Thermostable enzyme-immobilized magnetic responsive Ni-based metal-organic framework nanorods as recyclable biocatalysts for efficient biosynthesis of: S -adenosylmethionine

Article abstract of DOI:10.1039/c8dt04857f

A novel magnetic responsive Ni-based metal-organic framework material was developed to efficiently separate and immobilize thermal enzymes with high catalytic performance. Ni-based metal-organic framework nanorods (Fe₃O₄/Ni-BTC) with high magnetic responsiveness are prepared conveniently by a one-pot hydrothermal process. With the characterization, it was confirmed that Ni-based metal-organic framework nanocomposites were synthesized as nanorods mounted with magnetic Fe₃O₄ nanoparticles on the surface. Although Fe₃O₄/Ni-BTC showed a preference for histidine-tagged enhanced green fluorescent protein (His-eGFP), we found that a variety of forces played roles in enzyme immobilization, including affinity between Ni²⁺ and histidine tags, electrostatic attraction, hydrogen bonding and hydrophobic forces. After understanding the mechanism of the as-prepared nanocomposites, a new immobilization strategy for thermostable S-adenosylmethionine synthetase (SAMS) was further evaluated. As a result, SAMS from cell lysate achieved about 95% activity recovery in the biosynthesis of S-adenosylmethionine (SAM) under high temperature conditions (70 °C) with a simple mixing step. At the same time, the immobilized enzyme was more stable against temperature variation (by nearly 8-fold in an 80 °C water bath after 2 h) and extreme pH (by nearly 1.3-fold at pH 3) and exhibited excellent reusability after immobilization. This work indicates that magnetic responsive Ni-based nanorods are highly promising for thermostable enzyme immobilization with high efficiency and stability.

Full text of DOI:10.1039/c8dt04857f

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Journal of Separation Science
Separation of six compounds from pigeon pea leaves by elution–extrusion counter-current
chromatography
Xiaoai He, Huichen Zhang, Xianrui Liang*
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals,
College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014,
China
Running title: separation and identification of six compounds from pigeon pea leaves
*Correspondence: Dr. Xianrui Liang, Collaborative Innovation Center of Yangtze River
Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang
University of Technology, Hangzhou, 310014, China.
Email: liangxrvicky@zjut.edu.cn
Abbreviations: HSCCC, high speed counter-current chromatography; EECCC,
elution-extrusion counter-current chromatography; HepG2, human hepatoma cell; UPLC,
ultra-performance liquid chromatography; Q-TOF, quadrupole time-of-flight; CCK-8, cell
counting kit-8; IC50, half-maximum inhibitory concentration.
Received: 10 23, 2018; Revised: 01 10, 2019; Accepted: 01 11, 2019
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jssc.201801111.
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Keywords: antiproliferation activity; elution-extrusion counter-current chromatography;
pigeon pea leaves; structure identification
Abstract
A valid and reliable method was established to separate six compounds from pigeon pea
leaves via elution-extrusion counter-current chromatography. A solvent system composed
of n-hexane/methanol/formic acid aqueous solution with pH=3 (10:6:4, v/v) was screened
to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four
compounds including 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%)
and 25.2 mg of 5 (94.2%) were obtained by conventional elution from 100 mg of the
precipitation fraction, respectively. Two compounds including 14.4 mg of 3 (96.3%) and 28.1
mg of 6 (96.6%) with high K values were obtained by the subsequent extrusion procedure.
The structures of compounds 1-6 were identified as
3-methoxy-5-(2-phenylethenyl)-phenol, pinostrobin chalcone, pinostrobin,
2-hydroxy-4-methoxy-6-(2-phenylvinyl)-benzoic acid, longistylin C and cajaninstilbene
acid by quadrupole time-of-flight mass spectrometry, 1H and 13C NMR, respectively. The in
vitro antiproliferation activities of compounds 1, 5 and 6 against human hepatoma cell were
evaluated and the half-maximum inhibitory concentrations were acquired.
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1. Introduction
Pigeon pea (Cajanus cajan L.) is a multipurpose plant of the Leguminosae family belonged to
genus Cajanus. It acts as one of the major grain legume crops as well as a good supplement
of dietary essential minerals, such as vitamin and protein [1, 2]. Pigeon pea has highlighted
its great medicinal value for treating bronchitis, coughs, fevers, inflammation, ulcers,
diarrhea, jaundice, diabetes, dermatosis, blood disorders and plasmodium disease [3-5].
