Development of new Drugs from Natural extracts for the treatment of Periodontal Disease

Chung, Chong-Pyoung 경희대학교 동서의학연구소 1999 INTERNATIONAL SYMPOSIUM ON EAST-WEST MEDICINE Vol.1999 No.1

Chong-Pyoung Chung Dept.of Periodontology, College of Dentistry, Seoul National Universtiy, Seoul, Korea. Development of new Drugs from Natural extracts for the treatment of periodontal Disease. Proceedings of International Symposium on East-West Medicine, Seoul. 65-75, 1999. -Over the past decades, interesting progress has been made about the development of new drugs from natural extracts for the treatment of periodontal disease. Among many research directions regarding natural extracts, antimicrobial and antiinflammatory function on these have been investigated to evaluate the possibility of development of new drugs. The purpose of this study was to investigate the screening and tracking of a strong candidate for new drugs from several natural extracts for treatment of periodontal disease. The antimicrobial and antiinflammatory effects on Magnolia cortex and several natural extracts were evaluated. Their antimicrobial activity against serveral periodontopathic microflora including P. gingivalis, P. intermedis, As, etc,and also whether these agents showed cell cytotoxic effect, anticollagenolytic activity and inhibition of cytokine production on the gingival fibroblast was looked into. Among several natural extracts, Magnolia cortex were the strongest to the antimicrobial activity to several periodontopathic microflora. But, Magnolia cortex was shown to moderate form of cell cytotoxicity for gingival fibroblasts compared with other natural etracts. To investigate the anticollagenolytic and inhibitory effects on IL-1 and PGE2 production, Magnolia cortex, Zizyphus fructus and several extracts were tested with monocyte, crude collagenase, and human gingival fibroblast. Among the several natural extracts studied, Zizypus fructus was the strongest anticollagenolytic activity and also highly effective in inhibiting IL-1 and PGE2 production compared with other natural extracts. And its effects were comparable to that of Prednisolone. Magnolia cortex and Zizypus fructus showed synergistic strong antimicrobial, highly anticollagenolytic effect and strong inhibition of IL-1βand PGE2 production without any antagonistic effects. To evaluate the regenerative effects of several natural extracts including safflower extract in vitro, and in vivo, their stimulatory effects on PDL fibroblasts, osteoblasts, and rat calvarial defect were investigated. Magnolia cortex and safflower extracts significantly stimulated and increased the growth and survival rate of osteoblasts, PDL fibroblasts, and also significantly increased new bone formation in rat calvarial defect. In vivo, experimental studies on the regenerative effects of the mixture of Magnolia cortex with Safflower extract and Magnolia cortex with Zizypus fructus extract were performed on experimental periodontitis of beagle dogs. Magnolia cortex with safflower extract and Magnolia cortex with Zizypus fructus were highly effective on the inhibition of inflammation and disease progression of periodontal disease. These results suggested that Magnolia cortex and several related natural extracts might be used to prevent, inhibit periodontal disease progression.

Flurbiprofen 함유 키토산 제제가 치은 섬유아세포에 미치는 영향

정종평,박윤정,이승진,유인철,최상묵 梨花女子大學校 藥學硏究所 1997 藥學硏究論文集 Vol.- No.6

The main goal of periodontal regeneration is to be achieved by epithelial exclusion, periodontal ligament cell activation or alveolar bone regeneration. The purpose of this study was to investigate on the physico-chemical and biological characteristics of biodegradable chitosan beads. Chitosan beads were fabricated by ionic gelation with sodium tripolyphosphate and they had the size in 300㎛ diameter. As therapeutic agent, flurbiprofen was incorporated into the beads by 10, 20% loading contents. The release of drugs from the chitosan beads was measured in vitro. Also, biological activity tests of flurbiprofen loaded chitosan beads including cytotoxicity test, ihhibition of IL-1β production, suppression to PGE_2 production, collagenase inhibition test, the ability of total protein synthesis, and tissue response were evaluated. The amount of flurbiprofen released from chitosan was 33-50% during 7 days. Minimal cytotoxicity was observed in chitosan beads. Flurbiprofen released from chitosan beads significantly suppressed the IL-1β production of monocyte, PGE_2 production and markedly inhibited collagenase activity. Meanwhile, flurbiprofen released from this system showed increased ability for protein synthesis. Throughout 4-week implantation period, no significant inflammatory cell infiltrated around chitosan bead and also fibroblast like cell types at the beads-tissue interface were revealed with gradual degradation of implanted chitosan beads. From these results, it was suggested that flurbiprofen loaded chitosan beads can be effectively useful for biocompatible local delivery system in periodontal regeneration.

