Received: 16 April 2021 Revised: 28 July 2021 Accepted: 1 August 2021

DOI: 10.1002/pca.3086

RESEARCH ARTICLE

Screening and characterisation of potential antioxidant,

hypoglycemic and hypolipidemic components revealed in
Portulaca oleracea via multi-target affinity ultrafiltration LC–MS
and molecular docking

Hui Zhang1,2,3,4 | Guilin Chen1,3,4 | Jinpeng Yang5 | Chunlei Yang5 |

1,3,4
Mingquan Guo

1
CAS Key Laboratory of Plant Germplasm
Enhancement and Specialty Agriculture, Abstract
Wuhan Botanical Garden, Chinese Academy of Introduction: Portulaca oleracea is a commonly used nutritional vegetable and tradi-
Sciences, Wuhan, China
2 tional herbal medicine with plenty of nutrients and manifold pharmacological activi-
College of Life Sciences, University of
Chinese Academy of Sciences, Beijing, China ties. However, the potential active ingredients for its remarkable antioxidant,
3
Sino-Africa Joint Research Centre, Chinese hypoglycemic and hypolipidemic activities remain unexplored.
Academy of Sciences, Wuhan, China
4
Objectives: The present study aims to systematically evaluate the antioxidant activi-
Innovation Academy for Drug Discovery and
Development, Chinese Academy of Sciences, ties of different extracts of P. oleracea and screen bioactive ligands that can interact
Shanghai, China
with α-glucosidase, pancreatic lipase, and superoxide dismutase (SOD).
5
Tobacco Research Institute of Hubei
Province, Wuhan, China
Methods: In this research, the antioxidant activities of different parts of P. oleracea
and their corresponding total phenolic content (TPC) and total flavonoid content
Correspondence
Mingquan Guo, CAS Key Laboratory of Plant
(TFC) were systematically determined. Subsequently, a multi-target affinity ultrafiltra-
Germplasm Enhancement and Specialty tion method was developed using affinity ultrafiltration with SOD, α-glucosidase, and
Agriculture, Wuhan Botanical Garden, Chinese
Academy of Sciences, Wuhan, China.
pancreatic lipase coupled to liquid chromatography–mass spectrometry (UF-LC–MS).
Email: guomq@wbgcas.cn Later, molecular docking was used to further investigate the possible interaction
Chunlei Yang, Tobacco Research Institute of mechanism between these ligands and target enzymes.
Hubei Province, Wuhan, China.
Email: ycl193737@163.com Results: Among them, the ethyl acetate (EA) fraction showed the highest antioxidant
activity along with the highest TPC and TFC, and four compounds in the EA fraction
Funding information
Natural Science Foundation of Hubei Province, were quickly retrieved as potential SOD, α-glucosidase, and pancreatic lipase ligands,
Grant/Award Numbers: 2019CFB254, respectively. Molecular docking revealed that these potential ligands exhibited strong
2019CFB254 to G. Chen; Natural Science
Foundation of China, Grant/Award Number: binding ability and inhibitory activities on SOD, α-glucosidase, and pancreatic lipase.
81903791 to G. Chen Conclusion: The present study revealed that P. oleracea can be used as a functional
food with excellent antioxidant, hypoglycemic and hypolipidemic effects. Meanwhile,
the integrated strategy based on multi-target UF-LC–MS and molecular docking also
provided a powerful tool and a multidimensional perspective for further exploration
of active ingredients in P. oleracea responsible for the antioxidant, hypoglycemic and
hypolipidemic activities.

Hui Zhang, Guilin Chen and Jinpeng Yang contributed equally to this work.

Phytochemical Analysis. 2021;1–14. wileyonlinelibrary.com/journal/pca © 2021 John Wiley & Sons, Ltd. 1
2 ZHANG ET AL.
KEYWORDS
antioxidant, diabetes, obesity, pancreatic lipase, Portulaca oleracea, SOD, UF-LC–MS,
α-glucosidase
1 | I N T RO DU CT I O N
Diabetes mellitus (DM) is a metabolic disorder syndrome
characterised by hyperglycemia with high morbidity and high mortal-
ity, especially type 2 diabetes (T2DM), which accounts for more than
90% of the total amount.1 It is estimated that about five million
people died from diabetes with the financial outlay of $850 billion in
2017.2 Obesity is a metabolic disease associated with genes, environ-
3
ment, lifestyle, inflammation and other factors. The global epidemic
of obesity and diabetes has led to a significant increase in the number
of people worldwide with metabolic syndrome.4 Key enzymes
involved in the pathogenesis and the treatment of T2DM and obesity
have been studied extensively, among which α-glucosidase and lipase
have attracted worldwide attention. Effective α-glucosidase inhibi-
5
tors, such as acarbose,6 voglibose,7 and miglitol,8 served as the main
tactic in the field of diabetes and its complications.9,10 Pancreatic
lipase is also an important target enzyme for obesity. Orlistat is the
only lipase inhibitor drug registered for the treatment of obesity.11
Moreover, epidemiological and clinical research results disclosed that
increased oxidative stress in adipose tissue is both the instigator and
the consequence of obesity and its related metabolic syndrome.12
Excessive reactive oxygen species trigger oxidative stress, which
causes significant damage to the body by activating cellular stress
pathways, ultimately leading to diabetic, obesity, and cardiovascular
13
disease. Therefore, further exploration of the potential of
antioxidants in the earlier processes is necessary for the prevention
and treatment of diabetes, obesity and other metabolic diseases.
Although α-glycosidase and lipase have been revealed as
effective enzyme targets for diabetes and obesity, there is still few
enzyme inhibitors in clinical use, thus searching for new drug
candidates is always to be appreciated, especially from natural
sources for their tremendous structural and functional diversity.14
For example, Moringa oleifera leaf extracts significantly inhibited
the activities of α-glucosidase and pancreatic lipase, as well as
enhanced the glucose consumption of 3T3-L1 cells.15 Additionally,
α-lipoic acid, a typical natural antioxidant,16 is widely applied to
treat diabetes and its complications by reducing blood lipids,
protein oxidation products, and protecting pancreatic cells from
damage by ameliorating oxidative stress.17 To some extent, the rich
and diverse active ingredients of edible/medicinal plants bring us
more choices and hopes.
Portulaca oleracea, a kind of annual herb widely distributed all
over the world, is a well-received vegetable with abundant ω-3 fatty
acids, dietary minerals, vitamins, and other nutrients. Meanwhile,
P. oleracea is also a traditional herbal medicine and has not only been
listed as one of the most commonly used medicinal plants but also
named “Global Panacea” by the World Health Organisation.18 It has
been reported that P. oleracea is rich in variable active constituents
including polysaccharides, alkaloids, polyphenols, fatty acids, and
flavonoids,19 and possesses many biological activities, such as
antioxidant, antibacterial, anti-inflammatory, hypoglycemic, and
20,21
hypolipidemic, Traditionally, P. oleracea was widely used in the
treatment of cardiovascular and cerebrovascular diseases, diabetes,
and their correlated complications.22 A previous study revealed that
P. oleracea extracts played an hypoglycemic role by improving insulin
resistance in mice with type 2 diabetes.23 Besides, the aqueous
extracts of P. oleracea inhibited hyperglycemia and diabetic vascular
inflammation in db/db mice, prevented the development of diabetic
endothelial dysfunction, and thus delayed the development of
diabetes and vascular complications.24,25 Meanwhile, its seed extracts
have been reported to exhibit significant hypolipidemic activity in
diabetic rats,26 and extracts exerted excellent inhibitory activity
against α-glucosidase and pancreatic lipase.27,28 However, the respon-
sible bioactive compounds of P. oleracea against those enzymes and
their underlying mechanisms are far from known.
In this context, the development of efficient screening methods
and strategies to reveal the bioactive ingredients from natural prod-
ucts and their underlying mechanism has become an urgent need.
Ultrafiltration liquid chromatography–mass spectrometry (UF-LC–MS)
is a high-throughput screening method that combines ultrafiltration
and LC–MS. It has attracted numerous attention due to its potential
to quickly, efficiently, and sensitively screen bioactive ingredients
from complex mixtures.29,30 In the present study, different fractions
with diverse antioxidant activity, total phenolic content (TPC), and
total flavonoid content (TFC) were firstly evaluated, and the correla-
tion between the antioxidant activity of P. oleracea and its TPC
and TFC was explored through correlation analysis. Subsequently,
superoxide dismutase (SOD), α-glucosidase, and pancreatic lipase
were selected as the targets to screen potential ligands from the
fractions with the strongest antioxidant activity through UF-LC–MS.
Ultimately molecular docking was employed to revealing the interac-
tion mechanism between these potential ligands and enzymes. Hence,
this study provides practical guidance and a theoretical basis for the
in-depth exploration of the potential applications of P. oleracea as an
effective functional food for the treatment of diabetes, obesity and
other metabolic diseases.
2 | EX PE RI MENT AL
2.1 | Chemicals and reagents
α-Glucosidase from yeast was purchased from Kamai Shu Biotechnol-
ogy (Shanghai, China). Lipase from porcine pancreas and SOD from
ZHANG ET AL.
bovine erythrocytes were purchased from Yuanye Bio-Technology
Co., Ltd (Shanghai, China). Ultrafiltration membranes (0.5 mL, 10 and
30 kDa) were purchased from Millipore Co. Ltd (Bedford, MA, USA).
Chromatographic grade acetonitrile and formic acid for high-
performance LC–MS liquid phase, and 2,2-diphenyl-1-picrylhydrazyl
(DPPH), 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic
(ABTS), and 1,3,5-tri(2-pyridyl)-2.4.6-triazine (TPTZ) for antioxidant
analysis, were purchased from Sigma-Aldrich Group (Shanghai, China).
The water for the LC–MS analysis was prepared with a pure water
meter from Nanjing EPED Technology Development Co., Ltd (Nanjing,
China). Rutin was purchased from J&K Scientific Ltd (Beijing, China),
all other analytical solvents and chemicals were purchased from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
2.2 | Plant materials
Portulaca oleracea was collected from a farm in Zhengzhou, Henan
province, in August 2018. The specimen was identified by Prof.
Guangwan Hu, a botanist at Wuhan Botanical Garden, Chinese
Academy of Sciences. The fresh plant materials were dried in an oven
under 40 C, crushed with a granulator, and stored in refrigerators at

