DEPARTMENT OF PHARMACY DEPARTMENT OF PHARMACOGNOSY PHARMACOGNOSY LABORATORY MANUAL FIRST SEMISTER

AL ZAHARAWI UNIVERSITY COLLEGE DEPARTMENT OF PHARMACY DEPARTMENT OF PHARMACOGNOSY PHARMACOGNOSY LABORATORY MANUAL FIRST SEMISTER BY Dr. SUHAD S. HUMADI And Dr. AHMED KAREEM OBAID 2019-2020 1 CONTENT Page Safety Measurement and Students Instructions 3 Course Syllabus 7 Introduction to Glycosides 8 Cardio-active Glycosides ( Extraction) 14 Cardio-active Glycosides (Identification) 25 Anthraquinone Glycosides (Introduction and Extraction) 36 Anthraquinone Glycosides (Identification) 45 Saponin Glycosides (Introduction, Extraction and Identification) 49 Flavonoid Glycosides (Introduction, Extraction and Identification) 57 Tannins (Introduction, Extraction and Identification) 65 Volatile Oils (Introduction, Extraction and Identification) 73 List of Figures 82 List of Reagents and Reactions 84 List of Chemicals used in all Experiments 85 List of Plants Used in Extraction and Identification Procedures 87 Preparation of reagents used in Experiments 88 References. 89 2 Safety Measurement and Students Instructions In Laboratory It is the responsibility of every staff and student to ensure that the laboratory is a safe environment for everyone. The following points are important. 1. Personal Protection and Hygiene:  Laboratory coats must be worn at all times in the laboratory.  Coats should be washed in laundry regularly or whenever they are contaminated.  If you have very long hair, please tie it back in the laboratory.  Gloves must be worn when handling chemicals, toxic compounds or biohazardous substances as instructed.  Eye protection will be mandatory for all personnel working in the research laboratory where hazardous materials, such as corrosive, injurious, infectious materials are handled.  Any abrasions cuts or open wounds in the skin should be covered with an adhesive plaster before beginning work. 2. Food and Beverage Consumption  Eating, drinking and smoking are strictly forbidden in the laboratory.  Laboratory refrigerators must not be used for the storage of food and beverages.  Glassware or utensils, kettles and microwave oven used for laboratory operations must not be used for preparation of food and beverages. 3 3. Chemicals  All laboratory workers must be familiar with the potential hazards of chemicals, and follow recommended procedures for their use, handling, storage and disposal. If in doubt, workers should consult the Material Safety Data Sheets (MSDS) concerning the relevant chemicals before commencing their experiments.  All chemicals must be clearly labeled. In addition, warning signs should be posted if special precautions are required (e.g., if the chemical is inflammable, highly toxic or carcinogenic).  Experiments involving organic solvents or volatile chemicals should be performed in the fume cupboard.  Waste solvents must not be disposed into the sink. Instead, they should be poured into waste solvent bottles that have been specially set apart for each solvent. Where possible, different solvents should not be mixed. 4. General Housekeeping  Work areas, including walkways and passages should be kept clean and free of obstruction. The floor should also be kept dry at all times.  All samples and solutions must be labeled clearly with the name of the substance, the initials of the user and supervisor, and the date.  All laboratory reagents and chemicals must be returned to the appropriate shelves or special storage areas (e.g., refrigerators) immediately after use.  Always double-check the name of the reagent to be used and the name of the reagent you are using. Reagent bottles should remain stoppered, except when you are actually pouring solutions out of them. Always replace the stopper or lid of stock solutions or stains. Be sure to put them on the container they came 4 from. Take only as much as you need and never return leftover solutions to a reagent bottle. Discard leftovers in the proper container.  Spilled chemicals must be cleaned up immediately and contaminated materials properly disposed.  Put plant remains in the designated containers. Do not discard solids or plant materials down the sinks. They will clog up the sink. Use specified containers for such wastes.  If glassware accidentally becomes broken, carefully clean it up with a broom and dustpan. Dispose of the broken glass in containers labeled FOR BROKEN GLASS ONLY.  Laboratory benches and instruments must be thoroughly cleaned after use, and Wash all glassware and put it back where you found it. Place all dirty slides and cover slips in the designated containers.  Your instructor will review with you the location and, where applicable, use of the safety equipment in the laboratory including: MSDS files emergency phone first aid kit fire extinguisher eyewash 5 5. Laboratory Equipment  Before operating any laboratory instrument, please ensure that you are familiar with the operation of the instrument. If in doubt, either read the instruction manual or consult the relevant staff.  If a record book is placed next to a particular laboratory instrument, all users of the instrument must ensure that the relevant information is recorded in the book.  All faulty or damaged laboratory equipment must be reported to the personin-charge.  Laboratory equipment must not be re-positioned unless prior approval has been obtained from the person-in charge.  All users of laboratory instruments must remember to turn off their electrical switches after use (unless stated otherwise).  All laboratory items (including spatulas, pipettes etc.) must be returned to their original storage place after use. 6 Course Syllabus Laboratory Laboratory Title Number 1 Introduction to Glycosides 2 Cardio-active Glycosides ( Extraction) 3 Cardio-active Glycosides (Identification) 4 Anthraquinone Glycosides (Introduction and Extraction) 5 Anthraquinone Glycosides (Identification) 6 Saponin Glycosides (Introduction, Extraction and Extraction and Identification) 7 Flavonoid Glycosides (Introduction, Identification) 8 Tannins (Introduction, Extraction and Identification) 9 Volatile Oils (Introduction, Extraction and Identification) 7 Lab.1 1. Introduction to Glycosides Objectives: By the end of this lab you will be able to understand the following topics  Definition and General Knowledge of Glycosides.  Classification of Glycosides.  Physical and Chemical Properties of Glycosides.  Extraction and Isolation of Glycosides.  Practice Questions 8 1.1. Introduction and Definitions. Glycosides are organic compounds that yield one or more sugars among the products of hydrolysis. The sugar group is known as the glycone and the non-sugar group as the aglycone or genin part of the glycoside as shown in figure (1.1.1) GLYCONE + AGLYCONE → GLYCOSIDE The glycone can consist of a single sugar group (monosaccharide) or several sugar groups (oligosaccharide). The most frequently occurring sugar is D-glucose, although rhamnose, digitoxose, cymarose and other sugars can also exist . Figure 1.1.1 “Demonstration of the glycone and aglycone parts in methyl β-D-glucoside” Inside the body, the glycoside 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. 9 Chemically, Glycosides are formed when the anomeric (hemiacetal or hemiketal) hydroxyl group of a monosaccharide undergoes condensation with the hydroxyl group of a second molecule, with the elimination of water. Thus, the formation of glycosides is an example of acetal formation, which is a reaction between a hemiacetal group and another hydroxyl group as shown in figure (1.1.2). The linkage resulting from such a reaction is known as a glycosidic bond. Glycosides are named for the sugar that provides the hemiacetal group. Thus, if glucose provides the hemiacetal group, the resultant molecule is a glucoside; if galactose provides the hemiacetal group, the result is a galactoside. Figure 1.1.2 “Acetal and ketal formation” Both α and β- glycosides are possible depending on the stereo-configuration at the anomeric carbon atom, however the β- glycosides are the primarily form that occur in plants. Note: The anomeric carbon is the carbon derived from the carbonyl carbon (the ketone or aldehyde functional group) of the open-chain form of the carbohydrate molecule. 