,, Screening and application of antimicrobials, anticancers, antitumors and antioxidants from native biomaterials

Transcription

1 1999,,. : : : ( ) :

2 ,, Screening and application of antimicrobials, anticancers, antitumors and antioxidants from native biomaterials

3 ,, : : : 7 : : : : - 1 -

4 .,,. (WTO),..,,, know-how

5 . lysozyme. lysozyme. lysozyme 3, 13 lysozyme single-step..,. DNA

6 . -, ,,,

7 ,.,, 3. 1,, 1. (Caesalpinia sappan. L) 60,,. brazilin protosappanin A. CHCl3-5 -

8 , ascorbic acid. CHCl3 (500 ppm) (60 ) BHT(200 ppm), BHA(200 ppm) -tocopherol(200 ppm). (Rhus vernieifula STOKES) 10 75% ethanol Rancimat, oven test, DPPH. gallic acid, butin, butein, sulfuretin. BHA tocopherol.,. 75% ethanol AI 4.2 AI 1.44 chloroform.. 75% ethanol chloroform. Chloroform phenol (RCF) BHT BHA 5, 2. MASS, Spectrometer NMR RCF-11-1 ; gallic acid, RCF-11-2 ; 3,4,7-trihydroxy-flavenone (butin), RCF-11-3 ; 2,4, 3, 4 tetrahydroxychalcon(butein), RCF ; 3,4,6 trihydoxyaurone(sulfuretin). Lecithin liposome 200 ppm lipid peroxide inhibition(%) RCF tocopherol

9 2. (Glycyrrhiza uralensis Fisch) 70,,,. Listeria monocytogenes 5 Staphylococcus aureus, Pseudomonas fluorescens, Bacillus cereus, Escherichia coli. Kuwanon G, liquritigenin. 70 Listeria Bacillus,,,. chloroform Listeria, Bacillus, E. coli, Staphylococcus aureus Kushenol F. Kushenol F 30 ppm Gram(+) Gram(-). ethanol n-hexane Listeria monocytogenes ppm % ethanol. 75% ethanol hexane chloroform 30 ppm 72.. A8 silica gel column chromatography SBD-1 SBD-2 SBD-1, SBD-2 (9z)-heptadeca-1,9-dien

10 4,6-diyn-3,8-diol(falcarindiol). SBD-2 monoglyceride Listeria.. 75% ethanol 40 ppm Listeria monocytogenes 5 72, minimum bactericidal concentration(mbc) 40 ppm. Chloroform silica gel chromatography gravacridonediol., Listeria monocytogenes 4. 5 ca.107cfu/g 8. 2 food grade PM30 lysozyme 0.25%, 35, 30 psi, 300 rpm. 12 YM30 flux, flux PM30. PM30 lysozyme YM30, 2. 5 PM30 PM30 flux 30% flux, - 8 -

11 35 flux 70%. lysozyme 110 units/ml, 2,821 units/ mg protein, PM30 lysozyme. lysozyme PM30 99% PFS PMS purification factor 0.25%. (0.25%, w/v) ovalbumin, PMS lysozyme. 0.9 m, SEM 0.1 N NaOH., polysulfone PM30 lysozyme. PM30 lysozyme scale-up, pump 3, 20, 16 psi. flux 5 18 LMH flux. PN01E PN03C pump speed flux, PN03C flux., PM30 flux PN03C flux polysulfone lysozyme

12 %. DNA, DNA, DNA 20-40%,. 5-10% 12. In vitro foci,, 5-27%., Balb/c mouse,,. bleomycin, DNA, DNA DNA, nuleosome-sized DNA ladder.,., western blot MMS DNA p53 PD PT, p

13 cyclin E, cyclin D1, CDK2, PCNA., DNA GADD45 western blot PD, PD. RCF-11 SNP MTT assay, SNP RCF-11 RCF-11, RCF-11. SNP RCF-11 12, RCF SNP. FACS. DNA, RCF-11 DNA synthesis rate. RCF-11 RCF SNP SNP DNA.,,.,,..,

14 .. 1. o,,, o (,, ) 21 o, medical foods,, o o 2. o 18 o 20 o,,

15 Summary. Title of Research Screening and application of antimicrobials, anticancers, antitumors and antioxidants from native biomaterials. The Objective and Importance of Research In recent years, the form of World Trade Organization(WHO) has led to an urgent condition to domestic agricultural economy, as we have got to compete fiercely with foreign products for the weakened competitive edge in agricultural field by the imported agricultural products. Therefore, it is necessary to develope and commercialize the high value-added agricultural products to cope with an open trade policy and a foreign competition. Moreover, it is the fact that in the grim reality of international society the know-how about an investigation procedure of useful substances, especially antimicrobials, anticancers, antitumors and antioxidants with physiological active agent is thoroughly kept a secret. Consequently we are badly in need of the investigation of these new substances and the development of manufacturing technique and process in

16 order to strengthen the competitiveness of our native biomaterials from farm and forest products on international markets and increase wealth in agricultural community and mountain village.. The contents and category of research o Selection of edible animals and plants - Selection of the control group to examine antimicrobial and antioxidative activity from edible animals and plants and medicinal plants. - Experiment on effect of components and metabolic products of eggs. - Experiment on effect of components and metabolic products of animals and plants. - Experiment at the level of culture cell on effect of anticancers and antitumors of extracts from animals and plants. o The establishment of methods to isolate and refine active substances - Experiment on the optimum isolation method of selected substances. - Establishment of applicable methods to refine active substances. - Content measurement of active substances according to conditions - Establishment of isolation refining method and correlation between

17 the organic functional analysis for antimicrobials, anticancers, antitumors and antioxidants o Experiment on identification of active substance and its effect - Identification of active substances. - Isolation of active substances. - Experiment on effect of new substances. - Research on the change of functionality of new substances and reinforcing condition. o Application experiment on food and culture cell - Identification of antimicrobial substances and application on food for shelf life extension. - Experiment on effect of antioxidative substance and its food application. - Identification of anticancer and antitumor effect through animal and culture cell experiments. - Mass revelation of natural substances and genes, refining and safety evaluation

