Proposal of an HPLC/UV/FLD Screening Method for the Simultaneous Determination of Ten Antibiotics in Environmental Waters.

An HPLC-UV/FLD method for simultaneous detection of ten antibiotics in surface waters was developed. Antibiotics were extracted from water using solid phase extraction. An Atlantis T3 column was used with acetonitrile and 0.05% trifluoroacetic acid as a mobile phase for separation, with a total running time of 45 min. Signal detection was performed at 280 nm; fluoroquinolones were additionally quantified by fluorescence detection. Validation parameters such as linearity, recovery and precision were evaluated. The limits of detection (LOD) in river waters were in the range 0.1-1.3 µg/L for antibiotics detected by UV, and 0.039 and 0.073 µg/Lfor fluoroquinolones detected by FLD. LOD are sufficiently low to consider this method as a first alternative for HPLC-MS methods that will allow alerting for the presence of antibiotics in surface waters. This screening method is rapid, sensitive, reproducible and economical.

Anti-inflammatory and nephroprotective activity of Juglans mollis against renal ischemia-reperfusion damage in a Wistar rat model.

BACKGROUND: Oxidative stress and the inflammatory process are involved in ischemia-reperfusion (I/R) injury. Juglans mollis has been reported as having antioxidant activity, which could attenuate the damage caused by I/R. We evaluated whether a methanolic extract of Juglans mollis (JM) exhibits nephroprotective activity in a Wistar rat model of I/R injury. METHODS: Four groups of six rats were used: Sham, I/R, JM, and JM + I/R. Two groups were dosed with JM (300 mg/kg) for 7 days before I/R. I/R injury was induced by clamping the renal hilums for 45 min and then reperfusing the kidneys for 15 h. Blood samples were taken to evaluate the levels of alanine aminotransferase (ALT), blood urea nitrogen, creatinine, superoxide dismutase (SOD), malondialdehyde (MDA), interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α). RESULTS: The levels of creatinine, ALT, MDA, IL-1ß, IL-6, and TNF-α were lower in JM + I/R than in I/R rats, whereas SOD level only was higher in JM + I/R than in Sham rats. No biochemical or histological damage was observed in JM rats compared with Sham rats; however, less histological damage was observed in JM + I/R rats compared with I/R rats. CONCLUSIONS: To our knowledge, this is the first report of nephroprotective activity of J. mollis against damage induced by I/R. This activity may be related to decreased levels of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) and modulation of oxidative stress markers (SOD and MDA) observed in the present study.

Modeling Antibacterial Activity with Machine Learning and Fusion of Chemical Structure Information with Microorganism Metabolic Networks.

Predicting the activity of new chemical compounds over pathogenic microorganisms with different metabolic reaction networks (MRN s) is an important goal due to the different susceptibility to antibiotics. The ChEMBL database contains >160 000 outcomes of preclinical assays of antimicrobial activity for 55 931 compounds with >365 parameters of activity (MIC, IC50, etc.) and >90 bacteria strains of >25 bacterial species. In addition, the Leong and Barabàsi data set includes >40 MRNs of microorganisms. However, there are no models able to predict antibacterial activity for multiple assays considering both drug and MRN structures at the same time. In this work, we combined perturbation theory, machine learning, and information fusion techniques to develop the first PTMLIF model. The best linear model found presented values of specificity = 90.31/90.40 and sensitivity = 88.14/88.07 in training/validation series. We carried out a comparison to nonlinear artificial neural network (ANN) techniques and previous models from the literature. Next, we illustrated the practical use of the model with an experimental case of study. We reported for the first time the isolation and characterization of terpenes from the plant Cissus incisa. The antibacterial activity of the terpenes was experimentally determined. The more active compounds were phytol and α-amyrin, with MIC = 100 µg/mL for Vancomycin-resistant Enterococcus faecium and Acinetobacter baumannii resistant to carbapenems. These compounds are already known from other sources. However, they have been isolated and evaluated for the first time here against several strains of multidrug-resistant bacteria including World Health Organization (WHO) priority pathogens. Last, we used the model to predict the activity of these compounds versus other microorganisms with different MRNs in order to find other potential targets.

