Variation in steviol glycosides in stevia rebaudiana leaves harvested in different developmental stages.
- c2012.
- 52p.
Thesis Report.
ABSTRACT: Seeds of perennial herb Stevia rebaudiana Bertoni show a very low germination percentage which limits mass propagation through seed and also production of homogenous populations with important features. Thus, tissue culture is alternate technique for mass propagation of stevia and only viable method to preserve germ line with higher steviol glycosides (SGs) because these SGs are non caloric high potency sweeteners found in the stevia leaf. Thus, in the present study successful in vitro clonal propagation of high stevioside containing variety of S. rabaudiana was done. Furthermore, it is suggested that the germ line with high glycoside content can be preserved through callus culture. In stevia, SGs include more than 30 ent-kaurene type di-terpenoid molecules and most of them are identified in recent past only. These di-terpens are synthesized solely in the mesophyll cells of the leaves from common precursor molecule geranyl geranyl diphosphate via the plastid localized 2C-methyl D-erythritol- 4-phosphate (MEP) pathway. All SGs derived from steviol molecule with kaurene back bone are glucosylated at C-19 of the C-4 carboxyl group or C-13 of hydroxyl group. SGs are transported in the vacuole by the uncharacterized vacuolar transport mechanism and stored within. Among these SGs, the most abundant are stevioside and rebaudioside A. Stevioside has less potency in sweetness than rebaudioside A which is 300 times sweeter than the sucrose. In addition to SGs other several compounds such as non glycosidic labdane type sterebins, vitamins, unsaturated oils, flavonoids and pigments etc. have also been found in the stevia. Thus to use stevia leaves as effective sweetner these unrelated compounds which don't have sweetening potential have to be separated. Unless other plant constituents such as cellulosic mass, lipids and oils, pigments, flavonoids and non glycosidic diterpenoids such as labdane type sterebins are removed properly the after taste bitterness of the stevia extract would remain intact. Thus, Steviol glycosides separation is difficult and these other constituents should be separated from the SGs. Classical method is accompanied by the treatment with hot water and it is associated with long extraction time and high temperature which does not effectively separate these other constituents. Thus, extraction of rebaudioside A would require complex step such as pretreatment, separation, purification and refining. In the present study, we developed a protocol for extraction and purification SGs using two solvents system. De-ionized water is used as aqueous solvent and diethyl ether is used as organic solvent. In this study, we showed that ultrasound assisted water extraction followed by specific pore size silica chromatographic purification and UV spectophotometric quantification is an efficient method for assessing the concentration of SGs in the dry leaves of S. rebaudiana. The total amount of SGs in stevia leaves have been reported to be up to 20% of the dry leaf weight. However, the concentration of SGs varies widely depending on the genotype and cultivation parameters. Thus, to study the feasibility of cultivating stevia in high altitude, the tissue cultured plants were planted in plastic tunnel structure at 2000 meter from sea level. Although there are few literatures looking in the SGs concentration in different developmental stages of same genotype, to study the feasibility of multiple harvesting for commercial application, the SGs concentration at different stages of growth was determined. After extraction and purification from dry leaves by our own designed protocol we have been able to show that abundance of glycoside varies at different developmental stages of plant. The maximum concentration of steviol glycosides in stevia leaves is found just prior to flowering stage of the plant. The leaves harvested during growth phase showed lower concentration of SGs and leaves after maturity and flowering exhibited very low amount of SGs. In this study, we also discuss the possible mechanism for this variation in SGs concentration in stevia leaves at different developmental stage of the plant.