Sialoglycoprotein isolated from Carassius auratus eggs promotes osteogenesis by stimulating mesenchymal stem cells to commit to osteoblast differentiation.

In this study, we explore whether the pro-osteogenic effects of sialoglycoprotein from Carassius auratus eggs (Ca-SGP) involve mesenchymal stem cells (MSCs). Ovariectomized osteoporotic mice treated with Ca-SGP had increased bone formation and reduced bone marrow adipose tissue. As MSCs are common progenitors of osteoblasts and adipocytes, we isolated MSCs from Ca-SGP-treated mice and found that they tended to differentiate into osteoblasts over adipocytes confirmed by Alizarin red and Oil red O staining. This change was seen at the gene and protein level. To further explore the effect of Ca-SGP on MSCs, we isolated MSCs from healthy mice and treated them with Ca-SGP in vitro. We discovered that Ca-SGP promoted MSC differentiation to osteoblasts. In addition, Ca-SGP promoted osteogenesis and reduced the fat in marrow cavity of adolescent mice. For the first time, our results demonstrate that Ca-SGP promotes osteogenesis via stimulating MSCs to commit to osteoblasts. Graphical Abstract ᅟ.

A general gas-assisted three-liquid-phase extraction method for separation and concentration of puerarin, 3'-methoxydaidzin, puerarinxyloside, daidzin and daidzein from puerariae extract.

In this work, a general and novel separation technique gas-assisted three-liquid-phase extraction was established and applied in separating and concentrating isoflavonoids from the actual sample of puerariae extract by one step. For the gas-assisted three-liquid-phase extraction method, optimal conditions were selected: polyethylene glycol 2000 and ethyl acetate as the flotation solvent, pH 5, (NH4 )2 SO4 concentration 350 g/L in aqueous phase, N2 flow rate 30 mL/min, flotation time 50 min, and flotation twice. Five isoflavonoids compounds puerarin, 3'-methoxydaidzin, puerarinxyloside, daidzin and daidzein were separated with recoveries of 82, 84, 80, 88 and 89%, respectively. The separated products were purified by preparative high-performance liquid chromatography, and the purity of the final products was >96%. The established general gas-assisted three-liquid-phase extraction was used to separate anthraquinones from Cassiae Semen under the optimal conditions, and the recoveries were >75%. The experimental results showed that the established gas-assisted three-liquid-phase extraction method is a general technique for separating active compounds from herb extract.

A preliminary origin-tracking study of different densities urinary exosomes.

Based on density differences of different subpopulations of exosomes, two kinds of micro-vesicles with different densities were captured from urine by a modified sucrose density gradient ultracentrifuge separation method. Verified by transmission electron microscope (TEM) and western blot, the results showed these two kinds of micro-vesicles were all exosomes. And these two kinds of exosomes were analyzed by TEM, 2D electrophoresis (2DE), and capillary zone electrophoresis (CZE), respectively. The results of TEM showed these two exosomes with different densities have different morphological characteristics, and some tiny proteomic differences were shown in the results of 2DE of these two exosomes. At the same time, the CZE results displayed these two kinds of exosomes possessed different retention times, indicated that they may have different electrification property and particle weight. These results may attribute to their different origins. This work may provide a preliminary experience for the origin-tracking study for urinary exosomes, and would be more useful for future targeted biomarker discovery.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78 mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30 g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

A simple gradient equilibrium method for better separation in countercurrent chromatography.

The stationary phase retention is one of the most important parameters in countercurrent chromatography. In this work, a simple gradient equilibrium method was developed to further improve the stationary phase retention based on the optimized condition in the traditional equilibrium model. Meanwhile, this novel gradient equilibrium method was used to separate three flavone model compounds and compared with the conventional isocratic equilibrium method to evaluate the separation efficiency. The results show that better resolution or shorter separation time could be achieved with gradient equilibrium compared to isocratic equilibrium. So this novel equilibrium method has enormous potential for obtaining a better separation or saving the separating time in the preparative separation of target compounds.

Isolation and purification of alkaloids from lotus leaves by ionic-liquid-modified high-speed countercurrent chromatography.

An effective high-speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two-phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic-liquid-modified high-speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C4 mim][BF4 ] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N-nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic-liquid-modified high-speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.

