Inhibition Of Phospholipase A2 Activity Research Articles

Effect of flavonoid-rich fraction of Pleiocarpa mutica leaves on phospholipase A2 activity

Pleiocarpa mutica (Apocynaceae) is a pharmaceutical plant investigated to identify the usefulness and efficacy of the shrubs in the treatment of various ailments. Also, flavonoids are polypenolic compounds frequently found in nature and are divided into flavones, anthocyanidins, isoflavones, catechins, flavonols, chalcones, and flavonones based on the structure of their chemicals. Hence, fresh leaves of Pleiocarpa mutica were collected from Ugbene-Ajima, Uzo-Uwani Local Government Area, Enugu State, Nigeria. The extraction of a flavonoid-rich fraction of P. mutica leaves was conducted by dissolving a certain amount of crude extract in 20 mL of 10% H2SO4 in a tiny flask and then heating the mixture in a water bath for 30 minutes at 100 °C to initiate hydrolysis. However, quantitative phytochemical analysis of the P. mutica flavonoid-rich fraction was carried out by utilizing standard conventional protocols. Newly drawn human blood samples were centrifuged for 10 minutes at 3,000 rpm, with the supernatant (plasma) being disposed of. After being measured and reconstituted as a 40% (v/v) suspension with phosphate-buffered saline, the red blood cells were rinsed three times with an equivalent volume of normal saline. The fraction substantially (p < 0.05) reduced the activity of phospholipase A2 in a concentration-dependent approach when compared to the standard drug (prednisolone), with a range of 0.2 mL to 1.0 mL reducing the amount of the enzyme by 47.58% to 63.61%. These results showed that the flavonoid-rich fraction of Pleiocarpa mutica leaves is potent in curtailing the inflammatory response through inhibition of phospholipase A2 activity.

Green synthesis, density functional theory (DFT), molecular docking, molecular dynamics simulation and biological activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives as PLA2 and proteinase K inhibitors

In diseases like atherosclerosis, rheumatoid arthritis, and sepsis, phospholipase A2 (PLA2) and proteinase K play a role in inflammation by releasing arachidonic acid (AA). The crucial step in the inflammatory process is believed to be the release of prostaglandins after PLA2 mobilizes AA. Drugs obstructing the COX and LOX pathways in the arachidonic acid cascade, drugs that inhibit these enzymes can treat inflammatory processes. To combat against these inflammatory promoters,the authors herein report an effective method for the synthesis of a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives (4a–h) under the effect of TiO2 as a photocatalyst in ethanolic medium, and their effect against phospholipase A2, as well as proteinase K, was evaluated. The results confirmed that using TiO2 (20 mg) as a photocatalyst exhibits excellent performance in terms of yield and reaction time. The yield of all the synthesized compounds is between 79 and 91 % in just 60 min. After closely evaluating the SAR of the examined compounds against both enzymes, it was discovered that compounds (4b, 4f) with electron-donating substituents have higher PLA2 inhibitory (%) activity than those with electron-removing substituents (4a, 4e). Compounds (4c, 4d) containing heterocyclic rings showed significantly higher proteinase inhibitory (%) activity when compared to other electron-donating or withdrawing compounds. It was further confirmed that studied compounds against both enzymes exhibited dose-dependent behavior. We studied the intermolecular interactions of the compounds by molecular docking with human non-pancreatic secretory phospholipase A2 and proteinase K. We evaluated the dynamical stability of the docked complexes using a 100 ns molecular dynamics simulation, which revealed stable interactions based on root mean square deviation/fluctuation (RMSD/RMSF), radius of gyration, Gibbs free energy landscapes, and principal component analyses (PCA). Moreover, the ADME properties of the compounds align with Lipinski’s rule of five, suggesting their potential as viable candidates for the development of therapies against inflammatory diseases. All compounds tested inhibited PLA2 more than proteinase K. However, compounds 4b and 4f better inhibited phospholipase A2, whereas compounds 4c and 4d showed better activity against proteinase K.

Effect of Seaweed-Derived Fucoidans from Undaria pinnatifida and Fucus vesiculosus on Coagulant, Proteolytic, and Phospholipase A2 Activities of Snake Bothrops jararaca, B. jararacussu, and B. neuwiedi Venom.

