Practical Pharmacognosy and Phytochemistry Manual 2022-2023 Hashemite University PDF

Hashemite University Faculty of Pharmaceutical Sciences Department of Pharmaceutical Chemistry Practical Pharmacognosy & Phytochemistry Manual (131703314) Student Name: Student number: Prepared by: Dr. I...

Hashemite University Faculty of Pharmaceutical Sciences Department of Pharmaceutical Chemistry Practical Pharmacognosy & Phytochemistry Manual (131703314) Student Name: Student number: Prepared by: Dr. Iman Almansi & Dr. Tahani Alwidyan Updated by: M.Sc. Farah Hudaib 2022/2023 -i-  Course Description Understanding the definition and material of pharmacognosy science and its applications in therapy and pharmacy, acquainting knowledge of natural drug products, their classification, production, evaluation as well as their general chemistry, and understanding the qualitative and quantitative tests used to evaluate plant material in its crude and powdered status according to pharmacopeia and WHO guidelines for plant-based material. In addition, this practical course intends to acquaint students with the required practical skills of natural product analysis including herbal sample preparation (drying and grinding), extraction, separation (chromatography; TLC), and characterization. The applications covered include some selected medicinal herbs and their extractives that are rich in various phytochemical groups of primary and secondary metabolism such as sugars, fixed and volatile oils, alkaloids, phenols, terpenoids, etc., particularly covered by the theoretical courses Photochemistry. Course Objectives 1. Knowledge and understanding of the science of pharmacognosy and the use of natural products (e.g. medicinal plants and herbs) in pharmacy and therapy. 2. Identify natural products of plant primary metabolism and their applications in therapy, pharmacy and food industry. 3. Get practical skills needed to evaluate macroscopically and microscopically some selected medicinal plants, as crude drugs, covered by the theoretical course 4. Generally define and investigate in the different fields and disciplines related to study of natural drugs and pharmacognosy science including sample preparation and separation. 5. Define, identify and evaluate natural drugs derived from plant primary metabolism (carbohydrates, lipids, and proteins). 6. To provide the students with the appropriate knowledge and skills of the methods of separation of natural plant constituents. 7. To identify the groups of plant primary and secondary constituents. 8. Qualitative analysis of plant constituents chromatographically and spectroscopically. 9. Constituents and uses of the analyzed plants 10. Acquaint practical knowledge of methods of identification, classification, production, chemical and physical evaluation of natural drug products. -ii-  Intended Learning Outcomes (ILOs) Successful completion of the course should lead to the following outcomes: A. Knowledge and Understanding: 1. Apply the science of pharmacognosy and the use of natural products (e.g., medicinal plants and herbs) in pharmacy and therapy. 2. Plant primary metabolites as natural products. 3. Basic principles of separation 4. Basic principles of chemical identification 5. Basic principles of chromatography (PC, TLC, CC 6. Basic principles of some spectroscopic methods. B. Intellectual skills (cognitive and analytical): 1. Generally, define and investigate in the different fields and disciplines related to study of natural drugs and pharmacognosy science including sample preparation and separation. 2. Define, identify, and evaluate natural drugs derived from plant primary metabolism (carbohydrates, lipids, and proteins). 3. Theoretical and practical aspects of separation applied to plant drugs 4. Theoretical and practical aspects of identification applied to plant drugs 5. Theoretical and practical aspects of isolation C. Subject specific skills The student is expected to learn how to conduct chemical reactions within medicinal chemistry context this includes: 1. Acquaint practical knowledge of methods of identification, classification, production, chemical and physical evaluation of natural drug products. 2. Identify natural products of plant primary metabolism and their applications in therapy, pharmacy and food industry. 3. Chromatographic analysis of plant drugs using TLC 4. Identification of impurities and degradation products of plant drugs of known and unknown origin based on TLC spot Rf and color comparisons 5. Determination of the purity of plant drugs D. Transferable Skills 1. Work in a team as a workgroup and discuss results with other colleagues. 2. Know how to conduct a literature survey, access specific information about medicinal plants and natural products as well as how to collect data of others’ research to prepare a group common report. 3. Skill of different chromatographic procedures (PC, TLC 4. Use of different lab equipment Reading List / References: Supplementary Textbook(s) 1 Practical Manual Pharmacognosy (by: Tyler, Brady, and Robbers). 1990. 2 Trease and Evans’ Pharmacognosy (by W.C. Evans). Edition (year): 15th (2000). 3 Theoretical course (Phytochemical Analysis) material 4 Plant Drug Analysis-A TLC Atlas (by H. Wagner and S. Baldt) 5 Pharmacognosy, Phytochemistry, Medicinal Plants (by J. Bruneton -iii-  Course Contents Credit Date Week Exp. Topics Teaching Assessment methods Hours No# Procedure General instruction and safety rules - Class participation Lecturing discussion andlaboratory apparatus - Laboratory Report Practical work Quizzes 8-12/10 1 3 01 & Definitions, Plant Tissue and Lab work evaluation Structure of Microscope Lecturing discussion - Class participation Microscopically Identification of Different Practical work - Laboratory Report 15-19/10 2 3 02 Starch Types Quizzes Recorded video Lab work evaluation Lecturing discussion - Class participation Microscopical Identification of Different Practical work - Laboratory Report 3 3 03 Quizzes 22-26/10 Roots and Rhizomes Recorded video Lab work evaluation Lecturing discussion - Class participation Microscopical Identification of Different Practical work - Laboratory Report 4 3 04 Quizzes 29/10-2/11 Barks and Woods Recorded video Lab work evaluation 5 Off days 5-9/11/2022 (First exam duration) Lecturing discussion - Class participation Practical work - Laboratory Report Microscopical Identification of Different 6 3 05 Quizzes 12-16/11 Leaves and Herbs Recorded video Lab work evaluation Lecturing discussion - Class participation Practical work - Laboratory Report Microscopical Identification of Different 19-23/11 7 3 06 Quizzes Flowers and Fruits Recorded video Lab work evaluation 8 Practical mid exam 26-30/11/2022 Lecturing discussion - Class participation Determination of Fixed Oils from British Practical work - Laboratory Report 3-7/12 9 3 07 Quizzes Pharmacopoeia (BP) Recorded video Lab work evaluation Lecturing discussion - Class participation Extraction and Identification of volatile oils - Laboratory Report Practical work 10-14/12 10 3 08 by TLC Quizzes Recorded video Lab work evaluation Lecturing discussion - Class participation Extraction and Identification of Flavonoids - Laboratory Report Practical work 17-21/12 11 3 09 Quizzes Recorded video Lab work evaluation Lecturing discussion - Class participation Practical work - Laboratory Report 24-28/12 12 3 10 Quizzes Recorded video Lab work evaluation 14 Theoretical final exam -iv-  Grade Distribution Assessment Grade Date 1. Quiz 15% Weekly 2. Midterm exam 25% To be arranged 3. Report 10% Weekly 4. Evaluation 10% Weekly 5. Final Examination 40% To be arranged Important regulations  On average, students need to spend 3 hrs of study and preparation weekly.  Excellent attendance is expected. According to the university policy, students who miss more than 15% of the lecture hours with or without excuse will be dismissed from the course  At the beginning of the lab, be on time and don't leave before the end of the lab session without an accepted excuse  If you missed a lab session, it is your responsibility to find out about any announcements or assignments you have missed  For any clarification, please communicate your instructor at his posted office hours or by appointment  Switch off your mobile or keep it silent throughout the lecture  Listen well to the lab discussion and avoid side discussions, if you have a question, ask your instructor and not your colleague  If you have any information, document your reference, if you didn't, then you broke the intellectual property rights law, and the law will be applied.  