Screening of north-east Mexico medicinal plants with activities against herpes simplex virus and human cancer cell line.

The plants examined in this study have previous biological activity reports indicating the possibility of found activity against herpes and cancer cell. The aim of this contribution was to carry out a screening of Juglans mollis (Juglandaceae), Persea americana (Lauraceae), Hamelia patens (Rubiaceae), Salvia texana (Lamiaceae), Salvia ballotaeflora (Lamiaceae), Ceanothus coeruleus (Rhamnaceae), Chrysactinia mexicana (Asteraceae) y Clematis drummondii (Ranunculaceae), against HeLa cells, VHS-1 and VHS-2. The method MTT was used to determine the 50% cytotoxic concentration (CC50), in Vero and HeLa cell lines. To determine the 50% inhibitory concentration (IC50) against herpes, the plaque reduction method was used. Results showed that none of the plants exhibited activity against HeLa cells. About antiherpetic activity, J. mollis and S. ballotaeflora extracts present antiherpetic activity in terms of their SI, increasingly interest for further studies on the isolation of compounds with antiherpetic activity and about the mechanisms of action that produce this activity.

Biological Activities of Extracts from Aerial Parts of Salvia pachyphylla Epling Ex Munz.

The antioxidant, antimicrobial, antiproliferative, and enzyme inhibitory properties of five extracts from aerial parts of Salvia pachyphylla Epling ex Munz were examined to assess the prospective of this plant as a source of natural products with therapeutic potential. These properties were analyzed by performing a set of standard assays. The extract obtained with dichloromethane showed the most variety of components, as they yielded promising results in all completed assays. Furthermore, the extract obtained with ethyl acetate exhibited the greatest antioxidant activity, as well as the best xanthine oxidase inhibitory activity. Remarkably, both extracts obtained with n-hexane or dichloromethane revealed significant antimicrobial activity against the Gram-positive bacteria; additionally, they showed greater antiproliferative activity against three representative cell lines of the most common types of cancers in women worldwide, and against a cell line that exemplifies cancers that typically develop drug resistance. Despite that, other extracts were less active, such as the methanolic or aqueous; their results are promising for the isolation and identification of novel bioactive molecules.

Optimization and Validation of a Microscale In vitro Method to Assess α-Glucosidase Inhibition Activity.

BACKGROUND: Microscale in vitro assays are fast, simple, and inexpensive, with reduced reagent quantities, waste, and experimental animal use. However, they have low reproducibility and low correlation with the results of in vivo models, possibly due to differences in precision and accuracy in methodologies between laboratories. OBJECTIVE: The objective was the optimization and validation of an in vitro assay, carried out on microscale, to assess the inhibition of α-glucosidase activity, which is indicative of antihyperglycemic activity. METHODS: The optimization was carried out using a fractional factorial design taking into account the best inhibition percentage and the absorbance of the controls. With the optimized experimental conditions in hand, we carried out method validation. RESULTS: The optimized conditions were as follows: enzyme concentration, 0.55 U/mL; substrate concentration, 111.5 µM; and 17.5 min incubation at 37°C. A linear range between 100 and 310.2 µg/mL of acarbose (r2 0.994) was established. The RSD was <2% and the % error was <3%. The Z factor was >0.96. This method was applied to four plant extracts, one of which was found to be very active. CONCLUSION: The method was found to be accurate, precise, selective, linear, and reliable in evaluating the antihyperglycemic activity of natural extracts in vitro.

Optimization and Validation of Two High-Throughput Methods Indicating Antiradical Activity.

