Β-carotene Assay Research Articles

Helichrysum monizii Lowe: Phenolic Composition and Antioxidant Potential

In Madeira Archipelago there are four endemic Helichyrsum species and three of them are used in the traditional medicine. Helichrysum monizii is a rare endemism with very scarce information available concerning its uses in the local traditional medicine. The aim of this work was to study for the first time Helichrysum monizii in terms of its antioxidant capacity and the identification of the phenolic compounds to which that activity is due. Three different methods of extraction were performed and total phenolic and flavonoid contents of extracts were correlated to radical scavenging and antioxidant capacity by DPPH, ABTS, FRAP and β-carotene assays. An HPLC-DAD-ESI/MS(n) method was employed for the separation and identification of the phenolic and flavonoid components. The results revealed a high antioxidant potential mainly related to the phenolic profile of the plant. Polar components of methanol extracts of Helichrsyum monizii were detected by a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI/MS(n) ) method. Thirty-three compounds were identified and 19 of them were identified as quinic acid derivatives. The high antioxidant potential Helichrysum monizii was for the first time established. Dicaffeoylquinic acids are the main responsible for that activity.

Antioxidant, antimicrobial, anti-inflammatory and anticancer activities of Carthamus tinctorius flowers

Carthamus tinctorius L. (Asteraceae) is an aromatic and folkloric medicinal plant thanks to its multiple virtues. However, few scientific studies investigated its biological activities. For that, this study aimed to investigate antioxidant, antibacterial, anti-inflammatory and anticancer activities of methanolic flower extracts of Carthamus tinctorius in order to validate some of its ethnopharmacological claims. Antioxidant activity was assessed via the ABTS radical scavenging and β-carotene inhibition tests, the antibacterial capacity were tested against human pathogen strains. Whereas, anti-inflammatory activity were estimated using inhibition NO release in LPS-stimulated Raw 264.7 macrophages, in comparison with N(G)-nitro-L-arginine methyl ester (L-NAME) which used as a positive control. In addition, anticancer activity was evaluated against Human lung carcinoma (A-549) and Human colorectal adenocarcinoma (DLD-1) cell lines. Main results showed that the flowers exhibit interesting biological activities. Indeed, flower extract displayed an inhibition percentage against ABTS equal to 30% and over 40% for the β-carotene inhibition assay. Antimicrobial activities were important especially against M. luteus strains (100% inhibition). Concerning anti-inflammatory activity, methanolic extract was able to inhibit NO release by 80% at 160µg/ml. Furthermore, C. tinctorius extract showed an anticancer activity against tumor cell lines DLD-1 with an IC50 value of 72±9µg/ml. These findings demonstrate the interesting potentiality of Carthamus tinctorius flowers as valuable source of antioxidant compounds which exhibit novel biological activities as antibacterial, anti-inflammatory and anticancer capacities.

Antioxidant Bibenzyl Derivatives from Notholaena nivea Desv.

Four new bibenzyl derivatives were isolated, together with other known bibenzyls, by bioassay-guided fractionation of a CHCl3-MeOH extract of Notholaena nivea Desv. (Pteridaceae) aerial parts. The structures were elucidated by NMR, ESIMS and other spectral analyses. Their antioxidative effects towards superoxide, lipidic peroxidation and the 2,2’-azino-bis-3-ethilbenzothiazoline-6-sulfonic acid (ABTS) radical were assayed. Results showed that the compound 3,12-dihydroxy-5-methoxybibenzyl (6) is the most active compound in the ABTS free-radical scavenging test, while in the coupled oxidation of β-carotene and linoleic acid assay the compound 5,12-dihydroxy-3-methoxydibenzyl-6-carboxylic acid (1) exerted the highest activity after 1h. A superoxide anion enzymatic test was also carried out and the results were confirmed by an inhibition of xanthine oxidase activity assay. The putative protective role played by compounds 1 and 6 on the injurious effects of reactive oxygen metabolites on the intestinal epithelium, using a Caco-2 human cell line, was investigated. H2O2-induced alterations were prevented by preincubating the cells with compounds 1 and 6.

