Synthesis and Antioxidative Activity of Piperine Derivatives Containing Phenolic Hydroxyl

Article abstract of DOI:10.1155/2020/2786359

Piperine was used in this study in its raw form, and different steps, such as amide hydrolysis and amidation, were used to synthesize piperine derivatives containing a phenolic hydroxyl group. DPPH and ABTS free radical scavenging assays were used to asse

Full text of DOI:10.1155/2020/2786359

Hindawi
Journal of Chemistry
Volume 2020, Article ID 2786359, 9 pages
https://doi.org/10.1155/2020/2786359
Research Article
Synthesis and Antioxidative Activity of Piperine Derivatives
Containing Phenolic Hydroxyl
,
,
Bei Qin ,1 2 Kuan Yang,2 3 and Ruijun Cao1
1School of Science, Xi’an Jiaotong University, Xi’an 710049, Shaanxi, China
2Department of Pharmacy, Xi’an Medical University, Xi’an 710021, Shaanxi, China
3College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021,
Shaanxi, China
Correspondence should be addressed to Bei Qin; qinbei0526@163.com
Received 15 April 2020; Revised 16 June 2020; Accepted 23 June 2020; Published 21 July 2020
Academic Editor: Ioannis G. Roussis
Copyright © 2020 Bei Qin et al. )is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Piperine was used in this study in its raw form, and different steps, such as amide hydrolysis and amidation, were used to synthesize
piperine derivatives containing a phenolic hydroxyl group. DPPH and ABTS free radical scavenging assays were used to assess piperine
derivative antioxidant activities. We constructed an AAPH oxidative stress erythrocyte model to study the effect of piperine derivatives
on the hemolysis rate of oxidatively damaged erythrocytes as well as the hemoglobin oxidation rate. )is AAPH model was also used to
determine piperine derivative effects on antioxidant enzyme activity and malondialdehyde (MDA) content. Results showed that
spectroscopic methods could synthesize and identify piperine derivatives containing phenolic hydroxyl groups (H-1∼H-3). Moreover,
DPPH and ABTS assay results showed that piperine derivative free radical clearance rates were higher compared with the parent
compound. Additionally, piperine derivatives (H-1∼H-3) were found to provide protection to AAPH oxidatively damaged erythrocytes
in their ability to inhibit AAPH-induced erythrocyte lysis, while hemoglobin oxidation was higher compared with the parent compound.
Piperine derivatives may protect intracellular glutathione peroxidase (GSH-Px) antioxidant enzyme system activities, safeguarding
against oxidative damage. )is study synthesized novel piperine derivatives for use as potential antioxidant agent candidates.
Piperine is an active alkaloid that is extracted from
peppers, long peppers, and other plants. It produces a variety
of physiological and pharmacological effects to occur, such
as antioxidative [7], antilipid [8], immunoregulative, anti-
tumor [9], and anti-inflammatory effects [10]. One study
found that piperine inhibited oxidative damage levels that
could potentially be associated with its effect on reducing
free radicals and reactive oxygen species (ROS) [7]. )e
antioxidative effects of piperine are derived by the inhibition
of lipid peroxidation and the enhancement of glutathione
(GSH) synthesis or transport [11]. Studies have shown that
piperine antioxidant activities are associated with the
molecule’s carbon-oxygen five-membered rings and amide
structure. Piperine reportedly undergoes demethylation of
its carbon-oxygen five-membered rings during metabolism
in the human body, mainly producing metabolites with a
bisphenol hydroxyl structure [12]. )e consequent synthesis
1. Introduction
Free radicals refer to any molecule with one or more un-
paired electrons that is a product of normal body meta-
bolism [1]. Some free radicals participate in cellular activities
in a controlled manner, while others are unbounded and
interact with nucleic acids, lipids, proteins, and other im-
portant biological molecules [2], causing acute and chronic
dysfunctions to occur. Accordingly, it is widely believed that
free radicals, which induce oxidative stress, are the main
contributors in the occurrence and development of multiple
diseases, fueling mechanisms that lead to cere-
brocardiovascular disease (CVD), tumors, inflammation,
and neurodegenerative diseases [2–4]. However, antioxi-
dants can scavenge excess free radicals within the body. As a
result, the discovery of novel antioxidant drugs has attracted
scientific interest and has led to much research [5, 6].

