cistus laurifolius

Journal of Ethnopharmacology 103 (2006) 455–460
Effect of
L. leaf extracts and flavonoids on
acetaminophen-induced hepatotoxicity in mice
Esra Küpeli , Didem Deliorman Orhan, Erdem Yesilada
Received 26 April 2005; received in revised form 15 August 2005; accepted 19 August 2005
Available online 10 October 2005
In this study, the effect of the flavonoids quercetin-3-methyl-ether (isorhamnetin) ( ), quercetin-3,7-dimethyl-ether ( ) and kaempferol-3,7-
dimethyl-ether ( ) isolated from
L. (cistaceae) leaves was assessed on lipid peroxidation (liver and plasma), cellular glutathione
(GSH) level and plasma AST (aspartate aminotransferase), ALT (alanine aminotransferase) enzyme activities in acetaminophen-induced liver
damage in mice. At 114 mg/kg oral dose quercetin-3,7-dimethyl-ether was shown to possess potent antioxidative activity.
© 2005 Elsevier Ireland Ltd. All rights reserved.
; Cistaceae; Flavonoid; Hepatotoxicity; Lipid peroxidation; Serum transaminase enzymes; Cellular GSH
The genus is one of the characteristic genera of the
Mediterranean region, colonizing degraded areas (Attaguile et
al., 2000).
L. (cistaceae) is a common plant
in Anatolia and is used against various ailments in traditional
medicine. The plant leaves are used to treat rheumatic and related
inflammatory diseases, externally as a bath or poultice to reduce
pain in rheumatism, against fever in common cold or applied
externally as a plaster on the dorsal part of the body in a line of
the kidneys for urinary inflammations (Yeşilada et al., 1997a).
A tea prepared from the leaves is used as hypoglycaemic and the
flowers and buds decoction is used for the treatment of peptic
ulcers in Turkish folk medicine (Sezik et al., 1991; Yesilada et
al., 1995). In previous phytochemical studies,
have been reported to contain flavonoids including quercetin,
kaempferol, apigenin, luteolin and theirs methyl–ethers, the
coumarine scopoletin, diterpenoids, sesquiterpenoids, and sugars
(Demetzos et al., 1989; Vogt et al., 1987; Wollenweber and
Mann, 1984).
Due to the widespread utilization of the plant as a traditional
remedy, it is essential to investigate the potential effects of the
crude drug on hepatic tissue for the evaluation of potential health
Corresponding author. Fax: +90 312 223 50 18.
esrak@gazi.edu.tr (E. Küpeli).
risks to users as well as to disclose the possible antioxidant
effect in the folkloric use. In the present study, the antioxidant
effect of
ethanol extract and fractions
were investigated against acetaminophen-induced liver damage
on subacute administration in mice. In order to assess the
activity, some biochemical plasma and hepatic tissue parameters
including malondialdehyde formation (MDA), transaminase
levels in plasma; aspartate transferase (AST) and alanine transferase
(ALT), and cellular glutathione (GSH) level in hepatic
tissue were measured. Through bioassay-guided fractionation,
the active antioxidant constituent(s) were isolated and identified
by spectroscopy.
NMR spectra were acquired on a JEOL instrument. 1 H NMR:
500 MHz, 13 C NMR: 125 MHz, using TMS as internal standard.
FAB-MS were obtained on a JEOL HX-110A instrument.
The active compounds were isolated after extensive
chromatography using Sephadex LH-20 (25–100 m, Lot
124H0053, Sigma Chem. Co., column size 30 mm 500 mm).
Silica gel (Kieselgel 230–400 mesh, Merck Art. No. 1.09385,
column size 35 mm 750 mm) and reversed phase 18 (LiChroprep
RP-18, Merck, column size 18.5 mm 352 mm) columns.
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.08.038

456
Male Swiss albino mice (20–25 g) were purchased from the
animal breeding laboratories of Refik Saydam Central Institute
of Health (Ankara, Turkey). The animals left for 2 days for
acclimatization to animal room conditions were maintained on
standard pellet diet and water ad libitum. The food was withdrawn
on the day before the experiment, but allowed free access
of water. A minimum of six animals was used in each group.
Throughout the experiments, animals were processed according
to the suggested international ethical guidelines for the care of
laboratory animals.
