Phytochemicals from Pterocarpus angolensis DC and Their Cytotoxic Activities against Breast Cancer Cells

Pterocarpus anglonesis DC is an indigenous medicinal plant belonging to the Pterocarpus genus of the Fabaceae family. It is used to treat stomach problems, headaches, mouth ulcers, malaria, blackwater fever, gonorrhea, ringworm, diarrhea, heavy menstruation, and breast milk stimulation. Column chromatography of the stem bark extracts resulted in the isolation of eight compounds, which included friedelan-3-one (1), 3α-hydroxyfriedel-2-one (2), 3-hydroxyfriedel-3-en-2-one (3), lup-20(29)-en-3-ol (4), Stigmasta-5-22-dien-3-ol (5), 4-O-methylangolensis (6), (3β)-3-acetoxyolean-12-en-28-oic acid (7), and tetradecyl (E)-ferulate (8). The structures were established based on NMR, IR, and MS spectroscopic analyses. Triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7), and non-cancerous mammary epithelial cell lines (MCF-12A) were used to test the compounds’ cytotoxicity. Overall, the compounds showed either no toxicity or very low toxicity to all three cell lines tested, except for the moderate toxicity displayed by lupeol (4) towards the non-cancerous MCF-12A cells, with an IC50 value of 36.60 μM. Compound (3β)-3-acetoxyolean-12-en-28-oic acid (7) was more toxic towards hormone-responsive (MCF-7) breast cancer cells than either triple-negative breast cancer (HCC70) or non-cancerous breast epithelial (MCF-12A) cells (IC50 values of 83.06 vs. 146.80 and 143.00 μM, respectively).