2
Journal of Chemistry
of phenolic hydroxyl groups enhances piperine antioxidant
activity [13]; hence, this enhancement should effectually be
retained during structural modification. Several studies have
shown that the derivatization of amide components within
piperine could also improve its antioxidant activity capacity
[14]. For example, Kharbanda et al. [15] derivatized the
amide structure of piperine to synthesize a series of piperic
acid and benzothiazole conjugates, and they found that the
derivatives significantly reduced factors associated with
PPAYc activity, consequently regulating lipid peroxidation
[15]. Several studies on natural antioxidants have reported
that most antioxidants were polyphenols, except for gly-
cosides and proteins, indicating that the presence of phe-
nolic hydroxyl groups effectively improves the reducing
power of compounds. Accordingly, this study derivatized
the amide structure of piperine to introduce phenolic hy-
droxyl groups, consequently obtaining phenolic hydroxyl-
containing piperine derivatives with improved antioxidant
capacities.
small amounts of methanol, and 6 mol/L hydrochloric acid
was used to adjust the pH level to one. Following this,
suction filtration was conducted, after which the substance
was dried to obtain compound 2. )e product was a yellow
powder with a mass of 7.61 g and a yield of 93.1%. 1H NMR
(400 MHz, DMSO-d6): δ 12.21 (s, 1H, -COOH), 7.30 (ddd,
J � 15.3, 7.0, 3.4 Hz, 1H, CH�CH-CO), 7.24 (d, J � 1.7 Hz,
1H, Ar-H), 7.03–6.79 (m, 3H, Ar-H, Ar-CH�CH-), 6.93 (d,
J � 8.0 Hz, 1H, Ar-CH�CH-), 6.06 (s, 2H, -OCH2O-), 5.93
(d, J � 15.2 Hz, 1H, CH�CH-CO). 13C NMR (101 MHz,
DMSO-d6): δ 168.03, 148.56, 148.45, 145.01, 140.21, 131.00,
125.33, 123.51, 121.63, 108.97, 106.20, 101.82.
2.2.2. (2E, 4E)-5-(Benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoyl
Chloride(2, C12H9ClO3). Compound 2 (6.91 g, 31.7 mmol)
was dispersed in 15 mL of anhydrous DCM. An oxalyl
chloride-DCM solution (12 mol/L, 2.70 mL) was added and
stirred for 2 h at room temperature to obtain an orange
liquid. Vacuum distillation was used to remove oxalyl
chloride and DCM to obtain an orange-red amide. It is
important to note that this amide must be prepared fresh
before use.
2. Materials and Methods
2.1. Chemicals and Experimental Animals. Oxalyl chloride
(98%) was purchased from Saan Chemical Technology Co.,
Ltd (Shanghai, China). TCI Development Co., Ltd
(Shanghai, China), supplied 3-aminophenol (98.5%). J&K
Scientific Co., Ltd, supplied 4-aminophenol (97%) and 2-
aminophenol (99%). Piperine was purchased from Shaanxi
Ciyuan Biotechnology Co., Ltd. Dichloromethane (DCM),
petroleum ether (PE), methanol, and ethyl acetate (EA;
analytical grade) were all purchased from Tianjin Fuyu Fine
Chemical Co., Ltd. )e methemoglobin (Hb) reagent kit,
catalase (CAT) visible light reagent kit, total protein
quantitation (BCA assay) reagent kit, malondialdehyde
(MDA) reagent kit, glutathione peroxidase (GSH-Px) re-
agent kit, and total superoxide dismutase (T-SOD) reagent
kit were all purchased from the Nanjing Jiancheng Bioen-
gineering Institute. AAPH (2, 2′-azobis (2-amidinopropane)
dihydrochloride) was purchased from Saan Chemical
Technology Co., Ltd.
2.2.3. Synthesis of Piperine Derivatives Containing Phenolic
Hydroxyl Groups (H-1, H-2, and H-3). As shown in Figure 1,
the reaction solution (4-aminophenol (2-aminophenol, 3-
aminophenol; 50.2 mg, 0.46 mmol)) was added to a 25 mL
round-bottom flask along with 2 mL of DCM. After 5 min of
ultrasonic dispersion, a newly prepared saturated sodium
bicarbonate (NaHCO3) aqueous solution (0.4 mL) was
added, followed by the slow addition of a freshly prepared
piperonyloyl chloride-DCM solution (0.5 mmol). Next,
2 mL of DMF was added to clarify the reaction system. )is
was followed by stirring at room temperature for 3-4 h. After
thin-layer chromatography detected the 4-aminophenol (2-
aminophenol, 3-aminophenol), 10 mL of distilled water was
added under stirring action to quench the reaction. Fol-
lowing this, 15 mL of EA was added before washing with a
5% NaHCO3 aqueous solution (2 × 20 mL), 5% HCl
(2 × 20 mL), and distilled water (2 × 20 mL). )e solution was
washed, concentrated, and vacuum-dried to obtain a pale-
yellow crude product.