Snakebite envenomation (SBE) causes diverse toxic effects in humans, including disability and death. Current antivenom therapies effectively prevent death but fail to block local tissue damage, leading to an increase in the severity of envenomation; thus, seeking alternative treatments is crucial. This study analyzed the potential of two fucoidan sulfated polysaccharides extracted from brown seaweeds Fucus vesiculosus (FVF) and Undaria pinnatifida (UPF) against the fibrinogen or plasma coagulation, proteolytic, and phospholipase A2 (PLA2) activities of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom. The toxicity of FVF and UPF was assessed by the hemocompatibility test. FVF and UPF did not lyse human red blood cells. FVF and UPF inhibited the proteolytic activity of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom by approximately 25%, 50%, and 75%, respectively, while all venoms led to a 20% inhibition of PLA2 activity. UPF and FVF delayed plasma coagulation caused by the venoms of B. jararaca and B. neuwiedi but did not affect the activity of B. jararacussu venom. FVF and UPF blocked the coagulation of fibrinogen induced by all these Bothropic venoms. FVF and UPF may be of importance as adjuvants for SBE caused by species of Bothrops, which are the most medically relevant snakebite incidents in South America, especially Brazil.

One-Step Affinity Purification of Leucine-Rich α2-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A2-Inhibitory Activities as β-Type Phospholipase A2 Inhibitors.

Snakes contain three types of phospholipase A2 (PLA2)-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA2. PLIβ is the snake ortholog of leucine-rich α2 glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA2 and weak inhibitory activity against Gloydius brevicauda basic PLA2. Elaphe climacophora PLIβ had weaker inhibitory activity against G. brevicauda basic PLA2 than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA2. Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA2, PL-X, or G. brevicauda basic PLA2. Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA2, but, whether it actually functions as a PLA2-inhibitory protein, PLIβ, depends on the snake.

Evaluation of the Anti-Inflammatory and Antioxidant, Activities of Parinari Curatellifolia Planch. Ex Benth (Chrysobalanaceae) Methanol Stem Bark Extract

It is used as human food and beverage, and in traditional medicine it is used to treat pneumonia and fever. The aim of this was to investigate its anti-inflammatory and antioxidant activities of Parinari curatellifolia methanol stem bark extract. A 2kg of the dried and powdered plant material was extracted with 80% methanol through cold maceration to obtain the crude methanol extract. The crude methanol extract was sequentially fractionated using n-hexane, ethyl acetate and methanol to yield n-hexane, ethyl acetate and methanol fractions respectively. Acute toxicity study was conducted following the OECD guidelines. The anti-inflammatory activity of the fractions was evaluated was evaluated using inhibition of albumin denaturation, inhibition of egg albumin-induced paw edema in rats and phospholipase A2 (PLA2) inhibition assay models. In vitro antioxidant activity was evaluated using DPPH de-colourizing, Nitric oxide scavenging activity and Ferric Reducing Antioxidant Power (FRAP) assays. Oral acute administration of the extract revealed an LD50 dose > 5000 mg/kg in rats. The extract caused a dose- and time-dependent inhibition of rat paw edema, and a concentration-dependent inhibition of Phospholipase A2 activity. IC50 of the extract for Albumin Denaturation inhibition was 37.22 ug/ml. In the antioxidant assays the IC50 of the extract against DPPH was 25.91 ug/ml, while the Nitric oxide Equivalence (mM)/mg was 42.38.

Fucoidan may treat jellyfish dermatitis by inhibiting the inflammatory effect of jellyfish venom

Jellyfish dermatitis is a common medical problem caused by jellyfish stings. However, there are no targeted and effective medications for their treatment. Here, the biological activity of fucoidan for treatment of jellyfish dermatitis was investigated for the first time. 3 mg/mL Fucoidan attenuated the inflammatory effects of Nemopilema nomurai nematocyst venom (NnNV), including dermal toxicity and myotoxicity. Fucoidan may decrease the inflammatory effects of NnNV by downregulating MAPK and NF-κB pathways. This may be attributed to the inhibitory effect of fucoidan on metalloproteinases and phospholipase A2 (PLA2) in NnNV. 3 mg/mL fucoidan reduced the metalloproteinase activity in NnNV from 316.33 ± 20.84 U/mg to 177.33 ± 25.36 U/mg, while the inhibition of PLA2 activity in NnNV by 1 mg/mL fucoidan could reach 37.67 ± 3.42 %. Besides, external application of 3 mg/mL fucoidan can effectively alleviate the symptoms of jellyfish dermatitis. These observations suggest that fucoidan has considerable potential for treatment of jellyfish dermatitis and could be regarded as a novel medicine for jellyfish envenomation. This study provides new ideas for treatment of jellyfish envenomation and suggests evidence for the use of fucoidan in the treatment of jellyfish dermatitis as well as broadens the potential application of fucoidan in clinical practice.

