GENERAL METHODS OFISOLATION AND SEPERATION OF PLANT CONSTITUENTS RAHUL B S M PHARM PART 1
CONTENTSINTRODUCTIONEXTRACTION PROCESS * Types of extraction * Solvents used * Process of extraction * Types of extractsSEPERATION AND IDENTIFICATION OF PLANTCONTISTITUENTS* Fractional crystallization * Fractional distillation * TLC* Fraction liberation * Sublimation * Column chromatography* Counter - current extraction * Paper chromatographyQUALITATIVE REACTIONS FOR THE DETECTION OFPLANT CONSTITUENTSAPPLICATION OF GLCAPPLICATION OF HPLCCONCLUSIONREFERENCE
INDRODUCTION• A natural product is a chemical compound or substanceproduced by a living organism. They may be extracted fromtissues of terrestrial plants, marine organism or micro - organismfermentation.• In that respect any biological molecule is a natural product, butin general the term is reserved for secondary metabolites(carotinoids, phytosterines, saponines, phenolic compounds,alkaloids, glycosinates, terpenes etc).• The extracts from plant tissue are a rich source of leadcompounds for nutraceutical or pharmaceutical applications
Extraction PLANT SteamDistillation MATERIAL Distillation Pressing Methods for recovery of secondary metabolites.
EXTRACTION PROCESSExtraction may be defined as the treatment of the plant or animaltissues with solvent, whereby the medicinally active constituentsare dissolved, and most of the inert matter remains undissolved.The solvent used for extraction is known as Menstruum and theinert insoluble material that remains after extraction is calledMarc
The various process used for extraction are1. MacerationIn this process, the whole or coarsely powdered crude drug isplaced in a stoppered container with the solvent and allowedto stand at room temperature for a period of at least 3 dayswith frequent agitation until the soluble matter has dissolved.The mixture then is strained, the marc (the damp solidmaterial) is pressed, and the combined liquids are clarified byfiltration or decantation after standing.
2. InfusionFresh infusions are prepared by macerating the crude drugfor a short period of time with cold or boiling water. Theseare dilute solutions of the readily soluble constituents ofcrude drugs.3. DigestionThis is a form of maceration in which gentle heat is used duringthe process of extraction. It is used when moderately elevatedtemperature is not objectionable. The solvent efficiency of themenstruum is thereby increased.
4. DecoctionIn this process, the crude drug is boiled in a specified volume ofwater for a defined time; it is then cooled and strained or filtered.This procedure is suitable for extracting water-soluble, heatstableconstituents. The starting ratio of crude drug to water isfixed, e.g. 1:4 or 1:16; the volume is then brought down to one-fourth its original volume by boiling during the extractionprocedure. Then, the concentrated extract is filtered and used assuch or processed further.
5. PercolationPercolation is a continuous flow of the solvent through the bedof the crude drug material to get the extract. In this process, the powdered drug is moistened with anappropriate amount of the specified menstruum and allowed tostand for approximately 4 h in a wellclosed container, afterwhich the mass is packed and the top of the percolator isclosed.Additional menstruum is added to form a shallow layer abovethe mass, and the mixture is allowed to macerate in the closedpercolator for 24 h.
The outlet of the percolator then is opened and the liquidcontained therein is allowed to drip slowly.Additional menstruum is added as required, until the percolatemeasures about three-quarters of the required volume of thefinished product.The marc is then pressed and the expressed liquid is added tothe percolate.Sufficient menstruum is added to produce the requiredvolume, and the mixed liquid is clarified by filtration or bystanding followed by decanting.
MODIFIED PERCOLATIONThe conventional percolation process is modified to includeevaporation for the production of more concentrated products,especially when the solvent is dilute alcoholIn simple percolationDrug imbibition maceration percolation and collect the percolateIn conventional percolationDrug imbibition maceration percolation and collect the 1000 ml of percolate maceration percolation and collect the 1000 ml of percolate maceration percolation and collect the 1000 ml of percolateThe process is continued in case the drug is not completelyexhausted.
Hot Continuous Extraction (Soxhlet)In this method, the finely ground crude drug is placed in aporous bag or “thimble” made of strong filter paper, of theSoxhlet apparatus.The extracting solvent in flask is heated, and its vaporscondense in condenser . The condensed extractant drips into thethimble containing the crude drug, and extracts it by contact. When the level of liquid in chamber rises to the top of siphontube , the liquid contents of chamber siphon into flask. Thisprocess is continuous and is carried out until a drop of solventfrom the siphon tube does not leave residue when evaporated.The advantage of this method, compared to previously describedmethods, is that large amounts of drug can be extracted with amuch smaller quantity of solvent.
