Journal of Agriculture and Food Research 14 (2023) 100879
Available online 19 November 2023
2666-1543/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Comparative analysis of phenolic content and in vitro bioactivities of Bidens
pilosa L. flowers and leaves as affected by extraction solvents
Trang H.D. Nguyen
a
, Danh C. Vu
b
, Phan Q.P. Hanh
c
, Xuyen T. Vo
c
, Van Cuong Nguyen
d
,
Thanh Ngoc Nguyen
e
, Lien Le Phuong Nguyen
f
,
g
, Laszlo Baranyai
f
,
*
a
Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Viet Nam
b
Institute of Applied Technology, Thu Dau Mot University, Binh Duong Province, Viet Nam
c
Faculty of Applied Technology, School of Technology, Van Lang University, Ho Chi Minh City, Viet Nam
d
Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Viet Nam
e
School of Science, Engineering and Technology, RMIT University Viet Nam
f
Institute of Food Science and Technology, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary
g
Industrial University of Ho Chi Minh City, Ho Chi Minh City, Viet Nam
ARTICLE INFO
Keywords:
Bidens pilosa
Phenolics
Albumin denaturation
Glucosidase
Amylase
Xanthine oxidase
ABSTRACT
Bidens pilosa L., native to South America, is valued for its purposes as a food and medicine. The study aimed to
examine phenolics, antioxidant activity and inhibitory effects of B. pilosa flower and leaf extracts on albumin
denaturation,
α
-glucosidase,
α
-amylase, xanthine oxidase, and tyrosinase. The choice of extractants (water,
methanol, acetone, and ethyl acetate) greatly influenced the phenolic content and bioactivities of the extracts.
The results demonstrated the flower and leaf extracts differed significantly with respect to total phenolic content
and abilities to inhibit albumin denaturation,
α
-amylase, and xanthine oxidase. The ethyl acetate extracts may
show the strongest activity to scavenge DPPH radicals, to inhibit
α
-amylase and
α
-glucosidase, and to protect
albumin against denaturation. The aqueous extract possessed the strongest capacity to inhibit xanthine oxidase
while the acetonic extract was more effective in suppressing tyrosinase compared to the others.
1. Introduction
Bidens pilosa L., commonly known as black-jack, beggar’s tick, or
Spanish needle, is an herbaceous plant species in the Asteraceae family.
It is natively distributed in tropical and subtropical areas of South
America, but now is found growing across the world [1,2]. Bidens pilosa
has a long history of traditional use as both a food and a medicine in
South America and Africa [3,4]. As a food source, the plant is valued for
its edible leaves, young shoots, and tender stems. In countries like Brazil,
Colombia, and Peru, it is consumed as a leafy vegetable and added to
soups, stews, salads, and other dishes. In addition to its use as a food,
B. pilosa has a rich history of medicinal applications in South America.
Indigenous communities and traditional healers have utilized various
parts of the plant, including the leaves, flowers, and roots, to alleviate
conditions such as digestive disorders, respiratory ailments, skin in-
fections, and inflammatory conditions [5]. Research on B. pilosa’s po-
tential health promoting properties has gained attention in recent years.
Studies have revealed B. pilosa possesses multiple bioactivities of
importance to human health, such as antioxidant, anti-inflammatory,
antidiabetic, anticancer, hepatoprotective, and immunomodulatory ac-
tivities [6]. For instance, administration of B. pilosa ethyl acetate extract
to L-NAME-induced hypertensive rats (75 and 150 mg/kg/day) pre-
vented elevated blood pressure, combated oxidative stress, and pro-
tected cells from damage in liver and kidney [7]. In another animal
model, B. pilosa extract demonstrated significant protective effects
against the hepatotoxicity, nephrotoxicity, and intestinal damage
induced by carbon tetrachloride [8]. Evidence suggests that potential
health promoting properties of B. pilosa could be attributed to the
presence of flavonoids. For example, extracts from the plant comprise 5,
6,7,4
′
-tetramethoxyflavone, 5,3
′
,4
′
-trihydroxy-3,7-dimethoxyflavone
and quercetin, exerting inhibitory effects on the formation of free radi-
cals, and the growth of human colon cancer RKO cells [9]. Poly-
acetylenic compounds ((E)-7-phenyl-2-hepten-4,6-diyn-1-ol and
(Z)-7-phenyl-2-hepten-4,6-diyn-1-ol) purified from B. pilosa exerted
anti-metastasis activities on HGC-27 cells through the reversal of the
EMT process and the inhibition of the Wnt/β-catenin and Hippo/YAP
* Corresponding author.
