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Phytochemical Investigation and Thin Layer Chromatography of Aegle

marmelos Leaves Methanolic Extract

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Advances in Life Sciences 5(15), Print : ISSN 2278-3849, 5685-5690, 2016
Phytochemical Investigation and Thin Layer Chromatography of Aegle
marmelos Leaves Methanolic Extract
MORE YOGESHWAR, R. M. GADE*, A. V. SHITOLE AND S. H. WAVARE
Department of Plant Pathology,
Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra
*email: gadermg@gmail.com
ABSTRACT
The present study is focused on phytochemical screening
of methanol leaf extracts of A. marmelos and the
development of phytochemical parameters and to
investigate the antifungal substances present in
methanolic extract obtained from leaves of A. marmelos
plant. Preliminary phytochemical screening of the extracts
revealed the presence of flavonoids, tannins, alkaloids,
saponins and phenolic compounds. Petroleum ether: Ethyle
acetate (2:1) solvent system gives maximum numbers of
compounds from methanolic extract of A. marmelos and
these compounds were separated on TLC plates. TLC
resulted in identification of three spots found in the
methanolic extract. The Rf values of methanolic extract of
A. marmelos run under Petroleum ether: Ethyle acetate
(02:01) solvent system were 0.04, 0.07, 0.14, 0.18, 0.25,
0.35, 0.45, 0.61, 0.70 and 0.84. The study will provide
referential information for the correct identification of
the crude plant extract of A. marmelos. Methanol was the
best solvent for extraction of antifungal constituents from
A. marmelos leaves.
Key words Aegle marmelos, Phytochemical analysis,
Extraction, TLC, compounds, methanolic
extract
Medicinal plants would be the best source to obtain
a variety of newer herbal antifungal compounds. For
centuries plants have provided mankind with useful,
sometimes lifesaving drugs. Modern agriculture, in
cases where correlation between chemical structure and
biological activities were noted, empirical science began
to give way to rational antimicrobial design. This
emerging approach to identify and develop potential new
fungicides is largely successful, due to the intellectual
cooperation of chemistry (medicinal). Therefore such
plants should be investigated to better understand their
properties, safety and efficacy. The use of fungicides
derived from plants has been in practice for a very long
time.
A. marmelos (L) Correa belonging to family
Rutaceae and commonly known as Bael has been used
as a folklore medicine since ancient time to cure various
human diseases. This plant is indigenous to India and is
abundantly found in Himalayan tract, Bengal, Central
and South India. Almost every part of this tree viz.,
root, stem, bark, leaf, flowers and fruit at all stages of
maturity have medicinal virtues as in (Maity, et al.,
2009). In fact as per Charaka (1500 BC) no drug has
been longer or better known or appreciated by the
inhabitants of India. Among the other parts of the tree
fruit is reported to be valuable Ayurvedic medicine for
chronic diarrhea, tonic for heart and brain, anti-viral
activity, hypoglycemic activity, antibacterial activity,
antiproliferative activity and against parasites as in
(Sunita, et al., 2011). The rural population of India is
more disposed to traditional ways of treatment because
of easy availability and cheaper cost. Plant are the richest
resource of drugs of traditional systems of medicine,
modern medicines, food supplements, folk medicine,
pharmaceutical intermediates and chemical entities for
synthetic drugs as in (Hammer, et al., 1999). The
medicinal value of plants lies in some chemical active
substances that produce definite physiological action
on human body as in (Aiyelaaghe, and Osamudiamen,
2009).
Moreover pharmacological studies have
acknowledged the value of medicinal plants as potential
source of bioactive compounds. These bioactive
substances are normally accumulated as secondary
metabolites in all plant cells but then their concentration
varies according to the plant part, season, climate and
particular growth phase as in (Maji, et al., 2010).
Scientific techniques have now been developed to isolate
such natural constituents with desired biological activity
and exploit them in management of phytopathogenic
microorganisms. A large number of plant based
preparations are utilized for treating various plant
diseases in India but not much work has been done on
the scientific validation of the utility of these plants in
agrochemical industries. Thus there is a need to conduct
systematic and scientific investigation to justify the use
of these plants in management of plant diseases in India.