Introduction
Pterocarpus of the Fabaceae (Leguminosae) family comprises 41 species mostly of timber trees found in the tropical parts of the world and native to Africa, South America, and Asia [1].The Pterocarpus genus is known to produce secondary metabolites, including flavonoids, isoflavonoids, pterocarpans, auriones, lignans, stilbenes, sterols, sesquiterpenes, and triterpenes [2].Pterocarpus angolensis DC (commonly known as Kiaat in Afrikaans; African teak or wild teak, in English; Morôtô, in seSotho; Mokwa in SeTwana, Mutondo, in TshiVenda; Mukwa or Mubvamaropa in Shona and Umvangazi in isiZulu) grows in the warm, semi-arid areas in the northeast of South Africa, Mozambique, Zimbabwe, northern Botswana, and Namibia and other northern parts of Africa [3].It grows in subtropical woodlands where the rainfall is above 500 mm per annum [3].The tree can be found in eastern and southern Africa at elevations ranging from sea level to roughly 1650 m in all types of woodland and wooded savannah [4].P. angolensis is used for a variety of purposes in folk medicine.A cold infusion is used to treat stomach problems, headaches, and mouth ulcers [5].Its decoction of bark and infusion of roots are often used in the treatment of different ailments such as to cure malaria, blackwater fever, gonorrhea, heavy menstruation, blood in the urine, and schistosomiasis [5,6].The tree is a superb wood that is resistant to termites, marine borers, and beetles [7].This wood is mostly used for furniture, joinery, and decorative veneer [3,5].
The compounds from P. angolensis exhibited antibacterial properties with the minimum inhibition concentration (MIC) of epicatechin-3-O-gallate 50 µg/mL against Staphylococcus aureus and Acinetobacter calcaoceuticus and hexamer of epicatechin 25 µg/mL against Staphylococcus typhi and Kocuria kristinae [9].Additionally, the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) values of methanol and dichloromethane (DCM) extract against bacteria (S. aureus and S. aglaactiae) and fungus (Candida krusei) varied from 0.166 g/mL to 0.0417 g/mL, respectively [13].The (DCM/Methanol v/v, 50%) extract of P. angolensis inhibited Plasmodium falciparum chaperones (PfHsp70-z and PfHsp70-1), with IC 50 values of 13.87 and 0.20 µg/mL, respectively [14].The crude aqueous extracts of bark and root from P. angolensis enabled the healing of breached tissue and induced an increase in chondrogenesis [15].According to our knowledge, no anticancer activities have been reported on the extracts and isolated compounds of P. angolensis.Hence, this study focused on the cytotoxicity properties of the crude extract and its phytochemicals.Furthermore, there is no report on the geographical variation in metabolites present in P. angolensis ecotypes.
Compounds 1, 2, 3, 4, 5, 6, and 8 were isolated from the DCM crude extract of cultivated P. angolensis, while five compounds, 1, 2, 4, 6, and 8, were obtained from the wild crude DCM extract.Compounds 6 and 8 were also isolated from hexane crude extracts, both cultivated and wild, respectively.Compounds 5, 6, and 7 were also isolated from crude ethyl acetate extracts from both wild and cultivated sources.This variation in the number of isolated compounds from the cultivated plants (7) compared to the wild plants (5) could be attributed to the differences in the geological location and climate conditions of the two populations.Limpopo Province, where the wild populations were collected, is much warmer than the Gauteng Province, where cultivated specimens were collected.Geographical location is known to influence the secondary metabolites profile of plants [16].
The physiochemical properties of compounds 1-8, including the IR, optical rotation, melting points, and molecular mass, are presented in Table 1 below.S2) of compound 1 showed seven methyl singlet proton resonances and one methyl doublet proton resonance at δ 1.17 (3H, s), 1.04 (3H, s), 1.00 (3H, s), 0.99 (3H, s), 0.94 (3H, s), 0.86 (3H, s), 0.86 (3H, s), and 0.71 (3H, s) ppm, respectively.One methyl resonance doublet at δ 0.86 (3H, s) ppm, J = 6.0 Hz was seen.The 13 C NMR and DEPT spectra revealed the presence of 30 carbon resonances attributed to eight methyls, ten methylene, three methine, and nine quaternary carbon atoms, as indicated in Table S1.The downfield carbon resonance at δ 213.2 ppm is a typical ketone group complemented by the carbonyl stretch of ketone (-C=O) present in the IR spectrum displaying at 1715.2 cm −1 assigned to C-3.The spectral data suggested compound 1 to be Friedelan-3-one, which was confirmed by the comparison of 1 H NMR and 13 C NMR with the literature.The compound (1) was previously isolated from the stem bark of the Peritassa rompata plant [17].
3α-hydroxyfriedel-2-one (2) was isolated as a white powder and a pseudo molecular ion [M + H] + peak at m/z 443.3447 corresponding to the molecular formula of C 30 H 50 O 2 .The 1 H NMR spectrum showed seven methyl proton singlet resonances at δ 0.87 (3H, s), 0.94 (3H, s), 0.97 (3H, s), 0.98, 1.00 (3H, s), 1.05 (3H, s), and 1.17 (3H, s) ppm and a doublet methyl proton resonance at δ 1.03 (3H, s) ppm.The 1 H NMR spectrum also revealed a doublet oxy-methine (1H, dd J = 11.8, 2.98 Hz) proton resonance at δ 3.81 ppm (H-3).The 13 C NMR spectrum showed eight methyls, ten methylene, three methine, and nine quaternary carbon atoms, as indicated in Table S1.A downfield carbon resonance was observed in the carbon spectrum at δ 211.9 ppm; this supported the presence of a sharp absorption band observed at 1662.6 cm −1 in the IR spectrum.Another difference observed in compound 2 was the hydroxyl (OH) and its electronegative effect due to the carbonyl stretch of the ketone (at 1662.6 cm −1 ) that is lower than the typical ketone, which appears at 1715 cm −1 as detected in compound 1 [18].Based on the NMR spectral data as well as the comparison with the literature [19], compound 2 was identified as 3-hydroxyfriedel-2-one.
3-Hydroxyfriedel-3-en-2one (3) was isolated as a white crystalline needle-like solid with a pseudo molecular ion [M + H] + at m/z 441.2928 that corresponds to a molecular formula of C 30 H 48 O 2 (calculated for m/z 440.3651).3-hydroxyfriedel-3-en-2one (3) is a striking derivative of compound 2 (3-hydroxyfriedel-2-one), and the 13 C NMR of compound 3 showed downfield sp3 quaternary carbon resonance of an alkene at δ 142.6 ppm and δ 140.8 ppm, which were assigned to C-3 and C-4, respectively.This assignment was in agreement with the IR spectrum displaying a sharp band C=C vibration at 1466.3 cm −1 .The double bond is missing in compound 2 because the alkene resonance is absent in the carbon spectrum.More differences were depicted: the chemical shifts in ring A of compound 3.The presence of H-3 in compound 2 previously assigned at δ 3.81 (1H, dd J = 11.8, 2.98 Hz), and H-4 at δ 1.29 ppm were also missing in compound 3 in the 1 H-NMR spectrum (Table S1).Using spectral data, compound 3, also identified as 3-hydroxyfriedel-3-en-2-one, was previously isolated from the stem bark of Mallotus philippensis [20].The compound (3) was, however, isolated for the first time from P. angolensis in this study, which was supported by a comparison of spectroscopic data with values from the literature [19,21].

