Loss of Tannins and Other Phenolics from Willow Leaf Litter

A litter bag experiment was conducted to determine the fate of condensed tannins during the initial phase of willow leaf decomposition. Chemical methods and HPLC were used to identify and quantify the phenolics in aqueous acetone extracts of the leaves during the first eight weeks after leaf fall. Extractable phenolics and tannins were lost rapidly from the leaves and had half lives of about 2.4 weeks. Lower molecular weight tannins were lost more rapidly than higher molecular weight tannins, suggesting that the primary route for loss of tannins is leaching. There was no evidence for decomposition of the tannin during the leaf decay process. After leaching, the tannin apparently sorbs tightly to the soil.     

Effects of sample drying and storage,and choice of extraction solvent and analysis method on the yield of birch leaf hydrolyzable tannins

In this study, I investigated the effects of different methods of sample drying and storage, and the choice of extraction solvent and analysis method on the concentrations of 14 individual hydrolyzable tannins (HTs), and insoluble ellagitannins in birch (Betula pubescens) leaves. Freeze- and vacuum-drying of birch leaves were found to provide more reliable results than air- or oven-drying. Storage of leaves at –20C for 3 months before freeze-drying did not cause major changes in tannin content, although levels of 1,2,3,4,6-penta-O-galloylglucose and isostrictinin were altered. Storage of dried leaf material at –20C is preferred because 1 year storage of freeze-dried leaves at 4C and at room temperature decreased the concentration of the pedunculagin derivative, one of the main ellagitannins of birch. Furthermore, storage at room temperature increased the levels of isostrictinin and 2,3-(S)-HHDP-glucose, indicating possible HT catabolism. Of the extraction solvents tested, aqueous acetone was superior to pure acetone, or aqueous or pure methanol. The addition of 0.1% ascorbic acid into 70% acetone significantly increased the yield of ellagitannins, presumably by preventing their oxidation. By comparing the conventional rhodanine assay and the HPLC–ESI-MS assay for quantification of leaf galloylglucoses, the former tends to underestimate total concentrations of galloylglucoses in birch leaf extract. On the basis of the outcomes of all the method and solvent comparisons, their suitability for qualitative and quantitative analysis of plant HTs is discussed, emphasizing that each plant species, with its presumably unique HT composition, is likely to have a unique combination of ideal conditions for tissue preservation and extraction.     

Factors affecting levels of some phenolic compounds,digestibility, and nitrogen content of the mature leaves ofBarteria fistulosa (Passifloraceae)

Levels of total phenolics, condensed tannins, acid detergent fiber, pepsin/cellulase digestibility, and nitrogen in mature leaves of 26 individuals of the ant-plant,Barteria fistulosa, have been determined. Analysis of the results in terms of the presence or absence of ants and the position of the branch from which the leaves were collected showed no relationship with concentrations of phenolics or fiber and only a weak relationship with digestibility and nitrogen. By contrast, light intensity strongly influenced levels of phenolics, notably condensed tannins, so that mature leaves of individuals growing in direct sunlight were less digestible and appeared to be of lower quality as food for herbivores than did mature leaves of individuals in shaded positions. Possible reasons for the variation in condensed tannin levels are discussed.     

Extraction of tannin from fresh and preserved leaves

The extractability of tannin from fresh, lyophilized, and dried leaves collected at various times in the growing season was determined using the radial diffusion assay for protein-precipitating phenolics. The amount of tannin extracted depended on the method of leaf preservation and on the maturity of the leaf. Early in the season, more tannin was extracted from lyophilized leaves than from fresh leaves, but late in the season more tannin was extracted from fresh leaves. At all times, more tannin was extracted with aqueous acetone than with aqueous or acidic methanol.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Characterization and antioxidative properties of condensed tannins from the mangrove plant Aegiceras corniculatum

Freeze‐dried leaf, stem bark, and root bark powders of Aegiceras corniculatum were extracted with three different types of polar solvents: methanol, ethyl acetate, and water. The methanol extracts had the highest concentrations in total phenolics and extractable condensed tannins, followed by water and ethyl acetate extracts. Analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) suggested that condensed tannins from leaf, stem bark, and root bark contained prodelphinidins and procyanidins, with the predominance of prodelphinidins and high level of galloylation. Acid‐catalyzed degradation in the presence of benzyl mercaptan indicated that gallocatechin, epigallocatechin, epigallocatechin‐3‐O‐gallate, and epicatechin‐3‐O‐gallate occurred as the terminal units and (epi)gallocatechin, (epi)gallocatechin‐3‐O‐gallate, (epi)catechin, and (epi)catechin‐3‐O‐gallate occurred as the extension units. The mean degrees of polymerization (mDP) of condensed tannins from leaf, stem bark, and root bark were 13.5, 7.4, and 12.3, respectively. The condensed tannins from leaf and stem bark exhibited a higher DPPH radical scavenging activity and ferric reducing/antioxidant power compared to that of synthetic antioxidant butylated hydroxyanisole (BHA). © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Decomposition and nutrient release from pruning residues of two indigenous agroforestry species during the wet and dry seasons

