Experiment No.

1
EXTRACTION AND PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES

Submitted by:

VAL JASON G. LAGRADA

BS Chem – 4

Submitted to:

MS. AILEEN MAY G. ANG

Chem 52a Instructor

JANUARY 20, 2014

 INTRODUCTION
The importance of plants is known to us well. The plant kingdom is a treasure
house of potential drugs and in the recent years, there has been an increasing awareness
about the importance of medicinal plants. Drugs from the plants are easily available, less
expensive, safe, and efficient and rarely have side effects. The plants which have been
selected for medicinal use over thousands of years constitute the most obvious choice of
examining the current search for therapeutically effective new drugs such as anticancer
drugs, antimicrobial drugs, anti-hepatotoxic compounds.
In the Philippines, Sinaw-sinaw or Peperomia pellucida is widely used as herbal
medicines especially in remote areas where there is less access to synthetic medicine. Peperomia
pellucida is also known as shiny bush or silver bush belonging to family Piperaceae. The
species develops during rainy periods (often in the spring) and thrives in loose, humid soils under
the shade of trees. It grows in moist habitat and is found throughout the major parts of
Ind ia. In different parts of India it is known with different names like Lochi pata in
Bangali. Mashitandu chedi in Malayalam and Pononoa in Assamese etc.
Whole plant or parts of plant are used for different purposes. P. pellucida has a history
of ethnomedicinal uses which include treatment of abdominal pain, abscesses, acne, boils, colic,
fatigue, gout headache, renal disorders, rheumatic pain and to treat (Khan and Omoloso, 2002).
Plants are good source bioactive compounds that exhibits both medicinal and nutritive
properties. Secondary metabolites are linked to these properties. These include groups of
phytochemicals like flavonoids, tannins, alkaloids, glycosides and saponins. Flavonoids
modulate the estrogen level in the body and inhibit tumor growth while phenolics are potent
antioxidant and steroids are reduces the risks of coronary heart diseases.
Different qualitative tests are employed in determining the presence of the above
mentioned classes of secondary metabolites. These include the Culvenor-Fitzgerald Method for
Alkaloids, Frothing test for Saponins, Tests for Flavonoids, Tannins and Glycosides. The
experiment is aimed at preparing concentrated plant extract and perform qualitative
phytochemical test on P. pellucida leaves, in view of their importance in medicine.
 METHODOLOGY
The materials and apparatus used in the experiment are as follows: test tubes, test tube
holder, alcohol lamp, beaker, spatula, pipet and aspirator. While the reagents used are
ammoniacal chloroform, chloroform, ammonia, anhydrous sodium sulphate, distilled water,
mercuric iodide, potassium iodide, bismuth (III) nitrate, acetic acid and fehling’s reagent.

I. Extraction and Preparation of Stock Plant Extract

Fresh samples collected were first cleaned thoroughly with tap water and was rinsed with
distilled water. It was dried via air-dry or oven-dry method (40oC). After which, the air-dried
samples were cut with clean scissors to reduce its size. 50 g of the sample was placed in an
Erlenmeyer flask and was treated with sufficient 80% methanol and the volume used was
recorded. The flask was closed with rubber stopper and was set aside for 24-48 hours. After that
span of time, the mixture was filtered together with the methanol used in washing the flask,
discarding the residue. The filtrate was concentrated under vacuum at temperature below 50 oC to
about 20 mL. The exact volume and weight of the concentrated extract was recorded. The extract
was then stored in the refrigerator (0-5oC) and it was labelled with the following information:
name of plant, concentration of plant extract and date of extraction.

II. Phytochemical Screening of Secondary Metabolites

A. Field Test for Alkaloid: The Culvenor-Fitzgerald Method
This method is designed to screen alkaloid-containing plants at actual location slight.
With the aid of mortar and pestle and clean sand, two to four grams of freshly cut samples were
triturate with sufficient chloroform. It was followed by the addition of 10 mL ammoniacal
chloroform and the mixture was stirred. The mixture, then, was filtered into a test tube and 1 mL
sulphuric acid was added and was shaken. After which, the clear upper layer was pipette off into
two small test tubes. The first test tube was subjected to Dragendorff’s Test (addition of 2-3
drops of Dragendorff’s reagent while 2-3 drops of Mayer’s reagent was added on the second test
tube for the Mayer’s Test. The formation of orange and white precipitate indicates positive
results for Dragendorff’s and Mayer’s Tests, respectively.
 B. Laboratory Test for Alkaloids
One and a half millilitres of 10% HCl was added to 5 mL of the extract in a test tube. It
was then heated for 20 minutes and was allowed to cool to room temperature and was filtered.
The filtrate was then transferred into two separate test tubes. 5 drops of Maeyer’s Reagent and 5
drops of Draggendorff’s reagent were added to the first and second test tubes, respectively. The
results were then noted.

C. Test for Saponins (Frothing Test)

Two milliliters of extract was placed in a test tube and was shaken vigorously for 2
minutes. It was then subjected to warm water bath. The presence of frothing which persists for 5
minutes is an indication of the presence of saponins in the extracts.

D. Test for Flavonoids

Three pieces of magnesium turnings were added to 3 mL of extract in a test tube. After
which, the mixture was heated and 3 drops of concentrated HCl was added. An orange-pink
coloration is an indication of the presence of flavonoids in the extracts.

E. Test for Tannins

Three drops of 5% Ferric chloride solution was added to 1 mL of extract. A greenish
black precipitate is an indication of the presence of tannins in the extracts.

