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1.
Molecules ; 27(9)2022 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-35566296

RESUMEN

Edible bird's nest (EBN) is an expensive health food. There are many adulterants in the market. It remains challenging to discriminate EBN from its adulterants due to a lack of high-specificity markers. Besides, the current markers are confined to soluble fraction of EBN. Here, both soluble and insoluble fractions were analyzed by LC-QTOF-MS/MS. A total of 26 high-specificity peptides that were specific to EBN were selected as qualitative authentication markers. Among them, 10 markers can discriminate EBN from common adulterants, 13 markers discriminate white EBN from grass EBN/common adulterants, and 3 markers discriminate grass EBN from white EBN/common adulterants. Three of them, which showed high signal abundance (Peak area ≥ 106) and satisfactory linearity (R2 ≥ 0.995) with EBN references, were selected as the assay marker; and their peptide sequences were confidently identified by searching database/de novo sequencing. Based on these markers, a qualitative and quantitative analytical method was successfully developed and well-validated in terms of linearity, precision, repeatability, and accuracy. The method was subsequently applied to detect EBN products on the market. The results indicated that more than half of EBN products were not consistent with what the merchants claimed.


Asunto(s)
Aves , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Péptidos , Espectrometría de Masas en Tándem/métodos
2.
Molecules ; 27(14)2022 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-35889516

RESUMEN

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.


Asunto(s)
Gelatina , Espectrometría de Masas en Tándem , Animales , Biomarcadores/análisis , Bovinos , Equidae , Gelatina/análisis , Caballos , Péptidos/análisis , Porcinos , Espectrometría de Masas en Tándem/métodos
3.
Molecules ; 23(7)2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29958399

RESUMEN

Polysaccharides, which exert immunoregulatory effects, are becoming more and more popular as food supplements; however, certain components of ordinary foods could be reducing the polysaccharides beneficial effects. Quercetin, a flavonoid found in common fruits and vegetables, is one such component. This study investigated the effects of quercetin on Astragalus polysaccharide RAP induced-macrophage activation. The results show quercetin decreases the NO production and iNOS gene expression in RAW264.7 cells, and it inhibits the production of cytokines in RAW264.7 cells and peritoneal macrophages. Western blot analysis results suggest that quercetin inhibits the phosphorylation of Akt/mTORC1, MAPKs, and TBK1, but has no effect on NF-κB in RAP-induced RAW264.7 cells. Taken together, the results show that quercetin partly inhibits macrophage activation by the Astragalus polysaccharide RAP. This study demonstrates that quercetin-containing foods may interfere with the immune-enhancing effects of Astragalus polysaccharide RAP to a certain extent.


Asunto(s)
Planta del Astrágalo/química , Polisacáridos/farmacología , Animales , Activación de Macrófagos/efectos de los fármacos , Ratones , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Quercetina/farmacología , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos
4.
Molecules ; 21(6)2016 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-27314319

RESUMEN

A neutral α-glucan, named BP1, with a molecular mass of approximately 9.45 kDa, was isolated from Lobelia chinensis by hot-water extraction, a Q-Sepharose Fast Flow column and Superdex-75 column chromatography. Its chemical structure was characterized by monosaccharide analysis, methylation analysis and analysis of its FT-IR, high performance gel permeation chromatography (HPGPC) and 1D/2D-NMR spectra data. The backbone of BP1 consists of →6α-d-Glcp¹â†’6,3α-d-Glcp¹â†’(6α-d-Glcp¹)x-6,3α-d-Glcp¹-(6α-d-Glcp¹)y→. The side chains were terminal α-d-Glcp¹â†’ and α-d-Glcp¹â†’ (6α-d-Glcp¹)z→4α-d-Glcp¹â†’3α-d-Glcp¹â†’4α-d-Glcp¹â†’ (x + y + z = 5), which are attached to the backbone at O-3 of 3,6α-d-Glcp¹. The results of the effect of BP1 on mouse macrophage cell line RAW 264.7 indicate that BP1 enhances the cell proliferation, phagocytosis, nitric oxide production and cytokine secretion in a dose-dependent manner. Because the inhibitor of Toll-like receptor 4 blocks the BP1-induced secretion of TNF-α and IL-6, we hypothesize that α-glucan BP1 activates TLR4, which mediates the above-mentioned immunomodulating effects.


Asunto(s)
Glucanos/química , Interleucina-6/biosíntesis , Lobelia/química , Receptor Toll-Like 4/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucanos/administración & dosificación , Glucanos/aislamiento & purificación , Inmunomodulación , Metilación , Ratones , Monosacáridos/química , Monosacáridos/aislamiento & purificación , Fagocitosis/efectos de los fármacos , Células RAW 264.7/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier
5.
Food Chem ; 409: 135334, 2023 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-36586266

RESUMEN

Edible bird's nest (EBN) is a popular and expensive food material. The limited supply and great demand result in the use of adulterants. The authenticity concern is raised due to the lack of appropriate quality markers. Herein, this study aims to provide a specific oligosaccharide marker for rapid EBN authentication. Comparing the benzocaine (ABEE)-labeled saccharide profiles of multiple batches of EBN and adulterants indicates seven unique EBN oligosaccharides. The most abundant one, named BNM001, was selected as a marker and characterized to be Neu5Ac (2-3) Gal by MS and NMR spectra. This new oligosaccharide marker enables a rapid authentication of EBN within 10 min. ABEE labelling of this marker further upgraded the accuracy and sensitivity of the LC-qTOF-MS quantitative analysis. The relative marker content was associated with the quality of EBN products. These results suggest a specific and efficient quality marker for rapid authentication of EBN and related products.


