PHYTOCHEMICAL SCREENING.pptx- plant extraction procedures

PHYTOCHEMICAL SCREENING By: Kristel A. Honrado-Peralta, RPh Kristel A. Honrado- Peralta, RPh

Objectives To identify the different primary and secondary metabolites present in different medicinal plants. To acquire the skills in identifying different plant constituents. Kristel A. Honrado- Peralta, RPh

Discussion Phyto is a Greek word that means plant. Different methods can be used to determine the different constituents present in plants. One of the methods of identifying the constituents is through phytochemical screening. Kristel A. Honrado- Peralta, RPh

A method for use for phytochemical screening should be: Simple Rapid Designed for a minimum of equipment Resonably selective for the class of compounds under study Quantitative in so far as having a knowledge of the lower limit of detection is concerned; and if possible Should give additional information as to the presence or absence of the specific members of the group being evaluated. Kristel A. Honrado- Peralta, RPh

Phytochemical investigations of plants involves the following: Authentication and extraction of plant material Separation and isolation of the constituent of interest Characterization of isolated compounds Investigation of biosynthetic pathways to particular compounds Quantitative evaluation Pharmacological assessment of the separated components Kristel A. Honrado- Peralta, RPh

Extraction of plant material: Extraction depends on the nature of the plant material and the components to be isolated. Dried materials should be powdered before examination. Fresh materials can be homogenized or soaked to a solvent such as alcohol. Alcohol general solvent for extraction Light petroleum (essential, fixed oils, steroids) Ether and chloroform (alkaloids) Water immiscible solvent (alkaloids) Acidification (aromatic acids & phenols) Extraction Kristel A. Honrado- Peralta, RPh

Extraction of plants procedures Kristel A. Honrado- Peralta, RPh

Kristel A. Honrado- Peralta, RPh

Materials / Reagents Potassium hydroxide 0.5M Sodium chloride 0.9% Sodium chloride 10% Ammonia 28% Hydrochloric acid 2M Hydrogen peroxide 5% Ethyl alcohol 80% Acetic acid anhydride Agar powder anhydrous sodium sulfate Benzene Chloroform Hydrochloric acid 12M Conc. sulfuric acid Dichloromethane Distilled water Draggendorff’s reagent Fehling’s solution (A and B) Ferric chloride TS Gelatin powder Glacial acetic acid Hexane Nitric acid Magnesium turnings Mayer’s reagent Millon’s reagent Octyl alcohol Kristel A. Honrado- Peralta, RPh

Materials / Reagents Petroleum ether Soda solution Sodium chloride Tannic acid solution Wagner’s reagent Cheesecloth Cork/ stopper Freshly extracted cow’s blood Filter paper Gugo bark Hot plate Laboratory glassware / materials Medicinal plant Petri dish Reflux condenser Rotavapor Water bath Kristel A. Honrado- Peralta, RPh

Procedure Collection and preparation of samples Collect for phytochemical screening. Wash, air-dried and oven dried at 40 ° C and grind. Phytochemical screening of samples Preparation and extraction of samples 1. Weigh 100g of the powdered sample; place it in a 500mL Erlenmeyer flask. 2. Macerate it with sufficient amount of 80% ethyl alcohol to completely submerge the material. 3. Keep the material soaked for 24-48 hours. Reflux distillation could be used to further extract the constituents. 4. Filter and rinse the flask with fresh portions of 80% ethyl alcohol. Discard the residue. Kristel A. Honrado- Peralta, RPh

Procedure 5. Collect the filtrate and evaporate it using rotavapor to separate the solvent from the extract. 6. Further concentrate the extract to about 50mL at a controlled temperature of 40 ° C. 7. Measure the volume of the concentrated extract. 8. Compute for the concentration of the extracts in grams of dried plant material per mL extract. 9. Store the extract in a tightly closed container in a cool temperature. Label the container properly with the name of the sample, concentration of the plant extract in grams per mL, and the date of extraction. Kristel A. Honrado- Peralta, RPh

Procedure Note: To prevent fungal growth, the researcher should add small amount of chloroform. Formula Equivalent weight/mL Percentage yield x 100 Kristel A. Honrado- Peralta, RPh

