# 3,4-Dicaffeoylquinic Acid, a Major Constituent of Brazilian Propolis,

Authors: Tomoaki Takemura; Tomohiko Urushisaki; Mayuko Fukuoka; Junji Hosokawa-Muto; Taketoshi Hata; Yumiko Okuda; Sachie Hori; Shigemi Tazawa; Yoko Araki; Kazuo Kuwata
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic
acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus
(IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities
of these compoundsin vivo. PWE or PEE (Brazilian green propolis ethanol
extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had
been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were
significantly extended compared to the untreated mice. Moreover, oral administration of
3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than
PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly
increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly
decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique
mechanism of anti-influenza viral activity, that is, enhancing viral clearance by
increasing TRAIL.


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## Body

## 1. Introduction

Influenza is a common infectious disease, and various anti-influenza drugs are already on
the market. For example, M2 ion channel inhibitors (amantadine and rimantadine) and
neuraminidase inhibitors (zanamivir, oseltamivir, etc.) have been used to treat influenza
viral infections in many areas, including the United States, Europe, and Asia [1–3].
Although oseltamivir is one of the most popular anti-influenza drugs, many resistant strains
have already been discovered [4, 5]. Fortunately, the resistant strains that have been found to date are
not yet pandemic. However, we need to carefully monitor the emergence and prevalence of new
strains with pandemic characteristics, such as the H1N1 pandemic with drug resistance, which
may occur because of the high incidence of mutations [6] and genomic rearrangements [7]. To avoid
the worst, the discovery of novel anti-influenza agents that do not carry the risk of
creating resistant strains would be urgently required.Propolis, a resinous substance collected by honeybees from various plant sources, is used
by the bees to protect the nest entrance against intruders and bacteria. Since ancient
times, it has been used as a traditional folk medicine in many countries. Recent studies
have demonstrated that propolis has a wide range of pharmacological properties, including
antibacterial [8–10], antioxidant [11],
anti-inflammatory [12], and antitumor activities
[13]. The activity and constituents of propolis
vary with its geographical origin [14]. The
differences in its constituents arise from differences in the plants from which it is
sourced [15].The antiviral activity of propolis has been reported and includes anti-BBMV [16], anti-HSV [17–21], antipoliovirus [22], anti-IBDV [10], anti-reovirus [10], antipotato viruses
[23], and anti-HIV [24] activities [25, 26]. The anti-influenza activity of propolis has been also described in
several reports [27–30], but the details of its activity were different. The reason for this
may be differences in the plant origin of the propolis [14].Recently, we have demonstrated that Brazilian green propolis water extract (PWE) and its
components, particularly 3,4-dicaffeoylquinic acid (3,4-diCQA), have anti-IAV activity
[31]. Moreover, we found that a central mechanism
of these compounds is the enhancement of cellular viability via unknown cellular processes
rather than activity against the viral components [31].Here, we examined thein vivo effects of propolis extracts on the
lifetimes of mice infected with the IAV. To obtain insights into the mechanism of the
antiviral effects of PWE and PEE, we examined tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) and IAV hemagglutinin (HA) gene expression in the lungs,
because it has been reported that the ethanol extract of Polish propolis has anti-tumor
activity via TRAIL enhancement [32].


## 2. Methods

### 2.1. Reagents and Compounds

Oseltamivir phosphate (Tamiflu) was purchased from the Chugai Pharmaceutical Co., Ltd.
(Tokyo, Japan). Chlorogenic acid was purchased from the Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). All media and reagents for cell culture were purchased from Invitrogen
(Carlsbad, CA, USA), Sigma (St. Louis, MO, USA), and Wako Pure Chemicals (Osaka, Japan).
Water extracts of Brazilian green propolis (Minas Gerais State, Brazil) originating fromBaccharis dracunculifolia [33]
were obtained from the API Co., Ltd. (Gifu, Japan). 3,4-diCQA (99% or 82%
purity) was purified from 25% ethanol-extracted Brazilian green propolis via column
chromatography and HPLC (details not shown).


### 2.2. Mice

Female BALB/c mice (5 weeks old) were obtained from Japan SLC, Inc. (Hamamatsu, Japan)
and housed at room temperature (maintained at 23 ± 3°C) with a relative humidity
range of 32–64% and a regular 12 hr light/dark cycle. The mice were fed
a CE-2 rodent diet from CLEA Japan Inc. (Tokyo, Japan) and allowed free access to
water.


### 2.3. Viruses

The influenza virus wild-type strain A/WSN/33 (H1N1), generated from cloned cDNAs using
plasmid-based reverse genetics [34], was kindly
supplied by Dr. Yoshihiro Kawaoka (Division of Virology, Department of Microbiology and
Immunology, Institute of Medical Science University of Tokyo, Japan). The viruses were
stored at −80°C until use.


### 2.4. Cells

Madin-Darby canine kidney (MDCK) cells were a kind gift from Professor Hideto Fukushi
(United Graduate School of Veterinary Sciences, Gifu University) and were maintained inα-minimal essential medium (MEM) containing 10% fetal bovine
serum (FBS) and penicillin-streptomycin.


