Antibacterial property of isoflavonoids isolated from Erythrina variegata against cariogenic oral bacteria.
The antibacterial property of 7 compounds, isolated from Erythrina variegata (Leguminosae) by repeated silica gel column chromatography, against cariogenic oral bacteria was investigated. Extensive spectroscopic study revealed that all were isoflavonoids. Among them, 3,9-dihydroxy-2,10-di([gamma],[gamma]-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) showed the highest antibacterial activity against mutans streptococci, other oral streptococci, Actinomyces and Lactobacillus species with a minimum inhibitory concentration (MIC) range of 1.56-6.25 [micro]g/ml, followed by 3,6a-dihydroxy-9-methoxy-2,10-di([gamma],[gamma]-dimethylallyl)pterocarpan (erystagallinA) and 9-hydroxy3-methoxy-2-[gamma],[gamma]-dimethylallylpterocarpan (orientanol B) (MIC range: 3.13-12.5 [micro]g/ml). The antibacterial effect of erycristagallin to mutans streptococci was based on a bactericidal action. Erycristagallin (6.25 [micro]g/ml: MIC) completely inhibited incorporation of radio-labelled thymidine into Streptococcus mutans cells. Incorporation of radio-labelled glucose into bacterial cells was also strongly inhibited at MIC, and 1/2 MIC of the compound reduced the incorporation approximately by half. The findings indicate that erycristagallin has a potential as potent phytochemical agent for prevention of dental caries by inhibiting the growth of cariogenic bacteria and by interfering with incorporation of glucose responsible for production of organic acids.
Key words: Cariogenic bacteria, isoflavonoids, antibacterial activity, Erythrina variegata, glucose incorporation
Dental caries is an indigenous infection by oral bacteria and is one of the most prevalent diseases in human (Marsh and Martin, 1999). Different types of Gram-positive bacteria, all of which possess the ability to produce organic acids from sugars and to survive under acidic conditions, are closely linked to the development of dental caries (Loesche, 1986, Marsh and Martin, 1999). Additionally, Streptococcus mutans, the primary cariogenic agent in humans, has the ability to adhere to tooth surfaces through synthesis of extra-cellular polysaccharides from sucrose (Hamada and Slade, 1980, Hamada et al., 1984, Loesche, 1986). It subsequently metabolizes fermentable sugars in plaque to organic acids, resulting in demineralization of tooth enamel. Other oral bacteria such as Streptococcus sanguis, Actinomyces and Lactobacillus species are also associated with root surface and fissure caries (Loesche, 1986, Ximenez-Fyvie, 1999). Thus, the strategy for prevention of dental caries includes elimination of cariogenic bacteria from the oral cavity, prevention of organic acid production and inhibition of enzymes responsible for the synthesis of extra-cellular polysaccharides (Marsh, 1993). Among these strategies, inhibiting the growth of cariogenic bacteria is the most practical and reliable means for the prevention of dental caries. Many synthetic compounds, such as chlorhexidine, have widely been used for this purpose (Baker et al., 1987). However, unexpected side-effects, including brown staining of tooth, a predisposition to calculus formation, enhanced bacterial colonization, desquamation and soreness in the oral mucosa, due to such substances have also been reported (Flotra et al., 1971, Vaahtoniemi et al., 1994, Walton et al., 1994).
In recent years, secondary plant metabolites (phytochemicals) with antibacterial properties have actively been investigated as alternatives to synthetic compounds (Sakanaka et al., 1989, Walker, 1990, Tsuchiya et al., 1994, Sato et al., 1996a). Among these phytochemicals, flavonoids seem to be the most potent candidates because they show broad pharmacological activities and are widely distributed in many edible plants and beverages (Havsteen, 1983). Plants belonging to Leguminosae are abundant in flavonoids, some of which function in defense against pathogenic microorganisms (Ebel and Grisebach, 1988).
In the present study, isoflavonoids were isolated from Erythrina variegata (Leguminosae), their structures were determined, and then their antibacterial property against cariogenic oral bacteria was investigated.
