工學碩士學位請求論文 Clostridium difficile 에대한우슬 (Achyranthes japonica Nakai) 과 비피도박테리아 (Bifidobacteria) 의항균및상승효과 Synergic antimicrobial effects of Achyranthes japonica Nakai and Bifidobacteria against Clostridium difficile 2006 年 2 月 仁荷大學校大學院 生物工學科 ( 生物工學專攻 ) 丁先美
工學碩士學位請求論文 Clostridium difficile 에대한우슬 (Achyranthes japonica Nakai) 과 비피도박테리아 (Bifidobacteria) 의항균및상승효과 Synergic antimicrobial effects of Achyranthes japonica Nakai and Bifidobacteria against Clostridium difficile 2006 年 2 月指導敎授許泰鍊이論文을工學碩士學位論文으로提出함仁荷大學校大學院生物工學科 ( 生物工學專攻 ) 丁先美
이論文을丁先美의碩士學位論文으로認定함. 2006 年 2 月 主審 인 副審 인 委員 인
요 약 항생제오 남용으로인해 Clostridium difficile (C. difficile) 에의한장염이나설사의발생빈도가높아지고있으며, 이로인한항생제내성문제가크게대두되고있다. 따라서본연구에서는화학합성항생제약물치료를대체할수있는새로운천연항균성의약품및식품소재개발을목적으로항염증효과가있는 40여가지생약재의 C. difficile에대한항균활성을검색하였고, 그결과가장높은항균활성을보인우슬 (Achyranthes japonica Nakai) 에탄올추출물을항균활성시료로선정하였다. 우슬에탄올추출물로부터얻은에틸아세테이트분획물 (AJNEA) 은 C. difficile에대하여 90% 이상의높은항균활성을보였으며, 최소저해농도 (minimum inhibitory concentration: MIC) 625 µg/ml로나타났다. 또한 AJNEA가유산균의생장에미치는영향을조사한결과 Bifidobacterium longum, Lactobacillus helveticus 와 Streptococcus thermophilus을제외한실험에사용된나머지유산균에대해서는억제효과를나타내지않았다. 유산균배양상층액의항균효과및 AJNEA와의항균상승효과를알아보기위해유산균배양액의 C. difficile에대한항균활성을조사한결과 Lactobacillus rhamnosus GG, Lactobacillus bulgaricus 배양 I
상층액에서 50% 이상의항균성을나타냈다. 그러나 AJNEA를첨가한 Lactobacillus rhamnosus GG, Lactobacillus bulgaricus 배양상층액에서의상승효과는나타나지않았다. AJNEA (100 ppm) 를첨가한 Bifidobacterium infantis (68.8%), Bifidobacterium adolescentis (33.2%) 배양상층액에서높은상승효과 (synergic effect) 를보였다. 식품첨가물로서의효과를보기위하여 AJNEA를첨가하여제조한 yoghurt의상층액의제조하여 C. difficile에대한저해효과를측정하였다. 그결과 Lactobacillus acidophilus : Streptococcus thermophilus : Bifidobacterium adolescentis = 1:1:1 와 Lactobacillus acidophilus : Streptococcus thermophilus : Bifidobacterium infantis = 1:1:1을 starter로각각에 0.5% AJNEA를첨가하여제조한 yoghurt에서 C. difficile에대한높은항균활성 (60%) 이그대로나타냈다. AJNEA의 C. difficile 에대한성장저해효과뿐만아니라 C. difficile toxin A에대한저해활성을보기위해농도별로처리한결과, AJNEA 3,000 ppm 처리농도에서 toxin A band가관찰되지않았다. 따라서본연구는우슬이 C. difficile에의해유발된장염이나설사치료에있어화학합성항생제를대체할만한천연항균성제제및기능성식품소재로서의실용화가능성을시사하였다. II
ABSTRACT Clostridium difficile (C. difficile) is not only the most common infectious cause of nosocomial diarrhea, accounting for 20~25% of all antibioticassociated diarrhea, but also identified in 95% of pseudomembranous colitis (PMC) cases. In particular, there has been a recent increase in the number of children and infants suffering from C. difficile-related infections during antibiotics treatment. The overuse of antibiotics may alter the normal intestinal flora and increase the risk of developing C. difficile diarrhea. Although antibiotics (vancomycin, etc.) have various problems such as high medical costs, antibiotic resistance, and relapses or re-infections during treatment periods, new alternative agents for treatment of C. difficile associated diarrhea or infection diseases was not yet determined. Therefore, in this study, we assessed the antimicrobial effect of Achyranthes japonica Nakai (AJN) and Lactic acid bacteria (LAB) against C. difficile for development of new natural antimicrobials or functional materials. Among the screened 42 traditional herbal medicines, ethyl acetate soluble fractionate from AJN (AJNEA) against C. difficile was showed appreciable high inhibitory activity by minimum inhibitory concentration (MIC) test and MIC of AJNEA was 625 µg/ml. In addition, we investigated antimicrobial activity III
