Semi-preparative HPLC Research Articles

Sesquiterpenoids from Aquilariae Lignum Resinatum

Eight sesquiterpenoids were isolated from petroleum ether extract of Aquilariae Lignum Resinatum by various column chromatography techniques including silica gel, ODS, and semi-preparative HPLC. Their structures were identified on the basis of physicochemical properties, UV, IR, MS, and NMR spectroscopic data as(4S,5S,7R,10S)-5,7-dihydroxy-11-en-eudesmane(1),(7R,10S)-eudesma-4-en-11,15-diol(2),(2R,4S,5R,7R)-2-hydroxyeremophila-9,11-dien-8-one(3), 7α-H-9(10)-ene-11,12-epoxy-8-oxoeremophilane(4),(+)-9β,10β-epoxyeremophila-11(13)-en(5), 4(14)-eudesmene-8α,11-diol(6), 12,15-dioxo-selina-4,11-dien(7), and 2β,8 aα-dihydroxy-11-en-eremophilane(8). Compounds 1 and 2 are new compounds, and their absolute configurations were determined by calculating ECD. Compounds 1, 4, and 6-8 could significantly improve taurocholic acid(TCA)-induced gastric mucosal GES-1 cell injury at a concentration of 20 μmol·L~(-1), and the cell protection rates were 23.51%±2.79%, 16.10%±1.25%, 24.45%±4.89%, 17.48%±2.93%, and 21.44%±2.39%, respectively.

Purification, Identification, and Properties of a Novel Carotenoid Produced by Arthrobacter sp. QL17 Isolated from Mount Qomolangma.

The genus Arthrobacter is a source of many natural products that are critical in the development of new medicines. Here, we isolated a novel carotenoid from Arthrobacter sp. QL17 and characterized its properties. The carotenoid was extracted with methanol, and purified by column chromatography and semi-preparative HPLC. Based on micrOTOF-Q and NMR analyses, the pigment was chemically characterized as 2,2′-((((1E,3E,5E,7E,9E,11E,13E,15E,17E,19E)-3,7,14,18-tetramethylicosa-1,3,5,7,9,11,13,15,17,19-decaene-1,20-diyl)bis(2,2,4-trimethylcyclohex-3-ene-3,1-diyl)) bis(ethan-2-yl-1-ylidene))bi(propane-1,3-diol), and named arthroxanthin. The biological activities of arthroxanthin were evaluated with DPPH, ABTS and MTT assays. Arthroxanthin exhibited excellent radical scavenging properties, as shown for 2, 20-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-n-(3,2-ethyl-benzothiazole-6-sulfonic acid) ammonium salt (ABTS), respectively, with IC50s of 69.8 and 21.5 µg/mL. It also showed moderate anticancer activities against HepG2, Hela, MDAB-231, SW480, and MKN-45 with IC50 values of 107.6, 150.4, 143.4, 195.9, and 145.5 μg/mL, respectively. Therefore, arthroxanthin derived from Arthrobacter sp. QL17 may be a potent antioxidant and anticancer agent for food and pharmaceutical use.

Peri-Decoration of a Tetraazaperylene with Urea Units: Chiral Octaazaperopyrenedioxides (OAPPDOs) and Their Optical and Chiroptical Properties.

Octaazaperopyrenedioxides (OAPPDOs) are a new class of fluorescent polycyclic aromatic hydrocarbons based on a tetraazaperylene core that is formally condensed with N-substituted urea units in the two opposite peri positions. Here, we report the synthesis of series of substituted OAPPDO derivatives with different N-substitution patterns (H, alkyl, benzyl) in the peri positions, including bay-chlorinated OAPPDOs. Starting from the latter, a series of bay-arylated OAPPDOs was synthesized by Suzuki cross coupling, which resulted in the formation of helically chiral OAPPDO derivatives. The electrochemical and photophysical properties were investigated by UV/Vis and fluorescence spectroscopy as well as cyclic voltammetry. The P and M enantiomers of a phenylated OAPPDO were separated by semipreparative HPLC and further analyzed by CD spectroscopy. The frontier orbital energies, the mechanism of the isomerization, the electronic excitation and the CD spectrum (TD-DFT) were computed and compared to the experimental data. The reversible 1e- oxidation of the OAPPDOs generates the corresponding radical cations, one of which was characterized by EPR spectroscopy. The reversible oxidation process was also systematically investigated by spectro-electrochemistry.

