HPLC Peak Research Articles

Multifaceted toxicity assessment of Au, Ag, and TiO2 nanoparticles synthesized by quorum quenching bacterium Salmonella bongori: Impact on bacterial pathogens, cancer cells, mosquitoes, zebrafish, and brine shrimp

The present study pertained to evaluate biological properties of the nanoparticles synthesised using quorum quenching-QQ Salmonella bongori-SB. The QQ potential of SB was confirmed through streaking (halos) and diffusing cell-free-supernatant (decolored) against Chromobacterium violaceum-CV and significant antagonistic effect on Pseudomonas aeruginosa-PA and P. fluorescens-PF. HPLC peaks confirmed the degradation of 3-oxo-C12-HSL (3.185) by QQ-SB at 2.579 and 2.924. The morphological characteristics-(crystallite size; shape with size range), and significant antibacterial-ABA, antibiofilm-ABFA and anti-proliferative-APA (with IC50 values on HeLa and A549 cells) activities of different-NPs were recorded as follows: SB-AuNPs (31.61 nm; aggregated spherical (13.66–18.38 nm)) resulted in good ABA (on PF and Serratia marcescens), ABFA (on PF) and APA (IC50: 33.79 and 33.74 µgmL−1). Similarly, SB-AgNPs (19.83 nm; clustered spherical; 4.81–8.58 nm) showed the most-significant ABA (CV and PF), ABFA (PA, CV and PF) and APA (IC50: 24.57, and 22.7 µgmL−1). Whereas, SB-TiO2NPs (47.33 nm; spherical with 75.13 nm) did not produced significant activities. Overall, the first report on the QQ potential of SB and its NPs emerged to be good candidate that might combat the pragmatic difficulties against MDR strains and drug failures in cancer treatments. Therefore, standardizing its applications in quorum sensing inhibitory mechanisms and anti-proliferative effects is warranted.

Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.

The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Anti-microbial property of Trigonella foenum-graecum L. Methanol Extract [TFGME] on Pathogenic organisms

Methanol extract of Trigonella foenum-graecum L. and its role on pathogenic micro-organisms is the scope of this study. Initially, Trigonella foenum-graecum L. was treated with methanol and the obtained extract was termed as Trigonella foenum-graecum L. Methanol Extract [TFGME]. TFGME shows the presence of Polyphenols, glycosides, phytosterol, saponins and etc., when it subjected to initial preliminary screening. HPLC analysis of TFGME suggests that the presence of several organic molecules as it elutes 9 major peaks after the solvent peak in reverse-phase HPLC at 216nm. In addition, TFGME exhibit anti-microbial property by exhibiting zone of inhibition when it was incubated with pathogenic organisms such as E. coli, S. aureua, Pseudomonas, Salmonela and shigella. The obtained results were recorded in MIC (Minimum Inhibition Concentration) of TFGME values. Furthermore, when TFGME was subjected to analysis of minerals content by using ICP-OES instrument, it was confirmed that TFGME contains aluminium, copper, iron, manganese and zinc in the extract.

Phenolic profile, color parameters and antioxidant activity of walnut kernel extracts as influenced by different time and temperature during extraction

AbstractThe aim of the present study was to find the best extraction parameters to obtain the highest amounts of polyphenols and antioxidants from the walnut. Walnut kernels from ‘Alsószentiváni 117’ cultivar were used for extraction. The extraction methods were as the follows:Method 1: shaking water-bath at 50 °C for 30 min.Method 2: shaking water-bath at 50 °C for 30 min, then storing at 5 °C for 20 h.Method 3: shaking water-bath at 40 °C for 30 min.Method 4: shaking water-bath at 40 °C for 30 min, then storing at 5 °C for 20 h.According to our results Method 1 showed the highest FRAP value (34.43 mg AAE g−1), the DPPH value (52,94%) and the highest HPLC peaks for chlorogenic acid, epicatechin and rutin were also seen in extracts obtained using Method 1. TPC values of Method 3 were 26.06 mg GAE g−1 for Method 1 it was 25.65 mg GAE g−1. The results of color values, L* and ΔE* were similar in all extracts as well. In our experiments extraction Method 1 proved to be better than others.

