Journal of Pharmacognosy and Phytochemistry 2016; 5(3): 120-128

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2016; 5(3): 120-128 Antiviral and antioxidant activities of flavonoids of
Received: 18-03-2016
Accepted: 19-04-2016 Ficus virens: Experimental and theoretical
investigations
Mohamed AA Orabi
Faculty of Pharmacy, Al-Azhar
University, Assiut 71524, Egypt. Mohamed AA Orabi and Esam A Orabi
Esam A Orabi
A) Department of Chemistry, Abstract
Faculty of Science, Assiut A phytochemical investigation of Ficus virens leaves led to isolation of six flavonoids (1–6). On the basis
University, Assiut 71516, Egypt. of 2D and/or 1D NMR, and mass data, together with the comparison with the literature values, the
B) Department of Chemistry and flavonoids were identified as quercetin (1), quercetin-3-O-α-D-arabinopyranoside (2), quercetin-3-O-β-D-
Biochemistry, Concordia galactopyranoside (3), kaempferol-3-O-α-D-arabinopyranoside (4), kaempferol-3-O-β-D-
University, 7141 Sherbrooke galactopyranoside (5), and vogelin J (6). The procedures for simultaneous isolation of the glycosides 2–5
Street West, Montréal, Québec are described. The antioxidant activity of the isolated flavonoids were experimentally evaluated. The
H4B 1R6, Canada structural characteristics and structure-antioxidant activity relationship of these isolates are studied by
quantum mechanical calculations on the molecules and on the free radicals derived from each compound
by an abstraction of single hydrogen from the various aromatic OH groups. The calculations are
performed at the B3LYP/6-31+G(d,p) level of theory, and show that glycosylation of quercetin and
kaempferol distorts the planar geometry of the aromatic moiety of both the molecular and radical
compounds. The viral inhibitory effects of the isolated flavonoids on Coxsackie B4 (CVB4), and
hepatitis A virus (HAV) are also investigated. The flavonoids 1, 3, and 6 exerted mild anti CVB4
inhibitory effects, while 2, and 3 exerted mild inhibitory activities against HAV.

Keywords: Ficus virens, Flavonoid glycosides, DPPH, antioxidant, Quantum chemistry.

