Marine Natural Products

Metabolit sekunder dari biota laut

TOKSIN LAUT

Pendahuluan
✔ Toksin laut menarik perhatian akibat kasus-kasus
keracunan yang terjadi dan pengaruhnya terhadap sosio-
ekonomi. Beberapa hal yang menjadi titik perhatian utama
meliputi:
✔ Penentuan/pengembangan metode deteksi, determinasi
dan terapi.
✔ Pengetahuan atas mekanisme aksi di tingkat molekuler
✔ Banyak toksin ditemukan perangkat yang berguna dalam
menjelaskan fenomena biologis dan farmakologis (cth.
Pengunaan tetrodotoksin untuk mempelajari kanal sodium,
dan asam okadaik dalam mempelajari fosfatase protein
✔ Modifikasi kimia dari toksin yang diikuti dengan QSAR
menyediakan target yang menarik bagi ahli kimia dan
farmasi

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 Marine toxins
● Dalam pembahasan ini toksin laut dibedakan
atas 5 kelompok:
– Penyebab keracunan kerang yang berakibat
kelumpuhan
– Penyebab keracunan kerang yang meracuni
syaraf
– Penyebab keracunan kerang yang berakibat
diare
– Ciguatera (keracunan seafood)
– Penyebab keracunan lainnya

1. Paralytic shellfish poisoning

(PSP)
● Keracunan akut yang sering letal akibat makan kerang
dan kepiting
● Merupakan akibat dari fenomena RED TIDE: Pada
saat yang tdk dapat diperkirakan terjadi ledakan
populasi plankton-plankton merah (Gonyaulax
tamarensis, Pyrodinium bahamense, dll).Toksin yang
dihasilkan plankton tersebut (Saxitoxin (SXT) dan
congenernya) menyebabkan kematian masal ikan.
Kerang dan kepiting dapat bertahan tetapi menimbun
racun di dalam tubuhnya dan selanjutnya
menyebabkan keracunan pada manusia

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 Saxitoksin

Deteksi PSP
● Bioassay dengan mencit dan lalat
– Dipilih mencit BB 20 g di suntik ip dgn 1 ml larutan uji. Waktu
kematian dicatat dan toksisitas ditentukan dalam mouse units
(Mu) terhadap tabel standar dan dikoreksi dengan faktor yang
diperoleh dari mencit kontrol yang disuntik dengan standar
saxitoxin dihydrochloride dan dinyatakan sebagai mikrogram
equivalent dari saxitoxin dihydrochloride. Satu Mu adalah jumlah
racun yg dibutuhkan untuk membunuh satu mencit 20g dalam
waktu 15'. Uji ini akurat tetapi akan menghadapi kesulitan untuk
memperoleh keseragaman bobot hewan uji.
● Uji ikatan dengan kanal sodium
– Aktivitas farmakologis SXT dikembangkan sbg teknik deteksi.
Racun berikatan dgn kanal sodium di membran sel syaraf,
mencegah influx sodium dan depolarisasi sebagian dari
membran. Uji ini cocok utk menentukan sifat farmakologis racun
dan bukan untuk deteksi rutin.

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 Deteksi PSP
● Immunoassay
– Saxitoxin diikat oleh bovine serum albumin (BSA) dengan
perlakuan formaldehyde dan antibodi yang dipreparasi dari
antiserum kelinci. Uji ini kurang akurat tetapi cocok untuk
digunakan sebagai field rapid test.
● Uji kimiawi
– Metode fluorometri terhadap saxitoksin dengan berdasar
atas produk degradasinya akibat perlakukan dengan NaOH-
hidrogen peroksida. Metode ini sensitif tapi tdk bisa
mendeteksi neosaxitoksin. Kelemahan lainnya adalah
produk lain yang berfluoresensi akan mengganggu uji.

Kelompok racun PSP lainnya

● Tetrodotoksin (TTX)
– Merupakan racun laut yang paling dikenal karena sering
menimbulkan keracunan makanan, keunikan struktur dan
aktivitasnya.
– Mendapatkan namanya dari ikan buntal (pufferfish fam.
tetraodontidae)
– Aktivitas spesifik blocking kanal sodium dari membran yang
tereksitasi.

