Plant Stevia Rebaudiana Research Articles

Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana

For the purpose of enhancing the secondary metabolite content in micropropagated Stevia rebaudiana plants without inhibiting plant growth, node explants were cultured on woody plant medium (WPM) containing alginate (ALG), casein hydrolysate (CH), pectin (PEC), yeast extract (YE), methyl jasmonate (MeJA), salicylic acid (SA), or chitosan (CHI). The highest shoot number; shoot length, node number, and leaf number; leaf length; and stem diameter were observed on WPM containing 1.0 g/L YE; 100 µM CHI; 0.5 g/L CH; and 1.0 g/L ALG, respectively. The root regeneration frequency reached 100 % on WPM supplemented with 0.5, 1.0, or 2.0 g/L PEC, 1.0 g/L YE, or 50 µM CHI and the control. The highest root number was obtained on WPM containing 0.5 g/L PEC, while the longest root length was observed on WPM containing 1.0 g/L YE. The highest biomass accumulation was observed with treatment of 100 µM CHI. According to the high-performance liquid chromatography results, except for the treatments with 200 µM CHI, 100 or 200 µM MeJA, and 200 µM SA, the remaining elicitor treatments increased stevioside production compared to the control. The production of stevioside increased from 1.56 mg/g dry weight (DW) to 14.69 and 14.54 mg/g DW in the in vitro plantlets exposed to 0.5 g/L ALG and 2.0 g/L YE, respectively. Rebaudioside A was observed on only 0.5 g/L ALG-treated plants as 0.55 mg/g DW. The stevioside content of field-grown plants was identified as 15.06 mg/g DW. The present findings provide important information regarding the effect of elicitors on plant growth and secondary metabolite production of in vitro micropropagated S. rebaudiana.

Agronomical and phytochemical evaluation of Stevia rebaudiana genotypes

The agronomical potential and the phytochemical variability of 18 genotypes of the Paraguayan plant Stevia rebaudiana have been investigated in Switzerland in order identify the best genotype for local cultivation. Over a two years period, yields in dry leaves ranged from 10 to 170 g m -2 , with a percentage of leaves ranging from 53 to 75 %. HPLC analyses showed a notable variability in phytochemical composition, with stevioside content ranging from 0.3 to 7.9 % w/w and rebaudioside A from 0.3 to 6.5 % w/w. Cultivation of S. rebaudiana in Switzerland is feasible. With a density of 10 plants per m 2 , the potential yields of dry matter are approximately 1-2 t ha -1 . The most productive genotypes (Pharmasaat, Hem Zaden, Stepa and Mediplant 3 and 11) will be submitted to the industry for organoleptic evaluation.

Bioactivity and Enzyme Inhibition Properties of Stevia rebaudiana

Objective: The main study was to evaluate the anti- inflammatory, anti-oxidant, and the gastro-protective properties of extracts of Stevia rebaudiana. In addition the purpose of the study was to investigate new, natural inhibition of urease and xanthine oxidase enzymes, as well as to find the sugar contents of extracts from the plants. Method: The polyphenolic contents of methanol extracted samples were evaluated in three different ways: total phenolic contents, total flavonoid content and condensed tannin. The antioxidant activity was determined using ferric reducing antioxidant power and using the free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) tests. Phenolic compounds were determined by high-performance liquid chromatography. Results: Results of antioxidant activity gave a total pheneolic content of 12.303-8.477 mg/g gallic acid equivalent, total flavonoid content of 2.378-1.518 mg/g quercetin equivalent and condense tanin of 3.435-0.713 mg/g catechin equivalent in leaf and flower extracts. Ferric reducing antioxidant power values of the Stevia rebaudiana leaf and flower were discovered to be 202.499-116.826 M FeSO47H2O/g respectively. The radical scavenging activity values of the 2,2- diphenyl-1-picrylhydrazyl radical and the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) tests ranged between 0.533-0.398 and 6.637-17.382 mg/mL respectively. The inhibitory effect of the Stevia leaf extract on xanthine oxidase and urease was also investigated and the concentrations that gave 50% inhibition of maximal activity were found to be 23.51g/mL and 9.45g/mL respectively. Glucose and fructose were determined to be the sugars in the Stevia leaf. Conclusion: The results indicate that the Stevia rebaudiana plant is not only rich in steviol glycosides, but is also a powerful natural antioxidant and anti-inflammatory with gastro-protective properties. Keywords: Antioxidant, radical, phenolics compound, xanthine oxidase, urease.

