Comparison of Antioxidant and Physiological Activities of Various Solvent Extracts from Hizikia fusiformis

886

Copyright © 2020 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

서 론

현대의도시화와공업화로인한환경오염과서구화된식생활 변화로인하여고혈압

,

,

,

.

.

,

,

·

(Cho and Choi, 2010; Lee et al., 2016; 2020a).

구대상의한계와환경오염등으로인하여최근해양생물이육 상생물의대체자원으로주목받고있다

(Byun and Kim, 2005;

Lee, 2011).

독특한생육환경으로인하여다양한생리활성물질을가지고 있는것으로보고되었다

.

,

,

,

대로건강기능성식품및의약품소재로개발되고있다

(Kwon

and Youn, 2017).

,

소재개발을위한유용한자원중하나로평가되고있다

(Lee et

al., 2017).

(Hizikia fusiformis)

형태는원주상줄기로체장

20-100 cm

추출용매에 따른 톳(Hizikia fusiformis) 추출물의 항산화 및 생리활성 비교

이연지·전유진

·김용태*

군산대학교 식품생명공학전공, ¹제주대학교 수산생명의학전공

Comparison of Antioxidant and Physiological Activities of Various Sol- vent Extracts from Hizikia fusiformis

Yeon-Ji Lee, You-Jin Jeon¹ and Yong-Tae Kim*

Department of Food Science and Biotechnology, Kunsan National University, Gunsan 54150, Korea

1Department of Marine Life Sciences, Jeju National University, Jeju 63243, Korea

The seaweed Hizikia fusiformis is rich in protein, carbohydrates, vitamins, and minerals. This study investigated the antioxidant and physiological activities of H. fusiformis extracts prepared with 70% ethanol, 80% methanol, or distilled water. The extraction yields of these various solvent extracts were as follows: ethanol extract, 15.26%;

methanol extract, 17.95%; and water extract, 45.62%. The methanol extract showed the highest total polyphenol content (24.06 mg GAE/g), but total flavonoid content was similar in all extracts. ABTS (2,2'-Azino-bis(3-ethylben- zothiazoline-6-sulfonic acid)) radical scavenging activity was highest in the ethanol extract (IC

: 0.90 mg/mL), while the methanol extract exhibited the strongest DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (IC

: 8.09 mg/mL), reducing power (EC

: 0.40 mg/mL), and ferric reducing antioxidant power (0.28 mM). By contrast, tyrosinase and α-glucosidase inhibitory activities were higher in the ethanol extract than in the other extracts. The high BACE1 (β-secretase) inhibitory activity was observed in the ethanol extract (IC

: 1.03 mg/mL). These results indicate that H. fusiformis ethanol extracts may be useful for their antioxidant and functional properties in food and pharmaceutical materials.

Keywords: Antioxidative activity, Anti-dementia, BACE1, Hizikia fusiformis , Physiological activity

*Corresponding author: Tel: +82. 63. 469. 1824 Fax: +82. 63. 469. 7448 E-mail address: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received 15 October 2020; Revised 10 November 2020; Accepted 25 November 2020 저자 직위: 이연지(대학원생), 전유진(교수), 김용태(교수)

https://doi.org/10.5657/KFAS.2020.0886

Korean J Fish Aquat Sci 53(6), 886-893, December 2020

해조류이다

.

,

·

.

2010

21,133

; 2019

33,477

,

2-4

,

,

(Lee et al., 2020a; Statistics Korea, 2020).

,

,

(glutamic acid

aspartic acid)

,

,

,

(Lee et al., 2016).

,

(Siriwardhana et al., 2003; Kim et al., 2013),

(Kim et al., 1994),

(Kwon et al., 2015)

(Cui et al., 2019)

,

,

,

(Cui et al., 2019).

,

.

재료 및 방법

실험 재료 및 시약

본실험에사용된톳

(Hizikia fusiformis)

,

.

Folin-Ciocalteu’s reagent, gallic acid, quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-eth- ylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl, vitamin C, mushroom tyrosi- nase, 3,4-dihydroxy-L-phenylalamine (L-DOPA), kojic acid, angiotensin I-converting enzyme (ACE), hippuryl-his-leu (HHL) acetate salt, captopril, acetylcholinesterase (AChE), acetylthiocholine iodide (ATC), 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB)

Sigma-Aldrich Co.(St. Louis, MO, USA)

. yeast α-glucosidase, ρ-nitrophenol- α-glucopyranoside

Wako Chemical Co. (Kanagawa, Japan)

.

.

톳 추출물의 제조

건조된톳을분쇄기

(FM700SS, Hanil, Seoul, Korea)

분쇄한후

3

(

, 70% ethanol, 80% methanol)

.