L. leaves were collected from Bolu,
Dörtdivan in May 2002 and was identified by Prof. Dr. M. Vural
from the Department of Botany, Faculty of Science, Gazi University.
A voucher specimen is deposited in the Herbarium of
Faculty of Pharmacy, Gazi University (GUE-2300).
Some 2 kg of powdered material was extracted three times
with EtOH (10 L) by stirring in a 60 C water bath for 8 days
each. The combined ethanol extract was evaporated to dryness
under reduced pressure to yield ‘EtOH extract’ (315 g).
The EtOH extract was then resuspanded in 1500 ml of MeOH
and extracted with -hexane (7 500 ml). Combined hexane
extract was evaporated under reduced pressure to yield
‘Hexane fraction’ (64.8 g). MeOH was removed from the
remaining solution and diluted with distilled H 2 O to 2000 ml
and further fractionated by successive extractions with chloroform
(7 500 ml), ethyl acetate (5 250 ml) and watersaturated
-butanol (3 200 ml). The extracts as well as the
remaining aqueous phase were evaporated to dryness under
reduced pressure to yield the “CHCl 3 Fr.” (98.4 g), “EtOAc
Fr.” (28.3 g), “BuOH Fr.” (29.7 g) and “R–H 2 O Fr.” (84.5 g),
respectively.
Four grams of the CHCl 3 fraction was permeated on a
Sephadex LH-20 column using MeOH as eluent. Some 43
fractions of 15 ml each were collected. Fractions were compared
by TLC on silica gel using CHCl 3 /MeOH (8:2) and
toluene/ether (1:1) as mobile systems and combined as follows:
Fr.1–13 (1.23 g), Fr.14–19 (0.72 g), Fr.20–30 (0.91 g), Fr.31–43
(1.12 g). Some additional 5.36 g of the CHCl 3 fraction were
worked-up under identical conditions to obtain more material for
further purification. Vacuum-chromatography of the flavonoidenriched
fractions (silica gel; petroleum ether/EtOAc (1:1), (1:2)
and EtOAc/MeOH (8:2) as solvent system) and reverse-phase
(RP-18) chromatography using MeOH/H 2 O yielded three pure
compounds: ( ) (1.23 g), ( ) (0.95 g), ( ) (0.82 g). The structure
of compounds was elucidated by spectroscopic methods as
quercetin-3-methyl-ether ( ), quercetin-3,7-dimethyl-ether ( )
Scheme 1.
and kaempferol-3,7-dimethyl-ether ( ) (Scheme 1). The spectral
data are in agreement with the reported values (Barbera et
al., 1986; Guerrero et al., 2002; Smolarz et al., 2003; Stevens et
al., 1995).
The extract, fractions and pure compounds were suspended
in 0.5% CMC in distilled water prior to oral administration
to experimental animals. Test groups of mice were orally
treated with EtOH extract (500 mg/kg body weight), hexane
(206 mg/kg), CHCl 3 (312 mg/kg), EtOAc (90 mg/kg), -BuOH
(94 mg/kg) or R-H 2 O fractions (268 mg/kg) for 7 following
days, once a day by gastric gavage. The control group
(untreated) and acetaminophen group (positive control) were
administered in 0.5% CMC suspension for the same period.
The reference drug, ascorbic acid (Vitamin C) suspended in
0.5% CMC was directly administered to animals at 100 mg/kg
dose.
Some 60 min after the administration of the last dose on
seventh day, except the control group mice, each of the
acetaminophen group and test group animals was challenged
with the suspension of acetaminophen (800 mg/kg body weight)
in 0.5% CMC to induce hepatic injury (Fairhurst et al., 1982).
Four hours after acetaminophen administration, blood samples
were withdrawn by cardiac puncture and then the mice were sacrificed
by overdose of diethylether. Blood samples collected in
heparinized tubes were centrifuged at 3000 (4 C) for 10 min
to obtain plasma. Plasma samples were used to determine the
lipid peroxide levels and the enzyme (AST, ALT) activity. Liver
of each mouse was promptly removed and used to determine
the tissue levels of malondialdehyde (MDA) and cellular glutathione
(GSH).

