UPLC-ESI-Q-TOF-MS/MS based metabolomics investigation on chemical constituent consistency of Zhenwu Decoction before and after compatibility.

Zhenwu Decoction (ZWD), a classic formula from Zhang Zhongjing's "Treatise on Typhoid Fever" in the Han Dynasty, consists of five traditional Chinese medicines: Aconiti Lateralis Radix Praeparata (ALRP), Paeoniae Radix Alba, Poria Cocos, Ginger, and Rhizoma Atractylodis Macrocephalae. To evaluate the chemical constituent consistency of ZWD before and after compatibility, an ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry was established to comprehensively study the constituents of ZWD. By normalizing the peak area, the pairwise compatibility of ALRP and the other four medicinal herbs, as well as the compatibility of the entire formula were studied, respectively. Multivariate statistical analysis was used to identify the differences. The processed data were analyzed by principal component analysis and supervised orthogonal partial least squared discriminant analysis, and an S-plot was generated to compare the differences in the chemical composition of the two types of decoction samples. The results showed that during the decoction process of ZWD, a total of seven components were recognized as differential compounds before and after compatibility of ZWD, namely 6-gingerol, zingerone, benzoylhypaconine, hypaconitine, benzoylaconine, paeoniflorin and fuziline. The results of this study provide basic data reference for understanding the law of ZWD compatibility and are valuable for the compatibility study of other herbal medicines.

Improving Bioaccessibility and Bioavailability of Isoflavone Aglycones from Chickpeas by Germination and Forming ß-Cyclodextrin Inclusion Complexes.

Chickpea isoflavones have diverse pharmacological activities but with low water solubility and bioavailability. In this work, the isoflavone content in chickpeas was first increased by germination, and then the bioaccessibility and bioavailability of isoflavones in chickpea sprout extracts (CSE) were enhanced using ß-cyclodextrin (ß-CD) inclusion techniques. Firstly, the total content of isoflavones was increased by 182 times through sprouting, and isoflavones were presented mostly in the germ and radicle. Then, the chickpea sprout extract/ß-cyclodextrin (CSE/ß-CD) inclusion complex was prepared and characterized. The in vitro test showed that the cumulative release of two isoflavones, formononetin (FMN) and biochanin A (BCA), in the CSE/ß-CD was significantly increased in a simulated digestive fluid. The in vivo rat pharmacokinetics demonstrated that the inclusion of FMN and BCA by ß-CD effectively increased their bioavailability in rat plasma and tissues, especially in the liver. The study provides a feasible strategy for improving the bioavailability of isoflavones from chickpeas and is also beneficial to the utilization of other legume resources.

Mechanochemical preparation of red clover extract/ß-cyclodextrin dispersion: Enhanced water solubility and activities in alleviating high-fat diet-induced lipid accumulation and gut microbiota dysbiosis in mice.

Red clover (RC) extract is rich in isoflavones (formononetin and biochanin A) that have various biological functions. However, its low water solubility limits its bioavailability. In this study, an RC extract/ß-cyclodextrin (RC/ß-CD) dispersion was prepared by ball milling to enhance its water solubility and biological availability. The water solubility of formononetin and biochanin A was 34.45 and 13.65 µg/mL (increased to 3.11 and 2.14 times higher than that of RC alone), respectively. The alleviating effects of the dispersion on lipid accumulation and gut microbiota were evaluated in mice. The RC/ß-CD dispersion showed a better effect on inhibiting lipid accumulation, especially on total triglycerides. The dispersion group had a higher relative abundance of Akkermansia, Muribaculaceae, and Bacteroides than RC alone, along with a higher level of acetic and butyric acid. The study provides a feasible way for improving the bioaccessibility and bioactivity of RC isoflavones in red clover.

Components research on Tetrastigma hemsleyanum Diels et Gilg: Identification and effect of drying methods on the content of ten main constituents by targeting metabolomics method.

