Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation

Docosapentaenoic acid (DPA) is a unique fatty acid that exists in two isomeric forms (n-3 and n-6), which differ in their physiological behaviors. DPA can undergo free radical-mediated peroxidation via lipoxygenase (LOX). 15-LOX, one of the LOX isomers, has received much attention in cancer research because of its very different expression level in normal tissues compared to tumors and some bioactive fatty acid metabolites modulating the tumorigenic pathways in cancer. However, the mechanism linking 15-LOX, DPA metabolites, and their bioactivities is still unclear, and the free radicals generated in DPA peroxidation have never been characterized. In this study, we have studied radicals formed from both soybean and human cellular (PC3-15LOS cells) 15-LOX-catalyzed peroxidation of DPAs at various pH's using a combination of LC/ESR/MS with the spin trapping technique. We observed a total of three carbon-centered radicals formed in 15-LOX-DPA (n-3) stemming from its 7-, 17-, and 20-hydroperoxides, whereas only one formed from 17-hydroperoxide in DPA (n-6). A change in the reaction pH from 8.5 (15-LOX enzyme optimum) to 7.4 (physiological) and to 6.5 (tumor, acidic) not only decreased the total radical formation but also altered the preferred site of oxygenation. This pH-dependent alteration of radical formation and oxygenation pattern may have significant implications and provide a basis for our ongoing investigations of LOXs as well as fatty acids in cancer biology.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Poduction of free radicals from phenol and tocopherol by bleomycin-iron(II) complex.

Oxygen-bubbling of 1:1 bleomycin(BLM)-Fe(II) complex efficiently generates hydroxy radical which is inhibited by catalase. Hydroxy radical produced from BLM-Fe(II)-O₂ system oxidizes 2,6-di-tert-butyl-p-cresol and α-tocopherol to form the corresponding phenoxy and tocopheroxyl radicals, respectively. These free radicals were identified by the ESR hyperfine structures.     

Antioxidant potential of ferulic acid.

Ferulic acid is a ubiquitous plant constituent that arises from the metabolism of phenylalanine and tyrosine. It occurs primarily in seeds and leaves both in its free form and covalently linked to lignin and other biopolymers. Due to its phenolic nucleus and an extended side chain conjugation, it readily forms a resonance stabilized phenoxy radical which accounts for its potent antioxidant potential. UV absorption by ferulic acid catalyzes stable phenoxy radical formation and thereby potentiates its ability to terminate free radical chain reactions. By virtue of effectively scavenging deleterious radicals and suppressing radiation-induced oxidative reactions, ferulic acid may serve an important antioxidant function in preserving physiological integrity of cells exposed to both air and impinging UV radiation. Similar photoprotection is afforded to skin by ferulic acid dissolved in cosmetic lotions. Its addition to foods inhibits lipid peroxidation and subsequent oxidative spoilage. By the same mechanism ferulic acid may protect against various inflammatory diseases. A number of other industrial applications are based on the antioxidant potential of ferulic acid.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Reaction of biological phenolic antioxidants with superoxide generated by cytochrome P-450 model system

Scavenging of a superoxide and simultaneous formation of free radicals with phenolic antioxidants were investigated with cobalt and iron tetraphenylporphyrin-thiolate complexes as models of P-450 enzymes. The kinetics of decay of the superoxide and development of free radical ESR signal intensities were studied. Based on a molecular orbital calculation of the hyperfine splitting, the radical species generated were confirmed to be those of phenoxyl radicals. The biological implication of superoxide, including active oxygen forms, for the reaction was discussed.     

Plant senescence processes and free radicals

Free radicals acting at sensitive subcellular sites, appear to play a pivotal role in both the deleterious and beneficial effects of maturation and senescence of various plant organs--leaves, flowers, and fruit. As evidenced by ESR spectrometry, spin trapping, specific membrane phase transition studies and enzyme kinetics, an important factor in the above processes appears to be lipoxygenase activity producing polyunsaturated fatty acid (PUFA) hydroperoxides and subsequently several free radical species and senescence-promoting compounds such as ethylene, malondialdehyde and jasmonic acid. The most intensely investigated are the oxy-free radical species including O2-., .OH, RO., ROO., PUFA and semiquinone free radicals. Higher plants are equipped with ways and means to combat free radicals and these may be classified under two general headings; (a) direct scavengers including SOD, ascorbic acid, and alpha-tocopherol acting in concert (b) incipient preventative mechanisms against radical formation, these include xanthine oxidase inhibitors, strategies based on endogenous H2O2 disposal in the form of peroxidative enzymes and glutathione turnover, and Ca2+ channel blockers. The antisenescence phytohormone cytokinin appears to possess a dual effect and may act in both capacities. The special case of delayed free radical formation in comparatively dry biological systems such as seeds is detailed, and specific free radical-generating photosensitizer compounds are also discussed.     

Plant catechols prevent lipid peroxidation in human plasma and erythrocytes

The antioxidant activity of several plant catechol derivatives was tested in buffer, plasma, and human erythrocytes. In buffer, chlorogenic acid (CGA), caffeic acid (CA), and dihydrocaffeic acid (DCA) reduced ferric iron equally well in the ferric reducing antioxidant power (FRAP) assay. Low concentrations of the polyphenols enhanced the ability of plasma to reduce ferric iron by about 10%. In plasma, lipid hydroperoxide and F₂-isoprostane formation induced by a water-soluble free radical initiator were reduced by CGA at concentrations as low as 20 M. During incubation at 37°C, human erythrocytes took up DCA, but not CGA, and intracellular DCA enhanced the ability of erythrocytes to reduce extracellular ferricyanide. When intact erythrocytes were exposed to oxidant stress generated by liposomes containing small amounts of lipid hydroperoxides, extracellular CGA at a concentration of 5 M decreased both lipid peroxidation in the liposomes, and spared -tocopherol in erythrocyte membranes. These results suggest that the catechol structure of these compounds convey the antioxidant effect in plasma and in erythrocytes.     