18 . Results of research The research on Screening and application of antimicrobials, anticancers, antitumors and antioxidants from native biomaterials consists of 3 details research units and is summarized in terms of unit. The 1st research subject Screening and application of antimicrobials and antioxidants from native biomaterials. 1. Screening and effect measurement of antioxidative substances The antioxidative activities of more than 60 edible or medicinal plants that have not been experimented before(e.q. Caesalpinia sappan. L) were examined and extracts from medicinal plants such as Caesalpinia sappan. L, Dalbergia odorifera T.CHEN showed more effective antioxidative activity than commercial antioxidants. The antioxidative active compounds of Caesalpinia sappan. L were isolated and identified as brazilin and protosappanin A, which were a very effective antioxidative compounds as natural substances and revealed high antioxidative activity on edible oil. Chloroform fraction of Dalbergia odorifera T.CHEN extract showed strong antioxidative activity on lard and palm oil, and synergistic effect with ascorbic acid. In accelerated storage test on lard and palm oil at 60, chloroform fraction(500 ppm) of Dalbergia odorifera T.CHEN extracts showed

19 similar antioxidative activity to BHT(200 ppm) and stronger activity than BHA(200 ppm) and -tocopherol(200 ppm). Antioxidative activity of 75% ethanol extracts from more than 10 edible or medicinal plants such as Rhus verniciflua stokes etc. was examined by Rancimat, oventest, and DPPH method. The antioxidative active compounds of Rhus verniciflua STOKES, Caesalpinia sappan. L and Dalbergia odorifera T.CHEN were isolated and identified as gallic acid, brazilin and mucronulatol, respectively. The compounds showed more effective antioxidative activity than commercial antioxidants such as BHA and tocopherol, and was judged to show the possibility of a new natural antioxidant. These antioxidants exhibited thermal stability and retardation of oxidation in food application experiment, satisfying the commercial purpose. 75% ethanol extracts from Myristica fragrans Houttuyn were examined on lard and palm oil and its AI was 4.2 and 1.44,respectively. Its chloroform fraction exhibited the strong antioxidant activity. The identification of antioxidative components from Rhus verniciflua STOKES was carried out. 75% ethanol extracts of Rhus verniciflua STOKES Bark were examined on lard and palm oil, and showed comparatively strong antioxidative activity, chloroform fraction exhibited the strongest antioxidant activity. The free phenolic acid fraction of chloroform extract showed that the RCF was 5 times on lard and twice on palm oil as effective as commercial antioxidants such as BHT and BHA. The following chemical structure was identified through MASS, spectrometer and NMR. RCF-11-1 : gallic acid RCF-11-2 : 3',4',7-trihydroxyflavenone(butin)

20 RCF-11-3 : 2',4',3,4-tetrahydroxychalcone(butein) RCF-13-1 : 3',4',6-trihydroxyauron(sulfuretin) Liposome system was made with lecithin, each fraction 200 ppm was added to it, and lipid peroxide inhibition(%) was measured. In consequence, RCF-11-3 showed the stronge antioxidative activity but lower activity than -tocopherol. 2. Isolation and effect measurement of antimicrobial active substance Antioxidative activities of over 70 edible or medicinal plants such as Glycyrrhiza uralensis Fisch were examined and screening of Morus alba Linne, Glycyrrhiza uralensis Fisch, Sophora flavescens Ait and Dryopteris crassirhizama Nakai with strong effect was completed. Antimicrobial effect of tested plant extract on food poisoning bacteria (5 strains of Listeria monocytogenes and Staphylococcus aureus) and food spoilage bacteria (Pseudomonas fluorescens, Bacillus cereus, Escherichia coli) has been proved to be quite strong. Antimicrobial compounds of extracts of Morus alba Linne and Glycyrrhiza uralensis Fisch was identified as Kuwanon G and liquritigenin respectively and they showed far more effective antimicrobial activity than commercial antimicrobial agents. Myristica fragrans Houttuyn, Sophora flavescens Ait, Dystaenia takesimana kitagawa, and Heracleum mollendorffii Hance were selected from 70 species of edible medicinal plants, and they had growth inhibition effect on food poisoning bacteria Listeria. Chloroform fraction of Sophora flavescens

21 Ait. exhibited strong growth inhibition on Listeria monocytogenes, Bacillus, E.coli and Staphylococcus aureus. Its antimicrobial compound was identified as Kushenol F. Kuschnol F. from Sophora flavescens Ait showed strong growth inhibition on Gram(+) and Gram(-) bacteria at 30 ppm and showed far more effective antimicrobial activity than commercial antimicrobial agents. Ethanol fraction and n-hexane fraction of Myristica fragrans Houttuyn showed growth inhibition on 5 strains of Listeria monocytogenes and its compounds are being identified. Ethanol extract of Dystaenia takesimana lkitagawa(ulrung Island native plant) exhibited strong growth inhibition and bactericidal effect on food poisoning bacteria and spoilage bacteria at 10 ppm and its compounds are being identified. 75% ethanol extract from more than 90 species of edible or medicinal plants was screened antimicrobial activity. Fractionation of 75% ethanol extracts from Heracleum mollendorffii Hance showed that hexane and chloroform fraction had strong antimicrobial activity and exhibited growth inhibition for 72 hours even 30 ppm level. Identification of antimicrobial active substance from Dystaenia takesimana kitagawa was examined. After silica gel column chromatography, SBD-1 and SBD-2 were separated from A8 fraction. SBD-1 was identified as complex of fatty acid and SBD-2 was identified as (9z)-heptadeca-1,9- dien- 4,6-diyn-3,8-diol(falcarindiol). Addition SBD-2 to monoglyceride led to remarkable improvement of antimicrobial activity on Listeria. Antimicrobial activity of Ruta graveolens Linne was measured. 75% ethanol extracts of Ruta graveolens Linne root exhibited growth inhibition on 5 strains of Listeria monocytogenes for 72 hr even at low concentration 40 ppm