457
The methodology described by Kurtel et al. (1992) was
used. Briefly, 1 ml of plasma was mixed with 2.0 ml of
trichloroacetic acid (TCA; 15%, w/v)–thiobarbituric acid (TBA;
0.375%)–0.25 N HCl and mixed throughly and centrifuged at
10,000 for 5 min. The supernatant was mixed with 20 l
of butyl hydroxy toluene (BHT; 0.02% in 95% EtOH, w/v) to
prevent further oxidation and heated for 15 min in a boiling
water bath. After cooling under running water, the flocculent
precipitate was removed by centrifugation at 10,000 for
5 min. The absorbance of the sample was measured at 532 nm
against blank that contained all the reagents except plasma.
1,1,3,3-Tetraethoxypropan was used as standard for the curve
calibration.
with no homogenate. Results were expressed as
tissue.
mol GSH/g
Biocon standard kits and DAX-48 autoanalyzer were used to
measure AST and ALT activities in plasma samples according
to the method of Wilkinson et al. (1972).
Animals employed in the experiments were observed during
48 h and morbidity or mortality was recorded, if happens, for
each group at the end of observation period.
The method of Ohkawa et al. (1979) as modified by Jamall
and Smith (1985) was used to determine lipid peroxidation in tissue
samples. Mice were sacrified by an overdose of diethylether.
The liver of each mouse was immediately excised and chilled in
ice-cold 0.9% NaCl and then perfused via the portal vein with
ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 1.0 g of wet
tissue was weighted exactly and homogenized in 9 ml of 0.25 M
sucrose using a teflon homogenizer to obtain a 10% suspension.
The cytosolic fraction was obtained by a two-step centrifugation
first at 1000 for 10 min and then at 2000 for 30 min
at 4 C. A volume of the homogenate (0.20 ml) was transferred
to a vial and was mixed with 0.2 ml of a 8.1% (w/v) sodium
dodecyl sulphate solution, 1.50 ml of a 20% acetic acid solution
(adjusted to pH 3.5 with NaOH) and 1.50 ml of a 0.8% (w/v)
solution of TBA and the final volume was adjusted to 4.0 ml
with distilled water. Each vial was tightly capped and heated in
boiling water bath for 60 min. The vials were then cooled under
running water.
Equal volumes of tissue blank or test sample and 10%
TCA were transferred into a centrifuge tube and centrifuged at
1000 for 10 min. The absorbance of the supernatant fraction
was measured at 532 nm in a Beckman DU 650 spectrometer.
Control experiment was processed using the same experimental
procedure except the TBA solution was replaced with distilled
water due to the peroxidative effect of acetaminophen on tissue.
Livers of acetaminophen-treated mice were used as positive control
and 1,1,3,3-tetraethoxypropan was used as standard for the
curve calibration.
Some 200 mg of liver was homogenized in 8.0 ml of 0.02 M
EDTA in an ice bath. The homogenates were kept in the ice bath
until used. Aliquots of 5.0 ml of the homogenates were mixed
in 15.0 ml test tubes with 4.0 ml distilled water and 1.0 ml of
50% trichloroacetic acid (TCA). The tubes were centrifuged for
15 min at approximately 3000 . Some 2.0 ml of supernatant
was mixed with 4.0 ml of 0.4 M Tris–buffer, pH 8.9, 0.1 ml Ellman’s
reagent [5,5 -dithiobis-(2-nitro-benzoic acid)] (DTNB)
added, and the sample shaken. The absorbance was read within
5 min of the addition of DTNB at 412 nm against a reagent blank
The data obtained were analyzed by one-way of variance
(ANOVA) and Student–Newman–Keuls post hoc tests for the
significant interrelation between the various groups using Instat
computer software. < 0.05 was considered to be significant
different from the control.
In living systems, dietary antioxidants such as -tocopherol,
ascorbic acid, carotenoids, as well as flavonoids and related
phenolic compounds are suggested in protection from oxidative
damage of tissues in the body and eventually for a healthy
life (Haraguchi, 2001). Especially flavonoids have been shown
to scavenge various reactive oxygen species and implicated as
inhibitors of lipid peroxidation (Mora et al., 1990).
