Practical Pharmacognosy - Third Year PDF

Glycosides Glycosides are compounds that yield on hydrolysis, one or more sugar part and another non-sugar part. The sugar part is known as glycone , and the non-sugar part is the aglycone. In general there are two basic classes of glycosides: C- glycosides, in whi...

Glycosides Glycosides are compounds that yield on hydrolysis, one or more sugar part and another non-sugar part. The sugar part is known as glycone , and the non-sugar part is the aglycone. In general there are two basic classes of glycosides: C- glycosides, in which the sugar is attached to the aglycone through C-C bond, and the O- glycosides in which the sugar is connected to the aglycone through oxygen –carbon bond. Chemically the glycosides are acetals in which the hydroxyl group (OH) of the glycone is condensed with the hydroxyl group of aglycone. More simply the glycosides may be considered as sugar ether. Two forms of glycosides are present, the α-form and the β-form, but the β-form is the one that occur in plants, even the hydrolytic enzymes act on this type. O OH ORe Re C H Re C ORe Re C ORe H H Hemiacetal Acetal Inside the body the glycosides will be cleaved to glycone and aglycone parts, the glycone part confers on the molecule solubility properties, thus is important in the absorption and distribution in the body, while the aglycone part is responsible for the pharmacological activity. Generally all glycosides are hydrolyzed by boiling with mineral acids , on the other hand the presence of specific enzyme in the plant tissue, but in different cells from those that contain the glycosides, are able to hydrolyzed the glycosides, such as the emulsin enzyme which is present in the almond kernel, and the myrosin enzyme which is found in the black mustard seeds.  (Fig.1) General Structure of Glycosides Generally in the extraction of glycosides we have to consider the following points: 1. Apolar solvent, which is mostly alcohol, but not water, since water may induce fermentation, in addition water need high temperature due to its high boiling point. 2. Neutralization of the extract with base, since the presence of acid lead to hydrolysis of the glycoside. 3. Use of heat is to inhibit the activity of hydrolytic enzymes that present in the plant cell. The glycosides are classified according to the chemical structure of the aglycone to: 1. Cardioactive glycosides. 7. Alcohol glycosides. 2. Anthraquinone glycosides. 8. Aldehyde glycosides. 3. Saponin glycosides. 9. Lactone glycosides. 4. Cyanophore glycosides. 10. Phenol glycosides. 5. Isothiocyanate glycosides. 11. Miscellaneous glycosides. 6. Flavonoid glycosides.  Exp. No.1 [Lab.1] Cardioactive Glycosides They are named so, due to their action on the heart muscle. The aglycone part here is steroid, which is chemically cyclopentaphenanthrene. cyclopentaphenanthrene nucleus The steroidal aglycones are of two types: 1) Cardinolides(α-β unsaturated 5 – member lactone ring). 2) Bufadienolides (doubly unsaturated 6-member lactone ring). The more prevalent in nature is cardinolides type. For maximum activity of cardioactivce glycosides the following points are important: 1) 17 -β –lactone ring (cardinolide or bufadinolide). 2) 3 -β - OH. 3) 14 -β-OH. 4) CATSC (C= cis between two rings (A&B). A= Anti in one ring (5&19). T=Trans between two rings (B&C). S= Syn in one ring (8&18). As represented bellow: Cardenolide Bufadienolide Plants Containing CardioactiveGlycosides: 1) Digitalis (digitalis or foxglove) Digitalis purpurea of the family Scrophulariaceae. The name digitalis is from Latin digitus which means finger refers to finger – shaped, while purprea refers to purple color of their flower. This plant contains anumber of glycosides as digitoxin , gitoxin and getaloxine. 2) Digitalis lanata of the same family, from which the digoxin is obtained. 3) The plant used in our laboratory is Nerium oleander of the family Apocyanaceae. The main glycoside of which is oleandrin. Nerium oleander Isolation and Identification of the Cardioactive Glycosides: 1. Extraction: Aim: To isolate the cardioactive glycosides. Equipments:  Large beaker & two medium size beakers.  Two conical flasks.  Centrifuge & Centrifuge tubes.   Separatory funnel.  Water bath. Reagents:  70% ethanol.  Lead sub acetate.  10% sodium phosphate solution.  Chloroform: Ethanol (3:1 v/v).  Anhydrous sodium sulphate.  4N HCl acid.  