of LAB against C. difficile. Among the tested strains, Lactobacillus rhamnosus GG, Lactobacillus bulgaricus and Lactobacillus acidophilus were showed more than 40% high effect by turbidity method. However, culture media supernatant of Lactobacillus species with AJNEA not showed synergic effect against C. difficile. Culture media supernatant of Bifidobacterium infantis with AJNEA (68.6%) or Bifidobacterium adolescentis with AJNEA (33.2%) was presented clearly synergic effect when the compare to control which was added with only each organism or AJNEA (100 ppm). In yoghurt prepared with Lactobacillus acidophilus : Streptococcus thermophilus : Bifidobacterium adolescentis = 1:1:1 or Lactobacillus acidophilus : Streptococcus thermophilus : Bifidobacterium infantis = 1:1:1 as starter and 0.5% AJNEA powder, high synergic antimicrobial effects against C. difficile was showed 79% and 65.2%, respectively. In addition, we tested inhibitory effect of AJNEA against C. difficile toxin A using immunoassay. As the result, toxin A was not detected at the concentration of 3,000 ppm AJNEA. Therefore, these results demonstrated that AJN appears to be potential natural antimicrobial agent and expected to be useful in the development of functional source against C. difficile infection. IV
목 차 요약 I Abstract III 목차 V LIST OF SCHEMES AND TABLES Ⅷ LIST OF FIGURES IX ABBREVIATIONS XI 1. 서론 1 2. 재료및방법 4 2.1. 시료 4 2.2. 사용균주및배지 4 2.3. 시약 5 2.4. 시료추출물의조제 8 2.5. 활성물질의추출및분획 8 2.5.1. 우슬에탄올추출 8 V
2.5.2. 우슬용매분획 8 2.6. C. difficile에대한항균활성검색 11 2.6.1. 액체배지희석법 (broth dilution method) 11 2.6.2. 원통평판법 (cylinder agar plate method) 11 2.6.3. 최소저지농도측정 (minimum inhibitory concentration :MIC) 12 2.7. 우슬의유산균생장에대한영향 12 2.8. 유산균의 C. difficile에대한항균활성 12 2.9. 상승항균활성측정 13 2.10. 요구르트 (Yoghurt) 제조및항균상승효과 14 2.10.1. Yoghurt 제조 14 2.10.2. AJNEA 첨가 yoghurt의 C. difficile 항균활성 15 2.11. C. difficile toxin A 활성 15 3. 결과및고찰 16 3.1. 에탄올추출물수율 16 3.2. 생약재추출물의 C. difficile에대한항균활성 16 3.2.1. 생약재에탄올추출물의항균활성 16 3.2.2. AJNEA의농도별항균활성 17 3.3. AJNEA의유산균생장에대한영향 23 VI
3.4. 유산균의항균활성 26 3.5. AJNEA와유산균의상승효과 29 3.6. AJNEA 첨가 yoghurt의항균및상승효과 33 3.7. C. difficile toxin A 저해 35 4. 결론 37 5. 참고문헌 39 VII
LIST OF SCHEMES AND TABLES Scheme 1. Preparation of ethanol in extracts from traditional herbal medicines 9 Scheme 2. Preparation of solvent soluble fractionates from traditional herbal medicines 10 Table 1. List of herbal medicines used in the experiment for assessment of activity against C. difficile 6 Table 2. Kinds of yoghurt starter used for yoghurt preparation 14 Table 3. Screening of antimicrobial activity of herbal medicine extracts against C. difficile 18 Table 4. Antimicrobial effect of each with concentration of AJNEA against C. difficile 22 Table 5. Synergic effect of AJNEA and each of culture media supernatant of B. adolescentis and B. infantis against C. difficile 31 VIII
LIST OF FIGURES Figure 1. Antimicrobial effect of the organic solvent soluble fractionates separated fractionated from ethanolic extracts of medicinal plant 20 Figure 2. Antimicrobial activities of with different concentration of Achyranthis radix against C. difficile 21 Figure 3. Growth effect of AJNEA on Bifidobacterium spp. 24 Figure 4. Growth effect of AJNEA on Lactobacillus spp. 25 Figure 5. Antimicrobial activity of culture medium supernatants of Bifidobacterium spp. against C. difficile 27 Figure 6. Antimicrobial activity of culture medium supernatants of Lactobacillus spp. against C. difficile 28 Figure 7. Synergic antimicrobial effect of AJNEA and culture medium supernatants of Bifidobacterium spp. against C. difficile 30 Figure 8. Synergic antimicrobial effect of AJNEA and culture medium supernatants of Lactobacillus spp. against C. difficile 32 IX
Figure 9. Synergic antimicrobial effect of supernatants of yoghurt prepared with AJNEA and different starter species against C. difficile 34 Figure 10. Inhibitory effect of AJNEA with different concentration against C. difficile toxin A 36 X
ABBREVIATIONS AJN AJNEA ATCC G L H Kg KTCC LAB MIC min Mg ml mg/ml Achyranthes japonica Nakai Ethyl acetate soluble fractionate from AJN American type culture collection Gram Liter(s) Hour(s) Kilogram(s) Korean culture center of microorganisms Lactic acid bacteria Minimum inhibitory concentration Minute(s) Miligram(s) Mililiter(s) Miligram/ Miligram µl Microliter Μm Nm rpm O.D. ppm Spp Micrometer Nanometer Revolution per minute Optical Density Part per million Species XI
