[학위논문]Streptococcus mutans 와 Streptococcus sobrinus의 활성과 충치관련 독성요인에 대한 호장근의 억제효과 In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus원문보기
동의보감에 따르면 치과질환 예방 및 처치를 위해 호장근을 전통적으로 사용해왔다. 그러나 치의학 영역에서의 호장근에 대한 연구는 찾아보기 어렵다. 본 연구는 충치관련세균인 Streptococcus mutans 및 Streptococcus sobrinus에 대한 호장근 ...
동의보감에 따르면 치과질환 예방 및 처치를 위해 호장근을 전통적으로 사용해왔다. 그러나 치의학 영역에서의 호장근에 대한 연구는 찾아보기 어렵다. 본 연구는 충치관련세균인 Streptococcus mutans 및 Streptococcus sobrinus에 대한 호장근 메탄올 추출물의 항균효과와 충치관련독성요인에 대한 억제 효과에 대한 연구를 시행하여 호장근의 충치예방약물로써의 가능성을 탐색하였다. 먼저, 호장근 메탄올 추출물의 구강세균의 생활성에 대한 영향을 연구하기 위하여 S. mutans 및 S. sobrinus 외 다수의 그람 양성균과 그람 음성균의 최소성장억제농도와(MIC) 최소세균사멸농도를(MBC) 측정하였다. 특히, 충치발생과 관련성이 가장 높다고 알려진 S. mutans 및 S. sobrinus의 경우에는 time-kill assay와 최소성장억제농도 미만의 농도에서 세균성장속도에 대한 약물의 효과를 측정하였다. S. mutans 및 S. sobrinus에 대한 호장근 메탄올 추출물의 충치관련독성요인에 대한 억제 효과에 대한 연구를 시행하기 위하여 최소성장억제농도 미만의 농도에서 자당-의존성 부착, water-insoluble glucan formation, glycolytic acid production, acid tolerance 관련 실험을 시행하였다. 실험결과 호장근 메탄올 추출물은 구강세균에 대하여 광범위 항균효과를(MIC 0.5-4 mg/ml) 보였으며 MIC 보다 2-4배 높은 농도에서 최소세균사멸농도는 보였다. S. mutans와 S. sobrinus는 MBC 또는 MIC 2배 높은 농도에서 1시간 배양 후 유의한 수로 감소하였다. S. mutans와 S. sobrinus는 최소성장억제농도 미만의 농도에서 doubling time이 농도 의존적으로 각각 211%와 123% 증가하였다. 또한, 최소성장억제농도 미만의 농도에서 S. mutans와 S. sobrinus의 충치관련 독성요인을 전체적으로 억제하였다. 호장근 메탄올 추출물의 성분을 분석하기 위하여 시약분석 방법 및 high-performance liquid chromatography를 시행하였으며, 호장근 메탄올 추출물은 alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates 등으로 구성되어 있으며 주성분은 emodin, resveratrol, anthraglycoside B, physcion, polydatin 등으로 확인되었다. 결론적으로 호장근 메탄올 추출물은 emodin, resveratrol, physcion, polydatin 등을 주성분으로 하며 고농도에서는 구강세균 특히 충치와 관련된 S. mutans와 S. sobrinus의 생활성을 억제할 수 있고, 저농도에서는(최소성장억제농도 미만의 농도) S. mutans와 S. sobrinus의 증식과 충치관련 독성요인을 억제할 수 있기 때문에 향후 충치 예방약물로서의 가능성을 보여주고 있다.
