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Introduction
Abelmoschus manihot (L) medik is native to the Old World
tropics and has been naturalized in some wild New World
tropical areas. It is an edible hibiscus of the Malvaceae
(Mallow) family, and is also used as a staple in folk medicine
in Papua New Guinea, Vanuatu, Fiji, New Caledonia, or China
for a variety of purposes, including: the control of fertility, to
ease childbirth, to stimulate lactation, to help against
menorrhagia, to induce abortion, and to prevent
osteoporosis[1_3].
In recent studies, more researchers have been interested
in the total flavonoids in the flowers of A
manihot. Hypero-side, isoquercetin, and quercetin
3'-glucoside are important ingredients in total flavonoids. Among these, the content of
hyperoside is the highest. Hyperoside (hyperin),
quercetin-3-O-β-D-galactoside, is a flavonol glycoside which widely
exists in many traditional medicines, such as Semen
cuscutae[4], St John's
wort[5,6], hawthorn[7], Balbisia calycina[8], and Alchornea
cordifolia[9]. As an important bioactive
com-pound, hyperoside has been documented to possess
antiviral activity[10,11],
antinociceptive[12_14],
anti-inflammatory[15],
cardioprotective[7,16,17],
hepatoprotective[18_20], and gastric-mucosal-protective
effects[21,22].
In previous studies, it has been shown that hyperoside
demonstrates hepatoprotective properties in various
chemically-induced hepatocyte injury
models[18_20]. However, to our knowledge, the activity of hyperoside against viral
hepatitis has never been tested. Therefore, in this study we aimed
to evaluate the anti-hepatitis B virus (HBV) activity of
hyperoside extracted from A manihot.
Materials and methods
Plant material A manihot were collected from Xinghua,
Jiangsu province and identified by Prof Xian-rong WANG at
the Anhui Institute of Medical Science (Heifei, China).
Extraction of hyperoside Flower materials were extracted
with 80% ethanol and subsequently partitioned in ethyl
acetate. Ethyl acetate extracts were chromatographed over a
polyamide column using gradient mixtures of ethanol and
distilled water. Ethanol extracts yielded total flavonoid
following solvent removal under vacuum. The total flavonoid
was dissolved in ethanol and crystallized to obtain pure
hyperoside (about 97%). The structure of isolate was
determined by reverse phase high performance
liquid chromatography in comparison with authentic hyperoside (the National
Institute for the control of Pharmaceutical and Biological
Products, Beijing, China)
In vitro anti-HBV activity tests
Cell culture The HBV-producing 2.2.15 cells were
obtained from the Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences (Beijing, China). These
cultures were derived from HepG2 cells that were transfected
with a plasmid vector containing G418-resistance sequences
and 2 head-to-tail dimmers of the HBV genome. The cells
were found to produce elevated levels of HBeAg and HBsAg.
The 2.2.15 cells were cultured in complete MEM (containing
10% FBS, 100 kU/L benzylpenicillin, streptomycin, G-418,
L-glutamine 0.03%, pH 7.0) in 75 cm2 tissue culture flasks at 37
ºC in a humidified 5% CO2.
Cell toxicity studies The 2.2.15 cells were first seeded
into 96-well plates (Corning Inc, Corning, NY, USA) at a
density of 1.0×105 cells per mL and cultured in 200 µL
complete MEM containing 10% FBS. After 24 h of incubation,
the cells were washed 3 times with phosphate-buffered
saline (pH 7.0) and treated with different concentrations
(0.20, 0.10, 0.05, 0.025, and 0.0125 g/L) of hyperoside in
serum-free medium for 12 d. The medium was replaced every
4 d in MEM supplemented with various concentrations of
hyperoside. Untreated cells were used as the control.
Because the drug was fuscous, MTT assay could not be used
to measure the toxicity of this drug. Therefore, as an index of
toxicity, the cell pathological changes (CPE) were observed
by a microscope. The degree of CPE was graded as: all
positive cells (_), the number of negative cells <25% (+),
25%_49% (++), 50%_75%(+++), and >75% (++++). This test was
done 3 times under the same conditions.
Determination of HBsAg and HBeAg The 2.2.15 cells
were incubated in 24-well plates at a density of
1.0×105 cells per mL in 1000 µL MEM medium containing 10% FBS. After
24 h, the 2.2.15 cells were treated with different
concentrations of hyperoside (0.05, 0.025, 0.0125, 0.00625, and 0.003125
g/L) in serum-free medium. The cells were grown in the
presence of hyperoside for 9 d and the medium was replaced
every 3 d. After d 6 and d 9, the supernatant was collected
and performed at -20 °C. The HBsAg and HBeAg in the
culture medium were simultaneously measured by EIA kits
on d 6 and d 9. This test was done twice under the same
conditions.
