Antioxidative activities of phenylethanoid glycosides from Ligustrum purpurascens

Article abstract of DOI:10.1021/jf0100604

Tea and kudingcha (bitter tea) are the two most popular beverages consumed in China. Tea derived from the leaves of Camellia sinensis has been well studied for its various health benefits, but there are very limited data on the biological activities of bitter tea derived from the leaves of Ligustrum purpurascens (LP). The present study was carried out to characterize the antioxidants present in the bitter tea brewed from the leaves of LP. It was found that the crude glycoside fraction possessed strong protection against oxidation of human low-density lipoprotein (LDL). The column chromatographic separation led to the isolatation of five phenylethanoid glycosides, namely, acteoside, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B. When acteoside was heated in the boiling water, it was isomerized to form isoacteoside. Acteoside, isoacteoside, and ligupurpuroside A purified from LP were protective, whereas cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B exhibited no protection to human LDL from Cu²⁺-medicated oxidation. Acteoside, isoacteoside, and ligupurpuroside A were also effective in preventing the peroxyl free radical-induced oxidation of α-tocopherol in human LDL. The antioxidant activities of acteoside, isoacteoside, and ligupurpuroside A were comparable to that observed for a green tea antioxidant, (-)-epicatechin gallate. The inhibitory effect of these three phenylethanoid glycosides on oxidation of human LDL and α-tocopherol was dose-dependent at concentrations of 5-40 μM. The present results suggest that the bitter tea beverage derived from LP contains effective antioxidants that may have an equal benefit as a green tea beverage.

Full text of DOI:10.1021/jf0100604

J. Agric. Food Chem. 2001, 49, 3113−3119
3113
An tioxid a tive Activities of P h en yleth a n oid Glycosid es fr om
Ligu str u m pu r pu r a scen s
Ivan Yuen Fan Wong,† Zheng-Dan He,† Yu Huang,‡ and Zhen-Yu Chen*,†
Departments of Biochemistry and Physiology, The Chinese University of Hong Kong, Shatin,
New Territories, Hong Kong, People’s Republic of China
Tea and kudingcha (bitter tea) are the two most popular beverages consumed in China. Tea derived
from the leaves of Camellia sinensis has been well studied for its various health benefits, but there
are very limited data on the biological activities of bitter tea derived from the leaves of Ligustrum
purpurascens (LP). The present study was carried out to characterize the antioxidants present in
the bitter tea brewed from the leaves of LP. It was found that the crude glycoside fraction possessed
strong protection against oxidation of human low-density lipoprotein (LDL). The column chromato-
graphic separation led to the isolatation of five phenylethanoid glycosides, namely, acteoside,
ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B. When
acteoside was heated in the boiling water, it was isomerized to form isoacteoside. Acteoside,
isoacteoside, and ligupurpuroside A purified from LP were protective, whereas cis-ligupurpuroside
B, trans-ligupurpuroside B, and osmanthuside B exhibited no protection to human LDL from Cu2+
-
medicated oxidation. Acteoside, isoacteoside, and ligupurpuroside A were also effective in preventing
the peroxyl free radical-induced oxidation of R-tocopherol in human LDL. The antioxidant activities
of acteoside, isoacteoside, and ligupurpuroside A were comparable to that observed for a green tea
antioxidant, (-)-epicatechin gallate. The inhibitory effect of these three phenylethanoid glycosides
on oxidation of human LDL and R-tocopherol was dose-dependent at concentrations of 5-40 µM.
The present results suggest that the bitter tea beverage derived from LP contains effective
antioxidants that may have an equal benefit as a green tea beverage.
Keyw or d s: Acteoside; bitter tea; isoacteoside; kudingcha; ligupurpuroside A; Ligustrum purpura-
scens; phenylethanoid glycoside
INTRODUCTION
these activities. The chemical composition of LP has
been previously studied (6). Five phenylethanoid gly-
cosides from LP were isolated and identified (Figure 1).
