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Anti-influenza virus activity of green tea by-products in vitro and efficacy

against influenza virus infection in chickens

Article in Poultry Science · January 2012

DOI: 10.3382/ps.2011-01645 · Source: PubMed

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 IMMUNOLOGY, HEALTH, AND DISEASE

Anti-influenza virus activity of green tea by-products in vitro and efficacy

against influenza virus infection in chickens

H. J. Lee,* Y. N. Lee,* H.-N. Youn,* D. H. Lee,* J. H. Kwak,† B. L. Seong,‡ J. B. Lee,* S. Y. Park,*

I. S. Choi,* and C. S. Song*1

*College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701 Korea;
†College of Pharmacy, Sung-kyunkwan University, Cheoncheon-Dong 300, Jangan-gu, Suwon, Gyeonggi-do, 440-
746 Korea; ‡Department of Biotechnology, College of Life Science and Biotechnology,
Yonsei University, Seodaemun-gu, Seoul, 120-749 Korea

ABSTRACT Polyphenolic compounds present in green
tea, particularly catechins, are known to have strong
anti-influenza activity. The goal of this study was to
determine whether green tea by-products could func-
tion as an alternative to common antivirals in animals
compared to original green tea. Inhibition of viral cy-
topathic effects ascertained by neutral red dye uptake
was examined with 50% effective (virus-inhibitory)
concentrations (EC50) determined. Against the H1N1
virus A/NWS/33, we found the anti-influenza activity
of green tea by-products (EC50 = 6.36 μg/mL) to be
equivalent to that of original green tea (EC50 = 6.72
μg/mL). The anti-influenza activity of green tea by-
products was further examined in mouse and chicken
Key words: green tea, by-product, influenza, anti-influenza, chicken
influenza infection models. In mice, oral administra-
tion of green tea by-products reduced viral titers in the
lungs in the early phase of infection, but they could
not protect these animals from disease and death. In
contrast, therapeutic administration of green tea by-
products via feed or water supplement resulted in a
dose-dependent significant antiviral effect in chickens,
with a dose of 10 g/kg of feed being the most effective
(P < 0.001). We also demonstrated that unidentified
hexane-soluble fractions of green tea by-products pos-
sessed strong anti-influenza activity, in addition to eth-
yl acetate-soluble fractions, including catechins. This
study revealed green tea by-product extracts to be a
promising novel antiviral resource for animals.
2012 Poultry Science 91:66–73
doi:10.3382/ps.2011-01645

INTRODUCTION to livestock animals. These issues highlight the urgent

The influenza virus, a highly infective agent that
causes acute pulmonary diseases, continues to emerge
and re-emerge around the world. The ecology and epi-
demiology of influenza viruses involve various wild and
domestic avian species, as well as various mammalian
hosts, including humans, pigs, and horses. Furthermore,
the capacity for transmission among (or between) ani-
mals and humans has raised worldwide concern about
a possible pandemic. Although influenza vaccination is
the primary way to prevent the disease, antiviral drugs
are an important adjunct to vaccine for the control
and prevention of influenza. However, antiviral medica-
tions are not approved for use in animals because of
drug resistance concerns (Beard et al., 1987; Ilyushina
et al., 2005), and they are too expensive to administer
©2012 Poultry Science Association Inc.
Received May 31, 2011.
Accepted August 13, 2011.
1 Corresponding author: songcs@konkuk.ac.kr
need to discover alternative antiviral materials suitable
for animal use because it is important to minimize the
source and spread of influenza viruses and genes from
animals to humans.
Green tea is a historically popular beverage among
Asian populations, and is produced from the leaves of
the evergreen plant Camellia sinensis. The major active
ingredients in green tea are polyphenolic compounds
known as catechins (Balentine et al., 1997), which are
reported to possess antiviral and antimicrobial activi-
ties (Weber et al., 2003; Yan et al., 2004; Savi et al.,
2006). Furthermore, a recent study on catechins showed
that they could alter the infectivity of an influenza vi-
rus not only by specific interaction with viral hemag-
glutinin (HA), but also with viral RNA synthesis in
cells (Song et al., 2005). In addition, catechins inhibited
the endonuclease activity of viral RNA polymerase; the
galloyl group is important for viral RNA polymerase
function (Kuzuhara et al., 2009). Therefore, the poten-
tial use of green tea and its constituent catechins for
protecting against influenza have attracted attention.

