The Role of Tannins in Astringency, Precipitation, and Antioxidant Activity of Plant Extracts
The Role of Tannins in Astringency, Precipitation, and Antioxidant Activity of Plant Extracts
Tannins are important polyphenolic components in plant extracts and are widely present in tea, grape seeds, pomegranate peel, gallnut, cocoa, nut skins, oak, berries, and many medicinal and edible plants. Their structures contain abundant phenolic hydroxyl groups, which can impart astringency and a mouth-drying sensation to extracts, induce protein-, polysaccharide-, or metal ion-related precipitation, and play important roles in free radical scavenging, metal chelation, and regulation of oxidation reactions.
Keywords: tannins; plant extracts; astringency; precipitation; antioxidant activity; polyphenols; proanthocyanidins; hydrolyzable tannins; condensed tannins
1 Basic Concepts of Tannins
1.1 Structural Characteristics
(1) Polyphenolic hydroxyl structure
Tannins usually contain multiple phenolic hydroxyl groups and can interact with proteins, polysaccharides, alkaloids, and metal ions through hydrogen bonding, hydrophobic interactions, π-π stacking, and metal coordination. The greater the number of phenolic hydroxyl groups and the more suitable their spatial distribution, the stronger the binding capacity of the molecule toward exogenous substrates.
(2) Relatively high molecular weight
Compared with simple phenolic acids and some small-molecule flavonoids, tannins usually have higher molecular weights and more complex conformations. Higher molecular weight enables multipoint binding more readily, resulting in pronounced protein precipitation capacity, astringency, and colloidal instability.
(3) Structural heterogeneity
Tannins in plant extracts rarely exist as a single structure. Instead, they usually occur as mixtures with different degrees of polymerization, substituents, linkage types, and oxidation states. Therefore, tannin-related functions cannot be explained only by “content level”; structural type, degree of polymerization, and sample matrix should also be considered.
1.2 Main Types
Type | Structural Basis | Typical Sources | Main Research Focus |
Hydrolyzable tannins | Gallic acid or ellagic acid forms ester bonds with a sugar core | Gallnut, pomegranate peel, oak, chestnut wood | Acid/base hydrolysis, metal chelation, antioxidant activity, and astringent effects |
Condensed tannins | Flavan-3-ol units polymerize to form proanthocyanidins | Grape seeds, cocoa, tea, sorghum, apple peel | Astringency, protein precipitation, degree of polymerization, and antioxidant activity |
Complex tannins | Hydrolyzable tannin structures combined with flavanol structures | Some medicinal plants and woody plants | Structural identification, activity differences, and quality control |
2 Tannins and Astringency in Plant Extracts
2.1 Mechanism of Astringency Formation
(1) Binding to salivary proteins
Astringency is not a typical taste, but rather an oral tactile sensation and mouth-puckering feeling. After entering the oral cavity, tannins can bind to proline-rich salivary proteins to form soluble or insoluble complexes, reducing the lubricating effect of saliva and producing dryness, roughness, and astringency.
(2) Reduced mucosal lubrication
Normal salivary proteins form a lubricating layer on the oral mucosal surface. After tannins bind to these proteins, the stability of this lubricating layer decreases, increasing friction on the oral surface and producing a pronounced astringent sensation.
(3) Multipoint crosslinking effect
Multiple phenolic hydroxyl groups on tannin molecules can interact with multiple protein sites simultaneously, forming a crosslinked network. Tannins with higher degrees of polymerization are more likely to produce multipoint binding, and therefore usually show stronger and more persistent astringency.
2.2 Factors Affecting Astringency Intensity
(1) Degree of polymerization
Oligomeric tannins and tannins with moderate degrees of polymerization usually have pronounced astringency. When the degree of polymerization is too low, protein-binding sites are limited and astringency may not be strong. When the degree of polymerization is too high, solubility decreases, precipitation may occur more readily, but sensory release may slow down.
(2) Degree of galloylation
Galloyl groups can increase the number of phenolic hydroxyl groups and enhance protein-binding capacity. Therefore, galloylated catechins, galloylated proanthocyanidins, and some hydrolyzable tannins often show strong astringency.
