LWT - Food Science and Technology 162 (2022) 113467

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LWT
journal homepage: www.elsevier.com/locate/lwt

Exploring the phytochemicals and inhibitory effects against α-glucosidase

and dipeptidyl peptidase-IV in Chinese pickled chili pepper: Insights into
mechanisms by molecular docking analysis
Meiqi Li a, Xi Bao a, Xueting Zhang c, Hongbing Ren d, Shengbao Cai a, Xiaosong Hu a, b,
Junjie Yi a, *
a
Faculty of Food Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China
b
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
c
Wenshan Academy of Agricultural Sciences, Wenshan, 663000, China
d
Yunnan Hongbin Green Food Group Co. LTD, Yuxi, 652600, China

A R T I C L E I N F O A B S T R A C T

Chemical compounds studied in this article: The present work investigated the effect of 90 days’ traditional fermentation on phytochemicals and inhibition
Chlorogenic acid (PubChem CID: 1794427) towards α-glucosidase and dipeptidyl peptidase-IV (DPP-IV) of pickled chili pepper, and clarified their possible
Kaempferol-3-O-Rutinoside (PubChem CID: mechanisms based on molecular docking analysis. The results show that traditional fermentation significantly
5318767)
enhanced the inhibition for α-glucosidase but reduced the inhibitory effects towards DPP-IV (P < 0.05), espe­
Caffeic acid (PubChem CID: 689043)
Isoschaftoside (PubChem CID: 3084995)
cially fermented for 42 days. In pickled chili peppers, a total of 12 phytochemical compounds were characterized
O-Glucoside (PubChem CID: 5282102) and quantified, of which kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, apigenin, luteolin, and luteolin
Luteolin O-(Apiosylmalonyl) glucoside glycosides increased significantly after fermentation (P < 0.05). Among the compounds, luteolin showed the
(PubChem CID: 102130970) highest inhibition against α-glucosidase and isorhamnetin-3-O-glucoside exhibited the strongest inhibition ca­
Luteolin (PubChem CID: 5280445) pacity towards DPP-IV. Molecular docking results indicated luteolin, apigenin, and isorhamnetin-3-O-glucoside
Apigenin (PubChem CID: 5280443) could effectively interact with crucial amino acid residues of α-glucosidase, while isorhamnetin-3-O-glucoside
Rutin (PubChem CID: 5280805) and isoschaftoside could effectively interact with the key amino acid residues of DPP-IV.
Gallic acid (PubChem CID: 370)
Keywords:
Chili pepper
Fermentation
DPP-IV
α-Glucosidase inhibition
Molecular docking

1. Introduction
Type 2 diabetes mellitus (T2DM) is a chronic disease of carbohydrate
metabolism disorder characterized by hyperglycemia (Zhao et al.,
2020). In 2019, around 463 million people worldwide had diabetes and
it is estimated the number will increase to about 700 million by 2045,
90% of which are T2DM (Papoutsis et al., 2021). The main therapeutic
approaches for T2DM include targeting key glycemic regulation com­
ponents, such as digestive enzymes of carbohydrates and incretin hor­
mones (Cao et al., 2021). As known, α-glucosidase plays a key role in the
intestinal digestion of carbohydrates and its activity is one of the main
factors affecting postprandial glucose in the blood. (Yang et al., 2021).
The inhibition of α-glucosidase enables to delay in the production of
glucose hydrolyzed from carbohydrates, thereby controlling the post­
prandial glucose level in the blood (Zabidi, Ishak, Hamid, Ashari, &
Mohammad Latif, 2021). On the other hand, it is found that dipeptidyl
peptidase-IV (DPP-IV) can effectively degrade glucagon-like peptide 1
(GLP-1) and gastric inhibitory peptide (GIP), and the latter two peptides
stimulate the pancreas to produce insulin after a meal to lower blood
glucose (Imai et al., 2020). Therefore, the DPP-IV inhibitor can prevent

* Corresponding author. , Faculty of Food Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, PR China.
E-mail addresses: lmqkmust@163.com (M. Li), xibao19980127@163.com (X. Bao), 442988139@qq.com (X. Zhang), hblssp@hblssp.cn (H. Ren), caikmust2013@
kmust.edu.cn (S. Cai), huxiaos@263.net (X. Hu), junjieyi@kust.edu.cn (J. Yi).

