Cytotoxic and antibacterial xanthones from the roots of Maclura cochinchinensis

Abstract Three new furanoxanthones, macochinxanthones A–C (1–3) and sixteen known xanthones (4–19) were isolated from the roots of Maclura cochinchinensis. Their structures were elucidated by spectroscopic analysis including NMR, UV and IR, as well as mass spectrometry. Chiral-phase HPLC analysis of 1–3 revealed that they were scalemic mixtures with an enantiomeric excess (ee) of 0.05%, 36.8% and 8%, respectively. Most of the isolated xanthones exhibited potent cytotoxicity against four cancer cell lines (KB, HelaS3, A549 and HepG2) with IC50 values in the range of 1.29–90.15 µM. In addition, many of them displayed antibacterial activity against Gram-positive bacteria and Methicillin resistant Stephylococus aureus (MRSA) with MIC values in the range of 4–128 µg/mL. Graphical Abstract


Introduction
Maclura cochinchinensis (Lour.) Corner (Syn. Maclura amboinensis and Cudrania cochinchinensis) belongs to the Moraceae family. It is widely distributed in most of Asia districts such as India, Sri Lanka, China, Japan, Taiwan, Burma, Laos, Vietnam, Cambodia, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea, as well as Australia (Berg et al. 2011;Siripong et al. 2012). It has been used in folk medicines for hepatitis, rheumatism and neuralgia (Nakashima et al. 2015), wound healing, bruising, boils, scabies, oedema, blood statis, dysmenorrhea and contusions (Chien et al. 2019). In Thailand, M. cochinchinensis is known as 'Kae Lae' and can be found throughout the country, where in remote areas people use it for treating chronic fever, diarrhea, fainting, skin infection, abnormality of the lymph nodes (Bunyapraphatsara et al. 2000;Kongkiatpaiboon et al. 2017). This medicinal plant has been reported as a source of bioactive compounds such as xanthones (Chang et al. 1989a(Chang et al. , 1989bNakashima et al. 2015Nakashima et al. , 2017 and flavonoids (Chien et al. 2019;Polbuppha et al. 2021). Most of these compounds demonstrate a variety of biological activities including antifungal (Fukai et al. 2003), antimicrobial (Polbuppha et al. 2021), anticancer (Siripong et al. 2012;Chien et al. 2019), anti-inflammation (Nakashima et al. 2017), anti-lipid peroxidation (Chang et al. 1994) and antidiabetic (Chewchinda et al. 2021). To further search for bioactive compounds from the roots of M. cochinchinensis, the bioassay screening of n-hexane and EtOAc extracts showed antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 20-160 lg/mL, and cytotoxicity against KB and HelaS3 cell lines with IC 50 values in the range of 2.97-10.07 lg/mL. Herein the phytochemical study of the roots of M. cochinchinensis and their biological activities are presented.
Compound 1 was isolated as a pale yellow solid and its molecular formula C 19 H 18 O 6 was deduced from the 13 C NMR and HRESI-MS, m/z 343.1176 [M þ H] þ (calcd. 343.1182) implying 11 degrees of unsaturation. The UV spectrum showed absorption maxima at 253, 280 and 313 nm, corresponding with a 1,3,5,6-tetraoxygenated xanthone skeleton (Nakashima et al. 2015). The IR spectrum showed absorption bands of hydroxyl (3344 cm À1 ) and carbonyl (1623 cm À1 ) functionalities. The 1 H and 13 C NMR spectroscopic data of 1 ( 3. The HMBC correlations of H-4 to C-2, C-3 and C-9a; H-12 and H-13 to C-2; H-14 to C-2; and the methoxy protons to C-3 indicated that 2,3,3-trimethyl dihydofuran moiety fused with the A-ring at C-1 and C-2 ( Figure S1). Furthermore, the correlations of H-7 to C-5 and C-8a; H-8 to C-6, C-9 and C-10a confirmed two hydroxy groups were located at C-5 and C-6. The planar structure of 1 was similar to a known cudracuspixanthone C isolated from the roots of Cudrania tricuspidata (Jo et al. 2014), except for the absence of a prenyl unit at C-4 in 1.
It appeared that the chemical shift values for C-2 (d C 118.2) and OMe (d C 54.3) of 1 were more shielded than those of cudracuspixanthone, C-2 (d C 124.4) and OMe (d C 62.0), suggesting the effect of the isoprene group at C-4 in cudracuspixanthone C. This was supported by the chemical shift value for C-2 (d C 118.8) of its analogue, cudratrixanthone H isolated from Cudrania tricuspidata (Kwon et al. 2014). Thus, 1 was identified as a new furanoxanthone, and it has been named macochinxanthone A. In the proposed biosynthesis, compound 1 could be derived from the isolated precursor crudraniaxanthone (16) by a ring closure of the 1,1-dimethyl-2-propenyl group at C-2, to form a dihydrofuran ring fused with the A-ring at C-1 and C-2. It was found that the specific rotation value of 1 was almost zero [0.0198 (c 0.08, MeOH)] which corresponded with the absence of the Cotton effect signal in its ECD spectrum. This data suggests that 1 was a mixture of enantiomers. Further chiral-phase HPLC analysis of 1 revealed that it was a scalemic mixture with an enantiomeric excess (ee) of 0.05% ( Figure S2). However, the attempt to separate this enantiomer pair by chiral HPLC was unsuccessful.
Compound 2 was isolated as a light brown powder and its molecular formula C 23 H 24 O 8 was established from the 13 C NMR and HRESI-MS, m/z 429.1550 [M þ H] þ (calcd. 429.1549), revealing 12 degrees of unsaturation. The IR spectrum showed absorption bands of hydroxyl (3311 m À1 ) and carbonyl (1631 cm À1 ) functionalities. The UV spectrum showed absorption maxima of the xanthone core similar to that of 1. The 1 H and 13 C NMR spectroscopic data of 2 (Table S1) -14 and H-15, and H-16 and H-17, supporting the partial connection in the molecule. The HMBC spectrum of 2 displayed correlations of H-8 to C-6, C-16, C-10a and C-9; H-16 to C-6, C-7, C-8 and C-18; H-17 to C-16, C-18, C-19 and C-20; and H-19 to C-17 and C-20, indicating that the 2-(isopropyl-2-ol)-3-hydroxyhydrofuran moiety fused with the B-ring at C-6 and C-7. This moiety was similar to a known xanthone 1,2,5,4 0 -tetrahydroxy-4-(1,1-dimethyallyl)-5 0 -(2-hydroxypropan-2-yl)-4 0 ,5 0 -dihydrofurano(2 0 ,3 0 :6,7) xanthone isolated from Garcinia xanthochymus (Chen et al. 2010). Furthermore, correlations of H-4 to C-2, C-3 and C-9a; H-12 and H-13 to C-2; H-14 to C-2, C-3 and C-15; and H-15 to C-11 and C-14 indicated a 2,3,3-trimethyldihydrofuran unit fused with the A-ring at C-2 and C-3 ( Figure S1) similar to those of isolated gerontoxanthone G (7). The small coupling constant values (4.8 Hz) of vicinal protons, H-16 and H-17 suggested that the two protons were in cis orientation (Minch 1994). Thus, 2 was discovered as a new furanoxanthone and it has been named macochinxanthone B. Among isolated compounds, gerontoxanthone G (7) could be proposed as a precursor of 2 by ring closure involving oxidation of a prenyl unit at C-7 to form a hydroxy hydrofuran fused with the B-ring at C-6 and C-7 in the biosynthesis pathway. Since the specific rotation value of 2 was almost zero [-0.0003 (c 0.2, MeOH)], and the ECD spectrum showed no signal for a Cotton effect, the chiral HPLC analysis of 2 was performed and the result displayed as a scalemic mixture with an ee of 36.8% ( Figure S3). An attempt to separate this enantiomer pair by chiral HPLC was unsuccessful.
Compound 3 was isolated as a light brown solid and its molecular formula was established from the 13 C NMR and HRESI-MS, m/z 443.1708 [M þ H] þ (calcd. 443.1706) to be C 24 H 26 O 8 , having 12 degrees of unsaturation. The UV, IR and NMR spectroscopic data of 3 were similar to those of 2, except that a hydroxyl group at C-5 was replaced by a methoxy group (d H/C 4.08/d C 59.3) in 3. The HMBC correlation of methoxy protons to C-5 revealed that a methoxy group was substituted at C-5 ( Figure S1). Therefore, 3 was identified as a new furanoxanthone analogue and it has been named macochinxanthone C. The structure of 3 could be proposed to derive from the isolated precursor gerontoxanthon E (6) by involving oxidation and the ring closure of a prenyl group at C-7, the same as 2. Similar to that of 2, the specific rotation value of 3 was almost zero [-0.0003 (c 0.08, MeOH)], and the ECD spectrum showed no signal for a Cotton effect. Consequently, the chiral HPLC analysis of 3 ( Figure S4) indicated as a scalemic mixture with an ee of 8%, and this enantiomer pair could not be separated by chiral HPLC.