1. 서론 Clostridium difficile (C. difficile) 은혐기성아포형성그람양성균의중요한병원균으로 1978년 Bartlette 등에의해발견되었다. 우리나라에서는 1997년위막성대장염환자에게서 C. difficile의 toxin 이발견되면서항생제성대장염의원인이밝혀졌고, 특히, 생후 1개월이내의신생아에게서많이발견된다 15, 26). C. difficile의감염을일으키는주요원인으로서 ampicillin, amoxicillin, cepahlosporins, clindamycin을포함한광범위한항생제의사용은 C. difficile의과증식을유도하고증식된 C. difficile은대장내에존재하는유익균을억제하고과성장하여대장점막에염증을유도하고, 항생제유발설사 (antibiotic-associated diarrhea) 와위막성대장염 (pseudomembranous colitis), C. difficile에의한설사 (C. difficile-associated diarrhea : CDAD) 를유발시킨다 24). 특히 C. difficile이병독성인자로서생산하는장독소 (enterotoxin) 인 toxin A 와세포독소 (cytotoxin) 인 toxin B가설사와장염을유발시킨다 1, 4, 20). 이중 C. difficile toxin A는사람의대장상피세포를자극하여각종세포작용에관여한다 16). CDAD는 vancomycin이나 metronidazole 항생제를사용하여치료하는데이들항생제의사용으로인한재발율이급증하게된다. 이 1
러한문제를보안하기위해, 최근에는 C. difficile에의한설사를치료하기위해장내정상적균총의형성과건강증진효과가있는것으로알려진생균제 (probiotics) 또는 yoghurt를 CDAD 치료에사용하기도한다. Probiotics란살아있는균을말하는것으로사람이나동물이섭취하였을때위장관에머물러생존할수있는미생물로항균제나약물치료와같은요인에의한장질환발생을예방하거나치료하는데이용될뿐만아니라, 유당불내증을경감시켜주고, 혈중콜레스테롤을감소시켜주며, 암을예방시켜준다 32). 가장일반적으로사용되고있는 probiotics는유산균으로 Bifidobacterium spp. 와 Lactobacillus spp. 의유익한효과는오래전에인정되어왔다. Probiotics는사람이나동물에게서발병하는광범위한위장관질병을치료하기위해사용된다. 몇몇연구에서급성설사로병원에입원한영유아들에게유산균를처리하였더니설사발생율이감소한것으로나타났다 6, 10, 12, 13, 24, 30, 33). 특히, Saccharomyces boulardii, L. GG, Bifidobacterium breve, Bifidobacterium longum 과 Lactobacillus acidophilus와같은 probiotics가항생제성설사를예방하는효과가있는것으로보고되었다. 9, 28, 32). 우슬 (Achyranthes japonica Nakai : AJN) 은쇠무릎 (Achyranthes bidentata 또는 Achyranthes fauriei) 의뿌리를지칭하는생약제로한국, 중국, 일본등의아시아에광범위하게분포하는다년초식물로 2
비름과 (Amaranthaceae) 에속한다. 우슬은한방에서이뇨, 진통, 소종, 구어혈등의치료목적으로사용되고있으며 25), 우슬은항염증, 항암작용, 간보호, 항산화등의생물학적활성도보고되어있다. 우슬의활성성분으로서 oleanolic acid, saponin, metamorphosis hormone, inokosterone, ecdysone, polysaccharide, 20-hydroxyecdysone, steroid계열인 β-sitosterol, stigmasterol, rubrosterone 등이보고되어있다 25, 30, 31). 본연구는항생제내성장염유발균인 C. difficile에대한항균활성을갖고있으며동시에유산균에는생장저해를일으키지않는천연기능성식품및의약품소재개발을위한기초연구로서문헌조사를통해독성이없고항염증작용이알려져있는 40여가지의생약재의 C. difficile에대한항균활성을조사하였다. 가장유용할만한효과를보인우슬을활성시료로선정하였으며, 우슬추출물이 C. difficile에미치는항균효과및유산균의성장에미치는영향을조사한결과, 우슬은 C. difficile에대하여매우우수한항균활성을나타내었으며, 또한 Bifidobacterium adolescentis와 Bifidobacterium infantis에대해높은상승효과를보였고이는 yoghurt 제조후에도그활성을확인하였다. 따라서분연구는우슬이우수한 C. difficile 치료를위한항생제대체물질이며, 이를이용한기능성식품및의약품개발가능성을시사하였다. 3
2. 재료및방법 2.1. 시료본실험에사용한 40여종의한약재는서울경동시장약재도매상가에서원산지를확인한후구입하였으며, 모든원료는건조및마쇄하여사용하였다 (Table 1). 2.2. 사용균주및배지 본실험에사용한 Clostridium difficile 은한국미생물보존센터 (KTCC) 에서분양받아사용하였고, 이균주의생육배지는 Reinforced Clostridial Medium (RCM) broth (Difco, USA) 와 Brucella agar with hemin and Vitamin K1 (BA) (Sigma) 을사용하였고, 유산균은본실험실에서보유하고있던 Bifidobacterium adolescentis ATCC 15703, Bifidobacterium bifidum ATCC 29521, Bifidobacterium breve ATCC 15700, Bifidobacterium infantis ATCC 15697, Bifidobacterium longum ATCC 15707, Lactobacillus brevis ATCC 14869, Lactobacillus helveticus ATCC 15009, Streptococcus thermophilus KCTC 21185 및 Denmark 의 CHR. Hansen s laboratory 에서구입한 Lactobacillus bulgaricus CH-2, ABT YC-180 와 KTCC 에서분양받은 Lactobacillus acidophilus ATCC 4356, L. rhamnosus GG IFO 3863 을사용하였다. 배지로는 4
Bifidobacterium spp. 는 Trypticase-proteose peptone-yeast extract (TPY) broth 배지를사용하였고, Lactobacillus spp. 와 ABT 혼합균주는 Lactobacilli MRS broth (Difco, USA) 을사용하였다. 2.3. 시약 C. difficile toxin A를저해시키는지확인하기위하여 C. difficile toxin A kit (Oxoid, UK) 를사용하였으며, yoghurt 제조에사용한가는정백당 ( 제일제당 ), 생유 ( 서울우유 ), 탈지분유 ( 서울우유 ) 는시중에서구입하여사용하였다. 실험에사용한추출용매는시약용 1급 ethanol, n-hexane, chloroform, ethyl acetate, n-butanol을사용하였고, 그외의시약은일급또는특급을사용하였다. 5
