International Journal of Heat and Mass Transfer 143 (2019) 118584

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International Journal of Heat and Mass Transfer

journal homepage: www.elsevier.com/locate/ijhmt

Investigating the solid-liquid extraction process of puerarin

by molecular dynamics simulations
Yuefei Zhang a,⇑, Zhangli Zhou a, Lian Zou b, Mei Zhang c, Ruan Chi d
a
School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, China
b
School of Electronic Information, Wuhan University, Wuhan 430072, China
c
School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China
d
Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, China

a r t i c l e i n f o a b s t r a c t

Article history: The solid-liquid extraction is an efficient method to obtain bioactive components from natural medicinal
Received 10 April 2019 herbs. The diffusion of the bioactive molecule is the key to efficient solid-liquid extraction. The current
Received in revised form 11 July 2019 research aims to investigate the dissolution mechanism and the dispersal behavior of puerarin in the
Accepted 14 August 2019
solid-liquid extraction process by molecular dynamics simulations. A cluster including 20 puerarin mole-
cules immersed into the solvent box with 80 Å thicknesses and 5 ns of MD simulations are performed to
all systems. The effects of ethanol concentration and extraction temperature on the molecular dispersion
Keywords:
efficiency of puerarin are investigated. The simulation results show various dissolution behaviors in dif-
Molecular dynamics
Puerarin
ferent concentration of ethanol solution. Due to the H-bonding interaction between puerarin and sol-
Extraction vents, the puerarin molecules can move gradually out of the cluster. To quantitatively describe the
Dispersion diffusion process of puerarin cluster, the RDFs for oxygen-oxygen (O-O) pair of puerarin cluster, the dif-
Molecular cluster fusion coefficient of the cluster, and the number of hydrogen bonds and lifetime of hydrogen bonds
between puerarin molecules are calculated. The results show that the best condition of dispersion is at
60% of the ethanol concentration and 323.15 K of the system temperature, which can be fitted with
the experiment of extraction. This research provides clear images of the dissolution process of drug
molecular dispersions at the molecular level. Also, the MD can effectively predict the extraction condi-
tions of the actual experiment.
Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction
Radix Puerariae from the root of Pueraria lobata (Willd.) Ohwi or
Pueraria thomsonii Benth [1], which belongs to the Leguminosae
family, has been widely used as medicine for centuries in East Asia
countries. It is also utilized as food in China and as a dietary sup-
plement in the USA, Australia, and the United Kingdom. Radix
Puerariae containing a variety of useful isoflavonoids has been
widely used in the treatment of diarrhea, fever, inflammatory,
acute dysentery, cancers, cerebrovascular and cardiovascular dis-
eases in pharmacological and clinical studies [2–5]. Nowadays,
injection of puerarin, the major isoflavone form Radix Puerariae,
has become a common vasodilator for the treatment of angina
and myocardial infarction [6]. In the resent study, it was found that
puerarin could induce neuritogenesis and synapse formation in
cultured hippocampal neurons, and thus could improve memory
⇑ Corresponding author.
E-mail addresses: yuefeiz@wit.edu.cn (Y. Zhang), zoulian@whu.edu.cn (L. Zou).
functions [7]. Radix Puerariae has attracted much attention of sci-
entists due to its wide applications.
The acquisition of the natural active compounds mostly relies
on solid-liquid extraction from all kinds of natural sources [8].
The traditional solvent for extraction is organic solvents and their
mixtures with water, which include acetone, methanol, ethyl lac-
tate, and chloroform. For a long time, conditions of solid-liquid
extraction have been mostly optimized through a large number
of experimental data. Researchers can only judge the advantages
and disadvantages of extraction conditions through observation
of yield of the target extract, while few studies have been focused
on the microscopic process of the extraction process. From the
micro level, mass transfer during solid-liquid extraction may be
conceptualized as solute diffusion in a media. At present, molecu-
lar dynamics is an effective method to simulate the microscopic
process.
Molecular dynamics (MD) simulation, a unique technique in
studying the interactions between molecules, provides a direct
way to theoretically explore molecular behavior that is impossible

https://doi.org/10.1016/j.ijheatmasstransfer.2019.118584
0017-9310/Ó 2019 Elsevier Ltd. All rights reserved.
2 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584