)e H-1 crude product was then purified using column
chromatography on a silica gel (mixture eluent: EA : PE � 2 :1)
to obtain an 84 mg product (yield: 58%) as a pale-yellow solid.
1H NMR (400 MHz, DMSO-d6): δ 9.89 (s, 1H, -CO-NH-),
7.47 (d, J � 8.8 Hz, 2H, Ar-H), 7.31–7.23 (m, 2H, Ar-H,
CH�CH-CO), 7.03–6.90 (m, 4H, Ar-H, Ar-CH�CH-), 6.71
(d, J � 8.8 Hz, 2H, Ar-H), 6.26 (d, J � 14.9 Hz, 1H, CH�CH-
CO), 6.06 (s, 2H, -OCH2O-). 13C NMR (101 MHz, DMSO-d6):
δ 163.69, 153.86, 148.42, 148.26, 140.60, 138.78, 131.55, 131.31,
125.66, 125.26, 123.21, 121.31, 115.58, 108.91, 106.18, 101.74.
ESI: m/z 310.1 [M + 1]+.
Male Sprague Dawley laboratory rats (280 10 g) were
obtained from Chengdu Dossy Experimental Animals Co.,
Ltd. All experimental animals underwent routine feeding
and housing as well as ad libitum access to food and water.
)e experimental procedures used in this study were ap-
proved by the Ethics Committee of Xi’an Medical
University.
2.2. PiperineDerivativeSynthesis. Piperine was used as a raw
material to synthesize three structurally modified piperine
derivatives based on the pathways shown in Figure 1.
2.2.1. (2E, 4E)-5-(Benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic
Acid(1, C12H10O4). Piperine (10.69 g, 37.51 mmol) was
)e crude H-2 product was then purified using column
chromatography on a silica gel (mixture eluent: EA : PE � 2 :1)
to obtain a 102 mg product (4a) (yield: 71%) as a pale-yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 9.96 (s, 1H, Ar-OH),
9.60 (s, 1H, -CO-NH-), 7.77 (d, J � 8.0 Hz, 1H, Ar-H),
dispersed in
a potassium hydroxide (KOH) (237 g,
4.22 mol)/methanol solution (300 mL) and refluxed at 75°C.
After 24 h, the solution was cooled and subjected to suction
filtration to obtain a white solid. )e solid was dispersed in

Journal of Chemistry
3
O
O
(COCl)2
O
20% KOH in MeOH
75°C 24h
O
O
O
O
OH
N
1
Piperine
OH
OH
O
O
N
H
H-1
NH2
O
O
O
NaHCO3(aq)
RT 4h
N
H
O
O
Cl+
OH
H-2
HO
O
NH2
OH
2
O
O
N
H
OH
H-3
O
H2N
Figure 1: Schematic used in the preparation of piperine derivatives.
7.36–7.29 (m, 2H, Ar-H, CH�CH-CO), 7.04–6.87 (m, 6H, Ar-
H, Ar-CH�CH-), 6.79 (td, J � 7.6, 1.6 Hz, Ar-CH�CH-), 6.53
(d, J � 14.9 Hz, CH�CH-CO), 6.06 (s, 2H, -OCH2O-). 13C
NMR (101 MHz, DMSO-d6): δ 164.95, 148.51, 148.44, 148.40,
141.73, 139.52, 131.20, 126.97, 125.48, 125.39, 124.42, 123.37,
122.80, 119.55, 116.72, 108.94, 106.24, 101.78. ESI: m/z 310.1
[M + 1]+.
Finally, the H-3 crude product was purified using col-
umn chromatography on a silica gel (mixture eluent: PE :
AE : HAc � 2 :1 : 0.05) to obtain a 95 mg product (4a) (yield:
66%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 10.00 (s, 1H, -CO-NH-), 7.33–7.27 (m, 3H, CH�CH-CO,
Ar-H), 7.09–6.92 (m, 6H, Ar-H, Ar-CH�CH-), 6.45 (d,
J � 7.4 Hz, 1H, Ar-CH�CH-), 6.29 (d, J � 14.9 Hz, 1H,
CH�CH-CO), 6.06 (s, 2H, -OCH2O-). 13C NMR (101 MHz,
CDCl3): δ 164.20, 158.16, 148.43, 148.34, 141.23, 140.88,
139.22, 131.26, 129.75, 125.57, 125.05, 123.32, 110.88, 110.44,
108.93, 106.87, 106.22, 101.76. ESI: m/z 310.1 [M + 1]+.
absorbance (D0 and Dj) was then measured. VC was used as
the control. )e experiment was carried out in parallel
triplicate, and the following was used to calculate the DPPH
free radical clearance rate (IP) and IC50:
Di − Dj
IP � 1 −
× 100%.