https://doi.org/10.1016/j.lwt.2022.113467
Received 14 January 2022; Received in revised form 18 March 2022; Accepted 16 April 2022
Available online 20 April 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Li et al.
the GLP-1 and GIP from being degraded and thereby increasing insulin
secretion during the post-prandial phase to lower glucose level in the
blood (Nongonierma et al., 2019). Although there are clinical synthetic
α-glucosidase inhibitors (e.g., acarbose) and DPP- IV inhibitors (e.g.,
sitagliptin), the chemicals have many obvious side effects (diarrhea,
intestinal bloating) (Cam et al., 2020). Hence, it is important to explore
natural inhibitors with fewer side effects from the food matrix.
Previous studies have shown that numerous food components
exhibited good inhibitory activities towards α-glucosidase and DPP-IV.
For example (Jia et al., 2021), has proved that the polysaccharide
from wheat bran had great inhibitory activity on α-glucosidase. In
addition, it has been demonstrated that bioactive peptides from quinoa
protein hydrolysates displayed effective inhibitory properties towards
DPP-IV (Mudgil et al., 2020). In particular, studies have found that
phenolic extracts from many plants possessed much efficient hypogly­
cemic activity (Chung et al., 2011; Gandhi, Ignacimuthu, & Paulraj,
2011). For example, flavonoids isolated from rhizomes of Potentilla
anserina L. could effectively inhibit α-glucosidase activity (Yang et al.,
2021). Besides, the caffeoylquinic acids in aronia juice can inhibit both
DPP-IV and α-glucosidase activities (Imai et al., 2020).
Fermentation, as a common food processing method, can efficiently
increase the bioavailability of polyphenols in the food matrix (Du &
Myracle, 2018; Li et al., 2020). Chili peppers (Capsicum species),
belonging to Solanaceae family, are one of the most widely grown
economical crops around the world (Antonio, Wiedemann, & Veiga
Junior, 2018). Previous studies reported that the extracts from pepper
exhibited strong inhibitory activity against α-glucosidase (Kongstad,
Ozdemir, Barzak, Wubshet, & Staerk, 2015; Park, Zhu, Pae, & Park,
2016). However, studies about the influence of fermentation on the
inhibitory effects of pepper towards α-glucosidase and DPP-IV activities
are scarce. The traditional Chinese pickled chili pepper, named Paojiao
in Chinese, enjoys a high status in southwest China, especially in Yunnan
Province (Ye et al., 2020). Up to now, studies about Paojiao are mainly
related to its flavor, such as the influence of pepper variety, ripening
stages, and producing regions on its flavor properties (Ye et al., 2020,
2022). To the best of our knowledge, no studies have been done on the
bioactivities of Chinese pickled chili pepper, especially about the effect
of the fermentation process on hypoglycemic activity. Therefore, the
objective of the study is to investigate the changes of phytochemical
compounds and the inhibitory activities towards α-glucosidase and
DPP-IV of Chinese pickled chili pepper during traditional fermentation
(90 days). Furthermore, the underlying inhibitory mechanisms of the
main phytochemicals were further illuminated by molecular docking.
Results obtained in the present work could provide a scientific basis for
exploring the function and upgrading the economic value of Chinese
pickled chili pepper.
2. Materials and methods
2.1. Chemicals and materials
Folin-Ciocalteau reagent, AB-8 macroporous resin, acarbose, p-
nitrophenyl-α-D-glucopyranoside (pNPG) were purchased from Beijing
Solarbio Science & Technology Co., LTD. (Beijing, China). α-Glucosidase
(EC: 3.2.1.20, ≥50 units/mg protein) from Saccharomyces cerevisiae was
offered from Shanghai Ryon Biological Technology Co., LTD, (Shanghai,
China). DPP-IV inhibitor screening kit was purchased from Cayman
Chemical Company (Ann Arbor, MI, USA). LC-MS grade solvents
(acetonitrile and formic acid) were purchased from Merck (Darmstadt,
Germany). The standard phytochemical compounds including chloro­
genic acid, kaempferol-3-O-rutinoside, caffeic acid, isoschaftoside,
kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, luteolin, and
apigenin were obtained from Chengdu Must Bio-Technology Co., LTD.
(Chengdu, Sichuan, China).
2
LWT 162 (2022) 113467
2.2. Sample preparation
2.2.1. Preparation of crude extract
Pickled chili peppers used in the study were kindly provided by a
local company Yunnan Hongbin Green Food Group Co. LTD in October
2020 from Jianshui city, Yunnan Province, China. The texture properties
and microbial situations of all samples were acceptable. Raw chili
pepper (Capsicum frutescens L.) was fermented in an open industrial-
scale pickled tank for 90 days. The pickled tanks (10 m × 10 m × 4
m) were built by the production company without brand, which were
fully sank under the ground. During the fermentation process, the brine
(8.16% salt, 0.22% calcium chloride, and 0.63% glacial acetic acid) was
kept homogeneous through the circulation system. An aerobiosis spon­
taneous fermentation was conducted under 25–30 ◦ C by a temperature
control system. In this study, pickled Chinese chili pepper was sampled
at different fermentation moments, including 0, 7, 14, 21, 42, 56, and 90
days. During sampling, pickled chili peppers at each fermentation
moment were taken from two different pickled tanks. For each tank,
samples were collected at three points from top to bottom and then
mixed. The mixed samples from each tank were further extracted and
analyzed separately.
After sampling, all samples were lyophilized (Scientz-10N, Scientz,
China). The lyophilized samples were ground into powder (AQ-180E,
Nail, China) and passed through a 60-mesh sieve. The powder sample
(10 g) was subjected to ultrasonic-assisted extraction using 100 mL of
80% ethanol in an ultrasonic cleaning bath (200 W, 20 ◦ C, for 30 min).
The ultrasonic-assisted extraction procedure for each sample was
repeated three times. After centrifugation (4 ◦ C, 8000 g for 10 min), the
collected supernatant was concentrated by a rotary evaporator (Hei-
VAP, Heidolph, Germany) at 35 ◦ C to remove removed all organic sol­
vents. The obtained concentrated samples were lyophilized again to
obtain the crude extract.
2.2.2. Purification of crude extract
The crude extract was purified with AB-8 macroporous resin ac­
cording to the reported method (Xie, Chen, & Fu, 2021). Briefly, 2.0 g of
the crude pepper extract was completely suspended in 15 mL of distilled
water, and the sample suspension was loaded onto the resin by using a
peristaltic pump (BT100s, Lead fluid, China) at a flow rate of 0.25
mL/min. Then distilled water containing 0.1% formic acid was used to
elute with flow rate of 0.5 mL/min for 12 h at room temperature.
Thereafter, the AB-8 resin was eluted by using methanol containing
0.1% formic acid with flow rate of 0.5 mL/min for another 12 h. Finally,
the methanol eluate was collected, concentrated by the rotary evapo­
rator (Hei-VAP, Heidolph, Germany) at 35 ◦ C, and lyophilized to obtain
the absorbed sample, which was weighed and stored at − 40 ◦ C for
further analysis. The extraction ratios (w/w) of different samples were
6.111% (0-day), 4.760% (7-day), 6.153% (14-day), 5.053% (21-day),
4.523% (28-day), 5.426% (42-day), 4.873% (56-day), 3.840% (90-day),
respectively.
2.3. Determination of total phenolics content
The total phenolics content (TPC) of different fermented peppers was
determined using the Folin–Ciocalteu reagent as the previous study
(Zhou et al., 2019). In brief, the purified sample (equivalent 1.0 g of chili
pepper) was dissolved in 80% methanol (1.0 mL), which was used as the
sample solutions. The sample solution (50.0 μL) was mixed with 500.0
