Phytochemical screening and Polyphenol estimation by HPLC of Terminalia arjuna

1. ~ 471 ~
_______________________________________
* Corresponding author: Ramakrishna U.V
E-mail address: uv.ramakrishna@gmail.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 3 | 2013 Research article
Phytochemical screening and Polyphenol estimation by
HPLC of Terminalia arjuna
Ramakrishna U.V1,
*, Dinesh kumar B2
, Sukesh Narayan Sinha2
,
Ratna Priya2
*,1
Department of Pharmaceutical Analysis & Quality Assurance, Bapatla College of Pharmacy,
Bapatla, A.P. India.
2
Food and Drug Toxicology Research Centre, National institute of nutrition, Tarnaka,
Hyderabad, A.P., India.
ABSTRACT
Medicinal Plants, as a source of remedies, are widely used as alternative therapeutic tool for the prevention or
treatment of many diseases. Our main objective was to evaluate the components in Terminalia arjun samples
and to evaluate the amount of polyphenols in the Terminalia arjuna dosage form using High Performance
Liquid Chromatography.
KEYWORDS: Terminalia arjuna, Proximate Analysis, HPLC.
INTRODUCTION[1,2,3]
Arjuna consists of dried stem bark of Terminalia
arjuna, found as naturally growing plant in dense
forests.
The thick, white-to-pinkish-grey bark has been
used in India’s native Ayurveda for over three
centuries, primarily as a cardiac tonic. Clinical
evaluation of this botanical medicine indicates it
can be of benefit in the treatment of coronary artery
diseases, heart failure and possibly
hypercholesterolemia. It has also been found to be
antiviral and anti-mutagenic. Terminalia’s active
constituents include tannins, cardenolide,
triterpenoidsaponins (arjunic acid, arjunolic acid,
arjungenin, arjunglycosides), flavonoids (arjunone,
arjunolone, luteolin), gallic acid, ellagic acid,
oligomericproanthocyanidins (OPCs), phytosterols,
calcium, magnesium, zinc and copper.[4]
MATERIALS AND METHODS
Proximate Analysis of the Test Samples of
Terminalia arjuna [7, 8, 9, 10, 18]
The analysis of samples in 50% methanol and
water and moisture content were determined by
standard procedures:
SCIENTIFIC CLASSIFICATION
Kingdom Plantae
Division Magnoliophyta
Class Magnoliopsida
Order Myrtales
Family Combretaceae
Genus Terminalia
Species T.arjuna
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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Determination of Foreign matter
Foreign matter was visualized by unaided eye and
then separated, weighed and expressed as
percentage of total weight as per standard
procedure mentioned in WHO Library.
DRUG
FOREIGN MATTER
(%w/w)
TABLET 0.0
CAPSULE 0.0
POWDER-1 0.0
POWDER-2 0.2
CHOORNAM 0.0
BARK 0.1
Determination of Moisture Content
1gm of powdered drug was weighed into a
weighed flat and thin porcelain dish and dried
in oven at 100ºC. Porcelain dishes are cooled
in desiccators. The loss in weight is recorded
as moisture.
PRODUCT
MOISTURE
CONTENT (%w/w)
TABLET 0.01
CAPSULE 0.05
POWDER-1 0.02
POWDER-2 0.01
CHOORNAM 0.01
BARK 0.01
Determination of ash
Different ash values like total ash, water
soluble ash and acid insoluble ash were
determined as per standard procedure
mentioned in WHO library.
PRODUCT
WEIGHT OF ASH
(%)
TABLET 6%
CAPSULE 4.3%
POWDER-1 4.6%
POWDER-2 8.7%
CHOORNAM 8.3%
BARK 8%
Determination of extractable matter
Different extractive values like water soluble
extractive and alcohol soluble extractive value were
determined as per standard procedure mentioned in
WHO library.
Drug
Water soluble
extractives
(%w/w)
Alcohol soluble
extractives (%w/w)
TABLET 5.9 3.5
CAPSULE 5.4 3.2
POWDER-1 4.9 2.9
POWDER-2 5.8 3.6
CHOORNAM 5.3 3.3
BARK 5.0 3.1

