GLYCOSIDES Introduction

Overview Non-reducing organic compounds On hydrolysis with acids, alkalis or enzymes  A sugar moiety (= glycone, formed of one or more sugar units). A non-sugar moiety (= aglycone = genin). Widely distributed in nature Usually accompanied with enzymes used for their synthesis or hydrolysis.

Chemistry Acetals or sugar ethers –OH of anomeric carbon (e.g. at C-1 of aldose sugar) is replaced by a moiety having a nucleophilic atom e.g.: O, S, N or C  O-, S-, N- or C-glycosides

Most common is the -O- linkage connecting the reducing group of the sugar part & an OH (alcoholic or phenolic) of the aglycone  O-glycosides.

Chemistry Sugars exist in - or - forms & can  a - or -glycosides. Most naturally occurring glycosides are of -type.

Sugar moiety (= Glycone) Type of sugar Monosaccharide (e.g. glucose, rhamnose) Oligosaccharide (e.g. di-, tri- or tetrasaccharide) Most common is -D-glucose Others : pentoses & deoxy-hexoses (2-deoxy sugars) Sugar derivatives e.g. glucuronic acid Position of Attachment Mostly at only 1 position, sometimes at 2 different positions. Role of sugar moiety Helps in stabilization & solubilization of glycoside. Carries aglycone to site of action.

Non-sugar moiety (= Aglycone = Genin) Contains free -OH (in O-glycosides), free -NH (in N-glycosides) etc….  acetal link with sugar Type : belongs to different chemical groups e.g. phenols, alcohols, flavonoids, steroids etc……. Role: pharmacologically active part of glycosides.

Pharmacological activity Mainly associated with & due to aglycone part Anticancer, anti-inflammatory, antihepatotoxic, immunomodulating, antiviral, analgesic, diuretic antipyretic, hypoglycemic & cardiotonic activities. Examples: Arbutin: urinary antiseptic Salicin: anti-rheumatic & analgesic Rhein: anti-inflammatory Sennosides & barbaloin: laxatives Digoxin & lanatosides: cardiotonics

Function in the plant Energy producers Detoxifying agents Stabilizers for labile substances. Decorative or attractive (pigments). Protective

Stability & hydrolytic cleavage Glycosides may be subjected to acid, alkaline or enzymatic hydrolysis. Effect of acid hydrolysis Acetal linkage is cleaved before linkage between different sugars Glycosides containing normal sugars are more resistant to acid hydrolysis, than those containing 2-deoxy sugars. Drastic conditions may cause chemical changes in aglycone.

Stability & hydrolytic cleavage Effect of alkaline hydrolysis Being acetals, glycosides are relatively stable to alkaline hydrolysis. Alkalis may modify: Aglycone part e.g. strong alkalis open lactone ring of cardiac glycosides  loss of cardiotonic activity. Sugar part e.g. mild alkalis remove acetyl group of acetyldigitoxose in lanatosides.

Stability & hydrolytic cleavage Effect of enzymatic hydrolysis Generally, a specific enzyme is required to hydrolyze a particular glycoside. Certain enzymes hydrolyze a large number of glycosides e.g. Emulsin (all b-linked glycosides). Myrosin (all S-containing glycosides). Enzymes are usually named after the type of sugar : e.g. glycosides, which contain b-glucose, are hydrolyzed by b-glucosidase, & those containing rhamnose by rhamnosidase

Classification & Nomenclature According to: Chemical nature of aglycones : alcoholic, ester, phenolic, flavone, anthraquinone, steroid, lactone, monoterpenoid, diterpenoid & triterpenoid glycosides Presence of a special functional group: aldehydic (aldehyde group), cyanophore or cyanogenic (cyanide group) & thio- glycosides (thiol group, -SH) Therapeutic activity: cardiac, urinary antiseptic & laxative glycosides Natural form: primary & secondary glycosides Nomenclature Names are usually derived from botanical origin e.g.salicin from Salix, sennosides from Senna etc…...

Properties Colorless solids, mostly bitter in taste (except populin, stevioside & neoastilbin  sweet). Generally soluble in water & hydroalcoholic solutions. Non-reducing (do not reduce Fehling’s solution) except if aglycone part contains a reducing group (e.g. K-strophanthoside).

