Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule / 药学学报

The quality control of Chinese patent medicines containing animal-derived crude drugs is relatively difficult, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are relevant methods, they are usually qualitative, and quantitative indicators are either lacking or have poor specificity. This paper has proposed to use molecular quantitative technology to control the quality of Chinese patent medicines containing animal-derived crude drugs. In this study, a molecular quantitative method based on fluorescence quantitative PCR was established for the determination of Jinqian Baihua She in Jinlong Capsule. The method has good specificity, sensitivity, and repeatability. There is a good linear relationship between the content of DNA fragments and the CT (cycle threshold) value. The content of the Bungarus multicinctus-specific fragment in Jinlong Capsule is 24.1-46.6 IU·mg-1. It is suggested that the content of the specific fragment of Jinqian Baihua She should not be less than 19.3 IU·mg-1 as one of the quality control criteria of Jinlong Capsule. The study can provide a reference for the quality control of Chinese patent medicines containing animal-derived crude drugs.

Necrosis tisular local y trombocitopenia después de una intoxicación por mordedura de Bungarus multicinctusen una niña / Local tissue necrosis and thrombocytopenia following Bungarus multicinctus envenomation in a child

La intoxicación por mordedura de serpiente es un problema de salud pública global. En la población pediátrica, la intoxicación por mordedura de serpiente presenta características diferentes que en los pacientes adultos. La Bungarus multicinctus es una especie de elápido sumamente venenoso. Las presentaciones clínicas documentadas después de la intoxicación por mordedura de Bungarus multicinctus son reacciones locales mínimas, insuficiencia respiratoria, dolor generalizado e hiponatremia potencialmente mortal. Presentamos el caso de una intoxicación por mordedura de Bungarus multicinctus en una niña con manifestaciones clínicas atípicas, incluidas necrosis tisular grave y trombocitopenia con coagulopatía.

Snakebite envenoming is a global public health problem. The pediatric population poisoned by snakebite envenoming has different features than adult patients. Bungarus multicinctus is a highly venomous species of the elapid snake. The documented clinical presentations following Bungarus multicinctus envenoming are minimal local reactions, respiratory failure, general pain, and life-threatening hyponatremia. We present an uncommon case of Bungarus multicinctus envenomation in a girl with unusual clinical findings, including severe tissue necrosis and thrombocytopenia with coagulopathy.

Molecular phylogenetics of Black Cobra (Naja naja) in Pakistan

Background: Snakes are found on every continent in the world except Antarctica, and on smaller land masses. Being ecologically important, they also cause a large number of bites, leading to millions of deaths. Mitochondrial and nuclear gene sequences are being used to identify, characterize, and infer genetic biodiversity among different snake species. Furthermore, phylogenetics helps in inferring the relationships and evolutionary histories among these species. Black cobra is one of the four most venomous snakes in Pakistan. Four mitochondrial (ND4, Cytochrome b, 12S rRNA, and 16S rRNA) and four nuclear (C-mos, RAG-1, BDNF, and NT3) genes were used to trace diversity and infer the phylogenetic relationship of black cobra in Pakistan. Results: Almost similar phylogenies were obtained through maximum likelihood and Bayesian inference, showing two species of cobra in Pakistan, namely, black cobra (Naja naja) and brown cobra (Naja oxiana). All Naja species were divided into three clades: black cobra (N. naja) and brown cobra (N. oxiana) cladding with different species of Naja; N. naja (Pakistan) cladding with N. naja from Nepal; and N. oxiana showed close relationship with Naja kaouthia from Thailand and Naja siamensis from Thailand. Conclusion: It was confirmed genetically that there are two cobra species in Pakistan, i.e., black and brown cobras. This study will help in not only genetic conservation but also developing anti-venom against snake species.

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Background: Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 g/kg, i.m.) or 0.9% NaCl solution (50 L, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 g/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (1000.2 g/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 g/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 g/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom...

