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, 4(2), 123-140
ISSN: 0975-7538
Review Article
www.ijrps.pharmascope.org
ABSTRACT
Plants have been used as medicine in ancient. Now days Pharmaceutical companies start processing of medicinal
and aromatic plants in their formulation by using extraction of active components. Extraction of plant components
like volatile, Essential or ethereal oils and mixtures composed of volatile liquid and solid compounds depend upon
their composition and their boiling point. Now days there are several processes like distillation, enfleurage, mac-
eration, expression, solvent extraction and fluid extraction are available for extraction of plant components. This
review also summarizes the characters of phytoconstituents, choice of solvents, influence of solvents, extraction
strategy, procedures for extraction of herbal drugs and treatment of drug residue after extraction.
Keywords: Secondary metabolites; Phytoconstituents; Enfluerage
1. INTRODUCTION a very central stage in modern civilization as natural
source on chemotherapy as well as amongst scientist in
Natural products are secondary metabolites which are
search for alternative sources of drugs. About 3.4 bil-
derived from herb or animal sources. Natural products
lion people in the developing world depend on plant-
are chemical compounds found in nature and they
based traditional medicines. This represents about 88
have pharmacological and biological activity. Natural
per cent of the worlds inhabitants, who rely mainly on
products are generally used in drug discovery and drug
traditional medicine for their primary health care.
design. Separation of a single molecular entity is very
(Doughari et al., 2009; Yadav et al., 2003).
difficult from complex mixtures contain fats, oils, alka-
loid, tannins and glycoside. In 1803 the first alkaloid, According to the World Health Organization, a medici-
nicotine and then morphine, strychnine, emetine and nal plant is any plant which, in one or more of its or-
many others were separated. This review compiles the gans, contains substances that can be used for thera-
recent literature with special focus on various ap- peutic purposes, or which are precursors for chemo-
proaches for extraction. This review also summarizes pharmaceutical semi synthesis. Such a plant will have
choice of solvent, interfering compound and strategies its parts including leaves, roots, rhizomes, stems, barks,
involve in extraction. flowers, fruits, grains or seeds, employed in the control
or treatment of a disease condition and therefore con-
Medicinal plants have been the mainstay of traditional
tains chemical components that are medically active.
herbal medicine amongst rural dwellers worldwide
These non-nutrient plant chemical compounds or bio-
since antiquity to date. The therapeutic use of plants
active components are often referred to as phyto
certainly goes back to the Sumerian and the Akkadian
chemicals (phyto- from Greek - phyto meaning plant)
civilizations in about the third millenium BC. Hippo-
or phytoconstituents and are responsible for protect-
crates (ca. 460377 BC), one of the ancient authors
ing the plant against microbial infections or infesta-
who described medicinal natural products of plant and
tions by pests. (Liu, 2004) The study of natural prod-
animal origins, listed approximately 400 different plant
ucts on the other hand is called phytochemistry. Phy-
species for medicinal purposes. Natural products have
tochemicals have been isolated and characterized from
been an integral part of the ancient traditional medi-
fruits such as grapes and apples, vegetables such as
cine systems, e. g. Chinese, Ayurvedic and Egyptian
broccoli and onion, spices such as turmeric, beverages
(Daffre et al., 2008). Over the years they have assumed
such as green tea and red wine, as well as many other
sources. (Bohlin et al., 1993) The science of application
* Corresponding Author of these indigenous or local medicinal remedies includ-
Email: minnu_3k27@yahoo.com
ing plants for treatment of diseases is currently called
Contact: +91- 7893817339
Received on: 04-12-2012 ethno pharmacology but the practice dates back since
Revised on: 02-03-2013 antiquity. Ethno pharmacology has been the mainstay
Accepted on: 15-03-2013 of traditional medicines the entire world and currently
is being integrated into mainstream medicine.
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 123
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
Different catalogues including De Materia Medica, His- (MDR) strains of a number of pathogenic bacteria such
toria Plantarum, Species Plantarum have been vari- as methicillin resistant Staphylococcus aureus, Helico-
ously published in attempt to provide scientific infor- bacter pylori, and MDR Klebsiela pneumonia has re-
mation on the medicinal uses of plants. The types of vived the interest in plants with antimicrobial proper-
plants and methods of application vary from locality to ties. In addition, the increase in cases of opportunistic
locality with 80% of rural dwellers relying on them as infections and the advent of Acquired Immune Defi-
means of treating various diseases. For example, the ciency Syndrome (AIDS) patients and individuals on
use of bearberry (Arctostaphylos uva-ursi) and cran- immunosuppressive chemotherapy, toxicity of many
berry juice (Vaccinium macrocarpon) to treat urinary antifungal and antiviral drugs has imposed pressure on
tract infections is reported in different manuals of phy- the scientific community and pharmaceutical compa-
totherapy, while species such as lemon balm (Melissa nies to search alternative and novel drug sources.
officinalis), garlic (Allium sativum) and tee tree (Me-
2. CLASSES OF PHYTOCONSTITUENTS
laleucaalternifolia) are described as broad-spectrum
antimicrobial agents. A single plant may be used for 2.1 ALKALOIDS
the treatment of various disease conditions depending
These are the largest group of secondary chemical con-
on the community. Several ailments including fever,
stituents made largely of ammonia compounds com-
asthma, constipation, esophageal cancer and hyper-
prising basically of nitrogen bases synthesized from
tension have been treated with traditional medicinal
amino acid building blocks with various radicals replac-
plants. The plants are applied in different forms such as
ing one or more of the hydrogen atoms in the peptide
poultices, concoctions of different plant mixtures, infu-
ring, most containing oxygen. The compounds have
sions as teas or tinctures or as component mixtures in
basic properties and are alkaline in reaction, turning
porridges and soups administered in different ways
red litmus paper blue. In fact, one or more nitrogen
including oral, nasal (smoking, snoffing or steaming),
atoms that are present in an alkaloid, typically as 1, 2
topical (lotions, oils or creams), bathing or rectal (ene-
or 3 amines, contribute to the basicity of the alkaloid.
mas). Different plant parts and components (roots,
The degree of basicity varies considerably, depending
leaves, stem barks, flowers or their combinations, es-
on the structure of the molecule, and presence and
sential oils) have been employed in the treatment of
location of the functional groups (Daffre et al., 2008)
infectious pathologies in the respiratory system, uri-
They react with acids to form crystalline salts without
nary tract, gastrointestinal and biliary systems, as well
the production of water.
