Search results for: headspace gas chromatography

Authors: Samir Falhi, Neji Gharsallah, Adel Kadri

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Ruta chalpensis L. is a medicinal plant in the family of Rutaceae, has been used as an important traditional in the Mediterranean basin in the treatment of many diseases. The current study was devoted to investigate and evaluate the chemical composition, total phenolic, flavonoid and tannin contents, and in vitro antioxidant activities of ethyl acetate, ethanol and hydroalcoholic extracts and essential oil from the aerial parts of Ruta chalpensis from Tunisian Sahara. Total phenolic, flavonoid and tannin contents of extracts ranged from 40.39 ± 1.87 to 75.13 ± 1.22 mg of GAE/g, from 22.62 ± 1.55 to 27.51 ± 1.04 mg of QE/g, and from 5.56 ± 1.32 to 10.89 ± 1.10 mg of CE/g respectively. Results showed that the highest antioxidant activities was determined for ethanol extract with IC50 value of 26.23 ± 0.91 µg/mL for 2,2-diphenyl-1-picrylhydrazyl assay, and for hydroalcoholic extract with EC50 value of 412.95±6.57 µg/mL and 105.52±2.45 mg of α-tocopherol/g for ferric reducing antioxidant power and total antioxidant capacity assays, respectively. Furthermore, Gas Chromatography–Mass Spectrometry (GC-MS) analysis of essential oil led to identification of 20 compounds representing 98.96 % of the total composition. The major components of essential oil were 2-undecanone (39.13%), 2-nonanone (25.04), 1-nonene (13.81), and α-limonene (7.72). Spectral data of Fourier-transform infrared spectroscopy analysis (FT-IR) of extracts revealed the presence of functional groups such as C= O, C─O, ─OH, and C─H, which confirmed its richness on polyphenols and biological active functional groups. These results showed that Ruta chalpensis could be a potential natural source of antioxidants that can be used in food and nutraceutical applications.

Keywords: antioxidant, FT-IR analysis, GC-MS analysis, phytochemicals contents, Ruta chalpensis

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Authors: Soomal Hamza, Uzma Imran

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Organochlorine pesticides bio-accumulate into the fat of fish, birds, and animals through which it enters the human food cycle. Due to their persistence and stability in the environment, many health impacts are associated with them, most of which are carcinogenic in nature. In this study, the level of organochlorine pesticides has been detected in Keenjhar Lake and remediated using Rhizoremediation technique. 14 OC pesticides namely, Aldrin, Deldrin, Heptachlor, Heptachlor epoxide, Endrin, Endosulfun I and II, DDT, DDE, DDD, Alpha, Beta, Gamma BHC and two plants namely, Water Hyacinth and Slvinia Molesta were used in the system using pot experiment which processed for 11 days. A consortium was inoculated in both plants to increase its efficiency. Water samples were processed using liquide-liquid extraction. Sediments and roots samples were processed using Soxhlet method followed by clean-up and Gas Chromatography. Delta-BHC was the predominantly found in all samples with mean concentration (ppb) and standard deviation of 0.02 ± 0.14, 0.52 ± 0.68, 0.61 ± 0.06, in Water, Sediments and Roots samples respectively. The highest levels were of Endosulfan II in the samples of water, sediments and roots. Water Hyacinth proved to be better bioaccumulaor as compared to Silvinia Molesta. The pattern of compounds reduction rate by the end of experiment was Delta-BHC>DDD > Alpha-BHC > DDT> Heptachlor> H.Epoxide> Deldrin> Aldrin> Endrin> DDE> Endosulfun I > Endosulfun II. Not much significant difference was observed between the pots with the consortium and pots without the consortium addition. Phytoremediation is a promising technique, but more studies are required to assess the bioremediation potential of different aquatic plants and plant-endophyte relationship.

Keywords: aquatic plant, bio remediation, gas chromatography, liquid liquid extraction

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Authors: Nacéra Benoussaid, Lehalali Meriem, Benchabane Messaoud

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Our work focuses on the production of compound antibiotic effect of volatile nature namely hydrogen cyanide and the production and identification of molecules phénazinique by some strains of fluorescent Pseudomonas spp isolated from the rhizosphere of some trees for a possible use as bio pesticides antifungal effect and/or antibiotic. We tested the production of hydrogen cyanide of 21 strains of Pseudomonas spp. fluorescent among them 19 strains (90, 47%) showed a positive cyanogenesis.The antagonism test executed in vitro showed that Pseudomonas strains have a higher anti fungal effect relative to their antibacterial effect with diameters of inhibition zones up to 3, 9 cm recorded by the strain F48 against Coleosporiumsp compared with recorded results against bacteria with a maximum inhibition of 1, 26 cm among this antagonistic strain.Three strains were selected by testing for producing phénazines namely PI9, BB9 and F20. The effect of the antimicrobial activity was performed on different culture media (GN, King B, ISP2 and PDA). The results of our study allowed us to retain the King B medium as ideal medium for the production of secondary metabolite. The produced phenazinique compounds was extracted from various organic solvents, and after the results of antibiographie against germs - targets, the extracts of ethyl acetate gave the best results compared to dichloromethane and hexane.The Analysis of these compounds of antibiotic phenazinique effect within layer chromatography (CCM) and high performance liquid chromatography( HPLC) indicate that both strains PI9 and F20 are productive of phenazine-1-carboxylic acid (PCA). The BB9 strain is suspected to be productive of another phenazinique compound.

Keywords: Pseudomonas ssp. fluorescents, antagonism in vitro, secondary metabolite, phenazines, biopesticide.

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Authors: Ahmed Alkarimi, Kevin Welham

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Organic mixed mode monolithic columns were fabricated from; glycidyl methacrylate-co-ethylene dimethacrylate-co-stearyl methacrylate, using glycidyl methacrylate and stearyl methacrylate as co monomers representing 30% and 70% respectively of the liquid volume with ethylene dimethacrylate crosslinker and 2,2-dimethoxy-2-phenylacetophenone as the free radical initiator. The monomers were mixed with a binary porogenic solvent, comprising propan-1-ol, and methanol (0.825 mL each). The monolith was formed by photo polymerization (365 nm) inside a borosilicate glass tube (1.5 mm ID and 3 mm OD x 50 mm length). The monolith was observed to have formed correctly by optical examination and generated reasonable backpressure, approximately 650 psi at a flow rate of 0.2 mL min⁻¹ 50:50 acetonitrile: water. The morphological properties of the monolithic columns were investigated using scanning electron microscopy images, and Brunauer-Emmett-Teller analysis, the results showed that the monolith was formed properly with 19.98 ± 0.01 mm² surface area, 0.0205 ± 0.01 cm³ g⁻¹ pore volume and 6.93 ± 0.01 nm average pore size. The polymer monolith formed was further investigated using proton nuclear magnetic resonance, and Fourier transform infrared spectroscopy. The monolithic columns were investigated using high-performance liquid chromatography to test their ability to separate different samples with a range of properties. The columns displayed both hydrophobic/hydrophilic and hydrophobic/ion exchange interactions with the compounds tested indicating that true mixed mode separations. The mixed mode monolithic columns exhibited significant separation of proteins.