Acetaminophen (paracetamol), a frequently used analgesic
and antipyretic drug, is known to be hepatotoxic in high doses,
which is primarily metabolized by sulfation and glucuronidation
to unreactive metabolites, and then activated by the cytochrome
P-450 system to produce liver injury. It is established that
acetaminophen is bioactivated to a toxic electrophile, -acetyl-
-benzoquinone imine (NAPQI), which binds covalently to tissue
macromolecules, and probably also oxidizes lipids, or the
critical sulphydryl groups (protein thiols) and alters the homeostasis
of calcium (Lin et al., 1997). Post-mitochondrial supernatants
isolated from livers of rats given a single large oral dose
of acetaminophen (800 mg/kg) showed rapid rates of lipid peroxidation
when incubated in vitro. Lipid peroxidation probably
occurs simultaneously with the proposed covalent binding of
the active metabolite of acetaminophen. Since the former process
is known to cause severe and extensive membrane damage,
it may be a very important factor in acetaminophen-induced liver
necrosis (Fairhurst et al., 1982).
As shown in Table 1, in the liver and plasma of
acetaminophen-treated group, tissue and plasma lipid peroxidation
levels (219.2 and 1172%) as evidenced by MDA determination
increased significantly as compared to control group, however,
the content of GSH in liver decreased (8.7%). Additionally,
acetaminophen was found to cause several folds increases in
plasma AST and ALT levels (106 and 71.6%). Ethanol (EtOH)
extract of
leaves administered in 500 mg/kg

458
Table 1
Effect of
EtOH extract, subfractions and the isolated flavonoids on MDA and GSH levels against acetaminophen-induced liver damage in mice
Materials Dose (mg/kg) Plasma MDA level Liver homogenate MDA level Tissue GSH
Plasma (nmol/ml) % Change a Liver (nmol/g) % Change a mol/g % Change a
Control (0.5% CMC) 1.8 0.1 118.2 15.4 106.1 8.1
Acetaminophen b 800 11.4 2.1 *** +1172 377.8 29.6 *** +219.2 96.9 10.6 8.7
Ascorbic acid c 100 4.6 0.2 *** 59.5 183.7 17.9 ** 51.4 126.3 11.5 +30.3
EtOH extract c 500 5.8 0.9 *** 49.3 194.7 19.3 *** 48.5 135.6 7.9 ** +39.9
Hexane Fr. c 206 7.9 1.5 * 31.1 296.1 24.1 * 21.6 117.3 8.6 +21
CHCl 3 Fr. c 312 6.7 0.8 *** 41.3 260.8 16.7 ** 31.0 153.4 6.1 *** +58.3
EtOAc Fr. c 90 7.0 1.0 * 38.4 278.8 14.4 ** 26.2 117.8 7.2 +21.6
-BuOH Fr. c 94 8.7 2.2 23.4 307.8 21.6 18.5 128 8.4 * +32.1
R-H 2 O Fr. c 268 8.9 1.9 21.3 303.2 18.8 19.8 106 4.4 +9.4
Quercetin-3-methyl-ether c 147 6.8 0.6 * 40.3 316.6 12.3 * 16.2 109.7 13.1 +13.2
Quercetin-3,7-dimethyl-ether c 114 6.1 0.4 *** 46.8 300.8 14.8 ** 20.5 115.5 6.3 +19.2
Kaempferol-3,7-dimethyl-ether c 98 7.1 0.5 * 37.5 355.15 14.3 6.0 104.5 9.5 +7.8
Results are presented as mean S.E.M.
a (+) Represents percentage of increase and ( ) decrease when compared to either vehicle or acetaminophen.
b Compared to vehicle control (0.5% CMC).
c Compared to acetaminophen as hepatotoxin.
* Change to significance from control or acetaminophen; < 0.05.
** Change to significance from control or acetaminophen; < 0.01.
*** Change to significance from control or acetaminophen; < 0.001.
dose as well as ascorbic acid, as reference compound, showed
a significant effect at plasma AST, ALT, MDA and liver tissue
MDA, GSH levels (Tables 1 and 2). Besides, we observed that
EtOH extract was more effective than ascorbic acid in plasma
AST levels.
The active EtOH extract was fractionated through successive
solvent-solvent extractions and five fractions namely Hexane
Fr., CHCl 3 Fr., EtOAc Fr., -BuOH Fr. and R-H 2 O Fr. were
obtained.
