Therapeutic Use of Snake Venom Components: a Voyage from Ancient to Modern India

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Mini-Reviews in Organic Chemistry, 2014, 11, 45-54 45 Therapeutic Use of Snake Venom Components: A Voyage from Ancient to Modern India

Payel Bhattacharjee and Debasish Bhattacharyya*

Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mallick Road, Jadavpur, Kolkata-700032, India

Abstract: It is a time tested phenomenon that in spite of deadly toxins in snake venoms, they are used for curing certain ailments of human suffering. Historically this application appears to co-evolve in many countries where India has significant contribution. Before independence, few British surgeons and naturalists documented the profile of snakes in India and investigated their venoms. With the advancement of technology, it is now conclusively known that venoms are rich source of bioactive compounds that can be used for therapeutic purposes. Many venom components have been purified and their structures at molecular level have been derived. Since overproduction of peptides and proteins are now possible in vitro, venom components are likely to serve as templates or scaffold to provide information for developing drugs of discrete specificity and high potency. Venom components are used as diagnostic tool, e.g. Russell’s viper (Daboia russelli) venom factor V and X activator and ecarin from Saw scaled viper (Echis carinatus) venom for blood clotting test. Components from cobra (Naja naja) venom showed good results as chemotherapeutic agent in the clinical trials. In this review, the trend of investigations on snake venom in India from ancient time to modern period has been presented focus- ing on their therapeutic applications.

Keywords: Zootherapy, snake venom, Ayurveda, toxins, enzymes, non-enzyme toxins, pharmacotherapy.

1. INTRODUCTION venomous species of the families Elapidae and Viperidae. The Eastern part of India has 92 species under 34 genera of Nearly 80% of the world's population from nonaffluent which 15 are venomous [2]. Two such species have been countries depends on plant and animal-based medicines. In illustrated (Fig. ). In the strong belief that snakes are India, for the 65% of the population of 1.22 billion, 1 dangerous and threat to human society, there is little room to traditional medicine is the only source of healthcare. The consider that snakes possess an exceptional resource of healing of human ailments by using therapeutics isolated directly or indirectly from animal is known as zootherapy. In valuable medicines in their venoms. The venoms contain India, 15-20% of the traditional medicine is based on animal- numerous toxins that could be used to develop therapeutic derived substances [1]. It is certain that many animal species agents for the treatment and prevention of human diseases. have become extinct or under red alert because of Local inhabitant of Sierra Madre (California, USA) used to overexploitation. Apart from ecological imbalance and say ‘the more poisonous the animal, the more potent its cruelty involved, these practices are not sustainable. Thus, antipoison’ [3]. There is a similar proverb in India- ‘Poison unless cultivated, zootherapy remains questionable. kills poison’. However, with the advancement of chemical and biological The advancement of modern medicine depends largely sciences, it is now possible to extract the information related on the developments of synthetic organic chemistry where to healing of diseases from animal sources and multiply the the lead molecules originated from natural products. Latent product in vitro. This mini-review aims to decipher such phase for traditional medicine is now rapidly changing due information available from the components of snake venoms to the evolutions of drug-resistant microorganisms, side primarily available in India. effects of modern drugs and emerging diseases where no From ancient time, the remedial system of Ayurvedaa, medicines are available. Scientists are experiencing difficulty has employed various toxic substances like venoms from in discovering new structures, templates and scaffolds in the snake, scorpion, bee etc. India has a rich diversity of about finite world of chemical diversity. A number of synthetic 300 species of snakes of which 67 are front-fanged drugs have adverse and undesirable side effects whereas, natural product derived medicines are achieving impressive success. Bradykinin potentiating peptide (BPP), an inhibitor *Address correspondence to this author at the Division of Structural of angiotensin-converting enzyme (ACE), was first Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, identified from Bothrops jararaca venom in mid 1960s. This Raja S.C. Mallick Road, Jadavpur, Kolkata-700032, India; molecule provided the basis for the designing of the peptide- Tel: +91 33 2499 5764; Fax: +91 33 2773 0284; mimetic life-saving drugs, which has anti-hypertensive E-mail: [email protected] ______property [4]. The researchers, pharmaceutical companies and aAyurveda - Traditional medical system practiced by the Hindu ultimately the global community could assimilate the ancient communities of India. It is based on plant and animal products including knowledge to set their future steps in the discovery of safer, inorganic materials that sometimes involve rigorous processing. cheaper and effective drugs.

