重结晶法分离精制莱鲍迪甙A的研究

用甲醇、异丙醇和水不同比例的混合溶剂对甜叶菊糖以及分离出甜菊甙的母液浓缩物分别进行重结晶研究，考察了甜菊甙和莱鲍迪甙A的结晶分离及菜鲍迪甙A结晶纯化的最佳条件，得到了高纯度的莱鲍迪甙A，并对其进行了熔点测定和HPLC表征，为莱鲍迪甙A的分离与纯化提供了一种简便的方法。     

含酮基吸附剂对莱鲍迪甙A的吸附选择性研究

合成了一系列具有不同骨架结构的中极性含酮基大孔吸附树脂，研究了树脂极性与骨架结构的关系，讨论了树脂对甜菊糖中两种主要糖甙甜菊甙及莱鲍迪甙Ａ的吸附能力和吸附选择性，并设计通过树脂柱的动态色层法从甜菊甙含量高的甜菊糖中成功地分离出高莱鲍迪甙Ａ糖产品．     

Selectivity of polymer adsorbent in adsorptive separations of stevia diterpene glycosides

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Anti-Quorum Sensing Activity of Stevia Extract,Stevioside, Rebaudioside A and Their Aglycon Steviol

Governments are creating regulations for consumers to reduce their sugar intake, prompting companies to increase the ratio of artificial sweeteners in their products. However, there is evidence of some deleterious effects ascribed to the aforementioned synthetic agents and therefore consumers and food manufacturers have turned their attention to natural dietary sweeteners, such as stevia, to meet their sweetening needs. Stevia is generally considered safe; however, emerging scientific evidence has implicated the agent in gut microbial imbalance. In general, regulation of microbial behavior is known to depend highly on signaling molecules via quorum sensing (QS) pathways. This is also true for the gut microbial community. We, therefore, evaluated the possible role of these stevia-based natural sweeteners on this bacterial communication pathway. The use of a commercial stevia herbal supplement resulted in an inhibitory effect on bacterial communication, with no observable bactericidal effect. Purified stevia extracts, including stevioside, rebaudioside A (Reb A), and steviol revealed a molecular interaction, and possible interruption of Gram-negative bacterial communication, via either the LasR or RhlR receptor. Our in-silico analyses suggest a competitive-type inhibitory role for steviol, while Reb A and stevioside are likely to inhibit LasR-mediated QS in a non-competitive manner. These results suggest the need for further safety studies on the agents.     

Salt Stress Mitigation via the Foliar Application of Chitosan-Functionalized Selenium and Anatase Titanium Dioxide Nanoparticles in Stevia (Stevia rebaudiana Bertoni)

High salt levels are one of the significant and major limiting factors on crop yield and productivity. Out of the available attempts made against high salt levels, engineered nanoparticles (NPs) have been widely employed and considered as effective strategies in this regard. Of these NPs, titanium dioxide nanoparticles (TiO₂ NPs) and selenium functionalized using chitosan nanoparticles (Cs–Se NPs) were applied for a quite number of plants, but their potential roles for alleviating the adverse effects of salinity on stevia remains unclear. Stevia (Stevia rebaudiana Bertoni) is one of the reputed medicinal plants due to their diterpenoid steviol glycosides (stevioside and rebaudioside A). For this reason, the current study was designed to investigate the potential of TiO₂ NPs (0, 100 and 200 mg L⁻¹) and Cs–Se NPs (0, 10 and 20 mg L⁻¹) to alleviate salt stress (0, 50 and 100 mM NaCl) in stevia. The findings of the study revealed that salinity decreased the growth and photosynthetic traits but resulted in substantial cell damage through increasing H₂O₂ and MDA content, as well as electrolyte leakage (EL). However, the application of TiO₂ NPs (100 mg L⁻¹) and Cs–Se NPs (20 mg L⁻¹) increased the growth, photosynthetic performance and activity of antioxidant enzymes, and decreased the contents of H₂O₂, MDA and EL under the saline conditions. In addition to the enhanced growth and physiological performance of the plant, the essential oil content was also increased with the treatments of TiO₂ (100 mg L⁻¹) and Cs–Se NPs (20 mg L⁻¹). In addition, the tested NPs treatments increased the concentration of stevioside (in the non-saline condition and under salinity stress) and rebaudioside A (under the salinity conditions) in stevia plants. Overall, the current findings suggest that especially 100 mg L⁻¹ TiO₂ NPs and 20 mg L⁻¹ Cs–Se could be considered as promising agents in combating high levels of salinity in the case of stevia.     