E-mail address: Baranyai.Laszlo@uni-mate.hu (L. Baranyai).
Contents lists available at ScienceDirect
Journal of Agriculture and Food Research
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https://doi.org/10.1016/j.jafr.2023.100879
Received 31 July 2023; Received in revised form 10 September 2023; Accepted 14 November 2023
Journal of Agriculture and Food Research 14 (2023) 100879
2
signaling pathways [10]. Previous research has primarily focused on
tentative identification, semi-quantification, and isolation of phyto-
chemicals, particularly polyacetylenes and flavonoids, in the plant
[10–12]. In the present study, total phenolic content, individual
phenolic acids and flavonoids of various extracts of B. pilosa flowers and
leaves were determined. Free radical scavenging assays were performed
to evaluate antioxidant capacity of the extracts. Furthermore, inhibitory
activities against
α
-glucosidase,
α
-amylase, tyrosinase, xanthine oxidase,
and bovine albumin denaturation were assessed.
2. Materials and methods
2.1. Chemicals
Extraction solvents (HPLC-grade) were purchased from Fisher Sci-
entific (Pennsylvania, the United States). Phenolic acid analytical stan-
dards were obtained from Sigma-Aldrich (Missouri, the United States).
Rutin and quercetin were obtained from Chengdu Biopurify Phyto-
chemicals (Sichuan, China).
2.2. Samples
Bidens pilosa was supplied by an herbal products company located in
Luong Son ward, Hoa Binh province, Vietnam in December 2021. The
authentication of the plant was performed by the Institute of Southern
Ecology, Ho Chi Minh city. The plant’s flowers and leaves underwent
careful washing and drying until the moisture content dropped below
10%. Subsequently, the dried samples were placed in clean Ziploc bags
and stored in a refrigerator (4 ◦C, 65% relative humidity) until further
analyses.
2.3. Crude extract preparation
Crude extracts of B. pilosa were prepared by combining the dried
sample (10 g) with 100 mL of each solvent (water, methanol, acetone,
and ethyl acetate). The mixtures were subjected to shaking on an orbital
shaker at ambient temperature for 24 h, and then centrifuged at 5500
rpm for 10 min. The resulting supernatants were subjected to solvent
removal using a rotary evaporator. The obtained crude extracts were
then used for analyses of phenolics and in vitro bioactivities.
2.4. Determination of phenolic content
Total phenolic content (TPC) in B. pilosa flower and leaf extracts was
determined according to the method described by Vu (2022) [13]. For
the quantification of individual phenolics, a high-performance liquid
chromatography system coupled with a diode-array detector
(HPLC-DAD) was employed. The analytes were chromatographically
separated on a VertiSep GES C18 reverse-phase column (250 ×4.6 mm,
5.0
μ
m particle size) maintained at 30 ◦C. The mobile phase comprised
100% methanol (solvent A) and 1% formic acid in water (solvent B). The
elution of phenolics was carried out at a flow rate of 0.8 mL/min,
following a previously established elution gradient by Nguyen (2023)
[14]. In detail, the gradient profile was as follows: 25% A for 3 min,
25–40% A for 5 min, hold 40% A for 4 min, 40–60% A for 4 min, hold
60% A for 4 min, 60–80% A for 3 min, hold 80% A for 3 min, 80%–85%
A for 4 min and hold 80% for 2 min, then reduced to 25% A at the 35th
min. Phenolic acids were detected at a wavelength of 295 nm, while
flavonoids were detected at 360 nm. The quantification of phenolic
compounds was performed with external standards following the
method of Nguyen (2023) [14].
