Stevia rebaudiana Bertoni, source of a high-potency natural sweetener: A comprehensive review on the biochemical, nutritional and functional aspects

Stevia rebaudiana Bertoni, an ancient perennial shrub of South America, produces diterpene glycosides that are low calorie sweeteners, about 300 times sweeter than saccharose. Stevia extracts, besides having therapeutic properties, contain a high level of sweetening compounds, known as steviol glycosides, which are thought to possess antioxidant, antimicrobial and antifungal activity. Stevioside and rebaudioside A are the main sweetening compounds of interest. They are thermostable even at temperatures of up to 200 °C, making them suitable for use in cooked foods. S. rebaudiana has a great potential as a new agricultural crop since consumer demand for herbal foods is increasing and proximate analysis has shown that Stevia also contains folic acid, vitamin C and all of the indispensable amino acids with the exception of tryptophan. Stevia cultivation and production would further help those who have to restrict carbohydrate intake in their diet; to enjoy the sweet taste with minimal calories.     

Polyphenols from Stevia rebaudiana (Bertoni) leaves and their functional properties

The major polyphenol components from Stevia rebaudiana (Bertoni) leaves (PPS) are chlorogenic acids, a polyphenol family of esters, including hydroxycinnamic acids with quinic acid, which possesses excellent hydrophilic antioxidant activity and other therapeutic properties. As an abundant byproduct during production of steviol glycosides, the PPS would be a new antioxidantive food resource or additives applied in foods and drugs with antidiabetic function. Extracting PPS from S. rebaudiana (Bertoni) leaves together with steviol glycosides would be an economic process, which will change most operation process in current Stevia factories. The quantification of PPS needs to be unified for regulation. In view of the current regulation status of polyphenols and extracts from Stevia, the PPS would be ready to go to the market with few regulation barriers in the near future. This review will summarize the analysis, extraction, and some functional properties of PPS, such as antioxidant, antidiabetic, antimicrobial, anti-inflammatory, and anticancer.     

Stevia and sucrose effect on plaque formation

An in vivo study was done by measuring the accumulation of dental plaque after rinsing with a solution of 10% sucrose four times daily during 5 days and comparing it with a rinsing of 10% solution of Stevia rebaudiana Bertoni four times a day, during 5 days a week. The accumulation of dental plaque after rinsing with Stevia was 57, 82% less than under rinsing with sucrose as measured by the Silness-L?e index and 10, 40% less plaque when measured by O’Leary index of plaque.     

Production of steviosides in ex vitro and in vitro grown Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni plants grown in vitro and ex vitro were investigated for variations in the profile of steviosides in their leaves, shoots, roots and flowers. Steviosides were extracted by hydrolysis and esterification, evaporated to dryness and dissolved in methanol for quantitative analysis by high‐performance liquid chromatography (HPLC). The HPLC analysis and separation profiles indicated the presence of several different steviosides, predominantly eight known sweet diterpene glycosides. The most abundant compounds identified were steviolbioside, stevioside, rebaudiosides C and A and dulcoside A. The highest stevioside contents were recorded in 1‐month‐old greenhouse leaves (64.80 g steviolbioside kg^?1 dried plant material) and in vitro leaves (0.99 g rebaudioside A kg^?1 dried plant material). The recovery of steviosides from the leaves by methanol extraction was 90%. This is the first time that the eight predominant sweet diterpene glycosides in the various plant parts of S. rebaudiana have been investigated. Copyright © 2006 Society of Chemical Industry     

Quantitative analysis of stevioside in the leaves of Stevia rebaudiana by near infrared reflectance spectroscopy

Stevioside, a diterpenic glycoside extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, is a natural sweetener 300 times sweeter than sucrose. HPLC is routinely used to measure stevioside content in Stevia leaves. Our objectives were to investigate the use of near infrared reflectance spectroscopy (NIRS) to analyse stevioside concentration in Stevia leaves. The NIRS calibrations were developed from 64 samples covering the range normally found in Stevia leaves (4-13%). Another 30 samples were used for validation. Standard error of calibration and coefficient of multiple determination (R²) were 1.47 and 0.90 respectively, using the first derivative mathematical treatment. The validation results, although less accurate, suggest that NIRS is a precise and simple method for routine stevioside determination in Stevia leaves.     

Separation of Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography

Stevia rebaudiana Bertoni contains several steviol glycosides with sweet flavour. They all are sweeter than sucrose (up to factor 450). The various steviol glycosides are difficult to separate by reversed-phase chromatography. In this paper, five different hydrophilic liquid interaction chromatography columns are characterized using isocratic elution (5–20% water in acetonitrile with buffer or formic acid). Separation of the steviol glycosides is possible with all but one of the tested columns, but the robustness of the separation against changes of buffer concentration and percentage of water differ. Aqueous percentage and ion strength of the eluent are the main factors to be optimized in method development.     

