Rapid identification and quantification of Pseudostellaria heterophylla with its adulterants by HPLC-CAD fingerprint combined with improved quantitative analysis of multi-components by single marker (QAMS).

The P. heterophylla and its adulterants were identified by HPLC-CAD fingerprint of sucrose and oligosaccharides in P. heterophylla. The improved quantitative analysis of multi-components with a single marker (iQAMS) was further established for simultaneous determinations of sucrose and oligosaccharides in P. heterophylla. The HPLC-CAD fingerprint and similarity coefficients between P. heterophylla and its adulterants showed significant differences. The relative errors (REs) between iQAMS method and external standard method (ESM) were below 3.00%, but significant difference was shown between iQAMS (different marker for whole program with gradient elution) and QAMS (one marker for whole program with gradient elution), indicating that QAMS method should be improved, especially for gradient elution which influence the response of analytes. The accuracy, precision, reproducibility, and stability of this method were validated which exhibited satisfactory results, indicating that iQAMS method could be used for quantitative analysis of sucrose and oligosaccharides in P. heterophylla instead of ESM. The iQAMS combined with HPLC-CAD fingerprint could be used to determine the content of each oligosaccharide, and it can be used for quality control of P. heterophylla.

Atomic force microscopy based conformation and immunological activity of Lentinan injections.

The purpose of this study was to investigate the morphological characteristics of different brands of lentinan injections produced in China using atomic force microscopy (AFM) and their relationship to immunological activity. Based on AFM imaging, chain height could be used as characterizing the conformation of lentinan, and the heights of 95 % confidence interval for triple, double and single helix were 1.746 ± 0.039 nm, 1.564 ± 0.037 nm and 1.243 ± 0.031 nm, respectively, which were calculated using self-developed MATLAB protocol. AFM characters and their immunological activity of different lentinan injection were compared. In detail, two parameters, triple helix ratio 51.3 % and adhesion force 800 pN, of Jinling (JL) lentinan injection are much higher than samples of other four manufacturers. In addition, immunological activity of JL lentinan injection is also significantly higher than Yineng's. High performance size exclusion chromatography (HPSEC) profiles of different lentinans were also compared, and the data were in accordance with those from AFM. Molecular weight accumulation curves could be used for evaluation of quality consistence of different batches of lentinan from same manufacturer and/or different manufacturers. The results showed that quality consistence of lentinan from different manufactures is poor, which should be greatly improved.

Characterization and immunological activity of polysaccharides from two types of Dendrobium devonianum with different appearance.

Dendrobium devonianum, a precious species of Dendrobium, is also called "Zi-pi Shi-hu" in Chinese due to its purple-skinned appearance. In this study, polysaccharides from green (GDP) and purple (PDP) skin D. devonianum were isolated by homogenate assisted extraction, ethanol precipitation and ultrafiltration. Their difference in physicochemical properties and immunological activities were investigated and compared. The results suggested that both GDP and PDP mainly contained glycosidic linkages of ß-1,4-Manp and ß-1,4-Glcp, but their molecular weights, 2.044 × 105 (GDP) vs 3.563 × 105 (PDP), and compositional monosaccharides of mannose to glucose ratio, 16.46: 1.00 (GDP) vs 7.61: 1.00 (PDP), were significantly different. Especially, GDP showed stronger immunological potentiation activity in vitro. Although the extraction yield of GDP was lower than that of PDP, considering the production of raw material and immunological activity of its polysaccharides, green skin D. devonianum should be a good alternative of purple one. These findings are helpful to provide evidence for improving the cultivation and production of D. devonianum.

Quality Evaluation of Ophiopogon japonicus from Two Authentic Geographical Origins in China Based on Physicochemical and Pharmacological Properties of Their Polysaccharides.

