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1 Marine plants and their role in antiviral research Shakira Navsa

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                   Marine plants and their role in antiviral research

                                     Shakira Navsa



Abstract

Terrestrial plants with medicinal properties have long been used to effectively treat

viral infections or to palliate the effects of such infections. Their biological properties

have been well documented and sophisticated drugs have been developed from their

extracts. Unfortunately, marine plants have not enjoyed the same prestige. They have

been ‘historically disadvantaged’ because of their inaccessibility but recent advances

in marine technology has made it possible to explore the full potential of micro- and

macro algae. This review deals with the known antiviral activity of marine plants

against viruses such as mumps, herpes simplex virus (HSV), murine leukaemia virus,

etc. It also shows that marine plants have a role to play in antiviral research as they

exhibit certain anti-retroviral activity which could contribute to the elimination of the

Human Immunodeficiency Virus (HIV).



Introduction

Algae include a wide variety of plants that range from diatoms, which are

microscopic, unicellular organisms, to seaweeds extending over 30 m. Algae are

grouped into six main classes mainly on the basis of colour. The unicellular algae are

placed in the kingdom Protista and are classified as euglenoids, dinoflagellates and

diatoms. All have chloroplasts and carry out photosynthesis. Multicellular green algae

(division Chlorophyta), red algae (division Rhodophyta), and brown algae (division

Phaeophyta) are all seaweeds. Seaweeds are widely distributed in the ocean, occurring
                                            2


from the tide level to considerable depths, free-floating or anchored, and include kelp,

dulse, rockweed, and sea lettuce [3].


When compared with land plants and animals, the use of marine organisms in folk

medicine is very restricted, particularly outside Asia. Algae found along the ocean’s

shores have been accessible as a food resource but have been inaccessible as a drug

resource, mainly because algae with pharmaceutical potential are found at

considerable depths [1]. However, with recent advances such as the development of

scuba diving, the seabed is being scanned for any potential anti-virals that could be

harvested from algae.



Historically marine macroalgae or seaweeds have been used as crude drugs in the

treatment of goitre, hypothyroidism, Basedow’s disease, anaemia during pregnancy

and in intestinal disorders [1]. Evidently seaweeds do possess compounds with

pharmaceutical uses and therefore research into the full potential of their biological

activities is justifiable.



The important question, which this review will address, is, why is there now such a

need to explore the full medicinal potential of marine plants, when historically these

properties have been largely ignored? In dealing with this question, the role which

marine plants have to play in antiviral research will become clear.



Background on antiviral activity of marine algae

Studies of the growth, development and reproduction of marine red algae have shown

that in the fertilization process in these plants there is a specific union of spermatium

and trichogyne [3] followed by complex post-fertilization events involving very
                                            3


specific cell fusions. If the walls of the cells involved in these processes contain

chemically identifiable molecules that determine this specificity, similar to those in

antigen-antibody interactions, they could have significant therapeutic potential [2].



An early report on the antimicrobial property of seaweed was published in 1951 [5].

Since then, the antiviral effect of polysaccharides from marine algae to mumps virus

and influenza B virus was reported [6]. Subsequently, polysaccharides from extracts

of red algae were found to inhibit HSV and other viruses [7]. These observations did

not generate much interest because the antiviral activity of the compounds was

considered to be largely non-specific [8]. The isolation of sulphated polysaccharides

from algae with antiviral activity against enveloped viruses increased interest in algae

as a source of antiviral compounds. Enveloped viruses include HIV, HSV-1 and HSV-

2, influenza A virus, Simian immunodeficiency virus (SIV) and Human

Cytomegalovirus (HCMV)



Algal extracts with antiviral activity

With the discovery of the first antiviral drugs in 1950 and their initial clinical use in

1962, it became clear that it was possible to destroy a virus without destroying the

host cell [9]. With the development of the plaque inhibition test it became possible to

test large numbers of compounds for their antiviral activity [9]. It was soon

discovered that agar, a sulphated polysaccharide from marine red algae, used in

plaque assay was itself antiviral [10]. Agar was thought to inhibit viral attachment to

uninfected cells, rather than inhibiting the virus within the infected cell. However,

recent work on the specific carbohydrates such as carrageenans and other sulphated

polysaccharides such as dextran sulphate and heparin [11] suggested that these
                                            4


molecules might inhibit both DNA- and RNA- virus infections and may operate both

outside of, and within, infected cells.



The search for either useful phycocolloids or pharmacologically active substances in

marine algae usually begins with screening studies that focus on specific flora [12].

