共役ポリエン脂肪酸の特異的な生理作用とその作用機構

宮澤陽夫

（東北大学大学院農学研究科）

Chemical structures of LA, CLA, LnA and CLnA.

COOH9

11

Conjugated linolenic acidα-Eleostearic acid

(n-5, 18:3, c9t11t13)

COOH9

1113

α-Linolenic acid(n-3, 18:3, c9c12c15)

COOH9

12

15

Linoleic acid(n-6, 18:2, c9c12)

Conjugated linoleic acid (n-7, 18:2, c9t11, CLA)

in dairy products

COOH9

12

in Safflower oil

in Perilla oilin Tung oil

Chemical structures of conjugated fatty acids found in plant seed oil and algae.

α-Parinaric acid

COOH

(n-6, 20:5, c5c8t10t12c14)

Bosseopentaenoic acid(n-5, 18:4, c9t11t13c15)

in seed oil, Parinarium laurinum in red algae, Bassiella orbignana

COOH

Stellaheptaenoic acidConjugated EPA(n-3, 22:7, c4c7t9t11c13c16c19)

in green algae, Anadyonene stellata(n-3, 20:5, c5t7t9c12c17)in red algae, Plilota filicina

COOH

COOH

Conjugated Fatty Acids Found in Natural Resources

Structure Trivial name Location (Species)

2t , 4c -Decadienoic acid (10:2) Stillingic acid Seed oil ( Spapium sebiferum )3t , 5c -Tetradecadienoic acid (14:2) Megatomoic acid Female black carpet beetle

(Attagenus megatoma)

7t , 9c -Octadecadienoic acid (18:2)9c , 11t -Octadecadienoic acid (18:2)10t, 12c-Octadecadienoic acid (18:2)9t , 11 t -Octadecadienoic acid (18:2)10t , 12 t -Octadecadienoic acid (18:2)

Dairy products, Human milkDairy productsDairy productsDairy productsDairy products, Seed oil

(Chilopsis linearis)Seed oil ( Calendula officinalis )Seed oil ( Jacaranda mimosifoliaTung oil (Aleurites fordii )Tung oil (Aleurites fordii )Seed oil ( Catalpa ovata )Seed oil ( Punica granatum )

8t , 10 t , 12c -Octadecatrienoic acid (18:3)8c , 10t , 12c -Octadecatrienoic acid (18:3)9c , 11t , 13ｔ -Octadecatrienoic acid (18:3)9t , 11 t , 13ｔ -Octadecatrienoic acid (18:3)9t , 11 t , 13c -Octadecatrienoic acid (18:3)9c , 11t , 13c -Octadecatrienoic acid (18:3)

Calendic acidJacaric acidα-Eleostearic acidβ-Eleostearic acidCatalpic acidPunica acid

)

9c, 11 t , 13ｔ , 15c -Octadecatetraenoic acid (18:4) Parinarium laurinumParinaric acid Seed oil ( )

5c , 8c , 10t , 12t , 14c -Eicosapentaenoic acid (20:5)5c , 7t , 9t , 14c , 17c -Eicosapentaenoic acid (20:5)5t , 7t , 9t , 14c , 17c -Eicosapentaenoic acid (20:5)

Red algae ( Bassiella orbignianaRed algae ( Plilota filicina )Red algae ( Plilota filicina )

Basseopentaenoic acid )

4c , 7c , 9t , 11 t , 13c , 16c , 19c -Docosaheptaenoic acid (22:7) Stellaheptaenoic acid Green alge ( Anadyomene stellata )

Contents

1. Oxidation of conjugated fatty acids and its prevention

2. Metabolic conversion to CLA from conjugated trienoicfatty acids

3. Anti-cancer action of CEPA

4. Anti-angiogenic activity of CEPA and CDHA

5. Anti-obesity activity of conjugated PUFA

Oxidation of Conjugated and Nonconjugated PUFA.

18:2

18:3

20:5

22:6

18:2

22:620:5

18:3

CLACLnA

CDHA

CEPA

0

20

40

60

80

100

0 6 12 18 24

LA

CLA

LnA

CLnA

EPA

CEPA

DHA

CDHA

37℃Thin-film in test tube

Unoxi

diz

ed s

ubst

rate（％）

(n=3-6)Time（hr）

(n=3-6)days

Oxidation of Conjugated and Nonconjugated PUFA.

37℃Thin-film in test tube

Uno

xidi

zed

subs

trate（％）

18:2-TG

CLA-TG18:3-TGCLnA-TG

18:2-TG + VE

18:3-TG + VE

CLA-TG + VE

CLnA-TG + VE

CLnA-TG = Tung oil18:3-TG = Perilla oil18:2-TG = Safflower oil

+0.1% VE

0

20

40

60

80

100

0 2 4 6 8 10 12 14

300200 250150

333290

276262248234

222208

182

196

113

126100

0

M+

12mu 12mu

Relative %

182196

208

222

234

248 262

O

N

m/z

9, 11-Octadecadienoic acid (CLA) M+=333

GC of Liver Fatty Acid Methyl Esters &GC-MS 0f CLADimethyloxazoline Derivative

Fatty acid methyl esters in the liver of rat fad ESA diet.

0 20 40 60Retention Time (min)

Fatty acid methyl esters Std mixture.CLA(10t,12c)

18:3

20:4

22:6

20:020:1

α-ESA

β-ESA

CLA(9c,11t / 9t,11c)

Det

ecto

r Res

pons

eStd.