Ophiopogon japonicus is widely used as a tonic herb in China. According to the origins, MaiDong of Chinese materia medica can be classified as Zhe MaiDong (Ophiopogon japonicus in Zhejiang), Chuan MaiDong (Ophiopogon japonicus in Sichuan), Duanting Shan MaiDong (Liriope muscari), and Hubei MaiDong (Liriope spicata). In terms of quality control, polysaccharides-based evaluations have not yet been conducted. In this study, microwave-assisted extraction (MAE) was used for the preparation of polysaccharides from 29 batches of MaiDong. HPSEC-MALLS-RID and HPAEC-PAD were employed to investigate their molecular parameters and compositional monosaccharides, respectively. The ability to scavenge ABTS radicals and immune promotion abilities, in terms of nitric oxide releasing and phagocytosis on RAW 264.7 macrophages, were also compared. The results showed that polysaccharides in different MaiDong varied in molecular parameters. All polysaccharides mainly contained fructose and glucose with small amounts of arabinose, mannose, galactose, and xylose. For polysaccharides of Zhe MaiDong and Chuan MaiDong, the molar ratio of Fru to Glc was roughly 15:1 and 14:1, respectively. Zhe MaiDong exhibited better antioxidant and immune promotion activity, and so did that of fibrous roots. The pharmacological activity, however, did not account for the variation in growth years. Finally, indicators for quality control based on multivariate statistical analysis included: yield, antioxidant activity, the content of fructose, and RI signal. It was concluded that MaiDong's fibrous roots had similar components to the root, and their quality was not significantly affected by growth age. This may provide some guidance for the cultivation and use of MaiDong.

Effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping analysis and pharmacological activity assays.

BACKGROUND: Polygonatum cyrtonema, one of origins of Polygonata Rhizoma (HuangJing in Chinese), is traditionally steamed repeatedly before being used as herbal medicine in China. However, there has no standard for steaming of HuangJing. Therefore, a comprehensive study for effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary, which is helpful to explore the effect of steam on the physiochemical and biological activities of its polysaccharides and develop steaming standard of Polygonatum cyrtonema. METHODS: To explore the effect of steam on physiochemical and biological activities of P. cyrtonema polysaccharides (PCP), six polysaccharides named PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5 were extracted from the herb consecutively steamed for 0-5 times, respectively. Their molecular weight distribution, monosaccharide composition and PACE fingerprints were investigated through HPSEC-MALLS-RID, HPAEC-PAD and saccharide mapping based on polysaccharides analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, respectively. In addition, their antioxidant ability and immunostimulatory activities on RAW 264.7 cells in terms of NO production and phagocytosis were compared. RESULTS: Results suggested that molecular weights could be changed during steam, which increased by first steaming and then decreased with further steaming though all polysaccharides' molecular weights were 105-107 Da. They all showed irregularly spherical conformation in aqueous solution based on AFM imaging. Their monosaccharide composition and PACE fingerprints were significantly different after steaming, i.e., galactose increased while glucose and mannose decreased, and ß-1,4-Galp appeared while ß-1,4-Manp increased, after steaming. Steamed PCP significantly increased scavenging activity against ABTS radicals, while PCP0 had the best immunostimulatory effect on RAW 264.7 in terms of NO production and phagocytosis. CONCLUSIONS: In summary, steam significantly affected the chemical composition and bioactivities of polysaccharides from P. cyrtonema. Considering the balance beneficial effects of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuous steam is the optimal choice under the given conditions.

A quantitative method for polysaccharides based on endo-enzymatic released specific oligosaccharides: A case of Lentinus edodes.

