URDANETA CITY UNIVERSITY Pharmacognosy with Plant

Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 1
PREPRATATION OF CRUDE DRUG
Learning Objectives:
1. To research about a plant under study
2. To be able to prepare a crude drug.

Materials:
Plant sample, mortar and pestle, sieve No: 40, Jars and Bottles, Textbooks in Botany, Pharmacognosy and
Plant Chemistry.

Procedure:
A. Background of the Plant
Research on the Following information about your plant sample:
1. Official Name
2. Other Name (indicate whether English, Tagalog, etc.
3. Botanical Source
4. Description of the plant
5. Cultivation and Distribution of the plant
6. Reported constituents
7. Reported Uses

B. Collection of Plant Sample

 For herbs (non-woody plants), collect complete with roots, stems, leaves, flowers, fruits
and seeds
 For underground parts, excavate carefully and remove dirt thoroughly
 For large fruits, cut cross sectional in about 1’ thickness
 For woody and bigger plants, include small branches or twigs with reproductive
structures, healthy leaves, stipules when present and other features of the plant, also the
bark and wood samples.
 For infloresceneces and infructescences (clusters of fruits) borne on the trunk, carve out
these parts from the plant including a piece of the bark.

C. Preparation of the Crude Drug

 1. Collect the plant part and wash if necessary (to remove dust, dirt, etc)
2. Remove the extraneous parts (E.g Petiole). Wipe the sample with a dry, clean, cloth
3. Arrange on a pan and dry in the oven continuously for 5 hours at 105 degrees C. Turn the
sample every now and then to ensure uniform drying at the end of the period, test for the
brittleness of the sample.
4. Pulverize the dried sample using a mortar and pestle
5. Let it pass through a no. 40 Sieve and keep in a clean dry, and tightly covered bottle
NOTE: Divide the sample into 2 portions. One Portion will be used for moisture determination and ash
determination and other portion will be used for drug extraction.

Data:
Write the information on the Background of the Plant

Questions:
1. What is a crude drug?
2. When are underground Parts used as drugs collected?
3. What is the ideal weather for collecting flowers?
4. How are leaves used as drugs packaged and stored?
5. Differentiate Gums from Latex.
6. How and when are plant Exudates (gums, latex, and resins) collected?

References:
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 2
MOISTURE DETERMINATION

Objectives:
To determine the moisture content of a dried plant sample by gravimetric analysis
To study the significance of moisture content determination

Materials:
Dried plant sample from Experiment No. 1
Evaporating Dish
Dessicator
Oven
Digital Balance

Procedure:
1. Accurately weigh 5 grams of the sample. The sample should be air-dried before weighing
2. Pre-heat the oven at 105 degrees C
3. Dry the sample in a tared evaporating dish for 5 hours. Allow to cool in the dessicator and then
weigh.
4. Continue the drying and weighing at 1hr. intervals until the loss of weight is not more than 0.25%
in 1 hour drying
5. Calculate the Percent Moisture.
*Taring of the evaporating dish:
Wash, Dry and place the evaporating dish in an oven for 20 minutes. Cool then weigh. Return the
evaporating dish in the oven then cool and weigh again. Repeat the heating, cooling and weighing
processes until a difference of not more than 02. Mg between two (2) successive weights is obtained or
until constant weight is obtained.

Formulas:

Loss of Weight = Weight of the dried sample – Constant weight

%moisture = Loss in weight X100

Weight of dried sample
DATA AND RESULTS:
PARAMETERS
Weight of evaporating dish
Weight of dried sample
Weight of evaporating dish + Sample after 5 hours
of heating
Weight of sample after 5 hours of heating
Constant weight of sample after 1-hr interval
heating
%of Moisture

Computations:

Questions:
1. Define constant weight
2. Differentiate moisture content from water content
3. What is the importance of determining the moisture content of drugs
4. What is the official monograph limit for moisture content of drugs
5. Enumerate and describe/define the other USP/NF official methods of moisture determination.

References
:
URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 3
ASH DETERMINATION
Objectives:
To determine the total ash content and acid insoluble ash content of a dried plant sample.
To study and comprehend the relevance of ash content determination of drugs.

Materials:
Dried plant sample (from Expt No.1) Crucible with cover, incinerator/Rapid asher, Hot water, ashless filter
paper, alcohol, dilute HCl, dessicator, digital balance.

Procedure:
A. Total Ash
1. Accurately weigh 5 grams of the dried plant sample in a tared crucible.
2. Incinerate at low- temperature not exceeding very dull redness until free from carbon
3. If a carbon free ash cannot be obtained, exhaust the charred mass in 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 to dryness and heat the whole to
low redness.
4. If a carbon free ash still not obtained, cool the crucible, add 15ml of alcohol, gbreak the ash
with glass rod, burn off the alcohol and again heat the whole to low redness.
5. Cool then determine the weight of the ash
6. Calculate the %of the total ash from the 5 g sample.
*Taring of crucible
The crucible is heated to dull redness, cooled in the dessicator and weighed. The
processes are repeated until constant weight is obtained.

