SUPPLEMENTARY MATERIAL
RP-HPLC-DAD method for the identification of two potential antioxidants agents viz.
verminoside and 1-O-(E)-Caffeoyl-β-gentiobiose from Spathodea campanulata leaves.
Pone Kamdem Bonifacea,d, Manju Singhb, Surjeet Vermab, Aparna Shuklac, Feroz Khanc,
Santosh Kumar Srivastavab and Anirban Pald*.
a
CSIR-TWAS fellow, Department of Biochemistry, Faculty of Science, University of
Dschang, P.O. Box-67, Dschang, Cameroon.
b
Department of Analytical Chemistry, CSIR-Central Institute of Medicinal and Aromatic
Plants, Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow-226015, Uttar Pradesh, India.
c
Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and
Aromatic Plants, Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow-226015, Uttar Pradesh,
India.
d
Department of Molecular Bioprospection, CSIR-Central Institute of Medicinal and
Aromatic Plants, Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow-226015, Uttar Pradesh,
India.
*Corresponding author
Telephone: +91-522-2718644, Fax: +91-522-2342666
Email address: a.pal@cimap.res.in, drapaul@gmail.com
Abstract:
A simple and reliable High Performance Liquid Chromatographic (HPLC) method was
successfully developed for the study of fingerprint chromatograms of extract and fractions
from the leaves of Spathodea campanulata (SC) using verminoside (1) and 1-O-(E)Caffeoyl-β-gentiobiose (2) as marker compounds. Antioxidant activity of SC was performed
using free radical of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) as an experimental
model. The docking study of selected target, tyrosinase and ligands (ascorbic acid,
compounds 1&2) was performed through Autodock Vina v0.8. Fingerprints of methanol,
chloroform, ethylacetate, n-butanol and water extracts could resolve 13, 11, 22, 16 and 5
peaks respectively.
Extract, fractions and compounds 1&2 previously isolated from SC displayed
remarkable antioxidant activity with radical scavenging activity (RS50) ranging from 2.56.7µg/ml. In silico study identified compounds 1&2 as potential inhibitors of tyrosinase
correlating with the observed antioxidant activity in vitro.
Key words: Spathodea campanulata, HPLC, fingerprint, chromatogram, antioxidant,
docking.
4. Experimental
4.1. Chemical investigation of SC
4.1.1. Instrument and chromatographic conditions
Chromatographic analysis was performed using a Waters modular HPLC system (Waters,
Milford, USA) consisting of 2996 photo diode array detector, 600 E pump, 717 autosampler
and C18 column (150 mm × 4.6 mm i.d.; 4 µm) maintained at 27oC. Empower software was
also used in this study.
4.1.2. Chemicals
L-ascorbic acid, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and Dimethylsulphoxide
(DMSO) were obtained commercially from Sigma Aldrich, USA. The organic solvents
(Ethanol, ethylacetate, n-hexane, n-butanol and chloroform) were purchased from Merck,
India. Verminoside and 1-O-(E)-caffeoyl-β-gentiobiose were previously isolated from
Spathodea campanulata (Boniface et al. 2014).
4.1.3. Plant material
The fresh leaves of SC were collected in the month of February 2011 from the adjoining area
of Dschang, located at latitude 5.4500° N and longitude 10.0667° E in the northwest region
of Cameroon. The specimen was identified and deposited at the Central Institute of Medicinal
and Aromatic Plants, Lucknow, under voucher number 12542.
4.1.4. Extraction
The shade dried leaves (250g) were coarsely powdered and extracted three times with
methanol (3x 1.5L). The combined MeOH extract was concentrated under vacuum at 40°C,
which afforded 27.5g of MeOH extract.
4.1.5. Fractionation of the methanol extract
The crude methanol extract was suspended in 500 ml of water and successively fractionated
with 500ml of n-hexane, chloroform, ethyl acetate and n-butanol (Bibi et al. 2010) to afford
respective fractions.
