African Journal of Food Science and Technology (ISSN: 2141-5455) Vol. 4(1) pp. 1-8, January, 2013
Available Online http://www.interesjournals.org/AJFST
Copyright©2013 International Research Journals
Full Length Research Paper
HPLC/DAD/MS phenolic profile, antioxidant activities
and inhibitory action of struchium sparganophora
(Linn) and telfairia occidentalis (Hook. F) Against low
density lipoprotein oxidation
*
Sule Ola Salawu, David Morakinyo Sanni, Akintunde Afolabi Akindahunsi
Department of Biochemistry, Federal University of Technology, Akure, Nigeria
Accepted 4 December, 2012
The present study sought to investigate the phenolic composition of Struchium sparganophora and
Telfairia occidentalis extracts using HPLC/DAD/MS techniques, evaluate their radical scavenging
activities and inhibitory action against copper induced human Low Density Lipoprotein (LDL) oxidation.
The chromatographic analysis revealed the presence of apigenin derivatives, chlorogenic acid and
three dicaffeoyl derivatives in Strcuchium sparganophora while chlorogenic acid, rutin and three
Kaempferol derivatives were identified Telfairia occidentals. ABTS radical scavenging activities and
DPPH radical scavenging activities are higher in Telfairia occidentalis compared with Struchium
sparjanophora. Also, the total phenolic content was higher in Telfairia occidentalis compared with
Strcuchium sparganophora. Similarly, the inhibitory action of the vegetable extracts against copper
induced human LDL oxidation showed that Telfairia occidentalis posses a higher inhibitory action
(63%) compared with Struchium sparjanophora (55%). Therefore, the antioxidant activities and the
ability of the extract to inhibit the copper induced human LDL oxidation shows that the studied
vegetables could be harnessed in the management of some coronary and cardiovascular disorders.
Keywords: Phenolic content, antioxidant activities, LDL oxidation, leafy vegetables.
INTRODUCTION
Green leafy vegetables are important protective foods
and highly beneficial for the maintenance of health and
prevention of disease. The dark green leaves provide a
high amount of carotene, ascorbic acid and micro
minerals which play important roles in nutrient
metabolism and slowing down of degenerative diseases
(Yi-Fang et al., 2002). Africa is endowed with a variety of
traditional vegetables and different types are consumed
by the various ethnic groups for different reasons.
Vegetables are the cheapest and most available sources
of important proteins, vitamins, minerals and essential
amino acids in Nigeria (Okafor 1983).
Degenerative diseases such as cancer, cardiovascular
disease, osteoporosis, aging and other mutagenic
processes are caused by oxidative damage to cell
components and human DNA. Antioxidants present in
*Corresponding Author Email: sosalawu@yahoo.com
food can help limit this damage by disabling free radicals
and other harmful reactive species (thus minimizing
oxidation) and by stimulating the body’s own defense
systems to fight disease by using these groups of
protective chemicals (Scalbert et al., 2005).
Polyphenolic compounds are commonly found in both
edible and inedible plants, they have multiple applications
in food, cosmetic and pharmaceutical industries
(Kahkonen et al., 1999). The antioxidant capacity of
phenolic compounds is mainly due to their redox
properties, which allow them to act as reducing agents,
hydrogen donors, singlet oxygen quenchers or metal
chelators. In addition to their roles as antioxidants, these
compounds exhibit a wide spectrum of medicinal
properties, such as anti-allergic, anti-inflammatory, antimicrobial,
anti-thrombotic,
cardio-protective
and
vasodilatory effects (Balasundram et al., 2006).
Phytochemicals, including phenolics are suggested to
be the major bioactive compounds contributing to the
health benefits of vegetables and fruits (Yang et al.,
2 Afr. J. Food Sci. Technol.
2004; Sinelli et al., 2008). It was shown that the health
properties of these natural products depend on the
contents of bioactive compounds, mainly phenolic
compounds, and partly on dietary fibers (Chun et al.,
2005).
Green leafy vegetables are widely consumed in Nigeria
as source of important proteins, vitamins, minerals and
essential amino acids with emerging interest on their
health benefits. The aim of the present investigation
therefore is to analyze the phenolic composition,
antioxidant activities and the inhibitory effect of the
selected vegetables on copper-induced Human Low
Density Lipoprotein (LDL).
MATERIALS AND METHODS
Materials
The vegetables (Telfairia occidentalis and Struchium
sparganophora) were collected from local farms in Akure,
south-western Nigeria, and voucher specimens were
deposited at the Department of Biochemistry, Federal
University of Technology, Akure, Nigeria and Department
of Pharmaceutical Science, University of Florence, Italy.
