EXPERIMENT NO.

4 PHYSICAL PROPERTIES OF PLANT EXTRACT

The purity and quality of plant extract may be judged to some extent
by their appearance, odor, color, taste, consistency; but the information
gained from determination of the pH, and solubility tests serve as an
identifiable measure for the plant extract.
pH, measures the hydrogen ion concentration at 25°C. The pH of a
solution can be determined directly by measuring the electric potential arising
at special electrodes immersed in the solution with the aid of a pH meter.
The pharmacopoeia prescribes solubility tests in 1 ml of each solvent
such as carbon tetrachloride, chloroform, petroleum ether, ethyl alcohol,
normal saline solution and distilled water in 1mL of plant extract collected in
separate test tubes (1:1 ratio).

Materials:
50% methyl alcohol or Balance
80% ethyl alcohol Erlenmeyer flask 500mL
Carbon tetrachloride Graduated cylinder
Chloroform Aluminum foil
Petroleum Ether Funnel Wire gauze
Distilled water Filter paper 6 Test tubes
Ethyl alcohol Beaker 500mL Tripod
Normal saline solution evaporating dish
Bunsen burner

Procedure: Preparation of the crude extract

1. Weigh 50 g of the ground dried plant material (per group) in an
Erlenmeyer flask
2. Add sufficient amount of 50% methyl alcohol or 80% ethyl alcohol to
completely submerge the plant materials
3. Stopper and macerate the mixture for 72 hours
4. Filter through a Buchner funnel preferably with gentle suction. Rinse the
flask and the plant material with fresh portions of either methyl alcohol or
ethyl alcohol
5. Combine the washings with the first filtrate. Discard the plant residue.
6. Concentrate the filtrate or until there is no trace of the alcohol
7. Measure and weigh accurately the crude extract
8. Calculate the % yield
9. Store the extract in a tightly stoppered container, preferably in the cold
temperature.

Name: Gr. No.: Rating:

Yr & Sec: Date Performed: Date Submitted:

EXPERIMENT NO. 4 PHYSICAL PROPERTIES OF PLANT EXTRACT

Data and Results:

Name of Plant sample: _________________________ Scientific name:

_________________________
Common names: _________________________
_________________________
Family: _________________________
 Physical properties: Results
Color
Consistency
Odor
pH
Specific gravity
Taste
Solubility:
Carbon tetrachloride
Chloroform
Petroleum Ether
Distilled water
Ethyl alcohol
Normal saline
solution

Calculations:
Total Amount Yield =

Percentage Yield =

Conclusion:

EXPERIMENT NO. 5
PHYTOCHEMICAL ANALYSIS OF PLANT EXTRACT

Phytochemistry is concerned with an enormous variety of organic

substances that are elaborated and accumulated by plants and deals with
the chemical structures of these substances and biosynthesis, and their
natural distribution and biological function. Chemical constituents of plants
can be classified in different ways. Based on biosynthetic origin, solubility
properties, and presence of certain key functional groups, it is classified as
phenolic compounds, substances that are readily recognized by their
hydrophilic nature and by their common origin from aromatic precursor
shikimic acid. Terpenoids share lipid properties and a biosynthetic origin from
isopentyl pyrophosphate.
Organic acids, lipids, alkenes, and related hydrocarbons,
polyacetylenes, and sulfur compounds are derived biosynthetically from
acetate. Nitrogen compounds of plants like amino acids, amines, alkaloids,
cyanogenic glycosides, indoles, purines, pyrimidines, cytokinins, and
chlorophyll are basic substances recognized by their positive responses to
either ninhydrin or Dragendorff reagent. Monosaccharides, oligosaccharides,
sugar alcohols, and cyclitols are water–soluble carbohydrates and their
derivatives. Macromolecules of plants such as nucleic acids, proteins, and
polysaccharides are easily separated from other constituents by their high
molecular weights.

