Indo American Journal of Pharmaceutical Research, 2015
ISSN NO: 2231-6876
PHYTOCONSTITUENTS FROM AERIAL ROOTS OF FICUS BENGHALENSIS LINN
Jain Saloni Jayeshkumar*, Khan Tabassum
SVKM’s Dr. Bhanuben Nanavati College of Pharmacy,Gate No.1, Mithibai College Campus,V.M. Road ,Vile Parle (West),Mumbai
400 056.
ARTICLE INFO
Article history
Received 24/10/2015
ARTICLE
INFO
Available
online
Article
history
31/10/2015
Received
xxx
Available
Keywordsonline
xxx
Ficus Benghalensis,
Aerial Roots, Flavonoids,
Keywords
Column Chromatography
Ethyl Acetate, LC-MS.
ABSTRACT
The aerial roots of Ficus benghalensis Linn (Moraceae) have been reported to have
immunomodulatory,
anti-bacterial and hair growth promoting activities. There are no reports
ABSTRACT
on isolation and characterization of phytoconstituents from the aerial roots. The aim of
present study involved isolation and characterization of phyto constituents from the aerial
roots. Hence in this study an extract rich in flavonoids (i.e ethyl acetate rich extract) of aerial
roots was prepared and isolation of constituents was done using column chromatography. A
total of 7 compounds were identified and characterized from the ethyl acetate rich extract of
the aerial roots of Ficus benghalensis using LC-MS and HPLC-UV techniques. These
includes Kaempferol 3-O-acetyl-glucoside, Quercetin 3-arabinoside 7-rhamnoside, Quercetin
3-O-(6"-malonyl-glucoside) 7-O-glucoside, Naringenin-feruloyl hexose, Pelargonidin 3-Orutinoside, Epicatechin gallate, Cyanidin 3-O-rutinoside. The results of phytochemical
investigations indicate that flavonoids are major class of compounds present in this part of
plant which can be used as as potential antioxidants and anticancer agents.
Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
www.iajpr.com
Page
Please cite this article in press as Dr. Tabassum Khan et al. Phytoconstituents From Aerial Roots of Ficus Benghalensis Linn.
Indo American Journal of Pharmaceutical Research.2015:5(10).
3261
Corresponding author
Dr. Tabassum Khan
Department of Pharmaceutical chemistry
SVKM’s Dr. Bhanuben Nanavati College of Pharmacy,Gate No.1,
Mithibai College Campus,V.M. Road ,Vile Parle (West),Mumbai 400 056
[email protected]
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
INTRODUCTION
Ficus benghalensis Linn is commonly known as Banyan tree. This tree is considered to be a sacred tree in India. It is a large
evergreen tree distributed all over India from sub Himalayan region to the deciduous forest of Deccan and South India [1,2]. It is an
evergreen tree grows up to 30 meters, with spreading branches and many aerial roots. The leaves are stalked, ovate-corate, 3-nerved
entire, when young downy on both sides. The petioles are broad smooth greasy gland at the apex, compressed and downy. The fruits
are in auxiliary pairs as the size of a cherry. The external features of the bark are 12-18 mm thick, grey, closely adhered ashy white,
light bluish-green or grey patches, slightly curve, thickness varies with the age of the tree. The surface of bark is deeply fissured and
rough due to the presence of longitudinal and transverse row of lenticels, mostly circular and prominent, fracture short in outer 2/3 of
bark while inner portion shows a fibrous fracture[3,13,14].