as on the skin. (Cowan, 1999)
Majority of alkaloids exist in solid such as atropine,
Medicinal plants are increasingly gaining acceptance
some as liquids containing carbon, hydrogen, and ni-
even among the literates in urban settlements, proba-
trogen. Most alkaloids are readily soluble in alcohol
bly due to the increasing inefficacy of many modern
and though they are sparingly soluble in water, their
drugs used for the control of many infections such as
salts of are usually soluble. The solutions of alkaloids
typhoid fever, gonorrhoea, and tuberculosis as well as
are intensely bitter. These nitrogenous compounds
increase in resistance by several bacteria to various
function in the defence of plants against herbivores
antibiotics and the increasing cost of prescription
and pathogens, and are widely exploited as pharma-
drugs, for the maintenance of personal health. Unfor-
ceuticals, stimulants, narcotics, and poisons due to
tunately, rapid explosion in human population has
their potent biological activities. In nature the alkaloids
made it almost impossible for modern health facilities
exist in large proportions in the seeds and roots of
to meet health demands all over the world, thus put-
plants and often in combination with vegetable acids.
ting more demands on the use of natural herbal health
Alkaloids have pharmacological applications as anes-
remedies.
thetics and CNS stimulants. More than 12, 000-
Current problems associated with the use of antibiot- alkaloids are known to exist in about 20% of plant spe-
ics, increased prevalence of multiple-drug resistant cies and only few have been exploited for medicinal
Table 1: Methods for detection of alkaloids
Reagents Composition of the reagent Result
Meyers reagent Potassium mercuric iodide solution Cream precipitate
Wagners reagent Iodine in potassium iodide Reddish-brown precipitate
Tannic acid Tannic acid Precipitation
Hagers reagent A saturated solution of picric acid Yellow precipitate
Solution of potassium bismuth
iodide potassium chlorate, a drop Orange or reddish-brown
Dragendorffs reagent of hydrochloric acid, evaporated precipitate (except with
to dryness, and the resulting caffeine and a few other
residue is exposed to ammonia alkaloids
vapour
124 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
purposes. The name alkaloid ends with the suffix ine preparations. (Cowann, 1999) Amygdalin has been
an plant-derived alkaloids in clinical use include the used in the treatment of cancer (HCN liberated in
analgesics morphine and codeine, the muscle relaxant stomach kills malignant cells), and also as a cough sup-
(+) -tubocurarine, the antibiotics sanguinafine and ber- pressant in various preparations. Excessive ingestion of
berine, the anticancer agent vinblastine, the an- cyanogenic glycosides can be fatal. Some foodstuffs
tiarrythmic ajmaline, the pupil dilator atropine, and the containing cyanogenic glycosides can cause poisoning
sedative scopolamine. Other important alkaloids of (severe gastric irritations and damage) if not properly
plant origin include the addictive stimulants caffeine, handled. To test for O-glycosides, the plant samples
nicotine, codeine, atropine, morphine, ergotamine, are boiled with HCl/H2O to hydrolyse the an-
cocaine, nicotine and ephedrine. Amino acids act as thraquinone glycosides to respective aglycones, and an
precursors for biosynthesis of alkaloids with ornithine aqueous base, e. g. NaOH or NH4OH solution, is added
and lysine commonly used as starting materials. to it. For C-glycosides, the plant samples are hydro-
lysed using FeCl3/HCl, and and an aqueous base, e. g.
2.2 GLYCOSIDES
NaOH or NH4OH solution, is added to it. In both cases a
Glycosides in general, are defined as the condensation pink or violet colour in the base layer after addition of
products of sugars (including polysaccharides) with a the aqueous base indicates the presence of glycosides
host of different varieties of organic hydroxy (occa- in the plant sample.
sionally thiol) compounds (invariably monohydrate in
2.3 Flavonoids
character), in such a manner that the hemiacetal entity
of the carbohydrate must essentially take part in the Flavonoids re important group of polyphenols widely
condensation. Glycosides are colorless, crystalline car- distributed among the plant flora. Stucturally, they are
bon, hydrogen and oxygen-containing (some contain made of more than one benzene ring in its structure (a
nitrogen and sulfur) water-soluble phytoconstituents, range of C15 aromatic compounds) and numerous re-
found in the cell sap. Chemically, glycosides contain a ports support their use as antioxidants or free radical
carbohydrate (glucose) and a non-carbohydrate part scavengers. (Yadav et al., 2003) The compounds are
(aglycone or genin). Alcohol, glycerol or phenol repre- derived from parent compounds known as flavans.
sents aglycones. Glycosides are neutral in reaction and Over four thousand flavonoids are known to exist and
can be readily hydrolyzed into its components with some of them are pigments in higher plants. Quercetin,
ferments or mineral acids. Glycosides are classified on kaempferol and quercitrin are common flavonoids pre-
the basis of type of sugar component, chemical nature sent in nearly 70% of plants. Other group of flavonoids
of aglycone or pharmacological action. The rather older include flavones, dihydroflavons, flavanflavonols, an-
or trivial names of glycosides usually has a suffix in thocyanidins, proanthocyanidins, calchones and cate-
and the names essentially included the source of the chin and leuco anthocyanidins.
glycoside, for instance: strophanthidin from Strophan-
2.4 Phenolics
thus, digitoxin from Digitalis, barbaloin from Aloes,
salicin from Salix, cantharidin from Cantharides, and Phenolics, phenols or polyphenolics (or polyphenol
prunasin from Prunus. However, the systematic names extracts) are chemical components that occur ubiqui-
are invariably coined by replacing the ose suffix of tously as natural colour pigments responsible for the
the parent sugar with oside. This group of drugs are colour of fruits of plants. Phenolics in plants are mostly
usually administered in order to promote appetite and synthesized from phenylalanine via the action of
aid digestion. phenylalanine ammonia lyase (PAL). They are very im-
portant to plants and have multiple functions. The
Glycosides are purely bitter principles that are com-
most important role may be in plant defence against
monly found in plants of the Genitiaceae family and
pathogens and herbivore predators, and thus are ap-
though they are chemically unrelated but possess the
plied in the control of human pathogenic infections.
