Echinacea complex--chemical view and anti-asthmatic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthma-related cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. AIM OF THE STUDY: To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. MATERIAL AND METHODS: The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). RESULTS: Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. CONCLUSION: Pharmacodynamic studies have confirmed significant bronchodilatory and anti-inflammatory effects of Echinacea complex that was similar to effects of classic synthetic drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine as a supplementary treatment of allergic disorders of the airways, such as asthma.

Chemical and pharmacological profiles of Echinacea complex.

Echinacea purpurea has a long history in traditional medicine. To verify the pharmacological efficacy of active principles, a polysaccharide-phenolic-protein complex has been isolated from flowering parts of herb by alkaline extraction. It showed on GPC and HPLC one peak of molecular mass around 10 kDa. Chemical and spectroscopic analyses revealed carbohydrate, phenolic and protein contents in Echinacea complex. Pharmacological tests have shown its marked cough suppressing and bronchodilatory effects. The antitussive effect of Echinacea was similar to the narcotic drug codeine and the bronchodilatory effect was more significant than salbutamol, the antiasthmatic drug used in a clinical practice. Pharmacodynamic study shows the beneficial effects of Echinacea complex on the respiratory system and highlights the great potential for development of antitussive and bronchodilatory drugs from natural sources.

Radical scavenging and antioxidant effects of Matricaria chamomilla polyphenolic-polysaccharide conjugates.

Matricaria chamomilla L. (MC), a member of the Asteraceae family, is one of the oldest medicinal plants, widely used worldwide for a variety of healing applications. Its recommendations, derived from both traditional and modern medicine, include numerous disorders such as inflammation, ulcers, wounds, gastrointestinal disorders, stomach ache, pharyngitis, rheumatic pain, as well as the other ailments. This work is focused on another aspect of the biological activity of chamomile polyphenolic-polysaccharide conjugates--their antioxidant properties in the protection of blood plasma components against in vitro oxidative stress. Measurements of DPPH and ABTS radical scavenging indicated considerable anti-free radical action of MC. Pre-incubation of blood plasma with MC considerably diminished the extent of ONOO(-)-induced oxidative modifications such as protein carbonyl groups, SH groups, 3-nitrotyrosine, as well as the formation of lipid hydroperoxides. The analysis of the FRAP assay result shows a considerable increase of ferric reducing ability of blood plasma in the presence of MC. The results obtained in this study indicate that polyphenolic-polysaccharide conjugates isolated from M. chamomilla substances possess antioxidant properties. The M. chamomilla macromolecular glycoconjugates may be useful in the creation of new natural-based medications or dietary supplements, helpful in the prevention and treatment of oxidative stress-mediated disorders.

[Biosensors as a tool to use in the experiments on blood and selected blood proteins]. / Biosensory jako narzedzie do wykorzystania w badaniach krwi oraz wybranych bialek krwi.

Over last few years, biosensors have become increasingly used as a research tool. Using innovative techniques of detection and estimation of the strength of intermolecular bonds, is particularly important in biochemical processes, including the study of mechanisms of interactions between plasma proteins in the circulatory system. With the numerous enhancements biosensors have become extremely sensitive devices, allowing for analysis of diverse biological material. Moreover, the use of immobilization techniques enables to use sample repeatedly, which significantly reduces costs and the ability to monitor tests in real-time shorten the time of experiment. The presented work discusses examples of the usage of biosensors in the research on the mechanisms of the interactions of blood plasma proteins. The experiments on cancer biomarkers present in the blood circulation system, protein C deficiency causing non-controlled hemorrhagic accidents, and on the level of fibrinogen, as well as 20S proteasome concentration in plasma, are just some examples of biosensors usage in the analyses of blood. They are also applicable in the research work performed the project WROVASC--Integrated Cardiovascular Center, concerning the mechanisms of anticoagulant activity in blood plasma of the polyphenolic-polysaccharide macromolecules of plant origin.

[The plant anticoagulants with perspective to use in treatment of thrombosis]. / Antykoagulanty pochodzenia roslinnego z perspektywa wykorzystania w leczeniu zakrzepic.

The diseases of blood circulation system--cardiovascular diseases are the main causes of mortality in developing, low and middle-income countries all over the world. The specialists recommend the prophylaxis to avoid the dangerous complications connected with these diseases, what can reduce the final treatment costs. All over the world there is continuous research of novel, therapeutically better, more effective anticoagulant or anti-platelet agents, with multiple targets, without so many side effects. Plant material is a good source to do this kind of research. The authors show the results of their few years research on polyphenolic-polysaccharide plant conjugates, isolated from medicinal plants, popular in Poland, which is continuing in the framework of the project WROVASC--Integrated Center of Cardiovascular Medicine. This research group has been working on isolation, structure characterization and biological activity of these macromolecular compounds. Because of anticoagulant, antioxidant as well as anti-platelet properties of these plant structures they are promising to be a new source of the innovative therapeutic agent.

The influence of conjugates isolated from Matricaria chamomilla L. on platelets activity and cytotoxicity.