1. Introduction
The plant-derived medicines are mainly favored because of their low toxicity, rapid
degradation in the environment, and limited resistance [1]. Ficus virens W. T. Aiton
(Moraceae), synonym Ficus cunninghamii Miq., is a medium sized tree commonly known as
white fig [2]. Its syconia are edible, and the new foliage can also be eaten as a vegetable and
pickle [3]. In Egypt, many Ficus species are grown in streets, gardens, parks for the ornamental
and shade purposes. The use of Ficus species as health-improving food or as therapeutic agent
is known long ago. In folk medicine, many parts of F. virens such as bark, latex, leaves and
fruits are used in the treatment of blood diseases, apoplexy, vertigo, delirium, pain,
rheumatism, diabetes and also as antioxidants [4]. F. virens has good amount of phenolic
content, and exert strong free radical scavenging activity [5–9]. In this study, six known
flavonoids (1‒6) were isolated from leaves of F. virens. Among them, four flavonoid
glycosides, quercetin-3-O-α-D-arabinopyranoside (2), quercetin-3-O-β-D-galactopyranoside
(3), kaempferol-3-O-α-D-arabinopyranoside (4), and kaempferol-3-O-β-D-galactopyranoside
(5), which are usually reported in literatures as inseparable mixture [10], are isolated in high
purity conditions. The chromatographic procedures including preparative reversed-phase (RP)-
HPLC technique for simultaneous isolation of these glycosides are shown in this work. The
DPPH radical scavenging activities of the isolated flavonoid in comparison with that of
standard kaempferol are experimentally investigated. The results revealed that the
glycosylation process lowers the radical scavenging ability of the flavonoids compared to their
aglycone parts. To understand the structural-antioxidant activity of these compounds, we
performed quantum mechanical calculations to determine the structural and energetic
properties of these flavonoids and of their radicals.
Besides the protective effect of the flavonoids by scavenging oxidants from the biological
Correspondence: systems, in many folk medicines, part of the therapeutic effect was ascribed for the flavonoid
Mohamed AA Orabi class of compounds, because of their broad pharmacological activities. Some flavonoids-rich
Faculty of Pharmacy, Al-Azhar plant extracts as well as single pure flavonoids showed noticeable antiviral activity by in vivo
University, Assiut 71524, Egypt. and in vitro studies [1, 11].
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The CVB4 and HAV belong to Picornaviridae family of granular powder revealed four distinct peaks upon RPHPLC
viruses; small plus-strand RNA viruses that cause a wide range analysis. A preparative RPHPLC purification of the DCM-
of pathogenicity ranging from lethal and severe (encephalitis, insoluble part (57 mg) led to isolation of quercetin-3-O-α-D-
myocarditis) to mild (common cold and influenza-like arabinopyranoside (2, 4.6 mg), quercetin-3-O- β-D-
diseases) [12]. CVB4 is thought to cause diseases such as galactopyranoside (3, 19 mg), kaempferol-3-O-α-D-
myocarditis, pancreatitis, hepatitis, aseptic meningitis, arabinopyranoside (4, 2.8 mg), and kaempferol-3-O-β-D-
meningoencephalitis, gastroenteritis, necrotizing enterocolitis, galactopyranoside (5, 3.4 mg).
and pneumonia, and even death in neonates [13]. HAV is the
most common cause of acute viral hepatitis that transmitted 2.4 Spectroscopic analyses
through the fecal-oral route; it causes about 10 million Electrospray ionization mass (ESIMS) spectra were acquired
infections worldwide each year [14]. To investigate the antiviral on an API-4000 instrument (AB Sciex, Framingham, MA,
potency of the isolated compounds, we measured the USA). The solvent used was [CH3CN/H2O (1:1, v/v)]. The 1D
inhibitory effects of the isolated flavonoids on CVB4 and (1H and 13C) and 2D (1H–1H COSY, HSQC and HMBC) NMR
HAV viruses. spectra were recorded on a Varian INOVA AS 600 instrument
(600 MHz for 1H and 151 MHz for 13C; Agilent, Santa Clara,