Table 1. List of herbal medicines used in the experiment for assessment of activity against C. difficile Botanical origin b Scientific name Family name Used part a Achyranthis radix Achyranthes japonica Nakai Amaranthaceae Ra Aconitum koreanum Aconitum koreanum R. Raymond Ranunculaceae R Acorus gramineus Acorus gramineus Soland. Araceae Rh Angelicae gigantis radix Angelica gigas Nakai Umbelliferae Ra Angelicae koreanae radix Angelica koreana Max. Umbelliferae R Atractylodis rhizoma alba Atractylodes japonica Koidz. Compositae Rh Aurantii fructus Citrus aurantium L. Rutaceae Fc Bupleurum falcatum Bupleurum falcatum L. Umbelliferae Ra Celosiae semen Celosia argentea L. Amaranthaceae Se Chaenomelis fructus Chaenomeles sinensis Koehn. Rosaceae Fr Clematidis radix Clematis mandshurica Rupr. Ranunculaceae R Cnidium officinale Ligusticum chuanxiong Hort Umbelliferae Fr Coptidis rhizome Coptis chinensis Franch Ranunculaceae R Corni fructus Cornus officinalis Sieb.et Zucc. Cornaceae Fr Crataegi fructus Crataegus pinnatifida Bunge Rosaceae Fr Cyperi rhizoma Cyperus rotundus L. Cyperaceae Se Dictamni cortex Dictamnus dasycarpus Turcz. Rutaceae Ra Erycibeae caulis Erycibe obtusifolia Benth. Convolvulaceae C Eucommiae cortex Eucommia ulmoides Oliv. Eucommiaceae C Gastrodia elata Gastrodia elata Blume Orchidaceae R Gentianae radix Gentiana scabra Bunge Gentianaceae R Ginseng radix Panax ginseng C. A. Mey. Araliaceae R Hoelen (Poria) Poria cocos Wolf Polyporaceae W Kalopanacis cortex Kalopanax pictus Nakai Araliaceae C continued 6
Botanical origin b Scientific name Family name Used part a Ledebouriellae radix Ledebouriella seseloides Wolff Umbelliferae R Ligustri lucidi fructus Ligustrum lucidum Aiton Oleaceae Fr Liriope platyphylla Liriope plathyphylla WANGET et TANG Liliaceae Fr Lonicerae flos Lonicera japonica Thunb. Caprifoliaceae Fl Mori cortex Morus alba L. Moraceae Sb Moutan radicis cortex Paeonia suffruticosa Andr. Paeoniaceae C Paeoniae radix alba Paeonia lactiflora Pall. Paeoniaceae Ra Phellodendri cortex Phelloedndron amurense Ruprecht Rutaceae R Platycodi radix Platycodon grandiflorum ADC. Campanulaceae Ra Polygoni multiflori caulis Polygonum multiflorum Thunb. Polygonaceae Ra Poncirus trifoliate Poncirus trifoliata RAFIN Rutaceae Ra Puerariae radix Pueraria thunbergiana Benth. Leguminosae R Rubi fructus Rubus coreanus Miq. Rosaceae Fr Schisandrae fructus Schisandra chinensis Baill. Schisandraceae Fr Siegesbeckiae herba Siegesbeckia glabrescensmakino Compositae L Smilacis glabrae rhizoma Smilax glabra Roxb. Liliaceae Rh Sophorae flavescentis radix Sophora flavescens Ait. Leguminosae Ra Taraxacum mongolicum hand. Taraxacum platycarpum H. Taraxacum St Tetrapanacis medulla Tetrapanax papyriferus K.Koch Araliaceae St a C : cortex, Fl : flower, Fc : fruit cortex, Fr : fruit, L : leaf, R : root, Ra : radix, Rh : rhizome, Sb: stem bark, Se : seed, St : stem, T : tuber, W : wolf b Each ethanol extract of herbal medicines was treated at 1000 ppm in RCM broth as final concentration. 7
2.4. 시료추출물의제조건조및마쇄한시료 50 g에 10배용량의 95% 에탄올을가하여침지한뒤, 실온에서 24시간동안정지추출하고, 이를총 3회반복하여추출하였다 (Scheme 1). 추출액은여과지 (Watman No. 1) 를사용하여여과하고감압농축기 (EYLA, Japan) 를사용하여감압농축한후건조하여에탄올추출물시료로사용하였다. 에탄올추출물은 RCM 배지에적정농도로조제하여항균활성을측정에사용하였다. 2.5. 활성물질의추출및분획 2.5.1. 우슬에탄올추출우슬 2 kg을분쇄하여에탄올을가하여실온에서 24시간방치후여과하여, 다시잔사에에탄올을가하여침지한후여과하는과정을 3회반복실행하여얻은여액을감압농축하여추출물로제조하였다. 2.5.2. 우슬용매분획 Scheme 2. 의방법에따라먼저 95% 에탄올로추출한후극성에따라 n-hexane, chloroform, ethyl acetate, n-butanol, water 순으로분획하여감압농축하였다. 8
Herbal medicines Powder (50g) + 95% ethanol extracted for 24 h filtrated Repeated of 3 times Supernatant Residues evaporated in vacuum Extracts Scheme 1. Preparation of ethanol in extracts from traditional herbal medicines 9
