Author's Accepted Manuscript

Protective activity of medicinal plants and their isolated compounds against the toxic effects from the venom of Naja (Cobra) species Arham Shabbir, Muhammad Shahzad, Paul Masci, Glenda C. Gobe

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S0378-8741(14)00694-1 http://dx.doi.org/10.1016/j.jep.2014.09.039 JEP9046

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Journal of Ethnopharmacology

Received date: 11 April 2014 Revised date: 25 September 2014 Accepted date: 25 September 2014 Cite this article as: Arham Shabbir, Muhammad Shahzad, Paul Masci, Glenda C. Gobe, Protective activity of medicinal plants and their isolated compounds against the toxic effects from the venom of Naja (Cobra) species, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2014.09.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Table 1: Anti-venom activities of medicinal plants against various species of Naja genus Plant / Constituents

Family

Part

Naja species

Mucuna pruriens (L.) DC.

Fabaceae

Seeds

Naja sputatrix

Annona senegalensis Pers.

Annonaceae

Root

Naja nigricollis nigricollis

Rhizomes

Naja naja siamensis

Curcuma parviflora Wall.

Zingiberaceae

C. cf. zedoaria

Zingiberaceae

Flavone, tannic acid, quercertin, and curcumin

References

Prevented damage to heart and vessels, protected against respiratory paralysis, neuromuscular blockage, decline of BP, and suppression of atrial rate and contractility Increased survival time, decreased beginning and severity of toxic signs, and yeast induced pyrexia Enhanced diaphragm muscle contraction, blocked antibody attachment to venom, and protein destruction.

Neutralized activity

hyaluronidase

enzyme

Fung et al., 2009 Fung et al., 2011 Fung et al., 2012 Adzu 2005

et

al.,

Daduang et al., 2005 Girish and Kemparaju, 2005

Naja naja

Glycoprotein from Withania somnifera (L.) Dunal

Solanaceae

Root

Fagonia cretica L.

Zygophyllaceae

Twigs & leaves

Mimosa pudica L.

Fabaceae

Root

Areca catechu L.

Arecaceae

Seeds

Fabaceae

Bark

Tiliaceae

Bark

Pithecellobium dulce (Roxb.) Benth. Pentace burmanica Kurz

Anti-venom activity

Neutralized PLA2, Increased survival Machiah and Gowda, 2006 time, and LD50 dose of venom Naja naja karachiensis

Prevention against hemorrhage

Razi et al., 2011

Inhibited lethality, myotoxicity, Mahanta and acetylcholinesterase, plasma protease, and Mukherjee, PLA2, and decreased CPK levels 2001 Naja kaouthia

Inhibited lethality, necrosis, and Acetylcholinesterase

1

Pithayanukul et al., 2005

Quercus infectoria G. Olivier Lupeol acetate from Hemidesmus indicus (L.) R. Br. Ex Schult β-sitosterol and stigmasterol from Pluchea indica (L.) Less. Rediocides A and G from Trigonostemon reidioides (Kurz) Craib

Fagaceae

Nutgall

Apocynaceae

Root

Inhibited lethality, respiratory changes, Potentiated antiserum

Asteraceae

Root

Inhibited lethality, neurotoxicity, Gomes et al., cardiotoxicity, respiratory changes, and 2007 PLA2. Potentiated antiserum

Euphorbiaceae

Root

Increased survival time

2

cardiotoxicity, Chatterjee et al., and PLA2. 2006

Utsintonga et al., 2009

Protective activity of medicinal plants and their isolated compounds against the toxic effects from the venom of Naja (Cobra) species

Arham Shabbir 1, 2, Muhammad Shahzad 2, 4, Paul Masci 3 and Glenda C Gobe 4* 1.

