Structure-DPPH* scavenging activity relationships: parallel study of catechol and guaiacol acid derivatives

The scavenging behavior of a series of catechol and guaiacol acid derivatives toward DPPH(*) was examined having as a starting point the order of activity derived on the basis of theoretically calculated BDE values. The studied compounds were protocatechuic, homoprotocatechuic, dihydrocaffeic, and caffeic acids and also vanillic, homovanillic, dihydroferulic, and ferulic acids. Catechol and guaiacol were used as reference compounds. Observations from the parallel study were made with regard to structural features (number and position of hydroxyl groups and the side-carbon chain characteristics) that regulated the behavior of the compounds experimentally. The exceptional DPPH(*) scavenging behavior observed for homoprotocatechuic acid in ethanol and for caffeic acid in acetonitrile could not be supported by the respective BDE values. Ferulic was the most active among guaiacolic acids, whereas dihydroferulic exhibited the highest stoichiometry. Ionizable carboxylic groups seem to affect considerably the relative order of activity as was also evidenced using the ORAC assay. Questions raised about the validity of widely accepted views on criteria for SARs are discussed with regard to literature findings.     

Interaction of the Mixture of Phenolic Acids with Modified Kaolinite under Batch and Dynamic Conditions

Phenolic acids play an important role in the formation of soil profiles, however their cooperative sorption by mineral phases under environmentally relevant concentrations is poorly studied. In the present work the sorption of an equimolar mixture of phenolic acids by kaolinite modified with amorphous aluminum hydroxide has been studied under both batch and continuous-flow conditions. It has been found that the sorption of gallic and protocatechuic acids containing OH groups in the ortho position exceeds the sorption of p-hydroxybenzoic and methoxylated acids (vanillic, syringic, and ferulic) by an order of magnitude. The study of sorption under dynamic (continuous-flow) conditions has shown the competition of acids for binding sites, while the active centers of kaolinite-Al(OH)_x are being occupied. The sorbed gallic acid displaces the other acids, which pass into solution in the following order: p-hydroxybenzoic acid > vanillic acid > syringic acid ? ferulic acid > protocatechuic acid. The revealed regularities indicate potentially important role of ortho-substituted hydroxybenzoic acids in the formation of soil organic matter, while p-hydroxybenzoic, vanillic, and syringic acids can be more important for the composition of soil solutions and natural waters.     

Antioxidant properties of ferulic acid and its related compounds

Antioxidant activity of 24 ferulic acid related compounds together with 6 gallic acid related compounds was evaluated using several different physical systems as well as their radical scavenging activity. The radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) decreased in the order caffeic acid > sinapic acid > ferulic acid > ferulic acid esters > p-coumaric acid. In bulk methyl linoleate, test hydroxycinnamic acids and ferulic acid esters showed antioxidant activity in parallel with their radical scavenging activity. In an ethanol-buffer solution of linoleic acid, the activity of test compounds was not always associated with their radical scavenging activity. Ferulic acid was most effective among the tested phenolic acids. Esterification of ferulic acid resulted in increasing activity. The activity of alkyl ferulates was somewhat influenced by the chain length of alcohol moiety. When the inhibitory effects of alkyl ferulates against oxidation of liposome induced by AAPH were tested, hexyl, octyl, and 2-ethyl-1-hexyl ferulates were more active than the other alkyl ferulates. Furthermore, lauryl gallate is most effective among the tested alkyl gallates. These results indicated that not only the radical scavenging activity of antioxidants, but also their affinity with lipid substrates, might be important factors in their activity.     

