P. acnes (extracellular lipase) BHI broth
lipase activity assay unknown components (crude lipase) Pablo et al. (1974)
1.
(a) P. acnes
1 loop P. acnes 200 mL BHI broth
4 250 mL 16 250 mL
50 mL 200 mL 4
37 16 hr 50 m L 3000 rpm 10 min
( 100 L )
(b) S. epidermidis
1 loop S. epidermidis 200 mL TSB broth
4 250 mL 16
250 mL 50 mL 200 mL 4
37 24 hr 50 ml 3000 rpm 10 min
( 100 L )
2.
(1)
Pablo et al. (1974) (ammonium sulfate)
pH = 6
pI = 5 pH pI
pH = pI
(
)
( )
( 3-1) 3-1
2001
(2).
500 mL 60
(ammonium sulfate) (361×0.5 =180.5 gm) 10 min
50 mL 10 min 4000 rpm
PBS
PBS 100
L 100 L - 20 (Cat. EW-02899-42 ,
Cole-Parmer, 625 East Bunker Court, USA) 6
PBS ( PBS 1:100) 4 48 hr 24 hr
PBS PBS
Amicon® ultra-15 centrifugal filter devices (PL -10, Millipore, Concord Road, Billeri, CA, USA) 4 30 min
enpendorff - 20
(3)
(a) BSA BSA (Cat: 85041C, Sigma) d.d. water
0 200 400 600 800 1000 g/mL
噁 g/mL 0 200 400 600 800 1000
1000ug/ml BSA L 0 20 40 60 80 100
DDW噁 L 100 80 60 40 20 0
L 100
(b) Sample 5 L
(c) well 25 L bio-rad protein assay reagent A (Cat 500-0113, Bio-rad,
Hercules, CA, USA) 5 L standard sample 20櫞 L Bio-rad
protein assay reagent B (Cat 500-0114, Bio-rad) ELISA reader
( 750 nm) BSA
(4) SDS-PAGE
1櫞 separating gel
75 75
comb stacking gel comb
comb 5X
sample buffer 4:1 100 10 min
well 100
comassie stain solution ( ) 30 min
comassie stain solution distain solution (2櫞
methanol & 1櫞 acetic acid in milli Q water) ( ) distain solution
0.75 mm
(10 %) running gel (mL)
(4 %) Stacking gel (mL)
A (30 % acrylamide) 3.35 0.33
B
1.5 M Tris (base)
TEMED (sigma T-8133) 1.8 mL
2.5 -
C
TEMED (sigma T-8133) 0.4 mL
-- 0.62
Diaz et al. (1999) (a)
P. acnes S. epidermidis sample buffer (5X) 4:1 Sample buffer (5X)
125 mM Tris (base) 3.785 gm
2 mM EDTA 2Na 0.0185 g
2 % SDS 5 g
5 % β-mercaptoethanol 12.5 mL
20 mL HCl pH 6.8 50 mL
SDS-PAGE (b)
2.5 Triton X-100 15
min SDS Triton X-100 d.d. water 1 X PBS
1 min PBS ( 0.1 mM
4-MUB PBS) 30 sec 1 X PBS 30 sec
(ChemiDoc XRS+) (170-8265, bio-rad, Hercules, CA, USA)
ethidium bromide
(6) (fluorescent assay)
1. lipase
lipase activity tributyrine
(tributyrylglycerol) agar plate assay, titrimetry (Gupta et al., 2003)
(fluorescence assay) (Roberts et al.1985)
( 3-2) 4-methylumbelliferyl
butyrate (4-MUB) 4-MU 360 nm
3-2
Figure 3-2 Hydrolysis reaction on which the fluorescent lipase assay is based.
