A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway

Molecular Biology Reports - We herein report the preparation of a full-length raucaffricine-O-beta-d-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450)....     

Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni

A promising method of micropropagation of Stevia rebaudiana Bertoni has been developed with an aim to increase the biomass, survivability of the plantlets and stevioside production, using chlorocholine chloride (CCC). Microshoots transferred to the MS medium containing different combinations CCC and IBA were found to be most effective in terms of growth pattern, hardening ability of the plantlets and stevioside content, compared to MS medium containing either IBA or CCC. Among other combinations tested, MS medium supplemented with 3 mg/l CCC and 3 mg/l IBA was found most effective in inducing significant changes like reduced shoot length, increased number of roots, higher leaf size, increased biomass and chlorophyll retaining capacity, higher survival percentage and most importantly the elevated stevioside content. Collectively, the major observations of this research indicate that application of CCC in micropropagation of S. rebaudiana Bertoni is a promising approach and has commercial prospects.     

Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors

Isosteviol (ent-16-ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and related compounds from stevioside by microbial transformation and chemical conversion and assayed the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases (pols) and human DNA topoisomerase II (topo II), and IC50 value for pol alpha was 64.0 microM. This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I, and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. With pol alpha, isosteviol acted non-competitively with the DNA template-primer and nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD50 values of 84-167 microM, and 500 microg of the compound caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship between the structure of stevioside-based compounds and these activities were discussed.     

Stevioside

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.     

Dulcosides A and B,new diterpene glycosides from Stevia rebaudiana

New diterpene glycosides, dulcosides A and B were isolated from Stevia rebaudiana Bertoni and their structures were established as 19-O-β-glu     

甜菊良种的单株选育

经高效液相色谱分析及部分农艺性状的综合评价 ,从 3个甜菊 (SteviarebaudianaBertoni)优良品系 (中山一号、YS0 0 3和YS0 0 4)的自然并辅助人工授粉结实得到的 13株单株中筛选出 2株优良单株SZ990 0 40 0 3和SZ990 0 40 11,SZ990 0 40 0 3单株干叶总甙含量为 16 .6 4% ,R A甙 13.34 % ,R A甙 /总甙为 80 .17% ;SZ990 0 40 11单株干叶总甙含量 19.2 0 % ,R A甙 12 .30 % ,R A甙 /总甙为 6 4.0 6 %     

Characterization of bacterial mutagenicity mediated by 13-hydroxy-ent-kaurenoic acid (steviol) and several structurally-related derivatives and evaluation of potential to induce glutathione S-transferase in mice

Stevioside is a sweet-tasting diterpene glycoside that is derived from Stevia rebaudiana (Bertoni) Bertoni (Compositae). It is used commercially in Japan and other parts of the world as a sucrose substitute. Whereas stevioside demonstrates no mutagenic activity in a variety of test systems, the aglycone, steviol (13-hydroxy-ent-kaurenoic acid), is mutagenic toward Salmonella typhimurium strain TM677 in the presence of a metabolic activating system derived from the liver of Aroclor 1254-pretreated rats. The required activating component is localized in the microsomal fraction of rat liver, suggestive of a cytochrome P-450-mediated reaction. Partially purified epoxide hydrolase does not inhibit steviol-induced mutagenicity, indicating that an active metabolite is not an epoxide that serves as a substrate for this enzyme preparation. The 13-hydroxy group of steviol is required for the expression of mutagenicity since ent-kaurenoic acid is nonmutagenic, and acetylation of steviol at this position negates mutagenicity. Similarly, diterpenes bearing a strong structural resemblance to steviol, cafestol and kahweol, were found to demonstrate no mutagenic activity toward Salmonella typhimurium TM677, as were their respective acetates and palmitic acid esters. Conversely, 19-O-beta-D-glucopyranosyl steviol, a potential hydrolysis product of stevioside, is mutagenic and bactericidal in the presence of a metabolic activating system. Additionally, in contrast to the nonmutagenic diterpenes cafestol and kahweol that are effective as inducers of glutathione S-transferase activity, evaluation by administration to mice proved steviol, isosteviol and various steviol glycosides to be inactive in this process. Thus, structural differences among these naturally occurring and semi-synthetic diterpenes appear to impart major differences in biological activity that may relate to human health upon dietary ingestion.     

