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Biosci. Biotechnol. Biochem.

, 73 (5), 10601065, 2009

Marker Constituents of the Natural Antioxidant Eucalyptus Leaf Extract


for the Evaluation of Food Additives
Yoshiaki A MAKURA,1; y Morio Y OSHIMURA,1 Naoki S UGIMOTO,2
Takeshi Y AMAZAKI,2 and Takashi Y OSHIDA1
1

College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
Division of Food Additives, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku,
Tokyo 158-8501, Japan
2

Received November 26, 2008; Accepted February 9, 2009; Online Publication, May 7, 2009
[doi:10.1271/bbb.80832]

In order to establish the marker constituents of the


natural antioxidant food-additive Eucalyptus leaf extract, the UV-absorbing constituents of two eucalyptus
leaf extracts registered as food additives (eucalyptus A
and B) were investigated. Several major peaks on the
reversed-phase HPLC chromatogram of eucalyptus A
were characterized as gallic acid, ellagic acid, 3-O- -Dglucuronides of quercetin and kaempferol, and a hydrolyzable tannin dimer, oenothein B, by direct comparison
with authentic specimens isolated from Eucalyptus
globulus leaves. A new gallotannin was found in the
E. globulus leaf extract, and its structure was found
to be 1,2,3,6-tetra-O-galloyl- -D-galactose. Two major
peaks on the HPLC chromatogram of eucalyptus B
were identied as gallic acid and ellagic acid, indicative
of degradation products from hydrolyzable tannins in
the leaves. Considering the evaluation of antioxidant
activity by radical scavenging ability, a standardization
of eucalyptus leaf extract, including the antioxidative
polyphenol, oenothein B, is proposed.
Key words:

natural antioxidant; food additive; eucalyptus; Eucalyptus globulus; polyphenol

Natural antioxidant food additives, most of which are


extracts including numerous ingredients, are widely used
in food processing to prevent deterioration in the quality
of foods by oxidative damage. Among a variety of such
natural additives registered in the List of Existing Food
Additives in Japan,1) eucalytus leaf extract is dened as
an ethanol extract or steam distillate from the leaves
of Eucalyptus globulus Labill (Myrtaceae), and this
additive is characterized as containing -diketones as
the main constituents.
E. globulus has traditionally been used as a medicinal
plant for the treatment of enterocolitis, arthralgia, and
burns in China.2) Eucalyptus oils prepared by steam
distillation are also used as avoring and fragrance.3)
-Diketones, such as 16-hydroxy-18-tritriacontanone
and 4-hydroxy-tritriacontane-16,18-dione (wax constituents), as well as ellagic acid and its related compounds,
were reported as antioxidants of the leaves of E.
globulus by Osawa et al. in the 1980s.46) In addition
to the antioxidants, the chemical constituents of the
leaves have been investigated extensively to reveal the
y

presence of a wide array of compounds, including monoterpenes, such as 1,8-cineole, sesquiterpenephloroglucinol derivatives, avonoids, tannins, and
related polyphenols.3,713)
As part of studies evaluating ocial food additives
conducted by research groups of the Ministry of Health,
Labour and Welfare of Japan since 2003, eucalyptus
leaf extract was found to include gallic acid, quercetin
3-O--D-glucuronide, kaempferol 3-O--D-glucuronide,
globuluside, cryptmeridiol, 4-epi-cryptmeridiol, 3,13dihydroxyurs-11-en-28-oic acid, -eudesmol, and macrocarpal I, of which the rst three were the main
constituents.14) However, -diketones, dened in the
ocial list as the main constituents, were not detected.
Thus the standardized markers in eucalyptus leaf extract
are currently undened.
This paper describes a re-examination of the constituents of the commercial food additive eucalyptus leaf
extract based on a detailed characterization of the
antioxidant polyphenols of the raw material (E. globulus
leaves). It also proposes a new standard, including a
characteristic marker ingredient, for the quality of this
antioxidant food additive.

