Crystal Structure Elucidation and Anticancer Studies of (-)-Pseudosemiglabrin: A Flavanone Isolated from the Aerial Parts of Tephrosia apollinea

Tephrosia apollinea is a perennial shrublet widely distributed in Africa and is known to have medicinal properties. The current study describes the bio-assay (cytotoxicity) guided isolation of (-)-pseudosemiglabrin from the aerial parts of T. apollinea. The structural and stereochemical features have been described using spectral and x-ray crystallographic techniques. The cytotoxicity of isolated compound was evaluated against nine cancer cell lines. In addition, human fibroblast was used as a model cell line for normal cells. The results showed that (-)-pseudosemiglabrin exhibited dose-dependent antiproliferative effect on most of the tested cancer cell lines. Selectively, the compound showed significant inhibitory effect on the proliferation of leukemia, prostate and breast cancer cell lines. Further studies revealed that, the compound exhibited proapoptotic phenomenon of cytotoxicity. Interestingly, the compound did not display toxicity against the normal human fibroblast. It can be concluded that (-)-pseudosemiglabrin is worthy for further investigation as a potential chemotherapeutic agent.


Introduction
Chemotherapy can be defined as the use of chemicals to treat cancer by preventing cancer cells from dividing, proliferating and surviving. Chemotherapeutic agents can be divided into two major groups: natural products and synthetic. Currently more than 27% of all prescription drugs are derived from natural sources [1]. This is more significant with regard to anti-cancer drugs in which more than 80% are plant-derived compounds [2]. Synthetic chemotherapy can potentially cause serious side effects, the toxicity associated with the conventional cancer chemotherapy arises primarily from the lack of specificity for tumor cells. Most of the currently available anticancer drugs are designed to have selective toxicity towards rapidly dividing cells [3]. It is reported that natural compound(s) especially of plant origin, with selective cytotoxicity against specific tumor cell line, can offer either prophylaxis (chemoprevention) or a relatively safer treatment option with minimum side effect [4]. Chemopreventive agents derived from natural products or their synthetic derivatives have shown cell growth inhibition, antiproliferation and apoptosis in various cancer cell lines [5]. For example, retinoids and antiestrogens are known to block or delay the progression of transformed cells by modulating cell proliferation or differentiation, these agents are believed to promote cytostatic effects [6,7,8].
Tephrosia apollinea is a member of family Leguminosae and genus Tephrosia. T. apollinea is a perennial shrublet which is widely distributed in Africa. In Sudan the shrublet is abundantly distributed in the Nile Valley, in the sub-Saharan northern region and along the coast of the Red Sea. Several reports have indicated that the extract from some species of the genus Tephrosia possess dynamic pharmacological activities such as, piscicidal, insecticidal and anti-cancer properties [9]. Furthermore, a review of the literature indicates that a number of species of Tephrosia have been studied for their chemical compositions [10]. Studies analyzing their chemical composition revealed the presence of rotenoids, isolflavones, flavanones, chalcones, and flavones. The study by Abou-Douh et al. [11] reported the presence of complex prenylated flavones derived from 7-oxygenated compounds in the extracts of T. apollinea. The most recent work on T. apollinea describes the isolation of stereoisomers, (-)-semiglabrin and (-)pseudosemiglabrin [8]. Additionally, the study also explored the stereochemistry of (-)-semiglabrin using x-ray crystallography [11].
Using an in vitro model of anticarcinogenesis, the study [11] reported that, (-)-pseudosemiglabrin showed no significant anticarcinogenic activity in a cell and enzyme based in vitro assay against H4IIE rat hepatoma cells. The study [8] reported that (-)pseudosemiglabrin failed to inhibit the enzymes (cytochrome 1A and quinone reductase) involved in carcinogen metabolism and detoxification. The study [8] further reported that the compound did not show inhibitory effect on the enzymes (cyclooxygenase-1 and cyclooxygenase-2) actively involved in tumor-promoting mechanism.
In the present study, extracts of the aerial parts of T. apollinea were subjected to bioassay-guided fractionations, which resulted in isolation of (-)-pseudosemiglabrin (SSG). The structural and stereochemical features were confirmed by spectral and X-ray crystallographic techniques. The compound was evaluated for its potential antiproliferative effect against a panel of human cancer and normal cell lines. Furthermore, an attempt was made to understand the mode of cytotoxicity induced by SSG in cancer cells by performing Hoechst 33342 and rhodamine 123 fluorescence assays.

