Tannin notes

Hey,

I am a B.Pharma. student. This is my personal notes on the topic called Tannins, a topic from Unit 2 i.e. Secondary metabolites from the subject Pharmacognosy and Phytochemistry II from Semester 5th. Syllabus of this subject is according to GTU. Hope this will be much helpful for your reading.

Thank you.

1. TANNINS Degree : B.Pharma . Subject : Pharmacognosy and phytochemistry II Semester : 5 th Notes by Jahanvi Tankaria

2. Contents Introduction to Tannins Physical properties Chemical properties Classification Identification tests Medicinal properties and uses Importance of tannins Biosynthesis of tannins Isolation

3. Introduction Tannins are complex organic non-nitrogenous, polyphenolic plant products showing astringent and antioxidant property . The name ‘tannin’ is derived from the French ‘ tanin ’ (tanning substance) and is used for a range of natural polyphenols . The term ‘tannin’ was first used by Seguin in 1796 to denote substances which have the ability to combine with animal hides to convert them into leather which is known as tanning of the hide . This term was used to denote substances present in plant extract which react with protein of animal hide, prevent their putrefaction (decay or rotting) and convert hide/skin into leather.

4. They have a property to tan animal skin to convert to leather. Conversion imparts resistance to water, heat, and abrasives. They have property to precipitate gelatin & heavy metals. They can be extracted using water-acetone/alcohol mixture. Tannins are widely distributed in several plant species and are found in wood, bark, leaves and fruits. Natural polyphenols – contain sufficient hydroxyl group and other suitable group (such as carboxyl) to form strong complex with protein and other macromolecules. Astringent property – precipitate proteins (Latin – Astringere which means to bind fast) Mostly high molecular weight compound and are water soluble.

5. Physical properties State - Amorphous, non crystalline Molecular weight = 500 to >20,000 D Solubility - Soluble in – water, alcohol, dilute alkali, glycerin, acetone - Sparingly soluble in – Ethyl acetate - Insoluble in – Organic solvent except acetone Form colloidal solution with water Taste - Astringent taste Combine with skin/hide to form leather

6. Chemical properties 1) Precipitation Tannin precipitate with protein, gelatin, alkaloids, glycosides and heavy metals. Tannin precipitate by salt of copper, tin, lead. (e.g. copper acetate, lead acetate ) Precipitated by strong potassium dichromate or chromic acid solution. 2) Antioxidant property Antioxidants property (Free radicals which are highly reactive binds to cell and damages cell so to prevent damage antioxidants bind with those free radicals) – polyhydroxy phenolic compounds. Because of accumulation of OH group on small size nucleus, these agents have anti-oxidant nature.

7. 3) Astringent property Tannins have property to react with protein of mucous membrane, shrinks the cells there and cause precipitation. 4) Show acidic reaction due to phenolic group as they donate proton and form phenoxide ion (Aqueous solution of tannin is acidic in nature) 5) Carcinogenicity Prolong use of tannin containing plant material is hazardous because it causes cancer. Habitual use of Areca catechu can cause oral and esophageal cancer. 6) Reaction with iron/ferric salts Hydrolysable tannin + FeCl 3 → Blue black color precipitates Condensed tannin + FeCl 3 → Brownish green color precipitates 7) Reaction with potassium ferricyanide and ammonia Tannin + potassium ferricyanide /ammonia → Deep red color formation

8. Classification The tannin compounds can be divided into two major groups on the basis of Goldbeater’s skin test. A group of tannins showing the positive tanning test may be regarded as true tannins, whereas those, which are partly retained by the hide powder and fail to give the test, are called as pseudotannins .

9. 1) True Tannins: - Most of the true tannins are high molecular weight compounds. These compounds are complex polyphenolics , which are produced by polymerization of simple polyphenols . They may form complex glycosides or remains as such which may be observed by their typical hydrolytic reaction with the mineral acids and enzymes. Two major chemical classes of tannins are usually recognized based on this hydrolytic reaction and nature of phenolic nuclei involved in the tannins structure. The first class is referred to as hydrolysable tannins, whereas the other class is termed as condensed tannins. a) Hydrolysable Tannins: - These tannins are hydrolysable by mineral acids or enzymes such as tannase .

10. Their structures involve several molecules of polyphenolic acids such as gallic , hexahydrodiphenic , or ellagic acids, bounded through ester linkages to a central glucose molecule (β D-glucose). On the basis of the phenolic acids produced after the hydrolysis, these are categorized further into Gallotannins and Ellagitannins . Gallotannins which on hydrolysis produces Gallic acid. Ellagitannins on hydrolysis produces Ellagic acid ( Ellagic acid is formed by intraesterification of hexahydrodiphenic acid). Hydrolysable tannins are sometimes referred to as pyrogallol tannins as the components of phenolic acids on dry distillation are converted to pyrogallol derivatives. The hydrolysable tannins are soluble in water, and their solution produces blue colour with ferric chloride.

