Quality analysis of the medicines from the glycoside’s group.
Glycoside – a natural substances tha t contain carbohydrates where glycosidic part of the molecule (cyclic form of sugars) is connected with an organic radical that is not a sugar (aglycone or henin).
By the nature of the sugar glycosides are divided into two groups: piranosides and furanosides. There are also α-and β-glycosides depending on the configuration of carbon connected to the aglycone. Sugar part of the molecules may contain one or more interconnected sugars (monosaccharides, disaccharides, etc.).
Pentosides – pentose glycosides, hexosides – hexose glycosides, biosides – glycosides of disaccharides.
Bound of the sugar residue with henin is made through oxygen (O-glycosides) or nitrogen (N-glycosides: ATPh, antibiotics-aminoglycosides), or sulfur (thioglycosides: sinigrin from mustard).
O-glycosides by the nature of aglycone are divided into:
1) phenyl glycosides contain the phenyl radical in aglycone (bearberry glycosides – Arbutin);
2) Anthraquinone glycosides contain the aglycone, anthraquinone derivative (glycosides buckthorn, rhubarb, aloe);
3) flavone glycosides, which aglycone is derivative of flavone (catechins, rutin);
4) nitrogencontaining, cyanogenic glycosides (amigdaline);
5) glucoalkaloids, glycosides, in which the sugar component bound with the remainder of alkaloid (solasodine);
6) steroid glycosides (cardiac glycosides);
7) tannins (tannin);
8) saponins.
§ Cardiac glycosides (CG) – biologically active substances contained in some types of plants or secretions of some species of frogs; in small doses can show a specific effect on the heart muscle.
§ Sources of cardiac glycosides extraction: different types of digitalis, spring adonis, oleander, valley, erysimum, strofant, hellebore, and others.
§ In the plants primary (genuinic) glycosides are contained, which are very labile and down easily (under the action of enzymes, acids, alkalis) to form secondary glycosides. The latter may hydrolyse to aglycons and sugar components .
§ In 1875 y. from digitalis purpurea secondary glycoside are identified – digitoxin and in 40-th years of the twentieth century – from digitalis lanata – digoxin.
§ The first study of the chemical structure of cardiac glycosides Windaus completed in 1915 y., then in 30-th years Jacobs and Chesnet found that they contain steroid structure.
Current directions in the research of CG
Search of the new natural sources of CG and their extraction. Over the past 20 years the number of selected glycosides are doubled and is more than 2500. Shown, that biologically more active are ones from CG that are more unstable in conformation and thermodynamic relation, on the base of which the “alternatives” of natural strophanthin was synthesized – number of active 19-norcardenolides.
Chemical and biochemical transformations of the natural CG to improve their pharmacological properties. Thus, created nitroethers of CG combine cardiotonic, coronarolytic and respiratory effects .
Increasing of the CG content in cultured species by agrotechnical methods and introduction of the new species. New species of the digitalis lanata contains in 15-20 times more digoxin and lanatoside C than known species of the plant.
Developing of the new, more perfect methods of selection from the raw materials of CG used in medical practice. So, original ways of getting of digitoxin, digoxin, gitoxin, strofantidin, lanatoside C, erysimine, adenoside, cordigidin are developed. Cardiotonic drug corglycone was obtained.
Synthesis of some CG though conducted, but due to the presence of many stages and low output of the practical value is not received. The only industrial source of CG obtaining is plant material. The process of selection is difficult, because the plants contain enzymes that can change the chemical structure of glycosides and these changes are not reverse. Such changes can occur under the action of light, temperature, etc.. In the plants, usually a few CG are located and a number of related substances. To separate the mixtures of glycosides using chromatographic techniques.
By the chemical structure heart (and others) glycosides are esters in which aglycone and residues of mono-, di-, tri-or tetrasugars are bounded between each other by glycosidic connection. In some primary glycosides to the sugar component the balance of acetate acid attached.
