Standard Compounds Research Articles

Thioether-substituted Benzimidazolium Salts: Synthesis, Characterization, Crystal Structure, and Their Inhibitory Properties Against Acetylcholinesterase and Xanthine Oxidase

The sulfurous compounds are known as organosulfur, which has been associated with numerous biological activities in both natural products and synthetic organic compounds. In this work, we present the synthesis of a series of 4-(methylthio)benzyl substituted benzimidazolium salts. All compounds were characterized using NMR (1H and 13C) and FTIR spectroscopic methods as well as an elemental analysis technique. The molecular and crystal structures of the compound 1a were determined by X-ray crystallography revealing that the compound crystallized in the trigonal space group R-3. Enzyme inhibition studies demonstrated that a new series of sulfurous compounds precursors were highly potent inhibitors for xanthine oxidase (XO) and acetylcholinesterase (AChE) enzyme. The IC50 values were found in the range of 0.548 ± 0.033 to 0.725 ± 0.043 µM for XO promising strategy for the treatment from gout disease, while IC50 values were found in the range 0.813 ± 0.076 to 1.149 ± 0.072 µM toward AChE as the key enzyme promising strategy for the treatment of neurological disorders such as Alzheimer's disease (AD). Furthermore, pharmacodynamics studies prove the binding interaction patterns, structural orientations and drug potential of sulfide derivatives in the binding sites of xanthine oxidase (XO) and acetylcholinesterase (AChE) enzymes. Potential inhibitors (compounds 1d-f) were compared with standard compounds allopurinol (for XO) and donepezil (for AChE). Compared to the positive compound of target XO, the 4-vinylbenzyl group of potential compound 1f and the 4-methylbenzyl group of compound le more effectively formed electrostatic and hydrophobic interactions with the target's interaction site. While donepezil as standard compound interacts only at the peripheral anionic site of AChE, the related compounds interact with both regions (PAS and CAS sites) of the same target. These compounds were placed at the active sites of the respective targets by molecular docking method using AutoDock software. Binding energy, binding modes and interaction types were used to evaluate the series of 4-(methylthio)benzyl substituted benzimidazolium salts's ability to bind to each target. Binding energy lower than zero remarks spontaneous binding, and equal and/or lower than -5 kcal/mol remarks good binding. Besides XO, the related compounds show higher activity against the AChE enzyme. They can also be analyzed as strong candidate compounds in biological studies of related enzymes.

Efficacy of Biological Control Agents and Resistance Inducer for Control of Mal Secco Disease.

Mal secco, caused by Plenodomus tracheiphilus, is an economically important fungal vascular disease in citrus-growing countries of the Mediterranean basin. Preventing fungal infections usually requires a high number of copper treatments but European legislation imposes the minimization of their accumulation in soil. In our study, biological control agents (BCAs) and a plant resistance inducer (PRI), tested in four different experiments on citrus seedlings under controlled conditions, have resulted in promising strategies to control mal secco disease. Foliar (Experiment I) and soil (Experiment II) applications of two formulations of Bacillus amyloliquefaciens strain D747 (Amylo-X® LC and Amylo-X® WG) provided similar performances in reducing the disease amount (incidence and symptoms severity) over time compared to the untreated control, whereas copper hydroxide (Kocide Opti®) used as standard was the most effective treatment over time. In the third experiment, Pythium oligandrum strain M1 (Polyversum®) and Trichoderma asperellum strain ICC012 + Trichoderma gamsii strain ICC080 (Remedier®) were able to reduce disease incidence and symptoms severity compared to the untreated control. Remedier® provided the best performances in reducing the disease amount, whereas the Polyversum® application was the least effective treatment over time. The effectiveness of the Trichoderma spp. formulation in reducing P. tracheiphilus infections did not significantly differ from the standard copper compound (Kocide Opti®). Comprehensively, in the last experiment (IV), acibenzolar-S-methyl (ASM) alone and in mixture with metalaxyl-M proved as effective as B. amyloliquefaciens strain FZB24, with no dose-response relationships observed. These findings provide important insight for the integrated management of mal secco disease.

