Procedure
Content
Compiled Methods I. Ash Content A. Preparation of Sample The gross sample is quartered repeatedly to prepare the laboratory sample. Quartering consists of placing the sample, adequately mixed, as an even and square shaped heap and dividing it diagonally into four equal parts. The two opposite parts are then taken and carefully mixed. The process is repeated as necessary until the required quantity is obtained. B. Total Ash Accurately weigh a quantity of the test sample, representing 2 to 4 g of the air dried material, in a tared crucible and incinerate, gently at first, and gradually increase the temperature to 675 25° until free from carbon, and determine the weight of the ash. If a carbon free ash cannot be obtained in this manner, extract the charred mass with hot water, collect the insoluble residue on an ashless filter paper, incinerate the residue and filter paper until the ash is white or nearly so, then add the filtrate, evaporate it to dryness, and heat the whole to a temperature of 675 25°. If a carbon free ash cannot be obtained this way, cool the crucible, add 15mL of alcohol, break up the ash with a glass rod, burn off the alcohol, and again heat the whole sample to a temperature of 675 25°. Cool in a desiccators, weigh the ash, and calculate the percentage of total ash from the weight of sample taken. Formula: ( ) Where C= weight of ash, B= weight of sample C. Acid-Insoluble Ash Boil the Ash obtained as directed under Total Ash, with 25mL of 3N hydrochloric acid for 5 minutes, collect the insoluble matter on a tared filtering crucible or ashless filter paper, wash with hot water, ignite, and weigh. Determine the percentage of acid insoluble ash calculated from the weight of sample taken. Formula: ( ) Where D= weight of insoluble ash, B= weight of sample D. Water-Soluble Ash Boil the ash obtained from Total Ash with 25 mL of water for 5 minutes. Collect the insoluble matter with a sintered glass crucible or on an ashless filter paper. Wash with Hot water, and ignite for 15 minutes at a temperature not exceeding 450°. Subtract the weight of this residue, in mg, obtained under Total Ash, and calculate the percentage of water soluble ash with reference to the weight of sample as determined under Total Ash. Formula ( ) Where C= weight of ash (in total ash), E=weight of residue in water-soluble ash, B= weight of sample
II. Microbiological Tests A. KIRBY-BAUER/DISC-DIFFUSSION METHOD Materials: Nutrient agar (Mueller-Hinton) Petri dishes (at least 6) Test organism suspension Plant extract (from Phytochem screening) Whatman filter paper no. 1 Cotton swabs Forceps Procedure:
ALWAYS OBSERVE PROPER ASEPTIC TECHNIQUES.
1
Preparation of Mueller-Hinton agar 1. Müeller-Hinton agar should be prepared from a commercially available dehydrated base according to the manufacturer's instructions. o 2. Immediately after autoclaving, allow it to cool in a 45 to 50 C water bath. 3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed Petri dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm (60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm) 4. The agar medium should be allowed to cool to room temperature and, unless the plate is used o the same day, stored in a refrigerator (2 to 8 C).
Plates should be used within seven days after preparation unless adequate precautions, such as wrapping in plastic, have been taken to minimize drying of the agar.
Preparation of dried filter paper discs 1. Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter. (Use office puncher if available) 2. Placed discs in a Petri dish. 3. Sterilized in a hot air oven. Inoculation of Test Plates 1. Dip a sterile swab into the inoculum tube. 2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid. The swab should not be dripping wet. 3. Inoculate the dried surface of a MH agar plate by streaking the swab three times over the entire agar surface.
4. 5. 6. 7.
Rotate the plate approximately 60 degrees each time to ensure an even distribution of the inoculums. Rim the plate with the swab to pick up any excess liquid. Discard the swab into an appropriate container. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to the next step.
Application of Discs to Inoculated Agar Plates 1. Flame sterilize a pair of forceps until blue alcohol flame disappears. 2. Let cool for a few seconds. 3. Pick out one paper disc with forceps and immerse the paper disc into the plant extract for assay. 4. Lay the moistened filter disc gently on the cooled agar plate. 5. Press down disc on the agar surface. Never relocate a disc once it has come into contact with the agar surface (Distance between discs - 24 mm or more from center to center)
Plant extract from location 1 Plant extract from location 2 Plant extract from location 3 standard
blank
6. 7.
