Chap 3 (a).pdf

CHAPTER 3 EXTRACTION, IDENTIFICATION AND CHARACTERIZATION OF BIOACTIVE MOLECULES FROM NATURAL SOURCES BWK20803 Natural Product Chemistry ChM. Dr. Saliza binti Asman Semester 2 2023/2024 Screening of phytochemicals Methods of extr...

CHAPTER 3 EXTRACTION, IDENTIFICATION AND CHARACTERIZATION OF BIOACTIVE MOLECULES FROM NATURAL SOURCES BWK20803 Natural Product Chemistry ChM. Dr. Saliza binti Asman Semester 2 2023/2024 Screening of phytochemicals Methods of extraction and isolation of phytochemicals. Spectroscopic technique in structure elucidation of phytochemicals  Screening of phytochemicals Can you predict what type of phytochemicals presence in these plants? Scientific name: Elaies guineensis Scientific name: Cymbopogon citratus Common name: Oil palm Common name: Lemongrass Local name: kelapa sawit Local name: Serai  TYPE OF PHYTOCHEMICALS -Carotene – carotenoid (yellow, orange, red pigment)– carrot, pumpkin Terpenoid Citral - monoterpenoid (yellow) Lemongrass (Cymbopogon citratus) essential oil Flavonoid – e.g: flavanone Butterfly pea flower Naringenin Flavonoid – (bitter) anthocyanin/anthocyanidin (red, Naringin (bitter), Narirutin (sweet), orange blue, or purple colours) lemon juice e.g; Red/purple cabbage  PHYTOCHEMICALS SCREENING COLLECTION OF PLANT SAMPLES FRESH SAMPLE/S (authentication) DRIED SAMPLE/S Grinding BIOACTIVITY QUALITATIVE EVALUATION ANALYSIS OF Extraction PHYTOCHEMICALS (Infusion, maceration, decoction, soxhlet, distillation etc) Analysis of Analysis of QUANTITATIVE phytochemicals phytochemicals TOXICITY ANALYSIS OF Crude extract EVALUATION PHYTOCHEMICALS Determination/profiling Structure elucidation of Separation, isolation & of constituents by using unknown compounds by purification of constituents by chromatographic method different type of spectroscopy using different type of (Metabolomic methods (MS, UV, IR, NMR 1D- 1H, chromatographic methods 13C & 2D-HMQC, NOESY & HMBC, /chemometric study) (TLC, CR, PTLC, HPLC, GC, Optical rotation) [GC-MS, LC-MS/MS, etc] UHPLC, etc) Pure known compound  Screening of phytochemicals To carry out phytochemical screening the following points must be fulfilled: 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, H-NMR and C-NMR).  Screening of phytochemicals 1- Selection of promising plant materials. Before investing time, effort, and money in phytochemicals, SCREENING is very important to select a promising plant. The choice of the promising plant depends upon the following: 1. A plant that has biological activity. 2. A plant used in folk medicine. 3. A plant that shows particular toxicities. 8  Screening of phytochemicals 2- Proper collection of selected plants. Drug may be collected from: (1) Wild plants, (2) Cultivated plants Wild plant Cultivated plant Disadvantage Advantage 1- Scattered in large or Present in limited area. unlimited area 2- Difficult to reach Easy to reach 3- The collector must be highly The collector must not be skillful skilled botanists person 4- Deficiency may occur due to Continuous supply continuous collection 9  Screening of phytochemicals 2- Proper collection of selected plants. The following precautions should be considered during the stage of collection: i. The proper time of the day, time of the year, and maturity stage of the collection is particularly important because of the nature and quantity of constituents species according to the season and time of collection. (e.g: fruits) ii. The collected plant should be free from any contamination. iii. Collecting plants that are free from disease (i.e. which are not affected by viral, bacterial, or fungal infection). 10  Screening of phytochemicals 3- Authentication of plant material. This may be confirmed by: i. Establishing the identity by a taxonomy expert. ii. Collection of a common species in their expected habitat by a field botanist. iii. By comparing the collecting plant with a voucher specimen (herbarium sheet). 11  Screening of phytochemicals 4- Drying of plant materials. Aim of drying: i. Ease of transport ii. Ease of grinding iii. Inhibit the growth of microorganisms iv. Preservative of active constituents Method of drying: i. Shade and in sunlight (Natural drying) ii. Hot air drying or by freeze-drying (Artificial drying) 12  Screening of phytochemicals 4- Drying of plant materials. Changes may occur during the drying 1. Size and weight 2. Shape and appearance 3. Colour 4. Odour 5. Taste 6. Active constituent 13  Screening of phytochemicals 5- Grinding of the dried plants. Dried materials are usually powdered before extraction, whereas parts of plants (e.g. leaves, etc) can be soaked or macerated with a solvent such as alcohol. Homogenizing is particularly useful for stabilizing fresh leaves by dropping them into the boiling solvent. Dried biomass is ground into small particles using either a grinder or a mil. Plant material is milled first using a course mill and then a fine mill to generate a fine powder. The grinding process is important as effective extraction depends on the size of the biomass particles; large particles will be poorly extracted, whereas small particles have a higher surface area and will therefore be extracted more efficiently. 