66
 GREEN TEA BY-PRODUCTS AGAINST INFLUENZA
In the commercial green tea beverage industry, by-
products that remain after processing still contain
large amounts of proteins, carbohydrates, and phenolic
compounds. Several studies have reported on green tea
by-products used as potential sources of natural anti-
oxidants or functional materials (González-Paramás et
al., 2004; Lee et al., 2006; Potter et al., 2010). In addi-
tion, green tea by-products are recognized as a cheap
and effective nutritional feed supplement for use in the
livestock industry.
The objective of the present study was to investigate
the anti-influenza activity of green tea by-products in
comparison to that of original green tea using mouse
and chicken models in order to elucidate possible their
mechanisms of action on the influenza virus, and to
evaluate their potential as an alternative antiviral for
animals.
MATERIALS AND METHODS
Extraction and Isolation
Green tea leaves were collected at Bosung, Jeolla-
nam-do, Korea. Three types of green tea and green tea
by-product extracts were prepared as follows. Green
tea leaves (2.0 kg) were extracted 2 times with distilled
water (20 L) at 90°C for 5 h, and the extract was ly-
ophilized (at −50°C for 24 h) to obtain the green tea
water extract (30.1% yield). After the water extraction,
the green tea leaves were air-dried for the preparation
of the green tea by-products, which were subsequently
extracted with 60% ethanol (14 L) at 42°C for 5 h to
obtain the green tea by-product extract (20.5% yield).
The green tea extract was prepared by direct 60% etha-
nol extraction (at 42°C for 5 h) from original green tea
leaves (25.1% yield). In addition, green tea by-products
(1.4 kg) were extracted with methanol (twice with 14 L,
at 42°C for 5 h). The methanol extract was evaporated
to dryness under reduced pressure, and a portion of the
residue (150 g) was dissolved in methanol-H2O (9:1, 1.5
L). The resulting solution was partitioned with hexane
(1.5 L × 3), and then the aqueous methanol solution
was diluted with H2O to 30% of H2O in methanol and
extracted with dichloromethane (1.5 L × 3). The aque-
ous methanol solution was concentrated in vacuo, and
the aqueous residue was diluted with H2O to 1.5 L,
and then consecutively partitioned with ethyl acetate
(1.5 L × 3), and n-butanol (1.5 L × 3). Each of the
solvent fractions was evaporated under reduced pres-
sure to yield hexane (26.3 g), dichloromethane (31.8
g), ethyl acetate (59.5 g), n-butanol (15.1 g), and water
(16.0 g) extracts.
Virus and Cell
Human influenza virus A/NWS/33 (H1N1) and
avian influenza virus (AIV) A/chicken/Korea/310/01
(H9N2) were propagated in 9- to 11-d-old embryonated
chicken eggs for 48 h in an incubator at 37°C. Madin-
67
Darby canine kidney (MDCK) cells were obtained
from the American Type Culture Collection (Manas-
sas, VA) and cultured as monolayers in minimum es-
sential media supplemented with 8% heat-inactivated
fetal bovine serum.
Neutral Red Assay
The in vitro anti-influenza efficacy and toxicity of the
extracts and compounds were determined by measur-
ing the uptake of neutral red, as described previously
(Sidwell et al., 1995; Smee et al., 2001b; Sidwell et al.,
2005). To determine cytotoxicity, confluent MDCK
monolayers in 96-well plates were washed 2 times with
PBS, and then exposed to extracts or compounds in
200 µL of medium for 48 h at 37°C in 5% CO2. To
determine inhibitory efficacy, inoculums were aspirated
following viral adsorption, cells were washed 2 times
with PBS, and then they were incubated in 200 µL
of medium containing extracts or compounds for 48