(Roberts et al., 1985)
(1) sigma Aspergillus oryzae (Cat 62285,
BioChemika) lipase activity assay
well 2櫞 L 4 mM 2 mM 4 - MUB 2櫞 L
Aspergillus oryzae lipase (疱 4.5 2.25 1.125 0.56 0.2苷 0.14 0.07 mg/mL)
80 mL PBS 37 5 hr 0 5 10 15 20 30 45 60 120 180
240 300 min ( Fluorescence λex 360 nm; em 450 nm
4
mM 4-MUB
2. P. acnes S. epidermidis
Roberts et al.(1985) well 20 L crude lipase
solution 20 L 4-MUB (4 mM) 80 L PBS 5 hr 0 5
10 15 20 30 45 60 120 180 240 300 min ( Fluorescence λex 360 nm;
em 450 nm crude lipase (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 20 min S. epidermidis
Vmax 60 min P. acnes S.
epidermidis DMSO
DMSO
2.5 (V/V) DMSO stock
400 mg/mL 10 mg/mL
well 20 L 20 L 20 L
4 mM 4-MUB 6櫞 L PBS 37
( Fluorescence λex 360 nm em 450 nm)
(A.U.) Vmax
IC50 (C) (T)
酚
of inhibition=(1− )×100% C
T
1. DPPH
Tsai et al. (2008) DPPH
20 L (20 10 5 1 0.5 0.05 mg/mL) 20 L (DMSO
PBS) 96 well 200 L 1mM 2, 2-Di
(4-tert-octylphenyl)-1-picrylhydrazyl DPPH)(Cat. 257621, Sigma) 100 %
3 min 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) 20 mg/mL (DMSO
PBS) 96 well 50 L 10 mM sodium nitroprusside (SNP) (s0501,
Sigma) PBS( ) 90 min well 100 L 1:1
NO greiss reagent ( A B ) 1 min
OD 570 nm IC50 酚
% of scavenging activity =1-[Asample -Ablank of sample/Acontrol -Ablank of control]) x 100 %
3.
Tsai et al. (2008) (1)
(gallic acid) (G7384, Sigma) 40 mg 4 mL
stock 10 mg/mL d.d. water 4
2 1 0.5 0.2 0.1 0.05 0 mg/mL
(mg/mL) 0 0.05 0.1 0.2 0.5 1 2
10 mg/ml 噁 L) 0 5 10 20 50 100 200
DDW L 1000 995 990 980 950 900 800
L 1000
10 L 96 well 40 L
7.5% Na2CO3 50 L Folin & Ciocalteu’s phenol reagent (F9252, Sigma) (
) 30 min OD 765 nm
(mg Gallic acid equivalents/ g solid extract)
4.
Robak et al. (1988)
phenazine methosulfate (PMS) nicotainamide adenine
dinucleotide (NADH) nitroblue tetrazolium (NBT)
diformazam OD 560 nm
560 nm PBS 783 M
phenazine methosulfate (PMS) (P9625, Sigma) 600 Μ nicotainamide adenine
dinucleotide (NADH) (Cat N4505, Sigma) 201.8 M nitroblue tetrazolium (NBT)
(N6876, Sigma) ( ) 5櫞 L
(10 5 2 1 0.5 0.1 mg/mL) PMS NADH NBT
5 min 560 nm 50 L
50 L PBS phenazine methosulfate (PMS) 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 Fe2+ (4.6 M)-EDTA (2.3酚
M) H2O2 (24 mM) KH2PO4-NaOH (4.17 mM, pH 7.4) (P9791,
Sigma) 5櫞 L Fe2+ (4.6 M)-EDTA (2.3 M) 5櫞
L 4 mM luminal 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) (LCD 2084, ecom spol. s r.o.,
Americka, Praha, CZ) C18 (250 mm × 460 mm, Cat 720014.46, Macherey- nagel, Bethlehem, PA, U.S.A) A (Acetonitrile/water/acetic acid,
15:84:0.85) B (methanol) 90 min B 0%
100% 1 mL/min UV 284 nm
(4957s, extrasynthese,Genay, France) (C0609, sigma)
(SI-c9617, sigma) 400 mg/mL stock DMSO
d.d. H2O 10 mg/mL 0.45 m
mean ± SD SPSS 12.0
one way ANOVA vehicle
p .05
酚酚酚酚 酚酚酚酚
酚 酚酚酚酚
34 ( EA )
4-1 4-2 4-1
Table 4-1 Yield of ethanolic extracts of plants.
