Journal of Investigative and Clinical Dentistry (2010), 1, 120–125
ORIGINAL ARTICLE Oral Microbiology
Antibacterial activity of polysaccharide gel extract from fruit rinds of Durio zibethinus Murr. against oral pathogenic bacteria Pasutha Thunyakipisal1, Thatsanee Saladyanant1, Naulchavee Hongprasong2, Sunanta Pongsamart3 & Wandee Apinhasmit1 1 Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 2 Department of Periodontology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 3 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
Keywords Aggregatibacter actinomycetemcomitans, antibacterial activity, durian fruit rind, polysaccharide gel extract, Streptococcus mutans. Correspondence Dr Pasutha Thunyakipisal, Dental Biomaterial Sciences Program, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Bangkok 10330, Thailand. Tel: +66-2218-8885 Fax: +66-2218-8870 Email: [email protected] Received 22 February 2010; accepted 23 May 2010. doi: 10.1111/j.2041-1626.2010.00017.x
Abstract Aim: The polysaccharide gel (PG) extract from durian fruit rinds (Durio zibethinus Murr. ‘‘Monthong’’) is a pectic polysaccharide with antibacterial activity. This study aimed to investigate the in vitro antibacterial activity of PG against oral pathogens, Streptococcus mutans (S. mutans) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Methods: The inhibitory activity of PG at 50, 100, and 150 mg/mL against S. mutans (American Tissue Culture Collection 25175) and A. actinomycetemcomitans (American Tissue Culture Collection 43718) was determined after 1- and 5-min exposure by broth macrodilution susceptibility test and scanning electron microscopy. Normal saline or culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. Results: For 1-min exposure, 150 mg/mL PG or 0.1% chlorhexidine significantly possessed bactericidal activity against both tested bacteria (P = 0.037), while PG at 100 mg/mL possessed significant bactericidal activity against S. mutans (P = 0.037) and inhibitory activity against A. actinomycetemcomitans (P = 0.05). Blebs, irregular-shaped cells, and disrupted cells were found in bacteria treated with either 0.1% chlorhexidine or 50–150 mg/mL PG under scanning electron microscopy. Conclusion: The bactericidal activity of PG at 150 mg/mL against oral bacteria at 1-min exposure suggests its possibility to be used as a natural antibacterial ingredient in oral hygiene products.
Introduction Dental caries and periodontitis are common oral diseases. Streptococcus mutans (S. mutans) has been strongly implicated as the principal etiologic agent in human dental caries,1 whereas Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a bacterium associated with periodontitis.2,3 There is great interest in the use of antibacterial agents for the prevention and treatment of dental caries and periodontitis. The prevention of dental caries and periodontitis requires the control of these 120
pathogens that are in an oral biofilm known as dental plaque. Many attempts have been made to eliminate S. mutans and A. actinomycetemcomitans from the oral flora. Antibacterial agents, such as chlorhexidine, amoxicillin, cefuroxime, penicillin, sulfamethoxazole–trimethoprim, tetracycline, and erythromycin, have been effective in preventing dental caries.4 However, excessive and prolonged use of these chemicals can result in rearrangements of the oral and intestinal flora and cause undesirable side-effects, such as microorganism susceptibility, vomiting, diarrhea, and tooth staining.5 Therefore, ª 2010 Blackwell Publishing Asia Pty Ltd
P. Thunyakipisal et al.
it is important to develop alternative antibacterial agents from natural higher plants in a wide range of oral care products, such as toothpaste and mouthwashes. These natural agents have antibacterial activity to prevent dental caries and periodontitis and possess very low or no sideeffects. Some natural polysaccharides have antimicrobial properties, such as pectin,6 alginate,7 and chitosan.8 Recently, we isolated, purified, and characterized a novel natural polysaccharide from the fruit rinds of durian (Durio zibethinus Murr. of the Bombacaceae family) called ‘‘polysaccharide gel’’ (PG). Durian is the most consumed fruit in Thailand; it weighs approximately 2–4.5 kg and has large, sharp, spiny thorns. Following injury from harvesting, harvesters have been told to apply clear viscous exudates from the fruit rind or the cut-end of fruit stalk on the injured area in order to prevent infections and pus. PG is a pectic polysaccharide with immunomodulating activity.9,10 The PG extract from durian rinds has also been investigated for its antibacterial activity against a variety of bacteria.11 Wound dressing prepared from PG exhibits good activity for healing open wounds in the skins of pigs and dogs.12,13 PG has water-gelling property, contains a sugar component similar to those of high polymeric carbohydrates, and comprises two linear polygalacturonic acid chains, one of which is a neutral chain with side-chains of neutral sugars rich in galactose, rhamnose, and glucose in an area called the ‘‘hairy region’’; the other is an acidic polygalacturonan chain with lesser side-chains than the neutral chain.9 PG is an interesting substance for the development of a new antibacterial agent. It could possibly have the advantage of being a new type of water-soluble antibacterial agent and gives satisfactory results when utilized as an antibacterial polysaccharide. The in vitro antibacterial activity of PG against a variety of bacteria has been reported.11,14–16 Additionally, our previous study indicated that the minimum bactericidal concentration of PG against S. mutans and A. actinomycetemcomitans was 35 mg/mL within 24 h.17 However, this concentration of PG might take a longer time if used in oral hygiene products, such as toothpastes or mouth rinses against cariogenic and/or periodontopathogenic bacteria in humans, since toothbrushing or oral rinsing usually only takes a few minutes.18 Therefore, this investigation aimed at evaluating the in vitro antibacterial activity of the PG extract from durian fruit rinds against S. mutans and A. actinomycetemcomitas at 1 and 5 min in order to relate it to clinical applications in patients. Chlorhexidine gluconate at a 0.1% concentration, an effective antimicrobial chemical agent that suppresses S. mutans in dental plaque and saliva,19 was used as a positive control. ª 2010 Blackwell Publishing Asia Pty Ltd