Using the UPLC-QTOF-MS Fingerprinting Coupled with Clustering Analysis to Verify the Quality Control of Fritillaria przewalskii Maxim

Background: A method is proposed as follows to establish the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) fingerprinting and content determination for Fritillaria przewalskii Maxim. (FPM). Materials and Methods: The separation was developed using an Acquity UPLC CSH C18 column (2.1 mm × 100 mm, 1.7 µm). The gradient elution was carried out with acetonitrile solution with 0.1% acetic acid (containing 0.01 mol L−1 ammonium acetate) as the mobile phase, with the flow rate of 0.4 mL min−1, column temperature of 30°C, and injection volume of 2 µL. Fourteen batches of samples were analyzed under the above chromatographic conditions to ascertain the fingerprint of FPM sourced in different areas. A total of 45 common peaks were selected for analysis. Two chromatographic peaks were identified by comparison with the standard compound, and simultaneous content determination of the two compounds was carried out. These two compounds were identified as two alkaloids, peiminine and peimisine. SPSS 25.0 and SIMCA 14.1 were used for cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) on the peak area of the 45 common peaks of FPM. Results: Results from this showed that samples sourced from different regions could be successfully divided into three groups based on their origin. The contents of peiminine and peimisine in different batches of FPM ranged from 57.92 to 76.01 µg/g and 118.12 to 167.39 µg/g. A quick and convenient identification using UPLC fingerprinting combined with PCA was then established to differentiate among FPM samples from different growing regions. Conclusion: This method may prove to be helpful for the quality evaluation and control of FPM and related medicinal plants in the future.

New High-Throughput Screen Discovers Novel Ligands of Full-Length Nuclear Receptor LRH-1.

Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is a lipid-regulated transcription factor and an important drug target for several liver diseases. Advances toward LRH-1 therapeutics have been driven recently by structural biology, with fewer contributions from compound screening. Standard LRH-1 screens detect compound-induced interaction between LRH-1 and a transcriptional coregulator peptide, an approach that excludes compounds that regulate LRH-1 through alternative mechanisms. Here, we developed a FRET-based LRH-1 screen that simply detects compound binding to LRH-1, applying it to discover 58 new compounds that bind the canonical ligand-binding site in LRH-1 (2.5% hit rate), also supported by computational docking. Four independent functional screens identified 15 of these 58 compounds to also regulate LRH-1 function in vitro or in living cells. Although one of these 15 compounds, abamectin, directly binds LRH-1 and regulates full-length LRH-1 in cells, abamectin failed to regulate the isolated ligand-binding domain in standard coregulator peptide recruitment assays using PGC1α, DAX-1, or SHP. Abamectin treatment of human liver HepG2 cells selectively regulated endogenous LRH-1 ChIP-seq target genes and pathways associated with known LRH-1 functions in bile acid and cholesterol metabolism. Thus, the screen reported here can discover compounds not likely to have been identified in standard LRH-1 compound screens but which bind and regulate full-length LRH-1 in cells.

Fermented rapeseed meal enhances the digestibility of protein and macro- and microminerals and improves the performance of weaner pigs

Fermented rapeseed meal has the potential to replace enzymatic and pro- and prebiotic additives, organic acids, and zinc oxide in diets for piglets. The aim of the study was to analyse the effect of rapeseed meal fermented using Bacillus subtilis strain 87Y (FRSMb) in the amount of 8% of diets for weaners on growth performance, digestibility of nutrients and minerals, and the amount of biogenic substances excreted into the environment. A total of 288 pigs were used in the experiment. The study was conducted using on 288 pigs. The experiment was begun when the pigs were 28 days old. The piglets in the control group (C) were fed a standard compound feed. Piglets in groups FRA (8% fermented rapeseed meal + pro- and prebiotic additives, organic acids, and ZnO) and FR (8% fermented rapeseed meal) received a diet containing 8% FRSMb in place of some of the soybean meal in the standard feed. Additives, pro- and prebiotic additives, organic acids, and ZnO were removed from the diet for the piglets in group FR. Rapeseed meal was fermented using Bacillus subtilis strain 87Y. The experiment was carried out for a period of 60 days. Gain:Feed (G:F) between 28 and 88 days was lowest in group FR. Coefficients of apparent total tract digestibility (CATTD) and coefficients of apparent ileal digestibility (CAID) of crude protein (CP), crude fat, and crude fibre were higher in group FRA than in group C (P < 0.05). Coefficients of apparent total tract digestibility and CAID of Cu, Zn and Fe were increased (P < 0.05) in group FR relative to groups FRA and C. Absorption and retention of P and retention as a percentage of absorption were higher in the groups receiving 8% FRSMb than in group C (P = 0.001; P = 0.008 and P = 0.007, respectively). Most indicators of Ca balance were increased (P < 0.05) in the pigs in groups FRA and FR in comparison to group C. The level of ammonia released in the faeces of the piglets was lower in group FR than in the control group (23.28% reduction). The amount of Cu excreted in the faeces was higher in group C (P = 0.009), but the amount excreted in the urine was higher in group FRA (P < 0.001). The results of the research suggest that the inclusion of 8% FRSMb in diets for piglets has a beneficial effect by improving the digestibility of protein and minerals and thus reducing their excretion into the environment.

