Isolation of a new β-carboline alkaloid from aerial parts of Triclisia sacleuxii and its antibacterial and cytotoxicity effects

Abstract A new β-carboline alkaloid named sacleuximine A (1) together with known compounds palmatine (2), isotetrandrine (3), trans-N-feruloyltyramine (4), trans-N-caffeoyltyramine (5), yangambin (6), syringaresinol (7), sesamin (8), (+) epi-quercitol (9), 4-hydroxybenzaldehyde (10), β-sitosterol (11), quercetin 3-O-rutinoside (12) and myricetin 3-O-β-glucose (1→6) α-rhamnoside (13) have been isolated from methanol extract of Triclisia sacleuxii aerial parts. Compounds 1–10 were evaluated for their cytotoxicity against human adenocarcinoma (HeLa), human hepatocarcinoma (Hep3B) and human breast carcinoma (MCF-7) cells lines and also for antibacterial activities against both Gram-positive and Gram-negative bacteria. The cytotoxicity (IC50) values ranged between 0.15 and 36.7 μM while the minimum inhibitory concentrations were found to be in the range of 3.9 and 125 μM, respectively. This is the first report of antibacterial compounds and the isolation of lignans together with a β-carboline alkaloid from T. sacleuxii.


Introduction
The species Triclisia sacleuxii (Pierre) Diels of the Menispermaceae family is a tendril plant that grows in the lowland and riverside forests of many African countries (Jacques & De Franceschi 2007). In traditional medicine, the plant root is chewed and the sap swallowed to manage kidney problems, sore throat, schistosomiasis, intestinal worms, venereal diseases and root scrapings applied in topical scarifications to treat snakebites (Kokwaro 2009). Previous phytochemical studies reported the isolation of a series of tertiary bisbenzylisoquinoline alkaloids (Murebwayire et al. 2006), which displayed good antiplasmodial and antityrpanosomal activities in vitro against Plasmodium falciparum (Pollard 2008), Leshmania donovani promastigotes, Trypanosoma cruzei and Trypanosoma brucei (Murebwayire et al. 2008). Further biological activity studies using the same alkaloid fractions and isolates showed strong inhibition against acetylcholinesterase with lindoldhamine being reported as the most active compound, thus suggesting its potential in managing Alzheimer's disease (Murebwayire et al. 2009).