Polysaccharides exhibit multiple pharmacological activities, which are closely related to their structural characteristics. Therefore, quantitative quality control of polysaccharides based on chemical properties is of importance for their applications. However, polysaccharides are mixed macromolecular compounds that are difficult to separate, and the lack of standards made direct quantification more difficult. In this study, we proposed a new quantitative method based on the released specific oligosaccharides for polysaccharides from Lentinus edodes (shiitake) and other related fungi. Specific oligosaccharides were firstly released from polysaccharides using 1,3-ß-glucanase, then derivatized with 2-aminobenzamide (2-AB), which further separated by hydrophilic interaction chromatography (HILIC) and quantitatively determined by UPLC coupled with fluorescence detector (FLR). Laminaritriose was used as the universal standard for quantification of all the oligosaccharides. This method was validated according to linearity, limit of detection, limit of quantitation, precision, accuracy, repeatability and stability. In addition, the four specific oligosaccharides released from polysaccharides in L. edodes were qualitatively analyzed by extracted ion chromatogram (EIC) from UPLC-MS profiles, which were identified to be disaccharide, trisaccharide and tetrasccharide. The proposed strategy not only realized the quantitative analysis of polysaccharides by UPLC-FLR, but also could achieve the qualitative distinction of different polysaccharides.

Characterization and Comparison of Bioactive Polysaccharides from Grifola frondosa by HPSEC-MALLS-RID and Saccharide Mapping Based on HPAEC-PAD.

Grifola frondosa polysaccharides (GFPs) from different regions in China were characterized and compared using HPSEC-MALLS-RID and saccharide mapping based on HPAEC-PAD analysis for achieving and improving its quality control. The results showed that HPSEC chromatograms and molecular weight distributions of GFPs were similar. The average contents of each polysaccharide fraction (Peaks 1, 2, and 3) showed that Peak 3 was the main component and much higher than the other two polysaccharide fractions, which also contained protein. The result of saccharide mapping showed that α-1,4-glycosidic, ß-1,4-glycosidic and few ß-1,3-glycosidic linkages were existed in GFPs. The similarity result showed that HPAEC-PAD fingerprints of the oligosaccharide fragments after hydrolysis by endoglycosidase were certainly different, especially α-amylase with a mean similar index of only 0.781 ± 0.207. The result of hierarchical cluster analysis (HCA) showed that different batches of GFPs from China can be divided into different clusters. Furthermore, immune-enhancing activity based on RAW 264.7 cells showed significant differences among different GFPs. Based on grey relational analysis (GRA), the fractions of Peak 3 were regarded as the major contributors to its immuno-enhancing activity in GFPs. Overall, the implications from these results were found to be stable, comprehensive, and valid for improving the quality control of GFPs.

Quality evaluation of Salvia miltiorrhiza from different geographical origins in China based on qualitative and quantitative saccharide mapping and chemometrics.

Comparison of Salvia miltiorrhiza polysaccharides (SMPs) from different geographical origins in China (Henan, Hebei, Shandong, Sichuan, Shaanxi) was performed using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID), saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and combined with principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Based on the results of HPSEC-MALLS/RI, the relative content of SMPs showed a significant difference between different geographical origins, however, the molecular weight of SMPs showed almost no significance. SMPs can be discriminated as five regions after PACE coupled with OPLS-DA models analysis of endo-1,5-α-arabinanase hydrolysates. Moreover, all the PACE fingerprint indicated that 1,4-ß-D-Galp, 1,5-α-Araf, 1,4-α-D-GalAp and 1,4-ß-D-Glcp linkages existed in SMPs.

Abscopal effect of local irradiation treatment for thymoma: a case report.

INTRODUCTION: With the rapid development of immunotherapy in recent years, growing clinical evidence suggests that a combination of radiotherapy and immunotherapy could improve the abscopal response rates and increase survival. However, observations on abscopal effects in patients receiving radiotherapy alone are still very rare. This study reports a rare case of a patient with type B3 thymoma with multiple lung metastases, who received localized irradiation. CASE PRESENTATION: A 76-year-old Chinese woman was admitted to our hospital in November 2017, and her physical examination revealed a thymus mass with multiple lung metastases. Although the left lower lobe lesion was slightly larger than before, the patient presented with regression of the non-irradiated metastases after treatment with radiotherapy alone. CONCLUSION: This patient experienced an apparent regression of metastatic mass, suggesting a radiation-induced abscopal effect.