Isolation of the active ingredients of antimalarial activity of the stem bark of Pseudocedrela kotschyi (Dry zone cedar)

Natural products, either as pure compounds or as standardized extracts provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity. This study was carried out to isolate the active ingredients and determine the antimalarial activity of fractions of Pseudocedrela kotschyi stem bark following a scientific report of the antimalarial activity of crude extract of the plant material. Atotal of seven different fractions were obtained from 12g of the ethyl acetate crude extract using the flash chromatography method. GC-MS analysis of each of the seven ethyl acetate fractions was done and the identification of components present was based on a direct a comparison of the similarity index, retention times and mass spectral data with those for standard compounds. The compound prediction is based on NIST 05 Spectral library search programme. LD50 of 5 out of 7 fractions obtained was carried out using 45 mice of both sexes weighing 17-34g. The antimalarial (suppressive and curative) properties were evaluated in 40 mice in 3 different groups using NK 65 Plasmodium berghei berghei for each fraction. The plant treatments were compared against chloroquine and dimethyl sulfoxide. Results of GC-MS analysis of the seven ethyl acetate fractions of the plant extract showed the presence of forty-four different compounds, of which 3, 5-di-tert-butylphenol have been previously reported to have antimalarial properties. Acute toxicity (LD50 ) of fractions F1-4, F8, F9, F10-11, and F12-19 were 450mg/kg, 200mg/kg, 450mg/kg, 200mg/kg, and 450mg/kg respectively. All 7 fractions exhibited antimalarial activity. Remarkably, fractions F8 and F6-7 showed significant antimalarial activities for both suppressive and curative tests. These findings may benefit the antimalarial drug search in the current public health concerns of malaria disease.

LC-ESI-MS/MS Chemical Characterization, Antioxidant and Antidiabetic Properties of Propolis Extracted with Organic Solvents from Eastern Anatolia Region.

Geographic conditions (altitude, climate, and local flora) lead to significant differences in the chemical composition of propolis. Therefore, more research is needed for propolis in different geographical regions. So, the aim of this study was to evaluate the phenolic profile, total phenolic content, antioxidant, and antidiabetic properties of Pülümür propolis from Turkey. Methanol (MeOH), chloroform (CHCl3 ), and hexane extracts of propolis were analyzed. LC-ESI-MS/MS analysis of the extracts showed that the most abundant phenolic compound is caffeic acid in the MeOH extract (2943.12±11.12 μg phenolics/g extract), while on the other hand, CHCl3 extract had the highest total phenolic content (125.75±1.02 mg GAE/g extract). Antioxidant activity was measured using ABTS and DPPH assays, whereas CHCl3 extract (IC50 =6.35±0.11 and 28.84±0.10 μg/mL, respectively) and MeOH extracts (IC50 =5.04±0.07 and 28.80±0.09 μg/mL, respectively) showed relatively high antioxidant activity. The MeOH extract showed better antidiabetic activity than the standard compound, acarbose (IC50 =0.544 and 0.805 mg/mL, respectively).