μL of the Folin–Ciocalteu reagent and 450.0 μL of a 7.5% (w/v) of
Na2CO3 solution. The mixture was placed into hot water (70 ◦ C) for 10
min. Then, the absorbance of each mixture was measured at 765 nm by a
microplate reader (SpectraMax M5; Molecular Device). The TPC was
expressed as mg of gallic acid equivalents (GAE) per 100 g of dry pickled
chili pepper weight. For each purified sample, measurements were
conducted in triplicate.
M. Li et al.
2.4. Determination of total flavonoid content
The total flavonoid content (TFC) of different samples was deter­
mined using the aluminum trichloride colorimetric method (Zhou et al.,
2019). Briefly, 0.03 mL of 5% NaNO2 (m/v) was added to 0.2 mL of the
sample solutions (as prepared in Section 2.3), and the mixture was
allowed to stand for 5 min. Subsequently, 0.03 mL of 10% Al (NO3)3
(m/v) was added to the mixture, followed by adding 0.40 mL of 1 mol/L
NaOH after 6 min. Finally, the total volume was made up to 2.0 mL with
80% ethanol (v/v). The solution was placed at room temperature for
another 30 min, and the absorbance was measured at 510 nm with a
microplate reader. The TFC was expressed as mg of rutin equivalents
(RE) per 100 g of dry pickled chili pepper weight. For each purified
sample, measurements were conducted in triplicate.
2.5. Identification and quantification of phenolics
Phytochemical analysis of each extract was performed by using a
Thermo Fisher Ultimate 3000 ultrahigh-performance liquid chroma­
tography (UHPLC) system (Thermo Fisher Scientific, Germany) coupled
with a high-resolution mass spectrometer (HRMS). The analyses were
performed using an Agilent Zorbax SB-C18 column (2.1 mm × 100 mm,
1.7 μm) based on the previously reported approach (Barbosa,
Campmajó, Saurina, Puignou, & Núñez, 2020). The chromatographic
separation was carried out with the following conditions: the mobile
phases were 0.1% formic acid in water (A) and acetonitrile (B) at a flow
rate of 0.2 mL/min, and the injection volume of sample was 2 μL. The
gradient program was set as follows: 0–3 min 5% (B), 3–12 min 5–60%
(B), 12–16 min 60–80% (B), 16–20 min 80% (B), 20–21 min 5% (B), and
21–25 min 5% (B). The data were acquired in scan mode by using an m/z
range of 100–1500 in a negative mode. The mass working conditions
were as follows: capillary voltage, 3 kV; source temperature, 150 ◦ C;
cone gas flow rate, 80 L/h; desolvation gas flow rate, 800 L/h; and
desolvation temperature, 350 ◦ C. Nitrogen (>99% purity) and argon
(99% purity) were used as nebulizing and collision (product ion scan,
MS/MS) gases, respectively. Each sample was determined in six times.
Prior to UHPLC/MS analysis, equal volumes of each standard solu­
tion were mixed in methanol to prepare standard stock solution (50 μg/
mL). The working solutions of standards (0.78–50 μg/mL), used for
calibration curves, were made by appropriate dilution of the stock so­
lution in methanol. Phenolic compounds were identified by comparing
their retention time, [M-H]- and MS/MS fragment ions data with those of
standards or data on reported literature.
For the qualification, chlorogenic acid (1), kaempferol-3-O-rutino­
side (2), caffeic acid (3), isoschaftoside (4), kaempferol-3-O-glucoside
(6), isorhamnetin-3-O-glucoside (8), luteolin (9), and apigenin (12)
were quantified using their own standards, respectively. Luteolin C-
[pentosyl]-glucoside (5) and luteolin O-(apiosylmalonyl) glucoside (7)
were semi-quantified by their aglycone luteolin (9). Capsianoside (10)
and monomer capsianoside (11) were semi-quantified by isorhamnetin-
3-O-glucoside (8).
2.6. α-Glucosidase inhibitory activity
The α-glucosidase inhibitory activity was assayed followed by the
previously reported procedure (Justino et al., 2018) with some modifi­
cations. All samples were dissolved in 1% (v:v) DMSO. Briefly, 10 μL
intra-system concentrations of pickled chili peppers (i.e., 0.3125
mg/mL, 0.625 mg/mL, 1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL), stan­
dards (i.e., 5 μmol/L, 20 μmol/L, 80 μmol/L, and 320 μmol/L) or posi­
tive control (i.e., acarbose) were mixed with 100 μL phosphate buffer
(pH 6.8) and 10 μL of α-glucosidase (0.5 U/mL) at 37 ◦ C for 15 min,
respectively. Then, 40 μL of 10 mmol/L pNPG (dissolved in 200 mmol/L
phosphate buffer, pH 6.8) and 60 μL of 0.1 mol/L Na2CO3 were suc­
cessively added and the mixture was incubated at 37 ◦ C for 30 min. The
absorbance (OD) of the mixture was measured at 405 nm by SpectraMax
3
LWT 162 (2022) 113467
M5 microplate reader, and α-glucosidase inhibitory rate was calculated
with Eq (1).
( )
ODsample
α − glucosidase inhibitory(%) = 1 − × 100 (1)
ODcontrol
Where ODcontrol and ODsample represent the absorbance of the control
group and sample group. The IC50 value was obtained from the above
calculation results.
2.7. DPP-IV inhibitory assay
Determination of the inhibitory of DPP-IV of different fermented
samples and standards was conducted following an inhibitor screening
kit from Cayman (Ann Arbor, MI, USA) with the instructions. The intra-
system concentrations of samples were 0.3125 mg/mL, 0.625 mg/mL,
1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL; that of standards were 5 μmol/
L, 20 μmol/L, 80 μmol/L, and 320 μmol/L. In brief, the test samples were
redissolved in assay buffer, 10 μL sample solution mixed with 30 μL of
diluted assay buffer, 10 μL of diluted DPP-IV, and 50 μL substrate so­
lution. For the negative controls, the sample solutions were replaced by
the assay buffer solution. The plates were covered with plate cover and
incubated for 30 min at 37 ◦ C by a microplate reader under excitation
and emission wavelength at 355 nm, 455 nm, respectively. The DPP-IV
inhibition was calculated as Eq (2).
( )
Asample
DPP (IV) inhibitory(%) = 1 − × 100 (2)
Acontrol
Where Acontrol and Asample are the absorbance of the control group and
sample group, respectively. The IC50 value was calculated as the con­
centration of samples when DPP-IV inhibition had reached 50%.
2.8. Molecular docking analysis
The molecular studies for the interaction between the ligand and
receptor were performed by using SYBYL-X 2.1.1(Tripos, Inc., St. Louis,
MO, USA) (Fu, Liu, Ma, Yi, & Cai, 2021; Gao et al., 2020). Structures of
all standards were obtained from PubChem database (http://PubChem.
ncbi.nlm.nih.gov) and saved as PDB file format. The crystal structure of
α-glucosidase (PDB ID:3A4A) and DPP-IV (PDB ID:1X70) was obtained
from Protein Data Bank (http://www.rcsb.org/pdb). Based on previous
research, DPP-IV has three active sites (S1, S2, S3 pockets). S1 consists of
Tyr547, Ser630, Tyr631, Val656, Trp659, Tyr662, Tyr666, Asn710,
Val711, and His740. S2 consists of Arg125, Phe357, Arg358, Tyr547,
Pro550, and Asn710. S3 is Ser209, Arg358, and Phe357. To optimize the
structures of α-glucosidase and DPP-IV, water and other substructures
were removed and AMBER7FF99 charges and hydrogen atoms were
added prior to docking.
2.9. Statistical analysis
All experiment results were performed three times and presented as
mean (n = 3) values ± standard deviation (SD). Data were evaluated by
one-way ANOVA. The significance of the difference at P < 0.05 level was
evaluated via Tukey’s procedure. All analyses were conducted with
Origin 2021 software (OriginLab, Northampton, MA, USA).
3. Results and discussion
3.1. Identification and quantification of phytochemicals in pickled chili
pepper
The changes of phytochemical compounds in pickled chili pepper
extracts with different fermented time were determined by UHPLC/MS.
Fig. 1 shows the total ion chromatograms of the non-fermented sample
and 42-day fermented sample and the corresponding Table 1 involves
M. Li et al. LWT 162 (2022) 113467

