Isolation and identification of alkaloids from Macleaya microcarpa by UHPLC–Q-TOF-MS and their cytotoxic activity in vitro, antiangiogenic activity in vivo

Background Extensive bioactivities of alkaloids from the genus Macleaya (Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde) have been widely reported, as well as more and more concerned from the scientific communities. However, systematic research on the phytochemical information of M. microcarpa is incomplete. The aim of this study was to rapidly and conveniently qualitative analyze alkaloids from M. microcarpa by ultra-performance liquid chromatography/quadrupole-time-of-fight mass spectrometry (UHPLC–Q-TOF-MS) using accurate mass weight and characteristic fragment ions, furthermore separate and identify the main alkaloids, test antitumor activity in vitro and antiangiogenic activity in vivo. Results A total of 14 alkaloids from fruits of M. microcarpa were identified by UHPLC–Q-TOF-MS, including 5 protopines, 2 benzophenanthridines, 1 dimer, 1 dihydrobenzophenanthridines and 5 unknown structure compounds. Two major alkaloids were isolated by various column chromatographic methods. Their structures were determined by NMR data and related literatures. The two major alkaloids were evaluated for intro cytotoxic activities against HL-60, MCF-7, A-549, and in vivo antiangiogenic activity using transgenic zebrafish. Conclusions Current qualitative method based on UHPLC–Q-TOF-MS technique provided a scientific basis for isolation, structural identification, and in vitro or in vivo pharmacological further study of alkaloids from M. microcarpa in the future.


Introduction
The genus Macleaya contains two species, Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde, which mainly distributed in Japan, South and Northwest China, on slopes of grass or thickets at altitudes of 450-1600 m [1]. They are well-known for their very extensive application value and great exploitation foreground as well as pesticide, veterinary drug, medicinal ones in North America, Europe, China, which are directly associated with the multifarious alkaloids and their significant biological activities. A variety of alkaloids from M. cordata, such as sanguinarine, chelerythrine, protopine, allocryptopine, and others, exhibit anti-microbial, anti-inflammatory, insecticidal, analgesic, anticancer activity, have irreplaceable pharmacological Open Access BMC Chemistry *Correspondence: ren2323@163.com 1 College of Pharmacy, Jining Medical University, Rizhao 276826, Shandong, China Full list of author information is available at the end of the article effects [2][3][4]. However, systematic researches on the phytochemical composition of M. microcarpa are few.
UHPLC-Q-TOF-MS is widely used in qualitative compositions. The method can provide high resolution and accuracy data, as well as abundant structural information such as high-resolution second-stage mass fragment ions [5,6]. This study aimed to rapidly and conveniently characterize alkaloids from M. microcarpa by UHPLC-Q-TOF-MS using accurate mass weight and characteristic fragment ions, furthermore separate and identify the main alkaloids by chromatographic and spectral techniques, test antitumor activity in vitro using the trypan blue method and MTT method reported previously [7], and antiangiogenic activity in vivo using transgenic zebrafish [8].

Plant materials
The fruits of Macleaya microcarpa (Maxim.) Fedde were collected from Xiaguan Town, Neixiang County, Nanyang, Henan Province, China, in September 2017. (Notes: It was not a protective plant and was allowed to be collected). It was identified by Prof. Jian'an Wang (College of Pharmacy, Jining Medical University, Shandong, China). The voucher sample (XGBLH-20170918) was deposited in pharmaceutical experimental center, College of Pharmacy, Jining Medical University, Rizhao, China.

Sample preparation
The air-dried and crushed fruits of M. microcarpa (15.0 kg) were extracted with 95% EtOH (18 L) under cold maceration 3 times, each time for 7 days, respectively. The combined extracts were concentrated in vacuo to yield crude ethanol extracts, which was suspended in H 2 O, successively partitioned with Petroleum ether (PE), Methylene chloride (CH 2 Cl 2 ) and N-butyl alcohol (n-BuOH), to afford PE, CH 2 Cl 2 , n-BuOH and aqueous extracts (For further separation and purification).
The crude ethanol extracts (200 mg) was dissolved with 5 mL methanol by sonication at 200 W for 15 min. The solution was filtered with a 0.22 µm and then analyzed by UHPLC-Q-TOF-MS.
The MS conditions were set as follows: drying gas at a flow rate of 10 L/min; drying gas temperature, 350 °C; pressure of nebulizer gas pressure, 45 psig; capillary voltage (±) 3000 V positive and negative ion modes and the mass range from m/z 100 to 1200 Da. The MS/MS spectra were acquired with auto MS/MS mode at the acquisition rate of 2 spectra/s. The calculation of the elemental composition was acquired with Mass Hunter Workstation Software (Qualitative Analysis Version B.06.00) (Agilent Technologies, CA, USA).

Cytotoxic activity test in vitro
The method of cytotoxic activity test in vitro had based on our previously published work [9]. HL-60 (human leukaemia cell lines), MCF-7 (human breast cancer cell lines), A-549 (human lung adenocarcinoma cell lines), which were purchased from America Type Culture Collection, ATCC (Rockville, MD, USA) and cultured in RPMI-1640 medium (Gibco, New York, NY, USA) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mM glutamine and 10% heat-inactivated fetal bovine serum (Gibco) at 37 °C in humidified atmosphere with 5% CO 2 . Compounds 2 and 7 were evaluated for cytotoxic activities by the trypan blue method against HL-60, and MTT assay against MCF-7 and A-549 [10,11].
In the trypan blue method, cells in logarithmic growth were seeded at 5 × 10 4 cells/mL in 24-well microplates, 2 mL/well, and incubated with various concentrations of the compounds at 37 °C for 72 h. 50 μL suspension was taken from each well, and 50 μL 0.4% trypan blue was added to mix well, and observed under an optical microscope within 3 min. Trypan blue-stained (nonviable) cells and the total cell number were determined with a hematocytometer. The growth inhibition in cells after treatment with different concentrations was calculated comparing with control cells (5-Fluorouracil was used as a positive control), and a half growth inhibitory concentration (IC 50 ) was obtained by regression analysis of the concentration response data.
In the MTT assay, briefly, cells suspensions, 100 μL, at a density of 2.5 × 10 4 cells/mL, were plated in 96-well The high resolution mass spectra and secondary mass spectra for 5 unknown compounds microtiter plates and incubated for 24 h at 37 °C. Then the test compounds with different concentrations in DMSO, 100 μL, were placed into each microtiter plates and further incubated for 72 h. Finally, 50 μL of a 0.4% MTT solution was added to each well and incubated for 4 h. Then, the MTT was removed from the wells and the formazan crystals were dissolved in DMSO (200 μL) for 10 min with shaking. Then the plate was read immediately on a microtiter plate reader (Bio-RAD) at a wavelength of 570 nm to record the optical density (OD). The IC 50 value was defined as the concentration of the control in the MTT assay. 5-Fluorouracil (5-Fu) was used as a positive control. All the IC 50 results were expressed as average of three independent experiments.

Antiangiogenic activity test in vivo
Transgenic Tg (flk: EGFP) zebrafish was provided by drug screening laboratory, Biology Institute of Shangdong Academy of Sciences. PTK787 was provided by biochemistry laboratory, Biology Institute of Shangdong Academy of Sciences (20110902).
Angiogenesis plays a critical role in cancer growth and metastasis. Antiangiogenesis is an excellent target in cancer treatment. In recent assays, the zebrafish model is practical and efficient in vivo model in screening natural product and drug for anti-angiogenesis. [12,13].

Zebrafish embryo collection
Male and female transgenic Tg (flk: EGFP) zebrafish were fed separately, and regularly fed with artificial pellet bait and newly hatched Artemia nauplii, stocks were maintained in a controlled environment at 28.5 °C on a 14 h: 10 h light/dark cycle. Healthy sexual mature zebrafish were put into the same mating cylinder in 1:1 ratio of male and female. Fertilized eggs were obtained at am 9-10 the following day. After disinfection and washing, the fertilized eggs were transferred to embryo water (containing 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl 2 , 0.16 mM MgSO 4 ), and cultured at 28 °C.

Zebrafish embryo antiangiogenesis assay
Healthy and limpid embryos were picked out at 24 h postfertilization (hpf ) and distributed into a 24-well microplate (6-8 embryos/well) in 1 mL Holtfreter's solution and maintained at 28 °C. The sample solution was diluted with embryo water to different concentrations of 0.5, 1, 10, 100 μg/mL, and added into the well. The final volume of each well was 2.0 mL, and the content of DMSO in each well was adjusted to be consistent. 2.0 mL 0.1 μg/mL PTK787 solution served as positive controls. The embryo water or DMSO (0.5%, V/V) served as blank controls. Embryos were maintained in incubator at 28 °C for additional 48 h, placed onto a glass slide, photographed using SZX16 fluorescence stereomicroscope and DP2-BSW image acquisition system (Olympus, Japan) after anesthesia. Zebrafish somite intersegmental vessels (ISVs) were quantified using Image Pro Plus software. Anti-angiogenic effects were defined as decrease of SIVs length [14].
Statistical analysis SPSS 13.0 was used for statistical analysis, and independent sample t test was used to compare the differences among the groups. P < 0.05 was considered as significant difference. The qualifications and experience of the researcher met the experimental requirements, after the review by the ethics committee of Jining Medical University, and the research design conformed to the principles of scientific science and medical ethics (Ethical code 2019-YX-256).

Analysis of alkaloids from fruits of M. microcarpa by UHPLC-Q-TOF-MS
In this study, UHPLC-Q-TOF-MS technology was used to qualitative analyzed and identified alkaloids from fruits of M. microcarpa. The high resolution mass spectra and secondary mass spectra are shown in Figs. 1 and  2. These compositions are summarized along with their retention time, theoretical mass, molecular formula, observed mass, error and MS/MS fragments combined with literature reports [15,16] and previously isolated alkaloids from M. cordata [7,9,17,18]. A total of 14 possible alkaloids have been identified, of which 9 are known. The detailed data are listed in Table 1 and Fig. 3. The mass spectral fragmentation behaviors of 9 known and identified alkaloids are shown in Figs. 4, 5, 6 and 7.
The mass spectral cleavage behaviors of protopine alkaloids had the following rules (Fig. 4): The parent nucleus lost one molecule of H 2 O to form a closed fourmembered ring. Protopine alkaloids did not contain large π conjugate system, and the parent nucleus was prone to happen RDA cracking and α cleavage reactions, forming small fragment molecules, which would continue to lose hydroxyl or H 2 O moiety to form fragment peaks of [M A -17] + or [M A -18] + , respectively. The mass spectral cleavage rules of benzophenanthridine alkaloids were as follows (Fig. 5): Benzophenanthridine alkaloids were large π conjugate system, and the parent nucleus was difficult to fragment. If benzophenanthridine alkaloids contain methylenedioxyl, they would lose carbon monoxide to form a stable ternary oxygen ring. If O-dimethoxy group was present in benzophenanthridine alkaloids, the O-dimethoxy group would first lose a methyl moiety and then lose hydrogen to form a methylenedioxyl, and the methylenedioxyl will continue to lose one carbon monoxide to form a stable ternary oxygen ring. If benzophenanthridine alkaloids contain 5-or 6-methoxy or methyl groups, the methoxy or methyl groups would be directly lost to form [M-31] + or [M-15] + mass spectral fragments. The cleavage of the dimer mainly occurs at the junction of two alkaloids, to form corresponding precursory alkaloids (Fig. 6).
According to this cleavage rule and the MS/MS fragment peaks, compound 12 might be a dimer formed by sanguinarine and muramine by the C-C single bond, compound 13 might be a dimer formed by chelerythrine and muramine by the C-C single bond, compound 14 might be a dimer formed by muramine and a new To search the related molecular formula from the Scifinder database, 12-14 would be novel compounds. Their exact structures would be determined by NMR after separation and purification. Dihydrobenzophenanthridine alkaloids first lost 6-or 5-substituents to form relatively stable benzophenanthridine alkaloids, and the following fragment rule was consistent with that of benzophenanthridine alkaloids (shown in Fig. 7). The cleavage rules were helpful to identify the characteristic alkaloids in M. microcarpa by LC-MS method.

NMR data of compounds 2 and 7
Two major alkaloids, protopine and chelerythrine, were isolated and prepared from M. microcarpa for later activity testing.

Cytotoxic activity in vitro
Compounds 2 and 7 exhibited potent cancer cell growth inhibitory activities against HL-60, A-549, and MCF-7 cancer cell lines. The results are summarized in Table 2.

Antiangiogenic activity in vivo
In order to confirm that protopine and chelerythrine were associated with inhibition of tumor angiogenesis, we used a transgenic zebrafish model to evaluate the antiangiogenesis effects. Transgenic zebrafish embryos at 24 hpf treated with protopine and chelerythrine for 48 h showed a dose-dependent inhibition of ISV formation. The inhibitory effect of different doses of sample on intersegmental vessel (ISV) formation in zebrafish embryos were shown in Fig. 8 and Table 3. But there were no significant difference in the total length and morphology of ISVs between the treatment group and the control group. All the young fish in each 100 μg/mL sample group died. From these results, we could speculate that the antitumor effects of the samples were not achieved by inhibiting the vascular growth mechanism.

Conclusions
In this paper, we rapidly and conveniently qualitative analyze alkaloids from M. microcarpa by UHPLC-Q-TOF-MS using accurate mass weight and characteristic fragment ions, and combining with their cleavage rules. Some unknown compounds were discovered through this simple and sensitive method. The study on the cleavage rules of these alkaloids is helpful to identify the characteristic alkaloids by LC-MS method, and identify the structural types of alkaloids in M. microcarpa. The two major alkaloids of M. microcarpa, protopine and chelerythrine, exhibited potent cancer cell growth inhibitory activities in vitro, but they showed almost no antiangiogenic activity in transgenic zebrafish vivo model. However, the structure and bioactivity screening of new natural products from M. microcarpa still need further study, which is an integral part of drug discovery progress. QR guided the qualitative analysis alkaloids by UHPLC-Q-TOF-MS, and took charge in manuscript revision. HMH, FPZ and QR guided and designed experiments. All authors read and approved the final manuscript.

Funding
The collection, extraction, and separation of fruits of M. microcarpa were funded by the National Natural Science Foundation of China (Grant No. 31800282). The NMR data testing was supported by Postdoctoral Science