Inhibitory mechanisms and interaction of tangeretin, 5-demethyltangeretin, nobiletin, and 5-demethylnobiletin from citrus peels on pancreatic lipase: Kinetics, spectroscopies, and molecular dynamics simulation

Article abstract of DOI:10.1016/j.ijbiomac.2020.07.305

This study aimed to reveal the interaction and inhibitory mechanisms of tangeretin (TAN), nobiletin (NBT), and their acidic hydroxylated forms, 5-demethyltangeretin (5-DT) and 5-demethylnobiletin (5-DN) on porcine pancreatic lipase (PPL) using spectroscop

Full text of DOI:10.1016/j.ijbiomac.2020.07.305

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Inhibitory mechanisms and interaction of tangeretin,
5-demethyltangeretin, nobiletin, and 5-demethylnobiletin from
citrus peels on pancreatic lipase: Kinetics, spectroscopies, and
molecular dynamics simulation
Xin Huang, Junxiang Zhu, Li Wang, Huijuan Jing, Chaoyang Ma,
Xingran Kou, Hongxin Wang
PII:
S0141-8130(20)34064-2
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.07.305
BIOMAC 16353
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
25 April 2020
11 July 2020
29 July 2020
Please cite this article as: X. Huang, J. Zhu, L. Wang, et al., Inhibitory mechanisms
and interaction of tangeretin, 5-demethyltangeretin, nobiletin, and 5-demethylnobiletin
from citrus peels on pancreatic lipase: Kinetics, spectroscopies, and molecular dynamics
simulation, International Journal of Biological Macromolecules (2020), https://doi.org/
10.1016/j.ijbiomac.2020.07.305
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Inhibitory mechanisms and interaction of tangeretin,
5-demethyltangeretin, nobiletin, and 5-demethylnobiletin from citrus
peels on pancreatic lipase: Kinetics, spectroscopies, and molecular
dynamics simulation
Xin Huanga, b, Junxiang Zhuc, Li Wanga, b, Huijuan Jinga, b, Chaoyang Maa, b, Xingran
Koud*, Hongxin Wanga, b*
a
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu,
214122, People's Republic of China.
b
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu, 214122, People's Republic of China.
c
College of Food Science and Engineering, Qingdao Agricultural University,
Qingdao 266109, People's Republic of China.
d
School of Perfume and Aroma Technology, Shanghai Institute of Technology,
Shanghai 201418, People's Republic of China.
*Co-corresponding author: Tel: (086)510-85917795; Fax: (086)510-85329276;
E-mail: hxwang@jiangnan.edu.cn (H. Wang); kxran@163.com (X. Kou)

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Abstract
This study aimed to reveal the interaction and inhibitory mechanisms of tangeretin
(TAN), nobiletin (NBT), and their acidic hydroxylated forms, 5-demethyltangeretin
(5-DT) and 5-demethylnobiletin (5-DN) on porcine pancreatic lipase (PPL) using
spectroscopic techniques and molecular dynamics (MD) simulation. PPL inhibition
assay showed that the inhibitory activity of NBT (IC50 value of 3.60±0.19 μM) was
superior to those of three polymethoxylated flavones (PMFs), indicating it may be
related to the methoxy groups at the 3′-position in its molecular structure. Inhibition
kinetic analyses demonstrated that the inhibition types of the 4 PMFs were consistent
with the mixed inhibition model, which agreed well with the results from the
ultraviolet-visible (UV-Vis) spectroscopy, Circular dichroism (CD), fluorescence
spectroscopy, molecular docking, and MD simulation that PMFs could bind to the
PPL catalytic site and non-catalytic site, affecting the normal spatial conformation of
PPL and weakening its ability to decompose the substrate. All these findings suggest
that PMFs are a kind of natural lipase inhibitors, and NBT has the potential as a lipase
inhibition precursor because of its unique flavone skeleton structure.
Keywords
Lipase inhibition; Polymethoxylated flavones; Inhibition mechanism; Molecular
dynamics.

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1. Introduction
Obesity is a chronic metabolic disease resulting from excessive calorie intake and fat
metabolism disorders [1]. Obesity can induce a series of metabolic diseases, such as
hypertension, type 2 diabetes, fatty liver disease, obstructive sleep apnea,
cardiovascular diseases, and even certain cancers [2-4]. Currently, obesity has
replaced undernutrition as the gravest threat to public health and survival in the 21st
century [5]. The Lancet Commission reported that obesity affects more than two
billion people worldwide, and by 2025, nearly 124 million children and adolescents
are predicted to be obese [5]. Therefore, it is crucial to search for solutions for obesity
on a global scale. An important strategy for the treatment of obesity includes the
development of inhibitors of nutrient digestion and absorption [6]. Lipase (EC 3.1.1.3)
is a key enzyme responsible for breaking down dietary triacylglycerol into absorbable
monoglycerides and fatty acids [7]. Increasing evidence has demonstrated that
inhibition of lipase to reduce lipid adsorption is a potential approach to alleviate
obesity [8]. At present, the only clinically approved pancreatic lipase inhibitor is
Orlistat [9]. However, gastrointestinal side effects, such as flatulence and diarrhea,
may reduce patient compliance and limit its use [10]. Therefore, it is necessary to
search for effective and safe lipase inhibitors as alternatives for clinical use.
A number of studies have focused on natural plant materials, including polyphenols,
flavonoids, carotenoids, and polysaccharides, which exhibit weight loss and
lipid-lowering bioactivities with low toxicity and side effects in vitro and in vivo
[11-14]. Polymethoxylated flavones (PMFs) are a unique series of substances