* Materials and Methods
Isolation and structural determination of phytochemicals
The roots of Erythrina variegata L. (Leguminosae) were collected in Karachi, Pakistan in April 2000. A voucher specimen has been deposited at the Department of Natural Product Chemistry in the Faculty of Pharmacy, Meijo University, Japan. The naturally dried and finely powdered roots (470 g) were macerated with acetone. The chloroform-soluble fraction of the residue (19.26 g) was subjected to silica gel (230-400 mesh; Merck, Dermstadt, Germany) column chromatography followed by elution with methylene chloride (C[H.sub.2][Cl.sub.2]), C[H.sub.2][Cl.sub.2]-acetone (10:1 [right arrow] 1:1) and C[H.sub.2][Cl.sub.2]-methanol (10:1 [right arrow] 1:1) to give 42 fractions (frs. 1-42). Fr. 13 and frs. 16-18 were applied to silica gel column chromatography eluting with chloroform-acetone (40:1 [right arrow] 4:1) to yield compound 1 (13 mg) and 2 (212 mg), respectively. Fr. 20 was subjected to chromatography followed by elution with chloroform-acetone (10:1.5 [right arrow] 1:1) to provide compound 3 (101 mg). Fr. 21 was applied to silica gel column chromatography eluting with chloroformacetone (10:1.5 [right arrow] 4:1 [right arrow] 1:1) to yield 58 fractions (frs. 43-100). Frs. 48-52 were subjected to column chromatography on silica gel eluting with benzene-ethyl acetate (4:1) to furnish compound 4 (10 mg). Frs. 88-96 were subjected to silica gel column chromatography eluting with benzene-ethyl acetate (3:1 [right arrow] 1:1) and n-hexane-acetone (1.75:1) to afford compounds 5 (10 mg) and 6 (130 mg). The ethyl acetate-soluble fraction (9.22 g) was applied to silica gel column chromatography eluting with chloroform-acetone (40:1 [right arrow] 10:1.5 [right arrow] 1:1), n-hexane-acetone (1:1) and benzene-ethyl acetate (1:2) to yield compound 7 (4.2 mg). The chemical structures of the isolated compounds were determined by spectral analyses, including UV, IR, MS, ID- and 2D-NMR experiments and optical rotations as previously reported (Tanaka et al., 2001).
Preparation of bacterial cells
The cariogenic oral bacteria, including Streptococcus, Actinomyces and Lactobacillus species, were used. Mutans streptococci, including Streptococcus mutans OMZ175, ATCC25175, GS5, LM7, S. sobrinus ATCC33748, OMZ176, 6715, S. cricetus E49 and S. rattus BHI, and other oral streptococci, S. sanguis ATCC10556, S. gordonii ATCC10558, S. mitis ATCC903, ATCC9811, ATCC33399, S. oralis ATCC35037, S. salivarius ATCC 25975, Actinomyces viscosus ATCC15987, ATCC15988, ATCC19246 and Lactobacillus casei ATCC7469 were from laboratory stock cultures of the Department of Oral Pathology, Asahi University School of Dentistry, Japan. All strains were grown in Brain Heart Infusion (BHI; Difco, Detroit, MI) broth under anaerobic conditions at 37[degrees]C for 48 h. They were then diluted with the same medium to give a concentration of approximately 1.0 x [10.sup.8] colony forming unit (CFU)/ml.
Determination of minimum inhibitory concentration
Isolated compounds were dissolved in dimethyl sulfoxide (DMSO) and two-hold serial dilutions in DMSO were made. DMSO containing each compound was added to BHI agar plates (1% v/v) to give a final concentration range of 1.56-100 [micro]g/ml. Suspensions of above named bacterial strains were spotted onto agar plates using a bacterial planter (3GA 120K, Oriental Motor, Osaka, Japan) and incubated anaerobically at 37[degrees]C for 48 h. Minimum inhibitory concentration (MIC) was defined as the lowest concentration at which colonies failed to grow. BHI agar plate containing only DMSO (1% v/v) served as control.