Chemical Composition and Antioxidant Activity of Myrtus communis L. Floral Buds Essential Oil

The essential oil of Mytrus communis L. floral buds from Tunisia was analyzed by GC and GC/MS. Thirty-seven compounds were identified, representing 98.7% of the total oil. The major constituents were described as α-pinene (48.9%) and 1,8-cineole (15.3%). The antioxidant activity of the obtained essential oil was investigated by means of β-carotene bleaching (BCB) test and free radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Results showed that Myrtus communis L. floral buds essential oil has a significant antioxidant effect when tested by each method respectively. The strong antioxidant activity of the studied oil can be attributed to the high content in hydrocarbon monoterpenes and oxygenated monoterpenes.

Antioxidant constituents from Lawsonia inermis leaves: Isolation, structure elucidation and antioxidative capacity

The antioxidant potential of different fractions of Lawsonia inermis (Lythraceae) was investigated. The n-butanolic fraction showed the highest yield of extraction; it also exhibited a strong antioxidant activity in the DPPH assay and a potent capacity in preventing linoleic acid oxidation. Five phenolic glycosides were identified in this fraction. The structure of a new compound was established as 1,2,4-trihydroxynaphthalene-1-Ο-β- d-glucopyranoside. In addition, the known 2,4,6-trihydroxyacetophenone-2-Ο-β- d-glucopyranoside was described for the first time in this species. The three other compounds, lalioside (2,3,4,6-tetrahydroxyacetophenone-2-Ο-β- d-glucopyranoside), lawsoniaside (1,2,4-trihydroxynaphthalene-1,4-di-Ο-β- d-glucopyranoside) and luteolin-7-Ο-β- d-glucopyranoside, have been previously reported in L. inermis. The antioxidant activity of these glycosides was evaluated by DPPH and β-carotene assays, and compared to those of commercial standards. 1,2,4-Trihydroxynaphthalene-1-Ο-β- d-glucopyranoside was the most active in the DPPH free-radical scavenging test ( EC 50 = 6.5 μg/ml) and showed a moderate inhibition in the β-carotene bleaching assay. Chemical components of L. inermis have good antioxidant capacities and this species could be used as a potential source of new natural antioxidants.

Evaluation of antioxidant activities of various solvent extracts of Carissa opaca fruits

The chloroform and aqueous fractions of Carissa opaca fruit, a traditional medicinal fruit in Pakistan possessed a high amount of total phenolic and flavonoid contents as compare to other solvent fractions with potent antioxidant activities in scavenging DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radicals, and had strong iron chelating activity. On the other hand, the ethyl acetate fraction showed the highest inhibition of β-carotene linoleic acid peroxidation and phosphomolybdate assay. A high correlation coefficient existed between EC 50 values of DPPH, superoxides, hydroxyl, hydrogen peroxide, ABTS radicals, total phenolics and flavonoids, but a non significant correlation was found in the case of iron chelaters, β-carotene and phosphomolybdate assay. This study verified that the chloroform and aqueous fractions have strong antioxidant activities which were correlated with its high level of phenolics and flavonoids. These fractions can be used as a source of potential antioxidant or functional food material.

Bioactive substance contents and antioxidant capacity of raw and blanched vegetables

Five commonly consumed vegetables in Malaysia namely, four-angled bean (Psophocarpus tetragonolobus D.C.), French bean (Phaseolus vulgaris L.), long bean (Vigna sesquipedalis L.), snow pea (Pisum sativum var. macrocarpon L.) and snap pea (Pisum sativum) were blanched in boiling water for 10 min. The contents of total phenolics, ascorbic acid and β-carotene, and the antioxidant capacity as typified by β-carotene and free radical scavenging activity (DPPH) assays were determined for the raw and blanched vegetables. The study revealed that blanching caused a significant (p < 0.05) increase in β-carotene content [fresh (389–539 µg/100 g), blanched (510–818 µg/100 g)], except in snow pea. Conversely, there was a significant (p < 0.05) decrease in ascorbic acid content [fresh (1.2–7.8 mg/100 g), blanched (0.67–3.8 mg/100 g)]. After blanching, the total phenolic content and antioxidant activity either decreased or increased depending on the type of vegetables. The total phenolic content was positively correlated with the antioxidant activity of the studied vegetables to some extent, but not with ascorbic acid or β-carotene.

