RESUMO
Glycoside linkage analyses of medicine and food homologous plant polysaccharides have always been a key point and a difficulty of structural characterization. The gas chromatography-mass spectrometry (GC-MS) method is one of the commonly used traditional techniques to determine glycoside linkages via partially methylated alditol acetates and aldononitrile acetates (PMAAs and PMANs). Due to the simplicity of derivatization and the highly structural asymmetry of PMANs, reverse thinking is proposed using liquid chromatography-electrospray ionization-multiple reaction monitoring mass spectrometry (LC-ESI-MRM-MS) for the first time to directly determine the neutral and acidic glycosyl linkages of polysaccharides. The complete characterization of glycoside linkages deduced from PMANs was achieved using a combination of tR values, characteristic MRM ion pairs, diagnostic ESI+-MS/MS fragmentation ions (DFIs), and optimal collision energy (OCE). The DFI and OCE parameters were confirmed to be effective for the auxiliary discrimination of some isomers of the PMANs. The practicality of LC-ESI+-MRM-MS was further verified by analyzing the glycoside linkages of polysaccharides in five medicine and food homologous plants. This method can serve as an alternative to GC-MS for the simultaneous determination of neutral and acidic glycosyl linkages in polysaccharides.
Assuntos
Glicosídeos , Polissacarídeos , Espectrometria de Massas por Ionização por Electrospray , Polissacarídeos/química , Glicosídeos/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Acetatos/química , Nitrilas/química , Metilação , Cromatografia Líquida/métodos , Extratos Vegetais/química , Cromatografia Gasosa-Espectrometria de Massas/métodosRESUMO
BACKGROUND: Numerous studies indicate that natural polysaccharides have immune-enhancing effects as a host defense potentiator. Few reports are available on hormetic effects of natural polysaccharides, and the underlying mechanisms remain unclear. PURPOSE: AELP-B6 (arabinose- and galactose-rich pectin polysaccharide) from Aralia elata (Miq.) Seem was taken as a case study to clarify the potential mechanism of hormetic effects of natural polysaccharides. METHODS: The pharmacodynamic effect of AELP-B6 was verified by constructing the CTX-immunosuppressive mouse model. The hormetic effects were explored by TMT-labeled proteomics, energy metabolism analysis, flow cytometry and western blot. The core-affinity target of AELP-B6 was determined by pull down, nanoLC-nanoESI+-MS, CETSA, immunoblot and SPR assay. The RAW264.7Clec4G-RFP and RAW264.7Rab1A-RFP cell lines were simultaneously constructed to determine the affinity difference between AELP-B6 and targets by confocal laser scanning live-cell imaging. Antibody blocking assays were further used to verify the mechanism of hormetic effects. RESULTS: AELP-B6 at low and medium doses may maintain the structural integrity of thymus and spleen, increase the concentrations of TNF-α, IFN-γ, IL-3 and IL-8, and alleviate CTX-induced reduction of immune cell viability in vivo. Proteomics and energy metabolism analysis revealed that AELP-B6 regulate HIF-1α-mediated metabolic programming, causing Warburg effects in macrophages. AELP-B6 at low and medium doses promoted the release of intracellular immune factors, and driving M1-like polarization of macrophages. As a contrast, AELP-B6 at high dose enhanced the expression levels of apoptosis related proteins, indicating activation of the intrinsic apoptotic cascade. Two highly expressed transmembrane proteins in macrophages, Clec4G and Rab1A, were identified as the primary binding targets of AELP-B6 which co-localized with the cell membrane and directly impacted with immune cell activation and apoptosis. AELP-B6 exhibits affinity differences with Clec4G and Rab1A, which is the key to the hormetic effects. CONCLUSION: We observed hormesis of natural polysaccharide (AELP-B6) for the first time, and AELP-B6 mediates the hormetic effects through two dose-related targets. Low dose of AELP-B6 targets Clec4G, thereby driving the M1-like polarization via regulating NF-κB signaling pathway and HIF-1α-mediated metabolic programming, whereas high dose of AELP-B6 targets Rab1A, leading to mitochondria-dependent apoptosis.
