Effects of Microbial Utilization of Phenolic Acids and their Phenolic Acid Breakdown Products on Allelopathic Interactions

Reversible sorption of phenolic acids by soils may provide some protection to phenolic acids from microbial degradation. In the absence of microbes, reversible sorption 35 days after addition of 0.5–3 mol/g of ferulic acid or p-coumaric acid was 8–14% in Cecil A_p horizon and 31–38% in Cecil B_t, horizon soil materials. The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged from 0.12 to 0.25 in A_p and 0.65 to 0.85 in B_t horizon soil materials. When microbes were introduced, the r/s ratio for both the A_p and B_t horizon soil materials increased over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much more rapid in A_p than in B_t horizon soil materials. Reversible sorption, however, provided protection of phenolic acids from microbial utilization for only very short periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic and benzoic acids by soil microbes indicated that these processes can substantially influence the magnitude and duration of the phytoxicity of individual phenolic acids.     

Benefits of Citrate Over EDTA for Extracting Phenolic Acids from Soils and Plant Debris

The effectiveness of various citrate extractions to recover ferulic acid, a phenolic acid, from Cecil A_p and B_t soil and plant surface debris (crimson clover, rye, subterranean clover, and wheat) was compared with that of EDTA extractions. Citrate extractions were equivalent to or better than EDTA in recovering phenolic acids from soil and plant debris. Citrate, unlike EDTA, did not interfere with the Folin & Ciocalteu's phenol reagent when determining total phenolic acid content in soil and plant debris extracts. Care, however, must be taken when using Folin & Ciocalteu's phenol reagent to estimate total phenolic acid content, particularly in the presence of soil organic matter. Citrate was also more effective in extracting phenolic acids from A-horizon soils. Thus, citrate extractions overcome some of the major limitations observed for EDTA extractions.     

Mechanism by which noninhibitory concentrations of glucose increase inhibitory activity ofp-coumaric acid on morning-glory seedling biomass accumulation

Noninhibitory levels of glucose-C [ 72 µg carbon (C)/g soil] increased the inhibitory activity ofp-coumaric acid on morning-glory seedling biomass accumulation in Cecil B_t-horizon soil. The amount ofp-coumaric acid required for a given level of inhibition of shoot and seedling biomass accumulation decreased as the concentration of glucose increased. Soil extractions with neutral EDTA (0.25 M, pH 7) after addition of combinations ofp-coumaric acid and glucose (concentrations ranging from 0 to 1.25 µmol/g soil) to the soil showed that utilization ofp-coumaric acid by microbes decreased linearly as the concentration of glucose increased. The increased inhibitory activity of a given concentration ofp-coumaric acid in the presence of glucose was not due to a reduction in soil sorption ofp-coumaric acid or effects of nitrogen-limited microbial growth. Noninhibitory levels of phenylalanine andp-hydroxybenzoic acid slowed the utilization ofp-coumaric acid by microbes in a similar manner as glucose. The presence of methionine, however, did not affect the rate ofp-coumaric acid utilization by microbes. These observations suggest that differential utilization of individual molecules in organic mixtures by soil microbes can modify, and in this case increase, the effectiveness of a given concentration of an inhibitor such asp-coumaric acid on the inhibition of seedling growth such as morning-glory.The use of trade names in this publication does not imply endorsement by the United States government or the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Antioxidant and Biological Activities of Novel Structured Monoacylglycerol Derivatives with Phenolic Acids

Novel structured monoacylglycerol (MAG)-based phenolic lipids are synthesized from11-bromoundecanoic acid, phenolic acids, and solketal. Selected phenolic acids namely 4-hydroxy benzoic, vanillic, syringic, cinnamic, p-coumaric, sinapic, 4-fluorocinnamic, 4-hydroxyphenyl acetic acid, 3-(4-hydroxyphenyl) propanoic and dihydrocaffeic acids are employed for the synthesis of ten novel MAG-based phenolic lipids. The synthesized phenolic lipids are characterized by FT-IR, NMR, and mass spectra analysis. All the compounds are evaluated for antioxidant, antimicrobial, and cytotoxic activities. MAG derivative 8g of sinapic acid exhibits excellent antioxidant activity in both DPPH assay and inhibition of lipid oxidation assay. MAG derivative 8f bearing p-coumaric acid shows good antimicrobial activity against both Gram-positive and Gram-negative bacterial strains with a minimum inhibitory concentration (MIC) value of 6.25 µm mL⁻¹. All the synthesized compounds are found to exhibit cytotoxicity against B16, DU145, and CHO cell lines, while sinapic and p-coumaric acid derivatives exhibit better activities compared to other derivatives.     