SFC and CE—A Comparison of Two Orthogonal Methods for the Analysis of Dihydrochalcones in Apple Leaves

In recent years the analysis of natural products has been carried out using a range of approaches, but mainly utilizing liquid chromatography (LC) or gas chromatography (GC). However, alternative approaches with orthogonal selectivity like capillary electrophoresis (CE) and supercritical fluid chromatography (SFC) have increasingly been employed as well, even though they are often considered niche techniques only. In this study, we intended to confirm and compare their suitability as reliable state-of-the-art methods for the analysis of bioactive compounds by developing CE and SFC for the analysis of dihydrochalcones (DHCs) in apple leaves. The analytes were chosen as they have shown interesting pharmacological effects, such as anti-inflammatory, anti-tumor and immunomodulatory activities, and also present an interesting analytical challenge due to their structural similarity and polarity range. Both methods were well capable to separate the five standard compounds within short separation times and fulfilling the demands for an environmentally friendly “green” technology. CE as well as the SFC assay were validated for linearity, sensitivity, accuracy and precision according to ICH guidelines and met all respective requirements. Using the optimized methods, several Malus sp. samples were analyzed whereby a significant difference in the qualitative as well as quantitative DHC profile was revealed, with overall DHC concentrations ranging from 5.47% to 17.24%.

Discolored Components from the Black-streaked Heartwood Extracts of Teak

With regard to black-streaked discoloration of teak wood, the ethyl acetate extract from successive extraction was examined. By column chromatography, tectol and and two unknown compounds i.e. C-1 (molecular weight of 240) and C-2 (molecular weight of 210) were isolated. Discoloration tests, i.e. air oxidation, acidic (acetic acid), and alkaline (potassiun hydrogen carbonate) treatments were conducted to the isolated and other standard quinoine compounds (tectoquinone, lapachol, 2-tert-buthyl- anthraqunone, 2-hydroxy-methyl-anthraqunone). The results showed that tectol changes its color by the considerable decreasing in brightness whereas C-1 showed huge decrease in yellowness by air oxydation. Tectol did not change its color in pH value of 2.9 to 8.3 but C-1, C-2, and lapachol did change. Tectoquinone was hardly change color under alkaline or acidic treatment. The difference in quinone coloration might be due the structural features of a hydroxyl group and a double bond conjugated

Development of an automatic measurement system using atmospheric pressure photoionization ultrahigh-resolution mass spectrometry and application for on-line analysis of particulate matter

On-line chemical characterization of atmospheric particulate matter (PM) with soft ionization technique and ultrahigh-resolution Mass Spectrometry (UHRMS) provides molecular information of organic constituents in real time. Here we describe the development and application of an automatic measurement system that incorporates PM2.5 sampling, thermal desorption, atmospheric pressure photoionization, and UHRMS analysis. Molecular formulas of detected organic compounds were deducted from the accurate (±10 ppm) molecular weights obtained at a mass resolution of 100,000, allowing the identification of small organic compounds in PM2.5. Detection efficiencies of 28 standard compounds were determined and we found a high sensitivity and selectivity towards organic amines with limits of detection below 10 pg. As a proof of principle, PM2.5 samples collected off-line in winter in the urban area of Beijing were analyzed using the Ionization Module and HRMS of the system. The automatic system was then applied to conduct on-line measurements during the summer time at a time resolution of 2 hr. The detected organic compounds comprised mainly CHON and CHN compounds below 350 m/z. Pronounced seasonal variations in elemental composition were observed with shorter carbon backbones and higher O/C ratios in summer than that in winter. This result is consistent with stronger photochemical reactions and thus a higher oxidation state of organics in summer. Diurnal variation in signal intensity of each formula provides crucial information to reveal its source and formation pathway. In summary, the automatic measurement system serves as an important tool for the on-line characterization and identification of organic species in PM2.5.