11. Biosynthetically, gallic acid (= 3,4,5-trihydroxybenzoic acid) arises from the metabolism of shikimic acid. Hydrolysable tannin or pyrogallol -type tannin is a type of tannin that, on heating with hydrochloric or sulfuric acids, yields gallic or ellagic acids.

12. b) Condensed/Non hydrolysable Tannins: - Are not readily hydrolysable to simpler molecules with mineral acids and enzymes, thus they are also referred to as nonhydrolysable tannins. Also known as Proanthocyanidins . Contain only phenolic nuclei which are biosynthetically derived from flavonoids ( Flavanol , Catechin , Flavan-3,4-diol these are polymers of flavanoid ). When treated with acids or enzymes, they tend to polymerize (self condensate) yielding insoluble red coloured products known as phlobaphens . The phlobaphens give characteristic red colour to many drugs such as cinchona and wild cherry bark. On dry distillation, they yield catechol derivatives. Condensed tannins are also soluble in water and produces green colour with ferric chloride.

13. E.g. Cocoa bean, Pterocarpus , Cinchona, Cinnamon bark, Black catechu, Pale catechu, Wild cherry, Cinchona barks. They consist of flavan-3-ol units linked together by carbon-carbon bonds, most often 4→8 or 4→6, which result from coupling between the electrophilic C-4 of a flavanyl unit from a flavan-4-ol or flavan-3,4-diol and a nucleophilic position (C8, less commonly C-6) of another unit, generally a flavan-3-ol. Unlike hydrolysable tannins, these are not readily hydrolysable to simpler molecules and they do not contain a sugar moiety. Biosynthetically, flavonoids are derived from acetate and shikimate pathways. Polymers may include up to 50 monomer units. Condensed tannins occur due to polymerization (condensation) reactions between flavonoids . Non-hydrolysable tannins on heating with hydrochloric acid yield phlobaphenes like phloroglucinol .

14. Examples in detail Barks – Cinnamon, wild cherry, cinchona, willow, acacia, oak & hamamelis Roots and rhizomes – Krameria ( rhatany ) and male fern Flowers – Lime and hawthorn Seeds – Cocoa, guarana , kola Leaves – Hamamelis , hawthorn and tea, especially green tea Extracts and dried juices – catechu, acacia and mangrove cutches c) Complex Tannins These tannins are mixtures of both, hydrolysable and condensed tannins E.g. Tea, Quercus , Castanea

15. 2 ) Pseudo Tannins: - Pseudotannins are simple phenolic compounds of lower molecular weight. Do not give + ve Gold Beater's skin test. But responds most tests of the tannin. E.g. Chlorogenic acid – Nux vomica , Coffee, Mate Ipecacuanhic acid – Ipecac root/ Ipecacuanha Galllic acid – Rhubarb Catechins – Guarana , Cocoa Gallic acid, Chlorogenic acid, or the simple phenolics such as catechin are pseudotannins which are abundantly found in plants, especially in dead tissues and dying cells.

16. Identification tests 1) Goldbeater's skin test: - Goldbeater’s skin is a membrane produced from the intestine of Ox. It behaves just like untanned animal hide. Goldbeater skin soaked in 2% HCl (5 min) → Rinse with distilled water → Place in solution of tannin (5 min) → Wash with distilled water → Treat with 1% ferrous sulphate solution → Black brown colour appearance on skin indicates presence of tannins . Hydrolysable and condensed tannins both give the positive goldbeater’s test, whereas pseudotannins show very little colour or negative test.

17. 2) Gelatin test: - 1% gelatin sol n + 10% NaCl sol n + 1% tannin sol n → White buff colour precipitate Tannins cause precipitation of gelatin from solution . 3) Phenazone test: - 5 ml aq. tannin sol n + 0.5 gm Sodium acid phophate / sodium hydrogen phosphate → Warm → Cool & filter it → Filtrate + 2% phenazone sol n → Bulky coloured precipitates produced . 4) Test for Chlorogenic acid: - Aq. tannin sol n + aq. ammonia → Expose it to air → Green colour appears grdually due to presence of chlorogenic acid . 5) Vannilin HCl acid test: - Sample sol n + Vannilin HCl reagent (1 g vannilin + 10 ml alcohol + 10 ml dil. HCl sol n ) → Pink or red colour dur to formation of phloroglucinol