Durian fruit rind antibacterial activity
Materials and methods Extraction of polysaccharides The PG was isolated from the dried fruit rinds of durian (Durio zibethinus Murr. ‘‘Monthong’’). Freshly ground fruit rinds were dried in a hot air oven at 60C to obtain 19.6 ± 1.4% w/w dried material. The dried sample was extracted with hot water at 90–100C and adjusted to pH 4.5 with citric acid; the extract was filtered and purified, as described previously.20 Briefly, the water extract was concentrated under reduced pressure and added in the three volumes of acidified aqueous ethanol (4% HCl in 75% ethanol) to obtain the gel-like precipitate. The precipitate was collected by filtration, and then dried and pulverized. The dried precipitate was redissolved in water, filtered, and concentrated. To concentrate the solution, aqueous ethanol was added and the precipitate was recovered. The precipitate was washed twice with 75% ethanol and then 95% ethanol, and afterwards, dried, ground, and sieved to obtain a PG powder. Bacterial strains and culture conditions Bacterial strains, S. mutans (American Tissue Culture Collection [ATCC] 25175) and A. actinomycetemcomitans (ATCC 43718), were used in this study. Streptococcus mutans was cultured in trypticase soy broth (Difco; Becton Dickinson, Claix, France), and A. actinomycetemcomitans was cultured in Brain Heart Infusion (Difco; Becton Dickinson). All bacteria were incubated at 37C in a 5% CO2 atmosphere. To prepare the bacterial cell suspension for the antibacterial activity assay, bacterial cells were collected and resuspended with the sterilized 0.9% NaCl2. The turbidity was adjusted equivalent to a 0.5 McFarland standard and adjusted to a concentration of 1 · 106 CFU/mL before use. Broth macrodilution susceptibility test The antibacterial assay was determined by the number of surviving bacteria at 1- and 5-min exposure to PG (50, 100, and 150 mg/mL). PG was dispersed in distilled water and sterilized in an autoclave. The 0.5 mL of designated concentrations of PG were incubated with an equal volume of bacterial suspension (1 · 106 CFU/mL) obtained during the logarithmic phase of growth. After incubation for 1 or 5 min, the reaction was diluted by 10-fold serial dilution. The aliquot was subsequently spread onto a trypticase soy agar plate for S. mutans or brain heart infusion agar plate for A. actinomycetemcomitans. The plate was incubated for 48 h in an incubator with 5% CO2 at 37C. In this study, sterilized normal saline and 0.1% chlorhexidine were used as negative and positive controls, respectively. The experiment was repeated three times for each sample. 121
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Scanning electron microscopic evaluation Both bacteria were treated with PG (50, 100, and 150 mg/mL) at 37C for 1 and 5 min, washed with normal saline, and filtered through a polycarbonate sheet with a pore size of 0.4 lm. Bacteria on the polycarbonate sheets were then fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer (pH 7.4) at 4C for 24 h. After dehydration with graded ethanol, the samples were critically point dried with CO2, mounted on specimen stubs, and sputter coated with gold particles. Scanning electron microscopic examination was performed at 15-kV emission voltage and a sample tilt angle at 0 on a Jeol JSM-5410 (Jeol, Tokyo, Japan). The culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. Statistical analysis All statistical computations were performed by spss software (version 11.5; SPSS, Chicago, IL, USA). Data from the time-kill analysis were presented as the median, maximum, and minimum of the numbers of surviving bacteria (log10 CFU/mL). Kruskal–Wallis tests examined differences in viable bacterial counts among treated and control groups, followed by post hoc Mann–Whitney U-test. The chosen level of significance was P < 0.05. Results Broth macrodilution susceptibility test The results showed that the antibacterial activity of the PG, represented by the number of viable bacteria S. mutans and A. actinomycetemcomitans (in CFU/mL) depended on its concentration and the exposure duration (Table 1). At 1 min, PG at 150 mg/mL or 0.1% chlorhexidine possessed the bactericidal activity against both S. mutans and A. actinomycetemcomitans (P = 0.037), whereas
PG at 100 mg/mL possessed the bactericidal activity against S. mutans (P = 0.037) and exhibited slightly inhibitory activity against A. actinomycetemcomitans (P = 0.05). PG at 100 mg/mL possessed bactericidal activity against both S. mutans and A. actinomycetemcomitans after 5-min exposure to PG (P = 0.037). The exposure to PG at 50 mg/mL for 1 and 5 min decreased the number of S. mutans and A. actinomycetemcomitans, but they were not different from the negative control (P > 0.05). Scanning electron microscopic evaluation To investigate the effect of PG on the bacterial membrane, S. mutans and A. actinomycetemcomitans were incubated in the culture broth medium as negative controls, 0.1% chlorhexidine as the positive control, and PG at concentrations of 50–150 mg/mL for 1- or 5-min incubation. The results are shown in Figures 1 and 2. As a negative control, S. mutans incubated in the culture broth medium for either 1 or 5 min exhibited a round shape (cocci) with a smooth surface (Figure 1a). The surface alterations of S. mutans after 1- or 5-min incubation in 0.1% chlorhexidine showed similar results, but the longer time exhibited a higher degree of surface alterations. Various-sized blebs were found on bacterial cell surfaces (Figures 1b and 1c). In addition, irregular-shaped cells and collapsed cells were observed (Figure 1c). The pattern of surface changes of S. mutans treated in 50–150 mg/mL PG was similar to that in 0.1% chlorhexidine (Figures 1c, 1d and 1f), but the degree of changes depended on its concentration and the exposure duration. The surface appearance of A. actinomycetemcomitans incubated in the culture broth medium for 1 and 5 min as the negative control groups were shaped as short with a smooth surface (Figure 2a). The surface alterations of A. actinomycetemcomitans treated in 50–150 mg/mL PG and 0.1% chlorhexidine showed a similar pattern. The