4 C until use (voucher specimen number: ZH-2018-08-002).
2.3 | Preparation of different extracts from
P. oleracea
Initially, 4 L 90% (v/v) ethanol was added to 400 g dry powders of
P. oleracea, soaked at room temperature for 12 hours. After that, they
were ultrasonically extracted (200 W, 40 kHz) for 30 minutes, the
extracts were filtered. The earlier steps were repeated three times.
Then, the crude extract (CE) was concentrated, and the residue was
dissolved in water and successively partitioned by petroleum ether
(PE), dichloromethane (DCM), ethyl acetate (EA), and n-butanol (n-Bu)
until the organic phase was colorless. Finally, the extracts of each part
were collected and concentrated with a rotary evaporator at reduced
pressure, freeze-dried into dry powders under vacuum, and kept at

4 C till use.
2.4 | Evaluation of the antioxidant capacity of
P. oleracea
2.4.1 | Determination of DPPH assay
For the antioxidant activity of P. oleracea samples against DPPH free
radicals were determined according to a previous study reported by
Xu et al. 31
with slight modifications. Initially, 10 μL samples or the pos-
itive control solutions of vitamin C (31.25–1000 μM) were mixed with
190 μL of DPPH (100 μM) and incubated for 30 minutes in darkness
at room temperature. Then, the absorbance was measured at 517 nm.
The final results were expressed as the mean of three replicates (mean
3
± standard deviation). DPPH free radical scavenging ability was
expressed by inhibition rate (%) and half maximal inhibitory concentra-
tion (IC50) value, and calculated as follows:
DPPH  free radical scavenging effect ð%Þ ¼ ½ðAC  AS Þ=AC   100 ð1Þ
acid)
where AC and AS represent the absorbance value of the blank control
and the tested sample control.
2.4.2 | Determination of ABTS assay
A slightly improved method reported by Zhu et al. was used to con-
duct ABTS free radical scavenging experiments of the CE and differ-
ent extraction parts of P. oleracea.32 The ABTS solution (7mM in
water) was mixed with potassium persulfate (4.9 mM in water) in an
equal amount (v/v) and reacted in darkness for 12 to 16 hours as the
working solution (ABTS+). The ABTS+ solution was properly diluted
with methanol to ensure an absorbance of approximately 0.700
± 0.100 at 734 nm. Then, 190 μL ABTS+ solution was mixed with
10 μL samples, and the absorbance at 734 nm was recorded after
incubation in the dark for 30 minutes. Methanol and vitamin C
(31.25–1000 μM) were used as blank and positive controls, respec-
tively. The results of ABTS scavenging activity were calculated using
the earlier calculation (equation 1).
2.4.3 | Determination of ferric reducing antioxidant
power (FRAP) assay
The ferric reducing antioxidant power (FRAP) assay was conducted by
the method described by Xu et al. with slight modifications.31 The
FRAP reagent was produced by mixing acetate buffer (300 mM,
pH 3.6), TPTZ solution (10 mM) in hydrochloric acid (HCl, 40 mM),
and iron(III) chloride (FeCl3, 20 mM) at a ratio of 10:1:1 (v/v/v). Next,
10 μL suitably diluted samples, 30 μL ultrapure water, and 260 μL
FRAP reagent were added in turn and then incubated at 37 C for
10 minutes. The absorbance of the mixture was recorded at 593 nm.
Iron sulphate (FeSO4, 31.25–1000 μM) was used to establish a
calibration curve. The activity of FRAP was expressed as milligrams of
Fe2+ per gram of the sample (mg Fe2+/g sample).
2.5 | Determinations of total phenolic content
(TPC)
TPC was calculated using the Folin–Ciocalteu method reported by
Deng et al.33 Namely, 20 μL of the diluted sample, 20 μL Folin–
Ciocalteu (in pure water, 25%, v/v), 100 μL sodium carbonate
(Na2CO3, 1 M), and 20 μL ultrapure water were added into the reac-
tion system successively and incubated in the dark at room tempera-
ture for 1 hour. The absorbance was recorded at 760 nm. Gallic acid
(5–40 μg/mL) was used as the standard to establish a calibration
4 ZHANG ET AL.
curve. The results of TPC were expressed in milligrams equivalent of
gallic acid per milligram of dry sample (mg GAE/g sample).
2.6 | Determinations of total flavonoid content
(TFC)
The TFC of ethanol extracts and different fractions of P. oleracea was
determined by colorimetry, following the method described by Zou
et al. with minor modifications.34 Briefly, 60 μL of sample solution
were mixed with 360 μL of methanol and 20 μL of sodium nitrite
(NaNO2) solution (5%, w/v). After incubation for 6 minutes, 40 μL of
aluminium chloride (AlCl3, 10%, w/v) was added to the tubes and
shaken gently for 6 minutes. Next, 40 μL of 10% AlCl3 was added to
stand for another 6 minutes. Finally, 120 μL of sodium hydroxide
(NaOH) solution (4%, w/v) was added and reacted for 15 minutes, and
the resultant absorbance was measured at 510 nm. The results of TFC
were expressed in milligrams equivalent of rutin per milligram of dry
sample (mg RE/g sample).
2.7 | Procedures for screening and identifying
potential ligands based on UF-LC–MS
2.7.1 | Affinity ultrafiltration screening
The screening process of potential ligands was conducted based on a
method reported by Chen et al. with slight modification.15 Briefly,
180 μL EA fraction of P. oleracea was incubated with 20 μL SOD (2 U,
pH 7.8), α-glucosidase (6 U, pH 6.8), and pancreatic lipase (2 mg/mL,