Antibacterial action against S. mutans
S. mutans OMZ175 was grown in BHI broth for 36 h and washed 3 times with sterile phosphate buffered saline (PBS: 0.07 M, pH 7.0). Bacterial cells were resuspended in PBS at a concentration of approximately 6.2 x [10.sup.7] CFU/ml, and then the test compound (erycristagallin) dissolved in DMSO was added (1% v/v) to the suspension. The final concentrations of erycristagallin were 6.25 [micro]g/ml (MIC) and 12.5 [micro]g/ml (2 MIC). After incubation at 37[degrees]C (4 and 24 h), bacterial cells were vigorously mixed and ten-hold serial dilutions were made in PBS. Aliquots (100 [micro]l) were streaked onto Mitis Salivarius Agar (Difco) plates, followed by anaerobic incubation at 37[degrees]C for 48 h. The changes in viable cell numbers were determined by counting the number of colonies formed on each plate. The suspension containing only DMSO (1% v/v) served as control.
Incorporation of thymidine and glucose
The incorporation of radio-labelled thymidine and glucose into S. mutans OMZ175 cells was investigated as previously reported (Sato et al., 1996b). [Methyl-[sup.3]H]thymidine and [1-[sup.3]H]glucose were purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks., UK). Erycristagallin in DMSO (final concentration of 6.25 [micro]g/ml: 1% v/v) and thymidine (final radioactivity of 37 kBq/ml) were added to a cell suspension of S. mutans OMZ175 (approximately 2.0 x [10.sup.8] CFU/ml). In the study of glucose incorporation, radio labelled glucose (final radioactivity of 18.5 kBq/ml) and unlabelled glucose (1 mg/ml) were added to the suspension together with erycristagallin (final concentrations of 6.25 and 3.13 [micro]g/ml). The mixture was stirred at room temperature and aliquots (0.5 ml) were removed at specified time intervals (5, 10, 20 and 40 min) and mixed with 5.0 ml of ice-cold 10% trichloroacetic acid, then filtered (pore size of 0.45 [micro]m) to collect bacterial cells. Filters were washed twice with 5 ml of 5% trichloroacetic acid and subjected to an automatic sample combustion system (ASC-113; Aloka, Tokyo, Japan). The radioactivity recovered from the filters was measured by a liquid scintillation counter (LSC-900; Aloka). Four determinations were made for each measurement. Bacterial cell suspension containing DMSO (1% v/v) and thymidine or glucose served as control.
Spectroscopic study revealed that all isolates were isoflavonoids, and compounds 1-7 were identified as 9-hydroxy-3-methoxy-2-[gamma],[gamma]-dimethylallylpterocarpan (orientanol B), 3,6a-dihydroxy-9-methoxy 2,10-di([gamma],[gamma]-dimethylallyl)pterocarpan (erystagallin A), 3,6a-dihydroxy-9-methoxy-10-[gamma],[gamma]-dimethylallylpterocarpan (cristacarpin), 3,9-dihydroxy-2,10-di([gamma],[gamma]-dimethylallyl)coumestan (sigmoidin K), 3,9-dihydroxy-2,10-di([gamma],[gamma]-dimethylallyl)-6a, 11 a-dehydropterocarpan (erycristagallin), 3,6a,9-trihydroxy-2,10-di([gamma],[gamma]-dimethylallyl)pterocarpan (2-([gamma],[gamma]-dimethylallyl)-6a-hydroxyphaseollidin) and 6a-hydroxy-3- methoxy(3',4'-dihydro-3'-hydroxy)-2',2'-dimethylpyrano[5',6':9,10]pterocarpan (eryvarin A), respectively (spectral data available on demand). Their chemical structures are shown in Fig. 1. Two different versions of isoflavonoids, pterocarpans (cristacarpin, erycristagallin, erystagallin A, eryvarin A, orientanol B and 2-(gamma],[gamma]-dimethylallyl)-6a-hydroxyphaseollidin) and coumestan (sigmoidin K), were noticed among them. With the exception of eryvarin A, all compounds possessed [gamma],[gamma]-dimethylallyl group at C-2 and/or C-10 position in their molecules. In eryvarin A and orientanol B, a methoxyl group was substituted for the hydroxy group at position C-3.
