Prooxidative effect of cardols is involved in their cytotoxic activity against murine B16-F10 melanoma cells.

Cardols are resorcinolic lipids, available in many natural sources including cashew nut, pistachio, macadamia, and mango. Despite of several beneficial biological activities of cardols, cytotoxic activities of cardols are not fully understood. In preliminary studies, 5[8(Z),11(Z),14-pentadecatrienyl]resorcinol, known as cardol (C15:3) was found to inhibit tyrosinase-catalyzed melanin formation in cell-free system. In the case of cultured cell analysis, cardol (C15:3) showed intense cytotoxicity but not anti-melanogenic activity against B16-F10 melanoma cells. Subsequently, cardol (C10:0) and cardol (C5:0), containing shorter alkyl side chain, exhibited inferior cytotoxicity compared to cardol (C15:3). The cytotoxicity via cardol (C15:3) was reversed by the addition of antioxidants, indicating that intracellular prooxidative activity was involved. Furthermore, cardol (C15:3) produced significant levels of reactive oxygen species (ROS) while cardol (C5:0) generated lesser ROS levels. Our findings suggest the cytotoxic activity of cardols is their prooxidative effect depending on the length of alkyl side chain.

Effects of matsutake mushroom scent compounds on tyrosinase and murine B16-F10 melanoma cells.

Tyrosinase-catalyzed l-tyrosine oxidation is a key step in melanogenesis, and intense melanin formation is often a problem in chemotherapies or food preservation. Here we report that methyl cinnamate one of the constituents characterized from mycelium and sporocarp of American matsutake mushroom Tricholoma magnivelare inhibits both enzymatic and cellular melanin formation. Methyl cinnamate inhibits mushroom tyrosinase-catalyzed l-tyrosine oxidation while the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) was not inhibited. In subsequent cellular assays, methyl cinnamate significantly suppressed melanogenesis of murine B16-F10 melanoma cells without affecting cell growth. However, methyl 3-phenylpropionate, a dihydro-derivative of methyl cinnamate, did not possess melanogenesis, indicating that the double bond in the enone moiety is a key Michael reaction acceptor to elicit the activity. In addition, a rather rare chlorinated benzaldehyde derivative, 3,5-dichloro-4-methoxybenzaldehyde isolated from the same source, was found to show potent cytotoxicity, and the chlorine atom reduced a tyrosinase inhibitory activity but enhanced cytotoxicity. Our findings suggest that methyl cinnamate is a novel melanogenesis inhibitor from natural sources.

Anethole potentiates dodecanol's fungicidal activity by reducing PDR5 expression in budding yeast.

BACKGROUND: trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the mechanism underlying this synergistic effect of anethole has not been characterized. METHODS: We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux. RESULTS: Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5, in addition to a slight increase in PDR11, PDR12, and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δpdr5. In addition, Δpdr3 and Δlge1, lack transcription factors of PDR5 and PDR3, were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans. CONCLUSIONS: These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. GENERAL SIGNIFICANCE: Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters.

Suppression of superoxide anion generation catalyzed by xanthine oxidase with alkyl caffeates and the scavenging activity.

Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclear about the effect of caffeic acid and alkyl caffeates on superoxide anion (O2(-)) generation catalyzed by xanthine oxidase. Effects of caffeic acid and alkyl caffeates on the uric acid formation and O2(-) generation catalyzed by xanthine oxidase were analyzed. The scavenging activities of 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and O2(-) generated with phenazine methosulfate (PMS) and NADH were examined. Caffeic acid derivatives equally suppressed O2(-) generation, and the suppression is stronger than inhibition of xanthine oxidase. Scavenging activity of O2(-) is low compared to the suppression of O2(-) generation. Suppression of O2(-) generation catalyzed by xanthine oxidase with caffeic acid derivatives was not due to enzyme inhibition or O2(-) scavenging but due to the reduction of xanthine oxidase molecules. Alkyl caffeates are effective inhibitors of uric acid and O2(-) catalyzed by xanthine oxidase as well as antioxidants for edible oil.

