PEDIATRIC DENTAL JOURNAL 14(1): 29–35, 2004 29
In vitro antibacterial effects of the crude extracts of Sophora alopecuroides against oral microorganisms
Ulamnemekh Hulan, Tserensodnom Bazarragchaa, Michiko Nishimura and Tsutomu Shimono
Department of Behavioral Pediatric Dentistry, Graduate School of Medicine and Dentistry, Okayama University 2-5-1 Shikata-cho, Okayama 700-8525, JAPAN
Abstract Sophora alopecuroides is widely used in Mongolian traditional Key words medicine. The antimicrobial effects of the methanolic and aqueous extracts of its Growth inhibition, roots against Streptococcus mutans, Streptococcus sobrinus and Staphylococcus Mutans streptococci, aureus were examined in vitro. Four fractions were obtained from methanol Sophora alopecuroides, extract and labeled as Fractions-1, 2, 3, and 4. The MIC’s and MBC’s of the Staphylococcus aureus crude extracts of Sophora alopecuroides were determined, and bacterial cell growth curves in the presence of Fractions-1, 3, and 4 were drawn. Among the methanolic extracts, the extracts of Fractions-1, 3, and 4 possessed strong inhibitory effect on the growth of mutans streptococci and S. aureus. Fraction-2 and the aqueous extracts of S. alopecuroides had weak antibacterial activities. Thus, S. alopecuroides would be useful for the suppression of oral pathogens, and has potential for use in the prevention of dental caries and treatment of diseases caused by S. aureus.
Introduction traits such as sucrose-dependent adherence and lactic acid production that enable them to establish Sophora alopecuroides (L.), a plant native to the themselves in the oral cavity and initiate disease8). Gobi desert and nearby areas, is widely used in The nature of caries does not readily permit itself to Mongolian traditional medicine. The flowers and conventional antibiotic therapy9). Recently, the use leaves of S. alopecuroides are collected in the of herbal medicine as therapeutic agents for the summer, while its seeds and roots are gathered in prevention and treatment of dental caries and other the autumn. The roots are mostly used for medicinal oral diseases has been increasing10). Strategies to purposes. When taken internally, they are used for prevent caries include the inhibition of cell growth a variety of respiratory diseases, in reducing fever, of cariogenic bacteria and inhibition of plaque and for the treatment of skin infections. They are formation and acid production by plaque bacteria. also used externally as a poultice for swelling and Staphylococcus aureus is a common human patho- wounds, skin rashes and sores, and as a mouth rinse gen, which in addition to various skin and mucosal for treating tonsillitis, gingivitis and toothaches1). infections causes abscess formation, pneumonia, Dental caries is one of the most common chronic septicemia and gastroenteritis. In children, it causes diseases of humans. Mutans streptococci, in particular impetigo and the scalded-skin syndrome. It has been Streptococcus mutans and Streptococcus sobrinus, reported that 33% of children who had no diseases are considered to be the principal etiologic agents of other than dental caries were positive for S. aureus11). dental caries2–7). They possess a variety of virulence The objective of the present study was to inves- tigate the antibacterial effects of the crude extracts Received on July 23, 2003 of S. alopecuroides on the growth of S. mutans and Accepted on November 19, 2003 S. sobrinus, and also S. aureus.
29 30 Hulan, U., Bazarragchaa, T., Nishimura, M. et al.
