Farmakognozi Lab PDF

Laboratory Rules and Safety Lect. Eda AVCI Lect. Elif Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI Arrive at the lab on time. It is mandatory to wear a lab coat and keep it buttoned. Closed shoes should be worn at all times in the laboratory. Dangle jewelery should not be worn in the laboratory.  I...

Laboratory Rules and Safety Lect. Eda AVCI Lect. Elif Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI Arrive at the lab on time. It is mandatory to wear a lab coat and keep it buttoned. Closed shoes should be worn at all times in the laboratory. Dangle jewelery should not be worn in the laboratory.  It is forbidden to eat, drink and use laboratory materials for these purposes in the laboratory. Chewing gum should not be allowed in the laboratory.  Preliminary work should be done before coming to the laboratory. It should not be engaged in any other work in the laboratory except for the experiment. Experimental studies should be performed as described and shown. Telephones are prohibited in laboratory. Do not run or joke in the laboratory.  The labels of chemical materials should be read carefully. When using chemical substances, care should be taken not to deform the labels. Chemical substances should not be touched with bare hands. The laboratory should be left clean. Experiment reports must be written in legible and neat writing.  A pump should be used Chemicals should not be when drawing liquid with a inhaled. pipette. Never draw solvent by mouth!!! It should be worked with acids and alkalies in a fume hood. Even if the chemicals taken from the bottle are not used, they should never be put back in the original bottle. When chemicals and/or samples are spilled into the laboratory environment, they should be cleaned immediately.  Acids and alkalis should be added to the water little by little, no water should be added to them!! Volatile (ether, acetone, alcohol, etc.) and flammable substances should not be kept close to the flame.  When working with glass materials, care should be taken and excessive force should not be applied. Dirty and cracked glass materials should not be used. Materials should be washed after use. Gloves should definitely be worn.  Burners or electric heaters should always be turned off when not in use. Wooden tongs must be used during the heating process in the burner flame. The precision scale should be closed and unloaded when not in use. Chemicals should not be spilled on or around the precision scale. Spilled chemicals must be cleaned up. Solid substances should always be taken from bottles with a clean spatula. Do not insert the same spatula into another substance without cleaning it.  The glass of the fume hood should be kept closed as much as possible. When using electrical appliances, make sure that your hands are dry. Devices whose use is not fully known should not be used.  In case of splashing of chemicals on the skin or eyes, wash with plenty of water for at least 15 minutes and act in accordance with first aid rules. Ointment/spray should not be applied to the wound. If the burn is under the clothes, the clothes should never be tried to be taken off. The burn should never be touched by hand. If the clothes catch fire; It should never be run; the flame should be tried to be extinguished by rolling on the ground and help should be sought. The first thing to do when a fire breaks out is to report the fire. In cuts or bleeding; The wound and its surroundings are cleaned and covered. Pressure should be applied according to the severity of the bleeding. Chemical ingestion; If the person is conscious and able to swallow, drink water (fluid administration is not continued if he/she has a tendency to vomit). The person who has been exposed to the accident should be transported to the nearest health institution immediately. Pharmacognosy-1 Laboratory DEMO Lect. Eda AVCI Lect. E. Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI  Pharmacognosy, examines the identification, preparation, content, trade, storage, use and controls (purification, biological activity, active substance) of drugs of biological origin. Pharmacognosy-1 Laboratory will be conducted in two parts: 1. Microscopic Examination of Drugs 2. Phytochemical Analysis  In the pharmacognosy laboratory, a microscope will be used to examine the anatomical structure of drugs and drug powders.  Microscope Parts and Functions Light microscopes consist mainly of mechanical and optical parts. Mechanical Parts of Microscope Eyepiece Tube or Body Tube Nosepiece Stage Clips Stage Arm Coarse Focus Fine Focus Base Optical Parts of Microscope Objective Lenses Diaphragm Illuminator Eyepiece/Ocular Lens Condenser Diaphragm Magnification Power of Microscope Ocular magnification X Lens magnification  Points to Consider in Using Microscope The table on which the microscope is placed must be solid and free of shaking. The microscope should not be placed too close to the edge of the table. Unnecessary things on the table should be removed. Care should be taken not to crush the cables of the microscope.  Some agents (sartur and chloralhydrate) are effective in heat. In this case, the preparation must be heated on a small flame. Heating should be done 2 cm above the flame. The lens facing the preparation should neither strike the preparation nor touch the reagent.  The lenses of the microscopes should not be touched by hand. At the end of the study, the microscope should be left adjusted to the smallest objective.  Cleaning and Maintenance of Microscope Microscopes must be cleaned very carefully after use to obtain clear images. The microscope should be cleaned with a clean and soft cloth after each use.  The optical part should not be wiped with alcohol and hard tissue cloths!! Microscope lenses will deteriorate in a short time if left dusty, damp and at high temperatures. The microscope should never be left in the sun or in heat. After the study is completed and the cleaning of the microscope is completed, the cover of the microscope should be covered or stored in its container. Microscopic Examination of Drugs ✓Cell types Parenchyma, Sclerenchyma, Bundle sheath, Stone cell Cork tissue ✓Crystal types ✓Starch ✓Nonglandular and glandular trichomes ✓Stomata, leaf, flower ✓Bark, root, fruit, seed  REAGENTS Water Starch Chloralhydrate Solution FOLIUM, FLOS Act on the heat RADIX ,CORTEX, Sartur Reagent FRUCTUS Tile Ink MUCILAGE  SARTUR REAGENT Prof. Dr. Turhan Baytop Prof. Dr. Sarım Çelebioğlu SARTUR REAGENT CONTENT Lactic acid More than one Sudan III diagnosis on the same Aniline Iodine slice at once!! Potassium Iodide, Alcohol, Water  Sudan III is a dyestuff. It dyes oil, cutin and suberin in orange color. And it also dyes the cork tissue in a dark-red color. Lactic acid acidifies the environment and clarifies the tissues. Aniline dyes lignin yellow and therefore all lignified walls (wood pipes, sclerenchyma fibers, stone cells, stone fungus cells) become yellow. Iodine dyes starch blue-purple. Potassium iodide increases the solubility of iodine. Water and ethanol are solvents.  Preparation Microscope Object Slide Coverslip Preparate Coverslip Object Microscope Slide Water  Starch Analysis Lect. E. Ayça ALTINKAYA SAMİM Lect. Eda AVCI Lect. Reyhan ARICI  Introduction Strach is a Glycoside Bond carbohydrate consisting of many glucose units linked together by glycosidic bonds.  