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 µg/kg, i.m.) or 0.9% NaCl solution (50 µL, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 µg/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (100­0.2 µg/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 µg/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 µg/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom. Conclusions: This study found that Malayan krait venoms from both populations possess myotoxic, cytotoxic and nephrotoxic activities. These findings may aid in clinical diagnosis and treatment of envenomed patients in the future.(AU)

Fatal neurotoxic envenomation following the bite of a greater black krait (Bungarus niger) in Nepal: a case report

Neurotoxic envenomation following bites by kraits (Bungarus species) is a leading cause of snakebite mortality in South Asia. Over a long time, this had been attributed only to one species, the common krait (Bungarus caeruleus). However, recent research has provided increasing evidence of the involvement of several krait species. Here, we report a fatal case of neurotoxic envenomation following the bite of a greater black krait (Bungarus niger) in Nepal. Case presentation A 33-year-old man was bitten in the outdoor corridor of his home in the eastern hills of Ilam district while handling a snake he thought to be non-venomous. He subsequently developed severe abdominal pain, frequent vomiting, and signs of neurotoxic envenomation leading to respiratory paralysis. The patient did not respond to Indian polyvalent antivenom given 4 h after the bite and died under treatment 8 h after the bite. This is the second time that a B. niger was observed in Nepal, the first documented case of envenomation by this species in the country and the sixth reported case worldwide. Conclusions Previous distribution records from eastern India and western Nepal, from western hills in Nepal, and from lowland localities in India and Bangladesh indicate risk of envenomation by B. niger throughout the low and intermediate elevations of Nepal up to at least 1,500 m above sea level. As very few people in Nepal bring killed snakes to healthcare centers and because there is a general belief among local people that there are no kraits in the hills, bites by B. niger are likely to be misdiagnosed and underreported.

Fatal neurotoxic envenomation following the bite of a greater black krait (Bungarus niger) in Nepal: a case report

Background Neurotoxic envenomation following bites by kraits (Bungarus species) is a leading cause of snakebite mortality in South Asia. Over a long time, this had been attributed only to one species, the common krait (Bungarus caeruleus). However, recent research has provided increasing evidence of the involvement of several krait species. Here, we report a fatal case of neurotoxic envenomation following the bite of a greater black krait (Bungarus niger) in Nepal. Case presentation A 33-year-old man was bitten in the outdoor corridor of his home in the eastern hills of Ilam district while handling a snake he thought to be non-venomous. He subsequently developed severe abdominal pain, frequent vomiting, and signs of neurotoxic envenomation leading to respiratory paralysis. The patient did not respond to Indian polyvalent antivenom given 4 h after the bite and died under treatment 8 h after the bite. This is the second time that a B. niger was observed in Nepal, the first documented case of envenomation by this species in the country and the sixth reported case worldwide. Conclusions Previous distribution records - from eastern India and western Nepal, from western hills in Nepal, and from lowland localities in India and Bangladesh - indicate risk of envenomation by B. niger throughout the low and intermediate elevations of Nepal up to at least 1,500 m above sea level. As very few people in Nepal bring killed snakes to healthcare centers and because there is a general belief among local people that there are no kraits in the hills, bites by B. niger are likely to be misdiagnosed and underreported.(AU)

Indian common krait envenomation presenting as fulminant myocarditis and coma: a case report.

Fulminant myocarditis is an unusual manifestation of cardiotoxicity with severe elapid snake envenoming and is meagrely reported with snake bite due to Indian common krait. We report a 12-year-old boy who was admitted in complete locked-in state and hemodynamic instability after severe neurotoxic snake envenoming by Bungarus caeruleus (Indian common krait). His hospital course was complicated with recurrent episodes of sustained ventricular tachycardia requiring defibrillation; and cardiogenic shock requiring inotropes, vasopressors and intraaortic balloon counterpulsation. Severe heart failure features secondary to fulminant toxic myocarditis persisted even after full neurological recovery requiring prolonged standard medical heart failure therapy. Patient subsequently achieved full clinical recovery and regained normal left ventricular systolic function. We also reviewed the literature on cardiac manifestations, possible mechanisms and treatment of patients with cardiotoxicity due to elapid snake bites. The importance of anticipating severe cardiovascular complications is highlighted to help formulate appropriate therapeutic strategy.

Inhibition of toxic actions of phospholipase A2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds.

