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Rapid Isolation and Purification of Phorbolesters from Jatropha curcas by High-speed Counter-current Chromatography Wan Hua, Huiling Hu, Fang Chen, Lin Tang, Tong Peng, and Zhanguo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505655b • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Title: Rapid Isolation and Purification of Phorbolesters from Jatropha curcas by
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High-speed Counter-current Chromatography
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Author: Wan Hua†, Huiling Hu‡, Fang Chen*†, Lin Tang†, Tong Peng†, Zhanguo Wang *†
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National and Local Joint Engineering Laboratory for Energy Plant Bio-Oil Production and
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Application, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education,
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College of Life Science, Sichuan University, No. 24 South Section 1, First Ring Road, Chengdu
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610064, People’s Republic of China
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Pharmacy College, Chengdu University of Traditional Chinese Medicine, No.1166, Liutai road,
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Chengdu 611137, People’s Republic of China
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*
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E-mail address: [email protected] (Fang Chen); [email protected] (Zhanguo
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Wang).
13
†
Sichuan University
14
‡
Chengdu University of Traditional Chinese Medicine
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Corresponding Author:
Corresponding author. Tel.: +86 28 85416957, Fax: +86 28 85416957.
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Fang Chen, College of Life Sciences, Sichuan University, No.24 South Section 1, First Ring
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Road, Chengdu, 610064,P. R. China. Tel.: +86 28 85416957, Fax: +86 28 85416957, E-mail:
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[email protected]；Zhanguo Wang, College of Life Sciences, Sichuan University, No.24
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South Section 1, First Ring Road, Chengdu, 610064,P. R. China. Tel.: +86 28 85416957, Fax:
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+86 28 85416957, E-mail: [email protected]
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First Author: Wan Hua.
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Order of Authors: Wan Hua; Huiling Hu; Fang Chen; Ling Tang; Tong Peng; Zhanguo Wang.
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ABSTRACT: In this work, a high-speed counter-current chromatography (HSCCC) method was
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established for the preparative of Phorbolesters (PEs) from
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n-hexane—ethyl acetate—methanol—water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum
26
two-phase solvent system to separate and purify Jatropha factors C1 (JC1) with purity of 85.2 %
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determined by HPLC, and obtain a mixed compound containing 4-5 PE's. Subsequently, a
28
continuous semi-preparative HPLC was applied to further purify JC1 (99.8 %) as determined by
29
HPLC. In addition, a rapid and development UPLC-PDA and UPLC-MS were established and
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successfully used to evaluate the isolated JC1 and PEs-rich crude extract. The purity of JC1 was
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only 87.8 % by UPLC-UV. A peak (high similar compound to JC1) was indentified as the isomer of
32
JC1 by comparison the characteristic of UV absorption and MS spectrum. Meanwhile, this
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strategy was also applied to analyze the PEs-rich crude extract from J. curcas. It is interesting that
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there may be more than 15 analogues of PEs according to the same quasi-molecular ion peaks,
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high similar sequence-specific fragment ions and similar UV absorption spectrum.
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KEYWORDS: Jatropha curcas L.; Jatropha factor C1; HSCCC; 12-deoxy-16-hydroxyphorbol
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diesters; UPLC-MS.
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Jatropha curcas. The
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INTRODUCTION Jatropha curcas L. has become energy crop for the production of biodiesel 1-3. In addition, it
40 41
is widely investigated as animal feed 4, medicine and traditional medicine
5-10
42
attractively biological activities. However, the primary poisonous components, phorbol esters
43
(PEs) presented in J. curcas seed, prevent these comprehensive utilizations especially in the field
44
of the animal feed, medicine and traditional medicine 11.
for its highly
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PEs are diesters containing the same diterpene structure, 12-deoxy-16-hydroxyphorbol
46
(Figure 1), which are earliest known for its tumor promotion activity12. Six main PE components
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(Jatropha factors C1–C6, JC1-6) with the same molecular formula C44H54O8Na (ESI
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been isolated from J. curcas seed and designated as Jatropha factors C1, C2, C3, epimers C4 and
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C5, and C6 12-15.
+Na
) have
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Over a period of long time, PEs are considered to be merely toxic in the Jatropha biodiesel
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production chain. A large number of literatures have shown that J. curcas seed exhibits toxicity in
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a broad range of species, from microorganisms to higher animals by force-feeding seed oil, raw or
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defatted seed meals, and a variety of solvent extracts, which directly or indirectly related to PEs 11.
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For
55
phorbol-12-myristate-13-acetate, TPA) body weight in mice couple with acute injury mainly in liver,
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kidney, intestine and heart
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presence of less viable cell layers, partial tissue disintegration in epithelium and inflammatory
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response
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significant tumor promotion activity
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purposes without detoxification, a variety of physical, chemical and biological detoxification
the
purified
17
PEs,
which
16
have
an
LD50
of
28
mg/kg
(equivalent
to
; severe dermal and occular toxicity produced marked oedema,
; apoptosis-mediated inhibition in proliferation of Chang and Vero cell lines 12
18
;
. Due to Jatropha by-product cannot be used for edible
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methods have been investigated to detoxify the PEs such as solid-state fermentation19, enzymatic
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degradation20, heat treatment, adsorbing treatment21, light exposure
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micro-biological degradation24, 25.
64
22
, solvent extraction23,
As a matter of fact, apart from the toxicity, PEs demonstrate a myriad of biological activities in 14, 26
.
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pest-control, including against schistosomiasis, antimolluscicidal and antimicrobial activities
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Meanwhile, recently published papers have shown its potential anti cancer activities in triggering
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apoptosis, activating PKC-delta, caspase-3 proteins and down-regulating the proto-oncogenes in
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MCF-7 and HeLa cancer cell lines
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starting synthesize material of the most promising anti-AIDs drug prostratin 28, 29.
70
27
. It is particularly meaningful that PEs could be an important
Similar to the discovery in a recently published paper
14
, our previous published paper has
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realized that the total content level of PEs (equivalent to TPA) using the internal standard method
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at 280 nm is 20 times over the one using the external standard method
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absorbance of TPA is at 242 nm
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(equivalent to TPA), which is actually below 5%
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the high purity of PEs for revaluating its toxicity and bio-activity.
14, 15
15
, due to the λmax of UV
. Hence, the purity of PEs is generally regarded as 100% 16
. In this regard, it is very necessary to acquire
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At present, continuous liquid-liquid extraction, silica-gel column chromatography and
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semi-preparative HPLC are the conventional strategy of separation and purification PEs from J.
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curcas seed
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appropriately utilization of PEs (by-product of J. curcas seed) could be contributed to enhance the
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economic viability and sustainability of the J. curcas production chain. It is necessary to establish
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an effective and rapid method for separating the PEs with high recovery rate. In this work, PEs
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were successfully isolated and purified from J. curcas seed by HSCCC. In addition, combined
13-15, 17, 26
. Considering the rapid growth in Jatropha biodiesel industry, the official
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HSCCC and semi-HPLC method was established to purify the JC1. Furthermore, UPLC-PDA and
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UPLC-MS were also developed and established to analyze characteristics of the isolated JC1 and
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the PEs-rich crude extracts.
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MATERIALS AND METHODS
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Materials. Methanol and acetonitrile (HPLC grade) were purchased from Swell Scientific
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Instruments Co., Ltd (Chengdu, China).Formic acid (HPLC grade) was purchased from the
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Chengdu Kelong Reagent Co., Ltd (Chengdu, China). Water (HPLC grade) was prepared using
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an ultrapure water system (UPA, Chongqing, China). All of the other reagents used in the present
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study were of analytical grade. TPA (99%, CAS Registry No. 16561-29-8) was obtained from
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Sigma-Aldrich (Shanghai, China). J. curcas seed collected from Xichang (Sichuan, China) in 2013
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was identified by professor Tang Lin (Sichuan University).
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PEs-rich Crude Extract Preparations. Fine powder of dried J. curcas seed (3.10 kg) was
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extracted by ethyl acetate (20 L) at room temperature for two times (each for 24 h). All the filtrates
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were combined and concentrated under reduced pressure by a rotary evaporator at 45 oC to gain
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oily substance (985.2 g). 200 g of oily substance was dissolved with methanol (1000 mL),
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subsequently, the mixed solution was frozen at 4 oC for 24 h to produce the upper clarification
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methanol layer (PEs-rich solution) and precipitate (168.3 g). The upper methanol layer was
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concentrated under reduced pressure by a rotary evaporator at 55 oC to obtain PEs-rich crude
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extract (23.8 g).
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HSCCC Separation Apparatus. A TBE-300C (HSCCC, Shanghai Tauto Biotech Co., Ltd,
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Shanghai, China) is coupled with a set of three multilayer coils and a 20 mL sample loop. The
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solvents were delivered by a TBP 5002 (Shanghai Tauto Biotech Co., Ltd, Shanghai, China) pump.
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A DC-0506 constant-temperature circulating implement (Shanghai Sunny Hengping scientific
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instrument Co., Ltd, Shanghai, China) was used to control the separation temperature. The
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UV-absorbance of eluent was monitored by a UV-2000 detector (Shanghai sanotac scientific
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instrument Co., Ltd, Shanghai, China) at the wavelength of 280 nm. The data was collected by an
128
Easy Chrom 1000 workstation (Beijing Qingbohua Ltd., Beijing, China). The temperature of
129
separation column was maintained at 25 oC. A FB-36/7 air compressor pump was used to force
130
out the stationary phase.
