Title of Process: Evaluation of Drugs

To evaluate a drug means to identify it and to determine its quality and purity. The evaluation of a drug involves a number of methods which may be classified as follows:

1. Organoleptic 4. Chemical2. Microscopic 5. Physical3. Biological 6. Chromatographic

The macroscopic characteristics of a drug are the following:

1. shape and size2. color and fracture3. fracture and internal color4. odor and taste

Descriptions:Underground plant parts – rhizome, roots, bulbs, tubers

1. entire 4. cubical pieces2. longitudinal slices 5. broken pieces3. oblique/transverse slices

Shapes:

1. cylindrical 4. fusiform, ovid, pyriform2. cylindraceous 5. Terete (tapering gradually but nearly cylindrical)3. conical 6. Disc-shaped

External Marking:

Furrows – alternating ridges and valley which are more or less parallel, well defined, and usually due to the shrinkage of the internal parts caused by drying.

Wrinkles – are fine delicate furrowsAnnulations – transverse ring-like markingsFissures – furrows extended into the tissuesNodules – rounded outgrowths on the surfaceProjections – like roots/root hairs, stembases, buds, trichomes, spinesScars – found in the marks of the leaf, stem, roots, buds, flowers

Fracture refers to the way the plant parts break when subjected to sufficient pressure and this is of several types:

Complete – breaking clean acrossIncomplete – breaking only part way acrossShort – clear accompanied by resistance and characterized by projection of fibers

from the broken surfaceSplintery – breaking irregularly across into pieces with larger and smaller projecting

edges and splintersBrittle – easily broken, usually into many pieces when chopped unto hard surfaceTough – breaking with difficultyWeak – breaking with little effort

Fracture Surface

Even – smooth surfaceUneven – irregularly broken surfaceGranular – having grain-like appearanceHard – a compact surfaceMealy – surface characterized by powdering usually due to abundance of starchResinous – a smooth glossy surfaceConchodial – a resinous surface characterized by having the surface curved in convex

and concave fashionWaxy – exhibiting a dull wax-like surface

OdorAgreeable Disagreeable

1. Aromatic 4. Alliaceous2. Balsamic 5. Camphoraceous3. Spicy 6. Terebenthinate

Taste

1. Acid 4. Bitter2. Saline 5. Alkaline3. Saccharine 6. Metallic

TouchMucilaginous – producing a soft, slimy feelingOily – producing a bland, smooth feelingAstringent – producing a contraction of the tissues of the mouth having puckering feelingPungent – producing a warm biting sensationAcrid – producing an unpleasant irritating, tingling sensationNauseous – tending to induce vomiting

Title of Process: Phytochemical Screening of Plants

Materials:

Plant sample: fresh or dried Ammonium hydroxide 2NHClAlcohol 5% Hydrogen peroxide Mayer's reagentFehlings reagent Benzene Water bathAlpha-napthol Sodium carbonate SpatulaPhenylhydrazine Propanol Test tubesSodium acetate Millon's reagent Mortar and pestleAcetic acid Petroleum ether Analytical balanceSulfuric acid Lead acetate TS Hot plateResorcinol Lead subacetate BeakerHCl conc. Acetic anhydride Stirring rodFerric chloride Chloroform Magnesium ribbonSodium sulfate Valser's reagent KOHWagner’s reagent Erlenmeyer flask Erlenmeyer flaskDragendorff’s reagent Picric acid FunnelTannic solution NaCl solution MicroscopePicrate paper Gelatin solution Litmus paper2M HCl 1% HCl

Procedure:

A. Preparation of extract for evaluation

1 Preparation of aqueous extract

A plant sample (5-10 g) is warmed with about 25-50 ml of water on a water bath maintained at 50-60ºC, cooled and filtered.

2. Preparation of alcoholic extract

Weigh 50g of plant sample in an Erlenmeyer flask. Add 150 ml of sufficient Ethyl alcohol to completely submerge the plant. Cover with a funnel, act as condenser to minimize evaporation of the solvent. Place flask over a hot water bath and reflux for an hour. Filter the mixture while it is hot and rinse the flask and plant sample with alcohol. Include it with the extract. Discard plant residue and concentrate the extract to about 30 ml. Keep in tightly-stopper container.

Note: Add a small amount of Chloroform to prevent growth of fungi.

B. Chemical Evaluation of the prepared extract

1. Test for CarbohydratesUse aqueous extract for the following tests

Fehlings Test. To the heated solution of the substance, add drop by drop a mixture of equal parts Fehling’s solution in a water bath. A brick red solution indicates the presence of reducing sugars.

