Chapter 25
Isolation of Phytopharmaceuticals
INTRODUCTION
There is a revival of interest in the use of plants in pharmacy both from pharmaceutical industry as a source of new lead molecules find from the general public who are using plant extracts in many ways in conventional and complementary therapies. About one-quarter of all prescription drugs are of plant origin despite the fact that less than 5% of plant species have been investigated. Many of the synthetic medicine currently in clinical use have been from natural sources. It is a widely accepted fact that natural product chemistry surpasses the kind of chemistry that synthetic chemist can ever accomplish in the laboratory. Phytochemical diversity in terms of structural novelty is unprecedented in laboratory synthesis. Indeed the plant kingdom provides enormous chemical diversity. Advances in bioassay screening, isolation techniques and structural elucidation have greatly shortened and accelerated the process of drug discovery from medicinal plants. Nowadays it is a common practice among natural product chemists to use some type of bioassay to direct the progress of phytochemical investigation towards the discovery of new pure bioactive markers.
The increasing use of herbal preparations has highlighted need for adequate standards to ensure quality, safety and efficacy of such drugs and preparations. Many developing countries are becoming aware of the potential of their flora as a source of medicinally useful products. Some of the most important alkaloids, glycosides, aglycones, resin and essential oil components of commercial use have been presented here in respect to their isolation and identification.
ISOLATION OF ATROPINE
Atropine is a tropane alkaloid from the members of the Solanaceae family. It is present in Atropa belladona (Deadly Night shade), Datura stramonium (Thorn apple), and Hyoscyamus niger (Henbane), Other important solanaceous alkaloids are hyoscyamine, hyoscine (scopolamine), apoatropine,belladonine and norhyoscyamine. Atropine is an optically inactive laevorotatory isomer of hyoscyamine.
Isolation
Atropine is isolated from the juice or the powdered drug. Hyoscyamus muticus is the preferred source for the manufacture of atropine because of its high alkaloidal content, with D. stramonium next in order. The powdered drug material is thoroughly moistened with an aqueous solution of sodium carbonate and then extracted with ether or benzene. The alkaloidal free bases are extracted from the solvent with water acidified with acetic acid. The acid solution is then shaken with solvent ether to remove colouring matter. The alkaloids are precipitated with sodium carbonate, filtered off, washed and dried. The dried mass is dissolved in solvent ether or acetone and dehydrated with anhydrous sodium sulphate before filtration. The filtrate after concentration and cooling yields crude crystals of hyoscyamine and atropine from the solution. The crude crystalline mass is separated from the solution. The crude crystalline mass obtained after filtration is dissolved in alcohol, and sodium hydroxide solution is added and the mixture is allowed to stand until hyoscyamine is completely racemized to atropine which is indicated by the absence of optical activity.
The crude atropine is purified by crystallization from acetone. Atropine sulphate is the most important salt of atropine. It occurs in the form of colourless crystalline powder. It is soluble in water and alcohol but insoluble in ether and chloroform. Melting point: 115–116°C
Identification
- Dilute solution of atropine, when treated with concentrated nitric acid and the mixture evaporated to dryness on the steam bath, produces a pale yellow residue. The residue gives a violet colouration when a drop of freshly prepared solution of potassium hydroxide is added. This is known as Vitali–Marin reaction.
Thin Layer Chromatography of Atropine
One percentage solution of atropine dissolved in 2 N acetic acid is spotted over silica gel-G plate and eluted in the solvent system of strong ammonia solution; methanol (1.5:100). TLC plate is spread with an acidified iodoplatinate solution. Atropine gives the Rf value 0.18. Likewise atropine sulphate shows the Rf value 0.70, in the solvent system acetone : 0.5 M sodium chloride and spraying with Dragendorff ’s reagent.
ISOLATION OF ANDROGRAPHOLIDE
Andrographolide is a bitter diterpenoid lactone obtained from the dried herb of Andrographis paniculata; family Acanthaceae. It also consists of other bitters which includes neo andrographolide and andrographoside.
Isolation
The dried herb is cleaned to remove foreign matter and then crushed to coarse powder. The powder is exhaustively extracted with 1:1 mixture of methanol and ethylene dichloride. The total extract is concentrated and subjected to treatment with activated charcoal. The solution treated with charcoal is filtered, and the filtrate is concentrated to pasty mass and then dissolved in hot methanol. The methanolic solution is again treated with charcoal or passed through mixed charcoal and Hyflo bed. The resultant clear, light yellow coloured solution is reduced to about half of its volume and subjected to crystallization in crystallizer fitted with low-speed stirrer. The crystals obtained after about 24 h are filtered off and washed with chilled methanol. The filtrate and washings are processed for second crop after concentration. The crystalline product obtained is dried in vacuum dryer at temperature not more than 50°C. This procedure yields about 1.25–1.75% of andrographolide. Melting point: 218–220°C
Thin Layer Chromatography of Andrographolide
Dissolve about 1 mg of sample in 1 ml of methanol. Apply the spots over silica gel-G plate and elute in the solvent system chloroform-methanol (7:1). Spray the eluted plate with 20% sulphuric acid in methanol and heat at 120°C for 10 min. Andrographolide appears as a visible brownish spot at Rf value 0.70, while neoandrographolide appears as a pinkish spot at Rf value 0.39.
ISOLATION OF BACOSIDES
Bacosides are the triterpenic saponins obtained from dried, whole herb, preferably leaves and stems of Bacopa monnieri; family Scrophulariaceae. These are the tetracyclic triterpenic saponins which consist of the crystalline mixture of several saponins including bacosides A and B.
Isolation
The coarsely powdered drug is extracted with ethyl alcohol and the alcoholic extract is concentrated to dryness. Dry alcoholic extract is dissolved in 60% alcohol, and the solution is extracted in separating funnel with benzene to divide the constituents in benzene soluble and alcohol soluble fractions. Alcohol soluble part consists of slimy mass; it is further dissolved in alcohol and fractionally precipitated with ether and petroleum ether repeatedly leading to the separation of brown resinous material. Solution of alcohol– ether–petroleum ether mixture is concentrated and the residue is macerated with acetone and filtered. The acetoneinsoluble powder is partitioned between butanol and water. During concentration of butanol solution, a precipitate that settles down is separated and coded as A1 . The filtrate is again concentrated and precipitated with acetone and ether to afford a powder A2 . The mother liquor is concentrated to dryness to yield brownish powder A3 . Fraction A1 , A2 and A3 obtained consist mainly of bacoside A. The acetone soluble fraction is left in the cold for several days when a solid settles down. It is filtered, washed with acetone, yield major amount of bacoside B.
Thin Layer Chromatography of Bacoside
The extract or the isolated glycoside dissolved in methanol and spotted over silica gel-G plates. The plates are eluted in ethyl acetate–pyridine–water (4:1:1), and the TLC plates after drying are sprayed with trichloroacetic acid (25%) in chloroform. Bacoside A of A1 , A2 and A3 fraction gives Rf value 0.43 while bacoside B gives Rf value 0.09.
ISOLATION OF CAFFEINE
Caffeine or 1,3,7- trimethylxanthin is a purine base present along with other related bases like theophyline and theobromine in coffee, tea, cocoa, guarana, kola and mate. Although caffeine is largely produced synthetically, it is usually isolated from tea leaves or recovered from coffee seeds during decaffeination process. Tea leaves contain 1–4% of caffeine while coffee seeds contains 1–2% of caffeine. Caffeine was first discovered by Robiquet in coffee in 1821, and mid later in 1827, Oudry found it in tea leaves.
