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Tracer techniques, General techniques for biosynthetic studies, Pharmacognosy
- 1. General Techniques for Biosynthetic Studies Prepared by, Ms. M.S. Divya Sree, Assistant Professor, Department of Pharmacognosy, Sree Vidyanikethan College of Pharmacy, Tirupati, Andhra Pradesh
- 2. Introduction Living plants are considered as biosynthetic laboratory for production of primary as well as secondary metabolite. Various intermediate steps are involved in biosynthetic pathways in plants that can be investigated by means of following techniques: ‐ Using Tracer Techniques ‐ Using Isolated Organs / Tissues and Cells ‐ Grafting Method ‐ Using Mutant Strains . 2
- 3. TRACER TECHNIQUE ‐ It can be defined as technique which utilizes a labelled compound to find out or to trace the different intermediates and various steps in biosynthetic pathways in plants, at a given rate & time. ‐ Also this technique utilises the labelled compound which when introduced into plant system, they become part of general metabolic pool & undergo reactions associated with that particular plant system . 3
- 4. SIGNIFICANCE OF TRACER TECHNIQUE ‐ High sensitivity. ‐ Applicable to living system. ‐ Wide ranges of isotopes are available. ‐ More reliable, easy administration & isolation procedure. ‐ Gives accurate result, if proper metabolic time & technique applied. ‐ . 4
- 5. ‐ Different tracers can be used for different studies ‐ Location & Quantity of compound containing tracer 14C labelled glucose is used for determination of glucose in biological system. Ex. For studies on nitrogen and amino acid, Labelled nitrogen give specific information than carbon. . 5
- 6. CRITERIA FOR TRACER / ISOTOPE SELECTION Following points must be considered before selection 1. The starting concentration of tracer must be sufficient enough to withstand dilution in course of metabolism. 2. Physical & chemical nature of compound must be known for proper labelling. 3. Higher Half-life. 6
- 7. 5. Should actively participate during synthesis. 6. Should not damage the system (Harmless) 7. Specificity of radioactive tracer. 8. Tracer should be highly pure. 9. Binding of tracer during entire course of biosynthetic metabolism. 7
- 8. Two types of isotopes are generally used for labelling 1. Radioactive isotopes. 2. Stable isotopes. 1. Radioactive isotopes : - [e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I ] For biological investigation – C & H. For metabolic studies – S, P, and alkali and alkaline earth metals For studies on protein, alkaloids, and amino acid - labelled N-atom. 8
- 9. 2. Stable isotopes : - [e.g. 2H, 13C, 15N, 18O] Used for labelling compounds as possible intermediates in biosynthetic pathways. Usual method of detection - Mass spectroscopy [15N, 18O] - NMR spectroscopy [2H, 13C] 9
- 10. STEPS INVOLVED IN TRACER TECHNIQUE I. Preparation of labelled compound. II. Introduction of labelled compound into a biological system. III. Separation & determination of labelled compound in various biochemical fractions. 10
- 11. I. Preparation of Labelled Compound The labelled compound is produced by growing them in atmosphere of 14CO2. All carbon compounds get 14C labelled. The 3H (tritium) labelled compound is commercially available. Tritium is pure β – emitter of low intensity & its radiation energy is lower than 14C. By the use of organic synthesis: CH3MgBr + 14CO2 CH3 14COOHMgBr + H2O CH3 14COOH + Mg(OH)Br 11
- 12. II. Introduction of labelled compound into biological system ‐ Root feeding and Stem feeding ‐ Direct injection ‐ Floating method ‐ Spray technique ‐ Wick feeding 12
- 13. III. Separation & Detection Techniques ‐ GM Counters ‐ Liquid Scintillation Chamber ‐ Gas Ionization Chamber ‐ Mass Spectrophotometer ‐ NMR Spectrophotometer ‐ Auto-Radiography 13
- 14. Geiger–Muller (GM) Counters A Geiger–Muller counter, is a type of particle detector that measures ionizing radiation e.g. alpha, beta particles or gamma rays by the ionization produced in a low - pressure gas usually helium, neon or argon with halogens added in a Geiger–Muller tube. It briefly conducts electrical charge when a particle or photon of radiation makes the gas conductive by ionization. 14
- 15. This charge has been detected in form of current pulse. 15
- 16. Liquid Scintillation Chamber A scintillation detector or scintillation counter is obtained when a scintillator is coupled to an electronic light sensor such as a photomultiplier tube (PMT) or a photodiode. A scintillator is a material that exhibits scintillation — the property of luminescence when excited by ionizing radiation. Samples are dissolved or suspended in a "cocktail" containing a solvent (aromatic organics such as benzene or toluene), typically some form of a surfactant, and small amounts of scintillators. 16
- 18. Gas Ionization Chamber The ionization chamber is the simplest of all gas-filled radiation detectors, and is widely used for ionizing radiation; X-rays, gamma rays and beta particles. Conventionally, the term "ionization chamber" is used exclusively to describe those detectors which collect all the charges created by direct ionization within the gas through the application of an electric field. 18
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- 20. NMR Spectrophotometer It is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they contain. It relies on the phenomenon of nuclear magnetic resonance and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. 20
- 22. Mass Spectrophotometer Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. 22
