PHYTOCHEMICAL EXTRACTION

EXTRACTION PROTOCOLS

Dr. SINDHU K.

M. V. Sc. SCHOLAR,

DEPT. OF VPT,

COVAS,

POOKODE.

Extraction

• Is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures.

• The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or in dry powder form (after removing the solvent), & are intended for oral or external use.

Medicinal plants

• The Medicinal plants constitute an effective source of both traditional and modern medicines, herbal medicine has been shown to have genuine utility and about 80% of rural population depends on it as primary health care. [WHO, (2005)]

Medicinal plants are the richest bio-resource

drugs of traditional systems of medicine,modern medicines, nutraceuticals,food supplements, folk medicines, pharmaceutical intermediates,chemical entities for synthetic drugs.

galenicals

These include classes of preparations viz.,decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts,powdered extracts.

Evidence for Ethnopharmacology

• De Materia Medica, • Historia Plantarum, • Species Plantarum.have been variously published in attempt to provide scientific information on the medicinal uses of plants.

Standardized Extraction

The purpose of standardized extraction procedures for crude drugs(medicinal & aromatic plant parts)

To attain the therapeutically desired portions To eliminate unwanted material by treatment

with a selective solvent known as “menstrum”

The extract thus obtained, after standardization, may be used as medicinal agent • as such in the form of tinctures • fluid extracts • further processed to be incorporated in any

dosage form such as tablets and capsules

The general techniques of medicinal plant extraction

Maceration, Infusion, Percolation, Digestion, Decoction, Hot continuous extraction (Soxhlet), Aqueous-alcoholic extraction by fermentation, Counter-current extraction, Microwave-assisted extraction, Ultrasound extraction (sonication), Supercritical fluid extraction, Phytonic extraction (with hydrofluorocarbon solvents).

Extraction techniques For aromatic plants

Hydrodistillation techniques (water distillation, steam distillation, water and steam distillation),

Hydrolytic maceration followed by distillation, expression and enfleurage (cold fat extraction)

Headspace trapping, Solid phase micro-extraction, Protoplast extraction, Microdistillation, Thermomicrodistillation, Molecular distillation.

Choice of solvents

• Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure.

• The choice of solvent is influenced by what is intended with the extract.

Properties of a good solvent in plant extractions

low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of

the extract, preservative action, inability to cause the extract to complex or

dissociate.

Solvents used for active component extraction

Water

• Water is universal solvent.• used to extract plant products with antimicrobial

activity.• Traditional healers use primarily water &

consistent antimicrobial activity is obtained.Plant extracts: organic solvents >>> water extract.• Water soluble flavonoids (mostly anthocyanins)

have no antimicrobial significance.• only water soluble phenolics are important as

antioxidant compound.

Acetone

• Dissolves many hydrophilic and lipophilic components.

• a very useful extractant, especially for antimicrobial studies (phenolic group extract).

• extraction of tannins + phenolics: aqueous acetone >>> aqueous methanol • Both acetone and methanol were found to extract

saponins antimicrobial activity.

Alcohol

• The identified components from plants (antimicrobial) = aromatic or saturated organic compounds most often obtained through initial ethanol or methanol extraction.

• Ethanol, found easier to penetrate the cellular membrane to extract the intracellular ingredients(polyphenols) from the plant material.

• Methanol is more polar than ethanol but due to its cytotoxic nature.

• The higher concentrations of more bioactive flavonoid compounds were detected with ethanol

70% due to its higher polarity than pure ethanol.

Chloroform

• Used to obtain tannins and terpenoids.

• Terpenoid lactones successive extractions of dried barks with chloroform.

Ether

• Commonly used selectively for the

extraction of coumarins and fatty acids.

Dichloromethanol

• Specially used for the selective extraction of only terpenoids.

Steps Involved in the Extraction of Medicinal Plants

1. Size reduction2. Extraction3. Filtration4. Concentration5. Drying

1. Size ReductionObjective:• To rupture plant organ, tissue & cell structures so that its

medicinal ingredients are exposed to the extraction solvent.

• Size reduction maximizes the surface area, which in turn enhances the mass transfer of active principle from plant material to the solvent.