(3) Sample pH
pH affects the charge states of tannins and proteins. Under acidic conditions, some proteins are more likely to form complexes with tannins, so astringency may be more pronounced in fruit juices, fermented beverages, and acidic plant extracts.
(4) Coexistence of sugars, organic acids, and polysaccharides
Sugars can partially mask astringency. Pectin, polysaccharides, and proteins can bind tannins and reduce free tannin levels. Organic acids can alter flavor balance, making astringency more prominent or smoother.
2.3 Astringency in Different Extracts
(1) Tea extracts
Tea polyphenols, catechins, and some condensed tannins jointly affect the astringency of tea infusion. High concentration, polyphenol oxidation, and inappropriate extraction temperature may enhance bitterness and astringency.
(2) Grape seed and grape skin extracts
Grape seeds are rich in proanthocyanidins and show prominent astringency. In grape skins, tannins, anthocyanins, and other phenolics jointly affect the mouthfeel structure of wine, fruit juice, and extracts.
(3) Pomegranate peel and gallnut extracts
These extracts contain high levels of hydrolyzable tannins and show strong astringency. They have application value in functional foods or topical products, but oral products require careful control of dosage and taste modification.
3 Tannins and Precipitation Formation
3.1 Protein Precipitation
(1) Precipitation mechanism
After tannins bind to proteins, if the complex particle size increases beyond the stable range of the solution, turbidity or precipitation forms. Proline, hydrophobic amino acids, and basic amino acid residues in proteins often participate in tannin binding.
(2) Reversible and irreversible precipitation
Some tannin-protein complexes can redisperse after dilution, pH change, or salt concentration change. If further oxidation, crosslinking, or large-particle aggregation occurs, relatively stable precipitates may form.
(3) Application impact
In plant beverages, concentrated extracts, and fermentation broths, tannin-protein precipitation may cause turbidity, flocculation, and shelf-life instability. In clarification processes, this property can also be used to remove excess proteins or reduce astringency.
3.2 Polysaccharide and Colloidal Precipitation
(1) Polysaccharide bridging
Tannins can interact with pectin, gum arabic, starch degradation products, dietary fiber fragments, and other polysaccharides. Some polysaccharides can stabilize tannin colloids, while others may promote bridging flocculation.
(2) Concentration effect of extracts
After concentration, drying and reconstitution, or long-term storage of plant extracts, the equilibrium among tannins, polysaccharides, proteins, and inorganic salts may be disrupted, leading to increased turbidity or precipitation after reconstitution.
(3) Influence of processing conditions
Heating, low-temperature storage, pH adjustment, metal ion introduction, and changes in ethanol concentration can all affect tannin colloidal stability. Precipitation risk is especially important in beverages, oral liquids, and high-concentration plant extracts.
3.3 Metal Ion Chelation and Color Precipitation
(1) Iron ion chelation
Tannins have strong metal-chelating ability and readily form dark complexes with iron ions. If iron contamination or contact with metal containers occurs in plant extracts, color darkening, precipitation, or activity changes may result.
(2) Copper ion-catalyzed oxidation
Copper ions can promote polyphenol oxidation reactions, causing further tannin polymerization and resulting in browning and precipitation. In some antioxidant systems, tannins can chelate metals to inhibit oxidation, but under certain conditions, they may also participate in pro-oxidant reactions.
(3) Quality control significance
For high-tannin plant extracts, sources of metal ions should be controlled. When necessary, iron, copper, and other elements should be tested, and changes in color, turbidity, and precipitation during storage should be evaluated.
4 Tannins and Antioxidant Activity
4.1 Free Radical Scavenging
(1) Hydrogen- and electron-donating ability
Phenolic hydroxyl groups in tannin molecules can donate hydrogen atoms or electrons to free radicals, converting them into more stable forms. Free radical scavenging assays such as DPPH and ABTS can often reflect the antioxidant capacity of tannins.