22 and minimum bactericidal concentration was 40 ppm. Pure substance from chloroform fraction by silica gel column chromatography was identified as gravacidonediol. Food application experiment of Glycyrrhiza uralensis Fisch and Dystaenia takesimana kitagawa fractionate were carried out. Antimicrobial active fraction with growth inhibition effect on Listeria monocytogenes in liquid medium was added to cooked beef and it was stored at the normal temperature. In consequence, we found no effect of antimicrobial activity when plant extracts were applied to cooked beef. Because they had almost same bacteria numbers in control as well as in treatment. The 2nd research subject Development of continuous membrane separation process of antimicrobial enzyme which can be used as natural food grade preservatives Hen egg white lysozyme (HEWL) is very valuable as a natural preservative in food processing due to its selective bactericidal activity. HEWL which traditionally isolated by crystallization or freeze drying was simply separated from 13 different hen egg white (HEW) proteins by a single-step ultrafiltration. Freeze dried HEW (0.25%, w/v) dissolved in a citrate-phosphate buffer (ph 4.6) was ultrafiltered with a PM30 membrane under various operating

23 conditions, by changing concentration, temperature, transmembrane pressure ( PT), and stirring speed. Optimum separation conditions were decided when maximal flux was obtained. Under the optimum separation conditions, the effect of membrane material and fouling on flux as time passed as well as lysozyme concentration, protein concentration, specific activity (SA) in the permeate were measured. To determine the optimum separation concentration among the different hen egg white protein (HEWP) concentrations (0.25, 0.5, 1.0%, w/v), protein concentrations, lysozyme concentrations, specific activities (SA), and purification factors of prefiltered solution (PFS) and ultrafiltered solution(pms) were determined and compared. The purity of lysozyme separated at each step was analyzed and confirmed by gel chromatography and electrophoresis. The fouling deposits on membrane were observed by SEM. The optimum separation conditions of the scale-up process were to be determined based on the predetermined optimum conditions in bench-top scale. Using hollow fiber membrane made of polysulfone, the effects of feed flow rate, inlet pressure, and feed temperature on flux were measured. Under the optimum separation conditions, the change of flux as time was observed. The flux was measured by changing feed flow rate and inlet pressure when PN01E and PN03C membrane, which has different membrane material and pore size with PM30 membrane, were used. Best separation conditions of HEWL with PM30 membrane were sample concentration 0.25%, temperature 35, PT30 psi, and stirring speed 300 rpm. During the first 12 min, the flux of YM30 was higher, but at the steady-state it was lower than that of PM30. The SA of the PM30 permeate was over 2 times higher in spite of the lysozyme and protein

24 concentration being lower than that of YM30 permeate. The flux of 5 times used PM30 decreased 30% compared to a new PM30, but both had the same tendency in flux decrease when time passed. Both of them reached a steady-state after 35 min and remained at 70% of the initial flux. In the PM30 permeate, the lysozyme concentration and SA were 110 units/ml and 2,821 units/mg protein, respectively. Therefore, PM30 membrane separation was very effective for separation of antimicrobial lysozyme. The non-enzymatic proteins were removed over 99% by ultrafiltration(uf). The increased feed concentration did not contribute to the increase of SA. SA of PMS was 18 to 31 times higher than that of PFS. The optimum feed concentration was decided as 0.25% based on SA and purification factor. The non-enzymatic region of gel chromatogram was proved to be ovalbumin. The thickness of deposit on the UF membrane was approximately 0.9 m and removed by cleaning with 0.1 N NaOH. In scale-up separation process with PM30 hollow fiber membrane, the optimum separation conditions were pump speed 3, feed temperature 20, inlet pressure 16 psi. Although the severe decline in flux was observed in the initial filtration stage, the flux was maintained 18 LMH after 5 minutes. The increase in flux with PN01E and PN03C membrane showed different pattern as inlet pressure and pump speed increased. PN03C showed higher flux than PN01E under the optimum conditions. Since the flux of PM30 membrane was higher and the flux decline was much lower than PN03C, polysulfone hollow fiber membrane was the best in separation of natural antimicrobial lysozyme in large-scale process

25 The 3rd research subject Isolation of anti-tumor materials from Korean natural products and analyzes of their cellular and in vivo activities From the selected materials, the mulberry and ginesing aqueous extracts increased to about 10-40% of the cell membrane integrity and cell survival decrease induced by genotoxix treatment. Repair assays including DNA synthesis rate, UDS, DNA strand break also indicated that these extracts increased the repair activities indcued by genotoxix treatements. The presence of metaboliec activation system potentiated these activities. In vitro foci formation assay showed that these materials significantly decreased the frequencies of cancer cell induction. In vivo experiments using Balb/c mouse system also indicated their preventive effects on the formation of skin, lung, and liver cancers. The bleomycin-induced apoptosis of cancer cell was potentiated by these materials as judged by nuleosome-sized DNA ladder formation. Components of Rhus verniciflua and ginseng extracts were selected after screening of apoptosis-enhancing activities. In the case of ginseng, western blot analyses indicated that PD and PT saponin increased the expression of p53 and p21 after genotoxic treatment. in harmony with these results the expression of cell cycle-related proteins such as cyclin E, cyclin D1, CDK2, and PCNA was decreased. However, the expression of GADD45 was increased by PD saponin and decreased by PT saponin. RCF-11, the mixture of purified components from Rhus verniciflua, showed a increasing effect of cell survival induced by SNP when analyzed by MTT

26 assays. The effects were similar either when cotreated, post-treated, or pre-treated. When the RCF concentration was high, however, the effects were reversed. These results were also observed by cell morphology analysis and FACS ananlyses. Abnormal DNA synthesis induced by SNP was supressed by RCF. Various combination of these components with food-additives such as honey, fructose, etc. also showed the above bioactive effects and safeties were conserved during all processes. Thus these extracts and components showed sufficient bioactivities and safeties in concentrations applicable for food or drug applications.. Application plan of research results 1. Application plan o Application natural anticancers and antioxidants to functional beverage such as health beverage and antioxidants and antimicrobials to various kinds of processed food. o Careful attention must be paid to various food properties change in storage, senses, and functionality etc. and its problem solution caused by new substances use. Moreover, new techniques should be prepared for old age society and it should be developed. o It is necessary to research on method for application of medical

27 foods such as old age food and patient food and production on a commercial scale and development of functional ingredients-enriched foods. Then, there will be in great demand for healthful and lifeoriented processed food. o Research on application a manufacturing process of functional foods to products development of various medicinal plants, the subject of this study, is necessary. o Application a membrane separation manufacturing process to the other foods is necessary. 2. Make public through technical journals and mass media o Contribute to well-known scientific journal in domestic and abroad.(18 papers) o Presentation on conferences or meetings.(20 proceeding) o Submit to various company bulletins

