Lignosus rhinocerus, the tiger milk mushroom, is one of the most important medicinal mushrooms used by the indigenous people of Southeast Asia and China. It has been used to treat breast cancer. A cold water extract (LR-CW) prepared from the sclerotia of L. rhinocerus cultivar was found to exhibit antiproliferative activity against human breast carcinoma (MCF-7) and human lung carcinoma (A549), with IC(50) of 96.7 μg/mL and 466.7 μg/mL, respectively. In comparison, LR-CW did not show significant cytotoxicity against the two corresponding human normal cells, 184B5 (human breast cell) and NL 20 (human lung cell). DNA fragmentation studies suggested that the cytotoxic action of LR-CW against cancer cells is mediated by apoptosis. Sephadex G-50 gel filtration fractionation of LR-CW yielded a high-molecular-weight and a low-molecular-weight fraction. The high-molecular-weight fraction contains mainly carbohydrate (68.7%) and small amount of protein (3.6%), whereas the low-molecular-weight fraction contains 31% carbohydrate and was devoid of protein. Only the high-molecular-weight fraction exhibited antiproliferative activity against cancer cells, with IC(50) of 70.0 μg/mL and 76.7 μg/mL, respectively. Thus, the cytotoxic action of the LR-CW is due to the high-molecular-weight fraction, either the proteins or protein-carbohydrate complex.

PURPOSE: Pulmonary ischemia-reperfusion is a pathological process seen in several clinical conditions, including lung transplantation, cardiopulmonary bypass, resuscitation for circulatory arrest, atherosclerosis, and pulmonary embolism. A better understanding of its molecular mechanisms is very important. METHODS: Rat left lung underwent in situ ischemia for 60 min, followed by 2 h of reperfusion. The gene expression profiles and Src protein tyrosine kinase (PTK) phosphorylation were studied over time, and PP2, an Src PTK inhibitor, was intravenously administered 10 min before lung ischemia to determine the role of Src PTK in lung injury. RESULTS: Reperfusion following ischemia significantly changed the expression of 169 genes, with Mmp8, Mmp9, S100a9, and S100a8 being the most upregulated genes. Ischemia alone only affected expression of 9 genes in the lung. However, Src PTK phosphorylation (activation) was increased in the ischemic lung, mainly on the alveolar wall. Src PTK inhibitor pretreatment decreased phosphorylation of Src PTKs, total protein tyrosine phosphorylation, and STAT3 phosphorylation. It increased phosphorylation of the p85α subunit of PI3 kinase, a signal pathway that can inhibit coagulation and inflammation. PP2 reduced leukocyte infiltration in the lung, apoptotic cell death, fibrin deposition, and severity of acute lung injury after reperfusion. Src inhibition also significantly reduced CXCL1 (GRO/KI) and CCL2 (MCP-1) chemokine levels in the serum. CONCLUSION: During pulmonary ischemia, Src PTK activation, rather than alteration in gene expression, may play a critical role in reperfusion-induced lung injury. Src PTK inhibition presents a new prophylactic treatment for pulmonary ischemia-reperfusion-induced acute lung injury.

Envenomation by hump-nosed pit viper (Hypnale hypnale, Hh) in Sri Lanka has caused significant morbidity and mortality, attributed to 35% of total venomous snakebites. In Southwestern India (Kerala), H. hypnale was increasingly identified as a dangerous and common source of envenomation, second to the Russell's viper but ahead of the cobra bites. Unfortunately, there is still no specific antivenom to date. This study aims to investigate the immunological properties of the venom and to assess the feasibility of specific Hh antivenom production as well as the development of a diagnostic assay. Hh venom elicited satisfactory titers of anti-Hh IgG in rabbits after 3rd immunization. The anti-Hh IgG, isolated with caprylic acid precipitation method, was effective in neutralizing the venom lethality (potency=48 LD(50) per ml IgG) as well as its procoagulant, hemorrhagic and necrotic effects, indicating the possibility to produce the specific antivenom using the common immunization regime. Cross-reactivity studies using indirect ELISA showed that anti-Hh IgG cross-reacted extensively with several Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), presumably due to the presence of venom antigens common to both snakes. Levels of immunological cross-reactivity were vastly reduced with double-sandwich ELISA. Further work demonstrated that the assay was able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common local viperid) used to spike human sera at various concentrations. The assay hence may be a useful investigating tool for diagnosing biting species and studying the time course profile of venom concentrations in blood.

