Phytopharmaceuticals - By Dr.U.Srinivasa, Professor and Head, Srinivas college of pharmacy, Mangalore, Karnataka
– A LS IC UT ACE A RM PH TO NE SA HY INI NIVAP U RI Q .U.S DR
QUININE It is a quinoline alkaloid of cinchona bark.The other important alkaloids of this drugare quinidine, cinchonine, cinchonidine,cinchonamine etc., B.S. It consists of dried inner bark ofC.Calisaya, C.succirubra, C.officinalis,C.ledgeriana and hybrids of this. Family –Rubiaceae
Quinine and quinidine are stereo-isomers .Quinine is laevorotatory and quinidine is dextrorotatoryUses :Quinine is antimalarialQuinidine is a cardiac depressant therefore used in cardiac arrythmias.
ISOLATION1. The dry powder bark material is first well mixed with about 30% of its weight of calcium hydroxide or calcium oxide and sufficient quantity of sodium hydroxide solution to make a paste. It is allowed to stand for few hours.2. The mass is then transferred to a Soxhlet apparatus and extraction is carried out with benzene.
3.Subsequently the benzene extract is shaken with successive portions of 5% sulphuric acid.4.The aqueous acid extract is adjusted the pH 6.5 with dilute sodium hydroxide, cool. Crystals of neutral quinine sulphate are formed.5.These crystals are freed from cinchonine and cinchonidine by repeated recrystallization from hot water.
6. Colouring matter is removed by activatedcharcoal.7.Quinine sulphate crystals are dissolved indil sulphuric acid and made alkaline withammonia. Initially amorphous quinine isformed , which becomes crystalline . 8. Finally washed to remove sodium andammonium salts and dried to 45- 55 o C.
IDENTIFICATION1. Chemical tests2. Thin layer chromatography
T.L.C:Adsorbent : Silica gel -GMobile phase – Chloroform – diethylamine (9:1)Detection – Spray with methanolic KOH ; Sulphuric acid reagent or UV Light
AL– IC UT A CE RM HA E A OP RIN VAS YT D INIPH HE .SR EP .U R D
EPHEDRINEIt is an amino alkaloid ( Non –heterocyclic,proto-alkaloid or a typical alkaloid )present in various species of ephedra. Chemically , it is 1-phenyl,1- hydroxy,-2-methyl amino propane.
• B.S It consists of the dried aerial parts of ephedra sinica, ephedra nebrodensis and ephedra geridiana Family : Ephedraceae• The other important species are ephedra intermedia, ephedra major, ephedra alata.
USES :Ephedrine is a bronchodilator and useful inthe treatment of asthma and allergicconditions such as rhinitis, fever etc.Used as mydriatic
ISOLATION :1.Ephedra powder is first made alkaline with sodium carbonate and then macerated with benzene overnight.2.Benzene layer is separated by filtration and treated with Dilute HCL3.The aqueous acid layer is separated and made alkaline with solid potassium carbonate.
4. Then extracted with chloroform5.The chloroform extract is evaporated to dryness to get the residue. This is mixed with oxalic acid and warmed.6. Alkaloidal oxalates are formed (ephedrine oxalate and pseudo ephedrine oxalate)
• Ephedrine oxalate is less soluble in cold water , on cooling crystals of ephedrine oxalate is formed.• Pseudo-ephedrine oxalate is remains in solution• 7. ephedrine is liberated from its oxalate by shaking with alkali solution and then extracted with a mixture of chloroform and ether (1:3) to get ephedrine.
• Identification :• 1. A slightly alkaline solution of ephedrine on warming with Ninhydrine solution forms violet color.• 2. Ephedrine gives pink to red coloration with con.sulphuric acid and 3 to 4 drops of 40% formaldehyde solution.
• 3. Dissolve ephedrine in water and add dil Hcl and test separately with 10% copper sulphate solution and add 20% sodium hydroxide solution. A purple or voilet colour developed.• This is the ether extractable .On shaking with ether the organic layer is purple but aqueous layer is blue
• ESTIMATION :• 1 . Titration methods :• A. Back titration method• B. Non –aqueous titration method• 2. Colorimetry
1.Back titration method (I.P.1966) Principle : Ephedrine can be estimated byback titration method. In the procedure , the accurately weighedsample is treated with the known excessquantity of acid and the excess acid is backtitrated with alkali.
