Isolation, identification and analysis of phytoconstituents

1. Isolation, Identification and
Analysis of Phytoconstituents
MRS.REKHAAMIT BHALERAO
ASSISTANT PROFESSOR
PHARMACEUTICAL CHEMISTRY
PES MODERN COLLEGE OF PHARMACY, FOR LADIES

2. INTRODUCTION
 The increasing use of herbal preparations has
highlighted the need for adequate standards to
ensure quality, safety and efficacy of such drugs and
preparations. Many developing countries are
becoming aware of their flora as a source of
medicinally useful products.

3. TRITERPENOID
 Mono (C10) and sesquiterpenoid (C15) and certain
phenolic compounds comes under the consideration
of essential oil components. They are volatile in
nature and can be isolated using Clavenger's and
Karlsruhe apparatus. These apparatus are best for
quantitatively isolating small amount of such oils.
However, for a large scale industrial isolation, these
apparatus are suitably modified to serve the
purpose.
 Further isolation and purification of specific
components of the essential oil depends on the
amount of oil available and types of components
present in it. Fractional distillation and Crystallisation
are preferable if larger amounts are available.

4. Menthol
 Menthol is a terpene obtained from diverse mint oils
prepared synthetically by hydrogenation of thymol. It
occurs naturally in peppermint oil to the extent of 50-
60% along with methyl acetate, methyl
isovalerianate and small amounts of other terpenes.
 Peppermint oil refers to the steam distilled essential
oil isolated from the aerial parts of various species of
the mint family such as Mentha piperita, Mentha
arvensis and Mentha canadensis (Labiateae).

5. Menthol
 Menthol is used in foods, topical therapeutic
preparations, oral hygiene and dentifrice
formulations and tobacco products due to its
pleasant minty flavour and the cooling sensation it
imparts when in contact with the skin or oral
membranes.
 Menthol is combined with camphor and eucalyptus
oil in ointments, cough preparations, nasal sprays
and inhalants to relieve symptoms of bronchitis,
sinusitis and nasal congestion.

6. Menthol
 Menthol-produces the sensation of coolness in the
oral and olfactory regions only at low concentrations,
as higher concentrations induce a burning sensation
coincident with some modest degree of
desensitization. Menthol has local anesthetic and
counter-irritant qualities.
Structure of Menthol

7. Properties
 Menthol is 2-(2-Propyl)-5methyl-1-cyclohexanol.
Menthol occurs as white or colourless hexagonal or
needle like crystalline or granular solids, with
peppermint taste and odour.
 It has a melting point of 41-430 C and boiling point of
2120 C.
 It is slightly soluble in water,
 very soluble in alcohol, chloroform, ether, petroleum
ether and freely soluble in glacial acetic acid.

8. Isolation :
 The volatile oil of peppermint herb is separated by
water and steam distilled. The oil is then freed of
moisture by passing it through a bed of anhydrous
sodium sulphate. The dried oil is then taken in well-
sealed plastic containers and frozen to about - 60 0C
for about 7 days.

9. Isolation :
 Menthol in the oil separates as flaky crystals which
are filtered out. The mother liquor still contains some
menthol along with menthone and other terpenes.
Add 8 g boric acid for 100 ml of the dementholized
essential oil and further boil for 3 h to distill off
menthone. Convert the borate of menthol by
saponification with 50 ml of 15% sodium hydroxide
by heating under reflux for I h. Cool the resultant
solution to separate the remaining menthol. Collect
the flakes of menthol together, dry them in a
desiccator and note the yield.

10. Identification
 Heat a few crystals of menthol taken in a watch
glass over a water bath. The entire material
volatilizes without leaving any residue.
 To 0.5 g of menthol taken in a tube, add equal
quantity of camphor or thymol. Liquification of the
material indicates the presence of menthol.
 To 0.25 g of menthol, add 5 ml of sulphuric acid and
mix-well. There is formation of yellowish-red turbidity.
Allow to stand for 24 h. Formation of a transparent
oily layer with no odour of menthol indicates its
presence.

11. Analysis
 Weigh accurately 2 g of menthol in a 250 ml conical
flask with a ground glass jointed neck. Add 5 ml of a
mixture of 4 ml acetic anhydride and 1 ml of
anhydrous pyridine and boil under reflux for 30 min.
Through the condenser add 30 ml warm distilled
water and continue heating for 15 min.
 Remove from the heat source and cool completely.
Disconnect the condenser and rinse it with 15 ml of
distilled water. Titrate the contents of the flask with
0.5 M alcoholic potassium hydroxide (KOH) solution
using phenolphthalein as indicator.

