article_wjpps_1391274018

8/17/2019 article_wjpps_1391274018

1/13

www.wjpps.com Vol 3, Issue 2, 2014. 1899

Chaudhari

et al. World Journal of Pharmacy and Pharmaceutical Sciences

EXTRACTION AND PHARMACOGNOSTICAL EVALUATION OF

QUERCETIN FROM THE LEAVES OF FICUS BENGALENSIS

Sugandha G. Chaudhari1*

, Preetesh A. Mishra2, Harsha S. Chaudhari

2, Jigar S. Desai

2,

Aditya Adam2, Archana Shendkar

2

1Assistant Professor, Department of Pharmacology, Dr. L.H. Hiranandani College of

Pharmacy, Ulhasnagar.

2Student (M.pharm), Department of Pharmacology, Dr. L.H. Hiranandani College of

Pharmacy, Ulhasnagar.

ABSTRACT

Ficus bengalensis (FB) (Moraceae) is a very common plant species

found in India. Literature survey indicates the presence of some

important phytochemicals like, ketones, flavonoids, rutins, flavonols,

sterols, triterpenes and triterpenoids, furocoumarin and tiglic acid ester,

in the leaves. This research study was performed to obtain quercetin

from the hydro-alcoholic extract of leaves the of Ficus bengalensis

containing rutins. Presence of quercetin in the extract was confirmed

by using various phytochemical screening methods. Final extract was

also assessed quantitatively for the total amount of quercetin present

(% Yield).

KEYWORDS: Ficus bengalensis, Quercetin, rutins, leaf, hydro-

alcoholic, extraction.

INTRODUCTION

Ficus bengalensis (FB) (Moraceae) is commonly known as Banyan tree or Vata or Vada tree

in Ayurveda. There are more than 800 species and 2000 varieties of Ficus species, most of

which are native to the old world tropics.[1] It is endemic to Bangladesh, India and Sri Lanka.

It is also known as Bengal fig, Indian fig and East Indian fig, Indian Banyan or simply

Banyan (English), also borh, nyagrodha (Sansikrat), Bat, Bargad and Bar (Hindi). The

English name Banyan is given by the Britishers to this tree because under the tree Banias that

WWOORRLLDD JJOOUURRNN A A LL OOFF PPHH A A RRMM A A CC Y Y A A NNDD PPHH A A RRMM A A CCEEUUTTIICC A A LL SSCCIIEENNCCEESS

V V oolluummee 33,, IIssssuuee 22,, 11889999--11991111.. RReesseeaarrcchh A A rrttiiccllee IISSSSNN 2278 – 4357

Article Received on15 November 2013,

Revised on 21 December2013,Accepted on 24 January2014

*Correspondence for

Author:

Sugandha G. Chaudhari,

Assistant Professor,

Department of Pharmacology,

Dr. L.H. Hiranandani College

of Pharmacy, Ulhasnagar,

India.

8/17/2019 article_wjpps_1391274018

2/13


Chaudhari


is, the Hindu merchants used to assemble business. The triad Ganges, the Himalayas and the

Banyan tree symbolize the images of India, for this reason it is considered as National Tree.

Ficus means fig and bengalensis means belonging to or is of Bengal. [2] It is a member of four

sacred trees Nalpamara (Ksirivrksas) meant to be planted around the home and temples.[3]

Phytochemical investigation of F. bengalensis led to the exploration of a wide variety of

constituents which are responsible for its wide range of pharmacological activities. They

include ketones, flavonoids, rutins, flavonols, sterols, triterpenes and triterpenoids,

furocoumarin and tiglic acid ester. Rutin (quercetin-3-O-rutinoside), a glycoside of the

flavonoid quercetin, gives quercetin on hydrolysis.

METHODOLOGY

I.

Collection Of Plant Material

The leaves of plant Ficus bengalensis (Family- Moraceae) were collected from the botanical

garden of Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar, Maharashtra, India in the

month of September and October 2012. The leaves were authenticated at the Blatter

Herbarium (Blatter Herbarium Specimen Shah- 6555 of G. L. Shah), St. Xavier’s College,

Mumbai. The fresh leaves were washed to free them from any dust particles and

contaminants, and were subsequently dried at room temperature for 15-20 days. When the

leaves were sufficiently dry and easily crushable with hands, they were subjected to a grinder

to obtain a powdered form of the leaves. This powder was stored in an air tight container for

the further extraction process.