Test for alkaloid analysis Test Reagent/s used Positive Result/s Preliminary test 2-3 gtts of Mayer’s rgt ., Wagner’s rgt ., Draggendorf’s rgt . (+) slight turbidity (++) definite turbidity (+++) heavy turbidity Kristel A. Honrado- Peralta, RPh

Test for alkaloid analysis Test Reagent/s used Positive Result/s Confirmatory test 2-3 gtts of Mayer’s rgt ., Wagner’s rgt ., Draggendorf’s rgt . Presence of: 1 ° , 2° , 3° alkaloids Kristel A. Honrado- Peralta, RPh

Test for amines (video) Uses the same reagent for the test of alkaloids and yields precipitate indicated by +, ++, +++. Kristel A. Honrado- Peralta, RPh

Test for alkaloid analysis- Video Kristel A. Honrado- Peralta, RPh

Test for quaternary bases / amine oxide Video Kristel A. Honrado- Peralta, RPh

Important tests for alkaloids Chemical test Reagent Result / Observation Mayer’s Potassium mercuric iodide Creamy precipitate (ppt) Wagner’s Potassium triiodide sol’n Redish brown ppt Hager’s Picric acid Yellow ppt Dragendorff’s Potassium bismuth iodide Redish brown ppt Sonneschein’s Phosphomolybdic acid Formation of ppt Kristel A. Honrado- Peralta, RPh

Important tests for alkaloids Chemical test Reagent Result / Observation Scheibler’s Phosphomolybdic acid Formation of ppt Tannic acid N/A Formation of ppt Murexide test Caffeine + HCl + KCl 3 Purple color Von Urk (indole alkaloid) P- dimethylamminobenzaldehyde Blue color Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) Glycosides are organic compounds in which a hemiacetal linkage usually connects the anomeric carbon of a sugar ( glycone ) with an alcohol or phenolic hydroxyl of a second non-sugar molecule (aglycone) this type of linkage rise to the so-called o- glycosides (e.g. , salicin ) the most common type of glycosides found in plants. Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) T he sugar glucose may be the most commonly known hemiacetal. A hemiacetal is a carbon connected to two oxygen atoms, where one oxygen is an alcohol (OH) and the other is an ether (OR) . Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) Glycosides represent a large group of secondary metabolic products derived from plants, demonstrating several known functions, including growth regulation, allelopathy (inhibition of other plant growth), and defense mechanisms against damage induced by herbivores and pathogens [1]. They are composed of two bound portions, a sugar moiety, named glycone , and a second portion known as the aglycone or genin . The aglycone is bound to the anomeric carbon (C-1 carbon) of the glycone . Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) The breakdown of glycosidic linkages can be achieved by enzymes (such as β-glucosidases) or acids. The synthesis of glycosides by plants is based on the modification of secondary metabolites by glycosyltransferases , and some glycosides require additional reactions, including oxidation , acylation , and degradation [1,2]. Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) The glycone moiety allows the glycoside to be soluble in water. Usually, glucose is a monosaccharide , but could also be a di-, tri-, or tetrasaccharide . The most common glycone is glucose, and the formed glycosides are known as glucosides . Other typical glycones include l-rhamnose (forming rhamnosides ), l-fructose ( fructosides ), l-arabinose (arabinosides), and glucuronic acid (glucuronides). Based on the number of saccharides , glycones can be classified as monodesmoside or monoside (one), bidesmoside or bioside (two), and tridesmoside or trioside (three). Kristel A. Honrado- Peralta, RPh

Screening for glycosides ( heteroglycosides ) Kristel A. Honrado- Peralta, RPh

Chemical tests for glycosides Kristel A. Honrado- Peralta, RPh

Chemical tests for glycosides Test Reagent / chemical used Observation /result Steroids: Cardenolides & Bufadienolides Hexane Reddish- brown may turn blue / purple (indicates presence of: 2- deoxysugars ) Keller – killani: 2- deoxysugars FeCl 3, Conc. H 2 SO 4 Bluish color Kristel A. Honrado- Peralta, RPh

Chemical tests for glycosides Test Reagent / chemical used Observation /result Liebermann – Burchard test: Unsaturated steroids/ triterpenoid skeleton Na 2 SO 4 Dichloromethane (CH 2 Cl 2 ) Acetic acid Conc. H 2 SO 4 From blue  gree , red, purple, pink, violet Salkowiski’s : Unsaturated sterol/terpenes Chloroform (CCl 3 ) Red color Kristel A. Honrado- Peralta, RPh