Moreover, pigeon pea leaves revealed the notable therapeutic effect on femoral head
necrosis in Chinese folk medicine [6]. Modern phytochemical studies have demonstrated
that the extract of pigeon pea leaves exhibits remarkable anti-inflammatory [7],
antibacterial [8], antioxidant [9, 10] and antitumor activities [11]. Although diverse
pharmacological activities were observed from the pigeon pea leaves, the certain constituent
responsible for relevant bioactivity remained unclear. Considering the complex constituents
in pigeon pea leaves, an efficient isolation approach to prepare compounds from pigeon pea
leaves is warranted for the in-depth structural identification and bioactivity research.
High speed counter-current chromatography (HSCCC) was firstly reported by Ito in the
early 1970s based on the distribution of compounds between a pair of mutually immiscible
liquid phases [12]. The HSCCC exhibited superiorities including no irreversible adsorption,
support-free solid filler, less solvent consuming, rapid separation and larger throughput of
samples over the conventional separation techniques [13, 14]. As an efficient separation and
purification technique, it was successfully applied in the isolation of various natural
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compounds, such as flavones [15-17], terpenoids [18, 19], phthalides [20] and alkaloids
[21]. However, the multiple constituents in natural products usually possess distinct
polarity, which results in longer isolation time or poorer resolution in a single solvent system
for the classical HSCCC method. Elution-extrusion counter-current chromatography
(EECCC) combined of regular chromatographic elution and stationary-phase extrusion was
widely adopted since the capacity of using the liquid nature of the stationary phase to
greatly extend the hydrophobicity window [22, 23]. It was notable that EECCC mode could
cover a broad polarity range of solutes, highly promoting the separating power of
counter-current chromatography technique.
The EECCC method was established in this work to separate compounds in pigeon pea
leaves. The partition coefficient of the components in pigeon pea leaves was determined and
the preferred two-phase solvent system was screened. Six compounds (Fig. 1) were isolated
and their purities were analyzed by ultra-performance liquid chromatography (UPLC). The
structures were confirmed by quadrupole time-of-flight (Q-TOF) mass spectrometry, 1H
and 13C NMR technology. The in vitro cell antiproliferation activities of compounds 1, 5 and
6 were also preliminarily described in this study. To the best of our knowledge, there is no
report on the composition separation from pigeon pea leaves using EECCC technique.
2. Materials and Methods
2.1 Chemicals and reagents
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Pigeon pea leaves were collected from Yunnan Kunming and stored at room temperature
until analysis. Acetonitrile, methanol and formic acid (HPLC grade) were purchased from
Merck (Darmstadt, Germany). Penicillin, streptomycin, trypsin, phosphate buffered saline
and DMEM (high glucose) medium were acquired from Hyclone (Logan, Utah, USA). Fetal
bovine serum was bought from Zhejiang Tianhang Biotechnology Co., Ltd (Huzhou, China).
Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). Dimethyl
sulfoxide was obtained from Biofroxx (Germany). All cell culture plastics were from
Corning-Costar (NY, USA). Ultrapure water (18.2 MΩ) was purified by Barnstead TII super
Pure Water System (MA, USA). Other reagents (such as n-hexane, ethyl acetate and
ethanol) were all analytical grade obtained from Yongda Chemical Reagent Company
(Tianjin, China).
2.2 Sample preparation
Dry pigeon pea leaves were firstly ground to powder and sieved on an 80 meshes sifter. The
powder (100.0 g) was extracted with 75% ethanol solution (1:30, w/v) for half an hour under
ultrasonic bath at 30 °C. Next, the extract solution was concentrated by
decompression-evaporation to yield crude extract (13.4 g). The obtained extract was then
re-dissolved in water and centrifuged (7000 rpm, 15 min) for three times. The dried
precipitation (5.3 g) was obtained over a freeze dryer and used as the research object for
further experiments.
