咖啡和绿茶的抗氧化活性研究

茶和咖啡是全世界消费最多的饮料。该工作利用电子自旋共振(ESR)技术和ABTS〔2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)〕等方法,研究和比较了3种绿茶(龙井、碧螺春和毛峰)和3种咖啡的抗氧化活性,以及对ABTS 、DPPH、超氧阴离子、羟基自由基、单线态氧的清除作用和对脂质过氧化的抑制作用。利用高压液相色谱(HPLC)分析了绿茶中的抗氧化成分之一EGCG((-)-epigallocatechin gallate)和咖啡中的抗氧化成分之一绿原酸(chlorogenic acid5,CQA)的含量。结果发现,如果按每克剂量比较,对于清除ABTS 的能力而言,茶和咖啡水提取液对ABTS 清除能力各有千秋,咖啡水提取液的比龙井、毛峰水提取液的高,但与碧螺春无显著差异,咖啡B比咖啡A和C强;按每杯茶(3g/200mL)和咖啡(2.4g/200mL)对ABTS 的清除能力进行比较,咖啡的比龙井和毛峰的高但比碧螺春低,咖啡B比咖啡A和C强。对超氧阴离子、羟基和DPPH自由基及单线态氧的清除作用和对脂质过氧化的抑制作用的研究表明,如果按每克剂量比较,茶和咖啡水提取液没有明显区别;按每杯量进行比较时,茶水提取液比咖啡水提取液对自由基的清除能力稍强。HPLC结果表明,EGCG含量在毛峰中最高,5CQA含量在咖啡B中最高。该研究结果表明茶和咖啡中均含有丰富的抗氧化成分,并都具有较强的抗氧化活性和清除自由基能力。     

Microfloral contamination and hydrolytic enzyme differences between monsooned and non-monsooned coffees

AIMS: To analyse and compare, for the first time, fungal and bacterial populations and hydrolytic enzyme production in four monsooned and non-monsooned Arabica and Robusta coffee types. METHODS AND RESULTS: Overall, using serial dilution, the populations of bacteria (approximately 10(8) cfu g(-1)) were highest in monsooned coffees of both varieties. Fungal populations were lower (10(5) cfu g(-1)) and found predominantly on monsooned coffee beans. The major fungal species were Aspergillus terreus, A. restrictus and A. ochraceus. Of 19 semi-quantitative enzymes analysed, significantly higher concentrations of 2-naphthyl-butyrate, caprylate and 2-naphthyl-D-galactopyranosidase were present in Arabica monsooned coffee. CONCLUSIONS: The results suggest that speciality monsooned coffee has markedly different microbial and physiological characteristics from normally produced green coffees. SIGNIFICANCE AND IMPACT OF THE STUDY: Monsooned coffee may have a higher contamination with spoilage moulds, especially mycotoxigenic species.     

HEDONIC R-INDEX MEASUREMENT OF TEMPERATURE PREFERENCES FOR DRINKING BLACK COFFEE

R-index measures were obtained from judges who ranked black coffees for preferred drinking temperature. Preferred temperatures were above thermal pain and thermal damage thresholds. The least preferred temperature was below the oral thermal pain threshold. Similar results were obtained with a ranking for temperatures that judges felt were most likely to be served in a coffee shop. Observations of customers in coffee shops indicated that they began sipping coffee at similar high temperatures.     

Uptake of radiolabelled ochratoxin A from soil by coffee plants

[3H, 14C] Ochratoxin A, prepared biosynthetically, was applied in dilute NaHCO3 solution to the soil in which coffee plants had grown to four pairs of leaves. Three weeks later the compound, isolated from dilute NaHCO3 extract of leaves by immunoaffinity chromatography, was detected by scintillation counting as a 1-2 ppm component of leaf dry weight, greatly exceeding the trace (ppb) occurrence of ochratoxin A in some green coffees, which therefore might arise in the field directly from fungal activity in soil rather than from fungal infection of cherries or processed green coffee.     

The formation and reaction of methyl radicals produced by direct photolysis of aqueous solutions of N-acetyl substituted aliphatic amino acids at 77K.

