Introduction to secondary metabolites

INTRODUCTION TO SECONDARY
METABOLITES
GLYCOSIDE
RESIN
TANNIN
ALKALOID
FLAVANOID
VOLATILE OIL

Primary metabolites
• Primary metabolites are compounds that are commonly
produced by all plants and that are directly used in plant growth
and development.
• They do not possess biological or therapeutic activity.
• They are widely distributed in plants in large quantites.
• These are directly involved in growth and development of plant.
• Non expensive to isolate from plant.
• Examples : Carbohydrates, Proteins, and Lipids.

Secondary metabolites
• These are biosynthesized from the primary metabolites e.g-
alkaloids, glycosides, tannins, flavonoids, volatile oils and resin.
• They are limited in distribution.
• Restricted to taxonomic group
• Not directly involved in the growth and development
• Possess biological or pharmacological action on humans and
animals
• Serve as defensive, protective chemicals against microorganisms,
insects and higher herbivorous animals
• Present in small quantities
• Generally expensive to isolate from plant compared to primary
metabolites e.g- quinine form cinchona bark, Curcumin from
Turmeric.

• Glycosides are define as organic compound from plants and animal
source, which upon hydrolysis gives one or more sugar moieties along
with a non-sugar moiety.
• Sugar portion is called glycon
• non-sugar portion is called aglycon or genin.
• Sugar moiety can be glucose, xylose, galactose, mannose, rhamnose,
etc.
• While non-sugar moiety or aglycon moiety can be alcohol, phenol,
anthraquinone, sterol, flavanol, etc.
• Linkage between sugar and non-sugar portion is usually called as
glycosidic linkage.

Physicochemical properties of glycosides
• Colourless, solid, amorphous, non-volatile (flavonoid yellow,
anthraquinone-red or orange).
• Give positive reaction with Molisch's and Fehling's test (after
hydrolysis).
• They are water soluble compounds, insoluble in organic solvents.
• Most of them have bitter taste. (Except: glycyrrhizin, stevioside).
• Odorless except saponin (glycyrrhizin)
• Glycosides hydrolyzed by using mineral acids, alkali, temperature
or by using enzymes. And results into breakage of glycosidic
bond.
• Glycone part: water soluble, insoluble in the organic solvents.
• Aglycone part: water insoluble, soluble in the organic solvents.

Classification of glycosides :
1. BASED ON TE NATURE OF SUGAR MOITY :
• Glucoside: sugar portion is glucose
• Rhamnoside: sugar portion is rhamnose
• Pentoside sugar portion is pentose
• Fructoside sugar portion is fructose
• Arabinoside sugar portion is arabinose

2. BASED ON THE CHEMICAL NATURE OF NON
SUGAR MOIETY:

BASED ON LINKAGE BETWEEN GLYCON AND AGLYCON
PORTION:
• OH groups reacting with any of the following medicates like, OH, CH, SH, NH
product in aglycon part and forms different types of glycosidic bond.
• O-glycosides:
• Sugar molecule is combined with phenol or –OH group of aglycon, for
example, Salicin, cardiac glycosides, anthraquinone glycosides like
sennosides etc.
• N-glycosides:
• Sugar molecule is combined with N of the –NH (amino group) of aglycon,
for example, nucleosides
• S-glycosides:
• formed by the condensation of sulphohydryl (thiol - SH) group aglycon to
OH group of glycon.
• Sinigrin in black mustard.
• C-glycosides:
• Sugar molecule is directly attached with C—atom of aglycon, for example,
Anthraquinone glycosides like Aloin in Aloe vera, Barbaloin, Cascaroside
and Flavanoid glycosides, etc.

• BASED ON THERAPEUTIC NATURE OF GLYCOSIDE:
• Cardiac glycoside EX: Digitalis
• Laxative glycoside EX: Senna
• Anti-ulcer glycoside EX: Liquorice
• Bitter glycoside EX : Gentian
• Steroidal glycoside : Fenugreek seeds

Chemical test for glycosides
• Chemical tests for Anthraquinone glycoside :
A. Borntrager’s test :
• To 1 gm of drug add 5–10 ml of dilute HCl boil on water bath for 10 min
and filter.
• Filtrate was extracted with CCl4 / benzene and add equal amount of
ammonia solution to filtrate and shake.
• Formation of pink or red colour in ammonical layer due to presence of
anthraquinone moiety.
B. Modified borntrager’s test :
• To 1 gm of drug, add 5 ml dilute HCl followed by 5 ml ferric Chloride
• Boil for 10 min on water bath, cool and filter, filtrate was extracted with
carbon tetrachloride or benzene and add equal volume of ammonia
solution, formation of pink to red colour due to presence of
anthraquinone moiety.
• This is used C-type of anthraquinone glycosides.