Avenanthramides in oats (Avena sativa L.) and structure-antioxidant activity relationships

Eight avenanthramides, amides of anthranilic acid (1) and 5-hydroxyanthranilic acid (2), respectively, and the four cinnamic acids p-coumaric (p), caffeic (c), ferulic (f), and sinapic (s) acid, were synthesized for identification in oat extracts and for structure-antioxidant activity studies. Three compounds (2p, 2c, and 2f) were found in oat extracts. As assessed by the reactivity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH), all avenanthramides except 1p showed activity. Initially, the antioxidant activity of the avenanthramides decreased in a similar order as for the corresponding cinnamic acids, that is: sinapic > caffeic > ferulic > p-coumaric acid. The avenanthramides derived from 2 were usually slightly more active than those derived from 1. All avenanthramides inhibited azo-initiated peroxidation of linoleic acid. 1c and 1s were initially the most effective compounds. The relative order of antioxidant activities was slightly different for the DPPH and the linoleic acid assays run in methanol and chlorobenzene, respectively.     

Potential nitrite scavengers as inhibitors of the formation of N-nitrosamines in solution and tobacco matrix systems

The ability of 20 compounds, all but one tobacco constituents, to inhibit the formation of tobacco-specific N-nitrosamines (TSNA) was investigated in buffer and detergent solution and in tobacco midrib and lamina systems. In solution at pH 5.5, ascorbic acid and the phenolic acids caffeic and ferulic acid were the most potent inhibitors of the reaction between nornicotine and nitrite, with nearly complete inhibition at molar ratios test compound/nitrite > 1:1. Also, cysteine > dihydrocaffeic acid > protocatechuic acid approximately catechin acted as strong inhibitors with >90% inhibition at a ratio of 3:1. Lower inhibitions were observed with chlorogenic acid > p-coumaric acid > sclareol > serine. Rutin showed an inhibition of 34% at a ratio of 0.1:1. Sclareol, alanine, proline, and serine did not significantly affect the N-nitrosonornicotine (NNN) formation. alpha-Tocopherol and glutathione enhanced NNN formation at pH 5.5 but were inhibitors at pH 3. Cinnamic acid, vanillic acid, eugenol, and esculin enhanced NNN formation. Increased NNN formation was also observed for dihydrocaffeic acid, chlorogenic acid, protocatechuic acid, and catechin at a less-than-equimolar ratio of test compound to nitrite. The tobacco matrix experiments were performed with air-cured, ground tobacco midrib and lamina. Caffeic acid, ferulic acid, dihydrocaffeic acid and catechin were potent inhibitors of the formation of TSNA in the midrib as well as in the lamina. Also protocatechuic acid, glutathione, ascorbic acid, p-coumaric acid, chlorogenic acid and cysteine were inhibitors, while alpha-tocopherol and rutin inhibited the reaction in the midrib but not in the lamina. Cinnamic acid, vanillic acid, eugenol, alanine, proline and serine showed small effects only. The molar ratio of secondary alkaloid(s)/nitrite in the test systems were 0.1:1 (solution), approximately 0.25:1 (midrib), and approximately 1:1 (lamina) and is most likely the major contributor to the observed order of inhibition potency (solution > midrib > lamina) of the test compounds. The vicinal phenolic hydroxyl groups of polyphenols and the simultaneous presence of a phenol group and an olefinic bond in hydroxycinnamic acids were the most characteristic structural elements of the potent inhibitors.     

Effects of phenolic acids on human phenolsulfotransferases in relation to their antioxidant activity