Incubate the plates inverted for one week. Measure zone sizes.
REASONS FOR IMPORTANT STEPS: 1. Use of Mueller-Hinton Agar Medium The medium used in this test has to be the Mueller-Hinton agar because it is an agar that is thoroughly tested for its composition and its pH level. It is considered the best medium to be used for routine susceptibility testing of non-fastidious bacteria because it shows acceptable batch-tobatch reproducibility for susceptibility testing, it is low in sulfonamide, trimethoprim, and tetracycline inhibitors and it supports satisfactory growth of most non-fastidious pathogen. A large body of data and experience has been collected concerning susceptibility tests performed with 1 this medium.
2. 3. 4.
The swab should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. Each disc must be pressed down to ensure complete contact with the agar surface. A disc should never be relocated once it has come into contact with the agar surface because some of the drug diffuses almost instantaneously.
3
B. MINIMUM INHIBITORY CONCENTRATION DETERMINATION Materials: Diluted bacterial inoculum Plant extract (from phytochem screening) Test tubes with cap (13 per microorganism) Pipettes (5.0mL and 1.0mL)
Procedure: 1. Sterilize 13 screw capped test tubes and number each accordingly. 2. With a sterile 5.0mL serological pipette, introduce 1.0mL of Mueller-Hinton broth into tubes #2 to #11. To test tube #12, introduce 2.0mL of the broth. 3. Pipette 1.0mL of the plant extract into test tube #1 and #2, cap the tubes well. 4. Shake gently or vortex the contents of tube #2 for 5 seconds. 5. With a sterile 1.0mL serological pipette, aseptically withdraw 1.0 mL from the contents of tube #2 and transfer this to tube #3, cap the tube. Shake or vortex the contents of tube #3. 6. With another clean sterile 1.0mL pipette, aseptically withdraw 1.0mL from the contents of tube #3 and transfer this to tube #4, cap the tube. Shake or vortex the contents of tube #4. 7. Continue this process until 1.0mL has been withdrawn from tube #9 and subsequently added to tube #10, cap the tube. Shake or vortex the contents of tube #10. 8. Pipette off 1.0mL from the contents of tube #10 and discard this. 9. Introduce 1.0mL of the diluted bacterial inoculums into tubes #1 to #11 and to tube #13. 10. To tube #13, add 1.0mL of the antibiotic standard. 11. With all the tubes tightly capped, gently shake or vortex the contents of the tubes. o 12. Incubate tubes at 35 C for 18 to 24 hours. 13. After incubation period, examine the tubes for bacterial growth (turbidity in the tube or whitish pellet at the bottom of the tube). 14. The tube with the lowest concentration of plant extract at which no growth or turbidity is observed is reported as the MIC of the plant extract against the bacterial test organism.
Contents Volume of MuellerHinton Broth (mL) Concentration of extract (microg/mL) Volume of diluted inoculums (mL) Antibiotic standard Total volume (mL)
1 None 4000 1 None 2 Highest conc of plant extract
2 1 2000 1 None 2
3 1 1000 1 None 2
4 1 500 1 None 2
5 1 250 1 None 2
6 1 125 1 None 2
Test tube number 7 8 1 62.5 1 None 2 1 31.3 1 None 2
9 1 15.6 1 None 2
10 1 7.8 1 None 2 Lowest conc of plant extract
11 1 None 1 None 2 Negative control
12 2 None None None 2 Media control
13 None None 1 1 2 Positive control
III. Phytochemical Screening Preliminary Tests on Plant Materials: Preparation: Warm 5-10g with about 25-50 mL of water on a water bath 50-60⁰C for 15 minutes, cooled, and filtered.
Test pH Tannins Glycosides
Procedure Test the extract using pH paper or litmus paper Add a few drops of ferric chloride TS to 1mL extract Add a few drops of lead acetate TS to about 2 mL of extract. Filter the extact then add lead subacetate TS until weakly alkaline.