• Chemical Tests for Saponin Glycosides
• Haemolysis test
• A drop blood on slide was mixed with few drops of aq. Saponin solution,
RBC’s becomes ruptured in presence of saponins.
• Foam test
• To 1 gm of drug add 10–20 ml of water, shake for few minutes,
formation frothing in presence of saponins.
• Chemical Tests for Flavonoid Glycosides
• Ammonia test
• Filter paper dipped in alcoholic solution of drug was exposed to
ammonia vapor. Formation of yellow spot on filter paper.
• Shinoda test
• To the alcoholic extract of drug magnesium and dil. HCl was added,
formation of red colour indicates the presence of flavonoids.
• Vanillin HCl test
• Vanillin HCl was added to the alcoholic solution of drug, formation of
pink colour due to presence of flavonoids.

• Chemical tests for cardiac glycosides :
• Keller-kiliani test
• To the alcoholic extract of drug equal volume of water and 0.5 ml of
strong lead acetate solution was added, shaked and filtered. Filtrate was
extracted with equal volume of chloroform. Chloroform extract was
evaporated to dryness and residue was dissolved in 3 ml of glacial acetic
acid followed by addition of few drops of FeCl3 solution.
• The resultant solution was transferred to a test tube containing 2 ml of
conc. H2SO4. Reddish brown layer is formed, which turns bluish green
after standing due to presence of digitoxin.
• Legal test
• To the alcoholic extract of drug equal volume of water and 0.5 ml of
strong lead acetate solution was added, shaked and filtered. Filtrate was
extracted with equal volume of chloroform and the chloroform extract
was evaporated to dryness. The residue was dissolved in 2 ml of
pyridine and sodium nitropruside 2 ml was added followed by addition
of NaOH solution to make alkaline.
• Formation of pink colour in presence of glycosides.

• Baljet test
• Thick section of leaf of digitalis or the part of drug containing cardiac
glycoside, when dipped in sodium picrate solution, it forms yellow to
orange colour in presence of aglycones or glycosides.
• 3,5-dinitro benzoic acid test
• To the alcoholic solution of drug few drops of NaOH followed by 2%
solution of 3,5-dinitro benzoic acid was added. Formation of pink
colour indicates presence of cardiac glycosides.
• Chemical Tests for Coumarin Glycosides
• FeCl3 test
• To the concentrated alcoholic extract of drug few drops of alcoholic
FeCl3 solution was added. Formation of deep green colour, which turned
yellow on addition of conc. HNO3 indicates presence of coumarins.
• Fluorescence test
• The alcoholic extract of drug was mixed with 1N NaOH solution (one ml
each). Development of blue-green fluorescence indicates presence of
coumarins.

• Chemical Tests for Cynophoric (C Ξ N) Glycoside
• Sodium picrate test
• Powdered drug was moistened with water in a conical flask and few
drops of conc. Sulphuric acid was added. Filter paper moistened with
sodium picrate solution followed by sodium carbonate solution was
trapped on the neck of flask using cork. Formation of brick red colour
due to volatile HCN in presence of cynophoric glycosides takes place.
• Chemical Tests for Steroid and Triterpenoid Glycosides
• Libermann burchard test
• Alcoholic extract of drug was evaporated to dryness and extracted with
CHCl3, add few drops of acetic anhydride followed by conc. H2SO4 from
side wall of test tube to the CHCl3 extract. Formation of violet to blue
coloured ring at the junction of two liquid, indicate the presence of
steroid moiety.
• Salkowaski test
• Alcoholic extract of drug was evaporated to dryness and extracted with
CHCl3 add conc. H2SO4 from sidewall of test tube to the CHCl3 extract.
Formation of yellow coloured ring at the junction of two liquid, which
turns red after 2 min, indicate the presence of steroid moiety.

