Claudia Crestini 31. P-NMR Analysis of Tannins. May 8, 2014, Coimbra COST FP 1105

Size: px

Start display at page:

Download "Claudia Crestini 31. P-NMR Analysis of Tannins. May 8, 2014, Coimbra COST FP 1105"

Sharyl Dean
5 years ago
Views:

Crestini 31 P-NMR Analysis of Tannins Dipartimento di Scienze e Tecnologie Chimiche

1 May 8, 2014, Coimbra CST FP 1105 UNDERSTANDING WD CELL WALL STRUCTURE, BIPLYMER INTERACTIN AND CMPSITIN: IMPLICATINS FR CURRENT PRDUCTS AND NEW MATERIAL INNVATIN Claudia Crestini 31 P-NMR Analysis of Tannins Dipartimento di Scienze e Tecnologie Chimiche Università degli Studi di Roma Tor Vergata, ITALY. crestini@stc.uniroma2.it

2 2 Tannins are phenolic compounds that precipitate proteins Very diverse group of oligomers and polymers. Horvath (1981): "Any phenolic compound of sufficiently high molecular weight containing sufficient hydroxyls and other suitable groups (i.e. carboxyls) to form effectively strong complexes with protein and other macromolecules under the particular environmental conditions being studied ligomeric compounds with multiple structure units with free phenolic groups, Molecular weight ranging from 500 to >20,000, Soluble in water Bind proteins and form insoluble or soluble tanninprotein complexes.

3 ccurrence and Location Common in Gymnosperms and Angiosperms.

Monocotyledons. vacuoles or surface wax of the plants.

reduce palatability and, thus, protect against predators.

3 3 ccurrence and Location Common in Gymnosperms and Angiosperms. Within Angiosperms, tannins are more common in Dicotyledons than in Monocotyledons. vacuoles or surface wax of the plants. Bud tissues - as protection against freezing Leaf tissues - They serve to reduce palatability and, thus, protect against predators. Root tissues - chemical barrier to plant pathogens. Seed tissues -. maintainance of plant dormancy. Stem tissues - active growth areas

4 4 Tannins biological significance Protection of plants against insects/animals Bad Taste/Astringency (bitter taste) Animals: tannins reduce digestion of food Toxic to bacteria Pine calluses: created by fungal invasion Tree forms calluses as protective tissue Calluses contain high levels of tannins (Chinese 50-80%)

5 5 Biological activities Three mechanisms of bacterial toxicity have been identified enzyme inhibition and substrate deprivation, action on membranes, metal ion deprivation. In tissue-cultured cell assays tannins have shown antiviral, antibacterial and antiparasitic effects. Antimicrobial, apoptotic Antiinflammatory activity antioxidant

6 Uses 6 Tanning leather. Tannins have traditionally been the primary source of tannery tannin. Tannins produce different colors with ferric chloride. Iron gall ink is produced by treating a solution of tannins with iron(ii) sulfate. Tannin is a component in a type of industrial particleboard adhesive and of wood adhesives. Tannins, substitute a high proportion of synthetic phenol in phenol-formaldehyde resins. anti-corrosive primer, for treatment of rusted steel and rust inhibitor. Metals removal

7 7 Carbohydrates Starch-tannin interaction starch forms hydrophobic cavities that allow inclusion complexes with tannins Cellulose-tannin interaction - cellulose has a direct surface interaction with tannins. Cell wall carbohydrate-tannin interaction - this association is less understood. Hydrophobic and hydrogen linkages.

8 WHY TANNINS? 8 Tannins have form complexes with numerous types of molecules: Carbohydrates, Proteins, Polysaccharides, Bacterial cell membranes, Enzymes, Metals. Interactions of Tannins with other macromolecules: A source of inspiration

9 9 Tannins Classification Gallotannins Galloyl units or meta-depsidic derivatives bound to diverse polyol-, catechin-, or triterpenoid units. Ellagitannins At least two galloyl units are C C coupled. Condensed tannins Proanthocyanidins formed by linkage of C-4 of one catechin with C-8 or C-6 of the next catechin. Complex tannins A catechin unit is bound to a gallotannin or an ellagitannin unit.

