Indian Journal of Experimental Biology
Vol. 45, July 2007, pp. 579-593
Review Article
Bioactive molecules from amphibian skin: Their biological activities with
reference to therapeutic potentials for possible drug development
Antony Gomes1, Biplab Giri1, Archita Saha1, R Mishra1,
Subir C Dasgupta1, A Debnath2 & Aparna Gomes2
1
Laboratory of Toxinology and Experimental Pharmacodynamics
Department of Physiology, University of Calcutta
92 A. P. C. Road, Kolkata 700 009, India
2
Division of New Drug Development, Indian Institute of Chemical Biology
4 S. C. Mullick Road, Kolkata 700 032, India
The amphibian skin contains various bioactive molecules (peptides, proteins, steroids, alkaloids, opiods) that possess
potent therapeutic activities like antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep
inducing properties. Research on amphibian skin derived biomolecules can provide potential clue towards newer drug
development to combat various pathophysiological conditions. An overview on the bioactive molecules of various
amphibian skins has been discussed.
Keywords: Amphibians, Frog skin, Medicinal application, Skin bioactive molecules, Therapeutic potential, Toad skin
The amphibians are defenseless creatures that are
consumed preferably by a great variety of predators.
In order to protect themselves from the potential
predators, the amphibians have evolved different
morphological, physiological and behavioral features.
One such defense mechanism is the slimy glandular
secretion by the skin. Frogs and toads have two types
of skin glands–the mucous and the granular. The
mucous glands occur throughout the skin and their
secretion provides a moist coating that is necessary
for cutaneous respiration. The granular glands, also
called the serous or poison glands, may be distributed
across the body but are often concentrated around the
head or neck, and are usually activated by stress or
injury, and their secretions vary from species to
species from being slightly noxious to extraordinarily
toxic1.
In many ancient cultures, amphibians are believed
to possess medicinal properties. Frog potions are used
as aphrodisiacs, impotence and infertility preventions,
contraceptives and in many other illnesses. Also,
newts are often burned to ashes and then used in
medicinal formulas and concoctions. Even today, the
skins, bodies and body parts of salamanders are used
_______________
Phone: +91-33-2350-8386 (Extn 229);
Fax: +91-33-2351-9755; +91-33-2241-3288
E-mail: [email protected]
in traditional medicines. Torched newts are
sometimes sold in Asia as aphrodisiacs and the skin of
certain species are said to cure illnesses. Superstitions
and folklore as these may be, they were actually the
stepping-stones to modern biological sciences.
In fact, toad and frog skin extracts have been used
in Chinese medicine for treating various ailments.
Chan Su is a traditional Chinese medicine prepared
from dried white secretion of the skin glands of
Chinese toad and has been used as an oriental drug for
treating heart diseases, toothache, sinusitis,
haemorrhage of gums and other systemic illnesses.
Chinese doctors prescribed drinking of wine (in which
toad skin has been soaked) for the treatment of
leukemia. A well known, but somewhat toxic patent
medicine, Liu Shen Wan (containing toad secretion
and realgar), is sometimes recommended by Chinese
doctors for leukemia. Scientific research has
confirmed the presence of bufalin in Chan Su, which
is responsible for digoxin-like action2. Scientists have
also found that Chan Su possesses antineoplastic
properties3 and components of Chan Su are capable of
potentiating immune responses in experimental
animals4. Scientists throughout the world are now
exploring the therapeutic potential of various toad and
frog skin extracts and secretions. India, being a
tropical country with diverse geographical variations,
is home to wide species of toads, frogs and newts.
580
INDIAN J EXP BIOL, JULY 2007
There are about 200 species of frogs and toads in
India. However, not much work has been carried out
on toads and frog skin secretions in India as compared
to the rest of the world. Though, the jacket (coat) of
some of the most toxic frogs and toads might just be
the treasure house, storing some of the best
pharmaceutical promises. The present communication
is an effort to enlighten the importance of amphibian
skin – a future treasure house for development of
novel drugs against several pathophysiological
conditions.
Cardiotonic and anti-arrhythmic activity
Chan Su is prepared from skin secretion of Chinese
toad (Bufo bufo gargarizans), for treating arrhythmia
and other heart diseases. It had been introduced in
Europe in 17th century and was later replaced by
digitalis some 200 years ago5. Kyushin (Japanese
medicine similar to Chan Su) shows a beneficial
effect on congestive heart failure models in rabbits
due to its cardiotonic property6,7. The vasodilating
effect and positive inotropic action of Kyushin is
probably due to beta-adrenergic action8. Kyushin
significantly inhibits the aconitine-induced and
thyroxine-induced arrhythmia in guinea pigs. The
decrease in heart rate induced by electrical stimulation
to the parasympathetic nerve (vagus nerve) can be
restored by Kyushin, though Kyushin itself does not
affect the conduction system in Langendorff
preparation of rabbit hearts9. Kyushin dosedependently increases the left ventricular pressure and
mean aortic pressure and decreases the left ventricular
end-diastolic pressure in a dose-dependent manner,
but the myocardial oxygen consumption and heart rate
are not significantly affected in anaesthetized dogs10.
Bufalin and cinobufagin are the main components of
Chan Su, which are responsible for its digitalis-like
action. Although Kyushin itself does not affect the
conduction system, bufalin (0.3mg/ml) and
cinobufagin (1 mg/ml) inhibit conduction in
Langendorff preparation of isolated rabbit heart9.
Bufalin, cinobufagin and some other bufadienolides
like bufotalin, cinobufotalin, gamabufotalin and
resibufogenin⎯all show the cardiotonic effect in a
concentration-dependent manner in guinea pig
isolated heart preparations and cinobufagin possesses
the most cardiotonic action, similar to digitoxin, in
experimentally induced heart failure due to acute
local ischemia11. The cardiotonic steroids derived
from toad venom (bufalin, bufotalin, resibufagenin,
marinobufagenin, marinoic acid, marinosin) all inhibit
Na-K-ATPase activity12-17. The secretion from Bufo
viridis Laur. skin glands produces a marked
cardiotonic and vasotonic effect, increasing the
intraventricular and aortal pressure, the rate of
pressure growth in the ventricles and the contraction
index of the myocardium on experimental dogs under
anaesthesia18.
Antidiabetic activity
Many insulinotropic peptides have been isolated
from different toad and frog skins. Insulin-releasing
peptides (peaks 21, 22, 23, 24, 25) purified by reverse
phase HPLC from skin secretions of the toad
Bombina variegata have molecular masses of 1.64,
1.66, 1.68, 1.65 and 2.30 kDa, respectively19. They
significantly increase the insulin release by glucose
responsive BRIN-BD11 cells as compared to glucose
(5.6mM) alone. Peaks 21, 22 and 23 have sequence
homology to bombesin (Pyr-QRLGNQWAVGHLM)
originally isolated from Bombina bombina, while
peaks 24 and 25 are new. The mechanism underlying
their insulinotropic actions suggests possible
involvement of a cAMP dependent G-protein
insensitive pathway19. Fractions purified from skin
secretion of Rana palustris by reverse-phase HPLC
also increased the insulin-releasing activity of BRINBD11 cells. One such peptide (2.87 kDa) has a 27
amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC (having 48% homology with
antimicrobial peptide Brevinin-120. Insulinotropic
peptides are also present in the skin secretions of
Phyllomedusa trinitatis frog. One of them−a 28amino-acid peptide had 100% homology with the Cterminal of the 75-amino-acid dermaseptin BIV
precursor in the skin of the Phyllomedusinae
subfamily21. Insulinotropic peptides were also
secreted by the skin of Agalychnis litodryas22 and
Agalychnis calcarifer frogs23. An insulin-releasing
peptide isolated from Rana pipiens had 100%
sequence homology with an antimicrobial peptide
pipinin-124. Skin secretions of Rana saharica
possessed nontoxic insulinotropic peptides25. Two
were novel 1.89 and 2.93 kDa peptides. Other four
(2.67, 3.51, 4.92 and 4.8 kDa) were identical to
brevinin-1E,
brevinin-2EC,
esculentin-1
and
esculentin-1B, which belong to the group of
antimicrobial peptides isolated from skin secretions of
various Rana frog species. Contrast to the peptides
from Bombina, the mechanism underlying the
insulinotropic actions of esculentins-1 and -1B
possibly involves both cyclic AMP-protein kinase A
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
and C dependent G-protein sensitive pathways 26.
However, all the studies reported were done in vitro.
It would be interesting to see if such compounds
could increase the insulin release in vivo. These
insulinotropic peptides may be exploited as
antidiabetic agents in the long run.