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Measurement of partition coefficient. Similar to previous studies
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solvent systems are evaluated with partition coefficient (K) and separation factor (α) by HPLC
133
methods. The partition coefficient (K) is defined as Aupper/Alower of each analyte, in which, Aupper
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and Alower are the HPLC peak areas of objective compounds in the upper and lower phases,
135
respectively. The separation factor (α) is defined as the proportion between the two components
136
partition coefficients (α= K1/K2, K1 > K2). The HPLC analysis was performed on an Agilent 1200
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series system with a C18 column (4.6 × 250 mm, 5.0 µm) at 30 °C. The mobile phase was
138
acetonitrile-water (80:20, v/v) at the flow rate of 0.8 mL/min. The detection wavelength was set at
139
280
140
acetate–methanol–water=1:1:1:1, 3:1:3:1, 2:2:3:1, 3:3:3:1, 1.5:1.5:1.2:0.5, v/v) were screened
141
as follows: approximately 0.1 mL primary crude extract sample was placed in a 10 ml test tube,
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which was added 2 ml of each phase of the equilibrated two-phase solvent system; the tube was
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rigorously shaken for 1 min and left to stand at room temperature until equilibrium was obtained;
144
then 20 µL of the upper and lower phases were analyzed by HPLC at 280 nm, respectively.
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Preparation of Two-phase Solvent System and Sample Solution.
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The two-phase solvent system was performed by adding each solvent to a separatory funnel.
147
After shaking and thoroughly equilibrating tat room temperature, the upper phase and lower
148
phase were then separated and degassed by ultrasonic for 30 min. The upper phase was used as
nm.
Briefly,
a
series
of
two-phase
solvent
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30-34
, the optimal two-phase
systems
(n-hexane–ethyl
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stationary phase while the lower phase as mobile phase. The sample solution for HSCCC
150
separation was prepared by dissolving 2 g of PEs-rich crude extract in 20 mL of the lower phase.
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HSCCC Separation Procedure. The multilayer coil column was first entirely filled with the
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stationary phase at the flow rate of 30.0 mL/min. The apparatus was rotated at 800 rpm and then
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the mobile phase was pumped into the column at the flow rate of 10 mL/min in the head to tail
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elution mode. The mobile phase eluting at the tail outlet indicated hydrodynamic equilibrium has
155
been reached. Subsequently, 20 mL of sample solution was injected into the column. The peak
156
fractions were collected manually according to the elution profile and evaporated under reduced
157
pressure.
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Analysis and characterisation of HSCCC fractions. PEs-rich crude extract sample and each
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peak fraction obtained from HSCCC were analyzed by HPLC-UV using peak area calculation.
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The HPLC analyses were performed on an Agilent 1200 series system (Agilent Technologies)
161
equipped with a quaternary pump, a vacuum degasser, an auto injector, and an ultraviolet
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detector. The HPLC separation was performed on a C18 column (Ultimate, 4.6 × 250 mm, 4.7 µm,
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Welch Materials, Inc.) at 30 °C. The ultraviolet detector was set at 280 nm according to the λmax of
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UV absorbance of PEs. All of the samples were eluted at 0.80 mL/min using an isocratic elution of
165
acetonitrile and water containing 0.2 % formic acid (80/20, v/v). Agilent Chemstation version
166
B.04.01 was used for the system control and data acquisition.
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Purification and Identification of JC1. A continuous repeated semi-preparative HPLC method
168
was applied to further purify the JC1. The separation of JC1 was performed on a reversed-phase
169
C8 semi-preparative column (Welchrom-C8, 10 × 250 mm, 5 µm) with an isocratic elution of
170
acetonitrile-water (80:20, v/v) at a flow rate of 2.5 mL/min. The monitoring of the wavelength was
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set at 280 nm. The purity of JC1 was identified by HPLC. MS spectra of JC1 was measured in the
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positive mode ([M + Na] +) using a microTOF-QII mass spectrometer (Bruker Daltonics,
173
Germany).
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UPLC-PDA and UPLC-MS Analysis of Isolated JC1 and PEs-rich Crude Extract. UPLC-PDA
175
analyses were performed on an ACQUITY UPLC H-Class System (Waters, USA) equipped with
176
quaternary solvent manager QSM, auto sampler manager FTN and Photodiode Array eLambda
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detector. The chromatographic separation was performed on an Acquity UPLC BEH C18 column
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(1.7 µm, 150 mm × 2.1 mm, Waters, USA) at 40 °C. All of the samples were eluted at 0.40 mL/min
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using an isocratic elution of solvent A and B (80/20, v/v), in which, solvent A was composed of
180
acetonitrile-water (90/10, v/v) containing 0.1 % formic acid and 0.05 % ammonium formate,
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solvent B was the water containing 0.1 % formic acid and 0.05 % ammonium formate. The sample
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injection volume was 2 µL. The detection range of ultraviolet detector was set from 200 to 400 nm.
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Monitoring of the wavelength was set at 280 nm according to the λmax of UV absorbance of PEs.
184
Empower software version 3.0 was used for the system control and data acquisition.
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UPLC-MS analyses were performed on an ACQUITY UPLC I-Class-Xevo TQD System
186
(Waters, USA) interfaced with a Waters Quattro Premier XE triple quadrupole mass spectrometer
187
and equipped with electrospray ionization operated in the positive ionization mode. The
188
chromatographic separation condition of the UPLC-MS analyses was the same as the UPLC-PDA
189
analyses. The source dependent parameters maintained for the analytes were: cone gas flow,
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50 L/h; desolvation gas flow, 500 L/h; capillary voltage, 3.0 kV, source temperature, 120 °C;
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desolvation temperature, 500 °C. The optimum values for compound dependent parameters of
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cone voltage and collision energy were set at 30 V and 10 eV. Detection of the ions was
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performed in the multiple-reaction monitoring (MRM) mode, by monitoring the transition pairs
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(precursor to product ion) of m/z for PEs. Mass Lynx software version 4.1 was used to control all
195
parameters of UPLC and MS.
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RESULTS AND DISCUSSION
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Selection of the Two-phase Solvent System. Five main peaks (regarded as Jatropha Factors
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C1-5 according to their retention time appeared orderly in chromatograms, JC1-5) determined by
218
HPLC were used to select the appropriate two-phase solvent system. The results of the partition
219
coefficients (K) and separation factors (α) of these compounds in various two-solvent systems are
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listed in Table 1. According to the published papers
221
system requires the following considerations: (1) the equilibrating time should be considerably
222
short enough (< 30 s); (2) the partition coefficient (K) should be in a suitable range, usually
223
between 0.2 and 5; (3) the separation factor(α) should be greater than 1.5. After repeated attempt,
224
the
225
(1.5:1.5:1.2:0.5, v/v) was considered as an optimal proposal for its appropriate partition
226
coefficients (K) and separation factors (α).
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Isolation of PEs Fractions by HSCCC. The retention of the stationary phase was 56.7%.
228
Fractions Peak A and B were separated in 30 min with a total mobile elution volume of 300 mL
229
(Figure 2. A). Isolated fractions Peak A and B were analyzed by HPLC (Figure 2. C. c2 and c3).
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In the HSCCC separation, the expected result is five fractions (Figure 2. C. c4). It should be
231
noted, the main compound JC1 from PEs can be isolated fast, and the purity increased from
232
48.5% to 85.0 % (Figure 2. C. c3 and c4). Compared with the literatures
233
isolation methods of PEs including liquid-liquid extraction and silica-gel column chromatography
234
are time consuming, require a large amounts of organic solvents, and typically require numerous
235
steps resulting lower recovery and higher cost. In this work, the separation of PEs by HSCCC
236
shows an excellent sample recovery, low cost and high efficiency.
two-phase
solvent
system
composed
of
30-34
, the appropriate two-phase solvent
n-hexane/ethyl
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acetate/methanol/water
12-15
, the conventional
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Purification and Identification of JC1. The semi-preparative HPLC chromatogram of Peak A
238
was shown in Figure 2. B. b1. Subsequently, JC1 was further purified by using a continuous
239
semi-preparative HPLC through repeating sample injection at an interval of 5 minutes (Figure 2.
240
B. b2). The purity of JC1 measured by HPLC was 99.8 % (Figure 2. C. c1). HR-MS spectra of
241
JC1 showed in Figure 3. C, and the MS of molecular ion peak was 733.3695 ([M + Na] +).
242
According to the published papers during over 20 years, the JC1 is proved to be the one of the
243
highest quantity levels of PEs in J. Curcas seed 12-15, 17, 26. Therefore, it was obviously proofed that
244
the isolated compound was the JC1 in this work.
245
UPLC-PDA and UPLC-MS Analysis of Isolated JC1 and PEs-rich Crude Extract. It was
246
regrettable that the purity of JC1 was only 87.8% (Figure 3. A) by UPLC using the peak area
247
calculation method. It was easily to observe that there is a Peak S with the high similarity UV
248
absorption spectrogram to JC1 (Figure 3. A. a1 and a2). As we known, the purity of JC1 was
249
usually measured by conventional HPLC using the peak area calculation method in previous
250
published papers 13-15. The result of UPLC indicated that there was a Peak S (Figure 3. A), which
251
was difficultly separated with the JC1 using the conventional HPLC (Figure 2. C. c1).
