Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

Improving the antinutritional profiles of common beans (Phaseolus vulgaris L.) moderately impacts carotenoid bioaccessibility but not mineral solubility.

Common beans are a common staple food with valuable nutritional qualities, but their high contents in antinutritional factors (ANFs) can decrease the bioavailability of (i) fat-soluble micronutrients including carotenoids and (ii) minerals. Our objective was to select ANF-poor bean lines that would not interfere with carotenoid and mineral bioavailability. To achieve this objective, seeds of commercial and experimental Phaseolus vulgaris L. bean lines were produced for 2 years and the bean's content in ANFs (saponins, phytates, tannins, total polyphenols) was assessed. We then measured carotenoid bioaccessibility and mineral solubility (i.e. the fraction of carotenoid and mineral that transfer into the aqueous phase of the digesta and is therefore absorbable) from prepared beans using in vitro digestion. All beans contained at least 200 mg/100 g of saponins and 2.44 mg/100 g tannins. The low phytic acid (lpa) lines, lpa1 and lpa12 exhibited lower phytate levels (≈ - 80%, p = 0.007 and p = 0.02) than their control BAT-93. However, this decrease had no significant impact on mineral solubility. HP5/1 (lpa + phaseolin and lectin PHA-E free) bean line, induced an improvement in carotenoid bioaccessibility (i.e., + 38%, p = 0.02, and + 32%, p = 0.005, for phytofluene bioaccessibility in 2021 and 2022, respectively). We conclude that decrease in the phytate bean content should thus likely be associated to decreases in other ANFs such as tannins or polyphenols to lead to significant improvement of micronutrient bioaccessibility.

Intercropping with maize and sorghum-induced saikosaponin accumulation in Bupleurum chinense DC. by liquid chromatography-mass spectrometry-based metabolomics.

Bupleuri Radix is an important medicinal plant, which has been used in China and other Asian countries for thousands of years. Cultivated Bupleurum chinense DC. (B. chinense) is the main commodity of Bupleuri Radix. The benefits of intercropping with various crops for B. chinense have been recognized; however, the influence of intercropping on the chemical composition of B. chinense is still unclear yet. In this study, intercropping with sorghum and maize exhibited little effect on the root length, root diameter, and single root mass of B. chinense. Only the intercropping with sorghum increased the root length of B. chinense slightly compared to the monocropping. In addition, 200 compounds were identified by UHPLC-Q-TOF-MS, and metabolomic combined with the Venn diagram and heatmap analysis showed apparent separation between the intercropped and monocropped B. chinense samples. Intercropping with sorghum and maize could both increase the saikosaponins, fatty acyls, and organic acids in B. chinense while decreasing the phospholipids. The influence of intercropping on the saikosaponin biosynthesis was probably related with the light intensity and hormone levels in B. chinense. Moreover, we found intercropping increased the anti-inflammatory activity of B. chinense. This study provides a scientific reference for the beneficial effect of intercropping mode of B. chinense.

Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments.

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 µM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 µM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Optimizing the Vanillin-Acid Sulfuric Method to Total Saponin Content in Leaves of Yerba Mate Clones.

Yerba mate (Ilex paraguariensis) is a forest species consumed in the form of non-alcoholic beverages in South America, with applications in foods, cosmetics, and pharmaceutical industries. The species leaves are globally recognized for their important bioactive compounds, including, saponins. We adjusted the vanillin-acid sulfuric method for determining spectrophotometrically the total saponin in yerba mate leaves. Seeking to maximize the extraction of saponins from leaves, a Doehlert design combined with Response Surface Methodology (RSM) was used, considering ethanol:water ratios and ultrasound times. In addition, the same methodology was used for the analysis of times and temperatures in the vanillin-sulfuric acid reaction heating. The contents of total saponin in mature leaves were compared in four yerba mate clones. The extraction was maximized using 40 % ethanol:60 % water and 60 minutes of ultrasound assisted extraction (UAE) without heating. For the reaction conditions, 70 °C for 10 minutes heating is recommended, and UV/Vis reading from 460 to 680 nm. Using the optimized methodology, total saponin contents ranged from 28.43 to 53.09 mg g-1 in the four yerba mate clones. The significant difference in saponin contents between clones indicate great genetic diversity and potential for clones' selection and extraction of these compounds from yerba mate leaves.