The 30-40 mesh size is optimal.Hammer mill or a disc pulverizer which has built in sieves controlled by varying the speed of the rotor clearance

b/w the hammers & the lining of the grinder.

Extraction

Medicinal plants Aromatic plants

1. Cold aqueous percolation2. Hot aqueous extraction (decoction)3. Solvent extraction(cold / hot)

1. Essential oils2. Concretes3. Bsolutes4. Pomades5. Esinoids

parameters influencing the quality of an extract

• Plant part used as starting material • Solvent used for extraction • Extraction procedure

Effect of extracted plant phytochemicals depends on

• The nature of the plant material • Its origin • Degree of processing • Moisture content • Particle size

Selection of plant

• Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc.

• Plants are usually air dried to a constant weight before extraction.

• oven drying: every part were cut into pieces dried in an oven @ 60°C for 9 hrs. & pulverized.

• Other method for drying the plants is the oven drying at about 40°C for 72 h.

Filtration

• The extract so obtained is separated out from the marc (exhausted plant material) by allowing it to trickle into a holding tank through the built-in false bottom of the extractor, which is covered with a filter cloth.

• The marc is retained at the false bottom, and the extract is received in the holding tank.

• From the holding tank, the extract is pumped into a sparkler filter to remove fine or colloidal particles from the extract.

Concentration• The enriched extract from percolators or extractors, known

as miscella, is fed into a wiped film evaporator where it is concentrated under vacuum to produce a thick concentrated extract.

• The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free from solvent.

• The solvent recovered from the wiped film evaporator and vacuum chamber dryer is recycled back to the percolator or extractor for the next batch of plant material.

• The solid mass thus obtained is pulverized and used directly for the desired pharmaceutical formulations or further processed for isolation of its phytoconstituents.

Drying

• The filtered extract is subjected to spray drying with a high pressure pump at a controlled feed rate and temperature to get dry powder.

• The desired particle size of the product is obtained by controlling the inside temperature of the chamber and by varying the pressure of the pump.

• The dry powder is mixed with suitable diluents or excipients and blended in a double cone mixer to obtain a homogeneous powder that can be straight away used (for example, for filling in capsules or making tablets).

Variation in extraction methods

• Length of the extraction period, • Solvent used, • pH of the solvent, • Temperature, • Particle size of the plant tissues,• The solvent-to-sample ratio.

Parameters for Selecting an Appropriate Extraction Method

I. Authentication of plant material by botanist.II. Use the right plant part + the age of plant + the time,

season & place of collection.III. The nature of its chemical constituents.IV. Grinding methods & powdering techniques.V. Nature of constituents (polar/nonpolar).VI. The quality of water / menstruum.VII. The design & material of fabrication of the extractor.VIII.Analytical parameters of the final extract,

(TLC/HPLC).

The general techniques of medicinal plant extraction

maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, phytonic extraction (with hydrofluorocarbon

solvents).

Maceration• The whole / coarsely powdered crude drug is

placed in a stoppered container with the solvent.• Allow to stand @ room temperature for a period of

at least 3 days with frequent agitation until the soluble matter gets dissolved.

• The mixture then is strained, the marc (the damp solid material) is pressed,

• The combined liquids are clarified by filtration or decantation after standing.

• This method is best suitablefor use in case of the

thermolabile drugs.

Infusion

• Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water.

• These are dilute solutions of the readily soluble constituents of crude drugs.

Digestion

• This is a form of maceration in which gentle heat is used during the process of extraction.

• It is used when moderately elevated temperature is not objectionable.

• The solvent efficiency of the menstruum is thereby increased.

Image=microwave Digestion system

Decoction• In this process, the crude drug is boiled in a

specified volume of water (1;4) for a defined time,• Volume is reduced to 1/4th the original,• It is then cooled and strained / filtered.• This procedure is suitable for extracting water-

soluble, heat-stable constituents.• Typically used in preparation of Ayurvedic extracts

= “quath” / “kawath”

Percolation

• Used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts.

• The solid ingredients are moistened with an appropriate amount of the specified menstruum,

• Allowed to stand for approximately 4 hours in a well closed container, After stand time, the mass is packed & the top of the percolator is closed.

• the mixture is allowed• to macerate in the closed

percolator for 24 h.