• Resin can be defined as the complex amorphous product, solid or
liquid characteristics which on heating first sets softened then melt
and clear adhesive fluids.
• Resins produced and stored in the schizogenous cavities of the plants.
• Resins produced in plants normally or abnormally as a result of an
injury.
• If the resins are produced as normal products of metabolism without
any injury to the plant, the resins are called preformed, normal, or
physiological resins. e.g. Copaiba resin in some plants normally only a
few resin ducts are present and the plant produces very small quantity
of resin.
• As a result of wound, injury or abnormal circumstances the plant gets a
shock and in the newly developed secondary xylem and bark, a large
number of resin ducts are formed and resin is produced in large
quantity.
• Resin produced in this way is called abnormal, pathological or
traumatic resin. e.g. Benzoin, Tolu balsam, Storax.

Physical Properties:
• Resins are hard, transparent or translucent brittle materials
• Most of the resins have specific gravity more than one and are
therefore heavier than water.
• They are electrically nonconductive.
• It can be complex mixtures of acids, alcohols, phenols, esters,
glycosides.
• On being heated at a relatively low temperature resins first get
softened and ultimately melt down thereby forming either an adhesive
or a sticky massive fluid, without undergoing any sort of decomposition
or volatilization.
• On being heated in the air i.e., in the presence of oxygen, resins usually
burn readily with a smoky flame.
• They are practically insoluble in water, but frequently soluble in
ethanol.

Chemical properties of Resins :
• Majority of them undergo slow atmospheric oxidation whereby
their colour get darkened with impaired solubility.
• Resins are found to be a mixture of numerous compounds rather
than a single pure chemical entity.
• Their chemical properties are exclusively based upon the
functional groups present in these substances.
• Consequently, the resins are broadly divided into resin alcohols,
resin acids, resin esters, glucosidal resins and resenes.

Classification of Resins :
• Resins are classified mostly on the basis of their chemical nature and
secondly as per their association with the other group of compounds
like essential oils and gums.
1) Based on Chemical nature is given below
• Resin acids:
• Resin acids are the carboxylic acid group containing resinous substances
• These compounds are found in Free states or as the esters derivatives.
• Resins acids can be derivatized to their metallic salts known as
resonates which find their use in soap, paints, and varnish industries.
e.g. Abietic acid in Colophony, Commiphoric acid in Myrrh
• Resin esters:
• Resin esters are the esters of the resin acids or the other aromatic acids
like benzoic acid, cinnamic, salicylic acids, etc.
• They are sometimes converted to their free acids by the treatment with
caustic alkali. e.g. Benzoin

• Resin alcohols:
• Resin alcohols or resinols are the complex alcoholic compounds of high
molecular weight.
• Like resin acids they are found as free alcohols or as esters of benzoic,
salicylic, and cinnamic acid.
• They are insoluble in aqueous alkali solution but are soluble in alcohol
and ether. e.g. Benzoresinol in Benzoin, Storesinol in Storax
• Resin phenols:
• Resin phenols or resinotannols are also high molecular weight
compounds which occur in free states or as esters.
• e.g. Peruresinotannol in Balsam of Peru, Toluressinotannol in Tolu
Balsam, Siaresinotannols in Benzoin

• Glucoresins (Glycoresin):
• Resins get combined with sugars by glycosylation and produce
glucoresins.
• Glycoresin can be hydrolyzed by acidic hydrolysis to the aglycone
and glycone.
• e.g. Resins of Convolvulaceae like Jalap resin
• Resenes:
• Chemically inert resin products are generally termed as resenes.
• They are generally found in Free State and never form esters or
other derivatives.
• Resenes are soluble in benzene, chloroform and to some extent in
petroleum ether.
• Resenes are insoluble in water. e.g. Asaresene B in Asafoetida

On the basis of association of resin with gums and or
volatile oils is given below:
• Oleoresins : (resin + oil)
• Oleoresins are the homogenous mixture of resin with volatile
oils. e.g. Turpentine, Ginger, Copaiba, Turmeric
• Gum resin : (resin + gum)
• Gum resins are the naturally occurring mixture of resins with
gums. e.g. Ammioniacum, gamboge
• Oleo gum resin : (resin + gum + oil)
• Ole gum resins are the naturally occurring mixtures of resin,
volatile oil, gum. e.g. Gum myrrh, Asafoetida

• Balsams : (resin + aromatic acid)
• Balsams are the naturally occurring resinous mixtures which
contain a higher proportion of aromatic balsamic acids such
as benzoic acid, cinnamic acid and their esters.
• Balsams containing free acids are partially soluble in hot
water. e.g. Balsam of Peru, Balsam of Tolu,
• Chemical test for Resins :
• When triturated with water, it forms emulsion.
• When treated with 50 % Nitric acid, it gives green colour.