phytochemical information, including mass fragmentation data, reten­

Fig. 1. Base peak chromatograms of non-fermented (A) and fermented (42-
day) (B) chili pepper. The identified peaks and MS data are shown in Table 1.
tion time, and molecular formula. A total of 12 phytochemical sub­
stances were identified by comparing with the corresponding standards
or/and previously reported data. Among the compounds, ten of them
belonged to phenolic compounds that could be divided into phenolic
acid (compounds 1 and 3) and flavonoids (compounds 2, 4–9, and 13).
Compound 1 (peak 1, [M-H]- m/z = 353.0879) was identified as
chlorogenic acid, which was a typical hydroxycinnamic acid. Chloro­
genic acid is an ester of caffeic acid (Compound 3, peak 3, [M-H]- m/z =
179.0342) and quinic acid (Sato et al., 2011). In a previous report, the
two phenolic acids have been found in chili peppers (Capsicum frutescens
L.) (Dos Anjos et al., 2021). Among the phenolic substances, compound
9 (peak 9, [M-H]- m/z = 285.0456) only detected in the fermented
sample with a relatively high peak area was characterized as luteolin.
The compound 6 (peak 6, [M-H]- m/z = 579.1359) and compound 7
(peak 7, [M-H]- m/z = 665.1357) were identified respective luteolin
C-pentosyl-glucoside and luteolin O-(apiosylmalonyl) glucoside. Both
compounds belonged to luteolin glycosides (Guclu et al., 2021; Tsolmon
et al., 2021), which was also reported in a previous study of sweet
peppers (Jeong et al., 2011). In addition to phenolic compounds, com­
pound 10 (peak 10, [M-H]- m/z = 1083.5220) and compound 11 (peak
11, [M-H]- m/z = 1169.5220) were identified as capsianoside II and
monomer capsianoside, respectively, both of which belong to a type of
diterpene glycosides (Chilczuk et al., 2020; Macel et al., 2019). While
compound 12 (peak 12, [M-H]- m/z = 1007.5234), which had a rela­
tively high peak area in all samples, has not yet been described in the
literature. According to mass data and literature, compound 12 may be
formed by neutral loss of HCOOH, CH4, and H2O of compound 10 (Feng
et al., 2018). Thus, compound 12 was hypothesized to be a kind of
diterpene glycoside in pepper.
In Table 2, these eight compounds were quantified by the corre­
sponding standards, including chlorogenic acid, kaempferol-3-O-ruti­
noside, caffeic acid, isoschaftoside, kaempferol-3-O-glucoside,
isorhamnetin-3-O-glucoside, luteolin, apigenin were quantified by cor­
responding standards. Luteolin C-[pentosyl]-glucoside and luteolin O-
(apiosylmalonyl) glucoside were semi-quantified by their aglycone
luteolin. Capsianoside and monomer capsianoside were semi-quantified
by isorhamnetin-3-O-glucoside. The contents of kaempferol-3-O-ruti­
noside (2), luteolin C-[pentosyl]-glucoside (5), kaempferol-3-O-gluco­
side (6), luteolin (9), and apigenin (13) increased during fermentation
(P < 0.05), whereas chlorogenic acid (1), isoschaftoside (4) and luteolin
O − (apiosylmalonyl) glucoside (7) showed a decreasing trend with
prolonged fermentation time. Among them, kaempferol-3-O-rutinoside
(2) and kaempferol-3-O-glucoside (5) are derivatives of kaempferol,

Table 1
Identification of phenolic compounds in pickled chili pepper during fermentation by UHPLC/MS in negative mode.
Peak Compounds Rt [M-H]- m/z Molecular MS/MS Fragment Ions Reference
No. (min) formula