Molisch’s Test. This is the general test for carbohydrates. Add a few drops of alpha-Napthol and concentrated Sulfuric acid. A purplish solution indicates the presence of carbohydrates in the extract.

Osazone Formation. Heat the sample solution with Phenylhydrazine HCl, Sodium acetate and acetic acid. If yellow crystals are formed when examined under the microscope it is positive for certain sugar like glucose or fructose or glucosazone.

Selivanoff’s Test. Add crystal of Resorcinol to the sample solution and warm on a waterbath with an equal column of conc. HCl. Rose color is produced if ketone is present (fructose/inulin/hydrolyzed inulin).

Test for Pentoses. Heat sample solution in a test tube with equal volume of HCl containing a little Phloroglucinol. Red color means presence of pentoses.

Keller Killiani Test. Add Acetic acid containing a trace of Ferric chloride to the sample solution and transfer to the surface of conc. Sulfuric acid. The vapor should be a reddish brown solution which gradually turns to blue.

2. Test for Plant Acids

Reaction with Litmus paper. The basicity or acidity of the extract by use of litmus paper.

Sodium carbonate Test. Add few ml of Sodium carbonate T.S. to a boiled aqueous plant extract which froths poorly or not at all. The formation of a stable and dense froth indicates the presence of free acids (stearic, diterpene, triterpene dicarboxylic acid).

3. Test for Glycosides

Add to 2 ml of aqueous extract a few drops of Lead acetate T.S. Initial precipitation shows the presence of glycosides, mucins, tannins and proteins. Complete the precipitation and filter the mixture. To the filtrate, add Lead subacetate T.S. until the liquid is made neutral or weakly alkaline. Presence of glycosides is indicated by precipitation or turbidity.

4. Test for Saponins

Froth Test. Shake the aqueous extract vigorously for 30 seconds. Allow to stand in a vertical position and observe for 10 minutes. If a honeycomb froth greater than 3 cm rises above the surface of the liquid persists, this is presumed to contain saponins. Small amount of frothing may be due to proteins, certain plant acids or low concentration of saponins.

Differentiation of Saponins. Weigh about 10 g of the plant sample and slowly boil in 30 ml of alcohol for one hour in an Erlenmeyer flask covered with a funnel in a upright position. Filter the alcohol extract with 10 ml Petroleum ether extract. Repeat the extraction until all the color has been removed. Discard the petroleum ether extract. Extraction with Petroleum ether can be omitted if the dried extract is not much colored. To the dried extract is added 10 ml of Chloroform and stir for 5 minutes. Decant into a test tube containing about 100 mg of anhydrous Sodium sulfate or anhydrous Magnesium sulfate. Shake, filter and divide the collected filtrate into 3 clean, dry test tubes. Use one test sample as the control.

Liebermenn Burchard Test. Add three drops of Acetic anhydride to the first test tube. Mix gently. Observe the color changes within a period of one hour.

Salkowski Test. Hold the second test tube at 45ºC angle layer one with 2 ml of conc. Sulfuric acid by allowing it to run down the inside of the test tube. Note any immediate color changes at the junction of the extract and Sulfuric acid. Gently mix the Sulfuric and the extract and observe for immediate and/ or gradual change in color for over an hour period. A cherry red coloration is indicative of the presence of unsaturated steroids.

5. Test for Flavonoids (Using alcoholic extract).

Wilstater “Cyanidin” Test. To an alcoholic solution of the plant material and a small piece of Magnesium ribbon, followed by the addition of HCl. Colors will develop within one hour to two minutes following the addition of the acid subject variation intensity depending on the concentration of the flavonoid present in the sample. If flavonols are present the colors range from red to crimson.

Batesmith and Metcalf Test for Leucoanthocyanidins. Another test is digest few grams of the sample with 2N HCl in a Propanol for 15-30 minutes. Slow development of a strong red or violet color is indicative of a positive reaction.

Modified Borntragers Test. Take a portion of the alcoholic extract equal to 3 g of the sample and evaporate to dryness over a waterbath. Then add 10 ml of KOH and 1 ml of 5% Hydrogen peroxide. Heat for 10 minutes. Filter and

acidify the resulting filtrate with glacial Acetic acid. Extract twice with 5 ml Benzene and divide the resulting Benzene into two portions. Use one portion as control. Alkalinify one portion with Ammonia and compare with the control. Development of a pink color is positive indication.

