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.     

Selectivity of polymer adsorbent in adsorptive separations of stevia diterpene glycosides

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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．     

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。     

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.     

吸附树脂的色层吸附及其在甜菊甙分离纯化中的应用

研究了ADS－7等吸附树脂对甜菊甙各组分的吸附选择性及色层吸附性能,发现ADS－7吸附树脂不仅在吸附甜菊甙时对各个组分呈现出一定的选择吸附顺序,在水洗时还有一定的选择洗脱顺序,这两个顺序正好相反。利用ADS－7吸对脂的这一色层性能,制备了富含甜度最高、味质最好的Rebaudioside A及几科不含色素的高品质甜菊甙,具有很好的实际应用意义。     

PYR树脂对甜菊糖的吸附与洗脱性能研究

系统研究了PYR型树脂对甜菊糖的吸附与洗脱性能，并与目前国内应用于甜菊糖提取分离的某些商品化吸附树脂进行了比较。结果表明，PYR树脂比其它树脂具有更优的性能，其静态吸附容量比AB—8树脂提高25％，且吸附速度快，易于洗脱再生，是一种具有工业化应用前景的新型吸附剂。     

Kinetics and thermodynamics of rebaudioside A adsorption on a strongly acidic cation exchange resin

To explain the mechanism underlying the adsorption of stevia's polar component rebaudioside A in hydrophilic interaction liquid chromatography mode, the characteristics of rebaudioside A adsorption on various resins in an organic‐solvent‐rich system were studied. Among the tested resins, the strongly acidic cation resin FPC11 showed the best adsorption behavior for rebaudioside A. The factors affecting the adsorption kinetics of the resin for rebaudioside A are discussed. The results showed that the pseudo‐second‐order reaction model and intra‐particle diffusion model best described the adsorption kinetics of rebaudioside A on the resin. The adsorption rate was controlled by physical sorption, mainly via electron sharing or electron transfer between the adsorbent and the adsorbate. The adsorption process with multiple stages involved weak initial adsorption behavior. Thermodynamic studies showed that the adsorption of rebaudioside A on the resin was not an ideal monolayer adsorption, but mutual adsorption effects between the adsorbates. The adsorption was a spontaneous, entropy‐increasing endothermic process. The synergistic effect of hydrogen bonding and ion–dipole was a possible driving force.     

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.     

季铵基吸附树脂的合成及其在甜菊式甙提取分离中的应用

研究了由工业二乙烯苯聚合得到的大孔树脂进行氯甲基化的可行性，考察了聚合物孔结构催化剂用量、反应温度和时间等因素对氯甲基化反应的影响，由氯甲基化树脂制备了强极性季铵基吸附剂。并研究了合成的季铵基吸附剂对甜叶菊提取物中甜菊甙及色素的吸附－脱附性能，发现该吸咐剂对甜菊甙与色素的吸附－脱附性能完全不同，因而可用该吸附剂制备高品质的甜菊式。     

Detection of counterfeit stevia products using a handheld Raman spectrometer

Stevia is a highly appreciated natural sweetener because it can be consumed by diabetic patients. Due to the increasing popularity of stevia during the last years, counterfeit products have been making their way into the market. Raman spectroscopy is a versatile analytical technique that can be used for control tasks and handheld modern devices expand its possible applications to instant in situ measurements. The Raman spectra of six commercial stevia products (five purchased in Bolivia and one in Germany) were recorded and compared to the spectra of standards of rebaudioside A and stevioside as well as the spectra of the artificial sweeteners sodium cyclamate and sodium saccharin. Based on the Raman spectroscopic data, it was verified that three of the Bolivian products were counterfeit products and another one was rich in maltodextrin. The Raman spectra of one Bolivian product and the German one revealed rebaudioside A and stevioside as major components. Raman spectroscopy was capable of detecting contents as low as 5% (w/w) of sodium cyclamate during measurements of stevia-sodium cyclamate mixtures. The results show that Raman spectroscopy can successfully be used to detect counterfeit stevia and underline its high potential for the detection of food adulteration.     

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.     

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.     

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.     

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.     

