짚신나물추출물의생리활성과대사체분석 · 2020. 3. 17. · resveratrol, a natural...

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

https://en.wikipedia.org/wiki/Cannabinoid

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

https://en.wikipedia.org/wiki/Cruciferous_vegetables

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

https://science.sciencemag.org/content/275/5297

https://science.sciencemag.org/content/275/5297

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)


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


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.


Agrimonia pilosa Ledeb. Distribution

Reason of plant selection

Previous study for APL


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



MeO

HE

xt.

* * * *

NO inhibitory activity of the MeOH extract and its fractions of APL


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-


The phenolics profiles of the BuOH fraction of APLby HPLC-PDA-ESI-MS/MS


*,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)

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






C

A

B

The effect of the BuOH fraction of APL on production overexpressed inflammatory cytokines and enzymes


Isolation scheme of anti-inflammatory compoundsin the BuOH fraction of APL

25


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


[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


Alkyl group

Alkyl group

Alkyl group

34COSY NMR spectrum of 8 (Bold line)

A C

B


35HMBC NMR spectrum of 8 (Arrow line)

A C

B


26


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


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)


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.


*,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


Api-PG Api-BG ApiApi-PG Api-BG Api

Sample(μM) Sample(μM) Sample(μM)

Sample

(μM)

Sample

(μM)

**** **

******

* ***

**

****** ***

***

***

*

0


10 20 10 20 10 20

ApiPG Api-BG Api0 0


10 20 10 20 10 20

ApiPG Api-BG Api0 0


10 20 10 20 10 20

ApiPG Api-BG Api0


* **

*

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


Api-PG Api-BG Api


COX-2

Actin

010 20 10 20 10 20

0

Sample

(μM)B


ApiPG Api-BG Api



Sample (μM)

Sample (μM)

30

30

*

*

*

***

***

*****

*****

**

****

*

****

****

******

**

**

***

*

**** **

**

***

**

*



*,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


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


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.


*,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

******* **


**

** ** *

*

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


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


53


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.


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.



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


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.


Fresh and dry weights, and extraction rate of APL at growth stages

48


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


0

20

40

60

80

100

0 50 100

AB

TS

ra

dic

al s

cave

ng

ing

activity

(%, D

W)


0

20

40

60

80

100

0 50 100

AB

TS

ra

dic

al s

cave

ng

ing

activity

(%, D

W)


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


A

B C

Whole Stage 2 > Stage 1 > Stage 3 > Stage 4



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


Stage 1

1 dayafter transplanted

Stage 2

15 daysafter transplanted

Stage 3


Stage 4


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





Anti-oxidant activity



The phenotypic& functional characteristic of APL according to growth stages

짚신나물추출물의생리활성과대사체분석 · 2020. 3. 17. · resveratrol, a natural...

Documents