植物萃取物對痤瘡致病菌之生長、菌膜形成與脂解酶活性的抑制作用

全文

(1)Inhibitory effects of botanical extracts on the growth, biofilm and lipase activity of acne-causing bacteria. (Po-Jung Tsai, Ph.D.) (Yau Rong Liing).

(2) 酚酚. 酚. 酚. 酚. 酚. !. !. 酚. 酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚酚. 酚酚. 酚.

(3) (acne vulgaris) (Propionibacterium acnes). (Staphylococcus epidermidis). (pilosebaceous follicle) (acne-causing bacteria). (biofilm). P. acnes (superoxide anions). P. acnes. 34 officinalis ). (Rosmarinus (Salvia officinalis L. ). (Verbena officinalis Linn). (Camellia sinensis ). P. acnes (ethyl acetate) P. acnes. (biofilm). (lipase activity). HPLC P. acnes S. epidermidis. P.. acnes. S. epidermidis P. acnes S. epidermidis. P. acnes. S. epidermidis S. epidermidis P. acnes. (biofilm formation) 16 hr. 48 hr. biomass. (prevention of biofilm formation PBF). (removal of established biofilm. REB) EA. PBF. REB. I.

(4) P. acnes. S. epidermidis. (a). DPPH. DPPH (b) (c). (d) HPLC EA. EA. HPLC. II.

(5) Abstract Acne vulgaris is a chronic disorder of the pilosebaceous follicles of the skin. Acne-causing bacteria, such as Propionibacterium acnes and Staphylococcus epidermidis, play an important role in the pathogenesis of acne vulgaris. Both bacteria resided within the follicles grow as a biofilm and hydrolyzed triglyceride to free fatty acids by bacterial lipase has been proposed as a major factor in the pathogenesis of acne vulgaris. ROS, and especially superoxide anions, were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In addition, superoxide anions were generated by P. acnes stimulated keratinocytes and involved in the development of acne inflammatory lesions. The aim of this study was to investigate inhibitory effects of various botanical extracts on growth, biofilm formation, and extracellular lipase activity of acne-causing bacteria and their anti-oxidation properties. Aqueous, methanolic, ethanolic and EA extract of rosemary were analyzed and quantified by HPLC. The antimicrobial activity of extracts was assessed by determining of minimum inhibitory concentration (MIC) values obtained by a modified microdilution broth method. Results showed that ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea and black tea significantly inhibited the growth of acne-causing bacteria, including P. acnes and S. epidermidis. Aqueous extract of verbena significantly inhibited the growth of P. acnes. We also tested the inhibitory activity of botanical extracts on the biofilm-forming ability of P. acnes in a microtiter plate model. In a preliminary experiment, we determined the optimal conditions for P. acnes biofilm formation. A mature biofilm (i.e. a biofilm in which the biomass does not significantly increase any further) was established following 48 hr incubation. Following 16 hr incubation, ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea, and black tea significantly prevented biofilm formation of P. acnes. In addition, ethanolic, methanolic and aqueous extracts of sage and green tea could remove established mature biofilm of P. acne. Furthermore, we conducted experiments to investigate the inhibitory effect of botanical extracts on crude lipase activity of acne-causing bacteria. Results showed that. III.

(6) ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea and black tea significantly inhibited lipolytic activity of crude lipase purified from P. acnes and S. epidermidis. In the present study, the antioxidant activity of botanical extracts was determined by the scavenging activities of 2, 2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, superoxide and hydroxyl radical . We also evaluated the total phenolic contents of botanical extracts. The potent radical scavenging effects were observed in ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea, and black tea. Besides aqueous extract of rosemary, rosmarinic acid, carnosol and carnosic acid were determined and quantified by HPLC in the methanolic, ethanolic and EA extract of rosemary. In conclusion, extracts of rosemary, sage, verbena, green tea, and black tea had potent inhibitory effects on the growth, lipolytic activity and biofilm formation of acne-causing bacteria and possessed excellent anti-oxidative properties.These data suggested that extracts of rosemary, sage, verbena, green tea, and black tea may be potentially therapeutic agents for the treatment of acne vulgaris.. Key word: Propionibacterium acnes, Staphylococcus epidermidis, herbs, anti-acne, lipase, biofilm formation, anti-oxidation, HPLC. IV.

(7) .....................................................................................................................1 .....................................................................................................................1 .....................................................................................................................2 .............................................................................................................3 .............................................................................................3 .....................................................................................................5 ...................................................................................................................22 .......................................................................................................37 ...........................................................................................................37 ...................................................................................................................37 ...................................................................................................................39 ...................................................................................................................40 ...................................................................................................................52 ...................................................................................................................53 .......................................................................................................53. 1. (1) (2). (3). (4). (5) (6). ...............................................................................................................54 ......................................................................................................................55 P. acnes S. epidermidis ...............................................55 ..........................................................................56 .................................................................................................56 (a) (b) P. acnes .............................................................57 (prevention of biofilm formation PBF) a. .......................................................................................................................57 b (removal of established biofilm REB) ...............58 ......................................................................59 .........................................59 (a) (b) (lipase activity) ...............................................................................61 (c) .........................................................................62 (d) ................................................................63 a. P. acnes ...................................................63 b. S. epidermidis .........................................63 ....................................................................................65 (a) DPPH ........................................................65 (b) ....................................................65 (c) .................................................66 (d) ............................................................66 ............................................................................................67 HPLC ....................................................68 (a) .................................................................................................71 ................................................................................................71 (b). V.

(8) (c) 2. (1) (2) (a) (b) (3) (a). (b). (4) (a). (b). (c). 3. (1) (2) (a) (b) (3) (a) (b) (4) (a) (b) (c) (d) (5) 4. (1). .................................................................................................72 ..................................................................................................................73 P. acnes S. epidermidis .........................................73 P. acnes .............................................................74 (prevention of biofilm formation PBF) .......................74 (removal of established biofilm REB) ............................75 ................................................................76 P. acnes ..................................................76 ..................................................................................................76 a. b. ..................................................................................................76 S. epidermidis ........................................77 a. ..................................................................................................77 ..................................................................................................77 b. ....................................................................79 .....................................................................................79 a. DPPH ......................................................79 ..............................................................79 b. c. ..........................................................80 ......................................................................80 d. ........................................................................81 a. DPPH ......................................................81 ..................................................81 b. c. ..............................................82 d. ..........................................................82 .............................................................................83 a. ..............................................................................83 ..............................................................................83 b. ..........................................................................................................................84 P. acnes S. epidermidis .................................................84 P. acnes .....................................................................85 (prevention of biofilm formation PBF) .......................85 (removal of established biofilm REB) ............................86 ........................................................................87 P. acnes ..........................................................87 S. epidermidis ................................................87 ..........................................................................................89 DPPH ........................................................89 ................................................................89 .............................................................90 ........................................................................90 ....................................................................................91 ..........................................................................................................................92 P. acnes S. epidermidis .................................................92 VI.

(9) (2). P. acnes .....................................................................93 (a) (prevention of biofilm formation PBF) .......................93 (b) (removal of established biofilm REB) ............................94 ........................................................................95 (3) (a) P. acnes ..........................................................95 (b) S. epidermidis ................................................95 (4) ..........................................................................................97 (a) DPPH ....................................................................97 ................................................................97 (b) (c) .............................................................98 (d) ........................................................................98 (5) ....................................................................................99 .......................................................................................................100 ......................................................................................................................... 116 ................................................................................................................. 116 ................................................................................................................. 116. 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 2-11 2-12 2-13 2-14 2-15 2-16 2-17 2-18 3-1 3-2 4-1 4-2 4-3. ...........................................................................................................4 P. acnes .......................................5 .......................................................................................................7 Erythromycin ............................................................................................8 Clindamycin .................................................................................................8 .......................................................................................................8 Doxycycline .................................................................................................8 Benzoyl peroxide ..........................................................................................9 .........................................................................................................12 P. acnes IL-8 ............................................16 ...................................................................................................18酚 .......................................................................................19 ...................................................................................................21 .......................................................................................................................27 .......................................................................................................................27 .......................................................................................................................28 ...........................................................................................................................28 .......................................................................................................................29 Broth dilution method ..............................41 luminol .............................................52 P. acnes .............................................................................56 P. acnes .......................................................57 P. acnes .......................................................58. VII.

(10) 4-4 4-5 4-6 4-7 4-8 4-9 4-10 4-11 4-12 4-13 4-14 4-15 4-16 4-17 4-18 4-19 4-20 4-21 4-22 4-23 4-24 4-25 4-26 4-27 4-28 4-29 4-30 4-31 4-32 4-33 4-34 4-35 4-36 5-1 5-2. P. acnes (A) (B) ..................................59 S. epidermidis (A) (B) ........................60 (A) P. acnes (B) S. epidermidis ............61 (A) P. acnes (B) S. epidermidis ................62 (A) P. acnes (B) S. epidermidis ...........................................................................................................................63 (A) DPPH (B) NO ........................65 (A) (B) ................66 .......................................68 ...................................................................69 ...................................................................69 EA ....................................................................70 .......................................................................70 .......................................................................................71 .......................................................................................71 ........................................................................................72 (A) (B) P. acnes .......................................74 (A) (B) P. acnes .......................................75 (A) (B) P. acnes .......................76 (A) (B) S. epidermidis ..............77 DPPH .................................................79 ...............................80 DPPH .................................81 ...............................82 P. acnes ..........................................................85 P. acnes ..........................................................86 (A) P. acnes S . epidermidis .......87 DPPH .............................89 ...........................90 P. acnes ..........................................................93 P. acnes ..................................................................94 (A) P. acnes (B) S. epidermidis ...............................................................................................................................95 DPPH .............................97 ...........................98 .........................................................................108 theaflavins ........................................................................................112. VIII.