Fig. (1). Two deadly snakes of Indian subcontinent. Russell’s viper (Daboia russelli russelli) of Eastern India (left). The identifying features are: head is triangular with a ‘V’-shaped white line meeting at the tip of the snout. The dorsal surface of the body is brown and has three longitudinal rows of circular or oval shaped dark markings. Each spot has an inner black area and an outer white margin. The Indian green tree viper (right). Identifying features are: bright green color at the upper side and light yellowish green color at the ventral surface with a broad ‘V’-shaped head. Photographs were taken at Calcutta Snake Park and Sunderban Reserve forest, West Bengal, India respectively (credit Mr. Jyotirmoy Mitra).

2. HISTORICAL PERSPECTIVE the advice of ‘Rishis’ (sacred men) before the advent of the British people in India during 17th century. Subsequently ‘Agada-tantra’ is one of the eight branches of Ayurveda substantial changes took place in the modes of treatments. that deals with toxicology. Ancient India had the distinction With the advancement of allopathic system, the indigenous of having the advanced center of toxicological and herbal systems of medicine got defame and criticism. In the early research in Taxila (District of Rawalpindi, Punjab province, 20th century, traditional healers organized into professional Pakistan). As par Indian history of ‘Rasa Granthas’ (books associations and endorsed the case for national recognition dealing with mercury and snake venom), Hindu communities and funding. They were finally recognized after India’s were pioneers in the use of snake venom in medicine. independence in 1947, yet not been incorporated into the Earlier, it was known that snake venom at a very low dose national health care system and often termed as ‘comple- applied orally is a very powerful stimulants and if it is mixed mentary’, ‘alternative’ or ‘non-conventional’ medicine [6]. with bile of animals, its action becomes totally different. Few Recently it gained substantial importance in agriculture, important medicines like Suchikabharana, Ardhanarisvara, environmental management, biodiversity conservation and Brihat Suchikabharana, Aghorenrisingharana and Kalanala health care. rasa were prepared from snake venoms. Suchikabharana rasa, a dry mixture of cobra venom (Naja naja) containing metals, nonmetals and animal derived components, was 2.1. Impact and Use of Snake Venom in the Clinical administrated orally in small doses. It was effective in Treatment plague, fever, coma, tuberculosis etc. Ardhanarisvara was Our modern medical symbol of two snakes wrapped processed from Naja naja venom used for the treatment of around a staff, or 'caduceus', comes from ancient Greek obstinate fever. In Charak Samhita, Sushrata and Vagbhata mythology, according to which Asclepius discovered (great three classic doctrines in Ayurveda), the cobra venom medicine by watching as one snake used herbs to save life of has been mentioned for curing Dushyodara and Jalodara another snake. There are different interpretations for using (ascites) and Sannipatik Jwara (a type of fever) respectively. the serpent as a symbol in medicine. Few people believed In the Unanib system of medicine, cobra venom is referred as that casting off the skin by snakes symbolizes rejuvenation. a tonic, aphrodisiac, hepatic stimulant and for revival in Whereas, other thought that the serpent reflects the dual collapsed conditions. Some excellent homeopathy medicines nature of the work of physician in dealing with life and are prepared from venoms of Vipera russelli (Daboia death, sickness and health. In spite of being poisonous, snake russellic), Crotalus atrox, Naja naja and Lachesis muta. derived products are known to have medicinal properties Snake venoms have also been used in allopathic medicine from the time immemorial. The ambiguity of using serpent for more than a century to treat thrombosis, arthritis, cancer, as a symbol also indicates the ambiguity associated with the immune dysfunction, viral infections, delirium, use of drugs, which may be useful or harmful [7]. hallucinations, cholera and melancholia [5]. In this context, tribute should be paid to the remarkable Indians were solely dependent on traditional system of personality Constantin Hering (1800-1880) who collected medicines available from forests and herbal gardens under the venom of the ‘bushmaster’ Lachesis muta (then called Trigonocephalus lachesis) from Surinam (Northern South America, 1827-1833) for his investigations. Together with b Unani - Traditional medical system practiced by the Muslim communities Dr. William Wesselhoeft (1794-1858), he founded the North in South Asian countries. It is based on plant and animal products including inorganic materials that sometimes involve rigorous processing. American Academy of Homeopathic Medicine in Allentown, Pennsylvania (1835) and compiled data on Lachesis muta, cDaboia russelli - Scientific name of Russell’s viper. It is further classified Crotalus horridus, Vipera torva, Vipera redii and Naja naja as Daboia russelli russelli and Daboia russelli siamensis depending on their in a booklet. Hering's work is the main source of knowledge availability in the western and eastern part of the Bay of Bengal respectively. on the use of Lachesis muta and Crotalus horridus venom Therapeutic Use of Snake Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 47 still after 120 years. The medicinal properties of cobra physiological mechanisms by which they elicit their venom were first introduced in Western medicine by Hering pathological consequences. Biochemical characterization and through his book entitled ‘Effects of Snake Poison’ in 1837. separation of venom components was started in the middle In homeopathy, the venom of cobra is used to cure a number of the 20th century in many parts of the world especially in of health disorders which works primarily on the nerves, United States. Many of these laboratories collected venom heart, lungs and the ovary and it is especially prescribed to from South-East Asia for their investigations. Separation treat vascular problems of the heart accompanied by anginal techniques like electrophoresis and liquid chromatography pain. It is an effective medicine for curing migraine, dry have been extensively employed in comparative studies in cough, paralyzing diseases like polio [8]. the 1960s to isolate fractions of venoms. These techniques became more sophisticated with time. In India, purification In Indian subcontinent, Dr. Joseph Fayrer, a medical person first studied the comparative nature, physiological of snake venom components first appeared in 1982 [10]. action and mode of death by Naja naja, Daboia russelli, For the treatment of acute pain, it was practiced by Bungurus cueruleus and Ophiophugus hannah bites. He Indians to use cobra venom in combination with opium. In made contact with snake-catchers, who made the snake to the mid-1930s, David I. Macht pointed out that being more bite through a thin leaf stretched across a mussel shell; the powerful than morphine, cobra venom could be used as poison, like limpid syrup, ran down the grooved tooth into analgesic. The venom was believed to be an aphrodisiac and the shell where it was collected and preserved for used for the treatment of depression, back pain, menstrual experiments. Brunton and Fayrer initiated the comprehensive pain and headaches. The results from clinical trials indicated studies on the mode of action of cobra venom and found that 70% of patients receiving cobra venom as a drug got analogy with curare (arrow poisons). Dr. A.J. Wall (1881) in anti-inflammatory and analgesic benefits. Few cases Calcutta first distinguished that Daboia russelli and Naja published more than 50 years ago reported the use of small naja venoms have haemotoxic and neurotoxic action amounts of cobra venom for the treatment of pain related to respectively. Dr. Victor Ragotzi (1890) first reported that trigeminal neuralgia. Cobra venom has the potential to slow cobra venom impaired respiration by blocking the peripheral down the degeneration of joints as in Rheumatoid Arthritis. junction of somatic motor nerves [9]. But the side effects associated with cobra venom were headache, dry mouth, nausea, vomiting, dizziness, hemiplegia, 3. MODERN PERSPECTIVE palpitations and nystagmus. For its haemostatic property, viper venom was used in hemophilia and during major Patrick Russell (1726-1805), a Scottish surgeon and surgery to stop hemorrhages [11]. At the end of the 20th naturalist, initiated scientific classification of the snakes of century, these natural, highly evolved libraries of bioactive India. He classified 43 snake taxa on the basis of Linnean toxins and poisons gained interest as pharmaco-therapeutic principle. Claude Bernard (1813-1878), the father of modern molecules and in diagnostic applications (Box 1). Physiology and experimental medicine, first realized that the physiologically active components of snake venom that are otherwise toxins might have therapeutic potentials [10]. A 4. RECENT APPROACHES good deal of knowledge has been obtained about the The components of snake venom elicit dramatic syndromes associated with envenoming and poisoning and in physiological responses in animal models even when a little a number of cases, such agents promoted for therapeutic use. amount is injected. For this, pharmaceutical companies have In modern times, poisons and the toxins found in venomous systematically tested venoms for drug discovery [12]. A and poisonous organisms gained attention for research short description of those components is given below: primarily on the understanding of the biochemical and