동의보감에 따르면 치과질환 예방 및 처치를 위해 호장근을 전통적으로 사용해왔다. 그러나 치의학 영역에서의 호장근에 대한 연구는 찾아보기 어렵다. 본 연구는 충치관련세균인 Streptococcus mutans 및 Streptococcus sobrinus에 대한 호장근 메탄올 추출물의 항균효과와 충치관련독성요인에 대한 억제 효과에 대한 연구를 시행하여 호장근의 충치예방약물로써의 가능성을 탐색하였다. 먼저, 호장근 메탄올 추출물의 구강세균의 생활성에 대한 영향을 연구하기 위하여 S. mutans 및 S. sobrinus 외 다수의 그람 양성균과 그람 음성균의 최소성장억제농도와(MIC) 최소세균사멸농도를(MBC) 측정하였다. 특히, 충치발생과 관련성이 가장 높다고 알려진 S. mutans 및 S. sobrinus의 경우에는 time-kill assay와 최소성장억제농도 미만의 농도에서 세균성장속도에 대한 약물의 효과를 측정하였다. S. mutans 및 S. sobrinus에 대한 호장근 메탄올 추출물의 충치관련독성요인에 대한 억제 효과에 대한 연구를 시행하기 위하여 최소성장억제농도 미만의 농도에서 자당-의존성 부착, water-insoluble glucan formation, glycolytic acid production, acid tolerance 관련 실험을 시행하였다. 실험결과 호장근 메탄올 추출물은 구강세균에 대하여 광범위 항균효과를(MIC 0.5-4 mg/ml) 보였으며 MIC 보다 2-4배 높은 농도에서 최소세균사멸농도는 보였다. S. mutans와 S. sobrinus는 MBC 또는 MIC 2배 높은 농도에서 1시간 배양 후 유의한 수로 감소하였다. S. mutans와 S. sobrinus는 최소성장억제농도 미만의 농도에서 doubling time이 농도 의존적으로 각각 211%와 123% 증가하였다. 또한, 최소성장억제농도 미만의 농도에서 S. mutans와 S. sobrinus의 충치관련 독성요인을 전체적으로 억제하였다. 호장근 메탄올 추출물의 성분을 분석하기 위하여 시약분석 방법 및 high-performance liquid chromatography를 시행하였으며, 호장근 메탄올 추출물은 alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates 등으로 구성되어 있으며 주성분은 emodin, resveratrol, anthraglycoside B, physcion, polydatin 등으로 확인되었다. 결론적으로 호장근 메탄올 추출물은 emodin, resveratrol, physcion, polydatin 등을 주성분으로 하며 고농도에서는 구강세균 특히 충치와 관련된 S. mutans와 S. sobrinus의 생활성을 억제할 수 있고, 저농도에서는(최소성장억제농도 미만의 농도) S. mutans와 S. sobrinus의 증식과 충치관련 독성요인을 억제할 수 있기 때문에 향후 충치 예방약물로서의 가능성을 보여주고 있다.
Dental caries is one of the most common oral diseases worldwide.32 A widely adopted approach to reduce the incidence of the disease is the application of chemoprophylactic agents. These agents have attracted considerable attention and have played a crucial role in patient-directed approaches for pla...