Plant Extract and Isolation of Active Compound
Aerial parts of T. apollinea were sequentially extracted with nhexane, chloroform and ethanol to obtain three respective crude extracts ( Figure 1). Among all the extracts, chloroform extract showed most potent anti-proliferation activity against HL-60 (IC 50 19.2 mg/mL), K562 (14.8 mg/mL) and MCF-7 (16.4 mg/mL) cell lines. Chromatographic fractionation of chloroform extract yielded ten fractions (F1-F10). Among all the fractions, F5 was found to be the most active fraction against the proliferation of HL-60 (IC 50 13.6 mg/mL), K562 (26.1 mg/mL) and MCF-7 (11.4 mg/mL). Thus, F5 was further chromatographed using gradient elution of n-hexane-dichloromethane to yield SSG. A detailed procedure is described in the experimental part.

Spectroscopy
SSG was obtained as light green crystalline plates, M.P: 170-180uC. The molecular mass was determined by liquid chromatography-mass spectroscopy (LC-MS) and showed a molecular ion peak at 393.11. The ultraviolet (UV) spectrum showed absorption at l max 306, 256, (sh) and 215 nm indicating the flavone characteristics of the compound SSG [11,12,13]. The infrared (FT-IR) spectrum showed a strong and sharp vibrational band at 1740 cm 21 that indicates the presence of carbonyl (CO) moiety, more likely CO of an acetate group [14]. Also, a medium intensity band at 1640 cm 21 attributed the CO of pyranone ring [15,16]. Furthermore, a vibrational band at 1574 and 1604 cm 21 ascribed the benzene ring carbon-carbon stretch. These prominent characteristic features indicate the presence of flavones, a class of compounds based on a backbone of 2-phenylchromen-4-one [11,17,18,19]. The presence of alkyl groups was imputed by two vibrational bands at 2850, 2939 and 2974 cm 21 [20,21]. These three weak bands indicate the presence of alkyl groups attached to flavone backbone ( Figure 2).
Further, the title compound was characterized by 1 H and 13 C-NMR. The 13 C DEPT-135 and 145 NMR spectra recorded in CDCl 3 at 125.7 MHz at room temperature are shown in Figures S1 and S2, respectively in File S1. The 2D HSQC and HMBC NMR spectra of SSG are shown in Figures S3 and S4, respectively. Figures S5 and S6 in File S1 illustrate the characteristic diagonal component and cross peaks of SSG  obtained in 2D TOCSY and COSY NMR spectra, respectively. The data obtained from these spectral analyses were found to be comparable with that of the previous reports [11,22].