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belonging to the flavonoids family that are present in citrus fruit peels [15]. TAN and
NBT are the most abundant compounds in citrus peels, and they have been found to
possess physiological properties of anti-inflammation [16], anti-oxidation [17], and
improving diabetes and lipid metabolism [18, 19]. Many studies have shown that
PMFs can reduce fat and significantly prevent obesity by lowering cholesterol [20],
inhibiting stearoyl-CoA desaturase mRNA expression [21], regulating lipid
metabolism [22], and lowering blood glucose [23] in vivo. However, in recent years,
some studies have suggested that direct interaction of PMFs with lipase may be
another possible mechanism of their lipid-lowering activity. Zeng et al. investigated
the anti-lipase activity of the Citri reticulatae pericarpium in different harvest periods
and inferred that NBT might most likely act as the lipase inhibitor [24]. Our previous
study also demonstrated that TAN and 5-demethyltangeretin (5-DT) had good
antioxidant and lipase inhibitory effects in vitro [25], but their interaction and
inhibition mechanisms remain unclear. A recent study has reported the existence of 78
PMFs in citrus, and their bioactivity may be greatly influenced by the number and
position of methoxy groups [26]. Kawaguchi, Mizuno, Aida and Uchino [27] found
that the hydroxyl group and the methoxy group at the 3′ and 4′ position of the
flavonoid skeleton B ring of hesperidin could play an important role in the inhibitory
activity (IC50=32 μg/mL) of pancreatic lipase. However, the relationship between
structure of PMFs and their lipase inhibitory activity remains largely unknown.
To understand the relationship between the structural characteristics of PMFs and
their inhibition of lipase activity, we have isolated and purified TAN and NBT from

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citrus peels and obtained their corresponding hydroxylated products at 5-position,
including 5-DT and 5-demethylnobiletin (5-DN), by acid hydrolysis. Interestingly,
according to the order of compounds 5-DT (2), TAN (1) and 5-DN (4), and NBT (3)
(Fig. 1), the structure of the latter compound has one more methoxy group at 5 or 3′
positions than the former compound. In this study, the inhibition rates and
mechanisms of these PMFs on lipase were examined by lipase inhibition kinetic
experiments. Meanwhile, multispectral analysis and molecular dynamics (MD)
simulation techniques such as UV-visible analysis, CD spectrum, and fluorescence
spectroscopy were also conducted to confirm the interaction and mechanisms between
PMFs and lipase. Therefore, this study aims to analyze the key functional groups of
PMFs inhibiting lipase and reveal the molecular mechanism of their lipase inhibitory
activity.
2. Materials and methods
2.1 Chemicals and Materials
Porcine pancreatic lipase (PPL, type II), 4-methylumbelliferyl (4-MU) oleate,
and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO,
USA). Ethyl acetate, n-hexane, ethanol, phosphoric acid, citric acid, disodium
hydrogen phosphate, sodium dihydrogen phosphate, and 200–300 mesh (50–75 μm)
column separation of silica gel were all purchased from Sinopharm Chemical Reagent
Co., Ltd (Shanghai, China). Double-distilled water was used. Chromatographic grade
methanol, acetonitrile, and formic acid were purchased from Merck (Darmstadt,
Germany). The tangeretin and nobiletin, used as standards (purity of 95%), were

