Abstract: Background and Objective: India having a coastline of 7516.6 km2 enjoys a huge marine coastal biodiversity. Despite having a prevalent presence in the mangrove ecosystem scanty reports till date are available regarding composition and action of the venom of this anemone species. An attempt has been made to evaluate the toxicological and pharmacological constituents present in this venom. Materials and Methods: Pre-filtered venom was injected in the sub-plantar region of the rat right hind paw. The paw volumes were measured at different time intervals. Erythrocyte suspension was incubated with venom for different time intervals. Chicken lung, heart and liver tissues were pre-incubated with fixed concentration of the tentacle venom. Crude venom was applied on activated Thin layer chromatography(TLC ) plate and run in isopropanol: 0.1 N HCl (7:3) solvent system. Marked spots (Rf) were calculated and the spots were identified with standard acetylcholine, histamine and 5-HT (5-hydroxy tryptamine). Results: The venom has significant pro-inflammatory activity. In vitro assays signified the strong presence of Autacoids and PLA2 activity. The venom produces copious haemoglobin release with in vitro tissue damaging potential. Conclusion: The current observations elucidate that autacoids and PLA2 are together pharmacologically responsible for the site irritation and inflammation after stinging (envenomation) promoting pathophysiological manifestations. The species is a rich reservoir of biological amines (Acetyl choline and Histamine) and enzyme PLA2 which may serve as potent marine biomedical resources.
INTRODUCTION
The eastern, western and southern coast of peninsular India boasts of a highly productive estuarine ecology. In this deltaic complex, at the apex of Bay of Bengal is the place of India called " Sunderbans" the most renowned and happens to be the world’s largest unique mangrove ecosystem. This largest and complex ecosystem encompasses a wide array of floral and faunal biodiversity thus nurturing one of the most logically productive biome and taxonomically diverse niches for different inhabiting species. Accordingly different surveys time to time have had expressed the presence of at least 16 existing Cnidarian genera and among them the sea anemone species Pracondylactis indicus Dave has been the most prevalent and endemic species found till date1.
Among the venomous invertebrates Cnidarians are well known for their envenomation potentialities which had evolved among the earliest known metazoans discovered from Precambrian fossil records2. This phylum comprises benthic and aquatic animals including hard and soft corals and sea anemones (Class Anthozoa), hydroids and fire corals (Class Hydrozoa), jelly fishes (Class Scyphozoa) and box jelly fishes (Class Cubozoa). Nematocysts are the stinging capsules a unique characteristic feature of Class Anthozoa which is consisted of harpoon-like microscopic structures (Cnida) that sharply penetrates the surface layers of the victims and prays thus delivering the highly toxic mixture known as venom. Nevertheless, a handful number of sea anemone venoms have been investigated and some of them have been subjected to a battery of investigations and found to be acting as blockers of sodium and potassium channels3. Additionally a number of cytolysins had been potently lethal to vertebrates and crustaceans have been elucidated at the level of structural and functional units4,5. There had been a number of research works which have already elucidated that sea anemones are capable to produce at least 4 different groups of diverse peptides in their venoms. Based on their molecular characterization scientist could classify these diversified peptides chiefly into:
| • | 5-8 kilo Dalton (KDa) peptides with antihistaminic activity |
| • | 20 KDa pore-forming peptides inhibited by sphingomyelin |
| • | a putative group of proteins represented at present solely by an 80 kDa cytolysin6 |
With a coastline of 7516.6 km2 India7 occupies a unique position in the scenario of coastal biodiversity. Indian Sunderbans, the world’s largest mangrove ecosystem boasts of a range of floral and faunal assemblages which happened to be unique in their habit, habitat and biological properties. Despite having a prevalent presence in this coastal ecosystem till now only a very few scientific reporting had been done on the Cnidarian sea anemone Paracondylactis indicus Dave7. Skin contacts with the tentacles of this sea anemone (like other Cnidarian animals) caused itching, redness, pain and swelling in the contacted positions8. An adequate detailed knowledge about this venomous species was initiated and accordingly very interesting results have come out when systemic pharmacological studies were conducted.
Nematocysts are stinging capsules, a characteristic feature of Phylum Cnidaria. Nematocysts contain harpoon-like microscopic structures (Cnida) that penetrate the surface layers of victims and deliver a mixture of highly toxic substances i.e., venom. The present report had been the result of an attempt to elucidate the pharmacological manifestations of the active ingredients/compound(s), especially bioactives present in situ the venom of P. indicus (Dave).
