PHYTOCONSTITUENTS FROM AERIAL ROOTS OF FICUS BENGHALENSIS LINN

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1 Page3261 Indo American Journal of Pharmaceutical Research, 2015 ISSN N: PHYTCNSTITUENTS FRM AERIAL RTS F 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 ARTICLE INF Article history ARTICLE Received 24/10/2015 INF Article Available history online Received 31/10/2015 xxx Available Keywords online 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 ABSTRACT immunomodulatory, anti-bacterial and hair growth promoting activities. There are no reports 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--acetyl-glucoside, Quercetin 3-arabinoside 7-rhamnoside, Quercetin 3--(6"-malonyl-glucoside) 7--glucoside, Naringenin-feruloyl hexose, Pelargonidin 3-rutinoside, Epicatechin gallate, Cyanidin 3--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. 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 tabassum.khan@bncp.ac.in 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). Copy right 2015 This is an pen 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.

2 Page3262 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: INTRDUCTIN 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 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 rder :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 METHDS 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. 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].

3 Page3263 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: 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. Fraction number Solvent system Ratios of solvent used Amount of fraction collected 1 Toluene 100% 100 ml No peak 2 Toluene: ethyl acetate 90:10 100ml No peak 3 Toluene: ethyl acetate 80: ml No peak 4 Toluene: ethyl acetate 70: ml No peak 5 Toluene: ethyl acetate 60: ml No peak 6 Toluene: ethyl acetate 50: ml 2 peaks 7 Toluene: ethyl acetate 40: ml No peak 8 Toluene: ethyl acetate 30: ml No peak 9 Toluene: ethyl acetate 20: ml No peak 10 Toluene: ethyl acetate 10: ml No peak 11 Ethyl acetate 100% 100 ml 3 peaks 12 Ethyl acetate: chloroform 90: ml No peak 13 Ethyl acetate: chloroform 80: ml No peak 14 Ethyl acetate: chloroform 70: ml No peak 15 Ethyl acetate: chloroform 60: ml No peak 16 Ethyl acetate: chloroform 50: ml No peak 17 Ethyl acetate: chloroform 40: ml No peak 18 Ethyl acetate: chloroform 30: ml No peak 19 Ethyl acetate: chloroform 20: ml No peak 20 Ethyl acetate: chloroform 10: ml No peak 21 Chloroform 100% 100 ml No peak 22 Chloroform: methanol 90: ml No peak 23 Chloroform: methanol 80: ml 1 peak 24 Chloroform: methanol 70: ml 3 peaks 25 Chloroform: methanol 60: ml No peak 26 Chloroform: methanol 50: ml No peak 27 Chloroform: methanol 40: ml No peak 28 Chloroform: methanol 30: ml No peak 29 Chloroform: methanol 20: ml No peak 30 Chloroform: methanol 10: ml No peak 31 Methanol 100% 100 ml No peak No of peaks observed in HPLC

4 Page3264 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: All these column fractions as given table were subjected to HPLC analysis.ut 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. 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 V.

5 Page3265 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: RESULTS AND DISCUSSIN 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 Peak 1 (Rt=3.607 mins) of Column fraction 6. Figure 1: LC-MS of column fraction 6 in both positive and negative scan mode. Figure 2: HPLC spectra of peak 1 of Column fraction 6.

6 Page3266 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: 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--acetyl-glucoside, presents a λmax at 206nm, 257 and 285nm. The positive ES-API spectrum of Kaempferol 3--acetyl-glucoside exhibits the signals at m/z 285 which corresponds to aglycone part that is kaempferol and another signal at m/z which matches with actual molar mass of Kaempferol 3--acetylglucoside (m/z ). The difference between m/z-285 and m/z-489 is 204 which corresponds to M+H-acetylhexose that constitutes the glycone moiety [19]. H m/z-285 m/z-204 H 3 C Kaempferol 3--acetyl-glucoside.

7 Page3267 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Peak 2 (Rt= 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. Peak 1 of column fraction 6 at Rt= 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 with actual molar mass of Quercetin 3-arabinoside 7- rhamnoside (m/z ). 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].

