Glycosides (Lec 4) .ljkp[;,ft125drtgvcxdedytrryuoml;.,mnbvzsswegbbbbbbbbblp[,nbccfgv cxcll;;l kjkj PDF

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Summary

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Description

Glycosides • Definition: Organic natural compounds present in a lot of plants and some animals, these compounds upon hydrolysis give one or more sugars (glycone) β_form and non sugar (aglycone) or called genin. ➢ The sugar residue is in its cyclic form and the point

of attachment is the hydroxyl group of the hemiacetal function.

•  -Glycosides and  -glycosides are distinguished by the configuration of the hemiacetal hydroxyl group. • The majority of naturally-occurring glycosides are -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).

• 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”).

• Solubility: ➢ glycosides are water soluble compounds and insoluble in the organic solvents. ➢ Glycone part: water soluble, insoluble in the organic solvents. ➢ Aglycone part: water insoluble, soluble in the organic solvents. ➢ Some glycosides are soluble in alcohol.

Hydrolysis of Glycosides ❑Acid hydrolysis: The most characteristic chemical property of glycosides is their susceptibility to hydrolysis, to give the free sugar and the free genin. Hydrolysis of glycosides is effected in the laboratory by digestion with dilute acids (acid hydrolysis).

❑Enzymatic hydrolysis: Certain naturally occurring enzymes catalyze the hydrolysis of glycosides, and their action is usually specific, or selective. In many cases, the glycoside-bearing plants contain the specific enzymes, that are contained in different cells other than those containing the glycosides. Thus glycosides are not acted upon by the coexisting enzymes within the intact plant tissues. Upon processing (powdering, grinding, extraction etc.) the cell walls are destroyed and the glycosides are acted upon by the enzymes. Therefore, it is necessary to destroy (or deactivate) the enzymes which are present before attempting to isolate glycosidal constituents.

Separation between glycosides parts: Glycosides Hydrolysis glycone +aglycone +HcL +HcLdil Neutralization by G + A +salt+H2O Filtration Using alkaline (H2O+G)+A chloroform (H2O+G)+(chloroform+A) We can separate them by using separatory funnel. ➢Chloroform is usually used BUT The best solvent to extract aglycone is Ethylacetate because:

A- immiscible in water. B- always presents in the upper layer.

Note: Alcohol and acetone are water miscible solvent, so we can't use them as organic solvents for aglycone separation.

physico-chemical properties of glycosides(general) • Colorless, solid, amorphous, nonvolatile (flavonoid- yellow, anthraquinone-red or orange. • Give positive reaction with Molisch's and Fehling's solution test (after hydrolysis). • They are water soluble compounds, insoluble in organic solvents • Most of them have bitter taste (exception: glycyrrhizin).

Cont... • Odorless except saponin (glycyrrhizin).. • Glycosides hydrolyzed by using mineral acids and temperature or by using enzymes such as: a- Emolsin Bitter almond seeds. b- Myrosin or Myrosinase black mustard seeds. c- Rhamnase glycosides containing rhamnose as sugar part.

The function or the role of glycosides in the plant 1) Converting toxic materials to non or less toxic. 2) Transfer water insoluble substances by using monosaccharide. 3) Source of energy (sugar reservoir). 4) Storing harmful products such as phenol. 5) Regulation for certain functions(growth). 6) Some have beautiful colours (pollenation process).

Cont…

7) Some glycosides have antibacterial activity, so they protect the plants from bacteria and diseases. bacteria kill

Bitter almond

Amygdalin Eomlsin enzyme

hydrolysis

HCN

Classification of glycosides

I- Classifications of glycosides according to their therapeutic effects • CHF and cardiac muscles stimulators such as: a-Digitalis glycosides: digoxin, digitoxin, gitoxin (Fox glove leaves). b- Ouabain: Strophanthus gratus seeds. c- K-strophanthin -Strophanthus kombe seeds. d- Scillaren A,B which isolated from red and white Squill bulbs. e- Convolloside: Convallaria majalis.

Cont…

• Laxative group of glycosides: a- Sennoside A,B,C,D (Senna leaves and fruits). b- Cascaroside A,B (Cascara bark). c- Frangulin and glucofrangulin(Frangula bark). d- Aloin and barbaloin (Aloe vera and Aloe barbadensis juice).

•

Cont… Local irritant group: a-Sinigrin (Black mustered seeds_Brassica nigra) b-Sinalbin (White mustered seeds_Brasica alba)

• Analgesics and antipyretics: Salicin

hydrolysis

Salisylic acid (Salix bark).