Dental caries is one of the most common oral diseases worldwide.32 A widely adopted approach to reduce the incidence of the disease is the application of chemoprophylactic agents. These agents have attracted considerable attention and have played a crucial role in patient-directed approaches for plaque control.8,33 However, some of these agents, e.g. chlorhexidine and antibiotics, have undesirable side effects including tooth staining and the emergence of bacterial resistance. Recently, medicinal plants have shown promise as an alternative to chemoprophylactic agents for preventing dental caries. In this context, we have focused on P. cuspidatum, which has been used in traditional medicine for the control of dental diseases. Although the root of P. cuspidatum has been studied extensively for its biological activity,34,35 according to our knowledge, this is the first report on inhibitory effects of this plant against bacterial viability and the virulence factors of S. mutans and S. sobrinus. There has been continual controversy regarding whether a specific bacterial species or a non-specific mixture of bacteria is the agent responsible for the development of dental caries. Because many non-mutans bacteria are sufficiently acidogenic, acid tolerate, and numerous to produce the amount of acid needed for the development of dental caries,36,37 some researchers have accepted that mutans streptococci cannot, except in a few specific cases, be the bacterial cause of dental caries.37,38 However, the present study was based on the hypothesis that mutans streptococci are the principal cariogenic organisms, because previous epidemiological and immunological studies have provided evidence for the association of mutans streptococci with dental caries in humans.39 Among mutans streptococci, S. mutans is the dominant human type of mutans streptococci and overwhelmingly associated with human dental caries.2 However, it has been also reported that S. sobrinus is more acidogenic and cariogenic in animals than S. mutans.40,41 In addition, several epidemiological studies have shown that the presence of S. sobrinus is associated with high caries prevalence.42,43 Therefore, S. mutans and S. sobrinus were chosen as the main test bacteria. Lactobacilli and three Gram-negative bacteria, such as A. actinomycetemcomitans, were also selected because of their association with dental caries and periodontal disease, respectively. As shown in Figure 6, Anthraquinones (physcion, emodin), stilbenes (Resveratrol) and other phenolic compounds have been identified from methanolic extract of the roots of P. cuspidatum. Emodin is a predominant anthraquinone compound isolated from P. cuspidatum. A previous study showed that emodin demonstrated a chemopreventive effect on skin carcinogenesis.44 Jayasuriya et al.45 also reported that emodin is a potent tyrosinase inhibitor. Resveratrol is a phenolic compound found in many families of plants such as peanuts, grapes, wine, and Polygonum cuspidatum.20 The content of resveratrol in Polygonum cuspidatum was much higher than in grape and other plants.46 It was reported that resveratrol has many protecting properties to human health such as an antioxidant, modulator of lipoprotein metabolism, inhibitor of platelet aggregation, and vasorelaxing agent. The precise bioactive compounds of MEP that mediate the inhibitory effects against the viability of S. mutans and S. sobrinus are unknown. Based on the preliminary phytochemical analysis in the present study, the activity of MEP might be related to the presence of alkaloids, phenolics, and sterol/terpenes. Some studies reported that plant phenolics have a wide range of pharmacological activity such as antibacterial activity.47,48 According to Cowan,49 the mechanisms believed to be responsible for the phenolic antibacterial activity include enzyme inhibition by the oxidized compounds, possibly through a reaction with sulfhydryl groups or through more non-specific interactions with the proteins. It was also reported that alkaloids and terpenes are active against bacteria.50,51 The mechanism responsible for the action of alkaloids and terpenoids is not fully understood but is speculated to involve the ability of alkaloids to intercalate with DNA and membrane disruption by the lipophilic compounds.49 In the present study, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus. Because the assays for the inhibitory effects on the virulence factors were carried out at sub-MIC levels, the false positive results due to the bacteriostatic and bactericidal activity of MEP could be eliminated. Nevertheless, the inhibitory effects of MEP on the sucrose-dependent adherence could result from a decrease in the bacterial growth rate (Table 2). Since the doubling time of S. mutans and S. sobrinus was reduced by MEP, the total number of the bacteria that could adhere to the glass surfaces in the presence of MEP after 18 h incubation was lower than that of the controls. The inhibition of adherence could also related to the GTF inhibitory activity of MEP, whereby MEP prevents the synthesis of water-insoluble glucans. It is well