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LABORATORY MANUAL OF PHARMACOGNOSY

Experiment-1
Aim-To study the macroscopic characters of datura References- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Edition 41, Reprint 2008, Nirali Parkashan, Page no. 501-502 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 109-111 THEORY Synonym- Datura herb Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn. And other species of Datura belonging to Family Solanaceae MACROSCOPIC CHARACTERS:

Leaf
Color-Lower surface is slightly pale in color Upper surface is darker than lower surface. Size- the entire leaf has a length of 6-13 cm. and width of 8-13 cm. Shape- ovate and petiolate

Lamina- Glabrous and usually has 3 to 4 coarse teeth Apex-acute Base- unequal Texture- smooth and thin Midrib- Prominent

Seeds
Color-Brown Shape-Triangular Flowers- Reddish purple on outside and whitish on inner side Shape- Funnel Shaped

Fruits
Color- Green Shape-Oval with spines Size- Fruit has length of 4.5 cm and width of 3.5 cm Odour- Disagreeable and characteristic Taste- unpleasant bitter

Chemical constituentsDatura leaf mainly contains tropane alkaloids (upto 0.5%) of which hyoscine is main alkaloid. L-hyoscyamine and atropine are present in less quantity. Hyoscine is an ester of tropic acid ad scopine. Chemical tests: 1. Vitali-morin test-It is specific test for tropane alkaloids. Powdered sample is moistened with nitric acid followed by evaporation to dryness and addition of methanolic potassium hydroxide solution to the residue, a violet color is produced. 2. On addition of silver nitrate solution to the solution of hyosine hydrobromide, Yellowish white ppt. formed which is insoluble in nitric acid but soluble in dilute ammonia Uses1. Datura herb and its main alkaloid hyoscine are parasympatholytic with anticholinergic and central nervous system depressant effects. 2. It is used in cerebral excitement. 3. Along with morphine, it is used as preoperative medication. 4. It is used in treatment of ashma and cough

5. Hyoscine hydrobromide is used in motion sickness, gastric or duodenal ulcers Cautions:Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-2
Aim-To study the macroscopic characters of withania Reference- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2008, Nirali Parkashan, Page no. 13.79-13.81. 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 187-189 Theory Withania Synonym- Asgandh, Winter cherry, withania root Bio. Source:- It consists of dried roots and stem bases of Withania somnifera Dunal, belonging to family Solanaceae MACROSCOPIC CHARACTERS: Color- outer surface is Buff to grey-yellow with longitudinal wrinkles

Odour- Fresh roots have smell similar to urine of horse Taste- Bitter and acrid Shape- Straight, unbranched, conical and bears a crown. Fracture- smooth and powdery

Chemical constituentsMain constituents are alkaloids and steroidal lactones. is the main constituent. other Among alkaloids the various

alkaloids, withanine

are somnine,

somniferinine, withananine, pseudo-ithanine, tropine, pseudotropine, cuscohygrine, anferine andanhydrine. Two acyl steryl glucosides, sitoindoside VII and sitoindoside VIII, have been isolated from root. The leaves contain steroidal lactones, which are commonly called withanolides. The withanolides have C28 steroidal nucleus with C9 side chain and a six-membered lactone ring. Uses1. Sedative and Hypnotic 2. adaptogenic 3. chronic fatigue

4. dehydration 5. bone weakness 6. neurodegenerative disorders Cautions:Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-3
Aim-To study the macroscopic characters of Vinca Reference- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007, Edition 39th , Nirali Parkashan, Page no. 484-487. 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 184-186

Theory:
VINCA Synonym- Periwrinkle

Bio. Source- It consists of dried whole plant of Catharanthus roseus Don belonging to family Apocyanaceae MACROSCOPIC CHARACTERS: Leaves Color- Green Shape- Simple,petiolate, ovate or oblong, unicostate, reticulate, entire, brittle and oppositely arranged Apex- Acute and mucronate Lamina- glabrous somewhat shiny Margin- Entire with acute base Flowers Color- Violet, pink, white or carmine red in color Shape- Flowers are bractate, pedicillate, complete, hermaphrodite, normally 2-3 cm in cymarose axillary clusters. Fruits are follicles with several black seeds Odour- characteristics Taste-Bitter

Chemical constituentsIt mainly contains indole alkaloids (0.1-1.5%) of which vincristine and vinblastine are medicinally important. Vinblastine contains indole alkaloid part called catharanthine and dihydroindole alkaloid part called vindoline.Other alkaloids present are ajmalicine, serpentine and tetrahydroalstonine. Uses1. Vincristine and vinblastine are used in the treatment of cancer. 2. Vincristine sulphate is an antineoplastic agent which may act by arresting mitosis at the metaphase. 3. It si used in the treatment of Hodgkin disease and choriocarcinoma. 4. It also possess hypotensive and antidiabetic effects Cautions:- Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-4
Aim-To study the macroscopic characters of Rauwolfia Reference- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007, Edition 39th , Nirali Parkashan, Page no. 480-484.