Keywords: LC separation, proteins separation, monolithic column, mixed mode

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Authors: Su Mon Saw, Joe Rothnagel

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Short open reading frames (sORFs) located in 5’UTR of mRNAs are known as uORFs. Characterization of uORF-encoded peptides (uPEPs) i.e., a subset of short open reading frame encoded peptides (sPEPs) and their translation regulation lead to understanding of causes of genetic disease, proteome complexity and development of treatments. Existence of uORFs within cellular proteome could be detected by LC-MS/MS. The ability of uORF to be translated into uPEP and achievement of uPEP identification will allow uPEP’s characterization, structures, functions, subcellular localization, evolutionary maintenance (conservation in human and other species) and abundance in cells. It is hypothesized that a subset of sORFs are translatable and that their encoded sPEPs are functional and are endogenously expressed contributing to the eukaryotic cellular proteome complexity. This project aimed to investigate whether sORFs encode functional peptides. Liquid chromatography-mass spectrometry (LC-MS) and bioinformatics were thus employed. Due to probable low abundance of sPEPs and small in sizes, the need for efficient peptide enrichment strategies for enriching small proteins and depleting the sub-proteome of large and abundant proteins is crucial for identifying sPEPs. Low molecular weight proteins were extracted using SDS-PAGE from Human Embryonic Kidney (HEK293) cells and Strong Cation Exchange Chromatography (SCX) from secreted HEK293 cells. Extracted proteins were digested by trypsin to peptides, which were detected by LC-MS/MS. The MS/MS data obtained was searched against Swiss-Prot using MASCOT version 2.4 to filter out known proteins, and all unmatched spectra were re-searched against human RefSeq database. ProteinPilot v5.0.1 was used to identify sPEPs by searching against human RefSeq, Vanderperre and Human Alternative Open Reading Frame (HaltORF) databases. Potential sPEPs were analyzed by bioinformatics. Since SDS PAGE electrophoresis could not separate proteins <20kDa, this could not identify sPEPs. All MASCOT-identified peptide fragments were parts of main open reading frame (mORF) by ORF Finder search and blastp search. No sPEP was detected and existence of sPEPs could not be identified in this study. 13 translated sORFs in HEK293 cells by mass spectrometry in previous studies were characterized by bioinformatics. Identified sPEPs from previous studies were <100 amino acids and <15 kDa. Bioinformatics results showed that sORFs are translated to sPEPs and contribute to proteome complexity. uPEP translated from uORF of SLC35A4 was strongly conserved in human and mouse while uPEP translated from uORF of MKKS was strongly conserved in human and Rhesus monkey. Cross-species conserved uORFs in association with protein translation strongly suggest evolutionary maintenance of coding sequence and indicate probable functional expression of peptides encoded within these uORFs. Translation of sORFs was confirmed by mass spectrometry and sPEPs were characterized with bioinformatics.

Keywords: bioinformatics, HEK293 cells, liquid chromatography-mass spectrometry, ProteinPilot, Strong Cation Exchange Chromatography, SDS-PAGE, sPEPs

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Authors: Martin Cai, Khadijah B. Hashim, Leng Leo, Edmund F. Tian

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Analysis of traditional Chinese medicinal (TCM) supplements has always been a laborious task, particularly in the case of multi‐ingredient formulations. Traditionally, herbal extracts are analysed using one or few markers compounds. In the recent years, however, pharmaceutical companies are introducing health supplements of TCM active ingredients to cater to the needs of consumers in the fast-paced society in this age. As such, new problems arise in the aspects of composition identification as well as quality analysis. In most cases of products or supplements formulated with multiple TCM herbs, the chemical composition, and nature of each raw material differs greatly from the others in the formulation. This results in a requirement for individual analytical processes in order to identify the marker compounds in the various botanicals. Thin-layer Chromatography (TLC) is a simple, cost effective, yet well-regarded method for the analysis of natural products, both as a Pharmacopeia-approved method for identification and authentication of herbs, and a great analytical tool for the discovery of chemical compositions in herbal extracts. Recent technical advances introduced High-Performance TLC (HPTLC) where, with the help of automated equipment and improvements on the chromatographic materials, both the quality and reproducibility are greatly improved, allowing for highly standardised analysis with greater details. Here we report an industrial consultancy project with ONI Global Pte Ltd for the analysis of LAC Liver Protector, a TCM formulation aimed at improving liver health. The aim of this study was to identify 4 key components of the supplement using HPTLC, following protocols derived from Chinese Pharmacopeia standards. By comparing the TLC profiles of the supplement to the extracts of the herbs reported in the label, this project proposes a simple and cost-effective analysis of the presence of the 4 marker compounds in the multi‐ingredient formulation by using 4 different HPTLC methods. With the increasing trend of small and medium-sized enterprises (SMEs) bringing natural products and health supplements into the market, it is crucial that the qualities of both raw materials and end products be well-assured for the protection of consumers. With the technology of HPTLC, science can be incorporated to help SMEs with their quality control, thereby ensuring product quality.

Keywords: traditional Chinese medicine supplement, high performance thin layer chromatography, active ingredients, product quality

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Authors: S. G. Vasantharaju, Viswanath Guptha, Raghavendra Shetty

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Introduction: Aminophylline is a methylxanthine derivative belonging to the class bronchodilator. From the literature survey, reported methods reveals the solid phase extraction and liquid liquid extraction which is highly variable, time consuming, costly and laborious analysis. Present work aims to develop a simple, highly sensitive, precise and accurate high-performance liquid chromatography method for the quantification of Aminophylline in rat plasma samples which can be utilized for preclinical studies. Method: Reverse Phase high-performance liquid chromatography method. Results: Selectivity: Aminophylline and the internal standard were well separated from the co-eluted components and there was no interference from the endogenous material at the retention time of analyte and the internal standard. The LLOQ measurable with acceptable accuracy and precision for the analyte was 0.5 µg/mL. Linearity: The developed and validated method is linear over the range of 0.5-40.0 µg/mL. The coefficient of determination was found to be greater than 0.9967, indicating the linearity of this method. Accuracy and precision: The accuracy and precision values for intra and inter day studies at low, medium and high quality control samples concentrations of aminophylline in the plasma were within the acceptable limits Extraction recovery: The method produced consistent extraction recovery at all 3 QC levels. The mean extraction recovery of aminophylline was 93.57 ± 1.28% while that of internal standard was 90.70 ± 1.30%. Stability: The results show that aminophylline is stable in rat plasma under the studied stability conditions and that it is also stable for about 30 days when stored at -80˚C. Pharmacokinetic studies: The method was successfully applied to the quantitative estimation of aminophylline rat plasma following its oral administration to rats. Discussion: Preclinical studies require a rapid and sensitive method for estimating the drug concentration in the rat plasma. The method described in our article includes a simple protein precipitation extraction technique with ultraviolet detection for quantification. The present method is simple and robust for fast high-throughput sample analysis with less analysis cost for analyzing aminophylline in biological samples. In this proposed method, no interfering peaks were observed at the elution times of aminophylline and the internal standard. The method also had sufficient selectivity, specificity, precision and accuracy over the concentration range of 0.5 - 40.0 µg/mL. An isocratic separation technique was used underlining the simplicity of the presented method.

