The Evaluation of Chinese Herbal Medicine Effectiveness on Periodontal Pathogens

Transcription

1 The American Journal of Chinese Medicine, Vol. 31, No. 5, World Scientific Publishing Company & Institute for Advanced Research in Asian Science and Medicine The Evaluation of Chinese Herbal Medicine Effectiveness on Periodontal Pathogens You Chan, * Chern-Hsiung Lai, Hui-Wen Yang, Yuh-Yie Lin and Chi-Ho Chan * * Department of Microbiology and Immunology and Department of Oral Medicine Chung Shan Medical University, Taiwan, R.O.C. Department of Periodontology, School of Dental Medicine University of Pennsylvania, USA Abstract: The aim of this study was to evaluate the effects of herbal medicines in treating periodontal diseases. Three Chinese herbal composites [Conth Su (CS), Chi Tong Ning (CTN) and Xi Gua Shuang (XGS)], widely used for prevention and treatment of periodontal diseases, and the major components of these composites were tested for their ability to: (1) alleviate disease progression of experimental periodontitis in hamsters, (2) inhibit bacterial growth, and (3) induce mutations. Our results indicate that in treating experimental periodontitis, there were no significant differences between the animal groups with or without the use of Chinese herbal medicines in terms of the degree of inflammation, alveolar bone resorption, and rate of repair. However, hamsters treated with CS presented earlier regenerative epithelium. CTN demonstrated superior bacterial inhibition ability among all tested herbs (MIC g/ml); CS showed good anti-bacterial abilities at a concentration of 0.05 g/ml. It is interesting to note that while both CS and CTN were capable of inhibiting bacterial growth, none of the individual herb components showed comparable bacterial inhibition abilities. None of the tested herbal composites or their components showed signs of inducing cell mutations using the Ames test. These results indicated that traditional Chinese herbal medicines, which have been used to treat periodontal diseases for hundreds of years by Chinese people, can effectively inhibit bacterial growth without causing cell mutation. Further investigation into their possible clinical applications in periodontal therapy is encouraged. Keywords: Chinese Herbal Medicine; Conth Su; Chi Tong Ning; Xi Gua Shuang; Periodontal Therapy. Correspondence to: Dr. Chi-Ho Chan, Department of Microbiology and Immunology, Chung Shan Medical University, 110, 1st Section, Chien Kuo N. Road, Taichung, Taiwan, R.O.C. Tel: (+886) (ext. 1633), Fax: (+886) , chiho@csmu.edu.tw 751

2 752 Y. CHAN et al. Introduction Periodontal disease is one of the most prevalent diseases. Evidence of periodontal destruction has been found in humans living thousands of years ago (Slots, 1986). Before the development of modern periodontal therapy, our ancestors relied upon personal experience and natural medicine to treat the disease (Cao and Sun, 1998). Chinese people, through the teachings of their history, adopted various herbal medicines that were said to be effective or at least mitigatory in treating periodontal lesions. Periodontal diseases are characterized by loss of gingival and periodontal connective tissue attachment (Saglie et al., 1983). The tissue destruction in the periodontium caused by prolonged inflammation is initiated by bacterial colonization and invasion around teeth near the bottom of the periodontal pocket. Herbal composites may be effective in combatting the bacteria or mobilizing host cells in setting a good defense against these bacteria or accelerating the wound repair process (Nordland et al., 1978). Herbs have been used in treating periodontal diseases and reported by many studies (Parsons et al., 1987; Zhou et al., 1996; Song et al., 1997; Morimatsu et al., 1997). Some positive effects must exist to continue the use of these herbs. However, the lack of solid scientific evidence for Chinese herbal medicine effects in treating periodontal diseases is the major obstacle in proving ancestral Chinese wisdom (Savitt and Socransky, 1984). The aim of this study was to evaluate the influences of various locally applied herbal medicines on periodontal lesions and their effects on putative oral pathogenic bacterial growth and cell mutation. Three herbal medicines: Conth Sou (CS), Chi Tong Ning (CTN) and Xi Gua Shuang (XGS) were selected for this study. The function of CS is said to clear away stomachheat evil and wind-fire evil. The herbal action is mainly for cases involving a hot and foul breath, toothache, reddish and swelling of the gums, and ulcerative gingivitis. This herb is applicable to stomatitis and periodontitis because it prevents the formation of gum tartar and dental plaque. CTN is effective for improving the oral environment with a strong detoxification and swelling reduction. This herb is applicable to cases of chronic periodontal abscess and periodontitis and is especially good for the prevention and management of oral infections. XGS is indicated mainly for recurrent aphthus ulcers, tonsillitis, reddish and bleeding gums and tooth mobility due to flaming up of stomach-fire. These herbal composites are widely used by Chinese people for the prevention and treatment of periodontal diseases. In this study, three Chinese herbal medicines and their major components were tested for the abilities to: (1) alleviate the disease progress in an experimental periodontitis in hamsters, (2) inhibit bacterial growth, and (3) induce cell mutation. Materials and Methods Experimental Periodontitis Model Approximately eight-week-old male hamsters (National Laboratory Animal Breeding and Research Center) were fed with a high carbohydrate content diet (Diet 2000, USA). A sterile surgical stainless wire ligature was tied around the neck of the mandible right incisor teeth.