8 Page3268 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: m/z-302 H 3 C H H m/z-146 H H m/z-132 Quercetin 3-arabinoside-7-rhamnoside. Hence, peak 1 of column fraction 6 could be Kaempferol 3--acetyl-glucoside and peak 2 could be Quercetin 3-arabinoside- 7-rhamnoside. The LC-MS profile of Column fraction 11 is given below:

9 Page3269 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Peak 1 Peak 2 Peak 3 Peak 1 (Rt=2.182 mins) of Column fraction 11. Figure 8: LC-MS of column fraction 6 in both positive and negative scan mode. Figure 9: HPLC spectra of peak 1 of Column fraction 11.

10 Page3270 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: 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= mins could be Quercetin 3--(6"-malonyl-glucoside) 7--glucoside, presents a λmax at 210 nm and 369nm. The positive ES-API spectrum of Quercetin 3--(6"-malonyl-glucoside) 7--glucoside exhibits the signals at m/z 302 which corresponds to aglycone part that is Quercetin and another signal at m/z with actual molar mass of Quercetin 3--(6"-malonyl-glucoside) 7--glucoside (m/z ). (Mass spectrum compared to that obtained from CDRI MS database). H H H Quercetin 3--(6"-malonyl-glucoside) 7--glucoside.

11 Page3271 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Peak 2 (Rt= 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. Figure 14: MS spectra of peak 2 of Column fraction 11. Peak 2 of column fraction 11 at Rt= 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 which corresponds to aglycone part that is naringenin and another signal at m/z with actual molar mass of Naringenin-feruloyl hexose (m/z ). The difference between m/z-272 and m/z is 339 which corresponds to M+H-feruloyl hexose that constitutes the glycone moiety [19].

12 Page3272 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: H m/z Gluferuloyl m/z-339 Naringenin-feruloyl hexose. Peak 3 (Rt= mins) of Column fraction 11. Figure 15: HPLC spectra of peak 3 of Column fraction 11. Figure 16: HPLC-UV spectra of peak 3 of Column fraction 11.

13 Page3273 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Figure 17: MS spectra of peak 3 of Column fraction 11. Peak 3 of column fraction 11 at Rt= mins could be Pelargonidin 3--rutinoside m/z (Mass spectrum compared to that obtained from CDRI MS database). H + H 3 C H Pelargonidin 3--rutinoside. Hence peak 1 of column fraction 11 could be Quercetin 3--(6"-malonyl-glucoside) 7--glucoside, peak 2 could be Naringenin-feruloyl hexose and peak 3 could be Pelargonidin 3--rutinoside [19] The LC-MS profile of Column fraction 24 is given below:

14 Page3274 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Peak 1 Peak 2 Figure 18: LC-MS of column fraction 24 in both positive and negative scan mode. Peak 1 (Rt= mins) of Column fraction 24. Figure 19: HPLC spectra of peak 1 of Column fraction 24.

15 Page3275 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: 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= mins could be Epicatechin gallate presents a λmax at 206nm and 279nm (Mass spectrum compared to that obtained from CDRI MS database). H H Epicatechin gallate.

16 Page3276 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: Peak 2 (Rt= 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. Peak 2 of column fraction 24 at Rt= mins could be Cyanidin 3--rutinoside (Mass spectrum compared to that obtained from CDRI MS database).

17 Page3277 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: H H + H Cyanidin 3--rutinoside. Hence, peak 1 of column fraction 24 could be Epicatechin gallate and peak 2 could be Cyanidin 3--rutinoside.

18 Page3278 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: CNCLUSIN 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--acetyl-glucoside, Quercetin 3-arabinoside 7- rhamnoside,quercetin 3--(6"-malonyl-glucoside) 7--glucoside, Naringenin-feruloyl hexose, Pelargonidin 3--rutinoside, Epicatechin gallate, Cyanidin 3--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.

19 Page3279 Vol 5, Issue 10, Dr. Tabassum Khan et al. ISSN N: ACKNWLEDGEMENT I pay my sincere gratitude to my guide, teaching and non -teaching staffs and my institute Dr. Bhanuben Nanavati College f 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. ABBREVIATINS FULL FRM 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

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