Extraction and Fractionation

CHAPTER - 5

EXTRACTION AND
FRACTIONATION
 Extraction and Fractionation

5. EXTRACTION AND FRACTIONATION

INTRODUCTION
Literature survey of B. frondosa revealed that the aqueous and methanolic extracts of
leaves possess promising hepatoprotective activity^'. However the phytoconstituents
responsible for this activity have not been identified.

The roots of palash are reported for the treatment of ocular inflammation^*''*"''* V The roots
are not studied phytochemically in detail and have not been explored for other
pharmacological activities.

Thus it was thought worthwhile to isolate and identify the phytoconstituents and explore
their pharmacological activities from both these parts of the plant.

5.1 FRACTIONATION OF EXTRACTS

The total methanolic extracts of roots and leaves were subjected to fractionation to
separate the phytoconstituents according to their solubility.

5.\.\ Study of Roots:

The powdered roots (500 g) of B. frondosa were extracted in a Soxhlet apparatus
using methanol as solvent. The total methanolic extract (TM) thus obtained was
then fractionated using solvents of increasing polarity. The TM extract was first
fractionated in a separating furmel with Pet- ether 60° - 80°. The pet- ether fraction
so obtained was concentrated under vacuum using a rotary evaporator and then
dried on a water bath to complete dryness. The TM extract obtained after
fractionation with pet- ether was also concentrated at controlled temperature imder
vacuum in a rotary evaporator and adsorbed on silica gel 60- 120 by trituration. The
adsorbed methanolic extract was then successively extracted with solvents of
increasing polarity viz: chloroform, ethyl acetate, n- butanol, methanol and water.
The extracts were concentrated under vacuum and finally dried on steam bath and
stored in air- tight containers. Extractive values for each fraction were determined
and calculated on basis of total methanolic extract. The results are tabulated in
Table No. 5.1.

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 Extraction and Fractionation

5.1.2 Study of Leaves:

500 g of the powdered leaves of B. frondosa were extracted in Soxhiet apparatus
using methanol as solvent. The total methanolic extract (TM) so obtained was
subjected to fractionation using solvents of increasing polarity as given above in
Chapter No. 5.1.1. The extractive values for each extract were determined and
calculated with respect to the dried drug. The results are given in Table No.5.2.

5.2 PHYTOCHEMICAL SCREENING OF FRACTIONS:

The various successive fractions of TM extract of roots and leaves were subjected to
extensive phytochemical analysis, as described in Chapter No. 4.4, to determine the
nature of the phytoconstituents present, such as alkaloids, glycosides, flavonoids,
tannins and phenolic compounds, amino acids and gums and mucilages. The results for
the phytochemical screening of the successivefi-actionsof roots and leaves are given in
Table No. 5.3 and 5.4 respectively.

5.3 HPTLC STUDIES;

5.3.1 Study of Roots:
EXPERIMENTAL:
Dried successive fractions of the TM extract of roots were reconstituted in the
respective solvents and filtered to remove any undissolved particles. Each extract
was then spotted on precoated HPTLC plates using Camag Linomat FV applicator
with the spray on technique and developed in appropriate solvent systems to resolve
the phytoconstituents present in the extract as per conditions given below:

CHROMATOGRAPHIC CONDITIONS;

Applicator used : CAMAG Linomat IV applicator

Plate Material : Precoated HPTLC plates coated v^th Silica gel G 60 F254 on
aluminium support. (10 X 10 cm plates)
Solvent System : A) Chloroform : Acetone (8:2) for pet- ether and chloroform
fractions of TM extract.
B) n-Butanol : gl. Acetic Acid : Water (5:1:2) for ethyl acetate,
n-butanol, methanol and water extracts.