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2.3 Partition coefficient determination
Partition coefficient (K) of the target compounds was critical to the selection of the
two-phase solvent system. The determination procedure was as follows: approximately 2 mg
sample was added into a test tube containing 10 mL pre-equilibrated two-phase solvent
system (5 mL upper phase and 5 mL lower phase). The tube was shaken completely to
distribute the sample into the two phases thoroughly. The two-phase solvent system was
statically separated after the equilibrium was reached. Then 3 mL of each phase was
evaporated to dryness. The two samples were dissolved in 1 mL methanol and filtered
through a 0.22 µm PTFE membrane. The K was calculated by the following equation:
K = Au p p e r
p
/h
Aa s e
l o
(1)
where Aupper phase and Alower phase represented the peak area of the target compounds in the
upper phase and the lower phase, respectively. The separation factor expressed as α was
defined as the ratio of two K values:
α = K
1
/ K
2
(K
1
 K
2)
(2)
It was considered that an effective separation can be obtained with α >1.5 [24].
2.4 Preparation of the two-phase solvent system and sample solution
The two-phase solvent system utilized in this study was prepared by mixing
n-hexane/methanol/formic acid aqueous solution (pH=3) (10:6:4, v/v) in a separatory
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funnel at room temperature. After thorough equilibration, the two phases were separated
and degassed by ultrasonic for 30 min prior to use. The sample solution was prepared by
dissolving 30.0 mg sample in 5 mL upper phase and 5 mL lower phase.
2.5 Preparative separation by EECCC
The experiment was performed on a TBE-300A HSCCC (Tauto Biotechnique Company,
Shanghai, China) composed of a multilayer coil planet centrifuge equipped with a
polytetrafluoroethylene coil (tube diameter 1.6 mm) with a total capacity of 280 mL and a
manual injection valve with a 20 mL sample loop. The β value of the preparative column
varied from 0.5 at the internal terminal to 0.8 at the external terminal. The HSCCC system
was firstly filled with the upper phase (stationary phase) at a high flow rate (20.0 mL/min) by
a TBP-50A pump (Tauto Biotechnique Company, Shanghai, China). Hereafter, the lower
phase (mobile phase) was delivered at a flow rate of 2.0 mL/min in head-to-tail mode and
the apparatus was rotated at 850 rpm. Equilibration was established when a clear mobile
phase effluent appeared at the tail outlet. Then Sample solution was injected into the
injection valve and the effluent was monitored at 280 nm wavelength with a Model
UVD-200UV Monitor (Shanghai Jinda Biochemy Apparatus Co. Ltd., Shanghai, China),
temperature was set at 25 °C by an HX-1050 constant temperature circulating implement
(Beijing Boyikang Lab Instrument Co. Ltd., Beijing, China). After most compounds were
eluted out, the stationary phase (instead of the mobile phase) was pumped into the system
without changing the flow direction, flow rate, separation temperature and equipment speed.
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The phase change indicated the beginning of the extrusion procedure. The strong retention
substances were directly pushed out by the stationary phase in the extrusion step [25-27].
The data were collected with a V4.0 chromatogram workstation (Shanghai Jinda Biochemy
Apparatus Co. Ltd., Shanghai, China).
2.6 Operating conditions of UPLC, MS and NMR analysis
The UPLC assay was carried out on an ACQUITY UPLCTM system (Waters, Milford, MA,
USA) consisting of a binary solvent pump, an autosampler, a thermostated column
compartment, a photodiode array detector and Empower 2 software for data acquisition. The
chromatographic separation was operated on a BEH C18 column (100 mm×2.1 mm, 1.7 µm,
Waters) at 30 °C. The mobile phase consisted of 0.1% formic acid aqueous (v/v) (A) and
methanol (B) with the gradient: 0-8 min, 63-68% B; 8-15 min, 68-72% B; 15-20 min, 72-80%
B; 20-30 min, 80% B. The detection wavelength was set at 280 nm and flow rate was 0.2
mL/min. The injection volume was set at 3 µL.
The MS analysis was achieved using micrOTOF-Q II mass spectrometer (Bruker Daltonics,
Bremen, Germany) equipped with composite electrospray ionization in positive and negative
ion modes, micrOTOF control data acquisition system and Compass DataAnalysis data
processing system. The optimized conditions were as follows: Capillary voltage, 3500 V
under negative mode; Desolvation gas (N2), 4.0 L/min; Dry gas temperature, 180 °C. The
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m/z scan range from 50 to 1000 Da was selected. The 10 mM sodium formate was applied as
an internal calibration solution.