10 1.2. Classification of Glycosides. The glycosides can be classified on the basis of: 1.2.1. The Glycone: as mentioned previously ex. Glucoside, Glactoside. 1.2.2. The Glycosidic Linkage; there are four types:  O-glycosides: Sugar molecule is combined with phenol or –OH group of aglycon. For example cardiac glycosides  N-glycosides: Sugar molecule is combined with N atom of the – NH (amino group) of aglycon, for example, nucleosides  S-glycosides: Sugar molecule is combined with the S or SH (thiol group) of aglycon, for example, Sinigrin.  C-glycosides: Sugar molecule is directly attached with C-atom of aglycon, for example, Anthraquinone glycosides. 1.2.3. The Chemical Structure of the Aglycone  Cardioactive Glycosides.  Anthraquinone Glycosides.  Saponin Glycosides.  Cyanophore Glycosides.  Isothiocyanate Glycosides.  Flavonoid Glycosides.  Alcohol Glycosides.  Aldehyde Glycosides.  Lactone Glycosides.  Phenol Glycosides.  Miscellaneous Glycosides. 11 1.3. Physical Properties of Glycosides:  Glycosides are crystalline or amorphous substances.  Glycosides are soluble in water or alcohols and insoluble in organic solvents like benzene and ether.  The aglycone part is soluble in organic solvents like benzene, chloroform or ether. 1.4. Chemical properties of Glycosides:  Glycosides do not themselves reduce Fehling’s solution, the simple sugars which they produce on hydrolysis will do so with precipitation of red cuprous oxide.  Glycosides are hydrolysed into sugar and another organic compound by boiling with mineral acids (Except the C-Glycoside which requires more vigorous conditions as oxidative hydrolysis).  Glycoside are also hydrolysed by enzyme specific for the type of glycosidic linkage example: emulsin of almond kernels, and myrosinase of the black mustard. 1.5. Extraction and Isolation of Glycosides  Polar solvent, usually alcohol, is used in the isolation and extraction of glycosides.  Various enzymes present in plant parts are deactivated due to the use of heat.  The thermolabile glycosides, however, should be extracted at temperature preferably below 45°C. 12  The extract is treated with lead sub acetate to precipitate tannins and thus eliminate non-glycosidal impurities.  Neutral pH should be assured before and during extraction because as acidity may result in hydrolysis, and this is done by adding a base as CaCO3.  Defatting of fat-rich organs (e.g. seeds) before extraction is an important step as high amounts of lipids hinder glycoside extraction. The defatting process is usually carried out using petroleum ether 1.6. Practice Questions:  Define Glycosides.  Numerate the four types of the glycosidic linkage.  Numerate the different classes of glycosides according to the chemical nature of aglycone.  Explain the solubility of glycoside  Give the reason of : a) The Use of lead sub acetate in the extraction of glycosides. b) The use of petroleum ether as a preparative step in the extraction of glycosides from plant seeds. End of Lab.1 13 Lab.2 2. Cardio-active Glycosides (Introduction and Extraction) Objectives: By the end of this lab you will be able to understand the following topics  Definition and General Knowledge of Cardio-active Glycosides.  Chemical nature of Cardiac glycoside.  Maximum activity of Cardio-active glycosides  Plants containing Cardio-active Glycosides.  Extraction and Isolation of Cardio-active Glycoside  Report Sheet 14 2.1. Introduction Cardio-active Glycosides are an important class of naturally occurring drugs which acquired its name due to their action on the heart muscle. Cardiac steroids are widely used in the modern treatment of congestive heart failure and for treatment of atrial fibrillation and flutter; Yet their toxicity remains a serious problem. These drugs all act by affecting the availability of intracellular Ca +2 for myocardial contraction or increasing the sensitivity of myocardial contractile proteins 2.2. Chemical nature of Cardiac glycoside Cardiac glycosides are composed of two structural features: the sugar (glycone) and the non-sugar (aglycone–steroid) moieties. One to four sugars are found to be present in most cardiac glycosides attached to the 3β-OH group as shown in figure (2.2.1). Sugars most commonly present include L-rhamnose, D-glucose, D- digitoxose, D-digitalose, D-digginose, D-sarmentose, L-vallarose and D-fructose. These sugars predominantly exist in the cardiac glycosides in the β-conformation. The presence of acetyl group on the sugar affects the lipophilic character and the kinetics of the entire glycoside. Figure 2.2.1” attachment of the sugar moiety to the 3β-OH group of the steroid nucleus” 15 The steroid nucleus has a unique set of fused ring system that makes the aglycone moiety structurally distinct from the other more common steroid ring systems. This nucleus is called Cyclopentao-perhydrophenanthrene nucleus to which a lactone ring is attached; as shown in figure (2.2.2) Figure.2.2.2.”Cyclopentanoperhydrophenanthrene ring which consist of three six membered fully hydrogenated (perhydro) phenanthrene ring and five membered cyclopentane ring” The lactone ring of these glycosides are of two types as shown in figure(2.2.3):  Cardenolides: α-β unsaturated 5 membered lactone ring. These are the more prevalent glycosides.  Bufadienolides: Doubly unsaturated 6 membered lactone ring Figure 2.2.3 Cardenolide and bufadienolide chemical structures 16 2.3. Maximum activity of cardio-active glycosides For the optimum activity of cardia-active glycosides the following points are to be considered.  3  OH  14  OH  17 -unsaturated lactone ring.  The rings must comply with CATSC rule, that is: a) Rings A & B are cis to each other. b) Rings C & D are cis. c) Rings B and C in trans d) S: Syn in one ring (8 and 18) e) A: anti in one ring (5 and 19) 2.4. Plants containing Cardio-active Glycosides 2.4.1. Digitalis leaves (Purple Foxglove)  Digitalis purpurea F. Scrophulariaceae  Three important glycosides (digitoxin, gitoxin, gitaloxin) 2.4.2. Grecian Foxglove  Digitalis lanata F. Scrophulariaceae  Main glycoside is lanatoside A, B, C and D  Hydrolysis of Lanatoside C yields digoxin, a crystalline active glycoside 17 2.4.3. Nerium oleander  Nerium oleander F. Apocyanaceae (figure 2.4.3.1).  Main glycoside is oleandrin (figure 2.4.3.2).  Is the plant used in our laboratory as an example of cardioactive glycosides. Figure 2.4.3.1. “Nerium oleander plant” Figure 2.4.3.2. “Oleandrin structure” 18 2.5. Extraction and Isolation of Cardio-active Glycoside.  Aim: To extract cadrio-active glycoside  Plant used: Nerium oleander.  Part of plant used: dry leaves  Method of extraction: Maceration (see Figure 2.5.1).  