Magnetic nanofluid based on hydrophobic deep eutectic solvent for efficient and rapid enrichment and subsequent determination of cinnamic acid in juice samples: Vortex-assisted liquid-phase microextraction

A quick, environmentally friendly and easy approach for the determination of cinnamic acid in juice samples based on the creation and usage of a novel magnetic nanofluid (mixture of hydrophobic deep eutectic solvent and magnetic nanoparticles) has been reported. Response surface methodology was applied to justify the contribution of the efficient factors including pH, nanofluid volume, ionic strength and vortex time. Cinnamic acid concentrations were monitored and quantified based on their HPLC peak representing linear correlations under the best operational circumstances showing linearity between 3 and 550 ng mL−1. The LOD, LOQ, and enrichment factor for cinnamic acid were 0.8 ng mL−1, 2.7 ng mL−1 and 57.2, respectively. The proposed method was used for enrichment and subsequent determination of cinnamic acid from juice samples which suggests a potential alternative approach for cinnamic acid analysis in complicated food samples.

Inhibitory effect of Monascus purpureus pigment extracts against fungi and mechanism of action

The fungus Monascus produces several secondary metabolites of different pigment hues. These pigments have shown various biological activities. In this study, Monascus purpureus pigment extracts were tested (in vitro) against Penicillium expansum MTCC 4900, Rhizopus stolinfer MTCC 10595, and Aspergillus niger MTCC 8652 for antifungal activity. The UV–visible spectrum of M. purpureus fermented rice extracts showed λmax at 395, 425, and 500 nm. This indicated the solubility of yellow, orange, and red pigments in polar-based solvent extraction. The M. purpureus pigment extracts inhibited the radial growth and conidial germination of the test fungi. The fungi treated with pigment extract stained with DiBAC (a vital stain) emitted green fluorescence under a fluorescent microscope. These results indicated that the pigment extracts have affected the membrane potential of the treated fungi. Hence, the fungicidal activity of the pigment extracts is due to the disruption of the cell membrane. The HPLC analysis of the pigment revealed the presence of two major peaks. The UV–visible spectrum corresponding to the HPLC peak at 12-min retention time revealed the presence of orange pigment rubropunctatin. Apparently the rubropunctatin present in the extracts exhibited fungicidal activity. Further studies are warranted to assess the applications of M. purpureus pigments in preventing and treating fungus-related diseases.

A Method for Converting HPLC Peak Area from Online Reaction Monitoring to Concentration Using Nonlinear Regression

Online HPLC reaction progress monitoring provides detailed data-rich profiles; however, extracting kinetic information requires ultraviolet-visible response factors to determine concentrations from peak areas. If the reaction's overall mass balance is known and some analytical trend for all relevant species can be recorded, it is possible to estimate the absolute response factors of all species using a system of linear equations. We delineate a method using the Microsoft Solver plug-in to convert time course profiles to reagent concentrations without analytical standards.

Promising antimicrobial protein from <em>Klebsiella</em>

Increasing antibody resistance necessitates the use of new antimicrobial products. Antimicrobial peptides (AMP`s) And Proteins are a promising alternative to antibiotics currently being used due to their high degree of specificity & wide range of action Here we report a novel antimicrobial protein from iron reducing bacteria isolated from the coffee estate soils of Coorg district, Karnataka, India. Iron reducing bacteria are known to be antagonistic to others due to the presence of Pseuderophores. It makes them a good source of antimicrobial active principles. Fifteen isolates were obtained by selectively screening for iron reduction. These isolates were tested for antimicrobial activity against Gram positive [Staphylococcus aureus (MTCC3160), Bacillus cereus (MTCC6620)] & Gram negative [Escherichia coli (MTCC1650), Salmonella typhi (MTCC3224) and Pseudomonas aeruginosa (MTCC2453)] bacteria. Of the 15 isolates, one isolate, CBI-8, which showed good antimicrobial activity, was selected for further studies. The isolate was identified by MALDI-TOF & 16S rDNA sequencing as Klebsiella variicola/quasipneumonae (strain IS93) (genebank - MN814029). To induce and augment the production of antimicrobial molecules, CBI-8 was grown in the presence of Salmonella typhi and the spent medium was subjected to ammonium sulphate precipitation. The fractions which showed promising activity were further subjected to preparatory HPLC. The distinct bands obtained in the SDS-PAGE corresponding to the HPLC peak that showed antimicrobial activity from the induced sample was analyzed by Mass Spectrometry. Antimicrobial Peptide Database (APD3) calculator, Collection of Anti-Microbial Peptides (CAMPR3) software used to predict AMPs in the proteins identified from MS data.