252
Subsequently, A UPLC-MS was established and successfully used to identify the characteristics
253
of Peak S. Figure 3. B showed the TIC chromatogram of JC1. In the positive-ion mass spectra
254
intense, Peak S showed the same quasi-molecular ion peak ([M+H-H2O] +) at about 693.00 and
255
several similar sequence-specific fragment ions (at about 365.00, 311.00 and 292.90, m/z) as JC1
256
(Figure
257
(12-deoxy-16-hydroxyphorbol, Figure 1) in JC1 is 364.4327. In addition, the fragment ion at about
258
365.00 (m/z) was the mother nucleus ion peak ([M] +), moreover, the 311.00 (m/z) and 292.90
3.
D
and
E).
Because
the
molecular
mass
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mother
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+
and [M-4H20] + of mother nucleus, respectively. Thus, it
259
(m/z) were the fragment ions [M-3H20]
260
was easily deduced that Peak S was the isomer of JC1.
261
Furthermore, the sample of JC1 showed only one peak with HPLC but contained another
262
peak (Peak S) by UPLC, this result indicated it was doubtful the general five to seven PEs peaks
263
presented in J. curcas by conventional HPLC method 14-17, 35.Thus, it was imperative to establish a
264
high resolution method to confirm this doubt. In this work, an UPLC-PDA method was developed
265
and applied to analyze the PEs-rich crude extract. In our recently published paper, instead of the
266
conventional C18 column (4.5 µm), when using a high resolution C18 column (2.7 µm), the
267
HPLC-UV chromatogram at 280 nm of PEs-rich crude extract has shown more than 9 peaks
268
involving five main peaks 15. The content levels of PEs in J. curcas seed or their related products
269
are always calculated by the total amount equivalent to TPA using an internal standard method11,
270
16, 35
271
the PEs-rich crude extract. The choose UPLC-UV chromatograms at 240, 280 and 304nm of the
272
PEs-rich crude extract and TPA had been shown in Figure 4. A, B and C, respectively. It was
273
interesting that more than 17 peaks were separated out at 280nm (Figure 4.B). However, similar
274
to the reports
275
(Figure 2.C.c4). It is certified that the λmax of PEs always at about 280 nm, and the λmax of TPA at
276
about 242 nm
277
the λmax of more than 12 peaks were at about 280 nm, including peaks 1-8, 10-12 and 15, which
278
all similar to Peak 8 (JC1, Figure 4. B. a), at the same time, the secondary λmax of them were at
279
about 232 nm similar to TPA (Figure 4. B. c). It was easily deduced that there could exist more
280
than 12 homologous compounds of PEs. However, about 4 peaks (λmax at about 304 nm)
. In this study, TPA was selected as internal standard to establish the UPLC-PDA method for
14-17
, about 5-7 peaks were exhibited in the HPLC-UV chromatogram at 280 nm
14, 15
. Obviously, the results of the UV absorption spectrogram also demonstrated
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involving peaks 13, 14, 16 and 17 (Figure 4. B. b) were significantly different from the JC1. It was
282
questionable that whether these 4 peaks (λmax at about 304 nm) are the homologous of PEs.
283
UPLC-MS was used to further certify whether those 4 peaks belong to homologous of PEs.
284
The TIC chromatogram of PEs-rich crude extract was shown in Supporting Material Figure S1.
285
In the positive-ion mass spectra intense, more than 15 compounds showed the same
286
quasi-molecular ion peaks at about 693.00 ([M+H-H2O]+) in corresponding mass spectra
287
(Supporting Material Figure S1), and several similar sequence-specific fragment ions (at about
288
365.00, 311.00 and 292.90), which were all similar to JC1 (Figure 3. E). This indicates that these
289
15 compounds all contained the same mother nucleus. Therefore these results indicate that the
290
more than 15 compounds found in the UPLC-MS are all homologues of PEs.
291
In this work, a rapid and efficient HSCCC method was developed and successfully applied to
292
separate the PEs from J.Curcas seed. In addition, combining the HSCCC and semi-preparative
293
HPLC was developed and successfully applied to purify the JC1. Meanwhile, the PEs-rich crude
294
extract from J. curcas was also analyzed by UPLC-PDA and UPLC-MS, the results indicated
295
there may be more than15 analogues of PEs in J. curcas. This work will enhance the economic
296
viability and sustainability of the J. curcas production chain.
297
ACKNOWLEDGEMENTS
298
We kindly thank Xiaoyu Zhang (Ph.D., College of Life Science, Sichuan Normal University) and
299
Xueran Mei (M.S., College of Life Science, Sichuan Normal University) provided the help of the
300
HSCCC analysis. This work was supported by the National Natural Science Foundation of China
301
(Grant 31170312).
302
NOTES:
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The authors declare no competing financial interest.
304
ABBREVIATIONS USED
305
PEs, phorbolesters; TPA, phorbol 12-myristate-13-acetate; JC1-6, Jatropha factors C1-6; K,
306
partition coefficient; α, separation factor.
307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342
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Sudhakar Johnson, T.; Eswaran, N.; Sujatha, M., Molecular approaches to improvement of
Jatropha curcas Linn. as a sustainable energy crop. Plant Cell Rep. 2011, 30, (9), 1573-91. 2.
Abdulla, R.; Chan, E. S.; Ravindra, P., Biodiesel production from Jatropha curcas: a critical review.
Crit Rev Biotechnol. 2011, 31, (1), 53-64. 3.
Juan, J. C.; Kartika, D. A.; Wu, T. Y.; Hin, T. Y., Biodiesel production from Jatropha oil by catalytic
and non-catalytic approaches: an overview. Bioresour Technol. 2011, 102, (2), 452-60. 4.
King, A. J.; He, W.; Cuevas, J. A.; Freudenberger, M.; Ramiaramanana, D.; Graham, I. A.,
Potential of Jatropha curcas as a source of renewable oil and animal feed. J Exp Bot. 2009, 60, (10), 2897-905. 5.
Sharma, S. K.; Singh, H., A review on pharmacological significance of genus Jatropha
(Euphorbiaceae). Chin J Integr Med. 2012, 18, (11), 868-80. 6.
Devappa, R. K.; Makkar, H. P.; Becker, K., Nutritional, biochemical, and pharmaceutical potential
of proteins and peptides from jatropha: review. J Agric Food Chem. 2010, 58, (11), 6543-55. 7.
Thomas, R.; Sah, N. K.; Sharma, P. B., Therapeutic biology of Jatropha curcas: a mini review.
Curr Pharm Biotechnol. 2008, 9, (4), 315-24. 8.
Debnath, M.; Bisen, P. S., Jatropha curcas L., a multipurpose stress resistant plant with a potential
for ethnomedicine and renewable energy. Curr Pharm Biotechnol. 2008, 9, (4), 288-306. 9.
Paulillo, L. C.; Mo, C.; Isaacson, J.; Lessa, L.; Lopes, E.; Romero-Suarez, S.; Brotto, L.; Abreu, E.;
Gutheil, W.; Brotto, M., Jatropha curcas: from biodiesel generation to medicinal applications. Recent Pat Biotechnol. 2012, 6, (3), 192-9. 10. Sabandar, C. W.; Ahmat, N.; Jaafar, F. M.; Sahidin, I., Medicinal property, phytochemistry and pharmacology of several Jatropha species (Euphorbiaceae): a review. Phytochemistry. 2013, 85, 7-29. 11. Devappa, R. K.; Makkar, H. P.; Becker, K., Jatropha toxicity--a review. J Toxicol Environ Health B Crit Rev. 2010, 13, (6), 476-507. 12. Hirota, M.; Suttajit, M.; Suguri, H.; Endo, Y.; Shudo, K.; Wongchai, V.; Hecker, E.; Fujiki, H., A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol. Cancer Res. 1988, 48, (20), 5800-4. 13. Haas, W.; Sterk, H.; Mittelbach, M., Novel 12-deoxy-16-hydroxyphorbol diesters isolated from the seed oil of Jatropha curcas. J Nat Prod. 2002, 65, (10), 1434-40. 14. Roach, J. S.; Devappa, R. K.; Makkar, H. P.; Becker, K., Isolation, stability and bioactivity of Jatropha curcas phorbol esters. Fitoterapia. 2012, 83, (3), 586-92. 15. Wang, Z.; Tang, L.; Hu, H.; Guo, Y.; Peng, T.; Yan, F.; Chen, F., Metabolic profiling assisted quality control of phorbolesters in Jatropha curcas seed by high-performance liquid chromatography using a fused-core column. J Agric Food Chem. 2012, 60, (38), 9567-72.