Experimental design
Research duration and period: The research was started from February 2010 and completed within August 2012 with a duration of 2.5 years.
Collection of the specimen: Live sea anemones were collected from the coastal regions of West Bengal (Sunderban Mangrove). The tentacles were cut and immediately packed in sterile polythene packets, kept in freezing mixture and transported immediately to the laboratory for further extraction. All the pharmacological and biochemical investigations were carried out at the Molecular Pharmacology Laboratory, Division of Pharmacology, Department of Pharmacy Technology, Jadavpur University, Kolkata, India.
Preparation of the sea anemone venom (tentacle extract): The pre-freezed tentacles were homogenized in chilled phosphate buffer saline (pH-7.2). The extract was pooled, filtered and then centrifuged at 10,000 r.p.m., at 4°C for 30 min. The brownish supernatant was collected and hereafter mentioned as crude venom/tentacle extract (T.E.), kept in small aliquots and preserved at -70°C for future use.
Protein estimation: Protein estimation of the venom was done after the method of Bradford9, using bovine serum albumin (BSA) as standard. All the concentrations of the venom in various experimental models have been expressed in terms of protein equivalent.
Animals used: All pharmacological experiments were conducted using rats of Charles foster strain (each weighing 120-180 (g) and mice of Swiss Albino strain (each weighing 18-22 g). The animals (obtained from the Indian Institute of Chemical Biology (IICB), Kolkata 32) were acclimated to the laboratory environment for seven days. According to the ethical guidelines, the animals were housed in standard polypropylene cages (at 24±2°C ) and provided ad libitum access to food and water. The animals were used after an acclimatization period of at least 10 days in the laboratory environment. Control vehicle or test materials were administered intraperitoneally (i.p.) unless otherwise specified. All the experiments were performed according to the guidelines of the Institutional Animal Ethical Committee (Constituted under the guidelines Committee for the Purpose of Control and Supervision of Experiments on Animals, India). All the chemicals used during the performance of specific pharmacological and biochemical investigation were of AR grade (The standard Macron Fine Chemicals™ grade of analytical reagents).
Pharmacological tests done: The ability of P. indicus T.E. (crude venom) to induce oedema was studied in male Charles Foster rats (150-200 g). Two groups of rats (n = 5) were pre-treated with pre-filtered (0.22 μ, Millipore filter) extract (0.10 mg/paw) in normal saline, which was injected in the subplantar region of the right hind paw. The left paw received an equal volume of sterile saline (control). The volumes of both hind paws were measured using a plethysmometer at different time intervals i.e., 0.5, 1, 2, 3, 4, 5, 6 and 24 h following administration of the crude venom10. Oedema was expressed as the percentage increase in the volume of the treated (right paw) relative to that of the control (left paw), at each time interval, using the following formula:
| V 1 | = | Final volume displaced by the right paw |
| V2 | = | Initial volume displaced by the right paw |
| V3 | = | Final volume displaced by the left paw |
| V4 | = | Initial volume displaced by the left paw |
For in vitro tissue damaging activity fresh chicken liver, heart and lungs were washed with 0.9% NaCl solution, cut into small pieces of uniform size and dried with tissue paper. The tissues weighing 300±10 mg were pre-incubated (at 37°C) with 1 mL of 200 mM potassium-phosphate buffer (pH-7.4) for 45 min. The tissues were then washed twice with the same buffer and incubated (at 37°C) with fixed concentration of the tentacle extract in a volume of 3 ml (in 200 mM K-phosphate buffer, pH-7.4) for 5h. After incubation, the reaction mixtures were centrifuged for 5 min at 3000 r.p.m. and the absorbance of the supernatant was read at 540 nm, using a Hitachi U-2000 spectrophotometer. The percentage of haemoglobin released was calculated with respect to tissue samples incubated with 0.1% Triton X100 solution (considered to produce 100% lysis in the standard sets)11.
Biochemical assays: Indirect hemolytic activity of the tentacle extract was assayed using the method12, requiring the presence of lecithin to form lysolecithin and fatty acids necessary for the induction of haemolysis. The absorbance of distilled water induced spontaneous haemolysis was taken as 100% and the absorbance of the test samples were converted to corresponding haemolysis percentage.
