오미자 추출물의 Streptococcus mutans KCCM 40105 균주에 대한 항균 활성 물질 분리 및 동정 = Isolation and identification of antibacterial active substances against Streptococcus mutans KCCM 40105 strain of Schisandra chinensis extracts

다국어 초록 (Multilingual Abstract)

In this study, natural materials showing antibacterial activity against Streptococcus mutans (S. mutans) KCCM 40105 were selected, and the most efficient extraction conditions were confirmed. The antibacterial activity was stable under various treatment conditions. Of the 17 material groups tested, extracts of Sophora flavescens (SF) and Schisandra chinensis (SC) exerted antibacterial activity against the S. mutans strain. The SF and SC extracts showed antibacterial activity against five microorganisms (B. cereus, S. aureus, L. monocytogens, E. coil, and E. cloacae). Analysis revealed that the contents of crude fiber in the SF extract were higher than in the SC extract, whereas contents of crude fat were higher in the SC extract than in SF. The nitrogen-free extracts obtained were 75.04% and 66.09% in SF and SC, respectively. Total free amino acids were determined to be 1,1717.17 mg% in SF and 825.98 mg% in SC. Measurement of antibacterial activity according to the ethanol concentration revealed that the 30% ethanol extracts of SF and SC have the highest activity, as seen by the inhibition zones of 10.69 and 14.49 mm, respectively. The minimum inhibitory
concentrations (MIC) of the 30% ethanol extracts of SF and SC were 2.0 mg/mL and 5.0 mg/mL, respectively, against the S. mutans strain. The stability to heat was confirmed, but the 30% ethanol extract of SC was not active at pH 6.0 or higher. The antibacterial activities of the 30% ethanol extracts of SF and SC treated with nine enzymes were the same at 10 ppm and 100 ppm. However, the activity tended to decrease slightly in the 1,000 ppm concentration treatment group. Among the solvent partitions of 30% ethanol extracts from Schisandra chinensis, the butanol portion showed the highest antibacterial activity. A total of 24 fractions were
obtained from the butanol portion using silica-gel chromatography. Of these, fraction number (Fr) 7 showed the highest antibacterial activity. Among the 33 sub-fractions isolated from the Fr 7, sub-number 17 showed the highest antibacterial activity. A total of 5 peaks were obtained by pure separation of the fraction of sub-number 17 using HPLC, and peak 2 was identified as a substance exhibiting major antibacterial activity. The purified compound of peak number 2 was identified as tartaric acid by UV spectrometry, ^(13)C-NMR, ^(1)H-NMR, LC-MS, and HPLC.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 5
• 1. 재료 5
• 가. 실험재료 5
• 나. 사용균주 및 배지 7
• 다. 시약 8
• 2. 실험방법 9
• 가. 항균 활성 식품 소재 탐색 9
• 1) Streptococcus mutans 균주의 항균 활성 식품 소재 탐색 9
• 2) 병원성미생물 5종에 대한 고삼과 오미자 추출물의 항균 활성 검색 10
• 나. 고삼, 오미자에 대한 성분분석 및 항산화활성 11
• 1) 일반성분 분석 11
• 2) 유리아미노산 분석 12
• 3) Total polyphenol 함량 측정 14
• 4) Total flavonoid 함량 측정 15
• 5) DPPH free radical scavenging activity 측정 16
• 6) ABTS radical cation scavenging activity 측정 17
• 7) Superoxide dismutase-like Activity 측정 18
• 다. 항균 활성 검색 19
• 1) 추출 용매별 항균 활성 측정 19
• 2) Ethanol 농도별 추출 20
• 3) 순차적 용매 분획별 항균 활성 측정 21
• 4) 최소저해농도(MIC) 측정 23
• 5) 열 및 pH 안정성 24
• 6) 유기물 분해 효소 처리에 따른 안정성 평가 25
• 7) 산화효소와 과산화수소 처리에 따른 안정성 평가 26
• 8) 추출물 첨가 농도에 따른 구강 세정제 제형의 항균 활성 검색 27
• 라. 오미자 추출물의 항균 활성 물질 분리 및 활성 측정 28
• 1) 30% ethanol 추출물 제조 28
• 2) 용매 분획 29
• 3) Silicagel column chromatography에 의한 분리 31
• 가) 1차 silicagel column chromatography 31
• 나) 2차 silicagel column chromatography 34
• 4) High-performance liquid chromatography (HPLC)에 의한 순수분리 36
• 5) 오미자 지표 물질 schizandrin, gomisin A 및 kaempferol의 HPLC 크로마토그램과 항균 활성 확인 38
• 마. 항균 활성 물질 동정 39
• 1) UV spectrum 39
• 2) LC-MS에 의한 오미자 항균 활성 물질의 분자량 확인 40
• 3) Nuclear magnetic resonance(NMR)에 의한 구조 확인 42
• 4) HPLC를 통한 물질 검색 43
• 5) Tartaric acid와 오미자 항균 활성 물질의 UV spectrum 비교 45
• 바. 통계처리 46
• Ⅲ. 결과 및 고찰 47
• 1. 항균 활성 식품 소재 탐색 47
• 가. Streptococcus mutans 균주의 항균 활성 식품 소재 선발 47
• 나. 병원성미생물 5종에 대한 고삼과 오미자 추출물의 항균활성 51
• 다. 고삼, 오미자에 대한 성분분석 및 항산화활성 55
• 1) 일반성분 함량 55
• 2) 유리아미노산 함량 57
• 3) Total polyphenol 및 Total flavonoid 함량 59
• 4) DPPH free radical scavenging activity 62
• 5) ABTS radical cation scavenging activity 64
• 6) Superoxide dismutase-like activity 66
• 2. 항균 활성 검색 68
• 가. 추출 용매 및 ethanol 농도별 항균 활성 검정 68
• 나. 순차적 용매 분획물의 항균 활성 검정 72
• 다. 최소저해농도(MIC) 75
• 라. 열 및 pH 안정성 78
• 마. 유기물 분해 효소 처리에 따른 안정성 82
• 바. 산화효소와 과산화수소 처리에 따른 안정성 86
• 사. 추출물 첨가 농도에 따른 구강 세정제 제형의 항균 활성 89
• 3. 오미자 추출물의 항균 활성 물질 분리 및 동정 92
• 가. 1차 silicagel column chromatography에 의한 분획 및 검정 92
• 나. 2차 silicagel column chromatography에 의한 분획 및 검정 94
• 다. High-performance liquid chromatography(HPLC)에 의 한 순수분리 및 물질 확인 96
• 1) HPLC에 의한 1차 순수 분리물의 항균 활성 96
• 2) HPLC에 의한 2차 순수 분리물의 항균 활성 99
• 3) 오미자 지표 물질 schizandrin, gomisin A 및 kaempferol의 HPLC 크로마토그램과 항균 활성 확인 102
• 라. 항균 활성 물질 동정 105
• 1) UV spectrum 105
• 2) LC-MS에 의한 오미자 항균 활성 물질의 분자량 확인 107
• 3) Nuclear magnetic resonance(NMR)에 의한 구조 확인 109
• 4) HPLC를 통한 물질 확인 111
• 5) Tartaric acid와 오미자 항균 활성 물질의 UV spectrum 비교 113
• Ⅳ. 요 약 115