Photolysis of amino acids, peptides and their derivatives leads to the formation of free radicals in these substances. The electron-spin-resonance spectra obtained directly after irradiation at 77 K are not very well resolved. They are recognizable as the superposition of the spectra of different types of photoproduced radicals. CH3 radicals are formed by U.V. irradiation if methyl groups are present in the molecule. These radicals are easily detectable because of their four line spectrum. In this paper the formation of methyl radicals and their reaction with undamaged molecules of N-acetyl-substituted amino acids in investigated. The number of CH3 radicals present after a 30 min U.V. irradiation is higher if preceding U.V. irradiations and heat treatments are performed. The overall concentration of radicals is reduced only partially during this heat-treatment, while the CH3 radicals decay completely. Other experiments show that the rate of and the yield of CH3 radicals by U.V. irradiation increase with the dose of a preceding gamma-irradiation. The results suggest that there are substances present which are responsible for the higher production rate of methyl radicals after a preceding treatment. It is assumed that radicals are precursors of the fast-formed CH3 radicals     

Ethiopian coffee—its significance to world coffee problems

Recent studies seem to confirm that Ethiopia should definitely be considered as the native home ofCoffea arabica L. Good natural conditions make this country the main exporter ot this type of coffee in Africa and potentially one of the most important producers in the world. There is such a great genetical variation among the “wild” coffees that they constitute the best sources ot germ plasm now available for programs of improvement of the species. Already some trees have been found resistant or immune to all the cultures of the main rust organism with which they were inoculated.     

Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.     

The storage of green coffee (Coffea arabica): decrease of viability and changes of potential aroma precursors

BACKGROUND AND AIMS: When green coffee is stored for a prolonged time the coffee quality decreases distinctively. Apart from well-known 'off-notes' that arise from undesired oxidations of lipids, a typical 'flattening' of the cup quality is detectable. In order to elucidate the biological causes for this phenomenon, differentially processed coffees (wet, dry, semi-dry processing), were stored under standard conditions for 2 years and analysed comprehensively. METHODS: Wet-processed coffee was stored either as parchment coffee, where the endocarp remained around the beans or as hulled beans. Viability of coffee seeds was estimated using the tetrazolium-test of seed viability. Changes in concentration of free amino acids and soluble carbohydrates were analysed by HPLC. KEY RESULTS: Whereas all other coffees lost viability within the first 6 months of storage, coffee beans stored within the parchment remained viable for >1 year. Glucose and fructose decreased slightly in the course of storage and glutamine content declined significantly. However, the changes observed in sugar and amino acid content were not correlated with the viability of the coffee beans. Consequently, neither typical metabolic reactions occurring within living cells nor characteristic post-mortem reactions could be responsible for the observed changes. As a result of post-mortem reactions in re-imbibed seeds, a characteristic bluish-green colour developed, putatively due to the oxidation of chlorogenic acids and subsequent reactions with primary amino compounds. This coloration might be an appropriate marker to substantiate if coffee seeds had been stored for an expanded time and putative quality losses were not relevant so far. CONCLUSIONS: It is suggested that loss of viability is relevant for the aroma flattening. As neither metabolic nor post-mortem reactions were responsible for the observed changes, it is concluded that Maillard reactions that occur during storage might be the cause of the decrease in potential aroma precursors.     

Coffee consumption induces GSTP in plasma and protects lymphocytes against (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide induced DNA-damage: results of controlled human intervention trials

A number of animal studies indicate that coffee protects against chemical induction of cancer; also human studies suggest that coffee consumption is inversely related with the incidence of different forms of cancer. The protective effects were attributed to induction of glutathione-S-transferases (GSTs) and aim of the present human study was to find out if coffee causes induction of GSTs and protects against DNA-damage caused by (+/-)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), the DNA-reactive metabolite of benzo(a)pyrene. Ten participants consumed 1L unfiltered coffee/d over 5 days. Before and after the intervention, saliva and blood were collected and the overall GST activity was measured with 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, GSTP and GSTA were determined in plasma with immunoassays. In blood, only weak (p=0.042) induction of GST (CDNB) was found. Furthermore, pronounced (three-fold) induction of GSTP was observed in blood, whereas GSTA was not altered. No correlations were seen between induction of GST (CDNB) and GSTP activities and the GSTP1 genotypes of the participants. Also clinical parameters (creatinine, alanine, aminotransferase, aspartate aminotransferase, alkaline phosphatase), which are markers for organ damage, were monitored. None of them was altered by coffee, but serum cholesterol levels were slightly (not significantly) enhanced. In a second trial (n=7), GSTP induction by unfiltered and paper filtered coffees, differing in cafestol and kahweol contents, were compared. The participants consumed 1L coffee/d over 3 days. Again significant (three-fold) induction of GSTP was observed. The effects seen with the two coffees were identical, indicating that the diterpenoid concentrations are not responsible for the effects. In a further trial (n=7), the effect of coffee (unfiltered, 1L/d, 5 days) on BPDE induced DNA-migration was studied in comet assays. A 45% reduction effect was observed. Our findings show that coffee induces GSTP in humans and indicate that consumption may lead to protection towards polycyclic aromatic hydrocarbons.     