Sulfate conjugation by phenolsulfotransferase (PST) enzyme is an important process in the detoxification of xenobiotics and endogenous compounds. There are two forms of PST that are specific for the sulfation of small phenols (PST-P) and monoamines (PST-M). Phenoilc acids have been reported to have important biological and pharmacological properties and may have benefits to human health. In the present study, human platelets were used as a model to investigate the influence of 13 phenolic acids on human PST activity and to evaluate the relationship to their antioxidant activity. The results showed that chlorogenic acid, syringic acid, protocatechuic acid, vanillic acid, sinapic acid, and caffeic acid significantly (p < 0.05) inhibited the activities of both forms of PST by 21-30% at a concentration of 6.7 microM. The activity of PST-P was enhanced (p < 0.05) by p-hydroxybenzoic acid, gallic acid, gentisic acid, o-coumaric acid, p-coumaric acid, and m-coumaric acid at a concentration of 6.7 microM, whereas the activity of PST-M was enhanced by gentisic acid, gallic acid, p-hydroxybenzoic acid, and ferulic acid. The phenolic acids exhibited antioxidant activity as determined by the oxygen radical absorbance capacity (ORAC) assay and Trolox equivalent antioxidant capacity (TEAC) assay, especially gallic acid, p-hydroxybenzoic acid, gentisic acid, and coumaric acid, which had strong activity. The overall effect of phenolic acids tested on the activity of PST-P and PST-M was well correlated to their antioxidant activity of ORAC value (r = 0.71, p < 0.01; and r = 0.66, p < 0.01). These observations suggest that antioxidant phenolic acids might alter sulfate conjugation.     

Synergistic effects of thiols and amines on antiradical efficiency of protocatechuic acid

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid and its structural analogues (methyl protocatechuate, 3',4'-dihydroxyacetophenone, 3,4-dihydroxybenzaldehyde, and 3,4-dihydroxybenzonitrile) were examined in aprotic and protic solvents. In aprotic acetonitrile, all test compounds scavenged two radicals. In protic methanol, however, these compounds rapidly scavenged five radicals except for protocatechuic acid, which consumed only two radicals. The result indicated that higher radical scavenging activity in methanol than in acetonitrile was due to a nucleophilic addition of the methanol molecule on the oxidized quinones, which led to a regeneration of catechol structures. To investigate the importance of the nucleophilic addition on the quinones for the high radical scavenging activity, DPPH radical scavenging activity of protocatechuic acid and its analogues was examined in the presence of a variety of nucleophiles. The addition of a strong nucleophile such as a cysteine derivative significantly increased the radical scavenging equivalence. Furthermore, thiol adducts at C-2 and C-2,5 of protocatechuic acid and its analogues were isolated from the reaction mixtures. These results strongly suggest that the quinone of protocatechuic acid and its analogues undergo a nucleophilic attack at C-2 to yield 2-substituted-3,4-diols. Then, a regenerated catechol moiety of adducts scavenge two additional radicals by reoxidation into quinones, which undergo the second nucleophilic attack at the C-5. This mechanism demonstrates a possibility of synergistic effects of various nucleophiles on the radical scavenging ability of plant polyphenols containing a 3,4-dihydroxy substructure like protocatechuic acid and its analogues.     

Comparison of antioxidant activity, anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date (Phoenix dactylifera L.) varieties grown in Oman

Fresh and sun-dried dates of three native varieties from Oman, namely, Fard, Khasab, and Khalas, were examined for their antioxidant activity and total contents of anthocyanins, carotenoids, and phenolics, as well as free and bound phenolic acids. All results are expressed as mean value +/- standard deviation (n = 3) on a fresh weight basis. Fresh date varieties were found to be a good source of antioxidants (11687-20604 micromol of Trolox equiv/g), total contents of anthocyanins (0.24-1.52 mg of cyanidin 3-glucoside equiv/100 g), carotenoids (1.31-3.03 mg/100 g), phenolics (134-280 mg of ferulic acid equiv/100 g), free phenolic acids (2.61-12.27 mg/100 g), and bound phenolic acids (6.84-30.25 mg/100 g). A significant (p < 0.05) amount of antioxidants and carotenoids was lost after sun-drying of dates, whereas the total content of phenolics and free and bound phenolic acids increased significantly (p < 0.05). Anthocyanins were detected only in fresh dates. Date varieties had different levels and patterns of phenolic acids. Four free phenolic acids (protocatechuic acid, vanillic acid, syringic acid, and ferulic acid) and nine bound phenolic acids (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, and o-coumaric acid) were tentatively identified. Of the date varieties studied, Khalas, which is considered to be premium quality, had higher antioxidant activity, total carotenoids, and bound phenolic acids than other varieties. These results suggest that all date varieties serve as a good source of natural antioxidants and could potentially be considered as a functional food or functional food ingredient, although some of their antioxidant constituents are lost during sun-drying.     