for the experimental methods to attain in complex systems [9–11]. volume ratio of 25:75. The flow rate is 1.0 mL/min; the column
Compared to quantum chemical calculation, MD simulation can temperature is 25 °C; the injection volume is 5 lL, and the samples
handle systems with a huge number of molecules. In recent years, are analyzed at 250 nm.
MD simulation has been used in solvent systems for investigating
the dissolving process or other properties and characteristics of 2.4. Microwave-assisted extraction
specific substances in certain solvents, such as lithium nitrate in
aprotic ionic liquid, crystal of 3,4-bis(3-nitrofurazan-4-yl)furoxan A certain amount of crude powder is extracted with the ethanol
in H2O/AcOH and H2O/EtOH solvents, NaCl in water and aspirin solution. The influences of the MAE conditions on the yield of puer-
in water [12–15]. Also, Tan’s group explained the separation of arin are investigated by single factor experiment. The extraction
puerarin from daidzin with b-cyclodextrin by MD simulation yield is defined as follow:
[16]. They think that the solvent and media can change the confor-
CV
mation of b-cyclodextrin and the guest molecules selected can Y ð%Þ ¼  100% ð1Þ
mp
inside the cavity of b-cyclodextrin. However, simulations for the
process of solid-liquid extraction have rarely been reported. where C (g/mL) is the concentration of puerarin in the extract, V
In this work, microwave-assisted ethanol reflux extraction is (mL) is the volume of the extract, and mp (g) is the mass of the sam-
used to extract puerarin from Radix Puerariae, and the extraction ple weighed. The mass of puerarin in crude samples is determined
temperature, ethanol concentration, solid-liquid ratio and extrac- by HPLC analysis of samples which are extracted with aqueous
tion time are optimized by a single factor experiment. Then, to ethanol solution.
explore the micro process of the extraction for puerarin, the model
including 20 puerarin molecular clusters has been constructed. On 2.5. Models and simulation methods
this basis, a series of MD simulations on the dispersal process of
the puerarin cluster in ethanol solution with different concentra- In this study, the molecular dynamics simulation is performed
tions and temperatures are carried out. Some statistical parameters using GROMACS 2016.1 [17] simulation package with the GRO-
such as the radial distribution functions (RDF), the Root Mean MOS53A6 force field [18]. In the GROMOS force field, the bonded
Square Deviation (RMSD), the number of hydrogen bonds interactions are the sum of the bond, bond angle, torsional dihedral
(H-bonds) and the lifetime of H-bonds at different conditions have angle, and improper dihedral angle terms, which somewhat differs
been analyzed and are used to evaluate the dispersion degree of to other force fields:
puerarin molecules. Finally, through the comparison of simulation X X X X
and extraction experiment, we focus on an exploration of molecu- Ebonded ¼ Ebonds þ Eangles þ Edihedrals þ Eimproper ð2Þ
lar dynamics simulation that can guide the solid-liquid extracting
experiment at the molecular level. 1 b 2 2
Ebonds ðr Þ ¼ k r  r 20 ð3Þ
4
2. Experimental and simulation details
1 a
Eangles ðhÞ ¼ k ðcos h  cos h0 Þ2 ð4Þ
2.1. Materials and reagents 2

