Stevia Leaf to Stevia Sweetener: Exploring Its Science, Benefits, and Future Potential.

Steviol glycoside sweeteners are extracted and purified from the Stevia rebaudiana Bertoni plant, a member of the Asteraceae (Compositae) family that is native to South America, where it has been used for its sweet properties for hundreds of years. With continued increasing rates of obesity, diabetes, and other related comorbidities, in conjunction with global public policies calling for reductions in sugar intake as a means to help curb these issues, low- and no-calorie sweeteners (LNCSs, also known as high-potency sweeteners) such as stevia are gaining interest among consumers and food manufacturers. This appeal is related to stevia being plant-based, zero calorie and with a sweet taste that is 50-350 times sweeter than sugar, making it an excellent choice for use in sugar- and calorie-reduced food and beverage products. Despite the fact that the safety of stevia has been affirmed by several food regulatory and safety authorities around the world, insufficient education about stevia's safety and benefits, including continuing concern with regard to the safety of LNCSs in general, deters health professionals and consumers from recommending or using stevia. Therefore, the aim of this review and the stevia symposium that preceded this review at the ASN's annual conference in 2017 was to examine, in a comprehensive manner, the state of the science for stevia, its safety and potential health benefits, and future research and application. Topics covered included metabolism, safety and acceptable intake, dietary exposure, impact on blood glucose and insulin concentrations, energy intake and weight management, blood pressure, dental caries, naturality and processing, taste and sensory properties, regulatory status, consumer insights, and market trends. Data for stevia are limited in the case of energy intake and weight management as well as for the gut microbiome; therefore, the broader literature on LNCSs was reviewed at the symposium and therefore is also included in this review.

Oral subchronic and genotoxicity studies conducted with the amino acid, L-glutamine.

L-Glutamine is an abundantly occurring amino acid that serves numerous nutritional and physiological functions. It has current and potential applications as a therapeutic agent, dietary supplement, food ingredient, and in animal nutrition. To assess the safety of supplemental L-glutamine, a bacterial reverse mutation assay, in vitro chromosomal aberration assay, and a 13-week toxicity study were conducted. L-Glutamine showed no mutagenic activity in the bacterial reverse mutation assay, and did not induce chromosomal aberrations in Chinese hamster lung fibroblast cells in the in vitro chromosomal aberration assay. In the 13-week toxicity study, Sprague-Dawley rats (10/sex/group) were fed diets containing 0, 0.5, 2.5, or 5.0% L-glutamine. No deaths occurred, and no significant differences in body weights, body weight gains, ophthalmological findings, urinalysis parameters, or organ weights were observed between L-glutamine-fed rats and their respective controls. No toxicologically relevant effects on hematological or blood biochemical parameters were observed. Macroscopic and microscopic effects occurred at low frequency but were not associated with a dose-response relationship. Based on the results of the study, the no-observed-adverse-effect-level was determined to be 5.0% L-glutamine in the diet, the highest concentration tested (equivalent to 3832 and 4515 mg/kg body weight/day in male and female rats, respectively).

Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extracts.

Oligonol is a phenolic product derived from lychee fruit extract and green tea extract, containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. The safety of Oligonol was assessed in acute and subchronic studies and genotoxicity assays. In a single dose acute study of Oligonol, male and female rats were administered 2000mg/kg body weight (bw) Oligonol in water by gavage. Oligonol caused no adverse effects and body weight gain and food consumption were within normal range, thus the LD(50) of Oligonol was determined to be greater than 2000mg/kg. A 90 day subchronic study (100, 300 and 1000mg/kgbw/day, oral gavage) in male and female rats reported no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, haematology, gross pathology and histopathology. Similarly, no adverse effects were observed in mice fed diets providing 2, 20 or 200mg/kgbw Oligonol or 200mg/kgbw lychee polyphenol for 90 days. Oligonol did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA strains. Oligonol did not induce chromosomal aberrations in cultured Chinese hamster lung cells, but it showed increased polyploidy. In a micronucleus assay in mice, Oligonol did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations. The results from these safety studies and previous reports support the safety of Oligonol for human consumption.

Structure-function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth.