### 2.5. Virus Preparation in Cell Culture and Determination of the TCID50
Value

MDCK cells were cultured in T-75 culture flasks. Confluent cells were washed with
phosphate-buffered saline (PBS) and then incubated with the virus (0.1 multiplicity of
infection (MOI)) in 2 mL of viral growth medium (D-MEM (Dulbecco’s Modified
Eagle Medium) containing 0.625μg/mL of trypsin and 0.1% bovine serum albumin (BSA))
for 1 hr at 37°C. Then, 10 mL of viral growth medium was added, and the
cells were incubated for 24 hrs at 37°C. The culture supernatant was harvested
by centrifugation (3000 rpm at 4°C) and stored at −80°C until use.
TCID50 (50% tissue culture infective dose) values were determined by
infecting the MDCK cells with serial dilutions of the viral suspension in 96-well
microtiter plates, and the TCID50 values were calculated using the method of
Reed and Muench [35] after 6 days.


### 2.6. Mouse Adaptation and Preparation of Virus

Viruses that had been passaged 2 or 3 times in MDCK cells were further serially passaged
in 6-week-old female BALB/c mice. The first passaged virus lot was obtained by
intranasally inoculating a 20-μL suspension of the starting virus (5.1 ×
108 TCID50 per mL) into each animal, which had been lightly
anesthetized using diethyl ether. At 48 hrs postinoculation (hpi), the mice were
sacrificed. Their lungs were removed, placed into PBS (2 mL for the lungs of two
mice) supplemented with 0.1% BSA and penicillin-streptomycin, and homogenized using a
PRO200 homogenizer (PRO Scientific Inc.; Oxford, CT, USA). The suspension was further
centrifuged at 3000 rpm at 4°C, stored at −80°C, and termed MP1
(mouse passage 1). The subsequent passages were similarly performed by inoculating
40 μL of the viral solution of the previous passage into each
mouse. The second- and higher-passage virus lots were termed MP2, MP3, and so forth.


### 2.7. Anti-Influenza Therapeutic Efficacy in Mice

#### 2.7.1. Assessment of Survival Time

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 40μL of a mixture of MP6 to MP12 (i.e., mouse-adapted viruses
(8.3–53 × 104 TCID50/mouse, resp.)) once per day for 4
days via intranasal instillation. The mice were categorized into 6 groups: (1) control
(vehicle); (2) oseltamivir phosphate (0.5 mg/kg of body weight); (3) PWE
(100 mg/kg); (4) PEE (100 mg/kg); (5) 3,4-diCQA (99% purity;
50 mg/kg); (6) chlorogenic acid (50 mg/kg). The compounds (in a 5%
Arabic gum solution) were orally administered to the mice twice per day at intervals of
more than 6 hrs for 6 days (from 0 to 5 dpi (days postinfection)).
Administration began 4 hrs before viral inoculation. Mouse survival was observed
once per day for 12 days after viral inoculation.


#### 2.7.2. Assessment of mRNA Expression in the Lungs

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 20μL of MP14, a mouse-adapted virus (4.9 ×
105 TCID50/mouse) once per day for 2 days via intranasal
instillation. The mice were categorized into three groups: (1) control (vehicle); (2)
3,4-diCQA (82% purity; 50 mg/kg); (3) oseltamivir phosphate
(0.5 mg/kg). Each compound (in a 5% Arabic gum solution) was orally
administered to the mice twice per day at 6-hour intervals for 8 days (from 0 to
7 dpi). Administration began 4 hrs before viral inoculation. In the mRNA
expression study (n = 13–14 at 0 dpi), the 6 mice with
the lowest body weight in each group and the remaining surviving mice in each group were
killed at 4 and 7 dpi, respectively, and the mRNA expression in the lungs was
determined using quantitative real-time PCR (qPCR), as described below.Total RNA was extracted from the lung homogenate using the TriPure Isolation Reagent
(Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol.
Briefly, the mice were sacrificed, and their lungs were removed, placed into a solution
(1.5 mL for the lungs of one mouse) of TriPure, and homogenized using a PRO200
homogenizer. After centrifugation, the water phase of the lysate was recovered, and RNA
was precipitated with the ethanol and rinsed. The purified RNA was dissolved in
50–100μL of nuclease-free water. The amount of RNA was estimated
using a NanoDrop (Thermo Scientific, Wilmington, DE, USA). The cDNA from a 500-ng
aliquot was synthesized using a PrimeScript RT reagent kit (Takara Bio Inc., Shiga,
Japan) and oligo dT primers as RT (reverse transcription) primers, according to the
manufacturer’s protocol. Quantitative real-time PCR for the IAV or mouse gene was
then performed. Briefly, the aliquot of the RT reaction solution was amplified using
SYBR Premix Ex Taq II (Takara Bio Inc., Shiga, Japan) and a Real-Time PCR Thermal Cycler
(“Thermal Cycler Dice Real Time,” Takara Bio Inc., Shiga, Japan),
according to the manufacturer’s protocol. H1N1-specific primers were selected
based on HA (hemagglutinin) mRNA using Primer Express software (PE Applied Biosystems,
Waltham, MA, USA). The sequences of the HA primer sets were
5′-CAATGTATGCTTTCGCACTGAGTA-3′ for the forward primer and
5′-GACACTTCGTGTTACACTCATGCA-3′ for the reverse primer. The sequences of
the mouse TRAIL primer sets have been described elsewhere [36]. The mouse HPRT (hypoxanthine guanine phosphoribosyl transferase)
primer sets were purchased from Takara Bio Inc. (Shiga, Japan). The relative quantities
of H1N1 HA mRNA and mouse TRAIL mRNA were normalized to the mRNA expression of mouse
HPRT (a housekeeping gene).


