Isolation of Plant Compounds PDF

TERPENOIDS OCCURRENCE:  Naturally occurring chemicals in plant kingdom (leaves, stem, bark, roots, flowers)  Present in essential / volatile oils. Eg: oleoresin, ovinine, ergotamine ISOLATION:  Stem distillation – distillation of a liquid inner current of steam, used specially to pur...

TERPENOIDS OCCURRENCE:  Naturally occurring chemicals in plant kingdom (leaves, stem, bark, roots, flowers)  Present in essential / volatile oils. Eg: oleoresin, ovinine, ergotamine ISOLATION:  Stem distillation – distillation of a liquid inner current of steam, used specially to purify liquids that are not very volatile and are immiscible to H2O.  Solvent extraction – partial removal of substance from a solution mixture by dissolving it in another immiscible solvent  Enfleurage process- extraction of essential oils and perfumes from flowers using colourless animal or vegetable fats. STEAM DISTILLATION:  Plant  Maceration  Stem distillation  Distillates + purified solvent mixture  Dried  Solvent removed by evaporation  Oil purified SOLVENT EXTRACTION:  Plant  Maceration  MPM + solvent ( ether/benzene mixture)  Filtration  Solvent removed by evaporation  Oil ENFLEURAGE PROCESS: (for flowers)  Petals spread over molten fat layer  Essential oils absorbs on fat ( fat/ oil)  Oil+ fat+ solvent(pure ethanol)  Everything will dissolve  Fat is removed by coding below 20°c  Ethanol is evaporated at 0°c  Oil IDENTIFICATION TEST:  2ml of the organic extract was dissolved in 2ml of CHCl3  Evaporated to dryness  2ml of conc. H2SO4 was then added  Heated for about 2min.  Development of a greyish colour indicates the presence of terpenoids. THERAPEUTIC EFFECTS OF TERPENOIDS: Terpenoids have been found useful in the prevention and therapy of several diseases and cancer.  Anti-microbial  Anti-fungal  Anti-parasitic  Anti-viral  Anti-allergic PHARMACEUTICAL APPLICATION:  Used as active ingredient in agricultural pesticides.  Paclitaxel from pacific yew tree is used in cancer treatment. GLYCOSIDES Glycosides = sugar(glycon) + non sugar(aglycon) Sugar – glycon (monosaccharides) Glucose increases solubility, bioavailability of the sample. OCCURENCES:  Glycosides are mostly occurring in plants, animal tissue. Eg: digitalis, Apocyanaceae, Aracideae, Legumanceae. ISOLATION: The dried plant material is rendered into a moderately coarse powder. The powder is then extracted in a soxhlet apparatus with aqueous ethanol. The non-glycosidal impurities which get extracted along with glycosides are removed by precipitating them with legal acetate solution. The excess of lead acetate is then removed by passing H2S gas through the extract lead gets precipitated as lead sulphide which is filtered out. The filtrate contains the glycosides. The glycosides can be obtained by removal of the solvent under reduced pressure. Further, purification of the isolated glycosides is done by column chromatography. IDENTIFICATION TEST: 5ml of plant extract are treated with 2ml of glacial acetic acid containing 1 drop of ferric chloride solution. This is under layer with 1ml of conc. H2SO4. A brown ring at the interface indicates presence of carotenoid. A violet ring may appear below the brown ring while in the acetic acid layer. A greenish ring may appear from just gradually throughout this layer. THERAPEUTIC EFFECTS:  In plants, regularity function, protective function, sanitary function.  In animals, cardio active, local irritant in function.  It is used in synthesis of steroidal diuretics coffee, tea, sex hormones, oral contraceptives. PHARMACEUTICAL APPLICATIONS:  Flavouring agent  Colouring agent  Carrier for drug and medicine. ALKALOIDS OCCURRENCE:  90% alkaloids present in the plants such as Rubiaceae, Asteraceae, Paperceaeae.  It is also present in fungi, Pteridophytes, Gymnosperms.  They are basically crystalline in substances. ISOLATION: To isolate alkaloids the dried and powdered plant material is extract with petroleum ether or hexane. This removes fats, oils, terpenes, vax etc. This extract disorder the material now subjected to an alcohol extract with methanol, ethanol. The extract is evaporated to live crude alkaloids mixture. This extract then precipitates between dill aqueous tartaric acid solution and ethyl acetate. Other acid like citric acid can be used. The ethyl acetate layer contains neutral and weekly basic alkaloids. Evaporated the solvent to isolate them. The aqueous is neutralized with ammonia sodium carbonate and again extracted with ethyl acetate. Organic layer now contains basic alkaloids yield the aqueous layer contains quandary ammonia ions. IDENTIFICATION TEST: 5ml of plant extract are treated with 2ml of glacial acetic acid containing 1drop of ferric chloride solution. This is under layer with 1ml of conc. H2SO4. A brown ring at the interface indicates presence of carotenoids. A violet ring may appear below the brown ring while in the acetic acid layer. A greenish layer appear from just gradually throughout the third layer.  TANNINS OCCURRENCE: Oak, hemlock trees, legumes. Eg: hallo-tannins, condensed tannin. IDENTIFICATION TEST: 0.5g of the dried powdered sample is boiled in 20ml of water in a test tube and then filter few drops of ferric chloride is added and observed. Finally brownish green or blue black. Colour formed, so the presence of tannins. ISOLATION: Condensed tannins- the tannin preparation did are a mixture of condensed and hydrolysable tannins should not be used as a standard for the determination of condensed tannins in the plant extract. The biological activity of these isolated tannins would be expected to be similar to that of those present in the leaves. PHARMACEUTICAL APPLICATION:  Plants containing tannins have astringent, homeostatic antiseptic properties.  