BACKGROUND: The search for new natural or synthetic products with antioxidant activity is commonly based on methods that involve reduction of either 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 2-2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). However, the reported values of the effective concentrations are highly variable, even in controls. Herein, we optimize and validate both meth-ods of determining antiradical activity. METHODS: Optimization was carried out using both a fractionated factorial design and a basic sequential simplex method, by monitoring the reduction percentage. Quercetin or Trolox were used as positive con-trol. Furthermore, for each method, linearity, precision, accuracy, robustness, plate uniformity, signal variability, and Z factor, were established. RESULTS: The optimized conditions for the DPPH method were: DPPH 280 µM in ethanol and 15 min of reaction time in the dark. The linear range was between 7 and 140 µM with an R2 value of 0.9987. The optimized conditions for the ABTS method were: ABTS adjusted to 0.7 absorbance units, 70% concen-tration in ethanol, and a reaction time of 6 min in the dark. The linear range was found to be between 1 and 70% with an R2 = 0.9991. For both methods, the accuracy and precision were within limits and the Z factor value was higher than 0.89. The applicability of each method was assessed by analyzing eight plant extracts. CONCLUSION: The DPPH and ABTS reduction methods were optimized and validated on a microscale and could be expected to be implemented in any laboratory.

Diterpenes Synthesized from the Natural Serrulatane Leubethanol and Their in Vitro Activities against Mycobacterium tuberculosis.

Seventeen new derivatives of the natural diterpene leubethanol, including some potential pro-drugs, with changes in the functionality of the aliphatic chain or modifications of aromatic ring and the phenolic group, were synthesized and tested in vitro by the MABA technique for their activity against the H37Rv strain of Mycobacterium tuberculosis. Some compounds showed antimycobacterial selectivity indices higher than leubethanol.

HPLC method for the simultaneous analysis of fluoroquinolones and oxazolidinones in plasma.

A chromatographic method was implemented and validated for the simultaneous determination of antimicrobials proposed for the treatment of mycetoma: three fluoroquinolones: ciprofloxacin, moxifloxacin and sparfloxacin; two oxazolidinones: DA-7157 (DA2; torezolid) and its prodrug DA-7218 (DA1). Separation of analytes was achieved on an Atlantis dC18 column (150 × 4.6 mm i.d., 5 µm particle size) with a mobile phase composed of acetonitrile and trifluoroacetic acid 0.1% (v/v) using a gradient program. Total running time was 30 min. Quantification of sparfloxacin was carried out using a DAD at 278 nm; the oxazolidinones DA1 and DA2 and the quinolones ciprofloxacin and moxifloxacin were analyzed by fluorescence with an excitation wavelength of 292 nm and an emission wavelength of 525 nm. Intraday precision was in the range of 3.2 and 14.1%. Linearity range was from 0.3 to 10 µg/mL for sparfloxacin using DAD detector, and from 0.2 to 10 µg/mL for ciprofloxacin, 0.3 to 10 µg/mL for DA2, 0.4 to 10 µg/mL for DA1 and 0.04 to 10 µg/mL for moxifloxacin with fluorescence detector. Acetonitrile was used to precipitate proteins from plasma. Recoveries at low, medium and high concentration were between 80 and 120%. Limits of quantification were between 0.04 and 0.4 µg/mL in plasma. The method can be applied for individual or simultaneous determination of the antimicrobials in plasma.

Larvicidal and cytotoxic activities of extracts from 11 native plants from northeastern Mexico.

Of all mosquito-borne viral diseases, dengue is spreading most rapidly worldwide. Conventional chemical insecticides (e.g., organophosphates and carbamates) effectively kill mosquitoes at their larval stage, but are toxic to humans. Natural product-based insecticides may be highly specific. Herein, we report the insecticidal activities of 11 native Mexican plants against Aedes aegypti (L). Ether extracts of Ambrosia confertiflora De Candolle, Thymus vulgaris (L.), and Zanthoxylum fagara (L.), and both ether and methanol extracts of Ruta chalepensis L. were significantly larvicidal toward the dengue mosquito after 24 h of exposure. Of them, only the ether extract of A. confertiflora was toxic to Vero cells. In conclusion, the ether extracts of Thymus vulgaris, Z. fagara, and both ether and methanol extracts of Ruta chalepensis L., could be considered as potential bioinsecticides.

Chemical compositions and antimicrobial and antioxidant activities of the essential oils from Magnolia grandiflora, Chrysactinia mexicana, and Schinus molle found in northeast Mexico.