### 2.8. Statistics

The survival times were statistically compared using the Kaplan-Meier log-rank test and
pairwise multiple comparison procedures (the Holm-Sidak method) using StatView 3.5 (SAS
Institute, Cary, NC, USA). The body weights were compared using one-way analysis of
variance (ANOVA). When any significance was detected by ANOVA, Dennett’s
nonparametric test was then applied using Ekuseru-Tokei 2006 (Social Survey Research
Information Co., Ltd., Tokyo Japan). The relative mRNA expression of the target gene was
compared between treatment groups using a pairedt-test or
Student’s t-test. A P value of less than 0.01 (P< 0.01) was considered to be statistically
significant.


## 2.1. Reagents and Compounds

Oseltamivir phosphate (Tamiflu) was purchased from the Chugai Pharmaceutical Co., Ltd.
(Tokyo, Japan). Chlorogenic acid was purchased from the Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). All media and reagents for cell culture were purchased from Invitrogen
(Carlsbad, CA, USA), Sigma (St. Louis, MO, USA), and Wako Pure Chemicals (Osaka, Japan).
Water extracts of Brazilian green propolis (Minas Gerais State, Brazil) originating fromBaccharis dracunculifolia [33]
were obtained from the API Co., Ltd. (Gifu, Japan). 3,4-diCQA (99% or 82%
purity) was purified from 25% ethanol-extracted Brazilian green propolis via column
chromatography and HPLC (details not shown).


## 2.2. Mice

Female BALB/c mice (5 weeks old) were obtained from Japan SLC, Inc. (Hamamatsu, Japan)
and housed at room temperature (maintained at 23 ± 3°C) with a relative humidity
range of 32–64% and a regular 12 hr light/dark cycle. The mice were fed
a CE-2 rodent diet from CLEA Japan Inc. (Tokyo, Japan) and allowed free access to
water.


## 2.3. Viruses

The influenza virus wild-type strain A/WSN/33 (H1N1), generated from cloned cDNAs using
plasmid-based reverse genetics [34], was kindly
supplied by Dr. Yoshihiro Kawaoka (Division of Virology, Department of Microbiology and
Immunology, Institute of Medical Science University of Tokyo, Japan). The viruses were
stored at −80°C until use.


## 2.4. Cells

Madin-Darby canine kidney (MDCK) cells were a kind gift from Professor Hideto Fukushi
(United Graduate School of Veterinary Sciences, Gifu University) and were maintained inα-minimal essential medium (MEM) containing 10% fetal bovine
serum (FBS) and penicillin-streptomycin.


## 2.5. Virus Preparation in Cell Culture and Determination of the TCID50
Value

MDCK cells were cultured in T-75 culture flasks. Confluent cells were washed with
phosphate-buffered saline (PBS) and then incubated with the virus (0.1 multiplicity of
infection (MOI)) in 2 mL of viral growth medium (D-MEM (Dulbecco’s Modified
Eagle Medium) containing 0.625μg/mL of trypsin and 0.1% bovine serum albumin (BSA))
for 1 hr at 37°C. Then, 10 mL of viral growth medium was added, and the
cells were incubated for 24 hrs at 37°C. The culture supernatant was harvested
by centrifugation (3000 rpm at 4°C) and stored at −80°C until use.
TCID50 (50% tissue culture infective dose) values were determined by
infecting the MDCK cells with serial dilutions of the viral suspension in 96-well
microtiter plates, and the TCID50 values were calculated using the method of
Reed and Muench [35] after 6 days.


## 2.6. Mouse Adaptation and Preparation of Virus

Viruses that had been passaged 2 or 3 times in MDCK cells were further serially passaged
in 6-week-old female BALB/c mice. The first passaged virus lot was obtained by
intranasally inoculating a 20-μL suspension of the starting virus (5.1 ×
108 TCID50 per mL) into each animal, which had been lightly
anesthetized using diethyl ether. At 48 hrs postinoculation (hpi), the mice were
sacrificed. Their lungs were removed, placed into PBS (2 mL for the lungs of two
mice) supplemented with 0.1% BSA and penicillin-streptomycin, and homogenized using a
PRO200 homogenizer (PRO Scientific Inc.; Oxford, CT, USA). The suspension was further
centrifuged at 3000 rpm at 4°C, stored at −80°C, and termed MP1
(mouse passage 1). The subsequent passages were similarly performed by inoculating
40 μL of the viral solution of the previous passage into each
mouse. The second- and higher-passage virus lots were termed MP2, MP3, and so forth.