• Alkaloids are a basic, naturally occurring organic compounds that
contain at least one nitrogen atom and a complex ring structure.
• They occur naturally in seed bearing plants and are found in berries,
bark, fruit, roots and leaves.
• The name derives from the word alkaline; originally, the term was used
to describe any nitrogen-containing base (an amine in modern terms).
• Alkaloids are found as secondary metabolites in plants
• Alkaloids often contain one or more rings of carbon atoms, usually with
a nitrogen atom in the ring, sometimes nitrogen atom outside the ring
structure.
• The position of the nitrogen atom in the carbon ring varies with
different alkaloids and with different plant families.
• In some alkaloids, such as mescaline, the nitrogen atom is not within a
carbon ring.
• It is the precise position of the nitrogen atom that affects the properties
of these alkaloids.

Properties of Alkaloids
• In general, they are colourless, crystalline solids which are basic, have a
ring structure, and have definite melting points.
• They have a very bitter taste.
• Most alkaloids are also chiral molecules which mean they have
nonsuperimposable mirror images. This results in isomers that have
different chemical properties. For example, Quinine use as Anti-malarial
drug, while quinidine use as anti-arrhythmic agent..
• Generally free bases of alkaloids are soluble in organic solvents and
insoluble in water, where as alkaloidal salts are soluble in water and
partially soluble in organic solvents.
• For example, strychnine hydrochloride is much more soluble in water
than strychnine as a base.
• Usually alkaloids are derivatives from amino acids. Even though many
alkaloids are poisonous (e.g. strychnine or coniine), some are used in
medicine as analgesics (pain relievers) or anaesthetics, particularly
morphine and codeine.

Classification of Alkaloids
• Alkaloids are generally classified by their common molecular
precursors, based on the biological pathway used to construct the
molecule.
• There are three main types of alkaloids :
1. True alkaloids
2. Protoalkaloids
3. Pseudoalkaloids.
• True alkaloids and protoalkaloids are derived from amino acids, whereas
pseudoalkaloids are not derived from these compounds.

• True alkaloids:
• They derived from amino acids.
• They having nitrogen in heterocyclic ring.
• These alkaloids are highly reactive substances with biological activity
even in low doses.
• True alkaloids may occur in plants
• (1) in the free state
• (2) as salts
• (3) as N-oxides.
• The primary precursors of true alkaloids are such amino acids as L-
ornithine, L-lysine, L-phenylalanine/L-tyrosine, L-tryptophan and L-
histidine.
• Examples of true alkaloids include such biologically active alkaloids as
cocaine, quinine, dopamine and morphine, nicotine, atropine,
tubocurarine, strychnine.

• Proto alkaloids :
• They derived from amino acids.
• But they do not have nitrogen in heterocyclic ring.
• Such kinds of alkaloid include compounds derived from L-tyrosine and
L-tryptophan.
• Protoalkaloids are those with a closed ring, being perfect but
structurally simple alkaloids.
• They form a minority of all alkaloids.
• Eg: tyramine, histamine, ephedrine, yohimbine ,mescaline, choline.

• Pseudo alkaloids :
• Not derived from amino acids.
• They have nitrogen in heterocyclic ring.
• Pseudoalkaloids are compounds, the basic carbon skeletons of which
are not derived from amino acids.
• In reality, pseudoalkaloids are connected with amino acid pathways.
They are derived from the precursors of amino acids.
• They can also result from the amination and trans-amination reactions
of the different pathways connected with precursors of amino acids.
• These alkaloids can also be derived from non-amino acid precursors.
• Eg: coniine, capsaicin, ephedrine, solanidine, caffeine and theobromine.