Tetrastigma hemsleyanum Diels et Gilg (TH) is one of the new eight Genuine Medicinal Materials of Zhejiang. It has extensive biological activities, such as anti-inflammatory, anti-tumor and analgesic activities, etc. In this study, the chemical components of TH were systematically investigated by ultra high-performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (UPLC/Q-TOF-MS). Based on the MS spectrum, 39 compounds in TH extracts including 14 flavonoids, 10 fatty acids, 5 polyphenols and phenolic acids, 4 terpenes and other compounds were detected and tentatively identified. TH samples were treated under different drying methods (vacuum freeze drying, hot air drying, natural drying, light drying and vacuum drying). Besides, the effect of different drying methods on the content of 10 main chemical constituents in TH extracts including catechin, rutin, kaempferol-3-O-rutinoside and so on was also investigated by targeting metabolomics method with ultra high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) assisted by multivariate statistical analysis. Large differences were observed between vacuum drying and vacuum freeze drying with remarkable content changes. The contents of rutin, proanthocyanidin B1 and catechin were the most different among the various drying methods. The systematic identification of chemical constituents is helpful for the further medicinal development and application of TH. The effects of drying methods on the content of TH components were studied, which provided experimental data for the processing, storage and quality control of TH.

Study on Absorption, Distribution and Excretion of a New Candidate Compound XYY-CP1106 against Alzheimer's Disease in Rats by LC-MS/MS.

XYY-CP1106, a candidate compound synthesized from a hybrid of hydroxypyridinone and coumarin, has been shown to be remarkably effective in treating Alzheimer's disease. A simple, rapid and accurate high-performance liquid chromatography coupled with the triple quadrupole mass spectrometer (LC-MS/MS) method was established in this study to elucidate the pharmacokinetics of XYY-CP1106 after oral and intravenous administration in rats. XYY-CP1106 was shown to be rapidly absorbed into the blood (Tmax, 0.57-0.93 h) and then eliminated slowly (T1/2, 8.26-10.06 h). Oral bioavailability of XYY-CP1106 was (10.70 ± 1.72)%. XYY-CP1106 could pass through the blood-brain barrier with a high content of (500.52 ± 260.12) ng/g at 2 h in brain tissue. The excretion results showed that XYY-CP1106 was mainly excreted through feces, with an average total excretion rate of (31.14 ± 0.05)% in 72 h. In conclusion, the absorption, distribution and excretion of XYY-CP1106 in rats provided a theoretical basis for subsequent preclinical studies.

Investigation on Antioxidant Activity and Different Metabolites of Mulberry (Morus spp.) Leaves Depending on the Harvest Months by UPLC-Q-TOF-MS with Multivariate Tools.

The changes in active components in mulberry leaves harvested in different months and their antioxidant activities were investigated. Ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with multivariate statistical tools was used to investigate the chemical constituents in the extracts of mulberry leaves. The results indicated that mulberry leaves were rich in phenolic acids, flavonoids, organic acids, and fatty acid derivatives. In addition, 25 different compounds were identified in the different batches of mulberry leaves. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured to evaluate the in vitro antioxidant activities of mulberry leaves. Among the four batches, batch A, harvested in December, exhibited the strongest DPPH radical-scavenging activity, while batch B, harvested in March, showed the weakest activity. This was related to the total phenolic content in the mulberry leaves of each batch. The optimal harvest time of mulberry leaves greatly influences the bioactivity and bioavailability of the plant.

Removal of tetracycline from water by catalytic photodegradation combined with the microalga Scenedesmus obliquus and the responses of algal photosynthesis and transcription.

The antibiotic tetracycline (TC) and its degradation products (TDPs) in degradation solution present serious environmental problems, such as human health damage and ecological risk; thus further treatment is required before being released into the aquatic environment. Furthermore, their environmental impact on microalgae remains unclear. In this study, TC was degraded by photocatalysis using birnessite and UV irradiation, followed by biological purification using the microalga Scenedesmus obliquus. In addition, the photosynthetic activity and transcription of the microalgae were examined to evaluate the toxicity of TC and TDPs. The results show that photocatalytic degradation efficiency reached 92.7% after 30 min, and 11 intermediate products were detected. The microalgae achieved a high TC removal efficiency (99.7%) after 8 days. Exposure to the degraded TC solution (D) resulted in significantly lower (p < 0.05) biomass than the pure TC (T), and S. obliquus in the T treatment showed better resilience than the D treatment. Transcriptomic assays for different treatments revealed differential gene expression mainly involving the photosynthesis, ribosome, translation and peptide metabolic progresses. The up-regulation of photosynthesis-related genes and differential expression of chloroplast genes may be important for S. obliquus to acquire high photosynthetic efficiency and growth recovery when exposed to TC and TDPs. Our study provides a reference for TC removal using a combination of catalytic degradation and microalgal purification, and it is also helpful for understanding the environmental risk of TDPs in natural aquatic environments.