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 Mekanisme aksi racun PSP
● Tetrodotoxin and saxitoxin are the most widely studied marine
toxins by physiologists and pharmacologists. In spite of their
structural dissimilarities, both are known to inactivate the
sodium channel in the skeletal muscles and nerve tissues of
various animals. The effect of these toxins is specific as both
selectively block the transient Na+ current without any effect on
the steady state current by K+ ions. Owing to this specific action
of these toxins, many investigators are using them as tools in
the characterization of ion channels. These toxins have become
an extremely useful and popular chemical tool for the study of
neurophysiology and neuropharmacology. Tetrodotoxin binds to
the entrance part of the Na+ channel and inhibits Na+ channel
and Na+ influx, and generates an active potential, thus, causing
the blockade of nerve of muscle function. Tetrodotoxin is
commercially available and in carefully controlled doses is
being used as muscle relaxant and pain killer in neurogenic
leprosy and terminal cancer.

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 2. Neurotoxic Shellfish
Poisoning (NSP)
● The dinoflagellate Gymnodinium breve (Ptychodiscus
brevis) often forms blooms along the Florida coast and this
leads to mass mortality of fish. Large blooms of this
organism (red tide) can kill hundreds of tons of fish a day.
Sometimes the blooms cause human irritation in eyes and
throat in the coastal areas, and the contamination of
shellfish occasionally result in human intoxication. The
symptoms of neurotoxic shellfish poisoning (NSP) are mild
which generally subside within 36 hrs, and include tingling
and numbness in the mouth and digits, ataxias, hot cold
reversal of temperature sensation, myadriasis, reduced
pulse rate, diarrhea and vomiting.126 NSP is not a lethal
human intoxication.

Brevetoksin
● Brevetoxin B is of particular interest not only because it is
the most potent toxin of the family but also because it
uniquely binds to sodium channels on excited membrane.

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 Brevetoksin A &
Hemibrevetoksin B
● Hemibrevetoksin B

3. Ciguatera (Sea Food

Poisoning)
● The term ciguatera is used to food poisoning caused by ingestion
of toxic coral reef fish. Ciguatera not only endangers public health
but also hampers local, fisheries in tropical and subtropical regions
of the world. It is estimated that roughly 20,000 people suffer
annually from such poisoning. Two groups of compounds
implicated in the poisoning are ciguatoxin and maitotoxin. Both
groups of toxins are produced by the epiphytic dinoflagellate
Gambierdiscus toxicus and transferred to herbivorous fish and
subsequently to carnivores through the food chain.
● Ciguatoxin is regarded as the principal toxin responsible for human
illness. The clinical symptoms are diverse. Of these neurologic
disturbances are prominent. The most characteristic symptoms of
ciguatera are reversal of thermal sensation called “dry ice
sensation”. Other symptoms are joint pain, miosis, erethism,
cyanosis, and prostration. Gastrointestinal disorders are nausea,
vomiting and diarrhea. Cardiovascular disturbances are low blood
pressure and bradycardia.

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 Ciguatoksin
● The moray eel, which is placed near the top in the coral ecosystem
was found to contain more polar (more oxygenated) congeners,
whereas the dinoflagellate produces less polar ones. Ciguatoxin (70)
itself, the more oxygenated member of this class of toxin was absent
in the flagellates. These data suggested that less polar congeners
produced by G. toxicus were probably the precursors of the more
polar toxins found in fish. The latter appear to be formed by oxidative
enzyme systems in the fish. Interestingly, toxicity of more oxygenated
metabolite is often increased, as in the case with ciguatoxin which is
11 times more toxic than its plausible precursor

Mode aksi dari Brevetoksin dan

Ciguatoksin
● Pharmacological studies on brevetoxins and ciguatoxins had
revealed that the primary site of their action is voltage-sensitive
sodium channel (VSSC). Brevetoxin-B activated VSSC in
neuroblastoma cells in the presence of veratridine. The action
was blocked by tetrodotoxin. A binding assay using a radioligand
of brevetoxin, tritiated derivative of brevetoxin-B at 42-aldehyde
indicated that both toxins shared the same binding site on the
VSSC. Several studies have been carried out on ciguatoxin to
understand its mode of pharmacological and toxicological action
because its central role in human illness. Earlier it was thought
that CTX is a inhibitor of choline esterase. However, subsequently
it was revealed that CTX stimulated sodium ion influx into cells.

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 Ciguatera lainnya
● Maitotoksin
– Diverse actions of MTX have been reported. These are muscle
contractions, stimulation of hormones/ neurotransmitter release,
activation of phospholipase-C and A2 and activation of protein
kinases some of which do not appear to be directly linked with
simple elevation of intracellular Ca2+ concentration.
● Palitoksin
– Action: Membrane depolarization, Na+ or Ca2+ influx, stimulation of
arachidonic acid release, stimulation of neurotransmitter release,
inhibition of Na+/K+-ATPase, induction of contraction of smooth
muscles and tumor-promoting, studies had been carried out on
palytoxin, while it is proposed that palytoxin (PTX) acts through
Na+/K+-ATPase, detailed mechanism of its action is still largely
unknown.

