ACTIVITY OF CALOTROPIS GIGANTEA -...

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Volume- 1 Issue-4 (2014) ISSN: 2348 – 604X (Print); 2348 – 6058 (Online) © 2014 DAMA International. All rights reserved 15

STUDY OF PHYTOCHEMICAL CONSTITUENTS AND ANTIBACTERIAL

ACTIVITY OF CALOTROPIS GIGANTEA

Madhurima Dutta, Sarmistha Rej, Shahid Jamal,Sumanta Das and Sabyasachi Chatterjee*

Department of Biotechnology, The University of Burdwan, Rajbati, Burdwan - 713104, West Bengal, INDIA.

Email ID –[email protected]

ABSTRACT Plant materials are used throughout developed and developing countries as home remedies, over the counter drug

products and raw materials for the pharmaceutical industry and represent a substantial proportion of the global drug

market. It is therefore essential to establish internationally recognized guidelines for assessing their quality. Some

of quality control parameters of the leaves of Calotropis gigantea belonging to Apocynaceae family were

analyzed.Calotropis gigantea is an important Indian medicinal plant and widely used in Ayurveda for management

of various diseases.Different biochemical screening have been carried out to identify the important phyto-

constituents.A number of biological constituents in good yield and some have been shown to possess useful

biological actions belonging mainly to phenolics ,flavonoids,terpenoids.Extract of this plant possess useful

antimicrobial activities. The medicinal properties of this plant represent it as a valuable source of medicinal

compound. This study is collective information concerning the ethnobotany pharmacology, phytochemistry and

biological activities of the Calotropis gigantea.

KEY WORDS: Calotropis gigantea, Ethnobotany, Flavonoids, Medicinal plant, Phenolics, Phytoconstituents,

INTRODUCTION

Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide

variety of chemical compounds that are used to perform important biological functions, and to defend against attack

from predators such as insects, fungi and herbivorous mammals. At least 12,000 such compounds have been isolated

so far; a number estimated to be less than 10% of the total. Chemical compounds in plants mediate their effects on the

human body through processes identical to those already well understood for the chemical compounds in

conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work.

This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to

cause harmful side effects.(en.wikipedia.org/wiki/medicinal plant).

Plant materials remain an important resource to combat serious diseases in the world. The traditional medicinal

methods, especially the use of medicinal plants, still play a vital role to cover the basic health needs in the developing

countries. The medicinal value of these plants lies in some chemical active substances that produce a definite

physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids,

tannin, flavonoid and phenolic compounds. Within the recent years, infections have increased to a great extent and

antibiotics resistance effects become an ever-increasing therapeutic problem. Natural products of higher plants may

possess a new source of antimicrobial agents with possibly novel mechanisms of action. They are effective in the

treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated

with synthetic antimicrobials. Therefore, it is of great interest to carry out a screening of these plants in order to

validate their use in folk medicine and to reveal the active principle by isolation and characterization of their

constituents. Systematic screening of them may result in the discovery of novel active compounds. Calotropis

gigantea (Crown flower) is a species of Calotropis The giant milkweed, Calotropis procera, is native to the Old

World from the Arabian Peninsula to West, North and East Africa, also from Southern Asia to Malaysia. It has been

widely naturalized elsewhere including Australia, Hawaii, California, and the Caribbean and from Mexico to Brazil.

Plants are usually found scattered in dry coastal areas, on beaches, along roadsides, and in disturbed urban lots. The

plant is a soft-wooded, evergreen or semi-deciduous shrub or small tree. Leaves are concentrated near the growing

tips. Shrubs are often dense with leaves. As the plant becomes more treelike it produces an airy crown of a few

mailto:ID%20–[email protected]



twisted branches. The leaves are opposite, entire, and on extremely short petioles. The blades are broadly elliptic or

oblong to nearly orbicular. They have a short-pointed to blunt apex and a nearly clasping heart-shaped base. They are

3-8 inches long and 2-5 inches wide with a broad light yellow mid-vein and conspicuous main veins. The blades are

slightly leathery, light to dark green above and whitish green beneath being coated by fine soft hairs that are easy to

rub off. It is a large shrub growing to 4 m tall. It has clusters of waxy flowers that are either white or lavender in

color. Each flower consists of five pointed petals and a small, elegant "crown" rising from the centre, which holds

the stamens. The plant has oval, light green leaves and milky stem. The latex of Calotropis gigantea contains cardio

glycosides, volatile fatty acids and calcium oxalate. Calotropis is used as a traditional medicinal plant with unique

properties. Traditionally Calotropis is used alone or with other medicinals (Caius 1986) to treat common disease such

as fevers, rheumatism, indigestion, cough, cold, eczema, asthma, elephantiasis, nausea, vomiting, diarrhea (Das