The decomposition of leaves from Cordia africana Lam. and Albizia gummifera G. F.Gmel was investigated during the wet and dry seasons at Wondo Genet (Ethiopia). Litterbags of leaves were buried in soils under farmland and shaded-coffee agroforestry systems. Residual matter was recovered after 4, 8, 12, and 16 weeks and analysed for nitrogen (N), phosphorus (P), potassium (K), cellulose, lignin, soluble polyphenol and condensed tannin content. Mass-loss and release of N, polyphenols and condensed tannins were greater from Albizia leaves than from Cordia leaves, suggesting that a high polyphenol content does not necessarily retard decomposition. The rates of mass loss and release of the majority of leaf constituents were considerably faster during the wet season than during the dry season. Lignin decomposition, however, proceeded more rapidly during the dry season, and no significant seasonal differences were observed for polyphenol decomposition. The decomposition kinetics of most leaf components during the wet season were best described by a single-exponential model, but a quadratic model provided the best fit during the dry season. Initial leaf chemistry and season were important decomposition factors, while land-use effects were negligible. However, land-use effects showed distinct seasonal differences, with leaf litter decomposing more rapidly in soil under shaded-coffee than under farmland management, especially during the wet season. This study also demonstrated that polyphenol content does not show the predictive effects it has been attributed to have and that other constituents, such as condensed tannins, would be better suited for this purpose.     

Radical scavenging activity of canola hull phenolics

Possible use of canola hulls as a source of natural anti-oxidants was explored. Cyclone canola hulls were extracted with methanol (30 to 80%, vol/vol) and acetone (30 to 80%, vol/vol). The free radical-scavenging activity of phenolic extracts so prepared was evaluated using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical ion (ABTS^o−), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and chemiluminescence assays. The total content of phenolics in prepared extracts from canola hulls ranged from 15 to 136 mg sinapic acid equivalents per gram of extract. Higher levels of condensed tannins were detected in the acetone extracts than in the corresponding methanolic counterparts. Seventy and 80% (vol/vol) acetone extracts displayed markedly stronger antioxidant activity than any of the other extracts investigated. Statistically significant linear correlations were found between TEAC (Trolox equivalent antioxidant capacity) values (expressed in mM of Trolox equivalents per gram of extract) and total pehnolics, TEAC and total condensed tannins (i.e., determined using the modified vanillin and pronthocyanidin assays), as well as TEAC and protein precipitation activity of phenolic extracts (i.e., measured using the dye-labeled assay). The antioxidant activities of extracts as determined by the ABTS^o− radical ion assay correlated highly with those of the chemiluminescence and DPPH radical assays.     

Effect of Solvent,Preheating Temperature,and Time on the Ultrasonic Extraction of Phenolic Compounds from Cold‐Pressed Hempseed Cake

The effect of different solvents (aqueous methanol [70%, v/v], aqueous acetone [80%, v/v], and a solvent mixture [MA] of aqueous methanol [70%, v/v] and aqueous acetone [70%, v/v] in a ratio of 1:1 [v/v]), preheating temperatures (140, 160, and 180°C), and times of exposure (5, 15, and 30 min) on the ultrasonic extraction of the main phenolic compounds from hempseed cake (Cannabis sativa) was investigated. A simplified new high‐performance liquid chromatography (HPLC) method was developed to identify and quantify the main phenolics (namely, N‐trans‐caffeoyltyramine and cannabisin B) in the extracts. Two other main compounds, numbered 3 and 4 , were also detected. The results showed that the nature of the extracting solvent had a significant (P < 0.05) impact on the ultrasonic extraction of phenolic compounds. The acetone extracts exhibited the highest total phenolic content (TPC), followed by MA and methanol. The preheating temperature and time of exposure enhanced the TPC for all solvents examined. The main phenolics, N‐trans‐caffeoyltyramine, cannabisin B, and compound 3 , were positively affected by the temperature and time of exposure, irrespective of the solvents used. In sharp contrast, compound 4 appeared to be thermally sensitive: increasing preheating time and temperature decreased the yields of this compound. This study demonstrated that acetone was the most effective extracting solvent and that preheating enhanced the yield of the main phenolics.     

