In vitro cytotoxic effect of stigmasterol derivatives against breast cancer cells

Background Stigmasterol is an unsaturated phytosterol that belong to the class of tetracyclic steroids abundant in Rhoicissus tridentata. Stigmasterol is an important constituent since it has shown impressive pharmacological effects such as anti-osteoarthritis, anticancer, anti-diabetic, anti-inflammatory, antiparasitic, immunomodulatory, antifungal, antioxidant, antibacterial, and neuroprotective activities. Furthermore, due to the presence of π system and hydroxyl group, stigmasterol is readily derivatized through substitution and addition reactions, allowing for the synthesis of a wide variety of stigmasterol derivatives. Methods Stigmasterol (1) isolated from Rhoicissus tridentata was used as starting material to yield eight bio-active derivatives (2–9) through acetylation, epoxidation, epoxide ring opening, oxidation, and dihydroxylation reactions. The structures of all the compounds were established using spectroscopic techniques, NMR, IR, MS, and melting points. The synthesized stigmasterol derivatives were screened for cytotoxicity against the hormone receptor-positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12 A) cell lines using the resazurin assay. Results Eight stigmasterol derivatives were successfully synthesized namely; Stigmasterol acetate (2), Stigmasta-5,22-dien-3,7-dione (3), 5,6-Epoxystigmast-22-en-3β-ol (4), 5,6-Epoxystigmasta-3β,22,23-triol (5), Stigmastane-3β,5,6,22,23-pentol (6), Stigmasta-5-en-3,7-dion-22,23-diol (7), Stigmasta-3,7-dion-5,6,22,23-ol (8) and Stigmast-5-ene-3β,22,23-triol (9). This is the first report of Stigmasta-5-en-3,7-dion-22,23-diol (7) and Stigmasta-3,7-dion-5,6,22,23-ol (8). The synthesized stigmasterol analogues showed improved cytotoxic activity overall compared to the stigmasterol (1), which was not toxic to the three cell lines tested (EC50 ˃ 250 µM). In particular, 5,6-Epoxystigmast-22-en-3β-ol (4) and stigmast-5-ene-3β,22,23-triol (9) displayed improved cytotoxicity and selectivity against MCF-7 breast cancer cells (EC50 values of 21.92 and 22.94 µM, respectively), while stigmastane-3β,5,6,22,23-pentol (6) showed improved cytotoxic activity against the HCC70 cell line (EC50: 16.82 µM). Conclusion Natural products from Rhoicissus tridentata and their derivatives exhibit a wide range of pharmacological activities, including anticancer activity. The results obtained from this study indicate that molecular modification of stigmasterol functional groups can generate structural analogues with improved anticancer activity. Stigmasterol derivatives have potential as candidates for novel anticancer drugs. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-023-04137-y.


Introduction
Rhoicissus tridentata is a deciduous shrub in the Vitaceae family, widely distributed throughout the Eastern region of southern Africa [1].It is widely used in African medicinal systems to treat various ailments including erectile dysfunction, pains, swelling, cuts, wounds, kidney and bladder complications, stomach ailments, and livestock diseases, as well as for gynaecological purposes [2][3][4][5][6][7].The medicinal uses of R. tridentata were attributed to the presence of bioactive compounds such as terpenoids, alkaloids and flavonoids, which showed promising anti-cancer, antioxidant, and anti-inflammatory activities [7][8][9][10][11][12].The aqueous root extract of R. tridentata had the strongest antiproliferative effect, inhibiting HepG2 cell growth by 96.27%, while the methanol extract inhibited proliferation by 87.01%[2].Modification of natural product structures leads to the discovery of novel agents with improved cytotoxicity in resistant tumours, decreased toxicity, and improved solubility [13,14].In this study, the roots of R. tridentata were phytochemically investigated and afforded stigmasterol in good yield.Stigmasterol was then selected as a candidate for synthetic modification with the aim of enhancing the anticancer activity.
Stigmasterol is an unsaturated phytosterol that belongs to the steroids class.It is a secondary metabolite that was isolated for the first time in 1906 in Calabarbohne by Adolf Wind Form and A. Hauth [15], and has since been isolated as the common compound from various medicinal plants [16].Stigmasterol (stigmasta-5,22-dien-3β-ol, C 29 H 48 O), also known as Wulzen anti-stiffness factor or stigmasterin, is characterized by the presence of a hydroxyl group in position C-3 of the steroid skeleton.It also has double bonds in positions 5, 6 of the B ring, and in position 22, 23 in the alkyl substituent, as well as an isoprenyl tail (Fig. 1) [15,16].
Stigmasterol is used in various chemical manufacturing processes which are designed to generate numerous synthetic and semi-synthetic compounds for the pharmaceutical industry [15,17].It serves as a precursor in the synthesis of hormones such as progesterone as well as an intermediate in the biosynthesis of androgens, corticoids, oestrogens [18] and in the synthesis of vitamin D 3 [15].
Stigmasterol isolated from the Typhonium flagelliforme mutant plant was found to be more effective against the human breast cancer MCF-7 cell line with an IC 50 value of 0.1623 µM than cisplatin with an IC 50 value of 13.2 µM [32,33].Stigmasterol can induce oxidative stress in MCF-7 cells, which leads to apoptosis.A competitive activator with a single high-affinity binding site on FXR and hydrophobic interactions facilitates this [32].Li et al. [24] reported that stigmasterol suppressed proliferation and induced apoptosis in the human gastric cancer cell line SNU-1 through modulation of the JAK/STAT signalling pathway with an IC 50 value of 15 µM.According to Bae et al. [34], stigmasterol can reduce the growth of human ovarian cancer cells ES2 and OV90 by 50% at a treatment concentration of 20 µg/mL, dose-dependently inducing apoptosis of ovarian cancer cells by causing mitochondrial malfunction, ROS production, and calcium overload in the mitochondria and cytosol.