common property of an intensely bitter taste. The bit-
(Liu, 2004) They are classified into (i) phenolic acids
ters act on gustatory nerves, which results in increased
and (ii) flavonoid polyphenolics (flavonones, flavones,
flow of saliva and gastric juices. Chemically, the bitter
xanthones and catechins) and (iii) non-flavonoid poly-
principles contain the lactone group that may be diter-
phenolies. Caffeic acid is regarded as the most com-
pene lactones (e. g. andrographolide) or triterpenoids
mon of phenolic compounds distributed in the plant
(e. g. amarogentin). Some of the bitter principles are
flora followed by chlorogenic acid known to cause al-
either used as astringents due to the presence of tan-
lergic dermatitis among humans. Phenolics essentially
nic acid, as antiprotozoan, or to reduce thyroxine and
represent a host of natural antioxidants, used as
metabolism. Examples include cardiac glycosides (acts
nutraceuticals, and found in apples, green-tea, and
on the heart), anthracene glycosides (purgative, and
red-wine for their enormous ability to combat cancer
for treatment of skin diseases), chalcone glycoside
and are also thought to prevent heart ailments to an
(anticancer), amarogentin, gentiopicrin, androgra-
appreciable degree and sometimes are anti-
pholide, ailanthone and polygalin reported that ex-
inflammatory agents. Other examples include flavones,
tracts of plants that contain cyanogenic glycosides are
rutin, naringin, hesperidin and chlorogenic.
used as flavouring agents in many pharmaceutical
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 125
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
volatile oils or ethereal oils. They mostly contribute to process involves the use of heavy duty centrifugal de-
the odoriferous constituents or essences of the aro- vices so as to ease the separation of oil/water emul-
matic plants that are used abundantly in enhancing the sions invariably formed and with the advent of modern
aroma of some spices. Essential oils are either secreted mechanical devices the oil output has increased im-
either directly by the plant protoplasm or by the hy- pressively.
drolysis of some glycosides and structures such as di-
The extraction processes can be carried out with ei-
rectly Plant structures associated with the secretion of
thervolatile solvents (e. g hexane, petroleum ether or
essential oils include: Glandular hairs (Lamiaceae e. g.
benzene) resulting into the production of floral con-
Lavandula angustifolia), Oil tubes (or vittae) (Apiaceae
cretes- oils with solid consistency and partly soluble in
eg. Foeniculum vulgare, and Pimpinella anisum (Ani-
95% alcohol, or non volatile solvents (tallow, lard or
seed), modified parenchymal cells (Piperaceae e. g.
olive oil) which results in the production of perfumes.
Piper nigrum - Black pepper), Schizogenous or lysi-
Examples of volatile oils include amygdaline (volatile oil
genum passages (Rutaceae e. g. Pinus palustris - Pine
of bitter almond), sinigrin (volatile oil of black mus-
oil. Essential oils have been associated with different
tard), and eugenol occurring as gein (volatile oil of
plant parts including leaves, stems, flowers, roots or
Geum urbanum).
rhizomes. Chemically, a single volatile oil comprises of
more than 200 different chemical components, and 2.10 Steroids
mostly the trace constituents are solely responsible for
Plant steroids (or steroid glycosides) also referred to as
attributing its characteristic flavour and odour. (Liu,
cardiac glycosides are one of the most naturally oc-
2004) Essential oils can be prepared from various plant
curring plant phytoconstituents that have found thera-
sources either by direct steam distillation, expression,
peutic applications as arrow poisons or cardiac drugs.
extraction or by enzymatic hydrolysis. Direct steam
(Liu, 2004) The cardiac glycosides are basically steroids
distillation involves the boiling of plant part in a distilla-
with an inherent ability to afford a very specific and
tion flask and passing the generated steam and volatile
powerful action mainly on the cardiac muscle when
oil through a water condenser and subsequently col-
administered through injection into man or animal.
lecting the oil in florentine flasks. Depending on the
Steroids (anabolic steroids) have been observed to
nature of the plant source the distillation process can
promote nitrogen retention in osteoporosis and in
be either water distillation, water and steam distilla-
animals with wasting illness. Caution should be taken
tion or direct distillation. Expression or extrusion of
when using steroidal glycosides as small amounts
volatile oils is accomplished by either by sponge
would exhibit the much needed stimulation on a dis-
method, scarification, rasping or by a mechanical proc-
eased heart, whereas excessive dose may cause even
ess. In the sponge method, the washed plant part e. g.
death. Diosgenin and cevadine (from Veratrum veride)
citrous fruit (e. g., orange, lemon, grape fruit, berga-
are examples of plant steroids.
mot) is cut into halves to remove the juice completely,
rind turned inside out by hand and squeezed when the 3. ISOLATION OF NATURAL PRODUCTS
secretary glands rupture. The oozed volatile oil is col-
As time passes new separation techniques and analysis
lected by means of the sponge and subsequently
are being introduced to separate different nature of
squeezed in a vessel. The oil floating on the surface is
compounds like alkaloids, glycosides, steroids, sapon-
separated. For the the scarification process the appara-
ins, tannins, flavonoids etc. Carbohydrates, fats, and
tus Ecuelle a Piquer (a large bowl meant for pricking
proteins are considered more valuable as they are hav-
the outer surface of citrus fruits) is used. It is a large
ing dietetic importance. (Bohlin, 1998), (Rahman, 1989)
funnel made of copper having its inner layer tinned
Many starches and gums are used in pharmacy but lack
properly. The inner layer has numerous pointed metal
any marked pharmacological action; are used as
needles just long enough to penetrate the epidermis.
binder, viscosity builder and as hydrocolloid to increase
The lower stem of the apparatus serve two purposes;
the stability of emulsions and suspensions.
first, as a receiver for the oil; and secondly, as a handle.