B. Acid-insoluble ash
1. Boil the ash with 25ml dilute HCl for 5 Minutes
2. Filter using ashless filter paper
3. Wash with hot water then collect the insoluble matter (residue + Filter paper) in a tared
crucible
4. Ignite then allow to cool
5. Weigh the residue
6. Determine the % of acid insoluble ash.
Formulas:
% total ash= Weight of ash X100
Weight of plant sample

% Acid-insoluble ash = Weight of acid-Insoluble Ash X100

Weight of total Ash

Data and Results:

Computations:

Questions:
1. What is the importance of determining the total ash and acid insoluble ash content of drugs? Give
their possible composition
2. What temperature range does very dull redness indicate
3. Enumerate the approximate temperature equivalents when an electric furnace is used in ignition
4. What is the purpose of taring the crucible before using the incineration process?
5. What is the official monograph limit for acid- Insoluble ash content of drugs?
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 4
DRUG EXTRACTION: PERCOLATION METHOD

Objectives:
To be able to extract constituents from a given plant sample using the percolation method of extraction
To study the principles involved in different methods of drug extraction

Materials:
Powdered drug sample from experiment No.1, Percolator, cotton, sand, filter paper, alcohol,
evaporating dish, Electric Stove/Bunsen Burner, Thermometer, Tripod

Procedure:
The Powdered material is subjected to a given solvent (80% ethanol). The residue
obtained after evaporation of the menstruum will indicate the approximate amount of active
principles extracted.
1. Weigh the powdered sample accurately
2. Moisten the powdered sample with 80% Ethanol in order to permit the drug to absorb
the liquid and swell (the volume to be added must be enough to permit total swelling
without yielding a liquid extractive)
3. Leave the moistened plant material covered for 15 minutes
4. Pack the moistened plant material in the percolator. Observe proper procedure in
packing
5. Carefully add 80%ethanol (about 100ml) allow to macerate for 24 hours.
6. At the end of the maceration period, collect the percolate in a tared evaporating dish
7. Concentrate the extract to about 40ml.
8. Measure the volume of the extract or weigh if it is too viscous. Record the weight.
9. Determine the percentage of extractive obtained.
*For efficient and complete extraction, the menstruum should be divided into a
number of relatively small portions and used successively. Each portion should be in
complete intimate contact with all parts of the sample and then removed before the
addition of the next portion. The use of fresh menstruum favors the rapid and
thorough removal of the constituents.
ILLUSTRATION:
Draw and label the percolation Set-Up
DATA AND RESULTS

COMPUTATIONS

Questions:
1. What is the principle involed in percolation method of extraction
2. Define the following:
a. Menstruum
b. Marc
c. Percolate
3. Enumerate and define the other methods of drug extraction. Give the principle involved in each
method.
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

EXPERIMENT NO: 5
PRELIMINARY SCREENING OF PLANT CONSTITUENTS
Objectives:
To prepare plant extract for preliminary screening of particular plant constituents.
To identify the plant constituents present in the plant sample based on specific tests.
To be able to interpret preliminary and confirmatory tests for the presence of active plant constituents.

Materials:
Plant sample FeCl3 Solution Dil. H2O2
80/95%ethanol Concentrated Sulfuric acid Glacial HAc
Chloroform Anh. Na2SO4 Litmus paper
2M HCl Acetic anhydride Na2CO3
Dragendorff’s reagent Benzene Picric Acid
Mayer’s reagent Ammonia Solution Conc. HCl
28% ammonia Dichloromethane Mg. Ribbon
Hexane 0.5N KOH Octanol
Gogo extract Gelatin NaCL
Cassava Peeling Fehling’s Solution Soda Lime
HNO3 Millon’s Reagent Petri Dishes

Procedure:

