Lutein-stevioside nanoparticle attenuates H_(2)O_(2)-induced oxidative damage in ARPE cells

In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.

Evaluation of Uncertainty for Determination of Stevioside Content in Fermented Milk by HPLC

[Objectives]The paper was to establish an evaluation method for the uncertainty of stevioside(including stevioside,rebaudioside A,rebaudioside B,rebaudioside C,rebaudioside F,Dulcoside A,rubusoside and steviolbioside)content determination in fermented milk based on HPLC.[Methods]The mathematical model of stevioside content and the propagation rate of uncertainty were established,and the sources of uncertainty were analyzed.[Results]The uncertainty mainly came from four main aspects,including standard uncertainty u(C)introduced by solution concentration C,standard uncertainty u(V)introduced by sample volume V,standard uncertainty u(m)introduced by sample mass m weighing and standard uncertainty u(f_(rep))introduced by measurement repeatability of stevioside content after sample dissolution and constant volume.The uncertainty estimation table and fishbone chart of stevioside content X determination were established.The relative synthetic standard uncertainty of stevioside content was obtained,and the standard uncertainty was extended to form the measurement result of stevioside content and its uncertainty report.[Conclusions]The evaluation results can be directly applied to the daily practical detection work.

Improvement of the Assay Method for Steviol Glycosides in the JECFA Specifications

Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.

高效液相色谱法测定甜叶菊中Stevioside和Rebaudioside A的含量

建立高效液相色谱法测定甜叶菊中甜菊甙（Stevioside）和莱鲍迪甙A（Rebaudioside A）的含量。色谱条件：色谱柱为Agilent ZORBAX NH2 4．6×250mm 5μm；流动相为乙腈-pH3．0磷酸溶液（80：20）；流速1．0mL·min^-1；检测波长210nm；柱温40℃；进样量20μL。线性范围为0．1268～2．0456mg·mL^-1（r=1），加标平均回收率为98．16％，RSD为0．80％（n=6）。本方法准确度高、精密度高、重复性好、简捷易操作，可以作为甜叶菊中甜菊甙和莱鲍迪甙A含量的测定方法。

<i>In Vitro</i>Evaluation of Some Traits in <i>Stevia rebaudiana</i>(Bertoni) under Drought Stress and Their Relationship on Stevioside Content

The experiment was conducted to evaluate the differences of plant growth as well as sweetener material of two types of Stevia rebaudiana under conditions of absence and presence of drought stress. In vitro, the experiment was conducted as drought traits using four levels of polyethylene glycol (0.0, 10,000, 20,000, 30,000 ppm). It was observed that survival % and No. of shoots should be considered as the important factors associated with stevioside content in stevia plant. According to path coefficient analysis and positive direct effect of studied traits, survival % recorded the highest value (0.5386), whereas No. of shoots recorded the highest value (– 0.8827) as negative direct effect in related to stevioside content. The results showed that all traits were affected, especially under 30,000 ppm level of polyethylene glycol. Generally, the most of studies traits exhibited a recorded clearly difference between the two types and used drought levels on stevia plants. Therefore, selection based on survival % would be more effective to improving stevioside content of stevia plants in drought stress conditions.

Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of <i>Stevia rebaudiana</i>Bertoni

Stevioside, extracted from the leaves of Stevia rebaudiana Bertoni, is a natural, high intensity, lowcaloric sweetener with wide therapeutic activities. Conventional stevioside extraction methodologies involve the use of non-green solvents, supercritical fluids, microwaves, etc., however, all these processes are expensive, time-consuming and eco-unfriendly. Therefore an alternative process is desired for the isolation of stevioside. In this study, a novel enzyme-mediated extraction (EME) method has been developed. The dry stevia leaves were pre-treated with hydrolytic enzymes aided by transition metal salts (FeCl3). This was followed by pressurized hot water extraction (PHWE) to release stevioside. The crude extract was purified and clarified through multi-stage membrane filtration. The results confirm that metal salt-assisted cellulase pre-treatment enhanced the yield of stevioside to 72%, with 98% purity, which was higher in comparison to existing methods. Thus, the methodology developed establishes a simple, “green”, enzyme-mediated process for the efficient isolation of stevioside under economical and eco-friendly conditions.

The Extraction and Purification of Stevioside from Stevia

tevioside is a natural sweetener extracted from stevia,which is widely used in the food industry and the pharmacy.Centre on the extraction and purification of stevioside,using ADS-7 resin to extract stevioside through one single step,greatly shorten the process and solve the problems of lower yield and higher raw materials cost existed in the general process.