(2) Structural stabilization
Phenoxy radicals formed after tannin oxidation can be stabilized through resonance and intramolecular interactions, making them less likely to continue chain reactions. The number of aromatic rings, catechol structures, and galloyl groups all enhance this stabilization ability.
(3) Effect of polymerization degree
Oligomeric proanthocyanidins usually show good free radical scavenging capacity. Although polymeric tannins contain more phenolic hydroxyl groups, their reduced solubility and diffusibility may limit results in in vitro assay systems.
4.2 Metal Chelation-Based Antioxidant Activity
(1) Inhibition of Fenton reaction
Metal ions such as iron and copper can catalyze the generation of hydroxyl radicals from hydrogen peroxide. Tannins can reduce the probability of metal-catalyzed oxidation by chelating metal ions.
(2) Protection of lipid systems
In oils, emulsions, and cell membrane models, metal ions often promote lipid peroxidation. If tannins can effectively chelate metals and scavenge free radicals, they can reduce the formation of lipid oxidation products.
(3) Dual effects
Tannins do not act as antioxidants under all conditions. High tannin concentrations, free metal ions, and alkaline environments may promote polyphenol autoxidation, generating peroxides or quinone structures. Therefore, antioxidant evaluation should consider both dosage and system conditions.
4.3 Relationship with Antioxidant Assay Methods
Assay Method | Tannin Response Characteristics | Key Interpretation Points |
DPPH assay | Clear response to alcohol-soluble tannins and some proanthocyanidins | Affected by solubility and reaction time |
ABTS assay | Good compatibility with both water-soluble and alcohol-soluble tannins | Suitable for comparing total scavenging capacity of plant extracts |
FRAP assay | Strong response to highly reducing tannins | Reflects electron transfer capacity, not equivalent to free radical scavenging |
CUPRAC assay | Sensitive to polyphenols and metal reducing capacity | Suitable as a complementary method to FRAP |
ORAC assay | Can evaluate peroxyl radical absorbance capacity | Emphasizes dynamic protective capacity |
TBARS assay | Can evaluate inhibition of lipid oxidation | Closer to food and biomembrane oxidation scenarios |
5 Relationship Among Astringency, Precipitation, and Antioxidant Activity
5.1 Shared Structural Basis
Astringency, precipitation, and antioxidant activity are not isolated properties. They are all related to the phenolic hydroxyl groups, molecular weight, degree of polymerization, and binding capacity of tannins. Tannins that strongly bind proteins often also have strong reducing capacity and metal-chelating ability. However, this does not mean that stronger astringency necessarily indicates better antioxidant activity.
5.2 Functional Conflicts
(1) High antioxidant activity and high astringency
High tannin content can improve antioxidant capacity, but may also cause unacceptable astringency. In functional beverages, plant extract powders, and oral products, a balance between activity and sensory acceptability is often required.
(2) High activity and precipitation risk
The more readily tannins bind to proteins or polysaccharides, the more likely they are to cause colloidal instability. High-polyphenol plant extracts are especially prone to precipitation when combined with protein beverages, collagen peptide products, or mineral-containing formulations.
(3) Oxidative stability and color change
Tannins have strong antioxidant capacity, but they are also readily oxidized themselves. During storage, oxidative polymerization of tannins can lead to color darkening, increased precipitation, and activity changes.
5.3 Balancing Strategies in Product Development
(1) Control extraction intensity
Increasing ethanol concentration, prolonging extraction time, or raising temperature may increase tannin extraction yield, but may also increase astringency and precipitation risk. Extraction conditions should be adjusted according to product positioning.
(2) Select appropriate molecular weight fractions
Oligomeric tannins are more suitable for functional activity and absorption-related studies. Polymeric tannins are more likely to cause strong astringency and precipitation. Membrane separation, resin adsorption, or solvent fractionation can be used to regulate tannin fractions.
(3) Formula masking and stabilization
Sugars, acidulants, proteins, pectin, cyclodextrins, and some polysaccharides can regulate tannin taste and stability. However, some combinations may aggravate turbidity or precipitation, and should be verified through stability testing.