(1)
ꢀ
ꢁ
D0
2.3.2. ABTS Assay. )e ABTS (2, 2′-azino-bis(3-ethyl-
benzothiazoline-6-sulphonic acid) assay was conducted
according to a modified method by Re et al. [17]. )e ABTS
working cation solution (ABTS+) was produced through
reaction of the ABTS aqueous solution with potassium
persulfate (K2S2O8) and kept under darkened conditions for
12∼16 h. )e solution was then diluted to a concentration
with an absorbance of 0.6∼0.8 at 734 nm. Piperine (0.1 mL)
and its derivatives (1.25∼80 μmol/L) were added to 0.1 mL of
the ABTS solution. )e reaction solution was incubated
under darkened conditions at room temperature for 30 min.
Absorbance (Di) was measured at 734 nm. )e ethanol
solution (0.1 mL) was separately mixed with 0.1 mL of the
ABTS solution or 0.1 mL of the sample solution, and the
absorbance (D0 and Dj) was subsequently measured. VC was
used as a negative control. )e experiment was carried out in
parallel triplicate, and equation (1) was used to calculate the
ABTS free radical clearance rate (IP) and IC50.
2.3. In Vitro Antioxidant Activity Assessment of Target
Compounds
2.3.1. DPPH Assay. DPPH (2, 2-diphenyl-1-picrylhydrazyl)
is regarded as a generator of free radicals and is widely used
to quantify the antioxidative capacity of biological samples
and foodstuff. In this study, a microplate reader was used to
measure the in vitro antioxidative capacity of piperine and its
derivatives applying the DPPH assay. DPPH free radical
scavenging was tested according to the method reported by
Gao et al. [16]. Piperine (0.1 mL) and its derivatives
(0.078125∼5 mmol/L) were added to 0.1 mL of a 0.1 mmol/L
DPPH-ethanol solution. )e reaction solution was incu-
bated under darkened conditions at room temperature for
30 min. A microplate reader ()ermo Fisher, 1510) was used
to measure absorbance (Di) at 517 nm. An ethanol solution
(0.1 mL) was separately mixed with 0.1 mL of the DPPH-
ethanol solution or 0.1 mL of the sample solution, and
2.3.3. Protective Effects of Piperine Derivatives on Oxidatively
Damaged Erythrocytes. Centrifugation (5 min at 3500 rpm/
min) was used to separate erythrocytes and plasma with the
addition of an anticoagulant at 4°C. )e erythrocyte pellet
was collected and washed four times with phosphate-buff-
ered saline (PBS) (pH 7.4) at 4°C, followed by centrifugation
at 3500 rpm/min for 5 min. A suitable amount of PBS was
then added to evenly mix and dilute the pellet to obtain a
10% erythrocyte suspension.

4
Journal of Chemistry
)e erythrocyte suspension (100 μL) was added to the
statistical analysis of the data. One-way analysis of variance
(one-way ANOVA) was used for data analysis. Finally,
p < 0.05 was considered significant.
sample solution (50 μL) and incubated at 37°C for 30 min.
Following this, 100 μL of 200 mmol/L AAPH was added, and
the solution was incubated at 37°C for 1–4 h [18]. )e
erythrocyte reaction solution (200 μL) was then aspirated,
added to 1 mL of the PBS buffer, and centrifuged at 4°C and
3500 rpm/min for 5 min. Following this, 200 μL was added to
a 96-well plate. )e absorbance of the wells was measured at
a 540 nm wavelength and recorded as APBS. An identical
volume of the erythrocyte reaction solution was aspirated
and added to 1 mL of 4°C precooled double distilled water to
complete erythrocyte lysis. Centrifugation was carried out
under identical conditions, and the absorbance was mea-
sured at 540 nm and recorded as Awater. )e experiment was
carried out in parallel triplicate, and hemolysis rates were
calculated using
3. Results and Discussion
3.1. DPPH and ABTS Free Radical Scavenging Ability of
Piperine Derivatives. DPPH and ABTS are regarded as free
radical generators and widely used to quantify antioxidative
capacities of biological samples and foodstuff. In this study, a
microplate reader was used to measure the in vitro anti-
oxidative capacity of piperine and its derivatives using
DPPH and ABTS free radical clearance assays.
