Reverse-phase High-pressure Liquid Research Articles

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of β-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Molecular binding mechanism and novel antidiabetic and anti-hypertensive bioactive peptides from fermented camel milk with anti-inflammatory activity in raw macrophages cell lines.

The investigation was to determine the effect of camel milk fermented with Limosilactobacillus fermentum KGL4 (MTCC 25515) on ACE-inhibiting, anti-inflammatory, and diabetes-preventing properties and also to release the novel peptides with antidiabetic and anti-hypertensive attributes with molecular interaction studies. Growth conditions were optimised on the basis of total peptide production by inoculating the culture in camel milk at different rates (1.5, 2.0, and 2.5%) along with different incubation periods (12, 24, 36, and 48h). However, after 48h of fermentation with a 2.5% rate of inoculum, the highest proteolytic activity was obtained. Reverse phase high-pressure liquid chromatography (RP-HPLC) was used to calculate the % Rpa from permeates of 3kDa and 10kDa fractions. Molecular weight distributions of fermented and unfermented camel milk protein fractions were compared using SDS-PAGE. Spots obtained from 2D gel electrophoresis were separated on the basis of pH and molecular weight. Spots obtained from 2D gel were digested with trypsin, and the digested samples were subjected to RP-LC/MS for the generation of peptide sequences. The inhibition of tumour necrosis factor alpha, interleukin-6, and interleukin-1 during fermentation was studied using RAW 264.7 macrophages. In the study, fermented camel milk with KGL4 (CMKGL4) inhibited LPS-induced nitric oxide (NO) production and pro-inflammatory cytokine production (TNF-α, IL-6, and IL-1β) by the murine macrophages. The results showed that the peptide structures (YLEELHRLNK and YLQELYPHSSLKVRPILK) exhibited considerable binding affinity against hPAM and hMGA during molecular interaction studies.

Hindakia tetrachotoma ME03 (Chlorophyta) has high phenolic content, antioxidant capacity, and attenuates H2O2-induced oxidative stress and apoptosis in human cells

ABSTRACT Oxidative stress damages cellular components and may lead to cell death when it is not effectively counterbalanced by the endogenous antioxidant mechanisms. Exogenous sources of antioxidants are also necessary to cope with high levels of free radicals. Microalgae are rich sources of diverse metabolites with high antioxidant potential. In this study, metabolites of Hindakia tetrachotoma ME03, a green microalga previously isolated from thermal springs in Türkiye, were chemically fractionated using four solvents (ethanol-water, ethyl acetate, hexane and water). The extracts were evaluated for their antioxidant capacities, total phenolics, flavonoids and carotenoids. The highest antioxidant capacity, phenolic and carotenoid contents were obtained in the ethanol-water extracts. The antioxidant capacity in the ethanol-water extracts was measured as 12.42 ± 1.21 μmol Trolox g–1 DW and 67.98 ± 3.45 μmol Trolox g–1 DW by DPPH and FRAP assays, respectively. Twelve different phenolic compounds in the ethanol-water and ethyl acetate extracts were quantified by reverse phase high pressure liquid chromatography. Quercetin and rutin were the most abundant phenolics in the ethanol-water extracts, whereas the ethyl acetate extract was rich in phenolic precursor, benzoic acid. Finally, the cytoprotective effect of ethanol-water extracts of H. tetrachotoma ME03 against H2O2-induced oxidative damage was investigated using an in vitro cell line model of human breast adenocarcinoma (MCF7). Hindakia tetrachotoma ME03 extracts significantly decreased the intracellular reactive oxygen species levels and the percentage of apoptotic and necrotic cells in MCF7 cells. These findings suggest the strong cytoprotective activity of H. tetrachotoma ME03 extracts, which may be conferred by its antioxidative properties.