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16. Li, C. Y.; Devappa, R. K.; Liu, J. X.; Lv, J. M.; Makkar, H. P.; Becker, K., Toxicity of Jatropha curcas phorbol esters in mice. Food Chem Toxicol. 2010, 48, (2), 620-5. 17. Devappa, R. K.; Roach, J. S.; Makkar, H. P.; Becker, K., Occular and dermal toxicity of Jatropha curcas phorbol esters. Ecotoxicol Environ Saf. 2013, 94, 172-8. 18. Oskoueian, E.; Abdullah, N.; Ahmad, S., Phorbol esters isolated from jatropha meal induced apoptosis-mediated inhibition in proliferation of chang and vero cell lines. Int J Mol Sci. 2012, 13, (11), 13816-29. 19. Veerabhadrappa, M. B.; Shivakumar, S. B.; Devappa, S., Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98. J Biosci Bioeng. 2014,117(2),208-14. 20. Hidayat, C.; Hastuti, P.; Wardhani, A. K.; Nadia, L. S., Method of phorbol ester degradation in Jatropha curcas L. seed cake using rice bran lipase. J Biosci Bioeng. 2014,117(3),372-4. 21. Sadubthummarak, U.; Parkpian, P.; Ruchirawat, M.; Kongchum, M.; Delaune, R. D., Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product. J Environ Sci Health B. 2013, 48, (11), 974-82. 22. Yunping, B.; Ha, B. T.; Eunice, Y.; Chueng, L. L.; Yan, H., Light induced degradation of phorbol esters. Ecotoxicol Environ Saf. 2012, 84, 268-73. 23. Kumar, V.; Makkar, H. P.; Amselgruber, W.; Becker, K., Physiological, haematological and histopathological responses in common carp (Cyprinus carpio L.) fingerlings fed with differently detoxified Jatropha curcas kernel meal. Food Chem Toxicol. 2010, 48, (8-9), 2063-72. 24. Wang, X. H.; Ou, L.; Fu, L. L.; Zheng, S.; Lou, J. D.; Gomes-Laranjo, J.; Li, J.; Zhang, C., Detoxification of Jatropha curcas kernel cake by a novel Streptomyces fimicarius strain. J Hazard Mater. 2013, 260, 238-46. 25. Phengnuam, T.; Suntornsuk, W., Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation. J Biosci Bioeng. 2013, 115, (2), 168-72. 26. Devappa, R. K.; Rajesh, S. K.; Kumar, V.; Makkar, H. P.; Becker, K., Activities of Jatropha curcas phorbol esters in various bioassays. Ecotoxicol Environ Saf. 2012, 78, 57-62. 27. Oskoueian, E.; Abdullah, N.; Ahmad, S., Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-delta, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines. Molecules. 2012, 17, (9), 10816-30. 28. Wender, P. A.; Kee, J. M.; Warrington, J. M., Practical synthesis of prostratin, DPP, and their analogs, adjuvant leads against latent HIV. Science. 2008, 320, (5876), 649-52. 29. Devappa, R. K.; Malakar, C. C.; Makkar, H. P.; Becker, K., Pharmaceutical potential of phorbol esters from Jatropha curcas oil. Nat Prod Res. 2013, 27, (16), 1459-62. 30. Liang, J.; Ito, Y.; Zhang, X.; He, J.; Sun, W., Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave-assisted extraction coupled with high-speed counter-current chromatography. J Sep Sci. 2013, 36, (24), 3934-40. 31. Dai, X.; Huang, Q.; Zhou, B.; Gong, Z.; Liu, Z.; Shi, S., Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment. Food Chem. 2013, 139, (1-4), 563-70. 32. Regalado, E. L.; Tolle, S.; Pino, J. A.; Winterhalter, P.; Menendez, R.; Morales, A. R.; Rodriguez, J. L., Isolation and identification of phenolic compounds from rum aged in oak barrels by high-speed countercurrent
chromatography/high-performance
liquid
chromatography-diode
array
detection-electrospray ionization mass spectrometry and screening for antioxidant activity. J
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387 388 389 390 391 392 393 394 395 396 397
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Chromatogr A. 2011, 1218, (41), 7358-64. 33. Lopes-Lutz, D.; Mudge, E.; Ippolito, R.; Brown, P.; Schieber, A., Purification of alkylamides from Echinacea angustifolia (DC.) Hell. Roots by high-speed countercurrent chromatography. J Agric Food Chem. 2011, 59, (2), 491-4. 34. Degenhardt, A.; Engelhardt, U. H.; Lakenbrink, C.; Winterhalter, P., Preparative separation of polyphenols from tea by high-speed countercurrent chromatography. J Agric Food Chem. 2000, 48, (8), 3425-30. 35. Rakshit, K. D.; Darukeshwara, J.; Rathina Raj, K.; Narasimhamurthy, K.; Saibaba, P.; Bhagya, S., Toxicity studies of detoxified Jatropha meal (Jatropha curcas) in rats. Food Chem Toxicol. 2008, 46, (12), 3621-5.
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398
Table legend
399
Table 1.
400
Partition coefficients (K) and separation factors (α) of compounds in various solvent
401
systems. Partition coefficients （KU/L)
Separation factors (a)
Ratio of solvent system (n-hexane–ethyl acetate–methanol–water)
JC1
JC2
JC3
JC4
JC5
K2/K1
K3/K2
K4/K3
K5/K4
1:1:1:1
4.19
25.22
27.21
16.72
46.33
6.02
1.07
1.63
2.77
3:1:3:1
0.07
0.09
0.11
0.12
0.13
1.32
1.18
1.16
1.08
2:2:3:1
0.27
0.29
0.34
0.38
0.34
1.09
1.18
1.11
1.12
3:3:3:1
0.61
0.63
0.79
0.88
0.81
1.02
1.25
1.12
1.09
3:3:2:1
1.93
2.27
2.75
3.05
2.82
1.17
1.21
1.11
1.08
1.5:1.5:1.2:0.5
1.03
1.43
1.80
2.84
1.55
1.40
1.26
1.58
1.83
402
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Figures legends
404
Figure 1.
405
The structure of 12-deoxy-16-hydroxyphorbol.
406
Figure 2.
407
(A) Preparative HSCCC chromatogram of PEs-rich crude extract from J. curcas seed;
408
(B) Primary semi-preparative HPLC-UV chromatogram of isolated fraction Peak A by HSCCC
409
(b1), continuous semi-preparative HPLC-UV chromatogram of Jatropha factors C1 (JC1) using
410
repeating sample injection at an interval of 5 minutes (the English alphabet a-p (end-label of JC1)
411
represents the continuous repeating sample injection order, b2);
412
(C) HPLC-UV chromatograms of the Jatropha Factors C1 (c1), Peak B fraction isolated by
413
HSCCC (c2), Peak A fraction isolated by HSCCC (c3) and PEs-rich crude extract (c4).
414
Figure 3.
415
(A) UPLC-UV chromatogram of Jatropha factors C1 and Peak S at 280 nm, the UV absorbance
416
spectrogram of Peak S (a1) and Jatropha factors C1 (a2);
417
(B) TIC chromatogram of the Jatropha factors C1 and Peak S;
418
(C) MS spectrum of the Jatropha factors C1 and Peak S;
419
(D) Product ion MS spectrum of Peak S;
420
(E) Product ion MS spectrum of Jatropha factors C1.
421
Figure 4.
422
(A) UPLC-UV chromatograms of PEs-rich extract and TPA at 240 nm;
423
(B) UPLC-UV chromatograms of PEs-rich extract and TPA at 280 nm, UV absorbance
424
spectrogram of Peak 8 (a), UV absorbance spectrogram of Peak 17 (b), UV absorbance
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spectrogram of Peak 18 (c);
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(C) UPLC-UV chromatograms of PEs-rich extract and TPA at 304 nm.
427
TPA is phorbol-12-myristate-13-acetate,
428
Crude extract represents the main peaks detected from the PEs-rich crude extracts
429
1-18 represent the detected peak in the UPLC in their exist order;
430
Supporting Material Figure S1 Legend
431
(A-O) Product ion MS spectrum of the Peaks 3-17;
432
(P) UPLC–MS chromatogram in total ion current (TIC) of PEs-rich crude extract.
433
3-17 represent the detected peak in the UPLC-MS in their exist order.
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Figure 1. The molecule structure of 12-deoxy-16-hydroxyphorbol. 120x77mm (200 x 200 DPI)
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Figure 2. (A) Preparative HSCCC chromatogram of PEs-rich crude extract from J. curcas seed; (B) Primary semi-preparative HPLC-UV chromatogram of isolated fraction Peak A by HSCCC (b1), continuous semi-preparative HPLC-UV chromatogram of Jatropha factors C1 (JC1) using repeating sample injection at an interval of 5 minutes (the English alphabet a-p (end-label of JC1) represents the continuous repeating sample injection order, b2); (C) HPLC-UV chromatograms of the Jatropha Factors C1 (c1), Peak B fraction isolated by HSCCC (c2), Peak A fraction isolated by HSCCC (c3) and PEs-rich crude extract (c4). 113x156mm (300 x 300 DPI)
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Figure 3. (A) UPLC-UV chromatogram of Jatropha factors C1 and Peak S at 280 nm, the UV absorbance spectrogram of Peak S (a1) and Jatropha factors C1 (a2); (B) TIC chromatogram of the Jatropha factors C1 and Peak S; (C) MS spectrum of the Jatropha factors C1 and Peak S; (D) Product ion MS spectrum of Peak S; (E) Product ion MS spectrum of Jatropha factors C1. 160x139mm (300 x 300 DPI)
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Figure 4. (A) UPLC-UV chromatograms of PEs-rich extract and TPA at 240 nm; (B) UPLC-UV chromatograms of PEs-rich extract and TPA at 280 nm, UV absorbance spectrogram of Peak 8 (a), UV absorbance spectrogram of Peak 17 (b), UV absorbance spectrogram of Peak 18 (c); (C) UPLC-UV chromatograms of PEs-rich extract and TPA at 304 nm. TPA is phorbol-12-myristate-13-acetate, Crude extract represents the main peaks detected from the PEs-rich crude extracts 1-18 represent the detected peak in the UPLC in their exist order. 160x111mm (150 x 150 DPI)
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Article
Rapid Isolation and Purification of Phorbolesters from Jatropha curcas by High-speed Counter-current Chromatography Wan Hua, Huiling Hu, Fang Chen, Lin Tang, Tong Peng, and Zhanguo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505655b • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Title: Rapid Isolation and Purification of Phorbolesters from Jatropha curcas by
2
High-speed Counter-current Chromatography
3
Author: Wan Hua†, Huiling Hu‡, Fang Chen*†, Lin Tang†, Tong Peng†, Zhanguo Wang *†
4
National and Local Joint Engineering Laboratory for Energy Plant Bio-Oil Production and
5
Application, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education,
6
College of Life Science, Sichuan University, No. 24 South Section 1, First Ring Road, Chengdu
7
610064, People’s Republic of China
8
Pharmacy College, Chengdu University of Traditional Chinese Medicine, No.1166, Liutai road,
9
Chengdu 611137, People’s Republic of China
10
*
11
E-mail address: [email protected] (Fang Chen); [email protected] (Zhanguo
12
Wang).