Table 1. Effects of polysaccharide gel (PG) on bacterial number after exposure for 1 and 5 min No. surviving bacteria (log CFU/mL)
Bacteria Streptococcus mutans Aggregatibacter actinomycetemcomitans
Incubation time (min) 1 5 1 5
Negative control
50 mg/mL PG
Med
Max
Min
Med
Max
Min
Med
Max
6.05 6.00 6.01 6.18
6.22 6.27 6.20 6.70
5.87 5.83 5.85 5.85
5.57 4.98 6.01 5.71
5.65 5.12 6.08 5.94
5.47 4.85 5.91 5.34
0* 0* 2.53** 0*,**
0 0 2.90 0
150 mg/mL PG
0.1% chlorhexidine
Min
Med
Max
Min
Med
Max
Min
0 0 2.00 0
0* 0* 0* 0*
0 0 0 0
0 0 0 0
0* 0* 0* 0*
0 0 0 0
0 0 0 0
100 mg/mL PG
Data expressed as median (Med), maximum (Max), and minimum (Min) of the number of surviving bacteria (log CFU/mL). Sterilized normal saline and 0.1% chlorhexidine were used as negative and positive controls, respectively. *Compared with control groups (P = 0.037); **statistically significant difference between groups (P = 0.037). Kruskal–Wallis test, followed by post hoc Mann–Whitney U-tests.
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(a)
(b)
1 µm (c)
1 µm (d)
1 µm Figure 1. Scanning electron micrographs of Streptococcus mutans incubated in polysaccharide gel (PG) for 5 min. (a) Incubation in culture broth medium as a negative control, (b,c) incubation in 0.1% chlorhexidine as a positive control, (d) incubation in 50 mg/mL PG, (e) incubation in 100 mg/mL PG, and (f) incubation in 150 mg/mL PG. Various-sized blebs (white arrows), irregularly shaped cells (black arrow), and disrupted cells (white arrowheads) were observed in bacteria treated either with 0.1% chlorhexidine or 50–150 mg/mL PG.
(e)
degree of changes caused by PG depended on the concentration of PG and the exposure duration. There were various-sized blebs on the cell surfaces, irregularshaped cells, and disrupted cells found in the bacteria treated with 0.1% chlorhexidine (Figure 2b) and 50– 150 mg/mL PG (Figures 2c, 2d and 2e). Discussion Previous reports showed that after 24-h incubation of bacteria in PG, the PG possessed antibacterial activity against both Gram-positive and -negative bacteria, such as Staphylococcus aureus and Escherichia coli.14 The bactericidal activity of PG has also been studied against Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Lactobacillus pentosus, Escherichia coli, Staphylococcus aureus, Proteus vulgaris,11 streptococci isolated from bovine mastitis (Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Streptococcus bovis, and Streptococcus acidominimus),15 Vibrio harveyi,16 as well as ª 2010 Blackwell Publishing Asia Pty Ltd
1 µm (f)
1 µm
1 µm
S. mutans and A. actinomycetemcomitans.17 For the practical application in daily oral care activity, such as toothbrushing and oral rinsing, we further investigated the minimum bactericidal concentration of PG against S. mutans and A. actinomycetemcomitans as the representative of cariogenic and periodontopathogenic bacteria within 1- to 5-min exposure. The result exhibited that the antibacterial activity of PG against S. mutans and A. actinomycetemcomitans responded in time- and dosedependent manner. Although the precise antibacterial mechanism of PG has not been elucidated, our scanning electron microscopic data suggested that PG has at least affected bacterial membrane integrity. The cell surfaces of microorganisms are commonly composed of molecules of polysaccharides, such as lipopolysaccharides, capsular polysaccharides, and cationic/anionic polymers.21,22 The monosaccharide compositions of PG, which is similar to the sugar component that produces inhibitory activity in chitosan, might be involved with this mechanism.23,24 Approximately more than half of PG is composed of 123
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(a)
1 µm
(b)
(c)
1 µm
1 µm
(d)
(e)
1 µm
galacturonic acid,9 which is an acid sugar producing carbonyl anionic charge. In addition, the polyanionic polymer of PG might effectively bind and form a polyelectrolyte complex with the cationic side-chain of lipopolysaccharides present on the bacterial cell surface, resulting in the interference and disturbance of cell wall or cell membrane permeability. Therefore, the normal function of bacteria was inhibited.25 This investigation suggested that the bactericidal effect of PG might involve the interaction between acid and neutral sugars in PG and the outer surface of bacterial cells. For the consumptive safety of PG, it has been demonstrated that there were no toxic effects in the acute and subchronic toxicity tests in mice and rats by oral feeding with PG.26,27 From our previous studies, PG resistance bacteria (Staphylococcus aureus and Escherichia coli) were not observed during 30 days, therefore, it would be advantageous to use PG for antibacterial applications.11 The result supports a benefit of PG for its application as a topical antibactericide. This in vitro experiment has limitations, as it is considered a static system compared to an in vivo one, which 124
1 µm
Figure 2. Scanning electron micrographs of Aggregatibacter actinomycetemcomitans polysaccharide gel (PG) for 5 min. (a) Incubation in culture broth medium as a negative control, (b) incubation in 0.1% chlorhexidine as a positive control, (c) incubation in 50 mg/mL PG, (d) incubation in 100 mg/mL PG, and (e) incubation in 150 mg/mL PG. Various-sized blebs (white arrows), irregularly shaped cells (black arrow), and disrupted cells (white arrowheads) were observed in bacteria treated either with 0.1% chlorhexidine or 50–150 mg/mL PG.
