Indian Journal of Experimental Biology Vol. 39, August 2001, pp. 781-785

Isolation, purification and partial chemical characterization of a lethal factor from common Indian toad (Bulo melanostictus, Schneider) skin extract

Manika Das, S C Dasgupta*& A Gamest

Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, A.P.c. Road, Calcutta 700 009, India

Received 13 November 2000; revised II Apri/ 2001

Indian toad (BII/o me/al1os/ic/IIS, Schneider) skin extract (TSE) is pharmacologically potent and probably contains sev­eral bioactive compounds [Das e/. al., l11dial1 J Pharmacal, 28 (1996) 72). A lethal factor was isolated and purified by neu­tral alumina column chromatography followed by HPLC. Spectroscopic (UV, IR, FAB-MASS) study indicated that the le­thal factor (TSE-LF) was a 254 Da long chain compound with carbonyl, hydroxyl and ester as functional groups. LDso of TSE-LF was found to be 3.5 mg/kg (iv). Biological study showed that TSE-LF possesses hypotensive, cardiotoxic, neuro­tox ic activity and produced death by apnoea in experimental animal. Cyproheptadine antagonised TSE-LF induced contrac­tion of isolated smooth muscle indicating involvement of histamine/serotonin receptors. TSE-LF induced neurotoxic action on chick biventer cervices was mediated through Ca2

+ ion.

Generally, amphibians are viewed as rather defenceless creatures that are consumed readily by a great variety of predators. However, amphibians have evolved various morphological, physiological and behavioural features, which alone or in combination provide varying degrees of protection (defence) from potential predators. The basis for this defensive behavior is the presence of granular glands, the diverse secretions of which make many amphibians distasteful , toxic and noxious to predators). Different compounds such as bradykinn, caerulin, leptodactylin, physalaemin, phyllokin, suavagine and serotonin have been isolated from the skin of a wide variety of amphibians2

. The toad, Bufo melallos/ic/us (Schneider) is a very common amphibian in India and lhere is virtually no report on the biological 3.Ctivity/active constituents of its skin. Toad (Bllfo lIelallos/ictus Schneider) skin extract (TSE) is )harmacologically potent and probably contains ;everal bioactive compounds3

. TSE also possess mmunomodulatory and antineoplastic activit/ . An lttempt has now been made to isolate, purify and 'haracterize the lethal factor, present in TSE.

Ifaterials and Methods The following chemicals were used: alpha-

hloralose (Sigma, USA), 4-amino pyridine

Postgraduate Department of Zoology, Maulana Azad College, alcutta 700 013 India. ::orrespondent author: email : agomes@cucc.ernet.in

(E. Merck, Germany), cyproheptadine (MSD, India), EDT A (Qualigen, India), histamine (Sigma, USA), HPLC methanol (SRL, India), HPLC water (SRL, India), Neutral alumina (Qualigen, India), propylene glycol (Qualigen, India), urethane (E. Merck, Ger­many), verapamil (Sigma, USA).

Collection of toad skin and preparation of the ex­tract-Adult live toads (40-50 g) (B. melanostictus) were collected commercially during March to No­vember. Animals were pithed and their skins were separated from the body except parotid gland. The skin was kept in methanol at room temperature (RT) for 30 days. The supernatant was centrifuged and was pooled. It was evaporated to dryness by rotary evapo­rator and the extract was kept at RT (28°C) in a desic­cator. The toad skin extract (TSE) was dissolved at definite concentration in saline (0.9%) for further ex­periments.

I solation and pu rification of lethal factor( s}­

(a) neutral alumina column chromatography: The methanolic extract of toad skin (2 g) was di s­

solved in methanol. It was then centrifuged at 2000 rpm for 10 min. Neutral alumina was then added to the extract and evaporated to dryness. The residue was placed on the top of a neutral alumina column (336 cm2

) bed. The column was packed with petro­leum ether. The column was washed with petrol­benzene (3: I, I: I, 1000 ml each), benzene-chloroform (1: I, 1000 ml each) and chloroform-methanol (99: I ,

782 INDIAN J EXP BIOL, AUGUST 200 1

98 :2, 95:5, 1000 ml each). The fractions eluted was evaporated to dryness and biological tes t was per­formed to identi fy the lethal factor. (b) Preliminary studi es to identify lethal factor:

The different frac tion of TSE from column chro­matography was injected in to male albino mice (20-22 g) and letha lity observed up to 24 hr. Propylene glycol:saline ( I: 10) injected in mice considered as control. (c) Hi gh performance liquid chromatography

(HPLC): The fraction of TSE, which contained lethal factor,

was further purified by reverse phase HPLC using CI S column (Novapak, 60 A, 4 mm, 3.9x150 mM) at 254 nm wavelength . Here, methanol: water (45:55) sol­ven t system was used and the fl ow rate was main­tained at 0.4 ml/min in 30 min run . From this column, fractions (pure peak) were collected and the retention time of the pure compound was measured .