As shown in Figure 2(a), VC was used as a positive
control, and its ability to scavenge DPPH• free radicals was
the highest overall. Piperine had almost no scavenging effect
on DPPH• free radicals. After its structural modification, the
ability of piperine to scavenge DPPH• free radicals increased
and exhibited concentration dependence. Under identical
concentrations, the DPPH• free radical clearance rate of
piperine derivative 1 was higher than piperine derivatives 2
and 3. Greater than 80% of DPPH• free radicals were cleared
at 0.625 mmol/L of piperine derivative 1. )e IC50 value
reflects the 50% inhibitory concentration of the antioxidant
being measured. )erefore, lower IC50 values correspond to
the higher antioxidative capacity of the substance. As shown
in Table 1, the antioxidative capacity of the different de-
rivatives was as follows: VC > H-1 > H-2 > H-3 > piperine.
Figure 2(b) provides ABTS free radical clearance results
for piperine and its derivatives. Piperine did not exhibit any
clearance of ABTS free radicals. After structural modifica-
tion, however, the ability of piperine to clear ABTS free
radicals increased, and the clearance rate increased as
concentrations increased. At a 40 μmol/L concentration,
ABTS free radical clearance rates of H-1, H-2, and H-3 were
all above 90%. After IC50 calculations, the antioxidative
APBS
Awater
Hemolysis rate (%) �
× 100%.
(2)
ꢀ
ꢁ
)e erythrocyte suspension (100 μL) was added to the
sample solution (50 μL) and incubated at 37°C for 30 min.
Following this, 100 μL of 200 mmol/L AAPH was added, and
the oxidative damage treatment was carried out at 37°C for
1–4 h. )e erythrocyte reaction solution (200 μL) was then
aspirated, added to precooled double-distilled water
(100 μL), and centrifuged at 4°C and 3500 rpm for 5 min. )e
absorbance of the 200 μL supernatant was measured at
630 nm and 700 nm wavelengths and recorded as A630 and
A
700, respectively. Following this, 10 μL of a 5% potassium
ferricyanide solution was added, and the absorbance was
measured and recorded as AmetHB630 and AmetHB700, re-
spectively. )e hemoglobin oxidation rate was calculated
using
A
630 − A700
ꢂ
ꢃ
Hemoglobin oxidation rate(%) �
× 100%.
(3)
ꢄ
ꢅ
A
− AmetHB700
ꢂ
ꢃ
metHB630
capacity of the different derivatives was as follows: H-3 > H-
2 > H-1 > VC > piperine. However, there were no significant
statistical differences detected in the IC50 of H-2 and H-3.
2.3.4. Effect of Piperine Derivatives on AAPH-Induced Rat
Erythrocyte Antioxidant Enzyme System and MDA Content.
)e erythrocyte suspension (100 μL) was added to the
sample solution (50 μL) and incubated at 37°C for 30 min.
Following this, 100 μL of 200 mmol/L AAPH was added and
the solution was incubated at 37°C for 1–4 h. Precooled (4°C)
ultrapure water was added to the aforementioned erythro-
cyte reaction solution to complete erythrocyte lysis, and the
solution was centrifuged. )e pellet was collected and
washed thrice with PBS. )e erythrocyte pellet (10 μL) from
the previous wash was aspirated, added to 990 μL of 4°C
precooled double-distilled water, and stored in a freezer at
−80°C for subsequent experiments. SOD and MDA levels
and GSH-Px and CAT activities in erythrocytes were
measured following the manufacturer’s instructions.
3.2. Protective Effects of Piperine Derivatives on AAPH-In-
duced Erythrocyte Oxidative Hemolysis and Erythrocyte He-
moglobin Oxidation. AAPH is the most common free
radical inducer used in the construction of erythrocyte
oxidative damage models. Studies have shown that AAPH
has a strong ability to induce oxidative damage in eryth-
rocytes, resulting in hemolysis. For this study, we con-
structed an erythrocyte oxidative damage model. Firstly, we
assessed the protective abilities of piperine and its derivatives
on AAPH-induced erythrocyte oxidative hemolysis, while
further examining the effects of piperine derivatives on
hemoglobin oxidation rates in erythrocytes. Erythrocytes are
the best cellular model to study oxidative damage [19, 20].