Reducing substances
Alkaloids
Plant acids
Mix equal amounts of Fehling’s A and Fehling’s B, 1 mL of each and add to an equal amount of the extract. Heat to boiling. If there is no precipitate, add hydrochloric acid and heat to boiling. Add sodium hydroxide until neutral or alkaline and again add Fehling’s solution A & B. Heat to boiling. Acidify extract with 1% HCl and add one or two drops of Mayer’s reagent (mercuric-potassium iodide TS). Additional tests: Valser’s reagent (mercuric iodide TS) Wagner’s reagent (iodine TS) Dragendorff’s reagent (bismuth potassium iodide TS) Add a few mL of sodium carbonate TS to a boiled extract.
Positive Result Red litmus: acidic Blue litmus: basic Blue-black precipitate is indicative of tannins Precipitation shows the presence of glycosides, mucins, tannins, & proteins. Precipitation or turbidity shows the presence of glycosides Brick-red precipitate shows the presence of reducing sugars as hydrolytic product.
Precipitation is indicative of the presence of alkaloids
Saponins
Shake the extract vigorously for 30 seconds. Stand in a vertical position for 30 minutes.
A stable and dense froth is indicative of the presence of free acids (stearic, diterpene acids, tripterpenedicarboxylic acids) A honeycomb froth greater than 3 cm above the surface of the liquid indicates the presence of saponins
Saponins
Preparation: weight 15 g of the plant material and slowly boil in 50 mL alcohol for one hour in an Erlenmeyer flask covered with a funnel. Filter. Set aside ¼ of the alcoholic extract for test for flavonoids. Use the remaining ¾ of the alcoholic extract for tests 1 and 2. Differentiation test for saponins Evaporate the alcoholic extract to dryness. Cool. If colored, stir the dried extract for a few minutes with 10 mL of petroleum ether, repeat extraction until most of the color is removed. Discard the petroleum ether extract. This part can be omitted if the dried extract is not much colored. Add to the dried extract 10 mL of chloroform and stir for 5 minutes. Decant into a test tube containing about 100 mg anhydrous sodium sulfate. Shake, filter, and divide filtrate into three clean, dry, test tubes. Use one test tube as control. Liebermann-Burchard Test Add three drops acetic anhydride to the first test tube. Mix gently. Add one drop of concentrated sulfuric acid and mix gently. Observe color changes immediately and over a period of one hour. Result (+) blue, blue green, or green (+) red, pink, purple, or violet (-) pale yellow Substances Present Steroidal saponins Terpenoidalsaponins Saponins of saturated sterols or saturated triterpenes
Salkowski Test Hold the second test tube at 45⁰ angle, layer, 1-2 mL of concentrated sulfuric acid by allowing it to run down the inside of the test tube. Note any immediate changes at the junction of the extract and sulfuric acid. Gently mix the sulfuric acid and the extract and observe for immediate and/or gradual changes over one hour period. A cherry red color is indicative of the presence of unsaturated steroids. Test of Saponins and Sapogenins Take 5 mL of the alcoholic extract and add a few drops of a saturated alcoholic solution of cholesterol. Observe crystal formation. Test for Flavonoids 1. To an alcoholic solution of plant material add a small piece of magnesium ribbon, followed by the dropwise addition of concentrated HCl. Colors will develop within 1-2 minutes following the addition of the acid subject to variation in intensity depending on the concentration of flavonoid present in the sample. a. Colors ranging from orange to red (flavones) b. Colors ranging from red to crimson (flavanols) 2. Digest a few grams of the sample plant material with 2N HCl in 1-propanol for 15-30 minutes. A slow development of a strong red or violet color is indicative of a positive reaction for the presence of flavonoids.