After GATT there is a great surge to find out immense
Now, the freshly washed lemons are placed in the bowl
potential of traditional and other herbal drugs to pre-
and rotated repeatedly when the oil glands are punc-
vent and cure diseases. Several extraction, fractiona-
tured (scarified) thereby discharging the oil right into
tion/separation and isolation methods are developed
the handle. The liquid, thus collected, is transferred to
after which isolation of the active moiety and their
another vessel, where on keeping the clear oil may be
chemical examination is performed. Microwave extrac-
decanted and filtered. For the rasping process, the
tion, sonication, lyophilization, spray drying and vac-
outer surface of the peel of citrus fruits containing the
uum drying have also been employed with good re-
oil gland is skilfully removed by a grater. The raspings
sults. Generally the extraction is based on pharmacol-
are now placed in horsehair bags and pressed strongly
ogical activity rather than chemical nature of com-
so as to ooze out the oil stored in the oil glands. Ini-
pounds. Further studies are carried out after the isola-
tially, the liquid has a turbid appearance but on allow-
tion of the active moiety to confirm the compound.
ing it to stand the oil separates out which may be de-
(Abo et al., 1991) The phytochemical investigation of a
canted and filtered subsequently. The mechanical
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 127
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 129
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
130 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
al., 2009), (Fennell et al., 1994) According to the cus- There is no single perfect method for extraction, purifi-
tomary definition in this technology solvents are, under cation and isolation of compound. The first fractiona-
normal conditions, volatile, usually organic liquids ca- tion step is general extraction of secondary metabolite
pable of dissolving other gaseous, liquid or solid sub- which is done either by using single all-purpose sol-
stances without either themselves or the dissolved vents such as methanol or by using petroleum ether to
substance being chemically altered. Ln pharmaceutical defatted the material. Methanol dissolves most of the
technology, this is not always the case, as solvents such secondary metabolites and enhancing their release
as oils that are not volatile under normal conditions are from cellular matrix/cell surface. (Fennelll et al., 1994),
also used. (Yadav, 2003) The only inorganic solvents (Jakhetia, 2010) Filtration or centrifugation is the first
used in pharmacy are water and CO2, although liquids basic technique used to separate insoluble material
such as ammonia, sulphur dioxide and hydrocyanic acid and filtrate that contain dissolved secondary metabo-
solutions are used for special purposes in pharmaceuti- lite. The choice of extraction procedure and extraction
cal processing. (Vlietinck, 2010) Water, pure organic solvent depends on the physicochemical nature of sec-
liquids and mixtures of organic liquids with water or ondary metabolite, nature of plant material (fresh
with other organic\ liquids are used as extraction sol- parts, dried parts) and their physical state (particle
vents. These organic liquids are nearly always hydro- size). Fresh plants (leaves) are first homogenized or
carbons and their derivatives such as halogenated hy- macerated with alcohol prior to extraction. (Yadav et
drocarbons, alcohol, esters, ketones, ethers, oils, etc. al., 2003) An idea about properties of interfering com-
Selectivity, ease of handling, econorny, protection of pound provides best criteria for solvent selection for
the environment and safety are major factors to be extraction and to select method of extraction. So prior
considered in the choice of a suitable solvent or of a to extraction a major emphasis must be given on na-
mixture of several solvents. (Firn, 2010) The last two ture of interfering compounds.
factors in particular are becoming increasingly impor-
The 3 stages are basic
tant due to the legislators sharpened attention to
them. This has become so critical that many manufac- 1. First step of extraction involves solvent penetration
turers are making serious attempts to design extraction into herb cells/tissues, solubilization of secondary me-
processes so that the smallest possible number of tabolites and finally release the dissolved secondary
'safe' liquids, e. g. water, lower alcohol and in special metabolites in solvent of extraction. Solvents of vary-
areas, super critical gases are used. It must not be for- ing polarity are used alone or in combinations for ex-
gotten here that traditional plant extracts must satisfy traction depend on component. So a large proportion
the requirements of the pharmacopoeias. This is usu- of the unwanted material is removed. (Maceration,
ally possible only when the specified menstrum is used. Digestion, Decoction, Soxhlet extraction, Supercritical
A degree of choice is offered by using cheaper metha- chromatography, Hydro-distillation, Enflurage, Eculle,
nol instead of expensive ethanol (on which duties have Supercritical fluid chromatography).
to be paid) if its polarity is equal to that of the required
2. Second step is fractionation with subsequent analy-
ethanol-water mixture. As a rule of thumb, a 10%
sis: Either open silica column or counter current distri-
higher methanol concentration roughly corresponds to
bution/liquid-liquid extraction is used to separate or
the required ethanol concentration. (Heinrich et al.,
fractionation of components. (Distillation, Sublimation,
2004) One essential consideration in the choice of suit-
Evaporation, Fractional crystallization, fractional distil-
able solvents is the question of whether the menstrum
lation, Sublimation, Fractional crystallization, fractional
remains wholly or partly in the end product. lf this is
distillation, GC, CCD).
the case, physiologically harmless solvents must be
used. lf the end-product is free of solvent, as is the 3. The third final stage is achieved by HPLC or TLC
case with dried extracts, or if it still contains only harm- which involves separation of desired component in
less components from the originally used solvent mix- adequate purity (TLC, GC, GLC, Mass, NMR, UV,
ture after further processing of the miscella, then for Fluorimerty)
reasons of selectivity and economy the choice can be
Extraction
made without considering the physiological properties
of the mixture used. Extraction is followed either by
4. 1. 4 Interfering Compounds Powdered dried material is directly extracted to
achieve extract.
Many naturally occurring compounds may interfere
with the isolation and purification of a desired bioac- First defeat the material and then extraction of de-
tive plant constituent like lipids, pigments, tannins, sired component.
plasticizers, silicon etc.
Fresh plants (e. g. leaves) can be homogenized or
4. 2 EXTRCTION METHOD macerated with alcohol.
Extraction of plant material Selection of extraction method depends on
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 131
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
By changing the polarity of solvents Cleaner extracts and less thermal degradation as
compared to conventional solvent extraction and
During the extraction of the herbal drugs the rinsing of steam distillation methods,
extractive substances out of disintegrated plant cells,
swelling of the drug plant material in order to increase Super critical carbon dioxide (SCO2) - with its particu-
the permeability of the cell walls, Penetration of the larly attractive properties such as non-toxicity, non-
solvent into the plant cells and swelling of the cells, flammability, non-corrosiveness, chemical inertness,
Dissolution of the extractive substances, Diffusion of low critical temperature (304"K), moderately low criti-
the dissolved extractive substances out of the plant cell cal pressure (73 atm), easy availability, co-effectiveness
and finally the dissolution of extractive substances out and environmental acceptability is the preferred sol-
of intact plant cells by diffusion take place. It has been vent for many super critical extractions. Liquid carbon
found that the liquorice root when extracted with the dioxide is completely miscible with components of es-
solvent (0.25% ammonia solution) it penetrates into sential oils like aldehyde, ketones, esters and alcohols.