Keywords: Aminophyllin, preclinical pharmacokinetics, rat plasma, RPHPLC

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Authors: Patricia B. Cruz, Catrina Jean G. Valenzuela, Analyn N. Yumang

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Nearly a hundred per million of the Filipino population is diagnosed with Chronic Kidney Disease (CKD). The early stage of CKD has no symptoms and can only be discovered once the patient undergoes urinalysis. Over the years, different methods were discovered and used for the quantification of the urinary albumin such as the immunochemical assays where most of these methods require large machinery that has a high cost in maintenance and resources, and a dipstick test which is yet to be proven and is still debated as a reliable method in detecting early stages of microalbuminuria. This research study involves the use of the liquid chromatography concept in microfluidic instruments with biosensor as a means of separation and detection respectively, and linear regression to quantify human urinary albumin. The researchers’ main objective was to create a miniature system that quantifies and detect patients’ urinary albumin while reducing the amount of volume used per five test samples. For this study, 30 urine samples of unknown albumin concentrations were tested using VITROS Analyzer and the microfluidic system for comparison. Based on the data shared by both methods, the actual vs. predicted regression were able to create a positive linear relationship with an R² of 0.9995 and a linear equation of y = 1.09x + 0.07, indicating that the predicted values and actual values are approximately equal. Furthermore, the microfluidic instrument uses 75% less in total volume – sample and reagents combined, compared to the VITROS Analyzer per five test samples.

Keywords: Chronic Kidney Disease, Linear Regression, Microfluidics, Urinary Albumin

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Authors: Tsige Reda

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Essential oil of Thymus vulgaris was extracted by means of hydro-distillation. This study was done to investigate the chemical composition, antibacterial and antioxidant activities. The chemical composition of the essential oils was determined using gas chromatography coupled to mass spectroscopy (GC-MS). Using disc diffusion assay the antibacterial activity was assessed on one Gram-positive bacteria and one Gram-negative bacteria. The percentage oil yield of the essential oil was found to be 0.97 ± 0.08% (w/w) with yellow color. The physicochemical constants of the oil were also noted. The phytochemical screening of the plant extract revealed the presence of tannins, saponins, phenol, flavonoids, terpenoids, steroids and alkaloids. A total of 18 chemical constituents were identified by Gas Chromatography-Mass Spectroscopy analysis representing 100% of the total essential oil of Thymus vulgaris, with thymol (31.977%), o-cymene (29.992%), and carvacrol (14.541%). Previous studies have revealed that the thymol, o-cymen and carvacrol components of Thymus vulgaris are responsible for their biological activities. Thymus vulgaris have been used traditionally to treat a wide variety of infections. Based on the extensive use and lack of scientific evidence, a study was embarked upon to determine its bioactivity. The essential oil of Thymus vulgaris leaves exhibited higher activity towards the Gram-positive bacteria (Staphylococcus aurous) than the Gram-negative bacteria (Escherichia coli) and also has good antioxidant activity, and can be used medicinal and therapeutic applications. This activity may be due to the high amount of thymol, o-cymen and carvacrol.

Keywords: hydro-distillation, Thymus vulgaris, essential oil composition, phytochemical screening, physicochemical constants, antioxidant activity, antibacterial activity

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Authors: Yılmaz Uçar, Fatih Ozogul, Mustafa Durmuş, Yesim Ozogul, Ali Rıza Köşker, Esmeray Kuley Boğa, Deniz Ayas

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The aim of this study was to purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), that are essential oils from trout oil, using high-performance liquid chromatography (HPLC) method, bioconverted EPA and DHA into bioconverted EPA (bEPA), bioconverted DHA (bDHA) extracts by P. aeruginosa PR3. Moreover, in vitro antibacterial activity of bEPA and bDHA was investigated using disc diffusion methods and minimum inhibitory concentration (MIC). EPA and DHA concentration of 11.1% and 15.9% in trout oil increased in 58.64% and 40.33% after HPLC optimisation, respectively. In this study, EPA and DHA enriched products were obtained which are to be used as valuable supplements for food and pharmaceutical purposes. The bioconverted EPA and DHA exhibited antibacterial activities against two Gram-positive bacteria (Listeria monocytogenes ATCC 7677 and Staphylococcus aureus ATCC 29213) and six Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC700603, Enterococcus faecalis ATCC 29212, Aeromonas hydrophila NCIMB 1135, and Salmonella Paratyphi A NCTC 13). Inhibition zones and MIC value of bEPA and bDHA against bacterial strains ranged from 7 to 12 mm and from 350 to 2350 μg/mL, respectively. Our results suggested that the crude extracts of bioconversion of EPA and DHA by P. aeruginosa PR3 can be considered as promising antimicrobials in improving food safety by controlling foodborne pathogens.

Keywords: High-Performance Liquid Chromatography (HPLC), docosahexaenoic acid, DHA, eicosapentaenoic acid, EPA, minimum inhibitory concentration, MIC, Pseudomonas aeruginosa PR3

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Authors: Thidarat Duangyod, Chanida Palanuvej, Nijsiri Ruangrungsi

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Pentace burmanica Kurz., belonging to the Malvaceae family, is commonly used for anti-diarrhea in Thai traditional medicine. A method for quantification of (+)-catechin and (-)-epicatechin in P. burmanica stem bark from 12 different Thailand markets by reverse-phase high performance liquid chromatography (HPLC) was investigated and validated. The analysis was performed by a Shimadzu DGU-20A3 HPLC equipped with a Shimadzu SPD-M20A photo diode array detector. The separation was accomplished with an Inersil ODS-3 column (5 µm x 4.6 x 250 mm) using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as mobile phase at the flow rate of 1 ml/min. The isocratic was set at 20% B for 15 min and the column temperature was maintained at 40 ºC. The detection was at the wavelength of 280 nm. Both (+)-catechin and (-)-epicatechin existed in the ethanolic extract of P. burmanica stem bark. The content of (-)-epicatechin was found as 59.74 ± 1.69 µg/mg of crude extract. In contrast, the quantitation of (+)-catechin content was omitted because of its small amount. The method was linear over a range of 5-200 µg/ml with good coefficients (r2 > 0.99) for (+)-catechin and (-)-epicatechin. Limit of detection values were found to be 4.80 µg/ml for (+)-catechin and 5.14 µg/ml for (-)-epicatechin. Limit of quantitation of (+)-catechin and (-)-epicatechin were of 14.54 µg/ml and 15.57 µg/ml respectively. Good repeatability and intermediate precision (%RSD < 3) were found in this study. The average recoveries of both (+)-catechin and (-)-epicatechin were obtained with good recovery in the range of 91.11 – 97.02% and 88.53 – 93.78%, respectively, with the %RSD less than 2. The peak purity indices of catechins were more than 0.99. The results suggested that HPLC method proved to be precise and accurate and the method can be conveniently used for (+)-catechin and (-)-epicatechin determination in ethanolic extract of P. burmanica stem bark. Moreover, the stem bark of P. burmanica was found to be a rich source of (-)-epicatechin.