Sugars that are a part of cardiac glycosides, except glucose and ramnose are specific for these group of compounds. These sugars are 6-deoxyhexoses (L-ramnose), 2,6-deoxyhexoses (D-digitoxose) or 3-O-methyl ethers (D-cymarose, L-oleandrose). From the cardiac glycosides more than 50 carohydrates are allocated.
Aglycons (henins) of the cardiac glycosides have a steroid structure that are derivatives of cyclopentane perhydro-phenanthrene.
Monosaccharides, which more often are a part of cardiac glycosides
By the chemical structure aglycones can be divided into two groups, which are different according to the structure of attached in the position 17 lactone cycle. Cardiac glycosides containing fivemember lactone ring are called cardenolides and, as such, containing sixmember lactone ring with two double bonds – bufadienolides:
In position 3 to aglycones the sugar component is attached. Radical R – СН3 or СОН, and Х1, Х2, Х3 – Н or ОН.
Cardenolides are found in the various types of digitalis (Digitalis), strophanth (Strophanthus), valley (Convallaria), erysimum (Erysimum), oleander (Nerium oleander), spring adonis (Adonis vernaris).
Bufadienolides are a part of hellebore (Helléborus), squill (Scillae bulbus), and also are found in frogs (their venom contains bufohenins having steroid structure with sixmember lactone cycle).
Connect between the structure and action of cardiac glycosides
Carrier of biological activity is aglycone. The sugar component that is attached in position 3 to the aglycone influences on the rate of absorption, respectively, on the duration of action. The bigger quantity of sugar residues in the glycoside molecule cases their bigger activity.
Specific action of glycoside on the heart is caused by the presence of lactone cycle in the aglycone molecule in position 17 and OH-group in position 14. On the cardiotonic action substitute in position 10 has a great influence. For the most of aglycones this is methyl or aldehyde group. Aldehyde group oxidation to carboxylic group significantly weakens the effect on the heart muscle.
Replacing of the aglycons steroid cycle by the derivatives of benzene, naphthalene, and also like lactone cycle by other radicals and even the change of character of the connection between steroid nucleus and lactone, leads to the loss of physiological activity. This indicates the specificity of the molecular structure of cardiac glycosides and aglycones and on the complexity of obtaining them by synthetic way.
The chemical structure and obtaining of CG
l In medical practice use different galeeogalen preparations (extracts, tinctures), which contain CG and preparations of individual CG, which by their chemical structure are cardenolides.
l In Addition 1 to SPhU digitoxin, digoxin, quabaine are included.
l From the leaves of various kinds of digitalis extract:
Celanide (Сеlanidum) (digilanide or lanatoside С) – primary glycoside of the Digitalis lanata. Tabl. by 0,00025 g (0,25 mg); 0,05 % solution in fl. by 10 ml (0,5 mg in 1 ml) for internal usage and 0,02 % solution in amp. by 1 ml.
Digitoxin and digoxin – secondary glycosides in digitalis purpurea and lanata (Digitalis purpurea L., Digitalis lanata). Digoxin: tabl. by 0,25 mg and by 0,1 mg for children; 0,025% solution in amp. by 1 ml. Digitoxin – tabl. by 0,1 mg and suppositories by 0,15 mg.
Digalen-neo. Neo-galenic preparation from the leaves of Digitalis ferniginea. Amp. by 1 ml f/in.; flacons by 15 ml of preparation, for internal usage.
Chemical content of the primary digitalis glycosides
Secondary digitalis glycosides of both species after the loss of these products of hydrolysis consist of aglycones and sugar part, which is the same in all three kinds of secondary glycosides
From the seeds of various kinds of strophant extract:
Strophanthin-К (Strophanthinus К) –mixture of cardiac glycosides isolated from strophanth Kombe (Strophanthus Kombe Oliver). Issue: amp. by 1 ml of 0,025% solution for i/v or i/m injections.
