Isolation, Identification and Analysis of Phytoconstituents Terpenoids: Menthol, Citral, Artemisin

1. B.PHARM. 5th SEMESTER
PHARMACOGNOSY ANDPHYTOCHEMISTRY-II
BP504T.
UNIT-III
Isolation, Identification and Analysis of
Phytoconstituents
Terpenoids: Menthol, Citral, Artemisin

2. What is terpenoids
Any of a large class of organic compounds including terpenes,
diterpenes, and sesquiterpenes. They have unsaturated molecules
composed of linked isoprene units, generally having the formula
(C5H8) n.
Isoprene unit are 5 carbon unit
For having isoprene unit they are also called isoprenoids

4. Physical properties of terpenoids
Odorous
Volatile at room temperature
They represents essence or active constituents of plants
hence called essential oil
Lighter than water
Have high refractive index
Are optically active

5. Chemical Properties of teprenoids
Terpenoids are the hydrocarbones of plant origin of the general formula
(C5H8)n as well as their oxygenated, hydrogenated & dehydrogenated
derivatives.
Terpenoids are regarded as derivatives of polymers of isoprene,
CH3H2C=C-CH=CH2 (i.e.C5H80)
They consist of a complex mixture of terpenes or sesquiterpenes, alcohols,
aldehydes, ketone, acid & esters.
They are unsaturated compounds.
They undergo addition reaction with hydrogen, halogen, halogen acids to
form addition products
They undergo polymerization and dehydrogenation in the ring.
On thermal decomposition, terpenoid gives isoprene as one of the product.

6. Chemical Properties of teprenoids
Terpinoids are modified terpenes, where in methyl groups
have been moved or removed, or oxygen atoms added.
Sometimes terpinoids are added to proteins, e.g., to enhance
their attachment to the cell
membrane.
The steroids and sterols in animals are biologically produced
from terpinoid precursors.

8. MENTHOL
CH3
CH3
H3C
OH

9. Menthol
• Biological source: Menthol a monoterpene alcohol
obtained from oils of Mentha piperita var. Vulgaris
(Black peppermint) & Mentha piperita var. officinalis
(White peppermint),
• Family: Labiatae.
Mentha species contains about 1-3% of volatile oils & oil
contains not less than 44%.
G.S.: Asia, Europe, North America, Cultivated in India,
japan, Germany, France, Italy.
Menthol is isolated by 2 methods
Isolation
Method 1: Hydro-
distilation
Method 2: Steam
distillation

10. Hydro-distillation
Required quantity of coarse powder leaves
of Mentha piperita.
The peppermint oil is extracted by
hydrodistillation method by using clevenger
apparatus.
The oil is separated from water and allow to
cooling. After cooling crystals of (-) menthol
will separate out.
The crystals are collected by centrifugation
and re- crystallized the menthol by acetone
or any other low boiling point solvent.

11. 1. Hydro-distillation by Clevenger apparatus system

12. 2. Steam distillation
Required quantity of air dried mentha
plant is charged into stainless steel still
having perforated bottom.
The steam under pressure is generated
with the help boiler and steam is passed
through the drug.
The mixture vapour (water and volatile oil) are
passed through the condenser where vapour is
cooled and back to liquid form.
The mentha oil is collected in separating
can. Mentha oil is floated on top of the
water due to lighter than water.
The oil is then decanted and filtered.

13. 2. Steam distillation

14. 3. Method of Isolation
Pippermint volatile oil- separated by steam & water distillation.
To make oil free of moisture- it is passed through a bed of Na2SO4
The moisture free oil is frozen to -60°C for 7days by packing in tightly sealed plastic
container.
Menthol in oil separates as flaky crystals, separated by filtration.
Mother liq. Containing some menthol along with other terpenes.
The mother liq is treated with boric acid & further boiled for 3hr, followed by distillation
for removal of menthone.
Borate of menthol subjected to saponification with 50ml of NaOH by heating reflux for
1hr
Resultant sol. Is allowed to cool for separation of remaining menthol crystals and mixed,
dried in dessicator.

