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Phenolic constituents from Platycerium bifurcatum and their antioxidatant properties

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Semi-preparative HPLC separation of the ethyl acetate fraction of the leaf extract of Platycerium bifurcatum (Cav.) C. Chr. lead to the isolation of a polyphenolic compound- quercetin 3-O-β-D-glucopyranoside (1) and a carboxylic acidchlorogenic acid (2).The structures of the compounds were elucidated by 1D (1H, 13C), 2D NMR (COSY, HSQC, and HMBC); mass spectroscopy (HPLC/ESI-MS) and by comparism with the reported data. The antioxidant activities of the isolated compounds (1–2) were evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. These secondary metabolites were isolated for the first time from Platycerium bifurcatum. Keywords: P. bifurcatum; Polyphenolic; Structure elucidation; Antioxidant activities.
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M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
48
ISSN 0974 –
5211
Phenolic constituents from Platycerium bifurcatum and their
antioxidatant properties
M.O. Agbo
1, 2*
, C.O. Nnadi
2
, N.N. Ukwueze
3
, F.B.C. Okoye
4
1
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine Universität,
Düsseldorf 40225, Germany.
2
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, University of Nigeria, Nsukka 41001, Enugu State, Nigeria.
3
Department of Pure & Industrial Chemistry, University of Nigeria, Nsukka. Enugu State,
4
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria
*Corresponding Author
(Received 16 November 2013; Revised 01 December 2013-30 January 2014; Accepted 02 February 2014)
ABSTRACT
Semi-preparative HPLC separation of the ethyl acetate fraction of the leaf extract of
Platycerium bifurcatum (Cav.) C. Chr. lead to the isolation of a polyphenolic
compound- quercetin 3-O-β-D-glucopyranoside (1) and a carboxylic acid-
chlorogenic acid (2).The structures of the compounds were elucidated by 1D (
1
H,
13
C), 2D NMR (COSY, HSQC, and HMBC); mass spectroscopy (HPLC/ESI-MS) and
by comparism with the reported data. The antioxidant activities of the isolated
compounds (1–2) were evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
assay. These secondary metabolites were isolated for the first time from Platycerium
bifurcatum.
Keywords: P. bifurcatum; Polyphenolic; Structure elucidation; Antioxidant activities.
INTRODUCTION
Platycerium bifurcatum (Cav.) or ''staghorn fern'' is an epiphyte, growing naturally on
branches and trunks of trees in the tropical, subtropical jungles and rain forest (Bode
and Oyedapo, 2011; Hemipman and Roos, 1982). Platycerium bifurcatum is a lower
plant since it does not have roots and produce spores to reproduce, rather than
flowers. It belongs to the family Polypodiaceae and is propagated from its spores.
Apart from its ornamental uses, it has been reported to have wide medicinal uses. In
Nigeria, young leaves of Platycerium bifurcatum are prescribed as a common anti-
ulcer remedy (Pemberton, 2003). The leaf extract of Platycerium bifurcatum is
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M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
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49
reported to have diverse uses such as preventing miscarriages in women when taken
two months after conception (Flora and Ubah, 2006), treating oedema, coughs and
hypertension (Mensah, et al., 2006). Antibacterial potency of methanol extract of P.
bifurcatum using agar dilution method has been reported (Ojo, et al., 2007). The
isolation and characterization of polysaccharides from P. bifurcatum has also been
reported (Omeje, et al., 2007). Currently, there is great interest in finding antioxidants
from natural sources to minimize oxidative damage to cells. Oxidative stress has been
linked to cancer, aging, atherosclerosis, ischemic injury, inflammation and
neurodegenerative health conditions such as Parkinson’s and Alzheimer’s diseases.
