activity of the extracts were performed according to the National Committee for Clinical Laboratory Standards (NCCLS). For the disk diffusion assay, the direct method was used. S. mutans suspension was prepared inoculating bacterial colonies in sterile saline. The suspension was adjusted visually to match the turbidity of a McFarland 0.5 scale. The bacterial suspension was inoculated in a plate containing Mueller-Hinton agar, previously submitted to the sterility test, using the pour plate method. Disks were prepared with a 6 mm diameter and 2 mm thickness and impregnated with 20 lL of the CEx, obtained fraction, positive (chlorhexidine [CHX]-0.12%) and negative controls (DMSO), placed onto the inoculated agar plate and incubated at 36C – 1C under microaerophilic conditions for 24 h (Fig. 1). Zones of inhibition of microbial growth around the samples were measured and recorded after the incubation time. The diameters of inhibition zones
FIG. 1. Schematic representation of the disk diffusion test. Color images available online at www.liebertpub.com/ jmf
3
MELIA AZEDARACH EXTRACTS AGAINST S. MUTANS
FIG. 2. Schematic representation of the minimum inhibitory concentration determination.
were measured in millimeters using a millimeter scale. Tests were performed in triplicate.
sions were incubated in a bacteriological incubator at 36C – 1C for 24 h. Tests were performed in duplicate.
Determination of MIC Based on the results of the disk diffusion assay, the MIC test was conducted. Therefore, MIC was performed with the CEx and petroleum ether fraction of M. azedarach, as they showed inhibition zones in the disk diffusion assay. Eleven sterile test tubes were used, each containing 1 mL of brain heart infusion (BHI) broth. In tube 1, 1 mL of the extract fraction of M. azedarach was added, which previously showed an inhibition zone in the disk diffusion assay at a concentration of 0.1 g/mL. The solution was homogenized and 1 mL was transferred to tube 2. The dilution process continued until tube 7, where 1 mL of the solution was discarded. Tube 8 remained empty without inoculum, extract, or culture medium. In tube 9, 200 lL of 0.12% chlorhexidine 0.1 g/mL (0.02 g/mL) was added and homogenized, and 1 mL was transferred to tube 10. In tube 11, 1 mL of BHI was added (control) (Fig. 2). In each tube, with exception of tube 8, 200 lL of bacterial inoculum was added. After shaking the tubes, the suspen-
RESULTS Table 1 shows that the CEx of M. azedarach, presented a bactericidal potential similar to 0.12% CHX. The PE fraction presented a positive result, yet the bactericidal potential was lower than the value shown by the positive control (CHX). Based on the results, these two fractions were tested for MIC. The CF, EA, BT, and Aq fractions showed no bactericidal activity and therfore were not further tested for MIC. The disk diffusion results are summarized in Table 1, indicating which fraction of M. azedarach extracts showed bactericidal activity based on the inhibition zone. Tables 2 and 3 show, respectively, the test results of MIC related to PE and CEx. A MIC value of 0.1 g/mL was found for the PE fraction of M. azedarach, and a value of 0.025 g/mL was found for the CEx of M. azedarach. The positive control (CHX) showed no bacterial growth in the concentrations of 0.004 and 0.02 g/mL.
Table 1. Disk Diffusion Test Values for Tests 1–3 and Mean Value (in mm) and the Bactericidal Effect for the Experimental Groups Groups-substance CHX DMSO CEx PE CF BT EA Aq
Inhibition zone Test 1 (mm)
Inhibition zone Test 2 (mm)
Inhibition zone Test 3 (mm)
Inhibition zone Mean (mm)
Bactericidal effect
9.2 — 8.0 3.1 — — — —
9.5 — 8.0 3.1 — — — —
9.5 — 8.3 3.1 — — — —
9.4 — 8.1 3.1 — — — —
Yes No Yes Yes No No No No
CHX, chlorhexidine; DMSO, dimethylsulfoxide; CEx, crude extract; PE, petroleum ether; CF, chloroform; BT, butanol; EA, ethyl acetate; Aq, aqueous fractions of Melia azedarach. Yes indicates the presence of bactericidal effect.
