Chemical constituents of the stem of Marsypopetalum modestum and their bioactivities

Abstract Phytochemical investigation of Marsypopetalum modestum (Annonaceae) led to the isolation of a new phenylpropanoid glycoside, lyciumphenylpropanoid B (10), along with nine known compounds (1-9) from an aqueous methanolic extract of the stem. Most compounds are reported from this genus for the first time. The structures of the isolated compounds were elucidated using spectroscopic methods including NMR spectroscopy, high-resolution mass spectrometry, and quantum chemical electronic circular dichroism (ECD) calculations. Cytotoxic and antitubercular activities of several isolated compounds were evaluated. Dipyrithione (1) displayed anti-mycobacterial (MIC = 0.23 μM) and cytotoxic (IC50 = 0.8 μM in Hep G2 cells; 4.1 μM in HCT 116 cells) activities. Kelampayoside A (8) showed moderate cytotoxic activity against cancer cells. Graphical Abstract


Introduction
Marsypopetalum modestum (Pierre) B. Xue and R.M.K. Saunders (Annonaceae) is a small tree native in Southeast Asia (Elkington et al. 2014; Plants of the World Online 2022).Previously known as Polyalthia modesta (Pierre) Finet and Gagnep., this plant has been nomenclaturally transferred to Marsypopetalum modestum based on molecular phylogenetic evidence (Xue et al., 2011).Since then, the name has been adopted by the taxonomic community including the International Plant Name Index (www.ipni.org),Plants of the World Online (www.powo.science.kew.org), the Global Biodiversity Information Facility (www.gbif.org)and Turner (2015).Parts of the tree have been used in traditional medicine in Laos to treat tuberculosis and cancer-like conditions (Gupta et al. 2017;Pumiputavon et al. 2017).Bioassay-guided fractionation of M. modestum extract led to the isolation of an anti-mycobacterial constituent, dipyrithione (Elkington et al. 2014).Previous studies also demonstrated the cell cycle arrest and apoptotic activities of a methanolic leaf extract of M. modestum.Although phytochemical screening revealed the presence of flavonoids, tannins, alkaloids, anthraquinone glycosides, and sterols (Pumiputavon et al. 2017(Pumiputavon et al. , 2019)), little is known about the chemical constituents of this plant.In a continuation of a search for bioactive constituents from medicinal plants, we report herein the isolation and identification of ten secondary metabolites, including a new phenylpropanoid glycoside.Most of these metabolites have not been reported from this plant species before.In addition, cytotoxic activity against cancer cells and inhibitory activity against Mycobacterium tuberculosis were observed from several individual metabolites.

Cytotoxic and antitubercular activity
Compounds 1, 3, 5, 6 and 8 were tested for cytotoxic activity against HCT 116 colorectal carcinoma and Hep G2 hepatoma cells, and for inhibitory activity against M. tuberculosis.Dipyrithione (1), currently used as a pesticide and fungicide, was the only compound that showed antitubercular activity with an MIC value of 0.23 lM and is in agreement with previous findings (Elkington et al. 2014).When tested against noncancerous Vero cells, dipyrithione displayed weak cytotoxicity (IC 50 ¼ 12.9 lM).However, when tested in Hep G2 and HCT 116 cancer cells, compound 1 exhibited stronger cytotoxic activity with IC 50 values of 0.8 lM and 4.1 lM, respectively.Interestingly, the dipyrithione analog (3) did not exhibit any cytotoxic or antitubercular activity, suggesting the importance of the oxygen atoms in these compounds' structures for any activity.Another isolated compound, kelampayoside A (8), displayed cytotoxic activity against Hep G2 and HCT 116 cells with IC 50 values of 4.8 lM and 7.7 lM, respectively.The new compound (10) was not tested due to low isolation yield.The bioassay results are summarised in Table S2 of the supplementary material.

General experimental procedures
Technical details are provided in the supplementary material.

Plant material
Stems of M. modestum (Pierre) B. Xue and R.M.K. Saunders were collected in December 2010 from Champasak Province in Laos after an ethnobotanical interview with a local renowned healer.The plant material was authenticated by Professor D.D. Soejarto at the University of Illinois at Chicago College of Pharmacy, based on both genetic and phenotypic analysis.A voucher specimen has been deposited at the Field Museum of Natural History, Chicago, IL, USA, under the accession number 2301502.

ECD calculations
Two plausible stereoisomers of 10 (2 R-10 and 2S-10) were arbitrarily selected and the systematic random conformational analyses were performed in the SYBYL-X-2.1.1 program by using MMFF94s molecular force field, with an energy cutoff of 10 kcal/mol to the global minima.All of the obtained conformers were further optimised using DFT at the b3lyp/6-31 þ g(d) level in gas phase by using Gaussian09 software (Frisch et al. 2009).The optimised stable conformers were used for ECD calculations at the b3lyp/6-31 þ g(d) level with the consideration of the first 30 excitations.The overall ECD curves values were all weighted by Boltzmann distribution.The calculated ECD spectra values were subsequently compared with the experimental ones.The ECD spectra were produced by SpecDis 1.71 software with the UV correction of À25 nm (2 R-10) and À15 nm (2S-10), respectively (Bruhn et al. 2013).

Cytotoxicity assay
The cancer cells lines Hep G2 and HCT 116 were cultured at 37 C in 5% CO 2 in DMEM and McCoy's medium, respectively, supplemented with fetal bovine serum (10%) and penicillin/streptomycin (1%).Cells in log phase growth were harvested by trypsinisation, followed by centrifugation to remove the enzyme.10,000 HCT 116 and 20,000 Hep G2 cells were seeded per well in a 96-well plate (Microtest 96V R , Falcon) and incubated for 24 hours (37 C in 5% CO 2 ).Isolated compounds dissolved in DMSO were then added to the appropriate wells at concentrations of 1 lM, 5 lM, 10 lM, 25 lM, 50 lM, 75 lM, 100 lM; 0.2% DMSO was used as a control.The cells were incubated at 37 C in the presence of the test substance.After 24 hours, the test media was replaced with media containing MTT reagent (thiazolyl blue tetrazolium bromide, Alfa Aesar) at a final concentration of 0.5 mg/mL.After 4 hours of incubation, the media was discarded, DMSO was added to the formazan crystals and the plate was put on a plate shaker for 10 min, protected from light.The reading was taken at a wavelength of 570 nm and the viability was calculated with respect to DMSO control.Etoposide was used as a positive control.IC 50 values were calculated using GraphPad Prism software (San Diego, CA, USA).

Antitubercular assay
Inhibitory activity against Mycobacterium tuberculosis was assessed using microplate Alamar Blue assay, as previously described (Shetye et al. 2021).Rifampicin was used as a positive control.The active compound was further tested for its toxicity against mammalian Vero cells (Zhou B et al. 2020).

Conclusions
In conclusion, ten compounds were identified from a stem extract of M. modestum.Disulfide derivatives of pyrithione were found in the plant, along with other nitrogen-containing compounds.Three glycosides were isolated from the butanol fraction, including one with a new structure (10).Most of the isolated natural products are reported here from M. modestum for the first time.Dipyrithione and kelampayoside A showed cytotoxic activity against cancer cells.Additionally, antitubercular activity was confirmed for dipyrithione.Therefore, this study identified new and bioactive components from M. modestum and supported its use in traditional medicine.