in vitro the antioxidant activity of lignophenol from
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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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