GLYCOSIDES PHARMACOGNOSY DR. KIBOI

Glycosides Glycosides • Glycosides consist of a sugar residue covalently bound to a different structure called the aglycone

• The sugar residue is in its cyclic form and the point of attachment is the hydroxyl group of the hemiacetal function. The sugar moiety can be joined to the aglycone in various ways:

1.Oxygen (O-glycoside)

2.Sulphur (S-glycoside)

3.Nitrogen (N-glycoside)

4.Carbon ( Cglycoside) • α-Glycosides and β-glycosides are distinguished by the configuration of the hemiacetal hydroxyl group. • The majority of naturally-occurring glycosides are β-glycosides. • O-Glycosides can easily be cleaved into sugar and aglycone by hydrolysis with acids or enzymes. • Almost all plants that contain glycosides also contain enzymes that bring about their hydrolysis (glycosidases ). • Glycosides are usually soluble in water and in polar organic solvents, whereas aglycones are normally insoluble or only slightly soluble in water.

• It is often very difficult to isolate intact glycosides because of their polar character.

• Many important drugs are glycosides and their pharmacological effects are largely determined by the structure of the aglycone. • The term 'glycoside' is a very general one which embraces all the many and varied combinations of sugars and aglycones.

• More precise terms are available to describe particular classes. Some of these terms refer to: 1.the sugar part of the molecule (e.g. glucoside ). 2.the aglycone (e.g. anthraquinone). 3.the physical or pharmacological property (e.g. saponin “soap-like ”, cardiac “having an action on the heart ”). • Modern system of naming glycosides uses the termination '-oside' (e.g. sennoside).

• Although glycosides form a natural group in that they all contain a sugar unit, the aglycones are of such varied nature and complexity that glycosides vary very much in their physical and chemical properties and in their pharmacological action. Types of Glycosides

• Anthracene glycosides • Saponin glycosides • Coumarin glycosides • Flavonoid glycosides • Cyanogenetic glycosides (Cyanide glycosides) 1. Anthracene glycosides

Anthracene

• A number of glycosides in which the aglycones are anthracene derivatives occur as the pharmacologically active constituents of several cathartics of plant origin; e.g. cascara, rhubarb, aloe and senna. • anthracene glycosides are sometimes referred to as the anthraquinone glycosides or the anthraglycosides. • anthraquinone derivatives are glycosides, often glucosides or rhamnosides.

• The presence of the sugar residue is a prerequisite for the pharmacological effects.

• Anthraquinones are colored substances and many of them are used technically as dyes e.g. alizarin.

• Reduced forms of anthraquinones, which exhibit keto-enol tautomerism, are often encountered. • The anthracene derivatives occur in vegetable drugs in different forms at different oxidation levels; like anthraquinones, anthrones, anthranols, or oxanthrones. Inter-relationship of anthraquinone derivatives

OH

Anthranol (enol form)

Taut.

O O 8 1 7 9 2 4 H ABC 6 10 3 5 4 Anthrone (keto form) O Anthraquinone − 2 H

2 H O

O

H OH

Oxanthrone O Dianthrone • Anthracene compounds occur in these drugs or plant materials in some cases as the aglycones of O-glycosides (e.g. frangulin), and in other cases as the aglycones of C-glycosides (e.g. aloin). • Biosynthesis ; natural anthraquinones are synthesized either via the acetate-malonate pathway (like the medicinally important purgative anthraquinones), or they are derived from shikimate and mevalonate (like alizarin).

OH O OH O OH

OH

HO CH3

O O

Frangula emodin Alizarin A. Anthraquinones

• Although anthraquinone is not used extensively in medical practice, it is the starting material for the preparation of several synthetic laxatives and represent the basic structure of a number of important laxatives and dyestuffs. • Borntrager ’s test is often used for their detection. • The derivatives of anthraquinone present in purgative drugs may be dihydroxy phenols such as chrysophanol, trihydroxy phenols such as emodin or tetrahydroxy phenols such as carminic acid. • Bontrager's test is a chemical test for the identification of anthraquinone glycosides. • boil test sample with 1ml of sulphuric acid in a test tube for 5min,filter while hot. • Cool the filtrate and shake with equal volume of dichloromethane or chloroform then separate the lower layer of chloroform and shake it with half volume of dilute ammonia. • A rose pink to red colour is produced in the ammonical layer. O

