Pharmacognosy
First Year Diploma in Pharmacy (PH)
Padmashree Dr. D. Y. Patil Institute of Pharmacy, Akurdi, Pune - 411 044. November 2005toMay 2006 Prof. K. R. Deshmukh Principal,D.Y.P.I.O.P.Akurdi, Pune - 411 044. Mr. Yogesh. S. Bafana D.Y.P.I.O.P. Akurdi, Pune - 411 044. 1. 2.
3.
Project Coordinator
Subject Experts
Mr. Pravin V. Buge D.Y.P.I.O.P., Akurdi, Pune. Mr. Ravi B. Chintamani D.Y.P.I.O.P., Akurdi, Pune. Ms. R. S. Bende Shree Fattechand Jain College Of Pharmacy, Chinchwad, Pune.
O 2006, Maharashtra State Board of Technical Education, 49, Kherwadi, Aliyawar Jung Road, Bandra (East), Mumbai - 400 051 Maharashtra State, India. No part of this Laboratory Manual be reproduced, in any form or by any means, without permission in writing from MSBTE Mumbai.
of First Year Diploma in Pharmacy has completed the term work satisfactorily in Pharmacognosy (0807) for the academic year 200 -to 200 as prescribed in the curriculum.
Place : Date :
( Subject Teacher
Principal
Seal of Institution
Pharrnacognosy
LEARNING OVERVIEW
IMPORTANCE OF THE SUBJECT
Man knows disease since origin of human being, but causes of them were not known. They were assumed evil spirits. So people tried to cure them with plants, which are easily available. In that way ancestors accumulated knowledge of plants and preserved them in literature such as, 1. Rigveda 2. Ayurveda (ancient science of life)
3. Materia rnedica
4. Ebers papyrus
In all the old texts, preference has been given to description of plant as compared to other characteristics. The word 'Pharmacognosy' was coined in 1815 by C. A. Seydler. Pharmacognosy initially known as materia medica may be defined as scientific study of those substances ( plant and animal origin )which are used or have been used in medicine and pharmacy. Pharmacognosy can be considered as a valuable part of the cultural heritage of pharmacy. The name Pharmacognosy is formed from two Greek word 'Pharmakon' meaning drug and 'gnosis' meaning knowledge. The modern aspect of Pharmacognosy includes not only the crude drug but also covers their chemical scrutiny, leading to isolation of active principles. This subject deals with biological, biochemical, therapeutic and economic features of natural drugs and their chemical constituents. Even we are movirlg with great pace in the 21st century, Herbal medicine, cosmetics, Ayurvedic dosage form and research in the field of herbal formulation are of great interest. Therefore it is need of time to explore into area of systematic knowledge about herbal drugs. In recent year, popularity of natural drug is considered as an important contribution of Pharmacognosy in modern medicine.
Pharmacognosy
LINK 1 BLOCK DIAGRAM SHOWING INTER RELATIONSHIP OF SUBJECT AREAS, CURRICULUM 0B.IECTIVE AND JOB PROFILE.
CORE TECHNOLOGY 1) Human Anatomy and Physiology. 2) Bio-Chemistry and Clinical Pathology. 3) Health Education and Community Pharmacy. 4) Pharmacognosy.
TECHNOLOGY SUBJECTS
1) Pharmacologyand Toxicology. 2) Hospital and Clinical Pharmacy. 3) Pharmaceutics - II 4) Drug Store and Business Management 5) Pharmaceutical Jurisprudence 6) Practical Training.
1/
1 5)
5) Pharmaceutics - I
CURRICULUM 0B.IECTIVES 1) Develop attitude for personal development. 2) Develop social skills for social development. 3) Develop continued learning skills for life long learning. 4) Gain basic knowledge of human body and various illness 1 disorders. 5) Understand various drugs, their formulations and counseling to patients for their appropriate use. Develop communication skills.
1 6)
ii
Pharmacognosy
:..
.
PROCEDURE
Handlirlg Microscope, Preparationof sample for section cutting, Micro chemical test and chemical test, etc.
PRINCIPLES
CONCEPT
FACTS
iii
Pharmacognosy
DEVELOPMENT OF SKILLS
The objective of curriculum is to develop the desired skills in the students so that they can solve the field problems. After undergoing through the laboratory experiments, it is expected that the following skills shall be developed in the students.
intellectual Skills :
1. 2.
3.
4.
1. 2.
Logical thinking. ( I-,) Identification of different stages I crude drug with the help of morphology. ( I-, ) Interpretation from transverse section I chemical and Microchemical test.( I-, ) Develop the creativity in section cutting and staining. ( I-, ) To handle and observe instrument and crude drug correctly. ( M-, ) To follow step by step sequence of chemical test I operation. ( M-, ) Ability to prepare thin transverse section. ( M-, ) Labeling different component of T.S. ( M-, )
Motor Skills : 3.
4.