Vanillin-HCl method for condensed tannins: Effect of organic solvents used for extraction of tannins

Tannins are generally extracted using aqueous organic solvents, mainly methanol and acetone. The presence of various concentrations of methanol in the sample containing catechin or tannins did not alter the kinetics of their reaction, but the absorbance depended on the amount of methanol in the sample; the higher the methanol, the higher was the absorbance. Acetone reacted with acidified vanillin to produce a chromogen with _max at 548 nm, which produce a substantial error in the determination of condensed tannins. In the presence of acetone, the time courses of the reaction for catechin and tannins were different, which depended on the acetone concentration and the reaction temperature. Reaction conditions for catechin and tannins that enable their measurement in the presence of acetone are presented.     

Extraction of Condensed Tannins from Cervid Feed and feces and Quantification using a Radial Diffusion Assay

A radial diffusion assay was employed to quantify condensed tannins (CT) in feed and feces of mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus). This biological assay measures the precipitation of bovine serum albumin (BSA), with the area of the precipitation ring being proportional to the amount of extracted CT applied to the well. CT extracted from the bark of white spruce (Picea glauca) was used as the standard. CT were extracted with 70% (v/v) aqueous acetone and precipitated with 50% (v/v) aqueous methanol or 70% (v/v) aqueous acetone. Functional range of CT weights for suitable ring measurement was 0.5–4.0 mg, and equilibrium was achieved within an incubation period of 24 hr. Methanol (50%) was a more effective precipitation solvent than acetone (70%) having 13 ± 4% greater specific activity (P < 0.05) and superior capabilities for predicting CT content. Precipitation rings were evaluated on images magnified on a photocopier. Ring diameters measured on a 200% enlarged photocopy provided the most precise estimate of ring area (R² = 0.98). This convenient method reduced analysis times and enhanced accuracy and precision of tannin quantification. Analytical consequences and future research requirements are considered.     

Patterns and sources of leaf tannin variation in yellow birch (Betula allegheniensis) and sugar maple (Acer saccharum)

Leaves from forest-grown sugar maple (Acer saccharum Marsh) and yellow birch (Betula allegheniensis Britt.) trees were analyzed for four tannin measures (hydrolyzable and condensed tannins, total phenolics, and protein binding) at three times during the growing season. Fifteen-year-old half-sib sugar maples from four provenances, representing the geographical extremes of the sugar maple range and growing in a common garden, were examined for the same traits. We found no significant geographic or seed source component to variation in three of the four tannin measures. We found significant seasonal changes in both birch and maple leaf tannins. Withincanopy leaf tannin variation tended to obscure differences between trees in maple, but in birches between-tree differences in leaf tannin content were more readily found. We also found a significant negative correlation between leaf protein binding capacity and leaf wet weight.     

Fate of Tannins in Corsican Pine Litter

Tannins are ubiquitous in higher plants and also in litter and soils where they affect many biogeochemical processes. Despite this well-recognized role, their fate in litter and mineral soils is hardly known, as often only trace amounts, if any, are measured. In this study, we conducted an incubation experiment with Corsican pine litter to which known amounts of tannic acid (TA) or condensed tannins (CTs) from Corsican pine were added. Using Folin–Ciocalteu as a measure for total phenolics and HCl–butanol as an assay specific for CTs, acetone/water extractable phenolics and tannins decreased with time towards very low levels. Application of thermally assisted hydrolysis and methylation to litter before and after acetone/water extraction revealed that TA concentration decreased. By contrast, CTs remained to a great extent in the litter and could not be extracted suggesting that they were tightly bound.     

Evaluation of Methods for Measuring Polyphenols in Conifer Foliage

There is a resurgence of interest in the quantification of polyphenols in plant tissues because of their presumed ecological importance in plant–litter–soil and plant–animal interactions. The influence of sample preparation, extracting solvent, foliage quality, and assay method was investigated for the quantification of total phenols and condensed tannins in conifer foliage. Our results suggest that it is not possible to recommend a single optimal protocol for quantification of total phenol and condensed tannin fractions from plant materials. In general, the use of aqueous acetone (50–70% v/v) with freeze-dried materials gave the highest recovery. The Folin-Ciocalteau method for total phenols and the butanol–HCl hydrolysis method for condensed tannins appear superior to other common assays tested. There were large differences (1.4–2.2 times) in the reactivity of purified condensed tannins among species, indicating the importance of an appropriate standard for polyphenol quantification. A solid-state ¹³C NMR method with an improved "interrupted decoupling" pulse sequence yielded the highest concentrations for condensed tannins. Assuming that ¹³C NMR provides an accurate measure of total condensed tannin, the other extraction/assay methods used in this study recovered 50–86% of the condensed tannin fraction. The recovery rate is correlated with the nitrogen content of the foliage, which suggests that the formation of protein–tannin complexes may limit the extractability of condensed tannins. While ¹³C NMR condensed tannin values may give the best value for total condensed tannin concentrations, the water-soluble fraction may have the greatest physiological and/or ecological significance.     