,

• Additional menstruum is added as required, until the percolate measures about three-quarters of the required volume of the finished product.

• The marc is then pressed and the expressed liquid is added to the percolate.

• Sufficient menstruum is added to produce the required volume.

• The mixed liquid is clarified by filtration orby standing followed by decanting.

Hot Continuous Extraction

(Soxhlet)• The finely ground crude drug is placed in a

porous bag or “thimble” made of strong filter paper, which is placed in chamber of the Soxhlet apparatus.

• The extracting solvent in flask is heated, and its vapors condense in condenser.

• The condensed extractant drips into the thimble containing the crude drug & extracts it by contact.

Soxhlet apparatus• When the level of liquid in chamber rises to the

top of siphon tube, the liquid contents of chamber siphon into flask

• This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated.

Aqueous Alcoholic Extraction by Fermentation

• Some medicinal preparations of Ayurveda (asava & arista) adopt the technique of fermentation for extracting the active principles.

• The extraction procedure involves soaking the crude drug, [powder / a decoction (kasaya)], for a specified period of time

• Undergoes fermentation & generates alcohol in situ.• This facilitates the extraction of the active

constituents contained in the plant material.• The alcohol thus generated also serves as a

preservative.

,

• Some examples of Ayurvedic preparations:karpurasava, kanakasava, dasmularista.

• If the fermentation is to be carried out in an earthen vessel, it should not be new: water should first be boiled in the vessel.

• In large-scale manufacture, wooden vats, porcelain jars or metal vessels are used in place of earthen

vessels.

Counter-current Extraction

• Wet raw material is pulverized using toothed disc disintegrators to produce a fine slurry.

• Material to be extracted is moved in one direction generally in the form of a fine slurry within a cylindrical extractor where it comes in contact with extraction solvent.

• The further the starting material moves, the more concentrated the extract becomes.

• Complete extraction is thus possible when the quantities of solvent & material. Their flow rates should be optimized.

• sufficiently concentrated extract comes out at one end of the extractor while the marc, practically free of visible

solvent falls out from the other end.

Ultrasound Extraction (Sonication)

• The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz.

• This increases the permeability of cell walls & produces cavitation.Eg: extraction of rauwolfia root.

• Deleterious effect: Ultrasound energy (>20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.

Supercritical Fluid Extraction• Cylindrical extraction vessels are used.• The collection of the extracted analyte following SFE is

another important step: significant analyte loss can occur during this step.

• CO2 as the extracting fluid.• Organic solvents are frequently added to the CO2

extracting fluid to alleviate the polarity limitations• The component recovery rates generally increase with

increasing pressure/temperature.• The highest recovery rates in case of argon:

@ 500 atm & 150° C.

Phytonics Process

• A new solvent based on hydrofluorocarbon-134a, a new technology to optimize the extraction of plant materials.

• Advanced Phytonics Limited (Manchester, UK) has developed patented technology termed “phytonics process”.

• The products are fragrant components of essential oils & Biological/phytopharmacological extracts.

• The core of the solvent is 1,1,2,2-tetrafluoroethane, (HFC-134a).

• HFC-134a developed as a replacement for chlorofluorocarbons. (Boiling Point -25° C).

• the solvents can be customized: by using modified solvents with HFC-134a.

• The process can be made highly selective in extracting a specific class of phytoconstituents.

Aromatic Plant Extracts

• essential oils,• concretes,• absolutes, • pomades• resinoids.

Essential oils

• Used in a wide variety of consumer goods viz., detergents, soaps, toilet products, cosmetics, pharmaceuticals, perfumes, confectionery food products, soft drinks, distilled alcoholic beverages (hard drinks) and insecticides.

• Production technology is an essential element to improve the overall yield & quality of essential oil.

Presence of Essential Oils in plant parts.

Hetreogenous chemical group present in essential oils

The traditional methods for essential oil extraction

• Water distillation,• Water and steam distillation,• Steam distillation, • Cohobation, • Maceration,• Enfleurage.

methods

1. Distillation: Originally introduced by Von Rechenberg.• water distillation.• water and steam distillation.• direct steam distillation.2. Hydrolytic maceration distillation. 3. Expression.4. Cold fat extraction / Enfleurage.