6 Detection and Characterization of Tannins in Plant Extracts
6.1 Total Tannin Determination
(1) Protein precipitation method
The ability of tannins to bind and precipitate proteins can be used to indirectly evaluate total tannin levels. This method is strongly related to astringency and precipitation risk, but its responses to different tannin structures are not completely consistent.
(2) Folin-Ciocalteu method
This method can evaluate total reducing phenolics but is not tannin-specific. When used for tannin samples, it should be interpreted together with total phenols, non-tannin phenols, and protein precipitation methods.
(3) Vanillin assay and DMACA assay
These methods are commonly used to detect flavan-3-ols and oligomeric proanthocyanidins and are frequently applied in condensed tannin research. Results are affected by monomer composition, degree of polymerization, and reaction conditions.
6.2 Hydrolyzable Tannin Analysis
(1) Detection of gallic acid and ellagic acid
After acid, alkaline, or enzymatic hydrolysis, hydrolyzable tannins can release structural units such as gallic acid and ellagic acid. HPLC or LC-MS can be used to quantify these markers.
(2) Evaluation of tannic acid-type compounds
Tannic acid is often used as a model hydrolyzable tannin for studies of protein precipitation, antioxidant activity, and astringency. However, hydrolyzable tannins from different plant sources vary greatly in structure and cannot be fully represented by tannic acid.
(3) Structural complexity
Hydrolyzable tannins can form polygalloyl glucose, ellagitannins, and complex tannins. Structural elucidation usually requires high-resolution mass spectrometry, NMR, or comparison with standards.
6.3 Condensed Tannin Analysis
(1) Proanthocyanidin content
Condensed tannins often exist as proanthocyanidins and can be measured by the acid-butanol method, DMACA assay, or dedicated OPC assay kits.
(2) Polymerization degree analysis
Thiolysis, phloroglucinolysis, or LC-MS analysis can be used to determine mean degree of polymerization, extension units, and terminal units. Degree of polymerization is an important indicator for explaining differences in astringency, precipitation capacity, and antioxidant activity.
(3) A-type and B-type linkages
A-type and B-type proanthocyanidins differ in linkage mode, which may affect conformation, antioxidant activity, and biological activity. A-type proanthocyanidins are often emphasized in materials such as cranberries, while B-type proanthocyanidins are common in grape seeds and cocoa.
7 Application Characteristics of Tannins from Different Plant Sources
7.1 Tea
Catechins, galloylated catechins, and some polymerized polyphenols in tea jointly affect astringency, bitterness, and antioxidant capacity. Green tea extracts often have strong antioxidant activity, but astringency is pronounced at high concentrations. In fermented tea, polyphenol oxidation and polymerization alter taste and precipitation behavior.
7.2 Grape Seeds and Grape Skins
Grape seeds are rich in proanthocyanidins and have strong protein-binding capacity and astringency. Grape skins contain anthocyanins, tannins, and phenolic acids, affecting color, astringency, and storage stability in wine and fruit peel extracts.
7.3 Pomegranate Peel
Pomegranate peel is rich in hydrolyzable tannins and ellagitannins, with strong antioxidant capacity and pronounced astringency. Its extract is suitable for functional activity research, but astringency and precipitation control should be emphasized when used in foods or oral products.
7.4 Gallnut
Gallnut contains high levels of hydrolyzable tannins, shows strong astringency, and has prominent protein precipitation capacity. Its extract is representative in tannin research, astringent effect studies, and antibacterial research, but sensory acceptability is low at high dosage.
7.5 Cocoa and Sorghum
Proanthocyanidins in cocoa are closely related to bitterness, astringency, antioxidant activity, and processing flavor. Condensed tannins in sorghum can affect feed value, digestibility, and food mouthfeel, and are also associated with antioxidant activity and seed coat defense.
8 Effects of Processing and Storage on Tannin Properties
8.1 Heat Treatment
Moderate heating can promote tannin release from cell walls and increase extraction yield. Excessive heating may cause tannin oxidation, polymerization, or degradation. Heat treatment can also alter the binding mode between tannins and proteins or polysaccharides, affecting precipitation and astringency.
