Monoterpenoid Indole Alkaloids from Alstonia rupestris with Cytotoxic, Anti-Inflammatory and Antifungal Activities

Phytochemical investigation of the 70% EtOH extract of the leaves of Alstonia scholaris afforded seven new monoterpenoid indole alkaloids: scholarisins I-VII (1-7), and three known compounds: (3R,5S,7R,15R,16R,19E)-scholarisine F (8), 3-epi-dihydro- corymine (9), and (E)-16-formyl-5α-methoxystrictamine (10). Structural elucidation of all the compounds was accomplished by spectral methods such as 1D- and 2D-NMR, IR, UV, and HRESIMS. The isolated compounds were tested in vitro for cytotoxicity against seven tumor cell lines, anti-inflammatory activities against Cox-1 and Cox-2, and antifungal potential against five species of fungi. Compounds 1, 6, and 10 exhibited significant cytotoxicities against all the tested tumor cell lines with IC50 values of less than 30 μM and selective inhibition of Cox-2 comparable with the standard drug NS-398 (>90%). Additionally, 1, 2, 3 and 8 showed antifungal activity against two fungal strains (G. pulicaris and C. nicotianae).


Introduction
The genus Alstonia, which belongs to the family Apocynaceae, is widely distributed throughout the tropical areas of the World, including Central America, Africa, Indo-Malaya, Australia and Asia [1][2][3].
The genus Alstonia comprises about 60 species, eight of which grow in China [4]. Several of these species are used in Traditional Chinese Medicine, for example in the treatment of malaria, dysentery, defervescence, antitussive, and to arrest hemorrhages [5][6][7][8][9][10]. Monoterpenoid indole alkaloids occur abundantly in the family Apocynaceae [11][12][13][14][15][16][17], and to date, more than 300 such monoterpenoid indole alkaloids have been reported from the plants of this genus [18][19][20][21][22]. This type of alkaloids originates from the condensation of tryptophan with secologanin to give strictosidine and then further elaboration gives an impressive array of structural variants [23]. Monoterpenoid indole alkaloids were reported to have anticancer, antibacterial, antifertility, and anti-tussive activities [24][25][26][27][28]. Alstonia rupestris Kerr. is usually endemic in the western part of Guangxi Province of China. To the best of our knowledge, the phytochemistry of the A. rupestris has been rarely reported previously, which prompted the present study. Present investigation on chemical constituents of the EtOH extract of the leaves of A. rupestris led to seven new monoterpenoid indole alkaloids: scholarisin I-VII (1-7) together with three known compounds: (3R,5S,7R,15R,16R,19E)-scholarisine F (8), 3-epi-dihydrocorymine (9), and (E)-16formyl-5α-methoxystrictamine (10) (Figure 1). The structures of these compounds were elucidated mainly by NMR spectroscopic and mass spectroscopic methods. Furthermore, all the alkaloids were in vitro evaluated for their cytotoxic, anti-inflammatory and antifungal activities.   3. On the basis of the observation of NOESY data similar to those of 1, the stereochemistry of 2 was expected to be the same. Accordingly, the structure of 2 was established as scholarisin II and the structure was showed in Figure 1.  with the acyl carbon (δ 171.3) of the acetyl group. The stereochemistry of 3 was expected to be the same as 2 on the basis of the NOESY data. Thus, compound 3 was elucidated as scholarisin III and the structure was as shown in Figure 1.
Compound 4, a white amorphous powder, gave one quasimolecular ion peak at m/z 409.1735 [M+Na] + in its HRESIMS, accounting for a molecular formula of C 21 H 26 N 2 O 5 . The IR spectrum showed absorption peaks at 3430 (NH) and 1740 (C=O) cm −1 . In the 1 H-NMR spectrum, four aromatic proton signals at δ H 7.12 and 6.56 (each, 1H, dd, J = 8.2, 1.8 Hz), 6.78 and 7.06 (each, 1H, dt, J = 8.2, 1.8 Hz)] showed an ortho-disubstituted benzene ring, two singlet peaks at δ H 3.10 and 3.70 were assigned to the protons of a methoxy and a carbomethoxy group, respectively. Its 13 C-NMR displayed a pattern similar to that of scholarisine C [28], except that the double bond of 19/20 was substituted by a methine at δ C 58.4 (d, C-19) and a quaternary carbon at δ C 61.5 (s, C-20). The molecular formula C 21 H 26 N 2 O 5 displayed 10 unsaturation degrees, which indicated the presence of 19,20-epoxide combined with the appropriate NMR data. The NOE correlation of H-3/H-14α indicated the α configuration of C-3 ( Figure  3). On the basis of the NOE correlations of H-15/H-19 and H-18/21, the configurations of C-19 and C-20 was elucidated as S and R, respectively. The R configuration of C-5 was determined by the coupling constant of H-5 (d, J = 5.2 Hz) compared with that of scholarisine C (d, J = 5.4 Hz) [28]. Therefore, compound 4 was determined as scholarisin IV, with the structure as shown in Figure 1.