Separation of four flavonol glycosides from Solanum rostratum Dunal using aqueous two-phase flotation followed by preparative high-performance liquid chromatography.

Aqueous two-phase flotation followed by preparative high-performance liquid chromatography was used to separate four flavonol glycosides from Solanum rostratum Dunal. In the aqueous two-phase flotation section, the effects of sublation solvent, solution pH, (NH4 )2 SO4 concentration in aqueous solution, cosolvent, N2 flow rate, flotation time, and volumes of the polyethylene glycol phase on the recovery were investigated in detail, and the optimal conditions were selected: 50 wt% polyethylene glycol 1000 ethanol solvent as the flotation solvent, pH 4, 350 g/L of (NH4 )2 SO4 concentration in aqueous phase, 40 mL/min of N2 flow rate, 30 min of flotation time, 10.0 mL of flotation solvent volume, and two times. After aqueous two-phase flotation concentration, the flotation products were purified by preparative high-performance liquid chromatography. The purities of the final products A and B were 98.1 and 99.0%. Product B was the mixture of three compounds based on the analysis of high-performance liquid chromatography at the temperature of 10°C, while product A was hyperoside after the identification by nuclear magnetic resonance. Astragalin, 3'-O-methylquercetin 3-O-ß-d-galactopyranoside, and 3'-O-methylquercetin 3-O-ß-d-glucopyranoside were obtained with the purity of 93.8, 97.1, and 99.2%, respectively, after the further separation of product B using preparative high-performance liquid chromatography.

Separation of phenolic acids from sugarcane rind by online solid-phase extraction with high-speed counter-current chromatography.

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid-phase extraction with high-speed counter-current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid-phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid-phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two-phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid-phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p-coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution-extrusion counter-current chromatography and back-extrusion counter-current chromatography were compared.

[Comparative analysis on industrial standardization degree of Chinese and Korean ginseng].

Panax ginseng is a well-known medicinal plant all over the world. It has high nutritional value and medicinal value. China and South Korea are the major countries in the world for ginseng cultivation, production and exportation. China's ginseng production accounts for more than half of the world, but the output value is less than that of Korea. The standardization process of ginseng industry plays an important role. This paper makes a detailed analysis of the Chinese and Korean ginseng national standards and the standardization process, and makes a detailed comparative analysis of the categories, standard contents, index selection, age, implementation and promotion status of the Chinese and Korean ginseng standards. The development disadvantages of ginseng industry standardization were displayed. And we give our advises on the standard revision, implementation of China's ginseng industry standardization, hoping to enhance the competitiveness of China's ginseng industry.

Isolation and purification of arctigenin from Fructus Arctii by enzymatic hydrolysis combined with high-speed counter-current chromatography.

Enzymatic hydrolysis pretreatment combined with high-speed counter-current chromatography for the transformation and isolation of arctigenin from Fructus Arctii was successfully developed. In the first step, the extract solution of Fructus Arctii was enzymatic hydrolyzed by ß-glucosidase. The optimal hydrolysis conditions were 40°C, pH 5.0, 24 h of hydrolysis time, and 1.25 mg/mL ß-glucosidase concentration. Under these conditions, the content of arctigenin was transformed from 2.60 to 12.59 mg/g. In the second step, arctigenin in the hydrolysis products was separated and purified by high-speed counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (10:25:15:20, v/v), and the fraction was analyzed by HPLC, ESI-MS, and (1)H NMR spectroscopy. Finally, 102 mg of arctigenin with a purity of 98.9% was obtained in a one-step separation from 200 mg of hydrolyzed sample.

Non-Destructive Screening of Sodium Metabisulfite Residue on Shrimp by SERS with Copy Paper Loaded with AgNP.

