Semi-purified Fractions Research Articles

Antibacterial, Antibiofilm and Anti-Virulence Activity of Biactive Fractions from Mucus Secretion of Giant African Snail Achatina fulica against Staphylococcus aureus Strains.

Staphylococcus aureus is an important etiological agent that causes skin infections, and has the propensity to form biofilms, leading to significant mortality and morbidity in patients with wounds. Mucus secretion from the Giant African snail Achatina fulica is a potential source of biologically active substances that might be an important source for new drugs to treat resistant and biofilm-forming bacteria such as S. aureus. This study evaluated the effect of semi-purified fractions from the mucus secretion of A. fulica on the growth, biofilm formation and virulence factors of S. aureus. Two fractions: FMA30 (Mw >30 kDa) and FME30 (Mw 30−10 kDa) exhibited antimicrobial activity against S. aureus with a MIC50 of 25 and 125 µg/mL, respectively. An inhibition of biofilm formation higher than 80% was observed at 9 µg/mL with FMA30 and 120 µg/mL with FME30. Furthermore, inhibition of hemolytic and protease activity was determined using a concentration of MIC20, and FME30 showed a strong inhibitory effect in the formation of clots. We report for the first time the effect of semi-purified fractions of mucus secretion of A. fulica on biofilm formation and activity of virulence factors such as α-hemolysin, coagulase and proteases produced by S. aureus strains.

Chemical Evaluation, Antioxidant, Antiproliferative, Anti-Inflammatory and Antibacterial Activities of Organic Extract and Semi-Purified Fractions of the Adriatic Sea Fan, Eunicella cavolini.

Due to sedentary lifestyle and harsh environmental conditions, gorgonian coral extracts are recognized as a rich source of novel compounds with various biological activities, of interest to the pharmaceutical and cosmetic industries. The presented study aimed to perform chemical screening of organic extracts and semi-purified fractions obtained from the common Adriatic gorgonian, sea fan, Eunicella cavolini (Koch, 1887) and explore its abilities to exert different biological effects in vitro. Qualitative chemical evaluation revealed the presence of several classes of secondary metabolites extended with mass spectrometry analysis and tentative dereplication by using Global Natural Product Social Molecular Networking online platform (GNPS). Furthermore, fractions F4 and F3 showed the highest phenolic (3.28 ± 0.04 mg GAE/g sample) and carotene (23.11 ± 2.48 mg β-CA/g sample) content, respectively. The fraction F3 inhibited 50% of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS (2,2′-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid) radicals at the concentrations of 767.09 ± 11.57 and 157.16 ± 10.83 µg/mL, respectively. The highest anti-inflammatory potential was exhibited by F2 (IC50 = 198.70 ± 28.77 µg/mL) regarding the inhibition of albumin denaturation and F1 (IC50 = 254.49 ± 49.17 µg/mL) in terms of soybean lipoxygenase inhibition. In addition, the most pronounced antiproliferative effects were observed for all samples (IC50 ranging from 0.82 ± 0.14–231.18 ± 46.13 µg/mL) against several carcinoma cell lines, but also towards non-transformed human fibroblasts pointing to a generally cytotoxic effect. In addition, the antibacterial activity was tested by broth microdilution assay against three human pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The latter was the most affected by fractions F2 and F3. Finally, further purification, isolation and characterization of pure compounds from the most active fractions are under investigation.

IN VITRO ANTILEISHMANIAL ACTIVITIES OF THREE MEDICINAL PLANTS: ARGEMONE MEXICANA, MURRAYA KOENIGII AND CINNAMOMUM TAMALA AGAINST MILTEFOSINE RESISTANT PROMASTIGOTES OF LEISHMANIA DONOVANI PARASITES

