Industrial Crops and Products 74 (2015) 271–278

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Industrial Crops and Products

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Bioactive components and properties of ethanolic extract and its

fractions from Gynura procumbens leaves
Niwat Kaewseejan a , Sirithon Siriamornpun b,∗
a
Department of Chemistry, Faculty of Science, Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand
b
Research Unit of Process and Product Development of Functional Foods, Department of Food Technology and Nutrition, Faculty of Technology,
Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: The bioactive components and properties of various extracts from Gynura procumbens leaves were inves-
Received 12 January 2015 tigated. The leaves of G. procumbens were initially extracted with 95% ethanol and then sequentially
Received in revised form 8 May 2015 fractionated according to the solvent polarity of chloroform, ethyl acetate and n-butanol. Total phenolic
Accepted 9 May 2015
and total flavonoid contents of the crude ethanolic extract and its fractions ranged from 0.8–24.4 mgGAE/g
and 0.1–17.3 mgCE/g, respectively. The ethyl acetate fraction (EAF) showed the most potent radical scav-
Keywords:
enging activity on DPPH• , ABTS+ • , • OH and H2 O2 , with IC50 values of 0.22, 0.06, 0.01 and 0.03 mg/ml,
Phenolic acids
respectively. Lipid peroxidation inhibition and oxidative protein damage protecting activities were supe-
Protein damage
Peroxidation
rior for EAF compared to the other fractions, including ascorbic acid. All extracts possessed strong
Glycation anti-glycation activity with IC50 values between 0.04 mg/ml in the EAF and 0.15 mg/ml in the chloro-
Flavonoids form fraction. Correlation analysis showed the phenolic and flavonoid contents were highly positively
Antioxidant correlated with the antioxidant and anti-glycation activities. The individual bioactive compounds, as
determined using HPLC, were remarkably different, especially gallic, p-coumaric and ferulic acids for
phenolic acids, and myricetin, quercetin and kaempferol for flavonoids. These findings suggest that the
strongest bioactive properties of EAF from G. procumbens were because of these phenolics and flavonoids.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction as health products that can prevent the risk of degenerative dis-
eases caused by oxidative damage.
The oxidative processes in the human body produce free radi- Nowadays, research on natural antioxidants is increasing sig-
cals as byproducts, especially reactive oxygen species (ROS). Over nificantly, in many countries around the world, as a result of their
production of free radicals in the body can attack the various health benefits when part of drugs in the pharmaceutical industry.
biomolecules including DNA, protein and lipid, which eventually Previous studies have suggested that fruits, vegetables and cere-
leads to chronic diseases such as atherosclerosis, hypertension, dia- als are potential sources of natural antioxidants such as phenolics,
betes, cancer and other non-communicable diseases (Valko et al., flavonoids and other phytochemicals (La Vecchia et al., 2001; Butsat
2007). The antioxidants are generally composed of synthetic and et al., 2009; Fu et al., 2011). The best benefits for human health can
natural antioxidants that have been widely used as nutraceutical be realized not only from the consumption of vegetables and fruits
and pharmaceutical products to prevent or inhibit oxidative dam- with strong antioxidant properties, but also from that of medic-
age in human. However, the utilization of synthetic antioxidants, inal plants. Medicinal plants are considered another important
such as butylated hydroxytoluene (BHT) and butylated hydrox- alternative as a new source of natural antioxidants. Many studies
yanisole (BHA), must be strictly controlled due to their toxicity demonstrated that medicinal plants have potent antioxidant capac-
and potential carcinogenicity (Kahl, 1984; Botterweck et al., 2000). ity and contained high amounts of pharmaceutical compounds
Therefore, natural antioxidants play important roles for use as new (Barros et al., 2010; Li et al., 2013; Skotti et al., 2014). Medicinal
sources of non-toxic and inexpensive antioxidants to be developed plants have long been used for the treatment of human diseases
in many regions, particularly Asia. People are increasingly playing
attention to medicinal plants because of their potential therapeutic
actions and safely as well as high nutritional value (Fernandes et al.,
2014; Farzaneh et al., 2015).
∗ Corresponding author. Tel.: +66 857 474136; fax: +66 437 43135.
E-mail address: sirithons@hotmail.com (S. Siriamornpun).

http://dx.doi.org/10.1016/j.indcrop.2015.05.019
0926-6690/© 2015 Elsevier B.V. All rights reserved.
272 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278