13
†
Sichuan University
14
‡
Chengdu University of Traditional Chinese Medicine
15
Corresponding Author:
Corresponding author. Tel.: +86 28 85416957, Fax: +86 28 85416957.
16
Fang Chen, College of Life Sciences, Sichuan University, No.24 South Section 1, First Ring
17
Road, Chengdu, 610064,P. R. China. Tel.: +86 28 85416957, Fax: +86 28 85416957, E-mail:
18
[email protected]；Zhanguo Wang, College of Life Sciences, Sichuan University, No.24
19
South Section 1, First Ring Road, Chengdu, 610064,P. R. China. Tel.: +86 28 85416957, Fax:
20
+86 28 85416957, E-mail: [email protected]
21
First Author: Wan Hua.
22
Order of Authors: Wan Hua; Huiling Hu; Fang Chen; Ling Tang; Tong Peng; Zhanguo Wang.
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ABSTRACT: In this work, a high-speed counter-current chromatography (HSCCC) method was
24
established for the preparative of Phorbolesters (PEs) from
25
n-hexane—ethyl acetate—methanol—water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum
26
two-phase solvent system to separate and purify Jatropha factors C1 (JC1) with purity of 85.2 %
27
determined by HPLC, and obtain a mixed compound containing 4-5 PE's. Subsequently, a
28
continuous semi-preparative HPLC was applied to further purify JC1 (99.8 %) as determined by
29
HPLC. In addition, a rapid and development UPLC-PDA and UPLC-MS were established and
30
successfully used to evaluate the isolated JC1 and PEs-rich crude extract. The purity of JC1 was
31
only 87.8 % by UPLC-UV. A peak (high similar compound to JC1) was indentified as the isomer of
32
JC1 by comparison the characteristic of UV absorption and MS spectrum. Meanwhile, this
33
strategy was also applied to analyze the PEs-rich crude extract from J. curcas. It is interesting that
34
there may be more than 15 analogues of PEs according to the same quasi-molecular ion peaks,
35
high similar sequence-specific fragment ions and similar UV absorption spectrum.
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KEYWORDS: Jatropha curcas L.; Jatropha factor C1; HSCCC; 12-deoxy-16-hydroxyphorbol
37
diesters; UPLC-MS.
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Jatropha curcas. The
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INTRODUCTION Jatropha curcas L. has become energy crop for the production of biodiesel 1-3. In addition, it
40 41
is widely investigated as animal feed 4, medicine and traditional medicine
5-10
42
attractively biological activities. However, the primary poisonous components, phorbol esters
43
(PEs) presented in J. curcas seed, prevent these comprehensive utilizations especially in the field
44
of the animal feed, medicine and traditional medicine 11.
for its highly
45
PEs are diesters containing the same diterpene structure, 12-deoxy-16-hydroxyphorbol
46
(Figure 1), which are earliest known for its tumor promotion activity12. Six main PE components
47
(Jatropha factors C1–C6, JC1-6) with the same molecular formula C44H54O8Na (ESI
48
been isolated from J. curcas seed and designated as Jatropha factors C1, C2, C3, epimers C4 and
49
C5, and C6 12-15.
+Na
) have
50
Over a period of long time, PEs are considered to be merely toxic in the Jatropha biodiesel
51
production chain. A large number of literatures have shown that J. curcas seed exhibits toxicity in
52
a broad range of species, from microorganisms to higher animals by force-feeding seed oil, raw or
53
defatted seed meals, and a variety of solvent extracts, which directly or indirectly related to PEs 11.
54
For
55
phorbol-12-myristate-13-acetate, TPA) body weight in mice couple with acute injury mainly in liver,
56
kidney, intestine and heart
57
presence of less viable cell layers, partial tissue disintegration in epithelium and inflammatory
58
response
59
significant tumor promotion activity
60
purposes without detoxification, a variety of physical, chemical and biological detoxification
the
purified
17
PEs,
which
16
have
an
LD50
of
28
mg/kg
(equivalent
to
; severe dermal and occular toxicity produced marked oedema,
; apoptosis-mediated inhibition in proliferation of Chang and Vero cell lines 12
18
;
. Due to Jatropha by-product cannot be used for edible
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methods have been investigated to detoxify the PEs such as solid-state fermentation19, enzymatic
62
degradation20, heat treatment, adsorbing treatment21, light exposure
63
micro-biological degradation24, 25.
64
22
, solvent extraction23,
As a matter of fact, apart from the toxicity, PEs demonstrate a myriad of biological activities in 14, 26
.
65
pest-control, including against schistosomiasis, antimolluscicidal and antimicrobial activities
66
Meanwhile, recently published papers have shown its potential anti cancer activities in triggering
67
apoptosis, activating PKC-delta, caspase-3 proteins and down-regulating the proto-oncogenes in
68
MCF-7 and HeLa cancer cell lines
69
starting synthesize material of the most promising anti-AIDs drug prostratin 28, 29.
70
27
. It is particularly meaningful that PEs could be an important
Similar to the discovery in a recently published paper
14
, our previous published paper has
71
realized that the total content level of PEs (equivalent to TPA) using the internal standard method
72
at 280 nm is 20 times over the one using the external standard method
73
absorbance of TPA is at 242 nm
74
(equivalent to TPA), which is actually below 5%
75
the high purity of PEs for revaluating its toxicity and bio-activity.
14, 15
15
, due to the λmax of UV
. Hence, the purity of PEs is generally regarded as 100% 16
. In this regard, it is very necessary to acquire
76
At present, continuous liquid-liquid extraction, silica-gel column chromatography and
77
semi-preparative HPLC are the conventional strategy of separation and purification PEs from J.
78
curcas seed
79
appropriately utilization of PEs (by-product of J. curcas seed) could be contributed to enhance the
80
economic viability and sustainability of the J. curcas production chain. It is necessary to establish
81
an effective and rapid method for separating the PEs with high recovery rate. In this work, PEs
82
were successfully isolated and purified from J. curcas seed by HSCCC. In addition, combined
13-15, 17, 26
. Considering the rapid growth in Jatropha biodiesel industry, the official
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HSCCC and semi-HPLC method was established to purify the JC1. Furthermore, UPLC-PDA and
84
UPLC-MS were also developed and established to analyze characteristics of the isolated JC1 and
85
the PEs-rich crude extracts.
86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
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MATERIALS AND METHODS
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Materials. Methanol and acetonitrile (HPLC grade) were purchased from Swell Scientific
107
Instruments Co., Ltd (Chengdu, China).Formic acid (HPLC grade) was purchased from the
108
Chengdu Kelong Reagent Co., Ltd (Chengdu, China). Water (HPLC grade) was prepared using
109
an ultrapure water system (UPA, Chongqing, China). All of the other reagents used in the present
110
study were of analytical grade. TPA (99%, CAS Registry No. 16561-29-8) was obtained from
111
Sigma-Aldrich (Shanghai, China). J. curcas seed collected from Xichang (Sichuan, China) in 2013
112
was identified by professor Tang Lin (Sichuan University).
113
PEs-rich Crude Extract Preparations. Fine powder of dried J. curcas seed (3.10 kg) was
114
extracted by ethyl acetate (20 L) at room temperature for two times (each for 24 h). All the filtrates
115
were combined and concentrated under reduced pressure by a rotary evaporator at 45 oC to gain
116
oily substance (985.2 g). 200 g of oily substance was dissolved with methanol (1000 mL),
117
subsequently, the mixed solution was frozen at 4 oC for 24 h to produce the upper clarification
118
methanol layer (PEs-rich solution) and precipitate (168.3 g). The upper methanol layer was
119
concentrated under reduced pressure by a rotary evaporator at 55 oC to obtain PEs-rich crude
120
extract (23.8 g).
121
HSCCC Separation Apparatus. A TBE-300C (HSCCC, Shanghai Tauto Biotech Co., Ltd,
122
Shanghai, China) is coupled with a set of three multilayer coils and a 20 mL sample loop. The
123
solvents were delivered by a TBP 5002 (Shanghai Tauto Biotech Co., Ltd, Shanghai, China) pump.
124
A DC-0506 constant-temperature circulating implement (Shanghai Sunny Hengping scientific
125
instrument Co., Ltd, Shanghai, China) was used to control the separation temperature. The
126
UV-absorbance of eluent was monitored by a UV-2000 detector (Shanghai sanotac scientific
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instrument Co., Ltd, Shanghai, China) at the wavelength of 280 nm. The data was collected by an
128
Easy Chrom 1000 workstation (Beijing Qingbohua Ltd., Beijing, China). The temperature of
129
separation column was maintained at 25 oC. A FB-36/7 air compressor pump was used to force
130
out the stationary phase.