pH 7.4) in phosphate-buffered saline PBS buffer at 37 C for 1 hour,
respectively. The mixed solution was then transferred to an ultrafiltra-
tion membrane of 30 kD (for SOD and α-glucosidase) or 10 kD (for
pancreatic lipase), centrifuged at 10000 rpm for 10 minutes and washed
three times with appropriate PBS buffer to remove the non-specific
binding components. Then, 200 μL acetonitrile (90%, v/v) was added
and incubated for 20 minutes to release compounds bound to the
enzyme, followed by the centrifugation at 10000 rpm for 10 minutes.
Finally, the filtrates were collected, lyophilised, and redissolved with
40 μL methanol for high-performance liquid chromatography ultraviolet
electrospray ionisation tandem mass spectrometry (HPLC-UV/ESI-MS/
MS) analysis. The denatured enzyme which was incubated in boiling
water for 10 minutes was served as negative control.
2.7.2 | HPLC-UV/ESI-MS/MS analysis
The chemical composition of EA fraction from P. oleracea was not
clear. In this part, Thermo Accela 600 HPLC system coupled with a
TSQ Quantum Access MAX MS was acquired for the analysis and
identification. The separation of EA fraction was conducted with a
HPLC system equipped with Waters Sunfire RP-C18 column (4.6 mm
 250 mm, 5 μm; Waters, Wexford, Ireland). The mobile phase used
for HPLC elution consisted of 0.1% (v/v) formic acid aqueous solution
(mobile phase A) and acetonitrile (mobile phase B). The gradient con-
ditions for the EA fraction were as follows: 5–20% (B) in 0–
15 minutes; 20–22% (B) in 15–24 minutes, 22–24% (B) in 24–
26 minutes, and 24–26% (B) in 26–46 minutes. Furthermore, 15 μL
sample was injected at the flow rate of 0.8 mL/min. The chromato-
gram of EA fraction was obtained at the wavelength of 320 nm. The
ESI-MS/MS analysis was performed in the negative ion mode by a
TSQ Quantum Access MAX MS equipped with ESI interface under
the following conditions: the temperatures of vaporiser and capillary
were 350 C and 250 C, respectively; the gas pressure of sheath gas
(nitrogen gas, N2) and axu gas (helium, He) was 40 psi and 10 psi,
respectively; the source voltage was 3000 V and the cone voltage and
collision energy were 40 V and 10 V, respectively; mass range (m/z)
was from 150 to 1500, spray voltage was 3 kV. MS data (mass range
from m/z 100–1000) was obtained in the full-scan mode. The
preliminary identification of compounds in the EA fraction of
P. oleracea was carried out by comparing the parent ions, MS
fragments, and retention times with the references.
2.8 | Molecular docking analysis
According to the paper published by Chen and Guo,35 SOD (PDB:
2C9V), α-glucosidase (PDB: 3A4A), and pancreatic lipase (PDB: 1LPB)
were simulated with corresponding potential ligands in P. oleracea by
molecular docking. Initially, the three-dimensional (3D) structures of
the three enzymes were downloaded from the RCSB protein databank
(www.rcsb.org), and ChemBio3D Ultra (version 12.0) was used to
establish the 3D structure of the ligands with the lowest energy. The
molecular docking process was simulated and optimised as follows.
The structure of the enzymes and their ligands were prepared by
removing the water molecules and adding the hydrogen atoms, and
the energy is then minimised in the MMFF94 force field. The grid box
was centred on the active site of the enzyme with dimensions size of
60  60  60 points, and the coordinate axes were center_X: 22.346,
center_Y: 15.739, and center_Z: 15.492 (SOD); center_X: 21.519,
center_Y: 7.702, and center_Z: 23.554 (α-glucosidase); center_X:
5.998, center_Y: 18.427, and center_Z: 37.754 (pancreatic lipase).
Then, AutoDock Tools 1.5.6 was used to dock the ligands to the
active sites of enzymes to create usable conformations. Subsequent
docking calculations were carried out through 2.5  106 energy
assessments coupled with 30 independent runs of lamarckian genetic
algorithms. AutoDock Tools 1.5.6 and the Discovery Studio 4.1
software were employed for the analysis and visualisation of the
docking results.
2.9 | Statistical analysis
All assays were repeated at least in triplicate, and the results were
expressed as mean ± standard deviation. SPSS data analysis software
version 24 was used for regression analysis of IC50 values, and Origin
ZHANG ET AL.
2021 software was used for significant difference analysis with the
one-way analysis of variance (ANOVA). Difference was considered
significant at P ≤ 0.05.
3 | RESULTS AND DISCUSSION
3.1 | In vitro antioxidant activity of P. oleracea
Considering the diverse scavenging methods of reactive oxygen spe-
cies and the complexity of natural phytochemicals, a series of
methods have been developed to comprehensively evaluate the anti-
oxidant activity of phytochemicals.31 In this work, three methods
were applied to evaluate and compare the antioxidant activities of the
CE from P. oleracea together with its PE, DCM, EA, n-Bu, and water
fractions. The results are shown in Figure 1. Compared with CE, EA
fraction showed significantly stronger radical scavenging capacity
against DPPH with the IC50 value of 142.70 ± 6.48 μg/mL, and also
stronger than the other four fractions, respectively. Ascorbic acid
(Vc) was used as positive controls in these assays with the IC50 values
of 3.68 ± 0.12 μg/mL. Meanwhile, the EA fraction also showed the
strongest potential antioxidant activities in FRAP assay with the mg
Fe2+/g of 11.37 ± 1.40 mg Fe2+/g, which was not only significantly
higher than that of CE (3.50 ± 0.25 mg Fe2+/g), but also better than
the other four fractions. Besides, ABTS assays showed that DCM frac-
tion had the strongest scavenging activity with IC50 at 24.49
± 1.52 μg/mL, followed by the EA fraction with IC50 at 45.16
± 1.67 μg/mL. Other fractions along with Vc (4.55 ± 0.25 μg/mL) also
showed considerable scavenging activities. Based on the results from
these three methods, the EA fraction showed the strongest antioxi-
dant capacity as compared with the other four fractions. Thus, the EA
fraction was selected for further studies in this work.
3.2 | Total phenolic and flavonoid contents of
P. oleracea
Antioxidant compounds, mainly polyphenols and flavonoids, serve as
the first line of defense against free radicals. These compounds
F I G U R E 1 Antioxidant activities of the crude extract (CE), petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), and n-butanol (n-
Bu) and water (H2O) fractions of Portulaca oleracea. (A) The half maximal inhibitory concentration (IC50) values of 2,2-diphenyl-1- picrylhydrazyl
(DPPH) radical scavenging assay; (B) the IC50 values of 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assay;
(C) ferric reducing antioxidant power (FRAP) assay. ** P < 0.01, && P < 0.01 as compared with the CE and ascorbic acid (Vc), respectively
5
interact with free radicals before they attack the cells to prevent fur-
ther damages.36 Portulaca oleracea is rich in polyphenolic compounds,
and its content fluctuates greatly in different growing stages37 and
different extraction processes.38 Flavonoids are also a major compo-
nent of P. oleracea.39 Just as shown in Figure 2A, The EA fraction con-
tained the most abundant phenolics (46.67 ± 0.99 mg GAE/g dw),
which was more than four times higher than that of CE (P < 0.01), and
water fraction showed the lowest total polyphenols of 7.35 ± 0.21 mg
GAE/g dry weight (DW). Similarly, the content of total flavonoids in
different fractions also showed a certain diversity. As shown in
Figure 2B, the EA fraction remained the highest of 86.28 ± 9.21 mg
RE/g DW, significantly lower than that of the CE fraction (36.30
± 2.57 mg RE/g DW), while water fraction was still the lowest (13.22
± 0.95 mg RE/g DW). These results further validate our findings
earlier., and provide a clear clue for us to explore the potential active
ingredients for its remarkable antioxidant and hypoglycemic activities
from EA fraction.
3.3 | Correlation analysis between antioxidant
activities and phytochemical components
To evaluate the relationships between antioxidant activities (DPPH,
ABTS, and FRAP) of P. oleracea and its TPC and TFC, the correlation
analysis was conducted and the results are shown in Table 1. On the
one hand, significant correlations were found among the three experi-
mental results for the determination of antioxidant activities, the
Pearson correlation coefficients (R2) between DPPH with ABTS and
FRAP were 0.887 (P < 0.05), 0.831 (P < 0.05), the R2 between ABTS
and FRAP was 0.842 (P < 0.05), indicating that these three methods
were reliable and interchangeable. On the other hand, there were also
strong correlations between antioxidant activities and chemical com-
positions. The DPPH and ABTS values were highly negative correlated
with TPC with the R2 at 0.843 (P < 0.05) and 0.941 (P < 0.01),
respectively. While the FRAP values showed an extremely significant
(P < 0.01, R2 0.956) positive correlation with the TPC. The R2 between
the total flavonoids with three antioxidant methods and the total
polyphenol content were 0.786, 0.576, 0.809, and 0.707, respec-
tively. This indicated that the overall trend was consistent, but not
6 ZHANG ET AL.

