Specific immunomodulatory and secretory activities of stevioside and steviol in intestinal cells

Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM) decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alpha-mediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alpha-induced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB --> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30 min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-) secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects only Cl(-) secretion.     

Photostability of rebaudioside A and stevioside in beverages

The Coca-Cola Company and Cargill, Inc. have initiated the development and commercialization of the Stevia rebaudiana (Bertoni) derived sweetener rebaudioside A. Efforts were focused on high purity rebaudioside A (>97% by HPLC), commonly known as rebiana. In the course of the development program, extensive stability studies were carried out on rebiana, all supporting good stability for use in all food and beverage applications, including conditions where rebiana-sweetened beverages were exposed to light. Our findings on rebiana light stability refute those of an earlier study that suggested rebaudioside A to be unstable to sunlight exposure, while the structurally homologous stevioside is stable. We replicated the earlier study and found no significant photodegradation for either rebaudioside A or stevioside.     

The leaves of Stevia rebaudiana (Bertoni), their constituents and the analyses thereof: a review

The plant Stevia rebaudiana is well-known due to the sweet-tasting ent-kaurene diterpenoid glycosides. Stevioside and rebaudioside A are the most abundant and best analyzed, but more than 30 additional steviol glycosides have been described in the scientific literature to date. Most of them were detected in the last two years. This paper reviews these new compounds and provides an overview about novel trends in their determination, separation, analysis, detection, and quantification. The detection and analysis of further constituents such as nonglycosidic diterpenes, flavonoids, chlorogenic acids, vitamins, nutrients, and miscellaneous minor compounds in the leaves of Stevia rebaudiana are reviewed as well. A critical review of the antioxidant capacity of Stevia leaves and its analysis is also included. These different aspects are discussed in consideration of the scientific literature of the last 10 years.     

Metabolism of stevioside by chickens

In intubation experiments (643-1168 mg per animal), most of the stevioside administered to chickens was recovered unchanged in the excreta, and only about 2% was converted into steviol. Neither stevioside nor steviol could be found in the blood. In chronic studies (667 mg of stevioside/kg of feed) with laying hens and meat-type chickens, no significant differences were found in feed uptake, weight gain, and feed conversion as the result of stevioside administration. The egg production and egg composition of laying hens were not influenced. Most of the stevioside taken up was found untransformed in the excreta, and about 21.5% or 7.3% was converted to steviol by meat-type chickens or laying hens, respectively. No stevioside or steviol could be detected in the blood or in the eggs of the different groups of animals. In anaerobic incubation experiments with chicken excreta, only a 20% conversion of stevioside into steviol was found. No harmful effects were observed in the chronic stevioside supplementation experiments nor in the intubation experiments in which very high stevioside doses were given.     

Effect of stevioside and steviol on the developing broiler embryos

At day 7 of incubation, fertile broiler eggs were injected with different amounts of stevioside and steviol of 0.08, 0.8, or 4 mg stevioside/egg and 0.025, 0.25, or 1.25 mg steviol/egg. At hatch (day 21) and 1 week later, not any influence of the different treatments could be found on embryonic mortality, body weight of the hatchlings, deformations (e.g., bone, beak, and head malformations, abnormal feathering, open vent), or abnormal development of the gonads. No stevioside or steviol could be detected in the blood of the hatchlings. The hatchlings developed normally. It is concluded that prenatal exposure to stevioside and steviol is not toxic for the chicken embryo.     

甜叶菊中9种甜菊醇糖苷积累与其生物合成关键基因表达量的相关性

为了研究甜叶菊中主要甜菊醇糖苷积累特点与其生物合成关键基因表达量的关系,本研究选取3个品种甜叶菊(普赛科3号、中山2号、同心)为试验材料,定期在幼苗期、营养生长期、花蕾期采收植株的上位3对叶片,采用液相色谱-质谱法(LC-MS)检测9种甜菊醇糖苷,即莱宝迪苷A、B、C、D、E、F(RA、RB、RC、RD、RE、RF)、...     

Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program searching for plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column (250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between 1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).     