might reflect the influence of various dynamic factors, such as systemic conditions, salivary flow, diet, and dental anatomy. One way of circumventing the limitations of in vitro studies evaluating the antibacterial activity of PG against microorganisms of the dental biofilm is to make reference to animal and clinical studies assessing the efficacy of PG. In conclusion, the bactericidal test showed that PG, at a concentration of 150 mg/mL, completely inactivated S. mutans and A. actinomycetemcomitans only at 1 min. The effectiveness of PG against oral pathogenic bacteria strongly suggests that it could be employed as a natural antibacterial agent in oral hygiene products to control caries and periodontal disease. The improvement of PG properties and clinical application need to be further investigated. Acknowledgments This research project was supported by a grant from the Dental Research Fund, Faculty of Dentistry, Chulalongkorn University, Research University Fund, and The Thailand Research Fund (RGD 5020001), Thailand. The authors would like to thank staff at the Oral Biology ª 2010 Blackwell Publishing Asia Pty Ltd
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Center, Faculty of Dentistry, Chulalongkorn University for providing the research facilities. Special thanks to Mrs. Rujiporn Prateepasen, Scientific and Technological
References 1 Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986; 50: 353–80. 2 Slots J, Genco RJ. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res 1984; 63: 412–21. 3 Zambon JJ. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 1985; 12: 1–20. 4 Ja¨rvinen H, Tenovuo J, Huovinen P. In vitro susceptibility of Streptococcus mutans to chlorhexidine and six other antimicrobial agents. Antimicrob Agents Chemother 1993; 37: 1158–9. 5 Chen CP, Lin CC, Namba T. Screening of Taiwanese crude drugs for antibacterial activity against Streptococcus mutans. J Ethnopharmacol 1989; 27: 285–95. 6 Petronio MG, Mansi A, Gallinelli C, Pisani S, Seganti L, Chiarini F. In vitro effect of natural and semisynthetic carbohydrate polymers on Chlamydia trachomatisinfection. Chemotherapy 1997; 43: 211–7. 7 Song JW, Ghim HD, Choi JH, Ko SW, Lyoo WS. Preparation of antimicrobial sodium alginate with chito-oligipolysaccharide side chain. J Polym Sci 2001; 39: 1810–6. 8 Jung BO, Kim CH, Choi CH, Lee YM, Kim JJ. Preparation of amphilic chitosan and their antimicrobial activities. J Appl Polym Sci 1999; 73: 1713–9. 9 Hokputsa S, Gerddit W, Pongsamart S et al. Water-soluble polysaccharides with pharmaceutical importance from Durian rinds (Durio zibethinus Murr.): isolation, fractionation, characterisation and bioactivity. Carbohydr Polym 2004; 56: 471–81.
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Research Equipment Centre, Chulalongkorn University, Bangkok, Thailand, for the sample preparation used for the scanning electron microscopy.
