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Antiangiogenic compounds are gaining more and more interest as a new approach in the prevention and treatment of cancer and inflammatory diseases. The objective of this study was the evaluation of the antiangiogenic effect of a red clover extract (RCE) used in food supplements for menopausal complaints as well as of its main isoflavones in an in vivo system, the chorioallantoic membrane assay of fertilized hen's eggs. At a dosage of 250 microg/pellet the red clover extract showed excellent inhibition of angiogenesis. The antiangiogenic activity of the non-methylated isoflavones daidzein and genistein was higher than that of the methylated compounds formononentin and biochanin A. The results demonstrate that RCE is not only suitable for menopausal complaints, but might also be a powerful chemopreventive agent against chronic diseases e.g. which have a high incidence especially in elderly female. [ABSTRACT FROM AUTHOR]

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage-oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti-oxidative potential. Using the dextran sulphate sodium (DSS)-induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 microg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti-CD3 antibody in the presence of interleukin (IL)-2 (final concentration 10 U/ml). After incubation for 24 h, IL-1beta, IL-6, IL-12 tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels in supernatants were analysed by the beadlyte cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3.2 with acteoside versus 5.2 with phosphate-buffered saline (PBS) (P < 0.02). In chronic colitis both 120 microg (3.3 versus 5.2) or 600 microg acteoside (3.0 versus 5.2) significantly ameliorated colitis (both P < 0.02). Stimulated MLN from mice with chronic DSS-induced colitis treated with acteoside showed a significant down-regulation of IFN-gamma secretion (195 pg/ml with 600 microg acteoside versus 612 pg/ml with PBS, P < 0.02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.

Potent and specific inhibitors of hyaluronidases, a group of enzymes preferentially catalysing the hydrolysis of hyaluronic acid, are not known so far. Such compounds could be useful as pharmacological tools for studying the physiological and pathophysiological role of both hyaluronan and hyaluronidases. The effects of sulphated and non-sulphated structurally different oligosaccharides on bovine testicular hyaluronidase, hyaluronidase from bee venom and hyaluronate lyase from Streptococcus agalactiae (hylB (4755)) were studied with the Morgan-Elson reaction. Several active compounds were identified within a series of sulphated beta-(1,4)-galacto-oligosaccharides. The determined IC (50) values of these sulphated oligosaccharides ranged from 4 microM to 630 microM for all hyaluronan-degrading enzymes. Sulphated oligosaccharides like verbascose, planteose and neomycin showed comparable inhibition on all hyaluronidases, thereby possessing 100 - 500 times the activity of the widely accepted hyaluronidase inhibitor apigenin.

The antiinflammatory effects of ethanol and aqueous extracts from Drosera rotundifolia and from Drosera madagascariensis were compared in vivo in the HET-CAM assay. Both extracts from D. rotundifolia and the ethanol extract from D. madagascariensis showed remarkable efficacy at doses of 500 microg/pellet. The inhibition of the inflammation by the extracts was stronger than that by 50 microg hydrocortisone/pellet. In contrast, there was only a very weak effect observed at a dose of 500 microg/pellet of the water extract from D. madagascariensis. The chemical analyses of the extracts showed that the effect cannot be attributed to naphthoquinones, but might be due to flavonoids. Ellagic acid obviously plays an important role in the antiangiogenic effect of the Drosera extracts., ((c) 2005 John Wiley & Sons, Ltd.)

The essential oil of Betula pendula Roth. buds was obtained using both hydrodistillation and microdistillation techniques and their chemical compositions were analyzed using both gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Overall, more than 50 compounds were identified representing 80% and 92% for hydrodistillation and microdistillation, respectively. The main components (by hydrodistillation and microdistillation, respectively) found were alpha-copaene (12% and 10%), germacrene D (11% and 18%) and delta-cadinene (11% and 15%) in the analyzed essential oils. The microdistillation technique proved to be a useful tool and compliant alternative when compared to hydrodistillation.