Keywords
Furthermore, because of the pharmacophores present, such as alkene bonds and the hydroxyl group, Stigmasterol is readily derivatized through substitution and addition reactions, allowing for the synthesis of a wide variety of stigmasterol derivatives.As a result, stigmasterol was selected as a candidate for synthetic modification.Eight derivatives of Stigmasterol (1) were successfully synthesized via acetylation, oxidation, epoxidation and ring-opening and dihydroxylation of the epoxide, to enhance their anticancer activity.The generated products were characterized using spectroscopic techniques and screened for anticancer activity against the MCF-7 and HCC70 breast cancer cell lines in comparison to the MCF-12 A non-cancerous breast epithelial cell line.

General experimental procedures
Column chromatography was performed using silica gel (Kieselgel 40-63 μm, Macherey-Nagel, Germany).Thin Layer Chromatography (TLC) was carried out on Kieselgel-60 F 254 (Merck) 20 × 20 cm aluminium sheets for monitoring the compounds.1D and 2D NMR data were recorded on a Varian Gemini 400 spectrometer at 400 MHz and 100 MHz for 1 H and 13 C nuclei, respectively at room temperature.All compounds were dissolved in deuterated chloroform (CDCl 3 ).Chemical shifts for signals were reported in parts per million (ppm) on the delta scale (δ), referenced to tetramethyl silane as the internal standard.The chemical shifts were referenced at δ H 7.26 ppm in 1 H and δ C 77.04 ppm in 13 C NMR for CDCl 3 .Spin-spin coupling constants (J) were calculated in Hertz (Hz) and the splitting patterns were recorded as follows: s = singlet; d = doublet; t = triplet; q = quartet; dd = doublet of doublets; m = multiplet and b = broad.
Melting points (MP) were determined using a digital melting point apparatus (Stuart® SMP 20, Cole-Parmer, Staffordshire, UK), operated at 50 Hz and 75 W power and optical rotation was recorded on a Jasco P-2000 Polarimeter (JASCO, Germany).Infrared spectroscopy (IR) was carried out on a Fourier transform Spectrophotometer (PerkinElmer UATR Two, USA), in the range 400 cm − 1 to 4000 cm − 1 with a resolution of 4 cm − 1 and 32 scans.The high-resolution electron spray ionization mass spectroscopic (HR-ESI-MS) data were acquired on a Bruker Daltonics Compact QTOF mass spectrometer in positive mode (ESI + ) using an electrospray ionization probe.The mass spectrometer was coupled to a thermal scientific ultimate 3000 Dionex UHPLC system consisting of an RS Auto Sampler WPS-3000, Pump HPG-3400 RS and detector DAD-3000 RS, using an Acclaim RSLC 120 C18, 2.2 μm, 2.1 × 100 mm (P/N 068982) column at 40 ºC, flow rate 0.2 ml/min, solvent: Water-Acetonitrile (10:90, v/v) each solvent containing 0.1% of formic acid, isocratic condition, 5 min run.Processed spectra of all reported compounds are shown in supplementary materials 1.