8.2 Fermentation
During fermentation, microbial enzymes can degrade some tannins or transform phenolic structures, reducing astringency and improving mouthfeel. Some microorganisms can also release bound polyphenols and improve antioxidant capacity. Fermentation effects depend on strain, substrate structure, and fermentation conditions.
Tannins can undergo oxidative polymerization under the action of oxygen, metal ions, or polyphenol oxidase, resulting in color darkening, increased molecular weight, and increased precipitation. Such changes may occur during storage of tea, fruit juice, wine, and plant extracts.
8.4 Drying and Reconstitution
Spray drying, freeze drying, and hot-air drying can affect tannin distribution, oxidation degree, and reconstitution stability. Turbidity after reconstitution of high-tannin extract powders is often related to reaggregation of tannin-protein, tannin-polysaccharide, or tannin-mineral complexes.
9 Quality Control in Research and Application
9.1 Sensory Control
(1) Evaluation of astringency intensity
Astringency intensity can be evaluated by sensory assessment, electronic tongue, salivary protein precipitation experiments, or tribological methods. For functional foods and beverages, sensory control is as important as activity evaluation.
(2) Taste modification
Sweetness, acidity, polysaccharide thickening, and aroma modification can reduce perceived astringency, but cannot completely eliminate the protein-binding properties of tannins. In formulation development, both taste and stability should be considered.
9.2 Precipitation Stability Control
(1) Centrifugation and turbidity detection
Plant extracts can be evaluated for precipitation risk through turbidity, centrifuged sediment amount, particle size distribution, and storage stability tests.
(2) Protein compatibility testing
If extracts are used in protein beverages, peptide products, or dairy products, protein compatibility testing should be performed to evaluate the effects of pH, ionic strength, and heat treatment on precipitation.
(3) Metal ion control
Introduction of metal ions such as iron and copper should be avoided as much as possible. When necessary, chelators, filtration purification, or packaging material optimization can be used to reduce color and precipitation changes.
9.3 Antioxidant Activity Control
(1) Combination of multiple methods
Tannin-rich extracts should be evaluated using at least one free radical scavenging method and one reducing power method, such as DPPH/ABTS combined with FRAP/CUPRAC.
(2) Blank subtraction
High-tannin samples are often dark-colored and turbid, which can interfere with absorbance. Sample blanks and solvent blanks must be included in antioxidant assays.
(3) Component association
Antioxidant results should be correlated with total phenols, total tannins, proanthocyanidins, or hydrolyzable tannin markers to avoid judging product quality by a single activity value.
10 Reagent and Material Selection for Tannin Research
10.1 Tannin Standards, Colorimetric Detection Reagents, and Functional Evaluation Reagents
Product Module | Product Name | CAS No. | Role in the System | Applicable Scenario |
Hydrolyzable tannin standard | Tannic Acid | Representative hydrolyzable tannin standard | Methodological control for total tannins, protein precipitation, astringency mechanism, and antioxidant evaluation | |
Hydrolyzable tannin structural unit | Gallic Acid | Standard for galloyl structural units | Analysis of hydrolyzable tannin hydrolysis products and total phenol standard curve | |
Hydrolyzable tannin structural unit | Ellagic Acid | Ellagitannin-related marker | Analysis of hydrolyzable tannins in pomegranate peel, gallnut, nut skins, and related materials | |
Total condensed tannin standard | Proanthocyanidins | Representative condensed tannin standard | Proanthocyanidin content determination and antioxidant activity evaluation | |
Oligomeric proanthocyanidins | Grape Seed Oligomeric Proanthocyanidins | Representative OPC material | Grape seed extracts and functional evaluation of oligomeric tannins | |
Flavan-3-ol monomer | (+)-Catechin | Basic structural unit of condensed tannins | Flavan-3-ol composition analysis and HPLC quantification | |