Solvent-free lipase-catalyzed preparation of long-chain alkyl phenylpropanoates and phenylpropyl alkanoates

An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenylpropenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such as oleyl p-coumarate and oleyl ferulate. The various alkyl cinnamates were formed in high to moderate yield by lipase-catalyzed esterification of cinnamic acid and its analogues with fatty alcohols in vacuo at moderate temperatures in the absence of drying agents and solvents. Immobilized Candida antarctica lipase B was the most effective biocatalyst for the various esterification reactions. The relative esterification activities were of the following order: dihydrocinnamic > cinnamic > 3-methoxycinnamic > dihydrocaffeic approximately 3-hydroxycinnamic > 4-methoxycinnamic > 2-methoxycinnamic > 4-hydroxycinnamic > ferulic approximately 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With respect to the position of the substituents at the phenyl moiety, the esterification activity increased in the order meta > para > ortho. Rhizomucor miehei lipase demonstrated moderate esterification activity. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate, were also obtained in high yield by esterification of fatty acids with the corresponding 3-phenylpropan-1-ols.     

Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748(T)

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.     

Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: structure-activity relationship.

The structure-activity relationships of synthetic caffeic acid amide and ester analogues as potential antioxidants and free radical scavengers have been investigated. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH.) scavenging activity of the test compounds was N-trans-caffeoyl-L-cysteine methyl ester (5) > N-trans-caffeoyldopamine (4) > N-trans-caffeoyltyramine (3) > N-trans-caffeoyl-beta-phenethylamine (2) > Trolox C (8) > caffeic acid phenethyl ester (1) > caffeic acid (6) > ferulic acid (7). This established that the radical scavenging activity of the compounds increased with increasing numbers of hydroxyl groups or catechol moieties and also with the presence of other hydrogen-donating groups (-NH, -SH). The antioxidative activity of the compounds was also investigated in an emulsified linoleic acid oxidation system accelerated by 2,2'-azobis(2-amidinopropane) dihydrochloride. The order was 1 > 2 > 4 > 3 > or = 5 > 6 > 8 > 7. Therefore, in the emulsion system, the antioxidative activity of the test compounds depends not only on the hydroxyl groups or catechol rings but also on the partition coefficient (log P) or hydrophobicity of the compounds. This supports the concept that hydrophobic antioxidants tend to exhibit better antioxidative activity in an emulsion system.     

Evaluation of antioxidant capacity of cereal brans

Several oat brans (crunchy oat bran, oat bran alone, and oat breakfast cereal) and wheat brans (wheat bran alone, wheat bran powder, wheat bran with malt flavor, bran breakfast cereal, tablet of bran, and tablet of bran with cellulose) used as dietary fiber supplements by consumers were evaluated as alternative antioxidant sources (i) in the normal human consumer, preventing disease and promoting health, and (ii) in food processing, preserving oxidative alterations. Products containing wheat bran exhibited higher peroxyl radical scavenging effectiveness than those with oat bran. Wheat bran powder was the best hydroxyl radical (OH*) scavenger. In terms of hydrogen peroxide (H2O2) scavenging, wheat bran alone was the most effective, while crunchy oat bran, oat bran alone, and oat breakfast cereal did not scavenge H2O2. The shelf life of fats (obtained by the Rancimat method for butter) increased most in the presence of crunchy oat bran. When the antioxidant activity during 28 days of storage was measured by the linoleic acid assay, all of the oat and wheat bran samples analyzed showed very good antioxidant activities. The Trolox equivalent antioxidant capacity (TEAC) assay was used to provide a ranking order of antioxidant activity. The wheat bran results for TEAC (6 min), in decreasing order, were wheat bran powder > wheat bran with malt flavor > or = wheat bran alone > or = bran breakfast cereal > tablet of bran > tablet of bran with cellulose. The products made with oat bran showed lower TEAC values. In general, avenanthramide showed a higher antioxidant level than each of the following typical cereal components: ferulic acid, gentisic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, vanillic acid, vanillin, and phytic acid.     