• Procedure :• Weigh accurately about 0.5 gms of sample and dissolve in 5ml of ethanol (95%). Add 50ml of 0.1M Hcl and titrate with 0.1M NaoH, using methyl red as indicator until a yellow colour is produced.
• 2. Non –aqueous titration• Procedure :• Weigh accurately about 0.17 gms of sample and dissolve in 10ml of Mercuric acetate solution, by warming gently . Add 50 ml of Acetone and titrating with 0.1M Perchloric acid , determining the end point by Potentiometrically
• 3. By Colorimetry :• PRINCIPLE :• Being a secondary amine , ephedrine forms a colored complex (400nm ) with Picryl chloride in benzene
• Procedure :• Take 1,2,3,4,5 and 6ml of 0.01% solution of pure ephedrine in benzene into test tubes and adjust the volume to 90ml with benzene.• Add 1ml of 0.3% solution of Picryl chloride to each test tube and close tightly for 2 minutes for exactly.
• Place them in a water bath at 75-77 o C for 20 minutes.• Remove the tubes from the water bath and allowed to stand for 3 minutes and measure the absorbance at 400nm against the blank prepared by the same procedure by taking 90ml of benzene and 1ml of 0.3% solution of Picryl chloride.
• Estimation of the sample :• The isolated ephedrine sample is prepared in benzene and treated with 1ml of 0.3% solution of picryl chloride its absorbance is measured by keeping in the water bath at 75-77 o C for 20 min. and then allowing to stand for 3 min at room temperature.
• Calculation:• The concentration of the ephedrine in the sample is obtained by interpolation of the standard curve
L – TICA .D PH EU E R M , AC LID H A RM HO M . P HA AP A , OP GR VA S YT RO I N IPH D S R AN R . U . D
ANDROGRAPHOLIDE• It is the bitter principle of Kalmegh. It is chemically a bicyclic diterpenoid lactone .• Neo- andrograpolide and andrographisside are the other important active constituents of the plant
• Biological source : Kalmegh consists of dried leaves of the plant known as Andrographis paniculata• Family : Acanthaceae• Uses : Hepatoprotective , bitter tonic
ISOLATION• PRINCIPLE :• Isolation is based on solubility of andrographolide, it is soluble in methanol and ethanol
PROCEDURE• 1.Extract the dried coarse powder successively with petroleum ether (60- 80 o C) ,chloroform and methanol• 2. Collect the methanol extract and concentrate and treat his with charcoal for 24hrs
• 3.Filter and collect the filtrate , keep this filtrate for overnight for crystallization• 4.Then reflux the recovered charcoal with methanol , filter and add filtrate to the mother liquor . Keep it for overnight for crystallization.
• 5.Purify the combined crystals from methanol to get andrographolide ( M.P 228-229 o C )
IDENTIFICATION• Chemical test :• Sample and few drops of solution of copper acetate gives emerald green color• By T.L.C :• Adsorbent – Silica gel G• Mobile phase –Chloroform: Methanol (7:1)
• Reference standard- Andrographolide 1mg in 1.5ml of methanol• Test sample – Extract in methanol• Detecting reagent –• 20% sulphuric acid in methanol, heat at 120 o C for 10 minutes
• Rf value of Andrographolide – 0.70, brown color spot.• ESTIMATION METHODS :• 1.Gravimetric method• 2.Colorimetric method• 3.High Performance liquid chromatography (HPLC)
• 1.Gravimetric method :• Known weight of the drug is taken, the active constituents are isolated, dried to a constant weight , the weight is determined .• 2.Colorimetric method :• Andrographolide gives red color with alcoholic KOH, which is measured at 400- 800 nm
• 3.High Performance Liquid Chromatography (HPLC)• Column – 5µm Spherical silica gel (3mm X 15cm)• Mobile phase – Chloroform – Methanol (9:1)• Flow rate – 0.7 ml/ minutes• Detector – UV at 254nm• Standard preparation – Known
• Sample preparation –• Drug extracted with methanol, evaporated and residue dissolved in methanol ( 50µg/ml )• PROCEDURE : 10µg/ml of standard and test preparations are subjected to HPLC , record the chromatogram and calculate the percentage (%) of andrographolide from the respective peak areas.