15. Properties:
• Appearance: White crystalline substances, which is solid at room
temperature and melts slightly above (m.p. 41 to 43°C).
• Odor: Characteristic and pleasant
• Taste: Pungent followed by cooling sensation
• Solubility: Soluble in 70% alcohol, ether and chloroform, insoluble
in water
• Identification by chemical test:
• Few drops of sample is mixed with 5ml of nitric acid and heated on
water bath. Blue color is developed within 5 minutes, after some
time it becomes yellow which indicate the presence of menthol.
• Small qty of menthol in TT + equal qty of thymol or camphor →
liquifaction of contents in TT→ presence of Menthol
• Crystals of Menthol in watch glass+ heat on water bath → material
evaporates

16. Analysis
Take 10gm drug, add 10ml of acetic anhydride & 2gm of sodium
acetate to the flask. Reflux for 1 hr.
After cooling add 30ml water, boil it for 15 min. with continuous
stirring. Then transfer in separating funnel.
The oil layer is washed with water until it become neutral.
Add 2 gm of sodium sulphate & shake it. Allow it for 30 min. &
filter it.
Take both layer in different flask, add 5 ml ethanol and
Phenolphthelin indicator by adding 25 ml KOH ethanol solution.
(for neutralize it)
Again reflux for 1 hr. cool it titrated with HCl.

17. • % Menthol = 7.814 x (c-a) {0.021 x (c-b)/U}
A– 0.021 x (c-a)
• Where,
“a” & “b” = ml of HCl for oil
“c” = ml of HCl for blank
“U”= Un-acetylated oil
“A”=Acetylated oil

18. Analysis by TLC
• Sample preparation: 1mg of Menthol is dissolved 1ml of methanol
• Standard sample: Menthol
• Stationary phase: Silica gel 60 F 254
• Mobile phase: Pure Chloroform OR hexane: EtOAc :: 8: 2
• Detecting agent:
1% vanillin-sulphuric acid reagent and heat the plate at
110°C for 10 minutes
Anisaldehyde-sulphuric acid reagent, heat at 115°C-10min.
• RF Value: 0.34, 0.48-0.62
Utilization
• It is used as in various dosage forms for its cooling sensation,
flavoring property,
• carminative, antispasmodic and antipruritic
Storage condition
• It should be store in well closed and air-tight containers protected
from light and in cool place.

19. CITRAL

20. Citral
Biological source: Citral is a monoterpene aldehyde obtained
from oils of lemon grass Cymbopogon flexuosus and C. martini.
Family: Graminae.
It also present in Lemon oil (Citrus limonus) and Orange oil
(Citrus aurantium).
Family: Rutaceae.
Lemon oil: Lemony, sweet smell, yellow in color.
It contains not less than 75% of aldehyde calculated as Citral.
Citral is isolated by following methods
Isolation
Method 1: Hydro-
distillation
Method 2: Fractional
crystallization Method

21. Hydro-distillation
Required quantity of coarse powder leaves of
lemon grass or other powder.
The lemon grass oil is extracted by hydro
distillation method by using Clevenger
apparatus.
The oil is separated from water and allow to
cooling. After cooling of lemon grass will
separate out.

22. Citral
The fresh plant material is hydro-distilled
to obtain lemon grass oil.
It is purified by fractional crystallization.
To the total oil, first Sodium sulphite is added,
the citrals get converted into its sulphite salt.
The salt crystallizes out of the solution.
The crystals are filtered and washed with
ether or chloroform.

23. Properties:
• Appearance: Clear pale yellow liquid
• Odor: Strong lemon like odour
• Taste: Lemon like taste
• Solubility: Soluble in 3 parts of 70% alcohol,
chloroform and fixed oil. Insoluble in water.
• Boiling point: 224-228 0C
• Identification by chemical test:
• Alcoholic solution of Sudan red III is added to the
sample. Red colour is appeared which indicate
presence of citral.
• Tincture alkane is added to the sample. Red colour
is appeared which indicate presence of citral.

24. • Analysis by TLC
• Sample preparation : 1mg of Citral is dissolved 1ml of methanol
• Standard sample : Citral
• Stationary phase : Silica gel –G
• Mobile phase : Pure Chloroform
• Detecting agent : 2, 4, dinitrophenyl hydrazine reagent
• Color spots : Yellow to orange
• RF Value : 0.51
• Utilization:
• It is used as a flavouring agent and perfumery. Commercially citral
is act as precursor for the synthesis of ß-ionone. ß-ionone is used
as starting material for the synthesis of Vitamin A
• Storage condition:
• It should be store in well closed and air-tight containers protected
from light and in cool place.