In order to prompt the appearance of the shrimp color, sodium metabisulfite is frequently added in shrimp processing, which is, however, prohibited in China and many other countries. This study aimed to establish a surface-enhanced Raman spectroscopy (SERS) method for screening sodium metabisulfite residues on shrimp surfaces, in a non-destructive manner. The analysis was carried out using a portable Raman spectrometer jointly with copy paper loaded with silver nanoparticles as the substrate material. The SERS response of sodium metabisulfite gives two fingerprint peaks at 620 (strong) and 927 (medium) cm-1, respectively. This enabled unambiguous confirmation of the targeted chemical. The sensitivity of the SERS detection method was determined to be 0.1 mg/mL, which was equal to residual sodium metabisulfite on the shrimp surface at 0.31 mg/kg. The quantitative relationship between the 620 cm-1 peak intensities and the concentrations of sodium metabisulfite was established. The linear fitting equation was y = 2375x + 8714 with R2 = 0.985. Reaching an ideal balance in simplicity, sensitivity, and selectivity, this study demonstrates that the proposed method is ideally suitable for in-site and non-destructive screening of sodium metabisulfite residues in seafood.

HPTLC-Bioluminescent Bioautography Screening of Herbal Teas for Adulteration with Hypolipidemic Drugs.

Teas based on nutraceutical herbs are an effective tool against hyperlipidemia. However, the adulteration with chemical drugs is frequently detected. By coupling bioluminescent bioautography with high performance thin-layer chromatography (HPTLC), we developed a facile method suitable for screening hypolipidemic drugs (ciprofibrate and bezafibrate) adulteration in five different herbal teas (lotus leaf, Apocynum, Ginkgo biloba, Gynostemia and chrysanthemum). First, the sensitivity of a bioluminescent bacteria to the analyte was evaluated on different HPTLC layer materials, revealing that the best performance was achieved on the silica gel layer. On this basis, sample extracts were separated on silica gel plates via a standardized HPTLC procedure, forming a selective detection window for the targeted compound. Then, the separation results were rapidly visualized by the bioluminescence inhibition of bacteria cells within 6 min after dipping. The observed inhibition displayed an acceptable limit of detection (<20 ng/zone or 2 mg/kg) and linearity (R2 ≥ 0.9279) within a wide concentration range (50-1000 ng/zone). Furthermore, the optimized method was performed with artificially adulterated samples and the recovery rates were determined to be within the range of 71% to 91%, bracing its practical reliability. Showing superiorly high simplicity, throughput and specificity, this work demonstrated that the analytical method jointly based on HPTLC and bioautography was an ideal tool for screening bioactive compounds in complex biological matrix.

HPTLC screening of saccharin in beverages by densitometry quantification and SERS confirmation.

As a widely used artificially synthesized sweetener, saccharin faced numerous disputes associated with food safety. Therefore, its fast analysis in food is of crucial importance. In this study, an analytical method for the fast and reliable screening of saccharin in various beverages was established and validated, by combining HPTLC with densitometry and surface enhanced Raman spectroscopy. The diluted sample liquid was directly sprayed and separated on a silica gel plate using a mixture of ethyl acetate and acetic acid in the ratio of 9 : 1 (v/v) as the mobile phase. The separation realized full isolation of the analyte from background noises. Then, a densitometry analysis in the absorption-reflection mode (working wavelength 230 nm) was optimized to obtain quantitative data, showing a good linearity in the range of 40-200 ng per band (R 2 = 0.9988). The limits of detection and quantification were determined to be 6 and 20 ng per band, respectively, which were equal to 6 and 20 mg kg-1. The quantitative results also displayed satisfactory accuracy and precision, with a spike-recovery rate within 87.75-98.14% (RSD <5.13%). As a cost-efficient tool for confirmation, surface enhanced Raman spectroscopy was employed to profile the molecular fingerprint of the analyte eluted from the plate layer. Under optimized conditions (785 nm laser as the excitation light and silver nanoparticle loaded glass fiber paper as the active substrate), the elution of the saccharin band exhibited stable and sensitive surface enhanced Raman spectroscopy signals. This study demonstrated that HPTLC could be a versatile platform for food analysis, with outstanding simplicity and cost-efficiency.

A Simple and Standardized Method for the Determination of Total Solanesol in Potato Leaves and Its Extracts Based on HPLC-MS.