Isolation
Variety of methods is in use for the isolation of caffeine from different sources. Some important processes are described below
- The coarse powder of tea leaves is extracted with boiling water and the aqueous extract is filtered while hot. The warm extract is treated with lead acetate to precipitate tannins and filtered. The excess of lead acetate present in the solution is precipitated as leadsulphate with dilute sulphuric acid. The filtered solution is boiled with charcoal to remove colouring matter if any, and filtered to remove charcoal. The filtered decolourized solution is extracted with chloroform. The combined chloroform extract after evaporation affords caffeine as a white material. It is re-crystallized with alcohol.
- Finely or coarsely powdered tea leaves are extracted with ethanol in soxhlet extractor. The caffeine so extracted in ethanol is then adsorbed on magnesium oxide. Caffeine is then disorbed after treatment with 10% H2 SO4 . It is then extracted with chloroform and re-crystallized.
- Caffeine is extracted from coffee beans by the process of leaching with water. The highest yield up to about 90% was obtained when the coarse coffee powder is extracted with water at 75°C. The extraction takes about half an hour with water/coffee ratio of 9:1.
- Decaffeination of coffee using super-critical fluid extraction. Super-critical fluid extraction has been efficiently used for the decaffeination of coffee. The process was first developed by K. Zosel using liquefied carbon dioxide. The super-critical medium in a pressure vessel is circulated through moist coffee where it becomes charged with caffeine. It is then passed through second pressurized vessel containing an adsorbing medium such as activated carbon, resin or water which retains caffeine. Adsorbed caffeine is then separated by extraction with chloroform.
Melting point: 235–237°C
Thin Layer Chromatography of Caffeine
Dissolve 1 mg of caffeine in 1 ml of chloroform or methanol. Spot the sample on TLC plate and elute it in ethyl acetate–methanol–acetic acid (80:10:10). Visualize the dried TLC plate by exposure to iodine vapour. Caffeine develops a spot at Rf value 0.41.
ISOLATION OF CAMPHOR
Camphor is a bicyclic monoterpene ketone obtained from Cinnamomum camphora; family Lauraceae. It is natural camphor. Camphor occurs in all parts of the camphor tree. Camphor is also produced synthetically from pinene. It is in the form of optically inactive mixture.
Isolation
Camphor oil is obtained by steam distillation of the wood of camphor tree C. camphor. The main constituent of the crude oil is camphor up to about 50%, which can be separated by cooling and centrifugation. Fractionation of mother liquor gives two types of oils. The first known as white camphor oil is the first distillation fraction with cineol like odour, containing 35% of cineol. The brown camphor oil is a fraction with a higher boiling range than that of camphor. It is a pale yellow liquid containing about 80% of safrol. The production of natural camphor and camphor oils was formerly several thousands of tons per year but has declined as a result of the production of synthetic camphor. Melting point: 179–180°C
Thin Layer Chromatography of Camphor
Dissolve 1 mg of camphor in 1 ml of methanol. Apply the spots over silica gel-G plate and elute it in benzene–ethyl acetate–glacial acetic acid (90:5:5). Spray the dried TLC plate with 1% vanillin-sulphuric acid reagent and heat at 110°C for 10 min. Camphor gives the spot with Rf value 0.33.
ISOLATION OF CAPSAICIN
Capsaicin is the pungent principle known as capsicum oleoresin obtained from the dried, ripe fruits of Capsicum annum var. minimum and small-fruited varieties of C. fruitescens; family Solanaceae. Capsaicin is mostly present in the dissepiment of fruit. Chemically it is 8-methylN-vanillyl non-6-enamide. It is present to the extent of 0.02–0.14%.
Isolation
Dried, ripe fruits of capsicum are coarsely powdered for the extraction of oleo-resin. It is extracted with hot acetone or alcohol (90%). The extract obtained is concentrated and dried. The dried residue is further extracted with cold alcohol (90%) and the alcohol is removed by evaporation. Capsicum oleoresin thus obtained contains not less than 8% of capsaicin. Melting point: 57–66°C
Thin Layer Chromatography of Capsaicin
The oleo-resin 1 mg/ml is dissolved in alcohol and spotted on silica gel-G plate. The plate is eluted in the solventsystem containing a mixture of benzene-methanol (9:1). Spray the dried plate with a 0.5% solution of 2,6-dibromoquinone-chlorimide in methanol and allow to stand in a chamber containing ammonia fumes. Blue colour and the Rf value 0.31 of the principal spot corresponds to the spots of the standard solution.
ISOLATION OF COLCHICINE
Colchicine is an alkaloid obtained from the corms or seeds of Colchicum autumnale; family Liliaceae and also from other species of Colchicum. It is a tropolone group of alkaloid. It is an active ingredient of one of the 18 plants still in use, of the approximately 700 listed in the Papyrus Ebers of ancient Egypt.
Isolation
Colchicum corms or seeds are exhaustively extracted with ethanol. Alcoholic extract is concentrated and dried to syrupy residue. The residue is dissolved in water to precipitate the insoluble fats and resins. The filtered aqueous extract is then repeatedly extracted with chloroform or digested with lead carbonate. It is refiltered, evaporated to a small volume and further extracted with chloroform. Colchicine is recovered as a crystalline complex with chloroform. The chloroform is distilled off and the amorphous colchicine is recovered after the evaporations of the residual solvent. Amorphous colchicine may be crystallized from ethyl acetate as pale yellow needles. Melting point: 142–150°C
Thin Layer Chromatography of Colchicines
The dilute solution of colchicine in methanol is spotted on silica gel-G plates and eluted with chloroform-diethyl amine (9:1). Colchicine is detected by spraying with Dragendorff’s reagent. It gives Rf value of 0.41.
ISOLATION OF CURCUMIN
Curcumins or curcuminoids are the diaryl heptanoid compounds obtained from the dried rhizomes of Turmeric, Curcuma longa family, Zingiberaceae. It is a major colouring principle present up to 5% in the rhizomes, which constitutes about 50–60% of the mixture of three maincurcuminoids namely curcumin, desmethoxycucumin and bisdesmethoxycurcumin. The standardized extract of turmeric contains major proportion of the above curcuminoids. Commercial curcumin isolated from turmeric rhizome contains up to 97% pure product.
Isolation
Curcumin can be obtained by different processes. Turmeric powder is extracted with alcohol in soxhlet extractor. The alcoholic extract is concentrated under reduced pressure and dried. In another procedure, turmeric powder is first extracted with hexane followed by acetone. The acetone extract is concentrated and dried to yield curcumin. The most efficient way of isolating curcumin was found to be to extract with hot ethanol, concentrate the filtrate, throw the concentrate into superior grade kerosene, when a solid mass separates. The mass is stripped off kerosene with petroleum ether and re-crystallized from ethanol. The final product obtained is re-crystallized from hot ethanol to yield orange-red needles.
Melting point: Curcumin 183°C, desmethoxycucumin 168°C, and bisdesmethoxycurcumin 224°C
Thin Layer Chromatography of Curcumin
Dissolve 1 mg of curcumin in 1 ml methanol. Apply the spots on silica gel-G plate and elute the plate in the solvent system chloroform–ethanol–glacial acetic acid (94:5:1). Dry the eluted plate and visualize under 366 nm light. Curcumin exhibits a bright yellow fluorescent spot at Rf value 0.79. The other spots appearing at Rf values 0.60 and 0.43 correspond to desmethoxycurcumin and bisdesmethoxycurcumin.
