Final

ALKALOIDS ATROPINE

Isolation: Atropine is isolated from the juice or the powdered drug. Hyoseyamus 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 m 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 coloring matter. The alkaloids are precipitated with sodium carbonate, filtered off, washed and dried. The dried mtSl 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 seperated from the solution. The crude crystalline mass obtained after filtration is dissolved in alcohol and sodium hydroxide solution is added and the mixture 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.

Test: 1% 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). (1.5:100) . TLC plate is spread with an acidified iodoplatinate acidified iodoplatinate solution. Atropine gives the Rf value 0.18 likewise atropine sulphate shows the Rf value 0.70, in the solvent system acentone-0.5 M sodium chloride and spraying with Dragendroff’s reagent. Identification: Dilute solution of atropine, when treated with concentrated nitric and the mixture evaporated to dryness on the steam bath, produce a pale yellow residue. The residue gives a violet colouration when a drop of freshly prepared solution of potassium hydroxide is added. This is know as Vitali-Marin reaction.

Quinin and Quinidine Isolation:Cinchona Isolation:Cinchona bark is dried to 12-15% moisture and then ground o fine powder of about 60 mesh in disintegrator. It is mixed with slaked lime containing over 60% calcium hydroxide and water. During mixing, lime fixes the organic organi c acids as calcium salt. The mixture is left for 24 hrs. and then charged and extracted with benzene at a temperature of 190°f using steam. The hot extracted extracte d mineral solvent containing alkaloid is shaken up with dilute sulphuric acid to transfer the alkaloid to the acid. The aqueous acidic extract containing alkaloid is boiled and filtered through filter press while the solvent freed from alkaloid is recycled further for extraction. The filtered hot clear acidic extract is neutralized with hot sodium carbonate solution at pH 6.5 and cooled. Crude quinine sulphate seperates on cooling. It is centrifuged and refined by boiling with water and activated carbon to yield pure, white quinine sulphate. The mother liquor containing other cinchona alkaloids is treated with sodium carbonate and sodium hydroxide till it precipitates completely as the free bases. It is then extracted with ether to separate quinidine and cinchonidine. The dried ether extract is dissolved in dilute hydrochloric acid. The resulting mixture is neutralized with ammonia solution. Sodium potassium tartarate (Rochelle salt) is added which precipitates cinchonidine as a tartarate salt. The filtrate containing soluble quinidine tartarate is treated with potassium iodide solution where quinidine hydrogen hydrogen iodide is precipitated. It is collected and decomposed with ammonia to yield free quinidine alkaloid.lt is washed with water, dried, powdered and used for production of quinidine sulphate. Schematic diagram for the isolation of major cinchona alkaloids is predented in scheme 9.2.

Test: Dissolve pure cinchona alkaloids or the extract in methanol. Apph the spots over silica gel -G plate and elute in the given solvent system Spray the dried plate with Dragendorffs reagent. Some of TLC solveir. systems and Rf values of quinine and quinidine are given below Quinine

1) Chloroform-diethyl amine (9:1) 2) Chloroform-acetone-diethyl amine (5:4:1)

0.17 0.17

Quinidine

0.28 0.26

Identification: The solution of cinchona alkaloid when treated with bromine and ammonia produces emrald green colour. It is known as Thalleioquin test.

Morphine and Codeine

Properties: Morphine and its related alkaloids occurs as white crystalline powder. Morphine is laevorotatory, bitter alkaloid. It is sparingly soluble in water, ether and chloroform but soluble in alcohol and alkali solution. Morphine has been named after 'Morpheus" the Greek god of dreams. Isolation of morphine was first attempted by Derasne in 1803 and named it has salt of opium. In 1905 Seturner isolated morphine in pure form and referred to it as morphine acid

Quinin and Quinidine Isolation:Cinchona Isolation:Cinchona bark is dried to 12-15% moisture and then ground o fine powder of about 60 mesh in disintegrator. It is mixed with slaked lime containing over 60% calcium hydroxide and water. During mixing, lime fixes the organic organi c acids as calcium salt. The mixture is left for 24 hrs. and then charged and extracted with benzene at a temperature of 190°f using steam. The hot extracted extracte d mineral solvent containing alkaloid is shaken up with dilute sulphuric acid to transfer the alkaloid to the acid. The aqueous acidic extract containing alkaloid is boiled and filtered through filter press while the solvent freed from alkaloid is recycled further for extraction. The filtered hot clear acidic extract is neutralized with hot sodium carbonate solution at pH 6.5 and cooled. Crude quinine sulphate seperates on cooling. It is centrifuged and refined by boiling with water and activated carbon to yield pure, white quinine sulphate. The mother liquor containing other cinchona alkaloids is treated with sodium carbonate and sodium hydroxide till it precipitates completely as the free bases. It is then extracted with ether to separate quinidine and cinchonidine. The dried ether extract is dissolved in dilute hydrochloric acid. The resulting mixture is neutralized with ammonia solution. Sodium potassium tartarate (Rochelle salt) is added which precipitates cinchonidine as a tartarate salt. The filtrate containing soluble quinidine tartarate is treated with potassium iodide solution where quinidine hydrogen hydrogen iodide is precipitated. It is collected and decomposed with ammonia to yield free quinidine alkaloid.lt is washed with water, dried, powdered and used for production of quinidine sulphate. Schematic diagram for the isolation of major cinchona alkaloids is predented in scheme 9.2.

Test: Dissolve pure cinchona alkaloids or the extract in methanol. Apph the spots over silica gel -G plate and elute in the given solvent system Spray the dried plate with Dragendorffs reagent. Some of TLC solveir. systems and Rf values of quinine and quinidine are given below Quinine

1) Chloroform-diethyl amine (9:1) 2) Chloroform-acetone-diethyl amine (5:4:1)

0.17 0.17

Quinidine

0.28 0.26

Identification: The solution of cinchona alkaloid when treated with bromine and ammonia produces emrald green colour. It is known as Thalleioquin test.

Morphine and Codeine

Properties: Morphine and its related alkaloids occurs as white crystalline powder. Morphine is laevorotatory, bitter alkaloid. It is sparingly soluble in water, ether and chloroform but soluble in alcohol and alkali solution. Morphine has been named after 'Morpheus" the Greek god of dreams. Isolation of morphine was first attempted by Derasne in 1803 and named it has salt of opium. In 1905 Seturner isolated morphine in pure form and referred to it as morphine acid

meconate. More than twenty structures were proposed for morphine and other related alkaloids. The currently accepted structure is that proposed in 1925 by Gulland and Robinson. Morphine in the prototype of opiate analgesic drugs which act at several sites in the central nervous system to produce analgesia. Codeine and its salts are narcotic analgesics and antitussive agents. They are used as sedatives speci ally in allaying cough. Isolation : Several methods have been used for the isolation of morphine and codeine from opium. One of these methods commonly used.

Powdered opium is extracted with warm water till exhaustion. The aqueous extract is concentrated under vacuum and treated with a solution of calcium chloride (1:1). It is kept for 48 hours a nd then filtered. The filtrate containing the hydrochlorides of t he alkaloids is concentrated when hydrochloride salt of morphine and codeine deposit in the form of double compound known as 'Gregory salt'. This salt is dissolved in warm water and neutralized with dilute ammonia (pH 9). Morphine precipitates while codeine remains in solution as ammonium codeine chloride. Precipitated morphine is filtered through charcoal. Tin' filtrate is neutralized with ammonia and some amount of alcohol is added to precipitate morphine. Morphine is dissolved in dilute Hydrochloride acid to a saturated solution which on cooling crystallizes out as morphine hydrochloride. For purification of codeine, the solution of ammonium codeine chloride is concentrated, treated treate d with 30% sodium hydroxide hydroxi de and codeine is extracted with chloroform. Chloroform layer is extracted further with dilute sulphuric acid and the aqueous layer is decolorized with carcoal and subjected to filtration. The filtrate is rendered alkaline with sodium hydroxide solution and extracted with benzene. Benzene extract after evaporation to dryness afford codeine. Codeine phosphate is formed by neutralizing codeine with phosphoric acid and precipitating the salt from aqueous solution with alcohol.

Test : The Rf value of morphine and codeine in different solvent systems are listed below.

Emetine

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 the 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 aqeous phase. The ether extract is concentrated and redissolved in methanol and converted to emetine hydrobromide with a

methanolic solution of hydrobromic acid. Test : 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 modiied Dragendorff s reagent.

Solvent system 1) Chloroform-methanol(8 Chloroform-methanol(85:15) 5:15) 2) Benzene-Toulene-ethyl acetate diethyl amine-methanol amine-methano l (35:35:10:2) 3) Methyl ethyl ketone-ethanol-ammonia(5:4: ketone-ethano l-ammonia(5:4:l) l)

Rf 0.3-0.5 0.54 0.70

Strychnine and Brucine

Properties : Strychnine occurs as brilliant, colourless cubes or crystalline powder. It is very slightly soluble in water and forms water soluble salt in nitric and sulphuric acid. It is freely soluble in chloroform and dissolves in alcohol, benzene and pyridine. Brucine I occurs as crystalline powder soluble in chloroform and alcohol. Both alkaloids are very bitter in taste. Brucine isreadily distinguished from strychnine by being readily oxidized by dilule nitric acid with the formation of an intense red colour. Strychnine and brucine both are laevorotatory and both are generally used in resolution of dl acids. Strychnine is extremely toxic and functions as a central stimulant. Brucine is less toxic than strychinine. It is used as an alcohol denaUirant due to its extremely bitter taste. Isolation : Strychnine and brucine occurs together in several species of Strychnous especially in S. nux-vomica and S. ignatii. The dried ripe seeds of nux-vomica are treated with hot water or steam to soften and C then grinded and made into a paste with slaked lime. The pasty mass is subsequently extracted with chloroform till exhaustion. The chloroform extract is concentrated to 1/6 of its volume and then t hen successively extracted in seperating funnel with dilule sulphuric acid. Total aqueous acidic extract consists of strychnine and brucine in the form of their sulphate salts. The combined acid extract is concentrated. Strychnine and brucine can be separated from this acid extract by one of the following procedure.

(1) Concentrated acid extract is made alkaline with excess of ammonia solution, where both alkaloids precipitates together. The piri tpitatc is further extracted with 25% alcohol which dissolves Inm trie and leaves strychnine as insoluble mass. This residue of fiiiyehnine is iltered off, dried and recrystallized from ethanol.

(2) Brucine bisulphate is less soluble in concentrated acid extract and therefore it comes out from the solution which is removed by filtration. Mother liqnor is neutralized to yield crystallized material of strychnine Sulphate. It is then converted to its free base by precipitation with ammonia solution.

Test : Dissolve strychnine and brucine alkaloids in methanol and spot over silica gel G plates. Elute the plates with benzene-chloroform- dicthylamine (9:4:1) solvent system and spray the dried plate with dragendorff’s reagent. Strychnine and brucine corresponds to Rf respectively. Identification : Strychnine when treated with potassium dicromate and sulphuric acid gives violet colour which turns to red and finally yellow.

Vinblastine and Vincristine Properties : Vinblastine and vincristine both are soluble in alcohol and benzene. Its sulphate salt is freely soluble in water but slightly soluble ethanol. Both the alkaloids are white to slightly yellow crystalline or amorphous, odourless powder. These alkaloids in their pure form are unstable, very hydroscopic and highly toxic compounds. Vinblastine sulphate is used for the treatment of wide variety of neoplasms and in Hodkin's disease. Vincristine sulphate is recommended for the treatment of acute haemocytic leukemia and in other types of sarcomas.

