ORYZA OIL & FAT CHEM ICAL CO., LTD.

WALNUT POLYPHENOL

Hepatoprotective & Anti-oxidative Extract
For Metabolic Syndrome

■

WALNUT POLYPHENOL-P10,P30

(Powder，Food Grade)

■■■■

WALNUT POLYPHENOL-WSP10

(Water-soluble Powder，Food Grade)

WALNUT POLYPHENOL-PC10,PC30

（Powder，Cosmetic Grade）

WALNUT POLYPHENOL-WSPC10

（Water-soluble Powder，Cosmetic Grade）

WALNUT POLYPHENOL-LC

（Water-soluble Liquid，Cosmetic Grade）

■

WALNUT SEED OIL

（Oil，Food&CosmeticGrade）

ORYZA OIL & FAT CHEMICAL CO., LTD

ver. 1.0 HS
W ALNUT POLYPHENOL ver.1.0 HS

WALNUT POLYPHENOL

Hepatoprotective & Anti-oxidative Extract For Metabolic Syndrome

1. Introduction

Recently, there is an increased awareness on metabolic syndrome – a condition characterized by a group of metabolic risk factors in one person. They include abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, insulin resistance, prothrombotic state & proinflammatory state. The dominant underlying risk factors appear to be abdominal obesity and insulin resistance. In addition, non-alcoholic fatty liver disease (NAFLD) is the most commonly associated “liver” manifestation of metabolic syndrome which can progress to advance liver disease (e.g. cirrhosis) with associated morbidity and mortality. Lifestyle therapies such as weight loss significantly improve all aspects of metabolic syndrome, as well as reducing progression of NAFLD and cardiovascular mortality.
Walnut (Juglans regia L. seed) is one the most popular nuts consumed in the world.
It is loaded in polyunsaturated fatty acids – linoleic acid (LA), oleic acid and α-linolenic acid (ALA), an ω3 fatty acid. It has been used since ancient times and epidemiological studies have revealed that incorporating walnuts in a healthy diet reduces the risk of cardiovascular diseases. Recent investigations reported that walnut diet improves the function of blood vessels and lower serum cholesterol. Nevertheless, walnut is rich in micronutrients (vitamins & minerals), plant sterols and polyphenols.
A joint research project was conducted between Oryza Oil & Fat Chemical Co., Ltd. and Pola Chemical Industries, Inc. to study the physiological effects of testa of walnut. Various hydrolysable polyphenolic compounds were identified which are potent antioxidants and liver protective. Correspondingly, Walnut Polyphenol is believed to be beneficial in the treatment of metabolic syndrome.

Outer pericarp

Seed

Seed coat

Endocarp

Fig. 1. Walnuts（Juglans regia L.）

1

W ALNUT POLYPHENOL ver.1.0 HS

2. WALNUT POLYPHENOLS

Most polyphenolic compounds of walnut are loaded in the testa of walnut fruit as illustrated in Fig. 2. These are hydrolysable polyphenolic compounds as shown in Fig. 3 and its principle constituents are as listed in Table 1.

Table 1. Principle Polyphenolic

Total polyphenol （%）

Constituents and Their Respective Distribution in Walnut

Seed coats

(%)

Seeds

Pedunculagin (1) Ellagic acid (5)

Tellimagrandin I (6)

Casuarictin (7)

16.0 15.8
6.6

Dried whole seeds
Whole seeds

• 0
• 1
• 2
• 3
• 4

4.1
Tellimagranin II (10)

Rugosin C (11) Casuarinin (12)

Fukuda T. et al., BioFactors, 21,

251-253 (2004).
1.2 1.8 1.0
Fig. 2. The Content of Polyphenols in Walnut (%) （Fukuda T. Polyphenols from walnuts: Structures and functions. IFT 2006 Annual Meeting, Orland, Florida, June 24-28, 2006）

• OH
• HO CH₂

O

O
HO
HO
OH

• O
• OH

OH
HO CH₂
OH
HO

• O
• HO

HO
O

• OC
• CH₂
• CO
• HO

OC

HO

• OH
• O
• O
• OC

OH
OO
O

• OC
• HO

HO
O

• OC
• CH₂

OH OH OH

• O
• OH
• O

CO

• OH
• HO

HO

• O
• O

• OC
• OC

HO

• HO
• O OC
• HO

CO

• OH
• OH

OC

• OH
• HO

HO
OH
HO

2,3-O-(S)-HHDP-D- glucopyranose (2)

OH

Strictinin (4)

OH
HO HO
OH OH
OH
HO OH

Isostrictinin (3)

OH
HO

Pedunculagin (1)

OH
OH

• OH
• OH
• HO

OH
OH

OH
HO HO

• O
• OC

• CH₂
• HO

HO

• O
• O

O
OC

• O
• OH

OH OH
CO
HO

HO

• O
• O
• OC

O
OH OH
CH₂
HO

HO
O
OC
CH₂

• O
• OH

OH
O

• HO
• OC

• O
• OC
• O

O

• O
• OC

O OC

OH

• O
• H

• OC
• OH

HO
O
HO

HO
O

• HO
• OH

HO
CO

• CO
• OH

• OC
• OH
• HO

OH OH
OH
O

• OC
• OH
• OH

OH
O
HO

HO
HO HO

Tellimagrandin I (6)
Ellagic acid (5)