Immunomodulatory activity
The water-soluble, non-dialyzable fraction from
crude Chan Su can activate proliferation of
lymphocyte. It increases IL-2 and IL-12 level in the
supernatant of spleen cell culture, and increased the
natural killer activity of the C3H/HeN mice. These
results
show
that
Chan
Su
contains
immunopotentiating substances that may serve as an
immunomodulator in an organism4. Cinobufagin, the
second major component of Chan Su, has been used
successfully in high doses in attenuation and
treatment of infection and granulocytopenia during
combined chemotherapy. In patients with malignant
blood disease, after treatment with high dose of
cinobufocini, infection was significantly decreased
without a significant change in the number of
granulocytes before and after the treatment 28. In
experimental animals, TSE - skin extract of the Indian
common toad (Bufo melanostictus) presently known
as Duttaphrynus melanostictus, significantly increases
blood lymphocyte, splenic lymphocyte and
macrophage count, strongly suggesting the possible
involvement of toad skin component in first line of
defense through immunomodulation of lymphoid
cell28,29. It is assumed that TSE contained a variety of
immunomodulators, which might be involved in
immunopotentiation as judged by its positive
chemotactic property, negative T-cell rosette
formation and macrophage migration inhibition
property29.
An
octadecapeptide
pLR
(LVRGCWTKSYPPKPCFVR) was purified by
reverse-phase HPLC from the skin extract of the
Northern Leopard frog (Rana pipiens). Synthetic pLR
produces rapid, noncytolytic histamine release that
has a 2-fold greater potency when compared with one
of the most active histamine-liberating peptides,
melittin. pLR can permeabilize negatively charged
unilamellar lipid vesicles but not neutral vesicles, a
finding that is consistent with its nonhemolytic action.
pLR inhibits the early development of granulocyte
macrophage colonies from bone marrow stem cells
but does not induce apoptosis of the end stage
granulocytes, i.e. mature neutrophils30. An
octadecapeptide pYR (YLKGCWTKSYPPKPCFSR)
581
from the skin secretions of the dusky gopher frog
(Rana sevosa), shares 77.8% homology with pLR, can
release histamine from rat peritoneal mast cells and
inhibits the early development of granulocyte
macrophage colonies from bone marrow stem cells
but does not induce apoptosis of the end stage
granulocytes31.
Antimicrobial activity
Ambhibian skin has probably been the most
exploited for their antimicrobial components.
Amphibian granular glands produce certain secretions
that might be effective against microbial and fungal
infections1. A 6.7 kDa thermostable protein purified
from Bombina variegata pachypus was found to be
antimicrobially active32. Magainins, a family of
peptides isolated from the skin of African clawed toad
Xenopus laevis exhibit wide spectrum antibiotic
activity, inhibiting the growth of both Gram positive
and Gram negative bacteria, fungal species such as
Candida albicans, Cryptococcus neoformans and
Saccharomyces cerevisiae33. Two other peptides
isolated from Xenopus laevis skin secretions − PGLa
and a peptide released from the xenopsin precursor,
exhibit antimicrobial properties comparable to the
magainins34. It has also been reported that certain
sequences of the magainins are relevant for increased
antimirobial activity and for decreased haemolytic
activity. Amino acid omissions in the N-terminal
region (residues 1-14) result in the complete loss of
antimicrobial activity in both Magainin 1 and 2 and
also shows very low hemolytic activity against human
erythrocytes. But analogs with omissions in the Cterminal region, especially residues alanine-15,
glycine-18 or glutamic acid-19, while having equal or
increased antimicrobial activity relative to the original
magainin 1 or magainin 2 forms, have variable
hemolytic action. With omission of glutamic acid 19,
both magainin 1 and magainin 2 have equal activity
against E. coli and increased activity against S.
epidermis, while having lower hemolytic activity than
the original sequences. When glycine18 was omitted
from magainin-2-amide, the resulting analogue had
equal antimicrobial activity, but significantly
increased hemolytic activity. The C-terminal carboxyl
form of magainin 1, however, showed equal
antimicrobial activity, but substantially decreased
hemolytic action35. The magainins, PGLa and
magainin-2-amide dissipate the electric potential
across various energy-transducing membranes and
thus uncouple respiration from other free-energy-
582
INDIAN J EXP BIOL, JULY 2007
requiring processes and that is likely to be the
mechanism for the antimicrobial effects of these
compounds36. Central American tree-frogs in
Phyllomedusa genus secrete antimicrobial peptides
known as dermaseptins (28-34 residues) that are
cationic molecules which permeabilize the membrane
of Gram-positive and Gram-negative bacteria, yeasts,
and filamentous fungi, but show negligible hemolytic
activity. Some of the potent antimicrobial
dermaseptins are dermaseptin I, dermaseptin b,
dermatoxin, DS0137-40. Adenoregulin (33 amino acid)
and PS-1 (phylloseptins) isolated from Phyllomedusa
skin secretions also show antimicrobial activity41,42.
An antimicrobial peptide fraction isolated from the
skin of Bombina variegata contains a number of 27
amino acid residue peptides that are all related though
not identical to bombinin43. Bombinin-like peptides
(BLP1, BLP2, BLP3) isolated from the Asian toad
Bombina orientalis show strong antimicrobial activity
and are found to be more potent than magainin 2 in
their ability to kill bacteria44. Two groups of
antimicrobial peptides have been isolated from skin
secretions of Bombina maxima. One group has been
named maximins (maximins 1, 2, 3, 4 and 5), which
are structurally related to bombinin-like peptides
(BLPs). The other group named maximin H (H1, H2,
H3 and H4), are homologous with bombinin H
peptides45. Recently, a bombesin-like peptide PRbombesin derived from the skin of the Chinese red
belly toad, Bombina maxima, showed antimicrobial
activity, which is likely due to the proline rich
sequence46. Two antimicrobial peptides brevinin-1
and brevinin-2 were isolated from Rana brevipoda
porsa47. Three antimicrobial peptides have been
isolated from skin secretion of the European frog
Rana esculenta, out of which, two have similarities
with brevinin-1 and brevinin-2 and the third named
esculentin (46 residues) represents a different type of
peptide48. Six brevinin family antimicrobial peptides
were also identified from the Tsushima brown frog
Rana tsushimensis Stejneger49. Ranalexin, from Rana
catesbeiana (Bullfrog), is structurally related to the
bacterial antibiotic polymyxin50. Antimicrobial
peptide families, named gaegurins and rugosins were
isolated from the skin of a Korean frog Rana
rugosa51,52. Temporins, isolated from the skin of Rana
temporaria, are the smallest antibacterial peptides
found in nature and are active against gram-positive
bacteria53. Nine peptides (ranatuerins 1-9) isolated
from Rana catesbeiana show antimicrobial activity
towards Staphylococcus aureus. Among them,
ranatuerin
1
(SMLSVLKNLGKVGLG
FVACKINKQC) exhibits the broadest spectrum of
antimicrobial action with inhibitory activity against S.
aureus, Escherichia coli and Candida albicans54.
Ranatuerin 1T isolated from European brown frog
Rana temporaria also possesses growth-inhibiting
activity toward Staphylococcus aureus55. Ten peptides
with differential growth-inhibitory activity against the
Gram-positive bacterium Staphylococcus aureus, the
Gram-negative bacterium, Escherichia coli, and the
yeast Candida albicans were isolated from an extract
of the skin of a North American green frog Rana
clamitans56. Broad-spectrum antimicrobial peptides
named tigerinins (Rana tigrina)57, nigrocin 1 and 2
(Rana nigromaculata)58, japonicin-1, japonicin-2
(Rana japonica)59, temporin-1Ja (Rana japonica)59
have also been identified. Six antimicrobial peptides
isolated from skin of the Japanese mountain brown
frog Rana ornativentris were found to be related to
the brevinin-2 and temporin family60. Eight peptides
with antimicrobial and hemolytic activity belonging
to the previously identified brevinin-1, temporin-1,
palustrin-2, palustrin-3, esculentin-1 (two peptides),
and ranatuerin-2 (two peptides) families have been
isolated and characterized from skin glands of the
crawfish frog, Rana areolata61. Two peptides with
antimicrobial and cytolytic properties were purified
from an extract of the skin of Tago's brown frog Rana
tagoi, one of which is temporin related and the other
is similar to melittin from honeybee venom. Two new
antimicrobial cyclic 17-residue peptides named
ranacyclins E and T have been isolated from Rana
esculenta and Rana temporaria skin, respectively62.
Five antimicrobial peptides belonging to the brevinin2 family have been identified from the skin extract of
Hokkaido frog Rana pirica. The most abundant
peptide, brevinin-2PRa (680 nmol/g weight of dry
skin) shows high potency against a range of clinical
isolates of Pseudomonas aeruginosa63. Antimicrobial
peptides from the skin of the Yunnanfu Kunming frog
Rana grahami are structurally related to nigrocins,
brevinins and esculentins64,65.
