Hala El-Adawi etal ,J Biosci Tech, Vol 2 (1),2011,223-231 ISSN:0976-0172

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SOME MEDICINAL PLANT EXTRACTS EXHIBIT POTENCY

AGAINST VIRAL HEPATITIS C
*Hala El-Adawi , Maha El-Demellawy and Abeer Abd El-Wahab
Medical Biotechnology Dept., GEBRI, Universities and Research Center District, New
Borg El-Arab, Egypt, P.O. BOX: 21934. Tel: 203-4593422, Fax: 203-4593407,
*E-mail address: Halaeladawi@hotmail.com
ABSTRACT
Background: Hepatitis C virus (HCV) infection is gaining increasing attention as
a global health crisis. HCV may lead to a substantial health and, consequently,
economic burden over the next 10-20 years. Aim of the work: This research
seeks to demonstrate antiviral activity of naturally derived extracts on HCV. Keywords:
Aqueous extracts of Milk thistle (MSE) as well as lyophilized juice of ginger Reverse transcriptase- PCR,
were tested in-vitro at different concentrations (100, 200, 300, and 400μg/ml) Antiviral drug,
using the HepG2 cell line infected with HCV. Inhibition of viral replication was Hepatitis C,
detected by the amplification of viral RNA using the reverse transcriptase (RT)- Medicinal plants
PCR technique. Both the MSE and juice were considered active upon inhibiting
the viral replication inside the HCV-infected HepG2 cells, as evidenced by the
disappearance of the (+) and/or (-) strands of viral RNA- amplified products
detected by RT-PCR (compared with the positive control). Results: The effective
anti-viral dose was 100 μg /ml for Ginger and 300 μg /m l for MSE. Conclusion:
This study provides results justifying preclinical evaluation of the tested extracts
as antiviral therapy for HCV infections.

1. INTRODUCTION replicate HCV has prevented this trial for a

Hepatitis C virus (HCV), first identified in
1989 [1], is the major aetiological agent of
non-A non-B hepatitis. The infection occurs
principally through blood or blood- derived
products and affects globally more than 270
million people [2]. Egypt reports the highest
prevalence of HCV worldwide, ranging
from 6% to more than 40% among different
regions and demographic groups [3]. HCV
frequently leads to chronic hepatitis and
cirrhosis, in addition to being associated
with the development of hepatocellular
carcinoma [4]. In spite the highly vigorous
and extensive research in this field, a
protective vaccine and effective treatment
are not yet available.
The mainstay of anti-HCV therapy,
interferon (IFN-α) along with ribvirin leads,
at best, to viral clearance for about 40-50%
of patients infected with HCV [5].
Therefore, exploration for new anti-HCV
principles is urgently needed. However, the
absence of an efficient cell culture system to
long time. Recently, a model for in-vitro
anti-HCV screening was established [6-10].
This circumstance prompted us to engage in
search for unprecedented HCV invasion
inhibitors from medicinal plants. Many
herbal medicines possess antioxidant
properties, which may play an important
role in therapeutics [11-16]. Two of the most
beneficial medicinal plants are Ginger and
Milk thislte. Ginger (Zingiber officinale) has
long been used in traditional medicine as a
cure for some diseases including
inflammatory diseases [17]. Ginger contains
active phenolic compounds such as gingerol,
paradol and shogoal which possess
antioxidant,[18] anti-cancer,[19] anti-
inflammatory,[20&21] anti-angiogenesis [22] and
anti-artherosclerotic properties[23]. Milk
thistle has been reported to have protective
effects on the liver and to greatly improve its
function. It is typically used to treat liver
cirrhosis, chronic hepatitis (liver

