Tannin Fractions Research Articles

Pyrolysis of pre-treated tannins obtained from radiata pine bark

Water soluble and insoluble fractions of tannins obtained from radiata pine bark were pyrolyzed at the bench-scale in a continuous entrained flow pyrolysis reactor at 500°C. The tannin fractions were pre-treated with both calcium hydroxide and calcium formate to assist in continuous feeding and promote deoxygenation during pyrolysis. Oil yields and qualitative GC–MS were used to characterize the oil products. Pre-treatment assisted in continuous feeding, ameliorating some issues associated with the thermoplastic properties of tannins. A comparison of the pyrolysis of tannins with and without pretreatment is presented.

Evaluation of tannins interactions in grape (Vitis vinifera L.) skins

Tannins have a central role in grapevine berries both for their physiological and enological implications. In the skin tissue they can be in vacuolar solution, or associated to the cell walls through weak or strong physicochemical interactions. The present work aims to separate vacuolar, non-covalently and covalently bonded tannins fractions. A specific extraction procedure was developed. A first extraction in ethanol at low temperature allowed the quantification of vacuolar tannins. An urea treatment followed by an ethanol extraction at room temperature was able to separate non-covalently bonded compounds. Finally an acid catalysis was used to break down proanthocyanidin covalent bonds. The method was validated on ripe grape samples of three cultivars, on berries developed in two sun exposure conditions. The Ethephon treatment effect was also evaluated. Beside the method development, a preliminary evaluation of the cultivar, exposition and Ethephon treatment effects are discussed.

Nutritional and fermentation quality of ensiled willow from an integrated feed and bioenergy agroforestry system in UK

Agroforestry, the integration of trees and agriculture, is valued as a multifunctional land use approach that balances the production of commodities (food, feed, fuel, fibre etc.) with non-commodity outputs such as environmental protection and cultural and landscape amenities. In this study, the possibilities for fodder production from a short rotation coppice of willow (Salix viminalis) used for wood chips to produce energy were investigated. The nutritional value and ensilability were assessed from first year regrowth of willow harvested on 29 June 2011 at Wakelyns Agroforestry, Suffolk, UK. The willow branches with a stem diameter less than 8 mm were manually harvested from 4 plots in two replicates. From 2 plots, another sample was prepared including leaves only. Both dried raw material and silage samples ensiled in evacuated polyethylene bags were analyzed. The crude protein concentration was relatively high in leaf + stem silage (182 g/kg dry matter (DM)) and even higher in leaf only silage (219 g/kg DM) and the fibre concentration was relatively low. However, the organic matter digestibility determined by in vitro pepsin-cellulase method was low (0.421 for leaf + stem silage and 0.511 for leaf only silage) and it cannot be considered as a suitable feed for lactating dairy cows. However, it might be suitable for other animal groups with lower energy requirements. The appearance and smell of the silage samples at opening of the vacuum plastic bags was rather pleasant with minor deteriorations (probably yeasts) visible. The extent of fermentation was low and pH high (5.79) for a rather low DM material (DM concentration 276 g/kg). The water soluble carbohydrates of the raw material (35 g/kg DM) and the residual water soluble carbohydrate concentration in silages was relatively low, which at least partly explains the restricted production of fermentation acids. The fermentation profile was heterofermentative (acetic acid dominated instead of lactic acid). The concentrations of total condensed tannins fractions were almost twice as high in the leaf only silage compared to leaf + stem silage. Although the feed values of willow were low, it may have a role in multifunctional systems, where it can provide additional values in grazing situations such as self-medication and microclimate benefits. For easy and efficient use in animal production, controlled browsing might be used; otherwise methods for harvesting and preservation need to be developed. There seems to be some scope for ensiling willow material. This work is part of an EU FP7 funded project “Sustainable organic and low input dairying” (SOLID, KBBE.2010.1.2-02). For more information on the project, see www.solidairy.eu

Tannin-Rich Fraction from Pomegranate Rind Damages Membrane ofListeria monocytogenes

