Column Chromatography Research Articles

Synthesis of Polysubstituted Pyridines via Nitrogen-doped Graphene Catalyzed One-Pot Multicomponent Reaction under Solvent-Free Conditions

Abstract: Polysubstituted pyridine derivatives were produced with high to excellent yields in the presence of nitrogen-doped graphene (NDG) as a dual acid-based catalyst. NDG efficiently catalyzes the multicomponent reaction between arylaldehydes, diethyl-acetylene dicarboxylates, malononitrile, and ammonium acetate under solvent-free conditions at 80°C to afford the polysubstituted pyridines in short reaction times. The structures of the synthesized pyridines were established by Ft-IR, 1H, and 13C NMR spectroscopic analysis. The advantages of this method include the in-situ oxidation of prepared 1,4-dihydropyridines, one-pot procedure, solventless system, operational simplicity, and no column chromatography. Additionally, neither toxic solvents nor catalysts are needed, and the procedure can be very reliable among the reported methodologies. The yields and reaction times in the presence of four times recycled catalyst are in comparable to the fresh catalyst.

Effect of a phthalate derivative purified from Bacillus zhangzhouensis SK4 on quorum sensing regulated virulence factors of Pseudomonas aeruginosa

Pseudomonas aeruginosa causes life-threatening diseases and is resistant to almost all conventional antibiotics. The quorum sensing (QS) system of P. aeruginosa contributes to many pathogenic factors some of which are pigment production, motility, and biofilm. The disruption of quorum sensing system may be an impactful strategy to deal with infections. The present study investigates the anti-quorum sensing property of a bioactive molecule extracted from marine epibiotic bacteria present on the surface of seaweeds. Among all the isolates tested against monitor strain Chromobacterium violaceum (MTCC 2656), the one with the highest activity was identified as Bacillus zhangzhouensis SK4. The culture supernatant was extracted with chloroform which was then partially purified by TLC and column chromatography. The probable anti-QS compound was identified as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl ester) by GC-MS and NMR analysis. The treatment of P. aeruginosa MCC 3457 with the lead compound resulted in the reduced production of pyocyanin, rhamnolipids, exopolysaccharide, biofilm, and motility. The observations of light and scanning electron microscopy also supported the biofilm inhibition. The lead compound showed synergism with the meropenem antibiotic and significantly reduced MIC. The molecular docking and pharmacokinetics study predicted 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl ester), a phthalate derivative as a good drug candidate. The molecular dynamics study was also performed to check the stability of the lead compound and LasR complex. Further, lead compounds did not exhibit any cytotoxicity when tested on human embryonic kidney cells. As per our knowledge, this is the first report on the anti-QS activity of B. zhangzhouensis SK4, indicating that epibiotic bacteria can be a possible source of novel compounds to deal with the multidrug resistance phenomenon.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Botanical Extracts and Compounds of Castanea Plants and Methods of Use: US20190125818A1 - The United States Patent Evaluation.

