Hepatoprotective and antioxidant effects of Cymbopogon citratus Stapf (Lemon grass) extract in paracetamolinduced hepatotoxicity in rats

Purpose: To investigate the protective effect of Cymbopogon citrates Stapf. (CS, lemongrass) extract on paracetamol (PCM)-induced hepatotoxicity in rats.Methods: The rats were orally administered CS extract (1000 mg/kg/day) for 30 days prior to induction of hepatotoxicity by a single oral administration of PCM (3 g/kg). Hepatoprotection was assessed by measuring the level of hepatic markers including aspartate transaminase (AST), alanine transaminase (ALT) and oxidant/antioxidant markers including Malondialdehyde (MDA), protein carbonyl, and glutathione (GSH) in liver homogenate and serum. Phytochemical screening of the CS extract was also performed.Results: Phytochemical screening of the extract indicate the presence of tannins, flavonoids, and phenolic compounds. Elevation of serum AST, ALT, and MDA levels along with depletion GSH in the liver were observed in rats treated with PCM alone compared with control (p < 0.05). Pre-treatment of the animal with CS extract reduced the levels of hepatic markers (AST and ALT). Pre-treatment with CS extract also significantly reduced oxidative stress induced by PCM as shown by an increase in GSH level and reduction of MDA compared to rats treated with PCM alone (p<0.05).Conclusion: The results indicate that CS possesses antioxidant activity and it exerts its effect by reducing lipid peroxidation and restoring GSH. Pre-treatment with CS extract reduces oxidative stress and ameliorates hepatic injury induced by PCM.Keywords: Cymbopogon citratus, Antioxidant, Oxidative stress, Hepatotoxicit

In Vitro neurotoxicity and myotoxicity of Malaysian Naja sumatrana and Naja kaouthia venoms: Neutralization by monovalent and Neuro Polyvalent Antivenoms from Thailand

Naja sumatrana and Naja kaouthia are medically important elapids species found in Southeast Asia. Snake bite envenoming caused by these species may lead to morbidity or mortality if not treated with the appropriate antivenom. In this study, the in vitro neurotoxic and myotoxic effects N. sumatrana and N. kaouthia venoms from Malaysian specimens were assessed and compared. In addition, the neutralizing capability of Cobra Antivenom (CAV), King Cobra Antivenom (KCAV) and Neuro Polyvalent Antivenom (NPAV) from Thailand were compared. Both venoms produced concentration-dependent neurotoxic and myotoxic effects in the chick biventer cervicis nerve-muscle preparation. Based on the time to cause 90% inhibition of twitches (i.e. t90) N. kaouthia venom displayed more potent neurotoxic and myotoxic effects than N. sumatrana venom. All three of the antivenoms significantly attenuated venom-induced twitch reduction of indirectly stimulated tissues when added prior to venom. When added after N. sumatrana venom, at the t90 time point, CAV and NPAV partially restored the twitch height but has no significant effect on the reduction in twitch height caused by N. kaouthia venom. The addition of KCAV, at the t90 time point, did not reverse the attenuation of indirectly stimulated twitches caused by either venom. In addition, none of the antivenoms, when added prior to venom, prevented attenuation of directly stimulated twitches. Differences in the capability of antivenoms, especially NPAV and CAV, to reverse neurotoxicity and myotoxicity indicate that there is a need to isolate and characterize neurotoxins and myotoxins from Malaysian N. kaouthia and N. sumatrana venoms to improve neutralization capability of the antivenoms

Neurotoxic, cytotoxic and cardiovascular effects of some Australasian elapid venoms