h at 37°C in 5% CO2. After 48 h, neutral red dye was
added to the medium in each well at a concentration
of 0.034%, and incubated for 2 h at 37°C. After incu-
bation, the wells were rinsed, dried, and stored in the
dark. Dye was extracted for 30 min in the dark at room
temperature in absolute ethanol buffered with Soren-
son citrate buffer. The absorbance was measured spec-
trophotometrically at 540 nm with an ELISA micro-
plate reader (Sunrise; Tecan Trading AG, Saltzburg,
Austria). The cytotoxic concentration for 50% of cells
(CC50) and effective (virus-inhibitory) concentration
for 50% of infected cells (EC50) values were estimated
from concentration–effect curves after linear regression
analysis with r2 ≥ 0.9, representing the mean values of
3 independent experiments. The ratio between CC50
and EC50 was calculated as the selectivity index (SI).
Hemagglutination Inhibition Assay
Green tea by-product solutions (25 µL) at the in-
dicated concentrations were added to 96-well plates,
followed by 25 µL of virus with 200 hemagglutinat-
ing units/25 µL. The plates were incubated for 60 min
at 4°C. After this incubation period, 50 µL of chicken
erythrocyte suspension (0.5% in PBS) was added to
each well. The plates were further incubated for 60 min
for 4 h at 4°C. The wells were visually inspected for the
presence or absence of hemagglutination. Positive and
negative controls without treatment or without virus
were included. To assay possible hemagglutination by
green tea by-product solution itself, 50 µL of solution
in PBS at the indicated concentrations was incubated
with 50 µL of chicken erythrocyte suspension for 60 min
for 4 h at 4°C. All assays were performed in triplicate.
Neuraminidase Inhibition Assay
A neuraminidase inhibition assay was conducted to
test the effect of samples on the neuraminidase activ-
68 Lee et al.
ity of the tested viruses, as described previously (Shin
et al., 2010). Briefly, 2-fold dilutions (25 µL) of green
tea by-product extracts and selected compounds rang-
ing from 31.25 to 4,000 µg/mL in reaction buffer (150
mM sodium acetate with 1 mM calcium chloride, pH 7)
were mixed with equal volumes (25 µL) of influenza A/
NWS/33(H1N1) virus. After 1 h of incubation at room
temperature, equal volumes (50 µL) of the substrate so-
lution (4-MU-NANA; 2′-(4-methylumbelliferyl)-α-d-N-
acetylneuraminic acid sodium; Sigma, St. Louis, MO)
were added, and the mixture was further incubated and
protected from light at 37°C for 2 h. The optical den-
sity was then measured by fluorescence plate reader
(Gemini EM; Molecular Devices,Sunnyvale, CA) at an
excitation wavelength of 365 nm and an emission wave-
length of 450 nm. Standard curves were made by plot-
ting the percentage of fluorescence inhibition relative to
the activity of virus controls against the logs of inhibi-
tor concentrations. The 50% inhibitory concentration
(IC50) was calculated by regression analysis with r2
≥ 0.9, representing the mean values of 3 independent
experiments.
Quantitative Reverse-Transcription
PCR Analysis
The MDCK cells (approximately 90% confluent) were
infected with influenza A/NWS/33 virus [0.1 multiplic-
ity of infection (MOI)] and cultured in the presence of
green tea by-product solutions at the indicated concen-
trations. After 16 h of incubation, cells were scraped off
and collected by centrifugation (500 × g for 5 min at
4°C). Total cellular and viral RNA were isolated from
pellets using the RNeasy mini kit (Qiagen Inc., Valen-
cia CA) according to the manufacturer’s instructions.
First-strand cDNA synthesis and subsequent PCR re-
actions were carried out as described previously (Song