O Anthraquinone

OH O OH OH O OH

CH3 HO CH3 O O Chrysophanol Emodin

HO

O OH O CH3 OH COOH HO OH

HO OH

OH O

Carminic Acid B. Anthrones & Anthranols • These reduced anthraquinone derivatives occur either free or combined as glycosides. • They are isomeric and one may be partially converted to the other in solution. • Anthranols are converted upon oxidation into anthraquinones. Oxidation takes place in the crude drug during storage especially if powdered. • Schonteten’s test is often used for anthranols (green fluorescence). • Anthranols and anthrones are the main constituents of chrysarobin, a mixture of substances. OH OH OH OH O OH

CH3 CH3

Chrysarobin

(1,8-dihydroxy-3-methyl-9-anthrone; 3-methyl-1,8,9-anthracenetriol) C. Oxanthrones OH O OH

H OH

• These are intermediate products between anthraquinones and anthranols. • They give anthraquinones on oxidation with hydrogen peroxide. • An oxanthrone has been reported as a constituent of cascara bark. D. Dianthrones β-D-glucose–O O OH

These are compounds derived from • COOH

two anthrone molecules, which may COOH be identical or different. • They are important aglycones in β species of Cassia , Rheum and -D-glucose–O O OH Rhamnus . Sennosides • One of the best known is sennoside derived from two molecules of glucose and two molecules of rhein- anthrone. • On hydrolysis, sennoside yields the aglycone sennidin. E. Aloin-type or C-glycosides OH O OH • Aloin (Barbaloin) was obtained

from species of Aloe . OH • It is strongly resistant to normal HO

acid hydrolysis. O • In aloin, the sugar is joined to OH Aloin aglycone with a direct C-C linkage HO OH (a C-glycoside). • Two aloins (A and B) are known and arise from the chiral centre at C-10. 2. Saponin glycosides • A group of plant glycosides known as saponins share in varying degrees, two common characteristics: (a) They foam in aqueous solution. (b) They cause haemolysis of red blood cells.

• The aglycones of the saponins are collectively referred to as Sapogenins . The more poisonous saponins are often called Sapotoxins . • Plant materials containing saponins have long been used in many parts of the world for their detergent properties for example, in Europe, the root of Saponaria officinalis (Fam. Caryophyllaceae ) and in South America, the bark of Quillaia saponaria (Fam. Rosaceae). Such plants contain a high percentage of the glycosides known as saponins (Latin Sapo, means Soap) which are characterized by their property of producing a frothing aqueous solution. Properties: • Saponins form colloidal solution in water (hydrophilic colloids) which froths upon shaking. These substances modify and lower the surface tension and therefore foam when shaken. This has led to their use to increase the foaming of beer.

• Practical industrial applications of saponins include their use in cleaning industrial equipment and fine fabrics and as powerful emulsifiers of certain resins, fats and fixed oils. • In general, they have a bitter, acrid taste and drugs containing them are usually sternutatory (causing or producing sneezing ) and irritating to the mucous membranes of eyes and nose. • Characteristic for all saponins is their ability to cause haemolysis of red blood corpuscles and to destroy them. When injected into the blood stream, they are highly toxic. • When taken by mouth, Saponins are comparatively harmless, being not absorbed from the intestinal tract. Sarsaparilla , for example, is rich in saponins but is widely used in the preparation of nonalcoholic beverages. • Saponins are toxic especially to cold-blooded animals e.g. frogs. Many are used as fish-poisons. • The actual cause of the haemolysis : The red blood cells carry sterols in their membranes, and when brought into contact with saponins, the sterols of the RBCs are precipitated and the colloidal chemical properties of the membrane are so altered as to give hemoglobin passage to the surrounding medium.