A range of toxic components found in Australasian elapid venoms have been postulated to account for the clinical outcomes of envenoming. Depending on the species of snake, these outcomes may include neurotoxicity with flaccid paralysis, coagulopathy with spontaneous systemic hemorrhage, sudden cardiovascular collapse, and myotoxicity with skeletal muscle breakdown. In the present study, we investigated the toxins and mechanisms behind these often life-threatening symptoms from the venom of four species of Australasian elapid snakes (Acanthophis praelongus, Acanthophis rugosus, Oxyuranus scutellatus and Pseudonaja textilis) with emphasis on their neurotoxic, cytotoxic and cardiovascular activities. Previously, post-synaptic neurotoxins were postulated to be the primary components responsible for neurotoxicity following death adder envenoming in humans. However, neurotoxicity is often poorly reversed by antivenom or anticholinesterase suggesting that death adder venoms may contain pre-synaptic neurotoxins that do not respond, as well as post-synaptic neurotoxins, to antivenom. In this study, phospholipase A2 (PLA2) neurotoxins, P-EPTX-Ap1a and P-EPTX-Ar1a, were isolated from the venoms of Acanthophis praelongus (Northern death adder) and Acanthophis rugosus (Irian Jayan death adder), respectively. P-EPTX-Ap1a (20-100 nM) and P-EPTX-Ar1a (20-100 nM) inhibited indirect twitches of the chick biventer cervicis nerve-muscle preparation without affecting contractile responses to nicotinic receptor agonists. Pre-incubation of 4-bromophenacyl bromide (1.8 mM) markedly reduced the effect of both toxins on twitch height suggesting that PLA2 activity plays an important role to induce pre-synaptic neurotoxicity. Some snake pre-synaptic PLA2 neurotoxins have been reported to also possess myotoxic activity and promote cell death. Therefore, pre-synaptic neurotoxins from Oxyuranus scutellatus (Papuan taipan) and A. rugosus venoms, cannitoxin and P-EPTX-Ar1a, respectively, as well as the whole venoms, were examined for myotoxic and cytotoxic activities. Based on size-exclusion high performance liquid chromatography (HPLC) analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. A. rugosus venom induced significantly higher myotoxic activity than that of O. scutellatus venom in the chick biventer cervicis nerve-muscle preparation. In a rat skeletal muscle cell line (L6), A. rugosus venom and P-EPTX-Ar1a induced significantly greater cytotoxicity than O. scutellatus venom and cannitoxin. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. These activities were independent of the proportion of pre-synaptic neurotoxin and PLA2 activity in the whole venoms. The cytotoxicity and disruption in neurotransmitter (i.e. acetylcholine) release induced by snake pre-synaptic neurotoxins have been postulated to be due to an increase in intracellular Ca2+. In the current study, P-EPTX-Ar1a, a pre-synaptic neurotoxin from A. rugosus venom, caused an increase in cytoplasmic calcium and membrane depolarization in primary dorsal root ganglion (DRG) neurons. Influx of Ca2+ did not occur in Ca2+-free Hank’s solution with EGTA. Ca2+ influx was also significantly reduced in the presence of nifedipine or agatoxin indicating that L-type and P/Q-type voltage-gated calcium channels, respectively, are involved in Ca2+ influx. Patch clamp studies in whole cell mode demonstrated that P-EPTX-Ar1a evoked inward currents in DRG neurons which were blocked by SKF96365, a cationic channel blocker, suggesting that P-EPTX-Ar1a induced-Ca2+ influx may be related to membrane depolarization through the activation of cationic channels. Sudden cardiovascular collapse following envenoming by some Australasian elapids (e.g. Oxyuranus spp. or Pseudonaja spp.) is a poorly understood cause of mortality and morbidity. Previous studies showed that administration of O. scutellatus or Pseudonaja textilis (eastern brown snake) venoms cause sudden cardiovascular collapse in anaesthetized animals. In the current study, O. scutellatus venom failed to affect force of contraction and conductivity in rat isolated heart preparations. In anaesthetized rats, sub-lethal doses of O. scutellatus venom (5-10 µg/kg, i.v.) produced transient hypotension while 20 or 50 µg/kg (i.v.) of venom produced cardiovascular collapse in all animals tested. The administration of P. textilis venom (10 or 20 µg/kg, i.v.) to anaesthetized rats also induced sudden collapse. Interestingly, cardiovascular collapse induced by O. scutellatus or P. textilis venoms was attenuated by prior administration of small ‘priming’ doses of some Australasian elapid venoms (i.e. O. scutellatus, P. textilis or A. rugosus venoms) or a venom from an exotic snake i.e. Daboia russelii limitis. Prior administration of polyvalent snake antivenom or heparin also protected against sudden collapse induced by O. scutellatus or P. textilis venoms. A prothrombin activator-like compound, PTV3, was partially purified from P. textilis venom. Protein bands of PTV3 displayed homology to catalytic and non-catalytic subunits of prothrombin activator ‘pseutarin C’ of P. textilis venom. Administration of PTV3 (10 and 20 µg/kg, i.v.) induced rapid cardiovascular collapse which was abolished by prior administration of small priming doses of PTV3 (2 and 5 µg/kg, i.v.) or heparin. This indicates that the prothrombin activator-like compound, PTV3 may contribute to sudden cardiovascular collapse in anaesthetized rats. In isolated rat mesenteric arteries, P. textilis venom but not PTV3 induced endothelium-dependent relaxation. O. scutellatus venom also induced both endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric arteries which were inhibited by indomethacin, IbTX or Rp-8-CPT-cAMPs suggesting that vascular relaxation induced by the venom may be due to a combination of release of dilator autacoids and a direct relaxing effect on vascular smooth muscle involving the cAMP/protein kinase A (PKA) cascade. We subsequently isolated a PLA2 fraction (OSC3) from O. scutellatus venom and examined the hypotensive and vascular relaxant responses. OSC3 displayed high PLA2 activity and caused endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric artery rings. Indomethacin and Rp-8-CPT-cAMPs markedly attenuated vascular relaxation induced by OSC3 on endothelium-denuded mesenteric arteries. Reverse-phase HPLC analysis of OSC3 indicated the presence of 2 major components, i.e. OSC3a and OSC3b. Both components induced a hypotensive effect in anaesthetized rats which was attenuated by prior administration of indomethacin. The amino acid sequencing indicated that the active components of OSC3 showed homology to PLA2 toxins from O. scutellatus (coastal taipan) venom. This finding indicates that PLA2 of O. scutellatus venom contains indirect relaxant and hypotensive effects that involve other vasoactive compounds e.g. PGI2 or PKA. It can be concluded that Australasian elapid induced-early cardiovascular collapse involves a combination of mediator-induced relaxation and prothrombin activator-like compound. In conclusion, this study examined the mechanisms behind snake pre-synaptic neurotoxin-induced neurotoxic and cytotoxic activities and the contributing factors to early cardiovascular collapse following Australasian elapid envenoming. These data provide useful insights for the clinical management of snake envenomed patients