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Determination of tannins
0.5 ml of extract solution, 1-2 drops of ferric
chloride solution were added. Depending on the
colour obtained the presence or absence of tannins
(gallic/catecholic) was determined (Iyengar, 1995).
Determination of Swelling Index
The swelling index was the volume in ml taken up
by the swelling of 1g of plant material under
specific conditions. The index was determined with
method described by WHO.
DRUG SWELLING INDEX
TABLET 1.3
CAPSULE 1.8
POWDER-1 1.0
POWDER-2 1.8
CHOORNAM 0.6
BARK 0.6
Determination of foaming index
About 1gm of the plant material was reduced to a
coarse powder, weighed accurately and transferred
to 500ml conical flask containing 100ml of boiling
water. It is maintained at moderate boiling for
30min, cooled and filtered into 100ml volumetric
flask. Sufficient water was added through filter
paper to dilute to volume. The decoction was
poured into 10 stoppered test tubes in successive
portions of 1ml, 2ml, 3ml, etc. up to 10ml. the test
tubes are stoppered and shaken in length wise
motion for 15sec, 2 shakes per second. It was
allowed to stand for 15 min, and height of the foam
measured and foam index calculated. The index
was determined with the method which described
by WHO.
DRUG HEIGHT OF FOAM
TABLET 4.0 cm
CAPSULE 4.0 cm
POWDER-1 3.5 cm
POWDER-2 2.5 cm
CHOORNAM 2.0 cm
BARK 3.0 cm
Determination of microorganism
The presence or absence of pathogenic bacteria was
assessed using selective media such as MacConkey
Agar and CLED agar. No pathogenic bacteria were
detected in test substances using the above media.
Test for Phenols
1gm of drug is taken and 10 ml of water is added
and shaken vigoursly. 1ml of solution is taken in a
test tube and few drops of dilute nitric acid solution
is added. Colour changes from reddish to yellow
colour showing presence of phenols.
Test for Alkaloids
0.5gm of sample was wormed with 10ml of 2%
sulphuric acid for 2minutes and filtered 1ml portion
was treated with few drops of Dragendroff’s
reagent, orange brown precipitate was
observedshowing presence of alkaloids.
Test for Saponins
Ethanolic and water extracts are diluted to 1ml
separately with distilled water to 20ml and shaken
in a graduated cylinder for 15min and subjected to
foam test.
Thin-layer chromatography
Sample Preparation
To 200mg of test sample, 10ml of water was added
and the solution was stirred using a stir bar for
2min. Centrifugation was done at 1500 rpm for 15
minutes. The top layer collected was used as
sample for TLC.
TLC Analysis
Silica Gel-G was used for TLC analysis. Solvent
system employed consisted of Chloroform : Ethyl
Acetate : Formic acid (5:4:1). System suitability
factors such as reproducibility were performed. The
mobile phase was optimised using silica gel – G
and then on precoated plates. After complete run,
Rf values of different pigments were determined.

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PRODUCT Rf VALUE
TABLET
0.054
0.103
0.309
0.484
0.563
0.660
0.806
0.993
CAPSULE
0.060
0.121
0.484
0.757
CHOORNAM
0.054
0.115
0.296
0.575
0.993
PRODUCT Rf VALUE
POEDER-1
0.060
0.115
0.303
0.569
0.666
0.818
0.993
POWDER-2
0.060
0.121
0.327
0.490
0.575
BARK
0.060
0.115
0.303
0.484
0.581
0.660
0.993