Gynura procumbens, a fast growing herbaceous plant that is an then filtered using a sterilized cotton filter and Whatman No. 1 filter
annual evergreen shrub with a fleshy stem, belongs to the Aster- paper. The solvent was completely removed by a rotary evaporator
acea family, and it is an important medicinal plant in Southeast at 35 ◦ C in vacuo and the crude ethanolic extract (CEE) was obtained.
Asia, especially Malaysia, Indonesia and Thailand. The plant has The CEE was redissolved in distilled water and then the aqueous
been widely used as a traditional medicine to treat cancer, kid- solution was successively partitioned three times with chloroform,
ney disease, migraines, hypertension and diabetes (Perry, 1980). ethyl acetate and n-butanol to fractionate the non-polar and polar
Extracts of this plant have pharmacological activities such as anti- compounds. Non-polar or less polar compounds such as aglycone
hyperglycemic (Akowuah et al., 2002), anti-inflammatory (Iskander flavonoids could be fractionated by chloroform, while ethyl acetate
et al., 2002) and anti-hypertensive effects (Kim et al., 2006). Other was used to fractionate medium polar compounds like phenolics,
studies have also reported that G. procumbens showed anticancer flavonoids and glycosides. Polar compounds such as glucosides and
and antiproliferative actions (Kim et al., 2011; Nisa et al., 2012; sugar could be dissolved in n-butanol and water. Each resulting
Shwter et al., 2014). These actions have been attributed to the pres- solvent fraction was concentrated and dried by a rotary evapo-
ence of saponins, flavonoids and terpenoids (Akowuah et al., 2002). rator at 35 ◦ C in vacuo to give the chloroform fraction (CF), ethyl
However, few studies have focused on the fractionation of bioac- acetate fraction (EAF) and n-butanol fraction (BF). The rest fraction
tive compounds contributing to the potent biological activities of G. or aqueous fraction was also concentrated as described earlier and
procumbens leaves. The content of bioactive compounds is affected freeze–dried. As pre-experiment results, the bioactive properties in
by the method of extraction, which indicates that the solvent aqueous fraction as well as phenolic content were detected in very
used and step of extraction should be considered when needing low levels compared to other solvent-partitioned fractions; it was
to increase the bioactive compounds along with enhanced biolog- not selected for further investigation. This finding is also similar to
ical activities (Jun et al., 2014). Therefore, we aimed to fractionate the previous study that reported by Jun et al. (2014). The four frac-
bioactive compounds, especially phenolics and flavonoids using tions, including CEE, CF, EAF and BF were selected for evaluation of
multiple steps according to solvent polarity and to evaluate the bioactive compounds that contributing to the strong antioxidative
bioactive properties from G. procumbens leaves. The fractionation and anti-glycation activities.
steps were optimized and developed for the sequential extraction
of phenolic compounds to provide high recovery and high biologi-
cal activity. This study will provide valuable information about the 2.4. Determination of total phenolic content (TPC)
possible bioactive compounds that contributing to the excellent
bioactivity for pharmaceutical industrial application. The TPC of the CEE and its fractions were determined using the
Folin–Ciocalteau method (Bakar et al., 2009). Briefly, 200 ␮l of each
2. Materials and methods extract was mixed with 1 ml of 10% (v/v) Folin–Ciocalteau reagent.
After 5 min incubation, 800 ␮l of 7.5% (w/v) sodium carbonate was
2.1. Chemicals and reagents added and the mixture was left to stand for 30 min at room tem-
perature. The absorbance of the solution was reached at 765 nm
The compounds 2,2-diphenyl-1-picrylhydrazyl (DPPH), buty- by a spectrophotometer. The TPC was reported as mg gallic acid
lated hydroxyanisole (BHA), 2,2 -azino-bis-3-ethylbenzothia equivalents per gram dry weight (mgGAE/g DW).
zoline-6-sulphonic acid (ABTS), gallic acid, catechin and
Folin–Ciocalteau’s reagent were purchased from Sigma Chem-
ical Co. (St. Louis, Mo., U.S.A.). Bovine serum albumin (BSA),
2.5. Determination of total flavonoid content (TFC)
ascorbic acid, glucose, sodium azide, potassium persulfate, 6-
hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox)
The TFC of the CEE and its fraction were determined using a
and linoleic acid were obtained from Fluka (Buchs, Switzerland).
modified colorimetric method as described previously (Bakar et al.,
Hydrogen peroxide (H2 O2 ), ethanol, ethyl acetate, chloroform and
2009). Each extract solution (250 ␮l) was mixed with 1.25 ml of dis-
n-butanol were purchased from BDH Chemical Ltd. (Dagenham,
tilled water and 75 ␮l of 5% NaNO2 solution. The mixture was left
U.K.). All chemicals used in the present study were of analytical
to stand for 6 min and then 150 ␮l of 10% AlCl3 was added to the
grade.
solution. After 5 min, 500 ␮l of 1 M NaOH was added to the mixture
and made up to a total volume of 2.5 ml with distilled water. The
2.2. Plant material
absorbance of the solutions was measured at 510 nm using a spec-
trophotometer. The TFC was expressed as mg catechin equivalent
The fresh green leaves of the medicinal plant G. procumbens were
per gram dry weight (mgCE/g DW).
collected from Khon Kaen Province, Thailand. The plant was iden-
tified by a taxonomist from the Department of Biology, Faculty of
Science, Khon Kaen University, Thailand. Fresh leaves of G. procum-
bens (5 kg) were washed three times with tap water and two times 2.6. DPPH• scavenging activity
with distilled water to remove surface contaminants. After draining
water, the sample was dried under hot air oven at temperature of The DPPH• scavenging activity of the CEE and its fractions were
60 ◦ C for 48 h (moisture content of 7% dry basis). The dried sample measured according to a previously published method (Brand-
was ground into a fine powder, passed through a 60 mesh sieve to Williams et al., 1995). One half milliliter of each extract solution
obtain a particle size of approximately 210 ␮m and then stored in containing different concentrations of compounds was added to
desiccators until analysis. 0.5 ml of a freshly prepared 0.1 mM DPPH solution dissolved in
methanol. The mixture was shaken and allowed to stand at room
2.3. Extraction and fractionation of bioactive compounds temperature for 30 min in the dark. The absorbance of the mixture
was detected at 517 nm using a spectrophotometer (GENESYSTM
Bioactive compounds were extracted and fractionated from 10, Thermo Scientific, U.S.A.) against a control. A decrease in the
G. procumbens leaves according to solvent polarity. The dried G. absorbance indicates an increase in the DPPH• scavenging activity.
procumbens leaves (180 g) were extracted three times with 95% Results were represented as an IC50 value, which is the concentra-
ethanol under stirring for 3 h at room temperature. The extract was tion of the extract that scavenges 50% of the DPPH• .
 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278 273

