Studies on the Alkaloids of the Bark of Magnolia officinalis: Isolation and On-line Analysis by HPLC-ESI-MSn

The bark of Magnolia officinalis is a well-known Traditional Chinese Medicine. In the present study, two new alkaloids, named (S)-4-keto-magnoflorine (6) and (R)-3,4-dehydromagnocurarine (11), together with seven known alkaloids: (S)-magnoflorine (5), trans/cis N-feruloylputrescine (7/8), (R)-magnocurarine (10), (S)-tembetarine (12), (R)-oblongine (14), and (R)-asimilobine (17) were isolated and their structures elucidated by spectroscopic methods, including 1D, 2D NMR, and HRESI-MS. The absolute configurations of the isoquinoline alkaloids 5, 6, 10–12, 14, and 17 were determined by CD. In vitro inhibitory activities against aldose reductase, lipase, α-glucosidase, DPP–IV and three cancer cell lines (A549, Bel-7402, and HCT-8) were evaluated for all isolated compounds. However, all compounds showed weak activities in all tests at the same concentration as the positive control drugs. An HPLC-ESI-MSn method has been established for screening of alkaloids in the bark of M. officinalis. A total of 23 alkaloids were identified or tentatively characterized; including 13 aporphines, eight benzylisoquinolines and two amides. Plausible fragmentation pathways of the representative compounds 6, 7/8, 11, and 17 were proposed in the present study.


Introduction
The bark of Magnolia officinalis (Magnoliaceae), called Houpu in Chinese, has a very wide range of applications. It's a common ingredient in the treatment of abdominal swelling of various causes and edema. Many of the prescriptions containing Houpu are also aimed at treatment of lung disorders (including cough and asthma) or intestinal disorders [1,2]. Besides the well-known lignans magnolol and honokiol being the active compounds of M. officinalis, alkaloids are a group of interesting secondary metabolites of this species, which produces mainly isoquinoline-type alkaloids, the majority of which are aporphine and benzylisoquinoline derivatives [3][4][5][6]. In a previous paper from our group, 11 polar compounds including one major alkaloid-magnoflorine-have been isolated from the stem bark of M. officinalis [7]. In connection with our interest in the alkaloids of this plant, a further chemical study focusing on an alkaloids-enriched fraction has been conducted. The strategy of this study includes two steps: (1) isolation of main components and complete structural characterization by NMR, MS, and CD spectroscopies. (2) on-line identification of alkaloids by comparing retention time and MS n data. Twenty three alkaloids were identified or tentatively characterized, including 13 aporphines, eight benzylisoquinolines and two amides. Two new alkaloids, named (S)-4-ketomagnoflorine (6) and (R)-3,4-dehydromagnocurarine (11), together with seven known alkaloids were isolated ( Figure 1).   H-NMR spectrum showed two adjacent aromatic protons at δ H 6.55 (1H, d, J = 8.0 Hz) and 6.40 (1H, d, J = 8.0 Hz), and one isolated aromatic proton at δ H 6.87 (1H, s), which were ascribed to the hydrogens of A and D ring of the aporphine nucleus. Singlets at δ H 3.75 (3H, s) and 3.70 (3H, s) attributed to methoxy groups. The signals at δ H 3.31 (3H, s) and 2.82 (3H, s) indicated the presence of two N,N-dimethyl groups in the molecule.
The 13 C-NMR data together with a HSQC experiment indicated the presence of 20 carbons in 6, comprising one ketone carbon at δ C 187.4, 12 aromatic carbons between δ C 165.2 and 107.2, four methyl groups at δ C 58.1, 58.0, 56.2, and 48.0, two methylenes at δ C 69.9 and 31.5, and one methine at δ C 71.4. The NMR data were similar to those of magnoflorine [8], except for the ketone carbon signal and the low field hydrogen signal at δ H 3.91 (2H, s). The long-range 1 H-13 C correlation observed between H-3 (δ H 6.87, s) and δ C 187.4 confirmed the substitution at C-4 ( Figure 2). The NOE correlations between H-3 and δ H 3.70 (3H, s), between H-9 and δ H 3.75 (3H, s) confirmed the location of methoxys. The absolute configuration of C-6a was established as S by the circular dichroism (CD) spectrum, which showed a positive Cotton effect at 235 nm [9]. Thus, the structure of 6 was elucidated as (S)-4-keto-magnoflorine.
Compound 11 was obtained as a white amorphous powder. Its molecular formula was determined as C 19  . On the basis of the presence of AA'BB' and AB 2 systems, the basic structure of the molecule was suggested as that of a benzylisoquinoline. In HMBC spectrum, δ H 3.52 (3H, s) and 3.22 (3H, s) were correlated with each other, which indicated the both methyl groups were connected to the same hetero atom. In addition, these two methyl groups were also correlated with δ C 76.1 and 131.0 which unveiled the presence of a quaternary amine in its structure ( Figure 2). Compared with magnocurarine, the NMR spectral data of 11 showed some differences, including the absence of two aliphatic carbon signals and the presence of two more olefinic carbons at δ C 131.0 and 127.6, all of which implied that 11 possessed one more olefinic bond. The location of the olefinic bond at C-3 and C-4 was inferred by the HMBC correlations of H-5/C-4, N-methyl/C-3, and H-1/C-3. Moreover, the NOE correlation between H-5 and δ H 3.87 (3H, s) confirmed the location of methoxy. The absolute configuration of C-1 was established as R by CD spectrum, which showed a negative Cotton effect at 230 nm [10]. Thus, the structure of 11 was elucidated as (R)-3,4-dehydromagnocurarine.
Positive ion mode of ESI was selected in the present study. In general, quaternary alkaloids gave [M] + ions, tertiary and secondary alkaloids gave [M+H] + ions (Table 1). Isolated standards 5 and 17 generated significant [M-(CH 3 ) 2 NH)] + or [M+H-NH 3 ] + fragment ions as the base peak in their respective MS 2 spectra, corresponding to quaternary and secondary aporphines, respectively [14]. Based on the mentioned characteristics, compounds 1, 2, 4, and 9 should be quaternary aporphines due to the significant [M-(CH 3 ) 2 NH] + fragment ions in their MS 2 spectra. Similarly, compounds 19, 20, and 22 should be secondary aporphines due to the significant [M+H-NH 3 ] + fragment ions in their MS 2 spectra. The MS 2 fragmentation of compounds 18, 21, and 23 provided significant [M+H-CH 3 NH 2 ] + ions in the product-ion spectra, suggesting one methyl group at the nitrogen atom of these compounds. The benzylisoquinoline alkaloids 10 and 12-16 showed obvious cleavages between C-1 and C-1a to produce the significant ion fragments at m/z 107 or 137, which were ascribed to hydroxybenzyl and 3-methoxy-4-hydroxybenzyl fragments, respectively [15]. However, isolated benzylisoquinoline alkaloid 11 gave an ion at m/z 205 as the base peak in the MS 2 spectrum, undergoing the loss of a hydroxybenzyl fragment. Similarly, an ion at m/z 221 in MS 2 spectra of compound 3 indicated the substituent type of ring C. In the MS 2 spectra, compounds 3, 10 and 12-14 showed a characteristic [M-(CH 3 ) 2 NH] + loss, whereas compounds 15 and 16 showed a characteristic [M−CH 3 NH 2 ] + loss indicating the number of methyl groups at the nitrogen atom. The ion peaks derived from loss of CH 3 OH, CH 3 O, H 2 O, CH 3 , CO and CH 2 O were characteristic of aporphine and benzylisoquinoline alkaloids, and served for their structural elucidation [15]. A plausible fragmentation pathway of the representative compounds 11 and 17 is shown in Scheme 1.

