Pycnogenol®, a Procyanidin-Rich Extract from French Maritime Pine,
(Received October 31, 2007. Accepted May 7, 2008)
Inhibits Intracellular Replication of HIV-1 as well as Its Binding to Host Cells
SUMMARY: A procyanidin-rich extract from French maritime pine, Pycnogenol® (PYC), is known as an anti-
these molecules (2-5). Thus, sulfated-carbohydrates might be
their glycosides. About 65-75% of PYC constituents are
context of other diseases and disease processes such as dia-
Wu Yu Feng, Ritsuko Tanaka, Yoshio Inagaki, Yasunori Saitoh, Myint Oo Chang1,
PYC inhibits not only human immunodeficiency virus type-1 (HIV-1) binding to host cells, but also its replication
Novel therapeutic approaches need to be developed for the
compounds in the plant world, hydrolysable tannin exhibits
of varying chain lengths (7). In addition to antioxidant ac-
PYC is a unique compound capable of inhibiting not only
Tohti Amet, Norio Yamamoto, Shoji Yamaoka and Yoshiyuki Yoshinaka2*
after entry in susceptible cells in vitro. Prominent biochemical alterations induced by PYC were the elevated
treatment of human immunodeficiency virus type-1 (HIV-
anti-HIV-1 activity by direct binding to virus particles and
tivities resulting from the hydroxyl groups of the polyphenol
virus adsorption to host cells, but also HIV-1 replication;
Department of Molecular Virology, Graduate School and 2Human Gene Sciences Center, Tokyo Medical and
expression of an intracellular antioxidant protein, manganese superoxide dismutase (Mn-SOD), and the inhibition
1) infection, which is a causative agent of acquired immuno-
subsequent inhibition of the virus binding to host cells (6).
components, procyanidins have also been shown to exert a
Dental University, Tokyo 113-5819, and 1Department of Life and Environmental Science,
of phosphorylation of the ribosomal S6 protein. Interestingly, ectopic expression of Mn-SOD inhibited HIV-1
deficiency syndrome (AIDS) (1).
Pycnogenol® (PYC) is a standardized bark extract of the
variety of biological activities on cells when added to cell
Chiba Institute of Technology, Chiba 275-0016, Japan
oxidant that exerts a variety of physiological activities and is widely used in human beings. We report here that
replication as well. Inhibition of HIV-1 replication associated with induced expression of Mn-SOD in cells
There have been many reports on the anti-HIV-1 activity
French maritime pine, Pinus pinaster (Pycnogenol®; Horphag
culture; inhibition of cell growth, induction of apoptosis (8),
treated with PYC suggests the potential of this natural antioxidant inducer as a new anti-HIV-1 agent.
of natural substances obtained from foods, medicinal plants,
Research Ltd., Geneva, Switzerland). It consists of a con-
and inhibition of NF-κB activation (9,10). PYC has been
*Corresponding author: Mailing address: Human Gene Sciences
Center, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-5819, Japan. Tel: +81-3-5803-5178, Fax:
+81-3-5803-0234, E-mail: email@example.com
in particular sea algae. Sulfated-carbohydrates are well known
centrate of pine bark constituents such as polyphenolic
also shown to exert ameliorative effects on cardiovascular,
to inhibit virus binding to host cells via the negative charges of
monomers, procyanidins, and phenolic or cinnamic acids and
skin, cognitive, and menstrual disorders, as well as in the
useful as “microbicides” (2). Among a number of polyphenol
procyanidins that contain catechin and epicatechin subunits
betes and inflammation. In this report, we demonstrate that
moreover, based on these findings, this inhibition appears to
Japan). 3´-Azido-3´-deoxythymidine (AZT) and MG132, a
Intenational Inc., Etobicoke, Tronto, Ontario, Canada) and
cytopathic effects observed following the infection of MT-4
be related to PYC-induced expression of manganese super-
proteosome inhibitor, was purchased from SIGMA-ALDRICH,
antibiotics (100 U/ml penicillin and 100 μg/ml streptomy-
cells, and virus stocks (approximately 5×105 TCID50/ml)
oxide dismutase (Mn-SOD) and suppressed-phosphorylation
Inc. (St. Louis, Mo., USA), and 3-(4,5-dimethylthiazol-2-yl)
cin) at 37°C in an atmosphere of 5% CO2 (5,11,12). The
were prepared and stored at –70°C (5). The TCID50/ml value
of S6 protein.
