JAC Advance Access originally published online on June 18, 2008
Journal of Antimicrobial Chemotherapy 2008 62(4):751-757; doi:10.1093/jac/dkn253
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Original research |
Novel anti-adherence activity of mulberry leaves: inhibition of Streptococcus mutans biofilm by 1-deoxynojirimycin isolated from Morus alba
1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India 2 Z. A. Dental College, Aligarh Muslim University, Aligarh 202002, India 3 Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
* Corresponding author. E-mail: asad.k@rediffmail.com/huzzi99{at}hotmail.com
Received 12 March 2008; returned 17 April 2008; revised 22 May 2008; accepted 23 May 2008
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Abstract |
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Objectives: The present study focused on isolation, characterization and evaluation of purified compounds from Morus alba against Streptococcus mutans biofilm formation.
Methods: The effect of crude extract from M. alba leaves was evaluated against oral pathogens, chiefly S. mutans. MICs were determined by the microdilution method. The compound was purified by employing silica gel chromatography and critically analysed with GC–MS, NMR and IR spectroscopy. The S. mutans traits of adherence and biofilm formation were assessed at sub-MIC concentrations of the crude extract and purified compound. Both water-soluble and alkali-soluble polysaccharide were estimated to determine the effect of the purified compound on the extracellular polysaccharide secretion of S. mutans. Its effect on biofilm architecture was also investigated with the help of confocal microscopy.
Results: The purified compound of M. alba showed an 8-fold greater reduction of MIC against S. mutans than the crude extract (MICs, 15.6 and 125 mg/L, respectively). The extract strongly inhibited biofilm formation of S. mutans at its active accumulation and plateau phases. The purified compound led to a 22% greater reduction in alkali-soluble polysaccharide than in water-soluble polysaccharide. The purified compound was found to be 1-deoxynojirimycin (DNJ). Confocal microscopy revealed that DNJ distorts the biofilm architecture of S. mutans.
Conclusions: The whole study reflects a prospective role of DNJ as a therapeutic agent by controlling the overgrowth and biofilm formation of S. mutans.
Keywords: dental caries , phytochemicals , glucosyltransferases
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Introduction |
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The human mouth, with its diverse niches and environmental changes, is well known for its unrestricted formation of natural microbial biofilms.1 Streptococcus mutans is a member of the endogenous oral microflora and a major contributor to biofilms in the oral cavity.2 It is the principal causative agent of dental caries, in which short chain carboxylic acids released as its fermentation by-products demineralize the enamel and lead to cavitation in the tooth.3 The biofilms of S. mutans are also involved in infective endocarditis, a serious disease with a mortality rate of up to 50% despite antibiotic treatment.4
The glucosyltransferase (GTF; EC 2.4.1.5) enzymes produced by S. mutans are recognized as the key factors in the development of a biofilm, as glucan synthesized by these GTFs may provide binding sites for the bacteria.5,6 At least three GTFs are secreted by S. mutans: GTF B, GTF C and GTF D.5 GTF B synthesizes a polymer of mostly 
-1,3-linked glucan; GTF C synthesizes a mixture of insoluble -1,3-linked glucan and soluble -1,6-linked glucan; and GTF D synthesizes -1,6-linked glucan.5 There is an escalating demand for compounds that may reduce biofilm formation by inhibiting the secretion or activity of GTFs. A number of phytochemicals have been explored in this regard. The most elaborate studies have been conducted on oolong tea polyphenols that inhibit the GTFs and on propolis that inhibits both the growth and GTFs of S. mutans.7,8
A plethora of Indian medicinal herbs are employed for the treatment of dental caries, albeit they lack sound scientific evidence. Traditionally, mulberry (Morus alba) is chewed in toothache to avoid further destruction or cavitation of the tooth. M. alba has garnered great attention for its antioxidative and antidiabetic effects and is an important ingredient of herbal tea.9
Leaves of M. alba contain flavonoids such as apigenin (42.7 mg/g) and quercetin (4.0 mg/g).10,11 But, till now, no reports have described any anticariogenic potential of the crude extract of the leaves of the mulberry. However, a flavonoid, kuwanon-G, which had been isolated from the root bark of M. alba, shows antibacterial activity against S. mutans only.12 The present study deals with the antimicrobial activity of M. alba against S. mutans, its anti-adherence effects and effects on biofilm formation. We have also isolated and characterized the inhibitory compound from M. alba. The results obtained were validated using commercially available analogous compounds. The biological potential of this compound corroborates the available literature.
