Three new cytotoxic stilbene dimers from paphiopedilum dianthum

Abstract Three new stilbene dimers, paphiodianthins A-C (1–3), and nine known stilbenes, lignan and flavonoids (4–12) were isolated from the roots and leaves of Paphiopedilum dianthum (Orchidaceae). The structures of new compounds were elucidated from their NMR, HRESIMS and IR spectroscopic data. Cytotoxic activity of all isolated compounds was evaluated by in vitro MTT assay against two human cancer cell lines (MCF-7, Caco-2), doxorubicin-resistant and mitoxantrone-resistant MCF-7 sublines, as well as a normal cell line (NIH/3T3). Stilbenes 1, 3, 10 and 11 were strongly cytotoxic to both cancer cell lines with IC50 values ranging from 0.50 to 4.51 µM. Compounds 1, 10 and 11 were also active against chemotherapy-resistant MCF-7 sublines. Graphical Abstract


Introduction
The genus Paphiopedilum of the family Orchidaceae is a genus of lady's slipper orchids comprising approximately 80 species which are valued as exotic ornamental plants and are highly endangered.Their natural habitats are in Southeast Asia, India, southern China and New Guinea (Parveen et al. 2012).These orchids usually have thick and fleshy roots, forming tight masses when grown in pots.Currently, only a few studies on their chemical constituents and biological activities have been performed.Stilbenoids and flavonoids were found in the roots of two Paphiopedilum species native to Thailand: P. godefroyae and P. callosum (Lertnitikul et al. 2016;Nwe et al. 2019).An arylbenzofuran-type stilbene from P. godefroyae was strongly cytotoxic to human small cell lung cancer (NCI-H187) cells (Lertnitikul et al. 2016).In addition, some constituents of P. callosum were able to inhibit topoisomerase activity in a screening model utilizing transgenic Saccharomyces cerevisiae (Nwe et al. 2019).
Paphiopedilum dianthum Tang & F.T.Wang (double flowered paphiopedilum) is a rare slipper orchid native to southwestern China, central Laos and Vietnam (Hou et al. 2018).The orchid is ornamentally cultivated and, under greenhouse condition, each flower can bloom for 45-55 days (Yang et al. 2021).Currently, there has been no report of its ethnomedicinal uses and phytochemical or biological study of this orchid has never been performed.In our continuing investigation on the chemical constituents of Paphiopedilum species, we report herein the isolation of three new stilbene dimers (1)(2)(3) and nine known compounds including four flavonoids (4,6,7,12), four stilbenes (5,8,10,11) and one lignan (9) from the roots and leaves of P. dianthum (Figure 1).Their cytotoxicity against human breast cancer (MCF-7) and colon cancer (Caco-2) cell lines, a normal fibroblast cell line (NIH/3T3), as well as doxorubicin-resistant and mitoxantrone-resistant MCF-7 sublines, was measured by the MTT assay.
The cytotoxic activity of all compounds (1-12) was evaluated against MCF-7, doxorubicin-resistant MCF-7 (MCF-7/DOX), mitoxantrone-resistant MCF-7 (MCF-7/MX), Caco-2 and NIH/3T3 cell lines by the MTT assay, with doxorubicin as the positive control (Table S2).The stilbene dimers 1 and 3 exhibited strong cytotoxicity against MCF-7 and Caco-2 cell lines.Compound 1 was also active against both drug-resistant MCF-7 sublines.Stilbenes 5 and 8, which were the two most abundant compounds isolated from the roots of this orchid, were moderately cytotoxic against all cell lines tested.Interestingly, stilbenes 10 and 11, both of which resemble compound 8 but having an additional para-hydroxybenzyl moiety, were strongly cytotoxic to all cell lines.Compound 11 appeared to be the most potent stilbene isolated in this study.Its cytotoxicity against MCF-7 cells was comparable to doxorubicin and remained constant even against the chemotherapy-resistant sublines.Although both 10 and 11 were firstly reported as constituents of three Phragmipedium orchids (Garo et al. 2007), this study is the first evidence of their potent cytotoxic activity.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Inc., Waltham, MA, USA).UV spectra were recorded on a Shimadzu UV-160A spectrophotometer (Shimadzu Corp., Kyoto, Japan) in MeOH.IR spectra were obtained on a Perkin-Elmer FT-IR 1760X spectrometer.ESIMS spectra were obtained on a Bruker Daltonics microTOF mass spectrometer (Bruker Corp., Billerica, MA, USA).NMR spectra were recorded on a Varian INOVA 500 MHz NMR spectrometer (Varian, Inc., Darmstadt, Germany).Chemical shifts were reported in ppm (d) with coupling constants (J) in hertz.The residual signal of CDCl 3 was used as reference.Column chromatography (CC) was performed using silica gel Merck,KGaA,Darmstadt,Germany) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden).Thin layer chromatography (TLC) was performed on precoated silica gel 60 F 254 aluminium plates (Merck).TLC spots were examined under UV light (254 or 365 nm), then sprayed with 10% sulfuric acid in alcohol and heated.All organic solvents were commercial grade and were redistilled prior to use.

