Mass spectrometry-guided isolation of two new benzoquinoline alkaloids from Macleaya cordata

Abstract Two new alkaloids, named 2,3-methylenedioxy-7,10-dimethyl-7,8,9,10-tetrahydro-benzoquinoline (1) and 2,3-methylenedioxy-7,10-dimethyl-8-carboxyl-benzoquinoline (2), were detected primarily from the fruits of Macleaya cordata by their different fragmentation pathways. And then isolation of the two compounds was performed by column chromatography and preparative HPLC under the guiding of mass spectrometry. Finally, their structures were determined by spectroscopic analysis. Graphical abstract


Introduction
Macleaya cordata (Wild.) R. Br. belongs to genus Macleaya of the Papaveraceae family and has been used for over a thousand year as a traditional folk medicine (Zeng et al. 2013). Isoquinoline alkaloids, including benzophenanthridine, dihydro-benzophenanthridine, protopine and protoberberine, are its major biological activities components (Kosina et al. 2010;Feng et al. 2012). These alkaloids have several biological activities including antibacterial , anti-inflammatory, antimicrobial (Li et al. 2015), antitumour, anti-HIV ABSTRACT Two new alkaloids, named 2,3-methylenedioxy-7,10-dimethyl-7,8,9,10-tetrahydro-benzoquinoline (1) and 2,3-methylenedioxy-7,10dimethyl-8-carboxyl-benzoquinoline (2), were detected primarily from the fruits of Macleaya cordata by their different fragmentation pathways. And then isolation of the two compounds was performed by column chromatography and preparative HPLC under the guiding of mass spectrometry. Finally, their structures were determined by spectroscopic analysis. and animal growth promotion (Qing et al. 2014). A set of products (e.g. BoLuoHui ZhuSheYe ® , BoLuoHui ZhongYangDing ® ) has been developed successfully in China due to those bioactive alkaloids. Meanwhile, sanguinarine and chelerythrine, two main alkaloids isolated from this plant medicine, have been used for promoting animal growth as a popular natural feed additive (e.g. Sangrovit ® ) (stiborova et al. 2008;Zdarilova et al. 2008). In addition to these well-known chemical components, a series of alkaloids, which may have potential biological activities, is still unknown and requires further isolated and identified.
To the best of our knowledge, traditional phytochemistry isolation provides a comprehensive chemical foundation in the discovery of new drug candidates from plant medicines. However, this method is time consuming and low efficiency. Therefore, bioactivity-guided isolation is applied for rapid excavating the chemical components which respond to the relevant biological activities (Kang et al. 2013). In this study, a liquid chromatography-mass spectrometry (LC-Ms)-guided isolation technology is also applied for rapid uncovering the alkaloids which have not been reported from M. cordata. Finally, two new benzoquinoline alkaloids, named 2,3-methylenedioxy-7,10-dimethyl-7,8,9,10-tetrahydro-benzoquinoline (1) and 2,3-methylenedioxy-7,10-dimethyl-8-carboxyl-benzoquinoline (2) (Figure 1), were rapidly isolated under the guiding of mass spectrometry, and then identified on the basis of numerous spectroscopic data.
The 1 H, 13 C, dePT and HMQC NMR spectra (see Figure s2) of alkaloid 2 demonstrated signals of a methylenedioxy group at δ H 6.30 (2H, s)/δ C 104.8, a N-methyl group at δ H 4.84 (3H, s)/ δ C 52.6, a methyl group at δ H 3.16 (3H, s)/δ C 18.0. The neutral loss of a Co 2 molecular and formed the fragment ion at m/z 252.1008 (Δppm-0.79) was appeared in the tandem mass spectrometry (Ms/Ms) of 2 (see Figure s3), which indicated the signal of a carboxy group. Meanwhile, the -CooH group was further confirmed by the 13 C NMR data (δ C 169.6). In the HMBC spectra (see Figure s2), -oCH 2 o-(δ H 6.30) group showed clear correlations with C-2 (δ C 152.9) and C-3(δ C 150.8), indicating the direct linkage of the methylenedioxy with C-2, C-3. due to correlations of -CH 3 to C-7, C-8, C-6a and N-CH 3 to C-9, C-10a in HMBC signals, the methyl and N-methyl group can be assigned at C-7 and position-10, respectively ( Figure 1). The chemical shift of C-9 (δ C 149.3) and H-9 (δ H 9.22) were moved to low field compared with other aromatic tertiary carbons and protons, which indicated C-9 and H-9 were influenced by electron withdrawing group. Meanwhile, in the HMBC spectra, the -CooH (δ C 169.6) group showed clear correlation with H-9 (δ H 9.22). Therefore, the carboxy group can be assigned at C-8. other significant HMBC correlations for the structural elucidation of compound 2 are given in Figure s2. Finally, the structure of compound 2 was determined and identified as 2, 3-methylenedioxy-7,10-dimethyl-8-carboxyl-benzoquinoline.

General experimental procedure
ultraviolet (uV) spectra were taken on a Hitachi uV-3310 spectrophotometer. IR spectra were obtained with a Nicolet Impact 410 infrared spectrometer with KBr disc technique. 1d and 2d NMR spectra were acquired using a Bruker ACF-400 spectrometer (the 1 H NMR spectra at 400 MHz and 13 C NMR spectra at 100 MHz). The mass spectra were determined by an Agilent 1290 HPLC system coupled with a 6530 Q-ToF/Ms accurate-mass spectrometry. Agilent preparative HPLC system including a binary pump, an injector and a uV detector was used for isolation the target compounds. The reversed-phase Xcharge C18 (10 μm, 20 × 250 mm i.d., da Lian, RP China) was employed as chromatographic column. silica gel (200-300 mesh) used for column chromatography (CC) and silica GF 254 for TLC was supplied by Qingdao Marine Chemical Factory, Qingdao, RP China.

Plant material
The M. cordata fruits were collected from Jinzhai country (Anhui province, RP China) in August, 2014, and were authenticated by Prof. Jian-Guo Zeng (Hunan university of Chinese Medicine). A voucher specimen (AHJZ20140824) was deposited in the herbarium of the school of Pharmacy, Hunan university of Chinese Medicine, Hunan, RP China.

Detection, extraction and isolation
The structures of different types of alkaloid in M. cordata fruits have been speculated and elucidated in detail by their mass spectrometry (Qing et al. 2014;. However, two compounds were different from the well-known alkaloids according to their fragmentation pathways (see Figure s3). Therefore, the two compounds were regarded as new type of alkaloid in M. cordata. In order to determine the structure and investigation of the potential biological activities, the two alkaloids were isolated by the preparative HPLC system under the guiding of mass spectrometry.
The air-dried M. cordata fruits (50 kg) were extracted three times (3 × 1 h) under conditions of reflux with 95% ethanol, and the ratio of solid and liquid is 1:20. After evaporation of the ethanol in vacuum, a viscous residue was obtained which was suspended in 450 L (3 × 150 L) hydrochloric acid solution (PH3), and the acid water layer was obtained after standing for 24 h. Then, NH 3 . H 2 o was added for adjusting the PH to 10 and the alkaline water layer and undissolved residue (650 g) were isolated after standing for 24 h.

Supplementary material
supplementary material relating to this article is available online, alongside Figures s1-s4.

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

Funding
This work was supported by The National Key Technology R&d Program [2011BAd34B02].