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.

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.

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.

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.

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.

TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in <i>Stevia rebaudiana</i>Bertoni: An Important Medicinal Plant and a Natural Sweetener

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.

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.

甜菊甙类的提取和利用

甜菊甙类是原产于南美巴拉圭的菊科多年生草本植物——甜菊(Stevia rebaudianaBertoni)的茎叶中所含的具甜味的一类成分,其主要成分为甜菊甙(stevioside)和莱鲍迪甙 A(rebaudioside A)。甜菊在原产地称为甜草,作甜味料使用。

Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i>Bert. Using ISSR Marker

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of? fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

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.