Cardiovascular diseases (CVD) remain the principal cause of death in both advanced and developing countries of the world. Blood platelets are involved in the pathogenesis of atherosclerosis and thrombosis. Platelet adhesion and aggregation are critical events that occur in unstable coronary syndromes. The current research is focused on the role of polysaccharide-polyphenolic conjugates isolated from chamomile (Matricaria chamomilla L.) at concentrations of 10, 25, 50 and 100 µg/mL on blood platelets (obtained from healthy donors and from patients received combined anti-platelet therapy complex with clopidogrel and acetylsalicylic acid) aggregation and experimentally induced cell toxicity. The treatment of PRP obtained from healthy donors with polyphenolic-polysaccharide conjugates from M. chamomilla (L.) (MC) resulted in a dose-dependent, decrease of platelet aggregation induced by multiple agonists (ADP, collagen and arachidonic acid). In this study we also observed that the MC reduced platelet aggregation in PRP obtained from patients with cardiovascular disorders. The result of testing the MC on human blood platelets, mouse fibroblast cultures L929 and human lung cells A549 did not show any cytotoxicity effects. Compounds obtained from M. chamomilla L. are potential composite to the development of a new anti-platelet agent, which could be an alternative to the currently used anti-platelet drugs.

Effects of extraction condition on structural features and anticoagulant activity of F. vesca L. conjugates.

From the air-dried Wild strawberry (Fragaria vesca L., family Rosaceae) leaves five water-soluble glycoconjugates Fv I-V by different extraction conditions have been isolated. Effects of extraction steps/agents on chemical composition and anticoagulant activity of Fv I-V were examined. Dark brown F. vesca conjugates Fv I-V were recovered in 4.5-8.4% yields, based on dry herb. Isolates were composed of carbohydrate, phenolic and protein components. Fv I-V displayed on HPLC broad molecule-mass distribution patterns with dominance of low molecule-masses 9-14 kDa. Their carbohydrate parts revealed high hexuronic acids content (35-60%) while the dominant neutral sugars - galactose, arabinose and rhamnose were found in lower amounts and indicated the presence of rhamnogalacturonans associated with arabinogalactans in all F. vesca preparations. In all Fv I-V isolates high polyphenolic contents were determined, whereas proteins were found in low amounts only. In in vitro experiments on human pooled plasma Fv I-V showed at higher concentrations complete inhibition of plasma clot formation and the most active conjugates in aPTT, PT and TT tests were shown to be Fv I and Fv III, containing the highest amounts of phenolics.

Anticoagulant and anti-platelet activity of polyphenolic-polysaccharide preparation isolated from the medicinal plant Erigeron canadensis L.

The polyphenolic-polysaccharide preparation from Erigeron canadensis L. was isolated by multi-step process, characterized by chromatographic and spectroscopic methods, and was subjected to anion-exchange chromatography. The whole preparation demonstrated in vivo anticoagulant activity, and the effect was neutralized by protamine sulfate. It had also anti-platelet activity, limited to the cyclooxygenase pathway, induced by arachidonic acid. The plant preparation was fractionated to receive the fraction of the highest anticoagulant activity - 7-9IU/mg of heparin standard, expressed in aPTT. The influences of the plant preparation as well as its the most active fraction on thrombin and factor Xa inactivation by antithrombin, and on thrombin inhibition by heparin cofactor II, were compared. The both tested plant preparations inhibited thrombin as well as factor Xa amidolytic activities in the presence of antithrombin, but much higher concentrations were required to obtain the same effects like for unfractionated heparin. The mechanisms of anticoagulant activity in the case of the plant preparation are based on interactions with heparin cofactor II, to inactivate thrombin. Chromatographic and spectroscopic methods revealed its macromolecular polyanionic non-sulfated polyphenolic-polysaccharide conjugate, with carboxylic groups. The polysaccharide part constituted 32% of the total mass and was homogenous, with molecular mass 38kDa, containing mainly hexuronic acids, and much smaller amounts of glucose, arabinose, galactose, as well as some traces of mannose, xylose and rhamnose. Polyphenolic part, with molecular mass >12.5kDa, was rich in hydroxylic rests as well as in carboxylic groups, free and esterified. The polyphenolic-polysaccharide preparation from E. canadensis may become a new source of anticoagulant compound potentially useful in anticoagulant and anti-platelet therapy.

The effect of polyphenolic-polysaccharide conjugates from selected medicinal plants of Asteraceae family on the peroxynitrite-induced changes in blood platelet proteins.

