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.     

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.     

Stevioside enhances satellite cell activation by inhibiting of NF-κB signaling pathway in regenerating muscle after cardiotoxin-induced injury

Stevioside, a noncaloric sweetener isolated from Stevia rebaudiana, exhibits anti-inflammatory and immunomodulatory effects through interference of nuclear factor (NF)-kappa B pathway. We investigated whether this anti-inflammatory property of stevioside could improve muscle regeneration following cardiotoxin-induced muscle injury. Adult male Wistar rats received stevioside orally at an accepted daily dosage of 10 mg kg?1 for 7 days before cardiotoxin injection at the tibialis anterior (TA) muscle of the right hindlimb (the left hindlimb served as control), and stevioside administration was continued for 3 and 7 days. TA muscle was examined at days 3 and 7 postinjury. Although stevioside treatment had no significant effect in enhancing muscle regeneration as indicated by the absence of decreased muscle inflammation or improved myofibrillar protein content compared with vehicle treated injured group at day 7 postinjury, the number of MyoD-positive nuclei were increased (P < 0.05), with a corresponding decrease in NF-κB nuclear translocation (P < 0.05). This is the first study to demonstrate that stevioside could enhance satellite cell activation by modulation of the NF-κB signaling pathway in regenerating muscle following injury. Thus, stevioside may be beneficial as a dietary supplementation for promoting muscle recovery from injury. However, its pharmacological effect on muscle function recovery warrants further investigation.     

Modulation of Inflammatory Gene Expression by a Bilberry ( Vaccinium myrtillus L.) Extract and Single Anthocyanins Considering Their Limited Stability under Cell Culture Conditions

Studies with nonintestinal models indicate that anthocyanin-rich extracts can modulate inflammatory gene expression and may help prevent development of inflammatory bowel diseases (IBD). This work investigated the influence of a bilberry ( Vaccinium myrtillus L.) extract (BE) and comprising anthocyanins on pro-inflammatory genes in IFN-γ/IL-1β/TNF-α stimulated human colon epithelial cells (T84) by qRT-PCR and cytokine arrays. Moreover, the stability of selected anthocyanins under cell culture conditions was examined to assess their anti-inflammatory properties. BE and single anthocyanins significantly inhibited the expression and secretion of IBD-associated pro-inflammatory mediators (TNF-α, IP-10, I-TAC, sICAM-1, GRO-α) in the stimulated cells. The anti-inflammatory activity thereby strongly depends on the aglycon structure (hydroxylation and methylation pattern) and the sugar moiety. In contrast to anthocyanidins, which were highly unstable in cell culture medium, suggesting that their degradation products might contribute to the inhibitory effects assigned to the parent compounds, anthocyanins have higher stability and may directly contribute to BE's effects.     

Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.     

Heat-killed cells of lactobacilli skew the immune response toward T helper 1 polarization in mouse splenocytes and dendritic cell-treated T cells

It is believed that probiotics play an important role for the health of the host, including modulation of immune responses. Most studies have focused on the immunomodulatory effects of viable cells of lactic acid bacteria; however, we investigated those of heat-killed cells of lactic acid bacteria in this study. We first observed the effects on immune functions via stimulating splenocytes with three heat-killed Lactobacillus strains. Furthermore, we also investigated the effect of mouse dendritic cells (DCs) treated with these heat-killed Lactobacillus strains on T cell responses. The results showed that these Lactobacillus strains were able to stimulate cell proliferation and interleukin (IL)-10, IL-12 p70, and interferon (IFN)-gamma production but not transforming growth factor (TGF)-beta in splenocytes. In addition, these heat-killed Lactobacillus strains also stimulated high-level secretion of IL-12 p70 in DCs and switched T cells to T helper (Th) 1 immune responses, as evidenced by the elevated secretion of IFN-gamma but not IL-5, IL-13, and TGF-beta. These results showed that lactobacilli play a potentially important role in modulating immune responses and allergic reactions.     

Effects of diarylheptanoids on the tumor necrosis factor-alpha-induced expression of adhesion molecules in human umbilical vein endothelial cells

Atherosclerosis is a chronic inflammatory disease that is characterized by infiltration of mononuclear lymphocytes into the intima through the expression of adhesion molecules on the arterial wall. In the present study, we report the inhibitory effects of two diarylheptanoids, 5-O-methylhirsutanonol (1) and oregonin (2), isolated from the methanolic extracts of Alnus japonica leaves, on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compounds 1 and 2 inhibited tumor necrosis factor (TNF)-alpha-induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which also prevented adhesion of monocytes to HUVECs, and slightly suppressed the mRNA expression of the inflammation-associated gene interleukin-1beta (IL-1beta). A further study demonstrated the inhibitory effect of compound 1 on DNA-binding of nuclear factor kappaB (NF-kappaB) and on the phosphorylation and degradation of inhibitory factor kappaBalpha (IkappaBalpha) in TNF-alpha-stimulated HUVECs. These results indicate that compounds 1 and 2 may be useful in the prevention and treatment of atherosclerosis through attenuation of adhesion molecule expression by inhibition of NF-kappaB activation.     