The Stevia Rebaudiana Plant as Natural Sweetener with Antioxidant Properties

The Stevia Rebaudiana Plant as Natural Sweetener with Antioxidant Properties

Meeting the challenge of stevioside production in the hairy roots of Stevia rebaudiana by probing the underlying process

Agrobacterium rhizogenes mediated “hairy root cultures” of the renowned bio-sweetener producing plant—Stevia rebaudiana, were generated to explore their yet undetected biosynthetic potentials concerning the low calorie diterpene glycoside—stevioside. Four stable rhizoclones were studied under light and dark conditions showing better growth under dark. Two of these rhizoclones revealed substantially higher photosynthetic pigment accumulation under light. Evidently, capitalization on the available inter-clonal variability first time showed the stevioside synthesizing exclusivity in the SRA4 rhizoclone under light condition, while the rest failed. The dualities of the glycoside synthesizing power amongst the two photosynthetically active rhizoclones were resolved through quantitative RT-PCR analysis of UGT85C2 gene showing positive expression in the stevioside producing rhizoclone. These findings elucidated the decisive role of UGT85C2 in combination with photosynthetic proficiency of the studied hairy root cultures in regulating the biosynthetic pathway of S. rebaudiana.

Stevia Glycosides: Chemical and Enzymatic Modifications of Their Carbohydrate Moieties to Improve the Sweet-Tasting Quality.

Stevia glycosides, extracted from the leaves of the plant Stevia rebaudiana Bertoni, display an amazing high degree of sweetness. As processed plant products, they are considered as excellent bio-alternatives for sucrose and artificial sweeteners. Being noncaloric and having beneficial properties for human health, they are the subject of an increasing number of studies for applications in food and pharmacy. However, one of the main obstacles for the successful commercialization of Stevia sweeteners, especially in food, is their slight bitter aftertaste and astringency. These undesirable properties may be reduced or eliminated by modifying the carbohydrate moieties of the steviol glycosides. A promising procedure is to subject steviol glycosides to enzymatic glycosylation, thereby introducing additional monosaccharide residues into the molecules. Depending on the number and positions of the monosaccharide units, the taste quality and sweetness potency of the compounds will vary. Many studies have been performed already, and this review summarizes the structures of native steviol glycosides and the recent data of modifications of the carbohydrate moieties that have been published to provide an overview of the current progress.

An Assessment of Major Nutritional Components and Some Secondary Metabolites of In-Vitro Propagated Stevia Rebaudiana (Cultured in Bangladesh) Plant Leaves Dry Powder

Stevia rebaudiana, belongs to the family of Asteraceae, is a perennial and medicinal shrub. It’s leaves dry powder widely used as a natural sweetener which has no calories but reported to be 50– 300 times sweetener as sweet as sugar, that are said to be having insulin balancing properties without any side effects. This research was first time conducted in Bangladesh where a quantitative analysis were performed for determining the major constituents of in vitro propagated Stevia rebaudiana plant leaves dry powder that was prepared through oven dry after sun dry. The analysis revealed the powder as mild acidic (PH 5.345). Total chlorophyll contents were found to be 0.845mg% where chlorophyll–a and -b were 0.088 and 0.761 mg% respectively. The major macro nutrients such as ash, crude fiber, total carbohydrate, total protein and fat were determined at the amount of 7.05, 10.5, 53.52, 13.13 and 3.55 gm% respectively where the micro nutrients like Iron (Fe), Sodium (Na), Potassium (K), Calcium (Ca), Magnesium (Mg), Phosphorus (P) and Chloride (Cl) were found at the amount of 34.2, 184.3, 2500, 534.43, 465.34, 304.7 and 49.6 mg% accordingly. Screening for secondary metabolites showed the presence of alkaloids, flavonoids, saponins and tannins as the amount of 2.83, 17.20, 6.69 and 5.84 mg% respectively in dry powder. These findings also have been compared with the other reported values on Stevia from different countries of the world. Int J Appl Sci Biotechnol, Vol 3(4): 721-726

Functionalization of the double bond in the glycoside of the Stevia rebaudiana plant steviolbioside, as a way to macrocyclic glycosides

The C16=C17 bond in the glycoside steviolbioside from the plant Stevia rebaudiana was oxidized for the first time. Ketone, thiosemicarbazone, and oxime of steviolbioside with a heptaacetylated sophorosyl fragment, as well as binuclear and tetranuclear macrocyclic derivatives of this rebaudioside were synthesized.

Design and synthesis of isosteviol triazole conjugates for cancer therapy.

One of the keys for successfully developing drugs against the broad spectrum of cancer cell types is structural diversity. In the current study, we focused on a family of isosteviol derivatives as potential novel antitumor agents. Isosteviol is a tetracyclic diterpenoid obtained by acid hydrolysis of steviol glycoside extracts isolated from abundant Stevia rebaudiana plants. In this work, we have designed and synthesized a panel of isosteviol triazole conjugates using “click” chemistry methodology. Evaluation of these compounds against a series of cancer cell lines derived from primary and metastatic tumors demonstrated that these conjugates exhibit cytotoxic activities with IC50 in the low μM range. In addition, their anti-proliferative activities are cancer cell type specific. Taken together, our studies underscore the importance of structural diversity in achieving cancer cell type specific drug development.