4
DELLA BONA AND NEDEL Table 2. Minimum Inhibitory Concentration Values (g/mL) for the PE Fraction of M. Azedarach
Tube No. 01 02 03 04 05 06 07 08 09 10 11
Final extract concentration (g/mL)
Control concentration (g/mL)
Bacterial growth Test 1
Bacterial growth Test 2
0.1 0.05 0.025 0.0125 0.00625 0.003125 0.0015625 — — — —
— — — — — — — — 0.02 0.004 —
No Yes Yes Yes Yes Yes Yes No No No Yes
No Yes Yes Yes Yes Yes Yes No No No Yes
Yes indicates the presence of bacterial growth.
DISCUSSION Bioactive phytocomponents can offer an effective alternative to antibiotics and represent a promising approach in the prevention and treatment strategy for dental caries and other oral infections.4 Dental products containing natural compounds have been reported to have good commercial prospective due to popular acceptance of herbal medicine.6 Indeed, according to the World Health Organization (WHO) in 2008, more than 80% of the world population relied on traditional medicine for their primary healthcare needs.9 Khan et al.21 reported that extracts obtained from the leaves, stem, and root barks of M. azedarach contained tannins, flavonoids, and triterpenoids.21 There has been evidence that the most important compounds synthesized in the secondary metabolism of plant are alkaloids, flavonoids, tannins, phenolic compounds, steroids, resins, fatty acids, and gums, which are capable of producing physiological action on the human body.22 Flavonoids belong to the class of polyphenols and are a group of heterocyclic organic compounds present in plants and related products. There are 14 classes of flavonoids in Table 3. Minimum Inhibitory Concentration Values (g/mL) for CEx of M. Azedarach Tube No. 01 02 03 04 05 06 07 08 09 10 11
Final extract concentration (g/mL)
Control concentration (g/mL)
Bacterial growth Test 1
Bacterial growth Test 2
0.1 0.05 0.025 0.0125 0.00625 0.003125 0.0015625 — — — —
— — — — — — — — 0.02 0.004 —
No No No Yes Yes Yes Yes No No No Yes
No No No Yes Yes Yes Yes No No No Yes
*Yes-indicates the presence of bacterial growth.
total, differentiated on the basis of the chemical nature and position of substituents on the A, B, and C rings. Most of the studies regarding the antibacterial properties of flavonoids can be attributed to flavone, chalcone, flavonol, flavan-3-ol (also known as catechin), flavanone, flavolan (also known as proanthocyanidin), or their isoflavonoid counterparts.23 However, flavonoids have been reported as bioactive compounds responsible for the antibacterial activity of propolis against S. mutans and S. sobrinus.24 Among polyphenols, flavan-3-ols, flavanols, and tannins, received most attention due to their wide spectrum and higher antibacterial activity in compression with other polyphenols. In addition, this compound shows synergism with antibiotics and is able to suppress a number of microbial virulence factors, such as inhibition of biofilm formation, reduction of host ligand adhesion, and neutralization of bacterial toxins.25 Tannins are complex polymerized molecules with relatively high molecular weight.26 Several mechanisms could explain the effect of tannins in the bacterial growth inhibition, such as the destabilization of the cytoplasmic membrane,27 the inhibition of extracellular microbial enzymes, direct actions on microbial metabolism, or the deprivation of the substrates required for microbial growth, especially essential mineral micronutrients, such as ion and zinc (via tannins chelation with the metals), whose depletion can severely limit bacterial growth.25,28 In the present study, the CEx of M. azedarach presented a bactericidal potential similar to 0.12% chlorhexidine. The petroleum ether fraction showed positive results, although