짚신나물추출물의생리활성과대사체분석 · 2020. 3. 17. · resveratrol, a natural...
TRANSCRIPT
짚신나물추출물의생리활성과대사체분석
경북대학교 농업생명과학대학
강 영 화
2019. 10. 25.
2019 한국원예학회 제111차 추계학술발표회
원예과학과 기능성식물학 연구실
Contents
Part 1. Introduction of Natural Product Research
Part 2. The functional study on Agrimonia pilosa Ledeb.
Natural Product Research
Discovery of New Materials From Natural Products
- Benefit
• Chemical diversity• Uniqueness• Possibility of
lead compounds• Traditional medicine• Safety
Drug Discovery from Natural Products
Morphine
해열, 진통, 소염 항혈전
Dual EffectsSAR
Drug Discovery from Natural Products
Shikimic acid
Paclitaxel
Taxol
Osetamivir
Tamiflu
1) 돼지 독감 치료제2) 인간 신종플루 치료제
스위스 로슈사
Bristol-Myers
Ephedrin마황 (Ephedra sinica)
팔각회향
주목나무
Domestic Natural Drug
위령선 (Clematis mandshurica)
괄루근 (Trichosanthes kirilowii)
하고초 (Prunella vulgaris)
생약복합제
진통, 소염작용, 위장 장해 극소화
황해쑥 Artemisia argyi Lev. et Vant
(SK제약)
위점막보호, 항산화, 항염증 등-> 개발에 9년간 180억 소요2009년 855억원 매출
Eupatilin
스티렌 (위궤양치료제)
조인스 (관절염치료제)
천연물신약연구개발촉진법 2000년 1월 12일제정
황련(Coptis chinensis)아이비(Hedera helix)
만성 염증성 기관지염치료제
우슬 (Achyranthes japonica)방풍 (Ledebouriellaseseloides )두충 (Eucommia ulmoides)
진통, 소염, 골관절증 등
당귀(Angelica gigas)모과(Chinese Quince)방풍(Ledebouriellaseseloides)속단(Phlomis umbrosa)
골관절염 등
시네츄라 시럽(기관지염 치료제)
신바로 캡슐 (진통 소염제)
레일라 정 (진통 소염제)
(안국약품)
(녹십자)
(한국피엠지제약)(동아제약)
Cannabidiol(CBD)
대마 Cannabis sativa
영국 제약사,GW Pharmaceuticals의대마 성분 의약품 판매 승인뇌전증치료제(2019.9.23)
특용작물로 부터 의약소재
Tetrahydrocannabinol (THC)
113종의 cannabinoids
약용작물 53종
당귀
인삼구기자
천궁감초
고본
대황
속단 행인
(농림축산식품부자료 2012)
오미자
암예방원예작물의 기능성
Horticultural Crops : Health Benefit
Vegetables FlowersFruits
Prevention, delay, or reversal of cancer
in human populations by ingestion of
dietary or pharmaceutical agents that
alter the process of carcinogenesis.
M.B. Sporn 1976
CHEMOPREVENTION
“An Ounce of Prevention is Worth a Pound of Cure”
Normal Initiated Preneoplastic Neoplastic
Cell Cell Cell Cell
(1-2 days) ( >10 years) ( >1 years )
Procarcinogen
Secretion
Detoxification
Metabolic Activation
Initiation Promotion Progression
Ultimate
carcinogen
Stages of Chemical Carcinogenesis
Cancer-
blocking agents
Flavonoids
Ellagic acid
Indole-3-
carbinol
Limonene
Isothiocyanates
Cancer-suppressing agents
Curcumin, b-Carotene, Capsaicin, Resveratrol
Chemopreventive Phytochemicals
Nature Reviews Cancer volume 3, pages768–780 (2003)
Cancer vs Phytochemicals
• Chemoprevention : Cancer Initiation
By oxidative stress, chemical carcinogen,
Inflammation, Virus(HBV)
Antioxidant, detoxifying enzyme inducer,
anti-inflammatory phytochemicals
Quinone reductase inducer
Cruciferous vegetables
Indole-3-carbinolfrom glucobrassicin
Sulforaphane
Cruciferous Vegetables Health Benefit
Cauliflower Broccoli
Allyl isothiocyanate
Phenethyl isothiocyanate (PEITC)
Benzyl isothiocyanate (BITC)
Cabbage
Isothiocyanates
Cancer Chemopreventive Activity of
Resveratrol, a Natural Product Derived
from Grapes
• Pezzuto, John M.
• Science 10 Jan 1997Vol 275, Issue 5297
• Grapes and health
• Polyphenols
• Antioxidant activity & chemoprevention
• Prevention of cardiovascular disease
CHEMOTHERAPY
from Medicinal crops
CHEMOPREVENTION
from Horticultural crops
Research Theme
Antioxidants
Detoxifying Enzymes
Anti-inflammatory
Anti-cancer & !!!