## 2.7. Anti-Influenza Therapeutic Efficacy in Mice

### 2.7.1. Assessment of Survival Time

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 40μL of a mixture of MP6 to MP12 (i.e., mouse-adapted viruses
(8.3–53 × 104 TCID50/mouse, resp.)) once per day for 4
days via intranasal instillation. The mice were categorized into 6 groups: (1) control
(vehicle); (2) oseltamivir phosphate (0.5 mg/kg of body weight); (3) PWE
(100 mg/kg); (4) PEE (100 mg/kg); (5) 3,4-diCQA (99% purity;
50 mg/kg); (6) chlorogenic acid (50 mg/kg). The compounds (in a 5%
Arabic gum solution) were orally administered to the mice twice per day at intervals of
more than 6 hrs for 6 days (from 0 to 5 dpi (days postinfection)).
Administration began 4 hrs before viral inoculation. Mouse survival was observed
once per day for 12 days after viral inoculation.


### 2.7.2. Assessment of mRNA Expression in the Lungs

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 20μL of MP14, a mouse-adapted virus (4.9 ×
105 TCID50/mouse) once per day for 2 days via intranasal
instillation. The mice were categorized into three groups: (1) control (vehicle); (2)
3,4-diCQA (82% purity; 50 mg/kg); (3) oseltamivir phosphate
(0.5 mg/kg). Each compound (in a 5% Arabic gum solution) was orally
administered to the mice twice per day at 6-hour intervals for 8 days (from 0 to
7 dpi). Administration began 4 hrs before viral inoculation. In the mRNA
expression study (n = 13–14 at 0 dpi), the 6 mice with
the lowest body weight in each group and the remaining surviving mice in each group were
killed at 4 and 7 dpi, respectively, and the mRNA expression in the lungs was
determined using quantitative real-time PCR (qPCR), as described below.Total RNA was extracted from the lung homogenate using the TriPure Isolation Reagent
(Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol.
Briefly, the mice were sacrificed, and their lungs were removed, placed into a solution
(1.5 mL for the lungs of one mouse) of TriPure, and homogenized using a PRO200
homogenizer. After centrifugation, the water phase of the lysate was recovered, and RNA
was precipitated with the ethanol and rinsed. The purified RNA was dissolved in
50–100μL of nuclease-free water. The amount of RNA was estimated
using a NanoDrop (Thermo Scientific, Wilmington, DE, USA). The cDNA from a 500-ng
aliquot was synthesized using a PrimeScript RT reagent kit (Takara Bio Inc., Shiga,
Japan) and oligo dT primers as RT (reverse transcription) primers, according to the
manufacturer’s protocol. Quantitative real-time PCR for the IAV or mouse gene was
then performed. Briefly, the aliquot of the RT reaction solution was amplified using
SYBR Premix Ex Taq II (Takara Bio Inc., Shiga, Japan) and a Real-Time PCR Thermal Cycler
(“Thermal Cycler Dice Real Time,” Takara Bio Inc., Shiga, Japan),
according to the manufacturer’s protocol. H1N1-specific primers were selected
based on HA (hemagglutinin) mRNA using Primer Express software (PE Applied Biosystems,
Waltham, MA, USA). The sequences of the HA primer sets were
5′-CAATGTATGCTTTCGCACTGAGTA-3′ for the forward primer and
5′-GACACTTCGTGTTACACTCATGCA-3′ for the reverse primer. The sequences of
the mouse TRAIL primer sets have been described elsewhere [36]. The mouse HPRT (hypoxanthine guanine phosphoribosyl transferase)
primer sets were purchased from Takara Bio Inc. (Shiga, Japan). The relative quantities
of H1N1 HA mRNA and mouse TRAIL mRNA were normalized to the mRNA expression of mouse
HPRT (a housekeeping gene).


## 2.7.1. Assessment of Survival Time

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 40μL of a mixture of MP6 to MP12 (i.e., mouse-adapted viruses
(8.3–53 × 104 TCID50/mouse, resp.)) once per day for 4
days via intranasal instillation. The mice were categorized into 6 groups: (1) control
(vehicle); (2) oseltamivir phosphate (0.5 mg/kg of body weight); (3) PWE
(100 mg/kg); (4) PEE (100 mg/kg); (5) 3,4-diCQA (99% purity;
50 mg/kg); (6) chlorogenic acid (50 mg/kg). The compounds (in a 5%
Arabic gum solution) were orally administered to the mice twice per day at intervals of
more than 6 hrs for 6 days (from 0 to 5 dpi (days postinfection)).
Administration began 4 hrs before viral inoculation. Mouse survival was observed
once per day for 12 days after viral inoculation.