A L– IC UT H. D A CE M A ,P A RM .P H AR PH ,M TO Y IN N I VA SAPH UT S R I R . U D R.
RUTIN• There are around 200 types of Quercetin, Flavanoid glycosides, among this the rutin is the one of most important type. It is chemically Quercetin-3- rutinoside . On hydrolysis , it yields the aglycone quercetin and the sugars glucose and rhamnose.
SOURCES OF RUTIN• 1. Buck wheat ( Fagophyrum esculentum- Family – Polygonaceae )• 2. Rhubarb – ( Rheum emodi- Family - Polygonaceae )• 3.Tobacco – (Nicotiana tobaccum – Family – Solanaceae )• 4. Ruta – (Ruta graveolens – Family – Rutaceae )• 5. Tea – Thea sinensis – Family – Theaceae )• 6. Eucalyptus macroryncha ( Myrataceae )
ISOLATION• Source -• Eucalyptus macroryncha ( Myrataceae )• Boil the powder drug with boiling water . Filter while hot and collect the filtrate . Cool for the precipitation of the rutin. Recrystallize it from boiling water , dry the product.
IDENTIFICATIONPaper chromatography -Solvent system –n- butanol- acetic acid – water - 5% acetic acidDetection – Visible, UV light Standard reference – RutinSample – Extract powdered drug with 70% alcohol Extract fresh drug with 90% alcohol
ESTIMATION :• By colorimetry :• Standard solution – Rutin in alcohol ( 100µg/ml)• Test sample – weigh sample equivalent to 10mg rutin and add 80ml of ethanol. Boil on water bath and cool to room temperature .make up the volume to 100ml with ethanol.
• PROCEDURE :• Take 1,2,3,4,5 and 6ml of solution of pure rutin in alcohol into test tubes and add alcoholic aluminium chloride, acetic acid and 1N Hcl adjust the volume to 25ml with distilled water. Measure the absorbance of the sample against sample ,blank and standard at 420nm. Calculate the amount by comparison.
L – TICA C EU P H. D A A. M RM AR IN . P H A PH TH A , M TO AN VA SPHY YLL I N I H .U.SR P R D
PHYLLANTHIN• Phyllanthin and Hypophyllanthin are the two major constituents of the drug phyllanthus. They are Lignans .• Chemically , Phyllanthin is a diaryl butane, where as hypophyllanthin is an aryl tetra- hydronaphthalein
• Lignans are a group of chemical compounds found in plants .Lignans are one of the major classes of phyto- estrogens, which are estrogen like chemicals and acts as antioxidants.• The other classes of phyto-estrogens are the isoflavones, they are polyphenolic in nature.
• Biological source : It consists of the aerial parts of the plant Phyllanthus amarus• Family – Euphorbiaceae• Uses :• Phyllanthin and Hypophyllanthin are reported to have Hepatoprotective activity.
• The drug (Phyllanthus) exhibits antiviral activity , particularly on Hepatitis B in humans• It exhibits Diuretic and Hypotensive effects in humans .• The plant possesses antibacterial and antifungal activities.
ISOLATION• PROCEDURE :• 1.Mix the powder drug with lime water , It is allowed to stand for overnight .• 2.The mass is then transferred to a Soxhlet apparatus and extraction is carried out with petroleum ether.
