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ORIGINAL ARTICLE
Year : 2012  |  Volume : 136  |  Issue : 1  |  Page : 40-45

Inhibition of toxic actions of phospholipase A 2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds


1 Department of Physiology, University of Calcutta, Kolkata, India
2 Drug Development Diagnostics & Biotechnology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India
3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Date of Submission28-Sep-2010
Date of Web Publication10-Aug-2012

Correspondence Address:
Antony Gomes
Scientist, Drug Development Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032
India
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Source of Support: None, Conflict of Interest: None


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   Abstract 

Background & objectives: Phospholipase A 2 (PLA 2 ) is one of the major constituents of krait venom associated with several pathophysiological actions like myotoxicity, cardiotoxicity, neurotoxicity, etc. As there was no specific antiserum available against Bungarus fasciatus venom, this study was done with synthetic herbal compounds, anti PLA 2 rabbit antiserum and commercial polyvalent snake venom antiserum to neutralize the PLA 2 induced toxicities in experimental models.
Methods: B. fasciatus venom phospholipase A 2 fraction 38 (BF-38) was isolated by ion exchange chromatography, molecular weight was determined by mass spectrometry and its N terminal amino acid sequence was identified. Monospecific rabbit antiserum was raised against the PLA 2 in presence of Freund complete adjuvant. The neutralization of PLA 2 induced toxicities was done in in vitro and in in vivo models using synthetic herbal compounds, anti PLA 2 rabbit antiserum and commercial polyvalent snake venom antiserum.
Results: A toxic PLA 2 (BF-38) was purified from the B. fasciatus venom by CM-cellulose and HPLC, of 13.17 kDa and a minor band of 7.3 kDa using ESI-MS. The 13.17 kDa PLA 2 sequence was NLYQFKNMIQC. The 7.3 kDa toxin sequence was RKCLTKYSQDNES and was found to be <10 per cent w/w. Anti PLA 2 rabbit antiserum produced faint precipitant band in immunogel diffusion and showed low titre value. The commercial polyvalent snake venom antiserum, anti PLA 2 rabbit antiserum and the synthetic herbal compounds neutralized the PLA 2 induced toxicities at different intensities.
Interpretation & conclusions: Our results suggested that synthetic herbal compound (BA) along with antiserum might provide effective protection against PLA 2 induced toxicities of B. fasciatus venom.

Keywords: Banded krait - Bungarus fasciatus - herbal antagonists - phospholipase A 2


How to cite this article:
Gomes A, Bhattacharya S, Mukherjee S, Ih, Gomes A. Inhibition of toxic actions of phospholipase A 2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds. Indian J Med Res 2012;136:40-5

How to cite this URL:
Gomes A, Bhattacharya S, Mukherjee S, Ih, Gomes A. Inhibition of toxic actions of phospholipase A 2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds. Indian J Med Res [serial online] 2012 [cited 2019 Aug 26];136:40-5. Available from: http://www.ijmr.org.in/text.asp?2012/136/1/40/99560

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 A 2 (PLA 2 s), which constitutes about 65 per cent of the total venom proteins [3],[4] . The PLA 2 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 PLA 2 s exert their pathophysiological actions such as myotoxicity, cardiotoxicity, alteration in blood pressure, oedema, haemolytic activity, platelet aggregation, and neurotoxicity, which are often fatal [6] .

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 envenomation [7] . In view of the limited information available, this study was undertaken to purify a PLA 2 from the eastern Indian B. fasciatus venom and to neutralize its toxicity and biological activity with synthetic herbal compounds, anti PLA 2 rabbit antiserum and commercial polyvalent snake venom antiserum in in vitro and in vivo animal models.


   Material & Methods Top


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 x 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 analysis [8] and PLA 2 activity assay [9] .

High performance liquid chromatography (HPLC): The PLA 2 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 x 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 PLA 2: The molecular weight of the PLA 2 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 PLA 2 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 PLA 2 into the tail vein of mice (20 ± 2 g, n=6), observed for 24 h and mortality was recorded [10] .

Hyperimmunization of rabbit: Antiserum was prepared against the PLA 2 by hyperimmunization of male albino New Zealand rabbit (1.5 ± 0.2 kg, n = 4). The PLA 2 (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 PLA 2 (without FCA) for another three weeks [11] .

Immunogel diffusion and indirect haemagglutination titre: Immunogel diffusion was done [12] . The commercial polyvalent snake venom antiserum (10 μl)/anti PLA 2 rabbit antiserum (20 μl) was applied in central well and the peripheral wells contained crude B. fasciatus venom (10 μl)/PLA 2 (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 assay [13] .