Studies of Californian red algae in a search for anti-herpetic substances have been

particularly interesting [13]. The use of these plants as botanical agents to treat viral

infections has resulted in four patents.



The first two patents (U.S. patent numbers 4,162,308 and 4,162,309) [14] used

aqueous extracts of Neodildea Americana and N. integra. The third patent involved

Cryptosiphonia woodii (U.S. patent number 4,522,814) [15] which showed clear

clinical efficacy. The fourth patent (U.S. patent number 4,783,466) [2] is for the use

of carrageenan and other sulphated polysaccharides for the treatment of diseases,

including HIV, caused by retroviral infection.



Red algal extracts: carrageenan, sulphated polysaccharides and

polysulphates used as antiviral agents

In a study conducted in 1990 by Neushul and his co-workers [2], 39 species of marine

red algae were screened for activity against the herpes simplex virus in a simple assay

where extracts were added prior to infecting the target cells. Burroughs Wellcome ran

further tests of extracts of some of these species for this study. The first studies

showed that there was potent anti-retroviral activity in an aqueous extract of

Schizymenia pacifica against a murine leukaemia virus (See table 1). Tests undertaken

in Japan, using a reverse-transcriptase inhibition assay showed that nearly all of the 39
                                           5


species collected contained an inhibitory substance to the murine leukaemia virus.

Most of the carrageenophytes studied were active and this suggested 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 [15a,b]. Studies of other

heparoids like dextran sulphate (DS) also supported the idea that sulphated

polysaccharides can have potent anti-retroviral effects in vitro [16].



Anti-HIV activity of extracts and compounds from marine micro- and

macroalgae

The process by which HIV evades the immune system is as yet not fully understood.

It is known that a glycoprotein on the viral outer coat latches onto the CD4 receptors

on the surface of the T-lymphocytes, and the complex which these two elements form,

blocks the antibody access [19]. So the “immune system essentially cannot ‘see’ these

prime targets and generate a response to them [20].”



The major problem is to identify compounds that target the virus but do not negatively

affect the host cell. Micro- and macroalgae are some of the first sources of natural

compounds with in vitro anti-HIV activity [4].



Important studies that supported the idea that sulphated polysaccharides could have

anti-retroviral effects in vitro were conducted by Witvrouw and De Clercq [18]. These

studies found that some natural polysulphates isolated from algae exhibit a differential

inhibitory activity against HIV strains, which suggest differences in the target

molecules with which these compounds interact. The polysulphates acted by
                                             6


inhibiting the cytopathic effect of HIV and also prevented HIV-induced syncitium

(giant cell) formation [18] (See table 1).



The results of Witvrouw and De Clercq’s study [8] showed that compounds extracted

from algae have in vitro or in vivo activity against a wide range of retroviruses

including, HSV-1, HSV-2, HCMV, togaviruses, rhabdoviruses and HIV. Compounds

isolated from algae that have been tested against HIV include steroids and

sulfoglycolipids, but most of the research has used the natural and synthetic sulphated

polysaccharides [18]. The full antiviral potential of algal steroids and sulfoglycolipids

has therefore not been fully explored.



If sulphated polysaccharides from red algae do inhibit many different viruses, then it

is essential to understand the molecular basis for virus-specific inhibition in each case.

This is not easy to do because diverse viruses, infecting different kinds of cells

provide many targets for inhibition that will have to be identified. The antiviral

molecule itself will be hard to find as there are various types of carrageenan and

bioactivity is likely to be produced by a specific, sulphated fraction [2].



Sulphated polyanions and HIV activity

Based on the results of competitive binding studies, it was concluded that “disruption

of the CD4-gp120 interaction is probably responsible for most observed antiviral

activity of sulphated polyanions toward HIV infection of lymphocytes [25]. However,

HIV infection and gp120 binding to monocytes was unaffected by sulphated

polyanions, probably because these compounds were unable to block the CD4-gp120

interaction in monocytes [25].”
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The anti-HIV activity of polysulfates is due to their “shielding off the positively

charged sites in the V3 loop of the viral envelope glycoprotein (gp120). The V3 loop

is necessary for virus attachment to cell surface heparin sulphate before more specific
                                            +
binding occurs to the CD4 receptor of CD4 cells. This general mechanism also

explains the broad antiviral activity of polysulfates against enveloped viruses [18].”



Human clinical trials

Natural and synthetic sulphated polysaccharides have been tested for their

prophylactic properties and some synthetic compounds have been tested for tolerance

when administered to humans. Clinical tests with Acquired Immune Deficiency

Syndrome (AIDS) patients given intravenous and enterically-coated DS at San

Francisco General Hospital, have been encouraging [17].