Thin layer chromatography: Crude venom (1 mg mL–1) was applied on activated thin layer chromatography (TLC) plate coated with silica gel. Standard acetylcholine (Ach), histamine and 5-hydroxytryptamine (5-HT) were also applied and the plates were run in isopropanol: 0.1 N HCl (7:3) solvent system. After the run was over, the movement of the solvent front was marked. Spots were then developed first with iodine vapour and finally with 0.1% ninhydrin in acetone. Spots were marked, Retention factor (Rf) values were calculated out and the colour of the spots were also noted.
Statistical analysis: Results were expressed as Mean±SE (n = 5). Statistical analyses were performed with one way analysis of variance (ANOVA) followed by Student’s t-test or Post-hoc Dunnet’s test, wherever applicable. The p<0.05 was considered to be statistically significant.
RESULTS
Effect of the crude venom on pro-inflammatory activity in rat hind paw: The P. indicus T.E. (0.10 mg) when applied into the rat paw produced severe swelling in comparison to the control. It was observed that the P. indicus T.E exhibited significant pro-inflammatory activity, which was found to be maximum at the 4th h after administration of the extract and thereafter it was found to decrease steadily (Fig. 1).
Effect of the crude venom on in vitro tissue damaging activity of animal tissues: The tissue damaging activity of the crude venom was found to vary with the tissue samples used in this experiment, where maximum damage in terms of haemoglobin release was observed in the chicken lung tissue strips followed by liver and heart (Fig. 2).
| Fig. 1: | Time course of rat paw oedema produced by P. indicus tentacle extract (0.10 mg/paw). Values are expressed as mean percentage increase in paw volume±S.E., (n = 5). The p-value (vs. respective control) by student t-test. ***p<0.001 |
| Fig. 2: | Effect of P. indicus tentacle extract on different tissue samples. In vitro tissue damaging activity was expressed as mean percentage hemoglobin release±S.E. (n = 5). The p-value (vs. respective control) by students t-test. ***p<0.001 |
Indirect haemolysis and Phospholipase A2 (PLA2)-like activity of the crude venom on washed rat erythrocytes: Different concentrations of the T.E. were incubated with the 1% erythrocyte suspension and lecithin. From the dose response curve of indirect hemolysis activity it was found that nearly 245 μg protein of the crude venom (T.E.) brought about 50% lysis of the RBC suspension (in vitro) at 37°C after 30 min of incubation. So the 1 HD50 (hemolytic dose unit) unit was found to be 245 μg protein/mL.
| Fig. 3: | Indirect hemolytic activity of P. indicus tentacle extract in presence of lecithin |
| Table 1: | Thin layer chromatographic separation and respective Rf values against colour developed for spottings of the standards and P. indicus crude venom |
| TLC: Thin layer chromatographic | |
Now one unit (U) enzyme activity (Phospholipase factor) was defined as the amount of enzyme required to bring about 1% erythrocyte suspension lysis in 1 min. So specific unit is 1.66 U/0.245 mg protein/mL = 6.77 U of PLA2/mg protein/mL = 6.77 U of PLA2/HD50 dose (Fig. 3).
Presence of autacoids in the crude venom by TLC: The immediate symptoms like itching, swelling, redness, oedema are quite common with P. indicus contacts. It may be assumed that the direct presence of histamine, serotonin and acetylcholine are quite likely in the extract. On the TLC plate, the crude venom produced 4 distinct spots (S1, S2, S3 and S4). The S1 spot was similar in color and Rf value to that of Ach and S2 and S4 had similarity in color and Rf values to that of histamine and 5-HT (Table 1).
DISCUSSION
The bioactive components of marine cnidarians had been reported to contain different types of bioactive components namely, neurotoxins, hemolysins, cardiotoxins, which would have been widely distributed in sea anemones and about 32 species of sea anemones secrete these compound in their deadly venoms5,6 . The biological activities in addition to the pharmacology of the active ingredients of the sea anemone species Paracondylactis indicus (Dave) (reported from Sunderbans) had been able to catch the attention and thereby being documented, although the species happened to be an endemic one found in abundance in this region, quite lately1,8.
