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Plants under different environmental regimes exhibit phenotypic plasticity, sometimes producing more secondary metabolites when microenvironmental conditions are manipulated but these responses may be species, cultivar and/or genotype dependent. To test the hypothesis of whether in vitro plants of S. rebaudiana Bertoni would accumulate higher amounts of steviol glycosides when plants were growing under salt and drought stress, cultivar ST2100 plants were used. We thus applied 25 to 100 mM NaCl and polyethylene glycol 6000 (PEG) at 2.5% to 10.0% (w/v) to generate different Murashige and Skoog (Physiol Plant 15:473–497, 1962) media. Microplant cultures were also profiled for stevioside, rebaudioside A and steviol via LC–MS. Essential oil chemicals and fatty acids were assessed using GC–MS. Finally, a chemometric analysis of ethanolic extracts produced from treated and control plants is presented from MSE fragmentation data and various phenolic acids were tentatively identified using ion fragmentation patterns. Increasing amounts of both NaCl and PEG led to poor growth and development in cultures of S. rebaudiana. For example, the 25 and 50 mM NaCl-treated plants had fewer roots in comparison to controls and at even higher concentrations (75 and 100 mM NaCl), plants did not to root. Poor in vitro organogenesis was more pronounced with PEG. For instance, when plants were placed on a 10% PEG-medium, the ability for shoot regeneration was lost and callus became more apparent. Increasing levels of NaCl and PEG were also correlated to lowered levels of rebaudioside A and stevioside. In relation to the control plants that had 0.054 mg g−1 FW of steviol, the 25 mM NaCl treatment group had highest levels of this compound, recorded at 0.156 mg g−1 FW. All other salt treatments led to trace amounts of this chemical (0.005–0.009 mg g−1 FW) and it was not detected in any of the PEG-treated plants, except for the controls. The PCA loadings plots exposed stevioside, rebaudioside E and a steviol glycoside derivative as the MS signals that contributed to discriminant clusters segregating controls from the NaCl-treated groups. For PEG, segregation in the PCA is mostly influenced by dicaffeoylquinic acid as a marker ion, separating the controls from the treatment groups. PEG-treatments caused more prominent changes to the essential oil chemistry of Stevia plants. This was evident when 7.5 or 10% PEG was applied as sabinene, α-terpinolene, n-amyl isovalerate, 7-octen-4-ol, α-bergamotene, junipene, (+)-calarene, α-cadinol, β-pinene, α-bergamotene, (+)-calarene and junipene became undetectable. Changes of this nature may be undesirable when aromatic oils of S. rebaudiana are targetted for commercial markets as our data suggest adjustment to stresses may negatively impact volatile compounds leading to a loss of bioactive aromatic compounds. This study reports, for the first time, the effects of salinity and drought conditions in vitro on changed essential oil profiles of S. rebaudiana, providing new insights into the effects of stress on the essential oil chemistry of S. rebaudiana. Key message: Stevia responses to salt and drought stresses in vitro lead to lowered measured steviol glycosides (i.e stevioside, rebaudioside A and steviol) and significantly changes the essential oil terpenoid profiles [ABSTRACT FROM AUTHOR]

The concentrations of steviol and its derivatives stimulating the growth of wheat plants were measured: 10–8 for stevioside and 10–9 М for steviol and isosteviol. It was found that stevioside increased the activity of amylolytic enzymes and protein content, as well as frost tolerance of the roots of wheat seedlings. Thus, stevioside can be recommended for the development of complex phytopreparations for stimulating the growth processes and increasing the resistance of wheat plants to low temperatures. [ABSTRACT FROM AUTHOR]

