Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

Spider venom is a rich cocktail of neuroactive compounds designed to prey capture and defense against predators that act on neuronal membrane proteins, in particular, acetylcholinesterases (AChE) that regulate synaptic transmission through acetylcholine (ACh) hydrolysis - an excitatory neurotransmitter - and beta-secretases (BACE) that primarily cleave amyloid precursor proteins (APP), which are, in turn, relevant in the structural integrity of neurons. The present study provides preliminary evidence on the therapeutic potential of Phlogiellus bundokalbo venom against neurodegenerative diseases. Methods Spider venom was extracted by electrostimulation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF-MS). Neuroactivity of the whole venom was observed by a neurobehavioral response from Terebrio molitor larvae in vivo and fractions were screened for their inhibitory activities against AChE and BACE in vitro. Results The whole venom from P. bundokalbo demonstrated neuroactivity by inducing excitatory movements from T. molitor for 15 min. Sixteen fractions collected produced diverse mass fragments from MALDI-TOF-MS ranging from 900-4500 Da. Eleven of sixteen fractions demonstrated AChE inhibitory activities with 14.34% (± 2.60e-4) to 62.05% (± 6.40e-5) compared with donepezil which has 86.34% (± 3.90e-5) inhibition (p > 0.05), while none of the fractions were observed to exhibit BACE inhibition. Furthermore, three potent fractions against AChE, F1, F3, and F16 displayed competitive and uncompetitive inhibitions compared to donepezil as the positive control. Conclusion The venom of P. bundokalbo contains compounds that demonstrate neuroactivity and anti-AChE activities in vitro, which could comprise possible therapeutic leads for the development of cholinergic compounds against neurological diseases.(AU)

Phlogiellus bundokalbo spider venom: cytotoxic fractions against human lung adenocarcinoma (A549) cells

Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from Phlogiellus bundokalbo and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells. Methods: Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa FluorTM 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A2 (PLA2) activity of the cytotoxic fractions were also measured. Results: We observed and isolated six fractions from the venom of P. bundokalbo collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA2 activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA2 and cytotoxicity. Conclusion: The venom of P. bundokalbo spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.(AU)

Acute oral toxicity study of the crude ethanolic leaf extract of ficus pseudopalma blanco (moraceae) in sprague dawley rats.

Ficus pseudopalma Blanco is an ornamental plant endemic to the Philippines, especially in the island of Luzon. It is commonly used to treat kidney stones and diabetes and used for edible fruits. The leaves are cooked and eaten as vegetable despite the absence of studies on its possible toxic effect. This study was conducted to assess any toxicity of its leaf extract. Acute oral toxicity of the crude ethanolic leaf extract of F. pseudoplama was performed according to the guidelines set by OECD 425 on six 8-12 week old female Sprague Dawley rats weighing from 160-210g. One rat was treated with normal saline solution that served as the control. Toxicological and pharmacological observations were completed for 14 days. On day 14, all test animals were sacrificed via cervical dislocation and subjected to gross necropsy; liver samples were subjected to histopathological examination. Gross examination of the rodent’s organs was all normal and regarded as unremarkable. Toxicological screening showed that the experimentally treated rats behaved almost normally as the control. Histopathological examination showed no area of hepatic zonal necrosis and tumor formation was identified, no cytological aypia and sinusoid congestion, intact and uninterrupted hepatic lobular architecture, and portal tracts and vessels were unremarkable. These findings strongly suggest that the leaf extract is non toxic and safe for consumption up to 2000 mg/kg BW and may therefore be used for future nutraceuticals and drug development.

Effect of Fenofibrate on Free Radicals in vitro, Lipid Peroxides, Antioxidant Enzymes and Liver Transaminases in Hyperlipidemic mice.

In this study, fenofibrate was examined both in vitro and in vitro. Its antioxidant activity in vitro was screened using DPPH assay; and specific antioxidant activity was observed using nitric oxide (NO•) assay, hydroxyl radical (•OH) assay and TBARS assay. The same substance was administered in mice to observe its antioxidant enzyme – GSH) and catalase - induction, inhibition of serum transaminases –SGPT and SGOT, and lipid lowering activities. It was observed in vitro that DPPH (IC50 > 0.38 mg/mL), NO• (IC50 = 22.81 μg/mL) and lipid peroxidation (IC50 = 30.37 mg/mL) was inhibited but not •OH (SC50 < 0.05 mg/mL). In vivo experimentation for lipid peroxidation showed that TBARS concentration was decreased by 24.79% while increasing concentrations of both GSH and catalase. Serum transaminases were reduced by 3.65% and 8.93%, respectively. Lipid profiling showed a decline in triglycerides and low density lipoproteins by 54.87% and 16.67%, correspondingly; while the amount of high density lipoproteins was augmented by 45.13%. Fenofibrate scavenges of free radicals, stimulates •OH production that may increase body’s defense against pathogens, boosts liver antioxidant enzyme against free radicals, and lowers serum transaminases. The results strongly suggest that fenofibrate, aside from its lipid-lowering activity may also provide antioxidant defenses.