Abstract 10726: Cardiomyocyte Targeting Peptide to Deliver Amiodarone

Introduction: Amiodarone remains underutilized as an anti-arrhythmic due to significant off-target toxicities with long-term use. We hypothesized that the required dose could be minimized by targeting amiodarone to cardiomyocytes by utilizing a cardiomyocyte targeting peptide (CTP). Methods: CTP (APWHLSSQYSRT) was conjugated to amiodarone via a disulfide bond, HPLC purified, characterized by MALDI/TOF, and stability at 37°C assessed using serial HPLCs over 21 days. Adult, male guinea pigs (GP; n=4/group) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), 1/10 th the molar dose of CTP-amiodarone (5 days), or CTP (5 days), followed by euthanasia, hearts excised, perfused on a Langendorff apparatus with Tyrode’s solution, and placed in a custom-designed chamber blebbistatin (5 μM) to minimize contractions. Bolus injections of voltage (RH237) and Ca 2+ -indicator dye (Rhod-2/AM) were injected to simultaneously map cardiac action potentials (APs) and Ca 2+ transients (CaT), at 610-750nm and 570-595nm, respectively with 2 CMOS cameras, followed by pacing at 250ms to measure conduction velocity (CV), AP and CaT durations (at 90% recovery). Results: CTP-amiodarone produced a single HPLC peak at 16mins that remained stable for 21 days. CTP-amiodarone and amiodarone decreased conduction velocities in myocytes significantly compared to control GPs (0.92±0.05, 0.87±0.08ms, vs. 1.00±0.03m/s respectively, p<0.001). Amiodarone increased AP duration (192±5ms vs. 175±8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157±16ms, p=0.0136), similar to CTP alone (155±13, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased CaTs compared to vehicle treated GPs. CTP-amiodarone and CTP decreased CaTs to an extent greater than amiodarone alone (p<0.001). Conclusions: Our data suggests that amiodarone can be stably covalently linked to CTP, which is able to deliver amiodarone to cardiomyocytes in vivo in GPs, at 1/10 th the dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP alone to decrease AP durations and CaTs was a surprise finding and merits further study to fully characterize the anti-arrhythmic actions of the CTP-amiodarone conjugate.

Fruit peels that unlock curative potential: Determination of biomedical application and bioactive compounds

• DMSO fruit peel extract of Citrus aurantium possess excellent antimicrobial activity. • This is the first study to report the MIC,MBC/MFC antimicrobial activities of Citrus aurantium . • Flavonoids, tannins, and phenolic compounds were present only in DMSO fruit peel extract of Citrus aurantium . • Eleven main peaks in HPLC and twelve compounds in GC–MS were detected. Control of multi drug resistant bacteria and their infection rate is a threat for biomedical and clinical research. Citrus fruits have proven effective against incurable bacterial infections suggesting alternate therapeutics to replace antibiotics in the near future. The antimicrobial potential of bitter orange ( Citrus aurantium ), citron ( Citrus medica ), and wood apple ( Limonia acidissima ) fruit peel extracts against pathogenic microorganisms was focused in this study. The antimicrobial activities and minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values were determined using the agar well diffusion and micro dilution method respectively against Gram-positive bacteria ( Staphylococcus aureus and Staphylococcus epidermis), Gram-negative bacteria ( Pseudomonas aeruginosa and Klebsiella pneumoniae ), as well fungi ( Candida albicans and Aspergillus niger ). HPLC, GC-MS, and FT-IR analyses were used to identify phytochemicals and functional groups in addition to cytotoxicity investigations using MTT assay. DMSO peel extract of C. aurantium has shown superior antimicrobial activity against the studied pathogens, particularly S. aureus (33 mm), followed by C. medica (DMSO extract – inhibition zone of 26 mm) against S. aureus . While the methanolic extract of C. aurantium had the lowest inhibitory zone against S. epidermis (12 mm). The MIC varied from 0.39 µg/mL to 6.24 µg/mL, while the MBC/MFC ranged from 0.78 µg/mL to 12.5 µg/mL.In all samples of studied fruit peel extracts, the phytochemical analysis revealed the presence of alkaloids, steroids, saponins, flavonoids, tannins, terpenes, phenolics, and cardiac glycoside. Only the DMSO extract of C. aurantium contained flavonoids, tannins, and phenolic compounds. The DMSO peel extract of C. aurantium exhibited eleven main peaks in HPLC and GC-MS analysis, revealing twelve chemicals in total. The FT-IR spectrum was used to determine the chemical composition of the peel extract. The DMSO peel extract of C. aurantium outperformed other extracts and even conventional medications in terms of antimicrobial activity. The results prove that this plant may be evaluated further to uncover active biomolecules in the development of antimicrobial therapeutics.