Screening and Research on Skin Barrier Damage Protective Efficacy of Different Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) may prevent skin barrier damage, although their protective mechanisms and active monomeric constituents remain unclear. Here, three MELs were extracted from Candida antarctica cultures containing fermented olive oil then purified using silica gel-based column chromatography and semipreparative HPLC. All three compounds (MEL-A, MEL-B, MEL-C) were well separated and stable, and reliable materials were used for NMR and HRESIMS chemical structure determinations and for assessing MELs’ protective effects against skin damage. Notably, MEL-B and MEL-C effectively protected HaCaT cells from UVB-induced damage by upregulating the contents of filaggrin (FLG) and transglutaminase-1 (TGM1), as determined via ELISA. Moreover, MEL-B treatment (20 μg/mL) of UVB-irradiated HaCaT cells led to the upregulation of both the expression of mRNA genes and the key proteins FLG, LOR, and TGM1, which are known to be decreased in damaged skin cells. Additionally, histopathological analysis results revealed a markedly reduced intracellular vacuolation and cell damage, reflecting improved skin function after MEL-B treatment. Furthermore, immunofluorescence results revealed that MEL-B protected EpiKutis® three-dimensional cultured human skin cells from sodium dodecyl sulfate-induced damage by up-regulating FLG, LOR, and TGM1 expression. Accordingly, MELs’ protection against skin barrier damage depended on MEL-B monomeric constituent activities, thus highlighting their promise as beneficial ingredients for use in skin-care products.

High-performance thin-layer chromatography – antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)

The present work introduces a high-performance thin-layer chromatography (HPTLC)–direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC–EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC–mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform – ethyl acetate – methanol 15:3:2, V/V/V) was changed (to n-hexane – isopropyl acetate – methanol – acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1–2 and 3–4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3–64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Antidepressant alkaloids from the rhizomes of Corydalis decumbens.

Two new alkaloids (1 and 2), named 1,7-dimethoxy-2'-prenyl-1',9-dihydropyrrolo-carbazole (1) and 1,7-dimethoxy-4',5'-dimethylcyclopenta-carbazole-1',3'-dione (2), along with thirteen known alkaloids (3-15) were isolated by means of silica gel, sephadex LH-20, and semi-preparative HPLC from the CHCl3 extraction of Corydalis decumbens for the first time. Their structures were determined by NMR, MS, IR, UV, and related references. Compounds (1-15) were evaluated for their antidepressant activities by measuring inhibition of monoamine neurotransmitter reuptake in vitro. Among them, compounds 1, 2, 4, and 6 showed certain antidepressant activities.

Chemical component and in vitro protective effects of Matricaria chamomilla (L.) against lipopolysaccharide insult

