Red Algae antiviral properties
Antiviral carbohydrates from marine red algae
Department of Biological Sciences, University of California, Santa Barbara,
93106, USA and Neushul Mariculture Incorporated, 475 Kellog Way, Goleta,
CA, 93117, USA
Key words: AIDS, antiviral, herpes, red algae, seaweed, sulfated
It is possible that heparin-like sulfated polysaccharides from red algae, or
fractions thereof, might be found to be low-cost, broad-spectrum antiviral
agents. The prevailing view among virologists has been that sulfated
polysaccharides inhibit viral action by acting only at the surfaces of cells.
This perception now is changing with the finding that both the herpes virus
(containing DNA) and immunodeficiency virus (containing RNA) are inhibited
by sulfated polysaccharides that act within the cell as well as external to it.
Aqueous extracts of many red algae are active against retroviruses.
Carrageenan, a common cell wall polysaccharide from red algae, is co-
internalized into infected cells with Herpes simplex virus (HSV), inhibiting
the virus. Carrageenan also interferes with fusion (syncytium formation)
between cells infected with the human immunodeficiency virus (HIV) and
inhibits the specific retroviral enzyme reverse transcriptase.
Our increasing scientific understanding of the world around us is gained
through a series of different, and seemingly unrelated discoveries. But
collectively these discoveries are related, in that they support the concept
of biological specificity. Silverstein (1989), in his very readable book on
the history of immunology, reviews the philosophical roots of the concept
of biological specificity and shows how it has influenced the development
of modern immunological thought.
Those who have studied the growth, development and reproduction of marine
red algae know that secondary pit connection formation involves the fusion of
very specific cells. In the process of fertiliztion in these plants there is a
specific union of spermatium and trichogyne (Fetter & Neushul, 1981), and
this is followed by complex post-fertilization events involving very specific
cell fusions. Is it possible that the walls of the cells involved in these
processes contain chemically-indentifiable molecules that determine the
specificity, as is the case in antigen-antibody interactions? Obviously food-
scientists find it useful that specific carrageenans bind to specific milk
proteins. It is possible that this specificity could be useful therapeutically as
well? If so, then in addition to the large-scale cultivation of marine red
algae for food and phycocolloids, we would see them cultivated as a source of
pharmaceuticals. This would be interesting particularly if they served to
palliate or even hopefully cure virally-caused diseases.
At one time it was thought that viruses were so closely associated with their
host cells that it would be difficult, if not impossible, to destroy one and
not the other. However with the discovery of the first antiviral drugs in 1950
and their first clinical use in 1962, it became clear that antiviral therapy
was possible (Bauer, 1985). With the development of the plaque inhibition test
(Rada et al., 1960), it became possibile to test large numbers of compounds
for their antiviral activity. It soon was discovered that agar (a sulfated
polysaccharide extracted from marine red algae) used in plaque assays was
itself antiviral (Gonzales et al., 1987). But agar was thought to inhibit
viral attachment to uninfected cells, rather than inhibiting the virus within
the infected cell. It also was found that heparainized blood plasma, and
heparin alone, inhibited some Rous Sarcoma Virus strains (Solomon et al.,
1966). The idea that viral-binding carbohydrates inhibit by acting outside the
cell has prevailed among most virologists. Consequently, the discovery that
agar and other aqueous extracts of marine red algae have antiviral activity
stimulated little interest. Recent work on these aqueous extracts, on specific
carbohydrates in them (carrageenans) and on other sulfated polysaccharides
like dextran sulfate and heparin (Baba et al., 1988; MItsuya et al., 1988; Ueno
& Kuno, 1987) suggests that these molecules may inhibit both DNA- and RNA-
virus infections and may operate both outside of, and within infected cells.
An antiviral compound usually is identified by first gaining an understanding
of a virus-specific process (cellular attachment, genome uncoating, the action
of viral enzymes) and then showing how the compound or drug inhibits the
process. Then by understanding the relationship between the molecular
structure of the drug and the molecules with which it interacts, one can
produce and test related compounds. In this way it was found that synthetic
nucleosides (first produced as anti-neoplastic agents) either inhibit viral DNA
or RNA polymerasees, or act as chain terminators after being incorporated
into nucleic acids (Wood & Geddes, 1987; Muller, 1979)
Antiviral nucleosides that have been found to be useful for the treatment of
herpes virus infections are Idozuridine (IDU), trifluridine (TBT), virabaine
(ara-A), bromovinyldozyuridine (BVDU), fluoriodoaracytosine (FIAC),
ganciclovir (DHPG), and acyclovir. Another synthetic nucleoside, Zidovudine
(AZT) has been found to suppress replication of the human immunodeficiency
virus, the causal agent of AIDS. Non-nucleoside antivirals include the
amantadine, rimantadine, phosphonoformate and ampligen.