후박 및 은행엽 추출물의 항균, 항염 및 세포활성도에 미치는 영향

정종평,구영,배기환 충남대학교 약학대학 의약품개발연구소 1995 藥學論文集 Vol.11 No.-

Periodontal therapy for treatment of periodontitis involves the elimination of bacterial plaque and elimmation of the anatomic defects by regenerative procedure. The purpose of this study was to evaluate on the biological effect of magnolia and Ginkgo biloba extract to the antimicrobial, antiinflammatory and cellular activity. Antimicrobial assay was performed with the diffusion method of the extract by measuring of growth inhibitory zone of B. cereus from blood agar plate. Effect of the extract to ce11ular activity of gingival fibroblast were examined using MTT method and measured the result with optical density on 570nm by ELISA reader. Inhibitory effects of PGE_2 production from gingival fibroblast was performed with the addition of IL-1β and the extract to the well and examined to the product of PGE_2 from ce1l by ELISA reader. In vivo anti-inflammatory effect was performed with injection examined with clinically and histologically for their extent of mecrosis and inflammation. Antimicrobial activity of Magnolia extract showed significantly higher activity than that of control. However, GBE did not showed significant activity to compare with control, and mixture of Magnolia and GBE extract showed significantly higher activity than that of control. The effect of cellular activity to gingival fibroblast showed no significant differences of between control and Magnolia extract. However, GBE showed significant higher rate of cellular activity to compare with control and even to PDGF-BB, and also showed same degree of cellular activity even though mixed with Magnolia extract. The inhibitory effect of PGE_2 production showed significantly reduction of PGE_2 production to compare with control, but its inhibitory effect was not much strong to compare with Indomethacin. In vivo, antiinflammatory effect of Magnolia extract to P. gingivalis injection of Hamster buccal check showed significantly reduction of inflammatory cell infiltration and tissue necrosis, but GBE showed no effect on the inhibition of inflammatory process. These results suggested that Magnolia and GBE extract possessed different kind of biological activity and also can be compensated on their activity with each other for elimination of bacterial plaque and anatomical defect.

황금(Scutellariae Radix)의 에타놀추출물과 플라보노이드 성분들의 독성평가

정종평,구영,배기환 충남대학교 약학대학 의약품개발연구소 1995 藥學論文集 Vol.11 No.-

Flavonoids from Scutellariae Radix possessed a dual function both as an anti-inflammatory agent and an enhancer of cel1ular activity in gingival fibroblast. The purpose of this study was to evaluate on the toxicity of ethanolic extract from the root of Scutellariae Radix Georgi and its flavonoids, Wogonin, Baicalein, and Baicalin were isolated and purified by the following method. The crude drug was extracted with ethyl acetate and the residue was dissolved in ethyl alcohol. The ethyl a1cohol soluble fraction was separated, concentrated, and men chromatogaphed on a silica gel column. The acute oral LD 50 in rats was determined for EtOH ex. of Scutellariae Radix and three compounds were evaluated with a single oral gavage at three graded dosage levels. The acute intravenous LD 50 was determined with a single intravenous injection via the jugular vein at three graded dosage levels. Groups of 5 male and 5 female rats, 6 week of age at the start of the study, were fed diets containing 3 graded dosage leve1s for 14 days. Groups of 5 male and 5 female hamster received 0.5㎖ of the test article at once in a day for 5 days to the buccal cheek pouch for two minutes each. The acute ora1 LD50 for EtOH ex. of Scutellariae Radix is 1430㎎/㎏, and for Wogonin 1320㎎/㎏, or Baicalein 1250㎎/㎏, for Baicalin 1330㎎/㎏. The acute intravenous toxicity of EtOH ex. of Scutellariae Radix and its extracts was found to be 27㎎/㎏ body weight No toxic effects were observed in rats fed up to 200㎎/㎏ of EtOH ex.of ScutelIariae Radix, Wogonin, Baicalein and Baicalin in the diet for 14 days. The acute Mucouse Membrane LD 50 in hamsters was found to be greater than 100㎎/㎏. These resu1ts suggested that EtOH ex. of Scutellariae Radix and its flavonoids are safe for oral care products using limited amount of extract.