2. Material and methods CA, USA). Chemical shifts are given in δ (ppm) values
2.1 Materials relative to that of the solvent signal [acetone-d6 (δH 2.04; δC
The DPPH (Sigma-Aldrich Chemicals Co, Germany). Organic 29.8), DMSO-d6 (δH 2.50; δC 39.5)] on the tetramethylsilane
solvents (El-Nasr Pharmaceuticals Chemicals Co., Cairo, scale. Reversed-phase (RP) HPLC was performed on a YMC-
Egypt). Minimum essential media (MEM) (Caisson / USA). Pack ODS-A A-303 (YMC, Japan) column (4.6 i.d. × 250
Trypsin-EDTA 1:250 U (Molekulu/UK). MTT mm) developed using acetonitrile – water (2:8, v/v) with 0.1%
(Biobasic/Canada). Penicillin, streptomycin, and ampicillin acetic acid, at a flow rate of 1 mL/min in an oven set at 40 °C.
(Chemical industries and development company, Egypt). Detection was performed based on UV absorption at 280 nm.
Doxorubicin, a standard anticancer agent, and ribavirin, a Preparative RP-HPLC was performed at 40 °C on a YMC-
standard anti-viral agent, (Sigma-Aldrich Chemicals Co, Pack ODS-A, A-324 column (10 i.d. × 300 mm) using
Germany). acetonitrile – water (2:8, v/v) with 0.1% acetic acid, at a flow
rate of 2 mL/min with UV detection at 280 nm.
2.2 Plant materials
Leaves from F. virens tree were collected at May 2011 from Quercetin (1): ESI-MS m/z 603 [2M−H]−; 1H NMR (600
the experimental station of ornamental plants, Faculty of MHz, DMSO-d6) δ 12.48 (1H, s, C-5 OH), 10.77 (1H, s, C-7
Agriculture, Assiut, Egypt. A voucher specimen (Fv 185011) OH), 9.57 (1H, s, C-3 OH), 9.33 (1H, s, C-4ʹ OH), 9.28 (1H, s,
was deposited at the same Department. C-3ʹ OH), 7.67 (1H, d, J = 2.4 Hz, H-2ʹ), 7.53 (1H, dd, J = 2.4,
8.4 Hz, H-6ʹ), 6.88 (1H, d, J = 8.4 Hz, H-5ʹ), 6.41 (1H, d, J =
2.3 Extraction and isolation procedures 1.8 Hz, H-8), 6.18 (1H, d, J = 1.8 Hz, H-6); 13C NMR (151
The air-dried, powdered leaves (200 g) were exhaustively MHz, DMSO-d6) δ 175.8 (C-3), 163.8 (C-7), 160.7 (C-5),
extracted using MeOH/H2O (7:3, v/v, 1L × 7) at room 156.1 (C-9), 147.7 (C-4ʹ), 146.8 (C-2), 145.0 (C-3ʹ), 135.7 (C-
temperature over a period of 7 days. The obtained extract was 3), 121.9 (C-1'), 119.9 (C-6ʹ'), 115.6 (C-2ʹ), 115.0 (C-5ʹ), 103.0
filtered, and MeOH of the extract was evaporated under (C-10).
vacuum at 50 °C. The resulted aqueous extract (400 mL) was
partitioned using n-hexane (4 × 400 mL), dichloromethane Quercetin-3-O-α-D-arabinopyranoside (2): ESI-MS m/z 433
(DCM) (4 × 400 mL), ethyl acetate (5 × 400 mL) and n- [M−H]−; 1H NMR [600 MHz, (acetone-d6–D2O, 7:3)] δ 7.85
butanol (4 × 400 mL) to obtain the respective, n-hexane (6 g), (1H, d, J = 2.4 Hz, H-2ʹ), 7.53 (1H, dd, J = 2.4, 8.4 Hz, H-6ʹ),
DCM (3 g), ethyl acetate (12 g), n-butanol (9 g) and aqueous 6.90 (1H, d, J = 8.4 Hz, H-5ʹ), 6.47 (1H, d, J = 1.8 Hz, H-8),
(13 g) fractions. A part (3 g) of the ethyl acetate fraction was 6.22 (1H, d, J = 1.8 Hz, H-6), 5.14 (1H, d, J = 6.6 Hz, H-1ʺ),
subjected to silica gel column chromatography with a gradient 3.92 (1H, dd, J = 6.6, 9.0 Hz, H-2ʺ), 3.83 (1H, ddd, J = 1.8,
of DCM/MeOH (from 1:0 to 0:1, v/v) as an eluent, collecting 3.6, 7.2 Hz, H-4ʺ), 3.73 (1H, dd, J = 3.6, 12.0 Hz, H-5ʺ), 3.66
100 mL fractions. Eluates were monitored by TLC using pre- (1H, dd, J = 3.6, 8.4 Hz, H-3ʺ), 3.39 (1H, dd, J = 1.8, 12.6 Hz,
coated Silica gel G60 F254 plates (Merck, Germany), and H-5ʺ); 13C [151 MHz, (acetone-d6–D2O, 7–3),] δ 178.7 (C-3),
spots were visualized under UV, and by spraying with 10% 165.2 (C-7), 162.0 (C-5), 157.8 (C-2), 157.6 (C-9), 149.3 (C-
aqueous H2SO4 followed by heating at 110 ºC for 5 min. 4ʹ), 145.2 (C-3ʹ), 134.9 (C-3), 122.3 (C-6ʹ), 122.0 (C-1ʹ), 117.2
Similar fractions were combined together and concentrated (C-2ʹ), 115.9 (C-5ʹ), 104.9 (C-10), 103.7 (C-1ʺ), 99.4 (C-6),
under reduced pressure to give five fractions (A–E). Fraction 94.5 (C-8), 73.2 (C-3ʺ), 72.0 (C-2ʺ), 68.0 (C-4ʺ), 66.2 (C-5ʺ).
B (300 mg), eluted with DCM/MeOH (9.5:0.5, v/v) was
subjected to silica gel column, eluted with a gradient of n- Quercetin-3-O--D-galactopyranoside (=hyperoside =