1996). According to Ayurveda, the dried whole plant is a good tonic, expectorant, depurative, and anthelmintic. The

dried root bark is a substitute for ipecacuanha. The root bark is febrifuge, anthelmintic, depurative, expectorant, and

laxative. The powdered root used in asthama, bronchitis, and dyspepsia. The leaves are useful in the treatment of

paralysis, arthralegia, swellings, and intermittent fevers. The flowers are bitter, digestive, astringent, stomachic,

anthelmintic, and tonic (Agharkar 1991; Warrier et al. 1996). Calotropis is also a reputed Homoeopathic drug (Ghosh

1988; Ferrington 1990). The milk obtained from the leaves and barks are useful in treating corn on skin permanently.

MATERIALS AND METHODS

Collection of sample: Calotropis gigantea was collected from Golapbag area of The Burdwan District of West Bengal.

Preparation of extract:

The leaves and stem of the plant were collected and washed under running tap water to remove dust. The plant

samples (2g each) were crushed in motor pestle and dissolved in four different solvents (water, methanol, ethanol,

ethyl acetate). The solution then filtered and used for further phytochemical analysis.

Phytochemical analysis:

Qualitative, quantitative tests were performed for detection of phytochemical constituents of Calotropis gigantea

.Standard procedures as described by Kapoor et al., (1969), Harbone (1973, 1984), Smolenski et al., (1974), Boham

and Kocipal- Abyazan (1974), Van-Burden and Robison (1981), Trease and Evans (1996), Sofowara

(2008),Singleton et al., (1999), Edeoga, (2005) were used with some modifications to detect and estimate the

phytochemicals present in the different extract fractions of the plant.

1. Qualitative tests:

Leaf extracts in four different solvents (water, methanol, ethanol, ethyl acetate) were used for experimental purpose.

1.1. TEST FOR FLAVONOIDS: Leaf extract (500µl) was heated (80⁰-90⁰C) with 10 ml of ethyl acetate over a

steam bath for 3 minutes. The mixture was filtered and 4 ml of filtrated was shaken with 1 ml of dilute ammonia

solution and few drops of concentrated H2SO4was added. Yellow coloration of the solution shows positive

result(Fig:1).

1.2. TEST FOR ALKALOIDS: Leaf extract (500µl) was treated with 5 ml of aqueous 1%HCl solution on boiling

water bath for 20 minutes. The solution was centrifuged for 10 minutes at 3000 rpm and1 ml of supernatant was

treated with few drops of WAGNER’S reagent.(2 g of iodine and 6 g of KI dissolved in 100 ml of water).Reddish

brown color of solution shows positive result.

1.3. TEST FOR PHENOL: Leaf extract (500µl) was boiled with 2 ml of distilled water on water bath and filtered.

In filtrate 10% of ferric chloride solution was added. Blue black color of solution shows positive result (Fig: 2).

1.4. TEST FOR ANTHRAQUINONE: Leaf extract (500 µl) was shaken with 10 ml of benzene and filtered. In

filtrate 5 ml of 10% ammonia solution was added and mixed. Formation of violet and red color shows positive result

(Figure 3).



1.5. TEST FOR TERPENOIDS: Leaf extract (2 ml) was treated with 1 ml of 2, 4 dinitrophenyl hydrazine (DNP)

dissolved in 100 ml of 2M HCl. Yellow orange coloration of solution shows positive results(Fig:4).

1.6. TEST FOR TANNINS: Leaf extract (500µl) was boiled with 10 ml of distilled water and filtered. In the filtrate

1M Fecl3 was added. Formation of blue black precipitate shows positive result(Fig:7).

1.7. TEST FOR STEROIDS: Leaf extract (1ml) was treated with 2 ml of acetic anhydride and cooled in ice. The

solution mixed with 0.5 ml of chloroform and 1 ml of concentrated H2SO4.Formation of reddish brown ring shows

positive result(Fig:8).