Study on chemical composition and biological activities of essential oil and extracts from Stevia rebaudiana Bertoni leaves

Essential oil (EO), water extract (WE), and methanol-water (MWE) (50/50 v:v) were prepared from Stevia rebaudiana Bertoni leaves. Their chemicals compounds, antioxidant, anti-inflammation and antimicrobial activities were evaluated. The EO was analyzed by gas chromatography/mass spectrometry. The WE, and MWE compounds were identified by RP-HPLC. In EO, carvacrol, caryophyllene, caryophyllene oxide, spathulenol, cardinol, α-pinene, limonene, isopinocarveol and ibuprofen were identified as major compounds. Furthermore, in the WE and MWE, the major compounds were, quercetin dihydrate, protocatechuic acid and quercetin glucosyl. These results show that S. rebaudiana EO and extracts possess high antioxidant, anti-inflammation and antimicrobial properties. The antimicrobial and antifungal activities of the extracts (EO, WE, MWE) were tested on Staphylococcus aureus; Bacillus subtillis; Escherichia coli; Pseudomonas aeruginosa; Aspergillus niger and Candida albicans, the lowest activity was founded on the EO extract.     

<Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni: health promoting properties and therapeutic applications

Stevia is a genus of approx. 200 species of herbs and shrubs from the family Astraceae. One of the representatives of the genus is sweetleaf Stevia rebaudiana, formerly called Eupatorium rebaudianum Bertoni (Yadav et al. in Can J Plant Sci 91:1–27, 2011). S. rebaudiana Bertoni has been known for many centuries by indigenous tribes of South America, who called it “kaa-hee” (sweet herb). South American Indians used stevia both as a sweetener and as a medicinal plant. Folk medicine of Paraguayan indigenous tribes recommends it particularly as a substance strengthening the heart, the circulatory system and regulating blood pressure. Sweetleaf is also used in liver disease, stomachache, gastric hyperacidity and as a mild agent stimulating the nervous system. It exhibits antibacterial, antifungal and anticaries properties. Nutritive and health-promoting properties of stevia are presently being investigated in many research centres. The aim of this paper is to present the health-promoting value of S. rebaudiana as stevia leaf powder, crude leaf extract or steviol glycosides (highly purified stevioside, rebaudioside A).     

Stevia and sucrose effect on plaque formation

An in vivo study was done by measuring the accumulation of dental plaque after rinsing with a solution of 10% sucrose four times daily during 5 days and comparing it with a rinsing of 10% solution of Stevia rebaudiana Bertoni four times a day, during 5 days a week. The accumulation of dental plaque after rinsing with Stevia was 57, 82% less than under rinsing with sucrose as measured by the Silness-Löe index and 10, 40% less plaque when measured by O’Leary index of plaque.     

Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana

A simple reversed-phase high-performance liquid chromatographic method has been developed for the determination of the major steviol glycosides, the diterpene sweeteners derived from Stevia rebaudiana. The method is based on a water extraction step and a solid-phase extraction (SPE) clean-up. Different SPE cartridges and two HPLC columns were tested in the separation of the main steviol glycosides stevioside and rebaudioside A. A good separation was performed on a Luna HILIC analytical column with a mobile phase consisting of acetonitrile/water (85:15 v/v). The calibration curves were linear from 10 to 800 μg/mL and the recoveries for stevioside and rebaudioside A were 99 ± 4.4 and 100 ± 5.0%, respectively. The applicability of this method was demonstrated in the analysis of stevioside and rebaudioside A from Stevia plants grown in two different areas in Germany. Stevioside and rebaudioside A contents showed statistically significant differences (F- and t-test) between the two harvests. Nevertheless, the total concentrations (>12%) and the ratio of stevioside to rebaudioside A (6:4) were similar to those found in the countries in which Stevia rebaudiana originates. Based on a comparison of yields from different harvests, we discussed whether Stevia rebaudiana can be economically grown in the temperate zones of the northern European hemisphere.     