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Materials and methods |
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Microorganisms
The ATCC analogues of UA159 strain of S. mutans [Microbial Type Culture Collection (MTCC) #497], Actinomyces viscosus (MTCC #7345), Lactobacillus acidophilus (MTCC #*447) and Lactococcus lactis (MTCC #3038) were purchased from the MTCC, IMTECH, Chandigarh, India. The clinical samples were collected from the Department of Conservative Dentistry, Dental College, AMU, Aligarh, India. CLSI guidelines were followed for the isolation and characterization of S. mutans from samples. The strains of S. mutans were grown in a CO2-rich environment provided by candle jar incubation. The isolates were confirmed by PCR amplification of conserved regions of the GTF B and GTF C genes.13 All the strains were grown in a brain heart infusion (BHI) broth or soyabean casein digest (TSB) purchased from Hi Media Laboratories, India. The cultures were stored at –80°C in BHI containing 25% glycerol. For column chromatography, Silica Gel 60 (Merck, Germany) was used. 1-Deoxynojirimycin-hydrochloride, propidium iodide and XTT were purchased from Sigma, St Louis, USA. All the other chemicals used were of analytical grade and purchased from Merck.
Preparation of the herbal extract and purification of the inhibitory compound
Leaves of M. alba were washed thoroughly and dried at 37°C. The dried leaves were pulverized and then suspended in 95% ethanol for 2–3 days. After filtration and evaporation, the crude extract was oven-dried at 60°C. It was re-dissolved in ethanol to give the desired concentration. The crude extract was refluxed in petroleum ether to separate the oily portion. The remaining extract was then chromatographed on a silica gel column (75 cm x 2.1 cm), with solvents increasing in polarity. The solvent system initially comprised 3 L of petroleum ether, followed by increasing concentrations of ether in petroleum ether. Separate fractions of 30 mL were collected at a flow rate of 2.5 mL/min. The aliquots of each fraction were subjected to thin-layer chromatography (glass slides coated with silica gel, 1 mm) and stained with iodine vapour. The pure fractions were dried in the oven at 45°C. The dried fractions were dissolved in DMSO–ethanol (1:4, v/v) to obtain a final concentration of 1000 mg/L.
Antimicrobial activity against oral microbes
MTCC strains of A. viscosus, L. acidophilus, L. lactis, S. mutans and five clinical isolates of S. mutans—SM4, SM5, U152, U153 and SM6—were inoculated into BHI broth in test tubes and grown to stationary culture for 24 h at 37°C. Aliquots of 100 µL culture were inoculated in 10 mL of fresh medium containing various concentrations (0–75 mg/L) of crude extract. A final concentration of 0.001% (v/v) Tween 80 was included to enhance the solubility of the extract. DMSO, ethanol and Tween 80 were also included in the negative controls. Cultures were incubated for 24 h at 37°C with continuous shaking. Turbidometric analysis was performed at 600 nm after 24 h. The viable cell count was obtained by spreading 100 µL of culture on blood agar plates after 10–4–10–8 dilutions. The plates were incubated for 48 h at 37°C, and cells were counted as cfu.
Determining the MIC of crude extract and purified compound against oral microbes
The six strains of S. mutans (MTCC *497 and clinical isolates), A. viscosus, L. lactis and L. acidophilus were inoculated into BHI in test tubes and grown to a stationary phase at 37°C up to 108–109 cfu/mL. Overnight growth culture (50 µL) diluted to 105–106 cfu/mL was inoculated into fresh BHI (50 µL) containing various concentrations of serially diluted (1000–0.97 mg/L) extract or isolated compound. A final concentration of 0.001% (v/v) Tween 80 was included to enhance the solubility of the extract. The MIC was recorded as the lowest concentration totally inhibiting visible bacterial growth on the polystyrene plate after 48 h of incubation at 37°C. The MIC represents the mean of three independent experiments.
