Inhibition of toxic actions of phospholipase A₂ isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds

Abstract

Kraits are one of the most common poisonous snakes of the eastern India. Their geographical distribution ranges from Pakistan through Southern Asia to Indonesia and Central China^1,2. There are about 12 species of krait in Asia, among those the golden banded krait (Bungarus fasciatus; or common name Shankhamuti) is very common in West Bengal, India. One of the major components of B. fasciatus venom is phospholipase A₂ (PLA₂s), which constitutes about 65 per cent of the total venom proteins^3,4. The PLA₂ exerts its biological effects by hydrolyzing an acyl group at sn-2 position of the glycerophospholipids leading to the release of fatty acids and lysophospholipids which in turn either act as a second messenger or as pro-inflammatory agent. Amino acid sequences of >280 different PLA ₂ enzymes from snake venom have been identified so far (http://sdmc.lit.org.sg/Templar/DB/snaketoxin _PLA2/index.html)⁵. Venom PLA₂s exert their pathophysiological actions such as myotoxicity, cardiotoxicity, alteration in blood pressure, oedema, haemolytic activity, platelet aggregation, and neurotoxicity, which are often fatal⁶.

In India, there is no specific antiserum available against B. fasciatus venom. The commercial polyvalent snake venom antiserum raised against Naja naja, B. caeruleus, Russell's viper, Echis carinatus is used for B. fasciatus snakebite treatment. In the traditional and folk medicine, several herbs (Andrographis paniculata, Leucas linifolia, etc.) have also been used to treat B. fasciatus envenomation⁷. In view of the limited information available, this study was undertaken to purify a PLA₂ from the eastern Indian B. fasciatus venom and to neutralize its toxicity and biological activity with synthetic herbal compounds, anti PLA₂ rabbit antiserum and commercial polyvalent snake venom antiserum in in vitro and in vivo animal models.

Material & Methods

Chemicals and reagents: Lyophilized Bungarus fasciatus venom (BFV) was purchased from Calcutta Snake Park, Kolkata, India. Carboxy methyl cellulose, Freund complete adjuvant (FCA), sodium azide (Sigma, USA), Protein pack 60 column, Waters, Division of Millipore (Tokyo, Japan), acrylamide, bis-acrylamide, sodium dodecyl sulphate, coomasie brilliant blue, tri sodium citrate, ammonium per sulphate, glycine, prestained molecular weight marker (Fermentas, USA), were purchased. Commercial polyvalent snake venom antiserum (Batch no. 926) was a gift from Haffkine Institute, Mumbai, India. Synthetic herbal compounds 2-hydroxy-4-methoxy benzoic acid (BA) (Aldrich Sigma, USA), p-anisic acid (AA) (SRL, India) and salicylic acid (SA) (Loba Chemie, India) were purchased.

Animals: The study was conducted in Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, Kolkata, Male albino New Zealand strain rabbits (1.5 ± 0.2 kg), BALBc mice (20 ± 2 g) and male toad (40 ± 5 g) were used. All the animals were kept in standard laboratory conditions except toads; 12/12 h light-dark cycle, food and water were given ad libidum. Animal ethical clearance of Department of Physiology, University of Calcutta, Kolkata was obtained for the study protocol. Each experimental group was matched with a parallel control group treated only with saline solution (0.9%). Experiments were carried out at a laboratory temperature (25 ± 3°C).

Ion exchange chromatography: B. fasciatus venom (25 mg) was dissolved in 0.02 M working phosphate buffer, pH 7.2 and applied to a column (5.6 × 1.6 cm) of carboxy methyl (CM) -cellulose equilibrated with the buffer. Different protein fractions were collected (5 ml each) with step-wise NaCl gradient of increasing molarities of (0-1 M) in 0.02 M phosphate buffer. All the fractions collected were subjected to protein analysis⁸ and PLA₂ activity assay⁹.

