Application of Bioassay-Guided Fractionation Coupled with a Molecular Approach for the Dereplication of Antimicrobial Metabolites

A systematically delineated dereplication approach was described based on genome mining and bioassay-guided fractionation using endophytic fungus Xylaria psidii FPL-52(S) isolated from leaves of Ficus pumila Linn., (Moraceae). A polyketide synthase gene-based molecular screening strategy by a degenerate oligonucleotide primer polymerase chain reaction technique coupled with a bioinformatic phylogenomic approach revealed the presence of an iterative polyketide synthase gene within the genome of Xylaria psidii FPL-52(S). Chemical dereplication of ethyl acetate extract derived from a submerged fermentation culture broth of Xylaria psidii FPL-52(S) by bioassay-guided chromatographic and hyphenated analytical spectroscopic techniques led to the identification of polyketide mycoalexin 3-O-methylmellein. Antimicrobial profiling and minimal inhibitory concentration values for 3-O-methylmellein were determined by disc diffusion and microbroth dilution techniques. Gram-positive bacteria, dermatophytic and phytopathogenic fungi were susceptible in terms of inhibition zone and minimum inhibitory concentration values when compared to co-assayed standards. Herein, we highlight and demonstrate an improved approach which facilitates efficient dereplication and effect-guided fractionation of antimicrobial metabolite(s). The present work flow serves as a promising dereplication tool to survey the biosynthetic potential of endophytic fungal diversity, thereby identifying the most promising strains and prioritizing them for novel polyketide-derived antimicrobial metabolite discovery.

     

MS/MS-Based Molecular Networking: An Efficient Approach for Natural Products Dereplication

Natural products (NPs) have historically played a primary role in the discovery of small-molecule drugs. However, due to the advent of other methodologies and the drawbacks of NPs, the pharmaceutical industry has largely declined in interest regarding the screening of new drugs from NPs since 2000. There are many technical bottlenecks to quickly obtaining new bioactive NPs on a large scale, which has made NP-based drug discovery very time-consuming, and the first thorny problem faced by researchers is how to dereplicate NPs from crude extracts. Remarkably, with the rapid development of omics, analytical instrumentation, and artificial intelligence technology, in 2012, an efficient approach, known as tandem mass spectrometry (MS/MS)-based molecular networking (MN) analysis, was developed to avoid the rediscovery of known compounds from the complex natural mixtures. Then, in the past decade, based on the classical MN (CLMN), feature-based MN (FBMN), ion identity MN (IIMN), building blocks-based molecular network (BBMN), substructure-based MN (MS2LDA), and bioactivity-based MN (BMN) methods have been presented. In this paper, we review the basic principles, general workflow, and application examples of the methods mentioned above, to further the research and applications of these methods.     

Bioassay-Guided Isolation of a Low Molecular Weight PHB from Burkholderia sp. with Phytotoxic Activity

This work reports on the bioassay-guided isolation and identification of the macrocyclic pentolide 1, a cyclic polyhydroxybutyrate (PHB) with low molecular weight. This metabolite is produced by Burkholderia sp. and it exhibited phytotoxic activity in a Lemna minor bioassay. Its structure was determined by ¹H and ¹³C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, IR, and electrospray ionization tandem mass spectrometry analyses. The period for maximum production of the pentolide was optimized and determined on the basis of multiple reaction monitoring experiments at 15 days. The potential of Burkholderia sp. as a producer of higher biopolymers of PHB was also investigated. The methodology employed here accelerated the isolation and characterization of a phytotoxic metabolite whose structure can serve as a model for the synthesis of new classes of herbicides.     

Application of the Numerical Fractionation Approach to the Design of Biofunctional PEG Hydrogel Membranes

A mathematical model is described for surface‐initiated photopolymerization of PEG‐DA forming crosslinked biofunctional PEG hydrogel membranes based on the NF technique. The model includes an additional monomer with biological functionality, which is a common experimental strategy for the design of ECM mimics in tissue engineering in order to direct signaling pathways, and considers concentration‐dependent VP propagation and reaction diffusion termination. The influence of these features on the crosslink density of the soluble and gel phases, the progression through gelation, sol/gel fraction, and molecular weight distribution of biofunctional PEG hydrogel are studied using the NF model. This model may be useful for specific applications of tissue engineering.

     

Metabolite Profiling and Bioassay-Guided Fractionation of Zataria multiflora Boiss. Hydroethanolic Leaf Extracts for Identification of Broad-Spectrum Pre and Postharvest Antifungal Agents

Hydroethanolic leaf extracts of 14 Iranian Zataria multiflora Boiss. populations were screened for their antifungal activity against five plant pathogenic fungi and metabolically profiled using a non-targeted workflow based on UHPLC/ESI-QTOFMS. Detailed tandem mass-spectrometric analyses of one of the most active hydroethanolic leaf extracts led to the annotation of 68 non-volatile semi-polar secondary metabolites, including 33 flavonoids, 9 hydroxycinnamic acid derivatives, 14 terpenoids, and 12 other metabolites. Rank correlation analyses using the abundances of the annotated metabolites in crude leaf extracts and their antifungal activity revealed four O-methylated flavones, two flavanones, two dihydroflavonols, five thymohydroquinone glycoconjugates, and five putative phenolic diterpenoids as putative antifungal metabolites. After bioassay-guided fractionation, a number of mono-, di- and tri-O-methylated flavones, as well as three of unidentified phenolic diterpenoids, were found in the most active subfractions. These metabolites are promising candidates for the development of new natural fungicides for the protection of agro-food crops.     

Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill

Terminalia leiocarpa is a medicinal plant widely used in ethnoveterinary medicine to treat digestive parasitosis whose extracts were shown to be active against gastrointestinal nematodes of domestic ruminants. The objective of our study was to identify compounds responsible for this activity. Column fractionation was performed, and the activity of the fractions was assessed in vitro on Haemonchus contortus and Caenorhabditis elegans as well as their cytotoxicity on WI38 fibroblasts. Two fractions were the most active on both nematode models and less cytotoxic. LC-MS/MS analysis and manual dereplication coupled to molecular networking allowed identification of the main compounds: ellagic acid and derivatives, gallic acid, astragalin, rutin, quinic acid, and fructose. Other potentially identified compounds such as shikimic acid, 2,3-(S)-hexahydroxydiphenoyl-D-glucose or an isomer, quercetin-3-O-(6-O-galloyl)-β-D-galactopyranoside or an isomer, and a trihydroxylated triterpenoid bearing a sugar as rosamultin are reported in this plant for the first time. Evaluation of the anthelmintic activity of the available major compounds showed that ellagic and gallic acids were the most effective in inhibiting the viability of C. elegans. Their quantification in fractions 8 and 9 indicated the presence of about 8.6 and 7.1 µg/mg ellagic acid and about 9.6 and 2.0 µg/mg gallic acid respectively. These concentrations are not sufficient to justify the activity observed. Ellagic acid derivatives and other compounds that were found to be positively correlated with the anthelmintic activity of the fractions may have additive or synergistic effects when combined, but other unidentified compounds could also be implicated in the observed activity.     

HPTLC Coupled with Bioluminescence and Mass Spectrometry for Bioactivity-Based Analysis of Secondary Metabolites in Marine Sponges

JPC – Journal of Planar Chromatography – Modern TLC - Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in...     

UPLC Coupled with a Post-column Derivatization Approach for Identification of Bioactive Compounds in Huanglian Jiedu Decoction

Chromatographia - A UPLC coupled with post-column derivatization (PCD) and diode array detector (DAD) collecting biochemical reaction signal analysis technique (UPLC–PDC–DAD) was...     

A Coupled HPLC/Radioimmunoassay for Analysis of Zidovudine Metabolites in Mononuclear Cells

Abstract

Reverse-phase HPLC, with the ion-pairing agent tetrabutyl ammonium phosphate, was used to separate zidovudine (ZDV) and its 5′-phosphorylated metabolites in extracts from peripheral blood mononuclear cells incubated with 2 μM ZDV. Because intracellular concentrations were too small to be visualized using UV detection, 1 ml fractions were collected and assayed for ZDV and metabolites using a commercial radioimmunoassay (RIA). Resolution of components was satisfactory, with a total chromatography time of 32 minutes. Interassay variability of peak areas was less than 14%. Comparison of UV detected chromatograms to RIA detected chromatograms from a standard mixture of ZDV and metabolites showed no significant difference between corresponding relative peak areas. The ion-pairing agent elevated baseline concentrations as measured by RIA. Quantitation was therefore performed by concurrent measurement of total phosphorylated ZDV using an established procedure, followed by comparison of relative peak areas. Results indicate that ZDV 5′-triphosphate, the active metabolite, is a major component of total phosphorylated ZDV, with peak heights significantly above baseline in extracts from less than 10⁷ mononuclear cells. Therefore, it should be possible to reliably quantitate this metabolite in cells from HIV-infected patients using only 10 to 20 ml of blood.     

固相微萃取-气/质联用法测定尿中的海洛因代谢物

Shima dzu QP5050AGC／MS仪,EI源(70eV),GC-17A AFC流量控制,分流／不分流进样口,GC Solution软件工作站。固相微萃取(SPME)手柄,7μm和100μm PDMS纤维头与85μm PA纤维头(Supelco公司)。吗啡、6-单乙酰吗啡、可待因、乙酰可待因及海洛因标准品均购自公安部物证鉴定中心,用甲醇配成lg／L标准储备液,冷藏备用。     

Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry for Rapid Determination of the Metabolites of Baicalin Produced by Human Intestinal Bacteria

Baicalin, mainly isolated from Scutllaria baicalensis, has been reported to possess a wide range of biological activities. However, the information about the metabolic route and metabolites of baicalin was limited to the role of the human intestinal bacterial mixture. In this paper, four strains of bacteria including Bacteroides sp. 33 and 56, and Veillonella sp. 23 and 71 were isolated from human intestinal bacterial mixture and studied for their abilities to convert baicalin to different metabolites. A highly sensitive and specific ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method combined with mass defect filtering (MDF) provides high throughput capabilities for drug metabolism study. The chromatographic separation was performed on a 1.7 µm particle size C 18 column using gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of 4 metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation, and deglycosylation were the major metabolic pathways of baicalin in vitro. The present study provides important information about the metabolism of baicalin which will be helpful for fully understanding the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry approach for a rapid, simple, reliable, and automated identification of metabolites of natural products.     