Pomegranate rind has been reported to inhibit several foodborne pathogens, and its antimicrobial activity has been attributed mainly to its tannin fraction. This study aimed to investigate the antimicrobial activity of the tannin-rich fraction from pomegranate rind (TFPR) against Listeria monocytogenes and its mechanism of action. The tannin-related components of TFPR were analyzed by high-performance liquid chromatography and liquid chromatography-mass spectrometry, and the minimum inhibitory concentration (MIC) of TFPR was determined using the agar dilution method. Extracellular potassium concentration, the release of cell constituents, intra- and extracellular ATP concentrations, membrane potential, and intracellular pH (pHin) were measured to elucidate a possible antibacterial mechanism. Punicalagin (64.2%, g/g) and ellagic acid (3.1%, g/g) were detected in TFPR, and the MICs of TFPR were determined to be 1.25-5.0 mg/mL for different L. monocytogenes strains. Treatment with TFPR induced a decrease of the intracellular ATP concentration, an increase of the extracellular concentrations of potassium and ATP, and the release of cell constituents. A reduction of pHin and cell membrane hyperpolarization were observed after treatment. Electron microscopic observations showed that the cell membrane structures of L. monocytogenes were apparently impaired by TFPR. It is concluded that TFPR could destroy the integrity of the cell membrane of L. monocytogenes, leading to a loss of cell homeostasis. These findings indicate that TFPR has the potential to be used as a food preservative in order to control L. monocytogenes contamination in food and reduce the risk of listeriosis.

Molecular weights and tanning properties of tannin fractions from the Acacia mangium bark

The bark of Acacia mangium was smashed and extracted by acetone solution, and the extract was then degreased by petroleum ether. The degreased solution was extracted by diethyl ether and ethyl acetate successively. In this way, the tannin of A. mangium was divided as diethyl ether fraction, ethyl acetate fraction, and water fraction, respectively. The molecular weight of these tannins was measured by gel permeation chromatography (GPC), and the particle sizes of tannin were examined by Zetasizer ZS instrument. The results showed that the ether fraction consisted of the smallest size molecule with average molecular weight of 415 Da, possessing weaker tanning ability but fast penetration rate. The molecular weight of ethyl acetate fraction was 1788 Da, and it showed a significant tanning ability. For the water fraction, the molecular weight was 2808 Da with better tanning ability, and the biggest particle size was shown in the tannin. The thermal stability of the hide powder and cowhide tanned by these tannins was as followed: Water fraction > ethyl acetate fraction > diethyl ether fraction; the penetration was: diethyl ether fraction > ethyl acetate fraction > water fraction. These results could provide a valuable reference for the use of the A. mangium tannin. Key words: Acacia mangium, extraction, molecular weights, tanning ability tannage.

Antioxidant and Anti-Inflammatory Activities of Tannin Fraction of the Extract from Black Raspberry Seeds Compared to Grape Seeds

Black raspberry seed extract and its tannin fraction were characterized and their antioxidant and anti-inflammatory activities were compared to grape seed extract and its tannin fraction. Tannin fraction of black raspberry seed extract was mostly composed of ellagitannins including pedunculagin, sanguiin H6 and H10 isomers and galloyl-bis-HHDP glucose isomer, while that of grape seed extract was composed of proanthocyanidins including proanthocyanidin dimers, trimers, tetramers, pentamers, their monogallates and hexamers. Antioxidant activities of the tannin fraction of black raspberry seed extract, measured by polyphenol content, FRAP, DPPH and ABTS assays, were significantly higher than those of grape seed extract. Anti-inflammatory activity of the tannin fraction of black raspberry seed extract, measured by NO assay, was also significantly higher than that of grape seed extract. The results indicate that black raspberry seeds have potential benefits as a source of natural antioxidant and anti-inflammatory agents comparable to grape seeds. Practical Applications Black raspberry and grape fruits are widely consumed in various ways including wine, juice or raw fruits. Many byproducts, including seeds and wine pomace, are an attractive source of functional compounds. Our results show that the tannins extracted from the seeds have potential antioxidant and anti-inflammatory activities. Thus, these results may help increase utilization of byproducts to make dietary supplements. This may also reduce the waste products generated by the industry and provide additional profit to farmers and manufacturers.