they were lower than the positive control group, probably due to a lower concentration of bioactive compounds in this fraction. Both fractions were further subjected to MIC analysis. Chloroform, ethyl acetate, butanol, and aqueous fractions showed no bactericidal activity and therefore were not included in the MIC assay. Khan et al.21 evaluated M. azedarach extracts against several human pathogenic bacterial strains. The leaf extracts showed bactericidal activity against gram-positive and gram-negative bacterial strains, where the Streptococcus group presented high sensitivity.21 In addition, the present study showed a MIC value of 0.1 g/mL for the petroleum ether fraction of M. azedarach, 0.025 g/mL for the CEx of M. azedarach, and 0.004 g/mL for the positive control. Even though the MIC values were greater for the control group (CHX), CEx of M. azedarach should be considered as an alternative bioactive compound against S. mutans, since CHX has several side effects such as dental staining and oral irritation. Data showed that the petroleum ether fraction of M. azedarach is not as promising as the CEx due to inferior MIC values and a significantly lower inhibition zone, compared to CHX. Since the CEx of M. azedarach presented the best results when compared with the extract fractions, we can speculate that the bactericidal effect against S. mutans could be mediated by the synergism between different components. When these components are separated using the solvent of different polarity, the bactericidal effect is lost. Within the limitation of this study, it can be concluded that the CEx and petroleum ether fraction of M. azedarach
MELIA AZEDARACH EXTRACTS AGAINST S. MUTANS
have a bactericidal effect against S. mutans. In addition, the CEx is more effective among the extracts, with the zone of inhibition similar to 0.12% chlorhexidine and MIC of 0.025 g/mL. These results confirm that the extracts from M. azedarach could be an effective antibiotic. AUTHOR DISCLOSURE STATEMENT
14.
15.
No competing financial interests exist. 16.
REFERENCES 1. Fraza˜o P: Epidemiology of dental caries: when structure and context matter. Braz Oral Res 2012;26 Suppl 1:108–114. 2. Garcia-Godoy F, Garcia-Godoy F, DeVizio W, Volpe AR, Ferlauto RJ, Miller JM: Effect of triclosan/copolimer/fluoride dentrifice on plaque formation and gengivitis. Am J Dent 1990;3: 515–526. 3. Svatur B, Saxton CA, van der Ouderaa F, Rolla G: The influence of a dentifrice containing a zinc salt and a nonionic antimicrobial agent on the maintenance of gingival health. J Clin Periodont 1987;14:457–461. 4. Singh J, Kumar A, Budhiraja S, Hooda A: Ethnomedicine: use in dental caries. Braz J Oral Sci 2007;6:1308–1312. 5. Palombo EA: Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2011;2011:680354. 6. Gebara ECE, Zardetto CGDC, Mayer MPA: In vitro study of the antimicrobial activity of natural substances against S. mutans and S. sobrinus. Rev Odont Univ Sa˜o Paulo 1996;10:251–256. 7. Riihinen K, Ryyna¨nen A, Toivanen M, Ko¨no¨nen E, To¨rro¨nen R, Tikkanen-Kaukanen C: Antiaggregation potential of berry fractions against pairs of Streptococcus mutans with Fusobacterium nucleatum or Actinomyces naeslundii. Phytother Res 2011;25: 81–87. 8. Vasconcelos LCS, Sampaio F, Sampaio M, Pereira M, Higino J, Peixoto M: Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S. mutans, S. mitis and C. albicans. Braz Dent J 2006;17:223–227. 9. Khan AV, Ahmed QU, Mir MR, Shukla I, Khan AA: Antibacterial efficacy of the seed extracts of Melia azedarach against some hospital isolated human pathogenic bacterial strains. Asian Pac J Trop Biomed 2011;1:452–455. 