They include acteoside, ligupurpuroside A, cis-ligupur-
puroside B, trans-ligupurpuroside B, and osmanthuside
B. Isoacetoside is an isomer of acteoside and formed
when the leaves of LP were heated in the boiling water.
The present study was designed to quantify these
phenylethanoid glycosides present in LP and examine
their antioxidant activities against the oxidation of
human low-density lipoprotein (LDL) and R-tocopherol.
There has been increasing interest in natural anti-
oxidants present in fruits, vegetables, herbs, and bever-
ages. Several studies have suggested that intake of food
rich in antioxidants is associated with a lower risk of
some chronic diseases (1, 2). Tea is the most popular
beverage consumed worldwide. It is brewed from the
leaves of Camellia sinensis. It is known that tea cat-
echins possess potent antioxidant and free radical
scavenging activities. Bitter tea refers to the brew
prepared from the leaves of 10 species in 5 different
families that are collectively called kudingcha (3). Like
green tea, bitter tea has also been a popular beverage
for a long time in southern China including the prov-
inces of Guangxi, Guizhou, Zhejiang, Yunnan, and
Sichuan. Unlike green tea, no kudingcha beverages
contain any caffeine. There has been no report to date
of the study of the antioxidants possibly present in
kudingcha.
MATERIALS AND METHODS
E xt r a ct ion , Isola t ion , a n d Id en t ifica t ion of P h en yl-
eth a n oid Glycosid es. The dried leaves of LP were purchased
from a local store in Zhaotong, Yunnan province, China. The
isolation and purification of phenylethanoid glycosides were
carried out according to the method previously described (6).
In brief, the dried leaves (520 g) were extracted with 6 L of
ethanol four times under reflux. The extract was concentrated
in a rotary evaporator and then dissolved in distilled water
(Figure 2). The extract was fractionated in a column packed
with Diaion HP-20 using 10 L of water first and then 4 L of
ethanol as eluting solvents; two factions, A1 and A2, were
subsequently obtained. Fraction A1 mainly contained sugar
and organic acid components, and fraction A2 contained crude
glycosides. Fraction A2 (42 g) was further chromatographed
in a silica gel column (2 kg) and eluted using chloroform/
methanol (CHCl3/MeOH). Four fractions (B1-B4) were sub-
sequently obtained; each fraction was then subjected to a silica
gel column chromatography using various ratios of CHCl3/
MeOH as an eluting solvent. Afterward, fraction B1 yielded
In the province of Yunnan, the species Ligustrum
purpurascens (LP) is used to brew bitter tea. It has been
claimed to act as a stimulant to the central nervous
system, a diuretic, a treatment for sore throat, an aid
to weight loss, and a relief of hypertension (4, 5).
However, there were no solid scientific data to confirm
* Corresponding author [telephone (852) 2609-6382; fax
(852) 2603 7246; e-mail zhenyuchen@cuhk.edu.hk].
† Department of Biochemistry.
‡ Department of Physiology.
10.1021/jf0100604 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/12/2001

3114 J. Agric. Food Chem., Vol. 49, No. 6, 2001
Wong et al.
F igu r e 1. Chemical structures of phenylethanoid glycosides present in a bitter tea beverage derived from LP.
132 mg of osmanthuside B, whereas fractions B1 and B2 gave
65 mg of cis-ligupurpuroside B and trans-ligupurpuroside B.
Fractions B3 nd B4 were subjected to a silica column eluted
with CHCl3/MeOH (7:3, v/v) and then purified in a commercial
column packed with Si 60 (Lobar, 40-63 µm, 250 × 25 mm,
Merck, Darmstadt, Germany) followed by further purification
in a glass column packed with Sephadex LH-20. Two glycosides
were obtained, namely, acteoside and ligupurpuroside A.
Isoacteoside was obtained after the incubation of acteoside
at 100 °C. In brief, 16.1 mg of acteoside was dissolved in 100
mL of distilled water and heated at 100 °C for 8 h. Ap-
proximately 80% of the acteoside was converted to isoacteoside.