Bacterial infections are increasingly difficult to combat, which makes them a threat to public health on a global level. Staphylococcus aureus is considered one of the main causes of infections in hospitals, as it has a variety of virulence factors, as well as is able to produce bacterial biofilms, which, consequently, bring numerous damages to public health as a result of increased resistance to conventional antibiotics and a longer hospital stay. Therefore, the use of compounds extracted from medicinal plants is a potential pharmaceutically acceptable target, as they do not have toxicity and the potential to disrupt biofilms produced by Staphylococcus aureus already evidenced, thus revealing their relevance to our study. The objective of this work was to perform a critical analysis of a patent with natural extracts against bacterial biofilms found in the United States Patent and Trademark Office (USPTO) database, to map the possible bioactive compounds that may serve as potential future antimicrobial drugs. A technological survey was carried out to verify existing patents using natural extracts with anti-biofilm potential. For this, it was searched with the keywords: Botanical extracts AND biofilms; which were performed in the United States Patent and Trademark Office (USPTO) database. Thus, the selected patent used a non-aqueous extract partitioned and vacuum-contracted, subsequently lyophilized for assays with antimicrobial potential. Because of this, a patent was analyzed regarding its chemistry, and biological activity, followed by a critical analysis of the technology proposed in the invention. When using the keywords Botanical extracts AND biofilms in the USPTO, it was possible to find twenty-two inventions; however, only four patents in the USPTO were in agreement with the proposal of the natural extract having antimicrobial activity and an anti-biofilm potential, of which two belonged to the same applicant with similar proposals. The key point of this invention was to enable the compounds of the Castanea sativa plant and its methods of obtaining the extract to present a significant antimicrobial action associated or not with antibiotics, promoting the development of new therapies against bacterial infections capable of disrupting biofilms. The invention developed a methodology for extracting Castanea sativa, in which pentacyclic triterpene compounds were found mostly in its leaves. Whereas for the extraction, the crude methanol extracts called extracts 224 from the ground leaves were made by maceration, filtered, combined, concentrated under pressure in rotary evaporators, and lyophilized. After that, they were resuspended in water and partitioned in succession with hexane, ethyl acetate, and butanol. The most active refined partition was the 224C extract with the solvent ethyl acetate, which was subjected to further fractionation using silica column chromatography. Resulting in the most refined extract, which was 224C-F2, capable of acting directly on the quorum sensing of bacteria, mainly Staphylococcus aureus, blocking the translation of RNAIII, including a series of exotoxins. Regarding the antimicrobial capacity against Staphylococcus aureus, it presented Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 1.56 μg/mL-1 and > 100 μg/mL -1, respectively. Given the analyzed patent, it was possible to verify the importance of alternatives to reduce the impact of bacterial biofilms, which causes damage to industries in general and to health. From this, the invention analyzed has a promising proposal with antimicrobial potential focusing on the great impact of bacterial biofilms. Therefore, natural extracts with antibiofilmic potential can help to minimize the economic losses caused to health due to these multidrug-resistant microorganisms with different virulence mechanisms.

Withanolides production by the endophytic fungus Penicillium oxalicum associated with Withania somnifera (L.) Dunal.

Withanolides are steroidal lactones with diverse bioactive potential and their production from plant sources varies with genotype, age, culture conditions, and geographical region. Endophytic fungi serve as an alternative source to produce withanolides, like their host plant, Withania somnifera (L.) Dunal. The present study aimed to isolate endophytic fungi capable of producing withanolides, characterization and investigation of biological activities of these molecules. The methanolic fungal crude extract of one of the fungal isolates WSE16 showed maximum withanolide production (219mg/L). The fungal isolate WSE16 was identified as Penicillium oxalicum based on its morphological and internal transcribed spacer (ITS) sequence analysis and submitted in NCBI (accession number OR888725). The methanolic crude extract of P. oxalicum was further purified by column chromatography, and collected fractions were assessed for the presence of withanolides. Fractions F3 and F4 showed a higher content of withanolides (51.8 and 59.1mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC50 of 23.52 and 17.39µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC50 of 39.42 and 38.71µg/ml, superoxide anion scavenging assay with IC50 of 41.10 and 38.84µg/ml, and reducing power assay with IC50 of 42.61 and 41.40µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC50 of 30.34 and 22.05µg/ml, respectively). The withanolides present in fraction 3 and fraction 4 were identified as (20S, 22R)-1a-Acetoxy-27-hydroxywitha-5, 24-dienolide-3b-(O-b-D-glucopyranoside) and withanamide A, respectively, using UV, FTIR, HRMS, and NMR analysis. These results suggest that P. oxalicum, an endophytic fungus isolated from W. somnifera, is a potential source for producing bioactive withanolides.

Anti-Inflammatory and Anti-Arthritis Activity of Quercetin: A Comprehensive Review.