The 1-D and 2-D NMR spectra were recorded at 297 K on a Bruker AVANCE III HD 600
MHz spectrometer (Bruker Daltonics, Bremen, Germany). The tetramethylsilane (TMS, δ
0.00 ppm) were used as the reference. All spectra were processed using MestReNova
software.
2.7 Cell antiproliferative assay
The antiproliferative activities of compounds 1, 5 and 6 against human hepatoma cell
(HepG2) were determined by the CCK-8 method. Cells were plated in 96-well microplates
at 1×104 per well and firstly incubated for 24 h at 5 % CO2, 37 °C. Different
concentrations of each compound were tested in triplicate in one assay. Each well was
treated with 10 µL test solution and continuously incubated for 48 h at the same condition,
only cell medium was used as a control group. Then removed the supernatants and added
new medium before 10 µL CCK-8 solution was joined in. After 3 h incubation, the
absorbance was measured at 450 nm in a microplate reader (Infinite 200 PRO NanoQuant,
Tecan, Switzerland). The CCK-8 solution in medium without cells was used as a blank
group. The calculation formula of inhibition rate:
i n h i b i t i o n r aAt e
c
=A[
s
( A-
c
A)
b
/(
-
(3)
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where As, Ac and Ab represented the absorbance of the test group, control group and blank
group, respectively. The whole assays were performed in triplicate experiments and the half
maximal inhibitory concentration (IC50) was calculated by software SPSS 19.0.
3. Result and discussion
3.1 The optimization of EECCC separation
Generally, partition coefficient K was thought to be the essential factor for HSCCC
separation and the ideal K value range was 0.5-2.0. It was accepted that the best solvent
system existed when the K value was infinitely close to 1.0 [14]. The selectivity of the
two-phase solvent system was usually based on the polarity of analytes [15]. The selection
of liquid phase was also affected by solvent physical properties including the density,
viscosity and interfacial tension [28, 29]. The moderately polar solvent systems with suitable
physical properties were chosen due to the less polar sample used in this study, including
n-hexane/ethyl acetate/methanol/water system, n-hexane/petroleum
ether/methanol/water system and n-hexane/methanol/water system. The primitively
solvent system screening showed that n-hexane/ethyl acetate/methanol/water (7:3:5:5,
v/v) system and n-hexane/petroleum ether/methanol/water (1:1:1:1, v/v) system could
provide the suitable K value for all the target compounds. However, the α in these two
systems were mostly less than 1.5, indicating a relatively poor resolution. The K values for
most compounds in n-hexane/ethyl acetate/methanol/water (10:2:7:5, v/v) system and
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n-hexane/petroleum ether/methanol/water (6:5:6:5, v/v) system were small which made it
difficult to efficiently separate the components from the solvent front [16].
The n-hexane/methanol/water system was closer to the polarity of the solutes and five
different constituent ratios were selected for preliminary exploration experiments. However,
there was only a big rectangular peak in the actual spectrum and the target compounds were
not separated entirely in the n-hexane/methanol/water system. Considering the acidic
buffer was used as mobile phase in UPLC analysis, it was supposed that the pH may also
influence the K values of analytes investigated in this study [24]. Hence the pH value of
water in n-hexane/methanol/water system was adjusted by formic acid. Three pH values of
formic acid solution were evaluated (Table 1). It could be seen that K values for compounds
3 and 6 were generally large, meaning required longer time in conventional HSCCC mode.
