Isolation of Fisetin from Elaeagnus indica Serv . Bull . ( Elaeagnaceae ) with antioxidant and antiproliferative activity

Introduction : Eleaegnus indica is a medicinal straggling shrub, belongs to Elaeagnaceae family and reported as potent antimicrobial, anticancer and larvicidal properties. The prime aim of this study was focused on the isolation and characterization of the most active anti-oxidant principle from the acetone leaves extracts of E. indica . Methods : The chromatographic and spectral studies were performed in isolation of most active compound ‘Fisetin’ (a flavonol group) from E. indica. The isolated pure compound was tested for its antioxidant and antiproliferative property (on U-937 and HT-60 cell lines) by adopting standard protocols. Results: The active compound was isolated as yellowish amorphous powder. The structure of the compound was identified by various spectral analysis like LC-MS, CHNS anlysis, UV, FT-IR, 1D ( 1 H and 13 C) and 2D NMR (HMBC and HSQC) analysis. The remarkable antioxidant activity was recorded in various assays like NO ● (IC 50 39.43 ± 0.28 µg/mL), ● OH (IC 50 43.91 ± 0.35 µg/mL), O 2 ●- (IC 50 48.30 ± 0.67 µg/mL), DPPH ● (IC 50 70.32 ± 0.89 µg/mL) and FRAP (EC 50 48.69 ± 1.05 µg/mL). The significant antiproliferative effect of the fisetin was noted on both U-937 (IC 50 46. 75 ± 3.53 µg/mg) and HT-60 (IC 50 59.46 ± 1.81 µg/mg) cell lines. Conclusion : The present investigation shows that isolated fisetin harbour high antioxidant and antiproliferative potential and provide strong scientific evidence for their medicinal uses, particularly antioxidant and anticancer properties.


INTRODUCTION
Free radicals lead to an oxidative stress and causes damage to several cellular macromolecules like proteins, lipids and DNA. 1 The majority of the degenerative diseases, cancer, cataracts, malfunction of the immune system, atherosclerosis, myocardial infarction, arthritis, anemia, asthma, liver diseases, diabetes mellitus, inflammation, renal failure, brain dysfunction and stress are mainly linked to oxidative stress due to free radicals. 2Cancer is the second leading cause of death, after heart disease, where one in four deaths occurs 3 and it is estimated that by the year 2020, the number of cancer patients will reach to 16 million per year. 4Antioxidant compounds are involved in the prevention of cellular damage, aging and a variety of diseases. 5Many plants were reported to harbor significant free radicals scavenging efficacy and produce various antioxidative compounds (phenols, alkaloids and terpenoids) which have many therapeutic potential.Bioactive plant based antioxidants were able to inhibit cancer cytogenesis by suppressing the tumor initiation, promotion and progression, which are being considered as potential biocompatible anticancer agents. 6Recently, there has been growing interest in research about the role of phytocompounds in antioxidant and antiproliferative activity. 7,41laeagnus indica Serv., Bull.belongs to the Elaeagnaceae family, found in the hills at 750-1550 m, forest border, cleared slopes and exposed to full sun.The extracts of E. indica possesses good antimicrobial, anticancer 8 and larvicidal potential. 9,10The present study deals with the isolation and characterization of potent antioxidant compound from acetone extract of E. indica.In addition, the isolated compound (fisetin) was tested for its proficiency in antiproliferative effect.

Preparation of extracts
Powdered plant material (2 kg) was successively extracted with various solvents (hexane, chloroform, ethyl acetate, acetone and methanol) in an increasing polarity method in a Soxhlet apparatus until the efflux solvent became colorless.All extractives were filtered through Whatman filter paper (No: 1) and concentrated in vacuo at 40°C which yield 1.67%, 3.48%, 4.68%, 5.33% and 6.21% (hexane, chloroform, ethyl acetate, acetone and methanol respectively) of dark green to brown color viscous extracts.