Crop Productivity,Steviol Glycoside Yield,Nutrient Concentration and Uptake of Stevia rebaudiana Bert. under Mediterranean Field Conditions

Stevia rebaudiana Bertoni (Asteraceae Family), characterized by a high content of steviol glycosides (SG) in its leaves, represents an interesting source of non-sucrose and no-calorie sweeteners. Stevia, which is native to Paraguay, is a relatively new crop for the Mediterranean region, where it is grown as an annual or poly-annual crop. In order to assess the crop’s environmental requirements, productive performance and nutrient uptakes, a two-year field experiment was carried out in north-eastern Italy, where the species had never previously been cultivated. The aim was to evaluate the effects of important pre-harvest factors, such as cultivation site, harvest time and their reciprocal interaction, on productivity, leaf SG yield and nutrient requirements. Results showed that the timing of the harvest and the pedo-climatic conditions of the cultivation sites had a significant effect on the main biometric traits, total and leaf dry yield, SG production, macronutrient concentration and uptake.     

Productivity of two stevia varieties under reduced irrigation and fertilization inputs

A 3-year field study was conducted in central Greece to determine the productivity of two stevia [(Stevia rebaudiana (Bertoni) Bertoni] varieties (‘Morita’ and ‘Candy-stevia’) under normal and reduced irrigation (100% and 75% of the evapotranspiration) and fertilization [1:0.8:1.1 or 1:0.4:0.8 N:P:K ratio in the first year and only N fertilization (100% or 74% of the recommended rate) in the second and third years] inputs. Averaged across years, stevia cv. Morita achieved greater dry leaf yield (3.48 t ha^?1) than the cv. Candy-stevia (2.85 t ha^?1). Irrigation and fertilization inputs did not significantly affect stevia cv. Morita dry leaf and steviol glycosides (stevioside plus rebaudioside-A) yields; however, decreasing irrigation and fertilization caused slight reduction of cv. Candy-stevia yields. In cv. Morita leaves, the concentrations of stevioside and rebaudioside-A ranged from 5.97% to 7.78% and 3.73% to 4.79%, respectively, while the corresponding concentrations in cv. Candy-stevia leaves were 8.21–9.36% and 3.89–6.33%. Conclusively, both stevia varieties could achieve satisfactory dry leaf biomass and steviol glycosides yield, even when grown under reduced irrigation (at 75% of evapotranspiration) and reduced N fertilization (74% of the recommended rate). Thus, stevia could represent an alternative crop to tobacco in the Mediterranean conditions.     

Preparative separation and purification of rebaudioside a from steviol glycosides using mixed-mode macroporous adsorption resins

Preparative separation and purification of rebaudioside A from steviol glycosides using mixed-mode macroporous adsorption resins (MARs) were systematically investigated. Mixed-mode MARs were prepared by a physical blending method. By evaluation of the adsorption/desorption ratio and adsorption/desorption capacity of mixed-mode MARs with different proportions toward RA and ST, the mixed-mode MAR 18 was chosen as the optimum strategy. On the basis of the static tests, it was found that the experimental data fitted best to the pseudosecond-order kinetics and Temkin-Pyzhev isotherm. Furthermore, the dynamic adsorption/desorption experiments were performed on the mini column packed with mixed-mode MAR 18. After one run treatment, the purity of rebaudioside A in purified product increased from 40.77 to 60.53%, with a yield rate of 38.73% (W/W), and that in residual product decreased from 40.77 to 36.17%, with a recovery yield of 57.61% (W/W). The total recovery yield reached 96.34% (W/W). The results showed that this method could be utilized in large-scale production of rebaudioside A from steviol glycosides in industry.     

The response of Stevia (Stevia rebaudiana Bertoni) to nitrogen supply under greenhouse condition

Stevia (Stevia rebaudiana Bertoni) is a perennial plant producing some natural sweeteners. Stevia is considered as a new crop in some countries. This study was conducted to find the stevia response to nitrogen fertilizer supply. Different levels of nitrogen (0, 30, 60, 90, 120, and 150 kg/ha from urea source) were used in a greenhouse condition and then the stevia growth and metabolites were assessed under different availability of nitrogen. Results showed that the optimum growth of stevia was obtained by 60 kg/ha nitrogen and more nitrogen supply did not enhance the stevia growth. It was observed that the total steviol glycosides (SVglys) content of Stevia was significantly increased by nitrogen fertilizer application just up to 30 kg/ha, while it decreased by more rates of nitrogen fertilizer. Our result clearly showed that SVglys yield reached to maximum value by application of 60 kg/ha of nitrogen fertilizer. Since the variation of SVglys content and shoot growth of the stevia were compromised by 60 kg/ha nitrogen, it can be concluded that 60 kg/ha of nitrogen fertilizer could be considered as an optimum rate of nitrogen for stevia and could also be recommended for greenhouse conditions.     