• Saponins have a high molecular weight and their isolation in a state of purity presents some difficulties. • Structure of Saponins: According to the structure of the aglycone or sapogenin, two kinds of saponin are recognized: 1.The steroidal type (commonly tetracyclic triterpenoids, C-27). 2.The triterpenoid type (pentacyclic triterpenoids, C-30). • Both of these have a glycosidal linkage at C-3 and have a common biosynthetic origin via mevalonic acid and isoprene units. 21

18 26 25 17 19 27 1

29 30 3 Steroid skeleton

25 26 28

1

3 27 Pentacyclic triterpenoid skeleton

23 24 A. Steroidal saponins

• The steroidal saponins are less widely distributed in nature than the pentacyclic triterpenoid type. • Steroidal saponins are of great pharmaceutical importance because of their relationship to compounds such as the sex hormones, cortisone, diuretic steroids, vitamin D and the cardiac glycosides. • Examples: Diosgenin ( Dioscorea sylvatica), Sarsapogenin ( Smilax sp.). B. Pentacyclic triterpenoid saponins • Triterpenoid saponins my be classified into three groups represented by α-amyrin, β-amyrin and lupeol. • Examples: Primulagenin ( Primula sp.), Quillaiac acid (Quillaia saponaria ) and Glycyrrhetinic acid ( Glycyrrhiza sp.).

20 19 E

13 17 D 14

ααα−−−Amyrin βββ−−−Amyrin Lupeol 3. Coumarin glycosides

5 4 6 3

2 7 O O 8 1 Coumarin (Benzo-α-pyrone)

• The coumarins are shikimate-derived metabolites. • The majority of the coumarins are oxygenated at position C7. • Coumarins have a limited distribution in the plant kingdom and have been used to classify plants according to their presence (chemotaxonomy). • Coumarins are commonly found in the plant families Apiaceae , Rutaceae , Asteraceae and Fabaceae. • Some coumarins are phytoalexins and are synthesized de novo by the plant following infection by a bacterium or fungus. • Phytoalexins: any of a group of compounds formed in plants in response to fungal infection, physical damage, chemical injury, or a pathogenic process. Phytoalexins inhibit or destroy the invading agent. • These phytoalexins are H3CO broadly antimicrobial; for example, scopoletin is synthesized by the potato HO O O (Solanum tuberosum) Scopoletin following fungal infection.

OCH3 O • Khellin is an isocoumarin (chromone) natural product from Ammi Visnaga O (Apiaceae) and has activity as O CH3 a spasmolytic and vasodilator. OCH3 Khellin • It has long been known that animals fed sweet clover OH OH (Melilotus officinalis, Fabaceae) die from haemorrhaging. The O O O O poisonous compound Dicoumarol responsible for this adverse effect was identified as dicoumarol . • A number of compounds have OH O been synthesized based on

the dicoumarol structure, e.g. CH3 warfarin , which is widely used O O as anticoagulant. Warfarin • The psoralens are coumarins that possess a furan ring and are sometimes known as furanocoumarins . e.g. O O O psoralen and bergapten. Psoralen

• These compounds may be produced by the plant as a OCH3 protection mechanism against high doses of sunlight and some coumarins are formulated into sunscreens O O O and cosmetics for this purpose. Bergapten 4. Flavonoid glycosides

• Biosynthesis: flavonoids are products from a cinnamoyl-CoA

(C6C3, precursor from the shikimate pathway) starter unit, with chain extension using three molecules of malonyl-CoA. • Flavonoids are therefore of mixed biosynthesis, consisting of units derived from both shikimate and acetate pathways.

• The triketide starter unit undergoes cyclization by the enzyme chalcone synthase to generate the chalcone group of flavonoids. Cyclization can then occur to give a pyranone ring containing flavanone nucleus, which can either have the C2- C3 bond oxidized (unsaturated) to give the flavones or be hydroxylated at position C3 of the pyranone ring to give the flavanonol group of flavonoids. The flavanonols may then be further oxidized to yield the anthocyanins, which contribute to the brilliant blues of flowers and the dark colour of red wine. • The flavonoids contribute to many other colors found in nature, particularly the yellow and orange of petals; even the colourless flavonoids absorb light in the UV spectrum (due to their extensive chromophores) and are visible to many insects. [A chromophore is the part (or moiety) of a molecule responsible for its color]. • It is likely that these compounds have high ecological importance in nature as colour attractants to insects and birds as an aid to plant pollination. • Certain flavonoids also markedly affect the taste of foods: for example, some are very bitter and astringent such as the flavanone glycoside naringin, which occurs in the peel of grapefruit (Citrus paradisi). Interestingly. the closely related compound naringin dihydrochalcone , which lacks the pyranone ring of naringin, is exceptionally sweet, being some 1000 times sweeter than table sugar (sucrose). • the flavonoids have important dietary significance because, being phenolic compounds, they are strongly antioxidant.