Plant material
Roots of Maclura cochinchinensis were collected from Pattani province, Thailand in April 2018. The specimens were identified by Prof. Dr. Pranom Chantaranothai, Department of Biology, Khon Kaen University, Thailand, where a voucher specimen (SK 17) was deposited.

Conclusion
Nineteen xanthones (1-19) were isolated from the roots of Maclura cochinchinensis. Their structures were determined by spectroscopic methods. Three of them were new furanoxanthones, macochinxanthones A-C (1-3), and each of them was analysed by chiral-phase HPLC to be a scalemic mixture of enantiomers. However, attempts to separate these enantiomer pairs by chiral-phase HPLC were not successful. The biosynthesis of 1-3 was proposed. This is the first report of xanthones 10, 11, 13, 14 and 19 from M. cochinchinensis and C. cochinchinensis. All tested compounds, except 2, were cytotoxic towards four cancer cell lines, KB, HelaS3, A549 and HepG2. Several of them displayed potent cytotoxicity towards KB, HelaS3 and A549 cancer cell lines with IC 50 values in the range of 1.29-4.58 lM. In addition, several of them showed moderate or potent antibacterial activity against Gram-positive bacteria, B.cereus, B. subtilis, S. aureus and MRSA with MICs values in the range of 4-128 lg/mL. These findings support that roots of M. cochinchinensis are a rich source of bioactive xanthones, especially a major isolate 2-deprenylrheediaxanthone (18).