Glycosides
Preparation: Place 50 g of the dried, powdered plant material into 500 mL Erlenmeyer flask. Add 150 mL of 80% ethanol; Fit into the neck of the flask a stopper with a piece of long glass tubing. The glass tubing will serve as an air-cooled reflux condenser. Place the set-up on a water bath and reflux for 1 hour. Cool to room temperature and filter the mixture. Wash the residue with two 25-mL portions of 80% alcohol. Express any solvent remaining in the plant material using a glass rod. Discard the exhausted marc. Cardiac Glycosides Presence of unsaturated sterols Preparation: Evaporate the equivalent of 10 g of plant extract to dryness using a water bath in the hood. Allow it to cool to room temperature – the extracts should be either dry or syrupy. Add 10 mL hexane, stir and allow to settle. Decant and discard the supernatant. Repeat until the hexane extract has removed most of the pigmented substances. Add 10 mL of chloroform to the residue and stir for 5 minutes. Decant into a test tube and dry with anhydrous sodium sulfate. Shake and filter into a clean dry test tube. Divide the filtrate equally into 3 separate test tubes. Set aside a test tube to serve as a color control for the following tests: test Libermann-Burchard test procedure Add 3 drops acetic anhydride. Mix gently by swirling the test tube. Add 1 drop sulfuric acid and mix gently. Observe for 1 hour. result blue, blue green, or green (+ for steroidal saponins) red, pink, purple, or violet (+ for terpenoidalsaponins) pale yellow (+ for saponins of saturated sterols or saturated triterpenes) Cherry red color (+ for unsaturated sterols)
Salkowski Test
Hold the test tube at 45⁰ angle, layer, 1-2 mL of concentrated sulfuric acid by allowing it to run down the inside of the test tube.
Presence of 2-deoxy sugars Test Keller-Killiani Test Procedure Place 10 mL of the 80% ethanolicextract in an evaporating dish and dry on a steam bath. Add 3 mL of freshly prepared ferric chloride reagent (0.3 mL of 10% ferric chloride with 50 mL glacial acetic acid) stir and transfer to a test tube. Perform a ring test by layering 1 mL of sulfuric acid at an angle. Result Purple ring (+ for 2-deoxy sugars)
Anthraquinone Glycosides test Borntrager test Procedure Evaporate the equivalent of 1 g of plant extract to dryness using a water bath. Dissolve the residue in 30 mL of distilled water and filter. Shake the filtrate with 10 mL benzene in a separatory funnel and allow the mixture to separate. Test the benzene layer with 5 mL ammonia TS and shake. Result Red alkaline layer (+ for anthraquione glycosides)
Cyanogenic Glycosides test Guignard test Procedure Moisten 2-5 g sample. Prepare sodium picrate strip by dipping filter paper strip into the solution. Dry, blot, add 1 mL chloroform to the flask containing the moistened sample. Insert the filter paper strip in the flask & fold over the rim. Stopper. Heat to 35⁰C for 2 hours Result Yellow color of the filter paper turns to red in 15 minutes (+ for cyanogenic glycosides)
Tannins
Preparation: Comminute the sample and add 20 mL of distilled water. Boil for 15 minutes. Use 5 mL portions In the following tests: Test General test for tannins Procedure Add 2 mL of potassium ferricyanide TS to 5 mL of the extract. Add concentrated ammonia to the resulting solution. Soak a small piece of goldbeater’s skin in 2% HCl. Rinse with distilled water and place in the extract for 5 minutes. Wash with distilled water and transfer to a 1% ferrous sulfate solution. Dip a matchstick (wooden end) in the extract. Dry. Moisten with conc. HCl and warm near a flame. Add a few mL of to the aqueous extract and expose the resulting solution to air for quite some time. Result Blue-black precipitate (+ for gallitannins and ellagitannins) Brownish-green (+ for condensed tannins) Brown or black color (+ for tannins)
Goldbeater’s Skin Test
Test for Catechin
Pink or red (+ for phologlucinol)
Test for Chlorogenic Acid
Green color (+ for chlorogenic acid)
II. Microbiological Tests A. KIRBY-BAUER/DISC-DIFFUSSION METHOD Materials: Nutrient agar (Mueller-Hinton) Petri dishes (at least 6) Test organism suspension Plant extract (from Phytochem screening) Whatman filter paper no. 1 Cotton swabs Forceps Procedure:
ALWAYS OBSERVE PROPER ASEPTIC TECHNIQUES.
1
Preparation of Mueller-Hinton agar 1. Müeller-Hinton agar should be prepared from a commercially available dehydrated base according to the manufacturer's instructions. o 2. Immediately after autoclaving, allow it to cool in a 45 to 50 C water bath. 3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed Petri dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm (60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm) 4. The agar medium should be allowed to cool to room temperature and, unless the plate is used o the same day, stored in a refrigerator (2 to 8 C).
Plates should be used within seven days after preparation unless adequate precautions, such as wrapping in plastic, have been taken to minimize drying of the agar.