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inflammation), toxin-induced liver damage
and gallbladder disorders [24&25].
The current work seeks to justify preclinical
evaluation of the extracts of these natural
products as antiviral therapy for HCV
infections.
2. MATERIALS AND METHODS
2.1 supplies
Bulk ginger rhizome (from Zingiber
officinale) was obtained from Metro
supermarket, washed through with tap
water, peeled and cross-sectional cut into
2±mm thickness in order to squeeze them to
get the juice. Milk thistle (Silybum
marianum) seeds (were obtained from a
local market at Alexandria, Egypt. The seeds
of Milk thistle were washed with water and
air dried away from direct sunlight. Seeds
were crushed in a coffee grinder for a total
of 4 min at 15 s on &off intervals (actual
grinding time: 2 min) to avoid heating the
sample. The crushed seeds were wrapped
and stored at -18°C until extraction [26].
2.2 HPLC analysis
The HPLC grade solvents, including
methanol and phosphoric acid were obtained
from Merck (Darmstadt-Germany) and
nanopure water was used. The phenolic
compounds standards were obtained from
Sigma –Aldrich (St. Louis, Mo) and used as
received. The analysis was done using a
Beckman-C18 column (100X4.6 mm, 5um
particle size), equipped with an auto
sampler, quaternary pump and UV/Visible
multi-wavelength detector. The components
of the ginger and MSE samples were
separated as described by National Science
Foundation (NSF). The eluent A and eluent
B for the three extracts was as follows:
For ginger, Eluent A: Water
Eluent B: acetonitrile
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For MSE, Eluent A: 200 mL of methanol
were mixed with 800 mL of water. 5 mL of
phosphoric acid (85%, ACS Reagent grade)
were mixed into 995 mL of the methanol:
Water (20:80).
Eluent B: Mix 800 mL of
methanol with 200 mL of water. Mix 5 mL
of phosphoric acid into 995 mL of the
methanol: Water (80:20).
2.3 Determination of phenol content
The classic technique employed in phenol
analysis is the 4-aminoantipyrine
colorimetric procedure [27]. The absorbance
of the samples was read against blank at 500
nm using spectrophotometer (PerkinElmer
Lambda EZ 201, USA). The concentration
of the sample was calculated from the
standard curve prepared previously.
2.4 Determination of antioxidant activity
The antioxidant activities of the herbs water
extracts were determined using the ferric
thiocyanate (FTC) method [28] with slight
modification. Sample (20 mg) was dissolved
in 4 mL of 95% (w/v) ethanol, then was
mixed with 4.1 mL of linoleic acid [2.51%
(v/v) in 99.5% (w/v) ethanol], 8 mL of 0.05
M phosphate buffer pH 7.0 and 3.9 mL
distilled water and kept in screw-cap
containers at 40°C in the dark. To 0.1 mL of
this solution was then added 9.7 mL of 75%
(v/v) ethanol and 0.1 mL of 30% (w/v)
ammonium thiocyanate. Precisely 3 min
after the addition of 0.1 mL of 20 mM
ferrous chloride in 3.5% (v/v) hydrochloric
acid to the reaction mixture, the absorbance
at 500 nm of the resulting red solution was
measured, and it was measured again every
24 h until the time when the absorbance of
the control reached the maximum value.
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The percent inhibition of linoleic acid (Gibco-BRL), PBMC were stimulated with
peroxidation was calculated as: 5,10,25,50,100,150,200, 250 and 300 µg/mL
of each extract. All samples were assayed in
(%)𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 triplicates. A positive control culture was
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒
= 100 − �� � 𝑋100� included, where PBMC was stimulated with
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
2 µg/mL phytohemaglutinin-L (PHA,
Sigma).
All tests were run in duplicate, and analyses
of all samples were run in triplicate and Proliferation was determined after
o
averaged. incubation for 3 days at 37 C, 5% CO 2 and
P P R R

95% humidity, by addition of 20 mL of

2.5 Cell Culture BrdU labeling reagent (Roche, Penzberg,
Germany) in the last 2 h of the culture. The
In the last few years, a number of cell
labeled cultures were harvested and Brdu
culture systems have been developed that
uptake was determined using Cell
support reliable and efficient progression of
proliferation ELISA, Brdu (colorimeteric)
this virus. Among several human hepatocyte
kit (Roche) following the manufactures
cell lines analyzed, the hepatocellular
instructions. Data were presented as
carcinoma HepG2 cell line was found to be
stimulation index (SI), where proliferation is
the most susceptible to HCV infection [29].
considered positive if SI is ≥ 2.
HepG2 cells were washed twice with
2.7 Qualitative in-vitro Anti-HCV
RPM11640 media supplemented with
Screening
200µM L-glutamine and 25µM HEPES
PBMC and HepG2 cell culture were
buffer; N-[2-hydroxyethyl] piperazine-N`-
prepared as discussed, then infected with 2%
[2-ethanesulphonic acid] (all chemicals and
HCV-infected serum in RPMI culture
media, Cambrex). The cells were suspended
medium containing 8% FBS. Aqueous
at 2X10 5 cells/ mL in RPM1 culture media
extract of MSE as well as lyophilized juice
P P