A Sephadex LH-20 column (100 µm, Pharmacia Fine Chemi-
cals Co., Ltd.) was then used to separate isoacteoside from
acteoside, and the purity was tested using HPLC equipped
with a C-18 column (Hypersil ODS, 250 × 4.6 mm, 5 µm,
Alltech, Deerfield, IL).
Six purified phenylethanoid glycosides were subjected to
verification of their chemical structures on the basis of their
spectral data of UV, LC-MS, FAB-MS, 1H NMR, and 13C NMR
and comparison with those of published data (6) and reference
compounds (Figure 1).
HP LC An a lysis of P h en yleth a n oid Glycosid es. Acteo-
side, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupur-
pruposide B, and osmanthuside B were analyzed using a
Shimadzu LC-10AD HPLC (Tokyo, J apan) equipped with a
ternary pump delivery system. In brief, 5 µL of samples (1 mg/
mL in methanol) was injected onto a C-18 column (Hypersil
ODS, 250 × 4.6 mm, 5 µm, Alltech) maintained in a cold water
bath of 4 °C, via a rheodyne valve (20 µL capacity, Shimadzu,
Tokyo, J apan). An initial eluting mixture of H2O and aceto-
nitrile (80:20, v/v) was used at a flow rate of 1 mL/min. From
15 to 43 min, the percentage of acetonitrile was increased to
100%. Separated glycosides were monitored using a UV
detector at 227 nm (Shimadzu SPD-10AV). A typical HPLC
chromatogram of the ethanol extract derived from LP is shown
in Figure 3.
LDL Isola tion . Fresh blood was collected from healthy
subjects at the Prince of Wales Hospital, The Chinese Uni-
versity of Hong Kong, Shatin, Hong Kong. To prevent lipo-
protein modification, EDTA (0.1%) and NaN3 (0.05%) were
added. LDL was isolated from plasma according to the method
described previously (7). To minimize the oxidation of LDL,
the centrifuge tubes containing plasma were flushed with
nitrogen gas. First, the plasma was centrifuged at 1500g for
15 min to remove cells and cell debris. A NaCl/KBr solution
(153 g of NaCl, 354 g of KBr, and 100 µg of EDTA dissolved in
1 L of water, 1.33 g/mL) was then added to increase the plasma
density to 1.019. The plasma was recentrifuged at 160000g at
4 °C for 20 h. After the top layer containing chylomicron and
very low-density lipoprotein (VLDL) was removed, the density
of remaining plasma fractions was increased to 1.064 and they
were recentrifuged at 160000g for an additional 24 h. The top
LDL fraction was collected and then flushed with nitrogen and
stored at -70 °C. The protein content of isolated LDL was
determined using Lowry’s method (8).
LDL Oxid a tion . The stock LDL fraction (5 mg of protein/
mL) was dialyzed against 100 volumes of the degassed dialysis
solution (pH 7.4) containing 0.01 M sodium phosphate, 0.9%
NaCl, 10 µM EDTA, and 0.05% NaN3 in the dark for 24 h.

Characterization of Antioxidants Present in Ligustrum purpurascens
J. Agric. Food Chem., Vol. 49, No. 6, 2001 3115
F igu r e 2. Separation and purification of phenylethanoid glycosides from LP.
and Cu2+ was examined according to the method previously
described by Brown et al. (11). In brief, stock solutions of
each tested phenylethanoid glycoside (1 mM) were pre-
pared in water. Then the solution was diluted to 250 µM in
a
cuvette, and the absorption spectra were recorded at
wavelengths between 200 and 350 nm. To detect any chelation
or interaction between phenylethanoid glycosides and Cu2+
scans with the addition of 250 and 500 µM CuSO4 were taken
and compared with the spectrum for each tested phenyl-
ethanoid glycoside alone. If a chelation reaction occurs,
changes in the spectra of phenylethanoid glycosides are
expected.
,
F igu r e 3. HPLC profile of phenylethanoid glycosides present
in an ethanol extract of LP. See text for the conditions and
Table 1 for percent composition.