10 Gallotannins H H H 10 Gallotannins are gallic acid esters of a core polyol, usually glucose The simplest proto-tannin is pentagalloyl glucose ( b-1,2,3,4,6-pentagalloyl--d-glucopyranose) (PGG) PGG presents different isomers in which one aliphatic hydroxyl group is free and one other presents a digalloyl ester Polygalloyl ester chains are formed by a meta- or para-depside bond involving a phenolic hydroxyl rather than an aliphatic hydroxyl group Simple gallotannins with up to 12 galloyl groups are present in tannins extracted from sumac or oak galls. Tannic acid is described as glucose pentagalloylgallate, but it is actually a mixture of different isomers and partially galloylated glucose.

11 H 11 Ellagitannins H Esters of hexahydrodiphenic acid (HHDP) Generated by oxidative crosslinking of two galloyl groups. Upon hydrolysis release ellagic acid. Coupling occurs between the C-4/C-6 (eugeniin) and C-2/C-3 (casuarictin). Molecular weight range:

12 Proanthocyanidins (condensed tannins) 12 ligomers of flavonoid units (flavan-3-ol). 2 to 50 flavonoid units 4-8 or 4-6 interflavanoid bonds A 2 3 B 5 4 H H H H n H n C4 C8 linkage C4 C6 linkage

13 13 Catechin tannins Tannin activity comparable to polymeric tannins despite the low molecular weight High antioxidant activity correlated with antiinflammatory and anti cancer activities.

14 Colorimetric assays 14 Folin-Dennis method Reduction of phosphomolybdic acid by phenols in alkali. total soluble phenolics It does not differentiate between tannins and other phenolics. verestimation. Vanillin-HCl assay Exo-type reaction - vanillin reacts with the meta-substituted A-ring of flavanols to form a chromophore Low molecular weight flavanols overreact and large polymers underreact. Butanol-HCl assay Endo-type reaction - HCl catalyzed depolimerization yields a red anthocyanidin product. Uncomplete reaction: underestimation. Rhodanine assay The sample is subjected to hydrolysis to release gallic acid. The reaction between gallic acid and the dye rhodanine produces an intense color that is measured spectrophotometrically. Specific to gallotannins. Wilson and Hagerman assay The sample is subjected to hydrolysis to release ellagic acid. The reaction between ellagic acid and the sodium nitrite produce a colored solution. Specific for ellagitannins.

15 15 Tannins Complex mixtures of diverse polyphenolics Biological activities and chemical properties are modulated by the amount and distribution of phenolic groups Classical techniques for tannins analysis Phenolic groups content Hydrolyzable tannins content Non-hydrolyzable tannins content A new analytical technique able to detect and quantify the different phenolic groups in tannins is needed!

16 16 DEVELPMENT F ADVANCED ANALYTICAL METHDS FR PLANT PLYPHENLS ANALYSIS CH 2 H Lignin R R Cl P CDCl 3 /Pyr (internal standard) P R CH 2 P Lignin R P In situ labeling of phenolic moieties with 31 P

17 17 31 P-NMR of tannins models! H! Hydrolizable tannins models Gallic acid Methyl gallate Ellagic acid PGG Condensed tannins models Complex tannin models CH 3 H H H H H H H H H H H

18 !!!!!! 18! 31 P-NMR of tannins models! H o o Hydrolizable tannins models Condensed tannins models o o o Catechin Epicatechin Quercetin H H o o Gallocatechin Epigallocatechin H o Complex tannins models H

19 !!!! 19! 31 P-NMR of tannins models! o o o Hydrolizable tannins models Condensed tannins models Complex tannins models H IS C4 - C5, C3,C5 - C3,C4 - C7- H IS C4 - C5, C3,C5 - C7- C3,C4 - o Catechin galloil gallate o Epicatechin galloil gallate o Gallocatechin galloil gallate o Epigallocatechin galloil gallate H IS C7- C5,C3,C5,, C3,C5 - H IS C4 - C4 - C5,C3,C5,, C3,C5 - C7- C4 - C4 -

20 31 P-NMR of Tannins models: signals assignment 20 signal Chemical shift (ppm) Aliphatic 150,0-145,5 o-disubstituted phenolic 144,0-140,24 Catechol 140,24-138,8 o-substituted phenolic 138,8-137,9 o-unsubstituted phenolic 137,9-137,4 C 135,5-134!

