PI3K/AKT/mTOR, NF-κB and ERS pathway participated in the attenuation of H2O2-induced IPEC-J2 cell injury by koumine.

ETHNOPHARMACOLOGICAL RELEVANCE: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H2O2 and its potential molecular mechanisms need larger studies. AIM OF THE STUDY: We established an IPEC-J2 cell damage model induced by H2O2 to explore the protective mechanism of koumine on intestinal injury. MATERIALS AND METHODS: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H2O2-induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H2O2-trigged IPEC-J2 cell damage. RESULTS: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H2O2-induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1ß, IL-6, IL-8, TNF-α and TGF-ß), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H2O2. Moreover, koumine dramatically attenuated H2O2-triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-ΚB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H2O2-stimulated IPEC-J2 cell inflammation. CONCLUSIONS: This in vitro experimental study suggests that koumine suppresses the H2O2-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases.

Phytochemical Screening, Antioxidant and Antibacterial Properties of Extracts of Viscum continuum E. Mey. Ex Sprague, a South African Mistletoe.

Viscum continuum E. Mey. Ex Sprague is a woody evergreen semi-parasitic shrub that grows on the branches of other trees. It is used by African traditional healers for post-stroke management. This study reports on the qualitative phytochemical screening and the antioxidant and antimicrobial activities of Viscum continuum's acetone, methanol, hexane and dichloromethane extracts. Standard protocols for the phytochemical screening of extracts were employed. TLC bio-autography was used for qualitative antioxidants analysis. Assays: 2,2-diphenyl-1-picrylhydrazyl, H2O2 free-radical scavenging and ferric chloride reducing power were carried out for quantitative antioxidant analysis. The antimicrobial potential of extracts was screened using disc diffusion, bio-autography and broth micro-dilution. The results indicate the presence of alkaloids, phenolics and tannins in all extracts. Acetone and methanol revealed significant amount of saponins, phenolics, tannins and terpenoids. The extracts exhibited significant antioxidant potential on TLC with positive compound bands at an Rf range of 0.05-0.89. DPPH, H2O2 and the reduction of Fe3+ to Fe2+ assays indicated that methanol extract has a strong antioxidant potential, followed by acetone, DCM and lastly hexane. The extracts of Viscum continuum show the potential to be antibacterial agents. It can be concluded that Viscum continuum extracts contain phytochemicals which are capable of mitigating against chronic health conditions such as cancer, stroke and stress-related and infectious diseases.

CD39+ Regulatory T Cells Attenuate Lipopolysaccharide-Induced Acute Lung Injury via Autophagy and the ERK/FOS Pathway.

Acute respiratory distress syndrome (ARDS) is characterized by an uncontrollable cytokine storm, which is associated with high mortality due to lack of effective treatment. Regulatory T cells (Tregs) play an indispensable role in maintaining immune homeostasis and CD39 is considered as a functional cell marker of Tregs. In this study, we aimed to evaluate the effect of CD39+ Tregs on acute lung injury (ALI) and investigate the frequency of CD39+ Tregs in ARDS patients. We found that after lipopolysaccharide (LPS) treatment, CD39-/- mice exhibited more severe inflammation and wild type (WT) mice exhibited a decreased frequency of CD39+ Tregs in the peripheral blood. Furthermore, CD39+ Tregs had a protective effect on LPS-induced inflammation in vitro and the adoptive transfer of CD39+ Tregs had a therapeutic effect on ALI in vivo. We further sought to explore the mechanisms that affect CD39 expression on Tregs. LPS-induced inflammation in the lung impaired the immunosuppressive effect of Tregs via the autophagy-mediated downregulation of CD39. In addition, CD39 induced the expression of itself in Tregs via activating the ERK1/2-FOS pathway. Consistent with this finding, the frequency of CD39+ Tregs was also decreased in the peripheral blood of ARDS patients and was positively correlated with disease severity. Our results suggested that the adoptive transfer of CD39+ Tregs may provide a novel method for the clinical prevention and treatment of ARDS.

Data on the inhibitory effect of endophytic fungi of traditional medicinal plants against pancreatic lipase (PL).