Anti-adherence activity of extract against S. mutans
Glass surface adherence assay was performed by the method of Hamada et al.,14 with slight modifications. The bacteria were grown for 24 h at 37°C at an angle of 30° in a glass tube with 10 mL of BHI with or without 5% sucrose and various concentrations of inhibitory compound. The solvent controls included BHI (with or without sucrose) and equivalent amounts of DMSO, ethanol and Tween 80. After incubation, planktonic cells were decanted, and the attached cells were removed by 0.5 M of sodium hydroxide. Adherence was quantified by reading at 600 nm.
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Biofilm formation was assessed by using the protocol of Loo et al.15 with few modifications. Briefly, 50 µL of overnight growth culture of S. mutans strain (MTCC *497) diluted to 105–106 cfu/mL was inoculated into fresh BHI (150 µL) with 5% sucrose containing various concentrations (0–75 mg/L) of crude extract or purified compound (0–5 mg/L), with respective controls. After incubation for 24 h at 37°C, media and unattached cells were decanted from the microtitre plates. The remaining planktonic cells were removed by gentle rinsing with sterile water. The wells with adhered biofilms were fixed with formalin (37%, diluted 1:10) plus 2% sodium acetate, and each well was stained with 200 µL of 0.1% Crystal Violet for 15 min at room temperature. After two rinses with distilled water, bound dye was removed from the cells with 100 µL of 95% alcohol. Plates were then set on a shaker for 5 min to allow full release of the dye. Biofilm formation was quantified by measuring optical density at 630 nm by a Qualigens ELISA reader. Separate biofilms were formed in the presence of crude extract (0–15 mg/L) for time-dependent effects. At 6, 12, 20 and 24 h, the biofilms were analysed by the above-stated protocol.
S. mutans (MTCC *497) was grown in the presence of 5 mg/L purified compound for 24 h at 37°C in BHI supplemented with 1% (w/v) glucose (total volume taken was 100 mL). After growth overnight, cells were harvested and a culture supernatant was used to isolate the secretory protein. The protein was precipitated by 80% saturated ammonium sulphate. The precipitate dissolved in 20 mM phosphate buffer (pH 6.8) was extensively dialysed against 20 mM phosphate buffer (pH 6.8). Protein concentration was estimated with Folin–Ciocalteau reagent by the method of Lowry et al.16
To measure total glucan, a reaction mixture containing 1.5 mg of total dialysed protein in 10 mL of 50 mM sucrose buffered to pH 5.7 with sodium acetate (0.05 M) and 50 µg of purified compound from M. alba was set for 6 h of incubation at 37°C. An equivalent reaction mixture without the purified compound was also set as a control. The reaction mixture was then centrifuged at 10 000 g for 10 min to separate water-soluble (part A, supernatant) and water-insoluble polysaccharide (part B, pellet). To part A, four parts of methanol were added to precipitate out the polysaccharide. After centrifugation, the precipitate was washed thrice with methanol to remove non-polymerized sugars and dried in air. The precipitate was resuspended in water, and the polysaccharide in it was measured by the phenol-sulphuric acid method.17 Part B was dissolved in 1 M NaOH (1 mg of pellet/0.3 mL of 1 M NaOH). The alkali-soluble polysaccharide was then precipitated using four parts of methanol. It was washed, dried, resuspended and estimated in a similar manner as part A.
Effect of oxidized-pure fraction on the biofilm of S. mutans
The purified component was incubated with 0.02 M sodium meta-periodate in 0.05 M sodium acetate buffer (pH 4.5) at 4°C overnight, and then the oxidized purified component was evaluated for its effect on biofilm formation on microtitre plates by the above-stated protocol. A solution with equivalent amount of sodium meta-periodate was used as a vehicle control.