Edihedrals ðuÞ ¼ k ½1 þ cos u0 cos ðnuÞ

d
Radix Puerariae (planted in the range of Dabie Mountains, ð5Þ
Anhui) were purchased from a local drugstore. The fine powder
of Radix Puerariae was prepared by milling dried Radix Puerariae 1 i
Eimproper ðnÞ ¼ k ðn  n0 Þ2 ð6Þ
with a mechanical grinder and sieving through a 60 mesh metal 2
sieve. Puerarin (HPLC
99%) was purchased from Shanghai Medi- where r0 , h0 , u0 , n0 are the equilibrium distance, angle, torsional
cine Standardization Research Center and used as a standard. dihedral angle, and improper dihedral angle between two atoms
Methanol is HPLC grade, and Ethanol for extraction is analytical b a d i
respectively, and k ,k ,k , and k are the constants. In our simula-
grade. Doubly-distilled deionized water was obtained from a Milli-
tions, all non-bonded interactions consist of Lennard-Jones (LJ)
pore Milli-Q water purification system (Millipore, USA).
and electrostatic interactions such that
" #
2.2. Apparatus   X C 12 ði; jÞ C 6 ði; jÞ
uij rij ¼  12   6
r i;j r i;j
The extraction of Radix Puerariae was carried out by a MAS-II "   #
microwave synthesis/extraction reaction workstation (2450 MHz, X qi qj 1 1=2C rf ri;j 2 1  1=2C rf
þ   ð7Þ
Sineo Microwave Chemistry Technology Co., Ltd, Shanghai, China). 4pe0 e1 r i;j R3rf Rrf
The extraction temperature can be controlled by dual-channel
temperature detection, including a thermocouple sensor and where r ij is the distance between sites i and j, qi and qj are partial
non-contact infrared ray. The microwave power can be automati- charges on sites i and j, C 12 ði; jÞ and C 6 ði; jÞ are the LJ length and
cally adjusted with the temperature. The variation range of power energy parameters, and e0 is the dielectric permittivity of vacuum,e1
is from 0 to 1000 W. The concentrations of puerarin in samples is the relative dielectric permittivity within the sphere of radius Rrf
were detected by an Agilent 1100 HPLC system with a VWD vari- (cutoff radius).
able wavelength UV/Vis detector. The model of the box used in simulations are built by PACKMOL
[19]: a cluster of 20 puerarin molecules in the center of a cubic box
2.3. Determination of puerarin in Radix Puerariae of 8 nm, encapsulated by solvent molecules. For the initial confor-
mational searches, a series of conformers were generated by the 50
For the determination by HPLC, all the samples, including stan- cyclic simulated annealing using the AMBER99SB-ILDN force field.
dard solutions and extracts, must be filtrated through 0.45 mm And then, the structure of puerarin was initially optimized by den-
microporous membrane. The samples are injected and separated sity functional theory (DFT) [20] at the level of B3LYP/6-311g++(d,
on a Hypersil ODS-C18 column (200  4.6 mm, 5 lm). The mobile p) by Gaussian 16 package [21]. The most stable geometry in this
phases are the component solvent of methanol and water with a work is obtained by comparing the zero-point energies of the fifty
 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584 3

Table 1 Table 2
The numbers of puerarin, ethanol and water molecules in a calculating box. The atomic charges in the puerarin molecule.

Volume fraction (%) Number of molecules Number Atom Charge Number Atom Charge
Ethanol Water Puerarin 1 H9 0.235 26 O1 0.598
2 O4 0.586 27 H7 0.456
0 0 14,800 20
3 C7 0.520 28 C4 0.309
20 700 9037 20
4 C8 0.472 29 H3 0.175
40 1500 7260 20
5 C16 0.348 30 C6 0.147
60 2450 5268 20
6 H10 0.038 31 H8 0.149
80 4000 3228 20
7 O8 0.434 32 C17 0.078
100 4200 0 20
8 C12 0.350 33 H11 0.063
9 H5 0.214 34 O5 0.645
10 C11 0.003 35 H15 0.437
initial designed and optimized geometries in the calculation 11 H4 0.133 36 C19 0.214
processes. 12 C10 0.406 37 H13 0.060
The number of solvent molecules is among 4200–14,800, 13 C9 0.495 38 O6 0.654
dependent on the concentration and density of ethanol solution 14 C13 0.842 39 H16 0.447
15 O3 0.593 40 C20 0.032
at 298.15 K (Table 1). Optimized structure and force field parame-
16 C14 0.338 41 H14 0.093
ters of puerarin and ethanol molecules calculated by Automated 17 O2 0.253 42 O7 0.664
Topology Builder (ATB) and Repository Version 2.2 [22]. The SPC/ 18 C15 0.113 43 H17 0.477
E model [23] for water molecules is applied. The conjugate gradi- 19 H6 0.140 44 C18 0.141
ent method (CG) [24] is used for energy minimization and leap- 20 C5 0.132 45 H12 0.074
21 C1 0.147 46 C21 0.205
flog method for dynamics. The electrostatic interaction for the 22 H1 0.149 47 H18 0.094
whole system is incorporated with the Particle-mesh Ewald 23 C2 0.309 48 H19 0.008
(PME) method [25,26] with 1.0 nm cutoff. Velocity-rescale method 24 H2 0.175 49 O9 0.657
[27] as the excellent thermostat method is adopted for tempera- 25 C3 0.403 50 H20 0.419
ture (293.15–343.15 K) control and the reference pressure
(1 atm) is incorporated with the Parrinello-Rahman method [28].
All simulations are started with 1 fs time step under the of H-bonds and the interaction energy. RDF can describe in the rel-
isothermal-isobaric ensemble (NPT) [29], an ensemble with con- ative distribution probability of a particle at a certain distance from
stant temperature and pressure. the center of another particle. the formula of RDF can be written as
To seek out the optimum conditions for the solvent extraction
of puerarin, we explore the dispersion of puerarin clusters in etha- X
N
wi ðrÞ=N
gðrÞ ¼   ð8Þ
nol solutions with different concentrations. Table 1 lists the speci- ðN  1Þ dVV r
i¼1
fic number of molecules in the calculating box. It is obvious that
different intermolecular interactions caused by different molecular where wi ðr Þ is a count of the number of atoms having their centers a
milieu can lead to discrepant dispersion effects of puerarin clus- distance between r  dr and r + dr from the center of the ith atom in
ters. The non-equilibrium model may lead to errors in subsequent the calculating box, N is the total number of atoms in the calculat-
calculations or increase the amount of calculation. Therefore, a ing box, V is the volume, and dV r is the volume of the generalized n-
200-ps’ pre-equilibrium-process (energy minimization) was car- dimensional shell between radii r  dr and r + dr.
ried out before the formal simulation. Fig. 1 shows the structure RMSD is one of the most significant criteria for measuring geo-
of puerarin and the pre-equilibrium cluster. Table 2 lists the atomic metric deviations that the formula is shown as
charges of puerarin molecule. vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u
u1 X N
RMSD ðtÞ ¼ t kri ðtÞ  ri ð0Þk2 ð9Þ
2.6. The analysis of simulation process N i

