extraction of phytochemicals

Extraction of Phytochemicals

Presented By :Mr. N. V. Thorat

Introduction

IntroductionDefinition Extraction is process of isolation of soluble material from an insoluble residue, which may be liquid or solid, by treatment with a solvent on the basis of the physical nature of crude drug to be extracted, i.e. liquid or solid, the extraction process may be liquid—liquid or solid—liquid extraction.

Mass transfer is a unit operation, which involves the transfer of mass of soluble material from a solid to a fluid..

Menstrum = Solvent usedMarc = ResidueMicella = solution containing extracted substances

Solvents used for extraction of drugsWater, Ethanol, Methanol, Chloroform, Pet.Ether, Benzene, Acetone etcSelection of suitable solvent- Capable of extracting the active chemical

constituents.Ideal solvent-Cheap, Physically & chemically inert, non toxic ,Selective.

Maceration

Maceration is process of extraction of drugs with solvent with several daily shaking or strring at room temperature.

It is based on the immersion of the powdered drugs in a bulk of the solvent or menstruum (1:10) . Solid drug material is taken in stoppered container (prevent evaporation of solvent) and allowed to stand for at least 3 to 7 days in a warm place with frequent shaking. The mixture of crude drug containing solvent is filtered until most of the liquid drains off. The filtrate and the washing are combined.

Multiple maceration-

Appreciable amount of active constituents may left in first pressing of marc.

May extracted in next maceration.

Total quantity of solvent (menstrum ) divided in 2 (double maceration) or 3 ( triple maceration) equal portion.

Same quantity of menstrum at each maceration

Marc (residue) is pres at the end of maceration.

Maceration

PercolationAs the term indicates, percolation is a continuous flow of the solvent through the bed of the crude drug material to get the extract.In this process, first the powdered drug is treated with sufficient menstrum to make it uniformly wet. Damp material is allowed to stand for about 15 min, and then transferred to a percolator winch is generally a V- shaped vessel open at both the ends.To it, sufficient menstrum is added to saturate the drug. Place the lid on the top. When the liquid starts dripping out from the outlet of the percolator, the lower opening is closed. The drug material is allowed to macerate in the vessel for 24 h .The percolation is continued gradually using sufficient menstruum till completion.

Test for completion of percolationte.i) Few drops of last percolate Dry No residueii)Sp.gravity of last few ml of percolate = Sp.gravity of menstrum Uncompleiii)Sp.chemical test for drug conent. (e.g. Sp.chem. Test for alkaloids)

Percolation

Soxhlet extractionSoxhlet extraction is the process of continuous extraction in which the same solvent can be circulated through the extractor for several times. This process involves extraction followed by evaporation of the solvent. The vapours of the solvent are taken to a condenser and the condensed liquid is returned to the drug for continuous extraction. Soxhlet apparatus

Soxhlet apparatus, designed for such continuous extraction, consists of a body of extractor attached with a side tube and siphon tube as shown in Figure. The extractor from the lower side can be attached to distillation flask and the mouth of the extractor is fixed to a condenser.The crude drug powder is packed in the soxhlet apparatus directly or in a thimble of filter paper or fine muslin. The diameter of the thimble corresponds to the internal diameter of the soxhlet extractor. Extraction assembly is set up by fixing condenser and a distillation flask. Fresh activated porcelain pieces are added to the flask to avoid bumping of the solvent.

Soxhlet extraction

The vapours pass through the side tube and the condensed liquid gradually increases the level of liquid in the extractor and in the siphon tube. A siphon is set up as the liquid reaches the point of return and the contents of the extraction chamber are transferred to the flask. The cycle of solvent evaporation and siphoning back can be continued as many times as possible without changing the solvent so as to get efficient extraction. This method, although a continuous extraction process, is nothing but a series of short macerations.Soxhlet extraction is advantageous in a way that less solvent is needed for yielding more concentrated products. The extraction can be continued until complete exhaustion of the drug. The main disadvantage is that this process is restricted to pure boiling solvents or to azeotropes.