치주질환 병인균간의 성장억제 활성도에 관한 연구

정해원,이상철,정종평,손성희 대한구강생물학회 1988 International Journal of Oral Biology Vol.12 No.1

In a complex ecosystem such as periodontal pocket, bacterial antagonism may be a determinant in oral colonization and compositional changes of periodontopathic microflora. This study was performed to examine the growth inhibitory activities among the periodontopathic and indigenous bacteria. The growth inhibitory activity was examined as both producers and indicators by stab culture method. Inhibitory activities of cell sonicate and culture supernatant of S. mutans OMZ 65, which showed broad and strong inhibition against periodontopathic bacteria, was examined by the diffusion method. The results were as follows. 1. B. gingivalis, B. intermedius and A. actinomycetemcomitans strains showed weak inhibition against homologous and heterologous strains. Two among three strains of B. intermedius showed moderate inhibition against one strain of B. gingivalis and all of A. actinomycetemcomitans strains showed inhibitory activity to B. gingivalis. 2. The strains of S. mutans showed broad and strong inhibition against B. gingivalis, A. actinomycetemcomitans strains and moderate inhibition against B. intermedius, but S. sanguis strains showed weaker growth inhibitory spectra than those of S. mutans strains. However, the pattern of inhibitory activity showed, to a certain extent, species-specific. 3. S. mutans strains showed broader inhibitory spectrum against A. a. Korean strains than that of S. sanguis strains. But A. actinomycetemcomitans Korean strains has rarely showed growth inhibition against S. sanguis. 4. A. actinomycetemcomitans Korean strains showed weak inhibitory activity to B. gingivalis and no inhibitory activity to B. intermedius. 5. S. mutans showed strong inhibitory activity to homologous strains and typically species-specific than these of S. sanguis. Further investigation should be needed to clarify a relationship between the presence of plasmids and bacteriocin activity.

한국인 연령에 따른 Actinobacillus actinomycetemcomitans의 발현빈도 및 항체 역가에 관한 연구

정종평,김강주,이상철,손성희 대한구강생물학회 1988 International Journal of Oral Biology Vol.12 No.2

In order to investigate on the frequency and antibody levels to Actinobacillus actinomycetemcomitans from Korean population, normal group were divided into 5 category by the related age : 3-6 years, 7-12 years, 13-20 years, 21-35 years and over 36 years, and also disease group were divided into the diagnostic condition : Localized juvenile periodontitis, post-localized juvenile periodontitis, rapidly progressive periodontitis, adult periodontitis. Each group was composed of 10 to 20 patients and plque sampling was also performed on the mesial pocket of mandibular lst molar with 3 paper points on anaerobic condition. Isolation and identification were done by using a selective medium(tryptic soy agar supplemented with 10% serum, 75ug of Bacitracin and 5ug of Vancomycin per ml) in 10% CO_2 incubator for 3 days with routine Gram staining, biochemical test and colony morphology. Serum antibody levels were also performed with isolated A. actinomycetemcomitans SNUDC 10-1 by using modified ELISA technique. The frequency of A. actinomycetemcomitans in normal healthy group was within 10% and no detection was revealed in 7 years group. Otherwise, the frequency of localized juvenile, rapidly progressive, adult perioduntitis and adult periodontitis associated with diabetes mellitus were 75%, 68%, 42%, and 25%, respectively. Serum IgG antibody levels to A.a SNUDC 10-1 in umbrical cord blood was similar to 3-6 years or 13-20 years group, and 3-6 months group were shown only trace amount of antibody levels. The antibody levels of 1 year and 7-12 years group were revealed 2/4-1/3 levels of 3-6 years group and this levels were gradually increased from 7-12 years group until over 36 years group. However, the antibody levels of localized juvenile, post-localized juvenile and rapidly progressive periodontitis were significantly higher than that of healthy group and no significant differences of the level were recognized between each disease group.

한국인 성인형 치주염의 실험실 표식자 개발에 관한 연구

임석중,신형식,김강주,정종평 대한구강생물학회 1992 International Journal of Oral Biology Vol.16 No.2

The significance of plaque bacteria and its toxin in the etiology of various periodontal diseases has been well established. The purpose of this study is to develop laboratory marker in adult periodontitis. Five patients with adult periodontitis were examined. Bacterial morphotype was observed by phase contrast microscope. Microflora was isolated by selective and nonselective media. Data was analysed between healthy sites and disease sites by paired T-test. Wolinella recta in the disease sites was isolated more frequently than that of healthy sites and the proportion of Actinomyces in the healthy sites were higher than that in the disease sites. These results suggested that Wolinella recta in the disease sites and Actinomyces in the healthy sites might be the marker of adult periodontitis in Koreans. Further study is needed to clarify the in vitro virulence of Wolinella recta.