Species-Specific Identification of Streptococcus based on DNA Marker in 16S-23S rDNA Internal Transcribed Spacer.

Streptococcus is closely correspondent to human. The accurate species-specific identification method of Streptococcus is important for the bacteria clinical diagnosis, molecular epidemiological analysis, and microecological study. In the last decades, DNA markers are widely utilized for identification of prokaryotic species. However, 16S rDNA, the most popular bacterial DNA marker, cannot properly distinguish closely related Streptococcus species. In present study, we employed 16S-23S rRNA gene internal transcribed spacer (ITS) sequence to explore the species-specific DNA marker. We predicted the secondary structure of Streptococcus ITS sequence transcribed products. Then we identified that the specific and consensus sequences in the primary structure can be found occupying an individual subunit in the secondary structure, which explained the foundation of the mosaic-like structure of ITS. We evaluated the specificity of ITS in Streptococcus, and found that the specificity can be detected by a further analysis of a BLAST result. Then, we developed an identification procedure based on the ITS sequence. We verified the procedure by 500 ITS sequence. The accuracy rate of this procedure was 100% for Streptococcus at genus level, and 99.3% at species level. It suggested that ITS can be utilized to accurately identify Streptococcus at the species level. This work suggests that further exploration of ITS could be applied in other bacterial genera for identification and classification, which may be a useful topic for future microbiology studies.

Determination of seven oligosaccharides and sucrose in Pseudostellaria heterophylla by pressurized liquid extraction and ultra-high performance liquid chromatography with charged aerosol detector and tandem mass spectrometry.

A simple, rapid, and sensitive ultra-high performance liquid chromatography with charged aerosol detector (UPLC-CAD) method was developed for firstly simultaneous determination of seven oligosaccharides, including two pairs of linear oligosaccharides isomers (DP3-1, DP3-2 and DP4-1, DP4-2) and 3 high branched oligosaccharides (DP 5, 6 and 7), as well as sucrose in Pseudostellaria heterophylla. The separation was performed on a Waters BEH Amide column (2.1 × 150 mm i.d., 1.7 µm) with gradient elution within 20 min. All calibration curves for the investigated analytes showed good linearity (R2 > 0.9992). Their limits of detection (LOD) and quantification (LOQ) were in the ranges of 0.24-1.35 µg/mL and 0.78-4.96 µg/mL, respectively. Repeatability of all investigated components detected in samples was less than 2.9%. All the recoveries of each analyte ranged from 99.2% to 104.3% were acceptable. An ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS) method was also applied to authenticate the oligosaccharides in different samples by comparing to the isolated standards. Oligosaccharides, especially linear oligosaccharides isomers and high branched oligosaccharides with different degree of polymerization (DP), were firstly identified and determined in P. heterophylla. Their contents in 33 samples of P. heterophylla from different locations was successfully compared, which is helpful to evaluate its quality.

Functional polysaccharides of carob fruit: a review.

Polysaccharides in carob fruit, including carob bean gum (also known as carob gum, locust bean gum) and carob fiber, are widely used in industries such as food, pharmaceuticals, paper, textile, oil well drilling and cosmetics. Carob bean gum is a galactomannan obtained from the seed endosperm of carob tree and the fiber is obtained by removing most of soluble carbohydrates in carob pulp by water extraction. Both the gum and fiber are beneficial to health for many diseases such as diabetes, bowel movements, heart disease and colon cancer. This article reviewed the composition, properties, food applications and health benefits of polysaccharides from carob fruit.

Relationship of diversity and the secondary structure in 16S-23S rDNA internal transcribed spacer: a case in Vibrio parahaemolyticus.