Synthesis of novel 3-chloro-1-(5H-dibenz[b,f]azepine-5yl)propan-1-one derivatives with antioxidant activity

A series of 3-chloro-1-(5H-dibenz[b,f]azepine-5yl)propan-1-one derivatives (2a–k) bearing different amino acids were synthesized by base condensation reaction. 3-Chloro-1-(5H-dibenz[b,f]azepine-5yl)propan-1-one(2) was obtained by N-acylation of 5H-dibenz[b,f]azepine (1). All the synthesized compounds were evaluated for their potential over antioxidant activities against inhibition of lipid peroxidation by β-carotene and linoleic acid assay and inhibition of human low-density lipoprotein (LDL) oxidation assay. Typically, compound 2 showed weak antioxidant activity, whereas coupling of different amino acids enhances the antioxidant activities based on the presence of different functional groups. Among the derivatives, compound 2d showed significant antioxidant activities followed by 2h, 2i, 2j and 2k.

Anti-neurodegenerative activity of photosensitizing dyes (3): The effect on a hereditary cerebellar ataxia

Chokeberries (Aronia melanocarpa (Michx.) Elliot have recently become one of the most popular and widely used berry fruits, and there are numerous studies dealing with the investigation of their chemical composition and biological activities. Unlike berries, chokeberry leaves were far less investigated. Therefore, the aim of this study was to conduct a bioactivity-based analysis of chokeberry leaves collected after berries were harvested. Chemical analysis of hydroethanolic chokeberry extract and its ethyl acetate, n-butanol, and water fractions was performed. Antioxidant, hypoglycemic, and antineurodegenerative activities of the obtained samples were assessed by in vitro tests. The highest total phenolics content was found in n-butanol fraction (221.5 mg gallic acid equivalents/g dw). Fourteen compounds (six phenolic acids and eight flavonoids) were identified in the samples. Caffeoylquinic acid derivatives and quercetin glycosides were dominant phenolic compounds. The highest content of phenolic acids, as well as flavonoids, was recorded in n-butanol fraction (80.75 mg/g dw and 47.79 mg/g dw, respectively). This fraction was the most active in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Also, the n-butanol fraction was the most effective inhibitor of acetylcholinesterase (AChE) and tyrosinase (TYR) enzymes. All samples showed a better inhibitory effect on α-glucosidase enzyme with inhibition concentrations IC50 = 4.21 - 9.22 μg/mL, than α-amylase enzymes (IC50 = 14.36 - 88.61 μg/mL). Pearson’s correlation between the content of individual phenolics and the results of biological tests was performed. Thus, hyperoside and isoquercitrin showed a significant correlation with DPPH and ABTS tests, caffeoylquinic acids with FRAP test, quercetin 3-O-vicianoside with β-carotene assay, hyperoside and isoquercitrin with α-amylase inhibition activity, and isorhamnetin 3-O-rutinoside with α-glucosidase inhibition assay. Considering the obtained results, chokeberry leaves represent a valuable natural source of phenolic compounds with promising potential for the development of new products with beneficial effects on human health.

Antioxidant and Free Radical Scavenging Properties of Marennine, a Blue-Green Polyphenolic Pigment from the Diatom Haslea ostrearia (Gaillon/Bory) Simonsen Responsible for the Natural Greening of Cultured Oysters

Among microalgae, the marine diatom Haslea ostrearia has the distinctive feature of synthesizing and releasing, into the surrounding environment, a blue-green polyphenolic pigment called marennine. The oyster-breeding industry commonly makes use of this natural phenomenon for the greening of oysters grown in the ponds of the French Atlantic coast. This article reports the in vitro antioxidant properties of pure marennine. Two kinds of evaluation systems were adopted to test the antioxidative activity of marennine: antioxidant capacity assays (beta-carotene and thymidine protection assays and iron reducing power assay) and free radical scavenging assays (DPPH*, O2*-, and HO*). In almost all cases, marennine exhibited significantly higher antioxidative and free radical scavenging activities than natural and synthetic antioxidants commonly used in food, as shown by comparing median effective concentration (EC 50) values, for each test independently. This medium molecular weight polyphenol (around 10 kDa) from microalgae is thus a potentially useful natural antioxidant. Because of its blue-coloring property and water solubility, it could also be used as a natural food-coloring additive.

