Protoberberine Isoquinoline Alkaloids from Arcangelisia gusanlung

HPLC-DAD-directed isolation and purification of the methanol extract of stems of Arcangelisia gusanlung H. S. Lo. led to the isolation of a new protoberberine alkaloid, gusanlung E (1), along with fourteen known derivatives 2–15, seven of which were obtained from the genus Arcangelisia for the first time. The structures and absolute stereochemistry of these compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, mass spectrometry, and CD analyses. Gusanlung E (1) expressed weak cytotoxic activity against the SGC 7901 cell line with an IC50 value of 85.1 µM.


Introduction
Arcangelisia gusanlung H. S. Lo (Menispermaceae) is a small shrub widely distributed in the south of China including the provinces of Guangdong, Guangxi, and Hainan. The stems of A. gusanlung have been clinically used in Chinese folk medicine as an anti-inflammatory, antipyretic, and detoxication reagent [1]. Previous phytochemical investigations of the plant revealed the presence of a series of protoberberine alkaloids [2][3][4] and megastigane glycosides [5] in its stems. Protoberberine alkaloids, which belongs to a isoquinoline alkaloid class, are widely distributed in many species of the Berberidaceae, Annonaceae, Fumariaceae, Papaveraceae, Ranunculaceae, Rutaceae, and other plant families, encompassing a diverse class of secondary metabolites with many pharmacologically active members, such as berberine and palmatine [6,7]. Over the last decade, these alkaloids have attracted considerable attention due to their wide range of biochemical and pharmacological actions, which have applications in various therapeutic areas such as cancer, inflammation, diabetes, depression, hypertension, and various infectious areas [8].
In order to further investigate the active components of A. gusanlung, HPLC-DAD-directed isolation was carried out on the CH 3 OH extract of the stems of A. gusanlung. As a result, 15 protoberberine alkaloids including a new one, gusanlung E (1), together with fourteen known derivatives 2-15, seven of which were obtained from the genus Arcangelisia for the first time ( Figure 1). Herein, we report the detailed isolation and structural characterization of these compounds, as well as cytotoxic activity of gusanlung E (1).

Structural Characterization
Gusanlung E (1) was obtained as yellow crystals, and its molecular formula was determined as C 19 13 C-NMR and HSQC spectroscopic data suggested the presence of 19 carbons. The 1 H-NMR spectrum showed four aromatic protons at δ 6.86, 6.71, 6.62 and 6.61, one aromatic methoxyl group at δ 3.87 (3H, s) and an N-methyl signal at δ 3.20 (3H, s). The signal at δ 3.26 (2H, m) was assigned as H-5, whereas the signals at 3.49 (1H) and 3.82 (1H) were assigned as germinal protons to H-6. Moreover, signals of a pair of methylene protons and an isolated -CH-CH 2moiety were found in the aliphatic region. The large coupling constant (15.0 Hz) of a pair of doublets at δ 4.71 and δ 4.52 suggested the existence of germinal protons, which was confirmed by HSQC. This is a typical characteristic of methylene group (C-8) of the protoberberine alkaloids [9]. The signals of an isolated -CH-CH 2 -moiety were assigned to C-13a and C-13. In addition, there were three exchangeable protons were observed at δ 9.13 in the proton NMR spectrum of DMSO-d 6 . Analysis of the 1 H-, 13 C-, and HSQC NMR spectroscopic data (Table 1) revealed that there were twelve aromatic carbon signals: four aromatic methylene (δ C :115.8, 114.5, 114.1, 113.3), eight aromatic quaternary (four oxygenated); four methylene; one methane; one aromatic methoxyl (δ C 57.6) and one N-methyl carbon (δ C 50.7). According to the above information, the structure of 1 was closely related to the 2,3,10,11-tetrasubstituted-N-methyltetrahydroprotoberberine skeleton [10,11]. The complete assignments were accomplished using 1 H-1 H COSY, HSQC, HMBC and NOESY spectra.  Interpretation of the 1 H-1 H COSY NMR data of 1 confirmed that two isolated proton spin-systems belong to C-5-C-5a and C-13-C-13a units, and the remaining connections were established by analysis of HMBC correlations. The HMBC correlations from -OCH 3 to C-1, C-3, and C-4, whereas correlations from H-1 to C-3, C-13a and C-4a, and from H-4 to C-1a, C-2 and C-5, indicated that A ring possessed 2-OH and 3-OCH 3 substitutions ( Figure 2). The result was further confirmed by NOESY spectrum, in which the NOE correlations between 3-OCH 3 and H-4, H-4 and H-5, H-1 and H-13a were observed. In the same way, the cross peaks of H-9 with C-8, C-12a, C-11 and H-12 with C-10, C-8a, C-13 in the HMBC spectrum suggested dihydroxyl substitutions at C-10 and C-11 in D ring. Moreover, H-9 was correlated with H-8 and H-12 with H-13 in the NOESY spectrum ( Figure 3). Therefore, the planar structure of 1 was characterized as 2,10,11-trihydroxy-3-methoxy-Nmethyltetrahydro-protoberberine.

Cytotoxic Activities
Gusanlung E (1) exhibited weak cytotoxic activity against cell line SGC 7901 with IC 50 value of 85.1 µM.

Plant Material
The

Cytotoxicity Testing
The cytotoxicity of the compounds was determined using the colorimetric methylthiazoletetrazolium (MTT) assay with taxol as the positive control (IC 50 value 0.15 µM). The human stomach cancer cell line SGC 7901 in logarithmic phase were seeded in 96 well flat bottom microtitre plates at a density of 1 × 10 4 cells per well. cells were washed and maintained with different concentrations of drug, 10 µL MTT was added to the culture medium to a final concentration of 0.5 mg/mL and incubated at 37 °C for 4 h. Formazan crystals dissolved in 100 µL DMSO was added and 10 min later the absorbance of the solution was measured at a wavelength of 570 nm. All assays were carried out in triplicate.

Conclusions
From the chemical investigation of stems of A. gusanlung, fifteen protoberberine alkaloids including a new one, named gusanlung E (1), were isolated and identified. Gusanlung E (1) showed weak cytotoxicity against cancer cell line SGC 7901. These analogues should be studied in more advanced models to establish in vivo efficacy.