2.7. ABTS+• scavenging activity damage was induced by hydroxyl radicals generated in the Fen-
ton reaction between CuSO4 and H2 O2 . Briefly, 100 ␮l of 5 mg/ml
The ABTS+• scavenging activity of the CEE and its fractions were BSA, 20 ␮l of 10 mM CuSO4 , 20 ␮l of 0.2 M H2 O2 and 160 ␮l of
determined using a modified method as described previously (Re each fraction were mixed in an eppendorf tube. Then, the mixture
et al., 1999). The ABTS+• was generated by oxidation of 7 mM ABTS was incubated at 37 ◦ C for 20 min and then 120 ␮l of 5× loading
solution with 2.45 mM potassium persulfate at the ratio 1:1, and dye was added. This mixture was shaken and heated to 100 ◦ C
the mixture was left to stand in the dark at room temperature for for 5 min. After cooling, 10 ␮l of each sample solution was loaded
12–16 h before use. The resulting blue–green colored ABTS+• solu- into a 12% SDS-PAGE separating gel. The gel was electrophoresed
tion was adjusted to an absorbance of 0.70 ± 0.02 at 734 nm. Briefly, at a voltage of 180 V in a running buffer at pH 8.3 for 45 min and
200 ␮l of each extract solution was introduced into the test tubes stained with coomassie brilliant blue G-250 to determine the pro-
after the addition of 800 ␮l of the ABTS+• solution. The mixture was tein bands. After destaining, the gel was photographed and the
incubated at room temperature for 6 min and the absorbance was intensity of the original-BSA band and damaged-BSA band were cal-
measured at 734 nm against a control. Data were reported as an culated using the 1D gel analysis software (ImageQuant TL version
IC50 value, which is the extract concentration that scavenges 50% 7.0, GE Healthcare Bio-Sciences, Tokyo, Japan). The percentage of
of the ABTS+• . oxidative protein damage prevention activity was calculated using
the following equation:
2.8. Hydroxyl radical (• OH) scavenging activity
Proteindamageprotectingactivity(%) = [(P s – P 0 )/(P 1 – P 0 )] × 100
The • OH scavenging activity was determined as described
by Zhang et al. (2011). The • OH was produced by the Fenton
where Ps is the high peak of the protein band containing plant
reaction between FeSO4 and H2 O2 . Briefly, 60 ␮l of 0.75 mM 1,10-
extracts; P0 is the high peak of the protein band containing CuSO4
phenanthroline, 40 ␮l of 0.75 mM FeSO4 and 600 ␮l of 50 mM
and H2 O2 without plant extracts (negative control); and P1 is the
phosphate buffer at pH 7.4 were introduced into the test tubes
high peak of the protein band without plant extracts, CuSO4 and
and mixed thoroughly. Then 100 ␮l of 0.5% H2 O2 and 200 ␮l of
H2 O2 (positive control).
the extract were added. After incubation at 37 ◦ C for 60 min, the
absorbance of the mixture was measured at 536 nm using a spec-
trophotometer (GENESYSTM 10, Thermo Scientific, U.S.A.). The
2.12. Evaluation of anti-AGEs formation activity
results were reported as an IC50 value, which is the extract con-
centration that scavenges 50% of the • OH.
The inhibitory capacities of AGEs formation of the CEE and its
fractions were measured using a method of Vinson and Howard
2.9. Hydrogen peroxide scavenging activity
(1996). The total volume of glycation reaction solution (2.5 ml)
was prepared by mixing 500 ␮l of each plant extract, 500 ␮l of
The hydrogen peroxide (H2 O2 ) scavenging assay was carried
20 mg/ml BSA in phosphate buffer, 500 ␮l of 0.5 M glucose in phos-
out according to the procedure of Gülçin et al. (2010). A solution
phate buffer and 1 ml of 0.1 M phosphate buffer at pH 7.4 containing
of 40 mM H2 O2 was prepared in 50 mM phosphate buffer at pH
0.02% (w/v) sodium azide. This mixture was incubated at 37 ◦ C
7.4. Then, 200 ␮l of each extract solution was added into 2.2 ml
for 5 days in the dark and then the amount of fluorescent AGEs
of 50 mM phosphate buffer at pH 7.4 in the test tubes. After that,
formed was determined using a fluorescent spectrometer (LS 50B,
600 ␮l of 40 mM H2 O2 solution was added and the mixture was
PerkinElmer, U.S.A.) with an excitation wavelength of 330 nm and
incubated at 25 ◦ C for 30 min. The absorbance of the mixture was
emission wavelength of 410 nm. The percentage of anti-AGEs for-
recorded at 230 nm using a UV–vis spectrophotometer (T80+ , PG
mation was calculated based on the resulted fluorescent intensity
Instruments, U.K.). Data were reported as an IC50 value, which is
(FI) using the following equation:
the extract concentration that scavenges 50% of the H2 O2 .

Inhibition(%) = [1–(FIsample – FIsampleblank )/(FIcontrol – FIcontrolblank )] × 100