• Many disease states are known to be exacerbated by the presence of free radicals such as superoxide and hydroxyl, and flavonoids have the ability to scavenge and effectively ‘mop up’ these damaging oxidizing species. • Foods rich in this group have therefore been proposed to be important in ameliorating diseases such as cancer and heart disease (which can be worsened by oxidation of low-density lipoprotein); quercetin , a flavonoid present in many foodstuffs, is a strong antioxidant. Components of milk thistle (Silybum marianum), in particular silybin , are antihepatotoxins; extracts of milk thistle are generally known as silymarin.

OH O OH

HO O HO O OCH3 OH O

OH OH OH

OH O OH O Silybin Quercetin Some action and therapeutic uses of flavonoids

• Many flavonoid containing plants are: 1.Diuretic. 2.Antispasmodic. 3.Diaphoretic. 4.Increase tensile strength of capillary walls. 5.Free radical scavengers. 5. Cyanogenetic glycosides (Cyanide glycosides)

• Cyanogenesis is the ability of certain living organisms, plants in particular, to produce hydrocyanic acid (HCN, prussic acid). • Cyanogenesis in plants is a chemical defense mechanism against organism damaging or feeding on plant tissues and lead to release of HCN gas, which is toxic. • They are distributed in over 2000 plant species belonging to 110 families.

• These compounds, in presence of enzymes such as β-glucosidase , lose their sugar portion to form a cyanohydrin which, in the presence of water and hydroxynitrile lyase , can undergo hydrolysis to give benzaldehyde and the highly toxic hydrogen cyanide (HCN). • The sugar portion of the molecule may be a monosaccharide or a disaccharide such as gentiobiose or vicianose. If a disaccharide, enzymes present in the plant may bring about hydrolysis in two stages, as in the case of amygdalin. R1 CN

R2 O−Sugar • They are derivatives of α-hydroxynitrile or 2- hydroxynitrile (cyanohydrins). • In all cases the first sugar attached to the aglycone is β-D-glucose.

• R1 and R 2 are often different residues resulting in pairs of C-2 epimers. (Epimers are diastereomers that differ in configuration at only one of their stereogenic centers). • Most cyanogenetic glycosides are biosynthetically derived from the amino acids: valine, leucine, isoleucine, tyrosine or phenylalanine. • Cyanogenetic glycosides are easy to detect with a strip of filter paper impregnated with reagents able to give a color reaction with the hydrocyanic acid released upon crushing the plant material (e.g., picric acid/sodium carbonate or benzidine/cupric acetate). • Although hydrocyanic acid is a violent poison, it is important to remember that oral intake of cyanogenetic drugs does not necessarily cause severe intoxication, this is because the range of dangerous concentrations (0.5-3.5 mg/kg) can only be achieved by rapid and massive ingestion of plant parts rich in cyanogenetic glycosides. • Examples: 1. Amygdalin in bitter almonds ( Prunus amygdalus ). It is biosynthetically derived from phenylalanine. 2. Linamarin in linseed ( Linum usitatissimum). It is biosynthetically derived from valine.

HO O CN HO HO HO O CH3 O OH O O HO CH3 HO O CN OH OH HO H OH Amygdalin Linamarin 6. Steroidal cardioactive glycosides

• Cardiac glycosides are a group of natural products characterized by their specific effect on myocardial contraction and atrioventricular conduction. • In large doses they are toxic and bring about cardiac arrest in systole, but in lower doses they are important drugs in the treatment of congestive heart failure . • They have a diuretic activity. Since, the improved circulation tends to improve renal secretion, which relieves the edema often associated with heart failure. Distribution in nature • Cardiac glycosides occur in small amounts in the seeds, leaves, stems, roots or barks of plants of wide geographical distribution, particularly of the Fam. Apocyanaceae (e.g. seeds of Strophanthus, roots of Apocynum and fruits of Acokanthera); others are found in the Scrophulariaceae (e.g. leaves of Digitalis sp. ), Liliaceae (e.g. scales of the bulbs of Urginea and Convallaria), and Ranunculaceae (Adonis). • Cardiac glycosides are also found in animals only in exceptional cases: Bufadienolides occur in toads ( Bufo). 18 Lactone ring Structure of glycosides 12 11 17 19 13 C D 16 1 14 9 2 15 10 8 A B 3 5 7 O 4 6 Sugar moiety