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purchased from Shanghai Aladdin Biotechnology Co., Ltd (Shanghai, China).
2.2 Conversion of TAN and NBT to 5-DT and 5-DN
5-DT and 5-DN were obtained by acid hydrolysis of TAN and NBT. The specific
method for converting TAN and NBT to 5-DT and 5-DN with acid hydrolysis has
been reported in our previous study [25]. The transformed products were further
purified by a silica gel column and eluted with n-hexane/ethyl acetate (1:1, v/v). The
eluent was monitored by HPLC and evaporated to dryness under vacuum. The
isolated products were identified using ultra-high performance liquid chromatography
coupled with mass spectrometry (UPLC-TOF-MS). The detailed method of
UPLC-TOF-MS was listed in Supplementary Materials.
2.3 Real time-inhibition of PPL
The inhibitory activity of PMFs against PPL, (Sigma type II) was monitored in
real-time according to the procedure described previously[28]. In the assay,
4-methylumbelliferone (4-MU) oleate was used as the substrate, which is hydrolyzed
by PPL to 4-MU, a fluorescent product that can be monitored at the excitation and
emission wavelength of 340 nm and 460 nm, respectively. A 50 μL PPL solution (0.01
mg/mL, final concentration) and 50 μL different concentrations inhibitors (1, 5, 10, 15,
25, 50, 100, and 200 μM) dissolved in 0.1 M citrate phosphate buffer (0.1 M
citrate-Na2HPO4, pH 8.0) were mixed and incubated at 37 ℃ for 10 min in a 96-well
plate. Then, 100 μL 4-MU oleate (10 μM, final concentration) was added to initiate
the reaction, with the final concentration of DMSO at 1% (v/v, without loss of the

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catalytic activity). The fluorescence signals of each well were measured every 60 s
using a microplate reader (SynergyTM2, BioTek Instruments Inc., Winooski, VT,
USA) within 35 min of incubation at 37 ℃. Citrate phosphate buffer with sample (no
PPL added) was used as the sample control group, citrate phosphate buffer with PPL
(no sample added) as the blank group, and citrate phosphate buffer only as the blank
control group. The PPL inhibitory rate (1) was calculated as follows:
Inhibition rate (%) = [1-(A1-A2)/(A3-A4)] × 100%
(1)
where A1, A2, A3, and A4 were defined as fluorescence signals of the sample
group, the sample control group, the blank group, and the blank control group,
respectively.
2.4 Inhibitory kinetic analysis
The inhibitory kinetics of TAN (1), 5-DT (2), NBT (3), and 5-DN (4) against
PPL were studied by the Lineweaver-Burk equation. Initial velocity (v) for various
substrate concentrations against different inhibitor concentrations was determined
[28]. The inhibition type of the PMFs was analyzed based on the Lineweaver-Burk
plot, and the corresponding inhibition constant (Ki) was calculated by GraphPad
Prism 7.5 software (San Diego, Canada). The following equations for competitive
inhibition Eq. (2), noncompetitive inhibition Eq. (3), or mixed inhibition Eq. (4) were
used to calculate the Ki values.
V  (Vmax S)/
V  (Vmax S)/

Km (I / Ki ) S

(2)
(3)
(Km  S)(1 I / Ki )


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V = (Vmax S) / [(1+I / Ki) Km+(1+I / αKi) S]
(4)
where V and Vmax are the enzymatic reaction velocity and maximum velocity,
respectively; S and I represent the substrate and inhibitor concentrations, respectively;
and Ki and Km is the inhibition constant and the Michaelis constant (substrate
concentration at 0.5 Vmax), respectively. For all inhibition kinetic analysis,
goodness-of-fit parameters were employed to identify the most appropriate inhibition
modes.
2.5 UV-visible absorption spectra
The UV-visible absorption spectra were recorded by an ultraviolet
spectrophotometer (Mapada Instrument Co., Ltd., China) according to the method of
Ren with modifications [29]. Briefly, 250 μL of citrate-Na2HPO4 buffer (pH 8.0) or
PPL (0.1 mg/mL, final concentration) was mixed with 250 μL of different
concentrations of the four inhibitors (final concentrations of 50 and 100 μM,
respectively) and then allowed to stand for 30 min at 25 ℃ before measurement.
Using citrate-Na2HPO4 buffer as a reference, the absorption spectra of the PPL
solution, inhibitor + lipase complex solution, and inhibitor solution were scanned in a
range of 200–400 nm.
2.6 Circular dichroism (CD) measurement
CD spectroscopy was used to determine whether the four PMFs affect the
secondary conformation of PPL, and the experiment was performed as described by

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Du et al [30]. Mixture solutions of 50 μM of the four inhibitors and 0.1 mg/mL PPL
were injected into a 0.1 cm path length quartz cuvette and kept at 25 ℃ for 30 min.
Then, each mixture was measured using a Chirascan V100 spectrophotometer
(Applied Photophysics, Surrey, UK) at 100 nm/min with a slit width of 1 nm and a
time constant of 1 s. Each CD spectra was the accumulation of three times scans. The
data were recorded ranging from 200 nm to 260 nm at 1 nm interval and analyzed by
CDpro software after deducting the buffer spectrum.
2.7 Fluorescence spectroscopic measurements
The fluorescence spectra were conducted as described by Liu's method with
appropriate modifications [31]. In the presence of different concentrations of inhibitor
(0, 5, 10, 25, 50, 100, 150 and 200 μM, final concentration), 0.1 mg/mL PPL solution
was mixed at room temperature. After 30 min, the mixture was injected into a quartz
cuvette and the fluorescence intensity was recorded from 290 nm to 450 nm at an
excitation wavelength of 280 nm using a fluorescence spectrophotometer (Hitachi,
Japan). The excitation and emission slits were both 5 nm. According to the Stern–
Volmer equation, the fluorescence quenching results were plotted as a plot of
fluorescence intensity versus inhibitor concentration [32]:
F F 1 Kq 0