BACKGROUND: Solanesol is an important pharmaceutical intermediate raw material, mainly used to synthesize coenzyme Q10, vitamin K2. It can be found prominent in potato stems and leaves. But now potato stems and leaves are always abandoned or discarded as they are not suitable for use as feed in aquaculture or other purposes. These agricultural waste resources can be reutilized as the corresponding extracts. OBJECTIVE: To develop a simple and standardized method for the detection of total solanesol in potato leaves and its extracts. METHODS: N-hexane was chosen as the extraction solvent for three times in the solanesol extraction from potato leaves. HPLC-MS was used for the detection. RESULTS: The LOQ was 0.3 µg/g and the linear range was from 0.1 to 50 µg/mL. The precision and stability were evaluated by the relative standard deviations (RSDs) of three samples (potato leaves, Extract-1, Extract-2) for interday and intraday. The accuracy of the method was evaluated by the recoveries of three different spiked concentrations of solanesol for three samples, and results showed it ranged from 80.7% to 99.0% with RSDs less than 8.7%. CONCLUSIONS: The method we established can provide a simple and standardized way for the extraction and detection of total solanesol. HIGHLIGHTS: The work laid a foundation for the resource reutilization of potato stem and leaf.

Rapid microwave-assisted green synthesis of guanine-derived carbon dots for highly selective detection of Ag+ in aqueous solution.

In this work, a simple and green synthetic approach of novel guanine decorated carbon dots (G-CDs) using guanosine 5'-monophosphate and ethylenediamine through a domestic microwave oven was established for the first time. The as-prepared fluorescent G-CDs were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, UV-vis spectroscopy, and fluorescence spectroscopy. The obtained fluorescent G-CDs with a uniform morphology had desirable functional groups and excellent optical performances. Furthermore, the fluorescence intensity of G-CDs was remarkably quenched by Ag+ than that of other nucleotides-derived CDs. The density functional theory calculations were performed to confirm that the strong interaction of guanine-Ag+ was responsible for the remarkable fluorescence response of G-CDs towards Ag+. In addition, as a label-free fluorescence probe, the G-CDs displayed a good linear detection for highly selective Ag+ sensing over the range of 0-80 µM with the low detection limit of 90 nM. Therefore, the proposed G-CDs had the capacity for Ag+ detection in the real samples.

[Identification of the impurity in auramine O by high performance liquid chromatography-ion trap-time of flight mass spectrometry and preparation of the auramine O reference standard by preparative high performance liquid chromatography].

A novel and effective method was established for the qualitative analysis of impurities in auramine O samples of illegal food additives using high performance liquid chromatography-ion trap-time of flight mass spectrometry (HPLC-IT-TOF-MS). An impurity was identified in the auramine O sample using the optimized HPLC-IT-TOF-MS method. According to the exact mass of each fragment ion measured by multistage MS, the structure of the impurity was determined to be that of 4-(imino (4-(methylamino) phenyl) methyl)-N,N-dimethylaniline hydrochloride. The synthetic route of the auramine O and the source of the identified impurity were proposed. Simultaneously, a preparative high performance liquid chromatography (prep-HPLC) technique was successfully applied for the purification of the auramine O from complex samples. Prep-HPLC columns with particle sizes of 10 µm and 5 µm were used for the separation and purification with injection volumes of 1 mL and 500 µL, respectively. Finally an auramine O reference standard with 99.52% purity was obtained by secondarily purification and determined by the analytical HPLC area normalization method. Deducting the 0.34% moisture content and 0.13% ash content, the final purity of the sample was 99.05%, as determined by mass balance method. The chemical structure was examined using UV, IR, LC-MS, and NMR. The developed method is simple and efficient, and can be applied for the preparation of reference standard materials for other illegal food additives.

[Isolation and preparation of polyphenols from grape seed by high-speed counter-current chromatography combined with preparative high performance liquid chromatography].

Eight polyphenols were separated from grape seed by high-speed counter-current chromatography (HSCCC). The separation was performed using a two-phase solvent system composed of 1-butanol-ethyl-water (1:14:15, v/v/v) and n-hexane-ethyl-water (1:10:10, v/v/v). The upper and lower phases were used as the stationary and mobile phases, respectively. A flow rate of 2.0 mL/min was employed for the separation. The apparatus was rotated at 900 r/min, and the detection wavelength was set at 280 nm. Under the selected conditions, procyanidins B1, procyanidins B2, gallic acid, epicatechin gallate, and catechin were obtained from the grape seed extracts with a purity of 98.5%, 97.2%, 98.3%, 98.9%, and 96.7%, respectively. Epicatechin, epicatechin gallate, and gallocatechin gallate were obtained by preparative high performance liquid chromatography with the purity of 99.2%, 99.3%, and 99.2%, respectively. The proposed method is simple and shows high separation efficiency, and will be of great importance for the comprehensive utilization of grape seed.