50 g

200 mL

,

(ISA-CIP- S30-200, Ilshin autoclave, Daejeon, Korea)

300 MPa

3

.

flask

300 mL

100°C

3

.

70%

ethanol

80% methanol

50 g

20

25°C

24

shaking incubator (120 rpm)

.

.

,

에

-20°C

.

색도 측정

각 추출물의 색도는 색차계

(JC801, Color Techno System Co., Ltd., Tokyo, Japan)

L (

), a (

)

b (

)

.

3

.

(calibration plate)

L

96.5, a

-0.13, b

-0.05

.

Total polyphenol 및 Total flavonoid 함량 측정

Folin-Denis

Shetty et al. (1995)

.

(1 mL)

95%

1 mL

5 mL

50% Folin-Ciocalteu reagent 0.5 mL

5

.

5% Na

CO

1 mL

∙

1

(Optizen Pop, KLAB, Seoul, Korea)

725 nm

.

gallic acid

.

총플라보노이드함량은

Moreno et al. (2000)

.

(0.5 mL)

1.5 mL, 95%

0.1 mL, 10% aluminum nitrate

0.1 mL, 1 M potassium acetate

3

2.8 mL

30

415 nm

. Quercetin

.

ABTS 라디칼 소거활성 측정

ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

diammonium salt, Sigma-Aldrich Co., St. Louis, MO, USA]

radical

ABTS

radical decolorization assay (Re et al., 1999)

. 7.4 mM

ABTS

2.6 mM potassium persulfate

∙

24

radical

ABTS

734 nm

1.000±0.030 (mean±SD)

phosphate-buffered saline (pH 7.4)

.

50 μL

ABTS

950 μL

소에서

10

734 nm

계산식

, ABTS

radical scavenging ability(%)=[(Control

– Sample

)/Control

]×100

. IC

value (mg/mL)

50%

ABTS

radical

(mg/mL)

.

DPPH 라디칼 소거활성 측정

시료의 산화방지 활성을 측정하기 위하여 자유라디칼인

DPPH

Blois (1958)

Lee et al. (2017)

.

(1.5 mL)

0.4 mM DPPH radical etha- nolic solution (1.5 mL)

, 37°C

30

,

516 nm

. DPPH

, DPPH radical scavenging ability (%)=[(Control

-Sample

)/Control

]×100

, IC

value (mg/mL)

50%

DPPH

(mg/mL)

.

,

(Control

)

.

환원력 측정

환원력

(reducing power)

Oyaizu et al. (1988)

Lee et al. (2020b)

.

(1 mL)

1 mL

0.2 M sodium phosphate

(pH 6.6)

1 mL

1% (w/v) potassium ferricyanide

, 50°C

20

.

1 mL

10% (w/v) trichloroacetic acid (TCA)

,

(1,890 g, 10

)

.

1.5 mL

1.5 mL

0.3 mL

0.1% (w/v) ferric chloride

, 10

,

700 nm

,

광도가높을수록환원력이큰것을의미한다

. EC

value

0.5

(mg/mL)

. FRAP value 측정

FRAP (Ferric reducing antioxidant power)

Benzie and Strain (1996)

. 300 mM acetate buffer (pH 3.6), 40 mM HCl

10 mM TPTZ (2,4,6-tripyridyl-s-triazine)

20 mM FeCl

· 6H

O

10:1:1 (v/v/v)

FRAP

였다

.

0.15 mL

3.0 mL

FRAP

37°C

5

593 nm

. FeSO

· 7H

O

FRAP valve (mM)

.

Tyrosinase 저해활성 측정

각시료용액의

tyrosinase

Iida et al. (1995)

. 300 μL

900 μL

mushroom tyrosinase (50 Unit/mL)

1.5 mL

50 mM Phosphate buffer (pH6.8)

30

, 300 μL

10 mM 3,4-dihy- droxy-L-phenylalamine (L-DOPA)

,

475 nm

20

1

dopachrome

. Tyrosinase

(%)

, Tyrosinase inhibitory activity (%)=[(Control

- Sample

)/Control

]×100

.

(Control

)

.

α-Glucosidase 저해활성 측정

각시료의

α-glucosidase

Watanabe et al. (1997)

chromogenic assay

ρ-nitrophenol glucoside

. Yeast α-glucosidase

(100 mM phosphate buffer, pH 7.0, 0.2% bovine serum albumin

0.02% NaN

)

0.7 U/mL

,

ρ-nitrophenyl-α-glucopyranoside (5 mM)

.

100 μL

20 μL

well

405 nm

(time zero)

.

5

incu- bation

,

100 μL

5

incubation

산하였다

.