Exploration of the profiles of steroidal saponins from Rhizoma Paridis and their metabolites in rats by UPLC-Q-TOF-MS/MS.

INTRODUCTION: Steroidal saponins characterised by intricate chemical structures are the main active components of a well-known traditional Chinese medicine (TCM) Rhizoma Paridis. The metabolic profiles of steroidal saponins in vivo remain largely unexplored, despite their renowned antitumor, immunostimulating, and haemostatic activity. OBJECTIVE: To perform a comprehensive analysis of the chemical constituents of Rhizoma Paridis total saponins (RPTS) and their metabolites in rats after oral administration. METHOD: The chemical constituents of RPTS and their metabolites were analysed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). RESULTS: A reliable UPLC-Q-TOF-MS/MS method was established, and a total of 142 compounds were identified in RPTS. Specifically, diosgenin-type saponins showed the diagnostic ions at m/z 415.32, 397.31, 283.25, 271.21, and 253.20, whereas pennogenin-type saponins exhibited the diagnostic ions at m/z 413.31, 395.30, and 251.20. Based on the characteristic fragments and standard substances, 15 specific metabolites were further identified in the faeces, urine, plasma, and bile of rats. The metabolic pathways of RPTS, including phase I reactions (de-glycosylation and oxidation) and phase II reactions (glucuronidation), were explored and summarised, and the enrichment of metabolites was characterised by multivariate statistical analysis. CONCLUSION: The intricate RPTS could be transformed into relatively simple metabolites in rats through de-glycosylation, which provides a reference for further metabolic studies and screening of active ingredients for TCM.

Identification of cyclooxygenase-II inhibitory saponins from fenugreek wastes: Insights from liquid chromatography-tandem mass spectrometry metabolomics, molecular networking, and molecular docking.

INTRODUCTION: This research explores sustainable applications for waste generated from fenugreek (Trigonella foenum-graecum), a plant with both nutritional and medicinal uses. The study specifically targets waste components as potential sources of nutrients and bioactive compounds. OBJECTIVES: The focus is to conduct detailed metabolic profiling of fenugreek waste, assess its anti-inflammatory properties by studying its cyclooxygenase (COX) inhibitory effect, and correlate this effect to the metabolite fingerprint. MATERIALS AND METHODS: Ethanolic extracts of fenugreek fruit pericarp and a combination of leaves and stems were subjected to untargeted metabolic profiling using liquid chromatography-mass spectrometry integrated with online database searches and molecular networking as an effective dereplication strategy. The study also scrutinized the COX inhibitory capabilities of these extracts and saponin-rich fractions prepared therefrom. Molecular docking was employed to investigate the specific interactions between the identified saponins and COX enzymes. RESULTS: The analysis led to the annotation of 81 metabolites, among which saponins were predominant. The saponin-rich fraction of the fruit pericarp extract displayed the strongest COX-II inhibitory activity in the in vitro inhibition assay (IC50 value of 81.64 ± 3.98 µg/mL). The molecular docking study supported the selectivity of the identified saponins towards COX-II. The two major identified saponins, namely, proto-yamogenin 3-O-[deoxyhexosyl (1 → 2)] [hexosyl (1 → 4)] hexoside 26-O-hexoside and trigofenoside A, were predicted to have the highest affinity to the COX-II receptor site. CONCLUSION: In the present study, we focused on the identification of COX-II inhibitory saponins in fenugreek waste through an integrated approach. The findings offer valuable insights into potential anti-inflammatory and cancer chemoprotective applications of fenugreek waste.

A novel strategy by combining foam fractionation with high-speed countercurrent chromatography for the rapid and efficient isolation of antioxidants and cytostatics from Camellia oleifera cake.