2,5-diphenyl tetrazolium bromide (MTT) was purchased from
viability of cultured cells was determined after cell staining
was calculated according to the method of Reed and Muench.
MATERIALS AND METHODS
Cells, viruses, and plasmids: MT-4, Molt-4, and Molt-4/
were performed using an automated cell imaging counter
Vesicular stomatitis virus glycoprotein (VSV-G) or HIV-1
Reagents: Pycnogenol® was kindly supplied by Horphag
strain of lymphocyte-tropic HIV-1) were cultured in RPMI-
viruses, (VSV-G)-HIV-1 (5) and IIIB-env-HIV-1 (13), were
Research Ltd. Sodium dextran sulfate (DS) (MW 5,000) was
1640 medium and 293T cells in DMEM supplemented with
The culture supernatants of Molt-4/IIIB cells were used to
prepared 48 h post-transfection. The titration of viruses was
purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
heat-inactivated 10% fetal bovine serum (FBS) (Cansera
infect MT-4 cells. Virus titers were determined based on the
Dojindo, Kumamoto, Japan.
with trypan blue (3), and cell counting and image acquisition
Plasmid NLE-deltaEnv was co-transfected to 293T cells with
IIIB cells chronically infected with HIV-1 IIIB (a laboratory
CYTORECON (GE Healthcare Bio-Sciences Co., Piscataway,
IIIB-envelope-pseudotyped, respectively, by FuGENE 6. The
carried out by counting the number of EGFP-expressing cells
Virus binding assay: The inhibitory effects of PYC, DS,
scription (RT)-PCR with primers and probes as described
1) at a MOI of 1 for 1 h at 37°C, and were washed twice with
TGCGT-3´; U5-gag/probe2, 5´-FAM-TGGCGCCCGAACA
sequence detector system (Applied Biosystems, Foster City,
Japan) (14). Approximately 100 μg/ml of total protein from
among the virus-infected MT-4 cells to determine the TCID50/
and AZT on virus binding to MT-4 cells were examined by
previously (16). In parallel experiments, the cells were treated
PBS (–), and then re-fed with the regular culture medium.
GGGACTT-TAMRA-3´; 2-LTR/F, 5´-CCCTCAGACCCTTT
Calif., USA) and ABsolute QPCR ROX mix (ABgene, Ltd.,
5×104 cells was loaded in each lane. The proteins were then
ml value. The construction of expression vectors for Mn-SOD
the infection of MT-4 cells with IIIB-env-HIV-1 at a MOI of
in the same manner, and were then cultured for an additional
Test compounds were added at different time points after in-
TAGTCAGTG-3´; 2-LTR/R, 5´-TGGTGTGTAGTTCTGCC
Epsom, UK) as follows: 15 min at 95°C, 45 cycles of 15 s at
transferred to polyvinylidene disulfide membranes (PVDFs)
and FuGENE 6 (Roche Molecular Biomedicals Strategene,
1 in the absence or presence of the compounds (15). To allow
48 h at 37°C in medium without treatment, and the HIV-1
fection (15). Thirty hours after infection, released viral
AATCA-3´; 2-LTR/probe2, 5´-FAM-TGTGGATCTACCAC
95°C, and 60 s at 60°C. Integrated HIV-1 DNA was quanti-
and processed with the respective antibodies as previously
Indianapolis, Ind., USA) has been described previously (14).
virus binding to the cells only, and to block further virus rep-
p24Gag released in the culture supernatant was quantified by
p24Gag was quantified by ELISA.
ACACAAGGCTACTTCC-TAMRA-3´; huBG/F, 5´-CAAG
fied as described previously (17).
Antiviral activity assay: MT-4 cells were infected with
lication, the cells were incubated on ice for 1 h, and were
automated enzyme-linked immunosorbent assay (ELISA)
Quantification of viral DNAs by real-time PCR: The
AAAGTGCTCGGTGCCTT-3´; huBG/R, 5´-CCTGAAGT
Immunoblot analysis: For the sodium dodecylsulfate
an HIV-1 IIIB strain at a multiplicity of infection (MOI) of
then washed three times with Ca2+- and Mg2+-free phosphate
(Fuji Rebio Inc., Tokyo, Japan) using a p24Gag antigen from
viral DNAs were further assessed after the completion of
TCTCAGGATCCACG-3´; β-globin/probe, 5´-FAM-ACA
(SDS)-polyacrylamide gel electrophoresis (PAGE), the cells
0.01 in the absence or presence of various concentrations of
buffered saline (PBS (–)), and lysed with Isogen (Nippon Gene
Zeptometrix (Buffalo, N.Y., USA) as the standard (5).