Gypsy Moth Caterpillar Feeding has Only a Marginal Impact on Phenolic Compounds in Old-Growth Black Poplar

Species of the Salicaceae produce phenolic compounds that may function as anti-herbivore defenses. Levels of these compounds have been reported to increase upon herbivory, but only rarely have these changes in phenolics been studied under natural conditions. We profiled the phenolics of old-growth black poplar (Populus nigra L.) and studied the response to gypsy moth (Lymantria dispar L.) herbivory in two separate field experiments. In a first experiment, foliar phenolics of 20 trees were monitored over 4 weeks after caterpillar infestation, and in a second experiment the bark and foliar phenolics of a single tree were measured over a week. Of the major groups of phenolics, salicinoids (phenolic glycosides) showed no short term response to caterpillar feeding, but after 4 weeks they declined up to 40 % in herbivore damaged and adjacent undamaged leaves on the same branch when compared to leaves of control branches. Flavonol glycosides, low molecular weight flavan-3-ols, and condensed tannins were not affected by herbivory in the first experiment. However, in the single-tree experiment, foliar condensed tannins increased by 10–20 % after herbivory, and low molecular weight flavan-3-ols decreased by 10 % in the leaves but increased by 10 % in the bark. Despite 15 % experimental leaf area loss followed by a 5-fold increase in foliar jasmonate defense hormones, we found no evidence for substantial induction of phenolic defense compounds in old growth black poplar trees growing in a native stand. Thus, if phenolics in these trees function as defenses against herbivory, our results suggest that they act mainly as constitutive defenses.     

Chemical correlates of rhesus monkey food choice: The influence of hydrolyzable tannins

Feeding behavior was quantified of free-ranging adult rhesus monkeys in the Himalayan foothills of northern Pakistan. Twenty-eight food and 63 nonfood plant samples were collected and analyzed for tannins, alkaloids, fiber, lignin, protein, amino acids, total nonstructural carbohydrates, and free glucose. Most major foods of the rhesus were low in tannins and high in nutrients. The rhesus ate mainly parts of herbaceous plants, although woody plants were more abundant, possibly because herbs were lower in condensed tannins than woody plants. Feeding was negatively correlated with the astringency of tannins, ellagitannins, and total phenolics among all samples. Among leaves of both herbs and woody plants, feeding was negatively correlated with measures of hydrolyzable tannins. Total protein, total nonstructural carbohydrates, and water were not related to food choice and may not have been limiting at the site.     

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCl and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides, and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.     

Comparison of Chemical Compositions and Antioxidant Activities of Condensed Tannins From Different Parts of Calliandra haematocephala

In this study, the condensed tannins in 70% acetone extracts of leaf, twig, and stem bark of Calliandra haematocephala were purified by a Sephadex LH-20 column, and their structures were identified using MALDI-TOF-MS and thiolysis-HPLC-ESI-MS analyses. The results showed that the condensed tannin structures varied significantly among C. haematocephala organs. The condensed tannins from leaf were essentially of prodelphinidins, consisting mainly of (epi)gallocatechin and (epi)gallocatechin-3-O-gallate units; the condensed tannins from twig and stem bark were predominately of procyanidins, with catechin, epicatechin, and (epi)catechin-3-O-gallate as the main constitutive units for the former and epicatechin as the main constitutive unit for the latter, respectively. Furthermore, in vitro antioxidant activities of these condensed tannins were evaluated using the ABTS^·+ and FRAP methods. All of the condensed tannins exhibited stronger antioxidant activities compared to those of synthetic antioxidant BHA. For all organs, the two tested activities were found to be the highest in leaf condensed tannins, followed by that in twig condensed tannins and stem-bark condensed tannins. The results suggested that the number of hydroxyl groups on the B-ring and the level of 3-O-galloylation in condensed tannins were closely related with their antioxidant activities, and that the condensed tannins from different parts of C. haematocephala could be used as a natural additive in the food and cosmetic industries.