Exams are scheduled to be given two times throughout the semester; you are expected to attend all. If not, make-up exams will be offered for valid reasons. It may be different from regular exams in content and format.  Cheating, academic misconduct, fabrication and plagiarism will not be tolerated, and the university policy will be applied.  Each student is expected to familiarize himself with laboratory rules and safety precautions. -v-  Index Lab. Experiment Page no. no. 00 General Instructions and Laboratory Safety Rules Vii Part 1: Pharmacognosy Introduction: Definitions, Plant Tissue, and Structure of 1 3 Microscope Experiment no.1: 2 10 Microscopical Identification of Different Starch Types Experiment no.2 3 17 Microscopical Identification of Different Roots and Rhizomes Experiment no.3 4 28 Microscopical Identification of Different Barks and Woods Experiment no.4 5 35 Microscopical Identification of Different Leaves and Herbs Experiment no.5 6 45 Microscopical Identification of Different Flowers and Fruits Part 2: Phytochemistry Experiment no.6 7 54 Determination of Fixed Oils from British Pharmacopoeia (BP) Experiment no.7 8 60 Extraction and Identification of volatile oils by TLC Experiment no.8 9 67 Extraction and Identification of Flavonoids Experiment no.8 10 ?? -vi-  General Instructions A. Lab Safety Precautions Gloves and Eye protection Goggles must be worn all the time in the lab, regardless of what is being done. Prescription glasses (not sunglasses) are acceptable. Contact lenses provide no protection. Familiarize yourself with the location of fire extinguishers, safety showers, fire blankets and eye wash fountains and know how and when to use these devices. If chemical are splashed in or near the eye, wash immediately with clean and cold running water for 10 -15 min (remove contact lenses in case you have been wearing them). Consult a physician afterwards. When inserting glass tubing or thermometer into rubber stoppers, always lubricate both the glass and the whole with glycerin and protect hands with a towel. Never taste any compound in the laboratory. To determine the odor of a compound, bring the stopper of the bottle cautiously toward the nose. Do not inhale. Avoid any contact of chemicals with the skin, especially the face. Wash your hands as soon as possible after making transfers or other manipulations. When heating a test tube or carrying out a reaction in one, never point the tube toward yourself or your neighbor. Never heat an organic liquid directly over a flame except under a condenser. When refluxing a liquid, be sure that the condenser is tightly fitted. If a temperature below 95oC is sufficient, use a steam bath rather than a burner. Before lighting a flame check to see that volatile liquids are not being poured or evaporated in your vicinity. Always turn a burner off as soon as you finish using it. As a general practice, and particularly if a burner is in use, avoid loose- fitting long sleeves and cuffs. Long hair should be tied back during laboratory work. Any experiment involving the use or production of objectionable (i.e. poisonous or irritating) liquids or gases, must be performed in the hoods. -vii- -viii-  B. Laboratory rules Each student is expected to attend each lab session and to be in thelaboratory on time. Those students, who come early, should wait inside the lab but never gather in the corridors. Each student must wear a clean and buttoned up lab coat with his\here tag name before entering the lab. Skirt, short clothe, sandal (open shoes) and head caps forbidden in lab. Smoking, Drinking, Eating or Chewing gum is prohibited in the lab. Each student is responsible for keeping the laboratory clean and in good order. Coats, book and personal belongings should be kept in your lockers. Do not bring them with you to the lab. All working areas and balances must be kept clean. Powdered drugs, greasy or waxy materials or any insoluble waste materials will block the sink if thrown into it. Thus, dispose them properly in a waste basket. Water immiscible organic solvents and other liquids should be discarded in a designated waste – solvent can but never poured into a sink. Chemicals that react vigorously with water, such as acid chlorides or alkali- metals should be decomposed in a hood in a suitable way. If students are assigned to work as groups, each group is expected to work quietly and independently. Do not carry reagent bottles to your desk. Never return excess materials to reagent bottles. Each student must bring:  Marker  Two hand towels  Pair of gloves (nitrile  Sponge for disk cleaning gloves is preferred)  Detergent  Calculator to each lab  Matches You Will Be Held Responsible for Each Rule Mentioned Above, Any Violation Will Expose You to Either Being Dismissed From The Lab Or Losing Evaluation Marks Of That Particular Experiment Without Prior Notification -ix- Labs common glassware -ii-  Introduction Definitions.  Pharmacognosy: It derives from two Greek words, "pharmakon" or drug, and "gnosis" or knowledge. Recently it has undergone significant change defined as: “a science that study the physical, chemical, biochemical and biological properties of drugs, drug substances, or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources”. It is one of five major areas of pharmaceutical education. Research problems in pharmacognosy include studies in the areas of phytochemistry, microbial chemistry, biosynthesis, biotransformation, chemotaxonomy, and other biological and chemical sciences.  Phytochemistry: “It is the branch of chemistry concerned with plants and plant products”. Phytochemistry is the study of phytochemicals, which are chemicals derived from plants, to describe the structures of the large number of secondary metabolic compounds found in plants, the functions of these compounds in human and plant biology, and the biosynthesis of these compounds. The compounds found in plants are of many kinds, but most are in four major biochemical classes, the alkaloids, glycosides, polyphenols, and terpenes. Phytochemistry can be considered sub-fields of botany or chemistry. Activities can be led in botanical gardens or in the wild with the aid of ethnobotany. The applications of the discipline can be for pharmacognosy, or the discovery of new drugs, or as an aid for plant physiology studies. -3- Plant Tissues and Tissues Systems. When cells are grouped together for an identical function, a tissue is formed. In the plant body the following three tissue system can be distinguished: -4- 1- Ground Tissue Systems (Basic Cell Types): It consists of simple cells and comprises the bulk of the primary plant body. It synthesizes organic compounds, supports the plant and provides storage for the plant. It is mostly made up of plant cells called parenchyma cells but can also include some collenchyma and sclerenchyma cells as well. Parenchyma cells synthesize and store organic products in a plant. Mostof the plant's metabolism takes place in these cells. Collenchyma cells have a support function in plants, particularly in young plants. These cells help to support plants while not restraining growth due to their lack of secondary cell walls and the absence of a hardening agent in their primary cell walls. Sclerenchyma cells also have a support function in plants, but unlike collenchyma cells, they have a hardening agent and are much more rigid. 2- Dermal Tissue System: The dermal tissue system consists of the epidermis and the periderm. 3- Vascular Tissue System: Xylem and phloem throughout the plant make up the vascular tissue system. They allow water and other nutrients to be transported throughout the plant. -5- I. Preliminary Tests (Macroscopical Character) of Powdered Drugs. 1. Organoleptic Tests: A. Note the color:  White: acacia.  Brown: cinnamon.  Dark brown: clove.  Green: senna.  Orange: rhubarb. B. Note the odor (the following are particularly characterized):  Ginger, Clove, Cinnamon, Thyme, Peppermint C. Note the taste:  Aromatic: cinnamon, clove.  Aromatic and pungent: ginger.  Bitter: quassia.  Sweet: liquorice. 2. Physical Tests: A. Water solubility: Mix a small quantity of powder with a few drops of water and allow to stand. Aqueous extracts dissolve almost completely, while the gummy or mucilaginous nature of drug, such as acacia and linseed, becomes apparent. B. Volatility: Press a small quantity of powder between two filter papers. An oily stain, spreading but persisting when the paper is heated in an oven, occurs with powders containing fixed oils. Volatile oils will give a stain, disappearing on heating in an oven. C. Frothing test (detects the presence of saponins): Shake a little powder in a half a test-tube-full of water, and if any marked frothing occurs, suspect saponin-containing drugs. 