RT and the 2-LTR viral DNA had been quantified with the
were lysed with SDS sample buffer after being washed with
PYC. After 5 days of incubation, viable cells were quantified
Co. Ltd., Tokyo, Japan) for total RNA extraction. HIV-1
Time of addition assay: MT-4 cells (3.5 × 106) were in-
following primers and probes: U5-gag/F2, 5´-GTAGTGTGT
Then, β-globin mRNA was quantified for normalization of
PBS (–) twice and then briefly sonicated with a Handy Sonic
by MTT assay (Dojindo) (5,11).
mRNA levels were determined by quantitative reverse tran-
fected with the recombinant strain of HIV-1 (IIIB-env-HIV-
GCCCGTCTGTTG-3´; U5-gag/R2, 5´-CAAGCCGAGTCC
the results. Amplification was performed with an ABI 7700
system (model UR-20P; TOMY SEIKO Co. LTD., Tokyo,
described (18). Proteins of interest were visualized by sub-
Tyr185) were purchased from Cell Signaling (Beverly, Mass.,
Statistical analysis: The variables accounting for p24 pro-
whether or not PYC affects cell viability, and we then carried
viability to 50%) was 49.59 μg/ml. The selective index (SI =
only a few dead cells were observed with trypan blue stain-
harmful to MT-4 cells (data not shown). In this study, the cell
jecting the membranes to coloring reactions with NBT/BCIP
USA). Rabbit polyclonal antibodies to IκBα, Bcl-2 and
duction and viral RNA amounts observed in different cell
out an MTT assay to characterize the cytopathic effects
CC50/EC50) was calculated to be 2.16. The SI value obtained
ing in the presence of 30 to 60 μg/ml of PYC, showing 40 to
number was reduced to about 50% of baseline, but most of
(Boehringer Mannheim GmbH, Mannheim, Germany).
polyADP-ribose polymerase (PARP), monoclonal antibodies
populations and under different conditions (i.e., infection or
following virus production in HIV-1 infected MT-4 cells.
by the conventional MTT assay was clearly low; the growth
90% growth retardation, respectively, after incubation for 5
the cells were alive and growing slowly without any damage,
The following antiserum and antibodies were used in this
to α-tubulin, ubiquitin, and alkaline phosphatase-conjugated
transfection) were assessed using a 2-tailed, unpaired
Simultaneous addition of the compound at the time of infec-
of mock-infected cells was reduced by 10, 5, and 3% at 60,
days (Fig. 1Ba and 1Bb). At concentrations of 70-100 μg/
as if in a “cytostatic” state. In this context, we concluded that
study. Rabbit polyclonal antibodies to Mn-SOD and HO-1
secondary antibodies were from Santa Cruz Biotech (Santa
Student’s t test. A P-value of 0.05 or less was considered sig-
tion resulted in a dose-dependent inhibition of cytopathic
80, and 120 μg/ml of PYC, respectively, as compared to
ml of PYC, cell growth was severely inhibited accompanied
(hemeoxygenase-1), goat antibodies to HSP90β, and mono-
Cruz, Calif., USA). Each protein expression was estimated
effects (Fig. 1Aa) as well as a dose-dependent reduction in
reductions seen with of PYC-untreated cells (Fig. 1Aa). How-
by some cell death, but viable cells still remained after 5 days
clonal antibodies to HSP72 were purchased from Stressgen
by measuring the band intensities using an Analytical Imag-
(Victoria, B.C., Canada). Rabbit antiserum specific to the
ing System (AIS; Pharmacia/Applied-Biosystem, Uppsala,
(EC50) of PYC (inhibition of cell death induced by HIV-1
the viability of the cells, i.e., most of the PYC-treated cells
5 days in the presence of 30-70 μg/ml of PYC reversibly
phosphorylated or unphosphorylated form of the S6 ribo-
Sweden) followed by normalization with the band intensity
somal protein (Ser235/Ser236) and those to JNK1 (Thr183/
Antiviral activity of PYC: We first attempted to determine
the cytotoxic concentration (CC50) of PYC (reduction of cell
5% of the PYC-untreated cells died due to over-growth, but
centrations in excess of 100 μg/ml of PYC were irreversibly
p24 production (Fig. 1Ab). The 50% effective concentration
ever, in our cell growth assay, PYC treatment did not affect
of incubation. Notably, the surviving cells in the culture after
infection to 50% of the control value) was 22.94 μg/ml, and
were not stained with trypan blue. In those cultures, less than
grew after the removal of PYC (data not shown). Higher con-
PYC inhibits HIV-1 growth at a concentration of 20-70 μg/
cells. Cells were kept on ice and exposed to IIIB-env-HIV-1
ml without inducng cell damage. Similar results were obtained
for 1 h in the absence or presence of AZT and DS, a typical RT
with a human promyeloid leukemia cell line, HL-60 (8).