Aqueous Alcoholic Extraction by Fermentation It involves soaking the crude drug, in the form of either apowder or a decoction for a specified period of time, duringwhich it undergoes fermentation and generates alcohol in situ;this facilitates the extraction of the active constituents containedin the plant material.The alcohol thus generated also serves as a preservative.Some examples of such preparations arekarpurasava, kanakasava, dasmularista..
COUNTER-CURRENT EXTRACTION In counter-current extraction (CCE), wet raw material ispulverized and produce a fine slurry. Here the material to be extracted is moved in one directionwithin a cylindrical extractor where it comes in contact withextraction solvent.The further the starting material moves, the more concentratedthe extract becomes. Complete extraction is thus possible when the quantities ofsolvent and material and their flow rates are optimized.Finally, sufficiently concentrated extract comes out at one end ofthe extractor while the marc falls out from the other end.
AdvantagesSmaller volume of solvent as compared to other methodslike maceration, decoction, percolation.CCE is commonly done at room temperature, whichspares the thermolabile constituents from exposure to heatwhich is employed in most other techniques.As the pulverization of the drug is done under wetconditions, the heat generated during comminution isneutralized by water. This again spares the thermo labileconstituents from exposure to heat. The extraction procedure has been rated to be moreefficient and effective than continuous hot extraction.
Ultrasound Extraction (Sonication)The procedure involves the use of ultrasound with requenciesranging from 20 kHz to 2000 kHz;this increases thepermeability of cell walls and produces cavitation. The process is useful in some cases, like extraction ofrauwolfia root, its large-scale application is limited due to thehigher costs.DisadvantageThe deleterious effect of ultrasound energy (more than 20 kHz)on the active constituents of medicinal plants throughformation of free radicals and consequently undesirablechanges in the drug molecules.
Supercritical Fluid ExtractionThe critical point represents the highest temperature andpressure at which the substance can exist as a vapour andliquid in equilibrium. The phenomenon can be easily explainedwith reference to the phase diagram for pure carbon dioxide
A super-critical fluid is a substance, mixture, or element,which under certain operative conditions of pressure andtemperature, and mechanical operations, is above its criticalpoint but below the pressure needed to condense it into a solid.Extraction via super-critical fluids is better for theenvironment than conventional methods of extraction, becauseit uses gases such as CO 2 at high pressure, in a liquid orsuper-critical state, instead of chlorinated solvents whichproduce toxic waste.carbon dioxide is the preferred fluid for SFE, They arepowerful solvents and have a great capacity of penetration insolids, which allows a rapid and almost complete exhaustionof extractable solids. They can easily be completely separated from extracts,simply by modifying pressure or temperature, up to the point
advantages:i) The extraction of constituents at low temperature, whichstrictly avoids damage from heat and some organic solvents.ii) No solvent residues.iii) Environmentally friendly extraction procedure.The main drawback is the time of extraction, which isusually long. In fact, in some cases, it can take as much as 24hours. With normal fluids, extraction can be speeded up bymechanical shaking, but this presents problems when usingsuper-critical fluids, which limits industrial use.
Types of solventThe solvents used in extraction are capable of penetrating thetissues of the drug and dissolve the active principles contained inits cell. The various solvents used are water, propene, butane,ethylacetate, ethanol, methanol, CO 2 , N 2 O, acetone etc.WaterIt is the cheap, non toxic, non inflammable and has wide solventaction. Eg; proteins, glycosides, enzymes, sugar etc.Disadvantages•Water may promote growth of mould and bacteria, hencerequires a preservative.•It may leads to hydrolysis.•Large amount of heat is required to concentrate the aqueouspreparations.•It promote fermentation or decomposition of the preparation
AlcoholIt is the important solvent for dissolves alkaloids, alkaloidalsalts, glycosides etc. it also dissolves many colouringmatter, tannins, etc. it doesn’t dissolve gums waxes, fats etc.MeritsIt doesn’t allow the growth of mould and bacteria in above 20%of alcoholIt is nontoxic in the concentration mostly present in thepreparations.Small amount of heat is requiered for concentrationDemeritsCostInflammable, volatile etcSolvents such as ether, chloroform, light petroleum are rarelyused.
factors considered when selecting a solvent• Solvent power (selectivity). Only the active, desired constituents should beextracted from the plant material, which means that a high selectivity isrequired.• Boiling temperature. The boiling point of the solvent is aslow as possible in order to facilitate removal of the solventfrom the product.• Reactivity. The solvent should not react chemically with theextract, nor should it readily decompose.• Viscosity. A low viscosity of the solvent leads to low pressuredrop and good heat and mass transfer.• Safety. The solvent should be non-fl ammable and non-corrosive, and shouldnot present a toxic hazard; its disposal should not imperil the environment.• Cost. The solvent should be readily available at low cost.• Vapor pressure. To prevent loss of solvent by evaporation, alow vapor pressure at operating temperature is required.• Recovery. The solvent has to be separated easily from theextract to produce a solvent-free extract.
