Column Chromatography Research Articles

Discovery of a novel nanomolar angiotensin-I converting enzyme inhibitory peptide with unusual binding mechanisms derived from Chlorella pyrenoidosa

Chlorella pyrenoidosa (C. pyrenoidosa) has been cultivated in large quantities, and was proven to be antihypertensive when consumed orally. However, the antihypertensive peptides derived from C. pyrenoidosa remains scarce. In this study, trypsin was chosen to prepare the hydrolysate of C. pyrenoidosa, which was then fractionated by column chromatography. And ninety-nine peptides were identified by LC-MS/MS, after which 10 peptides were chosen by docking-based virtual screening and demonstrated the ability to inhibit ACE. Among them, LVAKA (LV-5) had the lowest IC50 (26.66 μM). LV-5, LKKAP, and PGLRP were identified as non-competitive ACE inhibitory peptides that exhibited significant stability in the face of extreme pH and high temperatures. Insilico and in-vitro simulated gastrointestinal digestion revealed that these three peptides could release ACE inhibitory peptide fragments after digestion. The sequence optimization of LV-5 led to the identification of LRAKA (LR-5), which was recognized as a novel nanomolar ACE peptide with an IC50 of 350 nM in-vitro and a potent antihypertensive effect in-vivo. Moreover, molecular dynamic simulation indicated that LR-5 interacted with an unconventional binding site in ACE. These findings underscore the potential of Chlorella as a source of antihypertensive peptides and suggest a promising future for the use of Chlorella-derived peptides in the management of hypertension.

Antitrypanosomal and antileishmanial activity of compounds from some Nigerian plants

Ten compounds, six extracts and five fractions obtained from three Nigerian plants were assayed for their in vitro antitrypanosomal and antileishmanial activities. Each plant was extacted with hexane, ethyl acetate, and methanol. Isolated compounds were characterized and identified based on their NMR chemical shifts and comparison to literature reports. The crude extracts, fractions and isolated compounds were tested against the kinetoplastid parasites: bloodstream forms of Trypanosoma brucei Lister 427WT and the derived multi-drug resistant clone B48, and promastigote forms of Leishmania mexicana cas9/T7 and the derived clone cas9ΔNT1. Column chromatography of the extracts using silica gel yielded ten compounds identified as curzerenone, epi-curzerenone, chloranthene F, isofuranodienone, 8(17)-12E-labdadiene-15, 16-dial and 15-hydroxy-8(17),12E-labdadiene-16-al from Siphonochilus aetiopicus, lupeol, linalolic acid and spinasterone from Calliandra portoricensis, and abruquinone B from Abrus precatorius. The assay results showed that the Siphonochilus aetiopicus and Calliandra portoricensis crude extracts, fractions and compounds displayed moderate activity against the Trypanosoma brucei brucei but showed less activity against Leishmania mexicana. Abrus precatorius crude extract, fraction, and isolated compound exhibited only weak trypanocidal and leishmanicidal activities against both kinetoplastid parasites tested. These findings have provided evidence for the use of Siphonochilus aetiopicus and Calliandra portoricensis in traditional medicine relating to parasitic diseases.

Scale-up of Sodium Persulfate Mediated, Nitroxide Catalyzed Oxidative Functionalization Reactions.

Oxidation is a valuable tool in preparative organic chemistry. Oxoammonium salts and nitroxides have proven valuable as reagents and catalysts in this endeavor. The objective of this study is to scale up the oxidative amidation, ester formation, and nitrile formation using nitroxide as an organocatalyst. Oxidative functionalization reactions were scaled from the 1 mmol to the 1 mole level. Sodium persulfate was used as the primary oxidant, and a nitroxide was employed as a catalyst. The products of the reactions were isolated in analytically pure form by extraction with no need for column chromatography. The oxidative amidation and esterification of aldehydes can be scaled up from 1 mmol to 1 mole effectively, with comparable product yields being obtained at each increment. This work shows that conditions developed on a small scale can be transferred to a larger scale without reoptimization. The oxidative functionalization of aldehydes to prepare nitriles is not amenable to direct scale-up due to the concomitant formation of significant quantities of the corresponding carboxylic acid, thereby compromising the product yield. Two of the three oxidative transformations studied here can be scaled up successfully from the 1 mmol to the 1 mole level.