Diterpenoid steviol and its derivatives have gained a growing interest for their broad therapeutic benefits in recent years. In nature, steviol exists primarily in the form of glycosides which are subject to hydrolysis after oral injestion. The present study evaluated the production of steviol using a new commercially available β-glucosidase and its inclusion complex, namely γ-cyclodextrin/steviol (γ-CD/steviol). Firstly, steviol was prepared by sequential hydrolysis from stevioside via steviolbioside, with the highest conversion of stevioside at 100% and a total yield of 64.9% based on the results of single factor experiments. To enhance steviol's aqueous solubility, γ-CD/steviol was prepared by a co-evaporation method. Results showed that the solubility of steviol increased by a factor of 102 times using an 1:1 γ-CD/steviol inclusion complex. Physiochemical properties of the γ-CD/steviol complex were evaluated by HPLC, FT-IR, TGA, XRD, SEM and NMR. At ambient temperature, the γ-CD/steviol complex aqueous solution was extremely stable when assayed in the pH range from 4.01 to 9.18 up to 30 days. [ABSTRACT FROM AUTHOR]

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473-497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH4NO3 is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO3, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3-72.1 mM NH4NO3 and 9.4-65.8 mM KNO3 and generated six different media with altered nitrogen. Similarly, six different concentrations of KH2PO4, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH2PO4. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g−1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g−1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g−1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC-MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles. [ABSTRACT FROM AUTHOR]

Previously unknown conjugates of the diterpenoid isosteviol 1 and 1-O-methyl-2,3-di-O-acetyl-β-Darabinofuranoside in which the terpenoid and carbohydrate molecules were linked through polymethylene spacers of various lengths were synthesized. Isosteviol 1 and its conjugate 10 exhibited high bacteriostatic activity selectively against Staphylococcus aureus ATCC 209p. Conjugates 9-11 inhibited growth of Mycobacterium tuberculosis H37Rv at the level of the antituberculosis drug pyrazinamide. Removal of the acetyl protection from conjugate 9 increased the antituberculosis activity by eight times. [ABSTRACT FROM AUTHOR]

Objective: To purify and characterize a specific enzyme from a commercial pectinase for the production of steviol from stevioside (Ste) without adding organic solvent and to improve steviol production.Results: Commercial Sumizyme PX converted Ste to steviol with a yield of 98%. β-Glucosidase from Sumizyme PX (βglyPX) was purified in three steps with 12.5-fold purification and 51% yield. The specific activity of the purified βglyPX was 141 U/mg. The molecular weight of βglyPX was ~ 116 kDa on SDS-PAGE. Its optimum activity was at pH 3.5 and 65 °C. It was stable for 12 h up to 55 °C and for 24 h at pH 2–9.5. Km values of βglyPX for pNPGal, oNPGlc, lactose, and Ste were 2.4, 0.7, 18, and 7.8 mM, respectively. The optimum conditions for steviol production were 55 °C, 900 U/ml, 80 mg Ste/ml, 12 h.Conclusion: βglyPX contains great potential for industrial steviol production from Ste. [ABSTRACT FROM AUTHOR]

Aconitum heterophyllum is the only non-toxic species of Aconitum genus having immense therapeutic potential. To date, only roots of this plant species are used for the preparation of various herbal formulations while leaves are discarded due to lack of information on the presence of any therapeutically active compounds. To the best of our knowledge, this is the first report on the detection and quantification of diterpene steviol in the leaves of A. heterophyllum. Interestingly, the level of steviol is equivalent to Stevia rebaudiana leaves which is the richest source reported so far. Thus, A. heterophyllum leaves will not only provide a novel source for extraction of steviol but also benefit the harvesters to get additional economic returns on leaf biomass for this high-value non-toxic plant species. [ABSTRACT FROM AUTHOR]