An efficient prodrug-based nanoscale delivery platform constructed by water soluble eight-arm-polyethylene glycol-diosgenin conjugate.

Drug resistance in tumors is one of the reasons result in the low anticancer efficiency of numerous drugs. Combination therapy has been proven to be a valid way against drug-resistant cancers. However, simply mix the drugs will not only cause many side efforts but also decrease anticancer effect. Herein, a self-assembled nanoparticle platform based on eight-arm-polyethylene glycol-diosgenin (8armPEG-DGN) conjugate was produced for encapsulating another hydrophobic anticancer drug. The 8armPEG-DGN/HCPT NPs were prepared through a simple nanoprecipitation method. The 8armPEG-DGN/HCPT NPs possess suitable size (~107 nm) and high binary drug loading capacity (15.67 wt% of DGN and 14.72 wt% of HCPT). Laser confocal scanning microscopy revealed that 8armPEG-DGN/HCPT NPs significantly increased intracellular uptake toward B16 cells compared with free drugs. Cytotoxicity assay showed the IC50 of 8armPEG-DGN/HCPT NPs were lower than simply mixing DGN and HCPT. In vivo tumor transplantation assay indicated that 8armPEG-DGN/HCPT NPs exhibited superior tumor grown inhibition compared with free drugs and HCPT/DGN Mix. These studies showed that the prepared 8armPEG-DGN/HCPT NPs drug delivery system could serve as a promising candidate for cancer therapy.

pH-Sensitive nanogels based on the electrostatic self-assembly of radionuclide 131I labeled albumin and carboxymethyl cellulose for synergistic combined chemo-radioisotope therapy of cancer.

Development of biocompatible and biodegradable nanocarriers with multiple functionalities has attracted great interest in recent years. In this study, a hybrid hydrogel nanoparticle (nanogel) platform based on the self-assembly of carboxymethyl cellulose (CMC) and bovine serum albumin (BSA) is presented for the first time. It was facile to realize the efficient co-delivery of radionuclide 131I and chemotherapeutic drugs such as camptothecin (CPT) to achieve the combined chemo-radioisotope therapy of cancer. Notably, a nanogel was prepared by a simple and green electrostatic interaction approach, instead of chemical reaction, showing typical spherical shape with average size about 120 nm, high drug loading capacity, robust stability and low hemolysis. Interestingly, such nanogels exhibited pH-dependent drug release profile, leading to significant reduction of damage to normal tissues. Furthermore, the as-prepared nanogels could effectively promote intracellular uptake, prolong blood circulation time and enhance accumulation in the tumor tissues. As a result, an excellent therapeutic effect was achieved both in vitro and in vivo through combined chemo-radioisotope therapy. Collectively, this study presents the preparation of a novel green nanocarrier by a reliable and simple approach, and offers an effective strategy for the combination of chemotherapy and radiotherapy.

Synthesis of fluorescent ionic liquid-functionalized silicon nanoparticles with tunable amphiphilicity and selective determination of Hg2.

Fluorescent silicon nanoparticles have attracted much attention in recent years due to their superior optical properties, strong photostability, low toxicity, and favorable biocompatibility. In this paper, we have prepared imidazolium ionic liquid-functionalized silicon nanoparticles (IL@SiNPs) via a simple one-pot hydrothermal route from 1-(trimethoxysilyl)propyl-3-methylimidazolium chloride that was denoted as [SmIm]Cl and sodium citrate. The synthetic IL@SiNPs could be a novel label-free sensing probe for sensitive and selective detection of Hg2+ ions with energy transfer from IL@SiNPs to Hg2+. A good linear relationship was obtained from 0 to 40 µmol L-1 with a detection limit of 0.45 µM. Furthermore, the solubility of the IL@SiNPs can be facilely converted by anion exchange without complex chemical modification. The ionic liquid-modified SiNPs may show great promise in biological and environmental applications due to their excellent biocompatibility and optical performance.