• Keeping elasticity of blood vessels like: Rutin_Rutoside (Bitter orange peels, Lemon peels)

• Anti-inflammatory group: a- Aloin for: acne b-Glycyrrhizin

&

peptic ulcer

II- Classification of glycosides according to glycone part • Glucose _ glucoside group like in Sennoside. • Rhamnose _ Rhamnoside like in frangullin. • Digitoxose _ Digitoxoside like in digoxin. • Glucose and Rhammnose _ Glucorhamnoside _ glucofrangulin. • Rhamnose and glucose _ Rhamnoglucoside _ Rutin.

III-Classification of glycosides on the basis of the linkage between glycone and aglycone part • O-glycosides : in these glycosides the sugar part is linked with alcoholic or phenolic hydroxyl or carboxyl group. • S-glycosides : in these glycosides the sugar attached to a Sulfur atom of aglycone such as in sinigrin. • N-glycosides : in these glycosides the sugar linked with Nitrogen atom of (-NH2,-NH-)amino group of aglycone like in nucleosides DNA,RNA • C-glycosides : in these glycosides the sugar linked (condensed) directly to Carbon atom of aglycone like in aloin.

C-glycosides are not hydrolyzed by acids or alkalis or by enzymes mainly . N.B :

IV-Classification of glycosides according to aglycone part : 1- if aglycone part alcohol -this group called alcoholic group- like Salicin 2- if aglycone part aldehyde- this group called aldehydic group - like glucovanillin. 3- if phenol called phenolic- group like arbutin . 4-if cyanone called cyanogenic or cyanophoric or cyanoside- like amygdalin.

5-if thio called glycosides or isothiocyanate glycoside like sinigrin or sinalbin (-S=C=N-) (SCN) 6-anthracene -------> anthraquinone glycoside – sennoside-. 7-steroid ------→ → steroidal glycoside (cardiac) Digoxin 8-flavone ,flavonol, flavanone –flavonoid glycoside 9-triterpenoid –saponin glycoside –glycyrrhizin, melanthin (nigella seeds) or ginsenoside .

Nomenclature Of Glycosides Most of glycoside may be named according to the plant from which they isolated for example: 1-salicin –salix2-cascaroside _cascara

3-aloin- Aloe vera 4- sennoside – senna5-frangulin – frangula 6- glycyrrhizin – glycyrrhiza And others.

Always glycosides founded in the plant with the enzymes which hydrolyzed them, we must damage these enzymes firstly.

Extraction and Isolation of Glycosides

➢The fact that glycosides include a large number of chemically unrelated classes, makes the proposal of a common extraction and isolation procedure somewhat difficult. However, it is helpful to give some important notes that must be considered during extraction and isolation of glycosides. 1- Being generally more soluble in polar solvents (such as water, alcohols and dilute alcohols) than in non-polar solvents (such as petroleum ether and benzene), glycosides are preferentially extracted from vegetable tissues using the former group of solvents. Non-polar organic solvents are generally used for the preliminary defatting process, and in the preceding purification steps so as to eliminate the contaminating organic substances other than glycosides. 2- Glycosides are not precipitated from their aqueous solutions by the addition of such salts of heavy metals as lead acetate whereas many other organic substances are precipitated by this treatment and are thus eliminated from the glycosidal solution

So, the general methods of isolation of glycosides usually involve the following steps : (1) Destruction of the hydrolyzing enzymes. (2) Defatting or purification of the plant material by the use of non-polar solvents, in which glycosides show no appreciable solubility e.g. petroleum ether or benzene. (3) Extraction of the glycosidal constituents by either alcohol, water or dilute alcohols. (4) Concentration of the alcoholic extracts so as to get rid of the organic solvent, and subsequent addition of water (or hot water), and filtering off any precipitated matter. (5) Further purification of the aqueous solution may be accomplished by the extraction of non-glycosidal impurities by an immiscible organic solvents, and the precipitation of other water-soluble organic impurities by the addition of lead acetate solution. (6) Precipitation of the excess lead salts from the aqueous solution. (7) Isolation of the glycosides from the purified aqueous solution by crystallization.

Qualitative Tests

-There are no simple identification tests for glycosides, except for the characterization of the cleavage products in some cases. (1)

Tests depending on the chemical nature of the genin:

a- Steroidal or cardiac glycosides give positive Lieber-mann’s test, indicative of steroidal structure. b- Anthraquinone glycosides and/or aglycones give characteristic red color with alkalis. c- Flavonoidal glycosides and /or aglycones give characteristic color reactions with ammonium hydroxide, fixed alkalis, aluminum chloride or ferric chloride, according to their type. d- Cyanogenetic glycoside give upon hydrolysis hydrocyanic acid which can be easily tested for. e- Sulphur-containing glycosides give black precipitate of silver sulphide upon treatment with silver nitrate solution. In addition each individual glycoside may respond to any special test that depends upon the chemical structure of its own genin.

