Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms

Context: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery. Objective: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied. Materials and methods: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro. Results: The purification procedure resulted in the isolation of kaempferol-3-O-(2 '',4 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (1), kaempferol-3-O-(2 '',6 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 mu g/mL and 100 mu M. Discussion and conclusion: This is the first time that the presence of compounds 1-2 in S. pohlii and 3-4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Abstract Background Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicao) leaves: Application to chemical variability evaluation

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goias, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart, specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens. (C) 2012 Elsevier B.V. All rights reserved.

A VALIDATED HPLC ANALYTICAL METHOD FOR THE ANALYSIS OF SOLASONINE AND SOLAMARGINE IN IN VITRO SKIN PENETRATION STUDIES

To assess topical delivery studies of glycoalkaloids, an analytical method by HPLC-UV was developed and validated for the determination of solasonine (SN) and solamargine (SM) in different skin layers, as well as in a topical formulation. The method was linear within the ranges 0.86 to 990.00 mu g/mL for SN and 1.74 to 1000.00 mu g/mL for SM (r = 0.9996). Moreover, the recoveries for both glycoalkaloids were higher than 88.94 and 93.23% from skin samples and topical formulation, respectively. The method developed is reliable and suitable for topical delivery skin studies and for determining the content of SN and SM in topical formulations.

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS. Ursolic acid is a natural molecule that presents several pharmacological properties. In this work, an analytical method by RP-HPLC has been developed and validated for quantification of this drug in the solid dispersions, using PEG 6000 and Poloxamer 407 as polymers. The method was specific, linear in the range of 1.0-50.0 mu g mL(-1) (r<0.99), precise (CV < 5% for both inter-and intra-assays), accurate (maximum deviation of +/- 13%), and robust to the parameters evaluated. This method has proved to be simple and useful for ursolic acid determination in solid dispersions, enabling its determination in pharmaceutical dosage form.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min(-1) into a Phenomenex Gemini (R) C18, 5 mu m analytical column (150 x 4.6 mm id.). The calibration curve was linear over the range from 0.2 to 200 ng mL(-1) for dextromethorphan and doxylamine and 0.05 to 10 ng mL(-1) for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study. (C) 2012 Elsevier B.V. All rights reserved.

Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD

To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feijao River, which is the source of drinking water for the city of Sao Carlos, Sao Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides - ametryn, atrazine, diuron and fipronil - used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L-1 and 0.07 to 0.17 mg L-1, respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed.

A Validated Reverse Phase HPLC Analytical Method for Quantitation of Glycoalkaloids in Solanum lycocarpum and Its Extracts

Solanum lycocarpum (Solanaceae) is native to the Brazilian Cerrado. Fruits of this species contain the glycoalkaloids solasonine (SN) and solamargine (SM), which display antiparasitic and anticancer properties. A method has been developed for the extraction and HPLC-UV analysis of the SN and SM in different parts of S. lycocarpum, mainly comprising ripe and unripe fruits, leaf, and stem. This analytical method was validated and gave good detection response with linearity over a dynamic range of 0.77-1000.00 mu g mL(-1) and recovery in the range of 80.92-91.71%, allowing a reliable quantitation of the target compounds. Unripe fruits displayed higher concentrations of glycoalkaloids (1.04% +/- 0.01 of SN and 0.69% +/- 0.00 of SM) than the ripe fruits (0.83% +/- 0.02 of SN and 0.60% +/- 0.01 of SM). Quantitation of glycoalkaloids in the alkaloidic extract gave 45.09% +/- 1.14 of SN and 44.37% +/- 0.60 of SM, respectively.

Assessment of the stereoselective fungal biotransformation of albendazole and its analysis by HPLC in polar organic mode

A high-performance liquid chromatographic method using polar organic mode was developed to analyze albendazole (ABZ), albendazole sulfone (ABZSO(2)) and the chiral and active metabolite albendazole sulfoxide (ABZSOX, ricobendazole) that was further applied in stereoselective fungal biotransformation studies. The chromatographic separation was performed on a Chiralpak AS column using acetonitrile:ethanol (97:3, v/v) plus 0.2% triethylamine and 0.2% acetic acid as the mobile phase at a flow rate of 0.5 mL min(-1). The present study employed hollow fiber liquid-phase microextraction as sample preparation. The method showed to be linear over the concentration range of 25-5000 ng mL(-1) for each ABZSOX enantiomer, 200-10,000 ng mL(-1) for ABZ and 50-1000 ng mL(-1) for ABZSO(2) metabolite with correlation coefficient (r)> 0.9934. The mean recoveries for ABZ, rac-ABZSOX and ABZSO(2) were, respectively, 9%, 33% and 20% with relative standard deviation below 10%. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. This study opens the door regarding the possibility of using fungi in obtaining of the active metabolite ricobendazole. Nigrospora sphaerica (Sacc.) E. W. Mason (5567), Pestalotiopsis foedans (VR8), Papulaspora immersa Hotson (SS13) and Mucor rouxii were able to stereoselectively metabolize ABZ into its chiral metabolite. Among them, the fungus Mucor rouxii was the most efficient in the production of (+)-ABZSOX. (C) 2011 Elsevier B.V. All rights reserved.

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as estoraque do campo or cuia de brejo, has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts. Copyright (c) 2011 John Wiley & Sons, Ltd.