1 Chlorogenic acid 8.23 353.0879 C16H18O9 191.0553(100), 135.0441(16.13) Standard

2 Kaempferol-3-O-rutinoside 8.59 593.1517 C27H30O15 383.0775(53.69), 353.0669(100) Standard
3 Caffeic acid 8.73 179.0342 C9H8O4 134.0360(100) Standard
4 Isoschaftoside 8.94 563.1410 C26H28O14 353.0669(100) Standard
5 Luteolin C-[pentosyl]-glucoside 9.56 579.1359 C26H28O15 447.0925(1), 327.0506(0.88), 298.0804(0.25), 285.0403(100) Tsolmon et al.
(2021)
6 Kaempferol-3-O-glucoside 9.75 447.0934 C27H30O15 299.0561(100), 284.0362(80.02) Standard
7 Luteolin O-(apiosylmalonyl) 10.02 665.1357 C21H20O11 621.1464(9.86), 579.1340(3), 489.1049(5.61), 285.0400(100), Guclu et al.
glucoside (2021)
8 Isorhamnetin-3-O-glucoside 10.19 477.1038 C22H22O12 315,285.0394(7.34)163.0025(0.52) Standard
9 Luteolin 11.67 285.0406 C15H10O6 133.0285(100)107.0125(5.65) Standard
10 Capsianoside II 11.82 1083.5220 C50H83O25 1083.5223(100), 1084.5251(57.91), 921.4693(44.96), Macel et al.
775.4118(32.02) (2019)
11 Monomer Capsianoside 11.97 1169.5220 C53H86O28 1083.5219(100), 1065.5117(98.34), 1125.5328(58.69), Macel et al.
1066.5149(56.61) (2019)
12 Unknown 12.19 1007.4695 775.4119(100), 921.4696(80.75), 757.4013(70.49), 903.4590 –
(58.34)
13 Apigenin 12.5 269.0456 C15H10O5 121.0282(3.48), 118.0367(9.42), 117.0334(100), 105.0332 Standard
(1.50), 107.0126(4.39)

4
M. Li et al. LWT 162 (2022) 113467

Table 2
The quantity of identified compounds in pickled chili pepper at different fermentation moments by UHPLC/MS in negative mode.
Peak Compounds 0 day 7 days 14 days 21 days 28 days 42 days 56 days 90 days
No.

1 Chlorogenic acid 3553.02 ± 2810.10 ± 2651.23 ± 1165.12 ± 705.41 ± 752.77 ± 822.70 ± 556.57 ±
13.56a 138.47b 173.61b 38.58c 117.43d 162.37d 143.65d 85.25d
2 Kaempferol-3-O-rutinoside 149.88 ± 122.15 ± 632.53 ± 374.26 ± 649.47 ± 1095.74 ± 1048.77 ± 1047.91 ±
4.44d 8.60d 53.90b 108.17c 47.34b 46.70a 21.15a 25.21a
3 Caffeic acid 99.01 ± 1284.24 ± 1840.80 ± 1695.63 ± 690.66 ± 1047.54 ± 903.46 ± 572.96 ±
6.72e 67.15b 117.97a 116.57a 12.57d 53.57c 45.04c 20.41d
4 Isoschaftoside 614.46 ± 476.95 ± 188.44 ± 195.10 ± 172.57 ± 271.27 ± 288.37 ± 188.25 ±
19.30a 45.38b 11.39e 24.09de 8.99e 51.50cd 34.87c 30.50e
5 Luteolin C-[pentosyl]- 10.55 ± 46.42 ± 7.41c 77.74 ± 7.14b 86.66 ± 83.13 ± 137.25 ± 151.53 ± 134.68 ±
glucoside 0.87d 11.36b 9.64b 17.51a 9.23a 11.36a
6 Kaempferol-3-O-glucoside 19.29 ± 14.27 ± 0.29e 17.39 ± 17.94 ± 3.24d 19.76 ± 24.00 ± 27.76 ± 32.24 ±
0.39d 0.43de 0.87d 0.56c 0.89b 0.61a
7 Luteolin O- 267.08 ± 82.23 ± 6.89d 200.29 ± 172.20 ± 172.40 ± 170.15 ± 137.40 ± 71.62 ±
(apiosylmalonyl)glucoside 5.90a 29.07b 46.82bc 12.04bc 11.07bc 7.04c 6.77d
8 Isorhamnetin-3-O- 588.47 ± 152.20 ± 435.16 ± 427.38 ± 418.47 ± 465.15 ± 473.63 ± 354.58 ±
glucoside 25.07a 23.85d 48.68bc 50.17c 25.52bc 22.34ab 12.07ab 11.83bc
9 Luteolin 0.00 ± 0.00d 31.87 ± 1.18c 120.97 ± 105.45 ± 106.49 ± 159.17 ± 152.68 ± 157.64 ±
14.83b 22.97b 3.34b 2.51a 10.18a 10.89a
10 Capsianoside 87.25 ± 42.28 ± 3.53e 142.53 ± 132.31 ± 40.96 ± 60.34 ± 109.38 ± 113.95 ±
4.39c 0.50a 14.11a 3.84e 0.25d 1.24b 1.53b
11 Monomer capsianoside 1267.00 ± 854.95 ± 1126.26 ± 867.09 ± 507.98 ± 375.10 ± 535.01 ± 418.23 ±
21.38a 4.71c 1.46b 49.20c 11.59d 6.44e 8.21d 9.10e
12 Apigenin 15.55 ± 19.21 ± 0.37d 27.46 ± 0.74c 29.82 ± 1.63b 28.98 ± 35.26 ± 36.72 ± 35.71 ±
0.04e 0.20bc 0.36a 1.11a 0.83a
TPC 568.70 ± 425.53 ± 569.62 ± 473.15 ± 442.71 ± 546.70 ± 486.55 ± 382.83 ±
50.10a 25.35c 37.23a 49.23abc 41.97bc 20.53ab 33.61abc 21.86c
TFC 461.90 ± 460.83 ± 702.23 ± 540.90 ± 459.25 ± 649.16 ± 621.35 ± 459.41 ±
25.00d 11.24d 30.24a 18.23c 18.50d 7.40ab 30.44b 22.82d

Values are expressed as the mean ± S.D (n = 3) in μg/g of dry weight.