English

Many Nigerian plants have been hypothesized to have anticancer potentials. However, not many of them have been subjected to acceptable scientific evaluation for their potential anticancer effects. In this study, six of such plants were selected to evaluate the effects of their crude, hexane, chloroform, ethylacetate, detanninfied and tannin fractions for brine shrimp lethality assay and the most cytotoxic fractions of each plant were further tested on gene expressions of TP53 and retinoblastoma (Rb) genes in human breast cancer cell line (MCF-7). Gladiolus psittacinus (Gps), Icacina trichantha (Itr), Spilanthes filicaulis (Sfi),Curculigo pilosa (Cpi), Anthocleista djalonensis (Adj), and Tapinanthus bangwensis (Tba) medicinal plants were selected for this study. Crude extracts of 80% aqueous ethanol macerated plant materials were fractionated into hexane, chloroform and ethylacetate fractions. The resultant aqueous fractions were detanninfied to produce aqueous detanninfied fractions and tannin fractions. The 36 panel of plant fractions produced from all the plants were used for the study. From our findings, hexane fraction of S. filicaulis (Sfi-HF) showed the highest cytotoxic effect (LC50 21.30 µg/ml) on brine shrimps showing a low signal of p53 gene expression but a high intensity of Rb gene expression in MCF-7 cell lines. Moreso, crude extract of G. psittacinus (Gps-CE) showed a significant (P<0.05) increase in TP53 gene expression in comparison with the control group and also a high intensity of Rb gene expression. Our results demonstrates the modulatory potentials of Sfi-HF and Gps-CE on TP53 and Rb gene expressions in MCF-7 breast cancer cell lines suggesting a possible mode of action of Sfi-HF and Gps-CE amongst a panel of 36 extract fractions. Key words: TP53 gene, retinoblastoma (Rb) gene, brine shrimps, cytotoxicity, gene expression, medicinal plants

Anti-Candida Targets and Cytotoxicity of Casuarinin Isolated from Plinia cauliflora Leaves in a Bioactivity-Guided Study

In addition to the bio-guided investigation of the antifungal activity of Plinia cauliflora leaves against different Candida species, the major aim of the present study was the search for targets on the fungal cell. The most active antifungal fraction was purified by chromatography and characterized by NMR and mass spectrometry. The antifungal activity was evaluated against five Candida strains according to referenced guidelines. Cytotoxicity against fibroblast cells was determined. The likely targets of Candida albicans cells were assessed through interactions with ergosterol and cell wall composition, porosity and architecture. The chemical major component within the most active antifungal fraction of P. cauliflora leaves identified was the hydrolysable tannin casuarinin. The cytotoxic concentration was higher than the antifungal one. The first indication of plant target on cellular integrity was suggested by the antifungal activity ameliorated when using an osmotic support. The most important target for the tannin fraction studied was suggested by ultrastructural analysis of yeast cell walls revealing a denser mannan outer layer and wall porosity reduced. It is possible to imply that P. cauliflora targeted the C. albicans cell wall inducing some changes in the architecture, notably the outer glycoprotein layer, affecting the cell wall porosity without alteration of the polysaccharide or protein level.

HPLC Retention Thermodynamics of Grape and Wine Tannins

The effect of grape and wine tannin structure on retention thermodynamics under reversed-phase high-performance liquid chromatography conditions on a polystyrene divinylbenzene column was investigated. On the basis of retention response to temperature, an alternative retention factor was developed to approximate the combined temperature response of the complex, unresolvable tannin mixture. This alternative retention factor was based upon relative tannin peak areas separated by an abrupt change in solvent gradient. Using this alternative retention factor, retention thermodynamics were calculated. Van't Hoff relationships of the natural log of the alternative retention factor against temperature followed Kirchoff's relationship. An inverse quadratic equation was fit to the data, and from this the thermodynamic parameters for tannin retention were calculated. All tannin fractions exhibited exothermic, spontaneous interaction, with enthalpy-entropy compensation observed. Normalizing for tannin size, distinct tannin compositional effects on thermodynamic parameters were observed. The results of this study indicate that HPLC can be valuable for measuring the thermodynamics of tannin interaction with a hydrophobic surface and provides a potentially valuable alternative to calorimetry. Furthermore, the information gathered may provide insight into understanding red wine astringency quality.

Condensed conifer tannins as antifungal agents in liquid culture

Abstract In the last decades, many wood preservatives have been prohibited for their ecotoxicity. The present article is focusing on the conifer-derived condensed tannins as environment-friendly options for the substitution of artificial wood preservatives. Eight different tannin fractions were extracted from spruce cones, spruce barks, and pine cones. The parameters of tannin extraction, such as the methods of purification and concentration of active components in the extracts, have been investigated. The cone and bark extracts were tested for the growth inhibition of eight brown-rot fungi, three white-rot fungi, and four soft-rot fungi in liquid cultures. The cone tannins provided a more efficient fungal growth inhibition than bark tannins. Purification increased the antifungal properties of the extracts. The growth of brown-rot fungi was inhibited by the tannins already at low concentrations. However, the extracts were not effective against the white-rot or soft-rot fungi. More investigation is needed concerning the tannin source and the purification procedure of the extracts before tannins can be considered as an ecologically benign wood preservative.

