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

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

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

2. W h a t i s t e r p e no i d s 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 (C 5 H 8 ) n . Isoprene unit are 5 carbon unit For having isoprene unit they are also called isoprenoids

3. P h y s i c a l p r o pe r ti e s o f t e r p e no i d s 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

4. 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.

5. 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.

6. MENTHOL

7. 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

8. Hydro-distillation

9. 1. Hydro-distillation by Clevenger apparatus system

10. 2. Steam distillation

11. 2. Steam distillation

12. 3. Method of Isolation

13. 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

14. Analysis

15. % Menthol = 7.814 x (c-a) {0.021 x (c-b)/U} A – 0. 21 x ( c - a) Where, “a” & “b” = ml of HCl for oil “c” = ml of HCl for blank “U”= Un - acetylated oil “A ” = Ac e t y l a t e d oil

16. 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.

17. CITRAL

18. 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

19. Hydro-distillation

20. Citral

21. 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 C 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 .

22. 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.

23. ARTEMISIN

24. 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

25. Extraction

26. 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.

27. MICROWAVE-ASSISTED EXTRACTION AND TEST SAMPLE PREPARATION

28. PARTITION OF EXTRACT 1. hexane extracts (non-polar fraction) 2. methanol extracts 2.methanol-water extract (polar fraction) 1.ethyl acetate extract ( semipolar fraction)

29. 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 %

30. 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

31. 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

32. 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

33. 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.