Department

of

Pharmacy,

COMSATS

Institute

of

Information

Technology,

Abbottabad,

22060-Pakistan

([email protected]) 2. Department of Pharmacology, University of Health Sciences, Lahore, Pakistan ([email protected]) 3. Venomics Research Centre, Translational Research Institute, School of Medicine, The University of Queensland, Australia ([email protected]) 4. Centre for Kidney Disease Research, Translational Research Institute, School of Medicine, The University of Queensland, Australia ([email protected])

*

Author for correspondence:

Prof Glenda Gobe School of Medicine, University of Queensland Translational Research Institute, 37 Kent Street 3

Woolloongabba, Brisbane, Australia 4102 Phone 61 7 344 38011; Fax 61 7 344 37779; Email [email protected]

4

Abstract ETHNOPHARMACOLOGICAL RELEVANCE: Various medicinal plants have protective properties against the toxicities of the venom of cobra snake (Naja species). They may be used as local first aid for the treatment of snakebite victims, and can significantly inhibit lethality, cardio-, neuro-, nephro- and myotoxicity, hemorrhage, and respiratory paralysis induced by the cobra snake venom. The plants or their extracts may also complement the benefits of conventional anti-serum treatment. AIM OF THE REVIEW: This review provides information on the protective, anti-venom, properties of medicinal plants against snakebites from cobras. In addition, it identifies knowledge gaps and suggests further research opportunities. METHODS: The literature was searched using databases including Google Scholar, PubMed, ScienceDirect, Scopus and Web of Science. The searches were limited to peer-reviewed journals written in English with the exception of some books and a few articles in foreign languages. RESULTS: The plants possess neutralization properties against different cobra venom enzymes, such as hyaluronidase, acetylcholinesterase, phospholipase A2 and plasma proteases. Different active constituents that show promising activity against the effects of cobra venom include lupeol acetate, β-sitosterol, stigmasterol, rediocides A and G, quercertin, aristolochic acid, and curcumin, as well as the broad chemical groups of tannins, glycoproteins, and flavones. The medicinal plants can increase snakebite victim survival time, decrease the severity of toxic signs, enhance diaphragm muscle contraction, block antibody attachment to venom, and inhibit protein destruction. In particular, the cardiovascular system is protected, with inhibition of blood pressure decline and depressed atrial contractility and rate, and prevention of damage to heart and vessels. The designs of experimental studies that show benefits, or otherwise, of use of medicinal plants have some limitations: deficiency in identification and isolation of active constituents 5

responsible for therapeutic activity; lack of comparison with reference drugs; and little investigation of the mechanism of anti-venom activity. CONCLUSION: Despite some current deficiencies in experimental or clinical analysis, medicinal plants with anti-venom characteristics are effective and so are candidates for future therapeutic agents. We suggest that emphasis on identification and testing of active ingredients in research in the future will assist better understanding of their anti-venom activity.

Key words: Cobra, Naja genus, medicinal plants, anti-venom

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1. Introduction

Snakes of the Naja genus are commonly called cobras. They belong to the Elapidae family and typically occur in regions throughout Africa and Southern Asia. Naja species that most commonly pose threats to human health are Naja kaouthia, N. oxiana, N. naja, N. atra, N. sputatrix, N. siamensis, N. philippinesis and N. sumatrana (Warrell, 1996; 1999). Altogether, there may be over four million snakebites every year in Asia, approximately half of which are envenomed (poisonous), with many causing death (Kasturiaratne et al., 2008). Snake venom is a mixture of toxic substances with different toxic activities. Cobra venom, in particular, is a mixture of different toxic enzymes, such as phospholipase A2 (PLA2), acetylcholinesterase, hyaluronidase and plasma proteases (Yingprasertchai et al., 2003; Rucavado et al., 2004). The outcomes of venom toxicity include nephro-, neuro- and cardiotoxicity, respiratory and circulatory collapse, necrosis, haemorrhage and odema (Mahanta and Mukherjee, 2001; Machiah and Gowda, 2006; Gomes et al., 2007). Treatment of cobra bite victims warrants a medical emergency.

To date the most common specific therapy against snakebite is a polyvalent snake anti-serum (Pla et al., 2012). Snake venom antiserum is a sterile preparation containing immunoglobulin fragments from, for example, horse, goat or rabbit serum, made after the host animal is injected with the venom. The anti-serum is injected into snakebite victims. The anti-toxic immunoglobulins and their derivatives (called anti-venom, but sometimes referred to as anti-venin or anti-venene) are obtained from the serum of the healthy host animals immunized against venoms of different snake species. However, anti-serum may not protect sufficiently against systemic venom-induced toxicity, it may not reach the local area of snakebite where focal necrosis occurs, and often causes hypersensitivity 7

