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Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans.

Abstract

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. Preliminary antibacterial screening revealed that the extract of Myristica fragrans, widely cultivated for the spice and flavor of foods, possessed strong inhibitory activity against S. mutans. The anticariogenic compound was successfully isolated from the methanol extract of M. fragrans by repeated silica gel chromatography, and its structure was identified as macelignan by instrumental analysis using 1D-NMR, 2D-NMR and EI-MS. The minimum inhibitory concentration (MIC) of macelignan against S. mutans was 3.9 [micro]g/ml, which was much lower than those of other natural anticariogenic agents such as 15.6 [micro]g/ml of sanguinarine, 250 [micro]g/ml of eucalyptol, 500 [micro]g/ml of menthol and thymol, and 1000 [micro]g/ml of methyl salicylate. Macelignan also possessed preferential activity against other oral microorganisms such as Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei in the MIC range of 2-31.3 [micro]g/ml. In particular, the bactericidal test showed that macelignan, at a concentration of 20 [micro]g/ml, completely inactivated S. mutans in 1 min. The specific activity and fast-effectiveness of macelignan against oral bacteria strongly suggest that it could be employed as a natural antibacterial agent in functional foods or oral care products.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Antibacterial activity; Myristica fragrans; Macelignan; Streptococcus mutans; Dental caries

Introduction

Dental plaque plays an important role in the development of dental caries, which results in both tooth dysfunction and loss. Streptococcus spp. have been implicated as primary causative agents of dental caries in humans and experimental animals (Hamada et al., 1984). Especially, Streptococcus mutans and Streptococcus sobrinus have been isolated from human dental plaque and are known as the cariogenic oral bacteria (Loesche, 1986). Dental plaque is formed mainly in two stages; the initial and reversible attachment of various oral bacteria to the tooth surface, followed by sucrose-dependent glucosyltransferase activity to synthesize an insoluble glucan layer in which firm and irreversible adhesion of S. mutans occurs (Koo et al., 2002). Acid forming microorganisms, including Lactobacillus spp., in the plaque elute lactic acid by metabolizing fructose as their carbon source, which finally results in dental caries (Marsh, 1999).

Many attempts have been made to eliminate S. mutans from the oral flora. Antibiotics such as ampicillin, chlorhexidine, erythromycin, penicillin, tetracycline and vancomycin have been very effective in preventing dental caries (Jarvinen et al., 1993). However, excessive use of these chemicals can result in dearrangements of the oral and intestinal flora and cause undesirable side effects such as microorganism susceptibility, vomiting, diarrhea and tooth staining (Chen et al., 1989). Sanguinarine is an alkaloid isolated from the rhizome of Sanguinaria canadensis, which shows a broad spectrum against various oral bacteria (Dzink and Socransky, 1985). It has been used as an anticariogenic agent in a wide range of oral care products such as toothpastes and mouthwashes due to its strong antibacterial effectiveness (Eley, 1999). Its industrial application, however, had been greatly reduced as sanguinarine was reported to be associated with oral leukoplakia (Mascarenhas et al., 2001). These problems necessitate further search for natural antibacterial agents that are safe for humans and specific for oral pathogens.

Myristica fragrans Houtt. (Myristicaceae), known as pala in Indonesia, luk jan in Thailand, nikuzuku in Japan, and commonly nutmeg or mace, has been used traditionally for spice and medicinal purposes for carminative, hypolipidaemic, antithrombotic, antiplatelet aggregating, antifungal, aphrodisiac, anxiogenic, anti-ulcerogenic, antitumor and anti-inflammatory activities, etc. (Morita et al., 2003; Sonavane et al., 2002; Capasso et al., 2000; Park et al., 1998; Ozaki et al., 1989). M. fragrans Houtt has been reported to contain 25-30% fixed oils and 5-15% volatile oils such as camphene, elemicin, eugenol, isoelemicin, isoeugenol, methoxyeugenol, pinene, sabinene, safrol, etc., and also chemical substances such as dihydroguaiaretic acid, elimicin, myristic acid, myristicin and lignan compounds (Janssen et al., 1990; Kuo, 1989; Isogai et al., 1973; Forrest and Heacock, 1972). Among these compounds, eugenol is widely used in dentistry as root canal sealers and is very effective in their antibacterial activity against oral bacteria (Lai et al., 2001). It is also used as a local anaesthetic agent for treatment of postoperative pain after gingivectomy (Skoglund and Jorkjend, 1991).