Safety and tolerance to orally administered dextran sulphate (DS)

There exists a good deal of contention as to the toxicity and safety of administering

DS as a chronic drug for the treatment of HIV-infected patients. In 1989, Abrahms

and his co-workers [17] carried out a two part study using DS. Ten patients in each of

3 cohorts (AIDS and asymptomatic) were given 2700mg/day and ten other patients in

each cohort were given 5400 mg/day. It was concluded that oral DS “…is not without

toxicity despite the current lack of conclusive evidence of systemic absorption [17].”

In the second part of the study, DS was given orally 3 times daily for eight weeks in

total daily doses of 900-5400 mg [17]. There was no change in CD4 lymphocyte

numbers or HIV antigen levels and there was no evidence of systemic absorption of

the parent compound. However, “in view of the promising in vitro effects and
                                           8


acceptable toxicity, oral DS as a potential antiretroviral agent continues to be studied

[17].”



Subsequent studies however, seemed to dim hopes of the further potential clinical use

of DS in the treatment of HIV [28]. In a study conducted by Flexner and his co-

workers in 1991 [28], the maximally tolerated dose of DS was administered by

continuous intravenous infusion to 10 subjects with symptomatic HIV infection for up

to 14 days. The results of this study showed conclusive evidence that “Continuous

intravenous DS was toxic, producing profound but reversible thrombocytopenia in all

8 subjects who received the drug for more than 3 days and extensive but reversible

alopecia (baldness) in 5 of these subjects. Because of its toxicity and lack of

beneficial effect on surrogate markers, DS is unlikely to have a practical role in the

treatment of symptomatic HIV infection.



South Africa’s seaweed resources and their potential for use in antiviral

research.

South African agar-producing seaweed , collected from beaches in Saldanha Bay, is

being farmed on an experimental basis [29]. South African seaweed is dried and

exported, mainly to Japan, where the agar is extracted. There is potential for farming

seaweed on the Northern Cape coast, especially if agar were to be extracted locally

[29].


Large beds of kelp grow on Western Cape shores and are washed up on the beaches

Much of this material is collected and exported for the production of a seaweed

concentrate that has a good international market in agriculture. The local seaweed

industry is worth about R15 million annually, and has the potential to expand, both by
                                            9


greater use of existing natural resources and by farming of some of these marine

plants [30].


The known species of seaweeds in South Africa have risen from 545 to 803 , to over

850 currently [31] . South Africa has a rich biodiversity of red marine algae. Species

of red algae found on the west coast include: Epymenia obtusa, Neuroglossum

binderianum, Hymena venosa and Schizymenia obovata, among others [32].


Studies on South Africa’s red seaweeds have shown that their crud e extracts often

contain compounds with antiviral activity [26]. A few South African red seaweeds

have been assayed for their antimicrobial, cytokinin-like anti-inflammatory activities

[26]. A study conducted by Cameron and his co-workers at the University of the

Western Cape(UWC) [26] aimed at determining the activity of the extract

dichloromethane:methanol (2:1) extracted from some common red seaweeds from the

South African West Coast. These extracts were tested against specific bacteria and

antimicrobial activity was observed [26].


South Africa has a wealth of seaweed resources that are being explored for their

economic potential. It would also be worthwhile to explore the pharmaceutical

potential of these resources, as it could be of benefit to antiviral research.


Discussion:

Further exploration into the role of algal extracts in antiviral research


The majority of research conducted on the antiviral properties of algal extracts has

focused on the sulphated polysaccharides. However, none of the studies reviewed

here have specifically explored another class of algal compounds with antiviral

properties, namely the algal lectins. Lectins have specific anti-HIV properties as
                                            10


certain lectins are able to bind to the glycans present in the gp120 molecule in the

HIV envelope. This results in inhibition of both virus-cell fusion and HIV infectivity

[33] as well as syncitium formation [34]. The absence of research using algal lectins

seems to be a gap in the studies of antiviral compounds in algae rather than a

conclusion that algal lectins are inactive [4].



There are still many areas of uncertainty with respect to the antiviral properties of

algal extracts. The studies mentioned in this review have shown that certain algal

extracts exhibit antiviral activity, but it has only been postulated or suggested that the

biologically active components are sulphated polysaccharides [2]. Also, drugs such as

DS which have shown in vitro activity against certain viruses have not undergone

sufficient clinical trials to determine their efficiency and level of toxicity.



In order to develop advanced antiviral, and specifically anti-retroviral drugs, more

energy, time and money will have to be invested in marine plant antiviral research.