Chemoprevention : Cancer Initiation
짚신나물 추출물의 생리활성과대사체분석
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Screening of the extracts of 53 plants.
0
0.2
0.4
0.6
0.8
1
1.2
DM
SO
DM
SO
LF
T
TV
T
AD
L
AD
S
AD
R
AD
U
CZ
T
KS
T
AJT
PO
T
LB
F
LB
L
LB
S
LB
D
ES
T
ES
S
ES
R
SS
H
SS
B
SS
L
SS
S
SB
T
SB
U
LG
T
PA
T
PA
U
PA
R
SJT
SJU
SJD
TK
U
TK
L
EA
P
EA
L
EA
S
AS
T
TC
F
AG
R
AG
N
SD
T
EC
L
PJL
TR
R
TP
T
TP
L
RC
F
DB
F
AH
R
AR
L
AJT
CO
L
FV
S
AP
L
NO
re
lease
(Fo
ld o
f L
PS
tre
ate
d g
rou
p)
50ug/mL 100ug/mL
Cells were pre-treated with 50 and 100 µg·mL-1 of plants extract for 4 h and then treated with 1 µg·mL-1 LPS for 24 hr.
Bars represent mean values ± standard deviation, (n = 3)
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Cells were pre-treated with 50 µg·mL-1 of plant extract for 4 hr and then treated with 1 µg·mL-1 LPS for 24 hr.
Bars represent mean values ± standard deviation, n = 3
NO & COX-2 inhibitory activity of the extracts of 53 plants.
9
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Agrimonia pilosa Ledeb.
짚신나물선학초용아초낭아초
민간에서 지혈제, 위궤양, 장염, 지사제로 사용
동의보감, 북한 동의학 사전 : 위암, 식도암, 자궁암등 암 치료제
Order Rosales
Family Rosaceae
Genus Agrimonia
Species A. pilosa
1) High extraction rate
2) Absence of previous studies related to anti-inflammatory effect
Functional
activity
anti-oxidant (He et al, 2010), anti-allergic (Kim et al, 2012),
anti-obesity (Lee et al, 2012) and hypoglycemic activities (Liu et al, 2014)
Functional
component
quercitrin, tiliroside, vitexin, corosolic acid, maslinatic acid,
agrimonine and agrimonolide etc. (Liu et al, 2014)
Jirisan Mt.
Odaesan Mt.
Taebaeksan Mt.
Deogyusan Mt.
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Agrimonia pilosa Ledeb. Distribution
Reason of plant selection
Previous study for APL
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
The isolation of anti-inflammatory metabolites from anti-inflammatory plant and their structure identification
Purity 95% ↑
02
Selection of the anti-inflammatory plant
01
NF-kB
Pro-inflammatory cytokines
LPS
Inflammatory
enzymes
The structure-activity relationship (SAR) and mechanism study of active components
03
Phenotypic and functional characteristics investigation of anti-inflammatory plant by harvesting time
Phenotypic
characteristics
Functional
characteristics
Selection of optimal
harvesting time
Stage 1
1 day
after transplanted
Stage 2
15 days
Stage 3
30 days
Stage 4
45 days
04
Whole Procedure
생리활성 성분연구(추출 및 분리)
추출, 농축
활성실험(활성유무)
대량으로..
물질분리분리 및정제
구조 동정
Activity-Guided Isolation
F1(-)
F2
(-)
F3
(-)
F4
(-)
F5
(-)
F6
(+)
F7
(-)
F8
(-)
F9
(-)
F10
(-)
F11
(+)
F12
(-)
F13
(-)
F14
(-)
F15
(-)
F16
(-)
F17
(-)
F18
(-)
K1
(+)
F19
(-)K2
(+)
K3
(-)
F20
(-)
Active Extract
Fractions
(test
results)
Fractions
(test
results)
Structure elucidation
Column Chromatogrphy
천연물의생리활성평가
- In vitro bioassay :
HTS (high throughput screening)
- Mecanism study (mRNA & Protein)
0
0.2
0.4
0.6
0.8
1
1.2
DM
SO
DM
SO
Me
OH
Ext.
He
xa
ne F
r.
Eto
Ac F
r.
BuO
H F
r.
NO
pro
duction
(Fo
ld o
f L
PS
tre
ate
d g
rou
p)
5 µg/ml 10 µg/ml 20 µg/ml 40 µg/ml
0
0.2
0.4
0.6
0.8
1
1.2D
MS
O
DM
SO
Me
OH
Fr.
He
xa
ne F
r.
EtO
Ac F
r.
