76

CHAPTER 5
PHYTOCHEMICAL EVALUATION

Phytochemistry is mainly concerned with enormous varieties of

secondary plant metabolites which are biosynthesized by plants. The plant
kingdom represents a treasure trove of structurally diverse bioactive
molecules. Most of the best plant medicines are the sum of their constituents.
The beneficial physiological and therapeutic effects of plant materials
typically result from the combinations of these secondary products present in
the plant. The information on the constituents of the plant clarifies the uses of
the plants but only a small percentage have been investigated for their
phytochemicals

and

only

fraction

has

undergone

biological

or

pharmacological screening. As more phytoconstituents are being identified

and tested, traditional uses of the plants are being verified [44].
In phytochemical evaluation the powdered leaves were subjected to
phytochemical screening for the detection of various plant constituents,
characterized for their possible bioactive compounds, which have been
separated and subjected to detailed structural analysis.
5.1

MATERIALS AND METHODS

After authentification, the fresh, healthy plant leaves of Symplocos

cochinchinensis (Lour.) were properly dried in shade for 2-3weeks. It was

pulverized in a blender, sieved and used for further studies.

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5.1.1

Preparation of the Extracts

About 2 kg of air-dried plant material was extracted in soxhlet

assembly successively with n-hexane, chloroform, ethyl acetate and methanol

(order of increasing polarity). Each time before extracting with the next
solvent, the powdered material was dried.
Each extract was concentrated by using rotary vacuum evaporator.
The extract obtained with each solvent was weighed and the percentage yield
was calculated in terms of dried weight of the plant material. The colour and
consistency of the extract were also noted. All the solvents used for this entire
work were of analytical reagent grade (Merck, Mumbai).
5.1.2

Qualitative Chemical Tests [45, 46]

The n-hexane, chloroform, ethyl acetate, methanol extracts and the

leaf powder were subjected to qualitative chemical analysis.

Test for alkaloids
A small portion of the extract was stirred separately with a few
drops of dilute hydrochloric acid and filtered. The filtrate was carefully tested
with various alkaloidal reagents such as Mayers reagent, Dragondroffs
reagent, Hagers reagent and Wagners reagent.
Test for carbohydrates
The minimum amount of the extracts were dissolved in 5ml of
distilled water and filtered. The filtrate was subjected to test for
carbohydrates.

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Molischs test
The filtrate was treated with 2-3 drops of 1% alcoholic alpha
naphthol and 2ml of concentrated sulphuric acid was added along the sides of
the test tube.
Fehlings test
The filtrate was treated with 1 ml of Fehlings A and B and heated
in a boiling water bath for 5-10min. Appearance of reddish orange precipitate
shows the presence of carbohydrates.
Test for glycosides
Cardiac glycoside
Keller-Killani test- To 2 ml of extract, glacial acetic acid, one drop
5 % ferric chloride and concentrated sulphuric acid were added. Appearance
of reddish brown colour at the junction of the two liquid layers indicates the
presence of cardiac glycosides.
Anthraquinone glycosides
Borntragers Test To 3 ml extract dilute sulphuric acid was
added, boiled and filtered. To the cold filtrate equal volume benzene or
chloroform was added. The organic layer was separated and ammonia was
added. Ammonical layer turns pink or red.
c)

Saponin glycosides
Foam test The extract and powder were mixed vigorously with

water.

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d)

Coumarin glycosides
Alcoholic extract when made alkaline, shows blue or green

fluorescence.
Test for phytosterol
1gm of the extract was dissolved in few drops of dry acetic acid,
3ml of acetic anhydride was added followed by few drops of concentrated
sulphuric acid. Appearance of bluish green colour shows the presence of
phytosterol.
Test for fixed oils and fats
(a) Small quantity of the various extracts was separately pressed
between two filter papers. Appearance of oil stain on the paper
indicates the presence of fixed oil.
(b) Few drops of 0.5N alcoholic potassium hydroxide was added
to a small quantity of various extracts along with a drop of
phenolphthalein. The mixture was heated on a water bath for
1-2hrs. Formation of soap or partial neutralization of alkali
indicates the presence of fixed oil and fats.
Test for tannins and phenolic compounds
Small quantity of various extracts were taken separately in water
tested for the presence of phenolic compounds and tannins with
(a) Dilute ferric chloride solution (5%)