ISOLATION OF DIGOXIN
Digoxin or Lanoxin is the most widely used cardiac glycoside obtained from the leaves of Digitalis lanata; family Scrophulariaceae. It is a secondary glycoside which is produced from a primary glycoside Lanatoside C. Its hydrolysis yields three molecules of digitoxose sugar and digoxigenin. It is a highly potent drug and should be handled with exceptional care.
Isolation
Digoxin is obtained commercially from the fresh leaves of Digilatis lanata. Lanatosides are the naturally occurring primary glycosides of D. lanata which includes Lanatoside A, lanatoside B and lanatoside C. Unstable lanatoside A is the acetyl derivative of purpurea glycoside A, and lanatoside B is the acetyl derivative of purpurea glycoside B. Lanatoside C has no counter part in D. purpurea. Hydrolysis of lanatosides by enzyme splits off glucose and hydrolysis by mild alkalies splits off acetyl groups leaving digitoxin, gitoxin and digoxin as the residues from Lanatoside A, B and C, respectively.
To extract lanatosides, fresh leaves are ground with neutral salt to inactivate the enzymes, and the pulp is further extracted with ethyl acetate. If the leaves are first defatted with benzene prior to extraction, better yield of the glycoside is obtained. The ethyl acetate extract is concentrated, dried and further subjected to chromatographic purification to yield lanatoside A (46%), lanatoside B (17%) and lanatoside C (37%). The fractions obtained are crystallized from alcohol. Lanatoside C after subsequent hydrolysis affords digoxin. Digoxin on acid hydrolysis yields digoxigenin as an aglycone and three moles of digitoxose. Melting point: 230–265°C
Thin Layer Chromatography of Digoxin
Dissolve about 1 mg of the glycoside in 1 ml of alcohol. The sample is spotted over silica gel-G plates and eluted with cyclohexane-acetone-acetic acid (49:49:2). Dried TLC plates are sprayed with 50% aqueous sulphuric acid. Digoxin appears as a blue spot under 385 nm UV light.
Identification Test
Digoxin is dissolved and diluted in hot methanol. The aliquot of the solution is evaporated to dryness. Acid ferric chloride TS is added to the residue. A green colour develops that slowly changes to a deep green blue colour.
ISOLATION OF DIOSGENIN
It is obtained from the dried tubers of Dioscorea deltoidea Wallich and other species of Dioscorea (Dioscoreaceae).
Isolation of Diosgenin
Alcoholic extraction method
The diosgenin tubers are cut into small pieces and dried under sun. The dried tubers are powdered, extracted with ethanol or methanol twice for 6–8 hours. It is filtered and the filtrate is concentrated to a syrupy liquid. The concentrated liquid is then hydrolysed using an acid, hydrochloric acid or sulphuric acid for 2–12 h. About 85% of the crude diosgenin is precipitated. The precipitates are filtered, washed with water and purified with alcohol.
Acid hydrolysis method
The dried tubers are powdered to a mesh size of 100–200 meshes. It is then refluxed or heated in autoclave with 2–4 N mineral acid for 2–6 h. It is filtered and the crude hydrolyte is washed with water, until neutral. Dried and extracted again for 6 h with hydrocarbon solvent. The liquid is concentrated to about 25 ml. It is allowed to stand for some time in a refrigerator for 1 h. The crystals of diosgenin are filtered out and then it’s washed with acetone.
Fermentation cum acid hydrolysis method
The fresh green roots are collected and smashed in a hammer mill. The mesh is placed in the fermentation bin and allowed for fermentation for two days. The fermented mesh is dried in sun to reduce the moisture content to 7–8%. It is then subjected to hydrolysis with a mineral acid at reduced temperature. The resulting solution is extracted with heptane to obtain diosgenin.
Incubation cum acid hydrolysis method
The fresh plant material is incubated in water at 37°C for few days. It is later subjected to acid hydrolysis. The hydrolysed liquid is concentrated and extracted with hydrocarbon solvent to obtain diosgenin. The fresh roots are homogenized with equal weight of water, concentrated acid is added until the required strengthis obtained and then it is extracted with hydrocarbon solvent to obtain diosgenin.
Melting point: 204–207°C
Thin Layer Chromatography of Diosgenin
The sample dissolved in methanol is spotted in Silica gel plates and developed in Toluene: ethyl acetate (7:3). Dark green spot (Rf - 0.37) of diosgenin will appear when the dried plate is sprayed with anisaldehyde-sulphuric acid reagent.
ISOLATION OF EMETINE
Emetine is the major active constituent of the rhizomes and roots of Cephaells ipecacuanha and C. acuminata; family Rubiaceae. Emetine was first isolated in a crude form by Pelletier in 1817, and recognized as an alkaloid in 1823. Ipecacuanha also consists of some other related isoqunoline alkaloids which includes cephaeline, psychotrine and emetamine.
Isolation
The powdered ipecacuanha is extracted with about 70% ethanol or methanol. The extract obtained is concentrated and dissolved in water and the solution is made strongly basic with ammonia and extracted with di-isopropyl ether. Di-isopropyl ether extract is then treated with 10–15% aqueous potassium hydroxide to remove cephaeline. The extract is further evaporated to yield emetine. It is purified via dihydrobromide or dihydroiodide salt. These halide salts are converted to the hydrochloride by neutralizing the regenerated free base.In another process, ipecac powder is treated with ammonia and ether. The ether extracts is subjected to dilute sulphuric acid treatment to yield alkaloids. Dilute acid extract is then nearly neutralized and washed with ether and then made strongly alkaline and treated with ether. Emetine goes into ether while cephaeline remains in the aqueous phase. The ether extract is concentrated and redissolved in methanol and converted to emetine hydrobromide with a methanolic solution of hydrobromic acid.
Melting point: 70°C
Thin Layer Chromatography of Emetine
Emetine hydrochloride is dissolved in methanol or water and spotted on silica gel-G plates. TLC is eluted in the following solvent systems and the spot is visualized under UV light or by spraying with bromocresol green or modified Dragendorff ’s reagent.
ISOLATION OF ERGOMETRINE
Ergometrine or ergonovine is a naturally occurring indol alkaloid found in the sclerotia of Claviceps purpurea; family Clavicipitaceae developed on plants of rye, Secale cereale; family Gramineae. It is classified as one of the water-soluble, amine ergot alkaloids. It was discovered almost simultaneously in 1935 by five independent research groups. Ergot also contains ergotamine and ergotoxine groups of alkaloids which are water-insoluble groups.
Isolation
In the laboratory scale isolation technique, ergot powder is completely defatted with petroleum ether (60–80°) in soxhlet extractor. The petroleum ether extract removes about 30% fat and colouring matter. The residual marc dried below 40°C is transferred to a porcelain dish, made to semi-solid mass by adding sufficient solvent ether and dilute ammonia with stirring. The material is stirred to dryness and then packed in a soxhlet and extracted with solvent ether for about 5 h. The ether extract filtered and to it little acetone added and shaken in separating funnel with three volumes of 1% of tartaric acid. The total acidic extract is combined and dried under reduced pressure to yield total ergot alkaloid.