10. Saleem R, Rani R, Ahmed M, Sadaf F, Ahmad SI, ul Zafar N, Khan SS, Siddiqui BS, Khan SA, Faizi S: Effect of cream containing Melia azedarach flowers on skin diseases in children. Phytomedicine 2008;15:231–236. 11. Jafari S, Saeidnia S, Hajimehdipoor H, Ardekani MR, Faramarzi MA, Hadjiakhoondi A, Khanavi M: Cytotoxic evaluation of Melia azedarach in comparison with Azadirachta indica and its phytochemical investigation. Daru 2013;21:37–43. 12. Petrera E, Coto CE: Therapeutic effect of meliacine, an antiviral derived from Melia azedarach L., in mice genital herpetic infection. Phytother Res 2009;23:1771–1777. 13. Carpinella MC, Ferrayoli CG, Palacios SM: Antifungal synergistic effect of scopoletin, a hydroxycoumarin isolated from
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Melia azedarach L. fruits. J Agric Food Chem 2005;53:2922– 2927. Carpinella MC, Defago MT, Valladares G, Palacios SM: Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management. J Agric Food Chem 2003;51:369–374. Roop JK, Dhaliwal PK, Guraya SS: Extracts of Azadirachta indica and Melia azedarach seeds inhibit folliculogenesis in albino rats. Braz J Med Biol Res 2005;38:943–947. Defago´ M, Valladares G, Banchio E, Carpinella C, Palacios S: Insecticide and antifeedant activity of different plant parts of Melia azedarach on Xanthogaleruca luteola. Fitoterapia 2006; 77:500–505. Aoudia H, Ntalli N, Aissani N, Yahiaoui-Zaidi R, Caboni P: Nematotoxic phenolic compounds from Melia azedarach against Meloidogyne incognita. J Agric Food Chem 2012;60:11675– 11680. Kamaraj C, Rahuman AA, Bagavan A, Mohamed MJ, Elango G, Rajakumar G, Zahir AA, Santhoshkumar T, Marimuthu S: Ovicidal and larvicidal activity of crude extracts of Melia azedarach against Haemonchus contortus (Strongylida). Parasitol Res 2010;106:1071–1077. Akihisa T, Pan X, Nakamura Y, Kikuchi T, Takahashi N, Matsumoto M, Ogihara E, Fukatsu M, Koike K, Tokuda H: Limonoids from the fruits of Melia azedarach and their cytotoxic activities. Phytochemistry 2013;89:59–70. Kikuchi T, Pan X, Ishii K, Nakamura Y, Ogihara E, Koike K, Tanaka R, Akihisa T: Cytotoxic and apoptosis-inducing activities of 12-O-Acetylazedarachin B from the fruits of Melia azedarach in human cancer cell lines. Biol Pharm Bull 2013;36: 35–139. Khan MR, Kihara M, Omoloso AD: Antimicrobial activity of Horsfieldia helwigii and Melia azedarach. Fitoterapia 2001;72: 423–427. Dahiya P, Purkayastha S: Phytochemical screening and antimicrobial activity of some medicinal plants against multi-drug resistant bacteria from clinical isolates. Indian J Pharm Sci 2012;74:443–450. Cushnie TP, Lamb AJ: Recent advances in understanding the antibacterial properties of flavonoids. Int J Antimicrob Agents 2011;38:99–107. Uzel A, Sorkun K, Onc¸ag˘ O, Cogu˘lu D, Genc¸ay O, Salih B: Chemical compositions and antimicrobial activities of four different Anatolian propolis samples. Microbiol Res 2005;160:189– 195. Daglia M: Polyphenols as antimicrobial agents. Curr Opin Biotechnol 2012;23:174–181. Kandra L, Gye´ma´nt G, Zaja´cz A, Batta G: Inhibitory effects of tannin on human salivary alpha-amylase. Biochem Biophys Res Commun 2004;319:1265–1271. Hatano T, Kusuda M, Inada K, Ogawa TO, Shiota S, Tsuchiya T, Yoshida T: Effects of tannins and related polyphenols on methicillin-resistant Staphylococcus aureus. Phytochemistry 2005; 66:2047–2055. Heinonen M: Antioxidant activity and antimicrobial effect of berry phenolics—a Finnish perspective. Mol Nutr Food Res 2007;51:684–691.
FULL COMMUNICATION
Evaluation of Melia azedarach Extracts Against Streptococcus mutans Alvaro Della Bona1 and Fernanda Nedel 2 1
Post-Graduate Program in Dentistry, Dental School, University of Passo Fundo, Passo Fundo, Brazil. 2 Post-Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, Brazil.