The simulation of the extraction process is evaluated by several where N is the number of target molecules, ri(t) is the position of
important statistical parameters, namely, RDF, RMSD, the statistics molecule i at time t. RMSD represents the mobility of molecules

Fig. 1. Molecular geometry of puerarin (a) and pre-equilibrium cluster (b).

4 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584
in the systems. Moreover, according to the Einstein’s relation, the
diffusion coefficient of the system can be calculated by the relevant
Mean square displacement (MSD), as Eq. (10) shows
D E
6D ¼ lim kr i ðt Þ  r i ð0Þk2 =t ¼ lim MSDðtÞ=t ð10Þ
t!1 t!1
The number of H-bond in the simulation process helps to deter-
mine the dispersion of Puerarin clusters in solvents. The standards
of H-band are (a) the distance between donor and acceptor should
be less than 3.5 Å; (b) the angle of H atom-donor-acceptor should
be less than 30°. The lifetime of the H-bonds is calculated from the
average overall autocorrelation functions of the existing functions
(either 0 or 1) of all H-bonds:
C ðsÞ ¼ hsi ðt Þsi ðt þ sÞi ð11Þ
With si ðt Þ ¼ f0; 1gfor H-bond i at time t. The integral of C ðsÞ
gives a rough estimate of the average H-bond lifetime sHB :
Z 3.2. Simulation of the solid-liquid system with different ethanol
1
sHB ¼ C ðsÞds ð12Þ
0
Both the integral and the complete autocorrelation function
C ðsÞ were output, so that more hydrogen-bond kinetics can be
used to get better estimates for sHB .
The rate of relaxation to equilibrium is
D E dynamics study, a cluster with 20 puerarin molecular is estab-
kðt Þ ¼ dc=dt ¼  s ð0Þ½1  sðt Þ =hsi ð13Þ
This kðt Þis the average rate of change of hydrogen-bond pop-
ulation for those trajectories where the bond broke at a time t later.
According to the Luzar and Chandler analysis of H-bonds kinetics
[30,31], kðtÞ  mexpðmtÞ, where m is the rate constant for H-bond
breaking, and 1=m would be the average H-bond lifetime. So from
the average lifetime of H-bonds between puerarin molecules, the
H-bonds decay dynamics at the different system can be compared.
3. Results and discussion
3.1. Effect of extraction conditions on the yield of puerarin
The effect of related conditions on microwave-assisted extrac-
tion process is investigated through Fig. 2(a–d). The radiant power
of microwave varies with the temperature of the extraction solu-
tion. So the low power means slow heating. Therefore, it is not
selected as one variable in further research, and the microwave
irradiation power will be fixed at 600 W.
For study of the effect of extraction temperature on the yield of
puerarin, extraction process was carried out at a different temper-
ature from 30 °C to 65 °C, while the other extraction conditions are
fixed at 8 min of extraction time, 60% of the ethanol concentration
and 1:20 of solid-liquid ratio, respectively. As shown in Fig. 1(a),
the yield of puerarin is increased with enhancing of extraction
temperature increased from 40 °C to 50 °C. The maximum yield
of puerarin is observed when the temperature was 50 °C. When
extraction temperature is above 50 °C, the yield of puerarin has
not increased significantly or even slightly decreased.
The solid-liquid ratio is the main factor to influence the extrac-
tion efficiency. An overlarge solid-liquid ratio can lead to solute
saturation. The too small solid-liquid ratio can increase the cost.
In this extraction experiment shown in Fig. 2(b), when the solid-
liquid ratio is less than 1:20 (g: mL), the yields of puerarin are very
similar actually. Considering economic efficiency, 1:20 (g: mL) is
the optimal solid-liquid ratio in the following experiment.
The yield of puerarin varied with the extraction time is shown
in Fig. 2(c), while the other extraction conditions, including extrac-
tion temperature, solid-liquid ratio and the concentration of etha-
nol, is fixed at 50 °C, 1:20 (g: mL) and 60%, respectively. It shows
that the yield of puerarin increased with the increase of extraction
time from 4 to 10 min. The maximum yield of puerarin is observed
at 10 min. After this point, it is hard that the yield of puerarin
increases with the extraction time, and even decreases slightly