Phenolic profile of Asturian (Spain) natural cider

The polyphenolic composition of natural ciders from the Asturian community (Spain), during 2 consecutive years, was analyzed by RP-HPLC and the photodiode-array detection system, without previous extraction (direct injection). A total of 16 phenolic compounds (catechol, tyrosol, protocatechuic acid, hydrocaffeic acid, chlorogenic acid, hydrocoumaric acid, ferulic acid, (-)-epicatechin, (+)-catechin, procyanidins B2 and B5, phloretin-2'-xyloglucoside, phloridzin, hyperin, avicularin, and quercitrin) were identified and quantified. A fourth quercetin derivative, one dihydrochalcone-related compound, two unknown procyanidins, three hydroxycinnamic derivatives, and two unknown compounds were also found. Among the low-molecular-mass polyphenols analyzed, hydrocaffeic acid was the most abundant compound, representing more than 80% of the total polyphenolic acids. Procyanidins were the most important family among the flavonoid compounds. Discriminant analysis was allowed to correctly classify more than 93% of the ciders, according to the harvest year; the most discriminant variables were an unknown procyanidin and quercitrin.     

Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria

The in vitro antimicrobial activity of commercial coffee extracts and chemical compounds was investigated on nine strains of enterobacteria. The antimicrobial activity investigated by the disc diffusion method was observed in both the extracts and tested chemical compounds. Even though pH, color, and the contents of trigonelline, caffeine, and chlorogenic acids differed significantly among the coffee extracts, no significant differences were observed in their antimicrobial activity. Caffeic acid and trigonelline showed similar inhibitory effect against the growth of the microorganisms. Caffeine, chlorogenic acid, and protocatechuic acid showed particularly strong effect against Serratia marcescens and Enterobacter cloacae. The IC(50) and IC(90) for the compounds determined by the microtiter plate method indicated that trigonelline, caffeine, and protocatechuic acids are potential natural antimicrobial agents against Salmonella enterica. The concentrations of caffeine found in coffee extracts are enough to warrant 50% of the antimicrobial effect against S. enterica, which is relevant to human safety.     

Antioxidant activities of phenolic acids on ultraviolet radiation-induced erythrocyte and low density lipoprotein oxidation

The exposure of mammalian cells to UV light induces various deleterious responses. Some of the major harmful effects are DNA damage, cell membrane peroxidation, systemic immune suppression, and aging acceleration. Reactive oxygen species and free radicals are believed to be largely responsible for some of the deleterious effects of UV upon cells. Typical administration of antioxidants has recently proved to represent a successful strategy for protecting the cells against UV-mediated oxidative damage. The objective of this study was to investigate the inhibitory effect of phenolic acids (caffeic acid, ferulic acid, gallic acid, and protocatechuic acid) on oxidative damage in human erythrocytes and low-density lipoprotein (LDL) induced by UVB radiation. The results revealed that the thiobarbituric acids reactive substances induced by UVB were decreased from 2.78 to 0.12-0.89 nmol MDA/mg protein in erythrocyte ghost and from 0.72 to 0.14-0.43 nmol MDA/mg protein in LDL by the addition of phenolic acids (100 muM). Caffeic acid, ferulic acid, and gallic acid exhibited over 85 and 60% inhibitory effect toward UVB-induced oxidation in erythrocytes and LDL, respectively. Phenolic acids, especially gallic acid, could maintain the normal glutathione levels and glutathione peroxidase activity in hemolysate from erythrocytes that were exposed to UVB radiation in comparison with untreated control. The results indicate that the antioxidant activities of caffeic acid and ferulic acid play a potential role in protection against UVB oxidative damage to human erythrocytes and LDL.     