Isolation: Vinca leaves and herb first minced and extracted with aqueous-alcoholic acetic acid solution. Aqueous extract is concentrated and the residue further extracted with 2% hydrochloric acid. This acid extract is adjusted to pH 4 with sodium hydroxide solution and then extracted with benzene. Again the pH of aqueous solution is raised to 7 and again extracted with benzene to separate alkaloids. The pH 7 benzene extracts were mixed together, concentrated, dried and dissolved in benzene-methylene chloride (65:35) and further subjected to chromatographic fractionation on neutral alumina column with benzene-methylene chloride as an eluent using gradient elution technique. Vinblastine rich fractions are evaporated to dryness to yield vinblastine sulphate which is subsequently crystallized from alcohol.

Test : Thin layer chromatography is an efficient means in identifying and monitoring the separation of vinca alkaloids. The sample alkaloid is dissolved 1 mg in 0.1 ml of 25% water in methanol and spotted on the silica get G plate and eluted with the solvent system acetonitril-benzene (3D:70). Eluted plates are dried and sprayed with 1% solution of eerie ammonium sulphate in 85% phosphoric acid. Vincristine free base gives the Rr value 0.39 with the above spraying agent.

Reserpine 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 lew vigorous shakings. Fitter off the solution and extract the residue with 4 volumes of 0.5 NH S04 in seperating 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 iltered, 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.

Test: (1)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

Ergometrine

Properties : Ergometrine maleate is a white to greyish white or faintly yellow, odourless, crystalline powder. It darkens with age and on exposure to light. It is slightly hygroscopic in nature. Ergometrine maleate is readily soluble in water and alcohol but insoluble in chloroform and ether. Its 1% solution in water has a pH of 3 to 5. Ergometrine base is more soluble in water than the

other principal alkaloids of ergot. Ergometerine maleate is an oxytocic and produces much faster stimulation of the uterine muscles than do other alkaloids. However it shows a much less vasoconstrictor action. 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 semisolid mass by adding sufficient solvent ether and dilute ammonia with stirring. The material is stirred to dry- ness and then packed in a soxhlet and extracted with solvent ether for about 5 hrs. The ether extract filtered and to it little acetone added and shaken in seperating funnel with three volumes of 1% of tartaric acid. The total acidic extract is combined and died 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

Vasicine Properties : Vasicine is a white, crystalline powder with better taste. It sublimates in high vaccum. Vasicine is soluble in alcohol, acetone and chloroform but slightly soluble in water. It is responsible for the bronchodilatory and expectorant properties. It also

shows abortifacient and antimicrobial activity against gingival inflammation and pyrrhoea. Isolation : Vasaka leaves are dried, coarsely powdered and basiied 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 evapored to get the total alkaloid extract containing vasicine as a major alkaloid. Vasicine can be further purified from the dry extract by crystallization. Test : 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.

Nicotine Properties : Nicotine is a colourless or pale yellow, very hygroscopic oily liquid with an unpleasent pungent odour and a sharp burning persistant taste. It gradually becomes brownish on exposure to air or light. Weight per ml. is about 1 01 g. Nicotine is soluble in water, alcohol, chloroform, ether, kerosene, petroleum ether and fixed oils. Isolation : Nicotine is generally isolated from the tobacco waste as the main tobacco crop has always to enter the normal consumption channel iccause 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 IfOsene. The organic solvent extract of nicotine is then treated with dilute sulphuric acid to obtain nicotine sulphate solution. The product Hi 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 exchange: 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. Test : Dissolve 1mg of sample in 1ml 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 0.1M phosphate buffer. After drying the plate for 15 minutes, expose it to bromine cyanide vapour for visualization. Nicotine gives the spot corres pond ing to t he Rf va lue 0. 77. Re acti on in volve d in the V is u a l i z a t i o n o f n i c o t i n e i s k n o w n a s K o n i g r e a c t i o n .

Properties : Piperine is pale or whitish crystalline powder which is tasteless at first but gives burning aftertaste. It is neutral to pH. Piperine shows anticonvulsant. CNS depressant, antipyretic, antioxidant and hep at op ro te ct iv e p ro pe rt ie s. It in cr ea se s

the bioavailability of antitubercular drugs when given together. Isolation : Black pepper is coarsely powdered and extracted in soxhlet extractor with 95 % ethano l. The al coholi c extrac t is concen trat ed u nder reduced pressure. The concentrated black pepper extract is added to 10% solution of potassium hydroxi de in 9 5% ethanol. Heat the resulting solution and add water dropwise, a yellow precipitate forms. Add wat er unt il no m ore sol id appear s to f orm and the mixture is allowed to stand at least overnight. The solid is collected by the suction filtration and dried. Piperine is recrystallized from acetone or from hot acetone-hexane (3:2) solution. Crystallization of piperine is a slow process. The flask must be allowed to stand for at least 24 hours. Test : Dissolve about 1 mg of piperine in lml methanol. Apply the test, solu ti on on th e 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 254nm. Piperine appear s as a violet coloured spot at R value 0.48. If the TLC plate is sprayed with anisaldehyde-glacial acetic acid methanol-cone. Sulphuric acid reagent (0.5-10:85:5) and heated at ii0°C for 10 minutes, piperine appears as a yellow spot.

Colchicine Properties : Colchicine occurs as a pale yelL]w odourless, amorphous scales or powder that turns darker when exposed to light. It is soluble in water, alcohol, ether and chloroform. It lacks pronounced basicity and does not f orm a well defined salt like other alkaloids. However, it is precipitated by alkaloidal reagent and is conventionally considered as alkaloid. Colchicum has antimitotic activity. In presence of colch1cine chromosomes of the cell continues to divide without the formation of spindle fibre and subsequent cell division to form daughter cells. 1t is largely used as a polyploidic agent. It has been employed experimentally in the treatment of various neoplastic diseases. 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 iltered 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. Tests : The dilute solution of colchicine inmethanol is spotted on silica gel-G plates and eluted with chloroform-diethyl amine (9:1) Colchicine is detected by spraying with alkaloidal reagent. It gives R value 0.41.

Solasodine Properties : Solasodine is a crystalline powder. Its alcoholic solution is alkaline to litmus. It is freely soluble in benzene, pyridine and chloroform, moderately soluble in alcohol and acetone, slightly soluble in ether but practically insoluble in water. Solasodine is used as an importamt starting material for the synthesis of steroidal

drugs.

Isolation : The dried berries are first powdered and subjected to clefattying with petroleum ether to yield a 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. Concentraed hydrochloric acid is then added to it until the final concentration reaches 5-6%. The whole mass is refluxed for about six hours to attain complete hydrolysis of glycoalkoloid. The reaction mixture is then basified with ammonia and again refluxed for one hour. 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 vaccum. Test : Dissolve 1mg of sample and standard in 1ml 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 R. value 0.60, corresponding to solasodine is visible on both test and standard solution track.

Levodopa Properties : Levodopa is a whitish or almost white, odourless and almost tasteless, crystalline powder. It is soluble in aqueous solutions of mineral acids and alkali carbonates. It is practically insoluble in alcohol, chloroform and ether. Aqueous solution is neutral or slightly acidic in nature. Levodopa is readily oxidized in air and light. The drug is used in the treatment of Parkinsonism. It is believed to replace depleted brain dopamine in patients with Parkinson’s disease.

Isolation : Coarsely powdered Mucuna seeds are extracted with lefhineralized water containing 1% v/v of acetic acid at 50°C. Acetic acid extract is iltered. The iltered extract is concentrated by reverse osmosis and the liquid concentrate so obtained is kept at a low temperature (6° - 8°C) for about 24 hours for crystallization of L-dopa. The crystals are iltered through a centrifuge, washed with cold water and dried under vacuum at 50°C. The iltrate 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 to 2.5% of L-dopa which still consists of some impurities of amino acids. It is further purified either by recrystallization or by using liquid ion exchangers.

Tests : 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 Dragendorff s reagent shows the spot at Rf value 0.4.

Taxol Properties : Taxol is hydrophobic. An ester side chain at 13 position of the taxane ring is essential for its cytotoxic activity. The presence of 2' hydroxyl group of the side chain enhances the activity. Taxol is used in the treatment of metastatic carcinoma of the overy after failure of the first line treatment or subsequent chemotherapy. It is also used in the treatment of breast cancer. Isolation : The extraction of taxol from the dried and powdered bark' of T. brevifolia is done using alcohol as a solvent. The total alcoholic extract is concentrated, dried and subjected to sequential chromatography on silica gel and florisil, yielding a mixture of taxol and cephalomannine which is finally seperated by careful I chromatography on silica gel using dichloromethane/1-propanol as eluent. This procedure yields pure taxol in the first fractions, but later fractions consists of mixture of taxol and cephalomannine. A good separation of taxol from cephalomannine can however be obtained by converting cephalomannine to its diol using osmium tetroxide. As taxol is not affected by these conditions, it can be separated from the diol by simple flash chromatography. Taxol is also extracted by using super-critical fluid extraction from the bark of pacific yew. The use of carbon dioxide and ethanol mixtures at 318 K gives a recovery of about 50-85% of the total taxol present. It is furnished as an extract representing taxol upto 1.5% of the total extract as compared with 0.125% for an ethanol extract. The use of the super-critical fluid extraction is thus a promising technique for selective extraction of taxol from plant material.

Tests :The presence of taxol in plant extract can be detected by two dimensional TLC method using diphenyl modified silica gel plates. The eluting systems are hexane-isopropanol-acetone (15:2:3) and methanol water (7:3). The with other system uses a cyano modified silica gel plate dichloromethane-hexane-acetic acid (9:10:1) and water-methyl cyanide-methanol-THF (8:5:7:0.1) as eluting systems.

Caffeine Properties : Caffeine occurs as a white powder or white, glistering needles mated together. It is odourless and has a bitter taste. Caffeine may be sublimed at 178°C without decomposition. However, its faster sublimation is achieved at 160-165°C under 1mm pressure. Caffeine is anhydrous or it may contain one molecule of water of hydration. Anhydrous caffeine is soluble in chloroform and alcohol. It is sparingly soluble in water but the solubility increases in water at 80°C. Its solubility in water is markedly increased by the presence of citric acid, benzoates, salicylates and bromides. Being a weak base it does not forms stable salts with strong acids. Caffeine is a central nervous system Stimulant and diuretic. Isolation : Variety of methods are in use for the isolation of caffeine "from different sources. Some important processes are described below. (1) The course powder of tea leaves is extracted with boiling water and the aqueous extract is iltered while hot. The warm extract is treated with lead acetate to precipitate tannins and iltered. The excess of lead scutate present in the solution is precipitated as lead sulphate with dilute sulphuric acid. The iltered solution is boiled with charcoal to remove colouring of matter if any, and filtered to remove charcoal. The iltered decolourized solution is extracted with chloroform. The combined chloroform extract after evaporation affords caffeine as a white material. It is recrystallized with alcohol. (2) Finelly or coarsely powdered tea leaves are extracted with ethanol in ioxhlet extractor. The caffeine so extracted in ethanol is then adsorbed On magnesium oxide. Caffeine is then disorbed after treatment with 10% It is then extracted with chloroform and recrystallized. (3) Caffeine is extracted from coffee beans by the process of leaching with water. The highest yield upto 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. ( 4) Decaffeination of coffee using super-critical fluid extraction.

