Essential oil and plant extract of oregano as agents influencing the virulence factors of Candida albicans.

Candida albicans, a polymorphic yeast, is a physiological component of the human and animal commensal microbiome. It is an etiological factor of candidiasis, which is treated by azole antifungals. Growing resistance to azoles is a reason to look for other alternative treatment options. The pharmacotherapeutic use of plant extracts and essential oils has become increasingly important. In our experiment, C. albicans showed susceptibility to four observed plant extracts and essential oils from peppermint ( Mentha piperita), thyme ( Thymus vulgaris), sage ( Salvia officinalis), and oregano ( Origanum vulgare). Oregano plant extract and essential oil showed the highest antifungal activity, at MIC values of 4.9 mg/mL and 0.4 mg/mL respectively. Therefore, it was subjected to further research on the influence of virulence factors - biofilm formation, extracellular phospholipase production and germ tube formation. Oregano plant extract and essential oil showed an inhibitory effect on the observed C. albicans virulence factors at relatively low concentrations. The extract inhibited the adherence of cells at MIC 12.5 mg/mL and essential oil at MIC 0.25 mg/mL. Degradation of the formed biofilm was detected at MIC 14.1 mg/mL for plant extract and at MIC 0.4 mg/mL for essential oil. Extracellular phospholipase production was most effectively inhibited by the essential oil. In particular, the number of isolates with intensive extracellular phospholipase production decreased significantly. Of the 12 isolates intensively producing extracellular phospholipase, only 1 isolate (4.5%) retained intense production. Essential oil caused up to a 100 % reduction in germ tubes formation and plant extract reduced their formation depending on the concentration as follows: 2.6% (0.8 mg/mL), 21.2 % (6.25 mg/mL), and 64.5 % (12.5 mg/mL) compared to the control.

Phytochemical characterization and phospholipase A2 inhibitory effect of Vitex negundo L. root extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Snake bites are a critical health issue in many parts of the world particularly in Asian countries lacking efficient health facilities in rural areas. Cobra is the most common snake type in Asia and is responsible for a large number of mortalities particularly in rural areas. Plants are usually considered the most effective and easy-to-approach treatment for snake bites in rural areas of various countries. Vitex negundo L. is an important medicinal plant traditionally used to treat snake bite envenomation in many countries of Asia. AIM OF THE STUDY: From literature survey of plants traditionally used in the treatment of snake bites in Asian countries including India, Pakistan and Sri Lanka, roots of V. negundo were selected for the present study. Anti-snake venom potential of its roots was assessed through various in vitro assays targeting the phospholipase A2 enzyme. MATERIALS AND METHODS: V. negundo roots were sequentially extracted in different organic solvents to get fractions and in methanol to get total extract. The extracts were evaluated for phospholipase A2 (PLA2) inhibitory potential through inhibition of venom-induced hemolysis, ADP-induced platelet aggregation, PLA2-induced fatty acid hydrolysis and anticoagulant effect of cobra venom. Antioxidant power was determined using DPPH and superoxide radical scavenging assays. GC-MS and HPLC analysis was performed for the total methanol extract. RESULTS: Strong PLA2 inhibitory effect was observed for all the extracts. The ethyl acetate, acetone and methanol fractions significantly inhibited toxic effects of cobra venom under in vitro conditions. Radical scavenging potential of these fractions was also significantly high as compared to non-polar fractions in both DPPH and superoxide scavenging assays. Phytochemical analysis indicated high phenolic and flavonoid contents in these fractions. GC-MS and HPLC analysis of total methanol extract confirmed the presence of bis(2-ethylhexyl) phthalate, phenol, o-Guaiacol, palmitic acid-methyl ester, methyl stearate, quercetin and kaempferol in the plant. CONCLUSION: The study concluded that the roots of V. negundo, particularly their polar extracts, have strong PLA2 inhibitory effect against cobra venom confirming their traditional use to manage snake bites. The roots of this plant can be further studied for isolation of plant-based antisera.

Preparation of magnetic dialdehyde starch-immobilized phospholipase A1 and acyl transfer in reflection.