4.1.6. Preparation of the standard solutions
The marker compounds viz. verminoside and 1-O-(E)-caffeoyl-β-gentiobiose stock solutions
(1mg/ml) were separately prepared by dissolving 10 mg each in 5 ml methanol and diluted
with 5ml of the mobile phase. It was stored in a refrigerator at 4oC. A working solution was
prepared by pooling 50:50 of both standard solutions with mobile phase.
4.1.7. Sample preparation for HPLC analysis
Stock solution of defatted extract and fractions were prepared at concentration of 20 mg/ml in
methanol. The stock solution was subsequently diluted to 1mg/l. After filtering through 0.5
µm membrane filter, the diluted solution of extracts was injected on HPLC column in
triplicate.
4.1.8. Analytical procedure
Defatted samples and standards were diluted adequately with eluent prior to the injection.
The gradient elution was carried out by using solvent system comprising water:
trifluoroacetic acid (99.99:0.01, v/v) (Solvent-A), and solvent-B encompassing acetonitrile:
trifluoroacetic acid (99.99:0.01, v/v). A linear gradient programming was accomplished at
27oC with initial composition of 95% of A and 5% of B with a flow rate of 1.2ml/min
changing to 60% of A and 40% of B at 30min with flow rate of 0.6ml/min while the same
remained unchanged at 35 min. The initial conditions were restored beyond 37min with a
flow rate of 0.8 ml/min and detection was done at 240 nm.
4.1.9. Quantification of compounds1&2 in extract and fractions from Spathodea campanulata
The quantification of compounds1&2 in extract and fractions was performed based on their
peak areas on the HPLC chromatograms. Percent content of compounds1&2 in extract and
fractions were obtained through the formula: % content = (Peak area of sample/Peak area of
standard) X (Concentration of Standard/Concentration of sample) X 100.
4.2. Antioxidant activity of extract, fractions and compounds from SC
The antioxidant activities of plant extract, fractions and pure compounds eventually were
evaluated by DPPH radical scavenging method as described by Mensor et al. (2001) with
minor modifications. Briefly, the test samples, initially dissolved in DMSO were mixed with
a 20 mg/l of methanol based DPPH solution, to give final concentrations of 15.625, 31.25,
62.5, 125, 250, 500 and 1000 μg/ml. After 30 min at room temperature, the absorbance
values were measured at 517 nm and converted into percentage of antioxidant activity as
follows: % Inhibition = (Absorbance of control-Absorbance of test sample) ×
100/Absorbance of control. The RS50 (Concentration of sample in µg/ml scavenging 50% of
free radicals of DPPH) values were evaluated by plotting the curve of absorbance function of
the concentrations of each sample. L-ascorbic acid, a known antioxidant agent, was used as
positive control.
4.3. In silico study
The 2D structure of ascorbic acid, compounds 1&2 were drawn through ChemBio Office
suite Ultra v12.0 software (CambridgeSoft Corp., UK, 2010). They were subsequently
subjected to energy minimization using Discovery Studio v3.5 software (Accelrys Inc., USA)
by applying CHARMm forcefield to most of the small molecules. The 3D crystallographic
structure of tyrosinase was retrieved through Brookhaven Protein DataBank (PDB)
(http://www.pdb.org) (PDB ID: 1WX2). As a matter, structure 1WX2 was chosen due to its
better resolution and the presence of coordinated copper molecules in active site and bound
ligands. The docking study of selected target and ligands (ascorbic acid, compounds 1&2)
was done through Autodock Vina v0.8 (Molecular Graphics Lab at The Scripps Research
Institute, La Jolla, CA 92037, USA). Binding pose with the lowest docked energy belongs to
the top-ranked cluster was selected as the final model for post-docking analysis. The post
docking analysis was done by using UCSF Chimera v1.5.3 (NCRR, University of California,
San Francisco, supported by NIH P41 RR001081) and PyMOL (The PyMOL Molecular
Graphics System, Version 1.5.0.4 Schrödinger, LLC, USA).