Soxhlet extraction

Dean Stark assembly (Moisture content determination ) An excess of water in herbal materials will encourage microbial growth, the presence of fungi or insects, and deterioration following hydrolysis. Limits for water content should therefore be set for every given herbal material. Azeotropic distillation method (Dean stark apparatus)

The sample to be analyse placed in a flask together with a suitable water immiscible solvent (toluene, Xylene, carbon tetrachloride) and pieces of porous pot and is distilled.

The water in the sample co-distils with the solvent,condensing the distillate as an immiscible layer.

When the sample is distilled together with an immiscible solvent, such as toluene or xylene , the water present in the sample is absorbed by the solvent. The water and the solvent are distilled together and separated in the receiving tube on cooling.

Dean Stark assembly

Extraction of volatile oil

General methods of extraction

Volatile oils are extracted by

Distillation (Water distillation, water & steam distillation, and steam distillation, Distillation per se)

Solvent extraction

Mechanical method (Ecuelle and Enfleurage )

Extraction of Volatile oils

Hydro-Distillation

Hydro-distillation method used for extraction of volatile oil from herbal drugs. The fresh material is subjected to hydro distillation in case of the leaf drugs. Air-dried subterranean parts are extracted by steam distillation.

Material is completely immersed in water and is boiled by applying heat either by direct heatng or by steam jacket. The plant material and boiling water are in direct contact in this method.


Water and steam distillation

The plant material is lodged on perforated grid or screen inserted the above the bottom of still. The still is filled with water upto sufficient level and the water is heated. The saturated low pressure steam rises through the plant material.The plant material comes in contact with only steam and not with boiling water

Advantages over Hydrodistillation

Less decomposition of plant material

Les time ,Les fuel and high yield

Disadvantage

Large quantity of steam required for complete vaporisation of high boiling essential oils, and .hence more time required for distillation


Steam distillationThe plant material is supported on grid and saturated steam is injected through the charge by means of open or perforated coils placed below the grid

AdvantagesProduces best yield and purest oilEnables the operator to attain the end sought most economically and with simplest apparatus

Supersaturated steam distillation sometimes employed for distilling high boiling and chemically stable compound


Distillation per seDistillation per se or as such ,without use of any water or steam is employed in cases of separation of volatile fraction of turpentine. It becomes possible as the resin remains in molten liquid and support the separation of less volatile fraction.


Enfleurage method is used for extraction of delicate perfumes. The fresh flower petals are mechanically spread on layer of fatty material, allowed to imbibe and the exhausted petals are replaced by fresh material. The process is continued till the fatty layer is saturated with volatile principles which are then extracted with lipid solvent.

Ecuelle method is used for extraction of citrus oils, wherein oil cells in rind are ruptured mechanically using pointed projections by twisting raw material over them in clockwise direction either mechanically or manually.


Enfleurage method

Ecuelle method

Solvent extraction

The sophisticated perfume industry utilizes solvent extraction technique for production of volatile oils using lipid solvents like ether or benzene, as it is quick, quality assured and economical method. The deterioration of volatile oils is less as compared to hydro-distillation. The solvent distilled off may be re-used for extraction

1)Selection and purification of the solvent

2)The systematic extraction of the flowers

3)Evaporation & recovery of the solvent and production of vegetable wax saturated with perfume.


Newer techniques of extraction

Microwave assisted extraction

Principle of MAE

When plant material is immersed inside a microwave ,The heating up moisture inside the plant cell due to microwave effect, results in evaporation and generates tremendous pressure on the cell wall. The cell wall is pushed from inside due to the pressure and the cell wall ruptures. Thus the exudation of active constituents from the ruptured cells occurs, releases the content into solvent hence increasing the yield of phytoconstituents.

One of the most commonly used mixtures is hexane-acetone.

Advantages

Advantages of microwave heating compared to conventional methods include energy-saving rapid heating rates , short processing times, deep penetration of the microwave energy (which allows heat to be generated efficiently without directly contacting the work-piece), instantaneous and precise electronic control, & no generation of secondary waste.


Advantages of Microwave Assisted Extraction : It reduces solvent consumption, It has a shorter operational time, It possess moderately high recoveries, Has a good reproducibility & minimal sample manipulation for extraction process.

