日本食物繊維研究会誌Vol.7No.1(2003)

報 文

In vitro the Antioxidant Activity of Lignophenol from Beech

(Fagus crenata Blume) and Hinoki (Cryptomeria japonica D.Don)

Shuzo FUJITA1, Eriko OHMAE2, and Masamitsu FUNAOKA2

1 Faculty of Health Science, Aomori University of Health and Welfare, CREST JST

2 Faculty of Bioresources, University of Mie, CREST JST

We examined the antioxidant activity of lignin which is a kind of dietary fiber by using a new lignin derivative, 'lignophenol'. Lignophenol is close to natural lignin compared with any other lignin derivatives prepared with the

former methods. Twelve kinds of lignophenols were prepared from beech (Fagus crenata Blume) and hinoki

(Cryptomeria japonica D.Don) by a phase-separative reaction system composed of cresol and sulfuric acid. The

antioxidant activity of lignophenol against the oxidation of linoleic acid was determined by the peroxidative value

(POV) and the thiobarbituric acid (TBA) methods. All lignophenols effectively prevented the oxidation of lipids as

same as BHA, and the lignophenols treated with alkali and acetylated had high activity. The antioxidant activity of

lignophenol from hinoki that has guaiasyl (o-methoxyphenolic) groups tended to be higher than that of beech that has

guaiasyl and syringyl groups.

Key words: lignin, lignin derivative, lignophenol, antioxidant activity

Lignin is a kind of polymer constituting the plant cell

wall together with cellulose and hemicellulose, and also

functions as a dietary fiber. This polymer consists of

hydrocarbons that form a phenylpropane structure with a

side chain of three carbons in the aromatic nucleus,

although most dietary fiber is a carbohydrate. This

polymer contains a methoxyl group, and a polyphenol in

a broad sense with the basic skeleton combined in a

myriad. Lignin has been defined as the polymer

produced by dehydrogenase for p-hydroxy cinnamic

alcohol, and has methoxyl groups showing some

characteristic reactions"'. Lignin is, therefore, a unique

polyphenol polymer that has a function as a dietary fiber.

However, the research on lignin as a dietary fiber has

been limited. Lignin is insoluble and excreted in feces

without being digested by human digestive enzymes,

fermented or decomposed. Eastwood et al2 reported on

the bile acid adsorption property of lignin, and Morgan et

al3) on its hypocholesterolemic activity in blood. Story et

a1.4) examined the absorption of bile acid for dietary

fiber, and reported that bile acid adsorbed in

hydrophobic lignin better. Sakagami5) reported that

intravenous administration of lignin caused preferential

accumulation of lignin in the liver, stomach and lungs. In

these reports it was uncertain whether lignin was

contaminated by carbohydrates such as hemicellulose

during the preparation of lignin, or whether the chemical

structure is decomposed by strong alkali such kraftlignin

and/or acid treatment. In the meantime, Nakagawa6)

investigated the effect of green tea catechin, which

resembles the basic chemical structure on the lignin, on

antioxidant capacity of human plasma, and reported that

drinking green tea contributes to prevent cardiovascular

disease by increasing plasma antioxidant capacity in

humans.

There are several methods of lignin preparation from

1Mase,Hamatate,Aomori-city,Aomori032-8505,Japan(〒030-8505青 森 市 浜 館 字 間瀬58-1)

2Kamihama,Tsu,Mie514-8507,Japan(〒514-8507三 重 県津 市 上 浜 町1515)

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J. Jpn. Assoc. Dietary Fiber Res. Vol. 7 No.1 (2003)

wood products as follows: Dioxane extraction by

grinding according to Bjorkman is often used, but

whether the extract is really lignin is uncertain". The

periodic acid method is also popular, but about 20% of

the phenol nucleus of lignin is cleaved by the oxidation

and converted to quinone8). The method reported by

Klason the lignin is the most popular method of

modifying lignin in which the carbohydrate in the plant

is decomposed and removed by 65-72% sulfuric acid,

and as the result, the structure was polymerized by the

strong acid9). The separation of lignin in its native form is

difficult because it is complicatedly combined with the

cellulose and the other carbohydrates in the plant.

