Phytochemical screening and Polyphenol estimation by HPLC of Terminalia arjuna

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* Corresponding author: Ramakrishna U.V E-mail address: [email protected]

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.V

1,*, Dinesh kumar B

2, Sukesh Narayan Sinha2,

Ratna Priya2

*,1Department of Pharmaceutical Analysis & Quality Assurance, Bapatla College of Pharmacy,

Bapatla, A.P. India. 2Food 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

Ramakrishna U.V, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [471-482]

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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.



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.



POLYPHENOLS TABLET HPLC

TA

BL

ET

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

CA

PS

UL

E

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



PO

WD

ER

-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


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

PO

WD

ER

-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


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

CH

OO

RN

AM

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


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.



POLYPHENOLS BARK HPLC

BA

RK

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


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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Phytochemical screening and Polyphenol estimation by HPLC of Terminalia arjuna