Identification and analysis of the active compound
The potent fraction of M. alba extract was subjected to GC–MS chromatography. The GC–MS was obtained on a Shimadzu QP-200 instrument at 70 eV and 250°C. The ULBON HR-1 GC column, equivalent to an OV-1 fused silica capillary column, 0.25 mmx50 M, with a film thickness of 0.25 µm, was used for separation. Helium was used as the carrier gas. The initial temperature was 100°C for 5 min and then increased at a rate of 5°C/min to 250°C. 1H-NMR spectroscopy was used to identify the compound. The NMR spectrum was measured on a Bruker DRX-300 spectrometer at 300 MHz in CDCl3-containing tetramethyl silane (TMS) as the internal standard at 25°C. The IR spectroscopy was performed on a Shimadzu 8201 from 4000 to 450 cm–1 with KBr as the solvent.
Biofilms and confocal microscopy
Biofilms, for confocal analysis, were cultivated on glass coverslips (n = 3) as described by Lynch et al.,18 with slight modifications. One hundred microlitres of a 1.0 OD600 culture of S. mutans was inoculated in a nine-well microtitre plate seeded with glass coverslips, containing TSB supplemented with 0.25% sucrose. Concentrations of 1 and 5 mg/L purified compound were used to observe its effect on the formation of biofilms. The plates were incubated at 37°C for 24 h under anaerobic conditions. The coverslips were then removed, and non-invasive confocal analysis of fully hydrated biofilms was performed using a Leica Microsystems CLSM (Heidelberg, Germany) fitted with a water immersion dipping objective lens (60x) and a Kr–Ar laser. The specimens were stained for 1 h with propidium iodide (0.2 mg/mL) in a buffer containing 0.1% sodium citrate, 0.1 mg/mL RNAse and 0.3% brij-58. The excitation wavelength was 594 nm. A scan speed of 400 lines/s was used to ensure minimum dislocation due to the movement of cells. Images of the colour were adapted to the 8 bit range of the system. An HCX PL APO CS 63.0 x 1.32 Oil UV objective was used with an additional zoom of x4, resulting in a 512 x 512 image with a pixel size of 0.12 µm. The pinhole size was set at 1AIRY Disc. A series through the whole thickness of the biofilm was made with a step size of 0.04 µm. Each biofilm was scanned at five randomly selected positions away from the disc edge. In accordance with optimal settings described here, images were acquired using a x1 digital magnification, a pinhole setting of 1 Airy unit and a scan average of 2; the detector gain (500–550 arbitrary units) and amplifier offset (0–0.05 arbitrary units) were used to obtain adequately contrasted greyscale images based on the brightest region of the biofilm that was scanned. Each stack of an experiment was examined, and the threshold value that best fit all image stacks of a trial was chosen. The images of the control and in the presence of purified compound were averaged and compared.
Assessment of cellular viability
The concentration of the crude extract and of the purified compound showing a capacity to inhibit glucan and biofilm formation by S. mutans were tested for their effect on bacterial viability using tetrazolium sodium 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)-benzene sulphonic acid hydrate (XTT; Sigma) reduction assay.19
The values were calculated as the mean of individual experiments in triplicate and compared with those of the control groups. Differences between two mean values were calculated by Student's t-test. A one-way analysis of variance (ANOVA) was performed for comparison of multiple means. The thickness of 1-deoxynojirimycin (DNJ)-treated and -untreated biofilms was compared using SPSS 11.0.0 statistical software with one-way ANOVA and post hoc tests (LSD); statistically significant tests were set at a P value of less than 0.05.
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Antimicrobial activity of crude extracts
Figure 1(a) shows the effect of crude extract of M. alba on the growth of A. viscosus, L. acidophilus, L. lactis and S. mutans. The effect of S. mutans on five clinical isolates, SM4, SM5, U152, U153 and SM6, is shown in Figure 1(b). No significant inhibition of growth could be seen against A. viscosus, L. acidophilus and L. lactis in the concentrations used and conditions stated. The growth of S. mutans was inhibited by the crude extract of M. alba (P < 0.05) in a concentration-dependent manner. To confirm the activity, the cells were plated on 3% sheep blood agar plates after an overnight growth in the presence of the extract, with appropriate dilutions. The growth inhibitory activity was found to be bacteriostatic in nature.
