Herbs and herbal constituents active against snake bite

Indian Journal of Experimental Biology Vol. 48, September 2010, pp. 865-878 Review Article Herbs and herbal constituents active against snake bite Antony Gomes1*, Rinku Das1, Sumana Sarkhel1, Roshnara Mishra1, Sanghamitra Mukherjee1, Shamik Bhattacharya2 & Aparna Gomes2 1 Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92 A P. C. Road, Kolkata 700 009, India 2 Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India Snake bite, a major socio-medical problem of south east asian countries is still depending on the usage of antisera as the one and only source of treatment, which has its own limitations. In India, mostly in rural areas, health centres are inadequate and the snake bite victims mostly depend on traditional healers and herbal antidotes, as an alternative treatment. The present review has been focussed on the varied folk and traditional herbs and their antisnake venom compounds, which might be a stepping stone in establishing the future therapy against snake bite treatment and management. Keywords: Alternative medicines, Herbal compound, Snake bite, Snake bite treatment, Snake venom Introduction Snake bite, till date remains a public health hazard in tropical countries, especially in India. Accurate records to determine the exact epidemiology or even mortality in snake envenomation cases are inadequately available1. Hospital records fall far short of the actual number owing to the dependence on traditional healers and practitioners. In India, on an average 2,50,000 snake bites are recorded in a single year. There are 52 poisonous species of snakes available in India, of which majority of the bites and mortality are attributed to species like Ophiophagus hannah (king cobra), Naja naja (spectacled cobra), Daboia russelli (Russell's viper), Bungarus caeruleus (common krait) and Echis carinatus (saw-scaled viper). Snake venom, the most complex of all poisons is a mixture of enzymatic and non-enzymatic toxic compounds as well as other non-toxic proteins, non-proteins, including carbohydrates and metals that is stored in poison glands. There are more than twenty different enzymes including procoagulant enzymes, haemorrhagins, cytolytic or necrotic toxins, presynaptic and post-synaptic neurotoxins, phospholipases A2, B, C, D, hydrolases, phosphatases (acid and alkaline), proteases, esterases, acetylcholinesterase, —————— * Correspondent author Fax: 91 033 2351 9755 E-mail: [email protected]; [email protected] Telephone: 91 033 2350 8386 (Extn. 229) 094331 39031 (M) transaminase, hyaluronidase, phosphodiesterase, nucleotidase, ATPase and nucleosidases (DNA and RNA). Very few non-protein components have been isolated from snake venom, an anticonvulsant non-protein cardiotoxin is among them2. The pathophysiologic basis for morbidity and mortality is the disruption of normal cellular functions by these enzymes and toxins. The treatment for snake bite is as variable as the bite itself. The only available treatment is the usage of antivenom against snake bite. The first antivenom (called an anti-ophidic serum) was developed by Albert Calmette, a French scientist of the Pasteur Institute in 1895, against the Indian Cobra (Naja naja). Antivenom binds to and neutralizes the venom, stopping further damage, but do not reverses the damage already done. Some individuals may react to the antivenom with an immediate hypersensitivity reaction3. Other alternative treatment involves the usage of folk and traditional medicines in snake bites. Medicinal herbs are the local heritage with global importance. Various plants have been used against snake bite, in folk and traditional medicine. In Ayurvedic system of medicine different plants and their compounds are reported to possess antisnake venom activity. But they also possess their individual toxicities and most of the folk medicinal plants have no scientific validation. This review is an attempt to focus on the antivenom treatment of snake bite, its limitations, herbal antagonists and herbal constituents active against snake bite and its future. 866 INDIAN J EXP BIOL, SEPTEMBER 2010 Antivenom treatment of snake envenomation and its limitations The most common and effective method of treating snake bite victims is through antivenom, a serum made from the venom of the snake4. In India, polyvalent antivenom is prepared by Central Research Institute, Kasauli, Simla, and the Haffkine Corporation, Parel, Mumbai. The WHO has designated the Liverpool School of Tropical Medicine as the international collaborating centre for antivenom production and/or testing4. Antivenoms, in most countries are costly and may be in limited supply. Antivenoms for therapeutic use are often preserved as freeze-dried ampoules, but some are available only in liquid form and must be kept refrigerated. The majority of snake antivenoms are administered intravenously. The intramuscular route has been questioned in some situations as they are not uniformly effective. Antivenom should be given as quickly as possible so that the venom’s side effects can be managed. Antivenom should be given only if the range of specificity is stated which includes the species known or thought to have been responsible for the bite. Liquid antivenom that turned opaque should not be used because precipitation of