Shining light on chiral inorganic nanomaterials for biological issues.

The rapid development of chiral inorganic nanostructures has greatly expanded from intrinsically chiral nanoparticles to more sophisticated assemblies made by organics, metals, semiconductors, and their hybrids. Among them, lots of studies concerning on hybrid complex of chiral molecules with achiral nanoparticles (NPs) and superstructures with chiral configurations were accordingly conducted due to the great advances such as highly enhanced biocompatibility with low cytotoxicity and enhanced penetration and retention capability, programmable surface functionality with engineerable building blocks, and more importantly tunable chirality in a controlled manner, leading to revolutionary designs of new biomaterials for synergistic cancer therapy, control of enantiomeric enzymatic reactions, integration of metabolism and pathology via bio-to nano or structural chirality. Herein, in this review our objective is to emphasize current research state and clinical applications of chiral nanomaterials in biological systems with special attentions to chiral metal- or semiconductor-based nanostructures in terms of the basic synthesis, related circular dichroism effects at optical frequencies, mechanisms of induced optical chirality and their performances in biomedical applications such as phototherapy, bio-imaging, neurodegenerative diseases, gene editing, cellular activity and sensing of biomarkers so as to provide insights into this fascinating field for peer researchers.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Polyisoprenylated benzophenone derivatives from Garcinia cambogia and their anti-inflammatory activities.

Ten new polyisoprenylated benzophenone derivatives, 4,8-epi-uralione F (1), 4,8-epi-uralione G (2), uralione S (3), coccinone J (4), 6-epi-coccinone C (5), coccinone I (6), 36-hydroxy-guttiferone J (7), multiflorone I (8), garciniagifolone F (9) and 36-hydroxy-garciniagifolone F (10), were isolated from the fruits of Garcinia cambogia, along with seven known analogues. The structures of the new compounds were established based on the detailed analysis of 1D and 2D nuclear magnetic resonance (NMR) spectra and high resolution electrospray ionization mass spectrometra (HRESIMS), and their absolute configurations were determined from the electronic circular dichroism (ECD) spectra. All the isolates were tested for their inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The results indicated that compound 1 displayed a potent NO inhibitory effect with an IC50 value of 41.60 ± 0.17 µM. Furthermore, compound 1 suppressed inducible NO synthase (iNOS) expression in a dose-dependent manner through inhibiting the activation of nuclear factor-κB (NF-κB).

Characterization of a Bowman-Birk type trypsin inhibitor purified from seeds of Solanum surattense.

A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Diterpenoids with inhibitory activity of nitrite production from Croton floribundus.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton floribundus Spreng. (Euphorbiaceae), popularly known as Capixinguí, stands out due to its widespread use in traditional medicine to treat wounds, syphilis, hemorrhoids, eye diseases and as a purgative. AIM OF THE STUDY: To characterize clerodanes diterpenes from C. floribundus and to evaluate the effects of the fraction and diterpenes (1-5) on inhibition of nitrite production. MATERIALS AND METHODS: The hydroethanolic root extract of C. floribundus was fractionated on a solid phase extraction column to obtain the fraction named Fr80%. From this, five compounds were obtained and characterized. The absolute configuration of compound 1 was determined by a combination of electronic and vibrational circular dichroism spectroscopies. Additionally, compounds 1-5 were evaluated for their inhibitory effects on nitrite production induced by lipopolysaccharide (LPS) in RAW 264 macrophage cell. RESULTS: Five clerodane diterpenoids were characterized, and the absolute stereochemistry of 1 was established as 3R,4R,5R,8R,9R,10S,12S. The IC50 values obtained through inhibition of nitrite production were 28.52 ±â€¯2.21 µM (1), 40.26 ±â€¯2.79 µM (2), 25.47 ±â€¯2.16 µM (3), 35.78 ±â€¯2.93 µM (4) and 40.58 ±â€¯4.78 µM (5). In the tested concentrations, the samples presented low toxicity in macrophages. CONCLUSIONS: Four new diterpenes were characterized from C. floribundus, these being croflorins A-D (1-4) and a known halimane (5). These compounds exhibited inhibitory effect on nitrite production.

Preparation and Characterization of a Novel Polysaccharide-Iron(III) Complex in Auricularia auricula Potentially Used as an Iron Supplement.