Comprehensive review on agro technologies of low‐calorie natural sweetener stevia (Stevia rebaudiana Bertoni): a boon to diabetic patients

Stevia rebaudiana Bertoni is a low‐calorie natural sweetener plant native to Paraguay. The leaves of stevia have sweetening compounds called steviol glycosides (SGs), which contain different marker compounds, i.e. stevioside (St), rebaudioside (Rb) A, B, C, D and E, dulcoside A and steviol biosides, which are nearly 300 times sweeter than sugar. Stevia is a better alternative to sugar in formulating food products, reducing the harmful effect of sugar and improving the nutrient properties. We have tried to compile a literature on various agronomic and management aspects which are helpful in increasing the yield and quality of stevia to be grown as a crop that will benefit farmers and industrialists. The stevioside thus obtained can be used to make different food products for sweetening purposes, which could be a boon to diabetic patients. Incorporation of different agronomic techniques like propagation method, transplanting time, intercropping, irrigation, mulching, plant geometry, pinching and harvesting time not only improve the biomass but also increase the quality of stevia. Therefore, agronomic considerations are of high priority to utilize its maximum potential. © 2015 Society of Chemical Industry     

On-line monitoring of stevioside sweetener hydrolysis to steviol in acidic aqueous solutions

Stevioside, a potent sweetener obtained from leaves of Stevia rebaudiana (Bertoni), is the glycone of steviol. However, despite its natural origin, there has been concern about stevioside toxicity due to hydrolysis to the carcinogenic steviol. To approve it as an additive, the FAO/WHO committee on food additives has required further information about hydrolytic stability of the steviol glycoside in acidic foods and beverages. In this study, aqueous solutions of stevioside at different pH values were monitored in real time by direct infusion ESI-MS. Owing to the high speed and sensitivity of ESI-MS monitoring, fast hydrolysis of the stevioside molecule to steviol in aqueous acidic solutions was observed, particularly in acidic juices.     

Steviol glycosides are not altered during commercial extraction and purification processes

Steviol glycosides, sweet diterpenes extracted from the shrub Stevia rebaudiana Bertoni, are approved as sweeteners in many countries throughout the world. Former heat and pH‐investigations of these glycosides have established their stability. However, due to the complex purification process, the natural authenticity has still been discussed and challenged, recently. Thus, the objective of this work was to show that the steviol glycosides are not chemically modified during the commercial extraction and purification process. Therefore, samples of three independent commercial‐scale extraction and purification batches of steviol glycosides, each batch containing a sample of the untreated stevia leaves, the first water extract and the high‐purity end product, were analysed using HPLC‐UV and HPLC‐ESI‐MS/MS. The results show that the commercial powders of extracted steviol glycosides with an estimated purity of more than or equal to 95% contain the same steviol glycosides as the dried stevia leaves and their hot water infusions, demonstrating that steviol glycosides are not affected by the purification process.     

Effect of Stevia rebaudiana on Oxidative Enzyme Activity and Its Correlation with Antioxidant Capacity and Bioactive Compounds

The effect of different concentrations of Stevia rebaudiana Bertoni (Stevia) on polyphenoloxidase and peroxidase activities, antioxidant capacity and other bioactive compounds of a mixture of fruits, during 10 days of incubation at 10 and 37 °C, was studied. A significant decrease (p?<?0.05) in both enzyme activities was observed when Stevia was added, showing an inhibitory effect. Samples with a high Stevia concentration showed the highest total phenolic content and antioxidant capacity, whereas non-significant changes were found in the ascorbic acid and carotenoid contents of the samples with Stevia added. A negative significant dependence (p?<?0.05) during incubation was obtained between enzyme activity and antioxidant capacity, so that antioxidant capacity increases as enzyme activity decreases. Therefore, enzyme activity could be used as an indirect index of antioxidant capacity. The results clearly showed that Stevia had significant potential for use not only as a sweetener but also as a natural preservative agent.     

Preparative separation and purification of Rebaudioside A from Stevia rebaudiana Bertoni crude extracts by mixed bed of macroporous adsorption resins

The separation and purification of Rebaudioside A (RA) from Stevia rebaudiana Bertoni crude extracts (Steviosides) by macroporous adsorption resin (MAR) mixed bed were systematically investigated. MAR mixed bed of HPD750-LSA40-LSA30-DS401 was selected due to its better separation degree. Based on the kinetics/thermodynamics experiment of the mixed bed, it was found that the experimental data fitted better to the pseudo-second-order model, and intra-particle diffusion was rate-limiting step. The adsorption isotherm was consistent with IV equilibrium adsorption isotherm classified by Brunauer. Furthermore, the influencing factors for the separation of RA based on HPLC were also investigated. Under the optimal conditions, the separation degree for RA (D_AS) increased from 0.771 to 1.54. Moreover, the experimental results showed that the purity of the obtained product increased from 60% to 97%.     