Quabaine (Ouabainum) (Strophanthin-G) – glycoside from Strophanthus gratus. Ampoules by 1 ml of 250 mcg/мml solution for i/v injections.
l From the leaves of Convallaria majalis extract:
Corglycone (Corglусоnum) –the sum of not less than five glycosides. Ampoules by 1 ml of 0,06% solution for i/v injections.
Convallatoxin (Convallatoxinum) –the main glycoside of lilies. Ampoules by 1 ml of 0,03% aqueous solution of the substance for i/v njections.
l Adoniside (Аdonisidum) – neo-galenic preparation of spring adonis grass (Аdonis vernalis). Flacons by 15 ml.
l From Erysimum – erysimine, cardiovalene –the sum of glycosides.
Chemical content of the medical drugs from cardiac glycosides
Digitoxin (Digitoxinum) (SPhU)
Properties of the CG pharmacopoeial preparations
1. Digitoxin – white or almost white powder. Practically insoluble in water, easily soluble in the mixture of equail volumes of methanol and methylene chloride, few soluble in 96% alcohol and methanol.
2. Digoxin –white or almost white powder or colorless crystals. Practically insoluble in water, easily soluble in the mixture of equail volumes of methanol and methylene chloride, few soluble in alcohol.
3. Quabaine – crystalline white powder or colorless crystals. Moderately soluble in water and ethanol, practically insoluble in ether andethylacetate.
Assay
Spectrophotometry in the visible part of spectrum. The content of all three drugs in the substance is calculated, taking into account the results of measurements of optical density and concentration of the studied solution and solution of PhSE of the appropriate drug (standard method).
Identification of the pharmacopoeial preparations of CG
IR–spectroscopy (digoxin, digitoxin) –spectrum should be identical to the spectrum of PhSE drug.
TLC (digoxin, digitoxin, quabaine).
Cedde reaction (on the fivemember lactone cycle) with alkaline solution of 3,5-dinitrobenzoic acid (digitoxin, digoxin) – purple color.
Raymond’s reaction (on the fivemember lactone cycle) with alkaline solution of dinitrobenzene (quabaine) – blue color.
Keller-Kiliani reaction (on deoxysugars) (digoxin, digitoxin).
Solution of quabaine substance in H2SO4 conc. – pink color, which quickly becomes red, this solution in the UV-light gives a green fluorescence (steroid cycle).
Reaction on deoxysugars (quabaine). After the acidic hydrolysis with Fehling’s reagent – red sediment.
Purity test of the pharmacopoeial preparations of CG
Digitoxin. Impurities (gitoxin, other glycosides) – TLC.
Digoxin. Impurities (gitoxin, digitoxin and other glycosides) – TLC.
Quabaine. Impurities (gitoxin, digitoxin and other glycosides) – TLC. Alkaloids and Strophenthin-К – with solution of tannin acid no precipitate formed .
Storage
All three drugs remain in the protected dark place. Poisonous substances. List А.
General chemical reactions for CG identification
Reactions on steroid cycle
1. Liberman-Burkhard reaction:a small amount of substance is dissolved in a few drops of ice acetate acid and mixed with a mixture of acetic anhydride and concentrated sulfate acid. Slowly the coloration appears, which goes from pink to green or blue. This reaction glycosides give, which are in the processing by strong acids capable to dehydration (corglycone, strophenthin-К).
2. Rosenheim reaction:to the chloroform solution of substance add 96% trichloracetic acid – color appears, which gradually changes from pink to lilac and blue. This reaction is typical for steroids containing diene group or can form it under the influence of reagent.
3. Steroid cycle in cardenolides can be detected by fluorimetric method by the usage as a reagent mixture of phosphate and sulfate acids with iron (III) chloride; iron perchlorate solution in sulfate acid and so on.
Reactions on the fivemember lactone cycle of cardenolides
Legal reaction – the interaction in an alkaline medium with sodium nitroprusside red coloration appears and gradually fades.
Raymond’s reaction – in alkaline medium with m-dinitrobenzene reddish-purple coloration appears.