Isolation of antioxidant compound
Based on the preliminary results of phytochemicals and antioxidant properties, 11 the acetone extract was selected for isolation of antioxidant compound.E. indica acetone extract (50 g) was fractioned through

Isolation of Fisetin from Elaeagnus indica Serv. Bull. (Elaeagnaceae) with antioxidant and antiproliferative activity
Free Radicals and Antioxidants, Vol 6, Issue 2, Jul-Dec, 2016 a silica gel (60-120 mesh) column chromatography (column length 60 cm, diameter 4 cm) using hexane:ethyl acetate (100:0 to 0:100) solvent system (increasing 10% polarity) which yielded 58 fractions (F).All the fractions were tested for their DPPH radical scavenging potential.Among 58 fractions, only six fractions (F15, F28 F33, F35, F42 and F52) were showed considerable antiradical activity (Table 1).However, remarkable DPPH radical scavenging potential was observed in fraction F52 (hexane:ethyl acetate 40:60) with significant low IC 50 value (37.33 µg/mL).Hence, fraction F52 (2.88 g, brownish yellow color, solid in nature) was permeated through a silica gel column with hexane:ethyl acetate (100:0 to 70:30) in order of increasing 5% polarity which yielded 10 subfractions (EASF=Elaeagnus Antioxidant Sub-Fractions) and their DPPH radical scavenging potential was tested.Lowest IC 50 value (12.22 µg/ml) (Table 1) was noticed in EASF9 (hexane: ethyl acetate 45:55) which was renamed as EIA (E.indica antioxidant compound).The results of analytical TLC (pre coated silica gel F 254, Merck, Germany) of EIA fraction showed single spot with R f value of 0.75 (under UV light, iodine vapor and after spraying CAM stain) using n-Butanol-Acetic acid-Water (BAW) (4:1:5) solvent system as mobile phase.The purity of isolated EIA was confirmed by analytical LC (Thermo/ Finnigan Surveyor System) which eluted with methanol/water and LC column outlet was coupled to a Thermofleet (LCQ-Fleet) Ion Trap mass spectrometer equipped with an ESI ion source.Data acquisition and mass spectrometric evaluation were carried out in Qual Browser; Thermo Electron, San Jose, CA.Melting point (m.p.) of EIA was determined using a hot stage melting point apparatus (Leica GALEN III) equipped with microscope and are uncorrected.UV λ max of EIA was recorded on Perkin Elmer, Lambda-650 UV-Visible Spectrophotometer.The IR spectrum of the EIA was recorded in PerkinElmer, Spectrum RX-I spectrophotometer in the range of 400 to 4000 cm -1 wavelength with a resolution of 1 cm −1 .The elemental analysis, of EIA was carried out in PerkinElmer 2400 Series II.1D ( 1 H and 13 C) and 2D NMR (HMBC and HSQC) spectra were recorded (DMSO-d 6 ) using a Bruker AV-500 MHz NMR spectrometer with TMS as internal standard.

Antiproliferative Activity
The antiproliferative activity of isolated compound was determined by methylthiazolyl diphenyl-tetrazolium bromide (MTT) assay as described by Mosmann 17 on U-937 (human leukemic monocyte lymphoma) and HT-60 (human acute promyelocytic leukemia) cell lines.The U-937 and HT-60 cell lines were obtained from National Institute of Cell Sciences, Pune, India.The cells were treated with various concentrations of the isolated compound (3.13-1000 µM/ml).The effect of the isolated compound on the proliferation of U-937 and HT-60 cells was expressed as percentage of cell viability.IC 50 values are calculated graphically from the curve plotted for the percentage of cell viability against different concentrations of the compound.

Statistical analysis
All the experiments were carried out at least in triplicates.Data were represented as mean ± standard deviation (SD) of three determinations.The Inhibitory Concentrations (IC 50 ) were determined graphically from the curve fitted (nonlinear regression) to the mean values of quotients.The analyses were performed by logarithmically transforming the data to comply with analysis of variance (ANOVA) in a completely randomized design and Tukey's multiple range test (at p<0.05) by employing SPSS (16.0) software.