hexane/ethyl acetate (1:0 – 0:1, v/v), collecting 20 hyperin, 3): ESI-MS m/z 463 [M−H]−; 1H NMR [600 MHz,
mL/fraction, and yielded seven sub-fractions (B1–B7). (acetone-d6–D2O, 7:3)] δ 7.90 (1H, d, J = 2.4 Hz, H-2ʹ), 7.53
Crystallization from DCM/MeOH (1:1, v/v) of the sub-fraction (1H, dd, J = 2.4, 8.4 Hz, H-6ʹ), 6.90 (1H, d, J = 9.0 Hz, H-5ʹ),
B2 (70 mg), eluted with n-hexane/ethyl acetate (6:4, v/v), 6.41 (1H, d, J = 1.8 Hz, H-8), 6.21 (1H, d, J = 1.8 Hz, H-6),
afforded pure yellow crystals of vogelin J (11 mg, 6). The sub- 5.10 (1H, d, J = 7.8 Hz, H-1ʺ), 3.90 (1H, br. d, J = 3.6 Hz, H-
fraction B4 (100 mg), eluted with n-hexane/ethyl acetate (4:6, 4ʺ), 3.83 (1H, dd, J = 7.8, 9.6 Hz, H-2ʺ), 3.61 (1H, dd, J = 6.6,
v/v), was further purified on Sephadex LH-20 column (1 i.d. × 9.6 Hz, H-6ʺ), 3.60 (1H, dd, J = 3.6, 9.6 Hz, H-3ʺ), 3.48 (1H,
17 cm) eluted with DCM/MeOH (1:1, v/v) to afford quercetin br.t, J = 6 Hz, H-5ʺ), 3.44 (1H, dd, J = 6.6, 10.8 Hz, H-6ʺ); 13C
(9 mg, 1). The fraction D (300 mg), eluted by DCM/MeOH NMR [151 MHz, (acetone-d6–D2O, 7–3)] δ 178.6 (C-3), 165.2
(8:2, v/v), was dissolved in DCM. A DCM-insoluble yellow (C-7), 161.8 (C-5), 157.8 (C-2), 157.5 (C-9), 149.2 (C-4ʹ),
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145.0 (C-3ʹ), 134.4 (C-3), 122.2 (C-6ʹ), 121.9 (C-1ʹ), 117.4 (C- 5% CO2 incubator (Jouan CO2 incubator, France) at 37 °C.
2ʹ), 115.8 (C-5ʹ), 104.7 (C-10), 104.4 (C-1ʺ), 99.5 (C-6), 94.5 The 24 h cell monolayers were treated with each sample
(C-8), 76.0 (C-5ʺ), 74.0 (C-3ʺ), 72.3 (C-2ʺ), 68.6 (C-4ʺ), 60.7 dilutions (each dilution in triplicate), while cell control
(C-6ʺ). contained only medium. The cells were incubated for 48 h at
37 °C in a 5% CO2 incubator. The culture medium was
Kaempferol-3-O-α-D-arabinopyranoside (4): ESI-MS m/z removed, and 20 μL of MTT solution (5 mg/mL) was added to
417 [M−H]−; 1H NMR [600 MHz, (acetone-d6–D2O, 7:3)] δ cells in each well for 4 h at 37 °C. After removal of MTT, 200
7.8 (2H, d, J = 8.4 Hz, H-2ʹ/6ʹ), 6.93 (2H, d, J = 7.8 Hz, H- μL DMSO was added to solubilize the formazan crystal, an
3ʹ/5ʹ), 6.47 (1H, d, J = 1.8 Hz, H-8), 6.23 (1H, d, J = 1.8 Hz H- MTT metabolic product, and incubated for 30 min at 37 °C.
6), 5.14 (1H, d, J = 6 Hz, H-1ʺ), 3.90 (1H, dd, J = 6.0, 9.0 Hz, The optical densities (OD) were measured at 560 nm using an
H-2ʺ), 3.80 (1H, dd, J = 3.6, 7.8 Hz, H-4ʺ), 3.67 (1H, dd, J = ELISA reader (MRX microplate reader, Dynex technologies,
4.8, 12.0 Hz, H-5ʺ), 3.66 (1H, dd, J = 3.6, 7.8 Hz, H-3ʺ), 3.32 USA). The percentage of cytotoxicity is calculated as
(1H, dd, J = 2.4, 12.0 Hz, H-5ʺ). [(A−B/A) × 100], where A and B indicate the mean of three
optical densities of control, and treated cells, respectively. The
Kaempferol-3-O-β-D-galactopyranoside (= Trifolin, 5): 50% cytotoxic concentration (CC50) is defined as the
ESI-MS m/z 447 [M−H]−; 1H NMR [600 MHz, (acetone-d6– concentration (μg/mL) that can reduce 50% of cell viability
D2O, 7:3)] δ 8.07 (2H, d, J = 8.4 Hz, H-2ʹ/6ʹ), 6.91 (2H, d, J = compared to control cells. The maximum non-toxic
8.4 Hz, H-3ʹ/5ʹ), 6.45 (1H, d, J = 1.8 Hz, H-8), 6.22 (1H, d, J = concentration (MNTC) of each extract was determined and
1.8 Hz H-6), 5.1 (1H, d, J = 8.4 Hz, H-1ʺ), 3.86 (1H, dd, J = was used for assessing the viral inhibitory activities.
1.2, 3 Hz, H-4ʺ), 3.78 (1H, dd, J = 7.8, 9.6 Hz, H-2ʺ), 3.55
(1H, dd, J = 3, 9.6 Hz, H-3ʺ), 3.57 (1H, dd, J = 6.6, 9.6 Hz, H- 2.7 Antiviral assay
6ʺ), 3.44 (1H, ddd, J = 1.2, 6.6, 11.4 Hz, H-5ʺ), 3.40 (1H, dd, J The direct effect of the flavonoids on CVB4, and HAV
= 6,6 11.4 Hz, H-6ʺ); 13C NMR [151 MHz, (acetone-d6–D2O, infectivity was evaluated according to the protocols described
7:3)] δ 178.8 (C-3), 165.1 (C-7), 161.9 (C-5), 160.7 (C-2), previously [15] with little modification. Briefly, Vero cells
158.3 (C-4ʹ), 157.6 (C-9), 134.8 (C-3), 131.8 (C-2ʹ/C-6ʹ), grown in 96-well plates at concentration 1 × 103 cells/well
115.8 (C-3ʹ/C-5ʹ), 104.9 (C-10), 104.1 (C-1ʺ), 99.5 (C-6), 94.5 were incubated at 37 °C and 5% CO2 overnight to allow the