Iron deficiency anemia has been a widespread disease. As an effective and stable iron supplement, the physiochemical properties of the polysaccharide iron complex have been widely studied. In this study, we characterized a novel polysaccharide-iron(III) complex extracted in an edible fungal species Auricularia auricular (AAPS-iron(III)). The highest iron content (28.40%) in the AAPS-iron(III) complex was obtained under the optimized preparation conditions including an AAPS to FeCl3∙ 6H2O ratio of 2:3 (w/w), a pH value of 8.0 in solution, a reaction temperature of 50°C, and a reaction time of 3 h. The physical and chemical properties of the AAPS-iron(III) complex were characterized by qualitative and quantitative analyses using scanning electron microscope, particle size distribution, thermogravimetric analyzer, Fourier transform infrared spectroscopy, circular dichroism, and 1H nuclear magnetic resonance. Result showed that, although the iron was bound to the polysaccharide, it was released under artificial gastrointestinal conditions. The AAPS-iron(III) complex exhibited high stability (under 50-256°C) and water solubility. The AAPS-iron(III) complex also showed high antioxidant activity in vitro, demonstrating an additional health benefit over other typical nonantioxidant iron nutritional supplements. Furthermore, the AAPS-iron(III) complex showed high efficiency on the treatment of the iron deficiency anemia in the model rats. Therefore, the AAPS-iron(III) complex can be used as a nutritional fortifier to supply iron in industrial processing and to assist the treatment of iron deficiency anemia.

Physicochemical stability and antioxidant activity of soy protein/pectin/tea polyphenol ternary nanoparticles obtained by photocatalysis.

The ternary nanoparticles were fabricated by soy protein, pectin and tea polyphenol through photocatalysis. The particulate characteristics, including particle size, polydispersity index, and zeta potential were monitored for ternary nanoparticles formed under different photocatalysis time. Photocatalysis was favorable to form ternary nanoparticles with moderate particle size (310-370 nm), uniform distribution, spherical shape, and improved antioxidant activity. It was found that the fluorescence intensity of soy protein decreased with the increase in photocatalysis time in the ternary nanoparticles. Far-UV circular dichroism results indicated that increasing photocatalysis time could alter the secondary structure of soy protein with an increase in the proportion of ß-sheet and ß-turn structure at the cost of unordered coil and α-helix structure. According to FT-IR results, photocatalysis time could also modulate the conjugation between pectin and soy protein. In addition, photocatalysis could increase the binding affinities among the components, leading to better environmental stability of the ternary nanoparticles. The ternary nanoparticles in this study could be used as a good alternative to understand and consequently improve the physicochemical stability in food, pharmaceutical, and cosmetic matrices.

Binding Studies of Andrographolide with Human Serum Albumin: Molecular Docking, Chromatographic and Spectroscopic Studies.

BACKGROUND: Human serum albumin acts as a carrier protein to a variety of drugs and aids their transport. Andrographis paniculata, a herbal plant has been used as a source of traditional medicine in the Asian countries. Among the various constituents of this plant, andrographolide is the most active and is being used from centuries in the treatment of many chronic and infectious diseases. OBJECTIVE: The present study was designed to evaluate the interaction and binding affinity of andrographolide with HSA, by molecular docking, chromatographic and spectral studies. METHODS: Andrographolide was docked with crystal structure of human serum albumin (1AO6) using Auto Dock Vina software and the interactions were analyzed by a visualizing software py- MOL. For further characterization and confirmation, andrographolide (3x10-5 M) and HSA (0.001, 0.005, 0.01, 0.02, 0.04 M) sample mixtures were incubated at 37°C for 3h in a metabolic shaker, followed by centrifugation. The supernatant and the filtrate were analyzed by UV spectroscopy, HPLC, CD and FTIR spectral analysis. RESULTS: The docking studies revealed that andrographolide interacted with HSA and formed hydrogen bonds with Trp 214, Arg 218 and Lys 444 amino acid residues. The UV spectral analysis revealed a decrease in the absorption peak of HSA due to its interaction with andrographolide. A new peak was observed at retention time 7.45 min by HPLC analysis and the Bmax was found to be 7.5 ± 0.4 mg protein with a Kd value of 1.89 mM, indicating interaction of andrographolide with HSA. The CD spectra results suggested, a marginal decrease in the negative ellipticity without any significant shift in peak, indicating the stabilization of the HSA-andrographolide complex. The FTIR analysis of the andrographolide-HSA mixture showed a peak at wave number 1637 cm-1 (a shift of amide I groups from 1646 cm-1) and 1016 cm-1 which corresponded to the ligand, confirming the complex formation. CONCLUSION: The molecular docking studies demonstrated the interactions of andrographolide to the crystal structure of HSA. The chromatographic and spectroscopic analysis confirmed the binding of andrographolide with HSA and their complex formation. Overall the present studies conclude the binding of andrographolide to HSA protein, favoring its pharmacokinetics.

