Multi-level chemical characterization and anti-inflammatory activity evaluation of the polysaccharides from Prunella vulgaris.

Prunella vulgaris (PV) is a medicine and food homologous plant, but its quality evaluation seldom relies on the polysaccharides (PVPs). In this work, we established the multi-level fingerprinting and in vitro anti-inflammatory evaluation approaches to characterize and compare the polysaccharides of P. vulgaris collected from the major production regions in China. PVPs prepared from 22 batches of samples gave the content variation of 5.76-24.524 mg/g, but displayed high similarity in the molecular weight distribution. Hydrolyzed oligosaccharides with degrees of polymerization 2-14 were characterized with different numbers of pentose and hexose by HILIC-MS. The tested 22 batches of oligosaccharides exhibited visible differences in peak abundance, which failed to corelate to their production regions. All the PVPs contained Gal, Xyl, and Ara, as the main monosaccharides. Eleven batches among the tested PVPs showed the significant inhibitory effects on NO production on LPS-induced RAW264.7 cells at 10 µg/mL, but the exerted efficacy did not exhibit correlation with the production regions. Conclusively, we, for the first time, investigated the chemical features of PVPs at three levels, and assessed the chemical and anti-inflammatory variations among the different regions of P. vulgaris samples.

Fingerprinting and characterization of the polysaccharides from Polygonatum odoratum and the in vitro fermented effects on Lactobacillus johnsonii.

Polygonatum odoratum (Yu-Zhu) can be utilized to treat the digestive and respiratory illness. Previous studies have revealed that the underlying therapeutic mechanism of P. odoratum polysaccharides (POPs) is associated with remodeling the gut microbiota. However, POPs in terms of the chemical composition and fermentation activities have been understudied. Here we developed the three-level fingerprinting approaches to characterize the structures of POPs and probed into the beneficial effects on promoting the growth and fermentation of Lactobacillus johnsonii. POPs were prepared by water decoction followed by alcohol sedimentation, while trifluoroacetic acid under different conditions to prepare the hydrolyzed oligosaccharides and monosaccharides. POPs exhibited three main molecular distribution of 601-620 kDa, 4.12-6.09 kDa, and 3.57-6.02 kDa. Hydrolyzed oligosaccharides with degree of polymerization (DP) 2-13 got primarily characterized by analyzing the rich fragmentation information obtained by hydrophilic interaction chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (HILIC/IM-QTOF-MS). Amongst them, the DP5 oligosaccharide was characterized as 1,6,6-kestopentaose. The molecular ratio of Fru: Ara: Glc: Gal: Xyl was 87.72: 0.30: 11.56: 0.19: 0.23. In vitro fermentation demonstrated that 4.5 mg/mL of POPs could significantly promote the growth of L. johnsonii. Co-cultivated with 4.5 mg/mL of POPs, L. johnsonii exhibited stronger antimicrobial activity against Klebsiella pneumoniae. The concentrations of short-chain fatty acids in the POPs-lactobacilli fermented products, including acetic acid, isobutyric acid, and isovaleric acid, were increased. Conclusively, POPs represent the promising prebiotic candidate to facilitate lactobacilli, which is associated with exerting the health benefits.

Polygonatum odoratum polysaccharide attenuates lipopolysaccharide-induced lung injury in mice by regulating gut microbiota.

Polygonatum odoratum is appreciated for its edible and medicinal benefits especially for lung protection. However, the contained active components have been understudied, and further research is required to fully exploit its potential application. We aimed to probe into the beneficial effects of Polygonatum odoratum polysaccharide (POP) in lipopolysaccharide-induced lung inflammatory injury mice. POP treatment could ameliorate the survival rate, pulmonary function, lung pathological lesions, and immune inflammatory response. POP treatment could repair intestinal barrier, and modulate the composition of gut microbiota, especially reducing the abundance of Klebsiella, which were closely associated with the therapeutic effects of POP. Investigation of the underlying anti-inflammatory mechanism showed that POP suppressed the generation of pro-inflammatory molecules in lung by inhibiting iNOS+ M1 macrophages. Collectively, POP is a promising multi-target microecological regulator to prevent and treat the immuno-inflammation and lung injury by modulating gut microbiota.

