SUPPLEMENTARY MATERIAL
Isolation and characterization of methanol soluble fraction of Alternanthera philoxeroides
(Mart.) – evaluation of their antioxidant, α-glucosidase inhibitory and antimicrobial
activity in in vitro systems
Abhishek Bhattacherjee a # *1, Tanushree Ghoshb1, Rajarshi Sil c2 and Abhijit Datta a+3
a
Department of Botany, Presidency University, 86/1, College Street, Kolkata, West Bengal,
700073, India.
b
Department of Polymer Science and Technology, c Department of Biophysics, Molecular
Biology and Bioinformatics, University of Calcutta, 92, Acharyya Prafulla Chandra Road,
Kolkata- 700009, India.
#
Present address: Department of Biophysics, Molecular Biology and Bioinformatics, University
of Calcutta, 92, Acharyya Prafulla Chandra Road, Kolkata 700009, India.
+
Present address: Department of Botany, Jhargram Raj College, Jhargram, West Bengal 721507
Author’s contribution indicated by (1-3)
*
Corresponding author: Abhishek Bhattacherjee, Email: abhishek_raja67@rediffmail.com,
abhishekraja67@gmail.com.
Abstract
Alternanthera philoxeroides (Mart.) is a tropical weed commonly known as alligator weed. It
grows rapidly within a small span of time and easily available all over the world. The objective
of this work was to isolate and characterize the major phenolic components present in the
methanol soluble fraction (fraction X) of Alternanthera philoxeroides leaves and to explore the
biological activity (antioxidant, α-glycosidase inhibition and antimicrobial) of the fraction in in
vitro system. Chromatographic (HPLC) and spectroscopic (MALDI-TOF, 1H NMR) techniques
were used to purify and characterize the phenolics present in fraction X. Seven major phenolics
(safrole, myrcene, kaempferol, ferulic acid, salicylic acid, syringic acid and chlorogenic acid)
were found in fraction X. The fraction showed anti-oxidant property, dose dependent inhibition
of α-glycosidase activity and anti-microbial activity. Hence, isolated fraction X from the weed
has therapeutic potential in pathophysiological condition.
Experimental
Chemicals
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS), aluminium chloride (AlCl3), 2,5 dihydroxybenzoic acid (DHB), α-glucosidase and pNitrophenyl-α-D-glucopyranoside were purchased from Sigma-Aldrich Company, USA. HPLC
grade methanol, acetonitrile and acetic acid were purchased from Spectrochem, India. Other
chemicals were of analytical grade and purchased from Sisco Research Laboratory Pvt. Ltd.,
India.
Preparation of plant materials
Alternanthera philoxeroides (Mart.) Grisb were collected from the local ponds. Generally these
plants were found around ponds and they grow in a semi-aquatic condition. Collected plants
were cleaned under running tap water and then they were rinsed with double distilled water.
Cleaning is very essential to remove foreign particles present on the outer surface of these plants.
Plants were verified by their taxonomic classification and their herbarium specimens were also
prepared. A voucher specimen was deposited at the Central Herbarium of Department of Botany,
Presidency College with sample ID: PR-AB-AL21098. Leaves were detached and dried in oven
at 60 oC for 30 min. Dry leaves (50 g) were crushed in a mortar-pestle with methanol. Step wise
separation of the crude extract was done using a silica column (10 x 1 cm) (Scheme S1)
[Bhattachyerjee & Chakraborti, 2013]. After separation, resulting methanolic fraction was
sonicated for 1 h under cold condition and filtered through a Whatmann-1 filter paper. Fine powder
(23 mg) was obtained after lyophilization of the filtrate. This powder was used to prepare a
methanolic stock solution (1 mg/ml) (Fraction X). Three different concentration of fraction X (20,
40 and 60 µg/ml was used to evaluate its antioxidant, α-glucosidase inhibitory activity and antimicrobial activity.