The effect of different nitrogen levels on yield and quality of stevia (Stevia rebaudiana bert.)

In the present work, the efficiency of different nitrogen doses (0, 50, 100, 150, and 200 kg ha^?1) on growth, yield, and quality of stevia (Stevia rebaudiana Bert.) was investigated in 2011–2013. The study was conducted in Antalya located in the Mediterranean Region of Turkey. Terra rossa type soil (LVx, FAO) characteristics of the experimental field were clay loam, with high amounts of lime (33,9%) and slightly alkaline (pH 7.7). The experiment was carried out in randomized block design with four replications. All the results were summarized as mean of three years. The highest fresh and dry biomass yields (26.75 t ha^?1 and 7.5 ha^?1, respectively) were obtained from 150 kg ha^?1 N dose and followed by 100 kg ha^?1 N dose (26.29 t ha^?1 and 7.24 ha^?1, respectively). Whereas the highest fresh and dry leaf yields (13.27 t ha^?1 and 3.82 t ha^?1, respectively) were realized in 100 kg ha^?1 N dose. Actually, all nitrogen doses gave higher biomass and leaf yields compared to the control. On the hand, major steviol glycosides (stevioside and rebaudioside A) in the leaf were not influenced by nitrogen levels. In conclusion, 100 kg ha^?1 N dose was found to be suitable for cultivation of stevia under field conditions.     

甜菊不同层次叶片叶绿体超微结构观察及其糖苷含量变化

甜菊Stevia rebaudiana Bertoni不同层次叶片叶绿体超微结构的观察及其糖苷含量的测定表明:在上层幼嫩叶片中,叶绿体小,类囊体膜较简单,基粒片层和基质片层数量少,糖苷含量居中。在中层成叶的叶片中,叶绿体大,类囊体膜复杂,基粒片层多,基质片层密集,在基质中出现“淀粉区”结构,糖苷含量最高。在下层衰老的叶片中,叶绿体趋于解体,类囊体膜退化,糖苷含量最低。上述结构与功能密切相关,说明甜菊在现蕾期,中层叶最多,且糖苷含量也最高,是收获的最适时期。     

Identification of steviol glucuronide in human urine

Stevioside (250 mg capsules) was given three times daily to 10 healthy subjects. Steviol glucuronide (steviol 19-O-beta-D-glucopyranosiduronic acid; MM, 494.58; melting point, 198-199 degrees C) was characterized in the 24 h urine as the only excretion product of oral stevioside by MS, NMR, IR, and UV spectroscopy. This is the first report on the unambiguous identification of steviol glucuronide in human urine.     

Arbuscular mycorrhiza enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms

The sweet herb of Paraguay, Stevia rebaudiana (Bertoni), is becoming more important worldwide in herbal care for diabetes, as it produces the zero-calorie sweeteners steviol glycosides (SGs)—stevioside and rebaudioside-A. While arbuscular mycorrhizal fungi (AMF) have been shown to enhance production of secondary metabolites in many plant species, their effect on S. rebaudiana has not been studied. Moreover, relatively little is known about the mechanisms that may be involved in the increased accumulation of phytochemicals in mycorrhizal plants. Therefore, this study was performed to test the ability of Rhizophagus fasciculatus (Thaxt.) C. Walker & A. Schüßler to improve the yield of SGs in S. rebaudiana and to relate this with some AMF-induced physiological changes in addition to improved phosphorus (P) uptake. The performance of plants inoculated with R. fasciculatus was compared with that of non-mycorrhizal plants with similar P concentrations. Mycorrhizal (M) and non-mycorrhizal plants with P-supplementation (NM + P) produced higher concentrations of SGs compared with control plants. However, M plants had more SGs than did NM + P plants. The higher content of SGs in M plants is due to increased concentrations of SGs and to the enhanced biomass of the shoots. The increase in biomass is directly due to the improved uptake of nutrients (N, K, Mg, Cu, Fe, Mn and Zn), and chlorophyll and carbohydrate concentrations in M plants. Higher concentrations of total carbohydrates and jasmonic acid in M plants than in NM + P plants contribute to more biosynthesis of SGs via the methyl erythritol phosphate pathway. This study suggests that AMF-mediated increases in SGs involve both nutritionally and non-nutritionally linked mechanisms.     