Characterization and comparison of cardiomyocyte protection activities of non-starch polysaccharides from six ginseng root herbal medicines.

Ginseng is rich of polysaccharides, however, the evidence supporting polysaccharides to distinguish various ginseng species is rarely reported. Focusing on six root ginseng (e.g., Panax ginseng-PG, P. quinquefolius-PQ, P. notoginseng-PN, red ginseng-RG, P. japonicus-PJ, and P. japonicus var. major-PJM), the contained non-starch polysaccharides (NPs) were structurally characterized and compared by both the chemical and biological evaluation. Holistic fingerprinting at three levels (the NPs and the acid hydrolysates involving oligosaccharides and monosaccharides) utilized various chromatography methods, and the treatment of H9c2 cells with the NPs by OGD and H2O2-induced injury models was used to assess the protective effect. NPs from six Panax herbal medicines occupied about 20 % of the total polysaccharides, which were of the highest content in RG and the lowest in PN. NPs from six ginseng exhibited weak differentiations in the molecular weight distribution, while marker oligosaccharides were found to distinguish PN and RG from the others. Glc and GalA were more abundant in the NPs for PG and RG, respectively. NPs from PQ (100/200 µg/mL) showed significant cardiomyocyte protection effect by regulating the mitochondrial functions. This work further testifies the role of polysaccharides in quality control of herbal medicine, with new markers discovered beneficial to distinguish the ginseng.

Comparative Characterization of the Ginsenosides from Six Panax Herbal Extracts and Their In Vitro Rat Gut Microbial Metabolites by Advanced Liquid Chromatography-Mass Spectrometry Approaches.

Ginseng extracts are extensively used as raw materials for food supplements and herbal medicines. This study aimed to characterize ginsenosides obtained from six Panax plant extracts (Panax ginseng, red ginseng, Panax quinquefolius, Panax notoginseng, Panax japonicus, and Panax japonicus var. major) and compared them with their in vitro metabolic profiles mediated by rat intestinal microbiota. Ultrahigh-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) with scheduled multiple reaction monitoring (sMRM) quantitation methods were developed to characterize and compare the ginsenoside composition of the different extracts. After in vitro incubation, 248 ginsenosides/metabolites were identified by UHPLC/IM-QTOF-MS in six biotransformed samples. Deglycosylation was determined to be the main metabolic pathway of ginsenosides, and protopanaxadiol-type and oleanolic acid-type saponins were easier to be easily metabolized. Compared with the ginsenosides in plant extracts, those remaining in six biotransformed samples were considerably fewer after biotransformation for 8 h. However, the compositional differences in four subtypes of the ginsenosides among the six Panax plants became more distinct.

A multi-dimensional liquid chromatography/high-resolution mass spectrometry approach combined with computational data processing for the comprehensive characterization of the multicomponents from Cuscuta chinensis.