the roots more rapidly and this process is accelerated At same time, proteins, starches, mineral salts and wa-
by raising the temperature. The steeping and swelling ter are insoluble in liquid carbon dioxide. Essential oils
process is strongly influenced by particle size and is obtained by liquid carbon dioxide extraction are supe-
more evident radically. Upon penetration into the rior to that obtained through steam distillation and
plant material, the solvent becomes enriched with ex- solvent extraction. Extraction of several natural prod-
tractive substances and hence the highest content of ucts such as pyrethrins from chrysanthemum flower,
extractive substances is found on the solvent front. essential oils from anise, caraway, clove, star anise,
Some general points should be considered in terms of cinnamon and ginger are increasingly done by this
the extraction process, such as the overall characteris- process. (Heinrich et al., 2004), (Abo et al., 1991).
tics of the secondary metabolites to be extracted (e. g. Solid phase extraction
some glycosides are thermolabile or pH-sensitive). Al-
though the normal practice is 'to apply 2iitanOarO Solid phase extraction is process of separation of dis-
technique to obtain a crude extract from a plant mate- solve and suspended component from liquid mixture
rial, e. g. an acid-base shakeout to prepare an alka- by using another component in the mixture according
loidal extract, because of the structural diversity within to their physical and chemical property. (Fennell et al.,
a given natural product group and their possible special 1994).
requirements, it is advisable to consult specific review Distillation
papers, and books in order to prevent the avoidable
loss of desired bioactive metabolites caused by the use Distillation may be defined as separation of compo-
of an inappropriate extraction technique. The most nents of a mixture of two or more liquids by virtue of
simple extraction processes employed may be classi- difference in their vapor pressure. There are three sys-
fied as follows: extraction with organic solvents: perco- tems of distillation-
lation, maceration, and extraction using a Soxhlet ap- Hydro distillation
paratus; and extraction with water: infusion, decoc-
tion, and steam distillation. (Voravuthikunchai et al., Hydro-steam distillation
2003), The most popular method of extraction is to use Steam distillation
a liquid solvent at atmospheric pressure, possibly with
the application of heat. Other methods include steam Hydro distillation
distillation, supercritical fluid extraction and the use of Hydro distillation is the oldest method being used for
liquefied gases under moderate pressure. The choice of separation of essential oil. In this method plant mate-
method depends on the factors listed above as well as rial is contact with boiling water in a crude metallic
132 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
distillation unit. This process use principle of osmotic tracted with strong alcohol to yield extracts. This is
press principle of osmotic pressure to diffuse oil from very much the same technique used in solvent extrac-
the oil glands. The essential oil of a plant consists of tions, where solvents are used instead of the hot oil as
many compounds which generally boil between 150 used in maceration. (Heinrich et al., 2004) 0, (Kar,
to 300 C. The vapors pass through a coiled tube con- 2007).
tained in a water bath and condensate is obtained at
Enfleurage (Extraction with cold fat)
the bottom of the condenser tube. The disadvantages
are that the heat is difficult to control and hence the Enfleurage is the process of extraction of fragrance by
rate of distillation is variable. Also the possibility exists absorbing it from flowers in contact with cold fats. This
for local overheating and "burning" of the charge process is adopted for fragrant flowers of jasmine and
which can lead to poorer quality oil. tuberose, which continue to manifest their characteris-
tic fragrance even in plucked condition. Fats should be
Hydro-steam distillation
saturated and odorless to prevent entrance of fat
To overcome the drawback of water distillation, modi- odors. Refined lard or beef suet are preferred. Fat is
fications in techniques was developed. In this tech- thinly layered on both sides of a glass plate supported
nique plant material is supported on a perforated grid on a rectangular wooden frame or chassis. Fresh fra-
or screen inserted at some distance above bottom of grant flowers are lightly layered on fat coated chassis.
still. Water filled below the grid is heated which pro- Enfleurage gives a much greater yield of flower oil than
duce saturated and wet steam; produced steam pass other methods. Despite this advantage, enfleurage has
through plant material and vaporized essential oil. lately been replaced by extraction with volatile sol-
vents because enfleurage is a very delicate and lengthy
Steam distillation
process requiring much experience and labour. (Aded-
A process of extracting essential oils from plant prod- apo et al., 2005), (Doughari et al., 2009).
ucts through a heating and evaporation process is
Extraction with volatile solvents
known as steam distillation. Steam distillation is a
popular method for the extraction of volatile oils (es- Principle of extraction with volatile solvents is simple.
sential oils) from plant material. This can be carried out Fresh flowers are charged into specially constructed
in a number of ways. One method is to mix the plant extractors and extracted systematically at room tem-
material with water and to heat to boiling (distillation perature, with a carefully purified solvent usually pe-
with water). The vapors are collected and allowed to troleum ether. Solvent penetrates flowers and dissolve
condense, and the oil separated from the water.. It natural flower perfume together with some waxes and
resembles hydro-steam distillation except that no wa- albumins and colouring matter. Solution is subse-
ter is kept in bottom of still. This method is efficient quently pumped into an evaporator and concentrated
and gives higher yields. However, it is not generally at a low temperature. After the solvent is completely
employed to delicate flowers. To maximize the yields driven off in vacuum, concentrated flower oil is ob-
of the oils, precautions must be taken to ensure effi- tained. Thus, temperature applied during entire proc-
cient condensation of the steam and vaporized oil and ess is kept at a minimum; live steam as in case of distil-
collection of the condensate in such a way as to pre- lation, does not exert its action upon delicate constitu-
vent loss of the volatile material. However, to avoid ents of flower oil. Compared with distilled oils, ex-
risk of explosion, a completely closed system must not tracted flower oils more truly represent natural per-
be used. The advantages of this type of "dry" steam fume as originally present in flowers. (Jakhetia et al.,
distillation are that it is relatively rapid, therefore 2010).
charging and emptying the still is much faster and en-
Expression
ergy consumption is lower. The rapid distillation is also
less likely to damage those oils which contain reactive It is physical process in which pressure is applied to
compounds, e. g. esters. However, it cannot be used squeeze the oil out of the material or juice from plant.
where the oil contains hydrolysable components such This was usually achieved by a tincture press. This
as esters or those that are easily. (Doughari et al., method is employed when essential oils are thermo
2009), (Vlietinck, 2010). sensitive. It is used for isolating essential oils from
lemon and orange peels. In general, expression in-
Maceration (Extraction with hot fat)
volves squeezing any plant material at great pressures
Maceration is process of extraction with hot oil or fat. to press out oils or other liquids. The process is carried
In maceration, oil cells of fragrant flowers are ruptured out by hand-operated presses or crushes in isolated
by immersion in a hot fat or oil at 60-70C which in rural areas or by gigantic mechanical presses in indus-
turn absorbs essential oils. Fat is separated from spent trial centres. (Fennell et al., 1994), (Voravuthikurchai et
flowers and reused for absorbing fragrance from next al., 2003).
batch of fresh flowers. Fat retained by flowers is recov-
ered by hydraulic pressing. Resultant perfumed po-
made is frequently marketed as such but is often ex-
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 133
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
134 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
lar and can be extracted into petroleum ether. Xantho- separation using ultra filtration, gel filtration, ion-
phylls are more polar therefore be extracted into etha- exchange chromatogramphy or electrophoresis.
nol or mixtures of ethanol and less polar solvents such (Doughari et al., 2009), (Jakhetia et al., 2010).
as chloroform. (Fennell et al., 1994).