Materials and Methods


General. UV spectra were recorded on a Shimadzu UVmini-1240
(Shimadzu, Kyoto, Japan). Electrospray ionization (ESI)-MS and highresolution (HR) ESI-MS spectra were obtained using a micrOTOF-Q
(Bruker Daltonics, Billerica, MA) mass spectrometer using acetonitrile
as the solvent. 1 H- and 13 C-NMR spectra were recorded on a Brucker
AVANCE500 instrument (Bruker BioSpin, Billerica, MA) (500 MHz
for 1 H and 126 MHz for 13 C), and chemical shifts are given in ppm
values relative to those of the solvents (methanol-d4 (H 3.30; C 49.0)
and acetone-d6 (H 2.04; C 49.0)) on a tetramethylsilane scale. The
standard pulse sequences programmed for the instrument (Avance 500)
were used for the 2D measurements (COSY, HSQC, and HMBC). JCH
was set at 8 or 10 Hz in HMBC.
Reversed-phase (RP) HPLC conditions were as follows: column,
Cosmosil packed column 5C18-PAQ (5 mm, 150  4:6 mm i.d.)
(Nacalai Tesque, Kyoto, Japan); mobile phase, solvent A was 5%
acetic acid and solvent B was methanol (MeOH) (030 min, 050% B
in A; 3035 min, 5085% B in A; 3540 min, 85% B in A; 4050 min,
9590%; 5055 min, 90100% B in A; 5560 min, 100% B); column
temperature, 40  C; ow rate, 1.0 ml/min; detection, 200400 nm.
Normal-phase HPLC conditions were as follows: column, YMC-Pack
SIL A-002 (5 mm, 150  4:6 mm i.d.) (YMC, Kyoto, Japan); mobile
phase, n-hexane-MeOH-tetrahydrofuran-formic acid (55:33:11:1) and

To whom correspondence should be addressed. Fax: +81-89-926-7162; E-mail: amakura@cc.matsuyama-u.ac.jp

Constituents of the Natural Food Additive Eucalyptus Leaf Extract


oxalic acid 450 mg/l; column temperature, room temperature; ow
rate, 1.5 ml/min; detection, 280 nm.
Column chromatography was conducted using MCI Gel CHP-20P
(75150 mm) (Mitsubishi Chemical, Tokyo), Toyopearl HW-40 (ne
grade) (Tosoh, Tokyo), and YMC GEL ODS-AQ (AQ12S50) (YMC).
TLC was carried out on Silica gel 60 F254 plates (Merck, Darmstadt,
Germany), and the spots were visualized by heating the plates after
spraying with diluted sulfuric acid reagent or under a UV lamp
(254 nm). An absorbance microplate reader, SpectraMax M2 multimode reader (Molecular Devices, Sunnyvale, CA), was used.
Samples and reagents. Two commercial eucalyptus leaf extract
products (eucalyptus A: brown powder, eucalyptus B: brown liquid)
were obtained through the Japan Food Additives Association (JAFA)
(Tokyo). Three commercial eucalyptus oils were used as products
derived from E. globulus. The samples were dissolved in MeOH or
aqueous MeOH before HPLC and TLC. Fresh leaves of E. globulus
were collected from the medicinal plant garden of Matsuyama
University in April 2007. Dried leaves were kindly donated by
Nagaoka Perfumery Co., Ltd. (Osaka, Japan).
Extraction and isolation. Dried leaves of E. globulus (400 g) were
homogenized in 70% MeOH (MeOHH2 O 7:3) (10 liters), and a
concentrated solution (about 1 liter) was extracted successively with nhexane (5 liters), ethyl acetate (5 liters), and water-saturated n-butanol
(5 liters), to give the respective n-hexane (4.2 g), ethyl acetate (7.0 g),
n-butanol (15 g), and water (32 g) extracts. The ethyl acetate extract
(7 g), which showed many peaks on HPLC similar to those of the food
additive products, was chromatographed over Toyopearl HW-40, MCIGEL CHP-20P, and YMC GEL ODS-AQ with aqueous MeOH to give
a new compound, 1,2,3,6-tetra-O-galloyl--D-galactose (14) (2 mg),
together with gallic acid (1) (15 mg), oenothein B (2)15) (34 mg),
ellagic acid (3) (20 mg), quercetin 3-O--D-glucuronide (4)16) (86 mg),
kaempferol 3-O--D-glucuronide (5)16) (53 mg), chlorogenic acid (6)17)
(38 mg), 5-O-p-coumaroylquinic acid (7)18) (8 mg), phloridzin (8)19)
(26 mg), quercetin 40 -O--D-glucoside (9)20) (13 mg), tellimagrandin I
(10)21) (5 mg), tellimagrandin II (11)21) (18 mg), 1,2,3-tri-O-galloyl-D-glucose (12) (3 mg), 1,2,3,6-tetra-O-galloyl--D-glucose (13) (2 mg),
1,2,3,4,6-penta-O-galloyl--D-glucose (15)22) (12 mg), 8-demethylsideroxylin (16)23) (2 mg), sideroxylin (17)23) (2 mg), and 30 -O-methyl
ellagic acid 4-O--D-glucose (18)24) (2 mg). These compounds were
identied by direct comparison with authentic specimens or by
comparison of their spectral data with those reported in the literature.
The physical data of the new compound 14 were as follows:
1,2,3,6-Tetra-O-galloyl--D-galactose (14): UV max (MeOH) nm
("): 210 (4.05), 275 (3.73). 20 D +40 (c 0.5, MeOH). 1 H-NMR
(500 MHz, methanol-d4 ) H : 7.07, 7.06, 7.05, 6.95 (each 2H, s, galloyl
H), 6.08 (1H, d, J 8:5 Hz, gal H-1), 5.88 (1H, dd, J 8:5, 10 Hz, gal
H-2), 5.38 (1H, dd, J 3:5, 10 Hz, gal H-3), 4.50 (1H, dd, J 6:5,
12 Hz, gal H-6), 4.43 (1H, dd, J 5:5, 12 Hz, gal H-6), 4.36 (1H, brd,
J 3:5 Hz. gal H-4), 4.29 (1H, brt, J 6:5 Hz, gal H-5). 13 C-NMR
C : 168.0, 167.6, 167.5, 166.4 (galloyl C-7), 146.4 (4C), 146.3 (2C),
146.2 (2C) (galloyl C-3, 5), 140.4, 140.1, 139.9, 139.8 (galloyl C-4),
120.4 (2C), 120.2, 120.1 (galloyl C-1), 110.5, 110.4, 110.3, 110.2 (each
2C, galloyl C-2, 6), 94.3 (gal C-1), 75.1 (gal C-3), 75.0 (gal C-5), 69.8
(gal C-2), 68.0 (gal C-4), 64.0 (gal C-6). HR-ESI-MS m=z: 787.0974
(M  H , Calcd. for C34 H27 O22 : 787.1000).