Cedde reaction – in alkaline medium with 3,5-dinitrobenzoic caid purple color appears.
Baljet reaction (Balje) -with alkaline picric acid solution orange-red color appears.
The disadvantage of the above reactions is that almost all glycosides with close structure give the same color. Therefore, this reaction caot be used to identify individual glycosides. Therefore, Reichstein proposed for recognition of individual glycosides use concentrated or 84% sulfuric acid, which gives color reactions with various glycosides, which are held in time and color change.
Mechanism of the Baljet reaction (Balje)
Reaction on the sixmember lacton cycle of bufadienolides
Glycoside is dissolved in chloroform and add gradually saturated solution of antimony (III) chloride, at the heating dark-red color appears.
Reactions of sugar component
l After the acidic hydrolysis can be used inherent sugar reactions, based on their reductive properties:
a) With Fehling reagent – formation of the red sediment Сu2O;
b) With Tollense reagent – “silver mirror“reaction.
c) Specific for 2-deoxysugars contained in the molecules of most cardiac glycosides, are:
d) Keller – Kiliani reaction:glycoside solution in concentrated acetate acid containing iron (III) chloride thicken on concentrated sulfate acid. On the boundary of two layers dark red or brown ring appears, the top layer paints in blue or blue-green color. The reaction occurs only when deoxysugar is in the free state or takes extreme position in the molecule of glycosides.
b) Febba and Levy reaction (for those glycosides, in which 2-deoxysugars from both sides linked with other sugars) – with trichloroacetic acid and p-nitrophenylhydrazine.
c) Pezets reaction: at the heating of glycoside with xanthydrol or anthrone in the presence of concentrated acetic acid with the following addition of several drops of sulfate acid or phosphate acid red or green, blue-green coloring appears. During the reaction under the action of concentrated acids, sugar component forms furfural or its derivatives, which are condensed with xanthydrol or anthrone.
Identification of the medicinal substances from CG groups can be done by the usage of specific rotation. Perspective is also a technique, based on the building of chromatographic diagrams that show the dependence of Rf from the system of solvents. IR- and UV-spectroscopy are also used.
Investigation of the good quality of CG
Particular attention is drawn to the presence of impurities of extraneous glycosides. This applies primarily to the preparations derived from plants that contain more similar in structure CG. For the determination of impurities using TLC, determining of the related glycosides on the chromatogram by the Rf value and the nature of the fluorescence spots in UV-light after the treatment by appropriate reagents (chloramine B, m-dynitrobenzene).
Incompatibility of CG
Cardenolides are incompatible with acids and compounds, which give acidic reaction of the environment. Hydrolysis by glycoside bound takes place. Reaction goes without any visible changes (with ascorbic acid and other vitamins with acidic рН of environment).
Cardiac glycosides are incompatible with alkalis and compounds, which give alkaline reaction of the environment(NaHCO3, barbital–sodium and others).
Assay of CG
Perform by spectrophotometric, photocolorimetric methods by the products of interaction in an alkaline environment with nitroderivatives of aromatic series. Qualitative and quantitative assessment of CG also are conducted by the high effective liquid chromatography (HELC), which allow to determine not only the main but also related glycosides.
Biological control is used to determine the lowest doses of standard and investigated substances that cause systolic shutdown of the heart of experimental animals. Then calculate the content of frog (FUA), cat (CUA), pigeon (PUA) units in 1 g of envestigated substance, in one tablet or 1 ml.
Some of the CG and their dosage forms can be defined by polarographic method. The advantage of this method is that the determination made by the reduction of double bond, conjugate with the carbonyl group of lacton cycle. This system is known, is one of the factors that determine the biological activity of cardiac glycosides. Polarography also is combined with the previous chromatographic separation of CG.
Storage and aplication of CG
CG and their drugs are stored in tightly corked container that protect from light and moisture (corglycon – not more than +5 º C). Such conditions would prevent their hydrolytic cleavage.