[FIGURE 1 OMITTED]
Among 7 isoflavonoids, erycristagallin was the most potent inhibitor of bacterial growth in all strains tested. It inhibited the growth of mutans streptococci, other streptococci and L. casei within 6.25 [micro]g/ml, and A. viscosus, at 1.56 [micro]g/ml (Table 1). Orientanol B also showed high activity, however, it failed to inhibit the growth of some streptococcal strains at 6.25 [micro]g/ml. The growth inhibitory activity of erystagallin A and 2-([gamma],[gamma]-dimethylallyl)- 6a- hydroxyphaseollidin was nearly identical with MIC values of 6.25-25 [micro]g/ml against streptococci. The MIC values of cristacarpin against streptococci and Actinomyces were about 100 and 25 [micro]g/ml, respectively. Although sigmoidin K failed to inhibit mutans streptococci at 100 [micro]g/ml, it was active against S. mitis and Actinomyces at 1.56-25 [micro]g/ml. Eryvarin A only exhibited growth inhibitory potency against A. viscosus ATCC19246 at 100 [micro]g/ml.
When S. mutans OMZ175 (6.2 x [10.sup.7] CFU/ml) was incubated with erycristagallin of 6.25 [micro]g/ml for 4 and 24 h, the viable cell number reduced to approximately 3.8 x [l0.sup.4] and 1.0 x [10.sup.2] CFU/ml, respectively (control: 4.2 x [10.sup.7] and 2.2 x [10.sup.6] CFU/ml). At a concentration of 12.5 [micro]g/ml, viable cell number reduced to about 2.3 x [10.sup.3] CFU/ml at 4 h incubation and no colony was observed after 24 h incubation (data not shown).
The influence of erycristagallin on the incorporation of radio-labelled thymidine and glucose into bacterial cells is shown in Table 2. Incorporation of thymidine gradually increased during incubation time of 5 to 20 min and markedly increased at 40 min in the control. Glucose incorporation into bacterial cells also increased with incubation time in the control. Erycristagallin of 6.25 [micro]g/ml strongly interfered with the incorporation of thymidine throughout the experimental interval. Although a small increase of glucose incorporation was observed during the incubation time from 5 to 10 min, no further increase was observed under the presence of erycristagallin of MIC value. At 1/2 MIC, glucose incorporation reduced approximately by half of the control through the experimental interval.
Much attention have been focused on the exploration and application of phytochemicals with pharmacological activities as medicinal agents (Balandrin and Bollinger, 1985). In the dental field, polyhydroxyl flavonoids, such as gallocatechin and epigallocatechin, from green tea have been shown to possess inhibitory activity on S. mutans growth at a concentration range of 250-500 [micro]g/ml and an inhibitory effect on extra-cellular polysaccharide synthesis (Sakanaka et al., 1989, Otake et al., 1991). Ooshima et al. (1993) reported that polyphenols from oolong tea inhibit glucosyltransferase activity responsible for the synthesis of extracellular polyaccharides by S. mutans and the development of experimental dental caries in SPF rats. Tsuchiya et al. (1994) have isolated a flavanone (sophoraflavanone G) from Sophora exigua (Leguminosae) that exhibits antibacterial activity to cariogenic bacteria.
E. variegata has been used as a folk medicine in tropical and subtropical regions, and is known to possess pharmacological activities including inhibition of the [Na.sup.+]/[H.sup.+] exchange system, antibacterial and anti-inflammatory effects (Deshpande et al., 1977, Telikepalli et al., 1990, Hegde et al., 1997). The antibacterial potency of the 7 isoflavonoids, isolated from E. variegata, against cariogenic oral bacteria varied significantly among them. Although DMSO has antibacterial potency, 5% of DMSO never inhibited bacterial growth. Erycristagallin showed the most potent growth inhibitiory activity. Its potency was higher than or equivalent to those of previously reported flavanones (Tsuchiya et al., 1994, Sato et al., 1996a). In contrast, sigmoidin K (coumestan), the 6-oxygenated derivative of erycristagallin had much reduced activity. In addition to differences in core structure (pterocarpans and coumestan), it seems that the presence and the position of substituent groups in the molecules are important factor for their antibacterial potency. Further study concerning structure-activity relationship of related isoflavonoids would provide information to improve antibacterial potency.