This comprehensive exploration delves into the multifaceted attributes of quercetin, a flavonoid with extensive health-promoting potential. The review navigates through its fundamental properties, encompassing its chemical structure, classification as a flavonoid, and its natural prevalence in various sources. Addressing solubility, stability, and bioavailability challenges, the investigation delves into innovative isolation techniques, including solvent extraction, solid-phase extraction, natural deep eutectic solvents, supercritical fluid extraction, microwave-assisted extraction, column chromatography, and high-performance thin-layer chromatography. Transitioning into pharmacological implications, the study unveils quercetin's roles in anti-inflammatory pathways, antioxidant effects, and immune modulation, reflecting its versatile significance in health management. The review highlights its impact on wound healing processes and its potential to mitigate arthritis, elucidating its holistic contributions. Culminating in an exploration of recent studies, the analysis underscores quercetin's remarkable anti-inflammatory and anti-arthritis activities, reflecting its substantial potential across various ailments. The review concludes by projecting future trajectories, emphasizing prospects for an advanced understanding of quercetin's mechanisms, sustainable extraction techniques, clinical integration, and exploration of synergistic combinations. Collectively, this review investigation underscores quercetin's dynamic role at the intersection of natural compounds and medicinal applications, offering profound implications for well- being and health enhancement.

An extended Monte Carlo simulation code for modeling gas chromatography experiments with superheavy elements and their homologs

AbstractMonte Carlo simulations are commonly used to model the behavior of chemical species of the heaviest elements and their homologs in gas chromatography experiments. In this paper, we present an extension of the fundamental Monte Carlo simulation proposed by Zvara in 1985. While preserving the core functionality, our code features two enhancements: first, it allows simulating experiments in which a primary radioisotope decays into a daughter isotope belonging to a different element, hence exhibiting different chemical properties. Second, it allows modeling scenarios where conversion of an initial chemical species to a different one can occur at temperatures high enough to overcome an activation barrier, facilitating simulations of related physisorption and chemisorption processes. This Monte Carlo code is applicable to open tubular and rectangular chromatography columns.

Secondary Metabolites from the Mangrove-derived Fungus Trichoderma sp.

Background:: Secondary metabolites from the mangrove-derived fungi have great potential to produce natural products with novel structures and significant biological activities. Objective:: The objective of this study was to isolate and identify the secondary metabolites from the mangrove-derived fungus Trichoderma sp. WHUF0342, and evaluate their antibacterial activities. Methods:: The compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high-performance liquid chromatography (HPLC). Their structures were elucidated by comparing the NMR and MS spectroscopic data with those of literature. The antibacterial activity were evaluated by modified broth microdilution assay. Results:: Eight compounds were isolated from the fermented extracts of the fungus Trichoderma sp. WHUF0342 and identified as chaxine B (1), nafuredin (2), dichlorodiaportin (3), ferulaic acid (4), bis(2-ethylhexyl) benzene-1,2-dicarboxylate (5), methyl 4-hydroxyphenylacetate (6), 4- hydroxyphenyl acetate (7), and 4-hydroxyphenylethyl acetate (8). Chaxine B (1) showed antibacterial activity against the phytopathogenic bacterium Ralstonia solanacearum with a minimum inhibitory concentration (MIC) value of 16 μg/mL. The antibacterial activity against plant pathogen of compound 1 was reported for the first time in this study. Conclusion:: This study not only enriched the secondary metabolites of mangrove-derived fungi but also provided a valuable resource for the prevention of agricultural pathogen infections.

'Passivated Precursor' Approach to All-Alkynyl-Protected Gold Nanoclusters and Total Structure Determination of Au130.