As cardiotonic means at the acute and chronic circulatory failure or cardiovascular failure. They differ in strength, duration, speed of action, influence on the central nervous system.
Medical drugs of CG are more effective at the i/v introduction. For this the drugs are pre-dissolved in 20-40% glucose solution or isotonic solution to the concentration of 0,025%.
Higher single doses of individual CG are 0,5 mg, daily – 1,0 mg. Overdose causes severe dysfunction of cardiac activity. This requires them to be ascribed to toxic substances. You must consider the ability of CG gradually accumulate in the body (the degree of accumulation).
Glycosides are a distinct class of compounds that are either found iature or can be synthetically prepared. The natural glycosides are isolated from various plant species namely digitalis purpurea Linne, digitalis lanata Ehrhart, strophanthus gratus, or acokanthea schimperi. Therefore, these compounds are also named as digitalis, digoxin, and digitoxin. Currently, digoxin is the only cardiac glycoside commercially available in United States. Glycosides have a characteristic steroid (aglycone) structure complexed with a sugar moiety at C-3 position of the steroid through the p-hydroxyl group. Glycosides are mainly used in the prophylactic management and treatment of congestive heart failure and atrial fibrillation. They are known to relieve the symptoms of systemic venous congestion (right-sided heart failure or peripheral edema) and pulmonary congestion (left-sided heart failure). However, glycosides also find applications to treat and prevent sinus and supraventricular tachycardia and
symptoms of angina pectoris and myocardial infarction, but only in combination with p-adrenergic blocking agents and in patients with congestive heart failure. The exact mechanism of pharmacological action of glycosides has not been fully elucidated. However, glycosides exhibit a positive inotropic effect accompanied by reduction in peripheral resistance and enhancement of myocardial contractility resultingin increased myocardial oxygen consumption. They also inhibit the activities of sodium-potassium activated ATPase, an enzyme required for the active transport of sodium across the myocardial
cell membranes. Glycosides are normally administered either orally or by N injection and possess a half-life of 36 h to 5-7 days iormal patients, depending on the choice of drugs.
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. 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 soluble in alcohol.
Separation between glycosides parts:
Glycosides glycone +aglycone +HCL
G + A +salt+H2O
(H2O+G)+A (H2O+G)+(chloroform+A)
We can separate them by using separatory funnel
The best solvent to extract aglycone is Ethyl acetate because:
A- immiscible in water.
B- always presents in the upper layer.
physico-chemicalproperties 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
(except: populin, glycyrrhizin, stevioside).
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:Convallariamajalis – Lily of the Valley.
• 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).
• Local irritant group:
a-Sinigrin(Black mustered seeds_Brassica nigra)
b-Sinalbin(White mustered seeds_Brasica alba)
• Analgesics and antipyretics:
Salicin Salisylic acid – Willow or Salix bark.
• Keeping elasticity of blood vessels like:
Rutin_Rutoside (Bitter orange peels, Lemon peels)
• Anti-inflammatory group:
a- Aloin for 1)acne 2)peptic ulcer
b-Glycyrrhizin
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.
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 iucleosides DNA,RNA
• C-glycosides : in these glycosides the sugar linked (condensed) directly to Carbon atom of aglycone like in aloin
N.B C-glycosides are not hydrolyzed by acids or alkalis or by enzymes mainly .
Classification of glycosides according to a glycone part :
• 1- if a glycone part alcohol -this group called alcoholic group like Salicin
• 2- if a glycone part aldehyde- this group called aldehydic gr. 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 ——à steroidal glycoside (cardiac) Digoxin
• 8-flavone ,flavonol, flavanone –flavonoid glycoside
• 9-triterpenoid –saponin glycoside –glycyrrhizin ,melanthin (nigella seeds) or ginsenoside .
Classification of glycosides according to a glycone part.