P r otection of P h en yleth a n oid Glycosid e to r-Toco-
p h er ol in LDL. The protective role of each tested phenyle-
thanoid to R-tocopherol in human LDL was studied as we
previously described (12). Human LDL was isolated as previ-
ously described above. The stock LDL fraction (5 mg of protein/
mL) was dialyzed against 100 volumes of the degassed dialysis
solution (pH 7.4) containing 0.01 M sodium phosphate, 0.9%
NaCl, 10 µM EDTA, and 0.05% NaN3 in the dark for 24 h.
Oxidation of R-tocopherol in LDL was induced by 2,2′-azobis-
(2-amidinopropane) dihydrocholoride (AAPH). The LDL (150
µg of protein/mL) was incubated in sodium phosphate buffer
(pH 7.4, 10 mM) at 37 °C with constant stirring. The various
amounts of each phenylethanoid glycoside were added before
the addition of 1.0 mM AAPH. An aliquot of the incubation
solution (1 mL) was recovered and chilled in ice. One milliliter
of ethanol containing 0.5 mg of butylated hydroxytoluene as
an antioxidant and 1.0 µg of tocopherol acetate as an internal
standard was added and immediately extracted with 2 mL of
hexane. The hexane was evaporated under a gentle stream of
nitrogen, and the resulting extract was redissolved in 100 µL
of ethanol and subjected to HPLC analysis for determination
of R-tocopherol in LDL.
The dialysis solution was changed four times. Oxidation of
LDL was conducted as previously described by Puhl et al. (9).
LDL protein (150 µg) was incubated in a mixture containing
5 µM CuSO4 at 37 °C for up to 32 h. The oxidation was then
stopped by the addition of 25 µL of 1.0% EDTA and cooled at
4 °C. The degree of LDL oxidation was monitored by measur-
ing the production of thiobarbituric acid-reactive substances
(TBARS) as previously described (10). After the reaction was
stopped by the addition of EDTA at 4 °C, 2 mL of 0.67%
thiobarbituric acid and 15% trichloroacetic acid in 0.1 N HCl
solution were added to the LDL-incubated tube. The incuba-
tion mixture was then heated at 95 °C for 1 h, cooled on ice,
and centrifuged at 1000g for 20 min. TBARS was determined
by measuring the absorbance at 532 nm. Calibration was done
with a malondialdehyde (MDA) standard solution prepared
from tetramethoxypropane, although thiobarbituric acid reacts
with a number of lipid oxidation products including other
aldehydes and peroxyl radicals. The value was expressed as
nanomoles of MDA per milligram of LDL protein.
In ter a ction s of P h en yleth a n oid Glycosid es w ith Cu 2+
The chelation reaction between each tested phenylethanoid
.

3116 J. Agric. Food Chem., Vol. 49, No. 6, 2001
Wong et al.
Ta ble 1. Con ten t of Som e P h en yleth a n oid Glycosid es
P r esen t in L. pu r pu r a scen sa
content (g/100 g of dry leaves)
acteoside
2.1
0.7
0.1
0.2
0.5
ligupurpuroside A
cis-ligupurpuroside B
trans-ligupurpuroside B
osmanthuside B
a
Average of three separate extractions.
HP LC An a lysis of r-Tocop h er ol in LDL. R-Tocopherol
in LDL was determined using a Shimadzu LC-10AD HPLC
equipped with a ternary pump delivery system (12). In brief,
10 µL of the extract dissolved in ethanol was injected onto a
C-18 column (Microsorb MV, 250 × 4.6 mm, i.d., 5 µm, Rainin,
Woburn, MA) via a rheodyne valve (20 µL capacity; Cotati,
CA). Methanol was used as an eluting solvent at a flow rate
of 2 mL/min. R-Tocopherol was monitored by using a diode
array detector at either 200 or 280 nm and quantified
according to the amount of R-tocopherol acetate standard
added.