Materials and methods Assay for growth inhibition effect Methanol extract (ME) preparation The disk diffusion method was employed for One kg of ground, dried radices of S. alopecuroides investigating the growth inhibition effect of the was extracted with 10 liter of 80% methanol. The crude extracts of S. alopecuroides against the test mixture was magnetically stirred at room temperature microorganisms. for 1 week followed by filtration using a 0.45m One loop of each bacterial strain was inoculated cellulose filter (Toyo Roshi Co., Tokyo, Japan) into separate tubes with brain heart infusion (BHI, and evaporated at 40°C using a rotary vacuum Difco Laboratories, Detroit, MI, USA) broth and ,0.8סevaporator (EYELA, NE-1, Tokyo Rikakikai Co., grown to log phase for 7–8 h at 37°C (OD Ltd.) to give a dark brown oily residue. This extract 600 nm). Aliquots of 100l cell suspensions of was washed with warm distilled water and decanted. each microorganism were inoculated into BHI agar. The remaining water-insoluble phase was taken up Sterile paper disks (6 mm diameter) impregnated in 200 ml of 100% methanol, and was labeled as with 15l of the test extracts of S. alopecuroides Fraction-4. (750g/disk for the Fractions-1 and 2 and WE, The water-soluble phase was evaporated at 80°C 1.5l/disk for the Fractions-3 and 4 extracts) were to give an oily residue which could not be frozen. The placed on the inoculated plates. Ten % and 100% condensate was then washed and taken up in cold methanol and water served as the negative controls, distilled water. This water-soluble fraction consisted while 0.2% chlorhexidine gluconate was the positive of 2 phases, a liquid phase and sediment. After control. The plates were incubated at 37°C for 48 h. filtration through a cellulose filter, the sediment At the end of the incubation, the clear zone of was dried, and the resulting powder was labeled bacterial inhibition was measured. as Fraction-1. The supernatant was evaporated and freeze dried. It was labeled as Fraction-2. Solutions Determination of minimal inhibitory of both Fractions-1 and 2 were prepared in distilled concentration (MIC) and minimal water at 100 mg/ml, 50 mg/ml, and 10 mg/ml, bactericidal concentration (MBC) 20°C until use. The broth dilution method was used to determineמ respectively, and stored at The remaining water-insoluble phase was redis- the MIC. The lowest concentration of the crude solved in 100% methanol and labeled as Fraction-3. extract at which no visible bacterial growth could Solutions of Fractions-3 and 4 were prepared at be detected by visual and spectrophotometrical concentrations of 100l/ml, 50l/ml, and 10l/ml inspection (600 nm) was defined as the MIC. Test 20°C. microorganisms were inoculated into tubes withמ in distilled water and stored at BHI broth and grown to log phase for 7–8 h at 37°C nm). The cell suspension of each 600 ,0.8סWater extract (WE) preparation (OD Fifty grams of ground, dried radices of S. alopecu- microorganism was 100-fold diluted with sterile roides were suspended in 500 ml of distilled water saline. Aliquots of the 200l cell suspensions were at 90°C until one third of its original volume was inoculated at 105 to 106 CFU/ml into 2 ml of BHI evaporated. It was then centrifuged at 3000 rpm for broth containing the crude extracts of S.alopecuroides 20 min at room temperature, and the supernatant at 2-fold dilutions and incubated at 37°C for 18 h. was filtered through a cellulose filter. The filtrate The OD’s of the tubes were measured every 6 h. was evaporated by a rotary evaporator at 80°C. The At the same time, 100l was removed from each sticky condensate was then freeze-dried. Solutions tube, serially diluted and plated onto BHI agar to of the resulting lyophilizate were prepared in distilled determine the MBC. The plates were anaerobically water at concentrations of 100 mg/ml, 50 mg/ml, incubated at 37°C for 48 h. The bacterial growth 20°C. curves in the presence of the test extracts were thenמ and 10 mg/ml and stored at determined. Microorganisms Each experiment was carried out at least 3 times. Streptococcus mutans ATCC 25175, Streptococcus sobrinus ATCC 33478 and Staphylococcus aureus Statistical analysis IFO 13276 were used for testing the antibacterial The data for growth inhibition zones were analyzed effect of S. alopecuroides. by ANOVA and Bonferroni post hoc test. The ANTIBACTERIAL EFFECTS OF S.ALOPECUROIDES 31
Table 1 Growth inhibition effect of the crude extracts of S. alopecuroides Growth inhibition zone diameter in mm Solutions tested S. mutans S. sobrinus S. aureus
Fraction-1 extract of S. alopecuroides a 750g/disk 9–12 9–11 10–11 Fraction-2 extract of S. alopecuroides a 750g/disk NGI NGI NGI Fraction-3 ME of S. alopecuroides b 1.5l/disk 9–12 9–10 10–11 Fraction-4 ME of S. alopecuroides b 1.5l/disk 11–12 9–10 11–14 WE of S. alopecuroides a 750g/disk NGI NGI NGI 0.2% chlorhexidine gluconate 21–23 19–22 17–19 10% methanol NGI NGI NGI 100% methanol NGI NGI NGI Distilled water NGI NGI NGI NGI: no growth inhibition was observed a: Disks (6 mm) soaked with 15l of the 50 mg/ml extracts of Fractions-1 and 2 and WE (750g/disk) b: Disks soaked with 15l of the 100l/ml extracts of Fractions-3 and 4 (1.5l/disk)