As shown in Table 1, CHCl 3 Fr. (41.3% for plasma and 31%
for tissue) and, in a lesser degree, EtOAc Fr. (38.4% for plasma
and 26.2% for tissue) possessed a potent activity against both
plasma and tissue MDA levels. Of the five fractions tested, the
CHCl 3 Fr. (58.3%) appeared to be the most effective in increasing
the hepatic GSH levels, as compared to the acetaminophen
group. Additionally, only CHCl 3 (21.5%), EtOAc (10.1%) and
-BuOH (19.6%) fractions produced a decrease in plasma ALT
levels while all of the fractions caused reduction in plasma AST
Table 2
Effect of
in mice
EtOH extract, subfractions and the isolated flavonoids on plasma transaminase enzyme levels against acetaminophen-induced liver damage
Materials Dose (mg/kg) ALT AST
IU (L) % Change a IU (L) % Change a
Control (0.5% CMC) 3474 16 1683 57
Acetaminophen b 800 5960 251 *** +71.6 3468 219 *** +106.0
Ascorbic acid c 100 5864 188 1.6 2108 115 *** 39.2
EtOH extract c 500 4692 113 *** 21.3 2319 119 *** 33.1
Hexane Fr. c 206 6452 114 +8.3 2660 167 * 23.3
CHCl 3 Fr. c 312 4680 115 *** 21.5 2576 235 * 25.7
EtOAc Fr. c 90 5360 240 * 10.1 2194.4 161 ** 36.7
-BuOH Fr. c 94 4792 138 *** 19.6 2796 258 * 19.4
R-H 2 O Fr. c 268 6180 139 +3.7 2656 300 * 23.4
Quercetin-3-methyl-ether c 147 4704 124 ** 21.1 2289.6 121 ** 34.0
Quercetin-3,7-dimethyl-ether c 114 4932 147 *** 17.3 3120 167 10.0
Kaempferol-3,7-dimethyl-ether c 98 4904 124 *** 17.7 2288 150 ** 34.0
Results are presented as mean S.E.M.
a (+) Represents percentage of increase and ( ) decrease when compared to either vehicle or acetaminophen.
b Compared to vehicle control (0.5% CMC).
c Compared to acetaminophen as hepatotoxin.
* Change to significance from control or acetaminophen; < 0.05.
** Change to significance from control or acetaminophen; < 0.01
*** Change to significance from control or acetaminophen; < 0.001

459
levels (19.4–36.7%). The results showed that pretreatment with
CHCl 3 Fr. (in ALT) and EtOAc Fr. (in AST) represented the most
significant alleviation of the plasma enzyme activity induced by
acetaminophen. AST can be generally found in the liver, cardiac
muscle, skeletal muscle, kidneys, brain, pancreas, lungs,
leukocytes, and erythrocytes, whereas, ALT is present in highest
concentration in liver (Rej, 1978). Therefore, the CHCl 3
Fr. was considered to be more effective than the EtOAc Fr. on
acetaminophen-induced liver damage.
According to the biochemical test results, the following
experiments were directed to CHCl 3 Fr. On TLC analysis,
CHCl 3 Fr. was found to be rich in flavonoids. Through successive
column chromatographies three main flavonoids were
isolated, and their structures were elucidated as quercetin-3-
methyl-ether (isorhamnetin) ( ), quercetin-3,7-dimethyl-ether
( ), kaempferol-3,7-dimethyl-ether ( ) by spectral techniques.
Effect of the isolated flavonoid aglycones on acetaminopheninduced
liver damage was also studied using the same biochemical
methods on plasma and liver tissue.
Mice treated with flavonoid significantly reduced
acetaminophen-induced increases in plasma (46.8%) and liver
(20.5%) MDA production compared with acetaminophentreated
mice. On the other hand, flavonoid also caused reduce
of acetaminophen-induced increase in plasma (40.3%) and liver
(16.2%) MDA production. According to these results, flavonoid
is considered to have more inhibitory effect than that of
flavonoid on acetaminophen-induced lipid peroxidation.
Glutathione (GSH) plays an essential role in the detoxification
of acetaminophen and protects hepatocytes by uniting with
reactive metabolites of acetaminophen. Thus, it prevents them
from binding covalently to liver proteins. Intracellular decrease
of the reducted GSH exposes the cell to the destructive effects of
the oxidative stress (Lauterburg and Velez, 1988). In other word,
the hepatic GSH is non-protein reserve of the liver and responsible
in reducing the NAPQI-induced liver damage (Ischiropolus
et al., 1992). The GSH activities showed alleviation of 13.2, 19.2
and 7.8%, respectively, in liver of mice, received flavonoids ,
and , compared with the acetaminophen group.