6. Test for Cyanogenic Glycosides

The Guignard Test. Place 2 to 5 grams of the crushed plant sample in tube. Moisten it with enough water, then add a few ml of Chloroform to enhance enzyme activity make a slit on the test tube cork stopper and insert picrate paper on the slit to prevent it from touching the sides of the test tube at 35 to 40ºC or kept at room temperature for 3 hours. Observe the picrate paper for any color change. Positive result is indicated by a change from yellow to bright red within 15 minutes. Intensity of red color measures the concentrate of the cyanogenic glycosides.

7. Test for Tannins

Using 15 ml of Ethyl alcohol extract, evaporate to dryness over a water bath. Cool. Add 25 ml of hot distilled water. Cool. Add 5 gtts. of 10% NaCl solution to salt out undesirable constituent. Filter. Use for the following test:

Ferric Chloride Test. Add a few drops of Ferric chloride T.S. to a 2 ml extract. Formation of a greenish black, blue black to black precipitate indicates a positive result.

Gelatin Test. Treat a portion of the filtrate with the gelatin salt solution. Formation of a precipitate indicates a positive result. Negative result indicates the presence of polyphenolic compounds.

8. Test for Alkaloids

Preliminary Tests. Acidify the aqueous extract with 1% HCl and add one or two drops of Mayer’s reagent. A positive test is shown by precipitation. Samples of the plant which yield precipitate with at least four to five alkaloidal reagents such as:

Mayer’s Reagent (Mercuric-Potassium Iodide T.S.)Valser’s Reagent (Mercuric Iodide T.S.)Wagner’s Reagent (Iodine T.S.)Dragendorff’s Reagent (Bismuth Potassium Iodide T.S.)Tannic SolutionSaturated solution of Picric acid

Confirmatory Test. Alkalinify the acidified aqueous extract about 5 ml with 2% Ammonium hydroxide and extract the resulting solution with 10 ml portion

Chloroform. Combine the Chloroform extracts and evaporate it to dryness over steam bath. Set aside the alkaline layer for the test for quaternary bases. Take up the residue remaining after concentrating with 5 ml of 2M HCl and stir over results. Positive results indicate the presence of either primary, secondary or tertiary alkaloids.

Test for Quaternary Bases and or Amine Oxides. Acidify the alkaline aqueous layer from the confirmatory test above with 2M HCl. Filter and divide the filtrate separately with Mayer’s and Dragendorff’s reagents. Definite formation of a precipitate in both tests may be taken as indication for the presence quaternary and/or amine oxide basis. Turbidity of solution after the addition of the alkaline reagents may be the result of incomplete chloroform extraction thus, should be considered negative for quaternary bases.

9. Test for Fixed oil, Fats and Volatile oils

Stain Test. Weigh 2g of the plant sample and add 10 ml of Petroleum ether or Hexane. Boil. Place a few drops of the extract on a bond paper and observe any stain produced on the paper.

Title of Process: Isolation of Pectin

Materials:

Plant sample: pomelo (rind) Beaker Stirring rod3N Sulfuric acid Thermometer Chopping board Celite Funnel Water bathEthyl alcohol Cheese cloth SpatulaScissors or Knife Hot plate Test tubes

Procedure:

1. Weigh about 25 g of the pericarp of pomelo and, using a air of scissors or a knife, cut the tissue into cubes of about 1 cm.

2. Place the tissue in a beaker and add 100 ml of water and 1.5 ml of 3N Sulfuric acid.

3. Heat the mixture to 90ºC for 30 minutes with occasional stirring.4. Continue the digestion at 85 to 90ºC for 30 minutes.5. Strain the mixture through cheesecloth, expressing the tissue to collect the

solution.6. Repeat the digestion for 15 minutes.7. Strain and combine the extract.8. Add 2 g of Celite and filter, using a Buchner funnel.9. Collect 100 ml of the pectin solution.10.Add Isopropyl alcohol or Ethyl alcohol to a concentration of 55% by volume.11.Collect the precipitated pectin by filtration using a Buchner funnel.12.Dry the product.13.Wash the gummy precipitate with 25 ml of the Ethyl alcohol.14.Compute the percentage of pectin.

Title of Process: Isolation of Plant Acids

Materials:

Plant sample: gabi and calamansi Beaker Analytical balanceAcetic acid Knife Buchner funnelCalcium acetate Muslin cloth10% HCl Stirring rod5% Sulfuric acid Chopping board

Procedure:

Extraction of plant acids from gabi.1. Boil 100g of the plant sample (gabi) to be used with 200 ml of water until it

becomes soft.2. Squeeze the boiled mass through muslin cloth.3. Boil the filtrate again and while hot, using a Buchner funnel, acidify the filtrate

with Acetic acid and add concentrated solution of Calcium acetate until no more precipitate is formed.