In vivo anti-HBV activity tests
Animals and treatments Peking ducklings within 1 d of
hatching were used as the in vivo model system. The
animals were obtained from an animal breeding farm, Chinese
Academy of Medical Sciences, [SCXK-(Peking)2002_001,
Peking, China]. The animal quarters were maintained at 22±
2 °C and 50%±10% humidity with a 12 h light/12 h dark cycle.
The ducklings were inoculated intravenously with duck
hepatitis B virus (DHBV)-DNA-positive serum from the
Shang-hai ducks (0.2 mL/animal). Seven days after the
injection, the ducklings were divided into 5 groups: the
control group (normal saline); the positive drug group (lamivudine
or 3TC, 0.05
g·kg-1·d-1); and the hyperoside 0.02, 0.05, and
0.10 g·kg-1·d-1 groups. Drugs were administered orally twice
daily for 10 d. Sera were obtained before treatment (d 0), on
d 5 and d 10 during treatment, and d 3 (d 13) after the
cessation of treatment. The serum levels of DHBV-DNA were
detected by dot hybridization.
Detection of DHBV-DNA Fifty μL of serum was spotted
directly onto the nitrocellulose filters. DNA hybridization
was initiated by adding a recently prepared DHBV
32PDNA probe at 1.0×106
cpm/mL using the same prehybridization procedure over night. Filters were washed twice in 1×SSC
(20×SSC: 3 mol/L NaCl, 0.3 mol/L sodium citrate, pH 7.0),
0.1% SDS at 65 °C for 2 h, and 1×SSC at room temperature for
30 min with gentle, constant agitation. The filter was dried
and autoradiographed at -70 °C using X-ray film with an
enhancer screen. After an autoradiographic image had been
obtained, the filter was exposed in the phosphorimaging
screen for 1_2 h, and the samples were quantitated by
FujixBAS1000 (Fuji, Tokyo, Japan); the percentage density
of the phosphorimaging units was calculated.
Histopathological examination of hepatocytes On d
13, each duckling was laparotomized to obtain the liver
immediately after collecting blood from the leg vein.
Fragments of the ducklings liver were fixed in 10% formalin
solution, dehydrated with ethanol solution from 50% to 100%,
embedded in paraffin and cut into 5 µm sections, and stained
using hematoxylin-eosin dye for photomicroscopic observations.
Statistical analysis All data were expressed as mean±SD
and analyzed by one-way repeated-measure ANOVA and
t-test for comparisons between groups. Values were
considered significantly different at P<0.05.
Results
In vitro anti-HBV activities
Cytotoxicity of hyperoside in 2.2.15 cells
Hyperoside-induced cytotoxicity was observed by microscope. After 12
d of incubation with 0.05 g/L hyperoside, no significant
difference was found from that of the control. However, when
the hyperoside concentration increased, cell injury caused
by hyperoside was observed. According to the
Reed-Meuench equation, the 50% toxic concentration
(TC50) was 0.115 g/L, and the maximum nontoxic concentration
(TC0) was 0.05 g/L (Table 1).
Inhibitory effect of hyperoside on HBsAg and HBeAg
expression in 2.2.15 cells After 8 d of incubation, HBsAg
and HBeAg produced in the culture medium were measured.
The positive control drug was 3TC (0.05 g/L). The results
showed that HBeAg and HBsAg of the cells incubated with
hyperoside were less than that of the control cells, and the
median effective concentrations (IC50) were about 0.012 and
0.015 g/L, respectively, on d 4, 0.009 and 0.011 g/L,
respec-tively, on d 8 (Tables 2, 3). The therapeutic indices,
determined by IC50 vs
TC50, were about 9.58, 12.78 of HBeAg, respectively, on d 4 and d 8 and 7.67 and 10.45 of HBsAg on
d 4 and d 8, respectively (data not shown). The HBeAg
inhibition rates of 3TC were 60.54% on d 4 and 54.68% on
d 8, respectively (data not shown). The HBsAg inhibition
rates of 3TC were 58.23% on d 4 and 41.48% on d 8 (data not
shown). Table 2 shows that significant inhibition of HBeAg
by hyperoside was observed at 0.0125 g/L (P<0.01), and a
high inhibition was noted at the hyperoside concentration
equal to 0.05 g/L on d 8. Furthermore, hyperoside also showed
inhibitory activity on HBsAg excretion, about 82.27% at the
concentration of 0.05 g/L on d 8. The inhibition rate
percentages of both HBeAg and HBsAg were both time- and
dose-dependent. It could also be observed that hyperoside had a
relatively stronger inhibition on HBeAg than HBsAg.
In vivo anti-DHBV activities
Inhibitory effect of hyperoside on DHBV-DNA Next,
the anti-HBV activity of hyperoside was investigated
in vivo using the DHBV-DNA-infected duckling model. During this
experimental period, no significant side effects were observed
in animals receiving antiviral therapy or in control animals.
The levels of serum viral DNA were recorded in the 5 groups
before the experiment (d 0). As shown in Table 4, with the
exception of the control group, serum levels of DHBV-DNA
of each group decreased with different extents after
treatment with hyperoside and 3TC on d 5 and d 10, respectively.
