Pharmacognosy and Phytochemistry Extraction and Identification PDF

Pharmacognosy and Phytochemistry Extraction and identification of medicinal plants By Dr. Farah Al-Mamoori To carry phytochemical study the following points 1- Selection of promising plant materials. 2- Proper collection of selected plants. 3- Authentica...

Pharmacognosy and Phytochemistry Extraction and identification of medicinal plants By Dr. Farah Al-Mamoori To carry phytochemical study the following points 1- Selection of promising plant materials. 2- Proper collection of selected plants. 3- Authentication of plant material. 4- Drying of plant materials. 5- Grinding of the dried plants. 6- Garbling of the dried plants 7- Packing, storage and preservation 8- Extraction and fractionation of constituents. 9- Methods of separation and purification. 10- Methods of identification of isolated compounds(structure elucidation e.g. UV, IR, MS, 1H-NMR and 13C-NMR). Plant selection of plant material for study in drug discovery are performed by different approaches: -Random selection -Ethnopharmacology -Chemotaxanomy -Geographical -Computer-based selection -Literature information selection technique We discussed plant collection previously  Herb authentication is a quality assurance process that ensures the correct plant species and plant parts are used This may be confirmed by: 1- Establishing the identity by a taxonomy experts. 2-By comparing the collecting plant with a voucher specimen(herbarium sheet) We discussed the points 4-7previously Extraction, as the term is used pharmaceutically, involves the separation of medicinally active portions of plant from the inactive or inert components by using selective solvents in standard extraction procedures Extraction is process of isolation of soluble material from an insoluble residue, which may be liquid or solid, by treatment with a solvent on the basis of the physical nature of crude drug to be extracted, i.e. liquid or solid, the extraction process may be liquid— liquid or solid—liquid extraction. Mass transfer is a unit operation, which involves the transfer of mass of soluble material from a solid to a fluid. Solvents used for extraction Water, Ethanol, Methanol, Chloroform, Petrelum.Ether, Acetone etc Selection of suitable solvent: Capable of extracting the active chemical constituents. Selection of the solvent extraction approach is very important. If a plant is under investigation from an ethnobotanical perspective, then extraction should mimic the traditional use. For example; if indigenous people use a specific extraction protocol such as a water extract, a cold/heat tea then an identical or at least a very similar method should be used in the laboratory so that the same natural products are extracted. Failure to extract biomass properly may result in loss of access to active compounds. Additionally, using an inappropriate extraction method, such s strong heating of biomass with a solvent, may result in degradation of natural products and consequent loss of biological activity. The overall mechanism of solid-liquid extraction can be described as follows: first, the solvent is mixed with the plant material, diffused onto the solid surface, penetrated into the particles, and gradually dissolves the soluble ingredients. The subsequent processes can be viewed as a mass transfer from the interior of the cell to the surface (internal diffusion) and from the surface to the bulk solvent (external diffusion). Solvent - A solvent with a similar polarity to that of the analyte should be chosen. Ideally, the solvent should be nontoxic, nonreactive, nonflammable, environmentally friendly and affordable. Particle size – Grinding before solvent extraction promotes an increase of the contact area between the solvent and the solid matrix. Extraction efficiency increases with decreasing particle size. Sample humidity – the water in the solid material can compete with the extraction solvent for the solute’s dissolution, so in most cases the material is dried under conditions that do not cause degradation of the compounds. Temperature – the solubility and diffusivity of the material being extracted increases with increasing temperature, hence improving the rate of extraction. An upper temperature limit should be set to avoid undesirable degradation reactions and to ensure the solvent remains in a liquid state. Solvent agitation - Agitation of the solvent increases turbulent diffusion and material transfer rates. Non polar solvents (petroleum ether and hexane) will  dissolve non-polar compounds (fats and waxes). While polar solvents (methanol, ethanol and water)  dissolve polar compound (alkaloid salts and sugars). Rotary evaporator A rotary evaporator (sometimes abbreviated to rotavap) is a piece of equipment primarily used to remove solvent from a sample through ―evaporation under reduced pressure‖. The presence of reduced pressure in the apparatus causes the solvent (in the round bottom flask) to boil at a lower temperature than normal. The process of freeze-drying plant samples ensures high efficiency in removing water, while at the same time retaining bioactive ingredients. We discuss it previously In this process, the whole or coarsely powdered crude drug is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water. These are dilute solutions of the readily soluble constituents of crude drugs. In this process, the crude drug is boiled in a specified volume of water for a defined time; it is then cooled and strained or filtered. This procedure is suitable for extracting water- soluble, heat stable constituents. Digestion is an extractive method similar to maceration and uses slight warming in the extraction process. Care is, however, exercised to avoid the temperature altering the bioactive phytochemicals of given plant material. Therefore, there is increased efficiency in using the extraction solvent due to warming.  A percolator (a narrow, cone-shaped vessel open at both ends) is generally used.  The solid ingredients are moistened with an appropriate amount of the specified menstruum and allowed to stand for approximately 4 h in a well closed container, after which the mass is packed and the top of the percolator is closed.  Additional menstruum is added to form a shallow layer above the mass, and the mixture is allowed to macerate in the closed percolator for 24 h.  