Cytotoxic compounds from the leaves and stems of the endemic Thai plant Mitrephora sirikitiae

Abstract Context Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders (Annonaceae) is a plant endemic to Thailand. Its constituents and their biological activities are unknown. Objective Isolation and identification of the compounds in the leaves and stems of M. sirikitiae and determination of their cytotoxicity. Materials and methods Methanol extracts of the leaves and stems of M. sirikitiae were separated by chromatography, and spectroscopic methods were used to determine the structures of the components. The cytotoxicity of the extracts and pure compounds was evaluated using the sulforhodamine B assay with several cell lines. The cells were treated with the compounds at concentrations of 0.16–20 µg/mL for 48 or 72 h. Results The investigation of the extracts of M. sirikitiae leaves and stems resulted in the isolation of a new lignan, mitrephoran, and 15 known compounds. Among these compounds, 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, ciliaric acid, 6-methoxymarcanine A, and stepharanine were isolated from this genus for the first time. The alkaloids liriodenine and oxoputerine exhibited strong cytotoxicity against all tested cells (IC50 values of 6.59–11.02 µM). In contrast, magnone A, 3′,4-O-dimethylcedrusin, and 6-methoxymarcanine A inhibited the growth of some of the tested cells (IC50 values of 2.03–19.73 µM). Magnone A and 6-methoxymarcanine A showed low toxicity for Hek 293 cells (IC50 >20 µM). Discussion and conclusions M. sirikitiae is a source of cytotoxic lignans and alkaloids. Among the cytotoxic compounds, magnone A and 6-methoxymarcanine A are potentially useful lead compounds for the further development of anticancer agents because of their selective inhibitory effects on cancer cell lines.


Introduction
Cancer is one of the major causes of morbidity and mortality worldwide. The International Agency for Research on Cancer (IARC) has estimated the incidence of cancer, and its mortality rate to be 18.1 million new cases and 9.6 million deaths in 2018 alone. One-third of global cancer cases are lung, breast, and colorectal cancers, and these are the top five causes of cancerrelated deaths (Bray et al. 2018). Chemotherapy is the backbone of treatment for many cancers at different stages, complementing surgery and radiotherapy. However, cancer cells frequently develop resistance to chemotherapeutic drugs, limiting the effectiveness of treatment. Thus, the identification of new effective anticancer compounds is needed. A number of anticancer drugs are derived from natural sources including plants, microorganisms, and marine organisms (Bailon-Moscoso et al. 2017). Plants are important sources of biologically active compounds and promising chemotherapeutic agents. As part of our on-going study of bioactive compounds obtained from plants, we have investigated the cytotoxic compounds present in a variety of plants in Thailand. Interestingly, our in vitro screening of the cytotoxic activity of plant extracts indicates that those from Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders (Annonaceae), known as Mahaphrom Rachini in Thai, have substantial cytotoxic activity. M. sirikitiae is a plant endemic to northern Thailand. It was first discovered on a mountain peak at an altitude of 1100 m in the Mae Surin Waterfall National Park, Mae Hong Son Province in 2004 (Weerasooriya et al. 2004). However, to date, phytochemical and biological studies of this plant have not been reported.
In the present study, our cytotoxic activity screening of the crude methanol extracts and fractions of M. sirikitiae leaves and stems demonstrated that this plant exhibits moderate to potent cytotoxicity against many cancer cell lines. Therefore, we report herein the lignans, dihydrobenzofuran lignan, alkaloids, and diterpenoids isolated from the methanol extracts of leaves and stems of M. sirikitiae, together with their cytotoxic effects on the cancer cell lines P-388 (mouse lymphoid neoplasma), KB (human oral nasopharyngeal carcinoma), HT-29 (human colon carcinoma), MCF-7 (human breast carcinoma), A549 (human lung carcinoma), ASK (rat glioma), and non-cancerous Hek-293 cells (human embryonic kidney cells).

Plant materials
The leaves and stems of M. sirikitiae were collected from Mae Surin Waterfall National Park, Mae Hong Son Province, Thailand in September 2007. The plant was identified by Narong Nuntasaen and a voucher specimen (BKF144972) has been deposited at the Forest Herbarium, Royal Forestry Department, Bangkok, Thailand.

Extraction, isolation, and characterisation
The air-dried powdered leaves (1.0 kg) and stems (2.0 kg) of M. sirikitiae were extracted by maceration with distilled MeOH (9 Â 4 L), and the solvent was removed using a rotary evaporator, followed by freeze drying, to yield 102.0 g of crude MeOH leaf extract and 72.4 g of crude MeOH stem extract.