Determination of Rebaudioside A and Stevioside in Leaves of S.rebaudiana Bertoni Grown in Mexico by a Validated HPLC Method

Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Stevia, although an HPLC method is recommended by the FAO/WHO-JECFA. Varieties of Stevia have been recently grown in Mexico, with no previous report of glycosides by a validated method. The aim of this study was to validate an isocratic HPLC method for content determination of main glycosides in the leaves of Stevia cultivated in Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 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. Rebaudioside A and stevioside were determined in two Stevia varieties: Morita II and Criolla, and also validation parameters were calculated. Rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003;in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). The recoveries of fortified samples were 100% ± 10% and precision RSD was ≤6.27%. The criteria of validation showed accuracy, linearity (≥0.99), and precision;therefore, the determination of glycosides was performed with reliability.

Potassium deficiency inhibits steviol glycosides synthesis by limiting leaf sugar metabolism in stevia(Stevia rebaudiana Bertoni)plants

The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.

Simple extraction and membrane purification process in isolation of steviosides with improved organoleptic activity

In recent years there has been increasing demand for natural non-nutritive high intensity sweeteners with low-calorie value as an alternative to sucrose. Extracts of the leaves of Stevia rebaudiana (Bertoni), have been known for their sweet taste. Steviosides and rebaudioside-A are the two major diterpenoid glucosides components present in the leaf extracts of the stevia, these glycosides are 300 times sweeter than sugar and also exhibits wide therapeutic activity. The conventional methods of isolation of steviosides involve long extraction and purification procedures;therefore optimization of product yields is a challenging problem. The present study, establishes a new improvised process of extraction of steviosides from the stevia leaves in which the dry treated leaves were grounded, defatted, and extracted through pressurized hot water extractor (PHWE), followed by purification and concentration of the sweet glycosides through ultra (UF) and nano (NF) membrane filtration in obtaining high (98.2%) purity steviosides. This process established “green” method for isolation of high quality steviol glycosides, with improved final yield is 10.1% from 11% of crude leaf extract and observed the improved organoleptic and biological activity (antioxidant). Thus the method confirms a simple, inexpensive and eco-friendly process in obtaining pure steviosides.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method

The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.

Maternal stevioside supplementation improves intestinal immune function of chicken offspring potentially via modulating gut microbiota and down-regulating the promoter methylation level of suppressor of cytokine signaling 1(SOCS1)

The intestinal immune function of chickens is limited during the early growing stage.Maternal nutritional intervention has been suggested to affect the innate immunity of offspring.The present study aimed to investigate the effects of maternal stevioside supplementation on the intestinal immune function of chicken offspring.A total of 120 Jinmao yellow-feathered breeder hens were fed a basal diet or a diet supplemented with 250 mg/kg stevioside for 5 weeks.During the last week,200 breeding eggs from each group were collected for incubation.After hatching,80 male offspring(40 chickens from each group)were randomly selected and fed the same basal diet for 28 d.In addition,90 well-shaped fertile eggs of non-treated breeder hens were incubated for the in ovo injection experiment.Steviol dissolved in 20%glycerol was injected at 7 d of incubation.The results showed that maternal stevioside supplementation could improve embryonic development,jejunal integrity and proliferation in the jejunal crypt(P<0.05).Maternal stevioside supplementation could also increase the innate transcription levels of cytokines and endotoxin tolerance-related factors in the jejunum of chicken offspring(P<0.05).At 28 d of age,the offspring following maternal stevioside supplementation exhibited higher jejunal secretory immunoglobulin A and serum interferons levels(P<0.05).A higher abundance of Lactobacillales induced by maternal stevioside supplementation was positively correlated with intestinal immunerelated factors(P<0.05).The in ovo injection with steviol did not alter either embryonic development or intestinal immune function of hatching chickens(P>0.05).Furthermore,maternal stevioside supplementation could induce hypo-methylation on the promoter region of suppressor of cytokine signaling 1(SOCS1).In conclusion,maternal stevioside supplementation could improve the intestinal immune function of chicken offspring potentially via modulating the gut microbiota and downregulating the promoter methylation level of SOCS1.

甜菊糖苷的生物学功能及其在畜禽生产中的应用研究进展

甜菊糖苷是一类提取于甜叶菊的四环二萜类糖苷物质,被认为是一种天然、安全、低热量、高甜度的甜味剂。因其具有良好的抗氧化、抗炎、免疫调节及促生长的作用,可作为新型饲料添加剂应用于畜禽生产。本文主要综述了甜菊糖苷的化学结构、提取方法以及味觉评价、甜菊糖苷在机体的水解代谢过程、甜菊糖苷的生物学功能及其在畜禽生产中的相关应用进展,以期为甜菊糖苷在畜禽生产中的合理开发和应用提供参考。

THE STRUCTURE MODIFICATION OF STEVIOSIDE

I. INTRODUCTIONBecause most of the synthetic sweetners are toxic, people have turned to search natural substances and its anologs for nonnutritive sweetners.Stevioside is a natural substance extracted from a plant named Servia rebaudlana bertoni. Since it has potential dangert and is bitter after taste, people have tried to modify its structure to obtain safe, high-sweetness quality, nonnutritive sweetners.