Simultaneous Quantification RP-HPLC Method Development and Validation for Atorvastatin and Tadalafil in Bulk Form

Background: Tadalafil and Atorvastatin together have been shown to significantly enhance hemodynamics and sexual performance in middle-aged and older patients with hyperlipidemia. Despite of the number of techniques for estimating tadalafil and atorvastatin calcium, the Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) technique for simultaneous determination of Atorvastatin calcium and Tadalafil has not yet been disclosed. Purpose: A sensitive, simple, rapid, reverse phase high pressure liquid chromatography (RP-HPLC) method has been developed and validated for the simultaneous estimation of Tadalafil and Atorvastatin in bulk form. Methods: The method was carried out utilizing the C-18 column (Sapphirus, 5 x 250 mm) and mobile phase at 1 ml/min flow rate. 0.05M phosphate buffer (30:70 v/v gradient mode) and acetonitrile were the ideal mobile phase conditions. The pH was changed with glacial acetic acid to 4.0. Results: Tadalafil and Atorvastatin had retention times of 3.517 and 4.117, respectively, according to data collected concurrently. The linearity range for Atorvastatin and Tadalafil (15 µg/ml to 90 µg/ml) was established using an external standard calibration method. It was calculated that the recovery rate ranges from 98.09 to 101.44 percent, and it was discovered that all values fall within this range. The limits of detection for Atorvastatin and Tadalafil are 0.318 µg/ml and 0.672 µg/ml, respectively. Conclusion: The method was found to be robust, durable, accurate and linear for validated parameters. The % RSD observed for flow rate, wavelength, and analyst variation was found to be below 2, which was considered acceptable in the robustness investigation.

Cucurbita maxima Seeds Reduce Anxiety and Depression and Improve Memory.

The current study was planned to assess the neuropharmacological benefits of the Cucurbita maxima seed. These seeds have been conventionally used for the nutritional as well as amelioration of various diseases. However, there was a need to provide a pharmacological basis for such use. Four central nervous system-related functions, that is, anxiety, depression, memory, and motor coordination, were evaluated, and the levels of brain biogenic amines were also assessed. Anxiety was evaluated through selected experimental models, such as light and dark apparatus, elevated plus maze, head dip, and open field test. The head dip test was mainly used to assess exploratory behavior. Depression was assessed by two animal models, that is, the forced swim test and tail suspension test. Memory and learning ability were assessed by the passive avoidance test, stationary rod apparatus, and Morris's water maze test. Motor skilled learning was assessed by stationary rod and rotarod apparatus. Reversed phase high-pressure liquid chromatography was used to determine biogenic amine levels. Results reveal that C. maxima exhibited anxiolytic and antidepressant effects with memory improvement. There was a reduction in the weight of the animal following chronic administration. Furthermore, no remarkable effects were observed on motor coordination. Norepinephrine was found elevated, which may be linked to its antidepressant effects. These biological effects of C. maxima may be due to the presence of secondary metabolites, such as cucurbitacin, beta-sitosterol, polyphenolic compounds, citrulline, kaempferol, arginine, β-carotene, quercetin, and other antioxidants. The outcomes of the present study authenticate that the chronic use of C. maxima seeds reduces the intensity of neurological problems like anxiety and depression.

Comprehensive Evaluation of HPLC-MS/MS Method for Quantitation of Seven Anticoagulant Rodenticides and Dicoumarol in Animal Serum.

Anticoagulant rodenticides (ARs) are commonly utilized for controlling rodent populations, however non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone, and warfarin) and dicoumarol (a naturally occurring anticoagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-pressure liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS) using electrospray ionization (negative mode) combined with multiple reaction monitoring (MRM). In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. Inter-assay accuracy ranged 99-104% and relative standard deviation ranged 3.5-20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future.