Compound 5, a white amorphous powder, exhibited a molecular formula of C 21 H 26 N 2 O 5 , based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 387.1923 [M+H] + (calcd. 387.1920). The general features of NMR spectra closely resembled those of 4, except for the configuration of C-5. H-5 was observed as a doublet of doublets at δ H 4.90 (1H, dd, J = 7.2, 5.6 Hz) in the 1 H-NMR spectrum, which indicated the S configuration of C-5 [28]. This evidence indicated that compound 5 was an isomer of 4, and 5 was identified as scholarisin V, with the structure shown in Figure 1. . These data showed similarities to those of 3-epi-dihydrocorymine (9) [29]. Comparing the 1 H-and 13 C-NMR data of 6 with those of 3-epi-dihydrocorymine, the data were almost identical. The only significant difference was that the signals of one hydroxymethyl group was replaced by those of the formyl group (δ C 194.5; δ H 8.55), which was supported by the observation of the HMBC correlations of the proton signal at δ H 8.55 (H-17) with the carbon signals of C-7, C-15 and carbonyl group of carbomethoxy ( Figure 4). In the NOE experiment, the correlation of H-3/H-21β indicated the β orientation of C-3. The E-form of the double bond of 19/20 was determined on the basis of the NOE correlations of H-19/21 and H-18/15. On the basis of the observation of NOESY data similar to those of 9, the stereochemistry of 6 was expected to be the same. Accordingly, the structure of 6 was established as scholarisin VI ( Figure 1). The NMR data of 7 was almost identical with those of (E)-16-formyl-5α-methoxystrictamine (10) [30]. The only significant difference was that a hydroxymethyl group [δ H 3.68, 3.91 (each, 1H, d, J = 13.2)] in 7 instead of the formyl group at C-16 in 10, was confirmed by HMBC correlations of H-17 with C-7, C-15 and carbonyl group of carbomethoxy ( Figure 5). The relative configuration of compound 7 was determined by the NOESY experiment. Based on the similarity of NOE spectrum with that of 10, the NOE interactions of H-3/H-14α, H-15/H-14α, H-5/H-21, and H-15/H-18 indicated the H-3α, H-15α, and 19E configuration compared with that of (E)-16-formyl-5α-methoxystrictamine. From these data, 7 was named scholarisin VII. The in vitro cytotoxic activities of the isolated alkaloids were evaluated against seven tumor cell lines by using the revised MTT method as described in the Experimental. The results are summarized in Table  2. Alkaloids 1, 6, and 10 exhibited significant cytotoxicity (IC 50 < 30 μM) while 2, 3, and 7-9 showed weak cytotoxic activites (IC 50 > 40 μM) against all the tested tumor cell lines. Furthermore, alkaloids 4 and 5 without the linkage between C-5 and N-4 were non-cytotoxic (IC 50 > 80 μM). The results indicated that the linkage between C-5 and N-4 was essential for cytotoxic properties, while the formyl group on C-16 might strengthen the cytotoxic activities for this type of alkaloids. The compounds 1-10 were tested in vitro for their anti-inflammatory activities. The results of the anti-inflammatory assay were summarized in Table 3. Among the assayed compounds, only alkaloids 1, 6 and 10 with formyl group at C-16 displayed selective inhibition of Cox-2 (> 90%). Alkaloids 2-5 and 7-9 had no anti-inflammatory activities or selective inhibition of Cox-2 comparable to those of 1, 6 and 10 although they possess the same monoterpene indole skeleton. The observations indicated that the formyl group at C-16 should be essential for this type of alkaloids to possess the anti-inflammatory activity. All compounds were tested for their antifungal activities by the disc diffusion method by measuring the inhibition zones and for the most active compounds, minimum inhibitory concentration (MIC) values were also determined. Antifungal properties (Table 4)

