Chemotaxonomy of the ethnic antidote Aristolochia indica for aristolochic acid content: Implications of anti-phospholipase activity and genotoxicity study.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochia indica L. (Aristolochiaceae) is a common medicinal plant described in many traditional medicine as well as in Ayurveda used against snakebites. Besides, the plant has also been reported traditionally against fever, rheumatic arthritis, madness, liver ailments, dyspepsia, oedema, leishmaniasis, leprosy, dysmenorrhoea, sexual diseases etc. The plant is known to contain its major bioactive constituent aristolochic acid (AA) known for its anti-snake venom, abortifacient, antimicrobial and antioxidant properties. MATERIALS AND METHODS: This present work describes a validated, fast and reproducible high performance thin layer chromatography (HPTLC) method to estimate AA from the roots of 20 chemotypes of A. indica procured from 20 diverse geographical locations from the state of West Bengal, India. Further, an evidence-based approach was adopted to investigate the reported anti-venom activity of the aqueous extracts of the A. indica roots by assessing its phospholipase A2 (PLA2) inhibitory properties since PLA2 is a major component of many snake-venoms. Finally, the cytotoxicity and genotoxicity of the aqueous root extract of the Purulia (AI 1) chemotype were assessed at various concentrations using Allium cepa root meristematic cells. RESULTS: The highest amount of AA (7643.67 µg/g) was determined in the roots of A. indica chemotype collected from Purulia district followed by the chemotypes collected from Murshidabad, Jalpaiguri and Birbhum districts (7398.34, 7345.09 and 6809.97 µg/g respectively). This study not only determines AA in the plants to select pharmacologically elite chemotypes of A. indica, but it also identifies high AA producing A. indica for further domestication and propagation of the plants for pharmacological and industrial applications. The method was validated via analyzing inter-day and intra-day precision, repeatability, reproducibility, instrumental precision, limit of detection (LOD) and limit of quantification (LOQ) and specificity. Chemotypes with high AA content exhibited superior anti-PLA2 activity by selectively inhibiting human-group PLA2. Moreover, A. indica root extract significantly inhibited mitosis in Allium cepa root tips as a potent clastogen. CONCLUSIONS: The present quick, reproducible and validated HPTLC method provides an easy tool to determine AA in natural A. indica plant populations as well as in food and dietary supplements, a potential antivenin at one hand and a possible cause of aristolochic acid nephropathy (AAN) at another. Besides, the cytotoxic and mitotoxic properties of the root extracts should be used with caution especially for oral administration.

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.

In silico and in vitro neutralization of PLA2 activity of Daboxin P by butein, mimosine and bakuchiol.

Medicinal plants have always been used for snakebite treatment by traditional healers but they lack scientific evidence of action. However secondary metabolites of such plants have been explored and found to inhibit the toxic effect of venom proteins. Literature survey from 2003 to 2019 resulted in identification of 251 secondary metabolites with such properties. In silico docking studies of these metabolites with modelled structure of Daboxin P, a PLA2 from Indian Daboia russelii revealed that butein, mimosine and bakuchiol bind to Daboxin P with high affinity. Butein interacted with the catalytic triad but mimosine and bakuchiol interacted with the Ca2+ binding residues of Daboxin P. In vitro validation showed that the molecules inhibited the sPLA2 activity of Daboxin P. Interestingly, mimosine and bakuchiol could also neutralize the anti-coagulatory activity of Daboxin P. Further, it was observed that butein and mimosine could neutralize the PLA2 activity of Indian big four venoms dose dependently. On the other hand, mimosine and bakuchiol could also neutralize the pro/anti-coagulatory effect of big four crude venom. Thus, in this study, three molecules have been identified which can neutralize the PLA2 activity and pro/anti-coagulatory effect of Daboxin P as well as crude venom of big four.

Jabuticaba (Plinia jaboticaba) skin extracts as inhibitors of phospholipases A2 and proteases.