A polysaccharide from Periplaneta americana promotes macrophage M2 polarization, exhibiting anti-inflammatory and wound-healing activities

A miscellaneous polysaccharide, PAP55–3-1, with a molecular weight of 23.03 kDa, was isolated from Periplaneta americana through extraction with dilute alkali solution, ethanol precipitation, and column chromatography purification. Structural analysis shows that PAP55–3-1 is mainly composed of five monosaccharides: galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose. Its main glycosidic bonds are: Manp-(1→, Galp-(1→, →3)-Galp-(1→, →3,6)-Manp-(1→, →2,6)-Manp-(1→, →6)-Manp-(1→, →4)-Galp-(1→, →6-Glcp-(1→, →6)-Galp-(1→, →2)-Manp-(1 →, →3,4)-Glcp-(1→, →3,6)-Galp-(1→. In vitro experiments demonstrated that PAP55–3-1 can effectively inhibit reactive oxygen species (ROS) and O2− production following H2O2−induction. After H2O2−induction, HIF-1α (hypoxia-inducible factor) was translocated in mitochondria PAP55–3-1 increased localization of HIF-1α was located on mitochondria to maintain the stability of mitochondrial function stability, thereby effectively inhibiting H2O2-induced mitochondrial oxidative damage. Additionally, PAP55–3-1 inhibited the M1 polarization of macrophages stimulated by H2O2 and promoted the phenotype polarization of macrophages from M1 to M2, displaying anti-inflammatory and pro-repair properties. In vivo experimental results indicated that PAP55–3-1 promoted wound healing in mice. Immunohistochemical experiments revealed a reduction in CD68 expression and increase in CD206 expression in both positive and the high-dose polysaccharide group control group. This further demonstrated that PAP55–3-1 promotes the phenotype polarization of macrophages from M1 to M2, exerting anti-inflammatory and wound-healing activities.

Antibacterial Polyketides from the Plant Endophytic Fungus Fusarium sp.

Background: Endophytic fungi have been recognized as new sources of natural products with a variety of biological activities, providing lead compounds for drug discovery and development. Objective: The objective of this study is to isolate and identify the secondary metabolites from the plant endophytic fungus Fusarium sp. HJY2 and evaluate their antibacterial activities. Methods: The compounds were isolated and purified by the methods of silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high performance liquid chromatography (HPLC). The structures of the isolated compounds were elucidated by comparing the NMR and MS spectroscopic data with those of pieces of literature. The antibacterial activities were evaluated by the broth microdilution method. Results: Seven polyketides were isolated from the fermented extracts of the fungus Fusarium sp. HJY2 and identified as sydowinol (1), dihydrolateropyrone (2), 13-oxo-9Z,11E-octadecadienoic acid (3), (E)-ferulic acid (4), 4-hydroxyphenylacetic acid (5), methyl 2-(2-hydroxyphenyl)acetate (6) and 4-hydroxy-3-methoxyacetophenone (7). Compound 3 exhibited moderate antibacterial activities against Bacillus subtilis, Mycobacterium smegmatis, Ralstonia solanacearum, and Xanthomonas campestris pv. campestris with MIC values of 40, 40, 80 and 40 μg/mL, respectively. Conclusion: Seven compounds were isolated from the plant endophytic fungus Fusarium sp. HJY2. Compound 1 was isolated from the Fusarium genus for the first time. Compound 3 showed moderate antibacterial activities.

Macrocyclic anti-inflammatory diterpenes from the South China Sea soft coral Sinularia erecta and their isolation by combined column chromatography

Eleven new macrocyclic casbane-type diterpenes, namely sinueracasbanones E–O (1–11), have been isolated from the South China Sea soft coral Sinularia erecta using combined column chromatography. The structures of new compounds were elucidated by extensive spectroscopic data analysis, the modified Mosher's method, X-ray diffraction analysis and quantum mechanical calculation methods. In bioassay, compounds 2, 5, 10 and 11 displayed interesting anti-inflammatory activities by the inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α protein release with half maximal inhibitory concentration (IC50) values of 15.6, 7.1, 13.7 and 6.6 µM, respectively.