25. ARTEMISIN

26. Artemisin
Biological source: Artemisin is a sesquiterpenoid
lactone, obtained from the unexpanded flower- heads of
Artemisia cina Berg, Artemisia brevifolia Wall,
Artemisia maritime Linn. and other species
Family: Asteraceae.
It contains 2-3% of essential oil and two crystalline
substances i.e Santonin and Artemisin.
G.S: It is found in Italy, Iran, South Austrelia, In India
Gujarat, J&K, Karnataka, UP and Hariyana

27. Extraction
Extraction can be done
by 2 ways;-
Soxhlet extraction
Microwave-assisted extraction
REQUIREMENTS
• Raw material
• Solvents (methanol, n-hexane, n-heptane,
benzene, toluene, chloroform, methylene
dichloride, ethyl acetate, acetone, acetonitrile..)

28. SOXHLET EXTRACTION AND TEST SAMPLE
PREPARATION
100 mg of fine powder was placed into an extraction
thimble
extracted with 170 ml of solvent via hot soxhlet extraction
method for 6 hours over a water bath
The extract was evaporated and redissolved in 5 ml
methanol
10 μl of these test solutions was used for quantification
purpose.

29. MICROWAVE-ASSISTED EXTRACTION AND TEST
SAMPLE PREPARATION
10 μl of these test solutions was used for quantification purpose.
The extract thus obtained was evaporated and redissolved in 5 ml methanol
For microwave-assisted extraction (MAE) the same parameters should be followed for every
solvent
Extraction parameters (160 watts, 120s, 10 ml per extraction cycle, two extraction cycles,
and cleanup with 2 ml of corresponding solvent at the end of second cycle of extraction)
100 mg of fine powder was extracted under the influence of microwave energy using
solvents

31. Partition the extract using 50 ml hexane
Partitioning done many times until hexane layer become colorless
Two layers got from this process
The methanol extract obtained was added 10 ml of distilled water
Partitioned again using 50 ml of ethyl acetate
Partitioning done many times until the ethyl acetate layer is colourless.
Again we get 2 layers
PARTITION OF EXTRACT
1. hexane extracts (non-
polar fraction)
2. methanol extracts
Each extract was concentrated using a rotavapor at a temperature of 40˚c.
2.methanol-water
extract (polar fraction)
1.ethyl acetate extract
(semipolar fraction)

32. ESTIMATIONS
By 2 methods:-
• TLC Densitometer
• HPTLC method
TLC METHOD
• Mobile phase :ethyl Acetate: hexane (3:97)
• Stationary phase :60 F254 silica gel
• Detecting reagent :anisaldehyde-sulphuric acid reagent
• Spot volume :10 μL of test and standard sample spots
• Spot colour :Pink-colored spots of artemisinin
• Room temp. :25 +- 2˚C
• Relative humidity :45+- 2%

33. Extrapolating the graph to x axis give
concentration of test sample
Values are plotted in calibration curve (con
Vs spot area)
A simple TLC-densitometric technique has
been developed for the rapid and accurate
analysis of artemisinin in a at 540-610 nm
using tungsten lamp
large number of Artemisia annua plantlets

34. HPLC METHOD
•EQUIPMENT MODEL: -- WATERS 501
•COLUMN: -- WATERS C18
•DETECTOR: -- UV 260 nm
•MOBILE PHASE: -- phosphate buffer : methanol (6:4) – PH – 7.9
•FLOW RATE: -- 1ml/min
Calibration curve was plotted with diff conc

35. Description - - colour less needles /white crystalline powder,
odourless
Identification - - TLC/ HPLC with std
Molecular formula - - C15H22O5
Molecular wt - - 282.3
Solubility - - partially insoluble in water
very soluble in dichloromethane
MP - -
freely soluble methanol, ethanol
151-154 c
LOSS ON DRYING - - NMT 0.5%
STORAGE - - Tightly closed container
SPECIFIC SHEET

36. Identification by chemical test:
Sample is boiled with 10ml of alcohol and filtered. Sodium
hydroxide is added to filtrate and heated. Red colour is
appeared in liquid .
• Analysis by TLC
• Sample preparation: 1mg of Artemisin is dissolved
Chloroform
• Standard sample: Artemisin
• Stationary phase: Silica gel –G
• Mobile phase: Petroleum ether-Ethyl acetate (1:2)
• Detecting agent: p-dimethyl amino benzaldehyde and
heat at 80° C to produce color
• RF Value: Compare with standard Artemisin

37. Artemisnin
• Analysis by UV
• 1mg of Artemisin is mixed with 10ml of methanol
and analysed λ200-400nm.
• Utilization:
• It is used as antimalarial.
• It acts both against chloroquine sensitive and
resistant P. falciperum and P. vivax malarial
parasites.
• Storage condition:
• It should be store in well closed and air-tight
containers protected from light and in cool place.