2.10. Linoleic acid peroxidation inhibition

The inhibitory activities of linoleic acid peroxidation of the CEE

and its fractions were measured by the ferric thiocyanate method
(FTC) as described by Ismail et al. (2010). Briefly, 2 ml of each 2.13. Analysis of phenolic acids and flavonoids by RP-HPLC
extract, 2 ml of 2.51% linoleic acid solution dissolved in 95% ethanol,
4 ml of 50 mM phosphate buffer at pH 7.0 and 2 ml of distilled water The component and content of phenolic acids and flavonoids
were mixed in a 10 ml vial with a screw cap. Then, the mixture was from the CEE and its fractions were determined using RP-HPLC
kept at 40 ◦ C in the dark for several days; the oxidation of the lipid (LC-20AC, Shimadzu, Japan) according to a method of Butsat et al.
was measured after eight days of incubation. Thirty micro-litters (2009). Each extract was dissolved in ethanol, filtered through a
of the reaction mixture was added to 2.91 ml of 75% ethanol, 30 ␮l 0.45 ␮m membrane filter and injected onto an Inetsil ODS-3C18
of 30% ammonium thiocyanate and 30 ␮l of 20 mM FeCl2 dissolved column (4.6 mm × 250 mm, 5 ␮m; Hichrom Limited, Berks, U.K.)
in 3.5% hydrochloric acid. After 3 min, the degree of lipid oxidation with the injection volume of 20 ␮l. The mobile phases used were
was measured at an absorbance of 500 nm using a UV–vis spec- 1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) at
trophotometer (T80+ , PG Instruments, U.K.). The absorbance of the a flow rate of 0.8 ml/min. The bioactive components of the extract
mixture was measured every 24 h until a constant absorbance value were separated using gradient elution at 38 ◦ C as described previ-
was reached. ously by Butsat et al. (2009). The eluted bioactive compounds were
detected at 280 nm for phenolic acids and 370 nm for flavonoids
2.11. Protein damage protecting activity with UV-diode array detector (SPD-M20A, Shimadzu, Japan). Phe-
nolic compounds in the samples were identified by comparison
The in vitro protein damage protecting activity of the CEE and of retention times and UV spectra of authentic compounds. The
its fractions were determined by sodium dodecyl sulfate poly- content of phenolic compounds in the samples was detected using
acrylamide gel electrophoresis (SDS-PAGE). The oxidative protein external standard methods.
274 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278

Table 1
Extraction yield, TPC and TFC of CEE and its solvent fractions isolated from G. procumbens leaves.

Fractions Yield (%) TPC (mgGAE/g DW) TFC (mgCE/g DW) TFC/TPC (%)

CEE 27.83 ± 1.11a 16.08 ± 0.38b 10.33 ± 0.88b 64.24

CF 1.68 ± 0.70d 0.77 ± 0.20d 0.08 ± 0.00d 10.38
EAF 6.40 ± 0.93c 24.36 ± 1.11a 17.33 ± 1.39a 71.14
BF 8.64 ± 0.09b 5.92 ± 1.61c 4.92 ± 0.29c 83.11

Values are expressed as mean ± SD of triplicate measurements. Means with different letters in the same column represent significant differences at p < 0.05.

Table 2
Free radical scavenging activity of CEE and its fractions isolated from G. procumbens against DPPH, ABTS+ , OH and H2 O2 radicals.

Fractions IC50 A (mg/ml)

DPPH• scavenging ABTS+ • scavenging •

OH scavenging H2 O2 scavenging

CEE 0.47 ± 0.02 c

0.22 ± 0.00c
0.12 ± 0.01 c
0.27 ± 0.00c
CF 3.33 ± 0.06a 0.90 ± 0.01a 0.51 ± 0.01a 2.31 ± 0.03a
EAF 0.22 ± 0.01d 0.06 ± 0.00d 0.01 ± 0.00e 0.03 ± 0.00d
BF 0.71 ± 0.03b 0.49 ± 0.01b 0.23 ± 0.00b 0.88 ± 0.02b

TroloxB 0.04 ± 0.00e 0.01 ± 0.00e 0.01 ± 0.00e 0.02 ± 0.00d

BHAB 0.02 ± 0.00e 0.01 ± 0.00e 0.01 ± 0.00e 0.02 ± 0.00d
Ascorbic acidB 0.03 ± 0.00e 0.06 ± 0.00d 0.10 ± 0.00d 0.01 ± 0.00d
A
The concentration of the plant extract that scavenges 50% of free radical. Lower IC50 values indicate higher radical scavenging activity.
B
Standard synthetic antioxidants were used as a reference for radical scavenging activity. Means with different letters in the same column represent significant differences
at p < 0.05.