Solvents and Reagents used: Ethanol, Lead subacetate, chloroform, 10% sodium phosphate solution, anhydrous sodium sulphate, HCl,  Equipment: Large and medium sized beakers, conical flasks, separatory funnel, centrifuge and centrifuge tubes, Water bath. Figure 2.5.1 “ example of maceration extraction method” 19 2.5.1. Procedure Macerate 10 gm of the powdered leaf with 100 ml of 70% ethanol for 24 hours with intermittent shaking using large beaker (previously prepared ) (Repeat procedure twice) Filter Alcoholic Filtrate Marc Take 20 ml of the resultant filtrate in a conical flask Add 10 ml of lead sub acetate solution (mix and stand for 5 minutes) Centrifuge for 5 minutes Decant and take the supernatant (upper layer) Add 10 ml of 10 % sodium phosphate solution Centrifuge for 5 minutes Take the supernatant and divide into two divisions Fraction A and Fraction B 20 Fraction A Put the first division (Fraction A) in a separatory funnel Add 10 ml of Chloroform: ethanol (3:1v/v) Х 2 Shake and stand Take the organic lower layer and put in conical flask Add Small quantity of anhydrous sodium sulphate and allow to stand for few minutes until clear solution is obtained decant the Chloroform-ethanol extract and reduce the volume on water bath to get fraction A 21 Fraction B Place the other division of the extract in the conical flask Add 3 ml of 4 N HCl Boil in water-bath for 15 minutes Cool and transfer to separatory funnel Add 10 ml of chloroform Х 2 Take the lower chloroform layer Add small quantity of Anhydrous sod. Sulphate & allow standing for few minutes until getting a clear solution then decant the chloroform layer and concentrated on water bath to about 1ml to get fraction B 22 2.5.2 Discussion  Fraction A contains the glycoside  Fraction B contain the genin part.  Polar solvent is used for the extraction of glycoside thus Ethanol was used  Lead subacetate was used to precipitate tannins  Use of chloroform-ethanol in partition is due to the fact that the chloroform will take the genin part while the ethanol will take the glycoside there will be no loss in the glycoside  Excess of lead was removed from the filtrate by the use of 10% sodium phosphate solution, lead will be precipitated as lead phosphate as represented in the following equation. 3Pb(CH3COO)2 + 2Na3PO4 Pb3(PO4)2 +6Na(CH3COO)  Use of HCl in fraction B is to hydrolyse the glycosidic linkage and release of the aglycon part.  Chloroform organic solvent will extract the genin part (aglycone part)  Anhydrous sodium sulphate solution is used as an inert drying agent to remove excess water.  Type of Glycoside is O-glycoside. End of Lab 2 23 Report Sheet Experiment 1, Lab 2 Extraction of cardio-active glycoside Student Name: 1. Objective: 2. Botanical name of the plant used. 3. Principle. 4. Results and Discussion 24 Lab.3 3. Cardio-active Glycosides (Identification) Objectives: By the end of this lab you will be able to identify and understand  Chemical test used in the identification of cardio-active glycosides  Chemical tests for the identification of the steroidal nucleus (aglycone)  Identification of cardioactive glycosides by chromatography 25 3.1. Chemical tests used in the identification of cardio-active glycosides in general 3.1.1. Baljet’s Test. 3.1.1.1. Procedure  Take1 ml of Fraction A  Add 2 drops of picric acid  Make it alkaline with NaOH (sodium hydroxide solution) using litmus paper. 3.1.1.2. Results Turbid Yellow to orange color. 3.1.1.3. Discussion The colored product is made by the connection of cardenolide-anion and a nitro-group containing molecule. The lactone ring of the aglycone forms a complex with organic nitro compounds in a basic environment, where the intensity and the shade of the resulting colour depend on the concentration of the cardenolides (Solution B should be darker than Solution A). The reaction is presented by the following diagram with the color (Figure 3.1.1.3) Figure 3.1.1.3 “ Baljet’s test principle reaction and color result” 26 3.1.2. Keller-Killian’s Test 3.1.2.1. Procedure:  Take 1 ml of Fraction A in a test tube.  Add 2 ml of Glacial acetic acid  Add one drops of 0.1% of ferric chloride solution (FeCl3)  Take 1ml of concentrated H2SO4 and add it to the above mixture in drops so as to make two layers. 3.1.2.2. 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 (as shown in figure 3.1.2.2.1) Figure 3.1.2.2.1” color reaction of Keller-Killian Test 27 3.1.2.3. Discussion  Principle of the reaction is to convert 2-deoxysugars into characteristics coloured derivatives.  The use of glacial acetic acid is to hydrolyze the glycoside into glycone and aglycone parts. The addition of 0.1 % ferric chloride is an oxidizing agent needed in the reaction.  The H2SO4 has a high density therefore will be in the lower layer causing charring of the sugar and hence giving the reddish-brown color. 3.2. Chemical tests For the Identification of the steroidal nucleus (aglycone) 3.2.1. Raymond’s Reaction. 3.2.1.1. Procedure:  Take 1 ml of Fraction B in a test tube.  Add 1 to 2 drops of 10% sodium hydroxide solution (NaOH)  Add few drops of alcoholic solution of 1 % 3,5-dinitrobenzene. 3.2.1.2. Results Color reaction is pink 3.2.1.3. Discussion: The reaction between cardenolides and poly-nitroaromatic derivative in an alkaline solution, are based on the fact that the C-C coupling of the unsaturated lactone ring with nitrated aromatic derivatives produce dye complexes. The dye complexes are known as Meisenheimer compounds of the cyclohexadienate type (Figure 3.2.1.3.1) 28 Figure 3.2.1.3.1 “Meisenheimer dye complexes” 3.2.2. Kedde’s Reaction 3.2.2.1. Procedure:  Take 3 ml of Fraction B in a test tube  Add 2 ml of 1% 3,5-dinitrobenzoic acid  Add 1 ml of 0.5 N aqueouse methanolic KOH (50%) 3.2.2.2. Results The reaction mixture immediately turns purple-violet, which colour disappears after a few min. 3.2.2.3. Discussion first, a cardenolide anion is formed, which is converted to a purpleviolet anion by adding 3,5- dinitrobenzoic acid. Because of the hydrolysis of the lactone ring, the colour disappears in a few min. The reaction works with 5 member, α-, β- unsaturated γ-lactone ring, and it is based on the proton dissociation catalysed by alkali as shown in figure (3.2.2.3.1) Figure 3.2.3.1 “Kedde’s Reaction” 29 3.2.3. Lieberman’s Sterol Reaction 3.2.3.1. Procedure  Take 1 ml of Fraction B in a test tube.  Add 5 ml of anhydrus acetic acid and shake well.  Take 4 drops of the above mixture and place in a porcelain dish  Add one drop of Conc. H2SO4 3.2.3.2. Results A change of color from rose, through red , violet, and blue to green. The color is slightly different from compound to compound. 3.2.3.3. Discussion: This reaction is due to the steroidal part of the molecule and it is characteristic of the aglycone part, anhydrus acetic acid is as a dehydrating agent; the sulphuric acid is a dehydrating and oxidizing agent to give the reaction color. 3.2.4. Legal’s Reaction 3.2.4.1. Procedure:  Dissolve few mls of Fraction B in 2 mls of pyridine.  Add 1 mls of Sodium nitroprusside.  Followed by adding 1 ml of sodium hydroxide NaOH 3.2.4.2. Results A transient blood red color develops 3.2.4.3. Discussion This test is for the unsaturated lactone ring of the genin part 30 3.3 . Identification of Cardioactive Glycosides by Chromatography Thin Layer Chromatography TLC is used to investigate the presence of Oleandrin in the tested extract of both Fraction A and Fraction B. In this test the following will be needed: 3.3.1. Stationary Phase: This is represented by the use of Silicagel G 60 F254 3.3.2. Mobile Phase: Could be any of the following solvent mixtures  Chloroform:Ethanol:Water (7:3:1)  Ethyl acetate: Methanol:Water (75:10:5)  Butane: Xylene: Formamide (50:50:4)  Chloroform: Tetrahydrofuran: Formamide (50:50:6) 3.3.3. Standard Reagent: Oleandrin. 3.3.4. Spray Reagent: Liberman’s Reagent 3.3.5. Procedure:  Prepare 100 ml of the mobile phase and place in the glass jar.  Cover the tank with the glass lid and allow to stand for 45 minutes before use.  Use a pencile to draw the base line on the slica gel plate (the base line should be higher than the mobile solvent level see figure 3.3.5.1)  Apply the sample spots (fraction A and fraction B) and the standard spot, on the silica gel plates, on the base line.  Put the silica gel plate in the glass tank and allow the mobile phase to raise to two third of the plate  Remove the plate from the tank and mark the solvent front. 31 Figure 3.3.5.1. “TLC technique”  Allow drying and then detecting the spots by use of the spray reagent and heat the plate at 105-110c for 5-10 minutes in the oven.  Note the spots, and calculate the RF value for each spot.  