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Determination of MIC
Table 1 shows the MIC of crude ethanolic extract of M. alba against MTCC strains of different oral pathogens. The purified compound had an MIC of 15.625 mg/L against the MTCC strain of S. mutans. The decrease in the MIC by 
8-fold reflects the higher activity of the purified compound when compared with that of the crude extract. The MIC was also evaluated against clinical isolates of S. mutans. The clinical isolates showed a statistically similar inhibitory effect and MICs similar to that of the standard strain (P > 0.05). The MIC of purified compound against S. mutans is comparable with that of chlorhexidine.20
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Effect of anti-adherence extract to glass tubes
The inhibitory effects of different concentrations of crude extract on adherence of S. mutans (MTCC *497) to glass tubes are shown in Figure 2. The extract of M. alba inhibited slightly sucrose-independent adherence (r = –0.00 092). The extract of M. alba, however, inhibited sucrose-dependent adherence in a pronounced, dose-dependent manner (r = –0.02386). At a concentration of 25 mg/L of M. alba crude extract, the adherence of S. mutans was reduced by 50%.
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Biofilm formation by S. mutans
The crude extract of M. alba inhibited biofilm formation in a concentration-dependent manner [Figure S1, inset; see Supplementary data at JAC Online (http://jac.oupjournals.org/)]. The effect was tested at 6, 12, 20 and 24 h to visualize whether it could adversely affect S. mutans biofilms in each phase of biofilm growth: 6 h, adherent phase; 12 h, active accumulated phase; 20 h, initial plateau accumulated phase; and 24 h, plateau accumulated phase.21 This study showed that the effect of crude extract is concentration-dependent as well as biofilm phase growth-dependent (Table 2). The percentage of adherent cells under control conditions and at various concentrations of crude extract was found to be similar at 6 h of biofilm growth. But, at 12 h, the percentage of adherent cells at the maximum concentration of crude extract used was 72% less than that in the control group. The adherent cells were also reduced at 20 and 24 h of S. mutans biofilm growth. Thus, biofilm formation was inhibited by the crude extract during the active accumulated phase, initial plateau accumulated phase and plateau accumulated phase. A similar result was also obtained when biofilms of S. mutans were developed in the presence of the purified compound (Figure S1).
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Estimation of polysaccharide
The polysaccharides synthesized by the total secretory proteins of S. mutans were found to be lower in the cells grown in the presence of purified compound from M. alba when compared with its vehicle control. The amount of water-soluble polysaccharide formed in the presence of 5 mg/L purified compound was 23.2% lower with respect to control, whereas the amount of alkali-soluble polysaccharide was 44.8% lower than the control. The amount of protein isolated was, however, similar in the presence and absence of extract (data not shown). It is noteworthy that the present experiment was undertaken at concentrations ineffective for its antibacterial activity. XTT assay confirmed that the concentrations used did not affect bacterial viability. This implies that the purified compound inhibits the synthesis of polysaccharide by the bacteria, without affecting protein secretion. However, the inhibition was more pronounced on the alkali-soluble polysaccharide than on the water-soluble polysaccharide. The moderate polysaccharide synthesis in growing cells of S. mutans results in decreased cellular adherence to a glass surface.
Effect of oxidized pure fraction on S. mutans
The purified active fraction after oxidation by sodium meta-periodate was tested again for its effect on planktonic growth as well as on the biofilm of S. mutans, and we found that it lost its inhibitory effect on the growth of S. mutans at the concentrations tested. Biofilm formation was also unhindered, suggesting a complete loss of activity of the pure fraction upon oxidation. These data imply that the purified compound is a carbohydrate moiety.
Identification of the compound
The purified compound was obtained as a pale white amorphous compound. The GC–MS showed (m/z) signals at 87, 84, 82, 50, 49, 48 and 47. The base peak was observed at 83. The NMR spectrum showed peaks at 
5.4 (dd, NH), 5.1 (broad singlet), 4.7 (dd), 4.4 (m), 4.2 (dd), 4.0 (dd), 3.6 (dd) and 3.4 (dd). The IR spectrum shows bands 
(cm–1) at 3376, 1655, 1453, 1407, 1108 and 1027.