28 Contents Presentation note 1 Abstract(in Korean) 2 Summary(in English) 13 Contents(in English) 26 Contents(in Korean) 28 Isolation, identification and food application of antimicrobial and antioxidative substances from edible plant materials 31 Contents 33 Chapter 1. Introduction 35 Chapter 2. The contents and category of research 39 Chapter 3. Results of study 45 Chapter 4. Achievement of study and contribution 202 Chapter 5. Application plan of research results 206 Chapter 6. Reference 207 Development of continuous membrane separation process of antimicrobial enzyme which can be used as natural food grade preservatives 243 Contents 245 Chapter 1. Introduction 247 Chapter 2. The contents and category of research

29 Chapter 3. Results of study 260 Chapter 4. Achievement of study and contribution 312 Chapter 5. Application plan of research results 314 Chapter 6. Reference 315 Isolation of anti-tumor materials from Korean natural products and analyzes of their cellular and in vivo activities 321 Contents 323 Chapter 1. Introduction 325 Chapter 2. The contents and category of research 330 Chapter 3. Results of study 334 Chapter 4. Achievement of study and contribution 370 Chapter 5. Application plan of research results 373 Chapter 6. Reference

30 ,, food grade

31

32 ,, : : :

33 ,, Listeria monocytogenes (DryopteriscrassirhizomaNakai) Listeriamonocytogenes L. monocytogenes

34 8 Falcarindiol Listeria monocytogenes Bulk emulsion system Listeria monocytogenes (Glycyrrhiza uralensis Fisch) (Dystaenia takesimana kitigawa)

35 1 (WTO),.., know-how (, 1995) (, 1987)

36 ,,. (Beuchat, 1989), (Wills, 1956). allicin -SH group, (Al-Delainy, 1970). ( a, 1988). eugenol anethole Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Clostridium perfringens Escherichia coli. cinnamic aldehyde aflatoxin thymol (Buchanan, 1981). (, 1989), 14 5, (, 1990). (, 1979), (, 1965) (, 1968)., (, 1979), (Valsa ceratosperma) (, 1988) (, 1991). 28 Pythium ultimum (Bae, 1987),, 9 (, 1990)

37 Listeria monocytogenes ( c, 1994). (Harman, 1982) tocopherol. BHA, BHT (Ito, 1983) (Kasuga, 1988;, 1992). tocopherol (Haumann, 1990) (Coal, 1974).. caraway, sage, cumin, rose mary, thyme, clove (Faraga a, 1989) thyme clove (Faraga b, 1989) (, 1982). sesamolinol, sesaminol, sesamol (, 1990) (, 1981) methanol (, 1987). ethanol (, 1992) tocopherol synergistic effect. epigallocatechin epigallocatechin gallote,

38 (, 1993), (Yen, 1993) (Tsuda, 1993) tocopherol synergistic effect. rosemary (Eun, 1993) ( b, 1992). tocopherol rosemary.,

39 , synergist % ethanol 1 methanol hexane,.,,. 3 6,. hexane

40 .. chlorlform, ethyl acetate, butanol, ethanol silica gel column chromatography,. TLC, spot. GC-MS, NMR IR,. 3.. Rancimat ppm,,. antioxidant index(ai) - induction time induction time ,, ppm

41 60 POV, TBA, AV POV, TBA, AV.. Synergist, Rancimat. synergist ascorbic acid, palmitic acid, tocopherol, lecithin. 1.. L. monocytogenes ATCC, Disk method., seitz filter,, filter paper disk ( 7mm)

42 , aerobic, dist clear zone 15mm 1.. Minimum inhibitory concentration (MIC) ,000 ppm broth, broth control OD,. Broth 1. MIC..,. Chloroform, ethyl acetate, butanol, water,.,. MIC,.. broth,... L

43 monocytogenes 5 L. monocytogenes. 35 tryptone soy broth, 1,...,,.,. 2.., (,, ) TLC UV,.. IR Mass spectrophotometer NMR. Porarimeter CD..,,,,,.,,,

44 3..,....,

45 3 1.,, (1-5), (6-8) cashew apple(9), bacteriocin(10-14), chitosans(15,16). Streptococcus mutans (17) (18), 75% (19). Gram, (20-22) (23)., (24) Listeria monocytogenes (25-27) Listeria monocytogenes

46 (28), (29) Table 1. Table 1. List of medicinal herbs used for antimicrobial experiment Korean Part Botanical name (abbreviation) name used Terminalia chebula Retz (Tc) Fruit Pueraria thunbergiana Benth (Pt) Flower Euphorbia sieboldianus Hara (Es) Whole Crcuma longa L.(Cl) Root Zingiber officinale Rosc (Zo) Root Gallus gallus domesticus Brisson (Gd) Bark Angelica tenuissima Nakai (At) Root Davallia mariesii Moore (Dm) Root Sophora japonica L.(Sj) Flower Cibotium barmetz(l) J.Sm (Cb) Root Geoclemys reevessi Gray (Gr) Root Euonymus alatus(thunb)sieb (Ea) Stem Citrus unshiu Marc (Cu) Bark Rosa laevaigate Michx (Rl) Fruit Lycium chinense Miller (Lc) Root Arisaema japonicum Bl (Aj) Root