Introduction Starch formed as a result of the polymerization of glucose (monosaccharides); It is called reserve starch or storage starch. In leaves, starch produced as a result of photosynthesis in chloroplasts is called "assimilation starch".  Introduction The main reason why glucose is stored in the form of starch is to balance the intracellular osmotic pressure. Because glucose dissolves in water, it increases osmotic pressure. Starch is partially soluble in water. Osmotic Pressure Osmotİc Balance Turgor Pressure  Properties of Starch Starch is flavorless and odorless. It is a powder that is partially insoluble in water and insoluble in alcohol. It is used as a thickening agent in the food industry. It is used as an excipient (filler) in pharmacy.  Starch Storage Places Bark Root It is stored as a reserve within leucoplasts in Rhizome these tissues. Tuber Seed  Structure of Starch The place where starch first begins to form is called Hilum. It is observed as a point inside the starch grain. Starch grains become stratified starting from the hilum. These plates are called starch circles. According to the location of Hilum 1. Centric 2. Eccentric Hilum is in the Hilum is at the edge of center of starch starch. Centric Starch Grain in Beans Eccentric Starch Grains in Potatoes  Starch Types The typical starch grain in this type has one Simple Starch starch granule in a leucoplast There is more than one hilum and more Compound Starch than one starch grain in the structure Solanum tuberosum (Potato), Simple and Compound Starch Grains Simple Starch Compound Starch  Amylum Oryzae Amylum Phaseoli (Rice Starch) (Bean Starch) Amylum Tritici Amylum Solani (Wheat Starch) (Potato Starch)  Amylum Drugs Samples Plant: Solanum tuberosum (Potato) Drug: Amylum Solani (Potato Starch) Family: Solanaceae (Nightshade Family) Reagent: Distilled water Microscope Magnification: 10x40 Amylum Drugs Samples Plant: Triticum vulgare (Wheat) Drug: Amylum Tritici (Wheat Starch) Family: Poaceae (Graminae) Reagent: Distilled water Microscope Magnification: 10x40 Plant: Zea mays (Corn) Drug: Amylum Maydis (Corn/Maize Starch) Family: Poaceae (Graminae) Reagent: Distilled water Microscope Magnification: 10x40 Amylum Drugs Samples Plant: Oryza sativa (Rice) Drug: Amylum Oryzae (Rice Starch) Family: Poaceae (Graminae) Reagent: Distilled water Microscope Magnification: 10x40 Analysis of Cell Types Parenchyma, Cork tissue, Sclerenchyma, Conducting Tissue, Stone Cell Lect. Eda AVCI Lect. E. Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI  EPIDERMA Epiderma is a tissue that covers the single-layered epidermis outer surfaces of plants. cuticle upper epiderma It has protective properties against external factors. palisade parenchyma Its outward facing surface is covered with the cuticle layer. It plays a role in storing water and spongy parenchyma regulating substance exchange. Although it is normally single-layered, cuticle multi-layered epidermis is also rarely encountered.  PARENCHYMA Parenchyma is a living tissue composed of thin-walled cells. Parenchyma tissue; It has functions such as photosynthesis, respiration, secretion, ventilation, transmission, storage, wound repair and regeneration. Ventilation (airway) parenchyma In terms of function, parenchyma can be divided into 4 large groups: Photosynthetic parenchyma Storage parenchyma Conducting parenchyma Ventilation(airway) parenchyma  CORK TISSUE They are superficial and polygonal non-living cells formed as a result of the destruction of the epidermis on the outer part of the bark. The cell wall is thin, dark- colored and deepened. In the stone cork, which is a second type of cork tissue, the cell wall A. Cross section of Cinnamomi cassiae cortex is lignified, thickened and a thin-walled cork, b stone cork, c cork with thickened porous. walls in a horseshoe shape B. Thin-walled cork SARTUR !!! C. Stone cork Dark-Red color D. Stone cork with a thickened wall in the shape of a horseshoe in the cross-section of the Granati cortex  SCLERENCHYMA The cells of these tissues have lost their ability to grow. For this reason, sclerenchyma tissue is located mostly in mature organs. It has thick secondary wall that provide mechanical resistance in plants. It contains high amounts of lignin. Protects thin-walled cells from damage against bending, twisting, weight and pressure.  SCLERENCHYMA 1) Sclenchyma fibers 2) Sclerenchyma cells (sclereids)  CONDUCTING TISSUE It is the tissue formed by the transmission system that carries water and substances dissolved in water from the roots and compounds synthesized as a result of assimilation from the leaves to other organs of the plant. Conducting tissue consists of xylem and phloem.  CONDUCTING TISSUE Xylem; It consists of trachea, tracheid, xylem parenchyma and xylem sclerenchyma. Trachea is the tubes formed by the melting of the walls between the cells arranged on top of each other in a cylindrical shape. Independent cells with narrower diameters, elongated cylindrical or prism-shaped, and pointed at both ends form the tracheids. Xylem parenchyma is living cells with abundant cytoplasm, used to store and transmit various nutrients and are distributed throughout the xylem. Xylem sclerenchyma are long cells with lignified walls that support the conducting tissue. It transmits water and dissolved substances from the root to other organs of the plant.  CONDUCTING TISSUE Phloem is a composite tissue that transmits assimilation products. It consists of sieve tubes, companion cells, phloem parenchyma and phloem sclerenchyma. Sieve tubes are living cells with wide lumen, thin walls, that join each other end-to-end like pipes and whose transverse walls on their junction surfaces are perforated like sieves. The narrower, cytoplasm-rich parenchymatic cells located next to the sieve tubes are called companion cells. Phloem parenchyma is also parenchymatic cells. Phloem sclerenchyma, on the other hand, are cells that serve as support and whose walls are thickened by lignin accumulation. CONDUCTING TISSUE Xylem forms; A round, B,C spiral, D reticulated, E Trachea and tracheitis in Liquiritiae radix  STONE CELLS Stone cells are cells that are thickened by the accumulation of lignin in their cell walls. Idioblasts are defined by abnormal shapes, sizes and contents that are different from neighboring cells. Another name for the idioblast is the sleeved stone cell. a b c d a) Stone cells in the primary cortex of Cinnamomi cassiae cortex b) Idioblast in Theae folium c) Stone cells in Lini semen in top view d) Stone cells in the Rhamni purshianae cortex  Major Elements in a Microscopic Analysis-1 Folia elements: Stoma-bearing epidermis parts, spongy and palisade parenchyma parts, thin vascular bundles, secretory cells, trichomes (covering hairs). Flores elements: Corolla papillary epidermis cells, pollen, papillary stigma pieces, characteristic endothecium layer pieces of anthers (secretory and cover hairs, stoma, etc.). Semen elements: Aleurone, starch, fat and the nutrient tissue containing them (endosperm), cotyledon pieces, testa epidermis, small amounts of thin wood tube pieces.  