4. The precipitate is then allowed to settle for 12 hours.5. The clear liquid is decanted and the precipitate is dissolved with minimum

amount of 10% HCl.6. The mixture is boiled and allowed to cool in an iced bath.7. On cooling, characteristic calcium oxalate crystals will precipitate out.8. Weigh the crystals obtained and add a sufficient amount of 5% Sulfuric acid.9. Allow the mixture to stand for some time, filter and evaporate the filtrate on a

water bath allow to dry in air.10.Weigh and transfer the oxalic acid extract in an amber colored bottle.11.Compute for the percentage yield.

Perform the following tests:

1. Test for SolubilityTransfer a small amount of the crystals in a test tube. Add a few drops of Ammonium oxalate T.S. until a precipitate is formed. Divide the precipitate formed and transfer it into separate test tubes. Test the solubility of the precipitate in HCl and Acetic acid. (Plant acids are insoluble in Acetic acid but soluble in Hydrochloric acid).

2. Calcium chloride testTransfer a small amount of the crystals in a test tube. Add a small amount dilute HCl and warm the mixture. Add a few drops of Calcium chloride T.S. Test the solubility of the precipitate formed in Acetic acid and Hydrochloric acid.

3. Potassium permanganate test

Transfer a small amount of the crystals in a test tube. Add a small amount dilute HCl and warm the mixture. Add a few drops of KmnO4. (plant acid decolorize KmnO4).

Extraction of plant acids from Lemon1. Extract 90 ml of lemon juice by squeezing. Transfer the juice in a pre-

weighed beaker. Weigh the beaker and the lemon juice.2. Slowly add 10% NaOH solution while stirring until the mixture is slightly

alkaline.3. Strain the solution through a fine muslin cloth to remove large particles of pulp

and then filter. (use whatman filter paper)4. Measure the filtrate and place in a container and add 5 ml of 10% Calcium

chloride solution with constant stirring to each 10 ml filtrate.5. Heat to boiling and filter off copious precipitate of calcium citrate form hot

solution.6. Wash the precipitate collected twice with small quantities of boiling water.7. Re-suspend in a minimum quantity of cold water, heat to boiling and once

more collect the insoluble calcium citrate by filtration.8. Weigh the air-dried salt, place in a beaker and add 1N Sulfuric acid enough to

convert the salt to acid.9. Allow the mixture to stand for a few minutes, filter off the insoluble calcium

sulfate and concentrate the filtrate to a small volume on a steam bath.10.Weigh and calculate the % yield.

Perform the following tests:1. Organoleptic Evaluation.

Observe the color, odor, taste and appearance of the crystals.2. Solubility Test:

Test the solubility of the crystals in alcohol, water and ether.3. Chemical Test:

To mixture of 15 ml of pyridine and 5 ml of acetic anhydride, add 10 mg of crystals and observe the color formation.

Place 5 ml of citric acid solution in a test tube. Render it alkaline with dilute NaOH solution. Add 0.1% KMnO4 solution till a permanent purple is produced. Heat gently to boiling. Observe the color.

Title of Process: Ash Determination

Materials:

Dried plant sample: Ashless filter paper Analytical balanceEthyl acohol Crucible with cover Buchner funnelDessicator Crucible tongFurnace FunnelAnalytical balance Dilute acid

Procedure:

A. Determination of the Total Ash Content

1. Weigh accurately from 2 to 4 grams of dried plant material in tared crucible.2. Incinerate at low temperature until dull red, and until it is free of carbon.3. Cool and determine the weight of the ash.4. If the carbon-free ash content cannot be obtained, exhaust the charred mass with

hot water, collect the insoluble residue on an ashless filter paper, incinerate and filter until the ash is free of carbon.

5. Add the filtrate and evaporate to dryness.6. Heat the whole to a dull redness.7. If carbon-free cannot be obtained in this manner, cool the crucible and add 15 ml

of alcohol to break up the ash with the use of a glass rod.8. Burn off the alcohol and again heat to dull redness. Cool and weigh the ash.9. Compute the percentage of the total ash from the weight of the plant sample.