Ethnopharmacological relevanceChamomile (Matricaria chamomilla L.) is a popular herbal tea for the treatment of hepatitis and cholecystitis in traditional Uygur medicines. Aim of the studyTo investigate the anti-inflammatory activity and chemical composition of M. chamomilla, and clarify its molecular mechanism. Materials and methodsM. chamomilla was extracted with 75% ethanol and then extracted with different solvents to obtain five fractions, namely petroleum ether fraction (EOPE), dichloromethane fraction (EOD), ethyl acetate fraction (EOEA), n-butanol fraction (EOB), and water fraction (EOW). Cytotoxicity and the effect on the nitric oxide (NO) production of RAW264.7 cells induced by LPS of the five fractions were screened, and the most active one (EOD) was selected for further investigations. The components of EOD were identified by LC-MS/MS analysis in combination with comparison of retention time and UV absorption with authentic compounds by HPLC. In addition, five most abundant compounds of EOD were isolation by column chromatography and semi-preparative HPLC and their structures were further confirmed by HRMS and NMR data analysis and comparison with data in literatures. Then the underlying anti-inflammatory mechanism of EOD were predicted through Network pharmacology using the identified compounds from EOD, and further verified by Western Blot and ELISA experiments. ResultsEOD showed the most significant inhibition ratio against NO in RAW264.7 cells without toxicity among the tested five fractions. Thirty-seven compounds including flavonoid-O-glycoside, flavonoid aglycone, methylated flavonoid aglycone, phenolic acid, coumarin, sesquiterpene, and triterpene were identified from EOD by LC-MS/MS and comparison with authentic compounds. The five most abundant compounds in EOD were isolated and determined to be axillarin (26), tricin (30), chrysoeriol (31), centaureidin (33) and chrysosplenetin (35). IL-6, NF-κB, ERK1 and ERK2 cascade, TNF were the most important anti-inflammatory targets of EOD predicted by Network pharmacology. Western Blot and ELISA experiments revealed that EOD significantly decreased the protein expression levels of inflammatory factors (PGE2, MCP-1, IL-6, TNF-α), iNOS, COX-2, NF-κB (p-P65 and p-IκBα), MAPKs (p-p38, p-ERK and p-JNK), and increased the protein expression levels of Nrf2, HO-1 and CYP2E1. In addition, EOD blocked the p65 protein into the nucleus and promoted the nuclear translocation of Nrf2 in RAW264.7 cells induced by LPS. ConclusionM. chamomilla exerted anti-inflammatory effect via NF-κB, MAPK and Nrf2/HO-1 pathways. It could be further applied as a safe anti-inflammatory agent from natural source.

Abstract 1074: Cellular uptake and metabolism of MT1-MMP-activated taxane prodrugs

Abstract ICT3205 is a targeted cytotoxic agent, comprised of an MT1-MMP-specific peptide and a paclitaxel warhead. Paclitaxel, originally approved in 1992, is one of the most commonly used chemotherapies for various cancer types, but is often characterized by its dose-limiting toxicity and low aqueous solubility. Tumor-targeted prodrugs are a promising strategy for the selective delivery of taxanes to prostate tumors, especially given the results of STAMPEDE trial1. Previous work has shown that ICT3205 is selectively metabolized in tumor tissue over the normal tissue, and that this is specifically mediated by MT1-MMP2. In vivo studies have demonstrated that when administered at an equimolar dose to paclitaxel, ICT3205 has a superior anticancer effect, with an absence of systemic toxicity. In this study we aim to further investigate the cellular uptake and metabolism of ICT3205 and other taxane analogues in MT1-MMP +ve (HT1080) and MT1-MMP -ve (MCF-7) cells in vitro. The compound will be also studied in the PC3 prostate cancer cell line, since it has been demonstrated before that there is a good correlation between MT1-MMP protein and mRNA expression. ICT3205 and analogues have been synthesized, and purified by semi-preparative HPLC. We report the tumor-specific metabolism of ICT3205 in prostate tumors ex vivo, and we are currently studying its cellular uptake and metabolism in MT1-MMP +ve and MT1-MMP -ve cancer cell-lines using HPLC, mass spectrometry and immunofluorescent microscopy. Moreover, ex vivo metabolism studies are performed on the analogues, while they will be also assessed for their metabolism and cellular uptake. These data will provide a better understanding of the mechanism of uptake and metabolism of this type of peptide prodrugs, and will provide the basis for the future synthesis and optimization of these prodrugs. 1.James ND et al. Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomized controlled trial. Lancet. 2016;387(10024):1163-772.Loadman PM et al. Improved delivery of paclitaxel to prostate tumors: a membrane-type matrix metalloproteinase (MT-MMP)-targeted approach. Cancer Res. 2016;76:3 Citation Format: Athina Polykandritou, Alexandra Serre, Hannah Spencer, Goreti R. Morais, Amanda Race, Steven Shnyder, Paul Loadman, Robert A. Falconer. Cellular uptake and metabolism of MT1-MMP-activated taxane prodrugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1074.