Hence the EECCC mode was adopted for efficient separation. For EECCC mode, the
suitable K value range could be elongated to 0.4-16 [25]. The α values in
n-hexane/methanol/formic acid solution system with solvent volume ratio of 10:4:6 (pH=3),
10:6:4 (pH=3), 10:7:3 (pH=2) and 10:5:5 (pH=1) were all larger than 1.5 as shown in Table
1. However, K values for compounds 5 and 6 in n-hexane/methanol/formic acid solution
(pH=3) (10:4:6, v/v) system were too large to attain good separation within a reasonable
time. The K value for compound 2 in n-hexane/methanol/formic acid solution (pH=2)
(10:7:3, v/v) system was too small to efficiently isolate from the solvent front, leading to a
lower resolution. The HSCCC results displayed that the preferred solvent system was
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n-hexane/methanol/formic acid solution (pH=3) (10:6:4, v/v), which could supply suitable
α values and relatively ideal K values for compounds 1, 2, 4 and 5. Compounds 3 and 6 with
bigger K values were eluted successively in the extrusion process as shown in Figure 2. It
was obvious that there was a high peak adjacent to compound 2, which might affect the
purity of compound 2. If necessary, semi-preparative liquid chromatography could be
employed for the succeeding purification. Other target compounds were all appropriately
isolated in Figure 2. It demonstrated that the EECCC mode could serve as an effective
method to isolate the components in pigeon pea leaves which have a large difference of K
values.
It was found that the addition of acid in the solvent system would greatly improve the
separation effects, which could be related to the acidic properties of compounds in the
pigeon pea leaves extracts. The subsequent structural identification showed there were
carboxyl, phenol hydroxyl groups in the structure. The acidic conditions would keep these
compounds from dissociating to the ionic state. These findings provided useful ideas for
similar compound isolation by counter-current chromatography technology.
3.2 UPLC analysis and structure identification
The UPLC spectra of the precipitation fraction under optimum condition and purities of
compounds 1-6 were shown in Figure 3. A total of 9.2 mg of compound 1 (96.8%), 3.2 mg of
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2 (88.0%), 14.4 mg of 3 (96.3%), 6.2 mg of 4 (94.2%), 25.2 mg of 5 (94.2%) and 28.1 mg of 6
(96.6%) were obtained from 100 mg of precipitation fraction through EECCC mode.
The structural identification of six compounds were all performed with Q-TOF MS, 1H NMR
and 13C NMR. The MS and NMR spectra were provided in supporting information (Figure
S1-S6). The compounds feature, high-resolution MS and 1-D NMR results were listed as
follows:
Compound 1: Faint green solid, measured m/z 225.0925 [M-H]-, calculated formula
C15H13O2, err -1.7 ppm. 1H-NMR (CDCl3, δ /ppm): 7.52 (2H, d, J = 7.2 Hz, H-2′, 6′),
7.38 (2H, t, J = 7.2 Hz, H-3′, 5′), 7.30 (1H, m, H-4′), 7.09 (1H, d, J = 16.2 Hz, H-A),
7.02 (1H, d, J = 16.2 Hz, H-B), 6.67 (1H, s, H-4), 6.63 (1H, s, H-6), 6.37 (1H, s, H-2),
3.85 (3H, s, -OCH3). 13C-NMR (CDCl3, δ /ppm): 161.14 (C-3), 156.86 (C-1), 139.71
(C-5), 137.07 (C-1′), 129.42 (C-4′), 128.71 (C-3′, 5′), 128.32 (C-A), 127.80 (C-B),
126.60 (C-2′, 6′), 105.94 (C-6), 104.95 (C-4), 101.00 (C-2), 55.40 (-OCH3). The
above results were consistent with the literature [30] and the compound was identified as
3-methoxy-5-(2-phenylethenyl)-phenol.
Compound 2: Faint yellow solid, measured m/z 269.0826 [M-H]-, calculated formula
C16H13O4, err -2.6 ppm. 1H-NMR (CDCl3, δ /ppm): 10.58 (1H, s, broad peak, -OH), 8.09
(1H, d, J = 15.6 Hz, H-2), 7.83 (1H, d, J = 15.6 Hz, H-1), 7.64 (2H, m, H-2′, 6′), 7.41
(3H, m, H-3′, 4′, 5′), 6.00 (2H, s, H-6, 8), 3.83 (3H, s, -OCH3). 13C-NMR (CD3OD,
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δ /ppm): 194.28 (C-3), 167.71 (C-7), 165.79 (C-5, 9), 143.15 (C-1), 136.90 (C-1′),
131.10 (C-4′), 129.92 (C-3′, 5′), 129.33 (C-2′, 6′), 128.74 (C-2), 106.59 (C-4),
94.61 (C-6, 8), 55.87 (-OCH3). The above results were consistent with the literature [31,
32] and the compound was identified as pinostrobin chalcone.