- violet colour

(b) 1% solution of gelatin with 10%NaCl - white precipitate

(c) 10% lead acetate solution

- white precipitate

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Test for proteins

Various extracts were dissolved in few ml of water and treated with
(a) Millons reagent: Appearance of red colour shows the
presence of proteins and free amino acids.
(b)

Biuret test: Equal volume of 5% solution of sodium

hydroxide and 1% copper sulphate were added. Appearance of
pink or purple colour indicates the presence of proteins and
free amino acids.

Test for gums and mucilages

About 10ml of various extracts were added separately to 25ml of
absolute alcohol with constant stirring and filtered. The precipitate was dried
in air and examined for its swelling properties and for the presence of
carbohydrates.
Test for flavanoids
(a) With aqueous solution of sodium hydroxide blue to violet
colour (Anthrocyanins), yellow colour (Flavones), yellow to
orange (Flavonones).
(b) With concentrated sulphuric acid yellowish orange colour
(Anthrocyanins), orange to crimson colour (Flavonones).
(c) Shinodas test the extracts were dissolved in alcohol, to that
a piece of magnesium and followed by concentrated
hydrochloric acid was added drop wise and heated.
Appearance of magenta colour shows the presence of
flavonoids.

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Test for lignin

With alcoholic solution of phloroglucinol and concentrated
hydrochloric acid appearance of red colour shows the presence of lignin.
Test for terpenoids
Nollers test: The substance was warmed with tin and thionyl
chloride. Pink coloration indicates the presence of triterpenoids.
Test for steroids
Libermann Burchard Reaction: 2 ml extract was mixed with
chloroform. To this 1-2 ml acetic anhydride and 2 drops concentrated
sulphuric acid were added from the side of test tube. First red, then blue and
finally green colour appears.
5.1.3

Thin Layer Chromatography [47,48]

Of the various methods of separating and isolating plant

constituents, thin layer chromatography (TLC) is one of the most powerful

technique used for the separation, identification and estimation of single or
mixture of components present in various extracts.
Mechanism employed in this reliable technique is adsorption in
which solute adsorbs on the stationary phase according to its affinity.
Substances are separated by differential migration that occurs when a solvent
flows along the thin layer of stationary phase. The substance which is having
more affinity towards mobile phase moves faster when compared to the
substance which has less affinity leading to the separation of the compounds.

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TLC Plates
Precoated silica gel on aluminium plates were used as a stationary
phase.
Sample application
The extracts to be analysed were diluted with respective solvents
and then spotted with help of capillary tube just 2 cm above its bottom.
Selection of mobile phase
Solvent mixture was selected on the basis of the phyto constituents
present in each extract. Solvents were analysed as its order of increasing
polarity. Several mobile phases were tried for the separation of maximum
components. After several trials, the best solvent system was selected which
showed good separation with maximum number of components.
Solvent system
Hexane extract

Methanol :Chloroform(9:1)

Chloroform extract -

Methanol :Chloroform(9:1)

Ethylacetate extract -

Methanol : Ethyl acetate: Hexane: Acetic

acid (2:7:1: 0.5)

Methanol extract

Ethyl acetate: Water ( 6:3:1)

Rf values were noted down for each selected extracts after elution
by using different detecting agents such as Dragendroffs, Ninhydrin,
Libermann Burchard, concentrated sulphuric acid and ferric chloride.

83

5.1.4

High Performance Thin Layer Chromatography[49]

HPTLC method is a modern sophisticated and automated

separation technique derived from TLC. Pre-coated HPTLC graded plates and
auto sampler was used to achieve precision, sensitive, significant separation
both qualitatively and quantitatively.
High performance thin layer chromatography (HPTLC) is a
valuable quality assessment tool for the evaluation of botanical materials
efficiently and cost effectively. HPTLC method offers high degree of
selectivity, sensitivity and rapidity combined with single-step sample
preparation. In addition it is a reliable method for the quantitation of
nanograms level of samples. Thus this method can be conveniently adopted
for routine quality control analysis. It provides chromatographic fingerprint of
phytochemicals which is suitable for confirming the identity and purity of
medicinal plant raw materials.
Basic steps involved in HPTLC
Extracts used

n-hexane, chloroform, ethyl acetate and

methanol

Application mode

Development mode :

CAMAG Linomet IV.