Plant material
Fresh roots (800 g) and leaves (2.3 kg) of Paphiopedilum dianthum Tang & F.T.Wang were collected from an orchid nursery in San Pa Tong, Chiang Mai, Thailand (18 37 0 51ʺN 98 53 0 43ʺE), in February 2017.They were cut into small pieces and dried in hot-air oven at 50 C.The plant was authenticated by one of the authors (R. Suttisri) and a voucher specimen of the whole plant (RS17021) has been deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

Cytotoxicity assay
The cytotoxic activity of all isolated compounds against human breast adenocarcinoma (MCF-7), colon adenocarcinoma (Caco-2) and normal fibroblast (NIH/3T3) cell lines was measured by the MTT method (Twentyman and Luscombe 1987).In addition, their cytotoxicity towards doxorubicin-resistant (MCF-7/DOX) and mitoxantrone-resistant (MCF-7/MX) MCF-7 cell sublines was evaluated (Chaisit et al. 2017;Ma et al. 2013).Both sublines were developed by growing MCF-7 cells in RPMI-1640 complete medium containing low concentrations of doxorubicin (DOX) or mitoxantrone (MX).The resistant cells were selected and sub-cultured upon stepwise increment of the chemotherapeutic drug concentrations.In order to retain their drug resistance, MCF-7/DOX and MCF-7/MX sublines were maintained in RPMI-1640 medium containing 1.5 lM DOX or 0.7 mM MX, respectively.They were cultured in the medium without DOX or MX for three days prior to cytotoxicity assay.Cells were seeded in 96-well microplates at a density of 5 Â 10 3 cells/well and cultured for 24 h, then treated with the isolated compounds (0-100 lM) or DOX (positive control) for 72 h.After that, the cells were washed and further incubated in serum-free medium containing MTT reagent (0.83 mg/ml) for 3 h at 37 C to create formazan crystals.These crystals were dissolved with DMSO and the optical density was measured at 570 nm, using a Perkin-Elmer microplate reader.Cell viability was calculated according to formula: % cell survival ¼ [OD test/OD control] Â 100.Half maximal inhibitory concentration (IC 50 ) values were estimated from linear regression analysis of concentration-response curve (Chaisit et al. 2017).Each experiment was done in triplicate and repeated twice.The results were expressed as mean ± standard deviation (SD).

Conclusion
Phytochemical investigation of P. dianthum roots and leaves led to the isolation of twelve compounds including three new stilbene dimers (1)(2)(3) and nine known compounds (4-12).Stilbenes are quite abundant in the roots of this slipper orchid native to Asia.The new stilbene dimers 1 and 3 and the para-hydroxybenzyl-substituted stilbenes 10 and 11 exhibited strong cytotoxicity against MCF-7 and Caco-2 cancer cell lines.Although they were also cytotoxic to normal cell line (NIH/3T3), it is interesting that 1 and 11 could retain their potent antiproliferative activity against chemotherapyresistant MCF-7 sublines.