In vitro cytotoxic activity assay
The methanol extracts and the isolated compounds were evaluated for their cytotoxic activities against several cell lines, including murine lymphocytic leukaemia (P-388), human oral epidermoid carcinoma (KB), human colon carcinoma (Col-2 and HT-29), human breast cancer (MCF-7), human lung carcinoma (Lu-1 and A549), rat glioma (ASK), and noncancerous human embryonic kidney cell (HEK-293) by sulforhodamine B (SRB) assay in 96-well microtiter plates (Skehan et al. 1990). This method measures the cellular protein content of cultures in 96-well microtiter plates. The cell lines were seeded into 96-well microtiter plates and treated with the test compounds at concentrations of 0.16-20 mg/mL for 72 h, except for the P-388 cells, which were treated for 48 h. Then the cell cultures were fixed with 20% trichloroacetic acid (Merck) and stained with 0.4% SRB (Sigma-Aldrich) dissolved in 1% acetic acid (Merck) for 1 h. The cellular-protein-bound dye was extracted with 10 mM unbuffered Trisbase solution (pH 10.5) (Sigma-Aldrich) for the determination of optical density at 510 nm with a microtiter plate reader (Thermo Scientific TM Multiskan TM GO Microplate Spectrophotometer). The cytotoxic potency was expressed as median inhibition concentrations (IC 50 ), the concentration that inhibits 50% of cell viability (mg/mL and mM). The IC 50 values were determined from the nonlinear regression curve fit in GraphPad Prism software (version 5). Ellipticine (Sigma-Aldrich), a cytotoxic plant alkaloid causing topoisomerase II inhibition and DNA intercalation, was used as a positive control.
Mitrephoran (6) was isolated as colourless crystals. The HR-ESI-MS data revealed the parent ion at m/z 411.1420 [M þ Na] þ , suggesting a molecular formula of C 21 H 24 O 7 . Moreover, in the EI-MS spectrum of 6, the molecular ion and base ion peaks were observed at m/z 388 [M] þ (8) and 165 (100) which indicate benzylic and tetrahydrofuran ring fragments. The IR spectrum of 6 showed the characteristic absorption bands of a hydroxyl group and a carbonyl group conjugated with an aromatic ring at 3396 and 1672 cm À1 . In addition, its UV spectrum contained k maxima at 230, 276, and 305 nm, corresponding to the furanoid lignan skeleton. The 13 C and distortionless enhancement by polarisation transfer (DEPT) NMR spectra of 6 indicated the presence of 21 carbons atoms, including one carbonyl carbon, six aromatic methine carbons, six aromatic quaternary carbons, three methane carbons, two methylene carbons, and three methoxy carbons, as listed in Table 1. The 1 H-NMR spectrum of 6 showed three singlets at d 3.99, 4.01, and 4.02 ppm indicating the presence of three methoxy substituents on aromatic rings at C-3 0 , C-3 00 , and C-4 00 , respectively. The six signals in the low-field region of the 1 H NMR spectrum were assigned to six aromatic protons of two 1,3,4-trisubstituted benzene rings. The first set appearing as an ABX coupling system consisting of d 7.67 (1H, dd, 8.4, 2.0 Hz, H-6 00 ), 7.63 (1H, d, 2.0 Hz, H-2 00 ), and 6.98 (1H, d, 8.4 Hz, H-5 00 ), and these were assigned to the protons of a 3 00 ,4 00 -dimethoxyphenyl ring connected with a carbonyl carbon (C-4a). The broad singlets at d 7.08 (1H, s, H-2 0 ), 6.94 (1H, s, H-6 0 ), and 6.93 (1H, s, H-5 0 ) were identified to three aromatic protons on a 3 0 -methoxy-4 0 -hydroxyphenyl ring. The proton signals at d 3.83 (1H, dd, 10.9, 4.4 Hz, H-3a), 3.73 (1H, dd, 10.9, 5.6 Hz, H-3a), and 4.37 and 4.24 (each 1H, m, H-5) were assigned to non-equivalent methylene protons on carbon atoms bearing oxygen atoms of a hydroxymethylene substituent and a furan ring, respectively. The 1 H-NMR spectrum of 6 also displayed signals at d 4.73 (1H, d, 9.1 Hz, H-2), 2.96 (1H, m, H-3), and 4.24 (1H, m, H-4) assignable to the three methine protons at C-2, C-3, and C-4, respectively. Full assignment of the 1 D NMR spectra and the connectivities of compound 6 were established based on 1 H-1 H correlated spectroscopy (COSY), heteronuclear single quantum coherence spectroscopy (HSQC), and heteronuclear multiple bond correlation (HMBC) spectroscopic data analyses. Moreover, the absolute configuration and positions of three methoxy and one hydroxy functional groups were confirmed by single-crystal X-ray diffraction analysis. The NMR spectra of compound 6 are similar to that of forsylthialan B (Piao et al. 2008), but the HMBC spectroscopic and X-ray crystallographic data indicated that there is a hydroxyl group at C-4 0 of the phenyl ring, and stereocenters carbons C-2, C-3, and C-4 were assigned as R, S, and R configurations, respectively ( Figure 2).