Common name Botanical name Part examined Yield (%)
Mustard seed Sinapis alba L. seed 15.8
Galangal AlipiniaGalanga root 10.7
Lemon grass Cymbopogon citrat leaves 28.8
Garlic Ailium sativum L root 12.0
Ginger Zingiber officinale Roscoe root 2.0
Wild bitter gourd Momordica charantiaC fruit --
Osmanthus Osmanthus fragrans flower --
Mung bean Vigna (L.) Wilczek seed 2.2
Rangoon creeper Quisqualis indica Linn. fruit 2.3
Lilium Lilium formosanum flower 1.5
Ginkgo Ginkgo biloba L root 4.8
Chinese cedar Toona sinensis Juss Roem leaves --
Pricklyash peel Zanthoxylum bungeanum Maxim. seed 15.0
Black pepper Piper nigrum seed 9.0
Caraway Carum carvi seed 0.9
Peppermint Mentha piperata leaves 3.3
Basil Ocimum basilicum leaves 10.6
Hawthorn fruit Fructus Crataegi Pinnatifidae friuts 40.0
Ginkgo Ginkgo biloba fruits 4.8
Tang Kuei Anglica sinensis Didl root --
Perilla Perilla frutescens leaves 6.3
Dandelion Taraxacum formosanum Kitamura flower buds 10.0
Saffron Crocus Satovis flower buds 30.0
Greater burdock Arctium lappa L. seed 20.0
Houttuynia Houttuynia cordata leaves 6.0
Purslane Portulaca oleracea Linn. root --
Medicinal citron Citrus medica fruits 31.3
Longan Dimocarpus longgana Lour fruits 44.0
Aromatic madder Elsholtzia ciliata leaves 6.0
4-2
Table 4-2 Yield of botanical extracts.
Common
name Botanical name Part
examined Yield (%)
Ethanol EA Methanol Aqueous Rosemary Rosmarinus officinalis leaves 8.74 2.81 17.60 7.00
Sage Salvia officinalis L. leaves 19.30 1.00 21.30 13.50
Black tea Camellia sinensis leaves 3.94 1.24 13.50 6.21
Grean tea Camellia sinensis leaves 10.90 4.34 30.10 14.90 Verbena Verbena officinalis Linn leaves 10.50 1.58 19.40 7.90
酚
1.
(1)
P. acnes S. epidermidis
broth dilution assay 34 P. acnes
(Rosmarinus officinalis ) (Salvia officinalis L.) (Camellia sinensis) (Verbena officinalis Linn)
EA P. acnes S. epidermidis
(a) P. acnes and S. epidermidis 4-3
EA P. acnes MIC MBC 8 mg/mL 16
mg/mL P. acnes
S. epidermidis (MIC = 4 mg/mL) (MBC = 4 mg/mL)
EA (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 P. acnes 酚 酚 S. epidermidis
MIC MBC MIC MBC
Extracts酚 (mg/mL) 酚 (mg/mL)
Rosemary
Aqueous > 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.
酚
(2).
(a)
crystal violet staining P. acnes 12-16 hr 48-72
hr 16 hr 24 hr
48 hr 24 hr
0 50 100 150 200 250
12 16 24 48 72
Incubation time(h)
% of crystal violet staining compare to 12hr value
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.
酚
(b) P. acnes 4-2 4-3
a. (prevention of biofilm formation PBF)
(0.125 mg/mL) (0.5 mg/mL)
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
0
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.
酚
b. (removal of established biofilm REB)
P. acnes
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
0 50 100 150 200 250 300 350
Ethanolic extract Methanolic extract EA extract Aqueous extract
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.