Spectral characterizatioll of lethal facto/,- The ultraviolet (UV) spectrum (200-400 nm) of the pure lethal factor was taken in Perkin-Elmer Spectropho­tometer (Model 5503) using spectral methanol as a solvent. The IR spectrum of the pure compound was registered on a Perkin-Elmer Spectrophotometer (Model 732) using a thin film of pure compound. Mass Spectroscopy (FAB-MS) was performed to de­termine the molecular weight of the pure compound (this was done at Mass Spectrometry Centre, Indian Institute of Chemical Technology, Hyderabad, India). Biological activity of lethal factor-

Lethality: LDso was computed according to the method of Litchfield and Wilcoxons.

Effect 0 11 cardiovascular systeJII-Blood pressure: Arterial blood pressure was re­

corded via an indwelling arterial cannul a in the com­mon carotid artery of diethyl ether alpha-chloralose anaesthetized (80 mg/kg, iv) male cat (2.5 -3 .0 kg) using a mercury manometer on a rotating smoked drum. The drug was infused through the femoral vei n.

Isolated heart and auricle: Isolated guineapig heart (Langendorff)6 was prepared and perfused with oxy­genated (98% O2 and 5% CO2) Tyrode's solution at 37°±l oC [NaCI 137 mM, KCI 2.7 mM, CaCh 1.8 mM, MgCI2 I mM, NaHCO) 11 .9 mM, NaH2P04 0.4 mM and glucose II mM] and contractions were re­corded on a rotating drum using a heart lever. Isolated guineapig auricle was prepared as described by Burn 7

and suspended (5 ml bath) in oxygenated (95% O2

and 5% CO2) Ringer solution (29°± 1°C) [NaCI 15 mM, KCI 5.6 mM, CaCIz 2.2 mM, NaHCO) 6 mi\­glucose 11 .1 mM] . The spontaneous contraction ( auricle was recorded on a smoked drum using a heal lever.

Effect Oil respiratioll-Respiration was recorded il urethane (1.75 g/kg, ip) anaesthetised male albino rat ( 180-200 g), as per Gaddums.

Effect Oil isolated nerve lIIuscle preparatiolls-Ra phrenic nerve diaphragm (Btilbring)9 and chick bi venter cervices (Ginsborg and Warriner)lo were sus· pended in physiological salt solut ion [NaCI 135 mM KCI 5 mM, CaCIz 2 mM, MgCIz I mM, NaHCOJ 15 mM, Na2HP04 I mM, Glucose I 1 mM] gassed wi th carbogen (95% O2 + 5% CO2) at 29°± I °C and st i mu­lated with a square wave electronic stimulator (8 -12 V, 0.5 m sec duration, 0.2 Hz) . Contractions were re­corded wi th Brodie' s lever on a smoked kymograph paper.

E./fect Oil isolated slllooth lIIuscle-Guineapig iso­lated ileum (3 ml bath) and rat fundus (6 ml bath) were suspended in oxygenated Tyrodes solution [NaCI 137 mM, KCI 2.7 mM, CaCh 1.8 mM, MgCl2 I mM, NaHCO) 11 .9 mM, NaH2P04 0.4 mM, glucose 5.5 mM] at 37°± I °C and contractions were recorded with a frontal writing lever on a smoked kymograph paper.

All results were expressed as mean±SE. The sig­nificance of di fference between means was deter­mined by Student 's t-test.

Results Isolation alld purification of lethal factor-TSE

(2 g) applied on neutral alumina column (336 cm2)

was eluted with different solvent system and among all the fractions, the chloroform: methanol (99: I) sol­vent fraction produced protrusion of eye ball , hi gh respiratory rate and convulsion in mice, which lead to death within I ±0.5 min (n=6), whereas no other frac­tion produced such toxic manifestat ion and lethality. The yield of the substance in chloroform: methanol (99: I) solvent fraction was 9± 1.8%. To test the ho­mogeneity of this fraction, the chloroform: methanol (99: I) solvent fraction was again purified by HPLC to obtai n pure lethal factor.

The HPLC chromatogram of ch loroform-methanol (99: I) solvent frac ti on first produced a small hump followed by a sharp peak with a retenti on time of 2.1 min , 97.9% area and 92.9% height (Fig. 1). Biological testing, of HPLC fraction in male albino mice, indi-

DAS el al.: LETHAL FACTOR FROM TOAD SKIN 783

cated that the large peak possessed lethal activity. Here the fold of puri fication factor of the lethal com­ponent was found to be 114 times (Table I).

