Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana.

Plants respond to environmental stress by synthesizing a range of secondary metabolites for defense purposes. Here we report on the effect of chronic ultraviolet (UV) radiation on the accumulation of plant secondary metabolites in Arabidopsis thaliana leaves. In the natural environment, UV is a highly dynamic environmental parameter and therefore we hypothesized that plants are continuously readjusting levels of secondary metabolites. Our data show distinct kinetic profiles for accumulation of tocopherols, polyamines and flavonoids upon UV acclimation. The lipid-soluble antioxidant α-tocopherol accumulated fast and remained elevated. Polyamines accumulated fast and transiently. This fast response implies a role for α-tocopherol and polyamines in short-term UV response. In contrast, an additional sustained accumulation of flavonols took place. The distinct accumulation patterns of these secondary metabolites confirm that the UV acclimation process is a dynamic process, and indicates that commonly used single time-point analyses do not reveal the full extent of UV acclimation. We demonstrate that UV stimulates the accumulation of specific flavonol glycosides, i.e. kaempferol and (to a lesser extent) quercetin di- and triglycosides, all specifically rhamnosylated at position seven. All metabolites were identified by Ultra Performance Liquid Chromatography (UPLC)-coupled tandem mass spectrometry. Some of these flavonol glycosides reached steady-state levels in 3-4 days, while concentrations of others are still increasing after 12 days of UV exposure. A biochemical pathway for these glycosides is postulated involving 7-O-rhamnosylation for the synthesis of all eight metabolites identified. We postulate that this 7-O-rhamnosylation has an important function in UV acclimation.

Steviol glycosides: chemical diversity, metabolism, and function.

Steviol glycosides are a group of highly sweet diterpene glycosides discovered in only a few plant species, most notably the Paraguayan shrub Stevia rebaudiana. During the past few decades, the nutritional and pharmacological benefits of these secondary metabolites have become increasingly apparent. While these properties are now widely recognized, many aspects related to their in vivo biochemistry and metabolism and their relationship to the overall plant physiology of S. rebaudiana are not yet understood. Furthermore, the large size of the steviol glycoside pool commonly found within S. rebaudiana leaves implies a significant metabolic investment and poses questions regarding the benefits S. rebaudiana might gain from their accumulation. The current review intends to thoroughly discuss the available knowledge on these issues.

Alternatively activated macrophages engage in homotypic and heterotypic interactions through IL-4 and polyamine-induced E-cadherin/catenin complexes.

Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13-exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4-dependent, arginase-1/ornithine decarboxylase-mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4-dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4-driven macrophage fusion and heterotypic interactions with CD103(+) and KLRG1(+) T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13-exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.

The food additives inulin and stevioside counteract oxidative stress.

Prebiotics such as inulin (Inu)-type fructans and alternative natural sweeteners such as stevioside (Ste) become more popular as food ingredients. Evidence is accumulating that carbohydrates and carbohydrate-containing biomolecules can be considered true antioxidants, capable of scavenging reactive oxygen species (ROS). Here, we report on the ROS scavenging abilities of Inu and Ste in comparison with other sugars, sugar derivatives and arbutin. It is found that Inu and Ste are superior scavengers of both hydroxyl and superoxide radicals, more effective than mannitol and sucrose. Other compounds, such as 1-kestotriose, trehalose, raffinose and L-malic acid, also showed good reactivity to at least one of the two oxygen free radicals. The strong antioxidant properties of Inu and Ste are discussed. Within the plant vacuole, these compounds could play a crucial role in antioxidant defense mechanisms to help survive stresses. Addition to food assists in natural sweetening, food stabilization and maximizes health impact.

Steviol glucuronide, a metabolite of steviol glycosides, potently stimulates insulin secretion from isolated mouse islets: Studies in vitro.

