Single-Molecule Identification and Quantification of Steviol Glycosides with a Deep Learning-Powered Nanopore Sensor.

Steviol glycosides (SGs) are a class of high-potency noncalorie natural sweeteners made up of a common diterpenoid core and varying glycans. Thus, the diversity of glycans in composition, linkage, and isomerism results in the tremendous structural complexity of the SG family, which poses challenges for the precise identification and leads to the fact that SGs are frequently used in mixtures and their variances in biological activity remain largely unexplored. Here we show that a wild-type aerolysin nanopore can detect and discriminate diverse SG species through the modulable electro-osmotic flow effect at varied applied voltages. At low voltages, the neutral SG molecule was drawn and stuck in the pore entrance due to an energy barrier around R220 sites. The ensuing binding events enable the identification of the majority of SG species. Increasing the voltage can break the barrier and cause translocation events, allowing for the unambiguous identification of several pairs of SGs differing by only one hydroxyl group through recognition accumulation from multiple sensing regions and sites. Based on nanopore data of 15 SGs, a deep learning-based artificial intelligence (AI) model was created to process the individual blockage events, achieving the rapid, automated, and precise single-molecule identification and quantification of SGs in real samples. This work highlights the value of nanopore sensing for precise structural analysis of complex glycans-containing glycosides, as well as the potential for sensitive and rapid quality assurance analysis of glycoside products with the use of AI.

Non-target and target quantitative metabolomics with quantitative aroma evaluation reveal the influence mechanism of withering light quality on tea aroma and volatile metabolites evolution.

Withering is the first and key process that influences tea quality, with light quality being a key regulatory factor. However, effects of withering light quality (WLQ) on transformation and formation pathways of tea aroma and volatile metabolites (VMs) remain unclear. In the present study, four WLQs were set up to investigate their effects on tea aroma and VMs. The results showed that blue and red light reduced the grassy aroma and improved the floral and fruity aroma of tea. Based on GC-MS/MS, 83 VMs were detected. Through VIP, significant differences, and OAV analysis, 13 key differential VMs were screened to characterize the differential impacts of WLQ on tea aroma. Further analysis of the evolution and metabolic pathways revealed that glycoside metabolism was the key pathway regulating tea aroma through WLQ. Blue light withering significantly enhanced glycosides hydrolysis and amino acids deamination, which was beneficial for the enrichment of floral and fruity VMs, such as geraniol, citral, methyl salicylate, 2-methyl-butanal, and benzeneacetaldehyde, as well as the transformation of grassy VMs, such as octanal, naphthalene, and cis-3-hexenyl isovalerate, resulting in the formation of tea floral and fruity aroma. The results provide theoretical basis and technical support for the targeted processing of high-quality tea.

Untargeted metabolomic profiling of Prinsepia utilis Royle leaves by Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry.

Prinsepia utilis Royle, native to the Himalayas, is esteemed in Chinese and Indian folk medicine for its diverse medicinal benefits, targeting arthritis, pain relief, bone disorders, and joint discomfort. This study examined the 25% aqueous methanol extract of P. utilis leaves using UPLC-Q-TOF-MS/MS, identifying 78 metabolites, 76 of which were reported for the first time in P. utilis. These included 64 phenolics represented by 56 flavonoids, 5 phenolic acids, 3 phenolic glycosides, 4 terpenoids, 2 lignan glycosides, and 8 other compounds, expanding the knowledge of its chemical composition. These findings lay a foundation for further research, providing insights into potential bioactive compounds and opening avenues for applications in natural product drug discovery, traditional medicine, and nutraceutical development, leveraging the plant's established traditional uses.

Distribution of active ingredients and quality control of Forsythia suspensa with AP-MALDI mass spectrometry imaging.