Table 2 The MIC and MBC of the crude extracts of S. alopecuroides S. mutans S. sobrinus S. aureus Solutions tested MIC MBC MIC MBC MIC MBC
Fraction-1 extract of S. alopecuroides a 4.9 156 9.8 78 2.4 9.8 Fraction-2 extract of S. alopecuroides b 10 NT 10 NT 1.25 NT Fraction-3 extract of S. alopecuroides c 0.156 0.625 0.312 0.625 0.078 0.156 Fraction-4 extract of S. alopecuroides c 0.039 0.625 0.039 0.156 0.009 0.078 WE of S. alopecuroides b 10 NT 10 NT 2.5 NT MIC: minimal inhibitory concentration; MBC: minimal bactericidal concentration; WE: water extract; NT: not tested a: MIC’s and MBC’s of Fraction-1 extract expressed in g/ml b: MIC’s of Fraction-2 and WE expressed in mg/ml c: MIC’s and MBC’s of Fractions-3 and 4 expressed in l/ml
relationships between extract concentrations and crude extracts of S. alopecuroides against the above- bacterial numbers were evaluated with Pearson mentioned bacterial strains. Distilled water and 10% correlation coefficient. and 100% methanol, which served as the negative controls, showed no visible inhibition zones, whereas Results 0.2% chlorhexidine, used as the positive control, showed the greatest zone of inhibition. Crude extracts of Fractions-1, 3, and 4 of S. The MIC’s of test extracts are shown in Table 2. alopecuroides exhibited much stronger antibacterial The MIC of either WE or Fraction-2 extract of activities than either the water extract or Fraction-2 S. alopecuroides against both streptococci were extract against S. mutans, S. sobrinus and S. aureus. 10 mg/ml. While against S. aureus, the MIC of WE Table 1 shows the growth inhibition zones of the was 2.5 mg/ml, and that of the Fraction-2 extract 32 Hulan, U., Bazarragchaa, T., Nishimura, M. et al.
Fig. 1a Effect of Fraction-1 extract of S. alopecuroides Fig. 2a Effect of Fraction-3 extract of S. alopecuroides on the growth of S. mutans on the growth of S. mutans
Fig. 2b Effect of Fraction-3 extract of S. alopecuroides Fig. 1b Effect of Fraction-1 extract of S. alopecuroides on the growth of S. sobrinus on the growth of S. sobrinus
Fig. 1c Effect of Fraction-1 extract of S. alopecuroides Fig. 2c Effect of Fraction-3 extract of S. alopecuroides on the growth of S. aureus on the growth of S. aureus
was 1.25 mg/ml. The MIC of the Fractions-1, 3, and test extracts are shown in Figures 1a to 3c. Bacterial 4 were much more lower against all bacterial strains. number was expressed in the base-10 logarithm of The bacterial growth curves in the presence of the colony forming units (CFU) per ml of medium. The ANTIBACTERIAL EFFECTS OF S.ALOPECUROIDES 33
for 15 hours before cell growth started. While at concentration of 2.4g/ml, the CFU of S. aureus did not increase on the plates, and thus the growth of S. aureus was inhibited bacteriostatically at this concentration. At 2.4g/ml, cell growth of S. mutans (Fig. 1a) was inhibited for 6 hours and that of S. sobrinus (Fig. 1b) for 12 hours before bacterial cell growth started. The growth of S. mutans was inhibited bacteriostatically at 4.9g/ml and that of S. sobrinus at 9.8g/ml. At 9.8 to 39g/ml, the Fraction-1 extract bacteriostatically inhibited cell growth of S. mutans. At concentrations of 78 to 156g/ml, bacterial numbers of S. mutans decreased, Fig. 3a Effect of Fraction-4 extract of S. alopecuroides and thus, the growth of S. mutans was partially S. mutans on the growth of bactericidally inhibited. While at concentrations of 313g/ml to 5 mg/ml, the growth was completely blocked. After incubation with the Fraction-1 extract at 19 and 39g/ml, the growth of S. sobrinus was partially bactericidally inhibited. At 78g/ml to 5mg/ml, Fraction-1 completely inhibited cell growth of S. sobrinus. S. aureus was bactericidally inhibited at the concentrations of 9.8g/ml to 5 mg/ml. The Fraction-3 extract inhibited the growth of S. mutans bacteriostatically at 0.078l/ml, partially bactericidally at 0.156 and 0.313l/ml, and com- pletely bactericidally at concentrations of 0.625l/ml to 5l/ml (Fig. 2a). S. sobrinus was inhibited by Fraction-3 at 0.313l/ml, while at 0.626l/ml, Fig. 3b Effect of Fraction-4 extract of S. alopecuroides cell growth was completely blocked (Fig. 2b). The on the growth of S. sobrinus growth of S. aureus was inhibited bacteriostatically at 0.078l/ml and bactericidally at 0.156l/ml (Fig. 2c). The Fraction-4 extract bacteriostatically inhibited the growth of both S. mutans (Fig. 3a) and S. sobrinus (Fig. 3b) at 0.039l/ml, whereas that of S. aureus at 0.009l/ml (Fig. 3c). There were significant negative correlations between the concentrations of the test extracts of S. alopecuroides at 2-fold dilutions and bacterial numbers (PϽ0.05), and statistically significant positive correlations between the bacterial numbers at 6 h, 12 h, and 18 h (PϽ0.001).
