Active constituents of Zanthoxylum nitidium from Yunnan Province against leukaemia cells in vitro

Zanthoxylum nitidium (Roxb.) DC (Rutaceae) is well known for inhibiting the proliferation of human gastric, liver, kidney and lung cancer cells, though research on its potential use in treating leukaemia is relatively rare. Twenty-six compounds were isolated from the chloroform and petroleum ether extracts of the roots and leaves of Z. nitidium (Zanthoxylum nitidium). They were ( +)-9′-O-transferuloyl-5, 5′-dimethoxylaricriresinol (1), 8-(3′-oxobut-1′-en-1′-yl)-5, 7-dimethoxy-coumarin (2), 5, 7, 8-trimethoxy-coumarin (3), 5-(3′, 3′-dimethyl-2′-butenyloxy)-7, 8-dimethoxy-coumarin (4), 2-(5-methoxy-2-methyl-1H-indol-3-yl) methyl acetate (5), 2′-(5, 6-dihydrochleletrythrine-6-yl) ethyl acetate (6), 6-acetonyldi-hydrochelerythrine (7), 6β-hydroxymethyldihydronitidine (8), bocconoline (9), zanthoxyline (10), O-methylzanthoxyline (11), rhoifoline B (12), N-nornitidine (13), nitidine (14), chelerythrine (15), 4-hydroxyl-7,8-dimethoxy-furoquinoline (16), dictamnine (17), γ-fagarine (18), skimmianine (19), robustine (20), R-( +)-platydesmine (21), 4-methoxyl-1-methyl-2-quinoline (22), 4-methoxy-2-quinolone (23), liriodenine (24), aurantiamide acetate (25), 10-O-demethyl-12-O-methylarnottianamide (26). Four among them, compounds 4 – 6 and 16, were first confirmed in this study by UV, IR, 1D, 2D NMR and HR-ESI–MS spectra. Compounds 1 – 2 and 11 were isolated from Z. nitidium for the first time. Of the assayed compounds, 1, 2, 9, 10, 14, 15 and 24, exhibited good inhibitory activities in the leukaemia cell line HEL, whereas compound 14 (IC50: 3.59 µM) and compound 24 (IC50: 15.95 µM) exhibited potent inhibitory activities. So, to further investigate the possible mechanisms, cell cycle and apoptosis assays were performed, which indicated that compound 14 causes obvious S-phase arrest in HEL cells and induced apoptosis, whereas compound 24 only induced apoptosis. The present results suggested both compounds 14 and 24 are promising potential anti-leukaemia drug candidates. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-021-00771-0.


Introduction
Leukaemia is closely related to the haematopoietic system, which includes the bone marrow [1], and malignant tumours of the haematopoietic system pose a serious threat to human health and life. Although early highdose combination chemotherapies can achieve complete remission in many patients, the 5-year survival rate of these patients is still unsatisfactory [2], and the discovery of new anti-leukaemia drugs is very important.
Identifying candidate drug molecules in natural products is an important approach for discovering innovative drugs. Zanthoxylum nitidium (Roxb.) DC, locally called "liangmianzhen", belongs to the family Rutaceae [3]. The palnt is distributed in Guangdong, Fujian, Yunnan, and Taiwan provinces of China. The chemical components of Z. nitidium are diverse and complex, including alkaloids, flavonoids, lignans and coumarins. Research on Open Access BMC Chemistry *Correspondence: muzi0558@126.com 1 State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China Full list of author information is available at the end of the article active substances has mainly focused on alkaloids, especially benzophenanthridine, furanquinoline, quinolones, amides, and aporphine, and a much smaller number of non-alkaloids have also been reported [4]. To data, previous studies on the biological activity of Z. nitidium have examined inhibition of the proliferation of human gastric, liver, kidney, lung and nasopharyngeal carcinoma cells [5]. In contrast, the anti-leukaemia properties of this plant are comparatively unknown. High expression of Fli-1 gene plays an important regulatory role in the process of vascular endothelial cell generation and tumour cell proliferation, as well as in promoting tumorigenesis and development [6,7]. As the Fli-1 gene is a new target for drug screening, we sought to investigate the involvement of inhibitory effects on Fli-1 against leukaemia by active compounds of Z. nitidium.
In our previous work, ethanol extracts of Z. nitidium exhibited significant inhibitory effects on the proliferation of HEL cells (The human erythroleukemia lines), which highly express Fli-1, with no significant toxicity in vitro. To find a lead compound with a good effect on the Fli-1 gene, 26 compounds were isolated, purified and identified from the roots and leaves of Z. nitidium from Yunnan province, and their antitumour activities against HEL cells were evaluated. The chemical structures of compounds 4, 5, 6 and 16 were first characterized through spectroscopic analyses based on UV (Ultraviolet and visible spectrum), IR (Infrared spectroscopy), 1D and 2D NMR, and HR-ESI-MS spectra. Moreover, the antitumor activities of the 26 compounds in HEL cells were first evaluated, and the possible mechanism of two active compounds was investigated.