protein indicates loss of activity which is directly proportional to increased risk of reactions. In India, other centres which are involved in manufacturing of antivenom are Bharat Serums and Vaccines Ltd., Mumbai, Serum Institute, Pune, King Institute, Chennai, Vins Bio-products Ltd.; and Biological ‘E’ Ltd., Hyderabad, etc. Antivenom serum (AVS) manufacturers recommend skin sensitivity testing to predict adverse AVS reactions. But, the usefulness of skin testing is doubtful, as skin testing carries the risk of inducing an acute reaction and delays the initiation of AVS administration. Herbal antidotes active against snake bite In almost all parts of the world, where venomous snakes occur, numerous plant species are used as folk medicine to treat snake bite. Generally an aqueous, methanol or ethanol extract is prepared out of the plant parts. Topical application of the plant or its sap onto the bitten area, chewing leaves or barks or drinking plant extracts or decoctions or injecting the extrcts are some procedures intended to counteract snake venom activity. The roots of the plant Ophiorrhiza mungo, Peristrophe bicalyculata, Gymnema sylvestre Gloriosa superba, Cucumis colosynthis, Alangium salvifolium, leaves of Enicostemma axillare, Calycopteris floribunda, Calotropis gigantea, Aristolochia indica are used in Ayurvedic medicine. Ayurveda states the usage of specific plants against specific snake bites, e.g. root extract of Abrus precatorius is used against krait bite, leaf paste of Azadirachta indica with rock salt is used against viper bites. Leaves and bark of Casearia sylvestris, (guacotonga) are used as a standard Ayurvedic drug to treat snake bite in Columbia, India, etc. Aristolochia indica is used as a decoction for snake bite. Seeds of Psoralea corylifolia are used both in Ayurveda and Siddha against snake bite. Origanum dictamnus juice is consumed in wine to cure snake bite. Tea made from the leaves of Cecropia peltata is used as a cure for a wide variety of ailments including snake bite. Achyranthes aspera, is used in treatment of bleeding, renal complications, scorpion bite, snake bite, etc. The first scientific investigation regarding the herbal antidotes was from Knowles5. He screened several plants/plant constituents, used by local healers, but failed to report their efficacies against snake envenomation, either due to sublethal dose of venom or non lethal dose. Later, Mhaskar and Caius6 challenged the effectiveness of herbal antidotes by screening 314 plants and 184 combinations against venom induced lethality, ignoring the systemic changes induced by snake venom6. This pioneering theory was later contradicted by various reports on effectiveness of herbal antidotes against systemic toxicities as well as lethality. The ether soluble fraction of Aristolochia species, inactivates Naja naja venom and reduces haemorrhage caused by Trimeresurus flavoviridis and Vipera russellii venoms7,8. Eclipta prostrata L. (Asteraceae) is used as an anti-venom against snake bite in China and in Brazil. Schumanniophyton magnificum, Eclipta prostrata or Aristolochia shimadai, have the capacity to inhibit phospholipase A2, other enzymes (e.g. ATPase) along with other physiological and biochemical properties (such as effects on uterine tone or the protection of mitochondrial membranes). Antihaemorrhagic effect of persimmon tannin from Diospyros kaki is also well known. The survival time was prolonged after pretreatment with extracts of Diodia scandens and Andrographis paniculata9. GOMES et al.: HERBAL ANTIDOTE & SNAKE BITE TREATMENT Rhizomes of Curcuma Sp. inactivated postsynaptic neurotoxin of the Thai cobra (Naja naja siamensis) in mice10. Aqueous extracts of the bark of Schumanniophyton magnificum and the leaves of Mucuna pruriens var. utilis, Strophanthus gratus and Strophanthus hispidus have the ability to prolong the clotting time when administered along with a standardised dose of Echis carinatus venom11. The survival time of male abino mice was increased by the extract of the leaves of Guiera senegalensis when compared to the Echis carinatus and Naja nigricollis venom treated animals12. Bothrops atrox venom induced haemorrhage was completely neutralized by the extracts of the stem barks of Brownea rosademonte, Tabebuia rosea, the whole plants of Pleopeltis percussa, Trichomanes elegans, rhizomes of Heliconia curtispatha, leaves and branches of Bixa orellena, Phylodendrum tripartitum, Struthanthus orbicularis, Gozalagunia panamensis, the ripe fruit of Citrus limon leaves, branches and stems of Ficus nymphaeifolia13. Partial protection of haemorrhage was also shown by Aristolochia grandiflora, Columnea kalbreyeriana, Sida acuta, Selaginella sp., Pseudele- phantopus spicatus, rhizomes of Renealmia alpinia, stem of Strychnos xynguensis, leaves, branches and stem of Hyptis capitata, Ipomoea cairica, Neurolaena lobata, Ocimum micranthum, Piper pulchrum, Siparuna thecaphora, Castilla elastica, Allamanda cathartica, the macerated fruits of Capsicum frutescens, unripe fruits of Crescentia cujete, leaves and branches of Piper arboretum and Passiflora quadrangularis13. The stem barks of Brownea rosademonte, Tabebuia rosea; rhizomes of Renealmia alpinia, Heliconia curtispatha; the whole plants of Pleopeltis percussa, Trichomanes elegans; and the ripe fruits