GRID TABLE
Following table gives grid of the experiments and related intellectual and motor skills. Following table gives grid of the experiments and related intellectual and motor skills. Teacher shall ensure for development of generic skills during the practical. Students are expected to focus on acquiring specific skills mentioned therein. No. Experiment No. & Title Intellectual skills Motor skills MI
.
11
A. TO KNOW YOUR PHARMACOGNOSY LABORATORY
1. To study the compound microscope.
2. To understand technique of Section Cutting, Staining and Mounting.
12
13
14
M2
M3
M4
d d d d
d
d d
d
d d d
d
d
d
d d
d
d d d d d d
d d
d
d d d d
d d
d
d d
iv
Pharrnacognosy
No.
Intellectual skills
P
13
12
13
14
Mq
M2
M3
M4
d d
13. To study the Morphological and Microscopical characteristics - Leaf. - - - - - - of Senna C. MORPHOLOGICAL STUDY OF CRUDE DRUGS. 14. To study Morphological characters of carminatives (Ajowan, Black pepper, cardamom and Nutmeg.) and laxatives (Isapghula and Rhubarb ). 15. To study Morphological characters of Drugs acting on Central nervous System (Aconite, Ashwagandha, Ephedra) and Antitussive (Tulsi and Vasaka).
16. To study the Morphological characters of Anti tumor (Vinca), Antihypertensive (Rauwolfia) and Diuretic (Gokhru)
- d- - d
d d
17. To study the Morphological characters of Antiseptic (Curcuma and Neem ) , Vitamin (Amla) and Antirheumatics (Colchicum).
-
4
d
d
-
18. To study the Morphological characters of perfumes and Flavouring agents (Sandal wood) and F~bres (Cotton, Silk and Wool).
19. To study the Morphological characters of Antidiabetics drugs (Gymnema) and unorganized crude drugs (Asafoetida and Kaolin)
20. To study the Morphological characters of Miscellaneous drugs (Garlic, Liquorice, Shankpushpi and Shatawari) D. IDENTIFICATIONTEST FOR CRUDE DRUGS. 21. To identify unknown unorganized powder drug with the help of physical and chemical tests. a. Carbohydrates : Acacia, Agar, Algin, Honey, Tragacanth. b. Protein : Gelatin. 22. To identify unknown unorganized powder drug with the help of physical and chemical tests. a. Tannins : Pale and Black Catechu. b. Mineral : Kaolin. 23. To identify unknown unorganized powder drug with the help of physical and chemical tests. a. Lipids : Bees wax. b. Resin : Benzoin myrrh. 24. To identify unknown organized powder drug with the help of Physical and chemical tests. a. Senna b. Starch c. Termeric
Pharmacognosy
No.
Intellectual skills
11
12
13
Motor skills MI M 2 M 3 M 4
14
25 To identify unknown fibres with the help of Physical and chemical tests. a. Cotton b. Silk C. wool
vi
Pharrnacognosy
4. 5. 6.
7.
Teacher shall ensure that required equipment are in working condition before start of experiment, also keep operating instruction manual available. Explain prior concepts to the students before starting of each experiment. Involve the student's activity at the time of conduct of each experiment. While taking observation each student (from batch of 20 students) shall be given a chance to perform the experiment. List of questions given at the end of each experiment. Teacher shall instruct the students to attempt all questions given at the end of each experiment. Teacher shall ensure that each student writes the answers to the allotted questions in the laboratory manual after performance is over. If the experiment setup has variations in the specifications of the equipment, the teachers are advised to make necessary changes, wherever needed. Teacher shall assess the performanceof students continuously as per norms prescribed by MSBTE. Teacher is expected to share the skills and competencies to be developed in the students. Teacher may provide additional knowledge and skills to the students even though not covered in the manual but are expected from the students by the industries. Teachers shall ensure that industrial visits recommended in the manual are covered. Teacher may suggest the students to refer additional related literature of the technical papers, reference books, seminar proceedirlgs, etc. During assessment teacher is expected to ask questions to the students to tap their achievements regarding related knowledge and skills so that students can prepare while submitting record of the practical. Focus should be given on development of enlisted skills rather than theoretical knowledge. Teacher should enlist the skills to be developed in the students that are expected by the industry. Teacher should organized group discussion, brain storming sessions, seminars to facilitate the exchange of knowledge amongst the students. Teacher should ensure that revised CIAAN-2005 norms are followed simultaneously and progressively. Teacher should give more focus on hands on skills and should actually share the same. Teacher shall also refer to the circular No. MSBTEID-50lPharm. Lab. Manual /200613160 dated 4th May 2006 for additional guidelines.
8.
9.
17.
18.
19.
20. 21.
+ vii
Pharmacognosy
viii 6
Pharmacognosy
A. 1. 2. 3. B. 4.
To study the compound microscope. To understand technique of Section Cutting, Staining and Mounting. To study the Microchemical reagent.