Preparation of Stock Plant Extract

1. Place 100g of dried plant sample or 20g of fresh plant sample (cut into small pieces) in an Erlenmeyer
flask.
2. Add 100 mL of 80% ethanol (95% ethanol for fresh plant sample)
3. Cover with a funnel which will act as a condenser to minimize the evaporation of the solvent
4. Place the flash over a hot water bath and reflux for an hour
5. Pour the mixture while hot through a Buchner Funnel lined with filter paper and fitted to a suction
flask
6. Rinse the flask and plant material with fresh portions of 80 to 95% of ethanol. Include washings with
the extract and discard the plant residue.
7. Measure the volume of the extract. Weigh if it is too viscous for volume measurements.
NOTE: to prevent fungal growth, add a trace of toluene or chloroform. Any unused extract is saved and
placed in the refrigerator to be utilized in the other tests.
A. Screening for Alkaloids
1. Preliminary test
a. Take 5 ml of the stock plant extract and place in an evaporating dish
b. Evaporate to a syrupy consistency over a steam bath
c. Add 5ml of 2M HCl, heat with stirring for about 5 minutes then cool
d. Add about 0.5 g of NaCl, stir then Filter
e. Wash the residue with enough 2M HCl to bring the filtrate to a volume of about 5ml
f. Take 1 ml of the filtrate and test with 3 drops of Dragendorff’s reagent
g. Take another 1ml of the filtrate and test with 3 drops of Mayer’s reagent
h. Record the results observed
2. Confirmatory Test
a. To the remaining 3 mL of the filtrate, add dropwise enough 28% ammonia until the
solution is alkaline to litmus.
b. Extract the alkaline solution three times with small portions of less than 10mL of
chloroform. Combine the lower chloroform extracts and reserve the upper aqueous
layer for procedure A.3.
3. Test for quaternary bases and/Amine Oxide.
a. Acidity the alkaline aqueous layer obtained in procdure A.2.b with 2M HCl.
b. Filter and divide the filtrate into 2 portions
c. To one portion, add Dragendorffs reagent and to the other add Mayer’s Reagent.
d. Observe the result and record.
B. Screening of Steroids: Cardenolides and Bufadienoldes
1. Defatting the Plant Extract
a. Evaporate 10 mL of the stock plant extract to incipient dryness over a water bath. Cool
to room temperature
b. Defat by taking up the residue with 6mL of hexane and water (2:1 ratio)
c. Partition by gently shaking the mixture in a test tube
d. Pipette out the upper hexane layer
e. Repeat the treatment with hexane until most of the colored pigments have been
removed. Discard all the hexane extracts properly
f. Heat the defatted aqueous layer over a water bath to remove the residual hexane. Cool
to room temperature.
g. Divide to 2 portions.

2. Keller-Killiani test: test for 2-deoxysugars

a. To one portion of the defatted aqueous layer, add 3 mL of ferric chloride and stir
b. Incline the test tube then add cautiously 1mL of conc. Sulfuric Acid. Letting the acid
trickle down along the insides of the test tube
c. Observe for any coloration at the interface of the acid and aqueous layers. A reddish-
Brown Color, which may turn blue or purple. Indicates the presence of 2-deoxysugars.

3. Liebermann-Burchard test: For unsaturated Steroids

a. To another portion of the defatted aqueous layer, add 10mL of dichloromethane and
stir the mixture with a glass rod for a few minutes. Then allow to stand
 b. Pipette off the lower dichloromethane extract and pass this extract through about
100mg of Anhydrous Sodium Sulfate placed over dry filter paper in a funnel.
c. Divide the filtrate into 2 portions, use one for the control
d. Treat the other portion with 3 drops of acetic anhydride then 1 drop of concentrated
sulfuric acid.
e. Observe for any immediate color change
f. Let stand for an hour and observe for further color changes and compare with the
control. Positive results of colors ranging from blue to green, red, pink, purple or violet
indicates the presence of steroid/triterpenoid skeleton.

C. Screening for Anthraquinone Glycosides

1. Borntrager’s test
a. Evaporate 5ml of the stock plant extract to incipient dryness over a steam bath
b. Take up the residue with 10mL distilled water and filter
c. Extract the filtrate with 5ml benzene twice.
d. Divide the combined benzene extracts into 2 portions. Using 1 portion as the control
e. To the other portion, Add 5ml of ammonia solution
f. Shake and observe the alkaline layer for color changes. The formation of a red color
indicates the presence of anthraquinones.

2. Modified Borntrager’s Test

a. Evaporate 5ml of the stock plant extract to incipient dryness using a water bath
b. Take up the residue with 10ml of 0.5N KOH and 1ml of dilute Hydrogen Peroxide.
c. Heat on a water bath for 10minutes. Cool and filter. Add glacial acetic acid dropwise until
acidic to litmus paper
d. Extract with 5ml of benzene 2 times. Divide the combined extract into 2 portions using 1
portion as the control.
e. Alkalinify the other portion with 5 ml ammonia solution
f. Observe the alkaline layer for color changes. A pink color indicaties the presence of
anthraquinones.