• 3.Collect the petroleum ether extract and concentrate under reduced pressure.• 4. Mix the extract with methanol and boil , collect the dewaxed methanol extract , evaporate to dryness, collect the residue.• 5. Dissolve the residue in petroleum ether , concentrate and allow to stand (Yellow oil gets separated)
• 6. The residue is subjected to column chromatography on Alumina and elute with n- hexane: ethyl acetate (99:1)• 7. 99- 108 fractions corresponds to hypophyllanthin and 109- 137 fractions corresponds to phyllanthin• Subject them to chromatography further to yield pure phyllanthin and hypophyllanthin
IDENTIFICATION• BY T.LC :• Adsorbent – Silica gel GF254• Solvent system – n- hexane: ethyl acetate (2:1)• Detection – Vanillin in sulphuric acid reagent• Rf value – Phyllanthin – 0.20 ( Blue spot)• Hypophyllanthin – 0.25
ESTIMATION :• By HPLC :• Column - µ- Bondapak-18 ( 3.9mm X 30cm)• Mobile phase – Methanol – Water (66: 34)• Flow rate – 1.8ml/Minute• Detection – UV at 230nm
• Standard – Known concentration (0.05- 2µg)• Sample –• Macerate 1gm of the drug powder with lime water at room temperature for 18hrs .Reflux with 30ml methanol containing 3% KOH for 1hr .cool and filter .collect the filtrate• Reflux mark with methanol containing 3% KOH . Filter and collect the filtrate . Combine the extracts and concentrate under reduced pressure. Make up to 50ml.
• Sampling –• Apply 10µl of both standard and sample solutions• Determination –• Note the peak areas corresponding to phyllanthin and hypophyllanthin in both standard and samples and calculate their percentages accordingly.
- C AL HD U TI A ,P ACE RM HA ARM .P ,M PH N A S A TO XI N I V HY ITO R IP IG . S D R.U D
DIGITOXIN It is the primary active constituent ofDigitalis purpurea Family – Scrophulariaceae It is acardiac glycoside exert highly specificand powerful action on cardiac muscle.They make the heart to function moreefficiently.
• Thus they are used therapeutically to strengthens the weakened heart. as the aglycone part of these glycosides is the steroidal moiety, they are also called steroidal glycosides.
ISOLATION• 1.Macerate powdered drug with water at 45 o C for 4- 5 hrs and collect aqueous extract.• 2. Macerate mark with 20% methanol for 24hrs and collect methanol extract.• 3. Combine aqueous and methanol extracts
• 4.Make alkaline the above extract with sodium hydroxide solution ( Hydrolysis).• 5.Extract with chloroform.• 6.Collect the chloroform extract and evaporate to dryness.• 7.Subject the residue to column chromatography for isolation of digitoxin
Synopsis of column chromatography – Adsorbent – Silica gel GSolvent system –Follow gradient technique , initially with carbon tetrachloride, followed by ethyl acetate and methanol.
• Carbon tetrachloride fraction : Colouring matter (Pigments)• Ethyl acetate fraction : Flavones and anthraquinones• Methanol fraction : Digitoxin ( Rf value- 0.486)• Digitoxin is purified by recrystallization from alcohol and diethyl ether (1:1) mixture
IDENTIFICATION• 1. Legal test – Dissolve the extract in pyridine and add sodium nitroprusside solution – Pink to red colouration on making alkaline.• 2. Baljet test – Extract with few drops of sodium picrate solution – Yellow to orange colour
• 3.Keddes test –• Sample with few drops of 3,5- dinitrobenzoic acid in methanol and few drops of potassium hydroxide solution – Reddish / bluish colour.• 4. Raymonds test –• Sample with few drops of dinitrobenzene and few drops of methanol potassium hydroxide solution – Bluish voilet colour.
ESTIMATION• By Spectroscopic method• By Bioassay• By Spectroscopic method – ( I.P .1966)• Weigh accurately about 40mg of digitoxin and dissolve in 100 ml of ethanol. Dilute 5ml of this solution to 100ml with ethanol. Add 3ml of alkaline picric acid solution to 5ml of this diluted solution.
• Allow to stand for 30 minutes and measure its absorbance against blank at 495nm
• By Bioassay method-• Digitalis and its preparations can be biologically assayed for their potency.• Principle –• Comparison of the effect of a known dilution of the drug with that of the similar dilution of standard preparation forms the basis of bioassay.