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HPLC Chromatograms of Polyphenols in
various Dosage Forms of Terminalia arjuna
INSTRUMENT
Dionex (Rapid separation liquid chromatography)
COLUMN
Waters spherisorb, C18150mm x 4.6µm ODS2
SOFTWARE Chromeleon
DETECTORS DAD (Diode Array Detector)
MOBILE PHASE
Acetonitrile: Methanol: Dichloromethane
(700:100:200)
DILUENTS: Chloroform
FLOW RATE: 0.5ml/min
PROCEDURE [6, 21, 22]
Standard solutions were injected into the system
under the standard conditions mentioned above and
the chromatograms were recorded. Different
sample solutions were prepared and injected into
the system under the same chromatographic
conditions. The R.T of the peaks in the sample
chromatograms is compared with that of standard
chromatogram to identify the compounds. After
identifying the components, the amount of the
components as calculated. Amount of the
components present in the sample are calculated.
POLYPHENOLS CONTENT (mg/100gm)
SAMPLE
NAME
GALLIC
ACID
3,4, DI
HYDROXY
BENZOIC
ACID
CAAFFEIC
ACID
COUMARIC
ACID
ELLAGIC
ACID
CHLOROGENIC
ACID
TABLET 2.74 5.23 0.32 0.51 5.29 0.92
CAPSULE 8.09 15.09 1.00 0.87 10.91 8.58
POWDER-1 14.65 0.54 0.61 0.90 0.59 2.81
POWDER-2 7.65 20.61 2.06 0.09 0.22 17.98
CHOORNAM 52.37 14.53 0.66 0.15 20.19 10.04
BARK 52.64 13.87 2.04 1.36 19.46 13.69
The amount of Gallic acid was found to me higher
in Choornam and Bark followed by Powder-1.The
amount of 3, 4, DIHYDROXY BENZOIC
ACIDwas found to me higher in Bark and Powder-
2 followed by Capsule. The amount of CAAFFEIC
ACID was found to me higher in Bark followed by
Powder-2. The amount of COUMARIC ACID was
found to me higher in Bark followed by Powder-1.
The amount of COUMARIC ACID was found to
me higher in Bark followed by Powder-1. The
amount of ELLAGIC ACIDwas found to mehigher
in Choornam followed by Bark. The amount of
CHLOROGENIC ACID was found to me higher in
Powder-2 followed by Bark.

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POLYPHENOLS TABLET HPLC
TABLET
COMPONENT
RT
STANDARD
RT
SAMPLE
CONTENT
mg/100rm
Gallic Acid 1.263 1.937 2.74
3,4 Dihydroxy
Benzoic Acid
2.123 2.167 5.23
Caffeic Acid 3.383 3.400 0.32
Coumaeic Acid 4.860 3.117 3.117
Ellagic Acid 10.340 12.803 12.803
Chlorogenic
Acid
2.593 2.687 2.687
Tablet was found to have a decent amount of Ellagic Acid and all the other components were found to
be low.

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.POLYPHENOLS CAPSULE HPLC
CAPSULE
COMPONENT RT STANDARD RT SAMPLE CONTENT mg/100rm
Gallic Acid 1.263 1.820 8.09
3,4 Dihydroxy Benzoic Acid 2.123 2.110 15.09
Caffeic Acid 3.383 3.373 1.00
Coumaeic Acid 4.860 3.150 0.87
Ellagic Acid 10.340 12.797 10.91
Chlorogenic Acid 2.593 2.710 8.58
Capsule was found to have a good amount of 3,4, DIHYDROXY BENZOIC ACID and ELLAGIC ACID, a
decent amont of GALLIC ACID and CHLOROGENIC ACID and the other components were found to be
low.
POLYPHENOLS POWDER-1 HPLC

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POWDER-1
COMPONENT RT STANDARD RT SAMPLE CONTENT mg/100rm
Gallic Acid
1.263 1.040 14.65
3,4 Dihydroxy Benzoic Acid
2.123 2.120 0.54
Caffeic Acid
3.383 3.407 0.61
Coumaeic Acid 4.860 3.127 0.90
Ellagic Acid 10.340 12.847 0.59
Chlorogenic Acid
2.593 2.710 2.81
Powder-1 was found to have a good amount of GALLIC ACID and the other components were found to be low.
POLYPHENOLS POWDER-2 HPLC
POWDER-2
COMPONENT
RT
STANDARD
RT
SAMPLE
CONTENT
mg/100rm
Gallic Acid 1.263 1.887 7.65
3,4 Dihydroxy
Benzoic Acid
2.123 2.087 20.61
Caffeic Acid 3.383 3.350 2.06
Coumaeic Acid 4.860 3.053 0.09
Ellagic Acid 10.340 12.844 0.22
Chlorogenic
Acid
2.593 2.687 17.98