ABSTRACT Although the incidence of caries worldwide has declined in recent years, it is necessary to search for new means to overcome this disease and its microbiological agents. Phytochemistry can become an effective alternative to antibiotics, offering a promising strategy in the prevention and therapy of dental caries. This study aimed to evaluate in vitro the bactericide activity of a bioactive phytocomponent from Melia azedarach against Streptococcus mutans. The crude extract (CEx) from leaves and stem barks of M. azedarach in chloroform, petroleum ether, acetate ethyl, butanol, and aqueous fractions was evaluated using seven different concentrations. Disk diffusion and minimum inhibitory concentration assays were used to evaluate the antibacterial activity. 0.12% chlorhexidine was used as a positive control. The CEx and the petroleum ether fraction from M. azedarach showed significant antibacterial activity against S. mutans, confirming its antibiotic potential.
KEY WORDS: antibacterial activity bioactive phytocomponents caries M. azedarach S. mutans
tive to traditional antibiotics and represent a promising practice in the prevention and treatment strategy for various diseases, including dental caries.4 In a recent review on the potetial of plant extract and natural products to overcome oral bacteria, authors stated that natural products derived from medicinal plants have proven to be a rich source of biologically active compounds, many of which have been the basis for the development of new chemicals in the pharmaceutical industry. In addition, the authors emphasized that there are about 500,000 plant species worldwide and only 1% had their bioactive phytocomponents investigated.5 The antibacterial activity of mauve dyes, sage, cocoa, chamomile, thyme, and propolis has been studied against S. mutans and Streptococcus sobrinus. Only thyme, cocoa, and propolis showed antibacterial activity.6 Riihinen et al.7 suggested that the polyphenol constituent in the high molecular size fraction of bilberry, blackcurrant, lingonberry, and cranberry juice can affect the aggregation of oral bacteria.7 Vasconcelos et al.8 evaluated a herbal gel of Punica granatum Linn (pomegranate) against S. mutans, Streptococcus mitis, and Candida albicans, using the miconalzole (Daktarin oral gel) as a positive control. The pomegranate gel showed a higher efficacy than miconalzole in experiments with three and four associated microorganisms.8 Together, these results suggest the possibility of using natural substances as a controlling agent of plaque microorganism. Melia azedarach L., popularly known as Persian lilac or chinaberry, is native to tropical Asia. However, this plant has been widely spread, it was introduced and naturalized in Philippines, United State of America, Brazil, Argentine, and
INTRODUCTION
D
ental caries is considered a public health problem where Streptococcus mutans plays a key role in its etiology. In addition to local and systemic changes resulting from the cariogenic process, psychosocial changes are also a consequence of inadequate dentition. Dental caries development is linked to the accumulation of microorganisms in the biofilm formed on the surface of the teeth (dental plaque) associated with the frequent intake of sugar. Thus, the primary objective in terms of oral health is to maintain dental plaque control, by mechanical means, and regulate the intake of sugar. However, one should consider the difficulties for patients to maintain adequate plaque control and a balanced diet.1 Indeed, studies have demonstrated that after 3 months of complete plaque removal by professional prophylaxis and oral hygiene orientation, the plaque index reached 60–80% from the initial state. In conclusion, as a function of time, people feel discouraged regarding proper oral hygiene. These observations encouraged the use of substances as an attempt to make up for the lack of motivation and maintain a reduced plaque index.2,3 Although caries prevalence in Brazil has decreased in recent years, mainly due to the addition of fluoride to water and tooth pastes, it is necessary to search for new ways to overcome this disease and its microbiological agents. Bioactive phytocomponents can offer an effective alternaManuscript received 10 December 2013. Revision accepted 18 June 2014. Address correspondence to: Alvaro Della Bona, DDS, MS, PhD, Post-Graduate Program in Dentistry, Dental School, University of Passo Fundo, Campus I, BR285, km 171, Passo Fundo, RS 99001-970, Brazil, E-mail: [email protected]