Super-critical fluid extraction has been e fficiently 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. The types of medium and temperature pressure conditions are given in a table 9.2. Tests :(1) Dissolve 1mg of caffeine in 1ml of chloroform or methanol. Spot the sample on TLC plate and elute it in ethyl acetate-methanolacetic acid (80:10:10). Visualize the dried TLC plate by exposure to iodine vapour. Caffeine develops a spot at R value 0.41. (2) Dissolve about 5mg'in 1ml of HC1 in a porcelain dish. Add 50 mg. of potassium chlorate and evaporate on a steam bath to dryness. Invert the dish over a vessel containing a few drops" of 6N ammonium hydroxide. The residue acquires a purple colour, which disappears upon the addition of a solution of alkali.

A Coniine

Isolation : Coniine may be isolated by adopting the various following steps, namely: (i) The powdered unripe, fully grown died fruits of hemlock are mixed with a dilute solution of KOH and then subjected to stream distillation. The distillate is collected and neutralized carefully with dilute HC1 and evaporated to dryness preferably under vacuum. (ii) The residue obtained as stated in (i) above is extracted with alcohol, iltered and the alcohol evaporated to dryness under vacuum. The alcohol helps in extracting the alkaloidal salts that are dissolved in water; it is then rendered alkaline either with diluted KOH solution or with dilute NH4OH and inally extracted with ether successively. (iii) The ether from the combined ethereal layer is evaporated completely, when an oily liquid consisting of the free bases remains in the residue. (iv) Finally, the residue is subjected to ractional distillation in a current of H2-gas when the alkaloids could be broadly separated and a mixture containing coniine and y-coniceine shall pars over as the irst fraction at 171-172°C. These two alkaloids are consequently made to their corresponding hydrochloride salts, evaporated to dryness and extracted with acetone. Thus, coniine hydrochloride would be separated as an insoluble product, while the y-coniceine may be recovered by evaporating acetone under vacuum. 1. is a oouness alkaline liquid. 2. It darkens and polymerizes on being exposed to air and light. 3. It has a mousy odour. 4. It is steam volatile. 5. Solubility: 1 ml dissolves in 90 ml of water, less soluble in hot water. The base dissolves in about 25% water at room temperature. It is found to be soluble in alcohol, ether, acetone, benzene, amyl alcohol, and slightly soluble in chloroform.

C.Capsaicin Isolations : The capsicum fruits are crushed and extracted with either hot acetone or ethanol by using the method of percolation. The solvent i.e., hot acetone or ethanol is evaporated under vacuum. The residue is extracted once again with successive quantities of warm acetone or ethanol until and unless the marc is completely free from any pungent principles. It contains approximately not less than 8 % of capsaicin.

Identification Tests : 1. An alcoholic solution of capsaicin gives rise to a distinct bluish green colour upon adding a few drops of FeCl3 solution (0.5% w/v). 2. When capsaicin is dissolved in a few drops of concentrated H2S04 and a few crystals of sucrose is added, it yields a violet colour after a few hours.

Uses 1. It is used as a topical analgesic. 2.It is often employed as a tool in neurobiological research. 3.It is used in creams to counter neuralgia caused by herpes infections and in other pain-relieving formulations. Isolation : The alkaloid aconitine is subjected to hydrolysis which yields benzoyl aconine and acetic acid. The resulting benzoyl aconine is further hydrolyzed to yield aconine and benzoic acid. Aconine being very soluble in water may be separated easily rom the less water-soluble by product, i.e., benzoic acid.

Identification Tests : It forms two distinct derivatives as given below: (a) Aconine Hydrochloride Dihydrate (C 25H42C1NO9.2H20): It is obtained as crystals having mp 175-176°C and D-8°. (b) Aconine Hydrobromide Sesquihydrate ( C25H42BrNO9 .VA H20]: It is obtained as crystals from water with mp 225°C.

A. Tomatidine Isolation : It is obtained by the hydrolysis of tomatine to yield a molecule of tomatidine along -with 2_moles of D-glucose, 1-mole of D-xylose and 1-mole of D-galactose as depicted below:

Identification test : Its hydrochloride derivative (C27 H45 N02. HC1) is obtained as crystals from absolute ethanol having mp 265-270°C and [a]25 - 5° (methanol).

A.3 Solanidine Isolation : It is obtained by the hydrolysis of solanine which yields one mole each of L-Rhamnose, D-Galactose, and D-Glucose as shown below. Identification Tests: The same as described under A.l. earlier in this section. Besides it has the following specific features for the corresponding derivatives, namely:

(a) Hydrochloride Deivative: (C27H43NO.HCl): Prisms from 80% alcohol and gets decomposed (b) Methvliodide Derivative: (C27H43NO.CH3I): Crystals from 50% (v/v) ethanol and decomposes at 286°C. (c) Acetylsolanidine Derivative: (C29H45N02): Crystals obtained from ethanol having mp 208°C. Biosynthesis of Solasodine, Tomatidine and Solanidine Like the sapogenins the steroidal alkaloids are also derived rom cholesterol, with suitable side-chain modiication during the course of biochemical sequence of reactions as given under.

From the above biochemical sequence of reactions it is evident that: (i) L-arginine seems to be used as a source for N-atom through amination via a substitution process upon 26-hydroxycholesterol,

B.1.1 Protoveratrines Isolation : Protoveratrine A and B are usually exracted together and referred to as ‘protoveratrines’. About 2kg of dried rhizomes of V. album is powdered and then extracted and the extracted with benzene and ammonia. The total alkaloids are purified by extracting with acetic, re-extracted into benzene. The solvent is removed under vacuum, the residue is dissolved in ether from which the crystalline powder of the crude protoveratrines separates out. The crude product is recrystallized rom alcohol-acetic acid and upon subsequent alkalinization of the solution with dilute ammonia. By this method one may obtain 8-10 g of protoveratrine powder from 8 kg of If album rhizomes. Consequently, protaverine A and B may be separated by the help of counter current distribution of the "protaverine" between benzene and acetate buffer (pH 5.5) and ultimately subjected to column chromatography on acid aaluminium oxide (A1203).

B.1.2 Veratridine Isolation : Veratridine can be isolated as the commercial veratrine (mixture) i.e., the mixture of alkaloids cevadine, veratridine, cevadiline, sabadine and cevine obtained from the seeds of S. oficinale stated above, as its sparingly soluble nitrate derivative.

Identification Tests : 1. It

readily forms its nitrate derivative which is an amorphous powder and sparingly soluble in water. 2. Its sulphate salt is formed as its needles which happens to be very hygroscopic. 3. It readily forms its perchlorate derivative as long needles from water having mp 259-260°C (after drying at 120°C in Vacuo).

C. Lobelanine Isolation : The aqueous layefobtained in step (3), as stated under isolation of lobeline, is subjected column chromatography and the lobelanine is collected as one of the major fractions.

Identification Tests : 1. Lobelanine Hydrochloride (C22H25N02.HC1): The crystals obtained from dilute ethanol decomposes at 188°C; it is soluble in chloroform; and slightly soluble in absolute ethanol and cold water. 2. Lobelanine Hydrobromide (C22H25N02.HBr): The crystals do not give a sharp mp, but gets decomposed at 188°C. 3. Lobelanine Nitrate (C22H25N02.HN03): The crystals obtained from dilute ethanol has mp 153-154°C. 4. If differs from Lobeline in lacking OH moiety; and therefore, does not reac t with nitrous acid nor with benzyl chloride. 5. It being a diketonic compound-forms a dioxime. D.Lobelanidine Isolation: It is obtained as one of the fractions obtained from the column chromatography of the aqueous extract under isolation of lobeline. Identification Tests :

1.Lobelanidine Hydrochloride (C22H29N02. HC1): It is obtained as needles from ethanol having mp 135-138°C. 2. Lobelanidine Hydrobromide (C22H29N02.HBr): Its crystals have a mp 189°C.

Synthesis from Lobeline, Lobelanine and Lobelanidine First of all, lobelanine may be synthesized by the interaction of one molecule of glutaric dialdehyde, two moles of benzoyl acetic acid, and one mole of methylamine hydrochloride; allowing the reaction mixture to stand for 40 hours at 35°C and at pH 4.5. Thus, the resulting product lobelanine gives rise to: (a) Lobeline: When subjected to partial reduction, and (b) Lobelanidine: On being subjected to complete reduction.

B.Lupanine Isolation : The racemic and optical isomers of lupanine have been duly isolated from various species of Lupinus (Fabaceae/Leguminosae) as stated below:

Identification Test : Lupanine forms the corresponding lupanine hydrochloride dihydrate (C15H24N2O.HCl.2H2O) which is obtained as rhombic crystals from water having mp 127°C (dry).

B.Cocaine Isolation : Cocaine is extracted from the plant by digestion either with sodium carbonate solution or with lime water and by subsequent solvent extraction using petroleum ether (bp 160-180°C; or 200-220°C). The combined petroleum ether extract is shaken up with dilute HC1. The solution of hydrochloride thus obtained is concentrated carefully in a thin-film evaporator. In case, the leaves are rich in cocaine content, as in the Peruvian coca leaves, a major portion of cocaine gets separated as crystals.

1.

2.

3.

4.

Identification Tests Cocaine Permanganate: The addition of a drop of saturated solution of KMn04 to a solution of cocaine prepared in a saturated solution of alum gives rise to a violet crystalline precipitate due to the formation of cocaine permanganate. It clearly shows characteristic violet aggregates of plates when examined under the microscope. Cocaine Hydrochloride (C17H21N04.HC1) (Cocaine Muriate): It is obtained as granules, crystals, or powder. It has a slightly bitter taste and usually numbs lips and tongue. Its physical characteristics are: mp ~ 195°C; [a]D - 72° (C = 2 in aqueous solution); 1 g dissolves in 0.4 ml of water; 3.2 ml cold and 2 ml hot alcohol; 12.5 ml chloroform. It is also soluble in glycerol and acetone; and insoluble in ether or oils. Cocaine Nitrate Dihydrate (CI7H22N207.2H20): Its crystals have mp 58-63°C. It is freely soluble in water or ethanol; and slightly soluble in ether. Cocaine Sulphate (C I7H21N04.H2S04): It is obtained as white, crystalline or granular powder, which is soluble in ethanol and water.

C. Cinnamoyl Cocaine Isolation : Instead of the Peruvian leaves the Java leaves of E. coca are treated in the same manner and fashion as described under cocaine earlier (section 'B'). It has been observed that the mixed hydrochloride mostly-comprise of cinnamoyl cocaine which gets separated as fine needles. Identification Tests : 1. It reduces an acidic solution of KMn04 in cold i.e., at ambient temperature, which helps to detect the presence of this alkaloid in an admixture with cocaine. 2. It undergoes hydrolysis when warmed with HC1 to yield /-ecgonine, cinnamic acid and methanol.

D. Ecgonine Isolation : Ecgonine may be obtained by the hydrolysis of cocaine as given below:

Identification Tests 1. Ecgonine Hydrochloride (C9H15N03.HC1): It is obtained as the triclinic plates obtained from water having mp 246°C soluble in water and slightly in ethanol. 2. Ecgonine Trihydrate: It is obtained as plates from 90% ethanol having mp 93-118°C (anhydrous substance gets decomposed at 212°C).

E.Synonyms Isolation :Hyoscyamine may be isolated from the Belladona leaves by adopting the following "steps sequentially:

1. The finely powdered and sieved Belladona leaves is extracted with 95% (v/v) ethanol in a Soxhlet Apparatus till no more alkaloids come out from the marc. The ethanolic extract is concentrated to a syrupy residue under vaccuo and subsequently treated with dilute HC1. The resinous matter is separated by filtration and the resulting solution is further purified by shaking out with petroleum ether (40-60°C) several times. 2. The purified acidic solution thus obtained is made alkaline wit h ammonia solution (dilute) carefully and extracted with chloroform successively. The combined chloroform layer is once.