More than 100 phytochemical compounds have been
isolated from various parts of the plant, namely phenols,
flavonoids, alkaloids, cardiac glycosides, saponins,
terpenoids, steroids, and tannins. These compounds are
well known to possess biological and pharmacological
activity against various chronic diseases such as cancer
and cardiovascular and gastrointestinal disorders (Chew,
et al., 2009). Antioxidant, antiulcer, antidiabetic,
anticancer, antihyperlipidaemic, anti-inflammatory,
5686 Advances in Life Sciences 5(15), 2016
Table 1. Preliminary phytochemical analysis of
Methanolic extract of Aegle marmelos
alone
Test Aegle marmelos
Alkaloids +
Saponins +
Tannins and phenolics +
Fixed oils and fats +
Steroids and sterols +
Cardio glycosoides - In vitro evaluation of plant extracts by poisoned food
Flavonoids + technique on PDA medium: The efficacy of Acetone,
+ presence, - absence
antimicrobial, antispermatogenic effects have also been
reported on various animal models by the crude extracts
of this plant (Jyothi Saradha, and Subba Rao, 2010).
Every part of A. marmelos plant such as its fruits,
stem, bark, and leaves possesses medicinal property
and is used for treating various eye and skin infections.
Leaf is considered to be one of the highest accumulatory
parts of the plant containing bioactive compounds which
are synthesized as secondary metabolites (Cowan, 1999).
The present study was, therefore, aimed at
evaluating the phytochemical potential and antifungal
activity of A. marmelos methanolic leaf extracts. The
aim of the present paper is to determine the bioactive
substances contributing to the antifungal value of the
leaves of A. marmelos using methanol as solvent
systems.
MATERIALS AND METHODS
Evaluation of plant solvent extracts: The fresh leaves
of A. marmelos, were collected from various places of
Dr. P.D.K.V., Akola (M.S.), India. The area which was
selected for the collection of the plant materials for the
present study was 5 km2 around the district headquarter.
The identification and authentication of the plants was
carried out at Department of Botany Dr. P.D.K.V., Akola
(M.S.), India. Collected fresh plant leaves were
thoroughly washed under tap water to remove dust and
other impurities and once with distilled water and then
shade dried separately under shade with occasional
shifting for about 3 to 4 weeks. The dried leaves were
coarsely powdered with sample grinder and stored in
airtight container until further use (Thenmozhi, et al.,
2011).
Preparation of solvent extracts from tested leaves:
Acetone, Ethanol, Methanol and Chloroform were used
as solvent for preparation of leaves extracts. Forty
gram powder of each leaves was separately soaked in
200 ml of each solvent separately in 500 ml conical
flask and then plugged tightly with cotton and wrapped
with paper. All conical flasks were kept on rotary shaker
for four days and then allowed to stand for 5 hr to
settle the leaves material. Supernatant from each flask
was filtered separately through Whatman No. 1 filter
paper and evaporated at room temperature. Residual
portion of leaves was repeatedly extracted three times
to harvest maximum metabolites from leaves. Air dried
extracts were weighed separately and transferred into
small vials and kept in refrigerator at 50C until further
use.
Ethanol, Methanol and Chloroform extracts of A.
marmelos, at 250, 500, 750 and 1000 µl concentration
were tested against P. debaryanum under in vitro
condition following poisoned food technique on PDA
medium (Al-Rahmah, et al., 2013).
One gram crude extract of all the flowers extracted
with Acetone, Ethanol, Methanol and Chloroform were
diluted in 10 ml DMSO separately and from this 250,
500, 750 and 1000 µl suspension were poured separately
in conical flasks which containing 60 ml sterilized melted
PDA medium for 3 plates. The conical flask was shacked
well for uniform mixing of plant extract with media
and poured in plates then allowed for solidification. In
control set, only 250, 500, 750 and 1000 µl DMSO
were used. For each treatment, 3 replicates (plates)
were used. All the plates were inoculated individually
with 5 mm diameter discs of the test fungal cultures
and then incubated at 28±2°C, until the control plates
reached full growth. To know the effect of different
plant extracts. The per cent growth inhibition (I) of
test fungus was calculated by using formula suggested
by Vincent (1947).