Polysaccharides
Glycosides
Polysaccharides are polymers of sugars or sugar deriva-
Glycosides are relatively polar in nature and its polarity tives. Generally, there are three types of sugar poly-
depends on both number and type of sugar the struc- mers - those that are completely water soluble, those
ture of the aglycone. Most glycosides can be extracted that partially dissolve in water and swell to form gels
with polar solvents such as acetone, ethanol, metha- and lastly, those that are water insoluble. Polysaccha-
nol, water or mixtures of these. However, cardiac gly- rides that totally or partially dissolve in water can be
cosides have bulky steroidal aglycone, which shows extracted using cold or warm water.
appreciable solubility in chloroform. When extracting
Volatile Oils
into water, and sometime enzymatic breakdown also
possible. This will not occur if boiling water is used or if Volatile oils are the odorous principals found in various
significant proportions of alcohol or ammonium sul- plant parts. Because they evaporate in air at ordinary
phate are added to the extract. In some cases, it may temperatures, they are called volatile oils, ethereal oils
be the aglycone rather than the glycoside that is to be or essential oils.
extracted, and this requires hydrolytic separation of
4.3 ISOLATION METHODS
the aglycone and sugar before or after extraction. (Abo
et al., 1991). While different techniques have evolved for the extrac-
tion of the phytoconstituents so as to obtain crude
Phenolic Compounds
extracts (complex mixtures), fractions (simpler mix-
These can exist as free phenols or in glycosidic form. tures) and isolated (pure) components from natural
Due to the multiplicity of hydroxyl functions, phenols sources. These are usually then subjected to a number
tend to be relatively polar and dissolve in aqueous al- of further analytical investigations in order to obtain
cohols. As they are weak acids, they may also be ex- more information on the properties of their constitu-
tracted or partitioned into aqueous alkali as phenolate ent substances.
salts. A problem encountered with phenolic com-
Broadly, the nature of further investigations after ex-
pounds is that they can undergo extensive polymeriza-
traction of a crude drug is of three types:
tion a reaction by the action of polyphenol oxidizes.
This reaction will responsible for the development of 1. Qualitative chemical analysis - determination of the
brown coloration in damaged plant material when ex- nature of the constituents of a mixture or the structure
posed to the air and in certain extracts. The polymeri- of an isolated compound.
zation reaction is catalyzed by acid. (Kamboj, 2010).
2. Quantitative chemical analysis - determination of
Proteins the purity of an isolated substance or the concentra-
tion of a single substance or group of substances in a
Due to the presence of free carboxylic, amino and phe-
mixture
nolic groups on amino acid side- chains in proteins,
most can be ionized at high or low pH values. The pH at 3. Bioassays - determination of the biological or phar-
which no net charge is carried is known as the isoelec- macological activity of substances and the dose range
tric point and this will vary with each protein depend- over which they exert their effects.
ing on the constituent amino acids. At pH values above
The amount of fraction available is possibly the most
the isoelectric point, the protein carries a net negative
important factor in making decisions about its future
charge, and the pH values below the isoelectric point, a
treatment and analysis. Investigative methods can be
net positive charge is carried. As a result of this, most
either non-destructive or destructive. Non-destructive
proteins can be extracted with water, buffers, dilute
methods mean that the sample can be recovered and
acid or base or simple salt solutions. However, more
used for other tests. Some physico-chemical proce-
lipophilic proteins require the use of 70-80% alcohol.
dures, e. g. chemical tests, analytical chromatography,
Selective precipitation of groups of proteins in a crude
mass spectrometry and all biological testing proce-
protein extract can be achieved by gradual addition of
dures are destructive i. e. they ule up the sample and
acetone, ethanol or ammonium sulphate. Conversely,
generally it cannot be recovered. Another important
for proteins with a greater solubility in salt solutions
factor is that th6 different types of analysis require the
that water, e. g. globulins, a crude protein mixture can
sample to be present in different types of medium. lt is
be extracted in 10% sodium chloride solution and the
always desirable to supply the fractions as solids, which
globulins precipitates by addition of water. For prola-
can be weighed accurately and reconstituted in the
mines, extraction in 70-80% alcohol can be followed by
appropriate solvents. The solid forms are usually pro-
precipitation by dilution with water. The precipitation
duced from solutions by evaporation under reduced
step can be followed by resolubilization and further
pressure to minimize decomposition or, in the case of
136 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
aqueous solutions, by freeze-drying, sometimes called ing with a mixture of the acid salts, it is possible to
lyophilisation. (Heiurich, et al., 2004), (Kamboj, 2010). fractionally liberate the acids by addition of mineral
acids. Sodium salts of acids on treatment with dilute
Isolation techniques
HCl yield free organic acids. (Kar, A 2007).
1) Sublimation
Fractional crystallization
2) Distillation
The method is used on the difference in solubility of
3) Evaporation the components of a mixture in a particular solvent.
Frequently, derivatives of the particular components
4) Fractional liberation
are used (picrates of alkaloids, osazones of sugars).
5) Fractional crystallization (Yadav, et al., 2003).