Isolation of herbicidal compounds, quercetin and β-caryophyllene, from Digera muricata

Synthetic herbicides are available to control weeds but these herbicides have many concerns. Eco-friendly herbicides obtained from plants is a better alternative to synthetic ones. Despite that the herbicidal activity of Digera muricata extracts have been reported but there are no studies regarding the isolation and identification of herbicidal compounds from D. muricata. Herein, we are reporting the identification of two herbicidal compounds from chloroform extract obtained from D. muricata. The chloroform extract was initially tested on the germination and early growth of two weeds; Avena fatua and Melilotus indicus as well as wheat where a significant decline in % age germination and growth of both weeds was observed. Among the 8 different fractions obtained using different chromatographic techniques, fractions 2 and 7 were found to be phytotoxic to both test weeds. The herbicidal efficacy was tested at 200, 150, 100, 50, 25 μg/ml. These two fractions were further purified on Reversed Phase High Pressure Liquid Chromatography (RP-HPLC). Fraction 2 yielded 3 sub-fractions (2A, 2B & 2C), while fraction 7 yielded 2 sub-fractions (7A, 7B). Fraction 2B caused 43%, 53%, and 57% decline in seed germination, shoot dry weight, and root dry weight of A. fatua, while these values against M. indicus were 50%, 81% and 84%, respectively. Fraction 7A caused 25%, 36%, and 42% decline in seed germination, shoot dry weight, and root dry weight of A. fatua, while these values against M. indicus were 35%, 62% and 69%, respectively, at 200 μg/ml conc. Cyanazine caused 61% and 50% reduction in seed germination of A. fatua and M. indicus, respectively. The herbicidal effects of these two fractions were found nonsignificant against wheat. Fourier Transform Infrared Spectroscopy (FT-IR), elemental analysis (C,H) and Nuclear Magnetic Resonanace (NMR) analyses of these two fractions depicted the presence of quercetin (Fraction 2B) and β-caryophyllene (Fraction 7A). In post emergence bioassays, the isolated compounds caused significant decrease in the biomass of both weeds. Plasma membrane integrity assays revealed electrolyte leakage in treated leaf discs of both weeds. It was concluded that quercetin and β-caryophyllene isolated from D. muricata exhibited toxicity towards both test weeds without harming wheat.

Multivariate Analysis and Response Surface Modeling to Green Analytical Chemistry-Based RP-HPLC-PDA Method for Chromatographic Analysis of Vildagliptin and Remogliflozin Etabonate.

The fixed-dose combination (FDC) of vildagliptin (VDG) and remogliflozin etabonate (RGE) is used as antidiabetic medicine. Numerous reverse phase high-pressure liquid chromatographic (RP-HPLC) methods have been reported for the estimation of VDG and RGE using toxic organic solvents such as acetonitrile and methanol. These organic solvents are also hazardous to the environment. Hence, the robust and green analytical chemistry-based RP-HPLC-PDA method has been developed for chromatographic analysis of VDG and RGE for the safety of analysts and protection of the environment. The multivariate analysis has been carried out for the identification of critical method risk parameters (CMRPs) and critical method performance attributes (CMPAs) using principal component analysis (PCA). The identified CMRPs and CMPAs were linked with each other for optimization of the RP-HPLC-PDA method using DoE-based response surface modeling. The analytical design space (ADS) has been explored for robust chromatographic analysis of VDG and RGE. The chromatographic analysis of VDG and RGE has been carried out using Shim-Pack C18 column (250 mm L, 4.6 mm ID, 5.0 µm PS) and isopropyl alcohol-0.1% (v/v) formic acid (FA) in water (45 + 55, v/v, pH -3.5). The developed method has been validated in accordance with ICH Q2 (R1) guidelines. The method has been applied for the assay of VDG and RGE in their FDCs. The results of the assay were found in compliance with the labeled claims. The developed RP-HPLC-PDA method did not include any toxic or carcinogenic solvents. Hence, it is safe for analysts and the environment. The greenness profiles of the published and proposed RP-HPLC methods were evaluated by the national environmental method index (NEMI) scale, and the analytical greenness scores were calculated using the AGREE software. The developed method can be used as an eco-friendly tool in the pharmaceutical industry for routine analysis and quality control of FDCs of VDG and RGE. Development of a green and robust RP-HPLC method for the estimation of VDG and RGE using safe organic solvents. The analytical quality by design (AQbD) approach has been implemented in the development of a method to minimize solvent wastage. The method was applied for the assay of FDCs of VDG and RGE.

