A. M. Popov

Study of antioxidant and membrane activity of rosmarinic acid using different model systems

Rosmarinic acid is found in many species of different families of higher plants and its chemical structure is phenol propanoid with various biological activity. In this paper, we conducted a comparative study of antioxidant (radical-scavenging) properties of rosmarinic acid in systems of 2,2tāzo-bis(2-methylpropionamidin)dihydrochloride-luminol and hemoglobin-hydrogen peroxide-luminol, determined its protective potential in preventing peroxidation of linoleic acid, and evaluated the effect on the permeability of planar bilayer lipid membranes. Linoleic acid peroxidation was assessed by iron-thiocyanate method. In these studies, trolox was used as a reference antioxidant, and ascorbic acid, and dihydroquercetin were taken as standards. Rosmarinic acid is significantly superior to trolox, ascorbic acid and dihydroquercetin in the tests for antioxidant activity in the systems studied, as well as in inhibition of linoleic acid peroxidation. According to their activity the investigated substances can be arranged in the following order: rosmarinic acid > dihydroquercetin trolox > ascorbic acid. Rosmarinic acid does not cause significant changes in the permeability of planar bilayer membranes in a dose range of 0.5 to 10 μg/mL. Antioxidant activity of rosmarinic acid is due to the neutralization of reactive oxygen species and/or luminol radicals generated in model systems. The observed features of the antioxidant and membrane activity of rosmarinic acid, which may underlie the previously mentioned pharmacological effects are discussed.

A Comparative Study of the Hemolytic and Cytotoxic Activities of Triterpenoids Isolated from Ginseng and Sea Cucumbers

Specific features of cytotoxic (against tumor cells), hemolytic, and liposomal (effect on permeability) activities of triterpenoids isolated from sea cucumbers and ginseng roots were studied. It was shown that oleanolic acid, protopanaxatriol, and protopanaxadiol at 5 to 20 μg/ml inhibited the growth of tumor cells, while at doses up to 100 μg/ml, they did not induce hemolysis or changes in liposome permeability. Monoglucosides of protopanaxadiol, Rh2, Rg3, and substance K exerted moderate cytotoxic and membrane activities. The membrane sensitivity to these glucosides was inversely proportional to the membrane content of cholesterol. The cytotoxicity of the protopanaxadiol-active glycosides increased with a decrease of pH of the medium. All studied glycosides did not affect the cell and model lipid membranes. The activity of the oleanolic acid glycoside, ginsenoside Z-R1, depended to a great extent on the pH of the medium. The decrease of pH from 7.4 to 5.6 increased the membranolytic activities by more than one order of magnitude. Glycosides from sea cucumbers, echinosides A and B, holothurins A and B, holotoxin А1, and cucumarioside G1, had very high cytotoxic and liposomal activities. Addition of cholesterol to cell membranes enhanced the cytotoxic effects of these glycosides. The ginsenosides with two carbohydrate moieties (bisdesmosides), as well as all the panaxatriol glycosides we studied did not exhibit cytotoxic activities against tumor cells or alter the permeability of model lipid and lipid-sterol membranes. The triterpenoids studied were classified into four categories in accordance with their membranotropic activities. A possible protective role of these glycosides in the organism-producent is discussed.

Peculiarities of the corrective effects of polar lipids and bioantioxidants from sea hydrobionts in impairments of lipid and carbohydrate metabolism

A total mixture of phospho- and glycolipids from sea macrophytes Sargassum pallidum, Ulva fenestrata, Zostera marinawas separated and the fatty acid composition was determined. Biological activity of the mixtures of polar lipids and natural antioxidants (echinochrome A from the flat sea urchin Scaphechinus mirabilis and a polyphenolic complex from the sea grass Zostera marina) were studied under conditions of impairments of carbohydrate and lipid metabolism. Doses and compositions of mixtures of polar lipids and antioxidants possessing high corrective activity were optimized in mice with the experimental model of atherosclerosis and diabetes. Based on these results possible mechanisms of the effects of polar lipids containing various polyunsaturated fatty acids and the investigated antioxidants have been proposed. The developed compositions may be used for creation of new biologically active additives and drugs.

Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A2-2 and monogalactosyldiacylgycerol

A novel antigen carrier has been formulated on the basis of a cucumarioside-A2-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.