(2) Tests depending on the sugar part :

a- Some cardiac glycosides (e.g. most of the Digitalis glycosides) contain 2deoxysugars which are responsible for the keller-kiliani’s test which is given by these glycosides. b- The specificity of action of the hydrolyzing enzymes is often applied for the identification of the sugar moieties of glycosides, or even for the identification of the glycoside as a whole. For Example: The glycoside scillarin is acted upon by the enzyme scillarinase with the liberation of the genin and the disaccharide scillobiose. This disaccharide is selectively hydrolyzed by the enzyme scillobiase. The enzyme gentianase is capable of hydrolyzing gentianosides (gentianose containing glycosides). The enzyme prunase is the specific hydrolyzing enzyme for the glycoside prunasin and other (1ry) glycosides that give prunasin as secondary glycoside, e.g. amygdalin. The enzyme amygdalase is specific for the glycoside amygdalin. The enzyme myrosin (or myrosinase) is specific for thio-D-glucosides : e.g. sinigrin and sinalbin.

Distinction of the Different Glycosidic Linkages

1- Using universal enzymes:

Enzymatic hydrolysis is specific; i.e. for each glycoside (or a type of glycosides) there is a specific enzyme (or a class of enzymes) that exerts a hydrolytic action on it (or on them ). This specificity of action of enzymes is said to be due to the steric configuration of the co-enzyme portion, and hence the stereo-specificity of the enzymatic action. The same enzyme is, in some cases, capable to hydrolyze different glycosides; but α and β -stereo-isomers of the same glycoside are usually not hydrolyzed by the same enzyme. Emulsin (a mixture of β -enzymes) for example, has been found to hydrolyze most β -glycosidic linkages and, therefore, those glycosides which are attacked by emulsin are regarded as β -glycosides.

Maltase and invertase, on the other hand, are α -glycosidases i.e. capable of hydrolyzing α -glycosides only.

Distinguish if α or β glycoside : A drop of the aqueous solution of the glycoside under test is applied to a filter paper. After the solvent is evaporated, the glycosidal spot is sprayed by a solution of the suspected specific enzyme and the filter paper is kept in humid atmosphere for few minutes then dried, sprayed with aniline citrate solution and warmed. The appearance of a reddish brownish color in the spotted area is an indication of the liberation of a reducing sugars, which means that the sprayed enzyme is the specific one; and thus, the identity of the glycoside under test is established.

Alcoholic group of glycosides Such as: Salicin which obtained from Salix bark, Willow bark.

➢ Salicin hydrolyzed by: 1.Enzyme emulsin 2.Acid like HCl, HNO3 3.Alkaline solutions like NaOH

Salicin is an alcoholic β-glucoside. Salicin is an analgesic,anti-inflammatory agent and antipyretic. ➢ Salicin is closely related in chemical make-up to aspirin. When consumed, the acetalic etherbridge is broken down. The two parts of the molecule, glucose and salicylic alcohol, then are metabolized separately. ➢ By oxidizing the alcohol function the aromatic part finally is metabolized to salicylic acid.

.

Phenolic group of glycosides:Such as arbutin which isolated from bearberry leaves (Uva ursi). Uses: as antiseptic and antibacterial & mild diuretic

Uvi ursi also contains methylarbutin

Aldehydic group of glycoside Such as : Glucovanillin which obtained from : Vanilla pods OR from Coniferin ** Vanillin : volatile oil which used as flavoring agent ** vanillin : 1.phenolic group volatile oils 2.aldehyde group of volatile oils

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 -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 -hydroxynitrile or 2hydroxynitrile (cyanohydrins). • In all cases the first sugar attached to the aglycone is -D-glucose. • R1 and R2 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

CN HO

CH3 O

O

OH

CH3

HO HO

O O OH HO HO

O O OH

HO OH

Linamarin

Amygdalin

H

CN

General structure of thioglycosides

BLA BLACK CK MUST MUSTARD ARD ‫لسود‬฀‫بذر الخردل ا‬ Orig Origin in  Black mustard is the dried ripe seeds of Brassica nigra. Family Cruciferae Constituents ents  Constitu  - Mucilage;  - Fixed oil and Protein. Glycoside, sinigrin +myrosin enzyme in presence of water glucose+ potassium sulphate +allylisothiocyanate (volatile oil). 

On hydrolysis (myrosin enzyme): glucose+ potassium acid sulphate +allylisothiocyanate (volatile oil).

BRASSICA ALBA ‫لبيض‬฀‫بذر الخردلا‬ WHITE MUSTARD AND YELLOW MUSTARD

 Orig Origin in 

White mustard is the dried ripe seeds of Brassica alba Family Cruciferae.

CONS CONSTITUEN TITUEN TITUENTTS: -Fixed oil and protein.  -Mucilage. 



-Crystalline glycoside, sinalbin+ myrosin enzyme + water

glucose + K-

acid sinapine sulphate + acrinyl-isothiocyanate.



acrinyl isothiocyanate with a pungent taste and powerful rubefacient action as it is not volatile....