Taxonomic classification of Ficus benghalensis Linn[2]
 Kingdom : Plantae
 SubKingdom : Tracheobionta
 Super division : Spermatophyta
 Division :Magnoliophyta
 Class :Magnoliopsida
 Subclass : Hamamelidae
 Order :Urticales
 Family :Moraceae
 Genus :Ficus
 Species :F.benghalensis
Biological activities and chemical constituents reported in Ficus benghalensis Linn:
In the traditional system of medicine, this plant is used for various health problems and diseases. A literature search on Ficus
benghalensis indicated various therapeutic uses such as astringent, haemostatic, anti-inflammatory, antioxidant and anticancer agent
and also in the treatment of diarrhoea, dysentery and in the treatment of skin disorders, ulcers, vaginal disorders, leucorrhoea,
menorrhagia and in case of deficient lactation[4,5]. Preliminary phytochemical investigation of roots of Ficus benghalensis indicated
the presence of carbohydrates, flavonoids, amino acids/ proteins, steroids, saponins and tannins [6,13]. The bark of the Ficus
benghalensis has been reported to contain leucopelargonidin -3-0-x-L rhamnoside and leucocynidin. 3-0-x-D galactosyl cellobioside,
glucoside beta glucoside, 20-tetratria conthene-2- one, 6- hepatatria contene-10-one, pentatricentan -5-one, beta sitosterol- alpha –Dglucose and mesoinositol[7,8]. The aqueous and methanol extracts of the aerial roots have been reported to exhibit
immunomodulatory activity [16] and the n-hexane extract exhibited hair growth promoting potential[17]. The aqueous extract also
exhibited antibacterial activity [18]
There are no reports on phytoconstituents isolated from aerial roots. Hence this research study is undertaken in an aim to
determine some phytoconstituents in the aerial roots using chromatography and spectroscopy techniques and to identify and
characterize isolated phytoconstituents in the aerial roots of Ficus benghalensis Linn.
MATERIAL AND METHODS
Plant material:
The fresh aerial roots of Ficus benghalensis Linn were collected in and around of Mithibai college campus, during the period
from June-September 2014, were authenticated by Dr. Sunita Shailaijan at Department of Botany, Ramanarayan Ruhia college,
Mumbai. All reagents and solvents used were of analytical reagent grade and procured from S.D. Fine Chem.Limited.
Page
3262
Preparation of ethyl acetate rich extract:
An ethyl acetate extract of aerial roots of Ficus benghalensis containing flavonoids was prepared using the scheme given
below[15].
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Scheme for preparation of ethyl acetate rich extract [15].
Column chromatography for ethyl acetate rich extract of Ficus benghalensis aerial roots :
The ethyl acetate rich extract was subjected to column chromatography using silica gel G (60-120#) as stationary phase and
using the scheme below:
Table 1. Column chromatography results.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Toluene
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Toluene: ethyl acetate
Ethyl acetate
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Ethyl acetate: chloroform
Chloroform
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Chloroform: methanol
Methanol
Ratios of
solvent
used
100%
90:10
80:20
70:30
60:40
50:50
40:60
30:70
20:80
10:90
100%
90:10
80:20
70:30
60:40
50:50
40:60
30:70
20:80
10:90
100%
90:10
80:20
70:30
60:40
50:50
40:60
30:70
20:80
10:90
100%
www.iajpr.com
Amount of
fraction
collected
100 ml
100ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
No of peaks observed
in HPLC
No peak
No peak
No peak
No peak
No peak
2 peaks
No peak
No peak
No peak
No peak
3 peaks
No peak
No peak
No peak
No peak
No peak
No peak
No peak
No peak
No peak
No peak
No peak
1 peak
3 peaks
No peak
No peak
No peak
No peak
No peak
No peak
No peak
3263
Solvent system
Page
Fraction
number
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
All these column fractions as given table were subjected to HPLC analysis .Out of which peaks were observed in fraction
number 6, 11, 23, 24. Hence these fractions were subjected to hyphenated techniques like LC-MS and HPLC-UV analysis.
Page
3264
LC-MS of column fractions:
The LC-MS of column fractions(6, 11, 23, 24) ethyl acetate rich extract was carried out using an Agilent 1100 HPLC system
equipped with a degasser, binary pump, autosampler and column thermostat. For the separation of compounds, a reversed-phase
kromasil C18 analytical column (250 x 4.6mm i.d., 5 µm particles) was used. The column thermostat was operated at 24˚C. The
mobile phase used for the separation was a mixture of formic acid 0.1% (V/V) in water (A) and acetonitrile (B), in linear gradient
mode, as follows: until 5 min 65% B and 35% A, at 10 min 60% B and 40% A, 85% B and 15% A until 15 min. The flow rate was 0.4
mL/min. For detection and quantification, the HPLC system was coupled with an Agilent 1100 Ion Trap SL mass spectrometer,
operated with an electrospray (ESI) ion source in positive ion mode. The vaporization gas used by the mass spectrometer was
nitrogen, at 65 psi; the dry gas was also nitrogen at a flow rate of 12 L/min and heated at 360ºC. The capillary potential was set at
+2500 V.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
RESULTS AND DISCUSSION
LC-MS and HPLC-UV spectra of column fractions (6, 11, 24) of Ethyl acetate rich extract :
The LC-MS profile of Column fraction 6 is given below:
Peak 1
Peak 2
Figure 1: LC-MS of column fraction 6 in both positive and negative scan mode.