Chloroform. Procedure: Method of extraction: Maceration. Plant used: Nerium oleander. Part used: dry leaves. Maceration 10 gm of the powdered leaf in 100 ml of 70%ethanol for 24 hrs. (Prepared previously) Take 10 ml of alc. Extract in conical flask Add 10 ml of lead sub acetate solution (Mixing& standing for 5 mins.) Centrifuge (5 mins.) Decant and take the supernatant (upper layer)  Add 10 ml of 10%sodium phosphate solution Centrifuge (5 mins.) Take supernatant and divide in to two divisions ------------------------------------------------------------------- Fraction A Take one division and put in a separatory funnel Add [10 ml of Chloroform: Ethanol (3:1 v/v)] two times (Shake& stand) Combine the organic lower layer and put it in the conical flask Add Small quantity of Anhydrous sod. Sulphate & allow standing for few minutes until get a clear solution, decant the Chloroform-ethanol extract and reduce the volume on water bath to get: Fraction A Fraction B Place the other division of the extract in the conical flask Add 3 ml of 4N HCl Boiling in water bath (15 mins) Cool &transfer to a separatory funnel Add [10 ml of Chloroform] tow times Combine the chloroform extracts (lower layers) Add Small quantity of Anhydrous sod. Sulphate & allow standing for few minutes until get a clear solution then decant the chloroform layer and concentrated on water bath to about 1ml. and we get: Fraction B Results: Fraction A : Contain the whole glycosides. Fraction B : Contain the aglycone (genin) part only. [Lab.2] The Chemical Tests 1. Baljet's Test: Aim: The identification of the cardio active glycosides in general. Equipments& Reagents:  Test tube.  Picric Acid.  Sodium hydroxide solution. Procedure : Take 1ml of fraction A, add 2 drops of Picric acid then make it alkaline with Sod. Hydroxide solution.(litmus paper). Results: Turbid , yellow to orange in color. 2. Keller- Killian's Test Aim: The identification of the cardio active glycosides in general. Equipments& Reagents:  Test tube.  Glacial acetic acid  0.1 % of ferric chloride solution.  Conc. H2SO4. Procedure: Take1ml of fraction A, and 2ml of glacial acetic acid, add 1 drop of 0.1 % of ferric chloride solution.Take 1ml of conc. H2SO4 and add to the above mixture in drops so as to make two layers. Results: Two layers are formed; the upper one has light bright green color. The lower layer has transparent clear color (H2SO4 layer).The junction appears as a reddish –brown ring. Other Chemical Tests for the Identification of Sterol Glycosides: 1. Raymond 's Reaction: Aim: To identify the sterol nucleus. Equipmentsand Reagents:  Test tube.  10% sodium hydroxide solution.  1% m-dinitrobenzene. Procedure: To 1ml of fraction A add 1-2 drops of 10% sodium hydroxide and few drops of an alcoholic solution of 1%m-dinitrobenzene. Result: Pink color appears. 2. Kedde's Reaction : Aim : To identify the sterol nucleus. Equipments and Reagents:  Test tube.  1% 3,5-dinitrobenzoic acid.  0.5 N aqueous methanolic KOH (50 %). Procedure: To a solution of glycoside add a solution of 1% 3, 5-dinitrobenzoic acid in 0.5N aqueous methanolic KOH (50%).Report the color. 3. Lieberman's Sterol Reaction: Aim: To identify the sterol nucleus. Equipments and Reagents:  Test tube.  Porcelain dish.  Anhydrous acetic acid.  Conc.H2SO4. Procedure: Take 1ml of fraction A in a test tube then add 5ml of anhydrous acetic acid and shake well. Take 4 drops of the above mixture and place in a porcelain dish, and then add one drop of conc.H2SO4. Result: A change of color from rose, through red,violet and blue to green. The colors are slightly different from compound to compound. Discussion: This reaction is due to the steroidal part of the molecule and it is characteristic of the aglycone of the scillarenin type (unsaturated steroidal part). 4. Legal's Reaction: Few mls of the glycoside or the purified extract of the crude drug is dissolved in pyridine. When sodium hydroxide and sodium nitroprusside are added alternatively, a transient blood-red color develops. This is atest for the unsaturated lactone ring of the genin. The Identification of Cardio active Glycosides By Chromatography: By the use of thin layer chromatography (T.L.C)  The stationary phase = Silica gel G.  The mobile phase =Chloroform: Ethanol: Water (7:3:1) Or Ethyl acetate: Methanol: Water (75:10:5).   The standard compound = Oleandrin.  The spray reagent = Lieberman's reagent.  Mechanism of separation =Adsorption.  Developing = Ascending.  Other mobile phases : Butanone: Xylene: Formamide (50:5:4) Chloroform: tetrahydrofuran: Formamide (50:50:6). Procedure: 1) Prepare 100ml of mobile phase, and place it in the glass tank. 2) Cover the tank with glass lid and allow standing for 45 minutes before use. 3) Apply the sample spots (fraction A & fraction B), and the standard spot on the silica gel plates, on the base line. 4) Put the silica gel plate in the glass tank and allow the mobile phase to rise to about two-third the plate. 