GROSS ANATOMICAL STUDY OF CRUDE DRUGS.
01 06 14
To study the Morphological and lVlicroscopical characteristics of Cinchona Bark. To study the Morphological and Microscopical characteristics of Cinnamon Bark. To study the Morphological and Microscopical characteristics of Clove Buds. To study the Morphological and Microscopical characteristics of Coriander Fruit.. To study the Morphological and Microscopical characteristics of Datura Leaf. To study the Morphological and Microscopical characteristics of Fennel Fruit. To study the Morphological and Microscopical characteristics of Ginger Rhizome. To study the Morphological and Microscopical characteristics of To study the Morphological and Microscopical characteristics of Nux-Vomica Seed. To study the Morphological and Microscopical characteristics of Senna Leaf.
18
5.
24
6.
30
7.
36
8.
42
9.
48
10.
54
11.
12. 67
13.
73
+ ix
Pharrnacognosy
Sr. No.
Page No.
Date of Performance
'
C.
14.
15.
86
16.
93
17.
98
18.
104
19.
110
20.
115
D.
21.
22.
128
X 6
Pharmacognosy
Sr. No.
Page No.
Date of Performance
23.
To identify unknown unorganized powder drug with the help of physical and chemical tests. a. Lipids : Bees wax. b. Resin : Benzoin myrrh. To identify unknown organized powder drug with the help of Physical and chemical tests. a. Senna b. Starch c. Termeric. To identify unknown fibres with the help of Physical and chemical tests. a. Cotton b. S~lk c. Wool. Appendices Appendix-l (Reagents and solutions) Appendix-Il (Botanical terms) Appendix-Ill (Therapeuticalterms) Appendix-IV (Guidelines for Annual Practical Examination) Appendix4 (Colour diagrams)
133
24.
137
25.
142
Sr. No.
Examination
1. 2. 3.
First sessional Practical Examination Second sessional Practical Examination Third sessional Practical Examination
* To be transferred to Proforma of CIAAN - 2004 (Proforma I - 1) Note :- The guidelines for the conduct of Annual Practical Examination are enclosed at the end of page no.177.
+ xi
Pharmacognosy
Experiment No. 1
Experiment No. 1
1.0
2.0
TITLE :
To know the compound Microscope, the components and making it ready for working.
PRIOR CONCEPTS:
Simple microscope.
3.0
NEW CONCEPTS:
Proposition 1 : Illumination system For distinctive and proper viewing of field object, it provides light, which may be either plane or concave mirror or electrically illuminated by tungsten filament. Proposition 2 : Magnification system Magnified real image can be viewed by objective and eyepiece.
4.0
LEARNING OBJECTIVES :
Intellectual Skill : 1. Acquire skills of operating compound microscope. 2. Develop skills to get magnified real image. Motor Skill : To set the microscope for observations and operation. 1.
5.0
REQUIREMENTS :
Apparatus :
Experiment No. i
Pharmacognosy
Objective turret
.1-.,--
Fine Focus
illumination System
6.0
STEPWISE PROCEDURE :
6.1 Microscope : It is an optical instrument, comprising of a kns or a combination of lenses, which enables to view magnified images of a minute object. The Englishman Robert Hooke developed an instrument that was the true forerunner of the compound microscope used today. The compound microscope essentially consists of three major systems. Support system : It comprises of base, stage and body tube. Illumination system : It comprises of lightsource or mirror, iris diaphragm and condenser. Magnification system :This includes objective (is a set of lenses placed a near the object, it partially magnified the object) and eyepiece (a more magnified form of real image is observed) Facts and figures about microscope 1. Magnifying power (M.P.) M.P. = magnification of objective x magnification of eyepiece. e.g. M.P. = 15x X l o x = 150x thus, the object viewed is magnified 150 times. 2. Resolving power of objective (R.P.) Resolving power of an objective is defined as the ability to separate distinctly two small elements of an object, which are, situated a short distance apart. R. P can be . measured by Numerical Aperture (N.A) of an objective. Greater the N.A. greater is the resolving power. Working distance : The distance between the object and the objective is known as working distance. It decreases with increasing magnification. This means higher the power of the objective, lesser is the working distance. Focusing : Focusing an object while viewing through eyepiece means adjustment of working distance. This is done with the help of coarse and fine adjustment of knob.
6.2
3.
4.
Pharmacognosy
Experiment No. 1
5.
Field of view : The area of the object, which one can view through the eyepiece, is the field of view. The field of views narrows as magnification increases.
7.0
OBSERVATION :
(Subject teacher on the basis of permanent slide shall give one exercise to the students. In exercise he shall ask student to focus the slide and observe the particular tissue component. Subject teacher shall give idea about tissue components present in the permanent slide for confirmation of the learning. ) (Space for observations)
8.0
CONCLUSION :
From the observation it is concluded that the component of given permanent slide is found to be. .. ..... . ... . .. ... . . . ... ... . . . ... . .. . .... ... . ... . . . .. . ... . ..