Flavan-3-ol monomer | (-)-Epicatechin | Proanthocyanidin structural unit | Tannin composition analysis in tea, grape seed, and cocoa | |
Galloylated catechin | Epigallocatechin Gallate | Galloylated flavanol standard | Research on tea polyphenol astringency, protein binding, and antioxidant activity | |
A-type proanthocyanidin | Procyanidin A1 | A-type dimeric proanthocyanidin standard | A-type/B-type linkage differences and cranberry-type material analysis | |
A-type proanthocyanidin | Procyanidin A2 | A-type dimeric proanthocyanidin standard | Oligomeric tannin structural analysis and functional component quantification | |
B-type proanthocyanidin | Procyanidin B1 | B-type dimeric proanthocyanidin standard | Tannin composition analysis in grape seed, cocoa, and fruit peel | |
B-type proanthocyanidin | Procyanidin B2 | B-type dimeric proanthocyanidin standard | HPLC/LC-MS quantification and condensed tannin structural research | |
Trimeric proanthocyanidin | Procyanidin C1 | Trimeric proanthocyanidin standard | Polymerization degree analysis and functional evaluation of oligomeric tannins | |
Galloylated proanthocyanidin | Procyanidin B2 3′-Gallate | Galloylated proanthocyanidin standard | Analysis of esterified tannins in tea, grape seed, and cocoa | |
Galloylated proanthocyanidin | Procyanidin B2 3,3′-Di-O-gallate | Di-galloylated proanthocyanidin standard | Research on esterification degree, astringency intensity, and protein-binding capacity | |
Total phenol standard | Gallic Acid | Total phenol standard curve | Folin-Ciocalteu method and plant extract polyphenol evaluation | |
Condensed tannin color development | Vanillin | Chromogenic reagent for the vanillin assay | Preliminary determination of flavan-3-ols and oligomeric proanthocyanidins | |
Condensed tannin color development | 4-Dimethylaminocinnamaldehyde | Chromogenic reagent for DMACA assay | High-sensitivity colorimetric detection of proanthocyanidins and catechins | |
Condensed tannin structural analysis | Phloroglucinol | Phloroglucinolysis reagent | Analysis of proanthocyanidin extension units, terminal units, and mean degree of polymerization | |
Protein precipitation evaluation | Gelatin | Protein precipitation model material | Total tannin determination and evaluation of astringency-related precipitation capacity | |
Protein precipitation evaluation | Casein | Food protein model substrate | Compatibility evaluation between tannins and milk proteins/protein beverages | |
Astringency/precipitation control | Polyvinylpyrrolidone | Binds polyphenols and reduces free tannins | Removal of polyphenol interference and reduction of astringency and precipitation risk | |
Astringency/precipitation control | Polyvinylpolypyrrolidone | Adsorbs polyphenols and tannins | Beverage clarification, tannin removal, and extract stability optimization | |
Colloidal stability evaluation | Pectin | Polysaccharide interaction model | Evaluation of tannin-polysaccharide formulation, turbidity, and precipitation risk | |
Free radical scavenging evaluation | DPPH | Stable free radical probe | Determination of free radical scavenging capacity of tannin extracts | |
Free radical scavenging evaluation | ABTS | ABTS⁺ radical substrate | Antioxidant evaluation of water-soluble and alcohol-soluble tannins | |
Reducing capacity evaluation | TPTZ | FRAP chromogenic ligand | Determination of ferric reducing antioxidant power | |
Antioxidant standard | Trolox | Antioxidant equivalent standard | Standard curves for ABTS, FRAP, ORAC, and related methods | |
Lipid oxidation evaluation | Thiobarbituric Acid | TBARS chromogenic reagent | Lipid peroxidation inhibition and MDA equivalent analysis |
Tannins play three roles in plant extracts: sensory contribution, physicochemical stability regulation, and antioxidant function. Their polyphenolic hydroxyl structures provide free radical scavenging and metal-chelating capacity, while also causing protein binding, astringency, and precipitation risk. In the development and evaluation of plant extracts, tannin content, structural type, degree of polymerization, protein precipitation capacity, antioxidant activity, and storage stability should all be included in the analysis to achieve a balance between functional value and product acceptability.
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