Types of extractAqueous extractsThe medicinal preparations intended to be used immediately afterpreparation or to be preserved for use, solvent used is water. The methodsused for their preparation are decoction, infusion, and digestion.Hydro alcoholic or AlcoholicThese are prepared by the methods of maceration and percolation egtintures, here the solvent using is alcoholSoft extractsThey are extracts with semisolid or syrup consistency Can be used in avariety of dosage form like ointments and suppositoriesEg; glycerriza extractsDry extractsThey powdered extracts or dry powder Extract obtained from suitableprocess is filtered and get concentrated under vacuum, dried completelyby spray or tray drying.Eg; belladona used in dossage forms such as capsules, tablets etc.
SEPARATION AND ISOLATION OF CONSTITUENTSThe instrumentation for the structure for the structureelucidation of organic compounds becomes effective andallows the use of increasingly.The most difficult operation in phytopharmacetical researchis the isolation and purification of plant constituents.The physical methods used are chromatographic techniquesand methods such as fractional crystallisation, fractionaldistillation, fractional liberation.Chemical method is based on groups or moieties present inthe compound and chemical reactions.
FRACTIONAL CRYSTALLISATIONIt is an important method for the purification ofcompounds from mixture.It depends upon the compound which form crystals atthe point of super saturation in the solvent in which it issolubleMany natural products are crystaline nature even inmixture, process such as concentration, slowevaporation, refrigeration are using for crystalisation
FRACTIONAL DISTILLATIONThis method is used for the separation ofthe components from volatile mixturesLargely using in the separation ofhydrocarbons from oxygenated volatileoil eg citral, eucalyptolFRACTIONAL LIBERATIONIn this proces the groups of compoundshaving the tendency of precipitation fromthe solution.Incertain cases the compounds maymodified by converting to its salt form.This proces is often used in separation ofcinchona alkaloids, morphine etc.
SUBLIMATIONHere the compound isheated the solid statechanges to gaseous statewithout passing via liquidstate. Such compounds getdeposited in form ofcrystals or cake.This method is traditionallyused for the separation ofcamphor from chips ofcinnamomum camphora.
CHROMATOGRAPY Chromatography is widely used for the separation & identification of components of a mixture. Separation of chemical compounds is carried out by mobile phase and stationary phase.Chromatography can be classified according to mechanism ofseparation as:adsorption chromatography, partition chromatography, ion exchange chromatography,size exclusion chromatography and affinity chromatography.
PAPER CHROMATOGRAPHYThe principle is partitionMainly the stationary phase ismoisture present in the cellulosefibers and mobile vary as we using.The components separated basedon their solubilityThe ratio between the distance travelled on the paper by acomponent of the test solution & the distance travelled by thesolvent is termed the RF value. Under standard conditions, thisis a constant for the particular compound.In practise, however, variations of the RF value often occur & itis best to run a reference compound alongside the unknownmixtures.
ADVANTAGESi. Simple & inexpensiveii. Sensitive – gives good separation of very small amounts, of especially water-soluble compounds, e.g. sugars.DISADVANTAGESi. Fragile – chromatogram may be destroyed by chemicals used for visualizationii. May be time-consuming.
THIN LAYER CHROMATOGRAPHY (TLC)TLC is an e.g. of adsorption chromatography, the stationaryphase being a thin layer adsorbent held on a suitable backing.Separation of the compounds present in the plant extractdepends on the differences in their adsorptive/desorptivebehaviour in respect of the stationary phase.TLC involves a thin layer of adsorbent, mixed with a bindersuch as CaSo4, which is spread on a glass plate & allowed todry.The plant mixture to be separated is applied as a spot near thebase of the plate, which is then placed in a closed glass tankcontaining a layer of developing solvent.