2. Alkaloids are mainly divided into two categories on the basis of their
chemical structure, that is, heterocyclic rings.
• Atypical alkaloids
• These are also known as non heterocyclic alkaloids and contain
nitrogen in aliphatic chain.
• Do no have heterocyclic ring in their structure, instead they have simple
benzene ring with aliphatic chain attached to it.
• 1. phenyl ethyl amine alkaloid – Ephedra
• 2. Tropolone alkaloid – Colchicum
• 3. Modified diterpene - Taxus
• Typical alkaloids
• These are also known as heterocyclic alkaloids and contain nitrogen in
heterocyclic ring system.
• Two types of typical alkaloids :
1. Mononuclear heterocyclic alkaloids
2. Polynuclear heterocyclic alkaloids

Mononuclear heterocyclic alkaloids :
1. Pyridine alkaloids – Lobelia
2. Piperidine alkaloids – Piperine
3. Pyrrole - Coca
4. Pyrrolidone - Tobacco
5. Imidazole – Pilocarpus
Polynuclear heterocyclic alkaloids :
1. Isoquinoline alkaloids – Opium
2. Quinoline alkaloids – Cinchona
3. Quinazoline alkaloids – Vasaka
4. Indole alkaloids – Ergot, Rauwolfia, Nux vomica
5. Tropane alkaloids – Datura, Belladona
6. Purine alkaloids – Coffee , Tea

Chemical tests for alkaloids
1. Dragendorff’s Test : Drug solution + Dragendroff’s reagent (Potassium
Bismuth Iodide), formation of Orangish red colour.
2. Mayer’s Test : Drug solution + few drops of Mayer’s reagent
(potassium mercuric iodide), formation of creamy-white precipitant.
3. Hagers test : (Saturated aq. Solution of Picric acid), formation of
crystalline yellow precipitate.
4. Wagner’s Test : Drug solution + few drops of Wagner’s reagent (dilute
Iodine solution), formulation of reddish-brown precipitate.
5. Tannic Acid Test : Drug solution + few drops of tannic acid solution,
formation of buff coloured precipitate.
6. Caffeine and some other alkaloids do not give these precipitates. It is
usually detected by murexide test.
7. Colchicine gives yellow color with mineral acids.
8. Indole alkaloids give bluish-violet to red color when treated with
sulphuric acid and p-dimethyl amino benzaldehyde

• “Tannins are complex non-nitrogenous, polyhydroxy phenolic
compound that are very difficult to separate since they don't
crystallize, are called tannins.”
• These compounds comprise a large group of compounds that are widely
distributed in the plant kingdom.
• The term ‘tannin’ was first used by Seguin in 1796 to denote substances
which have the ability to combine with animal hides to convert them
into leather which is known as tanning of the hide.
• Since long they are known as the astringent substances because they
combine with tissue proteins and precipitate them, hence, they are
used in medicines as mild antiseptics, in the treatment of diarrhea, and
to prevent minor hemorrhages.
• Commercially, they find extensive application in leather industry.

CHARACTERISTICS OF TANNINS
1. Tannins are colloidal solutions with water.
2. Non crystalline substance.
3. Soluble in water (exception of some high molecular weight
structures), alcohol, dilute alkali, and glycerin.
4. Sparingly soluble in ethyl acetate.
5. Insoluble in organic solvents, except acetone.
6. Molecular weight ranging from 500 to >20,000.
7. Oligomeric compounds with multiple structure units with free
phenolic groups.
8. Can bind with proteins and form insoluble or soluble tannin—protein
complexes.

Classification of Tannins :
• Two major chemical classes of tannins are usually recognized based on
this hydrolytic reaction and nature of phenolic nuclei involved in the
tannins structure. The first class is referred to as hydrolysable tannins,
whereas the other class is termed as condensed tannins.
• 1. Hydrolysable tannins:
• These tannins undergo hydrolysis by acids or enzymes and produce
gallic acid or ellagic acid.
• According to acid produced they are known as Gallitannins or
ellagitannins.
• These tannins were formerly known as pyrogallol tannins as on dry
distillation gallic acid and other similar components yield pyrogallol.
• The hydrolysable tannins are soluble in water, and their solution
produces blue colour with ferric chloride.
• E.g Bahera, Arjuna, Amla

Nonhydrolysable or Condensed Tannins
• Condensed tannins, are not readily hydrolysable to simpler molecules
with mineral acids and enzymes, thus they are also referred to as
nonhydrolysable tannins.
• The term proanthocyanidins is sometimes alternatively used for these
tannins.
• The compounds containing condensed tannins contain only phenolic
nuclei which are biosynthetically related to flavonoid.
• When treated with acids or enzymes, they tend to polymerize yielding
insoluble red coloured products known as phlobaphens.
• The phlobaphen is responsible for red colour to many drugs such as
cinchona and wild cherry bark.
• On dry distillation, they yield catechol derivatives.
• Condensed tannins are also soluble in water and produces green colour
with ferric chloride.
• E.g Pale Catechu, Black Catechu, Ashoka

Pseudo tannins
• Pseudotannins are simple phenolic compounds of lower
molecular weight.
• They do not respond to the tanning reaction of Goldbeater’s skin
test.
• Gallic acid, Chlorogenic acid, or the simple phenolics such as
catechin are pseudotannins which are abundantly found in plants,
especially in dead tissues and dying cells.