Assuntos
Pectinas , Animais , Camundongos , Pectinas/farmacologia , Lectinas Tipo C/metabolismo , Células RAW 264.7 , Metabolismo Energético/efeitos dos fármacos , Polissacarídeos/farmacologiaRESUMO
A glucosyl-rich pectin, JMMP-3 (Mw, 2.572 × 104 g/mol, O-methyl % = 3.62%), was isolated and purified from the pericarp of the immature fruit of Juglans mandshurica Maxim. (QingLongYi). The structure of JMMP-3 was studied systematically by infrared spectroscopy, monosaccharide compositions, methylation analysis, partial acid hydrolysis, and 1/2D-NMR. The backbone of JMMP-3 possessed a smooth region (â 4GalA1 â) and a hairy region (â 4GalA1 â 2Rha1 â) with a molar ratio of 2: 5. The substitution of four characteristic side chains (R1-R4) occurs at C-4 of â 2,4)-α-Rhap-(1â, where R1 is composed of â 5)-α-Araf-(1â, R2 is composed of â 4)-ß-Galp-(1 â and ß-Galp-(1â, R3 is composed of α-Glcp-(1â, â4)-α-Glcp-(1 â and â 4,6)-α-Glcp-(1â, and R4 is composed of â 5)-α-Araf-(1â, ß-Galp-(1â, â 4)-ß-Galp-(1â, â 3,4)-ß-Galp-(1â, â 4,6)-ß-Galp-(1 â and â 2,4)-ß-Galp-(1 â . In addition, the antitumor activity of JMMP-3 on HepG2 cells was preliminarily investigated.
Assuntos
Frutas , Juglans , Pectinas , Juglans/química , Pectinas/química , Pectinas/isolamento & purificação , Humanos , Frutas/química , Células Hep G2 , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificaçãoRESUMO
Derivative spectroscopy is used to separate the small absorption peaks superimposed on the main absorption band, which is widely adopted in modern spectral analysis to increase both the valid spectral information and the identification accuracy. In this study, a method based on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) with first-order derivative (FD) processing combined with chemometrics is proposed for rapid qualitative and quantitative analysis of Panax ginseng polysaccharides (PGP), Panax notoginseng polysaccharides (PNP), and Panax quinquefolius polysaccharides (PQP). First, ATR-FTIR with FD processing was used to establish the discriminant model combined with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and linear discriminant analysis (LDA). After that, two-dimensional ATR-FTIR based on single-characteristic temperature as external interference (2D-sATR-FTIR) was established using ATR-FTIR with FD processing. Then, ATR-FTIR with FD processing was combined with PLS to establish and optimize the quantitative regression model. Finally, the established discriminant model and 2D-sATR-FTIR successfully distinguished PGP, PNP and PQP, and the optimal PLS regression model had a good prediction ability for the Panax polysaccharide extracts content. This strategy provides an efficient, economical and nondestructive method for the distinction and quantification of PGP, PNP and PQP in a short detection time.
Assuntos
Panax notoginseng , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise Discriminante , Análise dos Mínimos Quadrados , PolissacarídeosRESUMO
OBJECTIVES: Oligo-/polysaccharides from Cyathula officinalis Kuan (COPs) and Achyranthes bidentata Blume (ABPs) have attracted researchers' attention in the fields of healthy food supplements and traditional Chinese medicine (Niúxi) due to their multiple bioactivities combined with their nontoxic and highly biocompatible nature. The purpose of this paper was to provide a systematic and comprehensive overview of the extraction, purification, and structural analysis methods, chemical characteristics, biological activities, and structure bioactivity relationship. Furthermore, the possible development trends and perspectives for future research, and traditional uses of Niúxi are also summarized. METHODS: All the information was gathered from a library search and scientific databases. KEY FINDINGS: Although COPs and ABPs are derived from different plants, they have similar structural features in type, structure, and glycosidic linkage patterns and biological activities in vivo and in vitro. However, there are differences in monosaccharide compositions, which can be used as an identification mark. CONCLUSIONS: As traditional Chinese herbal medicine, C. officinalis and A. bidentata have similar pharmacological activities. The COPs and ABP possess wide pharmacological effects such as antitumor, antioxidant, anti-osteoporosis, and anti-inflammatory. Meanwhile, the biological activity and structure-activity relationship of purified COPs and ABPs are less studied, future research should focus on them.