Background & objectives: Phospholipase A2 (PLA2) is one of the major constituents of krait venom associated with several pathophysiological actions like myotoxicity, cardiotoxicity, neurotoxicity, etc. As there was no specific antiserum available against Bungarus fasciatus venom, this study was done with synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum to neutralize the PLA2 induced toxicities in experimental models. Methods: B. fasciatus venom phospholipase A2 fraction 38 (BF-38) was isolated by ion exchange chromatography, molecular weight was determined by mass spectrometry and its N terminal amino acid sequence was identified. Monospecific rabbit antiserum was raised against the PLA2 in presence of Freund complete adjuvant. The neutralization of PLA2 induced toxicities was done in in vitro and in in vivo models using synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum. Results: A toxic PLA2 (BF-38) was purified from the B. fasciatus venom by CM-cellulose and HPLC, of 13.17 kDa and a minor band of 7.3 kDa using ESI-MS. The 13.17 kDa PLA2 sequence was NLYQFKNMIQC. The 7.3 kDa toxin sequence was RKCLTKYSQDNES and was found to be <10 per cent w/w. Anti PLA2 rabbit antiserum produced faint precipitant band in immunogel diffusion and showed low titre value. The commercial polyvalent snake venom antiserum, anti PLA2 rabbit antiserum and the synthetic herbal compounds neutralized the PLA 2 induced toxicities at different intensities. Interpretation & conclusions: Our results suggested that synthetic herbal compound (BA) along with antiserum might provide effective protection against PLA2 induced toxicities of B. fasciatus venom.

Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE) activity. The enzyme showed optimum activity at alkaline pH (8.5) and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5) inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5) inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km) for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.(AU)

Severe Neurotoxic Envenoming and Cardiac Complications after the Bite of a ‘Sind Krait’ (<i>Bungarus </i>cf. <i>sindanus</i>) in Maharashtra, India

We report a case of severe envenoming with unusual complications and two anecdotal cases of fatalities following proven 17-scale-row ‘Sind krait’ (<i>Bungarus</i> cf. <i>sindanus</i>) bites on people sleeping in temporary huts at construction sites in Pune District, Maharashtra, India. A 25-yr-old male developed progressive neuromuscular paralysis, abdominal pain and autonomic disturbances complicated by four prolonged episodes of pulseless ventricular tachycardia requiring defibrillation, and followed by pulmonary edema secondary to impaired left ventricular systolic function and hyperfusion. There was no response to antivenom; mechanical ventilation was required for six days. Only one other case of fatal envenoming likely caused by this species had been reported previously in India. The distribution of <i>B. sindanus</i> sensu lato from eastern Afghanistan to India overlaps with that of the superficially very similar common krait (<i>Bungarus caeruleus</i>). Thus, <i>B.</i> cf. <i>sindanus</i> envenoming may be common but routinely overlooked or misdiagnosed.

Effects of Bungarus candidus (Malayan krait) venom on general circulation and renal hemodynamics in experimental animals.

Background: Many studies have reported the occurrence of lethal acute renal failure after snakebites. Bungarus candidus (Malayan krait) is a medically important venomous snake distributed widely throughout Southeast Asia. The best known features of systemic envenoming by B. candidus are neurotoxic. Objective: Obtain more information on effects of B. candidus venom on changes in systemic and renal hemodynamics in experimental animals. Methods: Twelve adult male New Zealand white rabbits were used to study the effect of B. candidus venom on general circulation and renal hemodynamics. An anesthetized animal was intravenously injected with B. candidus venom at a dosage of 50μg/kg bodyweight. All changes of parameters were observed after initial post venom injection and recorded at 30 min intervals until 150 minutes after envenomation. Results: After envenomation, cardiovascular responses showed a marked decrease in mean arterial pressure within two minutes, afterwards gradually returning closely to baseline values. There were stepwise decreases in heart rate and cardiac output, while total peripheral resistance was slightly increased. The renal hemodynamics significantly decreased by glomerular filtration rate, effective renal plasma flow and effective renal blood flow, while the filtration fraction significantly increased. Envenomed animals showed a reduction in renal fraction, while renal vascular resistance stepwise increased. The plasma potassium level tended to increase. Animals showed stepwise decreases in urinary excretion of Na+, K+ and Cl-. A marked decrease in plasma calcium level was apparent at 120 minutes, while plasma creatine phosphokinase and lactate dehydrogenase levels increased at 30-120 minutes. Conclusion: A significant drop in blood pressure was attributed to a sustained fall in cardiac output, which would be associated with a reduction in heart rate. Sustained hypotension would contribute to reduction of renal blood flow, which results in decreased GFR.