High performance liquid chromatography (HPLC): The PLA₂ fraction obtained from ion exchange chromatography was desalted by 1KD centripep (3000 rpm, 4°C, 3 h) and further purified by a Protein pack 60 column (7.8 × 300 mm). The column was eluted with 50 mM Na-K phosphate buffer, pH 7.2 containing 0.15 M NaCl, the flow rate was 0.8 ml/min and the peak were monitored at 280 nm.

Molecular weight and sequence determination of PLA₂: The molecular weight of the PLA₂ was determined through SDS-PAGE and confirmed through electron-spray-ionization mass spectrometry (ESI-MS) (Sciex API100, Perkin Elmer, USA) in Proteomics and Structural Biology Research (Academia Sinica, Taiwan). N-terminal amino acid sequences of the PLA₂ were determined by Edman-degradation with the sample on a gas-phase amino acid sequencer coupled with a henylthiohydantoin amino acid analyzer (Perkin Elmer, USA).

Lethality determination: The minimum lethal dose (MLD) was assessed by injecting different concentration of the PLA₂ into the tail vein of mice (20 ± 2 g, n=6), observed for 24 h and mortality was recorded¹⁰.

Hyperimmunization of rabbit: Antiserum was prepared against the PLA₂ by hyperimmunization of male albino New Zealand rabbit (1.5 ± 0.2 kg, n = 4). The PLA₂ (48 μg/kg) was mixed with FCA (1:1), and administered intramuscularly in the first, second and third week of immunization followed by booster dose of PLA₂ (without FCA) for another three weeks¹¹.

Immunogel diffusion and indirect haemagglutination titre: Immunogel diffusion was done¹². The commercial polyvalent snake venom antiserum (10 μl)/anti PLA₂ rabbit antiserum (20 μl) was applied in central well and the peripheral wells contained crude B. fasciatus venom (10 μl)/PLA₂ (10 μl). It was kept at 4°C for 48 h and the precipitant bands were visualized. The antiserum titre was determined with indirect haemagglutination assay¹³.

Neutralization technique: PLA₂ activity was determined by egg yolk coagulation time⁹. One PLA₂ unit was defined as the minimum amount of PLA₂ required to increase the clotting time of the egg yolk emulsion by 1 min against saline control. Neutralization was done by mixing different amount of PLA₂ with a fixed amount of anti-PLA₂ rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C × 1 h and the clotting time was recorded¹⁴.

The minimum oedema dose (MED) of the PLA₂ was assessed by injecting 0.01 ml of different concentrations of PLA₂, sub-plantar (s.p) in the paw of male albino mice and the oedema was recorded within 2 h of observation¹⁵. One MED unit was defined as the minimum amount of PLA₂ required to induce inflammation in mouse paw by 100 per cent within 30 ± 10 min, when administered subplantarly. Neutralization was done by mixing different amount of PLA₂ with a fixed amount anti PLA₂ rabbit antiserum / commercial polyvalent snake venom antiserum (10 mg/ ml) / synthetic herbal compounds (5 mg/ml) incubating at 37°C × 1 h and the decrease in inflammation was recorded by digital calipers (Mitutoyo, Japan)¹⁴.

Plasma recalcification time using goat plasma was determined¹⁰. One minimum plasma recalcification dose (MPRD) unit was defined as the minimum amount of PLA₂ required to increase the clotting time of the plasma by 100 per cent against saline control. Neutralization was done by mixing different amounts of the PLA₂ with a fixed amount of anti PLA₂ rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C × 1 h and the clotting time was recorded¹⁴.

One minimum cardiotoxic dose (MCTD) was defined as the minimum amount of PLA₂ required to cause the onset of cardiac arrhythmia within 10 ± 2 min upon its administration on isolated toad heart suspended in Ringer amphibian fluid at room temperature (28 ± 1°C). Neutralization was done by mixing different amount of the PLA₂ with a fixed amount of anti PLA₂ rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C × 1 h to study the inhibition of the cardiac arrhythmia¹⁶.