The fruits of Forsythia suspensa (F. suspensa) have been used as a traditional Chinese medicine for 2000 years. Currently, the quality control of F. suspensa strictly follows the instructions of Chinese Pharmacopeia, which mainly controls the content of forsythoside A, phillyrin, and volatile oil. In this study, air pressure MALDI mass spectrometry imaging (AP-MALDI MSI) was used to evaluate the quality of F. suspensa fruits and the distribution of dozens of active ingredients. The variation of active ingredients was measured for more than 30 batches of samples, regarding harvest time, cultivated environment, shelf-life, and habitat. Fifty-three active ingredients could be detected in F. suspensa fruits with AP-MALDI MSI. Seven active ingredients were upregulated, four ingredients downregulated, and 15 ingredients did not change in ripe fruits. A sharp variation of active ingredients in late September was observed for the Caochuan fruits harvested in 2019, which is closely related to the appearance of the ginger color of the pericarp under the microscope observation. The microscope observation is a reliable way to classify ripe and green fruits instead of outlook. Just considering forsythoside A and phillyrin, it is found that wild fruits are better than cultivated fruits, but cultivated fruits have high contents of other ingredients. The shelf-life of F. suspensa fruits is proposed to be 3 years, considering the 26 ingredients investigated. It was found that Luoning wild fruits are better than those from Caochuan with a new evaluation method. Mass spectrometry imaging is an easy, objective, and effective method to evaluate the quality of F. suspensa fruits.

Evaluation of exposure to cyanogenic glycosides and potential hydrogen cyanide release in commercially available foods among the Korean population.

The release of hydrogen cyanide (HCN) after food ingestion can pose a serious health risk to consumers. This study aimed to simultaneously quantify four cyanogenic glycosides (lotaustralin, prunasin, taxiphyllin, and dhurrin) using liquid chromatography-tandem mass spectrometry. The analysis scope extended beyond agricultural products to various consumer foods to estimate dietary exposure to cyanogenic glycosides and assess its risk levels. The major exposure sources are cassava chips (lotaustralin), apples (seeds) (prunasin and dhurrin), and Prunus mume axis (taxiphyllin). In addition to quantifying specific cyanogenic glycosides, this study proposed the development of a preliminary risk assessment framework based on the dietary exposure assessment and the calculation of theoretical levels of HCN derived from cyanogenic glycoside concentrations. In the absence of established guidelines for the permissible intake of foods containing cyanogenic glycosides, this study provides initial guidance for assessing the risks associated with a range of commonly consumed foods.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Rapid purification and scale-up of tilianin using counter-current chromatography with rectangular horizontal tubing.

In counter-current chromatography (CCC), linear scale-up is an ideal amplification strategy. However, when transferring from analytical to predictable preparative processes with high throughput, linear scale-up would be challenging due to limitations imposed by differences in instrument parameters, such as gravitational forces, tubing cross-section area, tubing length, column volume and flow rate. Some effective scale-up strategies have been studied for different instrument parameters, but so far, these scale-up works have only been tested on standard circular (SC) tubing. The previous research of our group found that rectangular horizontal (RH) tubing can double the separation efficiency compared with conventional SC tubing, and has industrial production potential. This paper used the separation of tilianin from Dracocephalum moldavica L. as an example to demonstrate how to scale up the optimized process from analytical SC tubing to preparative RH tubing. After systematic optimization of solvent systems, sample concentration and flow rate on the analytical CCC, the optimized parameters obtained were successfully transferred to the preparative CCC. The results showed that a crude sample of 2.07 g was successfully separated using a solvent system of n-hexane - ethyl acetate - ethanol - water (1:4:1:5, v/v/v/v) in reversed phase mode, and the three consecutive separations produced a total of 380 mg tilianin in 75 min with high purities of 98.3%, as analyzed by HPLC. The total throughput achieved from the analytical to semi-preparative scale was improved by 138 times (from 12 mg/h to 1.66 g/h), while the column volume was increased by only 46.5 times (from 15.5 mL to 720 mL). This is the successful application of CCC for the separation and purification of tilianin. Given that SC tubing is the traditional configuration for CCC columns, this study is a necessary step to prove the applicability of RH tubing columns for routine use and potential large-scale industrial applications.

Quantitative analysis of six sesquiterpene glycosides from Dendrobium nobile Lindl. under different growth conditions by high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry in MRM mode.