Up to date, the aril of M. fragrans, mace, has been reported to show antibacterial activity against cariogenic S. mutans (Hattori et al., 1986). However, few studies have been conducted using nutmeg, the seed kernels of M. fragrans, against oral bacteria. In this study, an active compound against cariogenic S. mutans was isolated from the nutmeg of M. fragrans, and its effectiveness was investigated in comparison with other well-known commercial antibacterial agents.

Materials and methods

Plant material

M. fragrans Houtt. (Myristicaceae) was collected in the year 2002 from the Biofarmaka Research Center of Bogor Agricultural University (Indonesia) and was identified by Dr. K. Latifah. The plant material was shade dried and ground to powder. A voucher specimen has been deposited at 4 [degrees]C in the Bioproducts Research Center, Yonsei University, Seoul, Korea. All reagents were purchased from Sigma Co. (USA), unless otherwise stated.

Bacterial strains and culture conditions

The following 9 oral microorganisms were used in this study: Actinomyces viscosus ATCC 15987, Porphyromonas gingivalis ATCC 53978, Staphylococcus aureus ATCC 12600, Streptococcus mutans ATCC 25175, Streptococcus sanguis ATCC 35105 and Streptococcus sobrinus ATCC 27351 from the American Type Culture Collection (Rockville, MD, USA) and Lactobacillus acidophilus KCCM 32820, Lactobacillus casei KCCM 35465 and Streptococcus salivarius KCCM 40412 from the Korean Culture Center of Microorganisms (Seoul, Korea). S. aureus, S. mutans, S. salivarius, S. sanguis and S. sobrinus were cultured in Brain Heart Infusion (BHI; Difco, USA) at 37 [degrees]C for 24 h aerobically. Yeast malt extract and LBS broth were used for the culture of A. viscosus and L. acidophilus at 37 [degrees]C for 24 h anaerobically. L. casei was cultured in beef extract (1%), 1 N NaOH (2.5%), trypticase (3%), yeast extract (0.5%), [K.sub.2]HP[O.sub.4] (0.5%), cysteine-HCl (0.05%), 0.025% resazurin solution (0.4%), hemin solution (0.1%) and menadione solution (0.002%) at 37 [degrees]C for 24 h anaerobically. P. gingivalis was cultured in PGB medium [BHI (1.85%), yeast extract (0.5%), cysteine (0.05%), hemin solution (1%) and menadione solution (0.1%)] at 37 [degrees]C for 24 h in an anaerobic jar with anaerogen (Oxoid Ltd., England).

Isolation of an antibacterial compound

A total of 100g of dried seed kernels of M. fragrans were ground and extracted twice with 75% aqueous methanol (400 ml, v/v) for 24h at room temperature. The methanol extract was concentrated, frozen, lyophilized (7g) and further fractionated successively with ethyl acetate, n-butanol and water. Each fraction was evaporated and dried under reduced pressure (ethyl acetate fraction 4.2g, butanol fraction 0.7g, water fraction 2.1g). Since the strongest activity against S. mutans was observed in the ethyl acetate fraction, further separation was performed using silica gel column chromatography (Merck Kieselgel 60, 70-230 mesh) by eluting with n-hexane:ethyl acetate solution (10:1, v/v), and 10 ml volumes of eluant were collected in test tubes. The collected tubes were divided into six fractions (Fr. I-Fr. VI) following silica thin layer chromatography (TLC; 60 [F.sub.254], Merck). Fr. III, providing considerable inhibitory activity against S. mutans, was further separated through a silica gel column using n-hexane:ethyl acetate (20:1, v/v), yielding Fr. III-B (0.52 g). Fr. III-B was eluted with 80% methanol using Rp-18 column chromatography (Merck LiChroprep[R], 25-40 [micro]m), and Fr. III-B-2 (0.5 g) was finally obtained as a single compound.

Instrumentation

NMR spectra were recorded on a Bruker Avance-500 spectrometer (Germany) at 600 MHz for [.sup.1]H and 150 MHz for [.sup.13]C in CD[Cl.sub.3] with TMS as an internal standard. Complete proton and carbon assignments were based on 1D ([.sup.1]H, [.sup.13]C, [.sup.13]C-DEPT) and 2D ([.sup.1]H-[.sup.1]H COSY, [.sup.1]H-[.sup.13]C HMQC, [.sup.1]H-[.sup.13]C HMBC) NMR experiments. Mass spectra (EI-MS) were measured using JMS-700 Mstation (JEOL Ltd., Japan).

