Antioxidative, Antimicrobial, and Anti-proliferative Activities of Floret and Stalk of Broccoli (Brassica oleracea L.)

브로콜리 꽃송이 및 줄기의 항산화, 항균 및 대장암 세포 생육억제효과

김미선¹, 이예슬¹, 권하영¹, 김종식², 손호용¹*

1안동대학교식품영양학과

2안동대학교생명과학과

Received: January 27, 2014 / Revised: March 3, 2014 / Accepted: March 6, 2014

서 론

브로콜리

(Brassica oleracea L.)

(Brassicaceae)

^,

^.

^,

^,

^.

^75%

^,

²⁰⁰⁰

한선호도증가와함께브로콜리재배와소비가증가되고있 다

^{[9, 21].}

^,

^[13],

^,

,

서는물에살짝데쳐먹는다

^[10].

한가공식품개발에관심이집중되고있는데

^,

분말을첨가하여제조한파운드케익

^[21],

연구

^[4]

^,

^[22]

^.

브로콜리는

ascorbic acid,

-carotene, rutin, selenium, glutathione, quercetin

^,

^,

Antioxidative, Antimicrobial, and Anti-proliferative Activities of the Floret and Stalk of Broccoli ( Brassica oleracea L.) Mi-Sun Kim

, Ye-Seul Lee

, Ha-Young Kwon

, Jong Sik Kim

, and Ho-Yong Sohn

*

1

Department of Food and Nutrition, Andong National University, Andong 760-749, Republic of Korea

2

Department of Life Science, Andong National University, Andong 760-749, Republic of Korea

In the course of study for a use for non-edible parts of broccoli (Brassica oleracea L), and the development of processed food utilizing these parts, edible floret and non-edible stalk were extracted with ethanol and different organic solvent fractions were prepared. With 10 different extracts and fractions, their useful components and various biological activities, such as antioxidant, antimicrobial and anti-proliferation activity, were investigated. The stalk has more abundant water soluble carbohydrate when compared with the floret, and floret has higher hexane-soluble pigments. Analysis of total flavonoid and total polyphenol con- tents showed that the floret has 1.5~1.99 times higher concentrations than the stalk. Among the fractions, ethylacetate (EA) fractions have the highest amount of total flavonoid and total polyphenol. The stalk and floret possessed 9.45 and 42.01 mg- total flavonoid/g, respectively. In the antioxidation activity assay, the EA fraction of floret showed strong radical scavenging activity and reducing power, while the n-hexane fraction of the stalk exhibited nitrite scavenging activity. In the antimicrobial activity assay, the EA fraction of floret showed a strong and broad-range of antibacterial activity, irrespective of gram positive or gram negative bacteria. In a while, the hexane and EA fractions revealed anti-proliferative effects against the human colorectal cancer cell HCT-116. Strong anti-proliferative activities were found in the hexane fraction of stalk (18.4% of cell viability), and the n-butanol fraction of floret (6.9% of cell viability). Our results suggest that the further study of the characterization of active fractions and the identification of active components from different parts of broccoli are needed to develop functional foods or novel plant-derived medicines.

Keywords: Brassica oleracea L floret, stalk, antioxidation, antimicrobial, anti-proliferation

*Corresponding author

Tel: +82-54-820-5491, Fax: +82-54-820-7804 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

서는브로콜리가신경을진정시키고간장의기능을활발하 게하는기능이있다고알려져있다

^.

^,

^,

^[17]

^[16]

^,

^.

^,

^[15],

^[7],

[11], ovalbumin

[23]

성

^[13]

^.

glucosynolate

^[14],

glucosynolate

sulforaphane [25]

[3, 6, 20], non- small lung cancer, chondrosarcoma

[18, 19, 24, 28],

^[3]

^.

sulforaphane

platelet adenylate cyclase

[5]

^.

본연구에서는브로콜리의비가식부위의효율적인이용 및브로콜리의가공식품개발연구의일환으로브로콜리의 주요가식부위인꽃부위

^(floret)

(stalk)

기용매분획물을조제하여항산화

^,

^.