II. Extraction- Rutin[4]

Around 15-20 grams of the leaf powder was taken in a 250 ml R.B.F (Round Bottom Flask)

and was suspended in 30% ethanolic solution. This mixture was refluxed and subjected to

agitated maceration at 60ºC for 2 hrs in order to extract rutin in the solvent. The extraction

procedure was carried out on the magnetic stirrer so as to assist the agitation with the help of

magnetic needle, while the plate of the stirrer provided the required heat. The powders were

extracted twice for efficient extraction of rutin. Filtration was done using vacuum in the

Buchner’s flask with whattmann filter paper. The filtrate was used for further steps of

extraction.

III. Extraction- Quercetin[5]

Rutin extracted in the hydro-alcoholic solvent (filtrate) was to be hydrolysed into quercetin.

The filtrate was therefore refluxed with 5% HCl for 2 hrs in order to perform Total Acid

8/17/2019 article_wjpps_1391274018

3/13


Chaudhari


hydrolysis of the rutin present within. The hydrolysate thus obtained, was further extracted

with ethyl-acetate in separating funnel to dissolve the flavonoid- quercetin in the organic

solvent. After vigorous shaking of the mixture (hydrolysate solution and ethyl acetate

solution), and settling of the ethyl-acetate layer (upper layer in the separating funnel), the

ethyl-acetate fraction was collected in the petri-plates and the volatile solvent was allowed to

evaporate. The dried extract left behind was the portion of the hydro-alcoholic leaf extract of

Ficus bengalensis supposed to be containing quercetin in it. This dried Hydro-alcoholic Ficus

bengalensis Leaf Extract (HFBLE) was stored in an air-tight container, and was further

subjected to phytochemical and pharmacological screening.

IV. Flow Chart-

Collection of fresh leaves of Ficus bengalensis

Washing, and drying of leaves at room temperature

Grinding of dried leaves to prepare powder

Powder suspended in hydro-alcoholic solvent (30% ethanolic), agitated maceration- 2 hrs,

60ºC.

Remaceration of the used powder under same conditions for efficient extraction of rutin

Filtration

Acid hydrolysis of the filtrate using 5% HCl for 2 hrs, to hydrolyze rutin into quercetin

Hydrolystae extracted with the ethyl acetate solution in a separating funnel

Quercetin containing ethyl acetate (upper layer) is collected in the petri-plate from the

separating funnel.

The volatile ethyl acetate is allowed to evaporate, and the remaining powder is dried and

collected

Dried hydro-alcoholic extract is stored in air-tight container for further use.

8/17/2019 article_wjpps_1391274018

4/13


Chaudhari


PHYTOCHEMICAL SCREENING

a. Test For The Presence Of Different Compounds

Test for Tannins[6]

Ferric Chloride Test

Procedure: A few drops of 1% neutral ferric chloride solution were added to the solution of

the extract, and observation was recorded.

Test for Saponins[7, 8]

Foam test

Procedure: Small amount of extract was shaken with little quantity of water, and the

observation was recorded.

Test for Flavonoids[9, 10]

Shinoda test

Procedure: To the alcoholic solution of extract a few fragments of magnesium ribbon and

concentrated hydrochloric acid was added. The solution was warmed in the hot water bath,

and the observation was recorded.

Ferric chloride test

Procedure: Few drops of neutral ferric chloride solution were added into the solution of

extract.

Lead acetate test

Procedure: To the extract, a few drops of aqueous basic lead acetate solution were added.

Alkaline reagent test/ NaOH test:

Procedure: To the alcoholic solution of extract a few drops of sodium hydroxide solution

were added.

Test for Sterols[11]

Salkowski test

Procedure: Few drops of concentrated sulphuric acid were added to the solution of extract,

shaken and allowed to stand.

Liebermann-Burchard test

Procedure: To the solution of extract, few drops of acetic anhydride was added and mixed

well. 1 mL of concentrated sulphuric acid was added from the sides of the test tube.

