Glycosides and Neutral Principles

Glycosides and Neutral Principles

• Experiment 5 Post Lab covers Glycosides and Neutral Principles

Objectives

• Define glycosides and classify them according to type.
• Differentiate tests used to identify saponin glycosides in samples.
• Identify the steps in isolating saponin glycosides from plant samples.

Glycosides Defined

• Glycosides are compounds yielding one or more sugars and a non-sugar component upon hydrolysis.
• They are also known as "sugar ethers."

Glycoside Functions

• Glycosides serve regulatory, protective, and sanitary functions in plants.
• They have various therapeutic uses, including cardiac glycosides, laxatives, local irritants, and analgesics.

Glycoside Components

• Glycosides have two components: glycone and aglycone.
• The glycone is the sugar component and is the common denominator and is inactive.
• The aglycone, or genin, is the non-sugar component, basis of classification and is active.

Glycone Classification

• If the glycone is glucose, the glycoside is a glucoside.
• If the glycone is fructose, the glycoside is a fructoside.
• If the glycone is galactose, the glycoside is a galactoside.
• If the glycone is glucuronic acid, the glycoside is a glucuronide.

Glycosidic Bond Types

• Glycosides are classified by glycosidic bond type: C-glycosidic, O-glycosidic, N-glycosidic, and S-glycosidic bonds.

Glycoside Classification by Aglycone Portion

• Glycosides can be classified based on their aglycone portion into categories like alcohol, aldehyde, anthraquinone, steviol, steroidal, saponin, phenol, isothiocyanate, iridoid, chromone, coumarin and cyanogenic etc.

Alcohol Glycosides

• The aglycone portion of alcohol glycosides is an alcohol derivative.
• Willow bark (Salix purpurea, S. fragilis) is a source of alcohol glycosides, containing Salicin.

Aldehyde Glycosides

• The aglycone portion of aldehyde glycosides consists of an aldehyde derivative.
• Vanilla (Vanilla planifolia, V. tahitensis) is a source of aldehyde glycosides.

Anthraquinone Glycosides

• Anthraquinone glycosides compose the largest group of naturally occurring quinone pigments.
• They are used as dyes, cathartics, and habit-forming stimulant laxatives.
• Derivatives are often orange-red, soluble in hot water and dilute alcohol.
• Characteristically give a red, violet, green, or purple color with a base.
• Bontrager's test indicates their presence with a red coloration test in the lower ammoniacal layer while a Modified Bontrager's test indicates their presence with a pink color.
• Sources include Cascara sagrada (Rhamnus purshianus), Frangula (Rhamnus frangula), Aloe species, Rhubarb species, Indian rhubarb, Senna (Cassia species), and Chrysarobin (Andira araroba)

Chromone Glycosides

• Chromone glycosides feature an aglycone portion derived from a benzo-δ-pyrone nucleus.
• Sources of these glycosides include Hypericum erectum, Japanese eurya (Eurya japonica), and Cassia multijuga.

Coumarin Glycosides

• Coumarin glycosides have an aglycone portion derived from a benzo-α-pyrone nucleus.
• Tonka bean (Dipteryx odorata), Meadowsweet (Filipendula ulmaria), Sweet clover (Melilotus officinalis), and Vanilla grass (Anthoxanthum odoratum) are sources

Cyanogenic Glycosides

• Cyanogenic glycosides yield hydrocyanic acid upon hydrolysis.
• They are often found in legumes, root crops, and grasses, usually coexisting with hydrolytic enzymes in plants.
• Most are lost after plant harvest due to spontaneous hydrolysis.
• Amygdalin, also known as laetrile, is thought to have antitumor properties.
• Guignard’s test gives a red color within 15 minutes when the tube is warmed, indicating their presence.
• They can be found in Wild cherry (Prunus serotina), Apricot (Prunus armeniaca), Almond (Prunus amygdalus), and Cassava (Manihot esculenta).