In this paper, using a coprecipitation method to prepare Fe3O4 magnetic nanoparticles (Fe3O4 MNPS), magnetic dialdehyde starch nanoparticles with immobilized phospholipase A1 (MDSNIPLA) were successfully prepared by using green dialdehyde starch (DAS) instead of glutaraldehyde as the crosslinking agent. The Fe3O4 MNPS was characterized by infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), the Brunauer-Emmett-Teller (BET) surface area analysis method, thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) et al. The results showed that the alkaline resistance and acid resistance of the enzyme were improved after the crosslinking of DAS. After repeated use (seven times), the relative activity of MDSNIPLA reached 56 %, and the magnetic dialdehyde starch nanoparticles (MDASN) had good carrier performance. MDSNIPLA was applied to enzymatic hydrolysis of phospholipids in the soybean oil degumming process. The results showed that the acyl transfer rate of sn-2-HPA was 14.01 %, and the content of free fatty acids was 1.144 g/100 g after 2 h reaction at 50 °C and pH 5.0 with appropriate boric acid. The immobilized enzyme has good thermal stability and storage stability, and its application of soybean oil improves the efficiency of the oil.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Optimization using response surface methodology of phospholipase C production from Bacillus cereus suitable for soybean oil degumming.

This work deals with the optimization of an extracellular phospholipase C production by Bacillus cereus (PLCBc) using Response Surface Methodology (RMS) and Box-Behnken design. In fact, after optimization, a maximum phospholipase activity (51 U/ml) was obtained after 6 h of cultivation on tryptone (10 g/L), yeast extract (10 g/L), NaCl (8.125 g/L), pH 7.5 with initial OD (0.15). The PLCBc activity, esteemed by the model (51 U) was very approximate to activity gutted experimentally (50 U). The PLCBc can be considered as thermoactive phospholipase since it showed a maximal activity of 50 U/mL at 60 °C using egg yolk or egg phosphatidylcholine (PC) as substrate. In addition, the enzyme was active at pH 7 and is stable after incubation at 55 °C for 30 min. The application of B. cereus phospholipase C in soybean oil degumming was investigated. Our results showed that when using enzymatic degumming, the residual phosphorus decrease more than with water degumming, indeed, it passes from 718 ppm in soybean crude oil to 100 ppm and 52 ppm by degumming using water and enzymatic process, respectively. The diacylgycerol (DAG) yield showed an increase of 1.2% with enzymatic degumming compared to soybean crude oil. This makes our enzyme a potential candidate for food industrial applications such as enzymatic degumming of vegetable oils.

Identification and characterization of the key lethal toxin from jellyfish Cyanea nozakii.

Jellyfish Cyanea nozakii venom is a complex mixture of various toxins, most of which are proteinous biological macromolecules and are considered to be responsible for clinical symptoms or even death after a severe sting. Previous transcriptome and proteome analysis identified hundreds of toxins in the venom, including hemolysins, C-type lectin, phospholipase A2, potassium channel inhibitor, metalloprotease, etc. However, it is not clear which toxin in the venom plays the most important role in lethality. Herein, we isolated the key lethal toxin (Letoxcn) from jellyfish Cyanea nozakii using anion exchange chromatography, size-exclusion chromatography, and cation exchange chromatography. The molecular weight of Letoxcn is ∼50 kDa with the N-terminal sequences of QADAEKVNLPVGVCV. Peptide mass fingerprinting analysis of Letoxcn shows that it may have some motifs of phospholipase, metalloproteinase, thrombin-like enzyme, potassium channel toxin, etc. However, only metalloproteinase activity but no hemolytic, PLA2, or blood coagulation activity was observed from in vitro toxicity analysis. Overall, this study uncovered and characterized the key lethal toxin in the venom of jellyfish Cyanea nozakii, which will not only help to reveal the molecule mechanism of the lethality, but also develop effective treatment like antivenom for this jellyfish sting in the future.

Characterization of a novel thermostable phospholipase C from T. kodakarensis suitable for oil degumming.