Phenolic acid content of soils from wheat-no till,wheat-conventional till,and fallow-conventional till soybean cropping systems

Soil core (0–2.5 and/or 0–10 cm) samples were taken from wheat no till, wheat-conventional till, and fallow-conventional till soybean cropping systems from July to October of 1989 and extracted with water in an autoclave. The soil extracts were analyzed for seven common phenolic acids (p-coumaric, vanillic,p-hydroxybenzoic, syringic, caffeic, ferulic, and sinapic; in order of importance) by high-performance liquid chromatography. The highest concentration observed was 4 g/g soil forp-coumaric acid. Folin & Ciocalteu's phenol reagent was used to determine total phenolic acid content. Total phenolic acid content of 0- to 2.5-cm core samples was approximately 34% higher than that of the 0- to 10-cm core samples. Phenolic acid content of 0- to 2.5-cm core samples from wheat-no till systems was significantly higher than those from all other cropping systems. Individual phenolic acids and total phenolic acid content of soils were highly correlated. The last two observations were confirmed by principal component analysis. The concentrations were confirmed by principal component analysis, tions of individual phenolic acids extracted from soil samples were related to soil pH, water content of soil samples, total soil carbon, and total soil nitrogen. Indirect evidence suggested that phenolic acids recovered by the water-autoclave procedure used came primarily from bound forms in the soil samples.The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Inhibition of growth ofPhytophthora parasitica var.nicotianae by aromatic acids and coumarins in a laboratory bioassay

Hydroxy-, dihydroxy-, trihydroxy-, methoxy-, dimethoxy-, hydroxy-+methoxy-, amino-, chloro-, and nitro-substituted benzoic, phenylacetic, phenylpropanoic, and phenylpropenoic (cinnamic) acids were evaluated for activity against the growth ofPhytophthora parasitica var.Nicotianae, Races 0 and 1, in a laboratory bioassay. Several substituted coumarins were also tested. In general, for Race 0, the phenylpropenoic acids were more active (on a millimolar basis), than the corresponding benzoic, phenylacetic, or phenylpropionic acids (9 of 14 series). Among the most active acids wereo-hydroxycinnamic and the chloro- and methoxycinnamic acids. The activities of unsubstituted benzoic and phenylpropionic acids were comparable to the most active compounds tested. Monohydroxyaromatic acids were more active than most dihydroxy acids of the same chain length. Dihydro-3,4-dihydroxycinnamic acid was slightly more active than the corresponding cinnamic acid, while the reverse was true for the mono-p-hydroxycinnamic acid versusp-hydroxyphenylpropionic acid. Coumarin was more active than its hydroxy, methyl, hydroxymethyl, or methoxy derivatives. In general, Race 1 was even more significantly affected by the aromatic acids. Glycosylated coumarins were inactive in the bioassay, compared to their aglycones.     

Effect of lipid unsaturation on the antioxidative activity of some phenolic acids

Antioxidative properties ofp-hydroxybenzoic, vanillic, syringic, 3,4-dihydroxybenzoic,p-coumaric, ferulic, sinapic and caffeic acids were studied in the concentration range 0.02–0.20 wt% during autoxidation at 100°C of lard and sunflower oil methyl esters (MEL and MESO, respectively). In both lipid systems, the derivatives of benzoic acid had weaker inhibiting properties than did the corresponding analogues of cinnamic acid. The effectiveness and strength of the antioxidative action were considerably lower in the lipid system MESO, which was rich in linoleic acid and was more easily oxidized. Thep-hydroxybenzoic, vanillic, syringic andp-coumaric acids in this system exercised no inhibiting effect. We established that the molecules of the investigated phenolic acids initiated the chain radical process of autoxidation, and the formed antioxidant radicals propagated the chains as a result of the reaction with the lipid substrate. These reactions proceeded at a higher rate in MESO than in MEL.     