F I G U R E 2 (A) Total phenolic content (TPC) and (B) total flavonoid content (TFC) of Portulaca oleracea. GAE/g DW: gallic acid equivalent per
gram of dry weight; RE/g DW: rutin equivalent per gram of dry weight. ** P < 0.01, as compared with the crude extract (CE)

T A B L E 1 Pearson correlation coefficients (R2) among the various oxidative stress damage induced by free radicals.43 Extracts of
antioxidant activities and phytochemical contents of Portulaca P. oleracea reduced postprandial blood glucose by effectively reducing
oleracea
α-amylase and α-glucosidase activities,28 and also showed dose-
ABTS FRAP TPC TFC dependent antioxidant and anti-inflammatory activities against lipo-
DPPH 0.887 * 0.831 * 0.843 * 0.786 polysaccharide (LPS) induced lung injury in rats.18 In addition, it can
ABTS 0.842 * 0.941 ** 0.576 also greatly inhibit the activity of lipase.29 The earlier analysis indi-

FRAP 0.956 ** 0.809 cated that it was of theoretical and practical significance to explore
the antioxidant, hypoglycemic, and hypolipidemic activities of
TPC 0.707
P. oleracea by targeting SOD, α-glucosidase, and pancreatic lipase.
Note: * and ** indicate the correlation is significant at the level of 0.05 and
Ultrafiltration can be applied to effectively and sensitively screen
0.01, respectively.
DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,20 -azino-bis- the specific bioactive ingredients from the complicated extracts. The
(3-ethylbenzothiazoline-6-sulphonic acid); FRAP, ferric reducing binding ability of the ligand to the enzyme was measured by compar-
antioxidant power; TPC, total phenolic content; TFC, total flavonoid ing the relative binding affinity (RBA), which was obtained by calculat-
content. ing the ratio of the peak area after incubation with active and
inactivated enzymes. Compound with an RBA greater than 1.5 was
considered as a potential ligand.44
significant. Taken together, the earlier results implied that polyphenols The RBA was calculated as follows:
might be the main contributors to the antioxidant capacity of
P. oleracea, which was consistent with the report of Voynikov et al.40 RBA ¼ AS =AC ð2Þ

where AS and AC represent the peak areas of samples incubated with

3.4 | UF-LC–MS analysis
3.4.1 | Screening for the potential superoxide
dismutase, α-glucosidase, and pancreatic lipase ligands
in P. oleracea
α-Glucosidase and pancreatic lipase are key enzymes that catalyse the
digestion of carbohydrates and fat,41 its inhibitors provide deeper
insights into the treatment of diabetes, obesity, and other metabolic
diseases. Free radicals (a group of harmful reactive oxygen species)
are highly reactive small molecules that can damage DNA, proteins,
carbohydrates, and lipids. These damages could structurally lead to
cell damage or apoptosis, which ultimately leads to chronic diseases
such as diabetes and atherosclerosis. 42
Exogenous antioxidant
enzymes, such as SOD, a major antioxidant metalloenzyme responsi-
ble for scavenging reactive oxygen species, can protect cells from
activated and inactivated enzymes, respectively. Just as shown in
Table 2 and Figure 3, for SOD, genistein-40 -O-glucoside (peak 18) and
N-acetyl-L-phenylalanine (peak 15) exhibited higher RBAs with SOD,
with the RBA values of 1.62 and 1.57, other peaks also show good
higher RBA values, but their configuration needs to be further deter-
mined. For α-glucosidase, three of the 17 compounds showed higher
RBA values (more than 1.5), including, feruloylmalic acid (peak 17),
N-(carboxyacetyl)phenylalanine (peak 14), genistein-40 -O-glucoside
(peak 18), and the corresponding RBA values were 1.70, 1.53, and
1.52, respectively. For pancreatic lipase, N-(carboxyacetyl)phenylala-
nine (peak 14) exhibited the highest RBA value of 2.08, followed by
the genistein-40 -O-glucoside (peak 18) of 1.86, dhurrin 60 -glucoside
(peak 19) of 1.77. On the whole, genistein-40 -O-glucoside (peak 18)
showed higher RBA values to SOD, α-glucosidase, and pancreatic
lipase, indicating that it may exert antioxidant, hypoglycemic, and
hypolipidemic activities through multiple targets. The SOD, pancreatic
ZHANG ET AL. 7