Chemical studies on soil humic acids

Seventeen samples of soil humic acids, two fractions of soil fulvic acid sample, and several related compounds such as lignin, tannin, flavonoid and artificial humic substances were decomposed in conc. KOH solution at 180°C. Succinic acid, glutaric acid, phloroglucin, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, 3,4-dihydroxy-5-methoxybenzoic acid, and gallic acid were detected in the degradation products of humic acids. The amounts of these degradation products were discussed in relation to the degree of humification or the sources of the humic acid samples. Succinic acid also resulted from glucose, polymaleic acid, and the humic acid and humin prepared from glucose, but glutaric acid resulted only from glucose humic acid and glucose humin but not from glucose and polymaleic acid. Succinic acid and glutaric acid were supposed to result from the same structural portions in humic acids because of the very significant positive linear correlation between their amounts. p-Hydroxybenzoic, vanillic, protocatechuic, and 3,4-dihydroxy-S-methoxybenzoic acids were presumed to result mainly from lignin structure in humic acids. Soil humic acids yielded small amounts of gallic acid although the yields by hydrolysable tannins were in large amounts. The yields of above-mentioned degradation products from humic acids decreased with increasing degree of humification. Phloroglucin resulting from ftavonoids including condensed tannins were also found in the degradation products of humic substances. Its yield showed no linear correlation with RF value of humic acid, and is presumed to be rather related to the vegetation at the sites of soil sampling.     

Phenolic composition of kiwifruit juice

Phenolic compounds in kiwifruit pulp were separated and characterized by reversed-phase HPLC, and the effect of juice processing on the phenolic composition was studied. Fractionation of phenolic compounds was achieved through selective elution from C-18 cartridges prior to preconcentration and subsequent separation by HPLC. Strongly acidic compounds were identified as derivatives of coumaric and caffeic acids, including chlorogenic acid, protocatechuic acid, and a derivative of 3,4-dihydroxybenzoic acid. The weakly acidic fraction contained epicatechin, catechin, and procyanidins (B3, B2, or B4 and oligomers). Flavonols were present as the glycosides of quercetin (glucoside, rhamnoside, and rutinoside) and kaempferol (rhamnoside and rutinoside). Phenolic compounds were present, at levels of <1-7 mg/L, in clarified juice. The concentration of phenolics was highest after high-temperature short-time treatment (HTST) of juice. Hydrolysis of hydroxycinnamic acids occurred after enzyme addition and HTST treatment. The flavonol glycoside composition is the best identifier of kiwifruit juice.     

Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection

A high-performance liquid chromatographic (HPLC) method with diode-array detection (DAD) was used to identify and quantify free and total phenolic acids (m-hydroxybenzoic acid, p-hydroxybenzoic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, chlorogenic acid, and ellagic acid) in plant foods. Free phenolic acids were extracted with a mixture of methanol and 10% acetic acid. Bound phenolic acids were liberated using first alkaline and then acid hydrolysis followed by extraction with diethyl ether/ethyl acetate (1:1). All fractions were quantified separately by HPLC. After HPLC quantification, results of alkali and acid hydrolysates were calculated to represent total phenolic acids. Ellagic acid was quantified separately after long (20 h) acid hydrolysis. The methods developed were effective for the determination of phenolic acids in plant foods. DAD response was linear for all phenolic acids within the ranges evaluated, with correlation coefficients exceeding 0.999. Coefficients of variation for 4-8 sample replicates were consistently below 10%. Recovery tests of phenolic acids were performed for every hydrolysis condition using several samples. Recoveries were generally good (mean >90%) with the exceptions of gallic acid and, in some cases, caffeic acid samples.     

Polyphenol oxidase from yacon roots (Smallanthus sonchifolius)

Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.