Allelopathy in agroecosystems: Wheat phytotoxicity and its possible roles in crop rotation

The germination rates of cotton and wheat seeds were significantly affected by various extracts of wheat mulch and soils collected from the wheat field. This toxicity was even more pronounced against seedling growth. Five allelochemics: ferulic,p-coumaric,p-OH benzoic, syringic, and vanillic acids, were identified from the wheat mulch and its associated soil. Quantitatively, ferulic acid was found at higher concentrations thanp-coumaric acid in the soil. Various concentrations of ferulic andp-coumaric acids were toxic to the growth of radish in a bioassay. The functional aspects of allelochemic transfer from decaying residue to soil and the subsequent microbial degradation within agroecosystems are discussed, particularly as they relate to wheat crop rotation, with wheat and cotton, in Pakistan.     

Relationships between Phenolic Acid Concentrations,Transpiration, Water Utilization,Leaf Area Expansion,and Uptake of Phenolic Acids: Nutrient Culture Studies

Phenolic acid treatments of cucumber seedlings (Cucumis sativus cv “Early Green Cluster”) inhibited transpiration, water utilization, leaf area, and absolute and relative rates of leaf expansion. The cinnamic acids, ferulic and p-coumaric acids, were two to five times more inhibitory than the benzoic acids, p-hydroxybenzoic acid and vanillic acid. When phenolic acid concentrations were maintained at inhibitory concentrations through multiple successive treatments, percent inhibition of water utilization remained relatively constant for a given concentration and phenolic acid, percent inhibition of leaf area initially increased and then leveled off to a constant percent, and percent inhibition of transpiration and rates of leaf area expansion declined over time. Subsequently, p-coumaric acid was chosen as the model compound for further study. When p-coumaric acid was inhibitory, percent inhibition of transpiration, water utilization, and rates of leaf area expansion of actively growing leaves rapidly declined (i.e., was lost) as p-coumaric acid concentrations surrounding roots decreased. Absolute and relative rates of leaf expansion, for example, declined approximately 12 and 14%, respectively, for every 0.1 mM decline in p-coumaric acid concentration. Uptake of p-coumaric acid by cucumber seedling roots was continuous over the 24- or 36-hr periods monitored, but was not consistently related to the initial p-coumaric acid treatment concentrations. However, declining p-coumaric acid concentrations monitored at 6- or 12-hr intervals over the 24- or 36-hr periods continued to be highly correlated to the initial p-coumaric acid treatment concentrations. A 25% depletion by 13-d-old cucumber seedlings took 8.5, 12, 19.5, 25, and 29.5 hr for 0.125-, 0.25-, 0.5-, 0.75-, and 1-mM treatments, respectively. Uptake during periods when phenolic acid concentrations and root uptake (depletion from solution) were related appeared to represent periods dominated by apoplastic movement into the intercellular spaces of roots. Uptake during periods without this relationship likely represented periods dominated by symplastic movement. The ability of cucumber seedlings to modify active phenolic acid concentrations surrounding their roots suggests that cucumber seedling can directly influence the magnitude of primary and secondary effects of phenolic acids through feedback regulation.     

Effects of mixtures of phenolic acids on phosphorus uptake by cucumber seedlings

To determine how individual phenolic acids in a mixture might affect phosphorus (P) uptake, 15-day-old cucumber seedlings grown in solution culture were treated with ferulic, vanillic,p-coumaric, or equimolar mixtures of these phenolic acids. Phenolic acid and P uptake were determined by solution depletion. The joint action of the mixtures of these phenolic acids on P uptake was primarily additive. Thus, as the number of phenolic acids increased in the mixture, the concentrations of the individual phenolic acids in the mixture required to bring about a given response declined. Seedling uptake of individual phenolic acids from solution mixtures of phenolic acids was reduced when compared to the uptake of phenolic acids from single phenolic acid solutions. The magnitude of the reduction varied with phenolic acid and concentration. The dose required for 50% inhibition of P uptake was approximately two to three times higher for vanillic acid (6.73 mM) than for ferulic (2.27 mM) andp-coumaric acids (3.00 mM) when dose was based on the initial treatment concentrations. The dose required for 50% inhibition of P uptake was not significantly different for the three phenolic acids (42 ± 5 mol/g root fresh weight) when dose was based on phenolic acid uptake. Potential reasons for these differences are discussed.Paper No. 12527 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned. This research was partially supported by the US-Spain Joint Committee for Scientific and Technological Cooperation project CCA-8309/166.     