Experimental results showed that, at a 200 mmol/L AAPH
concentration and incubation at 37°C for 2 h, the erythrocyte
hemolysis rate was >60%, comprising of greater than half the
number of erythrocytes. Moreover, no significant differences
were observed between 2 h and 4 h of incubation. )erefore,
2.4. Data Processing. )e experiments were carried out in
parallel triplicate. All data was expressed as the mean-
standard deviation (x̅ s). SPSS 22.0 software was used for

Journal of Chemistry
5
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
0.0
0.0
0
20
40
60
80
0
1
2
3
4
5
Concentration (mM)
Concentration (mM)
H
H-1
H-2
H-3
vc
H
H-1
H-2
H-3
vc
(a)
(b)
Figure 2: DPPH and ABTS free radical clearance curves of piperine and its derivatives. (a) )e DPPH free radical clearance rate (IP) of
piperine and its derivatives at different concentrations. (b) )e ABTS free radical clearance rate (IP) of piperine and its derivatives at
different concentrations.
Table 1: IC50 of the DPPH and ABTS free radical inhibition rates of the different piperine derivatives.
Compound
IC50 of DPPH radical oxidation inhibition rate (mM)
IC50 of ABTS radical oxidation inhibition rate (μM)
Piperine
H-1
—
—
0.21 0.43
8.70 0.092
6.15 0.066
6.04 0.34
9.80 1.46
H-2
1.25 0.14
7.82 2.52
0.00097 0.00078
H-3
VC
incubation at 37°C for 2 h was selected for our subsequent
experiments.
that piperine and its derivatives exhibit protective effects
against AAPH-induced erythrocyte oxidative hemolysis,
wherein the protective ability of H-1 was stronger compared
with the parent compound.
As shown in Figure 3(a), erythrocytes were stable after
1–4 h of incubation in PBS (the control group). After 1 h of
AAPH oxidative stress, no hemolysis occurred. Subse-
quently, as incubation time increased, the hemolysis rate
increased significantly, reaching a maximum hemolysis rate
at 2 h. When erythrocytes were preincubated with piperine
derivatives prior to 2 h of AAPH stress, their hemolysis rates
decreased compared to the AAPH model group (p < 0.05).
Moreover, damage to hemolysis rates after 2 h of AAPH
stress showed that percentages of H, H-1, H-2, and H-3 were
72.60%, 66.84%, 49.23%, 52.08%, and 64.07%, respectively.
)is confirmed that the compounds exhibited protective
effects towards AAPH-induced erythrocyte lysis and that
H-1 and H-2 activities were higher compared to H-0.
Figure 3(b) provides concentration effects of samples while
being protected against AAPH-induced erythrocyte lysis.
Results showed that when the hemolysis rates of H-1, H-2,
and H-3 were compared to piperine, H-1 had a significant
hemolysis rate reduction (p < 0.05). )e protective ability of
H-1 towards AAPH-induced erythrocyte lysis also exhibited
concentration dependence; namely, when concentrations
were 25, 50, and 100 μmol/L, H-1 hemolysis rates were
59.42%, 49.23%, and 20.0%, respectively. )ese results show
Erythrocyte oxidative damage causes hemoglobin oxi-
dation and formation of methemoglobin. )erefore,
quantifying methemoglobin levels in erythrocytes can reflect
the level of erythrocyte oxidative damage. As shown in
Figure 4(a), methemoglobin levels in the control group were
extremely low and stable. In the AAPH group, however,
methemoglobin levels significantly increased as incubation
time increased. )is showed that methemoglobin levels
increased in cells after AAPH damage to erythrocytes. After
erythrocytes were preincubated with piperine derivatives
followed by 1 h and 2 h of stress, piperine derivatives were
found to be ineffective in reducing increases in AAPH-in-
duced methemoglobin. However, after 4 h of stress, the H-1
derivative significantly reduced AAPH-induced methemo-
globin levels (p < 0.05). )is showed that the H-1 derivative
has protective effects against AAPH-induced hemoglobin
oxidation. Figure 4(b) shows concentration effects of pip-
erine derivatives on AAPH-induced erythrocyte hemoglobin
oxidation. Results showed that H-1 exhibited protective
effects against hemoglobin oxidation at 50 and 100 μmol/L
concentrations. )ese findings showed that the H-1

6
Journal of Chemistry
100
80
60
40
20
0
100
80
60
40
20
0
∗
#
#
#
∗
#
#
#
#
∗
#
25
50
100
1
2
3
4
Concentration of compounds (µM)
Incubation time (h)
Control
AAPH
AAPH + H0
AAPH + H1
AAPH + H2
AAPH + H3
Control
AAPH
H
H-1
H-2
H-3
(a)
(b)
Figure 3: Protective effect duration of piperine derivatives on AAPH-induced erythrocyte hemolysis and time curves. (a) Time curves of the
protective effects of piperine derivatives on AAPH-induced erythrocyte hemolysis at a concentration of 50 μM. (b) )e relationship between
the protective effects of piperine derivatives at different concentrations and AAPH-induced erythrocyte hemolysis after 2 h incubation.