Extraction Process
The choice of extraction procedure depends on the nature of the
plant material and the components to be isolated. Dried materials are usually
powdered before extraction, whereas fresh plants can be homogenized or
macerated with a solvent such as alcohol. The latter is also particularly
useful for stabilizing fresh leaves by dropping plant constituents and as such
may give problems in the subsequent elimination pigments, resins, etc. water
immiscible solvents are widely used-light petroleum and, ether and
chloroform. The extraction of organic bases usually necessitates basification
of the plant material if a water-immiscible solvent is to be used; for aromatic
acids and phenols acidification may be required. Extraction itself may be
performed by repeated maceration with agitation, percolation or by
continuous extraction. Special methods for volatile oils, such as the
enfleurage process. Ultrasound may enhance the extraction process for
some plant materials and the BP uses this in the preparation of 50%
ethanolic solution of opium for the assay of
alkaloids.(Trease and Evans, 2007)

Plant constituents
Alkaloids are group of molecules with a relatively large occurrence in nature
around the Globe. They are very diverse chemicals and biomolecules, but
they are all secondary compounds and they are derived from amino acids or
from the transamination process. Alkaloids are classified according to the
amino acids that provide their nitrogen atom and part of their skeleton.
Similar Alkaloids can have quite different biosynthetic pathways and different
bio-impacts. Alkaloids have been found to exhibit dramatic physiological
effects, making them valuable in the field of medicine. Although most
alkaloids have bitter taste, the taste test is by no means an infallible test to
detect the presence of alkaloids in fresh leaves or fruits. ( Aniszewski, 2007)
Anthraquinones from the largest group of naturally occurring quinine
pigments. They occur in plants usually as hydroxylated, methylated or
carboxylated derivatives of anthraquinones, anthrones, anthranal or
dianthrone. They also occur as O- and/or as C-glycosides, or as their
aglycones. They are commonly used as dyes and
cathartics or purgatives (Guevarra, 2008)
Carbohydrates or sugars, form an extensive class of natural organic
compounds that constitute the major fraction of the organic substance on our
planet. These compounds of carbon, hydrogen and oxygen include the
sugars formed in photosynthesis occurring as storage products and used up
in respiration; the starches which are also storage products; and the
celluloses which from the framework of the plant. (Momcilo, 2009)
Cyanogenic glycosides are chemical compounds that occur naturally in
many plants, including species of Prunus (wild cherry).
Sambucus(elderberry), Mannihot (cassava), Linum (flax), Bambusa
(bamboo), and Sorghum (sorghum). Chemically, they are defined as
glycosides of α- hydroxynitriles. These compounds are potentially toxic as
thay are readily broken down by enzamic hydrolysis to liberate hydrogen
cyanide when the plant suffers physical damage.(Guevarra, 2008)
Flavonoids are plant secondary metabolites with a diphenylpropane-
containing structure. Flavonoids are normal constituents of the diet,
as they are present in many fruits, vegetables and beverages. Numerous
preclinical studies have been shown that flavonoids have anticancer properties,
yet some studies have revealed that high doses of some flavonoids can
produce carcinogenic effects.(Shwabb, 2006)
Fixed Oils - These contain carbon, hydrogen and oxygen and are
also insoluble in ether and chloroform. These are the chief storage materials
of seeds. Other substances extracted with fats are waxes, phospholipids and
sterols. (Hulburd, 2007)
Glycosides are white crystalline substances when pure with bitter taste,
soluble in water and in most organic solvents except ether. They are non-
reducing substances which on hydrolysis brought about by reagents or
enzymes yield one or more sugars and the non-sugar part called aglycone.
Aglycones are of such varied nature and complexity that glycosides vary in
their physical and chemical properties and in their pharmacological
action.(Arias, 2006)
Proteins Any of a group of complex organic macromolecules that contain
carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of
one or more chains of amino acids. Proteins are fundamental components of
all living cells and include many substances, such as enzymes, hormones,
and antibodies, that are necessary for the proper functioning of an organism.
They are essential in the diet of animals for the growth and repair of tissue
and can be obtained from foods such as meat, fish, eggs, milk, and legumes.
(Wooldridge, 2009)
Resins any of a group of solid or semisolid amorphous compounds that are
obtained directly from certain plants as exudations. They are used in
medicine and in varnishes .They large number of synthetic, usually organic,
materials that have a polymeric structure, esp such a substance in a raw
state before it is moulded or treated with plasticizer, stabilizer, filler, etc.
(Wooldridge, 2009)
Saponins are highly molecular-weight glycosides, consisting of a sugar
moiety linked to a triterpene or steroid aglycone. The classical definition of
saponins is based on their surface activity; many saponins have detergent
properties, give stable foams in water, show haemolytic activity, have a bitter
taste and are toxic to fish (piscicidal). Such attributes, while not common to
all saponins, have frequently been used to characterized this class of natural
products. However, because of the numerous exceptions which exist,
saponins are now more conveniently defined on the basis of their molecular
structures, namely as triterpene or steroid glycosides. (Martson, 2006)
Steroids: Cardenolides and Bufadienolides are steroidal glycosides and
are referred to as cardiac glycosides since they have found a dramatic effect
on the heart muscle. These cardiac glycosides differ on the steroidal
skeleton, the cardenolides have 5-membered lactonering while the
bufadienolides have a 6-numbered lactone ring. Both types have an
unsaturated lactone ring and one or more 2- deoxysugars. ( Guevarra, 2008)
Tannins are a major class of secondary metabolites that are widespread in
plants (Mole and Kraus, 2003). They are water-soluble polyphenolics with
molecular weights typically ranging from 1000 to 3000. Tannins are capable
of complexing and subsequently precipitating proteins, and they can also
bind to other macromolecules.( Graca, Barlocher and Gessner, 2007).
Terpenoids (or isoprenoids) are a group of compounds in all living
organisms and the largest group of natural compounds existing in nature
with more than 40,000 structures (Bohlmann and Keeling, 2008). Terpenoids
possess many biological properties, which have made them widely used for
traditional and modern purposes as pharmaceuticals, flavors, fragrances,
pesticides, antineopalstic drugs, antibacterial agents, food additives (eg.
Vitamins, etc.) (Mahmoud and Croteau, 2002; Kintzios, 2006; Bohlmann and
Keeling, 2008).
Volatile Oils is a concentrated hydrophobic liquid containing volatile aroma
compounds from plants. Essential oils are also known as volatile oils,
ethereal oils or aetherolea, or simply as the "oil of" the plant from which they
were extracted, such as oil of clove. An is "essential" in the sense that it
carries a distinctive scent, or essence, of the plant. Essential oils do not form
a distinctive category for any medical, pharmacological, or culinary purpose.
(Panda, 2009)

Phytochemical test for the extract

Qualitative tests will be done on the extract to determine the
presence of the plant constituents:

Screening for Alkaloids Test for Alkaloid

content
Add 5 ml of 2M hydrochloric acid to 2 grams of crude extract. Heat the
mixture accompanied by stirring for 5 minutes then cool. Add about 0.5 gram
of sodium chloride in the mixture. Stir the solution and filter, then add enough
of the 2M hydrochloric acid to wash again the mixture, filter and bring to its
final volume of 5mL.

Dragendorff’s Test
To about 1 mL of the filtrate, add 5 drops of Dragendorff’s reagent.
Formation of orange precipitate indicates the presence of alkaloids.

Mayer’s Test
Take about 1 mL of the filtrate and add five drops of Mayer’s reagent.
Formation of white precipitate indicates the presence of alkaloids.

Screening for Glycosides

Saponins
a. Froth test: Dissolve 1 g of crude extract into 10 mL of purified water,
stopper and shake vigorously for 30 seconds. Allow to stand and observe
for 30 minutes. Formation of “honeycomb” froth greater than 3 cm from
the surface of the liquid persists after 30 minutes will indicate the
presence of saponin.
Deoxysugar
b. Keller-Killiani test: Defat 1 g of crude extract with 10 mL of hexane.
Decant off the hexane extract and repeat the treatment until most of the
colored pigments have been removed. Discard the hexane extract. Warm
the defatted residue over a water bath to remove the residual hexane.
Add 3 mL of ferric chloride reagent. Stir and transfer the mixture to a test
tube. With the test tube in an inclined position, add cautiously 1 mL of
concentrated sulfuric acid, letting the acid run along the sides of the tube.
Allow the mixture to stand and observe for any coloration at the interface.
Formation of reddish-brown color, which may turn to blue or purple, will
indicate the presence of 2-deoxysugar.

Steroidal group
c. Liebermann-Burchard Test: Defat 1 g of the crude extract by extracting
with 10 mL of hexane or petroleum ether. Discard the hexane or
petroleum ether extract. Repeat the treatment until most of the coloring
 material have been removed. Treat the resulting residue with 10 mL
chloroform and stir the mixture for about 5 minutes. Allow to stand and
pipette off the chloroform extract. Dry the chloroform extract by filtering
the mixture through about 100mg sodium sulfate over a dry filter paper.
Divide the filtrate into 2 portions. Use one for control. Treat the other
portion with 5 drops of acetic anhydride, then one drop of concentrated
sulfuric acid. Observe for any immediate color change. Let it stand for
an hour and observe for further color change. Compare with the control.
Unsaturated lactones
d. Kedde test: Dissolve 1 g of crude extract with 2 mL of chloroform. Collect
the chloroform layer (lower layer) and treat with 5 drops of kedde
reagent. Formation of violet color indicate the presence of unsaturated
lactone portion of the glycosides. Leucoanthocyanins
e. Bate-Smith and Metcalf test: Dissolve 1 g of the crude extract with 1 mL
concentrated HCl and observes for any color change. Warm the mixture
for 15 minutes. Observe for further color change within an hour.
Formation of red or violet color indicate the presence of
leucoanthocyanins. γ – benzopyrone nucleus
f. Wilstater cyanidin test: Dissolve 1 g of the crude extract with 1 mL
concentrated HCl. Add 5 pieces of magnesium turnings. Observe for any
color change within 10 minutes. If definite coloration occurs, dilute with
an equal amount of water and add 1 mL of octyl alcohol. Shake well and
allow to stand. Note the color in each layer.

Carbohydrates Analysis
Dissolve 1 gram of the extract in 10 ml of distilled water and filter. The
filtrate will be used to test for the presence of carbohydrates.

Molisch’s Test: treat the filtrate with 2 drops of alcoholic α-naphthol

solution in a test tube. Add 2 mL of sulfuric acid in the test tube in a slanting
position.
Formation of the violet ring at the junction indicates the presence of
Carbohydrates.

Benedict’s Test: treat the filtrate with Benedict’s reagent and heat
gently. Orange red precipitate indicates the presence of reducing sugars.

Fehling’s Test: Hydrolyse the filtrate with dil. HCl, neutralize with
alkali and heat with Fehling’s A & B solutions. Formation of red precipitate
indicates the
presence of reducing sugars. (Tiwari, 2011)

Test for Proteins

Add about 10 mL of water to 1 gram of the crude extract.

Xanthoproteic Test
In a test tube, place about 1 mL of the aqueous extract and add 10 drops of
nitric acid. Observe for the formation of the white precipitate.
 Millon’s Test
Place about 1 mL of the aqueous extract in a test tube, and add 10 drops of
Millon’s reagent. Place the test tube in a boiling water bath. Observe for color
change.

Ninhydrin Test: To the extract, add 0.25% w/v ninhydrin reagent and
boil for few minutes. Formation of blue color indicates the presence of amino
acid. (Tiwari,
2011)

Tannins Analysis
Take an equivalent of 10 g plant material from the stock plant extract
prepared and evaporate this to incipient dryness over a steam bath. Extract
the residue with 20 mL of hot distilled water. Add 5 drops of 10% sodium
chloride solution; filter and divide the filtrate into three test tubes. Take one
portion as the control; take an aqueous solution of tannic acid as a reference
standard.

Gelatin Test: treat one portion of the filtrate of the plant extract with three
drops of gelatin salt reagent. Do likewise to the tannic acid solution, the
reference standard; compare with the control and the reference standard.
Formation of jelly precipitate indicates the presence of tannins. (Guevara,
2005)

Ferric chloride Test: treat another portion of the plant filtrate with three
drops of ferric chloride reagent. Do likewise to the tannic acid solution;
compare with the control and the reference standard. A blue-black color
indicates the presence of hydrolyzable tannins, while a brownish- green color
may indicate the presence of
condensed tannins. (Guevarra, 2005)

Lead Acetate- Acetic Acid Test: In a test tube, place 5 mL of the

solution of the extract in distilled water in a mixture and add 4-5 mL of lead
acetate TS and 2mL acetic acid TS. Observe for the formation of precipitate,
which will be dissolved upon the addition of acetic acid.