Free Radical Scavenging by Myocardial Fatty Acid Binding Protein

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O^?₂,). hydroxyl radical (OH') and hypochlorite radical (OCl') which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O^?₂, OH' and OCl' as indicated by the FABP inhibition of O^?₂-dependent reduction of cytochrome c, OH'-dependent hydroxybenzoic acid formation and OCl'-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH' than O^?₂, and inhibited OH' mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.     

Antioxidative activities of histidine containing caffeic acid-dipeptides

Antioxidants have been utilized in both the food and cosmetics industries to neutralize the activities of reactive oxygen species (ROS) and free radicals. Histidine-containing peptides are powerful antioxidants that exist in nature. Additionally, hydroxycinnamic acid (HCA)-peptide conjugates exhibit a synergistically enhanced antioxidative activity. Thus, caffeic acid (CA), a natural antioxidant, was conjugated to histidine-containing dipeptides (His dipeptides) in order to develop better antioxidants. The antioxidative activities were measured using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and lipid peroxidation test with ferric thiocyanate method. Some of the CA-His dipeptides exhibited better radical scavenging activities than CA, and all of the CA-His dipeptides showed enhanced lipid peroxidation inhibitory activities. His dipeptide enhanced the antioxidative activity of CA, and the position of histidine also affected the antioxidative activity of the compounds. CA-proline-histidine amide (CA-Pro-His-NH₂) exhibited the highest activity in both the free radical scavenging test and the lipid peroxidation inhibition test.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Characterization of the structure and reactions of free radicals from serotonin and related indoles

The ESR spectra of the free radicals formed by the autoxidation of serotonin, 5-hydroxyindole, and 5-hydroxytryptophan in 1 N NaOH are presented. The analysis of the hyperfine splitting constants in H2O and D2O characterize these free radicals as semiquinone-imines, the one-electron oxidation product of the corresponding indole. At alkaline pH, autoxidation of these compounds ultimately leads to solid precipitate and unresolved ESR spectra characteristic of polymeric material. The reduction of cytochrome c at pH 7.4 by a wide variety of indoles correlates with the amplitude of the ESR signal in 1 N NaOH, as do other processes thought to be related to 5-hydroxyindole free radical formation. Relative to the rate of cytochrome c reduction, neither serotonin nor the serotonin free radical appears to react with oxygen to form superoxide. In the presence of NAD(P)H, the serotonin radical most probably oxidizes NAD(P)H to form the NAD(P). radical. The NAD(P). radical then reacts with oxygen to form superoxide, which ultimately reduces cytochrome c.     

Formation of Methemoglobin and Phenoxyl Radicals from P-Hydroxyanisole and Oxyhemoglobin

The reaction of p-hydroxyanisole with oxyhemoglobin was investigated using electron spin resonance spectroscopy (ESR) and visible spectroscopy. As a reactive reaction intermediate we found the p-methoxyphenoxyl radical, the one-electron oxidation product of p-hydroxyanisole. Detection of this species required the rapid flow device elucidating the instability of this radical intermediate. The second reaction product formed is methemoglobin. Catalase or SOD had no effect upon the reaction kinetics. Accordingly, reactive oxygen species such as hydroxyl radicals or superoxide could not be observed although the spin trapping agent DMPO was used to make these short-lived species detectable. When the sulfhydryl blocking agents N-ethylmaleimide or mersalyl acid were used, an increase of the methemoglobin formation rate and of the phenoxyl radical concentration were observed. We have interpreted this observation in terms of a side reaction of free radical intermediates with thiol groups.     

Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion

Abstract

Here, we determined the electron spin resonance (ESR) spectra of standard reaction mixtures (I) containing 25 μM flavin mononucleotide (FMN), 0.018% tea tree (Melaleuca alternifolia) oil, 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and 1.0 mM FeSO₄(NH₄)₂SO₄ irradiated with 436 nm visible light (7.8 J/cm²). Prominent ESR signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) were detected, suggesting that free radicals form in the standard reaction. In order to know whether singlet oxygen (¹O₂) is involved in the radical formation or not, ESR measurement was performed for the standard D₂O reaction mixture (I) which contained 25 μM FMN, 0.0036% tea tree oil, 1.9 M acetonitrile-d3, 20 mM phosphate buffer (pH 7.4), 0.1 M 4-POBN and 1.0 mM FeSO₄ in D₂O. The ESR peak height of the standard D₂O reaction increased to 169 ± 24% of the control. Thus, ¹O₂ seems to be involved in the formation of the radicals because D₂O increases the lifetime of singlet oxygen. High-performance liquid chromatography-ESR-mass spectrometry analyses detected 1-methylethyl and methyl radicals in the standard reaction. The radicals appear to form through the reaction of ferrous ion with α-terpinene endoperoxide (ascaridole), which generated from the reaction of α-terpinene with ¹O₂. The 1-methylethyl and methyl radicals may exert a pro-oxidant effect under these conditions.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: An ESR spin-trapping investigation

Abstract

In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a^N =14.86±0.03 G and a^H_β =2.48±0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl₃ (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2^?/^?), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.