Sta tistics. Data are expressed as means ( SD. The analysis
of variance (ANOVA) when applicable was used for statistical
evaluation of significant differences in protection to LDL
oxidation by each phenylethanoid glycoside using Sigmastat
(J andel Scientific Software, San Rafael, CA). Differences were
considered significant when p < 0.05.
F igu r e 4. Inhibitory effect of six phenylethanoid glycosides
(40 µM) isolated from LP on the production of TBARS in Cu2+
-
RESULTS
mediated oxidation of human LDL. The LDL (150 µg of protein/
mL) was incubated in sodium phosphate buffer (pH 7.4)
containing 5 µM CuSO4. The oxidation was conducted at 37
°C. Data are expressed as mean ( SD of n ) 6-8 samples.
ECG from green tea was used as a reference antioxidant.
Means at the same time point with different letters differ
significantly at p < 0.05.
The antioxidant activity of the water fraction (A1) and
the total glycoside fraction (A2) derived from the leaves
of LP (Figure 3) was first tested in LDL oxidation under
the same conditions. It was found that fraction A2
possessed strong protection against oxidation to human
LDL. It was therefore targeted for isolation and puri-
fication of possible antioxidants. According to He et al.
(6), the column chromatographic separations led to the
purification of five phenylethanoid glycosides (Figure 1).
As shown in Table 1, HPLC analysis found that acteo-
side was most abundant (2.1 g/100 g of dry leaves)
followed by ligupurpuroside A, osmanthuside B, and
trans-ligupurpuroside B.
of 10-20 µM acteoside, isoacteoside, and ligupurpuro-
side A extended the lag time to >10 h. Acteoside showed
a stronger antioxidant activity than isoacteoside (Figure
5).
The protective effect of six phenylethanoid glycosides
on R-tocopherol in human LDL was also examined
(Figure 6). cis-Ligupurpuroside B, trans-ligupurpuroside
B, and osmanthuside B showed no protection to R-to-
copherol. This was in agreement with that observed in
the TBARS test. In contrast, acteoside, isoacteoside, and
ligupurpuroside A were strongly protective to R-toco-
pherol in human LDL. These three phenylethanoid
glycosides also demonstrated a dose-dependent protect-
ing role to R-tocopherol in human LDL (Figure 7).
When the leaves of LP or pure acteoside was heated
in boiling water, HPLC analysis demonstrated that a
new peak following that of acteoside gradually became
bigger with heating. This peak was later identified as
isoacteoside, which is an isomer of acteoside. The
1
structure of isoacteoside was confirmed using H NMR
and 13C NMR. The 1H NMR spectrum of isoacteoside
was similar to that of acteoside, except for differences
in the chemical shifts of H-4 (acteoside, δ 4.81; isoac-
teoside, δ 3.41) and 2H-6 (acteoside, δ 3.63 and 3.84;
isoacteoside, δ 4.34 and 4.50) in their glucosyl moiety.
The 13C NMR chemical shifts of isoacteoside were close
to those of acteoside, but slight differences were ob-
served in the shifts at C-3, C-4, and C-6 in its glucosyl
moiety (acteoside, δ 81.66, 70.69, 62.49; isoacteoside, δ
84.45, 70.94, 65.20).
DISCUSSION
Green, oolong, and black tea beverages from the
leaves of the plant C. sinensis have been extensively
studied for their antioxidant, anticarcinogenic, and
hypolipidemic activities. However, there are very limited
data on bitter tea, especially the beverage from the
species LP cultured in the provine of Yunnan, China.
Many studies suggest that oxidation of human LDL is
one risk factors in the development of cardiovascular
disease (13-19). The present results demonstrated
clearly that the bitter tea derived from LP contained at
least two antioxidants, namely, acteoside and ligupur-
puroside A, which were effective in protecting human
LDL from oxidation.