problems (Corrigan et al., 1978; Stahel et al., 1985; Sutherland et al., 1992; de Silva et al, 2011). Note that the Australian-made horsederived anti-sera from Commonwealth Serum Laboratories are decomplemented by heating at 56oC for 30 min, and the Fc fragment removed using specific enzyme proteolysis, to reduce the incidence of anaphylaxis. This may also be the case for other commerciallyavailable anti-sera. The production of anti-serum may be problematic in other ways: for example, anti-serum generated against neurotoxins of various species of cobra is complicated by the low immunogenicity and low molecular size of the neurotoxins (Chotwiwatthanakun et al., 2001). In this review, the term “anti-venom” will be used more generally than referring to the beneficial use of an anti-serum against snakebite, that is we use the term to describe an outcome from the medicinal plants that is beneficial against the snakebite venom.

Medicinal plants which possess anti-venom properties might be helpful as first-line or complementary therapy in the treatment of snakebite victims, especially in those countryside areas where the anti-serum is not readily available (Pithayanukul et al., 2004; Pithayanukul et al., 2005). It is likely that the medicinal plants and their extracts could also aid in recovery from snakebites with, or after, anti-serum delivered to the snakebite victims, thereby making them useful before and after medical treatment in the conventional hospital setting. Various medicinal plants which are utilized in countryside areas for the treatment of snakebites are described in literature, and many studies have now been conducted to assess and clarify the effectiveness of these plants (Biswas et al., 1977; Ratanabanangkoon et al., 1993; Chatterjee et al., 2006; Gomes et al., 2007).

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This review describes the anti-venom potential of different medicinal plants against toxicities of various species of the Naja genus. The review also highlights the limitations in current experimental studies. Data were collected during January to February 2013 using Google Scholar, PubMed, ScienceDirect, Scopus and Web of Science software. Various search terms were used, for example, medicinal plants, ethnopharmacology, cobra, Naja, anti-venom, (or antivenom), anti-venin, anti-venene, snakebite and toxicity. The searches were limited to peer-reviewed journals written in English with the exception of books and a few articles in foreign languages.

2. Traditional uses of anti-venom medicinal plants

The plants we describe in this review as having anti-venom, or anti-toxic, properties against Naja snakebite have multiple other purposes in traditional medicine. For example, Hemidesmus indicus has been used as a tonic, diuretic, diaphoretic and demulcent in Unani and Ayurvedic systems of medicines (Chatterjee et al., 2006); Withania somnifera has been used in Ayurvedic medicine to prevent disease in the elderly, athletes and during pregnancy. It has been prescribed for longevity, arthritis and rheumatism, and as a tonic (Mishra et al., 2000); Mucuna pruriens is found in America, Asia, India, and Africa. The plant has been used in Indian and Ayurvedic medicines for Parkinsonism (Katzenschlager et al., 2004); Annona senegalensis is found in northern Nigeria and has been used traditionally as a pain reliever and stimulant (Mustapha, 2013); Fagonia cretica is found in desert regions of India, Pakistan, parts of Europe and Africa. Traditional practitioners have prescribed this plant for breast cancer, asthma, urinary discharge, toothache, liver trouble, vomiting, dysentery, fever, skin diseases and stomach troubles. The plant reduces fever and also possesses astringent properties (Hussain et al., 2007; Lam et al., 2012); Mimosa pudica is native to Central and South America. It has been prescribed by 9

practitioners of Ayurvedic medicine for the treatment of uterine and vaginal complications, fatigue, asthma, inflammation, leucoderma, dysentery, leprosy and blood diseases (Joseph et al., 2013); Seed powder of Pithecellobium dulce has been prescribed for the treatment of diabetes (Nagmoti and Juvekar, 2013); and Areca catechu is mentioned in Old Indian scripts as a remedial agent of anemia, obesity, leprosy, leucoderma, and has de-worming activities (Amudhan et al., 2012). Therefore the uses of medicinal plants are broad, and readers are encouraged to look up various compendia, for example, Compendium of Materia Medica or Compendium of Medicinal Plants, for extra reading.

