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Phytochemical Screening and Antioxidant Activity of The Leaves of Plant Casearia Tomentosa PDF
Phytochemical Screening and Antioxidant Activity of The Leaves of Plant Casearia Tomentosa PDF
2
Shukla, Abha1; Tyagi, Ritu1 and Shukla, Rishi Kumar
Received: October 05, 2015 Accepted: November 15, 2015 Online: December 31, 2015
Abstract Introduction
In the present study petroleum ether extract Search for drugs to improve the quality of
of the leaves of plant Casearia tomentosa life and cure diseases has been a part of
were investigated for phytochemical human life right from its beginning. In many
screening and antioxidant activity. Casearia of the well developed ancient civilizations
tomentosa leaves were extracted by soxhlet this knowledge was evaluated and formed an
apparatus and phytochemical screening was essential part of the texts of their traditional
evaluated using standard methods. The systems of medicine, such as Ayurveda,
antioxidant activity was performed by 1, 1- Siddha and Unani (Singh et al., 2009).
diphenyl-2-picrylhydrazyl (DPPH) free Ayurveda has enriched with numerous plants
radical scavenging method and Ascorbic acid introduction, their medicinal importance and
used as standard antioxidant. Phytochemical usage. Plants have been used and still are
screening revealed that the presence of using as a rich source of many natural
various medicinal active phytoconstituent products. In India most of which have been
such as terpenoids, steroids, phytosterol, fat extensively used for traditional human health
and oil etc. This extract shows good care system (Rex and Lyla, 2009).
antioxidant activity with IC50 value 280 The Indian Himalayan region alone supports
µg/ml. All these experimental analysis about 18,440 species of plants of which about
established a good support to the use of this 45% are having medicinal properties.
plant in herbal medicine and can be used to According to Samant et al., out of the total
prevent oxidative stress. species of vascular plants, 1748 species are
Keywords: Casearia tomentosa | medicinal. Uttarakhand is a storehouse of a
Phytochemical | Antioxidant activity | rich variety of herbs and medicinal and
Ascorbic acid | Terpenoids aromatic plant species (Samant, et al., 1998).
Medicinal plants are increasingly gaining
For Correspondence:
acceptance even among the literates in urban
1
Department of chemistry, Kanya Gurukula Campus, settlements, probably due to easy availability,
Gurukula Kangri Vishwavidyalaya, Haridwar (U.K.)
2
Department of chemistry, Gurukula Kangri
no side-effects, and better patient
Vishwavidyalaya, Haridwar (U.K.) compliance. These contain dozens of active
Email: abs.gkv@gmail.com
constituents such as alkaloids, flavonoids,
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Shukla et al./Vol. VI [2] 2015/141 - 147
glycosides, saponins, terpenoids, phytosterol, and finally grinded to powdered form and
steroids, tannins etc which combine to give stored in polythene bags for further use.
the plant its therapeutic value Chemicals and reagents
(Vijayaraghavan et al., 2013). Natural
(DPPH)1,1-diphenyl-2-picrylhydrazyl
antioxidants play a key role in health
(Sigma Aldrich), Ascorbic acid (Rankem,
maintenance and prevention and treatment of
India), Petroleum ether (Merck), Ethanol
complex diseases like atherosclerosis, stroke,
(Merck). All the other solvents and chemical
diabetes, Alzheimer’s disease and cancer
used were of analytical grade.
(Jayasri et al., 2009; Uddin et al., 2008). In the
last few decades, the demand for natural Extraction
antioxidants has been increased due to 150 gm air dry powderd leaves of Casearia
consumer concerns about the safety of tomentosa was treated with 1250 ml of
synthetic antioxidants (Shukla et al., 2014). petroleum ether by soxhlet extraction
One of such natural source is Casearia technique for 18 hr. It was concentrated to
tomentosa, it is a small tree up to 50-80 cm dryness under reduced pressure and
girth and 7 m tall belongs to the family controlled temperature using rotary
Salicaceae. Its common name is Chilla. evaporator. The petroleum ether extract
Different parts of Casearia tomentosa is yielded a greenish yellow waxy mass. The
traditionally claimed for its medicinal collected leaves extract was stored in a
importance like in ulcers, dropsy, fissures, refrigerator.
