Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

NP3 MS Workflow: An Open-Source Software System to Empower Natural Product-Based Drug Discovery Using Untargeted Metabolomics.

Natural products (or specialized metabolites) are historically the main source of new drugs. However, the current drug discovery pipelines require miniaturization and speeds that are incompatible with traditional natural product research methods, especially in the early stages of the research. This article introduces the NP3 MS Workflow, a robust open-source software system for liquid chromatography-tandem mass spectrometry (LC-MS/MS) untargeted metabolomic data processing and analysis, designed to rank bioactive natural products directly from complex mixtures of compounds, such as bioactive biota samples. NP3 MS Workflow allows minimal user intervention as well as customization of each step of LC-MS/MS data processing, with diagnostic statistics to allow interpretation and optimization of LC-MS/MS data processing by the user. NP3 MS Workflow adds improved computing of the MS2 spectra in an LC-MS/MS data set and provides tools for automatic [M + H]+ ion deconvolution using fragmentation rules; chemical structural annotation against MS2 databases; and relative quantification of the precursor ions for bioactivity correlation scoring. The software will be presented with case studies and comparisons with equivalent tools currently available. NP3 MS Workflow shows a robust and useful approach to select bioactive natural products from complex mixtures, improving the set of tools available for untargeted metabolomics. It can be easily integrated into natural product-based drug-discovery pipelines and to other fields of research at the interface of chemistry and biology.

Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals.

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250 mg/mL).

Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

Application of eco-friendly natural deep eutectic solvents (NADES) in HPLC for separation of complex natural products: Current limitations and future directions.

Natural deep eutectic solvents (NADES) have been used in chromatography as extraction media and HPLC mobile phase additives, but only once have they been used as HPLC major mobile phase component. This review illustrates current knowledge and major limitations on use of NADES in HPLC mobile phase as well as to propose possible NADES may be ready for use as HPLC mobile phases and the detectors they can be used with. High viscosity is one of the major roadblocks encountered when using NADES as a mobile phase component in HPLC regardless of detectors employed. A comprehensive review of published literature was conducted to identify articles that focused on using NADES as extraction solvents for natural products, particularly polyphenols or reported NADES viscosities to establish a database of NADES which could be used as HPLC mobile phases under various conditions. Other identified challenges that limit NADES application in HPLC mobile phase include low volatility, NADES wavelength cutoff (UV and Fluorescent detectors) and impurities. Methods for overcoming these limitations are discussed so that NADES may be more integrated into HPLC systems in the future.

Total Synthesis of (-)-Amovillosumin A and Structure Correction of (-)-Amovillosumin C Using Chemical Synthesis.

Norlignans are a rare class of natural products isolated from a diverse range of plant species, many of which have interesting biological activities including antibacterial, antioxidant, phytotoxic, platelet aggregation inhibitory effects, and more. Isolated from Amomum villosum (Amomi Fructus), amovillosumins A (1) and C (3) are norlignans which were of interest to synthesize, due to their interesting bioactivities, specifically their ability to increase stimulation of glucagon-like peptide-1 (GLP-1) secretion. In this research, key intermediate 15 was used to stereoselectively synthesize (7R,8R)-amovillosumins A (1) and C (3). The developed method includes a Mitsunobu coupling, a modified rhodium-catalyzed Miyaura arylation, and an acid-catalyzed cyclization in key bond-forming steps. After synthesis, the structure of 1 was confirmed, but it was revealed that the benzodioxane-containing structure of amovillosumin C (3) that had been proposed in the literature was incorrect. Thus, with further investigation a structure correction of 3 was achieved by synthesis, the correct structure being 8-O-4'-oxynorlignan.

Cytotoxicity sesquiterpenoids from the leaves of Datura stramonium L.

Four new sesquiterpenoids, dstramonins A-D (1-4), and one new natural product (5), together with three known compounds (6-8), were isolated from the leaves of Datura stramonium L. The structures of new compounds were elucidated by extensive spectroscopic analysis and comparison with the literature. The cytotoxicity of isolates against LN229 cells was assessed and compounds 2-4, and 7 displayed cytotoxic activity with IC50 values ranging from 8.03 to 13.83 µM.

Non-targeted chemical analysis of consumer botanical products labeled as blue cohosh (Caulophyllum thalictroides), goldenseal (Hydrastis canadensis), or yohimbe bark (Pausinystalia yohimbe) by NMR and MS.