ADVANTAGES OF TLC OVER PAPER CHROMATOGRAPHY Separation of compounds can be achieved more rapidly & with less plant material. -The separated spots are more compact & clearly demarcated from one another -Reagents such as concentrated H2SO4 would destroy a paper chromatogram, but ma be used to locate the separated substances on a TLC plate.
COLUMN CHROMATOGRAPHYIt is a method used to purifyindividual chemicalcompounds from mixtures ofcompounds the principle ofseparation is adsorption. The classical preparativechromatography column, is aglass tube with a diameter from5 mm to 50 mm and a height of5 cm to 1 m with a tap and somekind of a filter (a glass frit orglass wool plug – to prevent theloss of the stationary phase) atthe bottom.
GAS CHROMATOGRAPHY (GC)It is an analytical technique for separating compounds based primarily ontheir volatilities. GC provides both qualitative and quantitative information for individualcompounds present in a sample. Compounds move through a GC column as gases, either because thecompounds are normally gases or they can be heated and vaporized into agaseous state. The differential partitioning into the stationary phase allows the compoundsto be separated in time and space.
APPLICATIONSQuality controlcontamination of plant and plant based products with pesticides, herbicides and manyother materials that are considered a health risk, all such products on sale today mustbe carefully assayedIdentification of Source /OriginThe source of many plants (herbs and spices) can often be identified from thepeak pattern of the chromatograms obtained.Technique of fingerprint could really identify the false herbal products.The fundamental reason of quality control of herbal medicines is based onthe concept of phytoequivalence of herbs, and then to use this conception toidentify the real herbal medicine and the false one, and further to doquality control.Qualification and Quantification of PhytoconstituentsAlkaloidsCapillary gas chromatography (GC), often coupled with a mass spectrometeras a detector (GC-MS), is a well established technique for analyzing complexmixtures of alkaloids.
TerpenesA qualitative comparative study was performed for terpenesfrom volatile oils by GC and GC-MS techniqueFlavanoids and FlavonesFlavonoids receive considerable attention in the literature,specifically because of their biological and hysiologicalimportance. Gas Chromatography Coupled to MassSpectrometry GC-MS is established as a routine techniquefor the analysis of flavonoid aglycones.Essential Oils /Volatile oilsMany pharmacologically active components in herbalmedicines are volatile chemical compounds. Thus, theanalysis of volatile compounds by gas chromatography isvery important in the analysis of herbal medicines
High-performance liquid chromatography (HPLC)High performance liquid chromatography is a powerful tool inanalysis. This page looks at how it is carried out and showshow it uses the same principles as in thin layer chromatographyand column chromatography.
Application of HPLC1- Isolation and purification of biologically active natural products2- Control of synthetic reactions Identification of intermediates and target compound.3- Biosynthesis study Detection of biogenetic intermediates and enzymes involved.4-Control the microbiological processUsed for separation of antibiotic from broth mixture5- Pharmacokinetics study Pharmacokinetic study comprises the measurement of drug metabolitesconcentration in body fluids, absorption, bioavailability and elimination ofdrugsHPLC determines the drug and its metabolites in one step.6- Stability test Rapid method of analysis in stability test.7- Quality control HPLC is used to know the identity, purity and content of the ingredients(drugs, raw and pharmaceutical products,8- Drugs metabolisms
9- Purificationrefers to the process of separation or extraction the targetcompound from other compounds or contaminants10- Quantification of compounds by HPLCQuantitative (assay) and qualitative determination of naturalproducts11- Is the process of determination of the unknownconcentration of a compound in a known solution.12- Identification of compound by HPLC through :- Comparison of retention time with authentic- Comparison of UV spectrum of the compound with that ofthe authentic.- Comparison of the Mass spectrum with that of the authentic.
