preliminary phytochemical screening, antifungal and

www.wjpps.com Vol 4, Issue 08, 2015.

1431

Kaur et al. World Journal of Pharmacy and Pharmaceutical Sciences

PRELIMINARY PHYTOCHEMICAL SCREENING, ANTIFUNGAL AND

ANTHELMINTIC ACTIVITIES OF BARK AND LEAVES EXTRACTS

OF FICUS BENGALENSIS LINN

*Arvinder Kaur, Jasreen Kaur, Maninderjeet Kaur, Gurdeep Kaur,

Rajinder Kaur, Manpreet Kaur, Amit Kapoor

G.H.G Khalsa College of Pharmacy, Gurusar Sudhar, Ludhiana, India.

ABSTRACT

Nystatin, was used as a standard drug for antifungal activity. The bark

and leaves of F. bengalensis were subjected to successive Soxhlet

extraction using different solvents in an increasing order of their

polarity viz starting from petroleum ether (60-80oC), chloroform,

methanol and then distilled water. Weighed quantity of the various

extracts, were subjected to preliminary phytochemical screening.

Antifungal activity of crude extract on Candida albicans and

Aspergillus niger fungal strains were analyzed by Agar diffusion cup

plate method. All the extracts viz Petroleum ether (PE), Chloroform

(CE), Methanol (ME) and Water (WE) were used in range of 25 mg/ml to 100 mg/ml for

screening antifungal activity. Piperazine is used as standard drug for Anthelmintic activity.

Both aqueous and methanol extracts of F. bengalensis in range of 50 mg/ml to 100 mg/ml

were screened for Anthelmintic activity. Antifungal activity is not observed by pet ether,

chloroform, methanol and water extracts in concentration range from 25 mg/ml to 100

mg/ml. The methanol extract showed potent anthelmintic activity at dose of 25 mg/ml than

water extract. F. bengalensis possesses good anthelmintic activity. The antifungal activity is

may be due to the presence of flavonoids and phenolic compoundspresent in whole plant.

KEYWORDS: Anthelmintic activity, Antifungal activity, Piperazine, Candida albicans,

Aspergillus niger, Extracts, F. bengalensis.

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Article Received on

11 June 2015,

Revised on 02 July 2015,

Accepted on 23 July 2015

*Correspondence for

Author

Arvinder Kaur

G.H.G Khalsa College of

Pharmacy, Gurusar Sudhar,

Ludhiana, India.


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INTRODUCTION

Traditional medicinal plants have a long history of therapeutic uses as described in Ayurveda,

Sidha and Unani. Importance of such plants is more now a days.

F. bengalensis has been traditionally used in the treatment of mental disorders, hypertension,

insomnia, diabetes, common cold, fever, epilepsy and as anti-aging agent. Further, F.

bengalensis has been the centre of interest to pharmacologists as it exhibits a variety of

pharmacological activities viz. anti-inflammatory, anticonvulsant, analgesic, antidepressant,

antiasthmatic, etc. Keeping in mind the traditional/ alternative and complementary medicinal

uses and diverse activity potential, F. bengalensis seems to hold a great potential for in depth

investigation for various biological activities, especially antifungal activity.[1,2]

The rationale

behind the study gets further strengthen by the fact that it is used in Ayurveda for treatment

of diarrhoea, piles, teeth and skin disorders.[3]

Variety of phytoconstituents are present in

different parts of F. bengalensis such as the bark contains leucopelargonidin-3-0-α-L

rhamnoside and leuco cynidin 3-0-α-D galactosyl cellobioside, glucoside, beta glucoside,

pentatriacontan-5-one, beta sitosterolalpha- D-glucose. The whole plant contain, crude

protein, crude fibres, CaO, phosphorous, rutin, friedelin, taraxosterol, lupeol, β-amyrin along

with psoralen, bergapten and β-sisterol, quercetin-3-galactoside. It also contains

leucodelphinidin derivative, bengalenoside, aglucoside, leucopelargonin and leucocynidin

derivatives. F. bengalensis also contain taraxasterol.[2]

So this plant was selected for anti-

fungal and anthelmintic activity.

Therefore, in lieu of above deliberations it was thought worthwhile to investigate antifungal

and anthelmintic activities by implementing the following plan of work.

1. Subjecting F. bengalensis to detailed preliminary phytochemical screening

2. Screening F. bengalensis for antifungal and anthelmintic activities

MATERIAL AND METHODS

Plant material

The bark and leaves of F. bengalensis were collected from HARI OM HERBS of Santinagar

Chhutmalpur in july 2012. The plant was authenticated by Dr. K. MADHAVA CHETTY, Sri

Venkateswara University, TIRUPATI and the plant specimen is kept at the Herbarium of

G.H.G Khalsa College of Pharmacy, Gurusar Sudhar, Ludhiana


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Standard drug

Nystatin, was used as a standard drug for antifungal activity. The drugs obtained were free

gift sample from Jackson Pharmaceuticals Ltd., Amritsar, India.