Major Elements in a Microscopic Analysis-2 Fructus elements: In addition to those seen in the seed powder, parts of the pericarp (exocarp, mesocarp, endocarp), endocarp parts characteristic of each fruit, and thin xylem are seen. Radix elements: Abundant parenchymatic pieces, pieces bearing pith branches visible from different directions and if the drug is not peeled, pieces of cork tissue and xylem are seen. Cortex elements: While all Radix elements are seen, xylem is not found.  DRUGS TO BE EXAMINED Drug: Liquiritiae radix Drug: Cinnamomi cassiae cortex Drug: Theae folium (Çay (Meyan kökü-Liquorice root) (Tarçın kabuğu-Cassia bark) yaprağı-Tea leaf) Plant: Glycyrrhiza glabra (Meyan- Plant: Cinnamomum cassia Plant: Camellia sinensis (Çay- Liquorice) (Çin tarçını-Chinese cassia) Tea) Family: Fabaceae (Legumes) Family: Lauraceae (Laurel) Family: Theaceae (Tea family) Reagent: Sartur Reagent: Sartur Reagent: Sartur MM: 10x40 MM: 10x40 MM: 10x40  Content of Sartur Reagent 1. Sudan III is a dyestuff. It dyes oil, cutin and suberin in orange color. And it also dyes the cork tissue in a dark-red color. 2. Lactic acid acidifies the environment and clarifies the tissues. 3. Aniline dyes lignin yellow and therefore all lignified walls (wood pipes, sclerenchyma fibers, stone cells, stone fungus cells) become yellow. 4. Iodine dyes starch blue-purple. 5. Potassium iodide increases the solubility of iodine. 6. Water and ethanol are solvents. Liquiritiae radix A B C D A) Sclerenchyma fiber and starches B) Cork tissue C) Tracheitis D) Parenchyma in cork tissue Cinnamomi cassiae cortex A B C D A) Scleranchyma fiber and starch grains B) Stone cell C) Scleranchyma fiber, stone cell and starch grains D) Cork tissue Theae folium A) Stoma A B B) Spongy parenchyma and epiderma C) Vascular bundle D) İdioblast C D Types of Calcium Oxalate Crystals and Other Plant Elements Lect. Reyhan ARICI Lect. E. Ayça ALTINKAYA SAMİM Lect. Eda AVCI  What is crystal? The products of metabolism are called ergastic substances. The best known ergastic substances are carbohydrates (e.g. cellulose and starch), proteins and fats. Also crystalline minerals belong to this group. Some of these residual substances, which we call ergastic substances, are harmful to plants. They are either excreted or rendered harmless by changing their composition. Calcium oxalate crystals found in plants are called "crystal/billur". Crystals are usually found in the parenchyma cells of the bark, root and leaf (mesophyll). Sometimes they are also found in the cells around the sclerenchyma bundles. Oxalic acid, which is formed as a result of metabolism in the plant, combines with the calcium taken by the plant from the soil to form harmless calcium oxalate crystals.  Types of Crystals Simple Crystals Compound Crystals Raphide Twin Crystal Crystal Sand Druse Single Crystal Simple Crystal Array  Simple Crystals Raphide (Stiloid) Crystal Sand Single Crystal Long needle-shaped crystals It is called clusters formed It is not combined with any of calcium oxalate (e.g. by small crystals. Calcium other crystal. Ipecacuanhae radix, oxalate crystals are small (e.g. Allium cepa’s peel) Sarsaparillae radix, Scillae and amorphous. (e.g. bulbus). Belladonnae folium)  Compound Crystals Twin Crystal Druse Simple Crystal Array It is called crystals formed by It is called star-shaped Crystals in an array around the combination of two calcium oxalate crystals (e.g. sclerenchyma bundles (e.g. simple crystals (e.g. Stramonii folium). Sennae folium, Liquiritiae Hyoscyami folium). radix).  Drugs to be examined today Drug: Scillae bulbus Drug: Hyoscyami folium Drug: Sennae folium Drug: Rhei rhizoma (Squill Bulb) (Henbane Leaf, Plant: Cassia acutifolia (Rhubarb Rhizome) Plant: Urginea maritima Hyoscyamus Herb) (Senna/Sinameki) Plant: Rheum palmatum (Ada Soğanı/Scilla, White Plant: Hyoscyamus niger Family: Fabaceae/ (Çin raventi/Chinese Squill) (Kara Banotu/Henbane) Leguminosae (Legume Rhubarb) Family: Liliaceae (Lily Family: Solanaceae family/Baklagiller) Family: Polygonaceae family/ Zambakgiller) (Patlıcangiller) Reagent: Chloral hydrateReagent: Chloral hydrate Reagent: Chloral hydrate Reagent: Chloral hydrate (t°) (t°) (t°) (t°) Microscope Microscope Microscope Microscope Magnification: 10x40 Magnification: 10x40 Magnification: 10x40 Magnification: 10x40 Family: Liliaceae, Plant: Urginea maritima, Drug: Scillae bulbus A B A: Fragments of vessels with spiral and annular thickening. B: Epidermis. C C: Raphides. Family: Solanaceae, Plant: Hyoscyamus niger , Drug: Hyoscyami folium A: Epidermis. A B B: Stomata. C: Multicellular covering trichome. D. Solanaceae type glandular trichome: Stem and head cells one or several. C D E: Twin crystal. E Family: Fabaceae, Plant: Cassia acutifolia , Drug: Sennae folium B C A: Covering trichome with dotted cuticula B: Trachea C. Paracytic stoma in the epidermis. D D: Calcium oxalate crystals. Family: Polygonaceae, Plant: Rheum palmatum , Drug: Rhei rhizoma A: Druses B: Starch granules. A C C: Wood tubes B  Exam Preparation !!! Scillae bulbus Hyoscyami folium  Exam Preparation !!! Sennae folium Rhei rhizoma Flower Examination and Leaf Covering Trichome Examination PHARMACOGNOSY LABORATORY I Lect. Eda AVCI Lect. E. Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI What is Flower (Flos)? It is the reproductive organ of Angiospermae. In a complete flower, from outside to inside, there is a green calyx made of sepals, a colored corolla that forms the petals, an androcheum where the stamens are located and a gyneceum consisting of pistils. Microscopic analysis of powdered drugs reveals the tissues of the flower parts, which are different in appearance for each flower. Floral (Flores) Elements: Papillary epidermis cells of the corolla, pollen, papillary stigma pieces, characteristic endothecium layer pieces of the anthers (glandular and covering trichomes, stoma, etc.).  Leaf (Folium) They arise from the nodiums on the plant stem and lateral branches and have limited growth. It is the main organ in the plant where events such as photosynthesis and transpiration occur. Leaf anatomically consists of epidermis, mesophyll and vascular bundles. The epidermis is the outer part of the upper and lower surface of the leaf, made of a single row of cells, carrying the stomata. The mesophyll consists of palisate and spongy parenchyma containing chloroplasts. Palisate parenchyma consists of long or short cylindrical cells with very narrow intercellular spaces. Spongy parenchyma cells are round or sleeved cells with wide intercellular spaces. ❖ If the palisade parenchyma is only present on the upper side of the leaf and the spongy parenchyma on the lower side, such leaves are called "bifacial leaves" (Belladonnae folium). ❖ If there is palisate parenchyma in both epidermis (upper and lower) and there is sponge parenchyma between the palisate parenchymas, such leaves are called "monofacial leaves" (Sennae folium). Bifacial Leaf (Belladonnae folium) ue: upper epidermis le: lower epidermis pp: palisade parenchyma gt: glandular trichome sp: spongy parenchyma cs: crystal sand  Trichomes of Leaves They are the outward extensions of epidermal cells. There are 2 types of trichomes: Covering and Glandular. Covering trichomes can be single-celled (tooth-shaped trichome, cuticle dotted trichome) or multicelled (T-shaped trichome, candlestick-shaped trichome, star-shaped trichome, elbow-shaped trichome, whip-shaped trichome, gnarled trichome). Covering Trichomes a.Thymi herba (tooth-shaped trichome), b. Thymi herba (elbow-shaped t.), c. Sennae folium (cuticle dotted t.), d. Absinthii herba (T-shaped t.), e. Hyoscyami folium (gnarled t.), f. Digitalis folium (gnarled t.), g. Rosmarini folium (candlestick-shaped t.) h. Farfarae folium (whip-shaped t.) i. Malvae folium (star-shaped t.)  