(C-8), 76.0 (C-5ʺ), 74.0 (C-3ʺ), 72.1 (C-2ʺ), 68.6 (C-4ʺ), 60.7 cells to attach to the wells. The viral suspensions containing
(C-6ʺ). 100 TCID50 of the virus were either incubated with an equal
volume of MNTC of the flavonoids or with drug-free vehicle
Vogeline J (6): ESI-MS M/Z 335 [M−H]−; 1H NMR [600 at 4 °C for 1 h. The cells monolayers were treated with 100 µL
MHz, (acetone-d6–D2O, 7:3)] δ 6.63 (1H, s, H-3), 6.45 (1H, d, viral/sample suspension, and the plates were placed on a
J = 0.6 Hz, H-8), 7.88 (2H, d, J = 9.0 Hz, H-2ʹ/H-6ʹ), 6.98 (2H, shaking table (150 rpm) for 5 min. Plates were then incubated
d, J = 9.0 Hz, H-3ʹ/H-5ʹ), 5.72 (1H, d, J = 9.0 Hz, H-3ʺ), 6.6 at 37 °C in 5% CO2 atmosphere for 24 h to allow the virus to
(1H, dd, J = 0.6, 9.0 Hz, H-4ʺ), 1.42 (6H, s, H-5ʹ and 6ʹ); 13C take effect. The cell monolayers were rinsed carefully with
NMR [151 MHz, (acetone-d6–D2O, 7:3)] δ 183.12 (C-4), phosphate buffered solution. The veridical effect was
165.3 (C-2), 162.2 (C-4ʹ), 159.97 (C-7), 157.7 (C-9), 156.59 determined by an MTT assay following the procedures
(C-5), 129.4 (C-3ʺ), 129.14 (C-2ʹ), 129.14 (C-6ʹ), 122.47 (C- described above. The antiviral activity for a given compound
1ʹ), 116.75 (C-3ʹ), 116.75 (C-5ʹ), 115.48 (C-4ʺ), 105.93 (C-6), was calculated from the equation % antiviral activity =
105.61 (C-10), 103.72 (C-3), 95.7 (C-8), 78.71 (C-2ʺ), 28.34 [(A−B)/(C−B)] × 100). Where, A is the mean optical density
(C-5ʺ), 28.34 (C-6ʺ). of the treated cells, B is the mean optical density of virus
control, and C is the mean optical density of cell control.
2.5 Cell lines and viruses
VERO cell line, and the viruses HAV and CVB4 were 2.8 Practical assessment of the DPPH radical scavenging
obtained from center of viral research and studies, Faculty of activities
Medicine, Al-Azhar University, Cairo, Egypt. Vero cells were The DPPH radical scavenging activities was determined by a
grown in minimum essential medium (MEM). The media were modified method by Wang et al., 2007 [17]. DPPH radical was
supplemented with 10% heat inactivated fetal bovine serum prepared in MeOH as a 100 μM solution. A flavonoid was
(FBS), 100 units/mL penicillin, 100 μg/mL streptomycin and dissolved in MeOH, and two fold dilutions (final
1% HEPES (4-2-hydroxyethyl-1-piperazine ethane sulfonic concentrations 0.78 – 100 μg/mL) were prepared. To 270 μL
acid) and incubated in a humidified 5% CO2 atmosphere. The DPPH solution 30 μL of the different dilution (in triplicate) of
medium used for the cytotoxicity and antiviral assays the samples in MeOH was added. The mixture was shaken
contained 2% of fetal bovine serum. Viral stocks were vigorously and kept in the dark for 30 min. The absorbance
prepared in Vero cells. The virus titer was estimated from was measured on UV spectrophotometer at 517 nm and the
cytopathogenicity of cells induced by viral infection and scavenging activity was determined by the following equation:
expressed as 50% tissue culture infectious doses (TCID50) [15]. % Scavenging activity = [Acontrol ‒ Asample]/Acontrol × 100]. The
IC50 value for each sample was determined graphically by
2.6 Cytotoxicity assay plotting the percentage disappearance of DPPH as a function
Two-fold dilutions (1000 –1.9 μg/mL) from the pure of the sample concentration.
compounds were prepared in MEM (containing 2% antibiotics
and 2% FBS). The cytotoxic activity of the samples was 2.9 Quantum mechanical (QM) calculations
determined in Vero cells by using the colorimetric 3-(4,5- All QM calculations are performed in gas phase with Gaussian
dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide 09 software [18] using the B3LYP functional [19, 20] and the 6-
(MTT) method [16]. Briefly, Vero cells were grown in 96-well 31+G(d,p) basis set. The geometry of the six isolated
microtiter plates at concentration 5 × 10 3 cells/well for 24 h, in flavonoids is optimized starting with various initial conformers
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Journal of Pharmacognosy and Phytochemistry