3. Chemical Test: Test using colored chemical reactions -6- II. Structure of Microscope and Microscopical Technique:  Structure of microscope: 1. Eyepiece: where you look through to see the image. 2. Body tube: long tube that holds the eyepiece and connects it to the objectives. 3. Nosepiece: the rotating part of the microscope at the bottom of the body tube; it holds the objective lenses. 4. Objective lenses: they vary in length (the shortest has the lowest power of magnification; the longest has the highest power of magnification). 5. Arm-part: you carry the microscope with it. 6. Coarse adjustment knob: large, round knob on the side of the microscope. Used for focusing the slide. 7. Fine adjustment knob: small, round knob on the side of the microscope, used to fine-tune the focus after using the coarse adjustment knob 8. Stage: large, flat area under the objectives, where the sample or specimen is placed for examination. 9. Light source: usually found near the base of microscope. -7-  Microscopical technique and reagents: For microscopical observation a small sample is placed on a glass slide and dispersed in a suitable mountant (any substance in which a specimen is suspended between a slide and a cover glass for microscopic examination). This has a refractive index, which will give image contrast and may also clear the preparation by dissolving pigments or other substances. A cover slip is gently placed on top of the preparation thus trapping the particles. a) Reagents (mountants): 1. Chloral hydrate solution: particularly useful for the examination of dried plant materials as it acts as a clearing agent and also expands shrunken cells to restore their natural shape. This mountant removes certain characters such as: starch, but it does not affect calcium oxalate, oils or fats. 2. Glycerol: it is a non-drying mountant which has no solvent power. As it does not dissolve starch it is useful for the routine qualitative examination of starch. 3. Water: used for starch. 4. Phloroglucinol + HCL: used for lignified tissue. b) Magnification: Your microscope has 3 magnifications: Scanning, Low and High. Each objective lens has a written magnification. In addition to this, the ocular lens (Eyepiece) has a magnification of 10X. The total magnification = the ocular lens magnification x the objective lens magnification. -8- Self-reading:  Care of the microscope 1. Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example. 2. Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done. 3. Always make sure the stage and lenses are clean before putting away the microscope. 4. Never use a paper towel, your shirt, or any material other than good quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents may separate or damage the lens elements or coatings. 5. Cover the instrument with a dust jacket when not in use. 6. Focus smoothly; don't try to speed through the focusing process or force anything. For example, if you encounter increased resistance when focusing then you've probably reached a limit and you are going in the wrong direction.  How to use microscopes: 1. Place your microscope on a secure table, free from vibration, to begin. Try to have the microscope at least one foot away from any edge to avoid an accidental fall. 2. Turn on the lamp and set the intensity for comfortable viewing. 3. Place a specimen slide on the stage. 4. You should now begin to learn an important skill that will significantly increase your enjoyment of the instrument. You must learn to view through the eyepiece(s) with both eyes open! Whether you have a monocular microscope (one eyepiece,) or a binocular microscope (two eyepieces,) start from the beginning to use both eyes 5. Start from the beginning by low power (4 X). 6. If you wish to move to a higher power objective, it should take very little -9-  movement of the fine adjustment knob to bring the image into focus. Similarly, a particle in the image which is centered in the field of view should remain in the center as objectives are changed. 7. Initially, slowly focus back (turn the fine focus knob to raise the optical tube) while looking through the eye piece. Once the specimen comes into focus, you can make fine adjustments up or down with the fine focus knob without fear of damaging the slide or the microscope. 8. If the specimen does not come into view (does not focus), raise the tube a little with the coarse focus knob and attempt to focus again with the fine focus knob. Once the object is in focus, switching objective lenses (to a higher power) should be possible without any further coarse adjustments.  Preparation of the Specimen: 1. Add the plant material in a clean slide. 2. Add few drops of chloral hydrate. 3. Heat using Bunsen flame for few minutes (never let the slide dry). 4. Put the cover slip on the slide in an angle 45° and lower slowly to drive out any air bubbles. 5. Before examination be sure that the area not covered by the cover slip is clean and dry. -10-  Experiment (1) Microscopically Identification of Different Starch Types Starch (polysaccharide) is the main form in which plants store carbon. It occurs as semi- crystalline granules composed of two polymers of glucose, called amylose and amylopectin. Depending on the plant organ, it can act as a store of carbon for lengths of time as short as a day (e.g. in leaves) or as long as many years (e.g. in dormant seeds). Starch granules are characterized by internal growth rings. There is enormous variation in granule size and shape between plant organs, and between species. Starch is the major carbohydrate of nutritional importance in the diet: it is degraded to glucose by amylases in the mouth and small intestine. When cooked in water, starch forms gels or pastes that have a wide range of industrial applications in both food and nonfood industries.  The starch granule consists mainly of two components: 1. Amylose: long unbranched linear chain of glaucous residues, soluble in water. 2. Amylopectin: long branched chain of glaucous residues, insoluble in water. It is the major starch component and usually forming the outer layers of the granule.  Use of starch in pharmaceutical industry: 1. Dusting powder (in which adsorbent properties are important). 2. Antidote in treatment of iodine poisoning. 3. Skin emollient, basis for enemas. 4. Tablet disintegrant. 5. Lubricant for surgeon’s gloves. -11-  Macroscopical characters of starch: A) Organoleptic: Starch occurs in irregular, angular masses or as a white powder, smooth touch, odorless, and starchy taste. B) Physical properties: 1. Solubility:  Cold water  insoluble (suspension)  Hot water  soluble (gelatinization: breaking down the intermolecular bonds of starch molecules, the semi-crystalline structure is lost and the smaller amylo and increasing the mixture's viscosity).  Organic solvents ( alcohol, ether, benzene)  insoluble  Chloral hydrate  soluble 2. pH:  Corn and wheat starches have neutral pH  Rice starch has slightly alkaline pH  Potato starch has slightly acidic pH. C) Chemical test: I3- (I2/KI) + starch solution dark blue color (The strength of the resulting blue color depends on the amount of amylose present) Chemically all types of starch are the same; they can be differentiated only under microscope.  Microscopical character of different starch types: Starch can be identified by microscopical examination (they should be first mounted with water) by the observation of the following granules characteristics: I. Shape: the shape of the granules varies not only in the different plants but also in the different organ in the same plant. II. Size: the granules vary greatly in size even in the same plant. They could be very minute to such a large size. III. Hilum: (the starting point of the granules in the amyloplast) is another -12-  character that can be used to distinguish different types of starch. On microscopical examination, hilum takes the form of a rounded dot or simple or multiple clefts. IV. Striation: starch granules are built up of the deposition of successive layers around the hilum. Concentric rings or striation are often clearly visible in large granules as potato starch. V. Aggregation of the granule: starch granules may be simple or compound. Compound granules produced by the continuous deposition of starch around two or more centers.  