D. Screening For Flavonoids

1. Defatting the stock plant extract
a. Evaporate 10 mL of the stock plant extract to incipient dryness over a water bath and
cool to room temperature.
b. Take up the residue with 9 mL hexane and water (2:1 ratio) Discard the hexane extract
c. Dilute the defatted aqueous layer with 10 mL of 80% Ethanol
d. Filter and Divide the filtrate into 3 test tubes using one portion as the control
2. Bate- Smith and Metcalf Methods: Test for leucoanthocyanins
a. To one portion, add 0.5mL conc. HCl
b. Observe for any color change
c. Warm the solution in a water bath for 15 minutes. Observe for further color change
within an hour and compare with the control.
d. Positive result is the appearance of a strong red violet color
 3. Wilstatter “cyanannidin” test: test for benzopyrone nucleus
a. To another portion of the filtrate add 0.5 ml conc. HCl
b. Add 3 pieces of Magnesium ribbons
c. Observe any color changes within 10 minutes
d. If definite coloration occurs, dilute with an equal volume of water and add 1 ml octanol
e. Shake well and allow to stand
f. Note the color in each layer. Positive result is a color ranging from orange to red to
crimson and magenta and occasionally green or blue.

E. Screening for Saponins

1. Preparation of “gogo” extract
a. Cut 1 g of the bark of gogo into small strips and soak in 10 mL of 80 percent ethanol
b. Allow to stand for 30 minutes

2. Froth test
a. Place 5ml of the stock Plant extract in a test tube.
b. Place 5 mL of the gogo extract in a separate test tube to stand as the standard.
c. Add 10 ml of distilled water to each test tube stopper and shake the test tubes
vigorously for 30 seconds
d. Allow to stand for 10 minutes and observe for “honeycomb” froth. A positive result is
obtained when the honeycomb froth is greater than 2 cm height from the liquid’s
surface and persists after 10 mins.

3. Hemolysis test – Agar cup Semi quantitive Method

a. Obtain an agar Plate
b. Cleanly bore 3 equidistant Cups into the agar using the mouth of a small test tube.
Label each agar cup at the bottom of the inverted plate.
c. Fill one agar cup with the aqueous plant extract
d. Fill another agar cup with the googo extract to serve as the positive control
e. Fill the 3rd agar cup with distilled water to serve as the negative control.
f. Allow the covered plates to stand for one hour
g. Observe for any clear zones of hemolysis.

F. Screening for Tannins and Polyphenols

1. Preparation of the test solution
a. Evaporate 10 mL of the stock plant extract to incipient dryness over a water bath.
b. Extract the residue with 20 mL of hot distilled water then add 5 drops of 10% sodium
chloride solution
c. Filter and divide the filtrate into 3 portion using one portion as the control.
2. Preparation of gelatin-Salt Reagent
Mix an equal amount of 1%gelatin solution with 10% sodium Chloride solution
3. Gelatin Test
a. To one portion of the filtrate, add 3 drops of gelatin-salt reagent
b. Do the same to a solution of tannic acid to serve as the reference standard
c. Compare with the control and the reference standard.
 4. Ferric Chloride test
a. To Another portion of the filtrate, add 3 drops of ferric Chloride reagent
b. Do the same to a solution of tannic acid to serve as the reference standard
c. Compare the results obtained.

G. Screening for Cyanogenic Glycoside

1. Preparation of Sodium Picrate Paper
a. Dissolve 5 g of sodium carbonate and 0.5 g picric acid in enough water to make
100ml solution
b. Dip strips of filter paper (1 cm X 5 Cm) into a freshly prepared solution sodium
picrate
c. Dry the filter paper strips

2. Guignard Test
a. Place 5g of the crushed plant material in a test tube. Moisten with enough water
then add a few drops of chloroform to enhance enzyme activity
b. Do the same procedure using cassava peeling to serve as the reference standard.
c. Stopper the test tubes with a cork from which is suspended a strip of sodium
picrate paper, taking care that the strip does not come in contact with the inner
sides of the test tube.
d. Warm the tubes in a water bath or keep at room temperature for 3 hours.
e. Observe any color changes in the sodium picrate paper.

H. Screening for Carbohydrate

Evaporate 5ml of the stock plant extract. Dissolve the residue with 10ml of distilled water. Divide
the solution into 2 portions and perform the following.
1. Fehling’s test
a. Boil 5 mL of freshly prepared Fehling’s solution in a test tube then add an equal
quantity of the plant extract.
b. Boil again
c. Observe the color changes that occur

2. Moore’s Test
a. Boil 5 mL of the plant extract with an equal volume of soda lime
b. Record observation

I. Screening for Proteins

Evaporate 5 mL of the stock plant extract. Dissolve the residue with 10 mL of
distilled water. Divide the solution into 2 portions and perform the following:
1. Xanthoproteic test
a. To 1 mL of the aqueous extract, add 10 drops of Nitric acid
b. Watch for the formation of a white precipitate

2. Million’s test
 a. To 1 mL of the aqueous extract, add 10 drops of Millon’s Reagent
b. Place over a water bath and observe the color.