The essential oils from Magnolia grandiflora and Chrysactinia mexicana leaves, and from Schinus molle leaves and fruit, were characterized by gas chromatography/flame-ionization detection and gas chromatography/mass spectrometry. Twenty-eight compounds from M. grandiflora leaves were identified (representing 93.6% of the total area of the gas chromatogram), with the major component being bornyl acetate (20.9%). Colorless and yellow oils were obtained from the C. mexicana leaves with 18 (86.7%) and 11 (100%) compounds identified, respectively. In both fractions, the principal component was sylvestrene (36.8% and 41.1%, respectively). The essential oils ofS. molle leaves and fruit were each separated into colorless and yellow fractions, in which 14 (98.2) and 20 (99.8%) compounds were identified. The main component was alpha-phellandrene in all fractions (between 32.8% and 45.0%). The M. grandiflora oil displayed antifungal activity against five dermatophyte strains. The oils from S. molle and M. grandiflora leaves had antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes, which cause skin infections that potentially may lead to sepsis. However, the antioxidant activities of all oils were small (half maximal effective concentration values >250 microg/mL).

A new antimycobacterial furanolignan from Leucophyllum frutescens.

Bioassay-guided fractionation of the minor active fractions obtained from the root bark of Leucophyllumfrutescens (Berl.) I. M. Johnst. led to isolation from the n-hexane extract of a new compound with moderate activity against the H37Rv Mycobacterium tuberculosis strain (MIC 63 microg/mL), and low cytotoxicity, as shown by the IC5o against Vero cells. The compound was identified by 1D/2D NMR spectroscopy a s2',5"-dimethoxysesamin.

Multi-wavelength high-performance liquid chromatographic fingerprints and chemometrics to predict the antioxidant activity of Turnera diffusa as part of its quality control.

The determination of the antioxidant activity of Turnera diffusa using partial least squares regression (PLSR) on chromatographic data is presented. The chromatograms were recorded with a diode array detector and, for each sample, an enhanced fingerprint was constructed by compiling into a single data vector the chromatograms at four wavelengths (216, 238, 254 and 345 nm). The wavelengths were selected from a contour plot, in order to obtain the greater number of peaks at each of the wavelengths. A further pretreatment of the data that included baseline correction, scaling and correlation optimized warping was performed. Optimal values of the parameters used in the warping were found by means of simplex optimization. A PLSR model with four latent variables (LV) explained 52.5% of X variance and 98.4% of Y, with a root mean square error for cross validation of 6.02. To evaluate its reliability, it was applied to an external prediction set, retrieving a relative standard error for prediction of 7.8%. The study of the most important variables for the regression indicated the chromatographic peaks related to antioxidant activity at the used wavelengths.

Early administration of peroxisomicine A1 (T-514 extracted from K. parvifolia seeds) causes necrosis of implanted TC-1 cells without affecting target organs in a murine model / La administración temprana de peroxisomicina A1 (T-514 extraída de las semillas de K. parvifolia)causa necrosis de células TC-1 implantadas sin afectar órganos blanco en un modelo murino

Peroxisomicine A1 (PA1), one of the toxins isolated from seeds of plants of the Karwinskia genus, whose targets organs are the liver, kidney, and lungs. There is a selective toxicity in vitro to cancer-cell lines derived from the lungs, liver, and colon, compared to normal cell lines. PA1 caused apoptosis in several cancer-cell lines in culture. In toxic doses to rodents, it causes extensive apoptosis in the liver, kidney, and lungs. In our study we were interested in evaluating, for the first time, the morphological effects of administration of PA1 to implanted TC-1 cells and in the target organs in vivo. The TC-1 cells were cultured and injected into the hind limb of C57BL-6 mice. The animals were divided into 3 groups; those treated with four doses of 1 mg/kg each of PA1, the untreated control, and the vehicle-control groups. All mice were killed 10 days after cell implantation. Samples were obtained from TC-1 cells at the implantation site and from the liver, kidney, and lungs. The samples were processed for examination under light and electron microscopy. In the PA1-treated group, the TC-1 cells had necrosis, whereas in the control groups the tumor cells were undamaged. The target organs did not show any lesions. We demonstrated for the first time that there is a selective toxic effect of PA1 on the TC-1 cells in vivo.

