, 43(5), 437-444, 2010

Kor J Fish Aquat Sci 43(5), 437-444

Nannochloropsis
oculata

ACE,
-glucosidase

Tatsuya Oda
1

ACE, -Glucosidase and Cancer Cell Growth

Inhibitory Activities of Extracts and Fractions
from Marine Microalgae, Nannochloropsis oculata
Seon-Heui Cha, Min-Joo Kim, Hye-Young Yang, Chang-Beum Jin,
You-Jin Jeon1, Tatsuya Oda2 and Daekyung Kim*
2

Marine
Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Korea
1
School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Korea
Division of Biochemistry, Faculty of fisheries, Nagasaki University, Nagasaki 852-8521, Japan

Extracts of the marine microalgae Nannochloropsis oculata were obtained using 80% methanol (MeOH)
and water. The 80% MeOH extract was further fractionated with n-hexane, chloroform, ethyl acetate
(EtOAc), n-butanol (n-BuOH), and water to isolate the active fraction. Seven samples were prepared and
their angiotensin
converting enzyme (ACE), -glucosidase, and cancer cell growth inhibitory activities in vitro were
determined.
The most effects.
profoundByACE
inhibitory
activity
was observed
in theactivity
chloroform
the others
had
moderate
contrast,
greater
-glucosidase
inhibitory
was fraction,
found in while
the EtOAc
fraction,
n-hexane fraction, and water extract of N. oculata. The antiproliferative effects of the extracts and fractions
against HL-60, U937, CT-26, and SK-Hep1 cancer cells were also determined. The n-BuOH fraction had
the strongest antiproliferative effects on CT-26 cells in a time-dependant manner (P<0.05). These results
suggest that the extracts and fractions from N. oculata could be used as a potential functional food or
as pharmaceutical ingredients.
Key words: Microalgae, Nannochloropsis oculata, Angiotensin converting enzyme (ACE), -glucosidase, Cancer

Pendahuluan

.


(Frohlich, 1982).
angiotensin (angiotensin converting
enzyme, ACE) protease
(Miyoshi et al., 1991). Angiotensin 10
angiotensinogen renin
, ACE C dipeptide
(His-Leu) 8
angiotensin (Curtiss et al., 1978; Dzau, 2001).
angiotensin
, aldesterone
sodium .
ACE ,
bradykinin

.
.

, , , , , ,
. ,

.
1520%
, ,
Corresponding author: dkim@kbsi.re.kr

437

438

Tatsuya Oda

(Cushman and Cheung, 1971;

Sealey and Larahg, 1990). ACE
ACE
alacepril, benazepril, captopril, cilazapril, enalapril,
fosinopril, lisinopril, moexipril, perindopril, quinapril,
ramipril,
tandolapril zofenopril (Kato
and Suzuko, 1971; Atkinson and Rovertson, 1979,
Sawayama et al., 1990).
, (Ondetti,
1977)
ACE .
, ACE
captopril
,

ACE
.

,
,
,
, ()
. ,

,

,
,
ACE

Nannochloropsis oculata .
,
, , ,

10% ,
(Woo et al., 1998; Choi et al., 2008; DeFronzo,
1992). -glucosidase

brush border

maltose, sucrose, glucoamylase

-glucosidase ,

(Del Prato et al., 2007; Van de Laar et al.,
2009). -glucosidase acarbose,
voglibose ,

,

(Tsujimoto et al.,
2008).
,

.
, doxorubicin paclitaxel (taxol)
.

.
(algae)
. ,
,
,
,
1
(Choi
et al., 2000).
,
, ,
.


.
50% ,
-linolenic acid (GLA), phycocyanin, myxoxanthophyll,
zeaxanthin
.
.

. docosahexaenoic acid (DHA),
eicosapentaenoic acid (EPA)
(long-chain
polyunsaturated fatty acids, LCPUFAs) .
EPA 20 52 (20:5) LCPUFA.
Nannochloropsis, Nitzschia, Navicula, Porphyridium
EPA . EPA
, EPA .
,
toxin
.

Nannochloropsis oculata

Dulbecco's Modified Eagle's Medium

(DMEM), Fetal bovine serum (FBS), penicillin-streptomycin
Trypsin-EDTA Gibco/BRL (Gaitherburg, MD, USA)
. Bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-

Nannochloropsis oculata

2-yl)-2,5-diphenyltetrazolium bromide (MTT), glucosidase, p-nitrophenyl--D-glucopyranoside, DMSO

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

f/2-Si
1 L NaNO3 (75 mg), NaH2PO4H2O (5 mg),
FeCl36H2O (3.15 mg), Na2EDTA2H2O (4.36 mg),
MnCl2
4H2O (0.18 mg), CoCl26H2O (0.01 mg), CuSO45H2O (9.8
g),
Na2MoO42H2O (6.3 g), ZnSO47H2O (22 g), B12 (0.5
g),
biotin (0.5 g), thiamine HCl (0.1 mg)
,
pH 8 12115
.

f/2-Si 26, 60% , (50

2
mol/m /s) 12 . ,
f/2-Si 100 mL, 500 mL
.

439

,
. 4 80%
methanol (MeOH)
(rotary vacuum
evaporator, N-1000, EYELA, Japan) 40
. MeOH 10 80%
MeOH n-hexane n-hexane
80% MeOH . 80% MeOH
chloroform
chloroform . EtOAC n-BuOH
.
80% MeOH,
n-hexane, chloroform, EtOAC, n-BuOH

(Fig. 1).

ACE
ACE Cushman and Cheung (1971)
hippuryl-L-histidyl-L-leucine
(Hip-His-Leu) ACE

hippuric acid
.
300 mM NaCl (pH 8.35)

100 mM sodium borate buffer 5 mM Hip-His-Leu
, 80 L 37
3
ACE (100 mU/mL) 20 L 30

. 1 M
HCl 250 L
.