酚
(3)
(a)
P. acnes S. epidermidis 500 160
(mg protein/L of culture) SDS-PAGE ( 4-4)(
4-5) P. acnes S. epidermidis (molecular mass) 44 41.6
44 KD zymography
SDS-PAGE P. acnes 41 KD (Ingham et
al., 1981) S. epidermidis 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.
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.
酚
(b) (lipase activity)
P. acnes S. epidermidis
4 mM 4-MUB 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) Vmax 60min (18573 A.U.)
Time (minute)
(B) S. epidermidis
4-6 (A) P. acnes (B) S. epidermidis
Figure 4-6 Different concentrations of crude lipase from (A) P. acnes and (B) S.
epidermidis
酚
(c)
( )
(0.5 0.1 0.05 0.005 mg/mL) P. acnes S. epidermidis.
P. acnes IC50 0.13 mg/mL S. epidermidis IC50 0.22 mg/mL( 4-7)
concentration (mg/mL)
0.0 0.1 0.2 0.3 0.4 0.5
% of inhibition
0
% of inhibition
0
(B) S. epidermidis
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
酚
b. S. epidermidis
(IC50 = 0.37 mg/mL) > (IC50 = 0.78 mg/mL) (IC50 = 1.70 mg/mL) > EA (IC50 = 5.55 mg/mL)
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
Concentration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(A) P. acnes
(B) S. epidermidis
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.
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 P. acnes lipase 酚 酚 S. epidermidis lipase
IC50 (mg/mL) 酚 IC50 (mg/mL)
Rosemary 酚
Aqueous 0.68 0.37
Methanolic 1.31 0.78
Ethanolic 5.89 1.70
Ethyl-acetate 8.68 5.55
IC50 of tetracyclinewere 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
酚
% of scavenging activity
0 Methanolic extract EA extract Aqueous extract
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
% of scavenging activity
0
Fig 4-9 (A) DPPH and (B) NO radical scavenging activity of rosemary extracts (mean ± SD, n =3)
酚
% of scavenging activity
0
Ethanolic extract Methanolic extract EA extract Aqueous extract
concentration(ug/mL)
0 200 400 600 800 1000
% of scavanging activity
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(C) superoxide anion
(D) hydroxyl radical
4-10 (A) (B)
Fig 4-10 (c) superoxide anion and (B) hydroxyl radical scavenging activity of rosemary extracts (mean ± SD, n =3)
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
(mg GAE/g) Extracts
IC50
DPPH NO Superoxide
anion
Hydroxyl radical 酚
(mg/mL) (mg/mL) (mg/mL) (ug/mL)
酚
Rosemary
Aqueous 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 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.
酚
(6) HPLC
Cuvelier et al. (1996) HPLC
22.4 64.4 74.1 min 4-11
( 4-16 4-17 4-18 )
EA 4-12 4-13 4-14 4-15
4-11
Fig 4-11 HPLC profile of an experimental solution obtained by mixing three standard compound.
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.
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.
酚
HPLC EA
EA 4-16
(a)
(1.069 ± 0.17 mg/g) (0.28 ± 0.02 mg/g) EA (0.07 ± 0.00 mg/g)
4-16
Fig 4-16 standard curve of rosemarinic acid (b)
(3.26 ± 0.10 mg/g) (0.85 ± 0.09
mg/g) EA (0.63 ± 0.02 mg/g)
4-17
Fig 4-17 standard curve of carnosol
酚
(c)
EA (6.02 ± 0.39 mg/g) (2.61 ±
0.12 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
acid carnosol carnosic acid Extract
mg/g extract
Rosemary
Aqueous -- -- --
Methanolic 1.069 ± 0.17 3.26 ± 0.10 2.61 ± 0.12 Ethanolic 0.28 ± 0.02 0.85 ± 0.09 1.03 ± 0.11 Ethyl-acetate 0.07 ± 0.00 0.63 ± 0.02 6.02 ± 0.39
酚
epidermidis (MIC = 1 mg/mL) (MBC = 1 mg/mL) EA (MIC = 16 mg/mL) (MBC = 16 mg/mL)
P. acnes (MIC = 2 mg/mL) (MBC = 2
mg/mL) (MIC = 2 mg/mL) (MBC = 16 mg/mL) EA
(MIC = 4 mg/mL) (MBC = 16 mg/mL) (MIC = 4 mg/mL)
S. epidermidis S. epidermidis
(MIC = 1 mg/mL) (MBC = 1 mg/mL) EA (MIC =
16 mg/mL) (MBC = 16 mg/mL) 酚 酚
4-7 P. acnes S. epidermidis
Table 4-7 Anti-bacterial properties of Camellia sinensis extracts.