Frenatin 3 belonging to a group of frenatin peptides
isolated from the skin of giant tree frog Litoria
infrafrenata, exhibits wide spectrum antimicrobial
properties66. The largest group of antimicrobial
peptides isolated from amphibian skin is the caerin
group of peptides, with more than 30 identified
from the Australian frog species of Litoria
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
genus. All caerin 1 peptides have similar primary
structures
based
on
that
of
caerin1.1
(GLLSVLGSVAKHVLPHVVPVIAEHL-NH2, 2.58
kD), and are active mainly against Gram-positive
bacteria67,68. Four maculatin peptides isolated from the
skin of the tree frog Litoria genimaculata show
antibiotic activity, with maculatin 1.1 showing the
most pronounced activity, particularly against Grampositive organisms69. Two major citropin peptides
isolated from skin glands of the Blue Mountains tree
frog Litoria citropa, namely citropin 1.1 and citropin
1.2 show significant wide-spectrum antibacterial
activity70.
Kassinatuerin-1
(from
Kassina
senegalensis) and pseudins 1-4 (from the paradoxical
frog Pseudis paradoxa) possess broad spectrum
antimicrobial
activity71,72.
Fallaxin
(from
Leptodactylus fallax skin) and pentadactylin (from
Leptodactylus
pentadactylus
skin)
possess
antimicrobial activity, but potencies are relatively low
(MIC values in the range 25-200 microM)73,74.
Syphaxin 1.5 (1.58 kDa peptide) from Leptodactylus
syphax show antimicrobial activity on Staphylococcus
aureus and Escherichia coli75.
Though mostly antimicrobial peptides have been
identified from amphibian skin, a recent interesting
finding showed that two bufadienolides from the skin
secretions of the Brazilian toad Bufo rubescens,
named telocinobufagin (402.16 D) and marinobufagin
(400.15 D) are active against Staphylococcus aureus
and Escherichia coli76.
Antiprotozoal activity has been demonstrated by
many antimicrobial peptides. Dermaseptin (DS), a 34
amino acid residue cationic peptide isolated from
Phyllomedusa sauvagii skin, has been studied in vitro
on promastigotes of Leishmania mexicana.
Immunocytochemical, freeze fracture, label fracture
and electron microscopic observations showed that
the amphipathic peptide generates perturbations of the
lipid bilayer leading to altered permeability of the
surface membrane and death of the parasite77.
Temporins A and B secreted from the skin of
European red frog Rana temporaria show antileishmania activity at micromolar concentrations,
with no cytolytic activity against human erythrocytes.
They cause severe membranolysis of the parasite,
which is likely to make it difficult for the pathogen to
develop
resistance78.
Synthetic
dermaseptin
derivatives have been found to exert antimalarial
activity79. Recently, caerin 1 peptides have been
reported to be active against malarial parasite among
583
which caerin 1.8 is the most potent68. Dermaseptin
DS01 (from the skin of from Phyllomedusa oreades),
phylloseptins PS-4 and PS-5 is active against
Trypanasoma cruzi40,42.
Antiviral activity
Amphibian skin secretions contain certain
compounds having strong antiviral properties.
Brevenin1, a frog skin defensive peptide possesses
potent antiviral activity on Herpes simplex virus 1 and
2 and its antiviral activity is maintained even after
reduction and carboxamidomethylation procedures
that abolish its prominent hemolytic and cytolytic
effects80. Esculentin-2P (E2P) and ranateurin-2P
(R2P), two microbial peptides isolated from Rana
pipiens, can inactivate frog virus 3 and channel catfish
herpes virus. Antimicrobial peptides, E2P and R2P
can act within minutes and at temperatures as low as
0oC to inhibit viral infections. Moreover, these
compounds appear to inactivate the virus directly and
do not act by inhibiting replication in infected cells81.
Synthetic dermaseptins⎯dermaseptins (S1-S5) are
effective against herpes simplex virus type 1
(HSV1)82. Maximin 3 from Bombina maxima,
possesses significant anti-HIV activity45. A novel 63
kDa heme-containing protein BAS-AH isolated from
skin secretions of Bufo andrewsi shows dosedependent inhibition on HIV-1 infection and
replication83. The discovery that the frog peptides can
kill HIV virus even when it is hidden in dendritic cells
suggests that they could be developed as mucosal
preventives84.
Antineoplastic activity
Magainins, a class of antibiotic peptides, also
possess anti-tumor activity. Magainin 2 and its
synthetic analogues could rapidly and irreversibly
lyse hematopoietic tumor and solid tumor target cells
though the concentrations are relatively non-toxic to
well-differentiated cells85. Synthetic magainin A
(MAG A) and magainin G (MAG G) show in vitro
antitumor activity against small cell lung cancer cell
lines86. Bombina variegata cutaneous venom has been
shown to inhibit the proliferation of human leukaemic
cell line HL 60 in a dose-dependent manner87.
Citropin 1.1, another wide spectrum antibacterial
peptide, also shows anticancer activity by inhibiting
nNOS88. The concentration for anticancer activity is
significantly less compared to that required for lysis
of red blood cells. A4K14-citropin, a synthetic
modification of citropin 1.1 results in a 10-fold
584
INDIAN J EXP BIOL, JULY 2007
increase in the anticancer activity. Five 20,21epoxybufenolides, namely, 20S,21-epoxyresibufogenin, 20R,21-epoxyresibufogenin, 3-O-formyl20S,21-epoxyresibufogenin,
3-O-formyl-20R,21epoxyresibufogenin,
and
3-oxo-20S,21epoxyresibufogenin, isolated from Chan Su
significantly inhibit the leukemia MH-60 cell line89.
Bufalin, one of the major components of Chan Su,
has been shown to have anti-cancer properties in
leukemia as well as melanoma cells. It induces
differentiation in human erythroleukemia K562 cells
and also produces a strong differentiation-inducing
activity in three other human leukemia-derived cell
lines HL60, U937, ML1 to monocyte/macrophagelike cells90,91. Bufalin arrests the growth of ML1 cells
preferentially at the G2 phase and U937 cells at the S
and G2 phases of the cell cycle92. Bufalin induces
differentiation of ML1 cells through the modulation
of several protein kinase activities in a distinct way
from RA and 1 alpha, 25(OH) 2D3. This effect of
bufalin on the cell cycle of leukemia cells is similar to
that of topoisomerase inhibitors93. Bufalin reduces the
level of topoisomerase II in human leukemia HL60
cells and also increases the inhibitory effects of
anticancer drugs like cisplatin and RA on cell growth
and enhanced the induction of cell death. Na-KATPase inhibition by Bufalin initiates the process of
K562 cell differentiation94. Bufalin also shows that
growth inhibitory and differentiation inducing effects
on SSCC-1 cells95 and also decreases the rate of cell
proliferation of mouse melanoma clone B16-F10 cells
with a concomitant stimulation of expression of its
melanotic phenotype96. Bufalin or cinobufagin
increases the intracellular calcium concentration and
apoptosis in prostate cancer cell lines LNCaP,
DU145, and PC397. Bufalin significantly inhibits the
cell proliferation and DNA synthesis of cultured
ovarian endometriotic cyst stromal cells and induces
apoptosis and the G0/G1 phase cell cycle arrest of
these cells by down-regulation of the cyclin A, Bcl-2,
and Bcl-X(L) expression with the simultaneous upregulation of the p21 and Bax expression, and
caspase-9 activation98.
Recently, it has been reported that Chan Su induces
apoptosis in a human bladder carcinoma cell line, T24
in a concentration dependent manner, which is
associated with a down-regulation of anti-apoptotic
Bcl-2 and Bcl-X(S/L) expression and an up-regulation
of pro-apoptotic Bax expression. Chan Su treatment
induces the proteolytic activation of caspase-3 and
caspase-9, and a concomitant degradation of
poly(ADP-ribose)-polymerase
and
beta-catenin
protein. Chan Su also decreases the levels of COX-2
mRNA and protein expression without significant
changing the levels of COX-1, which is correlated with
an inhibition in prostaglandin E(2) synthesis3.
The Indian toad (Bufo melanostictus) skin
methanolic extract TSE possesses significant
antineoplastic activity on EAC cells and human
leukemic cell lines (U937 and K562)29,99,100. EAC
bearing mice when treated with TSE survive for a
longer period as compared to untreated mice. TSE
inhibits the proliferation of U937 and K562 cells by
apoptosis. Leukemic cells are much more susceptible
toward TSE as compared to normal lymphocytes. A
non-protein crystalline antineoplastic compound (BMANF1) has been isolated from TSE by alumina gel
column chromatography and HPLC and has been
found to be active against EAC cells and human
leukemic cell lines (K562 and U937) (unpublished
data)101.
Sleep inducing activity
Amphibian glandular secretions contain large
amounts of small peptides related to tryptophyllin
(tryptophan containing peptide), first discovered in the
South American hylid frog Phyllomedusa rhodei102.
One of the tryptophyllin (FPPWM-NH2) has been
found to be immunoreactive to a set of cells in the rat
adenohypophysis103. The tryptophyllins may play a role
as neurotransmitters or neuromodulators, and some of
them induce sedation and behavioural sleep in birds.