3 CHINESE HERBS AND PERIODONTITIS 753 The ligatures were left in the same position during the first 4 weeks and served as a retention device for oral microorganisms. At the end of that period, the ligatures were removed and experimental periodontitis had been successfully induced in all ligated teeth (American Dental Association, 1972). Five hamsters served as controls to evaluate the tissue repair from periodontitis. One animal in the control group was sacrificed on days 0 (the day that ligature was removed), 1, 2, 3 and 7, by intraperitoneal anesthesia. Labio-lingual gingival tissue section specimens were fixed with 4% formalin. After decalcification, dehydration and embedding in wax, the blocks were sectioned and stained with hematoxylin and eosin. Twenty-four hamsters, successfully induced with periodontitis were smeared daily with three different herbal composites over the periodontal lesions (CS, CTN and XGS for eight hamsters, respectively) (Table 1). Two animals were sacrificed on days 1, 2, 3 and 7 to retrieve periodontal tissue specimens. Another group of 24 hamsters were inoculated with a mixed culture of oral bacteria over the already existing experimental periodontitis lesions for two weeks. Chinese herb composites (CS, CTN or XGS) were then applied to these lesions (eight animals for each medicine). Tissue sections were made on days 1, 2, 3 and 7. Table 1. Three Chinese Herb Composites Tested in This Study and Their Components Herb Composites Components * Conth Sou (CS) (640 mg) Chi Tong Ning (CTN) (800 mg) Xi Gua Shuang (XGS) (1000 mg) Borneolum (30 mg) Cortex lycii radicis (240 mg) Rhizome cimicifugae (100 mg) Rosin (Kolophonia) (240 mg) Radix gentianae (30 mg) Borneolum (100 mg) Radix angelicae pahuricae (200 mg) Rhizoma cimicifugae (100 mg) Rizoma coptidis (200 mg) Piper longum (200 mg) Borneolum (30 mg) Exocarpium citrulli (700 mg) Radix scutellariae (100 mg) Rhizoma coptidis (100 mg) Radix sophorae tonkinensis (70 mg) *The powder extracts of CS, XGS, CTN and 11 individual herbal components were obtained from Sun Ten Pharmaceutical Co. Ltd., Taichung, Taiwan. The powder extract was standardized conforming to the Chinese Herbal Medicine Good Manufacturing Regulations imposed by the Committee on Chinese Medicine and Pharmacy, Department of Health, Taiwan to ensure strict quality control.