Sources of starches: a. Endosperm of the grain of: 1. Maize: Zea mays L. (Gramineae) 2. Wheat: Triticum aestivum L. (Gramineae) 3. Rice: Oryza sativa L. (Gramineae) b. Tubers of potato: Solanum tuberosum L. (Solanaceae) shown in the table below: Potato Wheat Maize Rice Grain or fruit of Tubers of Solanum Grain or fruit of Grain or fruit of Origin Oryza tuberosum L. Triticum aestivum L. Zea mays L. sativa L. Family Solanaceae Gramineae Gramineae Gramineae Polyhedral Rounded, oval. Polyhedral Shape Oval and pyramidal with sharp lenticular in side view with blunt angles angles Faint centric point, Cleft or fissured Hilum Eccentric pointed Not present Linear in side view (triangular,centric) Striation Present, concentric Faint Not present Not present Simple, semi- Mostly Aggregation Simple Mostly simple compound compound Rounded 10-35 Consist of large Consist of microns granules about 25-45 Consist of granules Size microns and smaller granules Or Ovoid 30-100 measuring 2- granules about 6-7 measuring 5-30 microns 10 microns microns microns -13- Potato Wheat Maize Rice -14-  Practical work 1. Observe the following physical characteristics of (maize, wheat, rice, and potato) starches: a. Color b. Taste c. Oder d. Texture 2. Determine the pH of starch: mix 0.2 g of starch with 5 ml water, dip in a piece of pH paper and read the pH. 3. Observe the solubility of starch in water before and after boiling. 4. Observe the microscopical appearance of four different types of starch:  On slide add few drops of water to a little of the powdered starch and examine under microscope. 5. Add few drops of iodine and notice the color produced. -15-  Report sheet (1) Microscopically Identification of Different Starch Types No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Observed physical and chemical characters: - Color: - Odor: - Taste: - Solubility - Iodine test result: 3. Microscopical Examination: 1. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: -16- 2. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used : - Magnification : - Description : 3. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: 4. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: -17-  Experiment (2) Microscopical Identification of Different Roots and Rhizomes Root: is the descending portion of the plant axis which usually grows below the soil which serves as anchor and absorb water and nutrient to the transport to the upper portion of the plant. Rhizome: a fleshy, elongate stem, often subterranean. Peeled & Unpeeled root and rhizomes: Peeled rhizomes and roots will have the outermost layer removed (cork layer will be absent), on the contrary of the unpeeled one which will preserve the outermost layer. Sclerenchymal cells: Sclerenchyma is a hard supporting tissue with heavy secondary thickening and divided into two categories according to their aspect ratio:  Scleride ( stone cells): are typically roughly isodiametric. Although elongated and branched forms also occur. They may be found singly, in-group or as a complete layer.  Fibers: are typified by high length to width ratio. They are usually thick walled and have a narrow lumen and pointed ends. Fibers are usually classified according to the area in which they occur. A crystal sheath is sometimes formed around sclerenchyma and this features is often of diagnostic significance. Cork: Cork tissues is built up of a compact masses of cells, the mature cork cellis dead, impermeable to water and often filled with dark reddish-brown content rich in tannins and related substances. Calcium oxalate: are found in plants as a result of interaction of oxalic acid (a metabolic product) with calcium salt. Different types of calcium oxalate crystals with various shapes can be used as a diagnostic element for plant identification  Rosettes (as in Rhubarb and clove).  Prisms (liquorice). -18-   Single acicular crystals (cinnamon)  Bundles of acicular crystals  Sandy crystals. We are going to examine three selected plants: Liquorice , Chinese rhubarb, and Turmeric rhizome I. Liquorice: The dried unpeeled roots and stolen of Glycyrrhiza glabra L. (Fabaceae).  Physical characters:  Color : yellowish brown  Taste: sweet  Constituents:  Glycyrrhizin (glycyrrhizic acid, glycyrrhizinic acid): a saponin responsible for the sweet taste of liquorice. Glycyrrhizin occurs as a mixture of potassium and calcium salts, on hydrolysis releases two molecules of D-glucuronic acid and the aglycone glycyrrhetic.  Flavonoids (responsible for the yellow color of liquorice).  Starch granules  Uses:  Flavoring agent.  Demulcent and mild expectorant.  Peptic ulcer healing: reduced symptoms and improved healing in 75% of the studied cases.  Gastric and duodenal ulcers: Deglycyrrhizinated liquorice (380mg, 3 times daily) to 169 patients with chronic duodenal ulcers was as effective as antacid or cimetidine.  Contraindications:  Hypertension, cholestatic disorders or cirrhosis of the liver, hypokalaemia, or chronic renal insufficiency, and during pregnancy.  Warnings:  Prolonged use of large doses (50g/day> 6weeks) may increase water accumulation, causing swelling of the hands and feet.  Sodium excretion is reduced, and potassium excretion is increased. Blood pressure may rise. -19-  Identification test for saponins: 1. Liquorice + 60% H2SO4: yellow to orange color produced due to the presence of saponins. 2. Frothing test: Liquorice + Water with shaking, a persistent foam for at least one minute.  Examination under microscope: -20- II. Chinese rhubarb: dried rhizomes of Rheum palmatum L. (Polygonaceae).  Physical characters:  Color : orange to brownish-yellowish  Odor: characteristic, aromatic odor  Constituents:  Anthraquinones (laxative): rhein, emodin, aloe-emodin  Tannin (astringent)  Uses:  Short term treatment of occasional constipation.  In cathartic preparations.  Identification test for Anthraquinones:  Rhubarb + KOH: pink to red color produced with the free oxidized form of anthraquinones.  Identification test of tannins:  Rhubarb + FeCl3: dark green color produced. -21-  Examination under microscope: -22- III. Turmeric rhizome: Dried rhizome of Curcuma longa L. (Zingiberaceae).  Physical characters:  Color : yellow to orange  Constituents:  Coloring materials (Curcuminoids) curcumin.  Volatile oil contains sesquiterpenes.  Uses:  In the treatment of acid.  Flatulent.  Atonic dyspepsia.  Knee osteoarthritis.  Inflammatory bowel disease.  Peptic ulcer.  Contraindications:  Obstruction of the biliary tract, in cases of gallstones, and hypersensitivity to the drug.  Identification test for lignin (organic substance binding the cells, fibers and vessels which constitute wood and the lignified elements of plants):  Turmeric powder + Phloroglucinol + conc. HCL: Pink color on the wallsof fibers and vessels. -23-  Examination under microscope: -24-  Practical work: 1. Observe the microscopical appearance of the three plants. 2. Do the chemical testing for plant identification as following:  Identification test for saponins: 1. Liquorice powder (in a test tube) + 1-2 drops of 60% H2SO4⟶yellow- orange color. 2. Liquorice powder (in a test tube) + 3 ml H2O ⟶shake vigorously⟶allow to stand, foam of 3 cm height that persist for at least one minute is a positive result.  Identification test for Anthraquinones: Rhubarb powder (in a test tube) + KOH ⟶ pink to red color.  Identification test of tannins: Rhubarb powder (in a test tube) + FeCl3 ⟶ dark green color produced.  Identification test for lignin: Turmeric powder (on slide) + Phloroglucinol-HCL⟶cover with coverslip and observe the pink color on the walls of fibers and vessels. -25-  Report sheet (2) Microscopical Identification of Different Roots and Rhizomes No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Plants Information: Plant Common Major Constituents Uses Main Physical Name Char. 3. Plant Identification Chemical Tests: Plant Identification Chemical Tests Plant Common Name Chemical Reaction Result -26- 4. Microscopical Examination: 1. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: 2. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: 3. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: -27-  Experiment (3) Microscopical Identification of Different Barks and Woods The layers of mature woody stem include (ordered from outside to the inside): 1. Cork 2. Cork cambium Bark 3. Cortex 4. Phloem 5. Vascular cambium 6. Xylem ⟶ Wood Xylem: One of the two types of transport tissue (vascular tissue) in plants, phloem being the other. It is the principal water-conducting tissue of the plant. Xylem is a compound tissue made up of parenchyma, fiber, and tracheary elements (tracheids and vessel members). Tracheids and vessel elements are distinguished by their shape; vessel elements are shorter, and are connected together into long tubes that have pores at each end, while trachieds have no pores. Phloem: Is a compound tissue and is responsible for the transport of food. It contains parenchyma, sclerenchyma and sieve element. The sieve tubes are the most highly specializedcell composed of a vertical series of elongated cells, interconnected by perforation in their walls in areas known as sieve plates. Sieve elements are usually broken during powdering while phloem scleride (stone cell) are important in the identification of certain barks. -28-  We are going to examine three selected plants: Cinnamon Bark and Quassia Wood I. Cinnamon Bark: Dried bark of Cinnamomum zeylanicum B. (Lauraceae).  Physical characters:  Color: reddish brown  Odor: pleasant and aromatic  Constituents:  Cinnamaldehyde (60-75 %)  Volatile oils (up to 4%)  Eugenol  Tannins  Uses:  Uses described in Pharmacopeias: Dyspeptic conditions, fullness, flatulence, and loss of appetite, also used to treat abdominal pain with diarrhea.  Experimental pharmacology: Antibacterial and antifungal (O-methoxy cinnamaldehyde). Carminative activity (essential oil). Antispasmodic (Cinnamaldehyde).  Identification test of tannins:  Cinnamon + FeCl3: dark green color produced. -29-   Examination under microscope: II. Quassia Wood: Dried wood of Picrasma excels P. (Simaroubacease).  Constituents:  Bitter quassinoids: quassin, isoquassin and neoquassin -30-  Uses:  Insecticide  Remedies for infestation of lice and warms (Anti Parasite)  Identification test for lignin:  Quassia powder + Phloroglucinol + conc.HCL: Pink color on the walls of fibers and vessels.  Examination under microscope: -31-  Practical work: 1. Observe the microscopical appearance of the studied plants. 2. Do the chemical testing for plant identification as following:  Identification test of tannins: Cinnamon powder (in a test tube) + FeCl3  dark green color produced.  Identification test for lignin: Quassia powder (on slide) + Phloroglucinol + conc.HCL  cover with cover slip and observe the pink color on the walls of fibers and vessels. -32-  Report sheet (3) Microscopical Identification of Different Barks and Woods No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Plants Information: Plant Common Major Constituents Uses Main Physical Name Char. 3. Plant Identification Chemical Tests: Plant Identification Chemical Tests Plant Common Name Chemical Reaction Result -33- 4. Microscopical Examination: 1. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used : - Magnification : - Description : 2. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used : - Magnification : - Description : -34-  Experiment (4) Microscopical Identification of Different Leaves and Herbs Epidermis: This is the outermost layer of the plant structure and is usually one cell thick. Important diagnostic features include the shape of the anticlinal (vertical) and the periclinal (horizontal) walls (e.g. straight or wavy), the presence of thickening (such as beading), and the occurrence of striations of the surface cuticle (waxy protecting film covering the epidermis of leaves). Distributed among the relatively non specialized epidermal cells are a number of highly specialized and characteristic structures mainly stomata and trichome. Trichomes: Most leaves and many herbaceous stems, flowers, fruits, and seeds possess hairs or trichomes of one kind or another. Many show hair of more than one type. -35-  The trichomes (hair) may be grouped into: - Non-glandular (protective) hairs: These may be unicellular which vary from small papillose outgrowth to large structures, or multicellular. - Glandular (secretory) hairs: A glandular hair usually has a stalk and a head. The stalk may be either unicellular or multicellular, and likewise, the head may be either unicellular or multicellular. Stomata: Stomata (Singular: stoma) most frequently occur on the young leaves and stems, but can also be found on other organs like flowers. Stomata allow gas exchange between the surrounding air and the photosynthetic cells inside the leaves. A stoma consist of a pore surrounded by two guard cells. The arrangement of epidermal cells around the stoma falls into one of four main types: Anomcytic stomata: the stomata are surrounded by cells resembling the other epidermal cells with no particular arrangement. Anisocytic stomata: the stoma is surrounded by three or four subsidiary cells one of which is smaller or larger than the others. Diacytic stomata: the stoma has two subsidiary cells with their long axis at right angles to the pore of the stoma. Paracytic stomata: the stoma has two subsidiary cells with their long axis parallel to pore of the stoma. -36-  We are going to examine three selected plants: Sage Leaf , Senna Leaf, and Absinthium Herb I. Sage Leaf: Dried leaf of Salvia officinalis (Labiatae).  Constituents:  Volatile oils (cineole)  Flavonoids  Terpenes  Tannins  Acids  Uses:  Antispasmodic activity  Gingivitis  Enhance memory (cineole)  Hypoglycaemic activity  Antimicrobial and Antiviral -37-   Examination under microscope: II. Senna Leaf: Dried leaves of Cassia acutifolia, Cassia angustifolia (Fabaceae).  Physical characters:  Color : grayish-green to yellowish-green  Taste : mucilaginous slightly bitter  Constituents:  Anthraquinones: dianthrone glycosides, primarily sennosides A and B.  Flavonoids.  Saponins. -38-  Uses:  Laxative.  Identification test for Anthraquinones:  Plant powder + KOH: yellow to red color  Examination under microscope: -39- III. Absinthium Herb (Wormwood): Dried leaves and flowering tops of Artemisia absinthium (Asteraceae, compositae)  Physical characters:  Color : dark green or blue  Taste : bitter acrid taste  Constituents:  Volatile oil: Thujone  Bitter glycoside: Absinthin  Uses:  Tonic  Antispasmodic  Febrifuge (treat fever)  Warning:  Classified as unsafe herb by FDA because of the neurotoxic potential (causes seizures) of thujone and its derivatives.  Identification test for lignin -40-  Examination under microscope: -41-  Practical work: 1. Observe the microscopical appearance of the three plants. 2. Do the chemical testing for plant identification as following:  Identification test for Anthraquinones: Senna powder (in a test tube) + KOH ⟶ Yellow to red color.  Identification test for lignin: Wormwood powder (on slide) + Phloroglucinol + conc.HCL⟶ cover with cover slip and observe the pink color on the walls of fibers and vessels. -42-  Report sheet (4) Microscopical Identification of Different Leaves and Herbs No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Plants Information: Plant Common Major Constituents Uses Main Physical Name Char. 3. Plant Identification Chemical Tests: Plant Identification Chemical Tests Plant Common Name Chemical Reaction Result -43- 4. Microscopical Examination: 1. Common name: Origin (Scientific name + part of plant examined): Family: Mounting reagent used: Magnification: Description: 2. Common name: Origin (Scientific name + part of plant examined): Family: Mounting reagent used: Magnification: Description: 3. Common name: Origin (Scientific name + part of plant examined): Family: Mounting reagent used: Magnification: Description: -44-  Experiment (5) Microscopical Identification of Different Flowers and Fruits Flower: a determinate axis with spore-bearing appendages (and usually sterile appendages) and short internodes occurring in the angiosperm. Pollen grain: a microspore wall containing a mature or immature male gametophyte in seed plant. Fruit: the mature ovary or ovaries of one or more flowers and sometimes associated structures. - Endocarp: the innermost layer of the ovary wall in the fruit. - Exocarp: the outermost layer of the ovary wall in the fruit. - Mesocarp: the layer in the fruit between exocarp and endocarp. We are going to examine three selected plants: Chamomile Flowers, Clove Flowers, and Anise Fruit I. Chamomile Flower: dried flower of Matricaria chamomilla L. (Asteraceae, Compositae) “German chamomile”.  Constituents:  Volatile oils: α-bisabolol (up to 50%) and chamazulene (1-15%), and Matricin.  Flavonoids: rutin.  Coumarins.  Uses:  Internal use: Dyspepsia, epigastric bloating, impaired digestion and flatulence. In the treatment of restlessness and in mild cases of insomnia due to nervous disorders.  