I

1 KSV
I
 
(5)
0
where F0 and F were the fluorescence intensities before and after the addition of
the quenchers (PMFs), respectively. Kq, KSV, τ0, and [I] were the bimolecular
quenching constant, the Stern–Volmer dynamic quenching constant, the fluorescence

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lifetime in the absence of the quencher (herein τ0 is 1.59 ns [33]), and the
concentration of the quenchers, respectively. Ksv could be calculated from the slope of
F0/F against [I], which indicated a dynamic balance of molecular diffusion and
collisions and measured the quenching efficiency of effectors on the enzyme.
The mechanism of fluorescence quenching can be divided into two categories:
static quenching owing to ground-state complex formation and dynamic quenching
owing to collisions between molecules [33]. For the static type, the binding constant
(KA) and the binding site (n) are calculated from the intercept and the slope of
lg[(F0−F)/F] against lg[I] with the linear regression Eq. (6) [32]:
lg
2.8 Molecular docking and MD simulation
TAN (ID: 68077), 5-DT (ID: 96539), NBT (ID: 72344) and 5-DN (ID: 358832)
were downloaded from the PubChem database


F  F

/ F

 lg KA  n lg

I

(6)
0
data
(https://pubchem.ncbi.nlm.nih.gov/). The crystal structure of pancreatic lipase (PDB:
1LPB) with a resolution of 2.46 Å was obtained from the Research Collaboratory for
Structural Bioinformatics (RCSB) Protein Data Bank (http://www.rcsb.org). The
PDBQT format files of all above models were generated by the AutoDockTools
1.5.6rc3 software [34]. Then, the AutoDock 4.2 program [34] was used to determine
the possible binding sites through a Lamarckian genetic algorithm (LGA). A docking
box centered on the Arg265 residue was used in a size of 50 × 30 × 30 Å3, which
covered the catalytic center of pancreatic lipase (His263, Ser152, and Phe77). The
number of alternative conformations was set to 100. All produced conformations with

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respective docking energies (kcal mol−1) were clustered with a tolerance of
root-mean-square deviation (RMSD) of 12.0 Å.
Base on the docking results, MD was used to obtain the stable conformation of the
pancreatic lipase-NBT complex by Gromacs 2018.3 program [35]. The Gromos96
54a7 force field was applied to a cubical periodic box with a 1.0 nm solute-wall
distance, which contained pancreatic lipase complexed with nobiletin filled with
SPC/E water and counterions. The energy of the box was optimized using the steepest
descent method, with a maximum step of 5000, up to a maximum force Fmax of no
more than 1000.0 kJ mol−1 nm−1. Then, the box was equilibrated under the canonical
and isothermal-isobaric ensembles. After each equilibration for 100 ps, production
runs of 50 ns were performed. The MD results were visualized using the Chimera
1.12 packages [36].
2.9 Statistical analysis
All experiments were implemented in triplicate, and the data are shown as mean ± SD.
The IC50 values (the concentration of inhibitor that reduces enzyme activity by 50%)
were evaluated by nonlinear regression using SPSS statistics software (version 17.0,
IBM SPSS Software, Inc., USA).
3. Result and discussion
3.1 Structural identification of the PMFs in citrus peel extract by UPLC-TOF-MS
In this study, TAN (1) and NBT (3) were obtained by extraction with ethyl
acetate. 5-DT (2) and 5-DN (4) were obtained by the method already described and

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illustrated in Fig. 1. The PMFs were identified by comparing the retention time of
UPLC diagram (Figs. 2a-d) and quasi-molecular ions of TOF-MS spectra (Figs. 2a’-d’)
based on positive ionization mode. The results in Fig. 2a’ exhibited molecular ion
[M+H]+ at m/z 373, [M+Na]+ at m/z 395, and an isotopic ion [M+H+1]+ at m/z 375; a
fragment ion [M+H-14]+ yielding at m/z 358 was attributed to the loss of methylene,
which were consistent with the mass spectrometry results reported for TAN [37]. The
results indicated in Fig. S1b’ displayed molecular ion [M+H]+ at m/z 359 and
[M+Na]+ at m/z 381, consistent with the molecular weight of 5-DT (358 Da), and
[M+H+1]+ ion at m/z 360, [M+H+2]+ ion at m/z 361, and [M+H+3]+ ion at m/z 362
were corresponding to the isotope atom present in 5-DT [38]. The spectrum shown in
Fig. 2c’ could be tentatively identified as NBT based on [M+H]+ ion at m/z 403,
[M+Na]+ ion at m/z 425, and isotopic ion clusters of [M+H+1]+ at m/z 404 and
[M+H+2]+ at m/z 405 [39]. The spectrum shown in Fig. S1d’ was possibly 5-DN with
[M+H]+ ion at m/z 389 and [M+Na]+ ion at m/z 411. [M+H+1]+ ion at m/z 390 and
[M+H+2]+ ion at m/z 391 were considered to be isotopic molecular ions [38].
Therefore, in combination with chromatograms and mass spectra, the four PMFs
could be identified in turn as TAN (RT 5.30 min, 372 Da), 5-DT (RT 5.99 min, 358
Da), NBT (RT 4.89 min, 402 Da), and 5-DN (RT 5.57 min, 388 Da), which were
further investigated for the PPL inhibition experiments.
3.2 Real-time inhibition of lipase by the four kinds of PMFs
Citrus fruits are widely distributed and consumed around the world and are an