Consumers have unprecedented access to botanical dietary supplements through online retailers, making it difficult to ensure product quality and authenticity. Therefore, methods to survey and compare chemical compositions across botanical products are needed. Nuclear magnetic resonance (NMR) spectroscopy and non-targeted mass spectrometry (MS) were used to chemically analyze commercial products labeled as containing one of three botanicals: blue cohosh, goldenseal, and yohimbe bark. Aqueous and organic phase extracts were prepared and analyzed in tandem with NMR followed by MS. We processed the non-targeted data using multivariate statistics to analyze the compositional similarity across extracts. In each case, there were several product outliers that were identified using principal component analysis (PCA). Evaluation of select known constituents proved useful to contextualize PCA subgroups, which in some cases supported or refuted product authenticity. The NMR and MS data reached similar conclusions independently but were also complementary.

The composition and mechanical properties of porcine placental ECM from three different breeds.

Biologic scaffolds are extensively used in various clinical applications such as musculotendinous reconstruction, hernia repair or wound healing. Biologic scaffolds used in these applications vary in species, breed and tissue of origin, and other variables that affect their properties. Decellularization and sterilization processes also determine the characteristics of these scaffolds. The goal of the present study is to compare the composition and mechanical properties of decellularized porcine placental scaffolds from three different porcine breeds: Landrace, York and Duroc. Placental extracellular matrix (ECM) scaffolds from the three porcine breeds preserved the amnion/chorion ECM structure and the basement membrane markers laminin and collagen type IV. ECM placental scaffolds showed similar contents of collagen, elastin and lipids, and minimal differences in glycosaminoglycans content. Mechanical properties from the three breeds ECM placental scaffolds were also similar and stable for 24 months. While this study serves as preliminary characterization of porcine ECM scaffolds, future studies will determine their compatibility and suitability for tissue engineering applications.

A practical strategy enabling more reliable identification of ginsenosides from Panax quinquefolius flower by dimension-enhanced liquid chromatography/mass spectrometry and quantitative structure-retention relationship-based retention behavior prediction.

To accurately identify the metabolites is crucial in a number of research fields, and discovery of new compounds from the natural products can benefit the development of new drugs. However, the preferable phytochemistry or liquid chromatography/mass spectrometry approach is time-/labor-extensive or receives unconvincing identifications. Herein, we presented a strategy, by integrating offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), exclusion list-containing high-definition data-dependent acquisition (HDDDA-EL), and quantitative structure-retention relationship (QSRR) prediction of the retention time (tR), to facilitate the in-depth and more reliable identification of herbal components and thus to discover new compounds more efficiently. Using the saponins in Panax quinquefolius flower (PQF) as a case, high orthogonality (0.79) in separating ginsenosides was enabled by configuring the XBridge Amide and CSH C18 columns. HDDDA-EL could improve the coverage in MS2 acquisition by 2.26 folds compared with HDDDA (2933 VS 1298). Utilizing 106 reference compounds, an accurate QSRR prediction model (R2 = 0.9985 for the training set and R2 = 0.88 for the validation set) was developed based on Gradient Boosting Machine (GBM), by which the predicted tR matching could significantly reduce the isomeric candidates identification for unknown ginsenosides. Isolation and establishment of the structures of two malonylginsenosides by NMR partially verified the practicability of the integral strategy. By these efforts, 421 ginsenosides were identified or tentatively characterized, and 284 thereof were not ever reported from the Panax species. The current strategy is thus powerful in the comprehensive metabolites characterization and rapid discovery of new compounds from the natural products.

Building Block Extractor: An MS/MS Data Mining Tool for Targeted Discovery of Natural Products with Specified Features.

The utilization of a building-block-based molecular network is an efficient approach to investigate the unknown chemical space of natural products. However, structure-based automated MS/MS data mining remains challenging. This study introduces building block extractor, a user-friendly MS/MS data mining program that automatically extracts user-defined specified features. In addition to the characteristic product ions and neutral losses, this program integrates the abundance of the product ions and sequential neutral loss features as building blocks for the first time. The discovery of nine undescribed sesquiterpenoid dimers from Artemisia heptapotamica highlights the power of this tool. One of these dimers, artemiheptolide I (9), exhibited in vitro inhibition of influenza A/Hongkong/8/68 (H3N2) with an IC50 of 8.01 ± 6.19 µM. Furthermore, two known guaianolide derivatives (16 and 17) possessed remarkable antiviral activity against influenza A/Puerto Rico/8/1934 H1N1, H3N2, and influenza B/Lee/40 with IC50 values ranging from 3.46 to 11.77 µM. In addition to the efficient discovery of novel natural products, this strategy can be generally applied to grab derivatives with specific fragments and enhance the annotation power of LC-MS/MS analysis.