B. Determination of Acid-Insoluble Content1. Boil the ash obtained in letter A with 25 ml of dilute acid (HCl) for 5 minutes.2. Collect the insoluble matter on an ashless filter paper and wash with hot water.3. Ignite, cool and weigh.4. Determine the percentage of acid-insoluble ash from the weight of the plant

sample taken.

Title of Process: Moisture Determination

Materials:

Plant sample: Analytical balanceMoisture determination balance

Mortar and Pestle (if needed)

Dessicator ScissorsOven

Procedure:

A. Using the Oven

1. Cut the plant sample into pieces (fresh), and accurately weigh 10g of the sample.2. Dry at 105 ºC for 5 hours (optional), cool and weigh.3. Continue heating, drying and weighing at one interval until the loss is not more

than 0.25% in one drying.4. Determine the moisture content from the weight of the plat sample taken.

C. Using the Moisture Determination Balance

1. Place a clean and dry aluminum pan on the balance and allow the instrument to warm for 15 minutes.

2. Weigh 10 g of the ground fresh plant sample and spread the sample evenly on the pan.

3. Set the desired temperature and start heating also adjust the timer to the desired number of minutes of exposure (5-15 minutes).

4. Observe. As the weight of the sample decreases the moisture content reading become steady.

5. Determine the percentage of moisture content of the plant sample.6. Clean the balance after using.

Title of Process: Identification and isolation of Glycosides

Materials:

Plant sample: senna, uva-ursi Ammonia T.S. Baryta waterChloral hydrate Ferric chloride T.S. MicroscopeHCl, diluted Ethyl alcohol Glass slide w/cover slipHydrogen sulfide Lead acetateChloroform Basic Lead acetate

Procedure I:

A. Leaves yielding glycosides

1. Leaflet-Senna

a. Cut small squares (2x2mm) from a leaflet of the sample.b. Gently heat with 5 ml Chloral hydrate T.S. in a small evaporating dish until

all the sections are cleared of color.c. Mount a few sections in Chloral hydrate T.S. on a slide.d. Examine microscopically and observe the epidermal cells, stomata and

trichomes.e. Draw the specimens.

2. Leaf-Uva ursi

a. Examine a sample Uva and prepare a labeled sketch (x5) of a single leaf.b. Place a small amount of the powdered drug in a watch glass.c. Moisten the powder uniformly with a small amount of dilute HCl (to

hydrolyze the arbutin to hydroquinone and glucose).d. Examine under microscope.

3. Extractive from Leaf – AloinPerform the NF XI ID TESTS:

a. Aloin is soluble in ammonia TS and in solutions of alkali hydroxides, having a green fluorescence.

b. To an alcohol solution of aloin, add a drop of ferric chloride TS, a brown to greenish green color is produced.

B. Bark

1. Cascara sagrada (or Glycyrrhiza glabra)Perform from the USP xix ID tests for Cascara or licorice

a. Add 1 g powdered Cascara or licorice to 10 ml hot water, shake the mixture occasionally until it is cold, filter, dilute the filtrate with water to 10 ml and add 10 ml of ammonia TS; an orange color is produced.

b. Macerate 10 g of powdered cascara or licorice with 1 ml of alcohol, add 10 ml of water, boil the mixture, then cool, filter and shake the filtrate 10 ml of ether: a greenish yellow ether solution separates. Shake 3 ml of the ether solution with 3 ml ammonia solution with 20 ml of water; a distinct orange pink – color remains.

c. Root – Sarsaparilla, Gensing, StrophantusPerform organoleptic examination of the available specimen.Tabulate the result.

C. Seed – Black and White mustard a. nose-bite test – black mustard b. taste-bite test – white mustard

Procedure II:Extraction of Saponins

1. Extract saponins from gogo bark by boiling about 10 g of the sample in water (cut the sample into small pieces and if needed extract first with Petroleum ether)

2. Filter and add neutral Lead acetate solution to the aqueous solution. (formation of precipitated lead salts indicate presence of acid saponins)

3. To the filtrate, add basic Lead acetate to precipitate neutral saponins.4. Wash the lead precipitate and suspend in water.5. Decompose by passing in hydrogen sulfide.6. Filter the Lead sulfide and absorbed impurities.7. Concentrate the filtrate and finally allow to evaporate to dryness.8. Purify the saponins by Chloroform and precipitate with Ether.9. Weigh the residue and compute the % yield.

Perform the following tests:1. Make 20 ml of 2% saponins solution and test with the following reagents.

a. Froth testShake about 10 ml of the solution to the test tube. Observe.

b. Reaction with Baryta water.Treat 5 ml concentrated aqueous solution of saponin with a saturated solution of baryta water. Observe.

c. Reaction with Lead acetate.Treat 5 ml concentrate solution of saponin with 5 ml Lead acetate solution. Observe.