Long-lived mutagenic radicals induced in mammalian cells by ionizing radiation are mainly localized to proteins

We have provided evidence that long-lived radicals, produced by ionizing radiation, are highly mutagenic and transforming in mammalian cells. Long-lived radicals are scavenged effectively by vitamin C or by epigallocatechin-3-O-gallate (EGCG). Long-lived radicals are not involved in lethality or in the induction of chromosome aberrations. We now report the results of experiments that define the relative amounts of long-lived radicals in DNA and proteins and identify the major protein radicals as sulfinyl radicals (R-CH2-S-O*). To make these assignments, yields of long-lived radicals in gamma-irradiated salmon sperm DNA and albumin were compared by ESR. ESR spectra of long-lived radicals produced in irradiated Syrian hamster embryo (SHE) cells were analyzed precisely and compared with ESR parameters obtained by density functional theory calculations. Long-lived radicals yields of 99.8% were produced in proteins. We also identified a new type of long-lived radical as H-added phenylalanine radicals. While our evidence does not rule out the possibility of important biological consequences of the low-level long-lived radicals created by radiation, it implicates radicals in proteins as playing a key role in genetic effects of ionizing radiation. We suggest that these novel radicals, wherever they reside, need to be considered in explanations of biological sequela of radiation.     

Free radicals in dicarboxylic acids: an e.s.r. study of gamma-irradiated single crystals of glutaric acid at 77 K

Electron spin resonance techniques were used to study the gamma-radiation-induced free radicals in single crystals of glutaric acid in the temperature range from 77 K to 300 K. Three different radicals are stabilized at 77 K. The decarboxylation radical is the dominant species and the other two radicals are assigned to the anion and to the substituted acetyl sigma-radical. When the temperature of the crystal is raised, these radicals disappear and the previously studied room temperature radicals appear. E.S.R.-data and the results from semi-empirical INDO-MO calculations were compared in order to elucidate the structures of the various radicals.     

Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.     

Inhibiting Effect of Browned Processed Foods on Trypsin

Inhibition of proteases was examined in browned foods including soy pastes, soy sauces, fish sauce, sauces, teas, coffees, and others in association with melanoidin, a Maillard product, which had been found to be a potent inhibitor of trypsin. Most of the foods were effective in restraining trypsin activity based on hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide, while being ineffective on chymotrypsin except for black tea and cola. Especially, teas were noted for their strong inhibitory effect on trypsin. The color intensity of the foods in terms of optical density at 400 nm appeared not to correlate with the extent of inhibition except for soy sauces.     

Inorganic radicals of relevance to biological systems

There is little doubt that the most important inorganic radicals involved in biological systems are those which are intermediates in the oxygen-water redox cycle, i.e. OH., O_2, and HO.2. Aspects of the structure and reactivities of these radicals are considered, together with methods of detection. In particular, the use of e.s.r. spectroscopy is outlined, including rapid-freeze and spin-trapping techniques. Attention is called to comparisons and contrasts between these radicals and corresponding sulphur-centered radicals, although these are not strictly "inorganic". The oxygen-centred radicals are usually generated in vivo by redox reactions, but they are also of importance in radiolytic processes because they are formed from water. Other radicals formed in this way whose structures and reactivities are considered include solvated electrons and hydrogen atoms.     

Radiation studies of aryl glycosides : Part VI. Radicals derived from Phenyl β-d-glucopyranosides

Radicals formed by γ-irradiation of o-, m-, and p-substituted-phenyl β-d-glucopyranosides have been studied in the polycrystalline state, in glassy methanol, and in frozen, aqueous solution. Substituted cyclohexadienyl radicals and radicals derived from the d-glucopyranosyl group are evident after irradiation of the compounds in the solid state and in frozen, aqueous solutions. Cyclohexadienyl radicals are the more stable during thermal annealing and are present in 2–3 times greater yield than the sugar radicals. p-Hydroxy- and methoxy-phenyl β-d-glucopyranosides yield phenoxy radicals, which can be transformed into substituted cyclohexadienyl radicals by thermal annealing. Hydrogen abstraction and inter- and intra-molecular hydrogen-transfer are the most likely processes leading to the radicals which have been identified.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).     

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO₂·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.     

Germination of coffee seeds and its significance for coffee quality

Besides genotypic characteristics, the crucial factor that determines coffee quality is the mode of post-harvest treatment, i.e., the wet and dry processing. Up to now, the resulting characteristic flavour differences between these differentially processed coffees were attributed exclusively to differences in starting material. However, as these quality differences are still evident, even when identical coffee samples were processed by the two methods in parallel, the differences must be created by metabolic processes in the coffee beans themselves. Based on expression studies of the germination-specific isocitrate lyase and the resumption of cell cycle activity, monitored by the abundance of beta-tubulin, we evidence that germination is initiated in coffee seeds during the course of standard coffee post-harvest treatments. The extent and nature of the germination processes depend on the processing method. The coherence of metabolic events, substantial differences in the chemical composition of the coffee beans, and the generation of specific coffee qualities establishes the basis for a quite novel approach in coffee research.