1.8. TEST FOR SAPONINS: Leaf extract (500µl) was boiled in 5 ml of distilled water and filtered. 2.5 ml of

filtrate was mixed with 1.5 ml of distilled water and shake vigorously. Generation of stable and persistent froth shows

positive result.

1.9.TEST FOR CARBOHYDRATE: Few drops of Molisch’s reagent was added to leaf extracts, shaken vigorously

and then concentrated H2SO4 was added. Formation of purple ring at the interface shows positive result(Fig:5).

1.10. TEST FOR GLYCOSIDES: Leaf extract (500µl) was dissolved in pyridine solution. In this solution few

drops of 20% sodium nitroprusides and few drops of NaOH were added. Pink to red color shows positive

result(Fig:6).

1.11. TEST FOR PROTEINS: Leaf extract (500µl) was taken and 0.1 ml of Millions reagent was added. Brown

coloration shows positive result(Fig:9).

2. Quantitative test: Leaf extracts in four different solvents (water, methanol, ethanol, ethyl acetate) were used for

experimental purpose.

2.1. PHENOL TEST: Leaf extract (1ml), 2ml of distilled water and 0.5 ml of Folin-ciocalteu’s reagent were taken in

a test tube. After 3 minutes 2 ml of 20% sodium carbonate solution was added. The tubes were kept in boiling water

bath for 1 minute and cooled .The solution was diluted one fold and O.D was taken at 680 nm (Jadhav.et al.,

2012)(Fig:10).

2.2. FLAVONOIDS TEST: Leaf extracts (1ml) was mixed with 4 ml of distilled water and 0.3 ml of 5%

NaNo2.After 5 minutes 0.3 ml of 10% Alcl3 was added. Then 2 ml of 1M NaOH was added. The solution was

diluted and O.D measured at 520 nm.(Fig:11)

3. Determination of antibacterial activity using agar cup method:

3.1. Antibacterial activity test by agar cup method: The crude extracts were screened for antibacterial activity

using agar well diffusion method described by Russel and Furr (1977).Agar cup method was followed in which

culture of Bacillus subtilis and E coli were spreaded out onto Muller Hinton agar plates. Wells were made on the

plates with a cork borer(diameter 1.2 cm) to which different extraction were added in specific volume (1ml) along

with controls (sterile water ,methanol,ethanol,ethyl acetate).All the plates were incubated for 24 hours at 370C.

3.2. Comparison of antibacterial activity of leaf and stem extract of Calotropis gigantea using agar cup assay:

The agar cup method was done to compare the antibacterial activity of leaf and stem extracts of Calotropis gigantea.

4.The DNA was isolated from plant leaves(Calotropis gigantea.) by BARC (BHABA ATOMIC RESEARCH

CENTRE) (2003) method, developed by E.Nalini, N.Jawali and S.G .Bhagwat.

RESULT AND DISCUSSION

This study has revealed the presence of phytochemicals considered as active medicinal chemical constituents.



Fig 1: Test for flavonoids Fig 2: Test for phenol

Fig 3: Test for Anthraquinone Fig 4: Test for terpenoid

Fig 5: Test for carbohydrate Fig 6: Test for Glycosides

Fig 7: Test for tannins Fig 8: Test for steroids

Fig 9: Test for Protein

Important medicinal phytochemicals such as terpenoids, phenol, flavonoids, carbohydrate, protein, alkaloids and

tannins were present in studied experimental plants. The result of the phytochemical analysis shows that the four

medicinal plants ( Calotropis gigantea, Clerodendrum infortunatum, Solanum nigrum and Ricinus communis) are



rich in at least one of alkaloids, flavonoids, terpenoids, phenol, steroids and tannins. The phytochemical screening

and qualitative estimation of four medicinal plants (Clerodendrum infortunatum Calotropis gigantea, Solanum

nigrum and Ricinus communis) showed that the leaves were rich in phenol, tannins, terpenoid, flavonoids, alkaloids

etc (Table no:1- 4).