Achyranthes japonica Nakai (AJN, 2 kg) extracted with 95% Ethanol filtered evaporated Ethanol extracts (44.67 g) Dissolved in water added Hexane partitioned between Hexane and Water Hexane Fr. (6.36 g) Aqueous layer added Chloroform partitioned between CHCl 3 and Water Chloroform Fr. (0.86 g) Aqueous layer added ethyl acetate partitioned between EtOAc and Water Ethyl acetate Fr. (1.16 g) Aqueous layer added Butanol partitioned between BuOH and Water Butanol Fr. (11.5 g) Water Fr. Scheme 2. Preparation of solvent soluble fractionates from traditional herbal medicines 10
2.6. C. difficile 에대한항균활성검색 2.6.1. 액체배지희석법 (broth dilution method) 각각의시료추출물을적정농도로첨가한후액체배지에활성화된균액을 O.D. 값이 0.3이되도록희석한후, 10 ml 액체배지에 1% 접종하였다. 37 에서 12시간배양한후배양액의 O.D. (600 nm) 를측정하여균의생장억제효과를아래와같은식에의하여산출하였다. 이때 blank는각시료만첨가하고, 균액을접종하지않은것으로하였다. Inhibitory effect (%) = [(control - control blank) - (treatment - treatment blank)] 100 (control - control blank) 2.6.2. 원통평판법 (cylinder agar plate method) BA 평판배지에활성화된균액을 O.D. 값이 0.3 이되도록희석하여 100 µl 도말후 0.45 µm microfilter 로제균한시료를원통에 100 µl 흡수시킨다. 12~24 시간배양한후원통 ( 직경 7 mm) 주위의억제환크기에따라항균활성을비교하였다. 11
2.6. 3. 최소저지농도측정 (minimum inhibitory concentration :MIC) 활성화된균액을 O.D. 값이 0.3 이되도록희석한후, 10 ml RCM broth 배지에 1% 접종한후 syringe filter (Φ 0.45 µm) 로제균된시료를농도별로처리하여 37 에서 12 시간배양한후, O.D. (600 nm) 값을측정하여탁도를나타내지않은최소농도를 MIC 로 결정하였다 2.7. 우슬의유산균생장에대한효과우슬의유산균생장에대해미치는영향은유산균을 10 ml TPY broth 또는 MRS broth 배지에 1% 접종하고시료를 syringe filter (Φ 0.45 µm) 로제균한후 500 ppm의농도로처리하여 37 에서 18시간동안배양한후분광광도계를이용하여 O.D. (600 nm) 를측정하였다. 이때대조군은시료를넣지않고 TPY broth 또는 MRS broth 배지에유산균 1% 를접종하여 37 에서 18시간동안배양한것으로하였다. 2.8. 유산균의 C. difficile에대한항균활성유산균을 10 ml TPY broth 또는 MRS broth 배지에 1% 접종하여 37 에서 18시간배양한후 3,000 rpm에서 10분간원심분리하여상승액을회수하였다. 회수한상층액은동결건조기 (EYELA Ltd., Tokyo, Japan) 을이용하여동결건조시킨후사용하였다. O.D. 값이 12
0.3이되도록희석한균을 RCM broth배지에 1% 분주하고유산균을배양한상층액을 syringe filter (Φ 0.45 µm) 로제균한다음, 500 ppm 농도로처리하여 37 에서 12시간배양한후배양액의 O.D. (600 nm) 를측정하여유산균의항균활성을측정하였다. 2.9. 상승항균활성측정 C. difficile에대해높은항균력을보인우슬에틸아세테이트분획물 (AJNEA) 을유산균배양시배지에혼합하여배양한후배양된배지상층액의항균력상승여부를확인하였다. 유산균배양시 TPY broth 또는 MRS broth 배지에 AJNEA를 100 ppm 농도로처리하여균액을 1% 접종하고 37 에서 18시간배양한후 3,000 rpm에서 10분간원심분리하여상승액을회수하여동결건조한후앞의항균활성측정과같은방법으로항균활성을측정하여항균력을비교하였다. 대조군은 AJNEA를넣지않고배양한유산균의배지상층액을각시료와동일한농도인 500 ppm씩분주하여검증하였다. 13
2.10. 요구르트 (Yoghurt) 제조및항균상승효과 2.10.1. Yoghurt 제조제품에서의항균활성을확인하기위해 AJNEA를첨가한 yoghurt 를제조하였다. 살균한 10% (w/v) skim milk를기본배지로하여 Table 2에나와있는균주 (starter) 를동등한비율로혼합하여 2% 접종하였다. 접종한배지를 37 에서 8 시간발효하여이를 yoghurt 원액으로사용하였다. 생유에탈지분유 5% 와가는정백당 1.75 % 첨가하여 90 에서 10분간살균한후, 37 로냉각시키고, AJNEA를 0.5% 가한다음 yoghurt 원액을전체 volume의 2% 가되도록접종하여 37 에서 8시간이상배양하여 yoghurt를제조하였다. 제조된 yoghurt는냉동보관하여실험에사용하였다. Table 2. Kinds of yoghurt starter used for yoghurt preparation NO. Yoghurt starter group 1 ABT (L. acidophilus + L. bulgaricus + S. thermophilus) 2 ABT : B. adolescentis = 1:1 3 ABT : B. infantis = 1:1 4 L. acidophilus : S. thermophilus : B. adolescentis = 1:1:1 5 L. acidophilus : S. thermophilus : B. infantis = 1:1:1 14
2.10.2. AJNEA 첨가 yoghurt 의 C. difficile 항균활성 AJNEA 를 0.5% 첨가하여제조한 yoghurt 를 3000 rpm 에서 15 분간 원심분리하여 yoghurt 상층액을회수한후동결건조하여실험을 수행하였다. RCM broth 배지에 yoghurt 상층액을 syringe filter (Φ 0.45 µm) 로제균시킨다음, 1000 ppm, 2000 ppm 농도로처리하여 O.D. 값이 0.3 이되도록희석한균을 1% 접종하여 37 시간배양한후 O.D. (600 nm) 값을측정하였다. incubator에서 12 2.11. C. difficile toxin A 측정활성화시킨 C. difficile 배양액을 13,500 rpm에서 10분간원심분리한후상층액을시료로사용하였다. C. difficile toxin A가분리된상층액과 AJNEA을각각 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm 농도로하여혼합한후상온에서 30분간반응시켰다. 반응시킨각각의용액을 125 µl씩 kit의 sample창에분주하고 30분후에 band를확인하였다. 15
3. 결과및고찰 3.1. 에탄올추출물수율에탄올추출물은중량을측정하기위하여동결건조후각시료의수율을구하였다 (Table 3). 그중강활, 금은화, 초용담등이 40% 이상을향부자, 황금, 산수유등도 20% 이상의높은수율을보였으나해동피와산약등의수율은매우낮았다. 3.2. 생약재추출물의 C. difficile 에대한항균활성 3.2.1. 생약재에탄올추출물의항균활성 40여종의생약재에탄올추출물에대하여 C. difficile의항균활성을검색한결과 1,000 ppm의농도에서, 희첨, 황백은 90% 이상의높은활성을보였고, 지각, 방풍, 길경등은 50% 이상의활성을나타냈으나, 위령선, 산사, 인삼, 금은화등은 C. difficile에대한항균활성을보이지않았다 (Table 3). 높은활성을나타낸우슬, 희첨, 황백에대하여 n-hexane, chloroform, ethyl acetate, n-butanol 순으로용매분획을실시하였고, 이들분획물들에대한항균활성을조사하였다 (Fig. 1). 그결과, AJNEA가 95.16±0.7% 로가장높은항균력을보였으며, 희첨클로로포름분획물에서 80% 이상의높은활성을보였 16