The samples were air-dried (5–7 days) and then oveno
dried at 30 C to constant weight. The dried samples were
then kept in sealed air-tight polythene bags until analysis.
The dried samples were finely powdered immediately
before extraction. All the standards used were purchased
from Extrasynthese (Geney, France), with the only
exception of rutin from Sigma–Aldrich (St. Louis, MO,
USA).
Extraction
A dried sample (1 g each) was extracted with 40 ml (20
ml x 2) of ethanol/water 7:3 (v/v) with water acidified by
formic acid (pH 2.5). The samples were filtered and the
clear solution directly analyzed by HPLC/DAD/MS.
HPLC/DAD/MS analysis
Analyses were performed using an HP 1100 liquid
chromatograph equipped with HP DAD and 1100 MS
detectors; the interface was an HP 1100 MSD API-electro
spray. All the instruments were from Agilent Technology
(Palo Alto, CA, USA). The MS analyses were carried out
in negative mode with a fragmentor range between 80150 V.
Method
A
C12
column,
150 x 4mm
(4µm) Synergi max
o
(Phenomenex- Torrance CA) maintained at 30 C and
equipped with a 10 x 4 mm pre-column of the same
-1
phase was used with a flow rate of 0.4 ml min . The
eluents were H2O acidified to pH 3.2 by formic acid (A)
and acetonitrile (B). The following linear solvent gradient
was applied: from 95% A to 85% A in 5 min, to 75% A in
8 min and a plateau of 10 min, to 55% A in 12 min and a
plateau of 5 min, to 10% A in 3 min, and a final plateau of
2 min to wash the column. The total time of analysis was
45 min.
Quantitative evaluation
The standards chlorogenic acid, rutin and luteolin 7-Oglucoside were used for the quantitative evaluation.
Three five-point calibration curves were prepared as
follows: chlorogenic acid at 330 nm (range 0.038–0.3
2
mg/ml and r of 0.9996) was used to evaluate all the
cynnamoyl compounds; luteolin 7-O-glucoside at 330 nm
2
(range 0.11–0.88 mg/ml and r of 0.9999) was selected to
apigenin derivatives,; rutin at 350 nm (range 0.13–1.02
2
mg/ml and r of 0.9999) was used to quantify all the
derivatives of Kaempferol
Antioxidant Activities
Determination of total phenolic content
The total phenolic content of the ethanol-water extract 7:3
(v/v) extracts was determined by the Folin-Ciocalteu
assay of Singleton and Rossi (1965), as described by
Waterman and Mole (1994). The ethanol-water extract
(0.25 ml), was placed in a 25 ml volumetric flask and 5 ml
distilled water was added. Folin-Ciocalteu’s phenol
reagent (1.25 ml) was added and mixed. After 2 min, 3.75
ml 20% (w/v) sodium carbonate solution was added. The
contents were mixed and distilled water was added to
volume and mixed. The mixture was left to stand for 2 h
after addition of the sodium carbonate after which the
absorbance of the mixture was measured at 760 nm
using a Lambda EZ150 spectrophotometer (Perkin
Elmer, USA). The standard used was catechin and the
results were expressed as mg Tannic acid equivalents/g
sample on a dry basis.
Ferric reducing antioxidant power
The reducing power of the extracts was determined by
assessing the ability of the extract to reduce FeCl3
solution as described by Oyaizu (1986). Briefly,
appropriate dilution of the extract (2.5 ml) was mixed with
2.5 ml 200 mM sodium phosphate buffer (pH 6.6) and 2.5
ml 1% potassium ferricyanide. The mixture was
o
incubated at 50 C for 20 min and then 2.5 ml 10%
trichloroacetic acid was added. This mixture was
Salawu et al. 3
centrifuged at 353 x g for 10 min. Five millilitres of the
supernatant was mixed with an equal volume of water
and 1 ml of 0.1% ferric chloride. The absorbance was
measured at 700 nm. The ferric reducing antioxidant
power was expressed as mg ascorbic acid equivalent/ g
sample, on dry weight basis.