4 754 Y. CHAN et al. Bacteria Strains The bacterial strains used in this study included Actinobacillus actinomycetemcomitans ATCC 29522, Campylobacter rectus ATCC 33238, Centipeda periodontii ATCC35019, Eikenella corrodens ATCC 23834, Escherichia coli ATCC 53323, Fusobacterium nucleatum ATCC 25585, Porphyromonas gingivalis FDC 381, Porphyromonas endodontalis ATCC 35406, Prevotella intermedia ATCC 25611, Pseudomonas aeruginosa ATCC 10145, Selenomonas sputigena ATCC 33150, Staphylococcus aureus ATCC 9144, and Streptococcus sanguis ATCC Bacterial Growth Inhibition Herb composites or components were diluted in brucella blood agar plate (BBAP) (Merck, Germany). The final concentrations tested in this study were 0.2 g/ml, 0.1 g/ml, 0.05 g/ml and g/ml. All of the tested bacteria were maintained using weekly subculture on BBAP enriched with hemin and menadione, and incubated in an anaerobic workstation (Electrotek, West Yorks, UK) at 37 C (Chan and Chien, 1994). After an overnight culture in brain heart infusion (BHI) broth (Oxoid, USA), each tested bacteria was taken and quantified with a colorimeter (Vitek Hazelwood Mo., USA). The bacteria were then diluted in solution to an optical density of McFarland N0.1 at 405 nm. Five microliters of the adjusted bacterial suspension were spread onto the plates. After inoculation, the media were incubated anaerobically for 48 hours in 35 C. Bacterial growth was measured by spectrophotometer (Dynatech, USA) at 630 nm and compared with the negative control (media only) and positive control groups (bacteria were inoculated without herbal medicines or components). Ames Test The Ames test was performed as a plate incorporation assay on minimal agar plates according to Maron and Ames (1983). S. typhimurium (TA98) was used as a tester strain to detect mutations. Briefly, bacteria cultures were grown overnight in a cultural medium containing histidine and 0.2 g/ml herb composites (CS, CTN or XGS). Control bacteria were grown in a cultural medium containing histidine only. Bacteria were then subcultured onto basal cultural agar plates containing no histidine. Colony forming units (CFU) on each agar plate were compared to the control. A positive result was defined by herb-treated bacteria CFU greater than ten-fold as that of the control. Results Effects of Herb Application on Experimental Periodontitis Experimental periodontitis produced in hamsters from surgical stainless wire ligatures tied around the incisors for 28 days showed signs similar to human periodontitis with gingival swelling and redness, gingival atrophy, and bleeding on probing (Figs. 1B and E), when

5 CHINESE HERBS AND PERIODONTITIS 755 compared with that of the normal control group (Figs. 1A and D). Once the surgical stainless wire ligatures were removed from the teeth, the inflammation subsided fairly quickly, and 4 7 days after removal the gingival tissue regained near normal appearance. Figure 1. Gross and histological changes (hematoxylin and eosin stain 200) of gingival tissue of hamster. Figures A and D show normal gingival tissue. On the other hand, swelling, redness and atrophy (arrows in Fig. B), inflammatory infiltrate of lamina propria (Fig. E) are observed in the gingival tissue of a hamster after 4 weeks experimental periodontitis induction. Reduction of inflammation of gingival tissue is observed in the herb-treated hamster (Figs. C and F).