Compound 3: Faint green solid, measured m/z 269.0828 [M-H]-, calculated formula
C16H13O4, err -3.3 ppm. 1H-NMR (CDCl3, δ /ppm): 12.02 (1H, s, -OH), 7.46 (2H, m,
H-2′, 6′), 7.41 (3H, m, H-3′, 4′, 5′), 6.08 (1H, d, J = 1.8 Hz, H-6), 6.07 (1H, d, J
= 1.8 Hz, H-8), 5.42 (1H, m, H-2), 3.81 (3H, s, -OCH3), 3.09 (1H, m, H-3a), 2.83 (1H, m,
H-3b). 13C-NMR (CDCl3, δ /ppm): 195.80 (C-4), 168.02 (C-7), 164.18 (C-5), 162.82
(C-9), 138.40 (C-1′), 128.90 (C-3′, 4′, 5′), 126.16 (C-2′, 6′), 103.17 (C-10),
95.17 (C-6), 94.30 (C-8), 79.26 (C-2), 55.72 (-OCH3), 43.41 (C-3). The above results
were consistent with the literature [31, 32] and the compound was identified as pinostrobin.
Compound 4: White solid, measured m/z 269.0822 [M-H]-, calculated formula C16H13O4, err
-1.1 ppm. 1H-NMR (CDCl3, δ /ppm): 7.88 (1H, d, J = 15.6 Hz, H-A), 7.51 (2H, d, J = 7.8
Hz, H-2′, 6′), 7.35 (2H, t, J = 7.8 Hz, H-3′, 5′), 7.26 (1H, m, H-4′), 6.83 (1H, d,
J = 15.6 Hz, H-B), 6.66 (1H, d, J = 2.4 Hz, H-5), 6.43 (1H, d, J = 2.4 Hz, H-3), 3.86 (3H,
s, -OCH3). 13C-NMR (CDCl3, δ /ppm): 173.43 (-COOH), 165.70 (C-2), 164.20 (C-4),
143.50 (C-6), 137.49 (C-1′), 130.77 (C-B), 130.07 (C-A), 128.63 (C-3′, 5′), 127.72
(C-4′), 126.78 (C-2′, 6′), 116.42 (C-1), 107.62 (C-5), 100.17 (C-3), 55.46 (-OCH3).
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The above results were consistent with the literature [30] and the compound was identified
as 2-hydroxy-4-methoxy-6-(2-phenylvinyl)-benzoic acid.
Compound 5: Green solid, measured m/z 293.1559 [M-H]-, calculated formula C20H21O2,
err -3.9 ppm. 1H-NMR (CDCl3, δ /ppm): 7.47 (2H, d, J = 7.8 Hz, H-2′, 6′), 7.35 (2H,
m, H-3′, 5′), 7.33 (1H, d, J = 4.2 Hz, H-F), 7.30 (1H, m, H-4′), 6.92 (1H, d, J = 15.6
Hz, H-G), 6.69 (1H, s, H-6), 6.37 (1H, s, H-2), 5.11 (1H, m, H-B), 3.79 (3H, s, -OCH3),
3.41 (2H, d, J = 6.6 Hz, H-A), 1.80 (3H, s, H-D),1.67 (3H, s, H-E). 13C-NMR (CDCl3, δ
/ppm): 158.57 (C-3), 154.54 (C-1), 137.92 (C-5), 137.59 (C-1′), 130.89 (C-C), 130.49
(C-G), 128.68 (C-3′, 5′), 127.61 (C-4′), 126.57 (C-2′, 6′), 126.54 (C-F), 123.51
(C-B), 120.95 (C-4), 104.12 (C-6), 98.58 (C-2), 55.72 (-OCH3), 25.78 (C-E), 24.45
(C-A), 17.97 (C-D). The above results were consistent with the compound longistylin C.