New environmentally-friendly antimicrobials and biocides from Andean and Mexican biodiversity.

Persistent application of pesticides often leads to accumulation in the environment and to the development of resistance in various organisms. These chemicals frequently degrade slowly and have the potential to bio-accumulate across the food chain and in top predators. Cancer and neuronal damage at genomic and proteomic levels have been linked to exposure to pesticides in humans. These negative effects encourage search for new sources of biopesticides that are more "environmentally-friendly" to the environment and human health. Many plant or fungal compounds have significant biological activity associated with the presence of secondary metabolites. Plant biotechnology and new molecular methods offer ways to understand regulation and to improve production of secondary metabolites of interest. Naturally occurring crop protection chemicals offer new approaches for pest management by providing new sources of biologically active natural products with biodegradability, low mammalian toxicity and environmentally-friendly qualities. Latin America is one of the world's most biodiverse regions and provide a previously unsuspected reservoir of new and potentially useful molecules. Phytochemicals from a number of families of plants and fungi from the southern Andes and from Mexico have now been evaluated. Andean basidiomycetes are also a great source of scientifically new compounds that are interesting and potentially useful. Use of biopesticides is an important component of integrated pest management (IPM) and can improve the risks and benefits of production of many crops all over the world.

Anti-Salmonella Activity of Volatile Compounds of Vietnam Coriander.

Essential oil derived from the fresh leaves of Polygonum odoratum Lour was tested for their effects on a foodborne bacterium Salmonella choleraesuis subsp. choleraesuis ATCC 35640 using a broth dilution method. This essential oil showed a significant antibacterial activity against S. choleraesuis at the concentration of 200 µg/mL. Twenty-five volatile compounds were characterized from this essential oil by GC-MS, and aldehyde compounds were found abundant and accounted for more than three-fourths of the essential oil. Among the compounds characterized, dodecanal (C12 ) was the most abundant (55.5%), followed by decanal (C10 ) (11.6%). Both alkanals were effective against S. choleraesuis with the minimum growth inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 100 µg/mL. The most potent antibacterial activity against this bacterium was found with two minor compounds, dodecanol (lauryl alcohol) and 2E-dodecenal, both with each MBC of 6.25 µg/mL. Their primary antibacterial action against S. choleraesuis provably comes from their ability to function as nonionic surface-active agents (surfactants), disrupting the native function of integral membrane proteins nonspecifically. Thus, the antibacterial activity is mediated by biophysical processes. In the case of 2E-alkenals, a biochemical mechanism is also somewhat involved, depending on their alkyl chain length.

Biopesticides from plants: Calceolaria integrifolia s.l.