A 'passivated precursor' approach is developed for the efficient synthesis and isolation of all-alkynyl-protected gold nanoclusters. Direct reduction of dpa-passivated precursor Au-dpa (Hdpa = 2,2'-dipyridylamine) in one-pot under ambient conditions gives a series of clusters including Au22(C≡CR)18 (R = -C6H4-2-F), Au36(C≡CR)24, Au44(C≡CR)28, Au130(C≡CR)50, and Au144(C≡CR)60. These clusters can be well separated via column chromatography. The overall isolation yield of this series of clusters is 40% (based on gold), which is much improved in comparison with previous approaches. It is notable that the molecular structure of the giant cluster Au130(C≡CR)50 is revealed, which presents important information for understanding the structure of the mysterious Au130 nanoclusters. Theoretical calculations indicated Au130(C≡CR)50 has a smaller HOMO-LUMO gap than Au130(S-C6H4-4-CH3)50. This facile and reliable synthetic approach will greatly accelerate further studies on all-alkynyl-protected gold nanoclusters.

Fractionation and Identification of Phenolic Compounds from Ethylacetate Fraction of Ageratum Conyzoides Methanolic Leaf Extract

Natural phenols are significant functional substances that are commonly present in plants and provide advantages for human health. Phenolics can be utilized for a variety of therapeutic applications, such as antioxidant, anti-inflammatory, antidiabetic, and antibacterial activities. This work aimed to fractionate and identify the phenolic compounds present in ethylacetate fractions of the methanolic leaf extracts of A. conyzoides. Fractionation and purification of the ethylacetate fraction using column chromatography and TLC, followed by FTIR and LC-MS for identification, were employed in this work. While the FTIR spectroscopy was used to identify the fractions with –OH functional group, LCMS was used to identify the phenolics. After fractionation and purification, six ethylacetate samples were obtained, each with different Rf values, of which three showed a pronounced presence of OH functional groups in phenolics. Subsequently, four phenolic compounds were identified: furocoumaric acid, liquiritin, isorhamnetin, and syringin. This research has demonstrated that ethylacetate fraction of A. conyzoides methanol leaf extract is a good source of the phenolic compounds: Furocoumaric acid, Liquiritin, Isorhamnetin, and syringin.

Anti-diabetic activity-guided isolation of α-amylase and α-glucosidase inhibitory terpenes from Capsella bursa-pastoris Linn.

Abstract The hypoglycaemic and hypolipidemic potential of ethanol extract of C. bursa-pastoris (ECbp) in streptozotocin (STZ)-provoked diabetic rats was evaluated, and compounds with their α-amylase and α-glucosidase inhibitory potential were isolated. Acute oral toxicity was evaluated in rats. Streptozotocin (STZ) (50 mg/kg body weight) was injected intraperitoneally into rats for diabetes induction. In diabetic rats, ECbp (0.2 g/kg b.w, p.o.) was administered orally for 21 days, and its outcome on blood glucose levels and body weight was observed on a weekly basis besides lipid profile. Compound isolation from ECbp was performed using column chromatography. Oral feeding of ECbp did not produce any toxic effects or death at a dose of 2,000 mg/kg body weight. A serum glucose reduction trend was observed in rats fed with glucose pre-treated with 200 mg/kg b.w. ECbp also appreciably (p < 0.001, p < 0.01, and p < 0.05) diminished raised blood glucose with decreased blood cholesterol levels and led to increased serum high-density lipoprotein levels in comparison to diabetic control rats. Body weight levels were considerably higher (p < 0.05) in diabetic rats treated with ECbp than in diabetic control rats. Isolation of two terpene derivatives (ECbp-1 and ECbp-2) was performed using ECbp, which exhibits significant α-amylase and α-glucosidase inhibition.

Determination of fraxetin, fraxin and dimethylfraxetin in rat plasma by UPLC-MS/MS