## 2.7.2. Assessment of mRNA Expression in the Lungs

Six-week-old female BALB/c mice were lightly anesthetized with diethyl ether and
inoculated with 20μL of MP14, a mouse-adapted virus (4.9 ×
105 TCID50/mouse) once per day for 2 days via intranasal
instillation. The mice were categorized into three groups: (1) control (vehicle); (2)
3,4-diCQA (82% purity; 50 mg/kg); (3) oseltamivir phosphate
(0.5 mg/kg). Each compound (in a 5% Arabic gum solution) was orally
administered to the mice twice per day at 6-hour intervals for 8 days (from 0 to
7 dpi). Administration began 4 hrs before viral inoculation. In the mRNA
expression study (n = 13–14 at 0 dpi), the 6 mice with
the lowest body weight in each group and the remaining surviving mice in each group were
killed at 4 and 7 dpi, respectively, and the mRNA expression in the lungs was
determined using quantitative real-time PCR (qPCR), as described below.Total RNA was extracted from the lung homogenate using the TriPure Isolation Reagent
(Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol.
Briefly, the mice were sacrificed, and their lungs were removed, placed into a solution
(1.5 mL for the lungs of one mouse) of TriPure, and homogenized using a PRO200
homogenizer. After centrifugation, the water phase of the lysate was recovered, and RNA
was precipitated with the ethanol and rinsed. The purified RNA was dissolved in
50–100μL of nuclease-free water. The amount of RNA was estimated
using a NanoDrop (Thermo Scientific, Wilmington, DE, USA). The cDNA from a 500-ng
aliquot was synthesized using a PrimeScript RT reagent kit (Takara Bio Inc., Shiga,
Japan) and oligo dT primers as RT (reverse transcription) primers, according to the
manufacturer’s protocol. Quantitative real-time PCR for the IAV or mouse gene was
then performed. Briefly, the aliquot of the RT reaction solution was amplified using
SYBR Premix Ex Taq II (Takara Bio Inc., Shiga, Japan) and a Real-Time PCR Thermal Cycler
(“Thermal Cycler Dice Real Time,” Takara Bio Inc., Shiga, Japan),
according to the manufacturer’s protocol. H1N1-specific primers were selected
based on HA (hemagglutinin) mRNA using Primer Express software (PE Applied Biosystems,
Waltham, MA, USA). The sequences of the HA primer sets were
5′-CAATGTATGCTTTCGCACTGAGTA-3′ for the forward primer and
5′-GACACTTCGTGTTACACTCATGCA-3′ for the reverse primer. The sequences of
the mouse TRAIL primer sets have been described elsewhere [36]. The mouse HPRT (hypoxanthine guanine phosphoribosyl transferase)
primer sets were purchased from Takara Bio Inc. (Shiga, Japan). The relative quantities
of H1N1 HA mRNA and mouse TRAIL mRNA were normalized to the mRNA expression of mouse
HPRT (a housekeeping gene).


## 2.8. Statistics

The survival times were statistically compared using the Kaplan-Meier log-rank test and
pairwise multiple comparison procedures (the Holm-Sidak method) using StatView 3.5 (SAS
Institute, Cary, NC, USA). The body weights were compared using one-way analysis of
variance (ANOVA). When any significance was detected by ANOVA, Dennett’s
nonparametric test was then applied using Ekuseru-Tokei 2006 (Social Survey Research
Information Co., Ltd., Tokyo Japan). The relative mRNA expression of the target gene was
compared between treatment groups using a pairedt-test or
Student’s t-test. A P value of less than 0.01 (P< 0.01) was considered to be statistically
significant.