Allelopathic activity in wheat-conventional and wheat-no-till soils: Development of soil extract bioassays

The primary objective of this research was to determine if soil extracts could be used directly in bioassays for the detection of allelopathic activity. Here we describe: (1) a way to estimate levels of allelopathic compounds in soil; (2) how pH, solute potential, and/or ion content of extracts may modify the action of allelopathic compounds on germination and radicle and hypocotyl length of crimson clover (Trifolium incarnatum L.) and ivyleaved morning glory (Ipomoea hederacea L. Jacquin.); and (3) how biological activity of soil extracts may be determined. A water-autoclave extraction procedure was chosen over the immediate-water and 5-hr EDTA extraction procedures, because the autoclave procedure was effective in extracting solution and reversibly bound ferulic acid as well as phenolic acids from wheat debris. The resulting soil extracts were used directly in germination bioassays. A mixture of phenolic acids similar to that obtained from wheat-no-till soils did not affect germination of clover or morning glory and radicle and hypocotyl length of morning glory. The mixture did, however, reduce radicle and hypocotyl length of clover. Individual phenolic acids also did not inhibit germination, but did reduce radicle and hypocotyl length of both species. 6-MBOA (6-methoxy-2,3-benzoxazolinone), a conversion product of 2-o-glucosyl-7-methoxy-1,4-benzoxazin-3-one, a hydroxamic acid in living wheat plants, inhibited germination and radicle and hypocotyl length of clover and morning glory. 6-MBOA, however, was not detected in wheat debris, stubble, or soil extracts. Total phenolic acids (FC) in extracts were determined with Folin and Ciocalteu's phenol reagent. Levels of FC in wheat-conventionaltill soil extracts were not related to germination or radicle and hypocotyl length of either species. Levels of FC in wheat-no-till soil extracts were also not related to germination of clover or morning glory, but were inversely related to radicle and hypocotyl length of clover and morning glory. FC values, solute potential, and acidity of wheat-no-till soil extracts appeared to be independent (additive) in action on clover radicle and hypocotyl length. Radicle and hypocotyl length of clover was inversely related to increasing FC and solute potential and directly related to decreasing acidity. Biological activity of extracts was determined best from slopes of radicle and hypocotyl length obtained from bioassays of extract dilutions. Thus, data derived from the water-autoclave extraction procedure, FC analysis, and slope analysis for extract activity in conjunction with data on extract pH and solute potential can be used to estimate allelopathic activity of wheat-no-till soilsThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, or criticism of similar ones not mentioned.     

Allelopathic effects ofChrysanthemum morifolium on germination and growth of several herbaceous plants

Aqueous extracts obtained from young green tops ofChrysanthemum morifolium inhibited the germination of six flowering plants, including chrysanthemum itself, provided for experiments. The same phenomenon was also clearly observed when powder made from young green tops and old leaves of chrysanthemum was used. Moreover, the growth of seedlings planted again in garden soil which was once used for the culture of chrysanthemum was greatly interrupted. Chrysanthemum cultured in used garden soil showed far less dry weight than that cultured in fresh garden soil. The weight of chrysanthemum cultured using its root exudates was also less than that cultured with water leachate of fresh garden soil, and therefore these results may be considered to indicate allelopathic effects. In order to find the allelochemicals related to this phenomenon, benzoic acid and phenolic acids such as salicylic,p-hydroxybenzoic, vanillic, gentisic, protocatechuic, syringic, gallic, ferulic, and caffeic acids were identified by gas chromatography.     