The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils' phospholipids. This enzymatic method reduces the gums' volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. KEY POINTS: • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.

Mass spectrometry and synchrotron-FTIR microspectroscopy reveal the anti-inflammatory activity of Bua Bok extracts.

INTRODUCTION: Bua Bok or Centella asiatica (CA) is an Asian vegetable with anti-inflammatory benefits. Asiaticoside, asiatic acid, madecassoside and madecassic have been characterised as major active ingredients with a wide range of pharmacological advantages. In manufacturing processes, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) are used to routinely determine the active compounds in raw materials. OBJECTIVES: This research aims to explore anti-inflammatory properties, characterise metabolites and observe the biochemical changes of the inflammatory induced macrophages after pretreatment with the potential extracted fractions. METHODS: Bua Bok leaf extracts were prepared. Macrophages were pretreated with non-toxic fractions to determine the anti-inflammatory action. Tentative metabolites of effective fractions were identified by LC-MS. Synchrotron fourier-transform infrared (S-FTIR) microspectroscopy was utilised to observe the biochemical change of the lipopolysaccharide (LPS)-induced cells after pretreatment with potential fractions. RESULTS: Fractions of ethyl acetate, 30% and 100% ethanol highly increased the nitrile scavenging and suppressed the function of phospholipase A2 . Fractions of 70% and 100% ethanol strongly decreased nitric oxide production. The comparison of 39 chemical compounds was presented. The change of proteins was improved after pretreatment of macrophages with fraction 70% ethanol. Fraction of 100% ethanol revealed the lipid accumulation was lower than 70% ethanol and diclofenac. CONCLUSION: While the anti-inflammatory actions of 70% and 100% ethanol were similar. S-FTIR expressed they inhibited inflammatory response with the distinct features of biomolecules. The S-FTIR, LC-MS and biological assay confidently provided the efficient strategies to inform the advantage of herbal extract on cellular organisation instead of a single compound.

A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization.

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab')2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25-0.5 µL of intravenous Apilic antivenom/µg honeybee venom. In vivo injection of 0.1-1 µg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 µg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

Identifying and revealing the geographical variation in Nemopilema nomurai venom metalloprotease and phospholipase A2 activities.

Venom geographical variation is common among venomous animals. This phenomenon presents problems in the development of clinical treatments and medicines against envenomation. The venomous giant jellyfish Nemopilema nomurai, Scyphozoan, is a blooming jellyfish species in the Yellow Sea and the East China Sea that causes numerous jellyfish sting cases every year. Metalloprotease and phospholipase A2 (PLA2) are the main components in Nemopilema nomurai venom and may activate many toxicities, such as hemolysis, inflammation and lethality. Geographical variation in the content and activity of these enzymes may cause different symptoms and therapeutic problems. For the first time, we verified metalloprotease and PLA2 geographical variation in Nemopilema nomurai venom by performing a comparative analysis of 31 venom samples by SDS-PAGE, analyzing protease zymography, enzymatic activity, and drawing contour maps. Band locations and intensities of SDS-PAGE and protease zymograms showed geographical differences. The enzymatic activities of both metalloprotease and PLA2 showed a trend of geographic regularity. The distribution patterns of these activities are directly shown in contour maps. Metalloproteinase activity was lower near the coast. PLA2-like activity was lower in the Southern Yellow Sea. We surmised that metalloproteinase and PLA2-like activities might be related to venom ontogeny and species abundance respectively, and influenced by similar environmental factors. This study provides a theoretical basis for further ecological and medical studies of Nemopilema nomurai jellyfish venom.

Mezoneuron benthamianum inhibits cell adherence, hyphae formation, and phospholipase production in Candida albicans.