of Citrus limon, demonstrated 100% neutralizing capacity of snake (Bothrops atrox) venom within 48 hours. Partial protection was also shown by the leaves, branches and stem of Costus lasius; the whole plant of Sida acuta; rhizomes of Dracontium croatii; leaves and branches of Bixa orellana and Struthanthus orbicularis14. Bothrops atrox venom induced haemorrhage was completely neutralized by stem barks of Brownea rosademonte and Tabebuia rosea; the whole plants of Pleopeltis percussa, Trichomanes elegans and Senna dariensis; rhizomes of Heliconia curtispatha; leaves and branches of Bixa orellana, Philodendron tripartitum, Struthanthus orbicularis and Gonzalagunia panamensis; ripe 867 fruits of Citrus limon; leaves, branches and stem of Ficus nymphaeifolia and moderate neutralization were shown by the whole plants of Aristolochia grandiflora, Columnea kalbreyeriana, Sida acuta, Selaginella articulata and Pseudoelephantopus spicatus; rhizomes of Renealmia alpinia; the stem of Strychnos xinguensis; leaves, branches and stems of Hyptis capitata, Ipomoea cairica, Neurolaena lobata, Ocimum micranthum, Piper pulchrum, Siparuna thecaphora, Castilla elastica and Allamanda cathartica; the macerated ripe fruits of Capsicum frutescens; the unripe fruits of Crescentia cujete; leaves and branches of Piper arboreum and Passiflora quadrangularis15. Mucuna pruriens var. utilis seed aqueous extract significantly inhibited the Echis carinatus venom induced myotoxic, cytotoxic and coagulation activities in experimental animals16. Aqueous and alcoholic extracts of dried roots of Mimosa pudica inhibited lethality, myotoxicity and toxic enzymes of Naja kaouthia venom17. Oral administration of garlic could be used as a prophylactic tool against cobra venom induced histological and histochemical patterns of the gastric and hepatic tissue changes in rats18. A water-methanol extract of Parkia biglobosa stem bark extract could neutralize two snake venoms (Naja nigricollis, and Echis ocellatus) in several experimental models19. Phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms was shown by crude aqueous extract of Mandevilla velutina20. The extract of the Brazilian plant Marsypianthes chamaedrys inhibited fibrinogen clotting induced by several Brazilian snake venoms, indicating that it affects thrombin-like enzymes21. Aqueous extract of Casearia sylvestris showed anti PLA2, hemorrhagic and myotoxic activities caused by crude snake venoms and toxins22. On the other hand, Casearia mariquitensis inhibited some hematological and systemic alterations induced by Bothrops neuwiedi pauloensis venom23. Mandevilla illustris inhibited phospholipase activity of Crotalus durissus terrificus snake venom along with prolongation of the survival time and a decrease in lethality24. Mimosa pudica has been reported to possess anti-hyaluronidase activity against Naja naja, Vipera russelli and Echis carinatus venom25. The butanolic extract of Mimosa pudica and Eclipta prostrata partially inhibited the hemorrhagic activity but displayed very low anti-phospholipase A2 activity and did not inhibit proteolytic activity of Malayan pit 868 INDIAN J EXP BIOL, SEPTEMBER 2010 viper venom26. Edema, defibrination and coagulation effects of Bothrops asper venom were neutralized by the leaves and branches of Bixa orellen, Ficus nymphaeifolia, Struthanthus orbicularis and Gonzalagunia panamensis; the stem barks of Brownea rosademonte and Tabebuia rosea; the whole plant of Pleopeltis percussa and Trichomanes elegans; rhizomes of Renealmia alpinia, Heliconia curtispatha and Dracontium croatii, and the ripe fruit of Citrus limon27. The ethanol root extract of Acalypha indica possesses potent snake venom neutralizing properties28. Aqueous extract of Tabernaemo ntana catharinensis inhibited the lethal activity of Crotalus durissus terrificus snake venom29 and partially neutralized myotoxic effect of Bothrops jararacussu venom and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity]30. The methanol extract of the root bark of Annona senegalensis Pers caused reduction in the Naja nigricotlis nigricotlis venom induced hyperthermia in rats31. Musa paradisiaca L. successfully neutralized viper venom actions in in vitro experiments only32. Pentaclethra macroloba exhibited full inhibition of hemorrhagic and nucleolytic activities induced by several snake venoms, along with partial inhibition of myotoxic, lethal, phospholipase and edema activities. It totally inhibited Bothrops jararacussu metalloprotease induced hemorrhage in in vivo model33. Aqueous extracts of Croton urucurana antagonized the hemorrhagic activity of Bothrops jararaca venom and proanthocyanidins were involved in this activity34. Aqueous extracts of fresh roots, stems or leaves of Mikania glomerata, efficiently neutralized different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes35. Cordia verbenacae inhibited paw edema induced by Bothrops jararacussu snake venom36. Aqueous extract from aerial parts of Bauhinia forficata is a promising source of natural inhibitors of serine-proteases involved in blood clotting disturbances induced by Bothrops and Crotalus crude venoms37. The methanol bulb extract of Crinum jagus significantly protected mice from death, myonecrosis and haemorrhage induced by the lethal effects of Echis ocellatus, Bitis arietans