Abstract 2463: 18F-Talazoparib: A novel potential PET imaging agent of PARP

Abstract Background: Poly-(ADP-ribose) polymerases (PARPs) are an enzyme family that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to an acceptor protein. PARP family members play fundamental roles in single-strand DNA break (SSB) repair, cell signaling of DNA damage and inflammation, cell death, and cellular replication. Various PARP inhibitors have been developed and PARP1 inhibitors are currently used to treat subsets of breast and ovarian cancer patients. Of all the PARP inhibitors studied, Talazoparib (Pfizer), appears to be the most stable in pre-clinical and clinical studies, affords nanomolar affinity to PARP1, and demonstrates the highest affinity trapping of PARP on the DNA-protein complex. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, therapy dose optimization and validation of target engagement. Several PARP imaging agents have been developed and studied as possible tracers, by modifying the structure of an original PARP inhibitor to accommodate an imaging or therapeutic isotope. Herein we used a copper-mediated 18F-radio-fluorination strategy with a novel aryl boronic ester precursor to access 18F-Talazoparib (18F-TZ), a 18F-radiolabeled form of the structurally identical Talazoparib. Methods: A novel boronic ester precursor was synthetized in six steps synthesis with an overall yield of 3.3% and characterized by NMR and mass spectrometry. Radiofluorination was performed using an automatic GE TracerLab system and analytical radioHPLC was used for tracer characterization and for stability analysis. A panel of tumor cell lines representing a variety of PARP expression levels (Hela, SUM149, SUM1315, MDA-MB-436, MDA-MB-231, PSN1, MiaPaca2, Jurkat) were tested for time-dependent uptake and specific binding, utilizing both 4 °C and excess cold TZ blocking. Results: 18F-TZ was synthetized in 5.0±0.80% (n=20) radiochemical yield and a molar activity of 18.9-98.0 GBq/µmol by a copper-mediated radiofluorination of the corresponding pinacolic boronic ester, followed by purification via semi-preparative HPLC and reformulation in ethanol. 18F-TZ was obtained in >99% radiochemical purity, displaying up to 4 hr shelf stability (25 °C and 4 °C) in PBS (10% EtOH). 18F-TZ was also stable in human plasma up to 4 hr at 37 °C. Both shelf and plasma stability tests showed a decrease of the radioHPLC peak consistent with decay half-life, but did not show the formation of secondary species. All cell lines selected for the in cellulo study showed uptake of radioactive 18F-TZ after 30 min of incubation at 37 °C. Cell uptake was blocked >98% by excess cold TZ or incubation of cells at 4 °C. Conclusion: 18F-Talazoparib was synthesized by copper-mediated radiofluorination from the corresponding pinacol ester and demonstrates both uptake and target specificity in PARP-expressing tumor cells. Citation Format: Riccardo Muzzioli, Federica Pisaneschi, Yi Rao, Seth Gammon, David Piwnica-Worms. 18F-Talazoparib: A novel potential PET imaging agent of PARP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2463.

Chemical constituents of Combretum dolichopetalum: Characterization, antitrypanosomal activities and molecular docking studies

Purpose: To evaluate the in vitro activities of a gallic acid ester and two apigenin flavone glucoside constituents of Combretum dolichopetalum against Trypanosoma brucei brucei s427 (Tbs427) and Trypanosoma congolense IL3000 (Tc-IL3000), and their interactions with a lysosomal papain-like cysteine protease (CP) enzyme in silico.Methods: Anti-trypanosomal activity-guided separation of ethyl acetate fraction using column chromatographic (CC) technique and purification of the CC sub-fractions with semi-preparative HPLC yielded three (1-3) compounds. The structures were characterized based on nuclear magnetic resonance (NMR) spectroscopic analyses and tested for activities against Tbs427 and Tc-IL3000. All the compounds were subjected to molecular docking studies for the inhibition of trypanosomal cathepsin B (TbCatB) CP.Results: An ester (1), a butyl gallate, and two positional isomeric apigenin flavone glucosides (2, 3) were identified. The compounds 2 (vitexin) and 3 (isovitexin) showed low in vitro IC50 against the tested parasites. However, 2 (IC50, 25 μM) was more potent than 3 (IC50, 68 μM) against Tbs427 while both were equipotent (IC50 = 2, 11.5 μM and 3, 10.8 μM) against Tc-IL3000. Compound 1 (butyl gallate) showed higher activity against Tc-IL3000 (IC50 = 0.80 μM) than to Tbs427 (IC50, 2.72 μM). The molecular docking study showed that all the compounds had minimum binding energies with a higher affinity towards the active pocket of TbCatB compared to the controls and native inhibitor (CA074).Conclusion: The relatively high in vitro activities and their strong affinity for TbCatB support the need for further optimization of the compounds towards lead identification against animal trypanosomiasis.