when the extraction time is too long.
The concentration of ethanol can influence the polarity of the
solution. The effect of ethanol concentration is shown in Fig. 2
(d). Various concentration of ethanol is from 40% to 80%, while
other extraction parameters are 50 °C of the extraction tempera-
ture, 10 min of the extraction time, and 1:20 of the solid-liquid
ratio, respectively. Fig. 2(d) indicated that the yield of puerarin
reaches the highest value when the concentration of ethanol is at
50%. The higher concentration of ethanol is not benefited for the
extraction of puerarin.
concentration
3.2.1. The snapshots of simulation process
Puerarin is one of the secondary metabolites of plant cells. After
the Radix Puerariae is dried, puerarin molecules will exist in the
form of small clusters in the plant cells. To facilitate molecular
lished, and the dispersion process of this cluster in ethanol solution
with different concentration has been simulated.
The simulation time is 5 ns and the simulation temperature is
323.15 K (50 °C). Some parameters (temperature, density, total
energy, and RMSD) are used to illustrate whether the simulation
achieves sufficient equilibrium. Fig. S-1 shows the variation ten-
dency of these parameters with the simulated time in the concen-
tration of 60% ethanol solution, which is similar to that in other
concentrations. It can be found from Fig. S-1 that the values of tem-
perature, density, total energy, and RMSD gradually become con-
stant at the end of the calculated time. These results illustrate
that the whole system has reached equilibrium after the simula-
tion of 5 ns.
For the exploration of the effect of ethanol concentrations on
dispersity of the puerarin cluster, six kinds of solvent boxes with
different concentration are built. The specific volume fractions
are 0% (in water), 20%, 40%, 60%, 80% and 100%. Fig. 3 shows the
snapshots of the cluster at the time of 5 ns in different ethanol pro-
portion. In pure water, it is difficult to observe the dispersion phe-
nomena of puerarin during the simulation period. This result of the
simulation is well fit to the character that puerarin is slightly sol-
uble in water. However, when the solution contains ethanol, more
or fewer molecules of puerarin will leave the cluster and go into
the solution. It can be seen obviously that the puerarin molecules
occurs in the form of complete dispersion in 60% ethanol solution.
The aromatic ring in the molecular of puerarin is hydrophobic, and
the glucoside group increases its polarity. The coexistence of
hydrophilic and hydrophobic groups in one molecule makes puer-
arin tend to be dispersed in a certain proportion of alcohol-
aqueous solution rather than in pure water and anhydrous ethanol.
3.2.2. The analysis of the H bonds
H-bond is an important factor that affects the dispersal process.
For quantitative evaluation of molecular dispersion degree of puer-
arin, the number and the lifetime of H-bonds between puerarin
molecules and between puerarin and ethanol molecules have been
counted. Each puerarin molecule has 6 H-bond donors (6 hydroxyl
groups) and 9 H-bond acceptors (9 oxygen atoms). The number
and the lifetime of H-bonds would change with the dispersion.
The statistical data of H-bond is shown in Fig. 4 and Table 3.
 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584 5

Fig. 2. The effect of extraction experimental conditions on the yields of the puerarin from Radix Pueraria.

41.11

53.63 62.49

0% 20% 40%

84.61
87.96
64.34

60% 80% 100%

Fig. 3. Snapshots of cluster at the time of 5 ns in different concentrations (red: water molecules, blue: ethanol molecules, green: puerarin molecules). The length values (unit:
Å) between the two atoms with the longest distance in the cluster are also flagged out. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
6 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584

Fig. 4. (a) The number of H-bonds between puerarin molecules in different ethanol concentrations with time. (b) The number of H-bonds between puerarin and ethanol
molecules in the 60% ethanol proportion with time.

Table 3
The properties of H-bonds and the diffusion constant in different concentrations at 323.15 K.