Flavonoid Glycosides

• Flavonoid glycosides are phenolic plant pigments containing a benzo-γ-pyrone nucleus.
• They include anthocyanins, leucoanthocyanins, catechins, aurones, and chalcones.
• They have reported antiviral, antifungal, anti-inflammatory, and cytotoxic activities.
• Anthocyanins yield colored aglycones known as anthocyanidins upon hydrolysis.
• Leucoanthocyanins have leucoanthocyanidin as their aglycone.
• The Bate-Smith & Metcalf test results in a strong red or violet color; the Wilstatter "cyanidin” test results in colors ranging from orange to red, to crimson and magenta, and occasionally to green or blue.
• These compounds are found in Hesperidin, Naringin, Rutin, and Quercitin

Iridoid Glycosides

• Iridoid glycosides have an aglycone portion made of the aglycone, iridoid, usually bound to glucose a glycone.
• Sources include Asterids (Aucuba japonica, Eucommia ulmoides) producing Aucubin, and Catalpa (Catalpa bignonioides, C. speciosa) making Catalpol.

Isothiocyanate Glycosides

• Isothiocyanate glycosides are derived via hydrolysis of glucosinolates which are sulfur-containing compounds found in cruciferous plants.
• They are important because of their anti-cancer properties.
• Black mustard (Brassica nigra) and white mustard (Brassica alba) are sources.

Phenol Glycosides

• Phenol glycosides contain a phenol group as the aglycone portion.
• Bearberry (Arctostaphylos uva-ursi) a a source.

Steroidal Glycosides

• Steroidal glycosides possess cardiac glycosides that effect the heat muscles
• The steroidal nucleus is a cyclopentanoperhydrophenanthrene nucleus.
• Cardenolide is more common, with optimum activity at C-23, having a 5-membered lactone ring.
• Bufadienolide is less common, less active, at C-24, having a 6-membered lactone ring.
• The Keller-killiani test indicates the presence of 2-deoxysugars; reddish-brown which may turn blue or purple.
• The Liebermann-Burchard test identifies unsaturated steroids and triterpenes; colors ranging from blue to green, red, pink, purple or violet.
• Kedde’s test tests for unsaturated lactones; blue-violet color
• Sources include Foxglove (Digitalis purpurea), Grecian foxglove (Digitalis lanata), Convallaria (Convallaria majalis), Pheasant’s eye (Adonis vernalis), Black hellebore (Helleborus niger), Dogbane (Apocynum cannabinum), Adelfa (Nerium oleander), Strophantus (Strophantus kombe), Ouabain (Strophantus gratus, Acokanthera quabaio), Squill and Red squill.

Steviol Glycosides

• Steviol glycosides have a steviol aglycone, and are used as natural sweetners as a substitute for sugar, being 40 to 300 times sweeter than sucrose.
• The source is Stevia (Stevia rebaudiana).

Saponins

• Saponins are steroidal/triterpenoid glycosides characterized by froth when agitated and used as detergent
• They exert a powerful hemolytic action on RBCs and are highly toxic when injected and can be used a fish poison.
• It contains sapotoxin a poisonous saponin which can be a spermicidal & molluscicidal activity.
• Saponins are harmless when taken orally.
• Two are steroidal found in monocot families in Dioscoreaceae species) and triterpenoidal (dicot; Agavaceae species).
• Neutral Lead acetate produces acid saponin if present are precipitated as lead salts
• Basic Lead Acetate precipitates neutral saponin
• Hydrogen Sulfide decomposes lead precipitate
• General characteristics - amorphous and soluble in water and alcohol
• It forms persistent froth with water.
• Causes hemolysis of RBC’s if reaching the blood.
• Bitter, acrid taste and drugs
• A froth test will indicates a honeycomb like froth greater than 2 cm persistent for 10 minutes
• Agar cup hemolytic test indicates clear zones of hemolysis (hemolytic halos)
• Fehling’s test indicate a brick red precipitate (presence of sugar, glycone portion)
• Baryta water test indicates the formation of precipitate, where Barium hydroxide will react with neutral saponins; neutral saponins have no acid-base groups.
• The Lead acetate test will indicates the formation of precipitate, where basic lead acetate reacts with neutral saponins; neutral saponins have no acid-base groups.

