The metabolism of [3-H]oleanolic acid and its monoglycosides in cytoplasm and vacuole of protoplasts isolated from Calendula officinalis leaves

Isolated protoplasts from C. officinalis leaves were supplied with [3-³H]oleanolic acid, its 3-O-monoglucoside and 3-O-monoglucuronide. Transformations of these compounds into two series of oleanolic acid glycosides, i.e. glucosides (derivatives of 3-O-monoglucoside) and glucuronides (derivatives of 3-O-monoglucuronide) in the extravacuolar space and the vacuole were investigated. In the cytoplasm free oleanolic acid is glycosylated to both monoglycosides and to higher glycosides. Monoglycosides are partly hydrolysed to free oleanolic acid and partly glycosylated to higher derivatives. The vacuole contains the same radioactive compounds as the extravacuolar space. However, it seems most likely that these compounds are transported there from the sites of their synthesis in the cytoplasm.     

Intracellular distribution and origin of sterols in Calendula officinalis leaves

In Calendula officinalis leaves 66% of all steryl forms are present in the ‘microsomal fraction’ (IV), 24% in the mitochondrial and Golgi membranes (III), 5% in the ‘chloroplast’ (II), 4% in the ‘cell wall and membrane’ (I) fraction and 1%. in the cytosol. Free sterols, their esters, glycosides and acylated glycosides are present in varying proportions in all cellular subtractions. Mevalonate-[2¹⁴C] labelling of sterols derived from various steryl forms showed that free sterols and all their derivatives, i.e. steryl esters and glucosides, are formed in fraction IV and are then translocated to other organelles. Fraction III is the main site of glycosylation of transported sterols as well as of acylation of steryl glycosides.     

The effect of pH and ATP on the transport of oleanolic acid monoglycosides into isolated vacuoles of Calendula officialis leaves.

The transport of oleanolic acid and its glycosides (3-O-monoglucuronide F and 3-O-monoglucoside I) into the vacuoles of C. officinalis leaves was studied. It was found that these monoglycosides are transported into the vacuolar space whereas free oleanolic acid only binds with the tonoplast. The transport of monoglycosides depends on pH of the medium, the optimum pH for monoglucoside I and monoglucuronide F being 6.0 and 7.0, respectively. Moreover, it was demonstrated that the transport of monoglucoside I, in contrast to that of monoglucuronide F, depends on ATP at 0.5-2.0 mM concentrations. The presented results indicate that different mechanisms underlie the transport of these two compounds into vacuoles.     

Oleanolic acid and ursolic acid affect peptidoglycan metabolism in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.     

Influence of elastin-derived peptides, glucose, LDL and oxLDL on nitric oxide synthase expression in human umbilical artery endothelial cells

Endothelial dysfunction plays an important role in the development of atherosclerosis. Elastin-derived peptides (EDP), hyperglycemia, hypercholesterolemia and oxidized LDL have a proven proatherosclerotic potential. Nitric oxide generated by endothelial nitric oxide synthase (eNOS; EC 1.14.13.39) is an important vasorelaxant. Here we studied the influence of those proatherosclerotic factors on eNOS gene and protein expression in artery-derived endothelial cells. Human umbilical artery endothelial cells (HUAEC) were incubated with or without: glucose (270 mg/dl), LDL (200 mg/dl), oxidized LDL (oxLDL 25 mg/dl) or κ-elastin (0.5 and 2.5 μg/ml). Gene expression was assessed by real time RT-PCR, whilst the eNOS protein by ELISA. In cells incubated with 2.5 μg/ml of κ-elastin, a 31 % increase of eNOS mRNA expression was observed, but the protein level remained unchanged. OxLDL, LDL and glucose decreased the eNOS protein level by 74 %, 37 % and 29 %, respectively. OxLDL decreased eNOS mRNA by 42 %. LDL non-significantly decreased eNOS mRNA expression. An elevated glucose level did not affect the eNOS mRNA expression. Hyperglycemia and an elevated level of LDL, particularly oxLDL, decreased the level of eNOS protein in endothelial cells. As κ-elastin did not decrease the expression of eNOS gene in HUAEC, the proatherogenic properties of elastin-derived peptides are unlikely to be due to their influence on eNOS.     

Metabolism of [3-<Superscript>3</Superscript>H]oleanolic acid in <Emphasis Type="Italic">Calendula officinalis</Emphasis> L. roots

The radioactive precursor, [3-³H]oleanolic acid was administrated to excised roots from four weeks old Calendula officinalis L. plants. Transformations of this compound into two series of its glycosides, i.e. glucosides and glucuronides were investigated. For the first time it has been shown that both series of oleanolic acid glycosides are synthesized in roots of young marigold plants. The pathway of their biosynthesis seems to be similar, although not identical, to the pathway occurring in green organs of C. officinalis.