F. Test for Glycosides

Ten milliliters of 50% HCl was added into 2 mL of extract in a test tube. Then, the
mixture was heated in a water bath for 30 minutes and 5 mL of the Fehling’s solution was added.
Finally, the mixture was boiled for 5 minutes. A brick-red precipitate is an indication of the
presence of glycosides in the extracts.
 RESULTS AND DISCUSSION
I. Extraction and Preparation of Stock Plant Extract
50 grams of air dried Peperomia pellucida (L.) leaves were cut into smaller pieces using
clean scissors. Extraction were done for 48 hours using 95% ethanol. After which, the solvents
were removed with the use of the rotary evaporator in the laboratory, yielding the corresponding
ethanol extract. The sample was then stored in the refrigerator for the preceding analysis.

Figure 1. Image of Peperomia pellucida

II. Phytochemical Screening of Secondary Metabolites

Table 1. shows the experimental and theoretical results on the secondary metabolites tested.
SECONDARY EXPERIMENTAL RESULTS THEORETICAL RESULTS
METABOLITES (Majumder, et. al. 2011)
Alkaloids
a. Dragendorff’s Reagent - +
b. Maeyer’s Reagent - +
Saponins - +
Flavonoids - +
Tannins + +
Glycosides - +
Key: (+) Positive; (-) Negative

Table 1 showed negative results for the following classes of secondary metabolites:
alkaloids, saponins, flavonoids and glycosides while only tannins showed positive results.
However, studies revealed that all secondary metabolites tested are present (Majumder, et. al.,
2011).
The photographic data for the results of Alkaloids, the Dragendorff’s and Maeyer’s Tests
are shown below.

Figure 1. Results for Dragendorff’s and Maeyer’s Tests

As shown in the picture, black precipitate which indicates the absence of alkaloids in the
plant extract. Alkaloids are pharmacologically active, complex organic compounds containing
one or more nitrogen atoms, characteristically as primary, secondary or tertiary amines, which
provide basicity to the alkaloid. However, it cannot be defined clearly because there are no
clear-cut boundary between alkaloids and naturally occurring complex amines.
In qualitative detection of alkaloid, it is often times precipitated from neutral or acidic
solution like potassiomercuric iodide solution (Maeyer’s reagent) and solution of potassium
bismuth iodide (Dragendorff’s reagent). Studies showed that alkaloids are present in all parts of
Peperomia pellucida (Majumdar et. al., 2011). Alkaloids have been associated with medicinal
uses for centuries and one of their common biological properties is their cytotoxicity
Saponins, on the other hand, are a class of chemical compounds that are amphipathic
glycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when
shaken in aqueous solutions. In terms of structure, have one or more hydrophilic glycoside
moieties combined with a lipophilic triterpene derivative.
Saponins are readily detected by the formation of froth or bubbles when agitated in
aqueous solution. Because of the presence of amphiphatic glycosides, it has the ability to form
soap-like foaming when shaken in aqueous solution. Their ability to foam is caused by the
combination of the non-polar sapogenin and the water soluble side chain.
 In the experiment, the sample gave negative results on saponins. Although studies
showed that saponins are present in all parts of Peperomia pellucida except for the stem.
Saponins are known to produce inhibitory effect on inflammation. Saponins has the property
of precipitating and coagulating red blood cells. Some of the characteristics of saponins
include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding
properties and bitterness (Yadav and Agarwala, 2011).
Next secondary metabolites tested was flavonoid. Flavonoids are plant phenols generally
containing 2 benzene rings and are derived from flavones and are known to have antioxidant
activity. Flavonoids are hydroxylated phenolic substances known to be synthesized by
plants in response to microbial infection and they have been found to be antimicrobial substances
against wide array of microorganisms in vitro. Their activity is probably due to their ability
to complex with extracellular and soluble proteins and to complex with bacterial cell wall.
Its presence can be detected by the formation of red precipitate after the addition of
magnesium turning and diluted HCl. The photos of the results are shown below in Figure 2.

Figure 2. Test for Flavonoids

The test tube labelled with letter A is the sample that was subjected to flavonoids test
while the other one is the original sample.
The image below in Figure 3 is the positive result for the qualitative test of tannins. Letter
A test tube is the original sample which is used as the basis in assessing the results. While letter
B test tube is the positive results for such analysis in the presence of black precipitate.
 Figure 3. Test for Tannins
By definition, tannins are complex organic, non-nitrogenous plant products, which
generally have astringent properties. These compounds comprise a large group of compounds
that are widely distributed in the plant kingdom. Tannins are known to bind to proline rich
protein and interfere with protein synthesis.
The last class of secondary metabolites analysed was the of the glycosides. When sugars
are present, it readily precipitated out into red brick with Fehling’s Reagent. Studies on the
phytochemical screening of Peperomia pellucida claimed that glycosides or sugars are present.
Glycosides are known to lower the blood pressure according to many reports.

CONCLUSIONS
Stock plant extract were prepared by soaking 50 grams of sample with 95% ethanol for
48 hours. It was concentrated under vacuum at around 40oC. Different phytochemical screening
were performed to determine the presence of important secondary metabolites. Based on the
aforementioned results, the following generalizations are drawn:
1. That the extract must be concentrated enough to detect the secondary metabolites present;
and
2. That tannins is present based on the experiment, although all secondary metabolites are
present on studies conducted.
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