CAMAG Twin Trough chamber.

Sample application
The samples were dissolved in same solvent and 10 l quantity of
sample was applied on the HPTLC silica merk 60F 254 graded plate sized
6cm x 10 cm as narrow bands using CAMAG Linomat 5 injector.

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Chromatogram Development
It was carried out in CAMAG Twin Trough chambers. Sample
elution was carried out according to the adsorption capability of the
component to be analysed. After elution, plates were taken out of the chamber
and dried.
Scanning
Plates were scanned under UV at 254nm. The datas obtained from
scanning were brought into integration through CAMAG software.
Chromatographic finger print was developed for the detection of
phytoconstituents present in each extract and Rf values were tabulated.
Mobile Phase
Hexane extract

Hexane:Chloroform: Acetic acid (7:3:0.5)

Chloroform extract -

Hexane:Chloroform: Acetic acid (7:3:0.5)

Ethyl acetate extract -

Butanol:Water:Acetic acid ( 4:5:1)

Methanol extract

Butanol:Water:Acetic acid ( 4:5:1)

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5.2

RESULTS

5.2.1

Extraction
The percentage yield of successive extractive values for leaves of

Symplocos cochinchinensis (Lour.) is tabulated in Table 5.1

Table 5.1 The percentage yield of successive extracts of the leaves of
Symplocos cochinchinensis (Lour.)

Extract

n-Hexane

Chloroform

Ethyl acetate

Methanol

Method of
extraction
Successive solvent extraction in
soxhlet apparatus

S.No

Colour

Physical
nature

Green/ Sticky

Waxy greasy

mass

semisolid

Greenish brown/
Sticky mass

Semisolid

2.0

Solid

5.7

Solid

8.8

Yellowish
green/ Thick

Yield
(%w/w)
2.0

solid mass
Brownish green/
Thick solid
mass

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5.2.2

Qualitative Chemical Analysis

Qualitative chemical analysis of phytoconstituents of the leaf

powder and various extracts of Symplocos cochinchinensis (Lour.)

is

tabulated in Table 5.2.

Table 5.2 Qualitative chemical analysis of phytoconstituents of the leaf
powder and various extracts of Symplocos cochinchinensis
(Lour.)
S.
No.

Test

Powder n-Hexane Chloroform

Ethyl
Methanol
acetate

1.

Alkaloids

2.

Glycosides

3.

Terpenoids

4.

Carbohydrate

5.

Proteins

6.

Steroids

7.

Flavonoids

8.

Phenols

9.

Tannins

10. Iridoid
glycoside

11. Quinones

12. Anthraquinone

13. Saponins

Note: + ve indicates positive result, whereas ve indicates negative result

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5.2.3

Thin Layer Chromatography

The TLC studies of the n-hexane, chloroform, ethyl acetate and

methanol extract are shown in Table.5.3

Table 5.3 The

TLC

studies

of

various

extracts

of

Symplocos

cochinchinensis (Lour.)

S.No

Extract

n-Hexane

Solvent system

No. of
Rf values
spots
2

Methanol : Chloroform(9:1)
2

Chloroform

0.62
0.66
0.37

0.43
0.48
0.27

Methanol : Ethyl acetate: Hexane

Ethyl acetate
: Acetic acid (2:7:1: 0.5)

0.33
0.88
0.20

Methanol

Methanol : Ethyl acetate:

Water (6:3:1)

0.45
0.52
0.72

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5.2.4

High Performance Thin Layer Chromatography

The HPTLC fingerprints of the n-hexane, chloroform, ethylacetate

and methanol extract are shown in Table 5.4 and Figures 5.1, 5.2, 5.3, and 5.4
Table. 5.4 The HPTLC fingerprints of various extracts of Symplocos
cochinchinensis (Lour.)

