DSpace at VNU: Semi-preparative HPLC separation followed by HPLC UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed

Accepted ManuscriptTitle: Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed Author: Santi Kongmany Truong Thi

Accepted Manuscript

Title: Semi-preparative HPLC separation followed by

HPLC/UV and tandem mass spectrometric analysis of phorbol

esters in Jatropha seed

Author: Santi Kongmany Truong Thi Hoa Le Thi Ngoc Hanh

Kiyoshi Imamura Yasuaki Maeda Luu Van Boi

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Semi-preparative HPLC separation followed by HPLC/UV and tandem mass

spectrometric analysis of phorbol esters in Jatropha seed

Santi Kongmanya, Truong Thi Hoab, Le Thi Ngoc Hanhc, Kiyoshi Imamurad,*, Yasuaki Maedad and Luu Van Boie

a Department of Chemistry, Faculty of Natural Science, National University of Laos,

Dongdok Campus, P.O Box 7322, Xaythany District, Vientiane, Laos

b Danang Environmental Technology Center, Institute of Environmental Technology, Vietnam Academy of Science and Technology, Tran Dai Nghia Road, Ngu Hanh Son District, Danang, Vietnam

c Graduate School of Engineering, Osaka Prefecture University, 1-2 Gaken-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan

d Research Organization for University-Community Collaborations, Osaka Prefecture

University, 1-2 Gaken-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan

e Faculty of Chemistry, Vietnam National University, Hanoi, 19 Le Thanh Tong St., Hanoi, Vietnam

* Corresponding author: +81-72-254-9863 Email: k_imamura@riast.osakafu-u.ac.jp

Highlights

 A group of phorbol esters (PEs) was extracted with MeOH from JCL seeds

 More than seven isomers of phorbol esters with mw of 710 were identified

 All of them were tigliane-type of diterpens with isomeric dicarboxylic moiety

 Main five PEs are isolated using semi-preparative HPLC analysis

 They were assigned as components of Jatropha factors as cited in the references

Abstract

Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas

Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components

contained in the PEs The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C1, C2, C3, and C4/5, but J6 was a mixture of Jatropha factor C6 and itsisomer based on the data of UV and LC-MS/MS, and J2 was identified using 1H-NMR analysis By characterization using LC-MS/MS analysis, all components of a series of PEs were

elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380

Keywords: preparative HPLC, reverse phase ODS-3 column, Jatropha curcas seeds, phorbol

ester isomers, tandem mass spectrometry characterization, 1H NMR spectrometry

1 Introduction

The non-edible seed oil from Jatropha curcas Linnaeus (JCL) has recently been reported

as a promising feedstock for the production of biodiesel [1,2] JCL is a flowering plant

belonging to the Euphorbiaceae plant family that is typically grown in tropical and

sub-tropical regions, where it is cultivated in free-draining sands and loams with no water logging [3] JCL is native to Mexico and Central America, and was introduced to Africa and Asia by Portuguese sailors in the 16th century [4] JCL seeds contain about 34% (w/w) of oil, which is lower than that of sun flower seeds (50%) or rapeseed (40–48%), but higher than that of soybean (18%) [5] However, JCL seed oil is non-edible because it contains numerous toxic phytochemicals, with PEs being identified as the main toxic compounds [6]

PEs have been reported to exhibit a wide range of interesting biological activities,

including the hyper-activation of protein kinase C (PKC), which can lead to abnormal signal transduction and negative responses, including tumorigenesis, skin irritation, inflammation, platelet aggregation and cell differentiation [7] PEs have also been reported to exhibit

molluscicidal, fungicidal and insecticidal activities, indicating that the extract of JCL seed oil could be used as a bio-control agent in agrochemical applications following its detoxification [8-10] Furthermore, a PE was used as an intermediate in the synthesis of the promising anti-HIV compound, prostratin [11] The concentrations of the different PEs in the seed kernels of JCL can vary from 0.87–3.32 mg/g, depending on the area of cultivation [6,12,13] The utilization of PEs as value-added by-products could be essential for promoting the

production of biodiesel from JCL

In 1988, Hirota et al [14] reported the isolation and structural characterization of a