Health-Promoting Compounds in Stevia: The Effect of Mycorrhizal Symbiosis,Phosphorus Supply and Harvest Time

This work aimed to establish the synergic role of arbuscular mycorrhizal fungi (AMF) symbiosis, phosphorus (P) fertilization and harvest time on the contents of stevia secondary metabolites. Consequently, steviol glycosides (SVglys) concentration and profile, total phenols and flavonoids as well as antioxidant assays, have been assessed in inoculated and no-inoculated plants, grown with or without P supply and collected at different growth stages(69, 89 and 123 days after transplanting).The obtained results suggest that the synthesis of stevia secondary metabolites is induced and/or modulated by all the investigated variability factors. In particular, AMF symbiosis promoted total SVglys content and positively influenced the concentration of some minor compounds (steviolbioside, dulcoside A and rebaudioside B), indicating a clear effect of mycorrhizal inoculation on SVglys biosynthetic pathway. Interestingly, only the mycorrhizal plants were able to synthesize rebaudioside B. In addition, P supply provided the highest levels of total phenols and flavonoids at leaf level, together with the maximum in vitro antioxidant activities (FRAP and ORAC). Finally, the harvest time carried out during the full vegetative phase enhanced the entire composition of the phytocomplex (steviolbioside, dulcoside A, stevioside, rebaudioside A, B, C. total phenols and flavonoids). Moreover, polyphenols and SVglys appeared to be the main contributors to the in vitro antioxidant capacity, while only total phenols mostly contributed to the cellular antioxidant activity (CAA). These findings provide original information about the role played by AMF in association with P supply, in modulating the accumulation of bioactive compounds during stevia growth. At the cultivation level, the control of these preharvest factors, together with the most appropriate harvest time, can be used as tools for improving the nutraceutical value of raw material, with particular attention to its exploitation as functional ingredient for food and dietary supplements and cosmetics.     

Sweeteners determination in table top formulations using FT-Raman spectrometry and chemometric analysis

A partial least squares (PLS) Fourier transform Raman spectrometry procedure based on the measurement of solid samples contained inside standard glass vials, has been developed for direct and reagent-free determination of sodium saccharin and sodium cyclamate in table top sweeteners. A classical 2² design for standards was used for calibration, but this system provides accuracy errors higher than 13% w/w for the analysis of samples containing glucose monohydrate. So, an extended model incorporating glucose monohydrate (2³ standards) was assayed for the determination of sodium saccharin and sodium cyclamate in all the samples. Mean centering spectra data pre-treatment has been employed to eliminate common spectral information and root mean square error of calibration (RMSEC) of 0.0064 and 0.0596 was obtained for sodium saccharin and sodium cyclamate, respectively. A mean accuracy error of the order of 1.1 and 1.9% w/w was achieved for sodium saccharin and sodium cyclamate, in the validation of the method using actual table top samples, being lower than those obtained using an external monoparametric calibration. FT-Raman provides a fast alternative to the chromatographic method for the determination of the sweeteners with a three times higher sampling throughput than that obtained in HPLC. On the other hand, FT-Raman offers an environmentally friendly methodology which eliminates the use of solvents. Furthermore, the stability of samples and standards into chromatographic standard glass vials allows their storage for future analysis thus avoiding completely the waste generation.     

Measurement of Stevioside by Square‐Wave Polarography

Steviol glycosides are electroactive compounds and can be detected by mercury electrode. The maxima of square‐wave polarograms of steviol and stevioside appear at ?1.3 V vs. Ag/AgCl in 1 M NaCl at approximately pH 11. The responses are quasireversible and probably caused by electroreduction of carboxyl, or ester groups, respectively. The net peak current is linearly proportional to steviol concentration between 1×10^?6 mol/L and 3.5×10^?5 mol/L. This calibration plot can be used for the determination of the concentration of stevioside in extracts of stevia (Stevia rebaudiana Bertoni) leaves. The proposed method is applicable for routine analysis of stevioside in commercial products.     

THE SYNTHESIS AND CHARACTERIZATION OF SOME POROUS RESINS AND THEIR ADSORPTION PROPERTY ON STEVIA GLYCOSIDES

A series of porous resins (PYR) with different content of pyridyl group were prepared,and their physical structure and adsorption properties on the stevia glycosides were studied in detail.After the selective adsorption of PYR resins,the rebaudioside A could be separated from the other components of the stevia glycosides,and a new method for obtaining pure rebaudioside A is reported.     

Steviosid aus Süßkraut

The diterpene glycoside stevioside is the most abundant among a group of sweet steviol glycosides, present in sweetleaf (Stevia rebaudiana), which is naturally occurring in Paraguay. It has already been used by the Guarani Indians as a sweetener. Since 2011 such “steviosides” have an EU approval as sweeteners E 960. They taste ca. 250 times sweeter than sucrose. Meanwhile, they have got a significant recognition, not least due to their use in Coca Cola life®. We describe the isolation of stevioside and rebaudioside A from dried leaves of the sweetleaf plant. The complete set of spectra for stevioside is reported. Based on students' laboratory work this project is a follow up of the book “Classics in Spectroscopy” by S. Berger und D. Sicker (Wiley‐VCH 2009).     