of each compound. Frequency calculations are performed on 3.2 Antioxidant activity

the optimized geometries to confirm that they are energy 3.2.1 DPPH radical scavenging activity
minima (no imaginary frequencies). Free radicals generated The F. virens leaves and stem bark are rich with phenolic
from the most stable conformer of each compound by components [7, 9]. An aqueous MeOH extract of leaves of F.
hydrogen abstraction from the OH groups in positions 3ʹ, 4ʹ, 5, virens, and the different fractions, EtOAc and n-BuOH,
or 7 are also optimized at the same level of theory. A relaxed obtained by successive fractionation of the MeOH extract
potential energy scan is performed by scanning the φ3-2-1’-6’ showed high anti-DPPH radical scavenging activities [5]. In the
dihedral angle in quercetin from 0° to 180° in 5° increment. present study, phytochemical investigation of EtOAc fraction
of an aqueous MeOH extract of leaves of F. virens led to
3. Results and discussion purification of six known flavonoids (1–6). In our knowledge,
3.1 Preparative HPLC purification and structural the antioxidant effect of vogelin J (6) was not reported
identification of the isolated flavonoids previously, whereas the antiradical activities of few or more of
An aqueous MeOH extract of leaves of F. virens was the other molecules 1–5, were studied extensively by various
fractionated by partitioning in n-hexane, DCM, ethyl acetate, methods. Since the antioxidant capacity depends on the
and n-butanol. The ethyl acetate fraction was subjected to concentration ratio between antioxidants and target, the
silica gel chromatographic fractionation with DCM/MeOH reaction conditions, and other factors [27], the anti-DPPH
gradients. Crystallization from [DCM/MeOH (1:1, v/v)] of the radical activities of the different isolated compounds, as well
early DCM/MeOH (9.5:0.5, v/v) eluate afforded pure sample as an authentic sample of the flavonoid kaempferol were
of 6, while repeated purification of the later eluate on evaluated under the same experimental conditions. The
Sephadex LH-20 afforded the pure flavonoid 1. Preparative concentration of antioxidant needed to decrease the initial
HPLC purification of the EtOAc polar part, eluted with DPPH concentration by 50% (IC50) was determined (see
DCM/MeOH (8:2, v/v), led to purification of four flavonoid experimental section). A lower IC50 value corresponds to a
glycosides (2–5). On the basis of spectroscopic experiments higher antioxidant power. On the basis of the obtained results,
including 1H and 13C NMR, COSY, HSQC and HMBC, quercetin [1, (IC50 = 14 ± 1.12)] is the most active, followed
together with ESI mass spectrometry and comparing of the by quercetin glycosides [2 (IC50 = 35 ± 1.21), and 3 (IC50 = 37
obtained data (Experimental section) with reported values, the ± 1.74)], kaempferol (IC50 = 40 ± 0.82), kaempferol glycosides
flavonoids (Fig. 1) were identified as quercetin (1) [21], [4, (IC50 = 54 ± 1.13) and 5 (IC50 = 59 ± 1.42], and vogelin J
quercetin-3-O-α-D-arabinopyranoside (2) [22, 23], quercetin-3-O- [6, (IC50 >100)].
β-D-galactopyranoside (3) [24]
, kaempferol-3-O-α-D-
[22]
arabinopyranoside (4) , kaempferol-3-O-β-D- 3.2.2 QM investigation of the structural-antioxidant
galactopyranoside (5) [25], and vogelin J (6) [26]. activity relationship
The most commonly occurring flavonols are those with
dihydroxylation in the 3ʹ and 4ʹ positions of the B ring, and to
a smaller extent, those with a single hydroxylation at the 4ʹ
position. The phenolic OHs are responsible for the antioxidant
activity of flavonoids. A number of studies have been focused
on understanding the structure-antioxidant activity relationship
of these compounds [28–38]. Although several factors, the most
important of which are lipophilicity, iron-chelation, and
scavenging of free radicals, determine the antioxidant power of
a compound [39], experiments are suggesting a relation between
the structure and the free radical scavenging ability of
flavonoids [35, 39]. Determination of the structural properties of
flavonoids and of their radicals is thus an important step
toward understanding their antioxidant potency. To the best of
our knowledge, computational investigations on flavonoids
have focused on the simple, sugar-free (aglycones) compounds
[29, 39]
. Previous experimental studies [38] and our herein
investigation show that the flavonoid glycosides are less active
as anti-DPPH radical than their non-glycosylated analogues.
Studying the influence of the sugar moiety on flavonoids
geometry aids in understanding the reason of reduction in
antioxidant activity upon glycosylation [40].