Identification and determination α and β-acids from Columbus hops using Reverse Phase High Pressure Liquid Chromatography

The present study has demonstrated the HPLC method for identification and determination a and b-acids in Columbus hops. the measurement conditions has been set up included the use of Reverse phase C18 column Chromolith, gradient elution with methanol (A) and Acetonitril acidified with phosphate buffer at pH 2.8. While the flow rate has been applied of 0.8 mL/min, and it is run for 25 minutes. The separation of standard and sample has been monitored using UV variable detector in the rang of 300-340 nm. Pretreatment sample has been done using SPE with the cartridge of C18 similar to the analyte column. The results found that a four predominant peaks for α and β-acids, namely cohumulone (6.6 min), the second peak represents a mixture of humulone and adhumulone (8.9 min), the third peak is colupulone (15.9 min) and the fourth is a mixture of lupulone and adlupulone (16.7 min). The quantity of sample has been determined according to multipoint calibration using ICE2 standard, the sample found are 0,209 g/L cohumulone, 1.087 g/L of humulone and adhumulone, 0,349 g/L of Colupulone and 0.630 g/L of mixture of lupulone and adlupulone. In addition it is high precision measurement that is indicated by the value of RSD less than 1%.

Antioxidant, Anti-Obesity, and Hypolipidemic Effects of Polyphenol Rich Star Anise (Illicium verum) Tea in High-Fat-Sugar Diet-Induced Obesity Rat Model.

Star anise (Illicium verum Hook. fil.) is commonly utilized as a culinary and medicinal fruit and is most famous in indigenous systems of medicine. The present research work aims to appraise and validate the potential of polyphenol-rich star anise tea (SAT) on oxidative stress, obesity and related biochemical parameters in high-fat-sugar-diet (HFSD)-induced obesity model in rats. SAT was prepared using the traditional method in warm water. The Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) analysis was performed for the simultaneous determination of phenolic acids and flavonoids in SAT. Two doses (250 and 500 mg/kg body weight) were selected to investigate the anti-obesity potential of SAT using HFSD-induced obese rat model. Major (>5 mg/100 mL) phenolic acids in SAT were p-coumeric acid, gallic aid, cinamic acid, chlorogenic acid and ferulic acid while catechin and rutin were the major flavonoids detected in the SAT. SAT exhibited 51.3% DPPH radical scavenging activity. In vivo study showed that higher doses of SAT (500 mg/kg body weight) significantly reduced the body weight increase (74.82%) and BMI (0.64 g/cm2). Moreover, significant reductions in the levels of serum total cholesterol, triglyceride, LDL and VLDL were recorded in all the treatment groups in comparison to the HFSDC group. Furthermore, SAT reduced the alterations in MDA, SOD and GSH levels of experimental groups thus showing the potential against oxidative stress. The SAT-500 group showed a significant decrease in the elevated kidney and liver weights and atherogenic index in comparison to the HFSDC group. The present study proved that SAT exhibited strong protective effects against obesity and oxidative stress, especially at higher doses.