Used to contain diarrhoea  Used to stop small haemorrhage. THERAPEUTIC EFFECTS:  Bind to protein enzymes  Block digestion  Anti oxidants VOLATILE OILS OCCURRENCE:  The odour, volatile principle of plant and animal source is known as volatile oil.  Lighter than air, critically active, high refractive index, poses distinct odours.  Glandular hair in Labiatae.  Modified parenchyma cells in Piperaceae.  Special tubes or oil tubes in pericarp of fruits of umbelliferae  Lysogenous and Schizogenous cavities in Rutaceae  In the conifers, volatile oils may occur in all tissues.  Petals of rose  Bark of cinnamon  Fruits of corriander  Leaves of peppermint. ISOLATION: 1.DISTILLATION BY WATER: It is applied to plants which are not injured upon boiling. Turpentine oil is obtained by this method. Crude turpentine is introduced into the distillation chamber along with water and subjected to heat below 4°c. A mixture of oil and water is obtained as a distillate which is collected in a Florentine flask. The distillate is separated into two layers oil being withdrawn through the upper outlet of the condenser and water from the lower outlet or vice versa in case of clove as clove oil is heavier than water. 2.WATER AND STEAM DISTILLATION: For substances which are usually injured by direct heat. Eg: clove, cinnamon, dried drug ground and covered with layer of water. Steam is passed through mixture, steam is generated by another chamber and piped out into container having drug. The vapour contain volatile oil are introduced in condensing chamber cooled and collected. 3.DESTRUCTIVE DISTILLATION WITHOUT AIR(VACUUM) : Members of Pinaceae are used. Tough materials such as barks, seeds and roots may be comminated to facilitate extraction. Broken pieces of wood are placed in chamber and heated with water without air in a vacuum. The decomposition of wood occurs and volatile oils generated are collected through the condenses. ECUELLE METHOD: Ecuelle method is used for the extraction of citrus oils where oils cells n rind is ruptured mechanically using pointed projection by twisting raw material over them in clockwise direction either by mechanical or manual. THERAPEUTIC APPLICATION:  As a counter irritant  Eucalyptus oil administered as an inhalant  Improves local circulation  As carminative, shows anti spasmodic  Thymol is used in mouth washes and gargles.  Local anaesthetic  Reduce secretion of lungs in cough and asthma.  Antiseptic, antibacterial, anti fungal  Anti helminthes  Also used in aroma therapy (eg: lavender, rosemary) IDENTIFICATION: Identification of volatile oil are done in these ways. 1. Physical test  2. Chemical test 3. Chromatogeaphic test PHYSICAL TEST: It involves the determination of relative density, refractive index, optical rotation, freezing points etc. Essential oils like clove oil have 1.030-1.063 relative density. 1.528-1.537 refractive index – 2°c to 0°c freezing point. CHEMICAL TEST: This includes acid value and peroxide value CHROMATOGRAPHIC METHODS: It is based on thin layer chromatographic and gas-liquid chromatography (GLC) to check the purity and composition of the material and to detect the impurity present. All these tests are used to confirm the present purity, quality of essential oils. RESINS OCCURRENCE: Resins are generally distributed in plants and rarely in insects. They are present in the ducts or cavities known as schizolysogenous ducts. Resins are of two types:  Normal or physiological resin  Abnormal or pathological resin 1.NORMAL OR PHYSIOLOGICAL RESIN: Resins that are performed in the plants and making injury to the plants can increase their yield. Resin of pinus is the example. 2.ABNORMAL OR PATHOLOGICAL RESIN: Resins that are found in the plants only when injury or incision is made. Bensoin, toles balsam are the example. Resins occur in secretary zone or structures. A few typical of such plant source along with their specific secretory structure are  Resin cells – ginger  Schizogenous ducts – pine wood  Glandular hairs – cannabis ISOLATION:   By extracting the drug with alcohol and precipitating resins present in concentrated extract by addition of water. Eg: jalap, ipomea, podophyllum  By distillation (for separation of oils) Eg: colophony  By heating the plant parts. Eg: Guaum  By making incision. Eg: Asfoetida, Balsams.  By processing incrustation. Eg: shellac IDENTIFICATION TEST: There are three types of identification test. PHYSICAL TEST: Properties such as solubility, taste, odour and examination of powder under the microscope can be helpful. CHEMICAL TEST: Identification of resin include:  Acid value  Iodine value  Saponification value Specific chemical test  Cinnamic acid  Benzoic acid and benzoin  Tolu balsam  Peru balsam THERAPEUTIC EFFECTS:  Resins are local irritant and hence and acts as local cathartic. Eg: jalap and ipomea  Used as anti cancer. Eg: podophyllum  In bronchial asthma. Eg: cannabin  Used in tinctures. Eg: benzoin  Used as ointments and plasters. Eg:terpentine and colophony  Used in the production of emulsions.