J. Screening for Fixed and Volatile Oil

Stain test/Spot test
1. Boil 2g of the plant sample in 10ml of hexane (NOTE: Do not use Open Flame)
2. Place 2 drops of the hexane extract on the surface of a piece of white paper
3. Note any stain produced on the paper

DATA AND RESULTS: (TABULAR FORM)

Questions:
1. What is the purpose of preliminary screening of plant constituents?
2. Why is 80% ethanol used in preaparing plant extracts?
3. Give the significance of each active plant constituent in pharmacy and medicine
4. What are primary, secondary, tertiary alkaloids?
5. What are cardiac Glycosides?
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 6

Production of Wine

Objective:
To be able to know the steps involved in the production of wine
Materials:

Yeast, ripe or slightly over-ripe fruits, bottle, cotton, knife, sugar, unbleached cloth, distilled
water.
Procedure:

1. Wash and peel the ripe or slightly over-ripe fruit. Blemished fruits or those no longer fit for table
consumption can be used provided they do not show any sign of decay.
2. Crush the peeled fruit and express the extract the juice then filter the juice through unbleached
muslin.
3. Add two parts distilled water to every part of the extracted juice then add one part sugar to every
four parts of the diluted juice.
4. Heat the mixture without boiling over a low fire for thirty minutes. Set aside to cool
5. Add 2 grams of commercial dry yeast to every liter of the mixture
6. Pour the mixture into any suitable bottle loosely stoppered with a cotton plug.
7. Put aside and allow to ferment for two or more weeks until all the air bubbles in the mixture have
been released.
8. After fermentation, pasteurize the bottle containing the mixture in a water bath for thirty
minutes, maintaining the temperature of the bath at 70-80 degrees Celsius.
9. Let the bottle stand to allow any sediments to settle then decant the clear liquid into another
bottle. Make sure that the bottle is filled to capacity.
10. Place an airtight cover and age for two to 3 months.
11. To obtain a clear wine, add well-beaten egg white to the aged wine in the proportion of one egg
white to every 2 to 3 liters of the mixture
12. Repeat pasteurization process until the egg white curdles
13. Strain the wine through unbleached muslin into the final bottle and cover tightly. The wine is
ready for drinking.
Calculations:
Questions:
1. Describe the principle behind wind production
2. Give 10 products of fermentation and the organism producing each.
3. Outline the Embden-Meyerhoff scheme for the formation of ethanol in yeast.
4. Why is the strength of alcoholic solutions directed by the USP/NF to be determined at 15.56
degrees Celsius?
5. What is meant by proof and gallon proof when referring to alcohol or alcoholic beverages?
6. If an alcohol beverage is 70% strong, what is the proof strength? Show your computations
7. Enumerate the types of wine and their respective sources.
8. Explain the physiologic action of alcohol both in small and large amounts.
9. Why is alcohol no anymore used as an anesthetic
10. Discuss the significance of fermentation in pharmaceutical processes. Cite examples

References:
 URDANETA CITY UNIVERSITY Pharmacognosy with
Plant Chemistry
College of Pharmacy
Laboratory Manual

Name: Date of Submission

Group No: Rating

Experiment No: 7
Carbohydrates
Objective:
1. To extract and test for the presence of starch and sugars in the assigned plant sample
and to observe the characteristics of its granules under the microscope
2. To extract and identify the sugar from sugar cane

Materials
Molisch’s reagent (α-naphthol solution) Phenylhydrazine hydrochloride Ethyl alcohol
Iodine T.S solution Sodium acetate Sulfuric acid
Fehling’s A (copper sulfate) Glacial acetic acid 1%sucrosesolution
Fehling’s B (Rochelle salt) Microscope Blender
Benedict’s reagent Glass slides and cover centrifuge
Barfoed’s reagent sweet Potato (group I) Cheese cloth
Seliwanoff’s reagent Squash (Group II) Oven
Bial’s reagent Sugar cane (Group I)
Apple (Group II)
Procedure:
B. Extraction of Starch
1. Pare your sample cut into small cubes. Weigh accurately about 100 grams
2. Add a small amount of water and homogenize or pound in a mortar with a pestle
3. Strain the pounded pulp through a piece of coarse cloth agitating the pulp vigorously with a
spatula.
4. Collect the filtrate in a large beaker. Express the pulp as completely as possible then add more
water to the residue on the cloth, straining and agitating and expressing as before
5. Repeat this washing, collecting all filtrate in the beaker. Allow the starch to settle for at least 30
minutes and then secant the supernatant liquid that contains cellular debris.
6. Add water, stir and allow the starch to settle. Decant once more. Filter if necessary to remove any
remaining water from the starch.
7. Transfer the product to a porcelain dish and dry in an oven at 50 degrees C. Weigh and determine
the percentage yield
 TEST FOR STARCH
1. Determine the color, odor, taste, appearance and solubility in water
2. Prepare a smooth mixture of a pinch of starch with 1ml of cold water. Stir it into 10ml. of boiling
water. Boil gently for 2 minutes and cool.
3. Test a slurry of starch with iodine T.S result:
4. Microscopic Test: Prepare wet mounts. Using a microscope, observe the typical shape and form
of a representative granules. Draw the Various shapes and forms of the granules

