Anthraquinones with potential antiproliferative activities from the fruits of Morinda citrifolia

Abstract The phytochemical investigation on the fruits of Morinda citrifolia led to the isolation and characterization of a new anthraquinone, moricitrifone (1), along with seven known anthraquinones (2–8). The chemical structure of 1 was elucidated by extensive spectral analyses. The known compounds (2–8) were identified by comparing their spectral data with those reported in the literature. The antiproliferative activities of all isolated anthraquinones (1–8) against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 were evaluated in vitro. Compounds 1–8 exhibited remarkable antiproliferative activities with IC50 values ranging from 0.26 ± 0.05 to 16.58 ± 0.18 μM, which were comparable to those of doxorubicin. Graphical Abstract


Introduction
The genus Morinda in the Rubiaceae family is composed of approximately 102 species, which is widely distributed in temperate, subtropical and tropical regions of the world. There are about 26 species, 1 subspecies as well as 6 varieties in China, which grows in South China, Southwest, Southeast and Central China (Editorial Committee of Flora of China 1999). Among them, several species had been used as folk medicines. Previous phytochemical studies on the plants belonging to the genus Morinda had caused the isolation and identification of various types of natural products, such as anthraquinones, lignans, iridoids, flavonoids, triterpenes and their glycosides, which showed a series of biological activities, such as anti-inflammatory activities, anti-tumor activities, antibacterial activities, lipoxygenase inhibitory activities hypoglycemic activities and tyrosine phosphatase 1B inhibitory activities (Sang et al. 2001;Su et al. 2005;Deng et al. 2007;Nguyen et al. 2013;Youn et al. 2016;Zhai et al. 2019;Yang et al. 2020;Zandi et al. 2020). Our preliminary research results exhibited that the 90% ethanol extract of the fruits of M. citrifolia displayed significant cell growth inhibitory activities against various human cancer cell lines with the IC 50 value of 8.42 ± 0.18 lg/mL. As a part of our ongoing research into natural products with diverse chemical structures and significant biological activities from tropical fruits and tropical medicinal plants distributed in China Liu et al. 2017Liu et al. , 2018Liu et al. , 2019aMa et al. 2018;Fu et al. 2019Fu et al. , 2020a, a phytochemical investigation on the fruits of M. citrifolia was thus carried out and had led to the isolation and characterization of a new anthraquinone, moricitrifone (1), along with seven known analogues. Their structures were elucidated by extensive spectral analyses. In addition, the antiproliferative activities of all isolated anthraquinones 1-8 against five human cancer cell lines were evaluated in vitro. Herein, we report the isolation, structure elucidation as well as antiproliferative activities of these isolated anthraquinones 1-8.

Results and discussion
The 90% ethanol extract of the fruits of M. citrifolia was suspended in distilled water and extracted successively with petroleum ether and ethyl acetate. The ethyl acetate extract fraction was repeatedly subjected to silica gel CC, Sephadex LH-20 CC, ODS gel CC and semi-preparative HPLC, to yield eight anthraquinones (1-8), including a new one, as shown in Figure 1.
16.58 ± 0.18 lM (as shown in Table 1). It is noteworthy that all isolated anthraquinones 1-8 exhibited significant antiproliferative activities against five human cancer cell lines with IC 50 values similar to those of doxorubicin, even stronger inhibitory activities than those of doxorubicin in some cancer cell lines.

General experimental procedures
IR spectra were measured using a Bio-Rad FTS-135 spectrophotometer in KBr discs. UV spectra were measured using a PharmaSpec UV-1700 spectrophotometer. NMR spectra were recorded on Bruker 400 MHz spectrometer using TMS as an internal standard, with chemical shifts recorded as d values. HR-ESI-MS spectra were recorded with an Acquity UPLC-Q-TOF Micro-mass spectrometer. Preparative HPLC was performed on a Thermo Fisher UltiMate 3000 LC series equipped with a MWD detector using a Waters XBridge C 18 column (250 Â 20 mm, 5 lm). Sephadex LH-20 (40-70 lm, Amersham Biosciences, Sweden), ODS gel (50 lm, Merck, Darmstadt, Germany), Silica gel H (10-40 lm, Qingdao Marine Chemical Inc., China) and Silica gel Qingdao Marine Chemical Inc.,China) were applied for column chromatography (CC).

Antiproliferative activity bioassays
The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW480. All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) in 5% CO 2 at 37 C. The cytotoxicity assay was performed using the MTT method in 96-well microplates Ma et al. 2018;Fu et al. 2019Fu et al. , 2020a. Briefly, adherent cells (100 lL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, and suspended cells were seeded just before drug addition with an initial density of 1 Â 10 5 cells/mL. Each tumor cell line was exposed to the tested compound at concentrations of 0.0625, 0.32, 1.6, 8.0, and 40.0 lM in triplicate for 48 h. Doxorubicin (Sigma, St. Louis, MO, USA) was used as the positive control. After treatment, cell viability was measured and the cell growth curve was plotted. IC 50 values were calculated by the Reed and Muench method Ma et al. 2018;Fu et al. 2019Fu et al. , 2020a.

Conclusions
In our current study, the chemical investigation on the fruits of M. citrifolia was carried out and had caused the isolation and characterization of a new anthraquinone, moricitrifone (1), as well as seven known anthraquinones (2-8). The discovery of 1 is not only a further addition to diverse and complex array of anthraquinones, but also, the existence of anthraquinones (2-8) as characteristic marker may be helpful in chemotaxonomical classifications. The antiproliferative activities of compounds 1-8 against several human cancer cell lines were also assessed, and confirmed to be very significant, which may be used as an explanation of the folk use of the fruits of M. citrifolia, which had been used to treat various tumors. These findings also indicated that these anthraquinones with notable anti-inflammatory effects isolated from the fruits of M.
citrifolia could be used for the research and development of new natural antitumor agents.

Disclosure statement
No potential conflict of interest was reported by the authors.