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Pharmacological studies of 'sapo' from the frog Phyllomedusa bicolor skin: A drug used by the Peruvian Matses Indians in shamanic hunting practices



The dried skin secretion from Phyllomedusa bicolor, 'sapo', is used by the Matses Indians of the Northern Peru, in shamanic rites mainly designed to improve luck in hunting. When rubbed into burned, exposed areas of the skin, the drug causes the prompt appearance of violent peripheral gastrointestinal and cardiovascular effects soon followed by remarkable central effects (increase in physical strength, heightening of senses, resistance to hunger and thirst, exalted capacity to face stress situations). All the peripheral and most of the central effects of 'sapo' can be ascribed to the exceptionally high content of the drug (up to 7% of its weight) in potently active peptides, easily absorbed through the burned, inflamed areas of the skin. The concentration in 'sapo' of the single peptides (phyllocaerulein, phyllomedusin, phyllokinin, demorphins and deltorphins) has been determined by bioassay, and peptide contents were correlated with the different symptoms of the 'sapo' intoxication.
Toxicon,Yol.3l, No.9, pp. 1099 1111, 1993.
Prìnted in Great Britain. 0041 0101/93 $6.00 + .00
Q 1993 Pergamon Press Ltd
vrrronro ERSpAMER,I GrurtaNr Fa.tcoNrnRt EnslaMnn,r cNzrl. SnwntNr,3 Ros,r Luts,q.
PorENZA,r DoNnrnne BARRA,2 GrusBpprNe MrcNocN,r2 and ANroNto BrlNcnra
rlnstitute of Medical Pharmacology III, 2Department of Biochemical Sciences, University of Rome .La
Sapienza', città Universitaria,00185 Rome, Italy; 3lnstitute of Neurobiology, cNR, v.1e-c.Marx, Rome,
Italy; and aDepartment of Anaesthesiology II, General Hospital of BÉgamo, Bergamo, Itaiy
(Received 17 February 1993; accepted 15 AprÌl 1993)
V. Ensp,q.N{ER, G. FALCoNTERT Ensr.nvrEn, C. SEvsnrNl, R. L. porENza, D.
BannL, G. MlcNocNA and A. BraNcHr. Pharmacological studies of 'sapo' from
the frog Phyllomedusa bicolor skin: a drug used by the Peruvian Matses
Indians in shamanic hunting practices. Toxicon 31, 1099-1 111, 1993.-The
dried skin secretion îrom Phyllomedusa bicolor,'sapo', is used by the Matses
Indians of the Northern Peru, in shamanic rites mainly designed to improve
luck in hunting. Vy'hen rubbed into burned, exposed areas ofthe skin, the drug
causes the prompt appearance of violent peripheral gastrointestinal and
cardiovascular effects soon followed by remarkable central effects (increase in
physical strength, heightening ofsenses, resistance to hunger and thirst, exalted
capacity to face stress situations). All the peripheral and most of the central
effects of 'sapo' can be ascribed to the exceptionally high content of the drug
(up to 7oh of its weight) in potently active peptides, easily absorbed through
the burned, inflamed areas of the skin. The concentration in'sapo'of the single
peptides (phyllocaerulein, phyllomedusin, phyllokinin, dermorphins and
deltorphins) has been determined by bioassay, and peptide contents were
correlated with the different symptoms of the 'sapo' intoxication.
OvBn the past few years we have had the opportunity to study the biogenic amines and
particularly the active peptides in the skin of several hylid frogs from Central and South
America belonging to the Phyllomedusinae subfamily (Rosrcnrm et al., 1986; Ensp.qùrsn
et al., 1986).
Particular attention was paid fo Phyllomedusa sauvagei and P. hypochondrialis from
Northern Argentina, P. rohdei and P. burmeisteri from CentralBrazil and P. bicolor from
the Peruvian and Brazilian Amazonas (Ensnarvmn et at., 1985).
We examined methanol extracts of both fresh and dried skins, with comparable results.
This material did not contain appreciable amounts of amines (except 10-20 pg bufotenine
per g dried skin in P. rohdei), nor bioactive alkaloids, but enormous amounts of a variety
of active peptides belonging to at least seven families: caeruleins, tachykinins, bradykinins,
V. ERSPAMER el a/.
Phyllomedusa bicolor spscwrnN, wITH DIvARICATED LEGS, BoUND To FouR SMALL srAKEs.
is ready for collection of sapo (bamboo stick). Photograph by Peter Gorman, with
permission (December 1990, Galvez River, near Angamos, Peru).
bombesins, sauvagine (corticotropin-releasing hormone-like peptide), tryptophyllins and
opioid peptides, selective for either p- or ó-receptors (dermorphins and deltorphins). The
pharmacological actions of these peptides have been described in a number of papers
(Enselrvrrn and MELCHToRRT, 1983; Enspalrnn et al., 1985).
In the course of our research on skin extracts of P. bicolor, our attention was attracted
by a report by GonuaN (1990), dealing with some shamanic practices by the Matses
Indians of the north-east corner of Peru, a subdivision of the Mayeruna tribe, in which the
use of the drugs of animal and vegetable origin played a prominent role.
One of these drugs, 'sapo', was of particular interest because it consists of a dry, resinish
substance obtained from a large tree frog which the Matses call 'dow kiet'. It is now well
established that the frog is actually P. bicolor (cf. Da.rv et al., 1992).
Sapo is employed in magic hunting rituals designed to improve the hunter's luck. To
collect sapo the Matses catch the frog and keep it for 3 days during which time its legs and
back are periodically and gently scraped with a stick of bamboo (Figs 1 and 2).
To dry the secretion coilected from the frog, the stick is placed in a leaf bag and hung
above the cooking fire between scrapings. The same stick is used over and over; by the end
of 3 days the stick is covered with a yellow substance and the dow kiet is set free. The sapo
retains its potency for at least a year (Gonru.tN, personal communication).
To use sapo, a Matses hunter first scrapes a bit of the dried resin from the stick, then
mixes it with saliva until it has a consistence somewhat like mustard. The recipient's arm
or chest is then burned with a smouldering twig, the burned skin is peeled away and the
liquified sapo introduced into the raw area, approximately the size of a matchhead.