Solvents

Petroleum ether (60-80°C), Chloroform and Methanol were employed for extraction of plant

material using soxhlet apparatus and finally the drug was boiled with distilled water.

Dimethyl sulphoxide, was used as solvent for dissolving different extracts. It is colourless

liquid with boiling point 189°C. It is miscible with water, chloroform, acetone, alcohol and

petroleum ether.

Chemicals

Sodium hydroxide, Chloral hydrate, Copper sulphate, Ferric chloride, Sulphuric acid, Iodine,

Lead acetate, Magnesium, Pottasium iodide, Picric acid, Mercuric chloride, Nitric acid,

Gelatin, Sodium chloride were used for phytochemical screening of the plant extracts.

Preparation of extracts

The bark and leaves of F. bengalensis were dried in shade and coarsely powdered. Five

hundred gram powder material was subjected to successive Soxhlet extraction using different

solvents in an increasing order of their polarity viz starting from petroleum ether (60-80oC),

chloroform, methanol and then distilled water for not less than 48 hours. After each

extraction the powdered material was dried in air at room temperature. Finally, marc was

digested with distilled water for 24 hours or more to obtain aqueous extract. Each extract was

concentrated in vaccum using Rotatory evaporator. Extracts were weighed subsequently and

the percentage yields were calculated of each extract obtained individually in terms of the air

dried weigh of plant material.


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Powdered Drug

Soxhlet extraction Pet ether

Marc Petrolium ether extract

Soxhlet extraction Chloroform

Marc Chloroform extract

Soxhlet extraction Methanol

Marc Methanol extract

Digestion Water

Marc Aqueous extract

Phytochemical Screening

Weighed quantity of the various extracts, were subjected to preliminary phytochemical

screening using standard methods.[4]

All the extracts of F. bengalensis were screened for

different classes of phytoconstituents using specific standard reagents.[5]

Antifungal activity studies

Examination of antifungal activity of bark and leaves extracts of F. bengalensis were

accessed by studying inhibition effect of extracts on two fungal strains. Antifungal activity of

crude extract on fungal strains is analyzed by Agar diffusion cup plate method.

Microorganisms (fungal strains)

The fungal strains were collected from Sanjay Biologicals, Amritsar, Punjab.

Fungus : Candida albicans , Aspergillus niger

Maintenance of culture

Nutrient agar slant were made for maintenance of cultures and preserved at 4ºC in an

incubator. For further use of cultures, sub-culturing is done at regular intervals.


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Media used

1. Potato dextrose agar (PDA) media (HI MEDIA Laboratories, Mumbai)

Ingredients Gm/L

Potato infusion 200

Dextrose 20

Agar 15

Preparation of inoculum

For preparing inoculums the cell suspension method is used. In this method a loopful of

fungal strain was taken and suspended in a nutrient broth which is further incubated in

incubator shake for 6 hrs at 30°C at 300 rpm. This cell suspension was used as inoculum.

Preparation of test samples from dried residues

Variable concentrations of extracts were prepared by dissolving dried residues in DMSO for

testing inhibitory efficacy against selected fungal strains. Stock solutions of extracts were

diluted in DMSO to produce concentrations ranging from 100 mg/ml to 25 mg/ml.

Antifungal screening

Diffusion of extract through the cavity or cylinder in petriplate is preceded and inhibition of

growth of microorganisms is called zone of inhibition which is measured in millimetre. From

the inoculums, prepared as above 0.1 ml of each of C. albicans and A. niger suspension was

spread uniformly on PDA medium petriplates. Cultured plates were incubated at 28°C for

seven days. A well 8 mm is prepared for both fugal strains by using a sterilized steel cork

borer which cuts the cultured plates in appropriate size and filled with aliquot volume of

extracts samples using sterilised micropipette.[6]

Then cultured plates were then incubated in BOD incubator at 28°C up to 48 hours for C.

albicans and for A. niger. Diameter of any resultant zone of inhibition including well size

was measured. For each combination of extract preparation and organism, the plates were

kept in triplicate (n=3). DMSO was used as control and Nystatin was used as standard

drug.[7]

Drugs (Test samples)

All the extracts viz Petroleum ether (PE), Chloroform (CE), Methanol (ME) and Water (WE)

were used in range of 25 mg/ml to 100 mg/ml for screening antifungal activity.


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Anthelmintic activity

Experimental animals

Indian adult earthworms were collected from Sanjay Biologicals Museum, Amritsar, Punjab.

The earthworms were washed with normal saline to remove all faecal matter. The

earthworms of 3-5 cm in length and 0.1-0.2 cm in width were used for all experimental

protocol.

Standard Drug

Piperazine (Glaxo Smithkline Pvt. Ltd.) is used as standard drug.