Chemical tests for Tannins :
1. Goldbeater’s skin test :
• Goldbeater’s skin is a membrane produced from the intestine of Ox.
• It behaves just like untanned animal hide.
• A piece of goldbeaters skin previously soaked in 2% hydrochloric acid
and washed with distilled water is placed in a solution of tannin for 5
minutes.
• It is then washed with distilled water and transferred to 1% ferrous
sulphate solution.
• A change of the colour of the goldbeater’s skin to brown or black
indicates the presence of tannin.
• Hydrolysable and condensed tannins both give the positive goldbeater’s
test, whereas pseudotannins give negative test.

• Phenazone Test :
• To 5 ml of aqueous solution of tannin containing drug, add 0.5 g of
sodium acid phosphate. Warm the solution, cool, and filter. Add 2%
phenazone solution to the filtrate.
• All tannins are precipitated as bulky, coloured precipitate.
• Gelatin Test :
• To a 1% gelatin solution, add little 10% sodium chloride. If a 1% solution
of tannin is added to the gelatin solution, tannins cause precipitation of
gelatin from solution.
• Test for Catechin (Matchstick Test) :
• Catechin test is the modification of the well-known phloroglucinol test
for lignin.
• Matchstick contains lignin.
• Dip a matchstick in the dilute extract of the drug, dry, moisten it with
concentrated hydrochloric acid, and warm it near a flame.
• Catechin in the presence of acid produces phloroglucinol which stains
the lignified wood pink or red.

• Test for chlorogenic acid :
• A dilute solution of chlorogenic acid containing extract, if treated
with aqueous ammonia and exposed to air, slowly turns green
indicating the presence of chlorogenic acid.
• Vanillin-hydrochloric acid test :
• Drug shows pink or red colour with a mixture of vanillin: alcohol :
dilute HCl in the ratio 1:10:10.
• The reaction produces phloroglucinol which along with vanillin
gives pink or red colour.

• The odorous, volatile principles of plant and animal sources are known
as volatile oils. They are liquid, lipophile and having a characteristic
smell.
• As they evaporate when exposed to air at ordinary temperatures, they
are also called as “ethereal oils”.
• They represent essence or active constituent of plant, hence they are
also known as “essential oils”
• Properties of volatile oil
• Volatile oils are nearly insoluble in water & soluble in alcohol, ether and
other lipid solvents.
• Volatile oils are usually lighter than water & their specific gravity is less
than one (except clove oil, have specific gravity more than one)
• Volatile oils have high refractive index and show optical rotation.

• Chemically, they are derived from terpenes and their oxygenated
compounds. thus they are known as Terpenoids or Isoprenoids.
• Terpenes are made up of isoprene units (C5H8)
• The chemical constituents present are complex & are mixtures of
terpenes as monoterpenes, sesquiterpenes or diterpenes & their
derivatives & phenyl propane derivatives.
• Some volatile oils show one or two components in higher percentage
e.g. Linalool in coriander, eugenol in clove and & carvone in caraway.
• This predominance of one or two components determines the odour &
use of a particular drug or its oil.

• Terpenoids are volatile substances which give plants and flowers their
fragrance.
• They are usually monoterpenes & sesquiterpenes.
• Monoterpenoids may be defined as molecules containing ten carbon
atom derived by dimerization of two molecules of isoprene unit usually
joined in a head to tail fashion.