6 756 Y. CHAN et al. The same rapid repair was also found in the herb-treated groups after applying herb composites, CTN, CS or XGS, daily onto the inflammation sites (Figs. 1C and F). There were no significant differences in inflammatory cell infiltration and bone destruction between the herb- and sham-treated groups. Interestingly, regenerative gingival epithelium seemed to appear earlier in the CS-treated groups than in the control, indicating a possible positive effect in treating periodontitis. There seemed to be no differences in the histological examinations between CTN-, CS- and XGS- treated groups. Smearing mixed oral bacteria on existing periodontitis exacerbated the gingival conditions, clinically reflected by severe inflammation and deeper periodontal pockets and histologically by heavier inflammatory cell infiltration. It was noted that the periodontal pathological lesions can persist without ligature retention. We also found an earlier appearance of regenerative epithelium after CS application, when compared to the CTN, XGS and the naturally healed groups (ligature removed without herb application). Bacterium Growth Inhibition by Chinese Herbs The effects of Chinese herbs on the growth of 13 species of bacteria against three different herb composite concentrations or each single component are shown in Tables 2 to 5. All three composites showed marked growth inhibition on the tested species at the concentration of 0.1 g/ml, with the exception of Pseudomonas aeruginosa and Staphylococcus aureus, which still grew well in the presence of 0.1 g/ml XGS (Table 3). With decreasing herbs concentrations, only CTN showed superior bacterium growth inhibition activities among these three tested composites (Tables 2 to 5). The microorganisms that grew on the CTN plates at g/ml (the lowest herb concentration used in this study) were P. aeruginosa and S. aureus (Table 5). None of them were putative periodontal pathogens or dental plaque forming bacteria. Interestingly, the individual CTN components only showed anti-bacterium effects at relatively high concentrations (0.1 g/ml) (Table 3). The CS showed bacterium inhibitory effects at concentrations of 0.05 g/ml, but could not inhibit bacteria growth at g/ml (Table 5). XGS only showed some anti-bacterium effects at its highest concentrations (0.2 g/ml), and revealed poor growth inhibiting activity on P. aeruginosa and S. aureus, which still grew well on BBAP containing 0.2 g/ml of XGS (Table 2). The antimicrobial activity of each herbal component, Borneolum, Rhizoma Coptidis, Radix angelicae pahuricae, Piper longum, Radix scutellariae and Rosin, had significant bacterium growth inhibition effects at concentrations of 0.1 g/ml (Table 3); while Cortex lycii radicis, Exocarpium, Gentain root and radix sophorae tonkinensis, were not particularly effective against the tested organisms, even at the highest concentration (0.2 g/ml) (Table 2). Mutagenesis Effects of Chinese Herbs None of the herb composites or any single herb components showed mutagenetic effects in the Ames tests (Data not shown). With tested concentrations of 0.2 g/ml, none of the herb composites or their components induced back mutation in Salmonella typhimurium TA98.

7 CHINESE HERBS AND PERIODONTITIS 757 Table 2. Bacterial Growth Inhibition by 0.2 g/ml Herbs Composite Compound* Bacteria Strains CS CTN XGS A. actinomycetemcomitans ± ± ± - C. periodontii ± ± - C. rectus ± + ± E. corrodens E. coli ± F. nucleatum ± ± P. gingivalis ± ± P. endodontalis ± + ± ± - P. intermedia ± - P. aeruginosa - - ± S. sputigena ± ± ± - S. aureus - - ± S. sanguis ± * Single component of Chinese herbs: 1, Borneolum; 2, Cortex lycii radicis; 3, Exocarpium citrulli; 4, Radix gentianae; 5, Piper longum; 6, Radix angelicae pahuricae; 7, Radix scutellariar; 8, Rhizoma cimicifugae; 9, Rhizoma coptidis; 10, Radix sophorae tonkinensis; and 11, Rosin. Table 3. Bacterial Growth Inhibition by 0.1 g/ml Herbs Composite Compound* Bacteria Strains CS CTN XGS A. actinomycetemcomitans ± C. periodontii ± ± ± - C. rectus ± E. corrodens ± E. coli ± F. nucleatum - - ± P. gingivalis ± P. endodontalis P. intermedia ± - P. aeruginosa ± ± - - ± + - S. sputigena ± ± ± S. aureus ± ± ± ± ± + ± S. sanguis ± + - * Single component of Chinese herbs: 1, Borneolum; 2, Cortex lycii radicis; 3, Exocarpium citrulli; 4, Radix gentianae; 5, Piper longum; 6, Radix angelicae pahuricae; 7, Radix scutellariar; 8, Rhizoma cimicifugae; 9, Rhizoma coptidis; 10, Radix sophorae tonkinensis; and 11, Rosin.