(11) 2-1 P. acnes 2-2 2-3 Phytochemical 2-4 2-4-1 2-4-2 2-4-3 2-4-4 2-4-5 2-4-5 3-1 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 4-11 4-12 4-13 4-14 4-15 5-1 5-2 5-3 HPLC 6-1 6-1-1. .........................................................................................6 .......................................................................11 ......................................................14 ...........................................................................................30 ( ) ....................................................................................31 ( ) ....................................................................................32 ( ) ....................................................................................33 ( ) ....................................................................................34 ( ) ........................................................................35 ( ) ........................................................................36 ...................................................................................................45 ...............................................................................53 .......................................................................54 P. acnes S. epidermidis .......................................55 P. acnes S. epidermidis ...........................64 ...................................................................................67 ...............................................................................72 P. acnes S. epidermidis ...................................73 P. acnes S. epidermidis ...................78 ...........................................................................83 P. acnes S. epidermidis .......................................84 P. acnes S. epidermidis ...........................88 ...................................................................................91 P. acnes S. epidermidis .......................................92 P. acnes S. epidermidis ...........................86 ...................................................................................99 .............................................................................................107 ............................................................. 112 theaflavins ...................................................................... 113 ..................... 118 ( ) ..............119. IX.

(12) (acne vulgaris). (pilosebaceous follicle) (abnormal hyperkeratinization). (increased sebum production). (hormones). (immunologic responses). (Toyoda & Morohashi, 2001). Danby (2008) ). dihydrotestosterone (DHT). germinative cell ( DHT. ). testosterone DHT. growth factor-1 (IGF-1). Grange et. al (2009). (. 5-α reductase glycemic load. insulin. testosterone. (Propionibacterium acnes). P. acnes. P. acnes. 1.

(13) P. acnes ROS. 2.

(14) (pilosebaceous unite). (infundibulum). (acne vulgaris). (Gollnick, 2003) (1). (androgen). (2) (3). (P. acnes). (4). (Toyoda &. Morohashi, 2001) (1) 5α-reductase (seborrhea) (2). (comedone). microcomedo (non-inflammatory lesions) (distended). (pustule) (Papules)(size. (Gollnick, 2003). 3. 5 mm ).

(15) 2-1 Figure 2-1 Current understanding of the development of acne lesions. :. (Gollnick, 2003). (3). P. acnes. P. acnes. (4) (papules). (pustules). 2001). 4. (nodules)(Toyoda & Morohashi,.

(16) (1) (coagulase – negative staphylococci (Propionibacterium acnes (diphtheroids). micrococci) (2). Propionibacterium granulosum ). (3). (Pityrosporum species). (Burkhart et al., 1999) P. acnes. P.. acnes P. acnes. P. acnes P. acnes. 0.4-0.7µm. 3-5µm. peptidoglycan. P. acnes. (Toyoda & Morohashi, 2001). P. acnes. 2-1. (Dessinioti & Katsambas, 2010). 2-2. P. acnes. Figure 2-2 Scanning electron photomicrograph of cultured P. acnes isolated from the follicular infundibulum of comedones. (Toyoda & Morohashi, 2001). 5.

(17) . 2-1 P. acnes Table 2-1 Data on the mechanism of action of Propionibacterium acnes in acne pathogenesis P. acnes produces lipase, proteases, hyaluronidase, and neutrophil chemotactic factors. P. acnes induces the production of TNF-α, IL-1α, and IL-8. P. acnes induces the expression of the proinflammatory cytokines IL-8, IL-1β, and TNF-α by human monocytes in acne patients and in controls. Inflammation triggered through TLR2 is important in the pathogenesis of acne, and P. acnes was shown to induce monocyte cytokine production (IL - 2, IL-8) through a TLR-2 dependent pathway. An increase in TLR2, TLR4, and MMP-9 expression by human keratinocytes occurred with incubation with P acnes fractions. P acnes extracts are directly able to modulate the differentiation of keratinocytes by inducing β1, α3, α6s, and αVβ6 integrin expression, and filaggrin expression on keratinocytes. P. acnes induces IL-8, and β-defensin-2 expression in keratinocytes via TLR2 and TLR4. P. acnes induces keratinocyte growth in vitro. P. acnes GroEL (a heat-shock protein) is able to upregulate the proinflammatory cytokine production of keratinocytes. P. acnes may be involved in the formation of the microcomedones. P. acnes biofilm may lead to the increased cohesiveness of corneocytes seen in acne. (Dessinioti & Katsambas, 2010). Staphylococcus epidermidis micrococci. staphylococci (acroinfundibulum). (Toyoda & Morohashi, 2001) S. epidermidis (persistent species) S. epidermidis (Farrell et al., 1993) Nishijima et al. (2000) P. acnes. S. epidermidis. S. epidermidis P. acnes. S. epidermidis. P. acnes. S. epidermidis. S. epidermidis. P. acnes. 6. S. epidermidis.

(18) 1. (antimicrobial agents) 2-3 (1) (3). (2). (4). 2-3 (Coates et al., 2002). (Worret (1). Fluhr, 2006). (topical antibiotics). a. Erythromycin macrolide group. 50s ribosome subunit erythromycin. P. acnes b. Clindamycin 50s ribosome subunit clindamycin. 7.

(19) A. tretinoin. BPO. P. acnes. 2-4 Erythromycin. 2-5. Clindamycin. (2). (systemic antibiotics) (doxycycline). minocycline. 30s ribosomal subunit hydrochloride. Minocycline. 2-6. 2-7. Doxycycline. (3). Benzoyl peroxide (BPO) chlorhydroxyquinolin. 8.

(20) BOP. BOP. benzoic acid. hydrogen peroxide. species). (reactive oxygen (Sagransky et al., 2009). 2-8 Benzoyl peroxide. 2 (Cowan, 1999). 2-2. (1) Phenolic and polyphenols a. Simple phenol and phenolic acids hydroxyl group. phenol group. phenolic sulfhydryl group (a). Quinones: quinone - hydroquinone pair ubiquinone quinones. nucleophilic amino. acid (adhesion). polypeptides. (b). Flavones, flavonoids, and flavonols: flavonoids. (c). Tannins:. isoflavan structure. catechins. polyphenolic compound theaflavins. isoflavanoids. thearubigins tannin. 9. simple.

(21) hydrogen bonding. b.. hydrophobic effects. polysaccharide. Terpenoids and essential oils essential oil fraction terpenes. tetraterpenes (C20, C30, and C40). isoprene structure C10H16. diterpenes triterpenes. hemiterpenes (C5) and sesqiterpenes (C15) terpenoids protozoa. Terpenenes. terpenoids. Terpenenes. lipophilic compounds c.. Alkaloids Berberine trypanosomes. berberine d.. alkaloid group. plamodia. harmane. alkaloids DNA. Lectins and polypeptides peptides Thionins peptides. 47. yeasts, gram-negativ. gram-positive bacterial e.. Polyacetylenes. 10.

(22) 2-2 Table 2-2 Major classes of antimicrobial compounds from plants Class Phenolics. Subclass Simple phenols Phenolic acids. Example Catechol Epicatechin Cinnamic acid. Quinones. Hypericin. Flavonoids Flavones. Chrysin Abyssinone. Mechanism Substrate deprivation Membrane disruption Bind to adhesins, complex with cell wall, inactivate enzymes Bind to adhesins Complex with cell wall Inactivate enzymes Inhibit HIV reverse transcriptase ? Bind to proteins Bind to adhesins Enzyme inhibition Substrate deprivation Complex with cell wall Membrane disruption Metal ion complexation. Flavonols Tannins. Totarol Ellagitannin. Coumarins. Warfarin. Interaction with eucaryotic DNA (antiviral activity). Terpenoids, essential oils. Capsaicin. Membrane disruption. Alkaloids. Berberine, Piperine. Lectins and polypeptides. Mannose-specific agglutinin. Polyacetylenes. Intercalate into cell wall and/or DNA Block viral fusion or adsorption Form disulfide bridges. Fabatin 8S-Heptadeca-2( Z ), ? 9( Z )-diene-4,6-diyne-1,8-diol. 酚 (Cowan, 1999). 11.

(23) (Biofilm) 1 (O’ Toole et al., 2000) (i) planktonic, (ii) attachment, (iii) microcolony formation, (iv) macrocolony exopolysaccharide matrix. (v) dispersal. macrocolony. macrocolonies. 2-9 (Mond & O’Toole, 2009). 12. DNA.

(24) 2 P. acnes P. acnes. gylcocalyx polymer (Burkhart. Burkhart, 2007). gylcocalyx polymer In vitro. P. acnes. (sessile cell). 50. 500. P.. acnes (Burkhart. 3. Burkhart, 2007). (phytochemical) (. ). quorum-sensing (QS) QS (Phytochemical) /. 2-3 (Simo˜es et al, 2009) (removal of biofilm) cross-linking interaction. ethylene glycol- bis -(b - aminoethyl ether)-N,N-tetra acetic acid (EGTA) P. aeruginosa. SDS, Triton X-100, Tween. 20. cross - linkage lysozyme (Chen & Stewart, 2000). 13. proteases.

(25) 2-3 Phytochemical Table 2-3 The effect of phytochemical on the formation of biofilm Phytochemical Biofilm effects Allium sativum extracts; Resveratrol. QS inhibition. Apigenin ; Farnesol. Strep. sobrinus biofilm inactivation. Brassica oleracea; Curcuma longa; Fragaria sp.; Ocimumbasilicum; Rosemarinus officinalis; Rubus. E. coli and P. aeruginosa quorum sensing. idaeus; R.eubateus, Vaccinium macrocarpon; V.. and swarming motility inhibition. angustifolium;. Zingiber officinale. Cinnamaldehyde. Autoinducer-2 and QS inhibition. Cinnamomum cassia essential oils. Inhibition of biofilm formation by E. coli. Cinnamomum nardus and C. zeylanicum essential oils Dihydroxybergamottin; Bergamottin. Improve E. coli biofilm formation potential Autoinducer-1 and 2 inhibition162 Prevention of antibiotic resistant Staph.. Diterpenoids from Salvia sclarea:. aureus and Staph. epidermidis adhesion. Salvipisone ;Aethiopinone. and inactivation of their biofilms Induction of biofilm formation by P.. Epigallocatechin gallate. syringae. Epigallocatechin gallate ; Ellagic acid; Tannic acid. Hamamelitannin from Hamamelis virginiana (Simões et al., 2009). 14. Inhibition of biofilm formation of Burkolderia cepacia, E. coli and P. putida QS inhibition of MRSA and Staph. epidermidis.