고, 황백은클로로포름과에틸아세테이트분획물에서 60% 이상의 활성을나타내었다. Kim 등은우슬이다양한진균성식물병해들에 대하여항진균력을보인다고보고한바있다 15). 3.2.2. AJNEA의농도별항균활성가장높은활성을나타낸 AJNEA의농도에따른항균활성을측정하였다. 이때, 일반적으로 C. difficile 유래설사치료제로사용되는 vancomycin 12.5 ppm을대조군으로사용하여 AJNEA의활성을비교하였다. 결과, AJNEA 500 ppm은 vancomycin과유사한활성 (95%) 을나타냈으며, 이때 MIC는 625 µg/ml 이었다 (Fig. 2). 그러나 50 ppm 농도에서는활성을나타내지않았다 (Table 4). 17
Table 3. Screening of antimicrobial activity of herbal medicine extracts against C. difficile Botanical prigin Yield (%) Inhibitory activity a Achyranthis radix 7.5 98.40.7 Aconitum koreanum 1.1 - b Acorus gramineus 3.4 30.1±7.8 Angelicae gigantis Radix 14.9 27.9±2.8 Angelicae koreanae Radix 43.0 - Atractylodis rhizoma Alba 7.7 36.2±6 Aurantii fructus 10.0 66.9±9.1 Bupleurum falcatum 5.0 12.4±4.5 Celosiae semen 6.6 13.9±9.1 Chaenomelis fructus 15.4 13.1±11.4 Clematidis radix 18.4 - Cnidium officinale 26.9 - Coptidis rhizoma 18.2 27.4±6.7 Corni fructus 28.8 - Cratagi fructus 28.6 - Cyperi rhizoma 19.9 15.4±6.7 Dictamni cortex 16.9 15.8±9.5 Erycibeae caulis 5.5 44.9±24.8 Eucommoae cortex 4.5 10.7±3.8 Gastrodia elata 10.3 22.6±5.4 Gentianae radix 44.0 12.0±3.3 Ginseng radix 3.3 - Hoelen (Poria) 6.3 13.3±9.4 Continued 18
Botanical prigin Yield (%) Inhibitory activity a Kalopanacis cortex 3.3 42.4±7.4 Ledebouriellae radix 3.3 59.5±9.6 Ligustri lucidi Fructus 39.3 18.1±9 Liriope platyphylla 5.6 27.3±5.7 Lonicerae flos 58.4 - b Mori cortex 7.4 - Moutan radicis Cortex 4.8 17.0±0. Paeoniae radix Alba 4.0 - Phellodendri cortex 11.1 90.0±3 Platycodi radix 1.5 57.6±14.9 Polygoni multiflori Caulis 17.0 10.8±3.6 Poncirus trifoliata 6.2 22.1±9.1 Puerariae radix 12.9 - Rubi fructus 10.4 17.1±6.4 Schisandrae fructus 27.4 - Siegesbeckiae herba 26.8 93.9±1.7 Smilacis glabrae Rhizoma 2.4 12.4±6.7 Sophorae flavescentis Radix 1.0 6.3±7.1 Taraxacum mongolicum Hand 18.2 28.5±16.4 Tetrapanacis medulla 6.3 17.7±10.9 a Anti-microbial effect was represented as % against C. difficile : Inhibitory effect (%)={(control - control blank) - (treatment - treatment blank)/ (control - control blank)} 100±S.D. b - : No effect 19
100 80 Inhibition (%) 60 40 20 0 Achyranthis radix Siegesbeckia glabrescensmakino Phelloedndron amurense Ruprecht Hexane Chloroform Ethyl acetate Buthanol Water Fig. 1. Antimicrobial effect of the organic solvent soluble fractionates separated fractionated from ethanolic extracts of medicinal plant Tested concentration of fractionates was 500 ppm. Data represent the means ± S.D. 20
100 Inhibition (%) 80 60 40 ` 20 0 100 200 300 400 500 vancomycin 12.5 Concentration (ppm) Fig. 2. Antimicrobial activities of with different concentration of Achyranthis radix against C. difficile Data represent the means ± S.D. (N=3) 21
Table 4. Antimicrobial effect of each with concentration of AJNEA against C. difficile Concentration (ppm) Clear zone Clostridium difficile 50 - a 100 + b 200 + 300 ++ c 400 ++ 500 +++ d Sample amount was 100 µl. a : No antimicrobial activity, C.Z.(clear zone) of sample < 10 mm b : C.Z. of sample 10~13 mm c : C.Z. of sample 13~16 mm d : C.Z. of sample >16 mm 22
3.3. AJNEA의유산균생장에대한영향 AJNEA가유산균의생장에미치는영향을조사한결과 (Fig. 3) 실험에사용한균주중 B. longum을제외한나머지 Bifidobacterium spp. 에대한생장저해효과는나타나지않았다. Lactobacillus spp. 중 L. acidophilus, L. brevis, L. GG에대해서는생장에영향을미치지않은것으로나타났고, L. bulgaricus는약간의증식효과를나타냈으나, L. helveticus와 S. thermophilus의생장에대해서는억제효과를보였다 (Fig. 4). 23
1.8 1.6 Bifidobacterium spp. Bifidobacterium spp. + AJNEA (500 ppm) 1.4 O.D. (600 nm) 1.2 1.0 0.8 0.6 0.4 0.2 0.0 B. adolescentis B. bifidum B. breve B. infantis B. longum Strains Fig. 3. Growth effect of AJNEA on Bifidobacterium spp. Data represent the means ± S.D. (N=3) 24
2.2 2.0 1.8 Lactobacillus spp. Lactobacillus spp. + AJNEA 500 ppm 1.6 O. D. (600 nm) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 L.acidophilus L. brevis L. bulgaricus L. helveticus L. GG S. thermophilus Strains Fig. 4. Growth effect of AJNEA on Lactobacillus spp. Data represent the means ± S.D. (N=3) 25