Page
Figure 2: HPLC spectra of peak 1 of Column fraction 6.
3265
Peak 1 (Rt=3.607 mins) of Column fraction 6.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Figure 3: HPLC-UV spectra of peak 1 of Column fraction 6.
Figure 4: MS spectra of peak 1 of Column fraction 6.
Peak 1 of column fraction 6 at Rt= 3.607mins could be Kaempferol 3-O-acetyl-glucoside, presents a λmax at 206nm, 257 and
285nm. The positive ES-API spectrum of Kaempferol 3-O-acetyl-glucoside exhibits the signals at m/z 285 which corresponds to
aglycone part that is kaempferol and another signal at m/z-489.3 which matches with actual molar mass of Kaempferol 3-O-acetylglucoside (m/z-490.13). The difference between m/z-285 and m/z-489 is 204 which corresponds to M+H-acetylhexose that constitutes
the glycone moiety [19].
HO
O
OH
m/z-285
O
OH
O
m/z-204
OH
O
O
OH
Page
H3C
3266
OH
O
Kaempferol 3-O-acetyl-glucoside.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Peak 2 (Rt=13.249 mins) of Column fraction 6.
Figure 5 : HPLC spectra of peak 2 of Column fraction 6.
Figure 6: HPLC-UV spectra of peak 2 of Column fraction.
Figure 7: MS spectra of peak 2 of Column fraction 6.
Page
3267
Peak 1 of column fraction 6 at Rt= 13.249 mins could be Quercetin 3-arabinoside 7-rhamnoside, presents a λmax at 210 nm
and 369nm. The positive ES-API spectrum of Quercetin 3-arabinoside 7-rhamnoside exhibits the signals at m/z 302 which
corresponds to aglycone part that is Quercetin and another signal at m/z-580.3 with actual molar mass of Quercetin 3-arabinoside 7rhamnoside (m/z-580.498). The difference between m/z-302 and m/z- 580 is 278 (arabinose-m/z 132+ rhamnose-m/z-146) that
constitutes the glycone moiety [19].
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
OH
m/z-302
H3C
O
O
O
OH
HO
OH
O
HO
OH
O
HO
O
m/z-146
HO
OH
m/z-132
Quercetin 3-arabinoside-7-rhamnoside.
Hence, peak 1 of column fraction 6 could be Kaempferol 3-O-acetyl-glucoside and peak 2 could be Quercetin 3-arabinoside7-rhamnoside.
Page
3268
The LC-MS profile of Column fraction 11 is given below:
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Peak 3
Peak 1
Peak 2
Figure 8: LC-MS of column fraction 6 in both positive and negative scan mode.
Page
Figure 9: HPLC spectra of peak 1 of Column fraction 11.
3269
Peak 1 (Rt=2.182 mins) of Column fraction 11.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Figure 10: HPLC-UV spectra of peak 1 of Column fraction 11.
Figure 11: MS spectra of peak 1 of Column fraction 11.
Peak 1 of column fraction 11 at Rt= 2.182 mins could be Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside, presents a
λmax at 210 nm and 369nm. The positive ES-API spectrum of Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside exhibits the
signals at m/z 302 which corresponds to aglycone part that is Quercetin and another signal at m/z- 712.5 with actual molar mass of
Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside (m/z-712.53). (Mass spectrum compared to that obtained from CDRI MS
database).
OH
OH
HO
O
O
O
HO
O
OH
OH
O
OH
O
OH
HO
Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside.
www.iajpr.com
Page
O
3270
OH
O
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Peak 2 (Rt=11.249 mins) of Column fraction 11.
Figure 12: HPLC spectra of peak 2 of Column fraction 11.
Figure 13: HPLC-UV spectra of peak 2 of Column fraction 11.