Rf value is calculated using the folrmula (figure 3.3.5.2 and 3.3.5.3.)  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 32 Figure 3.3.5.2 “ calaculation of RF value (solute represents spot)” Figure 3.3.5.3 “Diagram demonstating Rf value calculation End of Lab 3 33 Report Sheet (2) for Lab 3 Student Name: 1. Objective: 2. Record observations and results you obtained from the different chemical tests Chemical Principle Observations/Color notes test 34 3. Mention the mobile phase that you used in performing TLC for fraction A and B, and calculated RF values. 35 Lab Four 4. Anthroaquinone Glycosides (Introduction and Extraction) Objectives: By the end of this lab you will be able to identify and understand  Introduction and Chemistry Of Anthraquinone Glycosides  Medicinal use and Mechanism of action.  Plants containing Anthraquinone Glycosides  Extraction and Isolation of Anthraquinone Glycosides 36 4.1. Introduction and Chemistry Of Anthraquinone Glycosides Anthraquinones are glycosides that possess anthracene nucleus or their derivatives as aglycone in which 2 keto groups are attached to the benzen ring (anthraquinone) as represented in figure ( 4.1.1) . Figure 4.1.1 “Anthraquinone Nucleus with numbering system” The Glycosides upon hydrolysis, yeild aglycones that are di-, tri-, or tetrahydroxyanthraquinones or modifications of these compounds. Glycosides of anthranols, dianthrones and oxanthrones (reduced derivatives of anthraquinones ) also occure in plant materials and contribute to the theraputic activity as shown in figure (4.1.2). Figure 4.1.2”Anthraquinones derivatives” 37 The common aglycones related to these structures are aloe-emodin, emodin, rhein, chrysophanol and physcion as represented in figure 4.1.4. The sugars presents are usually arabinose, rhamnose and glucose. Figure 4.1.4 “Different anthraquinone structures” 38 4.2. Medicinal use and Mechanism of action. 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 hours 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. 4.3. Drugs containing anthraquinone glycosides include Cascara bark, Frangula, Senna leaves which are most widely used. Whereas Rhubarb and Aloe are not recommended as cathartics due to their irritating actions which increase the chnace for gripping effect . Senna Leaves ( Cassia acutifolia F. Leguminosae) are used as an example of the anthraquinone Glycosides. 39 Figure 4.3.1 “ Senna Leaves” The Principle active constituents of seena are dimeric glycosides whose aglyconees are compose of aloe-emodin and/or rhein. These are presented as Sennosides A, B, C and D , the greatest concentration being Sennoside A and B, as represented in figur ( 4.3.2) Figure 4.3.2 “Structures of Sennosides A,B, C and D” 40 4.4. Extraction and Isolation of Anthraquinone Glycoside.  Aim: To extract anthraquinone Glycoside  Plant used: Cassia acutifolia.  Part of plant used: dry leaves  Method of extraction: Decoction.  Solvents and Reagents used: chloroform, 60% Ferric Chloride, HCl,  Equipment: Large and medium sized beakers, conical flasks, separatory funnel, centrifuge and centrifuge tubes, round bottle flask, reflux, Filter papers and Water bath. 41 4.4.1. Procedure 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 (Aqueous layer) Divide into two portions Combine lower layer (Chloroform layer [Free aglycone (dianthrone)] Fraction B (Whole Glycosides) Fraction A Other part of the aq. Layer 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] two times Aqueous layer Glycone part Chloroform layer Aglycone part (monoanthrone) Fraction C 42 4.4.2. Results and Discussion  Fraction A contains the glycoside  Fraction B contain the genin part aglycone (dianthrone)  Fraction C contain the genin part aglycone (monoanthrone)  Polar solvent is used for the extraction of glycoside thus water was used.  Use of chloroform is to extract the aglycone part.  The O-glycoside is hydrolyzed to free anthraquinone by heating with dilute HCl acid, while the C- glycoside releases the free anthraquinone only after oxidative cleavage in the presence of FeCL3 in acidic medium releasing free anthraquinone (or rhein in the case of anthraquinone dimmers) and the sugar moiety  Type of Glycosides are C-glycosides and O-glycosides End of Lab 4 43 Report Sheet number 3 (Lab 4) Student Name: 1. Objective: 2. Botanical name of the plant used. 3. Principle. 4. Results 44 Lab 5 5. Anthraquinone Glycosides (Identification) Objectives: By the end of this lab you will be able to identify and understand  Chemical tests used for the identification of the anthraquinone glycosides specifically for aglycone.  Identification of anthraquinone glycosides by chromatography 45 5.1. Chemical tests used for the Identification of the anthraquinone glycosides specifically for aglycone 5.1.1. Borntrager test for anthraquinones.  Aim: To identify anthraquinone glycosides (specifically the aglycone part)  Principle: The principle of the test is based on the ability of free anthraquinone to form a colored product upon the addition of standard alkali (e.g. KOH, NaOH or ammonia). The reaction involves the formation of phenolate-type ions which are visibly colored. 5.1.1.1. Procedure:  Put 5 ml of each of the three fractions obtained in previous lab; fraction A, B and C in a test tube.  Add 2 ml of 10%Ammonia solution.  Observe the color of each test tube  Take 5 ml of chloroform in a test tube and add 2 ml of 10%ammonia, and used as control test to compare the different colors 5.1.1.2. Results and Discussion Pink color will be produced which is very clear with monoanthrones Fraction C than dianthrones which is Fraction B. Fraction A has the unhydrolyzed aglycone thus will give negative result 46 5.2. The Identification of anthraquinone glycosides by chromatography: This is carried out by the use of Thin Layer Chromatography TLC using the following:  Stationary phase = Silica gel G.  Mobile phase = n-propanol: Ethyl acetate: Water (60:30:30).  Standard compound =Sennoside.  The spray reagent = Alcoholic KOH 5%w/v.  Mechanism of separation = Adsorption.  Developing = Ascending. 5.2.1. Procedure:  Prepare 100ml of mobile phase, and place it in the glass tank.  Cover the tank with glass lid and allow standing for 45 minutes before use.  Apply the sample spots (fraction A, fraction B& fraction C), and the standard spot on the silica gel plates, on the base line.  Put the silica gel plate in the glass tank and allow the mobile phase to rise to about two-third of the plate.  Remove the plate from the tank, mark the solvent front on each plate and allow drying at room temperature, spray first with 25%nitric acid solution and heat for 10 minutes at 110 0C.  Allow to cool, and then spray with 5% w/v alcoholic KOH solution. Detect the spot formed and calculate the Rf values. 47 Report sheet number 4 (for lab 5)  Name of Student  Aim and objective:  Principle of chemical test used:  Results and discussion  Calculate and report the Rf values obtained in TLC 48 Lab 6 6. Saponin Glycosides Extraction and Identification Objectives: By the end of this laboratory , students will understand and identify the followings:  General introduction to saponin glycosides.  Chemistry of saponin glycosides.  Extraction of saponin glycoside.  Identification of saponin glycosides. 49 6.1. General introduction to Saponin Glycosides Saponins are glycoside compounds often referred to as a ‘natural detergent’ because of their foamy texture. They get their name from the soap wort plant (Saponaria), the root of which was used historically as a soap. Saponin glycosides are widely distributed in the higher plants; they form colloidal solutions in water that foam upon shaking. They have bitter, acrid taste and irritate mucus membranes. They destroy RBC by hemolysis and are toxic 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. 