Dereplication of cytotoxic compounds from different parts of Sophora pachycarpa using an integrated method of HPLC, LC-MS and 1H-NMR techniques

Abstract Sophora pachycarpa Schrenk ex C.A.Mey. is an annual plant belonging to the family Fabaceae. The cytotoxic activities of methanol-dichloromethane extracts (1:1) of different parts of S. pachycarpa were investigated on DU145 (prostate cancer cell line) and MCF-7 (breast cancer cell line) cell lines. The root extract of S. pachycarpa was the only extract that showed significant cytotoxic activity with IC50 values of 39.88 and 16.49 μg/mL on DU145 and MCF-7 cell lines, respectively. The root extract was then subjected to RP-HPLC for further fractionations. Among the isolated fractions from root extract, only one of them had remarkable cytotoxic effects with IC50 value of 26.43 on MCF-7 and 7.54 μg/mL on DU145 cell lines. Further purification led to isolation of a compound with IC50 values of 5.44 and 2.44 μg/mL on MCF-7 and DU145 cell lines, respectively. Based on 1H NMR and 13C NMR spectra, together with LC-MS, the structure of the purified compound was assigned as the flavonostilbene alopecurone A.


Introduction
Cancer is known as one of the most major causes of mortality in Iran and day by day more people are getting involved with this disease. As synthetic drugs which are used as the treatment, have shown a wide range of side effects and interactions, investigation on finding natural cytotoxic agents as sources of anticancer compounds in plants are attracting more attentions. Many important new drugs are obtained from natural sources by modifying the natural products or designing and synthesising new compounds using the natural compounds as the model (Gordaliza 2007 The genus Sophora from the family Fabaceae comprises 52 species around the world and most of them are distributed in Asia (emami et al. 2007). This genus is a rich source of flavonoids (Iinuma et al. 1995), prenylated flavonoids and quinolizidine-type alkaloids (Ruangrungsi et al. 1992;Atta-ur-Rahman et al. 2000). To date, either as a crude extract or as their purified compounds many species of genus Sophora showed cytotoxic activities (Jae et al. 2007;Lin et al. 2011;Vithya et al. 2012;Wang, Jin, et al. 2015;Yang et al. 2015).
Previous studies revealed the presence of quinolizidine alkaloids in Sophora pachycarpa (Abdusalamov et al. 1989). In addition, we reported isolation of prenylated flavonoids and flavonostilbenes from this plant (emami et al. 2007). In a study by Mousavi and colleagues, methanolic extract of S. pachycarpa induced apoptosis in A549, HeLa, HL-60, MCF-7, and PC3 cell lines. However, the active compounds have not been identified (Mousavi et al. 2014).
In this study, we aimed to identify cytotoxic compounds of S. pachycarpa methanolic extract using an integrated method of HPLC, LC-MS and 1 H NMR techniques. In this work, we evaluated cytotoxic properties of different parts of S. pachycarpa including leaves, seeds, flowers and roots on different cancer cell lines.