Frc. 1. A
The frog
Frog Skin Peptides
Ftc. 2. B,rMsoo srlcK wITH TIIE sApo MATERTAL usED IN THIS sruDy.
The effects elicited by the drug are described by GonulN (1990), in a rather journalistic
style, as follows:
"The effects are astounding: the moment the drug is placed upon your skin your body
begins to heat up. In moments you feel as though you're burning from the inside; you
begin to sweat. Your blood begins to race; your heart pounds. You became aware of every
vein and artery in your body and feel them opening to allow for the fantastic pulse ofyour
rushing blood. Your stomach cramps and you vomit violently. You lose the control of
your bodily functions; you may urinate and defecate and drool uncontrollably. You fall to
the ground and begin to lose consciousness; then suddenly, you may feel urges to do
things you've never done before. You might find yourself growling, barking or moving
about all fours. You feel as though animals are passing through you, trying to express
themselves through your body. But even this extraordinary feeling is secondary to the
speeding of your blood, a motion so fast that you think your heart would burst.
"For about fifteen minutes the rushing gets faster and faster, you are in agony. The pain
becomes so great that you wish you could die and get it over with, but you don't die. The
pounding slowly besomes steady and rhythmic and you gasp for air like a man saved from
drowning. Finally the pounding subsides and you're overcome with exhaustion. You
"There are no dreams or visions with 'sapo'; you may even wonder what it all was for
until you wake, then you are a god. Everything about you is larger than life, and your
physical strength is explosive. You can do without food for several days and run in the
jungle for hours without tiring. You can see in the dark effortlessly. You see animals
before they see you, and you sense which plants are benevolent and which are not. Every
TOX 31:9-8
V. ERSPAMER el a/.
sense you possess is heightened and somehow in tune with the jungle, as though the 'sapo'
put the rhythm of the jungle into your blood."
The violent visceral effects of sapo have been confirmed by GonuaN (personal com-
munication) in about ten self-applications of the drug, and correspond exactly to the
description by Dora and ClnNBmo (quoted by Funsr, 1974).
It is suggested that the drastic cleaning out of the body (vomiting, diarrhoea, urination,
sweating) observed in the first phase of the sapo application, with alleged elimination of
'toxin', may have some 'magic' effect in itself and may heighten the effects of other drugs
possibly taken prior to, or together with, sapo. Among other things, by cleansing the
body, Matses hunters would lose their'human' odour in the short time, making it easier to
approach and capture the prey.
According to the estimates by GonulN (personal communication) the amount of sapo
rubbed into the normal two or three burned skin areas may be 20 30 mg (10 mg per area)
with maximum doses of 100 mg per day. Sapo is used by the Matses not only to help on
long hiking trips or in tapir trap setting (in this last case sapo is applied, morning and
night, for at least 15 days, on four or five burns in the forearm and chest), but also to
produce abortion (application on two burns to the interior of the vaginal labia).
With regard to the central 'magic' effects of sapo, things are perhaps more complicated.
It is possible, and in some cases certain, that before or after sapo, the Matses (especially
the Amahuaca) take other drugs such as 'Banisteropsis vine' or 'nu-nu' snuff, which may
have more or less potent hallucinogenic effects. Thus it is difficult to decide how much of
the ecstatic trance experience may be ascribed to the frog poison and how much to other
drugs. It should be stressed, however, that Gorman has experienced central effects, "to
feel like god", after application of sapo alone.
The purpose of the present study, using an original sample of sapo from the Matses,
was to establish: (a) the actual amount of bioactive compounds in sapo, especially amines
and peptides; (b) the effects of the single active constituents or of a cocktail of them on the
cardiovascular system and viscera; and (c) the effects of the same constituents on the
central nervous system.
The results of this study would help us attempt to explain the two successive phases,
peripheral and central, of sapo intoxication.
Sapo materíal
A sample of sapo was placed at our disposal, in February 1991, by PsrEn GonulN. The skin secretion was
obtained from a P. bicolor specimen weighing 72 g, captured by the Matses near the Galvez River, Peru, on 22
December 1990.
The material, resinous in aspect, weighed 126 mg. It was ground in a small mortar and then extracted with
30 ml of 807o methanol. After 2 days the supernatant was removed and extraction with a similar quantity of
807o methanol repeated twice, again for 2 days. The liquids were combined, filtered and kept at 2'i.
The exhausted sapo material, weighing 52mg (41%o of the total weight), was extracied first with 5m1
petroleum ether and then, after removal of the solvent, boiled in 3 ml of 0.1 M HCl, for 20min. The methanol,
petroleum ether and acid extracts (the last two colourless) were submitted separately to bioassay.
Chromatography on alumina
An amount of the methanol extract corresponding to 50 mg sapo was evaporated and the residue, weighing
36 mg, dissolved in 50 ml of 957o ethanol. The liquid was loaded onto a chromatographic column packed with
45 g of alkaline alumina (Merck, Darmstadt, F.R.G.) and eluted using a stepwise gradient of aqueous ethanol:
19 steps from 95 to 10% ethanol. At each step two or three fractions of 50 ml were collected. The eluates were
keot at 2'C until used.
Frog Skin Peptides I 103
Thin layer chromatography and colour reacîions
Samples of the sapo methanol extract corresponding to 0.5 2 mg drug were applied to silica gel plates and
developed in 80% ethanol:acetic acid (10:1v/v). The developed chromatograms were then sprayed with
Dragendorff reagent (for alkaloids) and the Ehrlich reagent, p-dimethylaminobenzaldehyde (for
The occurrence and concentrations of the various active components of the sapo extracts were determined
using the following test preparations: caerulein-like peptides: guinea-pig gall bladder; bradykinin- and bombesin-
like peptides: rat uterus; tachykinins: guinea-pig ileum and rabbit colon; dermorphins: electrically stimulated
guinea-pig ileum; deltorphins: electrically stimulated mouse vas deferens; and sauvagine: rat diuresis.
All the above methods have been described in detail in previous papers (Ersra,urn and FalcoNrcnt Ensrllrrn,
1962; Ensrrr'rln et a1.,1972,1980; Bnoccnnoo et al., l98l).
The guinea-pig gall bladder, rat uterus, mouse vas deferens and rat antidiuresis tests gave reliable qualitative
and quantitative results even with the crude sapo extract. However, the quantitative evaluation of the
tachykinins and dermorphins was possible only after separation by chromatography.