6.2. Chemistry of Saponin Glycosides 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 as shown in figure (6.2.1)  Steroid saponins Steroid saponins are similar to the sapogenins and related to the cardiac glycosides. Diosgenin is the important steroid sapogenin (neutral C- atom is C27).  Triterpenoid Saponins They have a pentacyclic triterpenoid nucleus which is linked with either sugar or uronic acid. Glycyrrhizin, from licorice root, is an example of this group (acidic, and the C-atom is C30). 50 Figure 6.2.1 “Basic Sapogenin Structures” 6.3. Extraction of Saponin Glycoside.  Method of extraction: Decoction.  Plant used: Saponaria officinalis family Caryophyllaceae Figure 6.3.1.  Part used: Dry root  Solvent used: Water Figure 6.3.1” Saponaria officinali Plant” 51 6.3.1. Procedure  Add 1 gm of saponaria root in coarse powder to 25 ml distilled water in a beaker and boil gently for 2-3 minutes.  Filter hot and allow cooling.  Take 5ml of the resultant filtrate (sol.1) and dilute with water; shake vigorously, and observe the result.  Take 10 ml of the filtrate and add 5ml of dilute H2SO4 acid and boil gently for 3-5 mins. Make the filtrate obtained alkaline with NaOH, using litmus paper (sol.2). 6.3.2. Results and discussion Adding water to sol.1 and shaking will lead to the formation of foam which persist for 60 to 120 seconds, and is an indication of saponin glycoside. Saponin glycosides form colloidal solution in water that foam upon shaking as shown in figure (6.3.2.1), 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. Figure 6.3.2.1.” Saponification test” Addition of H2SO4 acid to the filtrate is to hydrolyse glycoside to the glycone and aglycone parts. NaOH is added as alkaline medium which is needed for the oxidation of the copper (II) ions by the sugars in Benedict’s Test 52 6.4. Identification of Saponin Glycosides 6.4.1. Benedict’s Test  Benedict's test is done on sol.2 as follows:  Take 5ml filtrate of sol 2 and add 2ml of Benedict's reagent, heat for 10mins on boiling water bath; record the results 6.4.1.1. Results and Discussion This test is used to identify the glycone sugar part .The color varies from green to dark red (brick) or rusty-brown, depending on the amount and type of sugar. When Benedict’s solution and simple carbohydrates are heated, the solution changes to orange red/ brick red. This reaction is caused by the reducing property of simple carbohydrates. The copper (II) ions in the Benedict’s solution are reduced to Copper (I) ions, which causes the color change. As mentioned earlier the addition of NaOH is to keep the media alkaline for the reduction reaction. 6.4.2. The Hemolytic Test This test is to identify Saponin glycosides 6.4.2.1. 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. 6.4.2.2. Results and Discussion The test tube containing 5ml of the extract of Saponaria will cause blood hemolysis, Which indicates the presence of saponin glycosides. The other test tube with normal saline is used as a control to compare the results. 53 6.4.3. Foam Index Foam index is a value which is used to express the quality of crude drug containing saponins. The method is based upon the property of saponin to form foam when shaken with water, and this ability to foam is caused by the combination of the non-polar sapogenin and the water soluble side chain. The foam index signifies the dilution of the substance or drug to be tested which gives a layer of foam 1 cm high, if the aqueous solution is shaken for 15 seconds and then allowed to stand for 15 minutes before reading is made. 6.4.3.1. Procedure  Prepare 0.1% decoction from the powdered drug. This is carried out by weighing 0.1 gm of the powdered material and adding 20 ml distilled water in a beaker and boiling for 2-3 minutes, then allow cooling.  Neutralize the extract by adding solution of 1% sodium carbonate drop wise using litmus paper and filter.  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 in all test tubes. Mark the level of solvent in all test tubes.  Shake the content of each test tube thoroughly for 15 seconds and allow to stand for 15 minutes.  After this time, the reading is made in the test tube containing the most dilute solution with a ring of foam1 cm height.(mark the level of foam in all test tubes and pick the one which has 1 cm height)  Results are interpreted as follows : a) If the height of the foam in every tube is less than 1 cm, then the foaming index is less than 100. 54 b) If a height of foam of 1 cm is measured in any tube, the volume of the plant material decoction in this tube (a) is used to determine the foaming index. c) If this tube is the first or second tube in a series, an intermediate dilution will be prepared in a similar manner to obtain a more precise result. d) If the height of the foam is more than 1 cm in every tube, the foaming index is over 1000. In this case a new series of intermediate dilution of the decoction will be prepared to obtain a result. e) The foaming index is calculated using the following formula: Foaming Index = 1000/a Where: a = The volume in ml of the decoction used for preparing the dilution in the tube where foaming to a height 1 cm is observed For example if the 1 cm foam was observed in test tube number 8, then the Foam index will be 1000/8 which is 125. 6.4.3.2. Results and Discussion  Results depend on your observation, and calculate the Foam index.  Addition of sodium carbonate is to convert the acidic saponins that may be present in the decoction, to salts, which are soluble in water.  Foam index is used to identify saponins presences and also used to compare between different test samples as which test sample has highest saponin content than the other. 55 Report sheet number 5 (for lab 6)  Name of Student  Aim and objective of this Lab:  Principle used for the extraction of Saponins  Methods and tests used for the identification of Saponins (mention each test and its principle)  Results and discussion for each test performed in this lab.  Explain the foam index and report your results and observations 56 Lab 7 7. Flavonoids Introduction, Extraction and Identification Objectives By the end of this laboratory , students will understand and identify the followings:  General introduction to flavonoid glycosides.  Chemistry of flavonoid glycosides.  Extraction of flavonoid glycoside.  Identification of flavonoid glycosides. 57 7.1. General introduction to Flavonoid Glycosides Flavonoids are among the most widely distributed natural product compounds in plants with over 2000 different compounds reported that occur both as aglycon free state and as glycosides (O -glycosides and C-glycosides). Many flavonoids are shown to have antioxidative activity, free radical scavenging capacity, coronary heart disease prevention, hepatoprotective, antiinflammatory, anticancer activities and other age-related diseases, while some flavonoids exhibit potential antiviral and anti bacterial activities. 7.2. Chemistry of Flavonoid Glycosides. Flavonoid chemical structures are based upon a C6-C3-C6 (phenylbenzopyran backbone) with a chroman ring bearing a second aromatic ring in position 2,3, or 4. The major general structural categories are flavones, flavanones, flavonols, anthocyandins, and isoflavones as represented in figure (7.2.1) In some cases the six-membered ring is replaced by a five membered ring or exist in a open chain isomeric form known as chalcones. 58 Figure 7.2.1 “General structural categories of Flavonoids” 7.3. Extraction of Flavonoid Glycoside.  Method of extraction: Maceration.  Plant used: Ruta graveolens family Rutaceae (figure 7.3.1.1)  Part used: Dry leaves. Figure 7.3.1.1 “Ruta graveolens family Rutaceae” 59 7.3.1 Procedure Macerate 10 gm. of the powdered leaves in 100ml of petroleum ether overnight. (Prepared previously) Filter and take the residue Macerate again the residue with 70 % aqueous methanol overnight ( prepared previously) Filter Concentrated the extract to small volume using rotary evaporator (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 60 7.3.2. Results and Discussion  Fraction A : Contain the whole glycosides.  