47 Table 1. Continued Korean Part Botanical name (abbreviation) name used Cynanchum atratume Bunge (Ca) Whole Chelidonium majus var. asiaticum(hara)ohwi (Cm) Santalum album (Sa) Imperate cylindrica var.kaengii(retz) Durand et Schinz (Ic) Poria cocos Wolf (Pc) Whole Root Root Root Amyda sinensis Wiegmann (As) Whole Curcuma zedoaria(berg)rosc (Cz) Root Belamcanda chinensis(l) DC (Bc) Root Panax notoginseng L.(Pn) Root Rhinoceros unicornis L.(Ru) Bark Dendrobium moni;iforme(l) Sw (Dmi) Whole Acorus graminens Soland (Ag) Root Asiasarum sieboldii Miq. var. seoulense Maeka (Asm) Stem & Leaves Caesalpinia sappan L.(Cs) Wood Artemisiae lavandulaefolia Dc (Al) Whole Alpinia officinarum Itance (Ao) Root Forsythia koreana Nakai (Fk) Bark Melandryum firmum Rohrb (Mf) Whole Arctium lappa L.(All) Seed Myristica fragrans Houtt (Mfh) Fruit Leonurus sibiricus L.(Ls) Whole Artenisiae capillaris Thunb (Ac) Whole Lithospermum erythorhizon S. et Z (Le) Whole Uncaria rhychophlla(miq) Jacks (Ut) Stem & Leaves Gentiana macrophlla Pall (Gm) Root Aconitum carmichaeli Debx (Acd) Root Asparagus cochinensis Merr (Acm) Root Manis pentadactyla L.(Mp) Root Celosia cristata L.(Cc) Seed Alpinia katsumadai Hayata (Ak) Fruit Morinda officinalis How (Mo) Root Po1ygonum aviculare L.(Pa) Whole Piper longum L.(Pl) Fruit

48 Table 1. Continued Korean Part Botanical name (abbreviation) name used Elipta prostrate L.(Ep) Whole Lscholtzia patrini Garck (Lp) Whole Piper nigrum L.(Pnl) Fruit Picrprrhiza kurrooa Royle ex Benth (Pk) Root Polygonatum sibirium Redout (Ps) Root Siegesbeckia orientalis L.var.(So) Whole 5 75% ethanol 85 3, rotary vaccum evaporator soluble solid 1 ml psychrotrophs Table 2. Slant 1 10 ml ml plate, 0.65 mm filter paper disc (Whatman N0. 2)

49 Table 2. List of strains and media used for antimicrobial experiment No Microorganism Media used 1 Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Bacillus cereus YUFE Staphylococcus aureus KFCC Pseudomons fluorescens KCTC Pseudomons fluorescens KCTC Leuconostoc mesenteroides KFCC Tryptic Soy Broth & Agar(Difco) Lactobacilli MRS Broth & Agar(Difco) disc plate 30, disc clear zone (mm) (30,31). disc. membrane filter (0.2 m, pore size), (ppm) 0.1 ml spectrophotometer (Cecil se 292, England) 620 nm (32). 75%, (33),

50 56 Table Clear zone diameter 15mm,,,,,,,,. (20,34) (35).. Table 3. Antimicrobial effect of each medicinal herbs ethanol extracts on various microbial strains Botanical Microorganism2) name1) SS3) Tc 163) Pt -4) Eo Cl Zo Gd At Dm Sj Cb Gr Ea

51 Table 3. Continued Botanical name1) Microorganism2) Cu Rl Lc Aj Ca Cm Sa Ic Pc As Cz Bc Pn Ru Dmi Ag Asm Cs Al Ao Fk Mf All Mfl Ls Ac Le Ur Gm SS3) Acd Acm Mp Cc

52 Table 3. Continued Botanical Microorganism2) name1) SS3) Ak Mo Pa Pl Ep Lp Pnl Pk Ps So )See Table 1 2)See Table 2 3)mg of soluble solid content of extract/disc 4)Clear zone diameter (mm) 5)No inhibition, (Table 3) 10 Fig. 1 Fig. 2. Fig. 1 L. monocytogenes ATCC 19114, B. cereus YUFE 2004, S. aureus KFCC P. fluorescens KCTC 1645, L. monocytogenes ATCC ,000 ppm. B. cereus YUFE ,000 ppm 36 2,000 ppm 72. S. aureus KFCC ,000 ppm

53 L. monocytogenes ATCC B. cereus YUFE 2004 S. aureus KFCC P. fluorescens KCTC 1645 Fig. 1. Antimicrobial effect of 75% ethanol extract Terminalia chebula Rets on several strains. control 100 ppm 500 ppm 1000 ppm 2000 ppm

54 L. monocytogenes ATCC L. monocytogenes ATCC L. monocytogenes ATCC L. monocytogenes ATCC Fig. 2. Antimicrobial effect of 75% ethanol extract Caesalpinia sappan L. on several strains. control 10 ppm 20 ppm 50 ppm 100 ppm

55 L.. monocytogenes ATCC B. cereus YUFE 2004 S. aureus KFCC P. fluorescenes KCTC 1645 Fig. 3. Antimicrobial effect of 75% ethanol extract Caesalpinia sappan L. on several strains (continued). control 10 ppm 20 ppm 50 ppm 100 ppm

56 2,000 ppm 3. P. fluorescens KCTC ,000 ppm. (Table 3) Fig. 2. Fig. 2 Table 3 clear zone diameter mm. L. monocytogenes ATCC ppm L. monocytogenes ATCC L. monocytogenes ATCC ppm. L. monocytogenes ATCC ppm 48. L. monocytogenes ATCC 19114, B. cereus YUFE 2004, S. aureus KFCC P. fluorescens KCTC ppm 48. (36) typhus (37).,

57 , Table 4. Table 4,. (38). Table 4. Growth inhibition of different solvent fractions on various microbial stains Microorganism1) Plant Solvent SS2) Terminalia CHCl3 203) chebula EtoAC ( ) BuOH Water Rosa CHCl laevaigate EtoAC ( ) BuOH Water sappan CHCl ) ( ) EtoAC BuOH Water Myristica CHCl fragrans EtoAC ( ) BuOH )See Table 2 Water )mg of soluble solid content of extract/disc 3)Clear zone diameter (mm) 4)No inhibition

58 56 75% 10, Listeria monocytogenes ATCC 15313, 19111, 19112, 19113, 19114, Bacillus cereus YUFE 2004, Staphylococus aureus KFCC 11764, P. fluorescens 1645, 2344 and Leuconostoc mesenteroides KFCC 12031,,,,,,,. 10 ppm L. monocytogenes ATCC 19114, B. cereus YUFE 2004, S. aureus KFCC P. fluorescens KCTC 1645.,,,,