^,

^hexane

ethylacetate

^,

^,

^.

재료 및 방법

실험재료

본실험에사용된브로콜리는

²⁰¹²

⁶

^H

^,

^(floret)

^(stalk)

^.

1 kg

^{10 L}

탄올

(Daejung Chemicals & Metals Co., Ltd. Korea)

²⁴

²

^,

(Whatsman No. 2)

⁵⁵

^C

(Eyela Rotary evaporator N-1000, Tokyo Rikakikai Co., Ltd., Japan)

^.

n-hexane, ethylacetate

n-butanol

^,

⁵⁵

^C

^.

^DMSO

in-vitro

^,

균및암세포생육억제활성평가에사용하였다

^.

Sigma Co. (USA)

^.

^(voucher specimen 2012-BO-F1~F5

2012-BO-S1~S5).

항산화 활성

브로콜리추출물및이의분획물의항산화활성은

^DPPH

(1,1-diphenyl-2-picryl hydrazyl) anion scavenging activity [DSA], ABTS [2,2-azobis(3-ethylbenzothiazoline-6-sulfonate)]

cation scavenging activity [ASA], nitrite scavenging activity [NSA]

^[8].

^DSA

^,

²⁰

^l

99.5% ethanol

²

¹⁰

^{M DPPH}

³⁸⁰

^l

37

C

³⁰

^{, 516 nm}

microplate reader (Asys Hitech, Expert96, Asys Co., Austria)

^{. DSA(%)}

^{[2]. ASA}

^{, 7 mM} ABTS (Sigma Co., USA) 5 ml

140 mM potassium persulfate 88 ml

¹⁶

ABTS

^,

^{414 nm}

^1.5

^ethanol

^.

¹⁹⁰

^l

¹⁰

^l

⁶

응시킨후

^{734 nm}

해

^ASA(%)

^[1].

ASA (%) = [(C

^S)/C]

¹⁰⁰

C: DMSO

S:

한편

^NSA

^,

^{(1 mM)}

^{0.1 N HCl}

^{pH 1.2}

^, 37

C

¹

Griess reagent (Sigma Co., USA)

^.

¹⁵

520 nm

^nitrite

^{. NSA(%)}

^[2].

NSA (%) = [1

^(A

^C)/B]

¹⁰⁰

A: 1 mM nitrite

¹

B: 1 mM nitrite

C:

환원력평가의경우

^Oyaizu

였다

[2]. Ethanol

^{2.5 ml}

0.2 M sodium

phosphate buffer (pH 6.6) 2.5 ml

10% potassium

ferricyanide 2.5 ml

⁵⁰

^C

²⁰

, 10% trichloroacetic acid 2.5 ml

^{4,000 rpm}

¹⁰

^.

²

^,

0.1% ferric chloride

5:1 (v/v)

고

^{700 nm}

^.

산화실험에서대조구로는

vitamin C (Sigma Co., USA)

^,

^DMSO

^.

³

^.

항균 활성

조제된브로콜리의추출물및이의분획물의항균활성은 기존의보고된방법과동일하게평가하였다

^[8].

Staphylococcus aureus KCTC1916, Listeria monocytogenes KACC10550, Bacillus subtilis KCTC1924

^,

Escherichia coli KCTC1682, Pseudomonas aeruginosa KACC10186, Proteus vulgaris KCTC2433, Salmonella

typhimurium KCTC1926,

Candida albicans KCTC1940

Saccharomyces cerevisiae IF00233

^.

^{, Nutrient} broth (Difco Co., USA)

³⁷

^C

24

^,

^OD

0.1

Nutrient agar (Difco Co., USA)

petri dish (90

15 mm, Green Cross Co., Ltd. Korea)

100

^l

^,

⁵

^l

disc-paper(

6.5 mm, Whatsman No.2)

^{, 37}

^C

²⁴

^[1],

Sabouraud dextrose (Difco Co., USA)

³⁰

^C

²⁴

^,

^[12].

ampicillin

miconazole (Sigma Co., USA)

¹

^g/disc

^,

^mm

^{, 3}

^.