In a further set of experiments the crude methanol sapo extract as well as the petroleum ether extract were
injected intracerebroventricularly in rats, in order to assess the direct central effects elicited by the drug.
(l) Crude sqpo methanol extract. The content of active peptides was as follows:
phyllocaerulein, expressed as caerulein (equiactive to the former) 30-35 pg per mg sapo;
phyllokinin 18 pg/mg; sauvagine 3 pglmg, finally, Ala-deltorphins, expressed as
Ala-deltorphin I5.3 pglmC. Phyllomedusin and the dermorphins, as already stated, could
not be determined in the crude extracts.
Intracerebroventricular injections in rats of the extract, in amounts corresponding to
20, 50, and 200 pg of drug per rat, produced only symptoms attributable to the opioid
peptides: increased locomotor activity with rearing, grooming and sniffing, characteristic
for the deltorphins (LoNcoNr et al., l99l), and analgesia, characteristic for the dermor-
phins (BnoccARDo et al., l98l). Threshold doses ranged from 20 to 50 prg; with 200 pg,
effects were intense. No other obvious neurological or behavioural manifestations could
be observed.
(2) Petroleum ether extract. The residue of the extract, taken up in water (slightly
opalescent) did not display appreciable effects in any of our test preparations. up to
amounts of 0.3 mg sapo/ml bath fluid. Similarly, the extract was completely inactive when
given intracerebroventricularly in doses up to 3 mg sapo.
(3) Acid extract. This extract displayed < 10 of the activity of the methanol extract in
our test preparations. Thus, only trace amounts of active compounds remained in sapo
after methanol extraction.
(4) Eluates from alumina column. Figure 3 presents the elution profile of the various
peptides from the alumina column. Sauvagine is retained by alumina and therefore
appears only in trace amounts in the eluates. (a) Phyllocaerulein was eluted by 40 to 10%
ethanol (26 pglmg sapo), with a recovery of 80%; (b) phyllokinin appeared in 60, to 50,
eluates (14 pglmg), with a recovery of 78oh; (c) phyllomedusin emerged in eluates 70, and
703 (18 p5lmù; assuming 80o recovery (as inferred from other experiments) the
amount occurring in the crude methanol extract would be 22 pClmg; (d) the ala-
deltorphins coeluted with phyllomedusin @.6 pglmg) with a 86olo recovery (Fig. a); (l)
finally, [Lys7]-dermorphin-OH was found in eluates 40 and 30 (0.2-0.25 p5lmù1' assuming
I 104 V. ERSPAMER el a/
70' 70' 70. 60' 602 50r 50' 40 30 20 10
Ftc. 3. Er-urroN pRoFILE FRoM AN ALUMINA coLUMN oF THE FouR MAIN pEprrDES occuRRrNG rN sApo
chromatography was obtained *nn fìrÍirritil ilt"ft;?)", aqueous ethanol, rrom e5 to 10%.
Numbers 1, 2, 3 (e.g. 70b702,70,) refer to the 50ml eluate fractions obtained successively at the
same ethanol concentration. [Lys7]-dermorphin-OH was eluted by 40 and 30o/o ethanol (not
shown). Sauvagine was retained by alumina. Peptides were quantitatively estimated by bioassay,
against their synthetic standards, using the smooth muscle preparations shown in Fig.5.
a 70 80o recovery from alumina, the actual content of the peptide in sapo would be
0.25-0.33 pclmc.
Methanol extracts of fresh P. bicolor skin also contain similar amounts of [Trpa.
AsnTl-dermorphin-OH. The peptide could not be detected in the sapo extract probably
because it was largely inactivated during drying of the cutaneous secretion. We have
demonstrated (unpublished observations) that [Trpa, AsnT]-dermorphin-OH is attacked
with exceptional rapidity by tissue endoproteases.
From data obtained in the bioassay of the crude sapo methanol extract and of the
eluates from the alumina column, it can be calculated that active peptides account
altogether îor '7.2oA of the sapo weight and for l2oA the weight of the methanol-
extractable constituents of the drus.
Thin layer chromatography
No Dragendorff- or Ehrlich-positive spots appeared on chromatograms following
application of 2 mg crude methanol extract, confirming the negative results obtained on
paper chromatograms of P. bicolor skin extracts, using amounts of up to 0.2 g fresh skin
(unpublished observations).
Sapo, the dried secretion obtained from P. hicolor skin, contains enormous amounts of
bioactive peptides, potently affecting the cardiovascular system and the visceral functions:
up to 32 pg caerulein per mg sapo, 18 pg phyllokinin, 22 pg phyllomedusin and 3 pg
sauvagine. In addition it contains 5.2 pg Ala-deltorphin (expressed as Ala-deltorphin I)
and 0.25-0.33 pg [Lys7]-dermorphin-OH. Sapo does not contain, like P. bicolor skin
extracts, detectable amounts of biogenic amines (especially indolealkylamines) and
Frog Skin Peptides
Itf lD.r-tur_ los '8or3oz-lor .ftDelElrúz *r loe Íllf '
lf "1;l#3i1""#Yì;i'Jà:;ililf x,T:",iìffi :Yil'Hll'"JÌ".1Yiil'ffi H#'J:'àH;
organ bath, 10ml. GPGB, guinea-pig -"ilîilià:: Responses elicited by amounts of eluates
corresponding to 2 pg sapo. For comparison l0 ng caerulein (CAER). Peak of caerulein-like
activity was found in 307o eluate. GPI, guinea-pig ileum. Responses elicited by amounts of eluates
corresponding to 3 pg sapo. Peak of phyllomedusin activity was found in 70r% eluate. MVD,
mouse vas deferens. Response elicited by amounts of eluates corresponding to l0 pg sapo. For
comparison 2 ng Ala-deltorphin I (DELT). Clear-cut peak of deltorphin activity was found in
70rok ehtafe. RU, rat uterus. Responses elicited by amounts ofeluates corresponding to l0pg
sapo. For comparison 100 ng phyllokinin (PHK). Peak of phyllokinin activity was found in 60,
and 50,7o eluates. Time marks, 5 min.