Fast and green universal method to analyze and quantify anthocyanins in natural products by UPLC-PDA.

This work developed a universal UPLC-PDA method based on safe reagents to analyze anthocyanins from different foods. Nine foods were studied by the developed chromatographic method, which was constructed using a solid core C18 column and a binary mobile phase composed of (A) water (0.25 molcitric acid.Lsolvent-1), and (B) ethanol. A total running time of 6 min was obtained, the faster comprehensive method for anthocyanins analysis. Mass spectrometry analysis was employed to identify a comprehensive set of 53 anthocyanins comprising glycosylated and acylated cyanidin, pelargonidin, malvidin, peonidin, petunidin, and delphinidin derivatives. Cyanidin-3-O-glucoside (m/z+ 449) and cyanidin-3-O-rutinoside (m/z+ 595) were used as standards to validate the accuracy of the developed method. The analytical parameters were evaluated, including intra-day and inter-day precision, robustness, repeatability, retention factor (k), resolution, and peak symmetry factor. The current method demonstrated excellent chromatographic resolution, making it a powerful tool for analyzing anthocyanins pigments.

Furanosesquiterpenes and Related Natural Products from Myoporum Species: Isolation and Semisynthesis.

Myoporum species are recognized as toxic plants. Essential oils from the leaves of these species contain furanosesquiterpenes, which comprise the active toxins. In this report, natural products isolation studies of three Myoporum species (M. insulare, M. parvifolium, and M. montanum) afforded two previously unreported furanosesquiterpenes (24 and 25) and three unprecedented γ-lactone-containing analogues (26-28), along with nine previously reported furanosesquiterpenes and five other natural products. Among the 14 furanosesquiterpenes and related compounds isolated in this study, we observed three distinct types of furanosesquiterpene structures isolated from each of these Myoporum species. Semisyntheses of four sesquiterpene natural products were completed from (-)-ngaione over two steps in each case. This included the synthesis of the lactam-containing sesquiterpene myoporumine A.

Raman spectroscopy provides valuable process insights for cell-derived and cellular products.

Two of the big challenges in modern bioprocesses are process economics and in-depth process understanding. Getting access to online process data helps to understand process dynamics and monitor critical process parameters (CPPs). This is an important part of the quality-by- design concept that was introduced to the pharmaceutical industry in the last decade. Raman spectroscopy has proven to be a versatile tool to allow noninvasive measurements and access to a broad spectrum of analytes. This information can then be used for enhanced process control strategies. This review article will focus on the latest applications of Raman spectroscopy in established protein production bioprocesses as well as show its potential for virus, cell therapy, and mRNA processes.

Structural diversification of fungal natural products by oxidative enzymes.

Ascomycota and basidiomycota fungi are prolific producers of biologically active natural products. Fungal natural products exhibit remarkable structural diversity and complexity, which are generated by the enzymes involved in their biosynthesis. After the formation of core skeletons, oxidative enzymes play a critical role in converting them into mature natural products. Besides simple oxidations, more complex transformations, such as multiple oxidations by single enzymes, oxidative cyclization, and skeletal rearrangement, are often observed. Those oxidative enzymes are of significant interest for the identification of new enzyme chemistry and have the potential to be biocatalysts for the synthesis of complex molecules. This review presents selected examples of unique oxidative transformations that have been found in the biosynthesis of fungal natural products. The development of strategies for refactoring the fungal biosynthetic pathways with an efficient genome-editing method is also introduced.

Chlorophyll Pigments of Olive Leaves and Green Tea Extracts Differentially Affect Their Antioxidant and Anticancer Properties.

Plant-based extracts possess biological potential due to their high content of phytochemicals. Nevertheless, photosynthetic pigments (e.g., chlorophylls) that are also present in plant extracts could produce undesirable pro-oxidant activity that might cause a negative impact on their eventual application. Herein, the phenolic content of olive leaf (OLE) and green tea (GTE) extracts was assayed, and their antioxidant and anticancer activities were evaluated before and after the removal of chlorophylls. Regarding phenolic content, OLE was rich in hydroxytyrosol, tyrosol as well as oleuropein, whereas the main compounds present in GTE were gallocatechin, epigallocatechin (EGC), epigallocatechin gallate (EGCG), gallocatechin gallate, and caffeine. Interestingly, fresh extracts' antioxidant ability was dependent on phenolic compounds; however, the elimination of chlorophyll compounds did not modify the antioxidant activity of extracts. In addition, both OLE and GTE had high cytotoxicity against HL-60 leukemic cell line. Of note, the removal of chlorophyll pigments remarkably reduced the cytotoxic effect in both cases. Therefore, our findings emphasize the remarkable antioxidant and anticancer potential of OLE and GTE and suggest that chlorophylls are of paramount importance for the tumor-killing ability of such plant-derived extracts.