18. 6) Test for Catechin (Matchstick test): - Catechin test is the modification of the well-known phloroglucinol test for lignin. Matchstick contains lignin. Dip the matchstick in aq. sol n of tannin/plant extract → Dry it near the flame → Moisten with Conc. HCl → Warm it near the flame → Matchstick turns Pink or Red in colour due to phloroglucinol formation. ( Catechin in the presence of acid produces phloroglucinol which stains the lignified wood pink or red) 7) Ferric chloride test: - Tannin sol n + FeCl 3 → Blue colour → Confirms presence of Hydrolysable tannins (Gallic acid, Ellagic acid) Tannin sol n + FeCl 3 → Brownish green colour → Confirms presence of Condensed tannins ( Catechol solution)

19. Medicinal properties and uses Tannins occur in crude drugs either as major active constituent as in oak bark, hammamelis leaves, and bearberry leaves, etc. or as a subsidiary component as in clove, cinnamon, peppermint, or garden sage. In many cases, they synergistically increase the effectiveness of active principles. Tannins are medicinally significant due to their astringent properties. They promote rapid healing and the formation of new tissues on wounds and inflamed mucosa . Tannins are used in the treatment of varicose ulcers, haemorrhoids , minor burns, frostbite, as well as inflammation of gums

20. Internally tannins are administered in cases of diarrhoea , intestinal catarrh, and in cases of heavy metal poisoning as an antidote. In recent years, these compounds have demonstrated their antiviral activities for treatment of viral diseases including AIDS. Tannins are used as mordant in dyeing, manufacture of ink, sizing paper and silk, and for printing fabrics. It is used along with gelatine and albumin for manufacture of imitation horn and tortoise shell. They are widely used in the leather industry for conversion of hide into leather, the process being known as tanning. Tannins are also used for clarifying beer or wine, in photography or as a coagulant in rubber manufacture. Tannins are used for the manufacture of gallic acid and pyrogallol , and sometimes as a reagent in analytical chemistry.

21. Importance of tannins Medicinal uses: Antidote Antiseptic Algicidals Astringents Anti-carcinogenic Antibiotic, Herbicides, Insecticides Industrial uses: Ink manufacute Vegetable tanning Preservatives Flavouring agent Perfumery agent Biological activities: Inhibition of lipid peroxidation Decrease in blood urea nitrogen content Inhibition of plasma Lipolysis in fat cells

22. Medicinal and biological properties: Tannin containing drugs precipitate proteins and have been traditionally used as styptics (stop hemorrhage) and internally for the protection of inflamed surfaces of mouth and throat. They play an important role in treatment of burns. They form a mild antiseptic protective layer on the surface of the injured skin below which regeneration of new tissue takes place. They act as anti-diarrheal, although not recommended in this respect as they usually delay elimination of bacterial toxins from the body. Tannins have been employed as antidote in poisoning by heavy metals, alkaloids and certain glycosides due to their precipitation as tannates . Recently tannins as most polyphenols were proved to have a potent antioxidant effect.

23. Biosynthesis of tannins Tannins belong to the phenolics class of secondary metabolites . All phenolic compounds; either primary or secondary are in one way or another formed through shikirnic acid pathway ( phenylpropanoid pathway). Other phenolics such as isoflavones , coumarins , lignins , and aromatic amino acids (tryptophan, phenylalanine, and tyrosine) are also formed by the same pathway. Hydrolysable tannins ( Hts ) and condensed tannins ( proanthocyanidins ) are the two main categories of tannins that impact animal nutrition.

24. Common tannins are formed as follows: Gallic acid is derived from quinic acid. Ellagotannins are formed from hexahydroxydiphenic acid esters by the oxidative coupling of neighbouring gallic acid units attached to a D-glucose core. Further oxidative coupling forms the hydrolysable tannin polymers . Proanthocyanidin (PA) biosynthetic precursors are the leucocyanidins (flavan-3,4-diol and flavan-4-ol) which on autoxidation , in the absence of heat, form anthocyanidin and 3-deoxyanthocianidin, which, in turn, polymerize to form PAs.

25. Isolation Both hydrolysable and condensed tannins are highly soluble in water and alcohol but insoluble in organic solvents such as solvent ether, chloroform, and benzene. Tannin compounds can be easily extracted by water or alcohol . The general method for the extraction of tannic acid from various galls is either with water-saturated ether, or with mixture of water, alcohol, and ether . In such cases, free acids such as Gallic and ellagic acid go along with ether , whereas true tannin gets extracted in water . If the drug consists of chlorophyll or pigment , it may be removed by ether . After extraction, the aqueous and ethereal layers are separately concentrated, dried, and subjected to further isolation and purification using various separation techniques of chromatography.

26. Thank you