Ethanol fraction (%)

0 20 40 60 80 100
Number of H-bonds 39.467 31.705 33.387 29.220 37.240 34.371
System H-bond lifetimea (ps) 3.60 7.33 8.45 9.91 12.22 15.40
p-w H-bond lifetimeb (ps) 17.49 19.53 23.11 23.29 31.46 0
p-e H-bond lifetimec (ps) 0 20.78 22.83 21.92 27.5 31.84
p-p H-bond lifetimed (ps) 67.1 66.34 57.61 50.62 76.83 78.84
mp - p e (ps1) 0.0149 0.0151 0.0174 0.0198 0.0130 0.0127
Df (105 cm2/s) 0.0677 0.0738 0.1316 0.3953 0.4105 0.1631
a
The average lifetime of H-bonds of whole system.
b
The average lifetime of H-bonds between puerarin and water molecules.
c
The average lifetime of H-bonds between puerarin and ethanol molecules.
d
The average lifetime of H-bonds between puerarin molecules.
e
The rate constant for H-bond breaking between puerarins.
f
The diffusion coefficient of puerarin cluster.

Fig. 4(a) shows the number of H-bonds between puerarin mole-
cules in different concentrations. The decline of the wave lines in
Fig. 4(a) reflects that puerarin molecules are leaving away from
each other as time goes by. The purple line shows a decline that
means puerarin molecules spread quickly in 60% ethanol solution.
The decline of the other colored lines is not more obvious than the
purple one, especially the red line, representing zero proportion of
ethanol, is almost at a high level throughout the whole simulation.
Comparing the number of H-bonds in Table 3, the lowest average
number of H-bonds between puerarin molecules is in the concen-
tration of 60%. So the effect of ethanol concentration on H-bonds
between puerarin and puerarin is well fitted with the snapshots
shown in Fig. 3. The number of H-bonds in the concentration of
60% varies with time is shown in Fig. 4(b). The red line represents
H-bonds between puerarin molecules while the blue line repre-
sents H-bonds between puerarin and ethanol molecules. The two
lines show opposite trends: the former decreases with time while
the latter rises. This change demonstrates that when the puerarin
dissolves in an ethanol solution, the H-bonds between puerarin
molecules will be broken, and the new H-bonds between puerarin
and ethanol will form.
H-bond lifetime has revealed the relaxation behavior of H-
bonds in different systems. The rate of H-bond relaxation is
kðt Þ ¼ - dc=dt  mexpðmt Þ, where m is the rate constant for H-
bond breaking, and 1=m would be the average H-bond lifetime. So
from the average lifetime of H-bonds between puerarin molecules,
the H-bonds decay dynamics at the different system can be com-
pared. As Table 3 shows, with the increase of ethanol concentra-
tion, the H-bond lifetime of the system, the H-bond lifetime
between puerarin and water and between puerarin and ethanol
all increased. This change indicate that the decay rate of H-bonds
become slower with the increase of H-bond lifetime. The increase
of the H-bond lifetime is related to the mobility of molecules.
Because water molecules are smaller and more mobile, the H-
bond lifetime mentioned above increases gradually with the
decrease of water, which can not present the dispersion degree
of puerarin cluster. However, the H-bond lifetime between puer-
arin molecules can reflect the dispersion degree of puerarin cluster.
The shorter the H-bond lifetime between puerarin molecules is, the
longer the average distance between puerarin molecules.
3.2.3. The analysis of the interaction energy
The non-bonded interaction energy includes Lennard-Jones and
Coulomb interaction energy. The non-bonded interaction energy
with time between puerarin and puerarin, puerarin and water,
puerarin and ethanol in different ethanol concentrations have been
shown in Fig. 5. As Fig. 5 shows, the interaction energy of puerarin
with water and puerarin with ethanol varies greatly under differ-
ent systems. So it doesn’t make sense to just compare absolute val-
ues of interaction energy between puerarin and water, puerarin
and ethanol, because different systems contain different numbers
of molecules. But the interaction energy between puerarin and
 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584 7

Fig. 5. The non-bonded interaction energy of puerarin-puerarin, water-puerarin and ethanol-puerarin in different ethanol concentrations with time.