Identification Tests : The various identiication tests for hyoscyamine are, namely: 1. Gerrard Reaction: Hyoscyamine (and also atropine) responds to the Gerrard Reaction wherein about 5-10 mg of it reacts with mereuric chloide solution (2% w/v) in 50% ethanol to give rise to an instant red colouration without warming. 2. Schaer's Reagent: A few mg of hyoscyamine when made to react with a few drops of the Schaer's Reagent i.e., 1 volume of 30% H202 mixed with 10 volumes of concentrated sulphuric acid, produces a distinct green colouration. C. Thebaine Isolation : Thebaine may be isolated from opium by means of the following steps, namely: Step-1: Opium (dried latex) is treated with calcium chloride solution and then extracted with warm water. Allow it to remain as such for 24 hours. Step-2: Filter the resulting product and collect the residue and iltrate separately. Residue— contains the salts of calcium as lactate, sulphate, resinate and meconate (To be discarded). Filtrate — contains the hydrochloride of various alkaloids present in opium. Step-3: Add dilute NaOH solution (2N) carefully to the resulting iltrate and allow it to stand for 46 hours. Filter the contents of the flask: Filtrate— contains morphine, codeine and narceine Residue—contains thebaine, papaverine and narcotine Step-4: Dissolve the residue or precipilate in dilute ethanol (50% v/v), make slightly acidic with the addition of dilute glacial acetic acid and finally add to it approximately three volumes of boiling distilled water.

Step-5: Filter the above reaction product: Filtrate— contains thebaine Residue—contains papaverine and narcotine Step-6: Concentrate the filtrate obtained in Step-5 under reduced pressure and add to it dilute NH4OH solution to make it alkaline; and extract the liberated alkaloid thebaine successively with Chloroform. The baine is obtained after evaportion of chloroform under vaccuo.

2.GLYCOSIDES AND AGLY CONES Sennosides Isolation : Various investigator have used variety of solvents such as 70i8©%'ethanol, 90% methanol or 80% acetone for t he extraction of sennosides. For the laboratory scale extraction and purification of sennosides coarsely powdered senna leaves or senna pods are irst extracted with benzene and 1-5% ethanol to remove pigments and resins. The dried marc is further extracted with ethanol and the alcoholic extract: is concentrated in vacuum below 40°C. Dry alcoholic extract is mixed with the solution of calcium chloride in methanol and the solution is filtered. To the filtrate methanolic ammonia is added until red brown colour disappears. The precipitated material is filtered and washed with methanol and dried. The precipitate containing calcium sennoside is suspended in methanol and acidified with gluconic acid at 40°C. The acidiied extract after filtration yield a precipitate containing yellow mass of sennoside A. The filtrate when treated with methanolic hydrobromic acid and subsequently evaporated affords sennoside B. Another industrial process for the isolation of calcium sennoside (30%) use 80-90% of methanol at pH 2-9 with organic acid such as citric or gluconic acid. The extract is filtered and then adjusted tc pH 3.7 to 3.9 with ammonia, stirred and allowed to stand for one hour The precipitate formed is removed by filtration. To the clear filtrate about 10% solution of methanolic calcium chloride is added and the contents are stirred for one hour, then 30% ammonia solution added with stirring until pH reaches to 6.5 To 6.8. The precipitation of calcium sennosides takes place within an hour. The precipitate is filtered, washed with methane!. The precipitate is quickly dried under vaccum at temperature not exceeding 50°C. The inal product yield calcium sennoside 30 35%, Tests : Dissolve about 1mg of sennoside in 1ml of solvent containing equal volumes of ethyl acetate, n-propanol and water (upper layer). The plate is silica gel-G plate is spotted with the sample and eluted in solvent system ethyl acetate-n propanol-water (4:4:3). The dried plate is expose to vapours of ammonia for 5 min till the colour develops. Cover the plates with glass and heat at 110 OC for 5-10 min Sennosides A and B develop two prominent spots corresponding to those of standard sennosides A and B.

Aloin Properties : Aloin is a pale or dull yellow crystalline powder odourless with an intensely bitter taste. Aloin A and B are soluble in water, but the later is labile in solution. It darkens on exposure to light. It is sparingly soluble in water but completely dissolve in alcohol and acetone. Aloin is incompatible with alkalis and oxidizing agents. Aloin elicits a drastic Cathartic action.

Isolation: Commercial aloe is obtained as a solid residue by evaporating the yellow exudates which drains from the leaves cut from various species of Aloe. The juice is concentrated by spontaneous evaporation or more often by boiling and then poured into tins or other suitable containers. Aloe solidifies on cooling to yield dark brown mass. Commercial aloes contains upto about 30% of aloin. Aloin h a crystalline substance obtained from commercial aloe mainly by extraction with hot acidified water and subsequently puified and crystallized. Aloin is separable by HPLC into aloin A and aloin B. Aloin A can be obtained by crystallization from methanol. Individual aloe anthraquinones are usually obtained in preparative amounts on polyamide columns, the purity being monitored by TLC. Test : Dissolve 1 mg of aloin in 1ml of methanol. Apply the spots over silica gel-G plate and elute the plate in solvent system ethyl-acelate-methanol-water (100:16.5:13.5). Dry the plate and observe visually as well as under UV light. Aloin gives the brownish spot at Rf value Q..45rwhich changes to yellow under UV light. Identifications : Aloin positively responds to the test for anthraquinone glyecosides-such as modified Borntrager test, borax test and bromine test explained under Anthraquinone glycosides (Part I).

Hypericin Isolation : Dried herb of St. John Wort containing foliage and flowering top is converted to coarse powder and extracted with 90% alcohol and then with 70% alcohol. The combined alcoholic extract || filtered and concentrated under reduced pressure at about 50"C to obtain dry extract known as St. John's Wort Dry Extract, It contains about 0.5% hypericin. Further purification of the extract by chromatographic fractionation yield hypericin.

Test : Dissolve 1mg of sample in methanol. Apply the spots on silica gel-G plate and elute in ethyl acetate-formic acid-glacial acetic acid-water (100; 11:11:26). Observe the dried TLC plate under US UV light. Hypericin appears as a spot with prominent reddish violet fluorescence at Rf value 0.78-0.8, corresponding with the spot of the standard.

Digoxin Properties : Digoxin accurs as colourless, crystals or white powder with a bitter

taste. It is practically insoluble in water dehydrated alcohol, chloroform and ether but soluble in pyridine, 80% ethanol and a mixture of equal volumes of

chloroorm and methanol. It is incompatible with acids and alkalies. Digoxin act in a similar way as that of digitalis in the treatment of congesitve heart failure. It is

preferred for its rapid absorption and lesser cumulative properties. 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. Hydrohysis 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 irst 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. Test : UDissolve about lmg of the glycoside in 1ml of alcohol. The sample is spotted over silica gel G plates and eluted with cyclohexane-acetoneacetic acid (49:49:2). Dried TLC plates are sprayed with 50% aqueous appears as a blue spot under 385 nm UV light. Identification: 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.

Bacosides Properties : Bacoside occurs as a white crystalline powder. It is soluble in alcohol, acetone, n-butanol but insoluble in petroleum ether. It increases the learning performance in experimental animals and humans. It is used as a brain tonic. Sedation is associated with the therapeutic doses of the drug.

Isolation : General process for the isolation of bacosids from B. monnieri is as follows. The coarsely powdered drug is extracted with ethyl ale hoi and the alcoholic extract is concentrated to diyness. Dry alcoholic extract is dissolved in 60% alcohol and the solution is extracted in seperating 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 seperation of brown resinous material. Solution of alcohol-ether-petroleum ether m xture is concentrated and the residue is macerated with acetone and Uttered. The acetone-insoluble powder is partitioned between butanol and water. During concentration of butanol solution a precipitate that settles down is seperated and coded as A,. The filtrate is again concentrated and precipitated with acetone and ether to afford a powder A,. The mother liquor is concentrated to diyness to yield brownish powder A,, fraction A,, A, and A, obtained consist mainly of bacoside A.

The acetone soluble fraction is let in the cold for several days when a solid settles down. It is iltered, washed with acetone, yield major amount of bacoside B Test : The extract or the isolated glycoside dissolved in methar.ol and spotted over silica gel G plates. The plates are eluted in ethyl cetatepyridine-water (4:1:1) and the TLC plates after drying are sprayed with trichoroacetic acid (25%) in chloroform. Bacoside A of A1, A2, and A3,. Fraction gives Rf value 0.43 while bacoside B gives R,. value 0.09

Diosgenin Properties : Diosgenin occurs as a white crystalline powder, soluble in the usual organic solvents and in acetic acid. Diosgenin is used as a phamaceutical aid for the synthesis of various steroidal drugs. Isolation : Dasgupta and Coworkers have reported 2.12% yield of diosgenin out of a total average of 3.86% of sapogenin present in the glycosidic form in the alcoholic extract. Dried rhizomes are powdered to mesh 20 sizes and first subjected to hydrolysis by refluxing with 5% hydrochloric acid for 2 hours. "I he hydrolyzed mass is filtered, washed twice with water and then twice with 5% sodium bicarbonate solution. It is finally washed with water till the washings are neutral. The residue thus obtained is dried and further extracted with toluene for 8 hours. The toluene extract is concentrated during which diosgenin gets precipitated. Diosgenin is filtered, washed with little hexane (40-60°C) and air dried to yield about 95%jpure product. Test : Dissolve about 1 mg of diosgenin in 1 ml of methanol. Silica gcl-G plates spotted with the sample are eluted in solvent system toluene-ethyl acetate (7:3). The dried plates are sprayed with anisaldehyde-sulphuric acid reagent and heated at 1 10°C for 10 minutes. Dark green spot is visualized in day light at R value 0.37 which corrgspetdslo reference-standard.

Guggulsterone (Gugulipid) Properties : Gugglsterone occurs as a white crystalline powder with aromatic odour. It is soluble in ethyl acetate, benzene, chloroform, petroleum ether and also in alcohol. It has been reported to possess significant hypolipidaemic and hypocholesterolaemic activity in experimental animals and in humans .

Isolation : Gum resin of C. mukul coarsely powdered and extracted With ethyl acelate. Tacelate. 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 3N 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 noncarbonyl and neutral ketonic fraction by following process. Neutral fraction is mixed with 10% semi-carbazide on silica gel and toluene, stirred and heated a 60-62°C for 14 firs, cooled and filtered. Silica gel is thoroughly washed with toluene to get toluene soluble noncarbonyl fraction. The above washed silica gel is mixed with aqueous 10% oxalic acid and toluene. Neutral ketonic fraction is chromatographed on silica ge! and eluted with benzene-ethyl acetate to yield ractions containing mixture of E-and Z-guggulsterone. E -and

Z-guggulsterone are further purified by rechromatography on silica gel. The scheme of separation of guggulsterone is given in scheme 9.1. Test : 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 died plate is sprayed with 1 % vanillin-sulphuric acid and heated at 110°C for 10-15 minutes. Guggulsterone gives bluish violet spots which correspond with the Rf value 0.45 of the standard.

Glyeyrrhetinie acid Properties : Glycyrrhetinic. acid is a white crystalline powder. It is insoluble in water but freely soluble in alcohol, chloroform, benzene, ether etc. It dissolves in fixed alkali solution due to the formation of water soluble salt. Glycyrrhizin is nearly 50 times sweeter than sucrose and its sweetness is perceptible in 1:20,000 dilution but glycyrrhetinic acid is not as sweet as glycyrrhizin. It is used in the treatment of noninfective inflammatory disorders of skin, mouth etc.