Gugo Extract

• The color is golden yellow to orange
• It is odorless
• Slightly bitter, acrid

Saponins: Sources

• Plants that contain saponins are Licorice (Glycyrrhiza glabra), Yam (Dioscorea floribuna, D. alata), Ginseng (Panax ginseng, P. quinquefolius) and Gugo (Entada phaseoloides).

Drugs Official in the USP 24/ NF 19

• Drugs official in the USP 24/ NF 19 include Aloe USP 24, Cascara Sagrada USP, Digitalis USP 24, Elm USP 24, Milk Thistle NF 19, Oriental Ginseng NF 19, Powdered Digitalis USP 24, Powdered Milk Thistle NF 19, Powdered Oriental Ginseng NF 19, Senna USP 24 and Sennosides USP 24.

Philippine Sources

• Saponins can be found in Philippine plants such as Adelfa, Akapulko, Campanero, Campanilla, Gugo, Kalumbibit, Lukban, Maguey, Pistula and Sabila.

Flavonoids

• Polyphenolic secondary metabolites
• antioxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties.

Flavonoid Subgroups

• Flavones, Flavonols, Flavanones, Flavanonols, Flavanols or catechins and Anthocyanins Chalcones

Drugs Official in the USP 30/NF 25 and BP/EP

• Birch leaf, BP/EP
• Horsetail, BP/EP
• Buckwheat Herb, BP/EP
• Motherwort, BP/EP
• Elder Flower, BP/EP
• Olive, BP/EP
• Ginkgo, USP/NF
• Passiflora, BP/EP
• Hawthorn Leaf with Flower, USP/NF
• Red Clover, USP/NF

Philippine sources of flavonoids

• Aratiles - Muntingia calabura Linn
• Atis – Annona squamosa Linn
• Balingbing - Averrhoa carambola Linn
• Bignay - Antidesma bunius
• Chico - Achras zapota Linn.
• Santol - Sandoricum koetjape
• Sampalok - Tamarindus indica Linn.
• Katmon - Dillenia philippinensis
• Kamias - Averrhoa bilimbi L.
• Duhat - Syzygium cumini (L.)

Extraction of Flavonoids

• Organoleptic test
• Physical test
• Chemical test
• Shinoda test
• Alkaline reagent test

Process for Extraction of Flavonoids

• Weigh 10g of the shade dried plant sample and chop into small pieces.
• Macerate with 200mL 70% ethanol for 3 days at room temperature (25°C).
• Filter the extracts in vacuum using Whatman filter.
• Evaporate to dryness
• Weigh the residue and compute the percentage yield.

Yield Percentage

• Percentage yield = (weight of the residue / weight of the plant sample) × 100

Note in Physical Test

-Color, odor and appearance of the extract as characteristics

• Example: Voacanga globosa leaf extracts is Greenish black smooth thin film with a strong fruity odor in Ethyl acetate

Glycones Solubility

• soluble in water an acetone and insoluble in other and chloroform
• It is dependent on the presence of glycoside and aglycone portion. Flavonoid glycosides are polar soluble and dissolves in water but are insoluble in organic solvents like ether. Flavonoid aglycones are soluble in organic solvents while they are sparingly soluble in water.

Shinoda Test

• Transfer About 3 mL of extract into test tube and few drops of concentrated hydrochloric acid (HCl)
• Add magnesium turnings and presence of a red to pink indicates the presence of flavonoids

Alkaline Reagent Test

• Add drops of sodium hydroxide to an ethanolic extract
• Appearance of an intense yellow that disappear after adding dilute HCl indicates the presence of flavonoids