(RPM1 supplemented media, 10% fetal

of ginger was added at concentrations of
bovine serum (FBS); Gibco-BRL). The cells
5,10,25,50,100,150,200, 250 and 300
were left to adhere on the polystyrene 6-well
µg/mL. Positive and negative control
plates for 4 h in an incubator (37◌C, ֯ 5%
cultures were included. After 96 h of
CO 2 , 95% humidity). The cells were washed
incubation at 37oC, 5% CO 2 and 95%
R R

twice from debris and dead cells by using

P P R R

humidity, another dose of the tested extract

RPM1 supplemented media.
was added. The cells were incubated for
another 96 h at 37oC, 5% CO 2 and 95%
P P R R

2.6 Proliferative activities

humidity, followed by total RNA extraction.
Peripheral blood mononuclear cells (PBMC)
The positive strand and its replicating form
were isolated from a healthy individual by
(negative strand) were detected by RT-PCR
Ficoll-Hypaque (Sigma, St.Louis,MO,USA)
using HCV specific primers to the 5`-
gradient centrifugation. The purified cells
untranslated region of the virus.
was cultured at 1.0X10 6 cell/mL in RPM1
P P

complete medium (Camprex, Verviers,

Belgium) supplemented with 25mM N-[2- 2.8 RNA extraction from PBMCs and
hydroxyethyl] piperazine-N`-[2- HepG2 cells
ethanesulphonic acid] (HEPES) (Sigma), Monitoring of the HCV viremia pre-and
4mM L-glutamine (Camprex), and 10% FBS post-antiviral therapy through the detection

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of viral (+) and /or (-) RNA strands by the
use of qualitative RT-PCR has become the
most frequently used, reliable and sensitive
technique. Recently, it has been reported
that the detection of the (-) strand HCV-
RNA using the RT-PCR is a very important
tool for understanding the life cycle of the
HCV. It provides a reliable marker for the
diagnosis of HCV and monitors the viral
response to antiviral therapy [6].
Based on these facts, the adopted method in
the present study contributes to the
simultaneous detection of the (+) and /or(-)
HCV-RNA strands isolated from peripheral
blood mononuclear cells (PBMC) of
infected patients as well as, HCV RNA
isolated from HepG2 cells. RNA was
isolated from PBMCs and HepG2 cells as
described by Lohr et al.1995 [30]. Briefly,
cells were precipitated and washed in the
same buffer to remove adherent viral
particles before lysis in 4 mol/L
guanidinium isothiocyanate containing
25mmol/L sodium citrate, 0.5% sarcosyl and
0.1 mol/L |β|-mercaptoethanol and 100 μL
sodium acetate. The lysed cells were
centrifuged at 12000 rpm for 10 min at 4ºC.
The aqueous layer was collected and mixed
with equal volume of isopropanol. After
incubation at 20ºC overnight, RNA was
precipitated by centrifugation at 12000 rpm
for 30 min at 4ºC and the precipitate RNA
was washed twice with 70% ethanol.
2.9 PCR of genomic and anti-genomic
RNA strands of HCV
Reverse transcription-nested PCR was
carried out according to Lohr et al.,1995
[30], with few modifications.
Retrotranscription was performed in 25 μL
reaction mixture containing 20 U of AMV
reverse transcriptase (Clontech, USA) with
either 400 ng of total HepG2 or PBMCs
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cells RNA, 40 U of RNAsin (Clontech,
USA), a final concentration of 0.2 mmol/L
from each dNTP (Promega, Madison,WI,
USA) and 50 pmol of the reverse primer
1CH (for plus strand) or 50 pmol of the
forward primer 2CH (for minus strand). The
reaction was incubated at 42ºC for 60 min
and denatured at 98 for 10 min.
Amplification of the highly conserved 5'-
UTR sequences was done using two rounds
of PCR with two pairs of nested primers.
First round amplification was done in 50 μL
reaction mixture, containing 50 pmol from
each of 2CH forward primer and P2 reverse
primer, 0.2 mmol/L from each dNTP, 10 μL
from RT reaction mixture as template and 2
U of Taq DNA polymerase (Promega, USA)
in a 1× buffer supplied with the enzyme.
The thermal cycling protocol was as
follows: 1 min at 94ºC, 1 min at 55ºC and 1
min at 72ºC for 30 cycles. The second round
amplification was done similar to the first
round, except for use of the nested reverse
primer D2 and forward primer F2 at 50 pmol
each. A fragment of 174 bp was identified in
positive samples. Primer sequences were as
follows: 1CH: 5'ggtgcacggtctacgagacctc-3',
2CH: 5'-aactactgtcttcacgcagaa-3', P2: 5'-
tgctcatggtgcacggtcta-3', D2: 5'-
actcggctagcagtctcgcg- 3' and F2: 5'-
gtgcagcctccaggaccc-3'. To control false
detection of negative-strand HCV RNA and
known variations in PCR efficiency, specific
control assays and rigorous standardization
of the reaction were employed: (1) cDNA
synthesis without RNA templates to exclude
product contamination; (2) cDNA synthesis
without RTase to exclude Taq polymerase
RTase activity; (3) cDNA synthesis and
PCR step done with only the reverse or
forward primer to confirm no contamination
from mixed primers. These controls were
consistently negative. In addition, cDNA
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Fig.1.
Resulting chromatogram and gradient elution schedule for
HPLC-UV analysis of (A): Ginger, (B): Milk thistle Extract