1061

scavenging assay, a methanolic solution of each test sample (2.0 ml)


was added to a solution (0.5 ml) of DPPH in MeOH (nal
concentration of DPPH, 0.2 mM). After mixing for 10 s, the solution
was left to stand for 30 min, followed by measurement of absorbance at
520 nm. The scavenging activity for the DPPH radical was expressed
as EC50 , the concentration of the test sample required for a 50%
reduction in absorbance relative to that of 0.2 mM DPPH in MeOH.
ORAC assay was performed in 75 mM phosphate buer (pH 7.4) with
a nal reaction volume of 200 ml. Trolox (20 ml) and uorescein
(120 ml; nal concentration 70 nM) solutions were pipetted into each
well of a 96-well microplate. The mixture was pre-incubated in a
microplate reader for 15 min at 37  C. A solution of 2,20 -azobis(2amidinopropane) dihydrochloride (AAPH) (60 ml: nal concentration,
12 mM) was added rapidly to the microplate, and after shaking for 15 s,
uorescence was recorded every min for 90 min at excitation and
emission wavelengths of 485 and 528 nm respectively. The area under
the curve (AUC) was calculated, and the net AUC was calculated by
subtracting the AUC of the blank (phosphate buer only) from that of
the sample. ORAC values were expressed as trolox equivalents
(mmol TE/g) using the calibration curve generated in each assay.

Results
HPLC analysis of eucalyptus leaf extract
Two commercial eucalyptus leaf extracts products
(eucalyptus A and B) used as antioxidant food additives
were analyzed. Figure 1 shows their HPLC chromatograms at 270 nm. Eucalyptus A showed an HPLC prole

34
2

Preparation of extracts of eucalyptus leaves. Chloroform (CHCl3 ),


ethanol (EtOH), and 50% aqueous ethanol extracts were prepared by
extracting dried leaves of E. globulus (10 g) with each solvent (50 ml),
and each extract was submitted to TLC and HPLC analysis.
Total polyphenol content. The total polyphenolic content was
determined by the Folin-Ciocalteu method25) in triplicate, using gallic
acid as the standard. The result was expressed as gallic acid equivalent
(mg) per weight of the eucalyptus sample (g).
Antioxidative assays. Antioxidant activities were estimated by freeradical scavenging assay 1,1-diphenyl-2-picrylhydrazyl (DPPH) method26,27) and the oxygen radical absorbance capacity (ORAC) method.28)
All tests were carried out in triplicate. Briey, in the DPPH radical