8/17/2019 article_wjpps_1391274018

5/13


Chaudhari


Test for glycosides[8]

Keller – Killiani test:

Procedure: 2 ml of the extract, 3 ml of glacial acetic acid and 1 drop of 5 % ferric chloride

was added. This solution was carefully transferred to the surface of 2 ml conc. H2SO4 and the

observation was noted down

Test for Alkaloids[8]

Wagner’s test:

Procedure: The extract was treated with few drops of Wagner’s reagent.

Mayer’s test

Procedure: The extract was treated with few drops of Mayer’s reagent.

Dragendorff’s reagent

Procedure: The extract was treated with few drops of Dragendorff’s reagent.

Hager’s test

Procedure: The extract was treated with few drops of Hager’s reagent.

Test for Carbohydrates:[8]

Small amount of extract was dissolved in little quantity of

distilled water and filtered separately. The filtrate was used to test presence of carbohydrates.

Molisch’s test

Procedure: The extract was treated with Molisch reagent and concentrated sulphuric acid was

added from the sides of the test tube to form a layer.

Fehling’s test

Procedure: Filtrates were hydrolyzed with dilute hydrochloric acid, neutralized with alkaliand heated with equal amount of Fehling’s A and B solutions.

Test for Resins[11]

Procedure: Dissolved the extract in acetone and pour the solution in to distilled water.

b. IR Profiling Of The Ficus Bengalensis Hydro-alcoholic Leaf Extract

The extract was assessed for its contents by using FTIR (Fourier Transform Infrared

Spectroscopy) Spectroscopy.

8/17/2019 article_wjpps_1391274018

6/13


Chaudhari


c.

HPTLC Profiling Of The Ficus Bengalensis Hydro-alcoholic Leaf Extract

I. Preparations

i. Standard Quercetin- 10 mg of standard quercetin was dissolved in 10 ml HPLC grade

methanol. 1 ml of this solution was diluted and made up to 10 ml using methanol (1:10). This

solution served as ‘Standard solution’ for HPTLC.

ii. Ficus bengalensis Hydro-alcoholic Leaf Extract (FBHLE) - 100 mg of the extract

powder was dissolved in 5 ml (Ethanol: Water, 4:1) solution, using a water-bath sonicator for

15 minutes. This solution served as ‘Sample solution’ for HPTLC.

II. Application Parameters

i. Solutions

-2 µl and 5 µl of Standard solution.

- 5 µl and 10 µl of FBHLE solution.

ii. Band length: 8 mm.

iii. HPTLC plate: 10X10 Silica Gel 60 F254 HPTLC Plate.

iv. Instrument: Hamilton Syringe and CAMAG Linomat 5 instrument.

v. Mobile Phase:[12] Ethyl Acetate: Formic acid: Glacial Acetic Acid: Water

(10:0.5:0.5:1.3).

vi. Visualizer: CAMAG Visualizer : 150503.

vii. Camera: Digital camera DXA252: 306921208, 16 mm scanner & Lens f4.0

viii. Wavelengths: 254nm, 366nm, and visible light.

QUANTIFICATION OF QUERCETIN IN THE FICUS BENGALENSIS

HYDROALCOHOLIC LEAF EXTRACT (FBHLE)

I. Dilutions

Standard quercetin- 10 mg of standard quercetin was dissolved in 10 ml HPLC grade

methanol. 1 ml of this solution was diluted and made upto 10 ml using methanol (1:10). This

solution served as ‘Standard solution’ for HPTLC.

Ficus bengalensis Hydro-alcoholic Leaf Extract (FBHLE) - 100 mg of the extract powder

was dissolved in 5 ml (Ethanol: Water, 4:1) solution. This solution served as ‘Sample

solution’ for HPTLC.

II. Calculations

Calculations were done in consideration with the total AUC (Area Under Curve) obtained

from the HPTLC plate analysis.

8/17/2019 article_wjpps_1391274018

7/13


Chaudhari


RESULTS & DISCUSSION

I. Phytochemical Tests

Table 1: Phytochemical Tests Results.