INTRODUCTION: The sesquiterpene glycosides (SGs) from Dendrobium nobile Lindl. have immunomodulatory effects. However, there are no studies on the growth conditions affecting its contents and quantitative analysis methods. OBJECTIVE: In the present study, a quantitative analysis method for six SGs from D. nobile was established. We explored which growth conditions could affect the contents of SGs, providing a basis for the cultivation and clinical application of D. nobile. METHODS: Firstly, based on the optimization of mass spectrometry parameters and extraction conditions for six SGs in D. nobile, a method for the determination of the contents of six SGs was established using high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Then, the methodology of the established method was validated. Secondly, the established method was applied to determine the contents of six SGs from 78 samples of D. nobile grown under different growth conditions. Finally, chemometrics analysis was employed to analyze the results and select optimal growth conditions for D. nobile. RESULTS: The results indicated significant variations in the contents of SGs from D. nobile grown under different growth conditions. The primary factors influencing SG contents included age, geographical origin, altitude, and epiphytic pattern. CONCLUSION: Therefore, the established method for determining SG contents from D. nobile is stable. In particular, the SG contents were relatively high in samples of 3-year-old D. nobile grown at an altitude of approximately 500 m on Danxia rocks in Chishui, Guizhou.

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS. The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

New insights into bee pollen: Nutrients, phytochemicals, functions and wall-disruption.

Bee pollen is hailed as a treasure trove of human nutrition and has progressively emerged as the source of functional food and medicine. This review conducts a compilation of nutrients and phytochemicals in bee pollen, with particular emphasis on some ubiquitous and unique phenolamides and flavonoid glycosides. Additionally, it provides a concise overview of the diverse health benefits and therapeutic properties of bee pollen, particularly anti-prostatitis and anti-tyrosinase effects. Furthermore, based on the distinctive structural characteristics of pollen walls, a substantial debate has persisted in the past concerning the necessity of wall-disruption. This review provides a comprehensive survey on the necessity of wall-disruption, the impact of wall-disruption on the release and digestion of nutrients, and wall-disruption techniques in industrial production. Wall-disruption appears effective in releasing and digesting nutrients and exploiting bee pollen's bioactivities. Finally, the review underscores the need for future studies to elucidate the mechanisms of beneficial effects. This paper will likely help us gain better insight into bee pollen to develop further functional foods, personalized nutraceuticals, cosmetics products, and medicine.

Chemical profile and difference analysis of four parts of Strobilanthes sarcorrhiza by ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and multivariate statistical analysis.

Strobilanthes sarcorrhiza (CTS) is a medicinal plant with various pharmacological effects such as tonifying kidney and anti-inflammatory. However, the chemical composition and difference of its four parts (leaves, stems, rhizomes, and root tubers) have been rarely reported. In this study, ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight MS was applied to analyze the chemical profile of CTS and identify 55 compounds, including terpenoids, phenylethanol glycosides, fatty acid derivatives, chain glycosides, flavonoid glycosides, and others. Among these compounds, 34 compounds were first identified in CTS. They were mainly terpenoids, phenylethanol glycosides, fatty acid derivatives, and so forth. Multivariate statistical analysis, such as principal component analysis and orthogonal partial least squares-discriminant analysis were also used to evaluate the difference in chemical compounds from the four parts of CTS. The results showed that phenylethanol glycosides were the main compounds of the underground parts, while terpenoids were the main compounds of the aboveground parts. This study revealed the chemical diversity and similarity of CTS and suggested that the rhizomes could be used as an alternative medicinal part to improve the resource utilization of CTS.

Quantitative and qualitative composition of proanthocyanidins and other polyphenols in commercial red wines and their contribution to sensorially evaluated tannicity.

The analysis of proanthocyanidins (PA) in red wine has typically been conducted using few key methods, such as phloroglucinolysis or precipitation assays. Here, the content of PAs and other common polyphenol groups in commercial red wines were analyzed with a group-specific liquid chromatography-tandem mass spectrometry method. Besides concentrations, the method provides qualitative information about the detected compound groups in the form of two-dimensional (2D) chromatographic fingerprints. The 2D fingerprints of PAs have not been utilized in analysis of red wine before. For instance, 2D chromatographic fingerprints revealed that the complex PA compositions were qualitatively notably similar between many wine types, even when there were considerable differences in concentrations. Finally, 201 commercial red wines had been categorized as either tannic or medium tannic based on their sensorial evaluations. The content of PAs and three different groups of oligomeric adducts of malvidin glycosides and PAs were measured from these wines. The compositional features of the PAs and PA-malvidin glycoside adducts were more important than concentrations in explaining the perceived tannicity.

Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Unraveling spatial metabolome of the aerial and underground parts of Scutellaria baicalensis by matrix-assisted laser desorption/ionization mass spectrometry imaging.

BACKGROUND: Scutellaria baicalensis Georgi, a traditional Chinese medicine, is clinically applied mainly as the dried root of Scutellaria baicalensis, and the aerial parts of Scutellaria baicalensis, its stems and leaves, are often consumed as "Scutellaria baicalensis tea" to clear heat, dry dampness, reduce fire and detoxify, while few comparative analyses of the spatial metabolome of the aerial and underground parts of Scutellaria baicalensis have been carried out in current research. METHODS: In this work, Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize the spatial imaging of the root, stem, and leaf of Scutellaria baicalensis at a high resolution of 10 µm, respectively, investigating the spatial distribution of the different secondary metabolites in the aerial and underground parts of Scutellaria baicalensis. RESULTS: In the present results, various metabolites, such as flavonoid glycosides, flavonoid metabolites, and phenolic acids, were systematically characterized in Scutellaria baicalensis root, stem, and leaf. Nine glycosides, 18 flavonoids, one organic acid, and four other metabolites in Scutellaria baicalensis root; nine glycosides, nine flavonoids, one organic acid in Scutellaria baicalensis stem; and seven flavonoids and seven glycosides in Scutellaria baicalensis leaf were visualized by MALDI-MSI. In the underground part of Scutellaria baicalensis, baicalein, wogonin, baicalin, wogonoside, and chrysin were widely distributed, while there was less spatial location in the aerial parts. Moreover, scutellarein, carthamidin/isocarthamidin, scutellarin, carthamidin/isocarthamidin-7-O-glucuronide had a high distribution in the aerial parts of Scutellaria baicalensis. In addition, the biosynthetic pathways involved in the biosynthesis of significant flavonoid metabolites in aerial and underground parts of Scutellaria baicalensis were successfully localized and visualized. CONCLUSIONS: MALDI-MSI offers a favorable approach for investigating the spatial distribution and effective utilization of metabolites of Scutellaria baicalensis. The detailed spatial chemical information can not only improve our understanding of the biosynthesis pathways of flavonoid metabolites, but more importantly, suggest that we need to fully exert the overall medicinal value of Scutellaria baicalensis, strengthening the reuse and development of the resources of Scutellaria baicalensis aboveground parts.

Secoiridoid glycosides from the fruits of Ligustrum lucidum and their in vitro anti-inflammatory activity.

Seven new secoiridoid glycosides (1-7), together with a known analogue (8), were isolated from the fruits of Ligustrum lucidum. Their structures with absolute configurations were determined by HR-ESI-MS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectroscopic analysis, as well as biogenetic consideration. Compounds 1 and 2 are the first examples of secoiridoid glycoside dimers featuring a rare rearranged oleoside-type secoiridoid moiety, and compounds 3-7 represent a new class of oleoside-type secoiridoid glycosides with unusual stereochemistry at C-1 position. A plausible biosynthetic pathway for this group of unusual secoiridoid glycosides was also proposed herein. In addition, the isolates were evaluated for their in vitro anti-inflammatory activity, and all tested compounds exhibited modest inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

[Heterocyclic compounds and phenolic glycosides from flowers of Dendrobium officinale].