Structural elucidation of major degradation products of milbemycin oxime drug substance using LC-MS and NMR

Milbemycin oxime (MO) drug substance is a 16-membered macrocyclic lactone that exhibits a broad spectrum of biological activity and high potency towards parasites. In this study, a comprehensive forced degradation study was carried out on MO drug substance to identify and characterize its major degradation products (DPs). MO drug substance was subjected to acid, base, oxidation (H2O2), heat (solid and solution state), and photolytic (solid and solution state) stress degradation as per the ICH guidelines. Chromatographic separation of the drug substance (MO A3 and MO A4) and its DPs was achieved using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water/acetonitrile (60/40, v/v) and mobile phase B consisted of ethanol/isopropanol (50/50, v/v). A total of twelve major DPs were observed for MO drug substance under various stress conditions. These DPs were further identified and characterized using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with MO A4 and MO A3 using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-dihydroperoxide MO A4) was isolated using semi-preparative HPLC and characterized by comparison of its nuclear magnetic resonance spectroscopy data with MO A4. The proposed structures of the DPs have been rationalized by appropriate degradation pathways for MO A4 and MO A3.

Two novel enantiomers from metarhizium flavoviride and their inhibitory activities against α-glucosidase

A pair of novel enantiomers metarrhiziumoids A (R-enantiomer, 14.3 mg) and B (S-enantiomer, 9.5 mg) were isolated from 3.0 g extract of Metarhizium flavoviride by silica gel column chromatography combined with preparative and semi-preparative HPLC, and identified with high resolution mass, 1D, 2D NMR and circular dichroism. Targets prediction showed that the two compounds had glycosidase inhibitory activity. Result of α-glycosidase inhibitory tests showed that the both enantiomers were competitive inhibitors of α-glycosidase, and the bioactivity of metarrhiziumoids B (IC50 = 174 μg/mL) was stronger than that of metarrhiziumoids A (IC50 = 259 μg/mL). Molecular docking results show that the two enantiomers have different optimal docking modes and the binding energy for the S-enantiomer (-7.8 kcal/mol) was lower than for the R-enantiomer (-7.20 kcal/mol). The docking mode selections and binding energy differences resulted in the inhibitory activity difference.

Indole Alkaloids and Chromones from the Stem Bark of Cassia alata and Their Antiviral Activities.

The Cassia (Leguminosae) genus has attracted a lot of attention as a prolific source of alkaloids and chromones with diverse structures and biological properties. The aim of this study is to screen the antiviral compounds from Cassia alata. The extract of the stem bark of this plant was separated using silica gel, MCI, ODS C18, and Sephadex LH-20 column chromatography, as well as semi-preparative HPLC. As a result, three new indole alkaloids, alataindoleins A–C (1–3); one new chromone, alatachromone A (4); and a new dimeric chromone-indole alkaloid, alataindolein D (5) were isolated. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Interestingly, alataindolein D (5) represents a new type of dimeric alkaloid with an unusual N-2−C-16’ linkage, which is biogenetically derived from a chromone and an indole alkaloid via an intermolecular nucleophilic substitution reaction. Compounds 1–5 were tested for their anti-tobacco mosaic virus (TMV) and anti-rotavirus activities, and the results showed that compounds 2–4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates were higher than those of the positive control (with inhibition rate of 32.8%). Compounds 1 and 5 also showed potential anti-TMV activities with inhibition rates of 26.5% and 31.8%, respectively. In addition, compounds 1–5 exhibited potential anti-rotavirus activities with therapeutic index (TI) values in the range of 9.75~15.3. The successful isolation and structure identification of the above new compounds provided materials for the screening of antivirus drugs, and contributed to the development and utilization of C. alata.