• HO
• OH

• OH
• HO
• OH

OH

OH
OH

Casuarictin (7)
Stenophyllanin A (8)

HO
HO
OH OH OH
OH OH OH
HO HO

• O
• OC
• O

HO HO

• OC
• CH₂
• OH

• CH₂
• OH

OH
OO

• O
• O

O
HO

• OC
• O
• O
• OC
• OC
• O OC

OH

OH OH

• O
• O

HO HO

• O
• OH

O
OC

• HO
• HOOC
• CH₂
• HO
• OC

O
OC
OC
OH

OH

• OC
• OH

OH
OO
OC

• OC
• OH

HO
OH H
HO

• O
• HO
• OH

• HO
• OH

• OH
• CO

OH

• OH
• OH

OH OH
HO HO

• O
• OH
• OC

• CH₂
• OC
• OH

O

O
OO
OC

Tellimagrandin II (10)

O OC OH

1,2,3,4,6-Penta-O-galloyl-β- D-glucopyranose (9)

HO HO
OH
HO

• OH
• CO
• HO

OH

• OC
• OH

HO HO

Casuarinin (12)

• OH
• HO

OH

Rugosin C (11)

Fig. 3. Chemical Structures of Hydrolysable Polyhenolic Compounds of Walnut
2

W ALNUT POLYPHENOL ver.1.0 HS

SOD like activity

SOD様活性

Casuarinin (12)

Csuarinin (12)

54.0

EC⁵⁰ (µg/mL)

Rugosin C (11)

50.0

Tellimagrandin II (10)

89.0

1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose (9)
Stenophyllanin A (8)

60.0
43.0

Casuarictin (7)

73.0

Tellimagrandin I (6)

42.0

Ellagic acid (5)

28.1

Strictinin (4)

31.0

Isostrictinin (3)

30.0

2,3-O-(HHDP)-D-glucopyranose (2)
Pedunculagin (1)

80.1
50.0

クルミポリフェノール

WALNUT POLYPHENOL

29.5

• 0
• 20
• 40
• 60
• 80
• 100

DPPH radical scavenging activity

DPPHラジカル消去能

Casuarinin (12)

Csuarinin (12)

0.7

50

(µg/mL)

EC
Rugosin C (11)

0.4 0.4

Tellimagrandin II (10)
1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose (9)
Stenophyllanin A (8)

1.0
0.5
0.4
0.6

Casuarictin (7)
Tellimagrandin I (6)
Ellagic acid (5)

1.2

Strictinin (4)

1.7

Isostrictinin (3)

1.1

2,3-O-(HHDP)-D-glucopyranose (2)
Pedunculagin (1)

2.1
3.7
26.2

WALNUT POLYPHENOL

クルミポリフェノール

• 0
• 1
• 2
• 3
• 4
• 5

• Fig. 4.
• (top) SOD Antioxidant Activity of Walnut Polyphenols

(bottom) DPPH Antioxidant Activity of Walnut Polyphenols
Fukuda T., et al., Antioxidative polyphenols from walnuts (Juglans regia L.).

Phytochemistry, 63, 795-801 (2003).

EC₅₀: 50% effective concentration of sample
3

W ALNUT POLYPHENOL ver.1.0 HS

3. Hepatoprotective Effect of WALNUT POLYPHENOLS

( I ) In vivo

Liver is the second largest organ in the human body. It plays a major role in metabolism of human body including glycogen storage, plasma protein synthesis and drug detoxification. It regulates a wide variety of high volume biochemical reactions and breaks down toxic substances (e.g. chemicals, viruses, alcohol etc) which may result in toxication (Fig. 5).

Fig. 5. Liver Functions and The Effect of Walnut Polyphenols on Liver Health

( a ) The effect of Walnut Polyphenols on CCl₄-induced liver damage in mice model
(detoxification)

The effect of Walnut Polyphenols on CCl₄-induced liver damage was studied. This

model is a simulated human hepatitis model as a result of oxidative stress and

toxication. Upon liver damage, the liver enzymes GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase) infiltrate into the blood stream resulting in elevated serum GOT & GPT levels. Walnut Polyphenols demonstrated a dose dependent lowering effect in GOT & GPT in CCl₄-induced liver damage in mice model (Fig. 6). Meanwhile, Walnut Polyphenols is more superior than Curcumin, a commonly used hepatoprotective agent.

10000

GOT

9000

GPT

8000 7000 6000 5000

*
**

4000 3000 2000 1000
0

*
*

**

• Control
• 100
• 200
• Curcumin 200

Dose （mg/kg）

Fig. 6. The Effect of Walnut Polyphenols on CCl₄-induced liver damage in mice model（n=8, mean±S.E., *: p<0.05, **: p<0.01）

4

W ALNUT POLYPHENOL ver.1.0 HS

【Method of Experiment】
Fasting mice (ddY, male, 5-wk old) were orally fed Walnut Polyphenols. One hour later, back of mice were subcutaneously injected with 10% CCl₄ previously diluted in olive oil (5ml/kg). Blood samples were collected 20 hours later for measurement of serum GOT and GPT.

( b ) The Effect of Walnut Polyphenols on D-galactosamine–induced liver damage in mice (detoxification)