Inhibition of proliferation of human carcinoma cell lines by phenolic compounds from a bearberry-leaf crude extract and its fractions

Phenolic compounds were extracted from the leaves of bearberry, a potential functional food ingredient, using 80% (v/v) aqueous ethanol after which the resultant crude extract was applied to a Sephadex LH-20 column. A fraction comprising low-molecular-weight phenolics (LMW fraction) and sugars was eluted from the column with 95% (v/v) ethanol. A tannin fraction was then obtained after switching the mobile phase to acetone/water (1:1, v/v). Phenolic compounds present in the crude extract and its two fractions showed antiproliferative activities in a concentration-dependent manner against five carcinoma cell lines, namely MCF-7 (estrogen receptor-positive breast carcinoma), DU-145 (androgen receptor-negative prostate carcinoma), HT-29 (colon carcinoma), SK-MEL-5 and MDA-MB-435 (melanoma; skin carcinoma). IC50 data revealed that the tannin fraction was best at retarding cell proliferation in the tested cancer cell lines. The greatest inhibition at 1.5μg fraction/mL assay was observed for the HT-29 colon carcinoma cell line. The proliferation of SK-MEL-5 skin carcinoma cells was also strongly inhibited by both the crude extract and LMW fraction. MDA-MB-435 cells were found to be the least sensitive for the materials tested, particularly for the LMW fraction.

Sensory Properties of Wine Tannin Fractions: Implications for In-Mouth Sensory Properties

Different molecular structures of grape tannins have been shown to influence astringency, however, the in-mouth sensory effects of different molecular structures in red wine tannins remains to be established. The objective of this research was to assess the impact of wine tannin structure on in-mouth sensory properties. Wine tannin was isolated from Cabernet Sauvignon wines of two vintages (3 and 7 years old) and separated into two structurally distinct subfractions with liquid-liquid fractionation using butanol and water. The aqueous subfractions had greater mean degree of polymerization (mDp) and contained a higher proportion of epigallocatechin subunits than the butanol-soluble subfractions, while the older wine tannin fractions showed fewer epicatechin gallate subunits than the younger tannin fractions. The red wine had approximately 3:1 mass ratio of the aqueous and butanol tannin subfractions which approximated an equimolar ratio of tannin in each subfraction. Descriptive sensory analysis of the tannin subfractions in model wine at equimolar concentrations revealed that the larger, more water-soluble wine tannin subfractions from both wines were perceived as more astringent than the smaller, more hydrophobic and more highly pigmented butanol-soluble subfractions, which were perceived as hotter and more bitter. Partial least squares analysis indicated that the greater hydrophobicity and color incorporation in the butanol fractions was negatively associated with astringency, and these characteristics are also associated with aged wine tannins. As the larger, water-soluble tannins had a greater impact on the overall wine astringency, winemaking processes that modulate concentrations of these are likely to most significantly influence astringency.

UPLC‐ESI‐MS study of the oxidation markers released from tannin depolymerization: toward a better characterization of the tannin evolution over food and beverage processing

Condensed tannins take an important part in the sensory quality of food and beverage. Sensory analyses are usually carried out with various tannin fractions isolated from food or beverage, and their interpretation are limited by the lack of knowledge in the fine and accurate molecular composition of the tannin fractions. Besides, the studies of the chemical reactivity conducted in model solutions with 'simple' flavanols allow a better understanding of their evolution pathways, but they cannot take into account their reactivity as polymers, specifically regarding oxidation. In particular, competition between intramolecular and intermolecular reactions may strongly impact on the tannin structures (size, branching and conformation) and consequently on their properties. An ultra-performance liquid chromatography-mass spectrometry electrospray ionization mass spectrometer analytical method was thus developed in order to identify oxidized tannins generated by autoxidation. Given the difficulties to separate and detect tannins with high DP, samples were depolymerized by chemical depolymerization prior to analysis. Since the linkages created by oxidation are not cleavable in the usual depolymerization conditions (contrarily to the original interflavanic linkages), specific oxidation residues are released from tannins structures after their autoxidation. Oxidation markers of both intermolecular and intramolecular mechanisms have been identified; these are mainly dimers and trimers, more or less oxidized, and some contain additional hydroxyl groups. Furthermore, the nature of the subunits (extension vs terminal) making up these dimers and trimers was clearly established.