Determination of three methylimidazole compounds in cosmetics by high performance liquid chromatography-tandem mass spectrometry

Methylimidazole compounds are byproducts formed during the caramel-coloring process and are used in various cosmetics. In addition metronidazole is an antibacterial and anti-inflammatory drug commonly used in modern medicine and is used in cosmetics to treat acne in the short-term. The illegal addition of metronidazole during cosmetics production can result in residual 2-methylimidazole (2-MEI), which, along with 4-methylimidazole (4-MEI), is a class 2B carcinogen. Therefore, establishing efficient, accurate, and sensitive analytical techniques for analyzing methylimidazole compounds in cosmetics is an urgent objective. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining 1-methylimidazole (1-MEI), 2-MEI, and 4-MEI in cosmetics was developed. Cosmetics samples were extracted via ultrasonication in acetonitrile and purified using a mixed cation-exchange (MCX) solid-phase extraction (SPE) column, with subsequent drying under a stream of nitrogen and redissolution in acetonitrile. The resulting solution was then filtered through a 0.22 μm organic filter membrane for further testing. The analytes were separated using an XBridge® shield RP18 chromatographic column (150 mm×4.6 mm, 3.5 μm) and isocratically eluted with 20 mmol/L ammonium formate solution (containing 0.1% formic acid)-acetonitrile (98∶2, v/v). The target compounds were ionized by electrospray ionization (ESI) source, analyzed in multi-reaction monitoring (MRM) mode, and quantified using the external standard method, with the peak area of the quantitative ion and the mass concentration of the compound taken as the longitudinal and transverse coordinates, respectively. Matrix-matching working curves were also constructed. 1-MEI exhibited good linear relationships in the range of 5-200 μg/L, with correlation coefficients (r2)≥0.9994, while 2-MEI and 4-MEI showed good linearities in the range of 2-100 μg/L with r2≥0.9984. The three methylimidazole compounds exhibited limits of detection (LODs) and quantification (LOQs) of 10-30 μg/kg and 25-100 μg/kg, respectively. Under three spiked levels (LOQ, 2LOQ, 10LOQ), the recoveries of three methylimidazole compounds were 80.9%-107.9%, with relative standard deviations (RSDs, n=6) of 1.2%-12.8%. The practicability of the method was examined using 48 cosmetic samples; 4-MEI was detected in nine samples at contents of 26-1000 μg/kg, while two samples contained 240 and 267 μg/kg of 2-MEI, respectively. 1-MEI was not detected in any of the 48 samples tested. The developed method is simple, fast, and highly sensitive, and provides methodological support for assessing risks and monitoring the three methylimidazole compounds in cosmetics through screening.

Determination of 42 antimicrobials in disinfection products by dispersive solid phase extraction-high performance liquid chromatography-tandem mass spectrometry