C. Extraction of Sugar
1. Weigh about 100 grams of sugar cane/Apple. Peel, cut into small pieces, add water, and
homogenize with a blender.
2. Squeeze out the juice, strain, and boil with the lime to neutralize free organic acids which cause
hydrolysis or inversion of sugar.
3. Remove the scum which rises to the top. Filter the liquid through a filter paper.
4. Concentrate by evaporating the solution at a much lower temperature (160 to 180 C) on a water
bath.
5. When the sugar has crystallized, separate the crystals from the syrupy molasses by centrifugation.
Dissolve in distilled water and purify by adding small amounts of charcoal.
6. Filter and again concentrate the solution by evaporating on a water bath.
7. Compute for the percentage yield.
Tests for Sugar
Physical Test
Determine the color, odor, taste, appearance and solubility in water.
Chemical Tests:
Molisch’s Test:

In a test tube, add 2 ml of the test carbohydrate solution and 2 drops of α-naphthol solution. Carefully
incline the tube and pour drop wise conc. H2SO4, using a dropper, along the sides of the tube. Observe the
violet color at the junction of the two liquids.

2) Fehling’s Test:

In a test tube, add 2 ml of the test carbohydrate solution and add equal volumes of Fehling
A & Fehling B and place it in a boiling water bath for few minutes.. When the contents of the
test tube comes to boiling, mix them together and observe any change in color or precipitate formation.
The production of yellow 'or brownish-red precipitate of cuprous oxide indicates the presence of reducing
sugars in the given sample.

3) Benedict’s Test:

In the test tube with 2 ml of Benedict's reagent, add 5-6 drops of the test carbohydrate solution and mix
well. Place the test tube in a boiling water bath for 5 minutes and observe any change in color or
precipitate formation. Cool the solution. Observe the colour change from blue to green, yellow, orange
or red depending upon the amount of reducing sugar present in the test sample.
4) Barfoed’s Test:

To 2 mL of the test solution add about 2-3 mL of Barfoed’s reagent. Mix it well and boil it for one minute
in the water bath and allow to stand for a few minutes. Formation of a red precipitate of cuprous oxide in
the bottom and along the sides of the test tube immediately, only monosaccharides answer this test. Since
Barfoed’s reagent is slightly acidic, this test is specific for monosaccharides.

5) Seliwanoff’s Test:

To 2 mL of Seliwanoff ’s reagent, add two drops of test solution. The mixure is heated to just boiling. A
cherry red condensation product will be observed indicating the presence of ketoses in the test sample.
There will be no significant change in colour produced for aldose sugar.

6) Bial’s Test:

To 5 mL of Bial’s reagent add 2–3 mL of test solution and warm gently in a hot water bath for 2minutes .
The formation of a bluish green product is indicative of pentoses. Hexoses generally react to form muddy
brown products.

7) Iodine Test:

Add 2 drops of iodine TS solution to about 2 mL of the carbohydrate containing test solution. A blue-
black colour is observed which is indicative of presence of polysaccharides.

8) Osazone Test:

To 0.5 g of phenylhydrazine hydrochloride add 0.1 gram of sodium acetate and ten drops of glacial acetic
acid. Add 5 mL of test solution to this mixture and heat under boiling water bath for about half an hour.
Cool the solution slowly and examine the crystals under a microscope. Needle-shaped yellow osazone
crystals will be observed for glucose and fructose, whereas lactosazone shows mushroom shaped and
maltose produces flower-shaped crystals.

DATA AND RESULT

Tabulate the following: a.Test performed, b. Expected positive result, Result with sample, d.
interpretation of result.
Questions:
1. Compare acid and enzymatic hydrolysis of starch.
2. Write the different stages in the acid hydrolysis of starch, state the color obtained with iodine at
each stage
3. What are dextrins? How are they prepared?
4. What is meant by Achromatic Point? What is its Significance?
5. Enumerate sources of sugars. Specify sugars present
6. Compare sugar with starch in terms of classification, color, taste, and appearance
7. Discuss the principle involved in each of the aforementioned chemical tests for carbohydrates.
Include equations when necessary.
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
College of Pharmacy Chemistry
Laboratory Manual

Name: Date of Submission

Group No: Rating

EXPERIMENT NO: 8
EXTRACTION OF CITRIC ACID FROM LEMON

Objective:
To be able to extract citric acid from Citrus Fruits like lemon, calamansi, or oranges
To prove its presence by performing identification test.
Materials:
Citrus Fruits, 10% NaOH Solution, 10% Calcium Chloride solution, Water bath, 1N sulfuric acid, Alcohol,
ether, Chloroform, tripod, Water bath, graduated cylinder, beaker, muslin cloth, funnel, filter paper,
stirring rod, test tubes, Bunsen Burner, wire gauze, Capillary tube, Evaporating Dish