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 Extraction and Fractionation

Application volume 5- 20 )il in a band size of 6 mm

[0.2 mg/ ml: Pet- ether and chloroform fractions
0.1 mg/ml: Ethyl acetate, n-butanol, methanol & water firacs.]
Development chamber CAMAG Twin Trough Chamber
Chamber Saturation 20 min.
Development Distance 90 mm
Development Time A) 20min & B)60 min.
Scanner CAMAG Scanner III
Detection 254imi (Aborption/ Reflection mode using Deuterium lamp) &
366nm (Fluorescence/ Reflection mode using Mercury lamp)
Integrator CATS V 4.06 Software
Spray reagents Vanillin sulphuric acid (VS), Ferric chloride (FeCls),
Dragendroff s reagent (DR), Liberman Burchard reagent (LB),
Natural Product Reagent (NP)

HPTLC fingerprints of the successive fractions of the TM extract were obtained using the
above mentioned parameters. The results are represented in Table No. 5.5 and the HPTL
fingerprints are given in Figure No. 5.1. The developed HPTLC plates were sprayed with
various spray reagents (viz.: VS, FeCb, DR, LB) to determine the nature of the
phytoconstituents resolved.

5.3.2 Study of Leaves:

EXPERIMENTAL
Dried successive fractions of TM extract of leaves of Butea frondosa were prepared as
described in section 5.3.1. Each extract was subjected to HPTLC studies to resolve the
phytoconstituents present. The results are given in Table No. 5.6. HPTLC chromatograms are
given in Figure No. 5.2. HPTLC plates were sprayed with various spray reagents (viz.: VS,
FeCla.DR, LB, NP) to determine the nature of the phytoconstituents present.

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 Extraction and Fractionation

RESULTS
Table No. 5.1: Extractive Values of Successive Fractions of TM extract of Roots

EXTRACTS YIELD (%wAv)

Total Methanolic Extract 4.8
Pet-ether (60"-80") fraction 0.36
Chloroform fraction 0.42
Ethyl Acetate fraction 0.45
n-Butanol fraction 0.86
Methanol fraction 1.04
Water fraction 1.89

Table No. 5.2: Extractive Values of Successive Fractions of TM extract of Leaves

EXTRACTS YIELD (%w/w)

Total Methanolic Extract 11.2
Pet-ether (60"-80") fraction 2.0
Chloroform fraction 1.45
Ethyl Acetate fraction 0.43
n-Butanol fraction 0.8
Methanol fraction 3.1
Water fraction 4.15

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 Extraction and Fractionation

Table No. 5.3: Phvtochemical Screening of Successive Fractions of TM extract of Roots

Test / Reagent used TM PE CH EA n-B Me Wt

ALKALOIDS
Dragendorff s reagent - - - - - - -

Mayer reagent - - - - - - -

Wagner reagent - - - - - - -

GLYCOSIDES
Bormtrager test - - - - - - -

Keller - Killani test - - - - - - -

Legal's test - - - - - - -

CARBOHYDRATES
Molisch reagent + - + + + - +
Fehling reagent + - + + + - +
FLAVONOIDS
Shinoda test + - - + - + -

TANNINS
Lead acetate reagent + - - + + + +
Ferric chloride reagent + - - + - + +
Gums & Mucilage + - - - - - +

TM: Total methanolic extract of roots ofB.frondosa.

PE: Pet-ether fraction of Total methanolic extract of roots of 5. frondosa.
CH: Chloroform fraction of Total methanolic extract of roots of B. frondosa.
EA: Ethyl acetate fraction of Total methanolic extract of roots of B. frondosa.
n-B: n- Butanol fraction of Total methanolic extract of roots of B. frondosa.
Me: Methanolic fraction of Total methanolic extract of roots of B. frondosa.
Wt: Water fraction of Total methanolic extract of roots of B. frondosa.

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 Extraction and Fractionation

Table No. 5.4: Phytochemical Screening of Successive Fractions of TM extract of Leaves

Test / Reagent used TM PE CH EA n-B Me Wt

ALKALOIDS
Dragendorff s reageant - - - -

Mayer reagent - - -

Wagner reagent
GLYCOSIDES
Bormtrager test - - - -

Keller - Killani test - - -

Legal's test
CARBOHYDRATES
Molisch reagent + - - - + +
Fehling reagent + + + +
FLAVONOIDS
Shinoda test + - - - + - -

TANNINS
Lead acetate reagent + - - - - + +
Ferric chloride reagent + + +
AMINO ACIDS
Ninhydrin reagent + - - - + - -

TM: Total methanolic extract of leaves of B. frondosa.