Preparation of test samples from dried residues

DMSO (Merck, 2001) is used to prepare various concentrations of extracts for experimental

work. DMSO is used as control.


All the earthworms were divided into four groups each group contains six earthworms. The

standard drug solution and extract doses were freshly prepared before starting the experiment.

All earthworms were released into 10 ml of each respective solution’s (8). One group is in

5% DMSO, one group in different conc. of methanol solutions as 25 mg/ml, 50 mg/ml and

100 mg/ml of F. bengalensis, one group in different conc. of aqueous solutions of F.

bengalensis as 25 mg/ml, 50 mg/ml and 100 mg/ml and one group is in 10 ml of piperazine

solution. Time of paralysis and death is recorded respectively.

Drugs (Test samples)

Both aqueous and methanol extracts of F. bengalensis in range of 50 mg/ml to 100 mg/ml

were screened for Anthelmintic activity.

RESULTS

Preliminary phytochemical screening

Table 1: Preliminary phytochemical screening observed for bark and leaves extracts of

F. bengalensis

S.

no Phytochemical constituents

Pet ether

extract

Methanol

extract

Chloroform

extract

Water

extract

1.

Alkaloids

1. Mayer’s reagent

2. Hager’s reagent

3. Wagner’s reagent

4. Dragendorff’s reagent

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-


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2.

Phenolic compounds and Tanins

1. Fecl3

2. Lead acetate test

3. Bromine water test

-

-

-

+

+

-

-

+

+

-

+

-

3. Saponin

1. Frothing test

-

+

+

+

4.

Carbohydrates

1. Molisch test

2. Fehling’s solution A

2. Fehling’s solution B

-

-

-

+

+

+

+

-

-

+

+

+

5.

Protein and Amino acids

1. Millon’s test

2. Biuret test

3. Ninhydrin test

-

-

-

-

-

-

-

-

-

-

-

-

6.

Glycosides test

1. Borntrager’s test

2. Legal’s test

-

-

-

-

-

-

-

-

7.

Flavonoids test

1. Alkaline reagent test

2. Shinoda test

-

-

+

+

+

+

-

+

8.

Phytosterols test

1. Liebermann’s test

2. Libermann Burchard test

-

-

-

-

+

+

-

-

+ = present, - = absent

Antifungal activity

Fig 1: A Fig 1: B


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Fig 1: C Fig 1: D

Fig 23: Antifungal activity of extracts on A. niger

A – Chloroform extract

B – Methanol extract

C - Water extract

D – Standard drug and control DMSO (5%)

Fig 2: A Fig 2: B

Fig 2: C Fig 2: D


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Fig 24: Antifungal activity of extracts on C. albicans

A - Chloroform extract

B - Methanol extract

C – Water extract

D – Standard drug and control DMSO (5%)

Table 2: Antifungal activity of extracts observed for 48 hrs at an interval of 12 hrs

Activity after 12 hrs

Aspergillus niger Candida albicans

DMSO 5% 0 0

Standard (50 µg/ml) 17.2 16

Standard (250 µg/ml) 18.5 18.2

Standard (500 µg/ml) 20 20

Methanol (25 mg/ml) 0 0




Chloroform (25 mg/ml) 0 0




Water (25 mg/ml) 0 0






DMSO 5% 0 0


















DMSO 5% 0 0


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DMSO 5% 0 0
















Fig 3: Antifungal activity of extracts after 12 hrs.


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Table 3: Anthelmintic activity represent time of paralysis and time of death.

Time of paralysis (min) Time of death (min)

Control (DMSO 5%) 0 0

Standard (10 mg/ml) 25±2.8 64±6.2

Methanol (25 mg/ml) 52±5.4 65.1±5.3

Methanol (50 mg/ml) 21.3±4.8 33.1±4.5

Methanol (100 mg/ml) 11.5±3.2 17.3±4.1

water (25 mg/ml) 94.3±8.9 187.5±17

water (50 mg/ml) 50.6±6.1 103.3±10.4

water (100 mg/ml) 23±2.7 52.8±5.5

Fig 7: Anthelmintic activity of extracts.

DISSCUSION AND CONCLUSION

The present study was designed to study the antifungal and anthelmintic activity of bark and

leaves extracts of F. bengalensis.

Antifungal activity is not observed by pet ether, chloroform, methanol and water extracts in

concentration range from 25 mg/ml to 100 mg/ml. The methanol extract showed potent

anthelmintic activity at dose of 25 mg/ml than water extract. As the concentration of

methanol extract was increased from 25 mg/ml to 100 mg/ml the paralyses and death time of

earthworm decreased as compared to water extract. It was observe that methanol extract at a

dose of 100 mg/ml showed more anthelmintic activity than standard drug piperazine at a

concentration of 10 mg/ml.

So, it is concluded that F. bengalensis possesses good anthelmintic activity. The antifungal

activity is may be due to the presence of flavonoids and phenolic compounds.


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preliminary phytochemical screening, antifungal and

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