The Indian toad (Bufo melanostictus) skin methanolic
extract TSE shows a significant potentiation of
sleeping time in mice104. A non-lethal sleep-inducing
factor SIF (880D conjugated aromatic compound with
a hydroxyl and carbonyl functional group) has been
purified from TSE105. EEG studies have shown that SIF
increases sleep and decreases awakening condition of
freely moving rats. SIF probably acts through
serotonergic/histaminergic receptors. It significantly
increases brain MAO and tryptophan hydoxylase
activity in mice.
Analgesic activity
Amphibian skin secretions are the potential source
of many powerful analgesics which also include many
of the bufogenins and bufotoxins. Dermorphin, a
heptapeptide with very potent opiate-like activity, has
been isolated from methanol extracts of the skin of the
South American frog Phyllomedusa sauvagei.
Surprisingly, it contains a D-amino acid residue in its
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
585
species world-wide, is thousand times more potent
analgesic than morphine and has been used during
gall-bladder operation117.
sequence (H-Try-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)
and perhaps this is of critical importance106.
Dermorphin and its analogue Hyp6-dermorphin
isolated from methanolic extracts of the skin of the
Brazilian frog Phyllomedusa rhodei are potent opioid
peptides occurring in amphibian skin107. Dermorphin
is much more potent than met-enkephalin, leuenkephalin, beta-endorphin, and morphine on the
guinea-pig ileum and vas deferens opiate receptors.
Dermorphin produces potent and long-lasting
analgesia in mice by intravenous injection,
and in rats by intracerebroventricular injection108.
Intracerebroventricular injections of dermorphin
result in analgesia. It was suggested that dermorphin
acted on the central receptor populations activated by
morphine and enkephalins109. The effect of
dermorphin on the spontaneous and evoked neuronal
activity by a nociceptive stimuli has been studied in
the nucleus lateralis anterior and ventrobasal complex
of the rat thalamus. The high firing frequency induced
by nociceptive stimuli is blocked by dermorphin
(1.5mg/kg, ip) under the same experimental
conditions110. Recently, it is proposed that κ and μ
opiod receptors are involved in the antinociceptive
activity of methanolic skin extract of Phyllomedusa
rhodei111.
Contraceptive activity
Synthetic magainins (Magainin A and G) show in
vitro spermicidal activity by disrupting the outer
plasma membrane of sperm cells and has the potential
to develop into a contraceptive agent118. In vitro and
in vivo studies on rats have shown that magainin-A,
other than having anti-bacterial, anti-viral, anti-fungal
action also shows spermicidal activities, and thus
could be a potent vaginal contraceptive. In vivo
studies on monkeys have indicated that magainin-A
may be used as an effective and safe intravaginal
contraceptive compound with additional protection
against sexually transmitted infection-causing
pathogens excepting HIV-1 and HIV-2119,120.
Magainin-2-amide is strongly embryotoxic and may
have a potential role as a contraceptive agent121. The
maximin group of antimicrobial peptides secreted
from the Chinese red belly frog Bombina maxima also
possess significant spermicidal action45. Synthetic
antimicrobial dermaseptin (DS4) exhibits potent
spermicidal action and could be developed to be a
potent vaginal contraceptive122.
A nonopioid analgesic epibatidine isolated from the
skin of the Ecuadorian poison frog Epipedobates
tricolor by Daly and co-workers112 has been found to
be a highly potent nicotinic analgesic and in tests of
analgesic properties it is about 120 times more potent
and has longer duration than nicotine in analgesia and
acts as a nicotine acetylcholine receptor agonist113.
The epibatidines have little or no activity at a variety
of other central receptors, including opioid receptors,
muscarinic receptors, adrenergic receptors, dopamine
receptors, serotonin receptors, and gammaaminobutyric acid receptors114. It has been suggested
that epibatidine is a potent agonist of ganglionic
nicotinic receptors and that the alkaloid elicits
cardiorespiratory effects similar to those of
nicotine115. Researchers at Abbott Laboratories in
North Chicago, Illinois synthesized up to 500
variations of epibatidine before developing a new
painkiller, ABT-594. In controlled studies of hepatic
cancerous pain, Bufalin exhibited analgesic effects
through increase in hepatic blood circulation116.
The majority of frogs of Litoria genus contain at
least one neuropeptide of caerulein group. Caerulein
1.1, a common neuropeptide found in many frog
Other activities
It should be mentioned that apart from the abovementioned therapeutic potential of amphibian skin
bioactive molecules, they may also be useful in
certain other clinical treatments. Amphibian skin
secretions have been shown to possess various
endocrine functions in mammals. The insulin
releasing activity of amphibian skin peptides have
already been discussed. Thyrotropin-releasing
hormone, a hypothalamic tripeptide that stimulates the
secretion of pituitary thyroid-stimulating hormone in
mammalian species is present in the skin of the frog
Rana pipiens123. Sauvagine, a polypeptide from the
skin of Phyllomedusa sauvagei, a frog of Central and
South America possesses pharmacological actions on
diuresis, cardiovascular system and endocrine
glands124. Dermorphin (having potent analgesic
activity), dose-dependently raises and decreases
prolactin (PRL) and luteinizing hormone (LH) levels,
respectively in ovarectomized rat125,126. Dermorphin
also inhibits gastrointestinal motility125 and
significantly stimulates TSH secretion in rats127. It
also significantly stimulates plasma glucagon as
well as decreases plasma glucose, both 10 and 30 min
586
INDIAN J EXP BIOL, JULY 2007
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
587
588
INDIAN J EXP BIOL, JULY 2007
given to salamander dermal secretions for exploring
their therapeutic potential. Fredericks and Dankert
following injection, suggesting an effect of
dermorphin on the endocrine pancreas128. TPH-7 and
TPH-13, (tryptophyllin peptides) isolated from the
skin of Phyllomedusa rohdei shows endocrine and
behavioural effects129. The 8kDa protein Bv8,
secreted by the skin of the frog Bombina variegata,
injected into the lateral ventricles of rat brain
suppresses diurnal, nocturnal, deprivation-induced
and neuropeptide Y-stimulated feeding and stimulates
diurnal drinking. Nocturnal drinking is increased only
in fasted rats. Bv8 injections into other brain areas
does not change rat ingestive behaviours130. Recently,
studies with skin extracts of Phyllomedusa rhodei
have shown that it could affect mice behaviour in
experimental models131.
Amphibian skin may be an effective candidate in
healing of wounds and in plastic surgery in the future.
The clue lies in the healing of wounds by toad skin.
Experimental wounds created on albino rat skin
healed faster when dressed with dorsal skin of freshly
sacrificed Indian frog (Rana tigrina) as compared to
control that were dressed with cotton gauze132. A part
of the healing efficacy of frog skin may be due to
collagen since proliferation, migration and
differentiation of epithelial cells are prime requisites
for a normal healing mechanism133. A novel two
domain trefoil factor family (TFF) protein named BmTFF2 purified and cloned from frog Bombina maxima
skin secretions can activate human platelets in a dosedependent manner and activation of integrin
alpha(IIb)beta(3) is involved134.
Other than frogs and toads, salamanders are also
important amphibians that are believed to have
medicinal implications. But little attention has been
have examined the skin from the tail region for the
production of antimicrobial peptides in the terrestrial
salamander, Plethodon cinereus. Fractionation of tail
skin extracts by cation-exchange chromatography and
reverse-phase HPLC eluted an antimicrobial fraction
that is active against Staphylococcus aureus, but not
against Escherichia coli135. Water-soluble skin
secretions of salamander Tylototriton verrucosus
display a wide spectrum of antimicrobial activities
and also contain both proteolytic activity and trypsin
inhibitory activity136.
Conclusion
This overview is an attempt to enlighten the fact
that amphibian skin is capable of providing a wide
variety of bioactive compounds that may be turned to
therapeutic advantage. Table 1 provides an insight
into the therapeutic potential of different frog and
toad skin biomolecules. Though many areas have
been explored, scientists may venture for therapeutic
activities of amphibian skin biomolecules towards
emerging pathophysiological conditions such as
stroke, depression, inflammation, seizures and
convulsion, memory and learning dysfunction and
other neurodegenerative diseases (Alzheimer’s,
Parkinson’s, etc.). Advanced knowledge on
biotechnology and chemistry may be put to use to
synthesize derivatives of the natural biomolecules,
which may turn out to be more effective than the
parent molecule. Thus, the multidimensional,
multifunctional components of the amphibian skin
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
warrant much more attention, which may lead to
newer and more effective drug development in the
near future.
Acknowledgement
Dedicated to Late Professor S C Lahiri (Calcutta
School of Tropical Medicine) and Late Professor A K
Nagchowdhury (Department of Pharmaceutical
Technology, Jadavpur University, Kolkata).
