Coumarins as antioxidants.

Coumarins, a well-known class of naturally occurring compounds, display a remarkable array of biochemical and pharmacological actions, some of which suggest that certain members of this group of compounds may significantly affect the function of various mammalian cellular systems. The development of coumarins as antioxidant agents has attracted much attention in recent years. Coumarins afford an opportunity for the discovery of new antioxidants with truly novel mechanisms of action. This review updates and expands the 2006 review by the same author. The review considers and incorporates the most recently published literature on coumarins as related to their antioxidant properties. A lot of coumarins have been identified from natural sources, especially green plants. These natural compounds have served as valuable leads for further design and synthesis of more active analogues. Beyond doubt, a deep understanding of the mechanisms of existing synthetic and natural coumarins will build the basis for the rational design.

DPPH Radical Scavenging Activity of Several Naturally Occurring Coumarins and Their Synthesized Analogs Measured by the SIA Method.

ABSTRACT Coumarins, naturally occurring compounds derived from benzopyran, have recently been studied extensively for their antioxidant properties. A lot of coumarins have been isolated and identified from natural sources and many others have been synthesized. It is also known that pharmacological and biochemical properties and thus also therapeutic application of simple coumarins depend upon the pattern of their substitution. As a part of studies of biological effects, four naturally occurring coumarins and 18 synthesized analogs of several compounds were assayed for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity. For this purpose the highly reliable DPPH test modified to be performed by sequential injection analysis (SIA) system was used. This in our laboratory-developed method was originally proposed for antioxidant screening of large series of plant extracts. In this assay, the DPPH test using the SIA method was used for fast and sensitive evaluation of EC(50) of coumarins. The evaluation of EC(50) of a single compound takes only 15 to 30 min. The structure-activity relationships of tested compounds are also established. The results verified 7,8-dihydroxy-4-methylcoumarins as excellent DPPH radical scavengers. Obtained results correspond with those of other studies and suggest the SIA procedure as a suitable method for fast and sensitive antioxidant analysis of various types of compounds.

Atractylis gummifera L. poisoning: an ethnopharmacological review.

Atractylis gummifera L. (Asteraceae) is a thistle located in the Mediterranean regions. Despite the plant's well-known toxicity, its ingestion continues to be a common cause of poisoning. The toxicity of Atractylis gummifera resides in atractyloside and carboxyatractyloside, two diterpenoid glucosides capable of inhibiting mitochondrial oxidative phosphorylation. Both constituents interact with a mitochondrial protein, the adenine nucleotide translocator, responsible for the ATP/ADP antiport and involved in mitochondrial membrane permeabilization. Poisoned patients manifest characteristic symptoms such as nausea, vomiting, epigastric and abdominal pain, diarrhoea, anxiety, headache and convulsions, often followed by coma. No specific pharmacological treatment for Atractylis gummifera intoxication is yet available and all the current therapeutic approaches are only symptomatic. In vitro experiments showed that some compounds such as verapamil, or dithiothreitol could protect against the toxic effects of atractyloside, but only if administered before atractyloside exposure. New therapeutic approaches could come from immunotherapy research: some studies have already tried to produce polyclonal Fab fragments against the toxic components of Atractylis gummifera.

Anti-proliferative effect on a prostatic epithelial cell line (PZ-HPV-7) by Epilobium angustifolium L.

Symptomatic benign prostatic hyperplasia (BPH) is a common condition in elderly men and has a significant impact on their daily lives. The drugs prescribed for treatment include alpha1-blockers, 5-alpha-reductase inhibitors and plant preparations. Epilobium angustifolium L. is deemed to be helpful in BPH therapy, although there is less information regarding the mechanism of its biological activity. The present study evaluated the effect of E. angustifolium extract on human prostatic epithelial cells (PZ-HPV-7). The exposure to E. angustifolium extract induced a marked inhibition of cell growth in all tested conditions. The anti-proliferative effect observed in in vitro systems clearly indicates a biologically relevant effect of compounds present in the extract. Considering these results, the use in traditional medicine of E. angustifolium extract against BPH seems to be justified. However, further experimental studies are needed to determine the biochemical mechanism of the action and the clinical value of the E. angustifolium extract.

Analgesic properties of Epilobium angustifolium, evaluated by the hot plate test and the writhing test.

The analgesic properties of Epilobium angustifolium (Ea), a plant containing flavonoids with anti-inflammatory activity, have not been sufficiently studied so far. Thus, we decided to evaluate, by the classical hot plate test and the writhing test, the analgesic effect of a dry extract of Ea obtained by evaporating a commercially available mother tincture. In the former assay, the effect of Ea (380 mg/kg) was slightly lower than that of morphine (10 mg/kg s.c.). In the writhing test, which is more sensitive for non-steroidal analgesics, the effect of Ea was already significant (P < 0.05) at 95 mg/kg while at doses > or = 190 mg/kg, its activity was similar to that of lysine acetylsalicylate (300 mg/kg). The LD50 of this dry extract of Ea was 1.4+/-0.1 g/kg. Further studies are necessary for the identification of the active principles and the elucidation of their mechanism of action.

Relaxant effects of Hydrastis canadensis L. and its major alkaloids on guinea pig isolated trachea.