The 16S-23S rDNA internal transcribed spacer (ITS) sequence, located in the rrn operon, has been analyzed and evaluated for use in phylogenetic analysis and the detection target of bacteria. The ITS region displays a high level of diversity, being present in multiple copies and displaying variability in both length and sequence, and it carries more phylogenetic information than 16S rDNA. However, appropriately identifying ITS regions to use in analyses is challenging. To solve this problem, we analyzed the ITS regions in Vibrio parahaemolyticus and predicted the secondary structure of each analogous rrn transcript. The genomic DNA of V. parahaemolyticus contains approximately 8-14 rrns, making it more complex than the sequences of most other bacterial species. We analyzed 216 ITSs, of which 206 ITSs come from 18 complete genomes, and 10 ITSs were identified in the present study. The subunits of each ITS were distinguished by their predicted secondary structures. We propose a refined backbone model of the V. parahaemolyticus ITS that can be applied to the sequences of other bacteria. The backbone includes C, V, tDNA and linker blocks. These blocks, which may represent true functional units, may be used as potential targets for phylogenetic analysis or molecular detection.

Explore the effects of Huang-Lian-Jie-Du-Tang on Alzheimer's disease by UPLC-QTOF/MS-based plasma metabolomics study.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with neither definitive pathogenesis nor effective treatment method so far. Huang-Lian-Jie-Du-Tang (HLJDT) is a classic formula of traditional Chinese medicine (TCM) proven to have ameliorative effects on learning and memory deficits of dementia. Morris water maze (MWM) test and pathology analysis have demonstrated that HLJDT could ameliorate learning and memory deficits in AD mouse model, which may act via its anti-neuroinflammation properties. According to our previous studies, an UPLC-QTOF/MS-based metabolomics approach was performed to explore the potential mechanisms of HLJDT on preventing AD. As a result, a total of 23 potential metabolites (VIP >1, |Pcorr| >0.58, CUFjk excludes 0, P < 0.05) contributing to AD progress were identified. The metabolic pathway analysis with MetPA revealed that glycerophospholipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, linoleic acid metabolism and tryptophan metabolism were disturbed in mouse model of AD. After HLJDT treatment, 14 metabolites were restored back to the control-like levels.

Metabolomics-based mechanisms exploration of Huang-Lian Jie-Du decoction on cerebral ischemia via UPLC-Q-TOF/MS analysis on rat serum.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian Jie-Du decoction (HLJDD), a traditional formula of Chinese medicine constituted with Rhizoma Coptidis, RadixScutellariae, CortexPhellodendri amurensis and Fructus Gardeniae, exhibits unambiguous therapeutic effect on cerebral ischemia via multi-targets action. Further investigation, however, is still required to explore the relationship between those mechanisms and targets through system approaches. MATERIALS AND METHODS: Rats of cerebral ischemia were completed by middle cerebral artery occlusion (MCAO) with reperfusion. Following evaluation of pharmacological actions of HLJDD on MCAO rats, the plasma samples from rats of control, MCAO and HLJDD-treated MCAO groups were prepared strictly and subjected to ultra-performance liquid chromatography quadrupole time of flight mass spectrometry for metabolites analysis. The raw mass data were imported to MassLynx software for peak detection and alignment, and further introduced to EZinfo 2.0 software for orthogonal projection to latent structures analysis, principal component analysis and partial least-squares-discriminant analysis. The metabolic pathways assay of those potential biomarkers were performed with MetaboAnalyst through the online database, HMDB, Metlin, KEGG and SMPD. Those intriguing metabolic pathways were further investigated via biochemical assay. RESULTS: HLJDD ameliorated the MCAO-induce cerebral damage and blocked the severe inflammation response. There were nineteen different biomarkers identified among control, MCAO and HLJDD-treated MCAO groups. Ten metabolic pathways were proposed from these significant metabolites. Incorporation with the biochemical assay of cerebral tissue, modulation of metabolic stress, regulation glutamate/GABA-glutamine cycle and enhancement of cholinergic neurons function were explored that involved in the actions of HLJDD on cerebral ischemia. CONCLUSION: HLJDD achieves therapeutic action on cerebral ischemia via coordinating the basic pathophysiological network of metabolic stress, glutamate metabolism, and acetylcholine levels and function.