참고문헌 (Reference)

1. Dental caries, Selwitz RH , Ismail AI , Pitts NB, 369 , 51-59, , 2007

2. Dental plaque formation, Rosan , B. and Lamont , R. J, 2 , 1599-1607, , 2000

3. The stephan curve revisited, Bowen WH, 101 , 2-8, , 2013

4. Trends in the utilization of enzyme in food industry, Oh SH, 9 , 1-9, , 2004

5. Antioxidant activity of extracts from the Lespedeza bicolor, Lee YS , Joo EY , Kim NW, 12 , 7-79, , 2005

6. Antioxidant determination by the use of a stable free radical, Blois MS., 191 , 1199-1200, , 1958

7. Changes of biologically active components in Pruns mume fruit, Seo KS , Huh CK , Kim YD, 15 , 269-273, , 2008

8. A new prenylated flavonone from the roots of Sophara flavescens, Kang SS , Kim JS , Son KH , Chang HW , Kim HP, 71 , 511-515, , 2000

9. Antimicrobial and anti-halitosis effects of Alnus firma extracts, Choi HY , Heo NS , Choi YW , Lee YG , Jeong YK , Joo WH, 22 , 1071-1076, , 2012

10. Antioxidant activities of ethanol extracts from barley sprouts ., Chae KS , Ryu EH , Kim KD , Kim YS , Kwon JW, 51 , 486-491, , 2019

11. Antimicrobial activity of Omija ( Schizandra chinensis ) extracts, Chung KH , Lee SH , Lee YC , Kim JT, 30 , 127-132, , 2001

12. Antibacterial effects of Sophora flavescens on Streptococcus mutans, Lee HO , Lee KH , Park NK , Jeong SI , Baek SH , Han DM, 13 , 539-546, , 2000