131
Measurement of partition coefficient. Similar to previous studies
132
solvent systems are evaluated with partition coefficient (K) and separation factor (α) by HPLC
133
methods. The partition coefficient (K) is defined as Aupper/Alower of each analyte, in which, Aupper
134
and Alower are the HPLC peak areas of objective compounds in the upper and lower phases,
135
respectively. The separation factor (α) is defined as the proportion between the two components
136
partition coefficients (α= K1/K2, K1 > K2). The HPLC analysis was performed on an Agilent 1200
137
series system with a C18 column (4.6 × 250 mm, 5.0 µm) at 30 °C. The mobile phase was
138
acetonitrile-water (80:20, v/v) at the flow rate of 0.8 mL/min. The detection wavelength was set at
139
280
140
acetate–methanol–water=1:1:1:1, 3:1:3:1, 2:2:3:1, 3:3:3:1, 1.5:1.5:1.2:0.5, v/v) were screened
141
as follows: approximately 0.1 mL primary crude extract sample was placed in a 10 ml test tube,
142
which was added 2 ml of each phase of the equilibrated two-phase solvent system; the tube was
143
rigorously shaken for 1 min and left to stand at room temperature until equilibrium was obtained;
144
then 20 µL of the upper and lower phases were analyzed by HPLC at 280 nm, respectively.
145
Preparation of Two-phase Solvent System and Sample Solution.
146
The two-phase solvent system was performed by adding each solvent to a separatory funnel.
147
After shaking and thoroughly equilibrating tat room temperature, the upper phase and lower
148
phase were then separated and degassed by ultrasonic for 30 min. The upper phase was used as
nm.
Briefly,
a
series
of
two-phase
solvent
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30-34
, the optimal two-phase
systems
(n-hexane–ethyl
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149
stationary phase while the lower phase as mobile phase. The sample solution for HSCCC
150
separation was prepared by dissolving 2 g of PEs-rich crude extract in 20 mL of the lower phase.
151
HSCCC Separation Procedure. The multilayer coil column was first entirely filled with the
152
stationary phase at the flow rate of 30.0 mL/min. The apparatus was rotated at 800 rpm and then
153
the mobile phase was pumped into the column at the flow rate of 10 mL/min in the head to tail
154
elution mode. The mobile phase eluting at the tail outlet indicated hydrodynamic equilibrium has
155
been reached. Subsequently, 20 mL of sample solution was injected into the column. The peak
156
fractions were collected manually according to the elution profile and evaporated under reduced
157
pressure.
158
Analysis and characterisation of HSCCC fractions. PEs-rich crude extract sample and each
159
peak fraction obtained from HSCCC were analyzed by HPLC-UV using peak area calculation.
160
The HPLC analyses were performed on an Agilent 1200 series system (Agilent Technologies)
161
equipped with a quaternary pump, a vacuum degasser, an auto injector, and an ultraviolet
162
detector. The HPLC separation was performed on a C18 column (Ultimate, 4.6 × 250 mm, 4.7 µm,
163
Welch Materials, Inc.) at 30 °C. The ultraviolet detector was set at 280 nm according to the λmax of
164
UV absorbance of PEs. All of the samples were eluted at 0.80 mL/min using an isocratic elution of
165
acetonitrile and water containing 0.2 % formic acid (80/20, v/v). Agilent Chemstation version
166
B.04.01 was used for the system control and data acquisition.
167
Purification and Identification of JC1. A continuous repeated semi-preparative HPLC method
168
was applied to further purify the JC1. The separation of JC1 was performed on a reversed-phase
169
C8 semi-preparative column (Welchrom-C8, 10 × 250 mm, 5 µm) with an isocratic elution of
170
acetonitrile-water (80:20, v/v) at a flow rate of 2.5 mL/min. The monitoring of the wavelength was
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set at 280 nm. The purity of JC1 was identified by HPLC. MS spectra of JC1 was measured in the
172
positive mode ([M + Na] +) using a microTOF-QII mass spectrometer (Bruker Daltonics,
173
Germany).
174
UPLC-PDA and UPLC-MS Analysis of Isolated JC1 and PEs-rich Crude Extract. UPLC-PDA
175
analyses were performed on an ACQUITY UPLC H-Class System (Waters, USA) equipped with
176
quaternary solvent manager QSM, auto sampler manager FTN and Photodiode Array eLambda
177
detector. The chromatographic separation was performed on an Acquity UPLC BEH C18 column
178
(1.7 µm, 150 mm × 2.1 mm, Waters, USA) at 40 °C. All of the samples were eluted at 0.40 mL/min
179
using an isocratic elution of solvent A and B (80/20, v/v), in which, solvent A was composed of
180
acetonitrile-water (90/10, v/v) containing 0.1 % formic acid and 0.05 % ammonium formate,
181
solvent B was the water containing 0.1 % formic acid and 0.05 % ammonium formate. The sample
182
injection volume was 2 µL. The detection range of ultraviolet detector was set from 200 to 400 nm.
183
Monitoring of the wavelength was set at 280 nm according to the λmax of UV absorbance of PEs.
184
Empower software version 3.0 was used for the system control and data acquisition.
185
UPLC-MS analyses were performed on an ACQUITY UPLC I-Class-Xevo TQD System
186
(Waters, USA) interfaced with a Waters Quattro Premier XE triple quadrupole mass spectrometer
187
and equipped with electrospray ionization operated in the positive ionization mode. The
188
chromatographic separation condition of the UPLC-MS analyses was the same as the UPLC-PDA
189
analyses. The source dependent parameters maintained for the analytes were: cone gas flow,
190
50 L/h; desolvation gas flow, 500 L/h; capillary voltage, 3.0 kV, source temperature, 120 °C;
191
desolvation temperature, 500 °C. The optimum values for compound dependent parameters of
192
cone voltage and collision energy were set at 30 V and 10 eV. Detection of the ions was
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performed in the multiple-reaction monitoring (MRM) mode, by monitoring the transition pairs
194
(precursor to product ion) of m/z for PEs. Mass Lynx software version 4.1 was used to control all
195
parameters of UPLC and MS.
196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214
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RESULTS AND DISCUSSION
216
Selection of the Two-phase Solvent System. Five main peaks (regarded as Jatropha Factors
217
C1-5 according to their retention time appeared orderly in chromatograms, JC1-5) determined by
218
HPLC were used to select the appropriate two-phase solvent system. The results of the partition
219
coefficients (K) and separation factors (α) of these compounds in various two-solvent systems are
220
listed in Table 1. According to the published papers
221
system requires the following considerations: (1) the equilibrating time should be considerably
222
short enough (< 30 s); (2) the partition coefficient (K) should be in a suitable range, usually
223
between 0.2 and 5; (3) the separation factor(α) should be greater than 1.5. After repeated attempt,
224
the
225
(1.5:1.5:1.2:0.5, v/v) was considered as an optimal proposal for its appropriate partition
226
coefficients (K) and separation factors (α).
227
Isolation of PEs Fractions by HSCCC. The retention of the stationary phase was 56.7%.
228
Fractions Peak A and B were separated in 30 min with a total mobile elution volume of 300 mL
229
(Figure 2. A). Isolated fractions Peak A and B were analyzed by HPLC (Figure 2. C. c2 and c3).
230
In the HSCCC separation, the expected result is five fractions (Figure 2. C. c4). It should be
231
noted, the main compound JC1 from PEs can be isolated fast, and the purity increased from
232
48.5% to 85.0 % (Figure 2. C. c3 and c4). Compared with the literatures
233
isolation methods of PEs including liquid-liquid extraction and silica-gel column chromatography
234
are time consuming, require a large amounts of organic solvents, and typically require numerous
235
steps resulting lower recovery and higher cost. In this work, the separation of PEs by HSCCC
236
shows an excellent sample recovery, low cost and high efficiency.
two-phase
solvent
system
composed
of
30-34
, the appropriate two-phase solvent
n-hexane/ethyl
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acetate/methanol/water
12-15
, the conventional
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237
Purification and Identification of JC1. The semi-preparative HPLC chromatogram of Peak A
238
was shown in Figure 2. B. b1. Subsequently, JC1 was further purified by using a continuous
239
semi-preparative HPLC through repeating sample injection at an interval of 5 minutes (Figure 2.
240
B. b2). The purity of JC1 measured by HPLC was 99.8 % (Figure 2. C. c1). HR-MS spectra of
241
JC1 showed in Figure 3. C, and the MS of molecular ion peak was 733.3695 ([M + Na] +).
242
According to the published papers during over 20 years, the JC1 is proved to be the one of the
243
highest quantity levels of PEs in J. Curcas seed 12-15, 17, 26. Therefore, it was obviously proofed that
244
the isolated compound was the JC1 in this work.
245
UPLC-PDA and UPLC-MS Analysis of Isolated JC1 and PEs-rich Crude Extract. It was
246
regrettable that the purity of JC1 was only 87.8% (Figure 3. A) by UPLC using the peak area
247
calculation method. It was easily to observe that there is a Peak S with the high similarity UV
248
absorption spectrogram to JC1 (Figure 3. A. a1 and a2). As we known, the purity of JC1 was
249
usually measured by conventional HPLC using the peak area calculation method in previous
250
published papers 13-15. The result of UPLC indicated that there was a Peak S (Figure 3. A), which
251
was difficultly separated with the JC1 using the conventional HPLC (Figure 2. C. c1).