Cytotoxicity Activity
The cytotoxicity of the pure compounds was evaluated using the in vitro cell culture against triple-negative breast cancer (HCC70) cell lines, hormone receptor-positive breast cancer (MCF-7), and non-tumorigenic epithelial cell lines derived from breast tissue (MCF-12A).A compound's ability to inhibit a particular biochemical function is determined by its half-maximum inhibitory concentration (IC 50 ).Etoposide was used as the positive control.The IC 50 for each compound against the various cell lines under investigation is listed in Table 2.

General Experimental Procedure
Column chromatography was performed on polyamide columns (Germany GmbH) over silica gel (Kieselgel 60 GF254, pore size 35-75 m particle size, Merck, Darmstadt, Germany), while thin-layer chromatography (TLC) was performed on Kieselgel 60 F254 (Merck) to a thickness of 0.25 mm.Under ultraviolet (UV) light, active spots on UV active silica gel were visualized (245 and 336 nm).All reagents and solvents (hexane, ethyl acetate, dichloromethane, and absolute ethanol) used in column chromatography were purchased from Merck and Sigma in South Africa and used exactly as received.

Plant Material
The natural (wild) habitat of P. angolensis in South Africa was spotted on the map and data of the South African Natural Biodiversity Institute (SANBI).The stem bark of mature Pterocarpus angolensis was collected from Nkowankowa (23.826923 • S, 30.313919 • E) in Limpopo province with an average temperature of 29 • C at the time of sampling.The plant was sampled from five different locations within a 500 m range of the area along the R71 roadside 55 km away from the town of Tzaneen towards Phalaborwa.The stem bark of mature cultivated P. angolensis was also collected from the Pretoria National Botanical Gardens (25.7395 • S, 28.2733 • E) in Gauteng province with an average temperature of 26 • C at the time of sampling.A voucher specimen of the plant (leaves, bark, and seeds) for identification with allocated number 18077 was deposited in the South African Natural Biodiversity Institute (SANBI).

Sequential Extraction
Pterocarpus angolensis plant material (stem bark of wild and cultivated trees) was cleaned with deionized water after being washed with tap water to remove dust.The stem bark was then cut into small pieces and placed in a laboratory room for two weeks at room temperature.The dried stem bark of the plant was ground into small pieces using a Retsch mill (Retsch SM 100, Haan, Germany).The small pieces of stem bark of P. angolensis were ground into a fine powder using a 1000 W Polymix PX-MFC 90D mill (Kinematica, Malters, Switzerland).The fine powdered plant material of 2.4 kg was weighed and added to 10 L round bottom flasks with 7500 mL organic solvents for extraction.The plant material was extracted sequentially with four analytical-grade organic solvents with increasing polarity order: Hexane (Hex) < dichloromethane (DCM) < ethyl acetate (EtOAc) < methanol (MeOH).The mixtures were shaken at room temperature for 48 h on an HS 501 horizontal shaker IKA (HS 501 digital IKA Werke, Staufen, Germany).A Whatman's No. 1 filter paper was used to filter the extracts.This step was carried out in duplicate for maximum extraction.All filtrates of extracts were concentrated using a G1 diagonal glassware Rotary Evaporator (Heidolph Hei-VAP, Heidelberg, Germany) set to a constant temperature of 37 • C. In a fume hood, the concentrated crude extracts of wild and cultivated P. angolensis were dried, the wild plant yielded 17.48 g (Hex), 39.02 g (DCM), 19.21 g (EtOAc), and 388.30g (MeOH), whereas the cultivated plant yielded 21.82 g (Hex), 42.45 g (DCM), 15.35 g (EtOAc), and 375.36 g (MeOH).The dried extracts of P. angolensis were stored at −10 • C to prevent any decomposition.

Nuclear Magnetic Resonance Spectroscopy
Moreover, 5.0-10.0mg sample of each pure isolated compound of P. angolensis was dissolved in deuterated chloroform, CDCI 3, and transferred into an NMR tube with a diameter of 5 mm, a precision of medium wall, a frequency limit of 400 MHz, and a length of 7 inches (Wilmad ® , Vineland, NJ, USA).Then, 1D and 2D NMR were acquired with gradient enhancement using a Varian 400 MHz premium-shielded NMR spectrometer (400/54/ASP, Santa Clara, CA, USA).Chemical shifts were measured relative to 7.2 ppm ( 1 H) and 77.0 ppm ( 13 C) NMR to deuterated chloroform, and structures of isolated compounds were proposed based on spectra interpretation [18].