5) Remove the plate from the tank, and allow drying, and then detecting the spots by the use of the spray reagent and heat the plates at 105 -110 0C for 5-10 mins in the oven. 6) Note the spots, and calculate the Rf value for each spot. Note/ the Rf value should be less than 1, because if the Rf value = 1, this means that there is no separation, and the sample moved with the solvent. Study problems: Q1. What is the meaning of CATSC, explain with structure? Q2.Give the reasons of: a) The addition of lead sub acetate, sodium phosphate and anhydrous sodium sulphate to the extract?  b) The use of chloroform: ethanol in partitioning of fraction A? c) The use of HCl in the extraction procedure of cardio active glycosides (F.B). d) The use of chloroform alone in partitioning of fraction B? e) The use of picric acid in the performance of Baljet's test? f) The use of glacial acetic acid in Keller Killian test procedure? g) The use of dinitrobenzene in Raymond and Kedde's reactions to identify the sterol nucleus? h) The addition of conc.H2SO 4 in Keller-Kellian test& Lieberman's sterol reactions? Q3.How can you identify an extract containing cardio active glycosides? Exp.No. 2 [Lab.3] Anthraquinone Glycosides Anthraquinone and related glycosides, are stimulant cathartics, and exert their action by increasing the tone of the smooth muscle in the wall of colon and stimulate the secretion of water and electrolytes into the large intestine. After the oral administration, the anthraquinone glycosides are hydrolyzed in the colon by the action of enzymes of the micro flora, to the pharmacologically active free aglycones which usually produce their effect in 8 -12 hrs. After administration, these agents are indicated for constipation in patient who do not respond to milder drugs and for bowel evacuation before investigational procedure or surgery. Stimulant laxative are habit forming so the long-term use may result in laxative dependence and loss of normal bowel function. The glycosides of anthranols and anthrones elicit a more drastic reaction than do corresponding anthraquinone glycosides and cause discomforting and gripping action. The drugs mostly used are cascara,frangula,hypericum and Senna. Aloe and Rhubarb are not recommended due to their irritating actions which increase the chance for gripping effect. The anthraquinone hydrolyzed to give aglycone which are di, tri, or tetra – hydroxyanthraquinone.Also there are antherone, dianthrones and oxanthrones. Fig (1): Chemical Structure of Anthraquinone Isolation and Identification of the Anthraquinone Glycosides: 1. Extraction: Aim: To isolate the anthraquinone glycosides. Equipments:  Large beaker & two medium size beakers.  Two conical flasks.  Centrifuge & Centrifuge tubes.  Separatory funnel.  Water bath.  Round bottom flask.  Filter paper.  Reagent bottle. Reagents:  Conc.HCl acid.  Chloroform.  60% w/v ferric chloride solution. Procedure: Method of extraction:Decoction. Plant used: Senna (Cassia acutifolia, Cassia angustifolia) Family: Leguminosaea. Part used: Dry leaves. Senna  Place 0.5 gm of powdered dry leaves of Senna in 50 ml of water Boiling (15 mins) Cool & filter Place the filtrate in separatory funnel and extract by shaking with [10 ml of Chloroform] two times Upper layer Combine lower layer (Aqueous layer) (Chloroform layer Devided into two portions [Free aglycone (dianthrone)] Fraction B (Whole Glycosides) Other part of the aq. Layer Fraction A Reflux (20mins) [Put in reagent bottle] Add 1) 3.5 ml of Ferric Chloride sol.(60 %w/v ). 2) 2ml of Conc. HCl acid. Cool Place in a separatory funnel and extracted with [10 ml of Chloroform] tow times Aqueous layer Chloroform layer Glycone part Aglycone part (monoanthrone) Fraction C Results: Fraction A : Contain the whole glycosides. Fraction B : Contain the aglycone (dianthrone). Fraction C: Contain the aglycone part (monoanthrone). [Lab.4] TheChemical Tests A.General Reaction: For the following tests, boil 1 gm of the crude drugs (Aloe) given with 100 ml of water, add a little of kieselghur, filter and use the solution for the following tests: 1. Schontetens Reaction (Borax test): Aim: Identification of the anthraquinone glycosides in general. Equipments& Reagents:  Test tube.  Small beaker.  Water bath.  Borax. Procedure: To 2ml of the Aloe extract, add 0.1gm of Borax and heat until dissolved. Pour a fewdrops of the liquid into test tube nearly full of water. Results: A green fluorescence is produced. Discussion : This green fluorescence is due to aloe- emodin anthranols liberated from barbaloin by hydrolysis with Borax giving this reaction. 2. Bromine Test for Aloin: Aim: Identification of the anthraquinone glycosides in general. Equipments& Reagents:  Test tube.  Bromine solution. Procedure: Take 2ml of the Aloe extract, add an equal volume or an excess of freshly prepared solution of bromine. Record the color. B.SpecificReaction: Borntrager's test : Aim: Identity test for aglycone part of anthraquinone glycosides. Equipments& Reagents:  Separatory funnel.  Test tube.  