Aconitum heterophyllum is an important component for various herbal drug formulations due to the occurrence of non-toxic aconites including marker compound, atisine. Despite huge pharmacological potential, the reprogramming of aconites production is limited due to lack of understanding on their biosynthesis. To address this problem, we have proposed here the complete atisine biosynthetic pathway for the first time connecting glycolysis, MVA/MEP, serine biosynthesis and diterpene biosynthetic pathways. The transcript profiling revealed phosphorylated pathway as a major contributor towards serine production in addition to repertoire of genes in glycolysis (G6PI, PFK, ALD and ENO), serine biosynthesis (PGDH and PSAT) and diterpene biosynthesis (KO and KH) sharing a similar pattern of expression (2-4-folds) in roots compared to shoots vis-a`-vis atisine content (0-0.37%). Quantification of steviol and comparative analysis of shortlisted genes between roots of high (0.37%) vs low (0.14%) atisine content accessions further confirmed the route of atisine biosynthesis. The results showed 6-fold increase in steviol content and 3-62-fold upregulation of all the selected genes in roots of high content accession ascertaining their association towards atisine production. Moreover, significant positive correlations were observed between selected genes suggesting their coexpression and crucial role in atisine biosynthesis. This study, thus, offers unprecedented opportunities to explore the selected candidate genes for enhanced production of atisine in cultivated plant cells. [ABSTRACT FROM AUTHOR]

Macrocycles consisting of two or four steviolbioside ( S. Rebaudiana glycoside) fragments linked at the carboxy groups by an octamethylene chain and at the 16-positions by alkanedihydrazide spacer derived from adipic, suberic, or sebacic acid have been synthesized. [ABSTRACT FROM AUTHOR]

In this review, we summarized and analyzed the literature data (from 1963 to March, 2014) on the synthesis and properties of quaternary ammonium derivatives of citronellal, menthol, camphor, lambertian acid, dehydroabietic acid, levopimaric acid, isosteviol, steviol, betulinic acid, dihydrobetulinic acid, platanic acid, oleanolic acid, ursolic acid, and polyprenols. Functionalization of terpenoids with ammnoium groups offers them new properties, such as cholinotropic, antiviral, and antimicrobial activities, or transforms them into chiral ionic liquids having a practical potential for application as chiral phasetransfer catalysts. The synthesis of ammonium derivatives of terpenoids possessing properties of cationic lipids or dimeric bis-quaternary ammonium surfactants was proved to have a promising outlook. A specific attention was paid to the method developed for transformation of any hydrophobic terpenoid or steroid into an amphiphilic water-soluble compound self-organized in the aqueous medium into vesicles (liposomes) forming cell-penetrating complexes with DNA. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana B. is a medicinal herb that accumulates low-caloric sweetening agents generally known as steviol glycosides among which stevioside and rebaudioside-A are predominant. These sweeteners are synthesized through the commonly known 2- C-methyl- d-erythritol-4-phosphate pathway common to steviol glycosides, gibberellic acid, pigments, and other isoprene-based secondary metabolites. The present investigation was designed to elucidate the role of steviol, an immediate precursor of sweetening agents, in the cultures of plants conducting stevioside biosynthesis in the leaf tissue. It was proposed that either the precursor or an elicitor was responsible for stimulating stevioside production in leaves. Transcript profiling of genes involved in stevioside biosynthesis was explored both in the roots and leaves of the plant and it was found that the transcript of the key enzyme kaurenoic acid hydroxylase, producing steviol, was the same in both tissue. However, when these plants were treated with the precursor steviol, most of the genes in the pathway were downregulated. Interestingly, it was found that very minute concentrations of the precursor were required to enhance the transcription level of genes of the pathway. [ABSTRACT FROM AUTHOR]

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and H- and C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas. [ABSTRACT FROM AUTHOR]

New nitrogen-containing derivatives of the diterpenoid isosteviol (16-oxo- ent-beyeran-19-oic acid) containing oxo, hydroxyimine, and thiosemicarbazone groups were synthesized. Isosteviol 15-oxo-16-thiosemicarbazone forms a 1: 1 complex with Cu II and inhibits the growth of Mycobacterium tuberculosis (H37R V, in vitro) at the minimum inhibitory concentration of 20 μg mL −1. [ABSTRACT FROM AUTHOR]