Drugs to be examined Drug: Rosmarini folium Drug: Malvae folium Drug: Sennae folium Drug: Tiliae flos Plant: Rosmarinus Plant: Malva sylvestris Plant: Cassia acutifolia Plant: Tilia cordata officinalis (Ebegümeci/Mallow) (Sinameki /Senna) (Ihlamur/Linden) (Biberiye /Rosemary) Family: Malvaceae Family: Fabaceae Family: Malvaceae Family: Lamiaceae (Ebegümecigiller/ Mallow (Baklagiller/Pea Family (Ebegümecigiller/ Mallow (Ballıbabagiller-Mint family) Reagent: Chloral hydrate family) Family) Reagent: Chloral hydrate (t°) Reagent: Chloral hydrate Reagent: Chloral hydrate (t°) MM: 10x40 (t°) (t°) MM: 10x40 MM: 10x40 MM: 10x40 MM:Microscope Magnification Family: Lamiaceae, Plant: Rosmarinus officinalis Drug: Rosmarini folium a) Candlestick-shaped b) Hypodermis c) Cuticle and epidermis trichome  Family: Malvaceae , Plant: Malva sylvestris Drug: Malvae folium a) Single-celled b) Star shaped trichome c) Druse covering trichome Family: Fabaceae Plant: Cassia acutifolia Drug: Sennae folium a) Single-celled covering b) Covering trichome with dotted cuticle trichome  Family: Malvaceae Plant: Tilia cordata Drug: Tiliae flos a) Curved, single-celled covering b) Triporate (3 pored) pollen trichomes c) Druse  Examination of the Leaf Element (Stomata, Covering and Glandular Trichomes) Pharmacognosy Laboratory I Lect. Reyhan ARICI Lect. E. Ayça ALTINKAYA SAMİM Lect. Eda AVCI Anatomy of The Leaf stoma Epidermis: They are living cells without intercellular space, usually single-row, equal in width and length. Cutin is deposited on the outer part of the epidermal cells and forms the cuticle layer. Cuticle can be smooth, dotted, striped, wavy or rough. The epidermis sometimes bulges inwards and sometimes to both sides. If this swelling is seen in every cell of the lower epidermis, it is called "papilla". The cell layers formed under the epidermis are called "hypoderma". Stoma: It is called the organs that provide gas exchange between the mesophyll and the external environment in the epidermis. While epidermis cells have not chloroplasts, stomatal cells have chloroplasts. Stomata, consisting of two kidney-shaped cells, are on both sides in monofacial leaves and only on the lower epidermis in bifacial leaves. The cells surrounding the stoma are called "guard cells". There are different stoma types according to the shape and number of guard cells.  Stoma Types According to European Pharmacopoeia 1 Anisocytic 3 Anomocytic Diacytic Paracytic (parallel- (unequal-celled) (irregular-celled) (opposite-celled) celled) Type Type Type 2 Type 4 1. Anomocytic (irregular-celled) Type: The stoma is usually surrounded by a variable number of cells that do not differ from the cells of the epidermis. Stomata in plants belonging to the Ranunculaceae family belong to this group. 2. Anisocytic (unequal-celled) Type: There are 3-4 neighbouring cells, one smaller than the others. Plants of the family Solanaceae belong to this group (Belladonnae folium, Stramonii folium, Hyoscyami folium). 3. Diacytic (opposite-celled) Type: There are 2 neighbouring cells at right angles to the stomatal axis. Plants of the Labiatae family belong to this group (Menthae folium). 4. Paracytic (parallel-celled) Type: Two neighbouring cells parallel to the stomatal axis (Cocae folium, Boldo folium, Sennae folium).  Number of Stoma: Average number of stomata in the epidermis per 1 mm² area. Number of Stoma = Number of lower epidermis cells/Number of upper epidermis cells Stoma Index: It is the percentage of the ratio of the number of stomata in the unit area of a leaf to the number of epidermis + stomata in the same unit area. Stoma Index = Number of Stoma x 100 / (Number of Stoma + Number of Epidermis Cells) The number of stoma may vary with leaf age but the stoma index is a constant value for a given species. Trichomes They are the outward extensions of epidermal cells. There are 2 types of trichomes: Covering and Glandular. Covering trichomes can be single-celled (tooth-shaped trichome, cuticle dotted trichome) or multicelled (T- shaped trichome, candlestick-shaped trichome, star- shaped trichome, elbow-shaped trichome, whip-shaped trichome, gnarled trichome). 1. Thymi herba (tooth-shaped trichome), 2. Thymi herba (elbow- shaped t.), 3. Sennae folium (cuticle dotted t.), 4. Absinthii herba (T-shaped t.), 5. Hyoscyami folium (gnarled t.), 6. Digitalis folium (gnarled t.), 7. Rosmarini folium (candlestick-shaped t.) 8. Farfarae folium (whip-shaped t.) 9. Malvae folium (star-shaped t.) Glandular Trichomes: They are trichomes, mostly composed of epidermal cells, which exude their secretions. They consist of stem and head. There are different types of glandular trichomes. 1: Rosmarini folium 2: Menthae folium 3, 4: Digitalis folium 5: Belladonnae folium 6: Hyoscyami folium 7, 8: Absinthii herba 9: Malvae folium 10, 11: Menthae folium 12, 13: Betulae folium 14: Cannabis herba  Types of Glandular Trichomes Labiatae (Lamiaceae)-type glandular trichome : It consists of one stem and eight head cells (10, 11). Solanaceae-type glandular trichome : The stem and head cells may be one or several (5, 6). Compositae-type glandular trichome : Glandular trichomes are two rows wide and several cells high (7, 8). Malvaceae-type glandular trichome : It consists of four cells positioned on a base cell (9). Kidney-type glandular trichome : It can be seen in two different forms; «the stem is unicellular and the head is composed of two cells side by side» or «the head and stem are unicellular» (3, 4).  Drugs to be examined Drug: Menthae folium Drug: Stramonii folium Plant: Mentha piperita Plant: Datura stramonium h (Mint/Nane) (Datura/Boru çiçeği) Family: Lamiaceae (Mint Family: Solanaceae family/Ballıbabagiller) (Nightshade family/Patlıcangiller) Reagent: Chloralhydrate (t°) Reagent: Chloralhydrate (t°) MM: 10x40 MM: 10x40 MM: Microscope Magnification Family: Lamiaceae, Plant: Mentha piperita, Drug: Menthae folium A: Lamiaceae-type glandular trichome B: Diacytic stoma C: Palisade parenchyma A B C Family: Solanaceae, Plant: Datura stramonium , Drug: Stramonii folium A: Solanaceae-type glandular A B trichome B: Anisocytic stoma C: Fragments of vessels with spiral and annular thickening D: Druse C D Microscopic Examination of Fruit and Seed Drugs PHARMACOGNOSY LABORATORY I Lect. Eda AVCI Lect. Elif Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI  What is FRUCTUS? It is the organ that forms with the development of the ovary and carries the seeds. It consists of 3 layers: exocarp, mesocarp and endocarp. The exocarp consists of a single row of cells, resembles an epidermis layer and carries a stoma. The structures of the mesocarp and endocarp layers, which consist of parenchymatic cells, vary depending on each fruit.  