AIMS: Steviol glycosides are the sweet components extracted from medicinal plant Stevia rebaudiana Bertoni, which have antihyperglycaemic effects. Steviol glucuronide (SVG) is the metabolite excreted in human urine after oral administration of steviol glycosides. We aimed to clarify whether SVG exerts direct insulin stimulation from pancreatic islets and to explore its mode of action. MATERIALS AND METHODS: Insulin secretion was measured after 60 minutes static incubation of isolated mouse islets with (a) 10-9-10-5 mol/L SVG at 16.7 mmol/L glucose and (b) 10-7 mol/L SVG at 3.3-16.7 mmol/L glucose. Islets were perifused with 3.3 or 16.7 mmol/L glucose in the presence or absence of 10-7 mol/L SVG. Gene transcription was measured after 72 hours incubation in the presence or absence of 10-7 mol/L SVG. RESULTS: SVG dose-dependently increased insulin secretion from mouse islets with 10-7 mol/L exerting the maximum effect in the presence of 16.7 mmol/L glucose (P < .001). The insulinotropic effect of SVG was critically dependent on the prevailing glucose concentration, and SVG (10-7 mol/L) enhanced insulin secretion at or above 11.1 mmol/L glucose (P < .001) and showed no effect at lower glucose concentrations. During perifusion of islets, SVG (10-7 mol/L) had a long-acting and apparently reversible insulinotropic effect in the presence of 16.7 mmol/L glucose (P < .05). Gene-transcript levels of B2m and Gcgr were markedly altered. CONCLUSION: This is the first report to demonstrate that SVG stimulates insulin secretion in a dose- and glucose-dependent manner from isolated mouse islets of Langerhans. SVG may be the main active metabolite after oral intake of steviol glycosides.

Effect of Cold Stress on Photosynthetic Traits, Carbohydrates, Morphology, and Anatomy in Nine Cultivars of Stevia rebaudiana.

Stevia rebaudiana Bertoni is a sweet medicinal herb that is cultivated worldwide. This study aimed to identify the genotypic responses and function of nine cultivars of S. rebaudiana (accession numbers 1-9 from the EUSTAS Stevia Gene Bank) to low temperature. Plants were grown in vitro and incubated under controlled conditions at 5° or 25°C for 1 month. Cold stress significantly decreased the maximum quantum yield of photosystem II (Fv/Fm) in all cultivars, which was more pronounced in cultivars 5, 6, 8, and 9. The efficiency of photosystems I and II (PIABS) also declined in cold-stressed plants and was accompanied by reductions in net photosynthesis (PN), intercellular CO2 (Ci), water use efficiency (WUE), and chlorophyll a, chlorophyll b and carotenoid contents, more so in cultivars 5, 6, 8, and 9. Regardless of the downregulation of photosynthetic capacity, the cold stress increased water-soluble carbohydrates in all cultivars, which was accompanied by an increase in fresh leaf mass and area, more so in cultivars 5, 6, 8, and 9. Furthermore, cold stress increased the stomatal index and density, epidermal cell density, stem diameter, xylem vessel width, phloem tissue width, and number of sclerenchyma in all cultivars. Even though the nine cultivars of S. rebaudiana had lower PSII efficiencies at low temperatures, the increase in carbohydrates and leaf mass suggests that damage to PSII is not responsible for the reduction in its efficiency.

Metabolism of stevioside by healthy subjects.

Stevioside (250-mg capsules) was given thrice daily for 3 days to 10 healthy subjects. Blood samples were collected and blood pressure measured after nocturnal fasting, before and at different time points during the third day of the administration of stevioside. No significant differences were found between the control and the stevioside condition for blood pressure and blood biochemical parameters. The 24-hr urinary volume and urinary excretion of electrolytes were not significantly different. Likewise, no significant difference was found for mean blood glucose and insulin between control and stevioside conditions. Thus, oral stevioside is not directly effective as a hypotensive or hypoglycemic agent in healthy subjects at the dose administered in this study. Stevioside, free steviol, and steviol metabolites were analyzed in blood, feces, and urine after 3 days of stevioside administration. No uptake was found of stevioside by the gastrointestinal tract or the amounts taken up were very low and below the detection limit of the UV detector. Stomach juice did not degrade stevioside. All the stevioside reaching the colon was degraded by micro-organisms into steviol, the only metabolite found in feces. In blood plasma, no stevioside, no free steviol or other free steviol metabolites were found. However, steviol glucuronide (SV glu) was found in maximum concentrations of 33 micro g/ml (21.3 micro g steviol equivalents/ml). In urine, no stevioside or free steviol were present, but SV glu was found in amounts of up to 318 mg/24-hr urine (205 mg steviol equivalents/24 hrs). No other steviol derivatives were detected. In feces, besides free steviol, no other steviol metabolites or conjugates were detected. Steviol was excreted as SV glu in urine.

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.

Gene transcription and steviol glycoside accumulation in Stevia rebaudiana under polyethylene glycol-induced drought stress in greenhouse cultivation.