2.14. Statistical analysis Flavonoids are the most common group of phenolic compounds-
rich plants and they are usually the great potential to scavenge
The data were reported as mean ± standard deviation (SD) and the free radicals (Gülçin et al., 2010). The results of the TFC of the
were analyzed by analysis of variance (ANOVA) using SPSS statisti- extracts are also presented in Table 1. A similar trend to the TPC
cal software (SPSS 11.5, SPSS Inc., IL, U.S.A). Significant differences results was observed in the TFC with the highest content found in
between the means were determined by the Duncan test at ˛ = 0.05 the EAF (17.3 mgCE/g DW) and the lowest content detected in the
and Pearson’s correlation coefficients were performed to compare CF (0.1 mgCE/g DW). Specifically, the TPC and TFC of EAF were 1.5
the correlations between various parameters. and 1.7 fold higher than that of the crude extract, indicating that
solvent fractionation purified and concentrated the phenolics and
flavonoids in EAF. The EAF could be the part that is rich in phenolic
compounds and that ethyl acetate is suitable to fractionate pheno-
3. Results and discussion
lics from G. procumbens leaves according to our results. In general,
the yield, composition and purity of phenolic compounds recov-
3.1. Extraction yield, total phenolic content (TPC) and total
ered from plant materials is dependent on the chemical nature,
flavonoid content (TFC)
sample size, extraction method and conditions, as well as the pres-
ence of interfering substances (Cheng et al., 2012). The relationships
The percentage yield of the CEE of G. procumbens leaves and its
between the TPC and TFC were shown as percentages of TFC/TPC.
different fractions are shown in Table 1. The results showed that the
The ratios of TFC to the TPC ranged from 10.4 to 83.1% (Table 1).
extraction yield of all extracts varied from 1.68 to 27.83% (w/w).
From these results, it can be understood that the EAF, CEE and BF of
The significant difference (p < 0.05) of the extraction yield for all
G. procumbens were composed of flavonoid compounds (64–83%)
extracts was observed. As shown in Table 1, the CEE had the highest
rather than other phenolic compounds.
percentage yield, followed by BF, EAF and CF. Extraction is the first
step in the recovery of bioactive compounds, especially phenolic
compounds from plant materials, which is an important process 3.2. Antiradical activity
and various techniques have been studied. It is well known that
the recovered yield of the extract from plant material is dependent The CEE and its fractions of G. procumbens leaves showed con-
on the type and polarity of solvent used, sample size, extraction centration dependent free radical scavenging activities as assayed
method and conditions (Cheng et al., 2012). by DPPH• , ABTS+• , • OH and hydrogen peroxide (H2 O2 ) (data not
Phenolic compounds have received increasing attention in shown). The free radical scavenging activities of all samples were
recent year, which are due to their biological activities and health reported as IC50 values in which a lower IC50 value indicates higher
benefits (Shahidi and Naczk, 2004). In the present study, the TPC antioxidant activity (Table 2).
of G. procumbens leaf extracts significantly varied according to the DPPH has been widely used as a free radical to evaluate antiox-
solvent used for fractionation and its polarity (Table 1). The high- idant compounds, which is able to reduce the DPPH• form to
est TPC was found in the EAF (24.4 gGAE/g DW), followed by CEE the DPPH non-radical by donating a hydrogen atom (Cho et al.,
(16.1 mgGAE/g DW), BF (5.9 mgGAE/g DW) and CF (0.8 mgGAE/g 2011). The DPPH• scavenging activity showed the highest value
DW). This finding was in agreement with previous studies that in the EAF (IC50 = 0.2 mg/ml), followed by CEE (IC50 = 0.5 mg/ml), BF
reported the phenolic content of the EAF was higher than other (IC50 = 0.7 mg/ml) and CF (IC50 = 3.3 mg/ml). The observed differen-
solvent fractions for Allium cepa (Singh et al., 2009) and purple per- tial scavenging activities of the fractions against the DPPH• system
illa (Jun et al., 2014). Previous studies reported that the content of could be due to the presence of different bioactive compounds in
phenolics in various plants is related with their antioxidant capaci- the fraction. The DPPH• scavenging activities of all fractions were
ties, probably due to their have the great potential to act as reducing less than (p < 0.05) those of the standard antioxidants tested includ-
agents. (Ismail et al., 2010; Jun et al., 2014). ing BHA, ascorbic acid and trolox (Table 2). However, the DPPH•
 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278 275

scavenging activity of EAF from G. procumbens was considerably

higher than the capacity of ethyl acetate fractions of other plants
reported from previous studies such as Nigella sativa (Mariod et al.,
2009). The DPPH• scavenging activity of all fractions showed a sim-
ilar trend with both the TPC and TFC, indicating that phenolics and
flavonoids could be responsible for antiradical activity.
ABTS+• scavenging activity of the CEE and other fractions
showed IC50 values in the ranging 0.06–0.90 mg/ml (Table 2).
A similar trend to the DPPH• results was found in the ABTS+•
with the strongest ABTS+• scavenging activity found in the
EAF (IC50 = 0.06 mg/ml). The ABTS+• scavenging activity of all
the tested samples was in the following descending order:
BHA ≈ trolox > ascorbic acid ≈ EAF > CEE > BF > CF (p < 0.05). This
finding was in agreement with that of Singh et al. (2009) who
reported that the extraction with ethyl acetate gave the high-
est antiradical activity. When compared to synthetic antioxidants,
there was no significant difference between the EAF and ascorbic Fig. 1. Linoleic acid peroxidation inhibitory activity of the CEE and its solvent frac-
acid (IC50 = 0.06 mg/ml) as indicated in Table 2. Deetae et al. (2012) tions from G. procumbens leaves and standards through the ferric thiocyanate test.
investigated the antioxidant activity of herbal teas from different
plants and verified that the phenolic compounds were important
shown in Fig. 1, all the fractions including commercial synthetic
scavengers of ABTS+• . This may be due to the presence of a high
antioxidants were able to inhibit the hydroperoxide formation
phenolic content in the EAF, as phenolic compounds play a vital
in the linoleic acid emulsion system throughout the incubation
role as antioxidants in living systems.
times when compared to the control. After three days of incuba-
The • OH scavenging activities of all fractions are in the
tion, the value of absorbance of the control was higher than the
following descending order: BHA ≈ trolox ≈ EAF > ascorbic
all samples tested and reached its maximum absorbance value
acid > CEE > BF > CF (Table 2). The results shown in Table 2
on the eighth day of incubation. The lipid peroxidation inhibitory
indicate that the highest • OH scavenging activity with an IC50
activity of all samples was arranged in the following descend-
value of 0.01 mg/ml was detected in the EAF. In addition, the • OH
ing order: trolox > BHA > EAF > ascorbic acid > CEE > BF > CF > control
scavenging activity of the EAF was higher than that of ascorbic acid
(p < 0.05). Among all the fractions, the EAF exhibited a higher lipid
as a standard reference (IC50 = 0.10 mg/ml) and it also exhibited
peroxidation inhibitory activity than the other fractions. Further-
the strongest capacity that was similar to the BHA and trolox. The
more, the lipid peroxidation inhibitory activity of EAF (73.44%)
present results indicated that the • OH scavenging activity of the
was higher than that of ascorbic acid (62.48%) during the eighth
EAF during the crude extract fractionation was comparable to or
day of incubation. This finding clearly indicates that EAF had an
even more effective than those of synthetic standard antioxidants.
effective and potent antioxidant activity in the FTC assay. Con-
The strong antioxidant activity of EAF through the • OH scav-
sequently, this result suggests that EAF may contain antioxidant
enging assay can be explained by the high contents of phenolic
substances that inhibit the hydroperoxide formation and terminate
compounds.
the radical–chain reaction (Shahidi and Wanasundara, 1992).
The H2 O2 scavenging abilities of CEE and its fractions are also
shown in Table 2, and compared to those of commercial synthetic
antioxidants such as BHA, ascorbic acid and trolox. Among all the 3.4. Protein damage protecting activity
fractions, EAF exhibited the strongest H2 O2 scavenging activity
(IC50 = 0.03 mg/ml), whereas CF showed the lowest H2 O2 scav- The oxidative protein damage protecting effect of CEE and it
enging activity (IC50 = 2.31 mg/ml). When comparing to synthetic fractions on hydroxyl radical-mediated BSA protein was deter-
antioxidants, there was no significant difference between the EAF mined using SDS-PAGE. The gel patterns of the BSA protein exposed
and three synthetic antioxidants tested. This finding suggests that to Fenton reagent in the presence and absence of plant extracts
the EAF exhibited potential to be a H2 O2 scavenger, which was sim-
ilar to those of the synthetic antioxidants tested. Since H2 O2 itself is
not very reactive, it can sometimes be toxic to cells because it may
give rise to • OH within the cells (Chai et al., 2003). Interestingly,
the IC50 values of EAF were 2.1, 3.7, 12 and 9 folds lower than those
of the CEE for DPPH• , ABTS+• , • OH and H2 O2 assays, respectively.
This phenomenon may be explained by the solubility of antioxi-
dant compounds that is dependent on the polarity of the extraction
solvent, had been selectively concentrated in the polar medium
of the ethyl acetate fraction during the crude extract fractionation
(Jun et al., 2014). According to the antioxidant assays, we demon-
strated that active compounds like phenolics and flavonoids with
the strongest antioxidant activity were more soluble in a medium
polar of ethyl acetate.

