Ecologically pure, high-quality raw material of Stevia (with a high content of biologically active substances), grown in Nagorno-Karabakh was harvested from August to October 2016. Stevia used in this study was botanically authenticated and voucher specimens (2779 A) were deposited in the Herbarium of Institute of Hydroponics (Experimental Hydroponic Station, outdoor hydroponic station with 60–100 m2 vegetation surface area (feeding scaffolds with 12–20 repetitions). All indices of safety of Stevia dry leaves and Stevia-based food are defined in Republican Veterinary and Phytosanitary Laboratory Services Center State non-commercial Organization (SNCO) (Ministry of Agriculture of the Republic of Armenia) (Yerevan, Erebuni 12, reg. N2780, 20.01.2014) and fully comply with the decision of the RA Government on “The approval of the technical regulation of the requirements for juices and juice products and the repeal of the decision N744 of the RA Government of 26 June 2009”.
Isolation and purification of suprol fraction was carried out using a previously developed method  with some modifications. In particular, after suprol (10 g) homogenization in water (100 ml) and centrifugation (for 10 min at 6000 g), the fraction of native suprol was precipitated adding 0.05 M HCI to pH 6.2. Further, the suprol precipitate was dissolved in 0.005 M potassium phosphate buffer (PPB) at pH 7.4 and after centrifugation was subjected to ion-exchange chromatography on a column with cellulose DE-52 (4 × 10 cm), balanced with 0.005 M PPB under the given conditions. Suprol does not precipitate on a column of DE-52 under these conditions, but was purified from traces of other accompanying proteins of acidic nature. Similarly, for the removal of the accompanying protein impurities of basic nature, suprol solution was subjected to ion-exchange chromatography on a glass column with cellulose KM-52 (suprol was not absorbed on a column). Thus, suprol fraction was purified from traces of protein impurities of basic nature. After concentration of suprol fractions by lyophilization, it was subjected to gel filtration on a column with Sephadex G-100 and balanced by 0.005 M PPB at pH 7.4. First fraction of native suprol was collected. This fraction was further passed through the Biogel P-150 at pH 9.5 and fraction with a symmetric elution diagram was collected (the solubility of suprol in water increases significantly at pH 9.5). After these purification procedures of suprol, value of the optical spectral index (А280/А430) was no longer increased and amounted to 8.1 ± 0.12 (p < 0.05, n = 6).
Removal of traces of Nox from suprol-Nox complex was carried out as follows. Suprol-Nox complex was precipitated from opalescent solution at pH 4.8 by centrifugation. Traces of KOH were removed and the sediment was washed twice with water (1:50). Hydrogen peroxide at a concentration of 0.05 M was added to a water solution of precipitate and incubated for 10 min at room temperature. Next, the precipitate was thoroughly washed with water to remove traces of hydrogen peroxide, which was determined by permanganometric titration. The sediment was homogenized in water at pH 9.5 and was subjected to ion-exchange chromatography on a column with cellulose DE-52 at рН 9.5.
Gel electrophoresis of suprol was performed in 15% polyacrylamide gel at pH 8.9 and 4.5 according to acidic and basic nature of the proteins associated with suprol. However, suprol under the influence of an electric current is aggregated prior to polyacrylamide gel electrophoresis. In the course of electrophoresis and protein staining, the presence of other water-soluble and suprol-associated proteins of acidic and basic nature was not observed on these gels.
The specific content of suprol (milligrams in 1 g of Stevia leaves-mg/g) was determined after desalination of suprol and lyophilization.
The process of producing O2− of suprol was revealed by 4 different and well-known methods: the reduction of potassium permanganate, nitrotetrazolium blue, Coomassie brilliant blue and the oxidation of adrenaline to adrenochrome (E480 = 750 M− 1 cm− 1) [16,17,18]. The amount of suprol was considered per unit of O2− -producing activity of suprol capable of enhancing the formation of adrenochrome to 50%. Specific O2− -producing activity of suprol was expressed in u/mg suprol.
The lipid composition of suprol (8 mg) from suprol was determined by thin-layer chromatography. However, suprol is poorly soluble in organic solvents, which creates certain difficulties in the application of thin-layer chromatography .
Ascorbate-dependent malonic dialdehyde (MDA, a product of lipid peroxidation) was determined according to the method of Y. A. Vladimirov and others  determining the specific content of MDA (mg) in 1 mg of suprol. In particular, 1 ml of suprol (8 mg) was mixed with 1 ml of thiobarbituric acid (0.75%) and ascorbic acid (0.8 mM), heated at 50–60 °C for 30 min, then trichloroacetic acid (30%) was added and incubated at 90 °C for 30 min. After freezing and centrifugation, the amount of MDA in the supernatant was determined by measuring the density of the characteristic optical absorption of MDA at 535 nm (molar extinction of MDA at 535 nm is 1.56 × 105 M− 1 cm− 1).
Statistical significance of differences in the results was assessed by student’s t-test (М ± m) with determination of criteria of reliability of obtained data. Significant differences between obtained values were analyzed using GraphPad Prism5.0 software (GraphPad Software, Inc., San Diego, CA).
Optical absorption and fluorescence spectra were recorded on a Hitachi-2000 (Japan) and Perkin Elmer (USA) spectrophotometer, respectively, at 20 in cuvettes with an optical path of 1 cm.
In the course of the research, K-70 and K-24 centrifuges (Janetzki, Germany) were used.