The phenolic extracts of jabuticaba skin flour (JSF) were characterized by HPLC, and evaluated for their modulating action upon phospholipases A2 and proteases of snake venom, aiming at their possible use in the treatment of the various diseases associated with the action of venom toxins. Two types of extracts were prepared from JSF: aqueous and methanolic. These extracts, evaluated at different ratios, (venom: extract, m/m), significantly inhibited the phospholipase activity induced by the venom of Bothrops moojeni and Crotalus durissus terrificus, except for Bothrops atrox venom. The greatest hemolysis inhibitory action was observed for the methanolic extract, when incubated with venoms of B. moojeni and C. durissus terrificus, with inhibitions between 21 and 100%. Thrombolysis induced by venoms of B. moojeni and C. durissus terrificus was inhibited by both extracts, ranging from 32 to 83% and 51 to 83% for the aqueous and methanolic extracts, respectively. Both extracts extended coagulation time, induced by the venoms of B. moojeni and Lachesis muta muta. Inhibitory actions are related to phenolic compounds, such as gallic, syringic and p-coumaric acids, besides catechin, epigallocatechin gallate, epicatechin; resveratrol and quercetin, present in the extracts of jabuticaba skin flour, confirming their potential for nutraceutical use.

High activity chimeric snake gamma-type phospholipase A2 inhibitor created by DNA shuffling.

The gamma-type inhibitor of snake venom phospholipase A2 (PLIγ) is expressed extensively in livers of both venomous and non-venomous snakes. It is not clear why PLIγs from different snake species possess diverse activities. To obtain high activity PLIγs and interpret the sequence-function relationships, we used DNA shuffling to hybridize the PLIγs of Sinonatrix annularis (saPLIγ) and Elaphe carinata (ecPLIγ). Chimera PLIγs (cPLIγ) of ∼550 bp were obtained by a series of gene manipulations including DNase I digestion, primer-free PCR, and PCR amplification with PLIγs primer pair. After successful insertion of cPLIγs into pCANTAB5e phage vector, the transformed TG1 strain of Esherichia coli was achieved. The cPLIγ phage library was produced and panned in a five-pace snake venom-coated immune tube. Three high affinity cPLIγ isoforms survived two rounds of panning. Prokaryote expression by the pET28c vector was employed for production of the three cPLIγs and the two parental PLIγs. These all showed anti-hemorrhage activity with cPLIγ 2 demonstrating superior inhibition to the parent PLIγs. Sequence alignment showed that the three kinds of cPLIγ were produced by gene splicing of S. annularis and E. carinata at different sites. Primary sequence changes brought regional changes in secondary and tertiary structure, which may explain the differences in PLIγ activity.

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.

Tetraglochin andina Ciald.: A medicinal plant from the Argentinean highlands with potential use in vaginal candidiasis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Argentinean medicinal plant Tetraglochin andina Ciald, formerly classified as T. cristatum (Britton) Rothm is used in traditional medicine by inhabitants from Argentinean northwestern highlands (Puna) to treat candidiasis and as anti-inflammatory. AIM OF THE STUDY: To assess the potential of the crude drug as an anti-Candida agent with anti-inflammatory properties. The bioactivity and phytochemical composition of a dry extract of the plant was investigated. MATERIAL AND METHODS: The pharmacognostic description of the crude drug is carried out for the first time, including macroscopic and microscopic examinations of the different organs, physicochemical and extractive values (petroleum ether-, ethanol- and water-soluble). The dry extract from T. andina was evaluated as antifungal against pathogenic Candida sp. and Saccharomyces cerevisiae isolated from vaginal infections and reference strains, by the macrodilution and microdilution assays. The normal vaginal microbiome in women is characterized by the dominance of lactic acid-producing bacteria, mainly Lactobacillus spp. The effect of T. andina extract on Lactobacillus strains was also assayed. The inhibitory effect on proinflammatory enzymes (cyclooxygenase, lipoxygenase and phospholipase A2) and antioxidant capacity was studied. The chemical profile was analyzed by HPLC-ESI-MS. RESULTS: The hydroalcoholic extract inhibited the growth of all yeasts with Minimal Inhibitory Concentration (MIC) values between 12.5 and 400 µg GAE/mL and the MIC values on Lactobacillus were higher than the MIC values against Candida isolates ( > 400 µg GAE/mL). These results indicate that the hydroalcoholic extract could be used without affecting the normal microbiota of vaginal fluid. The extract showed antioxidant activity and could modulate the inflammatory process by three pathways (sPLA2, COX-2, LOX). The plant extract contained high total phenolic levels (386.9±1.7 mg GAE/g dry extract) and flavonoid levels (260.4±2.7 mg GAE/g dry extract). Fifty phenolic compounds were identified by HPLC-ESI-MS. They were mainly hydrolysable and condensed tannins. The dry extract was chemically and biologically stable during one year at room temperature or 4 °C. CONCLUSIONS: The presence of anti-Candida and anti-inflammatory activities in Tetraglochin andina extracts give support to their traditional use for treating conditions associated with microorganism infections and inflammatory process in humans. This plant preparation could be used to design phytopharmaceutical preparations to inhibit yeast growth and moderate the inflammatory and oxidative process.