colic pain in the abdomen, malarial fever, Phytochemical screening
tonsillitis pain, wounds, and in severe bone
The phytoconstituents present in petroleum
fractures as a plaster (Rao et al., 2014);
ether extract was analysed by using standard
Adhikari et al., 2010; Maurya and Seth, 2014).
qualitative method (Evans, 2009; Harborne,
Therefore, the present investigation was
1998). The leaves extract was screened for
undertaken to study the antioxidant potential
the presence of biologically active
of this plant and to put forward the evidence
compounds like alkaloids, flavonoids,
of the fact that this plant is having good
glycosides, saponins, terpenoids, phytosterol,
antioxidant activity.
steroids, tannins, fat and oils etc.
Material And Methods
Alkaloids
Plant material
Five milligrams of extract was dissolved in
Casearia tomentosa leaves were collected twenty milliliters of dilute HCl and then
from Lachhiwala forest Dehradun, filtered.
Uttarakhand (India) in the month of August
Mayer’s test:- 5 milliliters of filtrate was
and September, identified and authenticated
treated with Mayer’s reagent. Yellow colour
by Botanical Survey of India, (BSI)
precipitate indicates presence of alkaloid.
Dehradun with accession No.115689. A
voucher specimen has been deposited in Wagner’s test:- 5 milliliters of filtrate was
medicinal plants herbarium in Department of treated with Wagner’s reagent. Brown
Chemistry, Kanya Gurukula Campus, reddish precipitate indicates presence of
Gurukula Kangri Vishwavidyalaya. The alkaloids.
collected leaves were washed, dried in shade
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Dragendroff’s test:- 5 milliliters of filtrate Molisch Test:- 5 ml filtrate was treated with
was treated with Dragendroff’s reagent. Red a drop of alcoholic naphthol solution in a test
precipitate indicates presence of alkaloids. tube. Formation of Violet ring at the junction
Hager’s test:- 5 milliliters of filtrate was indicates the presence of carbohydrates.
treated with Hager’s reagent. Yellow Fehling’s Test:- 5 ml filtrate was boiled on
precipitate indicates presence of alkaloids. water bath with I ml of each Fehling solution
Flavonoids A and B. A red precipitate indicates the
presence of sugar.
Alkaline Reagent test:- Extract was treated
with few drops of NaOH solution. Formation Bendict’s Test:- 5 ml filtrate was treated
of intense Yellow color which becomes with Bendict’s reagent and heated gently in
colourless on addition of dilute acid (HCl or water bath. An orange red precipitate
H2SO4) indicates the presence of Flavanoids. indicates the presence of reducing sugar.
Lead Acetate test:- Extract was treated with Barfoed’s Test:- 5 ml filtrate was treated
few drops of Lead Acetate solution. with Barfoed’s reagent and heated gently in
Formation of intense Yellow coloured water bath. An orange red precipitate
precipitates indicates the presence of indicates the presence of reducing sugar.
flavanoids. Glycosides
Shinoda’s test:- Small quantity of extract Five milligrams of extract was dissolved in
was dissolved in alcohol. To that few piece twenty milliliters of dilute HCl and then
of magnesium with concentrated filtered.
hydrochloric acid was added dropwise and Modified Borntrager’s test:- Extract was
heated. Appearance of magenta colour treated with 5 % Ferric Chloride solution and
indicates the presence of flavonoids. immersed in boiling water for about 5
Sulphuric acid test:- Extract was treated minutes. The mixture was than cooled and
with few drops of concentrated sulphuric acid extracted with equal amount of chloroform.
. Yellow orange colour indicates the presence The chloroform layer was separated and
of flavonoids. treated with Ammonia solution. Formation of
Tannins pink colour in the ammonical layer indicates
the presence of glycosides.