S.No

Extract

n-Hexane

Chloroform

Ethyl acetate

Methanol

Detection
Wavelength
(nm)
280

280

280

280

No. of
spots

Rf values
0.07, 0.49, 0.60, 0.75,
0.82, 0.90

11

0.07, 0.28, 0.32, 0.42,

0.46, 0.50, 0.58, 0.76,
0.82, 0.88, 0.91

0.12, 0.29, 0.36, 0.49,

0.57, 0.81

10

0.06, 0.15, 0.18, 0.24,

0.33, 0.39, 0.51, 0.58,
0.65, 0.71

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Figure 5.1 The HPTLC fingerprint of n-Hexane extract of Symplocos

cochinchinensis (Lour.)

90

Figure 5.2 HPTLC fingerprint of chloroform extract of Symplocos

cochinchinensis (Lour.)

91

Figure 5.3 HPTLC fingerprint of Ethylacetate extract of Symplocos

cochinchinensis (Lour.)

92

Figure 5.4 HPTLC fingerprint of methanol extract of Symplocos

cochinchinensis (Lour.)

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5.3

DISCUSSION
Most of the traditional knowledge about medicinal plant was in the

form of oral knowledge. There is no uniform or standard procedure for

maintaining the inventory of these plants and the knowledge about their
medicinal properties.
Therefore, it is necessary that such procedures to be documented
and studied for systematic regulation and widespread application. The leads
for a significant number of modern synthetic drugs have originated from
isolated plant ingredients since the search for new entities begins from either
derivatizing the existing drug or from traditional medicinal system. It is very
important to undertake phytochemical investigations along with biological
screening to understand therapeutic dynamics of medicinal plants and also to
develop quality parameters.
In the phytochemical analysis different polarity of phytoconstituents
were sorted out from the coarsely powdered leaves of Symplocos
cochinchinensis (Lour.) by using solvents like n-hexane, chloroform, ethyl
acetate and methanol by successive extraction using soxhlet apparatus.
Successive extractive values revealed the solubility and polarity
particulars of the metabolites in the plant. Methanolic extract showed high
extractive yield 8.8 %w/w when compared to other extracts.
Qualitative preliminary phytochemical analysis was performed initially
with different chemical reagents to detect the nature of phytoconstituents and their
presence in each extract and powder.

Hexane and chloroform extracts

showed the presence of steroids. Ethyl acetate extract was found to contain
flavanoids, glycosides, proteins, saponins, alkaloids and carbohydrates.
Methanolic extract showed the presence of carbohydrates, flavanoids,
phenols, saponins, proteins, glycosides and alkaloids.

94

Qualitative chromatographic analysis of these extracts using thin

layer chromatography was performed to separate and identify the single or
mixture of constituents in each extract. The hexane extract showed 2 spots
(Rf values 0.62, 0.66), chloroform extract showed one spot (0.37), where as 3
spots were found with ethyl acetate extract (0.27, 0.33, 0.88) and 4 spots
were found in methanol extract (Rf values 0.20, 0.45, 0.52, 0.72). TLC was
performed for the identification of different components in the extracts
qualitatively.
HPTLC was scanned at 280nm with the best solvent to detect the
maximum number of components and peak abundance qualitatively. The
finger print of hexane extract showed 6 spots in the solvent system and the R f
values were 0.07, 0.49, 0.60, 0.75, 0.82, 0.90. Chloroform extract showed 11
spots in the solvent system

and the Rf values were 0.07, 0.28, 0.32, 0.42,

0.46, 0.50, 0.58, 0.76, 0.82, 0.88, 0.91.

The ethyl acetate extract showed 6 spots in the solvent system
and the Rf values were 0.12, 0.29, 0.36, 0.49, 0.57, 0.81. The finger print of
methanol extract showed 10 spots and the Rf values were 0.06, 0.15, 0.18,
0.24, 0.33, 0.39, 0.51, 0.58, 0.65, 0.71. HPTLC fingerprint is one of the
versatile tool for qualitative and quantitative analysis of active constituents. It
is also a diagnostic method to find out the adulterants and to check the purity.
Since, secondary metabolites are responsible for biological activity,
this study would be the leading path way of information for selection of the
extract for pharmacological activity and isolation of constituents responsible
for the activity.