Analytical method development and validation of related substances by rphplc of sofosbuvir and velpatasvir tablets

The developed method was a simple, accurate, precise, specific and robust method for the validation of Sofosbuvir and Velpatasvir Tablets by reverse phase high pressure liquid chromatography. For Sofosbuvir and Velpatasvir Tablets Chromatography was performed on Agilent 1200 series, UV and PDA Detector, Waters X-bridge C18 (150 mm x 4.6 mm, 3.5 µm) by using Mobile Phase A contains Buffer solution (0.6% Trifluroacetic acid in water adjusted pH to 2.2±0.05) : Acetonitrile (95:5)%v/v and Mobile Phase B contains mixture of purified water, methanol and acetonitrile in the ratio of (20: 30: 50) % v/v/v. at a flow rate of 1.0 mL/min and at 263 nm for Sofosbuvir and 320 nm for Velpatasvir wavelength. The retention times of About 48.0 minutes for Sofosbuvir and About 78.8 minutes for Velpatasvir respectively. Methyl Uridine and Impurity at RRT 0.39 found linear over the range of LOQ - 150 % of target concentration. Method also found precise by spiking impurities at specification level. Accuracy was demonstrate at LOQ - 150 % level by preparing sample in triplicate for each level and found accurate. Hence, the method could be successfully used for the analysis Impurities in Sofosbuvir and Velpatasvir Tablets Tablet.

An Integrated Chromatographic Strategy for the Large-Scale Extraction of Ergosterol from Tulasnellaceae sp.

A reliable chromatographic strategy is crucial for the extraction of target compounds from natural sources as it is related to the preparation efficiency, as well as the purity of the compounds. In this study, medium-pressure normal-phase liquid chromatography and high-pressure reverse-phase liquid chromatography were combined to prepare and purify ergosterol from Tulasnellaceae sp. of Gymnadenia orchidis. First, Tulasnellaceae sp. was extracted three times (2.0 L and 2 h each time) with ethyl acetate, and the 6.0 L of extract solution was concentrated under reduced pressure to yield 2.2 g of crude sample. Then, the crude sample was pretreated utilizing silica gel medium-pressure liquid chromatography to enrich the target ingredient (586.0 mg). Finally, high-pressure reversed-phase liquid chromatography was used to purify the target compound, and the compound was characterized as ergosterol (purity > 95%) using spectral data. Overall, the simple and reproducible integrated chromatographic strategy developed in this study has the potential for the large-scale purification of steroids for laboratory and even industrial research. To the best of our knowledge, this is also the first report of ergosterol in Tulasnellaceae sp.

Distribution of astaxanthin in the spiny lobster Jasus lalandii: trends during biological cycles

ABSTRACT Astaxanthin, the dominant carotenoid pigment in the South African spiny lobster, Jasus lalandii, was quantified in haemolymph and tissue extracts by means of reverse phase-high pressure liquid chromatography and analysed with respect to growth and reproduction cycles of adults. Haemolymph, exoskeleton, muscle, gonads and hepatopancreas from both sexes, as well as egg parcels from berried females were collected within days of removing the animals from the ocean. The astaxanthin profile is, therefore, representative of spiny lobsters in the wild. Astaxanthin is significantly more in exoskeleton, ovaries and egg parcels, and is influenced by the ovarian cycle in females: it accumulated in growing oocytes and remained in the extruded eggs ostensibly for protection as antioxidant. Radioactive inulin was used to determine total haemolymph volume of a spiny lobster and the gravimetric contribution of body organs and various tissues to the total weight of these animals were measured: muscle tissue constitutes 33% of the total wet weight of J. lalandii, while haemolymph (22%) and exoskeleton (20%) are other major contributors. For maximal harvesting of astaxanthin from carcases, it would thus, be best to focus only on the exoskeleton with an emphasis on the carapace, which can be easily removed.