Isoquinoline alkaloids from two species of Thalictrum genus plants

The chemical constituents from the roots of Thalictrum cultratum and T. baicalense were investigated. By various isolation methods, such as silica gel, aluminium oxide, ODS, and Sephadex LH-20 column chromatographies, and semi-preparative HPLC, 11 simple isoquinoline alkaloids were isolated from the ethanol extract of the roots of these two plants, including a new compound, named dehydrothalflavine(1), and ten known ones(2-11): N-methylcorydaline(2), N-methylthalidaldine(3), thaliflavine(4), oxyhydrastinine(5), noroxyhydrastinine(6), dimethoxyisoquinolone(7), thalactamine(8), dehydronoroxyhydrastinine(9), 6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline(10), and isopicnarrhine(11). Their structures were elucidated on the basis of HR-ESI-MS and 1 D and 2 D NMR techniques. Compound 1 was a new isoquinoline alkaloid. Compound 11 was obtained from Tha-lictrum plant for the first time. All compounds did not show cytotoxic activities against HL-60, U937, HCT116, Caco-2, and HepG2 cancer cell lines.

Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea

Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC50 58.91 ± 1.35 μ M ), moderate anti-inflammatory activity (IC50 254.79 ± 1.41 μ M ), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58 μM), moderate tyrosinase inhibition (IC50 1713.69 ± 8.65 μ M ), and moderate cytotoxicity against oral cancer (IC50 13.65 ± 0.02 μ M ), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.

Identification and characterization of major degradation products of Eprinomectin drug substance including degradation pathways using LC-HRMS and NMR

Eprinomectin (EPM) is a semi-synthetic potent antiparasitic drug widely used in veterinary medicine. In this study, a comprehensive forced degradation study was carried out on EPM drug substance as per ICH guidelines. Generation of adequate quantities of major degradation products of EPM via forced degradation studies was necessary for identification, structure elucidation, and understanding its degradation mechanism and degradation pathways. EPM drug substance was subjected to acid, base, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation. The degradation products (DPs) formed in the stressed degraded samples were successfully separated using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water and mobile phase B consisted of ethanol/isopropanol (98/2, v/v). A total of six major DPs of EPM drug substance formed under various stress conditions. The chemical structures of DPs were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized through comparison of their fragmentation profile with EPM B1a using tandem mass spectrometry (MS/MS). Additionally, two solvates (methanol adduct B1a #1 and methanol adduct B1a #2) were observed during the acid-stressed degradation study of EPM in presence of methanol. To confirm the chemical structure, these products were isolated with semi-preparative HPLC and characterized by using a combination of LC-MS/MS and nuclear magnetic resonance spectroscopy. The elucidated chemical structure of the degradation products of EPM was also justified through mechanistic explanations. Identification and characterization of the DPs including degradation mechanism(s) of EPM should facilitate the understanding of the stability behavior of EPM drug substances as well as aid in the design of new formulations made with EPM.

Metabolites and novel compounds with anti-microbial or antiaging activities from Cordyceps fumosorosea

High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis revealed that there are 20 main components in spores and mycelia extract of Cordyceps fumosorosea strain RCEF 6672 including mannitol (1), uridine (2), adenine (3). N6-(2-hydroxyethyl)-adenosine (4). N6-(2-hydroxyethylacetate)-adenosine (5), fumosoroseanoside A (6) and B (7), ovalicin-4α-alcohol (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9) and its isomer (10), fumosoroseain A (11) and its isomer (12), 5 non-ribosomal peptides (13 to 17) and 3 fatty acids (18 to 20). The compounds 5, 6, 7, 9 and 11 were prepared with preparative and semi-preparative HPLC and identified with 1D and 2D NMR. Compounds 4 and 5 were the first time identified from C. fumosorosea. Compounds 6, 7 and 11 are novel compounds. Compounds 6 and 7 showed antibacterial and antifungal activities, and 11 showed antiaging activity. All the secondary metabolites (4 to 8 and 11 to 17) have strong bioactivities indicating that the metabolites have pharmaceutical development potentiality.Graphical

Production and Secretion of Isowighteone in Hairy Root Cultures of Pigeon Pea (Cajanus cajan) Co-Treated with Multiple Elicitors.