Minuscules represented significant differences among samples in the same line (P < 0.05).
TPC: total phenolic content, the results were expressed as mg gallic acid equivalent (GAE)/100 g of dry weight.
TFC: total flavonoid content, the results were expressed as mg rutin equivalent (RE)/100 g of dry weight.
*Chlorogenic acid, kaempferol-3-O-rutinoside, caffeic acid, isoschaftoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, luteolin, and apigenin standards
were used for quantification of compound 1, 2, 3, 4, 6, 8, 9, and 12, respectively; luteolin standard was used for semi-quantification of compound 5 and 7; iso­
rhamnetin-3-O-glucoside standard was used for semi-quantification of compound 10 and 11.

whose increased content may be due to fermentation. The finding was
consistent with a previous report that the contents of kaempferol de­
rivatives in orange juice increased obviously after fermentation (Oli­
veras-López et al., 2016).
3.2. Total phenolics content and total flavonoid content of pickled chili
pepper
Total phenolics content and total flavonoid content of pickled chili
pepper with different fermentation time are summarized in Table 2.
During the whole fermentation process (90 days), TPC decreased from
568.70 ± 50.10 mg GAE/100g to 425.52 ± 25.35 mg GAE/100g during
first 7 days’ fermentation, then increased and eventually decreased to
382.83 ± 21.86 GAE/100g, which decreased by about 32.68%. During
the first 7 days’ fermentation, there were no significant changes of TFC
in pickled chili pepper (P > 0.05). Nevertheless, TFC increased from
460.83 ± 11.24 to 702.23 ± 30.24 RE/100g at 14th day, which was the
maximum value in the whole fermentation process, and then declined at
next 14 days (21st and 28th day). Then, during fermentation period
from the 28th day to 56th day, the TFC of pickled chili pepper increased
again. However, in the end of fermentation (90 days), the TFC of pickled
chili pepper was similar with that of chili pepper without fermentation
(P > 0.05). Fermentation is a dynamic system of microorganism, the
decrease in TPC and TFC may be due to the degradation of phenolic
compounds by the polyphenol oxidase produced by the microorganisms
(Park et al., 2017). On the other hand, at certain points throughout the
fermentation process, there was an increase in TPC and/or TFC, which
probably since the cell walls of the pickled chili pepper were destroyed
by microorganism hydrolysis to make the phenolic substances easier to
be extracted (Ravisankar, Dizlek, & Awika, 2021).
3.3. Inhibition of α-glucosidase and DPP-IV
In the treatment of type 2 diabetes, on the one hand, the rate of
carbohydrate conversion to glucose can be delayed by inhibiting the
activity of α-glucosidase, which could effectively control postprandial
blood glucose (Fu et al., 2021). On the other hand, inhibiting DPP-IV can
delay the degradation of GLP-1 and GIP to promote insulin secretion
thereby enhancing the cellular absorption of blood glucose (Amini
Sarteshnizi et al., 2021).
3.3.1. α-Glucosidase inhibition
The different pickled chili pepper samples and the main phenolic
standards were evaluated for their inhibition against α-glucosidase and
the IC50 results are summarized in Table 3. According to the IC50 results,
fermentation process could significantly enhance the inhibitory effects
towards α-glucosidase, especially fermentation for 42 days, whose IC50
value reduced by about 76.82% when compared with that of non-
fermented chili pepper. It was also found in a previous study that the
inhibitory activity of soybean after 14 days’ fermentation on α-gluco­
sidase increased significantly when compared with that of unfermented
soybean (Ademiluyi, Oboh, Boligon, & Athayde, 2014).
According to the analysis result of phytochemicals in Tables 1 and 2,
phenolic compounds were the main substances contained in the 80%
ethanol extracts of pickled chili pepper. Thus, it may be reasonable to
speculate that phenolic compounds take responsibility to the inhibitory
effects of different samples on α-glucosidase. Previous study indicated
that most of flavonoids in fruits, such as kaempferol derivatives and
luteolin, have potential inhibitory effect towards α-glucosidase (Abd
Ghafar, Mediani, Maulidiani, Ramli, & Abas, 2018; Cheng, Yi, Wang,
Peng, & Zou, 2014). In this study, the inhibitory activities of main