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essential part of the Mediterranean diet [40]. Our research determined the inhibitory
potentials of TAN, 5-DT, NBT, and 5-DN against PPL by the fluorometric method
using 4-MU oleate as the substrate to evaluate the inhibitory activity of the PMFs on
lipase in vitro. The 3-dimensional (3D) surface heat map (Figs. 3a-3d) was adopted to
provide an intuitive and comprehensive presentation of the real-time inhibition trend
of the four PMFs on PPL at different concentrations within 35 min. In the range of
concentrations (0, 5, 10, 15, 25, 50, 100 and, 200 μM, final concentration)
investigated, the inhibition surface heat map gradually changed from blue (low
inhibitory activity) to red (high inhibitory activity), indicating that the catalytic
activities of PPL were inhibited by the PMFs in a dose-dependent manner. The same
results were shown in the residual activity curves (Fig. 3e), the residual activity of
PPL decreased with the increasing concentration of the four inhibitors. When the
inhibitor concentration increased to 50 μM, the effects of the four PMFs on PPL
activity gradually reached the plateau stage. As shown in Table 1, the inhibitory
capacity of NBT on PPL was 4.08 times higher than that of TAN, and the inhibitory
capacity of 5-DN on PPL was 3.15 times higher than that of 5-DT. The result may be
associated with the existence of one more hydrophobic methoxy group at the
3′-position of the B ring of NBT and 5-DN compared with TAN and 5-DT, which
improved their ability to bind to the hydrophobic region of the lipase surface [41] and
exerted better PPL inhibition effects, whereas, there was no significant difference in
PPL inhibitory activities between 5-DT or 5-DN and their precursor compounds
(p>0.05). This result may suggest that the group at the 5-position of the PMFs,

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whether it was a hydroxyl group or a methoxy group, had no significant effect on the
inhibition of lipase activity (p>0.05). As can be seen from Table 1 and Fig. 3e, NBT
had the most potent inhibitory capacity (IC50 value of minimum) against PPL among
the four PMFs, which may confirm the inference made in our previous study [24].
Although many naturally derived flavonoids have been investigated for their potential
anti-lipase activities [27, 42, 43], it was evident from our study that TAN, 5-DT, NBT,
and 5-DN displayed remarkably stronger inhibitory effects on PPL than that of the
other flavonoids (hesperidin, Nelumbo nucifera leaf flavonoids, apigenin and its
glycosides, etc.), which made them promising candidates for their application as
natural lipase inhibitors.
3.3 Inhibitory kinetics of the four PMFs on PPL
There are four types of enzyme inhibition mechanisms: competitive inhibition,
non-competitive inhibition, uncompetitive inhibition, and linear mixed inhibition [30].
To investigate the inhibitory mechanisms of the four PMFs on PPL, kinetic studies
were implemented with the initial reaction rates over different concentrations of
4-MU oleate in the absence or presence of TAN, 5-DT, NBT, and 5-DN (0, IC50, and
2IC50, final concentration). As shown in Figs. 4a-d, with an increase in substrate
concentration, the enzymatic reaction rate was firstly positively correlated with the
substrate concentration, and then gradually tended to the maximum reaction rate. The
Lineweaver-Burk double reciprocal plots in terms of 1/v versus 1/[S] were shown in
Figs. 4a’-4d’. These double reciprocal plots produced a set of straight lines that