1- phenolic group of glycosides:-
Such as arbutin which isolated from bearberry leaves
Uses: UTI as antiseptic and antibacterial & mild diuretic
Drugs :
-Esoterica :cream :age spots
-hydroquinone solution : wet hands
2- saponin group of glycoside
а. Important group of glycosides which widely distributed specially in the higher plants parts
b. Most of them are neutral compounds, soluble in water insoluble in the organic solvents.
d. Irritant compounds for mucous membranes
e. They form with hydrolysis glycone part which usually β-D-glucose or it’s acids (glucuronic acid) + Sapogenin
Cardiac glycosides: group of glycosides which has powerful action on cardiac muscles (cardiac muscle stimulant).
C.G glycone part consist of one , two, three monosaccharide units or more similar or different
Aglycone part has steroidal nucleus
cyclopentanoperhudrophenanthrene
B.glycone part of C.G always attached at C-3 position of aglycone part
classification
1. cardenolide (one double bond, lactone ring) :
Has five member lactone ring (unsaturated) attached at C17 B position of steroidal nucleus.
2. Bufadienolide: (contain two double bonds, lactone ring)
Has six member ( unsaturated ) lactone ring attached at C-17 alpha – position
What is important for activity of C.G :-
1) The presence of hydroxyl group in C14 position makes the glycoside very active and gives rapid action in the body, but if we change it to (H+) group the drug will be inactive or less active.
2) The presence of Alpha & Beta unsaturated lactone ring increases the activity of C.G, but if we make it saturated the C.G will lose its activity.
3) The ring junctions Cis, Trans, Cis make the nucleus very stable so more active.
4) The sugar part increases absorption and distribution of C.G in the body
S.P : Glucose, Rhamnose, Cymarose, Digitoxose
Examples of Cardenolides
1) Digitalis glycosides
Digoxin
Digitoxin
Gitoxin
2) Strophanthus gratus C.G :
Oubain
3) Strophanthus Kombe glycoside :
K- strophanthin
Bufadienolide example
Squill bulb glycoside
Scillaren
Chemical tests :-
1) Keller Kiliani test : C.G + CH3COOH + H2SO4 + FeCl3 brown
2) Legal test : C.G + pyridine sodium nitroprusside Red to pink colour
Uses : 1-CHF 2- arrhythmias
General properties of C.G : 1- Amorphous powder 2-bitter taste
3- sol. In H2O 4-Insol. In Org. solvents
5- Very toxic compounds 6- Odorless
Structure
Cardiac glycosides are composed of two structural features : the sugar (glycoside) and the non-sugar (aglycone – steroid) moieties. (figure below)
The R group at the 17-position defines the class of cardiac glycoside. Two classes have been observed in Nature – the cardenolides and the bufadienolides (see figure below). The cardenolides have an unsaturated butyrolactone ring while the bufadienolides have an a-pyrone ring.
Nomenclature
The cardiac glycosides occur mainly in plants from which the names have been derived. Digitalis purpurea, Digitalis lanata, Strophanthus grtus, and Strophanthus kombe are the major sources of the cardiac glycosides. The term ‘genin’ at the end refers to only the aglycone portion (without the sugar). Thus the word digitoxin refers to a agent consisting of digitoxigenin (aglycone) and sugar moieties (three). The aglycone portion (figure below) of cardiac glycosides is more important than the glycone portion.
Structure – Activity Relationships
The sugar moiety appears to be important only for the partitioning and kinetics of action. (see section on pharmacokinetics of cardiac glycosides) It possesses no biological activity. For example, elimination of the aglycone moiety eliminates the activity of alleviating symptoms associated with cardiac failure.
The “backbone” U shape of the steroid nucleus appears to be very important. Structures with C/D trans fusion are inactive.
Conversion to A/B trans system leads to a marked drop in activity. Thus although not mandatory A/B cis fusion is important.
The 14b-OH groups is now believed to be dispensible. A skeleton without 14b-OH group but retaining the C/D cis ring fusion was found to retain activity.
Lactones alone, wheot attached to the steroid skeleton, are not active. Thus the activity rests in the steroid skeleton.