Objective: Leishmaniasis is one of the neglected tropical diseases in terms of drug development and discovery. Non-responsiveness and resistance to the drug in Leishmania species need to develop new antileishmanial potentials; herbal medicines could be the alternative one. Methods: In the present study, semi-purified fractions were prepared from the traditionally used three medicinal plants of India: Argemone mexicana (aerial shoot), Murraya koenigii (stem), and Cinnamomum tamala (bark) by using multiple solvent systems (non-polar to polar, beginning with petroleum ether followed by n-hexane, benzene, and chloroform) and an effort was given to assess the leishmanicidal activities against Leishmania donovani miltefosine resistant HePC-R (Ld/MIL-30) promastigotes in vitro and the IC50 concentrations were estimated. Results: The study revealed that the semi-purified fractions of A. mexicana, M. koenigii, and C. tamala have effective antileishmanial activities and the 50% inhibitory concentrations (IC50) are 50 μg/ml, 98 μg/ml, and 200 μg/ml, respectively. At these (IC50) concentrations, these plant semi-purified fractions were found to interfere in lipid and protein biosynthesis, alter cell morphology, DNA content, mitochondrial membrane potential, generating ROS, and apoptosis in promastigotes. The semi-purified fractions were also found noticeably non-toxic towards host splenocytes. Conclusion: These results could suggest that A. mexicana, M. koenigii, and C. tamala could carry potential novel compounds for the development of new drugs against Leishmaniasis.

Antioxidant, Mineralogenic and Osteogenic Activities of Spartina alterniflora and Salicornia fragilis Extracts Rich in Polyphenols.

Osteoporosis is an aging-related disease and a worldwide health issue. Current therapeutics have failed to reduce the prevalence of osteoporosis in the human population, thus the discovery of compounds with bone anabolic properties that could be the basis of next generation drugs is a priority. Marine plants contain a wide range of bioactive compounds and the presence of osteoactive phytochemicals was investigated in two halophytes collected in Brittany (France): the invasive Spartina alterniflora and the native Salicornia fragilis. Two semi-purified fractions, prepared through liquid-liquid extraction, were assessed for phenolic and flavonoid contents, and for the presence of antioxidant, mineralogenic and osteogenic bioactivities. Ethyl acetate fraction (EAF) was rich in phenolic compounds and exhibited the highest antioxidant activity. While S. fragilis EAF only triggered a weak proliferative effect in vitro, S. alterniflora EAF potently induced extracellular matrix mineralization (7-fold at 250 μg/mL). A strong osteogenic effect was also observed in vivo using zebrafish operculum assay (2.5-fold at 10 μg/mL in 9-dpf larvae). Results indicate that polyphenol rich EAF of S. alterniflora has both antioxidant and bone anabolic activities. As an invasive species, this marine plant may represent a sustainable source of molecules for therapeutic applications in bone disorders.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme

Main conclusionA synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect.Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

Effect of Ethanolic Caesalpinia sappan Fraction on In Vitro Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major epidemic in pig production, leading to economic losses in the pig industry worldwide. The use of medicinal plants with antiviral properties might be useful help to prevent and control PRRSV outbreaks. Caesalpinia sappan (CS) heartwood is an important herbal ingredient used in Thai folk medicine, possessing various biological activities, including antiviral activity. The present study focuses on the in vitro antiviral activity against PRRSV of a semi-purified fraction of ethanolic CS crude extract using preparative high-performance liquid chromatography. Qualification of the fractions illustrating positive antiviral activity was carried out with liquid chromatography–quadrupole time-of-flight mass spectrometry. The preparative chromatography separated the crude extract into six consecutive fractions, among which the first fraction showed potential antiviral activity by inhibiting PRRSV replication in a MARC-145 monolayer (virus titer 2.75 median tissue culture infective dose (TCID50)/mL (log10) vs. 9.50 median log10 TCID50/mL of the control) at 72 h post-infection, and this fraction included byakangelicin, brazilin, naringenin, and brazilein. These results provide useful information for further study to effectively develop the CS bioactive antiviral compounds against PRRSV as a feed additive or veterinary drug in the pig industry.

Potential Anti-leishmanial Activity of a Semi-purified Fraction Isolated from the Leaves of Parthenium hysterophorus.

