Isolation Active Ingredients From Magnolia Officinalis And Their Anti-caries Mechanisms

Traditional herbal medicines, the only kind of health remedies available to all ancients, have been used for thousands of years. Recently, natural products have attracted increased interest as an alterative choice in health care. The use of natural products has been one of the successful strategies for the discovery of new medicines; 78% of new antibiotics and 61% of new anti-tumor drugs approved by the Food and Drug Administration from 1983 to 1994 were natural products or derived from natural products. However, before traditional herbal medicines can be applied for clinic, there are still many problems must be solved, such as unclear structure, unknowing target and ill-defined mechanisms.Magnolia officinalis, with a bitter taste and a gentle property, is the bark of the root and stem of Magnolia officinalis Rend et wils. This medicinal plant has been well known as an important component of Chinese traditional medicines recipes such as Hou-pu San Wu tang, Si Qi tang, Ping wei san, treating for many diseases. Moreover, M. officinalis was shown to inhibit the growth of cariogenic bacteria in vitro and reduce dental caries. But until now, the systematic study of M. officinalis inhibiting caries has not been carried out and its molecular mechanism of anti-caries activity has never explored. So we conducted it in this study. The objective of this study was to isolate active ingredients from M. officinalis and examine their effect on the principal causative factors in the formation development of caries, including growth, adherence of the cariogenic bacteria, glucosyltransferase, α-amylase and a-glucosidase. It is the first time to explore the anti-caries properties of M. officinalisboth in suspension and experimental dental plaque systematically; and it is the first time to investigate the effect of magnolol on the catalytic phases and functional domains of GTFs for the purpose of defining its anti-GTF activity mechanism.There were three parts in this study:Part one Extraction of effective section from Magnolia officinalis and its effect on the growth of cariogenic microorganismsThree fractions were obtained by extract procedures (HP1, HP2, HP3). It was found that HP2 possessed a significant growth inhibition of all strains while HP2 and HP3 was no significant difference compared to the control (P > 0.05) . The bacteriostatic effects of HP2 required concentrations of 0.39 to 0.78 mg/ml, and the bactericidal effects required sterilizing concentrations of 2.76~4.82 mg/ml. HP2 of lOmg/ml rapidly reduced the number of S. mutans MT8148R, S. sobrinus 6715 and S. sobrinus B13 within 30 min to 1 h of incubation (reduction of 0.2 in the values of OD at 610 nm). It exerted bactericidal effects (OD>0.4 decrease) between 2 and 4 h of incubation. After 8 h of incubation the growth of Streptococcus spp. strains tested was blocked by HP2 completely. HP2 displayed bactericidal effects on Actinomyces Spp. after 10 h of incubation (OD < 0.05) . Weak antibacterial activities against S. cricetus AHT, S. mutans MT8148R, S. mutans MT6219 and A. viscosu ATCC29525 could be detected for HP1, but it did not display any bactericidal effect at the concentrations tested in this study. In conclusion, HP2 was the effective section of M. officinalis that possessed potential inhibiting growth of the cariogenic microorganisms.Part two Isolation and identification of effective ingredientsfrom Magnolia officinalisHP2 was further subjected to chromatography with silica gel colum and effective ingredients (MH, crystal I and crystal II) of M. officinalis were isolated. Treatment of MH with a coloration reagent (2% FeC^) produced positive result, indicative of phenolic compounds. Developed by Thin-layer chromatography (TLC)and monitored under UV light (X=254), MH showed two spots (Rf 0.67, 0.58) which corresponded to those of magnolol and honokiol. IRv^cm"1: 3078, 1216 (-OH), 1608, 1498, 880, 821 (1,2,4-benzene), 1638, 1430, 990, 908 (-CH=CH2). Crystal I IRv^'cm"1: 3160, 1210 (-OH), 1610, 1496, 885, 820 (1,2,4- benzene), 1640, 1410, 990, 900 (-CH=CH2); TLC, Rf 0.65; UVA*°Hnm: 294. Crystal II IRv^r cm"1: 3280(-OH), 1610, 1496, 880, 820 (1,2,4-benzene), 1640, 1435, 987, 907(-CH=CH2); TLC, Rf 0.55; UV A *°H nm: 294. Analysis of these data verified that MH composedof magnolol and honokiol. Crystal I was identified as magnolol and crystal II was identified as honokiol.Part three Anti-caries mechanism of effective ingredientsfrom Magnolia officinalisThe effects of effective ingredients from M. Officinalis on the growth, adherence of S. mutans, glucosyltransferase, oc-amylase and a-glucosidase were evaluated in suspension and the experimental dental plaque model during four experiments.1. A experimental biofilm model mimicking the environmental conditions of the dental plaque was used to examine the growth inhibition. MH, magnolol or honokiol were treated 3 min at 16.5, 20.5 and 24.5hr after the information of biofilm. The results showed that MH, magnolol or honokiol significantly inhibited the growth of the cariogenic bacteria. There was no significantly difference among the three compounds (P > 0.05). Our results also indicated that higher concentrations of MH, magnolol or honokiol were required to elicit the same antibacterial effect on surface bound bacteria than in suspension.2. MH, magnolol and honokiol demonstrated effectiveness in discreasing both sucrose-dependent adherence and sucrose-independent adherence in vitro. Their impact were no significant difference compared with each other (P > 0.05). After saliva-coated hydroxyapatite (S-HA) was treated by MH, magnolol or honokiol at the concentration of 0.4~3.2 mg/ml, the numbers of S. mutans UA159 adhering toS-HA decreased obviously. In addition, after S. mutans UA159 was treated by MH, magnolol or honokiol at the concentration of 0.4~3.2 mg/ml, the numbers of the bacteria adhering to S-HA decreased remarkably (P < 0.05) .It was speculated that these active ingredients can inhibit sucrose-independent initial adherence through changing the surface properties of acquired pellicle or affecting the surface components of bacteria.3. GTF plays the most important role in the development of dental caries. MH, magnolol or honokiol reduced total glucan synthesis, depending on their concentrations, respectively. Honokiol had a weaker efficacy in inhibiting GTF than MH or magnolol (P < 0.05). Magnolol significantly inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. There were no significant differences in Km values between the presence and absence of magnolol as determined by Lineweaver-Burk plot and Grafit enzyme kinetical program, and Vm in the presence of magnolol was lower than that in its absence. These findings suggested that magnolol inhibits both two sequential reaction phases of GTF-I non-competitively by operatings on the glucan-binding domain of GTF.4. Both saliva a-amylase and bacterial a-amylase inhibition increased with increasing concentrations of MH, magnolol or honokiol. a-Amylase inhibition was greatest for MH, which gave 70.11% saliva enzyme or 40.02% bacterial enzyme inhibition at concentration of 100 ug/ml, respectively. MH, magnolol and honokiol inhibited enzyme activity of a-glucosidase hydrolyzing amylolytic products of starch. Among these three fractions, MH exhibited highest degrees of a-glucosidase inhibitory effectiveness (P < 0.05) . They showed different a-glucosidase inhibitory degrees on various substrates, and the rank order was maltose > maltotriose > maltoheptaose.In conclusion, these findings suggested that the effective ingredients of M officinalis were strong inhibitors of growth and adherence of the main cariogenic bacteria, GTF, a-amylase and a-glucosidase both in suspension and on dental plaque; magnolol inhibits both two sequential reaction phases of GTF-I non-competitively by operatings on the glucan-binding domain of GTF. Molecular mechanism ofanti-GTF activity of magnolol was made clear so that inhibitor efficiency and prevention of caries can be predicted.