Plant material
The roots and leaves of Zanthoxylum nitidum (Roxb.) DC. were collected in Mengla County, Xishuangbanna, Yunnan Province. The plant material was identified as

Extraction and isolation
Air-dried roots and leaves of Z. nitidum (20.0 kg) were extracted by refluxing in 95% EtOH (100 L) three times (4, 3, and 2 h). After filtration, the combined EtOH extracts were concentrated to remove the alcohol, and the residue was resuspended in an appropriate volume of water. The mixture was extracted three times with equal volumes of petroleum ether and chloroform to afford 180.0 g of petroleum ether extract and 190.2 g of chloroform extract. The chloroform extract (190.2 g) was separated on a silica gel (50-74 μm) column eluted with a gradient of chloroform-MeOH (volume ratio: 100⁚ 1 to 0⁚ 100) to obtain 15 fractions (Fr.1 ~ Fr.15). The Fr.2 fraction was recrystallized from the chloroform-MeOH solvent to afford compound 10 (1.3 g); Fr.4 was recrystallized to afford compound 24 (360 mg). Each fraction was repeatedly subjected to normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography and Sephadex LH-20 column chromatography (alternating the use of MeOH and chloroform-MeOH as the eluents) to afford compounds 1 (15 mg

CTG assay for antitumour activity
The human leukaemia cell line HEL was purchased from American Type Culture Collection, and the cells were cultured in DMEM. All media were supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin, and 100 units/mL streptomycin (Invitrogen). The cells were cultured at 37 °C in a humidified environment with 5% CO 2 and passaged once every 2 days for three generations. The cells were incubated in fresh cell culture medium and washed carefully to avoid false-positive results. Briefly, HEL cells (8 × 10 3 cells per well) were seeded into 96-well plates, and the plates were incubated for 24 h. Then, 10 μL of adriamycin were added as a positive control, and 10 μL of various concentrations (40, 20, 10, 5, 2.5, 1.25 μM) of compounds (5 × 10 -6 mol/L) were added as the test group, with 5 wells per group. After incubation for 72 h, 20 μL of CTG reagent were added, and the cells were incubated for 10 min. After centrifugation (1500 rpm, 15 min) the supernatant was poured off, 160 μL of DMSO were added to each well, and the plate was heated and shaken for 10 min. Finally, the chemiluminescence of each well were determined using a microplate reader. After the experiment were repeated three times, the IC 50 value was calculated from the curves generated by plotting the percentage of viable cells versus the tested concentration on a logarithmic scale using Sigma Plot 10.0 software.

Cell apoptosis analysis
Apoptosis was detected by flow cytometry using Annexin V-FITC according to the manufacturer's protocol (BD Biosciences). HEL cells were treated with compounds 14 and 24 for 36 h before Annexin V and propidium iodide staining. The cells were kept under dark conditions at room temperature for 15 min before being subjected to flow cytometry analysis.

Cell cycle analysis
Cell cycle analysis was conducted by propidium iodide (PI) staining after treatment with compounds 14 and 24 for 36 h. Briefly, cells were plated in culture dishes and cultured with prepared DMEM medium for 12 h, after which the cells were treated with compounds 14 and 24 for 36 h, and the supernatant was removed. The treated cells were fixed with 70% ethanol overnight before staining with PI mixed with RNase. The cells were kept under dark conditions at room temperature for 30 min before being subjected to flow cytometry analysis.

Statistical analysis
All measurements were made in triplicate, and all data are expressed as the means ± SEM of three independent experiments. Significant differences from the respective control for each experimental group were examined by one-way analysis of variance (ANOVA) using GraphPad Prism 5 software. P < 0.05 was considered statistically significant.