Infection, Inflammation, and Immunity in Sepsis.

Sepsis is triggered by microbial infection, injury, or even major surgery. Both innate and adaptive immune systems are involved in its pathogenesis. Cytoplasmic presence of DNA or RNA of the invading organisms or damaged nuclear material (in the form of micronucleus in the cytoplasm) in the host cell need to be eliminated by various nucleases; failure to do so leads to the triggering of inflammation by the cellular cGAS-STING system, which induces the release of IL-6, TNF-α, and IFNs. These cytokines activate phospholipase A2 (PLA2), leading to the release of polyunsaturated fatty acids (PUFAs), gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which form precursors to various pro- and anti-inflammatory eicosanoids. On the other hand, corticosteroids inhibit PLA2 activity and, thus, suppress the release of GLA, AA, EPA, and DHA. PUFAs and their metabolites have a negative regulatory action on the cGAS-STING pathway and, thus, suppress the inflammatory process and initiate inflammation resolution. Pro-inflammatory cytokines and corticosteroids (corticosteroids > IL-6, TNF-α) suppress desaturases, which results in decreased formation of GLA, AA, and other PUFAs from the dietary essential fatty acids (EFAs). A deficiency of GLA, AA, EPA, and DHA results in decreased production of anti-inflammatory eicosanoids and failure to suppress the cGAS-STING system. This results in the continuation of the inflammatory process. Thus, altered concentrations of PUFAs and their metabolites, and failure to suppress the cGAS-STING system at an appropriate time, leads to the onset of sepsis. Similar abnormalities are also seen in radiation-induced inflammation. These results imply that timely administration of GLA, AA, EPA, and DHA, in combination with corticosteroids and anti-IL-6 and anti-TNF-α antibodies, may be of benefit in mitigating radiation-induced damage and sepsis.

Phospholipase A2 inhibitor varespladib prevents wasp sting-induced nephrotoxicity in rats

This study aimed to clarify whether varespladib, a phospholipase A2 (PLA2) inhibitor, can be used as a therapeutic agent for wasp sting-induced acute kidney injury (AKI). Rats were divided into control, AKI, and AKI + varespladib groups. The AKI model was established by subcutaneously injecting wasp venom at five different sites in rats. Varespladib treatment showed a significant inhibitory effect on wasp venom PLA2in vitro and in vivo. Moreover, we observed that varespladib decreased the levels of rhabdomyolysis and hemolysis markers compared with that in the AKI group. Histopathological changes in the kidney decreased significantly, and rat serum creatinine levels were reduced after varespladib administration. The significantly regulated genes in the kidney of the AKI group were mostly involved in inflammatory response pathway, and the administration of varespladib remarkably attenuated the expression of these genes. Therefore, varespladib inhibited wasp sting-induced functional and pathological damage to the kidneys. We propose that the PLA2 inhibitor varespladib protects the kidney tissue in a wasp sting-induced AKI model by inhibiting PLA2 activity.

S1018 Rectal NSAIDs Decrease Post-ERCP Pancreatitis (PEP) in Unselected Patients - Comprehensive Meta-Analysis of Randomized Controlled Trials