Antioxidant activity of fisetin
The findings of antioxidant properties of fisetin show significant antiradical capacity and is directly related to the concentration of fisetin (Figure 3).Fisetin showed remarkable strong inhibition activity on DPPH radicals than others having IC 50 value (12.23 µg/mL) which was lower than the reference compounds (ascorbic acid and BHA).Fisetin showed good Fe 3+ reduction property in FRAP assay with minimal EC 50 value (14.20 µg/mL) which was lower than positive controls.Fisetin harbor excellent hydroxyl radical neutralization potential with least IC 50 value (30.17 µg/mL) which are quite comparable with standards.Fisetin was found to be powerful quenchers of nitric oxide radicals with significant IC 50 value (39.57µg/mL).Considerable superoxide radicals scavenging activity were detected in fisetin with sustainable IC 50 value (75.26 µg/mL).9][20] Wherein Phenolic hydroxyl groups are prone to donate a hydrogen atom to free radicals and extend conjugated aromatic system to delocalize an unpaired electron. 21PPH radical scavenging activity guided isolation of acetone extract of E. indica yielded a flavonol compound, i.e. fisetin.Similarly several researchers have reported the isolation and structural elucidation (through 1 H and 13 C-NMR studies) of fisetin from various parts of many plants such as, xylem sap of Hymenaea courbaril, 22 aerial parts of Tanacetum parthenium, 23 fruit and leaf of Vitex rotundifolia, 24 leaves of Mayodendron igneum, 25 roots of Boesenbergia rotunda 26 and Sanguisorba officinalis. 27he scavenging activity of flavonoids depends on the number of free hydroxyl groups in the molecule.The ortho-dihydroxy structure of the B ring, of a flavonoid has the best electron-donating properties and confers higher stability in the radical form and participates in electron delocalization.Similarly, the 2, 3-double bond with a 4-oxo function in the C ring, are responsible for electron delocalization from the B ring.The 3-and 5-hydroxyl groups with the 4-oxo function in A and C rings, are essential for maximum radical scavenging potential of flavonoids.The 3-glycosylation reduces flavonoids activity when compared with corresponding aglycones which support the present results of antiradical activity of fisetin and the possible mode of inhibition of radicals. 28

Antiproliferative activity of fisetin
The results of antiproliferative property of fisetin isolated from E. indica showed good inhibitory effect on the growth of both U-937 and HL-60 cells in a dose dependent manner (Table 2, Figure 4).Moreover, the present results revealed that U-937 cells (IC 50 =98.22µM/mL) were more susceptible to fisetin than HL-60 cells (IC 50 =121.61µM/mL).Fisetin expressed superior antiproliferative activity due to the high antioxidant potential.Agullo et al. 29 stated that the antiproliferative effects of compounds on cancer cells are mainly due to their antioxidant potential which strengthens the findings of the present study.The extensive studies have been conducted to show that fisetin could inhibit several molecular targets, including cyclin-dependent kinases, [30][31][32] DNA topoisomerases I and II, 33,34 urokinase, 35 actin polymerization 36 and androgen receptor signaling. 37Moreover, fisetin has been recently reported to possess interesting anticancer activity in mice lung carcinoma 38 and prostate tumours. 37,39Murtaza et al. 40 reported that fisetin inhibited the proliferation of various types of human cancer cells and induces the cell cycle arrest or apoptosis as depicted in this study.

CONCLUDING REMARKS
Elaeagnus indicia is a good source for a variety of phytoconstituents.DPPH radical scavenging activity guided isolation method resulted in isolation of fisetin.The structure of fisetin was established by spectroscopic analysis and it expresses significant antioxidant and antiproliferative activity on U-937 and HL-60 cell lines.