BuO
H F
r.
Ce
ll via
bili
ty
(Fo
ld o
f L
PS
tre
ate
d g
rou
p)
5 µg/ml 10 µg/ml 20 µg/ml 40 µg/ml
Each value is expressed as mean ± standard derivation (n=3).
*,p<0.05; **,p<0.01; ***,p<0.001 (A) versus the non-treated control group (B) versus control group
treated with LPS alone.
BA 5 µg·mL-1 10 µg·mL-1 20 µg·mL-1 40 µg·mL-1
**
**
*
*
**
** **
**
*
*
*
** **
*
With LPS (1µg·mL-1)
5 µg·mL-1 10 µg·mL-1 20 µg·mL-1 40 µg·mL-1
With LPS (1µg·mL-1)
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
MeO
HE
xt.
* * * *
NO inhibitory activity of the MeOH extract and its fractions of APL
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
HPLC-PDA chromatograms of BuOH fraction from APL
12
3456
7
8
min0.0 5.0 10.0 15.0 20.0 25.0 30.0
280 nm
284.90
284.95
299.95
284.95
268.95
284.95
268.95
466.95
269.00
Pe
ak 1
Pe
ak 2
Pe
ak 3
Pe
ak 4
Pe
ak 5
Pe
ak 6
Pe
ak 7
Pe
ak 8
O
OH
HO
OH O
O
OH
HO
OH O
O
OH
HO
OH O
O
OH
HO
OH O
OH
O
OH
HO
OH O
OH
O
OH
HO
OH O
OH
O
OH
HO
OH O
OH
O
OH
HO
OH O
OH
OH
447.00
(-162)
464.80
(-180)
609.00
446.00
(-146) (-163)
460.70
(-175)
445.00
(-176)
593.00
(-308)
(-198)
501.20
(-231)
913.00
(-446)
MS/MS patterns of compounds 1-8
m/z 162
m/z 146
O
OH
HO
OH O
O
O
OH
OH
HO
OH
m/z
162
O
OH
HO
OH O
O
O
OH
OHOH
O
O m/z 180
m/z 175
O
OH
HO
OH O
O
O
OH
OHOH
HO
O
O
OH
O
OH O
O
HO
O
OH
OHOH
m/z 176
O
OH
HO
OH O
O
O
O
O
OH
HO
HO
OHm/z 308
The phenolics profiles of the BuOH fraction of APLby HPLC-PDA-ESI-MS/MS
Peak
No.Compound
TR
(min)
M-
(m/z)
Percentage of
total area of all
peak (%)
MS/MS
(m/z)References
1kaempferol-O-(acetyl)
-β-D-glucopyranoside13.8 487.0 16.6
284.9,
151.0
(Mikulic-Petkovsek, Slatnar,
Stampar, &Veberic, 2012)
2kaempferol-O-β-D-
glucopyranoside
16.1 447.0 3.7 285.0 (Pan et al., 2008)
3quercetin-O-β-D-
glucopyranoside
16.4 609.0 6.8 300.0 (Farias &Mendez, 2014)
4kaempferol-O-β-D-
glucopyranoside
16.9 460.7 17.7 285.0(Mikulic-Petkovsek et al.,
2012)
5 apigenin-O-β-D-glucuronide 17.2 445.0 8.1 269.0 (Lin & Lee, 2014)
6 kaempferol-O-β-D-rutinoside 17.5 593.0 5.1 285.0 (Chen et al., 2015)
7 Unknown 18.2 913.0 34.8467.0,
269.0-
8 Unknown 21.3 501.0 6.5501.0,
269.0-
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
The phenolics profiles of the BuOH fraction of APLby HPLC-PDA-ESI-MS/MS
Each value is expressed as mean ± standard derivation (n=3).