Fig. 3c Effect of Fraction-4 extract of S. alopecuroides Discussion on the growth of S. aureus Medicinal plants have been used worldwide since the beginning of recorded time. Active components in herbs have been serving as models for the devel- growth of test strains was inhibited in an extract opment of new therapeutic agents. Thus, in the field concentration-dependent manner. At 1.2g/ml, of dentistry extensive screenings for biologically Fraction-1 inhibited the growth of S. aureus (Fig. 1c) active substances from herbal sources with anti- 34 Hulan, U., Bazarragchaa, T., Nishimura, M. et al. cariogenic effects has been carried out10). The green can inhibit the growth and virulence properties of tea catechins have been found to have an inhibitory cariogenic bacteria, and also the growth of S. aureus. effect on the cell growth and adhesion of mutans Thus, S. alopecuroides would be useful for the streptococci12–14), and the roots of licorice have been suppression of oral pathogens, and has the potential reported to significantly inhibit the plaque formation for use in the prevention of dental caries and treat- of S. mutans15). Flavanones isolated from Sophora ment of diseases caused by S. aureus. Furthermore, exigua have shown to have a growth inhibitory effect the effective substances of S. alopecuroides, which against cariogenic bacteria16). The bakuchiol of are contained in Fractions-1, 3, and 4, should be Psoralea corylifolia, a tree with various uses in purified, identified and screened for their antimicro- traditional Oriental medicine, has exhibited antimi- bial properties. crobial activity against S. mutans, lactobacilli and 17) other oral microorganisms . The aqueous extract References of Azadirachta indica (Neem) has been reported to inhibit plaque formation by mutans streptococci18,19). 1) Ligaa, U.: Sophora alopecuroides L. In: Use of The Azadirachta indica Neem bark acetonic extract Medicinal Plants in Traditional Mongolian Medicine. 20) Book 1. Shuvuun Saaral Publications, Ulaanbaatar, has shown a growth inhibition effect on S. sobrinus , Mongolia, 1996, pp.393–394. (in Mongolian) and the Neem bark aqueous extract has exhibited an 2) Bratthal, D. and Kohler, B.: Streptococcus mutans inhibitory effect on the adherence and acid production serotypes: Some aspects of their identification, by S. sobrinus in vitro21). Regretfully, some kinds of distribution, antigenic shifts, and relationship to plants began to disappear as a result of an ecological caries. J Dent Res 55(Spec Iss): C15–C21, 1976. 3) Hamada, S. and Slade, H.D.: Biology, immunology, unbalance. This is one of reasons why plants should and cariogenicity of Streptococcus mutans. J Bacteriol be more extensively investigated. Rev 44(2): 331–384, 1980. S. alopecuroides is called “a magic herb” by 4) Van Houte, J.: Bacterial specificity in the etiology of the Mongolian people because of its wide use and dental caries. Int Dent J 30(4): 305–326, 1980. effectiveness in the treatment of a wide variety 5) Tanzer, J.M.: Microbiology of dental caries. In: of human diseases. 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