The effects of persistent organic pollutants (POPs) on humans and biodiversity are multiple and varied. Nowadays environmentally-friendly pesticides are strongly preferred to POPs. It is noteworthy that the crop protection role of pesticides and other techniques, i.e. biopesticides, plant extracts, prevention methods, organic methods, evaluation of plant resistance to certain pests under an integrated pest management (IPM), could improve the risks and benefits which must be assessed on a sound scientific basis. For this directive it is crucial to bring about a significant reduction in the use of chemical pesticides, not least through the promotion of sustainable alternative solutions such as organic farming and IPM. Biopesticides are derived from natural materials such as animals, plants, bacteria, and certain minerals. Most of them are biodegradable in relatively short periods of time. On this regard, substances from Calceolaria species emerge as a strong alternative to the use of POPs. The American genus Calceolaria species are regarded both as a notorious weeds and popular ornamental garden plants. Some have medicinal applications. Other taxa of Calceolaria are toxic to insects and resistant to microbial attack. These properties are probably associated with the presence of terpenes, iridoids, flavonoids, naphthoquinones and phenylpropanoids previously demonstrated to have interesting biological activities. In this article a comprehensive evaluation of the potential utilization of Calceolaria species as a source of biopesticides is made. The chemical profile of selected members of the Chilean Calceolaria integrifolia sensu lato complex represents a significant addition to previous studies. New secondary metabolites were isolated, identified and tested for their antifeedant, insect growth regulation and insecticidal activities against Spodoptera frugiperda and Drosophila melanogaster. These species serve as a model of insect pests using conventional procedures. Additionally, bactericidal and fungicidal activity were determined. Dunnione mixed with gallic acid was the most active fungistatic and fungicidal combination encountered. Several compounds as isorhamnetin, combined with ferulic and gallic acid quickly reduced cell viability, but cell viability was recovered quickly and did not differ from that of the control. The effect of these mixtures on cultures of Aspergillus niger, Fusarium moniliforme, Fusarium sporotrichum, Rhizoctonia solani, and Trichophyton mentagrophytes, was sublethal. However, when fungistatic isorhamnetin and dunnione were combined with sublethal amounts of both ferulic and gallic acid, respectively, strong fungicidal activity against theses strains was observed. Thus, dunnione combined with gallic acid completely restricted the recovery of cell viability. This apparent synergistic effect was probably due to the blockade of the recovery process from induced-stress. The same series of phenolics (iridoids, flavonoids, naphthoquinones and phenylpropanoids) were also tested against the Gram-negative bacteria Escherichia coli, Enterobacter agglomerans, and Salmonella typhi, and against the Gram-positive bacteria Bacillus subtilis, Sarcinia lutea, and Staphylococcus aureus and their effects compared with those that of kanamycin. Mixtures of isorhamnetin/dunnione/kaempferol/ferulic/gallic acid in various combinations were found to have the most potent bactericidal and fungicidal activity with MFC between 10 and 50 µg/ml. Quercetin was found to be the most potent fungistatic single compound with an MIC of 15 µg/ml. A time-kill curve study showed that quercetin was fungicidal against fungi assayed at any growth stage. This antifungal activity was slightly enhanced by combination with gallic acid. The primary antifungal action of the mixtures assayed likely comes from their ability to act as nonionic surfactants that disrupt the function of native membrane-associated proteins. Hence, the antifungal activity of isorhamnetin and other O-methyl flavonols appears to be mediated by biophysical processes. Maximum activity is obtained when the balance between hydrophilic and hydrophobic portions of the molecules of the mixtures becomes the most appropriate. Diterpenes, flavonoids, phenylpropanoids, iridoids and phenolic acids were identified by chromatographic procedures (HPLC-DAD), ESI-MS, and NMR hyphenated techniques.

Protective effects of α-tocopherol and ascorbic acid against cardol-induced cell death and reactive oxygen species generation in Staphylococcus aureus.

Cardol (C15:3), isolated from cashew (Anacardium occidentale L.) nut shell liquid, has been shown to exhibit bactericidal activity against various strains of Staphylococcus aureus, including methicillin-resistant strains. The maximum level of reactive oxygen species generation was detected at around the minimum bactericidal concentration of cardol, while reactive oxygen species production drastically decreased at doses above the minimum bactericidal concentration. The primary response for bactericidal activity around the bactericidal concentration was noted to primarily originate from oxidative stress such as intracellular reactive oxygen species generation. High doses of cardol (C15:3) were shown to induce leakage of K⁺ from S. aureus cells, which may be related to the decrease in reactive oxygen species. Antioxidants such as α-tocopherol and ascorbic acid restricted reactive oxygen species generation and restored cellular damage induced by the lipid. Cardol (C15:3) overdose probably disrupts the native membrane-associated function as it acts as a surfactant. The maximum antibacterial activity of cardols against S. aureus depends on their log P values (partition coefficient in octanol/water) and is related to their similarity to those of anacardic acids isolated from the same source.

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor.

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 µM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 µM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.

Multifunctional cytotoxic agents from Anacardium occidentale.

The effects of anacardic acids and cardols isolated from the cashew nut and apple Anacardium occidentale (Anacardiaceae) on murine B16-F10 melanoma cells were tested. Although anacardic acids and cardols were found to inhibit tyrosinase, a key enzyme in melanin synthesis, melanogenesis in melanocytes was not suppressed in cultured cells but rather enhanced. Both anacardic acids and cardols exhibited moderate cytotoxicity.

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625 mM. A hyperosmotic condition (1.2 M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition.