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converged to a point in the third quadrant, indicating that the four PMFs exhibited
reversible mixed-type inhibition on PPL [44]. Namely, the four PMFs were able to
form enzyme-inhibitor and enzyme-substrate-inhibitor complexes regardless of
whether a substrate molecule could bind with the enzyme active site or not [45],
which was different from orlistat forming a covalent bond with a serine residue at the
lipase active site [46]. Previous research has reported lipase inhibitors of natural
polyphenols and flavonoids, such as cocoa procyanidins [47], blackberry polyphenols
[48], polyphenols of cortex mori radices [44], and ginkgo biloba flavonoids [28] also
had mixed inhibition mechanisms. This indicated that these natural lipase inhibitors
could bind to the active site and the inactive center of the enzyme to exhibit a dual
inhibitory ability. The Ki value, a dissociation constant of inhibitors binding with
enzyme, is an important parameter to evaluate the affinity between lipase and the
inhibitor [28]. In this research, the Ki values of TAN, 5-DT, NBT, and 5-DN were
calculated, which were listed in Table 2. Compared with the Ki values of TAN and
5-DT, the Ki values of NBT and 5-DN presented a significant difference (p<0.05).
According to the mixed inhibition Eq. (4), when the Ki value decreased, the initial rate
of the reaction decreased, and the binding of the enzyme to the inhibitor weakened the
binding of the enzyme to the substrate. Therefore, the smaller the Ki value, the
stronger the binding between the enzyme and the inhibitor, and the stronger the
inhibitory activity [49]. Therefore, our results further verified the real-time inhibition
rate of lipase, and NBT and 5-DN were potent mixed-type inhibitors against PPL,
with Ki values <4 μM.

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3.4 UV-visible absorption spectra
UV-visible spectroscopy was applied in this study to confirm the structural
changes in PPL when combined with the PMFs. UV absorption alterations of the
chromophore in constituent amino acids can reflect changes in the structure and
stability of the protein [50, 51]. Figs. 6a-6d showed the absorption spectra curves of
lipases in the presence of TAN, 5-DT, NBT, and 5-DN at different concentrations
(PMFs + Lipase) while curves shown in Figs. 6a’-6d’ were obtained by deducting the
PMFs UV absorbance from PMFs + Lipase spectra curves. Two absorption peaks of
PPL at 219 nm and 267 nm got stronger with the increasing inhibitor concentration.
The strong absorption peak at 219 nm reflected the frame structure of the lipase [29];
the weaker absorption peak at 267 nm was the UV absorption of the aromatic amino
acids of the lipase (mainly of Trp and Tyr). The change in the absorption peak
indicated conformation alterations in PPL. In the absence of interaction between the
four inhibitors and PPL, the PMF + Lipase curves should coincide with the PPL
curves. It can be seen from Figs. 6a’-6d’ that the absorption peak at 267 nm had a
significant change for varying degree of redshift occurred after the addition of
inhibitors. The result suggested that the PMFs and PPL formed a new complex and
this complex altered the structural conformation of PPL, thereby affecting the
microenvironment around its Trp and Tyr residues and increasing or decreasing the
UV absorbance.
3.5 Circular dichroism (CD) of PPL
CD is a sensitive optical technique that has been widely used to study the optical

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conformational properties of various biological and synthetic macromolecules [52].
CD spectroscopy is commonly employed for the analysis of protein secondary
structures [53]. The CD spectra of PPL in the presence of different inhibitors were
shown in Fig. 5. Two negative peaks can be seen near 208ꢀnm and 228ꢀnm in the
spectra of PPL without inhibitors, which could be attributed to the α-helix of PPL [54].
All the spectra showed great differences with the addition of the inhibitors although
the position of the negative peaks did not change significantly. The result indicated
that PPL still retained an α-helical structure, but its normal secondary structure was
affected.
The CDpro software was used to measure the secondary structure of PPL in the
presence and absence of inhibitors. As shown in Table 2, the main secondary
conformation of PPL without inhibitors was α-helix (10%), β-sheet (32%), β-turn
(23%), and unordered (36.6%). However, with the addition of TAN, 5-DT, NBT, or
5-DN, both the percentages of α-helix and β-turn of PPL decreased while the
percentage of β-sheet increased significantly. This indicated that the four PMFs had a
certain degree of binding with the active site of PPL comprising α-helix [55], resulting
in a decrease in structural stability and inhibition of substrate decomposition activity
[56]. It has been reported that the 449 amino acid residues of PPL were arranged in
the N-terminal domain with typical α/β structure and C-terminal domain with a
β-sandwich structure [55]. The decrease in α-helix and the increase in β-sheet shown
in Table 2 may represent the inhibition degree of PPL by the four PMFs. NBT showed
the greatest modification in the content of α-helix (5.1%), β-sheet (64.1%), and β-turn