Compound 6: White solid, measured m/z 337.1460 [M-H]-, calculated formula C21H21O4, err
-4.3 ppm. 1H-NMR (CDCl3, δ /ppm): 11.55 (1H, s, -OH), 7.88 (1H, d, J = 15.6 Hz, H-F),
7.56 (2H, d, J = 7.2 Hz, H-2′, 6′), 7.41 (2H, t, J = 7.2 Hz, H-3′, 5′), 7.32 (1H, m,
H-4′), 6.86 (1H, d, J = 15.6 Hz, H-G), 6.69 (1H, s, H-5), 5.25 (1H, m, H-B), 3.98 (3H, s,
-OCH3), 3.40 (2H, d, J = 7.2 Hz, H-A), 1.82 (3H, s, H-D), 1.71 (3H, s, H-E). 13C-NMR
(CDCl3, δ /ppm): 175.83 (-COOH), 162.47 (C-2), 162.32 (C-4), 141.96 (C-6), 137.31
(C-1′), 131.97 (C-C), 130.85 (C-G), 130.39 (C-F), 128.77 (C-3′, 5′), 127.90
(C-4′), 126.83 (C-2′, 6′), 121.93 (C-B), 116.80 (C-3), 103.36 (C-5), 103.08 (C-1),
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55.75 (-OCH3), 25.84 (C-E), 22.12 (C-A), 17.82 (C-D). The above results were consistent
with the literature [30] and the compound was identified as cajaninstilbene acid.
3.3 Antiproliferation activity
Six compounds including two flavonoids (compounds 2 and 3) and four stilbene compounds
(compounds 1, 4, 5 and 6) were isolated by HSCCC method. The stilbene compounds
(compounds 1, 5 and 6) were evaluated for their antiproliferation activity and compound 4
wasn’t investigated due to its small content in pigeon pea leaves.
The antiproliferation activities of the stilbene compounds 1, 5 and 6 against HepG2 cell were
summarized in Table 2. All these three displayed antiproliferation activities (shown in Figure
S7-S9 respectively). Compound 6 with an IC50 value of 21.2 µM exhibited the strongest
antiproliferation activity compared to compounds 1 and 5. Compounds 1 (with an IC50 value
of 148.7 µM) and 5 (with an IC50 value of 53.8 µM) showed weak antiproliferation activities.
The isopentenyl substituent at the site 4 of benzene ring in the structure of compound 5
increased its hydrophobic property which hence increased the amount of cellular uptake. As
a result, compound 5 showed higher antiproliferation activity than compound 1. Compared
to compound 5, the carboxyl substituent on the benzene ring of compound 6 increased its
acidic property. When it was uptake by the cell, it changed the pH conditions inside the cell
and led to the cell apoptosis. However, the mechanisms of the antiproliferation effect of
these stilbene compounds against HepG2 cell require more detailed study.
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4 Concluding remark
In this study, six compounds from pigeon pea leaves were successfully separated by
HSCCC. The EECCC mode was introduced to widen the application range in some cases
with bigger K values. The key solvent systems were screened and the
n-hexane/methanol/formic acid aqueous solution with pH=3 (10:6:4, v/v) system was
preferred to achieve satisfactory separation result. The purities and structures of all the
separated compounds were analyzed and characterized by UPLC, Q-TOF MS and NMR,
respectively. The in vitro antiproliferation activities of the stilbene compounds 1, 5 and 6
against HepG2 cell were also evaluated and the IC50 values were acquired. The separation
method established in this study could provide good preparation choice for the further
activity investigation of the individual compound from the natural herbs.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Conflict of interest statement
The authors have declared no interest conflicts.
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Figure captions
Figure 1 Chemical structures of compounds 1-6.
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Figure 2 The EECCC Chromatogram of compounds 1-6. The EECCC conditions: solvent
system: n-hexane/methanol/formic acid aqueous solution with pH=3 (10:6:4, v/v); sample
size: 30 mg precipitation fraction dissolved in the mixture of 5 mL upper phase and 5 mL
lower phase; stationary phase: upper organic phase; flow rate: 2.0 mL/min; rotation speed:
850 rpm; temperature: 25 °C; detection wavelength: 280 nm.
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Figure 3 The UPLC chromatogram of precipitation fraction (a) and the isolated compounds
1-6 (b-g) from EECCC. The UPLC conditions: column: ACQUITY UPLC® BEH C18
column (100 mm×2.1 mm, 1.7 µm, Waters); mobile phase consisted of 0.1% formic acid
aqueous (v/v) (A) and methanol (B) with the gradient: 0-8 min, 63-68% B; 8-15 min, 68-72%
B; 15-20 min, 72-80% B; 20-30 min, 80% B; detection wavelength: 280 nm; column
temperature: 30 °C; flow rate: 0.2 mL/min.