Estimation of total polyphenol and flavonoid content
Amount of total polyphenol in fraction X was determined according to the published method
[Wolfe et al., 2003]. Fraction X (1 ml) was mixed with 5 ml Folin-Ciocalteu reagent (diluted
1:10 v/v) and 4 ml (75 g/l) of sodium carbonate. The tubes were vortexed for 20 s and left to
stand for 30 min at 40°C for color development. Absorbance was then measured at 765 nm using
the Hitachi-U2800 spectrophotometer. Results were calculated as mg/g of gallic acid equivalent
using the calibration curve and expressed as %. Total flavonoid content was estimated according
to the standard method [Ordon et al., 2006]. 2% AlCl3 (500 µl) ethanolic solution was added to
500 µl fraction X. After 1 h at room temperature, the absorbance was measured at 420 nm. A
yellow color indicated the presence of flavonoids. Total flavonoid content was calculated as
mg/g of quercetin using the following equation based on the calibration curve. Results were
expressed as %.
Chromatographic separation polyphenols
High performance liquid chromatographic (HPLC) technique was used to characterize the
polyphenols present in fraction X. Fraction was subjected to HPLC (LC-10AT VP Liquid
chromatograph, Shimadzu) analysis. HPLC photodiode array detector was set at 254 nm for
detection of phenolics. C-18 column [Luna 5u C18 (2) 100A, size-250 x 4.60 mm, Phenomenex,
USA] was used for HPLC separation. Methanol/water/acetonitrile/acetic acid (89:9:1:1) mixture
was used as mobile phase (flow rate 1 ml/min and scan time 100 min) [Saito et al., 2006].
Phenolics present in the fraction were further characterized my MALDI-TOF and 1H NMR
studies. Antioxidant, α-glucosidase inhibitory activity and anti-microbial activity of fraction X
was evaluated after proper characterization of the components.
Determination of active constituents by MALDI-TOF and NMR spectroscopy
Different fractions were purified via a semi preparative HPLC system attached with automatic
fraction collector. Collected fractions were lyophilized and the resultant powders were further
dissolved in water/ethanol (1:1 v/v) solution. 2, 5 dihydroxybenzoic acid (DHB) was used as
matrix (10 g/l) in water/ethanol (9:1). Sample suspension (0.5 µl) was mixed with 2 µl matrix
solution and analyzed by mass spectroscopy (4800 proteomic analyzer, applied biosystems,
USA) [Madeira & Florêncio, 2009]. Purified fractions were dissolved in CDCl3 and hydrogen
mapping was done by NMR (Bruker ultrashield plus 500 MHz) analysis.
Antioxidant property
The antioxidant activity of fraction X was determined using DPPH and ABTS radical scavenging
activity. DPPH radical scavenging activity was measured according to the method of LiyanaPathiana and Shahidi, (2005). DPPH (1 ml, 0.135 mM) prepared in methanol was mixed with 1.0
ml of fraction X ranging from 20 - 60 µg/ml. The reaction mixture was vortexed thoroughly and
left in the dark at room temperature for 30 min. The absorbance was measured at 517 nm. The
scavenging ability of the plant extract was calculated using the equation: DPPH scavenging
activity (%) = [(Absorbance of control – Absorbance of sample)]/ (Absorbance of control)] ×
100, where absorbance of control is the absorbance of DPPH + methanol and Absorbance of
sample is the absorbance of DPPH radical + sample (sample or standard).
ABTS radical scavenging activity was measured according to the method of Adedapo et
al., (2008). ABTS (7 mM) and potassium persulfate (2.4 mM) in equal amounts were mixed to
prepare the working solution. The mixture was allowed to react for 12 h at room temperature in
dark. The After 5-7 min, resulting solution was further diluted by mixing 1 ml ABTS+ solution
with 60 ml methanol to obtain an absorbance of 0.706 ± 0.001 units at 734 nm. The percentage
inhibition of ABTS + by the extract was calculated from the following equation: % inhibition =
[(Absorbance of control – Absorbance of sample)]/ (Absorbance of control)] × 100.
Results were expressed as mean ± standard deviation (S.D.) obtained for n observations.