Genetic signature of differential sensitivity to stevioside in the Italian population

The demand for diet products is continuously increasing, together with that for natural food ingredients. Stevioside and other steviol glycosides extracted from the leaves of the plant Stevia rebaudiana Bertoni are the first natural high-potency sweeteners to be approved for consumption in the United States and the European Union. However, the sweetness of these compounds is generally accompanied by aversive sensations, such as bitter and off-tastes, which may constitute a limit to their consumption. Moreover, consumers' differences in sensitivity to high-potency sweeteners are well known, as well as difficulties in characterizing their aftertaste. Recently, TAS2R4 and TAS2R14 have been identified as the receptors that mediate the bitter off-taste of steviol glycosides in vitro. In the present study, we demonstrate that TAS2R4 gene polymorphism rs2234001 and TAS2R14 gene polymorphism rs3741843 are functional for stevioside bitterness perception.

Assessment of Heavy Metals Tolerance in Leaves, Stems and Flowers of Stevia Rebaudiana Plant

Stevia has become rather widespread over a wide range of climatic locations around the world and can apparently be successfully grown under different cultivation conditions. Heavy metal accumulation of Stevia extract is dependent on obtaining heavy metals from the soil and water. Heavy metals from plant sources may also vary from place to place because soil heavy metals content varies geographically, thus, they have become the subject of many research projects. Stevia plant is easily contaminated during growth, development and processing and for this, an extensive research is needed to explore the characteristics of the heavy metal produced by the plant. The heavy metals produced from the herb and its toxicity of Stevia plant is not well documented and scientific evidence is limited to establishing Stevia plant as a medicinal plant. The samples were collected from Malacca, Malaysia. The fresh leaves, stems and flowers of the Stevia rebaudiana plant were dried using oven equipment and were grinded until fine to make powder and then of each extracted using Microwave digester. The analysis of samples was carried out by using an Inductively Coupled Plasma Mass-Spectrophotometer (ICP-MS) with different mode equipment to compare results of heavy metals in Stevia rebaudiana plant. Heavy metal accumulation in Stevia rebaudiana from leaves, stems, and flowers extraction is reported. Heavy metals in leaves, stems and flowers of Stevia rebaudiana presented variety of elements such as As, Cd, Cr, Cu, Fe, Mg, Pb, Se, Zn, Al, Ag, Co, Ca, Mn and Ni. The high tolerance to heavy metals in leaves, stems, and flowers of Stevia rebaudiana were presented at fifteen parameters below the permissible limit in plant and can be used as food product or therapeutic agent in traditional medicine.

Gene transcription of steviol glycoside biosynthesis in Stevia rebaudiana Bertoni under polyethylene glycol, paclobutrazol and gibberellic acid treatments in vitro

This experiment was carried out to measure the transcript levels of some genes involved in biosynthesis of steviol glycosides. Stevia rebaudiana plants were treated with polyethylene glycol (PEG), paclobutrazol (PBZ) and gibberellic acid (GA). Using quantitative real-time poly- merase chain reaction, the transcript levels of kaurene synthase (KS), kaurene oxidase (KO), kaurenoic acid hydroxylase (KAH) and three UDP-dependent glycosyl- transferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The transcription of ent-KS1-1, ent-KAH and UGT74G1 were fairly stable under different treatments. The transcription of ent-KO, UGT85C2 and UGT76G1 significantly decreased by PBZ and PEG treatments. The results indicate that both PBZ and PEG treatments resulted in the same negative effect on genes transcription, which could not be reversed by GA treatment.

Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i> Bert. Using ISSR Marker

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

Functional and structural variation of uridine diphosphate glycosyltransferase (UGT) gene of Stevia rebaudiana–UGTSr involved in the synthesis of rebaudioside A

