Biochromatography: Phytochemical Screening

(Villarta, Feldan P.)

Abstract: The experiment is aimed at screening the ethanolic extract of Euphorbia hirta for the

presence of alkaloids, phenols, flavonoids, saponins and tannins. Thin-layer chromatography was

used to analyze the secondary metabolites in the plant. The extract contains phenols, saponins

and flavonoids.

Introduction

Phytochemicals are a large group of plant-

derived compounds hypothesized to be

responsible for much of the disease

protection conferred from diets high in

fruits, vegetables, beans, cereals, and plant-

based beverages such as tea and wine (Arts

and Hollman, 2005).

Phytochemicals have provided the basis for

numerous commercial medications used

today for the treatment of a wide range of

diseases such as high blood pressure, pain,

asthma, and cancers (Park, 2006). Thus,

whether a specific plant contains

phytochemicals needs to be investigated.

The experiment specifically aims to screen

the ethanolic extracts of Euphorbia hirta for

the presence of alkaloids, phenols,

flavonoids, saponins and tannins.

Experimental Methods

A. Materials, Chemicals and Apparatus

The following chemicals were used during

the experiment: 95% ethyl alcohol, silica gel,

chloroform, methyl alcohol, ferric chloride

solution, vanillin, sulfuric acid, hydrochloric

acid, formic acid, and distilled water.

The following apparatus were utilized during

course the experiment: capillary tube,

Erlenmeyer flask, rotary evaporator, TLC

plate, beaker, aspirator, pipet, sprayer,

spatula, graduated cylinder, analytical

balance, funnel, vial and grinder.

B. Collection of Plant Sample

Fresh plant samples of Euphorbia hirta were

collected at the vicinities of Central

Mindanao University. Only plant samples

showing no signs of infections were picked.

The samples were washed with tap water

and rinsed with distilled water. They were

dried in an oven at 40C.

C. Extraction

The dried plant samples were ground to

reduce the size. About 50 g of the ground

plant sample was weighed and treated with

sufficient 95% EtOH to completely submerge

the material in an Erlenmeyer flask. The flask

was stoppered and the material was kept

soaked for 24 hours.

The mixture was then filtered. The flask and

plant material were rinsed with fresh

portions of 95% EtOH. The washings and the

plant material were transferred into a

funnel. The plant residue was discarded. The

filtrate was then concentrated under vacuo

at temperatures not exceeding 40C to

dryness. The extract was then transferred in

a vial and stored in the refrigerator.

D. Preparation of TLC plates

Silica gel was used as the coating material. A

slurry of silica gel was prepared. The slides

were coated by dipping them onto the

container of slurry. The coated plates were

then placed in oven at 105C for drying

purposes.

E. Phytochemical Screening

The extract was screened for the presence of

alkaloids, phenols, flavonoids, saponins and

tannins.

Before each test, a dried coated plate was

spotted using a capillary tube. The capillary

tube was dipped in a container containing

the extract dissolved in ethanol. The spot

was about 1 cm from the bottom of each

plate.

Test for Alkaloids

The spotted plate was placed in a chamber

(beaker) containing chloroform and

methanol in a 5:1 ratio. After the solvent has

reached the top of the plate, it was removed

and air-dried. It was sprayed with

Dragendorffs reagent. The positive result

for this test is the formation of orange spots.

However, it was not seen during the

experiment implying a negative result as

shown in Figure 1. Yellow spots were seen.

Test for Phenols

The spotted plate was placed in a beaker

containing ethyl acetate, formic acid, acetic

acid and water in a 100:11:11:25 ratio

respectively. After the solvent has spread up

to the top of the plate, it was removed and

air-dried. It was sprayed with 2% ferric

chloride in ethanol solution. A positive result

was seen during the experiment in this test

which is the formation of bright blue spot as

displayed in Figure 2.

Test for Flavanoids

The spotted plate was placed in a beaker

containing ethyl acetate, glacial acetic acid

and water in a 90:10:20 ratio respectively.

The plate was removed and air dried after

the solvent has reached the top of the plate.

It was sprayed with 5% ferric chloride

solution. The formation of gray spot is taken

to be a positive result as shown in Figure 3.

Test for Saponins

A solution of chloroform and methanol in a

30:5 ratio was placed in a beaker. The

spotted plate was then placed in that

beaker. It was removed after the solvent has

reached the tip of the plate. It was air-dried.

A solution of vanillin sulfuric acid was placed

in the sprayer and the plate was sprayed

with it. The formation of violet spots as

displayed in Figure 4 was taken to be a

positive result in this test.

Test for Tannins

A solution of chloroform, methanol and

water in a 66:35:10 ration was prepared and

placed in a beaker. The spotted plate was set

in that beaker and it was removed and air-

dried after the solvent has spread up to the

top of the plate. A solution of 0.5% (w/w)

vanillin in 4% (w/w) HCl was used as the

spraying solution. After the spotted plate

was sprayed, it showed a negative result as

shown in Figure 5. Supposedly, the positive

result for this is the formation of blue spots.

Figure 1. Test for Alkaloids

Figure 2. Test for Phenols Figure 3. Test for Flavonoids

Figure 4. Test for Saponins Figure 5. Test for Tannins

Results and Discussion

Phytochemicals are secondary metabolites

in one or more parts of the medicinal plants.

These have the ability to produce a definite

physiological action on the human body. In

the experiment, the extract from Euphorbia

hirta plant were analyzed qualitatively. Table

1 presents the results of phytochemical

analysis.

Table 1. Phytochemical Screening

Test Result

Alkaloids Negative

Phenols Positive

Flavonoids Positive

Saponins Positive

Tannins Negative

Phenols, flavonoids and saponins were

found in the extract of Euphorbia hirta. This

is in accordance with the results of the study

of Patil and Magdum (2011). They have

found out that the ethanolic extract of

Euphorbia hirta showed a positive result for

phenols, steroids, cardiac glycosides,

saponins, carbohydrates and flavonoids.

The presence of these secondary

metabolites in Euphorbia hirta, produce

some biological activity in man and animals

and it is responsible for their use as herbs.

These compounds also serve to protect the

plant against infection by microorganisms,

predation by insects and herbivores (Ketkar

and Ketkar, 1995).

Conclusions and Recommendations

The goal of this experiment was to screen

the ethanolic extract of Euphorbia hirta for

the presence of various secondary

metabolites. It was found out that the

ethanolic extract of the plant contains

phenols, saponins and flavonoids.

Since the results are in accordance with

previous study conducted, the experiment

itself is successful in screening the ethanolic

extract of the plant. Further studies should

be conducted especially in isolating the

compounds found in the plant.

References

Arts, I.C. and Hollman P.C. (2005).

Polyphenols and disease risk in

epidemiologic studies. Am J Clin Nutr,

81(1 Suppl): p. 317S-325.

Ketkar, A.Y. and Ketkar, C.M. Various uses of

nem products: Medicinal uses inducing

pharmacology in Asia in H. Schmuterer,

Ed. 1995, p. 518-525.

Park, J. B. (2006). Phytochemical Database.

Retrieved from the world wide web:

http://www.ars.usda.gov/News/docs.h

tm?docid=8875

Pastil, S.B. and Magdum C.S. (2011).

Phytochemical Investigation and

antitumour activity of Euphorbia hirta

Linn. European Journal of Experimental

Biology, 1(1): 51-56.