What is SEED ? It is the organ formed by the fertilization and maturation of the seed draft (ovule) in the flower, which has the ability to germinate and form the plant. Testa consists of embryo and nutritional tissue. Testa is the coat of the seed. It has an epidermis on its outer surface and several rows of cell layers underneath. The anatomical structure is different in each seed. Nutrient tissue; They are thin-walled parenchymatic tissues that carry starch, fat and protein, and are examined under the names endosperm and perisperm. The embryo is the draft of the plant that will give rise to the new plant. FRUIT SEED  The main elements characterizing powdered drugs in a microscopic analysis are as follows: Folia elements: Stoma-bearing epidermis parts, spongy and palisade parenchyma parts, thin vascular bundles, secretory cells, covering trichomes. Flores elements: Papillary epidermis cells of the corolla, pollen, papillary stigma pieces, characteristic endothecium layer parts of the anthers (secretory and cover hairs, stoma, etc.). Semen elements: Aleurone, starch, fat and the nutritional tissue containing them (endosperm), cotyledon pieces, epidermis of testa, small amounts of thin wood tube pieces. Fructus elements: In addition the seed, parts of the pericarp (exocarp, mesocarp, endocarp), endocarp parts characteristic of each fruit, and thin wood tubes are seen. Radix elements: Abundant parenchymatic pieces, pieces bearing pith branches , if the drug is not peeled, pieces of cork tissue and xylem are seen.. Cortex elements: While all Radix elements are seen, xylems are not found.  Drug: Anisi fructus Plant (Lat.): Pimpinella anisum Plant (Tur.): Anason Plant (Eng.): Anise Family: Apiaceae  MICROSCOPE ANALYSIS Family: Apiaceae, Plant: Pimpinella anisum, Drug: Anisi fructus A. Vascular bundles B. Schizolysigenous oil glands C. Endosperm carrying druse and oil drops D. Epicarp with covering trichome and stoma E. Endocarp and secretory ducts F. Endocarp and secretory ducts G. Testa in top view H. Pollen Reagent: Sartur (t°) Microscope Magnification: 10 x 40  Drug: Lini semen Plant (Lat.): Linum usitatissimum Plant (Tur.): Keten Plant (Eng.): Flax Family: Linaceae  MICROSCOPE ANALYSIS Family: Linaceae, Plant: Linum usitatissimum, Drug: Lini semen A: Pigment layer and endosperm of testa in transverse section B: Pigment layer of the testa and testa parenchyma C: Epidermis in top view (top view; cuticle separates into plates as the epidermis swells) D: Parenchyma cells E: Pigment clusters F: Pigment layer of the testa G: Epidermis cells and numerous fat drops H: Thick-walled cells of the sclerenchyma layer in superficial view Reagent: Sartur (t°) Microscope magnification: 10 x 40  Identification Reactions of Oses and Holosides Lect. Eda AVCI Lect. E. Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI  GLUCIDES OSIDES OSES Oses consist of C, H, O, carry two or more –OH Holosides Heterosides groups and a reducing group, and are found in nature in many different forms and in It contains only It contains one or large numbers. ose molecules in more oses as well as its structure. a non-ose molecule (aglycone). Identification Reactions of Oses 1) Classical Identification Reactions 1.1) Reduction of Metal Salts FEHLING REACTION: Reducing ose+ CuSO4 + Alkali →Cu2O precipitate (red) BARFOED REACTION: Reducing ose + Cu(CH3COO)2 / CH3COOH → Yellow, red precipitate BENEDICT REACTION : Reducing ose + Na2SO4/CuSO4 → Yellow, red precipitate TOLLENS REACTION : Reducing ose + AgNO3 / NH3 → Silver (Ag°) mirror 1.2) Reduction of Aromatic Nitro Compounds PICRIC ACID TEST : Ose solution + picric acid → Aminonitrophenol (red) Identification Reactions of Oses 1.3) Reactions Based on the Formation of Furfural Derivatives Seliwanoff (for ketoses)... furfural + resorcinol → red Anthrone (for pentoses)... furfural + phloroglucinol → red Molisch... furfural + α-naphthol → purple-violet ring Bial (for pentoses)... furfural + orcinol → pink, violet Naphthoresorcinol... furfural + naphthoresorcinol → different colors occur Identification Reactions of Oses 1.4) Osazone Reaction It is the reaction of the carbonyl group of the substances with aromatic hydrazines. The shapes, melting points and solubility of osazone crystals formed as a result of the reaction are different for each osazone. Ose solution + phenyl hydrazine + sodium acetate → OSAZONE Osazone Aldose Ketose 2) Identification By Chromatography: Gas or paper chromatography can be used for this purpose. Identification Reactions of Oses FEHLING: 1 ml of the test solution is put into the test tube. 2 ml of FEHLING A and 2 ml of FEHLING B solution are put on it. With heating, red-colored copper (I) oxide (Cu2O) precipitates in the presence of oses. If the experiment is carried out in a water bath, it is necessary to wait for a long time. MOLISCH: 1-2 drops of 5% solution of a-naphthol in alcohol are added to 1 ml of test solution. While the tube is held horizontally, a violet ring is formed by concentrated sulfuric acid slowly leaking from its edge. SELIWANOFF: 1 ml of the sample solution is acidified and boiled by adding 1 ml of Seliwanoff reagent. After a while, a red color appears in the presence of ketoses, while aldoses react later and a lighter red color appears. Pentoses produce a blue-green color. OSIDES Osides that have at least two oses in their structure are called "holozides". If the number of ose in holozides is less than 10, "Oligoholozide"; If there are more than 10, the term "Polyholoside" is used. POLYHOLOSIDES Polyholosides are products that are partially soluble in water and form precipitate by adding ethanol or Ca, Mg, Ba salts to their aqueous solutions. Pharmaceutically important polyholosides are starch, dextran, gum, mucilage, cellulose, pectin and mucopolyholosides.  EXPERIMENTS 1) Identification Reactions of Oses 2) Identification Reactions of Holosides 1) Identification Reactions of Oses Family: Leguminosae/Fabaceae (Legume Family) Plant: Glycyrrhiza glabra L. (Liquorice) Drug: Liquiritiae radix (Licorice Root) Preparation of Ose Solution: Weigh the drug equal to the tip of 1 spatula. In a mortar, mix thoroughly with 20 ml (measured in graduated cylinder) of hot distilled water and crush. Filtering is done twice through pleated filter paper into a conical flask. Add 1.5 ml of 10% lead acetate solution to the remaining filtrate (until precipitate formation is completed). Thanks to the lead acetate solution, substances such as chlorophyll, flavonoids and tannin in the drug are precipitated and removed. After this process, the mixture is filtered through pleated filter paper. Add 2 drops of disodium hydrogen phosphate and filter again. Disodium hydrogen phosphate precipitates excess lead acetate and removes it from the environment. Finally, the resulting test solution is divided into 3. 1) Identification Reactions of Oses Fehling Test: 1 ml of the test solution is put into the test tube. 2 ml of FEHLING A (CuSO4+H2SO4+H2O) and 2 ml of FEHLING B (Na,K Tartrate+NaOH+H2O) solutions are added onto it. The experiment is kept in the water bath for a long time. If heated in a burner flame, the test period is shortened. At the end of the experiment, red copper (I) oxide (Cu2O) precipitates. Oses without a reducing group (aldehyde, ketone) have reducing properties. They reduce metal salts in alkaline environment. Therefore, red colored Cu2O precipitates from the Fehling reagent. 