et al., 2005).
By-Product Extract Inhibition of Influenza
By Oral, Diet, and Water Administration
All animal procedures performed in this study were
reviewed, approved, and supervised by the Institutional
Animal Care and Use Committee of Konkuk University
(Seoul, Korea).
Each group of 10 BALB/c mice (approximately 17–
19 g) was orally treated with various types of green tea
by-product extract (at doses of 100, 10, or 1 mg/kg per
d) before 4-h intranasal virus exposure [influenza A/
NWS/33 (H1N1), 103.0 50% tissue culture infectious
dose (TCID50)/90 µL per mouse], as described previ-
ously (Sidwell et al., 2001). The extracts were admin-
istered by oral gavage daily for 5 d. On d 3 and 6, 5
mice per group were killed for lung infection parameter
analysis. Lungs were collected, weighed, given a sever-
ity score based on lung consolidation, and frozen for
assay of infectious virus titer (Sidwell et al., 1998; Smee
et al., 2001a).
Each group of 10 three-week-old specific-pathogen-
free (SPF) chickens was intranasally challenged with
influenza A/chicken/Korea/310/01(H9N2) virus at a
dose of 106.0EID50/0.1 mL per bird where EID stands
for egg infectious dose. Treatment was initiated 4 h
before virus exposure, and a diet supplemented with
lyophilized green tea by-product extract (1, 4, or 10 g/
kg of feed) or drinking water containing green tea by-
product extract (10, 1, or 0.1 mg/mL) was provided for
5 d. On d 5 after the virus challenge, the tracheas and
cecal tonsils were collected for isolation of the challenge
virus (Swayne et al., 2000).
RESULTS
Inhibitory Effect of By-Product Extracts
and Solvent Fractions
For initial comparison of antiviral activity, each
green tea leaf extract was tested by neutral red assay
in MDCK cells at a fixed concentration. The EC50 of
samples is summarized in Table 1. As expected, green
tea extract from the original leaves exhibited strong
inhibitory effects against the influenza virus (EC50 =
6.72 µg/mL, SI = 7.87). In contrast, the green tea wa-
ter extract, representing the most consumed form of
green tea, had inhibitory effects on the influenza virus
at 5- to 6-fold higher concentrations (EC50 = 39.57
µg/mL) than that of the green tea extract, whereas
the green tea by-product extract had strong inhibitory
effects (EC50 = 6.36 µg/mL, SI = 9.22). These results
indicate that anti-influenza active ingredients seemed
to be extracted merely by water infusion, and thus, re-
mained largely present in by-products, strongly inhib-
iting influenza virus in vitro. To identify the effective
fractions of green tea by-products, 5 different solvent
fractions were tested, as mentioned previously. Among
them, the hexane-soluble fraction (EC50 6.3 µg/mL)
and the ethyl acetate-soluble fraction (EC50 6.69 µg/
mL) showed higher anti-viral activities than those of
the other 3 fractions (Table 1).
Mode of Inhibitory Effect of By-Product
Extracts and Fractions
Influenza virus replication is characterized by a com-
plex sequence of different steps, during which time anti-
viral agents can interfere. To investigate the inhibitory
effects on influenza virus in detail, green tea by-product
extract and 2 effective fractions (ethyl acetate soluble
and hexane soluble) were added at different stages of
viral infection.
Initially, we evaluated whether the green tea by-prod-
uct extract could inhibit the interaction between the
viral HA and a specific cellular sialic acid-containing
 GREEN TEA BY-PRODUCTS AGAINST INFLUENZA 69
Table 1. In vitro efficacy of various green tea extracts and solvent fractions against influenza viruses1
Sample EC50 (µg/mL) CC50 (µg/mL) SI