The total alkaloid is further dissolved in dilute ammonia and extracted with four volumes of ether. The combined total ether extract is washed thoroughly with five successive quantities of water. Water-insoluble ergotamine and ergotoxine alkaloids stay with ether while the water-soluble ergometrine tartarte remains with aqueous extract. The aqueous extract is made faintly alkaline with dilute ammonia and further saturated with ether. Ergometrine free base is shifted to ethereal solution. It is again washed with water to remove impurities of other alkaloids. The ether extract is again treated with three volumes of 1% w/w tartaric acid in water. The combined acid extract is concentrated under reduced pressure to yield water soluble alkaloid. It is further purified by the column chromatographic fractionation. Melting point: 162°C
Thin Layer Chromatography of Ergometrine
Dissolve about 1 mg/ml of alkaloid in methanol and apply on silica gel-G plates. Elute the plates in solvent system toluene-butanol NH4 Cl (saturated) (6:4) and spray the dried TLC plates with Dragendorff ’s reagent. Ergometrine maleate shows the Rf 0.30. The elution of the silica gel-G TLC plate in other solvent system chloroform–ethanol– acetone (6:4:4), shows the Rf value 0.23.
ISOLATION OF EUGENOL
Eugenol is a 4-allyl-2-methoxy phenol obtained from the essential oil of clove buds Eugenia caryophyllus; family Myrtaceae. It is also obtained from Cinnamon leaf oil obtained from Cinnamomum zeylanicum; family Lauraceae. Both of the oils contain 80–90% eugenol.
Isolation
Dried clove buds are hydrodistilled to yield the clove oil. Being heavier than water it makes a layer beneath water. The lower layer of clove oil is separated from water. For the separation of eugenol from clove oil, the oil is dissolved in solvent ether to make about 10% solution. It is shaken with three successive volumes of 10% potassium hydroxide solution. Eugenol being phenolic compound gets converted to phenoxide and becomes soluble in water. The total aqueous alkaline extract is combined and washed with fresh ether to remove other impurities. Eugenol isregenerated by acidifying the aqueous alkaline extract with excess of sulphuric acid. The acidified solution is extracted in separating funnel with three successive volumes of solvent ether. The combined solvent ether extract is then washed with water. Ether is removed by distillation at very low temperature to yield pure liquid eugenol. Boiling point: 255°C
Thin Layer Chromatography of Eugenol
Dissolve about 1 mg of eugenol in 1 ml of methanol and apply the spots over silica gel-G plate. Elute the plate with pure benzene as a solvent system. Spray the dried plate with 1% anisaldehyde-sulphuric acid reagent and heat the plate at 110°C for 10 min. Eugenol shows the spot with dirty green colour at Rf 0.40 in case of normal chamber saturation at 24°C.
ISOLATION OF GINGEROLS AND SHOGAOLS
Gingerols are the oleoresin constituents of Ginger, Zingiber officinalis; family Zingiberaceae. These are long chain phenolic compounds responsible for the pungent taste of the drug. Ginger resin also contains their corresponding dehydration products, which are known as shogaols. Total resinous matter containing gingerols and shogaols is about 5-8% in ginger.
Isolation
Dry ginger is crushed to a coarse powder and extracted with 95% ethanol from alcoholic extract. Solvent is evaporated by distillation to obtain thick pasty mass. The thick pasty mass is suspended in water. The ginger resin precipitates in water which is removed by filtration and the residue obtained is dried under vacuum. In some cases the suspended oleoresin is extracted with solvent ether and theether extract is evaporated to dryness at low temperature to yield total ginger oleoresin.
Identification Test
Add about 5 ml of 70% sulphuric acid and 5 mg of vanillin to the small quantity of ginger oleoresin. Allow to stand for 15 min, and then add equal volumes of water, the solution obtained turns azure blue indicating the presence of gingerol.
Thin Layer Chromatography of Gingerols
Dissolve extract or gingerol in alcohol. Apply the spots over silica gel-G plate and elute in the solvent system ether-n-hexane (7:3). Spray the dried TLC plate with 1% vanillin-sulphuric acid and heat the plate at 110°C for 10 min. Spots due to gingerols occur at Rf value 0.2.
ISOLATION OF GLYCYRRHETINIC ACID
Glycyrrhetinic acid is a pentacyclic triterpenic acid obtained from the roots and stolones of Glycyrrhiza glabra; family Leguminoseae commonly known as liquorice. A major component of liquorice root is a sweet triterpenic saponin glycoside glycyrrhizin, which is a potassium and calcium salt of glycyrrhizic acid about 6–14%. After hydrolysis, it affords two molecules of gluconic acid and an aglycone glycyrrhetinic acid.
Isolation
The crude drug is first extracted with chloroform. Chloroform extract is discarded. The marc is again extracted this time with 0.5 M sulphuric acid. The acid extract is cooled and shaken with chloroform. The combined chloroform extract is concentrated and dried to yield glycyrrhetinic acid. Glycyrrhizin is hydrolysed to glycyrrhetinic acid during extraction with sulphuric acid.
In another method of extraction, liquorice powder is extracted with boiling water to isolate glycyrrhizin. The aqueous extract is concentrated, dried and used as liquorice extract. The liquorice extract can be dissolved in water andacidified with hydrochloric acid to pH 3-3.4 to precipitate glycyrrhetinic acid. The precipitate is filtered, washed with water till neutral pH and then dried to yield glycyrrhetinic acid. Ammoniated glycyrrhizin, used in pharmaceutical trades is prepared by precipitating glycyrrhizic acid from liquorice extract, dissolving it in ammonia and drying the solution after spreading in a thin film on a glass plate to give shining dark brown flakes. Melting point: 300°C
Thin Layer Chromatography of Glycyrrhetinic Acid
Dissolve 1 mg of glycyrrhetinic acid in about 1 ml of methanol-chloroform (1:1) mixture. Apply the spots over silica gel-G plates and elute in the solvent system Toluene– ethyl acetate–glacial acetic acid (12.5:7.5:0.5). Spray the dried plates with 1% vanillin-sulphuric acid or anisaldehyde-sulphuric acid and heat for 10 min at 110°C. glycyrrhetinic acid gives purplish spot corresponding to the Rf value 0.41.
ISOLATION OF GUGGULSTERONE
Guggulsterone or gugulipid is a steroidal constituent present in the neutral fraction of the gum resin of Commiphora mukul; family Burseraceae known as Guggul or Indian bdellium. As guggulsterone is a major compound of the resin which comes in the lipid soluble fraction of the drug is also called as guggulipid. It is present in the form of stereoisomers, i.e., E-guggulsterone and Z- guggulsterone. The other constituent present in the neutral fraction are guggulsterol and other steroids.
Isolation
Gum resin of C. mukul coarsely powdered and extracted with ethyl acelate. The solvent is evaporated under vacuum at 50°C to yield dark brownish gummy material. It is further dissolved in ethyl acetate and extracted with 3 N HCl. The acid extract is basified to yield a basic fraction. The neutral ethyl acetate fraction obtained after treatment with acid is further divided into non-carbonyl and neutralketonic fraction by following process. Neutral fraction is mixed with 10% semi-carbazide on silica gel and toluene, stirred and heated at 60–62°C for 14 h, cooled and filtered. Silica gel is thoroughly washed with toluene to get toluene soluble non-carbonyl fraction. The above washed silica gel is mixed with aqueous 10% oxalic acid and toluene, stirred and refluxed for 2.5 h cooled and filtered. Silica gel residue is washed with ethyl acetate thrice. The combined ethyl acetate extract is further washed with water, and then concentrated and dried to yield neutral ketonic fraction. Neutral ketonic fraction is chromatographed on silica gel and eluted with benzene-ethyl acetate to yield fractions containing mixture of E-and Z-guggulsterone. E-and Z-guggulsterone are further purified by rechromatography on silica gel.