In-silico and In-vitro Evaluation of the Anti-diabetic Potential of p-Propoxybenzoic Acid

Background: p-propoxybenzoic acid (p-PBA) is reported as an active chemical constituent of medicinal plants that possess anti-diabetic activity. It is termed a Multiple-Designed Ligand (MDL) having the ability to block more than one enzyme. A molecular docking study justifies the binding ability of p-PBA with acarbose and NaVO4 which were considered standard compounds having the ability to block target enzymes. α-amylase inhibition assay was used as an in-vitro screening model to evaluate the activity of p-PBA against diabetes on an initial basis. Methods: For the molecular docking study, a PDB file of p-PBA was prepared and PDB files of α-amylase (1C8Q), α-glucosidase (5KZW) and PTP1B (5K9W) were procured. p-PBA was docked against the enzymes using the blind docking method. The binding score of p-PBA and standard with enzymes was obtained and compared. The percentage inhibition of an α-amylase enzyme by p-PBA was measured by using a DNS-modified α-amylase inhibition assay and half-maximal inhibitory concentration (IC50) was calculated. Results: p-PBA has a significant inhibitory effect against α-amylase, α-glucosidase, and PTP1B with docking scores of 8.43 ± 0.44 kcal/mol, 9.19 ± 0.49 kcal/mol, and 9.40 ± 0.47 kcal/mol respectively. IC50calculated from the results of α-amylase inhibition assay p-PBA was 56.59 μg/mL. Conclusion: A combination of in-silico and in-vitro methods assessed p-PBA’s anti-diabetic potential on an initial basis. A molecular docking study involving p-PBA concluded the affinity of p-PBA to α-amylase, α-glucosidase, and PTP1B was significantly correlated with the affinity of acarbose and NaVO4. In-vitro α-amylase assay validated the compound’s inhibitory action against the enzyme.

Anticancer Activity of Methanol extract of Limnophila repens and Argyeia cymosa by Using SRB Assay

Introduction: Plants have a special place in the treatment of cancer. It is estimated that plant-derived compounds constitute more than 50% of anticancer agents. In this present study I attempted an experiment to find out the anti-cancer activity of selected plants Limnophila repens and Argyeia cymosa by using SRB Assay. Material method: I have collected the plants, dried very well and extracted with methanol crude methanol extract of the both plants tested for its anti-cancer activity. Results and disscussionThe anticancer activity of methanolic extracts of Argyreia cymosa and Limnophila repens was determined by sulforhodamine B colorimetric assay. The results of the cytotoxicity of extracts from both plant extracts analysed and The Methanol extract of Limnophila repens extract showed comparable activity to the standard compound, i.e., Adriamycin on Ishikawa and SCC-29B cell lines, respectively. This extract showed TGI, and GI50 was 38.9 and &lt;10 µg/ ml on Ishikawa cell lines and 58.12, &lt;10 and &lt;10 µg/ ml of LC50, TGI and GI50 activity on SCC-29B cell lines respectively. The Methanol extract of Argyeia cymosa showed GI50 was &gt;80 µg/ ml on Ishikawa cell lines and &lt;10 and &lt;10 µg/ ml of, TGI and GI50 activity on SCC-29B cell lines .Estimations based on GI50 values shows that MELR was more active against Ishikawa and SCC-29B cell lines than MEAC on Ishikawa (human endometrial adenocarcinoma) and SCC-29B (human oral cancer) cell lines.

Ergosterol and polyphenol contents as rapid indicators of orchardgrass silage safety

The ergosterol (ERG) has been proposed as a potential indicator of fungal contamination, along with polyphenol content analysis to predict silage safety. Despite efforts in controlling fungal growth in silage, mycotoxin co-contamination represents a possible risk for animal and human health. Modern analytical techniques determine a multitude of fungal metabolites contaminating feed. Nonetheless, these methods require sometimes arduous sample pre-treatment, long separation times, and expensive standard compounds to identified contaminants. Thus, the goal of this study was to suggest a rapid analysis of ERG and polyphenol contents to assess silage hygienic quality in ten orchardgrass varieties ensiled without and with biological and chemical additives. The determination of ERG on samples was performed by high-performance liquid chromatography using UV detection and UV/Vis spectrophotometry to determine the polyphenol content. Statistically significant differences (P < 0.05) between varieties, years and silage additives were found. Bepro was the unique variety that did not present ERG in the first cut in 2012. ERG content increased in the first cut in 2013 using biological additives as well as ERG and polyphenol contents in the first cut in 2013 using chemical additives compared with untreated silage. In addition, biological and chemical additives used in this study did not satisfactorily reduce the content of ERG and polyphenols in silage grass. Consequently, our results provide fast information about the progressive fungal contamination of grass silage. To our knowledge, it is the first time that the presence of ERG and polyphenols is determined in ten different orchardgrass varieties treated without and with additives. In general, ERG and polyphenol contents showed to be good indicators of orchardgrass silage safety.