基于16SrRNA序列分析红耳龟肠道拟杆菌和厚壁杆菌菌群多样性

脊椎动物肠道内定植了大量的微生物群落,在正常肠道菌群中,拟杆菌菌群和厚壁杆菌菌群作为优势菌群存在,对宿主的生理健康起着重要的作用。为了更好地了解外来入侵物种红耳龟的肠道拟杆菌和厚壁杆菌菌群多样性组成,本研究提取3只成体红耳龟粪便的宏基因组DNA,利用最新的Roche454测序技术对样本菌群的16SrRNA基因V3～V5区域进行测序。测序结果显示拟杆菌菌群为2纲5科11属,其中拟杆菌纲（98．82±1．24）％是绝对优势菌群;厚壁杆菌菌群被鉴定为3纲11科38属,主要由梭菌纲（95．81±0．88）％构成。此外,测序还检测到大量未知的拟杆菌类群（4．38±1．64）％和厚壁杆菌类群（14．96±8．15）％。本研究表明成体红耳龟粪便中拟杆菌菌群和厚壁杆菌菌群具有多样性。基于16SrRNA基因的Roche454测序分析可以很好地揭示肠道菌群的组成结构。     

Steviol quantification at the picomole level by high-performance liquid chromatography

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.     

In vitro fermentation by human fecal microflora of wheat arabinoxylans

The fermentation of three arabinoxylan (AX) fractions from wheat by the human fecal microflora was investigated in vitro. Three AX fractions, with average molecular masses of 354, 278, and 66 kDa, were incorporated into miniature-scale batch cultures (with inulin as a positive prebiotic control) with feces from three healthy donors, aged 23-29. Microflora changes were monitored by the culture-independent technique, fluorescent in situ hybridization, and short chain fatty acid (SCFA) and lactic acid production were measured by high-performance liquid chromatography. Total cell numbers increased significantly in all treated cultures, and the fermentation of AX was associated with a proliferation of the bifidobacteria, lactobacilli, and eubacteria groups. Smaller but statistically significant increases in bacteroides and clostridia groups were also observed. All AX fractions had comparable bifidogenic impacts on the microflora at 5 and 12 h, but the 66 kDa AX was particularly selective for lactobacilli. Eubacteria increased significantly on all AX fractions, particularly on 66 kDa AX. As previously reported, inulin gave a selective increase in bifidobacteria. All supplemented cultures showed significant rises in total SCFA production, with a particularly high proportion of butyric acid being produced from AX fermentation. The prebiotic effect, that is, the selectivity of AX for bifidobacteria and lactobacilli groups, increased as the molecular mass of the AX decreased. This suggests that molecular mass may influence the fermentation of AX in the colon.     

Identification of urolithin a as a metabolite produced by human colon microflora from ellagic acid and related compounds

Dietary ellagic acid and related polyphenols are metabolized in humans to dibenzopyran-6-one derivatives, and the microbial origin of these metabolites has been suggested. However, this has not been demonstrated so far. Fecal samples donated by six volunteers were incubated under anaerobic conditions, and aliquots were used to evaluate the fecal metabolism of ellagic acid, the ellagitannin punicalagin, and an ellagitannin rich extract from walnuts. The isoflavone daidzein was also incubated with the same fecal samples to follow the production of the microbial metabolites previously reported (dihydrogenistein, O-demethylangolensin, and equol) as a positive control of the system and to evaluate similarities between isoflavone and ellagic acid fecal flora metabolism. After fermentation the metabolite "urolithin A" (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one) was produced from ellagic acid, punicalagin, and the ellagitannin extract in all the fecal cultures from different volunteers, but with very different production rates and concentrations. This large variability in the concentration of metabolite and kinetics of metabolite production is consistent with the large variability found in the excretion of these metabolites in urine in vivo after human consumption of ellagitannins, and with differences in the composition of the fecal microflora. No correlation between isoflavone and ellagic acid metabolism by fecal microflora was observed. The present study confirms the microbial origin of the recently reported in vivo generated hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives in humans and is a further step in the study of the bioavailability and metabolism of ellagic acid and ellagitannins.