known that GTFs of S. mutans and S. sobrinus produce water-insoluble glucans from sucrose to promote adherence to the teeth,3-5 which contributes to the formation of dental plaque. Water-insoluble glucans are mainly produced by CA-GTF of S. mutans and CF-GTF of S. sobrinus.27,52 However, because several studies have shown that GTFs of the bacteria are present in cell-associated or/and cell-free forms and most water-insoluble glucans are synthesized by CF-GTFs,26,53 we examined CA-GTFs and CF-GTFs at the same time. Our data suggest that MEP is effective in the prevention of water-insoluble glucan formation by both CF-GTFs and CA-GTFs (Fig. 3A and B). The possible biological active components of MEP that modulate the inhibition of GTF activity are unknown. Because plant polyphenols have the ability to bind strongly to proteins54 and a non-competitive inhibition effect on GTF activity,55 the GTF inhibition activity may result from tannin and flavonoid content of MEP. Acid production and acid tolerance are other important caries-inducing factors of both S. mutans and S. sobrinus that deserve attention during an examination of medicinal plants for the prevention of dental caries. MEP reduced the production of glycolytic acid by S. mutans and S. sobrinus. This inhibitory activity might result from effects on the bacterial glycolytic pathways, because resting bacteria were used for this assay and some plant secondary metabolites are known to influence the permeability of natural and synthetic membranes and inhibit enzymes.56 MEP also inhibited the acid tolerance. Although the mechanism associated with the inhibition of acid tolerance was not revealed in the present study, it is possible to suppose that flavonoids and tannins in MEP, which can bind or form precipitates with various proteins,50 affect the ability of S. mutans to remove protons from the cytoplasm by the action of a membrane-bound enzyme, the proton-translocating ATPase or F-ATPase. In conclusion, the results of the present study showed that MEP has bacteriostatic or bactericidal activity at higher concentrations (≥MIC) and inhibitory effects on the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for control of dental plaque formation and subsequent dental caries formation. However, more biochemical and phytochemical investigations will be needed to isolate the active components. Furthermore, more study will be needed to determine if the isolated anticariogenic components express their effects in in vivo models as well as in biofilm models.
Dental caries is one of the most common oral diseases worldwide.32 A widely adopted approach to reduce the incidence of the disease is the application of chemoprophylactic agents. These agents have attracted considerable attention and have played a crucial role in patient-directed approaches for plaque control.8,33 However, some of these agents, e.g. chlorhexidine and antibiotics, have undesirable side effects including tooth staining and the emergence of bacterial resistance. Recently, medicinal plants have shown promise as an alternative to chemoprophylactic agents for preventing dental caries. In this context, we have focused on P. cuspidatum, which has been used in traditional medicine for the control of dental diseases. Although the root of P. cuspidatum has been studied extensively for its biological activity,34,35 according to our knowledge, this is the first report on inhibitory effects of this plant against bacterial viability and the virulence factors of S. mutans and S. sobrinus. There has been continual controversy regarding whether a specific bacterial species or a non-specific mixture of bacteria is the agent responsible for the development of dental caries. Because many non-mutans bacteria are sufficiently acidogenic, acid tolerate, and numerous to produce the amount of acid needed for the development of dental caries,36,37 some researchers have accepted that mutans streptococci cannot, except in a few specific cases, be the bacterial cause of dental caries.37,38 However, the present study was based on the hypothesis that mutans streptococci are the principal cariogenic organisms, because previous epidemiological and immunological studies have provided evidence for the association of mutans streptococci with dental caries in humans.39 Among mutans streptococci, S. mutans is the dominant human type of mutans streptococci and overwhelmingly associated with human dental caries.2 However, it has been also reported that S. sobrinus is more acidogenic and cariogenic in animals than S. mutans.40,41 In addition, several epidemiological studies have shown that the presence of S. sobrinus is associated with high caries prevalence.42,43 Therefore, S. mutans and S. sobrinus were chosen as the main test bacteria. Lactobacilli and three Gram-negative bacteria, such as A. actinomycetemcomitans, were also selected because of their association with dental caries and periodontal disease, respectively. As shown in Figure 6, Anthraquinones (physcion, emodin), stilbenes (Resveratrol) and other phenolic compounds have been identified from methanolic extract of