Green tea extracts 6.72 52.88 7.87

Green tea water extracts 39.57 182.75 4.62
Green tea by-product extracts 6.36 58.67 9.22
Solvent fractions of green tea by-products
Hexane 6.3 40.58 6.44
Dichloromethane 24.44 77.53 3.17
Ethyl acetate 6.69 51.1 7.64
n-Butanol NE — —
Water 20.43 520.6 25.48
1CC : 50% cytotoxic concentration; EC : 50% effective concentration; SI: selectivity index (CC /EC ),
50 50 50 50
where a higher SI means a more selective compound; and NE: not effective.

receptor. Although a previous report had demonstrated
that green tea and related catechins exhibited hemag-
glutination inhibition (HI) activity (Song et al., 2005),
we did not observe HI activity of the green tea by-
product extract or the 2 selected fractions on the influ-
enza virus at any concentration tested (approximately
4,000–15.625 µg/mL, 2-fold dilution). These conflicting
results related to HI activity may be because of differ-
ences in the extraction method and preparation of the
crude extracts and their purity.
We then tested the inhibitory effects of the green tea
by-product extract and the 2 selected fractions on the
transcription of viral genes in infected cells by quanti-
tative reverse transcription (RT)-PCR of influenza vi-
rus-specific mRNA (Figure 1). Downregulation of viral
RNA synthesis was evident only at the high concentra-
tion of green tea by-product extract (>800 µg/mL). In
the selected fractions, the inhibitory effect of the ethyl
acetate-soluble fraction was observed over 0.4 µg/mL,
but it was not observed with the hexane-soluble frac-
tion at the highest concentration (1 µg/mL) tested in
this experiment.
The inhibitory effect of green tea and related cat-
echins on viral neuraminidase has been previously re-
ported (Song et al., 2005). Therefore, we tested the green
tea by-product extract and the 2 selected fractions in
a neuraminidase inhibition assay. We determined that
the activity of neuraminidase was decreased significant-
ly by the by-product extract (IC50 = 188.72 µg/mL)
and the ethyl acetate-soluble fraction (IC50<31.25 µg/
mL), but barely at all by the hexane-soluble fraction
(IC50 = 2,573.44 µg/mL) (Table 2).
Inhibitory Effect of By-Product Extracts
on Influenza in Mouse and Chicken
There was no beneficial effect of green tea-related
extracts on the survival and clinical signs of mice chal-

Figure 1. Effect of green tea by-product extracts and selected fractions on influenza viral RNA synthesis in infected cells. Cells approximately
90% confluent were infected with the virus at 0.1 multiplicity of infection (MOI) and cultured with minimum essential media containing different
concentrations of extracts and compounds. Cells were lysed at 16 h postinfection, and total cellular and viral RNA were isolated from the cell pel-
lets. Quantitative reverse-transcription PCR was performed using specific primers for viral RNA (NP) and cellular RNA (β-actin). Similar results
were obtained in 3 triplicate experiments.
70
Table 2. Inhibitory effects of green tea by-product extract and
selected fractions on viral neuraminidase activities1
Sample
Green tea by-product extracts
Hexane-soluble fractions of green tea by-products
Ethyl acetate-soluble fractions of green tea by-products
1IC :
50 50% inhibitory concentration.
lenged with lethal influenza (data not shown). How-
ever, we observed a significant reduction in lung virus
titer in mice treated with the highest concentration of
extract (100 mg/kg per d) 3-d postchallenge, regardless
of extract type (P < 0.05, green tea water extract; P <
0.01, green tea extract; P < 0.001, green tea by-product
extract; Table 3). Treatment with low concentrations of
extracts (10 and 1 mg/kg per d) failed to reduce lung
virus titers (P > 0.05, data not shown).
Green tea by-product extract administered as either
10 g/kg of feed or 1 mg/mL of water induced a signifi-
cant decrease in the number of SPF chickens that shed
AIV (H9N2) in cecal tonsils 5 d after the challenge
(Table 4). As seen in Figure 2, administration of the
green tea by-product extract as a feed additive and
in drinking water also significantly reduced the titer
of challenge virus recovered from cecal tonsils (100.35–
103.8 EID50/g) compared with that of the nontreated
controls (104.9 EID50/g). The reduction in virus titer
was greater in the high-dose treatment groups (10 g/
kg of feed, and 10 and 1 mg/mL of water) than in the
low-dose treatment groups (Figure 2).
DISCUSSION
The primary objective of the present study was to in-
vestigate the possibility of using green tea by-products
as anti-influenza agents for animals. According to a
previous study (Song et al., 2005), catechin compounds
were highly concentrated in the ethyl acetate-soluble
fraction of green tea leaf extract. Green tea catechins
have previously been shown to exert anti-influenza
effects by inhibiting various steps in the life cycle of
Lee et al.
viruses (Carr and Kim, 1993; Nakayama et al., 1993;
Imanishi et al., 2002; Song et al., 2005). In the present
IC50 (µg/mL) study, both green tea by-product extract and its ethyl
acetate-soluble fraction showed effective inhibitory ef-
188.72
2,573.44 fects against influenza at various virus lifecycle steps,
<31.25 including transcription and release. In agreement with
the low water solubility of catechin (Nwuha et al., 1999;
Chung et al., 2003), the anti-influenza activity of the
ethyl acetate-soluble fraction was considerable because
of the catechin compounds. Differences in inhibitory
activity between the extract and the fraction may be
associated with differences in the concentration of ac-
tive compounds as a result of the extraction process.
Although water extracts of green tea showed inhibitory
activity on influenza, green tea by-products can still
be a potent anti-influenza resource after the extrac-
tion process because of the large amount of remaining
catechins.
In mice, clinical symptoms and fatality induced by
influenza were not decreased by treatment with green
tea by-product extract, and lung virus titer only de-
creased in the early phase of infection. These results
are supported by a previous report where green tea
catechin inhibited initial entry of an influenza virus
(Song et al., 2005). In contrast, an anti-influenza effect
of green tea by-products was obvious in chickens (Table
4; Figure 2). Considering intestinal absorption and me-
tabolism of green tea catechins (Donovan et al., 2001;
Scalbert et al., 2002), natural catechin, rather than
catechin metabolites, is expected to interact effectively
with influenza. Because the organs most affected by
influenza differ according to host species (respiratory
tract of mice and intestinal tract of chickens), orally ad-
ministered green tea by-product extract was expected
to inhibit influenza more effectively in chicken intes-
tines than that of its metabolites in mouse lungs. This
explanation is in agreement with the apparent interfer-
ence of green tea by-product extracts, fractions (in this
study), and catechins (Song et al., 2005, 2007) on viral
neuramanidase and HA directly. Therefore, green tea
by-product is regarded as more profitable for poultry
usage than for usage in mammals, but other application
methods for the delivery of by-product into the lungs;