Challenges encountered in plant metabolites characterization by liquid chromatography/mass spectrometry can arise from the insufficient chromatography separation, the lack of specific database, and low reliability of identification because of the ubiquitous isomerism. Herein, we present an integral approach, by combining comprehensive off-line two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), automatic peak annotation, molecular networking, and collision cross section (CCS) prediction, aimed to improve the resolution and reliability in MS-oriented metabolites characterization. Using the seeds of Cuscuta chinensis as a case, the configuration of an XBridge Amide column of hydrophilic interaction chromatography (HILIC) and a Zorbax SB-Aq column of reversed-phase chromatography (RPC), in an off-line mode, showed the orthogonality of 0.73 and effective peak capacity of 4361. Data-independent high-definition MSE (HDMSE) in the negative mode could enable high-coverage MS2 data acquisition and enhance the ions resolution, while computational peak annotation workflows facilitated by UNIFITM and Global Natural Products Social Molecular Network (GNPS) could efficiently characterize the targeted and untargeted compound analogs. A total of 302 compounds were identified or tentatively characterized, and 109 thereof were unreported. Moreover, CCS prediction (www.allccs.zhulab.cn) provided more possibilities to distinguish 12 pairs of isomers in the lack of reference standards. The 2D-LC/IM-QTOF-MS approach enabled the collection of five dimension of data related to each component (tR by HILIC and RPC, CCS, m/z in MS1 and MS2), and the intelligent metabolites characterization with more reliable MS data. Conclusively, the established integral strategy can be utilized in metabolome analysis to support the quality control of herbal medicines.

An ion mobility-enabled and high-efficiency hybrid scan approach in combination with ultra-high performance liquid chromatography enabling the comprehensive characterization of the multicomponents from Carthamus tinctorius.

Liquid chromatography/mass spectrometry (LC/MS) is extensively applied for the untargeted/targeted analyses of the herbal components, utilizing data-dependent acquisition (DDA) or data-independent acquisition (DIA) to record the fragmentation information useful for the structural elucidation. A new trend recently has emerged by integrating DDA and DIA to render the hybrid scan, which, unfortunately, has rarely been reported. Herein, by using the Vion™ ion-mobility quadrupole time-of-flight mass spectrometer, a hybrid scan strategy (HDMSE-HDDDA) was presented and validated by the untargeted characterization of the multicomponents from Carthamus tinctorius (safflower), in combination with reversed-phase ultra-high performance liquid chromatography (RP-UHPLC). Good chromatographic separation was achieved on an HSS T3 column within 26 min, while HDMSE-MS/MS was used to acquire the collision-induced dissociation MS2 data in the negative mode. Automatic workflows (e.g., data correction, precursors/product ions matching, and peak annotation) were well established on UNIFI™ (incorporating an in-house library recording 261 known compounds) to process the obtained MS2 data. Compared with single DDA or DIA, the hybrid approach of HDMSE-HDDDA better balanced between the coverage and reliability, led to high-definition MS spectra, offered useful collision cross section (CCS) information, and showed satisfactory identification performance comparable to MSE. A total of 141 components (involving 41 quinochalcones, 66 flavanols/flavones, 11 flavanones, 6 organic acids, 1 polyacetylene, and 16 others) were characterized from safflower. Moreover, CCS prediction could assist isomers characterization, to some extent. Conclusively, this hybrid scan approach enables a dimension-enhanced MS data acquisition strategy providing the complementary structural information, which more suits the chemical characterization of complex samples.

Multi-level fingerprinting and cardiomyocyte protection evaluation for comparing polysaccharides from six Panax herbal medicines.

The role of polysaccharides in quality control of ginseng is underestimated. Large-scale comparison on the polysaccharides of Panax ginseng (PG), P. quinquefolius (PQ), P. notoginseng (PN), Red ginseng (RG), P. japonicus (ZJS), and P. japonicus var. major (ZZS), was performed by both chemical and biological approaches. Holistic fingerprinting at polysaccharide and the hydrolyzed oligosaccharide and monosaccharide levels utilized various chromatography methods, while OGD and OGD/R models on H9c2 cells were introduced to evaluate the protective effects on cell viability and mitochondrial function. Polysaccharides from six ginseng species exhibited remarkable content difference (RG > PG/ZZS/ZJS/PQ > PN), but weak differentiations in molecular weight distribution and oligosaccharide profiles, while Glc and GalA were richer for monosaccharide compositions of PG and RG polysaccharides, respectively. RG polysaccharides (25/50/100 µg/mL) showed significant cardiomyocyte protection by regulating mitochondrial functions. These new evidences may provide support for the supplementary role of polysaccharides in quality control of ginseng.