Antioxidant activities of some Greek wines and wine phenolic extracts

Antioxidant activities and phenolic composition of eight Greek wines were determined. Red wines scavenged 1,1′-diphenyl-2-picryl-hydrazyl radical (DPPH ) to a much greater degree than whites, in proportion to their phenolic contents. Red wines inhibited β-carotene bleaching, while white wines were almost inactive. Red wines were more active than quercetin in DPPH and less active in β-carotene assay. Xinomavro-red must exhibited significant activities in both assays. Roditis-white must scavenged DPPH , and exhibited some activity in β-carotene assay. Young Xinomavro and Roditis wines and their extracts were more active in scavenging DPPH than the respective aged wine and extracts. However, young Xinomavro wine and its extracts were less active than those of aged wine in β-carotene assay. Roditis wine extracts exhibited only limited activity in β-carotene assay. Xinomavro young and aged wine extracts rich in anthocyanins and flavanols were more active than the others in both assays used.

Mung beans processed by solid-state bioconversion improves phenolic content and functionality relevant for diabetes and ulcer management

Abstract Mung bean substrate was enriched with phenolic antioxidants and levo-dihydroxy phenylalanine ( l -DOPA) through solid-state bioconversion (SSB) by Rhizopus oligosporus, with the goal to enhance health-linked functionality. The alpha-amylase inhibition linked to diabetes management and Helicobacter pylori inhibition linked to peptic ulcer management were investigated in bioprocessed extracts. The protein content and β-glucosidase activity of the substrate which are indicators of effective fungal colonization, increased with growth. The phenolic content increase with growth was linked to fungal β-glucosidase activity, indicating phenolic mobilization. The antioxidant activity as measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) inhibition method were high (65%) initially and increased to 90% on day 12, than maintained higher levels during the rest of growth. The antioxidant activity measured by β-carotene assay was high between days 4–8 when phenolic levels increased. Fungal-linked superoxide dismutase (SOD) activity was high in late stages indicating quenching of reactive oxygen species. The l -DOPA content was low during early growth stage (0.6–0.7 mg/g DW) and gradually doubled to 1.2 mg/g DW in late stages. The α-amylase inhibition potential was moderately high during early stages (days 0–2) followed by higher inhibition during days 4–10 which correlate to higher phenolic content. The change in the antimicrobial activities of the extracts against H. pylori was high on day 4 and was associated with high antioxidant activity but not high phenolic content. Elevated inhibition on day 8 directly correlated with high total phenolics content suggesting the role of phenolic mobilization. The major implication from this research is that SSB is a good strategy to improve the phenolic content of mung beans for enhanced functionality with improved antioxidant activity that contributes to α-amylase inhibition relevant to potential diabetes management and H. pylori inhibition linked to peptic ulcer management. Industrial relevance Solid State Bioconversion (SSB) of mung bean by R. oligosporus is a good strategy to enhance ingredient functionality due to mobilization of phenolic antioxidants. Such enhanced phenolic antioxidant activity potentially contributes to health-relevant functionality such as amylase inhibition for diabetes management and H. pylori inhibition for peptic ulcer management. Optimization of such SSB systems at industrial scale can help large-scale low cost production of such health-relevant ingredients.

Screening of functional compounds in supercritical fluid extracts from Spirulina platensis

Supercritical fluid extraction and fractionation of Spirulina platensis were carried out in order to obtain functional extracts with antioxidant and/or antimicrobial activities. The β-carotene bleaching method and DPPH free radical-scavenging assay were used to determine the optimal extraction conditions for antioxidant compounds. The best antioxidant extract was obtained in the first fraction when using intermediate pressures and temperatures (220–320 bar, 55 °C), with CO 2 plus 10% ethanol as cosolvent, whereas higher pressures and temperatures (320 bar, 75 °C) were needed when pure CO 2 was used. Besides, antimicrobial activities of microalgae extracts were tested against four different microbial species, including a gram positive bacterium ( Staphylococcus aureus), a gram negative bacterium ( Escherichia coli), a yeast ( Candida albicans) and a fungus ( Aspergillus niger). The most active fraction against all the microorganisms tested, was the one collected in the second fraction in the experiment performed at 220 bar and 26.7 °C with 10% of ethanol.