Antitumoral effects of γCdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 breast cancer cell.

Phospholipases A2 (PLA2s) overexpression is closely associated with the malignant potential of breast cancers. Here, we showed for the first the antitumoral effects of γCdcPLI, a PLA2 inhibitor from Crotalus durissus collilineatus via PI3K/Akt pathway on MDA-MB-231 cell. Firstly, γCdcPLI was more cytotoxic to MDA-MB-231 breast cancer cells than other cell lines (MCF-7, HeLa, PC3 and A549) and did not affect the viability of non-tumorigenic breast cell (MCF 10A). In addition, γCdcPLI induced modulation of important mediators of apoptosis pathways such as p53, MAPK-ERK, BIRC5 and MDM2. γCdcPLI decreased MDA-MB-231 adhesion, migration and invasion. Interestingly, the γCdcPLI also inhibited the adhesion and migration of endothelial cells and blocked angiogenesis by inhibiting tube formation by HUVECs in vitro and sprouting elongation on aortic ring assay ex vivo. Furthermore, γCdcPLI reduced the production of vascular endothelial growth factor (VEGF). γCdcPLI was also able to decrease PGE2 levels in MDA-MB-231 and inhibited gene and protein expression of the PI3K/Akt pathway. In conclusion, γCdcPLI showed in vitro antitumoral, antimestatatic and anti-angiogenic potential effects and could be an attractive approach for futures studies in cancer therapy.

Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights.

Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A2 (PLA2) of Naja naja (Indian cobra) venom for PLA2 activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA2 were observed at the binding pocket of the PLA2 protein. Further, this protein-ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA2 inhibition activity.

Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Macropisthodon rudis snake serum.

A novel phospholipaseA2 (PLA2) inhibitory protein (PLI) was purified from the serum of Macropisthodon rudis, a non-venomous snake mainly found in southern China. The molecular mass of the purified PLI was 160 kDa as determined by Superdex 200HR; however, the PLI protein had only one subunit of 25.4 kDa as determined by 12% SDS-PAGE, indicating an oligomeric protein. PLI cDNA obtained by PCR from the liver of Macropisthodon rudis, revealed 549 bps coding for a mature protein of 183 amino acid residues. Based on an amino acid sequence alignment with venomous and non-venomous snakes, this inhibitor was determined to be in the γ type family of PLI. In vitro experiments showed that PLIγ inhibited enzymatic, inflammatory, and antibacterial activities of snake venom PLA2 isolated from Agkistrodon acutus.

Marine Natural Product Inhibitors of Neutrophil-Associated Inflammation.

Neutrophils are widely recognized to play an important role in acute inflammatory responses, and recent evidence has expanded their role to modulating chronic inflammatory and autoimmune diseases. Reactive oxygen species (ROS) and microbicidal compounds released from neutrophils that are recruited to the site of inflammation contribute to the pathogenesis of multiple inflammation-associated diseases such as chronic obstructive pulmonary disease, atherosclerosis, and hepatitis. Marine organisms are a valuable source of bioactive compounds with potential for industrial and pharmaceutical application. Marine natural products that inhibit neutrophil activation could be used as drugs for the treatment of inflammatory diseases. Numerous studies investigating marine natural products have reported novel anti-inflammatory agents. Nevertheless, the detailed mechanisms underlying their actions, which could facilitate our understanding of the molecular events occurring in neutrophils, have not been reported in most of the associated research studies. Therefore, in this review, we will present marine products that inhibit neutrophil-associated inflammation. Furthermore, we will be limiting the detailed discussion to agents with well-investigated molecular targets.

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure-activity relationship.

In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads.

Anti-inflammatory and anti-proliferative activities of the wild edible cruciferous: Diplotaxis simplex.