Asunto(s)
Aves , Oligosacáridos , Animales , Carbohidratos , Alimentos , Espectrometría de Masas
6.
J Pharm Biomed Anal ; 185: 113235, 2020 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-32182447

RESUMEN

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.


Asunto(s)
Química Farmacéutica/métodos , Medicamentos Herbarios Chinos/análisis , Oligosacáridos/análisis , Control de Calidad , Química Farmacéutica/normas , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Medicamentos Herbarios Chinos/química , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas
7.
Front Pharmacol ; 9: 1580, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30804792

RESUMEN

Purpose: The purpose of this study was to determine if an Astragalus polysaccharide (RAP) can protect immune cells from the toxic side effects of paclitaxel (Taxol), a powerful anti-tumor drug whose equally powerful side effects limit its clinical use. Methods: We hypothesized that RAP can reduce the toxic effects induced by Taxol. To test this hypothesis, we conducted a series of studies in vivo and in vitro. First, we confirmed RAP's effects in vivo utilizing BALB/c mice inoculated with 4T1 mouse breast cancer cells as the tumor model. Mice were treated with RAP and/or Taxol, and the differences in the life spans were recorded. Second, a co-culture cell model was used to study the protective effect of RAP on cells vis-a-vis Taxol. The cell cycle and apoptosis of RAW 264.7 cells that were treated with RAP with/without Taxol were checked by flow cytometry and Hoechst staining. Proteins involved in the cell cycle and apoptosis were also tested by Western blot to reveal the probable mechanism. Results: RAP prolonged the life span of tumor-bearing mice treated with Taxol. The in vitro experiments showed that Taxol suppressed the proliferation of RAW 264.7 cells while RAP protected the RAW 264.7 cells from Taxol-induced suppression. The protection is selective because RAP had no effect on 4T1 cells. Furthermore, Taxol clearly led to cell cycle arrest mainly at the G2/M phase and generated cytotoxicity against RAW 264.7 cells, while RAP blocked cell cycle arrest and protected cells from apoptosis. Taxol up-regulated the protein levels of P-H2A, PARP, Chk1, p53, and p21 and down-regulated Bcl-Xl and Mcl-1, and RAP reversed the expression of all these proteins. Conclusion: These results suggested that RAP can protect immune cells from Taxol-induced toxicity, by changing the cell cycle and apoptosis.

8.
J Agric Food Chem ; 64(4): 881-9, 2016 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-26752248

RESUMEN

A crude polysaccharide fraction (cDOP) has been determined to be the characteristic marker of Dendrobium officinale, an expensive tea material in Asia, but its chemistry and bioactivity have not been studied. In work reported here, cDOP was destarched (DOP, 90% yield) and separated into two subfraction polysaccharides, DOPa and DOPb, which were characterized by monosaccharide composition and methylation analyses and spectral analyses (FT-IR and (1)H and (13)C NMR). Both are composed of mannose and glucose at similar ratios and have a similar structure with a backbone of 1,4-linked ß-D-mannopyranosyl and ß-D-glucopyranosyl residues. Significant differences were observed only in their molecular weights. Bioassay using mouse macrophage cell line RAW264.7 indicated that DOP and its two subfractions enhance cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. They also induced the proliferation of lymphocytes alone and with mitogens. DOPa and DOPb are thus proven to be major, active polysaccharide markers of D. officinale.


Asunto(s)
Dendrobium/química , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Polisacáridos/química , Polisacáridos/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Factores Inmunológicos/aislamiento & purificación , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Fagocitosis/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Polisacáridos/aislamiento & purificación , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/inmunología
9.
J Ethnopharmacol ; 179: 243-52, 2016 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-26743224