1
2
DELLA BONA AND NEDEL
many African and Arab countries.9,10 Traditional medicine has used M. azedarach leaves, roots, fruits, flowers, and seeds for the treatment of a large spectrum of diseases. Leaves have been used in the treatment of leprosy, vitiligo, anthelmintic, as an antidote against toxins, diuretic, chronic intestinal obstruction, and purulent sores. Roots are effectively used as a resolvent and deobstruent. Fruits have been used in the treatment of leprosy, vitiligo, head wounds, phlegmatic fevers, and as cough relievers. The flower is used for brain obstructions, as a temperament normalizer, headaches, and as other head pain relievers. Seed oil is the most active medicinal product of the plant and used as an antiseptic for sores and ulcers that show no tendency to heal. It is also used for rheumatism and skin disease such as ringworm and scabies. Internally, the oil is useful in malaria fever and leprosy. Fresh leaf extract is applied externally for burns and as a mouthwash constituent for gingivitis.9,11 Bioactive phytocomponent investigations of M. azedarach have identified antiviral activity against several RNA and DNA viruses,12 antimalarial, antifungal,13 antifeedant activity on a variety of insects,14 rodenticidal,15 insecticide,16 nematotoxic,17 ovicidal and larvicidal of anthelmintic,18 antitumoral,19,20 and antibacterial activity.9 However, the literature has poorly explored the antibacterial activity of M. azedarach related to dental caries. Therefore, this study aimed to evaluate in vitro the bactericide activity of M. azedarach against S. mutans. Additionally, we compared the bactericide activity of M. azedarach to chlorhexidine, a broad-spectrum antibacterial agent commonly used in dental practice, testing the hypothesis that the extract of M. azedarach has a bactericidal action similar to 0.12% chlorhexidine. MATERIALS AND METHODS Plant material and extract preparation Leaves and stem barks of Melia azedarach L. (Meliaceae) were collected at S 28 130 35.700 ; W 52 230 14.500 .
They were dried and powdered. Samples were taken to the herbarium of the University of Passo Fundo for botanical identification and storage. The plant powder was refluxed with ethanol in the ratio 1:10 (w/v) for 30 min. The crude ethanol extract was cooled and filtered. A portion of it was reserved as crude extract (CEx) and the remaining was used to carry out liquid–liquid separation. The solvent was revaporated under reduced pressure in a rotary evaporator (BUCHI Rotavapor-R; Buch Laboratories, Flawil, Switzerland). After separation by a polarity gradient, fractions of petroleum ether (PE), chloroform (CF), ethyl acetate (EA), butanol (BT), CEx, and aqueous (Aq), residue were collected. A standard concentration of 0.1 g/mL was used for disk diffusion and minimum inhibitory concentration (MIC) assays. The solvent used was dimethylsulfoxide (DMSO; MERCK KGaA, Darmstadt, Germany). Disk diffusion test S. mutans strain (ATCC 25175) was obtained from the State University of Campinas. The assays to evaluate the antibacterial activity of the extracts were performed according to the National Committee for Clinical Laboratory Standards (NCCLS). For the disk diffusion assay, the direct method was used. S. mutans suspension was prepared inoculating bacterial colonies in sterile saline. The suspension was adjusted visually to match the turbidity of a McFarland 0.5 scale. The bacterial suspension was inoculated in a plate containing Mueller-Hinton agar, previously submitted to the sterility test, using the pour plate method. Disks were prepared with a 6 mm diameter and 2 mm thickness and impregnated with 20 lL of the CEx, obtained fraction, positive (chlorhexidine [CHX]-0.12%) and negative controls (DMSO), placed onto the inoculated agar plate and incubated at 36C – 1C under microaerophilic conditions for 24 h (Fig. 1). Zones of inhibition of microbial growth around the samples were measured and recorded after the incubation time. The diameters of inhibition zones
FIG. 1. Schematic representation of the disk diffusion test. Color images available online at www.liebertpub.com/ jmf
3
MELIA AZEDARACH EXTRACTS AGAINST S. MUTANS
FIG. 2. Schematic representation of the minimum inhibitory concentration determination.
were measured in millimeters using a millimeter scale. Tests were performed in triplicate.
sions were incubated in a bacteriological incubator at 36C – 1C for 24 h. Tests were performed in duplicate.