• Test animals –• I.P. Suggests the use of both pigeons and guinea pigs.• Procedure –• Adult guinea pig are anaesthetized lightly with ether. The jugular vein of immobilized guinea pig is exposed and cannulated. Definite volumes of diluted preparations are introduced at 5 minutes of interval until it dies from cardiac arrest.
A L T IC C EU A M AR N H. D H I P N RMA,MP HI L, P T O G E , M.PHA Y S H IOP D .P HAR M ,D SA - RI NI VA .S .U DR
DIOSGENINIDENTIFICATION – By thin layer chromatographySynopsis –Adsorbent – Silica gel 60Solvent system – Toluene : Ethyl acetate ( 7:3)Reference standard – 1mg/ml Diosgenin in chloroform
• Preparation sample –• Reflux the powder with 50ml of 10% hydrochloric acid for 2hrs , collect the residue. Wash the residue with dilute sodium carbonate solution , collect the residue. Extract the residue with solvent ether successively , combine the ether extracts and concentrate. Dissolve the residue in 2ml of chloroform .
• Procedure – Apply 20µl of test and standard solutions on prepared plate.• Detecting reagent – Spray with anisaldehyde sulphuric acid reagent• Rf value – 0.37 ( Dark green spot)
ESTIMATION – BY HPTLC• Standard preparation –• Prepare known concentration of solution in chloroform The amount of substance in the spot is 2- 25µg.• Sample preparation –• Reflux 1gm of drug with 2.5N Hcl for 4hrs, cool and filter through Whatman filter paper , collect the residue and dry at a temperature less than 80O in an oven . Extract with petroleum ether in a soxhlet for 4 hrs and evaporate the petroleum ether extract to dryness.
• Dissolve the residue in chloroform , make up the volume to 10ml with chloroform• Solvent system – Toluene – Ethyl acetate (7:3)• Procedure - Apply known volume of standard and sample preparations in triplicate on precoated HPTLC plates and develop the plate to a distance of 8 cm.• Detecting reagent – Liberman- Buchards reagent and heat at 120o
• Cool and scan in a densiometer in reflection mode at 600nm.• By comparing the areas corresponding to diosgenin in sample and standard preparations , the amount of diosgenin in the sample is estimated.
– AL IC UT ACE RM E HA ID P S H. D O O IL , P YT TIC A, ,M.PHPH IA NIVAS HARM AS .U.SRI M,M.P DR H AR P D.
ASIATICOSIDE• Asiaticoside and medecassoside are the important triterpenoidal saponins of Mandukaparni ( Brahmi).• Source : It consists of dried aerial parts preferably leaves of Centella asiatica.• Family: Apiaceae (umbelliferae)
• Uses :• Brain tonic• Nerve tonic• Sedative• Spasmolytic• Anti-anxiety• Anti-stress• In Ayurveda it is described as ‘Rasayan’.
ISOLATION -• Percolate the powdered drug with 90% alcohol. Collect the alcoholic extract and extract successively with petroleum ether, diethyl ether, ethyl acetate and n- butanol.• Collect the n- butanolic extract and concentrate and the residue is extracted with acetone. Filter while in hot, filtrate on cooling forms precipitate.
• Subject the precipitate for preparative HPLC with Methanol : water (1:3)• Isolation of Asiaticoside and Medecassoside
• Identification: Give test for triterpenoidal saponins• 1) Salkowski test.• 2) Lieberman Burchard reaction.• 3) Trichloro acetic acid test.
a) Salkaowski test: - A few drops of concentrated sulphuric acid were added to the chloroform solution, shaken and allowed to stand. Lower layer turned yellow.b) Lieberman Burchardt test: - To the chloroform solution a few drops of acetic anhydride and 1ml of concentrated sulphuric acid was added. A deep red color was produced.
c) Trichloro acid and Stannic Chloride test: - To the chloroform solution a few drops of thionyl chloride and a pinch of stannic chloride were added. A range of colors green, blue, purple and finally turning to red were obtained.
