Chemoenzymatic Synthesis of Complex Phenylpropanoid Derivatives by the Botrytis cinerea Secretome and Evaluation of Their Wnt Inhibition Activity

In this study, a series of complex phenylpropanoid derivatives were obtained by chemoenzymatic biotransformation of ferulic acid, caffeic acid, and a mixture of both acids using the enzymatic secretome of Botrytis cinerea. These substrates were incubated with fungal enzymes, and the reactions were monitored using state-of-the-art analytical methods. Under such conditions, a series of dimers, trimers, and tetramers were generated. The reactions were optimized and scaled up. The resulting mixtures were purified by high-resolution semi-preparative HPLC combined with dry load introduction. This approach generated a series of 23 phenylpropanoid derivatives, 11 of which are described here for the first time. These compounds are divided into 12 dimers, 9 trimers (including a completely new structural scaffold), and 2 tetramers. Elucidation of their structures was performed with classical spectroscopic methods such as NMR and HRESIMS analyses. The resulting compound series were analyzed for anti-Wnt activity in TNBC cells, with several derivatives demonstrating specific inhibition.

Phytochemical Analysis, Pharmacological Activities, Isolation and Characterization of Bioactive Compounds from the Roots of Sterculia urens Roxb.

The present study was intended to explore the pharmacological significance of the crude root extract of Sterculia urens Roxb. Further the bio-active compounds were isolated and characterized using chromatographic and spectroscopic techniques. Soxhlet extraction apparatus was utilized for isolation of the chemical constituents from the root using a series of solvents such as n-hexane, ethyl acetate, methanol and water. The pharmacological activities such as inhibition of DPPH radical, α-amylase enzyme activity, albumin denaturation along with antibacterial and thrombolytic activities. The isolation of purified bioactive constituents was carried using preparative HPLC technique and the purified compounds were characterized using spectroscopic techniques like NMR, IR and mass. Among the crude root extracts, methanolic extract shows high DPPH radical scavenging activity with IC50 concentration of 26.74 ± 0.08 μg/mL. The IC50 concentrations in α-amylase enzyme inhibition activity was 263.96 ± 0.90, 127.73 ± 1.23 and 223.54 ± 4.76 μg/mL, respectively for ethyl acetate, methanol and water extracts, respectively. The methanolic extract shows high albumin denaturation inhibition assay than other extracts with IC50 concentration as 137.09 ± 0.20 μg/mL, which is very close to standard ascorbic acid. The methanolic extract also shows high % clot lysis than other extracts and results were comparable with 100 μL of streptokinase standard. The preparative HPLC followed by spectral analysis confirm that two known alkaloids (Sterculinine I & II) and three known flavonoids (gossypetin, apigenin and 6-hydroxyluteolin) were purified and characterized from the root methanol of Sterculia urens Roxb. The purified and identified compounds were reported for the first time in Sterculia urens Roxb.

Isolation and Structural Elucidation of Impurity in Sulfonamide Intermediate of Glyburide

A reproducible isolation method by Reverse Phase (RP) preparative HPLC technique for the isolation of one crucial impurity at 1.65 RRT (Relative Retention Time) in sulfonamide stage of Glyburide API (Active Pharmaceuticals Ingredient) was developed. Preparative chromatography was done on Luna C8, 10µm (250 mm x 21.2mm) preparative HPLC column with acetonitrile: water in 70:30 % v/v proportion as a mobile phase and 8 ml/min as a flow rate. This impurity was detected at 300 nm UV-wavelength maximum. This impurity was isolated from synthesized crude impurity of sulfonamide stage of Glyburide substance by preparative HPLC by injecting 50 mg/ml concentration over 5 ml fixed loop. Isolated impurity was elucidated as N-methyl impurity of sulfonamide intermediate of Glyburide API by means of chromatographic and spectral data. Structural elucidation carried out by spectral data was reviewed. This impurity was analyzed by reverse phase HPLC for purity analysis. A Inertsil C8 (250 x 4.6) mm, 5µ particle size was employed for separation. The mobile phase consisted of Water: Acetonitrile: Methanol in the ratio of 60:15:25 % v/v. The flow rate was set at 1 ml/min. Detection was carried out at 300 nm. 10µL of 2 mg/ml concentration of sample in methanol was injected. The column oven temperature was at 25°C.

Separation and Identification of Antioxidants and Aldose Reductase Inhibitors in Lepechinia meyenii (Walp.) Epling

We previously reported that Lepechinia meyenii (Walp.) Epling has antioxidant and aldose reductase (AR) inhibitory activities. In this study, L. meyenii was extracted in a 50% MeOH and CH2Cl2/MeOH system. The active extracts of MeOH and 50% MeOH were subjected to fractionation, followed by separation using high-speed counter-current chromatography (HSCCC) and preparative HPLC. Separation and identification revealed the presence of caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and butyl rosmarinate. Of these, rosmarinic acid, methyl rosmarinate, and butyl rosmarinate possessed remarkable antioxidant and AR inhibitory activities. The other compounds were less active. In particular, rosmarinic acid is the key contributor to the antioxidant and AR inhibitory activities of L. meyenii; it is rich in the MeOH extract (333.84 mg/g) and 50% MeOH extract (135.41 mg/g) of L. meyenii and is especially abundant in the EtOAc and n-BuOH fractions (373.71–804.07 mg/g) of the MeOH and 50% MeOH extracts. The results clarified the basis of antioxidant and AR inhibitory activity of L. meyenii, adding scientific evidence supporting its traditional use as an anti-diabetic herbal medicine. The HSCCC separation method established in this study can be used for the preparative separation of rosmarinic acid from natural products.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