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Powder-2 was found to have a good amount of 3,4, Dihydroxy Benzoic Acid and Chlorogenic Acid, a decent
amount of Gallic Acid and the other components were found to be low.
POLYPHENOLS CHOORNAM HPLC
CHOORNAM
COMPONENT
RT
STANDARD
RT
SAMPLE
CONTENT
mg/100rm
Gallic Acid
1.263 1.897 52.36
3,4 Dihydroxy
Benzoic Acid
2.123 2.093 14.53
Caffeic Acid 3.383 3.360 0.66
Coumaeic Acid
4.860 3.183 0.15
Ellagic Acid
10.340 12.910 20.19
Chlorogenic
Acid
2.593 2.693 10.04
Choornam was found to have a higher amount of Gallic Acid, good amount of 3,4, Dihydroxy Benzoic Acid,
Ellagic Acid And Chlorogenic Acid, and the other components were found to be low.

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POLYPHENOLS BARK HPLC
BARK
COMPONENT
RT
STANDARD
RT
SAMPLE
CONTENT
mg/100rm
Gallic Acid 1.263 1.840 52.64
3,4 Dihydroxy
Benzoic Acid
2.123 2.120 13.87
Caffeic Acid
3.383 3.390 2.04
Coumaeic Acid
4.860 3.117 1.36
Ellagic Acid
10.340 12.710 19.46
Chlorogenic
Acid
2.593 2.723 13.69
Bark was found to have a higher amount of Gallic
Acid, good amount of 3,4, Dihydroxy Benzoic
Acid, Ellagic Acid and Chlorogenic Acid, and the
other components were found to be medially low.
RESULTS AND DISCUSSION
The Various Phytochemical Screening parameters
has been performed, Such as Test for Alkaloids,
phenols, Saponins, Tannins, Determination of
Foreign matter, Swelling Index, Ash values,
Extractive values, Foaming index, Moisture
content, Microorganisms and TLC has been
performed
HPLC of the polyphenols in various brands of
Terminalia arjuna were performed on Dionex
RPLC (Rapid Separation Liquid Chromatography)
instrument, which has chromelon software
installed. The column used for the analysis was
Waters SpheriSorb C18 150mm×4.6 5µm ODS2
Column with a flow rate of 0.5ml/min. The
detector used to detect the samples was DAD
(Diode Array Detector) 450nm. Chloroform was
used as the diluents and the mobile phase used was

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Acetonitrile:Methanol: Dichloromethane in the
ratio 700:100: 200.
Standard solutions were injected into the system
under the standard conditions mentioned above and
the chromatograms were recorded. Different
sample solutions were prepared and injected into
the system under the same chromatographic
conditions. The R.T of the peaks in the sample
chromatograms is compared with that of standard
chromatogram to identify the compounds. After
identifying the components, the amount of the
components as calculated. Amount of the
components present in the sample are calculated.
The amount of Gallic acid was found to me higher
in Choornam and Bark followed by Tablet. The
amount of 3,4, Dihydroxy Benzoic Acid was found
to me higher in Bark and Powder-2 followed by
Capsule. The amount of Caaffeic Acid was found
to me higher in Powder-2 followed by Bark. The
amount of Coumaric Acid was found to me higher
in Bark followed by Powder-1. The amount of
Coumaric Acid was found to me higher in Bark
followed by Powder-1. The amount of Ellagic Acid
was found to me higher in Choornam followed by
Bark. The amount of Chlorogenic Acid was found
to me higher in Powder-2 followed by Bark. After
all the analysis and calculations it was found that
BARK was found to be higher in most polyphenols
followed by Choornam.
SUMMARY AND CONCLUSION
The phytochemical screening of the samples has
been done and Rf values of the samples using TLC
(Thin Layer Chromatography) were determined.
HPLC of the polyphenols in various brands of
Terminalia arjuna were performed on Dionex
RPLC (Rapid Separation Liquid Chromatography)
instrument, which has chromelon software
installed. The column used for the analysis was
Waters SpheriSorb C18 150mm×4.6 5µm ODS2
Column with a flow rate of 0.5 ml/min. The
detector used to detect the samples was DAD
(Diode Array Detector) 450 nm. Chloroform was
used as the diluents and the mobile phase used was
Acetonitrile: Methanol: Dichloromethane in the
ratio 700: 100: 200. After all the analysis and
calculations it was found that BARK was found to
be higher in most polyphenols followed by
Choornam.
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