Extraction & Isolation Due to difference in physical & chemical properties, no common general method is used for isolation of glycosides Common solvents used for extraction: water & aqueous alcohols Precautions before extraction Deactivation of enzymes Maintenance of neutral conditions Removal of fats (defatting)

Deactivation of enzymes Specific hydrolytic enzymes present with glycosides should be deactivated before or during extraction, especially when using fresh plant material. Methods of enzyme deactivation: Drying plant material for 15-30 min at 100 oC then slow drying at a low temperature. Freeze-drying (lyophilization) of plant material before extraction. Dipping fresh plant material in boiling water or boiling alcohol for 10-20 min. Boiling fresh plant material with acetone. Carrying out extraction at very low temperature. Carrying out extraction in presence of (NH4)2SO4.

Maintenance of neutral conditions & Defatting Maintenance of neutral conditions before & during extraction since: Acidity may cause hydrolysis. Mild alkalinity may produce racemization. Defatting of fat-rich organs (e.g. seeds) before extraction using petroleum ether.

Purification of extracted glycosides Methods of purification: + Lead acetate solution  precipitate non-glycosidic constituents (tannins, proteins & coloring substances). Excess lead removed from filtrate by H2S or Na phosphate. Not used in case of flavonoid glycosides which may be precipitated. + Acetone or ether  precipitate or crystallize out glycosides from alcohol or aqueous alcohol solutions. Chromatographic techniques e.g. CC or TLC

Quantitative Determination Spectroscopic techniques: Colorimetric methods e.g. cardiac glycosides using Balget's or Kedde's reagents. Spectrometric methods e.g. measuring of UV absorption. Chromatographic methods: Applied on glycosides or after hydrolysis: Aglycone extracted with organic solvent (e.g. ethyl acetate) & determined by HPLC or GC. Sugar part (in aqueous layer) determined by GC after derivatization (silylation) or HPLC.

Phenolic glycosides

Classification Grouped according to chemical structure into: Simple phenolic glycosides e.g. Arbutin. Phenolic & ester glycosides e.g. Gaultherin & Monotropin. Phenolic & alcoholic glycosides e.g. Salicin & Populin. Anthracene glycosides e.g. Sennosides. Coumarin glycosides e.g. Aesculin & Daphnetin. Flavonoid glycosides e.g. Rutin & Hesperidin.

1-Simple phenolic glycosides - Arbutin Source Dried leaves of Uva ursi (Ericaceae). Hydrolysis Acid or enzymatic (emulsin)  hydroquinone + b-D- glucose. Chemical tests Arbutin+ FeCl3  Blue color (phenolic) Arbutin + HCl / heat  crystals of hydroquinone. Uses Diuretic & urinary antiseptic & Both hydroquinone and arbutin have skin bleaching action.

2- Phenolic & ester glycosides- Gaultherin & Monotropin Source Gaultheria procumbens (Wintergreen), & Monotropa & Betula spp. Gaultherin is a 1ry glycoside & monotropin a 2ry glycoside.

Gaultherin & Monotropin Hydrolysis Hydrolysis by acid or enzyme: Both  Volatile aglycone = methyl salycilate (oil of wintergreen) Sugar part: Gaultherin disaccharide primeverose = xylose + glucose Monotropin  glucose Uses Antipyretic, analgesic & anti-rheumatic

3-Phenolic & alcoholic glycosides- i-Salicin Source Salix fragilis (4-10%) & Populus spp. Hydrolysis Enzyme or mild acids  saligenin + glucose.

Salicin-Hydrolysis + HCl +Heat for a long time : 2 molecules of saligenin  (dehydration)  saliretin (water insoluble).

i-Salicin Isolation Extract from Salix bark with hot H2O & filter. + lead acetate solution (tannins ) & filter. Pass H2S gas (to remove excess lead) & filter. Neutralize, concentrate & cool  salicin crystals. Chemical tests + conc. H2SO4  red color + H2O  no color. + Froehd’s  violet color. + K2Cr4O7 + dilute H2SO4  benzaldehyde odor. Uses: analgesic, antipyretic & anti-inflammatory.

ii-Populin (6'-Benzoyl salicin) Source: Sweet glycoside isolated from Populus spp Synthesis: From salicin. Chemical tests: Populin + Froehd’s reagent  violet color. Hydrolysis: Uses: analgesic, antipyretic & anti-inflammatory.

iii-Coniferin Source Hydrolysis: Cambium tissues of pine trees & lignified tissues of beet root. Hydrolysis: Coniferin  coniferyl alcohol + glucose

iii-Coniferin Chemical tests Coniferin + concentrated HCl  blue color. Coniferin + concentrated H2SO4  red color. Uses Coniferin & its aglycone coniferyl alcohol are used as starting material for semisynthesis of vanillin.