Determination of MIC Based on the results of the disk diffusion assay, the MIC test was conducted. Therefore, MIC was performed with the CEx and petroleum ether fraction of M. azedarach, as they showed inhibition zones in the disk diffusion assay. Eleven sterile test tubes were used, each containing 1 mL of brain heart infusion (BHI) broth. In tube 1, 1 mL of the extract fraction of M. azedarach was added, which previously showed an inhibition zone in the disk diffusion assay at a concentration of 0.1 g/mL. The solution was homogenized and 1 mL was transferred to tube 2. The dilution process continued until tube 7, where 1 mL of the solution was discarded. Tube 8 remained empty without inoculum, extract, or culture medium. In tube 9, 200 lL of 0.12% chlorhexidine 0.1 g/mL (0.02 g/mL) was added and homogenized, and 1 mL was transferred to tube 10. In tube 11, 1 mL of BHI was added (control) (Fig. 2). In each tube, with exception of tube 8, 200 lL of bacterial inoculum was added. After shaking the tubes, the suspen-
RESULTS Table 1 shows that the CEx of M. azedarach, presented a bactericidal potential similar to 0.12% CHX. The PE fraction presented a positive result, yet the bactericidal potential was lower than the value shown by the positive control (CHX). Based on the results, these two fractions were tested for MIC. The CF, EA, BT, and Aq fractions showed no bactericidal activity and therfore were not further tested for MIC. The disk diffusion results are summarized in Table 1, indicating which fraction of M. azedarach extracts showed bactericidal activity based on the inhibition zone. Tables 2 and 3 show, respectively, the test results of MIC related to PE and CEx. A MIC value of 0.1 g/mL was found for the PE fraction of M. azedarach, and a value of 0.025 g/mL was found for the CEx of M. azedarach. The positive control (CHX) showed no bacterial growth in the concentrations of 0.004 and 0.02 g/mL.
Table 1. Disk Diffusion Test Values for Tests 1–3 and Mean Value (in mm) and the Bactericidal Effect for the Experimental Groups Groups-substance CHX DMSO CEx PE CF BT EA Aq
Inhibition zone Test 1 (mm)
Inhibition zone Test 2 (mm)
Inhibition zone Test 3 (mm)
Inhibition zone Mean (mm)
Bactericidal effect
9.2 — 8.0 3.1 — — — —
9.5 — 8.0 3.1 — — — —
9.5 — 8.3 3.1 — — — —
9.4 — 8.1 3.1 — — — —
Yes No Yes Yes No No No No
CHX, chlorhexidine; DMSO, dimethylsulfoxide; CEx, crude extract; PE, petroleum ether; CF, chloroform; BT, butanol; EA, ethyl acetate; Aq, aqueous fractions of Melia azedarach. Yes indicates the presence of bactericidal effect.
4
DELLA BONA AND NEDEL Table 2. Minimum Inhibitory Concentration Values (g/mL) for the PE Fraction of M. Azedarach
Tube No. 01 02 03 04 05 06 07 08 09 10 11
Final extract concentration (g/mL)
Control concentration (g/mL)
Bacterial growth Test 1
Bacterial growth Test 2
0.1 0.05 0.025 0.0125 0.00625 0.003125 0.0015625 — — — —
— — — — — — — — 0.02 0.004 —
No Yes Yes Yes Yes Yes Yes No No No Yes
No Yes Yes Yes Yes Yes Yes No No No Yes
Yes indicates the presence of bacterial growth.