3.3. Inhibition of linoleic acid peroxidation

The ferric thiocyanate (FTC) method measures the amount of Fig. 2. SDS-PAGE profile of CEE and its fractions and synthetic antioxidants on oxida-
tive protein damage protecting activity induced with the Fenton reagent. Lane 1 BSA
peroxide produced during the initial stages of lipid oxidation. Fig. 1 marker (positive control), Lane 2 BSA + Fenton reagent (negative control) and lanes
shows the linoleic acid peroxidation inhibitory activity of the CEE 3–9, BSA + Fenton reagent in the presence of CEE, CF, EAF, BF, BHA, ascorbic acid and
and its fractions as well as commercial synthetic antioxidants. As trolox at a concentration of 80 ␮g/ml, respectively.
276 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278

Table 3 values of 86.35 and 91.68%, respectively. The concentrations of

Inhibitory activity of CEE and its solvent fractions against oxidative protein damage
the fractions that inhibit 50% of the AGEs formation (IC50 val-
induced by hydroxyl radicals generated in the Fenton reaction.
ues) ranged from 41.91 to 145.86 ␮g/ml (Table 4). Previous studies
Fractions Protein damage protecting activity (%) have reported the anti-glycation activity of standard anti-glycation
Concentration (␮g/ml) agents, catechin (81%) at the concentration of 0.5 mg/ml (Wang
et al., 2011) and rutin (82.5%) at the concentration of 3 mM or
20 40 80
1.83 mg/ml (Mosihuzzman et al., 2013). In the results obtained,
CEE 11.67 ± 0.37c 15.97 ± 1.87c 21.35 ± 1.21d we revealed that the EAF, CEE and BF of G. procumbens showed
CF n.d. n.d. 5.02 ± 1.62e
stronger anti-AGEs formation capacities than the standards cate-
EAF 48.18 ± 1.88b 66.25 ± 0.81a 68.89 ± 1.13b
BF 13.23 ± 1.85c 21.37 ± 1.55b 22.11 ± 1.60d chin and rutin. Consequently, this result suggests that all fractions,
except CF had strong efficiency and potency to inhibit the forma-
TroloxA 14.67 ± 2.86c 16.34 ± 3.39c 48.72 ± 1.06c
tion of AGEs in the glucose-mediated protein glycation system. The
BHAA 61.80 ± 0.59a 65.42 ± 1.77a 71.60 ± 1.68a
Ascorbic acidA 7.61 ± 1.83d 13.72 ± 1.30c 23.07 ± 1.20d evidence provided in this study is useful for designing further stud-
ies to investigate these anti-glycation agents for the management
Values are expressed as mean ± SD of triplicate measurements. Means with different
letters in the same column represent significant differences at p < 0.05. of late diabetic complications in vivo.
A
Standard synthetic antioxidants were used as a references. n.d. = not detected.
3.6. Correlation analysis