lipase, and α-glucosidase inhibitory activities of these five compounds
have not been measured in any of the references so far.
3.4.2 | Characterisation of potential superoxide
dismutase, α-glucosidase, and pancreatic lipase ligands
based on LC–MS
The main peaks of EA fraction were identified and characterised
by HPLC-ESI-MS/MS. The MS and MS/MS data of these peaks,

such as retention time (RT), deprotonated molecular ion [M  H] ,
and their representative MS/MS spectra fragments are listed in
Table 2. In addition, the chemical structures of the compounds
identified in the EA fraction of P. oleracea were tentatively
identified as shown later, and part of them was also presented in
Figure 4.
Peak 1 displayed a deprotonated molecular ion [M  H] of m/z
195. Two major fragment ions at m/z 177 [M  H  H2O] and
m/z 159 [M  H  2H2O] indicated the continuous release of the
H2O group from [M  H]. The product ions observed at m/z
129 [M  H  H2O  CH2O] and m/z 99 [M  H  H2O  CO2]
were suggestive for the successive losses of CH2O and CO2 moieties

T A B L E 2 The mass spectrometry (MS) data and relative binding affinities (RBAs) of compounds identified from the ethyl acetate (EA) fraction
of Portulaca oleracea

RBA
a
Peak RT MS/MS Superoxide Pancreatic
numbera (min) [M  H] Formula fragments Name dismutase α-Glucosidase lipase
1 3.25 195 C6H12O7 195, 177, 159, Gluconic acid 1.44 — 1.31
129, 99, 87
2 4.67 133 C4H6O5 133, 115, 89, 71 Malic acid 1.38 — —
3 5.59 188 C10H7NO3 188, 170, 144, 2-Hydroxyquinoline- — —
128, 102, 59 3-carboxylic acid
4 6.48 117 C4H6O4 117, 99, 73, 55 Succinic acid — —
5 11.68 315 — 315, 153, 152, Unknown 1.58 1.36 0.95
109, 108
6 12.22 341 C15H18O9 341, 179, 135 Caffeic acid-hexose 2.29 — —
7 13.28 355 C16H20O9 355, 193, 178, Ferulic acid hexose-I 1.34 1.25 0.92
149, 134
8 15.14 353 C16H18O9 191, 173, 171 Chlorogenic acid 1.17 1.26
9 16.65 355 C16H20O9 193, 178, 149, Ferulic acid hexose-II 1.60 0.65 0.94
134
10 17.25 278 — 278, 216, 162, Unknown — —
132, 119
11 18.71 236 C11H11NO5 236, 192, 174, N-Benzoylaspartic acid — —
148, 120, 77
12 22.44 502 C24H26O11N 340, 296, 268, Oleracein A — —
194, 145
13 22.98 401 — 401, 341, 221, Unknown 1.24 —
177, 113
14 23.54 250 C12H13NO5 206, 164, 147, N-(Carboxyacetyl) 1.53 2.08
103, 91 phenylalanine
15 25.26 206 C11H13NO3 206, 164, 147, N-Acetyl-L- 1.57 1.38 1.12
103, 91 phenylalanine
16 25.64 163 C9H8O3 163, 119 p-Coumaric acid 1.29 — —
17 27.57 309 C14H14O8 193, 178, 149, Feruloylmalic acid 1.34 1.70 1.42
134, 117
18 30.37 431 C21H20O10 431, 269, 89 Genistein-40 -O- 1.62 1.52 1.86
glucoside
19 31.11 472 C14H17NO7 310, 161, 148 Dhurrin 60 -glucoside 1.31 1.24 1.77
20 39.18 456 C20H27NO11 294, 148, 145 4-Hydroxybenzyl 1.54 1.25 1.41
cyanide-Rha-Glc
21 41.09 486 — 324, 175, 148 Unknown 1.47 1.19 1.32
22 42.95 175 — Unknown 1.45 1.14 1.26

(Continues)
8 ZHANG ET AL.

F I G U R E 3 The ultrafiltration
high-performance liquid
chromatography (UF-HPLC)
chromatograms of the potential
superoxide dismutase (SOD),
α-glucosidase, and pancreatic
lipase ligands in Portulaca
oleracea. The black, red, and blue
lines represent the HPLC
chromatograms of P. oleracea
without (A), with activated and
inactivated SOD (B), with
activated and inactivated
α-glucosidase (C), with activated
and inactivated pancreatic lipase
(D), respectively [Colour figure
can be viewed at
wileyonlinelibrary.com]

at m/z 159. Peak 2 revealed the molecular ion [M  H] at m/z
133, similar fragmentations of [M  H  H2O] and [M  H  CO2]
were observed at m/z 115 and 89. Another major fragment ion m/z
71 was obtained by the loss of H2O from m/z 89. By comparing the
fragmentation information of MS with previous references, peak
1 and peak 2 can be tentatively identified as gluconic acid and malic
acid, which were previously isolated from P. oleracea.45 Peak 4 showed
product ions at m/z 117 [M  H], 99 [M  H  H2O], 73 [M  H 
CO2], 55 [M  H  CO2  H2O], all of which were 16 Da less than
the corresponding product ions of peak 2, indicating the loss of –OH
ZHANG ET AL.
group from malic acid. These fragments are consistent with those
reported in the literature,45 hence peak 4 was tentatively assigned as
succinic acid.
Peak 3 displayed a deprotonated molecular ion at m/z 188. Two
distinguished fragments at m/z 170 [M  H  H2O] and m/z
144 [M  H  CO2] were generated by the loss of H2O and CO2.
Hence peak 3 was tentatively assigned as 2-hydroxyquinoline-
3-carboxylic acid, which has been detected in the aerial parts of
P. oleracea.46
FIGURE 4 Chemical structures of the compounds identified in the ethyl acetate (EA) fraction of Poertulaca oleracea
9
Peak 6 produced a deprotonated molecular ion at m/z 341
[M  H], and the appearance of the major product ions at m/z
179 [M  H  C6H10O5] were due to loss of a hexose
residue. Another obvious fragment at m/z 135 [M  H  C6H10O5
 CO2] was due to the loss of the CO2 (44 Da) group from m/z
179. Peak 6 was supposed to be caffeic acid-hexose by comparing
the information on fragment ions in the reported reference.45
Peaks 7 and 9 showed the same deprotonated molecular ions at
m/z 355 [M  H], suggesting that they were structural isomers. The
10 ZHANG ET AL.

appearance of the major product ions at m/z 193 [M  H 
C6H10O5] was due to the loss of hexose residue, the fragment ions
at m/z 178 and 149 were due to the subsequent neutral losses of –
CH3 (15 Da) and CO2 (44 Da) group, respectively. Product ion at m/z