and Naja nigricollis venoms38. Tamarind seed extract inhibited the PLA2, protease, hyaluronidase, l-amino acid oxidase and 5'-nucleotidase enzyme (major hydrolytic enzymes) activities of Vipera russelli venom in a dose-dependent manner. Further, the extract neutralized the degradation of the B-beta chain of human fibrinogen and indirect hemolysis caused by venom. The extract exerted a moderate effect on the clotting time. Edema, hemorrhage and myotoxic effects along with lethality, induced by venom were neutralized significantly when different doses of the extract were preincubated with venom before the assays. On the other hand, animals that received extract 10 minutes after the injection of venom were protected from venom induced toxicity39. Dichloromethane extract of leaves of Artemisia campestris L neutralized the venom induced actions of viper Macrovipera lebetina40. Ethanol extract of leaves of Galactia glaucescens prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom41. Oedema, haemorrhage, myonecrosis and coagulation induced by Indian Echis carinatus (saw-scaled viper) venom were neutralized by the methanol seed extract of Vitis vinifera L42. The aqueous extract of Schizolobium parahyba showed potent antisnake venom activity43,44. The active fractions of Aristolochia indica, Hemidesmus indicus, Strychnos nux vomica, Gloriosa superba, Eclipta prostrata, and Andrographis paniculata neutralized rattle snake venom induced actions45. The animals that received orally the extract of Aristolochia odoratissima leaves were protected against Bothrops atrox venom as mortality of experimental animals decreased from 100 to 80%46. The methanol root extracts of Vitex negundo Linn. and Emblica officinalis significantly neutralized the Vipera russelli and Naja kaouthia venom induced lethal activity in vivo studies; Vipera russelli venom-induced haemorrhage, coagulant, defibrinogenating and inflammatory activity were significantly neutralized by both plant extracts47. Hemidesmus indicus root extracts effectively neutralized Viper venom-induced lethal, haemorrhagic, coagulant, anticoagulant and inflammatory activities48. An active compound from the Strychnus nux vomica whole seed extract, neutralised Daboia russelli venom induced lethality, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethality, cardiotoxicity, neurotoxicity, PLA2 enzyme activity, and it also inhibited viper venom induced lipid peroxidation in experimental animals49 (Table 1). GOMES et al.: HERBAL ANTIDOTE & SNAKE BITE TREATMENT 869 Table 1—List of plants used against snake envenomation Scientific name Parts of the plant used Scientific name Abrus precatorius Acalypha indica Achyranthes aspera Acorus calamus Linn Actaea racemosa Alangium salvifolium Allamanda cathartica Aloe barbadensis Andrographis piniculata Annona senegalensis Pers Antidesma bunius Linn. Arctium lappa Aristolochia grandiflora Aristolochia odoratissima Aristolochia shimadai Artemisia campestris L Azadirachta indica Biophytum sensitivum Bixa orellana Bombax ceiba Linn Brownea rosa de monte Buchnania lanzan Spr Calotropis gigantea Calycopteris floribunda Capsicum frutescens Casearia mariquitensis Casearia sylvestris Cassia occidentalis Castilla elastica Cecropia peltata Cinnamomum zeylanicum Citrus limon Columnea kalbreyeriana Cordia verbenacae Costus lasius Crescentia cujete Crinum jagus Croton urucurana Cucumis colocynthis Curcuma longa Diodia scandens Diospyros kaki Dracontium croatii Sida acuta Philodendron tripartitum Piper arboretum Piper pulchrum Pleopeltis percussa Prenanthes alba Pseudelephantopus spicatus Psoralea corylifolia Raphanus sativus Rauwolfia serpentine Renealmia alpinia Rhizoma paridis roots roots Echinacea angustifolia Echinacea pallida Echinacea purpurea Eclipta prostrata L. Emblica officinalis Enicostemma axillare Equisetum giganteum Ficus lacor Ficus nymphaeifolia Ficus religiosa Galactia glauscescens Gonzalagunia panamensis Guiera senegalensis Gymnema sylvestre Heliconia curtispatha Hemidesmus indicus Hyptis capitata Impatiens balsamina Ipomoea cairica Leucas linifolia Lysimachia nummularia L. Mandevilla illustris Marsypianthes chamaedrys Melianthus major Mikania glomerata Mimosa pudica Moringa oleifera Lamk Mucuna pruriens var. utilis Mucuna pruriens var. utilis Musa paradisiaca Nerium indicum Mill Nerium oleander Neurolaena lobata Ocimum micranthum Oldenlandia corymbosa L Ophiorrhiza mango Origanum dictamnu Papever somniferum Paris polyphylla Parkia biglobosa Passiflora quadrangularis Pentaclethra macroloba Peristrophe bicalyculata Siparuna thecaphora Strophanthus gratus Strophanthus hispidus Struthanthus orbicularis Strychnos nux vomica Strychnos xinguensis Tabebuia avellanedae Tabebuia rosea Tabernaemontana catharinensis Tamarindus indica Trichomanes elegans Ulmus rubra rhizomes roots leaves, branches, stem leaves root, bark leaves whole plant leaves leaves leaves whole plant leaves, branches, shoot shoot, leaves stem, barks stem, bark leaves, roots leaves fruit leaves & bark roots leaves, branches, stem leaves fruit leaves, branches, stem fruit bulb roots rhizomes rhizomes whole plant shoot, leaves leaves, branches leaves, branches, stem whole plant leaves whole plant seeds tuber whole plant rhizomes roots Parts of the plant used roots leaves rhizomes latex leaves, branches, shoot flower