Snakebite envenoming is a major issue in certain developing parts of the world, with tens of thousands of deaths and a multitude of lingering permanent medical conditions on a yearly basis as a result. Venoms consist of a plethora of biologically active compounds, mainly peptides and proteins, that can induce these effects. Snake venom characterisation, in terms of identifying and quantifying the venom toxin proteins in crude venoms, can aid in pinpointing the venom toxins responsible for pathologies observed in snakebite victims. It can also assist in developing a new generation of efficient antivenoms. To facilitate this characterisation process, several approaches have been developed, of which venomics, toxicovenomics and antivenomics are the most important ones and aim to map venom toxin composition, their toxic effects, and antivenom efficacy, respectively. Most venomics approaches include peptide-centric analyses of protein digests in combination with advanced separation and detection hardware and involve the use of generic and dedicated software tools for data processing. However, a shift towards protein-level, and even proteoform-level, analysis can be observed over the last few years. A variety of hyphenated intact protein separation techniques, including gel-eluted liquid fraction entrapment electrophoresis, OFFGEL, gel filtration and several (ultra)high pressure and nanoscale chromatographic separations can be named, which allow retention of intact or native states of toxin proteins with ever increasing resolutions. Similarly, rapid detection hardware advances on the mass spectrometry side, and labelling techniques, allow for increasingly sensitive and quantitative venom analysis and characterisation. Most venomics research includes several complementary analytical techniques performed in parallel, with each technique often leading to exclusive protein identifications but also showing significant overlap. The original venomics approach, developed by the Calvete group and termed “Snake venomics”, is used most frequently and is the most profound methodology that has been advanced over the years, thereby resulting in sequentially further identification of venom toxin proteins based on previous results using increasingly in-depth characterisation approaches. The latest advances in this regard nowadays allow for quantitative analysis of all venom toxins in crude venoms, named “Absolute venomics”. This review will focus on the analytical aspects of venomics for which sample preparation, separation, detection and quantitation techniques are discussed. Finally, a comparison between recently published venomics studies is made.

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA₂s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A₂ inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA₂ proteins. We, therefore, conclude that PLA₂s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

Objective: This study aims to synthesize a series of five new Mannich bases of dehydrozingerone (DHZ) derivatives and to evaluate for their antiinflammatory and antioxidant activity. Methods: The synthesis was performed by refluxing DHZ with formaldehyde and secondary amines, and the structures of the synthesized compounds were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, and HR-MS. The anti-inflammatory and antioxidant activity evaluations were done by inhibition of heat-induced albumin denaturation and a free-radical DPPH method, respectively. Results: All the synthesized compounds (2a-e) showed anti-inflammatory and antioxidant activity. The highest anti-inflammatory activity was shown by compound 2c. The activity was comparable to the that of diclofenac sodium as a standard. While, the highest antioxidant activity was demostrated by compound 2e. The compound showed moderate activity compared to that of quercetin as a standard. Mostly of Mannich base derivatives of DHZ compounds exhibited higher antioxidant activity than that of DHZ. Conclusion: A series of five new Mannich bases of DHZ (2a-e) was synthesized successfully. Compound 2c demonstrated anti-inflammatory activity which was comparable to diclofenac sodium, while compound 2e exhibited moderate antioxidant activity compared to quercetin as standard. Key words: Dehydrozingerone, Mannich Bases, Synthesis, Anti-inflammatory, Antioxidant.

This study aims to determine and obtain the total polyphenolic and caffeine content from coffee beans (Coffea arabica L.) with some combination of factors. The extraction process was performed using natural deep eutectic solvent-based ultrasonic-assisted extraction (NADES-UAE) method with the different condition including extraction time, NADES ratio, and liquid-solid ratio. The total polyphenolic content was calculated using a microplate reader 96 well. The caffeine content was examined using high-performance liquid chromatography. Based on the results demonstrated the effect of different NADES components (namely lactic acid-sucrose and citric acid-glucose) on the total polyphenolic and caffeine content. The highest total polyphenolic content was 87.01 mg GAE/g sample (2 g/g lactic acid-sucrose ratio and 10 mL/g liquid-solid ratio for 15 minutes) and 62.91 mg GAE/g sample (5 g/g citric acid-glucose ratio and 15 mL/g liquid-solid ratio for 15 minutes). Whereas, the highest caffeine content was 4.45 mg/g (4 g/g lactic acid-sucrose and 15 mL/g liquid-solid ratio for 35 minutes) and 1.30 mg/g (2 g/g lactic acid-sucrose and 30 mL/g liquid-solid ratio for 5 minutes), respectively. These results were obtained from the green extraction method with rapid, easy, inexpensive, and environmentally friendly.

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A₂ (PLA₂) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA₂s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA₂ inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.

This study presents, for the first time, the successful application of analyzing a whole gas chromatography (GC) chromatogram by nuclear magnetic resonance (NMR) spectroscopy using a continuous repeatable and stable (n = 280) high-resolution (HR) GC fractionation platform with a 96-well plate. Typically with GC– or liquid chromatography–mass spectrometry analysis, (isomer) standards and/or additional NMR analysis are needed to confirm the identification and/or structure of the analyte of interest. In the case of complex substances (e.g., UVCBs), isomer standards are often unavailable and NMR spectra too complex to achieve this. This proof of concept study shows that a HR GC fractionation collection platform was successfully applied to separate, purify, and enrich isomers in complex substances from a whole GC chromatogram, which would facilitate NMR analysis. As a model substance, a chlorinated paraffin (CP) mixture (>8,000 isomers) was chosen. NMR spectra were obtained from all 96 collected fractions, which provides important information for unravelling their full structure. As a proof of concept, a spectral interpretation of a few NMR spectra was made to assign sub-structures. More research is ongoing for the full characterization of CP isomers using multivariate statistical analysis. For the first time, up to only a few CP isomers per fraction were isolated from a highly complex mixture. These may be further purified and certified as standards, which are urgently needed, and can also be used for persistency, bioaccumulation, or toxicity studies.