Interactions oftrans-cinnamic acid,its related phenolic allelochemicals,and abscisic acid in seedling growth and seed germination of lettuce

Phenolic compounds have been identified as the most common allelochemicals produced by higher plants. Inhibitions of cinnamic acid, its related phenolic derivatives, and abscisic acid (ABA) on seedling growth and seed germination of lettuce were studied.trans-Cinnamic acid, ando-,m-, andp-coumaric acids inhibited the growth of etiolated seedlings of lettuce at concentrations higher than 10^–4 M and seed germination above 10^–3 M. Coumarin inhibited seedling growth and seed germination at 10^–5 M or above. Chlorogenic acid inhibited seedling growth above 10^–4 M, but did not inhibit seed germination at 10^–5–5×10^–3 M. Low concentrations (below 10^–3 M) of caffeic and ferulic acids promoted the elongation of hypocotyls, but higher concentrations (over 10^–3 M) inhibited seedling growth and seed germination. These phenolic compounds and abscisic acid had additive inhibitory effects both on seedling growth and seed germination. The inhibition on lettuce was reversed by caffeic and ferulic acids at concentrations lower than 10^–3 M except for the inhibition of germination by coumarin. These results suggest that in naturetrans-cinnamic acid,o-, m-, p-coumaric acids, coumarin, and chlorogenic acid inhibit plant growth regardless of their concentration. However, caffeic and ferulic acids can either promote or inhibit plant growth according to their concentration.     

Primary Metabolites and Polyphenols in Rapeseed (Brassica napus L.) Cultivars in China

Defatted meals of 10 rapeseed (Brassica napus L.) varieties were investigated for their total phenolic, phenolic acid (free, esterified, and insoluble-bound forms), and tannin contents. The antioxidant capacities (AC) of methanol extracts from samples were assessed using the 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), Folin–Ciocalteu method and ferric reducing antioxidant power (FRAP), and β-carotene–linoleic acid tests. In the fraction of free phenolic acids, sinapic, caffeic, ferulic, syringic, gallic, and p-coumaric acids were identified. In the fraction of esterified phenolic acids, sinapine, sinapoyl glucoside, and disinapoyl gentiobiose were identified. After basic hydrolysis, sinapic, ferulic, cinnamic, and 4-hydroxybenzoic acids were identified, and sinapic acid (SA) constituted 98.3% to 99.6% of the total esterified phenolic acids. Eleven components (sinapic, protocatechuic, p-coumaric, syringic, vanillic, gallic, caffeic, ferulic, salicylic, cinnamic, and 4-hydroxybenzoic acids) in the fraction of insoluble-bound phenolic acids were identified. The AC of the samples correlated with the total phenolic content. Overall, the total phenolics showed a better correlation with AC than the individual phenolic compounds. Moreover, SA, sinapoyl glucoside, and disinapoyl gentiobiose showed a highly significant and strong positive correlation with the AC of rapeseed meals, and the derivatives of cinnamic acid showed a higher correlation with AC than the derivatives of benzoic acid. The change in the canolol content in rapeseeds under microwave irradiation is discussed. The correlation of the canolol formed with SA and its derivatives is discussed.     

Induction and/or Selection of Phenolic Acid-Utilizing Bulk-Soil and Rhizosphere Bacteria and Their Influence on Phenolic Acid Phytotoxicity

Bulk-soil and rhizosphere bacteria are thought to exert considerable influence over the types and concentrations of phytotoxins, including phenolic acids, that reach a root surface. Induction and/or selection of phenolic acid-utilizing (PAU) bacteria within the bulk-soil and rhizosphere have been observed when soils are enriched with individual phenolic acids at concentrations 0.25 mol/g soil. However, since field soils frequently contain individual phenolic acids at concentrations well below 0.1 mol/g soil, the actual importance of such induction and/or selection remains uncertain. Common bacteriological techniques (e.g., isolation on selective media, and plate dilution frequency technique) were used to demonstrate in Cecil A_p soil systems: (1) that PAU bacterial communities in the bulk soil and the rhizosphere of cucumber seedlings were induced and/or selected by mixtures composed of individual phenolic acids at concentrations well below 0.25 mol/g soil; (2) that readily available carbon sources other than phenolic acids, such as glucose, did not modify induction and/or selection of PAU bacteria; (3) that the resulting bacterial communities readily utilize mixtures of phenolic acids as a carbon source; and (4) that depending on conditions (e.g., initial PAU bacterial populations, and phenolic acid concentration) there were significant inverse relationships between PAU bacteria in the rhizosphere of cucumber seedlings and absolute rates of leaf expansion and/or shoot biomass. The decline in seedling growth could not be attributed to resource competition (e.g., nitrogen) between the seedlings and the PAU bacteria in these studies. The induced and/or selected rhizosphere PAU bacteria, however, reduced the magnitude of growth inhibition by phenolic acid mixtures. For a 0.6 mol/g soil equimolar phenolic acid mixture composed of p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid, modeling indicated that an increase of 500% in rhizosphere PAU bacteria would lead to an approximate 5% decrease (e.g., 20–25%) in inhibition of absolute rates of leaf expansion. As far as we know, this is the first time that such a relationship has been quantified.     