C-T
Per cent inhibition (I) = ————x 100
C
Where,
C = Growth of fungus in control in mm
T = Growth of fungus in treatment in mm
Chromatography: Based on in vitro growth inhibition
assay, methanol extract of leaves of A. marmelos were
selected for chromatographic analysis. This method was
used to study the preliminary phytochemicals, Thin layer
chromatography (TLC) for separation of different
compounds present in crude methanolic extract.
Preliminary phytochemical screening of plant leaves
methanol extracts: Preliminary phytochemical analysis
of methanol extract of leaves of A. marmelos were
performed for analysis of different phytochemicals like
cardio glycosides, saponins, fixed oils and fats, alkaloids,
steroids and sterols, flavonoids, tannins and phenolic
YOGESHWAR et al., Phytochemical Investigation and Thin Layer Chromatography of Aegle marmelos Leaves Methanolic Extract 5687

Table 2. Standardization of solvent system for methanolic extract of Aegle marmelos

Sr. No. Solvent system Proportion Methanolic extract of A. marmelos

Rf Colour
1 Benzen : ethanol 09:01 Smear of compounds, no bands
2 Methanol : water 08:02 Smear of compounds, no bands
3 Ethyl acetate 100% 0.12 Dark blue
0.25 Pent blue
2.00 Light blue
4 Ethyl acetate : methanol 95:05 0.05 Brownish
0.14 Light green
0.23 Pent blue
0.82 Blue
0.94 Light Brown
0.97 Brown
5 Ethyl acetate : methanol 92:08 Smear of compounds, no bands
6 Ethyl acetate : methanol 90:10 Smear of compounds, no bands
7 Ethyl acetate : methanol 85:15 Smear of compounds, no bands
8 Ethyl acetate : methanol 70:30 0.6 Pent blue
0.72 Light blue
9 Ethyl acetate : methanol 50:50 Smear of compounds, no bands
10 Methanol 100% Smear of compounds, no bands
11 Ethyl Acetate : acetic acid : petroleumether 19:1:5 Smear of compounds, no bands
12 Ethyl Acetate : acetic acid : petroleumether 15:6:4 Smear of compounds, no bands
13 Ethyl Acetate : acetic acid : petroleumether 20:6:4 0.05 Brown
0.62 Light blue
0.85 Pent blue
14 Ethyl acetate : methanol : petroleumether : water 19:3:3 0.09 Blue
0.23 Pent blue
0.32 Light Brown
0.36 Brown
0.50 Green
0.72 Light red
15 Ethyl acetate : methanol : benzen 20:6:3 Smear of compounds, no bands
16 Ethyl acetate : methanol : butanol 19:1:6 Smear of compounds, no bands
17 Petroleumether : ethyle acetate 22:3:5 0.04 Brown
0.07 Pent blue
0.14 Brownish
0.18 Light blue
0.25 Light blue
0.35 Pent red
0.45 Light red
0.61 Red
0.70 Light brown
0.84 Brownish
5688 Advances in Life Sciences 5(15), 2016
compounds by following method given by Thenmozhi,
et al. (2011).
Test for cardio glycosides: A volume of 5 ml of the
plant extract was treated with 2 ml of glacial acetic
acid containing a drop of ferric chloride solution. Then
it was underplayed with 1 ml concentrated sulphuric
acid. A brown ring of the interface indicates a deoxy
sugar characteristic of cardio glycosides. A violet ring
may appear below the ring, while in the acetic acid layer,
a greenish ring may form just gradually throughout thin
layer.
Test for saponins: About 2 g of the powdered sample
was boiled in 20 ml of distilled water bath and filtered.
The 10 ml of the filtrate was mixed with 5 ml of distilled
water and shaken vigorously for a suitable persistent
froth. The frothing was mixed with 3 drops of olive oil
and shaken vigorously, and then the formation of
emulsion was observed.
Test for fixed oils and fats (spot test): A small quantity
of powder was pressed between two filter papers. Oil
strains on the filter paper indicated the presence of fixed
oils.
Test for alkaloids: The plant extract was mixed with
a few drops of acetic acid followed by dragendroff’s
reagent and mixed well. An orange red precipitate
formed indicated the presence of alkaloid.