Camellia oleifera cake is a by-product, which is rich in functional chemical components. However, it is typically used as animal feed with no commercial value. The purpose of this study was to isolate and identify compounds from Camellia oleifera cake using a combination of foam fractionation and high-speed countercurrent chromatography (HSCCC) and to investigate their biological activities. Foam fractionation with enhanced drainage through a hollow regular decahedron (HRD) was first established for simultaneously enriching flavonoid glycosides and saponins for further separation of target compounds. Under suitable operating conditions, the introduction of HRD resulted in a threefold increase in enrichment ratio with no negative effect on recovery. A novel elution-extrusion countercurrent chromatography (EECCC) coupled with the consecutive injection mode was established for the successful simultaneous isolation of flavonoid glycosides and saponins. As a result, 38.7 mg of kaemferol-3-O-[2-O-D-glucopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 98.17%, FI), 70.8 mg of kaemferol-3-O-[2-O-ß-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 97.52%, FII), and 560 mg of an oleanane-type saponin (purity of 92.32%, FIII) were separated from the sample (900 mg). The present study clearly showed that FI and II were natural antioxidants (IC50 < 35 µg/mL) without hemolytic effect. FIII displayed the effect of inhibiting Hela cell proliferation (IC50 < 30 µg/mL). Further erythrocyte experiments showed that this correlated with the extremely strong hemolytic effect of FIII. Overall, this study offers a potential strategy for efficient and green isolation of natural products, and is beneficial to further expanding the application of by-products (Camellia oleifera cake) in food, cosmetics, and pharmacy.

Mathematical models for predicting protodioscin in tropical forage grasses.

Protodioscin is a saponin present in grasses that can lead to losses in animal production. Our hypothesis was that mathematical models can accurately and precisely predict the protodioscin concentration in tropical grasses. We evaluated the ability of four mathematical models to describe the protodioscin concentration in Brachiaria and Panicum cultivars with different regrowth periods. Six cultivars of Panicum: Aruana, Massai, Mombaça, Tanzânia, Tamani, and Zuri; and five of Brachiaria-grass: Marandu, Paiaguás, Piatã, Xaraés and Basilisk. Protodioscin concentration evaluations were carried out at 51, 84, 110, and 111 days of age. Linear, Quadratic, Exponential, and Logarithmic models were evaluated, and the adequacy of the models was verified. The models were compared for accuracy and precision by pairwise mean squared error analysis and the delta Akaike information criterion. The models did not differ from each other in terms of accuracy and precision. The exponential model showed a high ability to explain the observed variability between protodioscin concentration and plant age for Brachiaria grasses. Panicum grasses have constant protodioscin concentration. Mathematical models are capable of predicting the protodioscin concentration in grasses of the genus Brachiaria based on plant age. We recommend Exponential model to predict the concentration of protodioscin in Brachiaria grasses.

Characterization and quality evaluation of QiXueShuFu Decoction based on fingerprint and ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry.

QiXueShuFu Decoction (QXSFD) modified from the Bazhen Decoction which was originally from the classic Ming Dynasty is a traditional folk formula that boosts the body's immune system. However, its ambiguous chemical components limited its quality control evaluation. In this study, ultra-performance liquid chromatography (UPLC) fingerprint combined with multivariate analysis was used to evaluate the quality of 15 batches of QXSFD, and UPLC quadrupole-orbitrap mass spectrometry was used to further examine the chemical components in QXSFD, after which representative compounds from each disassembled prescription were selected for comparison. Fifteen batches of samples had 33 common peaks in which 11 differential components could be used as a reference for subsequent quality control. One hundred forty-three components were identified from QXSFD. Saponins were mainly derived from the monarch, terpenes from the minister, and polysaccharides and glycosides from the assistant. In addition, quantitative assay revealed that the content of ferulic acid, chlorogenic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside and 3,6'-disinapoyl sucrose in the whole prescription were higher than the contents of each disassembled prescription. This is the first comprehensive quality report on the chemical components of QXSFD, which is important for pharmacodynamic material basis and quality control.

Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Derivatization-Based Novel Chromatographic and Spectrophotometric Methods for the Simultaneous Determination of Gymnemic Acid and Resveratrol in Antidiabetic Polyherbal Formulation.