Antimicrobial and phytochemical properties of stem bark extracts from Piptadeniastrum africanum (Hook f.) Brenan

Aqueous, methanolic and ethyl acetate extracts of Piptadeniastrum africanum (Hook f.) Brenan stem bark were tested against fungi and representatives of Gram-positive and Gram-negative bacteria. Aqueous and methanolic extracts showed a maximum activity against the fungus Pyricularia grisea, a weak activity against the bacteria Staphylococcus aureus and Streptococcus mutans, while no activity was observed against Gram-negative bacteria. Ethyl acetate extract resulted inactive against all the tested microorganisms. Phytochemical investigations of the most active methanolic extract highlighted the presence of tannins and saponins. After purification by means of Sephadex LH-20 and RP-18 the tannin fraction resulted more active against bacteria, while the saponin fraction showed a pronounced activity against Pyricularia grisea. Five different strains of Pyricularia grisea were used and results show that the saponin fraction is promisingly active on the whole population (MIC and MFC: 0.03–0.06mgmL−1).The saponin fraction fungicidal activity was similar to that of Flutriafol (0.01–0.04mgmL−1), a commercially available fungicide, indicating that P. africanum stem bark extract can be a good candidate for the control of Pyricularia grisea, the most diffused rice blast disease fungus of rice.

Antibacterial activity tannin-rich fraction from leaves of Anacardium humile

In vitro tests conducted with extracts rich in tannins have identified several biological activities of this class of substance. Thus, this paper intends to evaluate the antibacterial activity of tannin-rich fraction obtained from leaf extracts of Anacardium humile A.St.-Hil. Extracts of A. humile leaves in 70% acetone were semi-purified with ethyl acetate and butanol. We quantified the total tannins of the semi-purified fractions, of the crude extract and of aqueous residues and then performed tests of the antibacterial activity of the tannins against Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212. All positive extracts underwent tannin isolation using a Sephadex LH-20 column. The tannins isolated from the samples were quantified and tested for the minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The tannins from crude extracts, semi-purifications and residues showed inhibition of S. aureus growth with MIC=500 g mL-1. All tannin fractions showed MIC against all strains and MBC, except against E. faecalis. The tannin fractions from Anacardium humile have antibacterial activity against Staphylococcus aureus, Pseudomononas aeruginosa and Enterococcus faecalis and, therefore, may be promising for future synthesis of new antibacterial agents.

Hypocholesterolemic effect of an aqueous extract of the leaves of Sansevieria senegambica Baker on plasma lipid profile and atherogenic indices of rats fed egg yolk supplemented diet.

The effects of an aqueous extract of the leaves of Sansevieria senegambica on daily weight gain, lipid profile and atherogenic indices of rats fed egg yolk supplemented diet was studied. The control group was given normal feed while the other three groups received 50 g egg yolk/kg feed. The extract was orally administered daily at 150 and 200 mg/kg body weight; while the test control and control groups received appropriate volumes of water by the same route. On gas chromatographic analysis of the aqueous crude extract, the phytosterol and tannins fractions contained 100 % of β-sitosterol and tannic acid respectively. The mean daily weight gain of the test control group was higher though not significantly, than those of the other groups. The plasma total cholesterol levels, cardiac risk ratio and atherogenic coefficient of the test control group was significantly higher (P<0.05) than those of the test groups, but not significantly higher than that of the control group. The plasma low density lipoprotein and non-high density lipoprotein cholesterol levels of the test control group was significantly higher (P<0.05) than those of the control and test groups. The plasma high density lipoprotein cholesterol of the test control group was significantly lower (P<0.05) than that of the control group, but not significantly lower than those of the test groups. There were no significant differences in the plasma triglyceride and very low density lipoprotein cholesterol levels, and the atherogenic index of plasma of all the groups. These results indicate a dose-dependent hypocholesterolemic effect of the extract, thus suggesting a likely protective role of the extract against the development of cardiovascular diseases. It also revealed the presence of pharmacologically active agents in the leaves.