A comparative assessment of the effects of alkaloid tryptanthrin, rosmarinic acid, and doxorubicin on the redox status of tumor and immune cells

A comparative study of the effects of natural compounds with different biological activity spectra and mechanisms of action on the dynamics of the change in the redox-status of tumor and immune cells was carried out by measuring the intracellular level of reactive oxygen species depending on the dose and incubation time. The quinazoline alkaloid tryptanthrin, phenol propanoid rosmarinic acid, and the anticancer agent doxorubicin were tested. This study was performed on Ehrlich adenocarcinoma tumor cells and splenocytes after loading with the oxidant sensing fluorescent probe 2′,7′-dichlorofluorescein diacetate. It was shown that when rosmarinic acid influences tumor cells it has a pronounced antioxidant activity at a low dose (1 mg/mL), while a high dose of rosmarinic acid (10 mg/mL) exhibits prooxidant activity. Interestingly, in a splenocyte cell culture, rosmarinic acid reduced the level of reactive oxygen species at low and high doses. The combined application of doxorubicin with rosmarinic acid at a low dose reduced the prooxidant effect of doxorubicin, which is a potent inducer of reactive oxygen species in tumor cells. Tryptanthrin is also a potent inducer of reactive oxygen species with respect to tumor and immune cells; it is a more potent prooxidant than doxorubicin. In addition, tryptanthrin enhanced the doxorubicin-induced formation of reactive oxygen species by tumor cells in the combined use of doxorubicin with tryptanthrin. However, the prooxidant effect of tryptanthrin is short-term and decreases after a prolonged incubation. The effect of reactive oxygen species on the potent mechanisms of the biological activities of the individual and combined substances under study is discussed.

A study of the antioxidant and membranotropic activities of luteolin using different model systems

Luteolin, a water-insoluble 3′,4′,5,7-tetrahydroxyflavon, is one of the best-studied representatives of bioflavonoids. Luteolin is an essential food component for humans and other mammals that possesses a wide spectrum of biological activities by affecting the activities of various metabolic enzymes, target receptors, and signal transduction pathways. In this study, we conducted a comparative study of the antioxidant (free-radical scavenging) properties of luteolin in 2,2′-azobis(2-methylpropionamidine) dihydrochloride–luminol and hemoglobin–hydrogen peroxide–luminol systems and assessed its effect on the permeability of planar lipid bilayer membranes. Trolox was used as a reference antioxidant, while ascorbic acid and dihydroquercetin were taken as standards. Luteolin shows moderate antioxidant activity, exhibiting a higher antioxidant capacity than trolox and ascorbic acid, but it was less effective than dihydroquercetin in tests for antioxidant activity in the studied systems. The studied compounds can be ranked according to the effectiveness of their antioxidant action: dihydroquercetin > luteolin > trolox > ascorbic acid. It should be noted that the antioxidant activity of a water-soluble form of luteolin, luteolin disulfate, is comparable with that of luteolin. Luteolin does not cause significant changes in the permeability of planar bilayer membranes in the dose range from 1.5 to 30 μM. Our findings indicate the presence of a high level of free-radical scavenging activity and the absence of a primary membranotropic effect for luteolin. It can be assumed that the multiple pleiotropic nature of luteolin activity towards a variety of biological systems is associated not only with a neutralizing effect in regard to reactive oxygen species, but also with the ability of luteolin to block and modulate different cell-signaling processes and biochemical pathways. The presumed mechanisms of the biological activity of luteolin and luteolin disulfate are discussed.

[Elaboration of immune stimulating lipid-saponin subunit antigen carrier based on glycolipid monogalactosyldiacylglycerol from sea macrophytes and triterpene glycosides from Cucumaria japonica]