inhibitor and an inhibitor for virus adsorption, respectively
PYC inhibits intracellular HIV-1 replication as well as
(19), or PYC. Levels of cell-associated HIV-1 RNA were
virus-binding to host cells: HIV-1 binding experiments: To
determine at which stage(s) PYC affects the virus lifecycle,
we examined whether PYC inhibits virus binding to host cells.
To avoid multiple rounds of infection, we used a recombi-
nant HIV-1 NLE-deltaEnv pseudotyped with an HIV-1 IIIB-
envelope that cannot produce infectious virions from infected
determined by quantitative RT-PCR (Fig. 1C). DS strongly
twofold stronger than had been expected from the binding
inhibited HIV-1 binding to the cells, while AZT treatment
results (Fig. 1C). This finding suggested that PYC could in-
was associated with virtually no inhibition. PYC also effec-
hibit virus replication after attachment of the virus to the cells.
tively inhibited HIV-1 binding. We next allowed the adsorbed
Time of addition experiments: To further clarify the anti-
virus to replicate for 48 h and quantified the amount of p24Gag
released in the culture medium (Fig. 1D). As expected, DS
strongly inhibited p24Gag production from infected cells, but
AZT was not associated with any inhibition. In comparison
to DS, PYC exhibited somewhat different inhibition prop-
erties; the relative inhibition of p24Gag production was
Fig. 1. PYC protects MT-4 cells from HIV-1-induced cell death and inhibits virus binding to MT-4 cells. (Aa) The viability of MT-
mRNA was determined by quantitative RT-PCR as described in the text. Relative HIV-1 mRNA levels are shown as the percentage
4 cells was determined by MTT assay 5 days after mock-infection (open circles) or infection with HIV-1 IIIB at MOI of 0.01
to that of cells without drug treatment. (D) In parallel experiment as in (C), cells were incubated at 37°C for additional 48 h
(closed circles). Relative cell growth was shown as the percentage of that for cells mock-infected without PYC. (Ab) Production
without drugs and HIV-1 p24Gag released in the culture supernatant was quantified by ELISA. Relative p24Gag levels are
of HIV-1 p24 in the culture supernatants of MT-4 cells in the same condition as in (Aa). The results are mean values of three
expressed as the percentage of that for drug-untreated control (N.T.) as in (C). Data shown are mean ± SD values of three
independent experiments. (Ba) Growth of MT-4 cells was determined after the treatment of various concentrations of PYC by
independent experiments. * represents P < 0.05 and ** represents P < 0.01.
staining with trypan blue. (Bb) After 5 days of incubation, cell morphology was observed by automated cell imaging counter,
CYTRECON, by staining with trypan blue. The result showed the typical pictures of three independent experiments. Magnification
is ×380. (C) MT-4 cells (5×105) were incubated on ice with IIIB-env-HIV-1 in the absence (N.T.) or presence of PYC (30 μg/ml),
AZT (1 μM) or DS (10 μg/ml) for 1 h. Then, cells were washed three times with RPMI 1640 supplemented with 10% FBS. Viral
viral effects of PYC, we next performed time of addition
with those seen in the untreated control (Fig. 2A). When AZT
Interestingly, PYC still reduced HIV-1 replication when it
experiments in MT-4 cells infected with HIV-1 IIIB. We com-
was added at 1 h, 2 h, and 6 h post-infection, it inhibited or
was added at 12 h post-infection. These results suggest that
pared the inhibitory effects of PYC with those of AZT and
delayed virus replication, but no additional inhibitory effects
PYC is effective at inhibiting HIV-1 replication at different
DS. MT-4 cells were infected with HIV-1 IIIB, and then DS,
were observed when AZT was added at 12 h post-infection.