Crystallography
Crystals of title compound suitable for x-ray crystallographic study were obtained by slow evaporation of the compound in dichloromethane/n-hexane solvent system (1:3). The crystals appeared as light green plates.
The compound crystallized in orthorhombic space group P2 1 2 1 2 1 , with two crystallographically different molecules having slightly different geometric parameters. Each unit of the compound consists of two benzene, one pyranone and two tetrahydrofuran (THF) rings. In addition, an acetate group is attached with one of the THF groups. The crystal refinement data is shown in Table 1, whereas the selected bond lengths and angles   Table S1 and Table S2, respectively in File S1. The detailed aspects of the crystal structure are illustrated in Figure 3.
It is an interesting and rarely observed phenomenon [23,24] in crystallography when the molecules of the same compound pack with slightly different geometries. This phenomenon, however, might not alter the biological properties of the compound. Nevertheless, the stereoisomers have some significant differences regarding physical properties like melting point, crystal packing, crystal color etc [25,26,27] as well as biological efficacies [26,27]. For example, the crystal structure of (-)-semiglabrin, a diasteroisomer of title compound ( Figure 4) has been reported by Abou-Douh et al [11] where the single crystals appeared as colorless needles instead of light green plates for SSG in the current report.
The melting point of the crystals was about 258-260uC for needles whereas in the current study the observed melting points were in the range of 168-170uC. Also, a number of bond angles are slightly different (1.560.5u) than these angles in its stereoisomer, (-)-semiglabrin. For example, O4A-C22A-C23A in SSG is 110.29(16)u whereas in (-)-semiglabrin the same bond angle is oriented at 108.10(11)u. The other bond angles are C16A-O4A-C22A and C15A-O3A-C14A having angles 117.41(15)u and 110.58 (14)u in the current structure whereas in its isomer these are 118.8(10)u and 111.10(9)u, respectively (Refer to Table S1 in File S1). Furthermore, these crystallographically different units also have difference in bond angles compared to each other e.g., C21A-C15A-C20A and C15A-O3A-C14A in structure A have a difference of 60.5u compared to the same angles in structure B ( Figure 3). The variations in the bond angles and bond lengths between the two units are given in Tables S1 and S2 in File S1.
Moreover, in both the geometrically different molecules, the THF rings make the dihedral angles of 11060.5u and 11660.5u with each other at both ends. All the three rings (two benzene and one pyranone) are in the same plane with a benzene and pyranone ring connected through a single bond. Such bonding situations have been more likely found to have some orientations in horizontal plane [28,29,30,31,32] however, in the current case both the rings are in the same plane. This might be due to packing effects where the molecules have p-p stacking interactions that might keep these rings in the plane. Figure S7 in File S1 illustrates the crystal packing pattern, which shows that the molecules are connected through the C = O ---H bonding in a three dimensional network.

Effect of SSG on Proliferation of Cancer Cell Lines
Antiproliferative effect of SSG was tested against nine tumor cell lines and one normal cell line using MTT assay. The median inhibitory concentration (IC 50 ) values were calculated for each cell line [33] and the values are given in Table 2. The compound showed selective cytotoxicity against six cancer cell lines namely The median inhibitory concentrations (IC 50 ) were determined by nonlinear regression analysis of log-concentration-response curves of 3 different tests (n = 3  Figure 5 shows the graphical illustration of the dose-dependent antiproliferative effect of SSG on various human cancer cell lines. Apparently this finding is in contrast with that of the previous study in which SSG was reported to be inactive in a cell-enzyme based in vitro assay conducted using H4IIE rat hepatoma cells, as the compound failed to show inhibitory effects on the initiation, promotion, and progression stages of the assay [11]. Nevertheless, the results of the present study agree in part with the previous findings in the sense that SSG did not show anti-proliferation activity against human hepatic carcinoma (HepG2) cells as the IC 50 value was found to be more than 300 mM. Interestingly, the compound displayed selective cytotoxicity against prostate, breast, leukemia and colon carcinoma cell lines. Figure 6 shows the effect of SSG on various human cancer cell lines. The results revealed that SSG was nontoxic to normal colonic fibroblast (CCD-18Co) cells (IC 50 = 327.25 mM).
A study reports that c-Pyranone derivatives isolated from Erigeron annuus showed poor antiproliferative activity against human hepatoma (SMMC-7721), embryo liver (L-02) and leukaemia (HL-60) cell lines [34]. However, the study revealed that a c-Pyranone derivative with ester group attached to the long side chain exhibited improved cytotoxicity especially against HL-60. Similarly, SSG also contains an ester group attached to one of the tetrahydrofuran rings and showed a promising cytotoxicity against HL-60 (IC 50 = 15.7 mM). A number of studies have shown that compounds containing ester groups induce anticancer activity against cancer cells [35,36,37,38] especially against HL-60 cell line [39].
Furthermore, Kajimoto and coworkers isolated Sophoranone, a flavone, from a traditional Chinese medicine, namely Shan Dou Gen that induced apoptosis in human leukemia (U937) cells via formation of reactive oxygen species and opening of mitochondrial permeability transition pores (IC 50 = 21.5 mg/mL) [40]. In the current study, the tested compound showed even better cytotoxic effect with IC 50 = 5.76 mM. Moreover, comparing with some other flavones tested against U937 cell line [41,42,43], results of the present study revealed that SSG exhibited more pronounced cytotoxic efficacy. According to best of our knowledge this is the first report that describes anticancer potential of SSG against a panel of cancer cell lines, where it significantly showed selective anti-proliferative activity against leukemia, prostate, breast and colon cancer cell lines.