EVAPORATIVE LIGHT-SCATTERING DETECTOR FOR ANALYSIS OF NATURAL PRODUCTS

EVAPORATIVE LIGHT-SCATTERING DETECTOR FOR ANALYSIS OF NATURAL PRODUCTS. The interest in the use of evaporative light scattering detector (ELSD) for the analysis of different classes of natural products has grown over the years. This is because this detector has become an excellent alternative compared to other types of detectors, such as the refractive index detector and the ultraviolet (UV) detector. This review describes the basic principles of ELSD functioning and discusses the advantages and disadvantages in using an ELSD for the analysis of organic compounds. Additionally, an overview, covering the last 23 years, of ELSD applications in natural products analysis (saponins, terpenes, carbohydrates, glycosides, alkaloids, steroids, flavonoids, peptides, polyketides, coumarins and iridoids) is presented and discussed.

CHEMICAL DEGRADATION OF ESFENVALERATE AND HPLC-UV-PAD DETECTION OF INTERMEDIATES AND BY-PRODUCTS

The impact of pyretroids, their by-products and degradation products on humans and the environment is recognized as a serious problem. Despite several studies regarding esfenvalerate toxicity and its detection in water and sediments, there is still a lack of information about its degradation intermediates and by-products in water. In this work, an HPLC method was developed to follow up the degradation of esfenvalerate and to detect the intermediates and by-products formed during the chemical degradation process. The chemical degradation was performed using an esfenvalerate suspension and different concentrations of hydrogen peroxide, temperatures, and pH. The reaction was monitored for 24 hr, and during the kinetic experiments, samples were collected at several reaction times and analyzed by HPLC-UV-PAD. In the degradation process, eleven different compounds (intermediate and by-products) were detected, among them the metabolites 3-phenoxybenzoic acid and 3-phenoxybenzaldehyde. HPLC-UV-PAD proved to be a valuable analytical technique for the rapid and reliable separation and determination of esfenvalerate, its degradation intermediates, and by-products.

DETERMINATION OF DICLOFENAC SODIUM IN RABBIT PLASMA BY HPLC/UV: EVALUATION AND CHARACTERIZATION OF ITS CRYSTALLINE FORMS, ANHYDROUS AND HYDRATE, ON THE ANTIPYRETIC EFFECT

Diclofenac sodium (DS) is a non-steroidal anti-inflammatory drug that is widely prescribed for the treatment of rheumatoid arthritis and post-surgery analgesia. The active pharmaceutical ingredient is the anhydrous form; however, it can also exist in hydrate form. In this context, knowing the properties of the solid state is important and relevant in the pharmaceutical area because they have a significant impact on the solubility, bioavailability, and chemical stability of the drugs. In the present study, data from XRPD, FTIR spectroscopy, and thermal analysis were used for the identification and characterization of DS forms (anhydrous and hydrate). An HPLC method was optimized to evaluate the plasma concentration of DS in rabbits. The optimized method exhibited good linearity over the range 0.1-60 mu g/mL with correlation coefficients of >0.9991. The mean recovery was 100%. Precision and accuracy were determined within acceptable limits. Finally, to compare the pharmacological properties of anhydrous and hydrate DS forms, we investigated their effects in the febrile response induced by lipopolysaccharide from E. coli in rabbits. The results show that the antipyretic effect of anhydrous and hydrate DS forms are similar.

AN EFFICIENT HPLC-UV METHOD FOR THE QUANTITATIVE DETERMINATION OF CEFADROXIL IN HUMAN PLASMA AND ITS APPLICATION IN PHARMACOKINETIC STUDIES

Cefadroxil is a semi-synthetic first-generation oral cephalosporin used in the treatment of mild to moderate infections of the respiratory and urinary tracts, skin and soft tissue infections. In this work a simple, rapid, economic and sensitive HPLC-UV method is described for the quantitative determination of cefadroxil in human plasma samples using lamivudine as internal standard. Sample pre-treatment was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed with a mobile phase consisting of a mixture of sodium dihydrogen phosphate monohydrate solution, methanol and acetonitrile in the ratio of 90:8:2 (v/v/v) at a flow rate of 1.0mL/min. The proposed method is linear between 0.4 to 40.0 mu g/mL and its average recovery is 102.21% for cefadroxil and 97.94% for lamivudine. The method is simple, sensitive, reproducible, less time consuming for determination of cefadroxil in human plasma. The method can therefore be recommended for pharmacokinetics studies, including bioavailability and bioequivalence studies.

A Validated Chromatographic Method for the Determination of Flavonoids in Copaifera langsdorffii by HPLC

Hydroethanolic extracts of C. langsdorffii leaves have therapeutic potential. This work reports a validated chromatographic method for the quantification of polar compounds in the hydroethanolic extract of C. langsdorffii leaves. A reliable HPLC method was developed using two monolithic columns linked in series (100 x 4.6 mm - C-18), with nonlinear gradient elution, and UV detection set at 257 nm. A procedure for the extraction of flavonols was also developed, which involved the use of 70% aqueous ethanol and the addition of benzophenone as the internal standard. The developed method led to a good detection response as the values for linearity were between 10.3 and 1000 mu g/mL, and those for recovery between 84.2 and 111.1%. The detection limit ranged from 0.02 to 1.70 mu g/mL and the quantitation limit from 0.07 to 5.1 mu g/mL, with a maximum RSD of 5.24%. Five compounds, rutin, quercetin-3-O-alpha-L-rhamnopyranoside, kaempferol-3-O-alpha-L-rhamnopyranoside, quercetin and kaempferol, were quantified. This method could, therefore, be used for the quality control of hydroethanolic extracts of Copaifera leaves and their cosmetic and pharmaceutical products.