(26) (lipase) (P. acnes. S. epidermidis. Pityrosporum ovale). P. acnes. (Higaki, 2003. Jaeger et al., 1994). Marple et al. (1971). clindamycin erythromycin. P. acnes. benzoyl peroxide. P. acnes. P. acnes. P. acnes Puhvel et al. (1972) 500µg/mL. 70 - 80% P. acnes Ca2+. (Golub et. al, 1998) Kampo formulation (Glycyrrhizae radix). Jumi-haidoku-to (Platycodi radix). Kampo formulation P. acnes. (flavonoids) P. acnes. 2006). 15. (Falcocchio et al.,.

(27) 1 Grange et al. (2009). P. acnes (superoxide anions). CD36. P. acnes. TLR-2. IL-8. P. acnes. 2-10 P. acnes IL-8 Figure 2-10. Proposed molecular mechanisms through which P. acnes induces ROS and IL-8 production in keratinocytes. Surface components of P. acnes are recognized by both CD36 and TLR-2. CD36 triggers the production of O2 N2 through the NADPH oxidase pathway (NOX) and combines with NO to form peroxinitrites which, in turn, activate p38 and ERK MAPKs, thus contributing to IL-8 production. In parallel, IL-8 production is activated through the TLR-2 signalling pathway. : (Grange et al., 2009). 16.

(28) 2. (superoxide anion radical 1. (singlet oxygen radical. O2). (hydroxyl radical. ROO .). .. OH). (peroxyl. (alkoxyl radical RO .). H2O2 ) (Halliwell et al.. O2 .-). (hydrogen peroxide. 1995). DNA. .. OH, 1O2, H2O2,. NO.. peroxynitrile (ONOO-). (Halliwell & Gutteridge, 1990). 3 C. E. flavonoids ( tannins. hydroxylcinnamate ester. lignans (. ). (Rice-Evans et al., 1997) Manach et al. (2004). (. ). phenolic acids(. ) flavonoids (. ) stilbenes. (flavonoids). ( (. (flavonols) (anthocyanin). (lignans)(. C). (flavones). A. 2-11). B). C (isoflavones). (flavanols)(. (flavanones) )(. 17. 2-12).

(29) 2-11 Figure 2-11. Chemical structures of polyphenols. : (Manach et al., 2004). 18.

(30) 2-12 Figure 2-12. Chemical structures of flavonoids. : (Manach et al., 2004). 19.

(31) Vaya & Aviram (2001) ( ). ROS/RNS. (initiators)(. ). (1). (Ingold, 1968). C. E. (propagation) (glutathione peroxidase (GPx)) (ROOH, and PL-OOH). (ROH, PL-OH) (Thomas et al.,. 1990). (. ). ). (. (Vaya & Aviram, 2001). (2). UV (peroxidases) (NADPH oxidase) hydroxylase). (xanthine oxidase). NADPH β-. (dopamine. (lipoxygenases). (Van Acker et al., 1996) (3). (Co-antioxidants) C. E. (Doba et al.,1985) Bowry, et al (1995). 20.

(32) (bilirubin) (catechol derivatives) 2-13). (aminophenol derivatives) quinols. C(6-palmityl ascorbate)( C. 2-13 Figure 2-13. Structures of some co-antioxidants (Vaya & Aviram, 2001). .. 21.

(33) 1. (Ocimum basilicum L.) (Labiatae). (Suppakul et al., 2003). 2. (Ginkgo biloba) (terpenoids) (ginkgolides). (bilobalide) (Kleijnen. 3. Knipschild, 1992). (Toona sinensis M. Roem). (Chang et al., 2006) Chen et al. (2009) (3,4,5-trihydroxybenzoic acid. 4. gallic acid. GA). (Glycyrrhiza glabra L.). : triterpene saponins chalcones. glycyrrhizic acid. flavonoids. isoflavonoids (Asl &. Hosseinzadeh, 2008). 5. (Osmanthus fragrans Lour). (Lee et al., 2007). 6. (Zingiber officinale Roscoe) oleoresin. 6-gingerol (1-[ 4-hydroxy-. 22.

(34) 3-methoxyphenyl ]-5-hydroxy-3-decanone). vanilloid, 6 - paradol. (1-[4-hydroxy-3-methyoxyphenyl]-3-decanone. 6-gingerol. (Bode & Dong, 2004). 7. (Alliun stivum L.) allicin. disulfide. diallyl trisulfide. diallyl tetrasulfide. diallyl. diallyl monosulfide. (Lai & Roy, 2004). 8. (Crocus sativus L.). carotenoids. (Abdullaev,. 2002). 9. (Crataegus) epicatechin. isoquercitrin. protocatechuic acid. rutin. chlorogenic acid. hyperoside,. quercetin. (Ho et al., 2004). 10. (Opuntia ficus indica (L.) Mill) betacyanin betanin (5-O-glucose betanidine) betaxanthin. 11. (Livrea & Tesoriere, 2004). (Carum carvi ) carvone. limonene. (Cowan, 1999). 12. (Mentha piperata) (carminative) (antiemetic). (diaphoretic). (emmenagogue). (analgesic). menthol. al. 2001). 13. (antispasmodic). (Sinapis alba L.). 23. menthone. (timulant) (Shotipruk et.

(35) glucosinolates pseudo-glucosides. myrosinase (Eskin et al., 2007). 14. (Alpinia galanga) 1-acetoxychavicol acetate. (Latha et. al., 2009). 15. (Cymbopogon citratus) Adegoke酚. Odesola (1996 ). (glycosides). (terpenes). 16. (Momordica charantia) charantin vicine. polypeptide-p. (Dans et al., 2007). 17. (Piper nigrum) volatile oils. 25. 25 volatile oils. (Dorman & Deans, 2000). 18. (Phaseolus aureus) (Nagajyothi et al.,. 2010). 19. (Quisqualis indica Linn.) ellagitannins(. quisqualin A. quisqualin B). (Jahan et al., 2009). 20. (Anglica sinensis(Oliv.) Diels) coumarins. ligustilides. (antiarrhythmic) (Chao et al., 2010). 21. (Perilla frutescens) perilla aldehyde. limonene. 24.

(36) (Takano et al., 2004). 22. (Taraxacum formosanum Kitamura) taraxinic acid 1''-0-β-Dglucopyranoside. (Leu et al., 2003). 23. (Arctium lappa L.) caffeoylquinic acid derivative lignans (mainly arctiin) (antimutagenicity) (Ferracane et al., 2010). 24. (Houttuynia cordata) quercitrin isoquercitrin decanoylacetoaldehyde (antipyretic). 25. (antidotal). (Kwon et al., 2003). (Portulaca oleracea Linn.) Portulaceae (analgesic). coumarins. monoterpene glycoside. dopamine dopa. N-trans-feruloyltyramine. noradrenaline ferulic acid. adenosine (Kumar et al.,. 2008). 26. (Citrus medica) Rutaceae. limonene. 27. γ-terpinene. (Conforti et al., 2007). (Euphoria longana Lam.). corilagin (an ellagitannin ). ellagic acid. (TNF-α). noradrenaline (Rangkadilok et al.,. 2005. 28. (Elsholtzia ciliate (Thunb.) Hyland) Labiatae. hypericin. quinones. 25.

(37) 2,5,9,12-tetra-(carboxyethylthiomethyl) hypericin. (Wang, et al.. 2006). 29. (Eletteria cardamomum Maton) cardamom. citronellal. citral. camphene. citronellol. ascaridole. α-phellandrene. camphor. geranyl acetate bisabolene. famesol. (Bernhard et al. 1971). 30. (Rosmarinus officinalis). carnosol rosmanol. borneol. bornyl acetate. cereus Staphylococcus aureus Vibrio parahaem Shigella sonnei (Lai. Bacillus Shigella flexneri. Roy, 2004) Craig (1999). (Wargovich et al., 2001) Moreno et al. (2006) acid. caffeic acid. carnosic rosmarinic acid. 2-14. 31. (Salvia officinalis L.) thymol Bacillus cereus. eugenol. Staphylococcus aureus. 2004) Craig (1999). 26. Vibrio parahaemolyticus (Lai. Roy,.

(38) Tada et al. (2010) carnosic acid. carnosol. rosmanol. 2-15. 32. (Camellia sinensis). (catechins) theaflavins. thearubigins. theaflavins aromatic ring (tropolone). (Hamilton-Miller, 1995). 5%. caffeine acid (. 10%. ). isoflavanoids epicatechin (EC). epigallocatechin (EGC). epicatechin gallate (ECG). epigallocatechin gallate (EGCG) epigallocatechin gallate. (Sakanaka et al., 1989) (leukocytes),. (Otake et al.,1991). 27.

(39) 2-16. 33. 2-17. (Verbena officinalis Linn) (Verbenaceae) El-Hela & Bdullah, (2010) Verbena rigda S. epidermidis. S. aureus. Bacillus subtulis Alzheimer's disease (Lai et. β-sitosterol, ursolic acid. al., 2006) oleanolic acid acids. 3-epiursolic acid. 3-epioleanolic acid. triterpenoids (Deepak & Handa, 2000). 2-18. 28. ursolic acid. oleanolic.

(40) 2-4 Table 2-4 Chemical constituents of the botanical extracts Common name Botanical name. Major phytochemical component. Biological effects. Mustard seed. Sinapis alba L.. Glucosinolates. Inhibit tumorigenesis and Eskin et al., 2007 prevention of heart diseases. Galangal. Alipinia Galanga. 10-acetoxychavicol acetate. Anti-bacteria. Latha et al., 2009. Lemongrass. Cymbopogon citratus. Tannins, alkaloids and glycosides, terpenes. Antimicrobial activities. Adegoke et al., 1996. Garlic. Alliun stivum L.. Allicin, diallyl disulfide, diallyl trisulfide, Anticancer, antibacterial diallyl tetrasulfide and diallyl monosulfide. Ginger. Zingiber officinale Roscoe. Antibacterial, antioxidant 6-gingerol (1-[4-hydroxyproperties,anti-inflammatory Bode & Dong, 2004 3-methoxyphenyl]-5-hydroxy-3-decanone ) and antinausea. Wild bitter gourd. Momordica charantia. Charantin, vicine, and polypeptide-p. Antidiabetic agent. Reference. Lai et al., 2004. Dans, 2007.