6) Fractional distillation Chromatography
7) Chromatography Chromatography represents a group of techniques for
separation of compounds of mixtures by their continu-
Fractionation
ous distribution between two phases, one of which is
All separation processes involve the division of a mix- moving past the other.
ture into a number of discrete fractions. These frac-
Chromatographic fingerprinting and marker com-
tional may be physically discrete fraction. In which suc-
pound analysis
cessive partition into diethyl ether, chloroform and
ethyl acetate will often afford in turn flavonones and Chromatographic fingerprint of an Herbal Medicine
flavonols, methoxylated flavonoids and flavonoid (HM) is a chromatographic pattern of the extract of
monoglycosides. Di- and polyglycosylated flavonoids some common chemical components of pharmacologi-
will remain in the residual aqueous phase. Saponins are cally active and or chemical characteristics. This chro-
water soluble compounds, and the crude saponin frac- matographic profile should be featured by the funda-
tions are water soluble compounds, and the crude mental attributions of integrity and fuzziness or
saponin fractions may be obtained. The alkaloids are sameness and differences so as to chemically rep-
basic compounds may be extracted either into aqueous resent the HM investigated. It is suggested that with
acid solution after removal of neutral impurities with the help of chromatographic fingerprints obtained, the
an organic solvent, or by treating the groups wet plant authentication and identification of herbal medicines
material with an alkaline substance such as CaCO3 can be accurately conducted (integrity) even if the
powder and extracting with diethyl ether after stand- amount and/or concentration of the chemically charac-
ing overnight. Specific literature should be consulted teristic constituents are not exactly the same for dif-
for specific plant extraction problems. (Voravuthikur- ferent samples of this HM (hence, fuzziness) or, the
chai, et al., 2003). chromatographic fingerprints could demonstrate both
the sameness and differences between various
Sublimation
samples successfully.
Sublimation is used for isolation of caffeine from tea,
Thus, we should globally consider multiple constituents
for its purification of materials present in a crude ex-
in the HM extracts, and not individually consider only
tract and for separation of camphor. (Kamboj, 2010).
one and/or two marker components for evaluating the
Distillation quality of the HM products. However, in any HM and
its extract, there are hundreds of unknown compo-
Steam distillation is much used to isolate volatile oils
nents and many of them are in low amount. Moreover,
and hydrocyanic acid form plant material. The TAS
there usually exists variability within the same herbal
oven is used for steam distillation on a semi micro
materials. Hence it is very important to obtain reliable
scale for the direct transfer of volatile materials from a
chromatographic fingerprints that represent pharma-
powdered drug to thin layer plate. (Heinrich, et al.,
cologically active and chemically characteristic compo-
2004).
nents of the HM. In the phytochemical evaluation of
Fractional liberation herbal drugs, TLC is being employed extensively for the
following reasons:
Some compounds are separated by fractional libera-
tion from a mixture. A mixture of alkaloid salts in it enables rapid analysis of herbal extracts with
aqueous solution, when treated with aliquots of alkali, minimum sample clean-up requirement,
will give first the weakest base in the free salt followed
by base liberation in ascending order of basicity. If the it provides qualitative and semi quantitative infor-
mixture is shaken with an organic solvent after each mation of the resolved compounds and
addition, then a fractionated series of base will be ob- it enables the quantification of chemical constitu-
tained. A similar procedure is used for organic acids ents. Fingerprinting using HPLC and GLC is also car-
soluble in water immiscible solvents. In this case, start- ried out in specific cases.
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 137
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
In TLC fingerprinting, the data that can be recorded duced to the market as quickly as possible. Having
using a highperformance TLC (HPTLC) scanner includes available such a powerful purification technique makes
the chromatogram, retardation factor (Rf) values, the it possible to spend less time on the synthesis condi-
colour of the separated bands, their absorption spec- tions.
tra, max and shoulder inflection/s of all the resolved
b. Liquid Chromatography- Mass Spectroscopy (LC-
bands. All of these, together with the profiles on deri-
MS)
vatization with different reagents, represent the TLC
fingerprint profile of the sample. The information so In Pharmaceutical industry LC-MS has become method
generated has a potential application in the identifica- of choice in many stages of drug development. Recent
tion of an authentic drug, in excluding the adulterants advances includes electro spray, thermo spray, and ion
and in maintaining the quality and consistency of the spray ionization techniques which offer unique advan-
drug. HPLC fingerprinting includes recording of the tages of high detection sensitivity and specificity, liquid
chromatograms, retention time of individual peaks and secondary ion mass spectroscopy, later laser mass
the absorption spectra (recorded with a photodiode spectroscopy with 600 MHz offers accurate determina-
array detector) with different mobile phases. Similarly, tion of molecular weight proteins, peptides. Isotopes
GLC is used for generating the fingerprint profiles of pattern can be detected by this technique. (Cowan et
volatile oils and fixed oils of herbal drugs. Furthermore, al., 1999).
the recent approaches of applying hyphenated chro-
c. Liquid Chromatography- Nuclear Magnetic Reso-
matography and spectrometry such as High-
nance (LC-NMR)
Performance Liquid ChromatographyDiode Array De-
tection (HPLCDAD), Gas ChromatographyMass Spec- The combination of chromatographic separation tech-
troscopy (GCMS), Capillary Electrophoresis- Diode nique with NMR spectroscopy is one of the most pow-
Array Detection (CE-DAD), High- Performance Liquid erful and time saving method for the separation and
ChromatographyMass Spectroscopy (HPLCMS) and structural elucidation of unknown compound and mix-
High- Performance Liquid ChromatographyNuclear tures, especially for the structure elucidation of light
Magnetic Resonance Spectroscopy (HPLC NMR) could and oxygen sensitive substances. The online LC-NMR
provide the additional spectral information, which will technique allows the continuous registration of time
be very helpful for the qualitative analysis and even for changes as they appear in the chromatographic run
the on-line structural elucidation. automated data acquisition and processing in LC-NMR
improves speed and sensitivity of detection. The recent
Liquid chromatography
introduction of pulsed field gradient technique in high
a. Preparative high performance liquid chromatogra- resolution NMR as well as three-dimensional technique
phy improves application in structure elucidation and mo-
lecular weight information. These new hyphenated
There are basically two types of preparative HPLC. One
techniques are useful in the areas of pharmacokinetics,
is low pressure (typically under 5 bar) traditional HPLC,
toxicity studies, drug metabolism and drug discovery
based on the use of glass or plastic columns filled with
process.
low efficiency packingmaterials of large particles and
large size distribution. A more recent form PLC, Prepa- Gas chromatography
rative High Performance Liquid Chromatography (Prep.
a. Gas Chromatography Fourier Transform Infrared
HPLC) has been gaining popularity in pharmaceutical
spectrometry
industry. In preparative HPLC (pressure >20 bar), larger
stainless steel columns and packing materials (particle Coupling capillary column gas chromatographs with
size 10-30 m are needed. The examples of normal Fourier Transform Infrared Spectrometer provides a
phase silica columns are Kromasil 10 m, Kromasil 16 potent means for separating and identifying the com-
m, Chiralcel AS 20 m whereas for reverse phase are ponents of different mixtures.