9.0
QUESTIONS :
Write answers to Q Q from Group A, Q Q from Group B and Q Q from Group C (Questions shall be allotted by the subject teacher. Subject teacher shall also add few more relevant questions) GROUP :A 1. State the importance of graphical structure in understanding the scope of the subject. 2. List the parameters of the graphical structure in the hierarchy. (Refer the graphical structure) 3. State two motor skills to be developed through this subject. 4. Classify the curriculum in different groups of subject. 5. State the importance of job description in designing the curriculum. GROUP : B 1. What is the contribution of Robert Hooke in Microscope? 2. Mention mechanical and optical units of compound microscope. State the meaning of 'Field of View' in Microscope. 3. 4. State the meanirlg of a. Magnifying power (M.P.) Revolving power of objective (R.P.) b. c. Working distance. 5. Why CEDAR WOOD OIL is used in case of oil immersion lenses? Give reason. GROUP :C Which precautions should be taken while handling the microscope? 1. 2. State the condition where simple microscope is used in Pharmacognosy practical. When concave mirror is used in compound microscope? 3. 4. Name four types of microscope. 5. What is the role of inclination joint in compound microscope?
.... ....
.... ....
.... ....
Experiment No 1
Pharmacognosy
Pharmacognosy
Experiment No. 1
..
Experiment No. 2
Pharmacognosy
Experiment No. 2
1.0
TITLE :
To understand the method of Section Cutting Technique, Staining, Mounting and Observation of section under Microscope.
2.0
PRIOR CONCEPT
Preparation of sample for sectioning.
3.0
NEW CONCEPT
Proposition 1 : Transverse section Transverse section is obtained by cutting along the -radialplane of a cylindrical portion of the stem, root, stolen and perpendicular to long axis. Proposition 2 : Staining To distinguish the arrangement of various tissues in the samples of crude drug, chemicals, dyes or colorants are used to impart colour to various tissue in section of drug sample. Proposition 3 : Observation For observation of section, selection of place in a laboratory where sufficient light is available is important. The low power observation helps to draw a schematic diagram. For distinctive transverse section high power observation is used.
4.0
LEARNING OBJECTIVES :
Intellectual Skill :
1.
2.
3.
Develop creativity in section cutting using different attributes and materials. To discriminate different components of the section. Identification of different stages. To observe the section under microscope.
Motor Skill :
1.
5.0
REQUIREMENTS :
Apparatus :
Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc
Chemicals :
Subject teacher shall give any single drug to the students among the root, stem, seed, leaf, bark, fruit, rhizome, etc to see the understanding of the methods of section cutting, staining, mounting and its observation.
6.0
STEPWISE PROCEDURE :
6.1
Section of a stem, root, stolon :
Different section can be obtained from a stem, root stolon, depending on the plane of cutting, each section revealing details from a different angle.
Pharmacognosy
Experiment No. 2
6.2
Transverse section (T.S.) Transverse section is obtained by cutting along the radial plane of cylindrical portion of the stem, root, stolon and perpendicular to long axis.
Fig. 2.1 This section when prepared and observed under the microscope reveals the radial arrangement of tissues and shows concentric layers and vascular bundles.
Fig. 2.2 6.3 Section of leaf In case of leaf, the important aspect to study is a section through the midrib taken perpendicular to the midrib and Observation of a surface preparation.
Fig. 2.3 6.4 Section of bark In case of bark, transverse section is important as it reveals the horizontal section of cells and shows lenticels.
Experiment No. 2
Fharmacognosy
Fig. 2.4
6.5 Section of fruit and seed in case of fruit and seeds, generally T.S. of various parts are observed under the microscope. In case of fruit and seed drug, separate section technique is required for individual drug.
Fig. 2.4
6.6 Section cutting technique Following materials are required for Pharmacognosy laboratory work. 7. Test tube holder and stand 1. Napkin 2. Adropper 8. Needle 9. Camel hair brushes 3. Filter paper 4. Stains 10. Watch glass 5. Drug sample 11. A sharp razor blades 6. Forceps 12. Micro-slide 13. Cover slip.
Section cutting can be done by taking transverse as well longitudinal section. Mostly transverse easy section is taken for study of crude drugs. Section cutting is a skill, which fac~litate learning of the tissuecomponents. There are various techniques of section cutting depending on the part of crude drug used. Eg. Green unripe papaya or potato is used for easy sectioning of the leaves. Section cutting includes the following steps :
Preparation of Sample for Sectioning
Fig. 2.5
8
Pharmacognosy
Experiment No. 2
2. Section cuttiqg.
Fig. 2.6
Fig. 2.7
Staning : A stain is a chemical dye (colorant), which combines chemically or physically with a cell content to impart colour to it. e.g. Sudan red Ill dissolves in the fixed oil present in the oil seed to impart red color. Staining Process
1. Take a clean watch glass and add the staining solution to it.
Fig. 2.8
Fig. 2.9
3. Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting. Fig. 2.10
Mounting Process
1. Transfer the section to be mounted on the glass slide with the help of brush
Fig. 2.11
Experiment No. 2
Pharmacognosy
Fig. 2.12 3. Place a clean cover slip over the section with the help of a forceps and needle.