Peroxisomicina A1 (PA1), una de las toxinas aisladas de las semillas de plantas del género Karwinskia, cuyos órganos blanco son hígado, riñón y pulmón. Hay una toxicidad selectiva in vitro contra líneas celulares cancerosas derivadas de pulmón, hígado y colon, comparadas con líneas celulares normales. PA1 causa apoptosis en varias líneas celulares malignas en cultivo. En dosis tóxicas a roedores, causa extensa apoptosis en hígado, riñón y pulmón. En nuestro estudio, estuvimos interesados en evaluar por primera vez los efectos morfológicos de la administración temprana de PA1 sobre células TC-1 implantadas y los órganos blanco in vivo. Las células TC-1 fueron cultivadas e implantadas en la extremidad posterior de ratones C57BL-6. Los animales fueron divididos en tres grupos: tratado con cuatro dosis de 1 mg/kg de peso de PA1, control sin tratamiento y control vehículo. Todos los animales fueron sacrificados 10 días posterior al implante de las células. Se colectaron muestras del sitio del implante de las células TC-1 y de hígado, riñón y pulmón. Las muestras fueron procesadas para su análisis a microscopía de luz y microscopia electrónica de transmisión. En el grupo tratado con PA1, las células TC-1 presentaron necrosis, mientras que en los grupos control las células tumorales se observaron sin daño. Los órganos blanco no mostraron lesión alguna. Demostramos por primera vez que existe un efecto tóxico selectivo de PA1 sobre las células TC-1 in vivo.

Activity against Streptococcus pneumoniae of the essential oil and 5-(3-buten-1-ynyl)-2, 2'-bithienyl isolated from Chrysactinia mexicana roots.

The essential oil of Chrysactinia mexicana retrieved from the root bark was characterized by gas chromatography coupled to a mass detector. The compounds silphiperfol-5-ene, 7-epi- silphiperfol-5-ene, modheph-2-ene, alpha-isocomene, beta-isocomene and methyl-linoleate were identified. The principal compound (76.42%) could not be identified by the library and was further isolated through a reverse phase C-18 chromatography followed by silica gel chromatography and identified as 5-(3-buten-1-ynyl)-2,2'-bithienyl. Both the oil and the isolated compound were tested for their antimicrobial activity against two strains of Streptococcus pneumoniae resistant to beta-lactam antibiotics. MICs were 250 microg/mL and 125 microg/mL respectively. This is the first report about extraction of oil and compound 5-(3-buten-1-ynyl)-2, 2'-bithienyl from roots of Chrysactinia mexicana as well as the determination of antimicrobial activity against S. pneumoniae.

Protective effect of four Mexican plants against CCl4-induced damage on the Huh7 human hepatoma cell line.

BACKGROUND: Centaurea americana, Krameria ramosissima, Juglans mollis and Turnera diffusa are used by traditional healers in the northeastern region of Mexico to protect against liver damage. However, the hepatoprotective properties of these plants have not been investigated scientifically. This study reports on the protective effects of these plants using an in vitro assay. MATERIAL AND METHODS: Extracts of plants were tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The effects of extracts from these plants on a human hepatoma cell line (Huh7) were evaluated according to cell viability and aspartate aminotransferase and malondialdehyde levels before and after exposure of the cells to carbon tetrachloride (CCl(4)). RESULTS: All extracts reduced DPPH levels by more than 50%. C. americana flower and stem/leaf extracts, the aerial part of T. diffusa, and the nut, leaf and bark of J. mollis extracts were used to assess hepatoprotective activity. The extract of the aerial part of K. ramosissima was toxic. Pretreatment of Huh7 cells with extracts from the flower of C. americana (FCA), the stem/leaf fraction of C. americana (S/LCA), the leaf of J. mollis (LJM) and the bark of J. mollis (BJM) prior to the CCl(4) challenge, protected against CCl(4)-induced liver damage, as evidenced by a significant decrease in the activity of the medium enzyme. The FCA, S/LCA, LJM and BJM extracts showed significant antilipid peroxidant effects in vitro. In conclusion, the hepatoprotective effects of the FCA, S/LCA, LJM and BJM extracts observed in this study may result from their antioxidative properties.