134 [M  H  C6H10O5  CO2] was due to the continued loss of
the CO2 (44 Da) group from m/z 178. Thus, peaks 7 and 9 were
presumed to be isomers of ferulic acid hexose and were tentatively
identified as ferulic acid hexose-I and ferulic acid hexose- II. 45,47
Peak 8 displayed a deprotonated molecular ion at m/z
353 [M  H]. Its main fragment ions at m/z 191, and m/z 173 were
produced by quinic acid. By comparing the MS fragments with the liter-
ature, peak 8 was tentatively assigned as chlorogenic acid, previously
40
isolated from P. oleracea. Peak 16, displayed its deprotonated molecu-
lar ion at m/z 163 [M  H]. Further fragments appearing at m/z
119 showed a loss of CO2 (44 Da) group. Hence, peak 16 was assigned
as p-coumaric acid, which was previously isolated from P. oleracea.20
Peak 11 produced a deprotonated molecular ion at m/z 236
[M  H]. Two distinguished fragments at m/z 192 [M – H  44] and

m/z 148 [M  H  2CO2] were generated, which indicated the contin-
uous loss of CO2 (44 Da) group. The fragment at m/z 174 was caused
by the loss of the H2O group on m/z 192 [M  H  CO2]. Another
fragment ion at m/z 120 was obtained by losing a C2H4 from m/z
148 [M – H – 44  44]. Hence, peak 11 was tentatively identified as
N-benzoylaspartic acid. Peak 15 displayed its molecular ion at m/z
206 [M  H] together with fragment ion at m/z 164 [M – H  42],
owing to the loss of –COCH3 moiety. Fragment appearing at m/z
147 indicated the loss of NH3 moieties. The fragment at m/z
103 showed a loss of CO2 (44 Da) group at m/z 147. The fragment at
m/z 71 was due to the loss of CH2 (14 Da) group at m/z 103. Thus, peak
15 can be tentatively identified as N-acetyl-L-phenylalanine, which was
previously detected in P. oleracea.47 Likewise, peak 14 exhibited a
molecular ion at m/z 250 [M  H], which was 44 Da higher than that
of peak 15, meanwhile, showed the same fragment ions at m/z
206 [M – H  CO2], m/z 164 [M – H  CO2  COCH3], m/z
147 [M  H  CO2  COCH3  NH3], m/z 103 [M – H  CO2 
COCH3  NH3  CO2], m/z 91 [M – H  CO2  COCH3  NH3
 CO2  CH2], respectively. Consequently, peak 14 was annotated as
N-(carboxyacetyl)phenylalanine, which was previously reported in
P. oleracea.47 Peak 12 exhibited its molecular ion at m/z 502 [M  H]
and characteristic MS/MS fragment at m/z 340 for the respective losses
of glucoside moieties. The fragment ion at m/z 296 showed a loss of
CO2 (44 Da) group at m/z 340. Another characteristic MS/MS fragment
was observed at m/z 194 [M – H  C9H6O2], indicative of the loss of
p-coumaroyl. Consequently, peak 12 was annotated as oleracein A, pre-
viously isolated from P. oleracea.48
Peak 17 gave a deprotonated molecular ion signal [M  H] at
m/z 309, its characteristic product ion was obtained at m/z 193.
MS/MS product ions at m/z 178 and 149 inferred the loss of CH3
(15 Da) and CO2 (44 Da) moiety of m/z 193. Another fragment ion at
m/z 134 suggested the loss of CO2 (44 Da) moiety of m/z 178. The
presence of these ions suggested that Peak 17 was feruloylmalic acid,
previously isolated in P. oleracea.20
Peak 18 showed a deprotonated molecular ion [M  H] at m/z
431, which produced daughter ions at m/z 269 [M – H  C6H10O5]

TABLE 3 Molecular docking analysis of the potential superoxide dismutase (SOD), α-glucosidase, and pancreatic lipase ligands in Portulaca
oleracea

SOD α-Glucosidase Pancreatic lipase

Peak BE IC50 Hydrogen- BE IC50 Hydrogen- BE IC50 Hydrogen-

no. Name (kcal/mol) (μM) bond atoms (kcal/mol) (μM) bond atoms (kcal/mol) (μM) bond atoms
14 N- — — — 5.09 185.88 5.82 54.57 GLN244,
(Carboxyacetyl) LYS239,
phenylalanine LYS238
15 N-Acetyl-L- 5.46 99.57 LYS3 — — — — — —
phenylalanine
17 Feruloylmalic — — — 4.55 462.71 GLN279, — — —
acid HIS280,
SER157,
ASN415,
LYS156,
LEU313
18 Genistein-40 -O- 6.07 35.70 LYS136, 8.11 1.14 ASP69, 6.27 25.24 GLN244,
glucoside HIS63, TYR158, ASN88,
ASN65 ASP215, ASP249,
ARG442, SER333,
ASP352 ARG265
19 Dhurrin 60 - — — — — — — 4.32 678.29 ASP249,
glucoside ASP257,
ASP331,
ARG265,
LYS268

Note: BE, binding energy; IC50, half maximal inhibitory concentration.

ZHANG ET AL. 11

due to the loss of glucoside residue. Thus, peak 19 was tentatively
identified as genistein-40 -O-glucoside, which was previously reported
in P. oleracea.39
Peak 19 gave a deprotonated molecular ion at m/z 472, and
its MS/MS fragments ions at m/z 310 [M – H  Glc] and
148 [M – H – Glc  Glc] implied the presence of two glucoside
residues. Another obvious product ion m/z 161 may be due to the
rupture of O-linkage on the glucoside residue. By comparing with
the fragmentation data in previous literature, peak 19 was tenta-
tively identified as dhurrin 60 -glucoside.49 Similar fragmentation
patterns and fragments were observed at peaks 20 and 21. Peak
20 showed the molecular ion [M  H] at m/z 456, its product
ions at m/z 294 [M – H  Glc] and m/z 148 [M – H – Glc 
Rha] indicated the presence of rhamnose and glucose residue.

