A NEW METHOD OF QUANTITATIVE DETERMINATION OF PIGMENTS IN STEVIA LEAVES

Abstract

Background. It is known that stevia leaves of Stevia rebaudiana (Bert.) contain a different complex of biologically active compounds (BAC) (flavonoids, phenylpropanoids, saponins, organic acids). In view of the fact that it is currently advisable to take orally dried and powdered medicinal plant raw materials, it is also interesting to study other BAC contained in the plant, in particular pigments [1, 2]. Stevia leaves contain a complex of natural pigments, the main of which are chlorophyll derivatives and carotenoids, represented by β-carotene and chlorophyll a. One of the most important properties of chlorophyll is anti-inflammatory, wound healing and antibacterial action, it also has an antioxidant effect [3–5]. Carotenoids also have a wide range of pharmacological properties, including antioxidant, radioprotective and anti-carcinogenic activity, which together have a positive effect on the immune system [5]. In this case, stevia leaves containing these compounds are of great interest. Stevia Rebo is a poorly studied plant. In the scientific literature, there are conflicting data on the chemical composition of leaf stevia, and regulatory documents are not provided. The results of the study can be used for the development and improvement of standards. Objective – development of methods for quantitative determination of β-carotene and chlorophyll a content in stevia leaves. Materials and methods. The objects of the study were dried stevia leaves harvested in the Krasnodar territory, the Republic of Crimea, Penza and Tver regions). Qualitative analysis of the sum of carotenoids and chlorophyll was performed by thin-layer chromatography from the acetone fraction in the following solvent system: petroleum ether-acetone (6:4). For quantitative determination of carotenoids and chlorophylls in extracts from stevia raw materials, a spectrophotometric method was used. Quantitative determination of carotenoids in stevia raw materials was performed using 95 % ethyl alcohol, hexane, acetone, petroleum ether, and ethyl acetate. Determination of chlorophyll in stevia raw materials was performed using 95 % ethyl alcohol, hexane, and acetone. Optical density was measured at the maximum absorption of carotenoids (450 nm) and chlorophyll a (664 nm). Results. The method of thin-layer chromatography was used to study the qualitative content of pigments in stevia raw materials. Visually, 4 spots are identified, one of which is yellow with Rf = 0,93 refers to β-carotene, Rf = 0,76 α-cryptoxanthin and β-cryptoxanthin. Chlorophylls a and b appear as blue-green and yellow-green spots with Rf = 0,10 and 0,08 respectively. Conclusions. Samples of stevia grown in different regions of Russia were analyzed using the developed method for determining carotenoids and chlorophyll. It was found that the content of carotenoids in stevia leaves varies from 4,33 to 7,15 mg%, chlorophyll from 3,63 to 4,21 mg%.

Keywords

• stevia rebaudiana
• β-carotene
• chlorophyll a
• spectrophotometry
• thin-layer chromatography