4. Diarrheic Shellfish
Poissoning
● Diarrheic shellfish poisoning (DSP), a major public health problem
even though it is not lethal. The toxic symptoms are abdominal
cramps (1 hr elapse time), nausea progressing to diarrhea (2-7 hr),
and a “raw”, “burning” feeling in the stomach. Diarrhea is noted in
92% of all cases, nausea in 80%, vomiting in 79%, abdominal pain in
53%, and chills in 10%. Diarrheic shellfish poisoning was first
discovered in 1976 when a poisoning due to mussel occurred in
North Eastern Japan, although no “red tide” was seen during the
infestation period. The causative organisms of the illness have now
been identified as several dinoflagellates in the genus Dinophysis.
Even at a low cell density (200 cells per liter) of dinoflagellates leads
to toxification of shellfish. DSP is associated with eating bivalves such
as mussels, scallops or clams which have accumulated dinoflagellate
toxins. Lack of a proper method of detection of the toxins in the past
made it difficult to diagnose the suspected gastroenteritis as the
amount of toxin that can induce illness in man by oral intake is very
small, equivalent to 32 g of dinophysistoxin-1 (DTX-1). Causative
toxins that have been identified are okadaic acid and its analogs and
pectenotoxins.

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 Okadaic acid dan
Denophysistoxin
● Okadaic acid has been found to act as an inhibitor of the protein
phosphatases. Numerous biochemical and pharmacological
studies have been carried out using as a probe. The biological
activities of are now considered to be due to its inhibitory action
against protein phosphatases. Okadaic acid inhibited PP2A at the
lowest concentration (Ki of 30 pm) PPI inhibited at the next lowest
concentration, and PP2B at the highest concentration. Okadaic
acid showed no effect on PP2C. Okadaic acid and DTX1 had
been reported to be non-phorbol ester type cancer promoter.

Toksin DSP Lain

● Pectenotoxin
– It was isolated as one of the diarrheic shellfish toxins from the
digestive glands of the scallop, Patinopecten yessoensis
found Northeastern Japan.
● Yessotoxin
– Yessotoxin, a novel polyether toxin, had been isolated from
scallops Patinopecten yessoensis implicated in diarrheic
shellfish poisoning.

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 5. Toksin-toksin lainnya
● Screening of micro algae for toxin production led to the
isolation of a wide variety of bioactive metabolites from
dinoflagellates. Some of these compounds were possibly
implicated in poisonings.
– Amphidinolid
– Amphidinol
– Surugatoxin
– Macroalgaltoxin
– Peptida toksik

Amphidinolid
● Amphidinolid (suatu makrolid)
– Three groups of macrolides named amphidinolides
had been isolated from the dinoflagellate
Amphidinium spp. symbiotic to flatworm
Amphiscolops breviviridis.

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 Amphidinol
● It was isolated from cultured cells (440 L) of the
dinoflagellate Amphidinium klebsii, collected at Ishigaki
island, Japan.
● Amphidinol is exhibiting potent antifungal and hemolytic
activities.

Surugatoksin
● In 1965, intoxication occurred due to ingestion of a
carnivorous gastropod, Babylonia japonica. The patient
complained of visual defects, including amblyopia,
mydriasis, with thirst, numbness of lips, speech
disorders, constipation and dysuria.

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 Macroalgal toxin
● Although microalgae are frequently involved in various type of
seafood poisoning, however, the involvement of macro algae in
seafood poisoning is rare. Human intoxication due to ingestion
of the red alga Polycaverosa tsudai (formerly Gracilaria edulis)
occurred in Guam, Japan, in 1991. Thirteen people became ill,
three of whom died. As the alga had been eaten widely with no
previous record of potential risk, a toxic glycoside named
polycavernoside-A isolated from the alga has been assumed to
be the causative agent of the poisoning.

Peptida toksik
● The peptides from red, brown green and blue-green algae with
toxic properties are known. From the brackish water Nodularia
spumigena species, which caused problems in Baltic Sea and
New Zealand, cyclic pentapeptide, nodularin had been isolated.
The peptide is closely related to microcystin, a potent
hapatotoxin and protein phosphate 2 and 2A inhibitor isolated
from the blue-green alga Microcystis aeruginosa.

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