2.5. Evaluation of in vitro bioactivities
Antioxidant activity of the extracts was evaluated using ABTS (2,2’-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-
diphenyl-1-picrylhydrazyl) radical scavenging assays. Inhibitory effects
of the extracts on bovine albumin denaturation,
α
-glucosidase,
α
-amylase, xanthine oxidase, and tyrosinase were also determined. The
assays were detailed in Supplementary material (S1 – S6).
2.6. Statistical analysis
The results from multiple readings were combined to calculate the
mean ±standard deviation. Statistical significance was determined
using Tukey’s HSD test at a confidence level of 0.05. The Mann –
Whitney U test was applied to determine whether TPC and bioactivities
differed between flowers and leaves. The nonparametric Kruskal−Wallis
test was used to compare the four groups of extracts (water, methanol,
acetone, and ethyl acetate). Multiple pairwise comparisons were
determined using the Dunn test. XLSTAT 2016 software (Addinsoft,
Paris, France) was employed to conduct the statistical analyses.
3. Results and discussion
3.1. Phenolic content
The results showed that the methanolic extract of B. pilosa flowers
(ME-F) contained the greatest total amount of phenolics (128.19 ±3.94
mg GAE/g), followed by the aqueous extracts of the leaves (WA-L) and
flowers (WA-F). The acetonic and ethyl acetate extracts of the leaves
(AC-L and EA-L) comprised the lowest total amount of phenolics. It is
Table 1
Phenolic contents of B. pilosa flower and leaf extracts.
Phenolics Flowers Leaves
WA-F ME-F AC-F EA-F WA-L ME-L AC-L EA-L
Gallic acid mg/g 1.06 6.60 n.d. 1.42 1.90 2.20 n.d. n.d.*
Chlorogenic
acid
0.35 4.95 n.d. 0.28 0.75 0.26 0.24 0.57
Caffeic acid n.d. 0.08 0.05 n.d. 0.03 n.d. n.d. n.d.
p-Coumaric acid 0.07 81.98 0.13 13.89 0.85 7.85 0.25 1.05
Ferulic acid 0.29 16.89 n.d. 3.18 1.36 n.d. 0.15 0.76
Salicylic acid 0.72 11.00 72.63 6.86 0.48 1.90 6.70 3.25
Cinnamic acid n.d. 19.32 55.10 22.74 n.d. 1.65 1.77 1.81
Rutin n.d. 2.84 n.d. 0.88 n.d. 1.05 n.d. n.d.
Quercetin n.d. 8.77 25.90 11.51 n.d. 1.22 0.72 1.03
TPC mg GAE/
g
65.97 ±0.48
c
128.19 ±3.94
a
48.47 ±7.74
d
60.14 ±5.55
cd
80.69 ±6.47
b
55.97 ±0.48
cd
20.42 ±0.00
e
22.36 ±2.10
e
The results of individual phenolic compounds (mg/g extract) are shown as mean of duplicate measurements. TPC was expressed as mean and standard deviation (mg
GAE/g extract) of triplicate measurements. WA-F, ME-F, AC-F, and EA-F stand for the aqueous, methanolic, acetonic, and ethyl acetate flower extracts. WA-L ME-L, AC-
L, and EA-L represent the aqueous, methanolic, acetonic, and ethyl acetate leaf extracts. Different lowercase letters (a, b, c, d, e) indicate significant differences in TPC
among the extracts (p <0.05). n.d.: not detected.
T.H.D. Nguyen et al.
Journal of Agriculture and Food Research 14 (2023) 100879
3
also noted that no significant differences in TPC between the flower/leaf
extracts from these two organic solvents were observed. As seen in
Table 1, the flower extracts obtained using the organic solvents
exhibited TPC values that were more than double those observed in their
leaf counterparts. In contrast, TPC of the aqueous extract obtained from
the leaves was 20% higher than that from the flowers. Cort´
es-Rojas et al.
[15] revealed that TPC of B. pilosa flower and leaf extracts prepared from
aqueous ethanol ranged between 20 and 120 mg GAE/g followed by root
with significantly lower values while stem obtained the lowest TPC.