Antimicrobials inhibit the growth and reproduction of microorganisms thereby alleviating skin and other problems caused by microorganisms. Antimicrobials are classified into different categories, including antibacterials, antifungals, and antivirals, among others, and include azoles, sulfonamides, tetracyclines, quinolones, and many other classes of synthetic and natural compound. The inappropriate or excessive use of antimicrobials can damage skin and other human organs and increase antimicrobial resistance. Relevant regulations and standards clearly state that antimicrobials are prohibited for use as ingredients in disinfection products. However, since antimicrobials enhance the disinfection or antibacterial effect of a product, with a significant short-term effect, antimicrobials are occasionally illegally added to disinfectant products, including those intended for human use. Therefore, establishing testing methods that provide technical support for enforcing regulations is an urgent objective. Herein, a method was established for the analysis of 42 antimicrobials in disinfection products, that is applicable to common types of disinfection-product matrix, including creams, gels, and aqueous solutions, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with dispersive solid phase extraction. The 42 antimicrobials comprise antibacterials, antifungals, and antivirals, and include seven sulfonamides, ten quinolones, three lincosamides, five tetracyclines, three macrolides, eight azoles, three purine nucleoside analogs, one furan, one nonpolyene antifungal, and one steroid. Briefly, 0.2 g of a sample was first dispersed in 2 mL of water and then extracted with 10 mL of 0.5% formic acid in acetonitrile, with 3 g of anhydrous Na2SO4 added to remove water. After centrifugation, 5 mL of the supernatant was cleaned using dispersive solid phase extraction with EMR-Lipid as the adsorbent. Lipids, waxes, surfactants, and moisturizing lubricants are commonly used as cream and gel matrices. Matrix substances containing long carbon chains dissolved in acetonitrile were removed using the EMR-Lipid adsorbent. Target analytes were separated on a Poroshell 120 EC-C18 analytical chromatography column (150 mm×3.0 mm, 2.7 μm), with 0.1% formic acid in acetonitrile and 0.1% formic acid aqueous solution used as mobile phases under gradient-elution conditions. The target analytes in the test solution were detected in positive ionization (ESI+) and multi-reaction-monitoring (MRM) modes. Analytes were characterized in terms of their retention times and selected ions, and quantified using the external-standard method. The main factors affecting method response, recovery, and sensitivity, such as the extraction method and solvent, purification method and adsorbent, mobile phase, and MS conditions, were examined during sample pretreatment and instrumental analysis. The 42 antimicrobials were effectively separated under the optimized experimental conditions; the target compounds exhibited linear working curves in the 0.25-5.0 mg/kg concentration range, with correlation coefficients (r) greater than 0.99. Limits of detection (LODs) for the 42 antimicrobials were determined from the signal-to-noise ratios (S/N) of their chromatographic peaks. LODs of 0.03-0.10 mg/kg were determined for the three matrices using 0.2-g samples and 10-mL test solution. Recoveries of 80.3-109.8%, with relative standard deviations (RSDs) of less than 9.8%, were obtained by determining three levels of each target analyte added to the three blank matrices; this process was repeated for six parallel samples. The developed method was used to analyze antimicrobials in commercially available disinfection products, with two sample batches testing positive. The established method is simple, accurate, precise, and suitable for the rapid screening and quantification of antimicrobials in disinfection products. This study provides powerful technical support for regulating the illegal addition of related antimicrobials to disinfection products.

Thiazoplanomicin, a new thiazolyl peptide antibiotic from the leaf-litter actinomycete Actinoplanes sp. MM794L-181F6.

A new bioactive substance was identified from a leaf-litter actinomycete strain by screening for antibacterial activity against Neisseria gonorrhoeae. The thiazolyl peptide antibiotic, named thiazoplanomicin, was isolated from the secondary metabolites of the leaf-litter actinomycetes Actinoplanes sp. MM794L-181F6 by extraction with n-butanol, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Thiazoplanomicin was characterized by LC-HR-ESI-MS, NMR, and X-ray analyses, along with analysis of the degradation products and chemical derivatives, and determined to be a nocathiacin-like multiple macrocyclic thiazolyl peptide. Thiazoplanomicin showed potent antimicrobial activity against gonococcal strains, including those resistant to known anti-gonococcal compounds such as telithromycin, azithromycin, and ceftriaxone, with MIC values ranging from 0.0312 to 0.125 µg ml-1. Such anti-gonococcal activity has not been reported on nocathiacin-like thiazolyl peptide antibiotic so far. Similar to other thiazolyl peptide antibiotics, thiazoplanomicin also showed potent antibacterial activity against Gram-positive bacteria with MIC values ranging from 0.0005 to 0.0156 µg ml-1 but showed no antibacterial activity against Escherichia coli.