The in vivo test on the chorioallantoic membrane of the fertilized hen's egg (CAM assay) is a current method to determine antiangiogenic, antiinflammatory activity and toxic effects of individual compounds or complex plant extracts. The method is used for testing natural compounds in small amounts for revealing various modes of action and the complex mechanisms related to angiogenesis and inflammation. Furthermore, possible side effects such as membrane irritation, toxic, and anticoagulant properties of the investigated material in question can be detected. For the evaluation, the essential oil obtained by hydrodistillation of the aerial parts of Origanum onites L., a common spice and medicinal plant, was tested for its effect in the chorioallantoic membrane (CAM) assay. The essential oil composition was revealed by means of gas chromatography-mass spectrometry (GC-MS). Eighty three components were identified, representing 99.1% of the total oil. Carvacrol, thymol, p-cymene, and gamma-terpinene were found as major components and were also individually tested in the CAM assay. Along with the monoterpenes carvacrol and thymol, their methyl ether derivatives were also examined for comparison of their physiological action. Neither the essential oil nor its components showed any pronounced antiinflammatory or antiangiogenic property in the CAM assay, at 10-250 microg/pellet. However, the irritant effect of the essential oil was linked to thymol in a dose-response fashion, up to 10 microg/pellet, where it was still showing irritation.

Phlomis linearis Boiss. & Bal. of the Lamiaceae family growing in central, east and southeast Anatolia is an endemic species for Turkey. The essential oil obtained from the aerial parts by hydro distillation was subsequently analyzed by GC/MS. The main components of the oil were found as beta-caryophyllene (24.2%), germacrene D (22.3%) and caryophyllene oxide (9.2%), among 49 identified compounds, representing 94.5% of the total essential oil. The overall biological activity of the essential oil (100 microg/pellet) was tested on the chorioallantoic membrane (CAM) of the fertilized hen's egg in order to examine the anti-angiogenic and anti-inflammatory activity. None of the tests showed pronounced activity, toxicity or irritation at the tested concentration.

A sulfated emodin glucoside, emodin 8-O-beta-D-glucopyranosyl-6-O-sulfate (1), was isolated from the roots of Rheum emodi in an investigation of the active constituents of this Nepalese medicinal plant, and its structure was determined by spectroscopic and chemical methods. Additionally, two rare auronols, carpusin (2) and maesopsin (3), besides other anthraquinones and phenolics, were isolated and identified. Compounds 2 and 3 showed significant antioxidant activity in the DPPH assay, while chrysophanol, physcion, and emodin and their 8-O-glucosides were found to be inactive.

LaPSvS1, a highly sulfated branched (1-->3)-beta-galactan was prepared from the arabino-galactan from Larix decidua Miller by partial hydrolysis and subsequent sulfation with SO(3)-pyridine in DMF. The molecular weight was analyzed by GPC and the sulfate content was determined by ion chromatography. LaPSvS1 exhibited good antiangiogenic and antiinflammatory effects in two different modifications of the known CAM-assay. In vitro results obtained in the FGF-2-trypsin-assay and in fluorospectrometric experiments revealed that LaPSvS1 interacts with the fibroblast growth factor 2 system. This interaction is correlated with the in vivo effect of LaPSvS1 on FGF-2 induced angiogenesis.

A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

The influence of the triterpenoid saponins 1-10 has been investigated on murine spleenocytes in the lymphocyte transformation test and on murine macrophages in an phagocytosis assay. The lymphocyte transformation test and the phagocytosis assay showed that the tested compounds have no stimulating effect. However, a significant inhibition of lymphocyte proliferation by the triterpenoid saponins 2, 6 and 10 was demonstrated.

Sulfated beta-(1-->4)-galacto-oligosaccharides were prepared from an arabino-galacto-rhamno-galacturonan from Lupinus polyphyllus Lindl. by successive partial hydrolysis and SO3-pyridine sulfation in DMF. The resulting oligosaccharide polysulfates were analyzed by analytical GPC and the sulfate content was determined by ion chromatography. DP 5 and higher showed a pronounced antiangiogenic effect with scores of 0.9-1.2 for DP 7-9 using the CAM-assay. An interaction with the fibroblast growth factor FGF-2 was noticed for DP 4-12 depending on the degree of sulfation using the FGF-2-trypsin assay.