Plant material
The plant Rhoicissus tridentata subsp.cuneifolia was purchased from a wildflower nursery in Hartbeespoort, S 05°04.579′E 044°35.033′,North West province of South Africa.The plant specimen was identified by Erich Van Wyk and a voucher specimen was deposited at the South African National Biodiversity Institute (SANBI) in Pretoria (number 22,033).

Extraction and isolation
Dried and grounded roots plant material (1864.36g) of R. tridentata was extracted sequentially with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) for 48 h at temperatures between 15 and 25 °C, using a shaker.The extracts were filtered and concentrated under reduced pressure at 35 °C on a rotary evaporator to obtain a yield (2.59 g n-hexane, 3.61 g DCM, 4.55 g EtOAc and 71.28 g MeOH).Based on TLC analysis, the DCM crude extract was then subjected to repeated column chromatography (CC) using silica gel mixed with n-hexane and eluted with solvent systems, n-hexane: DCM (9:1, 8:2, 7:3 and 5:5 v/v).A total of 200 fractions were collected into 100 ml beakers and concentrated to dryness under a fume hood.Fraction F13D from the DCM crude extract showed interesting compounds with R f values of 0.110 and 0. 231 as major compounds.This fraction afforded colorless crystals of sitosterol and 73.4 mg white powder of stigmasterol (1).

In vitrocytotoxicity assay The resazurin assay
The cell viability after treatment with the Stigmasterol derivatives was assessed using the resazurin assay [37].The triple negative breast cancer cell line (TNBC), HCC70 cells [oestrogen receptor (ER)-, progesterone receptor (PR)-, human epidermal growth factor-2 (HER-2)-] (ATCC: CRL-2315), was maintained in culture in RPMI-1640 media supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 100 mg/ml streptomycin, 100 U/ml penicillin, 12.5 mg/ml amphotericin (PSA), 2 mM GlutaMAXTM and 2,5% (v/v) sodium bicarbonate.The hormone receptor-positive (ER + , PR + , HER-2 − ), MCF-7 breast cancer cell line (ATCC: HTB-22) was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) FBS, 100 U/ ml PSA and 2 mM GlutaMAX™.The non-tumorigenic breast epithelial cell line MCF-12 A (ATCC: CRL-10,782) was maintained in DMEM:Hams F12 (1:1 ratio) supplemented in 10% (v/v) FBS, 500 ng/ml hydrocortisone, 100 U/ml PSA, 100 ng/ml cholera toxin, 10 mg/ml insulin and 20 ng/ml human epidermal growth factor (hEGF).All cells were maintained at 37 °C in a humidified 9% CO 2 incubator.The HCC70, MCF-7 and MCF-12 A cells were seeded at 5000 cells/well in a 96 well plate and were left overnight in a 9% CO 2 incubator at 37 °C to adhere.The cells were then treated with the synthetic compounds at a concentration range from 0.16 to 500.00 µM or with a vehicle control [0.2% (v/v) DMSO] for 96 h at 37˚C in a 9% CO 2 incubator.Thereafter, into each well, a solution of 10 µL of a 0.54 mM resazurin was added, followed by incubation for 2-4 h.Solubilization solution (10% (w/v) SDS in 0.01 M HCl) was then added overnight.0.54 nM of resazurin solution was added and the cells were incubated for 2-4 h at 37˚C in a 9% CO2 incubator.The fluorescence was then measured on a Spectramax spectrophotometer with excitation and emission wavelength set at 560 and 590 nm respectively.The experiment was repeated in technical triplicate and the data was analysed using GraphPad Prism Inc, (USA) with half-maximal effective concentration (EC 50 values) determined by nonlinear regression.Selectivity index values for the compounds were calculated as follows: (EC 50 of compound against MCF12A cells) ÷ (EC 50 of compound against breast cancer cells) where a SI > 1 is indicative of selective toxicity towards cancer cells vs. non-cancerous cells.