In contrast to sophoraflavanone G (Tsuchiya et al., 1994), erycristagallin has proven to act on S. mutans in a bactericidal manner at MIC. This bactericidal action of erycristagallin would be advantageous in the prevention of dental caries by inhibiting the formation of dental plaque.
Erycristagallin at MIC completely suppressed the incorporation of thymidine and glucose into S. mutans cells. The compound may exert its antibacterial activity by interfering with the bacterial uptake of metabolites and nutrients. Liu et al. (2001) have demonstrated that the combinations between vancomycin and flavonoids show synergistic effects against vancomycin-resistant enterococci, and suggested that the effects are resulted from inhibition of disaccharide peptide production by the flavonoids. Thus, flavonoids may possess multi-targeting antibacterial properties, and the mechanism(s) of erycristagallin to affect bacterial cells is now under investigation. Glucose uptake was considerably reduced even at 1/2 MIC. Inhibition of glucose uptake may lead to reduced production of organic acids by S. mutans, because glucose is commonly utilized by the bacterium to produce organic acids (Hamada et al., 1984). When caries prevention agents are administrated orally, they will initially be present at high concentrations in the oral cavity, but thereafter will soon be desorbed (Tsuchiya et al., 1997). Since many interrelated factors are responsible for the initiation and progression of dental caries, the activity of erycristagallin at sub-MIC doses would prolong its effectiveness in preventing dental caries.
Erycristagallin also inhibited Staphylococcus aureus and Enterococcus faecalis at almost the same concentration range with streptococci. In spite of such intensive effects against Gram-positive bacterial species, it was unable to exert antibacterial effects on Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa), even at 100 [micro]g/ml. The different effects of erycristagallin on Gram-positive and -negative bacteria may be ascribed to differences in bacterial cell walls: the outer membrane of Gram-negative bacteria interferes with accessibility of the compound to protoplasmic membrane. Sophoraflavanone G, a flavanone that shows the same antibacterial pattern, has been shown to reduce membrane fluidity using model membranes (Tsuchiya and Iinuma, 2000). Thus, bacterial cell membrane may be one of the operative targets for erycristagallin.
The in vivo effectiveness, retention in the oral cavity and toxicity remain to be studied for this isoflavonoid, erycristagallin. However, based on the findings of the current paper, it appears that erycristagallin has potential as a potent phytochemical agent for the prevention of dental caries.
Table 1. Minimum inhibitory concentrations of isoflavonoids against cariogenic oral bacteria. Isoflavonoids eryvarin sigmoidin crista- A K carpin Mutans streptococci >100 (1) >100 100 S. mutans ATCC25175 >100 >100 100 S. mutans GS 5 >100 >100 100 S. mutans LM 7 >100 >100 100 S. mutans OMZ 175 >100 >100 100 S. sobrinus ATCC33748 >100 >100 100 S. sobrinus OMZ 176 >100 >100 100 S. sobrinus 6715 >100 >100 100 S. cricetus E49 >100 >100 100 S. rattus BHI >100 >100 100 Other streptococci S. sanguis ATCC10556 >100 100 100 S. gordonii ATCC10558 >100 25 50 S. mitis ATCC903 >100 6.25 50 S. mitis ATCC9811 >100 25 50 S. mitis ATCC33399 >100 25 100 S. oralis ATCC35037 >100 >100 100 S. salivarius ATCC25975 >100 >100 50 Actinomyces A. viscosus ATCC15987 >100 6.25 25 A. viscosus ATCC15988 >100 6.25 25 