One new limonoid with cytotoxicity against glioma cell lines from Cipadessa baccifera

Abstract Glioma is a common malignant tumor with a high incidence rate but a low cure rate. In this paper, one previously undescribed limonoid (1), along with two known cipadesin-type limonoids 2 and 3, were isolated from Cipadessa baccifera. Their structures were established based on a comprehensive analysis of NMR and MS spectra. Compound 1 exhibited moderate cytotoxicity against U251 and BT-325 cells with IC50 values of 7.32 ± 0.21 and 13.25 ± 0.35 μM, suggesting that 1 might be a promising leading compound for the treatment of glioma. Graphical abstract


Introduction
Cipadessa baccifera (Roth) Miq.(C.baccifera) is widely distributed in the southwest region of China, such as in Sichuan, Guizhou, Guangxi, and Yunan Provinces.Its roots and leaves were the medicinal parts in traditional Chinese medicine (Wang et al. 2014;Yu et al. 2015;Zhang et al. 2015) for the treatment of colds, dysentery, diarrhea, abdominal pain, and rheumatism.The chemical investigation has revealed that a series of natural components, such as flavonoids, triterpenes, and limonoids, are its bioactive constituents, exhibiting various pharmacology effects, including anti-tumor, anti-bacterial, anti-HIV, and anti-oxidant activities (Yuan et al. 2005;Di et al. 2007;Yuan et al. 2007;Fu et al. 2014;Yu et al. 2016).
Glioma is a common malignant tumor of the central nervous system, mainly derived from the neuroepithelium.Gliomas have a high incidence rate but a low cure rate (Liu et al. 2019).So far, drug treatment is still the main method for the treatment of glioma (Omuro and DeAngelis 2013).In our studies on natural cytotoxic products against tumor cells, one previously undescribed limonoid (1) from leaves of C. baccifera along with two known compounds (2, 3) were discovered (Figure 1).In this paper, the isolation, structure determination, and the cytotoxicity of 1 against two glioma cell lines U251 and BT-325 were described.

Result and discussion
Compound 1 was obtained as a white amorphous powder, with the molecular formula of C 31 H 42 O 10 as suggested by its HR-ESI-MS giving an ion at m/z 597.2673 [M þ Na] þ (calcd 597.2676) along with its 13 C NMR spectrum.Therefore, 11 indices of hydrogen deficiency were indicated for the structure of compound 1.In the 1 H and 13 C NMR spectra of 1, signals for three ester carbonyls (d C 169.4, 174.2, 177.7), one exocyclic double bond (d C 115.3, 142.2; d H 5.05 and 5.25, each 1H, br s), and one b-substituted furan ring (d C 109.8, 140.7, 143.0; d H 6.41, 7.41, 7.47) were revealed, accounting for seven of the 11 degrees of unsaturation.Thus, the presence of four ring moieties was deduced based on the existence of four remaining indices of hydrogen deficiency for 1.Additionally, one methoxyl [d H 3.69, (3H, s)] and four tertiary methyls (d H 0.96, 0.97, 0.98, 1.03, each 3H, s) signals were also observed in the 1 H and 13 C NMR spectrum of 1.All the proton and carbon signals were correlated through the HSQC spectrum.Subsequently, the skeleton of 1 was deduced as the angolensate-type limonoid based on the biosynthetic reasoning and the NMR feature resembling those of the angolensate-type limonoids (Yu et al. 2020).Furthermore, the aforementioned deduction was confirmed by HMBC correlations (Figure S1), which suggested the presence of rings A-D (Figure 1).In addition, HMBC correlation from H-2 (d H 4.88) to C-1 0 (d C 177.7) demonstrated that the isobutyryloxy was linked to C-2 of the skeleton while those from H-1 (d H 3.55) to C-3 (d C 73.3) verified that both C-3 was oxidised.Furthermore, the HMBC correlations from 19-CH 3 (d H 0.96) to C-9 (d C 59.2), from H-9 (d H 2.12) to C-11 (d C 67.8)/C-12 (d C 37.4), and from 18-CH 3 (d H 1.03) to C-12 (d C 37.4) substantiated the presence of a hydroxyl at C-11 (Figure S1).
The relative configuration of 1 was determined by investigation on the NOESY data.The correlation of H 3 -19/H-2/H 3 -28 indicated that they were cofacial.H-3 was assigned to be axially oriented due to both its large J 2,3 coupling constant of 11.2 Hz and the NOESY correlation of H-3/H-17/H 3 -29.The strong correlations of H-12a/H 3 -18 and H-9/ H 3 -19 indicated the spatial location of Me-18 and H-9.The correlation between H-3 and H-17 suggested the orientation of the furan ring moiety as shown in Figure S1.Therefore, the structure of 1 was established as shown in Figure 1.The absolute configuration of 1 was determined by comparing the CD data of 1 with that of 2 0 -epicibacciferin B (Figure S2), a similar angolensate-type limonoid whose absolute configuration was determined by the X-ray diffraction analysis (Yu et al. 2020).The result showed that both 1 and 2 0 -epi-cibacciferin B gave the positive cotton effect at 202 nm and the negative one at 208 nm, implying the absolute configuration of 1 R, 2 R, 3 R, 5S, 9 R, 10S, 11 R, 13S, 14S, 17S for 1.Finally, the structure of 1 was elucidated as shown.
The cytotoxicity of 1 was evaluated on glioma cell lines U251 and BT-325.The results showed that 1 possessed moderate cytotoxicity against U251 and BT-325 cells with IC 50 values of 7.32 ± 0.21 and 13.25 ± 0.35 lM, respectively (temozolomide as the positive control with IC 50 values of 2.15 ± 0.08 and 3.53 ± 0.11 lM for U251 and BT-325 cell lines, respectively).