3. Anti-venom (anti-toxic) activities of medicinal plants against various Naja species The anti-venom or protective activities of medicinal plants against various Naja species, and their taxonomy, are summarized in Table 1. Depending on the plants, the roots, rhizomes, leaves and twigs, seeds, bark or nutgall are used to produce the plant extracts. In the following section, a summary of some anti-venom, or anti-toxic, activities of plants against specific cobra types is presented.

3.1. Anti-venom activity against Naja kaouthia Aqueous and alcoholic extracts of dried roots of Mimosa pudica were tested for their inhibitory activity on lethality, myotoxicity and toxic enzymes of N. kaouthia venom (Mahanta and Mukherjee, 2001). These authors found that the LD50 (lethal dose for 50% of the test population) of crude N. kaouthia venom was approximately 0.6 mg/kg body weight of mice. To evaluate the inhibition of lethality, different extracts were preincubated with venom for 60 min and then injected intravenously into albino mice, in comparison with mice injected only with the venom. Aqueous and methanol extracts were prepared from 0.4 g of dried and powdered roots of M. pudica 10

suspended in 200 ml of the solvent. One of the aqueous extracts was prepared at room temperature and the other by boiling the powdered root for 5 min. Methanol extracts were prepared at room temperature. The dried residue of the preparations was resuspended in saline at a known concentration (usually 0.1 mg/ml). For the venom inhibition study, various doses of venom were preincubated with the extracts before being injected intravenously into the mice, compared with animals dosed with venom only. A myotoxicity assay was also utilized: 15 µg of venom protein was mixed with different doses (in µg) of root extracts and incubated for 60 min at 37°C followed injection into the right gastrocnemius of mice. Control animals received a similar injection of venom only. After 4 h, blood samples were obtained for measurement of plasma creatine phosphokinase activity. About 200 µg of normal water extract and hot water extracts of roots were able to neutralize 35 µg and 20 µg of crude venom, respectively. The myotoxic effect of the venom was significantly reduced by the water extracts. Methanol extracts were deficient of significant inhibitory activity against lethality and myotoxicity. All extracts neutralized acetylcholinesterase, plasma protease and PLA2 activity of N. kaouthia venom in in vitro assays. Highest inhibition of protease activity was produced by the hot water extract, while highest PLA2 and acetylcholinesterase enzyme neutralization was found with the room temperature water extract (Mahanta and Mukherjee, 2001).

Polyphenols from aqueous extracts of the seeds of Areca catechu (AC), bark of Pithecellobium dulce (PD) and Pentace burmanica (PB), and nutgalls of Quercus infectoria (QI) were evaluated for possible cobra venom neutralization properties, using an in vitro toxicity assay (Pithayanukul et al., 2005). Chopped plant material was thrice macerated with aqueous ethanol (50%) and the solvent was partially evaporated under reduced pressure. Hexane was used to further defat the extract and again dried at 80-90°C. The ferric chloride test was used to determine the presence of tannins: hydrolysable tannins produced a blue-black color; while a combination of 11

hydrolysable and condensed tannins generated a greenish color. Tannin content of these plant extracts was 6.45%, 7.80%, 9.93%, and 34.68%, respectively, in AC, PD, PB and QI. Extracts from AC, PD and PB contained a combination of both hydrolysable and condensed tannins, while QI possessed only hydrolysable tannins. All the plant extracts totally inhibited lethality to Swiss albino mice, using different tannin concentrations (80, 431, 220, and 604 µg/mouse, respectively, for AC, PD, PB and QI). Their ED50 (effective dose for 50% of the test population) was calculated as 62, 364, 185, and 510 µg/mouse, respectively. These analyses showed that plants with a combination of different tannin types possess greater anti-venom activity. In a separate experiment, AC, QI and PD, possessing at least 30 µg of tannins, entirely protected male Sprague-Dawley rats from the necrotizing activity of the venom, while comparable results were detected at 45 µg for PB extract. Acetylcholinesterase activity of venom was inhibited at the tannin content of 0.1% (w/v) for the PB, AC and PD extracts, and 1.0% (w/v) for QI extracts. This again displayed the usefulness of presence of condensed tannins, or a mixture of tannins, as anti-venom agents. It was also demonstrated that inhibition of the nicotine acetylcholine (ACh) receptor (nAChR), and precipitation of venom proteins by plant polyphenols, contribute to anti-venom properties (Pithayanukul et al., 2005).