Enzymatic and immunological properties of Bungarus flaviceps (red-headed krait) venom

Bungarus flaviceps (red-headed krait) venom presents an intravenous LD50 of 0.32 ìg/g and exhibits enzymatic activities similar to other Bungarus toxins. ELISA cross-reactions between anti-Bungarus flaviceps and a variety of elapid and viperid venoms were observed in the current study. Double-sandwich ELISA was highly specific, since anti-B. flaviceps serum did not cross-react with any tested venom, indicating that this assay can be used for species diagnosis in B. flaviceps bites. In the indirect ELISA, anti-B. flaviceps serum cross-reacted moderately with three different Bungarus venoms (9-18 percent) and Notechis scutatus venom, but minimally with other elapid and viperid toxins. The results indicated that B. flaviceps venom shares common epitopes with other Bungarus species as well as with N. scutatus. The lethality of the B. flaviceps venom was neutralized effectively by antiserum prepared against B. candidus and B. flaviceps toxins and a commercial bivalent elapid antivenom prepared against B. multicinctus and Naja naja atra venoms, but was not neutralized by commercial antivenoms prepared against Thai cobra, king cobra and banded krait. These data also suggested that the major lethal toxins of B. flaviceps venom are similar to those found in B. multicinctus and B. candidus venoms.(AU)

Purification of an L-amino acid oxidase from Bungarus caeruleus (Indian krait) venom

Snake venoms are rich in enzymes such as phospholipase A2, proteolytic enzymes, hyaluronidases and phosphodiesterases, which are well characterized. However, L-amino acid oxidase (LAO EC.1.4.3.2) from snake venoms has not been extensively studied. A novel L-amino acid oxidase from Bungarus caeruleus venom was purified to homogeneity using a combination of ion-exchange by DEAE-cellulose chromatography and gel filtration on Sephadex® G-100 column. The purified monomer of LAO showed a molecular mass of 55 ±1 kDa estimated by SDS-PAGE. The specific activity of purified LAO was 6,230 ± 178 U/min/mg, versus 230 ± 3.0 U/min/mg for the whole desiccated venom, suggesting a 27-fold purification with a 25% yield. Optimal pH and temperature for maximum purified enzyme activity were 6.5 and 37ºC, respectively. Platelet aggregation studies show that purified LAO inhibited ADP-induced platelet aggregation dose-dependently at 0.01 to 0.1 µM with 50% inhibitory concentration (IC50) of 0.04 µM, whereas at a 0.08 µM concentration it did not induce appreciable aggregation on normal platelet-rich plasma (PRP). The purified protein catalyzed oxidative deamination of L-amino acids while the most specific substrate was L-leucine. The purified LAO oxidizes only L-forms, but not D-forms of amino acids, to produce H2O2. The enzyme is important for the purification and determination of certain amino acids and for the preparation of α-keto acids.(AU)

Preliminary studies with a neurotoxin obtained from Bungarus caeruleus venom

The neurotoxin purified from the venom of Bungarus caeruleus causes a neuromuscular blockade on acetylcholine-induced muscle twitch response in isolated frog rectus abdominis muscle preparation. Neuromuscular blockade produced by d-tubocurarine on acetylcholine-induced muscle twitch response in an isolated frog rectus abdominis muscle preparation was reversed to normal muscle twitch response in presence of neostigmine. Whereas the purified neurotoxin produced an irreversible neuromuscular blockade in presence of the same strength of neostigmine. As it is already known, botulinum toxin, which also brings about neuromuscular blockade, is effectively used as a drug in the treatment of painful movement disorders. Since the purified toxin also causes paralysis of the muscle, we propose its possible efficacy in the treatment of neuromuscular disorders.(AU)