Table 3. Antiviral effects of orally administered green tea extract (100 mg/kg per d) against influenza A/NWS/33 (H1N1) virus
infection in BALB/c mice
Mean lung parameter

d3 d6

Score2 Lung weight Virus titer Score Lung weight Virus titer
Treatment1 ± SD (mg ± SD) (log10/g ± SD) ± SD (mg ± SD) (log10/g ± SD)

Green tea water extract 0.7 ± 0.1 124 ± 5 5.4 ± 0.9* 2.5 ± 0.4 170 ± 45 6.3 ± 0.2
Green tea by-product extract 0.6 ± 0.1 120 ± 7 5.0 ± 0.8*** 2.3 ± 0.3 164 ± 18 6.2 ± 0.6
Green tea extract 0.7 ± 0.1 120 ± 6 5.7 ± 1.1** 2.4 ± 0.1 182 ± 8 6.0 ± 0.3
Distilled water 0.7 ± 0.1 128 ± 8 6.6 ± 0.4 2.9 ± 0.2 174 ± 39 6.5 ± 0.1
Normal control 0.0 ± 0.0 116 ± 5 0.0 ± 0.0 0.0 ± 0.0 128 ± 18 0.0 ± 0.0
1Oraltreatments were given for 5 d, starting 4 h before virus exposure.
2Lungs were assigned a consolidation score ranging from 1 (normal) to 4 (maximal plum coloration).
*P < 0.05, **P < 0.01, ***P < 0.001 compared with saline-treated challenge controls by Student’s t-test.
 GREEN TEA BY-PRODUCTS AGAINST INFLUENZA 71
Table 4. Reduction of viral shedding in tracheas and cecal tonsils of specific-pathogen-free chickens
treated with green tea by-product extracts in feed or water after challenge with influenza A/chicken/
Korea/310/01(H9N2) virus
Virus isolation2