In vitro and in vivo studies of cardiovascular disturbance following envenomation by Malayan Krait (Bungarus Candidus)

Snake envenoming is a serious health problem that affect the population of developing and underdeveloped countries, causing morbidity and mortality. Malayan krait (Bungarus candidus) is an elapid species found in Southeast Asia. The venom contains highly potent neurotoxins that inhibit neurotransmission at the neuromuscular junction. Interestingly, cardiovascular symptoms such as hyponatraemia and blood pressure irregularities which not related to the known neuromuscular blockade activity of the venom have been reported in some victims. However, potential mechanism behind these cardiovascular symptoms has yet to be identified. The understanding mechanism of this cardiovascular disturbance would enable the clinical to anticipate the clinical prognosis and design a better management for Malayan krait envenoming treatment. Methods In this study, we have examined the cardiovascular effects of Bungarus candidus venoms from northeastern (BC-NE) and southern (BC-S) parts of Thailand using anaesthetized rat and isolated rat thoracic aorta preparation. Results A comparison of the reverse-phase-HPLC profiles of both venoms showed some differences with peak elution times. BC-NE and BC-S (50-100 μg/kg, i.v.) produced a significant dose-dependent hypotensive response in anaesthetized rats which was abolished by prior administrations of hexamethonium (10 mg/kg, i.v.) and recommended dose of Malayan krait monovalent antivenom. BC-NE and BC-S (3-100 μg/ml) also caused endothelium-dependent relaxation in isolated rat aortic rings. The pre-incubation of L-NAME (0.2 mM) prior to the addition of venom attenuated the aortic relaxation effect, suggesting the involvement of endothelium derived nitric oxide. Conclusion These finding indicate that the cardiovascular disturbance observed after envenoming by Malayan krait may involve autonomic reflex and vascular nitric oxide mechanisms