Acidic natural deep eutectic solvents as dual solvents and catalysts for the solubilization and deglycosylation of soybean isoflavone extracts: Genistin as a model compound.

This study investigated the possibility of using green solvent natural deep eutectic solvents (NADESs) as dual solvent-catalysts for the solubilization and deglycosylation of soybean isoflavones. The deglycosylation behavior of genistin as a model compound in NADESs was compared. Acidic NADESs showed moderate solubility for genistin and could hydrolyze it to form genistein. The onset temperature of deglycosylation in the choline chloride/malic acid (Ch-Ma) was 60 °C. The solubilities of genistin in the Ch-Ma system were modeled. The dissolution process was endothermic and mainly enthalpy-driven. The deglycosylation followed first-order kinetics with a half-life (t1/2) of 40 min at 90 °C. The method was validated using soybean isoflavone extracts as a substrate and the ratio of glycoside to aglycone in the extracts could be adjusted by changing the conditions. The methods have great potential in the extraction and preparation of ready-to-use isoflavone extracts from soybean and other legumes.

Metabolomics analysis of differential chemical constituents and α-glucosidase inhibiting activity of Phyllanthus urinaria L. root, stem, leaf and fruit.

The differential chemical constituents of the different Phyllanthus urinaria L. (PUL) parts were investigated by UPLC/Q-TOF MS-based metabolomics. A total of 69 compounds were tentatively identified in the whole plant extract and 35 of them were common to root, stem, leaf and fruit parts. And four compounds were selected as biomarkers for leaves, fruits, stems and roots, respectively. The four PUL parts all had good α-glucosidase inhibitory activities and the activities of fruit, root and stem extracts were about fivefold higher than the leaf part. The hierarchical cluster analysis and heat map were used to explore the relationship between the α-glucosidase inhibitory activities and chemical constituent differences of four PUL parts.

Effects of Tetracycline on Scenedesmus obliquus Microalgae Photosynthetic Processes.

Tetracycline (TC) antibiotics can be detected worldwide in the aquatic environment due to their extensive use and low utilization efficiency, and they may affect the physiological processes of non-target organisms. In this study, the acute and sub-acute toxicities of TC on the freshwater microalga Scenedesmus obliquus were investigated with an emphasis on algal photosynthesis and transcription alterations during an 8 d TC exposure. The results showed that the IC10, IC30 and IC50 values were 1.8, 4.1 and 6.9 mg/L, respectively. During sub-acute exposure, the microalgae of the IC10 treatment was able to recover comparable growth to that of the control by day 7, while significantly lower cell densities were observed in the IC30 and IC50 treatments at the end of the exposure. The photosynthetic efficiency Fv/FM of S. obliquus first decreased as the TC concentration increased and then returned to a level close to that of the control on day 8, accompanied by an increase in photosynthetic activities, including light harvesting, electron transport and energy dissipation. Transcriptomic analysis of the IC10 treatment (1.8 mg/L TC) revealed that 2157 differentially expressed genes were up-regulated and 1629 were down-regulated compared with the control. KEGG and GO enrichments demonstrated that 28 photosynthesis-related genes involving light-harvesting chlorophyll protein complex, photosystem I, photosystem II, photosynthetic electron transport and enzymes were up-regulated, which may be the factor responsible for the enhanced photosynthesis and recovery of the microalgae. Our work may be helpful not only for gaining a better understanding of the environmental risk of TC at concentrations close to the real levels in natural waters, but also for explaining photosynthesis and related gene transcription induced by antibiotics.