Ultra-fast microscale purification of chiral racemates and achiral pharmaceuticals with analytical supercritical fluid chromatography

Improving efficiency while maintaining high quality separations is a central theme for specialized analytical/purification groups supporting discovery chemistry programs. Supercritical fluid chromatography (SFC) has become the prevalent technique for chiral separation and a complementary technique to reverse phase high-pressure liquid chromatography (RP-HPLC). In this manuscript we demonstrate the successful micro-isolation of chiral racemates, small molecules, and peptides using a sub-minute method on an analytical SFC system. The addition of a custom gas liquid separator (GLS) and alterations to the fluidic pathways allow the fractionation of desired products on a micro-scale SFC platform, providing analytical method development, purifications, and purity confirmation on a single SFC system. This enables micro-purification of pharmaceuticals including chiral racemates at high speed and reduced cost of materials. The resulting small-quantity, high-purity products enable follow-up enantioselective isolations from racemic products of parallel synthesis libraries. The processes established here will be beneficial for the isolations of other desired products in complex crude mixtures.

SIMPLE QUANTIFIED AND VALIDATED STABILITY INDICATING STRESS DEGRADATION STUDIES OF ORAL ANTI-DIABETIC AGENT DAPAGLIFLOZIN BY RP-HPLC METHOD

Objective: This method is focused on developing a precisely simplified and more accurate Reverse Phase–High Pressure Liquid Chromatography (RP-HPLC) method for the determination of Dapagliflozin in bulk and pharmaceutical dosage form as per guidelines of International Council for Harmonization (ICH). Methods: Evaluation and validation carried out using the RP-HPLC ZORBAX (C18) column (250 x 4.6 mm, 5 μm particle size) with a mobile phase consisting of Phosphate Buffer: Acetonitrile: Methanol in a ratio of 55:40:05 (v/v/v) at a flow rate of 1 ml/min with an injection volume of 10 μl. Results: Dapagliflozin was eluted at 2.12±0.05 min and detected at 225 nm. The regression equation y = 55762 x-29679 found to be linear with correlation coefficient r2 value of 0.9997. The developed RP-HPLC method was conveniently validated as per the ICH guidelines and found method was robust, sensitive, accurate, selective, specific, precise and linear. Conclusion: The proposed method was found to be accurate, precise, and robust for API and pharmaceutical dosage form as per experimentation analysis. The above developed method was found to be satisfied for Active Pharmaceutical Ingredient (API) and pharmaceutical formulation of Dapagliflozin to study its degradation products.

Analytical quality by design approach for the control of potentially counterfeit chloroquine with some NSAIDS using HPLC with fluorescence detection in pharmaceutical preparation and breast milk

AbstractChloroquine phosphate (CQ) the antimalarial drug and suggested to treat the pandemic disease coronavirus (COVID-19) is often adulterated with some of the non-steroidal anti-inflammatory drugs (NSAIDs) such as paracetamol, aspirin (ASP), or both. The purpose of this study is to detect such counterfeited drugs, using a reversed phase high pressure liquid chromatography (RP-HPLC) method with fluorescence detection. Analysis was divided into three phases. In the first phase, a Plackett-Burman design (PBD) was used to screen five independent factors, namely, buffer pH, buffer concentration (mM), acetonitrile content (%), flow rate (mL/min) and triethylamine (TEA) content in the buffer preparation (%). The selected dependent variables were (resolution, symmetry of peaks and run time). The objective of the second phase was to optimize the method performance using Box-Behnken design (BBD) and desirability function for multiple response optimization to obtain the best chromatographic performance with the shortest run time. Optimal chromatographic separation was achieved on a YMC-pack pro C18 ODS-A column (15 cm × 4.6 mm, 5 µm) at room temperature The optimum mobile phase consisted of acetonitrile and 5 mM sodium dihydrogen phosphate buffer containing 0.5% triethyamine (30:70, v/v) with the pH adjusted to 3.5 using an orthophosphoric acid solution. The flow rate was maintained at 1 mL/min, and the detection was performed with a fluorescence detector fixed at 380 nm (λemission) after excitation at 335 nm (λexcitation). The third phase was method validation according to ICH guidelines, providing to be specific, precise, accurate, and robust. The method is linear over a range of 0.4–8 µg/mL for chloroquine and ASP, while for paracetamol it is linear over 16–48 µg/mL. The developed RP-HPLC method was used for quantitation of the three drugs in chloroquine dosage form samples. The method shows a great tendency in the classification between the genuine chloroquine and the adulterated ones in pharmaceutical preparations and breast milk.

