Wednesday, October 31, 2012

Phytochemical Screening

Phytochemical Screening or Preliminary Test is the first thing to be done before major discoveries of molecules or drug entities are known. It is used to provide concrete knowledge and research to what plant active constituents have potential to benefit mankind.

Materials to needed:

100g of plant sample                                                     0.5N Potassium Hydroxide
80% or 95% ethyl alcohol                                             Diluted Hydrogen Peroxide (5%)
Mayer's reagent                                                            Glacial Acetic Acid
Wagner's reagent                                                          10% NaOH Solution
Gogo extract (10%)                                                      Water Bath
5% Sodium Carbonate                                                  Solution Benzene or Chloroform
1% Ferric Chloride                                                        Solution Octyl Alcohol
10% Sodium Chloride                                                   Solution Petroleum Ether or Hexane
2M Hydrochloric Acid                                                  Separatory Funnel
1N Sodium Hydroxide                                                  Magnesium Oxide
Magnesium Turnings                                                      Kedde Reagent
1% Gelatin                                                                    Concentrated Sulfuric Acid
Gelatin Salt Reagent                                                      Anhydrous Sodium Sulfate
Ferric Chloride Reagent                                                Acetic Anhydride
Sodium Picrate paper                                                    Ammonia Solution

A. Preparation of Extact
Weigh the ground dried plant material, about 100 grams, in an Erlenmeyer Flask, and treat with sufficient amount of 80% ethyl alcohol to completely submerge the material. Note the volume of alcohol used. Put a stopper on the flask and keep the material soaked for one to two days. The mixture will be filtered, rinsing the remaining material on the flask with fresh alcohol, is to be filtered again and added to the first filtrate, this must be done to provide accurate extraction. Collect the plant extract and discard the plant residue. Concentrate the filtrate to about 20ml. Measure the exact volume of the concentrated extract. Record the concentration of the stock plant extract as grams of dried plant material per ml of the extract obtained. Store the extract tightly closed, preferably in the cold condition (0-5 C).
Note: Concentrated extracts should be stored with a trace of toluene or chloroform to prevent fugal growth if kept at room temperature.
  • Data and Calculation:

    Weight of the beaker
    Weight of the beaker + plant sample
    Weight of the plant sample
    Volume of the extract
    Equivalent of plant material per ml of the extact

Equivalent of plant material / ml   =    Weight of Sample
                                                              Volume of Sample

B. Test for Alkaloids

Preliminary Test

Take an equivalent of 2.0grams of stock plant extract. Evaporate to a syrupy consistency over a steam bath. Add 5ml of 2M HCl and heat while stirring for 5 minute then cool. Add about 0.5 g Sodium Chloride, stir and filter. Wash the residue with enough 2M HCl to bring the filtrate to a volume of about 5ml. Place 1ml each of the filtrate to two test tubes and test as follows:

a) One ml of the filtrate add a few drops of Mayer's Reagent.
(+) result is formation of turbid or white precipitate that indicates the presence of alkaloids.

b) One ml is to be added a few drops of Wagner's Reagent.
(+) result is reddish brown colored precipitate indicates the presence of alkaloids

Record the relative amount of precipitation observed as follows:
(+) Slight Turbidity
(++) Definite Turbidity
(+++) Heavy Precipitation

Confirmatory Test

To the remaining three ml of the filtrate, add dropwise enough 28% ammonia until the solution is alkaline to the litmus. Extract the alkaline solution three time with addition of ten ml portions of chloroform. Combine the lower chloroform extracts and reserve the upper aqueous layer. (The alkaline layer is reserved for the test for quaternary and/or amine oxide bases). Evaporate the chloroform extracts until it dries, under the hood and over a steam bath. Take up the residue with 5ml of 2M HCl, stir over a steam bath for two minutes and cool. Filter and divide filtrate into two equal portions and test as in Preliminary test. Positive results indicate the presence of secondary or tertiary alkaloids.

Test for Quaternary and amine oxide bases

Acidify the alkaline aqueous layer obtained above with 2M HCl. Filter and divide into two parts, then use the confirmatory test for alkaloids. A positive results will show that quaternary or amine oxide molecules are present. But a positive result will be resulted when incomplete chloroform extraction is done, thus, should be considered negative for quaternary bases.

C. Test for Saponin Gyclocides

Froth Test

Take an amount of the alcohol extract equivalent to two grams plant material and transfer this to a test tube. In a separate test tube, have one ml of gogo extract to use as a control or standard. Add 10ml of distilled water to each of the test tubes, close tightly and shake both test tubes vigorously for thirty seconds. Stand for thirty minute then observe for a “honeycomb froth”. Compare result of extact with the standard.