Evaluation of the antioxidant activity of extracts from buntan ( Citrus grandis Osbeck) fruit tissues

The goal of the present work was to evaluate the antioxidant properties of buntan ( Citrus grandis Osbeck) using various solvents, such as n-hexane, ethyl acetate (EtOAc), butanol and methanol. The antioxidant activities of crude extracts were evaluated by using the free radical scavenging β-carotene assay and total polyphenol. Ethyl acetate extracts of falvedo exhibited high antioxidative activities, followed by albedo and segment membrane extracts. Chromatography separation of EtOAc extract of flavedo using a silica gel column, yielded six fractions (A, B, C, D, E and F) using gradient elution with benzene and acetone (19:1, 14:1, 9:1, 5:1, 1:1 and 0:l). Among them, two fractions (C and D) showed strong antioxidant activities using the free radical scavenging activity (DPPH) antioxidant assay. These two fractions were further purified using silica gel column chromatography and preparative TLC. Their extracts could well be useful to prevent oxidation in fruit juices and essential oil food products as well as for health supplements. Identification of the responsible components is underway.

A new sesquiterpene from the fruits of Allophylus laevigatus

From the fruits of Allophylus laevigatus a new sesquiterpene, 11-acetoxy-4α-methoxyeudesmane, was isolated alongwith the known compounds carissone and apigenin-8-C-β-rhamnopyranoside. The flavone showed no antioxidant activity in the autoxidation of β-carotene assay.

The influence of beer with different antioxidant potential on plasma lipids, plasma antioxidant capacity, and bile excretion of rats fed cholesterol-containing and cholesterol-free diets

The aim of this investigation was to assess the influence of beers with different antioxidant potentials on plasma lipid metabolism, plasma antioxidant capacity, and bile excretion of rats fed cholesterol-containing and cholesterol-free diets. Four types of beers were investigated in vitro. Two of them (designated as BeerHigh and BeerLow) with the highest and lowest antioxidant potentials (34.5% and 21.4% and 2.07 mmol/L and 1.65 mmol/L according to β-carotene assay and Trolox equivalent antioxidant coefficient, respectively), were chosen for the experiment on rats. A total of 60 male Wistar rats were divided into 6 dietary groups of 10 rats each; the groups were designated as Control, BeerA, BeerB, Chol, Chol/BeerA, and Chol/BeerB. The rats in the Control group were fed a basal diet (BD) only, which included wheat starch, casein, soybean oil, vitamin, and mineral mixtures. To the BD of the other five groups were added the following: BeerHigh (BeerA), BeerLow (BeerB), 1% of cholesterol (Chol), 1% of cholesterol and BeerHigh (Chol/BeerA), and 1% of cholesterol and BeerLow (Chol/BeerB). After 4 weeks of feeding, diets supplemented with BeerHigh and, to a lesser degree, with BeerLow (Chol/BeerA and Chol/BeerB groups) hindered a rise in plasma lipids and a decrease in plasma antioxidant capacity, and increased the bile excretion indices. Supplementation with BeerHigh and, to a lesser degree, with BeerLow in rats fed cholesterol-free diets increased their plasma antioxidant capacity. No significant changes in the plasma lipid levels, antioxidant capacity, and bile excretion indices were observed in the Control group. In conclusion, beer was found to have a positively effect on plasma lipid profile and plasma antioxidant capacity, and to increase the bile excretion indices in rats fed cholesterol-containing diets. The degree of this positive influence is directly connected to the contents of the bioactive components and the related antioxidant potential of beer. It is suggested that to achieve the best results, beer with the highest antioxidant potential must be consumed.