Spectral characterization oj lethal Jactor-The ul­trav iolet (UY) spectrum of the lethal factor showed Amax at 250 nm. The infra red OR) spectrum of the lethal compound indicated the presence of hydroxyl group (2500-2900 cm·'), carbonyl group (1650-1600 cm-') and ester group (1250 cm-') (Fig. 2).

Molecul ar weight of lethal factor was found to be 254 Da as evident from j . \B-mass spectra. Mass spectra indicated that this lethal factor possess linear hydrocarbon as it suffer successive loss of 14 mass unit. The genesis o f significant ion peak at mlz (% of base peak) 254, 239, 225, 211, 197, 183 etc. has been shown in mass spectra (Fig. 2), indicating that possi­bly it is a long chai n compound.

Natllre oj cOli/pound: The spectroscop ic studies showed that the lethal factor was likely to be a long chain compound with carbonyl, hydroxy l and ester as functional groups and hav ing molecul ar weight 254 Da.

Nall/ing oj the cOli/pound: The lethal factor isolated from toad skin extract by column chromatography followed by HPLC was designated as "TSE-LF" (TSE=Toad skin extract, LF=Lethal-factor).

Biological activity oj lethal Jactor-

Lethality: Percent mortality in male albino mice were recorded at different doses of TSE-LF (iv). LDso of lethal factor (TSE-LF) was found to be 3.5 mg/kg (iv) in male albino mice.

Action 0 11 cardio-vascular sy.',tem-

Blood pressure: On cat blood pressure TSE-LF (50 /lg, kg, iv, bolus) did not produce any change of blood pressure. TSE-LF at doses of 100 and 200 /lg/kg (iv, bolus) produced a fall of blood pressure, i.e. 10.15±0. 19 mmHg (n=4) and 20.25±3.3 mmHg (n=4) respectively . Blood pressure changes returned to the normal level within 1.5±0.5 min of observation.

Isolated heart alld auricle: On isolated guineapig heart, TSE-LF (10 /lg) decreased the frequency of heart rate (12±2.1 %) and increased amplitude of con­traction (26±1 .2%). All changes returned to the nor­mal level within 20±0.5 min of observation (n=4). rSE-LF (50 /lg) decreased the frequency of heart rate :n=4) (42.5±2.5%) followed by complete cardiac ar­·est within 60±2.5 min. On isolated guineapig auricle : 10 /lg/ml) , TSE-LF decreased the rate and finally Jroduced 100% blockade within 20±2.5 min (n=4).

Action 011 respiration: On rat respiration, TSE-LF (1 mg/kg iv, bolus) decreased the respiratory rate(16.7±2.5%) within 1.0±0.5 min and normal respi­ratory rate resumed after 15±1.5 min (n=4). TSE-LF (2.5 mg/kg, iv, bolus) decreased the respiratory rate (70.3±2.2%) within 1O±0.5 min and produced 100% respiratory apnoea within 60±2.5 min (n=4), followed by death .

Action 01/. isolated smooth II/uscle: On isolated guineapig ileum and rat fundu s, TSE-LF (4 ).!g/ml ) did not produce any contractile response, whereas at a dose of 10 /lg/ml it produced a slow contractile re­sponse, which attained its peak wi thin 65±0.5 sec (n=4). Thi s slow contractile response was blocked by cyproheptadine (2x I 0-9 ).!g/ml).

Action OIL isolated nerve-muscle: On isolated rat phrenic nerve diaphragm preparation, TSE-LF (40 and 80 ).!g/ml ) did not produce any change of electri ­cally induced twitch response. On isolated chick bi­venter cervices, TSE-LF 20 ).!g/ml , produced irre­versible blockade of elec trically induced twitch re­sponse within 20±1.2 min (n=4). However, acetyl­choline (0.02 mNI) and potass ium ch loride (10 mM) induced muscul otropic action remained unaltered. Neuro-muscul ar blocking activity was studied in the presence of low Ca2

+ (1 mM), EDT A, 5 ).!g/ml (Ca2+

chelating agent) and verapamil , 10 ).!g/ml (Ca2+ chan­

nel blocker). Low Ca2+ and EDT A potentiated

(40±4% and 75±7.5%, n=4 respective ly), TSE-LF

Table I-Fold of puri fi ca tion of lethal fac tor

Puri fication steps LD50 Fold of Yie ld (mg/kg) purifi - (%)

catio Il ( iv) (ti mes)

I Crude TSE 400 100 2 ColumIl chromatography

purified lethal factor 5 80 9.0±1.8

3 HPLC purified lethal fac tor (TSE-LF)

> 50 E

o~ o 10

3.5 11 4.28

Wove- le-nqth - 254 nm

Solvtnt. Me-thanol : Water (45 : 55)