Dilute HCl.  Benzene.  Dilute ammonia (10%). Procedure: To5ml of the Senna extract (fraction A), add 5ml dilute HCl, then place the mixture in a separatory funnel and partitioing with 5ml of benzene for 1min. Take the upper benzene layer (free aglycone) and shake it with dilute ammonia (10%).Check the intensity of the color. Results: Pink color will be produced which is very clear with monoanthrones than dianthrones. Discussion: The benzene extracts the aglycone, and with ammonia, forms anthraqunone salts, which have pink color. The Identification of Anthraquinone Glycosides By Chromatography: By the use of thin layer chromatography (T.L.C)  The stationary phase = Silica gel G.  The mobile phase = n-propanol: Ethyl acetate: Water (60:30:30).  The standard compound =Sennoside.  The spray reagent = Alcoholic KOH 5%w/v.  Mechanism of separation = Adsorption.  Developing = Ascending.  NOTE/ for the best result spray first with 25%w/v nitric acid then heat in the oven after that spray with KOH reagent. This step is done to intensity the color of the spot.  Procedure: 1) Prepare 100ml of mobile phase, and place it in the glass tank. 2) Cover the tank with glass lid and allow standing for 45 minutes before use. 3) Apply the sample spots (fraction A, fraction B& fraction C), and the standard spot on the silica gel plates, on the base line. 4) Put the silica gel plate in the glass tank and allow the mobile phase to rise to about two-third of the plate. 5) Remove the plate from the tank, and allow drying at room temperature, spray first with 25%nitric acidsolution andheatfor 10 minutes at 110 0C. 6) Allow to cool, and then spray with 5% w/v alcoholic KOH solution. Detect the spot formed and calculate the Rf values. Study problems: Q1. What are the main differences between three fractions ( A, B and C) of anthraquenone glycosides? Explain with structures? Q2.Give the reasons of: a) The use of Borax in shontetens reaction? b) The use of 10% dilutes ammonia in Borntrager's test? Q3.How can you identify an extract containing anthraquinone glycosides? Exp.No. 3 [Lab.5] Saponin Glycosides This group of glycoside is widely distributed in higher plants. Saponin glycosides form colloidal solution in water that foam upon shaking, this is due to a decrease in the surface tension action done by saponin glycosides, as a result of the hydrophobic/ hydrophilic characteristics of the saponin, and due to this property the saponins are used in the manufacturing of beer, and soap. Saponins have a bitter, acrid taste, and drugs containing them are usually sternutatory and otherwise irritating the mucus membrane. They destroy red blood corpuscles by hemolysis and are toxic especially to cold blooded animals therefore many saponins are used as fish poisons. The more poisonous saponin is often called sapotoxin, many are toxic to insects and mollusks, and some are used to control schistosomiasis snails. Saponin upon hydrolysis yield an aglycone known as sapogenin, which are crystallized upon acetylation, therefore this process is used for purification. According to the structure of the aglycone, two kinds of saponin are recognized: 1. Pentacyclictriterpenoid saponins (acidic, and the C-atom is C30) 2. Steroidal saponins (neutral C- atom is C27).  Pentacyclictriterpenoid saponin Steroidal saponin Isolation &Identification of the SaponinGlycosides: Procedure: Method of extraction: Decoction. Plant used: Saponaria officinalis family Caryophyllaceae. Part used: Dry root.  Saponaria officinalis Add 0.1 gm of saponaria root in coarse powder to 20 ml distilled water in a beaker and boil gently for 2-3 minutes. Filter hot and allow cooling: a) Dilute 5ml of the filtrate with water and shake vigorously. b) To the remaining of the filtrate add 5ml of dilute H2SO4 acid and boil gently for 3-5 mins.The aglycones are obtained by acid hydrolysis and are insoluble in water but are soluble in 90% alcohol. c) Make the filtrate obtained from (b) alkaline with NaOH, (litmus paper) and then carry Fehling's test or Benedict's test (5ml filtrate + 2ml of Benedict's reagent heat for 10mins on boiling water bath ). Specific Reaction: 1.TheHemolytic Test Aim:Identity test (specific) for saponin glycosides Equipments& Reagents:  Two test tube.  10% solution of blood in normal saline.  Normal saline. Procedure: Take two test tubes and place in each one 5ml of a 10% solution of blood in normal saline. To one of them, add 5ml of normal saline solution and to the other one add 5ml of the extract of Saponaria root.Shake both tubes gently and notice the result. Results: The test tube containing 5ml of the extract of Saponaria will cause blood hemolysis. 