Chiroptical spectroscopy and metabolomics for blood-based sensing of pancreatic cancer.

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure-sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.

A Tri-O-Bridged Diels-Alder Adduct from Cortex Mori Radicis.

Sanggenon X, an unusual tri-O-bridged Diels-Alder adduct, was isolated from Cortex Mori Radicis. Its structure was established by spectroscopic analysis, including NMR and HR-MS (High Resolution Mass Spectrometry). Sanggenon X contained three O-bridged rings, where the oxygenated bridgeheads were all quaternary carbons. Chemical methylation was carried out to deduce the linkages of the three O-bridges. The absolute configuration was determined by calculating the ECD (Electronic Circular Dichroism) using the TDDFT (Time-Dependent Density Functional Theory) method. Sanggenon X showed significant antioxidant activity against Fe2+-Cys-induced lipid peroxidation in rat liver microsomes, and was as effective as the positive control, curcumin.

Aggregation and conformational change of mushroom (Agaricus bisporus) polyphenoloxidase subjected to thermal treatment.

This study investigated changes in the activity, conformation and microstructure of mushroom polyphenoloxidase (PPO) subjected to thermal treatment. The inactivation of PPO can be achieved by high temperature-short time or mild temperature-long time treatment. Circular dichroism and fluorescence spectra suggested that heating process induced the rearrangement of secondary structure and the disruption of tertiary structure. Red shifts of fluorescence spectra showed positive correlations with the inactivation rate of PPO. There were significant differences in the conformation and molecular microstructure among PPO samples with the same relative activity, which were obtained by treating PPO at 45, 55 and 65°C for different times. In summary, PPO molecules were deformed at mild temperature, while higher temperature induced the formation of large aggregates. PPO with the same relative activity might exist in different forms.

Vibrational Circular Dichroism: Recent Advances for the Assignment of the Absolute Configuration of Natural Products.

Vibrational circular dichroism (VCD) emerged during the last decade as a reliable tool for the absolute configuration (AC) determination of organic compounds. The principles, instrumentation, and methodology applied prior to early 2013 were recently reviewed by us. Since VCD is a very dynamic field, the aim of this review is to update VCD advances for the AC assignment of terpenoids, aromatic compounds, alkaloids, and other natural products for the 2013-2014 period, when VCD was applied to the AC assignment of some 70 natural products. In addition, although discovered in 2012, a brief introduction to the VCD exciton coupling approach and its applications in natural products AC assignment is presented.

Optimization of the Number of Considered Conformers for the Absolute Configuration Determination of Catechin and Epicatechin Peracetates by VCD.

Epimeric catechin and epicatechin peracetates offer the possibility to explore the number of conformers needed for the absolute configuration determination without affecting the reliability of the assignment. Comparisons between experimental and DFT B3PW91/DGDZVP calculated curves showed that, in molecules.where the conformational flexibility generates a large number of conformers, it is possible to significantly reduce the number of conformers needed to generate VCD results that provide secure absolute configuration assignment.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

STUDIES ON THE INTERACTION BETWEEN TRIPTOLIDE AND BOVINE SERUM ALBUMIN (BSA) BY SPECTROSCOPIC AND MOLECULAR MODELING METHODS.

BACKGROUND: Triptolide is a major active constituent isolated from Tripterygiumwilfordii Hook F, a Chinese herbal medicine. This study investigated the intermolecular interaction between triptolide and bovine serum albumin (BSA). MATERIALS AND METHODS: The fluorescence, circular dichroism (CD) and molecular docking methods were used to investigate the intermolecular interaction between triptolide and BSA. The binding constant, the number of binding sites, binding subdomain and the thermodynamic parameters were measured. RESULTS: The results of this experiment revealed that the intrinsic fluorescence of BSA was effectively quenched by triptolide via static quenching. The experimental results of synchronous fluorescence and CD spectra showed that the conformation of BSA was changed in the presence of triptolide. CONCLUSION: It indicated that triptolide could spontaneously bind on site II (subdomain IIIA) of BSA mainly via hydrogen bonding interactions and Van der Waals force.