Assuntos
Achyranthes , Amaranthaceae , Osteoporose , Achyranthes/química , Polissacarídeos/farmacologia , Polissacarídeos/químicaRESUMO
INTRODUCTION: Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. OBJECTIVES: To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. METHODOLOGY: A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. RESULTS: Using rhamnose as an internal reference standard, the contents of the other monosaccharide components in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. CONCLUSIONS: The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.
Assuntos
Polissacarídeos , Ramnose , Polissacarídeos/análise , Polissacarídeos/química , Monossacarídeos/análise , Monossacarídeos/química , GlucoseRESUMO
ETHNIC PHARMACOLOGICAL RELEVANCE: "Yin-Jing" medicine (YJM) has been widely used by both ancient and modern Chinese medicine practitioners during long-term clinical practice. However, it remains unclear how to best guide other medicines to the targeted organs in a traditional Chinese medicine (TCM) prescription. Here, in an attempt to explain the scientific connotation of the YJM property (YJMP) attributed to a basic TCM theory, Platycodon grandiflorum (PG) was chosen as a case study to reveal the mystery of YJMP theory. AIM OF THE STUDY: The main purpose of this study is to employ modern chemical and molecular biology methods to confirm the "Yin-Jing" effect of PG, and further clarify its material basis and related possible mechanism. MATERIALS AND METHODS: The ammonia-induced lung injury rat model was utilized to determine the optimal dosage of traditional prescription Hui Yan Zhu Yu decoction (HYZYD) using Wright Giemsa staining, HE staining, Masson staining, and TUNEL analysis. With the same way, PG was confirmed to have potentiating therapeutic effect (PTE) by comparison with HYZYD and [HYZYD-PG]. TMT proteomics was used to reveal the "Yin-Jing" mechanism of action. Western blot assay (WB) was employed for verification of differentially expressed proteins. Additionally, four non-crossing fragmentations (Fr. A-D) were characterized by RPLC/SEC-ELSD and HILIC-ESI--Q-OT-IT-MS techniques. The PTE and guidance property assays were utilized to evaluate "Yin-Jing" functions by a compatible combination of hydroxysafflor yellow A (HYA) using qPCR, FCM, WB, HPLC, high content cell imaging (HCI) and high-resolution live-cell imaging (HRLCI) techniques. RESULTS: The HYZYD-M (medium dose group) significantly improved the lung injury level in a pneumonia model of rats. PG enhanced the therapeutic effect of HYZYD ascribed to Yin-Jing PTE functions. TMT proteomics revealed a category of differentially expressed proteins ascribed to Golgi-ER between HYZYD and [HYZYD-PG]. Fr. C (i.e., saponins) and Fr. D (i.e., lipids) were determined as therapeutic fragmentations via the LPS-induced A549 cell injury model; however, Fr. B (fructooligosaccharides and small Mw fructans) had no therapeutic effect. Further compatibility PTE assays confirmed Fr. B significantly improved efficiency by a combination of HYA. The guidance assays showed Fr. B could significantly increase the uptake and distribution of HYA into lung cells and tissues. HCI assays showed that Fr. B increased uptake of HYA accompanied by significant activation of Golgi-ER. Unlike Fr. B, HRLCI showed that Fr. A, C and D were not only unobvious activations of Golgi-ER but also insignificant facilitation of colocalizations between HYA and Golgi-ER. CONCLUSIONS: Fr. B is believed to be a key YJMP material basis of PG attributed to Yin-Jing PTE with characteristic of lung-oriented guidance property, whereas another abound Fr. C was determined to have synergistic effects rather than Yin-Jing material basis.