Dragendorff-positive alkaloids. Other peptide peaks, in addition to those produced by the
above peptides, are present in sapo HPLC chromatograms. Their study is in progress, but
none of them displayed any activity in our systems.
At this point, before passing on to a detailed discussion of our results, a preliminary,
fundamental question must be answered, that of the rate of absorption of the sapo
constituents through the burned areas of human skin.
It is well known that exposed inflamed dermis (like inflamed mucosae) easily absorb
drugs, as a consequence of vasodilatation and increased capillary permeability. Moreover,
bradykinins and tachykinins, occurring in large amounts in sapo, are potent pro-
inflammatory agents, further increasing vascular dilatation and permeability produced by
the thermal injury.
Both bradykinin and eledoisin (a tachykinin related to phyllomedusin) are active,
following intradermal injection in man, at doses as low as l-5 ng (DE Cano, 1963).
As previously stated, it has been calculated that the sapo material applied to a burn has
an approximate weight of 10 mg. If so, the peptide content of a single application would
be as follows: caerulein 320 pg, phyllomedusít 220 pg, phyllokinin 180 pg, sauvagine
30 pg, Ala-deltorphins 53 pg and [Lys7]-dermorphin-OH 3 pg.
xtf lD.,Tr_ los
Frog Skin Peptides I 107
(d) Sauvagine. The amount of this peptide present in 20-30 mg sapo is of the order of
60 90 pg. Peripherally, sauvagine causes in dogs, rabbits and rats a long-lasting fall in
blood pressure, accompanied by intense tachycardia (Ensr.r.urn et al., 1980).In the dog,
pr".rrri. decrease is due mainly to an intense vasodilatation in the mesenteric vascular
àrea. In rats, the threshold hypotensive and heart rate-stimulating dose of sauvagine, by
s.c. injection, is 0.5 pg/kg (MEI-cHIoRRI and NrcnI, 1983).
From the above data it seems likely that the amount of sauvagine rubbed into the skin
with 20-30 mg sapo (corresponding to 1-1.5 pglkù may contribute to the cardiovascular
effects ("blood racing, heart pounding") seen in sapo intoxication, as well as, through its
potent vasodilator action on gut mucosal vessels, to diarrhoea. The effects of the
activation of the pituitary-adrenal axis produced by sauvagine will be discussed later.
1e) Opioid pepfides. No peripheral effects whatsoever can be expected from the amounts
of Ala-deltorphins (105-160 pg) and [Lys7]-dermorphin-oH (6-9 occurring in
20-30 mg sapo.
In summaiy, it may be reasonably concluded that the intense peripheral cardiovascular
and gastrointestinal symptoms observed in the early phase of sapo intoxication may be
entirely ascribed to the known bioactive peptides occurring in large amounts in the frog
material. A predominant role seems to be played by caerulein, with potentiation of the
caerulein intoxication by phyllomedusin, phyllokinin and sauvagine. Abortion ascribed to
sapo may be due either to direct effect of the peptide cocktail on the uterine smooth
muscle o., *or. likely, to the intense pelvic vasodilatation and the general violent physical
reaction to the drug.
Central aspects of the sapo intoxication
(a) Caerulein, given s.c. in rats, at a dose 2 pglkg, displayed clear-cut satiety and
analgesic effects (JunNl and ZBrrsn, l98l). In man' an analgesic effect was observed,
following s.c. administration of caerulein, at doses of 0.06 0.2 pglkg 9 12 pgl60 kg) in
patients iuffering from renal colic, rest pain due to peripheral vascular insufficiency and
èu.r "un"., pain (Ensr,tMER, 1981, 1983; Gna.rrr, 1990). It has been suggested that the
analgesic effect, independent of any action on smooth muscles, may be related to the
release of B-endorphin or to a direct effect of the peptide on the aminergic system in the
CNS. On the other hand, caerulein given by i.v. infusion at a rate 2 nglkglmin (total
7,4g) caused a significant reduction in hunger and food intake in human volunteers
(SracuEn et al.,1982).
The amount of caerulein rubbed into the skin with 20-30 mg sapo (l I 16 Pglkg) would
be largely sufficient to produce satiety and analgesic effects in man.
(b) Sauvagine, given to rats by the s.c. route, from threshold doses of 0.3 pg/kg, caused
the release of corticotropin from the pituitary, with consequent activation of the pituitary
adrenal axis and an increase in plasma corticosterone levels. With 0.5 and 2 pg/kg, levels
rose by 100% and 4000 , respectively. The effects lasted for 2 to 4 hr. Simultaneously,
there was a release of B-endorphin and an elevation of plasma catecholamine and glucose
concentrations ( et al., l98l Ensp.q.unn and MprcnIonnI, 1983)'
It should be remembered that according to Gonu.qN (personal communication), the
maximum amount of sapo rubbed into the skin over the course of a day could be as high
as 100 mg (corresponding to 3000 ,t/g caerulein and 300 pg sauvagine) and that Matses
hunters setting traps could apply 20 30mg sapo to their skin twice daily for l0 15
I 108 V. ERSPAMER er a/.
consecutive days' It is clear that this would cause a persistent activation of the pituitary
adrenal axis by sauvagine and full manifestation of the analgesic and satiety effects aue io
(c) No appreciable central effects are to be expected by the amounts of phyllomedusin
and phyllokinin present in 20 30 mg sapo, unless they in"r"ur" the blood brain barrier
permeability, thus facilitating access to the brain not only of themselves but also of the
other active peptides. This would perhaps allow the appearance of the anti-dipsogenic
effect of phyllomedusin, with consequent enhanced r"rirìun". to thirst (De Ctio ú at.,
l 988).
(d) we do not know wherher the Ala-deltorphins present in 20 30 mg sapo
(120-180 prg) could have any central action in man, when administered by the s.c. Àute. tt
seems very improbable. In rats, intracerebroventricular doses as high as 0. l-0.5 pglkgare
required to elicit an increase in locomotor activity and othei behaviourai ""nig.,
(ERsPa.lrnn, 1992). Volunteers, infused with a total of 5 mg of Ala-deltorphin I ou"i u
30min period, showed no symptoms whatsoever (DEcu Usrnrr etat., l9g9). The same
can be said for [Lys7]-dermorphin-oH present in 20-30 mg sapo in an amount of 5 g pg.