Fig. 2 13
4. With the help of blotting paper, wipe out excess of water present outside the cover slip. The slide is ready for observation
Fig. 2.14
Procedure described above is the routine laboratory technique, so there may be evaporation of water and slide prepared will not last long. Glycerin is used to avoid evaporation of water and drying of section. In order to prepare a permanent mount, a special process is adopted named as Double Staining Technique. A permanent preparation is useful for preservation of good sections for study and for preparation of standards, with which the sample can be compared. This process generally involves staining with two reagents, hence called as Double Staining Technique. One of the stain Imparts colour to the lignified tissue and the other to the cellulose part. Observation : 1. Select a place in the laboratory for microscope, where sufficient light is available. Set the microscope in a such a way that the C-Arm towards to you and the objective and mirror facing the light. Fig. 2.15 2. Open the diaphragm completely withthe help of the substage mirror, Adjustthe position so that the field of view issufficiently illuminated. Fig. 2.16 3. Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser. Fix the slide between the clips. Now the slide Can be moved forward, backward or sideways above the stage with the helpof two screws provided on the mechanical stage. Take observations. Fig. 2.17
I
ui - 4 ~ ~ .
10
Pharrnacognosy
Experiment No. 2
7.0
OBSERVATION :
(Subject teacher on the basis of permanent slide shall give two to three exercises to the student.) (Refer Exp. No.1) (Space for observations)
8.0
CONCLUSION :
From the observation it is concluded that the given crude drug is found to be a ....................... ( stem, root, leaf, bark, seed, fruit, rhizome etc.) containing tissue components.. .......................................................................................................
9.0
QUESTIONS :
Write answers to Q....Q....Q....Q.... (Questions to be allotted by the subject teacher. Subject teacher shall also add few more relevant questions) 1. How to focus the Transverse section in order to get fine image? 2. What is role of condenser and iris diaphragm in critical illumination? 3. State a role of a chemical dye during staining procedure. 4. Give two examples of staining agent with their reactions. 5. Write the reactions of cell wall and cell content with staining agent along with observation. 6. List the necessary material required for section cutting. 7. How to make a sample preparation for microscopic examination? 8. What is the purpose of taking transverse section of crude drug? 9. Mention special method for section cutting of leaf. 10. Why young leaves are preferred to get fine section? Give reason. Space for writing answers
11
.Experiment No. 2
Pharmacognosy
12
Pharmacognosy
Experiment No. 2
13
Experiment No. 3
Pharrnacognosy
Experiment No. 3
1.0
TITLE :
To study Microchemical reagent.
2.0
PRIOR CONCEPT :
Morphology or Sensory characters of crude drug.
3.0
NEW CONCEPT :
Preposition 1 : Microchemical Reagents Microchemical reagents are used in Pharmacognosy practical for diagnostic identification of crude drug, which stains the different components of T.S. of crude drug.
4.0
LEARNING OBJECTIVE :
Intellectual Skill : 1. To acquire skills of staining. 2. Ability to interpret from observation.
5.0
REQUIREMENT :
Apparatus : Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc Chemicals : Microchemical reagents and water. Crude Drug : Subject teacher shall give any single drug to the students among the root, stem, seed, leaf, bark, fruit, rhizome, etc to see the understanding of the procedure of staining.
6.0
STEPWISE PROCEDURE :
6.1.
Study Of Microchemical Reagents : Pharmacognosy includes study of crude drugs obtain from natural origin. This drug can be studied by their morphological or sensory characters and microscopical characters. For microscopical characters, they are treated with various reagents such as : 1. Cleansing Reagents : These reagents are used to make the tissue clear in appearance. e.g. Chloroform, Acetic acid, Chloral hydrate and water, 2. Dehydrating Reagents : These reagents are used to remove water from the section or tissue and to make tissue clear in appearance. e.g. Absolute alcohol.
3. Bleaching Reagents : Reagents are used to bleach the tissues or section and to make tissue clear in appearance. e.g. Hydrogen peroxide.
4. Mounting Reagents : These reagentsare used to mount the tissues or section and to prevent the drying of sections. e.g. Glycerin or mixture of glycerin and water.