Antimicrobial and antioxidant activities of plants from northeast of Mexico.

Traditional medicine has a key role in health care worldwide. Obtaining scientific information about the efficacy and safety of the plants from our region is one of the goals of our research group. In this report, 17 plants were selected and collected in different localities from northeast Mexico. The dried plants were separated into leaves, flowers, fruit, stems, roots and bark. Each part was extracted with methanol, and 39 crude extracts were prepared. The extracts were tested for their antimicrobial activity using three Gram-negative bacterial strains (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii), three Gram-positive bacterial strains (Enterococcus faecalis and two Staphylococcus aureus strains), and seven clinically isolated yeasts (Candida albicans, C. krusei, C. tropicalis, C. parapsilosis and C. glabrata); their antioxidant activity was tested using a DPPH free radical assay. No activity against Gram-negative bacteria was observed with any extract up to the maximum concentration tested, 1000 µg ml(-1). We report here for the first time activity of Ceanothus coeruleus against S. aureus (flowers, minimal inhibitory concentration (MIC) 125 µg ml(-1)), C. glabrata (MICs 31.25 µg ml(-1)) and C. parapsilosis (MICs between 31.25 and 125 µg ml(-1)); Chrysanctinia mexicana against C. glabrata (MICs 31.25 µg ml(-1)); Colubrina greggii against E. faecalis (MICs 250 µg ml(-1)) and Cordia boissieri against C. glabrata (MIC 125 µg ml(-1)). Furthermore, this is the first report about antioxidant activity of extracts from Ceanothus coeruleus, Chrysanctinia mexicana, Colubrina greggii and Cyperus alternifolius. Some correlation could exist between antioxidant activity and antiyeast activity against yeasts in the species Ceanothus coeruleus, Schinus molle, Colubrina greggii and Cordia boissieri.

Development and validation of an HPLC-DAD analytical procedure for quality control of damiana (Turnera diffusa), using an antioxidant marker isolated from the plant.

Damiana is a shrub used as a dietary supplement and as an additive in several foods. This report presents the first validated analytical method for QC of products made from damiana. Through bioassay-guided fractionation, the compound showing the best antioxidant activity, determined using 2,2-diphenyl-1-picryl-hydrazyl assay in TLC, was purified and identified as 8-C-beta-[6-deoxy-2-O-(alpha-1-rhamnopyranosyl)-xylo- hexopyranos-3-uloside] (Cl50 5.56 g/mL). This is the only natural source from which this compound has been reported, and has been found in all native damiana samples analyzed. Therefore, it was used as a biomarker to develop an HPLC-diode-array detection analytical procedure using the first-order derivative of the chromatograms. Separation was achieved using an AccQ Tag C18 150 x 3.9 mm (4 microm) column, with a gradient of methanol and 0.1% trifluoroacetic acid in water. The method was shown to be accurate, sensitive, and reproducible, providing a useful quantitative means to apply QC to damiana products.

2,2'-Dimethoxy-4,4'-[rel-(2R,3S)-2,3-di-methylbutane-1,4-diyl]diphenol.

The title mol-ecule, C(20)H(26)O(4), commonly known as meso-dihydro-guaiaretic acid, is a naturally occurring lignan extracted from Larrea tridentata and other plants. The mol-ecule has a noncrystallographic inversion center situated at the midpoint of the central C-C bond, generating the meso stereoisomer. The central C-C-C-C alkyl chain displays an all-trans conformation, allowing an almost parallel arrangement of the benzene rings, which make a dihedral angle of 5.0 (3)°. Both hydr-oxy groups form weak O-H⋯O-H chains of hydrogen bonds along [100]. The resulting supra-molecular structure is an undulating plane parallel to (010).

In vivo therapeutic effect of gatifloxacin on BALB/c mice infected with Nocardia brasiliensis.

In the present work, we evaluated the effect of gatifloxacin on the evolution of experimental murine infection with Nocardia brasiliensis using linezolid as a control. Gatifloxacin was injected subcutaneously at 100 mg/kg body weight every 8 h for 4 weeks. This compound was equally as efficient as linezolid in reducing the production of lesions.