2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 160-162 Theory Synonym- Rauwolfia root, serpentine root, chootachand, sarpagandha Bio. Source- it consists of dried roots and rhizomes of Rauwolfia serpentine Benth, belonging to Family Apocyanaceae MACROSCOPIC CHARACTERSColor- outer surface- dull greyish brown with faint longitudinal ridges. Internal surface- pale yellow Odour-Odourless Taste- bitter Size- About 8-15 cm long, 0.5-1.0 cm thick Shape- Roots are subcylindrical, slightly tapering, somewhat tortuous, rarely branched Fracture- Short and irregular Chemical constituentsRauwolfia contains atleast 30 indole alkaloids (0.7-2.4%).madicinally important indole alkaloids are reserpine, rescinnamine, ajmalicine and serpentine.

Use1. Antihypertensive 2. Tranquillising agent and used in certain neuro-psychiatric disorders Cautions:Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-5
Aim-To study the macroscopic characters of Nuxvomica.

Reference- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007, Edition 39th , Nirali Parkashan, Page no. 476-479. 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 151-153 Theory NUXVOMICA Synonyms- Kuchla, karaskara, crow fig, semen strychni Bio. Source- It consists of dried ripe seeds of Strychnos nuxvomica Linn. Belonging to family loganiaceae MACROSCOPIC CHARACTERS: Color-outer surface is grey to greenish grey Odour-None Taste- Intensly bitter Size- 10-30 mm in diameter and 406 mm in thickness Shape- Disc shaped somewhat flat or irregularly bent and concavo-convex. Margin- rounded or acute

Surface- silky due to presence o dense, closely oppressed radiating, outwardly directed trichomes Hilum is raised in the centre of flat surface. Chemical constituentsNuxvomica seed contains 1.5-5% of bitter indole alkaloids. Chief constituents are Strychnine and brucine while vomicine, - Colutrine, pseudostrychnine and strychnicine are also present. Seeds contain 3% of fat. Nuxvomica also contain a glycoside i.e. loganin, chlorogenic acid and fixed oil Uses1. Bitter stomachic and tonic 2. CNS stimulant 3. Respiratory stimulant. 4. It is used in CNS depressants poisoning such as Barbiturate poisoning Cautions:Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-6
Aim-To study the transverse section and powder microscopy of Rauwolfia References- 1.Kokate C.K., Practical Pharmacognosy, Edition 11th, Vallabh Parkashan, Page no. 76-78. 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 151-153 Requirements- sample (rauwolfia root entire & powder), microscope, Glass slide, Coverslip, glycerine (50%), phloroglucinol, HCl

Theory
Synonym- Rauwolfia root, serpentina root, choota chand, sarpagandha Bio. Source- It consists of dried roots and rhizomes of Rauwolfia serpentina Benth, belonging to Family Apocyanaceae MICROSCOPIC CHARACTERSTransverse section of root 1 CORK- stratified with 1-7 laters of small cells, 1-3 layers of larger cells, suberised, larger cells lignified. 2. PHELLODERM- 10-12 rows of cellulosic parenchyma

3. Phloem- sieve tubes and parenchyma alternating with medullary rays 4. Medullary rays2-4 cells wide 5. Xylem- tatrach with small vessels, parenchyma, fibers,large celled medullary ras 6. Parenchyma of xylem and phloem, both lignified containing prisms and conglomerate crystals of calcium oxalate and starch granules 7. Granular section staining yellow with iodine in phloem parenchyma Transverse section of rhizome showsIn addition to the above structure, cortex, pericycle with unlignified pericyclic fibres, a small pith and perimedullary phloem in the periphery of pith, stone cells absent POWDER MICROSCOPY 1 The powder is pale brownish-yellow in color with bitter taste. 2. Microscopic examination shows abundant starch grains, simple and compound (2-4 components), spherical, with a distinct hilum in the form of split. 3. Cork-stratified iso diametric cells sometimes lignified. Polygonal in surface view. 4. parenchyma- pitted, lignified 5. vessels- lignified, fairly narrow with bordered pits 6. Fibers are lignified, slightly pitted, found with or without attached vessels

Cautions:Do not taste the crude drug because it might be harmful to the human body in crude form.

Experiment-7
Aim-To study the transverse section of Datura and Vinca References - 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Edition 41, Reprint 2008, Nirali Parkashan, Page no. 501-502 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 109-111 Requirements- sample (Datura leaf and catharanthus leaf), microscope, Glass slide, Coverslip, glycerine (50%) THEORY Synonym- Datura herb Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn. And other species of Datura belonging to Family Solanaceae MICROSCOPIC CHARACTERS: Study of transverse section1. Datura leaf is dorsiventral i.e. having distinct upper and lower surfaces. 2. Stomata are of anisocytic type on both surfaces. 3. Epidermal cells are polygonal with thin cuticle, anticlinical walls, slightly straight on the upper surface, wavy on the lower surface.