8 758 Y. CHAN et al. Table 4. Bacterial Growth Inhibition by 0.05 g/ml Herbs Composite Compound* Bacteria Strains CS CTN XGS A. actinomycetemcomitans - - ± ± - + ± C. periodontii ± - ± ± - C. rectus - - ± ± ± ± + ± E. corrodens - - ± ± ± ± + ± + + ± E. coli ± ± ± ± F. nucleatum ± ± ± P. gingivalis - - ± ± ± ± + ± P. endodontalis - - ± ± ± P. intermedia - - ± ± ± P. aeruginosa S. sputigena - - ± ± ± ± ± + ± + ± S. aureus ± S. sanguis - - ± ± ± ± * Single component of Chinese herbs: 1, Borneolum; 2, Cortex lycii radicis; 3, Exocarpium citrulli; 4, Radix gentianae; 5, Piper longum; 6, Radix angelicae pahuricae; 7, Radix scutellariar; 8, Rhizoma cimicifugae; 9, Rhizoma coptidis; 10, Radix sophorae tonkinensis; and 11, Rosin. Table 5. Bacterial Growth Inhibition by g/ml Herbs Composite Compound* Bacteria Strains CS CTN XGS A. actinomycetemcomitans ± - + ± ± C. periodontii ± - + ± ± ± ± ± C. rectus ± ± E. corrodens ± ± ± ± E. coli F. nucleatum ± P. gingivalis ± - ± ± ± ± ± P. endodontalis ± - + ± ± ± P. intermedia ± ± ± P. aeruginosa S. sputigena ± - ± ± + + ± S. aureus S. sanguis ± * Single component of Chinese herbs: 1, Borneolum; 2, Cortex lycii radicis; 3, Exocarpium citrulli; 4, Radix gentianae; 5, Piper longum; 6, Radix angelicae pahuricae; 7, Radix scutellariar; 8, Rhizoma cimicifugae; 9, Rhizoma coptidis; 10, Radix sophorae tonkinensis; and 11, Rosin.

9 CHINESE HERBS AND PERIODONTITIS 759 Discussion The tissue destruction caused by periodontitis is believed to associate with Gram-negative bacterial endotoxins and the host s immune responses. To treat periodontal diseases, plaque control and eradicating putative periodontal pathogens have proven to be effective (van Winkelhoff and de Graaff, 1986; Norland et al., 1978). Chemotherapy in treating periodontal diseases, including antibiotics and antiseptics in dentifrices, has been proven clinically effective (Goumas et al., 1997; Slot and van Winkelhoff, 1993; Moran et al., 1988). Unfortunately, the increased drug resistance activity of microorganisms to chemotherapy has become a serious problem over the past two decades. Insufficient antibiotic concentration can induce resistance during a treatment course in general dental practice (Jones, 2001; Chang et al., 2000; Ho, 2000; Kiriyama et al., 2000; Kleinfelder et al., 1999; Kulekci et al., 2000). In addition, microorganism-producing enzymes that lower the activity of antibiotics have been reported in recent years (Lyobe, 1997). Herbs have been used in treating periodontal diseases for hundreds of years. Animal models such as ferrets, hamsters, rats, dogs and nonhuman primates have been used in the last two decades to objectively evaluate the pathogenesis of human periodontal diseases and its various treatment modalities (Weinberg and Bral, 1999). Indeed, hamsters are the most popular laboratory animals for studying human oral diseases including periodontitis (Levy, 1980; Mikx et al., 1984; Soukos et al., 1998). In this study, we constructed two types of experimental periodontitis. The first was established by tying surgical stainless wire sutures around the necks of teeth, which served as a bacteria retention device. Removal of the surgical stainless wire ligature and thus, the bacterial niche, led to quick tissue repair. The second model maintained existing lesions by applying an oral bacteria mixture. Both models stressed the important role that the microorganism plays in the pathogenesis of periodontitis. Although two out of three Chinese herbal composites (CTN and CS) showed a different ability to inhibit bacterial growth in vitro, only CS showed a more rapid healing effect in the animal test. In fact, the hamster in all groups can recover from periodontitis despite the effect of different Chinese herbal composites. One possible reason may be due to the duration of the experimental periodontitis which was too short to reflect the different effects of the tested Chinese herbal composites. Further in vivo or clinical studies are necessary. In both conditions, the herb composite applications (CS) caused early regenerative epithelium appearance, indicating that these herbs induced an expedited repair process, and possessed a fairly good bactericidal effect that inhibited the growth of several oral microorganism species, which might be putative periodontal pathogens. Furthermore, CS was ground in powder form and used as a toothbrush powder. People who brush their teeth with CS may benefit from this practice in two ways. In addition to removing the dental plaque mechanically, which is the main source of oral or periodontal infections, the ingredients in CS will also kill the plaque bacteria. Taken together, using CS powder will undoubtedly prevent the accumulation of the bacteria that form dental plaque, improve oral hygiene and reduce the incidence of oral and periodontal diseases. It is claimed that Xi Gua Shung can strengthen the gum and, when applied to an abscess in the upper palate, reduce gum swelling (flaming up of fire evil) and eliminate toxic material