Confocal laser scanning microscopy of biofilms
Confocal analysis of biofilms in the absence and presence of purified compound was performed to examine its effect on biofilm architecture; see Figure S2, available as Supplementary data at JAC Online (http://jac.oupjournals.org/). The concentrations used were first tested for their cytotoxic effects by XTT assay. None of the concentrations used affected the growth of S. mutans. The cells appeared red against the black background due to propidium iodide staining of DNA. Each panel of the image is a representative view of 141.145 µm by 141.145 µm along the xy axis. CLSM-xy analyses provide the biofilm surface coverage, and the z-section analyses demonstrate the thickness of the biofilm. The control cells have biofilms that are less spread along the xy lane and show a thickness of 18.5 ± 0.2 µm. The cells grown in the presence of 1 mg/L purified compound have more clumped cells, covering a larger surface area, with a thickness of 16.8 ± 0.1 µm. The cells grown in the presence of 5 mg/L purified compound show a complete absence of clumped cells and the thickness is 10.3 ± 0.4 µm. The cells were individually scattered over the surface rather than in any arrangement.
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This is the first report to provide evidence that the leaf extract of M. alba shows an anticariogenic potential on the basis of bacteriostatic effects and biofilm of S. mutans. The plant is a native of Asia and is widely exploited for its varied medicinal properties. However, there is a lacuna with regard to scientific validation of the effects. We found that the crude extract of M. alba shows conspicuous growth inhibitory effects against S. mutans (Figure 1b and Table 1).
The crude extract also shows strong inhibitory action against glass-dependent adherence of S. mutans in the presence of high sucrose concentrations, whereas sucrose-independent adherence was reduced to a lesser extent (Figure 2). Sucrose-independent adherence is mostly due to the hydrophobic interaction of the cells with the glass. The reduced adherence in the presence of extract could be an effect of oils from the plant extract, which reduce the hydrophobicity of the bacteria. Sucrose-dependent adherence was decreased to a marked level. The bacteria synthesize sticky exopolysaccharides (glucan) with sucrose as the substrate that mainly mediates the clumping of the cells. Thus, this implies that the extract can reduce polysaccharide-mediated adherence of the bacteria.
Biofilm formation by bacteria is also mediated by glucan. The sticking of cells on a polystyrene surface forms a model for the in vitro biofilm formation of bacteria.15 This approach was used to study the biofilm of S. mutans in the presence of mulberry leaf extract at various concentrations. The dye released by the bound cells is an indirect revelation of the bacterial monolayer formed on the wells. The OD at 630 nm decreases in the presence of crude extract or purified compound, as the case may be (Figure S1) in a concentration-dependent manner.
It was found that only after 6 h of incubation, the crude extract did not inhibit primary adherence to the polystyrene plates, reflecting the inhibitory role in biofilm development rather than primary attachment to the surface.22 The cells show reduced adherence after 12, 20 and 24 h, implying that the crude extract is inhibitory at the active accumulation and plateau phases (Table 2). These stages of accumulation are marked by the active synthesis of glucan.23 Thus, crude extract either inhibits glucan synthesis or reduces glucan binding of the cell.
The extract of the M. alba leaf was subjected to column chromatography to separate the components. The purified compound was analysed by GC–MS. The single peak in the chromatogram confirmed the purity of the compound. This compound exhibited both bacteriostatic (Table 1) and anti-adherence effects (Figure S1, inset).
The total proteins that comprised GTFs, secreted in the presence of purified compound from M. alba and control, were used for in vitro synthesis and estimation of water-soluble and alkali-soluble polysaccharide. Although the amount of secretory protein was found to be same, the polysaccharide formed was less in the presence of the purified compound. We conclude that the compound is not effective at the protein-secretion level, but it inhibits the formation of polysaccharide by the protein.