References
1
Duellman W E & Trueb L, in Biology of amphibians
(McGraw-Hill, New York) 1986, 257.
2
Bhuiyan M B, Fant M E & Dasgupta A, Study on mechanism
of action of Chinese medicine Chan Su: Dose-dependent
biphasic production of nitric oxide in trophoblastic BeWo
cells, Clin Chim Acta, 330 (2003) 179.
3
Ko W S, Park T Y, Park C, Kim Y H, Yoon H J, Lee S Y,
Hong S H, Choi B T, Lee Y T & Choi YH, Induction of
apoptosis by Chan Su, a traditional Chinese medicine, in
human bladder carcinoma T24 cells, Oncol Rep, 14 (2005)
475.
4
Shimizu Y, Inoue E & Ito C, Effect of the water-soluble and
non-dialyzable fraction isolated from Senso (Chan Su) on
lymphocyte proliferation and natural killer activity in C3H
mice, Biol Pharm Bull, 27 (2004) 256.
5
Clarke B T, The natural history of amphibian skin secretions,
their normal functioning and potential medicinal
applications, Biol Rev, 72 (1997) 365.
6
Morishita S, Shoji M, Oguni Y, Ito C, Noguchi K &
Sakanashi M, Congestive heart failure model in rabbits:
effects of digoxin and a drug containing toad venom, Jpn. J
Pharmacol, 56 (1991) 427.
7
Morishita S, Shoji M, Oguni Y, Ito C, Noguchi K &
Sakanashi M, Effects of "kyushin", a drug containing toad
venom, on experimental congestive heart failure in rabbits.
Am J Chin Med, 20 (1992) 83.
8
Ojiri Y, Chibana T, Noguchi K & Sakanashi M,
Cardiovascular effect of a senso (toad venom) - containing
drug in anesthetized dogs (2): Influence of propranolol, Am J
Chin Med, 20 (1992) 147.
9
Morishita S, Sugimoto C, Shoji M, Hirai Y, Oguni Y, Ito C,
Higuchi M & Sakanashi M, Pharmacological actions of
"kyushin," a drug containing toad venom (3): Effects on
experimentally induced arrhythmia, Am J Chin Med, 21
(1993) 139.
10 Sakanashi M, Noguchi K, Chibana T, Ojiri Y & Shoji M,
Effects of intraduodenal administration of "kyushin," a senso
(toad venom)-containing drug, on systemic hemodynamics,
cardiacfunction and myocardial oxygen consumption in
anesthetized dogs, Am J Chin Med, 21 (1993) 7.
11 Yamahara J, Tanaka S, Matsuda H, Sawada T & Fujimura H,
The mode of cardiac action of cardiotonic steroids isolated
from Toad Cake in perfused working guinea-pig heart and
effect of cinobufagin on experimental heart failure, Nippon
Yakurigaku Zasshi, 88 (1986) 413.
12 Pamnani M B, Chen S, Bryant H J, Schooley J F Jr, Eliades
D C, Yuan C M & Haddy F J, Effects of three sodiumpotassium adenosine triphosphatase inhibitors, Hypertension,
18 (1991) 316.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
589
Hirai Y, Morishita S, Ito C, & Sakanashi M, Effects of
bufadienolides and some kinds of cardiotonics on guinea pig
hearts, Nippon Yakurigaku Zasshi, 100 (1992) 127.
Yuan C, Manunta P, Chen S, Hamlyn J M, Haddy F J &
Pamnani M B, Role of ouabain-like factors in hypertension:
effects of ouabain and certain endogenous ouabain-like
factors in hypertension, J Cardiovasc Pharmacol, 22 (1993)
S10.
Bagrov A Y, Roukoyatkina N I, Pinaev A G, Dmitrieva R I
& Fedrova O V, Effects of two endogenous Na+,K(+)ATPase inhibitors, marinobufagenin and ouabain, on isolated
rat aorta, Eur J Pharmacol, 274 (1995) 151.
Matsukawa M, Akizawa T, Morris J F, Butler V P Jr &
Yoshioka M, Marinoic acid, a novel bufadienolide-related
substance in the skin of the giant toad, Bufo marinus, Chem
Pharm Bull (Tokyo), 44 (1996) 255.
Matsukawa M, Akizawa T, Ohigashi M, Morris J F, Butler V
P Jr & Yoshioka M, A novel bufadienolide, marinosin, in the
skin of the giant toad, Bufo marinus, Chem Pharm Bull
(Tokyo), 45 (1997) 249.
Orlov B N & Krylov V N, Effect of the venom of the toad
Bufo viridis Laur. on cardiac hemodynamics in dogs,
Nauchnye Doki Vyss Shkoly Biol Nauki, 4 (1986) 54.
Marenah L, Flatt P R, Orr D F, McClean S, Shaw C &
Abdel-Wahab Y H, Skin secretion of the toad Bombina
variegata contains multiple insulin-releasing peptides
including bombesin and entirely novel insulinotropic
structures, Biol Chem, 385 (2004) 315.
Marenah L, Flatt P R, Orr D F, McClean S, Shaw C &
Abdel-Wahab Y H, Brevinin-1 and multiple insulin-releasing
peptides in the skin of the frog Rana palustris, J Endocrinol,
181 (2004) 347.
Marenah L, McClean S, Flatt P R, Orr D F, Shaw C &
Abdel-Wahab Y H, Novel insulin-releasing peptides in the
skin of Phyllomedusa trinitatis frog include 28 amino acid
peptide from dermaseptin BIV precursor, Pancreas, 29
(2004) 110.
Marenah L, Shaw C, Orr D F, McClean S, Flatt P R &
Abdel-Wahab Y H, Isolation and characterisation of an
unexpected class of insulinotropic peptides in the skin of the
frog Agalychnis litodryas, Regu. Pept, 120 (2004) 33.
Abdel-Wahab Y H, Marenah L, Orr D F, Shaw C & Flatt P R
Isolation and structural characterisation of a novel 13-amino
acid insulin-releasing peptide from the skin secretion of
Agalychnis calcarifer, Biol Chem, 386 (2005) 581.
Marenah, L.; Flatt, P.R.; Orr, D.F.; Shaw, C.; Abdel-Wahab,
Y.H. Characterization of naturally occurring peptides in the
skin secretion of Rana pipiens frog reveal pipinin-1 as the
novel insulin-releasing agent, J Pept Res, 66 (2005) 204.
Marenah L, Flatt P R, Orr D F, Shaw C & Abdel-Wahab Y
H, Isolation and structural characterization of novel Rugosin
A-like insulinotropic peptide from the skin secretions of
Rana saharica frog, Peptides 26 (2005) 2117.
Marenah L, Flatt P R, Orr D F, Shaw C & Abdel-Wahab Y
H, Skin secretions of Rana saharica frogs reveal
antimicrobial peptides esculentins-1 and -1B and brevinins1E and -2EC with novel insulin releasing activity, J
Endocrinol, 188 (2006) 1.
Yue B B, High dose cinobufocini in attenuation and
treatment of infection and granulocytopenia during combined
590
28
29
30
31
32
33
34
35
36
37
38
39
40
41
INDIAN J EXP BIOL, JULY 2007
chemotherapy of malignant blood diseases, Chung-KuoChung-Hsi-I-Chieh-Ho-Tsa-Chih, 12 (1992) 145.
Das M, Dasgupta S C & Gomes A, Toad (Bufo
melanostictus,
Schneider)
skin
extract
induced
haematological and biochemical changes in rodents, Indian J
Pharmacol, 30 (1998) 68.
Das M, Dasgupta S C & Gomes A, Immunomodulatory and
antineoplastic activity of common Indian toad (Bufo
melanostictus, Schneider) skin extract, Indian J Pharmacol,
30 (1998) 311.
Salmon A L, Cross L J, Irvine A E, Lappin T R, Dathe M,
Krause G, Canning P, Thim L, Beyermann M, Rothemund S,
Bienert M & Shaw C, Peptide leucine arginine, a potent
immunomodulatory peptide isolated and structurally
characterized from the skin of the Northern Leopard frog,
Rana pipiens, J Biol Chem, 276 (2001) 10145.
Graham C, Irvine A E, McClean S, Richter S C, Flatt P R &
Shaw C, Peptide Tyrosine Arginine, a potent
immunomodulatory peptide isolated and structurally
characterized from the skin secretions of the dusky gopher
frog, Rana sevosa, Peptides, 26 (2005) 737.
Barberio C, Delfino G & Mastromei G, A low molecular
weight protein with antimicrobial activity in the cutaneous
'venom' of the yellow-bellied toad (Bombina variegata
pachypus), Toxicon, 25 (1987) 899.
Zasloff M, Magainins, a class of antimicrobial peptides from
Xenopus skin: isolation, characterization of two active forms,
and partial cDNA sequence of precursor, Proc Nat Acad Sci
USA, 84 (1987) 5449.