This review is devoted to ent-kaurane diterpenoids and related glycosides, which represent two naturally occurring groups of compounds with interesting structures and properties. Much useful information about the biogenesis and biological activities of these compounds is presented; however, the greatest part of the review covers various aspects of the chemical transformations of native ent-kaurane diterpenoids, namely, their reactions via COOH, C=O, and OH groups and double bonds. Special attention is given to rearrangements of the carbon skeleton, primarily the Wagner-Meerwein rearrangement of the well-known kaurane diterpenoid steviol into its isomer, the well-known ent-beyerane diterpenoid isosteviol. A separate section is devoted to the chemical transformations of isosteviol, which are used to demonstrate how the well-known reactions of COOH and C=O groups, as well as CH-C(O)R fragments, proceed in unusual ways when these moieties are attached to rigid hydrocarbon skeletons of naturally occurring compounds (or when they are parts of them). The final section considers ent-kaurane glycosides, focusing on those isolated from the plant Stevia rebaudiana, which is well known because they are 300-400 times sweeter than sucrose. The chemical transformations and, in some cases, biotransformations of these compounds (rebaudiosides) are described in detail, including the total synthesis of some rebaudiosides aimed at creating a production technology for low-calorie sweeteners. The review covers data published between 1964 and 2010, including original results obtained by the authors. [ABSTRACT FROM AUTHOR]

XRD is used to study the structures of inclusion compounds of diterpenoid 15-ene-steviol with chloroform and 16- S-dihydrosteviol with chloroform and ethyl acetate. In all three cases, the guest-host ratio is 1:2; by means of a branched system of hydrogen bonds, diterpenoid molecules form a three-dimensional associate with helical voids, in which solvent molecules are located. Crystals of all three complexes are isostructural, the ethyl acetate molecule being localized in the corresponding inclusion compound, while solvent molecules are disordered in complexes with chloroform. [ABSTRACT FROM AUTHOR]

A new laboratory method for isolating the glycosides stevioside and rebaudiosides A and C from leaves of Stevia rebaudiana was proposed. According to HPLC, the glycoside contents in plants grown in Russia (Voronezh Oblast’) and Ukraine (Crimea) were 5–6% (stevioside) and 0.3–1.3% (rebaudiosides A and C). [ABSTRACT FROM AUTHOR]

The molecular and crystal structure of isosteviol sulphite ( 1) is studied by means of the X-ray crystallographic analysis. It is demonstrated that this compound has a 'dimer' structure, i.e. two isosteviol molecules are bound with a sulphite bridge. The molecule takes a tweezers-like conformation stabilized by intramolecular hydrogen bonds of acid groups. The molecules in the crystal are bound by intermolecular C-H...O hydrogen bonds. [ABSTRACT FROM AUTHOR]

Recent studies have shown that transforming microglia phenotype from pro-inflammation of M1 phenotype to anti-inflammation and tissue-repairing M2 phenotype may be an effective therapeutic strategy for preventing ischemic stroke brain injury. Isosteviol Sodium (STV-Na) has shown promise as a neuroprotective agent in cerebral ischemia model, although its effect on microglial polarization and chronic recovery after stroke is not clear. Here, we demonstrated that STV-Na treatment significantly reduced cerebral ischemic damage at both acute and chronic time points. STV-Na has a profound regulatory effect on microglia response after stroke. It can promote M2 polarization and inhibit microglia-mediated inflammation (M1) response following stroke in vivo and in vitro. Furthermore, we also found that Growth Arrest-Specific 5 (GAS5) altered OGD/R-induced microglial activation by increasing Notch1 expression via miR-146a-5p, the mRNA level of GAS5 and the protein level of Notch1 in vivo and in vitro, were discovered that both downgraded with STV-Na. Taken together, the present study demonstrated that STV-Na exerted neuroprotective effects by modulating microglia/macrophage polarization in ischemic stroke via the GAS5/miR-146a-5p sponge. These findings provide new evidence that targeting STV-Na could be a treatment for the prevention of stroke-related brain damage. [ABSTRACT FROM AUTHOR]