PHYTOCHEMISTRY AND PHARMACOGNOSY II, UNIT III, ISOLATION IDENTIFICATION AND ANALYSIS OF TERPENOIDS

1. Prepared by : Snehal Uttam Kashid
(Asso. professor)
Department of Pharmacognosy,
SPMs college of pharmacy, akluj

2. Menthol occurrence
 Menthol is monoterpene alcohol found in mint oil/peppermint oil.
 B.S. – Mintha piperita (45%), M.arvensis, M.canadensis,
M.piperascence (70-90%).
 Family – piperaceae
 It contain 1-3 % volatile oils
 Other C.C. – neomenthol, isomenthol, menthofuran, methone,
methyl acetate cineol.

3. Isolation of menthol
 Two methods employed for isoation of menthol
1) Steam distillation

4. 2. Hydro-ditillation

5. Characterization of menthol
A. Chemical detection
 small piece of potassium hydroxide into a test tube + 1 ml of the plant
essential oil with heating.
 The solution was cooled and 1 ml of diethyl ether was added.
 A few drops of carbon disulfide were added to the solution
 forming a yellow residue that indicates the presence of menthol.

6. Characterization of menthol
B. Thin-layer chromatography (TLC)
 Stationary phase – Silica gel G
 Mobile phase - toluene: diethyl ether: 1.75 M acetic acid (1:1:1)
 Method – 1 mg menthol is dissolved in 1 ml methanol spot were applied
on activated TLC plate .
 For activation TLc plate is preheated at 110°C for 10 min
 5 µl of reference solution of menthol and 5 µl of investigated wild mint oil
were applied to silica gel plates, Merck (Germany) 20 × 20 cm, 0.25 mm in
thickness. Dried the spot for few min.
 Development of TLc plate in mobile phase. And dried
 separated zones were visualized using iodine chamber/ UV cabinate.
Standard menthol served as positive control
 RF value – 0.48-0.62

7. TLC of menthol

8. Identification test
 10 mg menthol crystal + 4drops of H2SO4 + vanillin sulphuric acid
reagent.
Orange yellow color changes to violet on addition of water.
 Drug +glacial acetic acid 3drops of H2SO4and 1 drop of HNO3
 Menthol does not gives green or blue green color (thymol gives green
color)

9. BIOLOGICAL PROPERTIES
 Menthol's ability to chemically trigger the cold-sensitive TRPM8
receptors in the skin is responsible for the well-known cooling
sensation, when inhaled, eaten, or applied to the skin.
 Menthol's analgesic properties are mediated through a selective
activation of κ-opioid receptors.
 Menthol also blocks voltage-sensitive sodium channels, reducing
neural activity that may stimulate muscles.
 Menthol also enhances the efficacy of Ibuprofen in topical
applications via vasodilation, which reduces skin barrier function.
 It used in oral hygiene products and bad-breath remedies, such as
mouthwash, toothpaste
 As an additive in certain cigarette brands, for flavor, to reduce the
throat and sinus irritation caused by smoking
 As an antispasmodic and smooth muscle relaxant in upper
gastrointestinal endoscopy.
 As an antipruritic to reduce itching

11. Citral occurrence
 Citral is an acyclic monoterpenoid
 Citral occurs abundantly in Lemon myrtle (cultivated in Australia ),
lemongrass (India, south east Asia and Africa) lemon tea-tree (Kenya,
South Africa, and Australia) Lemon balm (south-central Europe, North
Africa, the Mediterranean region, and Central Asia)
 B.S. - Lemongrass oil is obtained form Cymbopogon flexuosus, or
Cymbopogan citrates
 Family - Graminae.
 Lemon grass oil Contains about 75 % to 85% of citral.
 It also occurred in lemon, lime, orange, ginger root, etc.

12. Isomerism of citral
 Two geometrical isomers occur in nature. The cis-isomers is known as
Citral-a and trans-isomers Citral-b.
 - Ordinary citral obtained from lemongrass oil is, in fact, mixture of
Citral-a (90%) and Citral-b (10%).

13. Physical properties of citral
 Citral is a clear yellow colored liquid with a lemon-like odor.
 Less dense than water and insoluble in water
 Melting point of citral is <-10°C
 Density of citral is 0.9 g/cm³
 Citral is not stable to alkanes and strong acid
 When heated to decomposition it emits acrid smoke and irritating
fumes

14. Isolation of citral
 Citral is isolated from lemon grass oil which is obtained from lemon
grass by steam distillation.
Method:
 Lemon grass (chopped or un chopped) is filled in the distillation flask.
 Flask fitted tightly so that the vapors and oils was not leaked out.
 The steam is injected in to it so that the upcoming steam carries away
the essential oil from the plant material then the lemon grass oil as well
as the vapors are passed through the condenser where they condensed
as the oil
 Oil is lighter then water so it will float through the surface of water
 it is then easily separated now the thus obtained is the lemon grass oil
which contain 85%

15. Isolation of citral
 Lemongrass oil is shaken with 5% sodium bisulphate solution for 25 to
30 min.
 Resultant is separated with fennel and wash with solvent ether or
ethanol.
 The citral is regenerated by decomposing the sodium bisulphate with
dil sodium hydroxide solution.
 Citral A is free from citral B during regenration process from
bisulphate.
 Niral(citral B) is isolate from citral mixture by shaking it with alkaline
cynoacetic acid solution.
 Separation of citral A and B depending on melting point citral A having
M.P. 108°C to 110 °C.