Metabolomics revealed the photosynthetic performance and metabolomic characteristics of Euglena gracilis under autotrophic and mixotrophic conditions.

Photosynthetic and metabolomic performance of Euglena gracilis was examined and compared under autotrophic and mixotrophic conditions. Autotrophic protozoa (AP) obtained greater biomass (about 33% higher) than the mixotrophic protozoa (MP) after 12 days of growth. AP maintained steady photosynthesis, while MP showed a remarkable decrease in photosynthetic efficiency and dropped to an extremely low level at day 12. In MP, low light absorption and photosynthetic electron transport efficiency, and high energy dissipation were reflected by the chlorophyll (chl a) fluorescence (OJIP) of the protozoa. The values of ΨO, ΦEo, and ETO/RC of MP decreased to extremely low levels, to 1/15, 1/46, and 1/9 those of AP, respectively, while DIO/RC increased to approximately 16 times that of AP. A total of 137 metabolites were showed significant differences between AP and MP. AP accumulated more monosaccharide, lipids, and alkaloids, while MP produced more amino acids, peptides, and long-chain fatty acids including poly-unsaturated fatty acids. The top nine most important enriched pathways obtained from KEGG mapping were related to ABC transporters, biosynthesis of amino acids, purine metabolism, and carbohydrate metabolism. There were significant differences between AP and MP in photosynthetic activity, metabolites, and metabolic pathways. This work presented useful information for the production of high value bioproducts in E. gracilis cultured under different nutritional conditions.

Fabrication of colloidal stable gliadin-casein nanoparticles for the encapsulation of natamycin: Molecular interactions and antifungal application on cherry tomato.

Natamycin was encapsulated in gliadin-casein nanoparticles (G-C NPs) to control black rot in cherry tomato against Alternaria alternata. The G-C NPs with a mean particle diameter of 211 ± 4 nm were prepared using an anti-solvent method. The composite NPs showed better stability against neutral pH, ion, and storage than gliadin NPs. The quenching of gliadin by natamycin was static with a constant of 5.99 × 10-12 M-1∙S-1, and was spontaneous with a free energy of -23.5 kJ∙M-1 at 298 K. Both hydrophobic stacking and hydrogen bonds between natamycin and gliadin were found as the major driven force in the formation of the complex. The NPs kept the antifungal activity of natamycin with improved photostability. The NPs coatings exhibited better results than natamycin in controlling black rot on cherry tomato. This study shows the potential of the G-C NPs as all-natural delivery systems for natamycin in post-harvest treatments.

Effect of Freeze-Thaw Cycles on Juice Properties, Volatile Compounds and Hot-Air Drying Kinetics of Blueberry.

This paper studied the effects of freeze-thaw (FT) cycles on the juice properties and aroma profiles, and the hot-air drying kinetics of frozen blueberry. After FT treatment, the juice yield increased while pH and total soluble solids of the juice keep unchanged. The total anthocyanins contents and DPPH antioxidant activities of the juice decreased by FT treatments. The electronic nose shows that FT treatments significantly change the aroma profiles of the juice. The four main volatile substances in the fresh juice are (E)-2-hexenal, α-terpineol, hexanal and linalyl formate, which account for 48.5 ± 0.1%, 17.6 ± 0.2%, 14.0 ± 1.5% and 7.8 ± 2.7% of relative proportions based on total ion chromatogram (TIC) peak areas. In the FT-treated samples, the amount of (E)-2-hexenal and hexanal decreased significantly while α-terpineol and linalyl formate remained almost unchanged. Repeated FT cycles increased the ethanol content and destroyed the original green leafy flavor. Finally, the drying kinetics of FT-treated blueberries was tested. One FT treatment can shorten the drying time by about 30% to achieve the same water content. The Deff values of the FT-treated sample are similar, which are about twice as large as the value of the fresh sample. The results will be beneficial for the processing of frozen blueberry into juice or dried fruits.

On the mechanism behind enhanced antibacterial activity of alkyl gallate esters against foodborne pathogens and its application in Chinese icefish preservation.