Oxidative damage is caused by free radicals and reactive oxygen species, mostly
generated endogenously (Aniya, 2002). Free radicals are atoms or groups of atoms
that have at least one unpaired electron, which make them highly unstable and
reactive. Living organisms accumulate free radicals through both normal metabolic
processes and exogenous sources. Although free radicals have beneficial effect during
energy production and as antibacterial, excessively high levels of free radicals cause
damage to cellular proteins, membrane lipids and nucleic acids, and eventually cell
death (Asres, et al., 2006; Pham-Huy, et al., 2008). Plant polyphenols provide a
protection against these diseases, since they have high antioxidant properties. Here,
we isolated two pure secondary metabolites from the air-dried leaves of Platycerium
bifurcatum by the method of Agbo, et al. (2013). The anti-oxidative potentials of
these isolated compounds were assessed by using DPPH radical scavenging method
previously described by Tsevegsuren, et al. (2007). This is the first report of
polyphenols from the leaves of Platycerium bifurcatum and its antioxidant activities.
P. bifurcatum is widely distributed in Nigeria and this has given an insight into its
ethno medicinal uses.
MATERIALS AND METHODS
Instruments and materials: The optical rotation was recorded on a Perkin-Elmer 241
MC polarimeter. 1D and 2D NMR spectra were recorded using Bruker ARX 500
NMR spectrometer. LC-MS measurements were performed on a Thermofinnigan
LCQ DECA mass spectrometer coupled to an Agilent 1100 HPLC system. Analytical
HPLC analysis was performed with a HPLC system (Dionex, Munich, Germany).
Semi-preparative HPLC was performed on a MERCK HITACHI system equipped
with a UV Detector L-7400 and a Pump L-7100 connected to a Kipp & Zonen Flatbed
Recorder. Vacuum liquid chromatography (VLC) was performed on silica gel (230–
400 mesh, Merck) using a glass column (i.d. 3×30cm). Gel permeation column
chromatography (CC) was performed on Sephadex LH-20 (Merck, Germany) using a
glass column (i.d. 3×110cm). TLC was performed on TLC plates pre-coated with
silica gel 60 F
254
(0.20 mm thickness, Merck, Darmstadt, Germany) using various
solvent combinations as the mobile phase.
Plant Material: Platycerium bifurcatum leaves were collected from Orba in Enugu
State, Nigeria in January, 2012. The leaves were identified by Mr. A. O. Ozioko of
the Bio resources Conservation and Development Program (BDCP), Nsukka. A
voucher specimen (PB 2076) was deposited at the herbarium of the Institute.
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
50
Extraction, Purification and Isolation: The air dried powdered leaves (500g) were
macerated five times with 500ml of methanol and extracted at room temperature for
48h with agitation. The filtrate was evaporated in vacuum (40°C) to obtain the crude
methanol extract (20g), which was suspended in 400ml of 10% methanol (MeOH) in
water and the resulting mixture successively partitioned against n-hexane, ethyl
acetate (EtOAc) and butanol (n-BuOH) to obtain n-hexane (HF, 1.24g), EtOAc (EF,
9.83g), n-BuOH (BF, 5. 87g) and water (WF, 0.92g) fractions, respectively. The
EtOAc fraction (7.20g) was purified by vacuum liquid chromatography using silica
gel (230–400 mesh, 3.0×30 cm, 500g) as the stationary phase and eluted with a
gradient of n-hexane in EtOAc (10:0, 8:2, 6:4, 4:6, 2:8, 0:10, each 500ml) and of
dichloromethane (DCM) in methanol (9:1, 7:3, 9.5:5, 3:7,1:9, each 1000ml) to afford
11 sub-fractions (EF1-EF11). Fraction EF7 (233.7mg) was further fractionated on
Sephadex LH-20 (DCM/MeOH, 1:1) to afford nine sub-fractions (EF7A–EF7I).
Fraction EF7B (41.3mg) was separated by semi-preparative HPLC using MeOH–H
2
O
as mobile phase to give compound 1 (8.6 mg, t
R
= 20.2min). Similarly, fraction EF11
(162.3mg) was further fractionated on Sephadex LH-20 (100% MeOH) to afford four
sub-fractions (EF11A–EF11D). Fraction EF11C (43.1mg) was separated by semi-
preparative HPLC using MeOH–H
2
O as mobile phase to give compound 2 (10.0 mg,
t
R
= 13.2min). Structure elucidation of the isolated compounds was carried out by
using
1
H,
13
C-NMR,
1
H,
1
H-Correlation spectroscopy (COSY),
1
H,
13
C- Heteronuclear
Multiple Quantum Coherence (HMQC) experiment,
1
H,
13
C-Heteronuclear Multiple
Bond Correlation Spectroscopy (HMBC), mass spectroscopy (HPLC/ESI-MS) and
UV spectroscopy.