Box (1). Historical Evidence of the Therapeutic Applications of Snake Venoms.

Mysterious Agari doctors were recruited by Mithradates of Pontus, whose Black Sea Empire challenged Roman power in the first century BC. As the world’s first experimental toxicologist, Mithradates and his international team of investigators sought a universal antidote to neutralize all poisons, by ingesting a cocktail of tiny doses of toxins and antidotes. His regimen calls to mind the principles of immunization. An astonishing medical milestone, carried out by Mithradates’ Scythian doctors, was reported by Appian, a Greek historian of the Mithradatic Wars. Their secret knowledge of venom’s beneficial powers saved Mithradates’ life on the battlefield and anticipated modern scientific discoveries by more than 2,000 years. In 67 BC, Mithradates suffered a grievous sword slash to the thigh. Bleeding profusely, he hovered near death, but the Agari staunched his wound with serpent venom. Mithradates recovered and led his army to victory. This is the first documented account of using the coagulating effects of miniscule amounts of steppe viper venom to stop severe hemorrhage, an exciting discovery made only recently by scientists in the new field of “venomics.” The ancient Scythian healers would not be surprised to learn that crystallized venom of steppe vipers (Vipera ursinii) from their homeland is now a major export to emergency rooms around the world. - Excerpt from [33].