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

Compound III-B-2 and other antibacterial agents, dissolved in 1% dimethyl sulfoxide (DMSO), were added to the first tube containing 1 ml BHI broth and serially diluted by the two-fold method, resulting in a range of 1000-1 [micro]g/ml (Park et al., 2003). A bacterial suspension (0.1 ml) containing 2 x [10.sup.5] colony forming units (CFU)/ml was added to each tube and incubated for 24h at 37 [degrees]C. MIC was determined by judging visually the bacterial growth in the series of test tubes. MBC was the concentration at which microorganisms were totally unable to remain viable. The control included an inoculated growth medium without the test compounds, and some commonly available antibacterial agents used for caries control were employed as positive controls. All experiments were performed in duplicate.

Measurement of bactericidal activity

The viable cell count method was used to examine the bactericidal activity. S. mutans was cultured in BHI broth, washed twice with pH 7.0 phosphate-buffered saline (PBS) and diluted to give a final concentration of 2 x [10.sup.5] CFU/ml. Mixtures of 1 ml cell suspension with 4 ml of compound III-B-2 or essential oils were incubated at 37 [degrees]C for 48h, and the viability of the incubated mixtures were determined by the pour plating method (Jones et al., 2001).

Results and discussion

Identification of an antibacterial compound

Compound III-B-2 showed a molecular weight of 328 from the EI-MS spectrum (m/z, 329, [M + 1][.sup.+]). Through comprehensive analysis of the NMR data, the molecular formula of compound III-B-2 was determined to be (8R, 8'S)-7-(3,4-methylenedioxyphenyl)-7'-(4-hydroxy-3-methoxyphenyl)-8,8'-dimethybutane. This compound was isolated previously by Woo et al. (1987), and by referring to this literature the compound was confirmed as macelignan (Fig. 1).

Antibacterial activity of macelignan

Antibacterial activity of macelignan was investigated in terms of MIC and MBC in comparison with some commercially available antibacterial agents. As shown in Table 1, MIC and MBC of macelignan against S. mutans were 3.9 and 7.8 [micro]g/ml, respectively. Its MIC is much lower than those of essential oils such as 250 [micro]g/ml of eucalyptol, 500 [micro]g/ml of thymol and menthol, and 1000 [micro]g/ml of methyl salicylate. It is especially interesting to note that macelignan gives a significantly lower MIC value than 15.6 [micro]g/ml of sanguinarine. Sanguinarine, a natural compound isolated from Sanguinaria yanadensis, had been widely used in industrial toothpaste and mouthwash products (Southard et al., 1984). The antibacterial activity of macelignan against S. mutans was almost comparable to the antibiotic chlorhexidine.

[FIGURE 1 OMITTED]

As demonstrated in Table 2, macelignan also exhibited preferential antibacterial spectra against other oral bacteria. The MIC values were 2 [micro]g/ml for L., 3.9 [micro]g/ml for L. casei, 2 [micro]g/ml for S. sanguis, 15.6 [micro]g/ml for S. sobrinus and 31.3 [micro]g/ml for S. salivarius. Lactobacillus spp. produce acids using sucrose and glucose as substrates, which causes a drop in pH and progressively demineralization of the tooth surface (Badet et al., 2001). Since macelignan exhibited strong antibacterial activity against both Streptococcus and Lactobacillus spp., dental caries could be effectively blocked through the prevention of both biofilm formation and pH drop. In contrast, relatively weak activity was observed for A. viscosus, P. gingivalis and S. aureus.

Antibacterial agents widely used presently for prevention of dental caries include xylitol, tea extracts, essential oils, antibiotics, etc. Xylitol, a natural sweetener derived from xylose, is presently being applied broadly to chewing gums, toothpastes and mouthwashes. The anticariogenic efficacy of macelignan necessitates long term of application under specified oral conditions, since xylitol has been reported to inhibit the growth of S. mutans only under strict sugar-starvation conditions (Trahan, 1995). Frequent consumption of tea has been reported to reduce dental caries in humans and experimental animals (Wu and Wei, 2002). Tea extracts contain various polyphenols: catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, etc. (Otake et al., 1991). Their MIC values against S. mutans were reported to be 250-1000 [micro]g/ml (Sakanaka et al., 1989), indicating low anticariogenic activity of tea constituents. Numerous antibiotics, including chlorhexidine, have been used as antibacterial agents against S. mutans to reduce plaque-mediated diseases, especially dental caries. However, these chemicals have been reported to show various side effects (Jones, 1997). The fact that the nutmeg of M. fragrans has long been used safely as a spice material and that macelignan exhibited much stronger activity compared to other antibacterial agents supports its possibility as a new natural agent for the prevention of dental caries.