12-deoxy-16-hydroxyphorbol diester from the seed oil of JCL, which was subsequently assigned as Jatropha factor C1, as shown in Fig 1 In 2002, four additional components of 12-deoxy-16-hydroxyphorbol diester were isolated and named as Jatropha factors C2, C3, C4/5

(epimeric isomers) and C6 [15] From a structural perspective, PEs are essentially diterpenes consisting of a tetracyclic carbon skeleton (A, B, C and D), which are also known as

tigliane-type diterpenes The carbon skeleton of PEs contains two hydroxyl groups at the C13 and C16 positions, which are combined with a dicarboxylic acid In light of the large number

of potential variations of the carboxylic acid group, many different isomers could be formed, although only six components of the 12-deoxy-16-hydroxyphorbol diesters have been

isolated and identified to date

High performance liquid chromatography (HPLC) over a C18 (ODS) reversed-phase column with UV absorbance at 280 nm has been used for the quantitative analysis of the PEs

in JCL with phorbol 12-myristate 13-acetate (TPA) as an internal reference standard [16-19, 26] The PEs found in the seeds and leaves of JCL, as well as those found in numerous animal tissues, were recently separated using a modified ODS column This method allowed for the PEs to be separated into five peaks, which were subsequently quantified by HPLC tandem mass spectrometry [12, 13]

Preparative HPLC has been used for the purification of PEs using a normal phase column [19] This technique has also been applied for the purification and isolation of specific

compounds from JCL extracts using sequentially by a C18 reversed phase column, followed

by a normal phase silica gel column [15]

The use of HPLC in conjunction with tandem mass spectrometry is a powerful and highly specific analytical tool for the identification of the individual components found in plant

extracts [20, 22, 24, 25] MS/MS spectra can also provide useful information pertaining to the fragmentation of the individual components, which can be used to search for novel

metabolites with similar structural features/core scaffolds This level of detail can be

particularly useful for determining the presence of related components in plant materials and investigating the phytochemical degradation, metabolism and biosynthesis in a quantitative and qualitative manner [12, 13]

The purpose of the present study is to elucidate the structural feature of a series of PEs

extracted from Jatropha curcas oil with MeOH Using HPLC/PDA analysis, a series of PEs

of MeOH extract was separated into more than seven components of PE A reversed phase semi-preparative HPLC analysis of an ODS-3 column was revised for the isolation of five peaks of components (J2, J3, J5, J6 and J7) from the extracts, and their structures was

assigned on the basis of the UV, LC-MS/MS and 1H NMR analysis The characterization using by LC-MS/MS analysis was conducted to elucidate the structural specification of a series of PEs consist of more than seven components

2 Experimental

2.1 Materials

The JCL seeds used in this study were cultivated in Trang Bang, Vietnam MeOH,

n-hexane, chloroform and ethyl acetate were all purchased in the analytical grade from Wako Pure Chemical Industry (Kyoto, Japan) Acetonitrile (HPLC analytical grade), anhydrous sodium sulfate (analytical grade), activated silica gel (Wakogel®C-200, grade: column

chromatography analysis), and deuterated dichloromethane (NMR spectral grade) and

phorbol 12-myristate 13-acetate (TPA) were also purchased from Wako Pure Chemical

Industry The purified water used in the current study was produced in Osaka Prefecture University (Sakai, Japan)

2.2 Extraction and purification of PEs

The JCL seeds were manually cracked using a pair of pliers and the kernels were separated from the seeds The seed kernels were subsequently homogenized using a grinder A sample

of the homogenized material (116 g) was suspended in MeOH (150 mL) and the resulting mixture was extracted under ultrasonic irradiation for 30 min using an ASU-10 ultrasonic apparatus (As One, Tokyo, Japan) The mixture was then allowed to stand until the MeOH layer was clearly separated from the residue and the supernatant was transferred to a 1 L Erlenmeyer flask The residue was then extracted 3 times with MeOH (100 mL), and the combined extracts were filtered and concentrated under vacuum at 40 °C to approximately 50

mL The volume of the MeOH extract was adjusted to 100 mL with MeOH and transferred to

a 250 mL separating funnel The lower oily layer of the mixture was removed, and the

remaining MeOH layer was extracted three times with 50 mL of MeOH-saturated hexane The lower MeOH layer was then evaporated to dryness under vacuum at 40 °C to give an oily residue, which was dissolved in ethyl acetate (30 mL) and transferred to a 100 mL separating funnel Water (10 mL) was then added to the separating funnel, and the resulting mixture was vigorously shaken for a few min before being separated into two phases by centrifugation The ethyl acetate layer was subjected to washing process two times to give an ethyl acetate extract containing PEs, which is referred to hereafter as crude PEs