The rise in serum AST and ALT levels has been attributed
to the damaged structural integrity of the liver (Chenoweth
and Hake, 1962), because these are cytoplasmic in location
and are released into circulation after cellular damage (Sallie
et al., 1991). Pretreatment of mice with flavonoid , and
noticeably decreased the ALT activities (21.1, 17.3 and 17.7%)
compared with the acetaminophen group. Flavonoids (34%)
and 3 (34%) provided a significant reduction in AST activities,
whereas flavonoid (10%) exhibited an insignificant reduction.
The reversal of alleviation of plasma enzyme activity in
acetaminophen-induced hepatic damage by these flavonoids
could explain the prevention of leakage of the intracellular
enzymes by its membrane stabilizing activity (Thabrew et al.,
1987).
While quercetin-3,7-dimethyl-ether ( ) displayed identical
activity than the CHCl 3 fraction in MDA levels, it did not show
any comparable effect with CHCl 3 fraction in tissue GSH and
MDA levels. As to ALT levels, all three flavonoids had equal
activity than the CHCl 3 fraction, whereas quercetin-3-methylether
( ) and kaempferol-3,7-dimethyl-ether ( ) were less active
than the CHCl 3 fraction.The isolated flavonoids did not induce
any apparent acute toxicity during the 48 h observation period.
As shown in Tables 1 and 2, flavonoid was the most active
compound, as can be expected due to the presence of -
catechol group (3 ,4 -OH) in the B-ring, which is known to be
an important substituent pattern in the antioxidant activity of
flavonoids. Although the same functional group also exists in
flavonoid , the effect was somewhat lesser, probably due to the
weaker lipophilic nature of the compound.
Previously, Dok-Go et al. (2003) studied the neuroprotective
effects of three antioxidant flavonoids, including quercetin,
quercetin 3-methyl-ether ( ), and 1-dihydroquercetin isolated
from - var. and the results indicated
that quercetin 3-methyl-ether was the most potent and suggested
as a promising neuroprotectant. It is evident that due to the neuroprotective
activity of this flavonoid, it would be beneficial for
the prevention and treatment of oxidative stress-induced neurological
disorders (Dok-Go et al., 2003).
On the other hand, several studies have reported that quercetin
3-methyl-ether ( ) displayed a remarkable activity against a
wide range of human picornaviruses, platelet aggregation and
histamine-induced tracheal relaxation in vitro (Ko et al., 1999;
Lin et al., 1995; Vanden Berghe et al., 1986). Moreover,
quercetin 3-methyl-ether isolated from
has
been reported to be effective for inhibiting the aldose reductase
activity. Therefore, this compound could be used to treat
the complications associated with diabetes (Enomoto et al.,
2004). Yeşilada et al. (1997a) reported that the methanolic
extract and liposoluble fractions (hexane and
chloroform) were active on IL-1 when assayed high concentrations.
In a previous study, the polysaccharide mixture
obtained from the flowers and flower buds of
was found to be active against pylorus ligation-, absolute
ethanol-, indomethacin-, indomethacin plus HCl/EtOH-induced
gastric and cysteamine-induced duodenal lesions (Yeşilada
et al., 1997b). The CHCl 3 extract and the precipitate obtained
from
leaves showed significant analgesic
activity on the tail flick assay (Ark et al., 2004).
The in vivo antioxidant effect of the flavonoid aglycones isolated
from , quercetin-3,7-dimethyl-ether ( )
and kaempferol-3,7-dimethyl-ether ( ) is reported for the first
time.
According to the results of the present study, the extracts
and isolated flavonoids from
possess a potent
antioxidant activity. Since oxidation is known to be involved in
the pathogenesis of many diseases in which treatment with
is claimed to be effective, further studies should
be carried out on the active compound(s) using other in vivo
and in vitro antioxidant models in order to assess the role and
elucidate the action mechanism.
The authors are extremely thankful to Dr. Emi Okuyama of
Graduate School of Pharmacy Science, Chiba University, Chiba,
Japan, for NMR and MS measurements.

460
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