20

Minutes

> 1%

30

Fig. I-HPLC purification of lethal factor e luted in chloroform : methanol (99: 1) fraction of alumina. (Absorbance was determined at 254 nm. The peak containing lethal factor is marked with an arrow)

784 INDIAN J EXP BIOL, AUGUST 2001

50 250nm A C

-~ ~

UJ -100 0 ~

Z ~I

1~9 ",6'00 -I t- 57 I

« UJ ro 0 4J llJ

71 a: z 0 « 50 (f) ro ro a::

85 121

141 « 0

(f) I~ 5

ro « 0 J Tll'}i9Z5,

300 400 500 50 100 150 200 250 300 350 400

WAVELENGTH (nm) m/z

100 B

80

;? ~

z 60 Q (f) (f)

~ (f)

40 z « a:: f-

20

WAVENUMBER (cm1)

Fig. 2-Spectroscopy study of lethal factor, TS E-LF (A = UV spectrum; B = IR spectrum; C = FAB-MASS spectrum)

induced neuro-muscular blocking activity . Verapamil completely antagonized TSE-LF induced neuro­muscular blockade on isolated CBC.

Discussion A wide variety of toxins of relatively low molecu­

lar weight substances such as bradykinin , caerulin, leptodactylin , physalaemin, phyllokin, sauvagine and serotonin have been isolated from the skin of different variety of anuraus2

• In the present investigation, a le­thal factor (TSF-LF) was sllccessfully isolated from the skin extract of the common Indian toad B. mela-

Ilostictus by neutral alumi na column chromatography followed by HPLC. Spectroscopic studies (UV, IR, MASS) indicated that TSE-LF was 254 Da long chain compound with hydroxyl, carbonyl and ester func­tional group. LD50 of TSE-LF was found to be 3.5 mg/kg (iv) in male albino mice. On cat blood pres­sure, TSE-LF produced hypotension. On isolated guineapig heart, TSE-LF increased the force of con­traction and decreased heart rate. At high dose, TSE­LF decreased the heart rate followed by complete car­diac arrest within 60 min of observation. On isolated guineapig auricle, TSE-LF decreased the rate fol-

DAS et al.: LETHAL FACTOR FROM TOAD SKIN 785

lowed by complete blockade of contraction within 20 min. The above observation indicated that TSE-LF induced cardiotoxic activity was due to blocking of the impulse generating system. The toxic substance, bufotoxin, isolated from Bufo vulgaris, also possesses strong cardiotoxic activity. The bufotoxin improved the contractile power of heart, causing an increase of tonus and lowering of frequency 1 I. Atelopoid toxin, isolated from di fferent species of atelopus species, possesses hypotensive and cardiotoxic activiti 2

. Be­side these, batrachotoxin , isolated from dendrobatid frog (Phyllobate aerotaenia) of south and central America produced cardiac arrest by inhibiting trans­mi ssion of impulse through Purkinje fibres 13.

On rat respiration, TSE-LF produced apnoea fol­lowed by death, which indicated respiratory paralysis or failure . Like TSE-LF, batrachotoxin produce respi­ratory paralysis and also muscle poisoning l4 . On iso­lated nerve muscle preparation (CBC), TSE-LF pro­duced complete blockade of electrically induced twitch response although the musculotropic action of acety lcholine and potass ium chloride remain unal­tered. This observation indicated that the neurotoxic nature of TSE-LF, was probably presynaptic in origin . TSE-LF induced neurotoxic action was mediated through Ca2+. Like TSE-LF, batrachotoxin , isolated from the dendrobatid frog, also blocks neuro­muscular transmission irreversibly in an isolated nerve muscle preparation by acting on both pre­synaptic and post-synaptic membranes l4 . Further ex­peri ment showed that batrachotoxin produces its ef­fect mainly by increasing Na+ permeabili ty l3.

Thus, it may be concluded that TSE-LF possesses both neurotoxic and cardiotoxic activity. Lethality induced by TSE-LF was a multifactorial issue, and cardio-respiratory failure was one of the major causes of lethality. The pharmacological activity of batra­chotoxin is close to the action of TSE-LF, but their chemical identiti es are completely different. Batra­chotoxin is an aromatic compound, whereas TSE-LF is a long chain compound with hydroxyl, carbonyl

and ester functional group. Thus TSE-LF is a new lethal compound, isolated from the skin of Bufo me/Q­/lostictus , with neurotoxic and cardiotoxic properties.

Acknowledgement This investigation was supported partly by awards

of Junior and Senior Research Fellowships to one of the author (Manika Das) by the University of Cal­cutta, Calcutta, India.

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