Effects of mixtures of phenolic acids on leaf area expansion of cucumber seedlings grown in different pH portsmouth A1 soil materials

Cucumber seedlings growing in A₁horizon Portsmouth soil material adjusted to pH 5.2, 6.0, or 6.9 were treated with 0, 0.25 or 0.5, mol/ g soil ferulic acid,p-coumaric acid, vanillic acid, or an equal mixture (0.5 mol/g total) of two acids every other day. A total of five treatments was given starting with day 7 from seeding. Absolute rates of leaf expansion were determined for seedlings. The experiment was terminated when seedlings were 17 days old. All three phenolic acids inhibited leaf expansion. The dose required for 50% inhibition of absolute rates of leaf expansion increased as pH of the soil systems increased. The order of toxicity based on 50% dose and relative potency were as follows: ferulic acid > vanillic acid =p-coumaric acid. Effects of mixtures of phenolic acids on absolute rates of leaf expansion, when compared to the effects of individual phenolic acids, were found to be antagonistic for the ferulic-vanillic acid mixture and the ferulic-p-coumaric acid mixture in the pH 5.2 soil systems. Several phenolic acid treatments were required before antagonistic effects of mixtures were evident. In all other instances, when treatment effects were significant, the effects of individual phenolic acids were additive.Paper No. 11875 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601. The use of trade names in this publication does not imply endorsement by North Carolina Agricultural Research Service of the product named, nor criticism of similar products not mentioned. This research was partially supported by US-Spain Joint Committee for Scientific and Technological Corporation project CCA-8309/166.     

Allelopathic potential in bilberry-spruce forests: Influence of phenolic compounds on spruce seedlings

Regeneration failure ofPicea abies in a subalpine bilberry-spruce forest was studied in relation to phenolic compounds, their occurrence and toxicity. Germination bioassays with natural leachates of bilberry (Vaccinium myrtillus) and spruce showed negative effects on root elongation of spruce seedlings. Growth bioassays on litter and humus demonstrated inhibitory effects of these organic layers.p-Hydroxyacetophenone, a spruce-specific metabolite, was isolated in spruce throughfall (10^–6 M), in water extracts of litter (between 1 and 8 µg/g dry wt) and organic layer (less than 1 µg/g dry wt) in addition to tannins and several common phenolic acids. Potential relationships between vegetation cover and phenolic pattern of the soil are discussed, since organic layers under bilberry heath exhibited higher amounts of phenolic acids and tannins than those under spruce.p-Hydroxyacetophenone and caffeic acid reduced, even at 5 × 10^–5 M, spruce seedling growth, especially root development, with additive effects for these two monomers. Autotoxicity involving spruce trees and allelopathy of understory species, mediated byp-hydroxy-acetophenone and other phenolic compounds, including tannins, deserves further attention in regeneration studies.     