Test for anthraquinone glycosides The largest group of naturally occurring quinine substances are the anthraquinones. Although they have a widespread use as dyes, their chief medicinal value is dependent upon their cathartic action. They are restricted distribution in the plant kingdom and are found most frequently in members of Rhamnaceae , Polygonaceae , Rubiaceae , Leguminosae and Liliaceae. As found in plant, they are usually carboxyllated , methylated, or hydroxyllated , derivatives of anthracenes, anthrone , anthranol , anthraquinone, or dianthrone. Kristel A. Honrado- Peralta, RPh

Test for anthraquinone glycosides Test Reagent / chemical used Observation /result Borntrager’s Benzene Red color Modified Borntrager’s KOH, H 2 O 2, Benzene Pink color Kristel A. Honrado- Peralta, RPh

Test for flavonoids Flavonoids refers to the class of plant secondary metabolites based around a phenyl benzopyrone structure. Commonly known antioxidant. Commonly referred bioflavonoids in the media The flavonoid synthetic pathway begins with a product of glycolysis phosphoenolpyruvate entering into the shikimate pathway to yield phenylalanine. Kristel A. Honrado- Peralta, RPh

Test for flavonoids Test Reagent / chemical used Observation /result Leucoanthocyanins : Bate- Smith & Metcalf T.M. Conc. HCl Strong red/ violet color Y-Benzopyrone nucleus: Wilstatter “ Cyanidine ” Conc. HCl Mg turnings Octyl ROH Orange -> red, crimson, magenta Occasionally green -> blue Kristel A. Honrado- Peralta, RPh

Screening methods for saponins Test Reagent / chemical used Observation /result Froth Gugo extract Ethyl ROH “Honeycomb” froth > 2cm Hemolytic: Agar- cup method Gugo extract Cow’s blood Clear zones for hemolysis “ hemolytical halos” Kristel A. Honrado- Peralta, RPh

Screening for tannins Two group of phenolic constituents, hydrolysable & condensed, comprise the tannins, substances which are important economically as agents for the tanning of leather and for certain medicinal purposes. Potential value as cytotoxic neoplastic agents. Kristel A. Honrado- Peralta, RPh

Properties of tannins Hydrolysable tannins Yellow -brown amorphous substances Dissolve in hot water to form colloidal dispersions Esters that can be hydrolyzed by boiling with dilute acid to yield a phenolic compound – usually a derivative of gallic acid & a sugar referred as “pyrogallol tannins” Kristel A. Honrado- Peralta, RPh

Properties of tannins Condensed tannins Polymers of phenolic compounds related to the flavonoids & are similar in general prop. Of HT but are not very soluble in water Phlabapenes / tannins- red – formed from treatment with boiling dilute acid red-brown insoluble polymers Kristel A. Honrado- Peralta, RPh

Test for tannins Test Reagent / chemical used Observation /result Gelatin Salt solution (NaCl sol’n ) Jelly ppt Ferric chloride Ferric chloride (FeCl 3 ) Hydrolyzable : Blue > black color NHT/ Condesed T.: Brownish-green color Kristel A. Honrado- Peralta, RPh

Screening for carbohydrates Test Reagent / chemical used Observation /result Fehling’s Freshly prepared fehling’s sol’n Reducing sugars: Brick-red Molisch’s α - naphtol + H 2 SO 4 Carbohydrates / Glycosides: Bluish-violet zone Benedict’s Benedict’s rgt Reducing sugars: red ppt Kristel A. Honrado- Peralta, RPh

Screening for proteins Test Reagent / chemical used Observation /result Millon’s Millon’s rgt Flesh color Xanthoproteic HNO 3 Yellow ppt Kristel A. Honrado- Peralta, RPh

Screening for lipids Test Reagent / chemical used Observation /result Lipid’s Petroleum ether, Hexane Permanent greasy stain on paper Kristel A. Honrado- Peralta, RPh

Thank you for active listening! Watch videos on how these tests are done for further understanding. Keep the faith future pharmacists! -mam Tel  Kristel A. Honrado- Peralta, RPh

PHYTOCHEMICAL SCREENING.pptx- plant extraction procedures

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PHYTOCHEMICAL SCREENING.pptx- plant extraction procedures

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