Assuntos
Lesão Pulmonar , Platycodon , Ratos , Animais , Platycodon/química , Medicina Tradicional Chinesa , Cromatografia Líquida de Alta Pressão/métodos , PulmãoRESUMO
The limitations of current medications for treating rheumatoid arthritis (RA) emphasize the urgent need for the development of new drugs. This study aimed to investigate the potential anti-RA mechanism of amygdalin using tandem mass tag (TMT)-based quantitative proteomics technology. First, the anti-RA activity of amygdalin was evaluated in a Complete Freund's adjuvant (CFA)-induced rat model. Then, the roles and importance of proteins in the extracted rat joint tissue were evaluated using TMT-based quantitative proteomics technology. A bioinformatics analysis was used to analyze differentially abundant proteins (DAPs). A proteomics analysis identified 297 DAPs in the amygdalin group compared with the model group, of which 53 upregulated proteins and 51 downregulated proteins showed opposite regulatory trends to the DAPs produced after modeling. According to enrichment analyses of the DAPs, the signaling pathways with a high correlation degree were determined to be the complement and coagulation cascades. Furthermore, western blotting and molecular docking were used to further validate the key node proteins, e.g., complement C1s subcomponent (C1s), component C3 (C3) and kininogen 1 (Kng1). These results suggest that amygdalin may be a promising agent for treating RA by regulating the complement and coagulation cascades.
Assuntos
Amigdalina , Artrite Reumatoide , Ratos , Animais , Amigdalina/farmacologia , Proteômica/métodos , Simulação de Acoplamento Molecular , Proteínas do Sistema Complemento , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/tratamento farmacológicoRESUMO
A novel galacturonoglucan, named SCP-1, is isolated and purified from Schisandra chinensis fruits. The structure of SCP-1 is systematically investigated by a combination of monosaccharide compositions, absolute Mw, methylation analysis, partial acid hydrolysis, isoamylase degradations, and nuclear magnetic resonance spectroscopy. The structure of SCP-1 is theoretically described as follows: (i) Glc and GalA in a molar ratio of 17:3; (ii) â 4)-α-Glcp-(1â, â4,6)-α-Glcp-(1â, â3,4,6)-α-Glcp-(1â, α-Glcp-(1â, â4)-α-GalAp-6-OMe-(1â, α-GalAp-6-OMe-(1â, ß-Glcp-(1â, â6-)-ß-Glcp-(1 â and â3,4)-ß-Glcp-(1 â in a molar ratio of 48:5:3:3:10:5:12:5:9; (iii) a repeating unit of â4)-α-Glcp-(1 â as a backbone with branched points at C-3 and C-6, substituted by different types of acidic and neutral side chains to form multiple branches; and (iv) a rigid rod configuration deduced from α value of 1.26 in Mark-Houwink equation ([η] = kMα). Anti-tumor assay investigated the effects of SCP-1 on human HepG2 cancer cell lines in vitro. This is for the first time to report a galacturonoglucan in S. chinensis fruits.
Assuntos
Schisandra , Humanos , Schisandra/química , Frutas , Linhagem CelularRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort. (Chuanxiong, LC), as an important traditional Chinese medicine (TCM), can not only be used as a monarch herb but also be used as a classic "Yin-Jing" () medicine in compound prescriptions, e.g., Buyang Huanwu Decoction (BHD). Although LC has the effect of guiding components into the brain in BHD, there is still a lack of scientific evidence on this "Yin-Jing" effects. Herein, we used pharmacokinetics and tissue distributions to investigate "Yin-Jing" effects of LC. To simplify the study, four major constituents in BHD, i.e., Calycosin (CA), astragaloside IV (AI), paeoniflorin (PA), and amygdalin (AM) were combined to form a simple compound (abbreviated as CAPA here) to replace the original BHD in this paper. The Yin-Jing medical property of LC was confirmed by the compatibility of CAPA with LC or its different fractions (Fr. A â¼ Fr. F). AIM OF THE STUDY: To explore the "Yin-Jing" medical property of LC via pharmacokinetics and tissue distributions by ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS). MATERIALS AND METHODS: The contents of CA, AI, PA, and AM were simultaneously determined by the established and validated UPLC-QQQ-MS method in different rat tissues and plasma after administration of CAPA with the combination of LC or Fr. A â¼ Fr. F. The pharmacokinetic parameters, e.g., Tmax, Cmax, AUC0-t and MRT0-t, were calculated to evaluate the efficiency of "Yin-Jing". RESULTS: The Cmax and AUC0-t of CA, AI, PA, and AM were remarkably