80
60
40
20
0
80
60
40
20
0
∗
∗
∗
25
50
100
1h
2h
4h
Incubation time
Concentration of compounds (µM)
Control
AAPH + H1
AAPH + H2
AAPH + H3
Control
AAPH + H1
AAPH + H2
AAPH + H3
AAPH
AAPH
AAPH + H
AAPH + H0
(a)
(b)
Figure 4: Duration of the protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation in erythrocytes and time
curves. (a) Time curves of the protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation at a concentration of
50 μM. (b) )e relationship between the protective effects of piperine derivatives at different concentrations and AAPH-induced he-
moglobin oxidation after 4 h incubation.
derivative had protective effects against AAPH-induced
hemoglobin oxidation in erythrocytes.
maintain a certain balance to protect tissues and organs from
being attacked by oxidizing agents. Among the body’s en-
zymatic and nonenzymatic antioxidative systems, super-
oxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GSH-Px) are major antioxidant enzyme systems
[21, 22]. GSH-Px is an antioxidant enzyme that is ubiquitous
in different tissues and cells in the human body. GSH-Px can
reduce glutathione (GSH) and generate hydrogen peroxide
(H2O2) inside cells to scavenge peroxides in the body as well
as also having antilipid oxidation effects. SOD is a crucial
3.3. Effects of Piperine Derivatives on AAPH-Induced Eryth-
rocyte GSH-Px, SOD, and CAT Activity. Free radicals (i.e.,
ROS and reactive nitrogen species (RNS)) possess strong
chemical reactivity and have the potential to cause oxidative
damage to biological macromolecules. Under normal cir-
cumstances, oxidative and antioxidative systems in the body

Journal of Chemistry
7
200
150
100
50
20
15
10
5
#
#
#
#
∗
#
#
∗
#
∗
∗
0
0
1h
2h
Incubation time
4h
1h
2h
Incubation time
4h
Control
AAPH + H2
AAPH + H3
vc
Control
AAPH + H2
AAPH + H3
vc
AAPH
AAPH
AAPH + H
AAPH + H1
AAPH + H
AAPH + H1
(a)
(b)
5
4
3
2
1
0
∗
∗
1h
2h
Incubation time
4h
Control
AAPH
AAPH + H
AAPH + H1
AAPH + H2
AAPH + H3
vc
(c)
Figure 5: Effects of piperine derivatives on AAPH-induced GSH-Px levels, SOD activity, and CAT activity in rat erythrocytes.
enzyme that scavenges free radicals in the body, and its
activity can reflect the ability of the body to scavenge free
radicals. CAT, ubiquitous in erythrocytes, catalyzes H2O2
degradation into H2O and O2, thereby reducing the oxi-
dative damage caused by the oxidation product OH• pro-
duced by H2O2 and metal ions [23].
Figure 5(a) shows the effects of piperine derivatives on
AAPH-induced GSH-Px activity in erythrocytes. In the
control group (i.e., the non-AAPH stress group), GSH-Px
levels in erythrocytes were stable. In the AAPH stress group,
GSH-Px levels in erythrocytes gradually decreased as in-
cubation time increased. After 1, 2, and 4 h of AAPH stress,
GSH-Px levels in erythrocytes successively decreased, and
the differences were statistically significant.
significantly increased GSH-Px levels (p < 0.05). After 4 h,
only the H-3 group showed significant differences in GSH-
Px levels compared to the AAPH group (p < 0.05). )ese
results showed that both H-2 and H-3 can increase GSH-Px
activity in erythrocytes, and between the two, the effect of
H-3 was the most significant while not exhibiting any sig-
nificant differences compared to the positive control (i.e.,
VC) (p > 0.05).