13. A study on determining chemical compositions of Schizandra chinensis, Hyun KH , Kim HJ , Jeong HC, 15 , 1-7, , 2002

14. Antibacterial effect of oriental medicinal herbs on dental pathogens, Yu YE , Park EY , Jung DH , Byun SH , Kim SC , Park SM, 46 , 200-206, , 2010

15. Quantitative analysis of Lignans from fruits of Schisandra chinensis, Kim KS , Kang SS , Ryu SN, 33 , 272-276, , 2002

16. Antimicrobial activity of coriander ( Coriandrum sativum L. ) extract, Kim YD , Kang SK , Choi OJ, 30 , 692-696, , 2001

17. Antioxidant activity of medicinal plant extracts cultivated in Jecheon, Min SH , Lee BR, 22 , 336-341, , 2007

18. Contents of total flavonoid and biological activities of edible plants, Lee JM , Son ES , Oh SS , Han DS, 16 , 504-514, , 2001

19. Antimicrobial activities of edible plant extracts against oral bacteria, Lee J , Kim Y , Kim DC , Chae HJ, 63 , 61-67, , 2020

20. Antimicrobial activity of medicinal herbs against staphylococcus aureus, Choi IL , Cho JY , Lim SC, 19 , 491-496, , 2006

21. Antimicrobial activity of Streptococcus mutans by herbal medicine extracts, Jeun ES , Yoon SH , Han MD, 2 , 31-28, , 2002

22. Biological activities of extracts from Omija ( Schizandra chinensis baillon ), Kwon HJ , Park CS, 15 , 589-592, , 2008

23. Effects of Lignum sappan extract on the growth of Streptococcus mutans KCTC 3065, Jeon ES , Han MD, 4 , 127-131, , 2004

24. Antimictrobial effect of natural plant extracts against periodontopathic bacteria, Lee SH , Kim MJ, 19 , 242-255, , 2019

25. Acetic acid fermentation properties and antioxidant activity of lemongrass vinegar, Lee MR , Kang CH , Bu HJ, 24 , 680-687, , 2017

26. Antimicrobial activity of some medical herbs and spices against Streptococcus mutans, You YS , Park KM , Kim YB, 21 , 187-191, , 1993

27. Antimicrobial effects of Schisandra chinesis extracts against Liseteria monocytogens, Lee SH , Lim YS, 25 , 229-234, , 1997

28. Determination of oxymatrine in Sophora radix by high performance liquid chomatography, Yang SK , Yun YJ , Namgung MO, 17 , 289-294, , 2004

29. Physicochemical propeties of Salvia miltiorrhiza bunge following treatment with enzymes, Kim SH , Hwang IW , Chung SK , Seo YJ , Kim JS , Jeong YJ , Kim MY, 22 , 699-707, , 2015

30. The antioxidative effect and ingredients of oil extracted from Schizandra chinensis seed, Ryu IH , Kwon TO, 20 , 63-71, , 2012

31. Inhibitory effects of Enterococcus faecium isolated from Korean infants on oral pathogens, Jeong EG , Lee JC , Seo JY , Kim SY , Kim Wan SU , Yun WH , Kim YS , Pi SH , You HK , Shin HS, 38 , 31-40, , 2008

32. Screening of the natural plant extracts for the antimicrobial activity on dental pathogens, Baek DH, 43 , 227-231, , 2007

33. Chemical components composition on different parts of fruit in Schisandra chinensis baillon, Lee KS , Lee BH , Seong BJ , Km SI , Han SH , Kim GH , Park SB , Kim HH , Choi TY, 45 , 851-858, , 2016

34. Antibacterial activities of Et-OH extract from extruded white ginseng on tooth decay bacteria, Son HJ , Han MS , Ryu GH, 38 , 951-957, , 2009

35. Prenylated flavonoids from the roots of Sophara flavescens with tyrosinase inhibitory activity, Son JK , Park JS , Kim JA , Kim Y , Chung SR , Lee SH, 69 , 559, , 2003

36. Isolation of antimicrobial substance from Schizandra chinensis baillon and antimicrobial effect, Lee JY , Min YK , Kim HY, 33 , 389-394, , 2001

37. Screening of antibacterial agent against Streptococcus mutans from natural and medicinal plants, Kim KW , Baek JK , Jang YW , Kum EJ , Kwon YS , Kim HJ , Sohn HY, 15 , 715-725, , 2005

38. Antioxidant activity and nitrite scavenging ability of olive leaf ( Olea europaea L. ) fractions, Choi NY , Lee JH , Shin HS, 40 , 257-264, , 2008

39. Antioxidant and antimicrobial effects of lemon and eucalyptus essential oils against skin floras, Kim JH , Kim MJ , Choi SK , Bae SH , An SK , Yoon YM, 37 , 303-308, , 2011