16. Charaterization of citral
Thin-layer chromatography (TLC)
 Stationary phase – Silica gel G
 Mobile phase - cyclohexane: ethylacetate with 85:15, and hexane:
ethylacetate with 90:10 V/V.
 Method – 5 µl is dissolved in 1 ml methanol spot were applied on
activated TLC plate .
 For activation TLc plate is preheated at 110°C for 10 min
 5 µl of reference solution of menthol and 5 µl of investigated wild mint
oil were applied to silica gel plates, Merck (Germany) 20 × 20 cm, 0.25
mm in thickness. Dried the spot for few min.
 Development of TLC plate in mobile phase. And dried
 separated zones were visualized using iodine chamber/ UV cabinate.
Standard menthol served as positive control.
 RF value –0.45 -0.50

17. Thin-layer chromatography (TLC)
Sample Reference

18. Identification test
 Citral is very sensitive to oxidizing agent and yield linalool which have
intence yellow colour.
 Geranial with tollens reagent (ammonical silver nitrate) gives geranic
acid.
 Giranil upon hydrogenation with sodium amalgum in acidic solution
gives citronellol.
 Geranial gets converted into p-cymene on treatment with potassium
bisulphate or dil H2SO4.

19. uses
 It is used in perfumes and flavorings and in the
manufacture of other chemicals.
 Citral also has a significant role in the production of
vitamin A.
 Citral has an effect on some insects. It has a mild
repellent action for some species.
 It also appears to act as a sex pheromone for the green-
veined white butterfly, released by males to attract
mates
 Citral has strong antimicrobial qualities

21. Artimisnin occurrence
 It is sesquiterene lactone with prominant antimalerial activity.
 Synonyms : Sweet worm wood,sweet annie, sweet sagewort, annual
mugwort or annual wormwood
 Biological source: Leaves and the closed, unexpanded flower heads of
Artemisia annua
 Family : Asteraceae/ Compositae
 Active constituents: Artemisinin, dihydro artemisisnin, artemisin,
artemisic acid.

22. Artimisnin occurrence
 Cultivated varities having 1% of artimisnin while wild varities having
0.01 to 0.5 % artimisnin content.
 Artemisinin semi-synthetic derivatives are a group of drugs used
against plasmodium species (P. Falciparum, P. Malariae, P. Ovale and P.
Vivax.)
 Chemically, artemisinin is a sesquiterpene lactone containing an
unusual peroxide bridge.
 This is native of china but cultivated in kashmir

23. Isolation of artimisnin
Extraction can be done by 2 ways-
a. Soxhlet extraction
b. Microwave-assisted extraction

24. Isolation of artimisnin using Soxhlet

25. Isolation of artimisnin
a) SOXHLET EXTRACTION
 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.

26. MICROWAVE-ASSISTED EXTRACTION

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

28. Charaterization of citral
Thin-layer chromatography (TLC)
 Mobile phase – ethyl Acetate: hexane (3:97)
 Stationary phase -- silica gel G
 Detecting reagent -- anisaldehyde-sulphuric acid
 Method – 5 µl is dissolved in 1 ml methanol spot were applied
on activated TLC plate .
 For activation TLc plate is preheated at 110°C for 10 min
 5 µl of reference solution of menthol and 5 µl of investigated
wild mint oil were applied to silica gel plates, Merck (Germany)
20 × 20 cm, 0.25 mm in thickness. Dried the spot for few min.
 Development of TLC plate in mobile phase. And dried
 separated zones were visualized using iodine chamber/ UV
cabinate. Standard menthol served as positive control.
 RF value – 0.45-0.48

29. Thin-layer chromatography (TLC)

30. Identification of artimisnin
1gm finely powdered drug is boiled with 10 ml alcohol and filter
Add sodium hydroxide to filtrate heat again
Red color develop in liquid
Artimisnin present

31. USES
 Effective against malaria & cerebral malaria
 Hepatitis B Schistosomiasis (caused by schistosomes)
 Several blood parasitic protozoans
 Against a variety of cancer cell lines including breast
cancer like
 Human leukemia
 Colon
 Small-cell lung carcinomas
 Drug-resistant cancers

32. Bibliography
 Kokate, C.K., Purohit, A.P. and Gohkale, S.B.
(2019) Pharmacognosy. In Terpenoids, 57th
Edition, Nirali Prakashan, Pune.
 Pharmacognosy And Phytochemistry By Vinod
Rangari. 2009 2nd edition, Career Publications.