Eight heterocyclic compounds and twelve phenolic glycosides were separated from the water extract of Dendrobium officinale flowers through chromatographic techniques, such as Diaion HP-20 macroporous adsorption resin column chromatography(CC), silica gel CC, ODS CC, Sephadex LH-20 CC, and preparative high performance liquid chromatography(PHPLC). According to the spectroscopic analyses(MS, ~1H-NMR, and ~(13)C-NMR) and optical rotation data, the compounds were identified as dendrofurfural A(1), 2'-deoxyadenosine(2), 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid(3), 4-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid(4), 1-(2-hydroxyethyl)-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde(5), 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde(6), methyl 5-(hydroxymethyl)-furan-2-carboxylate(7),(S)-5-hydroxymethyl-5H-furan-2-one(8), 2-methoxyphenyl-1-O-ß-D-glucopyranoside(9), arbutin(10), isotachioside(11), 2,6-dimethoxy-4-hydroxyphenol-1-O-ß-D-glucopyranoside(12), orcinol glucoside(13), tachioside(14), gastrodin(15), 4-O-ß-D-glucopyranosylvanillyl alcohol(16), 2,6-dimethoxy-4-hydroxymethylphenol-1-O-ß-D-glucopyranoside(17), icariside D_2(18), 4-formylphenyl-ß-D-glucopyranoside(19), and vanillin-4-O-ß-D-glucopyranoside(20). Among them, compound 1 is a new furfural benzyl alcohol condensate, with the skeleton first found in Dendrobium. Compounds 2-9, 11, 13, and 19 are reported from Dendrobium for the first time, and compounds 14 and 18 are reported for the first time from D. officinale. Compounds 11 and 14 showed moderate DPPH radical scavenging capacity, and compounds 11-14 demonstrated potent ABTS radical scavenging capacity, possessing antioxidant activity.

Chemometrics-based investigation of non-volatiles/volatiles flavor of tencha (Camellia sinensis cv. Yabukita, Longjing 43 and Baiye 1).

The increasing demand for tea consumption calls for the development of more products with distinct characteristics. The sensory quality of tencha is significantly determined by innate differences among tea cultivars. However, the correlations between the chemical composition and sensory traits of tencha are still unclear. To enhance the understanding of the flavor formation mechanism in tencha and further to develop new cultivars resources, we investigated non-volatiles and volatile metabolites as well as sensory traits in tencha from different tea cultivars (Camellia sinensis cv. Yabukita, Longjing 43 and Baiye 1); the relationships between the flavor traits and non-volatiles/volatiles were further evaluated by partial least squares - discriminate analysis (PLS-DA), multiple factor analysis (MFA) and multidimensional alignment (MDA) analysis. A total of 64 non-volatiles and 116 volatiles were detected in all samples, among which 71 metabolites were identified as key flavor-chemical contributors involving amino acids, flavonol glycosides, flavones, catechins, ketones, alcohols, hydrocarbons, aldehydes, esters and acids. The levels of taste-related amino acids, flavonol glycosides and gallic acid varied significantly among the tencha samples made from different tea cultivars. All the samples exhibited typical quality characteristics of tencha. The tencha from Camellia sinensis cv. Longjing 43 and Camellia sinensis cv. Baiye 1 (cultivated in the open) exhibited higher levels of amino acids and gallic acid, which were associated with the umami taste and mellow taste of tea infusion. Abundant flavonol glycosides were related to the astringency, while partial tri-glycosides specifically quercetin-3-O-galactoside-rhamnoside-glucoside and total of flavonol galactoside-rhamnoside-glucoside were associated with mellow taste. The floral alcohols were identified as significant contributors to the refreshing aroma traits of tencha. The green, almond-like, acidic and fruity odorants were associated with a green and fresh aroma, while the green, cheesy and waxy odorants such as ketones, esters, acids and hydrocarbons were associated with seaweed-like aroma. This study provides insight into sensory-related chemical profiles of tencha from different tea cultivars, supplying valuable information on flavor and quality identification for tencha.

Phytochemical profiling, human insulin stability and alpha glucosidase inhibition of Gymnema latifolium leaves aqueous extract: Exploring through experimental and in silico approach.