치주염의 원인균 및 정상상주균간의 길항기전 규명 및 치주치료에의 응용

손성희,정종평,김각균,김강주 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.1

It has been reported that microbial distribution and colonization in oral cavity have a relation to antagonism, synergism or symbiosis among microorganisms and bacterial antagonism have a relation to bacteriocin, hydrogen peroxide and bacterial metabolic end-products. The purpose of this study is to investigate on the in vivo and in vitro ecological interrelation and to detect the growth inhibition subatance and to clarify the antagonistic mechanism of the periodontopathic bacteria. In vivo ecological interrelation was studied by bacterial isolation & identification and in vitro ecological was studied by stab culture method. Bacteriocin-like substance was examined, purified, electrophoresed, and stained with silver stain. After the producer microorganism was cultured in various conditions, the pH & bacterial end-products of it was examined and the susceptibility of indicator bacteria by pH was measured. The presence of plasmid DNA was investigated by hot-boiling method. In in vivo ecologic interrelationship, Actinobacillus actinomycetemcomitans(Aa) was not isolated, black-pig-mented Bacteroides was less in the presence of Actinomyces species than in the absence of it in refractory periodontitis. In in vitro ecologic interlationship, S. mutans showed strong growth inhibitory activity against Porphyromonas gingivalis and Aa strains and more strong growth inhibitory activity against Aa Korean isolates than S. sanguis. After bacteriocin-like substance was examined, purified, electrophoresed, and stained with silver stain, a band was appeared whose molecular weight was 36 kd, pH and the production rate of lactic acid was varied according to the presence and absence of oxygen and glucose. These results suggested that there exists in vivo and in vitro growth inhibition among oral microorganisms and growth inhibition was affected by bacteriocin-like substance, pH and bacterial end-products of producer strains and sensitivity of indicator strains. Animal study is needed by using strains of growth inhibition and growth inhibitory substance.

Magnolol과 Honokiol이 항균, 교원질 분해효소, 세포독성 및 Cytokine생산에 미치는 영향

장범석,손성희,정종평,배기환 충남대학교 약학대학 의약품개발연구소 1993 藥學論文集 Vol.9 No.-

The oral microbiota such as P. gingivalis, P. intermedia and A. actinomycetemcomitans play a primary role in the initiation and progression of the periodontal disease. The purpose of this study was to evaluate the antimicrobial effects and inhibitory effects of honokiol and magnolol on the bacterial collagenase activity, cytotoxicity and cytokine production of periodontopathic microorganisms. The antimicrobial activities of honokiol and magnolol was evaluted with minimum inhibition concentration. Honokiol was more active than magnolol, but less than chlorhexidine on antimicrobial activity. The inhibitory effects of magnolol and honokiol on the collagenolytic activity and cytotoxicity were evaluated using a Collagenokit CLN-100 and rapid colorimetric assay (MTT method) for cellular growth and survival of gingival fibroblast and periodontalligament cell and [^3H]-thymidine incorporation for the gingival epithelial cell. The inhibitory effects on the collagenolytic activity was the highest in chlorhexidine, and the lowest in magnolol. Magnolol had the lowest cytotoxic effect and chlorhexidine had the highest. The inhibitory effects on cytokine production was evaluated using interleukin-1β ELISA kit (Cistron Biotech.), IL-6, TNF-α ELISA kit (Genzyme) and inhibitory effects were higher than bacterial LPS and there is no difference among the honokiol, magnolol and chlorhexidine. From these results, the antimicrobial and antienzymatic activities of honokiol and magnolol were seemed to inhibit bacterial growth and enzyme activites with lesser cytotoxic activities. Therefore, it was suggested that honokiol and magnolol are very effective antimicrobial agents on periodontal pathogens.

생약 추출물이 세포성장 및 cytokine 생산에 미치는 영향

류인철,손성희,정종평,배기환 충남대학교 약학대학 의약품개발연구소 1993 藥學論文集 Vol.9 No.-

The native connective tissue attachment of the periodontium is known to be a complex consisting of gingival fibroblasts, periodontal ligament cells, gingival epithelial cells, cementum, alveolar bone and extensive extracellular matrix (collagen, glycoprotein and proteoglycans). The purpose of this study was to evaluate the effects of natural extracts on DNA, collagen and protein synthesis and inhibition of cytokine production in the gingival and periodontal ligament fibroblasts and gingival epithelial cells. Healthy gingibal tissue was obtained from orthodontic treatment patients, and gingival epithelial cells, gingival fibroblasts and periodontal ligament cells were isolated and cultured from the samples. After treated with Ginseng protein, Pluronic F-68, Scutellariae Radix, centella asiatica, PDGF, IGF, DNA synthesis, total protein and collagen synthesis, and cytokine production of gingival epithelial cell, gingival fibroblast and periodontal ligament cells were measured. MTT method for DNA synthesis, Peterkofsky and Dingerman method for total protein and collagen synthesis, and IL-1 ELISA kit for cytokine production were used. The proliferation of epithelial cells was enhanced in Centella asiatica, Ginseng protein, Pluronic F-68 and Scutellariae Radix. The activities of PDL cells were increased in PDGF, IGF, and Pluronic F-68. Higher collagen synthesis was observed in Scutellariae Radix and total protein synthesis was increased in Scutellariae Radix and PDGF. The inhibitory effects on IL-1, IL-6, TNF-α were observed in all exrracts.