Surface Molecularly Imprinted Cellulose‐Synthetic Hybrid Particles Prepared via ATRP for Enrichment of Flavonoids in Olive Leaf

AbstractSurface molecularly imprinted cellulose‐synthetic hybrid particles are prepared via atom transfer radical polymerization (ATRP). The two‐step process involves the immobilization of α‐bromoisobutyryl bromide in the pristine microcrystalline cellulose, to generate ATRP macroinitiator particles, and then the creation of a crosslinked‐imprinted shell in the particles surface considering ATRP of 4‐vinylpyridine (4VP) and ethylene glycoldimethacrylate (EGDMA) with quercetin as imprinting template. Among the polymerization recipes tested, a system with ethanol as solvent preserves a final size of the hybrid particles suitable for application as adsorbent, while also incorporating the 4VP/EGDMA co‐monomers. Testing of imprinted/non‐imprinted particles for sorption/desorption of standard phenolic compounds shows the modification of the surface of the pristine cellulose and also the achievement of molecular imprinting (imprinting factor ≈2.6). Particles are used for the enrichment of flavonoids in olive leaf extracts and the special features of the developed molecularly imprinted adsorbents are again highlighted with this complex mixture of phenolic compounds. It is shown that production of fractions rich in luteolin‐7‐O‐glucoside, apigenin‐7‐O‐glucoside, or quercetin, among other flavonoids is possible (estimated enrichment factors up to 4). These results point up for the usefulness of natural‐synthetic materials with processes to get high‐added value compounds in the framework of circular bio‐economy.

Pangenome profiling of novel drug target against vancomycin-resistant Enterococcus faecium

Enterococcus faecium is a frequent causative agent of nosocomial infection mainly acquired from outgoing hospital patients (Hospital Acquired Infection—HAIs). They are largely involved in the outbreaks of bacteremia, UTI, and endocarditis with a high transmissibility rate. The recent emergence of VRE strain (i.e. vancomycin resistant enterococcus) turned it into high priority pathogen for which new drug research is of dire need. Therefore, in current study, pangenome and resistome analyses were performed for available antibiotic-resistant genomes (n = 216) of E. faecium. It resulted in the prediction of around 5,059 genes as an accessory gene, 1,076 genes as core and 1,558 genes made up a unique genome fraction. Core genes common to all strains were further used for the identification of potent drug targets by applying subtractive genomics approach. Moreover, the COG functional analysis showed that these genomes are highly enriched in metabolic pathways such as in translational, ribosomal, proteins, carbohydrates and nucleotide transport metabolism. Through subtractive genomics it was observed that 431 proteins were non-homologous to the human proteome, 166 identified as essential for pathogen survival while 26 as potential and unique therapeutic targets. Finally, 3-dehydroquinate dehydrogenase was proposed as a potent drug target for further therapeutic candidate identification. Moreover, the molecular docking and dynamic simulation technique were applied to performed a virtual screening of natural product libraries (i.e., TCM and Ayurvedic compounds) along with 3-amino-4,5-dihydroxy-cyclohex-1-enecarboxylate (DHS) as a standard compound to validate the study. Consequently, Argeloside I, Apigenin-7-O-gentiobioside (from Ayurvedic library), ZINC85571062, and ZINC85570908 (TCM library) compounds were identified as potential inhibitors of 3-dehydroquinate dehydrogenase. The study proposed new compounds as novel therapeutics, however, further experimental validation is needed as a follow-up. Communicated by Ramaswamy H. Sarma

Investigation of the chemical components of Ciwujia injection using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry

Ciwujia injection is commonly used to treat cerebrovascular and central nervous system diseases in clinical practice. It can significantly improve blood lipid levels and endothelial cell function in patients with acute cerebral infarction and promote the proliferation of neural stem cells in cerebral ischemic brain tissues. The injection has also been reported to have good curative effects on cerebrovascular diseases, such as hypertension and cerebral infarction. At present, the material basis of Ciwujia injection remains incompletely understood, and only two studies have reported dozens of components, which were determined using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). Unfortunately, the lack of research on this injection restricts the in-depth study of its therapeutic mechanism.In the present study, a qualitative method based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) was developed to analyze the chemical components of Ciwujia injection. Separation was performed on a BEH Shield RP18 column (100 mm×2.1 mm, 1.7 μm) using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phases, and gradient elution was performed as follows: 0-2 min, 0%B; 2-4 min, 0%B-5%B; 4-15 min, 5%B-20%B; 15-15.1 min, 20%B-90%B; 15.1-17 min, 90%B. The flow rate and column temperature were set to 0.4 mL/min and 30 ℃ respectively. MS1 and MS2 data were acquired in both positive- and negative-ion modes using a mass spectrometer equipped with an HESI source. For data post-processing, a self-built library including component names, molecular formulas, and chemical structures was established by collecting information on the isolated chemical compounds of Acanthopanax senticosus. The chemical components of the injection were identified by comparison with standard compounds or MS2 data in commercial databases or literature based on precise relative molecular mass and fragment ion information. The fragmentation patterns were also considered. For example, the MS2 data of 3-caffeoylquinic acid (chlorogenic acid), 4-caffeoylquinic acid (cryptochlorogenic acid), and 5-caffeoylquinic acid (neochlorogenic acid) were first analyzed. The results indicated that these compounds possessed similar fragmentation behaviors, yielding product ions at m/z 173 and m/z 179 simultaneously. However, the abundance of the product ion at m/z 173 was much higher in 4-caffeoylquinic acid than in 5-caffeoylquinic acid or 3-caffeoylquinic acid, and the fragment signal at m/z 179 was much stronger for 5-caffeoylquinic acid than for 3-caffeoylquinic acid. Four caffeoylquinic acids were identified using a combination of abundance information and retention times. MS2 data in commercial database and literature were also used to identify unknown constituents. For example, compound 88 was successfully identified as possessing a relative molecular mass and neutral losses similar to those of sinapaldehyde using the database, and compound 80 was identified as salvadoraside because its molecular and fragmentation behaviors were consistent with those reported in the literature. A total of 102 constituents, including 62 phenylpropanoids, 23 organic acids, 7 nucleosides, 1 iridoid, and 9 other compounds, were identified. The phenylpropanoids can be further classified as phenylpropionic acids, phenylpropanols, benzenepropanals, coumarins, and lignans. Among the detected compounds, 16 compounds were confirmed using reference compounds and 65 compounds were identified in Ciwujia injection for the first time. This study is the first to report the feasibility of using the UHPLC-Q/Orbitrap HRMS method to quickly and comprehensively analyze the chemical components of Ciwujia injection. The 27 newly discovered phenylpropanoids provide further material basis for the clinical treatment of neurological diseases and new research targets for the in-depth elucidation of the pharmacodynamic mechanism of Ciwujia injection and its related preparations.

Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography

Different Senecio species, especially S. inaequidens – a neophyte native to South Africa – have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a Torus™ DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 °C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 ≥ 0.9994), precision (inter-day variance ≤ 3.67%, intra-day variance ≤ 3.92%) and recovery rates (96.3–104.1%), with detection limits typical for SFC-PDA (≤ 4.24 µg/mL). Furthermore, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).

Plant and microbial sciences as key drivers in the development of metabolomics research