Procedure:
1. Squeeze out the juice of a citrus fruit. Measure 90 ml of the citrus juice concentrate into a 250ml
Beaker.
2. Carefully add 10% NaOH solution while stirring until the mixture is slightlty alkaline. Strain the
solution through a fine muslin to remove large particles of pulp and then filter through paper on
a Buchner funnel.
3. Measure the filtrate, place in a beaker and add 5ml of 10% Calcium Chloride solution with
constant stirring to each 10ml filtrate
4. Heat to boiling and filter off the copious precipitate of calcium citrate from the hot solution using
a Buchner funnel.
5. Wash the precipitate collected twice with small quantities of boiling water
6. Resuspend in minimum quantity of cold water, heat to boiling and once more collect the insoluble
calcium citrate by filtration.
7. Weigh the air dried sailt. Place in a beaker and add the calculated amount of 1N sulfuric acid
required to convert the salt to acid. Allow the mixture to stand for few minutes
8. Filter off the insoluble calcium sulfate and concentrate the filtrate to a small volume on a steam
bath.
9. Weigh and calculate the percentage yield.

Reaction in the extraction of Citric Acid:

Reaction in the Isolation of Citric Acid:

Identification tests:
Organoleptic Evaluation: Determine the color, taste, appearance, and Physical state
Physical Test: Solubility test: Add a pinch of the crystals to 5ml of each of the following solvents.
Chemical Test:
1. To a mixture of 15ml of pyridine and 5ml of acetic anhydride, add 10mg of the crystals and
observe the color formation
2. Place 5ml of citric acid solution in a test tube. Render it alkaline with dilute NaOH Solution.
Add 0.1% KMno4, until a permanent purple color is produced. Heat gently to boiling and
observe the color.

Data and Results:

Calculations
Conclusions

Questions:
1. Differentiate the properties of citric acid and tartaric acid. Tabulate
2. Provide natural sources of both citric and tartaric acid
3. Explain the importance of citric acid and tartaric acid in pharmacy
4. Explain the significance of citric and tartaric acid in medicine.
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

EXPERIMENT NO: 9
GUMS AND MUCILAGE

Objective
To know the difference between gums and mucilage by performing the different identification
tests.

Materials
Acacia tears/powders, tragacanth, agar granules, strips, gelatin, iodine solution, HCl, Water bath,
Fehling’s reagent, Barium Chloride T.S, Soda Lime litmus Paper, tannic Acid Solution.

Procedure:
Identification tests:
1. Iodine test. Add a few drops of iodine solution on the following samples
a. Acacia Powder
b. Tragacanth powder
c. Agar
d. Gelatin

2. Solubility test. Place a pinch of the above samples above samples in about 5 ml of water. Observe
the results for each of the samples and record.
3. Test to distinguish Agar from Gelatin
a. Prepare a solution of agar (Place a few granules in water and heat to almost boiling and
add a few drops of tannic acid solution. Repeat with gelatin
b. Hydrolyze a small amount of agar and gelatin solution separately by adding a few drops
of HCl to the solution and boiling in a water bath.
Perform the following tests to about 2ml of the sample and describe the results:
With Fehlings’ reagent
With Barium Chloride T.S
c. Heat a small amount of agar gelatin separately in a test tube with pinch of soda lime over
a direct flame. While heating, expose a moistened litmus paper.

DATA AND RESULTS:

Tabulate the following: A. Test Performed, B. Result with sample, C. Interpretation/Implication
Questions:
1. Differentiate gums from mucilages
2. How are gums classified
3. Provide sources of gums and mucilage
4. Give the pharmaceutical uses of gums and mucilages.
 URDANETA CITY UNIVERSITY Pharmacognosy with Plant
Chemistry
College of Pharmacy Laboratory Manual

Name: Date of Submission

Group No: Rating

EXPERIMENT 10
GLYCOSIDES

Objective:
To be able to confirm the presence of specific glycosides in the assigned plant sample and to
classify the glycosides present based on the chemical tests performed.