Title of Process: Identification of Lipids

Materials:

Plant sample: Lycopodium HCl, diluted. MicroscopeAnhydrous lanolin Ethyl alcohol Glass slide w/cover slipSpermaceti Glycerin, diluted BeakerYellow and white wax Stirring rod Hot plateAmmonia T.S. Erlenmeyer flask

Procedure:

Lycopodium (fixed oil)

A. Identify test

1. Place a small amount of the powder in a beaker half full of water. The powder floats but when the water is boiled the powder will sink.2. When Lycopodium is thrown into a flame, it burns with a quick flash.

B. Make a mount of the spores in Ethyl alcohol on a slide. Add a drop of dilute Glycerin and cover glass. Observed microscopically. Illustrate what you see..

Anhydrous Lanolin (fat)

A. Place a small amount of the sample on a slide and cover with a cover slip.

The sample should be thinly spread on the slide. Examine microscopically. Can you detect any crystalline? Illustrate.

Spermaceti

A. Place a small fragment of spermaceti on a slide and melt by gently heating the slide over the flame. Tilt over the flame. Tilt the slide to permit the wax to form a thin layer and allow to cool. When the wax has again solidified, observe microscopically and make a representative drawing of the crystalline structure.

B. Perform the USP XIX test for stearic acid in spermacetiWarm a mixture of about 1 g of spermaceti and 10 ml Ammonia T.S. in a stoppered contain until the spermaceti is melted. Shake the mixture thoroughly for a few minutes, cool, filter and acidify the filtrate with dilute HCl the liquid may become turbid but yields no precipitate.

Yellow wax/White waxA. Treat a small fragment of each wax in a manner similar to no. 1 in the experiment for

spermaceti and sketch the crystal obtained/observe.

Title of Process: Identification of Tannins

Materials:

Plant sample: tea 10% Gelatin Activated charcoalEthyl alcohol Ether Evaporating dishCalcium hydroxide Mortar and pestle Hot plateIodine solution CorkFerric chloride Erlenmeyer flask

Procedure:

Extraction of Tannins:

Pound 25 g of dry tea leaves using a mortar and pestle. Put the powder in an Erlenmeyer flask and add enough alcohol to cover the tea. Cover the flask tightly using a cork and macerate for 24 hours. Add a small amount of activated charcoal. Filter the mixture and transfer the filtrate to an evaporating disk. Evaporate the filtrate over a steam bath to dryness. Collect the crystal and determine the percentage yield.

Test for Tannins:

A. Organoleptic Evaluation: determine the color, odor and appearance

B. solubility test. Using a small amount of the extract dissolve in alcohol, ether and water.

C. Chemical test. Dissolve 0.2 g of the crystal in 15 ml of water and divide. It equally into 4 test tubes. Test the solution with the following reagents:

1. ferrous chloride t.s. ________________

2. 10% Gelatin ________________

3. Calcium hydroxide ________________

4. Iodine solution ________________

Title of Process: Identification of Resins

Materials:

Plant sample: cayenne Percolator Evaporating dishEthyl alcohol Litmus paper Hot plateHCl Cork Knife and chopping boardPhloroglucinol Erlenmeyer flask

Procedure:

1. Weigh about 100 g of pepper, wash and oven dry.2. Cut into small pieces, pound in a mortar and pestle.3. Moisten the powdered material with enough solvent (Acetone, Ethyl Alcohol, or

Ether) and macerate for one hour.4. Pack the macerated material carefully and firmly in the percolator and cover the

top of the percolator with a filter paper and place weight of some kind on the top of the paper.

5. Macerate for another hour.6. Percolate slowly until the drug is exhausted.7. Collect the percolates and concentrate by evaporation using a water bath (until

solvent had beendissipated).8. Weigh the residue and calculate the % yield.

Perform the following tests:

1. Determinethe solubility of the residue using the following solvents.a. waterb. Alcoholc. Ether

2. Prepare a 0.5% alcoholic oleoresin extract and mix with a small amount of water. Test the reaction using litmus paper. Observe.

3. Smoke Test. A small amount of extract is placed on a wire loop and ignited. An empyreumatic odor will be produced with a characteristic smoky flame, which indicates the presence of resin.

4. Phloroglucinol Test. The residue is treated with 1 ml of phloroglucinol test solution and 3 drops of HCl. A red color produced indicates the presence of resin.