Sr. No. Test Observation Inference

1 Test for tannins Blackish blue

coloration

+

2 Test for Saponins Foam produced

persists for 10 min

+

3 Test for Flavonoids

Shinoda test Red to pink

coloration

+

Ferric chloride test Blackish green

coloration

+

Lead acetate test Yellow precipitate

formed

+

Alkaline reagent test/ NaOH

test

Intense yellow color

which disappearedafter adding dilute

HCl

+

4 Test for sterols

Salkowski test Appearance of red

color in lower layer+

Liebermann-Burchard test Appearance of

reddish brown ring

+

5 Test for Glycosides No colored ring at

the interface

-

6 Test for Alkaloids

Wagner’s test Formation of reddish

brown precipitate

+

Mayer’s test Formation of creamywhite precipitate

+

Dragendorff’s reagent Formation of reddish brown precipitate

+

Hager’s test Formation of yellow

precipitate

+

7 Test for carbohydrates Molisch’s test A reddish violet ring +

Fehling’s test No formation of

precipitate

+

8 Test for resins Turbidity observed +

Indications: (+) Present, (-) Absent

Thus, the hydro-alcoholic leaf extracts of Ficus bengalensis contain tannin, saponin,

Flavonoids, sterols, alkaloids, carbohydrates, and resins in it.

8/17/2019 article_wjpps_1391274018

8/13


Chaudhari


I.II. IR Profiling Of The Ficus Bengalensis Hydro-Alcoholic Leaf Extract

Table 2: Peak positions obtained in the FTIR (Fourier Transform Infrared

Spectroscopy) spectrum.

Sr. No. Peak Position (cm-

) Probable Inter-atomic bond[13]

Standard Extract

1 3404.36 3402.43 O-H stretching vibration of phenols

2 1662.64 1662.64 C=O aryl ketonic stretch

3 1610.56 1608.63 C-----C aromatic ring stretch

4 1379.10 1386.82 In plane O-H bending of phenols

5 1319.31 1319.31 In plane bending of C-H bond in

aromatic hydrocarbon

6 1261.45 1288.45 C-O stretch of aryl ether

7 1197.79 1203.58 C-O stretch of phenol

8 1168.86 1153.43, 1114.86 C-CO-C stretch and bending in ketone

9 941.26, 823.60,680.87

819.75, 677.01,599.86

Out of plane C-H bending of aromatichydrocarbon

I.III. HPTLC Profiling Of The Ficus Bengalensis Hydro-Alcoholic Leaf Extract

Table 3: Data obtained from the HPTLC plate.

INDICATION SOLUTIONVOLUME

(µl)

CONCENTRATION

(mg/ml)

AUC (Area

UnderCurve)

MAX

Rf

Q2 Quercetin 2 0.1 907.3 0.70

Q5 Quercetin 5 0.1 3853.5 0.71

E5 Extract 5 20 19574 0.72

E10 Extract 10 20 25263 0.73

Table 4: Appearance of the bands.

Sr. No. Wavelength Appearance

Standard Quercetin Quercetin in the Extract

1 254 Brownish black band Brownish black band

2 366 Greyish green band Reddish brown band

3 Visible light Yellowish band Yellowish band

Thus, Rf values and the images of the HPTLC plates confirms the presence of quercetin in

the Ficus bengalensis Hydro-alcoholic Leaf Extract (FBHLE).

II. Quantification of Quercetin in the Ficus Bengalensis Hydro-alcoholic Leaf Extracts

(FBHLE).

Calculations

Standard solution- 2 µl of Standard solution applied on the HPTLC plate contained 0.2 µg of

quercetin, and similarly, 5 µl of Standard solution applied on the HPTLC plate contained 0.5µg of quercetin in it.

8/17/2019 article_wjpps_1391274018

9/13


Chaudhari


Table 5: AUC of Standard quercetin solution and FBHLE solution.

SOLUTION VOLUME (µl)

CONCENTRATION

OF QUERCETIN

(µg)

AUC (Area Under Curve)

OBTAINED

Standard solution 5 0.5 3853.5

Sample solution 5 Unknown 19574

Table 6: Concentration of quercetin in the FBHLE extract.

Quantity of Extract Amount of Quercetin present

5 µl (0.005 ml) 2.539 µg

100 gms 2.539 gms

RESULT- Hence, Ficus bengalensis Hydro-alcoholic Leaf Extract (FBHLE) contains 2.539

% w/w Quercetin in it.