The objective of this study was to investigate antibacterial activities and action mode of alkyl gallates against three food-related bacteria. Results show that the length of the alkyl chain plays a critical role in eliciting their antibacterial activities and octyl gallate (GAC8) exhibited an outstanding bactericidal effect against these strains. A possible bactericidal mechanism of GAC8 against E. coli was fully elucidated by analyzing associated changes in cellular functions of E. coli, including assessments of membrane modification and intracellular oxidation state. Our data strongly suggested that GAC8 functions outside and inside the bacterial membrane and causes increased intracellular reactive oxygen species (hydroxyl radicals) and subsequent oxidative damage. We demonstrated that the hydroxyl radical formation induced by GAC8 is the end product of an oxidative damage cellular death pathway involving a transient depletion of NADH, the tricarboxylic acid cycle, intrinsic redox cycling activities, and stimulation of the Fenton reaction. Also, chitosan-based edible films containing GAC8 have unique superiorities for icefish preservation at 4 °C. This research highlights the effectiveness of GAC8 as an attractive antibacterial, which possesses both antioxidant and antibacterial activities and can be used as a multifunctional food additive combined with the benefit of active packaging for food preservations.

Formononetin/methyl-ß-cyclodextrin inclusion complex incorporated into electrospun polyvinyl-alcohol nanofibers: Enhanced water solubility and oral fast-dissolving property.

Improving solubility and administration route of isoflavone formononetin (FMN) are critical factors to improve its bioavailability in the oral cavity. This study fabricated fast-dissolving nanofibers containing FMN/methyl-ß-cyclodextrin (FMN/Me-ß-CD) inclusion complex. The solubility of FMN could be increased by approximately 50 times at 20 mM aqueous Me-ß-CD. Interactions and thermodynamic parameters of the host-guest inclusion complex were studied by a fluorescence quenching method. The structure and mechanisms of the complex were further studied by molecular docking and molecular dynamics. Finally, polyvinyl-alcohol (PVA) nanofibrous webs containing the FMN/Me-ß-CD inclusion complex were fabricated by electrospinning. The dissolution test demonstrated that the FMN/Me-ß-CD/PVA nanofibers can be dissolved in artificial saliva within approximately 2 s. This study shows the potential of Me-ß-CD inclusion and electrospinning to improve solubility and administration route of isoflavones.

Effect of Drying Methods on Volatile Compounds of Burdock (Arctium lappa L.) Root Tea as Revealed by Gas Chromatography Mass Spectrometry-Based Metabolomics.

Burdock (Arctium lappa L.) is one of the nutritional foods widely planted in many countries. Dried burdock root (BR) is available as a herbal tincture and tea in many Asian countries with good flavor and taste. In this study, the volatile components in dried BR were identified and the effects of different drying methods on the volatile components were investigated by HS-GC-MS method. A total of 49 compounds were identified. Different drying methods including hot-air drying (HD, at 50, 60, 70, and 80 °C), vacuum drying (VD, at 50, 60, 70, and 80 °C), sunlight drying (SD), natural drying (ND), and vacuum freeze drying (VFD) were evaluated by HS-GC-MS-based metabolomics method. Results showed that different drying methods produced different effects on the volatile compounds. It was observed that 2,3-pentanedione, 1-(1H-pyrrol-2-yl)-ethanone, furfural, and heptanal were detected at higher concentrations in HD 80 and VD 70. The traditional HD and SD methods produced more flavor substances than VFD. The BR treated by the VFD method could maintain the shape of the fresh BR pieces while HD50 and VD80 methods could maintain the color of fresh BR pieces. These findings could help better understand the flavor of the corresponding processed BR and provide a guide for the drying and processing of BR tea.

Components identification and isomers differentiation in pigeon pea (Cajanus cajan L.) leaves by LC-MS.

A valid and reliable method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry using electrospray ionization was established to identify chemical constituents in the ethanol extract of pigeon pea leaves. A total of 58 compounds were detected both in positive and negative modes. Among them, 42 compounds, including 16 flavones, 1 flavonol, 5 flavanones, 9 isoflavones, 1 coumarin, 1 lactone, 6 stilbenes, 2 chalcones, and 1 other compound, were unambiguously identified or tentatively assigned in view of the retention time, the molecular formula, as well as the fragmentation patterns. Moreover, eight sets of isomers were differentiated by the ion trap mass spectrometry based on the fragment ion differences or the abundance differences of the same fragment ions. The energy-resolved mass spectrometry in light of the relative abundance of characteristic fragment ions was adopted in the study.