4.4. Statistical analysis
The data, expressed as Mean±SE, were subjected to Kruskal–Wallis one way analysis of
variance (ANOVA) through Graphpad PRISM Software. Inter group comparisons were made
by Duns-test (non parametric) for only those responses which yielded significant treatment
effects in the ANOVA test. p< 0.05 was considered statistically significant.
Table S1: Anti-oxidant activity of extract, fractions, compounds 1 & 2 from Spathodea campanulata in terms of percentages of inhibition and
RS50 values.
In-vitro antioxidant activity
Extract, fractions and compounds
Inhibition of DPPH free radicals (%)
RS50 (µg/ml)
Methanol extract (250µg/ml)
90.36±0.016***
4.38
Hexane fraction (250µg/ml)
32.25±0.071**
6.07
Chloroform fraction (250µg/ml)
66.07±0.320***
4.95
Ethylacetate fraction (250µg/ml)
95.25±0.321***
3.41
Butanol fraction (250µg/ml)
90.86±0.333***
4.85
Last water fraction (250µg/ml)
70.73±0.133***
4.97
Compound 1 (62.5 µg/ml)
95.54±0.125***
2.5
Compound 2 (62.5 µg/ml)
93.48±0.013***
2.67
Ascorbic acid (62.5 µg/ml)
91.83±0.260***
3.25
Blank
00±0.00
-
**- P < 0.01, *** - P <0.001, test vs negative control (blank).
RS50: Concentration of extract, fractions, compounds 1 & 2 from Spathodea campanulata inhibiting 50% of DPPH free radicals.
RS50 values; From 0-5 µg/ml: Very active; From 5-10 µg/ml: Active; From 10-20 µg/ml: Moderately active; Above 20µg/ml: Not active.
Table S2 : Comparison of binding affinity of ascorbic acid, compounds1&2 in terms of docking energy and binding site residues against
receptor tyrosinase (PDB: 1WX2).
Compounds
Binding docking
Binding pocket residues within 4Å radius
energy (kcal/mol)
Ascorbic
acid
1
2
-5.4
-8.2
-7.7
Interacting residues and
length (4Å)
ILE-42, HIS-54, TRP-184, HIS-190, ASN-191, HIS-194, MET-201,
ALA-202, THR-203, SER-206
HIS-38, ILE-42, MET-43, ASP-45, ARG-55, TRP-194, HIS-190, ASN-
ASN-191 (2.6) and THR-203
(2.4)
ILE-42 (2.4, 2.5), ARG-55
191, HIS-194, MET-201, ALA-202, THR-203, GLY-204, SER-206
(3.2) and SER-206 (3.1)
ILE-42, ASP-45, HIS-54, ARG-55, GLY-183, TRP-194, ASN-188,
ILE-42 (2.2), ARG-55 (2.8,
HIS-190, ASN-191, HIS-194, VAL-195, MET-201, ALA-202, THR203, SER-206
3.1), ASN-188 (3.2) and THR203 (2.2)
Underlined residues indicate the key residues of the enzyme active site shared with ascorbic acid, compounds1&2.
Figure S1 : A representative chromatographic fingerprinting of methanol extract (A), chloroform (B),
ethylacetate (C), n-butanol (D) and water (E) fractions from Spathodea campanulata and assortment of
standards (F).
(a): 1-O-(E)-Caffeoyl-β-gentiobiose; (b): Verminoside; (*): Non identified peaks
Figure S2 : Docking pose of ascorbic acid (a), compounds 1 (b) & 2 (c) onto the receptor tyrosinase (PDB: 1WX2) with binding energies of 5.4, -8.2 & -7.7 kcal/mol respectively.
The orange balls represent coordinated-copper ions of the enzyme in the active site. The compounds and residues are represented in yellow and
white stick forms respectively. Hydrogen bonds (pink dot line) and their distances between two pairs of atoms are also indicated.
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