14  Screening of phytochemicals 6- Garbling of the dried plants. Garbling is the final step in the preparation of a crude drug. Garbling consists of the removal of extraneous matter. Such as other parts of the plant, dirt and added adulterants. Excessive dust can clog percolators and result in a turbid extract which is hard to clarify. 15  Screening of phytochemicals 7- Packing, storage and preservation. 16  Screening of phytochemicals 8- Extraction process There is no general (universal) method for the extraction of plant materials. The precise mode of extraction depends on: 1. The texture of the plant material. 2. The water content of the plant material. 3. The type of substances to be extracted or the nature of active constituents. Extraction is the process of separation and isolation of soluble material (a medicinally active portion of plant materials) through the use of selective solvents and suitable methods of extraction. 17  Screening of phytochemicals 8- Extraction process The principal methods of extraction are: 1. Maceration 2. Percolation 3. Infusion 4. Decoction 5. Digestion 6. Continuous hot extraction technique (Soxhlet extraction process) 7. Liquid-liquid extraction 8. Distillation 18  Screening of phytochemicals 8- Extraction process 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 in a cold/heat tea, alcohol or alcohol-water mixtures, then an identical or at least a very similar method should be used in the laboratory so that the same natural products will be extracted. 19  Screening of phytochemicals 8- Extraction process The choice of extraction procedure depends on the nature of the plant material and the components to be isolated. Alcohol is a general solvent for many plant constituents (most fixed oils are exceptions) and as such may give problems in the subsequent elimination of pigments, resins, etc. Example of solvents usually used: Water, is widely used; light petroleum (essential and fixed oil, steroids). Ether and chloroform (alkaloids, quinines). The extraction of organic bases (e.g. alkaloids) usually necessitates the basification of plant material if a water-immiscible solvent is to be used; for aromatic acids and phenols, acidification may be required. 20  Cold extraction Extraction itself may be performed by repeated maceration with agitation, percolation, or by continuous extraction (e.g. in a Soxhlet extractor). Numerous extraction methods are available, the simplest being cold extraction (in a large flask with the agitation of the biomass using a stirrer) in which the ground dried material is extracted at room temperature sequentially with solvents of increasing polarity: first hexane (or petroleum ether), ethyl acetate, methanol, and finally water). 21  Cold extraction The major advantage of this protocol is that it is a soft extraction method as the extract is not heated and there is little potential degradation of natural products. The use of sequential solvents of increasing polarity enables the division of natural products according to their solubility (and polarity) in the extraction solvents. This can greatly simplify an isolation process. Cold extraction allows most compounds to be extracted, although some may have limited solubility in the extracting solvent at room temperature. 22  Hot percolation In hot percolation, the biomass is added to a round-bottomed flask containing solvent and the mixture is heated gently under reflux. Typically, the plant material is “stewed” using solvents such as ethanol or aqueous ethanol mixtures. The technique is sometimes referred to as total extraction and has the advantage that with ethanol, the majority of lipophilic and polar compounds is extracted. Plant sample 23  Hot percolation Heating the extracts for long periods may also degrade labile compounds; therefore a pilot experiment should first be attempted and extracts assessed for biological activity to ascertain whether this extraction method degrades the bioactive natural products. Be remember, as extraction is never truly total; for example, some highly lipophilic natural products are insoluble in polar solvents (e.g. the monoterpenes). Plant sample 24  Decoction The decoction is used for active ingredients that don’t modify with temperature. In this process the drug is boiled in water for 15 to 60 minutes depending on the plant or the active ingredient to extract. 25  Soxhlet Extraction As fresh solvents enter the apparatus by a reflux condenser, extraction is very efficient and compounds are effectively drawn into the solvent from biomass due to their low initial concentration in the solvent. The method suffers from the same drawbacks as other hot extraction methods (possible degradation of products), but it is the best extraction method for the recovery of big yields of extract. Moreover, providing biological activity is not lost on heating, the technique can be used in drug lead discovery. 