“One of the best accredited doctrines of recent times is that of the unity or solidarity of folk-lore. The collective investigations of historians, ethnologists, archeologists, philologists and sociologists reveal the singular fact that the myths, superstitions, laws and social customs of primitive peoples all over the earth, as well as the cruder ethnic aspects of religions, converge inevitably to a common point of similarity or identity.” ……… “Human races and racial customs have changed as they became more highly specialized. The heart of man remains the same. It follows that, in different aspects of space and time, all phases of folk-medicine and ancient medicines have been essentially alike in tendency, differing only in important details.” Fielding H. Garrison, 1870-1935 [34]. 48 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Bhattacharjee and Bhattacharyya

Fig. (2). Human blood coagulation cascade and the steps in which the snake venom components interfere. RVV-V, Russell’s viper venom factor V activator; RVV-X, Russell’s viper venom factor X activator; SVTLE, snake venom thrombin like enzymes. Activation and inhibition steps are denoted by and respectively. The Roman numerals denote different factors of the blood coagulation cascade.

4.1. Biological Properties and Therapeutic Perspectives adjuvants that are in use. Recently the usefulness of snake venom preparation in the treatment of drug-resistant HIV Snake venoms are a complex mixture of proteins, was demonstrated in clinical practices in Saudi Arabia [13]. peptides, carbohydrates, lipids, metal ions and organic Similar opportunity remains open for Indian snake venoms. compounds that are primarily used for the immobilization and digestion of prey. About 26 groups of enzymes have 4.1.1. Enzymes been identified in snake venoms where enzymes of 10 groups are usually present in single venom. Therapeutic Two classes of proteases are common in majority of the perspectives of whole venom are also considered. The snake venoms that have markedly different specificities and anticarcinogenic activities of Naja kaouthia (Indian biological activities. Serine proteases dominate viperid and monocellate cobra) and Daboia russelli russelli venoms have colubrid venoms but less abundant in elapid venoms. been demonstrated on carcinoma, sarcoma and leukemia Metalloproteases are common in viperid and colubrid venom models. Sub-lethal doses of venoms showed cytotoxicity on as well [14]. Ehrlich ascites carcinoma (EAC) cells in vivo and increased Venom serine proteases regulate the vertebrate the lifespan of EAC mice. These venoms showed potent hemostasis system (Fig. 2). These are employed for testing cytotoxic and apoptogenic effects on human leukemic cells antithrombin III, fibrinogen - its breakdown products and its (U937/K562) [13]. The viper venoms showed potent dysfunctions, prothrombin and dysprothrombinaemiasd, antidepressant activity in animal models. Hydrophidae blood clotting factors V, VII and X, lupus anticoagulants, (Lapenis curtus) venom showed antitumor activity against protein C and its regulatory pathway, von Willebrand factor EAC mice in vivo and HeLa, HepG2 tumor cell lines in (vWF) and related syndromes [15]. An anticoagulant enzyme vitro. Naja naja and Bungurus caeruleus (Banded krait) was purified from Trimeresurus gramineus (green tree viper) venoms are cytotoxic to EAC tumor cells in mice. The venom that induces fibrinolysis without affecting the venom also regulates the superoxide dismutase activity. interaction between thrombin and fibrinogen. Rather it Gomes et al. (2010) reported anti-arthritic property of cobra inhibits activation of prothrombin by binding reversibly to venom as it modulates anti-inflammatory markers. An prothrombin activation factors. A thrombin-like serine Iranian group have prepared chitosan encapsulated Naja naja oxiana (Indian or speckled cobra) venom, a nanomedicine, to dDysprothrombinaemias Dysprothrombinaemia (type II deficiency) in evaluate the antigen delivery potential as an alternative to the which prothrombin activity is reduced but the antigen level remain normal. Therapeutic Use of Snake Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 49 protease, grambin was purified from the venom of daboiatoxin from Daboia russelli russelli were effective Trimeresurus gramineus that showed high homology to against Burkholderia pseudomallei. Separately, it was thrombin-like enzymes. It caused defibrinogenation and showed that the PLA2s from Daboia russelli russelli, Echis antiplatelet activity after intravenous administration to mice carinatus and Naja naja exhibited strong antimicrobial and prolonged the time of platelet-rich thrombus formation. effects against Staphylococcus aureus [17]. Small PLA2s Thus, grambin was proposed for the treatment of venous and (10-17 kDa) from venoms of Naja naja and Daboia russelli arterial thrombosis. The venom of Echis carinatus (saw- showed the potential antibacterial activities against gram scaled viper) contains procoagulants, e.g. ecarin. The negative bacteria Naja naja venom contains two toxic PLA2s activation of prothrombin to -thrombin by ecarin has been of which one showed myotoxicity, neurotoxicity and employed in the Ecarin Clotting Time (ECT) test, which is oedema-inducing effect in mice and cytotoxicity to EAC used to monitor anticoagulation and thrombin inhibition in tumor cells. Three other acidic PLA s from Naja naja venom e 2 venous and arterial thromboembolism . The toxin, VRR-73 are cytotoxic to EAC tumor cells but devoid of lethality, from Daboia russelii russelii, shows fibrinolytic and hemolytic and anticoagulant activities. PLA2 protects human esterolytic activities that are independent of its hemorrhagic primary blood leukocytes from the replication of various activity. Further Daboia russelli russelli venom can be fully macrophage and T cell-tropic HIV strains. PLA2 interacts detoxified by thermal denaturation with reversible with the host cell and blocks the entry of virus. Immunokine, restoration of the fibrinolytic activity. The serine protease an oxidized derivative of alpha-cobra toxin (Naja naja RVV-V from Daboia russelli is factor V (FV) activator of siamensis) inhibit the HIV and Feline immunodeficiency human blood coagulation cascade, which converts the single- virus (FIV) induced infection of lymphocytes. An acidic chain coagulation factor V into a light and a heavy chain PLA2 (EC-I-PLA2) of 16 kDa, purified from the Echis (factor Va, FVa) with higher coagulant activity. This carinatus venom, is non-lethal to mice and devoid of property of RVV-V has been employed for detection of FV neurotoxicity, myotoxicity, anticoagulant activity and level in blood. RVV-V and ecarin are commercially cytotoxicity. But it inhibits ADP, collagen and epinephrine available in the blood coagulation assay kits and marketed induced human platelet aggregation. A potent acidic PLA2 by Abcam, Pentapharm, and Sigma-Aldrich etc. A peptide has been purified from Trimeresurus gramineus venom, toxin ‘Hannahpep’ from Ophiophagus hannah venom which showed inhibitory effect on platelet aggregations shows fibrinolytic activity with the possibility to treat induced by adenosine diphosphate, collagen, sodium thrombosis [15]. arachidonate and ionophore A-23187 in rabbit platelet-rich Viperidae venom metalloproteases are used for lowering plasma. Crotoxin, a cytotoxic PLA2 from Crotalus durissus plasma fibrinogen and dissolving thrombus. RVV-X from terriflcus showed good success in phase 1 clinical trial for Daboia russelli is a P-IV metalloproteinase which activates cancer treatment in Argentina [18]. Similar attempts could be factor X (FX). Physiologically, FXa is the most effective performed with the cytotoxic PLA2s from the snake venom activator of prothrombin. The presence of prothrombinase of Indian origin. complex (FXa, FVa, Ca2+ and a suitable negatively charged phospholipid membrane) accelerates the conversion of L-Amino Acid Oxidase (LAAO), that oxidizes amino prothrombin to thrombin. FX deficiency results in variable acids yielding H2O2, is abundant in venoms. They are bleeding tendency. Thus venom RVV-X is used in multifunctional enzymes having edema-inducing, anti- diagnostics for quantitative conversion of pro-factor X into bacterial, anti-leishmanial, anti-coagulant and anti-HIV factor Xa. A fibrin(ogen)olytic metalloprotease NN-PF3 properties and regulate platelet aggregation, induce apoptosis from Naja naja venom is reported. The smallest etc. The apoptosis-inducing activity was inhibited by H2O2 fibrin(ogen)olytic metalloproteinase reported is Lahirin (6.5 scavengers. LAAOs from Ophiophagus hannah venom kDa) from Naja kaouthia venom. Many metalloproteinase showed reduction in cellular proliferation of murine anticoagulants showed hemorrhagic or hemolytic activities melanoma, fibrosarcoma, colorectal cancer and Chinese as well [16]. hamster ovary cell lines. LAAO from Ophiophagus hannah have antibacterial activity. Cathelicidin, from Ophiophagus 2+ Phospholipase A2 (PLA2) catalyzes Ca dependent hannah showed remarkable antibacterial activity. LAAO hydrolysis of L-phospholipids to produce lysophospholipids characterized from Trimeresurus stejnegeri, Crotalas atrox and fatty acids. Because of their abundance and patho- inhibited infection and replication of HIV [19]. physiological roles, they are well characterized. Two types Acetylcholinesterase (AChE) of snake venoms differs of PLA2s are present in the venom of Asiatic viperidae such as Daboia russelli and Echis carinatus. Asiatic crotalidae from vertebrate AChE. It is common in elapid venoms except Dendroaspis species. As snake venom AChEs have venom contains up to four PLA2 isoforms. The Glu6-PLA2s wide range of activities, it offer a good system for analyzing have antiplatelet activity, the Asn6-PLA2s are neurotoxic the mechanism of peripheral site inhibition. Peripheral and/or myotoxic. Many Arg6-PLA2s are anticoagulant, while anionic site (PAS) of AChE located at the active center the Lys49-PLA2s have myotoxic and edema-inducing activity. The therapeutic potentials of these enzymes are gorge entry encompasses overlapping binding sites for extensively studied in Bothrops venom. Anti-bacterial allosteric activators and inhibitors. However, AChE in venom is nontoxic. Phosphodiesterase which is common in property of Daboia russelli russelli venom PLA2 was characterized against pathogenic bacteria like Pseudechis viperid venoms results in the negative cardiac reactions in australis. Crotoxin B from Crotalus adamanteus and victims, particularly a rapid drop in blood pressure. Phosphatases have been detected in several colubrid venoms. These two enzymes were not well characterized in terms of eThromboembolism - The process by which blood clots (thrombus) forms and circulates through the blood vessels. their therapeutic potentials [20]. 50 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Bhattacharjee and Bhattacharyya