Rapid bactericidal activity of macelignan

Essential oils such as eucalyptol, menthol, thymol and methyl salicylate, which are presently being used in mouthwash products for prevention of dental caries (Yu et al., 2000), together with macelignan were tested for bactericidal activity against S. mutans. Fig. 2 shows the dose-dependent bactericidal activity of macelignan and a comparison of its activity with commercial essential oils. Macelignan, with a concentration as low as 5 [micro]g/ml, exhibited stronger activity than 20 [micro]g/ml of essential oils. Moreover, the same concentration (20 [micro]g/ml) of macelignan completely killed S. mutans in 1 min. Similar rapid bactericidal activity against S. mutans was also reported for xanthorrhizol isolated from Curcuma xanthorrhiza (Hwang et al., 2000). Considering this rapid and effective bactericidal effect against S. mutans, macelignan possesses the potential to be practically applied to toothpaste or mouthwash products since brushing or rinsing ordinarily takes only a few minutes. Furthermore, since M. fragrans has been widely consumed as food materials with safety for a long period of time, application of macelignan to such products would be easily accomplished. In this research, inactivation of oral microbial flora in dental biofilm by macelignan was clearly observed through confocal laser scanning microscopy (figure not shown).

In summary, the results strongly suggest that macelignan may prevent dental caries by inhibiting the growth of Streptococcus spp. The bactericidal activity of macelignan against S. mutans was much stronger than those of commercially used natural essential oils. Moreover, macelignan also presented very strong antibacterial activity against lactic acid-producing Lactobacillus spp. Accordingly, macelignan might be potentially employed as an anticariogenic agent to oral care products in the place of essential oils.

[FIGURE 2 OMITTED]

Acknowledgements

This work was supported by the Yonsei University Research Fund of 2003 and the National Research Lab Program through the Functional Biopolymer Lab at Yonsei University (2000-N-NL-01-C-299).

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J.Y. Chung (a), J.H. Choo (b), M.H. Lee (c), J.K. Hwang (a,b,*)

(a) Department of Biomaterials Science and Engineering, Yonsei University, Seoul, South Korea

(b) Department of Biotechnology, Yonsei University, Seoul, South Korea

(c) Bioproducts Research Center, Yonsei University, Seoul, South Korea

Received 20 November 2003; accepted 7 April 2004

*Corresponding author. Department of Biotechnology, Bioproducts Research Center, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul, Republic of Korea. Tel.: +82 2 2123 5881; fax: +82 2 362 7265.

E-mail address: jkhwang@yonsei.ac.kr (J.K. Hwang).
Table 1. MIC and MBC values of macelignan and other antibacterial
compounds against S. mutans

Compounds MIC ([micro]g/ml) MBC ([micro]g/ml)

Macelignan 3.9 7.8
Chlorhexidine 1 2
Sanguinarine 15.6 31.3
Eucalyptol 250 500
Thymol 500 500
Menthol 500 1000
Methyl salicylate 1000 1000

Table 2. MIC and MBC values of macelignan (ML) and chlorhexidine (CHX)
against oral microorganisms

 MIC ([micro]g/ml) MBC ([micro]g/ml)
Microorganisms ML CHX ML CHX

Streptococcus mutans ATCC 25175 3.9 1 7.8 2
Streptococcus sobrinus ATCC 27351 15.6 3.9 31.3 3.9
Streptococcus sanguis ATCC 35105 2 2 3.9 3.9
Streptococcus salivarius KCCM 31.3 2 31.3 2
 40412
Lactobacillus acidophilus KCCM 2 31.3 3.9 31.3
 32820
Lactobacillus casei KCCM 35465 3.9 31.3 7.8 62.5
Porphyromonas gingivalis ATCC 125 3.9 250 7.8
 53978
Staphylococcus aureus ATCC 12600 250 2 250 3.9
Actinomyces viscosus ATCC 15987 250 7.8 500 7.8
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Author:Chung, J.Y.; Choo, J.H.; Lee, M.H.; Hwang, J.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2006
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