stages. To further investigate at which stages PYC inhibits
AZT or PYC was added several hours after infection. The
On the other hand, PYC clearly inhibited HIV-1 replication
HIV-1 replication, viral DNA synthesis was monitored by
replication of HIV-1 was assayed by quantifying HIV-1
in a manner different from that of DS and AZT, and PYC
quantitative PCR analysis post-infection in the absence or
p24Gag released in the culture medium. HIV-1 replication
treatment was associated with rapid and lasting suppression
was greatly inhibited by DS only when DS was added at the
of HIV-1 replication. PYC-induced inhibition appeared to
time of infection (0 h), and post-infection treatment with DS
begin at a very early stage of infection, because p24 produc-
did not confer any unequivocal inhibitory effects compared
tion was reduced when it was added at only 1 h post-infection.
presence of AZT or PYC. As shown in Fig. 2B, PYC reduced
biochemical changes induced by PYC. Cells were either in-
observed in the case of proteosome inhibitor MG132-treated
viral DNA synthesis at 6 h post-infection, indicating that the
fected or not infected with HIV-1 in the presence or absence of
cells. The expression of HSP60, HSP72, and HSP90β, and
inhibition of HIV-1 replication occurred before or at RT stage.
PYC, and the cell cultures were analyzed by immunoblotting
the phosphorylation of JNK-1 remained unaffected after 24
Moreover, increased formation of closed circular DNA (2-
for several proteins which exhibit modified expression patterns
h of incubation with PYC. Levels of expression of HSP27,
LTR) indicated that PYC inhibited the integration of viral
in a context of cellular stress. As shown in Fig. 3A, the accu-
HSP32, HSP47, p38, thioredoxin, IκBα, and Bcl-2 were also
DNA. In conclusion, PYC also inhibits HIV-1 replication at
mulation of Mn-SOD as well as reduced phosphorylation of
examined, and no significant changes were observed (data
multiple steps in the replication pathway, including at the
ribosomal S6 (an indicator of protein synthesis inhibition)
virus-host cell binding and intracellular replication stages.
were both evident in the presence of 30 μg/ml PYC. We
PYC induces the accumulation of Mn-SOD and inhibits
also noticed an accumulation of ubiquitinated proteins in the
the phosphorylation of S6 protein: We further examined the
presence of PYC; this result was somewhat similar to that
Fig. 2. PYC inhibits intracellular HIV-1 replication. MT-4 cells were infected with IIIB-env-HIV-1 at MOI of 1 for 60 min at 37°C,
and washed three times with RPMI 1640 supplemented with 10% FBS. (A) PYC, AZT or DS were added at different time points
after infection as indicated. Concentrations of each compound used were 30 μg/ml for PYC, 1 μM for AZT and 10 μg/ml for DS.
HIV-1 p24Gag released in the culture supernatant was quantified by ELISA 30 h after infection. Relative p24Gag levels are
showed as the percentage to that of cells without drug treatment. The results are mean values of three independent experiments.
(B) PYC, AZT, or DS were added culture cells 1 h post-infection. DNA was extracted at the indicated time points and subjected
to real-time PCR to quantify viral DNAs. Viral DNA levels were shown as relative copy number. The results are mean values of
three independent experiments.
not shown). It should be noted that ADP-ribosyl polymerase
(PARP) was not degraded, even in the presence of 70 μg/ml
of PYC (Fig. 3A), suggesting that no activation of the caspase
pathway was induced within 24 h. The accumulation of
Mn-SOD and the inhibition of phosphorylation of S6 protein
(Fig. 3A and 3B) corresponded to the reduction in p24Gag
release from infected cells. Approximately 65-75% of PYC
is composed of procyanidins, which effectively inhibit HIV-
1 replication (Fig. 3C). The accumulation of ubiquitinated
proteins in the presence of PYC (Fig. 3A) suggests impaired
protein degradation, which was associated with an accumu-
suggesting the involvement of redox regulation in HIV-1 rep-
ence between the chemical nature of procyanidins and that
praline-rich regions on proteins (20), virus binding to host
lation of Mn-SOD.
