An oligosaccharide marker for rapid authentication of edible bird's nest.

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.

Recent advances in qualitative and quantitative analysis of polysaccharides in natural medicines: A critical review.

Polysaccharides from natural medicines, being safe and effective natural mixtures, show great potential to be developed into botanical drugs. However, there is yet one polysaccharide-based case that has fulfilled the Botanical Guidance definition of a botanical drug product. One of the reasons is the analytical methods commonly used for qualitative and quantitative analysis of polysaccharides fall far behind the quality control criteria of botanical drugs. Here we systemically reviewed the recent advances in analytical methods. A critical evaluation of the strength and weaknesses of these methods was provided, together with possible solutions to the difficulties. Mass spectrometry with or without robust chromatographic separation was increasingly employed. And scientists have made significant progress in simplifying polysaccharide quantification by depolymerizing it into oligosaccharides. This oligosaccharides-based strategy is promising for qualitative and quantitative analysis of polysaccharides. And continuous efforts are still needed to develop a standardized quality control method that is specific, accurate, repeatable, and applicable for analyzing individual components in natural medicine formulas.

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS.

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.

Qualitative and Quantitative Analysis of Edible Bird's Nest Based on Peptide Markers by LC-QTOF-MS/MS.

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.

A specific and bioactive polysaccharide marker for Cordyceps.

Cordyceps is one of the most expensive and widely used functional foods. But the authenticity is still a concern due to the lack of appropriate markers. By targeting polysaccharides, this study aimed to develop a specific, and bioactive marker for Cordyceps. Firstly, the results of screening tests of 250 samples by examining both genetic markers and polysaccharide profile showed that a unique polysaccharide fraction (named CCP) was particular to the caterpillar parts. Its potential as a marker was further demonstrated by its ability to induce NO and cytokine production in RAW 264.7 cells. CCP was characterized to be an α-1,4-glucan with a branch at C-6 by the conventional structure analyzing and de novo oligosaccharides sequencing. The content of CCP was closely correlated to the traditional classification criteria. Generally, CCP was a marker that simultaneously enables qualitative and quantitative analysis of Cordyceps.

Enhanced Stromal Cell CBS-H2S Production Promotes Estrogen-Stimulated Human Endometrial Angiogenesis.

Angiogenesis is a physiological process for endometrial regeneration in the menstrual cycle and remodeling during pregnancy. Endogenous hydrogen sulfide (H2S), produced by cystathionine-ß synthase (CBS) and cystathionine-γ lyase (CSE), is a potent proangiogenic factor; yet, whether the H2S system is expressed in the endometrium and whether H2S plays a role in endometrial angiogenesis are unknown. This study was to test whether estrogens stimulate endometrial H2S biosynthesis to promote endometrial microvascular endothelial cell (EMEC) angiogenesis. CBS messenger RNA/protein and H2S production significantly differed among endometria from postmenopausal (POM), premenopausal secretory (sPRM), and proliferative (pPRM) nonpregnant (NP) and pregnant (Preg) women (P < .05) in a rank order of POM approximately equal to sPRM is less than pPRM is less than Preg, positively correlating with angiogenesis indices and endogenous estrogens and with no difference in CSE expression. CBS and CSE proteins were localized to stroma, glands, and vessels in endometrium, and greater stromal CBS protein was observed in the pPRM and Preg states. Estradiol-17ß (E2) (but not progesterone) stimulated CBS (but not CSE) expression and H2S production in pPRM endometrial stromal cells (ESCs) in vitro, which were attenuated by ICI 182 780. The H2S donor sodium hydrosulfide promoted in vitro EMEC angiogenesis. Co-culture with sPRM, pPRM, and Preg ESCs all stimulated EMEC migration with a rank order of sPRM less than pPRM approximately equal to Preg. CBS (but not CSE) inhibition attenuated ESC-stimulated EMEC migration. E2 did not affect EMEC migration but potentiated ESC-stimulated EMEC migration. Altogether, estrogens stimulate specific receptor-dependent stromal CBS-H2S production to promote endometrial EMEC angiogenesis in women.

An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

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.

Oligosaccharide-marker approach for qualitative and quantitative analysis of specific polysaccharide in herb formula by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry: Dendrobium officinale, a case study.