10 760 Y. CHAN et al. build-up. In the bacterium growth inhibition test, no antibacterial activity was detected at 10% XGS concentration. But its use has been very popular with Chinese people for treating periodontal diseases for hundreds of years. Positive effects from these traditional Chinese medicines must exist for this practice to have continued for so long. The result indicates that at 1:40 concentration (2.5%), CTN has a broad-spectrum antimicrobial activity. It inhibited the growth of all tested species of periodontal pathogens representing Gram-positive and Gram-negative as well as anaerobic and aerobic microorganisms. Some of these organisms play an important role in the etiology of oral infection (Martin et al., 1997; Savitt and Socransky, 1984; Slot, 1986) and may cause systemic diseases (Li et al., 2000). CTN exhibits a synergistic action, because the single components in CTN did not present the antibacterial effect at 0.05 g/ml concentration. The killing of these bacteria may be explained by the synergism between the constituents in the herbal composites. In conclusion, our study indicated that the Chinese herbal medicines CTN and CS have a broad spectrum of antimicrobial activity. The synergistic or additive action in killing of microorganisms may minimize the development of bacteria resistance. The low toxicity and low cost of these herbs should encourage further investigation leading to a better understanding of traditional Chinese medicine for prevention of plaque formation and strengthening of the gums, as well as in reducing the incidence of dental or periodontal infections. References American Dental Association. Recommended standard practices for biological evaluation of dental materials. JADA 84: 382, Cao, C.F. and X.P. Sun. Herbal medicine for periodontal diseases. Int. Dent. J. 48: , Chan, Y. and R. Chien. Identification and analyses of periodontal pathogens in Taiwan by microbiology tests. Chin. J. Microbiol. Immunol. 27: 59 69, Chang, S.C., W.C. Hsieh and C.Y. Liu. High prevalence of antibiotic resistance of common pathogenic bacteria in Taiwan: the antibiotic resistance study group of the infectious disease society of the Republic of China. Diag. Microbiol. Infect. Dis. 36: , Goumas, P.D., S.S. Naxakis, D.A. Papavasiliou, E.D. Moschovakis, S.J. Tsintsos and A. Skoutelis. Periapical abscesses: causal bacteria and antibiotic sensitivity. J. Chemother. 9: , Ho, M. Taiwan seeks to solve its resistance problems. Science 291: , Jones, R.N. Resistance patterns among nosocomial pathogens: trends over the past few years. Chest 119: S397 S404, Kiriyama, T., T. Karasawa, K. Nakagawa, Y. Saiki, E. Yamamoto and S. Nakamura. Bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections. Oral Surg. Oral Med. Oral Patho. Oral Radiol. Endo. 90: , Kleinfelder, J.W., R.F. Máller and D.E. Lange. Antibiotic susceptibility of putative periodontal pathogens in advanced periodontitis patients. J. Clin. Periodontol. 26: , Kulekci, G., D. Inanc, H. Kocak and C. Kasapoglu. Bacteriology of dentoalveolar abscesses in patients who have received empirical antibiotic therapy. Clin. Infect. Dis. 23: S51 S53, Levy, B.M. Animal analogues for the study of dental and oral diseases. Dev. Biol. Standard 45: 51 59, 1980.

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