Histopathological Changes in the Liver, Heart and Kidneys Following Malayan Pit Viper (<i>Calloselasma rhodostoma</i>) Envenoming and the Neutralising Effects of Hemato Polyvalent Snake Antivenom

Calloselasma rhodostoma (Malayan pit viper) is a medically important snake species that is widely distributed across Southeast Asia. Systemic coagulopathy causing severe haemorrhage and local tissue injury is commonly observed following C. rhodostoma envenoming. However, nephrotoxicity and congestive heart failure were previously reported in a patient who had a long length of hospital stay. In this study, we determined the effect of C. rhodostoma envenoming on cardiovascular disturbances and the associated morphological changes in the liver, heart and kidneys using animal models. We also evaluated the efficacy of Hemato polyvalent antivenom (HPAV; Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) in neutralising the histopathological effects of C. rhodostoma venom. The intravenous (i.v.) administration of C. rhodostoma venom (1000 µg/kg) caused a rapid decrease in mean arterial pressure (MAP) followed by complete cardiac collapse in anaesthetized rats. Moreover, the intraperitoneal (i.p.) administration of C. rhodostoma venom (11.1 mg/kg; 3 × LD50) for 24 h caused cellular lesions in the liver and heart tissues. C. rhodostoma venom also induced nephrotoxicity, as indicated by the presence of tubular injury, interstitial vascular congestion and inflammatory infiltration in the whole area of the kidney. The administration of HPAV, at manufacturer-recommended doses, 15 min prior to or after the addition of C. rhodostoma venom reduced the extent of the morphological changes in the liver, heart and kidneys. This study found that experimental C. rhodostoma envenoming induced cardiovascular disturbances, hepatotoxicity and nephrotoxicity. We also highlighted the potential broad utility of HPAV to neutralise the histopathological effects of C. rhodostoma venom. The early delivery of antivenom appears capable of preventing envenoming outcomes

Hypotensive and vascular relaxant effects of phospholipase A₂ toxins from Papuan taipan (Oxyuranus scutellatus) venom

Phospholipase A₂ (PLA₂) toxins are common and abundant components of Australasian elapid venoms. These toxins are associated with a range of activities including neurotoxicity, myotoxicity and coagulation disturbances. We have recently reported that sudden cardiovascular collapse induced by Papuan taipan (Oxyuranus scutellatus) venom involves a combination of the release of dilator autacoids and a direct effect on the smooth muscle. In this study, we aimed to isolate PLA₂ components from Papuan taipan venom and investigate their contribution to the hypotensive action of this venom. O. scutellatus venom was fractionated using size-exclusion high performance liquid chromatography (HPLC), and fractions screened for activity in anaesthetized rats. Fraction three from O. scutellatus venom (i.e. OSC3, 14.2 ± 1.0% of whole venom) produced a 64% decrease in mean arterial pressure. Reverse-phase HPLC indicated that OSC3 consisted of two major components (i.e. OSC3a and OSC3b). OSC3a and OSC3b produced a significant hypotensive response in anaesthetized rats which were attenuated by prior administration of indomethacin or the combination of mepyramine and heparin. N-terminal analysis indicated that OSC3a and b displayed sequence homology to PLA₂ toxins isolated from coastal taipan (O. scutellatus scutellatus) venom. These findings indicate that PLA₂ components may play an important role in the development of hypotension and vascular relaxation which may contribute to the effects observed after envenoming by these Australasian elapids

In Vitro Toxic Effects of Puff Adder (Bitis arietans) Venom, and Their Neutralization by Antivenom

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P &lt; 0.05; n = 3–5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom