Laboratory Exercises in Pharmacognosy and Plant Chemistry

Exercise No. 1

THE PHYTOCHEMICAL INVESTIGATION

I. INTENDED LEARNING OUTCOMES:

1. To identify the different primary and secondary metabolites
present in different medicinal plants
2. To acquire the skills in identifying different plant constituents
II. DISCUSSION:
"Phyto" is a Greek word that means plant. Different methods can be
conducted to determine the different constituents present in plants. One of the
methods of identifying the constituents is through phytochemical screening.
A method for use in phytochemical screening should be: (a) simple; (b) rapid;
(c) designed for a minimum of equipment; (d) reasonably selective for the
class of compounds under study; (e) quantitative in so far as having a
knowledge of the lower limit of detection is concerned; and if possible (f)
should give additional information as to the presence or absence of specific
members of the group being evaluated.
Phytochemical investigation of plants involves the following:
1. Authentication and extraction of the plant material
2. Separation and isolation of the constituent of interest
3. Characterization of the isolated compounds
4. Investigation of the biosynthetic pathways of compounds
5. Quantitative evaluation
6. Pharmacological assessment of the separated components

The proper time in the collection of plant parts for phytochemical

screening is an important matter to consider. The plant part is best collected as
follows:
PLANT PART BEST TIME OF COLLECTION
Roots/Rhizomes After vegetative process
Stem/Bark Before vegetative process
Flowers When they are about to bloom
Seeds When matured
Leaves When photosynthesis is active
Fruits When ripe

Extraction of Plant Material

Extraction depends on the nature of the plant material and the
components to be isolated. Dried materials should be powdered before a. Fresh
materials can be homogenized or soaked to a solvent such as alcohol. Alcohol
general solvent for extraction, light petroleum (essential, fixed oils, steroids),
ether and chloroform (Alkaloids), water immiscible solvent (Alkaloids), and
acidification (aromatic acids & phenols)
III. MATERIALS/REAGENTS

Potassium hydroxide 0.5 M Nitric Acid

Sodium chloride 0.9% Magnesium turnings
Sodium chloride 10% Mayer’s reagent
Sodium chloride 10% Millon’s reagent
Ammonia 28% Octyl alcohol
Hydrochloric acid 2M Petroleum ether
Hydrogen peroxide 5% Soda solution
Ethyl alcohol 80% Sodium chloride
Acetic acid anhydride Tannic acid solution
Agar powder Wagner’s reagent
Anhydrous sodium sulfate Cheesecloth
Benzene Cork/Stopper
Chloroform Freshly extracted cow’s blood
Hydrochloric acid 12M Filter paper
Conc. sulfuric acid Gugo bark
Dichloromethane Hot plate
Distilled water Laboratory glassware/materials
Draggendorff’s reagent Medicinal plant
Fehling’s solution (A and B) Petri dish
Ferric chloride TS Reflux condenser
Gelatin powder Rotavapor
Glacial acetic acid Water bath
Hexane

IV. PROCEDURE

A. Collection and Preparation of Samples

 Collect plant for phytochemical screening. Wash air-dried and oven
o
dried at 40 C and grind.

B. Phytochemical Screening of Samples

Preparation and Extraction of the Samples

1. Weigh 100 g of the powdered sample; place it in a 500 mL Erlenmeyer

flask.
2. Macerate it with sufficient amount of 80% ethyl alcohol to completely
submerge the material.
3. Keep the material soaked for 24-48 hours. Reflux distillation could be
used to further extract the constituents.
4. Filter and rinse the flask with fresh portions of 80% ethyl alcohol. Discard
the residue.
5. Collect the filtrate and evaporate it using rotavapor to separate the
solvent from the extract.
6. Further concentrate the extract to about 50mL at a controlled
o
temperature of 40 C.
7. Measure the volume of the concentrated extract.
8. Compute for the concentration of the extracts in grams of dried plant
material per mL extract.
9. Store the extract in a tightly closed container in a cool temperature.
Label the container properly with the name of the sample,
concentration of the plant extract in grams per mL, and the date of
extraction.
Note: To prevent fungal growth, the researcher should add a small
amount of chloroform.

