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.     

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。     

Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program searching for plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column (250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between 1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).     

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)、...     

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.     

Isolation and structural elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein from marigold flowers, kale, and human plasma

(13Z,13'Z,3R,3'R,6'R)-Lutein has been isolated and purified from extracts of marigold flowers, fresh raw kale (Brassica oleracea var. Acephala), and human plasma and fully characterized by (1)H and (13)C NMR, UV/vis, and MS. While the concentration of (13Z, 13'Z)-lutein in kale and human plasma compared to (all-E,3R,3'R, 6'R)-lutein was found to be quite low, this compound was readily isolated by fractional crystallization of lutein from marigold extracts. Thus, the mother liquors from two consecutive crystallizations of lutein from a saponified extract of marigold flowers were enriched in (13Z,13'Z)-lutein (8.7% of total carotenoids) and employed for the isolation of this compound by HPLC. The identity of the di-Z-lutein in kale and human plasma has been established by comparison of the HPLC-UV/vis-MS profiles of the purified compounds with those of a fully characterized sample, isolated from marigolds. 3-Hydroxy-beta,epsilon-caroten-3'-one and 3'-epilutein have also been identified in extracts from marigolds.     

Novel application of square-wave adsorptive-stripping voltammetry for the determination of xanthohumol in spent hops

This paper reports the development of a novel electrochemical assay for xanthohumol (XN) by square-wave adsorptive-stripping voltammetry (SWAdSV) with a hanging mercury drop electrode. The method showed good repeatability (CV < 2%) and linearity (between 10 and 250 μg L(-1)), as well as suitable limits of detection (2.6 μg L(-1)) and quantification (8.8 μg L(-1)). The method was applied for the quantification of this compound in spent hops, and the results obtained were compared with the HPLC-UV method. XN contents determined by the SWAdSV method were 16 ± 1 and 100 ± 4 μg L(-1) for aqueous and methanolic extracts, respectively. The developed new methodology considerably reduces the analysis time, approximately from 25 min (HPLC-UV method) to 7 min, enabling a high sample throughput. In addition, the detection and quantification limits were approximately 5-fold lower than those obtained with the chromatographic method.     

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.     

NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways

Metabolite profiling has been carried out to assess the compositional changes occurring in potato tubers after genetic modifications have been made to different metabolic pathways. Most major features in the (1)H NMR and HPLC-UV profiles of tuber extracts have been assigned. About 40 GM lines and controls belonging to 4 groups of samples (derived from cv. Record or cv. Desirée and modified in primary carbon metabolism, starch synthesis, glycoprotein processing, or polyamine/ethylene metabolism) were analyzed. Differences were assessed at the level of whole profiles (by PCA) or individual compounds (by ANOVA). The most obvious differences seen in both NMR and HPLC-UV profiles were between the two varieties. There were also significant differences between two of the four Desirée GM lines with modified polyamine metabolism and their controls. Compounds notably affected were proline, trigonelline, and numerous phenolics. However, that modification gave rise to a very abnormal phenotype. Certain lines from the other groups had several compounds present in significantly higher or lower amounts compared to the control, but the differences in mean values amounted to no more than a 2-3-fold change: in the context of variability in the whole data set, such changes did not appear to be important.     

Stability evaluation of selected polyphenols and triterpene glycosides in black cohosh

Black cohosh ( Actaea racemosa L., syn. Cimicifuga racemosa L.) is rich in both triterpene glycosides and polyphenols, which have various biological activities that may be important to its medical use. To evaluate the stability of the polyphenolic constituents and triterpene glycosides of black cohosh, experiments were conducted using three sample types: plant material, extracts of black cohosh, and encapsulated commercial extract. The samples were stored at various temperatures and humidity conditions. Three triterpene glycosides and six major polyphenols in black cohosh were quantitatively measured with an HPLC-PDA method at 0, 3, 6, and 9 weeks. The triterpene glycosides were stable at the tested conditions, whereas the polyphenols were stable only at room temperature and low humidity and not stable at higher temperature and/or humidity due to hydrolysis and/or oxidation. The rate of compound decomposition depended upon the chemical structure of the individual polyphenols. Polyphenols in the extracts decomposed more readily than those in plant material.     