Title of Process: Identification of Fixed Oils

Materials:

Plant sample: castor seeds Petroleum etherEthyl alcohol Mortar and pestleEther CorkPhenolpthalein Erlenmeyer flask 0.1 N NaOH Filter paper

Procedure:

1. Weigh 250 g of castor seeds.2. Using a mortar and pestle pulverize the seeds until they form small particles.

Place the sample in an Erlenmeyer flask and add Petroleum ether.3. Macerate the sample for 2 days with occasional agitation.4. Filter the mixture through a filter paper and collect the filtrate.5. Evaporate the filtrate until the Petroleum ether is removed.6. Cool the extract and determine the % yield.

Test the extract:1. Organoleptic evaluation: Determine the color, odor and the state of the oil2. Solubility test: Determine the solubility of the extract using different solvents:

(use 0.5 ml of the sample dissolved in 0.5 ml of the solvent)a. Alcoholb. Etherc. Chloroformd. Acetone

3. Stain test: Place a few drops of the oil on a piece of paper. Note the presence of the stain. Place the paper on an evaporating dish and heat for a few minutes on a water bath. Observe if there is change.

4. Acid Value test. Dissolve 5 g of the sample oil in 25 ml of the alcohol-ether mixture. Add 1 ml pph. Indicator and titrate the solution with 0.1 N NaOH. Calculate the acid value.

Acid value = V x N mgNaOH/meq Wt. of sample

Title of Process: Identification of Volatile Oils

Materials:

Plant sample: Clove – dried flower buds, Sodium sulfateClavenger extractor PycnometerTLC RefractometerAnalytical balance

Procedure:

Distillation with steam or Clavenger Method

Weigh 250 g of the plant material assigned and still in the appropriate set-up provided for. Distillation is done for 3 – 4 hours or more, until the plant material had been exhausted of its constituent or until at least 5 ml of the volatile oil has been collected. The extracted oil is dried with anhydrous Sodium sulfate and stored in a suitable amber-colored bottle in a cool place prior to analyses.

Analysis of the Isolated oil:

1. Thin Layer Chromatography (TLC)TLC will be performed on pre-prepared silica gel plates with benzene as developing solvent and the spots will be detected through iodine vapors and vanillin-sulfuric acid solution as the detecting solvents. Outline the spots detected in your plates with a lead pencil and make a reproduction of the TLC chromatography.

2. Specific gravity determinationThe specific gravity of the oils will be determined by the pycnometer method.

3. Refractive indexThe refractive index of a substance is the ratio of the velocity of light in air to the velocity of light in the substance. This property is valuable for the identification or detection of impure substances. Use the Abbe refractometer for this determination.

Title of Process: Isolation of Caffeine from Coffee

Materials:

Plant sample: Coffee beans Litmus paper Stirring rodAlkaloidal reagent Pycnometer Graduated cylinderChloroform RefractometerCalcium carbonate Erlenmeyer flaskSodium sulfate Hotplate

Procedure:

A. Extraction of caffeineReflux 200 g of ground coffee with 20 g of Calcium carbonate in 100 ml of water

using 1 liter flask. Continue refluxing for 30 minutes, then allow the mixture to cool just below the boiling point. Filter the hot mixture and extract half of the resulting filtrate with 50 ml of Chloroform, shake gently after each addition. Let it stand and allow the two layers to separate. Drain the organic layer slowly into a receiver. Repeat the extraction with a fresh 50 ml portion of Chloroform. Repeat the above procedure using the other half of the aqueous filtrate and combine the chloroform extracts. Dry the combined chloroform extracts with anhydrous Sodium sulfate and filter out the dried extract. Transfer the filtrate to an evaporating dish and place in a sand bath until the crystals are formed. Weigh the crystals obtained.

B. Determination of the physical characteristics of the caffeine extractEvaluate the caffeine extract by testing its reaction to litmus paper and describing

its color.

C. Determination of the chemical properties of the extractTest the extract with Mayer’s , Valser’s, Wagner’s and Dragendorff’s reagent and

note the color of the precipitate obtained.

Title of Process: Separation of Plant Pigments

Materials:

Plant sample: San Francisco leaf Petroleum ether Stirring rodAcetone Cork FunnelEther Separatory funnel30% KOH Beaker92% Methyl alcohol Erlenmeyer flask

Procedure:

A. Extraction of Pigments

Transfer 10 g of chopped San Francisco (Codiaeum variegatum (Linne), Family: Euphorbiaceae) leaves to an Erlenmeyer flask and macerate with 50 ml of 85% Acetone for 30 minutes. Divide the resulting acetone extract into 3 portions. Use 1/3 of the extract for Petroleum ether extraction and the last 1/3 for column chromatography.