Classification of Volatile oils :
Chemically a volatile oil is a mixture of several constituents in which
certain types of constituents are more predominating than the others as
hydrocarbons, alcohols, acids, ethers, esters, aldehydes, ketones or oxides

Storage of volatile oils :
• Volatile oils are liable to oxidation on storage in presence of air,
moisture, and light.
• The oxidation is followed by the change in colour, increase in viscosity,
and change in odour.
• Hence, volatile oils must be stored in well-closed completely filled
containers and away from light in cool places.
• Chemical tests for volatile oil :
• Presence of volatile oil in natural drugs can be detected by following
tests :
1. To the thin section of drug, add alcoholic solution of Sudan-III. Red
color obtained by globules indicate the presence of volatile oil.
2. To the thin section of drug, add a drop of tincture of alkane. Red color
indicates the presence of volatile oil.

Pharmaceutical applications of volatile oils :
• Volatile oils are used as flavouring agent, perfuming agent in
pharmaceutical formulations, foods, beverages, and in cosmetic
industries.
• These are also used as important medicinal agent for therapeutic
purposes like:
1. Carminative (e.g. Umbelliferous fruits)
2. Anthelmintic (e.g. Chenopodium oil)
3. Diuretics (e.g. Juniper)
4. Antiseptic (e.g. Eucalyptus)
5. Counter irritant (e.g. Oil of winter green)
6. Local anaesthetic (e.g. Clove)
7. Sedative (e.g. Jatamansi)
8. Insect repellent (e.g. Citronella)
9. Source of vitamin A (e.g. Lemongrass)

• Flavonoids are the most abundant polyphenols in human diet and they
are the group of compounds comprising the derivatives of flavone,
flavanones, flavanonols, flavonols and isoflavones.
• The chemical structure of these compounds is derived from the
aromatic nucleus of flavone or 2-phenyl benzopyran.
• The flavonoids are found either in free state or as glycosides in a variety
of plants.
• In fruits and vegetables, they are usually found in the form of glycosides.
• They are water-soluble and accumulate in cell vacuoles.

Chemical structure of flavonoids :
• Their basic structure is a skeleton of diphenyl propane
• namely, two benzene rings (ring A and B) linked by a three carbon chain
that forms a closed pyran ring (heterocyclic ring containing oxygen, the
C ring) with benzenic A ring.
• Therefore, their structure is also referred to as C6-C3-C6.

• In most cases, B ring is attached to position 2 of C ring, but it can also
bind in position 3 or 4
• together with the structural features of the ring B and the patterns of
hydroxylation of the three rings, makes the flavonoids one of the larger
and more diversified groups of phytochemicals, in nature.
• Their biological activities, for example they are potent antioxidants,
depend both on the structural characteristics and the pattern of
hydroxylation.

Classification of flavonoids :
• They can be subdivided into different subgroups depending on the
carbon of the C ring on which B ring is attached, and the degree of
unsaturation and oxidation of the C ring.
1. Flavones: They have a double bond between positions 2 and 3 and a
ketone in position 4 of the C ring.
2. Flavonols: Compared to flavones, they have a hydroxyl group in
position 3 of the C ring.
3. Flavanones: Flavanones, also called dihydroflavones, have the C ring
saturated; therefore, unlike flavones, the double bond between
positions 2 and 3 is saturated and this is the only structural difference
between the two subgroups of flavonoids.
4. Flavanonols: Flavanonols, also called dihydroflavonols, are the 3-
hydroxy derivatives of flavanones.

5. Isoflavones: Isoflavones are a subgroup of flavonoids in which the B
ring is attached to position 3 of the C ring. They have structural
similarities to estrogens, such as estradiol, and for this reason they
are also called phytoestrogens.
6. Neoflavonoids: They have the B ring attached to position 4 of the C
ring.
7. Anthocyanidins: Chemically, anthocyanidins are flavylium cations
and are generally present as chloride salts. They are the only group
of flavonoids that gives plants colors (all other flavonoids are
colorless).
8. Chalcones: Chalcones and dihydrochalcones are flavonoids with open
structure; they are classified as flavonoids because they have similar
synthetic pathways.

Introduction to secondary metabolites

Chemical test for flavonoids :
• Shinoda test :
• In this test, four pieces of magnesium fillings (ribbon) are added to the
ethanolic extract followed by a few drops of concentrated hydrochloric
acid.
• A reddish colour indicates the presence of flavonoid.
• Ammonia test
• Filter paper dipped in alcoholic solution of drug was exposed to
ammonia vapor. Formation of yellow spot on filter paper.
• Vanillin HCl test
• Vanillin HCl was added to the alcoholic solution of drug, formation of
pink colour due to presence of flavonoids.

Introduction to secondary metabolites

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Introduction to secondary metabolites