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Abstract
Background: We sought to
assess whether GFS, a
proprietary preparation
of Tasmanian Undaria
pinnatifida, has effects
on healing or
re-emergence of Herpetic
infections, and
additionally, to assess
effects of GFS in vitro.
Undaria is the most
commonly eaten seaweed
in Japan, and contains
sulphated
polyanions and other
components with
potential anti-viral
activity. Herpes simplex
virus type 1
(HSV-1) infections have
lower reactivation rates
and Herpes type 2
(HSV-2) infections have
lower
incidence in Japan than
in the west.
Methods: Patients with
active (15 subjects) or
latent (6 subjects)
Herpetic infections
(HSV-1, 2,
EBV, Zoster) were
monitored for response
to ingestion of GFS. GFS
extract was tested in
vitro for
human T cell
mitogenicity and
anti-Herpes activity.
Results: Ingestion of
GFS was associated with
increased healing rates
in patients with active
infections. In addition,
patients with latent
infection remained
asymptomatic whilst
ingesting GFS.
GFS extract inhibited
Herpes viruses in vitro
and was mitogenic to
human T cells in vitro.
Conclusions: Ingestion
of GFS has inhibitory
effects on reactivation
and is associated with
increased rate of
healing after Herpetic
outbreaks. GFS extract
potently inhibited
Herpes virus in
vitro, and had mitogenic
effects on human T
cells.
Background
Herpes viruses are
important human
pathogens, causing
both primary and
secondary infections
that range from
trivial mucosal ulcers
to life threatening
disorders in immuno-
compromised patients
[1,2]. The Herpes group
includes
HSV-1, HSV-2, Herpes
Zoster (chicken pox/
shingles), HCMV (human
cytomegalovirus), EBV,
Herpes
6, 7 (Roseola, post
transplant infections)
and Herpes 8
(associated with Kaposi
sarcoma). The
conventional treatment
of these infections is
with drugs such as
acyclovir
(ACV) that target the
viral DNA polymerase.
Whilst these
drugs are undoubtedly
efficient, their
long-term use has
led to the development
of resistant viral
strains that now
comprise 5% of all HSV
infections in
immuno-compromised
patients [1]. Finding
non-toxic alternatives
to these
drugs is of interest to
patients and to drug
companies investigating
more effective remedies.
Whilst the epidemiology
and serotype of Herpes
infections varies widely
within
the world community, as
a result of differing
subpopulations
and childhood exposure
rates, it seems that the
incidence
of HSV2 in Japanese
women is substantially
less
than that of American
women. (e.g., 50%
seroprevalence
in one study of
African-American women
vs. 7% in Japan)
[3]. We also noted that
the reactivation of
Herpes simplex
I (HSV-1) as measured by
shedding [4] is
considerably less
in the Japanese
population than in the
west. However,
EBV, some strains of
which are associated
with malignancies,
affects over 90% of the
population world wide
[5],
and seropositivity for
CMV is higher in the
Japanese population
[6].
Several plants have been
shown to have anti-viral
activity,
but edible brown marine
algae are unique in that
they are
a regular part of the
Japanese daily diet. Key
anti-viral active
agents within brown
algae are the sulphated
polyanions
[7–9]. In recent years
both natural and
synthetic
sulphated polyanions
have been a research
focus for their
potential therapeutic
activity. They are
considered to act
principally by
inhibiting the entry of
coated viruses such
as Herpes into host
cells [7–9], by
competing for receptors
at the cell surface.
This activity is, in
part, due to the
similarities
of the algal polyanions
to mammalian heparin
sulphate
molecules. There are a
number of receptors
(including a heparin
sulphate receptor)
expressed differentially
on various cell types,
which provide entry
points
for Herpes viruses [10].
This mechanism of
inhibition
contrasts with that of
commonly used drugs such
as ACV
which act as nucleic
acid inhibitors,
preventing viral
replication
after the virus has
entered the cell.
The main seaweed in the
Japanese daily diet is
Undaria
pinnatifida, commonly
known as 'wakame' [11].
The major
sulphated polyanion
present in Undaria is
characterised
as a 'galactofucan
sulphate' [12,13]. Other
sulphated
polyanions such as the
synthetic dextran
sulphates, pentosan
sulphates, clinically
used heparins, and
seaweed-derived
carageenans have all
been investigated for
anti-viral
activity in humans,
either systemically or
topically [7–9].