Stevia rebaudiana is a sweet herb of the Astraceae family, which is cultivated for the natural sweeteners it contains. The aim of this study was to assess the effect of drought, simulated by the application of polyethylene glycol (5%, 10%, and 15% w/v), on the content of steviol glycosides (SVglys) and transcription levels of six genes involved in the biosynthesis of these natural sweeteners. The transcription levels of ent-kaurene synthase, ent-kaurene oxidase, ent-kaurenoic acid hydroxylase, and three UDP-dependent glycosyltransferases, UGT85C2,UGT74G1 and UGT76G1 were downregulated under polyethylene glycol treatment. Polyethylene glycol treatment significantly decreased the amount of stevioside, rebaudioside A, B, C and F, steviolbioside, dulcoside A, rubusoside, and total SVglys. These results strongly suggest a close relationship of SVglys content with the transcription of genes involved in the SVglys biosynthesis pathway. Comparing the observations of the present study with other reports provided the knowledge that the Stevia response to drought stress can be influenced by different environmental and experimental factors, in addition to intensity of drought stress. In conclusion, these results strongly suggest that polyethylene glycol-induced drought stress has a negative effect on the content of SVglys and transcription of SVglys biosynthetic genes and that this should be investigated further. We recommend that sufficient irrigation of Stevia is required to obtain a high content of SVglys.

Validation of an HPLC method for direct measurement of steviol equivalents in foods.

Steviol glycosides are intense natural sweeteners used in foods and beverages. Their acceptable daily intake, expressed as steviol equivalents, is set at 0-4 mg/kg body weight. We report the development and validation of a RP-HPLC method with fluorometric detection of derivatized isosteviol, formed by acid hydrolysis of steviol glycosides. Dihydroisosteviol was used as an internal standard. Using this method, the amount of steviol equivalents in commercial steviol glycoside mixtures and different foods can be directly quantified. The method was successfully tested on strawberry jam, low-fat milk, soft drink, yogurt and a commercial mixture of steviol glycosides. Calibration curves were linear between 0.01 and 1.61 mM steviol equivalents, with a quantification limit of 0.2 nmol. The % RSD of intra-day precision varied between 0.4% and 4%, whereas inter-day precision varied between 0.4% and 5%, for high and medium concentrations, and between 3% and 8% for low concentrations. Accuracy of the analysis varied between 99% and 115%.

Uptake and tissue-specific distribution of selected polychlorinated biphenyls in developing chicken embryos.

Fertilized chicken eggs were injected with high doses of individual polychlorinated biphenyl (PCB) congeners (0.5 microg of PCB 77, 9.8 microg of PCB 153, or 10.9 microg of PCB 180) before incubation to investigate the structure-specific uptake of these compounds by the embryo and their accumulation in brain and liver tissue. In accordance with earlier publications, a gradual uptake and accumulation of these compounds was observed during the last week of embryonic development. The PCB uptake and distribution to the specific tissues did not appear to be structure dependent. Wet-weight liver PCB concentrations (18, 266, and 278 ng/g at hatching for PCB 77, PCB 153, and PCB 180, respectively) were consistently two- to fourfold higher than carcass levels (7 ng/g of PCB 77, 117 ng/g of PCB 153, and 81 ng/g of PCB 180 at hatching). Whereas liver and carcass concentrations increased exponentially between day 13 of incubation and hatching, PCB levels in brain tissue remained unaltered (range, 0.6-1.0 ng/g of PCB 77 and 8-12 ng/g of PCB 153 and PCB 180 throughout the last week of incubation). Lipid analysis of the organs suggested that the lipid composition of brain may be an important factor explaining the low PCB accumulation in this tissue.

Stevioside.

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.

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.

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.

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.

Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol.

Stevioside orally administered to pigs was completely converted into steviol by the bacteria of the colon. However, no stevioside or steviol could be detected in the blood of the animals, even not after converting steviol into the (7-methoxycoumarin-4-yl)methyl ester of steviol, a very sensitive fluorescent derivative with a detection limit of about 50 pg. The intestinal transport characteristics of stevioside, rebaudioside A and steviol were also studied in the Caco-2 system. Only a minor fraction of stevioside and rebaudioside A was transported through the Caco-2 cell layer giving a Papp value of 0.16x10(-6) and 0.11x10(-6) cm/s, respectively. The Papp value for the absorptive transport of steviol was about 38.6x10(-6) cm/s while the Papp value for the secretory transport of steviol was only about 5.32x10(-6) cm/s suggesting carrier-mediated transport. The discrepancy between the relatively high absorptive transport of steviol and the lack of steviol in the blood may be explained by the fact that in the Caco-2 study, steviol is applied as a solution facilitating the uptake, whereas in the colon steviol probably is adsorbed to the compounds present in the colon of which the contents is being concentrated by withdrawal of water.