Isolation of Echimidine and Its C-7 Isomers from Echium plantagineum L. and Their Hepatotoxic Effect on Rat Hepatocytes.

Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L., a plant domesticated in many countries. Because of echimidine’s toxicity, this alkaloid has become a target of the European Food Safety Authority regulations, especially in regard to honey contamination. In this study, we determined by NMR spectroscopy that the main HPLC peak purified from zinc reduced plant extract with an MS [M + H]+ signal at m/z 398 corresponding to echimidine (1), and in fact also represents an isomeric echihumiline (2). A third isomer present in the smallest amount and barely resolved by HPLC from co-eluting (1) and (2) was identified as hydroxymyoscorpine (3). Before the zinc reduction, alkaloids (1) and (2) were present mostly (90%) in the form of an N-oxide, which formed a single peak in HPLC. This is the first report of finding echihumiline and hydroxymyoscorpine in E. plantagineum. Retroanalysis of our samples of E. plantagineum collected in New Zealand, Argentina and the USA confirmed similar co-occurrence of the three isomeric alkaloids. In rat hepatocyte primary culture cells, the alkaloids at 3 to 300 µg/mL caused concentration-dependent inhibition of hepatocyte viability with mean IC50 values ranging from 9.26 to 14.14 µg/mL. Our discovery revealed that under standard HPLC acidic conditions, echimidine co-elutes with its isomers, echihumiline and to a lesser degree with hydroxymyoscorpine, obscuring real alkaloidal composition, which may have implications for human toxicity.

Determination of optical density (OD) of oligodeoxynucleotide from HPLC peak area.

Oligodeoxynucleotides (ODNs) are typically purified and analysed with HPLC equipped with a UV-Vis detector. Quantities of ODNs are usually determined using a UV-Vis spectrometer separately after HPLC, and are reported as optical density at 260 nm (OD260). Here, we describe a method for direct determination of OD260 of ODNs using the area of the peaks in HPLC profiles. It is expected that the method will save significant time for researchers in the area of nucleic acid research, and minimize the loss of oligonucleotide samples.

Separation of eight phenolic compounds from the over-ground parts of Aconitum pendulum Busch by repeated injection high-speed counter-current chromatography

ABSTRACT In this study, eight phenolic compounds were obtained from the over-ground parts of the Aconitum pendulum (A. pendulum) Busch through an efficient high-speed counter-current chromatography （HSCCC）method. First, the column chromatography for macroporous resin was used for the treatment of crude extract of over-ground parts of A. pendulum Busch. And then, 30% ethanol elution fraction was transferred to HSCCC for separation using the classic ethyl acetate, n-butanol and water system with the solvent ratio of 7:3:10. To improve parting efficiency, a mode of repeated injection was developed. Two groups of compounds existing in one HPLC peak were effectively separated by HSCCC. The results revealed that HSCCC could effectively isolate compounds with the same or similar polarity. Preparative HPLC was used to perform further purification, and then eight phenolic compounds were separated from the over-ground parts of the A. pendulum Busch for the first time. The current investigation successfully provides a reference for phytochemical research and, comprehensive development and utilization of A. pendulum Busch in the future. Furthermore, HSCCC efficiently separated compounds with similar polarity and its combination with preparative HPLC could play a great role in the separation of natural materials.