Among these, the hyperoside 0.10
g·kg-1·d-1 group, the 0.05
g·kg-1·d-1 group, and the 3TC group showed a significant
decrease of DHBV-DNA (P<0.01). Three days after the
cessation of treatment with 3TC, the viral replication level
returned to the pretreatment baseline. In the ducks treated
with hyperoside, the effect of DHBV-DNA inhibition lasted
(Figure 1). The mean percentage inhibition of viral DNA
levels with hyperoside 0.10 and 0.05
g·kg-1·d-1 was 60.94%
and 56.24%, respectively, on the last treatment day (d 5).
Histopathological features Histopathological profiles
of the liver from the model group ducklings revealed necrosis,
steatosis, and often swelling of the hepatic cytoplasm. The
protective effect of hyperoside was confirmed by
histopathological examinations. Administration of hyperoside to the
experimental animals (0.10
g·kg-1·d-1) showed a significant
improvement of the hepatocellular architecture over the model
group, as evident from a considerable reduction in necrosis
and vacuolation (Figure 2).
Discussion
Hepatitis B is a major epidemic disease in South-East
Asia, China, and Africa, where approximately 10% of the
population are chronic carriers[23]. HBV replicates within
infected hepatocytes and expresses viral epitopes on them to
induce T-cell mediated immune responses to cause hepatitis.
Currently, there are 2 arms of therapy to manage chronic
active hepatitis B: direct antiviral therapy to inhibit
replication of HBV or indirect immunomodulatory therapy to
enhance cellular immunity to destroy the virus-infected
hepatocytes.
Due to the low efficiency and many limitations of
immunomodulatory therapy with IFN-α, direct antiviral
therapy could have increasing importance. However,
although direct antiviral therapy with lamivudine could
efficiently control chronic active hepatitis B, drug resistance
could develop progressively after 6_9 months of the
initiation of therapy[24,25]. These unsatisfactory therapeutic
results strengthen the need for new anti-HBV agents. In this
report, our results imply that hyperoside possesses
anti-HBV activity.
In our experiment, the data shows that the
TC50 of hypero-side was 0.115 g/L and the
TC0 was 0.05 g/L in 2.2.15 cells, which suggests that the inhibitory activity of hyperoside
had no cytotoxicity. In the nontoxic concentration,
hypero-side inhibited HBeAg and HBsAg in a dose- and time-
dependent manner. Nevertheless, hyperoside showed
stronger inhibition on HBeAg with IC50=0.012 g/L than HBsAg
with IC50=0.015 g/L on d 4. At 0.05 g/L, the inhibition rate
percentage of hyperoside on HBeAg and HBsAg in 2.2.15
cells were 86.41% and 82.27%, respectively. Recent research
showed that another flavonoid from Phyllanthus
urinaria ellagic acid effectively blocked HBeAg secretion
(IC50=0.07 mg/mL). However, compared to hyperoside, it had no effect
on HBsAg[26].
As an HBV-infected animal model, DHBV-infected
Peking ducks were also used to evaluate the effects of
hyperoside against HBV. At 0.05 and 0.10
g·kg-1·d-1,
hypero-side significantly decreased DHBV replication with the
inhibition rate percentage 65% and 70%, respectively, on d 10.
On d 13, 3TC showed a rebound effect because of drug
cessation like most antiviral drugs. However, in contrast with
3TC, the inhibition rate percentage of hyperoside on
HBV-DNA showed no rebound after cessation on d 13. It
indicated that hyperoside could maintain for a long time in
treating viremia of HBV, and the effect of DHBV-DNA inhibition
showed a concentration-dependent response. From
histopathological examination, we could also confirm the
protective effect of hyperoside on the livers of DHBV-infected
ducklings. In summary, our results show that hyperoside
possesses anti-HBV activity whether the tests were done
in vivo or in vitro.
It is necessary to reveal the possible mechanisms of the
anti-HBV activity of hyperoside. During the replication of
hepadnaviruses DNA, DNA polymerase was a target enzyme
of antihepatitis drugs. Therefore, further investigation of
the DNA polymerase level in HBV replication is required.
Xiong et al[18] used the in
vitro D-GalN/TNF-α model to confirm that many flavonoids, including hyperoside, protect
liver cells from TNF-α-induced hepatocyte apoptosis.
TNF-α has been found to be a very important pathogenic
mediator in patients with alcoholic liver disease and viral hepatitis,
as well as in many animal liver injury
models[27]. Recent research showed that the activation of the annexin A7 (Axn7)
gene and the expression of the Axn7-GFP fusion protein could
cause a decrease in HBsAg
secretion[28]. However, the precise mechanism of the anti-HBV activity of hyperoside needs
further investigation.
Acknowledgement
We thank Dr Li ZHUANG (the Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences,
Beijng, China) for his excellent technical supports in this
study.
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