10 Chansiripornchai N, Chansiripornchai P, Pongsamart S. A preliminary study of polysaccharide gel extracted from the fruit hulls of durian (Durio zibethinus Murr.) on immune responses and cholesterol reduction in chicken. Acta Hortic 2008; 786: 57–60. 11 Pongsamart S, Nanatawanit N, Lertchaipon J, Lipipun V. Novel water soluble antibacterial dressing of durian polysaccharide gel. Acta Hortic 2005; 678: 65–73. 12 Chansiripornchai P, Pongsamart S, Nakchat O, Pramatwinai C, Ransipipat A. The efficacy of polysaccharide gel extracted from fruit-hulls of durian (Durio zibethinus L.) for wound healing in pig skin. Acta Hortic 2005; 679: 37– 43. 13 Chansiripornchai P, Pongsamart S. Treatment of infected open wounds on two dogs using a film dressing of polysaccharide gel extracted from the hulls of durian (Durio zibethinus Murr.): case report. Thai J Vet Med 2008; 38: 55–61. 14 Lipipun V, Nantawanit N, Pongsamart S. Antimicrobial activity (in vitro) of polysaccharide gel from durian fruit-hulls. Songklanakarin J Sci Technol 2002; 24: 31–8. 15 Maktrirat R, Pongsamart S, Ajariyakhajorn K, Chansiripornchai P. Bactericidal effect of post-milking teat dip prepared from polysaccharide gel from durian rinds on streptococci causing clinical bovine mastitis. Acta Hortic 2008; 796: 33–9. 16 Pholdaeng K, Pongsamart S. Studies on the immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus monodon shrimp against Vibrio harveyi and WSSV. Fish Shellfish Immunol 2010; 28: 555–61. 17 Musikapong P, Thunyakitpisal P, Pongsamart S. Antimicrobial activity of polysaccharide gel from
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durian fruit-halls against Streptococcus mutans and Actinobacillus actinomycetemcomitans. CU Dent J 2005; 28: 137–44. Chung JY, Choo JH, Lee MH, Hwang JK. Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans. Phytomedicine 2006; 13: 261– 6. Emilson CG. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res 1994; 73: 682–91. Pongsamart S, Panmuang T. Isolation of polysaccharides from fruit-hulls of durian (Durio zibethinus L.). Songklanakarin J Sci Technol 1998; 20: 323– 32. Sharon N, Lis H. Carbohydrates in cell recognition. Sci Am 1993; 268: 82–9. McKane L, Kandel J. Microbiology: essentials and applications, 2nd edn. New York: McGraw-Hill, 1996: 76–85. Muzzarelli R, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo PE. Antimicrobial properties of N-carboxybutyl chitosan. Antimicrob Agents Chemother 1990; 34: 2019–23. Tsai GJ, Su WH. Antibacterial activity of shrimp chitosan against Escherichia coli. J Food Prot 1999; 62: 239–43. Neu TR, Dengler T, Jann B, Poralla K. Structural studies of an emulsionstabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain. J Gen Microbiol 1992; 138: 2531–7. Pongsamart S, Sukrong S, Tawatsin A. The determination of toxic effects at a high oral dose of polysaccharide gel extracts from fruit-hulls of durian (Durio zibethinus L.) in mice and rats. Songklanakarin J Sci Technol 2001; 23: 56–62. Pongsamart S, Tawatsin A, Sukrong S. Long-term-consumption of polysaccharide gel from durian fruit-hull in mice. Songklanakarin J Sci Technol 2002; 24: 649–61.
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ORIGINAL ARTICLE Oral Microbiology
Antibacterial activity of polysaccharide gel extract from fruit rinds of Durio zibethinus Murr. against oral pathogenic bacteria Pasutha Thunyakipisal1, Thatsanee Saladyanant1, Naulchavee Hongprasong2, Sunanta Pongsamart3 & Wandee Apinhasmit1 1 Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 2 Department of Periodontology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 3 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
Keywords Aggregatibacter actinomycetemcomitans, antibacterial activity, durian fruit rind, polysaccharide gel extract, Streptococcus mutans. Correspondence Dr Pasutha Thunyakipisal, Dental Biomaterial Sciences Program, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Bangkok 10330, Thailand. Tel: +66-2218-8885 Fax: +66-2218-8870 Email: [email protected] Received 22 February 2010; accepted 23 May 2010. doi: 10.1111/j.2041-1626.2010.00017.x
Abstract Aim: The polysaccharide gel (PG) extract from durian fruit rinds (Durio zibethinus Murr. ‘‘Monthong’’) is a pectic polysaccharide with antibacterial activity. This study aimed to investigate the in vitro antibacterial activity of PG against oral pathogens, Streptococcus mutans (S. mutans) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Methods: The inhibitory activity of PG at 50, 100, and 150 mg/mL against S. mutans (American Tissue Culture Collection 25175) and A. actinomycetemcomitans (American Tissue Culture Collection 43718) was determined after 1- and 5-min exposure by broth macrodilution susceptibility test and scanning electron microscopy. Normal saline or culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. Results: For 1-min exposure, 150 mg/mL PG or 0.1% chlorhexidine significantly possessed bactericidal activity against both tested bacteria (P = 0.037), while PG at 100 mg/mL possessed significant bactericidal activity against S. mutans (P = 0.037) and inhibitory activity against A. actinomycetemcomitans (P = 0.05). Blebs, irregular-shaped cells, and disrupted cells were found in bacteria treated with either 0.1% chlorhexidine or 50–150 mg/mL PG under scanning electron microscopy. Conclusion: The bactericidal activity of PG at 150 mg/mL against oral bacteria at 1-min exposure suggests its possibility to be used as a natural antibacterial ingredient in oral hygiene products.