## 3. Results

### 3.1. PWE, PEE, and 3,4-diCQA Increase the Survival Times of Mice Infected with the
Influenza Virus

In preliminary experiments, MDCK culture-derived WSN/33 (H1N1, IAV) was only moderately
lethal to mice, so we adapted the parent WSN/33 to mice using serial passages to enhance
its virulence [37–39], as described in the Section 2. Mouse-adapted WSN/33 was able to kill more than 80% of mice within 10
days when the mice inoculated daily for 3 to 4 days. The anti-influenza activities of PWE,
PEE, 3,4-diCQA, chlorogenic acid, and oseltamivir phosphate were evaluated using the
mouse-adapted WSN/33 strains.The vehicle treatment (control) group exhibited a significant decrease in body weight
from 0 to 7 dpi. Treatment with PWE, PEE, or chlorogenic acid did not affect this
phenotype compared to the control. However, 3,4-CQA (at 5 dpi alone,P< 0.01) and oseltamivir (at 5 dpi,
P< 0.05; at 6-7 dpi, P< 0.01) slightly and strongly, respectively,
counteracted the body weight loss caused by the viral infection (Figures 1(a) and 1(b)).Effects of orally administrated oseltamivir phosphate, PWE, PEE, 3,4-diCQA, and
chlorogenic acid on the body weight of BALB/c mice infected with the IAV. (a) control
(⚫), oseltamivir (0.5 mg/kg; ■), 3,4-diCQA
(50 mg/kg; ◆), (b) PWE (100 mg/kg; ▲),
PEE (100 mg/kg; ▾), or
chlorogenic acid (50 mg/kg; ⚫) was administrated twice
daily starting at 4 hours before the first viral infection until 5 days
postinfection (5 dpi). The data represent the mean ± SE for the mice that
survived. *P< 0.05 and **P< 0.01 versus vehicle control.
(a)
(b)In the same experiment, 88.9% of mice in the control group died by 12 dpi,
while all of the oseltamivir-treated mice survived. The survival time of the oseltamivir
group was significantly longer than that of the control group (Figure2(a), Table 1). Moreover,
although chlorogenic acid had no effect, PWE, PEE, or 3,4-CQA treatment increased the
survival rate (Figures 2(a) and 2(b), Table 1).Table 1
Survival of mice infected with IAV after the oral administration of various
substances.
Treatment
n
Dose
Duration (dpi)
Survival time (dpi, mean ± SE)
Statistical significance
5% Arabic gum
9
0–5
7.4 ± 0.7
oseltamivir
8
0.5 mg twice/kg/day
0–5
>12.0#
**
PWE
9
100 mg twice/kg/day
0–5
10.0 ± 0.9
*
PEE
9
100 mg twice/kg/day
0–5
10.1 ± 0.9
*
3,4-diCQA
7
50 mg twice/kg/day
0–5
10.3 ± 1.5
*
Chlorogenic acid
9
50 mg twice/kg/day
0–5
8.1 ± 0.8
N.S.
#standard error not determined; *P < 0.05; **P < 0.01; N.S. not significant.Survival curves of BALB/c mice infected with the IAV to assess thein
vivo effects of orally administrated (a) control, oseltamivir
(0.5 mg/kg), 3,4-diCQA (50 mg/kg), (b) PWE (100 mg/kg), PEE
(100 mg/kg), or chlorogenic acid (50 mg/kg). The colors correspond to
those used in Figures 1(a) and 1(b). The infected mice were observed for 12 days.
The statistical results are shown in Table 1.
(a)
(b)


### 3.2. 3,4-diCQA Increases TRAIL Expression but Decreases HA mRNA Expression in Mouse
Lungs Infected with IAV

We examined TRAIL and HA mRNA expression in the lungs of mice infected with IAV.
Treatment with 3,4-CQA or oseltamivir had no effect on TRAIL mRNA expression at
4 dpi (Figure3). At 7 dpi, the
3,4-CQA treatment group exhibited an increase in TRAIL mRNA expression
(P< 0.05), while the oseltamivir treatment group
exhibited no change in the expression of TRAIL mRNA (Figure 3).Figure 3
TRAIL expression in the lungs of BALB/c mice infected with the IAV. Vehicle,
3,4-diCQA (50 mg/kg), or oseltamivir (0.5 mg/kg) was administrated twice
daily starting at 4 hrs before the first viral infection until 7 days
post-infection (7 dpi). Total RNA was recovered at 4 dpi or
7 dpi, and relative mRNA expression was measured using real-time PCR. The
quantity of mouse TRAIL mRNA expression was normalized to the mRNA expression of mouse
HPRT. The quantities are shown as mean ± SE.
**P< 0.01.In the same experiment, the oseltamivir treatment group exhibited less HA mRNA expression
than the control at 4 and 7 dpi (P< 0.05; Figure 4). The 3,4-diCQA treatment group exhibited similar HA mRNA expression at
4 dpi and slightly less expression at 7 dpi than the control group (Figure
4).Figure 4
HA expression in lungs. IAV HA mRNA expression was normalized to the mRNA expression
of mouse HPRT. The quantities are shown as mean ± SE.
**P< 0.01 (Student’s
t-test).Overall, oseltamivir treatment decreased IAV HA mRNA expression rapidly after infection
and had no effect on TRAIL mRNA expression, whereas 3,4-diCQA treatment moderately
decreased IAV HA mRNA expression and increased TRAIL mRNA expression with a delay.