In rats an analgesic effect could be appreciated only after |-2mg/kg of the peptide, giuÀ
by s.c. injection.
In conclusion, some central effects of sapo (increase in physical strength, enhanced
resistance to hunger and thirst and, more generally, increase ìn th" "upu.ity to face stress
situations) may be explained by the presence of caerulein and sauvagine in the drug; the
role played by the other peptides is uncertain. Of course, it cannot be ruled out th;t the
cocktail of peptides occurring in sapo, owing to its effect on the vasculature, may increase
the permeability of the blood brain barrier, facilitating access of all peptides to ihe brain.
At any rate, no hallucinations, visions or 'magic' effects are produced by the known
peptide components of sapo. Similarly, biogenic amines are beyond consideration,
because P. bicolor skin, like that of other Phyllomedusinae, does not contain detectable
amounts of these compounds.
In theory, it is not possible to exclude that other unknown sapo constituents, not
detectable in our bioassay procedures, display some central effects in man. This, however,
seems highly improbable because intracerebroventricular injections of sapo (methanol and
petroleum ether extracts) in rats did not produce neurological or behavioural effects other
than those expected from their content in known peptides, especially opioid peptides.
Recently, D1.rv et al. (1992) have isolated from a sample p. bicolor skin secretion
(sapo) a 33-residue linear peptide, very rich in alanine and lysine (l I and 6 residues,
respectively) and possibly including in the sequence a o-amino acid residue:
The peptide, named adenoregulin, enhances binding of agonists to A,-adenosine
receptors in rat brain membranes. It is accompanied by peptides, as yet not sequenced,
that inhibit binding. It is difficult to conceive that uo.nàr.g,ríin has any importani
participation in the peripheral and central effects of sapo. TÀe enormous s.c. dose of
6 pg/mouse (approx. 2a0 ltglkg) caused only modest behavioural depression. The s.c.
injection into mice of methanol or aqueous extracts of P.bicolor dried skin secretion in
doses equivalent to 0.1-6mg/mice of the dried material (:4_24gmg/kg) resulted in a
dose-related reduction of spontaneous locomotor activity. At highei doses, mice were
completely inactive for several hours, although when touched they would respond briefly
and then reassume a lethargic state. According to Dltry etat. (tggZ) the inaòtive state is
Frog Skin Peptides
reminiscent of the inactive but responsive state caused in mice by injection of some non-
natural adenosine analogs.
Unfortunately no data are presented on the blood pressure situation of the injected
mice. Reduction of locomotor activity and lethargic-like state, with preservation of
responsiveness to stimuli are typical of severe hypotension, that may be produced by the
enormous amounts of vasoactive peptides (up to 70 pglmg) occurring in the dried skin
secretion. The Matses share the 'magic' use of frog poisons with other Peruvian Indians,
the Amahuaca (ClnNErno, 1970) and the Cashinaua (KnNNErr KENSTNGnR, quoted by
Funst, 1974) as well as with the Brazilian Indians Mayoruna (cf. Dnrv etal., 1992) and
Marubo (MoNracNER-MELArrI, 1985). The Cashinaua seem to employ a different frog
species, and to interpret the experience as one ofpurification, designed to expel a sickness-
like condition and bad luck, especially in hunting.
RorH (1915) has reported similar'magic'practices among the Indians of the Guineas.
The poisonous exsudation and spaw of certain frogs or toads are rubbed into cuts made in
the skin, or introduced into the eyes, nose, mouth and ears of the hunters, with the same
drastic symptoms experienced during sapo intoxication.
We have studied as many as 12 Phyllomedusinae frogs collected from Northern
Argentina to Mexico, and have shown that all the examined species contained the same
cocktail of active peptides (bradykinins, tachykinins, caeruleins, sauvagine, opioid
peptides) as P. bicolor skin (Ensrnranu et al., 1985). Hence, the skin secretion of all these
frogs could provoke an intoxication similar to that elicited by sapo. Several Phyllomedusa
species, e.g. P. tomoptera, P. palliata and P. tarsius, are sympatrid to P. bicolor. In
accordance with this assumption, the injection of a methanol extract from a skin of
P. lemur, that was equivalent to 100 mg of wet skin, produced in mice an inactive state
similar to that caused by extracts of dry P. bicolor secretion (Dlrv et al., 1992).
Acknowledgemenls-This work was supported by grants from the Consiglio Nazionale delle Ricerche, CNR,
Rome, and Fidia Research Laboratories, Abano Terme, Padua (Italy). We are indebted to Miss P. Tulnlol and
Miss C. Cnrrr.roNl of the same laboratories for linsuistic suoervision.
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... Sauvagine is the only peptide in the family of the corticotropin-releasing hormone-like peptides identified in P. bicolor (Erspamer et al., 1993). It has 40 amino acid residues in which the C-terminal residue is amidated, as well as the presence of pyroglutamic acid as the N-terminal residue (Table 1). ...
... the diversity of toxins present in the secretion. In vitro cytotoxic and antitumor effects on different cells have been identified, as well as antibacterial and antiprotozoal properties, besides substances with high affinity for µ-opioid receptors Erspamer et al., 1993;Leite et al., 2005;Hesselink and Schatman, 2018) and dilation and increased permeability of vessels and the blood-brain barrier (Erspamer et al., 1989;Erspamer et al., 1993;Daly et al., 2009;Van Zoggel et al., 2012;Li et al., 2018). Overall, frog secretions tend to cause severe adverse side effects, including death. ...
... the diversity of toxins present in the secretion. In vitro cytotoxic and antitumor effects on different cells have been identified, as well as antibacterial and antiprotozoal properties, besides substances with high affinity for µ-opioid receptors Erspamer et al., 1993;Leite et al., 2005;Hesselink and Schatman, 2018) and dilation and increased permeability of vessels and the blood-brain barrier (Erspamer et al., 1989;Erspamer et al., 1993;Daly et al., 2009;Van Zoggel et al., 2012;Li et al., 2018). Overall, frog secretions tend to cause severe adverse side effects, including death. ...