Treatment1 Concentration Trachea Cecal tonsil

By-product
Feed additive (g/kg of feed) 10 3/10 1/10*
4 5/10 6/10
1 5/10 4/10
Drinking water (mg/ML) 10 3/10 4/10
1 3/10 3/10**
0.1 6/10 6/10
Control3 — 5/10 9/10
1Oral treatments were given for 5 d, starting 4 h before virus exposure.
2The chickens were intranasally challenged with 0.1 mL of inoculums containing 106.0EID50 of virus: no. of birds
reisolated/no. of birds inoculated; EID = egg infectious dose.
3Control groups not treated after virus challenge.

*P < 0.05, **P < 0.001 compared with nontreated controls by Fisher’s exact test.

for example, aerosol treatment (Droebner et al., 2007) Interestingly, the anti-influenza activity of the hex-
or nasal inoculation, may be effective in preventing re- ane-soluble fraction was as potent as that of the ethyl
spiratory infection in mammals. acetate-soluble fraction in MDCK cells. This result sug-

Figure 2. Avian influenza virus (AIV) titers in specific-pathogen-free (SPF) chickens treated with green tea by-product extracts after challenge
with influenza A/chicken/Korea/310/01(H9N2) virus. The SPF chickens, 3 wk in age, were intranasally inoculated with A/chicken/Korea/310/01
(H9N2) at a dose of 106.0EID50/0.1 mL per bird, where EID = egg infectious dose. The birds were treated for 5 d with green tea by-product
as a feed additive or in drinking water. On d 5, tracheas and cecal tonsils were collected to isolate the challenge virus. The error bars indicate
standard deviations from virus shedding titers of 10 chickens per group. *P < 0.05, **P < 0.005, and ***P < 0.001 by t-test, compared with the
nontreated challenge controls.
72 Lee et al.
gests that green tea leaves might contain noncatechin
antiviral compounds. Similarly, a study by Song et al.
(2005) suggested that factors other than catechins are
more responsible for anti-influenza activity, especially
for HI. However, the hexane-soluble fraction failed
to inhibit viral absorption, viral RNA synthesis, and
neuraminidase activity at the highest concentration ex-
amined. Many of the molecular mechanisms have been
attributed to anti-influenza activities. The green tea
extract exhibited anti-influenza effects by the acidifica-
tion of intracellular compartments, such as the endo-
somes and lysosomes (Imanishi et al., 2002). Fukuda et
al. (2003) determined that the hexane-soluble fraction
of green tea, in particular chlorophylls and lutein, ef-
fectively suppressed transformation of the aryl hydro-
carbon receptor, which correlates with an increased
severity of influenza (Neff-LaFord et al., 2007; Jin et
al., 2010). The main compounds of the hexane-soluble
fraction of the green tea by-products, and the detailed
mechanism involved in anti-influenza activity should be
investigated further in future studies.
There is no specific treatment for influenza virus
infection in animals, especially in poultry. Although
vaccines have been used to control influenza in vari-
ous poultry and other birds, vaccinated birds can po-
tentially become infected, shedding contagious viruses
into the environment because of incomplete immunity
induced by the vaccines. We have shown that oral ad-
ministration of green tea by-products in feed or water
reduces influenza replication in chickens. With regard
to the role of birds in AIV transmission, reduction of
viral shedding in chicken feces by treatment with green
tea by-product extracts would be useful for minimizing
viral circulation among bird species. A synergistic effect
would also be obtained by adaptive immunity induced
by vaccines. Green tea is already approved for use in
both humans and animals as a food or feed additive,
and its by-product extracts are a renewable, natural
product that is inexpensive to produce. We believe that
green tea by-products hold great potential for use in
animals at a very affordable cost, not only for prevent-
ing viral infections, but also for reducing the spread of
influenza viruses in epidemic areas.
ACKNOWLEDGMENTS
This work was supported by grants No.109015-03-2-
CG000 from the Korea Institute of Planning and Eval-
uation for Technology in Food, Agriculture, Forestry
and Fisheries (Anyang, Korea).
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