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.     

Purple carrot (Daucus carota L.) polyacetylenes decrease lipopolysaccharide-induced expression of inflammatory proteins in macrophage and endothelial cells

Carrots ( Daucus carota L.) contain phytochemicals including carotenoids, phenolics, polyacetylenes, isocoumarins, and sesquiterpenes. Purple carrots also contain anthocyanins. The anti-inflammatory activity of extracts and phytochemicals from purple carrots was investigated by determining attenuation of the response to lipopolysaccharide (LPS). A bioactive chromatographic fraction (Sephadex LH-20) reduced LPS inflammatory response. There was a dose-dependent reduction in nitric oxide production and mRNA of pro-inflammatory cytokines (IL-6, IL-1beta, TNF-alpha) and iNOS in macrophage cells. Protein secretions of IL-6 and TNF-alpha were reduced 77 and 66% in porcine aortic endothelial cells treated with 6.6 and 13.3 microg/mL of the LH-20 fraction, respectively. Preparative liquid chromatography resulted in a bioactive subfraction enriched in the polyacetylene compounds falcarindiol, falcarindiol 3-acetate, and falcarinol. The polyacetylenes were isolated and reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. These results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti-inflammatory bioactivity.     

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.     

Modulation of cytokine secretion by garlic oil derivatives is associated with suppressed nitric oxide production in stimulated macrophages

We previously described that garlic oil derivatives differentially suppress the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in activated macrophages. In the present study, we investigated the effects of the garlic derivatives, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS), on cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and the association between modulation of cytokines and inhibition of NO production was also assessed. The results indicated that these garlic compounds had different effects on the secretion of activated cytokines, including proinflammatory tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, as well as the antiinflammatory, IL-10. DAS inhibited the production of all stimulated cytokines in a concentration-dependent manner, and the inhibition was closely associated with the suppression of NO and PGE(2) production. DADS repressed the production of stimulated TNF-alpha and IL-10 and increased the production of activated IL-1beta and, to a lesser extent, IL-6; but only the decreased IL-10 production was associated with DADS-induced NO inhibition. Yet, the DAS- and DADS-suppressed NO production was independent of TNF-alpha. AMS, on the other hand, slightly suppressed the stimulated TNF-alpha but enhanced IL-10 production, and such modulation was closely associated with the decrease in NO production.     

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.     

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.     

DATS reduces LPS-induced iNOS expression, NO production, oxidative stress, and NF-kappaB activation in RAW 264.7 macrophages

Diallyl trisulfide (DATS), diallyl sulfide (DAS), and diallyl disulfide (DADS) are the three major organosulfur compounds (OSCs) in garlic oil. In contrast to DADS and DATS, evidence of an anti-inflammatory effect of DATS is limited. In this study compares the efficacy of DATS with those of DAS and DADS on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. The NO production in LPS-activated RAW 264.7 macrophages was suppressed by both DADS and DATS in a dose-dependent manner. At 100 muM, the nitrite levels of DADS- and DATS-treated cells were 57 and 34%, respectively, of cells treated with LPS alone. DAS, however, had no influence on NO production even at a concentration of 1 mM. Western blot and Northern blot assays showed that DADS and DATS but not DAS dose-dependently suppressed LPS-induced iNOS protein and mRNA expression in a pattern similar to that noted for NO production. LPS-induced cellular peroxide production was significantly inhibited by DADS and DATS (P < 0.05) but not by DAS. Electrophoresis mobility shift assays further indicated that DADS and DATS effectively inhibited the activation of NF-kappaB induced by LPS. Taken together, these results indicate that the differential efficacy of three major OSCs of garlic oil on suppression of iNOS expression and NO production is related to the number of sulfur atoms and is in the order DATS > DADS > DAS. The inhibitory effect of DATS on LPS-induced iNOS expression is likely attributed to its antioxidant potential to inhibit NF-kappaB activation.     

Anti-inflammatory effects of phenolic compounds isolated from the fruits of Artocarpus heterophyllus

Artocarpus heterophyllus Lam is a large evergreen tree cultivated throughout Southeast Asia for its fruits. Its leaves and roots have been used for medicinal purposes. The aim of this work was to study the in vitro anti-inflammatory effects of phenolic compounds isolated from the ethyl acetate extracts of the fruits of Artocarpus heterophyllus. Three phenolic compounds were characterized as artocarpesin [5,7,2',4'-tetrahydroxy-6-(3-methylbut-3-enyl) flavone] ( 1), norartocarpetin (5,7,2',4'-tetrahydroxyflavone) ( 2), and oxyresveratrol [ trans-2,4,3',5'-tetrahydroxystilbene] ( 3) by spectroscopic methods and through comparison with data reported in the literatures. The anti-inflammatory effects of the isolated compounds ( 1- 3) were evaluated by determining their inhibitory effects on the production of proinflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. These three compounds exhibited potent anti-inflammatory activity. The results indicated that artocarpesin ( 1) suppressed the LPS-induced production of nitric oxide (NO) and prostaglandin E 2 (PGE 2) through the down-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions. Thus, artocarpesin ( 1) may provide a potential therapeutic approach for inflammation-associated disorders.     