3.2.3 Optimized geometries of molecular flavonoids

Fig 1: Structures of the compounds 1–6. Geometry optimization of the six isolated compounds as well
as of kaempferol revealed various stable conformers for each
molecule. We report in Fig. 2 the geometry of the global
minimum conformer of each compound.

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Journal of Pharmacognosy and Phytochemistry

Fig 2: Optimized global minimum structures of quercetin (a), quercetin-3-O-α-D-arabinopyranoside (b), quercetin-3-O-β-D-galactopyranoside
(c), kaempferol (d), kaempferol-3-O-α-D-arabinopyranoside (e), kaempferol-3-O-β-D-galactopyranoside (f), and vogelin J (g) calculated in gas
phase at the B3LYP/6-31+G (d,p) level. Atom color code: oxygen (red), carbon (gray) and hydrogen (white). Doted lines show intramolecular
O–H···O hydrogen bonding and O–H···π interactions (C–H···O hydrogen bonds are not shown).

The geometries of quercetin (Fig. 2, a) and kaempferol (Fig. 2,

d) are characterized by planar geometry, the torsion angle φ 3-2-
1’-6’ is 0°. This planarity results in full conjugation through the
molecule [39]. In contrast to quercetin and kaempferol, the
geometry of their glycosylation derivatives 2–5 is not planar
with φ3-2-1’-6’ = 24°–61°. The largest deviation in planarity
occurs for Quercetin-3-O-β-D-galactopyranoside, due to the
large rotation of ring B as to allow for hydrogen bonding
between its OH groups and the sugar group (Fig. 2, c). The
hydroxyl group of the CH2OH sugar group in kaempferol-3-O-
β-D-galactopyranoside interacts with ring B through O–H···π
interaction (Fig. 2, f). Such interaction mode is known to
provide stabilization energy of ~2.5 kcal/mol [41]. Vogalin J (6)
is also characterized by a nonplanar geometry, φ3-2-1’-6’ = 18°
(Fig. 2, g). The non-planarity of 6 is a consequence of the
absence of the OH group at position 3, which when present
stabilizes the planar conformation through the week C–H···O
hydrogen bonding. It can thus be substantiated that flavonoid Fig. 3. Relaxed potential energy scan of quercetin calculated
aglycones possess planar geometry only when possessing an by scanning the dihedral angle φ3-2-1’-6’ in gas phase at
OH group at position 3 and the glycosylated flavonoids are B3LYP/6-31+G (d,p) level. ΔE is the energy relative to the
nonplanar. Distortion in planarity results in ring B being not lowest energy conformer (φ3-2-1’-6’ = 180°).
totally conjugated to the rest of the molecule. To calculate the
energy barrier associated with rotation of the φ3-2-1’-6’ dihedral 3.2.4 Optimized geometries of radical flavonoids
angle, we performed a relaxed potential energy surface scan of One of the proposed mechanisms for the antioxidant activity of
the φ3-2-1’-6’ dihedral angle from 0° to 180° and the results are flavonoids is via scavenging free radicals and hence
shown in Fig. 3. Calculations show an energy barrier of 5.1– preventing their oxidation. The activity of flavonoids as
5.6 kcal/mol for a 180° rotation. antioxidants thus largely depends on the stability of their free
radicals. Toward understanding the experimental trend in the
antioxidant activity of the six isolated flavonoids, we perform
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geometry optimization on free radicals derived from these While radicals formed at C-4ʹ remain the most stable in both
flavonoids through abstraction of a hydrogen atom from the quercetin and its glycosides 2 and 3, results suggest that
different aromatic OH group. The optimized geometries quercetin radical is more stable. This is attributed to the planar
together with the total electronic energies of these radicals are geometry of quercetin radical, resulting in full conjugation
reported in Fig. 4. through the molecule. This indicates that compound 1 is a
The optimized geometries of quercetin radicals (Fig. 4, a–e) better radical scavenger and hence a better antioxidant than
possess planer geometry, φ3-2-1’-6’ = 0°. Conformers b, c, d, and compounds 2 and 3. This is in agreement with experimental
e, are 2.61, 6.99, 13.84, and 22.68 kcal/mol less stable than results. It is found that the antioxidant activity of 1 is ~14
conformer a, indicating that radical stability follows the order μg/mL compared to ~35 μg/mL for 2 and ~37 μg/mL for 3.
C-4ʹ > C-3ʹ > C-3 > C-7 > C-5. In order to investigate the influence of the sugar groups of
Radicals of quercetin-3-O-α-D-arabinopyranoside (2), are compounds 4 and 5 on the stability of the various possible free
reported in (Fig. 4, f–i). In these four radicals, ring B is non- radicals, we optimized the geometry of four free radicals of
planar with ring C (φ3-2-1’-6’ = 29°–34°). In respective, kaempferol (Fig. 4, n–q). Similar to quercetin, all radicals are
conformers g, h, and i are 1.30, 13.97, and 23.68 kcal/mol less planar. The trend in the stability of these four radicals follows
stable than conformer f. This suggests that glycosylation does the order C-3 ≈ C-4' > C-7 > C-5 (conformers o, p, and q are
not alter the relative stability of the different free radicals (C-4ʹ 0.03, 6.01, and 15.00 kcal/mol less stable than conformer n).
> C-3ʹ > C-7 > C-5). This indicates that the 6.99 kcal/mol higher stability of C-4ʹ
Free radicals derived from the most stable conformer of radical relative to C-3 radical in quercetin (1) is due to
quercetin-3-O-β-D-galactopyranoside (3) are shown in (Fig. 4, stabilization of the C-4ʹ radical by hydrogen bonding with the
j–m). Loss of H from the OH group at C-3ʹ or C-4ʹ breaks the OH group at C-3ʹ. In another mean, the radical formed at C-4'
hydrogen bonding between ring B and the sugar group. Similar in quercetin (1) is more stable than that of kaempferol.
to radicals of compound 2, these four radicals have nonplanar Quercetin (1) is hence a better radical scavenger and better
arrangements of rings B and C (φ3-2-1’-6’ = 30°–60°). Compared antioxidant than kaempferol. This matches the experimental
to the most stable radical j, the radicals k, l, and m are 0.93, finding of an antioxidant activity of ~14 μg/mL for quercetin
10.21, and 19.44 kcal/mol less stable, respectively. and ~40 μg/mL for kaempferol.