Protective effect of tannins from Ficus racemosa in hypercholesterolemia and diabetes induced vascular tissue damage in rats

ObjectiveTo evaluate the protective effect of tannins from Ficus racemosa (F. racemosa) on the lipid profile and antioxidant parameters in high fat meal and streptozotocin induced hypercholestremia associated diabetes model in rats. MethodsThe crude tannin fraction was separated from the acetone (70% v/v) bark extract of F. racemosa. Oral administration of tannin fraction (TF) (100 & 200 mg/kg body weight) to rats fed with high fat meal for 30 days (4% cholesterol, 1% cholic acid, 0.5% egg albumin) and injected with streptozotocin (35 mg/kg i.p. in citrate buffer on 14th day). ResultsThe administration of TF significantly reverse the increased blood glucose, total cholesterol, triglycerides, low density lipoprotein and also significantly restored the insulin and high density lipoprotein in the serum. In addition tannins significantly restored the activity of antioxidant enzymes such as superoxide dismutase, catalase and decreased the, glutathione peroxidase, and glutathione, thereby restoring the antioxidant status of the organs to almost normal levels. ConclutionsThe results of this study show that two different doses of tannin supplementation had a favorable effect on plasma glucose and lipid profile concentrations. It also had an influence on attenuating oxidative stress in diabetic tats.

Pentaclethra macroloba tannins fractions active against methicillin-resistant staphylococcal and Gram-negative strains showing selective toxicity

The ethanol extract of the vegetal species Pentaclethra macroloba (Willd.) Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea) fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp.) and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae) multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591) was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.

Geographical variation and quality assessment of Stryphnodendron adstringens (Mart.) Coville within Brazil

Stryphnodendron adstringens (Mart.) Co- ville belongs to the Mimosoideae subfamily of the Leguminosae, and is also known as Barbatimao. It is extensively used in the traditional Brazilian medicine for its biological properties, which are attributed to the tannin fraction found in the bark. To maintain the genetic diversity of S. adstringens we have accessed twelve populations to establish an ex situ germplasm bank for future collections of seeds. Records on geographical boundary of occurrence, fruits, seeds and levels of extractions were gathered as species descrip- tors. Aiming the tannin fractions, we have noticed extraction of the bark on 46% of the evaluated trees as a practice that partially removes the bark disrupting wood vessels and causing premature death of the trees. In addition, the quality of seeds was low due to the high incidence of insect attack and immature abortion which compromises the species reproductive pattern. The geographical variation on the tannin content, seed production, predation and abortion was studied in twelve distinct populations located in three different states Sao Paulo, Goias and Minas Gerais. Populations with the highest content of phenols and tannins were Campo Alegre and Luislandia. Our data demonstrated that there is a need for conservation of S. adstringens. Plants of the population located in Campo Alegre and Luislandia were considered elite for non-timber prod- ucts of S. adstringens.

Condensed Tannins and Flavonoids from the Forage Legume Sulla (Hedysarum coronarium)

The condensed tannin concentrations and composition and the characterization of the phenolic constituents in the leaves of the forage legume sulla (Hedysarum coronarium), a biennial forage legume found in temperate agricultural regions, were studied. The colorimetric butanol-HCl assay was used for the quantitation of the seasonal condensed tannin concentrations in the leaves of sulla. Fractionation of extracts on Sephadex LH-20 using step elution with aqueous methanol, followed with aqueous acetone or gradient elution with water, aqueous methanol, and aqueous acetone, gave condensed tannin and flavonoid fractions. The chemical characteristics of the purified condensed tannin fractions were studied by acid-catalyzed degradation with benzyl mercaptan and electrospray ionization mass spectrometry (ESI-MS). Thiolysis revealed that epigallocatechin was the major extender unit (15-75%) while gallocatechin was the major terminal unit (50-66%), thus indicating the extractable sulla condensed tannin fraction as the prodelphinidin type. Condensed tannin oligomers to polymers obtained from Sephadex LH-20 gradient fractions ranged between 2.9 and 46 mDP. The homo- and heterogeneous oligomer ions in condensed tannin gradient fractions detected by ESI-MS ranged from 2 to 10 DP and are consistent with the values obtained by thiolysis (2.9-6.9 DP). Lower molecular weight phenolics, including flavonoids and phenolic acids, were characterized by liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS) and ESI/MS/MS on a linear ion trap. The flavonoids extracted with aqueous acetone and methanol from sulla leaves and identified included kaempferol, rutin, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol-3-O-β-D-glucoside-dirhamnoside, genistein-7-O-β-D-glucosyl-6″-O-malonate, formononetin-7-O-β-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.