increased in rat brain tissues compared with those of the control group after compatibility of LC. This demonstrated that LC has the Yin-Jing effects on brain tissues. Additionally, Fr. B or Fr. C might be the material basis by specifically studying the distributions of CA, AI, PA, and AM in brain tissue based on mutual compatibility. The effects of Fr. B and Fr. C on distributions of these constituents in other tissues or plasma was also studied to verify the effects of Yin-Jing of LC. The results showed that the same upward trend is found in heart, liver and plasma, but the intensity is insignificant as that in brain tissue. Furthermore, the Cmax and AUC0-t of some analytes in the rat spleen, lung, and kidney were significantly decreased compared with the control group (P < 0.05 or 0.01). CONCLUSIONS: LC has the function of Yin-Jing, especially guiding the components into the brain tissue. Moreover, Fr. B and Fr. C is suggested to be the pharmacodynamic material basis for the effect of Yin-Jing of LC. These finding explained that it was recommended to add LC into some prescriptions for treating cardiovascular and cerebrovascular diseases caused by Qi deficiency and blood stasis. This has laid a certain foundation for the research on the Yin-Jing efficacy of LC to better clarify the theory of TCM and guide the clinical application of Yin-Jing drugs.
Assuntos
Medicamentos de Ervas Chinesas , Ratos , Animais , Distribuição Tecidual , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Cromatografia Líquida , Espectrometria de Massas , Medicina Tradicional Chinesa/métodosRESUMO
An unprecedent glucuronoxylogalactoglucomannan (GXG'Gâ³M), ME-2 (Mw, 2.60 × 105 g/mol; O-acetyl % = 16.7 %), was isolated and purified from water extracts of Auricularia auricula-judae (black woody ear). Firstly, due to much higher O-acetyl contents, we prepared its fully deacetylated products (dME-2; Mw, 2.13 × 105 g/mol) for convenient structure survey. The repeating structure-unit of dME-2 was readily proposed based on Mw determination, monosaccharide compositions, methylation analysis, free-radical degradation and 1/2D NMR spectroscopy. The dME-2 was identified as a highly branched polysaccharide with an average of 10 branches per 10 sugar backbone units. The backbone was only repeating â3)-α-Manp-(1â residues, substituted at the C-2, C-6 and C-2,6 positions. The side chains included ß-GlcAp-(1â, ß-Xylp-(1â, α-Manp-(1â, α-Galp-(1â and ß-Glcp-(1â. Secondly, the complex substituted positions of O-acetyl groups in ME-2 were determined to be at C-2, C-4, C-6 and C-4,6 in the backbone and at C-2 and C-2,3 in some side chains. Finally, the anti-inflammatory activity of ME-2 was preliminarily explored on LPS-stimulated THP-1 cells. The above date not only provided the first example for structural studies of GXG'Gâ³M type polysaccharides, but also facilitated development and application of black woody ear polysaccharides as medicinal agents or functional dietary supplements.
Assuntos
Basidiomycota , Basidiomycota/química , Frutas , Polissacarídeos/química , MonossacarídeosRESUMO
In order to establish structural-fingerprinting of polysaccharides for improvement of quality assessment, a sample preparation method based on microwave assisted free radical degradation (MFRD) of plant polysaccharides was proposed to produce oligosaccharides and small Mw polysaccharides. As a case study of Schisandra chinensis and S. sphenanthera fruit polysaccharides (SCP and SSP), the MFRD condition (i.e., 100 °C, 30 s and 80 W) was confirmed to be optimal. The potential structures of the MFRD products of SCP and SSP were further discussed by combinations of HILIC-ESI--QTOF-MSE and HILIC-ESI--Q-OT-IT-MS/MS. As followed, multivariable statistical analysis shows a clear separation of SCP and the SSP in PCA and OPLS-DA plots based HILIC-ESI--QTOF-MSE data. The VIP plot unveils several key Q-markers (e.g., peaks 3, 8, 9, 10, 15, 25, 26, 28, 29 and 30) with significant differences and stable emergences. Furthermore, a low-polymerization compositional fingerprinting was successfully constructed for SCP and SSP using a high-performance anion-exchange chromatography with pulsed amperometric detection. Compared to the conventional sample preparation methods, the MFRD took only a few thousandth of the time to accomplish degradations of plant polysaccharides. It significantly improves sample preparations and is generally applicable to various polysaccharide samples.