## 3.1. PWE, PEE, and 3,4-diCQA Increase the Survival Times of Mice Infected with the
Influenza Virus

In preliminary experiments, MDCK culture-derived WSN/33 (H1N1, IAV) was only moderately
lethal to mice, so we adapted the parent WSN/33 to mice using serial passages to enhance
its virulence [37–39], as described in the Section 2. Mouse-adapted WSN/33 was able to kill more than 80% of mice within 10
days when the mice inoculated daily for 3 to 4 days. The anti-influenza activities of PWE,
PEE, 3,4-diCQA, chlorogenic acid, and oseltamivir phosphate were evaluated using the
mouse-adapted WSN/33 strains.The vehicle treatment (control) group exhibited a significant decrease in body weight
from 0 to 7 dpi. Treatment with PWE, PEE, or chlorogenic acid did not affect this
phenotype compared to the control. However, 3,4-CQA (at 5 dpi alone,P< 0.01) and oseltamivir (at 5 dpi,
P< 0.05; at 6-7 dpi, P< 0.01) slightly and strongly, respectively,
counteracted the body weight loss caused by the viral infection (Figures 1(a) and 1(b)).Effects of orally administrated oseltamivir phosphate, PWE, PEE, 3,4-diCQA, and
chlorogenic acid on the body weight of BALB/c mice infected with the IAV. (a) control
(⚫), oseltamivir (0.5 mg/kg; ■), 3,4-diCQA
(50 mg/kg; ◆), (b) PWE (100 mg/kg; ▲),
PEE (100 mg/kg; ▾), or
chlorogenic acid (50 mg/kg; ⚫) was administrated twice
daily starting at 4 hours before the first viral infection until 5 days
postinfection (5 dpi). The data represent the mean ± SE for the mice that
survived. *P< 0.05 and **P< 0.01 versus vehicle control.
(a)
(b)In the same experiment, 88.9% of mice in the control group died by 12 dpi,
while all of the oseltamivir-treated mice survived. The survival time of the oseltamivir
group was significantly longer than that of the control group (Figure2(a), Table 1). Moreover,
although chlorogenic acid had no effect, PWE, PEE, or 3,4-CQA treatment increased the
survival rate (Figures 2(a) and 2(b), Table 1).Table 1
Survival of mice infected with IAV after the oral administration of various
substances.
Treatment
n
Dose
Duration (dpi)
Survival time (dpi, mean ± SE)
Statistical significance
5% Arabic gum
9
0–5
7.4 ± 0.7
oseltamivir
8
0.5 mg twice/kg/day
0–5
>12.0#
**
PWE
9
100 mg twice/kg/day
0–5
10.0 ± 0.9
*
PEE
9
100 mg twice/kg/day
0–5
10.1 ± 0.9
*
3,4-diCQA
7
50 mg twice/kg/day
0–5
10.3 ± 1.5
*
Chlorogenic acid
9
50 mg twice/kg/day
0–5
8.1 ± 0.8
N.S.
#standard error not determined; *P < 0.05; **P < 0.01; N.S. not significant.Survival curves of BALB/c mice infected with the IAV to assess thein
vivo effects of orally administrated (a) control, oseltamivir
(0.5 mg/kg), 3,4-diCQA (50 mg/kg), (b) PWE (100 mg/kg), PEE
(100 mg/kg), or chlorogenic acid (50 mg/kg). The colors correspond to
those used in Figures 1(a) and 1(b). The infected mice were observed for 12 days.
The statistical results are shown in Table 1.
(a)
(b)


## 3.2. 3,4-diCQA Increases TRAIL Expression but Decreases HA mRNA Expression in Mouse
Lungs Infected with IAV

We examined TRAIL and HA mRNA expression in the lungs of mice infected with IAV.
Treatment with 3,4-CQA or oseltamivir had no effect on TRAIL mRNA expression at
4 dpi (Figure3). At 7 dpi, the
3,4-CQA treatment group exhibited an increase in TRAIL mRNA expression
(P< 0.05), while the oseltamivir treatment group
exhibited no change in the expression of TRAIL mRNA (Figure 3).Figure 3
TRAIL expression in the lungs of BALB/c mice infected with the IAV. Vehicle,
3,4-diCQA (50 mg/kg), or oseltamivir (0.5 mg/kg) was administrated twice
daily starting at 4 hrs before the first viral infection until 7 days
post-infection (7 dpi). Total RNA was recovered at 4 dpi or
7 dpi, and relative mRNA expression was measured using real-time PCR. The
quantity of mouse TRAIL mRNA expression was normalized to the mRNA expression of mouse
HPRT. The quantities are shown as mean ± SE.
**P< 0.01.In the same experiment, the oseltamivir treatment group exhibited less HA mRNA expression
than the control at 4 and 7 dpi (P< 0.05; Figure 4). The 3,4-diCQA treatment group exhibited similar HA mRNA expression at
4 dpi and slightly less expression at 7 dpi than the control group (Figure
4).Figure 4
HA expression in lungs. IAV HA mRNA expression was normalized to the mRNA expression
of mouse HPRT. The quantities are shown as mean ± SE.
**P< 0.01 (Student’s
t-test).Overall, oseltamivir treatment decreased IAV HA mRNA expression rapidly after infection
and had no effect on TRAIL mRNA expression, whereas 3,4-diCQA treatment moderately
decreased IAV HA mRNA expression and increased TRAIL mRNA expression with a delay.