6.2
14
Pharmacognosy
Experiment No. 3
Name of Reagent Eosin Conc. Sulphuric Acid + Iodine Phloroglycenol + Conc. Hcl Safranin Rheuthenium Red Alcoholic Picric Acid Sudan Red - 111 Dilute Iodine Conc. Sulphuric Acid Ferric Chloride
Component Cellulose Colour Lignin Lignin Mucilage Protein Fixed Oils And Fats Starch Saponins I Stone Cells Tannins
0bservation
Red Or Pink Colour Pale Blue Colour Red Or Pink Colour Red Or Pink Colour Red Or Pink Colour Yellow Colour Yellowish Brown Colour Deep I Pale Blue Green Colour Blue I Black Colour
6.3 Procedure : 1. Issue the glass watches and add microchemical reagents to it. 2. Take section of crude drug and transfer section in to watch glass containing water. 3. Place section in to watch glass containing microchemical reagents. 4. Keep it for specified period. 5. Mount the slide and observe it.
7.0
OBSERVATION :
Subject teacher on the basis of permanent slide shall give two to three exercises to the student.) ) (Refer Exp. No.I (Space for observations)
15
Experiment No. 3
Pharmacognosy
8.0
CONCLUSION :
From the above microscopical tests, ........................................ (Name of the microchemical reagent) gives.. ............................ colour indicating presence of ...................................... .................................................................................... components in given crude drug.
9.0
QUESTIONS :
Write answers to Q....Q....Q....Q.... (Questions t o be allotted by the subject teacher. Subject teacher shall also add few more relevant questions) 1. Define micorchemical reagent and list out different types of it. 2. Why dehydrating agent is used in Microchemical test? Explain it with the help of example. 3. Which Microchemical reagent is used for detection of tannin in cell? 4. Which cell component is detected by ruthenium red? 5. Define cleansing and bleaching reagent with help of example. 6. Tabulate Microchemicaltests for fennel. 7. Which lblicrochemical reqgent is used for detection of Calcium oxalate crystal? 8. Write the stepwise procedure of staining and mounting? 9. Why glycerin is used as mounting reagent? Give reason. 10. Tabulate Microchemicaltest for Cinchona. (Space for answers)
16
Pharrnacognosy
Experiment No. 3
17
Experiment No. 4
Pharmacognosy
Experiment No. 4
1.0 TITLE :
To study Morphological and Microscopical characteristics of Cinchona Bark.
2.0
PRIOR CONCEPTS :
Section cutting technique, staining, mounting and observation of transverse section of Cinchona Bark.
3.0
NEW CONCEPT :
Proposition 1 : Morphological characters It includes organoleptic characters and extra features. Proposition 2 : Microscopical characters It includes observation of important tissue components of transverse section of Cinnamon bark. Proposition 3 :Adulterants It is debasement of genuine crude drugs, which proved harmful.
4.0
LEARNING 0B.IECTIVE :
Intellectual Skill : 1. Ability to interpret the tissue components. Motor Skill : 1. Ability to prepare thin transverse section of cinnamon bark. 2.
To handle and observe instrument and crude drug correctly. Labeling different component of cell.
3.
5.0
REQUIREMENT SSSS :
Apparatus :
Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc.
Chemicals :
Phloroglucinol, Conc. HCI, Iodine solution, Glycerin, etc.
Crude Drug :
Cinchona bark.
6.0
DIAGRAM :
18
MAHARASKTRA STNE
Pharrhacognosy
Experiment No: 4
Fig. 4.2 T.S. of Cinchona Bark (Refer colour diagrams given in Appendix-V)
7.0
STEPWISE PROCEDURE :
7.1 7.2
7.3
7.4
Synonyms : English : Jesuit's bark, Peruvian bark. Biological source : It consists of dried bark of the cultivated trees of Cinchona calisaya Wedd., C. ledgeriana. Mocns, C. officinalis Linn., C. succirubra Pav. ex. Klotzsch, or hybrids of either of the last two species with either of the first two. It contains not less than 6 percent of total alkaloids of cinchona. Family : Rubiaceae. Macroscopy : Organoleptic characters : Odour : Slight and Characteristic Taste : Intensely bitter and slightly astringent. Microscopy : I. PERIDERM : Cork : Several layers of thin walled, flat, polygonal cells with reddish brown content, impregnated with suberin. Phellogen : 2to 3 layers thin walled cells without any cellular content. Pheloderm : 6 to 8 layers of thin walled rectangular cells without any cellular content.
19
Experiment No. 4
Pharrnacognosy
2. CORTEX:
Several layers of thin walled tangentially elongated cells containing reddish brown matter. Calcium oxalate crystals : 2 to 6 m long, microsphenoidal crystals Starch grains : Rounded, 6 to 10 m in diameter. Sclerei&s are absent. Cavities (secretion canals) are present.