4.1.2. Non-enzymatic Proteins and in the diagnosis of myasthenia gravis. Peptide neurotoxins are used for the identification and characterization of These proteins play significant roles in the prey immobi- membrane bound ion channels and receptors in vertebrate lization. Their characterization led to the development of cells, including human neurons. Kini and his team (2006) powerful research tools, diagnostic techniques and drugs. identified a small, stable cysteine-rich protein, Denmotoxin These include 3FTXs, Kunitz-type serine protease inhibitors, from Boiga dendrophila (mangrove catsnake), which is a sarafotoxins, cysteine-rich secretory protein (CRISP), disin- species-specific toxin. Denmotoxin interacts with mouse tegrins, C-type lectins, waprins, veficolins, and vespryns. muscle 1-nAChRs and serves as a model for Venoms also contain few non-protein toxics lile KC-MMTx understanding the mechanisms of species specificity as well from Ophiophagus hannah. It significantly decreases the as reversible binding of venom neurotoxins. Another - body temperature and offered protection against amphet- neurotoxin, haditoxin from Ophiophagus hannah exhibits amine aggregate toxicity in mice but failed to protect unique structural, functional and pharmacological profile amphetamine stereotypy in rats. It also provides protection with antagonism toward muscle () and neuronal against drug (strychnine, pentylenetetrazole, yohimbine) nAChRs ( , , and ) having highest affinity for - induced convulsions in mice. Brain neurotransmitter levels 7 3 2 4 2 7 nAChRs. It is cited as a key molecule in developing new and mono amine oxidase were altered in mice. Thus, KC- drugs for Alzheimer and Parkinson’s disease, schizophrenia, MMTx is a potent molecule for drug development. A heat nicotine addiction, anxiety and depression [22]. stable 7.2 kDa protein toxin (drCT-I, from Daboia russelli russelli) showed sequence homology with cytotoxins from The cardiotoxins cause cardiac arrest by depolarization Naja naja. This toxin has anticancer activity against EAC and degradation of the plasma membrane of skeletal muscle cells in vivo and human leukemic cells (U937, K562) in cells. Naja naja cardiotoxins are useful probes in cellular vitro. It was discovered that cobra venom factor (CVF) could processes like lipid metabolism and calcium ion regulation. destroy the hemolytic and bactericidal properties of serum. Beta-blockers, which are antagonists of -adrenergic Since it is structurally and functionally analogous to the receptors (-Ars), are the drugs of choice for CVDs and also mammalian serum complement factor C3, it activates and for migraine, tremor, situational anxiety, alcohol with- depletes the mammalian immune-complement system. It is drawal, hyperparathyroidism, glaucoma, portal hypertension, used to study various aspects of the complement system, and and gastrointestinal bleeding. Rajagopalan et al. (2007) is useful as an immunosuppressant in tissue transplantation characterized f-cardiotoxin from Ophiophagus hannah that and in cancer therapy. A humanized chimeric form of CVF causes bradycardia (slow heart rate), which was mediated by was developed, which offered cardioprotective functions in its interaction with -Ars. This protein could serve as vivo in mice. This offers a novel therapeutic agent for the scaffold for designing of highly speciﬁc and effective beta- complement mediated diseases including myocardial blocking peptides of minimal side effects. Warprin isolated infarction. Two proteins Atroporin and Kaotree, were from Naja nigricollis (black-necked spitting cobra) showed isolated from the venoms of Crotalus atrox and Naja significant similarity to that of elafin, which is a proteinase kaouthia respectively, which showed anticancer property on inhibitor with potent antimicrobial activity [23]. various types of cancer cell lines, e.g. breast, colon, liver, Snake venom lectins (Snaclecs, carbohydrate binding ovary etc. Since the combinations of Atroporin and Kaotree proteins) comprise of two subclasses, C-type lectins (CTLs) showed enhanced anticancer effect, Lipps (1999) proposed and C-type lectin related proteins (CLRPs). CTLs are its application in chemotherapy. Atroporin and Kaotree were involved in hemagglutination and platelet aggregation while tested by the National Cancer Institute, Bethesda, MD, USA CLRPs function as anticoagulant, procoagulant and which showed positive results. Trigramin, a peptide from agonist/antagonist of platelet activation. Snake venom CTLs Trimeresurus gramineus venom, inhibits binding of human affect platelets by binding to specific glycoprotein (GP) vWF to the glycoprotein IIb/IIIa complex in thrombin- receptors, thus widely used to investigate platelet function activated platelets in a dose-dependent manner and thereby, and regulation. These therapeutic aspects of CTLs of Indian inhibiting the platelet aggregation [21]. snake venoms are yet to be ascertained. Snake venom disintegrins, a class of non-enzymatic protein which Among the well studied snake venom neurotoxins are the originates from metalloproteases, are used in the diagnosis of -neurotoxins that bind to nicotinic acetylcholine receptors cardiovascular diseases and cancer. Snake venom C-type (nAChRs). They are capable of reversible blocking of neural lectins and disintegrins are being used to study the platelet transmission by competitively binding to the nAChR located physiology and are promising therapeutic agents for the at the postsynaptic membranes of skeletal muscles and routine coagulation tests. Aggrastat (tirobifan) is used as an neurons, preventing neuromuscular transmission and thereby antiplatelet drug (glycoprotein Iib/IIIa inhibitors), which was leading to death by asphyxiation. The post-synaptic neuroto- developed from a compound in the venom of the Echis xins are classified into (a) short neurotoxin, (b) long neuroto- carinatus. Aggrastat (marketed as tirofiban HCL) has been xin, (c) -neurotoxins and (d) other unconventional neuroto- produced that blocks certain blood platelet receptors and xins or weak neurotoxins. Daboia russelii venom contains helps to cure the heart attack patients [24]. Vascular vipoxine, which targets adrenergic receptors while Naja naja endothelial growth factor (VEGF) from Daboia russelli venom neurotoxin activates central cholinergic pathways by have been characterized which showed activity greater than nicotine and nicotinic agonists. A novel neurotoxin, native mammalian VEGFs. They induce hypotension in rats hannalgesic from Ophiophagus hannah, produces analgesia and enhance proliferation of vascular endothelial cells. without causing any neurological or muscular deficit. - VEGFs are important molecule for studying angiogenesis bungarotoxin from Bungarus multicinctus (Taiwan banded and other physiological events under normal and diseased krait) is used to generate acetylcholine receptor antibodies conditions [25]. Therapeutic Use of Snake Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 51