SSG Induces Morphological Modifications and Nuclear Condensation in the Cancer Cells
During apoptosis, cell exhibits a series of characteristic morphological and biochemical events, such as nuclear condensation, DNA fragmentation, dissolution of chromatin, and alterations in cellular membrane [44]. To detect particular events of apoptosis and trace mechanisms responsible for the apoptotic cell death, we employed commonly used staining assays to detect changes in the nucleus and mitochondria of treated cells.
In the present study, adherent cells (PC3, MCF 7 and HCT 116) were selected to study morphological modifications and nuclear condensation using Hoechst 33342 stain. The typical apoptotic morphological changes were observed in the cells treated with SSG in a time dependent manner ( Figure 7A). However, untreated cells displayed prominent nuclei and intact cell membrane without significant changes in cellular morphology. After 6 hr of SSG (10 mM) treatment, the nuclei started to condense and the chromatin distributed irregularly throughout the cytoplasm. The cells revealed shrunken, crescent-shaped nuclei with condensed chromatin, which are the sings of early stage of apoptosis ( Figure 7A). After 12 hr treatment, several cells showed the discrete chromatin bodies which are the characteristic sign of karyorrhexis, a later stage of apoptosis. The apoptotic index ( Figure 7B) for untreated PC3 was 3.960.07%, for, MCF-7 the apoptotic index calculated was 1.3560.08% and that of HCT 116 cells was 3.360.6% which were increased to 61.462.3% (PC3), 28.362.1% (MCF-7) and 19.261.4% (HCT 116) following treatment with SSG for 12 hr (Figures 8A and 8B).