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- 24. Autoradiography Autoradiography is a method for investigating the distribution of radioactive material in a plant object, e.g. histological tissues, a chromatography plate. It locates radioactive isotopes in the biological specimens. This techniques uses a photographic film or emulsion as detector of ionizing radiation. It is adopted to study metabolic pathways and biogenetic experiments. 24
- 25. In this technique an autoradiograph / autoradiogram is obtained as an image of the distribution pattern of radiolabelled metabolite formed on X-ray film. These patterns are formed due to decay emissions of radiolabelled metabolite. The sample containing radiolabelled metabolite comes in close contact with emulsion for a certain period of time (exposure period). With the help of autoradiograph obtained the distribution pattern of radioactive substance is elucidated 25
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- 27. METHODS FOR AUTORADIOGRAPHY 1. Precursor Product Sequences 2. Double & Multiple Labelling 3. Competitive Feeding 4. Isotope Incorporation 5. Sequential Analysis 27
- 28. 1. PRECURSOR PRODUCT SEQUENCE ‐ In this technique, the presumed precursor of the constituent under investigation on a labelled form is fed into the plant. ‐ After a suitable time the constituent is isolated, purified and radioactivity is determined. Application: This method is applied to the biogenesis of morphine & ergot alkaloids 28
- 29. 2. DOUBLE & MULTIPLE LABELLING ‐ This method gives the evidence for nature of biochemical incorporation of precursor which arises double & triple labelling. ‐ In this method specifically labelled precursor and subsequent degradation of the recovered product are both employed. Application: ‐ This method is extensively applied to study the biogenesis of plant secondary metabolite. ‐ Used for study of morphine alkaloid. 29
- 30. 3. COMPETITIVE FEEDING ‐ This method provides the possible intermediates that plant normally used during biogenesis. Application: - ‐ This method is used for elucidation of biogenesis of propane alkaloids. ‐ Biosynthesis of hemlock alkaloids (conline, conhydrine etc) using 14C labelled compounds. 30
- 31. 4. ISOTOPE INCORPORATION ‐ This method provides information about the position of bond cleavage & their formation during reaction. E.g. Glucose-1-phosphatase cleavage as catalyzed by alkaline phosphatase reaction occur with cleavage of either C – O bond or P – O bond. 31
- 32. 5. SEQUENTIAL ANALYSIS ‐ The principle of this method of investigation is to grow plant in atmosphere of 14CO2 & then analyze the plant at given time interval to obtain the sequence in which various correlated compound become labelled. Application: - ‐ 14CO2 sequential analysis has been very successfully used in elucidation of carbon in photosynthesis. ‐ Determination of sequential formation of opium hemlock and tobacco alkaloids. 32
- 33. Using Isolated Organ / Tissues / Cells 33 ‐ This method is based on using isolated parts of plant ( eg. Stem, roots etc.) ‐ This technique is useful in determination of site of synthesis of particular compound. Ex: Roots and Leaves for study of Nicotiana and Datura, Petal disc for study of oil of rose, Tropane Alkaloids formed in roots of Solanaceae Family.
- 34. 34 ‐ In this method, the radio labelled precursor is fed to the culture media under sterile conditions. ‐ The developing plant material is analysed. Then the results are interpreted. ‐ This is done on the basis of distribution pattern of the radiolabelled intermediates. ‐ This method can be used to trace out the exact sites for the biosynthesis of primary and secondary metabolites.
- 35. 35 Plant parts used ‐ Isolated shoots ‐ Rooted leaves ‐ Petal discs ‐ Isolated roots
- 36. 36 Advantages ‐ Plants/plant materials used for analysis can be reproduced from cultures when required. ‐ Plant material present in sterile nutrient medium can be maintained for longer periods ‐ Presumed radiolabelled precursors selected for metabolite study can be fed to plant through nutrient medium. ‐ As plant materials are maintained under aseptic conditions, any modifications of precursor which is otherwise seen in the presence of microorganisms is eliminated.
- 37. Grafting Methods 37 ‐ This method is used for the study of alkaloid formation by grafted plants. ‐ In this method cut surface of stock is united with cut portion of scion / graft of another closely related species. ‐ It is very useful technique for the development of many commercial varieties of medicinal plants and also to determine the exact sites for the biogenesis of secondary metabolites. Ex: Tomato Scions grafted on datura accumulate alkaloids, while datura Scion grafted on tomato contained very small amount of Alkaloids. This suggests that main site for formation of datura alkaloids is root
- 38. Using mutant strains 38 ‐ In this method mutant strains of micro organisms are produced which lack certain enzymes. ‐ Non availability of the enzyme at the specific site hinders the biosynthetic pathway. ‐ This results in the accumulation of the intermediate compound. ‐ In order to continue the pathway, the intermediate compound is artificially supplied.
- 39. 39 Example: Gibberella fujikuroi mutant strains They have similarity between biosynthetic pathway of gibberellins in fungus Gibberella fujikuroi and higher plants. Hence the mutant strains of Gibberella fujikuroi can be used to study pathway of gibberellins.
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