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7kDa as determined by SDS-PAGE and 35.8kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerus (known locally as 'Tiger Milk mushroom') is the most important medicinal mushroom used by the indigenous communities of Malaysia to treat fever, cough, asthma, cancer, food poisoning and as a general tonic. The sclerotium of the mushroom is the part with medicinal value. Lignosus rhinocerus was hitherto unexploited commercially because of limited supply. Recently, the mushroom was successfully cultivated. AIM OF THE STUDY: To evaluate the sub-acute toxicity study of the sclerotia from the wild type and two cultivars (TM02 and TM03) of the mushroom, using animal studies. MATERIALS AND METHODS: Sprague Dawley rats (5 rats/group/sex) were fed orally with 250, 500 and 1000mg/kg TM02, 1000mg/kg TM03 as well as 1000mg/kg wild type Lignosus rhinocerus sclerotial powder. Sclerotial powder was orally administered once daily and consecutively for 28 days. Body weight of each animal was measured and any gross behavioral change was observed daily. Hematological and clinical biochemical parameters as well as histopathological analysis were carried out on 29th day. RESULTS: The results showed that oral administration of the sclerotial powder at daily dose of up to 1000mg/kg had no adverse effect on the growth rate, hematological and clinical biochemical parameters (including renal and liver function parameters). Histological studies showed that the treatments did not induce any pathological changes in the liver, kidney, heart, spleen and lung of the animals. CONCLUSION: In conclusion, our results show that there was no treatment-related sub-acute toxicity in rats following 28-days oral administration of 250, 500 and 1000mg/kg TM02, 1000mg/kg TM03 as well as 1000mg/kg wild type Lignosus rhinocerus sclerotial powder. As the highest tested dose of 1000mg/kg was not associated with any toxicity concern, the NOAEL dose is higher than 1000mg/kg.

A special class of self-assembling peptide (EAK16-II) has been found to stabilize the hydrophobic anticancer agent ellipticine in aqueous solution. In this study, the mechanism of such peptide-ellipticine complexes to enhance cellular delivery and anticancer activity was evaluated. Results revealed that EAK16-II can form nanoparticles with ellipticine, having an average size of ~100 nm. This nano-formulation had comparable cytotoxicity to ellipticine dissolved in DMSO to human lung carcinoma A549 cells. It enhanced ellipticine uptake drastically when comparing to the micro-formulation. Such enhanced uptake was significantly reduced by inhibitors specifically for caveolae-dependent pathway. We also found both protonated and neutral forms of ellipticine present in the cells. Interestingly, both were found in the cytoplasm, co-localized with LysoTracker, whereas only protonated ellipticine was seen in the nucleus. The promising therapeutic efficacy, specific delivery pathway and intracellular distribution pattern discovered in this work may help further develop ellipticine as nano-formulations for clinical applications.

Mucuna pruriens Linn.(velvet bean) has been used by native Nigerians as a prophylactic for snakebite. Rats pretreated with M. pruriens seed extract (MPE) have been shown to protect against the lethal and cardiovascular depressant effects of Naja sputatrix (Javan spitting cobra) venoms, and the protective effect involved immunological neutralization of the venom toxins. To investigate further the mechanism of the protective effect of MPE pretreatment against cobra venom toxicity, the actions of Naja sputatrix venom on spontaneously beating rat atria and aortic rings isolated from both MPE pretreated and untreated rats were studied. Our results showed that the MPE pretreatment conferred protection against cobra venom-induced depression of atrial contractility and atrial rate in the isolated atrial preparations, but it had no effect on the venom-induced contractile response of aortic ring preparation. These observations suggested that the protective effect of MPE pretreatment against cobra venom toxicity involves a direct protective action of MPE on the heart function, in addition to the known immunological neutralization mechanism, and that the protective effect does not involve action on blood vessel contraction. The results also suggest that M. pruriens seed may contain novel cardioprotective agent with potential therapeutic value.

Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.

The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.