AbstractObjectiveThe levels of fraxetin, fraxin, and dimethylfraxetin in rat plasma to be measured using an ultra-performance liquid chromatography tandem mass-spectrometry (UPLC–MS/MS) technique and applied to their pharmacokinetics and bioavailability.MethodsThe protein precipitation technique was applied to the plasma preparation using acetonitrile and methanol (9:1, v/v). At a flow rate of 0.4 mL min−1, the elution time was 6 min. The mobile phase consisted of acetonitrile-water with 0.1% formic acid, and the chromatographic column was UPLC HSS T3 (50 mm × 2.1 mm, 1.8 μm). Quantitative analysis was conducted using multiple reaction monitoring (MRM) mode and detection was performed using electrospray ionization (ESI) positive ion mode. In each group, six rats were treated with fraxetin, fraxin, and dimethylfraxetin either orally (5 mg kg−1) or intravenously (1 mg kg−1).ResultsThe calibration curves showed good linearity in the range of 2–4,000 ng mL−1, where r was greater than 0.99. The bioavailability of dimethylfraxetin, fraxin, and fraxetin was determinated to be 19.7, 1.4, and 6.0%.ConclusionThe established UPLC-MS/MS method for determining the levels of these three compounds in rat plasma was successfully applied to the pharmacokinetics of dimethylfraxetin, fraxin, and fraxetin, and the bioavailability was calculated.

Maclura cochinchinensis (Lour.) Corner Heartwood Extracts Containing Resveratrol and Oxyresveratrol Inhibit Melanogenesis in B16F10 Melanoma Cells

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

FERMENTATION, PURIFICATION AND IN-VITRO EVALUATION OF ANTIMICROBIAL, ANTITUMOR AND ANTIOXIDANT COMPOUNDS FROM A HIMALAYAN ACTINOBACTERIA STRAIN STREPTOMYCES SP. PU-AK14

This study reports the laboratory scale fermentation, compounds purification and bioactivity screening of the strain Streptomyces sp. PU-AK14 isolated from Himalayan mountains range, Pakistan, for its potential to produce antimicrobial, antioxidant and antitumor compounds. For this purpose, laboratory fermentation (20 L working volume) was performed followed by extraction of the compounds by XAD adsorbent gel using the solvents methanol and ethyl acetate. Further purification of the compounds was done by silica gel column chromatography, sephadex column and preparative TLC. A total of 18 partially purified compounds were retrieved from crude extract and were screened for biological activity. The crude extract was more active against gram negative bacteria with MIC of 0.1 mg/ml against Klebsiella pneumoniae and Escherichia coli. Eight of the partially purified compounds exhibited activity against MRSA whereas five fractions were active against gram negative bacteria. Seven partially purified fractions exhibited promising antioxidant activity with radical scavenging activity ranging from 78.2 to 42.34 percent. Cytotoxicity of partially purified compounds ranged from 75% to 100% larval mortality at 100mg/ml against Artemia salina and 50% to 52% cell mortality against HCT 116 colorectal cancer cell line at 100mg/ml. The best anti-tumor activity was exhibited by the fraction AK141111L. The study suggested that Streptomyces sp. PU-AK14 is a potential source of chemotherapeutically useful compounds.

Phospholanes via Twofold C‐H‐Addition to Phospholes

AbstractA synthetic route towards phospholanes via twofold C−H‐addition of a DMSO molecule to β‐H substituted phospholes is described. The resulting fused phospholane oxothiolane system in case of symmetric α‐substitution possesses four stereogenic centers or rather six in case of asymmetric α‐substitution. The reaction conditions are shown to be applicable for a variety of phospholes with different P‐ or α‐substituents always leading to stereoselective formation of the fused heterocyclic ring system. All phospholane derivatives with one exception are sufficiently air stable for purification with column chromatography and are fully characterized by NMR and elemental analysis and mass spectrometry. With the help of X‐ray crystallography, the different configurations of the isomers could be demonstrated. For one model system derivatization reactions are presented. When oxidizing or reducing the sulfoxide group only a single diastereomer is observed.