酚 P. acnes 酚 S. epidermidis
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.
酚
(2) P. acnes 4-19 4-20
(a) (prevention of biofilm formation PBF)
EA (0.06 mg/mL)
(0.06 mg/mL) EA (0.06 mg/mL)
(0.125 mg/mL)
Concentration (mg/mL)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
% of crystal violet staining compared to control
0
% of crystal violet staining compared to control
40 60 80 100 120
140 Ethanolic extract
Methanolic extract
(A) black tea (B) green tea
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.
酚
(b) (removal of established biofilm REB)
P. acnes
0.06 mg/mL P. acnes
Concentration (mg/mL)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
% of crystal violet staining compared to control
0
% of crystal violet staining compared to control
20
(A) black tea (B) green tea
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.
酚
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(B) Green tea
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract (A) Black tea
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
酚
(b) S. epidermidis
a.
% of inhibition
0
Ethanolic extract Methanolic extract EA extract Aqueous extract (A) Black tea
Concentration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0 (B) Green tea
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.
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 tetracyclinewere 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
酚
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
(A) DPPH (B) NO
4-23 DPPH
Fig 4-23 DPPH and NO radical scavenging activity of black tea extracts (mean ± SD, n
=3).
酚
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
Concentration(ug/mL)
0 200 400 600 800 1000
% of scavanging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
(C) superoxide anion (D) hydroxylradical
4-24
Fig 4-24 Superoxide and hydroxyl radical scavenging activity of black tea extracts (mean ± SD, n =3).
酚
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
(A) DPPH (B) NO
4-25 DPPH
Fig 4-25 DPPH and NO radical scavenging activity of green tea extracts (mean ± SD, n
=3).
酚
% of scavenging activity
0
160 Ethanolic extract Methanolic extract EA extract Aqueous extract
Concentration(ug/mL)
0 200 400 600 800 1000
% of scavanging activity
0
(C) superoxide anion (D) hydroxyl radical
4-26
Fig 4-26 Superoxide and hydroxyl radical scavenging activity of green tea extracts (mean ± SD, n =3).
酚
(c) 4-9
a.
(158.72±14.42 mg GAE/g) (120.80±4.48 mg
GAE/mg) > (99.18±13.65 mg GAE/g)>EA
(62.50±4.17 mg GAE/g) b.
(207.58±18.97 mg GAE/g) (187.46±8.85 mg GAE/g)
(189.65±2.37 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 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.
酚
3.
(1) P. acnes and S. epidermidis
4-10
P. acnes (MIC = 8 mg/mL) (MBC
= 8 mg/mL) EA (MIC = 8 mg/mL) (MBC = 16 mg/mL)
(MIC = 16 mg/mL) P. acnes
S. epidermidis (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 P. acnes 酚 S. epidermidis
MIC MBC MIC MBC
Extracts酚 (mg/mL) 酚 (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.
酚
(2) P. acnes 4-27 4-28
(a) (prevention of biofilm formation PBF)
(1 mg/mL)
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
0 50 100 150
200 Ethanolic extract
Methanolic extract EA extract Aqueous extract
*
*
*
4-27 P. acnes
Figure 4-27 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of sage 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.
酚
(b) (removal of established biofilm REB)
(4 mg/mL) (2 mg/mL) P.
acnes
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
20
4-28 P. acnes
Figure 4-28 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of sage 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.