In the present perspective, emergence of resistant strains of Leishmania donovani and severe side effects resulting from the use of conventional anti-leishmanial therapies present an urgent need for developing novel agents against this parasite. We have explored the effectiveness of secondary plant metabolites as alternative choices in the treatment for visceral leishmaniasis (vl). The plant Parthenium hysterophorus L. (Asteraceae) was collected from the West Bengal State University Campus, Barasat, West Bengal, India. The leaves of this plant were extracted by different solvents, such as ethyl acetate, water, petroleum ether and hexane. Gas chromatography-mass spectrometry (GC-MS) analysis was also carried out for the identification of compounds in the hexane soluble fraction (PHFd) with substantial anti-leishmanial activities. The antipromastigote activity and cytotoxicity of this fraction were evaluated by the tetrazolium MTT assay. Other biochemical and physiological parameters were studied by microscopic observation and flow cytometric analyses. PHFd showed considerable activity against L. donovani promastigotes (IC50: 20 µg/ml). The PHFd also inhibited in vitro growth of L. major LV39 promastigotes dose dependently with an IC50 of 40 µg/ml. The GC-MS studies of this particular fraction revealed the presence of four major compounds with different retention times (RT) of 26.08, 33.11, 36.41, and 41.20 min. In this study, we also established that PHFd could induce DNA damage and subsequent apoptosis of L. donovani promastigotes with a concomitant increase in generations of reactive oxygen species (ROS) in a time-dependent manner. This fraction was also found to be effective in nitric oxide-mediated inhibition of intracellular amastigotes (IC50:12.5 µg/ml) without any noticeable cytotoxicity towards murine splenocytes in vitro. This study provides the basis for additional phytochemical and pharmacological studies on the antiprotozoal applications of P. hysterophorus.

Postharvest application of partitioned plant extracts from Sinaloa, Mexico for controlling papaya pathogenic fungus Colletotrichum gloeosporioides

Anthracnose compromises papaya production and is caused by the fungus Colletotrichum gloeosporioides. New natural alternatives to synthetic fungicides are necessary to control anthracnose due to health and environmental concerns. In this regard, extracts of plants from Sinaloa, Mexico, have shown activity against molds and yeasts with medical and agricultural importance; however, their protective effect on papaya fruit infected with C. gloeosporioides is still unknown. This study aimed to assess the in vitro and in vivo antifungal activities of crude methanol extracts (ME) from nine plants from Sinaloa, Mexico, and their semi-purified fractions. In vitro assays showed that C. gloeosporioides was inhibited by 7 out the 16 extracts assessed; Psidium sartorianum (pulp), Echeveria kimnachii (leaf), and Vitex mollis (VM) (pulp) had the highest antifungal activity and the lowest toxicity against Artemia salina. When these extracts were fractionated, the activity increased. Hexane (HF-VM) and ethyl acetate (EAF-VM) fractions of V. mollis were the most effective fractions (MEF), with the lowest minimum inhibitory concentration (MIC) (20 and 30 mg/mL, respectively). The in vivo results showed that HF-VM at 40 mg/mL (HF40) was the best to delay the apparition and development of anthracnose symptoms. Coumarins, alkaloids and terpenes were detected on this fraction by tube assays or thin layer chromatography (TLC). Moreover, this treatment decreased water loss and did not affect any of the quality parameters assessed. Therefore, HF40 is a natural alternative to thiabendazole (TBZ) in the protection of papaya fruit against anthracnose.

In-vitro evaluation of antioxidant and antiradical potential of successive extracts, semi-purified fractions and biosynthesized silver nanoparticles of Rumex vesicarius