Infrared Spectroscopy
Approximately 1.0 mg of isolated pure compounds of P. angolensis were analyzed using a Spectrum Two Universal (FTIR) spectrophotometer (Perkin Elmer, Waltham, MA, USA) to determine the functional groups present.For each sample, 32 scans in the mid-IR band of 4000-550 cm −1 were performed, and a background spectrum was obtained before each sample spectrum.

Mass Spectroscopy
High-resolution LC-MS spectra were collected using a Bruker Daltonics Compact QTOF Mass Spectrometer (Billerica, MA, USA) with an electrospray ionization probe in the positive mode (ESI+).Thermo Scientific Ultimate 3000 Dionex UHPLC (Milford, CT, USA) system with an Acclaim RSLC 120, C18, 2.2 m, 2.1 100 mm (P/N 068982) column was used for separation.The system's components included the Detector DAD-3000 RS, Pump HPG-3400 RS, and Auto Sampler WPS-3000 RS (Milford, CT, USA) The solvent mixture was water-acetonitrile (10:90, v/v), with each solvent containing 0.1% of formic acid.Isocratic elution with a 5 min run time and injection volume of 3 µL was used.

In Vitro Cytotoxicity Assay The Resazurin Assay
The cell viability after treatment with the isolated pure compounds of P. angolensis was analyzed using the resazurin assay in commercially available established cell lines purchased from the American Type Culture Collection [37].Triple-negative breast cancer cell line (HCC70) (estrogen receptor (ER) − , progesterone receptor (PR), (HER-2) − , (ATCC-CRL-2315)), hormone receptor-positive (ER + , PR + , HER-2) breast cancer cell line (MCF-7) (ATCC: HTB-22), and non-tumorigenic breast epithelial cell line (MCF-12A) (ATCC: CRL-10782, a kind gift from Anna-Mart Engelbrecht, Stellenbosch University, Stellenbosch, South Africa) were maintained in culture in a 9% CO 2 incubator at 37 • C. The HCC70 cell line was cultured in RPMI-1640 media (Gibco, Trenton, MI, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (BioWest, Nuaillé, France), 1% (v/v) 100 U/mL penicillin, streptomycin, and amphotericin (PSA) (Lonza, Basel, Switzerland), 1% (v/v) GlutaMAX TM (Gibco) and 0.25% (v/v) sodium bicarbonate (Gibco).The MCF-7 cell line cell was cultured in Dulbecco's Modified Eagles Medium (DMEM) (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) PSA and 1% (v/v) GlutaMAX TM .The MCF-12A cell line was cultured in DMEM/Ham's F-12 (BioWest) supplemented with 10% (v/v) FBS, 1% (v/v) PSA, 20 ng/mL human epidermal growth factor (Sigma, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma), 500 ng/mL hydrocortisone (Abcam, Cambridge, UK) and 10 µg/mL insulin (Novo Nordisk, Bagsvaerd, Denmark).All cells were seeded into 96-well plates at a density of 5000 cells per well and allowed to adhere for the night in a 9% CO 2 incubator at 37 • C. The cells were subsequently treated for 96 h at 37 • C in an incubator with 9 percent CO 2 with the isolated compounds with six 5-fold dilutions at concentrations ranging from 15.63 to 500.00 µM or with 0.2% (v/v) DMSO (Sigma) as a vehicle control.Thereafter, 0.54 nM of resazurin (Glentham Life Sciences, Corsham, UK) solution was added, and cells were then cultured for two to four hours at 37 • C in an incubator with 9% CO 2 .A Spectramax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) was used to measure the fluorescence of the resulting solutions.The excitation and emission wavelengths were set at 560 nM and 590 nM, respectively.The experiment was repeated in technical triplicate, and the data were analyzed using GraphPad Prism (Boston, MA, USA).Half-maximal inhibitory doses (IC 50 values) were determined using non-linear regression with Etoposide serving as a positive control.The selectivity index (SI) value of each compound was calculated as the ratio of IC 50 of MCF-12A/IC 50 of HCC70 or MFC-7 of the same compound, where an SI > 1 implies toxicity that is preferentially toxic to cancer cells compared to non-cancerous cells.

Figure 1 .
Figure 1.Chemical compositions of isolated compounds from P. angolensis.

Table 1 .
Physiochemical properties of the isolated compounds.