The aim of this study was to evaluate the phytochemical constituents, antioxidant, antifungal, and anti-virulence activities of traditionally used Mezoneuron benthamianum leaves. Extracts were prepared using acetone and methanol, and the preliminary phytochemical screening was performed. The antioxidant activity was studied using the DPPH method. Anti-Candida albicans activity was established and the effect on the germ tube and phospholipase production, as well as on the host cell adherence was assessed. The extracts showed the presence of anthraquinones, cardiac glycosides, flavonoids, reducing sugars, saponins, steroids, tannins, and terpenoids. Gallic acid and trans-resveratrol were among the predominant phytochemicals found in M. benthamianum. The crude extracts presented significantly higher antioxidant activity than the ascorbic acid standard. At 0.39 mg/mL, acetone extract inhibited the growth of Candida albicans. At lower concentrations (200-50 µg/mL), it significantly inhibited the adherence ability (up to 51%), formation of hyphae (up to 65%), and the production of phospholipase. In conclusion, at high concentrations, M. benthamianum kills C. albicans, and at lower concentrations, it can inhibit the virulence properties of this pathogen. This study on crude extract validates the traditional use of this plant. However, further research is required to establish the anti-virulence activity of the two compounds and their therapeutic potential.

Secreted Phospholipase PLA2G2D Contributes to Metabolic Health by Mobilizing ω3 Polyunsaturated Fatty Acids in WAT.

Polyunsaturated fatty acids (PUFAs) confer health benefits by preventing inflammation and obesity and by increasing thermogenesis in brown and beige adipocytes. As well as being supplied exogenously as nutrients, PUFAs are largely stored in membrane glycerophospholipids and released by phospholipase A2s (PLA2s). However, the molecular identity of the PLA2 subtype(s) that supplies endogenous PUFAs for metabolic homeostasis remains unclear. Here we show that PLA2G2D, a secreted PLA2 isoform, is constitutively expressed in M2-type macrophages in white adipose tissue (WAT) and shows a reciprocal correlation with obesity. Studies using global and macrophage-specific Pla2g2d-deficient mice reveal that PLA2G2D increases energy expenditure and thermogenesis by facilitating adipocyte browning, thereby ameliorating diet-induced obesity, insulin resistance, and WAT inflammation. Mechanistically, PLA2G2D constitutively supplies a pool of PUFAs, ω3 in particular, in WAT. Thus, our present findings underscore the contribution of the macrophage-driven PLA2G2D-ω3 PUFA axis to metabolic health.

Nonspecific phospholipase C6 increases seed oil production in oilseed Brassicaceae plants.

Plant oils are valuable commodities for food, feed, renewable industrial feedstocks and biofuels. To increase vegetable oil production, here we show that the nonspecific phospholipase C6 (NPC6) promotes seed oil production in the Brassicaceae seed oil species Arabidopsis, Camelina and oilseed rape. Overexpression of NPC6 increased seed oil content, seed weight and oil yield both in Arabidopsis and Camelina, whereas knockout of NPC6 decreased seed oil content and seed size. NPC6 is associated with the chloroplasts and microsomal membranes, and hydrolyzes phosphatidylcholine and galactolipids to produce diacylglycerol. Knockout and overexpression of NPC6 decreased and increased, respectively, the flux of fatty acids from phospholipids and galactolipids into triacylglycerol production. Candidate-gene association study in oilseed rape indicates that only BnNPC6.C01 of the four homeologues NPC6s is associated with seed oil content and yield. Haplotypic analysis indicates that the BnNPC6.C01 favorable haplotype can increase both seed oil content and seed yield. These results indicate that NPC6 promotes membrane glycerolipid turnover to accumulate TAG production in oil seeds and that NPC6 has a great application potential for oil yield improvement.

Impact of water degumming and enzymatic degumming on gum mesostructure formation in crude soybean oil.

Phospholipid gum mesostructures formed in crude soybean oil after water degumming (WD) and enzymatic degumming (ED) were studied at a range of phospholipid and water concentrations. For ED, phospholipase C (PLC), phospholipase A2 (PLA2) and a mixture of phospholipases Purifine 3G (3G) were used. Both WD and ED resulted in lamellar liquid-crystalline phases, however, of different topology. The dependence of the bilayer spacings (as observed by SANS and SAXS) on the ratio between amount of water and amphiphilic lipids differed for WD and PLA2 ED vs PLC and 3G ED. This difference was also observed for dynamics at molecular scale as observed by time-domain (TD) NMR and attributed to partial incorporation of diglycerides and free fatty acids into gum bilayers after PLC and 3G ED. Feasibility of using TD-NMR relaxometry for quantification of the gum phase and estimation of degumming efficiency was demonstrated.