α-glycosidase inhibition assay
Briefly, α-glucosidase (0.6 U/ml) was dissolved in 100 mM phosphate buffer (pH 7.0)
(containing 2 g/L bovine serum albumin and 0.2 g/L NaN3) and was used as an enzyme solution.
p-Nitrophenyl-α-D-glucopyranoside (5 mM) in the same buffer (pH 7.0) was added as a
substrate solution. Enzyme solution (50 µl) and 10 µl fraction X was dissolved in dimethyl
sulphoxide (DMSO) and incubated for 5 min at room temperature.
Following 5 min
preincubation at room temperature, the substrate solution (50 µl) was added and incubated for an
additional 5 min at room temperature. After incubation period, absorbance of the reaction
mixtures was measured at 405 nm. The increase in absorbance from zero time was measured and
percentage of α-glucosidase inhibition was calculated [Li et al., 2009]. Results were expressed as
mean ± standard deviation (S.D.) obtained for n observations.
Anti-microbial activity
Anti-microbial activity of fraction X was measured according to the method of Murray et al.,
(1995). Mueller Hinton Agar was used to prepare the petriplates for bacterial cultures
(Escherichia coli and Mycobacterium luteus). Test cultures were swabbed on the solidify media
and allowed to dry for 20 min. Fraction X loaded discs were placed on the surface of the media
and left for 40 min at room temperature. Negative control was prepared using the respective
solvent while streptomycin (10 µg/ disc) was used for positive control. Plates were incubated at
37°C for 24 h. Zone of inhibition (in mm) was recorded from respective control and
experimental petriplates. Results were expressed as mean ± standard deviation (S.D.) obtained
for n observations.
NMR annotation of the isolated compounds:
Safrole (C10H10O2) [Phenylpropane] M.W: 162.19 Da: 1H NMR assignments for the molecule:
5.924 (3, 1H, d, J=10.707), 5.927 (3, 1H, d, J=10.707), 6.645 (5, 1H, dd, J=2.861, J=1.555),
6.691 (7, 1H, dd, J=8.459, J=1.555), 6.581 (9, 1H, dd, J=8.459, J=2.861), 3.115 (10, 2H, d,
J=6.470), 5.671 (11, 1H, ddt, J=17.149, J=10.556, J=6.470), 4.856 (12, 1H, dd, J=10.556,
J=2.412), 4.855 (12, 1H, dd, J=17.149, J=2.412) (Figure 2a).
Myrcene [Monoterpene] M.W: 136.23 Da. This molecule was confirmed by its MALDI-TOF
spectra (Data not shown).
Kaempferol (C15H10O6) [Flavonol] M.W: 286.23 Da. 1H NMR assignments for the compound:
6.437 (11, 1H, d, J=2.321), 6.274 (13, 1H, d, J=2.321), 7.474 (14, 1H, ddd, J=8.291, J=1.300,
J=0.000), 7.474 (15, 1H, ddd, J=8.291, J=1.323, J=0.000), 7.156 (17, 1H, ddd, J=8.291, J=1.323,
J=0.000), 7.156 (18, 1H, ddd, J=8.291, J=1.300, J=0.000) (Figure 2b).
Ferulic acid (C10H10O4) [Hydroxycinnamic acid] M.W: 194.18 Da. 1H NMR assignments for the
molecule: 6.452 (4, 1H, d, J=15.568), 7.716 (5, 1H, d, J=15.568), 7.250 (7, 1H, dd, J=1.943,
J=0.633), 7.684 (8, 1H, dd, J=8.449, J=1.943), 6.780 (10, 1H, dd, J=8.449, J=0.633), 3.782 (13,
3H) (Figure 2c).
Salicylic acid (C7H6O3) [Monohydroxy benzoic acid] M.W: 138.12 Da. 1H NMR assignments
for the molecule: 7.939 (6, 1H, ddd, J=8.073, J=1.969, J=1.424), 7.051 (8, 1H, ddd, J=8.324,
J=1.969, J=1.234), 7.256 (9, 1H, ddd, J=8.073, J=7.350, J=1.234), 7.515 (10, 1H, ddd, J=8.324,
J=7.350, J=1.424)(Figure 2d).
Syringic acid (C9H10O5) [O-methylated trihydroxybenzoic acid] M.W: 198.17 Da. 1H NMR
assignments for the molecule: 7.258 (5, 1H, d, J=0.000), 7.258 (6, 1H, d, J=0.000), 3.800 (12,
3H), 3.801 (14, 3H) (Figure 2e).