R－A型甜菊糖甙的研制

以甜菊（ＳｔｅｖｉａｒｅｂａｕｄｉａｎａＢｅｒｔｏｎｉ）的干叶为原料生产的甜菊糖甙是一种食用天然甜味剂。提高甜菊糖甙（Ｓｔｅｖｉｏｓｉｄｅｓ）中优质甜味成分Ｒ－Ａ（甜菊Ａ３甙：ＲｅｂａｕｄｉｏｓｉｄｅＡ）的含量比例是生产中急待解决的课题。本文以Ｒ－Ａ型和Ｓｔ（甜菊甙Ｓｔｅｖｉｏｓｉｄｅ）型甜菊叶为原料,采用Ａ和Ｂ两种方法进行甜菊糖甙的提取,通过Ｂ法获得Ｒ－Ａ含量比例高的甜菊糖甙即Ｒ－Ａ型甜菊糖甙,为甜菊糖甙产品优质化提供了新方法、新技术     

Downstream processing of stevioside and its potential applications

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana Bertoni, which is commercially produced by conventional (chemical/physical) processes. This article gives an overview of the stevioside structure, various analysis technique, new technologies required and the advances achieved in recent years. An enzymatic process is established, by which the maximum efficacy and benefit of the process can be achieved. The efficiency of the enzymatic process is quite comparable to that of other physical and chemical methods. Finally, we believe that in the future, the enzyme-based extraction will ensure more cost-effective availability of stevioside, thus assisting in the development of more food-based applications.     

Steviol Glycoside Content in Different Organs of Stevia rebaudiana and Its Dynamics during Ontogeny

The contents of three major steviol glycosides (SGs) (stevioside and rebaudiosides A and C) in vegetative and generative organs during ontogeny of Stevia rebaudiana Bertoni were analysed with HPLC. Plant organs contained different amounts of the SGs, which declined in the following order: leaves, flowers, stems, seeds, roots. The highest content of the SGs was found in upper young actively growing shoot sections, whereas lower senescent shoot sections exhibited the lowest amount of such compounds. During ontogeny a gradual increase in the SG content was observed in both mature stevia leaves and stems, and this process lasted up to the budding phase and the onset of flowering. This revised version was published online in July 2006 with corrections to the Cover Date.     

盐胁迫对甜菊细胞内游离Ca^2＋浓度的影响（简报）

Fluo-3/AM, a calcium indicator, was introduced by low temprature loading method into callus protoplasts of Stevia rebaudiana Bertoni. The microscope observation results showed that NaCl in different concentrations (30-200mmol/L) can elevate the intracellular calcium concentration in protoplasts. The elevation was related to the amount of salinity. The effect of salinity stress was inhibited by LiCl pretreatment but restored by inositol pretreatment. This suggested that salinity stress promoted the cytoplasmic calcium activity, perhaps by activating the phosphoinoditide system. As a result of salinity stress signals, elevated calcium activity may trigger corresponding metabolic changes, such as synthesis of enzyme via activating other members of calcium signal system, fit the cells for the change of the environment.     

Revision and cladistic analysis of the eumenine wasp genus Pseudodynerus de Saussure (Hymenoptera, Vespidae, Eumeninae)

Abstract.  The eumenine genus Pseudodynerus is revised, and the phylogenetic relationships among its species are reconstructed based on adult morphology. The cladistic analyses of the 21 terminal species (15 ingroup and six outgroup species) and 48 characters, from external morphology and male genitalia, produced one cladogram under equal weights and two under implied weights. The monophyly of Pseudodynerus was corroborated in all three hypotheses, although the internal relationships varied depending on the weighting scheme. Two major clades were recovered under both equal and implied weights, one containing mostly Central and North American species and the other only South American species. The main difference between the hypotheses involves the placement of P. luctuosus , which comes out either as sister to a clade formed by P. hallinani  + ( P. maxillaris  +  P. quadrisectus  +  P. crypticus ) or as sister to a clade comprising the remaining species. Fifteen species of Pseudodynerus are recognized, of which four are described as new: P. crypticus from Costa Rica, P. carpenteri from French Guiana and Suriname, P. obesus from Rondônia, Brazil, and P. singularis from Espírito Santo, Brazil. One new synonymy is proposed: Odynerus ( Odynerus ) aztecus de Saussure, 1857  =  Pseudodynerus quadrisectus ( Say, 1837 ) syn.n. Names treated as varieties by Bequaert (1941) are not recognized at any level herein, as follows: Monobia mina-palumboi Gribodo, 1891 , stat.rev. ; Odynerus ( Leionotus ) beelzebub Zavattari, 1912 , stat.rev. ; Stenodynerus anisitsii garleppi Schrottky, 1911 , stat.rev. ; and Stenodynerus mondaiensis Bertoni, 1918, stat.rev. Lectotypes are designated for Odynerus bellone Lepeletier, 1841 , Odynerus ( Odynerus ) aztecus de Saussure, 1857 and Odynerus ( Stenodynerus ) migonei Bertoni, 1926 .     