PE: Pet-ether fraction of Total methanolic extract of leaves of B. frondosa.
CH: Chloroform fraction of Total methanolic extract of leaves of B.frondosa.
EA: Ethyl acetate fraction of Total methanolic extract of leaves of B. frondosa.
n-B: n- Butanol fraction of Total methanolic extract of leaves of B. frondosa.
Me: Methanolic fraction of Total methanolic extract of leaves of B. frondosa.
Wt: Water fraction of Total methanolic extract of leaves of B. frondosa.

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 Extraction and Fractionation

Figure No. 5.1; HPTLC Chromatograms of Successive Fractions of TM extract of Roots

Ti: Pet-ether fraction of T. methanolic extract of roots of B. frondosa.

Ty. Chloroform fraction of T. methanolic extract of roots of B. frondosa.
Ty. Ethyl acetate fraction of T. methanolic extract of roots of B. frondosa.
T4: n- Butanol fraction of T. methanolic extract of roots of B. frondosa.
T5: Methanolic fraction of T. methanolic extract of roots of B. frondosa.
T(,: Water fraction of T. methanolic extract of roots of B. frondosa.

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 Extraction and Fractionation

1 T1 T!

Figure No. 5.2; HPTLC Chromatograms of Successive Fractions of TM extract of Leaves

Tl : Pet-ether fraction of T. methanolic extract of leaves of B. frondosa.

T2 : Chloroform fraction of T. methanolic extract of leaves of B. frondosa.
T3 : Ethyl acetate fraction of T. methanolic extract of leaves of B. frondosa.
T4 : n- Butanol fraction of T. methanolic extract of leaves of B. frondosa.
T5 : Methanol fraction of T. methanolic extract of leaves of B. frondosa.
T6 : Water fraction of T. methanolic extract of leaves of B. frondosa.

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 Extraction and Fractionation

Table No 5.5: HPTLC orofile of successive solvent fractions of TM extract of Roots

No. of Rf Fluorescence Spray reagent

spots value behaviour
254 nm 366nm VS FeCla DR LB NP
Pet ether Fraction
1 0.83 Violet Blue Violet Blue - - -
2 0.74 Violet Blue Pink - - - -

3 0.70 Violet Blue Blue - - - -

Chloroform Fraction
I 0.83 Violet Blue Violet Brown - - -
2 0.74 Violet Blue Pink - - - -
3 0.70 Violet Blue Blue Blue - - -
4 0.62 Violet - - - - - -
5 0.52 Violet - - - - - -
6 0.37 Violet - - - - Violet -
7 0.16 Violet - Violet Blue - - -
Ethyl acetate Fraction
1 0.84 Violet Yellow Red/ Blue Violet -
Brown
2 0.73 Violet Blue Violet Blue - Violet Yellow
3 0.68 Violet Blue Red/ Blue - Violet Yellow
Brown
4 0.62 Violet Blue Red/ - - - -
Brown
n- Butanol Fraction
1 0.54 Violet Yellow Violet Blue - Blue -
Methane>1 Fraction
1 0.35 - - Violet Blue - Black -

2 0.21 Violet/ Blue Violet Blue - Blue -

brown
Water Fraction
1 0.20 Violet Yellow Violet Blue - - -
2 0.12 Violet - - Blue - - -

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 Extraction and Fractionation

Table No. 5.6: HPTLC profile of successive solvent fractions of TM extract of Leaves

No. of Rf Fluorescence Spray reagent

spots value behaviour
254 nm 366nm VS FeCl3 Dr. L.B N.P.
Pet ether Fraction
1 0.96 Violet/ Orange - - - - -
brown
2 0.83 Violet/ Orange - - - - -
brown
3 0.79 Violet - Violet - - Violet -
4 0.36 - - Violet - - Violet -
Chloroform Fraction
1 0.99 Violet/ Orange - - - - -
brown
2 0.90 Violet/ Orange - - - - -
brown
3 0.76 Violet/ Blue Violet Blue - Violet -
brown
4 0.60 Brown Orange - - - - -
5 0.49 Violet Orange - - - - -
Ethyl acetate Fraction
1 0.89 Violet Orange - - - - -