α-glucosidase

acarobose

.

, α-Glucosidase inhibitory activity (%)=[1-(Sample

/Con- trol

)]×100

, control

.

BACE1 (β-Secretase) 저해활성 측정

각시료의

BACE1 (β-Secretase)

Lee (2017)

.

BACE1

Lee (2017)

,

(MO- CAc-SEVNLDAEFRK(Dnp)RR)

Peptron Co. (Daejeon,

Korea)

.

은

black 96-microwell plate

0.1 M sodium acetate buffer

(pH 4.0) 225 μL, BACE1 10 μL

10 μL

37°C

30

preincubation

다음

, 0.5 mM substrate 5 μL

37°C

15

.

,

microplate reader (Infinte F200, Tecan, Männedorf, Switzerland)

Ex 328/Em 393 nm

.

0.1 M sodium acetate buffer (pH 4.0) 10 μL

,

, BACE1 inhibi- tory activity(%)=[100-(SV/CV)]×100

. SV,

; CV,

. 통계처리

실험결과는

SPSS 22.0 package program (SPSS Inc., Chi- cago, IL, USA)

3

±

.

(ANOVA)

P<0.05

Duncan’s multiple range test

.

결과 및 고찰

톳 추출물의 수율 및 색도 분석

톳에함유되어있는생리기능성물질의대량획득및미량의 유용성분을추출하기위하여

70% EtOH, 80% MeOH

. EtOH

MeOH

24

shaking incubator

,

.

Table 1

. EtOH

11.40%, MeOH

17.95%,

45.62%

로나타났다

.

> 80% MeOH> 70% EtOH

,

2.5

.

Kwon and Youn (2015)

,

해조직과세포막의변형으로인해용매들이세포안으로쉽게 들어감으로서기존물질들의용출량이증가할뿐만아니라새 로운물질도용출되어수율이높게나타나는것으로생각된다

(Kwon and Youn, 2015).

톳추출물제조시추출용매를달리한각추출물의색도를측 정한결과

,

L (lightness)

EtOH

47.00

, MeOH

71.48

76.57

,

(P<0.05).

a (redness)

EtOH

17.02

, MeOH

4.48

.

b (yellowness)

45.69

, EtOH

MeOH

57.01

58.91

. Lee et al. (2016)

Chlorophyll A&C, β-carotene

fucoxanthin

, Kwon and Youn (2015)

.

,

. 톳 추출물의 Total polyphenol 및 Total flavonoid 함량 분석

톳추출물제조시추출용매를달리한톳추출물들의총폴리 페놀함량및총플라보노이드함량을측정한결과는

Table 2

.

70% EtOH

13.04 mg GAE/g

, 80% MeOH

24.06 mg GAE/g,

6.91 mg GAE/

g

.

,

MeOH>

EtOH>

. Lee (2013)

46.59 mg/

g

, Kim et al. (2015)

19.89 mg/g,

16.44 mg/g

.

,

,

.

,

Table 1. Comparison of the extraction yields and color values of various solvent extracts from Hizikia fusiformis

Sample Yield

(%) Color value

Lightness (L) Redness (a) Yellowness (b)

70% EtOH extract 11.40 47.00±0.17^c,1,2 17.02±0.05^a 57.01±0.05^b

80% MeOH extract 17.95 71.48±0.20^b 4.48±0.08^c 58.91±0.04^a

Water extract 45.62 76.57±0.06^a 10.62±0.05^b 45.69±0.06^c

1Value are mean±SD (n=3). ²Means with different letters in a column are significantly different at P<0.05 by Duncan's multiple range test.

이드함량은

7.99-8.43 mg QE/g

.

.

이드는주로

anthocyanidins, flavonols, flavones, cathechins

flavanones

이드는추출용매에따른용출양의차이는적은것으로생각된 다

(Middleton and Kandaswami, 1994).

톳 추출물의 항산화 활성

톳추출물제조시추출용매로

70% EtOH, 80% MeOH

ABTS

DPPH

Fig. 1

,

ABTS

DPPH

IC

Table 3

.

ABTS

.

0.1-1 mg/mL

EtOH

ABTS

, 2 mg/mL

MeOH

EtOH

. ABTS

IC

EtOH

, 0.90 mg/mL; MeOH

, 0.96 mg/mL;

, 1.64 mg/

mL

, ABTS

EtOH> MeOH>

증류수추출물순으로높은것으로확인되었다

.

각추출용매별톳추출물의

DPPH

Fig.

1B

는경향을보여준다

. 10 mg/mL

DPPH

80% MeOH

58.87%

, EtOH

28.50%, 27.57%

, MeOH

50%

에불과한것으로확인되었다

.