Preparation of dried filter paper discs 1. Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter. (Use office puncher if available) 2. Placed discs in a Petri dish. 3. Sterilized in a hot air oven. Inoculation of Test Plates 1. Dip a sterile swab into the inoculum tube. 2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid. The swab should not be dripping wet. 3. Inoculate the dried surface of a MH agar plate by streaking the swab three times over the entire agar surface.
4. 5. 6. 7.
Rotate the plate approximately 60 degrees each time to ensure an even distribution of the inoculums. Rim the plate with the swab to pick up any excess liquid. Discard the swab into an appropriate container. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to the next step.
Application of Discs to Inoculated Agar Plates 1. Flame sterilize a pair of forceps until blue alcohol flame disappears. 2. Let cool for a few seconds. 3. Pick out one paper disc with forceps and immerse the paper disc into the plant extract for assay. 4. Lay the moistened filter disc gently on the cooled agar plate. 5. Press down disc on the agar surface. Never relocate a disc once it has come into contact with the agar surface (Distance between discs - 24 mm or more from center to center)
Plant extract from location 1 Plant extract from location 2 Plant extract from location 3 standard
blank
6. 7.
Incubate the plates inverted for one week. Measure zone sizes.
REASONS FOR IMPORTANT STEPS: 1. Use of Mueller-Hinton Agar Medium The medium used in this test has to be the Mueller-Hinton agar because it is an agar that is thoroughly tested for its composition and its pH level. It is considered the best medium to be used for routine susceptibility testing of non-fastidious bacteria because it shows acceptable batch-tobatch reproducibility for susceptibility testing, it is low in sulfonamide, trimethoprim, and tetracycline inhibitors and it supports satisfactory growth of most non-fastidious pathogen. A large body of data and experience has been collected concerning susceptibility tests performed with 1 this medium.
2. 3. 4.
The swab should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. Each disc must be pressed down to ensure complete contact with the agar surface. A disc should never be relocated once it has come into contact with the agar surface because some of the drug diffuses almost instantaneously.
3
B. MINIMUM INHIBITORY CONCENTRATION DETERMINATION Materials: Diluted bacterial inoculum Plant extract (from phytochem screening) Test tubes with cap (13 per microorganism) Pipettes (5.0mL and 1.0mL)
Procedure: 1. Sterilize 13 screw capped test tubes and number each accordingly. 2. With a sterile 5.0mL serological pipette, introduce 1.0mL of Mueller-Hinton broth into tubes #2 to #11. To test tube #12, introduce 2.0mL of the broth. 3. Pipette 1.0mL of the plant extract into test tube #1 and #2, cap the tubes well. 4. Shake gently or vortex the contents of tube #2 for 5 seconds. 5. With a sterile 1.0mL serological pipette, aseptically withdraw 1.0 mL from the contents of tube #2 and transfer this to tube #3, cap the tube. Shake or vortex the contents of tube #3. 6. With another clean sterile 1.0mL pipette, aseptically withdraw 1.0mL from the contents of tube #3 and transfer this to tube #4, cap the tube. Shake or vortex the contents of tube #4. 7. Continue this process until 1.0mL has been withdrawn from tube #9 and subsequently added to tube #10, cap the tube. Shake or vortex the contents of tube #10. 8. Pipette off 1.0mL from the contents of tube #10 and discard this. 9. Introduce 1.0mL of the diluted bacterial inoculums into tubes #1 to #11 and to tube #13. 10. To tube #13, add 1.0mL of the antibiotic standard. 11. With all the tubes tightly capped, gently shake or vortex the contents of the tubes. o 12. Incubate tubes at 35 C for 18 to 24 hours. 13. After incubation period, examine the tubes for bacterial growth (turbidity in the tube or whitish pellet at the bottom of the tube). 14. The tube with the lowest concentration of plant extract at which no growth or turbidity is observed is reported as the MIC of the plant extract against the bacterial test organism.