SYNTHESIS OF THE POLYSTYRENE-TYPE ADSORBENTS CONTAINING POLAR GROUPS AND THEIR CAPACITIES IN THE ADSORPTION OF STEVIOSIDE AND GYPENOSIDES

Seven polystyrene-type adsorbents (Ⅰ—Ⅶ) were synthesized by the modification of styrene-divinylbenzene copolymer respectively with hydroxymethyl, aminomethyl, methoxymethyl, phenoxymethyl, (4-hydroxy phenoxy)methyl, (4-acetylphenoxy)methyl, and acetyl groups. Their structures were characterized with chemical methods and physical techniques. The adsorption capacities of the adsorbents to stevioside and gypenosides were determined via batch test in which the HPLC method was used for the analysis of Saponin concentration of solution. The results showed that all the adsorbents could adsorb these saponins to a certain extent, but Ⅵ and Ⅶ had large capacities owing to their moderate polarities.

青贮甜菊叶内甜菊糖苷提取行为研究

【目的】为促进甜菊叶收储方式革新,打造低成本甜菊糖原料叶生产体系。【方法】本研究对不同青贮时长甜菊叶内甜菊糖苷提取行为进行分析。【结果】当青贮时间为3、7、15、21、30、60 d,甜菊叶所含各甜菊糖苷的常温水浸泡压榨提取效率随青贮时间延长逐渐增高,提取后各甜菊糖苷残余量随青贮时间延长而下降;70℃水浴浸泡提取4次后,日晒干叶中甜菊苷(Stevioside, STV)和瑞鲍迪苷(Rebaudiosides)系列(RA、RF、RC和RB等)甜菊糖苷残余量为2.7%~6.3%,青贮6月后直接浸提的各甜菊糖苷残余量为8.2%~13.3%,青贮6月烘干后再浸提的各甜菊糖苷残余量为12.2%~30.8%。【结论】本文除对影响青贮甜菊叶内各甜菊糖苷提取效率因素进行了探讨外,还基于研究结果对甜菊湿法收贮必要性和可行性进行了分析,为建立甜菊低成本收储方式提供参考。

Steviol Glycoside Content Dynamics during the Growth Cycle of Stevia rebaudiana Bert

The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 - 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g&middot;m-2 was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.

甜叶菊主要功能性成分研究进展

甜叶菊是一种重要的中草药、新型糖料作物和具有极高价值的经济作物。其富含糖苷类化合物、黄酮类化合物、绿原酸类化合物和其他多种功能性成分,具有抗氧化、抗菌、抗癌症、降血压、降血脂、降血糖、防龋齿等多种药理活性,日益受到人们的关注,目前在全球范围内广泛应用于食品、药品、保健品和化妆品等多个领域,具有极高的经济价值和广阔的市场前景。本文对甜叶菊主要功能性成分糖苷类化合物、黄酮类化合物和绿原酸类化合物的组成、检测、提取、分离纯化、功能活性、开发利用等方面进行了综述,并对其目前存在的问题和发展前景进行了展望,以期为我国甜叶菊资源的进一步开发、研究和利用提供一定的参考和借鉴。

基于碳水化合物分子量的植物材料产地溯源体系初探——以甜叶菊叶为例

为探讨利用植物材料所含碳水化合物分子量信息进行产地溯源可行性。以甜叶菊叶为例,基于两点发现:(1)甜菊苷(STV)为各化学型甜叶菊叶内稳定存在的化合物之一;(2)各材料依某一化合物分子量大小的排序结论与同时依多组分的排序结论一致,以STV分子离子M/Z检出值信息为参照,分析收获时期、收贮方式、品系、施肥及产地等因素对碳水化合物分子量影响。结果表明:甜叶菊叶含STV分子离子负离子M/Z检出值波动幅度受产区影响明显,受收获时期、收贮方式、品系、施肥方式等影响小。相对于品系、田间施肥和收贮方式等引致的碳(δ^(13)C)同位素分馏,生产用水中氘(δD)、氧(δ^(18)O)相对含量对材料所含碳水化合物分子量影响大,因受雨水和灌溉水等影响具有区域特异性,故可利用所含碳水化合物分子量作为植物材料产地溯源标志。本研究为植物材料产地溯源提供了一种相对简捷有效的新方法。