Methods for the synthesis of stigmasterol derivatives
Acetylation procedure of stigmasterol Pyridine (0.14 mL) and acetic anhydride (6 mL) were added to compound 1 (Stigmasterol) (0.739 g, 1.7907 mmol) in a 50 mL round-bottomed flask.The mixture was left to stir for 24 h at room temperature.The reaction was quenched with deionized water (10 mL) and was extracted with DCM (3 × 10 mL).The organic phase was washed with 2 M HCl (3 × 10 mL) to wash away excess pyridine and dried over anhydrous magnesium sulphate, filtered, and concentrated in vacuo.The residue was purified by column chromatography using n-Hexane: ethyl acetate (EtOAc) (98:2%) to afford compound 2 [38,39].

Oxidation procedure of stigmasterol
To a solution of compound 1 (1.002 g, 2.4280 mmol) in acetone (24.28 mL) at 0 °C, Jones reagent (CrO 3 /H 2 SO 4 ) (1.94 mL) was added drop-wise.The reaction mixture was stirred at temperatures between 15 and 25 °C for 22 h.Isopropyl alcohol was added dropwise to remove excess Jones reagent, indicated by the appearance of a deep green colour.Upon completion, 5 mL of water was added before extraction with diethyl ether (2 × 30 mL).The combined ether layers were washed twice with sodium bicarbonate and brine, dried over anhydrous magnesium sulphate, and concentrated in vacuo [40].The residue was purified by column chromatography using n-Hex: EtOAc (80:20%) to give compound 3 [39].

Oxidation of compound 6 (stigmastane-3β,5,6,22,23-pentol)
To a solution of compound 6 (0.453 g, 0.9430 mmol) in acetone (9.4 mL) at 0 °C was added Jones reagent (CrO 3 /H 2 SO 4 ) (0.38 mL) in a drop-wise manner.The reaction mixture was stirred at temperatures between 15 and 25 °C for 22 h.Isopropyl alcohol was added dropwise to remove excess Jones reagent, indicated by the appearance of a deep green colour.Upon completion, 5 mL of water was added before extraction with ether (2 × 30 mL).The combined ether layer was washed twice with sodium bicarbonate and brine, dried over anhydrous magnesium sulphate, and concentrated in vacuo [40].The residue was purified by column chromatography using n-Hex: EtOAc (80:20%) to give compound 7, compound 8 and compound 9.

Results and discussion
Analogues of stigmasterol were synthesized with the aim of enhancing its anticancer activity through acetylation, epoxidation, epoxide ring opening, oxidation, and dihydroxylation reactions as shown in Scheme 1.The synthesis of the stigmasterol derivatives was possible because of the pharmacophores present in stigmasterol, such as alkene bonds in position C-5, C-6, and position C-22, C-23 in the alkyl substituent and the hydroxyl group in the position C-3.

Characterization of synthesized compounds
Eight stigmasterol derivatives were successfully synthesized namely; Stigmasterol acetate (2), Stigmasta-5,22-dien-3,7-dione ( 3 Compound 2 was confirmed by the shift of the oxymethine proton (H-3) from the upfield region at δ H 3.45 ppm (tt, J = 10.4,4.3 Hz, 1 H) to a slightly downfield region at δ H 4.52 ppm (m, 1 H) in the 1 H NMR spectrum assigned to H-3, confirming an acetate group.In the 13 C NMR spectrum a presence of downfield carbon resonance at δ C 170.56 ppm was observed, typical of an acetate group.The NMR data of the derivative is comparable to the literature values reported by Foley et al. [39] for stigmasterol acetate (2).
The 1 H NMR spectrum of the compound exhibited a sharp and long singlet at δ H 6.16 ppm, attributed to the olefinic methine proton H-6.There was a chemical shift of the methine proton at position 6 from δ H 5.28 ppm (d, J = 4.9 Hz, 1 H) to δ H 6.16 ppm.The absence of the oxymethine proton at δ H 3.45 ppm (tt, J = 10.4,4.3 Hz, 1 H) of stigmasterol, in the 1 H NMR spectrum confirmed the formation of a carbonyl at position C-3 displaying a carbon resonance at δ C 199.44 ppm.Moreover, the difference in the 13 C NMR spectra of the product and the starting material (stigmasterol) was the appearance of the signals at δ C 199.44 and δ C 202.26 ppm in the 13 C NMR spectrum of the product.The peaks were attributed to the keto carbonyl carbons at C-3 and C-7, respectively [39].The proton at δ H 2.48 ppm (H-4) was seen correlating to C-3 which confirmed the assignment of the carbonyl to C-3.These values are consistent with those reported by Donkwe et al. [35].The structure of compound 7 was confirmed by the absence of the oxymethine proton at δ H 4.09 ppm (dd, J = 11.5, 6.3 Hz, 1 H) (H-3) of compound 6 and the appearance of a sharp and long singlet at δ H 6.16 ppm attributed to an olefinic proton (H-6) in the 1 H NMR spectrum of compound 7. Furthermore, there was an appearance of signals at δ C 199.35 and δ C 202.06 ppm in the 13 C NMR spectrum attributed to the keto carbonyl carbons C-3 and C-7, respectively.This is the first report of compound 7.
The 1 H NMR spectrum of compound 8 showed a shift in the proton resonance of H-6 from δ H 3.53 ppm to δ H 3.16 ppm confirming the addition of a hydroxyl group at C-6.The absence of the oxymethine proton resonance at δ H 4.09 ppm (dd, J = 11.5, 6.3 Hz, 1 H) (H-3) of compound 6, confirmed the introduction of a carbonyl at position C-3 in compound 8. Furthermore, there was an appearance of signals at δ C 211.07 ppm and δ C 211.33 ppm in the 13  The 1 H NMR spectrum of compound 9 indicated that the oxymethine proton at δ H 3.53 ppm (H-6) of compound 6 changed to an olefinic proton δ H 5.80 ppm (H-6).There was also an appearance of olefinic carbon resonances at δ C 168.43 ppm (C-5) and δ C 126.30 ppm (C-6) in the 13 C NMR spectrum [39].