Test for Fixed Oils, Fats and Volatile Oils Stain Test
Boil about 2 grams of the crude extract in 10 mL of petroleum ether or
hexane in a test tube. Place 2 drops of the petroleum ether extract on the
surface of white paper.
Observe the stain produced in the paper.

Test for Lipids

Salkowski’s Test
Dissolve about 1 gram of the extract in 1 mL of chloroform, add sulfuric acid
to form a lower layer. A reddish-brown color at the interface indicates the
presence of steroidal ring.

Test for Resin

 Flame Test
Ignite about 1 gram of the extract using a wire loop. Production of yellow
flame with smoky odor indicates the presence of resin.

Name: Gr. No.: Rating:

Yr & Sec: Date Performed: Date Submitted:

EXPERIMENT NO. 5
PHYTOCHEMICAL ANALYSIS OF PLANT EXTRACT

Data and Results:

 Name of Plant sample: _________________________ Scientific name:
_________________________
Common names: _________________________
_________________________
Family: _________________________
Results of Phytochemical analysis:

Constituents Actual Positive Inference

Results Results
Alkaloids
LiebermannBurchard Formation of
Test orange precipitate
Dragendorff’s indicates the
Formation of ofviolet
presence
Test
Unsaturated color indicate the
alkaloids.
lactones presence
Formation of of
d. Kedde test: white precipitatelactone
unsaturated
portion
indicates of
the the
Mayer’s glycosides.
presence of
Test
e. Leuco- Formation of red or
alkaloids.
Anthocyanins violet color indicate
Glycosides
Bate- the presence of
leucoanthocyanins.
Smith and Metcalf
a. Saponins Formation of
test- Froth “honeycomb” froth
greater than 3 cm
test from the surface of
f. γ – the Observe for any
liquid persists
benzopyrone aftercolor
30change
minuteswithin
nucleus will indicate the If
10 minutes.
definite
presence coloration
of
- occurs, dilute with
saponin.
Wilstater cyanidin an equal amount
b. Deoxy- Formation of of
test water and add 1 mL
reddish-brown
sugar – of which
octyl mayalcohol.
color,
KellerKilian turn to blue or and
Shake well
i allowwill
purple, to indicate
stand. Note
the presence in ofeach
Test the color
layer.
2deoxysugar.
c.Carbohydrates
Steroidal immediate color
a. Molisch’s Formation
change. of violet
group test ring at the junction
indicates the
presence of
Carbohydrates.
b. Benedict’s Test Orange red
precipitate indicates
the presence of
reducing sugars.
c. Fehling’s test Formation of red
precipitate indicates
the presence of
reducing sugars.
Proteins
a. Xantho- proteic formation of white
test precipitate.
b. Millon’s test Observe for color
change.
c. Ninhydrin test Formation of blue
color indicates
the
 presence of
amino acid.
Tannins
a. Gelatin Formation of jelly
test precipitate indicates
the presence of
tannins.
b. Ferric A blue-black color
chloride indicates the
presence of
Test hydrolyzable tannins,
while a brownish-
green color may
indicate the
presence of
condensed tannins.
c. Lead formation of
Acetate- precipitate, which will
be dissolved upon the
Acetic
addition of acetic
Acid acid.
Test
Fixed oil
stain test Permanent grease
spot present
A reddish-brown
Salkowski’s color at the interface
Test indicates the
presence of steroidal
ring.
Volatile oil Absence of spot or
– spot test grease
Resins Production of yellow
flame with smoky
odor indicates the
presence of resin.

Conclusion:

Questions:
 1. What is the purpose of preliminary screening of plant constituents?
2. Why is 80% ethanol used in preparing plant extracts?
3. Give the significance of each active plant constituent in pharmacy
and medicine.
4. What are primary, secondary, and tertiary
alkaloids?
5. What are cardiac glycosides?

References:

Cantoria, Magdalena C. Pharmacognosy in Action and Practice. Manila

NCRP Research Foundation, Inc., 2003.

Guevara, Beatrice Q. (editor), A Guidebook to Plant Screening:

Phytochemical and Biological. Manila:
UST Publishing House, 2005.