10

20

30

40

50

min

Fig. 1. RP-HPLC Proles of Eucalyptus Leaf Extracts.


a, Eucalyptus A; b, eucalyptus B; c, 70% MeOH extract of dried
leaves of Eucalyptus globulus; d, eucalyptus oil (product B). 1,
Gallic acid; 2, oenothein B; 3, ellagic acid; 4, quercetin 3-O--Dglucuronide: 5, kaempferol 3-O--D-glucuronide; 6, chlorogenic
acid. HPLC conditions are described in Materials and Methods.

1062

Y. AMAKURA et al.
OH

HO

OH

HO

HO

COOH

O
H2 C

O
O

OH

HO

HO

HO

HO

OH

O
CH 2

OH

4: R=OH
5: R=H

OH

OH

6: R=OH
7: R=H

O
R

HOOC

OH

OH

O
OH

OH

HO

HO
OH

HO

OH

OH

HO

R
O

O
O

OH

HO

HO

HO COOH

OH

OH

HO

OH
OH

HO

OH

OH

HO

OH

OH

HO

HO

HO

HO

OH

OH

O
O

HO

OH

OH

HOH2 C

OH
OH

OH

HO

OH

HO

OH

HO

HO
O

OH
CH2 OH

H 2C

O
O

OH
O
OH

HO

OH

HO

OH

OH
OH

10

HO
OH

HO

HO
O

HO

HO
O
O

O
O

H2 C

HO
HO

O
O

HO

O
OH

OH

HO

OH

HO

OH
O

OH

OH

OH
OH

14
OH

HO

H 3C

CH2OH
O

HO

OH

OH

OH

12: R=R'=H
13: R=H, R'=G
15: R=R'=G

OH

HO

OH

OH

OH
HO
O
H2C
O
O
O
C
O

OH

OH

O
OH

OH

OH

11

OH

OH

OH

OH

H 3CO

OH

OH

HO

R'OH2C
RO
O
C

OH

O
HO

OCH 3

Galloyl (G) =

C
O

OH
OH

18

16: R=H
17: R=CH3

Fig. 2. Structures of Compounds 118.

(Fig. 1a) similar to that (Fig. 1c) of an aqueous MeOH


extract prepared from the dried leaves of E. globulus,
the raw material of eucalyptus A and B, while
eucalyptus B gave a much simpler chromatogram
showing only two main peaks (Fig. 1b). On the other
hand, eucalyptus oil did not show the same peaks
(Fig. 1d). In order to examine standard samples for
the characterization of each HPLC peak in Fig. 1, an
aqueous MeOH extract was separated by column
chromatography, and 18 compounds (118) were characterized, as shown in Fig. 2. Among them, compound
14 was a new compound and its structure was elucidated
as follows: The molecular formula of 14, C34 H28 O22 ,
was determined by HR-ESI-MS in negative mode (m=z
787.0974 M  H , calcd. 787.1000). The 1 H-NMR
spectrum showed signals attributable to four galloyl

groups ( 7.07, 7.06, 7.05, and 6.95, each 2H-singlet),


and aliphatic proton signals characteristic of a galactopyranose residue of C1 conformation. The constituent
units of 14 were determined by acid hydrolysis, yielding
galactose and gallic acid, the former of which was
proven to be a D-series by HPLC analysis of the
derivative of the sugar by the method reported by
Tanaka et al.29) The positions of the galloyl groups on
the galactose residue were assigned as O-1, O-2, O-3,
and O-6 based on the appearance of the H-1H-3 and
H-6 signals in the lower eld ( 6.084.43) than the H-4
signal ( 4.36). The -orientation of the O-1 galloyl
group was indicated by a large coupling constant (d,
J 8:5 Hz) of the anomeric proton signal. Based on
these spectroscopic data together with 13 C-NMR and 2D
NMR (HSQC and HMBC) spectra, 14 was characterized