Recently collaborator, Funaoka, suggested `a phase

separation system' which is composed of a phenol and

concentrated acid to convert the native lignin to highly

phenolic and functional polymers10) ,11). He reported that

the chemical structure of the derivative which is called `lignophenol' is close to natural lignin compared with

those prepared with the former methods. In addition, the

stepwise method has been developed to produce a

different size of lignophenol molecule, which is called `a

switching device' (Figure 1). Lignophenol is perfectly

biodegraded in nature, giving no undesirable influence to

the biocycle, although it is one of the most durable

biopolymers. Therefore lignophenol may be used to

establish the recycling system of lignin. Although wood

and woody plants are utilized as products, lignin could

also be utilized effectively by using lignophenols. The

physiological function of natural lignin may also be

clarified by using lignophenol.

The rancidity of a lipid is caused by autoxidation of

lipid molecules by oxygen in the air, being catalyzed by

light or metal. Many compounds harmful to the human

body are produced by the autoxidation and therefore the

antioxidant is used to prevent the oxidation of lipid. a-

tocopherol, butylated hydroxyanisole (BHA), and

butylhydroxy- toluene (BHT) are antioxidants approved

Phenol derivatives

Lignophenols

Figure 1. Several lignophenols derived from lignin by the phase separation system. In this experiment lignocresol was used as

lignophenol.

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日本食物繊維研究会誌　Vol.7No.1 (2003)

for use in Japan, It is suggested that the phenolic

hydroxyl group contained in these compounds is related

to the inhibition of lipid oxidation12). Breddon reported

that both antioxidant and plastic effects on rubber such

as automobile tires could be expected for kraftlignin13)

because lignin has a phenyl ether structure which is

effective for preventing the autoxidation of lipids. This

study aims to obtain the fundamental information on the

antioxidant activity of lignin, which may relate its

physiological function as a dietary fiber.

Materials and methods

1. Preparation of The lignin and Lignophenols

Lignocresol was used in this experiment as

lignophenol. Twelve kinds of lignophenols were

prepared by degreasing wood flour by a Two Step

Methods10,11) (Figure 2). Air-dried beech and hinoki were

successively ground in a Wiley mill and a vibrational

mill to pass an 80 mesh screen, and extracted to remove

the contaminant with ethanol-benzene (1:2, v/v) for 48 h.

p-Cresol (10mL/g wood) in acetone was added to wood

flour with stirring. After standing for few minutes, 72%

sulfuric acid was added to the mixture and vigorously

stirred at room temperature. The reaction mixture was

gradually separated into organic and aqueous phases.

Excess water was added to the organic phase with

stirring. The organic phase was repeatedly rinsed with

water until the pH of the solution became neutral.

The lignophenol was then decomposed into lower

molecular substances by the second functional control

(SFC) by heating after alkali treatment (0.5N sodium

hydroxide). Furthermore some of these products were

acetylated. The acetylation was carried out by addition of

pyridine and acetic anhydride to alkali-treated sample.

After standing for 48 hours, the sample was washed by

adding cold water in several times and freeze-dried. Six

samples prepared from beech lignophenol are as follows;

B-1 (original), B-2 (SFC, heat-treated at 140•Ž), B-3

(SFC, heat-treated at 170•Ž), B-4 (lignophenol-acetate),

B-5 (SFC, heat-treated at 140•Ž and acetylated), B-6

(SFC, heat-treated at 170•Ž and acetylated). Six samples

from hinoki lignophenol were designated H-1 to H-6.

Figure 2. Procedure of preparation of lignophenol by the phase separation system. In this experiment lignocresol was used as

lignophenol.

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J. Jpn. Assoc. Dietary Fiber Res. Vol. 7 No.1 (2003)

BHA (butylated- hydroxyanisol, 2,3-tert- butyl-4-

hydroxy-anisole, Sigma B-1253 ) and kraftlignin were

used as controls.

2. Infrared (IR) analysis

By adding potassium bromide, 1mg of lignophenol

was molded into a tablet form. The sample spectrum

within 4000-900cm-1 was observed with a Shimadzu FT-

IR8400 infrared analyzer.