(41) 2-4-1 ( ) Table 2-4-1 Chemical constituents of the botanical extracts (continued) Common name. Botanical name. Major phytochemical component Biological effects. Reference. Mung bean. Phaseolus aureus. phenolics and flavonoids. Nagajyothi et al., 2010. Rangoon creeper. Quisqualis indica Linn.. Flavonoids, ellagitannins, sterols Antibacterial and terpenes. Ginkgo. Ginkgo biloba. Flavonoid, terpenoids. Protection of the nervous and Kleijnen et al., 1992 circulatory system. Antioxidative activity. Jahan et al., 2009. Tang Kuei. Anglica sinensis (Oliv) Diels. Coumarins, ligustilides. Protective effect on endothelial cell damage, antioxidant properties and anti-inflammatory. Perilla. Perilla frutescens. Perilla aldehyde, limonene, rosmarinic acid. Antioxidant, antiinflammatory Takano et al., 2004 and antibacterial. 31. Chao et al., 2010.

(42) 2-4-2 ( ) Table 2-4-2 Chemical constituents of the botanical extracts (continued) Common name. Botanical name. Major phytochemical component Biological effects. Saffron. Crocus sativus L.. Carotenoids. Anti-carcinogenic and antitumor activities. Reference. Abdullaev, 2002. Greater burdock Arctium lappa L.. Antioxidant activity, Caffeoylquinic acid derivatives, antimutagenicity, lignans (mainly arctiin) and anticarcinogenicity and various flavonoids antiaging. Houttuy niae Herba. Houttuynia cordata thunb. Guercitrin,isoquercitrin, decanoylacetoaldehyde. Antipyretic, antidotal, anti-inflammatory and anti-constipation. Purslane. Portulaca oleracea Linn.. Flavonoids, coumarins and monoterpene glycoside. Diuretics, febrifuge, antiseptic, antispasmodic and Kumar et al., 2008 vermifuge. 32. Ferracane et al., 2010. Kwon et al., 2003.

(43) 2-4-3 ( ) Table 2-4-3 Chemical constituents of the botanical extracts (continue) Common name. Botanical name. Major phytochemical component. Biological effects. Reference. Longan. Euphoria longana Lam.. Gallic acid, corilagin (an ellagitannin ), and ellagic acid. Antifungal activity, antihypertension, antiHIV, inhibition the release of TNF-α. Rangkadilok et al., 2005. Aromatic madder. Elsholtzia ciliate (Thunb.) Hypericin, quinones Hyland. Antiviral. Conforti et al., 2007. Osmanthus. -linalool, beta-linalool, Neuroprotective, free Osmanthus fragrans Lour酚trans-linalool oxide, cis-linalool oxide, radical scavenging and alpha-lonone, beta-lonone, anti-oxidative effects. capraldehyde and decalactone. Chinese cedar. Toona sinensis M. Roem. Gallic acid (3,4,5-trihydroxybenzoic acid, GA). Anti-inflammatory & anticancer,. Licorice. Glycyrrhiza glabra L.. Glycyrrhizic acid, glycyrrhizin. Antibacteria, antioxidant, Asl et al., 2008 antitumor activities. 33. Lee et al., 2007. Chen et al., 2009.

(44) 2-4-4 ( ) Table 2-4-4 Chemical constituents of the botanical extracts(continue) Common name. Botanical name. Major phytochemical component. Biological effects. Reference. Black pepper. Piper nigrum. Volatile oils. Antimicrobial. Dorman et al., 2000. Caraway. Carum carvi. Carvone, coumarin, limonene. Diuretics, antibacterial Antibacterial, carminative, anti-inflammatory, antispasmodic, antiemetic, diaphoretic, analgesic, stimulant, emmenagogue, and anticatarrhal. Cowan, 1999. Shotipruk et al., 2001. Peppermint Mentha piperata. Menthol, menthone. Basil. Ocimum basilicum. Antibacterial, radioprotective, Linalool, methylchavicol, anticarcinogenic and antioxidant eugenol, and methyl cinnamate properties.. Suppakul et al., 2003. Medicinal Citron. Citrus medica. Monoterpenes and sesquiterpenes. Antifertility agent, antioxidant, hypoglycaemic and anticholinesterase. Conforti et al., 2007. Dandelion. Taraxacum Taraxinic acid formosanum Kitamura 1''-0-β-Dglucopyranoside. Anti -hepatitis and cancer. Leu et al., 2003. 34.

(45) 2-4-5 ( ) Table 2-4-5 Chemical constituents of the botanical extracts (continue) Common name Botanical name. Major phytochemical component. Eletteria cardamomum India cardamom Maton. Camphene, α-phellandrene, camphor, citronellal, citral, citronellol, ascaridole, Antioxidant activity geranyl acetate, bisabolene, famesol. Bernhard et al., 1971. Hawthorn Fruit Crataegus. Epicatechin, chlorogenic acid, Hypocholesterolemic, hyperoside, isoquercitrin, antioxidant and protocatechuic acid, rutin, and quercetin blood-vessel-relaxing activity.. Ho et al., 2004. Opuntia ficus indica Pricklyash Peel (L. ) Mill. Betacyanin betanin ( 5-O-glucose betanidine ) and the betaxanthin indicaxanthin. 35. Biological effects. Reference. Antioxidant, radical scavenging Gentile et al., 2004.

(46) 2-5 ( ) Table 2-5 Chemical constituents of the botanical extracts used in the present study Common name. Botanical name. Major phytochemical component. Rosemary. Rosmarinus officinalis. Carnosol, rosmanol, essential oils, borneol, bornyl acetate, carnosic acid. Lai & Roy, 2004. Sage. Salvia officinalis L.. Thymol, eugenol, carnosic acid, carnosol, rosmanol. Lai & Roy, 2004. Black tea. Camellia sinensis. catechins. Hamiltonmiller, 1995. Green tea. Camellia sinensis. catechins. Hamiltonmiller, 1995. Verbena. beta-sitosterol, ursolic acid, Verbena officinalis Linn oleanolic acid, 3-epiursolic acid, 3-epioleanolic acid. 36. Biological effects. Reference. Deepak & Honda et al, 2000.

(47) 34. ( (d.d. H2O methanol ethanol. ) EA). 4 hr methanol. ethanol. EA. (Cat. N-1000V-W, Eyela, St.. Koishikawa, Tokyo, Japan). DMSO (dimethyl. sulfoxide, Cat D8418, sigma, St Louis, MO, USA) mg/mL. stock solution. 400. (. ). PBS. stock solution(. 400. mg/mL). (Propionibacterium acne (Staphylococcus epidermidis Industry research. BCRC10723). BCRC10785). (Food. development Institute, Hsinchu). (Bioresource. Collection and Research Center, Hsinchu). 0.3 mL. 10 mL. 24~72 hr. 37. 20-25. -80. 37.

(48) (1) 1.. (P. acnes) 37 g brain heart infusion (BHI) broth (Cat. 237500, Becton Dickinson and. Company, St. Becton Drive, FL, U.S.A) 100 mL. 0.22. 900 mL. m filter. d.d. water. 10. (Cat. 158968,. Sigma) 2.. (S. epidermidis) 30 g tryptic soy broth (Cat. 237500, Becton Dickinson and Company) 1. d.d. water. 3.. (agar plate) 7.5 g. Laboratories, Detroit, MI, USA). agar. 1.2 kg/cm2. 121. (Difco. 30. (2) 1.. (P. acnes) Tsai et al. (2010) 10 mL. loop. 37. Company). P. acnes. (Cat. 260672, Becton Dickinson and. (Cat. 261205, Becton Dickinson and Company) 2 mL. 8 mL. 72 hr 2.. (S. epidermidis) 1 loop 37. S. epidermidis. 10 mL tryptic soy broth. (Cat.DB45, Deng Yang, Taipei, Taiwan) 8 mL. 24 hr. 2 mL. 24 hr. (3) 24-72 hr 10. OD600 10櫞. 38. L. L.

(49) 37 ). 24 hr. (. 30-300. 10. ). ( (CFU/mL). OD 600nm. CFU/mL. P. acnes. 72 hr. 1.7. 6 x 1010. S. epidermidis. 24 hr. 1.1. 8 x 108. 39.

(50) 1.. (. 3-1) (broth dilution method). (minimal inhibitory concentration. MIC) (Tsai et al., 2010) (Chomnawang et al.,. 2005). DMSO DMSO. stock. DMSO. 4 % (V/V). 400 mg/mL. DMSO. 16 mg/mL. 8 4 2 1 0.5 0.25 0.125 mg/mL. working solution well. 100. L broth 100. 100. L. well. 100. well. 72 hr (P. acnes). L. 1×107 CFU/mL. 24 hr (S. epidermidis). 1x108 CFU/mL. well DMSO. PBS. (Cat T 7660, Sigma). 48 hr (P. acnes). 24 hr (S. epidermidis). Winooski, USA) MIC. MIC. 37. OD600 nm < 0.05 loop. agar plate (colony). (minimal bactericidal concentration. vehicle. OD600 nm (SynergyΠ, Biotek, St Tigan, blank. (MIC). OD600 nm. 2 x107 CFU/mL (S. epidermidis). broth. control. L. L broth. 2 x108 CFU/mL (P. acnes) well. 100. MBC). 40. 96 well 37. 24 hr.

(51) MBC MIC. 24hr incubation. 3-1 broth dilution method Figire 3-1 The anti-bacterial properties of botanical extracts was evaluated by broth dilution method. P. acnes 1. P. acnes. (biofilm). P. acnes step one: conditioning step two: attraction (easy removed and killed) step three: attachment (polysaccharide secretion) step four: metabolism (protected colony). P. ances biofilm (mature biofilm). biomass. (i.e. a biofilm in which the. biomass does not significantly increase any further) (prevention of biofilm formation) (removal of established biofilm). 41.