The unsaturated 17-lactone plays an important role in receptor binding. Saturation of the lactone ring dramatically reduced the biological activity.
The lactone ring is not absolutely required. For example, using a,b-unsaturated nitrile (C=C-CN group) the lactone could be replaced with little or no loss in biological activity.
Digoxin
General Notices
(Ph Eur monograph 0079)
C41H64O14
ıı781ıı20830-75-5
Action and use
Na/K-ATPase inhibitor; cardiac glycoside.
Preparations
Digoxin Injection
Paediatric Digoxin Injection
Paediatric Digoxin Oral Solution
Digoxin Tablets
Ph Eur
DEFINITION
3b-[(2,6-Dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-
2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]-12b,14-dihydroxy-5b-card-20(22)-enolide.
Content
96.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white powder, or colourless crystals.
Solubility
Practically insoluble in water, freely soluble in a mixture of equal volumes of methanol and
methylene chloride, slightly soluble in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparisonıdigoxin CRS.
TESTS
Appearance of solution
The solution is clear (2.2.1) and colourless (2.2.2, Method I).
Dissolve 50 mg in a mixture of equal volumes of methanol R and methylene chloride R and
dilute to 10 ml with the same mixture of solvents.
Specific optical rotation (2.2.7)
+ 13.9 to + 15.9 (dried substance).
Dissolve
dilute to 25.0 ml with the same mixture of solvents.
Related substances
Liquid chromatography (2.2.29).
Test solutionıDissolve 50.0 mg of the substance to be examined in 100.0 ml of methanol R.
Reference solution (a)ıDissolve 10.0 mg of digoxin CRS in methanol R and dilute to 20.0 ml
with the same solvent.
Reference solution (b)ıDilute 1.0 ml of the test solution to 100.0 ml with methanol R.
Reference solution (c)ıDissolve 2.5 mg of digoxigenin CRS (impurity C) in methanol R and
dilute to 5.0 ml with the same solvent. Dilute 1.0 ml of the solution to 50.0 ml with methanol R.
Dilute 1.0 ml of this solution to 10.0 ml with methanol R.
Reference solution (d)ıDissolve 50.0 mg of lanatoside C R (impurity H) in methanol R and
dilute to 100.0 ml with the same solvent. To 1.0 ml of this solution, add 1.0 ml of the test
solution and dilute to 20.0 ml with methanol R.
Reference solution (e)ıDissolve 5.0 mg of digoxin for peak identification CRS in methanol R
and dilute to 10.0 ml with the same solvent.
Column:ı
ı— size: l =
ı— stationary phase: octadecylsilyl silica gel for chromatography R (5 μm).
Mobile phase:
ı— mobile phase A: acetonitrile R, water R (10:90 V/V);
ı— mobile phase B: water R, acetonitrile R (10:90 V/V);
Flow rateı1.5 ml/min.
DetectionıSpectrophotometer at 220 nm.
Injectionı10 μl of the test solution and reference solutions (b), (c), (d) and (e).
Identification of impuritiesıUse the chromatogram supplied with digoxin for peak identification
CRS and the chromatogram obtained with reference solution (e) to identify the peaks due to
impurities A, B, C, E, F, G, K.
Relative retentionıWith reference to digoxin (retention time = about 4.3 min): impurity C =
about 0.3; impurity E = about 0.5; impurity F = about 0.6; impurity G = about 0.8; impurity L =
about 1.4; impurity K = about 1.6; impurity B = about 2.2; impurity A = about 2.6.
System suitabilityıReference solution (d):
ı— resolution: minimum 1.5 between the peaks due to impurity H and digoxin.