Diabetes mellitus Type 2 (DM2T) is a rapidly expanding metabolic endocrine disorder worldwide. It is caused due to inadequate insulin secretion by pancreatic beta cells as well as development of insulin resistance. This study aimed to investigate the anti-α-glucosidase, insulin stabilization effect, and non-cytotoxic nature of Gymnema latifolium leaf aqueous extract (GLAE). FTIR analysis revealed the functional groups of compounds present in GLAE. Through LC/ESI-MS/MS analysis, about 12 compounds which belongs to different classes, triterpene glycosides, flavonoids, phenolics, stilbene glycosides and chlorophenolic glycosides were identified. GLAE showed in vitro antioxidant activity. GLAE stabilized insulin by increasing its α-helical content. GLAE inhibited the mammalian α-glucosidase (IC50 = 144 µg/mL) activity through competitive mode (Ki = 61.30 µg/mL). GLAE did not affect the viability of normal cell line (Vero cell line) which shows its non-toxic nature. Molecular docking of phytocompounds identified in GLAE was done with human α-glucosidase and insulin. The top 2 compounds [Gymnema saponin V (GSV) and quercetin 3-(2-galloylglucoside) (QGG) with α-glucosidase; GSV and Z)-resveratrol 3,4'-diglucoside (RDG) with human insulin] with low binding free energy were subjected to 100 ns molecular dynamics simulation to ascertain the stable binding of ligand with protein. The MM/GBSA analysis revealed binding free energy of GSV/α-glucosidase and QGG /α-glucosidase to be - 20.9935 and, - 30.9461 kcal/mol, respectively. Altogether GLAE is valuable source of anti-α-glucosidase inhibitors and insulin stabilizing compounds, suggesting potential lead for further exploration as complementary medicine against DM2T.

Neurotrophic phenolic glycosides from the roots of Armoracia rusticana.

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis (1H, 13C, 1H-1H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells.

DCTPP1, a reliable Q-biomarker for comprehensive evaluation of the quality of tripterygium glycoside tablets based on chemical references.

BACKGROUND: As first-line clinical drugs, tripterygium glycoside tablets (TGTs) often have inconsistent efficacy and toxic side effects, mainly due to inadequate quality control. Therefore, clinically relevant quality standards for TGTs are urgently required. PURPOSE: Based on chemical substances and considering pharmacological efficacy, we aimed to develop an effective quality evaluation method for TGTs. METHODS: Representative commercial samples of TGTs were collected from different manufacturers, and qualitative UHPLC/LTQ-Orbitrap-MS and quantitative UHPLC-MS/MS analysis methods were successfully applied to evaluate their quality similarities and differences based on their chemical properties. Then the anti-immunity, anti-inflammatory and antitumor activities of TGTs and related monomers were evaluated using Jurkat, RAW264.7, MIA PaCa-2, and PANC-1 as cellular models. Subsequently, we predicted and verified small molecule-DCTPP1 interactions via molecular docking using the established DCTPP1 enzymatic activity assay. Finally, we performed a gray relational analysis to evaluate the chemical characteristics and biological effects of TGTs produced by different manufacturers. RESULTS: We collected 24 batches of TGTs (D01-D24) from 5 manufacturers (Co. A, Co. B, Co. C, Co. D, Co. E) for quality evaluation. The chemical composition analysis revealed significant differences in the substance bases of the samples. The D02, D18-D20 samples from Co. B constituted a separate group that differed from other samples, mainly in their absence of diterpenoids and triterpenoids, including triptolide, triptophenolide, and triptonide. In vitro anti-immunity, antitumor and anti-inflammatory tests using the same TGT concentration revealed that, except for D02, D18-D20, the remaining 20 samples exhibited different degrees of anti-immunity, antitumor and anti-inflammatory activity. Our experiments verified that triptolide, triptophenolide, and triptonide were all DCTPP1 inhibitors, and that TGTs generally exhibited DCTPP1 enzyme inhibitory activity. Moreover, the inhibitory activity of D02, D18-D20 samples from Co. B was much lower than that of the other samples, with a nearly tenfold difference in IC50. Further comprehensive analysis revealed a high correlation between DCTPP1 enzyme inhibition activity and the anti-immunity and antitumor and anti-inflammatory activities of these samples. CONCLUSION: The established DCTPP1 enzymatic activity assay proved suitable for quantitative pharmacological and pharmaceutical analysis to complement the existing quality control system for TGTs and to evaluate their effectiveness.