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Phyllomedusa bicolor (Phyllomedusidae), popularly known as the kambô in Brazil, is a tree frog that is widely distributed in South American countries and is known for producing a skin secretion that is rich in bioactive peptides, which are often used in indigenous rituals. The biological effects of the skin secretion were observed in the first studies with indigenous communities. Over the last six decades, researchers have been studying the chemical composition in detail, as well as the potential pharmacological applications of its constituents. For this reason, indigenous communities and health agents fear the misuse of the kambô, or the inappropriate use of the species, which can result in health complications or even death of users. This article seeks to provide a transdisciplinary review that integrates knowledge regarding the biology of P. bicolor, ethnoknowledge about the ritual of the kambô, and the chemistry and pharmacology of the skin secretion of this species, in addition to medical aspects of the indiscriminate use of the kambô. Furthermore, this review seeks to shed light on perspectives on the future of research related to the kambô.
... The numerous biologically active peptides contained in the skin secretions of the Amazonian Giant Monkey Tree frog, Phyllomedusa bicolor, exert diverse physiological effects on the human body when administered via small epidermal burn wounds as is standard practice in both traditional Amazonian Indigenous and Western neoshamanic Kambô practice ( Figure 1). 1 The amounts of bioactive peptides present in Kambô have been measured to be approximately 7.5% by dry weight, which hence enabled estimation of the amounts entering the body via transdermal application. 2 For one burn (dot or gate, as it is known by Kambô practitioners), the amount of Kambô applied has an approximate weight of 10 mg, meaning a standard dose of total peptides would be 30-50 mg via 3-5 gates. A single application of five points typically might contain phyllocaerulein (30-35 µg/mg): 1600 µg, phyllomedusin (22 µg/mg): 1100 µg, phyllokinin (18 µg/mg): 720 µg, sauvagine (3 µg/mg): 150 µg, deltorphins (5.3 µg/mg): 165 µg, and dermorphins (0.3 µg/ mg): 15 µg, along with trace amounts of many other peptides. ...
... Furthermore, there might be differences in composition between Kambô harvested from a male or a female frog. 2 While there is a fair amount of literature on the biochemistry and physiological effects of various individual peptides isolated from the skin secretions of Phyllomedusa bicolor, there have been virtually no human or animal studies on the clinical outcomes of receiving Kambô in the traditionally applied way. Erspamer, one of the first researchers to isolate and categorize many of the peptides from P. bicolor, speculated that synergistic influence on physiology may be exerted by the combined administration of the peptides. ...
... The broad range of small (700-4600 Da) 14 biogenic peptides identified in the secretions of various cutaneous glands in frogs of the Hylidae family, including Phyllomedusa bicolor are believed to provide the frogs with defenses against predators in their natural habitat. 2,[14][15][16][17] Much of the existing literature describes these amphibian peptides in reference to several frog species or families within the Phyllomedusae genus and other genera in the Hylidae frog family. Very few papers comprehensively present information that is species-specific to the Phyllomedusa bicolor frog in the context of peptide exposure from Kambô. ...
Kambô is an Amazonian ritual which includes the application of the defensive secretion of the Phyllomedusa bicolor frog to superficial burns made on the skin of human participants. The secretion, which contains a range of biologically active linear peptides, induces a short purgative experience that is extensively reported by participants to leave them with positive physical, emotional and spiritual after-effects. Various peptides identified in the secretion exert analgesic, vascular, and gastric effects in vivo, and antimicrobial and anti-cancer effects, among others, in vitro. While there has been some investigation into the physiological effects of various individual peptides isolated from the P. bicolor secretion, very little is known about the putative synergistic effects of concurrent administration of the complete substance through the transdermal methods used traditionally in the Kambô ritual. In this review and commentary, the authors summarize the existing biological information from animal research on peptides from the P. bicolor secretion, then consider the evidence in the context of Kambô administration to humans. The presented information suggests that specific peptides are likely to contribute to analogous physiological effects of Kambô in humans. The possibility that beyond their physiological action, the experiential or phenomenological component of these effects may have therapeutic applications is discussed, concluding with a consideration of the feasibility of human clinical research.
... adenoregulin, bradykinin, ceruletide, deltorphin, dermorphin, phyllomedusin, phyllokinin, sauvagine and others) have been identified in the Phyllomedusa secretion (De Morais et al., 2018). Among these, it has been hypothesised that ceruletide and sauvagine might be responsible for some of the central effects, whereas ceruletide, phyllokinin, phyllomedusin, sauvagine and opioid peptides might mediate peripheral effects (Erspamer et al., 1993). ...
... In literature to date, it has been controversial if Kambô can occasion alterations of consciousness, and if so, the mechanisms of action would remain elusive. On the one hand, the peptide structure of the substances identified in the secretion (Erspamer et al., 1993) make it highly unlikely that any direct pharmacodynamic effects upon the central nervous system are present. On the other hand, visionary phenomena have been reported anecdotally (Gorman, 1993), but it remains unclear whether these might have been facilitated by the simultaneous use of other ritual plants. ...
... On the other hand, visionary phenomena have been reported anecdotally (Gorman, 1993), but it remains unclear whether these might have been facilitated by the simultaneous use of other ritual plants. Overall, previous studies mostly described acute physical phenomena following the application of Kambô which are corroborated by the findings from our study (Daly et al., 1992;Erspamer et al., 1993;Hesselink, 2018b). ...
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Background/aim: Kambô is a name for the secretion of the Giant Maki Frog (Phyllomedusa bicolor), which has been used by indigenous cultures from the Amazonas basin and has recently become popular in alternative healing circles in Western countries, with a certain overlap with psychedelic self-exploration. Methods: We carried out an online-based survey in English (54.92%) and German investigating motivations for using Kambô, settings in which rituals take place, and subjective experiences during and after the application. Results: Participants (n = 386, mean age: 38.08 years, (standard deviation = 9.95)) were well-educated individuals with an increased lifetime prevalence of the use of ayahuasca (67.88%). A plethora of motivations for using Kambô was reported, including general healing, detoxification and spiritual growth. Acute effects included severe physical reactions and mild psychoactive effects, most surprisingly, the feeling of being connected to the frog's spirit (41.97%), whereas predominantly positive persisting psychological effects were reported. Few participants reported long-lasting physical (2.85%) or mental (1.81%) health problems which they attributed to Kambô. Of the participants, 87.31% reported an increase in personal well-being or life satisfaction, and 64.26% considered Kambô to have been at least of 'very much' spiritual significance for their lives. Conclusions: The majority of users claimed beneficial effects including more health-orientated behaviors, whereas only very few participants complained about new health problems which they ascribed to Kambô. In retrospect, Kambô was given a high personal and spiritual significance by many participants. Additional research is needed to determine in how far reported effects are modulated by setting and subjective expectations.