Introduction: Endoscopic retrograde cholangiopancreatography (ERCP) is fundamental for treatment of pancreatico-biliary diseases, but results in PEP in up to 15% of patients. Phospholipase A2 (PLA2) is an important factor in the pancreatitis inflammatory cascade and NSAIDs, which inhibit PLA2 activity, prevent PEP in high-risk patients. We endeavored to identify and quantitatively synthesize the existing high-quality data to date on rectal NSAID use for the prevention of post ERCP pancreatitis in unselected patients without specific risk factors for PEP. Methods: In collaboration with a librarian, we searched the following databases: Ovid Medline (coverage 1946 – Present), Embase and Embase Classic (coverage 1947 – Present), Cochrane Library (coverage 1898-present), Web of Science (coverage 1900-present), Clinicaltrials.gov, and Google Scholar through April 2021. Using the Covidence system, two independent reviewers identified randomized controlled studies assessing rectal NSAIDs for PEP prevention in unselected patients. Our primary outcome was odds of developing PEP and our secondary outcome was odds of developing moderately severe or severe PEP. Pooled odds ratios were estimated using the random effects model. Results: After review of 504 abstracts and 234 full text documents, we identified 25 randomized controlled trials evaluating rectal NSAIDs for the prevention of PEP in unselected patients; 4071 patients received rectal NSAIDs and 4005 patients received a placebo. The most frequent NSAID used was diclofenac in 56% of studies, followed by indomethacin in 36%, ketoprofen in 4%, and naproxen in 4%. Unselected patients receiving rectal NSAIDs were less likely to develop PEP (OR 0.59, 95% CI 0.45-0.78) (Figure 1) as well as moderately severe and severe pancreatitis (OR 0.50, 95% CI 0.33-0.75). Conclusion: In unselected patients undergoing ERCP, rectal NSAID administration decreases the risk of post-ERCP pancreatitis and development of moderately severe or severe PEP. They should be considered for unselected patients undergoing ERCP, not simply those high risk for PEP.Figure 1.: Baseline characteristics and clinical outcomes in patients who has ERCP with glucagon (ERCP w glucagon, group 1) compared to individuals ERCP without glucagon (ERCP wo glucagon, group 2) Note: SD-Standard Deviation; € A 1:1 propensity score matching was done based on the following variables: age, gender, race, hypertension, diabetes mellitus, obesity, chronic kidney disease (CKD), Ischemic heart diseases (IHD), chronic obstructive pulmonary disease, indomethacin use, sphincter of Oddi dysfunction (SOD).

Inhibition of phospholipase A2, platelet aggregation and egg albumin induced rat paw oedema as anti-inflammatory effect of Peltophorun pterocarpus stem-bark

BackgroundMost medicinal plants presently employed in traditional medicine are used without scientific evidence, thereby suggesting a need to explore efficient and reliable investigations of their potential. We, therefore, conducted the present study to ascertain the efficacy of flavonoid-rich extract of Peltophorum pterocarpum sterm-bark in the treatment and management of inflammatory disorders as employed in folk medicine.Materials and methodsFlavonoid-rich extract of Peltophorum pterocarpum sterm-bark and a total of fifty-five (55) Wistar rats were used for this study. Eighteen (18) mice were used for toxicity testing, and the phytochemical analysis was done using the Trease and Evans method, while the acute toxicity was done using Lorke’s method. In vivo anti-inflammatory study was done using the egg albumin-induced paw oedema method, while the in vitro anti-inflammatory studies were performed for the extract using phospholipase A2 inhibition and calcium chloride-induced platelet aggregation assays.ResultsThe phytochemical analysis revealed that the extract of Peltophorum pterocarpum sterm-bark contains tannins, terpenoids, steroids, phenols, alkaloids, flavonoids, glycosides, and saponins ranging from 0.307 ± 0.02 to 1279.567 ± 149.868. The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. In the systemic oedema of the rat paw, scalar doses of the extract significantly (p < 0.05) suppressed the development of paw oedema induced by egg albumin, particularly with the Indomethacin (1.77 ± 0.41) when compared with the control (5.50 ± 0.26). However, varying doses of the extract significantly (p < 0.05) inhibited phospholipase A2 activity and CaCl2-Induced platelet aggregation in a concentration, dose, and time-dependent manner, in comparison to prednisolone.ConclusionThese results indicate that the extract exhibited anti-inflammatory potential, and the mechanism of this activity has a promising ability to inhibit phospholipase A2 activity and platelet aggregation in rats inflicted with paw oedema.

Understanding the binding mechanism of succinic acid against phospholipase A2 from bee venom.

Phospholipase A2 (PLA2 ) is responsible for the release of fatty acids from glycerophospholipids. PLA2 is commonly found in mammalian tissues. It is also found in venom from different animals ranging from insects, arachnid, and snakes. The release of arachidonic acid in large amount results in inflammation and pain. Identification of compounds that can inhibit the activity of PLA2 is of large scientific and medicinal interest as these compounds can act as antidotes toward snake bites and bee stings. Among the different compounds that have been tested for inhibition of PLA2 , a secondary metabolite succinic acid is identified to inhibit PLA2 activity. The inhibition was analyzed using an in vitro PLA2 inhibition assay and isothermal titration calorimetry (ITC) studies. The molecular mechanism of the mode of inhibition was studied using molecular docking and simulation studies.

