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.     

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.     

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。     

甜叶菊中莱苞迪苷D、莱苞迪苷A含量测定方法的优化及应用

为比较不同品系甜叶菊中甜味品质较好的莱苞迪苷D(RD)、莱苞迪苷A(RA)含量组成,在传统C18、HSS T3、Amide色谱柱中选择适宜固定相建立高效液相色谱(HPLC)方法进行测定与分析。结果表明,HSS T3柱对甜菊糖苷选择性较好,可同时分离RA、甜菜苷(ST)、莱苞迪苷F(RF)、莱苞迪苷C(RC)、甜茶苷(RBS)、莱苞迪苷B(RB)、甜菊双糖苷(SB),其HPLC分析参数为:流动相32%乙腈和68%磷酸水(0.01%),等度洗脱,柱温40℃,波长210 nm,进样量10 μL,流速1.0 mL·min^-1;Amide柱对RD分离能力最佳,其HPLC分析参数为:流动相76%乙腈和24%水,等度洗脱,柱温40℃,波长210 nm,进样量10 μL,流速0.8 mL·min^-1。 分析比较12个扦插培育的甜叶菊品系,以编号2甜叶菊中RD含量及其占比最高,提示以其为原材料可生产含RD较高的甜菊糖苷;编号3、5、7、11甜叶菊中具较有高含量的甜菊糖苷(主要为RA),提示这些品种富含RA且甜菊糖苷产量较高;编号1、8甜叶菊中RA+RD占比较高,提示以其为原材料的甜菊糖苷甜味品质较好。本研究所建立的HPLC法为甜叶菊中RD、RA分析研究提供了方法参考,含量分析结果可为实际应用中选择适宜甜叶菊品种提供依据。     

施钾对甜叶菊糖苷跃变期生理指标和产量品质的影响

为提高河西绿洲灌区甜叶菊产量和品质,提升该区甜叶菊市场竞争力。以谱星6号为试验材料,比较了不同施钾水平(K2O 0、60、90、120、150 kg/hm2)对甜叶菊糖苷跃变期生理指标以及产量品质的影响。结果表明,不同施钾处理下,甜叶菊整个糖苷跃变期叶片各生理指标含量达到峰值的时间有所不同,叶片SOD活力、含钾量均在现蕾前5 d达到最高,可溶性蛋白、SPAD值、含氮磷量、总苷含量以及叶片干物质量在现蕾初期达到最高,POD活力、可溶性糖含量在现蕾后5 d达到最高,以上各指标均以施用K2O 120 kg/hm2处理表现最佳。其中总苷含量最高,达128.4 g/kg,较不施钾肥增加14.19%;产量也最高,达7 007.64 kg/hm2,较不施钾肥增加57.24%;净收益达5.26万元/hm2,比不施钾多3.40万元/hm2。通过甜叶菊叶片理化指标与产量品质的相关性分析,以上各生理指标与产量品质均呈现显著或者极显著的正相关关系。在当地甜叶菊生产中施用K2O 120 kg/hm2,且在现蕾初期采摘叶片可获得较好收益。     

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.     

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)、...     

Retention of alkamides in dried Echinacea purpurea

Different drying methods, such as freeze-drying (FD), vacuum microwave drying (VMD), and air-drying (AD), were applied to fresh roots and leaves of Canadian-grown Echinacea purpurea to determine the optimal method for preserving alkamide levels. Using HPLC, six alkamide fractions (alkamides 1, 2, 3, 6a/6, 7, 8/9) were quantitated in dried roots, whereas four alkamide fractions (alkamides 1, 2, 3, 8/9) were measured in dried leaves. Different elution conditions used in HPLC for alkamide analysis did not affect the eluted fractions nor the quantitation of different alkamides. Individual alkamide concentrations in roots and leaves were affected by the drying methods used. To preserve higher levels of total alkamides, FD was found to be the best method, VMD was a superior method for drying roots than AD at 70 degrees C, while AD at 50 degrees C was the preferred method for drying leaves of E. purpurea.     

Oil and squalene in amaranthus grain and leaf

Amaranthus grain of 104 genotypes from 30 species was investigated for oil and squalene contents and fatty acid profiles. The overall average oil content in Amaranthus grain was 5.0%, ranging from 1.9 to 8.7%. Squalene concentration in extracted oils ranged from trace to 7.3%, with an average concentration of 4.2%. The average contents of three major fatty acids in Amaranthus grain were 22.2, 29.1, and 44.6% for palmitic, oleic, and linoleic, respectively. The average fat content in dried mature leaves of 45 Amaranthus genotypes was 1.63%, ranging from 1.08 to 2.18%. The squalene concentration in leaf lipid extracts averaged 0.26%, ranging from trace to 0.77%, which is much lower than that from seeds. The major fatty acids of leaf extracts were linolenic, linoleic, and palmitic. Linolenic ranged from 56.5 to 62.0% of total fatty acids; linoleic, from 15.5 to 24.7%; and palmitic acid, from 13.5 to 15.5%. As for the fatty acid compositions at different growth stages, fatty acid content in leaf lipid was lower in mature leaves than in young leaves. The saturated/unsaturated ratio decreased when the leaf grew to maturity. Principal component analysis (PCA) was carried out on compositional characteristics of grain. The first two components accounted for 70% of the total variance (38.3 and 21.7%, respectively). There was a positive correlation between oil content and squalene yield, and a negative correlations were found between linoleic and either of the other two major fatty acids, palmitic and oleic. The taxonomic relationship among the species was also elucidated by PCA.     