SSG Reduces Mitochondrial Membrane Potential in PC3 Cells
Rhodamin 123 is a cationic probe which can be readily absorbed and accumulated in mitochondria of a live cell [45]. A loss of mitochondrial membrane potential (DY) is a marked indication of early apoptotic events [46]. To investigate whether the apoptosis induced by SSG in PC3 cells involved the loss of mitochondrial integrity, the mitochondrial membrane potential in PC3 cells was evaluated by visualizing the uptake of the lipophilic cation dye rhodamine 123 by mitochondria. The treated and untreated cells were exposed to rhodamine 123 and the intensity of rhodamine in the cells was observed ( Figure 9A). When the mitochondrial membrane potential decreases, the rhodamine 123 uptake by the cells also decreases and consequently the florescent signal reduces exponentially. Results of the present study showed an obvious intensification of fluorescence in the untreated cells, whereas the signal significantly reduced in the cells treated with SSG (10 mM), which suggests the loss in mitochondrial membrane potential. Furthermore, the fluorescent intensity decreased with increasing the treatment duration ( Figure 9A). The apoptotic indices after 6 and 12 h treatment with SSG were 41.463.1 and 67.164.2%, respectively ( Figure 9B). This result reveals that, there is a remarkable reduction in the mitochondrial membrane potential of PC3 cell line could be due to the induction of apoptosis caused by SSG.
Flavonoids and their metabolic precursors such as flavanones and phenolics have shown to possess promising anticancer properties. Our previous study [47] reported that plant flavonoids particularly polymethoxylated flavonoids, such as rosmarinic acid, eupatorin, sinensetin, and 39-hydroxy-5,6,7,49-tetramethoxyflavone exhibit anticarcinogenic properties via suppressing oxidative  stress in the cells. However, in the present study the photomicrographs of the affected cells in Hoechst 33342 and rhodamin 123 staining assays revealed that the toxicity caused by SSG could probably due to the induction of apoptosis pathway, as the affected cells clearly showed the unique features of apoptosis such as membrane blebbing, nuclear condensation and apoptotic bodies in cytoplasm of the cells. Few cells also showed the crescent shaped nuclei which indicate the advanced stage of apoptosis.
Chemopreventive agents from natural products that inhibit the transformation of normal cells to premalignant cells or the progression of premalignant cells to malignant cells are believed to function by modulating processes associated with xenobiotic biotransformation, with the protection of cellular elements from oxidative damage, or with the promotion of a more differentiated phenotype in target cells. Nevertheless, there is an increasing number of chemopreventive agents (e.g., retinoids, nonsteroidal anti-inflammatory drugs, polyphenols, flavonoids and vanilloids) which demonstrate apoptosis in premalignant and malignant cells in vitro or in vivo [5,48,49].
The isolated SSG was dissolved in DMSO to obtain 10 mg/mL stock solution and stored at 4uC. For treatment, SSG was diluted in indicated cultured medium at the indicated concentrations in each experiment.

Extraction and Fractionation
The plant materials were dried at room temperature and ground to a fine powder. Initially, 50 g of material was extracted with 200 mL n-hexane overnight. The filtrate was collected and the residue was brought to dryness and extracted with 200 mL chloroform following the same methods as for n-hexane. The filtrate was collected and the residue was brought to dryness and extracted with ethanol following the same method as for n-hexane and chloroform. Chloroform extract was found to be more active against cancer cell lines (HL-60, K562 and MCF-7), thus it was subjected to fractionation. For large scale extraction, 400 g of the plant material was extracted with chloroform using Soxhlet apparatus.

Isolation of (-)-Pseudosemiglabrin (SSG)
Chloroform extract (7 g) of T. apollinea was subjected to vacuum liquid chromatography which was performed on column (1067 cm) packed with silica gel of particle size (0.04-0.06 mm, 60-120 mesh). Solvent mixtures of petroleum ether, dichloromethane, and methanol were used in sequence of increasing polarities. Eluents of 100 mL each were collected and monitored by thin layer chromatography (TLC). The eluents with similar TLC chromatogram pattern were pooled together to obtain totally ten fractions. Bioassay results showed that fraction 5 (1.3 g) was the most active against the cell proliferation, which was further applied to column (3663.5 cm) packed with silica gel (65 g) of particle size 0.063-0.200 mm by gradient elution, starting with hexane-chloroform combination (95:05), followed by increasing amount of chloroform and methanol. A total of 124 fractions (10 mL each) were collected. Fractions 20-93 were combined together and purified using pen column (1662 cm) by gradient elution starting with petroleum ether-dichloromethane mixture (90:10) followed by increasing amount of dichloromethane. A total of 60 fractions (10 mL) were collected. Fractions 11-52 were combined on the basis of TLC profile to give one spot (600 mg). TLC analysis of the substance in several different solvent systems revealed a single fluorescent spot under UV light, with faint red spot of chlorophyll. Subsequently, the SSG was purified from the chlorophyll by fractional crystallization in mixture of n-hexane and dichloromethane to produce light green crystals (400 mg).