Structure Base Virtual Screening for Identifying Inflammatory Inhibitors

Phospholipase A2 (PLA2) is an enzyme that induces inflammation, making PLA2 activity an effective approach to reduce inflammation. Therefore, investigating natural compounds for this PLA2 inhibitory activity has important therapeutic potential. The objective of this study was to investigate the potential inhibitors for inflammatory diseases through a virtual screening approach. Out of 10,000 compounds from zinc database, only five compounds were selected based on the lowest free energy binding and further used for molecular interaction analysis. These five compounds were Metacetamol (-11.43 kcal/mol), 7-Methoxybenzofuran-2-carboxylic acid (-10.22 kcal/mol), 6-nitro-4H-1,3-benzodioxine-8-carbaldehyde (-10.08kcal/mol), 4-(2-Amino-1,3-thiazol-4-yl)benzene-1,3-diol (-9.86 kcal/mol), and 1-Ethyl-1H-indole-3-carbaldehyde (-9.53 kcal/mol). These findings also provide insight on valuable implications for the use of these five compounds in treating inflammation, and may help researchers develop more natural bioactive compounds in daily foods as anti-inflammatory agent.

Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SignificanceP. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.

Molecular modeling approach to identify inhibitors of Rv2004c (rough morphology and virulent strain gene), a DosR (dormancy survival regulator) regulon protein from Mycobacterium tuberculosis

Being a part of dormancy survival regulator (DosR) regulon, Rv2004c (rough morphology and virulent strain gene) has been identified in earlier experimental studies as an indispensable protein required for the growth and survival of Mycobacterium tuberculosis. This protein was predicted to have a role in inhibition of phospholipase A2 activity related to immuno-defence and other membrane-related events. Thus, considering significance of Rv2004c protein, a structure-based drug designing strategy was followed to identify potential inhibitors to this novel target. Initially, to validate the target, absence of homologous proteins in the host was verified through sequence and structure similarity search against human proteome. Then, a potential ligand binding site on the target was identified and virtual screening against Zinc database molecules was carried out. The top scoring hits along with their analogs were taken for docking studies with Glide. The binding free energy of the docked complexes of the Glide hits were predicted by Prime program from Schrodinger and molecules ZINC57990006, ZINC33605742, ZINC71773467 and ZINC34198774 were recognized as potential hits against this target. Analyzing the predicted pharmacokinetic properties of the molecules from QikProp and admetSAR tool, ZINC34198774 was identified as a valid molecule. Molecular dynamics simulation studies ascertained that ZINC34198774 could be a potential inhibitor against Rv2004c. Thus, results acquired from this study could be of use to design new therapeutics against tuberculosis. Communicated by Ramaswamy H. Sarma.

Characterization of plasma secretory phospholipase A2 activity in the prairie rattlesnake (Crotalus viridis)

BackgroundPlasma phospholipase A2 (PLA2) enzyme activity is a key component of innate immunity in most vertebrates. We evaluated circulating secreted PLA2 activity of prairie rattlesnakes (Crotalus viridis) by incubation of plasma with bacteria labeled with fluorescent membrane lipids.ResultsIncubation of bacteria with increasing volumes of plasma resulted in volume-dependent lysis of fatty acids from bacterial membranes. The activity was rapid, with substantial activity recorded after only 5 min of incubation with labeled bacteria, and a linear response for 20 min. In addition, the lysis activity was temperature-dependent, increasing activities from 5 to 20 °C, peak activities at 25–30 °C, and then decreasing activities from 35 to 40 °C. Furthermore, the activity was inhibited in a concentration-dependent manner by p-bromophenacyl bromide, a specific inhibitor of PLA2 activity, which indicated that the observed activities were due to the presence of PLA2 in the plasma of C. viridis.ConclusionsThis study represents the first description of secretory PLA2 activity in the plasma of a snake. Our study shows that in addition to being an important component of snake venom, PLA2 enzymes play an important role in the snake’s immune response.

PLA2 Inhibitor Varespladib as an Alternative to the Antivenom Treatment for Bites from Nikolsky's Viper Vipera berus nikolskii.