Unfortunately, pharmaceutical companies have taken little interest in further research

into the antiviral potential of algae because of the difficulty involved in working with

carbohydrates and the research costs involved [2].



Conclusion

From the tests and research studies already mentioned in this review, it is evident that

some knowledge of the antiviral activity of marine plants exists. Algal extracts have

shown antiviral activity against murine leukaemia virus [17], mumps and influenza

virus [6], HSV [7] and HIV [18]. Recent interest in the potential of marine plants as
                                           11


antivirals and particularly as anti-retrovirals has been aroused in response to the

desperate need to control the global AIDS pandemic.



Despite the research already conducted, the toxicity of sulphated polysaccharides

prevents them from having any immediate application in the treatment of HIV.

However, “comprehension of the mechanism of action is a main strategy for the

construction of specific inhibitors of the enveloped virus. The research of new

sulphated polysaccharides in macroalgae, microalgae … is a challenge for the future

[35].”



Algae produce many other classes of compounds with known biochemical and

cellular actions, and it might be worthwhile to investigate their anti-HIV activity. If

we leave our terrestrial shores in search of new medicines, we might discover many

novel antivirals, and even perhaps a cure for AIDS, in the vast expanse of the ocean’s

garden.



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Evans’ Pharmacognosy , 14th ed. London :Saunders; 1989. p. 18.

[2] Neushul M. Antiviral carbohydrates from marine red algae. Hydrobiologia 1990;
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[3] Fetter R, Neushul M. Studies on developing and released spermatia in the red alga,
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[5] Pratt R, Mautner RH, Gardner GM, Sha Y, Dufenoy J. Report on antibiotic
activity of seaweed extracts. J.Am.Pharm.Assoc 1951; 40: 575-579.
                                           12


[6] Gerber P, Dutcher JD, Adams EV, Sherman JH. Protective effect of seaweed
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[15a] Nakashima H, Kido Y, Kobayashi N, Motoki Y, Neushul M, Yamamoto N.
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[15b] Nakashima H, Kido Y, Kobayashi N, Motoki Y, Neushul M, Yamamoto N.
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[16] Ueno R, Kuno S. Anti-HIV synergism between dextran sulphate and
zidavudine[letter].Lancet 1987; 2: 796-797.

[17] Abrams D, Pettinelli C, Power M, Kubacki VB, Grieco MH, Henry WK. A phase
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[18] Witvrouw M, De Clercq E. Sulfated polysaccharides extracted from sea algae as
potential antiviral drugs. Gen. Pharmacol. 1997; 29: 497-511.
                                         13



[19] Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson
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[26] Cameron D, Keats D, Leng H and Green I. Antimicrobial activity of South
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Technology for sustainable industry.

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[28] Flexner C, Braditch-Crovo PA, Kornhauser DM, Farzadegan H, Nerhood LJ,
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                                         14




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[35] Filalimouhim R, Hours M. Antiviral activities of sulphated polysaccharides. Acta
Botanica Gallica. 1995; 142: 125-130.
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Table 1:Marine algal extracts and their mode of viral inhibition

   Marine alga            Extract               Virus inhibited      Mode of action

Schizymenia          Polysaccharide         •    Avian             Inhibits reverse
pacifica             with sulphate               myeloblastosis    transcriptase (RT).
                     residues [15a,b]            virus(AMV)        Selectively inhibits
                                            •    Rauscher          HIV RT and
                                                 murine            replication in vitro.
                                                 leukemian virus   [15a,b]
                                            •    HIV [15a,b]
Fucus vesiculosus    Polysaccharides        •    HIV [21]          Inhibits HIV-
                     Polyphenols [21]                              induced syncitium
                                                                   formation and HIV
                                                                   RT activity [21].
F. vesiculosus       Fucoidan [22]          •    HIV [22]          Inhibits HIV RT in
                                                                   vitro. Compound
                                                                   competes with
                                                                   nucleic acid
                                                                   substrate [22].
Sargassum horneri    Sulphated              •    HSV-1             Mode of action not
                     polysaccharides        •    HCMV              yet identified [23]
                     (fucose as main        •    HIV-1 [23]
                     sugar) [23]
Agardhiella tenera   Galactan sulfate[8]    •    HIV-1, HIV-2      Unknown [8]
                                            •    HSV, RSV
Gigartina            Carrageenan [24]       •    HSV [24]          Potential RT
skottsbergii                                                       inhibitor.
                                                                   Anticoagulant
                                                                   activity
C. ramosa            Sulphated galactan     •    HSV-1             Inhibitory action on
                     [24]                   •    HSV-2 [24]        virus adsorption
                                                                   [24]

				
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