Results and discussion
According to Table 1 the concentrations of doxorubicin as positive control which are capable of killing 50% of the cells in MCF-7 and DU145 cell lines are 1.266 and 0.107 μg/mL, respectively.
The extracts of leaves, flowers and fruits did not show significant cytotoxic effect comparing to the control positive in both cell lines. However, the root extract ( Figure 1) showed remarkable cytotoxicity with IC 50 values of 39.88 μg/mL in MCF-7 and 16.49 μg/mL in DU145 cell lines (Table 1) which implied that this part of the plant had a considerable cytotoxic effect and further investigations were needed to be done in order to find the compound that is responsible for this feature.
For this aim, the root extract was divided into four different fractions according to their retention time using the semi-preparative high performance liquid chromatography (HPLC-DAD) as shown in Figure S4. To distinguish which fraction plays the role of cytotoxicity, same doses of each fraction (1, 10, 25, 50 and 100 μg/mL) were tested in both cell lines. Fr 4 was the only fraction that showed cytotoxicity and its IC 50 values (Table 1) for both cell lines were less than 100 μg/mL (26.43 μg/mL in MCF-7 and 7.546 μg/mL in DU145 cell lines). This fraction was the candidate for further investigations. Figure S1 presents the LC/MS diagram obtained from the cytotoxic fraction. The positive scan on the Fr 4, contains a peak at 651.10000 m/z which coincides with the molecular weight of alopecurone A (650.25 g/mol). Two major compounds were isolated from the cytotoxic fraction by preparative thin-layer chromatography (PTLC) and their purities were confirmed by thin-layer chromatography (TLC) and their cytotoxicity effects were investigated on the both cell lines. only one of the pure compounds showed cytotoxicity with IC 50 values less than 10 μg/ mL. Thus, the cytotoxic compound was purified with PTLC method in higher amounts to recognise the detailed molecular structure. For this purpose, the structure of the compound was investigated with LC/MS and NMR experiments. The LC/MS results are shown in Figure  S2 and as it can be seen, the diagram shows a sharp peak at m/z 651.10 in positive scanning and another sharp peak at m/z 649.20 in negative scanning; which can be related to alopecurone A (Figure 2) with molecular weight of 650.25 g/mol.
The data obtained from 13 C NMR and 1 H NMR spectra are shown in Table S1. In 1 H NMR spectrum, 7″ and 8″ protons showed two doublets at δ H 5.51 and 4.39 with the coupling constant of 4.5 Hz. Hydrogens of the 4-hydroxy phenyl group on the 7″ carbon were appeared at 6.87 and 7.24 ppm as doublet peaks with the same coupling constant of 8.8 Hz.
In 13 C NMR spectrum, 7″ carbon is appeared at 94.9 ppm which is assigned to an ether carbon. Also, two peaks were appeared at 158.3 ppm and 158.4 ppm which were related to 11″ and 13″ aromatic carbons. Chemical shifts of 158.4 and 107.2 ppm were assigned to 4′ and 5′ carbon, respectively.
Based on 13 C NMR, 1 H NMR and LC/MS data and those reported in the literature (Azimova & Vinogradova 2013) the structure of purified compound was assigned as alopecurone A.
In an in vitro study in 2014, cytotoxicity and apoptogenic effects of methanol extract of the S. pachycarpa plant was demonstrated on MCF-7 cell line and the results showed that this extract decreases cell viability in a concentration-related order with IC 50 value of 52.33 μg/ mL. This was the first investigation on cytotoxicity effects of S. pachycarpa (Mousavi et al. 2014). However, the active constituents have not identified. In this study, alopecurone A was identified as cytotoxic constituent of the methanolic extract of S. pachycarpa.
Many species of genus Sophora showed cytotoxicity in previous studies (Shu et al. 2014;Mathi et al. 2015;. However, alopecurone A was not the active constituent in these studies. In a recent study, alopecurone A showed potent inhibition of multidrug resistance associated protein 1 (Ni et al. 2014). However, no data are available in literatures for its cytotoxicity. Here, for the first time we reported as a remarkable cytotoxic compound and further studies should focus on its mechanism(s) of action.

Plant materials
S. pachycarpa whole plant was collected from fields near Gonabad in the Khorasan province, north-east of Iran. The plant was identified in Ferdowsi University of Mashhad Herbarium and a voucher specimen (No. 12604) was deposited in the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences. Leaves, flowers, fruits (seeds) and roots of the plant were separately air dried and powdered.

Preparation of extract
Different parts of the plant were powdered separately and macerated in a solution of 1:1 methanol-dichloromethane over 48 h and the extraction process was followed by condensation on a rotary-evaporator and then the extract was fully dried with a freeze-drier device.

