Spiralosides A–C, Three New C27-Steroidal Glycoalkaloids from the Fruits of Solanum spirale

Abstract Three new C27-steroidal glycoalkaloids, spiralosides A–C (1–3), were obtained from the total alkaloids of Solanum spirale by chromatographic methods. On the basis of spectroscopic evidence, spiralosides A–C were elucidated as (22R,25S)-22,26-epiminocholest-5-ene-3β,16α-diol-N-acetyl-3-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl (1), (22R,25S)-22,26-epiminocholest-5-ene-3β,16α-diol-N-acetyl-3-O-β-d-glucopyranosyl (2), (22R,25S)-22,26-epiminocholest-3β,16α-diol-N-acetyl-3-O-β-d-glucopyranosyl (3), respectively. The total alkaloids of S. spirale have been screened for their antitussive and expectorant effects in intact animal model. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-016-0103-9) contains supplementary material, which is available to authorized users.


Result and Discussion
Compound 1 was obtained as a white, amorphous powder; ½a 23 D -46.6 (c 0.03, MeOH). It displayed a positive reaction to Dragendorff's reagent and gave the molecular formula of C 41 H 67 NO 12 [16]. Likewise the other anomeric configuration of the rhamnopyranosyl was confirmed as a-orientated on the basis of the chemical shift values of C-3 00 (d C 72.2), C-5 00 (d C 70.6) with those of the corresponding carbons of methyl aand b-rhamnopyranoside [17].The identification of the sugar residues were continued by hydrolysis with 10 % HCl to afford D-glucose and L-rhamnose, which were confirmed by GC chromatographic analysis of their L-cysteine methyl ester-TMS derivates. Besides of two sugar units, 13 C-NMR and DEPT spectra also showed 29 carbons, five methyl groups, ten methylenes, ten methines, and four quaternary carbons ( Table 1). Comparison of above data with those of capsimine [18] and baikeine [19], pingbeinine [20] showed that the aglycone of 1 was similar to capsimine with exception of an additional acetyl group at d C 22.2 (q) and 172.4 (s) in 1 (Fig. 1 (Fig. 2). The glycositatic position was unambiguously ascribed to be at C-3 from the HMBC correlation of d H 4.36 (1H, d, J = 7.8 Hz, H-1 0 ) with C-3 (d C 79.9). The 1 H-1 H COSY spectrum of 1 displayed five partial fragments a-e (Fig. 2). The signals of fragments a and b were from d  (Table 1).
The total alkaloids of S. spirale have been screened for the protective anti-tussive effect against ammonia liquor induced cough and the protective expectorant activity used by phenol red secretion test in mice. The results showed that the total alkaloids exhibited an inhibited tendency on antitussive in mice (Tables 2, 3).
Compounds 1-3 were evaluated for their cytotoxicity against five human cancer cell lines using the MTT method as reported previously [23]. Cisplation (sigma, USA) was used as the positive control. Unfortunately, the results showed that all compounds were inactive (IC 50 values [40 lM).

Plant Material
Air-dried fruits of S. spirale were collected in December 2014 from Shuangbai county, Yunnan province, P.R china,

Extraction and Isolation
The air-dried fruits from S. spirale (5 kg) were crushed and extracted with 20 L 90 % MeOH for five times under reflux for a total 3 h, and then combined extract was concentrated under reduced pressure to afford an extract. The extract was partitioned between EtOAc and 0.5 % HCl solution, getting a nor-alkaloid fraction. The acidic water-soluble, adjusted pH to 9-10, was extracted with EtOAc to give total alkaloids and water-soluble fraction. The total alkaloid fraction (84 g) was subjected to silica gel column   Values expressed as mean ± SEM (n = 10) ** P \ 0.01 for comparison of treated groups with control

Acid Hydrolysis of compounds 1-3 and GC Analysis
Compounds 1-3 (each 3 mg) were refluxed with 2 M HCl (1,4 dioxane/H 2 O 1:1, 2 mL) on water bath for 2 h. After cooling, the reaction mixture was neutralized with 1 M NaOH. The reaction mixture was extracted with CHCl 3 (3 9 5 mL). The aqueous layer was evaporated to dryness. The dried residue was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60°C for 1 h. Trimethylsilylimidazole (1.0 mL) was added to the reaction mixtures, and they were kept at 60°C for 30 min. The supernatants (4 lL) were analyzed by GC, respectively, under the following conditions: H 2 flame ionization detector. Column: 30QC2/AC-5 quartz capillary column (30 m 9 0.32 mm). Column temperature: 180-280°C with the rate of 3°C/min, and the carrier gas was N 2 (1 mL/min) injector temperature: 250°C; and split ratio: 1/50. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of D-glucose and L-rhamnose after treatment with trimethylchlorosilane (TMCS) in pyridine. The absolute configurations of the compounds 1-3 were determined by comparison of the retention times of the corresponding derivatives with those of standard D-glucose and L-rhamnose giving a single peak at 19.01 and 15.43 min, respectively.

Animals
ICR mice of either sex (20-22 g) were purchased from Kunming Medical College (License number SYXK2014-0004). All animals were housed at room temperature (20-25°C) and constant humidity (40-70 %) under a 12 h light-dark cycle in SPF grade laboratory. The animal study was performed according to the international rules considering animal experiments and the internationally accepted ethical principles for laboratory animal use and care.
3.5 Anti-tussive Activity Assay ICR mice of either sex weighing 21-24 g were divided randomly, 10 mice per group. The negative control of animals was treated with distilled water orally, and the positive control was treated with codeine phosphate, the remaining groups treated were with test samples respectively. Anti-tussive activity was investigated on a classical mouse cough model induced by ammonia liquor [24]. Briefly, each mouse was placed in a 300 mL special glass chamber and exposed to 40lL 25 % NH 4 OH. The cough frequency produced during 2 min exposure period was counted. In the second assay for alkaloids, cough frequency and latent period of cough were recorded.

Expectorant Effect Assessment
The procedures were performed as described previously [25]. Male and female mice were randomly allotted and treated with a single dose 30 min before intraperitoneal injection of phenol red solution (5 % in saline solution, w/v, 0.1 mL/10 g body weight). Mice were sacrificed by cervical dislocation 30 min after application of phenol red. After dissected free from adjacent organs, the trachea was removed from the thyroid cartilage to the main stem bronchi and put into 2 mL normal saline immediately. After ultrasonic for 5 min, 0.1 mL of 1 M NaOH solution was added to the saline and optical density of the mixture were measured at 546 nm using enzyme standard instrument.

Cytotoxicity Assay
Five human cancer cell lines, lung cancer A-549, human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, breast cancer MCF-7, and colon cancer SW480 cells, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10 % fetal bovine serum (Hyclone, USA) in 5 % CO 2 at 37°C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method in 96-well microplates [26]. Briefly, 100 lL of adherent cells was seeded to each well of a 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 1 9 10 5 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.064, 0.32, 1.6, 8, and 40 lM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected, and the cell growth curve was graphed. The IC 50 value was calculated by Reed and Muench's method [27].