Keywords: pentace burmanica, (+)-catechin, (-)-epicatechin, high performance liquid chromatography

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Authors: Asavasereerat K., Teacharsripaitoon T., Tungyingyong P., Charupongrat S., Noppiboon S. Hochareon L., Kitsuban P.

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Third generation of vaccines is based on gene therapy where DNA is introduced into patients. The antigenic or therapeutic proteins encoded from transgenes DNA triggers an immune-response to counteract various diseases. Moreover, DNA vaccine offers the customization of its ability on protection and treatment with high stability. The production of DNA vaccines become of interest. According to USFDA guidance for industry, the recommended limits for impurities from host cell are lower than 1%, and the active conformation homogeneity supercoiled DNA, is more than 80%. Thus, the purification strategy using two-steps chromatography has been established and verified for its robustness. Herein, pVax1/lacZ, a pre-approved USFDA DNA vaccine backbone, was used and transformed into E. coli strain DH5α. Three purification process parameters including sample-loading flow rate, the salt concentration in washing and eluting buffer, were studied and the experiment was designed using response surface method with central composite face-centered (CCF) as a model. The designed range of selected parameters was 10% variation from the optimized set point as a safety factor. The purity in the percentage of supercoiled conformation obtained from each chromatography step, AIEX and HIC, were analyzed by HPLC. The response data were used to establish regression model and statistically analyzed followed by Monte Carlo simulation using SAS JMP. The results on the purity of the product obtained from AIEX and HIC are between 89.4 to 92.5% and 88.3 to 100.0%, respectively. Monte Carlo simulation showed that the pVAX1/lacZ purification process is robust with confidence intervals of 0.90 in range of 90.18-91.00% and 95.88-100.00%, for AIEX and HIC respectively.

Keywords: AIEX, DNA vaccine, HIC, puification, response surface method, robustness

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Authors: Ekere E. Kokonne, Isimi C. Yetunde, Okoh E. Judith, Okafor E. Ijeoma, Ajeh J. Isaac, Olobayo O. Kunle, Emeje O. Martins

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NIPRIMAL® is a mono herbal formulation of Nauclea latifolia used in the treatment of malaria. The stability of extracts made from plant material is essential to ensure the quality, safety and efficacy of the finished product. This study assessed the stability of the formulation under three different storage conditions; normal room temperature, infrared and under refrigeration. Differential Scanning Calorimetry (DSC) and Thin Layer Chromatography (TLC) were used to monitor the formulations. The DSC analysis was done from 0oC to 350oC under the three storage conditions. Results obtained indicate that NIPRIMAL® was stable at all the storage conditions investigated. Thin layer chromatography (TLC) after 6 months showed there was no significant difference between retention factor (RF) values for the various storage conditions. The reference sample had four spots with RF values of 0.47, 0.68, 0.76, 0.82 respectively and these spots were retained in the test formulations with corresponding RF values were after 6 months at room temperature and refrigerated temperature been 0.56, 0.73, 0.80, 0.92 and 0.47, 0.68, 0.76, 0.82 respectively. On the other hand, the RF values (0.55, 0.74, 0.77, 0.93) obtained under infrared after 1 month varied slightly from the reference. The sample exposed to infrared had a lower heat capacity compared to that stored under room temperature or refrigeration. A combination of TLC and DSC measurements has been applied for assessing the stability of NIPRIMAL®. Both methods were found to be rapid, sensitive and reliable in determining its stability. It is concluded that NIPRIMAL® can be stored under any of the tested conditions without degradation. This study is a major contribution towards developing appropriate stability monitoring parameters for herbal products.

Keywords: differential scanning calorimetry, formulation, NIPRIMAL®, stability, thin layer hromatography

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Authors: Leila Najafian

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Sarcoplasmic protein hydrolysates (SPHs) and myofibrillar protein hydrolysates (MPHs) from patin (Pangasius sutchi) were produced using two types of proteases: Papain and Alcalase. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activities and metal chelating activity assays for antioxidant activities were carried out on the SPHs and MPHs. The hydrolysates were isolated and purified by ultrafiltration, gel filtration and reverse phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) was used in identifying peptide sequences. The results showed that when the DH of MPHs increased, the protein solubility increased, while the highest amount of the protein solubility of SPHs was after 60 min incubation. The effect of DH on antioxidant activities of SPHs and MPHs was investigated. Among the hydrolysates, papain-MPH and Alcalase-SPH, which had the highest antioxidant activities, were purified. The potent fractions obtained from RP-HPLC of sarcoplasmic (SI 3 fraction) and myofibrillar (MI 4 fraction) hydrolysates showed the highest DPPH radical scavenging activity. The FVNQPYLLYSVHMK peptide for MPH and the LVVDIPAALQHA peptide for SPH exhibited the highest antioxidant activity. The presence of hydrophobic and hydrophilic amino acids, namely leucine (L), valine (V), phenylalanine (F), histidine (H) and proline (P), in the peptide sequences of SPH and MPH are believed to contribute to high antioxidant activity. Hence, SPH and MPH from patin have the potential as a natural functional ingredient in food and pharmaceutical industry.

Keywords: patin (Pangasius sutchi), protein hydrolysates, antioxidative peptides, mass spectrometry

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Authors: Dmitry Yu. Korulkin, Raissa A. Muzychkina

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In review the generalized data about different methods of extraction, separation and purification of natural and modify anthraquinones is presented. The basic regularity of an isolation process is analyzed. Action of temperature, pH, and polarity of extragent, catalysts and other factors on an isolation process is revealed.

Keywords: anthraquinones; isolation; extraction; polarity; chromatography; precipitation; bioactivity; phytopreparation; chrysophanol; aloe-emodin; emodin; physcion.

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Authors: Marinos Xagoraris, Iliada K. Lappa, Charalambos Kanakis, Dimitra Daferera, Christina Papadopoulou, Georgios Sourounis, Charilaos Giotis, Pavlos Bouchagier, Christos S. Pappas, Petros A. Tarantilis, Efstathia Skotti

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The aim of this work was to recover phenolic compounds from grape skins produced in Greek varieties of the Ionian Islands in order to form the basis of calculations for their further utilization in the context of the circular economy. Isolation and further utilization of phenolic compounds is an important issue in winery by-products. For this purpose, 37 samples were collected, extracted, and analyzed in an attempt to provide the appropriate basis for their sustainable exploitation. Extraction of the bioactive compounds was held using an eco-friendly, non-toxic, and highly effective water-glycerol solvent system. Then, extracts were analyzed using UV-Vis, liquid chromatography-mass spectrometry (LC-MS), FTIR, and Raman spectroscopy. Also, total phenolic content and antioxidant activity were measured. LC-MS chromatography showed qualitative differences between different varieties. Peaks were attributed to monomeric 3-flavanols as well as monomeric, dimeric, and trimeric proanthocyanidins. The FT-IR and Raman spectra agreed with the chromatographic data and contributed to identifying phenolic compounds. Grape skins exhibited high total phenolic content (TPC), and it was proved that during vinification, a large number of polyphenols remained in the pomace. This study confirmed that grape skins from Ionian Islands are a promising source of bioactive compounds, suggesting their utilization under a bio-economic and environmental strategic framework.