Statistical analysis: The results were expressed as mean of observations (n=6); MLD was expressed as mean ± SE.

Results

On ion exchange chromatography, B. fasciatus venom showed six protein peaks. Fraction 38 showed phospholipase activity (Fig. 1, Table I). Further purification of fraction 38 by HPLC showed a major sharp peak followed by a small peak at 280 nm, with retention time of 11.8 and 13.7 min, respectively (Fig. 1, inset A).

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Fig. 1

Purification of PLA₂ from Bungarus fasciatus snake venom by carboxy methyl-ion exchange chromatography. B. fasciatus venom (25 mg) was applied in a column (5.6 × 1.6 cm) packed with CM- cellulose. Elution was done with 0.02 M phosphate buffer and in a stepwise gradient of NaCl (0 – 1 M). = Fraction 38 was collected and used as ‘BF-38’. Inset A: HPLC Chromatogram of Fraction 38 using Protein pack 60 column (7.8 ° 300 mm). The column was run with 50 mM Na-K phosphate buffer containing 0.15 M NaCl (pH 7.2, flow rate 0.8 ml/min). Retention time was 11.8 min.

Table I

Summary of two step purification of Bungarus fasciatus snake venom PLA₂ (BF-38)

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SDS-PAGE of fraction 38 showed two bands corresponding to around 14 and 7 kDa mass in the ratio of 9:1 (densitometric analysis by ImageJ software, data not shown). The exact molecular weight was confirmed using the mass spectroscopy on ESI-MS found to be 13.17 and 7.3 kDa (Fig. 2). The N terminal sequence of the first 11 amino acids of the 13.17 kDa band was found to be NLYQFKNMIQC and first 13 amino acids of the 7.3 kDa band was found to be RKCLTKYSQDNES. The PLA₂ was named as BF-38 (Bungarus fasciatus fraction 38).

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Fig. 2

Determination of molecular weight of BF-38 by electron-spray-ionization mass spectrometry (ESI-MS). A. Mass of the PLA₂ was 13177 Daltons. B. Mass of the 3FTx was 7305 Daltons.

The minimum lethal dose of the PLA₂ was found to be 17.3 mg/kg i.v., in male albino mice. One PLA ₂ unit was found to be 8 μg, the minimum oedema dose (MED) was 6 μg, the minimum plasma recalcification dose (MPRD) was 12 μg and the minimum cardiotoxic dose (MCTD) was 38 μg (Table II). BA (5 mg/ml) neutralized PLA₂ activity up to 2 fold, MED up to 1.5 fold, plasma recalcification up to 1 fold and MCTD 1 fold. AA (5 mg/ ml) neutralized PLA₂ activity up to 2 fold, MED up to 1.5 fold, MPRD 1 fold, but gave no protection against PLA₂ induced cardiotoxicity. SA (5 mg/ml) neutralized the PLA₂ activity up to 1.5 fold, MED up to 1 fold, MPRD up to 1 fold and MCTD up to 1 fold (Table II).

Table II

Neutralization of PLA₂ induced activities by synthetic herbal compounds

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Anti PLA₂ rabbit antiserum developed low titre value (1:32) assessed through haemagglutination assay. The anti PLA₂ rabbit antiserum showed faint precipitant band in immune gel diffusion.

Commercial polyvalent snake venom antiserum neutralized PLA₂ activity up to 1 fold, it gave no protection against MED, MPRD and MCTD (Table III). Anti PLA₂ rabbit antiserum neutralized PLA₂ activity up to 3.5 fold, MED up to 1 fold, MPRD up to 2 fold and MCTD up to 1 fold (Table III).