are presented in Fig. 2. As shown in Fig. 2, all the fractions pos- The correlation coefficients (r) between the mean values
sessed significantly protective effects (p < 0.05) by restoring the obtained from each assay were analyzed by performing a Pearson
band intensity of the BSA protein when compared to the nega- test. The TPC and TFC were strongly positively correlated with the
tive control. The percentage of oxidative protein damage protection radical scavenging assays against DPPH• (r = 0.986 and r = 0.956,
was calculated using the intensity of BSA protein obtained from the respectively), ABTS+• (r = 0.918 and r = 0.984, respectively), • OH
1D gel analysis software and the results are shown in Table 3. The (r = 0.819 and r = 0.931, respectively) and H2 O2 (r = 0.858 and
inhibition activity of the protein damage for all samples increased r = 0.937, respectively), as well as anti-AGEs formation activity
with an increase in fraction concentration. At a concentration of (r = 0.856 and r =n0.940, respectively) (p < 0.01). This finding was in
80 ␮g/ml, the EAF showed the highest protein damage protection agreement with previous studies that reported a strongly positive
activity (68.89%), which was superior to ascorbic acid (23.07%) and correlation between phenolic content, antioxidant and anti-AGEs
trolox (48.72%). This results indicate that the EAF possesses strong formation (Deetae et al., 2012). The anti-AGEs formation capac-
efficiency and potency when protecting against oxidative protein ities were strongly positively correlated to DPPH• , ABTS+• , • OH
damage induced by • OH generated in the Fenton reaction. This is and H2 O2 scavenging activities with values of r = 0.887, r = 0.922,
the first report to date on the protective effect of G. procumbens r = 0.948 and r = 0.852, respectively (p < 0.01). Our findings indicated
leaves against protein damage, and it may have a positive role in that the potency of anti-AGEs formation depended on the capabil-
inhibiting several stress or toxicity induced-protein oxidations. ity of the antioxidants. Wu and Yen (2005) also demonstrated that
phenolic compounds inhibited AGEs formation through scaveng-
3.5. Anti-AGEs formation activity ing free radicals and antioxidant capacities. In the present study,
we demonstrated that phenolic compounds may be responsible for
Glycation of protein alters the biological activity of the protein antioxidant and anti-glycation potentials in G. procumbens leaves.
and initiates its degradation and conversion to AGEs. Endoge-
nous AGEs formation is known to contribute to the progression 3.7. Compositions of bioactive compounds
of pathogenesis under conditions associated with diabetic com-
plications, Alzheimer’s disease and aging (Mosihuzzman et al., The CEE and its derived fractions had their phenolic acid com-
2013). In this study, we first evaluated ability of CEE and its frac- positions determined by RP-HPLC to evaluate the presence of
tions from G. procumbens to inhibit the formation of AGEs in a hydroxybenzoic acids (HBA) and hydroxycinnamic acid (HCA).
glucose-mediated protein glycation system. The inhibition activity The results of content and composition of phenolic acids in dif-
of protein glycation of all the samples increased with an increase ferent fractions from G. procumbens are shown in Table 5. As
in the concentration of fractions (Table 4). At a concentration this result obtained, both HBA and HCA were significantly differ-
of 0.20 mg/ml, EAF exhibited the highest anti-glycation activity ent as the solvent used for fractionation. For HBA, five phenolic
(99.86%), followed by CEE (96.27%), BF (87.24%) and CF (66.02%). acids were identified, namely gallic acid, protocatechuic acid, p-
This finding was in agreement with Sun et al. (2010) who reported hydroxybenzoic acid, vanillic acid and syringic acid, which were
that the ethyl acetate fraction of Chlorella pyrenoidosa and Nitzschia found in CEE and EAF. However, vanillic acid was not detected in
laevis showed the highest anti-AGEs formation activity with the CF and BF, whereas protocatechuic acid and p-hydroxybenzoic acid

Table 4
Inhibitory activity of CEE and its solvent fractions on the fluorescent AGEs formation in a BSA-glucose system.

Fractions Inhibition of AGEs formation (%) IC50 A (␮g/ml)

Concentration (␮g/ml)

40 80 160 200

CEE 46.61 ± 6.59a 61.33 ± 3.12b 87.74 ± 2.42b 96.27 ± 0.45a 51.71
CF 14.76 ± 4.34c 36.73 ± 4.23c 52.56 ± 3.31d 66.02 ± 3.60c 145.86
EAF 50.28 ± 2.64a 73.35 ± 2.86a 94.48 ± 1.69a 99.86 ± 0.15a 41.91
BF 37.96 ± 1.11b 56.62 ± 4.09b 77.72 ± 3.31c 87.24 ± 2.04b 62.71

Values are expressed as mean ± SD of triplicate measurements. Means with different letters in the same column represent significant differences at p < 0.05.
A
The concentration of the plant extract that inhibits 50% of AGEs formation. Lower IC50 values indicate higher anti-AGEs formation activity.
 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278 277

Table 5
Phenolic acid and flavonoid compositions of ethanolic extract and its soluble fractions from G. procumbens leaves.

Phenolic compounds Individual phenolic content in fractions (␮g/g DW)

CEE CF EAF BF

Hydroxybenzoic acids Gallic acid 88.00 ± 2.27b 39.20 ± 1.35d 501.91 ± 1.28a 53.45 ± 0.98c
Protocatechuic acid 83.71 ± 3.78b n.d. 209.24 ± 3.81a 45.79 ± 2.15c
p-Hydroxybenzoic acid 292.47 ± 2.51a n.d. 132.40 ± 1.10b 20.11 ± 3.10c
Vanillic acid 76.77 ± 4.23a n.d. 77.15 ± 0.70a n.d.
Syringic acid 120.55 ± 1.68b n.d. 169.20 ± 1.40a 86.82 ± 2.64c
Total 661.50 ± 4.87b 39.20 ± 1.35d 1089.90 ± 4.69a 206.17 ± 3.80c

Hydroxycinnamic acids Chlorogenic acid n.d. n.d. n.d. n.d.