Another study has reported that an aqueous methanolic extract of
B. pilosa leaves was composed of 91.3 mg gallic acid equivalents per
gram [12]. Recent research has shown TPC of an ethanolic extract of the
whole plant averages 107.5 mg GAE/g [16]. Perhaps, the variations in
TPC among the investigations could be due to the methods employed to
extract phenolics in the plant, plant parts, and/or sampling locations.
Table 1 also shows the concentrations of nine phenolic acids and fla-
vonoids in the extracts. Notably, all the compounds monitored in the
study were present in the methanolic flower extract (Fig. S1, Supple-
mentary material). Besides, gallic acid, chlorogenic acid, caffeic acid,
p-coumaric acid, ferulic acid, and rutin were found at the highest con-
centrations in this extract. Of these, p-coumaric acid was detected in all
the sample, with concentrations ranging between 0.07 and 81.98 mg/g.
Chlorogenic acid was found in all the extracts, except for the acetonic
flower extract (AC-F). The concentration of this compound in the
methanolic extract of flowers was 19 times as high as that in the
methanolic extract of leaves (ME-L). Unlike the phenolics above, sali-
cylic acid, cinnamic acid, and quercetin were detected at the highest
levels in the flower extract obtained with acetone (72.63, 55.10, and
25.90 mg/g, respectively). Prior research has reported tentative iden-
tification and/or semi-quantification of various phenolics in B. pilosa
extracts, including chlorogenic acid, caffeic acid, rutin and quercetin
Fig. 1. Free radical scavenging activity of aqueous (WA), methanolic (ME),
acetonic (AC), and ethyl acetate (EA) extracts of B. pilosa (500
μ
g/mL).
Different letters (a, b, c, d) indicate significant differences (p <0.05).
Fig. 2. Inhibitory effect of aqueous (WA), methanolic (ME), acetonic (AC), and ethyl acetate (EA) extracts of B. pilosa and diclofenac on bovine albumin
denaturation.
T.H.D. Nguyen et al.
Journal of Agriculture and Food Research 14 (2023) 100879
4
using NMR and high resolution mass spectrometry [11,12,17]. In
another study, gallic acid and ferulic acid were detected at the con-
centrations of 33.30 and 0.58 mg/g of methanolic leaf extract [18].
Other than these, no data about phenolics in flowers of the plant have
been reported. The present study was the first work to provide evidence
of phenolics present in B. pilosa flowers.
3.2. Antioxidant activity
The present study evaluated the antioxidant capability of B. pilosa
extracts (500
μ
g/mL) by gauging their ability to trap ABTS and DPPH
radicals (Fig. 1). Among the extracts, ME-F and AC-L may exhibit the
strongest antioxidant activity as measured by the ABTS assay (72.17 and
68.07%, respectively), followed by AC-F (48.76%) and EA-F (46.92%).
Comparatively, the aqueous extracts (WA-F and WA-L) displayed the
weakest activity. No significant difference in ABTS antioxidant potential
was observed between these two aqueous extracts. Similarly, EA-F and
EA-L differed insignificantly with respect to their ability to scavenge free
ABTS radicals. The antioxidant activity determined by DPPH assay
showed a different trend in which WA-F, AC-F, EA-F, EA-L, and ME-L
exerted greater activity than the others, with the percentage of inhibi-
tion ranging from 76.38 to 83.36%. On the opposite end, WA-L, ME-F,
and AC-L showed no significant differences in the ability to remove free
DPPH radicals.
According to a previous study, the leaf extracts of B. pilosa, specif-
ically the acetonic and methanolic extracts at a concentration of 500
μ
g/
mL, exhibited a significant ability to inhibit the formation of ABTS and
DPPH radicals, displaying an inhibition percentage exceeding 80%.
However, the aqueous leaf extract demonstrated a slightly lower inhi-
bition percentage of 60% [19]. Another investigation demonstrated that
the methanolic leaf extract at a concentration of 400
μ
g/mL also
exhibited a strong capacity for scavenging ABTS and DPPH radicals,
with percentage of inhibition ranging between 70% and 80% [18].