Fraction B : Contain the aglycone part.  petroleum ether is used as a defatting agent.  70 % aqueous methanol is used as glycosides are soluble in water and alcohol.  5% HCl is used to hydrolyse the glycoside to glycone and glycone with the aid of heat.  Chloroform is used to extract the aglycone part. 7.4. Identification of Flavonoid Glycosides The following chemical tests are carried on both, Fraction A and Fraction B, record your observations. 7.4.1. Alkaline Reagent Test. 7.4.1.1. Procedure To 1 ml of test solution add 0.5 ml of 10% NaOH (sodium hydroxide solution); formation of an intense yellow colour will indicate presence of Flavonoids. 7.4.1.2. Results and Discussion. Record your observations on both Fractions. The principle of this reaction based on the formation of phenoxides in an alkaline media which gives a yellow color 61 7.4.2. Ammonia Test. 7.4.2.1. Procedure Filter paper dipped in test solution is exposed to ammonia vapor. Formation of yellow spot on filter paper indicate positive test. 7.4.2.2. Results and Discussion. Record your observations 7.4.3. Shinoda test. 7.4.3.1. Procedure To 1 ml of the test solution, add few magnesium turning and 0.5 ml of Conc. HCl, formation of red color indicate the presence of flavonoids. 7.4.3.2. Results and Discussion Observe the color in Fraction A and B. Formation of red color indicate positive test. The principle of Shinoda reaction is based on the reduction of Keto group in the presence of acidic environment as presented in figure 7.4.3.2.1. Figure 7.4.3.2.1” Shinoda Test” 62 7.4.4. Chelation formation reaction. 7.4.4.1. Procedure To 1 ml of the tested solution add 0.5 ml of 10% sodium acetate and 1 ml of 2.5% solution of ALCl3. Observe the color 7.4.4.2. Results and Discussion. Intensification of the yellow color is observed as a positive reaction. The principle of the reaction based on the fact of chelate formation with the flavonoid nucleus as shown in figure 7.4.4.2.1. Figure 7.4.4.2.1” Chelate formation reaction” End of Lab 7 63 Report sheet number 6 (for lab 7)  Name of Student  Aim and objective of this Lab:  Principle used for the extraction of Flavonoids  Methods and tests used for the identification of Flavonoids (mention each test and its principle)  Results and discussion for each test performed in this lab. 64 Lab 8 8. Tannins Introduction, Extraction and Identification Objectives By the end of this laboratory , students will understand and identify the followings:  General introduction to tannins.  Classification of tannins  Chemistry of tannins.  Extraction of tannins.  Identification of tannins. 65 8.1. General introduction to Tannins The name ‘tannin’ is derived from the French ‘tanin’ (tanning substance) and is used for a range of natural polyphenols. Tannins are complex organic, nonnitrogenous polyphenol plant products, which generally have astringent properties. These compounds comprise a large group of compounds that are widely distributed in the plant kingdom. The term ‘tannin’ was first used by Seguin in 1796 to denote substances which have the ability to combine with animal hides to convert them into leather which is known as tanning of the hide. According to this, tannins are substances which are detected by a tanning test due to its absorption on standard hide powder. The test is known as Goldbeater’s skin test. 8.2. Classification of Tannins The tannin compounds can be divided into two major groups on the basis of Goldbeater’s skin test. A group of tannins showing the positive tanning test may be regarded as true tannins, whereas those, which are partly retained by the hide powder and fail to give the test, are called as pseudotannins.. True Tannins are chemically divided into two major classes based on the identity of the phenolic nuclei involved and the way they are joined in the tannins structure. The first class is referred to as hydrolysable tannins, whereas the other class is termed as nonhydrolysable or condensed tannins. 66 8.2.1. Hydrolysable Tannins As the name implies, these tannins are hydrolyzed by mineral acids or enzymes such as tannase. Their structures involve several molecules of polyphenolic acids such as gallic Figure (8.2.1.1), hexahydroxydiphenic, or ellagic acids, bounded through ester linkages to a central glucose molecule. Figure 8.2.1.1. “Gallic Acid Structure” 8.2.2. Non Hydrolysable Tannins or Condensed Tannins Condensed tannins, unlike the previously explained group are not readily hydrolysable to simpler molecules with mineral acids and enzymes, thus they are also referred to as nonhydrolysable tannins. The term proanthocyanidins is sometimes alternatively used for these tannins. The most widely studied condensed tannins are based on flavan-3-ol, Epicatechin and catechin as shown in figure 8.2.2.1, and Figure 8.2.2.2 Figure 8.2.2.1 “ Catechin structure” Figure 8.2.2.2 “ Epicatechin structure” 67 8.3. General Characteristics of Tannins  Tannins are colloidal solutions with water.  Non crystalline substance.  Soluble in water, alcohol, dilute alkali, and glycerin.  Sparingly soluble in ethyl acetate.  Insoluble in organic solvents, except acetone.  Can bind with proteins and form insoluble or soluble tannin—protein complexes.  They cause precipitation of solution of gelatin as well as alkaloids  They form dark blue, greenish-black soluble compounds with ferric salts.  They are precipitated by salts of copper, lead and tin 8.4. Medicinal use of Tannins  Tannins as astringents can be used internally for the relief of diarrhea and also can be used externally for acne and also relieve minor skin irritations which results from superficial cuts, insect bites and fungal infection.  are also used in the treatment of burns as the proteins of the exposed tissue are precipitated and form a mildly antiseptic, protective coat under which the regeneration of new tissues may take a place.  Tannins also have been employed as antidotes in poisoning by heavy metals, alkaloids and glycosides  Anti-tumor and anti-HIV activity  Use in the process of vegetable- tanning which converts animal hides to leather (leather industry).  Ink industry 68 8.5 Extraction of Tannins  Method of Extraction: Decoction  Plant used: Hamamelis Leaf or Witch Hazel leaves; Hamamelis virginiana Linne. F. Hamamelidaceae figure 8.5.1.  Active constituent: Hamamelitannin (figure 8.5.2) , Gallitannin, proanthocyanidin, gallic acid.  Part used: Dried Leaves Figure 8.5.1 “Witch Hazel leaves” Figure 8.5.2. Hamamelitannin structure” 69  Procedure Place 0.5 gm of powdered dry leaves of dried plant in 50 ml of water and boil for 15 minutes. Allow the mixture to cool then filter.  Results and Discussion. The resultant filtrate will contain tannins. As tannins are soluble in polar solvents, water was used in its extraction. 8.5. Identification of Tannins Several test can be used for the identification of Tannins. These are:  Gelatin Test To a 1% gelatine solution, add little 10% sodium chloride. If a 1% solution of tannin is added to the gelatine solution, tannins cause precipitation of gelatine from solution (white ppt.)  Ferric Chloride solution To 2 ml of the aqueous sample extract add 1 to 2 drops of diluted ferric chloride solution. A dark green or blue green coloration indicates the presence of tannins.  Lead sub acetate sol To 2 ml of the aqueous sample extract add 1 ml of lead sub acetate, precipitation indicates the presence of tannins. 