4. A single layer of elongated palisade cells is present below upper epidermis. 5. Next to palisade layer is a single row of rectangular cells containing rosette calcium oxalate crystals and remainder of the mesophyll consists of spongy parenchyma. 6. Leaf bears both covering and glandular trichomes, covering trichomes, uniseriate, multicellular and conical, usually less than 35 in diameter at the base, glandular trichomes with unicellular stalk and uni- and multi-cellular head. 7. Midrib region shows collenchymatous cells on both surfaces. 8. Xylem bundles are arranged in an arc. 9. The space between collenchyma and vascular bundle is occupied by 6-8 rows of cortical parenchymatous cells some of which are filled with calcium oxalate

THEORY:
VINCA Synonym- Periwrinkle Bio. Source- It consists of dried whole plant of Catharanthus roseus Don belonging to family Apocyanaceae MICROSCOPIC CHARACTERS: Study of transverse section1. Vinca leaf is dorsiventral. 2. Stomata are of anomocytic type on both surfaces. 3. Epidermal cells are polygonal with thin cuticle; anticlinal walls are straight on the upper surface and wavy on the lower surface.

4. A single layer of elongated palisade cells is present below upper epidermis. 5. Mesophyll consists of 5-7 layers of spongy parenchymatous cells with intercellular spaces. 6. the midrib region shows collenchyma on both surfaces blow epidermis 7. A zone of vascular bundles occupies the central region.

Experiment-8
Aim-To study the transverse section and powder microscopy of Withania References- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2008, Nirali Parkashan, Page no. 13.79-13.81. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 187-189 THEORY Synonym- Asgandh, Winter cherry, withania root Bio. Source: - It consists of dried roots and stem bases of Withania somnifera Dunal, belonging to family Solanaceae Requirements- sample (Withania root entire & powder), microscope, Glass slide, Coverslip, glycerine (50%), phloroglucinol, HCl MICROSCOPIC CHARACTERS:

Study of transverse section (T.S.)1. T.S. of root shows thin cork, composed of several layers of uniformly arranged, thin-walled cells 2. Cork surrounds a broad cortex, composed of thin-walled, polygonal, irregular parenchymatous cells containing starch grains. 3. Below cortex is a large wood, which consists of vessels, tracheids and fibres transversed longitudinally by medullary rays. 4. Medullary rays are single celled and straight. 5. Longitudinal section shows vessels and tracheids with pitted thickening Powder microscopy1. The powder is greyish in colour with characteristic odour and bitter and acrid taste. 2. Microscopic examination shows abundant vessels and tracheids with pitted thickening. 3. Starch grains, nearly all simple, spherical-ovoid with distinct central hilum are found scattered or parenchymatous cells of cortex. 4. Cork cells are thin walled and polygonal in surface view.

5. Fibres are narrow, elongated with tapered ends.

Experiment-9
Aim-To study the transverse section and powder microscopy of Nuxvomica References- 1. Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007, Edition 39th, Nirali Parkashan, Page no. 476-479. 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 151-153 3. Kokate C.K., Practical Pharmacognosy, Reprint 2007, Edition 4th, Vallabh Parkashan, Page no. 66-67. Requirements- sample (Nux-vomica seed & powder), microscope, Glass slide, Coverslip, glycerine (50%), phloroglucinol, HCl

Theory
NUXVOMICA Synonyms- Kuchla, karaskara, crow fig, semen strychni Bio. Source- It consists of dried ripe seeds of Strychnos nuxvomica Linn. Belonging to family loganiaceae MACROSCOPIC CHARACTERS: Study of transverse section1. Epidermis- large, thick walled lignified cells with slit like pits. 2. Epidermal trichomes- thick walled, bent, twisted, and lignified with about 10 longitudinal ribs on the limb. 3. Inner layer of epidermis with collapsed cells. 4. Endosperm-thick walled cellulosic parenchyma of isodiametric cells. 5. embryo and endosperm both contain aleurone grains and fixed oil globules 6. Plasmodesma- communication of the protoplast of endosperm cells through the cell walls by means of very fine protoplasmic threads. Powder Microscopy1. The powder is yellowish-grey to greenish-grey in color.