Context The present study deals with new biological properties of the wild edible Diplotaxis simplex (Viv.) Spreng (Brassicaceae). Objectives The current study evaluates the antioxidant, the anti-inflammatory and the anti-cancer properties of ethyl acetate and ethanol extracts from D. simplex flowers. Materials and methods The anti-proliferative activity of the extracts (10-70 µg/mL) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human colon cancer cell line Caco-2. The anti-inflammatory potential was evaluated by the inhibitory effect of the extracts (1.5-7.5 mg/mL) on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. Extracts (200 mg/kg) or indomethacin (50 mg/kg) as positive control were injected intraperitoneally for albino mice prior to the induction of the oedema by carrageenan. Antioxidant activities were investigated using various complementary methods. Results Flower extracts contained a high level of polyphenolics (17.10-52.70 mg GAE/g) and flavonoids (74.20-100.60 mg QE/g), which correlate with its appreciable antioxidant potential in ß-carotene peroxidation (IC50 value: 12.50-27.10 µg/mL), DPPH(•) radical-scavenging (IC50 value: 0.20-0.40 mg/mL), Fe(3+ )reducing (EC50 value: 0.10-0.14 mg/mL) and Fe(2+ )chelating (IC50 value: 0.20-0.60 mg/mL) assays. These extracts were effective in inhibiting cancer cell growth (IC50 value: 62.0-63.25 µg/mL). Besides, the ethyl acetate extract inhibited phospholipase A2 activity (IC50 value: 2.97 mg/mL) and reduced the paw oedema in mice (from 0.38 ± 0.01 to 0.24 ± 0.01 cm), 4 h post-carrageenan challenge. Conclusion These data suggest that D. simplex may be useful as a candidate in the treatment of inflammation and the colon cancer.

rBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy.

Phospholipase A2 inhibitors (PLIs) are important targets in the search and development of new drugs. This study aimed at evaluating the potential of an alpha-type phospholipase A2 inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake in its recombinant form (rBaltMIP) to complement the conventional antivenom therapy. Biochemical experiments showed that rBaltMIP presented pI 5.8 and molecular masses of ∼21 kDa by SDS-PAGE and 19.57 kDa by MALDI/TOF MS. After tryptic peptides sequencing, the results were compared with other PLIs available in databases, showing 100% identity between rBaltMIP and its native inhibitor BaltMIP and from 92% to 96% identity with other inhibitors. Myotoxic activities of BthTX-I and BthTX-II toxins were measured via plasma CK levels, showing myotoxic effective concentrations (EC50) of 0.1256 µg/µL and 0.6183 µg/µL, respectively. rBaltMIP neutralized the myotoxicity caused by these two toxins up to 65%, without promoting primary antibody response against itself. Nevertheless, this recombinant PLI was immunogenic when standard immunization protocol with Freud's adjuvant was used. In paw edema assays, EC50 of 0.02581 µg/µL and 0.02810 µg/µL, respectively, were observed with edema reductions of up to 40% by rBaltMIP, suggesting its use as an additional antivenom. In addition, myotoxicity neutralization experiments with the myotoxin BthTX-I showed that rBaltMIP was more effective in inhibiting muscle damage than the conventional antivenom. Thus, considering the severity of envenomations due to Bothrops alternatus (Rhinocerophis alternatus) and the low neutralization of their local effects (such as myotoxicity) by the current antivenoms, rBaltMIP is a promising molecule for the development of novel therapeutic strategies for clinical applications.

Evaluating the inhibitory potential of Withania somnifera on platelet aggregation and inflammation enzymes: An in vitro and in silico study.

Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases.

Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis.

Phospholipase A2 (PLA2) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA2s (svPLA2s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA2 inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA2, however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L16(4)(5)] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-ß-D-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA2 isolated from D. acutus. Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom.

High-resolution hyaluronidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in Chinese plants used to treat snakebite.

Inhibition of the necrotizing hyaluronidase, phospholipase A2 and protease enzymes in four snake venoms by crude water and ethanol extracts of 88 plant species used against snakebites in traditional Chinese medicine was measured. High-resolution hyaluronidase inhibition profiles were constructed for the 22 plants showing highest hyaluronidase inhibition, and the results were used to guide subsequent structural analysis towards specific hyaluronidase inhibitors. Structural analysis was performed by high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR. This allowed identification of four non-tannin inhibitors, i.e., lansiumamide B (6) from Clausena excavata Burm.f., myricetin 3-O-ß-D-glucopyranoside (7) from Androsace umbellata (Lour.) Merr., and vitexin (8) and 4',7-dihydroxy-5-methoxyflavone-8-C-ß-D-glucopyranoside (9) from Oxalis corniculata L. Absolute configuration of 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1) was determined using the Mosher method, which revealed two enantiomers, i.e., (2S,3R)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide and (2R,3S)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide with a ratio of 7:3.

Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, ß and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved.