Glycosides - Introduction (Students)

2024-11-05 GLYCOSIDES ◼ Glycosides are organic compounds formed of a sugar and a non sugar parts. ◼ Many natural glycosides are known to have valuable therapeutic uses for the treatment of various diseases. ◼ Pharmaceutical products containing natural glyco...

2024-11-05 GLYCOSIDES ◼ Glycosides are organic compounds formed of a sugar and a non sugar parts. ◼ Many natural glycosides are known to have valuable therapeutic uses for the treatment of various diseases. ◼ Pharmaceutical products containing natural glycosides are available and are widely used in the medicinal field. ◼ The glycosides are formed by the combination of the reactive hemiacetal group of a sugar with the hydroxyl group of other compound. 1 O-Glycosides are mixed acetals CH O O OH hemiacetal H CH2OH ROH O OR H acetal 2 1  2024-11-05 ◼ The synthesis and hydrolysis of the glycosides in plants are done under the influence of more or less specific enzymes. ◼ Hydrolysis of the glycosides by dilute acids or enzymes (which co-exist with the glycosides in the same plant), yields sugar residue glycone and a non- sugar part aglycone or -genin. 3 ◼ Depending on whether the sugar involved in the glycoside is - or  -sugar, there are two types of glycosides ; - and -Glycosides. ◼ -Glycosides are those derived from -sugar (i.e. they yield upon hydrolysis -sugar besides the aglycone), they also can be attacked by - glycosidases. ◼ -Glycosides are those derived from -sugar (i.e. they yield upon hydrolysis -sugar besides the aglycone), they also can be attacked by - glycosidases. 4 2  2024-11-05 5 ◼ Itshould be noted that most of the naturally occurring glycosides are of the -type, which is often, the medicinally active form. 6 3  2024-11-05 III- According to the sugar moiety: 1-Glucosides: 2- Ribosides: 3- Rhamnosides: IV- According to the aglycone: 1-Cardiac glycosides 2- Flavonoid glycosides 3- Anthracene glycosides 7 4- Saponin glycosides 5-Thioglycosides 6- Cyanogenic glycosides 7- Phenolic glycosides possessing phenolic aglycones e.g. arbutin. 8- Alcohol glycosides, with alcoholic aglycones e.g. salicin. 9-Antibiotic glycosides e.g, Amino-glycoside antibiotics 8 4  2024-11-05 10- Ester glycosides, such as the glycosides of methyl salicylate e.g gaultherin 11- Aldehyde glycosides, with an aldehyde aglycone e.g. glucovanillin. 12- Terpene glycosides : a- Monoterpene glycosides e.g. gentiopicrin and loganin. b- Triterpene saponin glycoside. 9 13- Glycoalkaloids: These are the glycosidal alkaloids i.e. They possess a glycosidic linkage and a cyclic nitrogen in the aglycone. e.g steroidal alkaloids of Solanum species e.g. solanine. 10 5  2024-11-05 14- Complex glycosides: These include a number of complex secondary carbohydrates e.g. gums, mucilage & pectin; a number of glycosidal resins e.g. Jalap resin as well as a number of glycosidal tannins e.g. glucogallin in Rhubarb. 11 ◼ Types of Glycosidic Linkages: ◼ O-Glycosides :most of the glycosides are formed by combination of a sugar(s) with the alcoholic or phenolic -OH group of a second compound (aglycone) ◼ S-Glycosides: when the linkage involves a sulfur atom of the aglycone which confirms - (thiol, SH group( 12 6  2024-11-05 ◼ N-Glycosides: when the aglycone (containing - NH group) is linked to the sugar through the N atom. ◼ C-Glycosides: where the glycosidic linkage involves a covalent bond between the sugar and the aglycone, it is a rare type of glycosides. This class of glycosides resists the usual hydrolysis procedures ◼ Glycoside + H Sugar + aglycone (or genin) Glycoside + H +glucose + glycone (- genin) 13 ◼ The enzymes and their effects on the glycosides: ◼ The enzymes, which present with the glycosides in the plant, are responsible for the hydrolysis of these glycosides. ◼ This may take place during drying of the plant materials, grinding, extraction or isolation processes. ◼ For each glycoside, there is a specific enzyme that causes its hydrolysis. 14 7  2024-11-05 ◼ Examples: ◼ -Emulsin, which is a mixture of- enzymes, can hydrolyze only -- glycosides. So it is called- glycosidase. ◼ -Maltase and invertase are capable of hydrolyzing- glycosides. So these enzymes are called- glycosidases. ◼ -Myrosin (or myrosinase) can hydrolyze s-glycosides (e.g. sinigrin) and also some -glycosides. 15 ◼ Adverse enzymatic effects ◼ The specific enzymes that co-exist with the glycosides in the same plant cause hydrolysis of the natural glycosides only when they brought into contact (during grinding for instance). ◼ Other theories postulate that both the glycoside and its specific hydrolytic enzyme present in the same cells, but they are physically separated. ◼ In this case, hydrolysis takes place when the condition (viz. temperature, pH... etc.) are favorable. 16 8  2024-11-05 ◼ Enzymatic hydrolysis, which may occur during collection, drying or grinding of the plant materials or during extraction and isolation of the glycosides may cause some undesired effects. ◼ This includes hydrolysis of the medicinally active glycosides into their components (usually inactive); or formation of undesired artifacts (possibly by transferring the sugar moiety from the natural glycoside to another compound.) 17 ◼ Determination of the glycosidic linkages: ◼ Using - or- glycosidases :depending on the fact that glycosides which can be hydrolyzed by- glycosidases (e.g. emulsin), are - glycosides, while those attacked by -glycosidases (e.g. maltase) are- glycosides. In the method: ◼ A drop of the glycoside aqueous solution is spotted on a filter paper. ◼ Drying the spot then spray with specific enzyme (or  glycosidases ◼ The paper left for a few minutes in a humid atmosphere. ◼ The paper is dried and sprayed with aniline citrate solution and warmed. ◼ The development of a reddish or brownish color indicates the liberation of reducing sugar(s), so the used enzyme is the specific one. 18 9  2024-11-05 ◼ If the enzyme used is emulsin-( glycosidase), the linkage is therefore, is- linkage → the glycoside is a- glycoside. ◼ If the sprayed enzyme is- glycosidase (e.g. maltase) and hydrolysis occurs (a red color on the paper → )it is an- glycoside. ◼ Using optical rotation measurements ][D25: ◼ This method depends upon the fact that glycosides yield on acid hydrolysis, free sugars that can undergo mutarotation. ◼ For example- ,glycosides produce- glucose (with a high value of specific rotation + 112° , dextro), then by time it mutarotates in solution and gradually transformed to- glucose until an equilibrium occurs between the two forms (specific rotation about +52.7°). 19 ◼ On the other hand  glycosides give- glucose (specific rotation +18.7°), this value increases by time as a result of mutarotation to reach equilibrium point (value of +52.7°). ◼ This method includes acid hydrolysis of the glycoside, followed by measuring of the - D of the solution, ◼ Gradual- →  glycosides. ◼ Gradual- →  glycosides. 20 10  2024-11-05 Physical Characters: ◼ Solids either amorphous or crystalline. ◼ Non volatile. ◼ Usually bitter in taste. ◼ Soluble in water and polar organic solvents. ◼ Reduce Fehling’s solutions only after hydrolysis. 21 Stability of Glycosides: 1- Effect of acid hydrolysis: ◼ Acids split sugars from the aglycones. ◼ The acetal linkage is more readily cleaved than the linkage between the individual sugars of the sugar chain. ◼ C-glycosides are resistant to acid hydrolysis. 22 11  2024-11-05 2- Effect of alkaline hydrolysis: A- Strong alkalis: ◼ Hydrolysis of ester groups. ◼ Opening of lactone rings e.g. Cardiac glycosides. B- Mild alkalis: ◼ Hydrolysis of ester groups e.g. Lanatoside A to Purpurea A ◼ Opening of lactone rings e.g. Cardiac glycosides. 23 3- Enzymatic hydrolysis: – Split the sugars stepwise starting from the terminal sugars. – All plants producing glycosides have enzyme that can hydrolyze these glycosides. – Enzymes are specific for the type of glycosidic linkages: ◼ Emulsin can hydrolyze - glycosides ◼ Invertase can hydrolyze - glycosides ◼ Myrosin can hydrolyze s-glycosides. 24 12  2024-11-05 ◼ Properties of the glycosides: ◼ Glycosides represent a group of natural compounds of diverse chemical structures. They only share the presence of one or more monosaccharide units in the structure. ◼ However, the following general properties should be known: ◼ Glycosides do not reduce Fehling's solution, but the sugars they produce after hydrolysis reduce Fehling's solution (ppt of red cuprous oxide.) ◼ Glycosides show no mutarotation except after hydrolysis (C-glycosides are exceptions(. 25 ◼ Most glycosides are soluble in polar solvents such as water and alcohols, they are generally insoluble in non-polar solvents while the aglycones are soluble in ether chloroform, and insoluble in water. ◼ The presence of sugar increases the solubility of the glycoside in water, but depends on the number the sugar units. 26 13  2024-11-05 ◼ Extraction and Isolation of Glycosides ◼ It is quite difficult to suggest a general method for extraction of the glycosides. ◼ Simply, because they represent a large group of compounds with different chemical structures and different physical properties. ◼ However, the following steps should be generally considered in designing an extraction method for glycosides: 27 ◼ Collection and drying: An immediate extraction of the fresh plant material should be done. However, drying (if necessary) should be carried out using a suitable method (e.g. freeze-drying), to avoid enzymatic effects. ◼ Deactivation or destruction of the hydrolyzing enzymes which co-exist with the glycosides, may be achieved by : ◼ A- Treatment of the fresh plant material with boiling alcohol or acetone. 28 14  2024-11-05 B- Immediate grinding of the fresh plant material ◼ in presence of hot alcohol and saturated solution of ammonium sulfate. ◼ This technique is also called enzyme inhibition technique. C- Lyophilization ◼ (freeze-drying) is a good way to avoid the effect of hydrolyzing enzymes by removal of water from the plant material at very low temperature. D-Preliminary purification of the plant material from fats and other non-polar impurities by extraction with non-polar solvents such as benzene or light petroleum, Glycosides are generally insoluble in these solvents 29 E- Extraction of the glycosides is usually done by polar solvents including alcohol or water. F- Purification of the extract from non-glycosidic plant constituents (i.e. impurities) may be carried out by: ◼ 1- Use immiscible organic solvents (to remove organic solvent soluble impurities). 30 15  2024-11-05 ◼ 2- Precipitation of water-soluble organic impurities (e.g, tannins, colouring materials, resins..,etc.) by addition of heavy metals (e.g. lead acetate followed by filtration and the excess lead is removed as lead sulfide or sulfate. ◼ G- Separation of the individual glycosides is - often carried out using different chromatographic techniques. ◼ The pure glycosides are then, separately crystallized from suitable solvents. 31 ◼ Tests for identity: ◼ In addition to the previously mentioned general properties, glycosides can be recognized by identification of their hydrolytic products (sugars and aglycone), after hydrolysis by acids or enzymes. ◼ The nature of the aglycone may be determined by applying certain tests such as : ◼ Leibermann's test for steroidal glycosides. ◼ Thioglycosides give black precipitate of silver sulfide when treated with silver nitrates. ◼ Flavonoids give characteristic colors with alkalis. 32 16  2024-11-05 ◼ Anthraquinones give reddish color with alkalis. ◼ Some sugars (e.g. digitoxose, a deoxysugar) are rare sugars and can be detected by Keller Kiliani test. ◼ Using specific enzymes may also assist identification of some glycosides. ◼ For example: prunase enzyme is the specific hydrolyzing enzyme for the glycoside prunasin and hence is used for its identification, ◼ in a similar way, myrosinase is the specific enzyme for thioglycosides (e.g. sinigrin and sinalbin), amygdalase for amygdalhin, etc. ◼ Unambiguous identification of glycosides can be achieved by spectroscopic analysis (e.g. IR, UV, MS, 1H- and 13C NMR). 33 ◼ Quantitative determination of glycosides ◼ Quantitative determination may be carried out by; ◼ I -Hydrolysis of the glycoside by an acid or enzyme, followed by quantitative determination of certain hydrolytic products and then calculating the percentage of the original glycoside using reference glycoside. ◼ Amygdalin + HOH benzaldhyde + HCN + 2 Glucose. 34 17  2024-11-05 ◼ Examples : ◼ Determination of allylisothiocyanate resulting from sinigrin. - Determination of HCN resulting from cyanogenetic glycosides. ◼ Chromatographic methods such as quantitative PC or TLC in the traditional way or by the aid of densitometer can be employed. ◼ Advanced methods such as GC and HPLC are also widely used for such determinations with great degree of precision. 35 - Colormetric or Spectroohotpmetric procedures can be applied for the determination of colored or fluorescent glycosides, respectively. ◼ Glycosides that can develop a colored or fluorescent product with a particular reagent can also be measured by these methods using standards. ◼ Biological methods can be used to determine biologically active glycosides (e.g. determination of LD50 of cardiac glycosides.) 36 18  2024-11-05 ◼ Classification of Glycosides In some cases they are named after the botanical origin of the producing plants. ◼ Example: the glycosides of senna leaf are sennosides A, B, C..etc., those isolated from Digitalis purpurea leaves are purpurea glycosides A, B, C...etc. and salicin is obtained from Salix species. ◼ Glycosides may be named or classified according to the name of the glycones present in their structures. ◼ Glucosides is a term given to the glycosides having glucose as the sugar pant, and rhamnosides when the sugar is rhamnose or galactosides when the sugar is galactose and so on. 37 ◼ Depending on the number of sugar units attached to the aglycone, glycosides may also be termed monosides, biosides, triosides... etc. ◼ According to their pharmacological activities, glycosides can be named as cardiac glycosides, laxative glycosides, counter.. etc 38 19  2024-11-05 ◼ The type of the glycosidic linkage is also used to classify the glycosides into two major groups either- glycosides or- glycosides. ◼ Glycosides are quite often named or classified based on the particular functional group of the aglycone involved in the glycosidic linkage. ◼ Therefore, there are O-glycosides, S- glycosides, N-glycosides and C-glycosides when the aglycone and the sugar are directly liked by a C-C covalent bond. 39 ◼ The term primary glycoside is used to describe the mother glycosides which contain a number of monosaccharides, controlled or enzymatic hydrolysis may lead to elimination of the terminal sugar from the primary glycoside to give a secondary glycoside 2ry. ◼ If two terminal sugar units are eliminated the obtained glycoside is called 3ry glycoside. 40 20  2024-11-05 ◼ Classification of glycosides according to the aglycone. ◼ This system of classification of the glycosides has been used by many ◼ authors and is considered to be a convenient method from a chemical point of view. Based on the chemical structure or nature of the aglycones, glycosides are classified to; ◼ -Simple alcoholic and phenolic glycosides. ◼ - Aldehydic glycosides -.Psoralens. ◼ -Steroidal glycosides -.Anthraquinone glycosides. ◼ -Cyanogenetic glycosides -.Coumarin glycosides. ◼ - Alkaloidal glycosides or glyco-alkaloids ◼ -Thioglycosides (S containing glycosides.) ◼ -Antibiotic Glycosides. ◼ -Complex glycosides (tannins, polysaccharides, derived.) 41 Effect of the sugar portion on solubility of the glycoside: CH2OH Ex. salicin, with one glucose unit, is soluble O Glc in ether, while glycosides with a larger sugar portion are more soluble in alcohol or aqueous alcoholic solvents. Salicin 42 21  2024-11-05 Effect of the sugar on susceptibility of the glycoside to acid hydrolysis: Generally 2-deoxy sugars can be - hydrolyzed easier than 6- deoxysugars, and the latter more easily than normal sugars. - Thus milder conditions can be - chosen to hydrolyze the glycoside involving 2- deoxysugars, without affecting the others. 43 Solubility of glycosides : * The presence or absence of various polarity contributing functional groups in the structure of the aglycone portion, would contribute to the degree of solubility in a given solvent * e.g. thioglycosides are soluble in water partly because of the ionic sulfate r e s i d u e. 44 22  2024-11-05 * The number and kind of monosaccharide units present in the sugar portion would contribute a certain polar character and thus contributing to the degree of solubility in p o l a r s o l v e n t s. 45 Hydrolytic cleavage and stability 1-Acid Hydrolysis: Glycosides can be hydrolyzed by heating with a dilute acid, where by the glycosidic linkages are cleaved. Glycosidic linkages involving different kinds of sugars are hydrolyzed with different degrees by acid hydrolysis 46 23  2024-11-05 e.g. 2-deoxysugars are hydrolyzed easier than 6-deoxy-sugars, which is still easier than normal sugars. - If there is a di-or an oligosaccharide unit in the molecule, it is practically impossible to cleave one unit of sugar at a time with acid hydrolysis. 47 When stepwise cleavage of monosaccharide units is needed, specific enzyme hydrolysis must be used. C-glycosides are more resistant to acid hydrolysis than O-glycosides i.e. they do require more Vigorous conditions. 48 24  2024-11-05 ◼ I- Simple Phenolic Glycosides ◼ This group includes glycosides that yield upon hydrolysis, simple phenolic aglycone. ◼ glycosides having alcoholic, aldehydic, carboxylic or ether group beside the phenolic group are also included in this class. 49 ◼ Arbutin ◼ Arbutin and its methyl ether derivative (methyl arbutin) are found in the leaves of bearberry (Uva ursi), which is the dried leaves of Arctostaphylos uva ursi family Ericaceae. ◼ Arbutin can be hydrolyzed by acid or by the enzyme emulsion (indicates- glycosidic linkage) to yield hydroquinone and glucose. 50 25  2024-11-05 51 ◼ Isolation: ◼ Powdered leaves extracted with boiling water. ◼ to the aqueous extract add lead acetate and filter. ◼ excess lead is removed by passing H2S through the filtrate. ◼ Finally concentrate the purified aqueous extract where arbutin crystallizes as silky needles. ◼ Chemical Tests: ◼ Arbutin + FeCl3 → blue color. ◼ Hydrolysis : ◼ hydroquinone is sublimable compound into feathery crystals. 52 26  2024-11-05 O Glucose OH Acid hyd. + Glucose H2O OH OH Hydroquinone ◼ Uses: ◼ Diuretic used in cystitis and urethritis. ◼ Bactericidal due to hydroquinone given upon hydrolysis in the body. ◼ Methyl-arbutin, also has diuretic and urinary tract antiseptic action. ◼ Mild astringent. ◼ Arbutin Skin Whitening- ◼ The primary mechanism of action involved here is Tryosinase inhibition 53 ◼ Arbutin is glucosylated hydroquinone and may carry similar cancer risks ◼ Although there are also claims that arbutin reduces cancer risk. ◼ The German Institute of Food Research in Potsdam found that intestinal bacteria can transform arbutin into hydroquinone, which creates an environment favourable for intestinal cancer. ◼ it is not known why this substance plays a role in cancer development. 54 27  2024-11-05 ◼ Salicin CH2OH ◼ Salicin is a phenolic O Gluc. glycoside present in the barks and leaves of many species of Salix and Populus, Family Salicaceae. ◼ Salicin is regarded as the natural guide of aspirin. ◼ Hydrolysis of salicin: ◼ Salicin can be hydrolyzed to- glucose and the aglycone saligenin (salicyl alcohol) by the emulsin enzyme. 55 56 28  2024-11-05 CH2OH CH2OH O Gluc. OH + glucose hyd. Saligenin ◼ When hydrochloric acid is used for hydrolysis at high temperature, two molecules saligenin combine to give yellowish-white crystals of water insoluble compound called saliretin. ◼ Oxidation of saligenin, directly produces salicylic acid. 57 CH2OH OH CH2OH OH HO HOH2C heat H+ O Saliretin Saligenin ◼ Isolation: As in arbutin. ◼ Test for identity: ◼ on hydrolysis, it yields saligenin + FeCl3 → blue color. ◼ Salicin is heated + potassium dichromate + dilute H2SO4 → odour of salicylic acid. [Oxidative hydrolysis]. ◼ Salicin + conc. H2SO4 → a bright red color, disappears upon addition of water. ◼ Uses: ◼ Antipyretic and anti-rheumatic. ◼ Teatment of hyperpigmented skin. 58 29  2024-11-05 ◼ Salicin is helpful for mild feverish colds and infections (influenza). ◼ acute and chronic rheumatic disorders. ◼ mild headaches, and pain caused by inflammation. ◼ Does not have the dangerous side effects associated with Aspirin. ◼ Salicin is used in medicine for the same purposes as salicylic acid and the salicylates. ◼ It is also used as a bitter tonic, i.e. a gastric stimulant, 59 Image:Bayer Aspirin ad, NYT, February 19, 1917.jpg COOH OCOCH3 Aspirin CH2OH O Glucose COOH Salicin OH Salicylic Acid 60 30  2024-11-05 What is ASPIRIN? 61 ◼ Gaultherin. COOCH3 ◼ Gaultherin is phenolic O-gluc. glycoside found in the leaf of Gaultheria procumbens family Ericaceae. ◼ It is a methyl salicylate glycoside. ◼ Hydrolysis: ◼ Hydrolysis with gaultherase enzyme (which is the natural enzyme in the plant) yields the aglycone methyl salicylate (winter green oil) and glucose. 62 31  2024-11-05 Coniferin ◼ Coniferin is a phenolic glycoside found in coniferous trees and asparagus. ◼ Coniferin is m- methoxy-p-hydroxy cinnamyl alcohol glucoside. 63 Hydrolysis : Enzyme hydrolysis of coniferin yields glucose and coniferyl alcohol which was formerly used for the preparation of artificial vanillin by oxidation with chromic acid. CH=CH-CH2OH CH=CH-CH2OH emulsin OCH3 + Glucose OCH3 O gluc. OH Chromic Acid Coniferin [OX.] CHO Coniferyl alcohol vanillin OCH3 OH 64 32  2024-11-05 Tests for identity: ◼ Coniferin + phloroglucinol + conc. HCl →red colour. ◼ Coniferin + conc. H2SO4 →red color. ◼ Coniferin + conc. HCl + heat → blue color. ◼ Uses: ◼ It was used in the preparation of vanillin. 65 ◼ Vanilloside ◼ Vanilloside (glucovanillin) is the main constituent of vanilla pods. ◼ Vanilla is the fully-grown but unripe fruit of Vanilla planfolia fam Orchidaceae, which has been subjected to a fermentation process. ◼ Green vanilla contains vanilloside and vanilloloside (vanillyl alcohol glucoside). 66 33  2024-11-05 CHO OCH3 O gluc. 67 ◼ Glucovanillin is an odorless glucoside. ◼ During curing (fermentation) these glycosides are acted upon by an oxidizing and a hydrolyzing enzymes (which present in the plant tissues) and vanillin with its characteristic odour is set free. ◼ Glucovanillin can be considered as an example of aldehydic glucoside as well as a phenolic glycoside. 68 34  2024-11-05 Glucovanillin Vanillin CHO CHO Enzymatic Hydrolysis + Glucose OCH3 OCH3 O-glc OH Green vanilla pods Brown vanilla pods Bitter in taste Sweet in taste Odourless Vanilla odour 69 70 35  2024-11-05 Commercial Preparation of Vanillin CH2-CH=CH2 CH=CH-CH 3 Iso-eugenol Eugenol Vanillin KOH Oxidation OCH3 OCH3 OH OH Guaiacol CHCl3+ NaOH Vanillin OCH3 Reflux OH CH=CH-CH 2OH H2SO4/K2Cr2O7 Coniferin Vanillin OCH3 O-glc 71 ◼ Uses: ◼ Glucovanillin can be used for the production of vanillin that is used in confectionery and in perfume; ◼ it has been replaced by synthetic vanillin. 72 36  2024-11-05 Many Uses for Vanilla ◼ believed by both Aztecs and Europeans an aphrodisiac ◼ scents perfume, soap, air fresheners, candles, body lotion ◼ flavors ice cream, chocolate, animal food, baby food, and tobacco 73 Many Uses for Vanilla ◼ insect repellant ◼ calming/soothing herbalist remedy (MRI scans show it relieves 63% anxiety) ◼ used in medications for Parkinson’s disease and high blood pressure 74 37