General experimental procedures
NMR spectra were recorded on a Brucker AVANCE 600 FT-NMR spectrometer at 600 MHz for 1 H and 150 MHz for 13 C.The chemical shifts are expressed as d value in ppm and the coupling constants (J) are given in Hz.Molecular weights were analysed on an Agilent 6530 LC-QTOF-MS spectrometer system.CD spectra was measured on JASCO pu-2080.Optical rotations were determined on an JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan).Silica gel for chemical isolation was from Qingdao Marine Chemical Co., Ltd.(Qingdao, China), and octadecysilyl (ODS) was purchased from YMC Chemical Co., Ltd.(Japan).Semipreparative HPLC for chemical purification was carried on a Shimadzu LC-10A chromatograph apparatus equipped with a UV detector and an ODS column (YMC-Pack ODS-A, 5 lm).Organic solvents were from Yuwang Chemical Co.(Shandong, China).

Plant material
Dried leaves of C. baccifera were collected in Tongren city, Guizhou Province, P.R. China.The material was authenticated by Dr. Zheng Xiang from Liaoning University.A voucher specimen (NO.20191120) has been deposited at the school of pharmaceutical science, Liaoning University.

Cell viability assay
Cell lines of U251 and BT-325 were purchased from ATCC.The cells in the logarithmic phase were digested with trypsin and adjusted the cell concentration to 5 Â 10 4 /mL.The prepared cell suspension was evenly mixed and 100 lL cell suspension was pipetted into each well of a 96-well plate and cultured at 37 C for 48 h in an incubator containing 5% carbon dioxide with the treatment of various concentrations of compound 1 (0, 5, 10, 20, 50, 100, 200 lM) or temozolomide (0, 1, 2, 5, 10, 20, 50 lM), respectively.The purity (! 97%) of each compound was determined by HPLC analysis before use in the bioassay.After centrifugation to remove the culture medium, washed it with PBS 3 times, added 10 lL of MTT solution (0.2% MTT solution) (Beyotime, China) to each well, and continue to culture for 4 h.Centrifugation again to remove the culture medium, washed it with PBS 3 times, and added 100 lL of dimethyl sulfoxide to dissolve and crystallise.Finally, the optical density (OD) value was measured at 570 nm by a microplate reader (Thermo Fisher Scientific, USA) (Zhang et al. 2014).Data are expressed as means ± SEM.

Conclusion
In this paper, one previously undescribed limonoid along with two known cipadesintype limonoids 2 and 3 were isolated from C. baccifera.The cytotoxicity of 1 on two glioma cell lines U251 and BT-325 cells was investigated.Compound 1 exhibited moderate cytotoxicity against U251 and BT-325 cells with IC 50 values of 7.32 and 13.25 lM.These results suggest that 1 might be a promising leading compound for the treatment of glioma and further chemical and biological studies on C. baccifera should be performed.