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Table 1 Partition coefficient (K) and separation factor (α) of six compounds in
n-hexane/methanol/water system and n-hexane/methanol/formic acid solution (v/v)
system
Solvent
system
K1 α1
K2
α2
K3
α3
K4
α4
K5
α5
K6
α6
HMW 10:3: 0.0
-
-
-
-
1.56 1.5 4.48 1.5 0.99 33.0 5.28 1.1 2.81 1.8
a)
7
3
8
9
0
8
0
10:4: 0.0
0.88 1.5 9.39 1.1 0.58 14.5 8.37 3.6 2.28 2.5
6
4
2
2
0
7
9
10:5: 0.0
0.33 5.5 7.75 2.4 0.40 1.21 3.13 2.6 1.17 2.9
5
6
0
8
8
3
d)
10:6:
4
-
0.12
-
4.87 3.2 0.18 1.50 1.52 1.8 0.81 4.5
0
8
0
d)
10:7: 0.1 3.4 0.05
-
3.06 5.5
6
-
-
0.55 1.5 0.36 2.1
3
7
0
3
2
HMFb 10:3: 1.5
-
54.5 1.3 12.0 5.5 2.19 1.34 1.63 1.0 39.65 3.2
)
7
8
3
8
5
0
3
9
pHc)
=3
10:4: 2.5 3.0 0.83
-
6.53 2.5 11.7 1.79 20.9 1.7 54.93 2.6
6
5
7
6
0
4
9
2
10:5: 0.6 2.0 0.32
-
-
-
-
8.02 1.1 1.69 2.56 4.05 2.4 7.29 1.8
5
6
6
0
0
0
10:6: 0.3 1.5 0.19
10.3 1.5 0.86 2.87 1.88 2.1 6.74 3.5
4
0
8
6
4
9
9
d)
10:7:
3
-
-
0.07
2.62 1.9 0.20 2.86 0.58 2.9 1.37 2.3
1
0
6
HMFb 10:3: 2.4 1.2 1.99
37.6 1.1 18.7 7.67 30.9 1.6 33.91 1.1
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)
7
4
3
4
1
1
6
5
0
pHc)
=2
10:4: 1.8 2.0 0.92
-
-
-
-
-
-
-
-
-
10.9 1.6 6.59 3.49 11.7 1.0 12.30 1.0
6
9
5
3
6
6
8
5
10:5: 0.5 1.8 0.30
15.7 1.0 2.18 3.89 6.17 2.8 15.23 2.4
5
6
7
8
4
3
7
10:6: 0.1 1.3 0.12
6.57 1.1 0.70 4.38 1.80 2.5 5.66 3.1
4
6
3
6
7
4
10:7: 0.1 2.1 0.06
3.09 1.7 0.28 2.15 0.70 2.5 1.77 2.5
3
3
7
5
0
3
HMFb 10:3: 8.9 4.8 1.83
22.0 2.4 29.5 1.34 62.4 2.1 187.4 3.0
)
7
3
8
2
7
2
3
1
8
0
pHc)
=1
10:4: 2.2 2.5 0.90
26.9 4.8 5.57 2.48 29.4 1.0 77.80 2.6
6
5
0
6
4
3
9
4
10:5: 1.0 3.1 0.35
8.50 1.6 2.02 1.85 5.19 2.5 15.67 1.8
5
9
1
4
7
4
10:6: 0.3 3.1 0.11
3.78 2.9 0.62 1.77 1.28 2.0 4.41 1.1
4
5
8
5
6
7
10:7: 0.1 2.2 0.05
2.00 1.2 0.30 2.73 0.70 2.3 1.57 2.2
3
1
0
7
3
4
a) HMW: n-hexane/methanol/water.
b) HMF: n-hexane/methanol/formic acid solution.
c) pH value is only belonged to formic acid solution, not the entire solvent system.
d) represents the solutes entirely dissolved in upper phase or lower phase.
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Table 2 Antiproliferation activities of compounds 1, 5 and 6 against HepG2 cell
compounds
IC50 valuea) (µM, mean ± SD)
148.7 ± 2.1
1
5
6
53.8 ± 0.4
21.2 ± 1.8
a) IC50 values were evaluated in triplicate experiments and triplicate in one assay.
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