Isolation : Glycyrrhetinic acid can be extracted from the crude drug by "following procedure. The crude drug is first extracted with chloroform, Chloroform extract is discarded. The march is again extracted this lime 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 the another method of extraction, liquoice powder is extracted with boiling water to isolate glycyrrhizin. The aqueous extract is concentrated, dried and used as iiquorice extract. The liquorice extract, can be dissolved in water and acidified with hydrochloric acid to pi I 3-3.4 to precipitate glycyrrhetinic acid. The precipitate is iltered, 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 ext ract, dissolving it in ammonia and drying the solution after spreading in a thin film on a glass plate to give shining dark brown flakes. Test : Dissolve 1mg 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 minutes at 110°C, Glycyrrhetinic acid gives purplish spot corresponding to the Rf value 0.41. Properties : Boswellic acid is a very light almost white and shiny crystalline powder with characteristic odour. It is soluble in alcohol, ether, chloroform and petroleum ether but insoluble in water. The mixture of B-bowellic acid is used as anti-inflammatory and antiarthritic agent.

Isolation : The oleo-gum resin of B. serrata is first defatted with petroleum ether (60-80°). The dried marc of oleo-gum resin is further exhaustively extracted with methanol. The methanolic extract is concentrated and then treated with 1 0% potassium hydroxide to produce acid fraction and neutral fraction. The acid fraction ol methanolic extract is subjected to column chromatographic fractionation over silica gel using increasing amounts of ethyl acetate and hexane to afford first acetyl P-boswellic acid and then acetyl-11- keto p-boswellic acid, P-boswellic acid and lastly 11-keto-p-boswellic acid. Test : 1 mg of boswellic acid is dissolved in 1ml of methanol. The silica gel-G plates spotted with the sample are eluteded with chloroform- methanol (95:5). Acetyl P-boswellic acid and P-boswellic acid Correspond Rf values 0.49 and 0.45 respectively. Properties : Gymnemic acid is a yellow to brown amorphous, bitter powder. It is almost insoluble in water but soluble in alcohol. It completely obtunds taste for several hours for bitter or sweet, eg. quinine or sugar, but not for sour, astrigent or pungent substances.

Isolation : Symnema leaves are coarsely powdered and extracted with 55% alcohol. The alcoholic extract is concentrated and dried to yield Gymnema leaf dry extract containing about 25% gymnemic acid. Gymnemic acid is seperated from the dry extract by further solvent treatment.

Test : Dissolve gymnenic acid in methanol and appl y the spots over silica gel-G plate. Elute the plate in solvent system n-butanol-glacial acetic acid-water (12:8:4). Spray the dried plate with 1% vanillin- sulphuric acid or 1% anisaldehyde-sulphuic acid reagent and heat at 110°C for 10 minutes. Gymnemic acid gives purplish spot which corresponds with standard on TLC plate. If the unsprayed plate is observed under UV light at 254 nm it shows the pale spot of gymnemic acid.

Artimisinin _  Properties : Artemisinin is a whitish crystalline powder soluble in most organic solvents. It is slightly soluble in oil.

Isolation : leaves of A. annua are air died, coarsely powdered and; subjected to extraction with petroleum ether (40-60°). The extract is concentrated, dried and redissolved in chloroform, to which acetonitrile is added to precipitate inert plant constituents such as sugars and waxes. The chromatographic fractionation of the concentrate on silica gel by eluting with chloroform-ethyl acetate yields the fraction of artemisinin. The fractions containing artemisinin could be crystallized from cyclohexane or 50% ethanol. Chinese plant have been reported to yield as much as 0.9% of artemisinin, but elsewhere plant yield about 0.1%. The highest content of artemisinin is found in the leaves of the top 50 cm of the plant and the highest yield is obtained from the leaves just before flowering.

Test : Dissolve artemisinin or the extract in chloroform and apply the spots of the sample and standard on the silica gel-G plate, flute the plates in solvent system petroleum ether-ethyl acetate (1:2). Spray the died plate with p-dimethyiaminobenzaldehyde and heal at 80"C to produce colour. The spot of artemisinin corresponds to the standard which can be quantitated by densitometry at 600 nm. The use of 2% solution of vanillin-sulphuric acid produce colour that is measure, at 560 nm.

Rutin Properties : Rutin is a pale or greenish yellow crystalline powder which gradually darkens on exposure to light. It is tasteless and odourless. Rutin is sparingly soluble in water but its solubility increases in boiling water. It is soluble in methanol, pyridine, formamide and alkaline solutions. Rutin as well as its aglycone quercetin both are used as vitamin P. It is also used is category thrombosis and for purification of blood. Isolation : Number of solvents such as ethanol, hot dilute isopropanol have been used for the extraction of rutin but in certain cases single stage batch extinction with boiling water is recommended.

When alcohol is used as a solvent the ground plant material is extracted with ethanol 80%. The ethanolic extract is concentrated under and the filtered. The filtrate is extracted twice with solvent ether and the ethereal extract is discarded. The aqueous solution concentrated under vaccum to obtain crystalline solid material. The solid maas is tillered and the crystals are washed with water followed by ether to crude rutin. It is purified by fractionation on magnesium silicate column.

Test : 1mg of rutin is dissolved in 1ml of methanol. The silica gel-G plate is spotted with the sample and eluted in the solvent system 10% aqueous sodium chloride solution. Rutin gives the yellow spot with an Rf value 0.43.

Phyllanthin Properties : Phyllanthin occurs as white crystalline powder. It is soluble in alcohol, acetone, benzene, chloroform and petroleum ether. Phyllanthin is reported to have remarkable hepatoprotective activity in experimental animals. It is used in the form of whole herb or extract for the treatment of jaundice and other liver diseases.

Isolation : The aerial parts of P. amarus are coarsely powdered and mixed thoroughly with lime and water. The mixture is allowed to dry overnight. The died material is exhaustively extracted with petroleum ether (60-80°). The petroleum ether extract is concentrated and dried under reduced pressure. Residue obtained is extracted with methanol thrice and the combined methanolic extract is again concentrated to diyness and redissolved in petroleum ether. It is reduced in volume by evaporation, allowed to stand until yellow oily material seperates c it. The remaining part of petroleum ether is chromatographed over neutral alumina and eluted with n-hexane-ethyl acetate (99:1) to

seperate fractions of hypophyllanthin and phyllanthin. The fraction are monitored by TLC and similar fractions are mixed together and processed for crystallization. Test : Dissolve 1mg of sample in 1ml of methanol and make the solution of the standard in the same way Apply the spots over silica gel plates and elute in the solvent system n-hexane-ethyl acetate (2:1). Spray the dried TLC plates with sulphuric acid reagent and heat at 110°C for about 10 minute. Phyllanthin (blue spot) and hypophyllanthin (brown spot) corresponds with the reference standards showing R f values 0.20 and 0.25 respectively.

Psoralen Properties : Psoralen occurs as a white crystalline powder soluble in alcohol, chloroform and pyridine. It is practically insoluble in water and petroleum ether. Psoralen solution gives a dark blue fluorescence under UV light which can be detected by applying a spot of sample on Whatman No. filter paper and observing under UV light. Isolation : The died fruits of S. corylfolia are very hard to powder. The fruits are converted to coarse powder and defatted with petroleum ether to remove essential oil and resinous matter present on the outer coat of the fruits. The marc is dried and further extracted with methanol. The methanolic extract contains psoralen along with many other coumarin and flavonoid compounds. The extract is concentrated, died and chromatographed on silica gel using pure chloroform SS in eluent Psoralen is obtained in first few fractions which art: concentrated to yield psoralen. It is further crystallized from alcohol or solvent ether.

Test : 1 mg of psoralen is dissolved in 1ml of methanol and spotted on silica gel-G plates. The plates are eluted in benzene-chloroform (9S:2). the dried plate is observed under UV light. Psoralen appears as a blue luoresoent spot showing the R value 0.25.

Khellin Properties : Khellin occurs as white, odourless crystalline powder, sometimes with slight yellowish tinge. It is bitter in taste. It is insoluble in cold water but spaingly soluble in boiling water. It is soluble in alcohol, acetone, chloroform and glacial acetic acid. Khellin relaxes smooth muscle and also exhibit spasmolytic action. It is used in the treatment of heart diseases. The drug has a cumulative effect. Isolation : Dried, ripe fruits of A. visnaga are extracted with methanol till exhaustion. Methanolic extract is concentrated and dried to yield mixture of components along with khellin. In laboratory scale isolation of khellin, the dry extract is subjected to column ehromatographic fractionation on silica gel in petroleum ether-ethyl acetate to yield the fractions containing pure khellin. In cases of large industrial batches, khellin can be seperated by solvent treatment and lastly by crystallization.

Test : About 1mg of khellin is dissolved in 1ml of methanol. Apply the spots over silica gel-G plate and elute in the solvent system toluene -diethyl ether (1:1), saturated with 10% acetic acid. The eluted plate is dried and observed under 365nm. Khellin appears as a dirty green fluorescence spot with R f value 0.4-0.6.

Andrographolide Properties : powder with/an extremely bitter taste. It is sparingly soluble in water but freely dissolves in acetone, methanol, chloroform and ether. Andrographpolide is used as an antipyretic, anti-inflammatory and hepatoprotective agent in Asian traditional medicine. Isolation : The dried herb is cleaned to remove foreign matter and then crused 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 hrs are filtered off and washed with chilled methanol. The filtrate and washings are processed for 2nd 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 to 175% of andrographolide. Test : Dissolve about 1 mg of sample in lm! 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 120 OC for 10minuts.

Rubarb Chemical Tests. 1. The Rhu tar* powder on being treated with ammonia gives rise to a pink colouration. 2. Rhubarb gives a blood-red colouration with 5% potassium hydroxide. 3. It gives a positive indication with modified Borntrager's test (see under Aloes).  _  4.2.1.3 ascara Sagrada  Properties : The bark is collected, during the dry season (April to August) from the 8 to 9 years oidtrees that have gained a height of 16-18 meters with their stems having a diameter of 8 to 10 cm, by inflicting longitudinal incisions on the fully developed stems. In usual practice, the coppicing technique is employed for the collection of bark. The bark is carefully stripped off from the branches and the stems. They are subsequently allowed to dry in shade by putting their inner-surface facing the ground so as to permit the completion in the enzymatic conversion of the anthranol derivative i.e., glycosides (an emetic principle) to its anthraquinone derivative usually present in the fresh drug, thereby exerting a milder cathartic activity. During this span of one year the drug must be duly protected from rain or humid environment so as to check the growth of mould.

Chemical test : It gives a positive indication with Modified Borntrager's test because of the presence of C-glycosides. Substituents/Adulterants The barks of Rhamanus californica  and R. fallax  are generally used as a substitute for cascara sagrada bark. Sometimes the frangula  bark is also used as a substitute for this drug. However, the former types of barks (Rhamnus species) exhibit a more uniform coat of lichens along with broader medullary rays when compared to the original drug species.

4.2.1.4 Frangula

Preparation : The preparation of frangula bark resembles to that of cascara bark (see section 4.2.1.3) just like the cascara bark, the frangula bark must be aged for at least a period of one year before it is used therapeutically so as to permit the reduced forms of the glycosides with harsh action to be oxidised to comparatively milder forms.