Polyamines and fatty acids in sucrose precultured banana meristems and correlation with survival rate after cryopreservation.

Polyamines and fatty acids were studied in proliferating meristem cultures of 3 banana cultivars with high (Cachaco), medium (Williams Bronze free) and low (Mbwazirume) survival rates after cryopreservation. A 2-week preculture on medium containing 0.4 M sucrose which is essential to obtain survival after cryopreservation resulted in increased polyamine levels, especially putrescine. This increase in putrescine content was positively correlated with the survival rate after simple freezing or after vitrification. The total fatty acid content also increased after a 0.4 M sucrose pretreatment. However, only the ratio of unsaturated/saturated fatty acids correlated positively with the survival rate after cryopreservation. This is the first report showing a correlation of both putrescine increase and level of unsaturation of membrane lipids after sucrose treatment with survival rate after cryopreservation.

Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods.

As part of an ongoing study on the effects of photoperiodism on the metabolism of steviol glycosides (SVglys) in Stevia rebaudiana, the spatio-temporal variations of free steviol (SV) have now been evaluated. For its quantitation, an internal standard method was used, based upon a specific fluorometric detection of SV as its methoxycoumarinyl derivative. The level of free SV in leaves did not exceed 30 µg/g dry wt and was at least 1000-fold smaller than that of its glycosidic conjugates. In other organs, free SV was mainly measured in stem tissue and apices, with relatively large amounts measured in the latter. Similarly to SVglys, the content of free SV was influenced by photoperiod and genotype. In plants grown under long-days (LD) of 16 h, more spatial variations were seen compared to those under short-days (SD) of 8h. In the former, upper leaves contained almost four times more free SV compared to lower ones near the end of vegetative growth. In addition, the correlation between SV and its glycosidic conjugates was more linear under SD. Despite the variability of SV levels, a decrease was noted in all conditions after flower opening, which can be related a decreased transcription of the biosynthetic genes involved.

Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni).

The effect of photoperiodism on steviol glycoside (SVgly) accumulation was investigated in Stevia rebaudiana. Topped plants were cultivated to develop new branches under a 16h or 8h photoperiod. During different ontogenetic phases, leaves, stems, lateral shoots, roots and reproductive organs were collected and analysed for nine SVglys. Long-day (LD) conditions prolonged vegetative growth, significantly increasing leaf biomass and total SVgly content. In both photoperiods, declines in SVglys were observed during reproductive development, occurring mainly in mature leaves under LDs or young leaves under SDs. When lateral shoots were included in plants under LDs, total leaf and SVgly yield per branch significantly increased, indicating a harvest during flowering is possible. The ratio of rebaudioside A (Reb A) to stevioside (ST) amounts was influenced by ontogeny and daylength, with larger ratios during vegetative growth under SDs. Linear correlations were observed between dry matter and total SVglys and between the major SVglys individually. Minor SVglys showed larger fluctuations, especially under SDs. Under LDs, the Reb A to ST ratio was inversely correlated with both leaf dry matter and total SVglys. The highly dynamic nature of the observed patterns suggests a complex regulation of SVgly metabolism on molecular and biochemical level.

Diterpene glycosides from Stevia phlebophylla A. Gray.

The rare Mexican species Stevia phlebophylla A. Gray was long considered to be the only known Stevia species, beside the well-known S. rebaudiana, containing the highly sweet diterpenoid steviol glycosides. We report a re-evaluation of this claim after phytochemically screening leaves obtained from two herbarium specimens of S. phlebophylla for the presence of steviol glycosides. Despite extensive MS analyses, no steviol glycosides could be unambiguously verified. Instead, the main chromatographic peak eluting at retention times similar to those of steviol glycosides was identified as a new compound, namely 16ß-hydroxy-17-acetoxy-ent-kauran-19-oic acid-(6-O-ß-D-xylopyranosyl-ß-D-glucopyranosyl) ester (1) on the basis of extensive NMR and MS data as well as the characterization of its acid hydrolysate. Seven more compounds were detected by ESIMS which are possibly structurally related to 1. It can therefore be concluded that S. phlebophylla is unlikely to contain significant amounts of steviol glycosides, if any.