59 2 Listeria monocytogenes 6 11 (Morus. bombycis Koidzumi), (M. monglica C. K. Schen), (M. tiliaefolia Makino) (M. alba Linne) 4 (M. lhou Ser. Koidzumi),, (1). Kuwanon E (2) (3) (4). (5) Listeria monocytogenes (6) (7,8) (9)., Kuwanon (9,10). Listeria monocytogenes Kuwanon G (7) flavone (11). Listeria monocytogenes,

60 Listeria monocytogenes 5 (ATCC 19111, ATCC 19112, ATCC 19113, ATCC ATCC 15313) tryptic soy agar, tryptic soy agar, tryptic soy broth (Difco). (12) (Morus alba Linne) 5 75% ethanol 78 3 chloroform, ethyl acetate, n-butanol. chloroform n-butanol:chloroform (1:9, 2:8, 3:7), butanol, methanol:water (5:5) silica gel ( mesh, Merk) column(4 100 cm). 1 silica gel column chromatography methanol:acetic acid:chloroform = 1:0.5:6 2 column (silica gel 60, mesh, Merk, cm) chromatography. (fraction 5') HPLC (Spectraphsics SP 8800, C18column) 93%(peak area) bactericidal. (fraction 5') butanol:2m-nh4oh = 10:1 preparative layer chromatography (silica gel 60-F254) TLC (silica gel 60-F254)

61 - Disc diffusion method(13) 1 10 ml tryptic soy broth ml tryptic soy agar plate. plate 6.5 mm filter paper (Whatman No.2) disc clear zone (mm) (14). Bactericidal 1 10 ml broth, ml (F-5') 100 ppm 300 ppm broth ml (control). F-5' 7 mg 75% ethanol 0.7 ml 100 ppm 0.1 ml broth 9.9 ml 300 ppm 0.3 ml broth 9.7 ml. ethanol. Bactericidal L. monocytogenes ATCC F-5' 100 ppm 300 ppm (SEM, Hitachi S-4100, Japan)

62 (75%) chloroform silica gel column 1 Table 1. Table 1. Antimicrobial activities after silicalgel partition column chromatograph of chloroform fraction from ethanol extract of Morus alba Linne bark Fraction No. (elution volume) ATCC ATCC L. monocytogenes ATCC ATCC ATCC F-11)(100) -7) F-21)(100) F-31)(360) F-41)(240) F-52)(670) 10.58) F-63)(100) F-74)(1800) F-85)(800) F-95)(500) F-106)(500) )n-BuOH:CHCl3=1:9. 2)n-BuOH:CHCl3=1:9, 3:7. 3)n-BuOH:CHCl3=5:5. 4)n-BuOH:CHCl3=5:5, saturated BuOH. 5)Saturated BuOH. 6)MeOH. 7)No inhibition. 8)Clear zone diameter (mm)

63 Table 1 Fraction 5 (F-5), F-6, F-7 F-7. (4,8,14) butanol:chloroform = 5:5. Table 1 F-7 silica gel column Table 2. Table 2. Antimicrobial activities after silicalgel partition column chromatograph of F-7 from Table 1 L. monocytogenes Fraction No. ATCC ATCC ATCC ATCC ATCC (elution volume) F-1'9)(144) ) F-2'9)(54) -12) F-3'9)(108) F-4'9)(72) F-5'9)(198) F-6'9)(162) F-7'10)(130) )MeOH:HOAc:CHCl3=1:0.5:6. 10)MeOH. 11),12)See Table 1. Table 2 F-5' 5. PTLC TLC

64 Fig. 1. Rf 0.5 Fig. 1. Thin layer chromatogram of Kuwanon G. (BuOH:2M-NH4OH=10:1, Rf=0.58). F-5 tryptic soy broth Listeria monocytogenes 5 Fig Fig. 2 6 F-5' 100 ppm. Listeria monocytogenes Tin men chu Sin mao heung(15)

65 Listeria monocytogenes Scott A 5,000 ppm Bacillus cereus 500 2,000 ppm(16), grape fruit Bacillus subtilis Aspergillus oryzae 100 ppm(17) Bacillus subtilis ppm allylisothiocyanate(18). terpinen ppm (19). F-5' Listeria monocytogenes. (8). F-5 tryptic soy broth Listeria monocytogenes (control) (Fig. 2 Fig. 6) F-5' Listeria monocytogenes. F-5' 300 ppm Fig. 7. Fig. 7 (Fig. 7-A) (Fig. 7-B)

66 - 66 -

67 Fig. 7. Scanning electron micrographs( 20,000) of L. monocytogenes ATCC A: control, B: treated with 300 ppm of F-5' for 12 hours at 30.. F-5' Listeria monocytogenes grape fruit Salmonella choleraesuis (17)

68 75% Listeria monocytogenes column chromatography (F-5') tryptic soy broth ppm Listeria monocytogenes

69 3 lactoferin, lysozyme,, ester (1), (2,3), chitosan(4), bacteriocin(5 7) (8 12) (13 19).,, (20), (21,22), (23) essential oil, flavonoid, tannin terpenoid phenol (24,25). (Sophora flavescens Ait.) (26) (27). 75% Listeria monocytogenes (ATCC 19111, 19112, 19113, )

70 (28). (Sophora flavescens Ait.). 800 g 75% 3 L 78 3,,.. Silica gel column chromatography. (21g) silica gel (500 g, mesh, Merck) : : (20 : 3 : 1, v/v) column ( cm) ( : : = 20 : 3 : 1 --->, v/v) step-wise 15 (S-1 S-15) S S-10 (3 g) : : (8 : 5 : 1,

71 : 5 : 1, v/v) silica gel column chromatography (150 g, cm) 9 (S-10-1 S-10-9) S TLC UV (254 nm, 365 nm) (15 20%). IR Perkin Elmer 599B 1H-NMR ZEOL JNM-LA400 (400 MHz), 13C-NMR ZEOL JNM-LA400(100 MHz) CDCl3: CD3OD = 5 : 1. Table 1. Table 1. List of strains and media used for antimicrobial experiment Microorganism tested Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Bacillus subtilis ATCC Escherichia coli ATCC Staphylococcus aureus KFCC Media used Tryptic soy broth & agar (Difco) Tryptic soy broth & nutrient agar (Difco) Incubation temperature ( )

