Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations.

Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic. Tissue culture is the best method with high efficacy to propagate stevia. Abiotic stress has an impact on steviol glycoside contents in stevia. Therefore, we investigated the effect of mannitol on the expression of four genes involved in the biosynthesis of stevia including UGT74G1, UGT76G1, kaurene oxidase and kaurene synthase genes and steviol glycosides accumulation in stevia under in vitro conditions. The highest expression of UGT76G1 gene occurred in the plants grown under 20 g/l mannitol. While for the kaurene synthase gene, the highest amount of gene expression was observed at 40 g/l mannitol. The results were different about kaurene oxidase gene. As the highest and lowest gene expression were seen in 50 and 30 g/l mannitol conditions respectively. There were the same results for UGT74G1 that means the most appropriate and also the most inopportune treatment for the gene expression were same as the condition for the kaurene oxidase gene. Compared with control, adding mannitol to media in all concentrations increases the expression of UGT76G1 gene. Estimation of steviol glycosides contents under different treatments of mannitol carried out by HPLC. According to the results, the highest amount of stevioside was produced under 20 g/l mannitol treatment. However, rebaudioside A was accumulated in its maximum amounts under 30 g/l mannitol. It can be concluded that adding mannitol to media in the certain concentration increases steviol glycoside contents in the stevia.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Effect of Stevia rebaudiana Bertoni extract on sexual dysfunction in Streptozotocin-induced diabetic male rats.

Stevia rebaudiana Bertoni has been used locally as a non-calorie sweetener in medicine and diabetic diet which claimed to have aphrodisiac properties, although no scientific data of this function have been reported. The aim of this study was to investigate the effect of S. rebaudiana extract on sexual dysfunction, testosterone levels and number of Leydig cells in Streptozotocin (STZ)-induced diabetic male rats. A total of 28 diabetic male rats were randomly divided into 4 groups: diabetic group without any extract and 3 extract groups (5, 50 and 100 mg/kg). Seven normal control rats were treated with vehicle mount latency and frequency of (ML, MF), intromission latency and frequency (IL, IF), ejaculation latency and frequency of (EL, EF), the mount latency post ejaculation (MPE), the intromission latency post ejaculation (ILE), the intromission frequency post ejaculation (IFE) were recorded during 30 min on days 0, 14, 28. The serum testosterone levels, blood glucose, sex organs weight, number of leydig cells and histology of testicular tissue were measured. The stevia group (5 mg/kg) had a significant (p<0.05) increase in EF and IF. The number of Leydig cells in the diabetic group were significantly (p<0.05) reduced compared to the normal group and diabetic groups with extract (5 and 50 mg/kg). The serum testosterone levels and other sexual behaviors did not show any significant differences. The low- dose stevia extract with attention to antioxidant, vasodilator and anti-diabetic properties can be aphrodisiac in STZ- induced diabetic male rats.

Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

Effect of nitrogen sources on gene expression of Stevia rebaudiana (Bertoni) under in vitro conditions.

Stevia rebaudiana (Bertoni) is a non-caloric sweetener belonging to Asteraceae family. Stevia compounds such as steviol glycosides (SGs) are 200 times sweeter than sugar. Stevioside and rebaudioside A are the two major steviol glycosides. Nitrogen is an essential element for plant growth and development. In this study the effects of nitrogen influenced by different concentrations of NH4NO3 (0, 825 and 1650 mg/l) and KNO3 (0, 950 and 1900 mg/l) is examined in MS medium. To analysis the UGT74G1 and UGT76G1 genes expression, involved in the synthesis of SGs, RT-qPCR technique was performed. Data showed that there were significant differences between all media. The shoot length, seedlings dry weight and leaf fresh weight of stevia increased with applying NH4NO3 along with KNO3. The highest expression of UGT74G1 gene, was observed in plantlets grown on MS medium with 0 mg/l NH4NO3 and 950 mg/l KNO3 (1.291 total lab unit) but the highest expression of UGT76G1 gene, was observed in plantlets grown on MS medium added by 1650 mg/l NH4NO3 +950 mg/l KNO3 (1.08 total lab unit). Moreover, the lowest value of UGT74G1 gene expression were revealed in MS medium added by 1650 mg/l NH4NO3 +0 mg/l KNO3 (0.80 total lab unit) and the lowest values of UGT76G1 gene expression seen in MS medium with 0 mg/l NH4NO3 +950 mg/l KNO3 (0.85 total lab unit) concentrations. The results of this study could be valuable in stevia breeding programs through glycosides biosynthesis pathways.

Effects of different concentrations of mannitol on gene expression in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important herbal sweetener plants from Asteracea family that have a lot of Steviol glycosides. Among different methods, tissue culture is the best way with high efficiency that is useful for studying stress tolerance mechanisms to obtain drought tolerance of stevia. For this purpose, different concentrations of mannitol (0, 10, 20, 30, 40, 50 mg/l) were used as various treatments in the culture medium of stevia. According to the results, the highest level of UGT85C2 gene expression (1.181 Total lab unit) was seen in plants grown under 30 mg/l mannitol treatment and the lowest level of this gene expression (0.603 Total lab unit) was observed under 40 mg/l mannitol treatment. However, the highest level of KO gene expression (1.323 Total lab unit) was observed under 20 mg/l mannitol. It shows stevia growth is affected by osmotic stress. Water deficiency has a negative impact on Stevia. However, the expression of genes had increased by particular mannitol concentrations. Actually, stevia can survive under various abiotic stresses.

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Quantification of sofosbuvir in human serum by liquid chromatography with negative ionization mass spectrometry using the parent peak and its source-induced fragment: Application to a bioequivalence study.

In the mass spectrometry of sofosbuvir, a new orally administered antihepatitis C drug, a weak peak is detected at the m/z value of the parent ion (m/z 530) as a result of in-source dissociation and current methods to its quantification, is based on monitoring of the parent peak using ultra high-performance liquid chromatography with tandem mass spectrometry. With these methods serum concentration of the drug is quantifiable only up to 4-5 h postdose. However, the fragmentation of the molecule generates a more stable ion at m/z 287 (base peak) with a signal intensity of about tenfold compared to the parent ion. Our study was aimed to improve sensitivity of analysis by acquisition of the m/z value of the daughter ion from which it originated instead of the parent molecule. This novelty allows us to measure serum concentrations of the drug for a longer time postdose and provides more opportunity for pharmacokinetic studies of sofosbuvir. Our method was linear over the concentration range of 2-2560 ng/mL of sofosbuvir in human serum with a limit of quantification of 2 ng/mL compared to 10 ng/mL reported previously. The coefficient variation values of both inter and intraday analysis were less than 13.8%, and the percentage error was less than 6.3.