Reduced fibrous capsule elastic fibers from biologic ECM-enveloped CIEDs in minipigs, supported with a novel compression mechanics model.

BACKGROUND: Fibrous capsules (Fb) in response to cardiovascular implantable electronic devices (CIEDs), including a pacemaker (P) system, can produce patient discomfort and difficulties in revision surgery due partially to their increased compressive strength, previously linked to elevated tissue fibers. OBJECTIVE: A preliminary study to quantify structural proteins, determine if biologic extracellular matrix-enveloped CIEDs (PECM) caused differential Fb properties, and to implement a realistic mechanical model. METHODS: Retrieved Fb (-P and -PECM) from minipigs were subjected to biomechanical (shear oscillation and uniaxial compression) and histological (collagen I and elastin) analyses. RESULTS: Fb-PECM showed significant decreases compared to Fb-P in: low strain-loss modulus (390 vs. 541 Pa) across angular frequencies, high strain-compressive elastic modulus (1043 vs. 2042 kPa), and elastic fiber content (1.92 vs. 3.15 µg/mg tissue). Decreases in elastin were particularly noted closer to the implant's surface (Fb-PECM = 71% vs. Fb-P = 143% relative to dermal elastin at mid-tangential sections) and verified with a solid mechanics hyperelasticity with direction-dependent fiber viscoelasticity compression simulation (r2 ≥ 98.9%). CONCLUSIONS: The biologic envelope composed of decellularized porcine small intestine submucosa ECM for CIEDs promoted fibrous tissues with less elastic fibers. Novel compression modeling analyses directly correlated this singular reduction to more desirable subcutaneous tissue mechanics.

Natural Products Drug Discovery: On Silica or In-Silico?

Natural products have been the most important source for drug development throughout the human history. Over time, the formulation of drugs has evolved from crude drugs to refined chemicals. In modern drug discovery, conventional natural products lead-finding usually uses a top-down approach, namely bio-guided fractionation. In this approach, the crude extracts are separated by chromatography and resulting fractions are tested for activity. Subsequently, active fractions are further refined until a single active compound is obtained. However, this is a painstakingly slow and expensive process. Among the alternatives that have been developed to improve this situation, metabolomics has proved to yield interesting results having been applied successfully to drug discovery in the last two decades. The metabolomics-based approach in lead-finding comprises two steps: (1) in-depth chemical profiling of target samples, e.g. plant extracts, and bioactivity assessment, (2) correlation of the chemical and biological data by chemometrics. In the first step of this approach, the target samples are chemically profiled in an untargeted manner to detect as many compounds as possible. So far, NMR spectroscopy, LC-MS, GC-MS, and MS/MS spectrometry are the most common profiling tools. The profile data are correlated with the biological activity with the help of various chemometric methods such as multivariate data analysis. This in-silico analysis has a high potential to replace or complement conventional on-silica bioassay-guided fractionation as it will greatly reduce the number of bioassays, and thus time and costs. Moreover, it may reveal synergistic mechanisms, when present, something for which the classical top-down approach is clearly not suited. This chapter aims to give an overview of successful approaches based on the application of chemical profiling with chemometrics in natural products drug discovery.

Rapid screening of antioxidant from natural products by AAPH-Incubating HPLC-DAD-HR MS/MS method: A case study of Gardenia jasminoides fruit.

A new AAPH-Incubating HPLC-DAD-HR MS/MS method was developed for the rapid and high-throughput screening of antioxidants directly in natural products and applied to Gardenia jasminoides fruit. This method was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be significantly reduced or disappeared after incubating with the AAPH which can release ROO at physiological conditions (37 °C, pH 7.4). Additionally, the activity of antioxidants can be evaluated by comparing the peak reduction rates and the screened components can be further identified by HRMS/MS. Then, 17 potential natural antioxidants from the crude extract of GJF was screened. Among them, three major components including crocin I, crocin II and crocetin showed excellent ROO scavenging activity, which were further validated by the ORAC assay. In conclusion, our study provided a simple and effective strategy to rapidly screen antioxidants in natural products.