Synthetic study on the relationship between structure and sweet taste properties of steviol glycosides

The structure activity relationship between the C??-C?? methylene double bond on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has been reported here for the first time. It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C??-C?? methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.     

THE SYNTHESIS AND CHARACTERIZATION OFSOME POROUS RESINS AND THEIR ADSORPTIONPROPERTY ON STEVIA GLYCOSIDES

1.INTRODUCTIONFromtheleavesofSteviaRebaudianaBertoni,asweetherbindigenoustoelevatedterrainonthebordersofBrazilandParaguay,eightsteviaglycosideshasbeenisolatedinayieldof10~15%andutilizedasahigh-sweetness,low-calorie,nonnutritionsweetener[1-3].Stevioside,rebaudiosideAandrebaudiosideCarepresentinthelargestquantitiesoftheeightsteviaglycosides.Amongthesethreeglycosides,rebaudiosideAisknownasthebestsweetenerwithrespecttotheintensityofsweetnessaswellasthequalityoftaSte,whiletheothertwomajorsw…     

Methods for extracting biochemical information from bacterial Raman spectra: an explorative study on Cupriavidus metallidurans

In this study, we explore Raman spectra of bacteria for their biochemical information. Therefore, a database of biomolecules was used and several approaches were applied such as the study of difference spectra, the calculation dot products, the usage of coefficients obtained from an EMSC procedure and the application of 2D correlation spectroscopy. These methods were applied on a dataset containing Raman spectra of Cupriavidus metallidurans LMG 1195 in five stages of its growth, aiming to extract information about the evolution of cell components during growth. EMSC coefficients seemed to be most promising for tracking metabolic products and the results were often confirmed by difference spectra or by 2D correlation spectroscopy.     

Enrichment and separation of rebaudioside A from stevia glycosides by a novel adsorbent with pyridyl group

The adsorptive capacity and selectivity of a novel adsorbent with pyridyl group toward stevia glycosides were studied. The effect of polarity and physical structure of the sorbent on the selectivity was investigated in detail. Two separation methods were applied in the enrichment of rebaudioside A(RA). They were selective elution using methanol or ethanol solution as solvent, and dynamic chromatographic separation using pyridyl resin with high selectivity. Results show that the chromatographic separation method can effectively enrich RA from stevia glycoside with high content of stevioside.     

Ultraviolet resonance Raman spectroscopy as a robust spectroscopic tool for in situ sunscreen analysis

Current techniques being used for sunscreen analysis are incapable of direct determination of the active ingredients in sunscreen formulations. Therefore, the development of methodologies for rapid in situ analysis of sunscreens is desirable. This paper describes the application of ultraviolet resonance Raman spectroscopy (UVRRS) to the direct in situ analysis of sunscreen formulations. High-quality UV resonance Raman spectra were obtained for five sunscreen active ingredients (AIs), mixtures of the AIs and real sunscreen formulation samples. The spectra from the sunscreen formulations gave distinct spectral signatures indicative of the sunscreen AIs in each sample, with essentially no interference from the complex sunscreen matrix. Also, despite the fact that many of the AIs are fluorescent, no fluorescence interferences in the resonance Raman spectra were observed. Excitation wavelength-dependent studies throughout the 244-275 nm region demonstrate that the best discrimination of the AIs was achieved at an excitation wavelength of 244 nm. Thus, by tuning the excitation wavelength within the absorption bands of the AIs, complete identification of these analytes can be achieved in situ without any sample pretreatment or separation. The limit of detection found for a common AI in situ with this technique is 0.23% (w/w), the limit of quantitation is 0.78% (w/w), while the dynamic range is between 0.8% and 50% (w/w). The technique is fast, robust, lacks any major interference, and can be adapted for routine online quality control.     

Monitoring stevioside in soju by high-performance liquid chromatography and liquid chromatography/mass spectrometry

A method using high-performance liquid chromatography (HPLC) with UV absorption detection was developed to monitor stevioside in soju, a distilled spirits product that is commercially available. The method uses a single-step dilution for sample preparation. It completely eliminates the time-consuming process of solid-phase extraction. A method using HPLC/mass spectrometry was optimized to confirm the identities of stevioside and other related impurities, including rebaudioside A, rebaudioside C, and dulcoside. The method was validated. The validation parameters included range (10.1-1007.3 ppm), precision, linearity, accuracy, robustness, system suitability, and intermediate precision. Stevioside standard solutions at 6 concentration levels were prepared for the validation work, including the tests for precision, linearity, and accuracy. The solutions were prepared in triplicate for each concentration. The relative standard deviation for the precision test was <3% for all 6 concentration levels. The correlation coefficient for the linearity within the concentration range was determined to be > 0.999. The average recovery ranged from 95.7 to 101.1% for the soju samples spiked with stevioside standard. The detection limit for stevioside was estimated at 75 ppb. The method was used to screen several soju samples; no detectable stevioside was found in the samples.     