噻菌茂在烟叶和土壤中的残留消解动态及安全性评价

为明确噻菌茂在烟草上使用后的环境安全性,建立了烟叶和土壤中噻菌茂残留的检测方法,并在山东和湖南两地开展了为期两年的噻菌茂在烟叶及其土壤中的消解动态和最终残留研究。结果表明,采用甲醇/水（70：30,V：V）提取,石油醚、二氯甲烷萃取,弗罗里硅土净化,高效液相色谱（HPLC-UV）测定,在0.01～5.0mg.kg-1添加水平下,噻菌茂在鲜烟叶、干烟叶和土壤中的平均回收率分别为90.50%～93.84%、88.19%～91.90%和88.34%～93.04%,相对标准偏差（RSD）分别为1.72%～2.79%、1.83%～4.13%、2.00%～2.71%。噻菌茂的最小检出量为1.4×10-12g,最低检出浓度分别为：鲜烟叶0.01mg.L-1,干烟叶0.01mg.L-1,土壤0.005mg.L-1。田间试验结果表明,噻菌茂在烟叶和土壤中消解较快,半衰期分别为2.85～3.44d和2.77～3.26d。噻菌茂可湿性粉剂按有效成分250g.hm-2（推荐高剂量）和375g.hm-（21.5倍推荐高剂量）于烟草旺长期-成熟期兑水施药3～4次,烟叶中噻菌茂最终残留量随采收间隔时间的延长而呈递减趋势,距末次施药后间隔7d采收的烟叶中噻菌茂的残留量为1.102～4.230mg.kg-1,21d残留量降为0.082～1.813mg.kg-1;而土壤中噻菌茂最终残留量均未检出（≤0.005mg.kg-1）。     

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.     

Determination of vitamin B(6) in cooked sausages

A reverse-phase high-performance liquid chromatography (HPLC) method has been described for the determination of various active forms of vitamin B(6) in meat products. Different extracting agents were tested to solubilize fully the analyte for quantification. The best data were obtained by extracting the samples with 5% (w/v) metaphosphoric acid. Separation by HPLC was performed with fluorescence detection (excitation, 290 nm; emission, 395 nm), on a 10 cm x 0.46 cm i.d. Hypersil BDS C(18) 5 microm column using a mixture of 50 mM phosphate buffer (pH 3.2) and acetonitrile (99:1, v/v) as mobile phase. Precision of the method was 0.5% (within a day) and 4.3% (between days). The detection limits were 0.020 mg/100 g for pyridoxal and pyridoxamine, 0.017 mg/100 g for pyridoxamine phosphate, 0.500 mg/100 g for pyridoxal phosphate, and 0.033 mg/100 g for pyridoxol, with a signal-to-noise ratio of 3. The recovery ranged from 92.0 to 100.0%.     

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.     

Chemical composition of the essential oils of Juniperus from ripe and unripe berries and leaves and their antimicrobial activity

The composition of the essential oil from ripe and unripe berries and leaves of Juniperus oxycedrus L. ssp. oxycedrus, Juniperus phoenicea ssp. turbinata and Juniperus communis ssp. communis was analyzed by GC-MS, and microbiological assays were carried out. Samples were collected in different localities (Sardinia, Italy) and hydro distilled. The yields ranged between 2.54% +/- 0.21 (v\w dried weight) and 0.04% +/- 0.00. A total of 36 components were identified. The major compounds in the essential oils were alpha-pinene, beta-pinene, delta-3-carene, sabinene, myrcene, beta-phellandrene, limonene, and D-germacrene. Both qualitative and quantitative differences between species and between different parts of the plant were observed. The essential oils and their major compounds were tested against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the minimum inhibitory concentration and minimum bactericidal concentration were determined. The results obtained led to a nonsignificant inhibitory effect, although all the essential oils from Juniperus phoenicea ssp. turbinata and the essential oil from leaves of Juniperus oxycedrus ssp. oxycedrus exhibited rather good or weak activity against Candida albicans and Staphylococcus aureus.     