Contents Volume of MuellerHinton Broth (mL) Concentration of extract (microg/mL) Volume of diluted inoculums (mL) Antibiotic standard Total volume (mL)
1 None 4000 1 None 2 Highest conc of plant extract
2 1 2000 1 None 2
3 1 1000 1 None 2
4 1 500 1 None 2
5 1 250 1 None 2
6 1 125 1 None 2
Test tube number 7 8 1 62.5 1 None 2 1 31.3 1 None 2
9 1 15.6 1 None 2
10 1 7.8 1 None 2 Lowest conc of plant extract
11 1 None 1 None 2 Negative control
12 2 None None None 2 Media control
13 None None 1 1 2 Positive control
III. Phytochemical Screening Preliminary Tests on Plant Materials: Preparation: Warm 5-10g with about 25-50 mL of water on a water bath 50-60⁰C for 15 minutes, cooled, and filtered.
Test pH Tannins Glycosides
Procedure Test the extract using pH paper or litmus paper Add a few drops of ferric chloride TS to 1mL extract Add a few drops of lead acetate TS to about 2 mL of extract. Filter the extact then add lead subacetate TS until weakly alkaline.
Reducing substances
Alkaloids
Plant acids
Mix equal amounts of Fehling’s A and Fehling’s B, 1 mL of each and add to an equal amount of the extract. Heat to boiling. If there is no precipitate, add hydrochloric acid and heat to boiling. Add sodium hydroxide until neutral or alkaline and again add Fehling’s solution A & B. Heat to boiling. Acidify extract with 1% HCl and add one or two drops of Mayer’s reagent (mercuric-potassium iodide TS). Additional tests: Valser’s reagent (mercuric iodide TS) Wagner’s reagent (iodine TS) Dragendorff’s reagent (bismuth potassium iodide TS) Add a few mL of sodium carbonate TS to a boiled extract.
Positive Result Red litmus: acidic Blue litmus: basic Blue-black precipitate is indicative of tannins Precipitation shows the presence of glycosides, mucins, tannins, & proteins. Precipitation or turbidity shows the presence of glycosides Brick-red precipitate shows the presence of reducing sugars as hydrolytic product.
Precipitation is indicative of the presence of alkaloids
Saponins
Shake the extract vigorously for 30 seconds. Stand in a vertical position for 30 minutes.
A stable and dense froth is indicative of the presence of free acids (stearic, diterpene acids, tripterpenedicarboxylic acids) A honeycomb froth greater than 3 cm above the surface of the liquid indicates the presence of saponins
Saponins
Preparation: weight 15 g of the plant material and slowly boil in 50 mL alcohol for one hour in an Erlenmeyer flask covered with a funnel. Filter. Set aside ¼ of the alcoholic extract for test for flavonoids. Use the remaining ¾ of the alcoholic extract for tests 1 and 2. Differentiation test for saponins Evaporate the alcoholic extract to dryness. Cool. If colored, stir the dried extract for a few minutes with 10 mL of petroleum ether, repeat extraction until most of the color is removed. Discard the petroleum ether extract. This part can be omitted if the dried extract is not much colored. Add to the dried extract 10 mL of chloroform and stir for 5 minutes. Decant into a test tube containing about 100 mg anhydrous sodium sulfate. Shake, filter, and divide filtrate into three clean, dry, test tubes. Use one test tube as control. Liebermann-Burchard Test Add three drops acetic anhydride to the first test tube. Mix gently. Add one drop of concentrated sulfuric acid and mix gently. Observe color changes immediately and over a period of one hour. Result (+) blue, blue green, or green (+) red, pink, purple, or violet (-) pale yellow Substances Present Steroidal saponins Terpenoidalsaponins Saponins of saturated sterols or saturated triterpenes
Salkowski Test Hold the second test tube at 45⁰ angle, layer, 1-2 mL of concentrated sulfuric acid by allowing it to run down the inside of the test tube. Note any immediate changes at the junction of the extract and sulfuric acid. Gently mix the sulfuric acid and the extract and observe for immediate and/or gradual changes over one hour period. A cherry red color is indicative of the presence of unsaturated steroids. Test of Saponins and Sapogenins Take 5 mL of the alcoholic extract and add a few drops of a saturated alcoholic solution of cholesterol. Observe crystal formation. Test for Flavonoids 1. To an alcoholic solution of plant material add a small piece of magnesium ribbon, followed by the dropwise addition of concentrated HCl. Colors will develop within 1-2 minutes following the addition of the acid subject to variation in intensity depending on the concentration of flavonoid present in the sample. a. Colors ranging from orange to red (flavones) b. Colors ranging from red to crimson (flavanols) 2. Digest a few grams of the sample plant material with 2N HCl in 1-propanol for 15-30 minutes. A slow development of a strong red or violet color is indicative of a positive reaction for the presence of flavonoids.