Thin Layer Chromatography of Guggulsterone
Dissolve about 1 mg of extract or guggulsterone in 1 ml of ethyl acetate. The silica gel-G plate spotted with the sample is eluted in the solvent system toluene-ethyl acetate (80:20). The dried plate is sprayed with 1% vanillin-sulphuric acid and heated at 110°C for 10–15 min. Guggulsterone gives bluish violet spots which correspond with the Rf value 0.45 of the standard.
ISOLATION OF HESPERIDIN
Hesperidin was isolated from various citrus species like Citrus mitis, Citrus aurantium, Citrus sinensis, belonging to the family Rutaceae. Hesperidin decreases the fragility of blood capillaries. Hesperidin could be isolated by two methods;
Method I
Take 200 g sun-dried peel, powder and place it in a 2-litre round bottomed flask attached to a reflux condenser. One litre of petroleum ether (40–60°C) is added to the flask and heated on a water bath for 1 h. The contents of the flask are filtered while hot through a buchner funnel, and the powder is allowed to dry at room temperature. The dried powder is taken back to the flask, and 1 litre of methanol is added to the flask. The contents are heated under reflux for 3 h and then filtered hot and the marc is washed with 200 ml hot methanol. The filtrate is concentrated under reduced pressure, leaving a syrupy residue crystallized from dilute acetic acid, yielding white needles of hesperidin.
Method II
Take 200 g of chopped orange peel in a 2-litre Erlenmeyer flask and 750 ml 10% calcium hydroxide solution is added and thoroughly mixed. The flask and its contents are left overnight at room temperature. The mixture is filtered through a large buchner funnel containing a thin layer ofcelite on the filter paper. The obtained filtrate would be yellow orange colour and is acidified carefully to pH 4-5 with concentrated hydrochloric acid. Hesperidin separates as amorphous powder. It is collected on a buchner funnel, washed with water, and re-crystallized from aqueous formamide. Melting point: 252–254°C
Identification Test
- Ferric chloride test: Addition of ferric chloride solution to hesperidin produces a wine red color.
- Magnesium-hydrochloric acid reduction test: Dropwise addition of concentrated hydrochloric acid to an ethanolic solution of hesperidin containing magnesium develops a bright violet color.
ISOLATION OF LEVODOPA
Levodopa or L-Dopa is a dihydroxyphenyl alanine obtained from the dried mature seeds of Mucuna pruriens; family Fabaceae. Two types of seeds, i.e., black or spotted variety are generally found in the market which consists of about 2.0% of L-Dopa content.
Isolation
Coarsely powdered Mucuna seeds are extracted with demineralized water containing 1% v/v of acetic acid at 50°C. Acetic acid extract is filtered. The filtered extract is concentrated by reverse osmosis and the liquid concentrate so obtained is kept at a low temperature (6–8°C) for about 24 h for crystallization of L-dopa. The crystals are filtered through a centrifuge, washed with cold water and dried under vacuum at 50°C. The filtrate and water washings still contain the compound of interest, i.e., L-dopa. It is mixed, concentrated and processed for second crop. The crystalline product obtained in I and II crops combiningly yield about 2.0–2.5% of L-dopa which still consists of some impurities of amino acids. It is further purified either by re-crystallization or by using liquid ion exchangers. Melting point: 283°C
Thin Layer Chromatography of Levodopa
Thin layer chromatographic study using solvent system n-butanol–acetic acid–water-methanol (15:7.5:7.5:1.5) and spraying the plates with Dragendorffs reagent shows the spot at Rf value 0.4.
ISOLATION OF MENTHOL
Menthol is a monoterpene alcohol obtained from diverse types of mint oils or peppermint. The sources of mint oil include black peppermint. Mentha piperita Var. vulgaris; white peppermint, M. piperita Var. officinalis; M. arvensis; M. canadensis Var. piperascens etc. Peppermint contains about 1–3% of volatile oil. First two species contains not less than 45% of menthol while the later species contains menthol up to about 70–90%. Along with menthol the oil contains (+) neomenthol, (+) isomenthol, menthone, menthofuran, menthyl acetate and cineol. The menthol obtained from the natural sources is. Levorotatory (l-menthol) or racemic (dl-menthol). Menthol can be synthetically prepared by hydrogenation of thymol.
Isolation
Mentha oil is obtained from the hydrodistillation or steam distillation of fresh above-ground parts just before flowering. For (-) menthol isolation from peppermint oil the oil is subjected to cooling. The crystals of menthol crystallize out from the oil which is separated by centrifugation. Cornmint oil obtained from the steam distillation of the flowering herb Mentha arvensis contains about 70–80% of free (-) menthol. Cornmint oil is cooled and the crystals of menthol produced are separated by centrifugation. Since the crystalline product contains traces of cornmint oil, this menthol has a slightly herbaceous minty note. Pure (-) menthol is obtained by re-crystallization from solvents with low boiling points. Dementholized corn mint oil from which (-) menthol is removed by crystallization and which still contains 40–50% free menthol can also be reused for producing (-) menthol. Melting point: 41–44°C
Thin Layer Chromatography of Menthol
Dissolve about 1 mg of menthol in about 1 ml of methanol. Apply the spot on silica gel-G plate and elute it in pure chloroform. Spray the dried plates with 1% vanillinsulphuric acid reagent and heat the plate at 110°C for 10 min. Menthol gives Rf value 0.48–0.62 in case of normal chamber saturation at 24°C.
ISOLATION OF NICOTINE
Nicotine is a pyridine alkaloid obtained from the dried leaves of tobacco plant Nicotiana tabacum; family Solanaceae. Tobacco leaves contains 2–8% of nicotine combined as maleate or citrate.
Isolation
Nicotine is generally isolated from the tobacco waste as the main tobacco crop has always to enter the normal consumption channel because it is a very high revenue earning commodity; it is extremely costly to use it as a raw material for nicotine extraction. However, tobacco waste material with less than 2% nicotine content is uneconomical for nicotine recovery.
For the isolation of nicotine, tobacco waste is thoroughly mixed with lime and extracted with water. Nicotine present in the aqueous solution is further extracted with organic solvents like chloroform or kerosene. The organic solvent extract of nicotine is then treated with dilute sulphuric acid to obtain nicotine sulphate solution. The product is separated as a heavy layer and denicotinated solvent is recovered and recycled in the procedure of extraction.