The aim of the present study was to assess in vitro the antiradical and antioxidant activities of successive extracts and semi-purified fractions from Rumex vesicarius L. In the present work, three extracts (n-Hexane, ethyl acetate and methanol) and 22 column fractions of methanolic extract (as promising extract) were evaluated against 2,2-diphenyl- 1-picrylhydrazyl (DPPH•) and 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) radical scavenging methods as antiradical and antioxidant activities compared with Butylated hydroxytoluene (BHT) as synthetic standard and silver nanoparticles of methanolic extract (Ag-NPs-Me), in addition to analysis of chemical constituents of extract and fraction using Gas chromatography–mass spectrometry (GC-MS). The obtained results revealed that, both methods go parallel showing that the concentration of extract and incubation time are dependent and proportional with phenolic compounds concentration. Absolute methanol extract recorded the highest antioxidant activity when compared with the other crude extracts with 79.3 and 78.8% against DPPH and ABTS respectively when compared with BHT as synthetic standard (89.4 and 89.9%) against DPPH and ABTS respectively. Calculation of the antiradical activity units showed the highest values of methanolic extract and its promising fraction (No. 12) after 300 seconds (5 minutes) comparing with antioxidant activity (30 min). Also, the antioxidant activity increased with synthetic Ag-NPs-Me when compared with methanolic extract by (IC50= 53.9 and 74.6 µg/ml respectively). Thus, the GC-MS analysis of successive extracts of R. vesicarius L showed a highly complex profile, containing approximately 24 different components. One pure compound was identified from fraction No. 12. The identified compound was l-(+)- ascorbic acid 2, 6-dihexadecanoate. The data also revealed presence of closely similar antioxidant activities in methanolic extract or its pure compounds with BHT when mixed at different proportions. From the obtained results it could be concluded that R. vesicarius methanolic extracts and fractions can be extensively used in the production of potential antioxidant, antiradical and AgNPs-Me for biomedical application on the consumer’s health.

Aqueous Polyphenol Semi-Purified Fractions from Galls of Quercus infectoria Increase ALP Activity and Mineralisation of hFOB 1.19 Cells

The galls of Quercus infectoria (QI) have been reported to possess numerous medicinal values and give a positive effect on bone metabolism. This study investigated the effects of semi-purified fractions of QI gall extract on the proliferation, alkaline phosphatase (ALP) activity, and mineralisation of human foetal osteoblast cell line (hFOB1.19). The semi-purified fractions (fraction A and B) were prepared by column chromatography methods. MTT assay was used to measure cell proliferation activity to obtain half maximal concentration (EC50) of the cells treated with fraction A (phenolic components and contained amide), fraction B (phenolic components with the presence of alkene), and pamidronate (drug control). The most potent or lowest EC50 was further used to measure ALP activity in the treated and untreated cells at day 1, 3, 7, 10, and 14 by ELISA. Cell mineralisation was determined by von Kossa staining for phosphate depositions and Alizarin Red S staining for calcium depositions. The EC50 values for fraction A and B were 8.86 and 9.92 μg/mL, respectively, which showed a greater effect compared to the pamidronate (15.27 μg/mL). The ALPactivity of both fractions in the treated cells were also greater compared to the two control groups (cells treated with pamidronate and untreated cells), starting from day 3 onwards. The calcium depositions appeared as red spots, while phosphate depositions appeared as black spots. Interestingly, the calcium depositions of cells treated with both fractions were higher than those of the two control groups. In conclusion, semi-purified fractions of QI gall extract enhanced proliferation, improved mineralisation, and increased ALP activity of hFOB 1.19 cells.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections

ABSTRACT We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Plectranthus amboinicus (Spreng.) Semi-purified Fractions with Selective β-Glucuronidase Inhibition Elucidated with gas chromatography-mass spectrometry and in silico docking

Background: Plectranthus amboinicus (Spreng.) is an herb commonly used in folk medicine and food by several Asian countries. The bioactivities of this medicinal plant have so far not been linked to a specific enzyme target. Bacterial beta-glucuronidases (β-GUS) expressed by human gut microbiota affect xenobiotic processing by reactivating toxic substances (e.g., anticancer drugs, nonsteroidal anti-inflammatory drugs, and food carcinogens) in the gut lumen. Objectives: An approach to alleviating the toxic effects of these compounds is by inhibiting bacterial β-GUS. Materials and Methods: We determined the Escherichia coli β-GUS inhibitory activity of P. amboinicus leaves using a bioassay-guided purification approach. The P. amboinicus chloroform extract was purified using normal-phase column chromatography to produce several fractions. The fractions were screened for E. coli β-GUS inhibitory activity using the 4-Methylumbelliferyl glucuronide (4-MUG) assay. Fractions with high activity were assayed further to determine toxicity against E. coli and selectivity compared to human β-GUS. Highly-active and highly-selective fractions were further characterized using gas chromatography-mass spectrometry (GC-MS) and in silico docking to identify specific compounds. Results: Assay-guided purification of the crude chloroform extract with β-GUS inhibitory activity (IC50 = 57.8 μg/mL) yielded four fractions with high activity: Fraction-543W (IC50 = 16.24 μg/mL), Fraction-5231 (IC50 = 3.087 μg/mL), and Fraction-52335A (IC50 = 12.93 μg/mL). The crude extract and fractions exhibited high selectivity for E. coli β-GUS against human β-GUS (P