From the results it was observed that P. indicus crude venom demonstrated both direct and indirect haemolytic activity. The direct haemolytic activity (HD50 210 μg mL–1) was found to be more potent8 than that of indirect hemolysis (IHD50 245 μg mL–1). Moreover the both direct and indirect hemolytic property was effectively inhibited by different blockers (data not shown). It was also found that the venom could produce 100% lysis of the washed rat erythrocytes within 4 h of incubation. It had been known that direct hemolysis activity of different animal venoms brought forth via formation of membrane pores or through direct membrane lipid peroxidation8. It had also been well known that PLA2 (phospholipases) present in the animal venoms could impart their action on the cell membrane via indirect hemolysis activity12-14. However, majority of the PLA2 was also known to be lacking the property of direct hemolysis12. Therefore at present this finding strongly indicated that the P. indicus crude venom contained both direct and indirect haemolytic activities and the direct hemolysis was much stronger in comparison to the presence of some weak PLA2-like enzymatic components. It could be proposed that the crude venom was able to impart the indirect haemolytic activity in presence of lysolecithin in this experimental model.
Furthermore, from earlier reported studies it was observed that this venom exhibited moderate proteolytic activity8. Since proteases had been having a role to contribute greatly towards excessive protein degradation in intact cell membranes13, it might be corroborated that the observed proteolytic activity could be another chief contributing factor, amongst the reserve bioactive metabolites, behind the direct tissue damaging action of this very venom. The venom was found to release copious amount of haemoglobin after incubation with chicken lung tissue. NK-PLA2 isolated from Indian monocled cobra (Naja kauthia) venom had also been found to produce the same effect in chicken heart, lung and liver tissue strips11. Therefore this tissue rupturing and haemoglobin releasing activity of the venom could be attributed to the disruption of blood capillaries, which had been the primary factor behind the release of haemoglobin. The rupturing and disruption of small blood capillaries could be by the proteases present in the venom along with the presence of PLA2-like component. However the rate and amount of haemoglobin release was found to be varying and tissue specific (highest in chicken lungs followed by liver and heart tissue) which could be due to the variable amount of tissue specific phospholipids/cholesterol ratio along with vitamin E concentration of the specific tissues11,13. Anemone venom injection from Anthopleurea asiatica (collected from Mumbai Coast, India) had been reported to produce necrosis in mice brain followed by hemolysis in heart, occlusion with hemolysed blood in kidneys and necrosis coupled with hemolysis in liver tissue15. Reports had been available regarding the toxicological activities of Indian sea anemones viz., Heteractis magnifica, Stichodactyla haddoni, Anthopleura middori and Paracondylactis sinensis, all collected from Gulf of Munnar, Southeast India and the researchers had found that both the crude and fractionated proteins obtained so far had been neurotoxic, cardiotoxic, nephrotoxic and hepatotoxic to experimental mice16. Therefore it might be deciphered that anemone venoms could impart highly tissue specific damaging activity in vertebrate species17.
It was observed that the venom produced severe swelling in rat hind paw within 1 h following sub-plantar injection and the oedema was found to persist for several hours. These results confirmed the outcome with the available reports from recent times. Toxins extracted from four different species of sea anemones (viz., Paracondylactis indicus, Paracondylactis sinensis, Heteractis magnifica and Stichodactyla haddoni), had been found to be exhibit potent pro-inflammatory activity in mice foot pad. Crude extract from H. Magnifica found to produce 200% ER (Edema ratio) followed by P. indicus (ER 160%) and ER of 120% was observed in both P. sinensis and S. haddoni16. It was well established that paw oedema formation had been due to the involvement of mediators like histamine, serotonin and prostaglandins18 and it was also well documented that such oedemogenic activity could also be attributed to increase in capillary permeability followed by exudation of mast cells and proteins18. Therefore, the observed pro-inflammatory activity of the venom could be mediated following the release of the lipid mediators of inflammation. Since a number of sea anemone species being already known to contain phospholipases (particularly PLA2), therefore the possibility of breakdown of membrane phospholipids (triggering arachidonic acid breakdown) following extract injection (locally in the paw) could be a major reason for the oedemogenic activity19,20. Excess release of PLA2 promoted vascular inflammation and associated pharmacological manifestations21. It could be noteworthy to mention that the Cnidaria Adamsia palliata and Condylactis gigantea had been reported to secrete PLA2-rich toxin through the nematocyst causing inflammatory responses21,22. PLA2 toxin catalyzed the hydrolyis of 2-acyl ester bonds of 3-sn-phospholipids producing arachidonic acid and lysophospholipids23. This released arachidonic acid failed to form bi-layers leading to changes in lipid membrane conformation and ultimately blocking the release of neurotransmitters. In that altered physiological state inflammation and pain occurred at the site of contact24. On the back drop, released arachidonic acid prompted LOX (lipoxygenase) and COX (Cyclooxygenase) which, through some intermediates like PEG2 (prostaglandin E2), GPCR (G-protein coupled receptor), PLC (Phospholipase C) and HPETE (Hydroperoxyeicosatetraenoic acid), transformed into PIP2 (Phosphatidylinositol bisphosphate) that ultimately blocks the TRPV1 (Transient receptor potential vanilloid receptor 1) channel25, PKC (Protein Kinase C) and PKA (Protein Kinase A) are helpers in this process.