4.2.2.3 Salicin Preparation : The powdered bark is macerated with hot water for several hours whereby the glucosi e (salicin) and tannin are extracted collectively. The resulting liquid extract is filtered, concentrated under vacuum and treated with lead acetate to remove the tannins as a precipitate. It is subsequently treated with hydrogen sulphite to remove the excess of lead. The clear filtrate is neutralized with ammonia, allowed to concentrate, chilled to obtain the crystals of salicin. The crude salicin may be further purified by treating its solution with animal charcoal and concentrating followed by cooling. Chemical tests It gives an instant bright red colour with concentrated sulphuric acid that fades out on the addition of water. 2. Its hydrolysed product saligenin gives a blue colour with ferric chloride. 3. On oxidation with potassium dichromate and sulphuric acid and heating yields salicylaldehyde having a characteristic odour. 1.

4.2.2.4 Populin Preparation : It may be prepared either from salicin by melting with benzoic anhydride, or from sahcin and benzoyl chloride in the presence of KOH as shown below: Chemical test : It gives exactly identical reaction with conc. H2SO4 and Frachde’s Reagent as those with salicin.

4.2.3.1Digitalis Preparation : Good quality digitalis is grown specifically from the seeds of selected strains that invariably yield only leafy plants enriched with glycoside contents. Even the soil is usually sterilized by steam before commencement of sowing. Mostly it grows both appreciably and luxuriantly at an altitude ranging between 1600-300 meters preferably in a shady environment. In actual practice, the sowing of seeds is performed in autumn (October/November), and the seedlings are virtually transplanted in the fields in the following springs (March/ April). The leaves are normally handpicked in the afternoon  during August / September in the first and second year, when almost 2/3rd of the flowers have fully bloomed. The leaves collected in the first year are found to contain the highest percentage of glycosides. The basal leaves and the ones located at the top are collected at the end. The discoloured leaves are sorted out and rejected outright. The selected leaves are duly spread on perforated trays (usually a thin bed), the trays are stacked one above the other in a well-closed dark drying shed heated by a stream of hot air maintained strictly at a temperature not more than 60°C. The dried leaves having a mixture content not more than 5% are carefully packed in suitable air-tight containers charged with appropriate dehydrating agents and shipped for export. Chemical Test : A plethora of chemical colour reactions have been evolved to be used as the qualitative tests either for the various glycosides or their corresponding aglycones in the chemical laboratory. However, the exact positions of the respective glycosides or their aglycones may be detected either on the paper charomatograms or on the thin layer chromatographic plates by virtue of the production of specific colours or by exposing the chromatograms under UV light so that the components would be detcted by their fluorescence

4.2.3.3.1 European Squill Preparation : Normally the white squill yields fully grown and healthy bulbs that have a height ranging between 18-20 cm and a diameter varying between 12-15 cm. These bulbs are grown in partially submerged condition in sandy soil in the mediterranean coastal region. The bulbs are usually collected in late August soon after the flowering season. The roots and the thin external scaly layers are removed and discarded. While the central fleshly bulbs are collected separately. These bulbs are then cut into transverse slices and subsequently dried either in sun rays or by artificial heating devices. chemical Test Sciilaren A on interaction with acetic anhydride and H 2SO4, it gives rise to a red co our which changes gradually first to blue and finally to bluish green colour.

4.2.3.3.2 Indian Squill

Preparation : The method of preparation of dried slices of Indian squill is very much alike the European squill. It loses approximately 80% of its weight after sun drying. Description Colour : Yellowish to White Odour : Slight and characteristic Taste : Acrid, bitter and mucilaginous Site : Length = 30-60 cm; Breadth = 3-8 mm Shape : Usually 4 to 8 slices are placed one on the top of other and gives it a curved shape.

4.2.3.4 Strophanthus Preparation : ripe strophanthus fruit comprises of two fully developed follicles each about 30cm broad with tapering at both ends and consisting of a number of seeds. The ripe fruits are collected from the wild plants, the seeds are subsequently separated and freed from their awns. Description Colour : Greyish green to light yellowish brown Odour : Slight unpleasant Taste : Bitter Size : Length 1- 2 cm; Breadth = 3-5 mm; Thickness = 2 mm Shape : Lanceolate to linear-lanceolate, acute at the apex, rounded or blunt at the base Weight : For 100 seeds 3-4 g Specific : On treating with 80% H2SO4 the endosperm exhibits a deep feature Emerald green colour.

Chemical Tests : 1. 2.

3. 4.

Generally the strophanthus glycosides exhibit an emerald green colouration on the addition of sulphuric acid. Dissolve about 0.1g of strophanthin in 5 ml of water and add to it a few drops of ferric chloride solution followed by a 1-2 ml of concentrated sulphuric acid; the appearance of an initial red precipitate that finally turns green within a period of 1 -2 hours. To 50 mg of strophanthin add 5 ml of water, shake and add 2 ml of 2% tannic acid solution, the appearance of a distinct precipitate affirms its presence. It shows positive Baljet Test, Legal Test and Keller Killiani Test (see Section 4.2.3.1).

4.2.4.3.1 Hesperidin Preparation : The glucoside may be isolated by adopting the laid down detailed procedures as in Section4.2.4.2i It is also present in the dried orange peel upto 8% and it occurs in the highest concentration in the white portion of the peel usually termed as albedo.

4.2.5.2.1 Khellol Glucoside Preparation : The annual herb plant usually bears flowers from March to April. The harvesting is carried out when the ripening of first fertilized flowers takes place. The plants are cut and preserved in stacks, preferably in a dry place, whereby all the fruits are ripened.

4.2.5.2.3 Cantarides Beetles Preparation : The fully developed insects, that are brilliant green in apprearance with a distinct us re, are invariably collected in the early morning on a large spread cloth by vigorously shaking the branches of the shrubs. The beetles are sacrificed either by exposing them to the vapours of chloroform, sulphur dioxide and ammonia in a closed chamber or dipping them into vinegar. The dead beetles are dried artificially at a controlled temperature not exceeding 40°C. Description Colour : Brilliant green or Bronze green Odour : Characteristic odour

4.2.6.1 Bitter Almods Chemical Tests he general tests of the cyanogenetic glycosides by means of microchemical reactions in naturally occurring crude drugs are based on their hydrolysis to yield hydrocyanic acid. In fact, there are four speciifc and characteristic reactions to detect the presence of liberated HCN, namely: 1. Ferriferrocyanide Test: Macerate 1 g of the powdered drug with 5 ml of alcoholic KOH (5% w/v) for five minutes. Transfer it to an aqueous solution containing FeSO4 (2.5 %w/v) and FeCl3 (1% vv/v), and maintain at 60-70°C for 10 minutes. Now, transfer the contents to HC1 (20%) when the appearance of a distinct prussian blue colour confirms the presence of HCN. 2. Precipitation of fig from HgNO 3: The reduction of aqueous mercurous nitrate solution (3% v,/v) to metallic Hg by HCN being observed by an instant formation of black metallic Hg in the cells. 3. Grignard Reaction Test: First of all, dip a strip of white filter paper into a solution of

picric acid (1 % w/v in water) drain and then dip into a solution of sodium carbonate (10% w/v in water) and drain. Now, place the crushed and moistened drug material in a small Erlenmeyer flask, and subsequently suspend the strip of the prepared sodium picrate paper above the material and stopper the flask with an air tight cork. Maintain the flask in a warm place for 1 hour when the liberated HCN would turn the sodium picrate paper from its original yellow colour to brick red colour due to the formation of sodium isopurpurate (Grignard's Reaction). 4. Cuprocyanate Test: First of all, saturate pieces of filter paper in a freshly prepared solution of guaic resin dissolved in absolute ethanol and allow them to dry completely in air. Now, carefully moisten a piece of the above paper with a very dilute solution of CuSO4 and place it into contact with a freshly exposed surface of the drug. In case, HCN is generated, it will give rise to a distinct stain on the paper.

4.2.6.2 Wild Cherry Bark Preparation : has been established that the wild cherry bark possesses the highest potency only uring the autumn. Therefore, the bark is mostly collected during this period. As the inner layer of the bark contains a substantial amount of HCN, hence soonafter collection it is necessary to get rid of the inner layer of cork. Consequently, after the removal of cork as well as a portion of the cortex, the exposed surface of the bark exhibiting phloem more or less give rise to an uniform dark brown coloured product, which is commercially known as 'flossed Bark. The resulting rossed bark is dried in the shade and stored carefully in a dry place for onward trasmission to several countries as a valued export material. Description Colour Odour

: Dark-brown colour : Mostly very faint; but when slightly moisten it has an odour resembling to that of benzaldehyde (bitter almond like)

Taste : Bitter and astringent Size : Length = 10 cm; Width = 4 cm; Thickness = 3-4 mm Shap : Mostly curved or chanelled Fracture : Short and granular Inner Surface : Reddish brown and longitudinally striated Outer Surface : `Bossed Bark' - Rough with pale buff coloured lenticel scars; 'Unrossed  Bark' —Reddish brown to brownish black, smooth, glassy and exfoliating cork having prominent whitish lenticels. Chemical Tests The chemical tests are same as described under Section 4.2.6.1.

4.2.6.3 Linseed Preparation : The eyanogenetie glycoside linamarin is prepared from the defatted oil meal, seed- skins or embryos of flax by standard methods available for glycosides. Description Colour : Reddish brown Odour : Characteristic odour Shape : Oval and strongly flattened Size : Length = 4-6 mm; Width = 2-3 mm. Chemical Tests: The mucilage of linseed seed gives a distinct red colour on being treated with Ruthenium Red Solution.

4.2.7.1 Black Mustard Preparation : The thioglycoside sinigrin is obtained from the defatted black- mustard seed by mloying standard methods. It is usually present in the seeds to the extent of 4%. Black mustard seeds are powdered and defatted with petroleum ether. The defatted meal is boiled with ethanol to destroy the enzyme. The resulting marc is squeezed while hot, dried at 100°C and maceraed in cold water for 3-4 hours with constant stirring, since sinigrin is fairly soluble in cold water. The liquid content is decanted and maceration is repeated a number of times to ensure complete extraction of the thioglycoside. The combined aqueous extract is collected and treated with mild alkalies, such as : BaCO3, so as to neutralize any free acidity. The liquid is now concentrated under vaccum to a syrupy consistency. The resulting syrup is boiled with ethanol (95% w/v) for about 2-3 hours to allow sinigrin to dissolve and at the same time to precipitate the mucilageous components. The alcholoic extracts are filtered and allowed to cool slowly when sinigrin crystallizes out (approximately 4%). Description Colour Odour Taste Size Shape

: Black, dark brown or reddish brown : Whole seed-none; Crushed seed-pungent characteristic odour. : Bitter : Approx. 0.9-1.0 mm in diameter : Mostly spherical in shape

Chemical Tests : 1. IffTtie powdered black mustard seeds on being treated with sodium hydroxide solution yields bright yellow colouration 2. Chromatographic Evaluation a) Paper chromatography of the mustard oil in a solvent system consisting of butanol-acetic acid water; and subsequently sprayin g the chromatogram with 0.02

N silver nitrate solution, drying at 100°C and finally spraying with 0.02 N potassium dichromate produces yellow spots a gainst a red background of silver chromate thereby confirming the presence of sinigrin. b) The thiourea derivatives are used as reference compounds along with the mustard oil spots in paper chromatography using the solvent system consisting of water saturated chloroform or butanol-ethanol-water. The chromatogram is sparyed with Grote's Reagent (i.e., a mixture of sodium nitroprusside, hydroxylamine and bromine) which distinctly yields blue spots with thiourea derivatives as well as sinigrin. Preparation : The powdered white mustard seeds are defatted with a suitable solvent (e.g., petroleum ether, n-hexane) and the dried marc is extracted with boiling ethanol (95% v/v). The thioglycoside is purified by dissolving in warm water, decolourised with activated charcoal, filtered and the resulting filtrate is crystallied out.