B. Petroleum ether extraction

The acetone extract thus obtained is then poured into twice the quantity of Petroleum ether contained in a separatory funnel. An equal quantity of water is added this being poured gently down on the sides of the funnel in order to avoid the formation of emulsions. In the course of a few minutes, the petroleum ether layers separate out and now contain the pigments. The lower layer which is slightly green is run off. The addition of distilled water and the subsequent removal of the lower layer is repeated about four times in order to remove completely the acetone from the petroleum ether solution.

C. Ether extraction

Repeat the whole process using Ether in place of the Petroleum ether.

D. Demonstration of the presence of Chlorophylls a and b.

Shake ten (10) milliliters of the Petroleum ether solution obtained above with 10 ml of 92% Methyl alcohol. Observe the two layers formed of which the petroleum ether layer contains chlorophyll a and the methanol layer contain chlorophyll b. The solution of chlorophyll a is blue green while that of chlorophyll b is purer green, but the color difference between them is diminished owing to the presence of the yellow pigments, the carotene of which is in the petroleum ether and the xanthophylls in the methanol layer.

E. Separation of green and yellow pigments.

Shake 5 ml of an ether solution of the pigments obtained in pigment extraction, with 2 ml of 30% KOH in Methyl alcohol. After the green color has reappeared, slowly add 10 ml of water then add a little more of Ether. On shaking the test tube, two layers are produced, the lower aqueous alkaline layer contains the saponified green pigments, while the upper ethereal layer contains the carotene and xanthophylls.

F. Separation of the two yellow pigments.

Wash the Ether layer obtained above with water in a separatory funnel, and evaporate down to 1 ml. Dilute with 10 ml of Petroleum ether and mix with 10 ml of 90% Methyl alcohol. Remove the methanol layer and treat the petroleum ether layer with Methanol. Separate the methanol layer. The process is repeated until the methanol layer is no longer colored. The methyl alcohol contains the xanthophylls, and the petroleum ether is carotene.

Perform the following tests:

1. Organoleptic evaluation: Determine the appearance, color and physical state of the green and yellow pigments.

2. Solubility Test. Using the small amount of green and yellow pigments extracts dissolve in Petroleum ether, Methanol, Acetone, Ethyl alcohol and water.

BIBLIOGRAPHY

Cantoria, Magdalena. 1. Selected Topics in Pharmacognosy. National Academy of Science and Technology: APO Production Unit, Inc.,Quezon City, Philippines. 1994

Evans, William Charles..Trease and Evans Pharmacognosy. 13th ed.Oxford Great Britain: Alden Press. 1985

Fetrow, Charles W., Avila, Juan R.. The Complete Guide to Herbal Medicines.Springhouse, Corporation, Springhouse, Philadelphia, U.S.A. 2000

Genaro, Alfonso R.. Remington Pharmaceutical Sciences. 120th ed. Easton,Pennsylvania: Mark Publishing Co. 2000

Knevel, Adelbert M. and Frank E. Di Gangi, Frank E.. Jenkin’s Quantitative Pharmaceutical Chemistry. 7th ed. New York: Mc GrawHill Book Company. 1967-1977

Li, Thomas S. C. . Medicinal Plants: Culture, Utilization ad Phytopharmacology:Technomic Publishing Company, Pennsylvania, U.S.A. 2000

Padua, Ludivina S. . Handbook on Philippine Medicinal Plants. University of thePhilippines, Los Baños, documentation and Information Section officeOf the Director of Research. 1996

Quisumbing, Eduardo. . Medicinal Plants of the Philippines. Manila: Katha Publishing Co., Inc. 1978

Revision Committee. . Reagent and Solution, United States Pharmacopeia.23rd ed. and National Formulary. 18th ed. Easton, Philadelphia:Mark Publishing Co. 1995

Robinson, Trevor. . The Organic Constituents of Higher Plants. 5th ed. NorthAmherst, MA.: Cordus Press. 1983

Tyler, Varro E., Lynn R. Brady and James E. Robbers. . Pharmacognosy. 9th ed.Philadelphia: Lea and Febiger. 1988

Tyler, Varro E., Robbers, James E. and Speedie, Marilyn K…Pharmacognosy and Pharmacobiotechnology. Baltimore, Maryland:Williams and Wilkins. 1996