External use: Inflammation and irritations of the skin and mucosa, including irritation and infections of the mouth and gums and hemorrhoids. -45-   Inhalation: Symptomatic relief of irritations of the respiratory tract due to the common cold.  Identification test for lignin  Examination under microscope: -46-  II. Clove flower: dried flower of Eugenia Caryophyllus (Myrtaceae).  Constituents:  Volatile oil: Eugenol (60-95%), and caryophyllene (5-10%).  Tannins.  Uses:  Treatment of toothache and minor infections of the mouth.  Antiseptic for minor wounds.  Antioxidant.  Anti-inflammatory.  Antiviral.  Identification test for lignin  Identification of tannins \ -48-  Examination under microscope: -48- 2. Anise fruit: dried ripe fruit of Pimpinella anisum (Umbelliferae, Apiaceae)  Constituents:  Volatile oil: anethole (80-95%).  Coumarins.  Flavonoids.  Uses:  Antispasmodic.  Expectorant.  Antibacterial, antifungal, and insecticidal.  Examination under microscope: -50-  Practical work: 1. Observe the microscopical appearance of the three plants. 2. Do the chemical testing for plant identification as following:  Identification test for tannins: Clove powder (in a test tube) + FeCl3 ⟶ dark green color.  Identification test for lignin: Plant powder (on slide) + Phloroglucinol + conc. HCL ⟶ cover with cover slip and observe the pink color on the walls of fibers and vessels. -51-  Report sheet (5) Microscopical Identification of Different Leaves and Herbs No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Plants Information: Plant Common Major Constituents Uses Main Physical Name Char. 3. Plant Identification Chemical Tests: Plant Identification Chemical Tests Plant Common Name Chemical Reaction Result -52- 4. Microscopical Examination: 1. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: 2. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: 3. - Common name: - Origin (Scientific name + part of plant examined): - Family: - Mounting reagent used: - Magnification: - Description: -53-  Experiment (6) Determination of Fixed Oils from British Pharmacopoeia (BP) Fixed oil is a triglyceride that can be extracted from vegetable origins, such as nuts or seeds of the botanical sources (e.g., almond, olive, sunflower……etc.). Triglyceride is an ester derived from glycerol and three fatty acids. Fatty acid is a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. The characteristics of oils are related to the properties of the fatty acids that they contain. The larger the number of carbon atoms, the higher the melting point; the larger the number of double bonds, the lower the melting point. Physicochemical properties of oil like density, viscosity, boiling point, specific gravity, saponification value (SV), iodine value (IV), acid value (AV) and peroxide value (PV) were studied to evaluate the compositional quality of oils and their commercial importance. I. Determination of Acid value (free fatty acids): This value is a measure of the amount of free fatty acids which have been liberated by hydrolysis of triglycerides due to the action of moisture and temperature. It is a measure of hydrolytic rancidity which is used to determine the quality, age, edibility and suitability of oil for industrial use (Pharmaceutical oil must not have any acidity. Refined oils which referred to neutralized or low acidity oils are used in industry). Acid value is a relative measure of hydrolytic rancidity because raw and crude oils are naturally hydrolyzed and consequently have a high acid value and it may be overestimated if other acid components are present in the system e.g., amino acids.  The acid value is defined as the number of milligrams of potassium hydroxide required to neutralize the free fatty acids present in one gram of oil. -54-  The acid value is determined by directly titrating the oil with a standard solution of potassium hydroxide using Phenolphthalein as a color indicator.  To calculate the Acid Value: AV = (M *Mw * V) / W Mw = molecular weight of potassium hydroxide, 56.11. V = the end point (mL) M = molarity of the potassium hydroxide solution, 0.1. W = weight of the sample taken (g) II. Determination of Peroxide Value (Test of Rancidity): Fixed oils are odorless but some time it can emit a faint nutty aroma. When fixed oils are exposed to air they react with the oxygen or water vapor to form a series of breakdown products in stages; starting with primary oxidation products (Peroxides, Dienes, Free fatty acid), then secondary products (Carbonyls, Aldehydes, Trienes) and finally tertiary products. The short-chain acids are volatile and have unpleasant smells and tastes. This oxidation process is called Rancidification. Oxidation progresses at different rates depending on many factors such as: storage temperature, light exposure, availability of oxygen, and the presence of moisture and metals. Oils with a high degree of unsaturation are most susceptible to oxidation. The peroxide value is a parameter specifying the content of oxygen as peroxide in a substance. The peroxide value is a measure of the oxidation present. Therefore, it is useful for assessing the extent of oil spoilage and rancidity that occurred during storage.  The peroxide value: is defined as the amount of peroxide oxygen per 1 kilogram of fat or oil. Traditionally this was expressed in units of milliequivalents (mEq). The peroxide value can be determined by measuring the amount of iodine which is formed by the reaction of peroxides with iodide ion. ROOH + 2KI + H2O ⟶ ROH + 2KOH + I2 The liberated iodine can be titrated with sodium thiosulphate (Na2S2O3) using starch solution as an indicator (amylose forms a blue solution with iodine and is colorless where iodine is titrated). -55-  I2 (purple) + 2Na2S2O3 ⟶Na2S4O6 + 2NaI (colorless) POV of fresh oils are less than 10 mEq/Kg, when the PV is between 30-40 mEq/Kg a rancid taste is noticeable.  Calculate the Peroxide Value: POV = (E.P * Normality of Na2S2O3 *1000) / W E.P = volume of sodium thiosulfate required to titrate sample (mL). W = weight of the sample taken for the test (g). Note: Normality of S2O32-= Molarity of Na2 S2O3  Saponification value:  The Saponification Value is the number of mg of potassium hydroxide required to neutralize the free acids and saponify the esters contained in 1.0 g of the substance. Saponification is the process of breaking down a neutral fat into glycerol and fatty acid salt by treatment with strong base. Saponification value is a measure of the average molecular weight of the triglyceride in a sample. The smaller the SV the larger the average molecular weight of the triglyceride present. The long chain fatty acid found in fats have low SV because they have a relatively fewer number of carboxylic functional groups per unit mass of the fat and therefore high molecular weight.  Iodine value: Unsaturated fatty acids can be converted into saturated by the process of hydrogenation. Depending upon the degree of unsaturation, the fatty acids can combine with oxygen or halogens to form saturated fatty acids. The iodine value gives a measure of the degree of unsaturation of a lipid. An increase in IV indicates high susceptibility of lipid to oxidative rancidity due to high degree of unsaturation.  The Iodine Value represents the number of grams of iodine absorbed by 100 g of the substance. -56-  Practical work:  Fixed oil examples: Arachis oil, Caster oil, Olive oil, Corn oil, Palm oil …etc.  Determination of AV 1. Dissolve about 10.0 g of oil, accurately weighed, in 50 mL of a mixture of equal volumes of alcohol and ether (which has been neutralized to phenolphthalein with 0.1 M potassium hydroxide or 0.1 M sodium hydroxide) contained in a flask. 2. Titrate with 0.1 M potassium hydroxide using 0.5 mL of phenolphthalein until the solution remains faintly pink after shaking for 30 s.  Determination of PV 1. Place about 5 g of oil, accurately weighed, in a 250-mL conical flask fitted with a ground- glass stopper. 2. Add 30 mL of a mixture of glacial acetic acid and chloroform (3:2) to the flask and shake to dissolve and add 0.5 mL of saturated potassium iodide solution. 3. Shake for exactly 1 min. 4. Add 30 mL of water. Titrate with 0.01 M sodium thiosulfate, adding the titrant slowly with continuous shaking, until the yellow color is almost discharged. 