Chemical Tests: The hydrolysed product of sinalbin e.g.; sinapine acid sulphate and other salts are crystalline and give rise to a distinct bright yellow colouration in an alkaline medium.

4.2.8.1.2 Solanum Khasianum Preparation : The plant usually grows in various climatic and agricultural conditions. Almost after duration of six months the plants are normally harvested for the collection of berries. They are dried immediately either in an artificial environment at low temperature (50-60°C) or dried preferably in shade so as to bring down the initial large moisture content to enable its prolonged storage life. The dried berries are powdered by mechanical grinders and the oil is removed by solvent extraction. The defatted material (marc) is then extracted in a soxhlet assembly with ethanol (95% v/v) The resulting alcoholic extract is filtered, concentrated under vacuo, treated with HC1 (12N) and refluxed for at least six hours. The alcoholic extract thus obtained is made alkaline by the addition of ammonia and the eontents are again refluxed for duration of 1 hour. The contents of the flask is filtered and the residue is washed, dried and taken up in chloroform. The resulting mixture is filtered and the steroidal alkaloid solasodine is obtained as a solid residue soon after evaporating the solvent.

4.2.8.1.3 Shatayari Preparation : the roots usually occur in the form of a cluster or fascicle at the base of the stem. The leaves are mostly linear green and needlelike. The steriodal spanonin is extracted by the standard methods.

4.2.8.2.1 Ginseng Preparation : The plants are usually harvested 3 to 5 years after transplantation. It is usual practice to affect the actual harvesting between July to October.

White Ginseng : It is obtained by removing the outer layers of the roots. However, it has been e that the removal of outer layers may tantamount to serious loss of the active components. Red Ginseng : it is obtained by first subjecting the roots to steaming and after that they are dried in environment between 50-60°C. The two types of roots sare subsequently graded and packed.

4.2.8.2.2 Liquorice : Preparation : The roots are usually harvested after 3 to 4 years from it s plantation when they mostly display enough growth. The rhizomes and roots are normally harvested in the month of October, particularly from all such plants that have not yet borne the fruits. There by ascertaining maximum sweetness of the sap. The rootlets and buds are removed manually and the drug is washed with running water. The drug is first dried under the sun and subsequently under the shade till it loses almost 50% of its initial weight. The large thick roots of the Russian Liquorice are usually peeled before drying. It is an usual practice in Turkey, Spain and Israel to extract a substantial quantity of the drug with water, the resulting liquid is filtered and evaporated under vacuo and the concentrated extract is molded either into sticks or other suitable forms.

4.2.8.2.3 Senega Preparation : The root is collected from the wild plants normally in summer. The stems are promptly and the roots are sorted out, washed thoroughly and dried either in the shade or artificial environment between 50-60°C. Chemical Tests : When sulphuric acid (80%) is added to a thick section of the drug or powder, it instantly produced a deep yellow colour.

4.2.8.2.5 Quillaja Preparation : The bark is collected from the trunk of wild plants. Careful incisions are made on the turn an ask is stripped off. The bark is freed from the outer dark coloured cork, cut into small pieces, dried, graded and packed in polybags.

4.2.9.1 Vanilla Preparation : The full grown and unripe fruits are hand picked at that particular stage when their colour Changes from green to yellow. These fruits are allowed to undergo fermentation whereby a characteristic flavour and aroma develops gradually. However, the very important and critical process of fermentation essentially consists of slow drying in shades at a controlled temperature. Fermentation, in fact, helps in the conversion of vanilloside to vanillin and glucose respectively.

4.2.10.1 Picrorhiza Preparation : The plant is either propogated by seeds or by rhizomes. The rhizomes are collected le cultivated and naturally growing plants washed dried and packed.

4.2.10.2 Gentian Preparation : The long rhizomes and fully grown fleshly roots of 2 to 5 year aged plants are dug up carefully and collected preferably in autumn. The roots and rhizomes are washed thoroughly to get rid of the adhered soil and then sliced into a longitudianal fashion. The freshly sliced pieces of roots and rhizomes generally appear white in colour and do not have any odour. However, during the process of gradual drying in small heaps at a controlled temperature of 50-60°C fermentation commences which eventually turns them into dark or yellow coloured product that have a characteristc odour.

4.2.10.3 Chirata Preparation : The plant usually flowers from July to October. It is collected for medicinal utilities as and when the capsuls are fully formed. The dried plants are tied into bundles weighting approximately 1-1.25 kg.

RESINS Capsaicin

Isolation : Dried, ripe fruits of capsicum are coarsely powdered lor ciie extraction of oleo-resin. It is extracted with hot acetone or alcohol (90%) t he 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

.

Test : The oleo-resin 1mg/ml is dissolved in alcohol and spoiled on silica gel-G plate. The plate is eluted in the solvent system containing mixture of benzene-methanol (9.1). Spray the dried plate with 0.5% solution of 2,6-dibromoquinone-chlorimide in methanol and allow to stand in a chamber containing ammonia fumes. Blue colour and the H value 0.31 of the principal spot corresponds to the spots of the .standard solutions.

Curcumins Properties : Curcumin occurs as an orange yellow, crystalline powder. It is insoluble in water and ether but soluble in alcohol and glacial acetic acid. It gives brownish red colour with alkalies and a light-yellow colour with acids. Curcumin shows wound healing, anti-inflammatory, antiarthritic and antimicrobial properties. It is used against peptic ulcer and dyspepsia. Isolation : can be obtained by different processes. Turmeric ponder 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 seperates. The mass is stripped off kerosene with petroleum ether and recrystallizcd from ethanol. The final product obtained is recrystallized from hot ethanol to yield orange-red needles.

Test : Dissolve 1mg of curcumin in 1ml 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 visualized.

Podophyllotoxin

Isolation : Podophyllum resin is extracted from dried rhizomes and roots by percolation with alcohol and subsequent precipitation from the concentrated percolate by addition of acidiied water. It contains not less than 40% of podophyllum resin and not more than 50% of hcxane insoluble matter.

The powdered, dried rhizomes and roots of podophyllum are extracted with 90% ethanol in soxhlet extractor. The alcoholic extract is concentrated to syrupy consistency by removing alcohol with distilation. A mixture of water containing about 2% hydrochloric acid is poured into syrupy extract at 5°C with continuous stirring. The mixture is allowed to stand for tvyo hours below 5°C and then iltered under vacuum. Wash the residue with acidified water cooled below 5°C. Residue is dissolved in suficient quantity of 90% hot alcohol, iltered and alcohol is distilled off. The residue is dried further at 80 C to constant weight. Test : Dissolve podophyllum resin and podophyllotoxin seperately in methanol. Apply the spots over silica gel-G plate and elute it irst with chloroform-inethanol (90:10), 6cm. Only the glycosides are seperated but aglycone like podophyllotoxin remains in the region of the front. The same TLC plate is again eluted with more weakly polar solvent chloroform-acetone (65:35), this time upto 12cm. During the second stage elution-glycosides do not migrate but aglycone move. Spray the dried plate with sulphuric acid-acetic anhydride (1:3) followed by heating at 100°C for 15 minutes. The zones and spots of glycoside: and aglycone corresponds to their standards. Podophyllotoxin gives the R f value 0.65.

Gingerols and Shogaols 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 Alteration and the residue obtained is dried under vacuum. In some cases the suspended oleoresin is extracted with solvent ether and the ether extract is evaporated to dryness at low temperature to yield total ginger oleoresin.

Test : (1) Dissolve extract or gingerol in alcohol. Apply the spots over silica gel-g and elute in the solvent system ether-n-haxane (7:3). Spray the dried TLC plate with 1% vanillin-sulphuric acid and heat the plate at 110 OC for 10 minutes. Spots due to gingerols occur at Rf value 0.2. Identification : Add about 5ml of 70% sulphuric acid and 5 mg of vanillin to the small quantity of ginger oleoresin. Allow to stand for 15 minutes and then add equal voumes of water, the solution obtained turns azure blue indicating the presence of gingerol.

BENZOIN 1. Mode of Tapping the Trees Tapping commences when the trees are about 7 years old, and although growth is retarded, the trees continue to yield benzoin for upwards off 20 years. By means of a knife a small triangular wound is made to expose the wood. The wounds are made about 40 cm. apart in a vertical row — three rows being spaced equally around the stem. About two months later the strips of bark vertically between the wounds are removed, and in each row four new wounds are made, three about 4 cm. above the original ones and the fourth about 40 cm. above the highest. This process is repeated four times a year until the rows are practically continuous where upon thre e new rows are star ted bet been the old.

2. Products of Tapping The resinous exudation following the first incisions is yellowish and very sticky, but about 2 months later it becomes rather hard. It is then scraped off together with the strips off bark between the wounds, and the next series of incisions made. Subsequent exudation is considerably different in appearance and composition, being milky-white instead of yellowish. In its downward course it hardens on the denuded area, where there is gradually built up a thick incrustation of many layers the outer surface undergoes oxidation, and rapidly assumes a pale yellow colour. The dried inclustation is removed in three stages: (a) About 6 weeks from wounding, the outer layer is carefully cut off. It consists of fine white pieces, and is graded as first quality. (b) Two weeks later the remaining and lager portion of the incrustation is almost completely cut off. This portion contains white tears embedded in a brownish matrix, and becomes the second quality. (c)

About 4 weeks later, i.e., just before the next series of wounds are to be made, the area is scraped, yielding a few white tears mixed with much wood debris and brown matrix. This constitutes a third quality. The total annual yield per tree is 2.5 to 7.5 kilograms. The formation of benzo5in, i.e., the white exudation, is attributable not only to wounding, but also to wound infection by fungi. Doubtless the resin produced initially (and probably the hard transparent matrix) is caused by wounding, space but the subsequent product is the outcome of fungal invasion. None of the above three qualities is exported alone the benzoin of commerce being an admixture in varying proportions, prepared in following manner: The material is chopped into small pieces, which produces the white almond-shaped tears, and then stamped into tins or cotton-lined boxes. These open containers are exposed to the sun, the contents soften, and unite to form a hard brittle mass. Chemical tests

1. Coarsely powder about 0.5 g. of Sumatra benzoin, and warm gently in a test-tube with 10 ml. of a 1 per cent solution of potassium permanganate. Notice the development of the odour of benzaldehyde, formed by oxidation of the cinnamic acid. Repeat the experiment, using Siam Benzoin. In this case the percentage of cinnamic acid is small (about I per cent), and under the condition of the test does not yield sufficient benzaldehyde to be perceptible. 2. Coarsely powder about 0.2 g. of Sumatra benzoin and digest in 5 ml. of ether for about 5 min.; pour 1 ml. of the ethereal solution into a porcelain dish containing two or three drops of sulphuric acid; rotate and observe the development of a reddish-brown colour without any marked purplish tinge. Repeat the experiment with Siam benzoin, and notice the deep purplish-red colour. 3. Ferric chloride added to an alcoholic solution of Siamese benzoin, produced green colour. Sumatra benzoin gives negative test for this reaction.