Herbal medicine is widely used for the treatment and prevention of various ailments, highlighting the importance of ensuring its consistency and quality. This research focuses on the simultaneous detection of Gymnemic acid (GYM) and Resveratrol (RES) in an antidiabetic polyherbal formulation as no reported method exists for their simultaneously detection. The objective of this study is to develop and validate novel derivatization-based spectrometric and HPTLC methods for the simultaneous determination of GYM and RES. The spectrophotometric method involved derivatization of GYM with benzoyl chloride, followed by measurement of absorbance at 349 nm an isoabsorptive point. The HPTLC method utilized post derivatization with vanillin-sulfuric acid, and its separation was achieved on pre-coated silica gel 60GF254 using chloroform:methanol:glacial acetic acid (13:4:0.1, v/v/v) as mobile phase and estimated at 575 nm. The developed method exhibits linearity, accuracy, precision, LOD, LOQ, specificity and robustness in accordance with the ICH Q2 (R1) guideline. The percent assay of GYM and RES in the marketed capsule formulation was statistically compared using an unpaired t-test, resulting in a range of 99.51-102.65%. These indicate no significant difference between the proposed method and the marketed formulation. Therefore, both novel methods can be interchangeably used for quality control of GYM and RES in polyherbal formulations.

MATLAB language assisted data acquisition and processing in liquid chromatography Orbitrap mass spectrometry: Application to the identification and differentiation of Radix Bupleuri from its adulterants.

Comprehensive and rapid analysis of secondary metabolites like saponins remains challenging. This study aimed to establish a semi-automated workflow for filtration, identification, and characterization of saikosaponins in six Bupleurum species. Radix Bupleuri, a high-sales herbal medicine, is often adulterated, restricting its quality control and applications. Two authentic Radix Bupleuri species and four major adulterants were analyzed through UHPLC-LTQ-Orbitrap-MS for targeted saikosaponin analysis. To reveal trace saikosaponins and obtain quality fragment data, a MATLAB-based process automatically enumerating "sugar chain + aglycone + side chain" combinations and deduplicating generated a predicted saikosaponin database covering all possible saikosaponins as a precursor ion list for comprehensive targeted acquisition. To focus on informative ions and reduce MS analysis workload, we utilized MATLAB to automatically filtrate the false positive ions by MS1 and MS2 spectrometry. The newly established MATLAB-assisted data acquisition approach exhibited 50 % improvement in characterization of targeted saikosaponins. Furthermore, positive and negative ionization workflows were designed for accurate saikosaponins characterization based on fragmentation rules. In total, 707 saikosaponins were characterized, including over 500 potential new compounds and previously unreported C29 aglycones. We identified 25 saikosaponins present in both authentic species but absent in adulterants as potential markers. This unprecedented comprehensive multi-origin species differentiation demonstrates the promise of MATLAB-assisted acquisition and processing to advance saponin identification and standardize the Radix Bupleuri market.

Effects of Steaming Treatment on the Effective Components and Efficacy in Vitro of Ginseng Flowers.

The effective composition, antioxidant, enzyme inhibition and bile binding ability of Ginseng flowers after different steaming times were studied. The results showed that different steaming times affected the effective components of ginseng flower, the content of polysaccharide and total saponins reached the highest when steaming for 5 times, the total flavonoids and phenol increased with the times of steaming. Steaming treatment significantly induced the ability of antioxidant and inhibition of α-amylase; but reduced the inhibition of α-glucosidase and cholate binding ability. Steaming treatment improved the effective content of ginseng flower and facilitate the production of low polar saponins; steaming changes the composition of ginsenoside.

Exploring the in vitro anti-inflammatory activity of gross saponins of Tribulus terrestris L. fruit by using liquid chromatography-mass spectrometry-based cell metabolomics approach.

Our previous studies confirmed the efficacy of gross saponins of Tribulus terrestris L. fruit in treating cerebral ischemia. This study aimed to investigate the related mechanisms in vitro. The lipopolysaccharide-induced BV2 cells model was constructed and treated with gross saponins at different concentrations to explore its anti-inflammatory activity. The cell metabolite changes were tracked by liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, and the metabolic biomarkers and related metabolic pathways were analyzed. Molecular biochemistry analysis was further used to verify the relevant inflammatory pathways. The results showed that the saponins reduced nitric oxide release and the secretion of tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6 from lipopolysaccharide-induced BV2 cells. Metabolic perturbations occurred in lipopolysaccharide-treated BV2 cells, which could be reversed by drug treatment via mainly regulating glycerophospholipid metabolism, tryptophan metabolism, purine metabolism pathways, etc. The western blot analysis demonstrated that saponin could suppress the activation of the inflammatory-related signaling pathway. The present study explored the in vitro anti-inflammatory mechanism of gross saponins of Tribulus terrestris L. fruit using an LC-MS-based cell metabolomics approach, which confirms the great potential of LC-MS for drug efficacy evaluation and can be applied in other herbal medicine-related analyses.

