Four Prenylflavone Derivatives with Antiplasmodial Activities from the Stem of Tephrosia purpurea subsp. leptostachya

Four new flavones with modified prenyl groups, namely (E)-5-hydroxytephrostachin (1), purleptone (2), (E)-5-hydroxyanhydrotephrostachin (3), and terpurlepflavone (4), along with seven known compounds (5–11), were isolated from the CH2Cl2/MeOH (1:1) extract of the stem of Tephrosia purpurea subsp. leptostachya, a widely used medicinal plant. Their structures were elucidated on the basis of NMR spectroscopic and mass spectrometric evidence. Some of the isolated compounds showed antiplasmodial activity against the chloroquine-sensitive D6 strains of Plasmodium falciparum, with (E)-5-hydroxytephrostachin (1) being the most active, IC50 1.7 ± 0.1 μM, with relatively low cytotoxicity, IC50 > 21 μM, against four cell-lines.


Introduction
Tephrosia purpurea (family Leguminosae) is one of the most widely distributed Tephrosia species and is found in tropical, subtropical, and other arid parts of the world. It consists of the four subspecies purpurea, leptostachya, appolinea, and barbigera, and four varieties, namely under subsp. leptostachya var. leptostachya and var. pubescens, and under subsp. barbigera var. barbigera and var. rufescens [1][2][3][4][5]. In Africa, a decoction of roots, leaves, and fruits of Tephrosia purpurea is given as a diuretic, for blood purification, and for the treatment of a cough and cold [6]. Its macerated leaves are used for curing diarrhoea and whooping cough in children [6]. In East Africa, its roots are used against stomach pains, while its leaves are used to treat snake bites and headaches. A decoction of its leaves and roots is used as a purgative [7], whereas that of the roots of T. purpurea subsp. leptostachya is employed for the treatment of schistosomiasis [6].
Phytochemical studies on T. purpurea collected from different parts of the world have resulted in the isolation of a wide variety of flavonoids; flavones [8,9], rotenoids [10], chalcones [11], and flavanones [12]. The crude extracts and pure compounds obtained from T. purpurea have shown a wide range of biological activities including antiplasmodial [12,13], anticancer [14], antacid [15], antidiabetic [16], analgesic and anti-inflammatory [17], and hepatoprotective [18] activities, and were also shown to be applicable to treat Helicobacter pylori infection [19]. Despite the presence of several subspecies and varieties of the taxa T. purpurea, the ethnobotanical, bioactivity, and phytochemical reports available so far have not been specific on the particular subspecies and variety. In order to better understand the relationship between T. purpurea and other species, the chemical variability among its subspecies and varieties has to be documented. With this in mind, the first phytochemical and biological report on T. purpurea subsp. leptostachya is reported here.

Results and Discussion
Extraction of the air dried stem of T. purpurea subsp. leptostachya with CH 2 Cl 2 /MeOH (1:1) at room temperature, followed by a combination of chromatographic separations, gave four new (1)(2)(3)(4) and seven known (5-11) compounds ( Figure 1). flavanones [12]. The crude extracts and pure compounds obtained from T. purpurea have shown a wide range of biological activities including antiplasmodial [12,13], anticancer [14], antacid [15], antidiabetic [16], analgesic and anti-inflammatory [17], and hepatoprotective [18] activities, and were also shown to be applicable to treat Helicobacter pylori infection [19]. Despite the presence of several subspecies and varieties of the taxa T. purpurea, the ethnobotanical, bioactivity, and phytochemical reports available so far have not been specific on the particular subspecies and variety. In order to better understand the relationship between T. purpurea and other species, the chemical variability among its subspecies and varieties has to be documented. With this in mind, the first phytochemical and biological report on T. purpurea subsp. leptostachya is reported here.

Plant Material
The stems of Tephrosia purpurea subsp. leptostachya were collected in April 2015 from the Kilungu hills in Makueni County, Kenya. The plant specimen was identified by Mr. Patrick C. Mutiso of the Herbarium, School of Biological Sciences, University of Nairobi, where a voucher specimen (Mutiso-841/April 2015) was deposited.

In Vitro Antiplasmodial Activity
The pure compounds were assayed using a non-radioactive assay technique as described by Smilkstein et al., 2004 [32] with modifications given in the literature [12,33].

Cell Culture
A549, HepG2, and non-tumoral cells were all purchased from ATCC. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics penicillin (50 U/mL) and streptomycin (50 µg/mL; Invitrogen, Paisley, Scotland, UK). All cell cultures were incubated at 37 • C in a 5% humidified CO 2 incubator.

Cytotoxicity Assay
All tested compounds were dissolved in DMSO at a final concentration of 50 mmol/L and stored at −20 • C before use. Cytotoxicity was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5.0 mg/mL) assay as previously described [34]. Briefly, 4 × 10 3 cells per well were seeded in 96-well plates before drug treatments. After overnight culture, the cells were then exposed to different concentrations of selected compounds (0.039-100 µmol/L) for 72 h. Cells without drug treatment were used as the control. Subsequently, MTT (10 µL) solution was added to each well and incubated at 37 • C for 4 h followed by the addition of 100 µL solubilization buffer (10% SDS in 0.01 mol/L HCl) and overnight incubation. A 570 nm was then determined in each well on the next day. The percentage of cell viability was calculated using the following formula: Cell viability (%) = A treated /A control × 100. Data were obtained from three independent experiments and the standard error was calculated.

Conclusions
Four new prenylflavones with seven known compounds were isolated from the stem of Tephrosia purpurea subsp. leptostachya. The isolated flavones were tested for antiplasmodial activity against the D6 strain of Plasmodium falciparum. Among these, (E)-5-hydroxytephrostachin (1) showed good activity (IC 50 1.7 µM). The compounds were also tested for cytotoxicity against two non-tumoral and two cancerous cell-lines. Most of these did not show cytotoxicity (IC 50 > 100 µM), while compound 1 showed IC 50 between 21-100 µM.
Supplementary Materials: The Supplementary Materials are available online. NMR, UV and MS spectra for all new compounds and spectral data for the known compounds are available as Supporting Information.