Bioactivity-directed evaluation of fruit of Kigelia africana (Lam.) benth. Used in treatment of malaria in Iwo, Nigeria

Ethnopharmacological relevanceThe ancient people of Iwo communities consisting of Ile-Ogbo, Olupona, Iwo and Ogbagba continue to engage in the traditional use of medicinal plants for the treatment and management of common diseases especially malaria. Aims of this studyThis study conducted an ethnomedicinal survey of plants used to treat malaria and feverish conditions by the people of Iwo, Nigeria. It also evaluated the antiplasmodial activity of the morphological parts of Kigelia africana (Lam.) Benth., and isolated, as well as characterised pure compounds from the semi-purified fractions of the fruit extract. Materials and methodsThe ethnomedicinal survey was conducted using semi-structured questionnaires administered to only herb sellers in Iwo, Ile-Ogbo, Olupona, and Ogbagba areas of Osun State. Extracts of K. africana morphological parts; leaf, root, stem bark, and fruit were obtained by cold maceration in methanol, followed by assessment of acute toxicity (LD50) and antiplasmodial activity in Plasmodium berghei infected rats using the 4-day suppressive test model. The most active fruit extract was further subjected to activity-guided fractionation and purification using n-hexane, dichloromethane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and methanol (MeOH) in gradients to obtain the semi-purified fractions and two pure isolated compounds using various chromatographic and spectroscopic techniques. Results and discussionFrom the survey, thirty-one plant species were identified for treating malaria in Iwo area. Azadirachta indica leaf was the most frequently used (78.3% of the respondents) while Manihot esculenta leaf (3.33%) was the least. The identified plants are distributed among 24 families, with Anacardiaceae and Asteraceae (11.67% each) been the most occurring families. Kigelia africana (Bignoniaceae) ranked the 6th position with 60% frequency of occurrence. The LD50 values obtained for the extracts were greater than 5000 mg/kg (p.o). The chemo-suppression activity of the extracts at 125 mg/kg was in the order of stem bark (26.59%), leaf (41.75%), root (43.95%), and fruit (54.54%). The semi-purified methanol fraction of the fruit showed the most antiplasmodial activity with a percent chemo-suppression of 69.94 and yielded 4-(2,3-dihydroxypropoxy)-3,5-dihydroxy-5-methylfuran-2-one and sucrose. ConclusionThe use of herbs and medicinal plants either singly or in combination for the treatment of malaria among the people of Iwo community in Nigeria is still well practised. Lack of formal education among most of the respondents and use of same local name for different plants species or plant parts; which often lead to wrong plant collection were among the constrains encountered. Kigelia africana has antiplasmodial activity in the order of fruit > root > leaf > stem bark.

Development of a novel and rapid phenotype-based screening method to assess rice seedling growth

BackgroundRice (Oryza sativa) is one of the most important model crops in plant research. Despite its considerable advantages, (phenotypic) bioassays for rice are not as well developed as for Arabidopsis thaliana. Here, we present a phenotype-based screening method to study shoot-related parameters of rice seedlings via an automated computer analysis.ResultsThe phenotype-based screening method was validated by testing several compounds in pharmacological experiments that interfered with hormone homeostasis, confirming that the assay was consistent with regard to the anticipated plant growth regulation and revealing the robustness of the set-up in terms of reproducibility. Moreover, abiotic stress tests using NaCl and DCMU, an electron transport blocker during the light dependent reactions of photosynthesis, confirmed the validity of the new method for a wide range of applications. Next, this method was used to screen the impact of semi-purified fractions of marine invertebrates on the initial stages of rice seedling growth. Certain fractions clearly stimulated growth, whereas others inhibited it, especially in the root, illustrating the possible applications of this novel, robust, and fast phenotype-based screening method for rice.ConclusionsThe validated phenotype-based and cost-efficient screening method allows a quick and proper analysis of shoot growth and requires only small volumes of compounds and media. As a result, this method could potentially be used for a whole range of applications, ranging from discovery of novel biostimulants, plant growth regulators, and plant growth-promoting bacteria to analysis of CRISPR knockouts, molecular plant breeding, genome-wide association, and phytotoxicity studies. The assay system described here can contribute to a better understanding of plant development in general.