Bioactivity-Guided Isolation of Secondary Metabolites from Camellia fascicularis: Antioxidative Antibacterial Activities and Anti-Inflammatory Hypoglycemic Molecular Docking

Camellia fascicularis is a valuable ornamental, edible, and medicinal plant with promising prospects for bioactivity development. We screened the bioactivity of eight fractions (Fr. A−I) obtained from the ethyl acetate phase of C. fascicularis via silica gel column chromatography. The results indicated that the anti-inflammatory, antioxidative, and antimicrobial active components were mainly found in Fr. B*, E, A, and H; Fr. A–G; and Fr. D–I, respectively. Bioactivity-guided isolation identified 18 secondary metabolites. Compounds 1, 3–5, 7, and 15–18 were isolated from the genus Camellia for the first time in this study, whereas the other compounds were also isolated from this plant for the first time. The structures of these compounds were elucidated through comprehensive spectroscopic techniques. Compounds 1, 9−11, 28, 30, and 31 demonstrated antioxidative activities comparable to those of ascorbic acid, whereas the remaining compounds exhibited diminished antioxidative activity. In terms of antimicrobial activity, compounds 7, 18, 22, and 27 exerted inhibitory potency against Pseudomonas aeruginosa, similar to tetracycline (MIC: 125 µg/mL). Other compounds showed moderate to weak inhibitory effects against Staphylococcus aureus and Escherichia coli (MIC: 250–500 µg/mL). Molecular docking revealed that compounds 2, 36, 41, and 65 showed strong binding affinity for 8ET0, whereas compounds 2, 36, 38, 40, 63, and 65 showed strong binding affinity for 3A4A. This research further increased the diversity of the secondary metabolites of C. fascicularis, laying a foundation for the subsequent development and utilization of this species.

The Structure Characterization of Polysaccharides from Codonopsis pilosula and the Structure-activity Relationship with Immune-regulation on THP-1 Cells.

In recent years, polysaccharides from Codonopsis pilosula (CPs) have received increasing attention for their excellent behaviors in immune-regulation. However, the relationship between the structure and immunomodulatory activity has rarely been reported. In this work, four fractions purified from crude CPs (CPW, CPS0.2, CPS0.5, CPS1) by chromatographic column separation were explored with both structure and immunomodulatory effects by THP-1 cells. The results showed that the monosaccharide composition, chain conformation, molecular weight (Mw) and aggregated state of CPs were different. At the same time, the immunomodulatory effects of CPs were also varied depend on structure differences, as indicated by the released cytokines of TNF-α and IL-1β by LPS-induced THP-1 cells. Finally, we summarized and concluded that the spherical structure and smaller particle size of CPs were the key factors attributed to better immune-enhancing effects. The results showed that the monosaccharide combination differences of polysaccharides might lead to anti-inflammatory or pro-inflammatory activity. Moreover, the higher percentage of glucose, the relatively large particle size, as well as the extended chain conformation of CPs might be the key factors attributed to better immune-enhancing effects. This study aims to provide a theoretical basis for establishing the structure-activity relationship of CPs.

Chemical composition of anti-microbially active fractions derived from extract of filamentous fungus Keratinophyton Lemmensii including three novel bioactive compounds

Screening for new bioactive microbial metabolites, we found a novel okaramine derivative, for which we propose the trivial name lemmokaramine, as well as two already known okaramine congeners – okaramine H and okaramine J - responsible for antimicrobial activity of the recently described microscopic filamentous fungus, Keratinophyton lemmensii BiMM-F76 (= CCF 6359). In addition, two novel substances, a new cyclohexyl denominated lemmensihexol and a new tetrahydroxypyrane denominated lemmensipyrane, were purified and characterized. The compounds were isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structures were elucidated by NMR and LC-MS. The new okaramine (lemmokaramine) exerted antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and fungi and anticancer activity against different mammalian cell lines (Caco-2, HCT116, HT29, SW480, MCM G1, and MCM DLN). Furthermore, we found a significant antioxidant effect of lemmokaramine following H2O2 treatment indicated by activation of the Nrf2 pathway. This is the first report describing analysis and structural elucidation of bioactive metabolites for the onygenalean genus Keratinophyton.