Large-scale profiling of diterpenoid glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem mass spectrometry

The plant Stevia rebaudiana accumulates a suite of diterpenoid metabolites that are natural sweeteners finding increased use as sugar substitutes. To guide breeding of stevia plants that accumulate substances with desirable flavor in high yield, rapid and accurate methods are needed to profile these substances in plant populations. This report describes an 8-min ultrahigh performance liquid chromatography-tandem mass spectrometry method for separation and quantification of seven stevia glycosides including steviolbioside; stevioside; rebaudiosides A, B, and C; rubusoside; and dulcoside as well as aglycones steviol and isosteviol. This negative mode electrospray ionization/multiple reaction monitoring method yielded low limits of detection <1 ng/mL for steviol, 6 ng/mL for isosteviol, and <15 ng/mL for all stevia glycosides. Stevioside and Reb A, B, and C were quantified in more than 1,100 extracts from stevia leaves as part of a large-scale profiling exercise. Leaf tissue levels in this population spanned about two orders of magnitude for stevioside (2-125 mg/g dry weight), Reb A (2.5-164 mg/g), Reb B (0.5-50 mg/g), and Reb C (1.5-125 mg/g), but levels of individual metabolites exhibited independent variation. The wide spread of metabolite levels highlights the utility and importance of performing targeted metabolic profiling for large plant populations.     

The potential of Raman spectroscopy for characterisation of the fatty acid unsaturation of salmon

Raman spectroscopy has been evaluated for characterisation of the degree of fatty acid unsaturation (iodine value) of salmon (Salmo salar). The Norwegian Quality Cuts from 50 salmon samples were obtained, and the samples provided an iodine value range of 147.8-170.0 g I₂/100 g fat, reflecting a normal variation of farmed salmon. Raman measurements were performed both on different spots of the intact salmon muscle, on ground salmon samples as well as on oil extracts, and partial least squares regression (PLSR) was utilised for calibration. The oil spectra provided better iodine value predictions than the other data sets, and a correlation coefficient of 0.87 with a root mean square error of cross-validation of 2.5 g I₂/100 g fat was achieved using only one PLSR component. The ground samples provided comparable results, but at least two PLSR components were needed. Higher prediction errors were obtained from Raman spectra of intact salmon muscle, and this may partly be explained by sampling uncertainties in the relation between Raman measurements and reference analysis. All PLSR models obtained were based on chemically sound regression coefficients, and thus information regarding fatty acid unsaturation is readily available from Raman spectra even in systems with high contents of protein and water. The accuracy, the robustness and the low complexity of the PLSR models obtained suggest Raman spectroscopy as a promising method for rapid in-process control of the degree of unsaturation in salmon samples.     

Raman spectroscopic investigation of silver electrodes

Raman spectroscopy has been applied to the study of the reduction of carbon dioxide and of formate and carbonate ions at a silver electrode. Raman spectra of adsorbed intermediate species, which are as yet only partially identified, have been detected and show marked variations with electrode potential. These spectral variations are clearly correlated with the voltammetric features for carbonate solutions and suggest that these reduction products complicate most measurements on silver electrodes in the cathodic region. The interpretation of the previously reported spectra due to adsorbed pyridine at silver electrodes has been reconsidered; interactions with surface carboxy species may be significant.     

Detection and chemical profiling of medicine counterfeits by Raman spectroscopy and chemometrics

Raman spectroscopy combined with chemometrics has recently become a widespread technique for the analysis of pharmaceutical solid forms. The application presented in this paper is the investigation of counterfeit medicines. This increasingly serious issue involves networks that are an integral part of industrialized organized crime. Efficient analytical tools are consequently required to fight against it. Quick and reliable authentication means are needed to allow the deployment of measures from the company and the authorities. For this purpose a method in two steps has been implemented here. The first step enables the identification of pharmaceutical tablets and capsules and the detection of their counterfeits. A nonlinear classification method, the Support Vector Machines (SVM), is computed together with a correlation with the database and the detection of Active Pharmaceutical Ingredient (API) peaks in the suspect product. If a counterfeit is detected, the second step allows its chemical profiling among former counterfeits in a forensic intelligence perspective. For this second step a classification based on Principal Component Analysis (PCA) and correlation distance measurements is applied to the Raman spectra of the counterfeits.