(3 L) 4, 4,500 rpm 30
. 3
5

30

Fig. 1. The scheme for solvent fraction of Nannochloropsis oculata.

440

Tatsuya Oda

hippuric acid 1.7 mL ethyl acetate

(EtOAc) , (800 g, 15)
1 mL microtube 40
(VS802, Vision, Korea) EtOAc
1 mL
.
ACE

228 nm
,
3

SPSS (SPSS Inc., Version 12.0)

, P<0.05

analysis of variance Duncan's multiple range test

.

Alpha-glucosidase

1520%

, ,
.






.
N. oculata ACE
, chloroform

. Hip-His-Leu

in
vitroACE , N.
oculata chloroform 500 g/mL
60% .

n-hexane, chloroform, ethyl acetate (EtOAc) n-buthanol

(n-BuOH)
n-hexane
500 g/mL
40% ,

30%,

25%
, EtOAC
, n-BuOH

20%

(Fig. 2).

Alpha-glucosidase Watanabe et al. (1997)

, -glucosidase

, p-nitrophenyl--D-glucopyranoside
. Alpha-glucosidase 0.2% BSA 0.02% NaN3
100 mM phosphate buffer (pH 7.0) 0.7 g/mL

, p-nitrophenyl-
-D-glucopyranoside 100 mM phosphate buffer (pH 7.0) 5
mM . DMSO
sample microplate 10 L
, DMSO
10 L . Sample microplate 50
L , 5
TM
microplate reader (Packard spectrocount ,
Meriden, CT, Austria) 405 nm .
50 L
5 405 nm
.

.
3

.

ACE

DMEM 10% FBS, 100 units/ml penicillin
100 g streptomycin
37
CO2 incubator (5% CO2) .
80% phosphate-buffered saline (PBS, pH 7.4)
0.25% trypsin-2.65 mM EDTA

2 .

HL60, U937, CT-26

SK-Hep1
5
10% FBS

110 cells/well
96 well plate
. 16 1% FBS

(500
g/mL)
0, 1, 2, 3 MTT assay
(Denizot and Lang, 1986) .
3
.

meanSD .

Fig. 2. Angiotensin converting enzyme (ACE) inhibitory

activity of Nannochloropsis oculata extracts and fractions.
The results are presented as meansS.D. of
three
independent a,b,c
experiments.
Values having different superscripts are
significantly different at P<0.05.

10% . Cha et
al. (2006)
19,
10,
26

(Myagropsis myagroides), (Shige sinicola),

Nannochloropsis oculata

(Petrospongium rugosum), (Hizikia fusiforme),

(Undaria pinnatifida), (Laminaria ochotensis)
,
(Enteromopha linza),
(E. compressa) ,

(Lithophyllum okamurai), (Gracilaria textorii),

(Ahnfeltiopsis flabelliformis),
(Lomentaria
catenata), (Laurencia okamurae),
(Grateloupia lanceolate), (Gracilaria verrucosa)
. Athukoral and Jeon (2005)
(E. cava), (Ishige okamurai), (Sargassum
fulvellum), (S. horneri), (S.
coreanum), (S. thunbergii) (Scytosiphon
lomentaria) 7


, 7

.

.
AEC
,
.
,
ACE
. ,
ACE .

ACE

. N. oculata
ACE
chloroform n-hexane

-glucosidase
-glucosidase

,

.
-glucosidase
brush border

, ,

.
-glucosidase
acarbose voglibose

.
-glucosidase ,

, ,

.
N. oculata
-glucosidase ,
EtOAc
-glucosidase
. p-nitrophenyl-

441

-D-glucopyranoside
in vitro
-glucosidase , N. oculata EtOAc 500
g/mL
63% . nhexane, chloroform, EtOAc
n-BuOH

n-hexane
500 g/mL 55% 48% ,

40%, n-BuOH 20%


(Fig. 3).

Fig. 3. Alpha-glucosidase inhibitory activity of

Nanno- chloropsis oculata extracts and fractions. The
results are presented as meansS.D. of three independent
experiments.
a,b,c
Values having different superscripts are significantly
different at P<0.05.

, , ,
, ,

.
, ,
. aldose reductase
Lee et al. (2004)
in vivo
.
,

, , , ,

,

.


. N. oculata -glucosidase
EtOAc EtOAc
.

442

Tatsuya Oda

Fig. 4. Antiproliferative effect of Nannochloropsis oculata extracts and fraction on HL-60, U937, CT-26 and SK-Hep1
cell lines. The results are presented as meansS.D. of three independent experiments.

(Matsuda et al., 2010) (Qwak et al., 2006)

.

,
.


. ,


,

(Reddy et al., 2003; Doll and Peto,
1981; Sharma et al., 1994).
(Schwartsmann et al., 2001)
,

. carotinoids (Krinsky,
1991)
mycosporine-glycine
(Dunlap and Yamamoto, 1995), , .

,
phytochemicals
,


nitrosoamine


(Chiharu et al.,
1992).

sulfated polysaccharide
fucoidan sarcoma-180, L-1210,
Meth-A, B-16 melanoma cell
,
sarcoma-180 cell
(Ryu et
al., 1989).
N. oculata
4

N. oculata
24, 48 72 MTT assay

Nannochloropsis oculata

,
HL-60 (human
promyelocytic leukemia cell), U937 (human leukemic
monocyte
lymphoma cell) , SK-Hep1
(human hepatocellular carcinoma cell)
, CT-26
(murine colon carcinoma cell)
. , n-BuOH
34% 66%
. 40%
(Fig. 4). N. oculata

, n-BuOH



N.
oculata
.

(JDC)

, .

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