The outlet of the percolator then is opened and the liquid contained therein is allowed to drip slowly. Steam distillation is the most common way to extract aromatic compounds (essential oil) from a plant. As the vapor mixture flows through a condenser and cools, it yields a layer of oil and a layer of water. The essential oil rises to the top and is separated from the hydrosol (floral water) and collected. Liquid-liquid extraction, also known as partitioning, is a separation process consisting of the transfer of a solute from one solvent to another, the two solvents being immiscible or partially miscible with each other. Various novel techniques have been developed for the extraction of nutraceuticals from plants in order to shorten the extraction time, decrease the solvent consumption, increase the extraction yield, and enhance the quality of extracts. The traditional extraction methods of natural products are often considered to be a bottleneck for most analytical procedures Ultrasound-assisted Soxhlet extraction extraction(UAE) Microwave-assisted Maceration extraction(MAE) Supercritical fluid extraction Percolation Enzyme-Assisted Decoction Extraction Stem-distillation Ultrasound increases the surface contact between solvents and samples and permeability of cell walls. Physical and chemical properties of the materials are altered and disrupt the plant cell wall; facilitating release of compounds and enhancing mass transport of the solvents into the plant cells. *A lot of the cell damage increasing cell damage,and mass transfer for bioactive compounds were inferred  It combines microwave and traditional solvent extraction.  In MAE, the extraction occurs as a result of changes in the cell structure caused by electromagnetic waves  Microwaves are made up of two oscillating perpendicular field’s i.e. electric field and magnetic field Is a process based on the use of solvents above or near their critical temperature and pressure to In the supercritical state, the solvent has intermediary recover extracts from solid matrices. properties between gases and liquids, which makes it useful as solvent for several compounds. This technology uses renewable solvents, such as CO2.  Uses specific enzymes to disrupt the cell wall of source material to improve its extraction yield. This technique can be combined with various other techniques to enhance the overall recovery of bioactives from source materials. A standardized herbal extract is an herb extract that has one or more components present in a specific, guaranteed amount, usually expressed as a percentage. The intention behind the standardization of herbs is to guarantee that the consumer is getting a product in which the chemistry is consistent from batch to batch Ginkgo Biloba Extract 120 mg (Ginkgo biloba) (leaf) (Standardized to contain 24% Ginkgo Flavone Glycosides Extracts made directly from crude plant material show wide variation in composition, quality, and therapeutic effects. Standardized extracts are high-quality extracts containing consistent levels of specified compounds. They are subjected to strict quality controls during all phases of the growing, harvesting, and manufacturing processes. Standardization can be defined as a requirement to have a minimum amount of one or several compounds in the extract. When the active principles are unknown, marker substances are established for analytical purposes and standardization. Marker substances are chemically defined substances that are important for the quality of the finished product. Ideally, the chemical markers chosen would also be the active constituents. Column chromatography is the most effective technique used in the separation of plant extracts into its components in pure form. PRINCIPLE Adsorption mixture of components dissolved in the mobile phase is introduced in to the column. Components moves depending upon their relative affinities A compound attracted more strongly by the mobile phase will move rapidly through the column, and elute from, or come off, the column dissolved in the eluent. In contrast, a compound more strongly attracted to the stationary phase will move slowly through the column. All the spectroscopic methods are very helpful for the confirmation of structure of natural compounds and also structure of degradation products. The various methods for elucidating the structure of compounds are; UV Spectroscopy: In compounds containing conjugated dienes or α,β-unsaturated  ketones, UV spectroscopy is very useful tool. IR Spectroscopy: IR spectroscopy is useful in detecting group such as hydroxyl group  (~3400cm-1) or an oxo group (saturated 1750-1700cm-1). Isopropyl group, cis and trans also have characteristic absorption peaks in IR region. NMR Spectroscopy: This technique is useful to detect and identify double bonds, to  determine the nature of end group and also the number of rings present, and also to reveal the orientation of group. Mass Spectroscopy: for determining mol. wt., mol. formula, and nature of functional  groups present. Definition:  Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the compounds in the mixture.  TLC can be used to help determine the number of components in a mixture, the identity of compounds, and the purity of a compound. By observing the appearance of a product or the disappearance of a reactant, it can also be used to monitor the progress of a reaction TLC is performed using thin sheets of glass, aluminum or plastic coated with a layer of stationary phase, usually silica gel.  Using polar group like Silica provide chance of separation of polar and non-polar material by using non-polar solvent. Molecular structure of silica gel (SiO2.xH2O)n TLC consists of three steps – 1)spotting 2)development 3)visualization. Spotting: In TLC, the sample must be carefully applied to the plate to minimize spreading. Sample sizes from 0.1 mg to 50 mg are the best for TLC. Smaller amounts are difficult to visualize, while larger spots result in overloading and variable results. Application of sample Development: The spotted plate is placed in a sealed development tank filled with vapor of mobile phase its lower side immersed in solvent to a level below the applied sample spots. The solvent rises due to capillary flow in a process called development. Development times can range from 3-60 minutes. Basic model of TLC chromatography chamber Then the TLC plate is dried by drier.