Characterization Techniques
The purified crystalline material was characterized by FT-IR spectrophotometer (FTIR-2000, Perkin Elmer, USA) using potassium bromide (KBr) disc method. According to this method sample was mixed with oven dried IR grade KBr and ground to fine powder. A disc (12.7 mm diameter and around 1 mm thickness) was obtained using hydraulic press (capacity 15 tons max.) at 8 tons for about half minute. The spectrum was scanned at infrared region of 400-4000 cm 21 .
Furthermore, the purified compound was analyzed by FT-NMR spectrometer (Bruker 500 MHz) in deuterated chloroform (CDCl 3 ). The NMR peaks were labeled as singlet (s), doublet (d), triplet (t), and multiplet (m), chemical shifts were referenced with respect to solvent signals.
The molecular weight of the compound was determined by liquid chromatography-mass spectrometer (LC-MS). The sample was prepared in HPLC grade methanol (10 mg/ml) and was filtered through 0.45 micron filter.
The single crystals obtained were analysed by Bruker SMART APEX2 CCD area-detector diffractometer. The molecular graphics were constructed by Bruker SHELXTL software.

In Vitro Cytotoxic Assay
Cytotoxicity of SSG was evaluated using MTT assay [47,50] against a panel of cell lines. The assay plates were read using a microtiter plate reader (Hitachi U-2000, Japan) at 570 nm absorbance. DMSO (0.1%) was used as a negative control. Tamoxifen, betulinic acid, 5-fluorouracil and imatinib were used as standard references.

Determination of Nuclear Condensation by Hoechst 33342 Stain
Effect of SSG on nuclear chromatin condensation in PC3, MCF7 and HCT 116 cells was quantified by fluorescence microscopy using Hoechst 33258 stain [51]. Cells were treated with SSG (10 mM) and analysed separately at two different time intervals (6 and 18 hr). Betulinic acid (10 mM) and 0.1% DMSO were used as positive and negative controls, respectively. The cells were fixed in 4% paraformaldehyde for 20 min before staining with Hoechst stain 33342 (1 mg/mL in PBS) for 20 min. Nuclear condensation and cytoplasm shrinkage was examined under a fluorescent microscope. Cells with bright colored, condensed or fragmented nuclei were considered apoptotic. The number of cells with apoptotic morphology was counted in randomly selected fields per well. The cells were photographed at 206magnification, using a EVOS f1 digital microscope (Advanced Microscopy Group, USA). The apoptotic index was calculated as a percentage of apoptotic nuclei compared to the total number of cells and presented as the mean 6 SD (n = 8).

Detection of Mitochondrial Membrane Potential by Rhodamin 123 Stain
Detection of the changes in mitochondrial membrane potential in PC3 cells treated with SSG was assessed by the retention of rhodamine 123 [26]. The PC3 cells were plated in 6 well plates for overnight. The cells were treated with SSG at 10 mM for 6 and 18 hr intervals and then fixed by 4% paraformaldehyde for 20 min. Betulinic acid (10 mM) and 0.1% DMSO were used as positive and negative controls, respectively. The rhodamine 123 was added to cells at a final concentration of 5 mg/mL and incubated for 30 min to stain the mitochondria. The wells then were photographed using inverted EVOS f1 digital microscope at 206 magnification power to monitor for fluorescent signals.

Statistical Analysis
Statistical difference between the treatments and the control were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differences were considered significant at p,0.05, and p,0.01.

Conclusions
In the present work, the isolation of (-)-pseudosemiglabrin (SSG) from aerial parts of T. apollinea and its detailed stereochemistry and antiproliferative activity is reported. From the results, it can be concluded that SSG has strong cytotoxic properties selectively against prostate, leukemia, breast and colon cancer cells. Eventually the pro-apoptotic property could be the principle factor for the observed cytotoxicity of SSG. Further, the in vivo antitumor studies of title compound are in progress using Ncr-nu/ nu mice xenograft models and will be reported in due course.

Additional Information
Crystallographic data of the structures have been deposited with the Cambridge Crystallographic Data Center, CCDC 946618 for (-)-pseudosemiglabrin. This data can be obtained free of charge from CCDC via www.ccdc.cam.ac.uk/data_request/cif.