Antioxidative activity of amino acids on tissue oxidative stress in human intestinal epithelial cell model

The protective effects of amino acids against H 2O 2-induced oxidative stress were investigated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were pretreated with amino acids (1, 2, and 5 mM) for 2 h and then stimulated with 1 mM H 2O 2 for 6 h. The secretion of IL-8, a proinflammatory mediator, was determined by ELISA as an indicator of tissue oxidative stress. The inhibition of H 2O 2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment with Cys, Val, Ile, Leu, Trp, His, Lys, and Ala. Cys enhanced glutathione (GSH) biosynthesis enzyme activity and increased cellular GSH levels. Branched-chain amino acids such as Val, Ile, and Leu elevated activities of GSH S-transferase (GST) and catalase. Trp, His, and Lys caused increases in GST activity. Ala enhanced GSH reductase activity. These data suggest that specific amino acids exert protective effects against tissue oxidative stress in intestinal epithelial cells based on the structure.     

Caffeic acid disturbs monocyte adhesion onto cultured endothelial cells stimulated by adipokine resistin

Adipokines have been implicated in the pathogenesis of atherosclerosis via pro-inflammatory mechanisms contributing to insulin resistance. The adipokine resistin causes endothelium dysfunction, which plays an important role in sustaining atherogenesis. This study investigated whether resistin induced expression of cell adhesion molecules and integrins in endothelial cells and THP-1 monocytes and whether such induction was attenuated by 1-20 μM caffeic acid. Resistin enhanced endothelial expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule 1 (ICAM-1), and E-selectin and monocyte expression of β1, β2, and α4 integrins. The enhancement of these proteins was diminished by caffeic acid with reduced THP-1 cell adhesion on activated endothelium. Caffeic acid at ≤20 μM demoted resistin-stimulated interleukin 8 (IL-8) production responsible for ICAM-1 and β2 integrin induction. The endothelial up-regulation of IL-8 secretion by resistin entailed toll-like receptor 4 (TLR4) activation, but caffeic acid diminished IL-8 production and TLR4 induction. Furthermore, caffeic acid encumbered resistin-activated nuclear factor κB (NF-κB) signaling. These results demonstrate that caffeic acid blocked monocyte trafficking to resistin-activated endothelium via disturbing NF-κB signaling responsive to IL-8. Therefore, caffeic acid may have therapeutic potential in preventing obesity-associated atherosclerosis.     

5-caffeoylquinic acid and caffeic acid down-regulate the oxidative stress- and TNF-alpha-induced secretion of interleukin-8 from Caco-2 cells

Although chlorogenic acid (CHA) easily reaches a millimolar level in the gastrointestinal tract because of its high concentration in coffee and fruits, its effects on intestinal epithelial cells have been little reported. We investigated in this study the down-regulative effects of 5-caffeoylquinic acid (CQA), the predominant isomer of CHA, on the H(2)O(2-) or TNF-alpha-induced secretion of interleukin (IL)-8, a central pro-inflammatory chemokine involved in the pathogenesis of inflammatory bowel diseases, in human intestinal epithelial Caco-2 cells. After the cells had been pre- and simultaneously treated with CQA, the oversecretion of IL-8 and overexpression of its mRNA induced by H(2)O(2) were significantly suppressed in a dose-dependent manner in the range of 0.25-2.00 mmol/L. We further found that a metabolite of CQA, caffeic acid (CA), but not quinic acid, significantly inhibited the H(2)O(2)-induced IL-8 secretion and its mRNA expression in the same dose-dependent manner. Both CQA and CA suppressed the TNF-alpha-induced IL-8 secretion as well. Caffeic acid at 2.00 mmol/l was able to absolutely block the H(2)O(2)- or TNF-alpha-induced oversecretion of IL-8 in Caco-2 cells. However, CQA and CA did not suppress the TNF-alpha-induced increase in the IL-8 mRNA expression, indicating that the suppressive mechanisms are different between TNF-alpha-induced and H(2)O(2)-induced IL-8 production models. These results suggest that the habit of drinking coffee and/or eating fruits with a high CHA content may be beneficial to humans in preventing the genesis of inflammatory bowel diseases.     