대장암세포 생육억제 활성

인간유래대장암세포

^HCT-116

Dulbecco's Modified Egle Medium (Gibco-BRL Inc., USA)

, 10% fetal bovine serum (Gibco- BRL Inc., USA), 1% penicillin

streptomycin (WelGene Inc., Deageon, Korea)

^[26].

리꽃송이및줄기시료들의

^HCT-116

치는 영향은

One solution cell proliferation assay kit

(Promega, USA)

^.

, 96 well plate

^well

³

¹⁰

²⁴

1 mg/ml

하고

²⁴

3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxphenyl)-2-(sulfophenyl)-2H-tetrazolium (MTS; Promega, USA)

^well

²⁰

^l

37

C, 5% CO

incubator

⁴

^,

96 well plate reader (Expert 96 UV ASYS Hitech, Austria)

^{490 nm}

^[26].

⁴

well

^.

기타 분석

브로콜리꽃송이및줄기의수분함량은적외선수분측정 기

(HG53 Halogen Moisture Analyzer, Mettler-Toledo International Inc., Zurich, Switzerland)

^,

각각의추출물및분획물의총

^flavonoid

존의보고한방법

^[8]

^,

18

⁴⁰⁰

^l

^90%

diethylene glycol 4 ml

1 N NaOH 40

^l

³⁷

^C

¹

^{420 nm}

^.

^rutin

^.

polyphenol

⁴⁰⁰

^l

⁵⁰

^l

Folin-ciocalteau, 100

^l

^Na

CO

¹

^{725 nm}

^[27].

tannic acid

^.

^sucrose

phenol-sulfuric acid

^,

glucose

^DNS

다

^[2].

³

^.

통계분석

실험결과는

SPSS 21.0

^mean

^SD

^,

^ANOVA

^,

Duncan

였다

^.

p < 0.05

^.

결과 및 고찰

브로콜리 줄기 및 꽃송이의 성분 비교

브로콜리줄기및꽃송이의수분함량은각각

^90.9

^3.1%

와

^86.8

^2.5%

^,

^4.13%

^5.35%

^.

n-hexane, ethylacetate, n-

butanol

^,

^ethanol

2.69, 1.05, 21.87%

6.94, 1.93, 29.53%

^,

^74.15%,

^57.61%

^,

^(Table 1).

n-hexane

ethylacetate

^,

^2.4

^,

^1.3

^.

^.

^1.47-1.65

^,

노이드함량및총폴리페놀함량은꽃송이가줄기보다

^1.50-

1.99

^.

^,

^.

^,

ethylacetate

9.45 mg/g

42.01 mg/g

^,

ethylacetate

181.12 mg/g- extract

^,

ethylacetate

^4.83

(Table 1).

브로콜리의 줄기 및 꽃송이의 항산화 활성

브로콜리줄기및꽃송이의에탄올추출물및분획물들의 항산화활성을평가한결과는

^{Fig. 1}

^{Table 2}

^.

^{vitamin C}

²⁵

^g/ml

^62.8

^1.4%

^DPPH

^{, 84.9}

^0.4%

nitrite

^1.0

^{0.04 (OD}

)

^{, 12.5}

^g/ml

^90.6

^0.2%

^ABTS

^.

^{vitamin C}

^,

^.

조제된 브로콜리 시료들을 대상으로 다양한 농도에서

DPPH

^,

탄올 추출물 경우

⁵⁰⁰

^g/ml

^15.15%

17.18%

^,

ethylacetate

⁵⁰⁰

^g/ml

^65.9

^0.8

^51.0

^3.2%

로우수한

^DPPH

^.

n-butanol

^34.85

^0.69%

^,

^DPPH

(Fig. 1A). ABTS

^,

및줄기추출물

^,

^DPPH

우수한활성을나타내었으며

^,

(Fig. 1B).