Constituents of the Natural Food Additive Eucalyptus Leaf Extract

as 1,2,3,6-tetra-O-galloyl--D-galactose. Since most


gallotannins characterized to date have a D-glucose
core, the presence of a galactose core in 14 is a rare
example in nature.
Based on a comparison with the HPLC data of the
above isolates from E. globulus, the main HPLC peaks
of eucalyptus A were characterized as gallic acid (1),
oenothein B (2), ellagic acid (3), quercetin 3-O--Dglucuronide (4), kaempferol 3-O--D-glucuronide (5),
and chlorogenic acid (6). On the other hand, the two
main peaks in eucalyptus B were identied as 1 and 3.
Although 1 and 3 have been reported frequently as
natural products, they are considered to be mostly
artifacts produced from hydrolyzable tannins during the
drying and/or isolation procedures. In fact, upon treatment of eucalyptus A with hot water monitoring by
HPLC, 1 and 3 increased time-dependently (Fig. 3), and
the HPLC after 8 h was similar to that of eucalyptus B.
Eucalyptus B was thus considered to be a product
prepared by heat extraction of the leaves.
TLC analysis of eucalyptus leaf extracts
-Diketones in E. globulus have been reported to give
spots at Rf 0.7 0.8 on silica gel TLC developed with
n-hexane-ethyl acetate (3:1) under UV irradiation.46) In
accordance with the previous nding, neither eucalyptus
A nor B showed such spots on TLC. Extracts of
E. globulus leaves were prepared with CHCl3 , EtOH,
and 50% aqueous EtOH. -Diketones were not detected
by TLC with the 50% EtOH extract prepared from fresh
or dried leaves of E. globulus (Fig. 4 (1)), while the
CHCl3 and EtOH extracts of the dried leaves exhibited
spots at about Rf about 0.8 under UV light and after
spraying with dilute sulfuric acid followed by heating
the plate (Fig. 4 (2)).
Antioxidant properties of eucalyptus leaf extract
The antioxidant eects of the food additives (eucalyptus A and B) in comparison with those of the CHCl3 ,
EtOH, and 50% EtOH extracts from fresh and dried
leaves of E. globulus were assessed by DPPH radical

1063

0h

1h

8h

10

20

30

40

50

min

Fig. 3. RP-HPLC Prole after Partial Hydrolysis of Eucalyptus A


(after 0, 1, and 8 h).
1, Gallic acid; 3, Ellagic acid. HPLC conditions are described in
Materials and Methods.

scavenging assay and the ORAC method. As shown in


Table 1, eucalyptus A showed the most potent antioxidant activity in DPPH radical scavenging assay, comparable to that of the 50% EtOH extract. Conversely,
eucalyptus B exhibited extremely weak activity, similar
to that of the CHCl3 extract of the dried leaves, although
these eects might have been underestimated because
they are oily products. The ORAC values showed
similar results (Fig. 5).
Because the eucalyptus leaf extract registered as a
food additive in the List of Existing Food Additives in

s.f.

ori.

a'

b'

c'

A B

a b

a'

c'

Fig. 4. TLC Proles of Eucalyptus Leaf Extracts.


1, Detected using a UV lamp at 254 nm; 2, detected with H2 SO4 /heat. a, Chloroform extract (fresh leaf); b, EtOH extract (fresh leaf); c, 50%
aqueous EtOH extract (fresh leaf); a0 , Chloroform extract (dried leaf); b0 , EtOH extract (dried leaf); c0 , 50% aqueous EtOH extract (dried leaf). A,
Eucalyptus A; B, Eucalyptus B, s.f., solvent front; ori., origin. Solvent, n-hexane-ethyl acetate (3:1).

1064

Y. AMAKURA et al.
E. globulus (fresh leaves)
Chloroform extract
EtOH extract
50% aq. EtOH extract

E. globulus (dried leaves)


Chloroform extract
EtOH extract
50% aq. EtOH extract

Food additives
Eucalyptus A
Eucalyptus B
0

1000

2000

3000

4000

5000

mol TE/g

Fig. 5. ORAC Values of Eucalyptus Leaf Extracts.


TE, Trolox equivalent

Table 1. DPPH Radical Scavenging Activity of Eucalyptus Leaf


Extracts and Eucalyptus Oils
EC50 a
(mg/ml)

Total polyphenolsb
(mg/g)

Eucalyptus A
Eucalyptus B

19.0
390.0

360
29

E. globulus (fresh leaves)


Chloroform extract
EtOH extract
50%aq. EtOH extract

162.7
23.5
19.1

34
237
548

E. globulus (dried leaves)


Chloroform extract
EtOH extract
50%aq. EtOH extract

477.0
44.1
24.7

30
357
408

>100 mg/ml
>100 mg/ml
>100 mg/ml

29

Eucalyptus Oil A
Eucalyptus Oil B
Eucalyptus Oil C
a
b

EC50 , 50% eective concentration


These values are expressed as mg of gallic acid equivalent per g of samples.