ABTS antiradical assay
Antioxidant activity of the extracts was determined using
the 2, 2’-azinobis-(3- ethylbenzothiazoline-6-sulfonic acid)
•+
ABTS antiradical assay (Awika et al., 2003). The ABTS
(mother solution) was prepared by mixing equal volumes
of 8 mM ABTS and 3 mM potassium persulphate
(K2S2O8) (both prepared using distilled water) in a
volumetric flask, which was wrapped in foil and allowed to
react for a minimum of 12 h in a dark place. The working
solution was prepared by mixing 5 ml of the mother
solution with 145 ml phosphate buffer (pH 7.4). A range
of trolox (6-hydroxy-2, 5, 7, 8-tetramethylchromancarboxylic acid) standard solutions (100–1000 µM) were
prepared in acidified ethanol. The working solution (2.9
ml) was added to the ethanol-water extracts (0.1 ml) or
Trolox standard (0.1 ml) in a test tube and mixed with a
vortex. The test tubes were allowed to stand for exactly
30 min. The absorbance of the standards and samples
was measured at 734 nm with a Lambda EZ150
spectrophotometer. The results were expressed as µM
Trolox equivalents/g sample, on dry weight basis.
DPPH antiradical assay
The DPPH assay was done according to the method of
Brand-Williams et al., (1995) with some modifications.
The stock solution was prepared by dissolving 24 mg
DPPH with 100mL ethanol and then stored at -20oC until
needed. The working solution was obtained by mixing
10mL stock solution with 45mL ethanol-water (7/3, v/v) to
obtain an absorbance of 1.170.02 units at 515 nm using
the spectrophotometer. Vegetable extracts (150 mL)
were allowed to react with 2850 mL of the DPPH solution
for 6 h in the dark. Then the absorbance was taken at
515 nm. Results are expressed in µM Trolox Equivalentg
sample. Additional dilution was needed if the DPPH
value measured was over the linear range of the
standard curve.
LDL oxidation assay
The ability of the extracts to protect against LDL oxidation
was determined spectrophotometrically by measuring the
amount of thiobarbituric acid reactive substances
2+
(TBARS) produced after Cu -induced oxidation of LDL in
the presence of the extracts (Liu and Ng, 2000). Briefly,
170 µl of an LDL solution (50 µg/ml) in PBS was
2+
incubated with 100 µM final concentration Cu in the
presence or absence (control) of 20 µL of diluted
ethanolic extracts. The oxidation was performed in screw
o
capped 2 ml eppendorf tubes at 37 C in a shaking water
bath for 3 h in the dark. Oxidation reaction was stopped
by adding 10 mM EDTA (final concentration).
Trichloroacetic acid (TCA) (200 µl, 20% w/v) and 200 µl
of 0.67% (w/v) thiobarbituric acid (TBA) in 0.2 M NaOH
were added to the post-incubation mixture. The mixture
o
was heated at 80 C for 30 min and cooled. After
centrifugation at 1500 × g for 15 min to remove
precipitated proteins, the absorbance of the supernatant
was measured at 532 nm. Lipid peroxidation inhibitory
ratio was estimated as a function of the absorbance of
the positive control.
Statistical analysis
All analysis for HPLC phenolic composition and
antioxidative activity determination and LDL oxidation
were run in triplicate. The mean value and standard
deviation were calculated using the Microsoft Excel
software (Microsoft Corporation, Redmond, WA).
RESULTS AND DISCUSSION
Phenolic compounds are large and diverse group of
molecules and are suggested to be the major bioactive
compounds contributing to the health benefits of
vegetables (Yang et al., 2004; Sinelli et al., 2008). The
phenolic composition of the selected vegetables was as
shown in Table1 and Figure1. Partial characterization of
the selected vegetables were obtain using the information
-,
obtained from the spectra data (Retention time, [M-H]
Fragment ions).
The HPLC analyses revealed the
presence of apigenin derivatives, chlorogenic acid and
three dicaffeoyl derivatives in the hydro alcoholic extract
of Struchium sparjanophora while chlorogenic acid, rutin
and three Kaempferol derivatives were identified in
Telfairia occidentalis. Table 3 showed the quantitative
estimates of the identified phenolic compounds. The
quantitative estimation of each identified phenolic
compounds revealed chlorogenic acid as the highest
phenolic compound in Struchium sparjanophora (1.019 ±
0.0036 mg/g dry weight), while kaempferol rutinoside and
kaempferol rhamnoside were shown to be relatively high
in Telfairia occidentalis (2.767 ± 0.029, 1.387 ± 0.017).
Each of the apigenin derivatives (0.338± 00036, 0.463 ±
0.0034, 0.742 ± 0.0038) and dicaffeoyl derivatives (0.584
± 0.0045, 0.202 ± 0.0043) occurred in lesser quantity
compared
to
chlorogenic
acid
in
struchium
sparjanophora. Although, putting together of all the
apigenin derivatives, makes apigenin derivative to rank
higher in Struchium sparjanophora. However, the
4 Afr. J. Food Sci. Technol.
Table 1. List of the identified compounds in Structchium sparejanophora and Telfeiria occidentalis by HPLC/DAD and
HPLC/ESI/MS
Sample/ Peak no.