Assuntos
Schisandra , Schisandra/química , Espectrometria de Massas em Tandem , Micro-Ondas , Polissacarídeos/química , Radicais LivresRESUMO
Due to green and environment-friendly characteristics, ultra-high-performance supercritical fluid chromatography has been widely used in analytical fields in recent years, but until now few reports are available for monosaccharide compositional analysis of macromolecule polysaccharides. In this study, an ultra-high-performance supercritical fluid chromatography technology with an unusual binary modifier is used to determine the monosaccharide compositions of natural polysaccharides. Each carbohydrate herein is simultaneously labeled as 1-pheny-3-methyl-5-pyrazolone and acetyl-derivative via pre-column derivatizations aiming to increase UV absorption sensitivity and decrease water solubility. Ten common monosaccharides are fully separated and detected on ultra-high-performance supercritical fluid chromatography combined with a photo-diode array detector by systematic optimization of multiple relevant parameters, for example, column stationary phases, organic modifiers, additives, flow rates, and so on. Compared with carbon dioxide as a mobile phase, the addition of a binary modifier increases the resolution of analytes. Additionally, this method has the advantages of small consumption of organic solvent, safety, and being environmental-friendly. It has been successfully applied for full monosaccharide compositional analysis of heteropolysaccharides from Schisandra chinensis fruits. To sum up, a new alternative approach is provided for monosaccharide compositional analysis of natural polysaccharides.
Assuntos
Cromatografia com Fluido Supercrítico , Schisandra , Monossacarídeos/análise , Cromatografia com Fluido Supercrítico/métodos , Frutas/química , PolissacarídeosRESUMO
It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8 aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each carbohydrate still produced a single chromatographic peak in RPLC conditions due to the É- and ß- isomers merged together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as followed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex polysaccharides from edible plants and fungi.
Assuntos
Cromatografia de Fase Reversa , Plantas Comestíveis , Humanos , Acetilação , Polissacarídeos/química , Monossacarídeos/análise , Espectrometria de Massas em Tandem/métodos , Carboidratos , Fungos , Cetoses , Cromatografia Líquida de Alta PressãoRESUMO
The quality evaluation of nature polysaccharides is a tough nut to crack because of its high Mw distributions and larger polarity property. It is well-known that infrared spectroscopy and multiple regression modeling have been used for quantitative examinations in multiple fields, but it has not been applied to the compositional analysis of polysaccharides. In this study, attenuated total reflectance-fourier transform infrared spectroscopy is used to simultaneously quantify aldoses, ketose and uronic acids in Atractylodes polysaccharides by a combination of multivariate regressions. After experience of different data processing pretreatments, the resulting spectrum contains maximum amount of information of monosaccharide contents in Atractylodes polysaccharides. In this case, different smoothing points, derivatives, SNV and MSC are used in the pre-modeling spectrum processing and VIP screening is used to reduce the number of variables to simplify the calculation of the model. All the most optimal prediction models have both good prediction ability (R2 ≥ 0.9 and RPD > 3) and no over fitting (RMSEP/RMSEC < 3). This strategy has opened a new possibility for the nondestructive determination of complex monosaccharide compositions of natural polysaccharides in a short detection time, low equipment requirement and high experimental safety.