A. viscosus ATCC19246 100 1.56 25 Lactobacillus L. casei ATCC7469 >100 >100 100 Isoflavonoids 2-([gamma],[gamma]- erysta- dimethylallyl)- gallin A 6a-hydroxy- phaseollidin Mutans streptococci 12.5 12.5 S. mutans ATCC25175 12.5 12.5 S. mutans GS 5 25 12.5 S. mutans LM 7 12.5 12.5 S. mutans OMZ 175 25 12.5 S. sobrinus ATCC33748 12.5 12.5 S. sobrinus OMZ 176 12.5 12.5 S. sobrinus 6715 12.5 12.5 S. cricetus E49 12.5 12.5 S. rattus BHI 25 6.25 Other streptococci S. sanguis ATCC10556 12.5 12.5 S. gordonii ATCC10558 6.25 6.25 S. mitis ATCC903 12.5 6.25 S. mitis ATCC9811 12.5 6.25 S. mitis ATCC33399 12.5 6.25 S. oralis ATCC35037 12.5 12.5 S. salivarius ATCC25975 12.5 6.25 Actinomyces A. viscosus ATCC15987 3.13 3.13 A. viscosus ATCC15988 3.13 3.13 A. viscosus ATCC19246 3.13 3.13 Lactobacillus L. casei ATCC7469 12.5 12.5 Isoflavonoids orien- erycrista- tanol B gallin Mutans streptococci 6.25 6.25 S. mutans ATCC25175 12.5 6.25 S. mutans GS 5 6.25 6.25 S. mutans LM 7 6.25 6.25 S. mutans OMZ 175 6.25 6.25 S. sobrinus ATCC33748 6.25 6.25 S. sobrinus OMZ 176 6.25 6.25 S. sobrinus 6715 6.25 6.25 S. cricetus E49 12.5 6.25 S. rattus BHI 12.5 6.25 Other streptococci S. sanguis ATCC10556 6.25 6.25 S. gordonii ATCC10558 12.5 3.13 S. mitis ATCC903 6.25 6.25 S. mitis ATCC9811 6.25 3.13 S. mitis ATCC33399 12.5 6.25 S. oralis ATCC35037 6.25 6.25 S. salivarius ATCC25975 6.25 6.25 Actinomyces A. viscosus ATCC15987 6.25 1.56 A. viscosus ATCC15988 6.25 1.56 A. viscosus ATCC19246 6.25 1.56 Lactobacillus L. casei ATCC7469 6.25 6.25 (1) Minimum inhibitory concentration ([micro]g/ml) Table 2. Incorporation of radio-labelled thymidine and glucose into S. mutans. Incubation time (min) 5 10 Thymidine control (1) 386 [+ or -] 29 (4) 521 [+ or -] 60 MIC (20 474 [+ or -] 108 534 [+ or -] 45 Glucose control 2052 [+ or -] 109 3096 [+ or -] 144 1/2MIC (3) 1212 [+ or -] 107 2011 [+ or -] 181 MIC 780 [+ or -] 98 1165 [+ or -] 149 Incubation time (min) 20 40 Thymidine control (1) 587 [+ or -] 55 2756 [+ or -] 1067 MIC (20 543 [+ or -] 84 477 [+ or -] 125 Glucose control 5216 [+ or -] 285 8548 [+ or -] 357 1/2MIC (3) 2784 [+ or -] 196 4285 [+ or -] 234 MIC 1081 [+ or -] 122 1057 [+ or -] 96 (1) DMSO was added (1% v/v) to the cell suspension of S. mutans OMZ 175 (2 x [10.sup.8] CFU/ml); (2) Erycristagallin in DMSO was added at 6.25 [micro]g/ml; (3) Erycristagallin in DMSO was added at 3.13 [micro]g/ml; (4) mean of 4 separate determinations [+ or -] S.D. (d.p.m).
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M. Sato, DDS., Department of Oral Pathology, Asahi University School of Dentistry, 1851-Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan. Tel. and Fax: +81-58-329-1427; e-mail: firstname.lastname@example.org
M. Sato (1), H. Tanaka (2), S. Fujiwara (3), M. Hirata (2), R. Yamaguchi (4), H. Etoh (5), and C. Tokuda (1)
(1) Department of Oral Pathology, Asahi University School of Dentistry, Gifu, Japan
(2) Department of Natural Product Chemistry, Faculty of Pharmacy, Meijo University, Nagoya, Japan
(3) Department of Prosthetic Dentistry, Asahi University School of Dentistry, Gifu, Japan
(4) Institute of Radioisotope, Asahi University School of Dentistry, Gifu, Japan
(5) Laboratory of Marine Biochemical Science, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan
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|Author:||Sato, M.; Tanaka, H.; Fujiwara, S.; Hirata, M.; Yamaguchi, R.; Etoh, H.; Tokuda, C.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jun 1, 2003|
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