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(16.1%) in PPL among the four inhibitors, which may suggest that NBT had a better
inhibitory effect on PPL than that of TAN, 5-DT, and 5-DN. This result was consistent
with the inhibition rate and kinetics of NBT on PPL.
3.6 Fluorescence spectroscopy
Fluorescence spectroscopy is a promising method for studying the interaction
between small molecules and proteins and has been widely used in structural studies
of functional proteins to reflect changes in the microenvironment of protein
fluorophores [57]. Seven Trp residues (five at N-terminal domain, two at C-terminal
domain, among them, three Trp residues near the active site of the enzyme), 25 Phe
residues, and 16 Tyr residues may be responsible for the PPL fluorescence and
provide conformational information about the binding between small molecules and
PPL [58]. The fluorescence spectra of PPL in the presence of different concentrations
of the four PMFs (0, 5, 10, 25, 50, 100, 150, 200 μM, final concentration) were shown
in Figs. 7a-d. With the increase of PMF concentration, the fluorescence intensity of
PPL decreased, accompanied by a redshift of the maximum emission peak (357 nm).
Furthermore, the maximum absorption peak of NBT shown in Fig. 7c was the most
obvious. This result indicated that the interaction between NBT and the fluorophore
(such as tryptophan) at the active site of PPL or other sites was stronger than that of
the three other PMFs, thus changing the original conformation of PPL and the
microenvironment around the fluorophore and leading to fluorescence quenching
[33].

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The mechanism of PPL fluorescence quenching induced by these PMFs can be
analyzed by the Stern-Volmer plot equation. Either in dynamic or static quenching,
F0/F is a linear relationship to [I], while mixed quenching is an upward curve towards
the y-axis [59]. A good linear relationship (R2>0.98) of TAN, 5-DT, and 5-DN was
observed in Fig. 7e and Table 2, while an upward curvature towards the Y-axis of
NBT was observed when the concentration was higher than 100 μM. These results
suggested the existence of a mixed quenching process at high concentrations. In
addition, the fluorescence quenching constant (Ksv) and binding affinity (Kq) of NBT
were significantly higher than those of the other three compounds (p<0.01), indicating
that NBT had better PPL binding ability than that of the other three PMFs. It has been
reported that the maximum scattering collision quenching constant of different
quenchers and biopolymers for dynamic quenching was 2.0×1010 L·M-1·s-1 [60].
Therefore, the combination of these four inhibitors with PPL was not a dynamic
quenching process. Finally, the binding constants (KA) and binding sites (n) of the four
PMFs to PPL could be accurately calculated by the intercept and slope of the double
logarithmic Eq. (6) (Fig. 7f). From the results listed in Table 2, it can be seen that the
order of KA values of the four inhibitors on PPL was NBT>5-DN>TAN>5-DT>103 L
mol−1, which indicated their efficiency in binding with PPL. Noteworthy, the affinity
of NBT to PPL was much higher than that of the other three inhibitors. The data for
binding sites (n) indicated that the four inhibitors had one common binding site with
PPL. The result suggested that PMFs may form a ground-state complex with a
fluorescent group, such as tryptophan at the PPL active site, thereby diminishing the

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ability of PPL to decompose the substrate. Our results also illustrated that the binding
of the four PMFs to PPL caused a change in the structure and the catalytic activity of
the protein. These also further validated the results of inhibition kinetics, CD, and
UV-visible spectra.
3.7 Molecular docking
The molecular docking simulations were performed to investigate the inhibitory
behaviors of the four PMFs against PPL-mediated 4-MU oleate hydrolysis from the
view of ligand-enzyme interactions [61]. Fig. 8 showed the spatial conformation of
the inhibitor-enzyme complex after the docking of TAN, 5-DT, NBT, and 5-DN with
PPL. As it can be seen from Fig. 8, there were two sites on PPL for binding of the
PMFs: one near the active site (binding site 1), strongly interacting with the His263,
Ser152, Phe77, and Arg256 residues (Figs. 8a-d) and the other (binding site 2) far
away from the active center (Figs. 8a’-d’). This result was consistent with the
inhibitory kinetic analysis of the mixed inhibition type. As shown in Fig. 8, the four
PMFs interacted with Tyr114 and Phe77 at docking site 1 (a-b) and Tyr369 at docking
site 2 (a'-b'), which directly quenched the endogenous fluorescence of PPL. This
further confirmed the results of the PPL fluorescence quenching experiment.
Molecular docking energy corresponds to the affinity between small molecules and
proteins; the lower the energy, the more stable is the ligand-protein complex structure
[62, 63]. In terms of the docking energy (Table 4), the four compounds were
preferentially bound to the active center (site 1), with docking energies of -6.22, -6.26,

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-6.60, and -6.21 kcal mol-1, respectively. Among them, the affinity of NBT to PPL was
higher than that of the other three compounds in binding sites 1 and 2, which was
consistent with the IC50 value and the binding affinity constant (Kq) of fluorescence
quenching. Moreover, in addition to a few hydrogen bonds, there were a lot of
hydrophobic interactions between the four PMFs and the residues of the two active
sites in the lipase (Fig. 8). These results further confirmed that the inhibitory effect of
the PMFs on the lipase was related to its structural methoxy group [64]. In addition,
the molecular docking analysis again proved that the four PMFs mainly inhibited the
substrate decomposition activity of lipase by hydrophobic interactions with lipase
activation site (site 1) and inactivation site (site 2). Thus, the interaction between NBT
and PPL was stronger than that of the other three PMFs with PPL.
3.8 MD simulation
In order to further understand the mixed inhibitory effect of the PMFs on PPL,
the MD technique was used to explore the detailed structural information of the
NBT-PPL complex, using NBT as a representative compound. In the MD simulation,
the root mean square deviation (RMSD) of the protein structure as a function of time
is an important measurement to monitor the equilibrium process of the system and the
stability of protein structure upon the binding of a ligand, and the RMSD is used to
assess structural deviation from the initial protein structure [60, 65-67]. As shown in
Fig 9a, the fluctuation of RMSD was less than 0.2 nm, indicating that PPL had
reached a stable state in both NBT and water systems within 50 ns [68]. The radius of