3. Gel permeation chromatography (GPC) analysis

One mg of lignophenol was completely dissolved in

I mL of tetrahydroxyfuran. GPC was carried out at a flow

rate of 1.0mL/min and 50kg/cm2 pressure on a Shimadzu

LC- 10A analyzer equipped with Shodex-801,802,803

and 804 columns after filtration of the sample through a

COSMONICE membrane filter. The eluent was detected

at 280nm.

Wave numbers (cm-1)

Figure 3. IR Spectra of lignophenol from hinoki.

4. Determination of peroxides

Peroxides from linoleic acid (cis-9-cis-12-

octadecadienoic acid, free type) in 75% ethanol solution,

containing 4mg of sample and 0.1mL of linoleic acid

was measured periodically by a peroxidative value

(POV) and thiobarbituric acid (TBA) methods. By the

POV method, we measured quantitatively lipid

hydroperoxide which was created at the initial stage of

the oxidation and by the TBA method the amount of

carbonyl compounds such as malondialdehyde which

were created by oxidation"'.

Result and Discussion

1. Identification and molecular weight

The infrared analysis was carried out to determine

whether lignin was converted into lignophenol. After the

acetylation, the peak (at 3100-3500cm-1) of phenolic

hydroxyl group of H-6 disappeared and the peak (at

1700cm-1) of the ester was detected (Figure 3). Infrared

analysis of the other samples gave the expected results.

The molecular weights of B-1, B-2, B-3, B-4, B-5 and

B-6 prepared from beech were determined 6,700, 700,

690, 7,400, 700 and 520 by GPC, respectively, and those

of H-1, H-2, H-3, H-4, H-5 and H-6 were 21,000, 1,350,

1,030, 25,000, 1,430 and 1,040, respectively. These

molecular weights are the expected values.

2. Function of lignophenol as antioxidant evaluated

by POV and TBA

The antioxidant activity was determined by adding

4mg sample to the linoleic acid. By the POV method a

strong antioxidant activity such as that of BHA was

detected in the samples except for B-4, and a difference

in the activity among the samples was hardly seen

(Figure 4a, b). By the TBA method no difference was

observed between the samples (Figure 5a, b). These

results showed that lignophenol derivatives have a strong

antioxidant effect similar to BHA in. It is assumed that

the antioxidant mechanism of lignophenol resembled that

of BHA by judging from the chemical structure.

A long-term experiment was carried out to examine

the antioxidant activity of the 12 samples in detail. Two

mg of each sample which is a half quantity of that used

in the previous experiment was added to linoleic acid.

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日本食物繊 維研 究会誌　Vol.7No.1(2003)

Figure 4a. Antioxidant activity of lignophenol from beech

by POV.

Figure 4b. Antioxidant activity of lignophenol from hinoki

by POV.

Figure 5a. Antioxidant activity of lignophenol from beech

by TBA.

Figure 5b. Antioxidant activity of lignophenol from hinoki

by TBA.

Figure 6. Antioxidant activity of lignophenol, kraftlignin and BHA for a long term by POV.

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J. Jpn. Assoc. Dietary Fiber Res. Vol. 7 No.1 (2003)

The experiment was continued for about three months

(Figure 6). The antioxidant activity of advanced

polymerizing B-1, B-4, H-1 and H-4 gradually decreased

with time. The activity of acetylated B-4 and H-4 were

remarkably decreased, while B-1 and H-1 were relatively

effective such as the other samples in inhibiting of

linoleic acid oxidation for a long term. Lignophenol from

beech, a broad-leaved tree (dicotyledonous angiosperm)

tended to have lower antioxidant activity than that from

hinoki which is a conifer (gymnosperm). This is

probably because the lignophenol from broad-leaved

trees is composed of guaiasyl-propane and syringyl-

propane while that from conifers are composed only of

the guaiasyl propane. B-6 and H-6, acetylated after heat

treatment at 170•Ž with alkali, were the most active. It is

considered that the antioxidant activity of lignophenol

against linoleic acid may be expressed by a gradual

formation of a hydroxyl group in lignophenols by

hydrolyzing the ester bond. Kraftlignin was also as

effective as lignophenols and BHA for the autoxidation

of unsaturated oil, though the kraftlignin solution was

dark-brown in color.