COLOPHONY Collection and Preparation Description : Colophony usually occurs in amber or yellow colour to almost black colour. Light coloured resins are used medicinally and oftenly occurs as shiny, glassy masses with pieces are sharply angled conchoidal surfaces. Angular fragments are translucent, brittle, viterous and covered with yellowish dust. The pieces are sharply angled with conchoidal surfaces. The odour and taste are fainty terebinthinate. On heating it softens at 80°C and fuses at 100°C. The alcoholic solution of colophony becomes white when added to water. Identification tests 1) 0.1 g. powder dissolved in 10 ml. of acetic anhydride, added to one drop of sulphuric acid yields purple colour rapidly changing to violet and finally brown. 2) Treat small amount of colophony powders with light petroleum and filter. To the fi Iterate add twice its volume of dilute copper acetate solution. The petroleum layer becomes emerald-green, due to he formation of copper salt of abietic acid. 3) Shake about 0.1 g. of freshly-powdered colophony with 10 ml. of light petroleum, filter, and 5 ml. of the filtrate add 10 ml. of dilute solution of cupric acetate, shake for several minutes. On separating into two layers the upper (light petroleum) is bright bluish-green in colour.

GUAICUM RESIN Collection and preparation : The resin is collected from the trunk by crudeway. The dark-coloured heartwood of the tree contains 20-25 per cent of resin the almost-white sapwood none. The resin is not obtained from the living tree, nor is solvent extraction usually employed owing to the very hard character of the wood, which renders preliminary comminution very difficult. Crude separation is effected by setting fire to the log and collecting the resin which melts and runs out. Sometimes both ends of the horizontally-supported log are set on fire, and the melted resin escapes at deep transverse incisions made about midwa y. Alterna tively a deep groo ve is made throughout the full length, the tree is supported in an inclined position, and upon set ting fire t o the upper end the melted resin flows down to form large blocks.

Chemical tests 1. Add a drop of dilute ferric chloride solution to alcoholic resin solution, which results in the f ormation of deep blue colour. This deep blue colour is destroyed by reducing agents and restored by oxidising agents. 2. 2Adulteration with colophony is occasionally practised, and may be detected by

the cupric acetate test. Digest about I g. of the powdered sample in 5 ml. of light petroleum spirit for 3 or 4 min., and filter. Guaiacum resin is almost insoluble in this medium; freshly-powdered colophony is entirely, and old powder largely, soluble. On treating the filtrate with the cupric acetate reagent a green colour is formed if colophony is present. Formation, Collection and Preparation : After mating the wingless fecundated female, become attached to twins mentioned trees permanently by their proboscides. They increase in size and secrete a resinous matter. Artificially infection is produced by removing twigs with g ravid f emales and attachin g them to suitable t rees. The encrusted twigs about 7 mm are collected during May-July and October-November and dried in Sun, to yield stick-lac. The resinous crust is broken from the twigs by means of knife and colourin g , matter is extracted with water or dilute soda. The extracted solution is dried off to yield residue, that are pressed into cakes, known as lac-dye. The exhausted resin brownish in colour is known as seed-lac, which are latter melted in a long sausage-shaped bag suspended over a charcoal fire and the lac is squeezed off. The molten resin is poured into moulds to form thin sheets, which are broken up form shellac. Bleached shellac is formed by dissolving lac in hot soda solution, bleaching with chlorine or sulphurous acid, precipitating with acid, followed by washing and pulling to form sticks under water.

ASAFOETIDA Colle ctio n and Preparation : The plant possesses large schizogneous duct and lysigenous cavities containing a milky liquid, which upon exudation and drying constitutes asafoetida. These ducts and cavities are numerous in the cortex of bot h ro ot and stem , an d alth ough bo th yiel d asafoetida, the drug is obtained chiefly from the stem. Collection is effected by incising the stem, collecting the milky exudation in skins, and drying it to a paste by exposure to the sun. Each stem is incised three or four times at intervals during the summer months, the average yield being about 600 grams of asafoetida per plant. From the root, collection is made by exposing its crown and excising the stem whereupon the milky liquid wells out on the cut surfaces of the root, where it dries and hardens. This is scraped off and a fresh surface made by cutting off a transverse slice or root; removal of the exudation and exposure of fresh surface proceeds until the root is exhausted. The yield is usually soft enough to agglomerate into masses when packed.

MYRRH Collection : The small trees which yield myrrh contain in the phloem parenchyma, closely approximated ducts containing ducts contained a yellowish granular liquid. The tissue between these ducts often collapses, thereby producing large cavities similarly filled, i.e., schizogenous ducts become lysigenous cavities. Collection is effected by incision into the bark, whereupon the secretion of the ducts and cavities exudes, hardens, and dries at the surface, at the same time changing in colour to reddish brown. Chemical tests : Thoroughly triturate about 0-5 g. of official myrrh with 1 g. of sand in moderate y powder, and shake with 10 ml. of ether. Filter, divide the filtrate into two portions, and evaporate each in a porcelain dish. The residues consist of volatile oil and the ether-soluble portion of the resin. Pour the vapour from a bottle of bromine over the film in one of the dishes and observe the dark violet colour. Invert the ether dish over a similar-sized dish containing a few drops of fumings nitric acid. The myrrh film acquires a violet colour. Fadhli Myrrh gives a positive, yemen usually a negative and bedelliums always a negative result with test.

COPAIBA Collection and preparation : The physical and chemical characters of the oleo-resin vary, not only with the species but also according to the soil, environment, age and health of the tree and the method and intensity of the tapping. Copaiba of any single botanical or geographical origin is never exported but various copaibas are mixed in appropriate proportions to give a product meeting the standard requirements. The chief requirements considered are specific gravity, viscosity, percentage of volatile oil, colour and acid value. The oleo-resin is formed in schizolysigenous cavities in the wood and seems to be a metamorphosed product of the cell walls. The copaiba trees, which often attain 100 metres in height, normally secrete oleo-resin. In each zone of secondary wood there is an extensive system of oleo-resin ducts connected to those of adjacent zones by lateral ducts, which are found only where branches of the tree arise. The oleoresin ducts are formed normally in the young wood by separation of rows of cells to form a passage, but later large irregular cavities, also containing oleo-resin, are formed by complete absorption of cells. The trees are tapped or boxed at the centre close to the base of the trunk and the oleo-resin collected directly to the containers, similar to the collection of turpentine.

Tests : Dissolve 5 drops in 10 ml. of glacial acetic acid, and add about 5 drops of nitric acid. Genuine Copaiba remain clear, or becomes only slightly cloudy. Gurjun balsam yields a deep purple colour.

BALSAM OF PERU Collection and Preparation : Ducts present in young twigs disappears as it grows into tree and it seems probable that injury leads to the formation of new wood containing oleo-resin ducts, from which baIsam is exuded over the injured area. Balsam is a pure pathological product and injury to the tree is effected as described below. About December, at the conclusion of the rainy season, areas of bark about 30 cm. long and 15 cm. wide are beaten with a mallet, hammer, or stick. About a week later the beaten areas are scorched with torches, and after a similar interval the scorched outer layer is detached, if it has not already peeled off. These areas are then covered with rages (or rags inserted between the loose bark and the wood), which absorb the oleo-resin now freely exuding. From time to time the rags are renewed, and the process repeated for several weeks. The balsam is removed from the saturated rags by gentle boiling with water, whereupon the • balsam separates as a layer at the bottom. The balsam is separated by decanting the water, and then allowed to stand in gourds until the mechanically-mixed water separates, and is then transferred to tins for export. Inferior balsam is also obtained by boiling the separated bark with water, followed by method as described above.

Chemical tests 1. Light Petroleum Test : Shake 3 g. of balsam with 15 ml. of light petroleum (b.p. below 40°); the balsam partly dissolves the undissolved portion clings as a sticky mass to the sides of the tube, and no insoluble powder sinks to the bottom. 2. Odour of the Light Petroleum Extract :  Filter if necessary the solution from (1) and evapate 6 ml. in a small dish on a water-bath; the residue has no odour of benzaldehyde or of oil of turpentine. 3. Acelic Anhydride Test :  Dissolve 3 drops of residue from (2) in 10 drops of acetic anhytiride, and add 2 drops of sulphuric acid; genuine drug gives a greenish-brown to green colour, but not reddish-violet of bluish-violet. The test detects the presence of colophony, copaiba, gurjun balsam, Canada turpentine or storax. 4. Nitric acid Test: To 4 drops of the residue in the dish (2) add 2 drops of nitric acid; a yellow colour develops round the drops of acid, but there is no green or yellowish-green (fixed oils); purplish-red (gurjun balsam) or bluish-green colour (balsam of Tolu, Canada turpentine, copaiba, or colophorty).

RESIN AND RESIN COMBINATIONS

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BALSAM OF TOLU Collection and Preparation : schizogenous ducts are absent in the tree except young twigs. This balsam is a pathological pioduct secreted by oleo-resin duct in the new wood formed as a result of injury. Each tree is tapped at numerous points by making wide V-shaped incisions in the bark; the balsam which exudes collects in gourds or calabash cups fixed at the base of the incisions. From these receptacles the balsams transferred to hide bags for transport to the depots, where it is packed in tins for export. Tests : (a)Test for identity : Boil 1 g. with 5 ml. of water, fi lter and note the odour of the filtrate. 3 ml. of solution of potassium permanganate (1 per cent), warm and observe the odour of benzaldehyde formed by oxidation of the cinnamic acid dissolved by the boiling water. (b) Acidity estimation and alcohol insoluble matter  (1) Digest about 1 g. in 10 ml. of 'alcohol (90 per cent) with frequent shaking. The insoluble residue should be small (official maximum 5.0 per cent). Test the solution with litmus and observe the acid reaction caused by the free cinnamic acid. (2) To the-alcoholic solution from (a) add a few drops of test solution of ferric chloride; a green colour` is produced. (C) Test for colophony as adulterant : Sophistication of the drug is effected by removal of the cinnamic acid or by admixture with colophony. The former may be indicated by the absence of crystals upon lens examination, but quantitative estimation is the only reliable method.

PREPARED STORAX Collection : Storax is prepared by bruising ur pun::1,tring of intervening tissues from schiz'tysigenous cavities, results in the exudation of balsam in to the wounded bark. Storax is a pathological product secreted by oleo-resin ducts in the new wood formed as a result of injury. In April, three- or four-year old trees in full leaf are prepared by removing strips of the bark and outer sapwood from opposite sides of the trunk, thereby inducing formation of balsam in inner wood. A few weeks later the exposed surfaces are scraped with a knife having a small curved blade, and the process repeated each week for a month and then at increasingly frequent intervals until the onset of the rainy season in November. By this time a considerable quantity of the wood has been removed, but by resting the tree for three or four years it can again be worked in a similar manner. The day's scrapings from the trees are each evening boiled with water and then filtered through coarse cloth bags by the aid of a primitive press, to remove most of the vegetable debris. The resin and water which exude from the press are collected in tubs, where the balsam, rising to the surface, is separated. This crude storax contains small fragments of xylem and bark and about 25 to 30 per cent of water. It purified by solution in hot alcohol, filtration and evaporation of the solvent with gentle heat to minimize loss of the volatile components. The product constitutes prepared or purified storax, which is the official article.

Abietic Acid Prepration : aration 4t is a widely available organic acid, prepared by the isomerization of rosin. It may also be synthesized from dehydroabietic acid. The commercial grade of abietic acid is normally obtained by heating either, rosin alone or with mineral acids. The product thus achieved may be glassy or partly crystalline in nature. It is usually of yellow colour and has a mp 85°C i.e,. much lower than the pure product (mp 172-175°C). Identification : It readily forms the corresponding methyl ester as methyl abietate (C21 H32 0 2), which is colourless to yellow thick liquid bp360-365°C, D20 1.040, and n20D 1.530.