Inhibition mode of soybean lipoxygenase-1 by quercetin.

Quercetin noncompetitively inhibited the peroxidation of linoleic acid catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC(50) value of 4.8 microM (1.45 microg/ml). This inhibition is considered to proceed in sequential order, by initially reducing the ferric form of the enzyme to an inactive ferrous form and then, by chelating the iron of the active site of the enzyme. In the pseudoperoxidase assay, quercetin was slowly oxidized by hydroperoxides to a rather stable intermediate, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one, and this oxidized intermediate still inhibited the enzymatic oxidation, probably as a chelator. Rutin and kaempferol also exhibited lipoxygenase-1 inhibitory activity, but to a much lesser extent than quercetin.

Tyrosinase Inhibition by 4-Substituted Benzaldehydes with Electron-Withdrawing Groups.

The oxidation of 4-t-butylcatechol catalyzed by mushroom tyrosinase was inhibited by 4-bromobenzaldehyde, 4-chlorobenzaldehyde, 4-fluorobenzaldehyde, 4-cyanobenzaldehyde, and 4-nitrobenzaldehyde with 50% inhibitory concentrations of 114 µM, 175 µM, 387 µM, 822 µM, and 1846 µM, respectively. The inhibition kinetics were analyzed by Dixon plots, which indicated that a series of 4-hallogenated benzaldehydes acted as partial noncompetitive inhibitors in the same manner as benzaldehyde. Although ß values were decreased with an increase of the tyrosinase inhibitory activity, full inhibition could not be observed. In contrast, 4-cyanobenzaldehyde and 4-nitrobenzaldehyde acted as mixed and noncompetitive inhibitors, respectively. Full inhibition was particularly represented by 4-nitrobenzaldehyde. According to a previous report, 4-alkylbenzaldehyde and 4-alkoxybenzaldehyde with a bulky substituent acted as full inhibitors. Those results suggested that the steric factor at the 4-position triggered the alternation between partial or full tyrosinase inhibition irrespective of electronic or hydrophobic effects.

Chemoprotective and antiobesity effects of tocols from seed oil of Maqui-berry: Their antioxidative and digestive enzyme inhibition potential.

Maqui-berry (Aristotelia chilensis) is the emerging Chilean superfruit with high nutraceutical value. Until now, the research on this commodity was focused on the formulations enriched with polyphenols from the pulp. Herein, contents of tocols were compared in the seed oil of Maqui-berry obtained through three different extraction methods followed by determining their antioxidative and enzyme inhibitions in-vitro. Firstly, oilseed was extracted with n-hexane (Soxhlet method), chloroform/methanol/water (Bligh and Dyer method) and pressing (industrial). These samples were used to access their effects against DPPH, HORAC, ORAC, FRAP, Lipid-peroxidation (TBARS), α-amylase, α-glucosidase, and pancreatic lipase. All the isomers of tocopherol and tocotrienol were identified, and ß-sitosterol was the only sterol found in higher amounts than other vegetable oils. The Bligh and Dyer method could lead to the highest antioxidative capacity compared to Soxhlet and press methods likely because the latter have a higher amount of tocopherols. Further, seed oil from Maqui berry and their tocols (α, ß, γ, δ-tocopherols, tocotrienols, and ß-sitosterol) warrant clinical investigation for their antioxidative and antiobesity potential. Taken together, these findings provide relevant and suitable conditions for the industrial processing of Maqui-berry.

Benzonitriles as tyrosinase inhibitors with hyperbolic inhibition manner.

As a novel mushroom tyrosinase inhibitor, 4-methoxybenzonitrile (anisnitrile) was identified (IC50 = 111.1 µM) with hyperbolic inhibition manner. The calculated αKi (166.3 µM) was larger than Ki (66.5 µM) by Dixon plots, indicating that this nitrile acts as a competitive-noncompetitive mixed type inhibitor. Similarly, 4-isopropylbenzonitrile (cuminnitrile) partially inhibited the oxidation catalyzed by tyrosinase (IC50 = 121.5 µM, Ki = 88.8 µM, and αKi = 239.8 µM). Nine other benzonitriles also exhibited partial tyrosinase-inhibitory activity. In particular, 4-methylbenzonitrile (IC50 = 79.9 µM) is considered to be the most potent among the tested benzonitriles. Benzonitriles barely caused intermolecular amidine formation under physiologic conditions. Furthermore, they possibly coordinate copper at the active site of tyrosinase. Hence, benzonitriles exhibit different inhibition characteristics as compared with that exhibited by benzaldehydes.