Lots of plants belonging to Asteraceae family are very popular in folk medicine in Poland. These plants are also known as being rich in acidic polysaccharides, due to the presence of hexuronic acids or its derivatives. Our preliminary experiments have shown that the extract from Conyza canadensis L. possesses various biological activity, including antiplatelet, antiocoagulant and antioxidant properties. The aim of our study was to assess if macromolecular glycoconjugates from selected herbal plants of Asteraceae family: Achillea millefolium L., Arnica montana L., Echinacea purpurea L., Solidago virgaurea L., Chamomilla recutita (L.) Rauschert., and Conyza canadensis L. protect platelet proteins against nitrative and oxidative damage induced by peroxynitrite, which is responsible for oxidative/nitrative modifications of platelet proteins: the formation of 3-nitrotyrosine and carbonyl groups. These modifications may lead to changes of blood platelet functions and can have pathological consequences. The role of these different medicinal plants in the defence against oxidative/nitrative stress in human platelets is still unknown, therefore the oxidative damage to platelet proteins induced by peroxynitrite and protectory effects of tested conjugates by the estimation of carbonyl group level and nitrotyrosine formation (a marker of protein nitration) were studied in vitro. The antioxidative properties of the polyphenolic-polysaccharide conjugates from selected tested medicinal plants were also compared with the action of a well characterized antioxidative commercial polyphenol - resveratrol (3,4',5-trihydroxystilbene). The obtained results demonstrate that the compounds from herbal plants: A. millefolium, A. montana, E. purpurea, C. recutita, S. virgaurea, possess antioxidative properties and protect platelet proteins against peroxynitrite toxicity in vitro, similar to the glycoconjugates from C. canadensis. However, in the comparative studies, the polyphenolic-polysaccharide conjugates from selected tested medicinal plants were not found to be more effective antioxidant, than the solution of pure resveratrol.

An acidic glycoconjugate from Lythrum salicaria L. with controversial effects on haemostasis.

AIM OF THE STUDY: Lythrum salicaria L. belongs to the small Lythraceae family of 22 genera, which range in habit from herbs to shrubs and trees found with worldwide distribution (Heywood, 1993). The generic name of Lythrum derived from Greek "luthron"--blood, possibly referring to the color of the flowers or to the one of its herbal use as an astringent to stop bleeding (Thompson et al., 1987; Mountain, 1994; Pawlaczyk and Pacula, 2002). The flowering parts and the flowering branch tips are used in traditional medicine and pharmaceuticals internally in a form of decoctions or as extracts for treatment of diarrhea, chronic intestinal catarrhs, hemorrhoids and eczema, or externally to treat varicose veins, venous insufficiency and gums (Mantle et al., 2000; Rauha et al., 2000). The aim of this study was to isolate the plant glycoconjugate from flowering parts of Lythrum salicaria, and to verify its influence on blood coagulation process. MATERIALS AND METHODS: From the air-dried flowering parts of this plant a water-soluble glycoconjugate has been isolated by hot alkaline extraction followed by neutralization and purification by multi-steps extraction with organic solvents, dialysis and concentration. The plant isolate was tested in vitro on anticoagulant activity on human plasma, and on Wistar rats blood system in vivo as well as ex vivo. RESULTS: A dark brown isolate was obtained in the yield of 8% of starting material (w/w) as a macromolecular compound with M(w) approximately 12,500. Chemical analysis revealed the presence of carbohydrates (30%), phenolics (1g contained 1.2mM of gallic acid equivalent) and proteins (0.8%). The result of compositional analyses of carbohydrate part revealed the predominance of uronic acids (approximately 66%), galactose (approximately 12%), rhamnose (approximately 10%) and arabinose (approximately 9%) residues indicating thus the presence of pectic type of polymers, i.e. galacturonan and/or rhamnogalacturonan associated with arabinogalactan in Lythrum glycoconjugate. In vitro and ex vivo experiments showed complete inhibition of plasma clot formation, however, the application of Lythrum glycoconjugate in vivo showed controversial effect on animal blood system in comparison with in vitro ones, i.e. pro-coagulant activity. CONCLUSION: The in vivo results give a scientific explanation for the traditional use of Lythrum salicaria as a styptic agent. It seems that pro-coagulant activity of this complex could be probably connected with the other factors in blood circulation system, like platelets.

Antioxidant and antiaggregatory effects of an extract from Conyza canadensis on blood platelets in vitro.

The antioxidative activity of the polysaccharide extract from Conyza canadensis in blood platelets treated with peroxynitrite (ONOO-) was studied. Peroxynitrite as a strong biological oxidant has toxic effects on blood platelets and induces the oxidation of thiols, carbonylation and nitration of platelet proteins and lipid peroxidation. Therefore, the aim of our study was to assess if the natural extract from herbal plant, Conyza Canadensis, may protect platelet proteins against nitrative and oxidative damage induced by ONOO-. In our study we measured oxidative damage of platelet proteins induced by peroxynitrite and protectory effects of this extract by estimation of the level of carbonyl groups and nitrotyrosine (a marker of platelet protein nitration). We also used cytochrome c reduction method to test the ability of this extract to change O2-* generation in platelets. Moreover, we determined the effects of the extract on blood platelet aggregation induced by ADP. We observed that the extract from Conyza canadensis distinctly reduced oxidation and nitration of proteins in blood platelets treated with ONOO-(0.1mM) and O2-* production in these cells. The extract from Conyza canadensis also inhibited platelet aggregation. The ability of the extract to decrease O2-* generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. The present study suggests that the natural polysaccharide extract from Conyza canadensis has antiaggregatory and antioxidative activities, and therefore may be beneficial in the prevention of peroxynitrite-related diseases, such as cardiovascular or inflammatory diseases.