As kerosene is not an ideal solvent due to its very high distribution coefficient and undesirable odour that it leaves in the final product, ion exchange chromatography is used for recovery of nicotine from aqueous solution. Nicotine is absorbed on a cation exchanger and subsequently eluted with a suitable medium. The exchanger can also be regenerated by washing with dilute acid and reuse several times. This process yields good quality insecticide grade nicotine from tobacco waste. Boiling point: 247°C
Thin Layer Chromatography of Nicotine
Dissolve 1 mg of sample in 1 ml of methanol. Apply the spot over silica gel-G plate, and elute the plate in chloroform–methanol–ammonium hydroxide (60:10:1). Spray the dried TLC plate with a mixture of equal volumes of 2% p-aminobenzoic acid in ethanol and 0.1 M phosphate buffer. After drying the plate for 15 min, expose it to bromine cyanide vapour for visualization. Nicotine gives the spot corresponding to the Rf value 0.77. Reaction involved in the visualization of nicotine is known as Konig reaction.
ISOLATION OF OPIUM ALKALOIDS
Morphine and codeine are the two important isoquinoline alkaloids present in the air-dried milky exudates obtained by incision of unripe capsules of Papaver somniferum Linn (Papaveraceae).
Isolation
The powdered drug is extracted with boiling water and to the aqueous phase calcium chloride is added and concentrated to get salts of morphine and codeine as crystals. It is treated with chloroform. The soluble portion consists of codeine and the insoluble portion consists of morphine. The powered drug is shaken with calcium chloride and filtered. To the filterate add 10% of sodium hydroxide solution. It is filtered. The marc consists of narcotine, papaverine, thebain and the filtrate consists of morphine, codeine. The filtrate is extracted with chloroform. The chloroform layer is separated. It consists of codeine while the aqueous layer consists of morphine and narceine. The aqueous layer is first acidified and later on slightly alkalized with ammonia. Morphine is precipitated on the addition of ammonia and the aqueous layer consists of narceine. The marc consisting of narcotine, papaverine and thebain is dissolved in alcohol and then acidified with acetic acid. To the acidified solution, add three volumes of boiling water. Precipitates of narcotine and papaverine are formed and thebain still remains in the aqueous solution. Papaverine is separated from narcotine by the addition of 0.3% oxalic acid solution and allowed to cool. On cooling the papaverine crystals are obtained. The aqueous solution is made alkaline with ammonia for the precipitation of narcapine which is re-crystalized using water.
Melting point: Morphine 254°C, Thebaine 193°C, Codeine 154–156°C
Identification Tests
- Opium alkaloids when treated with ferric chloride solution, deep reddish purple colour is produced which persist even after the addition of few drops of hydrochloric acid.
- Morphine when treated with concentrated sulphuric acid and formaldehyde gives dark violet colour.
Thin Layer Chromatography of Opium Alkaloids
Opium alkaloids are spotted in Silica gel-G plates and developed with two different solvent systems separately, solvent system I, chloroform: acetone: diethylamine (5:4:1) and solvent system II, xylene: methyl ethyl ketone: methanol: diethylamine (20:20:3:1). The dried plates are sprayed with Dragendroff ’s reagent. The spots of the alkaloids will be reddish brown in colour and the Rf values of the alkaloids in both the solvent systems are:
- Solvent system I: Morphine (Rf -0.10), Codeine (Rf -0.38), Thebaine (Rf -0.65), Papeverine (Rf -0.67) and Narcotine (Rf -0.72).
- Solvent system II: Morphine (Rf -0.12), Codeine (Rf -0.26), Thebaine (Rf -0.45), Papeverine (Rf -0.59) and Narcotine (Rf -0.74).
ISOLATION OF PIPERINE
Piperine is isolated from unripe fruit (black pepper) and the kernel of the ripe fruit (white pepper) of Piper nigrum, from the fruit of aschanti (Piper clusii), from long pepper (Piper longum), seeds of Cubeba censii, Piper fainechotti and Piper chaba. The piperine content of black pepper varies from 6 to 9%.
Isolation of Piperine
Finely powdered 20 g of black pepper is extracted with 300 ml 95% ethanol in a Soxhlet extractor for 2 h. The solution is filtered and concentrated in vacuum on a water bath at 60°C. 20 ml of alcoholic potassium hydroxide is added to the filtrate residue and after it while decanted from the insoluble residue. The alcoholic solution is left overnight, whereupon yellow coloured needle shaped crystals are deposited. The yield of piperine is 0.3 g. Melting point: 125–126°C
Thin Layer Chromatography of Piperine
The extracted piperine is spotted on TLC plate made up of Silica gel-G and developed with benzene: ethyl acetate (2:1). When detected at UV365 piperine exhibits blue fluorescence. When sprayed with anisaldehyde sulphuric acid reagent and heated at 110°C for 10 min, Piperine appears as yellow spot at Rf 0.25.
Dissolve about 1 mg of piperine in 1 ml methanol. Apply the test solution on the silica gel-G plate and elute the plate with toluene–diethyl ether–dioxane (9.4:3.2:2.4). Visualize the dried plate under UV light of 254 nm. Piperine appears as a violet coloured spot at Rf value 0.48. If the TLC plate is sprayed with anisaldehyde–glacial acetic acid–methanol– conc. sulphuric acid reagent (0.5:10:85:5} and heated at 110°C for 10 min, piperine appears as a yellow spot.
ISOLATION OF PODOPHYLLOTOXIN
Indian podophyllum is the root and rhizome of Podophyllum hexandrum Royle (Berberidaceae).
Isolation
Commercial podophyllin is obtained by extraction of powdered rhizome/roots of P. emodii with methanol. Then it is reduced under vacuum. Semi-solid mass is put into acidulated water (10 ml HCl in 100 ml water). The precipitates are allowed to settle. Filtrate is decanted and then washed with cold water. Resin obtained is dried, and upon drying, it gives dark brown amorphous powder called podophyllin. The obtained powder is extracted with chloroform and further purification is done by repeated re-crystallization from benzene alone or alcohol benzene mixture followed by washing with petroleum ether/hexane yield podophyllotoxin.
Another method of extraction to obtain pure podophyllotoxin is by dissolving the CHCl3 soluble fraction in alcohol. Then it is refluxed with neutral aluminium oxide so that solution becomes light yellow. To alcoholic solution benzene is added which yielded podophyllotoxin of 95–98%.
Another method of isolating podophyllotoxin from crude (P. emodii roots/rhizome) podophyllin/crude podophyllotoxin involves extraction over a bed of neutral alumina with solvents like benzene, toluene, xylene, etc., for about 1.5–4 h. Re-crystallization from organic solvents such ashot benzene, toluene and xylene yields pure podophyllotoxin (95–97%). Podophyllotoxin is a tetrahydronapthalin derivative with OH and lactone groups. The attachment at cis-position is responsible for the purgative property and the attachment at trans-position corresponds for anti-cancer property of the drug. Melting point: 114–118°C
Thin Layer Chromatography of Podophyllotoxin
Podophyllotoxin is dissolved in methanol and is spotted on the TLC plate; the solvent used is Toluene: ethyl acetate (5:7) and detecting agent is sulphuric acid. Spot of Podophyllotoxin under day light has violet colour (Rf -0.39).
ISOLATION OF QUININE AND QUINIDINE
Cinchona is the dried bark of the stem or of the root of Cinchona calisaya Wedd, Cinchona ledgeriana Moens, Cinchona officinalis Linn and Cinchona sucirubra Pavon or hybrids of any of the first two species with any of the last two species (Rubiaceae). Quinine is laevorotatory while quinidine is dextrorotatory stereoisomer.