5
M. Li et al. LWT 162 (2022) 113467

Table 3 different from that of α-glucosidase. In general, the IC50 values showed
α-Glucosidase inhibitory activities of different samples and purified that fermentation process significantly decreased the inhibitory effect of
compounds. pickled chili pepper on DPP-IV when compared with non-fermented
Fermented time(day) IC50 (mg/mL) sample (P < 0.05). However, the IC50 value of the sample fermented
0 3.58 ± 0.17a
for 42 days had no significant difference with that of non-fermented
7 1.37 ± 0.09b sample (IC50, 1.33 ± 0.12 mg/mL vs 1.28 ± 0.17 mg/mL) (P > 0.05).
14 1.11 ± 0.14d The various inhibition rates at different fermentation stages may be due
21 1.21 ± 0.11c to the changes in phytochemicals, especially the phenolics, during the
28 1.31 ± 0.15b
whole fermentation period (Gao et al., 2015). reported that the
42 0.83 ± 0.04f
56 0.98 ± 0.07e composition of phenolic compounds in sample could profoundly affect
90 0.97 ± 0.08e the enzyme activity. Many studies had confirmed that different phenolic
Phenolic standards IC50 (μmol/L) substances exhibited different inhibitory effects on DPP-IV (Johnson &
Luteolin 49.31 ± 8.86b de Mejia, 2016; Syama et al., 2017). Further experiments using eight
Kaempferol-3-O-glucoside
phenolic compounds mainly contained in different pickled chili pepper
˃200
Kaempferol-3-O-rutinoside ˃200
Apigenin 173.16 ± 17.05d samples were performed to investigate the various inhibitory effects of
Isorhamnetin-3-O-glucoside 136.78 ± 15.02c the phenolic standards on DPP-IV. The IC50 results are summarized in
Caffeic acid ˃200 Table 4. Among eight phenolic standards, isorhamnetin-3-O-glucoside
Isoschaftoside ˃200
exhibited the highest inhibitory activity (P < 0.05), followed by iso­
Chlorogenic acid ˃200
Acarbose (Positive control) 0.80 ± 0.03a schaftoside, kaempferol-3-O-rutinoside, and kaempferol-3-O-glucoside.
Luteolin and chlorogenic acid showed moderate inhibitory effects to­
wards DPP-IV. A previous study found that the IC50 value of iso­
phenolic compounds contained in the pickled chili pepper towards rhamnetin-3-O-glucoside was lower than luteolin, suggesting that
α-glucosidase were evaluated. Since the standards of the compounds 5 isorhamnetin-3-O-glucoside possessed the stronger inhibition (Gao
and 7 were not available, the rest eight phenolic substances were used in et al., 2020). In addition, in the current study, apigenin and caffeic acid
the current work. As shown in Table 3, three phenolic substances exhibited low inhibitory effects towards DPP-IV, whose IC50 values were
(luteolin, apigenin, isorhamnetin-3-O-glucoside) exhibited high inhibi­ both more than 200 μmol/L. Sitagliptin as positive control showed a
tory activities against α-glucosidase. Among these phenolic compounds, high DPP-IV inhibition with IC50 value of 0.29 ± 0.02 μmol/L.
luteolin showed the lowest IC50 value with 49.31 ± 8.86 μmol/L, indi­
cating that luteolin possessed the highest α-glucosidase inhibition. The 3.4. Molecular docking analysis of α-glucosidase and DPP-IV
inhibitory effects of apigenin and isorhamnetin-3-O-glucoside on
α-glucosidase were both weaker than that of luteolin. However, in the It is well known that molecular docking is an efficient method to
current experiment, the in vitro inhibitory activities of all other five study the binding sites of phytochemical compounds and enzymes to
phenolic compounds (kaempferol-3-O-rutinoside, kaempfer­ reveal the underlying mechanisms (Xie et al., 2021). To further inves­
ol-3-O-glucoside, caffeic acid, isoschaftoside, chlorogenic acid) against tigate the interaction of the predominant substances in pickled chili
α-glucosidase were very weak, and the IC50 values were all more than pepper with α-glucosidase or DPP-IV, molecular simulations were per­
200 μmol/L. In addition, according to the results in Table 2, the contents formed using the identified phenolic substances in pickled chili pepper.
of both luteolin and apigenin increased significantly during the tradi­ In molecular docking simulation, the C score is an important reference
tional fermentation process. Therefore, those two phenolic compounds indicator for reliable molecular docking results. and T score is the af­
may be the main substances involved in the α-glucosidase inhibition in finity between the reaction ligand and the receptor to determine the best
pickled chili pepper. Acarbose as a positive control showed a good conformation (Gao et al., 2020). In the present work, the hydrophobic
inhibitory activity (IC50, 0.88 ± 0.03 μg/mL). effect of the best docking configuration was analyzed by software
Ligplot+ software (EMBL-EBI, Cambridge, UK).
3.3.2. DPP-IV inhibition
The inhibition capacity of different samples against DPP-IV is pre­ 3.4.1. Molecular docking results of α-glucosidase
sented as IC50 values in Table 4. The inhibitory trend of DPP-IV was The number and distance of hydrogen bonds play a crucial role in
inhibiting the catalytic power of α-glucosidase (Fu et al., 2021). The
Table 4 docking detailed parameters of receptor− ligand interactions were listed
DPP-IV inhibitory activities of different samples and purified in Table S1 and the interaction between phenolics and the optimal
compounds. docking conformation of α-glucosidase are shown in Fig. 2. The in­
Fermented time(day) IC50 (mg/mL)
teractions between the optimum docking conformations of luteolin,
kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, apigenin, iso­
0 1.33 ± 0.12c
rhamnetin-3-O-glucoside, and caffeic acid with the amino acid residues
7 2.44 ± 0.10a
14 1.97 ± 0.02b of α-glucosidase are shown in Fig. 2 a1− a4, b1− b4, c1− c4, d1-d4, e1-e4,
21 2.39 ± 0.10a and f1-f4, respectively. Luteolin formed four hydrogen bonds (dashed
28 1.79 ± 0.07b lines) with THR306, ASN350, and ASP352 amino acid residues of
42 1.28 ± 0.17c α-glucosidase (Fig. 2a3). The average hydrogen bond distance was
56 1.87 ± 0.10b
90 2.47 ± 0.18a
2.174 Å. Ten hydrogen bonds were formed in kaempfer­
Phenolic standards IC50 (μmol/L) ol-3-O-glucoside-α-glucosidase complex and the residues involved were
Luteolin 50.05 ± 2.34e TRY158, ASP215, GLU277, ASP307, THR306, ARG315, ASN350,
Kaempferol-3-O-glucoside 36.87 ± 0.46d GLN353, and ASN415. The average hydrogen bond distance of these
Kaempferol-3-O-rutinoside 35.72 ± 1.29d
amino acid residues was 2.175 Å (Table S1 and Fig. 2 b1-b4). Three
Apigenin ˃200
Isorhamnetin-3-O-glucoside 23.68 ± 1.47b hydrogen bonds (dashed lines) with an average length of 2.385 Å were
Caffeic acid ˃200 formed between kaempferol-3-O-rutinoside and α-glucosidase active
Isoschaftoside 30.05 ± 1.48c sites. The amino acid residues at interacting sites included PRO312,
Chlorogenic acid 78.85 ± 6.39f ASN350, and GLU411 (Fig. 2c3). In addition, a strong hydrophobic
Sitagliptin (Positive control) 0.29 ± 0.02a
interaction between kaempferol-3-O-rutinoside and α-glucosidase was

6
M. Li et al. LWT 162 (2022) 113467

Fig. 2. Molecular interactions of phenolic compounds luteolin (a1-a4), kaempferol-3-O-glucoside (b1-b4), kaempferol-3-O-rutinoside (c1-c4), apigenin (d1-d4),
isorhamnetin-3-O-glucoside (e1-e4) and caffeic acid (f1-f4) against α-glucosidase, including the optimum docking conformations of phenolic compounds with
α-glucosidase (a1-f1 and a2-f2), the hydrogen bonds formed between phenolic compounds and active site of α-glucosidase (a3-f3), and the hydrophobic interactions
of phenolic compounds with α-glucosidase (a4-f4).

observed in the present study. As shown in Fig. 2c4 and Table S1, three amino acid residues at interacting sites, namely, THR306,
kaempferol-3-O-rutinoside interacted with nineteen amino acid residues ASN350, and ASP352 (Fig. 2d3). In Fig. 2e3, SER157, ASP307, SER311,
of α-glucosidase to form a hydrophobic interaction. Apigenin formed PRO312, LEU313, and ASP352 were involved in the interaction with
four hydrogen bonds (dashed lines, average length of 2.173 Å) with isorhamnetin-3-O-glucoside, and six hydrogen bonds with an average