Keywords: antioxidant activity, grape skin, phenolic compounds, waste recovery

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Authors: Juni C. Kim, Anna R. Robuck, Douglas I. Walker

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The human exposome, which includes chemical exposures over the lifetime and their effects, is now recognized as an important measure for understanding human health; however, the complexity of the data makes the identification of environmental chemicals challenging. The goal of our project was to establish a computational workflow for the improved identification of environmental pollutants containing chlorine or bromine. Using the “pattern. search” function available in the R package NonTarget, we wrote a multifunctional script that searches mass spectral clusters from untargeted gas-chromatography high-resolution mass spectrometry (GC-HRMS) for the presence of spectra consistent with chlorine and bromine-containing organic compounds. The “pattern. search” function was incorporated into a different function that allows the evaluation of clusters containing multiple analyte fragments, has multi-core support, and provides a simplified output identifying listing compounds containing chlorine and/or bromine. The new function was able to process 46,000 spectral clusters in under 8 seconds and identified over 150 potential halogenated spectra. We next applied our function to a deidentified dataset from patients diagnosed with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and healthy controls. Twenty-two spectra corresponded to potential halogenated compounds in the PSC and PBC dataset, including six significantly different in PBC patients, while four differed in PSC patients. We have developed an improved algorithm for detecting halogenated compounds in GC-HRMS data, providing a strategy for prioritizing exposures in the study of human disease.

Keywords: exposome, metabolome, computational metabolomics, high-resolution mass spectrometry, exposure, pollutants

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Authors: Eleni Poulou-Sidiropoulou, Charalampos N. Bompas, Martina Samiotaki, Alexios Vlamis-Gardikas

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The glutaredoxin (Grx) and thioredoxin (Trx) systems keep the intracellular environment reduced in almost all organisms. In Escherichia coli (E. coli), the Grx system relies on NADPH+ to reduce GSH reductase (GR), the latter reducing oxidized diglutathione to glutathione (GSH) which in turn reduces cytosolic Grxs, the electron donors for different intracellular substrates. In the Trx system, GR and GSH are replaced by Trx reductase (TrxR). Three of the Grxs of E. coli (Grx1, 2, 3) are reduced by GSH, while Grx4 is likely reduced by TrxR. Trx1 and Grx1 from E. coli may reduce ribonucleotide reductase Ia to ensure a constant supply of deoxyribonucleotides for the synthesis of DNA. The role of the other three Grxs is relatively unknown, especially for Grx2 that may amount up to 1 % of total cellular protein in the stationary phase of growth. The protein is known as a potent antioxidant, but no specific functions have been attributed to it. Herein, affinity chromatography of cellular extracts on immobilized Grx2, followed by MS analysis of the resulting eluates, was employed to identify protein ligands that could provide insights into the biological role of Grx2. Ionic, strong non-covalent, and covalent (disulfide) interactions with relevant proteins were detected. As a means of verification, the identified ligands were subjected to in silico docking with monothiol Grx2. In other experiments, protein extracts from E. coli cells lacking the gene for Grx2 (grxB) were compared to those of wild type. Taken together, the two approaches suggest that Grx2 is involved in protein synthesis, nucleotide metabolism, DNA damage repair, stress responses, and various metabolic processes. Grx2 appears as a versatile protein that may participate in a wide range of biological pathways beyond its known general antioxidant function.

Keywords: Escherichia coli, glutaredoxin 2, interactome, thiol-disulfide oxidoreductase

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Authors: Suhas Kadam, Vishal Chandanshive, Niraj Rane, Sanjay Govindwar

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Fimbristylis dichotoma, Ammannia baccifera, and their co-plantation consortium FA were found to degrade methyl orange, simulated dye mixture, and real textile effluent. Wild plants of Fimbristylis dichotoma and Ammannia baccifera with equal biomass showed 91 and 89% decolorization of methyl orange within 60 h at a concentration of 50 ppm, while 95% dye removal was achieved by consortium FA within 48 h. Floating phyto-beds with co-plantation (Fimbristylis dichotoma and Ammannia baccifera) for the treatment of real textile effluent in a constructed wetland was observed to be more efficient and achieved 79, 72, 77, 66 and 56% reductions in ADMI color value, chemical oxygen demand, biological oxygen demand, total dissolve solid and total suspended solid of textile effluent, respectively. High performance thin layer chromatography, gas chromatography-mass spectroscopy, Fourier transform infrared spectroscopy, Ultra violet-Visible spectroscopy and enzymatic assays confirmed the phytotransformation of parent dye in the new metabolites. T-RFLP analysis of rhizospheric bacteria of Fimbristylis dichotoma, Ammannia baccifera, and consortium FA revealed the presence of 88, 98 and 223 genera which could have been involved in dye removal. Toxicity evaluation of products formed after phytotransformation of methyl orange by consortium FA on bivalves Lamellidens marginalis revealed less damage in the gills architecture when analyzed histologically. Toxicity measurement by Random Amplification of Polymorphic DNA (RAPD) technique revealed normal banding pattern in treated methyl orange sample suggesting less toxic nature of phytotransformed dye products.

Keywords: constructed wetland, phyto-bed, textile effluent, phytoremediation

Procedia PDF Downloads 459

Authors: Ragaa T. Darwish, Maha A. Demellawy, Haidy M. Megahed, Doreen N. Younan, Wael S. Kholeif

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The testing of drug abuse is authentic in order to affirm the misuse of drugs. Several analytical approaches have been developed for the detection of drugs of abuse in pharmaceutical and common biological samples, but few methodologies have been created to identify them from fingerprints. Liquid Chromatography-Mass Spectrometry (LC-MS) plays a major role in this field. The current study aimed at assessing the possibility of detection of some drugs of abuse (tramadol, clonazepam, and phenobarbital) from fingerprints using LC-MS in drug abusers. The aim was extended in order to assess the possibility of detection of the above-mentioned drugs in fingerprints of drug handlers till three days of handling the drugs. The study was conducted on randomly selected adult individuals who were either drug abusers seeking treatment at centers of drug dependence in Alexandria, Egypt or normal volunteers who were asked to handle the different studied drugs (drug handlers). An informed consent was obtained from all individuals. Participants were classified into 3 groups; control group that consisted of 50 normal individuals (neither abusing nor handling drugs), drug abuser group that consisted of 30 individuals who abused tramadol, clonazepam or phenobarbital (10 individuals for each drug) and drug handler group that consisted of 50 individuals who were touching either the powder of drugs of abuse: tramadol, clonazepam or phenobarbital (10 individuals for each drug) or the powder of the control substances which were of similar appearance (white powder) and that might be used in the adulteration of drugs of abuse: acetyl salicylic acid and acetaminophen (10 individuals for each drug). Samples were taken from the handler individuals for three consecutive days for the same individual. The diagnosis of drug abusers was based on the current Diagnostic and Statistical Manual of Mental disorders (DSM-V) and urine screening tests using immunoassay technique. Preliminary drug screening tests of urine samples were also done for drug handlers and the control groups to indicate the presence or absence of the studied drugs of abuse. Fingerprints of all participants were then taken on a filter paper previously soaked with methanol to be analyzed by LC-MS using SCIEX Triple Quad or QTRAP 5500 System. The concentration of drugs in each sample was calculated using the regression equations between concentration in ng/ml and peak area of each reference standard. All fingerprint samples from drug abusers showed positive results with LC-MS for the tested drugs, while all samples from the control individuals showed negative results. A significant difference was noted between the concentration of the drugs and the duration of abuse. Tramadol, clonazepam, and phenobarbital were also successfully detected from fingerprints of drug handlers till 3 days of handling the drugs. The mean concentration of the chosen drugs of abuse among the handlers group decreased when the days of samples intake increased.

