Immunobiological properties of sesquiterpene lactones obtained by chemically transformed structural modifications of trilobolide.

Our previous research on immunostimulatory properties of trilobolide and its structurally related natural analogues isolated from Laser trilobum (L.) Borkh., encouraged us to investigate structurally related guaianolides belonging to a specific group of sesquiterpene lactones with characteristic glycol moiety attached to the lactone ring. Ever increasing attention has been paid to certain guaianolides such as thapsigargin and trilobolide for their promising anti-inflammatory, anticancer, anti-infectious and SERCA inhibitory activities. However, due to their alkylation capabilities, they might be cytotoxic. Search for compounds with preserved immunobiological properties and decreased cytotoxicity led us to transform some of their structural features, particularly those related to their side chain functionality. For this reason, we prepared a series of over 20 various deacylated, acyl modified, or relactonized derivatives of trilobolide. The immunobiological effects were screened in vitro using the rat peritoneal cells primed with lipopolysaccharide. Secretion of interferon-γ (IFN-γ), interleukins (IL) IL-1ß, IL-6 and tumour necrosis factor-α (TNF-α) were determined by ELISA, and nitric oxide (NO) production by Griess reagent. Relation between the molecular structure and immunobiological activity was investigated. Acetylation at 7-OH and 11-OH positions of the lactone ring, or acyl modification of the guaianolide functionalities (including relactonization) of trilobolide, led to inability to stimulate secretion of cytokines and production of NO. Interestingly, minor structural changes achieved by catalytic hydrogenation or hydrogenolysis retained the original immunoactivity of trilobolide. It can be concluded that several new chemically transformed sesquiterpene lactones resembling the immunobiological properties of trilobolide or thapsigargin were prepared and identified. The implication of the lactone vicinal diol (glycol) moiety, combined with other structure functionality, was confirmed as essential for immune properties of the trilobolide or thapsigargin type of guaianolides.

Trilobolide and related sesquiterpene lactones from Laser trilobum possessing immunobiological properties.

Three new and five known sesquiterpene lactones were isolated from the roots of Laser trilobum (L.) Borkh. Chemical identity of the known compounds and structural analysis of the new ones were determined by HR MS and NMR spectroscopy. The two new sesquiterpene lactones: 2-acetoxytrilobolide and 2-hydroxy-10-deacetyltrilobolide belong to the guaianolide type, and the third one, eudeslaserolide, to the biogenetically related eudesmanolide type. Both types, together with their biogenetic precursor of germacranolide type (laserolide) are present in L. trilobum, as well as in the related Laserpitium species. Purposefully selected set of these native sesquiterpene lactones was tested for specific immunobiological properties. The obtained results demonstrate that trilobolide and its acetoxy analog are strong activators of cytokine secretion. On the contrary, the other L. trilobum and Laserpitium siler constituents are only very mild activators, or even inhibitors of the cytokine and nitric oxide production.

Sesquiterpene lactone trilobolide activates production of interferon-γ and nitric oxide.

Trilobolide (TB), a sesquiterpene lactone isolated from Laser trilobum is an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). We have found that upon the in vitro exposure to TB, rodent peritoneal cells and human peripheral blood mononuclear cells secrete high amounts of IFN-γ. The effect is associated with the stimulation of high output NO biosynthesis in rat cells. The stimulatory potential of TB depends on the activation of MAP kinases p38 and ERK1/2, and transcription factor NF-κB. BAPTA-AM, a chelator of the intracellular calcium, remained without any effect on the secretion of IFN-γ triggered by TB. These results demonstrate that TB is a potent immunostimulatory agent.

Inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase thapsigargin stimulates production of nitric oxide and secretion of interferon-gamma.

Thapsigargin is a sesquiterpene lactone of guaianolide type isolated from the Mediterranean plant Thapsia garganica L. It is widely used experimentally as a potent and selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) leading to rapid elevation of intracellular calcium [Ca2+]i. Several previous reports have shown that thapsigargin interferes with production of nitric oxide (NO) by mouse peritoneal macrophages and mouse macrophage cell lines. The present data confirm that thapsigargin is a modest inducer of NO in mouse macrophages, production of NO being slightly enhanced by lipopolysaccharide. However, thapsigargin on its own very potently induces NO in macrophages of rats under conditions in vitro. The highest effect was observed after the concentration of 0.25 microM thapsigargin, producing approximately 30 microM accumulation of nitrites in supernatants of cells cultured for 24 h. The aim of our experiments was to investigate immune mechanisms implicated in activation of high-output NO biosynthesis. It has been found that thapsigargin dose-dependently induces secretion of interferon-gamma (IFN-gamma) in macrophages of both rats and mice, and also in human peripheral blood mononuclear cells. The IFN-gamma production was rather low in macrophages of mice while relatively very high levels of IFN-gamma were found in cultures of rat macrophages and human peripheral blood mononuclear cells. The concentration of IFN-gamma produced by 5 microM thapsigargin within the interval of 24 h exceeded 3 ng/ml in rat macrophages and approached 2 ng/ml in cultures of human peripheral blood mononuclear cells. The effects are mediated by mitogen-activated protein kinases (MAPKs) such as p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinases 1/2 (ERK1/2), and by nuclear transcriptional factor NF-kappaB. In summary, the original findings demonstrate immunostimulatory potential of thapsigargin and warrant more detailed preclinical studies.

Additional minor ecdysteroid components of Leuzea carthamoides.

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.