Qualitative Reactions For The Detection Of Plant ConstituentsTest for alkaloids1. Dragendorff’s test1 ml of extract, add 1 ml of Dragendroff’s reagent (potassium bismuth iodidesolution). An orange-red precipitate indicates the presence of alkaloids.2.Mayer’s test1 ml of extract, add 1 ml of Mayer’s reagent (potassium mercuric iodidesolution). Whitish or cream colored precipitate indicates the presence ofalkaloids.3.Hager’s test1 ml of extract, add 3 ml of Hager’s reagent (saturated aqueous solution ofpicric acid). Yellow colored precipitate indicates the presence of alkaloids4. Wagner’s test1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium iodide).Reddish brown colored precipitate indicates the presence of alkaloids
Test for glycosidesBontragers testIn this test boil test sample with 1ml of sluphuric acid in a testtube for 5min,filter while hot. Cool the filterate and shake withequal volume of dichloromethane or chloroform then seperatethe lower layer of chloroform and shake it with half volume ofdilute ammonia. A rose pink to red colour is produced in theammonical layer.Modified Borntragor’s Test:To 1 gm of drug add 5 ml dilute HCl followed by 5 mlferricChloride (5% w/v). Boil for 10 minutes on water bath, cooland filter, filtrate was extracted withcarbon tetrachloride orbenzene and add equal volume of ammonia solution, formationof pink tored colour due to presence of anthraquinone moiety.This is used C-type of anthraquinoneglycosides
Chemical tests for steroid and triterpenoid glycosidesLibermann Bruchard test:Alcoholic extract of drug was evaporated to dryness andextracted with CHCl 3 , add few drops of acetic anhydridefollowed by conc. H2SO4 from sidewall of test tube to theCHCl3extract. Formation of violet to blue coloured ring atthe junction of two liquid, indicate the presence of steroidmoiety.Salkovaski test:Alcoholic extract of drug was evaporated to dryness andextracted withCHCl3, add conc. H2SO4 from sidewall of testtube to the CHCl3 extract. Formation of yellow colored ring atthe junction of two liquid, which turns red after 2 minutes,indicate the presence of steroid moiety
Chemical tests for cardiac glycosides Keller Killiani test:To the extract of drug equal volume of water and 0.5 ml of strong lead acetatesolution was added, shaked and filtered. Filtrate was extracted with equalvolume of chloroform. Chloroform extract was evaporated to dryness andresidue was dissolved in 3 ml of glacial acetic acid followed by addition of fewdrops of FeCl3 solution. The resultant solution was transferred to a testubecontaining 2 ml of conc. H2SO4. Reddish brown layer is formed, which turnsbluish green after standing due to presence of digitoxose. Legal test:Treat the test solution with 2ml of pyridine and sodium nitropruside 2 ml wasadded followed by addition of NaOH solution to make alkaline. Formation ofpink colour in presence of glycosides or aglycon moiety.Baljet test:Treat the test solution with picric acid or sodium picrate solution, it formsyellow to orange colour in presence of aglycones or glycosides
Tests for tanninsGoldbeater’s skin test: Goldbeater’s skin is a membrane produced from theintestine of Ox. It behaves just like untanned animal hide. A piece ofgoldbeaters skin previously soaked in 2% hydrochloric acid and washed withdistilled water is placed in a solution of tannin for 5 minutes. It is then washedwith distilled water and transferred to 1 % ferrous sulphate solution. A changeof the color of the goldbeater’s skin to brown or black indicates the presenceof tannin. Hydrolysable and condensed tannins both give the positivegoldbeater’s test while pseudo tannins show very little color or negative test.Phenazone Test: To 5 ml of aqueous solution of tannin containing drug, add0.5 g of sodium acid phosphate. Warm the solution, cool and filter. Add 2% phenazone solution to the filtrate. All tannins are precipitated as bulky,colored precipitate.Gelatin Test: To a 1 % gelatin solution, add little 10 % sodium chloride. If a1 % solution of tannin is added to the gelatin solution, tannins causeprecipitation of gelatin from solution.
Tests for flavonoids:Shinoda Test: To the test solution, and few drops of conc.HCl. To this solution 0.5 g of magnesium turnings wereadded. Observance of pink coloration indicated the presenceof flavonoids.With Lead Acetate: To the small quantity of test solution leadacetate solution was added. Formation of yellow precipitateshowed the presence of flavonoid.With Sodium Hydroxide: On addition of an increasingamount of sodiumhydroxide, the ethanolic extract showed yellow coloration,this decolorized after addition of acid.
Tests of proteinBiuret testOn adding 1% copper sulphite to alkaline solution (4% NaOH solution) ofprotein, a violet colour is developed. This test is due to the presence of peptidelinkage.Xanthoproteic Test:To 5ml test solution add 1ml of Con.HNO3 and boil, yellow ppt is formed.On addition of NH4OH, yellow ppt. turned orange.Nihydrin test When protein is boiled with a dilute solution of ninhydrin, a violet colour isproduced.
CONCLUSIONExtraction, as the term is used pharmaceutically, involves theseparation of medicinally active portions of plant or animaltissues from the inactive or inert components by usingselective solvents in standard extraction procedures.The products so obtained from plants are relatively impureliquids, semisolids or powders intended only for oral orexternal use.There are several techniques for the separation andidentification of natural products. Selection of method isimportant in result.