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.

Angiogenesis is a strictly controlled process in the healthy, adult human body. It is regulated by a variety of endogenous angiogenic and angiostatic factors. It is only switched on, e.g., during wound healing. Pathological angiogenesis occurs, for example, in cancer, chronic inflammation, or atherosclerosis. Angiogenesis inhibitors are able to interfere with various steps of angiogenesis, like basement destruction of blood vessels, proliferation and migration of endothelial cells, or the lumen formation. Among the known angiogenesis inhibitors compounds derived from natural sources, like flavonoids, sulphated carbohydrates, or triterpenoids are playing a prominent role.

The ethanolic extract of Ilex aquifolium L. (Aquifoliaceae) concentration-dependently inhibited leukotriene B4 biosynthesis in isolated bovine PMNL with an IC50 value of about 60 micrograms/ml, whereas the effect on epidermal 12(S)-HETE biosynthesis was much less pronounced. The extract also inhibited the non-enzymatic, peroxyl radical-stimulated lipid peroxidation in model membranes and was further a scavenger of the iron-dependent generation of hydroxyl radicals from hydrogen peroxide as determined by protection against deoxyribose degradation. While inhibition of leukotriene biosynthesis was not mediated by its known phenolic constituents such as hyperoside, rutoside, and chlorogenic acid, these compounds were responsible for the inhibitory effects of I. aquifolium against non-enzymatic lipid peroxidation and deoxyribose degradation.

We have evaluated a series of laminarin sulphates with different degrees of sulphation (0.3-2.3) as antagonists of basic fibroblast growth factor (bFGF) and as inhibitors of the bFGF-dependent endothelial cell line FBHE. Inhibition of binding of bFGF by the laminarin sulphates increased with increasing degree of sulphation. Binding of bFGF to low affinity sites on BHK cells was inhibited more strongly than binding to high affinity sites. IC50 values for inhibition of binding to low and high affinity sites by the most highly sulphated laminarin sulphate (LAM S5; degree of sulphation 2.31) were 12 +/- 8 micrograms/ml and 69 +/- 66 micrograms/ml, respectively. LAM S5 dissociated bFGF from low affinity sites on BHK cells but not from high affinity sites. LAM S5 increased the electrophoretic mobility of bFGF indicating that LAM S5 binds directly to bFGF. LAM S5 reduced uptake of bFGF by FBHE cells by 67%. Increasing the degree of sulphation of laminarin sulphates increased the inhibition of bFGF-stimulated DNA synthesis of the endothelial cell line FBHE (IC50 for LAM S5 approx. 1 microgram/ml). There was no inhibition of DNA synthesis of FBHE cells by LAM S5 in the presence of 1 microgram/ml bFGF indicating that bFGF antagonism is involved in the anti-proliferative activity of this compound. LAM S5 may be of value against diseases associated with bFGF-dependent cell proliferation.

l-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of l-carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the anti-proliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of l-carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC50 value), approx. 0.5 microgram/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of l-carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by l-carrageenan (IC50 value, 5.5 micrograms/ml) and the cells were arrested at the G1/S boundary. l-Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor alpha (TGF alpha) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody alpha IR3 enhanced the inhibitory activity of l-carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by l-carrageenan. l-Carrageenan had low anti-coagulant activity. l-Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.

A water extraction of crushed leaves of Nerium oleander yielded 2.3% of a crude polysaccharide. The main fraction (67%) represents a pectic polysaccharide mainly composed of galacturonic acid besides rhamnose, arabinose and galactose. The polysaccharide structure was characterized by NMR, mild acid- and pectinase treatment combined with GC-MS analyses. In vivo tests for a possible antitumor activity did not result in a significant action. Investigation of immunomodulating activity brought some indications for mitogenic activity and a weak macrophage-mediated cytotoxicity. Increase of phagocytosis could not be doubtlessly assigned to the polysaccharide due to the high amount of endotoxin in the homogenous fraction OLE 2.

In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5βH-pregnan-3βol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5βH-steroidaglycones at C-3. The respective glucosides of the 5βH-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5βH-steroidglucosides.