... Furthermore, Gorman (1993) also shared the results of his anthropological experience with John Daly et al. (1992) who have developed experiments in order to confirm the scientific soundness of the properties of the secreted substances, i.e., dermorphin, delthorphin, and dermaseptin. Erspamer et al. (1993Erspamer et al. ( , p. 1099) relate the Phyllomedusa bicolor to different ethnographic reports, including Gorman's. Erspamer et al. (1993Erspamer et al. ( , p. 1106 conclude that "the gastrointestinal symptoms (vomiting, diarrhea) observed in the early phase of sapo intoxication may be predominantly, if not entirely, an expression (sic) of caerulein intoxication", found in the frog's secretion. ...
... Erspamer et al. (1993Erspamer et al. ( , p. 1099) relate the Phyllomedusa bicolor to different ethnographic reports, including Gorman's. Erspamer et al. (1993Erspamer et al. ( , p. 1106 conclude that "the gastrointestinal symptoms (vomiting, diarrhea) observed in the early phase of sapo intoxication may be predominantly, if not entirely, an expression (sic) of caerulein intoxication", found in the frog's secretion. In addition, Lima (2012, p. 140) states that there are more than fifteen indigenous groups in Southwest Amazonia which make use of the Phyllomedusa bicolor's secretion. ...
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This article intends to analyze the possible structure of the appropriation of traditional knowledge through the patent rights system. The research question is whether the international legislation on patents and biodiversity may be contributing to the process of appropriating traditional knowledge and natural genetic resources associated with Brazilian biodiversity from the Amazon region. The aim is to cross-reference the empirical facts concerning the use of traditional knowledge and the effects of the legal treatment of patent rights. The theory to be constructed is that the international regulations on patent rights and biodiversity contribute to the process of appropriating traditional knowledge associated with Brazilian biodiversity. Therefore, empirical qualitative research is conducted, utilizing the rules of inference developed by Lee Epstein and Gary King and the grounded theory methodology. Finally, the case of Phyllomedusa bicolor (Kampô frog) was selected and studied to extract empirical data which can be used to enunciate the theory of appropriation of natural genetic resources and associated traditional knowledge from the South by the North.
... Today it is known that the secretions of Phyllomedusa bicolor are composed of molecules with different biological activities, including antimicrobial peptides such as dermaseptins, and peptides with opioid properties such as dermorphins and deltorphins. Also known are peptides such as phyllocaerulein (with action on smooth muscles of the gastrointestinal system and analgesic effect), phyllokinin (a bradykinin with hypotensive action), phyllomedusin (a tachykinin with vasodilating action), and sauvagine (a peptide with peripheral vasodilating action) [30,31]. ...
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Kambo is the name of a natural substance derived from the glandular secretions of the amphibian Phyllomedusa bicolor, a species native to regions in South America. The communities living in these areas administer the substance generally transdermally during rituals for religious-purifying purposes, producing small skin burns. The scientific literature has reported some cases of intoxication following the use of Kambo but this aspect is still poorly understood. In fact, no shared therapy protocols exist for these events nor any real legislation on Kambo. The purpose of this work was to examine all cases of acute intoxication resulting from the administration of Kambo and published over the last 10 years, illustrating clinical signs, laboratory findings, instrumental tests, and therapy. The several cases identified in our review confirm that acute Kambo intoxication can occur, with serious and life-threatening effects. We developed a protocol aimed at the early diagnosis of cases of suspected acute intoxication by creating a treatment algorithm. The study aims to investigate the pathophysiology of these events in humans, proposing a protocol for the diagnosis and treatment of these cases that can be used by healthcare professionals.
... Nowadays, these animals are getting a big spot in scientific research. Probably the best-known species is the Amazonian Phyllomedusa bicolor, which has been constantly associated with the indigenous ritual known as "Vacina do Kambô" (Erspamer et al., 1993). In this ritual, also called "Vacina do Sapo" (toad vaccine), the skin secretions of P. bicolor are used and, upon contact with the skin of a participant, it causes a series physiological signal and symptoms. ...
Amphibians represent one of the main natural sources of bioactive molecules of interest to biotechnological research. The Phyllomedusidae family has several species occurring in Brazil and some studies demonstrate the biological potential of poisons of these species, however many still need to be characterized. Phyllomedusa iheringii is endemic in Brazilian and Uruguayan Pampa Biome and has little data in the literature regarding the action of its poison on experimental organisms. Thus, the present work evaluates the biological activity of P. iheringii secretion on the central and peripheral nervous system of a vertebrate model. The skin secretions of P. iheringii (SSPI) were collected through manual compression and electrical stimulation of the animal's bodies. The resulting content was used in neurobiological tests searching for modulatory effects on the main pathways involved in the neurotoxicity mechanism of vertebrates. SSPI affected the contraction force of the chick biventer cervicis muscle (Gallus gallus domesticus) at some concentrations used (5, 10, and 12 μg/mL). In slices from the cerebral cortex of G. gallus domesticus an increase in cell viability was observed after treatment with SSPI (10 μg/mL) and a neuroprotective effect when treated simultaneously with hydrogen peroxide (H2O2), Neostigmine (NEO) and Trichlorfon (TRI). The cholinergic pathway is possibly the main pathway modulated by SSPI since assays with the cerebral cortex and biventer cervicis muscle demonstrated the increased activity of the enzyme acetylcholinesterase (AChE) (SSPI 10 μg/mL and 12 μg/mL, respectively). SSPI (10 μg/mL) also prevented the modulation of NEO and TRI, two recognized anticholinesterase agents, in AChE activity in slices of the cerebral cortex. Therefore, our results have demonstrated the unpublished biotechnological potential of P. iheringii over the vertebrate model and its modulation on the nervous system, with apparent action on the cholinergic pathway.
... All participants were requested to fill in demographic information and (1). Participants were asked to fill in (2) only if the exemplary Kambô session that they reported about in (1) happened between 2 and 3 weeks ago, as the questions on the acute effects addressed this period after the Kambô session. ...