Extraction, processing, and storage effects on curcuminoids and oleoresin yields from Curcuma longa L. grown in Jamaica

Aromatic diarylheptanoid compounds from Curcuma longa Linn grown in Jamaica were quantified by UV-vis spectrophotometry and high-performance liquid chromatographic (HPLC) analyses. The oleoresin yields from ethanolic extracts were quantified and evaluated with regard to the effects of the type of postharvesting process and the type of extraction method conducted on the plant material. Fresh samples that were hot solvent extracted provided the highest oleoresin yields of 15.7% +/- 0.4 ( n = 3), and the lowest oleoresin yields of 7.8% +/- 0.2 ( n = 3) were from the dried milled samples that were cold solvent extracted. Data from the ASTA spectrophotometer assay confirmed that dried samples contained the highest curcuminoid content of 55.5% +/- 2.2 ( n = 6) at the fifth month of storage, and the fresh samples showed a curcuminoid content of 47.1% +/- 6.4 ( n = 6) at the third month of storage. A modified HPLC analysis was used to quantify curcumin content. Data from the HPLC analysis confirmed that the dried treated, hot extracted, room temperature stored samples had the highest curcumin content of 24.3%. A novel high-performance thin layer chromatography (HPTLC) method provided a chemical fingerprint of the C. longa with the use of a commercial curcumin standard.     

Determination of thiamin in cooked sausages

A reliable and rapid high-performance liquid chromatography (HPLC) method has been set up for the determination of total thiamin in difficult sample matrices such as cooked sausages. Different hydrolysis conditions and enzymes were tested to release the vitamin from its phosphate ester. The best data in the enzymatic digestion were obtained by incubating the samples with 6% clara-diastase at 50 degrees C for 3 h. After oxidation of thiamin to thiochrome, the sample extracts were purified by using a C(18) Sep-Pak cartridge. Final determination was performed by reversed-phase HPLC with fluorescence detector (excitation 360 nm, emission 430 nm), on a low-cost 25 cm x 4 mm i.d. Spherisorb C(8) cartridge using a mixture of 5 mM phosphate buffer pH 7.0 and acetonitrile (70:30, v/v) as mobile phase. Precision of the method was 1.5% (within a day) and 5. 2% (between days). The detection limit was 0.015 mg/100 g. All the recoveries from the different cooked sausages were better than 90% of thiamin hydrochloride added to samples of meats. In the samples analyzed, the mean value for thiamin was between 0.039 and 0.508 mg/100 g fresh weight.     

Rapid and sensitive determination of phenol in honey by high-performance liquid chromatography with fluorescence detection

The objective of this research was to develop a novel high-performance liquid chromatographic (HPLC) method involving a simple sample preparation procedure for the rapid, low-cost, and sensitive quantitation of phenol in honey at levels of regulatory and practical importance. After proper dilution of honey with water, the samples were analyzed by a gradient HPLC system, using a reversed-phase column with fluorescence detection at excitation and emission wavelengths of 270 and 300 nm, respectively. The eluents applied were water-acetonitrile-85% orthophosphoric acid (10:10:0.01, v/v/v) and water-85% orthophosphoric acid (20:0.01, v/v). The retention time of phenol was found to be 14.1 min, and the limit of quantitation for phenol in honey was set at 5 microg/kg. Overall recovery was 98%. The proposed method has been successfully applied to real sample analysis.     

Chemical composition of volatiles in Sardinian myrtle (Myrtus communis L.) alcoholic extracts and essential oils

The chemical composition of the volatile fraction of myrtle (Myrtus communis L.) alcoholic extracts and essential oils from leaves and berries collected in different places in Sardinia (Italy) was studied. A simple and rapid liquid-liquid extraction method was used to isolate volatile compounds from myrtle alcoholic extracts followed by GC and GC-MS analysis allowing the detection of 24 compounds. The volatile fraction was characterized by the terpenes fraction corresponding to that of the essential oils and by a fatty acid ethyl esters fraction. The variation during extraction of the volatile fraction in alcoholic extracts of berries and leaves was evaluated. Essential oils were obtained by hydrodistillation, and the yields were on average 0.52 +/- 0.03% (v/w dried weight) and 0.02 +/- 0.00% for leaves and berries, respectively. The essential oils were analyzed by GC and GC-MS, and a total of 27 components were detected, accounting for 90.6-98.7% of the total essential oil composition. Strong chemical variability depending on the origin of the samples was observed. The major compounds in the essential oils were alpha-pinene (30.0 and 28.5%), 1,8-cineole (28.8 and 15.3%), and limonene (17.5 and 24.1%) in leaves and berries, respectively, and were characterized by the lack of myrtenyl acetate.     

Microwave-assisted rapid determination of vitamins a and e in beverages

A new rapid procedure for the determination of vitamins A and E in beverages has been developed and validated. Key steps include a microwave-assisted saponification of the sample and a single-step extraction of the vitamins prior to HPLC analysis. All sample preparation steps are carried out consecutively in the same vial. The vitamins are determined using normal-phase (Si-60) HPLC with fluorescence detection. The method is applicable to beverages with a content of all-trans-retinol >0.14 mg/L and/or a content of alpha-tocopherol >1 mg/L. Recoveries determined by spiking experiments ranged from 91.3 to 106.3%. The precision of the method is characterized by relative standard deviations of <2% for alpha-tocopherol and <5% for all-trans-retinol.