Isolation
The powdered chinchona bark is mixed with about 30%, of its weight of calcium hydroxide or calcium oxide and sufficient quantity of 5% sodium hydroxide solution. Make it into a paste and allow it to stand for few hours. The moistened mass is then transferred into soxhlet and extracted with benzene. To the benzene extract, add 5% sulphuric acid and mix well. The benzene layer is separated from that of the aqueous layer, the benzene layer is discarded and to the aqueous layer sodium hydroxide is added to adjust the pH to 6.5. Cool and on cooling precipitates of quinine sulphate is formed. The precipitate is filtered and separated. The separated precipitate is then re-crystalized from hot water to free the salts from cinchonine and cinchonidine. The colouring matter is removed by treating it with activated charcoal. The quinine sulphate obtained is dissolved in dilute sulphuric acid, and it is later made alkaline withammonia. Quinine precipitates become crystalline, which are washed and dried at 45–55°C. The mother liquor consisting of quinidine, cinchonine and cinchonidine are slightly made alkaline with ammonia, and the precipitate formed is again subjected to extraction with ether. Two portions are obtained: the first is ether insoluble fraction consisting of cinchonine crystals and the other is the ether extract with quinidine and cinchonidine. The ether soluble fraction consisting of quinine and cinchonidine is first stirred with dilute hydrochloric acid followed by the addition of 25% of solution of sodium potassium tartarate. The resulting solution is allowed to stand for some time, and on standing, precipitates of cinchonidine tartarate is formed. The cinchonidine is re-crystalized from alcohol. To the liquor obtained after the separation of cinchonidine tartarate add potassium iodide solution. Addition of potassium iodide results in the precipitation of quinidine hydroiodide. This on treatment with an alkali (ammonia) liberates a base, which is dissolved in acetic acid. The colouring matter is removed by the treatment with activated charcoal. The quinidine obtained is finally re-crystallized from alcohol.
Melting point: Quinine 177°C, Quinidine 174–l75°C
Identification Tests
Thalleioquin test: To the sample add one drop of dilute sulphuric acid and 1 ml of water. Add bromine water dropwise till the solution acquires permanent yellow colour and add 1 ml of dilute ammonia solution, emerald green colour is produced.
Thin Layer Chromatography of Quinine and Quinidine
Alkaloids are dissolved in methanol and spotted in Silica gel-G plate. The solvent system used are Chloroform: diethyl amine (9:1) and Chloroform: acetone: diethyl amine (5:4:1). Dried plates are sprayed with dragendroff ’s reagent, the Rf value of quinine and quinidine in the first solvent system are 0.17 and 0.28, respectively, and in solvent system second, 0.17 and 0.26.
ISOLATION OF RESERPINE
Reserpine is an indole alkaloid obtained from the roots of Rauwolfia serpentina, family Apocyanaeae and also from other different species of Rauwolfia, such as R. micrantha, R. vomiforia and R. tetraphylla. The material obtained from natural sources may contain closely related alkaloids, which includes ajmaline, ajmalicine, ajmalinine, rescinnamine, reserpinine, serpentine and yohimbine. In R. serpentina, reserpine and rescinnamine both respond to the extraction procedures and extracted as a mixture of both while in R. tetraphylla, reserpine and deserpidine are extracted together.
Isolation
Rauwolfia root powder is exhaustively extracted with 90% alcohol by suitable method of extraction such as percolation. The alcoholic extract is concentrated and dried under reduced pressure below 60°C to yield rauwolfia dry extract containing about 4% of total alkaloids. Rauwolfia dry extract is extracted further with proportions of ether– chloroform–90% alcohol (20:8:2.5). To the extract obtained, add little dilute ammonia with intermittent shaking. Alkaloid is converted to water-insoluble base. Add water and allow the drug to settle after few vigorous shakings. Fitter off the solution and extract the residue with 4 volumes of 0.5 N H2 SO4 in separating funnel. Combine the total acid extract which contains the alkaloidal salt. The extract is filtered, made alkaline with dilute ammonia to liberate alkaloid. Finally, it is extracted with chloroform. The total chloroform extract is filtered; chloroform is removed by distillation and the total alkaloidal extract is dried under vacuum to yield total rauwolfia alkaloids. Total rauwolfia alkaloid consists of the mixture of over 30 different components. It is subjected to column chromatographic fractionation for the seperation of reserpine.
Melting point: 270°C
Chromatographic Study
Paper chromatography: Dissolve about 1 mg of the sample of rauwolfia extract/standard reserpine in methanol. Immerse a 20 x 20 sheet of Whatman No. 1 filter paper in the immobile solvent formamide—acetone (3:10). Blot the paper between filter paper toweling and allow the acetone to dry. Apply the spots of the sample solution and standard on the filter paper and dry the spots. Immerse the spotted paper in the chromatographic chamber containing the mobile solvent isooctane–carbon tetrachloride–pyperidine– ter. butyl alcohol (90:60:4:2) and elute the paper by ascending chromatography. Remove the chromatogram when the mobile solvent has raised approximately 7/8th of the height of the paper. Dry the chromatogram at 90°C in a current of air, spray the paper evenly with 25% trichloroacetic acid in methanol and again dry at 90°C for 10 min. Samples yield spots corresponding in position and colour to those of the standard solution.
Thin layer chromatography: Dissolve 1 mg of rauwolfia alkaloidal extract or pure reserpine in methanol. Apply the spots over the TIC plate. In case of Silica gel-G plates elute the plate in solvent system chloroform–acetone–diethylamine (50:40:30). In case of Alumina-G plate, elute in the solvent system cyclohexane–chloroform (30:70). Dry the eluted plates and spray with Dragendorff ’s reagent. In both the cases orange spot is given by the alkaloidal components of rauwolfia and by reference standard. In cases of silica gel-G plate, reserpine gives Rf value 0.72 while in case of alumina G plate, it gives Rf value 0.35.
ISOLATION OF SENNOSIDES
Sennosides are isolated from Tinnevelley senna, consists of dried compound leaflets of Cassia angustifolia Vahl (Leguminosae).
Method I
The leaves are dried and powdered. The powdered drug is shaken with benzene for 2 h on an electronic shaker. Filter and distill off the solvent and marc is dried at room temperature and extracted with 70% methanol for 4–6 hours. The extract is filtered under vacuum and it is reextracted with 70% of methanol for 2 h, and filtered. The methanolic extract is combined and concentrated to l/8th portion of its original volume. The concentrated solution is acidified with hydrochloric acid to a pH of 3.2. The acidified solution is kept aside for 2 h at a temperature of 5°C. The solution is filtered and to the filtrate add anhydrous calcium chloride dissolved in 25 ml of denatured spirit with constant and vigorous stirring. The pH is again adjusted to 8 by ammonia and it is set aside for 2 h. The solution is filtered; the precipitate obtained is dried over P2 O5 in a dessicator.
Method II
Powdered drug is extracted by shaking with ethanolic chloroform (93 parts of chloroform and 7 parts of ethanol)for 30 min. Filtered and the leaves are again extracted with acidic methanol (1.2 g of oxalic acid per liter of methanol). Both the extracts are combined and concentrated. It was kept for whole night in room temperature. Sennoside A precipitates out, Sennoside B remains in solution. Sennoside A is re-crystalized using triethylamine. Sennoside B solutions is precipitated by 10% methanolic solution of CaCl2 . Further seperated by methanolic ammonia solution (40 ml ammonia + 60 ml methanol). Dried washed with water and kept for one day. It is then re-crystylized using glycolmonoethylether.