DPPH radical scavenging activity: The radical scavenging activity of the compounds
was determined by the DPPH assay (Tsevegsuren, et al., 2007). The compounds were
dissolved in methanol to give a concentration of 1mg/1000µl stock solution. Ten
microliter (10µl) of the test samples was added to 490µl DPPH solution
(4.5mg/100ml) in an ependorf vial. The mixture was incubated for 3 min and the color
change (from deep violet to light yellow) of the DPPH free radical was measured by
recording the absorbance using a UV/Visible spectrophotometer (Perkin Elmer,
Lambda 25) at 517nm. Prior to the measurement, the difference in absorption between
a DPPH blank solution and the positive control (propyl gallate, 76µM) was
determined. This difference was then taken as 100% antioxidative activity. The
percent antioxidative activity was determined from the difference in absorption
between the samples at 76 µM and the DPPH blank as follows:
aA=% antioxidative activity compared to the positive control, AB=absorption of the DPPH blank
solution, Ap = absorption of the sample, and Apos = absorption of the positive control (propylgallate).
Measurements were done in triplicates, and the IC
50
values were determined by linear
regression.
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
51
RESULTS
Semi-preparative HPLC of the EtOAc fraction of the leaves of Platycerium
bifurcatum resulted in the isolation a polyphenolic glycoside and a carboxylic acid.
These known compounds were identified as quercetin 3-O-β-D-glucoside and 5'-O-
caffeoyl qunic acid (chlorogenic acid) by spectroscopic (1D and 2D NMR, and mass
spectra) comparison with the reported values. The antioxidative properties of the
isolated compounds showed that the compounds (IC
50
= 31.27±0.12 and 47.21±0.11)
were more active compared to the positive control with IC
50
values of 68.46±0.16.
DISCUSSION
Compound 1 was obtained as a pink amorphous powder, [α]
D20
= -10.9 (c 0.100,
methanol). The UV spectrum showed maximum absorption bands of a flavonoid
system at λ
max
360.0, 256.0, and 220.0 nm. The HPLC-MS (positive ion mode) at m/z
465 [M+H]
+
was compatible with the molecular formula C
12
H
20
O
12
. The
1
H NMR
(Table 1) showed aromatic protons with signals at [δ
H
6.20 (1H, d, J=2.0 Hz), δ
H
6.39
(1H, d, J=2.1 Hz)] consistent with the meta-coupled protons H-6 and H-8 of ring-A
and an ABX system at [δ
H
7.71 (1H, d, J=2.2 Hz, H-2'), δ
H
7.59 (1H, dd, J=8.5, 2.3
Hz, H-6'), δ
H
6.87 (1H, d, J=8.5 Hz, H-5')] corresponding to the catechol protons on
ring-B (Ebada, et al., 2008). The spectroscopic data indicated that this compound was
a flavonoid glycoside. From the mass spectrum data, fragments at m/z 303 [M-162 +
H]
+
(loss of glucose), together with the occurrence of an anomeric proton at δ
H
5.25 d
(J =7.6 Hz) was indicative of a glucose unit in the molecule (Figure 2). The
1
H NMR
data also revealed that the aglycone part was glucose by comparison of spectroscopic
data with those of the reported values (Islam, et al., 2012). The rest of the protons in
the sugar moiety resonated between 3.42 and 3.71 ppm. The anomeric proton
coupling constant of compound 1 (H-1'', J=7.6 Hz) define the stereochemistry of
glycosidic linkage as β (Mina, et al., 2013; Guo et al., 2012).The
1
H NMR data
recorded on MeOH-d
4
were in good agreement with published values (Jan, 2003; Li,
et al., 2011). Thus, compound 1 was elucidated as quercetin 3-O-β-D-glucoside.