2.Foam Index (according to Kofler) Foam index is a value, which is used to express the quantity of the saponin glycosides in the crude drugs. The method is based upon the property of saponin to form foam when shaken with water. The foam index signifies the dilution of the substance or drug to be tested which gives alayer of foam 1cm high if the aqueous solution is shaken for 15 seconds, and then allow standing for 15 minutes before reading is made. Foam Index Aim:Identity test (specific) for saponin glycosides Equipments& Reagents:  10 Test tubes having the same diameter.  Graduated pipette.  0.1% decoction from the powdered drug.  1% solution of sodium carbonate. Procedure: 1. Prepare 0.1% decoction from the powdered drug, neutralized it by adding solution of 1% sodium carbonate drop wise (litmus paper) and filter. 2. Into 10 test tubes having the same diameter, 1 to 10 ml of this decoction is added respectively using a graduated pipette, complete the volume to 10 ml with distilled water. 3. Shake the content of each test tube thoroughly for 15 seconds and allow to stand for 15 minutes. 4. After this time, the reading is made in the test tube containing the most dilute solution with a ring of foam1 cm height. For example: When this is occur in the test tube number 8, which contains 8ml of the decoction and 2 ml of water, then 8 ml of 0.1% decoction corresponds to 0.008 gm of the drug and the dilution is calculated from the following calculation: gm ml 0.1 100 X 8 X= 0.008 gm. of saponin in 8ml of decoction. gm ml 0.008 10 1 X X= 10/0.008 X=1250 ml. That means the ring of foam 1cm high is formed by a solution diluted 1:1250. The foam index is therefore 1250. Note/The addition of sodium carbonate is to convert the acidic saponins that may be present in the decoction, to salts, which are soluble in water. Study problems: Q1. How many kinds of sapo genin are found in the medicinal plants? Explain with structures? Q2.Give the reasons of: a) The addition of H 2SO 4 and boiling during the extraction of Saponaria root? b) Alkalinization with NaOH in Fehling's or Benedict's tests? Q3.How can you identify an extract containing saponin glycosides? Exp.No. 4 [Lab.6] Tannins Tannins compromise a large group of complex substances that are widely distributed in the plant kingdom. Chemically tannins are complex substances; they usually occur as mixtures of poly hydroxyl phenols that are difficult to separate because they do not crystallize. Tannins are divided according to the identity of the phenolic nuclei involved, and on the way they are joined into two classes: 1.Hydrolysable tannins: This class consists of gallic acid and related polyhydroxy compounds (hexahydroxydiphenic acid) and theirderivatives esterified with glucose. They are termed hydrolysable tannins due to ease of esters to hydrolyze to phenolic acids and sugar. They were formerly known as pyrogallol tannins.  Gallic aci Hexahydroxydiphenic acid 2. Nonhydrolysable tannins or condensed tannins: This class contains onlyphenolic nuclei but frequently linked to carbohydrates or proteins. When treated with hydrolytic agents, these tannins tend to polymerize, yield insoluble usually red-colored products known as phlobaphenes. The name (condensed tannins) is due to the fact that on the treatment with hot acid some of C-C bonds are broken yielding anthocyanidin monomers.These tannins are sometimes called catechol tannins. Catechin Anthocyanins (Are glucosides of anthocyanidins) General properties of Tannins:  Tannins are non–crystallizable compounds that,with water.form colloidalsolution possessing acid reaction and sharp "puckering "taste.  They cause precipitation of solution of gelatin as well as alkaloids.  They form dark blue, greenishblack soluble compounds with ferric salts.  They produce deep red color with potassium ferricyanide and ammonia.  They are precipitated by salts of copper, lead and tin by strong aqueous potassium dichromate or 1%chromic acid solution. In alkaline solutions; many of their derivatives readily absorb oxygen.  Tannins precipitate proteins from solution and can combine with proteins, rendering them resistant to proteolytic enzymes.when applied to living tissue this action is known as an"astringent" action and form the basis for therapeutic application of tannins. Uses of tannins: 1. Astringents, used in the gastrointestinal tract and on the skin abrasion. 2. In the treatment of burns, the proteins of the exposed tissue are precipitated and form amildly antiseptic protective coat under which the regeneration of new tissue may take place. 3. Use in the process of vegetable- tanning which converts animal hides to leather (leather industry). 4. Antidote treatment of alkaloids poisoning. 5. Ink industry.  