However, to date,
Undaria ingestion has
not been clinically
assessed in a western
population for its
effects on common
coated viruses such as
HSV-1 and 2 which have
reduced incidence in the
Japanese population
[3,4]. Undaria
extracts have been shown
to have anti-viral
effects on
HSV-1 in vitro [14] and
also against the Herpes
group virus
Epstein Barr Virus or
EBV [15]. Specific
molecules were
not identified in these
studies, thus the
observed anti-viral
activity may arise from
either or both
polyanions and other
components. Undaria
aqueous extract (made by
boiling
Undaria in water) and a
partially purified
galactofucan
sulphate extract were
recently assessed for
activity against
clinical isolates of
Herpes I and Herpes II
in vitro [16,17].
Twenty of the viral
strains were sensitive
to ACV, and nineteen
were resistant. In the
test, both types of
extract were
active against all
strains, and more active
against strains of
HSV-2 than strains of
HSV-1. The mode of
action was
shown to be blocking
attachment and entry of
HSV into
the host cells.
Undaria fractions have
also been shown to have
immune
stimulating qualities in
vitro [18] and other
brown seaweed
fractions have been
shown to have
immunological
effects in vivo
[19,8,9].
The mechanism of viral
inhibition by polyanions
such as
galactofucan does not
generate resistant
strains at the
same rates as ACV [7].
Aside from the
inhibition of viral
infectivity, it is
possible that immune
stimulation via
polyanions or other
components, increases
anti-viral activity
[18,19]
We hypothesised that
Undaria ingestion may be
associated
with resolution of
Herpes infections in a
western population,
and that this would be
reflected by inhibition
of
HSV infectivity and
increased T cell
activity in vitro. In
this
study, a proprietary
Undaria preparation
'GFS' was assessed
for effects on the
healing of, and the
inhibition of
outbreaks of HSV
infections in otherwise
healthy patients.
In addition, whole
aqueous extracts of the
GFS were assessed
for effects on HSV
infectivity in vitro and
on human
T cell stimulation in
vitro.
Methods
A) GFS
GFS was prepared from
Tasmanian Undaria
pinnatifida
and supplied in 560 mg
capsules by Marine
Resources Pty
Ltd. It comprises solely
of the alga, processed
to complete
dryness in the dark
within a day of harvest,
in hygienic
conditions. Batches were
milled and mixed to
ensure uniformity.
B) Patient study
Seventeen patients were
recruited for the study
by health
practitioners. Patients
gave verbal informed
consent to the
study. Health
practitioners monitored
the patients'
health. There are no
known adverse effects
related to the
ingestion of Undaria. No
other anti-viral
medications were
taken at the same time
as GFS. The duration of
the study
was from one month to 24
months. Patient ages
were
from less than 10 years
up to 72 years.
In total, seven cases of
HSV-1, five cases of
HSV-2, three of
active Herpes zoster
(one chicken pox, two
shingles) and two of EBV
were assessed. Results
are presented in tables
1 and 2. Patients
suffering from HSVI
typically experienced
lesions lasting ten
days. Patients with
HSVII had experienced
recurrent events of
varying severity,
sometimes
overlapping, of up to
ten days in duration.
One patient
with shingles reported
'cycle' duration of over
ten days.
Others did not report on
duration.
Dosage levels were
chosen to fall within
the limits of normal
dietary seaweed
ingestion in Japan.
Fifteen patients
with active Herpetic
viral infections were
given four 560
mg capsules of GFS per
day for ten days as a
'therapeutic
dose'. All patients
except subject 14, table
1 (primary
zoster infection) were
suffering repeat
outbreaks of
known aetiology
Six patients with latent
HSV-1 or 2 were given
two capsules
per day as a
'maintenance dose'. Four
of these patients,
identified by an
asterisk *, were
continuations of
treatment after the
active infection phase.
One patient
(subject 3, table 2)
took four capsules per
day.
C) In vitro effects
on HSV
GFS was mixed 1:40 w/v
with distilled water and
boiled
for 5 minutes. The
liquid was filtered
through a 0.45 μM
filter for sterilization
and stored at -20
degrees Celsius. An
aliquot of the
preparation was dried
and the weight was
obtained to determine
the concentration. The
concentration
used was the dry weight
of the dissolved solids
present.
Immortalized human
fibroblasts, HF cells,
were grown in
Minimal Essential Media
supplemented with
glutamine,
antibiotics, and 10%
foetal bovine serum
(FBS). Maintenance
medium was supplemented
with 1% FBS. Laboratory
strains of HSV and HCMV
(American type culture
collection) were tested
in this study. A stock
of each virus
was grown in cultured HF
cells and aliquots were
frozen
at -70°C. The titre of
each virus was
determined by a
plaque assay using HF
cells in 24-well plates
with an agarose
overlay.