Although envenoming by a small East European species of viper is rarely severe, and only exceptionally fatal, lack of specific antivenom stocks in a few areas within this region and possible severe side effects of antivenom application leave most bites to be treated only with antihistamines and supportive therapy. Varespladib is an effective inhibitor of snake phospholipase, and, as such, it could be considered as first-line therapy. The Nikolsky’s viper venom contains an extremely high concentration of phospholipase A2 (PLA2), responsible for the toxic effects of the venom, as well as minor amounts of other toxins. If Varespladib can successfully inhibit PLA2 activity, the Nikolsky’s viper could be one of the first venomous snakes having an antitoxin-specific treatment regimen. To assess that, Varespladib was administered alone subcutaneously to adult male CD-1 mice (8 mg/kg) and compared to mice exposed to Vipera berus nikolskii crude venom (8 mg/kg = 10 LD50) or a combination of Varespladib and the same amount of the venom. Experimental animals were monitored for the presence of envenoming symptoms and mortality for 48 h after injection. Eighty percent of mice receiving both Varespladib and venom survived, while 100% of the control group receiving venom alone died within 4 h. Experimental results are consistent with Varespladib acting as an effective antitoxin in the mouse model against Nikolsky’s viper venom. Further studies are needed under experimental conditions that more closely resemble natural envenoming (i.e., delayed administration).

Detoxifying Action of Aqueous Extracts of Mucuna pruriens Seed and Mimosa pudica Root Against Venoms of Naja nigricollis and Bitis arietans.

The World Health Organization included snakebite envenomation among Neglected Tropical Diseases in 2017. The importance of natural products from plants is enormous, given that most prescribed drugs originate from plants. Among this is Mucuna pruriens and Mimosa pudica, with many registered patents asserting their health benefits. This study investigated the in vitro neutralizing effects of Mucuna pruriens seed and Mimosa pudica root extracts on venoms of Naja nigricollis and Bitis arietans. In mice, the LD50 and phytochemical analysis of M. pruriens and M. pudica plant extracts were carried out prior to the evaluation of their haemolytic and fibrinolytic effect. Their effects on the activities of phospholipase A2 (PLA2) were also assessed. At a concentration of 50 mg/ml, both plant extracts were found to neutralize the fibrinolytic activity of N. nigricollis, but 400 mg/ml was required to neutralize the fibrinolytic activity of B. arietans. In haemolytic studies, 50 mg/ml concentration of M. pruriens extract suppressed haemolysis caused by N. nigricollis venom by 70% but at the same concentration, M. pudica extract reduced haemolysis by 49.4%. M. pruriens, at 50 mg/ml concentration, only inhibited phospholipase A2 activity by 7.7% but higher concentrations up to 400mg/ml had no effect against the venom of N. nigricollis; at 200 mg/ml. M. pudica extract inhibited PLA2 activity by 23%. The results suggest that M. pruriens and M. pudica may be considered as promising antivenom agents for people living in a snake-bite prone environment.

Varespladib (LY315920) and Methyl Varespladib (LY333013) Abrogate or Delay Lethality Induced by Presynaptically Acting Neurotoxic Snake Venoms.

The phospholipase A2 (PLA2) inhibitor Varespladib (LY315920) and its orally bioavailable prodrug, methyl-Varespladib (LY333013) inhibit PLA2 activity of a wide variety of snake venoms. In this study, the ability of these two forms of Varespladib to halt or delay lethality of potent neurotoxic snake venoms was tested in a mouse model. The venoms of Notechis scutatus, Crotalus durissus terrificus, Bungarus multicinctus, and Oxyuranus scutellatus, all of which have potent presynaptically acting neurotoxic PLA2s of variable quaternary structure, were used to evaluate simple dosing regimens. A supralethal dose of each venom was injected subcutaneously in mice, followed by the bolus intravenous (LY315920) or oral (LY333013) administration of the inhibitors, immediately and at various time intervals after envenoming. Control mice receiving venom alone died within 3 h of envenoming. Mice injected with O. scutellatus venom and treated with LY315920 or LY333013 survived the 24 h observation period, whereas those receiving C. d. terrificus and B. multicinctus venoms survived at 3 h or 6 h with a single dose of either form of Varespladib, but not at 24 h. In contrast, mice receiving N. scutatus venom and then the inhibitors died within 3 h, similarly to the control animals injected with venom alone. LY315920 was able to reverse the severe paralytic manifestations in mice injected with venoms of O. scutellatus, B. multicinctus, and C. d. terrificus. Overall, results suggest that the two forms of Varespladib are effective in abrogating, or delaying, neurotoxic manifestations induced by some venoms whose neurotoxicity is mainly dependent on presynaptically acting PLA2s. LY315920 is able to reverse paralytic manifestations in severely envenomed mice, but further work is needed to understand the significance of species-specific differences in animal models as they compare to clinical syndromes in human and for potential use in veterinary medicine.