Chlorogenic acid (C9H10O5) [Ester of caffic acid and (-) quinic acid] M.W: 354.31 Da. 1H NMR
assignments for the molecule: 6.551 (4, 1H, d, J=15.685), 4.965 (5, 1H, q, J=2.790), 7.736 (6,
1H, d, J=15.685), 3.266 (7, 1H, t, J=2.790), 1.748 (8, 1H, ddt, J=14.329, J=10.260, J=2.790),
1.808 (8, 1H, dq, J=14.329, J=2.790), 3.718 (11, 1H, d, J=2.790), 1.950 (12, 1H, dt, J=13.506,
J=2.790), 1.671 (12, 1H, ddd, J=13.506, J=10.260, J=2.790), 7.281 (13, 1H, dd, J=1.943,
J=1.165), 7.688 (14, 1H, dd, J=8.446, J=1.943), 6.769 (18, 1H, dd, J=8.446, J=1.165) (Figure 2f).
References
Adedapo AA, Jimoh FO, Afolayan AJ, Masika PJ. 2008. Antioxidant activities and phenolic
contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia
gummifera. BMC Complement Altern Med. 8:54–60.
Li YQ, Zhou FC, Gao F, Bain JS, Shan F. 2009. Comparative evaluation of quercetin,
isoquercetin and rutin as inhibitors of a-glucosidase. J Agric Food Chem. 57:11463–11468.
Liyana-Pathiana CM, Shahidi F. 2005. Antioxidant activity of commercial soft and hard wheat
(Triticum aestivium L.) as affected by gastric pH conditions. J Agric Food Chem. 53:2433–2440.
Madeira PJ, Florencio MH. 2009. Flavonoid-matrix cluster ions in MALDI mass spectrometry. J
Mass Spectrochem. 44:1105–1113.
Murray et al. 1995. Manual of clinical microbiology. 6th ed. Washington (DC): ASM.
Ordon AA, Gomez JD, Vattuone JM, Isla MI. 2006. Antioxidant activities of Sechium edule
(Jacq). Food Chem. 97:452–458.
Prain D. 1903. Bengal plants (Rep. ed. 1963). Botanical Survey of India.
Saito ST, Welzel A, Suyenaga ES, Bueno F. 2006. A method for fast determination of
epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea
using HPLC. Cienc Tecnol Aliment Campinas. 26:394–400.
Wolfe K, Wu X, Liu RH. 2003. Antioxidant activity of apple peel. J Agric Food Chem. 51:609–
614.
Scheme S1
Figure S1: Chromatographic separation of active components of fraction X. Phenolics were
separated from the fraction by a HPLC (UV detector 254 nm) using C-18 column with
methanol/water/acetonitrile/acetic acid (89:9:1:1) mobile phase. The separated compounds were
identified by comparing the chromatograms (retention time) of the standard compounds (safrole,
myrcene, kaempferol, ferulic acid, salicylic acid, syringic acid and chlorogenic acid) subjected to
HPLC under similar condition. Different phenolics and their content in 100 µg of fraction X
were also documented here.
Figure S2: Characterization of different phenolics using 1H-NMR and mass spectrometry. 1HNMR and mass spectrometric (inset) analysis of a) sefrole, b) kaempferol, c) ferulic acid, d)
salicylic acid, e) syringic acid and f) chlorogenic acid purified from fraction X.
Figure S3: Antioxidant property of fraction X. Dose dependent (20, 40 and 60 µg/ml) radical
quenching property of fraction X was demonstrated in DPPH and ABTS systems. Results were
expressed as mean ± S.D of five different experiments.
Figure S4: Dose dependent (20, 40 and 60 µg/ml) inhibitory effect of fraction X on αglucosidase activity. Results were expressed as mean ± S.D of five different experiments.
Figure S5: Antimicrobial activity of fraction X. a) Increased zones of inhibition were observed
in Escherichia coli (a) and Mycobacterium luteus (b) culture plate suggest inhibitory effect of
fraction X.