The response of Stevia (Stevia rebaudiana Bertoni) to nitrogen supply under greenhouse condition

Stevia (Stevia rebaudiana Bertoni) is a perennial plant producing some natural sweeteners. Stevia is considered as a new crop in some countries. This study was conducted to find the stevia response to nitrogen fertilizer supply. Different levels of nitrogen (0, 30, 60, 90, 120, and 150 kg/ha from urea source) were used in a greenhouse condition and then the stevia growth and metabolites were assessed under different availability of nitrogen. Results showed that the optimum growth of stevia was obtained by 60 kg/ha nitrogen and more nitrogen supply did not enhance the stevia growth. It was observed that the total steviol glycosides (SVglys) content of Stevia was significantly increased by nitrogen fertilizer application just up to 30 kg/ha, while it decreased by more rates of nitrogen fertilizer. Our result clearly showed that SVglys yield reached to maximum value by application of 60 kg/ha of nitrogen fertilizer. Since the variation of SVglys content and shoot growth of the stevia were compromised by 60 kg/ha nitrogen, it can be concluded that 60 kg/ha of nitrogen fertilizer could be considered as an optimum rate of nitrogen for stevia and could also be recommended for greenhouse conditions.     

Chemical composition of shallot (Allium ascalonicum Hort.)

An extensive phytochemical analysis of the polar extracts from bulbs of shallot, Allium ascalonicum Hort., led to the isolation of two new furostanol saponins, named ascalonicoside A1/A2 (1a/1b) and ascalonicoside B (4), respectively, along with compounds 2a and 2b, most likely extraction artifacts. On the basis of 2D NMR and mass spectrometry data, the structures of the novel compounds were elucidated as furost-5(6)-en-3beta,22alpha-diol 1beta-O-beta-D-galactopyranosyl 26-O-[alpha-L-rhamnopyranosyl-(1-->2)-O-beta-D-glucopyranoside] (1a), its epimer at position 22 (1b), and furost-5(6),20(22)-dien-3beta-ol 1beta-O-beta-D-galactopyranosyl 26-O-[alpha-L-rhamnopyranosyl-(1-->2)-O-beta-D-glucopyranoside] (4). This is the first report of furostanol saponins in A. ascalonicum. High concentrations of quercetin, isorhamnetin, and their glycosides were also isolated and described.     

Characterization and quantification of hydroxycinnamate derivatives in Stevia rebaudiana leaves by LC-MSn

Stevia rebaudiana leaves are used as a zero-calorie natural sweetener in a variety of food products in Asian countries, especially in Japan. In this study, the hydroxycinnamate derivatives of S. rebaudiana have been investigated qualitatively and quantitatively by LC-MSn. Twenty-four hydroxycinnamic acid derivatives of quinic and shikimic acid were detected, and 19 of them were successfully characterized to regioisomeric levels; 23 are reported for the first time from this source. These comprise three monocaffeoylquinic acids (Mr 354), seven dicaffeoylquinic acids (Mr 516), one p-coumaroylquinic acid (Mr 338), one feruloylquinic acid (Mr 368), two caffeoyl-feruloylquinic acids (Mr 530), three caffeoylshikimic acids (Mr 336), and two tricaffeoylquinic acids (Mr 678). Cis isomers of di- and tricaffeoylquinic acids were observed as well. Three tricaffeoylquinic acids identified in stevia leaves are reported for the first time in nature. These phenolic compounds identified in stevia might affect the organoleptic properties and add additional beneficial health effects to stevia-based products.     

Isolation and structural elucidation of some glycosides from the rhizomes of smaller galanga (Alpinia officinarum Hance)

Glycosidically bound compounds were isolated from the methanol extract of fresh rhizomes of smaller galanga (Alpinia officinarum Hance). Nine glycosides (1-9) were finally obtained by reversed-phase HPLC and their structures were elucidated by MS and NMR analyses. They were the three known glycosides, (1R,3S,4S)-trans-3-hydroxy-1,8-cineole beta-D-glucopyranoside (1), benzyl beta-D-glucopyranoside (3), and 1-O-beta-D-glucopyranosyl-4-allylbenzene (chavicol beta-D-glucopyranoside, 4); and the six novel glycosides, 3-methyl-but-2-en-1-yl beta-D-glucopyranoside (2), 1-hydroxy-2-O-beta-D-glucopyranosyl-4-allylbenzene (5), 1-O-beta-D-glucopyranosyl-2-hydroxy-4-allylbenzene (demethyleugenol beta-D-glucopyranoside, 6), 1-O-(6-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl)-2-hydroxy-4-allylbenzene (demethyleugenol beta-rutinoside, 7), 1-O-(6-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl)-4-allylbenzene (chavicol beta-rutinoside, 8), and 1,2-di-O-beta-D-glucopyranosyl-4-allylbenzene (9). Compounds 2-9 were detected for the first time as constituents of galanga rhizomes.     