Hydrastis or goldenseal, one of the most popular medicinal herbs in the U.S.A., is used in mild pathological conditions like cold and flu, based on the pharmacological properties of its active components, berberine (anticholinergic, antisecretory, and antimicrobial) and beta-hydrastine (astringent). We previously reported the relaxant effect of a total ethanolic extract of hydrastis on carbachol precontracted isolated guinea pig trachea, and with the present study, using the same experimental model, we aimed at evaluating the contribution of its major alkaloids, berberine, beta-hydrastine, canadine and canadaline to the total effect. Furthermore, using specific pharmacological tools, like timolol and xanthine amine congener, we attempted to elucidate its mechanism of action. The EC50 of berberine, beta-hydrastine, canadine and canadaline, were 34.2+/-0.6, 72.8+/-0.6, 11.9+/-1.2 and 2.4+/-0.8 microg/ml, respectively. Timolol effectively antagonized the effect of canadine (EC50 = 19.7+/-3.0 microg/ml) and canadaline (EC50 = 17.1+/-1.2 microg/ml) but not that of berberine and beta-hydrastine, while xanthine amine congener antagonized the effect of beta-hydrastine (EC50 = 149.9+/-35.3 microg/ml) and canadaline (EC50 = 26.1+/-3.0 microg/ml) but not that of berberine and canadine. Besides, the hydrastis extract, at concentrations between 0.01 and 0.1 microg/ml, potentiated the relaxant effect of isoprenaline on carbachol-precontracted isolated guinea pig trachea. These data, which are insufficient to draw definite mechanistic conclusions, indicate that the aforementioned alkaloids may act by interacting with adrenergic and adenosinic receptors.

Antimicrobial investigation of semipurified fractions of Ginkgo biloba leaves.

A total methanolic extract of Ginkgo biloba leaves was fractionated by solvent partition using ethyl acetate (fraction A), n-butanol (fraction B) and water (fraction C). The antimicrobial activity of the three fractions was evaluated using a number of Gram-positive and -negative bacteria and yeasts. The apolar fraction A appeared to be the most interesting because of its activity against several microorganisms; this fraction was further separated by high performance liquid chromatography, and shown to contain substances with strong inhibitory activity against Enterococcus faecalis 31, different from the major known chemical components of G. biloba leaves.

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.

Stabilization of rat serum proteins following oral administration of fish oil.

The mechanism of action of fish oil (FO), currently used in different chronic inflammatory conditions such as rheumatoid arthritis (RA), is not completely understood, although it is thought that it could alter the metabolism of endogenous autacoids. In addition, we hypothesized that the known capability of fatty acids (FA) of stabilizing serum albumin and perhaps other proteins, may be of pharmacological relevance considering that it is shared by other anti-rheumatic agents (e.g. nonsteroidal antiinflammatory drugs). Thus, we studied the effect of oral administration of FO and corn oil (CO), a vegetable oil with a different composition, on the stability of rat serum proteins, evaluated by a classical in vitro method based on heat-induced protein denaturation. FO, and, to a lower extent, CO inhibited heat-induced denaturation of rat serum (RS): based on the inhibitory activity (EC50) of the major fatty acids against heat-induced denaturation of RS in vitro, it was possible to speculate that in vivo effects of palmitic acid (C16:0) and eicosapentaenoic acid (EPA, C20:5, n-3) may be more relevant than that of linolenic acid (C18:2). To better investigate this phenomenon, we extracted albumin from the serum of animals treated or not with FO with a one-step affinity chromatography technique, obtaining high purity rat serum albumin preparations (RSA-CTRL and RSA-FO), as judged by SDS-PAGE with Coomassie blue staining. When these RSA preparations were heated at 70 degrees C for 30 min, it was noted that RSA-FO was much more stable than RSA-CTRL, presumably due to higher number of long chain fatty acids (FA) such as palmitic acid or EPA. In conclusion, we provided evidences that oral administration of FO in the rat stabilizes serum albumin, due to an increase in the number of protein bound long chain fatty acids (e.g. palmitic acid and EPA). We speculate that the stabilization of serum albumin and perhaps other proteins could prevent changes of antigenicity due to protein denaturation and glycosylation, which may trigger pathological autoimmune responses, suggesting that this action may be involved in the mode of action of FO in RA and other chronic inflammatory diseases.

Development of an in vitro assay for the screening of substances capable of dissolving calcium oxalate crystals.

Despite the risk of kidney damage, lithotripsy is the usual way of treating calcium oxalate (CaOx) stones, the most common type of nephrolithiasis, because no effective chemolytic agents are available. However, the search of new calcium chelators, less toxic than the current ones, continues, and some of them could be tested in experimental models of nephrolithiasis, after their ability of dissolving CaOx crystals is verified. In this connection, we developed a simple assay that requires only inexpensive equipment available in most laboratories for the screening of substances potentially capable of dissolving CaOx crystals. In particular, we decided to investigate whether substances previously shown to inhibit CaOx precipitation were also capable of dissolving this salt. Briefly, CaOx tablets of highly reproducible weight (4.55 +/- 0.07 mg) were prepared by spinning, at high speed (16,000 g), microcentrifuge tubes in which 500 microl aliquots of 0.1 M sodium oxalate and 0.1 M calcium chloride at pH 6 were added. When these tablets were incubated overnight with solutions at different concentrations of EDTA, sodium citrate, manganese chloride, sodium sulfate, sodium chloride, malic acid, succinic acid and gluconic acid, a significant dissolving activity was observed for EDTA ( approximately 25% at 0.25 M), sodium citrate ( approximately 30% at 1 M) and manganese chloride ( approximately 20% at 0.5 M). A good linear correlation (r2 = 0.84, p < 0.05) was found between the affinity for calcium and the activity of EDTA, sodium citrate, sodium sulfate, malic acid, succinic acid and gluconic acid, indicating that these compounds act mainly by chelating the calcium ion. Instead, manganese was supposed to act by interacting with the oxalate ion.