This year marks the 25th anniversary of the coinage of the term metabolome [S. G. Oliver et al., Trends Biotech. 16, 373–378 (1998)]. As the field rapidly advances, it is important to take stock of the progress which has been made to best inform the disciplines future. While a medical-centric perspective on metabolomics has recently been published [M. Giera et al., Cell Metab. 34, 21–34 (2022)], this largely ignores the pioneering contributions made by the plant and microbial science communities. In this perspective, we provide a contemporary overview of all fields in which metabolomics is employed with particular emphasis on both methodological and application breakthroughs made in plant and microbial sciences that have shaped this evolving research discipline from the very early days of its establishment. This will not cover all types of metabolomics assays currently employed but will focus mainly on those utilizing mass spectrometry–based measurements since they are currently by far the most prominent. Having established the historical context of metabolomics, we will address the key challenges currently facing metabolomics and offer potential approaches by which these can be faced. Most salient among these is the fact that the vast majority of mass features are as yet not annotated with high confidence; what we may refer to as definitive identification. We discuss the potential of both standard compound libraries and artificial intelligence technologies to address this challenge and the use of natural variance–based approaches such as genome-wide association studies in attempt to assign specific functions to the myriad of structurally similar and complex specialized metabolites. We conclude by stating our contention that as these challenges are epic and that they will need far greater cooperative efforts from biologists, chemists, and computer scientists with an interest in all kingdoms of life than have been made to date. Ultimately, a better linkage of metabolome and genome data will likely also be needed particularly considering the Earth BioGenome Project.

Screening method toward ClbP-specific inhibitors

BackgroundColibactin is a genotoxin produced by Escherichia coli and other Enterobacteriaceae that is believed to increase the risk of colorectal cancer (CRC) of their symbiosis hosts, including human. A peptidase ClbP is the key enzyme for activation of colibactin. Inhibition of ClbP is considered to impede maturation of precolibactin into genotoxic colibactin. Therefore, ClbP-specific inhibitors could potentially prevent the onset of CRC, one of the leading causes of cancer-related deaths in the world. This study intends to establish an efficient screening system for identifying inhibitors that are specific to ClbP.MethodsTwo types of assays were applied in the screening procedure: a probe assay and an LC–MS assay. For the probe assay, we employed the synthesized probe which we described in our previous report. This probe can be hydrolyzed efficiently by ClbP to release a fluorophore. Hence it was applied here for detection of inhibition of ClbP. For the LC–MS assay, formation of the byproduct of precolibactin maturation process, N-myristoyl-D-asparagine, was quantified using a liquid chromatography–mass spectrometry (LC–MS) technique. The probe assay can be performed much faster, while the LC–MS assay is more accurate. Therefore, our method employed the two assays in sequence to screen a large number of compounds for inhibition of ClbP.ResultsA library of 67,965 standard compounds was evaluated by the screening method established in the current study, and one compound was found to show a moderate inhibitory activity against ClbP.ConclusionA simple screening method for ClbP-specific inhibitors was established. It was proven to be reliable and is believed to be useful in developing potential prophylactic agents for CRC.

Enhancement of Rabbit Meat Functionality by Replacing Traditional Feed Raw Materials with Alternative and More Sustainable Freshwater Cladophora glomerata Macroalgal Biomass in Their Diets.

Today's challenges in the animal husbandry sector, with customers' demand for more beneficial products, encourage the development of strategies that not only provide more sustainable production from the field to the table but also ensure final product functionality. Thus, the current research was aimed at replacing some traditional feed raw materials in rabbit diets with C. glomerata biomass to improve the functionality of meat. For this purpose, thirty weaned (52-d-old) Californian rabbits were assigned to 3 dietary treatments: standard compound diet (SCD), SCD + 4% C. glomerata (CG4), and SCD + 8% C. glomerata (CG8). At the end of the feeding trial, 122-d-old rabbits were slaughtered, longissimus dorsi (LD) and hind leg (HL) muscles were dissected post-mortem, and moisture, protein, and lipid profiles were determined. Results revealed that CG4 treatment can increase protein (22.17 g/kg), total (192.16 g/kg) and essential (threonine, valine, methionine, lysine, and isoleucine) amino acid levels in rabbit muscles. Both inclusions gradually reduced fat accumulation in muscles (CG8 < CG4 < SCD) but improved the lipid profile's nutritional value by decreasing saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) and increasing polyunsaturated fatty acids (PUFA). As the dose of C. glomerata increased, the level of lipid oxidation decreased. Biomass supplementation enhanced PUFA/SFA and h/H levels while decreasing thrombogenicity index (TI) and atherogenic index (AI) levels in rabbit muscles, potentially contributing to the prevention of heart disease. Overall, dietary supplementation with C. glomerata biomass may be a more beneficial and sustainable nutritional approach to functionally enhancing rabbit meat.