Bioassay‐Guided Extraction and Isolation of Natural Herbicides from Dried Zanthoxylum limonella Alston Fruits

Weeds are problematic plant species around the world. Various strategies exist for controlling weeds, but chemical treatment remains the preferred method, particularly when using natural substances. In this research, a crude aqueous‐methanol extract from dried Zanthoxylum limonella fruits was acid‐base partitioned into four fractions: neutral extract (NE), acid extract (AE), basic extract (BE), and aqueous extract (AQ). These fractions were further separated into seventeen subfractions: NEF1 to NEF7, AEF1 to AEF5, and BEF1 to BEF5, which were then tested for herbicidal activity against the growth of Chinese amaranth (Amaranthus tricolor) and barnyard grass (Echinochloa crus‐galli). Active subfractions were isolated via column chromatography and identified using spectroscopic methods, yielding seven active compounds: xanthoxyline (1), tambulin (2), atanine (3), prudomestin (4), skimmianine (5), p‐methoxybenzoic acid (6), and methyl caffeate (7). Compounds 2–7 had not been previously reported in Z. limonella. Xanthoxyline (1) was identified as the most potent botanical herbicide, fully inhibiting seed germination of Chinese amaranth and barnyard grass. This compound also decreased seed imbibition and α‐amylase activity in both species. Molecular docking studies on the α‐amylase enzyme (PDB ID: 1BG9) revealed that the aromatic, hydroxy, and carbonyl groups of xanthoxyline (1) interact with the enzyme's active sites.

Antimicrobial potential of Hippocratea Indica Willd. Acetone Leaf fractions against Salmonella Typhi: an in vitro and in silico study

Salmonella Typhi is a major global concern in many low- and middle-income countries. In addition, the emergence and persistence of drug resistant strains has increased the impact of this disease. Plant metabolites have been explored traditionally and scientifically as antimicrobial agents. Thus, this study was designed to investigate the antimicrobial potential of acetone leaf fractions of H. indica against S. Typhi. Dried pulverized leaves of H. indica were extracted using cold maceration with acetone after defatting with n-hexane. The leaf extract was concentrated and subjected to column chromatography and eight bioactive fractions were identified. The fractions were characterized using gas chromatography-mass spectrometry. The fractions were evaluated for antibacterial activity against Salmonella Typhi in-vitro and in-silico. The lowest MIC was observed in fractions 20 and 21 (0.375 mg/mL) while the lowest MBC was observed in all fractions except 7, 17 and 18 (0.375 mg/mL). A ligand from fraction 8 had the highest binding affinity to Type I dehydroquinase (-3.4) and a ligand from fraction 7 had the highest binding affinity to Gyrase B (-11.2). This study concludes that the overall antimicrobial activity of the acetone leaf extract of H. indica provided evidence that it contains drug-like compounds that can be further explored as a drug candidate against S. Typhi.

Preparation, identification and α-glucosidase inhibitory activity of a high-methoxyl HG-type pectin from Liangping pomelo (Citrus maxima cv. Liangpin Yu) peel.

The peel from Liangping pomelo (Citrus maxima cv. Liangpin Yu) is generally discarded as waste during post-harvest handling and process, resulting in environmental pollution and waste. Pectin is the major component in pomelo peels and yields significant economic advantages. Thus, developing pomelo peel pectin (PPs) might be a feasible strategy to reduce environmental pollution caused by pomelo peel. The optimized PPs yield was 156.5 ± 2.5 g kg-1 under the inoculum size of 100 mg g-1, liquid-solid ratio of 31 mL g-1, fermentation time of 64 h, and fermentation temperature of 39 °C. PPs-6Aa, a pectin fraction from PPs purified with DEAE-52 cellulose, Sephadex G-100 and Sephadex G-75 column chromatography, showed higher α-glucosidase inhibitory activity, with an IC50 of 0.12 ± 0.03 mg mL-1. It was a high-methoxyl HG-type pectin of 42.8 kDa, and its repeat unit was →4)-α-GalpA-6-OMe-(1→4)-α-GalpA-6-OMe-(1→. Additionally, its α-glucosidase inhibitory activity might be related to hydrogen bonds formed with Lys-156, Glu-277, His-280, Asp-307, Arg-315, Asn-350, Asp-352 and Glu-411, and to hydrophobic interactions formed with Ser-157, Tyr-158, Asp-233, Gln-239, Ser-240, Phe-303, Thr-306, Leu-313, Phe-314, Gln-353 and Arg-442. These findings provide structural and bioactivity information on pectin from Liangping pomelo peel, which could be beneficial for the development of functional foods and pharmaceuticals. © 2024 Society of Chemical Industry.