Melting point: Sennoside A 200–240°C, Sennoside B 180–186°C
Identification tests: To the crude extract, organic solvent like benzene, ether or chloroform is added and shaken. The organic layer is separated and to it ammonia solution is added, the ammoniacal layer produces pink to red colour indicating the presence of anthraquinone glycoside.
ISOLATION OF SOLASODINE
Solasodine is an aglycone of steroidal glycoalkaloid found in many species of Solanaceae family. The various sources commonly used for the preparation of solasodine include the berries of Solanum incanum (1.8–2%), S. khasianum (1–1.75%) and S. xanthocarpum. Solasonine is a steroidal glycoalkaloid which yields an aglycone solasodine and the sugar such as mannose, glucose and galactose on hydrolysis.
Isolation
The dried berries are first powdered and subjected to defecting with petroleum ether to yield greenish yellow oil which is rejected as it is devoid of the glycoalkaloid.
The defatted material is extracted thrice with ethyl alcohol; the extracts are combined and concentrated to 1/10th of its volume. Concentrated hydrochloric acid is then added to it until the final concentration reaches 5–6%. The whole mass is refluxed for about 6 h to attain complete hydrolysis of glycoalkoloid. The reaction mixture is then basified with ammonia and again refluxed for 1 h. The cooled reaction mixture is filtered and the residue obtained is thoroughly washed with water till neutral pH and dried. The dried material is then dissolved in chloroform. Solasodine goes into chloroform. The solution is filtered and the solvent is evaporated to yield the residue containing solasodine. It is further purified by crystallizing it from methanol or by sublimation in high vacuum.
Melting point: 200–202°C
Thin Layer Chromatography of Solasodine
Dissolve 1 mg of sample and standard in 1 ml of methanol separately. Apply the test solution and standard on the silica gel-G plate and develop the plate in solvent system toulene–ethyl acetate-diethyl amine (7:2:1). Spray the dried plates with modified Dragendorff ’s reagent. Allow the plate to dry and again spray with 10% sulphuric acid in methanol. An orange to red spot of Rf value 0.60, corresponding to solasodine is visible on both test and standard solution track.
ISOLATION OF STRYCHNINE AND BRUCINE
Strychnine and brucine are isolated from the seeds of Strychnos nuxvomica. Strychnine and Brucine are virulent poison and is used medicinally as a tonic and stimulant. Nuxvomica seeds contain about 3% alkaloids.
Isolation of Strychnine
100 g of powdered nux vomica seeds are mixed thoroughly with 100 ml of 10% calcium hydroxide in water and left overnight at room temperature. The air dried slurry is extracted with chloroform in a soxhlet extractor for 3 h. The chloroform solution is then extracted several times with 5% sulphuric acid solution and subsequently basified with 10% aqueous sodium hydroxide solution. Cooled and the crystals are filtered. Required amount of 50% ethanol is added, and the mixture is refluxed until most of the solid has dissolved. The solution is filtered after adding charcoal. The filtered crystals of strychnine are washed with a little 50% ethanol. The mother liquor and washings are used for the isolation of brucine.
The crude strychnine is dissolved in 9 volumes of boiling water, and 15% sulphuric acid solution is added slowly with stirring, until the reaction is slightly acid to Congo red. Activated charcoal is added to the solution and the solution is refluxed for 1 h and filtered hot. On cooling strychnine sulfate crystallizes out, filtered and washed with cold water. The obtained crystals are dissolved in 15 volumes of water, heated at 80°C and neutralized with 10% aqueous sodium carbonate: after addition of charcoal, the solution is filtered hot. Strychnine precipitates on addition of aqueous sodium carbonate and cooling. The precipitate is filtered and washed with cold water. It is re-crystallized using ethanol.
Melting point: 286–288°C
Isolation of Brucine
The mother liquor remaining after separation of strychnine is concentrated in vacuo on a water bath until most of the alcohol is removed. The residue is acidified to pH 6 with dilute sulphuric acid and then concentrated to a volume of 3–4 ml overnight. It is kept in a refrigerator and the product is filtered and washed with cold water. Adding 4.5 volumes of hot distilled water and boiling with a little charcoal for 1 h purify brucine sulfate. It is filtered while hot and kept in a refrigerator for several days. Brucine is recovered from the sulfate using the similar procedure used for strychnine. It is re-crystallized using aqueous acetone.
Melting point: 178°C
Identification Tests
- Strychnine when treated with sulphovanadic acid gives purple red colour.Strychnine when treated with sulphuric acid and crystals of potassium dichromate gives purple colour, which slowly changes to red, while brucine gives immediate red colour.
- Brucine when treated with nitric acid gives blood red colour, which is discharged by the addition of stannous chloride solution.
Thin Layer Chromatography of Strychnine and Brucine
Both the alkaloids are dissolved in methanol and spotted in silica gel-G plate. The solvent system used is benzene: Chloroform: diethylamine (9:4:1). After the development the plate is sprayed with Dragendroff ’s reagent, Rf values of both the alkaloids corresponds respectively.
ISOLATION OF VASICINE
Vasicine is a pyrrolazoquinazoline alkaloid obtained from the leaves of Adhatoda vasica; family Acanthaceae. A. vasica known as vasaka is a highly reputed ayurvedic medicinal plant used for the treatment of respiratory ailments, particularly for the treatment of cough, bronchitis, asthma and tuberculosis. Vasicine is present in vasaka upto about 1.3%. The other alkaloids present include vasicinone, vasicinol, vasicinolone, vasicol and adhatonine.
Isolation
Vasaka leaves are dried, coarsely powdered and basified to pH 9 with ammonia solution. It is further extracted with chloroform. The total chloroform extract is combined and washed with water and dried over anhydrous sodium sulphate. The solvent evaporated to get the total alkaloid extract containing vasicine as a major alkaloid. Vasicine can be further purified from the dry extract by crystallization. Melting point: 210°C
Thin Layer Chromatography of Vasicine
Dissolve 1 mg of vasicine in 1 ml of methanol with little warming. Apply the spots of test solution on the silica gel-G plate and elute with toluene–methanol–dioxane–ammonia (1:1:2.5:0.5). Spray the dried TLC plate with Dragendorff ’s reagent. Vasicine gives orange coloured spot.
ISOLATION OF VINCA ALKALOIDS
Vinblastine is isolated from the dried entire plant of Catharanthus roseus Linn (Apocynaceae).
Extraction and Isolation
The dried leaf material is taken and is extracted with a solution of hot ethanol–water–acetic acid in a ratio of 9:1:1. The solvent is removed and to the residue hot hydrochloric acid solution of 2% is added. The pH of the acidic extractis adjusted to 4, for the precipitation of the non-alkaloidal components, which can be separated by filtration. The pH of the aqueous acidic solution is now adjusted to 7 and then extracted with benzene. The benzene layer is evaporated to obtain vinblastine and other alkaloids.
in the ninth fraction. Further elution of the column results in separating the fractions of vincristine.Melting point: Vinblastine: 284–285°C, Vincristine: 273– 281°C
Thin Layer Chromatography of Vincristine
Vincristine dissolved in 25% water in methanol solution, spotted in Silica gel-G plate and developed using the solvent, acetonitrile: benzene (30:70). The dried plates are sprayed with 1% solution of ceric ammonium sulphate in 85% phosphoric acid. The Rf value of the appeared spot would be 0.39.
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