Isolation, characterization and quantification of a pentacyclic triterpinoid compound ursolic acid in Scabiosa palaestina L. distributed in the north of Iraq

Ursolic acid (UA, 3 ?-hydroxy-urs-12-en-28-oic acid) are isomeric triterpenic acids. The high quantities of pentacyclic triterpenoids in Scabiosa species seems to be obvious and there is an evidence that most of pentacyclic triterpenoids that have been isolated are saponins. This is one of the most important characteristic of the genus Scabiosa, the main aglycones are ursolic acid and oleanolic acid. In the current study, isolation from the aerial part and roots of Scabiosa palaestina L. was performed using Preparative HPLC. Furthermore, detection and quantitation of ursolic acid was performed by high performance thin layer chromatography (HPTLC). The identification of isolated triterpenoid involves two methods including FT-IR coupled with LC-MS/MS that have been used for the simultaneous determination of the isolated UA. Quantitative analysis of Ursolic acid content in chloroform fractions revealed that both of the aerial parts and roots contain comparable concentration of 0.052 and 0.054 mg/ml respectively. The FT-IR and LC-MS/MS spectra of the isolated compound shows good agreement with those reported in literatures of Ursolic acid. Quantitative concentration of UA in chloroform fraction revealed that aerial parts and roots contain comparable concentrations and the spectral data for the isolated unknown were in good agreement with those reported in literature of UA.

Phytosterols in hull-less pumpkin seed oil, rich in Δ7-phytosterols, ameliorate benign prostatic hyperplasia by lowing 5α-reductase and regulating balance between cell proliferation and apoptosis in rats

Background: Pumpkin seed oil is widely used to treat benign prostatic hyperplasia (BPH), a common disease in elder men. However, its active components and mechanism have remained to be elucidated. Objective: The objective of the present study was to investigate the active components of pumpkin seed oil and its mechanism against BPH. Design: Total phytosterol (TPS) was isolated from hull-less pumpkin (Cucurbita pepo L. var. Styriaca) seed oil and analyzed by gas chromatography/mass spectrometry (GC/MS). Three phytosterols were purified by preparative HPLC (high performance liquid chromatography) and confirmed by NMR (nuclear magnetic resonance). TPS (3.3 mg/kg body weight, 1 mL/day/rat) was administered intragastrically to the testosterone propionate-induced BPH rats for 4 weeks. The structure changes of prostate tissues were assessed by hematoxylin & eosin (H&E) staining. The expression of androgen receptor (AR) and steroid receptor coactivator 1 (SRC-1) was analyzed by immunohistochemistry, while that of 5α-reductase (5AR), apoptosis, or proliferation-related growth factors/proteins was detected by real-time quantitative polymerase chain reaction or western blotting. Results: The ∆7-phytosterols in TPS reached up to 87.64%. Among them, 24β-ethylcholesta-7,22,25-trienol, 24β-ethylcholesta-7,25(27)-dien-3-ol, and ∆7-avenasterol were confirmed by NMR. TPS treatment significantly ameliorated the pathological prostate enlargement and restored histopathological alterations of prostate in BPH rats. It effectively suppressed the expressions of 5AR, AR, and coactivator SRC-1. TPS inhibited the expression of proliferation-related growth factor epidermal growth factor, whereas it increased the expressions of apoptosis-related growth factor/gene transforming growth factor-β1. The proliferation-inhibiting effect was achieved by decreasing the ERK (extracellular signal-regulated kinase) phosphorylation, while apoptosis was induced by Caspase 3 activation through JNK (c-Jun N-terminal kinase) and p38 phosphorylation. Conclusion: TPS from hull-less pumpkin seed oil, with ∆7-phytosterols as its main ingredients, is a potential nutraceutical for BPH prevention.

Luteapyrone, a Novel ƴ-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea.

In the process of screening for new bioactive microbial metabolites we found a novel ƴ-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was 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 structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

Combination of Pseudo-LC-NMR and HRMS/MS-Based Molecular Networking for the Rapid Identification of Antimicrobial Metabolites From Fusarium petroliphilum

An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea (Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call “pseudo-LC-NMR,” and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.

Silver Ion-Complexation High-Speed Countercurrent Chromatography Coupled with Prep-HPLC for Separation of Sesquiterpenoids from Germacrene A Fermentation Broth

A silver ion high-speed counter-current chromatography ([Ag+]-HSCCC) was developed to separate and purify five sesquiterpenoids from germacrene A fermentation broth. The solvent system was consisted of n-hexane-methanol-silver nitrate (3 mol/L) solution (10:9.5:0.5, v/v). By employing this chromatographic protocol, five sesquiterpenoids named β-elemene (1; 54.1 mg), germacrene A (2; 28.5 mg), γ-selinene (3; 4.6 mg), β-selinene (4; 3.4 mg), and α-selinene (5; 1.3 mg) were obtained successfully from 500 mg extracted crude sample with purities of 97.1%, 95.2%, 98.2%, 96.3% and 98.5%, respectively, combined with preparative HPLC. The results reveal that the addition of metal ion in biphasic solvent system significantly improved the HSCCC separation factor of sesquiterpenoids. Meanwhile, our study also provided an alternate approach to separate the compounds with less polarity, also geometrical isomers and various natural product classes.