1) Identification Reactions of Oses Molisch Test: 1 ml of the test solution is put into the test tube. Add 2 drops of 5% solution of α-naphthol in alcohol. Then, holding the tube horizontally, 5-6 drops of concentrated sulfuric acid are slowly leaked from its edge. At the end of the experiment, a violet purple ring is observed between the two layers formed. The essence of this experiment is that furfural, formed from the extracts in an acidic environment, gives colored condensation compounds with phenol or amines. 1) Identification Reactions of Oses Seliwanof Test: 1 ml of the test solution is put into the test tube. Add 3 drops of concentrated sulfuric acid. Then, 1 ml of Seliwanof reagent (resorcinol in aqueous HCl) is added and boiled. After a while, a red color appears in the presence of ketose. Aldoses react later and a lighter red color appears. Pentoses produce a blue-green color. The essence of the experiment is that hydroxymethyl furfural derivatives formed by heating hexoses in an acidic environment give a dark red color with resorcinol. Ketohexoses are more sensitive to this reaction. 2) Identification Reactions of Holosides Family: Poaceae (Poaceae Family) Preparation of Starch Solution: Mix 1 spatula of Plant: Triticum vulgare (Wheat) starch with 3 ml of cold water and add 25 ml of boiling Drug: Tritici amylum (Wheat Starch) water. The resulting mixture is heated in a water bath for 5 minutes (2 minutes on a burner flame). Dark blue color is observed. Identification of Starch with Iodine: 2 drops of iodine solution (lugol) are added to 2 ml of sample solution. Dark blue color is observed. 2) Identification Reactions of Holosides Searching for Reducer Ose in Starch 1) 2 ml Fehling A and 2 ml Fehling B are applied to 2 ml sample solution. The blue color of the solution does not change. 2) 2 ml of diluted sulfuric acid is added to 2 ml of sample solution. It is heated in a water bath for 30 minutes and left to hydrolyze. Then, take the tube and add 2 ml of alcali. Add 2 ml Fehling A and 2 ml Fehling B reagents. The tube is heated in a water bath. A red precipitate forms.  Identification Reactions of Flavonoids and Anthocyanidins Pharmacognosy Laboratory I Lect. Reyhan ARICI Lect. Eda AVCI Lect. Elif Ayça ALTINKAYA SAMİM  Flavonoids Flavonoids are chromone derivatives. Chromone is benzo-γ-pyrone and has not been found free in plants so far. The colours of these substances, which make up many coloured compounds in plants, become darker the more hydroxyl groups they contain and the higher the pH of the medium. Chromon (benzo-γ-pyrone) 2-phenyl chromon=FLAVONE 3-hydroxy flavone=FLAVONOL FLAVANONE FLAVAN DIHYDROFLAVONOL ❖ Flavonosides are generally soluble in water and ethanol, insoluble in ether, chloroform and benzene. Their aglycones are soluble in ether but insoluble in water. ❖ Most of these substances are crystalline. ❖ They are light or dark yellow depending on their ring structure. ❖ Flavonoids give a dark yellow colour in alkaline media (with diluted NaOH or KOH solutions). If acidified, they lighten in colour and precipitate. ❖ A widely used reaction for the identification of flavonoids is the "cyanidin reaction". In this reaction, the aqueous-alcoholic solution of flavonoids is treated with magnesium powder in hydrochloric acid, in other words reduced with natural hydrogen. As a result of the reaction, an orange colour is obtained with flavones, cherry red with flavonols and violet-red with flavanones. Chalcones and isoflavones are inert for this reaction. This reaction is also suitable for colourimetric determinations. ❖ Flavonoids have vitamin P activity and this activity is due to two -OH groups at the 3', 4' position.  Identification Reactions of Flavonoids Family: Malvaceae (Mallow family) Plant: Tilia cordata (Linden) Drug: Tiliae flos (Linden flower) The sample is powdered and transferred into an erlenmeyer flask. Add 20 ml of 50% ethanol, cover with cotton and heat in a water bath. It is then filtered. Add 1-2 drops of 5% aqueous solution of iron (III) chloride to 3 ml of the extract and a greenish blue black colour is formed.  Identification Reactions of Flavonoids Adding 1-2 drops of 10% NaOH solution to 3 ml of the extract produces a dark yellow colour. Adding 1-2 drops of lead acetate and 2 drops of 10% NaOH solution to 3 ml of the extract produces a yellow-orange colour. Lead acetate+NaOH NaOH Identification Reactions of Flavonoids Cyanidin (Shinoda-Shibata) Reaction Boil 1 spatula tip sample with 5 ml 50% ethanol for 5 min and filter. Add 0.5 ml of concentrated HCl and 1 spatula tip of magnesium or zinc powder to the filtrate. Hydrogen gas is observed. At the appropriate concentration, orange colour is observed in flavone, red colour in flavonol and purple colour in flavanone.  Anthocyanidins They are pigments which are very common in plants and give colour to flowers, leaves and fruits. Anthocyanidins contain benzopyrylium instead of benzopyrone. Benzopyrylium has no ketone at carbon 4. Anthocyanidins usually form anthocyanins by bonding with ose from the -OH groups at the 3rd and 5th positions. When it was observed that some colourless substances besides the red pigments in plants were transformed into coloured compounds under the influence of HCl and it was understood that these substances were compounds close to anthocyanidins, such compounds were named proanthocyanidins. Anthocyanidol ring  Identification Reactions of Anthocyanidins Family: Malvaceae (Mallow family) Plant: Hibiscus rosa-sinensis (Hibiscus) Drug: Hibisci flos (Hibiscus flower) 2 spatules of sample are extracted with 20 ml of 50% ethanol in a water bath for 10 min. The extract is filtered and filtrate;  Identification Reactions of Anthocyanidins 1. To observe the colour change in acidic and basic media; ✓ Add dilute HCl to 1 ml of the filtrate, the purple colour starts to lighten. ✓ To 1 ml of the filtrate, add 10% NaOH solution, greenish yellow colour is obtained.  Identification Reactions of Anthocyanidins 2. In order to examine the solubility of (aglycone) in the glycoside state and after hydrolysis; ✓ To 1 ml of the filtrate, add 1 ml of amyl alcohol and shake. The amyl alcohol layer remains colourless. ✓ Boil 1 ml of the filtrate with 1 ml of concentrated HCl. After cooling, shake with 1 ml of amyl alcohol. The amyl alcohol layer is coloured. Initially anthocyanosides are heterosides and insoluble in amyl alcohol. Then they are hydrolysed with diluted H2SO4 and heterosides are broken down and anthocyanosides are released. Since anthocyans are soluble in amyl alcohol, they colour this layer.  Identification Reactions of Anthocyanidins 3. Add 1 ml of lead acetate solution to the filtrate. A green precipitate is formed with lead acetate (phenolic substances precipitate with heavy metals). Experiment results respectively;  Anthracene and Coumarin Derivative Heterosides Pharmacognosy Laboratory I Lect. Elif Ayça ALTINKAYA SAMİM Lect. Reyhan ARICI Lect. Eda AVCI  ANTHRACENE DERIVED HETEROSIDES Some drugs of various families contain heterosides whose aglycone is an anthracene derivative. Anthracene derivative compounds are found in 3 types in the plant: 1. Oxantron 2. Antron ANTHRAQUINONE RING 3. Anthraquinone The enol form of oxanthrone is called anthrahydroquinone; the enol form of anthrone is called anthranol. The most stable of these three types are anthraquinones. Free anthraquinones form a dark red colour with alkali hydroxides. (BORNTRÄGER Reaction)  OH OH 8 1 7 2 Most of them are cathartic drugs (Rhei rhizoma, Aloe, 6 3 Sennae folium, Rhamni purshianae cortex vs.). Some drugs 5 4 are used as dyes (Coccionella, Rubiae radix vs.). 