Ferric Chloride Test:- 100 mg of the extract
was boiled with 20 ml of 45% ethanol for 5 Legal test:- Five milligrams of extract was
minutes. The mixture was cooled and then treated with sodium nitroprusside in Pyridine
filtered. Filtrate was diluted with distilled and NaOH. Formation of red colour indicates
water and then 2 drops of ferric chloride the presence of glycosides.
solution was added. A transient greenish to Keller killiani test:- Five milligrams of
black colour indicates the presence of extract mixed with chloroform and evaporate
tannins. to dryness. Add 1 ml glacial acetic acid
Carbohydrate containing trace amount of ferric chloride.
Transfer it to test tube and add carefully 0.5
50 mg extracts were dissolved in 20 ml of
ml of concentrated H2SO4 by the side of the
distilled water and filtered. The filtrate was
test tube. Acetic acid layer shows blue colour
used to test for the presence of carbohydrates.
indicates the presence of glycosides.
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free radical according to the method of plant derived terpenoids possess activities
Brand-Williams et.al with some modification like antioxidant, anticancer, anti-
(Brand - Williams et al., 1995). A working inflammatory, sedative, cytotoxic activity
solution of 0.004% was freshly prepared by etc. Plant steroids also referred to as ‘cardiac
dissolving 4 mg of DPPH in 100 ml of glycosides’ are one of the most naturally
methanol. 1ml of each extract solution of occurring plant phytoconstituents and
different concentration (1, 5, 10, 50, 100, numerous reports support their use as cardiac
250, 500, 750, 1000 µg/ml) was added to 3 drugs and as antioxidant (Mooradian, 1993).
ml working solution of DPPH, Keep this Beside these phytosterols was also present in
reaction mixture in dark for 30 min. After 30 this plant extract, which is also responsible
min the absorbance of the preparations were for antioxidant activity[16]. Various reports
taken at 517 nm with an UV-VIS support that plants fixed oil have variety of
spectrophotometer which was compared with biological activity such as cytotoxic and
the corresponding absorbance of standard antioxidant etc (Muna et al., 2014)
ascorbic acid of similar concentrations (1- From above discussion, we can interpret that
1000 µg/ml). 1 ml of methanol with 3 ml of the presence of these phytochemicals in
working DPPH solution serves as blank. petroleum ether extract shows medicinal
Then the % inhibition or % anti radical importance of leaves of Casearia tomentosa.
activity was calculated by equation:
DPPH free radical scavenging assay
% inhibition = (Absorbance of control−
DPPH radical scavenging activity is one of
Absorbance of sample) / (Absorbance of
the most widely used method for evaluation
control) × 100
of the antioxidant activity of plant extract.
IC50 of extract and standard ascorbic acid The principle of DPPH method is based on
was calculated by graphical method by the reduction of DPPH in the presence of a
plotting % inhibition vs concentration. hydrogen donating antioxidant. Extracts
Results and Discussion reduce the colour of DPPH due to the power
Extractive yield of hydrogen donating ability (Blosis, 1958).
DPPH is one of the compounds that possess a
The extractive yield (in % w/w) of petroleum
proton free radical with a characteristic
ether extract was 1.515 %. After complete
absorption, which decreases significantly on
removal of the solvent its consistency is
exposure to proton radical scavengers
waxy and greenish yellow in colour.
(Yamaguchi et al., 1998). Petroleum ether
Phytochemical screening extract of leaves showes DPPH anion
The result for phytochemical screening of scavenging power. The IC50 280 μg/mL, and
Casearia tomentosa leaves are summarized 20μg/mL were evaluated for petroleum ether
in Table.1. The Preliminary phytochemical extract and ascorbic acid respectively (Fig.1).
screening of extract of this plant revealed the Antioxidants may guard against reactive
presence of active phytoconstituent such as oxygen species (ROS) toxicities by
steroids, phytosterol, terpenoids and fats and scavenging reactive metabolites and
oils etc. Out of which terpenoids are among converting them to less reactive molecules
the most widespread and chemically diverse (Shukla et al., 2014).
groups of natural products. It is reported that
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120
100
% Inhibition
80
60
Ascorbic acid
40
20
PE
0
0 100 200 300 400 500 600 700 800 900 10001100
Concentration of extracts in µg/ml
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