3.4. 유산균의항균활성 Bifidobacterium spp. 와 Lactobacillus spp. 의배양상층액을 500 ppm 의농도로 TPY broth 또는 MRS broth 배지에첨가하고, C. difficile을접종시켜 12시간배양후 O.D. 를측정한결과, B. breve와 B. adolescentis는 C. difficile에대하여각각 29% 와 3.9% 정도의약간의생장저해활성을나타냈으나나머지 Bifidobacterium spp. 는항균력을나타내지않았다 (Fig. 5). 반면, L. bulgaricus와 L. GG는 50% 이상의높은항균력을보였고, L. acidophilus은 40% 이상, L. brevis, L. helveticus와 S. thermophilus 또한약간의항균효과를나타냈다 (Fig. 6). 위의결과로부터, Bifidobacterium spp. 자체의 C. difficile에대한항균활성은거의활인할수없었으나, 이에반해 Lactobacillus spp. 는자체적으로도항균활성을가지고있는것으로나타났으며, 특히 L. GG의경우는이전보고된연구결과 32) 와유사한활성도를나타냈다. 26
0.8 0.7 0.6 O. D. (600 nm) 0.5 0.4 0.3 0.2 0.1 0 Control B. B. bifidum B. breve B. infantis B. longum Control B. adolescentis B. bifidum B. breve B. infantis B. longum Strains Fig. 5. Antimicrobial activity of culture medium supernatants of Bifidobacterium spp. against C. difficile Filtered culture media supernatant of Bifidobacterium spp. was 100 ppm. Data represent the means ± S.D. (N=3) 27
1.0 0.9 0.8 0.7 O.D.(600 nm) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control L. acidopilus L.brevis L. bulgaricus L. helveticus L. GG S. thermopilus Control L. acidophilus L. brevis L. bulgaricus Strains L. helveticus L. GG S. thermophilus Strains Fig. 6. Antimicrobial activity of culture medium supernatants of Lactobacillus spp. against C. difficile Filtered culture media supernatant of Lactobacillus spp. was 100 ppm. Data represent the means ± S.D. (N=3) 28
3.5. AJNEA와유산균의상승효과 AJNEA를첨가하여배양한유산균상층액에대한항균효과를측정하였다. 사용한 Bifidobacterium spp. 중에서 B. adolescentis와 B. infantis에 AJNEA를첨가하여배양한배지상층액을처리한경우 AJNEA를첨가하지않고균주만배양한배양상층액을처리한경우와비교하였을때높은상승효과를보였다 (Fig 7). 반면, B. breve 와 B. longum의경우상승효과를보이지않았다. 높은상승효과를나타낸 B. adolescentis와 B. infantis에대하여첨가된 AJNEA의농도에따른 C. difficile에대한항균활성변화를측정하였다. 결과, B. adolescentis와우슬 50 ppm 첨가하여배양한배양상층액에서 25.6%, 200 ppm 첨가시 45.4% 의항균활성증가및상승효과를나타냈으며 B. infantis의경우, 12.5 ppm의 AJNEA 첨가시 30.4%, 200 ppm 첨가시 80.5% 의높은상승효과를나타내었다 (Table 5). Lactobacillus spp. 는 L. helveticus와 S. thermophilus에서약간의상승효과를보였으나나머지 Lactobacillus spp. 에서는상승효과가나타나지않았다 (Fig 8). 29
1.0 0.9 Bifidobacterium spp. Bifidobacterium spp. + AJNEA (100 ppm) 0.8 0.7 O. D. (600 nm) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control AJNEA 100 ppm B. adolescentis B. bifidum B. breve B. infantis B. longum Control AJNEA 100 pp B. adolescentis B. bifidum B. breve B. infantis B. longum Strains Fig. 7. Synergic antimicrobial effect of AJNEA and culture medium supernatants of Bifidobacterium spp. against C. difficile The concentration of sample was 100 ppm. Data represent the means ± S.D. (N=3) 30
Table 5. Synergic effect of AJNEA and each of culture media supernatant of B. adolescentis and B. infantis against C. difficile Inhibitory Effect (%) Treatment groups AJNEA concentration (ppm) 0 12.5 25 50 100 200 AJNEA - b - - 1.5±0.1 a 5.1±1.2 11.8±0.5 B. adolescentis+ajnea - - - 25.6±0.4 38.3±0.4 45.4±0.6 AJNEA - 1.5± 0.1 1.5± 0.2 1.5± 0.1 5.1± 0.2 11.8±0.5 B. infantis+ajnea - 30.4±0.3 43.4±1.0 73.4±0.6 73.9±0.1 80.5±0.7 a Result was presented as mean ± S.D. (N=3) b no antimicrobial activity 31
1.0 0.9 Lactobacillus spp. Lactobacillus spp. + AJNEA 100 ppm 0.8 0.7 O.D. (600 nm) 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Control AJNEA100 ppm L. acidopilus L.brevis L. bulgaricus L. helveticus L. GG S. thermopilus Control AJNEA 100 ppm L. acidophilus L. brevis L. bulgaricus L. helveticus L. GG Strains S. thermophilus Fig. 8. Synergic antimicrobial effect of AJNEA and culture medium supernatants of Lactobacillus spp. against C. difficile Data represent the means ± S.D. (N=3) 32