Assuntos
Atractylodes , Monossacarídeos , Análise dos Mínimos Quadrados , Polissacarídeos/análise , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
In this study, a plant metabonomics technique, utilizing ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI--QTOF), was used to clarify the differences of various processed Bupleurum scorzonerifolium Willd. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to determine the differences in raw and different processed B. scorzonerifolium samples, including vinegar stir-fry, wine stir-fry, honey stir-fry, honey bran stir-fry and charcoal stir-fry. Thus, 39 significant compounds, e.g., saponins, free fatty acids, fatty acid pentitol glycosides, organic acid and linalool glycosides, were clearly or tentatively identified by UPLC-ESI--QTOF-MS/MS fragmentation pathways and by comparison with available reference standards. Most importantly, fatty acid pentitol glycosides were discovered and identified in B. scorzonerifolium for the first time. Furthermore, a HepG2 hepatitis model induced by TNF-α was used to measure the anti-hepatitis effect of raw and processed B. scorzonerifolium in vitro. Molecular docking was used to understand the interaction of key Q-markers with the active sites of the target protein. The results show that the UPLC-QTOF-MS metabolomics coupled with molecular docking is a powerful tool to quickly identify quality control characteristics of B. scorzonerifolium and its products.
Assuntos
Bupleurum , Saponinas , Bupleurum/química , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos , Metabolômica/métodos , Simulação de Acoplamento Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. (PG) is a common natural medicine with a history of thousands of years. The processing products were mainly recorded as raw, honey-processed, wine-fried, yellow-fried, and bran-fried PG, which were respectively used for different clinical purposes. Therefore, it is necessary to study the chemical composition and pharmacological activity of PG after processing. AIM OF THE STUDY: To explore the effects of different processing methods on the composition and biological activity of PG using metabonomics and pharmacologic design. MATERIALS AND METHODS: UPLC-QTOF-MS combined with multivariate statistical analysis was used to identify different metabolites before and after the processing of PG. Network pharmacology was used to construct the metabolite-target-disease network. CCK-8 assay, flow cytometry, and western blotting were used to detect cell viability, apoptosis, and the expression of related proteins, respectively. RESULT: A total of 43 differentially expressed metabolites (VIP >10) were detected and identified in the analyzed groups. Based on their chemical nature, these metabolites were divided into five categories, namely, saccharolipids, flavonoid glycosides, alkynes, saponins, and lipids (including fatty acids, phospholipids, fatty aldehydes, and sterols). The content of lipids in the five processed groups (CH, FC, JZ, MZI, and MZG) was found to be higher than that in raw PG. In particular, the processing approaches explored herein increased the contents of many phospholipids, such as, glycerophosphoinositols, phosphatidic acids, and lysophosphatidyle·thanolamines. The 8 metabolites were found by venn diagram to distinguish different processed products (metabolites 2, 6, 19, 20, 21, 26, 28, and 38). The results of network pharmacology analysis showed that the primary anti-cancer targets of 43 metabolites of PG processing products are PIK3CA, Akt, and STAT3, and based on CCK-8 assay, MZI has a significant killing effect on A549 cells, compared to other processing techniques. Moreover, flow cytometry analysis showed that the cells treated with MZI exhibit significantly increased cell apoptosis, and that the effect is dose-dependent. Finally, the western blots performed herein demonstrated that the MZI effectively inhibits the expression of p-Akt and p-STAT3, which is consistent with the network pharmacology results. CONCLUSION: Depending on the processing technique, the contents of 43 different metabolites in PG were varied significantly. Specifically, the contents of phospholipids and fatty acids increase, whereas the contents of large Mw saponins decrease. Compared to the other investigated processing methods, MZI increases the potential of PG in inducing cell apoptosis and inhibiting cell proliferation by affecting the Akt and STAT3 signaling pathways. The increased levels of 3-O-ß-glucopyranosyl polygalacic acid and platycoside F after honey-frying confirm these results.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Preparações de Plantas/farmacologia , Platycodon/química , Células A549 , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metabolômica , Farmacologia em Rede , Preparações de Plantas/química , Preparações de Plantas/metabolismo , Platycodon/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
An arabinan-rich acidic polysaccharide, named ALP4-2 ([α]20 D = +197.8 (c 1.0 mg/mL, H2O); and Mw = 5.59 × 103 g/mol), was obtained from Atractylodes lancea (Thunb.) DC. ALP4-2 is mainly comprised of Ara along with a small amount of GalA, Gal, Rha, Glc and Xyl. The structure was decorated by glycosidic linkages of α-Araf-(1â, â3)-α-Araf-(1â, â5)-α-Araf-(1â, â3,5)-α-Araf-(1â, â2,4)-α-Rhap-(1â, α-GalAp-(1â, â4)-α-GalAp-6-OMe-(1â, â4)-α-GalAp-6-OMe and ß-Galp-(1â with a ratio of 6:1:7:5:5:1:7:1:4. The structure, configuration and microstructure of ALP4-2 was proposed by comprehensive considerations of results from SEC-MALLS-RID, SEC-HRMS, GC-MS, and 1D/2D NMR spectroscopy. Except for a high methyl ester in full pectin regions, an abundant arabinan moiety is observed in ALP4-2 with highly complex and branched characteristics. The immunoactivity displayed that ALP4-2 can significantly promote phagocytosis of macrophage without cytotoxicity, and stimulate nitric oxide and cytokines (TNF-α, IL-6 and IL-10) release on RAW 264.7.