Polygonum odoratum essential oil inhibits the activity of mushroom derived tyrosinase.

Plant derived compounds are a source of long term research focus due to their applications in a variety of fields, particularly food preservation. One key way in which phytochemicals are crucial in this area is by disrupting enzyme functionality. In this work, essential oil was extracted by steam distillation from the fresh leaves of Polygonum odoratum (Polygonaceae), commonly known as Vietnamese coriander, and shown to effectively inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase (EC1.14.18.1). Using GC-MS analysis, twenty five compounds were identified in the essential oil. The most abundant compounds in the essential oil were Alkanals - dodecanal (55.49%), and decanal (11.57%) - followed by anisaldehyde (6.35%); these compounds were individually investigated for inhibitory activity by performing single-compound screening. Each of the top three most abundant compounds inhibited the tyrosinase-catalyzed oxidation of L-DOPA, as identified by UV-VIS spectroscopy and oxygen consumption assays. The inhibitory activity of the major compounds increased when pre-incubated with tyrosinase and without significant additional oxygen consumption, suggesting k cat -type inactivation is not involved. Interactions of the head and tail components of the major alkanals may disrupt the tertiary structure of the enzyme, presenting a potential inhibitory mechanism.

Design and evaluation of anacardic acid derivatives as anticavity agents.

On the basis of antibacterial anacardic acids, 6-pentadecenylsalicylic acids, isolated from the cashew apple, Anacardium occidentale L. (Anacardiaceae), a series of 6-alk(en)ylsalicylic acids were synthesized and tested for their antibacterial activity against Streptococcus mutans ATCC 25175. Among them, 6-(4',8'-dimethylnonyl)salicylic acid was found to exhibit the most potent antibacterial activity against this cariogenic bacterium with the minimum inhibition concentration (MIC) of 0.78 microg/ml.

Effects of cuminaldehyde on melanoma cells.

Cuminaldehyde (4-isopropylbenzaldehyde) suppressed melanin formation in cultured murine B16-F10 melanoma cells in a dose-dependent decrease up to 0.25 mm without affecting cell growth. Approximately 30% suppression in melanin production resulted when the cells were cultured with 0.25 mm of cuminaldehyde. This activity was not noticeable with cultured human A375 melanoma cells.

Antifungal activity of alkanols against Zygosaccharomyces bailii and their effects on fungal plasma membrane.

A series of aliphatic primary alkanols from C(6) to C(13) were tested for antifungal activity against a food spoilage fungus Zygosaccharomyces bailii using a broth dilution method and were compared for their effects against Saccharomyces cerevisiae and Z. rouxii. Decanol (C(10)) was found to be the most potent fungicide against Z. bailii at a minimum fungicidal concentration of 50 microg/ml (0.31 mM), whereas undecanol (C(11)) was found to be the most potent fungistatic at a minimum inhibitory concentration of 25 microg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. Octanol (C(8)) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The primary antifungal action of alkanols comes from their ability to disrupt the native membrane-associated function of integral proteins nonspecifically as nonionic surface-active agents (surfactants). The antifungal activity of decanol against Z. bailii was slightly enhanced in combination with anethole.

Effects of phenolic compounds isolated from Rabdosia japonica on B16-F10 melanoma cells.

Pedalitin isolated from the aerial part of Rabdosia japonica (Labiatae), exhibited cytotoxicity against the murine B16-F10 melanoma cell line with an IC(50) of 30 microm (9.5 microg/mL). As the cells were cultured with this flavone, melanin production was not suppressed, but rather enhanced. Quercetin isolated from the same source exhibited similar activities, but rutin showed neither activity.