Figure 5(b) shows the effect of piperine derivatives on
AAPH-induced SOD activity in erythrocytes. Compared to
the control group, SOD activity in erythrocytes under the
AAPH stress group gradually decreased with increasing
incubation time. 50 μM of piperine derivatives were added to
erythrocytes prior to AAPH oxidative stress and compared
with the AAPH group. No significant effects respective to
SOD activity were observed in erythrocytes after 1 h of
When 50 μM of piperine derivatives were added to
erythrocytes prior to AAPH oxidative stress and compared
to the AAPH group, no significant effects on GSH-Px levels
were observed in erythrocytes after 1 h. At 2 h, H-2 and H-3
reaction time (p > 0.05). However, at 2 h, H, H-1, and H-3
significantly increased SOD activity (p < 0.05). After 4 h,

8
Journal of Chemistry
10
8
6
4
2
0
1h
2h
4h
Incubation time
Control
AAPH + H2
AAPH + H3
vc
AAPH
AAPH + H
AAPH + H1
Figure 6: Effects of piperine derivatives on AAPH-induced MDA levels in rat erythrocytes.
piperine derivatives and the positive control (i.e., VC)
upregulation of MDA levels in cells. )ere were no signif-
icant differences in MDA levels in erythrocytes treated with
exhibited no significant effects on SOD activity in eryth-
rocytes (p > 0.05). )ese results showed that as the duration
piperine and its derivatives compared with the AAPH group
of stress increased to 2 h, piperine and VC increased SOD
(p > 0.05). )ese results showed that piperine and its de-
activity in erythrocytes (p < 0.05); however, the derivatives
were unable to do so (p < 0.05). When the stress duration
increased to 4 h, no increase in SOD activity was observed in
erythrocytes in any group compared with the AAPH group.
When oxidative damage levels were severe, piperine was
unable to effectively prevent SOD consumption in eryth-
rocytes. )ese results showed that piperine derivatives ex-
hibit no significant effects on SOD activity in AAPH-
damaged erythrocytes.
rivatives do not protect against AAPH-induced lipid
peroxidation.
4. Conclusions
In this study, naturally extracted piperine was used as a
raw material to synthesize piperine derivatives contain-
ing a phenolic hydroxyl group. We demonstrated that the
free radical clearance rates of piperine derivatives were
higher compared with the parent compound. Piperine
derivatives exhibited a potent antioxidant ability in vitro.
By constructing an AAPH-induced erythrocyte oxidative
damage model, we demonstrated that piperine deriva-
tives have protective effects on AAPH-mediated oxida-
tively damaged erythrocytes. )e ability of the H-1
derivative to inhibit AAPH-induced erythrocyte lysis and
hemoglobin oxidation was higher compared to the parent
compound. We further investigated whether the pro-
tective effects of piperine derivatives against AAPH-in-
duced oxidative damage may derive from the protective
activity of the intracellular antioxidant enzyme system.
Our results showed that piperine derivatives can increase
GSH-Px activity in erythrocytes. In particular, the pro-
tective effect of the H-3 derivative was shown to be better
than piperine. To summarize, we designed and synthe-
sized three piperine derivatives, which effectively in-
creased the antioxidative capacity of piperine. )is study
provides a valuable research foundation for the appli-
cation of piperine as an antioxidant.
Figure 5(c) shows the effects of piperine derivatives on
AAPH-induced CAT activity in erythrocytes. Compared
with the control group, CATactivity in erythrocytes from the
AAPH stress group gradually decreased with increasing
incubation time. After 50 μM of piperine derivatives were
added, piperine and its derivatives did not show any sig-
nificant difference in intracellular CAT levels compared with
the AAPH group (p > 0.05). )ese results showed that
piperine and its derivatives do not cause any significant
differences in AAPH-induced CAT activity in rat erythro-
cytes (p > 0.05).
3.4. Effects of Piperine Derivatives on AAPH-Induced MDA
Levels in Rat Erythrocytes. A free radical imbalance in the
body causes unsaturated fatty acid oxidation to occur,
subsequently resulting in the formation of lipid peroxides, of
which malondialdehyde (MDA) is the most classic reaction
end-product. MDA causes cytotoxicity as it can disrupt the
integrity of the phospholipid membrane and induce cell
death [24]. MDA levels can reflect the level of tissue and cell
damage. Compared to the control group, MDA levels in the
AAPH group significantly increased as incubation time
increased (Figure 6). )is showed that AAPH induces ROS
generation, which causes lipid peroxidation and the
Data Availability
)e data used to support the findings of this study are
available from the corresponding author upon request.

Journal of Chemistry
9
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Conflicts of Interest
)e authors declare no conflicts of interest regarding the
writing and publication of this study.
Acknowledgments
)is study was financially supported by scientific research
funds provided by Xi’an Medical University (2016YXXK09),
Xi’an and Weiyang District Science and Technology Fund
(201930), and Xi’an Science and Technology Bureau Project
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