Plant Material
The leaves of A. scholaris were collected in Yongning, Guangxi Province, China, in September 2011. The sample was identified by one of the authors (G. B. Shi). A specimen (201109001AS) was deposited in the Herbarium of Shengyang Medicine College, Shengyang, China.

Cytotoxicity Assay in Vitro
The isolated compounds 1-10 were subjected to cytotoxic evaluation against A-549 cells (lung cancer), BGC-823 cells (human gastric carcinoma), HepG2 cells (human hepatocellular carcinoma), HL-60 (human myeloid leukemia), MCF-7 cells (human breast cancer), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) by employing the revised MTT method as described in the literature [31]. Doxorubicin was used as the positive control. All tumor cell lines were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL −1 penicillin and 100 μg/mL streptomycin in 25 cm 3 culture flasks at 37 °C in humidified atmosphere with 5% CO 2 . For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 ×g for 3 min, then resuspended in fresh medium at a cell density of 5 × 10 4 cells per mL. The cell suspension was dispensed into a 96-well microplate at 100 μL per well, and incubated in humidified atmosphere with 5% CO 2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 μM). After 48 h of treatment, 50 μL of 1 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested in triplicate and the IC 50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. The 50% inhibition concentration (IC 50 value) was determined by curve fitting and was used as criteria to judge the cytotoxicity (active: IC 50 ≤ 20 μM; moderately active: 20 μM < IC 50 ≤ 80 μM; not active: IC 50 > 80 μM). All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).

Anti-Inflammatory Assay in Vitro
The anti-inflammatory activities were determined according to a literature method with minor modifications [32]. The reaction system was incubated at 25 °C for 5 min, by sequential addition of the buffer, heme, test compounds, and Cox-1 or Cox-2 into the system followed by mixing with TMPD and arachidonic acid. The absorbance value was recorded at a wavelength of 590 nm after another 15 min of incubation at 25 °C. SC-560 and NS-398 were used as positive controls, which gave the inhibition of Cox-1 (63.20%) and Cox-2 (97.13%) respectively (Table 3). All cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. (Shanghai, China).

Antifungal Activity Bioassay
All compounds (purity > 90%) were screened for their antifungal activity in vitro using the disk-diffusion method as described in the literature with minor modifications [33]. Strains including five species of fungi [Gibberella pulicaris (KZN 4207), Alternaria alternata (TX-8025), Colletotrichum nicotianae (SACC-1922), Phytophthora capsici (KACC-40157), Gonatopyricularia amomi (MB-9671)] were used. Nystatin were used as positive controls for antifungal activity. A disk containing only DMSO was used as the negative control. Agar medium was used in the antifungal activity. To each agar plate, an inoculum containing 10 7 bacteria/mL or a 0.5 optical density of the McFarland Scale was incorporated. The plates were solidified and sterile filter paper disks (6-mm diameter) were done on each one. Solution of each compound (5 mM) in DMSO, antifungal agents (nystatin 10 μM/mL), and control vehicles (DMSO) were added into too. The plates were aerobically incubated at 37 °C for the five species of fungi during 24 h. The diameter of the inhibition zone was measured for testing of antifungal activities. Experiments were performed in triplicate, and the results are presented as the mean values of the diameters of the inhibitory zones from three runs. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96-hole plates. The diameters of the inhibitory zones and the MIC value were used as criteria to judge the antimicrobial activity (active: the diameters of the inhibitory zones ≥ 16 mm, MIC ≤ 5 mM; moderately active: the diameters of the inhibitory zones are visible, MIC > 5 mM; not active: the diameters of the inhibitory zones are invisible). All fungal were purchased from the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).