Fig 4: Optimized geometries of free radicals of compounds 1 (a–e), 2 (f–i), 3 (j–m), kaempferol (n–q), 4 (r–t), 5 (u–w), and 6 (x, y) generated
by abstraction of a hydrogen atom from the aromatic OH groups. Numbers represent the total electronic energy (in a.u.) and doted lines show
intramolecular O–H···O hydrogen bonds and O–H···π interactions.
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Free radicals formed by the removal of hydrogen from the agents among flavonoids is of great interest being safely
aromatic OH groups at C-4ʹ, C-5, and C-7 of compound 4 are consumed with large quantities in fruits and vegetables [43].
reported in Fig. 4 (conformers r–t) and those formed of Some plant-derived extracts rich with flavonoids such as
compound 5 are shown in Fig. 4 (u–w). Free radicals of both quercetin glycosides showed high activity against viral
compounds are non-planar, φ3-2-1’-6’ = 27°–32° for radicals of 4 infection in in vivo and in vitro studies. Quercetin and
and φ3-2-1’-6’ = 12°–19° for radicals of 5. Calculations show that quercetin-3-O-glycosides showed anti-Mayaro virus activity
[1]
structures s and t are 5.28 and 14.80 kcal/mol less stable than . The flavonoid dihydroquercetin (taxifolin) exhibit strong in
structure r and that structures v and w are 4.42 and 14.13 vitro and in vivo antiviral activities against CVB4 [12] and HAV
[44]
kcal/mol less stable than structure u. This shows that . Some flavonoids have also showed antiviral activities
glycosylation of kaempferol does not alter the relative stability against influenza virus [45, 46], HSV-1 and HSV-2 [22, 47],
of its free radicals. Again the planarity of kaempferol radicals enterovirus 71 [48] and dengue virus [49]. In our efforts to
allow for full conjugation and thus kaempferol is expected to explore the potential of natural products as source of low
possess a higher antioxidant activity than 4 and 5. It is toxicity and high antiviral selectivity candidate, we examined
experimentally found that kaempferol has an antioxidant the in vitro antiviral activity of six pure flavonoids (1‒6) from
activity of ~40 μg/mL compared to ~54 μg/mL for compound F. virens. To avoid the toxic effect of the flavonoid on the
4 and ~59 μg/mL for compound 5. virus carrier cells, Vero cells, the maximum non-toxic
Two radicals are optimized for vogelin J (6) (Fig. 4, n and o). concentration (MNTC) and the 50% cytotoxic concentration
Radical x (formed at C-4ʹ) is only 0.29 kcal/mol more stable (CC50) of the flavonoids 1‒6 were first determined. The results
than radical y (formed at C-7). A structural comparison revealed noticeable low cytotoxic effect for the glycosylated
between kaempferol and vogelin J (6) shows that the higher flavonoid (2‒5) than the aglycones 1 and 6. Consequently, the
stability of the free radicals at C-4ʹ relative to that at C-7 in the inhibitory effect of each compound at its MNTC was then
case of kaempferol is mainly due to the OH at C-3. The examined against HAV and CVB4 viruses using the MTT
absence of this hydroxyl group in 6 seems to destabilize the assay. Results of the virus inhibitory effects of the flavonoids
radical at C-4ʹ by ~ 6 kcal/mol. This is likely because the 1‒6 ( Table 1) demonstrated that quercetin (1), quercetin-3-O-
electronegative oxygen atom at C-3 provides stabilization of α-D-arabinopyranoside (2), and vogelin J (6) exert mild
the free radical. Among the six isolated compounds and inhibitory (20.3, 10.4 and, 8.6%) activity on CVB4 at their
kaempferol, vogelin J (6) is thus expected to have the lowest MNTC, 15.6, 15.6, and 62.5 μg/mL, respectively. On the other
antioxidant activity. In fact our experimental measurements hand, only the quercetin glycosides 2 and 3 exhibited mild
have shown no antioxidant activity of vogelin J (6) up to 100 inhibitory effects (8.0 and 12.3% at their MNTC (62.5 and
μg/mL. 15.6) on HAV, respectively. Kaempferol-3-O-glycosides (4
and 5) didn’t produce antiviral activity at the experimental
3.3. Antiviral activities of the isolated flavonoids conditions. Our results demonstrate that leaves of F. virens is a
A wide variety of active phytochemicals have been found to cheap source of flavonoids with mild antiviral activity against
have therapeutic effects against different genetically and the CVB4 and HAV viruses.
functionally diverse viruses [42]. Searching for therapeutic

Table 1: Cytotoxicity on Vero cells, and the anti-CVB4 and anti-HAV effects of the flavonoids 1–6.
Cytotoxicity (μg/mL)a Virus inhibitory activities (%)a
Compound CC50 MNTC CVB4 HAV
1 199.3±2.3 15.6±1.2 20.3±0.7 1.2±0.05
2 360.3±3.7 62.5±3.2 8.6±0.11 8.0±0.04
3 313.3±2.11 15.6±2.1 0 12.3±0.8
4 329.9±5.3 62.5±2. 0 0
5 313.3±1.19 31.3±1.9 0 0
6 195.1±2.4 15.6±1.5 10.4±0.14 0.4±0.13
Doxorubicinb 2.5±0.14 – – –
Ribavirinc
– – 49.0±1.2 54.0±1.7
(1.25 μg/mL)
a The values represent the mean ± SD
b Doxorubicin as standard cytotoxic drug
c Ribavirin as standard antiviral drug

Although the low antiviral activity of quercetin and its 3-O- 4. Conclusion
glycosides at our experimental conditions, this is the first Flavonoids in fruits and vegetables make them favorable
report on the anti-CVB4 and HAV properties of these healthy foods because of their antioxidant properties
flavonoids. Our results are an addition to the known antiviral associated with protection against inflammation, cancer, and
properties of flavonoids shown in recent reviews [42, 50]. numerous other chronic diseases. The structure-antioxidant
However, further anti-CVB4 and HAV investigations by in relationships of flavonoids have been discussed in various
vivo methods are required to ascertain the antiviral effects of reports. However, the majority of these discussions were
these fairly common metabolites in plants. Exploring for almost descriptive rather than elucidative [51]. An explanation
naturally occurring flavonoids with antiviral activities has attempt based on a quantum chemical study was shown for the
started since the 1940s, however, efforts are still necessary to flavonoid aglycones [39]. Our calculations shown here
develop these natural compounds as therapeutic antiviral demonstrated that flavonoid aglycones containing OH at
agents [43]. position 3 are planar and that glycosylation distorts this
planarity. The calculations predict antioxidant activity of the
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Journal of Pharmacognosy and Phytochemistry

compounds that follow the trend 1 > 2 ≈ 3 > kaempferol > 4 ≈ 8. Niranjan S, Garg VK. Ficus virens (white fig): an
5 > 6, which is in agreement with experimental findings. overview of its phytochemistry and pharmacology. Global
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tested flavonoids, our data from the antiviral experiment are an between flavonol glycosides and biological activities of
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5. Conflict of interest dihydroquercetin, a flavonoid from Larix sibirica, against
The authors declare that there is no conflict of interest. coxsackievirus B4 in a model of viral pancreatitis.
Archives of virology. 2016, 1-10.
6. Acknowledgments 13. Hunt JC, Schneider C, Menticoglou S, Herath J, Del Bigio
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