The potential anticariogenic compound from M. alba was analysed by MS, NMR and IR spectroscopic techniques. The IR spectrum showed characteristic absorption bands for NH and OH groups as a broad hump centred at 3376 cm–1. NH and OH groups have some common properties and their absorption due to these groups is superimposed, making their identification difficult. The other IR peaks were indicative of 1655 (N-H bending), 1453 (C-H methylene), 1407 (C-N group), 1108 (>C-O) and 1027 (C-O), respectively.
The NMR spectra revealed the presence of glucose-like peaks in the region 4.7-3.5 ppm. The anomeric carbon (H-1') at 4.7 was coupled to a proton at d (3.4, H-2', J = 8 Hz). Proton with d (3. 6, H-3') also demonstrated coupling with H-4', which appeared as multiplets at 4.4, which in turn exhibited connectivity at H-5' at 3.6 and 4.0. The hydroxymethyl group at 4.0 and 4.2 revealed geminal coupling in addition to vicinal coupling, with 3.6 assignable to H-5'. The doublet at 5.36 is assignable to the proton attached to nitrogen.
The complete analysis of the entire spectra confirmed the compound to be 1,5 deoxy-1,5-imino-D-glucitol, commonly known as 1-deoxynojirimycin (DNJ) [see Figure S3, available as Supplementary data at JAC Online (http://jac.oupjournals.org/)]. Although the MS did not show the peak for M+1+ at 164, the fragments observed are characteristic of its structure. This compound is an iminosugar analogue of D-glucose, with the oxygen in the ring replaced by nitrogen, and was originally isolated from plants and microorganisms, although a considerable number of analogues have been synthesized during the past few years.24 The concentration of such sugar-mimicking alkaloids in mulberry leaves is 0.14%.25
This is the first report that shows the anti-adherence effect of DNJ with mulberry as the parent source. The inhibition of glucan formation by DNJ has been explored previously26,27 and is obvious, as the compound is a potent glucosidase inhibitor.28 The alkali-soluble glucans are of selectively more importance than water-soluble glucan in adhesive interactions by S. mutans.29 We found that inhibition of alkali-soluble polysaccharide by the purified compound was greater when compared with the inhibition of water-soluble polysaccharide. This result corroborates the findings of Wunder and Bowen that the activity of GTF B was inhibited to a higher extent by DNJ than that of GTF C and D.26
CLSM images depict the architecture of cells in a biofilm. The cells in control are embedded in a polysaccharide matrix that stimulates cell clustering. Microcolonies grow away from the substratum to resemble mushroom stalk and cap structures. Undoubtedly, the biofilm architecture is disturbed in the presence of DNJ. The presence of 1 mg/L DNJ results in biofilms consisting of shorter microcolony height and a more even distribution over the substratum. The classic biofilm architecture of mushroom cap and stalk seems to disappear completely. The presence of 5 mg/L DNJ results in a complete loss of aggregates. The cells are scattered individually along the substratum.
The MIC of DNJ is comparable with those of the compounds found in propolis.8 With the current background, DNJ is above par compared with natural essential oils that target cells mainly by disrupting the membranes and have a far higher MIC.20 Therefore, DNJ could be a promising compound for targeting biofilms of S. mutans. Mulberry leaves can be consumed raw, and this precludes the possibility of any serious toxic effects. Glucosidase inhibitors often cause intestinal discomfort and diarrhoea,30 hence their incorporation of compound in the toothpastes, gels and chewing gums may be a better choice. Research is in progress in our laboratory to evaluate the kinetics of glucan inhibition and various other aspects of DNJ–S. mutans interaction.
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This work was supported by the internal funds of the Biotechnology Unit, AMU and DST grant 100/IFD/5160/2007–2008 (to A. U. K.). B. I. and S. N. K. acknowledge CSIR for a Senior Research Fellowship.
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None to declare.
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Supplementary data |
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Figures S1–S3 are available as Supplementary data at JAC Online (http://jac.oupjournals.org/).
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Acknowledgements |
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We acknowledge RSIC, CDRI, Lucknow, India for providing the facilities for analysis of the compound.
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References |
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