F I G U R E 5 Three-dimensional (A) and two-dimensional (B) docking models of genistein-40 -O-glucoside with superoxide dismutase (SOD) and
three-dimensional (C) and two-dimensional (D) docking models of genistein-40 -O-glucoside with α-glucosidase [Colour figure can be viewed at
wileyonlinelibrary.com]
12
Product ion m/z 145 may be due to the rupture of O-linkage on
the rhamnose. Therefore, peak 20 was tentatively deduced as
4-hydroxybenzyl cyanide-Rha-Glc, which has not yet been
identified from P. oleracea and in current databases.
3.5 | Molecular docking studies
Molecular docking can be employed to quickly simulate the ligand–
target interaction mechanisms at the molecular level through
computer simulation, including the active site, binding mode, and
energy, and reveal the potential relationship between chemical
structures and activity, which has become a broad and powerful
approach in the field of new drug discovery and development. The
inhibitory activities of P. oleracea extracts on α-glucosidase50
and pan-
creatic lipase28 have been widely reported. In addition, five potential
SOD, α-glucosidase, and pancreatic lipase ligands were screened
separately by the earlier UF-LC–MS method, but the interaction
mechanisms between these ligands and enzymes remain unclear. In
this study, molecular docking was hired to investigate the binding
mode of SOD, α-glucosidase, and pancreatic lipase with its ligands.
The binding energy (BE), in silico estimated IC50 values, and hydrogen
bonds are shown in Table 3.
As shown in Table 3, those three potential α-glucosidase
ligands screened out exhibited low BE and estimated IC50 values,
basically confirming the higher RBA revealed by UF-LC–MS in
Table 2. In addition, according to the BE and IC50 values, the ranks
of the binding strength and enzyme inhibitory activity were in the
following order: genistein-40 -O-glucoside (peak 18), feruloylmalic
acid (peak 17), and N-(carboxyacetyl)phenylalanine (peak 14), which
were consistent with the corresponding rankings of the number of
hydrogen bonds, suggesting that more hydrogen bonds might con-
tribute to the interaction strength and enzyme inhibitory activity to
some extent. For example, genistein-40 -O-glucoside exhibited the
lowest BE of 4.90 kcal/mol and the lowest estimated IC50 value
at 1.14 μM. At the same time, a larger number of hydrogen bonds
were also observed between genistein-40 -O-glucoside and α-gluco-
sidase. It formed six hydrogen bonds with residues on ASP69,
TYR158, ASP215, ASP352, and ARG442. For pancreatic lipase, the
orders of the binding strength and enzyme inhibitory activity were:
genistein-40 -O-glucoside (peak 18), N-(carboxyacetyl)phenylalanine
(peak 14), and dhurrin 60 -glucoside (peak 19), and which were basi-
cally consistent with the corresponding rankings of the RBA
revealed by UF-LC–MS in Table 2. Among them, genistein-40 -O-
glucoside (peak 18) showed the most potential bonding ability to
pancreatic lipase by binding to the adjacent residue, and the
corresponding BE and estimated IC50 value were 6.27 kcal/mol
and 25.24 μM, respectively. The other two compounds bind to
pancreatic lipase mainly by van der Waals interaction, hydrophobic
interaction, and Coulomb interaction rather than hydrogen bonds.
The three-dimensional docking models of genistein-40 -O-glucoside
with the α-glucosidase and pancreatic lipase active site are shown
in Figure 5.
ZHANG ET AL.
It was worth mentioning that different hydrogen-bonds were
formed between three potential ligands and diverse amino acid resi-
dues of α-glucosidase. Among them, there were common amino acid
residues between three ligands and lipase, suggesting that the interac-
tions among those three bioactive components with lipase may be
competitive. Meanwhile, in the docking simulation process, other
forces such as van der Waals interaction, hydrophobic interaction,
and Coulomb interaction also provided vital role in the interactions
between the earlier potential bioactive components and enzymes,
especially pancreatic lipase. In this sense, the present study provided
a convenient method to explore the interaction mechanisms of SOD,
α-glucosidase, and pancreatic lipase with potential ligands in
P. oleracea, and is of considerable significance for the development
of active components from P. oleracea with hypoglycemic and
hypolipidemic activities.
AC KNOW LEDG EME NT S
This work was jointly supported by the Natural Science Foundation of
China (Grant no. 81903791 to G. Chen) and the Natural Science
Foundation of Hubei Province (Grant No. 2019CFB254 to G. Chen).
The funders played no roles in the study design, data collection and
analysis, and decision to publish. The authors would like to thank Prof.
Guangwan Hu for his assistance with the authentication of the plant
sample.
CONFLIC T OF INT ER E ST
The authors declare no conflict of interest.
DATA AVAILABILITY STAT EMEN T
No data are available
OR CID
Hui Zhang https://orcid.org/0000-0001-9166-570X
Mingquan Guo https://orcid.org/0000-0002-6627-5627
RE FE RE NCE S
1. Liu S, Li D, Huang B, Chen Y, Lu X, Wang Y. Inhibition of pancreatic
lipase, α-glucosidase, α-amylase, and hypolipidemic effects of the
total flavonoids from Nelumbo nucifera leaves. J Ethnopharmacol.
2013;149(1):263-269.
2. Cho NH, Shaw JE, Karuranga S, et al. IDF Diabetes atlas: global esti-
mates of diabetes prevalence for 2017 and projections for 2045. Dia-
betes Res Clin Pract. 2018;138:271-281.
3. Hamre K. Obesity: Multiple factors contribute. Nature. 2013;
493(7433):480.
4. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet.
2005;365(9468):1415-1428.
5. Peng X, Zhang G, Liao Y, Gong D. Inhibitory kinetics and mecha-
nism of kaempferol on α-glucosidase. Food Chem. 2016;190:
207-215.
6. Tsunosue M, Mashiko N, Ohta Y, et al. An alpha-glucosidase inhibitor,
acarbose treatment decreases serum levels of glyceraldehyde-derived
advanced glycation end products (ages) in patients with type 2 diabe-
tes. Clin Exp Med. 2010;10(2):139-141.
7. Chen XL, Zheng YG, Shen YC. Voglibose (Basen AO-128), one of the
most important alpha-glucosidase inhibitors. Curr Med Chem. 2006;
13(1):109-116.
ZHANG ET AL.
8. Campbell LK, Baker DE, Campbell RK. Miglitol: assessment of its role
in the treatment of patients with diabetes mellitus. Ann Pharmacother.
2000;34(11):1291-1301.
9. Liu X, Feng J, Li Y. Preparation of carbon-functionalized
magnetic graphene/mesoporous silica composites for selective
extraction of miglitol and voglibose in rat plasma. Talanta. 2018;
182:405-413.
10. van de Laar FA, Lucassen PL, Akkermans RP, van de Lisdonk EH,
Rutten GE, van Weel C. α-Glucosidase Inhibitors for Patients With
Type 2 Diabetes: Results from a Cochrane systematic review and
meta-analysis. Diabetes Care. 2005;28(1):154-163.
11. Shirai K, Tanaka M, Fujita T, et al. Reduction of excessive visceral
fat and safety with 52-week administration of lipase inhibitor
orlistat in Japanese: Long-term clinical study. Adv Ther. 2019;36(1):
217-231.
12. Furukawa S, Fujita T, Shimabukuro M, et al. Increased oxidative stress
in obesity and its impact on metabolic syndrome. J Clin Invest. 2004;
114(12):1752-1761.
13. Panigrahy SK, Bhatt R, Kumar A. Reactive oxygen species: Sources,
consequences and targeted therapy in type 2 diabetes. J Drug Target.