2. Microscopical examination shows epidermis of testa with attached trichomes. 3. Trichomes are narrow lignified rods running longitudinally. 4. Endosperm cells from central region are large and very thick walled while those from outer part, small and relatively thin walled. 5. A few fragments of endosperm show faint plasmodesma

Experiment-10
Aim-To perform the chemical test of Nuxvomica and datura References- 1. Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007, Edition 39th, Nirali Parkashan, Page no. 476-479.&501-502 2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 151-153 3. Kokate C.K., Practical Pharmacognosy, Reprint 2007, Edition 4th, Vallabh Parkashan, Page no. 66-67. Requirements- Sample (Nux-vomica powder), Test tubes, test tube stands and holders, watch glass, UV lamp, Bunsen burner , Conc. H2SO4, Conc. HNO3, Ammonium Vanadate, methanolic Potassium KOH, Acetone, K2Cr2O7,

Theory
NUXVOMICA Synonyms- Kuchla, karaskara, crow fig, semen strychni Bio. Source- It consists of dried ripe seeds of Strychnos nuxvomica Linn. Belonging to family loganiaceae THEORY Synonym- Datura herb Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn. And other species of Datura belonging to Family Solanaceae Chemical tests: The thin section of nuxvomica seed is defatted and performs the following tests1. A section of endosperm is strained violet by a drop of ammonium vanadate in sulphuric acid due to presence of strychnine. 2. A section of endosperm is strained crimson or orange-red by a drop of nitric acid due to presence of brucine. 3. A thick section of drug is treated with iodine solution and concentrated sulphuric acid. Endosperm cell walls stain blue (due to hemicellulose) THEORY

Synonym- Datura herb Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn. And other species of Datura belonging to Family Solanaceae Chemical tests: 1. Vitali-morin test-It is specific test for tropane alkaloids. Powdered sample is moistened with nitric acid followed by evaporation to dryness and addition of methanolic potassium hydroxide solution to the residue, a violet color is produced. 2. On addition of silver nitrate solution to the solution of hyoscine hydrobromide, Yellowish white ppt. formed which is insoluble in nitric acid but soluble in dilute ammonia.

Experiment-11
Aim-To perform the chemical test of Rauwolfia and Cinchona References- 1.Kokate C.K., Practical Pharmacognosy, Edition 11th, Vallabh Parkashan, Page no. 76-78.

2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas Page no. 97 &160-161 3. Kokate C.K., Practical Pharmacognosy, Reprint 2007, Edition 4th, Vallabh Parkashan, Page no. 36-37. Requirements- sample (rauwolfia powder), Test tubes, test tube stands and holders, watch glass, UV lamp, Bunsen burner Vanillin, acetic acid, Bromine water, ammonia, Conc. H2SO4

Theory RAUWOLFIA
Synonym- Rauwolfia root, serpentina root, chotta chand, sarpagandha Bio. Source- it consists of dried roots and rhizomes of Rauwolfia serpentina Benth, belonging to Family Apocyanaceae Chemical tests:1. Powdered drug is treated with sulphuric acid and

p-dimethylaminobenzaldehyde, a bluish-violet to red color is developed. The test is specific for indole alkaloids. 2. 1% tincture of drug shows fluorescence in UV light.

CINCHONA Synonym- Peruvian bark, Jesuits bark Bio. Source- It consists of dried stem or root bark of the plant Cinchona succirubra, C. ledgeriana, C. calisaya, C. officinalis and other species of Cinchona belonging to family Rubiaceae Chemical tests:1. Heat the cinchona powder with glacial acetic acid. Reddish brown fumes on the cooler part of the test tube are seen. 2. Coarse powdered bark is treated with lime and extracted with alcohol. The alcoholic extract is evaporated and residue is taken up with dilute sulphuric Acid, the liquid gives a blue fluorescence. 3. Thalleioquin test- To 10ml of the extract, add 0.25 ml of bromine water and 2 ml of dilute ammonia solution are added and heated if necessary, a bright green colouration is produced.

Experiment-12
Aim-To perform TLC of given sample.

References-Shah N. B., Nayak B. S., Experimental Pharmacognosy, First April,2008, S. Vikas Page no. 255-257

Edition

Requirements- TLC Plate & Chamber, Capillary, oven, spraying bottle, Silica gel G, Sample

Theory
Thin Layer chromatography Principle- TLC has been included under both adsorbtion and partition. Separation may result due to adsoption or partition or by both phenomenon depending upon the nature of adsorbents used on plates and solvent system used for development. Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as themobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. Thin layer chromatography can be used to:

Monitor the progress of a reaction Identify compounds present in a given substance Determine the purity of a substance

Procedure:-1. Prepare the TLC plate and keep it for the saturation. Make the extract of the powdered drug and the spot it on the saturated TLC plate. Make the solvent system and let it saturate it for the half an hour. The run the solvent system till 3/4th of the plate. Dry the plate on the hot plate and then spray it by the spraying agent. Let it get air dried and the mark the spot and trace the spot on the tracing paper. Results: - Report the Rf value. Rf Value = Distance travelled by solute Distance travelled by solvent Cautions:Do not misuse the chemicals and keep the plate properly in the solvent chamber.