*,p<0.05; **,p<0.01; ***,p<0.001 versus control group treated with LPS alone
0
20
40
60
80
100
120
IL-6/β-actin IL-1β/β-actin TNF-α/β-actin
m R
NA
le
ve
ls o
f IN
Fcyto
kin
es
(%of
LP
S t
reate
d g
roup)
0 μg/ml
0 μg/ml
5 μg/ml
10 μg/ml
20 μg/ml
40 μg/ml
With LPS (1µg·mL-1)
With LPS
(1µg·mL-1)With LPS (1µg·mL-1)
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
0
20
40
60
80
100
120
iNOs/β-actin COX-2/β-actin
m R
NA
le
ve
ls o
f IN
F e
nzym
es
% o
f LP
S t
reate
d g
roup)
0 μg mL-1
0μg mL-1
5 μg mL-1
10 μg mL-1
20 μg mL-1
40 μg mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
0
20
40
60
80
100
120
iNOs/β-actin COX-2/β-actin
Pro
tein
leve
ls o
f IN
F e
nzym
es
(%, o
f L
PS
tre
ate
d g
rou
p)
0 μg mL-1
0μg mL-1
5 μg mL-1
10 μg mL-1
20 μg mL-1
40 μg mL-1
Actin
iNOs
COX-2
0 0 5 10 20 40
With LPS (1µg mL-1)
(µg mL-1)
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
µg·mL-1
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
With LPS (1µg·mL-1)
With LPS (1µg·mL-1)
With LPS (1µg·mL-1)
With LPS (1µg·mL-1)
C
A
B
The effect of the BuOH fraction of APL on production overexpressed inflammatory cytokines and enzymes
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Isolation scheme of anti-inflammatory compoundsin the BuOH fraction of APL
25
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Isolation scheme of anti-inflammatory compoundsin the EtOAC fraction of APL
HPLC chromatogram of 8 (MeOH) UV spectrum of 8 (MeOH)
32
267 337
Benzoyl
Band Ⅱ=267nm
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
[M+Na]+
[M+2Na]+
503.1557
525.1380
HR-FAB-MS 8 (MeOH)
33
13C NMR spectrum of 8
1H NMR spectrum of 8
DEPT spectrum of 8
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Alkyl group
Alkyl group
Alkyl group
34COSY NMR spectrum of 8 (Bold line)
A C
B
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
35HMBC NMR spectrum of 8 (Arrow line)
A C
B
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
26
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
BE1 R1=OH, R2=H, R3=α-D-Rha, R4=H, R5=OH
BE2 R1=H, R2=H, R3=α-D-Glc, R4=H, R5=H
BE3 R1=OH, R2=H, R3=α-D-Glc, R4=H, R5=H
BE4 R1=H, R2=H, R3= α-D-6″-butyl-Glc, R4=H, R5=H
BE5 R1=OH, R2=H, R3=H, R4=H, R5=OH
BE6 R1=OH, R2=H, R3=H, R4=H, R5=H
BE7 R1=H, R2=H, R3=H, R4=H, R5=H
BE8 R1=H, R2=H, R3=α-D-6″-propyl-Glc, R4=H, R5=H
BE13 R1=H R2=H, R3=H, R4=H, R5=OH
BE14 R1=H, R2=H, R3= H, R4=α-D-Glc, R5=H
BE15 R1=H, R2=α-D-Glc, R3= H R4=H, R5=H
BE9 R1=H, R2=CH3
BE10 R1=β-D-Glc, R2=CH3
BE11 R1=H, R2=H
BE12 R1=β-D-Glc, R2=CH3
Rha : rhamnopyranosyl
Glu : glucopyranosyl
Anti-inflammatory Active Compounds from APL
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
2 Apigenin-7-O
-β-D- glucopyranoside3 Kaempferol- 7-O-β-D-glucoside
5 Kaempferol 6 Apigenin
7 Agrimonolide 8 Agrimonolide-6-O-β-D- glucopyranoside
9 Desmethylagrimonolide
10 Desmethylagrimonolide-6-O-β-D-glucopyranoside
11 Luteolin
12 Vitexin 13 Isovitexin
1 Quercetin-7-O-β-D- rhanmoside 4 Quercetin(Liu et al., 2016; Jung and Park 2007;
Kato et al., 2010)
Liu et al., 2016; Taira et al., 2009;
Jung and Park 2007; Kato et al., 2010)
(Liu et al., 2016) (Liu et al., 2016),
(Kato et al., 2010),
(Liu et al., 2016; Taira et al., 2009;
Kato et al., 2010)
(Kato et al., 2010)
(Liu et al., 2016; Kato et al., 2010)
Eight compounds reported in APL
Six compounds isolated from APL for the first time
Conclusions
New compound from
Apigenin-7-O-b-glucuronide pentylester
Apigenin-7-O-b-glucuronide butylester
Apigenin
Luteolin
Quercetin
Kaempferol
Apigenin-7-O-Glc
Luteolin-7-O-Glc
Quercetin-7-O-Glc
Kaempferol-7-O-Glc
NO inhibitory activity
(IC 50, μM)
3.69 ± 0.34
14.43 ± 0.23
19.50 ± 1.71
4.62 ± 0.43
8.03 ± 1.26
22.24 ± 2.14
31.27 ± 3.75
14.37 ± 1.84
NO inhibitory activity
(IC 50, μM)Flavone aglycone Flavone-7-O-glucose (Glc)
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
The structure-NO inhibitory activity relationship of flavonoids from APL
41
(A) Phospholated (P)-IKK (B) P-IκBα and (C)P-p65 in LPS-induced Raw 264.7 macrophages.