(52) mocrotiter plate test et al., 2007) plate. (Stepanovic´ et.al, 2000) (Coenye. sterile polystyrene round-bottom 96 well. well. 100. 37. 12. L 16. 1x108 CFU/mL. 72 hr 24. 48. 72 hr. 24 hr. crystal violet. 2.. P. acnes 16 hr. 48 hr 16 hr. biomass. hr. 48. (prevention of. biofilm formation. PBF). (removal of established biofilm. REB) (1x108 CFU/mL). P. acnes well plate 37 (10櫞. 16. sterile polystyrene round-bottom 96. 48 hr. L/well). sub MIC. 24 hr. crystal violet staining. Crystal violet staining. well. 99% methanol (Cat 9093-68, Sigma). 15 min. 0.05 % crystal violet solution (Cat C0775, Sigma). 160. L. 10櫞. L. 20 min. 96 well well. well. 20. well. L 33% acetic acid solution (Cat. 017-00256, Wako, St.. Doshomachi, Osaka, Japan) blank(B). 5 min (C). (T). % of control. % of control =. (T − B) × 100% (C − B). 42. OD590nm.

(53) P. acnes. P. acnes. S. epidermidis. (extracellular lipase). BHI broth. lipase activity assay unknown components (crude lipase). Pablo et al. (1974). 1. (a) P. acnes 1 loop. P. acnes 4. 200 mL BHI broth. 250 mL. 16. 50 mL 37. 16 hr. 200 mL. 50 m L. 4 3000 rpm. (. 100. 250 mL. L. 10 min. ). (b) S. epidermidis 1 loop. S. epidermidis 4. 250 mL. 200 mL TSB broth. 250 mL. 16. 50 mL. 37. 24 hr. 200 mL. 50 ml. 4. 3000 rpm. (. 100. L. 10 min. ). 2. (1) Pablo et al. (1974). (ammonium sulfate) pH = 6. pI = 5. pH. pI pH = pI. (. 43.

(54) ). ( (. ). 3-1). 3-1. 2001. (2). 500 mL. 60. (ammonium sulfate) (361×0.5 =180.5 gm) 50 mL. 10 min. 10 min. 4000 rpm PBS PBS. L. 100. L. - 20. (Cat. EW-02899-42 ,. Cole-Parmer, 625 East Bunker Court, USA) PBS (. PBS. 100. 1:100). 44. 6 4. 48 hr. 24 hr.

(55) PBS. PBS. Amicon® ultra-15 centrifugal filter devices (PL -10, Millipore, Concord Road, Billeri, CA, USA) 4 enpendorff. 30 min. - 20. (3) (a). BSA. BSA (Cat: 85041C, Sigma) 0. 噁. 400. 600. 800. 200. 400. 600. 800. 1000. L. 0. 20. 40. 60. 80. 100. 100. 80. 60. 40. 20. 0. L. 100. (b) Sample. 5. well. 25. L. L bio-rad protein assay reagent A (Cat 500-0113, Bio-rad,. Hercules, CA, USA). 5. L. standard. sample. 20櫞. protein assay reagent B (Cat 500-0114, Bio-rad) (. g/mL. 0. L. (c). 1000. g/mL. 1000ug/ml BSA DDW噁. 200. d.d. water. Bio-rad. ELISA reader. 750 nm). (4). L. BSA. SDS-PAGE 1櫞. separating gel. 75. 75. comb. stacking gel. comb. comb sample buffer well. 4:1. 5X. 100. 10 min. 100 comassie stain solution (. ). comassie stain solution. 30 min distain solution (2櫞. 45.

(56) methanol & 1櫞. acetic acid in milli Q water) (. ). distain solution. 0.75 mm. A (30 % acrylamide) B 1.5 M Tris (base) TEMED (sigma T-8133) 1.8 mL C TEMED (sigma T-8133) 0.4 mL 10% SDS d.d. water 10% APS solution Total volume. B 1.5 M Tris (base)酚 TEMED (sigma T-8133) 300 mL HCl C 0.5 M Tris (base) TEMED (sigma T-8133) 40 mL HCl Sample buffer (5X) 125 mM Tris (base) 2 mM EDTA 2Na 2 % SDS 5 % β-mercaptoethanol 20 mL HCl (5). pH. pH. pH. 8.8. (10 %) running gel (mL) 3.35 2.5. (4 %) Stacking gel (mL) 0.33 -. --. 0.62. 0.1 4 200 L 10.05. 0.025 1.475 10櫞 L 5. 90.8 gm 1.8 mL 500 mL. 6.8. 6 gm 0.4 mL 100 mL. 6.8. 3.785 gm 0.0185 g 5g 12.5 mL 50 mL. (Zymography) Diaz et al. (1999). (a) P. acnes. S. epidermidis. sample buffer (5X). 46. 4:1.

(57) SDS-PAGE (b) 2.5 min. SDS. Triton X-100. Triton X-100. d.d. water. 1 min 4-MUB. PBS). 15 1 X PBS. PBS 30 sec. 1 X PBS. (. 0.1 mM. 30 sec. (ChemiDoc XRS+) (170-8265, bio-rad, Hercules, CA, USA) ethidium bromide. (6) 1.. (fluorescent assay) lipase lipase activity. (tributyrylglycerol) agar plate assay,. tributyrine titrimetry. (Gupta et al., 2003) (fluorescence assay). (Roberts et al.1985) (. butyrate (4-MUB). 3-2). 4-MU. 4-methylumbelliferyl 360 nm. 3-2 Figure 3-2 Hydrolysis reaction on which the fluorescent lipase assay is based. (Roberts et al., 1985). 47.

(58) (1). sigma. BioChemika) well 2櫞. Aspergillus oryzae (Cat 62285,. lipase activity assay. L. 4 mM. 2 mM. Aspergillus oryzae lipase (疱. 4.5. 80 mL PBS. 5 hr. 37. 240 300 min. 2.25. 4 - MUB 1.125. 2櫞. 0.56. L. 0.2苷. 0.14. 0.07 mg/mL). 0 5 10 15 20 30 45 60 120 180. Fluorescence λex 360 nm; em 450 nm. (. 4 mM. 4-MUB. 2. P. acnes. S. epidermidis. Roberts et al.(1985) solution. 20. L. well. 4-MUB (4 mM) 80. 450 nm. crude lipase. L. L PBS. 10 15 20 30 45 60 120 180 240 300 min em. 20. crude lipase 5 hr. (. 0. 5. Fluorescence λex 360 nm;. (lipolytic activity. ∆RFU/min/mg. protein ) Vmax (Lipase activity) = (fluorescence intensity at T Vmax time fluorescence intensity at T0)/ min/mg protein = ∆RFU/reaction time/ mg protein 1. ∆RFU/reaction time/ mg protein. 1. arbitrary unit (A.U.). A.U.. 3. P. acnes Vmax. Vmax. 20 min S. epidermidis. 60 min. P. acnes DMSO. epidermidis. DMSO. 48. S..

(59) 2.5. (V/V). DMSO. stock. 400 mg/mL. 10 mg/mL. well. 20. L. 20. 4 mM 4-MUB. 6櫞. L PBS. (A.U.). 20. L. 37. Fluorescence λex 360 nm. (. L. em. 450 nm). Vmax. IC50. (C). (T). 酚 of inhibition = (1 −. 1.. T ) × 100% C. DPPH Tsai et al. (2008) 20. L. DPPH (20 10 5 1 0.5 0.05 mg/mL). PBS). 96 well. 200. (4-tert-octylphenyl)-1-picrylhydrazyl 3 min. 20. L. (DMSO. L 1mM 2, 2-Di DPPH)(Cat. 257621, Sigma). 100 %. 490nm. IC50 DPPH % of scavenging activity =1-[Asample -Ablank of sample/Acontrol -Ablank of control]) x 100 %. 2. Tsai et al. (2007) 50. (20 10 5 1 0.5 0.05 mg/mL) PBS). Sigma). 96 well. PBS(. 50 ). NO. 20 mg/mL. (DMSO. L 10 mM sodium nitroprusside (SNP) (s0501, 90 min. greiss reagent (. OD 570 nm. A. well B IC50. 49. ). 酚. 100. L. 1 min. 1:1.

(60) % of scavenging activity =1-[Asample -Ablank of sample/Acontrol -Ablank of control]) x 100 %. 3. Tsai et al. (2008) (1) (gallic acid) (G7384, Sigma) stock 2. 1. 0.5. 0.2. 10 mg/mL. 0.1. 0.05. d.d. water. 4. 0 mg/mL. 0. 0.05. 0.1. 0.2. 0.5. 1. 2. 噁. 0. 5. 10. 20. 50. 100. 200. 1000. 995. 990. 980. 950. 900. 800. L). L L. 7.5% Na2CO3. 4 mL. (mg/mL) 10 mg/ml DDW. 40 mg. 1000. 10. L. 50. L Folin & Ciocalteu’s phenol reagent (F9252, Sigma) ( ). 96 well. 30 min. 40. L. OD 765 nm. (mg Gallic acid equivalents/ g solid extract). 4. Robak et al. (1988) phenazine methosulfate (PMS) dinucleotide (NADH) diformazam. nicotainamide adenine. nitroblue tetrazolium (NBT) OD 560 nm. 560 nm. PBS. phenazine methosulfate (PMS) (P9625, Sigma). 50. 600. 783. Μ nicotainamide adenine. M.

(61) dinucleotide (NADH) (Cat N4505, Sigma) (N6876, Sigma). 201.8. (. ) 5櫞. (10 5 2 1 0.5 0.1 mg/mL). L. PMS NADH. 5 min 50. M nitroblue tetrazolium (NBT). 560 nm 50. L PBS phenazine methosulfate (PMS). NBT. L. nitroblue tetrazolium (NBT). blank. IC50. % of scavenging activity =1-[Asample -Ablank of sample/Acontrol -Ablank of control]) x 100 %. 5. Jiang et al. (2006). Cheng et al. (2003) (Cat 215422, Sigma). (Cat. 216763, Sigma). luminol (Cat 09253, Sigma). 3-2 luminol Figure 3-2 The mechanism of luminal-enhanced chemiluminescence by reactive oxygen species (ROS). (Jiang et al. 2006) 200. L. 4 mM luminol. 51. Fe2+ (4.6. M)-EDTA (2.3酚.