Materials:
Aloe plant, Ferric Chloride Conc. H2SO4
Senokot® Dilute HCl Antimony trichloride
Apple Seeds CCl4 20% acetic anhydride
Vanilla NH4OH Strong lead acetate solution
Yellow Bell Flower HNO3 pyridine
Beaker Sodium Nitrite crystals Sodium nutriprusside
Knife Acetic Acid NaOH
Test tubes Saturated CuSO4 Ferric Chloride
Droppers NaCl 1N NaOH
Distilledwater Ethanol Sodium Picrate
Borax Nitric Acid Sodium Carbonate Solution
Bromine Chloroform

Ammonia
Magnesium Ribbons
Zinc Turnings
Vanillin HCl

Procedure:
Preparation of the sample

A. Anthraquinone Glycosides
Samples: Aloe and Senokot®
For Aloe: Cut the leaves transversely at the base. Let stand in a closed vessel and allow the juice to
flow freely without compression until sufficient amount is collected. Evaporate until a
concentrated juice is produced and allow to cool and solidify. Scrape the residue and subject to
the following chemical tests.
 a. Identification test
Dissolve about 1 part of the drug residue to 100 parts of distilled water and perform the
following tests.

General tests:
a. Schonteten’s test: to 2ml of the sample, add 0.5g of borax and heat until it dissolves. Place a
few drops into a test tube nearly full of water. A green fluorescence is produced.
b. Modified Borntrager’s test: to 2ml of the sample, add a few drops of ferric chloride and dilute
HCl (to bring about oxidative hydrolysis). Extract using about 5ml of Carbon tetrachloride.
Separate the carbon tetrachloride layer and shake it with dilute ammonium hydroxide. A rose
pink to cherry red color is produced.

Specific test for Aloe

A. Nitric Acid test: to 2ml of the sample add 1 ml of nitric acid. Cape aloe gives brownish color rapidly
changing to green; Curacao aloe, a deep brownish red; Zanzibar, yellowish to brown color (nitric acid
may be applied on the powder and gives the same result).
B. Nitrous acid Test: To an aqueous solution of the sample, add a few small crystals of sodium nitrite
and little acetic acid. Curacao aloe gives a red pink to carmine, cape aloe gives faint pink color, Zanzibar
and socotrine aloe show a little change in color. The test is due to the presence of isobarbaloin
C. Klunge’s test. To 5ml of the sample, Add 1 drop of saturated copper sulfate solution followed by 0.2g
of sodium chloride and 2.5ml ethanol. With the curacao aloe, a wine red color is produced and persists
for 12 hours. With the cape aloe, lesser color develops which rapidly fades to yellow. Zanzibar and
Scotrine give no color. Red color may be hastened by heating.

For the Next Tests to be done, perform the following procedures prior to conducting the chemical tests:
Extraction and isolation of glycosides:
1. Pulverize the plant sample, then perform extraction using percolation method with alcohol being
the solvent.
2. The alcoholic extract is then treated with lead acetate solution to precipitate tannins, proteins,
coloring matter and non-glycosidal part.
3. Filtrate is evaporated to dryness on water bath and dried residue is used for further testing.

D. Test for Coumarin Glycosides:

Sample: Coumadin®
Chemical Tests:
a. Ferric Chloride test: Prepare an alcoholic extract of the sample, add a few drops of alcoholic
ferric chloride solution. Formation of deep green colour, which turns yellow on addition of
nitric acid indicates the presence of coumarins in the sample.
b. Fluorescence test: The alcoholic extract of your sample is mixed with 1N NaOH solution (1ml).
Development of blue-green fluorescence indicates presence of coumarins.

E. Test for Cyanophore Glycosides:

Sample: Apple Seeds
Chemical Tests:
 a. Sodium Picrate test: Powderize the seeds then, moisten with water in a conical flask and add
a few drops of conc. Sulfuric acid. A filter paper impregnanted with sodium picrate solution
followed by sodium carbonate solution is trapped on the neck of the flask using a cork. Note
for the formation of brick-red color due to volatile HCN present.

F. Test For Aldehyde Glycoside

Sample: Vanilla
Chemical Tests:
1. Ammonia test: A strip of filter paper dipped in alcoholic solution of your sample is exposed
to ammonia vapor. Note for the formation of yellow spot on filter paper.
2. Shinoda test:
a. To the alcoholic extract of your sample, magnesium turning and diluted HCl is added.
Formation of red color indicates the presence of flavonoids.
b. To the alcoholic extract of your sample, Zinc turning and dil. HCl is added, formation of
deep red to magenta color indicates the presence of dihydroflavonoids.
3. Vanillin HCl test: Vanillin HCl is added to the alcoholic solution of your sample, and note the
formation of pink color due to the presence of flavonoids.

Data and results:

Botanical Source of the sample
Tabulate the following:
a. Name of the test performed
b. Reagent used/ with chemical formula
c. Expected positive result
d. Experimental result (wiith sample)
e. Interpretation of the result

Conclusions:

Questions for research:

1. Give the complete botanical sources and origins of the different aloes
2. Aloe is an ingredient in Compound Benzoin Tincture. Give its formulation and use
3. How are glycosides classified? Characterize each type of glycosides.
4. Give examples of plants yielding glycosides. And specify the glycosides they contain.