Six purified phenylethanoid glycosides demonstrated
various antioxidant activities (Figure 4). cis-Ligupur-
puroside B, trans-ligupurpuroside B, and osmanthuside
B showed no or little antioxidant activity, whereas
acteoside, isoacteoside, and ligupurpuroside A were
protective to human LDL, comparable to the activity of
(-)-epicatechin gallate (ECG). These three phenyletha-
noid glycosides demonstrated a dose-dependent anti-
oxidant activity in Cu2+-mediated LDL oxidation (Fig-
ure 5). In most cases, LDL was oxidized significantly
within 4 h in the absence of any antioxidant. Addition
R-Tocopherol is a primary antioxidant protecting
human LDL from oxidative modification (20-22). Sev-
eral reports suggest that vitamin E supplementation is
associated with a lower risk of coronary heart disease

Characterization of Antioxidants Present in Ligustrum purpurascens
J. Agric. Food Chem., Vol. 49, No. 6, 2001 3117
F igu r e 6. Protective effect of six phenylethanoid glycosides
(10 µM) isolated from LP on R-tocopherol in human LDL. The
LDL (150 µg of protein/mL) was incubated in sodium phos-
phate buffer (pH 7.4), and the oxidation of R-tocopherol in LDL
was induced by 1 mM AAPH at 40 °C. Data are expressed as
mean ( SD of n ) 6-8 samples. ECG from green tea was used
as a reference antioxidant. Means at the same time point with
different letters differ significantly at p < 0.05.
LDL but also R-tocopherol from oxidation in vitro.
Acteoside and ligupurpuroside A as natural antioxidants
may be beneficial if they are absorbed and circulated
in the blood.
The biochemical mechanisms by which acteoside,
isoacteoside, and ligupurpuroside A act as antioxidants
remain unclear at the present time. Probably, they
protect human LDL from oxidation by one of the
following mechanisms: (i) they function as chelators to
inactivate Cu2+ used in the initiation of free radicals
(11); and (ii) they act as a primary antioxidant by
directly reducing the formation of free radicals mediated
by Cu2+. To test the first possibility, the interaction
between Cu2+ and each phenylethanoid glycoside was
examined. Addition of equimolar Cu2+ did not cause any
major spectral shift of acteoside, isoacteoside, and
ligupurpuroside at wavelengths between 200 and 400
nm, indicating that these three phenylethanoid glyco-
sides cannot chelate Cu2+ (data not shown). Instead, it
was believed that the antioxidant activity of these
phenylethanoid glycosides was related to their number
of hydroxyl groups, that is, their electron- or hydrogen-
donating potency. In fact, osmanthuside B, cis-ligupur-
puroside B, and trans-ligupurpuroside B showed no or
little protection to human LDL because they had only
two separate phenol moieties. In contrast, acteoside,
isoacteoside, and ligupurpuroside A strongly inhibited
the oxidation of human LDL and R-tocopherol because
they were characerized by having two catechol moieties.
Thus, they are more vulnerable to loss of a proton or
an electron due to resonance delocalization. The anti-
oxidant activities observed for acteoside, isoacteoside,
and ligupurpuroside A are most likely attributable to
their proton-donating capacities.
F igu r e 5. Dose-dependent inhibitory effect of acteoside,
isoacteoside, and ligupurpuroside A on the production of
TBARS in Cu2+-mediated oxidation of human LDL. The LDL
(150 µg of protein/mL) was incubated in sodium phosphate
buffer (pH 7.4) containing 5 µM CuSO4. The oxidation was
conducted at 37 °C. Data are expressed as mean ( SD of n )
6-8 samples. Means at the same time point with different
letters differ significantly at p < 0.05.
in both men and women (23, 24). The level of serum
R-tocopherol is inversely correlated with the mortality
rate of cardiovascular disease (25, 26). The present
results clearly show that acteoside and ligupurpuroside
A present in the leaves of LP protected not only human
The results obtained in the present study may have
some implications for individuals who regularly con-

3118 J. Agric. Food Chem., Vol. 49, No. 6, 2001
Wong et al.
two antioxidants, namely, acteoside and ligupurpuroside
A, which are comparable to those in green tea. We are
currently investigating the absorption, metabolism, and
excretion of acteoside and ligupurpuroside A and ex-
ploring other possible health benefits including choles-
terol-lowering and cardiovascular relaxing activities
associated with drinking the bitter tea derived from LP.