Formula
weight of the plant sample (g)
Equivalent weight/mL =
volume of the plant extract (mL)

weight of the extract

Percentage yield = x 100
weight of the plant sample

TEST FOR THE PRESENCE OF ALKALOIDS

Alkaloids are basic nitrogenous compounds of high pharmacologic
functions.
Alkaloids are present in plant tissues as
 a. water-soluble salts of organic acids (tartaric, acetic, oxalic, citric, malic,
and lactic acids),
b. esters (e.g., atropine, scopolamine, cocaine, aconitine),
c. combined with tannins (Cinchona bark)
d. sugars (e.g., the glycoalkaloids of Solanum species)

rather than as free bases. Most alkaloids are isolated from plant matrices in the
form of crystalline, amorphous, nonodorous, and nonvolatile compounds.
Majority of alkaloids are colorless with a bitter taste.
Free bases of alkaloids are soluble in nonpolar organic solvents
(chloroform, methylene chloride, ether), while their solubility in water is low
(exceptions include caffeine and ephedrine). Salts of alkaloids are soluble in
water or dilute acids, whereas they are insoluble or sparingly soluble in organic
solvents.
These differences in the solubility of alkaloids, depending on their form,
are used in the pharmaceutical industry for their purification from complex plant
matrices and to produce pharmaceutically acceptable products.

A. Preliminary test

1. Get an equivalent of 20 grams of the sample from the stock extract and
place it in an evaporating dish.
2. Evaporate to a syrupy consistency over a steam bath. Add 5 mL of 2M
hydrochloric acid (HCl) and stir and heat it for about 5 minutes in a water
bath and allowed to cool. Add 0.5g sodium chloride, stir, and filter.
3. Wash the residue with enough 2M HCl to bring the filtrate to a volume of
about 6 mL.
4. Get one mL of the filtrate and test with 2 to 3 drops of Mayer’s reagent,
Wagner’s reagent and, Draggendorrf’s reagent. Observe the relative
amounts of precipitation as follows:
(+) Slight turbidity 11 ml of 2M CHU)
(++) Definite turbidity 0.5g Sodium chloride
(+++) Heavy precipitation 2mi Mayer's reagent
Wagner's
B. Confirmatory test Draggendorrf 's .

? Ammonia
1. To the remaining 3 mL of the filtrate, add enough 28% ammonia until the
solution becomes alkaline to litmus (Caution: ammonia causes burns,
vapors extremely irritating.)
2. Extract the alkaline solution three times with small portions of 10 mL of
 chloroform (Caution: Carcinogenic).
3. Combine the lower chloroform extracts and reserve the upper aqueous
layer for the test for quaternary bases.
13mL of 2M CHU)
4. Evaporate the chloroform extract to dryness under the hood and over a
0.5g Sodium chloride
steam bath.
2mi Mayer's reagent 5. Add 5 mL of 2M hydrochloric acid.
Wagner's
Draggendorrf 's 6. Filter the solution and divide the filtrate into three portions.
? Ammonia 7. Test one portion with Mayer’s reagent, Dragendorff’s reagent and Wagner’s
10mL Chloroform reagent. Observe the result and record as (+), (++) or (+++).
8. Positive results indicate the presence of primary, secondary and tertiary
alkaloids.

C. Test for quaternary bases and/or amine oxide

1. Acidify the alkaline aqueous layer obtain (refer to number 3 above) with 2M
HCl.
2. Filter and divide the filtrate into three portions. Add Mayer’s reagent to one
portion, Dragendorff’s reagent and Wagner’s reagent to the other two
portions. Observe and record the result.
3. A score of (++) or (+++) in both Mayer’s and Dragendorff’s tests indicates
the presence of Quaternary and/ or amine oxide bases; a (+) score may
be the result of incomplete chloroform extraction; thus, it should be
considered negative quaternary bases.

SCREENING FOR GLYCOSIDES (HETEROSIDES)

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

A. TEST FOR STEROIDS: CARDENOLIDES AND BUFADIENOLIDES

1. Take an equivalent of 10 g sample from the plant extract stock solution
previously prepared.
2. Evaporate to incipient dryness over a water bath and set aside to cool at
room temperature.
3. Defat the residue with the use of 6 mL of hexane and water, 2:1 v/v;
observe a partition by gently shaking the mixture in a test tube; pipette out
the upper hexane layer; then, repeat the treatment with hexane until most
of the colored pigments had been removed.
4. Discard properly all the hexane extracts. Heat the defatted aqueous layer
over a water bath to remove the residual hexane; cool at room
temperature.
5. Divide into three portions for Keller-killiani test, Liebermann-Burchard test
and Salkowiski's test.
6. Positive result: A reddish-brown color, which may turn blue or purple,
indicates the presence of 2-deoxysugars.

Keller-killiani test: test for 2-deoxysugars

 1. Add three mL of Ferric chloride to one portion of the defatted aqueous layer
free from hexane and stir.
2. Incline the test tube and add 1 mL of concentrated sulfuric acid cautiously
using a pipette, (Caution: very corrosive), letting the acid flow down along
the inside of the test tube; allow the mixture to stand upright and observe
the coloration at the interface of the acid and the aqueous layer.

Liebermann-Burchard test: test for Unsaturated steroids

1. Add 10 mL dichloromethane to a portion of the defatted aqueous layer free

from hexane, stir with a glass rod for a few minutes and allow to stand.
The lower dichloromethane (CH2Cl2) extract was pippetted off.
2. Dry the dichloromethane (CH Cl ) extract by passing the extract through
2 2
about 100 mg anhydrous sodium sulfate (Na2SO4) place a dry filter paper
in a funnel.
3. Divide the filtrate into two portions; one will be used as the control and the
other portion will be treated with 3 drops of acetic acid anhydride (Caution:
Corrosive); then add one drop of concentrated sulfuric acid (Caution:
Highly corrosive). Observe for any immediate color change.
4. Allow to stand for an hour and observe for further color change. Compare
with the control.
5. Positive results: Observe colors ranging from blue to green, red, pink, purple
or violet because of the steroid/triterpenoid skeleton.

B. Salkowiski's test

To the second portion of chloroform filtrate an equal volume of sulfuric acid

is added. The appearance of a red color indicates the presence of unsaturated
sterol and /or triterpenes.

TEST FOR ANTHRAQUINONE GLYCOSIDES

The largest groups of naturally occurring quinine substances are the

anthraquinones. Although they have a widespread use as dyes, their chief
medicinal value is dependent upon their cathartic action.
They are restricted distribution in the plant kingdom and are found most
frequently in members of the Rhamnaceae,Polygonaceae, Rubiaceae,
Leguminosae and Liliaceae. As found in plant, they are usually carboxylated,
methylated or hydroxylated derivatives of the anthracenes, anthrone, anthranol,
anthraquinone, or dianthrone.
A. Borntrager’s test

1. Take an equivalent of 1 g sample from the prepared stock extract and

 evaporate to incipient dryness over a steam bath.
2. Take the residue with 10 mL distilled water, filter and the discard the residue;
extract the aqueous filtrate with 5 mL portions of benzene (Caution:
'

Carcinogenic) O twice and combine the two portions of the benzene

extracts.
3. Divide the combined benzene extracts into two portions. One portion serves
as the control. Treat the other portion with 5 mL ammonia solution and
shake. Compare the results with the control tube.
4. Positive result: A red coloration in the lower ammoniacal layer indicates the
presence of anthraquinones.

B. Modified Borntrager’s test

1. Take an equivalent of 1 g sample from the prepared stock extract and
evaporate to incipient dryness over a steam bath.
2. Add 10 mL 0.5 M Potassium hydroxide and 1 mL of 5% hydrogen peroxide
(H2O2) and stir. Heat the resulting mixture over a steam bath for 10
minutes. Filter and discard the residue.
3. Acidify the filtrate with glacial acetic acid. Collect the filtrate and extract it
with two 5 mL portions of benzene (Caution: Carcinogenic); combine the
benzene extracts and divide into two portions, one portion serves as the
control and alkalinify the other portion with ammonia and shake. Compare
the result with the control.
4. Positive result: A pink color indicates a positive result.