酚
(b) S. epidermidis
(0.78 mg/mL) > (1.14 mg/mL)
> (IC50 = 3.03 mg/mL) EA
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
Concentration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(A) P. acnes (B) S. epidermidis
4-29 (A) P. acnes S . epidermidis
Figure 4-29 Effect of sage extracts on the lipolytic activity of crude lipase from (A) P.
acnes and (B) S. epidermidis.
酚
4-11 P. acnes S. epidermidis
Table 4-11 Effect of sage extracts on the lipolytic activity of crude lipase from P. acnes and S. epidermidis.
P. acnes lipase 酚 酚 S. epidermidis lipase
Extracts IC50 (mg/mL) 酚 IC50 (mg/mL)
Sage
Aqueous 0.69 0.78
Methanolic 1.13 1.14
Ethanolic 3.37 3.03
Ethyl-acetate 7.95 Smax
IC50 of tetracyclinewere 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 which each extract was tested.
酚
% of inhibition
0
140 Ethanolic extract Methanolic extract
Ethanolic extract Methanolic extract EA extract Aqueous extract
(A) DPPH (B) NO
4-30 DPPH
Fig 4-30 DPPH and NO radical scavenging activity of sage extracts (mean ± SD, n =3).
酚
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
Concentration(ug/mL)
0 200 400 600 800 1000
% of scavanging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
(C) superoxide anion (C) hydroxyl radical
4-31
Fig 4-31 Superoxide and hydroxyl radical scavenging activity of sage extracts (mean ± SD, n =3).
酚
(5) 4-12
(102.60±4.39 mg GAE/g) (71.67±1.07 mg
GAE/g) > (64.55±4.11 mg GAE/g) >EA (53.46±1.88 mg GAE/g)
4-12
Table4-12 Antioxidation properties of sage extracts
Total phenolic contents Antioxidation
(mg GAE/g) Extracts
IC50
DPPH NO Superoxide
anion
Hydroxyl radical 酚
(mg/mL) (mg/mL) (mg/mL) (μg/mL)
酚
Sage
Aqueous 10.47 0.9 0.10 75.82 102.60±4.39d Methanolic 0.68 0.6 0.66 6.04 71.67±1.07c Ethanolic 3.09 1.0 1.45 6.79 64.55±4.11b Ethyl-acetate 2.99 0.9 0.82 7.31 53.46±1.88a 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.
酚
4.
(1) P. acnes and S. epidermidis
4-13
P. acnes (MIC = 8 mg/mL) (MBC = 8
mg/mL) EA (MIC = 8 mg/mL) (MBC = 16 mg/mL)
(MIC = 8 mg/mL) P. acnes
S. epidermidis (MIC = 4 mg/mL) (MBC
= 4 mg/mL) EA (MIC = 8 mg/mL) (MBC = 8 mg/mL)
(MIC = 16 mg/mL) (MBC = 4 mg/mL)
4-13 P. acnes S. epidermidis
Table 4-13 Anti-bacterial properties of verbena extracts.
酚 酚 P. acnes 酚 S. epidermidis
MIC MBC MIC MBC
Extracts酚 (mg/mL) 酚 (mg/mL)
Verbena
Aqueous 8 > 16 酚 > 16 > 16
Methanolic 8 8 16 4
Ethanolic 8 16 4 4
Ethyl-acetate 8 16 8 8
Posotive control: tetracycline. The MIC of tetracycline against P. acnes and S.
epidermidis are 0.8 g/mL and 12.5 g/mL, respectively.
酚
(2) P. acnes 4-32, 4-33
(a) (prevention of biofilm formation PBF)
EA (4 mg/mL) (2 mg/mL)
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
0 50 100 150 200
Ethanolic extract Methanolic extract EA extract Aqueous extract
* *
*
4-32 P. acnes
Figure 4-32 Effect of different concentration ethanolic, methanolic, EA, aqueous
extracts of verbena 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.