Test for steroids and sterols: Two ml of acetic
anhydride was added to 0.5 g of the plant extract of
each sample with 2 ml of H2SO4. The colour change
from violet to blue green in the sample indicates the
presence of steroids and sterols.
Test for flavonoids: Five ml of dilute ammonia solution
was added to the aqueous filtrate of the plant extract
followed by the addition of concentrated H2SO4. A yellow
coloration observed in the extract indicated the presence
of flavonoids. The yellow colour disappeared on
standing.
Test for tannins and phenolic compound: About 0.5
g of the dried powdered sample was boiled in 20 ml of
water in a test tube and then filtered. A few drops of
0.1% ferric chloride was added and observed for
brownish green or a blue-black coloration. A few drops
of alcohol and ferric chloride solution were mixed with
the plant extract. A blue green or red colour indicated
the presence of phenol.
Thin Layer Chromatography (TLC): Thin layer
chromatography was carried out to know the chemical
profile of methanolic extract of A. marmelos leaves.
Preparation of TLC plates: The TLC plates were
prepared as described by Harborne (1998). Briefly, 25
g of silica gel-G (Hi media, Manufactured, India) was
mixed with 50 ml of distilled water and the slurry formed
was uniformly spread over TLC plates with a thickness
of 0.25 mm using the spreader. The plates were allowed
to dry at room temperature and heated in an oven at
1000 C for 2 h.
Standardisation of solvent system: Each sample of
the crude extract of A. marmelos leaves was diluted in
distilled water. The prepared TLC plates were marked
1 cm from bottom and 10 µl each sample was applied
on TLC plates at equal distance with the help of capillary
tubes. For separation of maximum bands on TLC plates
different solvent systems were used according to
polarity and from that petroleum ether: ethyl acetate
(02:01v/v) was selected as standard solvent system.
All these methanolic extract of A. marmelos
were screened for preliminary phytochemical analysis
and thin layer chromatography of methanolic extract
of A. marmelos. Observations were recorded for
presence or absence of phytochemicals, number and
Rf values of bands (compounds) present in extract.
RESULT AND DISCUSSION
Natural products with pesticidal activity are being
explored in order to make available the pesticides, which
are easily biodegradable, selective and can be locally
produced, especially for the farmers, who cannot afford
expensive synthetic pesticides. The results revealed that
A. marmelos plant extracts at each concentration
inhibited the growth of P. debaryanum. The rate of
growth inhibition was corroborated with its
concentrations in case of all the tested plant extracts.
Results of poison food technique represent that, at 1000
µl concentration (C4), complete inhibition of mycelial
growth of test fungus was recorded in Methanolic
extract of A. marmelos plant. By using the waste
medicinal plant as raw material for plant-derived
fungicides, one could can manage the disease, and at
the same time might create economic uses for these
unwanted waste material.
Chromatography: Based on in vitro results, methanolic
extract of A. marmelos selected for further partial
purification by chromatographic analysis.
All these methanolic extract of A. marmelos were
screened for preliminary phytochemical analysis and
thin layer chromatography of methanolic extract of A.
marmelos was carried out. Observations were recorded
for presence or absence of phytochemicals, number
and Rf values of bands (compounds) present in extract.
Preliminary phytochemical analysis: Preliminary
phytochemicals presents in methanolic extract of A.
marmelos was analysed by following standard procedure
as explained under “Materials and Methods”.
Observations on presence or absence of
YOGESHWAR et al., Phytochemical Investigation and Thin Layer Chromatography of Aegle marmelos Leaves Methanolic Extract
phytochemicals namely, cardio glycosides, saponins,
fixed oils and fats, alkaloids, steroids and sterols,
flavonoids, tannins and phenolic compounds were noted
as + for presence and – sign for absence and are
presented in Table 1.
Results presented in Table 1 revealed that, from
all the tested phytochemicals, saponins, alkaloids,
steroids and sterols, fixed oils and fats, flavonoids,
tannins and phenolic compounds were observed in
methanolic extract of A. marmelos. Further, cardio
glycosides were only not observed in methanolic extract
of A. marmelos.
Similar findings also observed by Rhouma, et al.