This article describes isolation and pancreatic lipase (PL) inhibitory potential of 18 endophytic fungi isolated from the various parts of six indigenous medicinal plants. PL catalyzes absorption and hydrolysis of triglycerides into di-glycerides into mono-glycerides and free fatty acids. PL inhibitors are well-known for the disruption of pancreatic lipase activity. The quest for novel pancreatic lipase inhibitors is crucially important owing to their therapeutic potential in the treatment of obesity and related chronic diseases. The present dataset provides information about the presence of endophytic fungi in the internal tissues of selected plants and the PL inhibitory potential of their metabolites using bioassay based screening. Absence of the yellow zone surrounding the standard Orlistat and test extract indicated PL inhibition due to the cumulative effect of metabolites present in the extract. The data suggests that TLC bio-autographic method is simple, rapid and reproducible and therefore it could be effectively used for high throughput screening of PL inhibitors from natural sources.

Exploring Common Culinary Herbs and Spices as Potential Anti-Quorum Sensing Agents.

Quorum sensing controls bacterial pathogenesis and virulence; hence, interrupting this system renders pathogenic bacteria non-virulent, and presents a novel treatment for various bacterial infections. In the search for novel anti-quorum sensing (AQS) compounds, 14 common culinary herbs and spices were screened for potential antipathogenicity activity against Chromobacterium violaceum ATCC 12472. Extracts of Glycyrrhiza glabra (liquorice), Apium graveolens (celery), Capsicum annuum (cayenne pepper) and Syzygium anisatum (aniseed) demonstrated good AQS potential, yielding opaque halo zones ranging from 12⁻19 mm diameter at sub-minimum inhibitory concentrations (0.350⁻4.00 mg/mL). For the same species, the percentage reduction in violacein production ranged from 56.4 to 97.3%. Zones with violacein inhibitory effects were evident in a celery extract analysed using high performance thin layer chromatography-bio-autography. The major active compound was isolated from celery using preparative-high performance liquid chromatography-mass spectrometry and identified using gas chromatography-mass spectrometry (GC-MS) as 3-n-butyl-4,5-dihydrophthalide (sedanenolide). Potent opaque zones of inhibition observed on the HPTLC-bio-autography plate seeded with C. violaceum confirmed that sedanenolide was probably largely responsible for the AQS activity of celery. The bacteriocidal properties of many herbs and spices are reported. This study, however, was focussed on AQS activity, and may serve as initial scientific validation for the anti-infective properties ascribed to several culinary herbs and spices.

HPTLC Autography Based Screening and Isolation of Mushroom Tyrosinase Inhibitors of European Plant Species.

In the course of this project, 133 plants were evaluated on their ability to inhibit tyrosinase, a key enzyme in melanogenesis. The screening was performed by means of a HPTLC autographic assay, resulting in the selection of three plants, Asplenium trichomanes, Pinus uncinata, and Scutellaria altissima, with promising tyrosinase inhibiting activities. With the aid of the HPTLC assay, it was not only possible to select the most interesting plant extracts, but also to monitor the activity-guided fractionation which, in a relatively short time period, led to the isolation of active principles. Benzoic acid, roseoside, and dihydrovomifoliol-O-ß-d-glucopyranoside could be identified as tyrosinase inhibitors present in P. uncinata. Globularin turned out to be the active principle of S. altissima, and 4-ethenylphenyl 6-O-(6-deoxy-α-l-mannopyranosyl)-ß-d-glucopyranoside was detected as tyrosinase inhibitor of A. trichomanes. The pure compounds were tested also in a 96 well-plate assay in order to determine their IC50 values. The lowest IC50 value (42 µm) could be obtained for globularin, whereas the other compounds, e. g., benzoic acid exhibited a rather high IC50 value (IC50 =552 µm). This stood in clear contrast to the autographic assay, but is has to be taken into account that the outcome of the autography assay is not only depending on the IC50 value of a compound, but also on the content of the respective constituent in the extract.

Rapid autographic method for detection of enzymatic browning inhibitors based on enzyme immobilization.

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing l-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 µg of 4-hexylresorcinol, 0.025 µg of ascorbic acid, 0.5 µg of cysteine and 1 µg of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors.

In vitro inhibition potential of mono-n-octyl phthalate on Mycobacterium tuberculosis H37Ra: Possibility of binding to mycobacterial PknB-An in silico approach.