Soravia E, Martini G & Zasloff M, Antimicrobial properties
of peptides from Xenopus granular gland secretions, FEBS
Lett, 228 (1988) 337.
Cuervo J H, Rodriguez B & Houghten R A, The Magainins:
sequence factors relevant to increased antimicrobial activity
and decreased hemolytic activity, Pept Res, 1 (1988) 81.
Westerhoff H V, Juretic D, Hendler R W & Zasloff M,
Magainins and the disruption of membrane-linked freeenergy transduction, Proc Natl Acad Sci USA, 86 (1989)
6597.
Mor A, Nguyen V H, Delfour A, Migliore-Samour D &
Nicolas P, Isolation, amino acid sequence, and synthesis of
dermatoxin from frog skin, an antibacterial peptide encoded
by a novel member of the dermaseptin genes family, Eur J
Biochem, 267 (2000) 4583.
Mor A, Amiche M & Nicolas P, Structure, synthesis, and
activity of dermaseptin b, a novel vertebrate defensive
peptide from frog skin: Relationship with adenoregulin,
Biochemistry, 33 (1994), 6642.
Amiche M, Seon A A, Wroblewski H & Nicolas P, Isolation
of dermatoxin from frog skin, an antibacterial peptide
encoded by a novel member of the dermaseptin genes family,
Eur J Biochem, 267 (2000), 4583.
Brand G D, Leite J R, Silva L P, Albuquerque S, Prates M V,
Azevedo R B, Carregaro V, Silva J S, Sa V C, Brandao R A
& Bloch C Jr, Dermaseptins from Phyllomedusa oreades and
Phyllomedusa distincta. Anti-Trypanosoma cruzi activity
without cytotoxicity to mammalian cells, J Biol Chem, 277
(2002) 49332.
Amiche M, Ducancel F, Lajeunesse E, Boulain J C, Menez A
& Nicolas P, Molecular cloning of a cDNA encoding the
precursor of adenoregulin from frog skin. Relationships with
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
the vertebrate defensive peptides, dermaseptins, Biochem
Biophys Res Commun, 191 (1993) 983.
Leite J R, Silva L P, Rodrigues M I, Prates M V, Brand G D,
Lacava B M, Azevedo R B, Bocca A L, Albuquerque S &
Bloch C Jr, Phylloseptins: a novel class of anti-bacterial and
anti-protozoan
peptides
from
the
Phyllomedusa
genus.Peptides, 26 (2005) 565.
Simmaco M, Barra, D, Chiarini F, Noviello L, Melchiorri P,
Kreil G & Richter K, A family of bombinin-related peptides
from the skin of Bombina variegata, Eur J Biochem, 199
(1991) 217.
Gibson BW, Tang D Z, Mandrell R, Kelly M & Spindel E R,
Bombinin-like peptides with antimicrobial activity from skin
secretions of the Asian toad, Bombina orientalis, J Biol
Chem, 266 (1991) 23103.
Lai R, Zheng Y T, Shen J H, Liu G J, Liu H, Lee W H, Tang
S Z & Zhang Y, Antimicrobial peptides from skin secretions
of Chinese red belly toad Bombina maxima, Peptides, 23
(2002) 427.
Li J, Xu X, Yu H, Yang H, Huang Z & Lai R, Direct
antimicrobial activities of PR-bombesin, Life Sci, 78 (2006)
1953.
Morikawa N, Hagiwara K & Nakajima T, Brevinin-1 and -2,
unique antimicrobial peptides from the skin of the frog, Rana
brevipoda porsa, Biochem Biophys Res Commun, 189 (1992)
184.
Simmaco M, Mignogna G, Barra D & Bossa F, Novel
antimicrobial peptides from skin secretion of the European
frog Rana esculenta, FEBS Lett, 324 (1993) 159.
Conlon J M, Al-Ghaferi N, Abraham B, Sonnevend A,
Coquet L, Leprince J, Jouenne T, Vaudry H & Iwamuro S,
Antimicrobial peptides from the skin of the Tsushima brown
frog Rana tsushimensis, Comp Biochem Physiol C Toxicol
Pharmacol, 143 (2006) 42.
Clark D P, Durell S, Maloy W L & Zasloff, M, Ranalexin A novel antimicrobial peptide from bullfrog (Rana
catesbeiana) skin, structurally related to the bacterial
antibiotic, polymyxin, J Biol Chem, 269 (1994) 10849.
Park J M, Jung J E & Lee B J, Antimicrobial peptides from
the skin of a Korean frog, Rana rugosa. Biochem Biophys
Res Commun, 205(1994), 948. [Erratum in: Biochem Biophys
Res Commun, 209 (1995) 775].
Suzuki S, Ohe Y, Okubo T, Kakegawa T & Tatemoto K,
Isolation and characterization of novel antimicrobial
peptides, rugosins A, B and C, from the skin of the frog,
Rana rugosa, Biochem Biophys Res Commun, 212 (1995)
249.
Simmaco M, Mignogna G, Canofeni S, Miele R, Mangoni M
L & Barra D, Temporins, antimicrobial peptides from the
European red frog Rana temporaria. Eur J Biochem, 242
(1996) 788.
Goraya J, Knoop F C & Conlon J M, Ranatuerins:
Antimicrobial peptides isolated from the skin of the
American bullfrog, Rana catesbeiana, Biochem Biophys Res
Commun, 250 (1998) 589.
Goraya J, Knoop F C & Conlon J M, Ranatuerin 1T: An
antimicrobial peptide isolated from the skin of the frog, Rana
temporaria, Peptides, 20 (1999) 159.
Halverson T, Basir Y J, Knoop F C & Conlon J M,
Purification and characterization of antimicrobial peptides
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
57
58
59
60
61
62
63
64
65
66
67
68
69
70
from the skin of the North American green frog Rana
clamitans, Peptides, 21 (2000) 469.
Sai K P, Jagannadham M V, Vairamani M, Raju N P, Devi A
S, Nagaraj R & Sitaram N, Tigerinins: a novel antimicrobial
peptide isolated from Indian frog rana tigerina, J Biol Chem,
276 (2001) 2701.
Park S, Park, S H, Ahn H C, Kim S, Kim S S, Lee B J & Lee
B J, Structural study of novel antimicrobial peptides,
nigrocins, isolated from Rana nigromaculata, FEBS Lett, 507
(2001) 95.
Isaacson T, Soto A, Iwamuro S, Knoop F C & Conlon J M,
Antimicrobial peptides with atypical structural features from
the skin of the Japanese brown frog Rana japonica, Peptides,
23 (2002) 419.
Kim J B, Iwamuro S, Knoop F C & Conlon J M,
Antimicrobial peptides from the skin of the Japanese
mountain brown frog, Rana ornativentris, J Pept Res,
58(2001), 349.
Ali M F, Lips K R, Knoop F C, Fritzsch B, Miller C &
Conlon J M, Antimicrobial peptides and protease inhibitors
in the skin secretions of the crawfish frog, Rana areolata,
Biochim Biophys Acta, 1601 (2002) 55.
Mangoni M L, Papo N, Mignogna G, Andreu D, Shai Y,
Barra D & Simmaco M, Ranacyclins, a new family of short
cyclic antimicrobial peptides: biological function, mode of
action, and parameters involved in target specificity,
Biochemistry, 42 (2003) 14023.
Conlon J M, Sonnevend A, Patel M, Al-Dhaheri K, Nielsen P
F, Kolodziejek J, Nowotny N, Iwamuro S & Pal T, A family
of brevinin-2 peptides with potent activity against
Pseudomonas aeruginosa from the skin of the Hokkaido
frog, Rana pirica, Regul Pept, 118 (2004) 135.
Xu X, Li J, Han Y, Yang H, Liang J, Lu Q & Lai R, Two
antimicrobial peptides from skin secretions of Rana grahami,
Toxicon, 47 (2006) 459.
Conlon J M, Al-Ghaferi N, Abraham B, Jiansheng H, Cosette
P, Leprince J, Jouenne T & Vaudry H, Antimicrobial
peptides from diverse families isolated from the skin of the
Asian frog, Rana grahami, Peptides, 27 (2006) 2111.
Raftery M J, Waugh R J, Bowie J H, Wallace J C & Tyler M
J, The structures of the frenatin peptides from the skin
secretion of the giant tree frog Litoria infrafrenata, J Pept
Sci, 2 (1996) 117.
Steinborner S T, Currie, G J, Bowie J H, Wallace J C &
Tyler M J, New antibiotic caerin 1 peptides from the skin
secretion of the Australian tree frog Litoria chloris.
Comparison of the activities of the caerin 1 peptides from the
genus Litoria, J Pept Res, 51 (1998) 121.
Apponyi M A, Pukala T L, Brinkworth C S, Maselli V M,
Bowie J H, Tyler M J, Booker G W, Wallace J C, Carver J
A, Separovic F, Doyle J & Llewellyn L E, Host-defence
peptides of Australian anurans: structure, mechanism of
action and evolutionary significance, Peptides, 25 (2004)
1035.