Interference with VIP to distinguish between real and false VIPoma:national study from the French Endocrine Tumors Group

Abstract Background Vasoactive Intestinal Peptide-secreting tumors (VIPomas) are digestive neuroendocrine tumors in which the hormonal secretion is life threatening. Biological confirmation is obtained by demonstrating an elevation in plasma VIP, usually using radioimmunoassay (RIA) method. In some cases, an analytical interference is suspected. We developed three different techniques to detect the interference in VIP RIA. Methods Three techniques were used: RIA after Sephadex column chromatography separation, RIA after Polyethylene Glycol (PEG) precipitation and125I-labelled VIP binding test. We included patients with suspicion of false positive VIP (FPV) elevation. We then compared results with those of a group of ‘real’, proven VIPoma (RV). Results A total of 15 patients with FPV elevation and 9 RV patients were included. Interference was detected in all FPV patients versus none in RV. Clinical and biochemical parameters did not differ between FPV and RV patients, but VIP concentration in RIA was significantly higher in FVP patients than in RV patients (228pmol/l vs 66 pmol/l, p=0.038). Using 125I-labelled VIP binding test, median proportion of radioactivity in the pellet was significantly higher in FVP than in RV patients (53% vs 13%, p < 0.0001). A 20.5% threshold presented excellent performances (Sensitivity 100% [79.6-100], specificity 100% [70.1-100]). Conclusions We developed three different laboratory techniques to reveal interference in RIA VIP assays. The diagnostic performance of all three was excellent. These techniques must be employed in cases of discordance between VIP elevation and clinical presentation.

Onnamide A suppresses the severe acute respiratory syndrome-coronavirus 2 infection without inhibiting 3-chymotrypsin-like cysteine protease.

Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.

Development of UHPLC-Q-Exactive Orbitrap/MS Technique for Determination of Proanthocyanidins (PAs) Monomer Composition Content in Persimmon

The main units of persimmon proanthocyanidins (PAs) are composed of flavan-3-ols including epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). Precise quantification of GCG is challenging due to its trace amounts in persimmon. In this study, to establish the optimal UHPLC-Q-Exactive Orbitrap/MS technique for the determination of PAs monomer composition in persimmon fruit flesh of different astringency types, mass spectrometry and chromatographic conditions were optimized. The results showed that when operating in negative ion mode, using a T3 chromatographic column (a type of C18 column with high-strength silica), acetonitrile as the organic phase, a 0.1% mobile phase acid content, and a mobile phase flow rate of 0.2 mL/min, the chromatographic peak shape and resolution of the PAs monomer composition improved. Additionally, there was no tailing phenomenon observed in the chromatographic peaks. At the same time, the intra-day and inter-day precision, stability, and recovery of the procedure were good. The relative standard deviation (RSD) of stability was less than 5%. The intra-day precision was in the range of 1.14% to 2.36%, and the inter-day precision ranged from 1.03% to 2.92%, both of which were less than 5%. The recovery rate ranged from 94.43% to 98.59% with an RSD less than 5%. The results showed that the UHPLC-Q-Exactive Orbitrap/MS technique established in this study can not only be used for the quantification of EGCG and GCG in persimmon fruit flesh but also be suitable for analyzing other PAs monomer compositions, providing robust support for the related research on persimmon PAs.

Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line

BackgroundThe burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa.MethodsThe proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay.ResultsThe crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity.ConclusionThis study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Stereodivergent Synthesis of the Four Stereoisomers of Diethyl 4-Hydroxyphosphopipecolate from Ethyl (R)-4-Cyano-3-hydroxybutanoate

The stereodivergent synthesis of all four stereoisomers of diethyl 4-hydroxyphosphopipecolate from the commercial chiral building block ethyl (R)-4-cyano-3-hydroxybutanoate is described. Key steps in the synthesis of the target compounds involve the triethyl phosphite nucleophilic addition to chiral N-acyliminium ion easily obtained from (R)-4-hydroxypiperidin-2-one, giving diethyl (2R,4R)- and (2S,4R)-4-hydroxyphosphopipecolate diastereoisomers, easily separable by column chromatography followed by the 4-hydroxy epimerization through a sequential oxidation and highly diastereoselective reduction strategy, affording the diethyl (2R,4S)- and (2S,4S)-4-hydroxy-phosphopipecolate, respectively, consistent with our recently found results. All synthesized compounds were thoroughly characterized.