Bioassay-guided fractionation, phospholipase A2-inhibitory activity and structure elucidation of compounds from leaves of Schumanniophyton magnificum

Context Schumanniophyton magnificum Harms (Rubiaceae) is used traditionally in Nigeria for the treatment of snake bites. Snake venom contains phospholipase A2 (PLA2) which plays a key role in causing inflammation and pain. Objective To assess the anti-inflammatory effect of the methanol extract of Schumanniophyton magnificum (MESM) leaves through the inhibition of PLA2 and investigate the compounds responsible for the effect. Materials and methods PLA2-inhibitory activity of MESM was assessed at concentrations of 0.1–0.8 mg/mL using human red blood cells as substrate. Prednisolone was used as the standard control. MESM was subsequently partitioned using n-hexane, dichloromethane, ethyl acetate and aqueous-methanol (90:10 v/v), after which PLA2-inhibitory activity of the partitions was determined. The best partition was subjected to chromatographic techniques and the fractions obtained were assessed for PLA2 inhibition at 0.4 mg/mL. Compounds in the most active fraction were determined using Fourier-transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). Results MESM significantly inhibited PLA2 activity at 0.8 mg/mL (44.253%) compared to prednisolone (35.207%). n-Hexane partition (SMP1) proved more active with inhibition of 55.870% observed at 0.1 mg/mL. Fraction 1 (SMF1) showed the highest PLA2-inhibitory activity of 58.117%. FTIR studies revealed the presence of some functional groups in SMF1, and GC-MS confirmed the presence of 9 compounds which are first reported in this plant. Hexadecanoic acid, ethyl ester was identified as the major compound (24.906%). Discussion and conclusions The PLA2-inhibitory activity of MESM suggests that its compounds may be explored further in monitoring anti-inflammatory genes affected by the venoms.

Combining in silico and in vitro approaches to identification of potent inhibitor against phospholipase A2 (PLA2)

Phospholipase A2 (PLA2) is the main constituent of snake venom. PLA2 enzymes catalyze the Ca2+ dependent hydrolysis of 2-acyl ester bonds of 3-sn-phospholipids, releasing fatty acids and lysophospholipids. Inside the body of the victim, PLA2 from snake venom induces either direct or indirect pathophysiological effects, including anticoagulant, inflammatory, neurotoxic, cardiotoxic, edematogenic, and myotoxic activities. Therefore, there is a need to find the potential inhibitors against PLA2 responsible for snakebite. In this study, we employed in silico and in vitro methods to identify the potential inhibitor against PLA2. Virtual screening and molecular docking studies were performed to find potent inhibitor against PLA2 using Traditional Chinese Medicine Database (TCM). Based on these studies, Scutellarin (TCM3290) was selected and calculated by density functional theory calculation at B3LYP/6-31G**++ level to explore the stereo-electronic features of the molecule. Further, molecular docking and DFT of Minocycline was carried out. Quantum polarized ligand docking was performed to optimize the geometry of the protein–ligand complexes. The protein-ligand complexes were subjected to molecular dynamics simulation and binding free energy calculations. The residence time of a protein-ligand complex is a critical parameter affecting natural influences in vitro. It is nonetheless a challenging errand to expect, regardless of the accessibility of incredible PC assets and a large variety of computing procedures. In this metadynamics situation, we used the conformational flooding technique to deal with rank inhibitors constructions. The systematic free energy perturbation (FEP) protocol and calculate the energy of both complexes. Finally, the selected compound of TCM3290 was studied in vitro analysis such as inhibition of PLA2 activity, hyaluronidase activity and fibrinogenolytic activity. The TCM3290 had a more binding affinity compare to Minocycline, and interacted with the key residues of TYR63 and GLY31. DFT represented the highest HOMO and LUMO energy of 0.15146 eV. MD simulation with 100 ns proved that an inhibitor binding mode is more stable inside the binding site of PLA2. In vitro analysis shows that TCM3290 significantly neutralized by PLA2. The above observations confirmed that Scutellarin (TCM3290) had a potent snake venom neutralizing capacity and could hypothetically be used for therapeutic drives of snakebite envenomation.