Pyrogalloltannins(Nut gall) Catechole tannins(Hamamelis leaf) Tests on Tannins I.Catechole tannins: Plant used:Hamamelis leaf or witch hazel leaves: Is the dried leaf of Hamamelis virginiana of the family Hamamelidaceae. A. Microscopically Examination: Examine the powder drug microscopically for the trichomes and notice the type of stellate, the form-branched stellate trichomes consisting of 4-12 unicellular branches united by their bases. B. Chemical Tests: Aim: Identity test for Catechol Tannins. Procedure: Boil 5gm hamamelis leaf, coarsely powdered, with 50 ml of water. Cool and filter. To 2 ml portions add the following reagents and notice the results: 1. Few drops solution of ferric chloride.  2. 1ml solution of lead sub acetate. 3. 1ml solution of potassium dichromate. 4. 2ml solution of gelatin. 5. 2ml solution of quinine dihydrochloride.(or any alkaloid). 6. 0.5ml solution of sodium acid phosphate, warm, cool and filter. To the filtrate add solution of phenazone. 7. Bromine solution. 8. 5ml Mitchell's reagent (5 ml of a 0.2% solution of iron and ammonium citrate) and add 1gm sodium acetate. Boil, cool and filter. Results: Notice the colors and precipitates obtained. II. PyrogallolTannins: Plant used: Gulls, Nutgall. Is the excrescence obtained from the young twigs of Quercus infectoria of the family Fagaceae. A. Microscopically Examination: Examine the powdered drug microscopically and notice the following: 1. Fairly numerous sclerenchymatous cells. 2. Lignin bodies. 3. The presence of only a few small vessels. 4. A few starch grains. 5. Tannin flakes, visible in clove oil mount. 6. Thick -walled, pitted parenchyma with both cluster and prismatic crystals of calcium oxalate. 7. Occasional insect fragments.  B.Chemical Tests: Aim: Identity test for Pyrogallol Tannins. Procedure: Prepare 0.1% suspension of powdered nutgall in water and is treated with: 1. A saturated solution of potassium dichromate plus a trace of acetic acid. 2. A 1% solution of sodium carbonate. 3. A 5% ferric sulphate solution. 4. A 1% ferric acetate solution. 5. Shake 0.1gm powdered nutgall with 1ml of water, micro filter one drop into an evaporating dish. Add one drop of a 5% ferric chloride solution. 6. Repeat the test no.5 with bromine water. Results: Notice the colors and precipitates obtained. Study problems: Q1. How many kinds of Tannins according to the phenolic nuclei? Explain with structures? Q2.How can you identify an extract containing Pyrogallol tannins? Q3.How can you identify an extract containing Catechol tannins? Exp.No. 5 [Lab.7] Volatile Oils They are odorous principles found in various plant parts. Because they evaporate when exposed to the air at room temperatures, they are called volatile oils; they are also called essential or ethereal oils. Volatile oils are colorless as a rule, particularly when they are fresh, but on long standing they may oxidize and resinify, thus darkening in color, to prevent thisdarkening, they should be stored in a cool, dry place in tightly stoppard, preferably full, amber glass containers. As a rule, volatile oils are immiscible with water, but they are sufficiently soluble to impart their odor to water. They are soluble in ether, alcohol and most organic solvents. Many volatile oils consist largely of terpenes (terpenes are natural products whose structures may be divided into isoprene units). Isoprene unit Phenylpropanoids Another major group of volatile oil constituents are the phenylpropanoids. (Thesecompounds contain the C6 phenyl ring with an attached C3 propane side chain). Generally volatile oils and volatile oil-containing drugs are divided in to the following classes: 1. Hydrocarbons. 2. Alcohols. 3. Aldehydes. 4. Ketones. 5. Phenols. 6. Phenolic ethers. 7. Oxides. 8. Esters. Essential oils are derived from various sections of plants: Leaves- Rosemary, Basil, Eucalyptus. Flowers- Rose, Lavender, Clove. Seeds- Almonds, Anise, cumin. Bark- Cinnamon. Rhizome- Ginger. Pharmacological Uses of Volatile Oils: Carminative as for Rosemary oil. Antitussive as for Eucalyptus. Antiseptic as Clove oil. Aromatherapy, alternative medicine as Lavender Oil.  Anise Isolation and Identification of the Volatile Oils: Aim: Determination of the volatile content of crude drugs by steam distillation method. Equipment : Clavenger type as an apparatus. Clevenger Apparatus Clevenger Apparatus (Oil heavier than Water) (Oil lighter than water) Procedure: 1) Weigh out 20 gm of the plant material (coarse powder) and place into a distilling flask; add few pieces of porous earthenware. 2) Add 200 ml distilled water to the flask and shake well. Add another 200ml of water by rinsing the neck of the flask. 3) Connect the distilling flask with the still head of the apparatus. By the means of the pipette or washing bottle, fill the receiver with water until over flows. 4) Connect the condenser of the apparatus with the cooling water (from the tap). 