Identification of Dyes in Coptic Textiles from the Museum of Faculty of Archaeology, Cairo University

High Performance Liquid Chromatography coupled to a Diode-Array-Detector (HPLC-DAD) is used to investigate samples which were extracted from ancient Egyptian textiles (4th–5th c. AD) of the Museum of Faculty of Archaeology, Cairo University. Madder is identified in several samples. According to semi-quantitative results, which are obtained from HPLC peak areas measured at 254 nm, madder that is rich in purpurin and poor in alizarin is identified in samples which were treated (i) only with madder and (ii) with madder and either indigo/woad (Indigofera species and other/Isatis tinctoria L.) or weld (Reseda luteola L.). The madder dye used in these samples could have been originated from Rubia peregrina L. However, the possible use of Rubia tinctorum L. (or other plants of the Rubiaceae family) by the Egyptian dyers cannot be ruled out, particularly if methods were developed by the ancient dyers to affect and control the relative composition of madder dye. The HPLC peak area ratio of alizarin versus purpurin is very high (>2.2) for samples which were treated with madder (probably originated from R. tinctorum) and a tannin source. Finally, in some samples, only indigoid dyes (indigo/woad) are identified.

Comprehensive detection of 120 additives in food using nontargeted MS data acquisition.

The compliance assessment on the labeling of food additives is a hard job, because there are nearly thousand legal food additives can be used in food, and countless illegal additives must also deal with. This study developed a non-targeted data acquisition screening method based on liquid chromatography high resolution mass spectrometry (HRMS) in which a precursor ion and two product ions of each analyte are able to be recorded. The high throughput screening method worked as foodomics that characterized and identified every food components as long as they were ionized in terms of theory. The data acquisition method called data independent acquisition (DIA) was achieved by a full scan form m/z 70–1050, and then followed wide window fragmentations of product ions recording. A full scan and the followed fragmentations generated 21 spectra in 2.6 s contributed about 6 data points for a typical 0.2–0.3 min width peak in HPLC. A detection database list of 120 additives included 79 colorants, 13 sweeteners, 12 preservatives and 7 antioxidants was established. Thirty-three commercial samples including beverages, candies, and sauces were surveyed for testing additives. Sweeteners (rebaudioside A) and flavoring agents (malic acid and fumaric acid) were found the most under declared additives. HPLC column often do not provide adequate retention for highly polar compounds such as organic acids (flavoring agents). In this study they were coeluted, but were able to be separated and determined by HRMS worked as the secondary separation tool. The surveillance results showed there is still room for food manufacturers to improve the connection between their product information and consumers.

Determination of Mitotane (DDD) and Principal Metabolite by a Simple HPLC-UV Method and Its Validation in Human Plasma Samples

Mitotane (DDD) is prescribed in adrenocortical renal carcinoma. Its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues and from a toxicological point of view, is probably more interesting than the other metabolite dichlorodiphenylacetate (DDA). Therapeutic Drug Monitoring (TDM) of DDD plasma concentrations is required to combine therapeutic efficacy with acceptable toxicity. Therefore, we developed a simple and fast HPLC-UV method to monitor plasma concentrations after a liquid–liquid extraction of plasma calibration samples, quality controls, and anonymous plasma samples with unknown DDD and DDE concentrations. Samples were injected into an HPLC instrument and peaks of mitotane (DDD), DDE and aldrin (internal standard, IS) were resolved by a stationary phase C18 column (250 mm × 4.6 mm, 5 μm), maintained at 35 °C. Mobile phase, made by water/acetonitrile (10/90, v/v), was pumped at a flow of 1.0 mL/min, and absorbance was monitored at a wavelength of 226 nm. Average recovery was 95% for all analytes, and the method was linear for both DDD (r2 = 0.9988, range 1–50 mg/L) and DDE (r2 = 0.9964, range 1–40 mg/L). The values of limit of detection and quantitation were 0.102 and 0.310 mg/L for DDD and 0.036 and 0.108 mg/L for DDE, respectively. The retention time values of DDD, DDE and IS were 7.06, 9.42 and 12.60 min, respectively. The method was successfully validated according to FDA guidelines and finally adopted for routine TDM.

Stereorandomization as a Method to Probe Peptide Bioactivity

Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized (sr) peptides, containing up to billions of different stereoisomers, as well-defined single HPLC peaks, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane-disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane-disruptive, and antibiofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogues of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane-disruptive and lipopolysaccharide-neutralizing activity, pointing to the existence of additional targets.