## 4. Discussion

First, we must point out that there are some controversies regarding the anti-influenza
effects of various propolis-related substances, especially inex vivo
versus in vivo experiments. We have shown that PWE and PEE can increase the
lifetimes of mice with IAV infections. In a previous report, anti-influenza activity was
found for PWE [31] but not PEE (personal
communication, Urushisaki T.) when using MDCK cells. Here, we speculate that, in ex
vivoexperiments, some substances (as yet unidentified) in PEE are cytotoxic to
MDCK cells and may mask its anti-influenza activity. We have also shown that 3,4-diCQA can
increase the lifetimes of mice with IAV infections (Table 1). 3,4-diCQA, a natural product, is an ester of two polyphenolic caffeic acids
and one cyclitol (−)-quinic acid. While chlorogenic acid (a natural product, an ester
of one caffeic acid and one cyclitol (−)-quinic acid) does not have anti-influenza
activity in vivo, these two compounds have anti-influenza activity
ex vivo [31]. This may occur due
to differences in the pharmacokinetic properties of these compounds in
vivo.The major chemical ingredients in PWE and PEE are listed in Table2 [40, 41], and these show that the amount of 3,4-diCQA in PWE and PEE is
3.3–6.1% and 1.9–3.5%, respectively. It must be noted that the
anti-influenza activities of PWE and PEE can only be partially explained by 3,4-diCQA. Thus,
it is very likely that unknown compounds that efficiently act against the IAV are also
contained in PWE and PEE.Table 2
Concentrations and molecular weights of the constituents of Brazilian green propolis
[40, 41].
Components
Content (w/w%) in PWE
Content (w/w%) in PEE
Molecularweight  (g/mol)
Chlorogenic acid
2.7–3.6
0.6–0.8
354.3
Caffeic acid
0.2
0.1–0.6
180.2
3,5-dicaffeoylquinic acid
4.3–4.9
2.4–2.7
516.5
3,4-dicaffeoylquinic acid
3.3–6.1
1.9–3.5
516.5
4,5-dicaffeoylquinic acid
—#
—#
516.5
3,4,5-tricaffeoylquinic acid
0.2
0.6
678.6
Artepillin C
0.2–0.6
11.4–14.0
300.4
Baccharin
0.0
6.8
562.6
Drupanin
0.1
1.8
232.3
Isosakuranetin
—#
1.2
286.3
p-coumaric acid
3.7
2.3–2.5
164.2
Ferulic acid
0.1
0.0–0.1
194.2
Quinic acid
—#
—#
192.2
#Data not available.We have also shown that 3,4-diCQA increases the mRNA expression of TRAIL in the lungs of
IAV-infected mice (Figure4). Although TRAIL is an
apoptosis-inducing factor in tumor cells [42], it
also induces the apoptosis of influenza virus-infected cells in infected animals via the
TRAIL receptor (DR5), whose expression is induced by the virus. Ishikawa et al. [36] have shown that TRAIL mRNA expression is induced by
an influenza virus infection in mouse lungs, and an anti-TRAIL monoclonal antibody (mAb)
delays influenza virus clearance in mice. Moreover, Brincks et al. [43] have shown that morbidity and the influenza virus titer are increased
in TRAIL-knockout (TRAIL−/−) mice. Therefore, TRAIL is an important
anti-influenza factor with viral clearance activity.3,4-diCQA did not affect TRAIL expression at 4 dpi (Figure3). However, the infected animals were protected even at early stages of
the infectious period without increased TRAIL expression because 3,4-diCQA increases the
viability of infected cells, as suggested by previous ex vivo experiments
[31]. It must be noted that MDCK cells do not
express the TRAIL receptor [44]. 3,4-diCQA induced
TRAIL mRNA at 7 dpi relative to the control group (Figure 3). During the middle stage of infection, TRAIL expression increased,
thereby increasing the lifetimes of the mice.It has also been reported that the induction of TRAIL mRNA depends on
NF-κB [45]. However, at the
current stage, it is unclear whether the enhancement of TRAIL mRNA expression by 3,4-diCQA
is due to NF-κB activation.We confirmed that Brazilian green propolis extracts (PWE and PEE) have anti-influenza
activities. Here, we also hypothesized that their mode of action at least partially includes
two mechanisms: an unknown cytoprotective mechanism [31] and the enhancement of viral clearance via TRAIL overexpression. We
hypothesize that both are induced by 3,4-diCQA and/or unknown active constituents of
Brazilian green propolis.An important point regarding our hypothesis concerning the anti-influenza effects of
propolis or its constituent, 3,4-diCQA, is that it might trigger or enhance the self-defense
machineries of the host. Although viruses can easily become resistant to anti-influenza
drugs, such as oseltamivir, that directly target viral proteins [4, 5], it would be more difficult
for viruses to become resistant to antiviral agents whose target molecules are host
molecules or machineries. Therefore, health supplements such as propolis may be useful as an
alternative strategy for protection against influenza virus infections.Many traditional Chinese medicinal herbs appear to have potential anti-influenza effects
[46], but their mechanisms are not well understood.
Our findings show that PWE and PEE and their constituent, 3,4-diCQA, may be useful as a
potent lead compound for anti-influenza medicine. This may promote research into
anti-influenza medicine developed from traditional substances.



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*Source: 946867-2011-08-25.xml*