• Distillation methods are good for powdered almonds, rose petals and rose blossoms.

• Maceration is adaptable when oil yield from distillation is poor.

• Solvent extraction is suitable for expensive, delicate & thermally unstable materials like jasmine, tuberose, and hyacinth.

• Water distillation is the most favored method of production of citronella oil from plant material.

1. Headspace trapping techniques- Static headspace technique- Vacuum headspace technique- Dynamic headspace technique2. Solid phase micro-extraction (SPME)3. Supercritical fluid extraction (SFE)4. Phytosol (phytol) extraction5. Protoplast technique6. Simultaneous distillation extraction (SDE)7. Microwave distillation8. Controlled instantaneous decomposition (CID)9. Thermomicrodistillation10. Microdistillation11. Molecular spinning band distillation12. Membrane extraction

Modern (Non-traditional) Methods of Extraction

Concrete• This is an extract of fresh flowers, herbs, leaves and the

flowering tops of plants obtained by the use of a hydrocarbon solvent such as butane, pentane, hexane and petroleum ether.

• Concrete is rich in hydrocarbon soluble material & devoid of water-soluble components.

• It is generally a waxy, semisolid, dark-colored material free from the original solvent.

• concretes are produced in static extractors.

• It is a normal practice to circulate freshsolvent through a battery of extractors.

• The enriched solvent from the extractor is pumped into an

evaporator for solvent recovery & the solvent content is reduced to about 1/10th the original volume.

Absolutes

• To make an absolute, the concrete is mixed with absolute alcohol & agitated thoroughly in a vessel with an agitator.

• During agitation, the temperature is kept at 40°-60° C and the concrete is immersed in the solution.

• The solution is cooled down to -5° to -10° C to precipitate out the wax, since waxes are normally insoluble in alcohol below -1° C.

• The precipitated wax is removed by passing the solution through a rotary filter.

• The filtrate from the rotary filter is pumped into a primary evaporator, where it is concentrated to about 10% alcohol content.

• Finally, the concentrated extract is pumped into an agitating-type evaporator, where the alcohol

is carefully removed under high vacuum.

Resinoids

• Resinoid is an extract of naturally resinous material, made with a hydrocarbon solvent.

• Resinoids are usually obtained from dry materials.• The extraction process is same as that of concrete

production, except that perforated discs are not used for stacking the material;

• instead powder from dry plant material is fed into the extractor.

Pomades• Pomades are obtained by a process known as enfleurage, which is a cold fat extraction method.• The fat is spread out on glass plates contained in wooden

frames, leaving a clear margin near the edges. • The absorptive surface of the fat is increased by surface

grooves made with a wooden spatula.• Fresh flowers are spread out on the surface of the fat and the

frames are stacked in piles.• After the perfume oils have been absorbed from the flowers,

the spent flowers are removed by hand.• Fresh flowers are again spread on the fat surface.• This is repeated until the fat surface is completely enriched

with perfume oils.• The pomade so obtained is ready for cold alcoholic extraction

major constraints in sustainable industrial exploitation

• Poor agricultural practices for MAPs,• Unscientific and indiscriminate gathering practices from the wild, poor

postharvest & post-gathering practices leading to poor quality raw material,

• Lack of research for the development of high-yielding varieties of medicinal and aromatic plants-MAP’s.

• Poor propagation methods.• Inefficient processing techniques.• Poor quality control procedures.• Lack of research on process

& product development.• Difficulty in marketing.• Non-availability of trained personnel.• Lack of facilities & tools to fabricate equipment locally.• Finally lack of access to the latest technologies & market information.

Conclusion

• The process of extracting MAP’s determines how efficiently we add value to MAP bio-resources.

• In the case of essential oils, the extraction process affects the physical as well as internal composition.

• Variations in the chemical constituents of the extracts of medicinal plants may result by using non-standardized procedures of extraction.

• Efforts should be made to produce batches with quality as consistent

as possible.

References

S. S. Handa. 2008. An Overview of Extraction Techniques for Medicinal and Aromatic Plants.(© United Nations Industrial Development Organization and the International Centre for Science and High Technology, 2008).

Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur. 2011. Phytochemical screening and Extraction: A Review. INTERNATIONALE PHARMACEUTICA SCIENCIA.

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