2.3 Clean-up using column chromatography

The oily residue (3.1 g) obtained from the MeOH extraction process was fractionated by column chromatography The oily residue was previously coated on a powder of anhydrous

Na2SO4 (4–5 g) by dissolving in MeOH and then removing the solvent using a rotary

evaporator under a reduced pressure A silica gel column was prepared by packing 17 g of silica gel (C-200), which had been activated overnight at 130 °C, into a glass column (30 × 1.5 cm i.d., Pyrex glass, glass fritted at the bottom, with a Teflon stop cock) as a slurry in n-hexane The top of the column was then packed with the Na2SO4 coated with the oily residue The column was then eluted sequentially with the following solvents at a flow rate of 2.5 mL/min: 20 mL of n-hexane, 100 mL of n-hexane/chloroform (50/50, v/v), 100 mL of chloroform, 100 mL of MeOH/chloroform (5/95, v/v), 100 mL of MeOH/chloroform (35/65, v/v) and 50 mL of MeOH The eluents were fractionated into 200 drops/tube (about 2 mL) using an SF2120 fraction collector (ADVANTEC, Tokyo, Japan) The fractionation process was initiated from chloroform (100%) and continued until MeOH (100%) The target PE compounds were collected in fractions 52–63, corresponding to the use of 5% MeOH in chloroform as the eluent The PE-containing fractions were subsequently combined and concentrated to give residues, which were purified by preparative HPLC and analyzed by LCMS/MS

2.4 Isolation of PEs using semi-preparative HPLC

The individual PEs were isolated from the PE-containing fractions described above by preparative HPLC using a GL-7480 HPLC system (GL Science Inc., Tokyo, Japan) equipped with a PDA detector (GL-7452, GL Science Inc.), an auto-sampler (GL-7420, GL Science Inc.) and a column oven (GL-7430, GL Science Inc.) The HPLC system was fitted with a preparative Inertsil® ODS-3 column (modified C18, 5 m, 7.6  250 mm, GL Science Inc.), which was operated at 40 C with a flow rate of 2 mL/min Water (A) and acetonitrile (B) were used as the mobile phases, and the system was subjected to the following gradient elution process: 50% B (0 min), 80% B (0–10 min), 80% B (10–25 min), 100% B (25–30

min), 100% B (30–45 min), 50% B (45–50 min) and 50% B (50–65 min) An 80 l aliquot of sample was repeatedly injected onto the system

The eluted compounds were monitored at wavelengths of 220, 250 and 280 nm, and the eluents themselves were collected using a CHF122SC fraction collector (ADVANTEC,

Tokyo, Japan), which was operated in the timer mode (i.e., collecting the eluent in a tube

every 20 seconds) The fraction collector was connected to an in-line HPLC analysis system and the fractionation process was initiated 10 min after the sample injection

An 80 l aliquot of PE-containing fractions (1.5 mL) described in section 2.3 was

repeatedly purified by preparative HPLC and fractionated into five portions containing J2, J3, J5, J6 and J7

2.5 HPLC/PDA analysis

The PE sample solutions were analyzed by high performance liquid chromatography

(HPLC) on a Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with a degasser unit (DGU-20A), auto-sampler (SIL-20A), column oven (CTO-20A) and photo diode array (PDA) detector (SPD-M20A) The resulting data were acquired and analyzed using version 1.2 of the Lab Solution software (Shimadzu) Solvent A (10 mM aqueous ammonium acetate) and solvent B (acetonitrile, CH3CN) were used as the mobile phases The HPLC system was fitted with an analytical Inertsil® ODS-3 column (modified C18, 4.6 m, 5.0  150 mm, GL Science Inc., Tokyo, Japan), which was eluted at a flow rate of 0.5 mL/min with the