LITERATURE CITED
(1) Sterinmetz, K. A.; Pottern, J . D. Vegetable, fruit and
cancer prevention: A review. J . Am. Diet. Assoc. 1996,
96, 1027-1039.
(2) Gey, K. F. 10-year retrospective on the antioxidant
hypothesis of ateriosclerosis: threshold plasma levels
of antioxidant micronutrients related to minimum car-
diovascular risk. J . Nutr. Biochem. 1995, 6, 206-236.
(3) He, Z. D.; Ueda, S.; Akaji, M.; Fujita, T.; Inoue, K.; Yang,
C. R. Monoterpenoic and phenyethanoid glycosides from
Ligustrum pedunculare. Phytochemistry 1994, 36, 709-
716.
(4) Ouyang, M. A.; Wang, H. Q.; Chen, Z. Y.; Yang, C. R.
Triterpenoid glycosides from Ilex kudingcha. Phytochem-
istry 1996, 43, 443-445.
(5) Chen, Y.; Li, K. S.; Xie, T. G. Hypotensive action of the
extract of kudingcha dongqingye. Chin. Tradit. Herb.
Drugs 1995, 26, 250-252.
(6) He, Z. D.; Liu, Y. Q.; Yang, C. R. Glycosides from
Ligustrum purpurascens. Acta Bot. Yunnanica 1992, 14,
328-336.
(7) Havel, R. J .; Eder, H. A.; Bragdon, J . H. The distribution
and chemical composition of ultracentrifugally separated
lipoproteins in human serum. J . Clin. Invest. 1995, 34,
1345-1353.
(8) Lowry, O. H.; Rosenbrough, N. J .; Farr, A. L.; Randall,
R. J . Protein measurement with Folin phenol reagent.
J . Biol. Chem. 1951, 193, 265-270.
(9) Puhl, H.; Waege, G.; Esterbauer, H. Methods to deter-
mine oxidation of low-density lipoproteins. Methods
Enzymol. 1994, 233, 425-441.
(10) Buege, J . A.; Aust, S. D. Microsomal lipid oxidation.
Methods Enzymol. 1978, 52, 302-310.
(11) Brown, J . E.; Khodr, H.; Hider, R. C.; Rice-Evans, C. A.
Structural dependence of flavonoid interaction with Cu+2
ions: implications for their antioxidant properties. Bio-
chem. J . 1998, 330, 1173-1178.
(12) Zhu, Q. Y.; Huang, Y.; Tsang, D.; Chen, Z. Y. Regenera-
tion of R-tocopherol in human low-density lipoprotein
by green tea catechin. J . Agric. Food Chem. 1999, 47,
2020-2025.
(13) Brown, M. S.; Goldstein, J . Lipoprotein metabolism in
the macrophage. Annu. Rev. Biochem. 1983, 52, 223-
261.
(14) Haberland, M. E.; Fong, D.; Cheung, L. Malodialdehyde-
altered protein occurs in atheroma of Watannable
heritable hyperlipidemic rabbits. Science 1988, 241,
215-218.
(15) J ialal, I.; Devaraj, S. Low-density lipoprotein oxidation,
antioxidants, and atheroscelerosis: a clinical biochem-
istry perspective. Clin. Chem. 1996, 4, 498-506.
(16) Palinski, W.; Rosenfeld, M. E.; Yla-Herttuala, S.; Gurter,
G. C.; Socher, S. S.; Butler, S. W.; Parthasarathy, S.;
Carew, T. E.; Sterberg, D.; Witztum, J . L. Low-density
lipoprotein undergoes oxidative modification in vitro.
Pro. Natl. Acad. Sci. U.S.A. 1989, 86, 1372-1376.