Simultaneous analysis of depurinated nucleic acid stem-loop and free adenine for ricin toxicity assay by hydrophilic interaction liquid chromatography-high-resolution mass spectrometry (HILIC-HRMS).

A simple, accurate method for measuring ricin activity was developed by detecting depurinated nucleic acid stem-loops and adenine using a commercially available hydrophilic interaction liquid chromatography (HILIC) column and a quadrupole-Orbitrap tandem mass spectrometer. Ricin in beverages was isolated using magnetic beads conjugated with ricin B-chain antibodies, and then incubated with a 14 mer RNA or a 12 mer RNA/DNA chimera, in which adenosine at the depurination site of RNA was replaced by deoxyadenosine. The adenine and depurinated nucleic acids were separated by HILIC and both analytes were detected by high-resolution mass spectrometry. The depurinated RNA was detectable at concentrations as low as 100 pM (6.5 μg mL-1) in orange juice and coffee, and 10 pM (0.65 μg mL-1) in milk and sake after incubation with the RNA substrate for 4 h. Free adenine was detectable at 10 pM in all matrices, although free adenine was also detected in all blanks and could not be distinguished from the coffee and orange juice blanks at 10 pM. When using the chimera as the substrate, the depurinated chimera and adenine were detected up to concentrations of 10 pM as larger peaks. However, since the depurinated chimera and adenine were also detected in blanks, careful judgment was needed to determine whether they were active. Following the assay, the captured ricin could be analyzed by enzymatic digestion and nano liquid chromatography-high-resolution mass spectrometry. The ricin A chain-specific T7A peptide was detectable at 10 pM for sake and at 100 pM for milk, orange juice, and coffee. Using the present method, a toxicity assay and qualitative analysis of ricin were feasible with a 0.2 mL beverage sample.

Cleaning Method Validation for Determination of Residues in Anti-Cancer Drug product for Injection by Using RP-HPLC Technique

Cleaning method validation is a part of good practices in pharmaceutical sector and need to seek the contamination present in production area. OBJECTIVE: An RP-HPLC method for the cleaning samples analysis for Anti-Cancer Drug product Melphalan Hydrochloride for Injection was develop and validated. The method was developed and validated for quantitation of Cleaning of residue left in the vessels of the production equipment’s. The analytical method used to determine the complete cleaning for Melphalan Hydrochloride for Injection by HPLC method. METHOD: The Cleaning validation was performed on Inertsil ODS,C18,(150mm×4.6mm,5μm) chromatographic column temperature at 40°C, and the detection wavelength was set at 260nm. Mobile Phase-A:10mL ammonium acetate, 88mL Purified water, 10mL acetic acid, 2mL Triethylamine, and Mobile Phase-B: 1mL Buffer, 10mL acetonitrile,190mL Purified water, and at a flow rate of 1.5 mL/min, and the injection volume was 10μL. RESULTS: The validation results of Melphalan Hydrochloride were linear over the concentration from 0.10 µg/mL to 0.75µg/mL (r = 1.00) and the recoveries from LOQ to 150% and precision ranged from 50% to 76% (RSD < 30%, n =9). CONCLUSION: This method has been validated successfully and all the results met the acceptance criteria. Hence method of Cleaning Validation is suitable for testing of residue sample of Melphalan Hydrochloride for Injection.