酚
(b) (removal of established biofilm REB)
P. acnes
Concentration (mg/mL)
0 1 2 3 4
% of crystal violet staining compared to control
40 60 80 100 120 140 160 180 200 220
Ethanolic extract Methanolic extract EA extract Aqueous extract
4-33 P. acnes
Figure 4-33 Effect of different concentration ethanolic, methanolic, EA, aqueous extracts of verbena 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.
酚
(3) 4-34
(a) P. acnes
P. acnes (IC50 = 0.33 mg/mL) >
(IC50 = 0.37 mg/mL) > =EA (IC50 = 0.56 mg/mL)
(b) S. epidermidis
S. epidermidis (IC50 =
0.18 mg/mL) > (IC50 = 0.23 mg/mL) > (IC50 = 0.25 mg/mL) >EA (IC50 = 0.38 mg/mL)
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(A) P. acnes
Concentration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11
% of inhibition
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
(B) S. epidermidis
4-34 (A) P. acnes (B) S. epidermidis
Figure 4-34 Effect of verbena extracts on the lipolytic activity of crude lipase from (A) P. acne and (B) S. epidermidis.
酚
4-14 P. acnes S. epidermidis
Table 4-14 Effect of verbena extracts on the lipolytic activity of crude lipase from P.
acnes and S. epidermidis
Extracts P. acnes lipase 酚 酚 S. epidermidis lipase
IC50 (mg/mL) 酚 IC50 (mg/mL)
Verbena
Aqueous 0.56 0.25
Methanolic 0.37 0.18
Ethanolic 0.33 0.23
Ethyl-acetate 0.56 0.38
IC50 of tetracyclinewere 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
酚
% of scavenging activity
0
140 Ethanolic extract
Methanolic extract EA extract Aqueous extract
concnetration (mg/mL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
% of scavenging activity
0
140 Ethanolic extract Methanolic extract EA extract Aqueous extract
(A) DPPH (B) NO
4-35 DPPH
Fig 4-35 DPPH and NO radical scavenging activity of verbena extracts (mean ± SD, n
=3).
酚
% of scavenging activity
0
% of scavanging activity
0
(C) superoxide anion (D) hydroxyl radical
酚 4-36
Fig 4-36 Superoxide and hydroxyl radical scavenging activity of verbena extracts (mean
± SD, n =3).
酚
(5) 4-15
(125.18±4.27 mg GAE/g) (113.41±16.26 mg GAE/g)
> (96.85±10.57 mg GAE/g) EA (82.89±7.23 mg GAE/g)
4-15
Table4-15 Antioxidation properties of verbena extracts
Total phenolic contents Antioxidation
(mg GAE/g) Extracts
IC50
DPPH NO Superoxide
anion
Hydroxyl radical 酚
(mg/mL) (mg/mL) (mg/mL) (μg/mL)
酚
Aqueous 4.5 4.37 0.41 57.21 113.41±16.26bc Methanolic 0.64 0.63 0.69 5.68 125.18±4.27c Ethanolic 0.73 0.66 0.7 5.52 96.85±10.57ab Ethyl-acetate 3.37 0.69 2.61 7.40 82.89±7.23a 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.
酚
(Burkhart et al., 1999)
Cowan (1999) (aromatic compound)
(terpenoids) (quinones)
terpenoids capsaicin( )
(1) (2)
(3) (4) Lectins ( ) (5) Polyacetylenes( )
( 5-1) (polar)
34 P. acnes
48 hr P. acnes
( ) P.
acnes (Cowan, 1999)
EA
EA P. acnes S. epidermidis
HPLC
酚
5-1
Table 5-1 Solvents used for active component extraction
Aqueous Ethanol methanol Chloroform Dichloromethanol Ether Acetone Anthocyanins Tannins Anthocyanins Terpenoids Terpenoids Alkaloids Flavonols
Aqueous Ethanol methanol Chloroform Dichloromethanol Ether Acetone Anthocyanins Tannins Anthocyanins Terpenoids Terpenoids Alkaloids Flavonols