Anti-biofilm effect of Nerium oleander essential oils against biofilm forming Pseudomonas aeruginosa on urinary tract infections

Based on the untreated biofilm formation in urinary tract infections (UTIs), current study was focused on the inhibition of Pseudomonas aeruginosa (P. aeruginosa) biofilm formation using Nerium oleander (N. oleander) essential oils. Initially, the LC-MS analysis of antibacterial potential of hydro distillated N. oleander essential oils was detected by LC-MS analysis. Initially, the antibacterial nature of essential oils was detected by agar well diffusion method against biofilm forming P. aeruginosa. Then, the possible anti-bacterial fractions of the essential oils were purified by preparative HPLC, and confirmed by analytical HPLC peaks. Possible anti-bacterial components of the fractions were merged together and confirmed by agar well diffusion method. Consecutively, the and minimum biofilm inhibition concentration experiment was proved that the concentration of 200 µg/mL of purified essential oils was very effective against P. aeruginosa biofilm formation. The decrease survival rate of the MTT assay result was also supported the minimum biofilm inhibition concentration result. Finally, all the results were more evident; the HPLC purified N. oleander essential oils as a promising anti-biofilm factor against P. aeruginosa biofilm formation.

Rapid Screening for EGFR Inhibitor in Rhei Radix et Rhizoma by HTRF Assay Coupled with HPLC Peak Fractionation.

In this paper, an HPLC peak fractionation approach combined with homogeneous time-resolved fluorescence analysis is proposed for screening epidermal growth factor receptor inhibitors from Rhei Radix et Rhizoma. With this approach, the amount of sample used for a single HPLC run is sufficient for performing a multiple assay due to the miniaturization ability of the homogeneous time-resolved fluorescence technology. This allows for improving the stability and repeatability of the activity assay for each fraction. From a total of 26 fractions collected from the Rhei Radix et Rhizoma extract, 13 fractions exhibit inhibitory activity against the epidermal growth factor receptor. The structures of activity compounds were determined by HPLC-LTQ-Orbitrap MS, revealing the presence of gallic acid, rhein, and emodin with IC50 values of 21.5, 5.29, and 10.2 µM, respectively. The ligand epidermal growth factor receptor interactions were explored by molecular docking simulations, and the inhibitory effects of the three compounds on A549 cell growth were tested in vitro by an MTT assay. This study demonstrates the suitability of the present screening method for drug discovery in natural products.

Acute and Sub-Acute Toxicity Studies and Pharmacodynamic Studies of Standardized Extract of Trachyspermum ammi (L.) Sprague (Fruits) Against Chemically Induced Inflammation in Rats.

Nowadays, researchers have been attempting to use herbal products as medicines which have proven to cause lesser side effects. The fruit part of Trachyspermum ammi (L.) - Ajwain has been an integral part of the Indian medicine system with much importance in Ayurveda and Unani medicine system and is prescribed by Vaidya gurus and Hakim in raw form or as a major constituent in the powdered formulations. This research aimed to evaluate acute and sub-acute toxicity of standardized T. ammi fruit and its anti-inflammatory property using experimental models. The extract of herbs was spectroscopically analyzed for the estimation of the number of bioactive compounds. Then acute and sub-acute toxicity analysis of the herbal extract was performed to ensure the toxic effects, if any. Biochemical parameters like ALT, AST, ALP, etc. and histopathological analysis were determined to study the toxicity of the extract. Then, the anti-inflammatory activity of the T. ammi fruit extract employing Carrageenan and formalin-induced edema model in rats was studied. Ajwain seeds have a pungent smell and a characteristic odor. The powder microscopy clearly showed endosperm, unicellular warty trichomes, striated cuticle in surface view, vittae, endodermis, and vascular strand. Phytochemical tests reported the presence of carbohydrates, alkaloids, tannins, etc. and characteristic peaks in UV, Mass, NMR, FTIR and HPLC were observed for the extract. Acute and sub-acute toxicity studies did not report any toxicity, and significant anti-inflammatory action was recorded. The spectroscopic and pharmacognostic analysis has shown the strong presence of flavonoids, mineral matter, protein, phenols, saponins, carbohydrates, volatile oils, fiber, glycosides and fat. Spectroscopic study interpretations have shown the presence of compounds like thymol, para-cymene, γ-terpinene, α- and β-pinene, carvone, limonene, saponins,, β-phellandrene, βfenchyl alcohol, α-thujene, β-phellendrene, α-thujene, etc. No signs of toxicity were recorded in acute and sub-acute toxicity studies assessing the relative weight and histopathological analysis. Significant anti-inflammatory potential of T. ammi fruit extract was found and LD50 was found to be beyond 3000 mg/kg. The results of this study could be useful; in setting the quality parameters for further identification of the crude herb and preparation of the monograph.