Isolation and structural elucidation
Dried roots and leaves (20 kg) of Z. nitidium were heated and refluxed in 95% EtOH. The resulting extract was concentrated and then partitioned between petroleum ether and chloroform. The extracts were further separated by recrystallization and various forms of column chromatography (CC) to afford compounds 1 -26 (Fig. 1).

Chemical Structure of compound 16
Compound 16 was obtained as tawny solid with a molecular formula of C 13 H 11 O 4 N deduced from its HR-ESI-MS spectrum (m/z 246.0760 [M + H] + ). The UV profile of 16 revealed λ max values of 249, 201 and 316 nm, which are similar to those of quinoline [11]. The IR spectrum displayed absorption bands for an aromatic ring (1516 and 1443 cm −1 ) and an ether (1151 and 1046 cm −1 ). As indicated in Table 4 03 (s, 1H)]. Based on the above nuclear magnetic resonance data, compound 16 is consistent with 4-hydroxy-7, 8-dimethoxy-furoquinoline, which has been previously reported in the literature [14].

Biological activities of the isolated compounds
To analyse the effects of the 26 compounds on leukaemia cells (HEL cell lines), their IC 50 values against HEL cells proliferation were determined by the CTG method, using adriamycin (IC 50 : 0.021 µM) as a positive control. As presented in Table 5

Compounds 14 and 24 induced cell cycle arrest
To confirm the effects of compounds 14 and 24 with different structures on the cell cycle, the cell cycle distribution of HEL cells was examined after treatment with the compounds for 36 h. As illustrated in Fig. 6, significant S-transition arrest was observed in HEL cells treated with compound 14, which provided the most significant effect. Indeed, the fraction of cells in the S-phase was dose-dependently increased by treatment with 14, and the population of cells in S-phase was markedly increased to 52.04% in cells treated with 8 μM compared to 37.92% in untreated cells. Conversely, compound 24, with a different structure, had no obvious effect on the HEL cell cycle.

Compounds 14 and 24 induced apoptosis of HEL cells
To determine whether the antiproliferative activity of 14 and 24 is accompanied by enhanced leukaemia cell apoptosis, flow cytometry and an Annexin V-FITC apoptosis detection kit were used to detect apoptosis. Compared with untreated cells, cells treated with compounds 14 and 24 displayed significant dose-dependent increases, as shown in Fig. 7. At the same time, compound 24 at 7.5 μM and 15.0 μM induced significant increases in apoptosis compared with the control group (DMSO). Compound 24 at concentrations of 7.5, 15 and 30 μM promoted apoptosis from 6.11% and 17.34% to 25.81% in a dose-dependent manner. Hence, compounds 14 and 24 caused obvious apoptosis in HEL cells in a concentration-dependent manner.

Conclusions
In summary, four compounds (4 -6 and 16) with incomplete spectra and 22 known compounds were isolated and identified from the chloroform and petroleum ether extracts of the roots and leaves of Z. nitidium. The chemical structures of compounds 4 -6 and 16 were elucidated by thorough spectroscopic analyses, and compounds 1, 2 and 11 were isolated from Z. nitidium for the first time. Among the isolated compounds, 1, 2, 9, 10, 14, 15 and 24, which are alkaloids, exhibited good inhibitory activities in the leukaemia cell line HEL, whereas compound 14 (IC 50 : 3.59 µM) and compound 24 (IC 50 : 15.95 µM) exhibited potent inhibitory activities. To clarify the effect of different compound structure 14 and 24 in HEL cells, apoptosis and cell cycle assays showed that compound 14 possesses antiproliferative activity, and induces S-phase cell cycle arrest and apoptosis in HEL cells. In contrast, compound 24 only induced apoptosis in HEL cells. These results indicated that benzophenanthridine alkaloids had significant inhibition activities in leukaemia cells, providing new ideas for the structural modification and mechanism involved. It was worth mentioning that two compounds (14 and 24) were firstly found as the potential lead compounds with a good effect on the Fli-1 gene in leukaemia. The changes in corresponding protein expression levels were quantified using Image J. Each bar represents the mean ± SEM (n = 3). P < 0.05, **P < 0.01 or ***P < 0.001 was considered statistically significant compared with the corresponding control values