21 21 Quantitative 31 P-NMR: A novel analytical tool for tannins analysis

22 31 P-NMR of hydrolyzable Tannins : Tannic acid (CHINA) A B C, D C D I.S. A B 22

23 31 P-NMR of hydrolyzable Tannins : Tannic acid (Turkey) aliphatic o-disubstituted phenolic o-substituted phenolic unsubstituted phenolic Internal Terminal C I.S. gallic 23

24 31 P-NMR of hydrolyzable Tannins : ellagitannin from chestnut wood ellagic acid Ar-P A: ppm Ar-P B: ppm o-disubstituted phenolic o-substituted phenolic C aliphatic unsubstituted phenolic I.S. B A 24

25 31 P-NMR of hydrolyzable Tannins : ellagitannin from oak wood ellagic acid Ar-P A: 139.

25 25 31 P-NMR of hydrolyzable Tannins : ellagitannin from oak wood ellagic acid Ar-P A: ppm Ar-P B: ppm aliphatic o-disubstituted phenolic o-substituted phenolic C unsubstituted phenolic I.S.

26 26 31 P-NMR of condensed Tannins: grape peel N -DISUBSTITUTED SIGNALS aliphatic o- disubstituted o- substituted C4 C8 linkage C4 C6 linkage

27 31 P-NMR Acacia Mearnii bark extract H H IS Gallocatechin/epigallocatechin units

28 31 P NMR, Acacia catechu wood and bark extract IS H catechin/epicatechin units

29 31 P NMR, Quebracho o wood extract IS H catechin/epicatechin units

30 30 31 P-NMR green tea extract H H IS Epigallocatechin galloil gallate

31 Conclusions P-NMR is a novel analytical tool to determine and quantify phenolic groups in tannins Avoiding contaminants interferences 31 P-NMR of suitably phosphitylated tannins allows detailed chemical characterization: Identification of ellagic, gallotannins and proantocyanidines Fingerprint of each tannin Proantocyanidins and gallotannins Evaluation of the regiochemistry of polymerization J. Agric. Food Chem., 2013, 61(39), Melone F, Saladino R, Lange H, Crestini C. J. Agric. Food Chem., 2013, 61(39), Melone F, Saladino R, Lange H, Crestini C.

32 32 utcomes & pportunities Mother nature offers a diverse set of materials whose structural diversity offers opportunities for further manipulation for the creation of novel functional materials. Modern 31 P NMR methods offer the foundations for a facile & in depth understanding of the structure of the diverse class of materials opening the doors for : - Novel high value materials that base their functionality on documented beneficial physiological effects of tannins, manipulating them at a molecular & and at the nanoscale - Functional foods - Personal care/cosmetic applications - New materials that take advantage of the natural chelating propensity of tannins

$Heiko Lange Lignin fractionation and tailoring Lignin based sensors development Dr.$

33 33 Thank you for your attention! Dr. Heiko Lange Lignin fractionation and tailoring Lignin based sensors development Dr. Mario Caruso tannins Elisavet Bartzoka Nanocapsules

34 34 Gravimetric assays Gravimetric method with Ytterbium Determines only soluble tannins. Selectively precipitates polyphenols from plant extracts. underestimation. Gravimetric method with PVP Determines only soluble tannins.pvp irreversibly binds tannins. underestimate tannins. Gravimetric method based on the detergent system Includes soluble and insoluble tannins. Measurement of the acid-detergent residue of the NDF (NAD) and the neutral-detergent residue of the ADF (AND). many soluble tannins are not measured.

35 35 Protein precipitation assays Radial diffusion assay complexes between tannins and bovine serum albumin. The most commonly used standard is tannic acid and the results are expressed in tannic acid equivalents. Problem: less useful for quantification than the colorimetric procedures.

Tannin Structural Elucidation and Quantitative 31 P NMR Analysis. 1. Model Compounds

pubs.acs.org/jafc Tannin Structural Elucidation and Quantitative 31 P NMR Analysis. 1. Model Compounds Federica Melone,, Raffaele Saladino, Heiko Lange, and Claudia Crestini*, Department of Chemical Sciences