Keywords: drugs of abuse, fingerprints, liquid chromatography–mass spectrometry, tramadol

Procedia PDF Downloads 94

Authors: Basanti Ekka, Talis Juhna

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Dairy industrial effluents originated by the typical processing activities are composed of various organic and inorganic constituents, and these include proteins, fats, inorganic salts, antibiotics, detergents, sanitizers, pathogenic viruses, bacteria, etc. These contaminants are harmful to not only human beings but also aquatic flora and fauna. Because consisting of large classes of contaminants, the specific targeted removal methods available in the literature are not viable solutions on the industrial scale. Therefore, in this on-going research, a series of coagulation, electrochemical, and catalytic methods will be employed. The bulk coagulation and electrochemical methods can wash off most of the contaminants, but some of the harmful chemicals may slip in; therefore, specific catalysts designed and synthesized will be employed for the removal of targeted chemicals. In the context of Latvian dairy industries, presently, work is under progress on the characterization of dairy effluents by total organic carbon (TOC), Inductively Coupled Plasma Mass Spectrometry (ICP-MS)/ Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), and Mass Spectrometry. After careful evaluation of the dairy effluents, a cost-effective natural coagulant will be employed prior to advanced electrochemical technology such as electrocoagulation and electro-oxidation as a secondary treatment process. Finally, graphene oxide (GO) based hybrid materials will be used for post-treatment of dairy wastewater as graphene oxide has been widely applied in various fields such as environmental remediation and energy production due to the presence of various oxygen-containing groups. Modified GO will be used as a catalyst for the removal of remaining contaminants after the electrochemical process.

Keywords: catalysis, dairy wastewater, electrochemical method, graphene oxide

Procedia PDF Downloads 114

Authors: Marinos Xagoraris, Elisavet Lazarou, Eleftherios Alissandrakis, Christos S. Pappas, Petros A. Tarantilis

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Strawberry tree (Arbutus unedo L.) and autumn heather (Erica manipuliflora Salisb.) are important beekeeping plants of Greece. Six monofloral honeys (four strawberry tree, two autumn heather) were analyzed by means of Solid Phase Micro-Extraction (SPME, 60 min, 60 oC) followed by Gas Chromatography coupled to Mass Spectrometry (GC-MS) for the purpose of assessing the botanical origin. A Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) fiber was employed, and benzophenone was used as internal standard. The volatile compounds with higher concentration (μg/ g of honey expressed as benzophenone) from strawberry tree honey samples, were α-isophorone (2.50-8.12); 3,4,5-trimethyl-phenol (0.20-4.62); 2-hydroxy-isophorone (0.06-0.53); 4-oxoisophorone (0.38-0.46); and β-isophorone (0.02-0.43). Regarding heather honey samples, the most abundant compounds were 1-methoxy-4-propyl-benzene (1.22-1.40); p-anisaldehyde (0.97-1.28); p-anisic acid (0.35-0.58); 2-furaldehyde (0.52-0.57); and benzaldehyde (0.41-0.56). Norisoprenoids are potent floral markers for strawberry-tree honey. β-isophorone is found exclusively in the volatile fraction of this type of honey, while also α-isophorone, 4-oxoisophorone and 2-hydroxy-isophorone could be considered as additional marker compounds. The analysis of autumn heather honey revealed that phenolic compounds are the most abundant and p-anisaldehyde; 1-methoxy-4-propyl-benzene; and p-anisic acid could serve as potent marker compounds. In conclusion, marker compounds for the determination of the botanical origin for these honeys could be identified as several norisoprenoids and phenolic components were found exclusively or in higher concentrations compared to common Greek honey varieties.

Keywords: SPME/GC-MS, volatile compounds, heather honey, strawberry tree honey

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Authors: Gaurav Bhanjana, Ganga Ram Chaudhary, Sandeep Kumar, Neeraj Dilbaghi

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Main sources of endocrine disrupting compounds in the ecosystem are hormones, pesticides, phthalates, flame retardants, dioxins, personal-care products, coplanar polychlorinated biphenyls (PCBs), bisphenol A, and parabens. These endocrine disrupting compounds are responsible for learning disabilities, brain development problems, deformations of the body, cancer, reproductive abnormalities in females and decreased sperm count in human males. Although discharge of these chemical compounds into the environment cannot be stopped, yet their amount can be retarded through proper evaluation and detection techniques. The available techniques for determination of these endocrine disrupting compounds mainly include high performance liquid chromatography (HPLC), mass spectroscopy (MS) and gas chromatography-mass spectrometry (GC–MS). These techniques are accurate and reliable but have certain limitations like need of skilled personnel, time consuming, interference and requirement of pretreatment steps. Moreover, these techniques are laboratory bound and sample is required in large amount for analysis. In view of above facts, new methods for detection of endocrine disrupting compounds should be devised that promise high specificity, ultra sensitivity, cost effective, efficient and easy-to-operate procedure. Nowadays, electrochemical sensors/biosensors modified with nanomaterials are gaining high attention among researchers. Bioelement present in this system makes the developed sensors selective towards analyte of interest. Nanomaterials provide large surface area, high electron communication feature, enhanced catalytic activity and possibilities of chemical modifications. In most of the cases, nanomaterials also serve as an electron mediator or electrocatalyst for some analytes.