72 1 10 ml, ml 10 ml ml membrane filter (0.2 m) (ppm) ( 48 ) 24 ( 12 ) 620 nm ml, ml ml.. 75% 50, 100, 500 1,000 ppm L. monocytogenes (Table 2) ppm

73 Table 2. Minimum inhibitory concentration of ethanol extract and solvent fraction of Sophora flavescens Ait. on Listeria monocytogenes L. monocytogenes EtOH extract Minimum inhibitory concentration (ppm) CHCl3 fraction EtOAc fraction BuOH fraction ATCC <MIC1)<100 MIC<50 100<MIC 100<MIC ATCC <MIC<500 MIC<50 100<MIC 100<MIC ATCC <MIC<500 MIC<50 100<MIC 100<MIC ATCC <MIC<500 MIC<50 100<MIC 100<MIC ATCC MIC<50 MIC<50 100<MIC 100<MIC 1)Minimum inhibitory concentration(ppm) 50 ppm. 2,000 ppm (28). (28). silica gel column ppm L. monocytogenes ATCC Table

74 Table 3. Minimum inhibitory concentration and yield of fraction obtained after silica gel column chromatography of chloroform fraction of Sophora flavescens Ait. ethanol extract on L. monocytogenes ATCC Fraction Elution solvent (hexane : EtOAc : MeOH) MIC (ppm) Yield2)(%) S-1 20 : 3 : 1 50<MIC1) S-2 20 : 3 : 1 50<MIC S-3 15 : 3 : 1 50<MIC S-4 15 : 3 : 1 50<MIC S-5 15 : 3 : 1 12 : 3 : 1 MIC< S-6 12 : 3 : 1 MIC< S-7 12 : 3 : 1 10 : 3 : 1 MIC< S-8 10 : 3 : 1 MIC< S-9 10 : 3 : 1 MIC< S : 3 : 1 9 : 3 : 1 MIC< S-11 8 : 3 : 1 50<MIC S-12 6 : 3 : 1 50<MIC S-13 6 : 3 : 1 50<MIC S-14 6 : 3 : 1 50<MIC S-15 4 : 3 : 1, MeOH 50<MIC )See footnote on Table 2 2)Fraction weight(g)/dried plant(g) : 3 : 1 9 : 3 : 1 S-5, S-6, S-7, S-8, S-9 S ppm 50 ppm. S

75 silica gel column ppm L. monocytogenes ATCC Table 4 S-10-6 S-10-7 S S-10-6 (Fig. 1). Table 4. Minimum inhibitory concentration and yield of fraction obtained after silica gel column chromatography of G-10 isolated from chloroform fraction of Sophora flavescens Ait. ethanol extract on L. monocytogenes ATCC Fraction Elution solvent (Hexane : EtOAc : BuOH) MIC (ppm) Yield2)(%) S : 5 : 1 30<MIC1) S : 5 : 1 30<MIC S : 5 : 1 30<MIC S : 5 : 1 30<MIC S : 5 : 1 30<MIC S : 5 : 1 6 : 5 : 1 10<MIC< S : 5 : 1 MIC< S : 5 : 1 30<MIC S : 5 : 1 30<MIC )See footnote on Table 2 2)See footnote on Table

76 Fig. 1. Thin layer chromatogram of fraction of S-10 isolated from chloroform fraction of Sophora flavescens AIT. ethanol extract (Hexane : EtOAc : MeOH = 6 : 5 : 1). S-10-6 S ppm Fig. 2. L. monocytogenes ATCC 19111, B. subtilis ATCC S. aureus KFCC S ppm L. monocytogenes ATCC 19112, ATCC ATCC E. coli ATCC ppm

77 L. monocytogenes ATCC B. subtilis ATCC E. coli ATCC S. aureus KFCC Fig. 2. Growth inhibition by G-4-4 isolated from chloroform fraction of Sophora flavescens Ait. ethanol extract on several strains of bacteria. - : control, - : 10 ppm, - : 30 ppm, - : 50 ppm, +-+ : 100 ppm

78 S-10-6 L. monocytogenes (28) G-4-4 (29) S. aureus Corilagin tannin (30) phloroglucinol (13) ppm S S-10-6 S ppm L. monocytogenes ppm CFU/mL S-10-6 (Fig. 3). L. monocytogenes ATCC ppm CFU/mL 72, 50 ppm, CFU/mL (29). ATCC ppm 103CFU/mL, 50 ppm 104 CFU/mL (29) (101). ATCC ppm 24, 50 ppm CFU/mL

79 ATCC ATCC ATCC ATCC Fig. 3. Bactericidal effect of G-4-4 isolated from chloroform fraction of Sophora flavescens Ait. ethanol extract on L. monocytogenes. - : control - : 30 ppm - : 50 ppm

80 . ATCC ppm CFU/mL ppm CFU/mL CFU/mL (29)., 50 ppm L. moncytogenes ATCC (ATCC 19113, ) (ATCC 19111)(29) S-10-6 L. moncytogenes ATCC S S MS spectrum (M+) peak H-NMR(Fig. 4) 7.22(1H, d, J=8.5 Hz, H-6 ), 6.33(1H, dd, J=8.5, 2.4 Hz, H-5 ), 6.27(1H, d, J=2.4 Hz, H-3 ) signal benzene, 6.0(1H, s, H-8 ), 5.50(1H, dd, J=11.1, 5.0 Hz, H-2) flavanone 2 proton 4.90(1H, t, J=6.7 Hz, H-7 ) methylene proton. geminal coupling exo-methylene 4.50(1H, d, J=21.1 Hz, H-4a ), 4.49(1H, d, J=21.1 Hz, H-4b ) signal 3 singlet methyl signal 1.56(3H, s), 1.51(3H, s), 1.40(3H, s) (H-5, 9, 10 ). S-10-6 A 3, B 2 flavanone isoprenoid

81 Fig. 4. 1H-NMR and 13C-NMR spectrum of S-10-6 isolated from chloroform fraction of Sophora flavescens Ait. ethanol extract (CDCl3 : CD3OD = 5 : 1)