5) Heat the distilling flask until the boiling starts. Record the time of the beginning of distillation, and continue the distillation for one hour. 6) Switch off heating. Allow the graduated receiver to cool. Read off the volume of the volatile oil (count all small divisions in the receiver of the layer of oil). 7) Calculate the %v/w of the volatile oil content of drug. Identification of Volatile Oils By Chromatography: By the use of thin layer chromatography (T.L.C).  The stationary phase = Silica gel G.  The mobile phase = Chloroform: Benzene (3:1).  The standard compound = Peppermint Oil.  The spray reagent =Vanilline _Sulphuric acid / Ethanol (10%v/v).  Mechanism of separation = Adsorption.  Developing = Ascending. Procedure: 1) Prepare 100ml of mobile phase, and place it in the glass tank. 2) Cover the tank with glass lid and allow standing for 45 minutes before use. 3) Apply the sample spot and the standard spot on the silica gel plates, on the base line. 4) Put the silica gel plate in the glass tank and allow the mobile phase to rise to about two-third the plate. 5) Remove the plate from the tank, and allow drying and then detecting the spots by the use of the spray reagent and heat the plates at 120◦C until the spot’s color intensity is reached in the oven. Detect the spot and calculate the Rf value. Determination of the Refractive Index of the Volatile Oils: Refractive index: When a ray of light passes from a less dense to a denser medium, it will bend or refract toward the normal. If (e) represents the angle of refraction and (i) the angle of incidence according to law of refraction: Sin i/ Sin e =N/n Where (n) is the index of refraction of the less dense and (N)is the index of the refraction of the denser medium. Procedure: 1) Connect the abbe refractometer with the cooling water system and record the temperature. 2) Wash the prism of the apparatus with absolute alcohol and dry it. 3) Illuminate the field so that the cross is clear. 4) Introduce 2 drops of volatile oil carefully on the surface of the prisms and cover it with another prism. 5) Turn the knob so that the line of the dark field reaches the center of the intersection bars of the cross. 6) Read off the refractive index (nD) on the scare at a given temperature. 7) Calculate the refractive index at 200C (nD20) corrections for the temp. Study problems: Q1. How many kinds of Volatile-oil according to the basic nuclei? Explain with structures? Q2.How can you determine the volatile content of crude drugs? Q3.How can you identify an extract containing Volatile oils? Q4. Define the Refractive index? How it can be measured? Exp.No. 6 [Lab.8] Flavonoid glycosides Flavonoids (from the Latin word flavus meaning yellow, their color in nature) are polyphenols of plant origin that are among the most important compounds in human diet due to their widespread distribution in foods and beverages. They can occur both in the free form (aglycones) and as glycosides, and differ in their substituents (type, number and position) and in their instauration. The most common classes are the flavones, flavonols, flavanones, catechins, isoflavones and anthocyanidins, which account for around 80 % of flavonoids. All flavonoids share a basic C6-C3-C6 phenyl-benzopyran backbone. The position of the phenyl ring relative to the benzopyran moiety allows a broad separation of these compounds into flavonoids (2-phenyl-benzopyrans), isoflavonoids (3-phenyl-benzopyrans) and neoflavonoids (4-phenyl-benzopyrans). Division into further groups is made. Fig.1. Structure of the structural backbones of the main flavonoid groups (flavan, isoflavan and neoflavan) and of relevant flavonoid classes. Atom numbering and ring nomenclature are also included.  They have been used extensively as chemotaxonomic markers and are abundant in the Polygonaceae, Rutaceae, Legaminosae, Umbellifereae and Compositae. They occur both in the Free State and as glycosides; most are O-glycosides but a considerable number of flavonoid C-glycosides are known. The glycosides are generally soluble in water and alcohol, but insoluble in organic solvents; the genins are only sparingly soluble in water but are soluble in ether. Flavonoids dissolve in alkalis, giving yellow solutions which on the addition of acid become colorless. Pharmacological activity of flavonoids: A number of flavonoid-containing herbs have now been included in the BP/ EP, examples are Birch leaf, Calendula Flower and Elder flower.The group is known for its anti-inflammatory and anti- allergic effects, for antithrombotic and vasoprotective properties, for inhibition of tumor promotion and as protective for the gastric mucosa. Some of these pharmacological properties can be explained on the bases of antioxidant activity. Many flavonoid -containing plants are diuretic (e.g. buchu and broom) or antispasmodic (e.g. liquorice and parsley). Some flavonoids have antitumour, antibacterial or antifungal properties. Isolation and Identification of Flavonoids: Aim: Isolation method of flavonoids from Ruta. Equipments and reagents:-  Large beaker.  