^ethanol Table 1. The yields of ethanol extraction and organic solvent fractions of broccoli and their components analysis of extracts and fractions.

Samples Extract/fraction Yield (%)

Content (mg/g) Total flavonoid

(RE)

Total polyphenol (TE)

Total sugar (SE)

Reducing sugar (GE)

Stalk ethanol extract 4.13 2.76 ± 0.00

16.87 ± 0.35

189.51 ± 2.38

172.96 ± 2.61

n-hexane fr.

2.69 4.82 ± 0.48

22.37 ± 0.92

44.90 ± 0.69

20.65 ± 0.82

ethylacetate fr. 1.05 9.45 ± 1.05

37.47 ± 0.79

39.99 ± 0.18

12.17 ± 0.16

n-butanol fr. 21.87 4.25 ± 0.32

26.45 ± 0.88

258.19 ± 13.16

232.69 ± 3.26

water residue. 74.15 1.62 ± 0.16

7.88 ± 0.48

205.86 ± 1.46

129.95 ± 0.98

Floret ethanol extract 5.35 5.79 ± 0.40

25.34 ± 6.25

128.83 ± 0.09

104.71 ± 0.98

n-hexane fr. 6.94 4.02 ± 0.48

19.92 ± 0.70

31.21 ± 0.91

27.10 ± 0.65

ethylacetate fr. 1.93 42.01 ± 1.21

181.12 ± 2.64

132.84 ± 1.37

51.32 ± 2.61

n-butanol fr. 29.53 11.85 ± 1.70

44.89 ± 0.84

194.61 ± 2.19

165.12 ± 1.30

water residue. 57.61 5.85 ± 0.38

11.74 ± 1.01

123.79 ± 1.64

93.06 ± 0.65

1)

fr.: fraction.

2)

RE: rutin equivalent.

3)

TE: tannic acid equivalent.

4)

SE: sucrose equivalent.

5)

GE: glucsoe equivalent.

6)

Values are means ± SD of triplicate determinations.

Different letters within a column differ significantly (p < 0.05).

추출물

, n-hexane

, ethylacetate

, n-butanol

^ABTS

^IC

^235.4

^g/ml, 190.4

g/ml, 45.6

g/ml, 178.9

^g/ml

^431.8

^g/ml

ABTS

(Table 2).

^{vitamin C}

^ABTS

IC

^4.0

^g/ml

^{. Nitrite}

^,

n-hexane

^nitrite

^,

^n-hexane

ethylacetate

^.

^n-hexane

^nitrite

^IC

^24.1

^g/ml

^,

^{vitamin C}

IC

17.6

^g/ml

^,

^ethanol

, n-hexane

ethylacetate

^nitrite

^IC

^99.1

g/ml, 80.2

^g/ml

Fig. 1. Comparison of antioxidant and nitrite scavenging activities of the ethanol extracts and their organic solvent fractions of

broccoli. Symbols for Vitamin C: , 0 µg/ml; , 3.1 µg/ml; , 6.2 µg/ml; and , 12.5 µg/ml, respectively.

58.9

^g/ml

^nitrite

^(Fig.

1C, Table 2).

시료에서미약한활성을나타내었으나

^,

ethylacetate

n-butanol

⁵⁰⁰

^g/ml

^0.74

0.20

^0.55

^0.02

^(OD

)

Table 2. The radical scavenging activities (IC

s) of the ethanol extracts and their organic solvent fractions of different parts of broccoli.

Samples/

Chemicals Extract./fr.

Radical scavenging activity: IC

(µg/ml)

DPPH ABTS Nitrite

Stalk ethanol extract >500 454.5 ± 5.7

>100

n-hexane fr. >500 >500 24.1 ± 0.8

ethylacetate fr. 489.3 ± 5.2

159.7 ± 11.2

>100

n-butanol fr. >500 448.8 ± 3.2

>100

water residue. >500 >500 >100

Floret ethanol extract >500 235.4 ± 5.2

99.1 ± 3.1

n-hexane fr. >500 190.4 ± 4.1

80.2 ± 2.2

ethylacetate fr. 214.7 ± 1.9

45.6 ± 1.5

58.9 ± 3.0

n-butanol fr. >500 178.9 ± 7.5

>100

water residue. >500 431.8 ± 5.2

>100

Vitamin C - 15.2 4.0 17.6

1)

fr.: fraction.