• The structure comprise a steroidal aglycone of

the (C23 ) cardenolide type or of the (C24 ) bufadienolide type, and a sugar moiety, most often an oligosaccharide. A. Structure of the aglycones

• All of the aglycones have in common the classic, tetracyclic, steroidal nucleus. • The A, B, C and D rings normally have a cis-trans- cis configuration or less often, a trans-trans-cis configuration. • Also common to all the aglycones is the presence of two hydroxyl groups: one is a 3β secondary alcohol, the other is a 14 β tertiary alcohol. • All of the aglycones have a β constituent at C-17: an α,β-unsaturated lactone. O

24 O O 23 O 23 21 21 22 22 20 20

17 17 16 16

14 14 15 15

Lactone ring of Lactone ring of Cardenolide Bufadienolide • The size of the lactone ring distinguishes two groups of aglycones: the C23 cardenolides with an α,β- unsaturated γ-lactone (= butenolide) and the C24 bufadienolides with a di-unsaturated δ-lactone (= pentadienolide). B. Structure of the sugar moiety • The sugar moiety is generally linked to the aglycone through the hydroxyl group at C-3. • The majority of the saccharides found in cardiac glycosides are highly specific: 1. 2,6-dideoxyhexoses, e.g. D-digitoxose 2. 2,6-dideoxy-3-methylhexoses, e.g. D-diginose 3. 6-deoxyhexoses, e.g. L-rhamnose 4. 6-deoxy-3-methylhexoses, e.g. D-digitalose 5. Hexose, e.g. glucose ( when these is a glucose unit, it is always terminal ). • The sugars can modify the activity (potency, toxicity), the solubility, the diffusion through membranes, the rate of absorption and transportation of the glycosides. C. Structure-Activity Relationships (SAR) • The cardiac activity is linked to the aglycone. • The sugar moiety does not participate directly in the activity, but its presence enhances the activity and modulates it by modifying the polarity of the compound. • The presence of a certain number of structural elements is required for, or at least favorable, to the activity: 1. The lactone at C-17, and it must be in the β configuration. 2. The configuration of the rings. The activity is maximized when the A, B, C and D rings are in the cis , trans , cis configuration. The C and D rings must be cis fused. 3. The substituents. The inversion of the configuration at C- 3 diminishes the activity, but 3-deoxy compounds are not completely inactive. O O

CD

A B OH

HO O

CH3 O CH3 H

OH H H H HO Digitoxigenin

A/B cis - B/C trans - C/D cis H Biosynthetic origin

• Aglycone of the cardiac glycosides are derived from mevalonic acid but the final molecules arise

from a condensation of a C21 steroid with a C2 unit (the source of C-22 and C-23). Bufadienolides are condensation products of a

C21 steroid and a C3 unit. Color reactions

• They can be due to the sugars or to the aglycone: A.Color reactions of the sugars . The only color reactions of the sugars that are of interest are those specific to 2- deoxyhexoses. e.g. Keller-Kiliani test .

B. Color reactions of the aglycones (steroidal nucleus). These are positive with any compound containing a steroidal nucleus including cardenolides or bufadienolide:

1) Antimony trichloride (SbCl 3) 2) Liebermann's test (for bufadienolides) C. Color reactions of the aglycones (lactone ring). These are characteristic for cardenolides having a five-membered lactone ring: 1.Legal's test 2.Raymond's test 3.Kedde's test 4.Baljet's test Pharmacological properties • Cardiac glycosides increase the force and speed of contraction of the heart. In patients with cardiac insufficiency, this positive inotropic effect translates into 1an increase in cardiac output, 2an increase in cardiac work capacity without any increase in oxygen consumption, 3a decrease in heart rate, and, indirectly, 4a decrease in arterial resistance. ( MOA) The glycosides are thought to act at the membrane level, by inhibition of the Na- K ATPase , which would result in an increase of the intracellular calcium ion concentration. Therapeutic indications

• Cardiac glycosides are currently indicated for: 1.Cardiac insufficiency with low output (generally in combination with diuretics), particularly when there is atrial fibrillation.