2 0.83 Violet Orange - - - - -

3 0.75 Violet Yellow Violet Blue - Yellow
4 0.56 Violet Blue Violet - - Yellow
n- Butanol Fraction
1 0.79 Violet - Violet Blue - Red/ -
Brown
2 0.69 Violet - Violet - - Yellow
3 0.38 - - Violet Yellow
Methano Fraction
1 0.78 Violet/ Blue - Blue - Violet -
brown
2 0.61 Violet/ Blue Violet - - - -
brown
3 0.46 Violet Blue Violet - - - Yellow
4 0.30 Violet Blue - - - Violet -
Water Fraction
1 0.40 Violet - Violet Blue - - -
2 0.12 violet - - - - - -

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 Extraction and Fractionation

DISCUSSION:
The total methanolic (TM) extract of roots when fractionated with solvents of increasing
polarity yielded highest extractive value for water fraction (1.89%) followed by methanolic
fraction (1.04%), n- butanol fraction (0.86%), ethyl acetate fraction (0.45%), chloroform
fraction (0.42%)) and pet- ether fraction (0.36%) indicating more of polar constituents.

The fractions of total methanolic extract of roots when studied phytochemically showed the
presence of carbohydrates in the ethyl acetate, n- butanol and water fractions, tannins & other
phenolic compounds were present in ethyl acetate, n- butanol, methanol and water fractions,
flavonoidal compounds were present in ethyl acetate and methanolic fractions and gums were
present in water fraction.

HPTLC studies of successive extracts of the roots indicated that the pet ether fraction gave
presence of 3 spots having Rf values of 0.83 (triterpenoid in nature), 0.74and 0.70 and were
of steroidal nature. Chloroform fraction gave seven spots at Rf values of 0.83, 0.74, 0.70,
0.62, 0.52, 0.39 and 0.16 of which the spots at Rf values 0.83 was triterpenoid in nature. Ethyl
acetate fraction resolved into four spots of which the spots at Rf values 0.84 (steroidal in
natxire), 0.73, 0.68 (flavonoid in nature) and 0.62. n-Butanol fraction showed one spot at Rf at
0.54 (triterpenoid in natiu-e). Methanolic extract gave 2 spots of Rf values 0.35 and 0.21
which gave positive test for steroidal and terpenoidal compounds. The water extract gave two
spot at Rf values of 0.20 and 0.12.

HPTLC studies of successive extracts of the roots totally revealed the presence of 9 spots.
From their fluorescence behavior and reactions with spray regents it could be concluded that
6 compounds of triterpenoid, 2 spots were that of steroidal nature and 2 spots were flavonoid
in nature.

The total methanolic (TM) extract of leaves when fractionated with solvents of increasing
polarity yielded highest extractive values for water fraction (4.15% w/w) followed by
methanolic fraction (3.1%) w/w), pet- ether fraction (2.0%o w/w), chloroform fraction (1.45%)
w/w), n- butanol fraction (0.8%) w/w) and ethyl acetate fraction (0.43%) w/w) indicating more
of polar constituents.

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 Extraction and Fractionation

Phytochemical screening of the fractions of total methanolic extract of leaves of B. frondosa

revealed the presence of carbohydrates and tannins & other phenolic compounds in the
methanolic and water fractions, flavonoids and amino acids were present in n- butanol
fraction.

HPTLC studies of successive extracts of the leaves indicated that the pet ether extract gave
the presence of 4 spots having Rf values of 0.96, 0.83 (chlorophyll), 0.79 and 0.36
(terpenoidal in nature). Chloroform fraction gave five spots of which the spots at Rf value of
0.76 was terpenoidal/ steroidal in nature. Ethyl acetate extract resolved into foiir spots of
which spots at Rf value of 0.75 and 0.56 were flavonoidal in nature, n- Butanol fraction
showed three spots at Rf of 0.79 (steroid compound), 0.69 and 0.38 (flavonoid in nature).
Methanolic extract gave 4 spots having Rf values of 0.78, 0.61, 0.46 and 0.30. of which the
spot at Rf values of 0.61 and 0.46 were of terpenoidal nature. The water extract gave two spot
at Rf values of 0.40 (terpinodal/ phenolic in nature) and 0.12.

HPTLC studies of successive extracts of the leaves revealed the presence of 12 spots. From
their flurosence behaviour and spray reagents it can be concluded that 5 spots were that of
chlorophyll, 2 of terpenoid compounds, 3 spots were flavonoid in nature and 2 of phenolic
compounds.

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