following gradient: 20% A (0–2 min), 18% A (2–2.20 min), 13% A (2.20–13.50 min), 8% A (13.50–18 min), 0% A (18–22 min), 0% A (22–25 min), 20% A (25–25.01 min) and 20% A (32 min) The column temperature was maintained at 35 C and the eluent was scanned at wavelengths in the range of 190–400 nm The PEs eluted from the column were monitored at

a wavelength of 280 nm The concentration of (purified) extract used for analysis was 40 mg/L (diluted solution) The concentration of each isolated compound used for analysis was 6.3 mg/L (J2), 15 mg/L (J3), 11 mg/L (J5), 12 mg/L (J6), 17 mg/L (J7) The injection volume was 20 µL

2.6 LC/MS/MS analysis

All of the compounds were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an 8030 LCMS system (Shimadzu), which was connected to an in-line HPLC-DAD system through a valve unit (FCV-12HAH) The injection volume was 20 µL This system was used to estimate the molecular weight and structure of the individual

components The LC-MS/MS experiments were conducted under the following MS

conditions: ionization interface (electrospray ionization (ESI)) voltage, +4.5 kV; nebulizer gas, N2; nebulizer gas flow rate, 2.0 L/min; drying gas, N2; drying gas flow rate, 15 L/min; heat block temperature (HB), 120 C; and desolvation temperature (DL), 250 C The

samples were separately analyzed in two different modes, including (i) the precursor-ion-scan

mode (product ion, m/z 311; mass range, m/z 300–800; collision energy, −17 V; scan speed, 0.2 sec/scan) and (ii) the production-ion-scan mode (precursor ion, m/z 728; mass range, m/z

200–800; collision energy, −10 V; scan speed, 0.2 sec/scan) The argon gas pressure for the collision was set at 230 kPa

2.7 Characterization by NMR

A sample of the J2 component of the PEs (ca 4 mg), which was isolated from the Jatropha

seed, was dissolved in 0.7 mL of deuterated dichloromethane (CD2Cl2) and analyzed by 1H NMR (400 MHz) using a JNM-GX400 FT-NMR spectrometer (JEOL, Tokyo, Japan) Each sample was scanned for more than 1 h and the resulting data were analyzed using the Delta NMR software (ver 5.0.4.4) (JEOL)

3 Results and discussion

310 and 320 nm in the UV contour view, which corresponded to the conjugation of the four double bonds with a molar absorption coefficient (ε) of approximately 8 × 104 [23]

Unfortunately, however, the precise nature of these components could not be determined by HPLC analysis because of their low concentrations These results therefore indicated that the group of PEs consisted of more than seven components with an isomeric dicarboxylic acid moiety

conjugated double bonds (i.e., an ε value of 3.6 × 104 for max 250–280 nm) [23] The

components of J7 exhibited three high intensity absorption maxima with max values of 291,

304 and 318 nm, which were attributed to the chromophore of the four conjugated double bonds, as described in section 3.1.1 In contrast, the spectra of the components of J4 and J6 were merged with two chromophores discussed above and gave a series of broad UV bands with max values of approximately 280 nm and three maximum bands with max values of 290,

305 and 320 nm These results suggested that J4 and J6 consisted of two components with one containing a chromophore consistent with three conjugated double bonds and the other containing a chromophore consistent with four conjugated double bonds

3.2 Isolation of PEs using preparative ODS-3 HPLC column

3.2.1 Isolation of components

Using the methods described in section 2.4, the five components of PEs were individually isolated from the crude mixture of PEs by semi-preparative HPLC using a reversed-phase CDS-3 column The HPLC chromatograms of the isolated components (J2, J3, J5, J6 and J7) are shown in Fig 3 A small amount of J1 was contained in the fraction J2, and a small

amount of J4 in the fraction J3, respectively Using by the semi-preparative column, the J1 was a shoulder peak of J2, the biggest one of phorbol esters and the J4 was almost overlapped with the J3 because of its lower resolution They were hardly isolated each other using by the semi-preparative column Based on the HPLC chromatograms at a wavelength of 280 nm, the purity of each component was estimated to be more than 90%

