Protein Constituent from Plants of the Genus Crambe and its Hypoglycemic Activity

Abstract

Crambe kotschyana Boiss. and C. orientalis L. (Cruciferae) are herbaceous perennial feed plants that are endemic and broadly distributed in Uzbekistan [1]. Young shoots of the plants are used in food. Dried roots are processed into flour. The starch content in roots is almost the same as in potatoes. The green plant is consumed by several animals (camels and sheep). The nutritional value of C. kotschyana exceeds that of many currently cultivated plants, especially the contents of essential amino acids and macroscopic and trace elements [2]. Alkaloids were first found by us in these plant species [3]. The S-containing alkaloids goitrin and goitridin [4] in addition to the known compound N-(benzimidazolyl-2)-O-ethylcarbamate [5] were previously isolated from the CHCl3-soluble total alkaloids in the EtOH extract of the aerial part and roots of two Crambe species. In continuation of this research, the alkaline aqueous solution remaining after extraction of total alkaloids from C. kotschyana and C. orientalis, which showed a reaction for N-containing compounds, was investigated for amino-acid content. Several plant species are known to accumulate free amino acids characteristic of this species. Individual nitrogenexchange steps and plant food values are commonly studied by determining free and bound amino acids. Therefore, we isolated and determined quantitatively the protein content in the alkaline aqueous solution remaining after extraction of total alkaloids. It contained 10% protein [6]. Free and bound amino acids were determined quantitatively on a T339 amino-acid analyzer. Free amino acids were isolated by heating a weighed portion (100 mg) of protein in distilled H2O (10 mL) on a water bath for 5 min. The extract was evaporated in a rotary evaporator and prepared for analysis on the amino-acid analyzer [7]. Bound amino acids were determined after acid hydrolysis in HCl (5.7 N) for 24 h at 110°C in vacuo [8]. Table 1 presents the analytical amino-acid composition. Table 1 shows that the amino acids were found mainly in the bound state. The total amino acids made up 2.12% whereas the total bound amino acids made up 16.9%. All essential amino acids, i.e., valine, threonine, isoleucine, leucine, lysine, phenylalanine, histidine, and arginine, which are not synthesized in vivo and are assimilated from food, were found in the amino-acid composition of Crambe protein. Glutamine, leucine, glycine, alanine, arginine, and lysine dominated the free and bound amino acids. HPLC was used to determine the molecular weights (MWs) of the isolated proteins. The MWs were determined by constructing a calibration curve using the logarithm of the protein MWs as a function of their retention times. The MW of the protein (peptide) was 10 kDa. The observed hypoglycemic effect of the C. kotschyana proteins was interesting. We studied the effect of the protein component mainly in untreated animals. The investigations showed that blood sugar was reduced by 14% (at the most effective dose of 50 mg/kg) 3 h after a single administration of the drug (the activity peaked at this time) (Table 2). However, these data were statistically insignificant. The hypoglycemic activity of the drug with exogenic administration of glucose showed that glucose loading increased the blood glucose level by 58.4% in control rats after 30 min (peak activity). The blood sugar level of test rats, which received C. kotschyana protein component at doses of 25 and 50 mg/kg 2.5 h before the start of the experiment, increased at this time by only 34.0 and 28.0%, respectively. The difference from the control was in all instances statistically significant (Table 3).