Limits:
ı— impurities E, K: for each impurity, not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.0 per cent);
ı— impurity L: not more than 0.3 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent);
ı— impurity G: not more than 0.8 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.8 per cent);
ı— impurities A, B: for each impurity, not more than 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b) (0.5 per cent);
ı— impurity F: not more than 2.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (2.5 per cent);
ı— impurity C: not more than 5 times the area of the corresponding peak in the
chromatogram obtained with reference solution (c) (1.0 per cent);
ı— any other impurity: for each impurity, not more than 0.2 times the area of the principal
peak in the chromatogram obtained with reference solution (b) (0.2 per cent);
sum of impurities other than A, B, C, E, F, G, K, L: not more than 0.7 times the area of
the principal peak in the chromatogram obtained with reference solution (b) (0.7 per cent);
ı— total: not more than 3.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (3.5 per cent);
ı— disregard limit: 0.05 times the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.05 per cent).
The thresholds indicated under Related Substances (Table 2034.-1) in the general
monograph Substances for pharmaceutical use (2034) do not apply.
Loss on drying (2.2.32)
Maximum 1.0 per cent, determined on
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on the residue obtained in the test for loss on drying.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances with the
following modification.
InjectionıTest solution and reference solution (a).
Calculate the percentage content of C41H64O14 from the declared content of digoxin CRS.
STORAGE
Protected from light.
IMPURITIES
Specified impurities
A, B, C, E, F, G, K, L.
Other detectable impurities (the following substances would, if present at a sufficient level, be
detected by one or other of the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or by the general monograph
Substances for pharmaceutical use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. Control of impurities in substances
for pharmaceutical use): D, H, I, J.
ıA. R1 = R2 = R3 = R4 = H: digitoxin,
ıB. R1 = R3 = R4 = H, R2 = OH: 3b-[(2,6-dideoxy-b-D –ribo-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]-14,16b-
dihydroxy-5b-card-20(22)-enolide (gitoxin),
ıE. R1 = R2 = OH, R3 = R4 = H: 3b-[(2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]- 12b,14,
16b-trihydroxy-5b-card-20(22)-enolide (diginatin),
ıH. R1 = OH, R2 = H, R3 = CO-CH3, R4 = Glu: 3b-[(b-D-glucopyranosyl-(1®4)-3-O-acetyl-2,6-
dideoxy-b-D –ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl)oxy]-12b,14-dihydroxy-5b-card-20(22)-enolide (lanatoside
C),
ıI. R1 = OH, R2 = R4 = H, R3 = CO-CH3: 3b-[(3-O-acetyl-2,6-dideoxy-b-D-ribo-hexopyranosyl-
(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]-
12b,14-dihydroxy-5b-card-20(22)-enolide (a-acetyldigoxin),
ıJ. R1 = OH, R2 = R3 = H, R4 = CO-CH3: 3b-[(4-O-acetyl-2,6-dideoxy-b-D-ribo-hexopyranosyl-
(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]-
12b,14-dihydroxy-5b-card-20(22)-enolide (b-acetyldigoxin),
ıK. R1 = OH, R2 = R3 = H, R4 = Dig: 3b-[(2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl)oxy]-12b,14-dihydroxy-5b-card-20(22)-enolide (digoxigenin
tetrakisdigitoxoside),
ıC. R = H: 3b,12b,14-trihydroxy-5b-card-20(22)-enolide (digoxigenin),
ıD. R = Dig: 3b-(2,6-dideoxy-b-D-ribo-hexopyranosyloxy)-12b,14-dihydroxy-5b-card-20(22)-
enolide (digoxigenin monodigitoxoside),
ıF. R = Dig-(1®4)-Dig: 3b-[(2,6-dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribohexopyranosyl)
oxy]-12b,14-dihydroxy-5b-card-20(22)-enolide (digoxigenin bisdigitoxoside),
ıG. R = Gdd-(1®4)-Dig-(1®4)-Dig: 3b-[(2,6-dideoxy-b-D-arabino-hexopyranosyl-(1®4)-2,6-
dideoxy-b-D-ribo-hexopyranosyl-(1®4)-2,6-dideoxy-b-D-ribo-hexopyranosyl)oxy]-12b,14-
dihydroxy-5b-card-20(22)-enolide (neodigoxin),
ıL. unknown structure.