Glycosides
Preparation: Place 50 g of the dried, powdered plant material into 500 mL Erlenmeyer flask. Add 150 mL of 80% ethanol; Fit into the neck of the flask a stopper with a piece of long glass tubing. The glass tubing will serve as an air-cooled reflux condenser. Place the set-up on a water bath and reflux for 1 hour. Cool to room temperature and filter the mixture. Wash the residue with two 25-mL portions of 80% alcohol. Express any solvent remaining in the plant material using a glass rod. Discard the exhausted marc. Cardiac Glycosides Presence of unsaturated sterols Preparation: Evaporate the equivalent of 10 g of plant extract to dryness using a water bath in the hood. Allow it to cool to room temperature – the extracts should be either dry or syrupy. Add 10 mL hexane, stir and allow to settle. Decant and discard the supernatant. Repeat until the hexane extract has removed most of the pigmented substances. Add 10 mL of chloroform to the residue and stir for 5 minutes. Decant into a test tube and dry with anhydrous sodium sulfate. Shake and filter into a clean dry test tube. Divide the filtrate equally into 3 separate test tubes. Set aside a test tube to serve as a color control for the following tests: test Libermann-Burchard test procedure Add 3 drops acetic anhydride. Mix gently by swirling the test tube. Add 1 drop sulfuric acid and mix gently. Observe for 1 hour. result blue, blue green, or green (+ for steroidal saponins) red, pink, purple, or violet (+ for terpenoidalsaponins) pale yellow (+ for saponins of saturated sterols or saturated triterpenes) Cherry red color (+ for unsaturated sterols)
Salkowski Test
Hold the test tube at 45⁰ angle, layer, 1-2 mL of concentrated sulfuric acid by allowing it to run down the inside of the test tube.
Presence of 2-deoxy sugars Test Keller-Killiani Test Procedure Place 10 mL of the 80% ethanolicextract in an evaporating dish and dry on a steam bath. Add 3 mL of freshly prepared ferric chloride reagent (0.3 mL of 10% ferric chloride with 50 mL glacial acetic acid) stir and transfer to a test tube. Perform a ring test by layering 1 mL of sulfuric acid at an angle. Result Purple ring (+ for 2-deoxy sugars)
Anthraquinone Glycosides test Borntrager test Procedure Evaporate the equivalent of 1 g of plant extract to dryness using a water bath. Dissolve the residue in 30 mL of distilled water and filter. Shake the filtrate with 10 mL benzene in a separatory funnel and allow the mixture to separate. Test the benzene layer with 5 mL ammonia TS and shake. Result Red alkaline layer (+ for anthraquione glycosides)
Cyanogenic Glycosides test Guignard test Procedure Moisten 2-5 g sample. Prepare sodium picrate strip by dipping filter paper strip into the solution. Dry, blot, add 1 mL chloroform to the flask containing the moistened sample. Insert the filter paper strip in the flask & fold over the rim. Stopper. Heat to 35⁰C for 2 hours Result Yellow color of the filter paper turns to red in 15 minutes (+ for cyanogenic glycosides)
Tannins
Preparation: Comminute the sample and add 20 mL of distilled water. Boil for 15 minutes. Use 5 mL portions In the following tests: Test General test for tannins Procedure Add 2 mL of potassium ferricyanide TS to 5 mL of the extract. Add concentrated ammonia to the resulting solution. Soak a small piece of goldbeater’s skin in 2% HCl. Rinse with distilled water and place in the extract for 5 minutes. Wash with distilled water and transfer to a 1% ferrous sulfate solution. Dip a matchstick (wooden end) in the extract. Dry. Moisten with conc. HCl and warm near a flame. Add a few mL of to the aqueous extract and expose the resulting solution to air for quite some time. Result Blue-black precipitate (+ for gallitannins and ellagitannins) Brownish-green (+ for condensed tannins) Brown or black color (+ for tannins)
Goldbeater’s Skin Test
Test for Catechin
Pink or red (+ for phologlucinol)
Test for Chlorogenic Acid
Green color (+ for chlorogenic acid)