5. BIOACTIVE MOLECULES WITH IMPORTANT effective recovery of the victim with least side effects. THERAPEUTIC POTENCY Enzyme-linked immunosorbent assay (ELISA) based snake venom detection kits are gaining interest worldwide for its Evolution of venoms follows a complex dynamic sensitivity. Snake Venom Detection Kit (SVDK) was process. Recently, Nicholas Casewell, a distinguished developed by Commonwealth Serum Laboratories (CSL) in toxinologist said “The venom gland of snakes appears to be a Australia. It is used for in vitro detection and immunotyping melting pot for evolving new functions for molecules, some of snake venom at the bite site, in urine, blood or other of which are retained in venom for killing prey, while others bodily tissues in snakebites in Australia and Papua New go on to serve new functions in other tissues in the body.” Guinea for the venom of Tiger snake, Brown snake, Black According to him, venom seemed to evolve a lot of new snake, Death Adder snake and Taipan snake. It is a rapid, functions, possibly to overcome resistance in prey [26]. sandwich enzyme immunoassay technique used to immuno- Venom components are attractive models for drug discovery type the venom for application of suitable monovalent for their higher catalytic efficiency, heat stability and antivenom therapy [28]. An optical immunoassay (OIA) kit resistance to proteolysis. Unraveling the repertoire of venom for species identification and venom quantification of four toxins at the genomic, transcriptomic and proteomic levels is common venomous land snakes of South Vietnam required for complete understanding of these molecules as (Trimeresurus albolabris, Calloselasma rhodostoma, Naja biotechnological tool. Identification of a ‘target’ is necessary kaouthia and Ophiophagus hannah) has been developed by along with the structural and functional elucidation of toxins. Gopalkrishnakone and his colleagues (2004) [29]. Similar The role of venom in producing ‘antivenom’ should not attempts have been made in India at the State Inter- be underestimated as it saves the life of a snake bite victim. University Centre for Excellence in Bioinformatics In addition, regional variation of venom compositions and (SIUCEB), University of Kerala, who are involved in the identification of attacking snake are major concerns. Though designing of a biosensor for identification of snake venom India pioneers in the production of Anti-Snake Venom that could help to reduce the mortality rate of snake bite (ASV), it is not adequate as par demand. Besides, the victims [30]. Suitable test kits are lacking in India, due to available polyvalent ASV contain antibody against cobra, narrow focus on regional varieties of snakes and cross- Russell’s viper, common krait and saw scaled viper. The reactivity between venoms. wide variation of snake species across the Indian sub- To limit the problem of cross-reactivity, identification of continent may lead to differences in efficacy of the anti- a protein in venom that is unique of that species is required venom across the region leading to hypersensitive reactions to raise specific antibody against that signature protein. in the victim’s body [27]. Therefore, production of species Therefore, ELISA analysis of the victim’s body fluid against specific antibody is required. This in turn, requires accurate the specific monovalent antibodies should yield one positive identification of the inflicting snake. This would aid in the result leading to the identification of the biting snake. administration of monovalent antivenom for rapid and Success of this method will also depend on the stability of