26  Soxhlet Extraction The most widely used method to extract plants is the Soxhlet extraction technique. The biomass is placed in a constructed filter paper and it is continuously refluxed. The solvent used is placed in a round bottom flask. Plant sample 27  Supercritical Fluid Extraction Supercritical fluid extraction utilizes the fact that some gases behave as liquids when under pressure and have solvating properties. The most important example is carbon dioxide which can be used to extract biomass and has the advantage that once the pressure has been removed, the gas boils off leaving a clean extract. Carbon dioxide is a non-polar solvent but the polarity of the supercritical fluid extraction solvent may be increased by the addition of modifying agent, which is usually another solvent (e.g. methanol or dichloromethane). 28  Removal of Solvent Removal of solvent should be carried out immediately after extraction, as natural products may be unstable in the solvent. Organic solvent extract usually has been removed by using a rotary evaporator, while aqueous extracts are generally freeze- dried using a lyophilizer. Freeze drying is widely applied whenever a thermolabile substance is of interest. It forms part of the extraction process of several products, for example; antibiotics, antioxidants, hormones, etc. Dried extracts should be stored at -20 °C prior to screening for biological activity as this will decrease the possibility of degradation of bioactive natural products. 29  Removal of Solvent Rotary Evaporator 30  Distillation Fractional distillation has been traditionally used for the separation of the components of volatile mixtures; in phytochemistry, it has been widely used for the isolation of components of volatile oils. On a laboratory scale it is not easy by this method to separate minor components of a mixture in a pure state and gas chromatography is now routinely used. Hydro and steam distillation is much used to isolate volatile oils from plant material. 31  Distillation Special method for volatile oil extraction, is the Clevenger method, and sometimes the enfleurage method is used. Enfleurage is the process of extracting fragrance from flowers by using odorless fats or oils to cap the essential oils. The perfumes of plants like jasmine could only be extracted by the enfleurage method. Steam distillation Hydro distillation enfleurage method 32  Distillation Microwave assisted essential oil extraction (modern technique of extraction process). Further reading; Belwal, et al (2018). A Critical Analysis of Extraction Techniques Used for Botanicals: Trends, Priorities, Industrial Uses and Optimization Strategies. Trends in Analytical Chemistry. 33  Phytochemicals analysis/screening Qualitative Quantitative Steroids, Reducing sugars, Determination of total alkaloids, Triterpenoids, Total flavonoids, Sugars, Total phenolics, Alkaloids, Total saponins, Phenolic compounds, Falvonoids, Total tannins, Saponins, Total glycosides. Tannins, Anthroquinones, Amino acids. 34  Qualitative Standard procedures Sofowara (1993) Trease and Evans (1989) Harbone (1973) 35  Phytochemicals analysis/screening Byadgi, S.A 36  Detection of alkaloids The individual extract is dissolved in dilute hydrochloric acid and filter. The filter is further tested with following reagents for the presence of alkaloids. 37  Detection of carbohydrates The individual extract is dissolved in distilled water and filter. The filter is further tested with Molisch’s Test: 38  Detection of reducing sugars 39  Detection of saponins 40  Detection of phytosterols 41  Detection of phenolic compounds 42  Detection of tannins 43  Detection of flavonoids 44  Detection of proteins and amino acids 45  Detection of terpenoids 46  Quantitative Total phenols determination: Hagerman A., Muller I., Makkar H. (2000) Total alkaloid determination: Harbone. J. (1973) Total flavonoids determination: Kumaran A., Karunakaran R. (2006) Total tannins determination: Van-Burden T, Robinson W. (1981) Total saponins determination: Obdoni B, Ochuko P. (2001) 47  Total phenols determination: Hagerman A., Muller I., Makkar H. (2000) 48  Total alkaloid determination: Harbone. J. (1973) 49  Total flavonoids determination: Kumaran A., Karunakaran R. (2006) 50  Total tannins determination: Van-Burden T, Robinson W. (1981) 51  Total saponins determination: Obdoni B, Ochuko P. (2001) 52  TASK 7 – Individual activity Observe any one (1) of the plant sample a. Search the related article from Google b. Write the main phytochemical class/pure compounds’ name of each sample c. State the method of analysis used d. Present your finding 53  SUMMARY ✓How to screen phytochemicals? ✓How to extract phytochemicals? ✓How to analyze the phytochemicals? Thank you