求解非弹性散射中耦合孔道方程组的一种混合方法

给出了求解原子-分子散射理论中耦合孔道方程组的一种混合方法,该法在强相互作用区域为Johnson的LOGD算法,在其余区域用参照势能具有V=-(A²/16)+Be^-AR形式的势能追踪方法,并以Lester-Bernstein的刚性转子问题为例进行了验证。     

Bioassay-Guided Fractionation of Erythrostemon yucatanensis (Greenm.) Gagnon & GP Lewis Components with Anti-hemagglutinin Binding Activity against Influenza A/H1N1 Virus

Erythrostemon yucatanensis (Greenm.) Gagnon & GP Lewis is a legume tree native to and widely distributed in southeast Mexico, where its branches are used in traditional medicine. An in vitro evaluation of the antiviral activity of extracts and fractions from the leaves, stem bark and roots against two strains of the AH1N1 influenza virus was performed, leading to the identification of bioactive compounds in this medicinal plant. In a cytopathic effect reduction assay, the fractions from the leaves and stem bark were the active elements at the co-treatment level. These were further fractionated based on their hemagglutination inhibition activity. The analysis of spectroscopy data identified a combination of phytosterols (β-sitosterol, stigmasterol and campesterol) in the stem bark active fraction as the main anti-hemagglutinin binding components, while 5-hydroxy-2(2-hydroxy-3,4,5-trimethoxyphenyl)-7-metoxi-4H(chromen-4-ona), which was isolated from the leaf extracts, showed a weak inhibition of viral hemagglutinin. Time of addition experiments demonstrated that the mixture of sterols had a direct effect on viral particle infectivity at the co-treatment level (IC50 = 3.125 µg/mL). This effect was also observed in the virus plaque formation inhibition assay, where the mixture showed 90% inhibition in the first 20 min of co-treatment at the same concentration. Additionally, it was found using qRT-PCR that the NP copy number was reduced by 92.85% after 60 min of co-treatment. These results are the first report of components with anti-hemagglutinin binding activity in the genus Erythrostemon sp.     

Molecular Recognition of the Fungicide Carbendazim by a Molecular Imprinted Polymer Obtained through a Mimic Template Approach

Abstract

A molecularly imprinted polymer recognizing the fungicide carbendazim was prepared using a mimicking template approach. Methyl-3-propylcarbamoyl-1H-benzimidazol-2-ylcarbamate was synthesized and used as a mimic template for polymer preparation. Selectivity of this polymer for carbendazim and structurally related substances (the template, fluberidazole, rabenzazole, thiabendazole, and benomyl and its two degradation products) was evaluated by liquid chromatography. Results demonstrate that imprinted polymer shows significant recognition properties for carbendazim, whereas the mimic and other carbendazim-related molecules are not recognized. This peculiar selectivity pattern can be explained as an imprinting effect due to the in situ formation of carbendazim from mimic degradation during the polymerization process.     

Thermal Field-Flow Fractionation as a Powerful Tool for the Fractionation of Complex Synthetic Polymers: A Perspective

Chromatographia - Synthetic polymers have complex molecular structures with distributions in molar mass, chemical composition, functionality and molecular topology. For the comprehensive analysis...     

Bioassay-Guided Fractionation of Siparuna glycycarpa n-Butanol Extract with Inhibitory Activity against Influenza A(H1N1)pdm09 Virus by Centrifugal Partition Chromatography (CPC)

Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water (9:1:10, v/v) was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL⁻¹ after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity.     

采用分子动力学模拟方法评价人类VDAC通道蛋白晶体结构的生物活性

电压依赖阴离子选择性通道（Voltage dependent anion-selective channel, VDAC）是线粒体外膜上重要的通道蛋白,转运线粒体和细胞质之间的代谢物,对于线粒体发挥生理功能具有决定性作用. 为研究VDAC分子的调控机制,在电场存在和不存在的情况下,我们对I型人类VDAC分子（hVDAC1）分别进行了分子模拟研究. 比较结果显示：外加电场的有无与方向变化对hVDAC1分子的运动模式和离子通过过程影响不明显,hVDAC1分子仍然保持以N端α螺旋在β桶内横向运动为主的运动模式. 综合模拟结果和相关的实验结果,我们推测,发表的hVDAC1晶体结构可能不是其发挥生理功能的结构状态.