(+) result is persistent froth (1 cm height)was observed for 1 hr. indicates the presence of saponins.

Hemolytic Test

Obtain a blood agar plate. Scoop three minicup from blood agar from three areas of the plate with equidistant from one another using the small test tube. Number each agar cup at the bottom of the inverted plate with a marking pen. With a small pipette, put enough extract to one of the agar cups to fill it. Fill the second with the same volume of gogo extract as positive control. Add the last cup with distilled water as negative control. Allow the the plate to be covered and stood undisturbed. After an hour, observer the agar for any zone clearing with the three agar cups. Mesure the diameter of the halo in millimeter.

(+) result is a homolytic zone is observed with the control and the plant extract due to rupture of red blood cells.

Procedure for removing interfering tannins and other plant polyphenolic compounds

Take a volume of the plant extract equivalent to ten grams plant material and evaporate this to incipient dryness over a steam bath. Extract the residue with twenty ml of hot distilled water. Add five drops of 10% Sodium Chloride solution and filter. Subject the saline filtrate with two grams of magnesium oxide to form a slurry and heat over a steam bath for about 10 minutes. Extract the slurry with hot 80% ethanol, then filter. Subject the detannated aqueous ethanol filtrate to the hemolytic test.

Liebermann-Burchard Test

Evaporate an equivalent of ten grams of plant extract until dryness using a water bath. Add about ten ml of hexane or petroleum ether to the cool residue. Stir for a few minutes, allowing to settle and decant off the supernatant. Repeat until all the color has been removed. To the residue, add about ten ml of chloroform and stir for five minutes. Decant into a test tube containing about 100mg of Anhydrous Sodium Sulfate. Shake and filter, dividing the filtrate into two clean dry test tubes. To one portion, add three drops of Acetic Anhydride. Mix gently. Then add one drop of Concentrated Sulfuric Acid, mixing it gently. Observe color changes immediately over a period of one hour. Use other test tube as reference.

(+)  results is reddish violet colour at the junction of the two layers and a bluish green colour in the Acetic Anhydride layer indicates the presence of unsaturated sterols and or/triterpenes.

D. Test for Cardenolides and Bufadienolides

Keller-killiani Test

Have an 80% alcohol extract equivalent to ten grams of plant. Evaporate until dryness over a water bath. Defat extraction by trituration with hexane to remove as much of the color pigments as possible. Discard the hexane solution. Warm the defatted residue over a water bath to remove the residual hexane solvent. Add three ml of FeCl Reagent. Stir to mix well and transfer to a test tube. Hold the test tube in an slant position, and carefully add an ml of Concentrated Sulfuric Acid that allows the acid to roll inside the walls of the test tube. Allow the mixture to stand for a while. Determine any change of color in the junction.

(+) result is presence of reddish brown color which may gradually become bluish or purplish color indicate the presence of two deoxysugars.

Salkwoski Test

Evaporate the equivalent of ten grams of plant extract to dryness using a bath water. Addition of about ten ml hexane or petroleum ether to the cool residue. Stir for a few minutes allowing to settle and decant off the supernatant. Repeat until all the color has been removed. To the residue add about ten ml of Chloroform and stir for five minutes. Decant into a test containing about 100mg of Anhydrous Sodium Sulfate. Shake and filter. Divide the filtrate into equal part. One part is to add one ml of Concentrated Sulfuric Acid by allowing it to run down the inside of the test tube. Hold the test tube at a 45o angle. Note that immediate color changes is formed at the junction of the extract and Sulfuric Acid. Gently mix. Observer the color that immediately changes over a period of an hour. Use the other test tube as standard.

(+) result is the appearance of a red color indicated the presence of unsaturated sterol and /or triterpenes.

Kedde Reaction

To five ml of the 80% ethanolic extract in an evaporating dish. Add five ml of Kedde Reagent and mix well with a stirring rod. To the mixture, add about two ml of 1N Sodium Hydroxide. Mix and determine color development. 

(+) result: blue-violet to purple color is positive result indicating of the presence of the unsaturated lactone ring.

F. Test for Flavonoids

Evaporate to equivalent of ten grams of alcohol plant extract to incipient dryness over a water bath. Cool to room temperature. Triturate the residue with ten ml of pertoleum ether and decant. Repeat triturating of the residue with fresh volumes until the equivalent is almost colorless. Discard the petreoleum ether extract. Dissolve the defatted residue in twenty ml of 80 percent ethyl alcohol. Filter off any insoluble residue. Divide into 4 parts, labeling A,B,C and D. Keep test tube A as control.