(17) Shaikh, M.; Martini, S.; Quiney, J . R.; Baskerville, P.;
La Ville, A. E.; Brows, N. L.; Duffield, R.; Turner, P. R.;
Lewis, B. Modified plasma-derived lipoproteins in hu-
man atherosclerosis plaques. Atherosclerosis 1988, 69,
165-172.
F igu r e 7. Dose-dependent protective effect of acteoside,
acteoside, and ligupurpuroside A on R-tocopherol in human
LDL. The LDL (150 µg of protein/mL) was incubated in sodium
phosphate buffer (pH 7.4), and the oxidation of R-tocopherol
in LDL was induced by 1 mM AAPH at 40 °C. Data are
expressed as mean ( SD of n ) 6-8 samples. Means at the
same time point with different letters differ significantly at p
< 0.05.
sume bitter tea derived from LP. It is known that green
tea contains four major catechin antioxidants, namely,
(-)-epicatechin (EC), (-)-epigallocatechin (EGC), ECG,
and (-)-epigallocatechin gallate (EGCG). To our best
knowledge, this is the first study to demonstrate that
the bitter tea beverage derived from LP also contains
(18) Steinberg, D.; Parthasarathy, S.; Carew, T. W.; Knoo,
J . C.; Witztum, J . L. Beyond cholesterol: modification
of low-density lipoprotein that increase its atherogenic-
ity. N. Engl. J . Med. 1989, 320, 915-924.

Characterization of Antioxidants Present in Ligustrum purpurascens
J. Agric. Food Chem., Vol. 49, No. 6, 2001 3119
(19) Witztum, J . L.; Steinberg, D. Role of oxidized low-density
lipoprotein in atherosclerosis. J . Clin. Invest. 1991, 88,
1785-1792.
(20) J essup, W.; Dean, R. T.; de Whalley, C. V.; Rankin, S.
M.; Leak, D. S. The role of oxidative modification and
antioxidants in LDL metabolism and the atherosclerosis.
Adv. Exp. Med. Biol. 1990, 264, 139-142.
(21) Knipping, G.; Rothneder, M.; Striegl, G.; Esterbauder,
H. Antioxidants and resistance against oxidation of
porcine LDL subfractions. J . Lipid Res. 1990, 31, 1965-
1972.
(22) Hodis, H. N.; Mack, W. J .; La Bree, L.; Cashin-Hemphill,
L.; Sevanian, A.; J ohnson, R.; Azen, S. P. Serial coronary
angiographic evidence that antioxidant vitamin intake
reduce progressing of coronary artery atherosclerosis.
J . Am. Med. Assoc. 1995, 273, 1849-1854.
Heiss, G. Dietary antioxidants and carotid artery wall
thickness: The ARIC Study. Circulation 1995, 92,
2142-2150.
(25) Pricen, H. M. G.; van Poppel, G.; Vogelezang, C.;
Buytenhek, R.; Kok, F. J . Supplementation with vitamin
E but not â-carotene in vivo protects low-density lipo-
protein from lipid oxidation in vitro: Effect of cigarette
smoking. Arterioscler. Thromb. 1992, 12, 554-562.
(26) Reaven, P. D.; Khou, A.; Beltz, W. F.; Parthasarathy,
S.; Witztum, J . L. Effect of dietary antioxidant combina-
tions in humans: Protection of LDL by vitamin E but
not â-carotene. Arterioscler. Thromb. 1993, 13, 590-600.
Received for review J anuary 16, 2001. Revised manuscript
received April 3, 2001. Accepted April 4, 2001. This project is
supported partially by a grant from the Hong Kong Research
Grant Council, Hong Kong Special Administrative Region,
People’s Republic of China.
(23) J ialal, I.; Grundy, S. M. Effect of dietary supplementa-
tion with R-tocopherol on the oxidative modification of
low-density lipoprotein. J . Lipid Res. 1992, 3, 899-906.
(24) Kritchevsky, S. B.; Shimalawa, T.; Tell, G. S.; Dennis,
B.; Carpenter, M.; Echfeldt, J . H.; Peacher-Ryan, H.;
J F0100604