Fig. 2.
Inhibition of linoleic peroxidation by ascorbic acid as a standard and ginger (A),
and MSE (B), as measured by the FTC method. Absorbance values represent
means of triplicates of different samples analyzed

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Fig.3.
Dose response curve of tested extract effect on lymphocyte proliferation of healthy individuals.
The data expressed as the Optical density of the BrdU in corporation assay as mentioned
in materials and methods. All reading above dashed line are positive

Fig.4.
RT-PCR amplification products on gel electrophoresis: A, represents the PCR product of HCV RNA (+) and (―)
strands isolated from HepG2 cells at different concentrations of ginger (1), and MSE (2). B, represents the PCR
product of HCV RNA (+) and (―) strands isolated from peripheral blood mononuclear cells (PBMC) of infected
patients at different concentrations of ginger (1) and MSE (2).

synthesis was carried out using only one
primer present followed by heat inactivation
of RTase activity at 95ºC for 1 h, in an
attempt to diminish false detection of
negative-strand prior to the addition of the
second primer.
3. RESULTS
3.1 HPLC
We adopted the HPLC method for analyzing
the isolated compounds as well as screening
the extracts. Fig.1. showed high sensitivity
and reproducibility, which agrees with the
standard chromatograms produced by NSF.
The resulting chromatogram A which
illustrated in Fig.1 shows that the three
major compounds in ginger were 6-
gingerdiol, 6-gingerol and 10-gingerol. In
the chromatogram C there were two major
compounds taxifolin and silychristin.
Taxifolin derivative is also present in a
significant amount, as well, low amount of