Fatty acids of specific chain lengths have been shown to inhibit the growth of Mycobacterium tuberculosis. In the present study, specific synthetic aromatic derivatives of n-octyl esters were investigated for their property to inhibit the growth of M. tuberculosis H37Ra. Agar well diffusion assay indicated that the crude synthetic derivatives obtained by the esterification of phthalic acid (PA) and n-octanol exhibited antimycobacterial activity. Further, the activity was authenticated with the Miroplate Alamar Blue Assay (MABA). Subsequently, the active component was purified by bioactivity guided chromatographic fractionation. The structure of the synthetic derivative was deduced by UV-Vis, FT-IR, LC-MS, GC-mass spectrometry, and NMR spectroscopy. Molecular docking and molecular dynamic simulation (MDS) were performed with Autodock 4.0 and GROMACS 5.1.2 softwares, respectively. It was found that mono-n-octyl phthalate (MOP) exhibited antimycobacterial activity with a MIC of 20 µg/mL, and not by any other related compounds, including di-n-octyl phthalate, PA, phthalic anhydride, and n-octanol. Binding of MOP with protein kinase B can participate in the binding cavity region, which was previously reported. Subsequently, we authenticate the stability with MDS. This is first report on the inhibition of M. tuberculosis growth by MOP.

Tracking leading anti-Candida compounds in plant samples; Plumbago europaea.

BACKGROUND AND OBJECTIVES: Due to the importance of finding new and more effective antifungal and antibacterial compounds against invasive vaginitis strains, this study was conducted for fast screening of plant samples. MATERIALS AND METHODS: Thirty Iranian plant samples were successively extracted by n-hexane, ethyl acetate and methanol to obtain a total of 90 extracts. Each extract was prepared in six concentrations and evaluated for antifungal activity via a micro-broth dilution method. Further phytochemical study of the aerial parts of Plumbago europaea, as the most promising source of anti-Candida compounds (with minimum inhibitory concentration of about 7µg/ml), was carried out and antifungal activity in the ethyl acetate extract was tracked using a combination of HPLC time-based fractionation and Thin Layer Chromatography-Bioautography via a bioassay-guided fractionation procedure. The compounds in the active region of the chromatogram were purified by a combination of column chromatography and preparative TLC, and then structure elucidation was achieved by 1D and 2D NMR, mass spectrometry and UV spectra. RESULTS: Seven compounds were isolated and identified: (1) plumbagin, (2) isoplumbagin, (3) 5, 8-dihydroxy-2-methyl-[1, 4] naphthoquinone, (4) droserone, (5) 7-methyljuglone, (6) Isozeylanone, and (7) methylene-3, 3'-diplumbagin. Antimicrobial activity of the purified compounds were also evaluated against C. albicans (MIC values ranging from 2 to 2500 µM) and Gardnerella vaginalis (MIC values ranging from 20 to 2500 µM). CONCLUSION: These naphthoquinone compounds could be surveyed for finding new and more effective anti-vaginitis agents via drug design approaches.

HPTLC-profiling of eleutherosides, mechanism of antioxidative action of eleutheroside E1, the PAMPA test with LC/MS detection and the structure-activity relationship.

Human body is constantly generating free radicals, which causes oxidative stress. Despite naturally occurring antioxidant systems in human body, free radicals cause lipid, proteins and DNA oxidation. New antioxidants are still urgent as well as their mechanisms of action should be explained. In this study, we investigated the mechanism by which eleutherosides B, E and E1 may act as antioxidants, identified eleutherosides in Eleutherococcus lasiogyne and Eleutherococcus giraldii, and explained in vitro the absorption of eleutheroside E1 based on passive transport. The DPPH∗ and DB-HPTLC tests were used to assess the antioxidant activity. Of the three eleutherosides, only eleutheroside E1 exhibited a strong anti-DPPH∗ activity (EC50 37.03 µg/mL; 63 mMol) compared to the raw extracts (EC50 170 and 180 µg/mL for E. lasiogyne and E. giraldii). This activity was also confirmed by the DB-HPTLC autography technique. According to Zaluski's hypothesis, the antioxidant mechanism of eleutheroside E1 is based on the complexation of DPPH∗ molecule with its aryl radical. During this reaction, the aryl radical of eleutheroside E1 (E1∗) and DPPHH are created. Next, the aryl radical (E1∗) is complexed with another DPPH∗ molecule. Additionally, the aryl radical can be stabilized by the presence of the methoxy groups in the aromatic ring, which increases its antioxidative action. The HPTLC-identification of extracts showed the presence of eleutherosides B, E and E1 in both species. The PAMPA test coupled with LC/MS detection showed a low permeability of eleutheroside E1 across artificial membrane. Because eleutherosides belong to the polyphenols, the TPC and TFC were quantified. The TPC and TFC varied from 51.4 to 49.3 mg/g dry extract for TPC, and from 5.73 to 4.91 mg/g dry extract for TFC, for E. giraldii and E. lasiogyne, respectively. In conclusion, eleutheroside E1 in its pure form could be a chemopreventive ingredient of new pharmacological or dietary products, stimulating the GALT. These findings can explain partially the adaptogenic activity of eleutheroside E1 on the GALT, which has been still unknown.