40. Effects of Schizandra chinensis extract on the growth of intestinal bacteria related with obesity, Jeong EJ , Lee WJ , Kim KY, 41 , 673-680, , 2009

41. Screening of aqueous extracts of medicinal herbs for antimicrobial activity against oral bacteria, Yim NH , Jung YP , Cho WK , Kim TS , Kim AY , Im MJ , Ma JY, 2 , 18-24, , 2013

42. Antimicrobial activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts ., Jeon ES , Han MD , Kim HD, 3 , 39-44, , 2003

43. Antioxidant activities and quality characteristics of Omija ( Schisandra chinensis Baillon ) cookies, Kim MK , Lee JM , Do JS , Bang WS, 24 , 931-937, , 2015

44. Schizandrin , oil compounds , and their extraction yield in fruits of Schisandra chinensis Baillon ., Kim KS , Park CG , Ryu SN , Bang JK , Lee BH, 45 , 158-162, , 2000

45. Antibacterial and antibiofilm activities of Diospyros malabarica stem extract against Streptococcus mutas, Kim HS , Lee SW , Sydara K , Cho SJ, 29 , 90-96, , 2019

46. Antimicrobial activity of Coptis chinensis and Sophora flavescens against Streptococcus mutans ATCC 25175, Eum JS, 16 , 384-389, , 2012

47. Antimicrobial activity of Psoralea corylifolia , Schisandra chinensis , and Spatholobus suberectus extracts, Choi EJ , Jang SR , Kang OJ , Bang WS, 45 , 495-500, , 2013

48. Comparison of the nutrient composition and quality of the root of Allium hookeri Grown in Korea and Myanmar, Park JY , Yoon KY., 46 , 544-548, , 2014

49. The antimicrobial potential of 14 natural herbal dentifrices : Results of an in vitro diffusion method study., Lee SS , Zhang W , Li Y, 135 , 1133-1141, , 2004

50. Comparison of the composition of free sugars , amin acids and minerals in black Omija ( schizandra niger max. ), Shin SC , Hyun KH , Lee KY, 11 , 47-50 (, , 1998

51. Investigation of antimicrobial activity of brown algae extracts and the thermal and pH effects on their activity, Lee SY , Kim JH , Song EJ , Kim KBWR , Hong YK Lim SM , Ahn DH, 18 , 506-512, , 2009

52. The antioxidative , antimicrobial and nitrite scavenging effects of Schizandra chinensis Ruprecht ( Omija ) seed, Jung GT , Ju IO , Choi JS , Hong JS, 32 , 928-935, , 2000

53. Effects of methanolic extract from Salvia miltiorrhiza bunge on in vitro antithrombotic and antioxidative activities, Yang SA , Im NK , Lee IS, 39 , 83-87, , 2007

54. Changes of physicochemical haracteristics of Schizandra chinensis during postharvest ripening at various temperatures, Jeong PH , Kim YS , Shin DH, 38 , 469-474, , 2006

55. Growth characteristics , main constituents and antioxidant activities in local accessions of Sophora flavescens AIT ., Kwon AR , Kim SI , Seong BJ , Jee MG , Lee KS , Kim HH , Doh ES ., 28 , 360-370 (, , 2020

56. Effects of drying methods on quality characteristics and antioxidative effects of Omija ( Schisandra chinensis baillon ), Lee S , Moon HK , Lee SW , Moon JN , Kim JK, 21 , 341-349, , 2014

57. Component analysis and comparison of biological activities of Salvia miltiorrhiza bunge from different cultivation regions, Yang EJ , Seon YK , Seo YS , Sing BY, 46 , 929-936, , 2017

58. Inhibitory effect of the Korean herbal medicine , dae-jo-whan , on platelet activating factor-induced platelet aggregation, Chang GT , Kang SK , Kim JH , Chung KH , Chang YC , Kim CH, 102 , 430-439, , 2005

59. Effects of Caesalpinia sappan extracts on the growth , acid production , adhesion , and insoluble glucan synthesis of Streptococcus mutans, Yu YO , Yu HH , Kim YJ , Yu MS , Seo SJ , Lee L , Lee HS, 27 , 277-288, , 2003

60. Global , regional , and national incidence , prevalence , and years lived with disability for 354 diseases and injuries for 195 countries and territories , 1990-2017 ; A systematic analysis for the Global Burden of Disease Study 2017, James SL , Abate D , Abate KH , Abay SM , Abbafati C , Abbasi N ,, 392 , 1789-1858, , 2018