, DPPH

IC

MeOH

, 8.09 mg/mL;

, 20.93 mg/

mL; EtOH

, 23.20 mg/mL

DPPH

Sample Total polyphenol

(mg GAE/g) Total flavonoid (mg QE/g) 70% EtOH extract 13.04±0.53^b,1,2 7.99±0.22^a 80% MeOH extract 24.06±0.50^a 8.34±0.39^a Water extract 6.91±0.05^c 8.43±0.09^a

1Values are mean±SD (n=3). ²Means with different letters in a column are significantly different at P<0.05 by Duncan's multiple range test.

Fig 1. ABTS (A) and DPPH radical scavenging activities (B) of various solvent extracts from Hizikia fusiformis. ABTS, 2,2'-azi- no-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt;

DPPH, 2,2-diphenyl-1-picrylhydrazyl.

(A)

(B)

Table 3. Antioxidant activities of various solvent extracts from Hizikia fusiformis

Sample ABTS (IC₅₀, mg/mL)¹ DPPH (IC₅₀, mg/mL) Reducing Power (EC₅₀, mg/mL)² FRAP value (mM)

70% EtOH extract 0.90 23.20 0.64 0.21

80% MeOH extract 0.96 8.09 0.40 0.28

Water extract 1.64 20.93 1.30 0.13

1The 50% inhibitory concentration (IC₅₀) values (mg/mL) were calculated from a log dose inhibition curve. ²EC₅₀ value for reducing power is expressed as the effective concentration at which the absorbance is 0.5.

라디칼소거활성은

MeOH>

> EtOH

. Kim et al. (2015)

1 mg/mL

ABTS

30.89%

38.69%

, DPPH

물모두

0-5%

,

Siriwardhana et al. (2003)

,

ABTS

DPPH

.

EtOH, MeOH

(re-

ducing power)

FRAP value

Table 3

.

(MeOH, EtOH,

)

(

),

(EC

, mg/mL)

MeOH

0.40 mg/mL

, EtOH

0.64 mg/mL,

1.30 mg/mL

.

,

MeOH> EtOH>

.

FRAP value

MeOH

0.28 mM, EtOH

0.21 mM,

0.13 mM

.

MeOH> EtOH>

FRAP value

.

FRAP value

,

FRAP value

Lee et al. (2014)

.

Tyrosinase 및 α-glucosidase 저해 활성

추출용매에따른톳추출물의

tyrosinase

(%)

α-glucosidase

·

Table 4

. Tyrosi- nase

ty-

rosine

의활성억제는피부미백과노화방지에매우중요하다

(Choi et al., 2011).

tyrosinase inhibitor

.

5 mg/mL

tyrosinase

EtOH

51%, MeOH

24%,

20%

,

3

EtOH

tyrosinase

. Choi et al. (1998)

1 mg/

mL

24%

tyrosinase

, Jeon et al. (2012)

0.1 mg/mL

44%

tyrosinase

, Kwon and Youn (2017)

1 mg/mL

40%

tyrosinase

,

.

tyrosinase

,

,

tyrosinase

.

당뇨병은고혈당상태가지속되는질환으로이환율이높고고 혈당상태의지속에따른다양한합병증으로인한사망률이높 은만성퇴행성대사질환이다

(Ali et al., 2017). α-Glucosidase

brush-border membrane

(Kim et al., 2014).

당뇨병을억제하기위하여다양한추출용매를사용한톳추출 물의

α-glucosidase

. EtOH, MeOH

5 mg/mL

α-glucosidase

EtOH

37.20%, MeOH

4.07%

이었으나

,

0.33%

.

Kim et al. (2015)

α-glucosidase

4%, EtOH

40%

.

톳 추출물의 항치매 효과

치매는신경세포의노화에의한퇴행성뇌질환으로뇌혈관성 치매와알츠하이머병으로분류한다

.

,

이머병은 정확한발병기전과원인이밝혀져있지않다

(Park

et al., 2014).

β-amyloid

40-42

,

. β-Amyloid

amyloid precursor protein (APP)

β-secretase

γ-secretase

(Park et al., 2014).

기위하여톳추출물의

APP

BACE1

(β-secretase)

.

BACE1

IC

Fig. 2

ties of various solvent extracts (5 mg/mL) from Hizikia fusiformis

Sample Inhibitory activity (%)

Tyrosinase α-Glucosidase BACE1 70% EtOH extract 51.00±1.37¹ 37.20±0.17 100.13±0.23 80% MeOH extract 24.33±12.11 4.07±0.03 68.10±0.10 Water extract 20.36±3.49 0.33±0.49 110.00±0.90

1Values are mean±SD (n=3).