Struchium sparejanophora
1
2.
3.
4.
5
6
7
Telfairia occidentalis
1.
2.
3.
4.
5.
-
Fragment ions
Compounds
Rt (min)
χmax (nm)
[M-H]
Chlorogenic acid
Apigenin derivative
Apigenin derivative
Apigenin derivative
Dicaffeoyl derivative
Dicaffeoyl derivative
Dicaffeoyl derivative
11.2
15.0
15.4
16.5
18.4
18.8
20.4
240/326
268/332
268/332
270/328
242/328
244/328
242/328
353
755.2
593
456
722
515
515
191
269
269
755, 531, 339, 269
179, 191, 353, 515
353, 191, 135
353, 191, 135
Isomer of chlorogenic acid
Rutin
Kaempferol glu. Rhamnoside
Kaempferol rutinoside
Kaempferol derivative
11.0
14.6
15.6
16.2
17.2
242/326
256/354
266/348
266/348
266/348
353
609
593
593
471
191,707
303, 633
287, 179, 617
287
287
B
Figure 1. Chromatographic profile at 330nm of the hydro alcoholic extracts: (A) Structchium sparejanophora (B) Telfeiria
occidentalis at 330nm. The profile was obtained with synergy max column and elution method 1
bioactivities of the phytochemicals are usually by additive
or
synergistic
interaction
of
the
constituent
phytochemicals, therefore the biological properties of
phenolics in plant food is the combining effect of the
major and the minor compounds (Liu, 2003; Haidari et al.,
2009).
Chlorogenic acid, as a phenolic acid, occurs ubiquitously
in food and posses series of biological effects in vitro and
in vivo, such as antioxidant capacity, radical scavenging
activity, antimutagenic/anticarcinogenic effect, and
inflammation inhibiting and endothelial protective
properties (Morishita
Salawu et al. 5
Table 2.Quantitative estimates of phenolic compounds in Struchium sparjanophora and Telfairia occidentalis
Sample/Peak no Struchium sparjanophora
1.
2.
3.
4.
5
6
7
Telfairia occidentalis
1.
2.
3.
4.
5.
Compounds
Chlorogenic acid
Apigenin derivative
Apigenin derivative
Apigenin derivative
Dicaffeoyl derivative
Dicaffeoyl derivative
Dicaffeoyl derivative
Isomer of chlorogenic acid
Rutin
Kaempferol glu. Rhamnoside
Kaempferol rutinoside
Kaempferol derivative
mg/g (Mean ± SD)
1.019 ± 0.0036
0.338 ± 0.0036
0.463± 0.0034
0.742 ± 0.0038
0.362± 0.00
0.584 ± 0.0045
0.202 ± 0.0043
0.482 ± 0.012
0.463 ± 0.019
1.387 ± 0.017
2.767 ± 0.029
0.483 ± 0.0063
Values represent Mean ± SD (n=4)
Table 3. Total Phenolic content and reducing power of Struchium sparjanophora and Telfeiria
occidentalis
Sample
Total Phenol (mg TAE/g)
Reducing Power (mg Vit. C Equiv./g)
SS
5.72 ± 0.72
14.92 ± 1.3
TO
13.04 ± 0.86
22.88 ± 0.63
Values represent Mean ± SD (n=4); SS- Struchium sparjanophora, TO-Telfairia occidentalis
and Ohnishi, 2001). Our previous investigation on the
phenolic composition of some tropical green leafy
vegetables also revealed the presence of chlorogenic
acid in Vernonia amygdalina and Corchorous olitorius
(Salawu, et al., 2009). This by implication is that
struchium sparjanophora will be a good source of this
important phyto-constituents and might therefore
contribute to body’s health promotion to some extent and
hopefully provide new ways for chronic disease
prevention. Apigenin has also been shown to possess
anti-inflammatory effect, free radical scavenging
properties and anti-carcinogenic effects (Nagaraja et al.,
2009). Apigenin has also been reported in vernonia
amygdalina commonly consumed in Nigeria (Salawu et
al., 2009) and this phenolic containing vegetable extract
have been reported to posses antimalarial (Abos and
Raseroka, 2003), antimicrobial (Erasto, Grierson and
Afolayan, 2006), and anticancer activities (Izevbigie
2003). Previous report on the flavonoid content of some
green leafy vegetables also revealed the presence of
Kaempferol (Chu et al., 2000; Hertog et al., 1992).