The earlier report7 had shown that various PLA2 inhibitors (Zn2+ or p-bromophenacyl bromide, a PLA2 inhibitor) had strongly inhibited the indirect haemolytic activity and EDTA (a protease inhibitor) had markedly attenuated the membranolytic activity of the crude venom. Therefore, it could be strongly promoted that the augmentation of this strong pro-inflammatory activity of the crude venom could be a combined effect of strong proteases, PLA2-like enzymatic components and different autacoids present in varying proportions, acting directly on the tissue lipid membrane surfaces, via release of arachidonic acid, which in turn induced cyclooxygenase forming prostanoids including Prostaglandin E2. Prostaglandin E2 had been an inflammatory mediator26, which increased vascular permeability and vasodilation, thereby rupturing of thin capillary beds triggering lipid mediators for acute inflammation. The venom happened to be the storehouse of different autacoids (Acetylcholine and histamine as revealed by TLC, Table 1) which pretended to be pivotal role playing/contributing factors behind the pronounced pro-inflammatory activity. The presence of these autacoids especially, histamine could play a significant role to play during the systemic envenomation resulting inflammation upon contact with the crude venom bringing forth the issues of redness, itching and swelling on exposed dermal surface. Additionally, Prostaglandin E2 upon releasing increased vascular permeability, triggered vasodilatation local itching and inflammation22-26. Therefore, on the basis of the present investigations, Paracondylactis indicus (Dave) was found to contain a variety of interesting biological activities and compounds, therefore warranted further molecular evaluation for the probable mechanism of action behind this pro-inflammatory activity.
CONCLUSION
Throughout the world, the new trend in drug discovery from marine natural resources had emphasized judicious investigations and exploitation as " possible future drugs of 21st century" for human welfare. Thus the need of the hour is to explore all marine life as possible reservoir of novel chemical entities. Sea anemones, like other coelenterates are the warehouse of numerous biologically active polypeptides and proteins including neurotoxins, cytolysins, phospholipases and proteinase inhibitors stored in their nematocysts. These peptide toxins are responsible for a wide array of harmful effects such as cardiotoxicity, dermatitis, local itching, swelling, erythema, pain and necrosis. The present study and data confirmed and extended the knowledge base of these above-mentioned findings and presence of PLA2-like enzymatic activity had been established. The venom was found to produce copious haemoglobin release from chicken lung strips followed by liver and heart tissues. The venom brought forth strong pro-inflammatory activity in experimental rat, immediately following injection at rat hind paw which was found to maximum at the 4th h after injection. The crude venom contained substantive amount of free histamine, serotonin and acetylcholine, which could serve as therapeutic resource for future experimental tool, especially to elucidate the molecular pharmacology of TRPV1 receptor kinetics.
SIGNIFICANCE STATEMENTS
Just like other sea anemone species already reported from different parts of coastal India, P. indicus is an endemic venomous species potentially capable to produce different toxicological manifestations in experimental assays. Paracondylactis indicus venom might make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of over activity of this receptor during a number of pathological processes especially in elucidation of mechanism of pain sensation during and after envenomation. In addition to its haemolytic, pro-inflammatory and histopathological symptoms, this very species demands the attention of the scientific community to be exploited as a reservoir of bioactive components/leads in addition to the presence of autacoids (free histamine, serotonin and acetylcholine), which in near future might serve as substantive potential bio-resources as intermediary experimental prop/aid in complex investigational assay models to elucidate the molecular pharmacology and toxicology of envenomation.
ACKNOWLEDGMENT
This study is dedicated in memory of our two beloved mentors and guides Late Profs. Dr. Asis Nag Choudhury and Dr. Tuhinadri Sen, whose constant support and unremitting encouragement paved our way to explore the wonderful world of Pharmacology. We sincerely and gratefully acknowledge the financial assistance accorded by the UGC, UPE-II and DST-SERB,(SERB/F/5359/2013-14 dt 19-11-13) Govt. of India.