Effects of topping on rhizome, and analysis of chemical composition, antioxidant activity and α-amylase and α-glucosidase inhibition of the aerial parts in Polygonatum cyrtonema.

Polygonatum cyrtonema is a perennial plant, and it has long been used in traditional Chinese medicine for food and medicine. The medicinal part of P.cyrtonema is the rhizome; however, the aerial part has not been studied. To understand the effect of the topping of aerial parts on the yield and chemical components of rhizomes, as well as the chemical constituents, antioxidant, and in vitro hypoglycemic activities of the aerial stem, leave, and flower parts of P.cyrtonema, the present study was conducted. The results showed that compared to the control (CK) treatment, the topping of the aerial part increased rhizome weight gain coefficient (3.43) and the total saponin content (37.60 mg/g) significantly (P<0.01) than the CK treatment. The contents of total phenols and total flavonoids in PCL and PCF were significantly (P<0.01) higher than those in rhizomes; however, the polysaccharide content (10.47%) in PCR (whole rhizome) was higher than that in PCS (3.65%), PCL (5.99%), and PCF (4.76%) content. The protein and amino acid contents in PCS, PCL, and PCF were higher than those in rhizomes. The protein and amino acid contents in PCS, PCL, and PCF were higher than those in rhizomes. PCS, PCL, and PCF showed strong antioxidant activity (DPPH, ·OH, ABTS, and FRAP), which were better than traditional medicinal parts (the rhizome).In vitro hypoglycemic results showed that PCS, PCL, and PCF had certain inhibitory activities on α-amylase and α-glucosidase (66.25% and 52.81%), which were close to the hypoglycemic activity of rhizomes (67.96% and 52.22%). The leaf extracts also showed better inhibitory activity. To sum up, the topping measures can improve yield and total saponin content of the rhizomes from P.cyrtonema, which can be applied to improve production. The stems, leaves, and flowers had a much stronger antioxidant and hypoglycemic activities and higher the total polyphenols, flavonoids, proteins, and amino acid content. Therefore, stems, leaves, and flowers of Polygonatum can be fully developed according to different needs. they are typically used in animal feed, food storage and cosmetics.

Tracking the effect of roasting and fermentation on the metabolites of licorice root (Glycyrrhiza glabra L.) using UPLC-MS analysis combined with multivariate statistical analysis.

BACKGROUND: Roasting, honey-roasting and fermentation are the most common pre-processing procedures of licorice roots. They were shown to noticeably change the composition of extracts. In this work, the common alterations in licorice secondary metabolites by processing were interpreted. Comprehensive metabolic profiling of different studied samples was undergone. METHODS: UPLC-QqQ-MS/MS analysis coupled to various chemometric analysis models was implemented to unravel the effect of different pre-processing procedures on the chemical profile of licorice samples. RESULTS: UPLC-QqQ-MS/MS analysis designated 133 chromatographic peaks with saponins, flavonoids, chalcones and pterocarpans being the most abundant groups. Triterpene saponins dominated the secondary metabolites in the aqueous extracts, with fermented samples showing the highest relative amounts. Meanwhile the ethanol extracts showed significant amounts of chalcones. Melanoidins were only detected in roasted and honey roasted samples. Multivariate models indicated that roasting of samples induced a greater effect on the polar metabolites rather than nonpolar ones. Variable of importance (VIP) plot indicated that glycyrrhizin and its hydrolysis product glycyrrhetinic acid, trihdroxychalcone diglycoside, glabrone and glabridin are the main chemical features responsible for the discrimination of samples. CONCLUSION: Coupling UPLC-MS/MS to multivariate analysis was a successful tool that unveiled the significant effect of different pre-processing methods on the chemical profile of processed and unprocessed licorice samples. Moreover, such coupling unraveled the discriminatory chemical compounds among tested samples that can be employed as markers for the processing procedure of licorice.

[New steroidal saponins from aerial parts of Paris polyphylla var. chinensis].