3.6. AJNEA 첨가 yoghurt의항균및상승효과앞에서실시한 AJNEA와유산균과의상승효과실험에서높은상승효과를보인 B. adolescentis와 B. infantis가포함된 5 group의 starter를제조한후, AJNEA을함유한 yoghurt를제조하여항균활성의변화도를확인하였다 (Fig. 9). ABT균주를 starter로하고, 0.5% AJNEA가첨가된 yoghurt의상층액을 1,000 ppm의농도로 C. difficile 에처리하였을때 0.5% AJNEA를첨가하지않은 yoghurt 상층액보다 50% 이상의상승효과를보였다. ABT : B. infantis = 1:1과 L. acidophilus : S. thermophilus : B. infantis = 1:1:1을 starter하여 0.5 % AJNEA를첨가한 yoghurt 상층액을 2,000 ppm 처리하였을때는 60% 이상의상승효과를보였으며, L. acidophilus : S. thermophilus : B. adolescentis = 1:1:1 starter로 0.5% AJNEA를첨가한 yoghurt 상층액또한 2000 ppm 을처리했을때 70 % 이상의높은상승효과를보였다. 반면, ABT : B. adolescentis = 1:1를 starter로한분획물첨가 yoghurt 상승액은 30 % 가량의낮은상승효과를나타냈다. 33
Inhibition (%) 0 100 80 60 40 Supernatant of Yoghurt (1000 ppm) Supernatant of Yoghurt adding 0.5% AJNEA (1000 ppm) Supernatant of Yoghurt (2000 ppm) Supernatant of Yoghurt adding 0.5% AJNEA (2000 ppm) `` 20 0 1 2 3 4 5 Fig. 9. Synergic antimicrobial effect of supernatants of yoghurt prepared with AJNEA and different starter species against C. difficile (1;ABT (L. acidophilus + L. bulgaricus + S. thermophilus), 2;ABT : B. adolescentis = 1:1, 3;ABT : B. infantis = 1:1, 4;L. acidophilus : S. thermophilus : B. infantis = 1:1:1, 5;L.acidophilus : S. thermophilus : B. adolescentis = 1:1:1) Data represent the means ± S.D. (N=3) 34
3.7. C. difficile toxin A 저해 AJNEA 가 C. difficile 의생장저해뿐만아니라, C. difficile toxin A 도 저해하는지농도별로조사하였다 (Fig. 10). 그결과 AJNEA 를 3,000 ppm 으로처리하였을때 toxin A 를나타내는 band 가관찰되지않은것으로보아 AJNEA 의농도가 3,000 ppm 일때 C. difficile toxin A 의생성을억제하는것으로사료된다. C. difficile toxin A 는장관투과성이높고, 세포막의손상을유도하여염증세포를점막내부로유입시켜염증반응을촉진시키는역할을하는것으로 보고되어 있는며 18), 우슬에서분리한 20-hydroxy ecdysone 의 cathepsin B 라는세포외막에존재하는효소에대한저해 활성을보고되어있다 19). 35
Fi g. 1 0. I n hi bi to r y ef fect of AJNEA with different concentration against C. difficile toxin A 36
4. 결론 항생제의광범위한사용으로항생제에내성을가지고있는 C. difficile에의한위막성대장염및항생제성설사가많이발생하고있으며, 현재사용된항생제에의한재발위험과막대한항생제치료비용을대체할수있는새로운천연기능성제제의개발이시급하다. 따라서본연구에서는병원성세균의성장을억제하고, 또한장내유익균총인유산균생장에는영향을미치지않는천연기능성소재를탐색하여 C. difficile 에대한항균효과및유산균과의상승효과에대해조사하였으며다음과같은결론을얻을수있었다. 1. 항균및항염증효과가있는 40여가지생약재의에탄올추출물에대하여 C. difficile에대한항균활성을측정한결과, 우슬 (Achyranthis radix), 희첨 (Siegesbeckiae herba), 황백 (Phellodendri cortex ) 은 90% 이상의높은항균성을보였다. 2. 90% 이상의높은항균활성을보인 3가지생약재에탄올추출물에대하여 n-hexane, chloroform, ethyl acetate, n-butanol 순으로용매분획을실시한후항균활성을측정한결과, 우슬에틸아세테이트분획물 (AJNEA) 은가장높은활성을나타내었고, 500 ppm에서 vancomycin 12.5 ppm에서의활성과유사한높은항균활성을나타 37
내었으며, 3,000 ppm에서는 C. difficile toxin A에대한저해활성도보였다. 3. AJNEA는 B. longum, L. helveticus 와 S. thermophilus를제외한나머지유산균에대해서거의생장억제효과를나타내지않았다. 4. L. acidophilus, L. bulgaricus와 L. GG의배양상층액은 C. difficile에대해 40 % 이상의높은항균활성을나타내었으나, Bifidobacterium spp. 는자체적인항균활성은나타내지않았다. 5. AJNEA를 100 ppm 첨가하여배양한 B. infantis 와 B. adolescentis 상승액은 C. difficile 생장에대하여각각 68.8% 와 33.2% 의상승항균효과를보였다. 6. L. acidophilus : S. thermophilus : B. adolescentis = 1:1:1 와 L. acidophilus : S. thermophilus : B. infantis = 1:1:1을 starter로 AJNEA를 0.5% 첨가하여제조한 yoghurt 상층액을 2,000 ppm 농도로처리하였을때 C. difficile에대하여 60 % 이상의항균상승효과를나타내었다. 7. 우슬추출물이 C. difficile 치료를위한천연항생제대체물로서의제제화가능성과천연기능성식품제조실용화가능성을시사하였다. 38
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감사의글 뭣도모르고대학원이라는곳을입학하여실험실에들어온날이엊그제같은데벌써 2년이라는시간이흘러이렇게감사의글을쓰게되었습니다. 대학원에들어와생활하는 2년동안길게만느껴졌던시간들이지금에와서는짧게만느껴집니다. 먼저그동안부족한저에게많은가르침을주시고, 논문을완성할수있도록지도해주신허태련교수님께진심으로감사의말씀을드립니다. 항상생물공학과의발전에여념이없으신생물공학과교수님들께도감사의말씀을드립니다. 제가힘들때옆에서위로도많이해주시고, 많은도움주신수임언니께감사드리고, 졸업진심으로축하드립니다. 우리실험실궂은일도맡아해줬던재원오빠, 뒷정리잘해줬던상민오빠와많은조언을해주신이기용박사님께도감사의말씀전합니다. 오빠들남은 1년더욱열심히하셔서좋은결실맺으셨으면좋겠어요. 그땐제가축하해드릴께요. 대학원동기, 선후배여러분덕분에저의대학원생활이외롭지않았던것같습니다. 감사합니다. 앞으로새로운대학원생활을하게될실험실의차석호교수님과진오언니께도항상좋은일만있길기원합니다. 내소중한친구들은정, 지연, 연수, 혜진, 민경, 현정, 정심, 보
라, 숙영, 명숙, 선진모두들바쁘다는핑계로친구로서잘챙겨주지못한점미안하고, 고맙다는말전합니다. 내년에대학원에입학하는윤선언니열심히연구해서좋은결과있길바라며, 조카힘들까봐많이챙겨주신외삼촌께도감사드립니다. 평택에서항상열심히하라며응원해준군자언니, 예쁜조카다이, 형부께도고마운마음전합니다. 하나밖에없는나의동생은영이의졸업또한축하하고, 이언니가많이사랑한다고전하고싶습니다. 마지막으로부족한점많은큰딸뒷바라지해주신어머니, 아버지께진심으로감사드립니다. 오래도록건강하시고, 행복하게사셨으면하는바람입니다. 저를응원해주고도와주신모든분들께도감사의말씀전합니다. 2005 년 12 월 27 일 정선미