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both silybinin A and B were also recorded.
All compounds were identified by their
retention times against standard samples.
Other peaks in both chromatograms were
not identified due to the lack of standards,
they are most probably, phenolic compounds
in a sense they contributed significantly to
the total phenolics in the extract because
total phenolics measured were much higher
than the sum of the individual phenolic
concentration identified and quantified by
HPLC.
3.2 The phenolic content
Both extracts recorded high phenolic
content. It was 40.1% for ginger and 77.3%
for MSE.
3.3 The antioxidant activity
Figure 2 shows the inhibition of linoleic
peroxidation by ginger and MSE
respectively in comparison with ascorbic
acid as a standard
3.4 The immuonomodulatory effect
The overall immuonomodulatory profiles of,
ginger and MSE enhanced lymphocyte
proliferation at concentrations up to 200
µg/ml, Figure 3. These results demonstrate
that not only do the extracts show no
cytotoxicity effect but they also augment
lymphocyte proliferation (immuno-
stimulatory effect) in a dose dependant
manner.
3.5 Anti-viral effect
Inhibition of viral replication was detected
by amplification of the viral RNA segments
using RT-PCR technique. The test extract is
considered to be active when it is capable of
inhibiting the viral replication inside The
HCV-infected cells, as evidenced by the
disappearance of the (+) and /or (-) strands
RNA amplified products detected by the
RT-PCR (compare to positive control).
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Out of the tested extracts, lyophilized juice
of ginger proved to inhibit HCV replication
completely at concentration 100μg/ml, while
MSE inhibited HCV replication at
concentration 300μg/ml as shown in fig. 4.
4. DISCUSSION
Usually, screening of antiviral compounds
has been snagged due to the lack of
representative smaller animal model.
However, in the case of HCV infection in
humans, vector-based assay techniques have
been very helpful in screening a variety of
antiviral agents [31], but measuring the viral
RNA synthesis in the absence and presence
of the agent in an efficient cell culture
system remains important. Real time PCR
has proven to be accurate for determination
of antiviral drug susceptibility of herpes
viruses [32&33]. The real time PCR assay
was used in the current study to determine
whether the HCV–RNA replication is
inhibited in infected cells (PBMC and
HepG2) by the plant extract or not.
Our data showed that lyophilized juice of
ginger caused complete inhibition of
intracellular viral replication at 100μg/ml. This
confirms the previous investigation done by
Sookkongwaree et al, 2005 [34], where they
reported the inhibition of HCV NS3 protease
by ginger extract. They expressed the full
length of HCV NS3 protease and then they
purified and started the assay at enzymatic
level. The second extract tested in the
current study was the MSE, showing an
inhibitory effect at 300μg/ml. We have two
records about the antiviral effect of MSE, the
first is the clinical study done by Ferenci et al,
2008 [35], where they stated that silibinin ( one
of the main constituents of MSE) is a potent
antiviral agent in patients with chronic HCV not
responding to pegylated interferon/ribavirin
therapy. The second record is the in vitro study
done by Bonifaz et al., 2009 [36]. They
concluded that the silymarin (MSE)
significantly downregulated HCV core
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mRNA (by 20%–36%) and protein (by
30%–60%) in CNS3 cells. Our current study
confirms those previous studies, and
emphasizes the importance of screening of
several herbs aiming identifying an anti-
infectious agent offer a great potential in the
field of pharmaceutical developments.
5. CONCLUSION
It can be fairly concluded that lyophilized
juice of ginger and MSE have a great