The sweetness of honey leaf plant Stevia rebaudiana is attributed to steviol glycosides or steviosides, accumulated in the leaves. Steviol glycosides are diterpenoids derived from steviol as the final step of glycosylation by the marker enzyme Uridine diphosphate glycosyltransferase (UGT). Out of the eight different steviol glycosides, rebaudioside A was detected as the sweetest glycoside with reduced bitter aftertaste. The pattern of glycosylation of steviol has a crucial role in maintaining the sweetness as well as the taste perception of stevioside. Within the 12 UGTs of S. rebaudiana so far elucidated, the functional genomics of three UGTs-UGT76G1, UGT74G1 & UGT85C2 in stevioside synthesis were studied. In the present study a UGT gene of S. rebaudiana named UGTSr showing resemblance with UGT76G1 was structurally analyzed and the functional role of the recombinant UGTSr in the synthesis of rebaudioside A was ascertained. The relative expression of UGTSr by qPCR showed a higher level of expression in mature leaves than in tender. Despite the similarity of nucleotide with UGT76G1, the gene UGTSr exhibits 48 SNPs and 39 associated amino acid substitutions with remarkable variation in the secondary and tertiary structure of the protein. The helical changes, the presence of a new amino acid, novel substitutions of amino acids and the hydrogen bond in the conserved histidine and aspartame residues observed in UGTSr support its functional stability and specificity from that of other UGTs of S. rebaudiana. Based on these features UGTSr exhibits a novel status from other UGTs of S. rebaudiana.

Large-scale profiling of diterpenoid glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem mass spectrometry

The plant Stevia rebaudiana accumulates a suite of diterpenoid metabolites that are natural sweeteners finding increased use as sugar substitutes. To guide breeding of stevia plants that accumulate substances with desirable flavor in high yield, rapid and accurate methods are needed to profile these substances in plant populations. This report describes an 8-min ultrahigh performance liquid chromatography-tandem mass spectrometry method for separation and quantification of seven stevia glycosides including steviolbioside; stevioside; rebaudiosides A, B, and C; rubusoside; and dulcoside as well as aglycones steviol and isosteviol. This negative mode electrospray ionization/multiple reaction monitoring method yielded low limits of detection <1 ng/mL for steviol, 6 ng/mL for isosteviol, and <15 ng/mL for all stevia glycosides. Stevioside and Reb A, B, and C were quantified in more than 1,100 extracts from stevia leaves as part of a large-scale profiling exercise. Leaf tissue levels in this population spanned about two orders of magnitude for stevioside (2-125 mg/g dry weight), Reb A (2.5-164 mg/g), Reb B (0.5-50 mg/g), and Reb C (1.5-125 mg/g), but levels of individual metabolites exhibited independent variation. The wide spread of metabolite levels highlights the utility and importance of performing targeted metabolic profiling for large plant populations.

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.

Callus induction studies on Stevia rebaudiana and antibacterial screening of leaf extracts

The medicinal plant Stevia rebaudiana (Asteraceae family) produces sweetsteviol glycosides in the leaves which can be used as a natural alternative toartificial sweetener. The propagation by seeds or other methods is not viabledue to the low rate of seed germination. The present study was focused oninduction and maintenance of callus from leaf explants of Stevia by usingdifferent concentrations of 2, 4 dichlorophenoxy acetic acid . Surface sterilizedleaf explants were inoculated in MS medium with vitamins, sucrose (30g l –1), agar (0.8% w/v) and supplemented with various concentrations of 2, 4‐D (0.5, 1.0, 1.5, 2.0, 2.5 and 3 mg l –1). Medium supplemented with 2.5 mg l–12, 4 ‐D showed best callus induction. Leaf extracts in different solvent systemswere screened for antimicrobial activity against some Gram ‐positiveand Gram ‐negative bacteria by disc diffusion method. The chloroform andmethanol extract of leaves exhibited a concentration dependent antibacterialinhibition.

Synthesis and antituberculosis activity of derivatives of Stevia rebaudiana glycoside steviolbioside and diterpenoid isosteviol containing hydrazone, hydrazide, and pyridinoyl moieties

Conjugates of antitubercular drug Isoniazid (hydrazide of isonicotinic acid), nicotinic and alpha-picolinic acid hydrazides and glycoside steviolbioside from the plant Stevia rebaudiana as well as the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. Besides, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties connected by dihydrazide linker were also obtained. Both initial compounds and their synthetic derivatives inhibit the growth of Mycobacterium tuberculosis (H37Rv in vitro). The minimum concentration at which the growth of M. tuberculosis was inhibited by 100% (MIC) for stevioside and steviolbioside equals 7.5 and 3.8 microg/mL, respectively. MIC values for conjugates of the hydrazides of pyridine carbonic acids and steviolbioside as well as isosteviol are in the ranges 5-10 and 10-20 microg/mL, respectively. Maximum inhibitory effect against M. tuberculosis showed the conjugates of isosteviol and adipic acid dihydrazide (MIC values ranged from 1.7 to 3.1 microg/mL). Antitubercular activity of the compounds studied is higher than the activity of antitubercular drug Pyrizanamide (MIC = 12.5-20 microg/mL) but lower than the activity of antitubercular drug Isoniazid (MIC = 0.02-0.04 microg/mL).

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

Ent-kaurane diterpenoids and glycosides: Isolation, properties, and chemical transformations

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 CH2-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.