7
M. Li et al. LWT 162 (2022) 113467

distance of 2.167 Å were established. Moreover, the hydrophobic
interaction between α-glucosidase and isorhamnetin-3-O-glucoside
involved ten amino acid residues. It was observed that the binding site
for caffeic acid was close to the active pocket (Fig. 2f3). Caffeic acid was
surrounded by the residues, namely, Asp69, Asp352, Arg442. The
average bond length above was 1.99 Å. Some previous studies pointed
out that some key amino acid residues in catalytic active sites, such as
Asp 69, Asp 215, ASP 352, ARG 442, and Glu 411, was responsible for
the catalytic action of α-glucosidase (Cai, Wu, Lin, Hu, & Wang, 2020;
Hua et al., 2018). In the present work, luteolin, apigenin, and iso­
rhamnetin-3-O-glucoside, all of which showed a good inhibition towards
α-glucosidase, bound with the crucial active amino acid residue ASP352,

Fig. 3. Molecular interactions of phenolic compounds luteolin (a1-a4), kaempferol-3-O-glucoside (b1-b4), kaempferol-3-O-rutinoside (c1-c4), chlorogenic acid (d1-
d4), isorhamnetin-3-O-glucoside (e1-e4), and isoschaftoside (f1-f4) with DPP-IV, including the optimum docking conformations of phenolic compounds with DPP-IV
(a1-f1 and a2-f2), the hydrogen bonds formed between phenolic compounds and active site of DPP-IV (a3-f3), and the hydrophobic interactions of phenolic com­
pounds with DPP-IV (a4-f4).

8
M. Li et al.
which may explain to a certain extent why those three phenolic com­
pounds possessed a better inhibition against α-glucosidase than the
other phenolic compounds.
3.4.2. Molecular docking results of DPP-IV
Molecular docking analysis was also performed to analyze the DPP-
IV inhibition mechanism of major phenolic compounds including
luteolin, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, chloro­
genic acid, isorhamnetin-3-O-glucoside, and isoschaftoside. Fig. 3a3
shows that one H-bond (dashed line) was formed between the active site
of DPP-IV and luteolin. The amino acid residue was Glu205 and the
hydrogen bond length is 1.947 Å. Kaempferol-3-O-glucoside formed four
H-bonds with the active sites of two amino acid residues (Ser630 and
ARG125) with an average distance of 2.170 Å (Fig. 3b3). Kaempferol-3-
O-rutinoside interacted with five amino acid residues (Arg125, His126,
Glu206, Ser630, and Tyr547) of DPP-IV to form six hydrogen bonds. The
average hydrogen bond distance was 2.253 Å (Fig. 3c3). In this study,
the interacting amino acid residues between chlorogenic acid and DPP-
IV included Ser209, Tyr547, and Tyr631, and they established four H-
bonds with an average distance of 2.136 Å (Fig. 3c3). In addition, iso­
rhamnetin-3-O-glucoside interacted with Glu206, Ser209, and Tyr547 to
form four hydrogen bonds (Fig. 3e3). The average hydrogen bond dis­
tance was 2.179 Å. In the current study, isorhamnetin-3-O-glucoside can
interact with eight amino acids in a hydrophobic manner, suggesting
that the hydrophobic interaction may contribute to the inhibition of
DPP-IV (Fig. 3e4). Moreover, Glu205, Ser209, His126, Tyr631, and
His740 were involved in the interaction with isoschaftoside, and six
hydrogen bonds with an average distance of 2.116 Å were established as
shown in Fig. 3e3. Fu et al. (2021) found that Glu206, Ser209, and
His126 play a key role in the formation of enzyme-ligand complex,
which are crucial sites in the inhibition of DPP-IV activity by the
phenolic compounds. Both isorhamnetin-3-O-glucoside and isoschafto­
side interacted with the amino acid residue Ser209 of DDP-IV, which
may explain why the two phenolic compounds possessed a better inhi­
bition against DPP-IV than the other phenolic compounds.
4. Conclusion
In this study, the effects of traditional fermentation on phytochem­
icals and inhibitions towards α-glucosidase and DPP-IV of pickled chili
pepper were investigated. A total of 12 phytochemical substances were
characterized and quantified in all samples by UHPLC-MS, including two
phenolic acids, eight flavonoids, and two diterpene glycosides. Among
the compounds, the contents of kaempferol-3-O-rutinoside (2), luteolin
C-[pentosyl]-glucoside (5), kaempferol-3-O-glucoside (6), luteolin (9),
and apigenin (13) increased, but chlorogenic acid (1), isoschaftoside (4),
and luteolin O- (apiosylmalonyl) glucoside (7) decreased during
fermentation. The inhibition towards α-glucosidase of pickled chili
pepper increased significantly after fermentation, especially fermenta­
tion for 42 days. Based on the in vitro analysis, luteolin showed the
highest inhibition against α-glucosidase and isorhamnetin 3-O-glucoside
exhibited the strongest inhibition capacity towards DPP-IV. The mo­
lecular docking analysis indicated it might be due to forming hydrogen
bonds and hydrophobic interactions with crucial amino acid residues of
α-glucosidase and DPP-IV. The outcome of the study could provide sci­
entific support for exploring the function of pickled chili pepper and
enhancing its economic value. However, the gastrointestinal digestion
process may affect the bioavailability and bioactivities of the phenolic
compounds in pickled chili pepper, which needs to be further investi­
gated in the future.
Funding
The present work was financially supported by the Young Elite Sci­
entists Sponsorship Program of China Association for Science and
Technology (YESS20200123), the Yunnan Provincial Natural Science
9
LWT 162 (2022) 113467
Foundation (202101BE070001-054), the Excellent Youth Funding of
Yunnan Province (YNQR-QNRC-2018-109), the Science and Technology
Project of Yunnan Province (202102AE090050), the Expert Workshop
of Wenshan Prefecture (KKH0202123001), and the National Charac­
teristic Vegetable Industry Technology System Post Expert Project
(CARS-24-G-21).
CRediT authorship contribution statement
Meiqi Li: Conceptualization, Investigation, Data curation, Writing –
original draft. Xi Bao: Data curation, Investigation. Xueting Zhang:
Resources. Hongbing Ren: Resources, Funding acquisition. Shengbao
Cai: Methodology, Supervision, Writing – review & editing. Xiaosong
Hu: Supervision, Funding acquisition. Junjie Yi: Conceptualization,
Supervision, Project administration, Writing – review & editing.
Declaration of competing interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2022.113467.
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