3. SECONDARY PHLOEM :
Sieve Tubes : The compact cells being about 200 m long and 15 to 20 m wide and having narrowcompanioncells; most of the sieve tubes are compressed and collapsed. Fibres : Nemerous, large, fusiform, lignified phloem fibres, having striated walls and conspicuous tubular or funnel-shaped pits, mostly isolated, some times in groups of 2 to 3 fibres. Pholem parenchyma : Thin, dark-reddish brown walls, somewith micro-priims of calcium oxalate. Medullary rays : One to three seriate, extended up to cortex cells, redially elongated and contain starch grains. Chemical constituents : Alkaloids : Quinine, Quinidine, Cinchonine, Chichonidine Uses : 1. Antimalarial 2. Antipyretic. Allied drugs : 1. Cuprea bark, 2. Colombian bark. Marketed preparations : 1. Cinchona extract, 2. Compound cinchona tincture. Procedure 1. Clean the platform and issue the apparatus. 2. Issue the sample of crude drug. 3. Preparation of sample for sectioning. Boding of the sample. Section cutting. Transfer the section in to Watch glass containing water. (If crude drug is too hard, or in any case where subject teacher may feel then the preparation of sample for sectioning is done before one hour or a day of the practical or may be varied in certain cases) 4. Staining Process. Take a clean watch glass and add the staining solution to it. With the help of brush, transfer the section taken from watch glass containing water to stain solution and keep it for 2 - 3 minutes. Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting. 5. Mounting Process. Transfer the section to be mounted on the glass slide with the help of brush. Add 1 - 2 drops of water on the section with the dropper. Place the clean cover slip over the section with the help of a forceps and needle. With the help of blotting paper, wipe out excess of water present outside the cover slip. The slide is ready for observation. 6. Observation. Select a place in the laboratory for microscope, where sufficient light is available. Set the microscope in such a way that the C-Arm towards to you and the objective and mirror facing the light. Open the diaphragm completely with the help of the sub stage mirror. Adjust the position so that the field of view is sufficiently illuminated. Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser.
. . . . .
. . . . . . .
20
Pharmacognosy
Experiment No. 4
. .
7.10
Fix the slide between the clips. Now the slide can be moved forward, backward or sideways above the stage with the help of two screws provided on the mechanical stage. Take observations. Staining :
1. T.S. + Phloroglucinol + Conc. HCI (1:1) Pink colour. Lignified Phloem fibres Fig. 4.3 2. T.S. + Iodine Blue colour. Starch.
8.0
OBSERVATIONS :
8.1 Observation table for Macroscopy : Sr. No. 1. Test Colour Odour Taste Observation
2.
3.
8.2 Observation table for Staining : Sr. No. 1. 2. 3. Test Observation Inferences
9.0
CONCLUSION :
The given sample of crude drug is found to be ... ... ... ... ... ... ...... ... ... ... ... ... ... . (Cinchona bark)
10.0 QUESTIONS :
Q Q Q Q (Questions to be allotted by the subject teacher. Write answers to Q .... .... Subject teacher shall also add few more relevant questions) 1. Write two external characters of Cinchona. 2. Differentiate between stem, root and bark of Cinchona with the help of external characters. 3. The name 'Cinchona' is derived from which incident? 4. Which staining test is used to detect starch? 5. What will be the inferences after treatment of T.S. of Cinchona with Dil. Hydrochloric acid? 6. Which microscopic component is present in periderm of Cinchona bark?
21
Experiment No. 4
Pharmacognosy
7. 8. 9. 10.
Draw well labeled diagram of Cinchona bark. Which microscopical test is used for identification of Lignified phloem and fibres? Mention four species of Cinchona bark. Which parasites are responsible for infection of Malaria? Give two names. (Space for answer)
22
Pharmacognosy
Experiment-No.4
23
Experiment No. 5
Pharmacognosy
Experiment No. 5
1.0 2.0 TITLE :
To study lblorphological and lMicroscopical characteristics of Cinnamon Bark.
PRIOR CONCEPTS :
Section cutting technique, staining, mounting and observation of transverse section of Cinnamon Bark seed.
3.0
NEW CONCEPTS :
Proposition 1 : Morphological characters It includes organolepticcharacters and extra features. Proposition 2 : Microscopical characters It includes observation of important tissue components of transverse section of Cinnamon bark. Proposition 3 :Adulterants It is debasement of genuine crude drugs, which proved harmful.
4.0
LEARNING 0B.IEC'I'IVES :
Intellectual Skill : 1. Ability to interpret the tissue components. Motor Skill : 1. Ability to prepare thin transverse s e d i n of cinnamon bark. 2. To handle and observe instrument and crude drug correctly. 3. Labeling different component ofcell.
5.0
REQUIREMENTS :
Apparatus :
Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc.
Chemicals :
Phloroglucinol, Conc. HCI, Iodine solution, Glycerin, Ruthenium Red solution, etc.
Crude Drug :
Cinnamon Bark.