Table1: Presence of phytochemical constituents in aqueous extraction

phytochemicals Calotropis

gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

Communis

Flavonoids -ve -ve -ve -ve

Alkaloids +ve +ve +ve +ve

Phenol +ve +ve +ve ++ve

Anthraquinone +ve +ve +ve +ve

Terpenoids +ve +ve +ve ++ve

Tannins +ve +ve +ve +ve

Steroids +ve +ve -ve +ve

Saponins +ve +ve +ve +ve

Carbohydrate +ve +ve +ve +ve

Glycosides +ve +ve +ve +ve

Protein +ve +ve +ve +ve

++ve: Strong positive, +ve: Positive,-ve: Negative

Table2: Presence of phytochemical constituents in methanolic extraction

Phytochemicals Calotropis

gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

Communis




Anthraquinone -ve -ve -ve -ve


Tannins +ve +ve +ve +ve

Steroids +ve +ve +ve +ve

Saponins -ve -ve -ve -ve



Proteins +ve +ve +ve +ve

++ve: Strong positive, +ve: Positive, -ve: Negative

Table 3: Presence of phytochemical constituents in ethanolic extraction




gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

Communis

Flavonoids +ve +ve +ve +ve


Phenol ++ve ++ve ++ve ++ve


Terpenoids ++ve ++ve ++ve ++ve

STannins +ve +ve +ve +ve

Steroids -ve +ve +ve +ve

Saponins -ve -ve -ve -ve




++ve: Strong positive, +ve: Positives,-ve: Negative

Table4: Presence of phytochemical constituents in ethyl acetate extraction


gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

Communis






Tannins -ve -ve -ve -ve

Steroids -ve -ve -ve -ve

Saponins +ve +ve +ve +ve




++ve: Strong positive, +ve: Positive, -ve: Negat

Table: 5 Quantitative estimation of phenol



Fig10: Determination of concentration of Phenolic compound

Table 6: O.D. Value at 680 nm

EXTRACON O.D VALUE AT 680 nm

Calotropis

gigantea

Cleorodendrum

infortunatum

Solanum

nigrum

Ricinus

communis

Aqueous 0.17 0.15 0.63 0.71

Methanolic 0.18 0.36 0.63 0.65

Ethanolic 0.12 1.01 0.23 0.77

Ethyl acetate 0.13 0.06 0.40 0.27

0

0.05

0.1

0.15

0.2

0.25

0.3

100

300

500

700

Aqueo

us

Met

han

oli

c

Eth

anoli

c

Eth

yl

acet

ate

O.D at

680nm

Concentration of

phenolic

compound(µg/ml)

O.D

at

680nm

100 0.168

300 0.198

500 0.229

700 0.267

Aqueous 0.17

Methanolic 0.18

Ethanolic 0.12

Ethyl acetate 0.13



Fig 11: Quantitative estimation of phenolic compounds

Table7: Quantitative estimation of flavonoids

Concentration of

flavonoids(µg/ml)

O.D at

520nm

500 0.125

1000 0.236

1500 0.375

2000 0.449

Aqueous NIL

Methanolic 0.03

Ethanolic 0.03

Ethyl acetate 0.15

Fig 12: Determination of concentration of flavonoids

00.20.40.60.81

1.2 Calotropis

gigantea

Clerodendrum

infortunatum

Solanum nigrum

Ricinus

communis

0

0.1

0.2

0.3

0.4

0.5

O.D at 520nm



Effect of leaf extract of Calotropis gigantea on bacteria (Bacillus subtilis):

Fig14: Zone of inhibition of leaf extract Fig15: Zone of inhibition of leaf extract

(ethanol) (water)

Fig16: Zone of inhibition of leaf extract Fig17: Zone of inhibition Of leaf extract

(methanol) (ethylacetate)

Fig 18: Zone of inhibition of ethanolic leaf extract against E.coli.

Table 8: O.D. Value at 520 nm



EXTRACTON O.D VALUE AT 520 nm

Calotropis

gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

communis

Aqueous NIL NIL 0.02 0.01

Methanolic 0.03 0.04 0.10 0.02

Ethanolic 0.03 0.17 0.2 0.07

Ethyl acetate 0.15 0.07 0.19 NIL

Fig13: Quantitative estimation of flavonoids

Table9: DETERMINATION OF ANTIBACTERIAL ACTIVITY OF LEAF EXTRACTS BY AGAR CUP

METHOD

Table: 10. Zone of Inhibition c.m.

0

0.05

0.1

0.15

0.2

Calotropis gigantea

Clerodendrum

infortunatum

Solanum nigrum

Ricinus communis

EXTRACTION Bacterial

culture

ZONE OF INHIBITION(cm)

Bacillus

subtilis

Calotropis

gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

communis

Aqueous 1.5 1.25 4 3.5

Methanolic 1.4 1.2 1.

6

1.5

Ethanolic 2.0 2.2 1.