General
Optical rotations were measured on a P2000 automatic digital polarimeter. UV spectra were obtained in MeOH on a Jasco V-650 spectrophotometer. IR spectra were recorded in KBr pellets on a Thermo Nicolet 5700 infrared spectrometer. CD spectra were measured on a Jasco J-815 spectropolarimeter. HRESI-MS were obtained on an Agilent 6520 Accurate-Mass Q-TOF LC-MS. NMR spectra were taken on Varian Mercury-400 spectrometer using DSS as references. Strong cation ion exchange resin 001 × 7 was a product of Tianjin Nankai Hecheng S & T Co., Ltd. (Tianjin, China). MCI CHP-20P (Toshiba, 75-150 μm) and RP-C18 (YMC, 40-60 μm) were used for column chromatographic separation. MPLC was performed on an EZ Purifier II flash chromatography system (Shanghai Li Sui E-Tech Co. Ltd. Shanghai, China). Analytical HPLC was conducted on a Waters 2695 pumping system equipped with a Waters 2996 photodiode array detector. The preparative HPLC was performed using a Waters 600 pump, a Waters 2487 detector, and an ODS column (250 mm × 20 mm, 5 μm; YMC). HPLC-MS n analysis was performed with an Agilent G2451AA 6320 Ion Trap LC/MS system. HPLC grade MeCN from Fisher (Fair lawn, NJ, USA) was used for LC-MS/MS analysis. Strata X-C strong cation resin (500 mg/6 mL, Phenomenex, Torrance, CA, USA) was used for SPE. Water was purified with a Milli-Q water purification system (Millipore, Bedford, MA, USA). Ammonium acetate and NH 4 OH were analytical grade.

Extraction and Isolation
The dried and powdered stem bark of M. officinalis (25 kg) was extracted with 70% ethanol (v/w = 7) under reflux for three times. The aqueous alcohol solutions were combined and concentrated in vacuo to afford a suspension (5 L). The solution obtained was adjusted to pH 1 using HCl and then partitioned three times with 2.5 L of chloroform. After centrifugation, the supernatant of the H 2 O layer was applied to an ion exchange column (001 × 7; H + form). The column was washed with 50% EtOH (10 L) and then the eluate of 2M NH 4 OH (50% EtOH) was collected. The solvent was evaporated under vacuum to yield the alkaloid fraction (73.6 g). The alkaloid fraction was initially chromatographed on a MCI column, eluted with a gradient of EtOH-H 2 O (0:100 to 95:5), to obtain nine fractions (Fr.1-Fr.9). Fr.2 (15 g, 5% EtOH eluent) was subjected to MPLC over an ODS column, eluted with a MeOH-H 2 O gradient (10:90 to 50:50) to give 10 (3.9 g) and a mixture of 7 and 8 (5 mg). Fr.3 (11 g, 10% EtOH elute) chromatographed on MPLC over an ODS column, eluted with a MeOH-H 2 O gradient (10:90 to 50:50) to give compound 5 (5.0 g) and a mixture (100 mg). This mixture was further separated by preparative HPLC (MeCN:0.1% TFA = 12:88, 10 mL/min) to give compounds 11 (14 mg) and 12 (8 mg

Sample Preparation
Compounds 5-8, 10-12, 14, and 17 were isolated by the authors as described above. Their purities were above 95%, as determined by HPLC analysis.