Anti-mycobacteria potential and synergistic effects of combined crude extracts of selected medicinal plants used by Bapedi traditional healers to treat tuberculosis related symptoms in Limpopo Province, South Africa.

BACKGROUND: Tuberculosis is an infectious communicable disease and the causative agent of the disease has over the years developed resistance to streamline chemotherapeutic agents with dire consequences and there is a need for development of new and more potent alternatives. METHODS: Constituents of leaves material of Combretum heroroense, Citrus lemon and Apodytes dimidiata were serially extracted using solvents of varying polarity. TLC finger print profile of the different extracts were determined by spraying eluted plates with vanillin sulphuric acid and 2, 2- diphenylpicryl hydrazyl (DPPH) for the presence of antioxidant constituents. Presence of different phytochemicals was determined using standard chemical test. Bioautography was used to determine the number of compounds present in sub-fractions active against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values extract and sub-fractions were determined using serial microplate dilution method against M. smegmatis (ATCC 1441), M. tuberculosis (ATCC H37Rv) and multi-drug resistant TB (MDR-TB) field strain. Synergy of the crude extracts of the three plants was determined using microplate dilution method against M. smegmatis. RESULTS: Mass extracted by different solvents was less than 6% dry weight for all the plants. Phlobatannins were not detected in A. dimidiata, C. heroroense and C. lemon as well as cardiac glycosides in C. lemon and A. dimidiata, and saponins in C. heroroense. Sub-fractions of the different plants were shown to contain constituents with antioxidant activity with the highest number detected in C. heroroense. Bioautography results reveal the presence of a compound(s) in the ethyle acetate sub-fraction of C. heroroense and butanol, methanol/water, ethyl acetate and water no.2 subfractions of A. dimidiata, active against M. smegmatis that were not shown to have antioxidant capacity. MIC results for different crude extracts of the three plants against M. smegmatis ranges from 0.1 to 3 mg/ml. The average MIC for the synergistic effect of the plants ranged from 0.04 mg/ml to 1.25 mg/ml. An activity greater than that obtained for the reference drugs was shown for the butanol and hexane fractions of A. dimidiata (0.47 mg/ml) against the field strain of MDR-TB while that obtained for the M.TB (ATCC H37Rv) was 0.31 mg/ml. CONCLUSION: A significant finding shown in this study reveals the potent anti-mycobacteria potential of sub-fractions of A. dimidiata against MDR-TB field strain that can lead to the isolation of compounds that can be used to counter resistant strains of tuberculosis.

Bioactivity of Diterpens from the Ethyl Acetate Extract of Kingiodendron pinnatum Rox. Hams.