D) T cell stimulation in
vitro
T cell mitogenicity was
evaluated by chromium
uptake.
Whole T cell
preparations were
obtained from buffy
coats
from pooled human blood
samples. They were
incubated
in RPMI supplemented
with 10% heat
inactivated foetal
calf serum, 5 mM
L-glutamine, 5 × 10 -5 M
2-mercaptoethanol
and 30 U/ml gentamycin.
Incubation for 72 hours
was at 5%CO2, 37°C in 24
well plates. Cells were
incubated
with either GFS whole
extract (at 25, 125 and
250 mcg/
ml as 1%, 5% or 10% of
total culture volume
prepared
from a stock solution at
2.5 mg/ml) or with the
known
mitogens (PHA) (1
mcg/ml) or Concanavalin
A (ConA)
(1 mcg/ml). Each
concentration was
assessed in triplicate
(n = 3)
Results
Patient study
Results are presented in
tables 1 and 2.
Active infection-table 1
All fifteen patients
with active Herpetic
viral infections
experienced
significant lessening or
disappearance of
symptoms.
No adverse side effects
were noted during the
study.
Two patients (subjects 4
and 5, table 1) with
noncompliant
dosage regimes resolved
infections in normal
time,
but noted no spread of
lesions (as occurred
during previous
outbreaks). Reduction in
lesion severity and
rapid
clearance were noted in
two patients (subjects
6,7, table
1), and pain reduction
as compared to previous
events
was noted by two
patients (subjects 2,14,
table 1). Two females
with genital HSV-2 had
persistent lesions which
resolved
during the course of
treatment (subjects
8,10, table
1).
In two cases of
diagnosed EBV, one clear
at four and the
other by ten days. In
the latter patient a
chronic sinus condition
also cleared (subjects
11,12, table 1)
Over ten days, faster
drying of zoster lesions
and increased
speed of normal cycle as
compared to previous
outbreaks
was noted by a male
patient (subject 15,
table 1) although no
reduction in pain was
reported. In an adult
male suffering
primary zoster (chicken
pox) lesions of whole
body
(subject 14, table 1),
pain reduction and rapid
healing of
lesions were noted. An
elderly female patient
noted a reduction
in pain and visible skin
lesions during the ten
day
course, and had no
recurrence of symptoms
whilst on a
lower dose for the next
two months.
Latent infections-table
2
All six patients on
maintenance doses noted
inhibition of
further outbreaks of
infection. No adverse
side effects
were noted during the
study.
HSV-1 outbreaks were
inhibited in two
patients taking a
maintenance dose over
three months and two
years respectively
(subjects 1 and 2 in
table 2). Low grade
HSV-1
associated
keratoconjunctivitis in
the former patient was
also inhibitedGFS ingestion correlated
with inhibition of a
previously
persistent HSV-2
infection for three
months in subject 4,
table 2. In this
patient, the infection
was ACV resistant and
outbreaks had been
apparent on a two weekly
basis for
over a year.
HSV-2 outbreaks at the
genital site were
inhibited in two
other female patients
whilst taking a
maintenance dose of
two capsules per day,
for one month (subjects
5,6, table
2).
Low grade recurrent
Herpes zoster (shingles)
lesions of
the torso were inhibited
for two months in an
elderly patient
whilst maintaining a
dose of four capsules
per day
(subject 3, table 2).
C) In vitro effects on
HSV
Herpes viruses were
assessed for infectivity
of human fibroblasts
cells in vitro.
Inhibition by GFS
extract was noted
as shown in table 3.
D) T cell stimulation in
vitro
GFS extract was assessed
for effects on whole
human T cell
preparation in vitro.
After incubation with
GFS extract or
mitogens PHA and ConA,
for 72 hours the
relative uptake
of chromium was assessed
as a measure of
mitogenicity.
The lowest concentration
of GFS extract tested
(25 mcg/
ml) exerted a four fold
mitogenic effect on T
cells, over
50% of the mitogenic
potency of the known
mitogens
PHA (six fold) and ConA
(seven fold).
Paradoxically, increased
concentrations of the
whole extract showed
decreasing
effects on mitogenic
activity. This may be
accounted for by the
increasing physical
inhibition due to
increased viscosity in
the culture media, or
the increasing
concentration of
unidentified inhibitory
components
present in the extract.