Studies on the enzymatic synthesis of lipophilic derivatives of natural antioxidants

The esterification of some natural antioxidants such as cinnamic acid derivatives and ascorbic acid in non-aqueous media, catalyzed by immobilized lipases from Candida antarctica and Rhizomucor miehei, was investigated. The alcohol chain length affected the rate of esterification of cinnamic acids by both lipases. Higher reaction rates were observed when the esterification was carried out with medium- or long-chain alcohols. The rate also depended on aromatic acid structure. The reactivity of the carboxylic function of the cinnamic acids was affected by electron-donating substituents in the aromatic ring. Higher yields were observed for the esterification of p-hydroxyphenylacetic acid (97%) catalyzed by C. antarctica lipase and for the esterification of cinnamic acid (59%) catalyzed by R. miehei lipase. Candida antarctica lipase was more suitable for producing ascorbic acid fatty esters, catalyzing with a relatively high yield (up to 65% within 24 h) the regioselective esterification of ascorbic acid with various fatty acids in 2-methyl-2-propanol. The reaction rate and yield depended on the fatty acid chain length and on the molar ratio of reactants. All ascorbic acid fatty esters produced by this procedure exhibited a significant antioxidant activity in a micellar substrate composed of linoleic acid.     

Effects of mixtures of four phenolic acids on leaf area expansion of cucumber seedlings grown in Portsmouth B1 soil materials

Cucumber seedlings growing in a 12 mixture of soil (Portsmouth B₁) and sand adjusted to pH 5.2 were treated every other day five times with 0, 0.0625, 0.125, 0.25, or 0.5 mol/g soil of ferulic, caffeic,p-coumaric,p-hydroxybenzoic, protocatechuic, sinapic, syringic, or vanillic acids. Treatments began when seedlings were 8 days old. The effects on mean absolute rates of leaf expansion were used to estimate the relative potencies of these phenolic acids to ferulic acid. Based on the results of this experiment, ferulic,p-coumaric,p-hydroxybenzoic, and vanillic acids were chosen for further study. Materials and procedures were identical in the second study, but treatments consisted of mixtures of the four phenolic acids at concentration combinations designed to achieve 40 % or 60 % inhibition of absolute rates of leaf expansion. Using joint action analysis, a model describing the action of the phenolic acid mixtures was developed. A model involving only two factor terms was sufficient to describe the observed responses of cucumber leaf area to the phenolic acid mixtures. The action ofp-hydroxybenzoic acid on absolute rates of leaf expansion was inhibited by the presence of the other three phenolic acids. No other antagonisms or synergisms existed among the four compounds.This research was partially supported by the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643 and by the US-Spain Joint Committee for Scientific and Technological Cooperation project CCA-8309/166.     

Why Phenolic Acids Are Unlikely Primary Allelochemicals in Rice

Allelopathy in rice (Oryza sativa, L.) effective against weeds has been found in about 3.5% of tested rice germplasm in both laboratory and field experimentation. However, the allelochemicals responsible for growth inhibition of rice-associated weeds have not yet been identified. In the literature, phenolic acids are often mentioned as putative allelochemicals. If phenolic acids commonly reach growth inhibitory concentrations in rice ecosystems, it must be expected that the degree of tolerance to phenolic acids will vary among traditional rice cultivars or plant species adapted to rice environments having inherently different phenolic acid concentrations. Phenolic acids concentrations are normally greater in submerged than in aerobic soils. A dose–response study, however, showed that seedlings of rice cultivars adapted to submerged anaerobic soils did not have higher level of tolerance against p-hydroxybenzoic acid than did seedlings of varieties adapted to aerobic upland soils. Moreover, traditional rice cultivars had no greater tolerance than did improved cultivars that were recently bred for traits other than tolerance of phenolic acids. Similarly, there were no differences in tolerance of p-hydroxybenzoic acid between two Echinochloa weed species adapted to either anaerobic or aerobic growth conditions. Thus, neither the rice cultivars nor weed species had evolved different tolerance levels against the phenolic acid. However, all rice cultivars had significantly greater tolerance of p-hydroxybenzoic acid than did either weed species. In a second experiment, the rates at which rice plants released phenolic acids into solution cultures were measured for at least one month, the time period of greatest allelopathic activity following planting under field conditions. The maximum release rate of phenolic acids during the first month of growth was approximately 10 g/plant/day. At a conventional plant density, the release rate of phenolic acids would be approximately 1 mg/m²day. This order of release rate cannot provide concentrations remotely close to phytotoxic levels determined for these rice cultivars and weed species. The results presented in this paper do not preclude the possibility that phenolic acids might be one component in a mixture of chemicals that, when present simultaneously, are allelopathic.