Different letters within a column differ significantly (p < 0.05).

Table 3. Antimicrobial activity of the ethanol extracts and their organic solvent fractions of different parts of broccoli against pathogenic and food-spoilage bacteria and fungi.

Samples/

Chemicals Extract/fr.

Growth inhibition zone (mm)

Gram positive Gram negative Fungi

SA

LM BS EC PA PV ST CA SC

Stalk ethanol extract 8.0 -

8.0 - - - - - -

n-hexane fr. 14.0 - 9.0 - - 8.0 - - -

ethylacetate fr. - - - - - - - - -

n-butanol fr. 9.0 - 8.0 - - - - - -

water residue. - - - - - - - - -

Floret ethanol extract - - 9.0 - - - - - -

n-hexane fr. - - 8.0 - - - - - -

ethylacetate fr. 11.0 10.0 13.0 15.0 12.0 18.0 13.0 - -

n-butanol fr. - 8.0 10.0 - - - - - -

water residue. - - - - - - - - -

Ampicillin - 25.0 24.0 26.0 10.0 13.0 38.0 16.0 - -

Miconazole - - - - - - - - 20.0 21.0

1)

fr.: fraction.

2)

SA: Staphylococcus aureus, LM: Listeria monocytogenes, BS: Bacillus subtilis, EC: Escherichia coli, PA: Pseudomonas aeruginosa, PV: Proteus vulgaris, ST: Salmonella typhimurium, CA: Candida albicans, SC: Saccharomyces cerevisiae.

3)

-: No inhibition.

The concentrations of the extract/fractions and antibiotics used were 500 µg/disc and 1 µg/disc, respectively. The growth inhi-

bition zone expressed was included size of disc-paper (6.5 mm of diameter). The data represent a classical result of three

independent determinations.

원력을나타내었다

^.

^,

ethylacetate

^DPPH

거능

^{, ABTS}

한활성을나타내었으며

^{, nitrite}

n-hexane

^.

^,

^,

^.

브로콜리 줄기 및 꽃송이의 항균활성 비교

브로콜리줄기및꽃송이의에탄올추출물및이의순차

적유기용매분획물의항균활성평가결과는

^{Table 3}

타내었다

^.

ampicillin

miconazole

¹

^g/disc

^,

^.

및 꽃

^-

^Bacillus

amyloliquefaciens

^, Staphylococcus aureus

^[13]

^,

⁵⁰⁰

^g/disc

Bacillus subtilis

^.

^n- hexane

n-butanol

Staphylococcus aureus

Bacillus subtilis

^,

^.

ethylacetate

^,

ethylacetate

^.

ethylacetate

sulforaphane

isothiocyanate

, sulforaphane

^spectrum

^,

ethylacetate

^.

브로콜리 줄기 및 꽃송이의 대장암세포 생육억제 활성 브로콜리줄기및꽃송이의에탄올추출물및이의순차 적유기용매분획물들의인간대장암세포생육억제활성결

과는

^{Table 4}

이추출물

^{(1 mg/ml)}

^77%

^,

^6.9-86%

^.

n-hexane

ethylacetate

었으며

^,

n-hexane

^18.4%

를나타내어가장강력한활성이나타났으나

^,

n-butanol

^.

sulforaphane

isothiocyanate

n-hexane

ethylacetate

^[25],

이의

n-butanol

세포성장억제물질이존재하리라예측된다

^.

브로콜리 줄기 및 꽃송이의 유용성분과 생리활성과의 상관관계 브로콜리줄기 및꽃송이의추출물및분획물들의

^total polyphenol

total flavonoid

^,

^{Table 5}

^.

ethylacetate

^.