Page
Peak 2 of column fraction 11 at Rt= 11.249 mins could be Naringenin-feruloyl hexose, presents a λmax at 225 nm and
279nm. The positive ES-API spectrum of Naringenin-feruloyl hexose exhibits the signals at m/z 272.1 which corresponds to aglycone
part that is naringenin and another signal at m/z- 611.5 with actual molar mass of Naringenin-feruloyl hexose (m/z-611.32). The
difference between m/z-272 and m/z-611.5 is 339 which corresponds to M+H-feruloyl hexose that constitutes the glycone moiety
[19].
3271
Figure 14: MS spectra of peak 2 of Column fraction 11.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
OH
HO
O
OH
m/z-339
m/z-272.1
O-Gluferuloyl
OH
O
Naringenin-feruloyl hexose.
Peak 3 (Rt=12.893 mins) of Column fraction 11.
Figure 15: HPLC spectra of peak 3 of Column fraction 11.
Page
3272
Figure 16: HPLC-UV spectra of peak 3 of Column fraction 11.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Figure 17: MS spectra of peak 3 of Column fraction 11.
Peak 3 of column fraction 11 at Rt= 12.893 mins could be Pelargonidin 3-O-rutinoside m/z- 579.3 (Mass spectrum compared
to that obtained from CDRI MS database).
OH
+
HO
O
O
OH
OH
O
OH
O
H3C
OH
O
OH
HO
OH
Pelargonidin 3-O-rutinoside.
Hence peak 1 of column fraction 11 could be Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside , peak 2 could be
Naringenin-feruloyl hexose and peak 3 could be Pelargonidin 3-O-rutinoside [19]
Page
3273
The LC-MS profile of Column fraction 24 is given below:
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Peak 1
Peak 2
Figure 18: LC-MS of column fraction 24 in both positive and negative scan mode.
Peak 1 (Rt=10.236 mins) of Column fraction 24.
Page
3274
Figure 19: HPLC spectra of peak 1 of Column fraction 24.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Figure 20: HPLC-UV spectra of peak 1 of Column fraction 24.
Figure 21: MS spectra of peak 1 of Column fraction 24.
Peak 1 of column fraction 24 at Rt= 10.236 mins could be Epicatechin gallate presents a λmax at 206nm and 279nm (Mass
spectrum compared to that obtained from CDRI MS database).
OH
O
HO
O
O
OH
HO
OH
Epicatechin gallate.
3275
OH
Page
OH
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Peak 2 (Rt=14.646 mins) of Column fraction 24.
Figure 22: HPLC spectra of peak 2 of Column fraction 24.
Figure 23: HPLC-UV spectra of peak 2 of Column fraction 24.
Figure 24: MS spectra of peak 2 of Column fraction 24.
Page
3276
Peak 2 of column fraction 24 at Rt= 14.646 mins could be Cyanidin 3-O-rutinoside (Mass spectrum compared to that
obtained from CDRI MS database).
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
HO
+
HO
O
HO
OH
O
OH
OH
O
OH
O
OH
Cyanidin 3-O-rutinoside.
Page
3277
Hence, peak 1 of column fraction 24 could be Epicatechin gallate and peak 2 could be Cyanidin 3-O-rutinoside.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
Page
3278
CONCLUSION
Phytochemical studies have led to the discovery of enormous number of natural products, their chemical diversity is unique
and many of them possess a variety of biological activities. A total of 7 compounds were identified from the ethyl acetate rich extract
of the aerial roots of Ficus benghalensis. These includes Kaempferol 3-O-acetyl-glucoside, Quercetin 3-arabinoside 7rhamnoside,Quercetin 3-O-(6"-malonyl-glucoside) 7-O-glucoside, Naringenin-feruloyl hexose, Pelargonidin 3-O-rutinoside,
Epicatechin gallate, Cyanidin 3-O-rutinoside. The phytoconstituents isolated are mostly flavonoids and phenolics which can be used
as potential antioxidants and anticancer agents in future uplifting the area of therapeutic approach.
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
ACKNOWLEDGEMENT
I pay my sincere gratitude to my guide, teaching and non -teaching staffs and my institute Dr. Bhanuben Nanavati College
Of Pharmacy for their contribution in my research work.
My great pleasures are in extending my thanks to Dr. Sunita Shailaijan ( Department of Botany, Ramanarayan Ruhia college,
Mumbai) for providing assistance in carrying out botanical verification of the plant for my research work.