Disadvantages of Microwave Assisted Extraction: An additional filtration or centrifugation is necessary to remove the solid residue during MAE. Furthermore, the efficiency of microwaves can be very poor when either the target compounds or the solvents are non-polar, or when they are volatile.

Applications of Microwave-Assisted Extraction: MAE can extract nutraceuticals products from plant sources in a faster manner than conventional solid–liquid extractions. MAE (80% methanol) could dramatically reduce the extraction time of ginseng saponin from 12 h using conventional extraction methods to a few seconds.


Counter current extraction

Counter current extraction is a liquid liquid extraction process which work on the principle of partition chromatography. It consist of series of contacting tubes called Craig Counter current distribution tubes ,each containing two different chambers, as shown in fig. The lower chamber A contains stationary phase in all tube while upper chamber B filled with mobile phase.

The mixture to be fractionated is introduced in chamber A of first tube in series. The apparatus is subjected to shaking by rocking back and front and then layers are allowed to separate. The component of the mixture are distributed between two layers according their partition coefficient. After phase separation the assembly is tiled 90° as shown in fig. The upper mobile phase flows through tube C into chamber B, Again the assembly rotated back to original position .The mobile phase of the chamber B flows out through tube D into the next tube in series which contain stationary phase without sample. The process is repeated several times depending upon partition efficiency or the number of tube.

Counter current extraction

Supercritical fluid extraction

Certain gases behave like a free flowing liquids or supercritical fluids at the critical point of temperature and pressure. Such supercritical fluids have a very high penetration powers and extraction efficiency.

The gases like carbon dioxide are held as a supercritical fluid at the critical point of 73.83 bar pressure and 31.06°C temperature. At this critical point CO2 behaves as a liquefied gas or free-flowing liquid and assists the extraction of the phytochemical constituents from the crude drugs.

Advantages

The advantages of CO2 in supercritical fluid extraction are that it is sterile and bacteriostatic. It is noncombustible and nonexplosive. No waste products are generated during the process, and it is available in large amount under favourable condition.

Supercritical fluid extraction

Alkaloids

General methods of extraction

Alkaloids

A) Stas Otto methodThe powdered drug moistened with water + alkali (to get free alkaloidal base )

Alkaloidal free base + Org.solvent

Filter

Filtrate

Evaporate

Alkaloidal free base

Alkaloids

B) Small scale extraction /Monske’s processStep I) Defatting with petroleum ether (especially in seed form of drug)

Alkaloid tested for solubility in petroleum etherInsoluble Soluble (e.g.-Extraction of ergotamine from ergot)

Alkaloid drug + Org. Acid Alkaloidal Salt

Drug +Pet. Ether (Defatting )

Step II ) Marc + Polar solvent (Water ,Ethanol,Methanol)

Filtrate Concentrated (by evaporation to thick syrup)

Partition between aqueous acid solution and an organic solvent (Sep.fennel)

Aq. phase + sodium carbonate / ammonia (To separate alkaloidal base)

Above extract + Org.solvent

Alkaloidal free base

Filter

(Purification of crude extract of alkaloids )

The drug containing glycoside

Finely powdered drug + AlcoholSoxhlet apparatus Enzymes present in plant parts are also

deactivated due to heating. Alcoholic extract + lead acetate (precipitate tannins )

FilterFiltrate + hydrogen sulphide gas

Precipitated of lead sulphideFilter Concentrate

Crude glycosides.Further purification by –

Fractional solubility, Fractional crystallization Chromatographic techniques – TLC etc

The thermolabile glycosides, however, should be extracted at temperature preferably below 45°C.

The characterization of isolated purified compounds is done by IR, UV, visible, NMR and mass spectrometry and elemental analysis.

Glycosides

Glycosides

Tannins

Both hydrolysable and condensed tannins are highly soluble in water and alcohol but insoluble in organic solvents such as solvent ether, chloroform, and benzene.

Tannin compounds can be easily extracted by water or alcohol

Crude drugs + water-saturated ether / mixture of water, alcohol, and ether

Separate aqueous and ethereal layers

Concentrate & drying

Isolation and purification

( Various separation techniques of chromatography)

Tannins

Gallic, ellagic acid, chlorophyll or pigments extracted in ether

Tannin gets extracted in water

extraction of phytochemicals

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