Chatterjee et al. (2006) investigated the usefulness of lupeol acetate (LA), from a methanol extract from roots of Hemidesmus indicus. The dried and powdered root (100 g) of H. indicus was extracted in methanol, centrifuged (2000 rpm) then the suspension was evaporated to dryness. The residue was placed on top of a silica gel column and eluted with petroleum ether:chloroform (1:1; 10 fractions), then chloroform:methanol (1:1; 10 fractions). The fraction eluates were evaporated to dryness and tested for venom neutralization in experimental animals (Swiss albino mice, Wistar rats and guinea pigs). They found that LA could neutralize the 12

activity of intravenously-delivered venom of N. kaouthia as well as the deadly viper Daboia russelii. LA significantly neutralized N. kaouthia venom-induced lethality, cardiotoxicity, neurotoxicity and respiratory injury in experimental animals. It inhibited the lethality of cobra venom at an ED50 = 59.8 µg, and respiratory changes at an ED50 = 66.3 µg, when infused through the jugular vein. It also neutralized 4 units of PLA2 activity, and one fold of minimal cardiotoxic dose (in an in vitro experiment using isolated guinea pig auricle). LA did not demonstrate any protective effect against venom-induced neurotoxicity. When delivered with a polyvalent snake venom anti-serum, LA also provided increased protection (protective ratios of up to 4.8 LD50s) compared with the anti-serum alone (protective ratios of up to 2 LD50s), compared with untreated snake venom (Chatterjee et al., 2006).

In a similar rodent study by Gomes et al. (2007), β-sitosterol and stigmasterol were identified as active components from the methanol root extract of Pluchea indica. Silica gel chromatography was used to purify the active constituents and spectroscopy was used for structure determination. In a study using Swiss albino mice and tail vein injections, the combination of these sterols (3:1, respectively; 100 µg) protected against lethality of N. kaouthia venom (ED50 = 49.5 µg), respiratory dysfunction (ED50 = 55.9 µg), neurotoxicity (ED50 = 54.4 µg), and cardiotoxicity (ED50 = 50.3 µg), and also neutralized 8 units of PLA2 activity (ED50 = 56.2 µg). The sterol mixture also increased anti-serum activity up to 355%, thereby demonstrating the benefit of extracting the active ingredients from the medicinal plants for use in a clinical situation where the anti-serum is available.

The rediocides A and G found in the roots of Trigonostemon reidioides are other medicinal plant extracts of interest against the effects of the venom of N. kouthia. Alpha-cobratoxin, the neurotoxin isolated from the venom of N. kaouthia, causes paralysis by preventing 13

ACh from binding to nAChRs (Utsintonga et al., 2009). Rediocides A and G from T. reidioides increased the survival time of mice when administered intravenously half an hour before the alpha-cobratoxin. SDS-polyacrylamide gel electrophoresis revealed that the rediocides were able to bind ACh binding protein with alpha-cobratoxin, thereby blocking its nAchR binding site (Utsintonga et al., 2009). Whilst this knowledge has limited application in a field snakebite situation, the results again emphasize the benefit of identifying the active components of plant extracts, and help explain the pathophysiological pathways of protection that might also be analyzed in other plant extracts.

3.2. Anti-venom activity against Naja sputatrix Mucuna pruriens is used by native Nigerians as a prophylactic for snakebite (Tan et al., 2009). Aqueous extracts of seeds of M. pruriens (MPE) were evaluated for their protective effect against the toxic properties of N. sputatrix (Javan spitting cobra). Rats were pre-treated with MPE seed extract (20 mg/kg; 7, 14 or 21 days prior to venom) and then challenged with an intravenous injection of the venom. The effectiveness of anti-MPE antibody to neutralize the lethalities of the venom was investigated by in vitro neutralization. MPE pre-treatment conferred effective protection against lethality of N. sputatrix venom. Indirect enzyme-linked immunosorbent assay (ELISA) showed 324% cross reaction between the venom of N. sputatrix and rabbit IgG anti-MPE, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. The ELISA technique is typically used to determine a presence and/or concentration of a certain antigen by binding with an immobilized specific antibody and detecting, for example, a subsequent color change.