Each value is expressed as mean ± standard derivation (n=3).
*,p<0.05; **,p<0.01; ***,p<0.001 (A)versus the non-treated control group (B) versus control group treated with LPS alone
0
20
40
60
80
100
120
0 0 10 20 10 20 10 20
Pro
tein
exp
ressio
n
(% o
f L
PS
tre
ate
d R
aw
26
4.7
ce
lls)
Sample
(μM)
B
P-pIKK
0
20
40
60
80
100
120
0 0 10 20 10 20 10 20
Pro
tein
exp
ressio
n
(% o
f L
PS
tre
ate
d R
aw
26
4.7
ce
lls)
0
20
40
60
80
100
120
0 0 10 20 10 20 10 20
Pro
tein
exp
ressio
n
(% o
f L
PS
tre
ate
d R
aw
26
4.7
ce
lls)With LPS (1μg·mL-1)
P-IκBα
Actin Actin
P- p65
Actin
Api-PG Api-BG Api
With LPS (1μg·mL-1)
CA
With LPS (1μg·mL-1)
Api-PG Api-BG ApiApi-PG Api-BG Api
Sample(μM) Sample(μM) Sample(μM)
Sample
(μM)
Sample
(μM)
**** **
******
* ***
**
****** ***
***
***
*
0
With LPS (1μg·mL-1)
10 20 10 20 10 20
ApiPG Api-BG Api0 0
With LPS (1μg·mL-1)
10 20 10 20 10 20
ApiPG Api-BG Api0 0
With LPS (1μg·mL-1)
10 20 10 20 10 20
ApiPG Api-BG Api0
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
* **
*
The effect of APD on inflammatory mechanism
42
(A) iNOs mRNA and protein expression ratio and (B) Cox-2 mRNA and protein expression ratio in
LPS-induced Raw 264.7 macrophages. Each value is expressed as mean ± standard derivation
(n=3). *,p<0.05; **,p<0.01; **,p<0.001 versus control group treated with LPS alone
0
20
40
60
80
100
120
0 0 5 10 20 40
CO
X-2
/β-A
ctn
mR
NA
exp
ressio
n
(% o
f L
PS
tre
ate
d g
rou
p)
Api Api-G Api-PG Api-BG
0
20
40
60
80
100
120
0 0 5 10 20 40
iNO
S/β
-Actn
mR
NA
exp
ressio
n
(% o
f L
PS
tre
ate
d g
rou
p)
Api Api-G Api-PG Api-BG0
10 20 10 20 10 200
iNOS
Actin
Sample
(μM)A
With LPS (1μg mL-1)
ApiPG Api-BG Api
0
20
40
60
80
100
120
0 0 10 20 10 20 10 20
iNO
S/β
-Actin
pro
tein
exp
ressio
n
(% o
f L
PS
tre
ate
d g
roup)
0
20
40
60
80
100
120
0 0 10 20 10 20 10 20
CO
X-2
/β-A
ctin
pro
tein
exp
ressio
n
(% o
f L
PS
tre
ate
d g
roup)
Api-PG Api-BG Api
With LPS (1μg mL-1)
Api-PG Api-BG Api
With LPS (1μg mL-1)
COX-2
Actin
010 20 10 20 10 20
0
Sample
(μM)B
With LPS (1μg mL-1)
ApiPG Api-BG Api
With LPS (1μg mL-1)
With LPS (1μg mL-1)
Sample (μM)
Sample (μM)
30
30
*
*
*
***
***
*****
*****
**
****
*
****
****
******
**
**
***
*
**** **
**
***
**
*
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Each value is expressed as mean ± standard derivation (n=3).
*,p<0.05; **,p<0.01; ***,p<0.001 versus control group treated with LPS alone
*
**
** * *
* * **
* * * *
**
*
* *
**
* *
Api > Api-BG > Api-PG > Api-G
Sample (μM)
Sample (μM)
The effect of APD on production of inflammatory enzymes
54
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Kaempferol derivatives
Apigenin derivatives
Quercetin derivatives
BuOH Fr. of APL
LPS P65 P50
P
HPLC-PDA-ESI-MS/MS analysis
Cyto
kin
es
Cytokines
Pro INF enzyme
TNF-α
IL-6
IL-1β
COX-2
iNOS
TNF-α
IL-6
IL-1β
Pro
IN
F
en
zym
es COX-2
iNOS
APLB
APLB
Conclusions
Anti-inflammatory mechanism of APL BuOH fraction
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
P65 P50
IκBα
P65 P50
IκBα
P P P
P65 P50
IκBα
degradation
IKKα/β
Cyto
kin
es
Nuclear
DNA
Inflammatory
Response
Oxidative stress
P P P
NucleusCytoplasm
P
P
[Extracellular stimuli]
P65 P50
P
LPS
Oxidative stress IKKα/β
Cytokines
Pro INF enzyme
Api
Api > Api-BG > Api-PG
TNF-α
IL-6
IL-1β
COX-2
iNOS
Api > Api-BG > Api-PG> Api-G
Api
TNF-α
IL-6
IL-1β
Pro
IN
F
en
zym
es COX-2
iNOS
Api > Api-BG > Api-PGApi > Api-BG > Api-PG> Api-G
Api
Api > Api-G > Api-BG≥Api-PG
Conclusions
Anti-inflammatory mechanism of Aigenin derivatives on IKK/IkBa/NFkB pathway
(A) The effect of APM on HepG2 cell viability was evaluated using the MTT assay. (B) Intracellular ROS levels in H2O2-
stimulated HepG2 cell was estimated by 2´,7´-dichlorofluoresce in diacetate (DCF-DA) using fluorescence
spectrophotometer, (C) the FACS CalibursystemTm and (D) fluorescence microscopy.