Anthocyanins are potent antioxidants and may be chemoprotective. However, the structure-function relationships are not well understood. The objectives of this study were to compare the chemoprotective properties of anthocyanin-rich extracts (AREs) with variable anthocyanin profiles to understand the relationship between anthocyanin chemical structure and chemoprotective activity, measured as inhibition of colon cancer cell proliferation. Additionally, the chemoprotective interaction of anthocyanins and other phenolics was investigated. AREs with different anthocyanin profiles from purple corn, chokeberry, bilberry, purple carrot, grape, radish, and elderberry were tested for growth inhibition (GI 50) using a human colorectal adenocarcinoma (HT29) cell line. All AREs suppressed HT29 cell growth to various degrees as follows: purple corn (GI 50 approximately 14 microg of cy-3-glu equiv/mL) > chokeberry and bilberry > purple carrot and grape > radish and elderberry (GI 50 > 100 microg of cy-3-glu equiv/mL). Anthocyanins played a major role in AREs' chemoprotection and exerted an additive interaction with the other phenolics present. Statistical analyses suggested that anthocyanin chemical structure affected chemoprotection, with nonacylated monoglycosylated anthocyanins having greater inhibitory effect on HT-29 cell proliferation, whereas anthocyanins with pelargonidin, triglycoside, and/or acylation with cinnamic acid exerted the least effect. These findings should be considered for crop selection and the development of anthocyanin-rich functional foods.

Protective effect of anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) against myelotoxicity induced by 5-fluorouracil.

The toxicities associated with 5-fluorouracil (5-FU), a potent broad-spectrum chemotherapeutic agent, can not only affect the morbidity and the efficacy of chemotherapy but also limit its clinical use. The objective of this study is to investigate the effects of a commercial anthocyanin-rich extract from bilberry (AREB) against 5-FU-induced myelotoxicity in vivo, and against chemosensitivity to 5-FU in vitro. A single injection of 5-FU at 200 mg/kg induced severe peripheral erythrocytopenia, thrombocytopenia and leucopenia as well as hypocellularity of the spleen and bone marrow in C57BL/6 mice. Oral administration of 500 mg/kg of AREB for 10 days significantly increased the number of red blood cells, neutrophils, and monocytes in peripheral blood to 1.2-fold, 9-fold, and 6-fold, respectively, compared with those seen after treatment with 5-FU alone (p< 0.05-0.001). The hypocellularity of the spleen and bone marrow caused by 5-FU was also distinctly alleviated in the AREB-treated group. Furthermore, AREB treatment with 50 and 100 microg/ml as a monomeric anthocyanin did not interfere with, but rather enhanced the chemotherapeutic efficacy of 5-FU in vitro. These results suggest that AREB may have protective potential against 5-FU-induced myelotoxiciy and/or the ability to enhance the chemotherapeutic effectiveness of 5-FU.

Pharmacokinetics and distribution of [35S]methylsulfonylmethane following oral administration to rats.

Methylsulfonylmethane (MSM) is a sulfur-containing compound found in a wide range of human foods including fruits, vegetables, grains, and beverages. More recently, it has been marketed as a dietary supplement worldwide. The objective of this study was to evaluate the pharmacokinetic profile and distribution of radiolabeled MSM in rats. Male Sprague-Dawley rats were administered a single oral dose of [35S]MSM (500 mg/kg), and blood levels of radioactivity were determined at different time points for up to 48 h. Tissue levels of radioactivity at 48 and 120 h and urine and fecal radioactivity levels were measured at different time points for up to 120 h following [35S]MSM administration to rats. Oral [35S]MSM was rapidly and efficiently absorbed with a mean tmax of 2.1 h, Cmax of 622 microg equiv/mL, and AUC0-inf of 15124 h.microg equiv/mL. The t1/2 was 12.2 h. Soft tissue distribution of radioactivity indicated a fairly homogeneous distribution throughout the body with relatively lower concentrations in skin and bone. Approximately 85.8% of the dose was recovered in the urine after 120 h, whereas only 3% was found in the feces. No quantifiable levels of radioactivity were found in any tissues after 120 h, indicating complete elimination of [35S]MSM. The results of this study suggest that [35S]MSM is rapidly absorbed, well distributed, and completely excreted from the body.

Intact anthocyanins and metabolites in rat urine and plasma after 3 months of anthocyanin supplementation.

Anthocyanins are polyphenols responsible for most red to purple colors in plants. Human consumption of these pigments is increasing because of their potential health benefits and use as natural colorants. With more than 600 different anthocyanins found in nature, the impact of chemical structure on their absorption and metabolism needs to be investigated. Urine and plasma samples were collected from 32 rats receiving control diet or chokeberry-, bilberry-, and grape-enriched (3.85 g cyanidin 3-galatoside equivalent/kg) diet for 14 wk. Below 2 micromol/l of anthocyanins and relatively higher levels of presumable metabolites were detected by high-performance liquid chromatography-photodiode array in the plasma. In the urine the total concentration of intact anthocyanins and methylated derivatives ranged from 17.4 (bilberry) to 52.6 (chokeberry) nmol/l. The type and number of anthocyanin glycosylations affected the absorption remarkably. Detection of an acylated anthocyanin in plasma and urine suggests bioavailability of these anthocyanin derivatives that are commonly found in commercially available colorants.

Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats.

The aim of the present study was to investigate the chemoprotective activity of anthocyanin-rich extracts (AREs) from bilberry (Vaccinium myrtillus L.), chokeberry (Aronia meloncarpa E.), and grape (Vitis vinifera) by assessing multiple biomarkers of colon cancer in male rats treated with a colon carcinogen, azoxymethane. Fischer 344 male rats were fed the AIN-93 diet (control) or AIN-93 diet supplemented with AREs for 14 wk. Biomarkers that were evaluated included the number and multiplicity of colonic aberrant crypt foci (ACF), colonic cell proliferation, urinary levels of oxidative DNA damage, and expression of cyclooxygenase (COX) genes. To assess the bioavailability, levels of anthocyanins in serum, urine, and feces were evaluated. Total ACF were reduced (P<0.05) in bilberry, chokeberry, and grape diet groups compared with the control group. The number of large ACF was also reduced (P<0.05) in bilberry and chokeberry ARE-fed rats. Colonic cellular proliferation was decreased in rats fed bilberry ARE and chokeberry ARE diets. Rats fed bilberry and grape ARE diets had lower COX-2 mRNA expression of gene. High levels of fecal anthocyanins and increased fecal mass and fecal moisture occurred in ARE-fed rats. There was also a significant reduction (P<0.05) in fecal bile acids in ARE-fed rats. The levels of urinary 8-hydroxyguanosine were similar among rats fed different diets. These results support our previous in vitro studies suggesting a protective role of AREs in colon carcinogenesis and indicate multiple mechanisms of action.

Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth.

Commercially prepared grape (Vitis vinifera), bilberry (Vaccinium myrtillus L.), and chokeberry (Aronia meloncarpa E.) anthocyanin-rich extracts (AREs) were investigated for their potential chemopreventive activity against colon cancer. The growth of colon-cancer-derived HT-29 and nontumorigenic colonic NCM460 cells exposed to semipurified AREs (10-75 microg of monomeric anthocyanin/mL) was monitored for up to 72 h using a sulforhodamine B assay. All extracts inhibited the growth of HT-29 cells, with chokeberry ARE being the most potent inhibitor. HT-29 cell growth was inhibited approximately 50% after 48 h of exposure to 25 microg/mL chokeberry ARE. Most importantly, the growth of NCM460 cells was not inhibited at lower concentrations of all three AREs, illustrating greater growth inhibition of colon cancer, as compared to nontumorigenic colon cells. Extracts were semipurified and characterized by high-pressure liquid chromatography, spectrophotometry, and colorimetry. Grape anthocyanins were the glucosylated derivatives of five different anthocyanidin molecules, with or without p-coumaric acid acylation. Bilberry contained five different anthocyanidins glycosylated with galactose, glucose, and arabinose. Chokeberry anthocyanins were cyanidin derivatives, monoglycosylated mostly with galactose and arabinose. The varying compositions and degrees of growth inhibition suggest that the anthocyanin chemical structure may play an important role in the growth inhibitory activity of commercially available AREs.

Anthocyanin-rich extract from Aronia meloncarpa E induces a cell cycle block in colon cancer but not normal colonic cells.

Anthocyanin-rich extracts, potent antioxidants and commercially available food coloring agents, have been reported to inhibit growth of various cancer cell lines. We investigated the effect of semipurified anthocyanin-rich extract from fruits of Aronia meloncarpa, on normal colon and colon cancer cell lines. A 24-h exposure to 50 mg monomeric anthocyanin/ml of Aronia extract resulted in 60% growth inhibition of human HT-29 colon cancer cells. The treated cells showed a blockage at G1/G0 and G2/M phases of the cell cycle. The cell cycle arrest coincided with an increased expression of the p21WAF1 and p27KIP1 genes and decreased expression of cyclin A and B genes. Prolonged exposure to the extract resulted in no further change in the cell number, indicating a cytostatic inhibition of cell growth. NCM460 normal colon cells demonstrated <10% growth inhibition at the highest concentration of 50 mg/ml extract. A 35% decrease in the cyclooxygenase-2 gene expression was observed within 24 h of exposure of HT-29 cells but did not translate into decreased protein levels or protein activity. These results support the need for further research to identify the specific component(s) in this extract that suppress cancer cell growth and the genes affected by these natural compounds.