252
Subsequently, A UPLC-MS was established and successfully used to identify the characteristics
253
of Peak S. Figure 3. B showed the TIC chromatogram of JC1. In the positive-ion mass spectra
254
intense, Peak S showed the same quasi-molecular ion peak ([M+H-H2O] +) at about 693.00 and
255
several similar sequence-specific fragment ions (at about 365.00, 311.00 and 292.90, m/z) as JC1
256
(Figure
257
(12-deoxy-16-hydroxyphorbol, Figure 1) in JC1 is 364.4327. In addition, the fragment ion at about
258
365.00 (m/z) was the mother nucleus ion peak ([M] +), moreover, the 311.00 (m/z) and 292.90
3.
D
and
E).
Because
the
molecular
mass
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mother
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+
and [M-4H20] + of mother nucleus, respectively. Thus, it
259
(m/z) were the fragment ions [M-3H20]
260
was easily deduced that Peak S was the isomer of JC1.
261
Furthermore, the sample of JC1 showed only one peak with HPLC but contained another
262
peak (Peak S) by UPLC, this result indicated it was doubtful the general five to seven PEs peaks
263
presented in J. curcas by conventional HPLC method 14-17, 35.Thus, it was imperative to establish a
264
high resolution method to confirm this doubt. In this work, an UPLC-PDA method was developed
265
and applied to analyze the PEs-rich crude extract. In our recently published paper, instead of the
266
conventional C18 column (4.5 µm), when using a high resolution C18 column (2.7 µm), the
267
HPLC-UV chromatogram at 280 nm of PEs-rich crude extract has shown more than 9 peaks
268
involving five main peaks 15. The content levels of PEs in J. curcas seed or their related products
269
are always calculated by the total amount equivalent to TPA using an internal standard method11,
270
16, 35
271
the PEs-rich crude extract. The choose UPLC-UV chromatograms at 240, 280 and 304nm of the
272
PEs-rich crude extract and TPA had been shown in Figure 4. A, B and C, respectively. It was
273
interesting that more than 17 peaks were separated out at 280nm (Figure 4.B). However, similar
274
to the reports
275
(Figure 2.C.c4). It is certified that the λmax of PEs always at about 280 nm, and the λmax of TPA at
276
about 242 nm
277
the λmax of more than 12 peaks were at about 280 nm, including peaks 1-8, 10-12 and 15, which
278
all similar to Peak 8 (JC1, Figure 4. B. a), at the same time, the secondary λmax of them were at
279
about 232 nm similar to TPA (Figure 4. B. c). It was easily deduced that there could exist more
280
than 12 homologous compounds of PEs. However, about 4 peaks (λmax at about 304 nm)
. In this study, TPA was selected as internal standard to establish the UPLC-PDA method for
14-17
, about 5-7 peaks were exhibited in the HPLC-UV chromatogram at 280 nm
14, 15
. Obviously, the results of the UV absorption spectrogram also demonstrated
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281
involving peaks 13, 14, 16 and 17 (Figure 4. B. b) were significantly different from the JC1. It was
282
questionable that whether these 4 peaks (λmax at about 304 nm) are the homologous of PEs.
283
UPLC-MS was used to further certify whether those 4 peaks belong to homologous of PEs.
284
The TIC chromatogram of PEs-rich crude extract was shown in Supporting Material Figure S1.
285
In the positive-ion mass spectra intense, more than 15 compounds showed the same
286
quasi-molecular ion peaks at about 693.00 ([M+H-H2O]+) in corresponding mass spectra
287
(Supporting Material Figure S1), and several similar sequence-specific fragment ions (at about
288
365.00, 311.00 and 292.90), which were all similar to JC1 (Figure 3. E). This indicates that these
289
15 compounds all contained the same mother nucleus. Therefore these results indicate that the
290
more than 15 compounds found in the UPLC-MS are all homologues of PEs.
291
In this work, a rapid and efficient HSCCC method was developed and successfully applied to
292
separate the PEs from J.Curcas seed. In addition, combining the HSCCC and semi-preparative
293
HPLC was developed and successfully applied to purify the JC1. Meanwhile, the PEs-rich crude
294
extract from J. curcas was also analyzed by UPLC-PDA and UPLC-MS, the results indicated
295
there may be more than15 analogues of PEs in J. curcas. This work will enhance the economic
296
viability and sustainability of the J. curcas production chain.
297
ACKNOWLEDGEMENTS
298
We kindly thank Xiaoyu Zhang (Ph.D., College of Life Science, Sichuan Normal University) and
299
Xueran Mei (M.S., College of Life Science, Sichuan Normal University) provided the help of the
300
HSCCC analysis. This work was supported by the National Natural Science Foundation of China
301
(Grant 31170312).
302
NOTES:
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303
The authors declare no competing financial interest.
304
ABBREVIATIONS USED
305
PEs, phorbolesters; TPA, phorbol 12-myristate-13-acetate; JC1-6, Jatropha factors C1-6; K,
306
partition coefficient; α, separation factor.
307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342
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Sudhakar Johnson, T.; Eswaran, N.; Sujatha, M., Molecular approaches to improvement of
Jatropha curcas Linn. as a sustainable energy crop. Plant Cell Rep. 2011, 30, (9), 1573-91. 2.
Abdulla, R.; Chan, E. S.; Ravindra, P., Biodiesel production from Jatropha curcas: a critical review.
Crit Rev Biotechnol. 2011, 31, (1), 53-64. 3.
Juan, J. C.; Kartika, D. A.; Wu, T. Y.; Hin, T. Y., Biodiesel production from Jatropha oil by catalytic
and non-catalytic approaches: an overview. Bioresour Technol. 2011, 102, (2), 452-60. 4.
King, A. J.; He, W.; Cuevas, J. A.; Freudenberger, M.; Ramiaramanana, D.; Graham, I. A.,
Potential of Jatropha curcas as a source of renewable oil and animal feed. J Exp Bot. 2009, 60, (10), 2897-905. 5.
Sharma, S. K.; Singh, H., A review on pharmacological significance of genus Jatropha
(Euphorbiaceae). Chin J Integr Med. 2012, 18, (11), 868-80. 6.
Devappa, R. K.; Makkar, H. P.; Becker, K., Nutritional, biochemical, and pharmaceutical potential
of proteins and peptides from jatropha: review. J Agric Food Chem. 2010, 58, (11), 6543-55. 7.
Thomas, R.; Sah, N. K.; Sharma, P. B., Therapeutic biology of Jatropha curcas: a mini review.
Curr Pharm Biotechnol. 2008, 9, (4), 315-24. 8.
Debnath, M.; Bisen, P. S., Jatropha curcas L., a multipurpose stress resistant plant with a potential
for ethnomedicine and renewable energy. Curr Pharm Biotechnol. 2008, 9, (4), 288-306. 9.
Paulillo, L. C.; Mo, C.; Isaacson, J.; Lessa, L.; Lopes, E.; Romero-Suarez, S.; Brotto, L.; Abreu, E.;
Gutheil, W.; Brotto, M., Jatropha curcas: from biodiesel generation to medicinal applications. Recent Pat Biotechnol. 2012, 6, (3), 192-9. 10. Sabandar, C. W.; Ahmat, N.; Jaafar, F. M.; Sahidin, I., Medicinal property, phytochemistry and pharmacology of several Jatropha species (Euphorbiaceae): a review. Phytochemistry. 2013, 85, 7-29. 11. Devappa, R. K.; Makkar, H. P.; Becker, K., Jatropha toxicity--a review. J Toxicol Environ Health B Crit Rev. 2010, 13, (6), 476-507. 12. Hirota, M.; Suttajit, M.; Suguri, H.; Endo, Y.; Shudo, K.; Wongchai, V.; Hecker, E.; Fujiki, H., A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol. Cancer Res. 1988, 48, (20), 5800-4. 13. Haas, W.; Sterk, H.; Mittelbach, M., Novel 12-deoxy-16-hydroxyphorbol diesters isolated from the seed oil of Jatropha curcas. J Nat Prod. 2002, 65, (10), 1434-40. 14. Roach, J. S.; Devappa, R. K.; Makkar, H. P.; Becker, K., Isolation, stability and bioactivity of Jatropha curcas phorbol esters. Fitoterapia. 2012, 83, (3), 586-92. 15. Wang, Z.; Tang, L.; Hu, H.; Guo, Y.; Peng, T.; Yan, F.; Chen, F., Metabolic profiling assisted quality control of phorbolesters in Jatropha curcas seed by high-performance liquid chromatography using a fused-core column. J Agric Food Chem. 2012, 60, (38), 9567-72.