70  Phenazone Test To 5 ml of aqueous solution of tannin containing drug, add 0.5 g of sodium acid phosphate. Warm the solution, cool, and filter. Add 2% phenazone solution to the filtrate. All tannins are precipitated as bulky, coloured precipitate End of lab 8 71 Report sheet number 7 (for lab 8)  Name of Student  Aim and objective of this Lab:  Principle used for the extraction of Tannins  Methods and tests used for the identification of tannins (mention each test and its principle)  Results and discussion for each test performed in this lab. 72 Lab 9 Volatile Oils Introduction, Extraction and Identification Objectives By the end of this laboratory , students will understand and identify the followings:  General introduction to Volatile Oils.  Chemistry of Volatile Oils.  Classification of Volatile Oils.  Physical Properties of Volatile Oils  Medicinal Use of Volatile Oils  Extraction of Volatile Oils.  Identification of Volatile Oils. 73 9.1. Introduction Volatile oils are odorous volatile principles of plant and animal source, evaporate when exposed to air at ordinary temperature, and hence known as volatile or etheral oils. They are also known as essential oils as they represent essence of active constituents of the plant and are essential for the plant. Aromatherapy: is a holistic healing treatment that uses natural plant extracts to promote health and well-being. Sometimes it’s called essential oil therapy. Aromatherapy uses aromatic essential oils medicinally to improve the health of the body, mind, and spirit. It enhances both physical and emotional health Volatile oils are liable to oxidation on storage in presence of air, moisture, and light. The oxidation is followed by the change in colour, increase in viscosity, and change in odour. Hence, volatile oils must be stored in well-closed completely filled containers and away from light in cool places. 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 9.2. Chemistry of Volatile Oils Chemical constituents of volatile oils maybe divide into two broad classes based on their biosynthetic origin:  Terpenoid derivatives formed via the acetate-mevalonic acid pathway.  Aromatic compounds formed via the shikimic acid-phenylpropanoid route 74 Terpenoids form a group of naturally occurring compounds majority of which occur in plants, they are volatile substances which give plants and flowers their fragrance. The term ‘terpene’ was given to the compounds isolated from terpentine, a volatile liquid isolated from pine trees. The simpler mono and sesquiterpenes is the chief constituent of the essential oils obtained from sap and tissues of certain plant and trees. The di- and triterpenoids are not steam volatile. They are obtained from plant and tree gums and resins. Tetra-terpenoids form a separate group of compounds called ‘Carotenoids’ as shown in diagram ( 9.2.1). The term ‘terpene’ was originally employed to describe a mixture of isomeric hydrocarbons of the molecular formula C10H16 occurring in the essential oils and the “ene” related to the unsaturated double bound Terpenoids Mono and Sesquiterpene Volatile oils Tetraterpenoid Di and Triterpenoid Nonvolatile oils steroids Carotenoids Diagram 9.2.1.” Classification of terpenoids” 75  Special Isoprene Rule It states that the terpenoid molecules are constructed of two or more isoprene units joined in a ‘head to tail’ fashion as shown in figure 9.2.2 and figure 9.2.3. Figure 9.2.2 “Joining Isoprene Units” Figure 9.2.3 “ Head to tail rule of isoprene units” 76 9.3. Classification of Volatile Oils The most acceptable classification whereby volatile oils and volatile-oil containing drugs may be grouped together on the basis of functional groups are : Hydrocarbons, Alcohols, Aldehydes, Ketones, Phenols, Phenolic ethers, Oxides and Esters. 9.4. Physical Properties of Volatile Oils Volatile oils are freely soluble in ether and in chloroform and fairly soluble in alcohol; they are insoluble in water. The volatile oils dissolve many of the proximate principles of plant and animal tissues, such as the fixed oils and fats, resins, camphor, and many of the alkaloids when in the free state. Volatile oils possess characteristic odour, have high refraction index, and most of them are optically active. They are colourless liquids, but when exposed to air and direct sunlight these become darker due to oxidation. Unlike fixed oils, volatile oils neither leave permanent grease spot on filter paper nor saponified with alkalis. 9.5. Medicinal Use of Volatile Oils Volatile Oils are used as important medicinal agent for therapeutic purposes like carminative, antiseptic, anthelmintic, diuretic, counter irritant, local anesthetic, sedative and insect repellent. Moreover they are also used as flavouring agent, perfuming agent in pharmaceutical formulations, foods, beverages, and in cosmetic industries 77 9.6. Extraction Of Volatile Oils  Aim: Determination of the volatile content of crude drugs by water distillation method.  Equipment : Clevenger type as an apparatus see figure 9.6.1and figure 9.6.2. Figure 9.6.1 “ Clevenger apparatus” Figure 9.6.2.” different parts of Clevenger according to the density of volatile oil in regards to water” 78  Method of Extraction: water distillation  Plant used: Peppermint; Mentha piperita Family Lamiaceae (figure 9.6.3)  Active constituent: Volatile oil: peppermint oil  Part used: Fresh Leaves Figure 9.6.3 “ Peppermint”  Procedure a) 20 g of sample is filled to the boiling flask of Clevenger apparatus, and then 200 ml of distilled water is added. b) The boiling flask is heated on the burner. c) Water vapour and the volatile oil dragged with water vapor are condensed on the cooler and collected in the apparatus’ burette. The loss of water returns to the boiling flask through curved pipe. d) The quantity of volatile oil is read from the burette as ml and recorded. e) The tap of the apparatus is opened and water from burette is taken till volatile oil level. f) Then the volatile oil is transferred to a little Erlenmeyer and dried with a tip of a spatula anhydrous sodium sulphate (Na2SO4). g) The clarified volatile oil is transferred to a dry little Erlenmeyer and the %v/w of the volatile oil content of drug is calculated. 79  Results and observations Record your observations and percent of volatile oil obtained 9.7. Identification of Volatile Oils 9.7.1. I dentification by TLC.  The stationary phase = Silica gel G.  The mobile phase = Chloroform: Benzene (3:1) or Toluene- Ethyl acetate (97:3)  The standard compound = Peppermint Oil.  The spray reagent =Vanilline _Sulphuric acid / Ethanol (10%v/v).  Mechanism of separation = Adsorption.  Developing = Ascending. 9.7.1.1. Procedure  Prepare 100ml of mobile phase, and place it in the glass tank.  Cover the tank with glass lid and allow standing for 45 minutes before use.  Apply the sample spot and the standard spot on the silica gel plates, on the base line.  Put the silica gel plate in the glass tank and allow the mobile phase to rise to about two-third the plate.  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. 80 9.7.1.2. Results and Discussion.  