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rotation (Rg) is an important parameter to investigate the compactness of the protein
structure in the process of simulation. The larger the Rg value of the system, the
larger the radius of the rotation range of the protein structure in a certain space, and
the looser the protein structure [60]. The Rg value obtained (Fig. 9b) showed that the
binding of NBT increased the Rg of PPL, indicating that NBT could unfold the
enzyme to loose the whole conformation. In other words, the greater the binding
energy of PMFs to lipase, the more unstable its structure, thereby leading to the loss
of catalytic activity of the functional site of lipase [69]. This also confirmed the
results of UV-Visible and CD spectra. Fig. 9e showed the solvation stable
conformation of PPL and NBT-PPL complexes obtained by MD analysis. From the
conformational diagram in Fig. 9e, it can be seen that NBT had two binding sites on
PPL, which was consistent with the results of the kinetic studies and molecular
docking. In addition, the results obtained from Figs. 9c and 9d revealed that the
hydrophobic forces dominated the interaction between NBT and residues at the
catalytic center (site 1) and non-catalytic center (site 2). This result further confirmed
our previous inference that the inhibitory effect of PMFs on lipase could be related to
the location and number of methoxy groups (hydrophobic groups) in its structure [64].
4. Conclusion
Obesity has become a global public health problem. The development of food-borne
small molecule compounds as effective inhibitors of pancreatic lipases can not only
replace chemical drugs, avoiding adverse reactions, but also provide theoretical

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support for reasonable adjustment of dietary habits. In this study, UV-visible
spectroscopy, CD spectroscopy, fluorescence spectroscopy, molecular docking, and
MD simulation, among other methods, were employed to investigate the inhibitory
mechanism and structure-activity relationship of TAN, 5-DT, NBT, and 5-DN on PPL.
The results showed that these PMFs may be able to change the spatial structure of
PPL through hydrophobic bond to form a complex, which affected the original
conformation and active site of PPL. Our results suggest that the inhibition of lipase
by the PMFs was mainly due to the methoxy group (hydrophobic group) at the
3′-position of the B ring. Furthermore, compared with the three other PMFs, NBT had
the best inhibitory effect on PPL and is expected to be a novel PPL inhibiting drug
precursor. Overall, this study provides basic data supporting the development of
anti-obesity functional foods.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgments
This work was financially supported by national food science and engineering
first-class discipline construction project (NO.JUFSTR20180204), The national key
research and development plan in the 13th Five-Year (NO.2017YFD0400803).
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Author statement
Xin Huang: Investigation, Data Curation, Writing-Original draft preparation;
Junxiang Zhu: Software, Methodology; Li Wang: Formal analysis; Huijuan Jing:
Visualization, Writing-Review & Editing; Chaoyang Ma: Methodology; Xingran
Kou: Writing-Review & Editing; Hongxin Wang: Resources, Conceptualization,
Funding acquisition.

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Table 1 The IC50 values, Ki values and the inhibition types of four PMFs against
PPL-mediated 4-MU oleate hydrolysis.
The type of
inhibition
Goodness
of fit (R2)
Compounds
MW
IC50 (μM)
Ki (μM)
TAN
5-DT
372.37
358.37
14.69±0.82
13.18±0.44
8.53±1.77
Mix
Mix
0.995
0.996
10.65±3.59
NBT
402.39
388.39
3.60±0.19
4.18±0.26
9.51±1.63
Mix
Mix
0.991
0.988
5-DN
18.65±5.12
PMFs, polymethoxylated flavones; PPL, porcine pancreatic lipase; 4-MU,
4-methylumbelliferyl; MW, molecular weight.

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Table 2 Secondary structural contents of PPL (0.1 mg/mL) in four kind of PMFs (50
μM).
α-helix
(%)
Compounds
β-sheet (%) β-turn (%)
Unordered (%)
PPL
TAN
5-DT
NBT
5-DN
10
6.4
5.5
5.1
5.4
32
23
36.6
32.2
37.5
36.8
37.2
53.2
63.2
64.1
61.6
18.9
17.2
16.1
17.2
PMFs, polymethoxylated flavones; PPL, porcine pancreatic lipase.