of sulfated compounds is properties of binding to substrates,
cells and growth inhibition in infected cells could both be
The monomer, dimmer, and trimer of flavan-3-ol in
i.e., procyanidins bind to substrates such as ODS (octadecyl
caused by direct non-specific interactions between proline-
procyanidins neither induced an accumulation of Mn-SOD
nor inhibited virus replication (unpublished observation). The
Here, we demonstrated that PYC inhibits not only the bind-
pounds bind to substrates such as anion exchange resins
Although the EC50 of PYC for the inhibition of HIV-1 rep-
accumulation of Mn-SOD coupled with an inhibition of virus
ing of the HIV-1 virus to host cells, but also inhibits intra-
through ion bonding. The distinct effects of these compounds
lication was not as high as that of AZT, the inhibition of virus
replication was observed only when the cells were treated
cellular HIV-1 replication after virus entry. The latter type of
on HIV-1 replication might be due to their distinct chemical
replication using natural substances is an important concept
with procyanidins comprised of more condensed oligomers
inhibition is a distinctive property of procyanidins compared
properties. Since it has been proposed that different mecha-
than tetramers (data not shown). Interestingly, overexpression
to sulfated compounds that inhibit HIV-1 replication only at
nisms involving hydrogen bonds and hydrophobic inter-
of Mn-SOD hampered p24Gag release in 293T cells (Fig. 4),
the stage when the virus binds to host cells. One major differ-
actions might be responsible for the binding of tannin with
silica gel) via hydrophobic interactions, while sulfated com-
rich proteins on virus and cell surfaces.
Fig. 4. Overexpression of Mn-SOD inhibits HIV-1 production. Cells
(293T) were transfected with the control vector, pTargeT, or pTargeT-
Mn-SOD expressing Mn-SOD. Forty-eight hours later, cells were
infected with VSV-G-HIV-1 and cultured for additional 48 h. (A)
Expression of Mn-SOD was verified by immunoblotting. (B) Relative
HIV p24-Gag levels per total cellular proteins in the culture superna-
tant are shown as the percentage of that for the control transfection.
Data shown are mean ± SD values of three independent experiments.
* represents P < 0.05 and ** represents P < 0.01.
Fig. 3. PYC induces Mn-SOD expression and inhibits S6 phosphoryla-
HIV-1 p24Gag released in the culture supernatant was quantified
tion. (A) Immunoblot analyses of virus-infected or uninfected MT-4
simultaneously by ELISA. Relative p24Gag amounts are expressed
cells in the absence or presence of increasing concentrations of PYC.
as the percentage of that for PYC-free uninfected cells. Data shown
MT-4 cells were mock-infected or infected with HIV-1 IIIB for 60
are mean ± SD values of three independent experiments. * represents
min at 37°C, washed three times with PBS (–), and then the indicated
P < 0.05 and ** represents P < 0.01.
concentrations of PYC was added. After 24 h of incubation, samples
for immunoblot analysis were prepared and analyzed with antibodies
indicated on the left. (B) Shown are the Mn-SOD, S6 and pS6 protein
band intensities relative to that for PYC-free uninfected cells. (C)
for the development of clinical applications. In general, virus
that the removal of reactive oxygen species (ROS) from virus-
pathway for efficient replication. Viral replication is often
from that for inflammatory cytokines. Immunoblot results
cellular membranes. However, it is possible that PYC affects
novel therapeutic strategies for the prevention and/or treat-
infection causes oxidative stress in infected cells (21). Al-
infected cells may interfere with virus replication. Along these
enhanced by inflammatory cytokines, which also induce
revealing the phosphorylated form of S6 suggested that the
the function of numerous proteins bearing the ATPase cata-
ment of virus-related diseases.