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16. Li, C. Y.; Devappa, R. K.; Liu, J. X.; Lv, J. M.; Makkar, H. P.; Becker, K., Toxicity of Jatropha curcas phorbol esters in mice. Food Chem Toxicol. 2010, 48, (2), 620-5. 17. Devappa, R. K.; Roach, J. S.; Makkar, H. P.; Becker, K., Occular and dermal toxicity of Jatropha curcas phorbol esters. Ecotoxicol Environ Saf. 2013, 94, 172-8. 18. Oskoueian, E.; Abdullah, N.; Ahmad, S., Phorbol esters isolated from jatropha meal induced apoptosis-mediated inhibition in proliferation of chang and vero cell lines. Int J Mol Sci. 2012, 13, (11), 13816-29. 19. Veerabhadrappa, M. B.; Shivakumar, S. B.; Devappa, S., Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98. J Biosci Bioeng. 2014,117(2),208-14. 20. Hidayat, C.; Hastuti, P.; Wardhani, A. K.; Nadia, L. S., Method of phorbol ester degradation in Jatropha curcas L. seed cake using rice bran lipase. J Biosci Bioeng. 2014,117(3),372-4. 21. Sadubthummarak, U.; Parkpian, P.; Ruchirawat, M.; Kongchum, M.; Delaune, R. D., Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product. J Environ Sci Health B. 2013, 48, (11), 974-82. 22. Yunping, B.; Ha, B. T.; Eunice, Y.; Chueng, L. L.; Yan, H., Light induced degradation of phorbol esters. Ecotoxicol Environ Saf. 2012, 84, 268-73. 23. Kumar, V.; Makkar, H. P.; Amselgruber, W.; Becker, K., Physiological, haematological and histopathological responses in common carp (Cyprinus carpio L.) fingerlings fed with differently detoxified Jatropha curcas kernel meal. Food Chem Toxicol. 2010, 48, (8-9), 2063-72. 24. Wang, X. H.; Ou, L.; Fu, L. L.; Zheng, S.; Lou, J. D.; Gomes-Laranjo, J.; Li, J.; Zhang, C., Detoxification of Jatropha curcas kernel cake by a novel Streptomyces fimicarius strain. J Hazard Mater. 2013, 260, 238-46. 25. Phengnuam, T.; Suntornsuk, W., Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation. J Biosci Bioeng. 2013, 115, (2), 168-72. 26. Devappa, R. K.; Rajesh, S. K.; Kumar, V.; Makkar, H. P.; Becker, K., Activities of Jatropha curcas phorbol esters in various bioassays. Ecotoxicol Environ Saf. 2012, 78, 57-62. 27. Oskoueian, E.; Abdullah, N.; Ahmad, S., Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-delta, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines. Molecules. 2012, 17, (9), 10816-30. 28. Wender, P. A.; Kee, J. M.; Warrington, J. M., Practical synthesis of prostratin, DPP, and their analogs, adjuvant leads against latent HIV. Science. 2008, 320, (5876), 649-52. 29. Devappa, R. K.; Malakar, C. C.; Makkar, H. P.; Becker, K., Pharmaceutical potential of phorbol esters from Jatropha curcas oil. Nat Prod Res. 2013, 27, (16), 1459-62. 30. Liang, J.; Ito, Y.; Zhang, X.; He, J.; Sun, W., Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave-assisted extraction coupled with high-speed counter-current chromatography. J Sep Sci. 2013, 36, (24), 3934-40. 31. Dai, X.; Huang, Q.; Zhou, B.; Gong, Z.; Liu, Z.; Shi, S., Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment. Food Chem. 2013, 139, (1-4), 563-70. 32. Regalado, E. L.; Tolle, S.; Pino, J. A.; Winterhalter, P.; Menendez, R.; Morales, A. R.; Rodriguez, J. L., Isolation and identification of phenolic compounds from rum aged in oak barrels by high-speed countercurrent
chromatography/high-performance
liquid
chromatography-diode
array
detection-electrospray ionization mass spectrometry and screening for antioxidant activity. J
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Chromatogr A. 2011, 1218, (41), 7358-64. 33. Lopes-Lutz, D.; Mudge, E.; Ippolito, R.; Brown, P.; Schieber, A., Purification of alkylamides from Echinacea angustifolia (DC.) Hell. Roots by high-speed countercurrent chromatography. J Agric Food Chem. 2011, 59, (2), 491-4. 34. Degenhardt, A.; Engelhardt, U. H.; Lakenbrink, C.; Winterhalter, P., Preparative separation of polyphenols from tea by high-speed countercurrent chromatography. J Agric Food Chem. 2000, 48, (8), 3425-30. 35. Rakshit, K. D.; Darukeshwara, J.; Rathina Raj, K.; Narasimhamurthy, K.; Saibaba, P.; Bhagya, S., Toxicity studies of detoxified Jatropha meal (Jatropha curcas) in rats. Food Chem Toxicol. 2008, 46, (12), 3621-5.
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398
Table legend
399
Table 1.
400
Partition coefficients (K) and separation factors (α) of compounds in various solvent
401
systems. Partition coefficients （KU/L)
Separation factors (a)
Ratio of solvent system (n-hexane–ethyl acetate–methanol–water)
JC1
JC2
JC3
JC4
JC5
K2/K1
K3/K2
K4/K3
K5/K4
1:1:1:1
4.19
25.22
27.21
16.72
46.33
6.02
1.07
1.63
2.77
3:1:3:1
0.07
0.09
0.11
0.12
0.13
1.32
1.18
1.16
1.08
2:2:3:1
0.27
0.29
0.34
0.38
0.34
1.09
1.18
1.11
1.12
3:3:3:1
0.61
0.63
0.79
0.88
0.81
1.02
1.25
1.12
1.09
3:3:2:1
1.93
2.27
2.75
3.05
2.82
1.17
1.21
1.11
1.08
1.5:1.5:1.2:0.5
1.03
1.43
1.80
2.84
1.55
1.40
1.26
1.58
1.83
402
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Figures legends
404
Figure 1.
405
The structure of 12-deoxy-16-hydroxyphorbol.
406
Figure 2.
407
(A) Preparative HSCCC chromatogram of PEs-rich crude extract from J. curcas seed;
408
(B) Primary semi-preparative HPLC-UV chromatogram of isolated fraction Peak A by HSCCC
409
(b1), continuous semi-preparative HPLC-UV chromatogram of Jatropha factors C1 (JC1) using
410
repeating sample injection at an interval of 5 minutes (the English alphabet a-p (end-label of JC1)
411
represents the continuous repeating sample injection order, b2);
412
(C) HPLC-UV chromatograms of the Jatropha Factors C1 (c1), Peak B fraction isolated by
413
HSCCC (c2), Peak A fraction isolated by HSCCC (c3) and PEs-rich crude extract (c4).
414
Figure 3.
415
(A) UPLC-UV chromatogram of Jatropha factors C1 and Peak S at 280 nm, the UV absorbance
416
spectrogram of Peak S (a1) and Jatropha factors C1 (a2);
417
(B) TIC chromatogram of the Jatropha factors C1 and Peak S;
418
(C) MS spectrum of the Jatropha factors C1 and Peak S;
419
(D) Product ion MS spectrum of Peak S;
420
(E) Product ion MS spectrum of Jatropha factors C1.
421
Figure 4.
422
(A) UPLC-UV chromatograms of PEs-rich extract and TPA at 240 nm;
423
(B) UPLC-UV chromatograms of PEs-rich extract and TPA at 280 nm, UV absorbance
424
spectrogram of Peak 8 (a), UV absorbance spectrogram of Peak 17 (b), UV absorbance
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425
spectrogram of Peak 18 (c);
426
(C) UPLC-UV chromatograms of PEs-rich extract and TPA at 304 nm.
427
TPA is phorbol-12-myristate-13-acetate,
428
Crude extract represents the main peaks detected from the PEs-rich crude extracts
429
1-18 represent the detected peak in the UPLC in their exist order;
430
Supporting Material Figure S1 Legend
431
(A-O) Product ion MS spectrum of the Peaks 3-17;
432
(P) UPLC–MS chromatogram in total ion current (TIC) of PEs-rich crude extract.
433
3-17 represent the detected peak in the UPLC-MS in their exist order.
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Figure 1. The molecule structure of 12-deoxy-16-hydroxyphorbol. 120x77mm (200 x 200 DPI)
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Figure 2. (A) Preparative HSCCC chromatogram of PEs-rich crude extract from J. curcas seed; (B) Primary semi-preparative HPLC-UV chromatogram of isolated fraction Peak A by HSCCC (b1), continuous semi-preparative HPLC-UV chromatogram of Jatropha factors C1 (JC1) using repeating sample injection at an interval of 5 minutes (the English alphabet a-p (end-label of JC1) represents the continuous repeating sample injection order, b2); (C) HPLC-UV chromatograms of the Jatropha Factors C1 (c1), Peak B fraction isolated by HSCCC (c2), Peak A fraction isolated by HSCCC (c3) and PEs-rich crude extract (c4). 113x156mm (300 x 300 DPI)
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Figure 3. (A) UPLC-UV chromatogram of Jatropha factors C1 and Peak S at 280 nm, the UV absorbance spectrogram of Peak S (a1) and Jatropha factors C1 (a2); (B) TIC chromatogram of the Jatropha factors C1 and Peak S; (C) MS spectrum of the Jatropha factors C1 and Peak S; (D) Product ion MS spectrum of Peak S; (E) Product ion MS spectrum of Jatropha factors C1. 160x139mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 4. (A) UPLC-UV chromatograms of PEs-rich extract and TPA at 240 nm; (B) UPLC-UV chromatograms of PEs-rich extract and TPA at 280 nm, UV absorbance spectrogram of Peak 8 (a), UV absorbance spectrogram of Peak 17 (b), UV absorbance spectrogram of Peak 18 (c); (C) UPLC-UV chromatograms of PEs-rich extract and TPA at 304 nm. TPA is phorbol-12-myristate-13-acetate, Crude extract represents the main peaks detected from the PEs-rich crude extracts 1-18 represent the detected peak in the UPLC in their exist order. 160x111mm (150 x 150 DPI)
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Journal of Agricultural and Food Chemistry
119x45mm (300 x 300 DPI)
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