Chromasil C18, Chromasil C8, YMC C18. The aim is to
b. Gas Chromatography-Mass Spectroscopy
isolate or purify compounds, whereas in analytical
work the goal is to get information about the sample. Gas chromatography equipment can be directly inter-
Preparative HPLC is closer to analytical HPLC than tra- faced with rapid scan mass spectrometer of various
ditional PLC, because its higher column efficiencies and types. The flow rate from capillary column is generally
faster solvent velocities permit more difficult separa- low enough that the column output can be fed directly
tion to be conducted more quickly In analytical HPLC, into ionization chamber of MS. The simplest mass de-
the important parameters are resolution, sensitivity tector in GC is the Ion Trap Detector (ITD). In this in-
and fast analysis time whereas in preparative HPLC, strument, ions are created from the eluted sample by
both the degree of solute purity as well as the amount electron impact or chemical ionization and sortored in
of compound that can be produced per unit time i. e. a radio frequency field; the trapped ions are then
throughput or recovery are important. This is very im- ejected from the storage area to an electron multiplier
portant in pharmaceutical industry of today because detector. The ejection is controlled so that scanning on
new products (Natural, Synthetic) have to be intro- the basis of mass-to-charge ratio is possible. The ions
138 JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140
trap detector is remarkably compact and less expen- difficult to separate into individual components. Com-
sive than quadrapole instruments. GC-MS instruments plex distillation procedure is used for solvent recovery.
have been used for identification of hundreds of com- An adequate care must be taken in disposing of sol-
ponents that are present in natural and biological sys- vents in a way that causes least damage to the envi-
tem (Madsen, et al., 1995). ronment. Most scientific establishments have standard
procedures for the disposal of organic solvents. (Kam-
Supercritical Fluid Chromatography (SFC)
boj, 2010).
Supercritical fluid chromatography is a hybrid of gas
Treatment of Drug Residue after Extraction
and liquid chromatography that combines some of the
best features of each. This technique is an important Further treatment of the drug residue is very essential
third kind of column chromatography that is beginning and more important in the context of extraction of
to find use in many industrial, regulatory and academic herbal drugs. This is necessary for several reasons as
laboratories. SFC is important because it permits the follows:
separation and determination of a group of com-
The drug residue contains considerable quantities of
pounds that are not conveniently handled by either gas
absorbed solution with valuable extractive sub-
or liquid chromatography. These compounds are either
stances
non-volatile or thermally labile so that GC procedures
are inapplicable or contain no functional group that The solvents used should be recovered.
makes possible detection by the spectroscopic or elec-
trochemical technique employed in LC. SFC has been There will be further uses of the drug residue, which
applied to a wide variety of materials including natural necessitate removal of the solvents which must not
products, drugs, foods and pesticides. be equated with recovery of the solvents)
Other Chromato-Spectrometric studies During extraction, the drug material, depending on its
nature and the species of plant from which it is ob-
The NMR techniques are employed for establishing tained, absorbs varying quantities of solvent and hence
connectivity between neighbouring protons and estab- swells up to varying degrees. The drug residue has a
lishing C-H bonds. INEPT is also being used for long species-dependent retention capacity for solvents,
range hetero nuclear correlations over multiple bond- which is known as the 'absorption capacity'. The sol-
ing. The application of Thin Layer chromatography vent can, in principle, be removed in two ways,
(TLC), High Performance Chromatography (HPLC) and
HPLC coupled with Ultra violate (UV) photodiode array (1) Expression of the drug residue
detection, Liquid Chromatography- Ultraviolet (LC-UV), (2) Expulsion of the solvent by warming with or with-
Liquid Chromatography-Mass Spectrophotometry out pressure reduction. (Voravuthikunchai et al., 2003)
(LCMS), electrospray (ES) and Liquid Chromatography-
5. CONCLUSION
Nuclear Magnetic Resonance (LC-NMR) techniques for
the separation and structure determination of antifun- Natural product drug discovery program a long term
gal and antibacterial plant compounds is on the in- capital-intensive program. Several extraction, frac-
crease frequently. Currently available chromatographic tionation/ separation and isolation methods are devel-
and spectroscopic techniques in new drug discovery oped after which isolation of the active moiety and
from natural products Currently, computer modelling their chemical examination is performed. Microwave
has also been introduced in spectrum interpretation extraction, sonication, lyophilization, spray drying and
and the generation of chemical structures meeting the vacuum drying have also been employed with good
spectral properties of bioactive compounds obtained results. Generally the extraction is based on pharma-
from plants. The computer systems utilise 1H, 13C, 2D- cological activity rather than chemical nature of com-
NMR, IR and MS spectral properties. Libraries of spec- pounds. Further studies are carried out after the isola-
tra can be searched for comparison with complete or tion of the active moiety to confirm the compound.
partial chemical structures. Hyphenated chroma- The phytochemical investigation of a plant involves the
tographic and spectroscopic techniques are powerful selection, collection, identification and authentication,
analytical tools that are combined with high through- extraction of the plant material (first fractionation),
put biological screening in order to avoid re-isolation of fractionation/separation (second fractionation) and
known compounds as well as for structure determina- isolation (third fractionation) of the constituents, char-
tion of novel compounds. Hyphenated chroma- acterization of the isolated compounds and investiga-
tographic and spectroscopic techniques include LC tion of the biosynthetic pathways of particular com-
UVMS, LCUVNMR, LCUVESMS and GCMS. pound, quantitative evaluations and pharmacological
activities. Thus, this review compiles the recent litera-
4. 4 Solvent Recycling
ture with special focus on various approaches for ex-
For both economical and ecological reasons the recov- traction& isolation methods of phytoconstituents pre-
ery of solvent is important. Use of single solvent is sent in plants.
much better rather than azeotropic mixture which is
JK Welfare & Pharmascope Foundation | International Journal of Research in Pharmaceutical Sciences 139
Charishma Yalavarthi and Thiruvengadarajan V. S (2013) Int. J. Res. Pharm. Sci., 4(2), 123-140