potential as sources for novel lead
compounds with specific antiviral
properties. Further bioassays guiding
separation and isolation of the active
compounds is therefore of interest. Finally,
it is also of interest to determine whether the
inhibitory effects are due to a synergistic
effect of more than one effective compound
or due to the sole effect of a highly potent
compound. Such studies require more
extensive kinetic studies using the pure
compounds.
6.AKNOWLEGEMENTS
The work has been recieved the financial
support from Mubarak City for Scientific
Research and Technology Applications
(MUCSAT).The authors thank Dr. Marwa
Ali for her excellent technical assistance
7. REFERENCES
[1] Choo Q.L, Kuo G, Weiner A.J, Bradley
L.R.D.W and Houghton M. 1989. Isolation of a c
DNA clone derived from a blood- brone non-
non-B viral hepatitis genome. Science 244, 359-
362
[2] World Health Organization, 2009. Sixty-second
world health assembly, Viral Hepatitis Report, A
62/22, 1-5
[3] Lehman, E. M. and Wilson, M. L. 2009.
Epidemic hepatitis C virus infection in Egypt:
estimates of past incidence and future morbidity
and mortality. J Viral Hepat., 16(9): 650-658
[4] Saito I, Miyamura T, Ohbayashi A, Harada H,
Katayama T, Kikuchi S, Watanabe Y, Koi S,
Onji M and Ohta Y, 1990. Hepatitis C virus
infection is associated with the development of
ISSN:0976-0172
Journal of Bioscience And Technology
www.jbstonline.com
hepatocellular carcinoma. Proc Natl Acad Sci U
S A. Sep;87(17):6547-6579
[5] Fried MW, Shiffman ML, Reddy KR, Smith C,
Marinos G, Gonçales FL Jr, Häussinger D,
Diago M, Carosi G, Dhumeaux D, Craxi A, Lin
A, Hoffman J and Yu J. 2002. Peginterferon
alfa-2a plus ribavirin for chronic hepatitis C
virus infection. N Engl J Med. 26; 347(13):975-
982.
[6] El-Awady M.K., Ismail S.M, El-Sagheer M,
Sabour Y.A and Amr K.S. 1999. Assay for
hepatitis C virus in peripheral blood
mononuclear cells enhances sensitivity of
diagnosis and monitoring of HCV-associated
hepatitis. Clin. Chim. Acta 283: 1-14
[7] El-Awady M.K, Tabll A.A,, Redwan E.M, ,
Youssef S.S, Omran M.H and El-Demellawy
M. (2005). Flow Cytometric Detection of
Hepatitis C Virus Antigens in Infected
Peripheral Blood Leukocytes: Binding and
Entry.World J Gastroenterol. 11:5203-5208
[8] El-Awady M.K, Tabll A.A, El-Abd Y.S, Bahgat
M.M, Shoeb H.A, Youssef S.S, Bader El Din
N.G, , Redwan E.M, El-Demellawy M, Omran
M.H, El-Garf W.T., Goueli S. A (2006). HepG2
cells support viral replication and gene
expression of hepatitis C virus genotype 4 In
Vitro.World J Gastroenterol. 12, 4836-4842.
[9] Al-Sohaimy S.A, Hafez E.E, Abd El-Wahab A
and El-Saadani M.A.2007. Anti-HCV lectin
from Egypatian Pisum sativum. Australian
Journal of Basic and Applied Sciences, 1(3):
213-219
[10] El-Fakharany E.M, Tabll A.M, Abd El-Wahab
A.E, Haroun B.M and Redwan E.M. 2008.
Potential activity of camel milk-amylase and
lactoferrin against HCV infectivity in HepG2
and lymphocytes. Hepatitis Monthly. 8(2): 57-64
[11] Yamaguchi T, Sano K, Takakura K, Saito I,
Shinohara Y, Asano T and Yasuhara H. 1998.
Ebselen in acute ischemic stroke: a placebo-
controlled, double-blind clinical trial, Ebselen
Study Group. Srtoke 29(1): 12-17
[12] Cadenas E and Packer L. 2002. Handbook of
Antioxidants, 2 nd ed., review and expanded,
New York: Marcel Dekker
[13] Wilson J and Gelb A. 2002. Free radicals,
antioxidants and neurologic injury: possible
relationship to cerebral protection by
anesthestics. J Neurosurg Anesthesiol. 14(1):
66-79
[14] Rao A and Balachandran B. 2002. Role of
oxidative stress and antioxidants in
neurogegenerative disease. Nutr Neurosci.
5(50): 291-309
230
Hala El-Adawi etal ,J Biosci Tech, Vol 2 (1),2011,223-231
[15] Di Matteo V and Esposito E. 2003.
Biochemical and therapeutic effects of
antioxidants in the treatment of Alzheimer,s
disease, parkinson,s disease, and amyotrophic
lateral sclerosis. Curr. Drug Targets CNS
Neurol Disord. 2(2): 95-107