Bate-Smith and Metcalf Test for Leucoanthocyanins 
Test tube B is to add half a ml of Concentrated HCl. Observe changes in color. Record the data. Warm the test tube on a water bath for fifteen minutes. Observe any color change within an hour, record results. Gradual development of a strong red or violet color is indicative of the precense of Leucoanthocyanins. Color formation may be dramatically slow. If the color is not immediately apparent, allow the test solution to stand at room temperature for about an hour before getting the result as negative.

Wilstatter “Cyanidin Test”

To test tube C, add a half ml of Concentrated HCl, and three or four pieces of magnesium ribbons. Take note of any color change (green, red ….) within ten minutes. Compare with test tube that serves as control. NOTE: Should definite appearance of coloration occur, cool. Dilute with an equal volume of water and add one ml of Octyl Alcohol. Shake and allow to separate. Note color in each layer.

(+) result is orange to red or crimson and magenta or greenish-blue color, which is reduction of magnesium metal

Test For Anthraquinone

Borntrager Test

Steam bath one gram of alcoholic plant extract to evaporate into dryness. Have a residue in ten ml of distilled water and filter. Discard the residue. Extract the filtrate with five ml of benzene extracts into two portions. Dividing the two combined benzene extracts into two equally portions. Reserve one as control. To the second portion add five ml of ammonia solution, then shake. Observe the alkaline layer for color changes.

(+) result is presence of red coloration.  because of sublimation yellow crystals of anthraquinones reacts with KOH. 

Modified Borntrager Test

Evaporate the equivalent of one gram plant extract to the incipient dryness using a water bath. The residue is taken up with ten ml of 0.5N KOH and a ml of diluted (5%) Hydrogen Peroxide and stir. Heat on a water bath for ten minutes. Cool and filter. Discard the residue. Add Glacial Acetic Acid dropwisely until acidic to litmus paper. Extract with five benzene, two times. Divide the combined benzene extracts into two portions. Reserve one portion as control. Two to five ml of ammonia solution is to be added on second portion until alkaline. Observer the alkaline layer for color changes.

(+) result is pink color, because of sublimation yellow crystals of anthraquinones reacts with KOH. 

Test For Cyanogenic Glycosides

Guignard Test

Place two to five grams of crushed plant sample in a test tube. Moisten with water and add a few drops of Chloroform to enhance enzyme activity. One ml of one percent emulsion solution may also be added to insure hydrolysis of the glycoside. For a firm stopper on the tube, use cork from which is suspended a piece of picrate paper. The paper strip must not be touched the inner sides of the test tube. Warm the tube at 35-40 C or keep it at room temperature for two to three hours. Observe any change in color of the paper. 

(+) result is the appearance of various shades of red within fifteen minutes is a measure of relative concertration of cyanogenic glycosides. If no color is observed after three hours, absence of glycoside is indicated.

Test for Tannins and Polyphonels

Take the etanolic extract equivalent to ten grams of plant material. Evaporated to incipient dryness over a water bath and cool. Add twenty ml of hot distilled water to the residue. Mix well with stirring rod and allow to cool at room temperature spontaneously. Add five drops of 10% Sodium Chloride solution to salt out undesirable constituents. Filter and divide the filtrate into four test tubes A,B,C and D. Reserve the contents of test tube A as standard.

Gelatin Test

To test tube B, add three drops of one percent Gelatin Solution. To test tube C, add three drops of gelatin-salt reagent. Observe any formation of a precipitate. 

(+) result: Jelly precipitate of the gelatin indicates the presence of tannins.

Ferric Chloride Test

To test tube D, add five drops of Ferric Chloride Solution. Observe any color change or formation of precipitate. 

(+) result: A blue-black color may indicate the presence of hydrolysable tannins, while brownish-gree color or greenish blue or greenish black may indicate condensed tannins, if the gelative is positive. Polyphenolic compounds give a negative test.

Test for Resin

Phloroglucino Test

Evaporated the one gram equivalent plant extract to incipient dryness using a water bath. Add few drops of phloroglucinol solution and hydrochloric acid to the residue and observe. 

(+) result is that appearance of a heavy bloody red color.

Test for Fixed Oils and Volatile Oils

Stain Test

Boil two grams of the plant sample in ten ml of petroleum ether. Take a piece of a white paper, put two drops of petroleum ether extract. Note any stain produced. 

(+) result is permanent oily stain or spot is observed.

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