The shortage of Paridis Rhizoma promotes comprehensive utilization and development research of waste aerial parts of the original plant. The chemical compositions of the aerial parts of Paris polyphylla var. chinensis were clarified based on the ultrahigh performance liquid chromatography tandem quadrupoles time of flight mass spectrometry(UPLC-QTOF-MS/MS) in the previous investigation, and a series of flavonoids and steroidal saponins were isolated. The present study continued the isolation and structure identification of the new potential compounds discovered based on UPLC-QTOF-MS/MS. By using silica gel, ODS, flash rapid preparation, and other column chromatography techniques, combined with prepared high performance liquid chromatography, five compounds were isolated from the 75% ethanol extract of the aerial parts of P. polyphylla var. chinensis, and their structures were identified by spectral data combined with chemical transformations, respectively, as(23S,25R)-23,27-dihydroxy-diosgenin-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→3)]-ß-D-glucopyranoside(1),(25R)-26-O-ß-D-glucopyranosyl-furost-5-en-3ß,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside(2),(25R)-27-O-ß-D-glucopyranosyl-5-en-3ß,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside(3),(25R)-27-O-ß-D-glucopyranosyl-5-en-3ß,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→3)]-ß-D-glucopyranoside(4), and aculeatiside A(5). Among them, compounds 1-4 were new ones, and compound 5 was isolated from P. polyphylla var. chinensis for the first time.

Residue of Chlormequat and Regulatory Effects on the Specialized Metabolites of Astragali Radix.

Presently, the utilization of chlormequat in Astragalus mongholicus Bunge (Leguminosae) cultivation is prevalent for augmenting rhizome (Astragali Radix) yield. However, indiscriminate and excessive chlormequat employment can detrimentally influence Astragali Radix quality and safety. This research aimed to comprehensively comprehend chlormequat risks and its influence on Astragali Radix metabolites. Diverse chlormequat concentrations were employed in Astragalus mongholicus cultivation, with subsequent analysis of residual chlormequat levels in Astragali Radix across treatment groups. Astragali Radix metabolic profiling was conducted through UPLC-QTOF-MS, and thirteen principal active components were quantified via UFLC-MS/MS. Findings revealed a direct correlation between chlormequat residue levels in Astragali Radix and application concentration, with high-dose residue surpassing 5.0 mg/kg. Metabolomics analysis identified twenty-six distinct saponin and flavonoid metabolites. Notably, the application of chlormequat led to the upregulation of seven saponins (e.g., astragaloside I and II) and downregulation of six flavonoids (e.g., methylnissolin-3-O-glucoside and astraisoflavan-7-O-ß-d-glucoside). Quantitative analysis demonstrated variable contents of active ingredients due to differing chlormequat concentrations, leading to astragaloside I increase (14.59-62.55%) and isoastragaloside II increase (4.8-55.63%), while methylnissolin-3-O-glucoside decreased (22.18-41.69%), as did astraisoflavan-7-O-ß-d-glucoside (21.09-47.78%). In conclusion, chlormequat application influenced multiple active components in Astragali Radix, causing constituent proportion variations. Elevated chlormequat concentrations led to increased active components alongside heightened chlormequat residues in Astragali Radix. Consequently, prudent chlormequat application during Astragali Radix production is imperative to avert potential detriments to its quality and safety.

An online preparative high-performance liquid chromatography system with enrichment and purification modes for the efficient and systematic separation of Panax notoginseng saponins.

In this study, an online preparative high-performance liquid chromatography (prep-HPLC) system based on the combination of the enrichment and purification modes for the efficient and systematic separation of Panax notoginseng saponins (PNS) was achieved. Five separation columns were used for the first and second separation of target components, eighteen trap columns were used to capture the effluents from the first separation or loading the trapped sample effluents, and a two-position eight-port valve was used to switch between the first and second separations. The conditions for the first and second separation of PNS were simulated and optimized with the online prep-HPLC system. Then, the PNS were separated using optimized chromatographic conditions. Notably, 14 monomer compounds with >90% purity (11 compounds with purity >97%) were simultaneously isolated from PNS using the above self-developed device, and their chemical structures were identified. Moreover, the separation time was less than 33.0 h. After 6 repeated enrichment and purification, the weight of each compound obtained was more than 5.0 mg, with compound 2 weighing over 900 mg. In brief, the self-developed prep-HPLC system, which integrated enrichment and purification, is suitable for the efficient and systematic separation of PNS and has broad application prospects, especially for the separation of complex chemical components in natural products.