Background Stevia rebaudiana Bertoni has various pharmacological actions, which includes antidiabetic, antioxidant, anti-inflammatory activities. The superoxide and consequently NADPH oxidase (Nox) are relevant targets involved in biological effects of Stevia. The presence of NADPH-containing superoxide-producing lipoprotein (suprol) in Stevia leaves has not yet been tested. The mechanism of producing superoxide radicals (O 2 − ) by suprol was determined in vitro, which is associated with the electron transfer from NADPH in the composition of suprol by traces of transition metal ions (Fe 3+ or Cu 2+ ) to molecular oxygen, turning it into O 2 − . It is expected that the therapeutic efficacy of Stevia leaves is caused by specific activity of superoxide-producing lipoprotein fraction. Methods For the first time, from the dry leaves of Stevia the NADPH-containing superoxide-producing lipoprotein was isolated and purified. The specific content of suprol (milligrams in 1 g of Stevia leaves- mg/g) was determined after desalination of suprol and lyophilization. Results According to the method provided, the specific content of the isolated suprol from Stevia’s leaves was up to 4.5 ± 0.2 mg / g (yields up to 68.5 ± 4.7%, p < 0.05, n = 6). Nox forms a stable complex with suprol. The optical absorption spectrum of the Nox-suprol complex represents the overlapping suprol and Nox spectra, with a certain background increase and characteristic features of optical absorption for Nox. Due to O 2 − producing capacity suprol-Nox complex discolors KMnO 4 solutions, Coomassie brilliant blue, restores nitrotetrazolium blue to formazan and oxidizes epinephrine to adrenochrome. The oxidation activity of adrenaline is 50.3 ± 5.1 U / mg / ml ( p < 0.05, n = 6). Conclusion Superoxide-producing lipoprotein fraction-Nox complex from Stevia leaves (membranes) can modulate redox regulated signaling pathways and may play a positive role in type-2 diabetes by means of adrenaline oxidation mechanism.

It is known that FAD is an important cofactor for Nox isoform, as an electron carrier from NADPH to Fe(III) of the heme group then to molecular oxygen reducing it up to О2 [1, 6]. On the other hand, the thermostable Nox is isolated from thermostable microorganisms [5]. However, the role of FAD as a factor in changing the conformation and thermostability of Nox isoform has not yet been determined. The aim of the present work was to determine the corresponding conditions under which the thermostability and production of superoxide radicals are also observed in the absence of NADPH.

From Armenian plant food – tomato (Solanum lycopersicum esculentum), carrot (Daucus carota subsp. sativus), green bean (Phaseolus vulgaris) and potato (Solanum tuberosum), the isoforms of superoxide (О2) producing complexes between NADPH containing protein component (NPC) and Fe(III) – NPC-Fe(III), were isolated and purified for the first time. At the same time, the fractionation of these complexes at pH 9,5 and 4,8 were carried out [1]. The isoforms of NPC-Nox are high thermostable biosystems (up to 100oC). The immediately mechanism of the production of О2 by these complexes, due to the transfer of the electron from NPC to the Fe(III), then to О2 for its reduction up to О2 was conditioned. As a substrate for NPC-Fe(III) is not free NADPH, but NADPH is connected with the protein component (PC). The forms of optical absorption spectra of these complexes in visible region essentially were differed, although, in UV region the characteristic for the proteins maximal optical absorption at 260-280 nm were observed. The higher specific content (mg/g) for complexes from tomato, than from carrot, green bean and potato were observed. The direct proportional dependence between the content of the NADPH in the composition of NPC and stationary concentration of produced О2 in homogenous phase (in solution) and gas phase were presented. The isolated NPC from indicated above complexes, at the expense of NADPH, only the reductive (antioxidant) effect was indicated and was activated the NADPH oxidase (Nox) in vitro, in particular from erythrocytes membranes (ЕМ) аnd leukocytes membranes (LM). By the blowing of the slightly opalescent aqueous solutions of these complexes with oxygen the gas phase О2 were produced, which were transferred oxygen through the silicone or glass tubes.