Antiangiogenic potential of three triterpenic acids in human liver cancer cells

Three triterpenic acids, oleanolic acid, ursolic acid and maslinic acid, at 2 or 4 μmol/L were used to study their antiangiogenic potential in human liver cancer Hep3B, Huh7 and HA22T cell lines. The effects of these compounds upon the level and/or expression of hypoxia-inducible factor (HIF)-1α, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL)-8, urokinase plasminogen activator (uPA), reactive oxygen species (ROS), nitric oxide (NO) and cell invasion and migration were examined. Results showed that these triterpenic acids at 4 μmol/L significantly suppressed HIF-1α expression in three cell lines (P < 0.05); and these compounds at test doses failed to affect bFGF expression (P > 0.05). Three triterpenic acids dose-dependently decreased production and expression of VEGF and IL-8, retained glutathione level, lowered ROS and NO levels, and declined cell invasion and migration in test cell lines (P < 0.05). These compounds also dose-dependently reduced uPA production and expression in Hep3B and Huh7 cell lines (P < 0.05); but these agents only at 4 μmol/L significantly suppressed uPA production and expression in HA22T cells (P < 0.05). These findings suggest that these triterpenic acids are potent antiangiogenic agents to retard invasion and migration in liver cancer cells.     

Mass production of rubusoside using a novel stevioside-specific β-glucosidase from Aspergillus aculeatus

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific β-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other β-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 μL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.     

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.     

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.     

Antioxidative stress activity of oligophosphopeptides derived from hen egg yolk phosvitin in Caco-2 cells

The protective effects of hen egg yolk phosvitin phosphopeptides (PPPs) against hydrogen peroxide (H2O2)-induced oxidative stress were evaluated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were stimulated with 1 mM H2O2 for 6 h, and the secretion of IL-8, a proinflammatory mediator, was determined by ELISA as a biomarker of oxidative stress. The inhibition of H2O2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment for 2 h with PPPs, but not with phosvitin. PPPs also suppressed the formation of malondialdehyde in H2O2-treated Caco-2 cells. Furthermore, intracellular glutathione levels and glutathione reductase activity were elevated by the addition of PPPs. The protective effects of PPPs against H2O2-induced oxidative stress were almost the same as that of glutathione, and PPPs with a high content of phosphorus exhibited higher protective activity than PPPs without phosphorus; however, phosphoserine itself did not show any significant antioxidative stress activity. These findings suggest that oligophosphopeptides from hen egg yolk phosvitin possess novel antioxidative activity against oxidative stress in intestinal epithelial cells and that phosphorus and peptide structure seem to have a key role in the activity.     

Effect of yak milk casein hydrolysate on TH1/TH2 cytokines production by murine spleen lymphocytes in vitro

Yak casein hydrolysate was derived from the enzymatic alcalase-hydrolysate of a typical northwestern China milk product called Qula. An in vitro study was conducted to examine their immunoregulatory effects on murine T cells, including Con-A-induced lymphoproliferation and splenocyte cell cycle, production, and messenger RNA (mRNA) expression of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and interleukin-4 (IL-4). The results showed that yak casein hydrolysate had lymphoproliferation activity on murine splenocytes and induced their cell cycle from the G1 to the S phase. It could increase Con-A-induced IL-2 and IFN-gamma production in spleen cells, but a very weak or no effect was observed in the absence of Con-A. The present study also showed that it could markedly increase the production and mRNA expression of IFN-gamma and IL-2, which are key cytokines for T helper 1 cell (Th1) cell development, in a dose-dependent manner. However, their effects on IL-4 secretion were not obvious, and the enhancement was much lower than that of IFN-gamma and IL-2. All of these demonstrated that yak casein hydrolysate could increase type 1 cytokine production, thereby shifting the Th1/T helper 2 cell (Th2) balance toward a Th1-dominant phenotype, which meant that yak casein hydrolysate could indeed not only modulate the differentiation of helper T cell but also has the capacity to modulate the Th1/Th2 balance. Therefore, yak casein hydrolysate may be useful for the treatment of cell-mediated immune diseases.     

Inhibitory effect on activator protein-1, nuclear factor-kappaB, and cell transformation by extracts of strawberries (Fragaria x ananassa Duch.)

The inhibitory effects of strawberry (Fragaria x ananassa Duch.) antioxidant enzymes on tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B (UVB) induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) were studied. The inhibitory effects of strawberry extracts on the proliferation and transformation of human and mouse cancer cells were also evaluated. Strawberries had high activities of glutathione peroxidase, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase. Strawberry extracts inhibited the proliferation of human lung epithelial cancer cell line A549 and decreased TPA-induced neoplastic transformation of JB6 P+ mouse epidermal cells. Pretreatment of JB6 P+ mouse epidermal cells with strawberry extract resulted in the inhibition of both UVB- and TPA-induced AP-1 and NF-kappaB transactivation. Furthermore, strawberry extract also blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs) and UVB-induced phosphorylation of ERKs and JNK kinase in JB6 P+ mouse epidermal cell culture. These results suggest that the ability of strawberries to block UVB- and TPA-induced AP-1 and NF-kappaB activation may be due to their antioxidant properties and their ability to reduce oxidative stress. The oxidative events that regulate AP-1 and NF-kappaB transactivation can be important molecular targets for cancer prevention. The strawberries may be highly effective as a chemopreventive agent that acts by targeting the down-regulation of AP-1 and NF-kappaB activities, blocking MAPK signaling, and suppressing cancer cell proliferation and transformation.