IMAGES

Figure 1: IR spectra of the Ficus bengalensis Extract:

8/17/2019 article_wjpps_1391274018

10/13


Chaudhari


Figure 2: IR spectra of the Standard Quercetin

Figure 3: Overlapped IR spectra of the Ficus bengalensis Extract + and the Standard

8/17/2019 article_wjpps_1391274018

11/13


Chaudhari


Quercetin

HPTLC Plate Images-

Figure 4 Figure 5 Figure 6

In all the HPTLC Plate images, Q2: Quercetin 2 µl, E5: Extract 5 µl,

Q5: Quercetin 5 µl, E10: Extract 10 µl

Figure 4: HPTLC Plate under the wavelength 254 nm.

Figure 5: HPTLC Plate under the wavelength 366 nm.

Figure 6: HPTLC Plate under the visible light.

CONCLUSION

As indicated by the literature and previous research works, present study too confirms the

presence of Flavonoids in the leaves of Ficus bengalensis. The extraction was mainly planned

to obtain the quercetin from the leaves of the Ficus bengalensis, present in the bound form

(rutins). The phytochemical results and (Table 1), IR (Table 2, Figure 1-3) and HPTLC

(Table 3-6, Figure 4-6) profiling of the extract confirmed the success of the method employed

for obtaining quercetin.

8/17/2019 article_wjpps_1391274018

12/13


Chaudhari


ACKNOWLEDGEMENT

The authors are thankful to Hyderabad (Sind) National Collegiate Board and Dr. P.S. Gide,

Principal, H(S)NCB’s Dr. L. H. Hiranandani College of Pharmacy for their constant support

and Guidance.

REFERENCES

1. Manoj A, Urmila A, Bhagyashri W, Meenakshi V, Akshaya W, Kishore NG.

Anthelmintic activity of Ficus bengalensis. Int. J. Green Pharm., 2008; 2(3): 170-172.

2. Patil VV, Pimprikar RB, Patil VR. Pharmacognostical studies and evaluation of anti-

inflammatory activity of Ficus bengalensis. J. Young Pharm., 2009; 1: 49-53.

3. The Wealth of India, Volume- (F-G). In: A Dictionary of Indian Raw Materials and

industrial products. Vol. 4. New Delhi: Council of Scientific and Industrial Research,

1999: 24- 26.

4. Marian Naczk, Fereidoon Shahidi. Phenolics in cereals, fruits and vegetables:

Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis,

2006; 41: 1523–1542.

5. Monika Olszewska. Flavonoids From Prunus Serotina Ehrh. Acta Poloniae

Pharmaceutica-Drug Research, 2005; 62(2): 127-133.

6.

CK Kokate. Practical Pharmacognosy. 4th edition, New Delhi, India; Vallabh Prakashan:

1994.

7. A Sofowara. Medicinal plants and traditional medicine in Africa. New York; Chichester

John Wiley & Sons: 1993.

8. GE Trease, WC Evans. Pharmacognosy, 15th edition, London; WB Saunders Publishers:

2002.

9. K Peach, MV Tracey. Modern methods of plant analysis, Vol 3. Berlin, Springer Verlag:

1956.10. AM Rizk. Fitoterapia, 1982, 52(2), 35-42.

11. RD Gibbs. Chemotaxonomy of Flowering Plants, Vol.1. Montreal and London, McGill

Queen’s University Press: 1974.

12. Tania Paul and Suchitra Banerjee; Invitro evaluation of α-amylase inhibitory activity &

antioxidant potential of Pteris vittata with special reference to its HPTLC profile;

International Journal of Pharma and Bio Sciences; 2013 Apr; 4(2): (P) 494 – 503.

8/17/2019 article_wjpps_1391274018

13/13


Chaudhari


13. A. Chourasiya, A. Upadhayay and R.N. Shukla, Isolation of quercetin from the leaves of

Azadirachta indica and anti-diabetic study of the crude extracts, J Pharm Biomed Sci.

2012, December; 25 (25); 179-181.

Date post:	06-Jul-2018
Category:	Documents
Upload:	bindu
View:	217 times
Download:	0 times

article_wjpps_1391274018

Documents