Development of a high‐performance liquid chromatography procedure to identify known and detect novel C‐13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W

As an aid for structure elucidation of new steviol glycosides, reversed‐phase C18 high‐performance liquid chromatography method was developed with several previously characterized diterpene glycosides, to identify known and detect novel aglycone‐C13 oligosaccharide moieties and indirectly identify C‐19 interlinkages. Elution order of several diterpene glycosides and their aglycone‐C13 oligosaccharide substituted with different sugar arrangements were also summarized. Comparison of the retention time of a product obtained after alkaline hydrolysis with the aglycone‐C‐13 portions of known compounds reported herein allowed us to deduce the exact positions of the sugars in the C‐13 oligosaccharide portion. The elution position of several steviol glycosides with an ent‐kaurene skeleton was helpful to describe an identification key. Two previously uncharacterized diterpene glycosides together with two known compounds were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13‐[(2‐O‐β‐d ‐xylopyranosyl‐ 3‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl) ester (rebaudioside V), whereas the other was determined to be 13‐[(2‐O‐β‐d ‐xylopyranosyl‐ 3‐O‐β‐d ‐glucopyranosyl‐β‐d‐ glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐α‐l ‐rhamnopyranosyl‐3‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl) ester (rebaudioside W). Previously reported compounds were isolated in gram quantities and identified as rebaudioside J and rebaudioside H. In addition, a C‐19 sugar‐free derivative was also prepared from rebaudioside H to afford rebaudioside H₁. Chemical structures were partially determined by the high‐performance liquid chromatography method and unambiguously characterized by using one‐dimensional and two‐dimensional nuclear magnetic resonance experiments.     

A silica gel orthogonal high-performance liquid chromatography method for the analyses of steviol glycosides: novel tetra-glucopyranosyl steviol

A silica gel orthogonal method using acetonitrile: water was developed for the analyses of fractions rich in very polar steviol glycosides and resolve regions of co-elution of these compounds in reversed-phase. Additionally, we also used this normal phase analytical method to scale up the purification process of steviol glycosides. Using these approaches, one novel minor tetra-glucopyranosyl diterpene glycosides together with three known compounds were purified from a commercial Stevia rebaudiana leaf extract. Compound 1 was unambiguously elucidated as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (rebaudioside Y) based on high-performance liquid chromatography retention times, tandem mass spectrometry dissociation pattern and 1D and 2D NMR experiments. Known compounds were isolated in gram quantities and identified as rebaudioside D, E and M.     

大孔吸附树脂结合酶解法分离纯化虎杖中白藜芦醇的研究

研究了大孔吸附树脂结合酶解法提取和纯化虎杖中白藜芦醇的方法,采用HPLC法测定虎杖中白藜芦醇的含量,考查了β-糖苷酶对虎杖药材酶解前后白藜芦醇含量的变化,并经静态吸附考察了4种树脂,最后确定以H1020作为提取分离白藜芦醇的树脂.此树脂吸附量较高,脱附容易,有利于得到质量较好的白藜芦醇产品,经该树脂吸附解吸,饱和吸附量可达51.4mg/g,解吸率达92.5%.大孔树脂分离纯化白藜芦醇的含量可达71.5%,而上柱前粗提物中白藜芦醇含量为8.71%,说明采用本法分离纯化虎杖中白藜芦醇是可行的.     

A Novel Application of Capillary Electrophoresis for Evaluating the Mutation Effects of Stevia Induced by Ion Implantation

Stevia rebaudiana is a member of the Compositae, native to Paraguay. It produces a number of high-potency low-carlorie sweeteners in its leaf tissue. The sweeteners are diterpene glycosides and range between 30 and 320 times sweeter than sugar. Therefore, Stevia is likely to become a major source of high-potency sweetener for the growing natural food market in the future. Generally, techniques of ion implantation have been used to treat plant seeds and induce mutation for increasing the produce of stevia sweeteners. Various methods for analysis products of stevia rebaudiana have been reported, including gas-liquid chromatography, thin-layer chromatography and HPLC etc. However, these methods are time consuming. The separation and determination of stevia sweeteners by capillary electrophoresis was successfully demonstrated recently^[1,2].     

Validation of HPLC‐UV method for determination of minor glycosides contained in Stevia rebaudiana Bertoni leaves

Leaves of Stevia rebaudiana contain glycosides with sweetness and biological activity. However besides the major glycosides, there are other glycosides within extracts that may contribute to its activity, and therefore it is important to quantify them. In this work, an isocratic HPLC method was validated for determination of dulcoside A, steviolbioside, rebaudioside C and rebaudioside B. An HPLC method was performed using a C₁₈ column (250 × 4.6 mm, particle size 5 µm) and a UV detector set at 210 nm. The mobile phase consisted of a 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The calibration curves were linear over the working range 25–150 µg/mL, with coefficient of correlation of ≥0.99 and coefficient of determination of ≥0.98. The LOD was 5.68–8.81 µg/mL, while the LOQ was 17.21–26.69 µg/mL. The percentage recoveries of fortified samples were 100 ± 10% and precision, relative standard deviation, was <10%. The method validation showed accuracy, linearity and precision; therefore this method can be applied for quantitative analysis of minor steviol glycosides in S. rebaudiana leaves. Copyright © 2014 John Wiley & Sons, Ltd.