Water bath.  Two medium size beakers. Reagents:  Petroleum ether.  70 % aqueous methanol.  5 % HCl.  Chloroform. Procedure: Method of extraction: Maceration. Plant used: Ruta graveolens family Rutaceae. Part used: Dry leaves. Ruta graveolens  SCHEME FOR ISOLATION OF FLAVONOID GLYCOSIDE Maceration 10 gm.of the powdered leaves in 100ml of petroleum ether overnight. (Prepared previously) The residue is dried Macerated again with 70 % aqueous methanol overnight ( prepared previously) Filter Concentrated the extract to small volume (15ml) Divided into two parts 5ml 10ml Fraction A Add 5ml of 5% HCl Boil for 20min. Cool& transfer to a separatory funnel Shake with (15ml of Chloroform) two times Aqueous layer Chloroform layer (Concentrated) Fraction B Results: Fraction A : Contain the whole glycosides. Fraction B : Contain the aglycone part. [Lab.9] TheChemical Tests A.General Reaction: 1-Cyanidin reaction with magnesium powder. Flavanonones and dihydroflavanols at the presence of HCl produce bright red colour; isoflavonoids and flavanes develop yellow, sometimes red colour, flavonoles do intensive red colour. 2-Reaction of flavonoids with two o-oxygroups in B cycle with lead acetate causes precipitation. Flavones produce intensive yellow, aurones - red, anthocyanes red or blue precipitates. 3-Wilson’s reaction: 5-oxyflavones and 5-oxyflavonoles with Wilson's reagent (boric and citric acids in anhydrous acetone) develop brightly yellow colour with yellowish-green fluorescense. 4-alkaline solution: In alkaline solutions flavanones produce uncoloured or yellow precipitates, that for some time become brightly yellow or yellow (isomerization to formation of chalkones); chalkones and aurones develop red or purple (it's their specific reaction), flavones and flavonoles produce yellow coloured precipitates. 5-Mineral acids: Flavones and flavonoles with mineral acids form oxonic (flavic) salts of brightly yellow or red colour, chalkones and aurones produce intensive colour of raspberry or red. 6-Other methods: Other methods of identification include chromatography,.colorimetric or spectrophotometric analysis after reaction with aluminium chloride. A.Special Reaction: 1-Shinoda test Four pieces of magnesium fillings (ribbon) are added to the ethanolic extract followed by few drops of concentrated hydrochloric acid. A pink or red colour indicates the presence of flavonoids. Colours varying from orange to red indicated flavones, red to crimson indicated flavonoids, crimson to magenta indicated flavonones. 2-Sodium hydroxide test About 5 mg of the compound is dissolved in water, warmed and filtered. 10% aqueous sodium hydroxide is added to 2 ml of this solution. This produces a yellow coloration. A change in color from yellow to colorless on addition of dilute hydrochloric acid is an indication for the presence of flavonoids. 3-p-Dimethyl amino cinnamaldehyde test A colorimetric assay based upon the reaction of A-rings with the chromogen p-dimethylaminocinnamaldehyde (DMACA) has been developed for flavanoids in beer that can be compared with the vanillin procedure. Identification of Flavonoids By Chromatography: 1-By the use of Paper chromatography (P.C):  The stationary phase = Filter paper ( Whatman no.1).  The mobile phase = n-BuOH:HOAc:H 2O (4:1:5).  The standard compound = Rutin.  The spray reagent = 5% alcoholic KOH.  Mechanism of separation = Partition.  Developing = Ascending. 2-By the use of Thin layer chromatography (T.L.C):  The stationary phase = Silica gel G.  The mobile phase = Ethyl acetate -formic acid - glacial acetic acid - water(100:11:11:26).  The standard compound = Rutin.  The spray reagent = flavonoids spot on TLC plates produce a yellow-brown Spots when reacted with Iodine vapor.   Mechanism of separation = Adsorption.  Developing = Ascending.  Detection: Flavonoids may appear as dark spots on a green background fluoresce when observed in UV light at 254 nm UV-plates containing fluorescent indicator (such as silica gel F254). If under 365 nm UV light, spot colors depending on the structure of flavonoids, can be yellow, green or blue fluorescent. It would be more clear and intense after being sprayed with the reagent.  Colors can be observed at 365 nm UV light are as follows: Quercetin, myricetin, and 3 & 7-O-glycosides: orange-yellow kaempferol, isorhamnetin, and 3 & 7-O-glycosides: yellow-green Luteolin and 7-O-glycosides: orange Apigenin and 7-O-glycosides: yellow-green Study problems: Q1. How many types of flavonoids are their according to the conjugation of basic nuclei? Explain with structures? Q2.How can you identify an extract containing flavonoids?