The selection of an effective antiviral compound is complicated by the fact
that most antiviral agents have been studies by many different investigators
using different virus strains, cell culture systems and assay procedures.
Consequently, their relative potency usually is not known. Comparative
efficacy studies, using the same cell culture system, have been conducted
for the herpes simplex virus by De Clercq et al. (1980), and data for the
effects of antivirals on human immunodeficiency virus have been assembled
by Abrams et al. (1988).
Algal extracts with antiviral activity
The search for either useful phycocolloids or pharmacologically active
substances in marine algae usually begins with screening studies that focus
on a specific flora (Blunden et al., 1981; Whyte et al., 1984; Ragan, 1984;
Pesando & Caram, 1984; Hodgson; 1984, Untawale et al., 1977; Hornsey
& Hide, 1974 Dhargalkar et al., 1980). Studies of California red algae by
Ehresmann et al. (1977, 1979), Hatch et al., (1979) and Richards et al.
(1978) in a search for anti-herpetic substances have been particularly
interesting. The use of these plants as botanical agents to treat viral
infection resulted in four patents. The first two (U.S. patent numbers
4,162,308 and 4,162,309) (Calvin & Ellis, 1979) used aqueous extracts
of Neodilsea americana and N. integra. The third patent is particularly well
documented, and involves the use of Cryptosiphonia woodii (Nonomura,
1985) (U.S. patent number 4,522,814). In this instance clinical efficacy
was clearly shown. The fourth patent (Neushul, 1988) (U.S. patent number
4,783,446) is for the use of carrageenan and other sulfated polysaccharides
for the treatment of diseases (including AIDS) caused by retroviral infection.
The pharmaceutical industry has been slow to become interested in these
discoveries, because of the technical difficulties involved in working with
carbohydrates and the research costs involved. Hopefully recent screening
stuides may stimulate future interest (Alarcon et al.,1984), as will our
growing appreciation of the immunomodulating effects of natural and synthetic
heparinods (Dziarski, 1989).
Carrageenan as a bioactive molecule
There is considerable evidence that carrageenans bind to and modulate cell-cell
interactions of various kinds including sperm-egg fusion in the brown alga
Fucus, fertilization in sea urchins, hamsters and guinea pigs, embryogenesis
in the green alga Volvox, aggregation of isolated sponge cells, the inhibition
of lymphocyte recirculation, lymphocyte binding to high endothelial venules
and autorosetting (Chong & Parish, 1987).
Degraded carrageenan has long been known to be a potent inflammatory agent
in vivo (Abraham et al., 1985; Selin & Oyarzabal, 1988). Hydrolyzed
carrageenans have been used to experimentally induce intestinal inflammation
in many animal models of inflammatory bowel diseases (Delahunty et al.,1987).
Carrageenans can have long-lasting effects on the immune system (Thomas &
Fowler, 1981). The extensive studies of carrageenan-induced colitis by
Onderdonk and his co-workers are particularly interesting in that carrageenan
did not cause intestinal inflammation and ulceraton in germ-free guinea pigs,
but when specific strains of the intestinal bacterium, Bacteriodes, and
carrageenan both were present in the lumen, inflammation and ulceration
was induced. In this instance it acts as a adjuvant (Onderdonk, 1985).
Red algal extracts and carrageenan as antivral agents
Because of the severity of the present AIDS epidemic and the debilitating
effects of herpes virus infections, it was important to re-examine the
antiviral effects of red algal extracts, even if they have been of little
interest to virologists. In attempting to confirm the findings of others
who have studied the antiviral effects of aqueous extracts of seaweeds,
we collected and screened 39 Californian marine red algal species for
activity against Herpes simplex virus in a simple assay where extracts
were added prior to infecting the target cells. Further tests of extracts
of some of these species were run for us by Burroughs Wellcome. These
first studies showed that there was potent anti-retroviral activity in an
aqueous extract of Schizymenia pacifica against a murine leukemia virus.