3.3. Inhibition of bovine albumin denaturation
The presence of heat stress or chemicals can cause the denaturation
of proteins, resulting in modifications to their biological, chemical, and
physical characteristics. Consequently, the denaturation of tissue pro-
teins could serve as an indicator for inflammatory conditions. In the
present investigation, we evaluated the inhibitory activity of extracts
obtained from the flowers and leaves of B. pilosa on the denaturation of
bovine albumin. This assay aimed to gain insights into the anti-
inflammatory activity of this plant species. Inhibition curves were con-
structed, and IC
50
values were determined, as illustrated in Fig. 2. The
ethyl acetate extract from the flowers (EA-F) exhibited the strongest
inhibitory effect on albumin denaturation, displaying the lowest IC
50
value of 192.50
μ
g/mL. Comparatively, the inhibitory effects of AC-F
and WA-F were less potent, with IC
50
values of 319.95
μ
g/mL and
320.35
μ
g/mL, respectively. The methanolic flower extract (ME-F)
demonstrated the weakest ability to suppress albumin denaturation, as
indicated by its higher IC
50
value of 508.49
μ
g/mL among the flower
extracts.
As regards the leaf extracts, EA-L exhibited the most potent protec-
tive effect on bovine albumin against denaturation, as indicated by its
IC
50
value of 448.42
μ
g/mL, followed by WA-L and ME-L. Conversely,
the acetonic leaf extract demonstrated the highest IC
50
value, indicating
weaker activity in protecting against denaturation. The results also
provided evidence suggesting that ethyl acetate may possess a greater
capacity for extracting potential anti-inflammatory constituents from
B. pilosa. When compared to diclofenac (IC
50
=84.19
μ
g/mL), which
served as the positive control in the assay, all the extracts exhibited
lower efficacy in protecting bovine albumin from denaturation induced
by heat. The specific mechanism through which the extracts were able to
inhibit the denaturation of albumin under heat stress conditions remains
unclear. However, it is possible that this phenomenon may be attributed
to the interactions between the phenolic compounds present in the ex-
tracts and the aliphatic regions surrounding the lysine residue on the
surface of the albumin molecules. Further investigation is needed to
elucidate the underlying molecular mechanisms involved in this process.
Previous studies have been performed to explore in vitro anti-
inflammatory potential of B. pilosa extracts. Reportedly, aqueous
extract of the plant had a capacity to suppress interleukin-1β-induced
COX-2 expression and PGE
2
release in normal human dermal fibroblasts
[20]. In an animal study, ethyl acetate fraction of methylene chlor-
ide/methanol (1:1) extract of B. pilosa leaves at doses of 50, 100 or 200
mg/kg was used to evaluate anti-inflammatory activity of the plant [21].
It was shown that the extract significantly reduced the edema formation
of the rat paw after treatment with carrageenan, dextran, and histamine.
Furthermore, the study suggested that the presence of quercetin and
isookanin could play a role in the anti-inflammatory activity observed.
3.4. Enzyme inhibitory activities
In vitro bioactivities related to the inhibition of glucosidase, amylase,
xanthine oxidase, and tyrosinase play significant roles in human health.
α
-Glucosidase and
α
-amylase inhibitors are particularly relevant for
managing blood glucose levels and preventing or controlling diabetes.
These enzymes are involved in carbohydrate digestion and their inhi-
bition can help decelerate the absorption of glucose, thereby regulating
postprandial blood sugar levels [22]. Xanthine oxidase, on the other
hand, is responsible for the production of uric acid, and its excessive
activity can lead to conditions like gout. Inhibiting xanthine oxidase can
help reduce uric acid levels, preventing the formation of painful crystals
[23]. Tyrosinase inhibitors are important for various cosmetic and
dermatological applications, as they can regulate melanin production
and help in treating hyperpigmentation disorders [24]. By inhibiting
tyrosinase, the enzyme responsible for melanin synthesis, these in-
hibitors can contribute to skin lightning and the management of con-
ditions such as melasma or age spots. Overall, understanding and
utilizing the in vitro bioactivities of these enzyme inhibitors are crucial
for the development of therapeutics, nutraceuticals, and cosmetic
products aimed at promoting human health.