甜叶菊红外-热风联合干燥工艺优化

为探究甜叶菊红外-热风干燥特性,以江西甜叶菊守田3号为试验材料,在研制的红外-热风联合干燥样机基础上,通过开展热风温度(90、100、110、120℃)、排湿功率(0、140、240、340 W)和辐射距离(140、150、160、170 mm)条件下的单因素和正交试验,探究甜叶菊红外-热风干燥特性曲线及干燥速率曲线,优化甜叶菊干燥工艺参数。结果表明,甜叶菊红外-热风联合干燥过程包含预热加速干燥阶段和降速干燥阶段;影响甜叶菊红外-热风联合干燥生产效率的影响因素顺序为:热风温度 > 辐射距离 > 排湿速率;最佳干燥工艺参数:热风温度120℃,排湿功率240 W、辐射距离140 mm,此时甜叶菊干燥时长6.57 min,能耗1.25 kW·h。本研究可为研制甜叶菊干燥装置和研究甜叶菊干燥特性提供参考。     

Application of tailor-made membranes in a multi-stage process for the purification of sweeteners from Stevia rebaudiana

In this paper the performance of a three stage process with commercial as well as tailor-made polyethersulphone (PES) membranes for the purification of sweeteners from Stevia rebaudiana Bertoni was evaluated. Retentions of the sweeteners for a synthetic mixture and plant extract in combination with flux decline measurements indicated that, in contrast with the laboratory-made membranes, on most commercial membranes a foulant layer was formed that influenced the separation performance negatively. For the plant extract, the best commercial membrane (PW010) had a selectivity and flux similar to the best laboratory-made membrane (27% PES), but the laboratory-made membrane was preferred because it showed a slightly lower retention of the sweeteners, as desired. Starting from an extract purity of 11% with the overall process (microfiltration, ultrafiltration, nanofiltration) a purity of 37% and a yield of 30% could be reached.     

Stevia – Ein natürliches Sü?ungsmittel mit gesundheitlichem Zusatznutzen?

Eine Besonderheit der natürlichen Sü?stoffe aus Stevia rebaudiana soll ihre vorteilhafte pharmakologische Wirkung sein. In der wissenschaftlichen Literatur wird immer wieder ein Einfluss auf den menschlichen Blutzuckergehalt und den Blutdruck genannt. Dies w?re ein Novum: Ein Sü?ungsmittel mit Heilwirkung.     

Preparative purification of rebaudioside A from aqueous extracts using chromatography: a process idea

An innovative chromatographic process idea for preparative purification of rebaudioside A from aqueous extracts of Stevia rebaudiana Bertoni leaves is developed. The process can handle high concentrated aqueous extracts, needs less equipment and has a lower solvent consumption compared to the state-of-the-art purification process of rebaudioside A. The innovative process consists of a cascade of three preparative chromatographic columns. The first column separates steviol glycosides from the plant matrix using water as eluent in reversed phase mode. The fraction of steviol glycosides is then concentrated on a second column which works as a capture column with a more non-polar adsorbent. The last column separates rebaudioside A from steviol glycoside under normal phase conditions using a mixture of acetonitrile and water as eluent. With a total yield of 97.5?±?1.5?% and a purity of 97.1?±?1.5?% the process fulfills the legal requirements for the purity of rebaudioside A.     

Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves

A fast isocratic HPLC method for analysis of steviol glycosides from Stevia rebaudiana leaves was developed with a high selectivity for nine known steviol glycosides and low eluent consumption. The analytical method was performed on a Purospher^? STAR RP-18 endcapped 3???m Hibar^? RT 250-4.6 column at 50?°C with an eluent composition of water (65 vol.?%) and acetonitrile (35 vol.?%). With a flow rate of 1?mL?min^?1, nine known steviol glycosides were detected selectively after 15?min. Method validation for rebaudioside A showed a LOD of 0.0004?mg?mL^?1 and a LOQ of 0.0038?mg?mL^?1. Particularly with regard to moderate solubility, the method is linear up to a concentration of rebaudioside?A of 4.8?mg?mL^?1. The linear calibration curve was obtained with a coefficient of determination of 0.9997?±?0.0002 and a total error of 2.01?% RSD (n?=?12). The accuracy of the method was determined by the percentage mean recovery rate to 100.99?±?2.01?%. The intra-day precision was in a range of 0.12 to 1.96?%?RSD and the inter-day precision varies from 0.02 to 1.89?% RSD. Small changes in operating conditions like eluent composition (65?±?2 vol.?%), temperature (50?±?10?°C) or flow rate (1?±?0.2?mL?min^?1) do not affect the performance of the analytical method. The reliable and robust proposed HPLC method can be applied for quantification of steviol glycosides in Stevia rebaudiana Bertoni leaves in laboratories and quality control in industry.