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The MPE work has been continued in recent years and investigators have now demonstrated effective improvement in neuromuscular, respiratory and cardiovascular disorders induced from N. sputatrix venom (Fung et al., 2011; 2012). Pretreatment with MPE (250 µg/mg) for three weeks prevented 2 out of 3 rats from death by venom injection, by protecting against vascular damage in the heart and liver (Fung et al., 2011). Crude venom injection of N. sputatrix decreased mean blood pressure (30%) within the first minute of administration, and then no change in blood pressure was observed for a 30 min period. After 30 min, further progressive reduction in blood pressure occurred. Pretreatment with MPE inhibited the progressive decline in blood pressure after 30 min but failed to change the initial fall in blood pressure. Pretreated animals also retained 85% of the baseline respiratory rate, demonstrating significant protection by MPE against respiratory paralysis. Pretreatment with MPE also partially protected against the neuromuscular blocking effect of the venom, but long lasting protection was not observed (Fung et al., 2011).

MPE pretreatment (250 µg/ml) against N. sputatrix venom toxicity was investigated in vitro using spontaneously-beating atria and aortic rings isolated from rats (Fung et al., 2012). MPE was prepared by soaking M. pruriens seed meal in distilled water for 1 day followed by centrifugation at 1000 g for 20 min. The supernatant was dried then resuspended to a known concentration. N. sputatrix venom-induced depression of atrial contractility and rate in the isolated atrial preparations was protected by MPE, but the extract had no effect on the venom-induced contractile response of the aortic ring preparations. These observations suggest that the protective effect of MPE involves a direct protective action on the heart function, and that protective effect does not involve action on blood vessel contraction. The authors also suggest that MPE may contain a novel cardioprotective agent with potential therapeutic value. This obviously warrants further study. 15

3.3. Anti-venom activity against Naja naja A modified ELISA was developed to screen for plants with inhibitory activity to N. naja siamensis cobra venom. Plant extracts were incubated with the venom, immobilized on 96-well microtiter plates, for 60 min before the subsequent addition of anti-serum. Loss of affinity of antigens, in venom, to their specific antibodies, in anti-serum, was predicted. Western immunoblotting analysis was used to reveal target molecules in venom, to which inhibitors in plant extracts reacted (Daduang et al., 2005). Extracts of Curcuma parviflora, C. longa and C. cf. zedoaria rhizomes blocked the attachment of anti-N. naja siamensis antibody to cobra venom antigen. C. cf. zedoaria maximally inhibited the binding, while the activity of C. parviflora was significantly decreased. In animal experiments (mice, delivered snake venom intravenously), C. cf. zedoaria extract enhanced the diaphragm muscle contraction time significantly and inhibited protein destruction by the venom. C. parviflora was less potent compared with C. cf. zedoaria in increasing contraction time and was not able to block protein destruction. C. longa did not display any encouraging results. The authors suggested that the modified ELISA could classify plants into an inhibition range of (in this study, 0 to 86%), and recommended the assay as a preliminary screening method for large numbers of plant samples.

Different plant-derived bioactive compounds, such as flavones, tannic acid, quercertin, aristolochic acid and curcumin, dosedependently inhibited the activity of hyaluronidase, which was purified from the venom of N. naja. Among all compounds, quercetin and aristolochic acid showed complete inhibition of the enzyme activity (Girish and Kemparaju, 2005) thereby demonstrating the benefit of identifying the bioactives in medicinal plant specimens. A glycoprotein was isolated from the extract of Withenia somnifera 16

roots. This glycoprotein neutralized most of the PLA2 isoforms in N. naja venom, dose-dependently. Maximal inhibitory activity was observed against a PLA2 isoform (NN-XIa-PLA2) at a mole-to-mole ratio of 1:2 (NN-XIa-PLA2:glycoprotein). The glycoprotein was not able to inhibit neurotoxic symptoms, but it decreased the lethality of the venom, exhibited by a 10-fold increase in survival time of Swiss wistar mice, and the increased LD50 for NN-XIa-PLA2 (Machiah and Gowda, 2006). The purified glycoprotein from W. somnifera, at an IC50 of 52 µg (IC50 = the amount needed to inhibit a given biological process), also completely inhibited the hyaluronidase activity of N. naja venom at a concentration of 1:1 (w/w) (Machiah et al., 2006).