Lack of interference of common phytoecdysteroids with production of nitric oxide by immune-activated mammalian macrophages.

Effects of selected common phytoecdysteroids on immunobiological responses triggered by lipopolysaccharide and interferon-gamma (IFN-gamma) were tested under in vitro conditions using murine resident peritoneal macrophages. Namely, production of nitric oxide was investigated. The series of test agents encompassed ecdysteroids occurring often as major components of the ecdysteroid fraction in numerous plant extracts: 20-hydroxyecdysone (20E), polypodine B, ajugasterone C, ponasterone A and inokosterone. Their structural variability concerns only variation in the number and position of hydroxyls. Two additional side-chain modified ecdysteroids: makisterone A (with a methyl substituent at position 24) and carthamosterone (with a cyclic side-chain lactone), and three ecdysteroid analogs: poststerone, rubrosterone and dihydrorubrosterone (devoid of side chains) were included into the test series. All test compounds, except of ponasterone A, represent natural substances isolated from the medicinal plant Leuzea carthamoides and are supposed to be significant for the often reported pharmacological activities of preparations derived from this species. However, the tested ecdysteroids did not interfere with the immunobiological activity of the immunocompetent cells. Our results thus differ from the so far reported information.

Development of a procedure for the sequential extraction of substances binding trace elements in plant biomass.

This work investigates how the amounts of some important substances in a plant, and their behaviour inside the plant, depend on the levels of stress placed on the plant. To this end, model plant spinach (Spinacia oleracea L.) was cultivated on soil treated with sewage sludge. The sewage sludge contained various trace elements (As, Cd, Cu, Zn), and the uptake of these trace elements placed the plant under stress. Following this, a sequential extraction procedure was employed to determine the levels and distributions of trace elements within the most important groups of compounds present in the spinach plants. Since the usual five-step sequential extraction procedure provides only general information on the distributions of elements within individual groups of organic compounds, due to the wide range of organic compounds within the individual fractions, this scheme was extended and improved through the addition of two solvent extraction steps-a butanol step (between the ethyl acetate and methanol solvent steps) and an H(2)O step (after the methanol+H(2)O solvent step). The distributions and levels of the trace elements within the main groups of compounds in spinach biomass was investigated using this new seven step sequential extraction (water free solvents: petroleum ether (A) --> ethyl acetate (B) --> butanol (C) --> methanol (D) --> water solvents: methanol+H(2)O (1+1; v/v) (E) --> H(2)O (F) --> methanol+H(2)O+HCl (49.3+49.3+1.4; v/v/v) (G)). The isolated fractions were characterized using IR spectroscopy and the trace element contents were determined in the individual fractions. Lipophilic compounds with low contents of Cd, Cu and Zn were separated in the first two fractions (A, B). Compounds with higher As contents (11.5-12.8% of total content) were also extracted in the second fraction, B. These two fractions formed the smallest portion of the isolated fractions. Low molecular compounds from secondary metabolism and polar lipids were separated in the third (C) and fourth (D) fractions, and high molecular compounds (mainly polypeptides and proteins) separated in the fifth and sixth fractions (E, F). The addition of the H(2)O solvent step was particularly useful for separating compounds that have a significant impact on trace element bounds. The methanol fraction was dominant for all treatments, and a significant decrease in the spinach biomass separated in this fraction was observed when the soil was treated with sewage sludge. Most of the As (35.5-38.8% of total content), Cu (45.0-51.6%) and Zn (39.8-47.2%) was also determined in this fraction. The G fraction (obtained after acid hydrolysis) contained polar compounds. Most of the Cd was also found in this fraction, as was a significant amount of Zn. Non-extractable residues formed the last fraction (polysaccharides, proteins).

High-performance liquid chromatographic analysis and separation of N-feruloylserotonin isomers.

The N-feruloylserotonin containing fraction was isolated from seeds of Leuzea carthamoides (Willd.) DC by solvent extraction followed by column chromatography on silica gel or on Sephadex LH-20. Nuclear magnetic resonance spectroscopic analysis of the isolated fraction showed the presence of four structurally related compounds. These compounds were identified as four isomers of N-feruloylserotonin: N-(Z)-feruloylserotonin, N-(Z)-isoferuloylserotonin, N-(E)-feruloylserotonin and N-(E)-isoferuloylserotonin. They were analyzed by HPLC on Separon SGX C18, Separon SGX and Separon SGX phenyl, using various mobile phases. Separon SGX phenyl phase was found the most efficient for a rapid analysis and for the final separation of the N-feruloylserotonin isomers.

Photochemical transformation of 20-hydroxyecdysone: production of monomeric and dimeric ecdysteroid analogues.

Structural modification of 20-hydroxyecdysone (20E) based on photochemical transformation yielded dimeric ecdysteroid 7alphaH,7'alphaH-bis-[(20R,22R)-2beta,3beta,20,22,25-pentahydroxy-5beta-cholest-8(14)-en-6-one-7-yl] as a main product. Its structure was determined by detailed NMR analysis. Furthermore, two new monomeric analogues: 14-epi-20-hydroxyecdysone and 14-deoxy-14,18-cyclo-20-hydroxyecdysone were identified in addition to the earlier described 14-deoxy and 14-hydroperoxy derivatives of 20E. Formation of the specific and so far unique ecdysteroid dimer has not been observed in earlier photo-transformation studies. The transformed dimeric analogue of 20-hydroxyecdysone retained the high agonistic activity on the ecdysone receptor in the B(II)-bioassay compared with the original 20E.