Stimulation of phenolics, antioxidant and antimicrobial activities in dark germinated mung bean sprouts in response to peptide and phytochemical elicitors

The phenyl propanoid pathway (PPP) was stimulated in mung bean sprouts through the pentose phosphate and shikimate pathways, by natural elicitors such as fish protein hydrolysates (FPH), lactoferrin (LF) and oregano extract (OE). Elicitation significantly improved the phenolic, antioxidant and antimicrobial properties of mung bean sprouts. The optimal elicitor concentrations were 1 ml/l FPH, 250 ppm LF and 1 ml/l OE for the highest phenolic content that was approximately 20, 35 and 18% higher than control, respectively, on day 1 of dark germination. The antioxidant activity estimated by β-carotene assay in mung bean sprouts was highest on day 1 of germination for all treatments and control. In general, higher antioxidant activity was observed in the elicited sprouts compared with control. In the case of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay the antioxidant activity for all treatments and control was highest on day 2. Among the different elicitor treatments, OE elicited mung bean sprouts showed the highest antioxidant activity of 49% DPPH inhibition on day 2. This increased activity correlates with high guaiacol peroxidase (GPX) activity indicating that the polymerizing phenolics required during lignification with growth have antioxidant function. For all elicitor treatments a higher glucose-6-phosphate dehydrogenase (G6PDH) activity was observed during early germination following the high phenolic content. This is due to the general mobilization of carbohydrates to the growing sprouts in response to elicitation. In general the GPX activity steadily increased with germination for treatments and control. The higher phenolics produced on day 1 was utilized for GPX-mediated polymerization to form polymeric phenolics and lignin required during germination. The late stage polymerization linked to GPX activity preceded stimulation of G6PDH. This indicated that as phenolics were polymerized by GPX in late stages, G6PDH linked precursors such as NADPH 2 and sugar phosphates were being made available. Antimicrobial activity against Helicobacter pylori was observed in the mung bean sprout extract from control, LF and OE treatments from the day 1 stage. Both the LF and OE elicited extracts showed high antimicrobial activity, which correlated to high antioxidant activity on day 1. The higher antimicrobial activity was also observed with the higher stimulation of G6PDH and GPX activity during early stages of germination. This leads to the hypothesis that enhanced mobilization of carbohydrates (as indicated by G6PDH activity on days 2 and 4), enhanced polymerization of simple phenols (as indicated by GPX activity on day 3) contributed to high antioxidant activity producing intermediary metabolites (day 2).

Antioxidant activity of extracts from Gevuina avellana and Rosa rubiginosa defatted seeds

Chilean hazelnuts (Gevuina avellana Mol) and mosqueta rose (Rosa aff. Rubiginosa L.) meals were extracted with methanol, ethanol, acidified water, acetone, butanol, diethyl ether and ethyl acetate. Ethanol and methanol extracted the highest amount of soluble substances from both seed meals. The highest concentration of total polyphenols was found in the ethanolic extracts, although that of acetone from R. rubiginosa presented similar values. The antioxidant activity of the extracts evaluated by the β-carotene assay and with as hydrogen radical scavenging ability showed that the activity of the butanol and methanol extracts from G. avellana was comparable to those of synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). The 2,2,-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging activity of the G. avellana methanol, ethanol and water extracts was 2–3 times lower than those of BHT and BHA, respectively. The activity of R. rubiginosa extracts ranged from that of BHT for the water extracts to 80% inhibition in respect to control achieved with the ethanol extracts.

In vitro evaluation of antioxidant activity of essential oils and their components

Clove and nutmeg essential oils were analysed by GC and GC–MS. These oils, together with 16–18 components found to be present, were tested for antioxidant properties at final concentrations of 0.05–2.5×104 ppm in an egg yolk-based thiobarbituric acid reactive substances (TBARS) assay and also undiluted in a β-carotene agar diffusion assay. Both the essential oils and the components tested in the TBARS assay demonstrated some degree of antioxidant activity. Only the clove oil, the nutmeg oil, eugenol and terpinolene demonstrated any ability to inhibit the oxidative bleaching of the β-carotene agar. The ability of the oil components to inhibit malondialdehyde formation, and therefore lipid peroxidation, in the TBARS assay, yet apparently to possess no activity in the β-carotene agar diffusion assay, demonstrates the importance in the screening of plant material for bioactivity of using a bank of assays in vitro before assigning bioactivities. By using a number of assays, not only should the number of false positives and negatives be greatly reduced, but evidence pertaining to the mechanism of action may be obtained. Copyright © 2000 John Wiley & Sons, Ltd.