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Polysaccharides of Radix Astragali (Astragalus membranaceus (Fisch) Bge.; Huangqi) are able to induce cytokine production of macrophages and are considered the main active ingredient for the immune-enhancing effect of this commonly used medicinal herb. AIM OF STUDY: To investigate the molecular mechanism of immunomodulating activities of a reported Astragalus polysaccharide, RAP, which is a hyperbranched heteroglycan with average molecular weight of 1334kDa. MATERIALS AND METHODS: The cytokine production of RAW264.7 cells were analyzed by using ELISA assays while cell viability was assessed by MTT method. Western blot analysis was used for determining protein contents of mitogen-activated protein kinases (MAPKs). In addition, the level of IL-6, iNOS, and TNF-α mRNA was determined by RT-PCR. RESULTS: It has been found that RAP itself did not have any cytotoxic effect on mouse mammary carcinoma 4T1 cells, but it significantly enhanced cytotoxicity of the supernatant of RAW264.7cells on 4T1 cells. Furthermore, RAP enhanced the production of NO and cytokines in RAW264.7 cells, and significantly up-regulated gene expressions of TNF-α, IL-6, iNOS. All these bioactivities were blocked by the inhibitor of TLR4 (Toll-like receptor 4), suggesting that TLR4 is a receptor of RAP and mediates its immunomodulating activity. Further analyses demonstrated that RAP rapidly activated TLR4-related MAPKs, including phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, and induced translocation of NF-κB as well as degradation of IκB-α. These results are helpful to better understand the immunomodulating effects of Radix Astragali. CONCLUSIONS: RAP may induce cytokine production of RAW264.7 cells through TLR4-mediated activation of MAPKs and NF-κB.


Asunto(s)
Planta del Astrágalo , Citocinas/metabolismo , Polisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Fosforilación/efectos de los fármacos , Polisacáridos/aislamiento & purificación , Receptor Toll-Like 4/antagonistas & inhibidores
10.
PLoS One ; 9(8): e105502, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25171171

RESUMEN

We have recently demonstrated that the greatly increased immunological activities of recombinant murine calreticulin (rCRT) are largely attributed to its self-oligomerization. Although native CRT (nCRT) can also oligomerize under stress conditions in vitro, whether this phenomenon could occur inside cells and the immunological activity difference between nCRT monomers and oligomers remained unclear. In this study, we illustrated the formation of CRT oligomers in tranfectant cells under "heat & low pH" (42°C/pH 6.5) condition. The mixture of nCRT oligomers and monomers (OnCRT) was obtained after 3 hr treatment of murine monomeric nCRT (MnCRT) under similar condition (42°C/pH 5.0) in vitro. The OnCRT thus obtained was better recognized by 2 monoclonal Abs from mice that had been immunized with oligomeric rCRT. Unlike MnCRT, OnCRT was able to elicit CRT-specific IgG production in mice. OnCRT also stimulated bone-marrow derived dendritic cells (BMDCs) to secrete significantly higher levels of TNF-α, IL-6 and IL-12p40 than did MnCRT in vitro. We postulate that oligomerization of soluble CRT may occur under certain pathophysiological conditions (e.g. ultrahyperpyrexia) and the resultant oligomers may exhibit exaggerated immunostimulating activities, thereby affiliating the inflammatory responses in vivo.


Asunto(s)
Anticuerpos/inmunología , Calreticulina/química , Calreticulina/inmunología , Multimerización de Proteína/inmunología , Animales , Anticuerpos/sangre , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Calreticulina/farmacología , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Epítopos/farmacología , Calor , Concentración de Iones de Hidrógeno , Inmunización/métodos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Subunidad p40 de la Interleucina-12/inmunología , Subunidad p40 de la Interleucina-12/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
11.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(3): 270-3, 2011 Mar.
Artículo en Zh | MEDLINE | ID: mdl-21419046

RESUMEN

AIM: To establish the transgenic cell strains expressing recombinant adenovirus vector of human Oncostain M(hOSM)gene which is supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34(+) hematopoietic stem/progenitor cell (HSPC) and compare its migration capacity before and after amplification in vitro. METHODS: Establish the transgenic cell strains expressing recombinant adenovirus vector of hOSM gene, and the objective gene was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34(+) HSPC separated by magnetic-activated cell sorting (MACS) was detected by the FCM. After culturing with feeder layer cells, detect the rate of proliferation by flow cytometry (FCM). To compare the homing ability of HSPC after amplification in vitro, detect the spontaneous migration rate and migration rate induced by SDF-1 using transmembrane migration assay (Transwell experiment). RESULTS: The green fluorescence was observed by fluorescence microscope in the transgenic cell strains, and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34(+) HSPC separated by MACS could reach(96.8 ± 2.28)%. After culturing with feeder layer cells for 7 days, the CD34(+) cells were 15.73 times in group containing hOSM more than in group without hOSM. The expression rate of adhension molecules on the surface of CD34(+) cells were also higher in the group containing hOSM than without hOSM. After using Transwell assys to detect the homing ability of culturing cells, the induction migration rate of stem cells clturing on transgenic cell strains was (40.68 ± 1.35)%, significantly higher than the control, which reveals a better homing ability. CONCLUSION: Recombinant adenovirus vector of hOSM gene as feeder layer cells can effectively proliferate umbilical cord blood CD34(+) HSPC in vitro and delay it differentiate, what's more, the stem cells retain a high homing ability after culturing on transgenic cell strains in vitro.


Asunto(s)
Sangre Fetal/citología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Oncostatina M/genética , Oncostatina M/metabolismo , Células Madre/metabolismo , Adenoviridae/genética , Antígenos CD34/inmunología , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Femenino , Técnicas de Transferencia de Gen/instrumentación , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Receptores CXCR4/metabolismo
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