leaves leaves, branches leaves roots rhizomes roots leaves, branches, stem flower leaves, branches, stem flower roots, stems, leaves roots, whole plant shoot, leaves seeds leaves leaves leaves, branches, stem leaves, branches, stem roots seeds roots leaves, branches roots leaves, branches, stem leaves leaves leaves, branches, shoot seeds stem, leaves, branches bark stem barks whole plant INDIAN J EXP BIOL, SEPTEMBER 2010 870 Table 1—List of plants used against snake envenomation—Contd. Scientific name Schizolobium parahyba Schumanniophyton magnificum Selaginella articulata Senna dariensisida acuta Vitex negundo Linn Vitis vinifera L Woofordia fruitcosa Xanthium sibiricum Parts of the plant used leaves bark whole plant whole plant roots seeds leaves Herbal constituents active against snake envenomation Acids—Aristolochic acid, contained in Aristolochia produces increase in immune response and it also inhibits the lytic activity and the edematose properties of some phospholipases of snake venoms50. 2-OH-4methoxy benzoic acid, isolated and purified from Hemidesmus indicus, possessed potent antiinflammatory, antipyretic and antioxidant properties of viper venom51. Lethality induced by Viper venom was maximally neutralized with 2-hydroxy-4-methoxy benzoic acid and anisic acid, both in in vitro and in vivo studies. The compound 2-OH-4-methoxy benzoic acid also showed adjuvant effects and antiserum action potentiation against Vipera russelli venom52. The exact mechanisms of venom neutralization were not established, except for 2-hydroxy-4-methoxy benzoic acid, where the functional groups, methoxy and hydroxy were partly responsible for the neutralization of the lethal effect and haemorrhagic activity52. The venoms of common Indian snakes Vipera russelli, Echis carinatus, Naja kaouthia and Ophiophagus hannah were taken to evaluate the lethal, haemorrhagic and defibrinogenation action neutralization with four compounds (2-hydroxy-4-methoxy benzoic acid from Hemidesmus indicus, anisic acid from Pimpinella anisum, salicylic acid from Filipendula ulmaria, Salix alba and aspirin from Salix alba) in experimental animals. Lethal action of venom were maximally neutralized with 2-hydroxy-4-methoxy benzoic acid and anisic acid, both in in vitro and in vivo studies. Haemorrhagic activity of Viper and Echis venom was neutralized with salicylic acid53. Rosmarinic acid, a new antidote against snake (Bothrops jararacussu) venom, was isolated which possessed phospholipase A2 (from Cordia verbenacea) inhibitor activity; it also inhibited most of the myotoxic activity with partial inhibition of edema along with the ability to potentiate commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in experimental models. Alkaloids—Atropine is found in some of the Solanacae family members and it exerted inhibitory action against the venom of green and black mamba (Dendroaspis angusticeps and D. polylepsis); these venoms are mainly responsible for neuro-transmitter release at cholinergic nerve terminals, therefore it was suspected that a cholinergic blocker like atropine may reduce their effects. AIPLAI (Azadirachta indica PLA2 inhibitor) was purified from the methanol leaf extract of Azadirachta indica (Neem); it inhibited the cobra and Russell's viper venoms (RVVs) phospholipase A2 enzymes in a dosedependent manner. AIPLAI significantly inhibited PLA2 enzymes, higher in cobra venom (Naja naja and Naja kaouthia) compared to that of crude RVV (Daboia russelli) when tested under the same condition54. Coumestan and steroids—Viper and cobra venom neutralization was shown by beta sitosterol and stigmasterol, isolated from the methanol root extract of Pluchea indica55. The active fraction could also antagonize venom-induced changes in lipid peroxidation and superoxide dismutase activity. Wedelolactone has been identified as a coumestane contained in Eclipta prostrata L. and was suggested to be an active component in fighting against snake venoms56. Wedelolactone, sitosterol and stigmasterol inhibits the effect of South American rattle snake57. Enzymes, peptides and pigments—Snake venom molecule are composed of three-dimensional proteins and some non-protein components. These proteins could be dissolved with natural solvents like bromelain and papain. Bromelain is found in pineapple (Ananas comosus) and papain is present in papaya fruit (Carica papaya). Thus these two natural proteolytic enzymes could be used to neutralize snake venom proteins. A peptide compound with a molecular weight of 6000 Da, reported to possess anticardiotoxic activity against cobra venom was isolated from the plant Schumanniphyton magnificum58. Turmerin, a protein from turmeric (Curcuma longa L.) inhibits the enzymatic activity and neutralizes cytotoxicity, oedema and myotoxicity of multitoxic phospholipase A2 of cobra (Naja naja)59. Melanin extracted from black tea was reported for the first time to possess antivenom activity against Agkistrodon contortrix laticinctus, Agkistrodon halys blomhoffii and Crotalus atrox snake venoms60. GOMES et al.: HERBAL ANTIDOTE & SNAKE BITE TREATMENT Glycoprotein and glycosides—A multiform glycoprotein (whose oligosaccharide chains were functional) was isolated from Mucuna pruriens seeds, neutralized Echis carinatus venom induced actions61. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera62. The WSG inhibited the phospholipase A2 activity of NN-XIaPLA2 isolated from the cobra venom (Naja naja), completely at a mole-to-mole ratio of 1:2 (NN-XIaPLA2: WSG)62 but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA2 isoforms from the venom to varying extent. Hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelli) venoms were inhibited by WSG. It also inhibited hyaluronidase activity of Indian cobra (Naja naja) venom63,64. Benzoylsalireposide and salireposide isolated from Symplocos racemosa inhibited phosphodiesterase I activity against snake venom. The methanol extract of the stem bark of Schumanniophyton magnificum and schumanniofoside, a chromone alkaloidal glycoside was isolated which reduced the lethal effect of black cobra (Naja melanoleuca) venom in mice. This effect was maximum when the venom was mixed and incubated with the extract or schumanniofoside. It is thought that the mode of action is by oxidative inactivation of the venom. Phenols—PLA2 activity of Bothrpos asper venom was neutralized by 4-nerolidylcatechol isolated from Piper umbellatum and Piper peltatum65. The ethanol extract from seed kernels of Thai mango (Mangifera indica L.) and its major phenolic principle (pentagalloyl glucopyranose) exhibited dose-dependent inhibitory effects on phospholipase A2, hyaluronidase and L-amino acid oxidase of Calloselasma rhodostoma and Naja naja kaouthia venoms in in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of seed kernal against both venoms were clearly supported by in vivo tests66. The plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria block non-selectively the nicotinic acetylcholine receptor by precipitation of Naja kaouthia venom67. Pterocarpanes—The aqueous alcohol extract of the root of a South-America plant called Cabeca de Negra is used against snake venom. From this 871 source, cabenegrin A-I and cabenegrin A-II were isolated, which have been found to be an anti-snake oral antidote68. Similarly the extract of a South American plant, Harpalyce brasiliana Benth, commonly called Portuguese Snake Herb, also yielded cabenegrin A-II (phenolic pterocarpan in nature). Edunol, a pterocarpan isolated from Harpalyce brasiliana, used in the northeast of Brazil against snake bites and roots of two Mexican 'snakeweeds', Brongniartia podalyrioides and Brongniartia intermedia (Leguminosae), reduced the expected mortality of mice previously treated with Bothrops atrox69 venom along with antimyotoxic, antiproteolytic and PLA2 inhibitor properties70. Tannins—The tannin from persimmon, a fruit from Diospyrus kaki inhibits edema in mice, induced by sea snake and also improved the survival rate in mice injected with Laticauda semifasciata and Trimeresurus flavoviridis venom71. Ellagic acid, a compound isolated from the aqueous extract of Casearia sylvestris has been reported to possess anti snake venom activity, mainly against Bothrops genus72. Terpenoids—Glycyrrhizin, a natural triterpenoid saponin extracted from the root of Glycyrrhiza glabra (licorice), with a molecular mass of 840 Da, has been characterized as a thrombin inhibitor73. This compound is known for its anti-inflammatory activity and Glycyrrhizin also exhibits in vivo antithrombotic properties against snake venom; it prevents both in vitro and in vivo venom-induced changes in hemostasis, suggesting a potential antiophidic activity74. A potassium salt of gymnemic acid, which is a triterpenoid glycoside obtained from Gymnema sylvestre inhibits ATPase in Naja Naja venom75,76. Lupeol acetate, isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R.Br. could significantly neutralize lethality, haemorrhage, defibrinogenation, edema, PLA2 activity induced by Daboia russelli venom. It also neutralized Naja kaouthia venom induced lethality, cardiotoxicity, neurotoxicity and respiratory changes in experimental animals77. Bothrops neuwiedi and Bothrops jararacussu venom induced hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases were neutralized by neo-clerodane diterpenoid, isolated from Baccharis trimera78. Oleanolic acid inhibited sPLA (2) activities of Vipera russelli and Naja naja snake venoms in a concentration-dependent manner. Inhibition of in vitro and in vivo sPLA2 activity by oleanolic acid 872 INDIAN J EXP BIOL, SEPTEMBER 2010 explains the observed anti-inflammatory properties of several oleanolic acid-containing medicinal plants79. The pentacyclic triterpenes (free or as glycosides) are found widely in several antisnake venom plants (Aegle marmelos, Centipeda minima, Aloe barbadensis, Phyllanthus niruri, Alstonia scholaris, Phyllanthus emblica, Elephantopus scaber, etc.) and provide nearly 20% protection against snake venom80. Quinovic acid-3O-alpha-L-rhamnopyranoside81, quinovic acid-3-O-betaD-fucopyranoside, and quinovic acid-3-O-beta-Dglucopyranosyl (1-->4)-beta-D-fucopyranoside isolated from the ethyl acetate extract of Bridelia ndellensis barks and Mitragyna stipulosa showed significant inhibitory activity against snake venom phosphodiesterase-I82,83. Triterpenoid saponin from Pentaclethra macroloba, inhibited antiproteolytic and antihemorrhagic actions induced by Bothrops snake venoms. These inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from Bothrops neuwiedi and Bothrops jararacussu venoms84. Ursolic acid a common