Based on the aforementioned information, the present study aimed
to investigate the inhibitory activities of B. pilosa extracts against en-
zymes, including xanthine oxidase,
α
-glucosidase,
α
-amylase, and
tyrosinase. The results of the assessment are shown in Table 2.
Table 2
Enzyme inhibitory effects of the B. pilosa extracts.
a
.
Extracts Xanthine
oxidase,
mg AE/g
α
-Glucosidase,
mg ACAE/g
α
-Amylase,
mg ACAE/g
Tyrosinase,
μ
g KAE/g
Flowers WA-
F
1428.08
±29.19 a
11.03 ±2.71
bcde
2.56 ±0.88
c
225.68 ±
13.90 a
ME-
F
445.58 ±
8.04 f
30.67 ±3.96 a 9.31 ±0.91
ab
n.i.
AC-
F
555.58 ±
21.84 e
3.87 ±2.77 e 10.12 ±
0.36 a
223.95 ±
18.92 a
EA-
F
425.58 ±
21.26 f
18.78 ±1.72 b 7.20 ±1.37
b
88.21 ±
13.31 c
Leaves WA-
L
801.42 ±
61.25 d
12.95 ±2.48
bcd
4.45 ±1.14
c
n.i.
ME-
L
847.25 ±
25.00 cd
4.67 ±1.07 de 10.59 ±
0.39 a
86.95 ±
11.90 c
AC-
L
985.58 ±
3.82 b
7.89 ±2.79 cde 10.39 ±
0.42 a
225.44 ±
19.40 a
EA-
L
912.25 ±
34.37 bc
13.59 ±4.53 bc 10.34 ±
0.19 a
179.60 ±
16.42 b
a
The concentration of the extracts was 4000
μ
g/mL. WA-F, ME-F, AC-F, and EA-F
stand for the aqueous, methanolic, acetonic, and ethyl acetate flower extracts.
WA-L ME-L, AC-L, and EA-L represent the aqueous, methanolic, acetonic, and
ethyl acetate leaf extracts. Different lowercase letters indicate significant dif-
ferences in bioactivities among the extracts (p <0.05). n.i.: no inhibition.
T.H.D. Nguyen et al.
Journal of Agriculture and Food Research 14 (2023) 100879
5
Regarding the xanthine oxidase assay, WA-F exerted the highest activity,
followed by AC-L and EA-L. The flower extracts obtained from methanol
(ME-F) and ethyl acetate (EA-F) displayed the lowest activity. As for the
inhibition of
α
-glucosidase, ME-F exhibited the highest potency while
AC-F and ME-L showed the weakest activity. With respect to the
α
-amylase assay, the aqueous extracts possessed significantly lower
inhibitory activity compared to the other extracts. The results also
revealed WA-F, AC-F, and AC-L were much stronger at inhibiting
tyrosinase than the other extracts.
Previous research reported a caffeoylquinic derivative and a flavo-
noid purified from B. pilosa leaves had a capacity to inhibit
α
-glucosidase
[25]. Studies also indicated the plant exhibited antidiabetic potential
due to the presence of polyynes in its chemical composition [26].
Beyond these, to our knowledge, there is a paucity of information about
inhibitory effects of B. pilosa extracts on the aforementioned enzymes
available in the literature. The findings of this study will enhance our
comprehension of the health benefits of B. pilosa.
3.5. Comparisons between solvent extraction methods
As discussed above, the extracts of B. pilosa may show different
enzyme inhibitory effects between the flowers and leaves due to their
varied chemical composition. In this section, box-and-whisker plots
were constructed to help visualize the differences. The use of the Mann –
Whitney U test showed the flower and leaf extracts varied significantly
with respect to TPC, inhibitory effects albumin denaturation,
α
-amylase,
Fig. 3. Total phenolic content and in vitro bioactivities of flower and leaf extracts from all the experiments.
Fig. 4. Total phenolic content and in vitro bioactivities of the aqueous (WA), methanolic (ME), acetonic (AC), and ethyl acetate (EA) extracts.
T.H.D. Nguyen et al.