In vitro cytotoxic activity
The cytotoxicity testing of the methanol extracts from the leaves, and stems of M. sirikitiae revealed that the leaf extract exhibited moderate to potent cytotoxicity against P-388, KB, Col-2, MCF-7, and Lu-1 cell lines, having IC 50 values of 0.7, 5.1, 12.0, 2.6, and 3.9 mg/mL, respectively. In contrast, the stem extract showed lower cytotoxicity against P-388, MCF-7, and Lu-1, having IC 50 values of 7, 13, and 17 mg/mL, respectively. Therefore, the extracts were further investigated with respect to the component cytotoxins. The isolated compounds (1-16) were subjected to cytotoxic activity assays against cancer cell lines: P-388, KB, HT-29, MCF-7, A549, and ASK and non-cancerous Hek-293 cells, and the results are presented in Table 2. Among those compounds, the alkaloids liriodenine (5) and oxoputerine (11) exhibited potent cytotoxic activity against all tested cell lines, having IC 50 values in the range of 6.59-11.02 mM. 6-Methoxymarcanine A (15) inhibited the growth of four tested cell lines, P-338, HT-29, MCF-7, and A549, having IC 50 values ranging from 8.33 to 12.30 mM but showed low cytotoxicity for KB and ASK cancer cell lines and the non-cancerous Hek-293 cell line. The lignan magnone A (3) showed specific cytotoxicity against P-388 and MCF-7, having IC 50 values of 8.96 and 4.40 mM, respectively. 3 0 ,4-O-Dimethylcedrusin (7) exhibited moderate to strong cytotoxic effects on KB, MCF-7, and A549 cells, having IC 50 values of 2.03, 3.77, and 10.32 mM, respectively, but it was also toxic to the Hek-293 non-cancerous cell line. Therefore, magnone A (3) and 6-methoxymarcanine A (15) are potentially useful anticancer agents because of their specific inhibitory activities in some tested cancer cell lines and non-toxic effects on non-cancerous cells. In contrast, the other compounds were inactive against all tested cell lines.
Some types of lignans have been reported to have cytotoxic and anticancer activities. For example, sesamin is a furofurantype lignan and is used as a dietary supplement because of its pharmacological activity, including protective effects against oxidative stress and cancer. The anticancer properties of sesamin are associated with its anti-proliferative, pro-apoptotic, antiinflammatory, anti-metastatic, anti-angiogenic, and pro-autophagocytic effects (Majdalawieh et al. 2017). In addition, aryltetralin lactone lignans such as podophyllotoxin and its derivatives have been shown to possess anticancer and antitumor activities because of their tubulin polymerisation and DNA topoisomerase II inhibitory activities (Ardalani et al. 2017). Moreover, 2,3-naphtalide lignans (Fukamiya and Lee 1986;Jin et al. 2014), butanetype lignans (Lambert et al. 2005;Wukirsari et al. 2014), and dihydrobenzofuran neolignans (Huang et al. 2013;Fukui et al. 2018) have been found to be cytotoxic against many cancer cell lines. The natural dihydrobenzofuran neolignan 3 0 ,4-O-dimethylcedrusin (7) has been shown to possess anticancer activity by inhibition of cell proliferation (Pieters et al. 1993). It has also been revealed that synthetic benzofuran lignan derivatives can mediate cell death by the induction of G2/M cell cycle arrest via a p53-dependent pathway (Manna et al. 2010). Furthermore, in our study, the tetrahydrofuran lignan 3 showed cytotoxicity against some tested cancer cell lines. A comparison of the activity of compounds 3 and 6 indicates that the methoxy group at C-4 0 of the aromatic ring is important to the activity of these compounds.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
We are grateful to the Thailand Research Fund [MRG5580047], the Centre of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation, Mahidol University under the National Research Universities Initiative and Junior Research Fellowship Programme from the French Embassy for the financial support.