In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans

An acidic polysaccharide (APS) was isolated from the extract of Cordyceps militaris grown on germinated soybeans. Analyses of sugar composition indicated that APS consisted of d-galactose, L-arabinose, D-xylose, L-rhamnose, and D-galacturonic acid. On the basis of the result of methylation analysis, APS was considered to be mainly composed of Araf-(1-->, -->5)-Araf-(1-->, -->4)-Galp-(1--> and -->4)-GalAp-(1--> residues. When the polysaccharide was intranasally administered, it decreased virus titers in the bronchoalveolar lavage fluid and the lung of mice infected with influenza A virus and increased survival rate. Furthermore, APS increased TNF-alpha and IFN-gamma levels in mice when compared with those of untreated mice. APS enhanced nitric oxide (NO) production and induced iNOS mRNA and protein expressions in RAW 264.7 murine macrophage cells. The induction of mRNA expression of cytokines including IL-1beta, IL-6, IL-10, and TNF-alpha was also observed. These results demonstrated that APS might have beneficial therapeutic effects on influenza A virus infection at least in part by modulation of the immune function of macrophages.     

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.     

Miyabenol A inhibits LPS-induced NO production via IKK/IkappaB inactivation in RAW 264.7 macrophages: possible involvement of the p38 and PI3K pathways

The anti-inflammatory effect of miyabenol A, a stilbene isolated from Vitis thunbergii, on lipopolysaccaride (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages was studied. Miyabenol A inhibited NO production (EC 50: 2.7 muM) and iNOS protein and mRNA expression in a parallel concentration-dependent manner. LPS-evoked NF-kappaB nuclear translocation and associated IkappaB degradation were abrogated by miyabenol A treatment. Phosphorylations of IKKalpha/beta, ERK1/2, JNK p38 MAPK, and Akt were observed in LPS-stimulated cells; nevertheless, miyabenol A selectively blocked IKKalpha/beta, p38, and Akt phosphorylation. Furthermore, LPS-stimulated IKKalpha/beta and Akt phosphorylation was abolished by p38 inhibitor SB203580. Wortmannin (a PI3K inhibitor) also attenuated LPS-induced IKKalpha/beta phosphorylation, although to a less extent than SB203580, but failed to affect p38 phosphorylation. These observations suggested that PI3K/Akt might lie downstream of p38 MAPK to coregulate LPS-induced IKKalpha/beta phosphorylation. Taken together, miyabenol A acted via interfering with p38 MAPK-related signal pathways to down-regulate IKK/IkappaB activation and NO production.     

Characterization of Aloeride, a new high-molecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity

We have characterized a new immunostimulatory polysaccharide called Aloeride from commercial aloe vera (Aloe barbadensis) juice. Aloeride is between 4 and 7 million Da, and its glycosyl components include glucose (37.2%), galactose (23.9%), mannose (19.5%), and arabinose (10.3%). At 0.5 microg/mL Aloeride increased NF-kappa B directed luciferase expression in THP-1 human monocytic cells to levels 50% of those achieved by maximal concentrations (10 microg/mL) of LPS. Aloeride induced the expression of the mRNAs encoding IL-1beta and TNF-alpha to levels equal to those observed in cells maximally activated by LPS. Acemannan, the major carbohydrate component from aloe, used at 200 microg/mL in the macrophage assay resulted in negligible NF-kappa B activation. Analysis of acemannan and Aloeride using size-exclusion chromatography suggests that the low activity of acemannan is due to trace amounts of Aloeride. Although Aloeride comprises only 0.015% of the aloe juice dry weight, its potency for macrophage activation accounts fully for the activity of the crude juice.     

Lotus corniculatus regulates the inflammation induced by bradykinin in a murine model of pleurisy

This study evaluated the anti-inflammatory efficacy of the crude extract (CE), the fractions derived from hexane (HEX), ethyl acetate (AcOEt), n-butanol (BuOH), and aqueous (Aq) and isolated compounds (oleanolic acid or kaempferitrin) obtained from the aerial parts of Lotus corniculatus var. Sa?o Gabriel in mice with bradykinin-induced pleurisy. Swiss mice were used for the In Vivo experiments. Inflammatory parameters [leukocytes; exudate concentrations; myeloperoxidase and adenosine-deaminase activities, and nitric oxide and interleukin-17 levels] were evaluated 4 h after pleurisy induction. The crude extract of Lotus corniculatus, its derived fractions, and isolated compounds inhibited leukocytes and the exudate. This inhibitory effect was associated with decreased of myeloperoxidase and adenosine-deaminase activities, nitric oxide products, and IL-17A levels. Lotus corniculatus presented important anti-inflammatory action by inhibiting leukocyte influx and exudate concentrations. This effect was directly related to the inhibition of nitric oxide and interleukinin17 levels. Oleanolic acid and kaempferitrin can account for these anti-inflammatory effects.     

Antiinflammatory properties of the muscadine grape (Vitis rotundifolia)

The muscadine grape possesses one of the highest antioxidant levels among fruits; yet, the effect of this fruit on mammalian metabolic systems has not received significant attention. To examine the antiinflammatory properties of the muscadine, grape skins were dried, pulverized, and extracted (10% w/v) with 50% ethanol. The extract was then tested in two different assays: the release of superoxide in phorbol myristate acetate-activated neutrophils and the release of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-beta), and interleukin-6 (IL-6)] by lipopolysaccharide-activated peripheral blood mononuclear cells. The release of superoxide and cytokines was inhibited by increasing concentrations of the extract. A 1:100 dilution of the extract inhibited superoxide release by approximately 60% while the release of TNF-alpha and IL-1beta was reduced at a dilution of 1:200 by approximately 15 and 90%, respectively (all P < 0.05). The inhibition pattern on the release of IL-6 was similar to that seen with TNF-alpha. In a related in vivo study, rats were fed a diet containing 5% (wt/wt) dried muscadine grape skins for 14 days and then were injected with carrageenan in the foot pad. After 3 h, paw edema was measured and the rats on the grape skin diet had approximately 50% less paw edema than controls (P < 0.05). These results demonstrate that the muscadine grape skin powder possesses significant in vitro and in vivo antiinflammatory properties.