constituent of many medicinal plants, inhibited PLA2 enzymes purified from Vipera russelli, Naja naja venom85. Quinonoid xanthene—Ehretianone, a quinonoid xanthene isolated from the root bark of Ehretia buxifolia. Roxb. has been reported to possess anti snake (Echis carinatus) venom activity86. Resveratrol—Hong Bei Si Chou is a herbal medicine used to treat snake bite in Guangxi province of China. It was found that resveratrol (3,4',5-trihydroxytransstilbene) isolated from the ethyl acetate part of Hong Bei Si Chou could antagonize snake toxins both in vivo and in vitro87. Alkaloid (12-methoxy-4-methylvoachalotine) extract of Tabernaemontana catharinensis inhibited lethality induced by Crotalus durissus terrificus snake venom88. Miscellaneous chemical groups and compounds— Several plant constituents like flavonoids, quinonoid, xanthene, polyphenols and terpenoids possesses protein binding and enzyme inhibiting properties and also inhibit snake venom phospholipase A2 (PLA2) activities of both Viper and Cobra venom89. Total inhibition of hemorrhage was observed with the ethanol, ethyl acetate and aqueous extracts of Bursera simaruba, Clusia torresii, Clusia palmana, Croton draco, Persea americana, Phoebe brenesii, Pimenta dioica, Sapindus saponaria, Smilax cuculmeca and Virola koschnyi83. Chemical analysis of these extracts identified catequines, flavones, anthocyanines and condensated tannins, which may be responsible for the inhibitory effect observed, probably owing to the chelation of the zinc required for the catalytic activity of Bothrops asper venom's hemorrhagic metalloproteinases. Plant-derived, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibited the hyaluronidase enzyme activity90. Further, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time. Medicinally important herbal compounds (acalyphin, chlorogenic acid, stigmasterol, curcumin and tectoridin) were screened against Russell's viper PLA291. These compounds showed favorable interactions with the amino acid residues at the active site of Russell's viper PLA2, thereby substantiating their proven efficacy as anti-inflammatory compounds and as antidotes. An active compound (SNVNF) was isolated and purified from the whole seed extract of Strychnos nux vomica, which could effectively antagonise Daboia russelli venom induced lethality, haemorrhage, defibrinogenating, oedema, PLA2 enzyme activities and Naja kaouthia induced lethality, cardiotoxicity, neurotoxicity, PLA2 enzyme activities. Hexane extract of Curcuma longa rhizomes, ar-turmerone92 also inhibited the proliferation and the natural killer cell activity of human lymphocytes. This compound has anti lethal activity against venom of Crotalus durissimus terrificus. Moreover when it was injected in mice it showed anti- hemorrhagic activity against Bothrops jararaca venom. (Table 2). Mechanism of snake venom neutralization by herbal compounds Herbal compounds that possess snake venom neutralization properties in experimental animal models (in vivo and in vitro) usually follows three protocols—(1) venom- herbal compounds mixed together, (2) herbal compounds followed by venom, and (3) venom followed by herbal compounds. Among these, the third technique is similar to clinical conditions. The venom dose is one of the critical factors, on which the herbal compounds could show their neutralizing effects. Higher the venom dose, less the fold of neutralization. So, what is desirable is that the venom should be tried from lower to higher dose. For this, a huge number of animals are required, which is sometimes very difficult from animal ethical issues. GOMES et al.: HERBAL ANTIDOTE & SNAKE BITE TREATMENT 873 Table 2—List of herbal compounds active against snake envenomation Compound Acids Aristolochic acid 2-OH-4-methoxy benzoic acid 2-hydroxy-4-methoxy benzoic, anisic acid, salicylic acid, aspirin Rosmarinic acid Alkaloids Atropine AIPLAI (Azadirachta indica PLA2 inhibitor) Coumestan and Steroids Beta sitosterol and stigmasterol Wedelolactone Enzymes, Peptides and Pigments Bromelain Papain Peptide Turmerin Melanin Glycoprotein and Glycosides Glycoprotein WSG Benzoylsalireposide and salireposide Phenols 4-nerolidylcatechol Pentagalloyl glucopyranose polyphenols Plant source Aristolochia sp. Hemidesmus indicus Hemidesmus indicus, Pimpinella anisum, Filipendula ulmaria, Salix alba Cordia verbenacea Dendroaspis angusticeps and D. polylepsis Azadirachta indica Pluchea indica Eclipta prostrata L. Ananas comosus papaya Schumanniphyton magnificum Curcuma longa L Black Tea Mucuna pruriens Withania somnifera Symplocos racemosa Piper umbellatum and Piper peltatum Mangifera indica L Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria Pterocarpanes Cabenegrin A-I and cabenegrin A-II Edunol Cabeca de negro Harpalyce brasiliana Tannins Tannin Ellagic acid Diospyrus kaki Casearia sylvestris Terpeinoids Glycyrrhizin Potassium salt of gymnenic acid Lupeol acetate Neo-clerodane Oleanolic acid Pentacyclic triterpenes Glycyrrhiza glabra Gymnema sylvestre Hemidesmus indicus R.Br Baccharis trimera Aegle marmelos, Centipeda minima, Aloe barbadensis, Phyllanthus niruri, Alstonia scholaris, Phyllanthus emblica, Elephentopus scaber Bridelia ndellensis Mitragyna stipulosa Quinovic acid-3-O-beta-D-fucopyranoside, and quinovic acid-3-O-beta-D-lucopyranosyl (1-->4)-beta-D-fucopyranoside Triterpenoid saponin Pentaclethra macroloba Ursolic acid Eriobotrya japonica Quinonoid xanthene Ehretianone Ehretia buxifolia. Roxb. Resveratrol Resveratrol(3,4',5-trihydroxytransstilbene) Hong Bei Si Chou Alkaloid (12-methoxy-4-methylvoachalotine) Tabernaemontana catharinensis Reference 50 51, 52 53 54 55,57 56,57 58 59 60 61 62, 63 64 65 66 67 68 69, 70 71 72 73, 74 75, 76 77 78 79 80 82, 83 84 85 86 87 88 (Contd.) INDIAN J EXP BIOL, SEPTEMBER 2010 874 Table 2—List of herbal compounds active against snake envenomation—Contd. Compound Miscellaneous Chemical Groups and Compounds Flavonoids, quinonoid, xanthene, polyphenols and terpenoids Aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone Acalyphin, chlorogenic acid, stigmasterol, curcumin and tectoridin SNVNF Ar- turmerone Plant source Reference Bursera simaruba, Clusia torresii, Clusia. palmana, Croton draco, Persea americana, Phoebe brenesii, Pimenta dioica, Sapindus saponaria, Smilax cuculmeca and Virola koschnyi. 89 90 Lonicera japonica, Hemidemus indicus, Curcuma longa 91 Strychnos nux vomica Curcuma longa 92 93 How the herbal compounds neutralize the toxic venom constituents with in the body? Till date, there is no definite answer or mechanism. Many hypothesis have been proposed such as (1) protein precipitation hypothesis44, (2) enzyme inactivation hypothesis93, (3) chelation hypothesis83, (4) adjuvant action hypothesis52, (5) anti-oxidant hypothesis77, (6) protein folding hypothesis, (7) combination hypothesis52 and many more. The above hypothesis have their own limitations. Among these, protein precipitation-inactivation hypothesis is more acceptable. However, more emphasis should be focused on this area in the near future. Future of antivenom and herbal therapy The limitations of AVS are well known and the world is looking for an alternative for snake bite treatment. Till date no suitable alternative measures are available, except the natural herbal remedies, which are showing promising expectations. The advantages of herbal compounds are that, they are cheap, easily available, stable at room temperature and could neutralize a wide range of venom antigen. In many cases, the whole herbal extracts are more powerful than the individual herbal compounds. The herbal compounds could effectively neutralize snake venom in presence of AVS, which is another advantage of herbal compounds. It may be opined that the identified herbal compounds having AVS potentiating actions might be selected for further trials. It is now obvious that the future of AVS is lying on the shoulder of herbal compounds and combination of these two antidotes may find a suitable alternative to the snake bite treatment in the near future. Combination therapy is an old practice of Ayurvedic medicine. Various therapeutic, ayurvedic formulations are available commercially, eg., Articulin-F comprising of a fixed combination of Boswellia serrata, Withania somnifera, Curcuma longa, Zinc for treating osteoarthritis, Trikatu comprising of black pepper, long pepper and ginger, for treating digestive ailments, etc. In ayurveda there could be combination ranging from two to twenty formulations, in fixed doses. Thus it is evident that ayurveda not only uses herbal components, but along with it various metal ions and spices are also important part of combination formulation. It is well recognized in ayurveda that a medicinal plant may need to be administered with other plants, that is in combination, in order to exert its therapeutic effect. The second plant may potentiate the action of the first, while the third might help to prevent the toxicity of the second plant. Recently emphasis has been given to the age old concept of combination therapy in several pathophysiological condition like cancer94, tuberculosis, malaria95 and AIDS. We are also hopeful that snake bite treatment may be benificial by this application and gradually the herbal compounds may find an alternative to the AVS. Conclusions Recently, the World Health Organization estimated that 80% people worldwide rely on herbal medicines for some aspect or other for their primary healthcare96. World Health Organization has shown great interest in documenting the use of medicinal plants used by tribals from different parts of the world. Many developing countries have intensified their efforts in documenting the ethnomedical data and scientific research on medicinal plants. Once these local ethnomedical preparations are scientifically evaluated and disseminated properly, people will be better informed regarding efficacious herbal drug treatment and improved health status in several pathophysiological conditions including snake bite in the near future. It is our responsibility to identify, cultivate and culture these eco-friendly herbs for the alleviation of human suffering and death against snake bite. GOMES et al.: HERBAL ANTIDOTE & SNAKE BITE TREATMENT Acknowledgement The first author AG acknowledge the partial financial help for plant and snake venom research from ICMR, New Delhi, India and CSIR, New Delhi, India. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Philip E, Snake bite & scorpion sting, in Pediatric & neonatal emergency care edited by Srivatava R N, (Jaypee Brothers, New Delhi) 1994, 227. 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