Results and discussion
Compound 1 was isolated as white amorphous powder with a molecular formula of C 42 H 64 N 2 O 3 as determined by HR-eSI-MS [M + Na] + at m/z = 667.4917 (calcd. 667.4920). The UV spectrum absorption peaks at 242 (4.12), 260 (3.32) and 280 (3.60) nm suggested the presence of hydroxy indole and hydroxy benzyl chromophores (Wu et al. 1989). Its IR spectrum showed significant absorption bands at 3414 (NH), 3316 (OH) and 1653 (C=O) cm −1 . The 1 H NMR spectrum data (Table S1) revealed two sets of aromatic signals, a 3H AMX system {δ 7.00 (d, J = 8.6 Hz), 6.67 (dd, J = 8.6, 2.4 Hz) and 6.86 (d, J = 2.4 Hz)} and a 4H AA'XX' system {δ 7.15 (d, J = 8.4 Hz) and 6.82 (J = 8.4 Hz)} suggesting the presence of two aromatic rings (Manguro et al. 2003). In addition, the presence of a relatively upfield methylene at δ 2.35 (t, J = 7.5 Hz), a methylene multiplet at δ 1.25 and a terminal methyl group 0.88 (t, J = 6.7 Hz) together with a NH signal at δ 6.90 (s) suggested the presence of a long aliphatic chain having a CH 2 -C = O connected to the carboline nucleus (Koskinen et al. 2014;Zhang et al. 2015). Furthermore, the presence of mutually coupled methylene protons δ 4.07 (d, J = 13.7, 4.1 Hz) and 3.38 (dd, J = 13.0, 4.6 Hz) signified a tetrahydro-β-carboline skeleton (Koskinen et al. 2014;Ngoc et al. 2016;Yang et al. 2016). The 13 C NMR spectrum (Table S1) displayed 14 aromatic carbon signals and an amide carbonyl carbon (δ 177.1) as evidenced by 135 DePT NMR spectrum. The methylene peaks at δ 3.14 (H-1'a) and 2.93 (H-1'b) exhibited 1 H-1 H COSY correlation with a pair of the aromatic protons at δ 7.15 (H-3' and H-7') and in turn with H-1 indicating the presence of a benzyl moiety connected to a methine carbon. Analyses of the 1 H-1 H COSY, HSQC and HMBC spectra ( Figure S9) established the presence of molecular features >CH-CH=CH-CH 2 -N< and >N-CO-(CH 2 ) 22 CH 3 , which on comparison with spectral data of shepherdine and oldhamiaines (Salmoun et al. 2002;Zhang et al. 2015) and further supported by HMBC correlations between H-1/C-2' , and H-3'/C-4' , deduced the presence of 4-hydroxybenzyl group bonded to a methine carbon attached to an SP 3 hybridised nitrogen atom and an SP 2 hybridised carbon (Camacho et al. 2002;Jain 2016). The NOSY correlation between the aromatic proton at δ 7.00 (H-8) and the indolic NH proton at δ 6.90 coupled with the HMBC cross peaks observed between H-1 and C-2'and in turn with C-4a allowed the allocation of the benzyl moiety to C-1. Similarly, the HMBC correlation between H-1 and the carbonyl carbon (C-1''') confirmed the attachment of the aliphatic chain on the second nitrogen. The aliphatic chain was deduced to be a tetracosan-1-one based on the eSIMS fragmentation (  (Salmoun et al. 2002;Zhang et al. 2015) and the NOeSY correlation between the benzylic methylene protons and the indolic proton NH suggested the configuration at C-1 as a β for H-1. This was further supported by the spin decoupling experiments which showed that the 4-hydroxylbenzyl moiety was linked in a =C-CH eq (C 6 H 4 OH ax )-N< requiring it to be at C-1 as α. Thus, on the basis of spectroscopic data and comparison with literature data, compound 1 was deduced to be 1-(6-hydroxy-1-(4-hydroxybenzyl)-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)tetracosan-1one, a new alkaloid named sacleuximine A. The cytotoxicity of the isolated compounds was evaluated by MTT method (Alley et al. 1988). The alkaloids (1-3) and the lignans (6-8) showed strong cytotoxicity effects (Fig. S11) against the three cancer cell lines while trans-N-feruloyltyramine (4) and trans-N-caffeoyltyramine (5) showed better activities. The latter compound was more active than compound 4 which may be attributed to the presence of ortho dihydroxyl groups. The activities of the alkaloids were in agreement with the previously reported cytotoxicity of β-carboline alkaloids against several human cancer cell lines with IC 50 values ranging from 0.3 to 1.2 μg/mL (Cao et al. 2007). Compounds (1-10) were also evaluated for their antibacterial activities against Gram-positive and Gram-negative bacteria strains (Table S2). Compound 1 exhibited moderate to low antibacterial activities while compound 3 showed significant (MIC 7.8 μM) inhibition against Gram-positive strains compared to compound 2 and the two ferrulamides (4 and 5). Lignans showed better antibacterial activity compared to the other compounds against the tested strains with 7 displaying higher activity (minimum inhibitory concentration [MIC] 3.9 μM) against the Gram-positive bacteria (Staphylococcus epidermis and Staphylococcus aureus).