(62) M). H2O2 (24 mM). KH2PO4-NaOH (4.17 mM, pH 7.4) (P9791,. Sigma). 5櫞. L 4 mM luminal. L Fe2+ (4.6. M)-EDTA (2.3. M) 5櫞. H2O2. 5櫞. L. (1 0.5 0.1 0.01 0.001 mg/mL). KH2PO4-NaOH. luminol. blank. DMSO. KH2PO4-NaOH. IC50. % of scavenging activity =1-[Asample -Ablank of sample/Acontrol -Ablank of control]) x 100 %. HPLC Cuvelier et al. (1996). (HPLC) Americka, Praha, CZ). (LCD 2084, ecom spol. s r.o., C18. (250 mm × 460 mm, Cat 720014.46,. Macherey- nagel, Bethlehem, PA, U.S.A) 15:84:0.85). A (Acetonitrile/water/acetic acid,. B (methanol). 100%. 1 mL/min. 90 min B UV 284 nm. (4957s, extrasynthese,Genay, France). (C0609, sigma). (SI-c9617, sigma) d.d. H2O. 400 mg/mL stock. 10 mg/mL. 0.45. one way ANOVA. SPSS 12.0 vehicle. .05. 52. DMSO. m. mean ± SD. p. 0%.

(63) 酚. 酚. 酚. 酚 34. ( 4-1. EA. ). 4-2. 4-1 Table 4-1 Yield of ethanolic extracts of plants. Common name Mustard seed Galangal Lemon grass Garlic Ginger Wild bitter gourd Osmanthus Mung bean Rangoon creeper Lilium Ginkgo Chinese cedar Pricklyash peel Black pepper Caraway Peppermint Basil Hawthorn fruit Ginkgo Tang Kuei Perilla Dandelion Saffron Greater burdock Houttuynia Purslane Medicinal citron Longan Aromatic madder. Botanical name Sinapis alba L. AlipiniaGalanga Cymbopogon citrat Ailium sativum L Zingiber officinale Roscoe Momordica charantiaC Osmanthus fragrans Vigna (L.) Wilczek Quisqualis indica Linn. Lilium formosanum Ginkgo biloba L Toona sinensis Juss Roem Zanthoxylum bungeanum Maxim. Piper nigrum Carum carvi Mentha piperata Ocimum basilicum Fructus Crataegi Pinnatifidae Ginkgo biloba Anglica sinensis Didl Perilla frutescens Taraxacum formosanum Kitamura Crocus Satovis Arctium lappa L. Houttuynia cordata Portulaca oleracea Linn. Citrus medica Dimocarpus longgana Lour Elsholtzia ciliata. 53. Part examined seed root leaves root root fruit flower seed fruit flower root leaves seed seed seed leaves leaves friuts fruits root leaves flower buds flower buds seed leaves root fruits fruits leaves. Yield (%) 15.8 10.7 28.8 12.0 2.0 --2.2 2.3 1.5 4.8 -15.0 9.0 0.9 3.3 10.6 40.0 4.8 -6.3 10.0 30.0 20.0 6.0 -31.3 44.0 6.0.

(64) 4-2 Table 4-2 Yield of botanical extracts. Common Part Botanical name name examined Rosemary Sage Black tea Grean tea Verbena. Rosmarinus officinalis Salvia officinalis L. Camellia sinensis Camellia sinensis Verbena officinalis Linn. leaves leaves leaves leaves leaves. 54. Yield (%) Ethanol 8.74 19.30 3.94 10.90 10.50. EA 2.81 1.00 1.24 4.34 1.58. Methanol 17.60 21.30 13.50 30.10 19.40. Aqueous 7.00 13.50 6.21 14.90 7.90.

(65) 酚. 1. P. acnes. (1) broth dilution assay. S. epidermidis. 34. P. acnes. (Rosmarinus officinalis ) (Camellia sinensis). (Verbena officinalis Linn). EA. (a). (Salvia officinalis L.). P. acnes. S. epidermidis. P. acnes and S. epidermidis EA. mg/mL. P. acnes. 4-3 MIC. MBC. 8 mg/mL. 16. P. acnes. S. epidermidis EA. (MIC = 4 mg/mL) (MBC = 4 mg/mL) (MIC = 8 mg/mL) (MBC = 8 mg/mL). S.. epidermidis. 4-3 P. acnes S. epidermidis Table 4-3 Anti-bacterial properties of rosemary extracts. 酚酚酚酚 P. acnes Extracts酚 Rosemary Aqueous. MIC MBC (mg/mL). 酚. S. epidermidis MIC MBC (mg/mL). > 16 > 16 > 16 > 16 Methanolic 8 16 4 4 Ethanolic 8 16 8 16 EA 8 16 8 16 Posotive control: tetracycline. The MIC of tetracycline against P. acnes and S. epidermidis are 0.8 g/mL and 12.5 g/mL, respectively.. 55.

(66) 酚. (2). (a) crystal violet staining hr. P. acnes. 12-16 hr. 16 hr. 48-72 24 hr. 48 hr 24 hr. % of crystal violet staining compare to 12hr value. 250 200 150 100 50 0 12. 16. 24. 48. 72. Incubation time(h). 4-1 P. acnes Figure 4-1 The growth curve of P. acnes biofilm in different culture times. The culture times are 16, 24, 48, 72 hr respectively. P. acnes biofilm was measured by crystal violet staining.Values are expressed relative to 12 hr. Error bars indicate standard error of the mean.. 56.

(67) 酚. (b). P. acnes. a.. 4-2. (prevention of biofilm formation (0.125 mg/mL). 4-3 PBF). (0.5 mg/mL). 300. Ethanolic extract Methanolic extract EA extract Aqueous extract. % of crystal violet staining compared to control. 250. 200. 150. * ** *. *. 100. ** *. *. *. *. *. *. 50. *. *. *. 1. 2. 0 0. * 3. 4. Concentration (mg/mL). 4-2 P. acnes Figure 4-2 Effect of different concentration of (A) ethanolic, (B) methanolic, (C) EA, (D) aqueous extracts of rosemary on the formation of P. acnes biofilm. Values are expressed relative to vehicle control following 16 hr of biofilm formation, as measured by crystal violet staining. *Significantly different from control group (0) analyzed by one way ANOVA.. 57.

(68) 酚. b.. (removal of established biofilm. REB). P. acnes. 350 Ethanolic extract Methanolic extract EA extract Aqueous extract. % of crystal violet staining compared to control. 300. 250. 200. 150. 100. 50. 0 0. 1. 2. 3. 4. Concentration (mg/mL). 4-3 P. acnes Figure 4-3 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of rosemary on P. acnes biofilm removal. Values are expressed relative to vehicle control following 48 hr of biofilm formation, as measured by crystal violet staining. *Significantly different from control group (0) analyzed by one way ANOVA.. 58.

(69) 酚. (3) (a) P. acnes. S. epidermidis. (mg protein/L of culture) 4-5) P. acnes 44 KD. 500 SDS-PAGE. S. epidermidis. (molecular mass). (. 4-4)(. 44. 41.6. 160. zymography. SDS-PAGE. P. acnes. al., 1981) S. epidermidis. 41 KD (Ingham et. 43 KD (Farrell et al., 1993). 4-4 P. acnes (A) (B) Figure 4-4 The SDS-PAGE gel of crude lipase from P. acnes. (A) Gel stained with coomassie blue with marker (B) Zymogram demonstrated lipase activity towards 4-MU butyrate.. 59.

(70) 酚. 4-5 S. epidermidis (A) (B) Figure 4-5 The SDS-PAGE gel of crude lipase from S. epidermidis. (A) Gel stained with coomassie blue with marker. (B) Zymogram demonstrated lipase activity towards 4-MU butyrate.. 60.

(71) 酚. (b). (lipase activity) P. acnes 4 mM. 4-MUB. S. epidermidis. 37. 5 hr. (. Fluorescence λex 360 nm; λem 450 nm (Lipase activity) = (fluorescence intensity at TVmax time fluorescence intensity at T0)/min/mg protein = ∆RFU/reaction time/mg protein 1 ∆RFU/reaction time/ mg protein. 1 arbitrary unit (A.U.). A.U. ( 4-6). P. acnes. 50 mg/mL (1 mg protein) Vmax 20min (273.87 A.U.) S. epidermidis 0.5 mg/mL (0.01 mg protein). 600. 1200. (A) P. acnes. 4.25 mg/mL 42.5 mg/mL 50 mg/mL 80 mg/mL. 500. (B) S. epidermidis. 0.5mg/mL 1mg/mL 5mg/mL 10mg/mL 20 mg/mL. 1000. 400. 800 RFU/min. RFU/min. Vmax 60min (18573 A.U.). 300. 600. 200. 400. 100. 200. 0. 0 0. 50. 100. 150. 200. 250. 300. 350. 0. 50. 100. 150. 200. 250. Time (minute). Time (minute). 4-6 (A) P. acnes (B) S. epidermidis Figure 4-6 Different concentrations of crude lipase from (A) P. acnes and (B) S. epidermidis. 61. 300. 350.

(72) 酚. (c) (. ). (0.5 0.1 0.05 0.005 mg/mL). P. acnes P. acnes. S. epidermidis. IC50. 120. 0.13 mg/mL. 0.22 mg/mL( 4-7). 120. (B) S. epidermidis. (A) P.acnes 100. 100 % of inhibition. % of inhibition. IC50. S. epidermidis.. 80. 80. 60. 60 40. 40. 20. 20. 0. 0 0.0. 0.1. 0.2. 0.3. 0.4. 0.0. 0.5. concentration (mg/mL). 0.5. 1.0. 1.5. 2.0. Concentration (mg/mL). 4-7 (A)P. acnes (B) S. epidermidis Figure 4-7 Effect of tetracycline on the lipolytic activity of crude lipase from (A) P. acne and (B) S. epidermidis. 62. 2.5.