Neutralization technique: PLA 2 activity was determined by egg yolk coagulation time [9] . One PLA 2 unit was defined as the minimum amount of PLA 2 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 PLA 2 with a fixed amount of anti-PLA 2 rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C x 1 h and the clotting time was recorded [14] .

The minimum oedema dose (MED) of the PLA 2 was assessed by injecting 0.01 ml of different concentrations of PLA 2 , sub-plantar (s.p) in the paw of male albino mice and the oedema was recorded within 2 h of observation [15] . One MED unit was defined as the minimum amount of PLA 2 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 PLA 2 with a fixed amount anti PLA 2 rabbit antiserum / commercial polyvalent snake venom antiserum (10 mg/ ml) / synthetic herbal compounds (5 mg/ml) incubating at 37°C x 1 h and the decrease in inflammation was recorded by digital calipers (Mitutoyo, Japan) [14] .

Plasma recalcification time using goat plasma was determined [10] . One minimum plasma recalcification dose (MPRD) unit was defined as the minimum amount of PLA 2 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 PLA 2 with a fixed amount of anti PLA 2 rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C x 1 h and the clotting time was recorded [14] .

One minimum cardiotoxic dose (MCTD) was defined as the minimum amount of PLA 2 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 PLA 2 with a fixed amount of anti PLA 2 rabbit antiserum/commercial polyvalent snake venom antiserum (10 mg/ ml)/synthetic herbal compounds (5 mg/ml) incubating at 37°C x 1 h to study the inhibition of the cardiac arrhythmia [16] .

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


   Results Top


On ion exchange chromatography, B. fasciatus venom showed six protein peaks. Fraction 38 showed phospholipase activity [Figure 1], [Table 1]. 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 ([Figure 1], inset A).
Figure 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 x 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 x 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.

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Table 1: 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 [Figure 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 PLA 2 was named as BF-38 (Bungarus fasciatus fraction 38).
Figure 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.

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The minimum lethal dose of the PLA 2 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 2]. BA (5 mg/ml) neutralized PLA 2 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 PLA 2 activity up to 2 fold, MED up to 1.5 fold, MPRD 1 fold, but gave no protection against PLA 2 induced cardiotoxicity. SA (5 mg/ml) neutralized the PLA 2 activity up to 1.5 fold, MED up to 1 fold, MPRD up to 1 fold and MCTD up to 1 fold [Table 2].
Table 2: Neutralization of PLA2 induced activities by synthetic herbal compounds

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Anti PLA 2 rabbit antiserum developed low titre value (1:32) assessed through haemagglutination assay. The anti PLA 2 rabbit antiserum showed faint precipitant band in immune gel diffusion.

Commercial polyvalent snake venom antiserum neutralized PLA 2 activity up to 1 fold, it gave no protection against MED, MPRD and MCTD [Table 3]. Anti PLA 2 rabbit antiserum neutralized PLA 2 activity up to 3.5 fold, MED up to 1 fold, MPRD up to 2 fold and MCTD up to 1 fold [Table 3].
Table 3: Neutralization of PLA2 induced toxicities by commercial polyvalent anti-snake venom antiserum and anti PLA2 rabbit antiserum

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   Discussion Top


PLA 2 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 PLA 2 enzyme inhibition. The antagonists of PLA 2 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 PLA 2 from the Indian banded krait venom and to neutralize the PLA 2 (BF-38) induced actions through synthetic herbal compounds and anti PLA 2 rabbit antiserum in in vivo and in vitro models.

Previously, eight variants of PLA 2 have been identified from the B. fasciatus venom samples from different regions [3],[4] including a homodimeric PLA 2 [19] . 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 PLA 2 , five three finger toxins (3FTx) from cDNAs [4] . In the present study in BF-38, the PLA 2 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, respectively [4] . We failed to separate this small amount of 3FTx combination from the PLA 2 . The separation of such multimeric toxins (PLA 2 and 3FTx) is largely unexplored and represents an exciting area of toxin molecular evolutionary research as stated earlier by Possani et al[20] . They identified similar multimeric 'Taicatoxin' from the Oxyuranus scutellatus snake venom, which is composed of an alpha-neurotoxic 3FTx, beta PLA 2 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 PLA 2 + 3FTx lethality was more than the other PLA 2 purified from B. fasciatus venom. This unique association between these two toxic molecules (PLA 2 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 antidote [22] . Alam et al[23] 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 Gomes [24] and Chatterjee et al[16] , 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 PLA 2 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 PLA 2 rabbit antiserum. Combination studies with salicyclic acid, anisic acid and commercial polyvalent snake venom antisera did not produce greater neutralization of PLA 2 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 Gomes [11] 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 Top


Authors thank the Council of Scientific & Industrial Research (CSIR), New Delhi for financial support.

 
   References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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