Immunotoxicological effects triggered by the rattlesnake Crotalus durissus cumanensis, mapanare (Bothrops colombiensis) venoms and its purified fractions on spleen and lymph nodes cells

Purpose: The snakes in Venezuela vary in their different venom composition amid the species. In this sense, studies have been carried out elucidating mechanisms related to their immunostimulatory and/or immunosuppressive effects in vitro, measuring inhibition or stimulation on the mice spleen and lymph nodes lymphocytes under the rattlesnake (Crotalus durissus cumanensis) (Cdc) and mapanare (Bothrops colombiensis) crude venoms actions, and also its purified fraction crotoxin (CTX) (Cdc) and a semi-purified fraction (SPF) (Bc) activities. Material and methods: The stimulation of lymphocyte proliferation was carried out in the presence or absence of Concanavalin A (ConA) and lipopolysaccharides (LPS). Results: The lymphocyte response was measured by the Alamar Blue® (Resazurin) assay, observing that the Crotalus crude venom increased basal proliferation in the spleen and lymph nodes, being also increased with ConA and LPS. CTX slightly decreased the proliferative response in the presence of mitogens. Both Bc venom and its SPF fraction had no significant effect on basal proliferation in the spleen and lymph nodes, but a decrease in the response with ConA was observed. These results suggest that CTX has an inhibitory action on lymphocyte proliferation, while Cdc crude venom has a stimulatory action on T and B cell populations. Bothrops colombiensis venom had no effect on these two types of cell populations. As it is known, lymphocytes are cells of enormous flexibility and can operate in diverse aspects, warranting that the correct immune response persists controlled. Conclusions: These results suggested that these different toxins can modulate lymphocyte functional activation toward an inhibitory or stimulatory state.

Antibacterial Activity and Identification of Metabolites from the Semi-Purified Fraction of Chrysophyllum albidum Leaf (African Star Apple)

Antibacterial Activity and Identification of Metabolites from the Semi-Purified Fraction of Chrysophyllum albidum Leaf (African Star Apple)

Fungal-derived extracts induce resistance against Botrytis cinerea in Arabidopsis thaliana

Development of a sustainable agriculture is a current trend searching for alternative and more environmentally friendly strategies to control crop diseases. The necrotrophic fungus Botrytis cinerea, the causal agent of grey mould, affects crops pre- and post-harvest and cause huge economical losses. Microorganisms and microbially-derived elicitors are known to induce plant defences. Here, we study two fungal extracts derived from Colletotrichum acutatum (isolate M11). The crude extract, called CF (culture filtrate) and the semi-purified fraction ACF (axenic culture filtrate) consist in a blend of fungal-derived elicitors. Their activities were evaluated in the plant-pathogen system Arabidopsis thaliana-B. cinerea. Both CF and ACF induced local and systemic resistance, rendering A. thaliana resistant against grey mould disease. The extracts showed no antifungal activity since no inhibition of mycelium growth, changes in hyphal ultrastructure or damage in cell membrane permeability were observed. These findings would indicate that the protective effect was indirect and it was found that CF and ACF induced up-regulation of the defence-related genes PR1, WRKY70 and ERF6, as well as giving local callose deposition in treated leaves. Both fungal extracts have the potential to be used as a main component of a biologically based disease control product with activity against grey mould, with the advantage that the purification process of CF is simpler than for ACF.

Determining the Targets of Fluopsin C Action on Gram-Negative and Gram-Positive Bacteria.