(73) 酚. (d). 4-8 50 mg/mL 0.5 mg/mL P. acnes. epidermidis. 4 mM 4-MUB. 10 15 20 30 45. 60 min. (A.U.). 37. 櫞 5. 2 hr. 0 15 30 60 100 min. IC50. a.. P. acnes (IC50 = 0.68 mg/mL) >. 1.31 mg/mL) >. (IC50 = 5.89 mg/mL) > EA. b.. (IC50 =. (IC50 = 8.68 mg/mL). S. epidermidis (IC50 = 0.37 mg/mL) >. 0.78 mg/mL). (IC50 = 1.70 mg/mL) > EA. 140. Ethanolic extract Methanolic extract EA extract Aqueous extract. (A) P. acnes 120. 140. (IC50 =. (IC50 = 5.55 mg/mL). Ethanolic extract Methanolic extract EA extract Aqueous extract. (B) S. epidermidis. 120. 100. % of inhibition. 100. % of inhibition. S.. 80. 60. 80. 60. 40. 40. 20. 20. 0. 0 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. concnetration (mg/mL). 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Concentration (mg/mL). 4-8 (A) P. acnes (B) S. epidermidis Figure 4-8 Effect of rosemary extracts on the lipolytic activity of crude lipase from (A) P. acne and (B) S. epidermidis.. 63. 11.

(74) 酚. 4-4 P. acnes S. epidermidis 酚 Table 4-4 Effect of rosemary extracts on the lipolytic activity of crude lipase from P. acnes and S. epidermidis. Extracts. Rosemary. P. acnes lipase. 酚酚. S. epidermidis lipase. IC50 (mg/mL). 酚. IC50 (mg/mL). 酚. Aqueous. 0.68. 0.37. Methanolic. 1.31. 0.78. Ethanolic. 5.89. 1.70. Ethyl-acetate. 8.68. 5.55. IC50 of tetracycline were 0.13 mg/mL and 0.23 mg/mL as positive control of crude lipase from P. acnes and S. epidermidis, respectively. IC50: concentration of inhibitor yielding a lipase inhibition of 5櫞 (IC50). The assays were performed by fluorescent assay (37 and pH 7), using 4 mM 4-MUB as substrate. Smax: the highest concentration at wich each extract was teasted. 64.

(75) 酚. (4). 4-9. (a). 4-10. DPPH DPPH. = 0.77 mg/mL). EA. (IC50 = 0.69 mg/mL) >. (IC50 = 0.89 mg/mL) >. (IC50. (IC50 = 7.83 mg/mL). (b) (IC50 = 0.62 mg/mL) > EA (IC50 = 0.84 mg/mL) >. (IC50 = 0.91 mg/mL). (IC50 = 0.92mg/mL). 140. % of scavenging activity. 100. Ethanolic extract Methanolic extract EA extract Aqueous extract. 120 100. 80 60 40. (B) NO. 140. (A) DPPH. % of scavenging activity. 120. 20. Ethanolic extract Methanolic extract EA extract Aqueous extract. 80 60 40 20 0. 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. concnetration (mg/mL). concnetration (mg/mL). 4-9 (A) DPPH (B) NO Fig 4-9 (A) DPPH and (B) NO radical scavenging activity of rosemary extracts (mean ± SD, n =3). 65.

(76) 酚. (c) (IC50 = 0.28 mg/mL) > (IC50 = 0.61 mg/mL) > EA. (IC50 = 1.26 mg/mL )>. (IC50 = 1.53. mg/mL) (d) (IC50 = 26.79 (IC50 = 32.79. g/mL) > EA. g/mL) >. (IC50 = 34.00 g/mL) >. (IC50 = 34.58. g/mL). Ethanolic extract Methanolic extract EA extract Aqueous extract. 140. 140. 120. % of scavanging activity. % of scavenging activity. 120. (C) superoxide anion. Ethanolic extract Methanolic extract EA extract Aqueous extract. (D) hydroxyl radical. 100. 100 80 60 40 20. 80. 60. 40. 20. 0. 0. 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 0. 200. 400. 600. 800. concentration(ug/mL). concnetration (mg/mL). 4-10 (A) (B) Fig 4-10 (c) superoxide anion and (B) hydroxyl radical scavenging activity of rosemary extracts (mean ± SD, n =3). 66. 1000.

(77) 酚. (5).. 4-5 (114.78±6.04 mg GAE/g). (98.76±8.79 mg GAE/g) EA. >. (63.18±2.07 mg GAE/g ). (59.9±2.26. mg GAE/g). 4-5 Table4-5 Antioxidation properties of rosemary extracts Total phenolic contents. Antioxidation. Extracts. (mg GAE/g). IC50 DPPH. NO. Superoxide anion. Hydroxyl radical. (mg/mL). (mg/mL). (mg/mL). (ug/mL). 7.83. 0.92. 0.28. 34.58. 114.78±6.04c. Methanolic. 0.69. 0.62. 0.61. 26.78. 98.76±8.79b. Ethanolic. 0.77. 0.91. 1.53. 32.79. 59.90±2.26a. Ethyl-acetate. 0.89. 0.84. 1.26. 34.00. 63.18±2.07a. 酚 Rosemary Aqueous. 酚. Data given as mean ± standard deviation of triplicate tests. Samples followed by the same letter are not significantly different according to Ducan’s multiple comparison test at P = 0.05. NF=Not found. IC50, concentration causing 5櫞 inhibition. Total phenolics contents were expressed as mg gallic acid equivalents g-1 of solid extract.. 67.

(78) 酚. (6). HPLC. Cuvelier et al. (1996). HPLC 22.4 64.4. ( EA. 4-16 4-12. 4-17 4-13. 74.1 min. 4-18 4-14. 4-11. ) 4-15. 4-11 Fig 4-11 HPLC profile of an experimental solution obtained by mixing three standard compound.. 68.

(79) 酚. 4-12 Fig 4-12 HPLC profile of an experimental solution obtained by plant ethanolic extract of rosemary.. 4-13 Fig 4-13 HPLC profile of an experimental solution obtained by plant methanolic extract of rosemary.. 69.

(80) 酚. 4-14 EA Fig 4-14 HPLC profile of an experimental solution obtained by plant EA extract of rosemary.. 4-15 Fig 4-15 HPLC profile of an experimental solution obtained by plant aqueous extract of rosemary.. 70.

(81) 酚. HPLC. EA EA 4-16. (a) (1.069 ± 0.17 mg/g) EA. (0.28 ± 0.02 mg/g). (0.07 ± 0.00 mg/g). 4-16 Fig 4-16 standard curve of rosemarinic acid (b) (3.26 ± 0.10 mg/g) mg/g). EA. (0.63 ± 0.02 mg/g). 4-17 Fig 4-17 standard curve of carnosol. 71. (0.85 ± 0.09.

(82) 酚. (c) EA 0.12 mg/g). (6.02 ± 0.39 mg/g) (1.03 ± 0.11 mg/g). 4-18 Fig 4-18 standard curve of carnosic acid. 4-6 Table 4-6 the contents of potent compound from rosemary extracts Rosmarinic carnosol carnosic acid acid Extract mg/g extract Rosemary Aqueous ---Methanolic 1.069 ± 0.17 3.26 ± 0.10 2.61 ± 0.12 Ethanolic 0.85 ± 0.09 1.03 ± 0.11 0.28 ± 0.02 Ethyl-acetate 0.07 ± 0.00 0.63 ± 0.02 6.02 ± 0.39. 72. (2.61 ±.

(83) 酚. 2. (1). P. acnes. S. epidermidis. 4-7 P. acnes mg/mL). (MIC = 1 mg/mL) (MBC = 1. (MIC = 2 mg/mL) (MBC = 16 mg/mL) (MIC = 4 mg/mL) (MBC = 16 mg/mL). epidermidis. EA S.. (MIC = 1 mg/mL) (MBC = 1 mg/mL) EA. (MIC = 16 mg/mL) (MBC = 16 mg/mL) P. acnes. mg/mL). (MIC = 2 mg/mL) (MBC = 2. (MIC = 2 mg/mL) (MBC = 16 mg/mL). (MIC = 4 mg/mL) (MBC = 16 mg/mL) S. epidermidis. (MIC = 4 mg/mL) S. epidermidis. (MIC = 1 mg/mL) (MBC = 1 mg/mL) 16 mg/mL) (MBC = 16 mg/mL). EA. EA. (MIC =. 酚. 酚 4-7 P. acnes S. epidermidis Table 4-7 Anti-bacterial properties of Camellia sinensis extracts. 酚 P. acnes 酚 S. epidermidis MIC MBC MIC MBC Extracts (mg/mL) 酚 (mg/mL) Black tea Aqueous 2 16 1 1 methanolic 1 1 16 16 ethanolic 4 16 16 16 Ethyl-acetate 4 16 16 16 Green tea Aqueous 4 > 16 1 1 Methanolic 2 2 16 16 Ethanolic 2 16 16 16 Ethyl-acetate 4 16 16 16 Posotive control: tetracycline. The MIC of tetracycline against P. acnes and S. epidermidis are 0.8 g/mL and 12.5 g/mL, respectively. Smax: the highest concentration at which each extract was tested.. 73.

(84) 酚. (2). P. acnes. (a). 4-19. (prevention of biofilm formation EA. 4-20. PBF). (0.06 mg/mL) (0.06 mg/mL) EA. (0.06 mg/mL). (0.125 mg/mL). 180. 140. (B) green tea. 140. % of crystal v iolet staining com pared to control. % of crystal v iolet staining com pared to control. Ethanolic extract Methanolic extract EA extract Aqueous extract. (A) black tea. 160. Ethanolic extract Methanolic extract EA extract Aqueous extract. 120. 120 100. 100. *. 80 60 40. *. *. *. *. *. 80. *. *. *. 60. * *. 20 0. *. * * *. *. 40 0.0. 0.1. 0.2. 0.3. 0.4. 0.5. 0.6. 0.0. Concentration (mg/mL). 0.1. 0.2. 0.3. 0.4. 0.5. Concentration (mg/mL). 4-19 (A) (B) P. acnes Figure 4-19 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of (A) black tea and (B) green tea on the formation of P. acnes biofilm. Values are expressed relative to vehicle control following 16 hr of biofilm formation, as measured by crystal violet staining. *Significantly different from control group (0) analyzed by one way ANOVA.. 74. 0.6.