Table III

Neutralization of PLA₂ induced toxicities by commercial polyvalent anti-snake venom antiserum and anti PLA₂ rabbit antiserum

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Discussion

PLA₂ is one of the most common abundant toxic enzymes present in several snake venoms including kraits. Several natural (aristolochic acid, betulinic acid, etc.)¹⁷ and synthetic compounds (isoxazolines, diazepinones, acenaphthenes, etc.)¹⁸ are responsible for the PLA₂ enzyme inhibition. The antagonists of PLA₂ vary with their origin, and hence generate a poor immune response in the body and are not always effective. The present study was aimed to identify a toxic PLA₂ from the Indian banded krait venom and to neutralize the PLA₂ (BF-38) induced actions through synthetic herbal compounds and anti PLA₂ rabbit antiserum in in vivo and in vitro models.

Previously, eight variants of PLA₂ have been identified from the B. fasciatus venom samples from different regions^3,4 including a homodimeric PLA₂¹⁹. Apparently, different B. fasciatus geographic samples or different batch of the venom may show variations. Earlier, from Kolkata B. fasciatus snake venom (KBF), we have purified seven PLA₂, five three finger toxins (3FTx) from cDNAs⁴. In the present study in BF-38, the PLA₂ was identified from BFV which was combined with 3FTx in the ratio 9:1. Both could be identified as previously found PLA-X and 3FTx-RK, respectively⁴. We failed to separate this small amount of 3FTx combination from the PLA₂. The separation of such multimeric toxins (PLA₂ and 3FTx) is largely unexplored and represents an exciting area of toxin molecular evolutionary research as stated earlier by Possani et al²⁰. They identified similar multimeric ‘Taicatoxin’ from the Oxyuranus scutellatus snake venom, which is composed of an alpha-neurotoxic 3FTx, beta PLA₂ and gamma Kunitz peptide in the ratio 1:1:4. In the present study, we found that BF-38 was lethal (17 mg/kg, i.v, bolus) in male albino mice. The LD₅₀ for similar isoforms (PLA-X-1 and XI-2) from pooled B. fasciatus venom (purchased from Miami Serpentarium Laboratory, USA) was >50 μg/g mice^21,3. The present PLA₂ + 3FTx lethality was more than the other PLA₂ purified from B. fasciatus venom. This unique association between these two toxic molecules (PLA₂ and 3FTx) providing complex lethal features of B. fasciatus envenomation may complicate therapeutic management further.

Krait snakebite management is a problem in South-East Asian countries especially in India, since no specific anti- krait venom antiserum is available. The rural people often use herbal preparations as an antidote²². Alam et al²³ identified 2- hydroxy -4-methoxy benzoic acid from the root extract of Hemidesmus indicus, which neutralized Russells’ viper venom induced lethal and haemorrhagic effects in experimental animals. Alam and Gomes²⁴ and Chatterjee et al¹⁶, also confirmed that some synthetic acids (2-hydroxy-4-methoxy benzoic acid, salicylic acid, p-anisic acid) are beneficial in neutralizing snake venom actions in in vitro and in vivo animal models. Probably due to its low molecular weight, the BF-38 showed poor antigenic property denoted by its low titre (1:32) against anti PLA₂ rabbit antiserum and faint immunogel diffusion bands. In the present study, the fold protection by the three synthetic herbal compounds was in the order BA > AA > SA. However, the folds of protection offered by the synthetic herbal compounds were greater than the commercial polyvalent snake venom antiserum, but it is less effective than anti PLA₂ rabbit antiserum. Combination studies with salicyclic acid, anisic acid and commercial polyvalent snake venom antisera did not produce greater neutralization of PLA₂ induced activities. However, 2-hydroxy-4-methoxy benzoic acid and commercial polyvalent snake venom antiserum produced a marginally by higher fold of protection (data not shown).

Earlier, Alam and Gomes¹¹ suggested that combination therapy (herbs and commercial polyvalent snake venom antiserum) may provide better protection against snake envenomation. The findings of the present study also suggest that in case of banded krait envenomation the combination therapy with the right choice of herbal antagonist and commercial polyvalent snake venom antiserum may provide better management.

Acknowledgment

References