^,

^DPPH

^ABTS

total polyphenol

^total flavonoid

0.846-0.912

으며

^,

^DPPH

서

total polyphenol

total flavonoid

0.858-0.936

^.

Table 4. Anti-proliferative activity of the ethanol extracts and their organic solvent fractions of different parts of broccoli against HCT-116 human cancer cell line.

Samples Extract/fr.

Relative cell proliferation (%)

DMSO - 100.0 ± 5.2

Stalk ethanol extract 77.9 ± 3.0

n-hexane fr. 18.4 ± 1.2

ethylacetate fr. 23.0 ± 2.5

n-butanol fr. 86.1 ± 1.6

water residue. 82.5 ± 2.6

Floret ethanol extract 77.0 ± 7.5

n-hexane fr. 22.5 ± 3.9

ethylacetate fr. 30.0 ± 1.9

n-butanol fr. 6.9 ± 2.3

water residue 77.5 ± 6.5

1)

fr.: fraction.

2)

Values are means ± SD of triplicate determinations.

The samples (1 mg/ml) were used to treat into HCT-116

human colorectal cell in 96 well microplate for 24 h, respec-

tively, and the cell proliferation was determined using One-

solution cell proliferation assay kit. Different letters within a

column differ significantly (p < 0.05).

었다

^.

^nitrite

^{, nitrite}

polyphenol

^.

^,

ethylacetate

n-butanol

^nitrite

의

n-hexane

필요하며

^,

^.

^.

요 약

본연구에서는브로콜리의비가식부위의효율적인이용 및브로콜리의가공식품개발연구의일환으로브로콜리의 주요가식부위인꽃송이와비가식부위인줄기부분을대상 으로각각에탄올추출물및이들의순차적유기용매분획 물을조제하여이들의유용성분분석및항산화

^,

^,

암세포생육억제능을평가하였다

^.

^,

^,

1.50-1.99

^.

ethylacetate

^{9.45 mg/g}

42.01 mg/g

^,

ethylacetate

181.12 mg/g

^.

^,

ethylacetate

^DPPH

거능

^{, ABTS}

^,

nitrite

^n-hexane

^.

^,

ethylacetate

^.

^,

n-hexane

ethylacetate

며

^,

^n-hexane

^18.4

^1.2%,

n-butanol

^6.9

^2.3%

Table 5. Correlation analysis between the content of total polyphenol and bioactivity or between the content of total flavonoid and bioactivity of different parts of broccoli .

Samples X Y Calculation equation

(X versus Y) Correlation coefficient

Stalk Total polyphenol DPPH SA

Y = 0.227X + 9.10 0.912

ABTS SA Y = 0.147X + 45.5 0.876

Reducing power Y = 2.839X + 0.15 0.200

Nitrite SA Y = -0.063X + 49.5 0.221

Anti-Proliferation Y = -1.813X + 97.8 0.348

Total flavonoid DPPH SA Y = 0.977X + 8.17 0.891

ABTS SA Y = 0.638X + 44.9 0.846

Reducing power Y = 1.378X + 0.15 0.241

Nitrite SA Y = -0.316X + 50.3 0.283

Anti-Proliferation Y = -8.286X + 95.5 0.539

Floret Total polyphenol DPPH SA Y = 0.309X + 11.23 0.905

ABTS SA Y = 0.080X + 69.3 0.262

Reducing power Y = 2.306X + 0.15 0.858

Nitrite SA Y = -0.227X + 9.10 0.578

Anti-Proliferation Y = -0.153X + 51.4 0.110

Total flavonoid DPPH SA Y = 1.389X + 9.41 0.936

ABTS SA Y = 0.335X + 69.2 0.232

Reducing power Y = 0.010X + 0.13 0.876

Nitrite SA Y = 0.552X + 43.8 0.490

Anti-Proliferation Y = -0.628X + 51.5 0.095

1)

SA : scavenging activity .