DISCUSSION Bioactive phytocomponents can offer an effective alternative to antibiotics and represent a promising approach in the prevention and treatment strategy for dental caries and other oral infections.4 Dental products containing natural compounds have been reported to have good commercial prospective due to popular acceptance of herbal medicine.6 Indeed, according to the World Health Organization (WHO) in 2008, more than 80% of the world population relied on traditional medicine for their primary healthcare needs.9 Khan et al.21 reported that extracts obtained from the leaves, stem, and root barks of M. azedarach contained tannins, flavonoids, and triterpenoids.21 There has been evidence that the most important compounds synthesized in the secondary metabolism of plant are alkaloids, flavonoids, tannins, phenolic compounds, steroids, resins, fatty acids, and gums, which are capable of producing physiological action on the human body.22 Flavonoids belong to the class of polyphenols and are a group of heterocyclic organic compounds present in plants and related products. There are 14 classes of flavonoids in Table 3. Minimum Inhibitory Concentration Values (g/mL) for CEx of M. Azedarach Tube No. 01 02 03 04 05 06 07 08 09 10 11
Final extract concentration (g/mL)
Control concentration (g/mL)
Bacterial growth Test 1
Bacterial growth Test 2
0.1 0.05 0.025 0.0125 0.00625 0.003125 0.0015625 — — — —
— — — — — — — — 0.02 0.004 —
No No No Yes Yes Yes Yes No No No Yes
No No No Yes Yes Yes Yes No No No Yes
*Yes-indicates the presence of bacterial growth.
total, differentiated on the basis of the chemical nature and position of substituents on the A, B, and C rings. Most of the studies regarding the antibacterial properties of flavonoids can be attributed to flavone, chalcone, flavonol, flavan-3-ol (also known as catechin), flavanone, flavolan (also known as proanthocyanidin), or their isoflavonoid counterparts.23 However, flavonoids have been reported as bioactive compounds responsible for the antibacterial activity of propolis against S. mutans and S. sobrinus.24 Among polyphenols, flavan-3-ols, flavanols, and tannins, received most attention due to their wide spectrum and higher antibacterial activity in compression with other polyphenols. In addition, this compound shows synergism with antibiotics and is able to suppress a number of microbial virulence factors, such as inhibition of biofilm formation, reduction of host ligand adhesion, and neutralization of bacterial toxins.25 Tannins are complex polymerized molecules with relatively high molecular weight.26 Several mechanisms could explain the effect of tannins in the bacterial growth inhibition, such as the destabilization of the cytoplasmic membrane,27 the inhibition of extracellular microbial enzymes, direct actions on microbial metabolism, or the deprivation of the substrates required for microbial growth, especially essential mineral micronutrients, such as ion and zinc (via tannins chelation with the metals), whose depletion can severely limit bacterial growth.25,28 In the present study, the CEx of M. azedarach presented a bactericidal potential similar to 0.12% chlorhexidine. The petroleum ether fraction showed positive results, although they were lower than the positive control group, probably due to a lower concentration of bioactive compounds in this fraction. Both fractions were further subjected to MIC analysis. Chloroform, ethyl acetate, butanol, and aqueous fractions showed no bactericidal activity and therefore were not included in the MIC assay. Khan et al.21 evaluated M. azedarach extracts against several human pathogenic bacterial strains. The leaf extracts showed bactericidal activity against gram-positive and gram-negative bacterial strains, where the Streptococcus group presented high sensitivity.21 In addition, the present study showed a MIC value of 0.1 g/mL for the petroleum ether fraction of M. azedarach, 0.025 g/mL for the CEx of M. azedarach, and 0.004 g/mL for the positive control. Even though the MIC values were greater for the control group (CHX), CEx of M. azedarach should be considered as an alternative bioactive compound against S. mutans, since CHX has several side effects such as dental staining and oral irritation. Data showed that the petroleum ether fraction of M. azedarach is not as promising as the CEx due to inferior MIC values and a significantly lower inhibition zone, compared to CHX. Since the CEx of M. azedarach presented the best results when compared with the extract fractions, we can speculate that the bactericidal effect against S. mutans could be mediated by the synergism between different components. When these components are separated using the solvent of different polarity, the bactericidal effect is lost. Within the limitation of this study, it can be concluded that the CEx and petroleum ether fraction of M. azedarach
MELIA AZEDARACH EXTRACTS AGAINST S. MUTANS
have a bactericidal effect against S. mutans. In addition, the CEx is more effective among the extracts, with the zone of inhibition similar to 0.12% chlorhexidine and MIC of 0.025 g/mL. These results confirm that the extracts from M. azedarach could be an effective antibiotic. AUTHOR DISCLOSURE STATEMENT
14.
15.
No competing financial interests exist. 16.
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