2.Supraventricular rhythm abnormalities: to slow down or decrease atrial fibrillation or flutter. Examples

• Strophanthus glycosides The name Strophanthus is derived from the Greek strophos (a twisted cord or rope) and anthos (a flower) . e.g. Strophanthus kombe The principle glycosides are: 1. K-strophanthoside 2. K-strophanthin-βββ 3. Cymarin O O

CH3 O CH H

H OH

O OH

Cymarose − β -D-glucose − α -D-glucose

Cymarin

K-strophanthin-βββ

K-strophanthoside • Squill glycosides Urginea maritima (L.) 0.1% − 2.4% total bufadienolides, ∼15 glycosides • White variety: average 0.2%-0.4% proscillaridin A, scillaren A, glucoscillaren A (aglycone: scillarenin) scilliphaeoside, scilliglaucoside • Red variety: < 0.1% scilliroside and glucoscilliroside (aglycone: scillirosidin); proscillaridin A and scillaren A as in the white variety

Pharmacological properties of squill

• White squill: it is an expectorant, but it also possesses emetic, cardiotonic (proscillaridin A), and diuretic properties. • Red squill: it is used as a rat poison (scilliroside), because rodents lack the vomiting reflex, which makes red squill particularly lethal to these animals. O

O

CH3

CH3 OH

H OH

β-D-glucose–O Scilliroside

O CH3

O (3β,6β)-6-(Acetyloxy)-3-(β-D-glucopyranosyloxy)-8,14-dihydroxybufa-4,20,22-trienolide • Digitalis glycosides Several species of Digitalis yield pharmacologically active principles. The most important of these species are Digitalis purpurea and Digitalis lanata . 1. Digitalis purpurea folium (Red foxglove leaves) 0.15% − 0.4% total cardenolides, ∼ 30 glycosides Purpurea glycosides A and B ( ∼60%), digitoxin ( ∼12%), gitoxin ( ∼10%) and gitaloxin ( ∼10%). 2. Digitalis lanata folium (White foxglove leaves) 0.5% − 1.5% total cardenolides, ∼ 60 glycosides Lanatosides A and C ( ∼50%), lanatosides B, D, E as well as digoxin and digitoxin .

• Digitoxin is a cardiotonic • Digoxin is the most widely glycoside obtained from D. used of the cardiotonic purpurea, D. lanata. glycosides, and it is • It is the most lipid-soluble of obtained from the leaves of the cardiac glycosides used in D. lanata. therapeutics. • It is a highly potent drug and • The major pharmacokinetic should be handled with parameters for digitoxin exceptional care. include complete oral • Digoxin tablets are 60 to absorption, which 80% absorbed. distinguishes it from other • Digoxin is indicated when the cardiac glycosides. risk of digitalis intoxication is • Digitoxin may be indicated in great, since it is relatively short- patients with impaired renal acting and rapidly eliminated when compared with digitoxin. function. Digitalis purpurea 7. Tannins

• Historically, the importance of tannin-containing drugs is linked to their tanning properties, in other words their ability to transform fresh hides into an imputrescible material: leather.

• Tannins are "phenolic natural products that precipitate proteins from their aqueous solutions". • The consequence of tanning is the formation of bonds between the collagen fibers in the hide, which imparts resistance to water, heat, and abrasion. This capability of tannins to combine with macromolecules explains why they precipitate cellulose, pectins, and proteins; it also explains their characteristic astringency and tartness: by precipitating the glycoproteins contained in saliva, tannins make the latter lose its lubricating power. • Most true tannins have molecular weights from about 1000 − 5000. Pseudotannins

• They are compounds of lower molecular weight than true tannins and they do not respond to the goldbeater's skin test . • Examples of drugs containing Pseudotannins are: »Gallic acid: Rhubarb »Catechins : Guarana, Cocoa »Chlorogenic acid: Mate, Coffee » Ipecacuanhic acid: ipecacuanha OH COOH

HO O OH

HO OH OH

OH OH Gallic acid Catechin

HOOC OH

OH O

HO O OH

OH Chlorogenic acid Function of tannins in plants

1.Tannins are considered the source of energy through their oxygen content. 2.They serve as a protective to the plant (plant antiseptics). 3.They may have function in respiratory activity , i.e. in the mechanisms of hydrogen transfer in plant cells. 4.Tannins play an important part in the acceptance of many foods and beverages by consumers e.g. tea, cocoa. Classification of tannins

• In higher plants, two groups of tannins are generally distinguished, which differ by their structure, as well as their biosynthetic origin: hydrolysable tannins and condensed tannins.

• Hydrolysable tannins Hydrolysable tannins are esters of a sugar (or related polyol) and of a variable number of phenolic acid molecules. • The sugar is most generally glucose. • The phenolic acid is either gallic acid, in the case of gallitannins, or Ellagic acid, in the case of the tannins conventionally referred to as ellagitannins. • Ellagic acid can arise by lactonization of hexahydroxydiphenic acid (= HHDP) during chemical hydrolysis of the tannin. • Hydrolysable tannins were formerly known as pyrogallol tannins , because on dry distillation gallic acid and similar components are converted into pyrogallol . • Biosynthetically , gallic acid (= 3,4,5- trihydroxybenzoic acid) arises from the metabolism of shikimic acid. • Examples of drugs containing Hydrolysable tannins: Gallitannins: rhubarb, cloves, Chinese galls, Turkish galls, hamamelis, chestnut and maple. Ellagitannins: pomegranate rind, pomegranate bark, eucalyptus leaves, and oak bark. OH OH

HO HO COOH

O HO COOH O

HOOC OH O HO OH O OH Gallic acid OH OH OH OH Hexahydroxydiphenic acid Ellagic Acid

HO OH

OH Pyrogallol • Condensed tannins (proanthocyanidins) Condensed tannins or proanthocyanidins are polymeric flavans. 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 (C-8, less commonly C-6) of another unit, generally a flavan-3-ol. • Unlike hydrolysable tannins, these are not readily hydrolyzed to simpler molecules and they do not contain a sugar moiety . OH OH

HO O HO O OH OH

OH OH

OH OH OH

(+) Catechin (catechol) Flavan-3,4-diol structure

OH

HO O OH

OH OH OH HO O OH

OH

OH A dimeric structure • Biosynthetically , flavonoids are derived from acetate and shikimate pathways. • Condensed tannins occur due to polymerization (condensation) reactions between flavonoids. • The polymers may include up to 50 monomer units. • On treatment with acids or enzymes condensed tannins are converted into red insoluble compounds known as phlobaphenes . Phlobaphenes give the characteristic red colour to many drugs such as red cinnamon bark. • Examples of drugs containing Condensed tannins: Some drugs (e.g. tea, hamamelis leaves and hamamelis bark) contain both hydrolysable and condensed tannins. The following are rich in condensed tannins. (1) Barks: cinnamon, wild cherry, cinchona, willow, acacia, oak and hamamelis (2) Roots and rhizomes: krameria (rhatany) and male fern (3) Flowers: lime and hawthorn (4) Seeds: cocoa, guarana, and kola (5) Leaves: hamamelis, hawthorn and tea, especially green tea (6) Extracts and dried juices: catechu, acacia and mangrove cutches

Properties and tests of tannins • Tannins are soluble in water, dilute alkalis, alcohol, glycerol and acetone, but generally only sparingly soluble in other organic solvents. • Solutions precipitate heavy metals, alkaloids, glycosides and gelatin. • With ferric salts, gallitannins and ellagitannins give blue- black precipitates and condensed tannins brownish- green ones. If a very dilute ferric chloride solution is gradually added to an aqueous extract of hamamelis leaves (which contains both types of tannin), a blue colour is produced which changes to olive-green as more ferric chloride is added. Other useful tests are the following: 1. Goldbeater's skin test Soak a small piece of goldbeater's skin in 2% hydrochloric acid; rinse with distilled water and place in the solution to be tested for 5 min. Wash with distilled water and transfer to a 1% solution of ferrous sulphate. A brown or black colour on the skin denotes the presence of tannins. Goldbeater's skin is a membrane prepared from the intestine of the ox and behaves similarly to an untanned hide. 2.Gelatin test Solutions of tannins (about 0.5-1 %) precipitate a 1% solution of gelatin containing 10% sodium chloride. Gallic acid and other pseudotannins also precipitate gelatin if the solutions are sufficiently concentrated. 3.Phenazone test 4.Test for catechin 5.Test for chlorogenic acid Medicinal and biological properties The applications of tannin-containing drugs are limited, and result from their affinity for proteins. • Tannin-containing drugs will precipitate protein and have been used traditionally as styptics and internally for the protection of inflamed surfaces of mouth and throat. • They act as antidiarrhoeals and have been employed as antidotes in poisoning by heavy metals, alkaloids and glycosides. sumac