Keywords: electrochemical, endocrine disruptors, microscopy, nanoparticles, sensors

Procedia PDF Downloads 252

Authors: Patrícia T. Souza, Marina Ansolin, Eduardo A. C. Batista, Antonio J. A. Meirelles, Matthieu Tubino

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Lipid oxidation is a major cause of the deterioration of the quality of the biodiesel, because the waste generated damages the engines. Among the main undesirable effects are the increase of viscosity and acidity, leading to the formation of insoluble gums and sediments which cause the blockage of fuel filters. The auto-oxidation is defined as the spontaneous reaction of atmospheric oxygen with lipids. Unsaturated fatty acids are usually the components affected by such reactions. They are present as free fatty acids, fatty esters and glycerides. To determine the oxidative stability of biodiesels, through the induction period, IP, the Rancimat method is used, which allows continuous monitoring of the induced oxidation process of the samples. During the oxidation of the lipids, volatile organic acids are produced as byproducts, in addition, other byproducts, including alcohols and carbonyl compounds, may be further oxidized to carboxylic acids. By the methodology developed in this work using ion chromatography, IC, analyzing the water contained in the conductimetric vessel, were quantified organic anions of carboxylic acids in samples subjected to oxidation induced by Rancimat. The optimized chromatographic conditions were: eluent water:acetone (80:20 v/v) with 0.5 mM sulfuric acid; flow rate 0.4 mL min-1; injection volume 20 µL; eluent suppressor 20 mM LiCl; analytical curve from 1 to 400 ppm. The samples studied were methyl biodiesel from soybean oil and unsaturated fatty acids standards: oleic, linoleic and linolenic. The induced oxidation kinetics curves were constructed by analyzing the water contained in the conductimetric vessels which were removed, each one, from the Rancimat apparatus at prefixed intervals of time. About 3 g of sample were used under the conditions of 110 °C and air flow rate of 10 L h-1. The water of each conductimetric Rancimat measuring vessel, where the volatile compounds were collected, was filtered through a 0.45 µm filter and analyzed by IC. Through the kinetic data of the formation of the organic anions of carboxylic acids, the formation rates of the same were calculated. The observed order of the rates of formation of the anions was: formate >>> acetate > hexanoate > valerate for the oleic acid; formate > hexanoate > acetate > valerate for the linoleic acid; formate >>> valerate > acetate > propionate > butyrate for the linolenic acid. It is possible to suppose that propionate and butyrate are obtained mainly from linolenic acid and that hexanoate is originated from oleic and linoleic acid. For the methyl biodiesel the order of formation of anions was: formate >>> acetate > valerate > hexanoate > propionate. According to the total rate of formation these anions produced during the induced degradation of the fatty acids can be assigned the order of reactivity: linolenic acid > linoleic acid >>> oleic acid.

Keywords: anions of carboxylic acids, biodiesel, ion chromatography, oxidation

Procedia PDF Downloads 447

Authors: Muhammad Naveed Aslam, Rida Fatima, Anam Moosa, Muhammad Taimoor Shakeel

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Bacillus strains produce antifungal secondary metabolites making them potential candidates for suppressing Fusarium wilt of chickpea disease. In this study, eighteen Bacillus strains were evaluated for their antagonistic effect against Fusarium oxysporum f. sp. ciceris causing Fusarium wilt of chickpea disease. In a direct antifungal assay, thirteen strains showed significant inhibition zones while the remaining five strains did not produce inhibition zones of FOC. Bacillus thuringiensis CHGP12 was the most promising strain exhibiting the highest inhibition of FOC. Antifungal lipopeptides were extracted from CHGP12 strain which showed significant inhibition of the pathogen. Liquid chromatography mass spectrometry (LCMS) analysis revealed that CHGP12 was positive for the presence of iturin, fengycin, surfactin, bacillaene, bacillibactin, plantazolicin, and bacilysin. CHGP12 was tested for biochemical determinants in an in vitro qualitative test where it showed the ability to produce lipase, amylase, cellulase, protease, siderophores, and indole 3-acetic acid (IAA). Furthermore, in a greenhouse experiment CHGP12 also showed a significant decrease in the disease severity in treated plants compared to control. Moreover, CHGP12 also exhibited a significant increase in plant growth parameters viz, root and shoot growth parameters, stomatal conductance, and photosynthesis rate. Conclusively, our findings present the promising potential of Bacillus strain CHGP12 to suppress Fusarium wilt of chickpea and to promote plant growth.

Keywords: liquid chromatography mass spectrometry, growth promotion, antagonism, hydrolytic enzymes, inhibition, lipopeptides.

Procedia PDF Downloads 105

Authors: Alma Gómez-Loredo, José González-Valdez, Jorge Benavides, Marco Rito-Palomares

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Fucoxanthin is a carotenoid that exerts multiple beneficial effects on human health, including antioxidant, anti-cancer, antidiabetic and anti-obesity activity; making the development of a whole process for its production and recovery an important contribution. In this work, the lab-scale production and purification of fucoxanthin in Isocrhysis galbana have been studied. In batch cultures, low light intensities (13.5 μmol/m2s) and bubble agitation were the best conditions for production of the carotenoid with product yields of up to 0.143 mg/g. After fucoxanthin ethanolic extraction from biomass and hexane partition, further recovery and purification of the carotenoid has been accomplished by means of alcohol – salt Aqueous Two-Phase System (ATPS) extraction followed by an ultrafiltration (UF) step. An ATPS comprised of ethanol and potassium phosphate (Volume Ratio (VR) =3; Tie-line Length (TLL) 60% w/w) presented a fucoxanthin recovery yield of 76.24 ± 1.60% among the studied systems and was able to remove 64.89 ± 2.64% of the carotenoid and chlorophyll pollutants. For UF, the addition of ethanol to the original recovered ethanolic ATPS stream to a final relation of 74.15% (w/w) resulted in a reduction of approximately 16% of the protein contents, increasing product purity with a recovery yield of about 63% of the compound in the permeate stream. Considering the production, extraction and primary recovery (ATPS and UF) steps, around a 45% global fucoxanthin recovery should be expected. Although other purification technologies, such as Centrifugal Partition Chromatography are able to obtain fucoxanthin recoveries of up to 83%, the process developed in the present work does not require large volumes of solvents or expensive equipment. Moreover, it has a potential for scale up to commercial scale and represents a cost-effective strategy when compared to traditional separation techniques like chromatography.

Keywords: aqueous two-phase systems, fucoxanthin, Isochrysis galbana, microalgae, ultrafiltration

Procedia PDF Downloads 397

Authors: Naveeda Akhtar Qureshi, Wajiha

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Coccidiosis is a protozoan parasitic disease of genus Eimeria. It is an avian infection causing a great economic loss of 3 billion USD per year globally. A number of anticoccidial drugs are in use however many of them have side effects and cost effective. With increase in poultry demand throughout the world there is a need of more drugs and vaccines against coccidiosis. The present study is based upon the use of F. racemosa a medicinal plant to be a potential source of anticoccidial agents. The methanolic leaves extract was fractionated by column and thin layer chromatography and got nineteen fractions. Each fraction different concentrations was evaluated for its anticoccidial properties in an invitro experiment against E. tenella, E. necatrix and E. mitis. The anticoccidial active fractions were further characterized by spectroscopy (UV-Vis, FTIR) and GC-MS analysis. The in silico molecular docking of active fractions identified compounds were carried out. Among all fractions significantly maximum sporulation inhibition efficacy was shown by F-19 (67.11±2.18) followed by F-15 (65.21±1.34) at concentration of 30mg/ml against E. tenella. The significantly highest sporozoites viability inhibition was shown by F-19 (69.23±2.11) followed by F-15 (67.14±1.52) against E. necatrix at concentration 30mg/ml. Anticoccidial active fractions 15 and 19 showed peak spectrum at 207 and 202nm respectively by UV analysis. Their FTIR analysis confirmed the presence of carboxylic acid, amines, phenols, etc. Anticoccidial active compounds like Cyclododecane methanol, oleic acid, Octadecanoic acid, etc were identified by GC-MS analysis. Identified compounds in silico molecular docking study showed that cyclododecane methanol of F-19 and oleic acid of F-15 showed highest binding affinity with target S-Adenosylmethionine synthase. Hence for further authentication in vivo anticoccidial studies are recommended.