Each value is expressed as mean ± standard derivation (n=3).
*,p<0.05; **,p<0.01; ***,p<0.001 (A) versus the non-treated control group (B) versus control group treated with LPS alone.
Control
(Non-treated)
H2O2 (1mM) H2O2 (1mM)
+ Quercetin (25 µg ·mL-1)
H2O2 (1mM)
+ APM (50 µg·mL-1)
Quercetin
Contro l
H2O2 (1mM) + Quercetin (25μg·mL-1)
H2O2(1mM)
H2O2 (1mM)+
APM (50µg·mL-1)
(DCF)
M1 M2
0
1
2
3
0 0 25 50 100 25
Inte
rcellu
lar
RO
S le
ve
l
(fo
ld o
f co
ntr
ol)
With H2O2 (1mM)
APM
(μg·mL-1)
Quercetin
0
0.2
0.4
0.6
0.8
1
1.2
0 25 50 100 200 25
Ce
ll via
bili
ty
(fo
ld o
f co
ntr
ol)
APM
(μg·mL-1)
B CA
D
******* **
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
**
** ** *
*
ROS scavenging activity of the MeOH extract of APL in situ
gallic acid (1), (+)-catechin (2), agrimonolide-6-O-β-D-glucopyranoside (3), vitexin (4), isovitexin (5), rutin (6), hyperoside (7), quercitrin (8), apigetrin (9), cynaroside (10), agrimonolide (11), quercetin (12), luteolin (13) and apigenin (14).
16The representative HPLC-PDA chromatograms of APM
The representative HPLC-PDA chromatograms of mixed standard
Chemical structures of mixed standard 1-14
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Metabolites Analysis of APM by HPLC-PDA analysis
CompoundContents
(mg·g-1, DW)
Free radical scavenging activity
ABTS3)
(RC502), μg·mL-1)
DPPH4)
(RC50, μg·mL-1)
Gallic acid (1) 1.28 ± 0.911) 5.21 ± 0.57 32.16 ± 1.69
Catechin (2) 26.98 ± 0.87 1.83 ± 0.08 26.19 ± 1.45
Agrimonolide-6-O-gluciside (3) 21.79 ± 0.46 >100 >100
Vitexin (4) 9.41 ± 0.25 5.56 ± 0.15 20.74 ± 1.18
Isovitexin (5) 0.89 ± 0.72 13.54 ± 1.46 36.97 ± 4.80
Rutin (6) 7.15 ± 0.49 5.76 ± 0.37 18.42 ± 1.85
Hyperoside (7) 14.68 ± 0.51 3.73 ± 0.38 28.43 ± 0.58
Quercitrin (8) 9.55 ± 0.51 6.48 ± 0.58 26.84 ± 0.23
Apigetrin (9) 46.49 ± 0.82 17.13 ± 0.66 43.64 ± 1.07
Cynaroside (10) 54.26 ± 2.60 10.59 ± 0.26 29.84 ± 0.58
Agrimonolide (11) 8.34 ± 1.60 26.42 ± 0.24 64.32 ± 1.87
Quercetin (12) 47.38 ± 3.79 0.09 ± 0.01 1.89 ± 0.21
Luteolin (13) 31.93 ± 3.33 2.69 ± 0.06 19.42 ± 0.26
Apigenin (14) 3.46 ± 1.49 5.70 ± 0.07 20.71 ± 1.14
ABTS - -
DPPH - -
1) Data is expressed as a mean ± SD of triplicate determinations2) RC50 values represent half the maximum radical scavenging concentration.3) ABTS; the activity 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity4) DPPH; the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity5) Pearson’s correlation analysis was conducted using averaged values of each variable (n=28).