Record your results and calculate the Rf value End Of Lab 81 Report sheet number 8 (for lab 9)  Name of Student  Aim and objective of this Lab:  Principle used for the extraction of Volatile oils  Methods and tests used for the identification of volatile  Results and discussion for each test performed in this lab. 82 List of Figures Lab. Figure Page Figure 1.1.1 “Demonstration of the glycone and aglycone parts in methyl β-D-glucoside” Figure 1.1.2 “Acetal and ketal formation” 9 No. 1 1 10 15 2 Figure 2.2.1” attachment of the sugar moiety to the 3β-OH group of the steroid nucleus” Figure.2.2.2.”Cyclopentanoperhydrophenanthrene ring which consist of three six membered fully hydrogenated (perhydro) phenanthrene ring and five membered cyclopentane ring” Figure 2.2.3 “ Cardenolide and bufadienolide together with their numbering system” Figure 2.4.3.1. “Nerium oleander plant” 2 Figure 2.4.3.2. “Oleandrin structure” 18 2 Figure 2.5.1 “ example of maceration extraction method” 19 3 26 3 Figure 3.1.1.3 “ Baljet’s test principle reaction and color result” Figure 3.1.2.2.1” color reaction of Keller-Killian Test 3 Figure 3.2.1.3.1 “Meisenheimer dye complexes” 29 3 Figure 3.2.3.1 “Kedde’s Reaction” 29 3 Figure 3.3.5.1. “TLC technique” 32 3 Figure 3.3.5.2 “ calaculation of RF value (solute represents spot)” Figure 3.3.5.3 “Diagram demonstating Rf value calculation” Figure 4.1.1 “Anthraquinone Nucleus with numbering system” 33 2 2 2 3 4 16 16 18 27 33 37 83 4 Figure 4.1.2”Anthraquinones derivatives” 37 4 38 4 Figure 4.3 “ Emodin :structural relationships of its derivatives” Figure 4.1.4 “Different anthraquinone structures” 4 Figure 4.3.1 “ Senna Leaves” 40 4 Figure 4.3.2 “Structures of Sennosides A,B, C and D” 40 6 Figure 6.2.1 “Basic Sapogenin Structures” 51 6 Figure 6.3.1” Saponaria officinali plant” 51 6 Figure 6.3.2.1.” Saponification test” 52 7 Figure 7.2.1 “General structural categories of Flavonoids” 59 7 Figure 7.3.1.1 “Ruta graveolens family Rutaceae” 59 7 Figure 7.4.3.2.1” Shinoda Test” 62 7 Figure 7.4.4.2.1” Chelate formation reaction” 63 8 Figure 8.2.1.1. “Gallic Acid Structure” 67 8 Figure 8.2.2.1 “ Catechin structure” 67 8 Figure 8.2.2.2 “ Epicatechin structure” 67 8 Figure 8.5.1 “Witch Hazel leaves” 69 8 Figure 8.5.2. “Hamamelitannin structure” 69 9 Diagram 9.2.1.” Classification of terpenoids” 75 9 Figure 9.2.2 “Joining Isoprene Units” 76 9 Figure 9.2.3 “ Head to tail rule of isoprene units” 76 9 Figure 9.6.1 “ Clevenger apparatus” 78 9 Figure 9.6.2.” different parts of Clevenger according to the density of volatile oil in regards to water” Figure 9.6.3 “ Peppermint” 78 9 38 79 84 List of Reagents and Reactions Reagent and Reaction Identification use Baljet’s Cardio-active Glycosides Kedde’s Cardio-active Glycosides Keller-Killian’s Cardio-active Glycosides Raymond’s Cardio-active Glycosides Legal’s Cardio-active Glycosides Lieberman’s Steroidal Nucleus Borntrager Anthraquinone Glycosides Benedict’s Saponin Glycosides Hemolytic Saponin Glycosides Alkaline Test Flavonoid Glycosides Ammonia Test Flavonoid Glycosides Shinoda Test Flavonoid Glycosides Chelation Formation Test Flavonoid Glycosides Gelatin Test Tannin Ferric Chloride Test Tannin Lead sub acetate Test Tannin Phenazone Test Tannin 85 List of Chemicals NO Compound Name 1 Ethanol 2 Lead sub Acetate 3 Chloroform 4 Sodium Phosphate 5 Anhydrous Sodium Sulphate 6 Hydrochloric acid 7 Picric Acid 8 Sodium Hydroxide 9 Glacial Acetic Acid 10 Ferric Chloride (Fecl3) 11 Sulphuric Acid 12 3,5-Dinitrobenzene 13 3,5-Dinitrobenzoic Acid 14 Potassium Hydroxide 15 Anhydrus Acetic Acid 16 Pyridine 17 Ammonia 18 Ethyl Acetate 19 Methanol 20 Butane 21 Xylene 22 Formamide 23 Tetrahydrofuran 86 24 N-Propanol 25 Copper Sulphate 26 Sodium Carbonate 27 Sodium Citrate 28 Petroleum Ether 29 Magnesium 30 Sodium Acetate 31 Aluminum chloride 32 Gelatine 33 Sodium Chloride 34 Phenazone 35 Benzene 36 Toluene 87 List of Plants Name of Plant Bioactive Compounds Nerium oleander Cardio-active Glycoside Cassia acutifolia Anthraquinone Glycoside Saponaria officinalis Saponin Glycoside Ruta graveolens Flavonoid Glycoside Hamamelis Tannins Mentha piperita Volatile oil 88 Preparation of reagents used in Experiments  Benedict’s reagent Benedict’s Solution prepared from 1 g of anhydrous sodium carbonate, 1.73 g of sodium citrate and 0.2 g of copper(II) sulfate pentahydrate, 100 m distilled water. Benedict’s solution is blue in color. Constituent Functions Copper sulphate Furnishes cupric ions (Cu++) Sodium carbonate Makes medium alkaline Sodium citrate Complexes with the copper (II) ions so that they do not deteriorate to copper(I) ions during storage Distilled water Solvent  10% Sodium Phosphate weigh 10gm of sodium phosphate and dissolve in Distilled water then complete the volume to 100 ml with same solvent.  10% Ammonia Solution To prepare 10% of ammonia solution check the concentration of ammonia solution that you have in your lab then depending on the concentration you want to prepare, you can use the dilution formula M1V1 = M2V2. 89 Assuming you need 100 mL of 10% ammonia and the concentration of ammonia in your lab is 25g 25 * V1 = 100 * 10. Solving the equation, V1 = 40. Therefore you need to add 40 ml of 25% ammonia to 60 ml of water to get 100 ml of 10% ammonia.  10% Sodium Hydroxide To prepare 10% sodium hydroxide solution dissolve 10gm of sodium hydroxide in Distilled water (D.W) and then complete the volume to 100 ml with the same solvent.  10% sodium acetate 10% sodium acetate solution is prepared by dissolving10gm of sodium acetate in D.W and then completing the volume to 100 ml by the same solvent.  2.5% AlCl3 To prepare 2.5% AlCl3 solution, dissolve 2.5gm of AlCl3 in D.W then complete the volume to100 ml with D.W.  25% nitric acid solution To prepare 25% nitric acid solution check the concentration of nitric acid solution that you have in your lab then depends on the concentration you want to prepare. You can use the dilution formula M1V1 = M2V2. Assuming you need 100 mL of 25% nitric acid and the concentration of nitric acid in your lab is 68% 90 68 * V1 = 100 * 25. Solving the equation, V1 = 36.8 ml. Therefore you need to add 36.8 ml of 68% nitric acid to 63.2 ml of water to get 100 ml of 25% nitric acid solution  60%FeCl3 To prepare 60%FeCl3, Dissolve 60gm of FeCl3 in D.W then complete the volume to 10 ml to get the 60%FeCl3 solution.  5% Alcoholic KOH 5% Alcoholic KOH is prepared by dissolving 5 gm of KOH in alcohol (methanol) then completing the volume to 100 ml with same solvent  1% 3,5- dinitrobenzene To prepare 1% of 3,5-dinitrobenzene, dissolve 1 gm of 3,5-dinitrobenzene in D.W then complete the volume to 100 ml with D.W.  4N HCl To prepare 4N HCl Use formula: C1 * V1 = C2 * V2 C1: Concentration of final solution = 4 N V1: Volume of the final solution = 100 ml C2: Concentration of stock solution = 12.18 N V2: Amount stock solution required = ??? *C2 = Normality of a 37% HCl solution which has density 1.2 g/ml, is 12.18 N. 4 * 100 = 12.18 * V2 91 V2 = 4 * 100 / 12.18 = 32.8 ml of HCl in your lab Amount of water = 100 – 32.8 = 67.2 ml Take 67.2 ml water in a measuring cylinder and add 32.8 ml concentrated HCl  10%sodium chloride To prepare 10% of sodium chloride solution, dissolve 10 gm of sodium in D.W then complete the volume to 100 ml with D.W.  1%sodium carbonate To prepare 1% of sodium carbonate solution, dissolve 1 gm of sodium carbonate in D.W then complete the volume to 100 ml with D.W.  Vanillin sulphuric acid To prepare vanillin sulphuric acid reagent dissolve 1 gm of vanillin in absolute ethanol with 1.5 ml concentrated sulphuric acid then complete the volume to 100 ml with ethanol.  Liebermann–Burchard spray reagent For preparation carefully dilute 1 mL of concentrated H2SO4 and 20 mL acetic anhydride with chloroform (or absolute ethanol) complete the volume to 100 ml with the same solvent. Spray or dip the plate and dry. 92 References 1. James E. Robbers, Marilyn K. Speedie, Varro E. Tyler “Pharmacognosy and Pharmacobiotechnology” 1996. 2. Biren N Shah, A.K Seth “ Text Book of Pharmacognosy and Phytochemistry” First Edition 2010, ELSEVIER.. 3. M. Bartnik, P.C. Facey, in Pharmacognosy “ Fundamentals, Application and Strategies, 2017 Elsevier Inc. 4. Kurt Greeff “Cardiac Glycoside: Part one experimental pharmacology” Springer- Verlag 2011 . 5. Shashank Kumar and Abhay K. Pandey “Chemistry and Biological Activities of Flavonoids: An Overview” The Scientific World Journal, Volume 2013, Article ID 162750, 16 pages; http://dx.doi.org/10.1155/2013/162750. 6. Nadia Abdalla Guied Saeed “ Phytochemical screening and quantification of saponins in the leaves of three Zizyphus species “B.Sc. ( Agric ) Hons. (1981) University of Khartoum. 7. Kokate C K, Khandelwal K R, Pawar A P, Gokhale S B, (2000). Practical Pharmacognosy, Nirali prakashan, Pune, 45-46. 8. Intisar S. 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