Isowighteone (3’-isoprenyl genistein) is a prenylated flavonoid derivative that exhibits antibacterial, pro-apoptotic and anti-inflammatory properties. To establish a bioproduction system for this bioactive compound, hairy root cultures of pigeon pea (Cajanus cajan (L.) Millsp.) were developed via Agrobacterium rhizogenes-mediated transformation. The cultures were co-treated with methyl jasmonate, cyclodextrin, hydrogen peroxide, and magnesium chloride to enhance the production of isowighteone. The amount of isowighteone that accumulated in the culture medium upon elicitation varied with the period of elicitation. Isowighteone was purified from extracts of the culture medium by semi-preparative HPLC, and its identity was confirmed by tandem mass spectrometry. After 144 h of elicitation in 12-day-old hairy root culture, the total yield of isowighteone was 8058.618 ± 445.78 μg/g DW, of which approximately 96% was found in the culture medium. The yield of isowighteone in the elicited hairy root culture was approximately 277-fold higher than in the non-elicited root culture. The difference between the phenotypes of the elicited and non-elicited pigeon pea hairy roots was studied using scanning electron microscopy. The non-elicited hairy roots had uniform surfaces whereas the elicited roots had non-uniform shapes. Pigeon pea hairy roots provide a sustainable platform for producing and studying the biosynthesis of isowighteone.

A comprehensive study to identify and characterize major degradation products of Ivermectin drug substance including its degradation pathways using LC-HRMS and NMR

Ivermectin (IVM) drug substance is a semi-synthetic macrocyclic lactone that exhibits a broad spectrum of activity and high potency towards endo- and ectoparasites. In this study, a comprehensive forced degradation study was carried out on IVM drug substance (under the conditions recommended in the ICH guidelines) to identify and characterize its major degradation products (DPs). IVM drug substance was subjected to acidic, alkaline, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradations. Chromatographic separation of the drug substance and its DPs was achieved using a gradient elution on a HALO C18 column (150 × 4.6 mm, 2.7 µm). A total of five major DPs were observed for IVM drug substance under various stressed conditions. Additionally, ivermectin API lots exhibited instability when stored under room temperature and at 45% relative humidity for two years. These DPs were identified and characterized using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and a comparison of their fragmentation profile with IVM H2B1a using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-epoxide H2B1a) was isolated using semi-preparative HPLC and its structure was elucidated comprehensively using LC-HRMS and nuclear magnetic resonance spectroscopy. The proposed structures of the DPs have been rationalized by appropriate degradation pathways of IVM H2B1a. Comprehensive degradation profile of IVM drug substance should facilitate the understanding of the stability profile of IVM drug substance, setting the specification of DPs in finished products as well as aid in the design of generic formulation made with IVM.

An Integrated Strategy for Rapid Screening of Multi-target Lead Compounds for the Treatment of Alzheimer's Disease from Traditional Chinese Medicines by UHPLC Combined with High-throughput Screening: A Case Study on Salviae miltiorrhizae Radix et Rhizoma

Background: Salviae Miltiorrhizae Radix et Rhizoma (Red Sage root) is widely used in traditional Chinese medicine (TCM) for the treatment of Alzheimer’s disease (AD) with demonstrated curative effects, based on the concept of "one drug with multiple therapeutic targets," which appears to be a good strategy for AD treatment. Objective: This study aimed to develop of high-throughput screening (HTS) method for multitherapeutic target components found in complex TCMs, which are active against AD, using Red Sage root as the case study. Method: Acetylcholinesterase (AChE) inhibitors (AChEIs) from Red Sage root extracts were pre-screened by ultrafiltration-HPLC (UF-HPLC) analysis, in which AChE was added to the extract and then ultrafiltered to remove non-binding compounds. Potential AChEIs were identified by HPLC analysis of compounds bound to AChE. A microplate-based HTS was then used to quantify the AChE inhibitory activity and antioxidant activity of the pre-screened compounds. Results: Pre-screening found ten potential inhibitors, which were identified by ESI-TOF/MS; six of these were purified by semi-preparative HPLC: Oleoyl neocryptotanshinone (1), Dihydrotanshinone Ⅰ (2), Cryptotanshinone (3), Tanshinone Ⅰ (4), Tanshinone ⅡA (5) and Miltirone (6). All six compounds had good AChE inhibitory activity and weak DPPH scavenging capacity. Conclusion: This study provides a platform and technology support for the rapid discovery of multi-target components, potentially active against AD, from complex TCMs and with strong potential for adaptation to the discovery of treatments for other diseases.