6.0
DIAGRAM :
24
Pharmacognosy
Experiment No. 5
STEPWISE PROCEDURE :
7.1 Synonyms : English : Cinnamon Bark Hindi : Kalmi - Dalchini Biological source : It consist of dried inner bark of the shoots of coppiced trees of Cinnamomum zeylanicum Nees. It contains not less than 1.O% vlw of volatile oil, belonging to family Lauraceae. Macroscopy : Organoleptic characters : Colour : Outer surface, dull yellowish-brown; Inner surface darker in colour Odour : Fragrant, Taste : Warm, sweet and aromatic Extra features : Bark is free of cork, siqgle or double, closely packed compound quills. Fracture : Splintery. Microscopy : 1. PERICYCLE (stone cell layers) : Produce the light coloured wavy, longitudinal lines on the outside of the bark.
7.2
7.3
7.4
25
Experiment No. 5
Pharmacognosy
Pericyclic fibres : Small groups of about 6 to 15 pericyclic fibres (lignified) occur at intervals. Sclerides : 3 to 4 layers of pitted sclerides, thickened lignified walls, isodiametric,slightly elongated tangentially (U-shaped thicknening ), with starch grains. 2. SECONDARY PHLOEM : Parenchymatous: few cells contains acicular calcium oxalate crystals and starch grains (diameter upto 10 p). Medullary rays : Biseriate, narrow at innersight, wider in thescleride band side, contains starch, acicular raphides. Pholem fibres :single, isolated, circular, lignified with stratification, being above 12 to 22 to 35 p wide and 200 to 500 to 600 p long Mucilage cells : can be identified after staining with Ruthenium red (shows pink / red colour). Oil cells : big, isolated. Cork and cortex are absent. 7.5 Chemical constituents : Volatile oil (0.5 to 1%), cinnamic aldehyde (55 to 65%), eugenol(4 to lo%), terpenes, mucilage, starch, calcium oxalate, tannins. Uses : 1. Carminative, 3. Mild astringent, Allied drugs : 1. Cassia bark or Chinese Cinnamon, 3. Java Cinnamon, 5. Oliver bark.
7.6
7.7
7.8 7.9
Marketed preparation : 1. Cinnamon is an ingredient of Compound-cardamom tincture I.P. Procedure 1. Clean the platform and issue the apparatus. 2. Issue the sample of crude drug. 3. Preparation of sample for sectioning. Boiling of the sample. Section cutting. Transfer the section in to Watch glass in to Watch glass containing water. (If crude drug is too hard, or in any case where subject teacher may feel then the preparation of sample for sectioning is done before one hour or a day of the practical or may be varied in certain cases) 4. Staining Process. Take a clean watch glass and add the staining solution to it. With the help of brush, transfer the section taken from watch glass containing water to stain solution and keep for 2 - 3 minutes. Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting. 5. Mounting Process. Transfer the section to be mounted on the glass slide with the help of brush. Add 1 - 2 drops of water on the section with the dropper. Place the clean cover slip over the section with the help of a forceps and needle. With the help of blotting paper, wipe out excess of water present outside the cover slip. The slide is ready for observation. 6. Observation. Select a place in the laboratory for microscope, where sufficient light is available. Set the microscope in such a way that the C-Arm towards to you and the objective and mirror facing the light. Open the diaphragm completely with the help of the sub stage mirror. Adjust the position so that the field of view is sufficiently illuminated.
. . .
. . . . .
.
. .
26
Pharrnacognosy
Experiment No. 5
Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser. Fix the slide between the clips. Now the slide can be moved forward, backward or sideways above the stage with the help of two screws provided on the mechanical stage. Take observations.
7.10 Staining: Subject teacher shall ask student to draw diagrams of staining in the space provided below.
1.
T.S. + Phoroglucinol + Conc. HCI (1:l) Pink colour Lignified cells: Pericyclic fibres, stone cells, cork cells. T.S. + Iodine Blue colour Starch
2.
3.
4.
8.0
OBSERVATIONS :
8.1 Observation Table for Macroscopy Sr. No. Test Observation
1. 2.
3.
4.
5.
8.2 Observation Table for Staining : Sr. No. Test Observation Inferences
1. 2.
3.
9.0
CONCLUSION :
The given crude drug is found to be ... ... ... ... ...... ...... ... ... ... ... ... ...... ... . (Cinnamon bark)
27
Experiment No. 5
Pharmacognosy
10.0 QUESTIONS :
Write answers to Q Q Q Q ....(Questions to be allotted by the subject teacher. Subject teacher shall also add few more relevant questions)
Give biological source of Cinnamon bark. Draw neat labeled macroscopical diagram of Cinnamon bark. Which microscopic character is detected by Ruthenium Red in case of Cinnamon bark? Write two crude drugs, which contain eugenol as main active chemical constituent. Mention four allied drugs of Cinnamon bark. Give the process of chemical test by which tannins are detected from Cinnamon bark. Write three regional names of Cinnamon bark other than title. Which volatile oil constituents are present in Cinnamon bark? Write four Therapeutic uses of Cinnamon bark. Why is Cinnamon presented in compound quill form? Give reason.
(Space for answers)
28
Pharmacognosy
Experiment No. 5
29