Soil quality effects on Chenopodium album flavonoid content and antioxidant potential

Antioxidant capacity, total phenolic content and flavonoid glycosides profile were compared in C.album samples grown in intensively cultivated (IC) and nondisturbed (ND) soils to evaluate differences in their nutraceutical potential. Petroleum ether, methanol, and aqueous extracts were sequentially obtained from C. album dried samples. Methanol crude extract exhibited the highest antioxidant potential and phenolic content, which were significantly enhanced by soil deterioration. This feature was enhanced in its ethyl acetate/n-buthanol subextract that also yielded higher amounts of the fraction containing flavonoid glycosides in samples grown in IC soils. Compounds were isolated by activity guided fractionation, and chemical structure-antioxidant activity relationships were established. Chemical structures were elucidated by chemical and spectroscopic methods. Six known flavonoid glycosides were isolated, and their antioxidant activity was determined by DPPH assay. 1, quercetin-3-O-(2",6"-di-O-R-L-rhamnopyranosyl)-beta-D-glucopyranoside; 2, kaempferol-3-O-(2",6"-di-O-R-L-rhamnopyranosyl)-beta-D-glucopyranoside; 3, quercetin-3-O-beta-D-glucopyranosyl-(1'-->6")-beta-D-glucopyranoside; 4, rutin; 5, quercetin-3-O-beta-D-glucopyranoside; and 6, kaempferol-3-O-beta-D-glucopyranoside. Triosides 1 and 2 were identified for the first time in C. album. Our results suggest that this edible weed, ubiquitously present in cultivated fields, should be considered as a nutraceutical food and an alternative source for nutrients and free radical scavenging compounds, particularly when collected from cultivated fields that seem to increase some of its advantages.     

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。     

甜菊醇糖苷生物合成途径关键酶基因KA13H的克隆及序列分析

本研究利用RT-PCR方法从甜叶菊叶片中分离了一个与甜菊醇糖苷生物合成密切相关的基因KA13H,对该基因的ORF、编码产物结构、同源性比对及次级结构等进行了生物信息学预测分析,同时初步分析该基因的组织表达和原核表达。经DNAMAN6.0软件分析,该基因编码一条分子量为54.476kD的由476个氨基酸残基组成的多肽,含有典型的细胞色素P450的血红素结合位点FXXGXXXCXG,TMPRED程序预测其C-端含有一个明显的跨膜区MIQVLTPILLFLIFFVFWKVY,是一个典型的细胞色素P450基因。推断的KA13H编码产物与其它生物合成相关细胞色素P450同源比对和系统发生分析表明,该蛋白与苜蓿(Medicago truncatula)CYP716A12和云杉(Sitka spruce)CYP720B1的一致性较高,分别为51%和46%,推测KA13H可能与CYP716A12和CYP720B1具有类似的功能。KA13H次级结构分析结果表明,KA13H与CYP74A2的一致性仅为15%,但是它们却有着类似的次级结构,都含有6个基质识别位点(SRSs)。半定量RT-PCR分析表明:KA13H在根、茎、叶和花中...     

MALDI-TOF MS analysis of food flavonol glycosides

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that is having a great impact on food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify flavonol glycosides in food samples. 2',4',6'-Trihydroxyacetophenone was chosen as the best matrix because it worked for crude sample extracts and ionized flavonol glycosides in both positive and negative MALDI-TOF MS modes. In the positive mode, multiple ion forms were observed for flavonol glycosides, including [M + H](+), [M + Na](+), [M + K](+), and [M - H + Na + K](+), with further fragmentation through loss of glycosidic residues. The negative mode for all flavonol glycosides resulted in [M - H](-) ion formation without detectable fragmentation. The multiple ions in the positive mode gave more information on individual flavonol glycoside structures than the negative mode. Flavonol glycosides showed similar intensities or responses in the positive mode, while kaempferol glycosides exhibited much less response than quercetin glycosides in negative mode.