Cytotoxic activities of stigmasterol derivatives
Stigmasterol and all synthesized stigmasterol derivatives were screened in vitro against breast cancer cells including, the triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7) and non-tumorigenic epithelial cell lines established from breast tissue (MCF-12 A) cell lines.Camptothecin was used as the positive control.The cytotoxicity data for all the synthetic derivatives are summarized in Table 1.

Conclusion
Eight derivatives (2-9) of stigmasterol (1) were successfully synthesized via acetylation, oxidation, epoxidation, ring-opening and dihydroxylation of the epoxide.The structures of all the compounds were determined using spectroscopic techniques and melting points.Stigmasta-5-en-3,7-dion-22,23-diol (7) and Stigmasta-3,7-dion-5,6,22,23-ol (8) are reported for the first time.Stigmasterol and all the synthesized derivatives were screened against non-tumorigenic epithelial cell lines established from breast tissue (MCF-12 A), hormone receptor-positive breast cancer (MCF-7) and triple-negative breast cancer (HCC70) cell lines.The synthesized stigmasterol analogues demonstrated increased cytotoxic activity overall compared to the stigmasterol (1), which was not toxic to the cancer cell lines (EC 50 ˃ 250 µM).In particular, 5,6-epoxystigmast-22-en-3β-ol (4) and stigmast-5-ene-3β,22,23-triol (9) showed improved cytotoxic activity and a degree of selectivity against MCF-7 breast cancer cells, with EC 50 values of 21.92 and 22.94 µM, respectively, and selectivity indices of 2.14 and 2.26, respectively.On the other hand, stigmastane-3β,5,6,22,23-pentol (6) demonstrated increased cytotoxicity against the triple negative breast cancer HCC70 cell line, with an EC 50 value of 16.82 µM, although this compound was not selective.Structural modification has proven to be a valid strategy to increase the anticancer activity of stigmasterol since all the synthesized derivatives displayed greatly improved activity.This represents a solid starting point in the development of novel anticancer drugs.Stigmasterol derivatives have potential as candidates for novel anticancer drugs hence further study of these compounds is needed.

5 =
C NMR spectrum of compound 8 attributed to the keto carbonyl carbons C-3 and C-7.This is the first report of compound 8. Stigmast-5-ene-3β,22,23-triol (9) was obtained as a white solid (0.102 g, 0.2285 mmol, 24%) with a melting point of 181-183 °C.The specific optical rotation was recorded as [α] D 19.-6.5° (c = 1.81,CHCl 3 ).The Q-TOF mass spectrum showed a molecular ion [M-H] − peak at m/z 445.3581 with the molecular formula of C 29 H 50 O 3 .The IR spectrum showed a broad (-OH) peak appearing at 3403.3, the absorption band at 2870.3 cm − 1 was attributed to C-H stretching and the (C = C) stretch was observed at 1683.4 cm − 1 .