1. Levinson, W. (2004): Human immunodeficiency virus. p. 313-321. In
though the importance of virus-induced oxidative stress for
lines, we found that PYC induces Mn-SOD expression and
oxidative stress. On the other hand, the expression of Mn-SOD
rate of cellular protein synthesis was reduced in the presence
lytic unit (ecto-ATPase) on the surface of virus-infected cells
virus replication is not yet well understood, a number of bio-
inhibits HIV-1 replication.
is inducible in TNF-α-, IL-1β-, and IFN-γ-treated cells (24-
of PYC. This finding might reflect altered bioenergetics in
(29). Indeed, inhibition of virus replication by PYC was also
chemical events induced by virus infection (e.g., inhibition
It was recently reported that MnTBAP (manganese [III]
26). Inflammatory cytokines, oxidative stress, and Mn-SOD
cells treated with PYC. Recent reports have shown that small
observed in cells infected with sindbis virus, a togavirus, and
Lipincot, New York.
of macro-molecule synthesis of host cells, but not of the
tetrakis [4-benzoic acid] porphyrin chloride), a synthetic
expression could all be associated with the modulation of
molecular weight polyphenols such as resveratrol and related
in SARS-related coronavirus-infected cells (unpublished ob-
virus; enhanced protein-tyrosine phosphorylation; activa-
peroxynitrite decomposition catalyst, inhibits HIV-1 replica-
several human SOD1 promoter binding sites for transcrip-
compounds bind directly to mitochondrial F1-ATPase and
servations). Further studies are clearly necessary to elucidate
This work was supported by grants from the Ministry of Education, Cul-
2. Donglei, Y., Susan, L., Natschke, M., et al. (2007): New developments
tion of NF-κB, and modification of activity of ion channels)
tion via the suppression of peroxynitrite production in virus-
tion factors (e.g., including AP-1 and NF-κB) by the redox
inhibit its activity (27,28). PYC has been reported to contain
the molecular mechanisms that yield the biological activity
ture, Sports, Science and Technology of Japan and the Ministry of Health,
in natural products-based anti-AIDS research. Med. Res. Rev., 27, 108-
are based on superoxide-induced oxidative responses, which
infected macrophages (23). This observation is consistent with
status of the cell. The mechanism of Mn-SOD induction by
high molecular weight oligomers comprise with the cathechin
of procyanidins present in commonly consumed foods. It is
Labour and Welfare of Japan, and the Human Health Science of Japan.
favor virus replication (22). Thus, it is reasonable to assume
our results showing that the virus required an oxidative stress
PYC remains to be studied, but it could fundamentally differ
unit, which may render it difficult for the protein to penetrate
hoped that such studies will contribute to the development of
Levinson, W. (ed.), Medical Microbiology & Immunology. 8th ed.
event. Proc. Natl. Acad. Sci. USA, 12, 5286-5290.
macology. Int. J. Clin. Pharmacol. Ther., 40, 158-168.
extract (GSPE) and antioxidant defense in the brain of adult rats. Med.
13. Langlade-Demoyen, P., Michel, F., Hoffenback, A., et al. (1988): Immune
of broad-spectrum antiviral agents. Mol. Pharmacol., 42, 1109-1117.
Sindbis virus induces phospholylation and intracellular translocation
22. Klotz, L.-O., Brivia, K. and Sies, H. (2000): Signaling by singlet oxygen
of manganese superoxide dismutase in rat glioma cells. Cell Growth
3. Nakashima, H., Kido, Y., Kobayashi, N., et al. (1987): Purification
5. Zhong, Y., Yoshinaka, Y., Takeda, T., et al. (2005): Highly potent anti-
8. Huang, W.W., Yang, J.S., Lin, C.S., et al. (2005): Pycnogenol induces
Sci. Monit., 12, 124-129.
recognition of AIDS virus antigens by human and murine cytotoxic T
16. Ryo, A., Suzuki, Y., Arai, M., et al. (2000): Identification and charac-
of heat shock protein HSP27 and activation of p38 MAP kinase signaling
in biological systems. p. 3-20. In C.K. Sen, H. Sies, and P.A. Baeuerle,
Differ., 7, 1175-1186.
and characterization of an avian myeloblastosis and human immuno-
HIV-1 activity isolated from fermented Polygonum tinctorium aiton.
differentiation and apoptosis in human promyeloid leukemia HL-60 cells.
11. Kameoka, M., Kitagawa, Y., Utachee, P., et al. (2007): Identification of
lymphocytes. J. Immunol., 141, 1949-1957.
terization of differentially expressed mRNA in HIV type 1-infected
pathway. Biochem. Biophys. Res. Commun., 253, 59-64.
(ed.), Antioxidants and Redox Regulation of Genes. Academic Press.
25. Harris, C.A., Derbin, K.S., Hunte-McDonough, B., et al. (1991): Man-
deficiency virus reverse transcriptase inhibitor, sulfated polysaccharides
Antiviral Res., 66, 119-128.