The antibiotic activity of metalloantibiotic compounds has been evaluated since the 90s, and many different modes of action were characterized. In the last decade, the effects of secondary metabolites produced by Pseudomonas aeruginosa LV strain, including a cupric compound identified as Fluopsin C, were tested against many pathogenic bacteria strains, proving their high antibiotic activity. In the present study, the bactericidal mechanisms of action of Fluopsin C and the semi-purified fraction F4A were elucidated. The results found in electron microscopy [scanning electron microscopy (SEM) and transmission electronic microscopy (TEM)] demonstrated that both Fluopsin C and F4A are affecting the cytoplasmatic membrane of Gram-positive and Gram-negative bacteria. These results were confirmed by fluorescence microscopy, where these bacteria presented permeabilization of their cytoplasmatic membranes after contact with the semi-purified fraction and pure compound. Using electronic and fluorescence microscopy, along with bacterial mutant strains with marked divisional septum, the membrane was defined as the primary target of Fluopsin C in the tested bacteria.

Identification of α-Glucosidase Inhibitors from Ipomoea alba by Affinity-Directed Fractionation-Mass Spectrometry

Size-exclusion chromatography, in conjunction with electrospray ionization mass spectrometry, exemplifies a screening methodology to identify α-glucosidase inhibitors from natural extracts. This approach could be used to speed the time of targeting active principles from complex matrices, as a substitute for the established in vitro enzymatic assays which depend on spectrophotometric methods where disadvantages including sensitivity to turbidity and substance interferences—extract pigmentation, or absorption in the visible light region of tested samples—represent factors that affect the chromogenic screenings. Gel permeation through a spin size-exclusion column allowed separating high-affinity molecules bounded to α-glucosidase in solution. After acid hydrolysis of the enzyme-ligand complex, electrospray ionization mass spectrometry was used as a direct detector for the ligand. This affinity-directed fractionation was illustrated by using a crude extract, semi-purified fractions, and pure active glycolipids from moon vine seeds, Ipomoea alba L., Convolvulaceae, in addition to acarbose (positive control), which led to the identification of albinosides VI (ESI-MS m/z 991 [M + Na]+) and VII (ESI-MS m/z 975 [M + Na]+), two tracked high-affinity glycolipids, which demonstrated an in vitro inhibitory potential of α-glucosidases from yeast and rat intestine. Molecular docking predicted that these inhibitors bind to the enzyme catalytic site as acarbose.

Antioxidant properties of extracts and SPE fractions from Annona muricata leaves

Annona muricata L. (Magnoliales: Annonaceae) is a tropical plant species known for its edible fruit and well known for the medicinal value. In this study, the antioxidant properties of A. muricata leaves is evaluated for different extracts and fractions. The airdried leaves of A. muricata leaves were successively extracted with n-hexane and methanol. The methanolic crude extract was further semi-purified by utilizing solid-phase extraction (SPE) in a mixture of mobile phase aqueous: methanol to methanol and resulted in four semi-purified fractions (ALM 1 – ALM 4). Antioxidant properties of extracts and fractions were evaluated by free radical scavenging activity against 1,1-Diphenyl-2- picrylhydrazyl (DPPH) radicals comparing with standard ascorbic acid, quercetin, and catechin. Results indicated that methanolic extracts showed exceptionally higher radical scavenging activity compared with n-hexane crude extract with IC50 44.21 µg/mL and 302.41 µg/mL, respectively. Meanwhile, fractions ALM 2 (aqueous: methanol; 50: 50) exhibited the highest scavenging activity with the IC50 of 12.46 µg/mL followed by ALM 1 (aqueous: methanol; 80: 20) with 51.44 µg/mL, ALM 3 (aqueous: methanol; 20: 80) with 54.39 µg/mL and ALM 4 (100% methanol) with 89.02 µg/mL, respectively. In conclusion, ALM 2 could be potentially applied as a natural antioxidant agent in both food, nutraceuticals or pharmaceuticals fields for health promotion. However, ALM 2 has to be further purified to study the constituents which possibly condensed mainly with phenolic and flavonoids that known to possess the highest antioxidant effects.