the roots of P. cuspidatum. Emodin is a predominant anthraquinone compound isolated from P. cuspidatum. A previous study showed that emodin demonstrated a chemopreventive effect on skin carcinogenesis.44 Jayasuriya et al.45 also reported that emodin is a potent tyrosinase inhibitor. Resveratrol is a phenolic compound found in many families of plants such as peanuts, grapes, wine, and Polygonum cuspidatum.20 The content of resveratrol in Polygonum cuspidatum was much higher than in grape and other plants.46 It was reported that resveratrol has many protecting properties to human health such as an antioxidant, modulator of lipoprotein metabolism, inhibitor of platelet aggregation, and vasorelaxing agent. The precise bioactive compounds of MEP that mediate the inhibitory effects against the viability of S. mutans and S. sobrinus are unknown. Based on the preliminary phytochemical analysis in the present study, the activity of MEP might be related to the presence of alkaloids, phenolics, and sterol/terpenes. Some studies reported that plant phenolics have a wide range of pharmacological activity such as antibacterial activity.47,48 According to Cowan,49 the mechanisms believed to be responsible for the phenolic antibacterial activity include enzyme inhibition by the oxidized compounds, possibly through a reaction with sulfhydryl groups or through more non-specific interactions with the proteins. It was also reported that alkaloids and terpenes are active against bacteria.50,51 The mechanism responsible for the action of alkaloids and terpenoids is not fully understood but is speculated to involve the ability of alkaloids to intercalate with DNA and membrane disruption by the lipophilic compounds.49 In the present study, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus. Because the assays for the inhibitory effects on the virulence factors were carried out at sub-MIC levels, the false positive results due to the bacteriostatic and bactericidal activity of MEP could be eliminated. Nevertheless, the inhibitory effects of MEP on the sucrose-dependent adherence could result from a decrease in the bacterial growth rate (Table 2). Since the doubling time of S. mutans and S. sobrinus was reduced by MEP, the total number of the bacteria that could adhere to the glass surfaces in the presence of MEP after 18 h incubation was lower than that of the controls. The inhibition of adherence could also related to the GTF inhibitory activity of MEP, whereby MEP prevents the synthesis of water-insoluble glucans. It is well known that GTFs of S. mutans and S. sobrinus produce water-insoluble glucans from sucrose to promote adherence to the teeth,3-5 which contributes to the formation of dental plaque. Water-insoluble glucans are mainly produced by CA-GTF of S. mutans and CF-GTF of S. sobrinus.27,52 However, because several studies have shown that GTFs of the bacteria are present in cell-associated or/and cell-free forms and most water-insoluble glucans are synthesized by CF-GTFs,26,53 we examined CA-GTFs and CF-GTFs at the same time. Our data suggest that MEP is effective in the prevention of water-insoluble glucan formation by both CF-GTFs and CA-GTFs (Fig. 3A and B). The possible biological active components of MEP that modulate the inhibition of GTF activity are unknown. Because plant polyphenols have the ability to bind strongly to proteins54 and a non-competitive inhibition effect on GTF activity,55 the GTF inhibition activity may result from tannin and flavonoid content of MEP. Acid production and acid tolerance are other important caries-inducing factors of both S. mutans and S. sobrinus that deserve attention during an examination of medicinal plants for the prevention of dental caries. MEP reduced the production of glycolytic acid by S. mutans and S. sobrinus. This inhibitory activity might result from effects on the bacterial glycolytic pathways, because resting bacteria were used for this assay and some plant secondary metabolites are known to influence the permeability of natural and synthetic membranes and inhibit enzymes.56 MEP also inhibited the acid tolerance. Although the mechanism associated with the inhibition of acid tolerance was not revealed in the present study, it is possible to suppose that flavonoids and tannins in MEP, which can bind or form precipitates with various proteins,50 affect the ability of S. mutans to remove protons from the cytoplasm by the action of a membrane-bound enzyme, the proton-translocating ATPase or F-ATPase. In conclusion, the results of the present study showed that MEP has bacteriostatic or bactericidal activity at higher concentrations (≥MIC) and inhibitory effects on the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for control of dental plaque formation and subsequent dental caries formation. However, more biochemical and phytochemical investigations will be needed to isolate the active components. Furthermore, more study will be needed to determine if the isolated anticariogenic components express their effects in in vivo models as well as in biofilm models.
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