Page
3279
ABBREVIATIONS
FULL FORM
FB
Ficus Benghalensis
EA
Ethyl acetate
DCM
Dichloromethane
UV
Ultraviolet spectroscopy
HPLC
High Performance liquid Chromatography
LC-MS
High performance Liquid chromatography-Mass spectroscopy
mg/kg
milligram/kilogram
µg/gm
microgram/gram
ml
millilitre
 No conflict of interest
www.iajpr.com
Vol 5, Issue 10, 2015.
Dr. Tabassum Khan et al.
ISSN NO: 2231-6876
REFERENCES
1. The Wealth of India, Volume-(F-G): In-A Dictionary of Indian Raw Materials and industrial products, Council of Scientific and
Industrial Research, New Delhi ,1999,vol 4, 24- 26.
2. Parrotta JA. Healing Plants of Peninsular India USA, CABI Publishing, 2001:517.
3. Koilpillai B, Sabasan GS, Sadananda S.Comparative pharmacognostic studies on the barks of four Ficus species. Turk.J.Bot.
2010;34: 215-224.
4. Chopra RN, Chopra IC, Handa KL, Kapur LD. Indigenous drugs of India, U.N. Dhur and sons Pvt.Ltd Calcutta, 1958 : 673- 675.
5. Khare CP. Encyclopedia of Indian Medicinal Plants, Springer publication. 2004. 216-217.
6. Aswar M, Aswar U, Wagh A, Watkar B, Vyas M, Gujar KN.Antimicrobial activity of Ficus benghalensis. Pharmacology on line.
2008 ;2 :715-725.
7. Mousa O, Vuorela P, Kiviranta J, Hiltunen R. Bioactivity of certain Egyptian Ficus species. J Ethanopharmacol. 1994; 41: 71-76.
8. Jaiswal R, Ahirwar DA. Literature Review On Ficus bengalensis, International Journal of Advances in Pharmaceutical
Research,2013; 5: 57-61.
9. Kokate CK, Purohit AP and Gokhale SB. Practical Pharmacognosy, Nirali Prakashan ,2nd ed,1994.
10. Khandelwal KR.Practical Pharmacognosy,1st ed ,Nirali Publications,1995, 9-20.
11. Sailor GU, Parmar G, Seth NR, Seth AK. Pharmacognostical and Preliminary Phytochemical Investigation of Leucas cephalotes
(Roth),International Journal of Pharmaceutical Research. 2010;1-6
12. Aiyegoro OA, Okoh AI.Prelimniary phytochemical screening and In vitro antioxidant activities of the aqueous extract of
Helichrysum longifolium DC.BMC Complementary and Alternative Medicine.2010;10:1-8
13. Hossain, Mohammad S. In vitro Antimicrobial Activity of Ethyl Acetate Extract of Ficus bengalensis Linn. International Journal
of Pharmacognosy and Phytochemical Research. 2014; 6: 56-58.
14. Shukla R, Gupta S, Gambhir JK, Prabhu KM, Murthy PS. Antioxidant effect of aqueous extract of the bark of Ficus bengalensis
linn in hypercholesterolaemic rabbits.J Ethnopharmacol.2004;92:47–51.
15. Manimozhi DM, Sankaranarayanan S.Evaluating the antibacterial activity of flavonoids extracted from Ficus benghalensis linn,
International Journal of Pharmaceutical and Biological Research.2012; 3:7-18.
16. Gabhe SY, Tatke PA, Khan TA.Evaluation of immunomodulatory activity of methanol extract of Ficus benghalensis linn in rats,
Indian J Pharmacol. 2006; 38:271-275.
17. Armani A, Armani S, Seneviratne C, Nazari R. Extracts and Compounds from Ficus Benghalensis for Increasing Hair Growth
and Decreasing Hair Loss, US20120128796, 24 May, 2012.
18. Singh KR, Watal G.Antimicrobial potential of Ficus bengalensis aerial roots, International journal of Pharma and Bio sciences.
2010;1:1-9
19. Cuyckens, F.; Ma, Y.L.; Pocsfalvi, G. & Claeys, M. Tandem mass spectral strategies for the structural characterization of
flavonoid glycosides. Analusis. 2000; 28:888-895.
Page
3280
54878478451151038
www.iajpr.com