• ESTIMATION : By HPLC method.• Column - µ - Bondapack C 18 (8mm X10 cm)• Mobile phase - Acetonitrile : water (1:3)• Flow rate - 1.5 ml/minute• Detection - UV at 205nm• Standard - known concentration of Asiaticoside: 0.02 to 0.4 mg/ml in methanol, Madecasosside: 0..2 to 4mg/ml.
• Sample preparation –• 2 gm of the drug , reflux with 90% methanol on water bath , cool and filter, collect the filtrate, concentrate under vaccum, make up to 50ml , dilutions can be made as per the requirements.
• Procedure: Inject 10ul sample and standard. Record the peak areas of asiaticoside and madecassoside of test and standard. Accordingly, the percentage of asiaticoside and madecassoside present in the sample can be calculated.
L – TICA C EU HD A ,P M RM A R S PHA PH IDE A, M TO OS VAS HY N INIP N R SE R.U.S D
• Sennosides are the active constituents of Senna. They are Sennoside A,B,C and D. They are dimeric anthraquinone glycosides.• Biological source: It consists of dried leaflets of cassia angustitolia and cassia acutifolia.• Family: Leguminosae.
• Uses:• Senna is a purgative drug, it is irritant purgative due to presence of Anthraquinone derivatives.
Methods of Isolation:Various methods are available for isolation of sennosides. But essentially, they are isolated as calcium sennosides because of better stability.The various methods are: 1.Extraction of Sennosides as their Calcium salts 2.Ahmed and Samia method 3. Stoll and samia method,
4. Modification of stoll and Becker method5. Notherman and lick method6. Mauzaram et.al method.
EXTRACTION – AS CALCIUM SENNOSIDES• 1. Extract the powder drug with benzene for 2hrs on an electric shaker.• 2. Filter and collect the marc. And dry the marc.• 3.Extract the dried marc with 70% methanol for 4hrs and collect the methanol extract .
• 4. Re- extract the marc with methanol.• 5. Combine the above( 3 & 4) extracts and reduce the volume to 1/8 of original volume .• 6. Acidify the concentrated extract to pH 3.2 with dilute hydrochloric acid and set aside for 2hrs at 50 o C .• 7. Filter and add anhydrous calcium chloride in spirit to the filtrate.
• 8. Adjust the pH to 8 with the help of ammonia and set aside for 2hrs.• 9.Finally collect and dry the precipitate (Calcium sennosides)
IDENTIFICATION -• Borntragers test –• Boil powdered leaves with dilute sulphuric acid . Filter and collect the filtrate. Cool it.• Now shake with an organic solvent like benzene /chloroform. Separate the organic layer and add equal quantity of ammonia
• Ammonia layer becomes pink or red indicating the presence of anthraquinone glycosides.• By Thin layer chromatography –• Adsorbent – Silica gel 60F254• Solvent system – n- propanol: ethyl acetate: water (4:4;3)
• Detection – Nitric acid in potassium hydroxide reagent or visible / UV- 366• Test sample –• Extract 1gm of powdered drug with 5ml methanol by heating on a water bath for 15minutes . Filter and use filtrate as sample.
• Visualization –• UV 366nm – Lemon yellow or Light blue.• Rf values – Sennoside A –0.4• Sennoside B - 0.2• Sennoside C - 0.7• Sennoside D - 0.5
ESTIMATION -• It is estimated by Calorimetrically ( IP 1996)• 1. The drug is powdered and extracted with water or hydro- alcoholic solution. The aqueous phase is extracted with chloroform or ether ( Eliminates free anthraquinones)• 2. The aqueous solution is oxidized with Ferric chloride and hydrolyzed with Hcl
• 3. The resulting anthraquinones are extracted with organic solvent chloroform or ether. The solvent is evaporated and the residue is redissolved in a methanolic solution of magnesium acetate, whose absorbance is measured at 515nm (Red colour)• Standard solution–1:8 dihydroxy anthraquinone
• By Bioassay –• The bioassay is carried out on mice or rats by counting the number of wet faecus after administration of the drug according to the weight of animal for determining the activity.