Components characterisation of Berchemia lineata (L.) DC. by UPLC-QTOF-MS/MS and its metabolism with human liver microsomes.

Investigations were performed on the determination of the main components in Berchemia lineata (L.) DC. (BL) and its metabolism with human liver microsomes (HLM). A total of 35 compounds were detected in BL extracts and 25 of them including 6 naphthopyrones, 10 flavonoids, 2 phenolic acids, 2 phenols, 4 fatty acids and 1 quinone were unambiguously or tentatively identified by UPLC-QTOF-MS/MS. Among them, naphthopyrones were first identified in BL extracts and labelled in chromatography. In addition, the weak inhibitory effects of BL extracts (IC50＝149.25 µg/mL) and rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glu-copyranside (the main component of BL extracts, M0; IC50＝82.14 µM) on CYP3A4 were also proved using testosterone as specific probe drug. The main metabolic pathway of M0 by HLM was hydroxylation in its aglycone, the metabolite was tentatively identified as 10-hydroxy-rubrofusarin-6-O-α-L-rhamnosyl-(1-6)-O-ß-D-glucopyranside. Components characterisation and the metabolism with HLM could help the further development and application of BL.

Influence of polysorbates (Tweens) on structural and antimicrobial properties for microemulsions.

Polysorbates (Tweens) are one of the most used excipients for essential oils encapsulation. In this work, the polysorbate based microemulsions (PMEs) for R-(+)-limonene (LMN) encapsulation were investigated for the structural and antimicrobial properties. PMEs were constructed using the pseudoternary phase diagrams, and then characterized for electrical conductivity, rheology, size distribution and particle geometry. Conductivity and rheological measurement results showed that Tween 80 and Tween 60 based microemulsions have identical phase transitions. Dynamic light scattering demonstrated that hydrodynamic diameters of oil-in-water microemulsions decreased from 30 nm to 25 nm during the dilution, while small-angle X-ray scattering indicated that their spherical geometries were maintained. PMEs exhibited enhanced antimicrobial efficiencies in vitro against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Interestingly, when Tween 80 was replaced by Tween 60, PME was observed more effective against S. aureus. The two PMEs structural analogues exhibited different antimicrobial efficiencies corresponding to the bioactivity of polysorbates. In conclusion, PMEs can be considered as a desirable system for LMN encapsulation to enhance its solubility and antimicrobial efficiency. Furthermore, the difference in the antimicrobial efficiency suggested that the choice of emulsifiers should be concerned to improve further applications.

Development of Starch-Based Antifungal Coatings by Incorporation of Natamycin/Methyl-ß-Cyclodextrin Inclusion Complex for Postharvest Treatments on Cherry Tomato against Botrytis cinerea.

The application of natamycin as a natural fungicide in edible coatings is challenging because of its low aqueous solubility. In this study, the natamycin/methyl-ß-cyclodextrin (N/ME-ß-CD) inclusion complex was fabricated and incorporated into waxy corn starch-based coatings for postharvest treatments. The phase solubility of natamycin in the presence of ME-ß-CD at 293.2 K, 303.2 K, and 313.2 K is determined and used to calculate the process thermodynamic parameters. The N/ME-ß-CD inclusion complex was confirmed and characterized by FTIR and 1H NMR spectroscopy. The results indicated that the inclusion complex was formed and the hydrophobic part (C16-C26) of natamycin might be partially inserted into the cavity of ME-ß-CD form the wide rim. The effects of N/ME-ß-CD incorporated starch-based coatings (N/ME-ß-CD S coatings) on postharvest treatments of cherry tomatoes were evaluated in vivo. The N/ME-ß-CD S coatings could reduce weight loss, delay fruit ripening, and inhibit fruit decay caused by Botrytis cinerea in tomato fruit during storage.