Leuk. Res., 29, 685-692.
the suppressive factors for immunodeficiency virus type-1 replication
14. Yoshinaka, Y., Takahashi, Y., Nakamura, S., et al. (1999): Induction of
human T cells. AIDS Res. Hum. Retroviruses, 16, 995-1005.
19. Clercq, D.E. (2002): New developments in anti-HIV chemotherapy.
ganese superoxide dismutase is induced by IFN-gamma in multiple cell
extracted from sea algae. Antimicrob. Agents Chemother., 10, 1524-
6. Nakashima, H., Murakami, T., Yamamoto, N., et al. (1992): Inhibition
9. Grimm, T., Chovanova, Z., Muchova, J., et al. (2006): Inhibition of NF-
using the siRNA mini-library directed against host cellular genes.
manganese-superoxide dismutase in MRC-5 cells persistently infected
17. Yamamoto, N., Tanaka, C., Wu, F-Y., et al. (2006): Analysis of human
Biochem. Biophys. Acta, 1587, 258-275.
23. Aquaro, S., Muscoli, C., Ranacci, A., et al. (2007): The contribution of
types. Synergistic induction by IFN-gamma and tumor necrosis factor
of human immunodeficiency viral replication by tannins and related
κB activation and MMP-9 secretion by plasma of human volunteers
12. Yamamoto, N., Yang, R., Yoshinaka, Y., et al. (2004): HIV protease
and quantitative assay based on real-time polymerase chain reaction.
teins. Crit. Rev. Oral Biol. Med., 13, 184-196.
ages. Retrovirology, 4, 76-80.
4. Clercq, D.E., Yamamoto, N., Pauwels, R., et al. (1992): Potent and
compounds. Antiviral Res., 18, 91-103.
after ingestion of maritime pine bark extract (Pycnogenol). J. Inflamm.,
inhibitor nelfinavir inhibits replication of SARS-associated coronavirus.
15. Yamamoto, N., Schols, D., Clercq, D.D., et al. (1992): Mechanism of
Virus Genes, 32, 105-113.
21. Schwarz, K.B. (1966): Oxidative stress during viral infection: a review.
24. Zhong, W.L., Oberley, W.L., Oberley, T.D., et al. (1996): Inhibition of
selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-
7. Rohdewald, P. (2002): A review of the French maritime pine bark
Biochem. Biophys. Res. Commun., 318, 719-725.
anti-human immunodeficiency virus action of polyoxometalate, a class
18. Nakatsue, T., Katoh, I., Nakamura, S., et al. (1998): Acute infection of
Free Rad. Biol. Med., 21, 641-649.
cell growth and sensitization to oxidative damage by overexpression
2 replication by a class of bycyclams interacting with a viral uncoating
extract (Pycnogenol), an herbal medication with a diverse clinical phar-
10. Devi, A., Jolitha, A.B. and Ishii, N. (2006): Grape seed proanthocyanidin
Biochem. Biophys. Res. Commun., 359, 729-734.
with an alphavirus, Sindbis. Bioche. Biophys. Res. Commun., 261, 139-
immunodeficiency virus type 1 integration by using a specific, sensitive
20. Bennick, A. (2002): Interaction of plant polyphenols with salivary pro-
peroxynitrite generation in HIV replication in human primary macroph-
285 Download full-text
or IL-1. J. Immunol., 147, 149-154.
26. Masuda, A., Longo, D.L., Kobayashi, Y., et al. (1988): Induction of
mitochondrial manganese superoxide dismutase by interleukin 1. FASEB
J., 15, 3087-3091.
27. Gledhill, J.R., Montgomery, M.G., Leslie, A.G.W., et al. (2007):
Mechanism of inhibition of bovine F1-ATPase by resveratrol and related
polyphenols. Proc. Natl. Acad. Sci. USA, 104, 13632-13637.
28. Lupas, A.N. and Martin, J. (2002): AAA proteins. Curr. Opin. Struct.
Biol., 12, 745-753.
29. Lin, S.H. and Guidotti, G. (1989) Cloning and expression of cDNA
coding for rat liver plasma membrane ecto-ATPase, The primary structure
of the ecto-ATPase is similar to that of the human biliary glycoprotein
I. J. Biol. Chem., 264, 14408-14414.