cistus laurifolius

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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.