This later was verified by colleagues in England and Japan. Stuides as early
as that of Solomon et al.,(1966) and other work in the 1970s (Schaffrath et
al., 1976) showed that sulfated oligo- or poly-saccharide compounds
suppressed retroviral replication and inhibited viral reverse transcriptases.
Tests run in Japan, using a reverse-transcriptases inhibition assay, showed
that nearly all of the 39 species we had collected contained an inhibitory
substance (Table 1).
It is intersting that although at least one carrageenophyte (Ahnfeltia
gigartionides) was only mildly active, and some agarophytes (Gracilaria,
Laurencia) was active, most of the carrageenophytes were active. This
suggests that a common immunomodulatory cell wall carbohydrate, like
carrageenan, was likely to be the active component. Further studies of the
aqueous extract and fractions thereof showed that carrageenan was indeed
the active antiviral component (Nakashima et al., 1987a, 1987b). Studies
of other heparinoids, like dextran sulflate, also supported the contention that
sulfated polysaccharides can have a potent anti-retroviral effect in vitro
(Ueno & Kuno, 1987; Nagumo & Hoshino, 1988; Mitsuya et al., 1988; Baba et al.
, 1988). Clinical tests with AIDS patients given intravenous and
enterically-coated dexran sulfate at San Francisco General Hospital also
have been encouraging (Abrams et al., 1988).
If sulfated polysaccharides from red algae do inhibit many different viruses,
then it will be essential to understand the molecular basis for virus-specific
inhibition in each case. This will not be an easy task. Diverse viruses,
infecting different kinds of cells, provide many targets for inhibition that
will have to be indentified. The antiviral molecule also will be hard to find
since there are a number of different kinds of carrageenan, and bioactivity
likely is produced by a specific, sulfated fraction. The extensive, often
contradictory literature on the inflammatory effects of carrageenans
illustrates the problems that can be anticipated. Those studying carrageenan-
induced inflammation almost always use off-the-shelf samples of carrageenan
purchased from those who extract it for use as a food additive. Attempts to
repeat the first experiments, using other carrageenan samples, often fail,
probably because of differences in this carrageenan. The life-history stage
of a red algal member of a family Gigartinaceae determines whether it
produced lambda or kappa carrageenan; consequently extracts from harvests
containing both life-history stages (gamete- and spore-producing) will have
varying amounts of these two carrageenan types. Secondly, the ratio of
kappa to iota carrageenan in a plant can vary greatly, even among species
of the same genus. The only way to be sure that one is obtaining a specific
carrageenan type is to carefully sort and identify the species and life-history
stage of the source alga. Because of taxonomic uncertainties it also is
important to file voucher specimens. If one starts from a botanically-
indentified source plant, and then chemically identifies the polysaccharide and
fractions thereof being screened, there is some likelihood that one's results
can be repeated by others. There is also an opportunity to gain an appreciation
of the molecular interactions that produce the antiviral activity. Attempts
should be made to determine the cellular site of action. As noted earlier,
dextran sulfate has been reported to inhibit syncytium formation (Baba et al.,
1988), acting external to the cell. Preliminary tests, using a new syncytium-
formation assay (Nara & Fischinger, 1988), showed that iota carrageenan also
inhibits syncytium formation by HIV-infected cells. There is some evidence
that dextran sulfate may be of some use clinically (Abrams et al., 1988).
Carrageenans, as noted earlier, may be taken up by infected cells.
The suggestion that heparinoid-receptors may be involved in interactions
between lymphocytes and macrophages might exlain some of the
immunodulatory effects reported for carrageenan (Chong & Parish, 1987;
Dziarski, 1989). Preliminary studies of degraded carrageenan have shown
that in combination with IL-4, carrageenan stimulates cell division by cultured
murine mast cells (K. Smith, unpublished). Clearly it will be important to
identify and characterize carrageenan and other heparnoid receptors to see
if binding to such receptor triggers a specific cellular response sequence.
It seems worthwhile to determine if red algal polysaccharides and other
heparinoids are clinically useful broad-spectrum antiviral agents, or if
they might be used clincially to enhance the generation of tumor-specific
cytotoxic cells, or serve in other ways as immunomodulatory agents.
This research was supported by the National Institutes of Health (SBIR
program, awards to Neushul Mariculture Incorporate) and was under
the able direction of R. J. Lewis, who also provided a helpful review
of this paper.
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