Gluten Protein Compositional Changes in Response to Nitrogen Application Rate

Field trials were conducted to evaluate the effect of nitrogen level on wheat protein content and composition in 16 cultivars over two years at three locations. The nitrogen treatment comprised two nitrogen levels, 0 kg ha−1 as low and 100 kg ha−1 as high nitrogen, applied as top dressings of 50 kg nitrogen per ha at tillering and stem extension growth stages. Increased nitrogen level generally enhanced grain protein by 11.3% (11.5% vs. 12.8%). Considering protein composition determined by reversed phase–high-pressure liquid chromatography, higher nitrogen supply generally enhanced the proportion of total gliadins, α-gliadins, γ-gliadins and high-molecular-weight glutenin subunits by 1.1%, 2.0%, 3.7%, 0.6% and 0.9%, respectively, and reduced albumins and globulins, ω-gliadins, total glutenins and low-molecular-weight glutenin subunits by 1.1%, 1.7%, 1.9% and 3.2%. Under a high nitrogen level, the historical cultivars Libellula, San Pastore and U-1 had a higher protein content (13.1–15.2%) with significantly higher total gliadins, which resulted in a significantly higher gliadin/glutenin ratio (1.68–1.92). In the modern cultivars, protein content varied between 11.4% and 14.6% with a well-balanced gliadin/glutenin ratio (1.08–1.50), except for cultivar MV Nemere which had a high gliadin/glutenin ratio at both nitrogen levels (1.81 vs. 1.87). In summary, increasing nitrogen level enhanced grain protein content while the composition of gliadin and glutenin fractions was changed to a lesser extent and was largely cultivar specific and therefore should be considered for wheat baking quality assessment and breeding purposes.

Extracted Trans-Resveratrol from Arachis hypogaea Enhances Expression of Sirtuin Gene and Replicative Life Span in Saccharomyces cerevisiae

Aims: Biotic stress given by Aspergillus niger enhances trans-resveratrol production in Arachis hypogaea plant. This plant extract increases sir2 gene expression and Replicative Life Span in Saccharomyces cerevisiae.
 Design of Study: Peanut plant was grown in aseptic environment, infected by Aspergillus niger. Plant extract used for quantification of trans-resveratrol by RP-HPLC. Yeast culture was grown in Potato dextrose media along with plant extract. Sir2 gene expression fold calculated by real time pcr. Replicative Life Span of yeast was measured by spectrophotometer.
 Place and Duration of Study: Allele Life Sciences Pvt. Ltd., Department of Biotechnology between February 2017 to March 2020.
 Methodology: Biotic stress in Arachis hypogaea plant was induced by wounding the leaves and introducing Aspergillus niger to enhance trans-resveratrol production. Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). Two methods conducted to check reverse ageing, first one epigenetic based, when extracted trans-resveratrol from infected Arachis hypogaea plant extract added to Saccharomyces cerevisiae culture, it enhanced expression of Sir2 gene in Saccharomyces cerevisiae measured by qPCR, ABI applied biosystem. Process included RNA isolation, cDNA synthesis and thereafter qPCR. Enhanced expression of sirtuin responsible for gene silencing as sirtuin (Sir2 gene product) is a class of Histone deacetylase transferase enzyme. Second method, Replicative Life Span of Saccharomyces cerevisiae culture increased when Aspergillus niger infected peanut plant extract added to yeast culture which was measured through spectrophotometer at 600nm and showed high absorbance value.
 Results: Tran-resveratrol was quantified by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) and yield was 2.24 mg/g. Sir2 gene expression increased by 1.56 fold in yeast grown in infected peanut plant extract. Absorbance of yeast culture grown in infected peanut plant extract was 0.522±0.008 which was higher than control.
 Conclusion: Sir2 gene expression enhances along with replicative life span in yeast in presence of peanut plant extract.