17
The correlation coefficients between the contents of the compounds in APM and their free radical scavenging activities
-1.0 1.00.0
Pearson’s correlation coefficient
ABTS DPPH
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
53
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Free radical scavenging activity of the MeOH extract of APL
Activity-guided isolation of polyphenols with α-glucosidase inhibitory activity from Agrimonia pilosa Ledeb.
4 Isocoumarin compounds : agrimonolide (1), agrimonolide-6-O-glucoside (2), desmethylagrimonolide (3), desmethylagrimonolide-6-O-β-D-glucopyranoside (4)
4 Flavonoids : luteolin (5), quercetin (6), vitexin (7) and isovitexin (8).
Compounds 3 and 8 were isolated from AP for the first time
AGI inhibitor : compounds 3, 6 and 1 showed high AGI activities, with IC50 values of 2.4, 2.9 and 3.7 µg/mL, respectively.
α-Glucosidase inhibitorfrom Agrimonia pilosa Ledeb.
α-Glucosidase inhibitorfrom Agrimonia pilosa Ledeb.
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
The seedling of APL was purchased from
Siloam herb garden .
The variety of APL was identified by Dr. Lee
Chun Gil, Okcheon Medicinal Plant Growth
Experiment Station.
Plants were transplanted into plastic pots (∅
270 mm) filled with a mixture of 100g of
fertilizers (Osmocote Plus, Everris,
Newzland) in 30 L of soil (plant world,
Nongwoobio, Korea); light feeding.
Air temperature inside the greenhouse was
maintained under 25°C.
Stage 1
1 day
after transplanted
Stage 2
15 days
after transplanted
Stage 3
30 days
after transplanted
Stage 4
45 days
after transplanted
Stage 5
60 days
after transplanted
A
B
46
The temperature of the greenhouse was measured at 8 am.A: Total height from the root up to top leaf.
B: From the root and stem boundaries up to top leaf.
C: Number of total leaves of whole plant.
D: Number of leaves of one stem.
Each value is expressed as mean ± standard derivation (n=6).
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Stage 1
1 day
after transplanted
Stage 2
15 days
after transplanted
Stage 3
30 days
after transplanted
Stage 4
45 days
after transplanted
Temper
ature
(℃)�
Humi
dity
(%)
Plant height (cm)No. of
stems
No. of leaveslength of
leaves
Width of
leavesA B B/A C D
Stage 1 13.8 26 14.3 ± 0.8 5.2 ± 0.2 0.36 ± 0.02 6.3 ± 0.0 35.3 ± 4.0 7.0 ± 0.2 1.5 ± 0.1 1.3 ± 0.1
Stage 2 14.1 26 25.2 ± 1.4 15.0 ± 0.2 0.59 ± 0.02 7.3 ± 0.5 41.2 ± 6.2 7.1 ± 1.4 2.9 ± 0.1 1.8 ± 0.1
Stage 3 14.6 26 27.4 ± 0.9 16.7 ± 0.1 0.61 ± 0.02 9.3 ± 0.5 75.4 ± 8.3 8.6 ± 0.8 4.4 ± 0.2 2.9 ± 0.1
Stage 4 15.1 26 41.3 ± 4.8 18.5 ± 0.4 0.5 ± 0.06 11.3 ± 0.5 89.8 ± 6.6 8.5 ± 1.0 6.3 ± 0.6 3.4 ± 0.3
Phenotype of APL according to growth stages
47
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Stage Part Fresh weight (g) Dry weight (g)/ Dry matter ratioExtraction
rate (%)
Stage 1
Whole 1.1 ± 0.2 0.3 ± 0.1/ 0.3 ±0.2 33.1 ± 0.2
Shoot 0.5 ± 0.1 0.1 ± 0.0 / 0.2 ± 0.1 33.3 ± 0.3
Root 0.5 ± 0.1 0.2 ± 0.0/ 0.4 ± 0.0 17.8 ± 0.1
Stage 2
Whole 5.0 ± 0.9 1.2 ± 0.1/ 0.2 ± 0.5 25.8 ± 0.3
Shoot 3.2 ± 0.2 0.9 ± 0.1/ 3.6 ± 0.2 30.3 ± 0.8
Root 2.1 ± 0.1 0.4 ± 0.0/ 0.2 ± 0.0 17.7 ± 0.4
Stage 3
Whole 9.5 ± 0.6 2.1 ± 0.4/ 0.2 ± 0.5 10.5 ± 0.2
Shoot 5.2 ± 0.7 1.0 ± 0.3/ 0.2 ± 0.5 24.0 ± 0.3
Root 4.3 ± 0.1 1.1 ± 0.1/ 0.3 ± 0.1 18.2 ± 0.3
Stage 4
Whole 13.0 ± 1.9 2.7 ± 0.4/ 0.2 ± 0.0 9.5 ± 0.2
Shoot 8.3 ± 0.7 1.5 ± 0.1/ 0.2 ± 0.0 23.3 ± 0.4
Root 4.8 ± 2.0 1.2 ± 0.5/ 0.3 ± 0.1 16.7 ± 0.1
Whole part: total plant from the root up to top leaf.