Non-specific phospholipases C, NPC2 and NPC6, are required for root growth in Arabidopsis.

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non-specific phospholipases C, NPC2 and NPC6, whose double knock-out causes a gametophyte-lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock-down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non-specific phospholipase C (NPC) -catalyzed phosphatidylcholine hydrolysis. The expression of phospho-base N-methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock-down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT-catalyzed pathway, which highlights a tissue-specific role of NPC enzymes in vegetative growth beyond the gametophyte-lethal phenotype.

Effects of high pressure treatment on lipolysis-oxidation and volatiles of marinated pork meat in soy sauce.

To investigate the effect of high pressure (HP) treatment (150 and 300 MPa for 15 min at 20 °C) on lipolysis-oxidation and volatile profile of marinated pork meat in soy sauce, the changes of lipase, phospholipase and lipoxygenase (LOX) activities, TBARS, free fatty acids and volatiles composition in control and HP treated samples were analyzed. Acid and neutral lipase activities and free fatty acids content decreased, while LOX activity and TBARS increased after HP treatment. Phospholipase had well stability under HP. The levels of volatile compounds from lipid oxidation and brine increased under HP and then contributed 73.16-78.25% of the typical aroma, while volatile compounds from carbohydrate fermentation, especially acetic acid, decreased with the pressure increasing. The decrease of free fatty acids during pressurization was probably attributed to the decline of lipase activity and the increase of LOX activity. These findings indicated that HP (150-300 MPa/15 min) promoted lipid oxidation and the permeation of brine, but inhibited carbohydrate fermentation.

The phospholipase DDHD1 as a new target in colorectal cancer therapy.

BACKGROUND: Our previous study demonstrates that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability, and that this effect is associated with the downregulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on the contribution of DDHD1 in neurological disorders, there is no information on its role in cancer. This study investigates the role of DDHD1 in colon cancer. METHODS: DDHD1 siRNAs and an overexpression vector were transfected into colorectal cancer and normal cells to downregulate or upregulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics using SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. RESULTS: The results indicate that DDHD1 supports colon cancer cell proliferation and survival, since its downregulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumors, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Additionally, we found that functional categories, significantly affected by DDHD1 silencing, were specifically related to cancer phenotype and for the first time associated to DDHD1 activity. CONCLUSIONS: In conclusion, this study provides the first evidence confirming the role of DDHD1 in cancer, providing a possibility to define a new target to design more effective therapies for colon cancer patients.

Evaluation of anti-enzyme properties of Origanum vulgare essential oil against oral Candida albicans.

This study aimed to evaluate the anti-enzymatic activity of Origanum vulgare (oregano) essential oil against 15 strains of Candida albicans. Candida albicans samples were isolated from the oral mucosa of patients with denture stomatitis treated in a Dentistry school on a public university. Preparation of the inoculum was performed with a suspension of C. albicans reactivated 24h earlier in 5mL of sterile phosphate buffer saline (PBS) adjusted to a 0.5-turbidity on the MacFarland scale (1,5×108UFC/mL). The essential oil was obtained by hydrodistillation in a Clevenger-type machine and analyzed by gas chromatography. Enzymatic assay was performed to test phospholipase anti-enzymatic properties. Chromatography analysis revealed that the main compounds present in the essential oil were 4-terpineol (41.17%), thymol (21.95%), γ-terpinene (5.91%) and carvacrol (4.71%). For the anti-enzymatic test, the statistical analysis showed that there was found statistically significant interactions between the factors time and concentration (P≤0,001). Thus, essential oil of oregano at 1%, 5% and 10% presented significant reductions in the production of the phospholipase enzyme produced by Candida albicans strains. However, the longer the incubation time of the essential oil, there is a relatively moderate reduction in its anti-enzymatic activity.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.