Compound 2 was obtained as a yellow amorphous powder, [α]
D20
= -21.4 (c
0.100, methanol). The UV spectrum showed absorption maxima λ
max
325.7, 239.9 and
217.0 nm. The molecular formula C
16
H
18
O
9
was deduced from the ESI-MS with ion
peaks at m/z 355.1 [M+H]
+
(Figure 3). Thus the molecular mass of compound 2 was
found to be 354 g/mol with nine double-bond equivalent. The
1
H and
13
C NMR
assignments were based on the DEPT and 2D-NMR (COSY, HMQC and HMBC)
experiments. The
1
H NMR spectrum of 2 in MeOH-d
4
exhibited signals in aliphatic
and aromatic range. In aromatic range, the
1
H-NMR spectrum (Table 2) revealed an
ABX system at [δ
H
7.03 (1H, d, J=2.0 Hz, H-2'), 6.91 (1H, dd, J= 8.2, 2.1 Hz, H-6'),
6.73 (1H, d, J=8.0 Hz, H-5')] characteristic for 1, 3, 4-trisubstituted benzene. The
1
H
NMR spectrum also showed a set of trans-olefinic protons at [δ
H
(7.57 and 6.25 (each
d, J = 15.8 Hz, H-7' and H-8')], indicating the presence of an (E)-caffeic acid moiety
(Satake, et al., 2007; Sri, et al., 2007). The presence of a quinic acid moiety was
further confirmed by the signals from two methylene protons at [δ
H
2.10 (d, J = 7.8
Hz), 2.16 (dd, J = 14.7,3.0 Hz) and 2.00 (d, J =11.8 Hz), 1.96 (d, J = 6.3 Hz)] for H-2
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
52
and H-6 respectively, with three methine protons at [δ
H
3.68 (dd, J= 9.9,3.2 Hz, H-3),
4.12 (d, J = 3.1Hz, H-4), and 5.38,m (H-5)] together with the corresponding carbon
resonances at δ
C
39.48, 41.14, 75.54, 73.54, 72.88 for C-2, C-6, C-4, C-3, and C-5
respectively.In the COSY spectrum, H-2 was found to correlate with H-3, which
further coupled to H
2
-2 and H-4. The latter proton showed correlation to H-5 which in
turn correlated to H
2
-6 thus revealing a typical coupling pattern attributable to a
quinic acid residue. The trans-olefinic protons were seen to correlate with the ester
C=O (δ
C
168.75) as was observed in the HMBC spectrum (Pauli et al., 1999
)
. The
13
C
NMR spectrum for 2 (Table 2) displayed 16 carbon signals differentiated as two CH
2
at δ
C
41.14 and 39.48, eight CH (including two olefins at δ
C
147.71 and 114.88, three
oxygenated at δ
C
75.54, 73.54, and 72.88, and three sp
3
carbons at δ
C
123.61, 117.05,
and 114.96), and six C [(including two oxygenated at δ
C
148.11, and 147.71, one keto
carbonyl at δ
C
169.85, one ester carbonyl at δ
C
168.75, one quaternary carbon at δ
C
78.18, well as one further quaternary carbon whose carbon signal was not detected
(C-1')] corresponding to C
16
H
18
O
9
. The HMQC spectroscopic data were used to
assign protons attached to their corresponding carbons (Table 2). The complete
structure was established by the HMBC spectrum, which shows correlation between
protons and carbons for two bonds. On the basis of these spectroscopic data, the
structure of 2 was identified as 5'-O-caffeoyl qunic acid (chlorogenic acid)
(Saracoğlu, et al., 2002).
The antioxidative potential of the isolated compounds of Platycerium
bifurcatum was investigated by DPPH free radical scavenging method. The isolated
compounds showed stronger antioxidative potential than Propyl gallate (positive
control) (Table 3). Some structure-activity relationships were observed with the anti-
oxidative properties of compound 1.These include the 3-glucosylation of the quercetin
nucleus (Williamson, et al., 1996); the presence of an ortho-dihydroxyl group in the
ring-B of the quercetin nucleus together with the 2,3-double bond in conjugation with
the 4-oxo functional groups. These functional groups help in the delocalisation of
electrons on the quercetin nucleus (Procházkova, et al., 2011). The anti-oxidative
properties of chlorogenic acid has been reported by Li et al., (2011) with an IC
50
of
0.1135mg/ml which compares favourably with our own value of IC
50
= 0.1175mg/ml
(49.21µM).