5. Add 5 mL of starch, and continue the titration, shaking vigorously, until the blue color is discharged. -57-  Report sheet (6) Determination of Fixed Oils from British Pharmacopoeia (BP) No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. Calculate the AV for the used oil (oils) showing detailed calculation: 3. Calculate the PV for the used oil (oils) showing detailed calculation: ** If more than one oil type have been tested and according to the results you obtained, which oil do you think has a better quality? Explain you answer: -58-  Experiment (7) Extraction and Identification of volatile oils by TLC Essential oils contain some highly volatile organic substances that can be isolated from odoriferous plants by various physical processes. The oils are usually concentrated in the seeds or flowers but may exist in other parts of the plants as well. Such oils were called essential because they were thought to represent the very essence of odor and flavor. Most essential oils are primarily composed of terpenes and their oxygenated derivatives (terpenoids). Chemical constituents of volatile oils can be divided into two broad classes: 1.) Terpene derivatives formed via acetate mevalonic acid pathway and 2.) Aromatic compounds formed via shikimic acid phenyl propanoid route. Several points of differentiation exist between volatile oils and fixed oils. Volatile oils can be distilled from their natural sources; they do not consist of glyceryl ester of fatty acids. Hence, they do not leave a permanent grease spot on paper and cannot be saponified with alkalies. Volatile oils do not become rancid, as do fixed oils, but instead, on exposure to light and air, they oxidize and resinify. Unlike essential oil, fixed oils are not volatile. Meaning, they do not evaporate rapidly even when under normal temperature or pressure. It acts as a carrier oil and diluent for essential oil to be easily absorbed. Essential oils can be obtained from plants by a number of processes such as mechanical pressing and grinding, maceration, solvent extraction, distillation and concentration. In many cases a combination of processes are required for an efficient and effective isolation. The specific extraction method employed is dependent upon the plant material to be distilled and the desired end-product. Most essential oils are extracted by steam distillation. -59- Distillation: Distillation is a method of separating liquids in a solution by using the differences in their boiling points. Several laboratory scale techniques for distillation exist, steam distillation is preferred for essential oils extraction.  Steam distillation: Steam distillation is a method for distilling compounds which are heat- sensitive. This process involves bubbling steam through a heated mixture of the raw material. Many organic compounds tend to decompose at high sustained temperatures. Separation by normal distillation would then not be an option, so water or steam is introduced into the distillation apparatus. By adding water or steam, the boiling points of the compounds are depressed, allowing them to evaporate at lower temperatures, preferably below the temperatures at which the deterioration of the material becomes appreciable. Chromatographic Techniques: One of the problems continually facing biochemists is the separation and purification of one or more biological compounds from a mixture of such compounds. One of the most convenient methods for achieving such separation is the use of chromatographic techniques. The selection of a particular form of chromatography to achieve a separation is dependent on the material to be isolated, and often several chromatographic methods may be used sequentially to achieve the purification of a compound. The term partition or distribution coefficient is normally used to describe the way in which a compound distributes itself between two immiscible phases. The distribution of compound can be described in term of its distribution between any two phases, such as liquid/liquid, solid/liquid, or gas/liquid phases. This concept of the distribution coefficient is the basic principle of chromatography. All chromatographic system consists of two phases. One is the stationary phase (SP), which may be solid, liquid or mixture. The second is the mobile phase (MP) may be liquid or gaseous and flows through the stationary phase. The choice of stationary or -60- mobile phases is made so that the compounds to be separated have different distribution coefficients. This can be achieved by setting up an equilibrium between SP and MP.  Thin layer chromatography (TLC) It is an adsorption chromatographic technique in which the two phases are: a solid stationary phase and a liquid mobile phase. The solid stationary phase is a thin layer of finely divided solid, such as silica gel or alumina, supported on glass, plastic or aluminum, and the mobile phase is a solvent. As the solvent moves past the sample spot that was applied on the solid phase, equilibrium is established for each component of the mixture. The more polar molecules, the higher the affinity it will have for the more polar silica plate and will therefore spend less time in the mobile phase. As a result, it will move up the plate more slowly. Conversely, a less polar molecule will spend more time in the mobile phase and will therefore move up the plate more quickly. The speed at which the molecules will move up the plate thus depends on the polarity of the solute, solvent, and adsorbent. The difference each molecule travels along the adsorbent in relation to how far the mobile phase has traveled is called the Retention factor (Rf) and can be used to identify molecules, as the value is molecule specific. The retention factor for any given molecule will vary depending on the mobile and stationary phases used.  TLC is a simple, quick, and inexpensive procedure that commonly used in the separation of substances from mixture, identification of a compound by comparing it with known substances, checking the purity, and in following the progress of a chemical reaction. -61-   How to Run a TLC Plate: Step 1: Prepare the developing container The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top (the latter is used in the undergrad labs at CU). Pour solvent into the chamber to a depth of just less than 0.5 cm. To aid in the saturation of the TLC chamber with solvent vapors, you can line part of the inside of the beaker with filter paper. Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate. Step 2: Prepare the TLC plate TLC plates are usually 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be. Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty. Measure 0.5 cm from the bottom of the plate. Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will spot the plate. Take care not to press so hard with the pencil that you disturb the adsorbent. Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together; about 4 samples on a 5 cm wide plate is advised. Step 3: Spot the TLC plate If the sample is not already in solution, dissolve about 1 mg in 1 mL of a volatile solvent. As a rule of thumb, a concentration of 1% usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; ifit is not concentrated enough, you will see nothing on the plate. Sometimes you will need to use trial and error to get well-sized, easy to read spots. -62-  Dip the capillary tube into the solution and then gently touch the end of it onto the origin line location on the TLC plate. Don't allow the spot to become too large - if necessary, you can touch it to the plate, lift it off and blow on the spot. After each spot is dry, you will repeat the entire process 2 more time, so that each solution has been spotted 3 times. Step 4: Develop the plate Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. The solvent will rise up the TLC plate by capillary action. Make sure the solvent does not cover the spot. Allow the plate to develop until the solvent is about half a centimeter below the top of the plate. Remove the plate from the beaker and immediately mark the solvent front with a pencil. Allow the plate to dry. Step 5: Visualize the spots If there are any colored spots, circle them lightly with a pencil. Most samples are not colored and need to be visualized with a UV lamp after spraying.  If the TLC plate runs samples, which are too concentrated, the spots will be streaked and/or run together.  If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate. -63-  Practical work: 1. Volatile oil extraction:  Plants to be used and their suitable reference:  Clove flower: Eugenol as a ref.  Anise fruit: Anethole as ref.  Thyme leaves: Thymol as ref.  Coriander: Linalol as a ref.  Procedure: 1. Weigh 1.0 g of powdered plant and put it with 10 ml distilled water in 50 ml Erlenmeyer flask, add few glass beads. 