Cell cultures and viability tests
DU145 and MCF-7 cell lines were obtained from Pasteur institute (Tehran, Iran) and separately maintained in flasks containing RPMI1640 growth medium enriched with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere (90%) containing 5% Co 2 . Also for each concentration and each time course study, a control sample was prepared which remained untreated but received equal volume of the growth medium. For measuring the cell viability in presence of doxorubicin as control positive for DU145 and MCF-7 cell lines, same concentrations of doxorubicin (0.5, 2.55 and 10 μg/mL) were used in both cell lines. To measure the cell viability in presence of the extracts of different parts of the plant, same concentrations of each part of the plant (5, 25, 50 and 100 μg/mL) were used in both cell lines and the viability percentages were measured by alamarBlue® test. Resazurin (alamarBlue®) is a blue weakly fluorescent dye which is reduced irreversibly to red and highly fluorescent resorufin in living cells. It is effectively reduced in mitochondria which makes it useful to assess mitochondrial activity and cell viability. About 10 4 cells of each MCF-7 and DU145 cell lines were seeded in each well of a 96-microwell plate and treated with various concentrations of extracts, fractions and pure compounds. After 72 h incubation, alamarBlue® was added to each well (10% w/w from stock solution of 140 μg/mL) and after 4 h of incubation, the absorbance was read at 570 and 600 nm in a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, U.S.A). IC 50 values for extracts, fractions and pure compounds isolated from cytotoxic fraction were calculated by excel 2013 software. Data were presented as mean ± SeM of three independent experiments with three replicates for each concentration of the extracts.
The same procedure was done for fractions and purified compounds to evaluate their IC 50 values.

Chromatographic conditions
Semi-preparative HPLC-DAD was performed on a KNAUeR liquid chromatograph system consisting of a quaternary pump (Smartline Pump 1000). Detection was carried out using diode array detector (Smartline DAD 2800), and data were processed using eZ Chrom elite software. The methanol-dichloromethane extract and fractions were subjected to reversephase HPLC using a gradient method of 20-100% methanol in water as the eluent including 0.05% trifluoroacetic acid. The separation was done on ACe 5 C18 (Advanced Chromatography Technologies Limited, Aberdeen, Scotland; 5 μM, 250 × 21.2 mm) at a flow rate 9 ml/min. The injection volume was 0.5 mL. A standard solution containing uracil, 4-hydroxymethyl benzoate, 4-hydroxy ethyl benzoate and benzophenone was injected into the HPLC device in order to validate the reliability of the system (Figure 1).
For further purifications of cytotoxic compounds, cytotoxic fraction obtained from HPLC was dissolved in methanol and loaded on preparative TLC plates with ethyl acetate:petroleum ether (70:30) as the eluent.
The 1 H NMR spectra of the isolated compounds were recorded at 30 °C on a Varian 600 1 H NMR spectrometer. Samples were dissolved in DMSo-d 6 . Chemical shifts at 2.50 ppm were referenced to the solvent peaks. LC-MS data was generated using a Waters Alliance 2790HPLC equipped with a Waters 996 photodiode array detector that was connected to a Waters ZQ mass spectrometer. A phenomenex analytical Luna C18 column (5 μm 100 Å, 4.6 × 50 mm) was used for LC-MS.
Molecular weights of the compounds in cytotoxic fraction were determined by LC/MS method and the obtained data were compared with Dictionary of Natural Products (DNP, version 20:2, CRC press).
Also the molecular weight of the cytotoxic pure compound isolated from the cytotoxic fraction was determined with the same method as mentioned above and was compared with DNP.

Statistical analysis
A one-way analysis of variance was used for testing overall group differences. If the p value was less than 0.05, differences between means were considered statistically significant.

Conclusion
In the present study, S. pachycarpa was investigated for its cytotoxic effect and the roots of the plant were recognised to be significantly cytotoxic. Further purification using an HPLC device led to isolation of alopecurone A as cytotoxic constituent with IC 50 values of 5.4 and 2.4 μg/mL on MCF-7 and DU145 cell lines, respectively. Regarding of the data obtained from this study, alopecurone A can be considered as a new potential anticancer compound for further investigation.