Fig. 6. (a) The RDF for O–O pair of puerarin cluster with different concentrations of ethanol at 5th ns. (b) MSD of the cluster with time in ethanol solution with different
concentrations.

puerarin was relatively stable under different systems. The interac-
tion energy of ethanol with puerarin decreased with time in differ-
ent systems. The interaction energy of water with puerarin
increased with time in 0–40% ethanol system, and then decreased
with time in 60–80% ethanol system. The interaction energy of
puerarin with puerarin decreased with time in 0–40% system and
increased with time in 60–100% ethanol system.
3.2.4. The analysis of RDF and MSD
To study the structure-property of puerarin cluster in different
concentration of ethanol, we have calculated the RDFs for oxygen-
oxygen (O-O) pair of puerarin cluster. The effect of the ethanol con-
centration on the RDFs of puerarin cluster is shown in Fig. 6(a). It
can be seen that the first peak of g(r) is located about at
r = 0.336 nm, which corresponds to the distent of H-bond between
8 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584

51.33 62.08
69.54

297.15 K 303.15 K 313.15 K

87.96
83.93
74.72

323.15 K 333.15 K 343.15 K

Fig. 7. Snapshots of cluster at the time of 5 ns in 60% ethanol solution at different temperatures. The longest distance (unit: Å) between the two atoms in the cluster are
flagged out.

Fig. 8. The number of H-bonds between puerarin molecules in 60% concentration at
different temperatures varies with time.
puerarins. Generally, the RDF curves in liquid state all tends rapidly
to be unity after the second neighbor peak, indicating the short-
range order and long-range disorder as expected in liquid state.
The puerarin molecule is a large molecule with 50 atoms compared
to the solvent molecule. So the every RDF curve of puerarin cluster
has several peaks. The trend of the RDF curve for O-O pair of puerarin
cluster is that the g(r) of the maximum peak gradually decreases
with the increase of ethanol concentration, and the RDF curve in
60% ethanol solvent has the lowest peak, indicating larger distence
between puerarin molecules in the 60% ethanol solution system.
The MSD of puerarin cluster is calculated, which can be used to
describe the mobility of puerarin molecules in the cluster. In Fig. 6
(b), the purple line, representing the MSD of puerarin molecules in
60% and 80% concentration, increases steeply. This trend illustrates
that the coordinates of puerarin molecules change greatly in 60%
and 80% concentration. The curves in the other concentrations,
especially in pure water, rise slowly. The diffusion coefficient of
the cluster depands on the slop of MSD. So according to the Ein-
stein’s relation, the diffusion coefficient of the system can be calcu-
lated in Table 3. Therefore, the best dispersal effect appears in 60–
80% concentration.

Table 4
The properties of H-bonds and the diffusion constant in 60% concentration at different temperatures.

Temperature (K)
293.15 303.15 313.15 323.15 333.15 343.15
Number of H-bonds 37.277 31.525 34.455 29.220 35.443 31.473
System H-bond lifetimea (ps) 15.37 15.37 12.97 11.19 9.91 8.88
p-w H-bond lifetimeb (ps) 42.88 36.02 30.69 23.29 21.46 19.37
p-e H-bond lifetimec (ps) 40.15 31.01 29.16 21.92 20.25 18.44
p-p H-bond lifetimed (ps) 71.70 74.67 53.01 50.62 59.47 57.58
mp - p e (ps1) 0.0139 0.0134 0.0189 0.0198 0.0168 0.0174
Df (105 cm2/s) 0.0953 0.0814 0.1123 0.3041 0.1878 0.4727
a–f
The notes refer to Table 3.
 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584 9

Fig. 9. The non-bonded interaction energy of puerarin-puerarin, water-puerarin and ethanol-puerarin at different temperature in 60% ethanol solution.

Fig. 10. (a) The RDF for O-O atom of puerarin cluster at the time of 5 ns in 60% concentration at different temperatures. (b) Mean square displacement of the cluster in 60%
concentration at different temperatures varies with time.