Assuntos
Atractylodes , Pectinas/química , Pectinas/farmacologia , Fagocitose , Polissacarídeos/química , Polissacarídeos/farmacologiaRESUMO
Carbohydrate analysis has always been a challenging task due to the occurrence of high polarity and multiple isomers. Aldoses are commonly analyzed by gas liquid chromatography (GLC) following aldononitrile acetate derivatization (AND). However, the GLC technique cannot be applied for the simultaneous determination of aldoses, ketoses, and uronic acids. In this study, a new method based on the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and AND is developed for the complete characterization of monosaccharide composition (i.e., aldoses, ketoses, alditols, amino sugars, and uronic acids) in plant-derived polysaccharides. In addition to discussing the possible byproducts, the study optimizes the multiple reaction monitoring (MRM) parameters and LC conditions. The final separation of 17 carbohydrates is performed on a BEH Shield RP18 column (150 mm × 2.1 mm, 1.7 µm) within 25 min, without using any buffer salt. Notably, the complex polysaccharides extracted from Ligusticum chuanxiong, Platycodon grandiflorum, Cyathula officinalis Kuan, Juglans mandshurica Maxim, and Aralia elata (Miq.). Seem bud can be successfully characterized using the developed method. Overall, the results demonstrated that the newly established LC-MS/MS MRM method is more effective and powerful than the GLC-based methods reported previously, and it is more suitable for the analysis of highly complex natural polysaccharides, including complex pectins, fructosans, and glycoproteins.
Assuntos
Acetatos , Monossacarídeos , PolissacarídeosRESUMO
A hydrophilic interaction liquid chromatography-negative electrospray-mass spectrometry (HILIC-ESI--MS) coupled with microwave assisted mild acid (MAMA) depolymerization is proposed here for unusual discrimination and characterization of plant polysaccharides: a case study of fruit polysaccharides in Schisandra chinensis and S. sphenanthera (SCP and SSP). The optimized MAMA hydrolysis procedure was proposed for sample preparations of low-polymerization saccharides (Mw < 5000 Da) released in SCP and SSP. In addition, HILIC-MS/MS was employed for elucidation of isomeric glycosidic linkages in terms of 18O labelling. The MAMA hydrolysates showed that the amount of neutral â(4Hex1)nâ moiety is confirmed to be more bigger than that of acidic â(4HexA1)n â in SCP, whereas the amount of acidic â(4HexA1)nâ moiety seems to be more bigger than that of neutral â(4Hex1)nâ in SSP. The resulting low-polymerization compositional fingerprinting (LCF) showed the performance on rapid visualization of SCP and SSP by HILIC-MIM-MS. Principal components analysis (PCA) and hierarchical cluster analysis (HCA) further unveils several key Q-markers (e.g., m/z 503, 369, 665, 827, 989, 1151 and 735) for rapid discrimination of SCP and SSP. This practical study showed that the LCF with PCA and HCA could effectively reflect structural differences and could rapidly achieve discrimination of SCP and SSP.