[16] Warner D, Sheng H and Batinic-Haberle I.
2004. Oxidants, antioxidants and the ischemic
brain. J Exp Biol 207 (18): 3221-3231
[17] Afzal M, Al-Hadidi D, Menon M, Pesek J,
Dhami MS. (2001). Ginger: an ethnomedical,
chemical and pharmacological review. Drug
Metabol Drug Interact.18: 159-90.
[18] Jeyakumar SM, Nalini N and Menon VP. 1999
Antioxidant activity of ginger (Zingiber
officinale) in rats fed a high fat diet. Med Sci
Res. 27:341-44.
[19] Shukla Y and Singh M. 2007: Cancer
preventive properties of ginger: A brief review.
Food Chem Toxicol.; 45: 683-90.
[20] Hudson, E.A., Fox, L.H., Luckett, J.C.A and
Manson, M.M. 2006: Ex vivo cancer
chemoprevention research possibilities.
Environmental Toxicology and
pharmacology.21: 204-214.
[21] Habib SH, Makpol S, Abdul Hamid NA, Das S,
Ngah WZ and Yusof YA. 2008. Anti-
inflammatory effect of ginger extract on
hepatocarcinoma 813. CLINICS, 63(6):807-813
[22] Huang S, DeGuzman A, Bucana CD and Fidler
IJ. 2000. Nuclear factor-kappaB activity
correlates with growth, angiogenesis, and
metastasis of human melanoma cells in nude
mice. Clin Cancer Res. 6: 2573-2581.
[23] Coppola G and Novo S. 2007: Statins and
peripheral arterial disease: effects on
claudication, disease progression, and
prevention of cardiovascular events. Arch Med
Res.; 38: 479-488.
[24] Greenlee H, Abascal K, Yarnel E, Ladas E.
2007. Clinical applications of Silybum
marianum in oncology. Integrative Cancer
Therapies 6:158-165.
[25] Tamayo C and Diamond S. 2007. Review of
clinical trials evaluating safety and efficacy of
milk thistle (Silybum marianum [L.] Gaertn.).
Integrative Cancer Therapies.6:146-157
[26] Palma M and Taylor L.T. 1999. Extraction of
polyphenolic compounds from grape seeds with
near critical carbon dioxide. J. of
Chromatography A. 849(1): 117-124
ISSN:0976-0172
Journal of Bioscience And Technology
www.jbstonline.com
[27] Ettinger, M. B.; Ruchhoft, C. C. and Lishka, R.
J. (1951): Sensitive 4-aminoantipyrine method
for phenolic compounds. Anal. Chem., 23: 1783
[28] Osawa, T. and Namiki, M. (1981): A novel type
of antioxidant isolated from leaf wax of
Eucalyptus leaves. Journal of Agricultural and
Biological Chemistry, 45: 735-739.
[29] Kato N, Ikeda M, Mizutani T, Sugiyama K,
Noguchi M, Hirohashi S and Shimotohno K.
1996. Replication of hepatitis C virus in
cultured non-neoplastic human
hepatocytes.Jpn.J.Cancer Res. 87:787-792
[30] Lohr HF, Goergen B, Meyer zum Buschenfelde
KH, Gerken, G. 1995. HCV replication in
mononuclear cells stimulates anti-HCV-
secreting B cells and reflects nonresponsiveness
tointerferon-alpha. J Med Virol; 46: 314-20.
[31] Eismone C.O, Grunwald T, Wildner O,
Nchinda G, Tippler B, Proksch.P Uberla K.
2005. In viro pharmacodynamic evaluation of
antiviral medicinal plants using a vector- based
assay technology. J.Appl. Microbiol. 99(6):
1346-1355.
[32] Stránská R., Van Loon A.M, Polman M and
Schuurman R. 2002. Application of real-time
PCR for determination of antiviral drug
susceptibility of Herpes simplex virus.
Antimicrob. Agents Chemother.46: 2943-2947
[33] Sergerie Y and Boivin G. 2003. Evaluation of
susceptibility of human herpesvirus 8 to
antiviral drugs by quantitative real-time PCR.
J.Clin Microbiol. 41: 3897-3900
[34] Sookkongwaree K, Geitmann M, Roengsumran
S, Petsom A and Daielson U H. 2006.
Inhibition of viral proteases by Zingiberaceae
extracts and flavones isolated from Kaempferia
parviflora. Pharmazie, 61: 717-721
[35] Ferenci P, Scherzer TM, Kerschner H, Rutter
K, Beinhardt S, Hofer H, Schöniger- Hekele M,
Holzmann H, Steindl-Munda P.2008. Silibinin
is a potent antiviral agent in patients with
chronic hepatitis C not responding to pegylated
interferon/ribavirin therapy. Gastroenterology,
135(5): 1561-1567
[36] Bonifaz V, Shan Y, Lambrecht RW, Donohue
SE, Moschenross D, Bonkovsky HL. 2009:
Effects of silymarin on hepatitis C virus and
haem oxygenase-1 gene expression in human
hepatoma cells. Liver Int. ,29(3):366-373.
231