Introduction Dental caries and periodontitis are common oral diseases. Streptococcus mutans (S. mutans) has been strongly implicated as the principal etiologic agent in human dental caries,1 whereas Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a bacterium associated with periodontitis.2,3 There is great interest in the use of antibacterial agents for the prevention and treatment of dental caries and periodontitis. The prevention of dental caries and periodontitis requires the control of these 120
pathogens that are in an oral biofilm known as dental plaque. Many attempts have been made to eliminate S. mutans and A. actinomycetemcomitans from the oral flora. Antibacterial agents, such as chlorhexidine, amoxicillin, cefuroxime, penicillin, sulfamethoxazole–trimethoprim, tetracycline, and erythromycin, have been effective in preventing dental caries.4 However, excessive and prolonged use of these chemicals can result in rearrangements of the oral and intestinal flora and cause undesirable side-effects, such as microorganism susceptibility, vomiting, diarrhea, and tooth staining.5 Therefore, ª 2010 Blackwell Publishing Asia Pty Ltd
P. Thunyakipisal et al.
it is important to develop alternative antibacterial agents from natural higher plants in a wide range of oral care products, such as toothpaste and mouthwashes. These natural agents have antibacterial activity to prevent dental caries and periodontitis and possess very low or no sideeffects. Some natural polysaccharides have antimicrobial properties, such as pectin,6 alginate,7 and chitosan.8 Recently, we isolated, purified, and characterized a novel natural polysaccharide from the fruit rinds of durian (Durio zibethinus Murr. of the Bombacaceae family) called ‘‘polysaccharide gel’’ (PG). Durian is the most consumed fruit in Thailand; it weighs approximately 2–4.5 kg and has large, sharp, spiny thorns. Following injury from harvesting, harvesters have been told to apply clear viscous exudates from the fruit rind or the cut-end of fruit stalk on the injured area in order to prevent infections and pus. PG is a pectic polysaccharide with immunomodulating activity.9,10 The PG extract from durian rinds has also been investigated for its antibacterial activity against a variety of bacteria.11 Wound dressing prepared from PG exhibits good activity for healing open wounds in the skins of pigs and dogs.12,13 PG has water-gelling property, contains a sugar component similar to those of high polymeric carbohydrates, and comprises two linear polygalacturonic acid chains, one of which is a neutral chain with side-chains of neutral sugars rich in galactose, rhamnose, and glucose in an area called the ‘‘hairy region’’; the other is an acidic polygalacturonan chain with lesser side-chains than the neutral chain.9 PG is an interesting substance for the development of a new antibacterial agent. It could possibly have the advantage of being a new type of water-soluble antibacterial agent and gives satisfactory results when utilized as an antibacterial polysaccharide. The in vitro antibacterial activity of PG against a variety of bacteria has been reported.11,14–16 Additionally, our previous study indicated that the minimum bactericidal concentration of PG against S. mutans and A. actinomycetemcomitans was 35 mg/mL within 24 h.17 However, this concentration of PG might take a longer time if used in oral hygiene products, such as toothpastes or mouth rinses against cariogenic and/or periodontopathogenic bacteria in humans, since toothbrushing or oral rinsing usually only takes a few minutes.18 Therefore, this investigation aimed at evaluating the in vitro antibacterial activity of the PG extract from durian fruit rinds against S. mutans and A. actinomycetemcomitas at 1 and 5 min in order to relate it to clinical applications in patients. Chlorhexidine gluconate at a 0.1% concentration, an effective antimicrobial chemical agent that suppresses S. mutans in dental plaque and saliva,19 was used as a positive control. ª 2010 Blackwell Publishing Asia Pty Ltd
Durian fruit rind antibacterial activity
Materials and methods Extraction of polysaccharides The PG was isolated from the dried fruit rinds of durian (Durio zibethinus Murr. ‘‘Monthong’’). Freshly ground fruit rinds were dried in a hot air oven at 60C to obtain 19.6 ± 1.4% w/w dried material. The dried sample was extracted with hot water at 90–100C and adjusted to pH 4.5 with citric acid; the extract was filtered and purified, as described previously.20 Briefly, the water extract was concentrated under reduced pressure and added in the three volumes of acidified aqueous ethanol (4% HCl in 75% ethanol) to obtain the gel-like precipitate. The precipitate was collected by filtration, and then dried and pulverized. The dried precipitate was redissolved in water, filtered, and concentrated. To concentrate the solution, aqueous ethanol was added and the precipitate was recovered. The precipitate was washed twice with 75% ethanol and then 95% ethanol, and afterwards, dried, ground, and sieved to obtain a PG powder. Bacterial strains and culture conditions Bacterial strains, S. mutans (American Tissue Culture Collection [ATCC] 25175) and A. actinomycetemcomitans (ATCC 43718), were used in this study. Streptococcus mutans was cultured in trypticase soy broth (Difco; Becton Dickinson, Claix, France), and A. actinomycetemcomitans was cultured in Brain Heart Infusion (Difco; Becton Dickinson). All bacteria were incubated at 37C in a 5% CO2 atmosphere. To prepare the bacterial cell suspension for the antibacterial activity assay, bacterial