Rozek T, Waugh R J, Steinborner S T, Bowie J H, Tyler M J
& Wallace J C, The maculatin peptides from the skin glands
of the tree frog Litoria genimaculata: a comparison of the
structures and antibacterial activities of maculatin 1.1 and
caerin 1.1, J Pept Sci, 4 (1998) 111.
Wabnitz P A, Bowie J H, Wallace J C & Tyler M J, The
citropin peptides from the skin glands of the Australian Blue
71
72
73
74
75
76
77
78
79
80
81
82
83
84
591
Mountains tree frog Litoria citropa. Part 2: sequence
determination using electrospray mass spectrometry, Rapid
Commun Mass Spectrom, 13 (1999) 1724.
Mattute B, Knoop F C &Conlon J M, Kassinatuerin-1: A
peptide with broad-spectrum antimicrobial activity isolated
from the skin of the hyperoliid frog, Kassina senegalensis,
Biochem Biophys Res Commun, 268 (2000) 433.
Olson L 3rd, Soto A M, Knoop F C & Conlon J M, Pseudin2: An antimicrobial peptide with low hemolytic activity from
the skin of the paradoxical frog, Biochem Biophys Res
Commun, 288 (2001) 1001.
Rollins-Smith L A, King J D, Nielsen P F, Sonnevend A &
Conlon J M, An antimicrobial peptide from the skin
secretions of the mountain chicken frog Leptodactylus fallax
(Anura:Leptodactylidae), Regul Pept, 124 (2005) 173.
King J D, Al-Ghaferi N, Abraham B, Sonnevend A, Leprince
J, Nielsen P F & Conlon J M, Pentadactylin: an antimicrobial
peptide from the skin secretions of the South American
bullfrog Leptodactylus pentadactylus, Comp Biochem
Physiol C Toxicol Pharmacol, 141 (2005) 393.
Dourado F S, Moreira K G, Brand G D, Melo J T, Leite J R,
Silva L P, Bloch C Jr & Schwartz, E.F, In Syphaxin 1.5, a
novel antimicrobial peptide isolated from the skin secretions
of the anuran Leptodactylus syphax. I5th World Congress on
Animal, Plant and Microbial toxins, held at Glasgow,
Scotland, July 23-28, 2006, edited by Harvey, A. (Elsevier:
Amsterdam) 2006.
Cunha Filho G A, Schwartz C A, Resck I, Murta M M,
Lemos S S, Castro M S, Kyaw C, Pires O R Jr, Leite J R,
Bloch C Jr & Schwartz E F, Antimicrobial activity of the
bufadienolides marinobufagin and telocinobufagin isolated
as major components from skin secretion of the toad Bufo
rubescens, Toxicon, 45 (2005) 777.
Hernandez C, Mor A, Dagger F, Nicolas P, Hernandez A,
Benedetti E L & Dunia I, Functional and structural damage
in Leishmania mexicana exposed to the cationic peptide
dermaseptin, Eur J Cell Biol, 59 (1992) 414.
Mangoni M L, Saugar J M, Dellisanti M, Barra D, Simmaco
M & Rivas L, Temporins, small antimicrobial peptides with
leishmanicidal activity, J Biol Chem, 280 (2005) 984.
Krugliak M, Feder R, Zolotarev V Y, Gaidukov L, Dagan A,
Ginsburg H & Mor A, Antimalarial activities of dermaseptin
S4 derivatives, Antimicrob Agents Chemother, 44 (2000)
2442.
80Yasin B, Pang M, Turner J S, Cho Y, Dinh N N, Waring A
J, Lehrer R I & Wagar E A, Evaluation of the inactivation of
infectious Herpes simplex virus by host-defense peptides,
Eur J Clin Microbiol Infect Dis, 19 (2000) 187.
Chinchar V G, Wang J, Murti G, Carey C & Rollins-Smith
L, Inactivation of frog virus 3 and channel catfish virus by
esculentin-2P and ranatuerin-2P, two antimicrobial peptides
isolated from frog skin, Virology, 288 (2001) 351.
Belaid A, Aouni M, Khelifa R, Trabelsi A, Jemmali M &
Hani K, In vitro antiviral activity of dermaseptins against
herpes simplex virus type 1, J Med Virol, 66 (2002) 229.
Zhao Y, Jin Y, Wang J H, Wang R R, Yang L M, Lee W H,
Zheng Y T & Zhang Y, A novel heme-containing protein
with anti-HIV-1 activity from skin secretions of Bufo
andrewsi, Toxicon, 46 (2005) 619.
Bradbury J, Frog skin hope for HIV prevention, Drug Discov
Today, 10 (2005) 1489.
592
INDIAN J EXP BIOL, JULY 2007
85
Cruciani R A, Barker J L, Zasloff M, Chen H & Colamonici
O, Antibiotic magainins exert cytolytic activity against
transformed cell lines through channel formation, Pro Nat
Acad Sci USA, 88 (1991) 3792.
86 Ohsaki Y, Gazdar A F, Chen H C & Johnson B E Antitumor
activity of magainin analogues against human lung cancer cell
lines, Cancer Res, 52 (1992) 3534.
87 Balboni F, Bernabei P A, Barberio C, Sanna A, Rossi Ferrini P
& Delfino G, Cutaneous venom of Bombina variegata
pachypus (Amphibia, Anura): effects on the growth of the
human HL 60 cell line, Cell Biol Int Rep, 16 (1992) 329.
88 Doyle J, Brinkworth C S, Wegener K L, Carver J A, Llewellyn
L E, Olver I N, Bowie J H, Wabnitz P A & Tyler M J, nNOS
inhibition, antimicrobial and anticancer activity of the
amphibian skin peptide, citropin1.1 and synthetic
modifications, Eur J Biochem, 270 (2003) 1141.
89 Kamano Y, Nogawa T, Yamashita A, Hayashi, M, Inoue M,
Drasar P & Pettit G R, Isolation and structure of a 20,21epoxybufenolide series from "Ch'an Su", J Nat Prod, 65
(2002) 1001.
90 Zhang L S, Nayaka K, Yoshida T & Kuroiwa Y, Bufalin as a
potent inducer of differentiation of human myeloid leukaemia
cells, Biochem Biophys Res Comm, 178 (1991) 686.
91 Zhang L Nakaya K, Yoshida T & Kuroiwa Y, Induction by
bufalin of differentiation of human leukemia cells HL60,
U937, and ML1 toward macrophage/monocyte-like cells and
its potent synergistic effect on the differentiation of human
leukemia cells in combination with other inducers. Cancer
Res, 53 (1993) 934.
92 Jing Y, Watabe M, Hashimoto S, Nakajo S & Nakaya K, Cell
cycle arrest and protein kinase modulating effect of bufalin on
human leukemia ML1 cells, Anticancer Res, 14(1994), 1193.
93 Hashimoto S, Jing Y, Kawazoe N, Masuda Y, Nakajo S,
Yoshida T, Kuroiwa Y & Nakaya K, Bufalin reduces the level
of topoisomerase II in human leukemia cells and affects the
cytotoxicity of anticancer drugs, Leuk Res, 21 (1997) 875.
94 Numazawa S, Shinoki M A, Ito H, Yoshida T & Kuroiwa Y,
Involvement of Na+,K(+)-ATPase inhibition in K562 cell
differentiation induced by bufalin, J Cell Physiol, 160(1994),
113.
95 Akiyama M, Ogura M, Iwai M, Iijima M, Numazawa S &
Yoshida T, Effect of bufalin on growth and differentiation of
human skin carcinoma cells in vitro, Hum Cell, 12 (1999) 205.
96 Zhang L, Yoshida T & Kuroiwa Y, Stimulation of melanin
synthesis of B16-F10 mouse melanoma cells by bufalin, Life
Sci, 51 (1992) 17.
97 Yeh J Y, Huang W J, Kan S F & Wang P S, Effects of bufalin
and cinobufagin on the proliferation of androgen dependent
and independent prostate cancer cells, Prostrate, 54 (2003)
112.
98 Nasu K, Nishida M, Ueda T, Takai N, Bing S, Narahara, H &
Miyakawa I, Bufalin induces apoptosis and the G0/G1 cell
cycle arrest of endometriotic stromal cells: a promising agent
for the treatment of endometriosis, Mol Hum Reprod, 11
(2005) 817.
99 Giri B & Gomes A, Antineoplastic activity of Indian toad
(Bufo melanostictus, Schneider) skin extract on EAC cells and
Human leukemic (U937 & HL60) cell line, Indian J
Pharmacol, 36 (2004) S83.
100 Giri B, Gomes A, Debnath A, Saha A, Biswas A K, Dasgupta,
S C & Gomes A, Antiproliferative, cytotoxic and apoptogenic
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
activity of Indian toad (Bufo melanostictus, Schneider) skin
extract on U937 and K562 cells, Toxicon, 48 (2006), 388.