1,8-dihydroxyanthraquinone 1,8- 1,2- dihydroxyanthraquinone dihydroxyanthraquinone derivative derivative OH 8 1 7 2 3 6 5 4 PURGATIVE COLOURANT 1,2-dihydroxyanthraquinone Coccus cacti - Coccionella  Drugs to be used in today's Anthracene Derivative Heterosite Experiments Family: Polygonaceae Family: Fabaceae (Legume family) Plant: Rheum sp. (Rhubarb) Plant: Cassia angustifolia/acutifolia (Senna) Drug: Rhei rhizoma (Rhizome of rhubarb) Drug: Sennae folium (Senna leaf)  1. MICROSCUBLIMATION EXPERIMENT Rhei rhizoma is powdered and placed on the slide as much as the tip of a spatula. Cover with another slide, leaving a distance of 1-2 mm in between. Start the heating process with a small flame. Sublimate is accumulated on the bottom surface of the upper slide. When 1 drop of sodium hydroxide solution is added to the sublimate, a red colour is formed.  2. BORNTRÄGER REACTION Shake 1 full spatula of Sennae folium in a tube with 2 ml toluene and decant*. When 1 ml of 10% ammonia solution is added to the filtrate, the aqueous part turns pink. Boil the remaining drug in the tube with 5 ml diluted sulphuric acid for 2 min. When hot, filter and shake with toluene. When the toluene part is separated and 1 ml of 10% ammonia solution is added, the aqueous part turns pink-red colour. *Decant: Transferring the liquid on top to another tube when a precipitate forms at the bottom of the liquid.  COUMARIN DERIVATIVE HETEROSIDES Coumarin a- pyrone γ- pyrone Coumarin heterosides are heterosides whose aglycone is in the coumarin structure. Coumarin is the lactone of orthohydroxy cinnamic acid, in other words benzo a - pyrone. Hydroxylated coumarins show blue or blue-green fluorescence under ultraviolet light. By taking advantage of this feature, it is possible to identify coumarins in chromatography and quantification can also be made based on this. Some drugs containing coumarin: Rutaceae species, Apiaceae species  Benzo a-pyrone -Coumaric acid Coumarinic acid Phenolic -OH gives blue-green fluorescence at UV 366 nm  Experimental Procedure Familya: Rutaceae Bitki (Lat.): Ruta graveolens Plant (Eng): Rue Bitki (Tur): Sedef otu Drog: Rutae herba  Preparation of Test Solution: Take 1 spatule of drug. Extract it with 10 ml 50% ethanol in a water bath for 10 minutes, filter it and take the filtrate into a capsule. After evaporation in a water bath, 3 drops of 1 N sodium hydroxide is added. Meanwhile, the coumarin ring turns into coumarinic acid and yellow color occurs. Coumarinic acid gives bluish-green fluorescence at UV 366 nm due to the phenolic hydroxyl.  Identification of Saponosides and Tannins Farmacognosy Laboratory I Lect. Reyhan ARICI Lect. E. Ayça ALTINKAYA SAMİM Lect. Eda AVCI  Saponosides Aqueous solutions of some heterosides found in plants form permanent foam when shaken. This type of heteroside is called saponoside (saponin). Saponosides are amorphous, odourless, colourless, optically active substances with an irritating taste. Usually soluble in boiling methanol and ethanol, precipitates on cooling. Most saponosides have haemolysis ability. This effect is not seen when taken orally. Because saponosides are not absorbed in the intestine. The aglycone released by hydrolysis of saponosides is called sapogenol. Sapogenols are polycyclic substances. There are two types of saponosides according to their sapogenols : 1) Steroidal saponosides (Neutral Saponins) (C27) 2) Triterpenoid saponosides (Acidic Saponins) (C30) Spirostane ring (C27) β-amyrenol (C30)  To identify saponosides, the drug is extracted with water, methanol or ethanol. A filter paper soaked in this extract is dried and then brought into contact with the blood-containing gelatine jelly. If a transparent zone forms around the filter paper, haemolysing saponoside is present (Some saponosides are not capable of haemolysis). Haemolysis Index: It is the dilution degree of the amount of saponoside sufficient to completely haemolise erythrocytes in a total solution of 2 ml. Foam Index: The dilution degree (inverse of concentration) of 10 ml saponin solution which forms a permanent foam at 1 cm height of in a tube of 16 mm in diameter after being shaken for 15 seconds and left for 15 min. Some Drugs Containing Saponosides Saponariae albae radix Liquiritiae radix Trigonella fenugraeci semen Identification Reactions for Saponosides Steroidal Saponins Triterpenoid Saponins Salkowski’s Test Brieskron-Briner’s Test Lieberman-Burchard’s Rosenthaler’s Test Test Anisaldehyde’s Test  Steroidal Saponins Salkowski’s Test: Add 1-2 drops of concentrated sulfuric acid to 1 ml of chloroformed extract and layer formation is observed. First, yellow color occurs. Then the colour gradually changes to red. Liebermann Burchard’s Test: 1 ml acetic acid anhydride and 1 drop of concentrated sulfuric acid are added to 2 ml of extract. Either the green color occurs directly, or the color turns green after the red and blue tones. Triterpenoid Saponins Brieskron-Briner’s Test: A few drops of chlorosulfonic acid are added to 2 ml of extract. The red color that occurs in 5-10 minutes is characteristic for triterpenes. Rosenthaler’s Test: 1% solution of vanillin in hydrochloric acid is added to 1 ml of extract. Red color occurs. Anisaldehyde’s Test: 2 ml of the extract is taken into a capsule and evaporated in a water bath. Freshly prepared anisaldehyde-sulfuric acid reagent is dropped onto it. First a pink, then a purple colour appears.  Experiments of Saponins Family: Caryophyllaceae (Karanfilgiller-Carnation family) Plant: Gypsophila arrostii (Çöven-Soapwort) Drug: Saponariae albae radix (Çöven kökü-Root of Soapwort) Preparation of Test Solution: Extract 1 spatula of the drug with 10 ml of 50% ethanol. Filter through pleated filter paper. Add 2 ml of 10% HCl to the filtrate and heat on a water bath for 10 min. After cooling, extract with 10 ml chloroform in a separatory funnel. The chloroformed extract remains at the bottom due to its higher concentration. The chloroform extract is taken and diagnostic tests are carried out. The reason for continuing the experiment with the chloroform extract is that it is the aglycone that will provide a positive result in the tests. The aglycone part is nonpolar so it is attracted to the nonpolar chloroform phase. For steroidal saponins, Salkowski’s Test: Take 1 ml of extract into a test tube. Add 2 drops of concentrated sulfuric acid and stratify. First yellow and then red colour is formed. For triterpenoid saponins, Brieskron-Briner’s Test: To 2 ml of the extract add a few drops of chlorosulfonic acid. The red colour is formed in 5-10 min is characteristic for triterpenoids. Salkowski Brieskron-Briner Foaming Test 0.03 g of the powdered drug is extracted with 100 ml of water and boiled for 30 min. It is then filtered. Put into a volumetric flask (balloon). It is completed to 100 ml. A series of 10 tubes 16 cm in length and 16 mm in diameter is prepared. Add 1, 2, 3,......10 ml of the decoction to the tubes respectively and fill each tube with 10 ml of distilled water. The tubes are closed with thumb, shaken horizontally for 15 seconds and waited for 15 minutes. At the end of the time, the foam heights in the tubes are measured. The degree of the dilution of the tube where the foam is 1 cm at height is recorded. If the foam height in all tubes is more than 1 cm, more diluted decoctions should be prepared. Calculation of Foam Index F.I.