cells were collected and resuspended with the sterilized 0.9% NaCl2. The turbidity was adjusted equivalent to a 0.5 McFarland standard and adjusted to a concentration of 1 · 106 CFU/mL before use. Broth macrodilution susceptibility test The antibacterial assay was determined by the number of surviving bacteria at 1- and 5-min exposure to PG (50, 100, and 150 mg/mL). PG was dispersed in distilled water and sterilized in an autoclave. The 0.5 mL of designated concentrations of PG were incubated with an equal volume of bacterial suspension (1 · 106 CFU/mL) obtained during the logarithmic phase of growth. After incubation for 1 or 5 min, the reaction was diluted by 10-fold serial dilution. The aliquot was subsequently spread onto a trypticase soy agar plate for S. mutans or brain heart infusion agar plate for A. actinomycetemcomitans. The plate was incubated for 48 h in an incubator with 5% CO2 at 37C. In this study, sterilized normal saline and 0.1% chlorhexidine were used as negative and positive controls, respectively. The experiment was repeated three times for each sample. 121
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Scanning electron microscopic evaluation Both bacteria were treated with PG (50, 100, and 150 mg/mL) at 37C for 1 and 5 min, washed with normal saline, and filtered through a polycarbonate sheet with a pore size of 0.4 lm. Bacteria on the polycarbonate sheets were then fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer (pH 7.4) at 4C for 24 h. After dehydration with graded ethanol, the samples were critically point dried with CO2, mounted on specimen stubs, and sputter coated with gold particles. Scanning electron microscopic examination was performed at 15-kV emission voltage and a sample tilt angle at 0 on a Jeol JSM-5410 (Jeol, Tokyo, Japan). The culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. Statistical analysis All statistical computations were performed by spss software (version 11.5; SPSS, Chicago, IL, USA). Data from the time-kill analysis were presented as the median, maximum, and minimum of the numbers of surviving bacteria (log10 CFU/mL). Kruskal–Wallis tests examined differences in viable bacterial counts among treated and control groups, followed by post hoc Mann–Whitney U-test. The chosen level of significance was P < 0.05. Results Broth macrodilution susceptibility test The results showed that the antibacterial activity of the PG, represented by the number of viable bacteria S. mutans and A. actinomycetemcomitans (in CFU/mL) depended on its concentration and the exposure duration (Table 1). At 1 min, PG at 150 mg/mL or 0.1% chlorhexidine possessed the bactericidal activity against both S. mutans and A. actinomycetemcomitans (P = 0.037), whereas
PG at 100 mg/mL possessed the bactericidal activity against S. mutans (P = 0.037) and exhibited slightly inhibitory activity against A. actinomycetemcomitans (P = 0.05). PG at 100 mg/mL possessed bactericidal activity against both S. mutans and A. actinomycetemcomitans after 5-min exposure to PG (P = 0.037). The exposure to PG at 50 mg/mL for 1 and 5 min decreased the number of S. mutans and A. actinomycetemcomitans, but they were not different from the negative control (P > 0.05). Scanning electron microscopic evaluation To investigate the effect of PG on the bacterial membrane, S. mutans and A. actinomycetemcomitans were incubated in the culture broth medium as negative controls, 0.1% chlorhexidine as the positive control, and PG at concentrations of 50–150 mg/mL for 1- or 5-min incubation. The results are shown in Figures 1 and 2. As a negative control, S. mutans incubated in the culture broth medium for either 1 or 5 min exhibited a round shape (cocci) with a smooth surface (Figure 1a). The surface alterations of S. mutans after 1- or 5-min incubation in 0.1% chlorhexidine showed similar results, but the longer time exhibited a higher degree of surface alterations. Various-sized blebs were found on bacterial cell surfaces (Figures 1b and 1c). In addition, irregular-shaped cells and collapsed cells were observed (Figure 1c). The pattern of surface changes of S. mutans treated in 50–150 mg/mL PG was similar to that in 0.1% chlorhexidine (Figures 1c, 1d and 1f), but the degree of changes depended on its concentration and the exposure duration. The surface appearance of A. actinomycetemcomitans incubated in the culture broth medium for 1 and 5 min as the negative control groups were shaped as short with a smooth surface (Figure 2a). The surface alterations of A. actinomycetemcomitans treated in 50–150 mg/mL PG and 0.1% chlorhexidine showed a similar pattern. The
Table 1. Effects of polysaccharide gel (PG) on bacterial number after exposure for 1 and 5 min No. surviving bacteria (log CFU/mL)
Bacteria Streptococcus mutans Aggregatibacter actinomycetemcomitans
Incubation time (min) 1 5 1 5
Negative control
50 mg/mL PG
Med
Max
Min
Med
Max
Min
Med
Max
6.05 6.00 6.01 6.18
6.22 6.27 6.20 6.70
5.87 5.83 5.85 5.85
5.57 4.98 6.01 5.71
5.65 5.12 6.08 5.94
5.47 4.85 5.91 5.34
0* 0* 2.53** 0*,**
0 0 2.90 0
150 mg/mL PG
0.1% chlorhexidine
Min
Med
Max
Min
Med
Max
Min
0 0 2.00 0
0* 0* 0* 0*
0 0 0 0
0 0 0 0
0* 0* 0* 0*
0 0 0 0
0 0 0 0
100 mg/mL PG
Data expressed as median (Med), maximum (Max), and minimum (Min) of the number of surviving bacteria (log CFU/mL). Sterilized normal saline and 0.1% chlorhexidine were used as negative and positive controls, respectively. *Compared with control groups (P = 0.037); **statistically significant difference between groups (P = 0.037). Kruskal–Wallis test, followed by post hoc Mann–Whitney U-tests.