Gomes A, Giri B, Kole L, Saha A, Debnath A & Gomes A, A
crystalline compound (BM-ANF1) from the Indian toad (Bufo
melanostictus,
Schneider)
skin
extract,
induced
antiproliferation & apoptosis in leukemic & hepatoma cell line
involving cell cycle proteins, Toxicon (2007) (Unpublished).
Montecucchi P C, Gozzini L, Erspamer V & Melchiorri P, The
primary structure of tryptophan containing peptides from skin
extracts of Phyllomedusa rhodei (tryptophyllins), Int J Peptide
Res, 24 (1984) 276.
Renda T, D'Este L, Buffa R, Usellini L, Capella C, Vaccaro R
& Melchiorri P, Tryptophyllin-like immunoreactivity in rat
adenohypophysis, Peptides, 6 Suppl 3 (1985) 197.
Das M, Auddy B & Gomes A, Pharmacological study of the
toad skin extract on experimental animals. Indian J
Pharmacol, 28 (1996) 72.
Das M, Malllick B N, Dasgupta S C & Gomes A, A sleep
inducing factor from common Indian toad (Bufo melanostictus,
Schneider) skin extract, Toxicon, 38 (2000) 1267.
Montecucchi P C, de Castiglione R, Piani S, Gozzini L &
Erspamer V, Amino acid composition and sequence of
dermorphin, a novel opiate-like peptide from the skin of
Phyllomedusa sauvagei, Int J Pept Protein Res, 17 (1981) 275.
Montecucchi P C, de Castiglione R & Erspamer V,
Identification of dermorphin and Hyp6-dermorphin in skin
extracts of the Brazilian frog Phyllomedusa rhodei, Int J Pept
Protein Res, 17 (1981) 316.
Broccardo M, Erspamer V, Falconieri Erspamer G, Improta G,
Linari G, Melchiorri P & Montecucchi P C, Pharmacological
data on dermorphins, a new class of potent opioid peptides
from amphibian skin, Br J Pharmacol, 73 (1981) 625.
Puglisi-Allegra S, Castellano, C, Filibeck, U, Oliverio A &
Melchiorri P, Behavioural data on dermorphins in mice, Eur J
Pharmacol, 82 (1982) 223.
Braga P C, Tiengo M, Biella G, Dall'Oglio G & Fraschini F,
Dermorphin, a new peptide from amphibian skin, inhibits the
nociceptive thalamic neurons firing rate evoked by noxious
stimuli, Neurosci Lett, 52 (1984)165.
Zanoni C I S, Molska G R, Zaharenko A J, Paraventi C,
Jimenez R S, Paula-Freire L I G, Parada C A, Fereira S H,
Malpezzi-Marinho E L A & Marinho E A V, In - κ and μ
opiod receptors are involved in the antinociceptive activity of
methanolic skin extract of Phyllomedusa rhodei on the hot
plate model. I5th World Congress on Animal, Plant and
Microbial toxins, held at Glasgow, Scotland, July 23-28, 2006,
edited by Harvey, A. (Elsevier: Amsterdam), 2006.
Spande T F, Garraffo H M, Edwards M W, Yeh H J C, Panel L
&
Daly
J
W,
Epibatidine:
A
Novel
(Chloropyridyl)azabicycloheptane with potent analgesic
activity from an ecuadoran poison frog, J Am Che. Soc, 114
(1992) 3475.
Qian C, Li T, Shen T Y, Libertine-Garahan L, Eckman J, Biftu
T & Ip S, Epibatidine is a nicotinic analgesic, Eur J
Pharmacol, 250 (1993) R13.
Badio B & Daly J W, Epibatidine, a Potent Analgesic and
Nicotinic Agonist, Mol Pharmacol, 45 (1994) 563.
Fisher M, Huangfu D, Shen T Y & Guyenet P G, Epibatidine,
an alkaloid from the poison frog Epipedobates tricolor, is a
powerful ganglionic depolarizing agent, J Pharmacol Exp
Ther, 270 (1994) 702.
GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES
116 Wang G, Sun G, Tang W & Pan X, The application of
traditional Chinese medicine to the management of hepatic
cancerous pain, J Tradit Chin Med, 14 (1994) 132.
117 Doyle J, Llewellyn L E, Brinkworth C S, Bowiem J H,
Wegener K L, Rozek T, Wabnitz P A, Wallace J C & Tyler M
J, Amphibian peptides that inhibit neuronal nitric oxide
synthase. isolation of lesuerin from the skin secretion of the
Australian Stony Creek frog Litoria lesueuri, Eur J Biochem,
269 (2002) 100.
118 Edelstein M C, Gretz J E, Bauer T J, Fulgham D L, Alexander
N J & Archer D F, Studies on the in vitro spermicidal activity
of synthetic magainins, Fertil Steril, 55 (1991) 647.
119 Reddy K V, Shahani S K, Meherji P K, Spermicidal activity of
Magainins: In vitro and in vivo studies, Contraception, 53
(1996) 205.
120 Clara A, Manjramkar D D & Reddy V K, Preclinical
evaluation of magainin-A as a contraceptive antimicrobial
agent, Fertil Steril, 81 (2004) 1357.
121 Mystokowa E T Niemierko A Komar A & Sawicki W,
Embryotoxicity of magainin-2-amide and its enhancement by
cyclodextrin, albumin, hydrogen peroxide and acidification,
Hum. Reprod, 16 (2001) 1457.
122 Zairi A Belaid A, Gahbiche A & Hani K, Spermicidal activity
of dermaseptins, Contraception, 72 (2005) 447.
123 Jackson I M & Reichlin S, Thyrotropin-releasing hormone:
Abundance in the skin of the frog, Rana pipiens, Science, 198
(1977) 414.
124 Montecucchi P C & Henschen A, Amino acid composition and
sequence analysis of sauvagine, a new active peptide from the
skin of Phyllomedusa sauvagei, Int J Pept Protein Res, 18
(1981) 113.
125 Rossi A, di Salle E, Briatico G, Arcari G, de Castiglione R &
Perseo G, Antinociceptive, prolactin releasing and intestinal
motility inhibiting activities of dermorphin and analogues after
subcutaneous administration in the rat, Peptides, 4 (1983) 577.
126 Cocchi D, Degli Uberti E C, Trasforini G, Salvadori S,
Tomatis R, Torpia R & Perelli-Cippo R, Prolactin releasing
and luteinizing hormone inhibiting activity of dermorphin
593
shorter homologues in the rat, Life Sci, 36 (1985) 1707.
127 Gullner H G & Kelly G D, Dermorphin: Effects on anterior
pituitary function in the rat, Arch Int Pharmacodyn Ther, 262
(1983) 208.
128 Gullner H G, Owen W W, Harris V & Unger R H,
Dermorphin: An opioid peptide from amphibian skin which
affects endocrine pancreatic function, Arch Int Pharmacodyn
Ther, 266 (1983) 155.
129 Montecucchi P C, Isolation and primary structure
determination of amphibian skin tryptophyllins, Peptides, 6
(1985) 187.
130 Negri L, Lattanzi R, Giannini E, De Felice M, Colucci A &
Melchiorri P, Bv8, the amphibian homologue of the
mammalian prokineticins, modulates ingestive behaviour in
rats, Br J Pharmacol, 142 (2004) 181.
131 Silva R W, Zaharenko A J, Paula-Freire L I G, Almeida E,
Frussa-Filho R, Malpezzi-Marinho E L A & Marinho E A V,
In - Activity of extracts from Phyllomedusa rhodei (Amphibia,
Anura) skin on the mice behaviour in the open field and rota
rod. I5th World Congress on Animal, Plant and Microbial
toxins, held at Glasgow, Scotland, July 23-28, 2006, edited by
Harvey, A. (Elsevier: Amsterdam) 2006.
132 Sai K P, Reddy P N & Babu M, Investigations on wound
healing by using amphibian skin, Indian J Exp Biol, 33 (1995)
673.
133 Kumar K V, Sai K P & Babu M, Application of frog (Rana
tigerina Daudin) skin collagen as novel substrate in cell
culture, J Biomed Mater Res, 61 (2002) 197.
134 Zhang J, Zhang Y, Wan S G, Wei S S, Lee W H & Zhang Y,
Bm-TFF2, a trefoil factor protein with platelet activation
activity from frog Bombina maxima skin secretions, Biochem
Biophys Res Commun, 330 (2005) 1027.
135 Fredericks L P & Dankert J R, Antibacterial and hemolytic
activity of the skin of the terrestrial salamander, Plethodon
cinereus, J Exp Zool, 287 (2000) 340.
136 Lai R, Yang D M, Lee W H & Zhang Y, Biological activities
of skin secretions of the salamander Tylototriton verrucosus, J
Nat Toxins, 11 (2002) 245.