Identification of attractant and repellent plants to coffee berry borer,Hypothenemus hampei

Colombia is one of the world's largest producers of coffee [Coffea arabica L. (Rubiaceae)]. The coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), is the main pest of coffee. This insect is controlled through an integrated pest management program that includes cultural, biological, and chemical control strategies. Despite research seeking CBB attractants and repellents, these potential management tools have not been successfully incorporated into control programs. This work proposes the use of plant functional diversity for CBB management, for which a number of plants related to coffee and weeds were selected. CBB preference to these plants was determined by olfactometry and volatile compounds emitted by them were identified. Field trials were performed to test CBB preference under field conditions. These trials determined the olfactory preference of CBB to coffee berries accompanied by material of the plants Crotalaria micans Link (Fabaceae), Lantana camara L. (Verbenaceae), Nicotiana tabacum L. (Solanaceae), Artemisia vulgaris L., Calendula officinalis L., Stevia rebaudiana (Bertoni) Bertoni, and Emilia sonchifolia (L.) DC. (all four Asteraceae). Under laboratory conditions N. tabacum, L. camara, and C. officinalis were identified as repellents for CBB in olfactometer assays, whereas E. sonchifolia functioned as attractant. Controlled field trials corroborated CBB repellency of N. tabacum and L. camara; both release volatile sesquiterpenes. Selected candidate attractants included E. sonchifolia plants, for showing attraction in the laboratory. The potential use of these plants in agroecological management of coffee plantations is discussed.     

盐胁迫对甜菊细胞内游离Ca2+浓度的影响

钙离子不仅是植物生长发育所需的一种大量元素,而且起了偶联细胞外刺激与胞内反应第二信使的作用,是多种受体激动后信息传递过程的中心环节。近年的研究表明:高温、干旱、触摸、低渗、低温、风、机械刺激、病原菌感染等多种环境胁迫均能引起胞质Ca~(2+)水平的改变。这种变化被认为是植物细胞感受环境胁迫的原初反应之一,它通过启动基因表达和胞内一系列生理生化过程调节细胞对环境改变的适应反应。胞质Ca~(2+)水平的改变有两种来源:胞外Ca~(2+)跨膜内流和胞内钙库如内质网中的Ca~(2+)释放。其中由肌醇磷     

Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light

The aim of this study was to determine whether steviol glycoside accumulation is under phytochrome control. The results indicate that Stevia rebaudiana Bertoni plants grown under short-day conditions showed precocious flowering and stagnation of steviol glycoside accumulation. Long night interruption by red LED light stimulated and sustained the vegetative growth as well as the accumulation of steviol glycosides in the leaves. After 7 weeks of treatment, steviol glycoside content was about two-fold higher in LED-treated plants than in the short-day control group. The effects of red LED light were measured both in a greenhouse and in a phytotron, irrespective of cultivar-specific differences. Therefore, it can be concluded that a mid-night interruption by red LED light during short photoperiods provides an easy and inexpensive method to increase vegetative leaf biomass production with an increased steviol glycoside yield.     

Bactericidal Activity of a Fermented Hot-Water Extract from Stevia rebaudiana Bertoni towards Enterohemorrhagic Escherichia coli O157:H7 and Other Food-Borne Pathogenic Bacteria

A fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157:H7. The colony-forming ability of the food-borne pathogenic bacteria tested so far was reduced to < 10^?7 when exposed to ?40% (v/v) solutions of the fermented extract at 37 C for 2 hr. Secretion of verocytotoxin 1 and 2 by enterohemorrhagic E. coli was also diminished by fermented extract at a concentration of ?10% (v/v). In contrast, the fermented extract did not significantly kill Bifidobacteria or Lactobacilli. The active principle(s) of the fermented Stevia extract were bactericidal under acidic conditions.     

甜叶菊RAPD反应体系的优化

以甜叶菊为试材,对影响其RAPD反应体系的7个因子进行优化.结果表明,20 μL的优化体系包括:双蒸水13.6 μL,10×Buffer(含15 mmol/L MgCI2)溶液3μL,2.5 mmol/L的dNTPS 1.2 μL,10 μmol/L的引物1μL,20 ng的模板DNA 1μL,1UTaq聚合酶;热循环...     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

甜菊叶愈伤组织诱导过程中叶绿体的超微结构变化

观察了甜菊(Stevia rebaudiana Bertoni)叶外植体愈伤组织诱导过程中叶绿体的超微结构变化。结果表明,当叶外植体转移到培养基上培养后,叶绿体的片层结构逐渐退化。在叶绿体发生退化的过程中伴有叶绿体出芽和原质体的形成。推测新产生的原质体来自叶绿体产生的芽状体。而叶绿体本身最后完全解体消失。叶绿体超微结构的这种变化与高度液泡化的叶肉细胞脱分化至分生状态是平行的。随着培养的进行,分生状态的细胞发生液泡化变为薄壁细胞时,在愈伤组织表层的细胞中,质体重新形成片层结构,而内部细胞的质体则充满淀粉粒。