Japan includes a product by steam distillation, three


commercially available eucalyptus oils (AC) were also
examined for antioxidant properties by DPPH radical
scavenging assay. As expected, these oils showed no
antioxidant eect (EC50 > 100 mg/ml, Table 1).

Discussion
Besides the previously reported major constituents
(1, 4, and 5) of the food additive eucalyptus leaf extract,
compounds 2 and 3 were newly detected as other major
components of eucalyptus A, which showed a potent
antioxidative eect in this study. Oenothein B (2) is a
unique hydrolyzable tannin dimer with a macrocyclic
structure that is known to exhibit diverse biological
activities, such as anti-tumor, antiviral, and a restoration
eect of antibiotics toward methicillin-resistant Staphylococcus aureus (MRSA) as well as antioxidant

eects.30) Although 2 has been found in some species


of Eucalyptus, Eugenia, and Melaleuca genera of
Myrtaceae,3133) this study is the rst report of its
isolation from E. globulus.
The antioxidant potencies of eucalyptus A and B as
well as the other extracts with various solvents were
clearly explained by their total polyphenol contents, as
seen in Table 1. The HPLC and TLC chromatograms of
eucalyptus A together with the relation between antioxidant eect and total polyphenols were similar to
those of the aqueous alcohol extract of the dried leaves
of E. globules (Figs. 1 and 4), implying that eucalyptus A is a product produced by aqueous EtOH extraction
at ambient temperature. On the other hand, the similarity
of the HPLC proles between eucalyptus B and the
partial hydrolyzate from the 50% EtOH extract suggests
that eucalyptus B was produced by heat extraction
(Figs. 1b and 3).
The present study reconrms that -diketones were
not detected in the eucalyptus leaf extracts, in agreement
with a previous paper.14) Although CHCl3 extracts of the
dried leaves indicated distinct spots (Rf about 0.8)
assignable to -diketones or related non-polar substances on TLC (Fig. 4 (2)), these substances might not
contribute to the antioxidative eect, because the CHCl3
extract had extremely weak antioxidant properties on
both radical scavenging and ORAC assay. Hence, it is
suggested that polyphenols should be dened as the
major components of the antioxidant food additive
eucalyptus leaf extract in the List of Existing Food
Additives in Japan rather than -diketones.
Based on the results of the present study, eucalyptus
leaf extract, as prepared by extraction with 50% EtOH
but not by steam distillation, can be characterized as
a polyphenol-rich antioxidant additive with potency
stronger than that of ascorbic acid (ORAC, 5001,000
mmol TE/g).34,35) Among polyphenols, including avonol glycosides and hydrolyzable tannins characterized
in the extract, bioactive oenothein B (2) might be a
signicant marker characteristic of the E. globulus leaf,
because it is a rare natural product with a unique

Constituents of the Natural Food Additive Eucalyptus Leaf Extract

structure and can be detected easily without interference


from other peaks on a reversed-phase HPLC chromatogram in a gradient mode with 5% AcOH and MeOH
(Fig. 1). In addition, 2 is fairly stable, as was observed
upon heating eucalyptus A (Fig. 3). Hence, compound 2
might be a good standard constituent for evaluating
eucalyptus leaf extract. On the ORAC assay, which is
widely used in the USA to evaluate the antioxidant
eects of foods and related natural products, eucalyptus
leaf extract was found to be a long-lasting antioxidant
with a potency of about 4,000 mmol TE/g (Fig. 5),
which conrms its usefulness as a natural antioxidant
food additive.

Acknowledgments
The authors thank Ms. Mie Tokuhara of Matsuyama
University, for technical assistance. Thanks are also
extended to the Japan Food Additives Association for
providing eucalyptus leaf extracts, and to Nagaoka
Perfumery Co., Ltd., for donating dried leaves of
E. globulus. This work was supported by a Health and
Labour Sciences Research Grant from the Ministry of
Health, Labour and Welfare of Japan.

10)
11)
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23)
24)

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