2. Connect the Erlenmeyer flask to a bent tube through rubber stopper into a receiving test tube. 3. Heat slowly on direct flame with caution until you collect in the tube about 2 ml distillate. 4. Add the distillate to a small test tube, then add a small amount of petroleum ether and shake to extract the oil. 2. TLC:  Solvent system: (sho uld be place in the chamber before plate preparation to allow the chamber to become saturated) - Tolunene: Ethylacetate (93:7), prepare 100ml.  Spraying reagent: - Vanillin / Sulfuric acid  Procedure: 1. Prepare your TLC plate as instructed and develop in the solvent system. 2. After the solvent had reached 10 cm, dry your TLC and spray it with the spraying reagent. 3. Analyze your results and calculate the Rf values. -64-  Report sheet (7) Extraction and Identification of volatile oils by TLC No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. According to the TLC results obtained: - Calculate the retention factor for all the separated sample spot, showing detailed calculations. - Attached the TLC plate obtained after spraying. - Determine the type of essential oil found in each of the plants you tested. - Fill your results in the following table: Plant name Essential oil type Color of spot after Retention spraying factor 3. Calculation details -65-  Experiment (8) Extraction and Identification of Flavonoids Flavonoids are an important class of natural products; particularly, they belong to a class of plant secondary metabolites having a polyphenolic structure. They constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognized as flower pigments in most angiosperm families. However, their occurrence is not restricted to flowers but are found in all parts of plants. Flavonoids are based on a C15 skeleton with a Chromane ring bearing a second aromatic B ring in position 2, 3 and 4. Flavonoids can be subdivided into different subgroups depending on the carbon of the C ring on which the B ring is attached and the degree of unsaturation and oxidation of the C ring. Flavonoids in which the B ring is linked in position 3 of the C ring are called isoflavones. Those in which the B ring is linked in position 4 are called neoflavonoids, while those in which the B ring is linked in position 2 can be further subdivided into several subgroups on the basis of the structural features of the C ring (e.g.: flavones, flavonols, flavanones, etc.). In plants, flavonoids have long been known to be synthesized in particular sites and are responsible for the color (yellow or red/blue pigmentation) and aroma of flowers, and in fruits to attract pollinators. Flavonoids protect plants from different biotic and abiotic stresses and act as unique UV filters. Also, function as signal molecules, detoxifying agents and antimicrobial defensive compounds. -66-  Flavonoids are associated with a broad spectrum of health-promoting effects and are an indispensable component in a variety of pharmaceutical, medicinal and cosmetic applications. This is because of their anti-oxidative, anti- inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme functions. They are also known to be potent inhibitors for several enzymes, such as xanthine oxidase (XO), cyclo- oxygenase (COX), lipoxygenase and phosphoinositide 3-kinase. Examples of plants with a high flavonoid content include parsley, onions, berries, green tea, and all citrus fruits. I. Extraction Extraction: is the separation of medicinally active portions of plant using selective solvents through standard procedures. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity. The choice of the solvents will determine the type of compound extracted from the samples.  Common extraction procedures  Maceration: In maceration, whole or coarsely powdered plant-drug is kept in contact with the solvent in a stoppered container for a defined period (minimum 3 day) with frequent agitation until soluble matter is dissolved. This method is best suitable for use in case of the thermolabile drugs.  Infusion: It is a dilute solution of the readily soluble components of the crude drugs. Fresh infusions are prepared by macerating the solids for a short period of timewith either cold or boiling water. -67-   Decoction: This method is used for the extraction of the water soluble and heat stable constituents from crude drug by boiling it in water for 15 minutes, cooling, straining and passing sufficient cold water through the drug to produce the required volume.  Soxhlet extraction (hot continuous extraction): Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compoundfrom the insoluble substance. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. This method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds.  The basic parameters influencing the quality of an extract are: 1. Plant part used as starting material 2. Solvent used for extraction 3. Extraction procedure  Variation in extraction methods usually depends upon: 1. Length of the extraction period. 2. Solvent used. 3. pH of the solvent. 4. Temperature. 5. Particle size of the plant tissues. 6. The solvent-to-sample ratio. -68-  Practical work: 1. TLC:  Plants to be used:  Arnica flowers  Grapefruit peels  Orange peels  Calendula flowers  References: Rutin, Hesperidin, Quercetin  Solvent system: Ethylacetate: Formic acid: Glacial acetic acid: Water (100:11:11:24)  Detection: 1- UV 365 2- 2- Spraying agent: Ferric chloride solution.  Procedure: 1. Extract 1g of the plant with 10 ml of ethanol by heating on a water bath for 5 minutes, then filter. 2. Prepare your TLC plate as instructed and develop in the solvent system. 3. Spray your TLC with the spraying reagent. 4. Analyze your results and calculate the Rf values. 2. Chemical tests: - Shinoda’s test: to 1ml of the alcoholic solution of plant extract, add Mg metal and then add HCL drop wise, detect the color. - To 1ml of alcoholic solution of hesperidin, add few drops of AlCl3 solution and detect the difference in the intensity of the yellow color. - Repeat the tests for Rutin solution. -69-  Report sheet (8) Extraction and Identification of Flavonoids No. Student name Section Group no. 1 2 3 4 5 1. Objective: 2. According to the TLC results obtained: - Calculate the retention factor for all the separated sample spot, showing detailed calculations. - Attached the TLC plate obtained after spraying. - Determine the type of Flavonoids found in each of the plants you tested. - Fill your results in the following table: Plant name Flavonoid type Color of spot after Retention spraying factor 3. Calculation details: 4. Chemical tests results: -70-  ‫اﻟﺠﺎﻣﻌﺔ اﻟﮭﺎﺷﻤﯿﺔ‬ ‫ﻛﻠﯿﺔ اﻟﻌﻠﻮم اﻟﺼﯿﺪﻻﻧﯿﺔ‬ ‫ارﺷﺎدات اﻟﺴﻼﻣﺔ اﻟﻌﺎﻣﺔ ﻟﻠﻤﺨﺘﺒﺮات‬ ‫ﻋﻠﻰ ﺟﻤﯿﻊ اﻟﻄﻼب ﻣﺮاﻋﺎة اﻟﺘﻌﻠﯿﻤﺎت اﻟﺘﺎﻟﯿﺔ‪ ،‬ﻟﻤﺎ ﻟﮭﺎ ﻣﻦ أھﻤﯿﺔ ﻛﺒﯿﺮة ﻋﻠﻰ ﺳﻼﻣﺔ اﻟﻌﺎﻣﺔ ﻓﻲ‬ ‫اﻟﻤﺨﺘﺒﺮات‪:‬‬ ‫ﻓﻲ ﺣﺎل وﺟﻮد اي ﺣﺎﻟﺔ طﺎرﺋﺔ ﯾﺮﺟﻲ اﻻﺗﺼﺎل ﺑﺎﻷرﻗﺎم اﻟﺘﺎﻟﯿﺔ‪.4790, 4791, 4666 :‬‬ ‫ﻋﺪم دﺧﻮل اﻟﻤﺨﺘﺒﺮ ﻗﺒﻞ اﻟﻮﻗﺖ اﻟﻤﺤﺪد ﻓﻲ اﻟﺒﺮﻧﺎﻣﺞ‪ ،‬وﻋﺪم اﻟﻌﻤﻞ ﻓﻲ اﻟﻤﺨﺘﺒﺮ ﺑﺪون وﺟﻮد‬ ‫‪.1‬‬ ‫اﻟﻤﺸﺮف‪.‬‬ ‫ارﺗﺪاء ﻣﺮﯾﻮل اﻟﻤﺨﺘﺒﺮ ﻣﻊ )‪ (tag name‬ﻗﺒﻞ دﺧﻮل اﻟﻤﺨﺘﺒﺮ‪ ،‬وارﺗﺪاء واﻟﻨﻈﺎرات اﻟﻮاﻗﯿﺔ‬ ‫‪.2‬‬ ‫ﻟﻠﻌﯿﻨﯿﻦ طﯿﻠﺔ ﻓﺘﺮة ﺗﻮاﺟﺪ ﻓﻲ اﻟﻤﺨﺘﺒﺮ‪.‬‬ ‫ﻣﻦ اﺟﻞ ﺳﻼﻣﺘﻚ اﻟﺮﺟﺎء ﻋﺪم ارﺗﺪاء اﻟﻘﺒﻌﺎت واﻟﻤﻼﺑﺲ اﻟﻘﺼﯿﺮة ﻛﺎﻟﺸﻮرت واﻟﺘﻨﻮرة واﻷﺣﺬﯾﺔ‬ ‫‪.3‬‬ ‫اﻟﻤﻜﺸﻮﻓﺔ )ﻣﺜﻞ اﻟﺼﻨﺪل(‪.‬وﻋﺪم ارﺗﺪاء اﻟﻌﺪﺳﺎت اﻟﻼﺻﻘﺔ ﻟﻠﻌﯿﻨﯿﻦ‪.‬‬ ‫ﻣﻦ اﺟﻞ ﺳﻼﻣﺘﻚ ﯾﻔﻀﻞ ارﺗﺪاء اﻟﻘﻔﺎزات اﻟﻤﺨﺒﺮﯾﺔ أﺛﻨﺎء ﻋﻤﻞ اﻟﺘﺠﺎرب‪.‬‬ ‫‪.4‬‬ ‫ﻣﻦ اﺟﻞ ﺳﻼﻣﺘﻚ ﻋﻠﻰ اﻟﻄﺎﻟﺐ اﻋﻼم اﻟﻤﺪرس ﻓﻲ ﺣﺎﻟﺘﮫ اﻟﺼﺤﯿﺔ ﺑﺸﻜﻞ ﺳﺮي اذا ﻛﺎن ﯾﻌﺎﻧﻲ‬ ‫‪.5‬‬ ‫ﻣﻦ ﺑﻌﺾ اﻻﻣﺮاض اﻟﺘﻲ ﺗﺴﺘﺪﻋﻲ اﻧﺘﺒﺎه اﻟﻤﺪرﺳﺔ ﻛﺎﻷﻣﺮاض اﻟﻤﻨﺎﻋﯿﺔ او ﺗﻨﺎوﻟﮫ ﻷدوﯾﺔ‬ ‫ﺗﻀﻌﻒ ﺟﮭﺎز اﻟﻤﻨﺎﻋﺔ او ﻓﻲ ﺣﺎل ﻛﺎﻧﺖ اﻟﻄﺎﻟﺒﺔ ﺣﺎﻣﻞ‪.‬‬ ‫اﻟﺘﻌﺮف ﻋﻠﻰ اﻻﻣﺎﻛﻦ وﺟﻮد اﻟﻄﻔﺎﯾﺎت اﻟﺤﺮﯾﻖ وﻣﻌﺮﻓﺔ ﻛﯿﻔﯿﺔ اﺳﺘﺨﺪاﻣﮭﺎ وﻛﺬﻟﻚ ﻣﻜﺎن وﺟﻮد‬ ‫‪.6‬‬ ‫اﻟﺪش وﺑﻄﺎﻧﻲ اﻟﺤﺮﯾﻖ‪.‬‬ ‫اﻟﻤﺤﺎﻓﻈﺔ ﻋﻠﻰ ﺟﻮ ﻣﻦ اﻟﻤﺴﺆوﻟﯿﺔ واﻟﺠﺪ واﻟﻨﻈﺎم‪.‬‬ ‫‪.7‬‬ ‫اﻻﻣﺘﻨﺎع ﻋﻦ اﻻﻛﻞ واﻟﺸﺮب واﻟﺘﺪﺧﯿﻦ داﺧﻞ اﻟﻤﺨﺘﺒﺮ‪.‬‬ ‫‪.8‬‬ ‫ﻋﺪم ﺷﻢ او ﺗﺬوق اﻟﻤﻮاد اﻟﻜﯿﻤﺎﺋﯿﺔ‪.‬‬ ‫‪.9‬‬ ‫ﻋﺪم اﻻﺳﺮاف ﻓﻲ اﺳﺘﻌﻤﺎل اﻟﻤﻮاد اﻟﻜﯿﻤﯿﺎﺋﯿﺔ‪.‬‬ ‫‪.10‬‬ ‫ﻋﺪم ﺗﻐﯿﺮ أﻣﺎﻛﻦ وﺟﻮد اﻟﻤﻮاد اﻟﻜﯿﻤﯿﺎﺋﯿﺔ ﺧﺼﻮﺻﺎ اﻟﻤﻮاد اﻟﻤﻮﺿﻮﻋﺔ ﻓﻲ ﺧﺰاﻧﺔ اﻻﺑﺨﺮة‪.‬‬ ‫‪.11‬‬ ‫ﯾﺠﺐ اﺿﺎﻓﺔ اﻟﺤﺎﻣﺾ اﻟﻲ اﻟﻤﺎء وﻟﯿﺲ اﻟﻌﻜﺲ‪.‬‬ ‫‪.12‬‬ ‫ﻋﺪم ﻓﺘﺢ ﺣﻨﻔﯿﺔ اﻟﻐﺎز ﻗﺒﻞ اﺷﻌﺎل ﻋﻮد اﻟﺜﻘﺎب‪.‬‬ ‫‪.13‬‬ ‫ﻋﺪم ﺟﻌﻞ ﻓﻮھﺔ أﻧﺒﻮب اﻻﺧﺘﺒﺎر ﺑﺎﺗﺠﺎه اﻟﻮﺟﮫ‪ ،‬او ﺑﺎﺗﺠﺎه ﺷﺨﺺ اﺧﺮ اﺛﻨﺎء ﻋﻤﻞ اﻟﺘﺠﺎرب‪.‬‬ ‫‪.14‬‬ ‫ﯾﺠﺐ ﻋﺪم ﺗﺴﺨﯿﻦ اﻟﻤﻮاد اﻟﻤﺘﻄﺎﯾﺮة او اﻟﻘﺎﺑﻠﺔ ﻟﻼﺷﺘﻌﺎل ﻋﻠﻰ اﻟﻠﮭﺐ اﻟﻤﺒﺎﺷﺮ‪ ،‬وﻋﺪم رج‬ ‫‪.15‬‬ ‫اﻟﺰﺟﺎﺟﺎت اﻟﺘﻲ ﺗﺤﺘﻮي ﻋﻠﯿﮭﺎ‪.‬‬ ‫ﯾﺠﺐ اﺟﺮاء اﻟﺘﻔﺎﻋﻼت اﻟﺘﻲ ﺗﻨﺘﺞ ﻋﻨﮭﺎ ﻏﺎزات اﻟﺴﺎﻣﺔ ﻓﻲ ﺧﺰاﻧﺔ اﻻﺑﺨﺮة‪.‬‬ ‫‪.16‬‬ ‫ﯾﺠﺐ ﺳﻜﺐ ﻛﻤﯿﺔ ﻛﺎﻓﯿﺔ ﻣﻦ اﻟﻤﺎء ﺑﻌﺪ ﺳﻜﺐ اﻻﺣﻤﺎض واﻟﻘﻮاﻋﺪ ﻓﻲ اﻟﻤﻐﺴﻠﺔ‪.‬‬ ‫‪.17‬‬ ‫اﻟﺘﻌﺎﻣﻞ ﻣﻊ اﻻدوات اﻟﺰﺟﺎﺟﯿﺔ ﺑﺤﺬر ﻻن ﺟﺮوﺣﮭﺎ ﻋﺎدة ﻣﺎ ﺗﻜﻮن ﺑﻠﯿﻐﺔ‪ ،‬ووﺿﻊ ﻣﺎدة اﻟﺘﺸﺤﯿﻢ‬ ‫‪.18‬‬ ‫ﻋﻨﺪ وﺻﻞ أداﺗﯿﻦ ﺑﺒﻌﻀﮭﺎ اﻟﺒﻌﺾ‪.‬‬ ‫ﻋﺪم إﻟﻘﺎء اﻟﻨﻔﺎﯾﺎت اﻟﺼﻠﺒﺔ ﻓﻲ اﻟﻤﻐﺎﺳﻞ أو وﺿﻊ ﻓﺮاﺷﻲ اﻟﺘﻨﻈﯿﻒ ﻓﻲ اﻟﻤﺤﺎﻟﯿﻞ اﻟﺤﻮاﻣﺾ أو‬ ‫‪.19‬‬ ‫اﻟﻘﻮاﻋﺪ‪.‬‬ ‫ﻋﺪم وﺿﻊ اﻻﻏﺮاض اﻟﺸﺨﺼﯿﺔ واﻟﻤﻼﺑﺲ ﻋﻠ?