(2009) found tannins, flavonoids and alkaloids as
preliminary phytochemicals in leaf extract of Pistacia
sp.
Thin Layer Chromatography (TLC): Thin layer
chromatography was used for separation of different
chemical constituents present in methanolic extract of
A. marmelos as described under ‘Materials and
Methods’.
Standardization of solvent system: Various solvent
systems were screened for efficient separation of bands
according to polarity. Total 2 solvent systems were used
in present investigation to know most suitable solvent
system for separation of compounds in methanolic
extract of A. marmelos. The Rf values and colour of
separated bands in different solvent systems under UV
trans illuminator are summarized in Table 2.
It is observed from data presented in Table 2, that,
different solvent systems showed differences in number
of bands and their Rf values in methanolic extract of A.
marmelos. Among all the tested solvent systems, Benzen
: Ethanol (09:01), Methanol : Water (08:02), Ethyl acetate
: Metanol (92:02), Ethyl acetate : Metanol (90:10), Ethyl
acetate : Metanol (85:15), Ethyl acetate : Metanol
(50:50), Methanol (100%), Ethyl Acetate: Acetic acid :
Petroleum ether (19:01:05), Ethyl Acetate: Acetic acid:
Petroleum ether (15:06:04), Ethyl acetate : Metanol:
Benzen (20:06:03). Ethyl acetate : Metanol: Butanol
(19:01:06) showed smearing of compounds and did not
produce any visible separation of compounds in
methanolic extract of A. marmelos, while most
promising solvent system produced good results on TLC
plate was Petroleum ether : Ethyle acetate (02:01). In
this solvent system maximum number of compounds
from methanolic extract of A. marmelos were separated
on TLC plates.
The Rf values of methanolic extract of A. marmelos
run under Petroleum ether: Ethyl acetate (02:01) solvent
system were 0.04, 0.07, 0.14, 0.18, 0.25, 0.35, 0.45,
0.61, 0.70, 0.84.
5689
Jawdah, et al. (2004) observed improved
separation of compounds in chloroform: methanol (9:1)
and ethyl acetate: petroleum ether from various plant
extracts. Masuduzzaman, et al. (2008) observed one
and three separated compounds of allamanda leaf
aqueous extract on TLC plate eluted with Hexane:
Benzeen (1:1) and Benzene: Ethyl acetate, respectively.
Subramaniam, et al. (2010) observed three bands from
jatropha biowash on TLC plates eluted with DCM:
MeOH (8:1) with Rf values 0.95, 0.90 and 0.70.
It is concluded that methanol extracts from leaves
of A. marmelos showed variable broad spectrum
antimicrobial activities. Although the exact active
components of these extracts that showed these effects
were not identified, yet the positive presence of
antimicrobial active principles such as phenols, sterols,
flavonoids, tannins, triterpenoids and coumarins seems
to cause these activities. The ability of the leaf extracts
of A. marmelos to inhibit growth of fungi is an indication
of its broad spectrum antimicrobial activity, which may
be employed as a source to develop new antimicrobial
agents. Methanol was found effective to give maximum
extraction yield of selected plant. Methanol was the best
solvent for extraction of antifungal constituents from
tested medicinal plant leaves.
Phytochemical analysis of methanolic extracts of
plants revealed that, from all the tested phytochemicals,
saponins, alkaloids, steroids and sterols, fixed oils and
fats, flavonoids, tannins and phenolic compounds were
observed in methanolic extract of A. marmelos. Further,
cardio glycosides were only not observed in methanolic
extract of A. marmelos. TLC (Thin layer
chromatography) of methanolic extract of A. marmelos
revealed that among 17 different solvent systems,
Petroleum ether: Ethyl acetate (02:01) was effective for
good separation of compounds on TLC plates.
Antifungal activity of plant extract in present study
may be due to presence of antifungal compounds like
glycosides, saponins, fixed oils, alkaloids, steroids,
flavonoids, tannins, phenolic, oils and fats were present
in respective plant extracts. Petroleum ether: Ethyl
acetate (02:01) solvent system was found effective for
separation of compounds on TLC plate.
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Received on 06-07-2016 Accepted on 10-07-2016