Isolated compounds
(-)- Quercetin 3-O-β-D-glucopyranoside (1): Pink amorphous powder, C
12
H
20
O
12
,
LC-MS: m/z 464 [M]
+
, 465 [M+H]
+
, 950 [2M + Na]
+
(Figure 2); UV λ
max
(MeOH)
nm: 360.0, 256.0, 220.0; [α]
D20
= -10.9 (c 0.100, MeOH);
1
H-NMR (CD
3
OD, 500
MHz): Table 1.
Chlorogenic acid (2): Yellow amorphous powder, C
16
H
18
O
9
, LC-MS: m/z 354 [M]
+
,
355 [M+H]
+
, 163 [caffeoyl-OH]
+
, 164 [caffeoyl-O]
+
(Figure 3); UV λ
max
(MeOH):
325.7, 239.9, 217.0; [α]
D20
= -21.4 (c 0.10, MeOH);
1
H-NMR (CD
3
OD, 500 MHz)
and
13
C-NMR (CD
3
OD, 125.76 MHz): Table 2.
Statistical analysis: The results were expressed as Mean±SEM of three
measurements. Analysis of variance was performed using one-way ANOVA.
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
53
Significant differences between means were determined by student’s t-test, and P
values<0.05 were regarded as significant.
CONCLUSION
Thus isolated compounds had good antioxidative activity in the DPPH assay. The
structures of the isolated polyphenols were obtained by comparism with reported
values. These secondary metabolites were isolated for the first time from Platycerium
bifurcatum.
Acknowledgements: The authors are grateful to Prof Dr Peter Proksch of the Institute of
Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Düsseldorf, Germany for
allowing us to use his Laboratory for this study. We are also grateful to Mr. Abugu, Alphonsus for
proof-reading the manuscript.
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55
Table -1:
1
H-NMR Data of Quercetin 3-O-β-D-glucoside (1).
Position δ
H
(ppm) J (Hz) COSY
6 6.20,d 2.0
8 6.39,d 2.1
2' 7.71,d 2.2
5' 7.59,m 6'
6' 6.87,dd 8.5,2.3 5'
1'' 5.25,d 7.6 2''
Sugar protons 3.71-3.42,m
Table -2:
1
H-NMR and
13
C-NMR Data of Chlorogenic acid (2).
Position δ
C
(ppm) δ
H
(ppm) J (Hz) COSY HMBC (HC)
1 78.18
2 39.48 2.10,d 7.8 3 3
2.16, dd 14.7,3.0 3 4
3 73.54 4.12,d 3.1 2, 4
4 75.54 3.68, dd 9.9,3.2 3, 5
5 72.88 5.38, m 4, 6
6 41.14 2.00, d 11.8 5
1.96,d 6.3 5
7 169.85
1' nd*
2' 114.96 7.03,d 2.0 6'
3' 147.71**
4' 148.11
5' 117.05 6.73,d 8.2 6'
6' 123.61 6.91, dd 8.2,2.1 2', 5'
7' 147.71** 7.57, d 15.8 8' 9'
8' 114.88 6.25, d 15.8 7' 9'
9' 168.75
*Not detected. **Interchangeable peaks.
Table- 3: Median Inhibitory Concentration (IC
50
) of Compounds 1-2
a
.
Compounds IC
50
(µM)
Quercetin 3-O-β-D-glucoside (1) 31.27±0.12
Chlorogenic acid (2) 47.21±0.11
Propyl gallate 68.46±0.16
Values expressed are Mean ± SEM; *P ˂ 0.05; n= 3
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
56
Figure-1: Structures of the isolated Compounds (1-2).
M.O. Agbo, et al. /Journal of Natural Products, Vol. 7(2014): 48-57
Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved
57
Figure -2: HPLC Analysis of Compound 1.
1 = HPLC Chromatogram of Compound 1; 2 = UV Spectrum of Compound 1
3 = Positive ion (3) and Negative ion (4) Modes HPLC–MS spectra of Compound 1
Figure-3: HPLC Analysis of Compound 2.
1 = HPLC Chromatogram of Compound 2; 2 = UV Spectrum of Compound 2
Positive ion (3) and Negative ion (4) Modes HPLC–MS spectra of Compound 2
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