Results are shown in
Figure 1.
Additional studies
illustrated little
effect on NK cell
activity
and no effects on L929
fibroblast growth over
24 or 72
hours (results not
shown). There was no
bacterial contamination
of the GFS (results not
shown), thus the
presence
of bacterial
lipopolysaccharides
(which may also act as
mitogens) was ruled out.
Discussion
This study was carried
out to assess the
effects of GFS in
patient studies and in
vitro.
GFS was ingested by
patients suffering
active or latent Herpes
infections. Results
indicated firstly, a
positive effect on
healing, and secondly,
inhibition of outbreaks
in cases of
HSV-1, HSV-2, ACV
resistant HSV-2, and
zoster. There
were no adverse side
effects noted, and GFS
was well tolerated
by all subjects. Reduced
pain levels were noted
in
some cases. A
particularly noteworthy
result in this study was
inhibition
of an ACV resistant case
of HSV-2. HSV-2 is a
sexually
transmitted disease of
increasing incidence
[4]. In part,
this is due to the fact
that partner
transmission may occur
during asymptomatic
shedding [4] or
unrecognised minor
outbreaks. Suppressive
therapies such as ACV
have
been tested for their
ability to inhibit
shedding [20]. However,
for long-term use,
non-toxic alternatives
such as GFS
may be preferred by
patients, who perceive
long-term conventional
drug use as detrimental.
In addition, GFS may
reduce the generation of
resistant strains which
arise
through prolonged use of
drugs such as ACV.
GFS extract potently
inhibited HSV and HCMV
infection
of human cells, and
stimulated human T cell
proliferation
in vitro. In other
studies [16,17] extracts
were active
against clinical strains
of HSV. These mechanisms
may be
relevant to the observed
clinical response.
Human T cell
proliferation in vitro
observed here concurs
with observations by
Shan et al [18]. Shan
found that in
the presence of various
seaweed extracts
including Undaria,
lymphocyte proliferation
and cytotoxic T cell
activity
was enhanced, but that
NK cell activity was
not. Cytotoxic
T cells play a prominent
role in the immune
response in
the local environment of
HSV-2 infections [21],
and have
been shown to be key
elements of immune
defence
against HSV-I and other
viruses [22,23]. Thus
the observed
T cell mitogenic effect
caused by GFS in vitro
may
be related to enhanced
immunity in vivo.
Sulphated molecules with
similarities to
sulphated galactofucan,
such as dermatan
sulphate have been shown
to be present in plasma
after oral ingestion
[24]. Macromolecules
are known to pass
through the gut into
plasma
and into lymph via the
extensive gut lymphoid
system
[25] where they may
affect immune function.
The gut
lymphoid system or
'GALT' comprises Peyers
patches and
gut cryptopatches where
different types of T
cells and other
immune cells participate
in uptake or contact
with gut
contents. T cell uptake
or contact with GFS by
either Peyers
patch or gut cryptopatch
dwelling cells may
result in
the proliferative
response noted here in
vitro. However,
immune indicators were
not measured in this
study.
Whilst further research
is called for, the
results obtained in
this study show that
ingestion of GFS is
associated with
resolution, reduced pain
and outbreak inhibition
of Herpes
virus infections.
Studies regarding
uptake,
immunostimulation
and anti-viral effects
of GFS are in progress.
Conclusions
The rise in nucleic acid
inhibitor resistant
strains of HSV-
2, and the need for less
aggressive anti-viral
therapies, indicates
a role for alternative
therapeutic routes such
as that
demonstrated here for
GFS.
Ingestion of GFS has
inhibitory effects on
reactivation of
HSV and positively
affects the resolution
of active Herpes
infections. GFS has
inhibitory effects on
Herpes infectivity
in vitro and a T cell
mitogenic effect in
vitro.
The mechanism of
inhibition of Herpes
infections may
be, in part, mediated by
galactofucan present in
the GFS.
Competing interests
Russell Cooper, Ken
Thompson, John Godwin,
Kate Elliot
have no declared
interests. JH Fitton and
Charles Dragar
are employed Marine
Biomedical Research
Tasmania Australia.
Authors' contributions
RC, JG, KE and JHF
participated in clinical
studies and JHF
drafted the manuscript.
KT carried out the in
vitro studies.
JHF and CD conceived of
the study, and
participated in its
design and coordination.
All authors read and
approved
the final manuscript.
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