BACKGROUND: Kingiodendron pinnatum Rox. Hams. is an endangered medicinal plant used in gonorrhoe, catarrhal conditions of genito-urinary and respiratory tracts. The scientific and pharmacological formulation of K. pinnatum has not been established so far though it is being traditionally used by tribes of the region. OBJECTIVE: P hytochemical screening and identification of the bioactive compounds from the ethyl acetate extract of Kingiodendron pinnatum Rox. Hams. MATERIALS AND METHODS: Chromatographic separation was carried out by thin layer chromatography and column chromatography. Bio-autography of the column fractioned extract and TLC chromatogram were evaluated in vitro for antibacterial activity. The PTLC, HP TLC were used for crude extract and HPLC, LCMS, FTIR, 1HNMR and 13CNMR were employed for the isolated compound in the ethyl acetate extract of K. pinnatum. RESULTS: Evaluation of solvent system for chromatographic separation revealed that ethyl acetate: petroleum ether in the ratio of 7:2.5 ml was the most appropriate one for the separation of diterpene compounds. The antibacterial bio-autography screening of TLC separated compound showed positive activity with Staphylococcus aureus and negative activity with Escherichia coli. Spectroscopic analysis of the isolated compound from the ethyl acetate extract of K. pinnatum revealed the presence of diterpene compound. CONCLUSION: It is evident from the present study that the ethyl acetate extract of K. pinnatum is rich in diterpene compounds and having potential antibacterial activity. SUMMARY: Novel extraction method for phytochemicls from Kingidendron pinnatum at RTAntibacterial property of diterpens extracted from Kingiodendron pinnatum Rox. Hams aganist S. aureus Abbreviations Used: TLC: Thin Layer Chromatography, PTLC: Preparatory Thin Layer Chromatography, HPTLC: High perormence Thin Layer chromatography, HPLC: High Performance Liquid Chromatography, LC-MS: Liquid chromatography Mass Spectra, FTIR: Fourier Transform Infrared Chromatography, NMR: Nuclear Magnetic Resonance.

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate.

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP(+)/NBT/PMS (ß-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 µg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.

Anti-bacterial and anti-oxidant activities of leaf extracts of Combretum vendae (Combretecacea) and the isolation of an anti-bacterial compound.

BACKGROUND: Combretum vendae A.E. van Wyk (Combretaceae) is used for the treatment of bacterial related infections and oxidative related diseases by indigenous people of South Africa. Dried leaves extracts of C. vendae were investigated for bioactivity against a variety of bacterial strains and their antioxidant potential evaluated. MATERIALS AND METHODS: Constituents of leaf material were serially extracted using solvents of varying polarities, TLC chromatograms of the fractions were sprayed with 2,2 diphenyl-1-picrylhydrazyl (DPPH) to determine the presence of antioxidant compounds. Bio-autography was used to determine the number of antibacterial compounds active against Staphylococcus aureus, Enterococcus faecalis, Eschericha coli and Pseudomonas aeruginosa. Minimum inhibitory concentration (MIC) values were determined using serial microplate dilution method. The chloroform fraction was subjected to bio-assay guided column chromatography to isolate the active compound. RESULTS: The mass extracted by different solvents was below 10% dry weight. MIC values for different extracts against different pathogens ranges from 0.08 to 0.64 mg/ml. The compound isolated was identified as acacetin having an Rf value of 0.28 following elution in the Ethanol: Methanol: Water [E: M: W (10: 1.35: 1 v/v). Acacetin had MIC values ranging from 0.16 to 0.35 mg/ml. CONCLUSION: We report for the first time the isolation of acacetin as the main antibacterial compound from the leaves of Combretum vendae.

Jean-Bertrand Pontalis: um poeta da psicanálise / Jean-Bertrand Pontalis: a poet of psychoanalysis / Jean-Bertrand Pontalis: un poeta del psicoanálisis

O artigo propõe uma aproximação à obra de Jean-Bertrand Pontalis. Com uma singular trajetória na psicanálise e na cultura francesa a partir da década de 50, Pontalis desenvolve uma forma própria de transmitir suas ideias e vivências. Percorrendo os caminhos dos sonhos, da linguagem e da clínica, podemos ter uma ideia aproximada do que seja a obra desse original psicanalista.

This paper attempts to approach the work of Jean-Bertrand Pontalis. Having a unique path in French psychoanalysis and culture from the 1950s onwards, Pontalis developed a way of his own to convey his ideas and experiences. Following the paths of dreams, language and the clinic, it is possible to develop an idea of what this creative psychoanalyst's work consists of.

El artículo propone una aproximación a la obra de Jean-Bertrand Pontalis. Con una trayectoria singular en el psicoanálisis y en la cultura francesa a partir de la década del 50, Pontalis desarrolla una manera propia de expresar sus ideas y experiencias. Recorriendo los caminos de los sueños, del lenguaje y de la clínica, podemos tener una idea aproximada de la obra de este original psicoanalista.