Procyanidin-Rich Extract from Grape Seeds as a Putative Tool against Helicobacter pylori.

Strains of Helicobacter pylori (H. pylori) resistant to various antibiotics have increased in recent years. In this context, the search for new therapeutic approaches is crucial. The aim of the present study was to demonstrate the antibacterial activity of a procyanidin-rich extract obtained from food-grade winery grape seeds against 14 H. pylori strains and elucidate its phenolic composition. Ten strains (71.4%) showed resistance to at least some of the tested antibiotics, while four isolates (28.6%) were susceptible to all antibiotics. Resistance to more than one class of antibiotics was observed in six strains (42.9%). The extract was able to inhibit the growth of all H. pylori strains in a range of a minimum inhibitory concentration (MIC) from 0.015 mg/mL to 0.125 mg/mL, confirming also the existence of a strain-dependent effect. The phenolic composition determined by reverse phase high pressure liquid chromatography, photodiode array, and mass spectrometry detection (RP-HPLC-PAD-MS) analysis revealed the presence of 43 individual compounds and allowed the quantification of 41 of them, including seven procyanidin tetramers, seven procyanidin pentamers, and six galloylated procyanidin dimers, trimers, and tetramers. The extract was composed mainly by catechin and procyanidin oligomers with a total amount of 5801 mg/100 g, which represent 92% of the total individual phenolic content. Among them, the most abundant were catechins (2047 mg/100 g), followed by procyanidin dimers (1550 mg/100 g), trimers (1176 mg/100 g), tetramers (436 mg/100 g), and pentamers (296 mg/100 g) that represent 35, 27, 20, 8, and 5%, respectively of the total flavanol constituents. The composition profile information may help to improve the production process of useful antibacterial extracts against H. pylori.

The Effect of Acetic Acid and Acetate Ions on Sorption–Desorption of a Mixture of Phenolic Acids by Modified Kaolinite

The sorption and desorption of a mixture of phenolic acids by the kaolinite modified with aluminum hydroxide was studied in the presence of acetate buffer. The experiments were performed in a 1-mL flow-through microcolumn in 5 and 50 mM sodium acetate buffer (pH 4.5) at a flow rate of 0.5 mL/min. The total concentration of phenolic acids was 0.01 mg/mL (0.06 mM); the concentration of each acid was 0.01 mM. Sodium acetate buffer (5 and 50 mM, pH 4.5) and 50 mM sodium acetate buffer supplemented with 0.1 mM oxalic acid were used for desorption. The concentration of phenolic acids was determined by reverse phase high-pressure liquid chromatography. The following order of sorption was observed: gallic > protocatechuic $$ \gg $$ p-hydroxybenzoic ~ vanillic ~ ferulic ~ syringic acid. The sorption of phenolic acids in 50 mM buffer comprised 18–35% of their sorption in 5 mM buffer, suggesting the competition of acetate ions and phenolic acids for binding sites on the mineral. The order of desorption of phenolic acids was opposite to the order of sorption and generally correlated with the stability constants of the phenolic acid complexes with aluminum hydroxide. All acids, except for gallic and protocatechuic acids, were weakly bound to the mineral and were almost completely (88–98%) desorbed with 5 mM acetate buffer. The total desorption of gallic and protocatechuic acids by all eluents comprised 25 and 45% of their sorbed amount, respectively. Desorption was significant only in 50 mM acetate buffer (12 and 23%) and in the same buffer with 0.1 mM oxalic acid (10 and 15%). Thus, it has been shown that the distribution of phenolic acids between the solid phase and solution is largely determined by the presence of competing aliphatic compounds. Based on desorption experiments, we propose possible types of complexes of phenolic acids and aluminum hydroxide on the surface of the mineral.