Qualitative and quantitative analysis of polysaccharides in herb formula remain challenge due to the limited choices of analytical methods concerning the intrinsic characteristics of large molecular mass. Herein, an oligosaccharide-marker approach was newly developed for quality assessment of polysaccharides in herbal materials, using Dendrobium officinale as a case study. This method involved partial acid hydrolysis of D. officinale polysaccharide (DOP) followed by p-aminobenzoic ethyl ester (ABEE) derivatization. Two ABEE-labeled oligosaccharides namely, Te-Man-ABEE and Pen-Man-ABEE, were selected as chemical markers due to their high specificity in herb formula. The linear relationship between the content of these two markers and the content of DOP was then successfully established respectively. The linear relationship was further transformed to that between peak area of chemical markers and DOP content so that chemical markers were not necessary to be isolated for analysis. This linear relationship was systemically validated in terms of precision and accuracy. The results showed that these two oligosaccharide-markers presented a good linear relationship with DOP (R2 ≥ 0.997) in the range of 0.68-16.02 µg. These markers also demonstrated satisfactory precision (RSD < 7.0%), and recovery (91.41%-118.30%) in real sample determination. Additionally, there was no significant difference between the results given by the two chemical markers as the RSD values were not more than 7.0%. While concerning the results given by the oligosaccharide-markers and the previously-published polysaccharide marker, the RSD value was not more than 6.4%. These suggest that the oligosaccharide-marker approach is a simple, quick, and reliable method to qualitatively and quantitatively determine of specific polysaccharide in herb formula.

Interference of Quercetin on Astragalus Polysaccharide-Induced Macrophage Activation.

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.

Comprehensive comparison of polysaccharides from Ganoderma lucidum and G. sinense: chemical, antitumor, immunomodulating and gut-microbiota modulatory properties.

Both Ganoderma lucidum (GL) and G. sinense (GS) are used as Lingzhi in China. Their functions are assumed to mainly derive from triterpenes and polysaccharides; however, the two species have very different triterpenes profiles, if this was the case, then the bioactivity of these two species should differ. Instead, could the polysaccharides be similar, contributing to the shared therapeutic basis? In this study, two main polysaccharide fractions from different batches of GL and GS were systematically compared by a series of chemical and biological experiments. The results showed that the polysaccharides from two species shared the same structural features in terms of mono-/oligo-saccharide profiles, molecular size, sugar linkages, and IR/NMR spectra. In addition, these polysaccharides showed similar tumor-suppressive activity in mice. Further study on RAW264.7 cells indicated that these polysaccharides exhibited similar inducing effects to macrophages, as evaluated in the phagocytosis function, NO/cytokines production, inhibition against the viability and migration of cancer cells. Mechanistic investigation revealed the identical activation via TLR-4 related MAPK/NF-κB signaling pathway and gut-microbiota modulatory effects. In summary, GL and GS polysaccharides presented similar chemical features, antitumor/immunomodulating activities and mechanism; this establishes polysaccharides as the active principles and supports the official use of both species as Lingzhi.

Astragalus Polysaccharide RAP Selectively Attenuates Paclitaxel-Induced Cytotoxicity Toward RAW 264.7 Cells by Reversing Cell Cycle Arrest and Apoptosis.

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.

Salidroside induces neuronal differentiation of mouse mesenchymal stem cells through Notch and BMP signaling pathways.

Salidroside (p-hydroxyphenethyl-ß-D-glucoside, SAL), a phenylpropanoid glycoside isolated from a popular traditional Chinese medicinal plant Rhodiola rosea L., possesses multiple pharmacological actions. Previous study showed that SAL could induce rat mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons and induce mouse MSCs D1 to differentiate into neuronal cells. However, the mechanisms of SAL-induced neuronal differentiation of MSCs still need investigation. In this study, we observed the effects of SAL on neuronal differentiation of D1 cells and the possible involvement of Notch and BMP signaling pathways. SAL inhibited the proliferation, induced neuronal phenotypes, and upregulated the expressions of neuronal-specific marker molecules, such as neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (MAP2), and beta 3 class III tubulin (Tubb3/ß-tubulin III) in D1 cells. SAL not only downregulated the expressions of Notch1 and hairy enhancer of split 1 (Drosophila) (Hes1) but also upregulated the expression of Smad1/5/8 and its phosphorylation (p-Smad 1/5/8). The neuronal differentiation effects of SAL on D1 cells were promoted by a Notch signaling antagonist, DAPT, but attenuated by a BMP signaling pathway antagonist, Noggin. Our findings suggest that SAL might be promising in inducing neuronal differentiation of mouse MSCs mediated by both Notch signaling pathway and BMP signaling pathway.