3.3. Simulation of the solid-liquid system at different temperatures
Temperature is another important factor which makes a notable
influence on the extraction efficiency. In this section, the different
temperature of the system (293.15 K, 303.15 K, 313.15 K, 323.15 K,
333.15 K, and 343.15 K) are investigated in 60% ethanol solution,
the optimal ethanol proportion obtained in the last section.
Fig. 7 shows the snapshots of the cluster at 5 ns in 60% ethanol
solution at different temperatures. In these snapshots, the solvent
molecules in the box are hidden given the same concentration of
ethanol. According to Fig. 7, higher temperatures are beneficial to
the dispersion, and the puerarin molecules disperse more thor-
oughly, which is a good agreement with experience. Fig. 8 shows
the number of H-bonds between puerarin molecules with time.
almost all curves are decreasing as time goes by except the red
curve at 293.15 K. this change indicate that the puerarin cluster
is gradually disintegrating, leading to the decrease of the number
of H-bonds between puerarin molecules. Besides, it is noteworthy
that the lowest curve, the minimum average number of H-bonds
between puerarin molecules are all appeared at 323.15 K. There
10 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584
(a)
0 ns 0.1 ns
39.8 41.41
2.0 ns 3.0 ns
68.32 78.44
(c)
Fig. 11. (a) The model of system (red: water molecules, blue: ethanol molecules, green: puerarin molecules). (b) Average RDF of the mass-center distances between puerarin
molecules. (c) Snapshots of cluster at different points in the 60% ethanol solution during the dispersion process. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
are the shortest H-bond lifetime (Table 4) at 323.15 K. So the decay
rate of H-bonds is faster at 323.15 K than at the other temperature.
The non-bonded interaction energy with time between puer-
arin and puerarin, puerarin and water, puerarin and ethanol in dif-
ferent ethanol concentrations have been shown in Fig. 9. As Fig. 9
shows, the interaction energy between puerarin and puerarin
increase slowly with time. The higher the temperature is, the faster
the interaction energy increases, while the interaction energy of
puerarin with water and the interaction energy of puerarin with
ethanol decrease.
The RDF and the MSD have evaluated the diffusion process.
Fig. 10(a) shows the RDF of the pair O–O with temperature for
describing the local structures of puerarin cluster. Based on the
comparison of the peak height of the RDFs for different tempera-
ture systems, the higher the temperature is, the fewer O-O pair dis-
tance of puerarin molecules is. However, when the temperature is
higher than 323.15 K, the decline of the peak is not obvious. As
Fig. 10(b) shows, the slopes of MSD curves at 293.15 K, 303.15 K,
and 313.15 K are close each other, which are lower than the slopes
of the other curves at higher temperatures. When the temperature
(b)
0.5 ns 1.0 ns
52.07
49.4
4.0 ns 5.0 ns
83.05 87.96
is above 323.15 K, the slope of MSD increase significantly, the max-
imum slope is at 343.15 K, likewise the average diffusion coeffi-
cient of 343.15 K is also highest. The incipient slope of the MSD
curve at 323.15 K is lower than at 343.15 K. However, the slope
of the MSD curve at 323.15 K increase with the increase of simula-
tion time. When the simulation time reaches 5 ns, the value of the
MSD at 323.15 K is close with the value of MSD at 343.15 K. There-
fore, the diffusion constant of 343.15 K and 323.15 K could be sim-
ilar each other in macro time scales. This is also the reason why the
optimal temperature of the actual experimental extraction process
is at 323.15 K.
3.4. The description of the dispersion process
For describing dissolution process of the puerarin clusters in the
ethanol solution form the molecular level, the whole system is
simulated by MD at the optimal conditions, i.e., the ethanol con-
centration of 60% and the temperature of 323.15 K. Along with
the simulation time, puerarin molecules are gradually out of the
cluster due to the interaction with solvent molecules. To quantita-
 Y. Zhang et al. / International Journal of Heat and Mass Transfer 143 (2019) 118584
tively describe the diffusion process of puerarin cluster, we display
the RDF of the barycenter of the cluster around puerarin molecules
varies at different time points [26]. The decline of three major
peaks with the increase of simulated time indicates that puerarin
molecules are separating from each other. Snapshots of the system
at different times show the dispersal process in Fig. 11(c) more
intuitively. The longest distances (unit: Å) between the two atoms
of the cluster at different time points are also flagged out in these
snapshots. At the beginning of the simulation (<0.1 ns), the cluster
has little change due to the strong interaction between puerarin
molecules. When more and more solvent molecules interact with
puerarin molecules, the cluster starts to split into several smaller
pieces at about 0.5 ns, and then puerarin molecules begin to depart
from these small pieces. At the time of 4.0 ns, the cluster disinte-
grates completely.
4. Conclusions
Through MD simulations, we have explored the dispersal pro-
cess of puerarin in ethanol solution and investigated the optimum
concentration and temperature for the dispersion. Through the
analysis of RDF, MSD, the number of H-bond, the lifetime of
H-bond and snapshots, the optimal conditions of simulated disper-
sion are the concentration of 60% and the temperature of 323.15 K,
which are well agreeable with the optimal extraction experimental
conditions from our group and literature [32]. This study provides
better insight into the dissolution mechanisms of puerarin. The
result from MD can guide the solid-liquid extraction of puerarin
in practice.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgment
This work is supported by the National Natural Science Founda-
tion of China (grant number 21006075); and the Natural Science
Foundation of Hubei Province, China (grant number 2014CFA115).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ijheatmasstransfer.2019.118584.
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