General experimental procedure
Column chromatography and thin-layer chromatography were performed using silica gel 0.035-0.070 mm, 60A and 60F 254 , respectively. Optical rotation was measured on a Jasco P-1020 Polarimeter (Jasco Corporation, Tokyo, Japan). Melting points were determined on a Gallenkamp apparatus (Manchester, UK). UV spectra were done on a Shimadzu UV-2401 A spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR data were recorded on a Bruker Tensor 27 FTIR spectrophotometer (Bruker Corporation, Bremen, Germany) as KBr pellet. The 1 H, 13 C and 2D-NMR spectra were recorded on Bruker AVANCe III-600 MHz spectrometer (Bruker Corporation, Zurich, Switzerland) equipped with a 5-mm TCI cryogenic probe head (z-gradient) using standard pulse sequences. Semi-preparative RP-HPLC was performed on a Hitachi Chromaster system (Hitachi Corporation, Japan) equipped with an YMC-Triart C18 column (250 × 10 mm i.d., 5 μm, YMC Corporation, Tokyo, Japan) using a flow rate of 0.5 mL/min, and eluent of 1:4-mixture of H 2 O + 0.1% HCO 2 H and H 3 CCN + 0.1% HCO 2 H. LR-MS was performed using an Agilent Technology 1200 series (Agilent Corporation, Bobingen, Germany) apparatus. LR-MS and HR-eSI-MS data were recorded on a Q-TOF ULTIMA-III (Waters Corporation, Manchester, UK) equipped with a LockSpray Interface.

Plant material
The aerial parts of T. sacleuxii were collected in August 2012 from Cha-Simba Rocks, Kilifi County, Kenya. The plant material was identified by Mr. Mutiso of Botany Department, Nairobi University, and a voucher specimen (eAH/2012/608) was deposited at the east African herbarium, Nairobi, Kenya.

Extraction and isolation of compounds
The air-dried ground aerial parts of T. sacleuxii (2 kg) were extracted three times with 95% aqueous methanol (each 5 L), each extraction lasting three days with occasional swirling. The extracts were combined and solvent removed under vacuo leaving behind a green residue, 60 g. The extract (56 g) was suspended in water (0.3 L), then partitioned successively into dichloromethane (1 L × 4) and n-butanol (0.5 L × 4), each yielding 26.0 and 19.0 g of extracts, respectively. The remaining H 2 O portion was freeze-dried affording ≈ 6 g of dark green extract.

Cell culture and cytotoxicity tests
Human cervical adenocarcinoma (HeLa), human hepatocarcinoma (Hep3B) and human breast carcinoma (MCF-7) cells were obtained from American Type Culture Collection (Manassas, VA). HeLa and Hep3B cells were cultured in Dulbecco's modified essential medium, and MCF-7 cells were maintained in a RPMI-1640 medium in a humidity of 5% CO 2 atmosphere at 37 °C. All media were supplemented with penicillin (100 ug/mL), streptomycin (100 μg/mL) and 10% heat-inactivated foetal bovine serum (FBS). Cytotoxicity assays were carried out according to Alley et al. (1988) method. The cancer cells seeded in 96-well plates at concentration of 1 × 10 4 cells/well were treated with various concentrations of test compounds and incubated in a humidity of 5% CO 2 atmosphere at 37 °C. After 72-h incubation, 10 μL of 5 mg/mL MTT was added to each well and incubated for another 4 h. After removal of the supernatant, formazan crystals were dissolved in 100 μL DMSO and the absorbance measured at 570 nm using a microplate reader. Doxorubicin was used as a positive control and the IC 50 value cancer cells against the 10 compounds (1-10) were obtained using the Log probit analysis.

In vitro antibacterial activity
The compounds were evaluated for their in vitro antibacterial activities against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), S. epidermis ATCC 25924) and S. aureus (ATCC 25923) according to the procedure of National Committee for Clinical Laboratory Standards (NCCLS) microdilution assay format (Murray et al. 1999). The test compounds were dissolved in 5% DMSO at an initial concentration of 1 mg/mL and serially diluted in plate to provide decreasing concentrations. Maximum tested concentration was 500 μg/mL while the minimum was 3.9 μg/mL. each well was then inoculated with 2-5 × 10 5 bacterial cells and incubated at 37 °C for 24 h. One well containing the micro-organisms and 5% DMSO without test compounds was for control while the other containing only growth medium was used for sterility control. Tetracycline was used as a positive control. The MIC was evaluated as the lowest concentration of the test substances that inhibited the growth of the bacteria strains.

Conclusion
The results of the study showed that compound 1 exhibited both cytotoxicity and antibacterial activities.