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(a)
(b)
1 µm (c)
1 µm (d)
1 µm Figure 1. Scanning electron micrographs of Streptococcus mutans incubated in polysaccharide gel (PG) for 5 min. (a) Incubation in culture broth medium as a negative control, (b,c) incubation in 0.1% chlorhexidine as a positive control, (d) incubation in 50 mg/mL PG, (e) incubation in 100 mg/mL PG, and (f) incubation in 150 mg/mL PG. Various-sized blebs (white arrows), irregularly shaped cells (black arrow), and disrupted cells (white arrowheads) were observed in bacteria treated either with 0.1% chlorhexidine or 50–150 mg/mL PG.
(e)
degree of changes caused by PG depended on the concentration of PG and the exposure duration. There were various-sized blebs on the cell surfaces, irregularshaped cells, and disrupted cells found in the bacteria treated with 0.1% chlorhexidine (Figure 2b) and 50– 150 mg/mL PG (Figures 2c, 2d and 2e). Discussion Previous reports showed that after 24-h incubation of bacteria in PG, the PG possessed antibacterial activity against both Gram-positive and -negative bacteria, such as Staphylococcus aureus and Escherichia coli.14 The bactericidal activity of PG has also been studied against Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Lactobacillus pentosus, Escherichia coli, Staphylococcus aureus, Proteus vulgaris,11 streptococci isolated from bovine mastitis (Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Streptococcus bovis, and Streptococcus acidominimus),15 Vibrio harveyi,16 as well as ª 2010 Blackwell Publishing Asia Pty Ltd
1 µm (f)
1 µm
1 µm
S. mutans and A. actinomycetemcomitans.17 For the practical application in daily oral care activity, such as toothbrushing and oral rinsing, we further investigated the minimum bactericidal concentration of PG against S. mutans and A. actinomycetemcomitans as the representative of cariogenic and periodontopathogenic bacteria within 1- to 5-min exposure. The result exhibited that the antibacterial activity of PG against S. mutans and A. actinomycetemcomitans responded in time- and dosedependent manner. Although the precise antibacterial mechanism of PG has not been elucidated, our scanning electron microscopic data suggested that PG has at least affected bacterial membrane integrity. The cell surfaces of microorganisms are commonly composed of molecules of polysaccharides, such as lipopolysaccharides, capsular polysaccharides, and cationic/anionic polymers.21,22 The monosaccharide compositions of PG, which is similar to the sugar component that produces inhibitory activity in chitosan, might be involved with this mechanism.23,24 Approximately more than half of PG is composed of 123
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(a)
1 µm
(b)
(c)
1 µm
1 µm
(d)
(e)
1 µm
galacturonic acid,9 which is an acid sugar producing carbonyl anionic charge. In addition, the polyanionic polymer of PG might effectively bind and form a polyelectrolyte complex with the cationic side-chain of lipopolysaccharides present on the bacterial cell surface, resulting in the interference and disturbance of cell wall or cell membrane permeability. Therefore, the normal function of bacteria was inhibited.25 This investigation suggested that the bactericidal effect of PG might involve the interaction between acid and neutral sugars in PG and the outer surface of bacterial cells. For the consumptive safety of PG, it has been demonstrated that there were no toxic effects in the acute and subchronic toxicity tests in mice and rats by oral feeding with PG.26,27 From our previous studies, PG resistance bacteria (Staphylococcus aureus and Escherichia coli) were not observed during 30 days, therefore, it would be advantageous to use PG for antibacterial applications.11 The result supports a benefit of PG for its application as a topical antibactericide. This in vitro experiment has limitations, as it is considered a static system compared to an in vivo one, which 124
1 µm
Figure 2. Scanning electron micrographs of Aggregatibacter actinomycetemcomitans polysaccharide gel (PG) for 5 min. (a) Incubation in culture broth medium as a negative control, (b) incubation in 0.1% chlorhexidine as a positive control, (c) incubation in 50 mg/mL PG, (d) incubation in 100 mg/mL PG, and (e) incubation in 150 mg/mL PG. Various-sized blebs (white arrows), irregularly shaped cells (black arrow), and disrupted cells (white arrowheads) were observed in bacteria treated either with 0.1% chlorhexidine or 50–150 mg/mL PG.
might reflect the influence of various dynamic factors, such as systemic conditions, salivary flow, diet, and dental anatomy. One way of circumventing the limitations of in vitro studies evaluating the antibacterial activity of PG against microorganisms of the dental biofilm is to make reference to animal and clinical studies assessing the efficacy of PG. In conclusion, the bactericidal test showed that PG, at a concentration of 150 mg/mL, completely inactivated S. mutans and A. actinomycetemcomitans only at 1 min. The effectiveness of PG against oral pathogenic bacteria strongly suggests that it could be employed as a natural antibacterial agent in oral hygiene products to control caries and periodontal disease. The improvement of PG properties and clinical application need to be further investigated. Acknowledgments This research project was supported by a grant from the Dental Research Fund, Faculty of Dentistry, Chulalongkorn University, Research University Fund, and The Thailand Research Fund (RGD 5020001), Thailand. The authors would like to thank staff at the Oral Biology ª 2010 Blackwell Publishing Asia Pty Ltd
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Center, Faculty of Dentistry, Chulalongkorn University for providing the research facilities. Special thanks to Mrs. Rujiporn Prateepasen, Scientific and Technological
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