=10/a a: The amount of the drug in the tube where the foam is 1 cm at height.  Tannins Tannins are nitrogen-free polyphenolic compounds existing in plants. They dissolve in water, ethanol and acetone easily; in lipophilic solvents such as ether and chloroform, poorly. They have bitter taste and astringent property. They bind the skin and solidify it. Tannins are present in plants as complex molecules named tannoid. When they united with oses, is refered to tannozid. Tannins are found in the cell vacuole and often in combination with some other substances such as alkaloids, proteins and oses. It is possible to examine tannins in two groups: 1-Hydrolysable Tannins 2-Condensed (Catechic) Tannins 1-Hydrolysable Tannins: They are esters of acid phenols with ose. They were formerly also called "pyrogallic tannins" because of the formation of pyrogallol in dry distillation. Hydrolysable tannins can be analysed in two classes according to the type of acid phenol in their structure: a) Gallic Tannins: (gallic acid+ose) b) Ellagic Tannins: (ellagic acid+ose) 2-Condensed (Catechic) Tannins: Non-hydrolysed condensed tannins are also called catechic tannins. These substances are not hydrolysed by acids or tannase (enzyme). When heated with strong acids or oxidation agents, they form red or brown coloured compounds which are called flobafen and are insoluble in most solvents. Condensed tannins give pyrocatechol by dry distillation.  Some Drugs Containing Saponins Hamamelidis Rosae flos cortex Rhei rhizoma Quercus cortex Theae folium Salicis cortex  Identification Reactions for Tannins Aqueous solution of tannins can be precipitated with heavy metal salts (Cu, Fe, Hg, Pb, Zn). They precipitate with reagents such as barite water, lime water, ammonium molybdate, sodium tungstate, gelatine solution. Most alkaloids also precipitate tannins. Tannin + % 5 FeCl3 ➔ brown-green color (catechic tannins), blue-black color (hydrolysable tannins) Tannin + saline gelatine solution ➔ cream coloured precipitate Catechic tannin + brominated water ➔ yellow-cream colour Catechic tannin + Stiasny reagent (Formalin + HCl) ➔ precipitate in pieces  Experiments of Tannins Family: Theaceae (Çaygiller-Tea family) Plant: Camellia sinensis (Çay-Tea) Drug: Theae folium (Çay yaprağı-Tea leaf) Preparation of Test Solution: Put 1 spatula of tea into a erlenmeyer, add 10 ml of water, boil on the water bath and filter. Identification reactions are performed with the prepared decoction. ❖ Add 1 drop of 5% iron (III) chloride aqueous solution to 1 ml of decoction. Green colour is observed for condensed tannins. Blue-black colour is observed for hydrolyzable tannins. ❖ 1 ml of 1% saline gelatin solution is added to 1 ml of decoction. Cream coloured precipitate is formed in the presence of tannin. It is sufficient even if it appears blurry. ❖ Add 1 ml of stiasny reagent to 1 ml of decoction. Heat in a water bath. In the presence of catechic tannins a fragmentary precipitate is formed. Cardioactive Heterosides Pharmacognosy Laboratory I Lect. Reyhan ARICI Lect. E. Ayça ALTINKAYA SAMİM Lect. Eda AVCI  GLUCIDES OSES OSIDES Holosides Heterosides It contains only It contains one or ose (glycone) more ose as well as molecules in its a non-ose molecule structure. (aglycone).  Cardioactive Heterosides Cardenolide (23 C) Bufadienolide (24 C) The aglycones of the cardioactive heterosides have a steroid structure and consist of cyclopentanoperhydrophenanthrene ring. Those with 23 carbons are called "cardenolide" and those with 24 carbons are called " bufadienolide".  Cardioactive Heterosides Depending on the plane formed by the A and B rings in the cyclopentanoperhydrophenanthrene structure, the substituents at the 5th and 10th C atoms can be cis or trans. Of these two configurational isomers, the cis ones are cardioactive. Cis isomer digitoxygenol → Cardioactive Trans isomer uzarigenol -> Antidiarrheic The lactone ring is also important for activity. If the ring is saturated or opened, cardioactivity disappears. Identification Reactions of Cardioactive Heterosides LEGAL :The 5-membered unsaturated lactone ring has reducing character. Due to this feature, it reduces ammoniacal silver nitrate and gives a red color with sodium nitroprussium in the presence of sodium hydroxide in a pyridine environment. Those containing a 6-membered lactone ring do not give color in the Legal experiment. LIEBERMANN: They give positive results for those whose aglycones are common to sterols. In chloroform and an acetic acid anhydrous environment give first violet, then blue, and then green color with sulfuric acid. It's not specific. Identification Reactions of Cardioactive Heterosides KELLER-KILIANI: The substance is dissolved in glacial acetic acid and after adding FeCl3 it is layered with concentrated sulfuric acid. A brown-purple ring forms on the surface between two liquid layers. This is characteristic for cardioactive heterosides (2-dezoxy ose). BALJET: Baljet reagent and 6% sodium hydroxide solution are added. Orange color occurs. Experimental Procedure Drug: Scillae bulbus (Sea onion Plant: Urginea maritima (Sea squill) Family: Liliaceae (Lily family) Extract Preparation: Boil 1 spatula full of the drug with 20 ml 50% ethanol in a boiling water bath for 5 minutes and then filter. 5 ml of 10% lead acetate solution is added to the filtrate. The precipitate formed is separated by filtration. The filtrate is shaken with 5 ml of dichloromethane in the separatory funnel. The lower phase (dichloromethane) is taken into the erlenmeyer flask. The remaining phase in the separatory funnel is shaken again with 5 ml of dichloromethane. The lower phase is taken again. And these two phases (dichloromethane phases) are combined and identification reactions are carried out.  Identification Reactions 1) Keller Kliani Reaction: 5 ml of dichloromethane extract is evaporated in the capsule. Add 1 ml of 3.5% iron (III) chloride solution in glacial acetic acid. The resulting solution is carefully drip from the edge of the tube into 2 ml of concentrated sulfuric acid solution in the test tube (all the solution in the porcelain capsule is added and waited for 10-15 minutes to observe the color change). A brown-purple ring forms between the two layers. 2) Baljet Test: 5 ml of dichloromethane extract is evaporated in the capsule. Then, it is dissolved in 1 ml of 50% ethanol and 1 ml of Baljet reagent and 2 drops of 6% sodium hydroxide solution are added. Orange color occurs. 2) Baljet Test: 5 ml of dichloromethane extract is evaporated in the capsule. Then, it is dissolved in 1 ml of 50% ethanol and 1 ml of Baljet reagent and 2 drops of 6% sodium hydroxide solution are added. Orange color occurs.