12. Analysis
 Let the titred value be 'a' ml. Carry out a blank
determination to determine the number of millilitres
of the titrant required to neutralize the acetic acid
produced by 5 ml of the acetylizing mixture. Let the
titred value be 'b' ml.
 Difference between b and a gives the number of
millilitres of titrant equivalent to the acetic acid used
up for the acetylation of menthol. Determine the
percentage of free menthol from the equivalent
factor of 0.5 M alcoholic KOH, which is 0.0781 g.

13. Citral
 Citral is present in the oils of several plants like
lemon grass (Cymbopogon citrates), family
Cymbopogon. Lemon grass is a good source of citral
(65-85%).
 citral is used in the synthesis of vitamin A, ionone
and methyl ionone and to mask the smell of smoke.
Citral is an exarnple of a very large group of natural
products called terpenes. Citral is used as a flavor
and for fortifying lemon oil. Citral has strong
antimicrobial qualities

14. Structure of Citral

15. Properties
 Citral is 3,7-dimethyl-2,6-octadienal, is either a pair
or a mixture of terpenoids with the molecular formula
C10H16O. The two compounds are double bond
isomers. The E-isomer is known as geranial or citral
A. The Z-isomer is known as neral or citral B.
 Citral is a pale yellow liquid, with a strong lemon
odour, that occurs in the essential oils of plants.
 It is insoluble in water but soluble in ethanol (ethyl
alcohol), diethyl ether and mineral oil.

16. Isolation
 Measure about 1.0 ml of lemongrass oil into a pre-
weighed clean and dry 50 ml round bottom flask. Add
about 30 ml of distilled water to the flask. Assemble
simple distillation setup and start heating record the
temperature at which distillation is Observed Collect the
distillate in a 25 ml graduated cylinder until the distillate is
no longer oily (about 15-20 ml distillate should be
collected). Pour distillate in separatory funnel and extract
with two 10 ml portions of methylene chloride(dichloro-
methane); combine the methylene chloride layer in one
Erlenmeyer flask. Dry the methylene chloride solution
with sodium sulfate (or magnesium sulfate). Remove the
drying agent through gravity filtration. Remove methylene
chloride using rotary evaporator.

17. Identification
 1 mg of citral is dissolved in 1ml of methanol and
the spots are applied on silica gel-G plate. TLC
plates are eluted in pure chloroform. The dried plates
are sprayed with 2,4-dinitrophenyl hydrazine
reagent. Citral gives yellow to orange spots with Rf
value 0.51.

18. Analysis
 To determine the amount of citral isomers, 1 g of
lemongrass oil was accurately weighed, dissolved in
100 ml ethanol and mixture was sonicated to furnish
a clear solution. Centrifuge this solution at 3500 rpm
for 15 minutes. Dilute I ml supernatant liquid with 10
ml of ethanol.

 Apply 2 µl of this solution to TLC plate and analyze
for trans and cis citral content by using toluene :
ethyl acetate (8.5:1.5) as a mobile phase. The sharp,
compact and well defined peaks at Rf 0.55 and 0.34
for trans and cis citral confirms the citral.

19. Analysis
 Artemisinin is the active antimalerial constituent of
traditional Chinese medicinal herb Artemisia annua,
family compositae. Chemically, artemisinin is a
sesquiterpene lactone containing an unusual
peroxide bridge. The endoperoxide-1,2,4-trioxane
ring is responsible for the drug's mechanism of
action.

 Atentisinin and its derivatives have been used for the
treatment of malarial and parasitic worm (helminth)
infections. They have the advantage over other
drugs in having an ability to kill faster and kill all the
life cycle stages of the parasites.

20. Structure of Artemisinin

21. properties
 Artemisinin is a whitish crystalline powder, soluble in
most organic solvents. It is slightly soluble in oil.

22. Isolation
 The leaves of A. annua are air dried, coarsely
powdered and subjected to extraction with petroleum
ether (40-600C ). The extract is concentrated, dried
and redissolved in chloroform, to which acetonitrile is
added to precipitate inert plant constituents such as
sugars and waxes.
 The chromatographic fractionation of the concentrate
on silica gel by eluting with chloroform-ethyl acetate
yield fraction of arternisinin. The fractions contains
artemisinin could be crystallised from cyclohexane or
50 percent ethanol.

23. Analysis
 Artemisinin or the extract is dissolved in chloroform
and spots of the sample and standard are applied on
the silica gel-G plate. The plates are eluted in
solvent system petroleum , ether-ethyl acetate (1:2).
The dried plate is sprayed P-dimethyl
aminobenzldehyde and heated at 800C to produce
colour.
 The spot of the artemisinin corresponds to the
standard which can be quantitated by densitometry
at 600 nm. The 2% solution of vanillin-sulphuric acid
produces colour that measured at 560 nm.