The ability of some triterpene glycosides of holothurians: holotoxin A1 from Apostichopus japonicus and a mixture of monosulfated triterpene glycosides from Cucumaria japonica called cucumarioside (CD) to form supramolecular complexes with cholesterol (Chol) and monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC) was studied. A transmission electron microscopy method was used to observe supramolecular lipid-saponin complexes formed by holotoxin A1 and CD with cholesterol in the presence of membrane lipids. The observed supramolecular complexes are tubular nanoparticles with a length of 100–300 nm, an external diameter of 10–16 nm and an internal diameter of 2–6 nm. The formation of tubular nanoparticles was more effective in the presence of MGDG than with PC. Nanoparticles forming in the presence of MGDG are shaped as a tubule, have a constant diameter and a strongly pronounced internal channel. In contrast, PC has no such properties; this lipid is unable to fully integrate in tubular nanoparticles. Based on electron-microscopy data the range of weight ratio of MGDG-Chol-CD was determined as a 1–10: 2: 3 that provided most effective formation of tubular nanoparticles. Different methods of incorporation of model antigens in complex MGDG-Chol-CD were studied. Influenza hemagglutinin and neuraminidase from commercial vaccine “Influvac” and pore forming protein YompF from Yersinia pseudotuberculosis were used as model antigens. From 54 to 72% of protein of “Influvac” vaccine and 88–92% of YompF were incorporated in supramolecular complexes depending on the method of incorporation. The loss of functional activity of hemagglutinin of vaccine “Influvac” was the result of applying ultrasonic disintegration for incorporation of this protein in complex MGDG-Chol-CD. YompF incorporation in MGDG-Chol-CD complex led to the increased diameter of tubular particles, in the same time incorporation of vaccine “Influvac” antigens produced the “cap” formation at the end of tubules. The possibility of a described supramolecular complex MGDG-Chol-CD to be a carrier for subunit bacterial and viral antigens is shown.

Pharmacological activity of echinochrome a alone and in the biologically active additive Timarin

Pharmacological activity of echinochrome A (EchA) alone and in the biologically active additives (BAA) Timarin, administered per os has been investigated in volunteers. Blood hematological, immunological, and biochemical parameters were investigated before and after administration of the substances used. EchA decreased serum glutatione (GSH) and increased catalase activity 1 h after treatment. Later (3 h after administration) catalase activity normalized, while GSH exceeded the initial level. Changes in blood lipids suggest decreased risk of atherogenesis. Changes found in blood sex hormone levels indicate that Timarin may influence sex gland functioning. Changes of hematological and immunological parameters have been interpreted as the result of a mild stressor effect of both EchA alone and in the BAA Timarin increasing adaptation reactivity of the body.

The complexation between holothurian triterpene glycosides and Chl as the basis for lipid-saponin carriers of subunit protein antigens

The ability of holothurian triterpene glycosides (cucumarioside A2-2 from Cucumaria japonica, cucumarioside G1 from C. fraudatrix, frondoside A from C. frondosa, and holotoxin A1 from Apostichopus japonicus) to form supramolecular lipid-saponin complexes was studied. TEM demonstrated that all the studied compounds form supramolecular cholesterol-saponin complexes (nanoparticles) in aqueous medium. The complexes formed by cucumarioside A2-2, holotoxin A1, and frondoside A had a tubular structure and fundamentally differed in the structure from the particles produced by cucumarioside G1. The morphology of the nanoparticles formed by cucumarioside A2-2, holotoxin A1, and cucumarioside G1 changed depending on the fraction of cholesterol in the lipid-saponin system; however, this pattern was not observed for frondoside A. At the same molar fraction of cholesterol in the lipid-saponin system, cucumarioside A2-2 formed the particles with the most pronounced tubular structure; the cholesterol-saponin complexes of holotoxin A1 had a less pronounced tubular structure, whereas the structure of frondoside A particles was extremely heterogeneous. Comparative analysis of the morphology of the described supramolecular complexes and specific structural features of the glycosides demonstrated that the structure of the corresponding nanoparticles depended on the degree of branching of the carbohydrate moiety in the glycoside molecule and the complexation with cholesterol was determined by the specific features of aglycone structure. Thus, the feasibility of producing new generation antigen carriers using the complexes in question was proved.

The development of a new adjuvant lipid-saponin complex and its use at experimental immunization by bacterial antigen

Immunostimulating complexes (ISCOM) have been modified by replacement of structural components of ISCOM; phosphatidylcholine has been replaced, by glycolipid of monogalactosyldiacylglycerol from sea macrophytes, saponin QuilA has been replaced by triterpene glycoside, Cucumarioside A2-2. The resultant complex includes morphological structures of two types: the ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of about 40 nm and length of 150–400 nm. We have defined these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of the experimental immunization of mice with a weak immunogen (subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund’s adjuvant. Thus, the TI-complexes represent a new type of adjuvant carriers for antigens.