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Kambô, the secretion of the Amazonian Giant Leaf Frog (Phyllomedusa bicolor) contains a plethora of bioactive peptides and was originally used by indigenous communities from the Amazon basin as medicine for improving hunting capacities. In the last 20 years, Kambô has spread to Western urban healing circles. To date it is still controversial whether the acute effects of Kambô include alterations of consciousness similar to known psychoactive substance like serotonergic psychedelics. Here we retrospectively assessed psychological effects of Kambô in a sample of anonymous users (n = 22, mean age: 39 years, ± 8.5; 45.5% female), administering standardized questionnaires for the assessment of altered states of consciousness (ASC), including the Altered States of Consciousness Rating Scale, the Phenomenology of Consciousness Inventory (PCI), the Mystical Experience Questionnaire (MEQ), the Challenging Experience Questionnaire (CEQ) for acute effects and the Persisting Effects Questionnaire (PEQ) and a scale assessing connectedness for subacute effects. The intensity of retrospectively reported acute psychological effects remained on a mild to moderate level, with no psychedelic-type distortions of perception or thinking. Conversely, persisting effects were predominantly described as positive and pleasant, revealing high scores on measures of personal and spiritual significance.
... All participants were requested to fill in demographic information and (1). Participants were asked to fill in (2) only if the exemplary Kambô session that they reported about in (1) happened between 2 and 3 weeks ago, as the questions on the acute effects addressed this period after the Kambô session. ...
Full-text available
Kambô is the name for the secretion of the Giant Leaf Frog (Phyllomedusa bicolor) containing a plethora of bioactive peptides. Originally, it is ritually used by different ethnicities from the Amazon basin as a remedy against bad luck in hunting. In the last twenty years, Kambô has spread to Western urban centers, often associated with the use of ayahuasca. Anecdotal reports claim beneficial effects on wellbeing and different medical and mental health conditions. However, to date it has been controversial if Kambô elicits altered states of consciousness. Here we retrospectively investigated acute and subacute psychological effects of Kambô in a sample of n = 22 anonymous users (n = 22, mean age: 39 years, ± 8.5; 45.5% female), administering standardized questionnaires for the assessment of psychoactive effects. Acutely, participants reported psychological effects which remained on a mild to moderate level, but no psychedelic-type distortions of perception or thinking. In contrast, persisting effects were predominantly described as positive and pleasant, revealing surprisingly high measures of personal and spiritual significance. Subacute and long-term effects showed some overlap with the ″afterglow″ phenomena that follow the use of serotonergic psychedelics.
The ‘kambô’ ritual involves application of Phyllomedusa Bicolor poison to superficial burns made on the user’s skin. Existing kambô literature describes case reports of severe harms. The objective of the current study was to better characterize typical observed responses to kambô administration. We performed a retrospective review of a kambô practitioner’s practice. Statistical analysis was descriptive, and characterized the number of kambô points or location of points administered as well as physical responses such as facial swelling, diaphoresis, immediate bowel movement, syncope, and occurrence of emesis. There were 147 unique participants that underwent 241 kambô rituals included. The mean number of points administered was 4.2 ± 2.2, while the median was 3 (range 1–11). Common physical responses included facial swelling (70.5%), diaphoresis (53.3%), bowel movement (45.2%), and syncope (10.4%). Higher number of points administered and receiving kambô at multiple locations appeared to increase facial swelling and diaphoresis. Responses to kambô are likely to be dose-dependent. No severe adverse effects were recorded in the sample, although some responses or components of the kambô ritual may promote risks of harm due to hyponatremia, asphyxiation from emesis, and injury due to syncope.
Research and new tools in biomaterials development by using peptides are currently growing, as more functional and versatile building blocks are used to design a host of functional biomaterials via chemical modifications for health care applications. It is a field that is attracting researchers from across soft matter science, molecular engineering and biomaterials science. Covering the fundamental concepts of self-assembly, design and synthesis of peptides, this book will provide a solid introduction to the field for those interested in developing functional biomaterials by using peptide derivatives. The bioactive nature of the peptides and their physical properties are discussed in various applications in biomedicine. This book will help researchers and students working in biomaterials and biomedicine fields and help their understanding of modulating biological processes for disease diagnosis and treatments.
When injected into the brain (periaqueductal gray (PAG), caudate nucleus, ventromedial thalamus) or subarachnoidal space of the lumbar spinal cord of rats, caerulein and CCK-8 produced a long-lasting (2 h) inhibition of the tail-flick response to thermal stimulation. The effective dose range for caerulin was from 1 to 5 ng per rat, for CCK-8 from 10 to 40 ng, and for morphine from 1 to 20 μg. On a molar basis and dependent upon the site of administration, caerulein was 4000–7000 times and CCK-8400-700 times more potent than morphine. Unsulphated caerulein (injected into the PAG) was less active than caerulin by a factor of 9. Sedation was seen after intracerebral, but not after spinal administration of the peptides; it was not observed after morphine or unsulphated caerulin. Naloxone (0.5 mg/kg i.p.) abolished the antinociceptive but not the sedative effect. It is concluded that caerulin and CCK-8 are very potent in reducing nociception by acting at both spinal and supraspinal levels.
1. The occurrence of sauvagine, a new polypeptide from amphibian skin, and its actions on rat blood pressure and diuresis were studied. 2. Sauvagine was found to be present in the skin of all the 10 Phyllomedusa species so far studied, amounts ranging from a few micrograms to 240 g per g fresh skin. 3. The polypeptide displayed in the rat an intense, long-lasting hypotensive action accompanied by tachycardia. Hypotension was not modified by either atropine or propranolol, excluding the participation of the autonomic nervous system in its production. Tachycardia, on the contrary, was partially inhibited by propranolol. 4. Hypotension is probably the main cause of the intense antidiuresis seen in hydrated rats following sauvagine administration. Reduction in urina volume was accompanied by a decrease in GFR and an increase in tubular Na+ reabsorption.
Eledoisin, physalaemin, and 29 synthetic polypeptides related to eledoisin and physalaemin were screened for their hypotensive effect in man. Experiments demonstrated that it was possible to modify the eledoisin and physalaemin molecules without any strong reduction of activity. Influence of structural variations on hypotensive effect is discussed.