•@ Lignophenol which is derived from lignin may be

useful as a dietary fiber, and also as an antioxidant.

Dietary fiber composed of carbohydrates, do not have

such antioxidant activity. Further studies are needed to

obtain the functional information of lignophenol and to

contribute to a research of dietary fiber.

Acknowledgment

•@ We are thankful for the financial support from

CREST JST.

•œ References

1) Nakano J ed: "Chemistry of lignin", pp2-9 (1982)

Uni Press Co., Tokyo, Japan (in Japanese)

2) Eastwood MA and Hamilton D: Studies on the

adsorption of bile salts to non-absorbed components

of diet. Biochim Biophys Acta 152:165-73 (1968).

3) Morgan B, Heald M SD, Atkin SD, Green J, and

Chain EB: Dietary fibre and sterol metabolism in

the rat. Br J Nutr 32:447-455(1974)

4) Story JA and Kritchvsky D: Dietary fiber and lipid

metabolism. " Fiber in Human Nutrition" (Ed. By

Spiller GA and Amen RJ, p180, Plenum Press, New

York (1976)

5) Sakagami H, Asano K, Yoshida T, and Kawazoe Y:

Organ Distribution and Toxicity of Lignin, in vivo.

13:41-44 (1999)

6) Nakagawa K, Ninomiya M, Okubo T, Aoi N, Juneja

LR, Kim M, Yamanaka K, and Miyazawa T: Tea

catechin supplementation increases antioxidant

capacity and prevents phospholipid hydroperoxida

tion in plasma of humans J Agric Food Chem

47:3967-3973 (1999)

7) Nakano J ed: "Chemistry of lignin", pp37-42 (1982)

Uni press Co., Japan

8) Nakano J ed: "Chemistry of lignin". pp43-44 (1982)

Uni press Co., Japan

9) Nakano J ed: "Chemistry of lignin". pp44-46 (1982)

Uni press Co., Japan

10) Funaoka M and Fukatsu S: Characteristics of lignin

structural conversion in a phaseseparative reaction

system composed of cresol and sulfuric acid.

Holzforschung 50:245-252(1996)

11) Funaoka M, Matsubara M, Seki N, and Fukatsu S :

Convertion of native lignin to highly phenolic

functional polymer and its separation from

lignocellulosics. Biotechnology and Bioengineering

46:545-552 (1995)

12) Hudson BJF ed: "Food antioxidants", pp79-85

(1990), Elsevier Sci. Publishers, N.Y.

13) Braddon DV and Falkehag SI: Torsional braid

analysis of the lignin derived rubber stabilizers. J

Polymer Sci. 40:101-104 (1973)

14) Osawa T and Namiki M: A novel type of

antioxidant isolated from leaf wax of Eucaliptus

leaves. Agric Biol Chem 45:735-740 (1981)

(平成15年3月17日 受理)

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日本食物繊 維研究 会誌Vol.7No.1(2003)

ブ ナ(Fagus crenata Blume)お よ び

ヒ ノ キ(Cryptomeria japonica D.Don)か ら 調 製 し た

リグ ノ フ ェ ノ ー ル のin vitroで の 抗 酸 化 性 に つ い て

藤 田修三1,大 前江利子2,舩 岡正光2

1青森県立保健大学健康科学部,CREST JST2三 重大学生物資源学部共生環境学科,CREST JST

　食物繊維である リグニ ンの抗酸化性について、新規 リグニン誘導物質であるリグノフェノールを調製 して評価

した。 この物 質はクラフ トリグニ ン等、既存の リグニ ン誘導体に比べて天然の リグニ ンに極 めて近い立体構造 お

よび性質を有 しているのが特徴である。ブナおよびヒノキの リグニ ンから相分離 システム法 によって調製 された

12種 類の リグノフェノール誘導体についてPOV法 およびTBA法 で抗酸化性 を測定 した。その結果、全ての リ

グノフェノール に合成酸化防止剤BHAに 匹敵する高い抗酸化活性が示 され、さらにはグアイアシル基を有する

ヒノキ リグノフェノールは、グアイアシル基 およびシリンギル基 をもつ ブナ リグノフェノール より抗酸化効果の

高いことがわかった。

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