Keywords: ficus racemosa, cluster fig, column chromatography, anticoccidial fractions, GC-MS, molecular docking., s-adenosylmethionine synthase

Procedia PDF Downloads 47

Authors: Dian Mayasari, Yosi Bayu Murti, Sylvia Utami Tunjung Pratiwi, Sudarsono

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Melastoma malabathricum L. is an Indo-Pacific herb that has been traditionally used to treat several ailments such as wounds, dysentery, diarrhea, toothache, and diabetes. This plant is common across tropical Indo-Pacific archipelagos and is tolerant of a range of soils, from low-lying areas subject to saltwater inundation to the salt-free conditions of mountain slopes. How the soil and environmental variation influences secondary metabolite production in the herb, and an understanding of the plant’s utility as traditional medicine, remain largely unknown and unexplored. The objective of this study is to evaluate the variability of the metabolic profiles of M. malabathricum L. across its geographic distribution. By employing high-performance liquid chromatography-diode array detector (HPLC-DAD), a highly established, simple, sensitive, and reliable method was employed for establishing the chemical fingerprints of 72 samples of M. malabathricum L. leaves from various geographical locations in Indonesia. Specimens collected from six terrestrial and archipelago regions of Indonesia were analyzed by HPLC to generate chromatogram peak profiles that could be compared across each region. Data corresponding to the common peak areas of HPLC chromatographic fingerprint were analyzed by hierarchical component analysis (HCA) and principal component analysis (PCA) to extract information on the most significant variables contributing to characterization and classification of analyzed samples data. Principal component values were identified as PC1 and PC2 with 41.14% and 19.32%, respectively. Based on variety and origin, the high-performance liquid chromatography method validated the chemical fingerprint results used to screen the in vitro antioxidant activity of M. malabathricum L. The result shows that the developed method has potential values for the quality of similar M. malabathrium L. samples. These findings provide a pathway for the development and utilization of references for the identification of M. malabathricum L. Our results indicate the importance of considering geographic distribution during field-collection efforts as they demonstrate regional metabolic variation in secondary metabolites of M. malabathricum L., as illustrated by HPLC chromatogram peaks and their antioxidant activities. The results also confirm the utility of this simple approach to a rapid evaluation of metabolic variation between plants and their potential ethnobotanical properties, potentially due to the environments from whence they were collected. This information will facilitate the optimization of growth conditions to suit particular medicinal qualities.

Keywords: fingerprint, high performance liquid chromatography, Melastoma malabathricum l., metabolic profiles, principal component analysis

Procedia PDF Downloads 132

Authors: Cristian Moisa, Andreea Lupitu, Adriana Csakvari, Dana G. Radu, Leonard Marian Olariu, Georgeta Pop, Dorina Chambre, Lucian Copolovici, Dana Copolovici

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Green synthesis of nanoparticles (NPs) represents a promising, accessible, eco-friendly, and safe process with significant applications in biotechnology, pharmaceutical sciences, and farming. The aim of our study was to obtain silver nanoparticles, using plant wastes extracts resulted in the essential oils extraction process: Thymus vulgaris L., Origanum vulgare L., Lavandula angustifolia L., and in hemp processing for seed and fibre, Cannabis sativa. Firstly, we obtained aqueous extracts of thyme, oregano, lavender, and hemp (two monoicous and one dioicous varieties), all harvested in western part of Romania. Then, we determined the chemical composition of the extracts by liquid-chromatography coupled with diode array and mass spectrometer detectors. The compounds identified in the extracts were in agreement with earlier published data, and the determination of the antioxidant activity of the obtained extracts by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assays confirmed their antioxidant activity due to their total polyphenolic content evaluated by Folin-Ciocalteu assay. Then, the silver nanoparticles (AgNPs) were successfully biosynthesised, as was demonstrated by UV-VIS, FT-IR spectroscopies, and SEM, by reacting AgNO₃ solution and plant extracts. AgNPs were spherical in shape, with less than 30 nm in diameter, and had a good bactericidal activity against Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens).

Keywords: plant wastes extracts, chemical composition, high performance liquid chromatography mass spectrometer, HPLC-MS, scanning electron microscopy, SEM, silver nanoparticles

Procedia PDF Downloads 161

Authors: M. Umar Dahot, G. S. Mangrio

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In this study Agro-industrial waste was used as a carbon source, which is a low cost substrate. Along with this, various sugars and molasses of 2.5% and 5% were investigated as substrate/carbon source for the growth of A.niger and Pectinase production. Different nitrogen sources were also used. An overview of results obtained show that 5% sucrose, 5% molasses and 0.4% (NH4)2SO4 were found the best carbon and nitrogen sources for the production of pectinase by A. niger. The maximum production of pectinase (26.87units/ml) was observed at pH 6.0 after 72 hrs incubation. The optimum temperature for the maximum production of pectinase was achieved at 35ºC when maximum production of pectinase was obtained as 28.25Units/ml.Pectinase enzyme was purified with ammonium sulphate precipitation and dialyzed sample was finally applied on gel filtration chromatography (Sephadex G-100) and Ion Exchange DEAE A-50. The enzyme was purified 2.5 fold by gel chromatography on Sephadex G-100 and Four fractions were obtained, Fraction 1, 2, 4 showed single band while Fraction -3 showed multiple bands on SDS Page electrophoresis. Fraction -3 was pooled, dialyzed and separated on Sephdex A-50 and two fractions 3a and 3b showed single band. The molecular weights of the purified fractions were detected in the range of 33000 ± 2000 and 38000± 2000 Daltons. The purified enzyme was specifically most active with pure pectin, while pectin, Lemon pectin and orange peel given lower activity as compared to (control). The optimum pH and temperature for pectinase activity was found between pH 5.0 and 6.0 and 40°- 50°C, respectively. The enzyme was stable over the pH range 3.0-8.0. The thermostability of was determined and it was observed that the pectinase activity is heat stable and retains activity more than 40% when incubated at 90°C for 10 minutes. The pectinase activity of F3a and F3b was increased with different metal ions. The Pectinase activity was stimulated in the presence of CaCl2 up to 10-30%. ZnSO4, MnSO4 and Mg SO4 showed higher activity in fractions F3a and F3b, which indicates that the pectinase belongs to metalo-enzymes. It is concluded that A. niger is capable to produce pH stable and thermostable pectinase, which can be used for industrial purposes.

Keywords: pectinase, a. niger, production, purification, characterization

Procedia PDF Downloads 395