2017;25(2):93-101.
14. Harvey AL. Natural products in drug discovery. Drug Discov Today.
2008;13(19-20):894-901.
15. Chen GL, Xu YB, Wu JL, Li N, Guo MQ. Hypoglycemic and hypo-
lipidemic effects of Moringa oleifera leaves and their functional chemi-
cal constituents. Food Chem. 2020;333:127478.
16. Choi SW, Ho CK. Antioxidant properties of drugs used in type 2 dia-
betes management: Could they contribute to, confound or conceal
effects of antioxidant therapy? Redox Rep. 2018;23(1):1-24.
17. Ghibu S, Craciun CE, Rusu R, et al. Impact of alpha-lipoic acid chronic
discontinuous treatment in cardiometabolic disorders and oxidative
stress induced by fructose intake in rats. Antioxidants. 2019;
8(12):636.
18. Miao L, Tao H, Peng Y, et al. The anti-inflammatory potential of Portu-
laca oleracea L. (purslane) extract by partial suppression on NF-κB and
MAPK activation. Food Chem. 2019;290:239-245.
19. Zhou Y, Xin H, Rahman K, Wang S, Peng C, Zhang H. Portulaca
oleracea L.: A review of phytochemistry and pharmacological effects.
Biomed Res Int. 2015;2015:925631.
20. Nemzer B, Al-Taher F, Abshiru N. Phytochemical composition and
nutritional value of different plant parts in two cultivated and wild
purslane (Portulaca oleracea L.) genotypes. Food Chem. 2020;320:
126621.
21. Hozayen W. Effects of aqueous purslane (Portulaca oleracea) extract
and fish oil on gentamicin nephrotoxicity in albino rats. Nature Sci.
2011;9:47-62.
22. Rahdar P. The studying effect of drought stress on germination, pro-
line, sugar, lipid, protein and chlorophyll content in purslane (Portulaca
oleracea L.) Leaves. J Med Plant Res. 2012;6(9):1539-1547.
23. Lee JH, Park JE, Han JS. Portulaca oleracea L. extract reduces hyper-
glycemia via PI3k/Akt and AMPK pathways in the skeletal muscles of
C57BL/Ksj-db/db mice. J Ethnopharmacol. 2020;260:112973.
24. Lee AS, Lee YJ, Lee SM, et al. Portulaca oleracea ameliorates diabetic
vascular inflammation and endothelial dysfunction in db/db mice. Evid
Based Complement Alternat Med. 2012;2012:741824.
25. Zidan Y, Bouderbala S, Djellouli F, Lacaille-Dubois MA,
Bouchenak M. Portulaca oleracea reduces triglyceridemia,
cholesterolemia, and improves lecithin: Cholesterol acyltransferase
activity in rats fed enriched-cholesterol diet. Phytomedicine. 2014;
21(12):1504-1508.
26. Nazeam JA, El-Hefnawy HM, Omran G, Singab AN. Chemical profile
and antihyperlipidemic effect of Portulaca oleracea L. seeds in
streptozotocin-induced diabetic rats. Nat Prod Res. 2018;32(12):
1484-1488.
13
27. Park JE, Han JS. Portulaca oleracea L. extract lowers postprandial
hyperglycemia by inhibiting carbohydrate-digesting enzymes. J Life
Sci. 2018;28:421-428.
28. Conforti F, Perri V, Menichini F, et al. Wild mediterranean dietary
plants as inhibitors of pancreatic lipase. Phytother Res. 2012;26(4):
600-604.
29. Chen GL, Huang BX, Guo MQ. Current advances in screening for bio-
active components from medicinal plants by affinity ultrafiltration
mass spectrometry. Phytochem Anal. 2018;29(4):375-386.
30. Fan MX, Chen GL, Sun BQ, et al. Screening for natural inhibitors of
human topoisomerases from medicinal plants with bio-affinity ultrafil-
tration and LC-MS. Phytochem Rev. 2020;19(5):1231-1261.
31. Xu YB, Chen GL, Guo MQ. Antioxidant and anti-inflammatory activi-
ties of the crude extracts of Moringa oleifera from kenya and their cor-
relations with flavonoids. Antioxidants (Basel). 2019;8(8):296.
32. Zhu MZ, Liu T, Zhang CY, Guo MQ. Flavonoids of lotus (Nelumbo
nucifera) seed embryos and their antioxidant potential. J Food Sci.
2017;82(8):1834-1841.
33. Deng J, Liu R, Lu Q, et al. Biochemical properties, antibacterial and
cellular antioxidant activities of buckwheat honey in comparison to
manuka honey. Food Chem. 2018;252:243-249.
34. Zou Y, Chang SKC, Gu Y, Qian SY. Antioxidant activity and phenolic
compositions of lentil (Lens culinaris var. Morton) extract and its frac-
tions. J Agric Food Chem. 2011;59(6):2268-2276.
35. Chen GL, Guo MQ. Rapid screening for α-glucosidase inhibitors from
Gymnema sylvestre by affinity ultrafiltration-HPLC-MS. Front
Pharmacol. 2017;8:228.
36. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and
functional foods: impact on human health. Pharmacogn Rev. 2010;
4(8):118-126.
37. Saffaryazdi A, Ganjeali A, Farhoosh R, Cheniany M. Variation in
phenolic compounds, α-linolenic acid and linoleic acid contents and
antioxidant activity of purslane (Portulaca oleracea L.) during
phenological growth stages. Physiol Mol Biol Plants. 2020;26(7):
1519-1529.
38. Gallo M, Conte E, Naviglio D. Analysis and comparison of the antioxi-
dant component of Portulaca oleracea leaves obtained by different
solid–liquid extraction techniques. Antioxidants (Basel). 2017;6(3):64.
39. Liang X, Li L, Tian J, et al. A rapid extraction and analysis method for
the simultaneous determination of 26 bioflavonoids in Portulaca
oleracea L. Phytochem Anal. 2014;25(6):537-543.
40. Voynikov Y, Gevrenova R, Balabanova V, Doytchinova I, Nedialkov P,
Zheleva-Dimitrova D. LC-MS analysis of phenolic compounds and
oleraceins in aerial parts of Portulaca oleracea L. J Appl Bot Food Qual.
2019;92:298-312.
41. Sun H, Zhang Y, Ding W, et al. Inhibitory activity evaluation and
mechanistic studies of tetracyclic oxindole derivatives as alpha-
glucosidase inhibitors. Eur J Med Chem. 2016;123:365-378.
42. Abeyrathne N, Huang X, Ahn DU. Antioxidant, angiotensin-
converting enzyme inhibitory activity and other functional properties
of egg white proteins and their derived peptides – a review. Poult Sci.
2018;97(4):1462-1468.
43. Sheng Y, Abreu IA, Cabelli DE, et al. Superoxide dismutases and
superoxide reductases. Chem Rev. 2014;114(7):3854-3918.
44. Munigunti R, Mulabagal V, Caldero  n AI. Screening of natural com-
pounds for ligands to PfTrxR by ultrafiltration and LC-MS based bind-
ing assay. J Pharm Biomed Anal. 2011;55(2):265-271.
45. Fernández-Poyatos MDP, Llorent-Martínez EJ, Ruiz-Medina A. Phy-
tochemical composition and antioxidant activity of Portulaca oleracea:
Influence of the steaming cooking process. Foods. 2021;10(1):94.
46. Yan J, Sun LR, Zhou ZY, et al. Homoisoflavonoids from the medicinal
plant Portulaca oleracea. Phytochemistry. 2012;80:37-41.
47. Yang Y, Zhao XJ, Pan Y, Zhou Z. Identification of the chemical compo-
sitions of ponkan peel by ultra performance liquid chromatography
14 ZHANG ET AL.

coupled with quadrupole time-of-flight mass spectrometry. Anal

Methods. 2016;8(4):893-903. How to cite this article: Zhang H, Chen G, Yang J, Yang C,
48. Farag MA, Shakour ZTA. Metabolomics driven analysis of 11 Portulaca
Guo M. Screening and characterisation of potential
leaf taxa as analysed via UPLC-ESI-MS/MS and chemometrics. Phyto-
chemistry. 2019;161:117-129.
antioxidant, hypoglycemic and hypolipidemic components
49. Selmar D, Irandoost Z, Wray V. Dhurrin-60 -glucoside, a cyanogenic revealed in Portulaca oleracea via multi-target affinity
diglucoside from Sorghum bicolor. Phytochemistry. 1996;43(3): ultrafiltration LC–MS and molecular docking. Phytochemical
569-572. Analysis. 2021;1-14. doi:10.1002/pca.3086
50. Sicari V, Loizzo MR, Tundis R, Mincione A, Pellicano TM. Portulaca
oleracea L. (purslane) extracts display antioxidant and hypoglycaemic
effects. J Appl Bot Food Qual. 2018;91:39-46.