TEST FOR FLAVONOIDS

The term flavonoids refers to a class of plant secondary metabolites based

around a phenyl benzopyrone structure. Flavonoids are most commonly known
for their antioxidant activity. Flavonoids are also commonly referred to as
bioflavonoids in the media –these terms are equivalent and interchangeable
since all flavonoids are biological in origin. The flavonoid synthetic pathway
begins with a product of glycolysis phosphoenolpyruvate entering into the
shikimate pathway to yield phenylalanine.

1. Take an equivalent of 10 g sample from the prepared stock extract and

evaporate to incipient dryness over a steam bath.
2. Cool at room temperature; defat by taking up the residue with 9 mL hexane

① a-
and water (2:1) or with petroleum ether (Caution: solvents are flammable).
3. Discard the hexane or petroleum ether extract.
4. Dilute the defatted aqueous layer with 10 mL of 80% ethyl alcohol; filter and
divide the filtrate into three test tubes.
5. Use one portion as the control and the 2 test tubes for test for

leucoanthocyanin and test for g-benzopyrone nucleus .

A. Test for Leucoanthocyanins: Bate-Smith and Metcalf Test Method

1. Treat one portion of the above alcohol filtrate with 0.5 mL concentrated
 hydrochloric acid (12 M); and observe for any color change.
2.Warm the solution for 15 minutes in a water bath. Observe for further color
change in color within an hour and compare with the control. The results
are recorded.
3. Positive result: A strong red or violet color indicates the presence of
leucoanthocyanins.

B. Test for y-benzopyrone nucleus: Wilstatter “cyanidin” test

1. Take another portion of the alcohol filtrate and treat it with 0.5 mL
concentrated hydrochloric acid (12 M).
2. Place three to four pieces of magnesium turnings and observe any color
change within 10 minutes; compare the results with the control tube.
o
3. If definite coloration occurs, dilute it with an equal volume of water and 1 mL
octyl alcohol. Shake the solution and allow to the solution to stand for few
minutes. Note the color in each layer. Record the results.
4. Positive result: Observe colors ranging from orange to red, crimson, and
magenta and occasionally to green or blue.

SCREENING METHODS FOR SAPONINS:

Saponins are glucosides with foaming characteristics. Saponins consist of
a polycyclic aglycones attached to one or more sugar side chains. The aglycone
part, which is also called sapogenin, is either steroid (C27) or a triterpene (C30).
The foaming ability of saponins is caused by the combination of a hydrophobic
(fat-soluble) sapogenin and a hydrophilic (water-soluble) sugar part. Saponins
have a bitter taste. Some saponins are toxic and are known as sapotoxin.

A. Froth Test

Preparation of the “gugo” extract

1. Take about 1 g of gugo Entada phaseoloides (L.) Merr. bark. Cut into
strips and soak in 10 mL of 80% ethyl alcohol.
2. Allow to stand for 30 minutes. Take a volume of the prepared sample
extract equivalent to 2 g sample and transfer into a test tube.
3. In a separate test tube, take 1 mL of the “gugo” extract and served as
the standard; Add ten mL of distilled water to each of the test tubes;
place a stopper and shake both test tubes vigorously for 30 seconds
and allow to stand for 10 minutes; observe for “honeycomb” froth;
compare the results of the extract with those of the standard.
4. Positive result: If the “honeycomb” froth greater than 2 cm height from
the surface of the liquid persists after 10 minutes, consider the sample
positive for saponins.

B. Hemolytic test: Agar cup method

 Preparation of Blood agar plates:

Defibrinated blood

1. Take about 200 mL blood of freshly slaughtered cow. Stir vigorously until
fibrin agglutinates.
2. Filter-off the gelatinous residue through cheesecloth. The filtrate is the
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defibrinated blood. Keep for 1-2 days at 3-4 C.

Blood agar plate

1. Add about 100 mL of 0.9% sodium chloride solution to 1.8 g agar

powder or as stated in the label on how to prepare blood agar.
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2. Allow to stand for 30 minutes then heat at 80 C on a water bath with
stirring.
o
3. Cool to 40 C; Add 1 mL of defibrinated cow’s blood and stir.
4. Pour about 30 mL in each petridish and allow to set.
5. Obtain a blood agar plate; using a cork borer of 10 mm. diameter or a
small test tube, bore the blood agar plate cleanly at a three equidistant
cups into the agar.
6. Number each agar cup at the bottom of the inverted plate; use a marking
pen.
7. Fill with a small pipette, one agar cup with the aqueous sample extract;
the other cup with “gugo” extract serving as positive control and the
third cup with distilled water and served as negative control; allow the
covered plates to stand undisturbed for one hour.
8. Observe any clear zone for hemolysis or “hemolytical halos”.
9. Record the diameter of the halo in millimeter as observed around the
entire agar cups.

SCREENING FOR TANNINS

Two groups of phenolic constituents, hydrolysable and condensed,

comprise the tannins, substances which are important economically as agents for
the tanning of leather and for certain medicinal purposes. More recently,
evidence has been presented in support of their potential value as cytotoxic
neoplastic agents.
Properties of tannins
Hydrolysable tannins are yellow-brown amorphous substances which
dissolve in hot water to form colloidal dispersions. They are astringent and could
be used in tanning industry. They are esters which can be hydrolyzed by boiling
with diluted acid to yield a phenolic compound, usually a derivative of gallic acid,
and a sugar. These are often referred to as pyrogallol tannins.
Condensed tannins are polymers of phenolic compounds related to the
flavonoids and are similar in general properties to the hydrolyzed tannins but are
not very soluble in water and following treatment with boiling dilute acid red-
brown insoluble polymers known as phlobaphene or tannins-red are formed.
Test for Tannins
 1. Take an equivalent of 10 g sample from the prepared extract and evaporate
to incipient dryness over a steam bath; extract the residue with 20 mL of
hot distilled water and add 5 drops of 10% sodium chloride solution.
2. Filter the solution and divide the filtrate into three test tubes. One portion
serves as the control; take an aqueous solution of tannic acid as reference
standard.

A. Gelatin test

1. Treat one portion of the filtrate of the sample extract with three drops of
gelatin salt solution (mix an equal amount of 1% gelatin solution and
10% sodium chloride solution) which serves as the reference standard.
Then compare the control and the reference standard.
A
2. Positive result: The formation of a jelly-precipitate indicates the presence
of tannins.

B. Ferric chloride test

1. Treat another portion, the sample extract with three drops of ferric
chloride reagent and likewise to the tannic acid solution. Compare the
results with the control and the reference standard.
2. Positive result: A blue-black color indicates the presence of hydrolyzable
tannins, while a brownish-green color may indicate the presence of
nonhydrolyzable/condensed tannins.

SCREENING FOR CARBOHYDRATES

Carbohydrates are carbon compounds that contain large quantities of
hydroxyl groups. The simplest carbohydrates also contain either an aldehyde
moiety (these are termed polyhydroxyaldehydes) or a ketone moiety
(polyhydroxyketones). All carbohydrates can be classified as either
monosaccharides, oligosaccharides or polysaccharides. Anywhere from two to
ten monosaccharide units, linked by glycosidic bonds, make up an
oligosaccharide. Derivatives of the carbohydrates can contain nitrogens,
phosphates and sulfur compounds. Carbohydrates also can combine with lipid to
form glycolipids or with protein to form glycoproteins

Procedure

Evaporate about 2 grams equivalent of 80% alcoholic extract; take the

residue with 10 mL distilled water and divide it into three equal parts; then
perform the following tests:

A. Fehling’s Test
 1. Boil about 5 mL of freshly prepared Fehling’s solution (2.5 mL each of
Fehling’s A & B) using a test tube. Add an equal quantity of plant
extract and boil again.
2. Positive result: brick red precipitate indicates the presence of reducing
sugars.

B. Molisch's test:
1. Mix two mL of the prepared filtrate with 0.2 mL of alcoholic solution of α-
naphthol 10% in addition to 2 mL of sulfuric acid.
2. Positive result if: bluish violet zone is formed this indicates the presence
of carbohydrates and/or glycosides.

C. Benedict's test:
1. Add to 1 mL of the filtrate, 5 mL of Benedict's reagent. Heat the mixture.
2. Positive result: appearance of red precipitate indicates the presence of
reducing sugars.

SCREENING FOR PROTEINS

Proteins contain a wide range of functional groups. These functional groups
include alcohols, thiols, thioethers, carboxylic acids, carboxamides, and a variety
of basic groups. When combined in various sequences, this array of functional
groups accounts for the broad spectrum of protein function. For instance, the
chemical reactivity associated with these groups is essential to the function of
enzymes, the proteins that catalyze specific chemical reactions in biological
systems

1. Evaporate 2 g equivalent of the plant extract.

2. Take up the residue with 10 mL of water. Perform the following tests:

A. Millon’s Test

1. Add to 1 mL of the aqueous extract, 10 drops of Millon’s reagent and

place in a boiling water bath.
2. Positive result: Observe for the presence of flesh color.

B. Xanthoproteic Test

1. Add to 1 mL of the aqueous extract, 10 drops of nitric acid.

2. Positive result: Formation of yellow precipitate.

SCREENING FOR LIPIDS

Lipids in biological systems include fats, sterols, fat soluble vitamins,
phospholipids, and triglycerides. They serve as structural components of
biological membranes. They provide energy reserves, predominantly in the form
of triglycerides (TGs; also called triacyglycerols, TAGs). Lipids and lipid
derivatives serve as biologically active molecules exerting a wide range of
functions. Lipophilic bile acids aid in emulsification, digestion and absorption of
dietary lipids as well as being a form of bioactive lipids

Procedure
1. Boiled about 2 g equivalent of plant sample and add 10 mL of petroleum
ether or hexane.
2. A piece of white paper and on its surface, place two drops petroleum ether
extract.
3. Positive result: a permanent greasy stain on paper.

THIN LAYER CHROMATOGRAPHY

TLC is a type of planar chromatography. It is routinely used by researchers
in the field of phyto-chemicals, biochemistry, and so forth, to identify the
components in a compound mixture, like alkaloids, phospholipids, and amino
acids. It is a semi quantitative method consisting of analysis.

Principle
Like other chromatographic methods, thin layer chromatography is also
based on the principle of separation.
The separation depends on the relative affinity of compounds towards
stationary and the mobile phase.
The compounds under the influence of the mobile phase (driven by
capillary action) travel over the surface of the stationary phase. During this
movement, the compounds with higher affinity to stationary phase travel slowly
while the others travel faster. Thus, separation of components in the mixture is
achieved.
Once separation occurs, the individual components are visualized as
spots at a respective level of travel on the plate. Their nature or character are
identified by means of suitable detection techniques.

System Components

TLC system components consists of:

1. TLC plates, preferably ready made with a stationary phase: These are
stable and chemically inert plates, where a thin layer of stationary phase is
applied on its whole surface layer. The stationary phase on the plates is of
uniform thickness and is in a fine particle size.
2. TLC chamber. This is used for the development of TLC plate. The chamber
maintains a uniform environment inside for proper development of spots. It
also prevents the evaporation of solvents, and keeps the process dust free
3. Mobile phase. This comprises of a solvent or solvent mixture The mobile
phase used should be particulate-free and of the highest purity for proper
development of TLC spots. The solvents recommended are chemically
inert with the sample, a stationary phase.
4. A filter paper. This is moistened in the mobile phase, to be placed inside
the chamber. This helps develop a uniform rise in a mobile phase over the
 length of the stationary phase.

Procedure

1. With a pencil, a thin mark is made at the bottom of the plate to apply the
sample spots.
2. Then, samples solutions are applied on the spots marked on the line in
equal distances.
3. The mobile phase is poured into the TLC chamber to a leveled few
centimeters above the chamber bottom. A moistened filter paper in mobile
phase is placed on the inner wall of the chamber to maintain equal
humidity
4. Now, the plate prepared with sample spotting is placed in TLC chamber so
that the side of the plate with the sample line is facing the mobile phase.
Then the chamber is closed with a lid.
5. The plate is then immersed, such that the sample spots are well above the
level of mobile phase (but not immersed in the solvent — as shown in the
picture) for development.
6. Allow enough time for the development of spots. Then remove the plates
and allow them to dry. The sample spots can now be seen in a suitable UV
light chamber, or any other methods as recommended for the said sample.

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