82 . 13C-NMR(Fig. 5) 25 signal ppm ketone, ppm benzene, ppm, ppm olefinic methine, ppm exomethylene, ppm methine, 17, 18, 25 ppm 3 methyl signal, ppm 4 methylene methyl. S-10-6 flavanone B 2,4 A 5,7 hydroxy 6 Wu (31) Shirataki (32) (24) kushenol F. 75%. Listeria monocytogenes (ATCC 19111, 19112, 19113, ) ppm 50 ppm. silica gel column chromatography 2 S-10-6 L. monocytogenes 5, Bacillus subtilis Staphylococcus aureus 10 ppm L. monocytogenes ppm E. coli 100 ppm. (S-10-6) IR, 1H-NMR 13C-NMR flavanone kushenol F

83 4,,,,, (1).,, (2,3)., (4).,. (Glycyrrhiza uralensis FISCH) glycyrrhizin 6 14%

84 (5,6). 75% L. monocytogenes (7), column chromatography. (Glycyrrhiza uralensis FISCH). 310 g 75% 1.6 L 78 3,,,.. Silica gel column chromatography(8,9). (20g) silica gel (700 g, mesh, Merck) : (30 : 1), column ( cm)

85 (30 : 1-->2 : 1, v/v) step-wise 13 (G-1 G-13) G-4 G-11. G-4(1g) : : (12 : 3 : 0.5, 10 : 3 : 0.5, v/v) silica gel column chromatography (55 g, cm) (G-4-1 G-4-7) G-4-4. G-11(2g) : : (15 : 3 : 1, 10 : 3 : 1, v/v) silica gel column chromatography (160g, 5 20 cm) 8. TLC UV (254 nm, 365 nm) (15 20%). IR Perkin Elmer 599B 1H-NMR ZEOL JNM-LA400 (400 MHz), 13C-NMR ZEOL JNM-LA400(100 MHz) CD3OD. Table 1. (7,10) 1 10 ml ml 10 ml ml membrane filter (0.2 m) (ppm) ( 48 ) 24 (

86 Table 1. List of strains and media used for antimicrobial experiment Microorganism tested Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Listeria monocytogenes ATCC Escherichia coli ATCC Staphylococcus aureus KFCC Media used Tryptic soy broth & agar (Difco) Tryptic soy broth & nutrient agar (Difco) Incubation temperature ( ) ) 620 nm ml, ml ml

87 75% (7) (50<MIC<100) 50, ppm L. monocytogenes 5. Table ppm, ppm 300 ppm. L. monocytogenes 5 (minimum inhibitory concentration : ppm). Table 2. Minimum inhibitory concentration of ethanol extract and solvent fraction of Glycyrrhiza uralensis FISCH L. monocytogenes EtOH extract MIC(ppm) CHCl3 fraction EtOAc fraction BuOH fraction ATCC <M1)<100 M<50 M<50 300<M CHCl3 fration Yield2)(%) EtOAc fraction ATCC <M<100 M<50 M<50 300<M ATCC <M<100 50<M<100 M<50 300<M ATCC <M< <M<300 50<M< <M ATCC <M<100 M<50 50<M< <M 1)Minimum inhibitory concentration(ppm) 2)Fraction weight(g)/dried plant(g)

88 silica gel column chromatography L. monocytogenes ATCC (Table 3) G-4, G-5, G-6, G-8 G-11 G-4 G-11 silica gel column chromatography. Table 4 G-4 G g TLC (Fig. 1). 6 : 1 4 : 1 G-4 G-11 L. monocytogenes ATCC ppm G-11 ( ). G-4-4 G ppm Fig. 2. L. monocytogenes ATCC G ppm ppm. L. monocytogenes ATCC 19112, ATCC ATCC G-4-4 L. monocytogenes ppm. S. aureus KFCC G ppm ppm

89 Table 3. Growth inhibition at 50 ppm of the fraction obtained after column chromatography of ethyl acetate fraction of Glycyrrhiza uralensis FISCH ethanol extract on L. monocytogenes ATCC Fraction Elution solvent (CHCl3: MeOH) Antimicrobial activity Yield1)(%) G-1 30 : 1-2) 0.05 G-2 30 : 1 +3) 0.05 G-3 G-4 30 : 1 15 : 1 15 : 1 10 : 1 ++4) ) 0.35 G-5 10 : G-6 10 : G-7 10 : 1 8 : G-8 8 : G-9 8 : G-10 G-11 G-12 G-13 1)See footnote on Table 2 2)- : no inhibition 3)+ : weak inhibition 4)++ : strong inhibition 8 : 1 6 : 1 6 : 1 4 : 1 4 : 1 3 : 1 3 : 1 2 : 1 5)+++ : very strong inhibition

90 Table 4. Growth inhibition at 50 ppm of the fraction obtained after column chromatography of G-4 isolated from ethyl acetate fraction of Glycyrrhiza uralensis FISCH ethanol extract on L. monocytogenes ATCC Fraction Elution solvent (Hexane : EtOAc : BuOH) Antimicrobial activity Yield1)(%) G : 3 : ) 0.04 G : 3 : G : 3 : G : 3 : G : 3 : G : 3 : G : 3 : 0.5, MeOH -3) )See footnote on Table 2 2),3)See footnote on Table 3 Fig. 1. Thin layer chromatogram of fraction of G-4 isolated from ethyl acetate fraction of Glycyrrhiza uralensis FISCH ethanol extract (Hexane : EtOAc : BuOH= 8 : 3 : 0.5)

91 L. monocytogenes ATCC L. monocytogenes ATCC E. coli ATCC S. aureus KFCC Fig. 2. Growth inhibition by G-4-4 isolated from ethyl acetate fraction of Glycyrrhiza uralensis FISCH ethanol extract on several strains of bacteria. - : control, - : 10 ppm, - : 30 ppm, - : 50 ppm, - : 100 ppm

92 , E. coli ATCC G ppm. sodium benzoate potassium sorbate L. monocytogenes 500 ppm (11), Artesmisa flavone (12), horminone(13) Arnica 4 sesqiterpenoid (14) S. aureus E. coli ppm G-4-4. G-4-4 L. monocytogenes G <MIC<50 G ppm , (Fig. 3), CFU/mL CFU/mL. 102 CFU/mL (ATCC 19112), (ATCC ) (ATCC 19111). G ppm L. monocytogenes, 100 ppm ATCC CFU/mL CFU/mL 104CFU/mL

93 ATCC ATCC ATCC ATCC