(85) 酚. (b). (removal of established biofilm. REB). P. acnes 0.06 mg/mL. 200. 200 Ethanolic extract Methanolic extract EA extract Aqueous extract. 150. 100. 50. 180. % of crystal v iolet staining com p ared to control. (A) black tea % of crystal v iolet staining com pared to control. P. acnes. (B) green tea. 160 140 120 100. *. 80. *. *. *. 60. *. *. *. 40 0. Ethanolic extract Methanolic extract EA extract Aqueous extract. *. 20 0.0. 0.1. 0.2. 0.3. 0.4. 0.5. 0.6. 0.0. Concentration (mg/mL). 0.1. 0.2. 0.3. 0.4. 0.5. Concentration (mg/mL). 4-20 (A) (B) P. acnes Figure 4-20 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of (A) black tea and (B) green tea on the removal of P. acnes biofilm. Values are expressed relative to vehicle control following 48 hr of biofilm formation, as measured by crystal violet staining. *Significantly different from control group (0) analyzed by one way ANOVA.. 75. 0.6.

(86) 酚. (3). 4-21 4-22. (a). P. acnes. a. P. acnes mg/mL) >. (IC50 = 0.37. (IC50 = 0.96 mg/mL)>. (IC50 = 1.48 mg/mL) >EA. (IC50 = 3.36 mg/mL) b. P. acnes >. (IC50 = 5.14 mg/mL). 120. 140. Ethanolic extract Methanolic extract EA extract Aqueous extract. (B) Green tea. 120. 100. 100 % of inhibition. % of inhibition. EA. Ethanolic extract Methanolic extract EA extract Aqueous extract. (A) Black tea. 140. (IC50 = 1.49 mg/mL). 80 60. 80 60. 40. 40. 20. 20. 0 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. concnetration (mg/mL). 0 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. concnetration (mg/mL). 4-21 (A) (B) P. acnes Figure 4-21 Effect of (A) black tea and (B) green tea extracts on the lipolytic activity of crude lipase from P. acne. 76. 10. 11.

(87) 酚. (b). S. epidermidis. a. S. epidermidis mg/mL) >. (IC50 = 1.61. (IC50 = 2.61 mg/mL) >. (IC50 = 5.34 mg/mL) EA. b. S. epidermidis mg/mL) >. (IC50 = 5.41 mg/mL). Ethanolic extract Methanolic extract EA extract Aqueous extract. (A) Black tea. 140 120. 60. Ethanolic extract Methanolic extract EA extract Aqueous extract. (B) Green tea. 120. 100 80. EA. 140. % of inhibition. % of inhibition. (IC50 = 3.32. 40. 100. 80. 60. 40. 20. 20. 0 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Concentration (mg/mL). 0 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. Concentration (mg/mL). 4-22 (A) (B) S. epidermidis Figure 4-22 Effect of (A) black tea and (B) green tea extracts on the lipolytic activity of crude lipase from S. epidermidis.. 77. 10. 11.

(88) 酚. 4-8 P. acnes S. epidermidis Table 4-8 Effect of black tea and green tea extracts on the lipolytic activity of crude lipase from P. acnes and S. epidermidis 酚 P. acnes lipase 酚 S. epidermidis lipase Extracts IC50 (mg/mL) 酚 IC50 (mg/mL) Black tea Aqueous 0.96 1.61 Methanolic 0.37 2.61 Ethanolic 1.48 5.34 Ethyl-acetate 3.36 Smax Green tea Aqueous 1.49 3.32 Methanolic 5.14 5.41 Ethanolic Smax Smax Ethyl-acetate Smax Smax IC50 of tetracycline were 0.13 mg/mL and 0.23 mg/mL as positive control of crude lipase from P. acnes and S. epidermidis, respectively. IC50: concentration of inhibitor yielding a lipase inhibition of 5櫞 (IC50). The assays were performed by fluorescent assay (37 and pH 7), using 4 mM 4-MUB as substrate. Smax: the highest concentration at wich each extract was teasted. 78.

(89) 酚. (4) (a). 4-23. a.. 4-24. DPPH DPPH. (IC50 = 0.59 mg/mL) >EA. (IC50 = 0.51 mg/mL) >. (IC50 = 2.52 mg/mL) >. (IC50 = 16.20. mg/mL) b. (IC50 = 0.04 mg/mL) (IC50 = 0.60 mg/mL) >EA. >. (IC50 = 0.69 mg/mL)>. (IC50 = 0.95. mg/mL). % of scav enging activ ity. 120. Ethanolic extract Methanolic extract EA extract. (A) DPPH. 140. Aqueous extract. 120. % of scavenging activ ity. 140. 100 80 60 40 20. Ethanolic extract Methanolic extract EA extract Aqueous extract. (B) NO. 100 80 60 40 20. 0. 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. concnetration (mg/mL). concnetration (mg/mL). 4-23 DPPH Fig 4-23 DPPH and NO radical scavenging activity of black tea extracts (mean ± SD, n =3).. 79.

(90) 酚. c. (IC50 = 0.06 mg/mL) > (IC50 = 0.26 mg/mL) >EA=. (IC50 = 0.33 mg/mL). d. (IC50 = 3.88 = 4.38. Ethanolic extract. 140. (IC50 = 7.57. g/mL) >. (C) superoxide anion. Methanolic extract EA extract. % of scavanging activity. (IC50. (IC50 = 9.49. Ethanolic extract Methanolic extract EA extract Aqueous extract. 140. 120. Aqueous extract. 120 % of scavenging activity. g/mL) >EA. g/mL)>. g/mL). (D) hydroxylradical. 100. 100 80 60 40 20. 80. 60. 40. 20. 0. 0. 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 0. 200. 400. 600. 800. Concentration(ug/mL). concnetration (mg/mL). 4-24 Fig 4-24 Superoxide and hydroxyl radical scavenging activity of black tea extracts (mean ± SD, n =3).. 80. 1000.

(91) 酚. (b). 4-25. a.. 4-26. DPPH DPPH. 0.55 mg/mL) >EA. (IC50 = 0.53 mg/mL)>. (IC50 = 3.57 mg/mL) >. (IC50 =. (IC50 = 11.63 mg/mL). b. (IC50 = 0.05 mg/mL) = 0.62 mg/mL) >EA. 120 100 80 60 40. Ethanolic extract Methanolic extract EA extract Aqueous extract. (IC50 = 0.69 mg/mL) >. (A) DPPH. 140 120 % of scav enging activity. % of scavenging activity. 140. >. (IC50. (IC50 = 7.45 mg/mL). Ethanolic extract Methanolic extract EA extract Aqueous extract. (B) NO. 100 80 60 40 20. 20 0. 0. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 concnetration (mg/mL). 4-25 DPPH Fig 4-25 DPPH and NO radical scavenging activity of green tea extracts (mean ± SD, n =3).. 81.

(92) 酚. c. (IC50 = 0.07 mg/mL) > (IC50 = 0.27 mg/mL) >. (IC50 = 0.28 mg/mL) >EA. (IC50 = 1.54. mg/mL) d. (IC50 = 4.80 6.71. Ethanolic extract Methanolic extract EA extract Aqueous extract. 160. (IC50 = 7.12. g/mL) >. (C) superoxide anion 140. 120 100 80 60 40. (IC50 = 9.08 Ethanolic extract Methanolic extract EA extract Aqueous extract. 160. % of scavanging activity. 140 % of scavenging activity. g/mL) >. g/mL) >EA. (IC50 = g/mL). (D) hydroxyl radical. 120 100. 20. 80 60 40 20. 0. 0. 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 0. concnetration (mg/mL). 200. 400. 600. 800. Concentration(ug/mL). 4-26 Fig 4-26 Superoxide and hydroxyl radical scavenging activity of green tea extracts (mean ± SD, n =3).. 82. 1000.

(93) 酚. (c). 4-9. a. (158.72±14.42 mg GAE/g) GAE/mg). >. (120.80±4.48 mg. (99.18±13.65 mg GAE/g)>EA. (62.50±4.17 mg GAE/g) b. (207.58±18.97 mg GAE/g) (189.65±2.37 mg GAE/g). (187.46±8.85 mg GAE/g) EA. (100.41±5.60 mg. GAE/g). 4-9 Table4-9 Antioxidation properties of black tea and green tea extracts. Total phenolic contents Antioxidation Extracts (mg GAE/g) IC50 DPPH. 酚. NO. 16.2. 酚. (mg/mL). (μg/mL). 0.95. 0.06. 4.38. 120.80±4.84c. (mg/mL) (mg/mL) Black tea Aqueous. Superoxide Hydroxyl anion radical. Methanolic. 0.51. 0.04. 0.26. 3.88. 158.72±14.42d. Ethanolic. 0.59. 0.6. 0.33. 9.49. 99.18±13.65b. Ethyl-acetate. 2.25. 0.69. 0.33. 7.57. 62.50±4.17a. 11.63. 7.45. 0.07. 9.08. 189.65±2.37a. Methanolic. 0.53. 0.05. 0.27. 4.80. 187.46±8.85a. Ethanolic. 0.55. 0.62. 0.28. 7.12. 207.58±18.97a. Ethyl-acetate. 3.57. 0.69. 1.54. 6.71. 100.41±5.60b. Grean tea Aqueous. Data given as mean ± standard deviation of triplicate tests. Samples followed by the same letter are not significantly different according to Ducan’s multiple comparison test at P = 0.05. IC50, concentration causing 5櫞 inhibition. Total phenolics contents were expressed as mg gallic acid equivalents g-1 of solid extract.. 83.

(94) 酚. 3. (1). P. acnes and S. epidermidis 4-10 P. acnes. = 8 mg/mL). EA. (MIC = 8 mg/mL) (MBC = 16 mg/mL). (MIC = 16 mg/mL) S. epidermidis. (MIC = 8 mg/mL) (MBC. P. acnes (MIC = 4 mg/mL) (MBC = 4 mg/mL). (MIC = 8 mg/mL) (MBC = 16 mg/mL) EA. (MIC = 16. mg/mL) (MBC = 16 mg/mL). 4-10 P. acnes S. epidermidis Table 4-10 Anti-bacterial properties of sage extracts. 酚酚酚 P. acnes S. epidermidis MIC MBC MIC MBC 酚 Extracts酚 (mg/mL) (mg/mL) Sage Aqueous 16 > 16 4 4 Methanolic 8 8 4 4 Ethanolic 8 16 8 16 Ethyl-acetate 8 16 16 16 Posotive control: tetracycline. The MIC of tetracycline against P. acnes and S. epidermidis are 0.8 g/mL and 12.5 g/mL, respectively.. 84.