In a study that aimed to determine the anti-venom potential of methanol extract from the aerial parts (leaves and twigs) of Fagonia cretica on haemorrhage induced by venom from N. naja karachiensis, the vitelline veins of fertilized hens’ eggs in their shells were studied.The haemorrhagic response of the vitelline veins to the venom was dose-dependent from 0.1 to 4.0 µg per 1.5 µl phosphate buffered saline (PBS). The extract effectively eliminated and neutralised, in a dose-dependent manner, the haemorrhagic activity of the venom. The minimal effective neutralising dose of F. cretica extract was found to be 15 µg per 1.5 µl PBS. This hemorrhagic test may have advantages in terms of saving time, eliminating the need for costly test equipment, and also reducing the need for small animal toxicity studies (Razi et al., 2011).

3.4. Anti-venom activity against Naja nigricollis nigricollis Annona senegalensis is used traditionally to treat victims of snakebite (Adzu et al., 2005). The potency of the methanol extract of the root bark of the plant was tested against N. nigricollis nigricollis venom delivered intravenously (10 mg/ml) in Wistar rats. The brine 17

shrimp (Artemia saline) lethality test was used to investigate the activity of the extract and to check its toxicity against zoologic systems. The extract dose-dependently enhanced the survival of rats compared with the venom alone, but was not able to eliminate lethality completely. The toxic signs that were recorded were: dyspnea and/or coma; spontaneous neurological abnormalities, for example, convulsion and tremors; and respiratory or movement impairment. The extract decreased early toxic signs and also lessened their severity. However it failed to restore the activities of the liver enzymes serum glutamate-oxalate-transaminase and serumpyruvate-transaminase, indicating some ineffectiveness against liver toxicity. The brine shrimp lethality test indicated an LD50 of 232.7 µg/ml for the extract itself (Adzu et al., 2005).

4. Limitations of medicinal plant anti-venom studies Critical evaluation of experimental studies for testing the therapeutic potential of medicinal plants has revealed some limitations. A major limitation is the use of experimental animals, mainly rats and mice, and in vitro models to study anti-venom properties. In these cases, the plant extract is typically delivered intravenously prior to, or concurrent with, the delivery of the snake venom, a scenario unlikely to be possible in the field where the medicine would be delivered after the snakebite. We found the differences in describing toxicities in the reports (as LD50, ED50, IC50, or a fold change on LD50) also limited interpretation and we suggest some sort of standardization of toxicity and lethality reporting is needed. Another limitation is the lack of evaluation of pharmacological activities that are of potential clinical importance, due to a shortage of clinical data. Comparison of pharmacological activities of medicinal plants with reference drugs or known anti-sera is suggested in future studies. Identification and isolation of active constituents

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responsible for any protection against venom toxicity is also essential. The development of effective therapeutic agents relies on a better understanding of anti-venom activity, and this should be emphasized in future studies.

5. Conclusion Many experimental investigations have shown that medicinal plants and their extracts significantly protect against lethality, cardiotoxicity, renal failure, neurotoxicity, myotoxicity, hemorrhage and respiratory paralysis induced by cobra snake (Naja spp) venom. Presently, systemic anti-sera, developed commercially against snake venom, are the treatment of choice. These anti-sera, however, provide little defense against local tissue damage due to their ineffectiveness in reaching the envenomed site, and many are associated with anti-serum reactions that in themselves may be harmful. Treatment with medicinal plants may provide an alternative to anti-serum treatments, or may be used to complement the activity and effectiveness of available snake venom anti-serum. The plants or their extracts can neutralize various toxic enzymes found in the venom, such as hyaluronidase, PLA2, acetylcholinesterase and plasma proteases. Anti-venom medicinal plants could possess several constituents that account for venom neutralization. These include lupeol acetate; sterols such as β-sitosterol and stigmasterol; rediocides A and G; quercertin, aristolochic acid, and curcumin; as well as the broad chemical groups such as tannins, glycoproteins, and flavones. Whilst there has been some progress in identifying the plantderived bioactive compounds in traditionally used medicinal plants, more research is needed for the development of plant-derived therapeutic antagonists against snakebite for the community in need.

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Graphical Abstract (for review)