Table 1. Some Important Therapeutic Molecules from Indian Snake Venoms

Therapeutic Molecules Subgroup Snakes Activity/ Therapeutic Use Group [Ref]

VRR-73 Daboia russelii russelli Fibrinolytic,esterolytic, hemorrhagic. Serine protease RVV-V Daboia russelii Blood coagulation test [15] Ecarin Echis carinatus Ecarin clotting time (ECT) test

RVV-X Daboia russelli Blood coagulation test Metalloprotease [16] NN-PF3 Naja naja Fibrin(ogen)olytic

Potential antibacterial activities against Small PLA (10-17 kDa) 2 Naja naja, Daboia russelli gram negative bacteria Crotoxin B and daboia- PLA2 [17,18] Crotalus adamanteus, Daboia russelli Gram-negative Burkholderia pseudomallei toxin Daboia russelli, Echis carinatus and Naja naja Antimicrobial effects against Antimicrobial protein Staphylococcus aureus

Platelet aggregation regulating, apoptosis LAAO [19] Abundant in all snake venoms inducing as well as anti-bacterial, anticoagulant and anti-HIV effects

Protect against drug induced increase in KC-MMTx Ophiophagus hannah body temperature and convulsion Non enzymatic protein drCT-I Daboia russelli Anticancer activity toxins [21-23] CVF Naja naja Immunosuppressant in tissue transplantation and in cancer therapy Atroporin and Kaotree Crotalus atrox and Naja kaouthia Anticancer property

Non protein toxin [31] OH-NPT1 Ophiophagus hannah Cardiotoxic 52 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Bhattacharjee and Bhattacharyya the ELISA kit in remote areas where conditions for and Ashokan Period), 16-17 April 2002. Indian Veterinary preservation of chemicals are nonexistent. A collaborative Research Institute, Izatnagar, Uttar Pradesh, India; 2002, p. 7. c) research has been proposed from different laboratories of Hayman, M.; Macht, D.I. Clinical and biochemical studies in cobra venom therapy. Med. Rec., 1940, 7, 67-69. Eastern India to make this hypothesis a success. [6] a) Banerji, D. Medical Practice in India and its Sociological It is noteworthy that compared to the information on Implications. Antiseptic, 1962, 59, 125-129. b) Barnes, P.M.; Bloom, B.; Nahin, R.L. 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Stoddart & Co., American Homoeopathic Publishing Society, therapeutic molecules from Indian snake venoms [32]. Philadelphia; 1879, vol. 6, pp. 559-650. b) Hering, C. Appletons' Cyclopædia of American Biography. Ed.; D. Appleton and Company, New York. 1892. c) Reid, P.F. Cobra venom: A review 6. CONCLUSION of the old alternative to opiate analgesics. Alt. Ther. Health Med., 2011, 17, 58-71. In summary, facilities are now available to view the [9] a) Hawgood, B.J. Sir Joseph Fayrer MD FRS (1824-1907) Indian structure and mode of interaction of the toxins at the Medical Service: snakebite and mortality in British India. molecular level. High resolution x-ray analysis of several Toxicon, 1996, 34, 171-182. b) Brunton, T.L.; Fayrer, J. On the venom enzymes of Indian origin are now available [33]. It is nature and physiological action of the poison of Naja expected that similar results will decipher meaningful and tripudians and other Indian venomous snakes. Proc. R Soc. Lond. 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Received: November 30, 2012 Revised: April 12, 2013 Accepted: June 21, 2013