Analysis of the major chiral compounds of Artemisia herba-alba essential oils (EOs) using reconstructed vibrational circular dichroism (VCD) spectra: En route to a VCD chiral signature of EOs.

An unprecedented methodology was developed to simultaneously assign the relative percentages of the major chiral compounds and their prevailing enantiomeric form in crude essential oils (EOs). In a first step the infrared (IR) and vibrational circular dichroism (VCD) spectra of the crude essential oils were recorded and in a second step they were modelized as a linear weighted combination of the IR and VCD spectra of the individual spectra of pure enantiomer of the major chiral compounds present in the EOs. The VCD spectra of enantiomer of known enantiomeric excess shall be recorded if they are not yet available in a library of VCD spectra. For IR, the spectra of pure enantiomer or racemic mixture can be used. The full spectra modelizations were performed using a well known and powerful mathematical model (least square estimation: LSE) which resulted in a weighting of each contributing compound. For VCD modelization, the absolute value of each weighting represented the percentage of the associate compound while the attached sign addressed the correctness of the enantiomeric form used to build the model. As an example, a model built with the non-prevailing enantiomer will show a negative sign of the weighting value. For IR spectra modelization, the absolute value of each weighting represented the percentage of the compounds without of course accounting for the chirality of the prevailing enantiomers. Comparison of the weighting values issuing from IR and VCD spectra modelizations is a valuable source of information: if they are identical, the EOs are composed of nearly pure enantiomers, if they are different the chiral compounds of the EOs are not in an optically pure form. The method was applied on four samples of essential oil of Artemisia herba-alba in which the three major compounds namely (-)-α-thujone, (+)-ß-thujone and (-)-camphor were found in different proportions as determined by GC-MS and chiral HPLC using polarimetric detector. In order to validate the methodology, the modelization of the VCD spectra was performed on purpose using the individual VCD spectra of (-)-α-thujone, (+)-ß-thujone and (+)-camphor instead of (-)-camphor. During this work, the absolute configurations of (-)-α-thujone and (+)-ß-thujone were confirmed by comparison of experimental and calculated VCD spectra as being (1S,4R,5R) and (1S,4S,5R) respectively.

Evaluation of DNA methyltransferase activity and inhibition via chiroplasmonic assemblies of gold nanoparticles.

Circular dichroism spectroscopy has been explored for detection of methyltransferase activity and inhibition based on DNA-induced chiroplasmonic assemblies of gold nanoparticles and endonuclease HpaII. Good accuracy, precision and sensitivity are obtained in complex matrices such as human serum samples, which is significant for clinical diagnosis and drug development.

Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions.

Three-dimensional structural imaging of starch granules by second-harmonic generation circular dichroism.

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best-known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second-harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second-harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second-harmonic generation, as a second-order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser-scanning microscope. In this work, we combine the axial resolving power of second-harmonic generation and chiral sensitivity of second-harmonic generation circular dichroism to realize three-dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second-harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second-harmonic generation circular dichroism response in complicated three-dimensional biological systems. The sample we use is starch granules whose second-harmonic generation-active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second-harmonic generation for right-handed circularly polarized excitation is significantly different from second-harmonic generation for left-handed one, offering excellent second-harmonic generation circular dichroism contrast that approaches 100%. In addition, three-dimensional visualization of second-harmonic generation circular dichroism distribution with sub-micrometer spatial resolution is realized. We observed second-harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second-harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three-dimensional structures of starch granules. The second-harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.

The interaction of a polymeric persimmon proanthocyanidin fraction with Chinese cobra PLA2 and BSA.

To elucidate the anti-venom mechanism of persimmon tannin, the interaction between a polymeric persimmon proanthocyanidin fraction (PT40) and phospholipase A2 (PLA2) or bovine serum albumin (BSA) were studied using a competitive binding assay and spectroscopic methods including Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), and resonance light scattering (RLS) spectroscopy. The results revealed that PT40 has a higher affinity for PLA2 than for BSA at physiological pH and induced greater conformational changes in PLA2 than in BSA. PT40 covalently bound to PLA2 in a reaction probably involving Lys residues. We propose that the high affinity of PT40 for PLA2 and the covalent modification of PLA2 by PT40 may be responsible for the ability of the tannin to irreversibly inhibit PLA2 catalytic activity, to prevent edema, and to neutralize the lethality of Chinese cobra PLA2 in vivo.