Shoot part: the plant from the root and stem boundaries up to top leaf.
Root part : the plant part from the root and stem boundaries up to bottom root.
The ratio between the original weight and the dry weight.
Each value is expressed as mean ± standard derivation (n=3).
Fresh and dry weights, and extraction rate of APL at growth stages
48
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
0
1000
2000
3000
4000
Shoot Root Whole
To
tal p
he
no
lic c
on
tent (g
GA
E·K
g-1
)
Stage 1 Stage 2 Stage 3 Stage 4
Whole Stage 2 > Stage 1 > Stage 3 > Stage 4
Shoot Stage 1 > Stage 2 > Stage 3 > Stage 4
Root Stage 3 > Stage 2 > Stage 4 > Stage 1
Changes of total phenolic contents of APL affected by growth stages
49
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
0
20
40
60
80
100
0 50 100
AB
TS
ra
dic
al s
cave
ng
ing
activity
(%, D
W)
Stage 1 Stage 2 Stage 3 Stage 4
0
20
40
60
80
100
0 50 100
AB
TS
ra
dic
al s
cave
ng
ing
activity
(%, D
W)
Stage 1 Stage 2 Stage 3 Stage 4
0
20
40
60
80
100
0 50 100
AB
TS
ra
dic
al scave
ng
ing
activity
(%, D
W)
Stage 1 Stage 2 Stage 3 Stage 4A B C
(µg·mL-1)(µg·mL-1) (µg·mL-1)
Stage 1 > Stage 2 > Stage 3 > Stage 4 Stage 3 > Stage 4 > Stage 1 > Stage 2 Stage 2 > Stage 1 > Stage 3 > Stage 4
Shoot Root Whole
Changes of antioxidant activities of APL affected by growth stages
51
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
A
B C
Whole Stage 2 > Stage 1 > Stage 3 > Stage 4
Shoot Stage 1 > Stage 2 > Stage 3 > Stage 4
Root Stage 3 > Stage 2 > Stage 4 > Stage 1
Shoot (µg·mL-1)
Stage 1 > Stage 2 > Stage 3 > Stage 4
0
0.5
1
1.5
Stage
1
Stage
2
Stage
3
Stage
4
Stage
5
Stage
1
Stage
2
Stage
3
Stage
4
Stage
5
Stage
1
Stage
2
Stage
3
Stage
4
Stage
5
Shoot Root Whole
Ce
ll via
bili
ty
(Fo
lds o
f L
PS
tre
ate
d g
rou
p)
2.5 μg·mL-1 5 μg·mL-1 10 μg·mL-1 20 μg·mL-1
0
5
10
15
20
Shoot Root Whole
NO
in
hib
itory
activity
(% a
t 5
μg
·mL
-1)
Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
0
10
20
30
40
0 10 20 30 40
NO
in
hib
itory
activity (
%)
Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
2.5 μg·mL-1 5 μg·mL-1 10 μg·mL-1 20 μg·mL-1
Changes of anti-inflammatory effect of APL affected by growth stages
Conclusions57
Anti-inflammatory Metabolites of Agrimonia pilosa Ledeb. and Their Mechanism
Stage 1
1 dayafter transplanted
Stage 2
15 daysafter transplanted
Stage 3
30 daysafter transplanted
Stage 4
45 daysafter transplanted
Phenotypic characteristics Functional characteristics
Plant height : 5.2-18.5 cm
Number of stems in one plant : 6-11
Number of leaves in one stem : 7-9
Leaf shape : odd-pinnate leaf
Small leaves form : egg-shaped ellipse
denticulated edge
Length of leaves : 1.5-6.3 cm
Width of leaves: 1.3-3.4 cm
Hairs grow on the front and back of the
leaves after Stage 3
Total phenolic content
Anti-inflammatory activity
Shoot Stage 1 > Stage 2 > Stage 3 > Stage 4
Root Stage 3 > Stage 4 > Stage 2 > Stage 1
Shoot Stage 1 > Stage 2 > Stage 3 > Stage 4
Root Stage 3 > Stage 4 > Stage 2 > Stage 1
Anti-oxidant activity
Shoot Stage 1 > Stage 2 > Stage 3 > Stage 4
Root Stage 3 > Stage 4 > Stage 2 > Stage 1
The phenotypic& functional characteristic of APL according to growth stages