3

2.5

Ethyl acetate NIL NIL NI

L

NIL



Fig19:Comparison of zone of inhibition of leaf and stem extract (ethanol) of Calotropis gigantea

Table 11: Comparative study of ethanolic leaf and stem extracts

PLANT

NAME

Bacterial

culture


ETHANOLIC

STEM

EXTRACT

ETHANOLIC

LEAF

EXTRACT

MIXTURE OF

STEM LEAF

EXTRAC(1:1)

Calotropis

gigantea

Bacillus

subtilis

1.7 1.8 1.6

Clerodendrum

infortunatum

1.5 1.7 2.2

Solanum

nigrum

1.5 1.7 0.0

Ricinus

communis

2.1 2 2.8

EXTRACTION Bacterial

culture


E coli

Calotropis

gigantea

Clerodendrum

infortunatum

Solanum

nigrum

Ricinus

communis

Ethanolic 2.8 3.3 4.5 3



Fig 20: Agarose gel electrophoresis (0.8%)

By qualitative analysis of leaf extract of Calotropis gigantea was shown that the extract contain alkaloids, phenol,

terpenoid, tannins, carbohydrate. But, all the crude extracts from leaf samples of Calotropis gigantea ,Clerodendrum

infortunatum, Solanum nigrum and Ricinus communis. did not show any colour change for flavonoids test (Table

no:1, Fig no:1), only ethanolic leaf extracts of Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and

Ricinus communis shown positive result for flavonoid test(Table no:3,Fig no:1) However, methanol crude extracts

from fresh leaves showed negative result for flavonoids, saponin and anthraquinone (Table no:2, Fig no:3) but

showed positive test for terpenoid, phenol, steroids, alkaloids (Table no:2, Fig no:4,2,8).The ethanolic leaf extract of

Calotropis gigantea shows positive results in flavonoids,phenol,alkaloids,terpenoids,tannins, and carbohydrate test

except anthraquinone,steroids,saponins test(Table no:3).

It was observed from the result that the methanolic leaf extract contain higher amount of phenol compared with other

extracts of Calotropis gigantean (Fig no. 10)According to standard graph we can determine concentration of phenol

in methanolic leaf extract and it was near about 300 µg/ml (Table no.5). In the quantitative test of flavonoids it was

observed from the result that the ethyl acetate leaf extract contain higher amount of flavonoids compared with other

solvent extracts of Calotropis gigante (Fig no:12)According to the standard graph of flavonoids compounds we can

determine the concentration of flavonoid in ethanolic leaf extract which was greater than 500 µg/ml (Table no:7).

From the above study it can be concluded that the studied experimental plants showed strong antibacterial activity

against some bacteria. The antibacterial activity of (water, ethanolic, methanolic, ethyl acetate) extracts of

Calotropis gigantea are given in(Table no: 9 and 10) (Fig no 14-17).From the results it was found that ethanolic

extracts exhibited significant antibacterial activity compare to the other extraction of Calotropis gigantea,

Clerodendrum infortunatum and, Solanum nigram, Ricinus communis against Bacillus subtilis (Table no:9).Aqueous

extract of Calotropis gigantea showed more inhibitory zone as compared to aqueous extracts of Clerodendron

infortunatum(Table no:9). Ethanolic extract of Calotropis gigantea also exhibited significant antibacterial activity



against E.coli (Table no:10, Fig no:18).When the leaf and stem extracts were mixed together in equal concentration

(1:1), it shows less inhibitory zone as compared to other individual extracts (table no:11). From these results we can

conclude that some of the component from the mixture of leaf and stem extracts exhibit the synergistic action against

bacteria.

The DNA was isolated and confirmed by agarose gel electrophoresis (Fig no: 20). The extracted DNA sample were

kept for further amplification and sequencing to check the homogeneity and phylogenetic relationship among the

different species of the same plant from the Apocynaceae family. Different bioinformatical tools such as BLAST

,FASTA, ClustalW were used to find out phylogenetic relationship. By column Chromatography partial purification

of ethanolic leaf extract was done and collected fraction was kept for further experiment like HPLC/ Gas

Chromatography, Mass spectroscopy etc.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the Department of Biotechnology, Department of Environmental Science, The

University Science Instrumentation Centre, The University of Burdwan for support and providing infrastructural

facility. The authors also acknowledge DBT, Govt. of India for funding this work.

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