Kaempferol glycosides that were identified in Telfairia
occidentalis have been reported to be a natural plant
product with potentially useful pharmacological and
nutraceutical activities common in vegetable, fruits, plant
and herbal medicines. Kaempferol is known for its health
promoting effect. Studies have shown that it reduces
cancer, arteriosclerosis, cardiovascular disorder, and
serve as antioxidant and anti-inflammatory (Yoshida et
al., 2008; Kowalski et al., 2005a, 2005b). Rutin which
was also identified in Telfairia occidentalis , is known for
its anti-inflammatory and vasoactive properties, as well
as for its capability to diminish capillary permeability and
to reduce the risk of arteriosclerosis, whereby reducing
coronary heart disease, possibly through the diminishing
of platelet aggregation (La Casa et al., 2000; Jiang et al.,
2007).
The total phenolic content (TPC) and the ferric reducing
antioxidant power (FRAP) of the vegetable extracts were
shown on Table 3. The result revealed that total phenolic
content (mg Tannic acid equivalent/ g of the sample) and
Ferric Reducing Properties (mg Ascorbic acid equivalent
/g of the sample) were high in Telfairia occidentalis
compared (TPC: 13.04 ± 0.86 and FRAP: 22.88 ± 0.63)
with Struchium sparjanophora (TPC: 5.72 ± 0.72, FRAP:
14.92 ± 1.30). Similarly, the same trend was observed in
ABTS and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activities of both phenolic extract with higher
radical scavenging activities in Telfairia occidentalis. The
result showed a direct relationship between the phenolic
content and the evaluated antioxidant activities. This is in
agreement with previous studies that reported a direct
correlation between phenolic content and antioxidant
capacity (Yang et al., 2002). Some studies have reported
the antioxidant activities of Telfairia occidentalis (Oboh
and Akindaahunsi, 2004; Oboh, 2005; Oboh et al., 2006)
Radical Scavenging Activities
(Micro-MolarTrolox Eq./g )
6 Afr. J. Food Sci. Technol.
4.00E-05
3.50E-05
3.00E-05
2.50E-05
2.00E-05
1.50E-05
1.00E-05
5.00E-06
0.00E+00
SS
TO
ABTS
DPPH
Figure 2. ABTS and DPPH radical Scavenging Activities (µM Trolox Equivalent/ g sample) of Struchium
sparjanophora (SS) and Telfairia Occidentalis (TO)
120
% Inhibition
100
80
60
40
20
TO
(0
.4
m
g/
m
l)
l)
(0
.2
m
g/
m
TO
SS
(0
.4
m
SS
(0
.2
m
g/
m
g/
m
l)
l)
PC
0
Figure 3. Inhibition of copper induced LDL oxidation by phenolic extracts of Struchium sparjanophora and
Telfairia occidentalis
and Struchium sparjanophora (Oboh, 2006), with dearth
of information on their phenolic composition.
Polyphenolic compounds in the diet enhance the
stability of low-density lipoprotein (LDL) to oxidation, and
evidence exists that LDL oxidation plays a significant role
in atherosclerosis and coronary heart disease (Steinberg
et al., 1989). The inhibitory activity (%) of the phenolic
extract of the studied vegetable is as presented in Figure
3. The result showed a good inhibitory activity at the two
selected concentration (0.2 and 0.4 mg/ml) for the two
Salawu et al. 7
studied vegetables with respect to the control. The
inhibitory activities of both vegetal materials against
human LDL oxidation will possibly be linked with their
phenolic constituents.
CONCLUSION
The phenolic extract of the studied leafy vegetables
(Struchium sparjanophora and Telfairia occidentalis)
showed the presence of some phenolic compounds
(phenolic acids and flavonoids) and demonstrate some
level of antioxidant activities. Therefore, the consumption
of these vegetable will be of immense benefit in the
prevention of a number free radical mediated diseases.
Also, the inhibitory action against the human LDL
oxidation
will make the vegetable rank among the
league of arsenals in ameliorating cardiovascular,
coronary heart diseases and other diseases that are
associated with lipid oxidation.
ACKNOWLEDGMENTS
We wish to equally acknowledge Professor Nadia
Mulinacci, of the department of Pharmaceutical science,
University of Firenze, Italy who provided some technical
expertise on the HPLC/DAD/MS instrumentation and
also the International Center for Theoretical Physics in
conjunction with International Agency for Atomic Energy
for their financial support.
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