Caffeic acid 111.72 ± 0.86c 123.02 ± 0.55b 136.34 ± 0.20a 109.72 ± 0.46d
p-Coumaric acid 826.15 ± 4.22c n.d. 2701.75 ± 8.64a 844.13 ± 4.09b
Ferulic acid 99.08 ± 0.36d 113.16 ± 0.72b 280.95 ± 0.50a 106.79 ± 0.57c
Sinapic acid 387.99 ± 3.65a 113.31 ± 1.00d 188.85 ± 2.56c 228.38 ± 3.32b
Total 1424.94 ± 2.32b 349.49 ± 3.28d 3307.89 ± 8.75a 1289.02 ± 1.52c

Flavonoids Rutin 42.56 ± 0.36c 36.88 ± 0.69d 84.38 ± 0.24a 53.26 ± 3.65b
Myricetin 251.10 ± 3.67b n.d. 261.18 ± 1.65a n.d.
Quercetin 135.87 ± 0.40b 122.79 ± 0.59d 193.22 ± 1.47a 129.78 ± 1.31c
Apigenin 49.92 ± 0.73b n.d. 85.92 ± 1.45a n.d.
Kaempferol 464.53 ± 1.81a 240.27 ± 0.63b 192.60 ± 0.67d 232.34 ± 1.80c
Total 943.98 ± 9.91a 399.94 ± 3.66d 817.30 ± 6.69b 415.38 ± 8.17c

Values are expressed as mean ± SD of triplicate measurements. Means with different letters in the same raw represent significant differences at p < 0.05.
n.d. = not detected.

were detected in all fractions except for CF. For HCA, four pheno- All individual flavonoids were found to be dominant in the EAF,
lic acids, including caffeic acid, p-coumaric acid, ferulic acid and except for kaempferol, which was found to be the most predomi-
sinapic acid were detected in all the fractions except CF. Chloro- nant flavonoid in the CEE. Kim et al. (2011) found that kaempferol
genic acid was not identified in all the fractions, whereas syringic was the dominant flavonoid present in the crude extract of G.
acid and p-coumaric acid were not only found in CF. The major phe- procumbens grown in Korea, which agrees with the present result.
nolic acids in all fractions were HCA bearing ortho-dihydroxyl or Interestingly, we found that after fractionation with ethyl acetate,
4-hydroxy-3-methoxyl groups, which contributed about 70–90% the contents of rutin and apigenin were 2 fold higher than that
to the total amount. Previous studies have reported that HCA of CEE and the amounts of myricetin and quercetin were slightly
shows higher antioxidant activity than HBA. The presence of a increased, whereas the content of kaempferol decreased by up
CH CHCOOH group in the HCA derivatives, which is more active to 2.4 fold from CEE (Table 5). The increases in antioxidant and
than the COOH group in HBA derivatives, leads to the greater anti-glycation activities of EAF from G. procumbens could because
antioxidant activity (Eom et al., 2012; Sánchez-Maldonado et al., of these flavonoids, especially rutin, myricetin, quercetin and
2011). Interestingly, fractionation with ethyl acetate enhanced the apigenin. Base on RP-HPLC analysis, the phenolic compounds iden-
total phenolic acid (HBA + HCA) of CEE from 2086 to 4398 ␮g/g DW tified and quantified in our study were the major contents and
in EAF (Table 5). The total phenolic acids of EAF were 2, 3 and 11 components present in all the fractions. In addition, the unknown
fold higher than those of the CEE, BF and CF, respectively. Gallic acid, compounds, which were not identified in this study, were found
protocatechuic acid, p-coumaric acid and ferulic acid were deter- to be the minor components; thus the LC–MS should be further
mined to be the most dominant phenolic acids in the EAF (Table 5). investigated. As the results obtained, it can be concluded that the
According to the bioactivity assays, the EAF also exhibited the high- fractionation using solvent polarity purified and concentrated most
est antioxidant and anti-glycation activities, indicating that EAF of the phenolic compounds responsible for biological activities.
could be composed of the phenolic acids that provide significant
activities. Many studies have demonstrated that the number and
position of the hydroxyl (OH) groups available on the structure of
the phenolic compounds influenced the antioxidant activity, with
an increase in the OH groups on the aromatic ring indicating higher 4. Conclusions
activities. Additionally, the presence of OH groups in the ortho
or para position and methoxy (OCH3 ) substituent groups in the Our study has demonstrated that the EAF derived from the CEE of
structure of phenolics increased the antioxidant activity (Sánchez- G. procumbens had the highest total phenolic and flavonoid contents
Moreno et al., 1998; Eom et al., 2012). Thus, these results suggest and exhibited the highest antioxidative activities, especially ABTS+•
that the differences in activities of the extracts are due to differ- scavenging, • OH scavenging, H2 O2 scavenging, lipid peroxidation
ences in structure, the type of substituent groups and number or inhibition and oxidative protein damage protecting activities. In
position of OH groups on the structure of the phenolic compounds. addition, EAF showed the strongest anti-AGEs formation capacity.
Our findings were in agreement with those previously reported by The differences in the antioxidant and anti-AGEs formation activi-
Jun et al. (2014), who found that the solvent fractionation of the ties of the derived fractions are due to differences in the individual
crude extract from purple perilla leaves increased total phenolic phenolic acids and flavonoids, as well as to the chemical struc-
content and antioxidant activity in the ethyl acetate fraction. tures of phenolic compounds. These findings suggest that the EAF
In the present study, we also identified five flavonoids, rutin, of G. procumbens may serve as a potential source of natural antiox-
myricetin, quercetin, apigenin and kaempferol, by RP-HPLC. The idant and anti-glycation agents for pharmaceutical and medicinal
quantifications of the five flavonoids were based on calibra- applications. However, further studies need to be conducted on the
tion curves of authentic standards (Table 5). The results showed evaluation of the antioxidant and anti-glycation activity by in vivo
that rutin, quercetin and kaempferol were found in all fractions, experiments, and to investigate their clinical effects in the human
whereas myricetin and apigenin were not detected in CF and BF. body.
278 N. Kaewseejan, S. Siriamornpun / Industrial Crops and Products 74 (2015) 271–278

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