3.2.2 Assignment of the isolated components

Based on the results of the UV absorption and 1H NMR analyses discussed in section 3.3,

J2 (λmax 283 nm, tR 15.3 min) was assigned as Jatropha factor C1 (λmax 284 nm in MeOH, tR

12.2 min) [15] Based on their UV absorption spectra and comparison of elution order on two kinds of C8 reversed-phase column, J3 (λmax 281 nm, tR 16.5 min), J5 (λmax 275 nm, tR 17.6 min) and J7 (λmax 291 nm, 304 nm, and 318 nm, tR 19.1 min) were assigned as Jatropha factors C2 (λmax 280 nm in MeOH, tR 13.2 min), C3 (λmax 272 nm in MeOH, tR 14.2 min) and

C4/5 (λmax 290, 303 and 317 nm in MeOH, tR ca 15.8 min) [15], while J6 (λmax ca 280, 292,

304, and 320 nm, tR 18.6 min) was assigned as a mixture of Jatropha C6 (λmax 273 nm, tR ca 15.8 min) [15] and a component containing four conjugated double bonds as discussed in 3.1.2 From the aspect of normal C8-reversed phase HPLC analysis, the elution order

observed by ODR-3 column were J2 < J3 < J5 < J6 < J7 in the order of increase, and

similarly that by Spherisorb S5 Octyl [15] were Jatropha factor C1 < C2 < C3 < C6 ≈ C4/5 These results indicated that the ODR-3 column showed the more superior separation ability than the Spherisorb S5 Octyl column, and its performance was sufficient to separate partially

a series of PEs consist of the complex phorbol ester isomers such as J6 components

3.3 Structural features of the PEs by NMR analysis

The isolated component of J2 was submitted for 1H NMR analysis The NMR details of the 12-deoxy-16-hydroxyl-phorbol and alkyl dicarboxylic acid moieties of J2 are shown in Tables 1 and 2 For comparison, the 1H NMR details of the Jatropha factor C1 isolated from Jatropha seed oil by Hirota et al [14] and Haas et al [15] have also been cited The chemical

shifts of the different hydrogen atoms of the 12-deoxy-16-hydroxyl-phorbol and alkyl

dicarboxylic acid moieties of J2 were consistent with those of Jatropha factor C1 and the structure of J2 was therefore assigned as Jatropha factor C1 Although J3, J5, J6 and J7 were also successfully isolated in the same way as J2, the concentrations of these materials were

too low to be analyzed by 1H NMR spectroscopy Furthermore, the observed values for these components in Fig 3 were overestimated as a consequence of using TPA as an external standard during their quantification by HPLC analysis [10]

3.4 Characterization of the PEs by LC-MS/MS Analysis

3.4.1 The ESI precursor ion (m/z 293) scan analysis

The purified PEs by column chromatography was subjected to the LC-MS/MS analysis The analytical parameters of the apparatus were optimized using by J2 fraction as a standard described in section 3.2.1 Tigliane-type PEs typically gives two diagnostic ions of the

diterpene skeleton with m/z values of 311 and 293 [22] The precursor ion scan

chromatogram (m/z 293) and the precursor ion spectra of the different peaks in Fig 5 were

analyzed in order to elucidate their quasi-molecular ions (parent ions) The precursor ion spectra of J2, of which the structure was identified by 1H-NMR spectrum was shown in Fig 5-J2 The two highly abundant mass ions, including an ammonia adducted quasi-molecular mass ion, [M+NH4]+ with m/z value of 728 and a fragment ion, [M-OH2+H]+ with m/z value

of 693 were obtained, accompanying with much weaker hydrogen adducted molecular ion [M+H]+ with m/z value of 711 The other components of PEs (J1, J3, J4, J5, J6 and J7) also

showed the almost similar patterns of the precursor ion spectra with that of J2 These results indicated that all components of PEs were the 12-deoxy-16-hydroxyphorbol diesters with same molecular weight of 710

3.4.2 The ESI product ion (m/z 728) scan analysis

The ESI product ion (m/z 728) scan chromatogram and the product ion spectra of the

different peaks identified in the chromatogram were shown in Fig 7 The ESI product ion chromatogram contained seven peaks, which were the same as those observed in the HPLC