A New LC-MS/MS-Based Method for the Simultaneous Detection of α-Tocopherol and Its Long-Chain Metabolites in Plasma Samples Using Stable Isotope Dilution Analysis

Background: Our study presented a novel LC-MS/MS method for the simultaneous quantification of α-tocopherol (α-TOH) and its phase II metabolites, α-13′-COOH and α-13′-OH, in human serum using deuterium-labeled internal standards (d6-α-TOH, d6-α-13′-COOH, d6-α-13′-OH). Methods: The method addresses the analytical challenge posed by the significantly different concentration ranges of α-TOH (µmol/L) and its metabolites (nmol/L). Previous methods quantified these analytes separately, which caused an increase in workflow complexity. Results: Key features include the synthesis of stable isotope-labeled standards and the use of a pentafluorophenyl-based core-shell chromatography column for baseline separation of both α-TOH and its metabolites. Additionally, solid phase extraction (SPE) with a HybridSPE® material provides a streamlined sample preparation, enhancing analyte recovery and improving sensitivity. By utilizing deuterium-labeled standards, the method compensates for matrix effects and ion suppression. This new approach achieves precise and accurate measurements with limits of detection (LOD) and quantification (LOQ), similar to previous studies. Calibration, accuracy, and precision parameters align well with the existing literature. Conclusions: Our method offers significant advantages in the simultaneous analysis of tocopherol and its metabolites despite concentration differences spanning up to three orders of magnitude. In contrast to earlier studies, which required separate sample preparations and analytical techniques for tocopherol and its metabolites, our approach streamlines this process. The use of a solid-phase extraction procedure allows for parallel sample preparation. This not only enhances efficiency but also significantly accelerates pre-analytical workflows, making the method highly suitable for large-scale studies.

In Vitro Antitumor, Antioxidant, and Hemolytic Activities of Chlorella sorokiniana Methanol Extracts and Collective Fractions

Chlorella species are fast-growing microalgae with significant industrial applications. The aim of the present study was to investigate the antitumor, antioxidant, and hemolytic activities of Chlorella sorokiniana UTEX 1230 crude methanol extracts and fractions. Ch. sorokiniana crude methanol extracts and collective fractions (CFs) were obtained from lyophilized biomass by maceration and column chromatography. Antitumor assays against murine lymphoma L5178Y-R and human breast cancer MCF-7 cells were performed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction technique, using human peripheral blood mononuclear cells (PBMC) as the control group. Antioxidant and hemolytic activities were evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH) and erythrocyte hemolysis, respectively. We showed that crude methanol extracts (IC50) increased L5178Y-R and MCF-7 cell growth inhibition, without affecting PBMC. In addition, all evaluated CFs showed significantly higher antioxidant activity than the positive control (ascorbic acid). CF3 and CF4 showed the highest cytotoxicity against L5178Y-R, whereas CF3, CF4, and CF5 caused the highest antitumor activity against MCF-7 cells. CF3, CF4, and CF5 induced significantly higher hemolytic activity compared with all other fractions. CF characterization revealed loliolide, cinnamic acid, methyl dihydrojasmonate, salsalvamide A, 1-monolinolenin, cryptophycin 29, costunolide, riboflavin lumicrome, and germicidin B, which have been related to antitumor and antioxidant activities. In conclusion, we demonstrated that Ch. sorokiniana extracts and fractions possess antitumor and antioxidant potential, without affecting human erythrocytes and PBMC.

Isolation and Characterization of Stigmasterol from Psidium guajava Leaves: A Promising Bioactive Compound with Therapeutic Potential

This study aimed to isolate and characterize bioactive compounds from the leaves of Psidium guajava Linn. The hydroethanolic extract yielded 32% (80 g) from the crude material, which was subjected to column chromatography for fractionation. Structural elucidation of the isolated compound was performed using 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy, including COSY, HSQC, and HMBC experiments. The spectral data confirmed the presence of stigmasterol, characterized by its distinct chemical shifts and long-range correlations between carbon and proton atoms. Stigmasterol was isolated as a white, crystalline solid and identified by its unique physio-chemical properties, such as solubility in organic solvents, a melting point of 163-164°C, and molecular formula C29H48O. This compound is known for its significant biological activities, including antibacterial, antimalarial, anti-inflammatory, antioxidant, and anticancer effects. By employing advanced techniques like 1D and 2D NMR spectroscopy, the study provides precise structural elucidation of stigmasterol, contributing to natural product research and reinforcing the medicinal potential of guava leaves as a source of pharmacologically active compounds. This adds to the existing knowledge of the plant’s bioactive components and their applications in medicinal chemistry