Std.

16:0

17:0

18:0

18:1

18:2

Conclusion

α-Eleostearic acid(CLnA)Conjugated triene

9

11

13

COOHΔ13 saturation reaction

9

11

In vivo

Conjugated linoleic acid(CLA)Conjugated diene

9

11

COOH11

9

These data suggested the existence of delta 13 saturation reaction to form CLA from alpha-eleostearic acid in rodents.

Fluorescence Dye Staining and TUNEL Analysis of DLD-1 Cells Supplemented with CEPA and CDHA

Control CEPA CDHA

Fluorescence dye staining analysis

TUNEL analysis

The cells added with CEPA and CDHA showed nuclea condensation and DNA fragmentation, which are characteristic for the apoptosis.

BBRC 2000

Anticarcinogenic action of conjugated PUFA

Conjugated trienes

CEPA, CDHA

Tumor cells

Growth inhibition by modification of fatty acid

metabolism

Apoptosis via lipid peroxidation

Normal growth

Conjugated dienes

CLA

Nonconjugated acids

M. Igarashi & T. Miyazawa. (2000) Biochem. Biophys. Res. Commun., 270, 649-656.

CEPA privents the growth of transplanted DLD-1 cells in nude mice.

Before

EPA CEPA

After 4 weeks

Control

*

(g)

*

**

CEPA privents the growth of colon cancer cells in nude mice.

0

5

10

15

20

25

30

Control

EPA

CEPAControl

EPA

CEPA

Body w.t. (g) Cancer / b. w. (g / 100g) Liver / b. w. (g / 100g)

*P<0.05 from Control. **P<0.01 from Control. Mean ±SD (n=5)

0

10

20

30

40

50

60

PCOOH PEOOH

ControlEPACEPA

PLO

OH

(nm

ol /

g t

issu

e)

*

CEPA Intake Causes Membrane Phospholipid Hydroperoxidation Leading to Apoptosis of Cancer Cells, Serum and Liver in Mice.

PCOOH PEOOH PCOOH PEOOHGroupSerum Liver

Cancer cells

0.30±0.03 0.01±0.010.29±0.09 0.05±0.010.32±0.04 0.09±0.01

nmol/ml16.1±1.9 11.6±2.117.7±3.4 12.8±3.216.1±3.2 13.7±5.8

nmol/g tissue Mean ±SD (n=5) *P<0.05ControlEPACEPA

Stimulated DNA fragmentation of cancer cells in mice supplemented with CEPA.

0

5

10

15

20

Control EPA CEPA

DN

A f

ragm

enta

tion (

%)

Mean ±SD (n=5) *P<0.05 compared with Control group.Fluorescence method

*

*

CEPAControl After 6 weeks

Anti-Angiogenesis with CEPA?Angiogenesis

Cancer and Angiogenesis

Blood vessel

VEGF, FGF e.t.c.Angiogenesis Factor

Tumor cells

Blood vessel rebirth

Multiplication Promotion Transition

Blood vessel

Angiogenesis

Adipose tissue

Adipocytokine

Leptin

FFA

TNF-α

Insulin resistance

Liver

Fatty acid synthesis

Erasion organization

Glucose metabolism

Blood sugar

Obesity

InsulinDiabetes

Lipids

0

5

10

15

20

LA CLA LnA CLnA EPA CEPA DHA CDHA

Mean±S.D.(n=6-8)*P < 0.05 from other groups.a,b,c, P < 0.05.

Leptin (

ng/m

l)

* *

Leptin

0

0.2

0.4

0.6

0.8

1

LA CLA LnA CLnA EPA CEPA DHA CDHA

Free

Fat

ty A

cids

(mEq/l

)

* *

FFA

0

1

2

3

4

5

6

7

8

LA CLA LnA CLnA EPA CEPA DHA CDHA

TN

F-α

(pg/m

l) a

ab

a

b

bb

cc

TNF-α

Plasma adipocytokine concentration

Adipose tissue

Adipocytokine

Leptin

FFA

TNF-α

Insulin resistance

Liver

Fatty acids synthesis

Erasion organization

Glucose metabolism?

Blood sugar

Anti-Obesity effect of CEPA & CDHA

InsulinDiabetes

CEPACDHA

Triacylglycerol concentration in 3T3-L1 cells.

0

20

40

60

80

100

120

0 2 4 6 8

OμM

EPA 5μM

EPA 10μM

CEPA 5μM

CEPA 10μM

DHA 5μM

DHA 10μM

CDHA 5μM

CDHA 10μM

Mean ±SD (n=10).

Incubation time (day)

Via

ble

cel

ls (

% o

f co

ntr

ol)

Mean ±SD (n=10). *P < 0.05 from Control.

0

100

200

300

400

500

-2 0 2 4 6 8

ControlEPACEPADHACDHA

TG

(m

g/

g p

rote

in)

Incubation time (day)

*

**

*

10μM

総括

・共役PUFA (共役EPA) の生物機能

1. In vitroで酸化されやすい。ビタミンEで効果的に抑制。

2. 共役PUFA (共役トリエン) は癌細胞の脂質過酸化を誘発。

3. 共役EPAは癌細胞にアポトーシスを誘発。

4. 共役EPAは担癌マウスの癌組織を退縮。

5. 共役トリエンの一部は動物体内で共役ジエンに代謝。

6. 共役トリエンに血管新生抑制作用。

7. 共役トリエンに抗肥満作用。