New Triterpenoid Saponins from the Herb Hylomecon japonica

Background: Hylomecon japonica, a plant of the Papaveraceae family which is well-known for the alkaloids they produce, is a perennial plant widely distributed in the northeast, central and east regions of China. Although a variety of chemical constituents, including alkaloids, flavonoids, and megastigmoids, have been isolated from H. japonica, the investigation of saponins in H. japonica has not been reported until now. Methods: Various separation techniques, including polyporous resin column chromatography, silica gel column chromatography and hemi-preparative HPLC were applied to the isolation of triterpenoid saponins, and chemical methods such as acid hydrolysis and spectroscopic methods including HRESIMS and NMR were applied to their structure elucidation, and the XTT reduction method was used to assay cytotoxicity. Results: Two new triterpenoid saponins, named hylomeconoside A (1) and B (2) which were identified as 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-quinovopyranoside (1) and 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (2), and two known triterpenoid saponins identified as dubioside C (3) and lucyoside P (4) on the basis of spectroscopic and chemical evidence, were isolated from H. japonica. Compound 1 exhibited moderate cytotoxicity on MGC-803 and HL-60 cells, with IC50 values of 43.8 and 32.4 μg·mL−1, respectively. Conclusions: Compounds 1 and 2 are new saponins, and 1 is considered to be one of the antitumor principles in this plant. This is the first time that triterpenoid saponins have been isolated from plants of the Papaveraceae family.


Introduction
Hylomecon japonica (Thunb.) Prantl et Kundig (Papaveraceae) is a perennial plant widely distributed in the northeast, central and east regions of China. Its roots have been used as a traditional Chinese medicine for the treatment of rheumatism and injuries [1]. Although a variety of chemical constituents, including alkaloids, flavonoids, and megastigmoids, were previously isolated from H. japonica, and pharmacological studies on its anti-inflammatory properties, antibacterial activities, and antitumor action have been reported [2][3][4][5][6][7], the investigation of saponins in H. japonica has not been reported until now. As part of our study on the plants of Papaveraceae family, we have been carrying out an  (2), and two known triterpenoid saponins identified as dubioside C (3) [8], and lucyoside P (4) [9] (Figure 1) on the basis of spectroscopic and chemical evidence.

Results and Discussion
The crude saponins prepared from the 70% EtOH extract of H. japonica herbs through a D101 polyporous resin column were subjected to silica gel chromatographies and semi-preparative HPLC to yield two new triterpenoid saponins, named as hylomeconoside A (1) and B (2), and two known triterpenoid saponins identified as dubioside C (3) [8] and lucyoside P (4) [9] on the basis of spectroscopic and chemical evidence.

Results and Discussion
The crude saponins prepared from the 70% EtOH extract of H. japonica herbs through a D101 polyporous resin column were subjected to silica gel chromatographies and semi-preparative HPLC to yield two new triterpenoid saponins, named as hylomeconoside A (1) and B (2), and two known triterpenoid saponins identified as dubioside C (3) [8] and lucyoside P (4) [9] on the basis of spectroscopic and chemical evidence.
The The β-anomeric configurations for D-xylopyranosyl, D-quinovopyranosyl, D-glucuronopyranosyl and D-galactopyranosyl moieties were determined by their large 3 J H1-H2 coupling constants of 6-8 Hz and the α-anomeric configuration for L-rhamnopyranosyl unit was determined by its small 3 J H1-H2 coupling constant.
One Xyl unit, Xyl a , identified starting from anomeric signals at δ H 5.08 (H-1 of Xyl a ) and δ C 105.9 (C-1 of Xyl a ), was identified to be in the terminal position, as observed by its 13 C-NMR chemical shifts. Another Xyl unit, Xyl b , identified starting from anomeric signals at δ H 4.94 (H-1 of Xyl b ) and δ C 106.9 (C-1 of Xyl b ), was substituted at the position of C-3 of Xyl b based on the deshielding of C-3 of Xyl b (δ C 87.2), and the Xyl a unit was attached to this position based on the long-range correlation observed in the HMBC experiment between signals at δ H 5.08 (H-1 of Xyl a ) and δ C 87.2 (C-3 of Xyl b ). The methyl doublet at δ H 1.66 (3H, d, J = 6.4 Hz) and the typical broad single of anomeric proton at δ H 6.34 (br s) were characteristic of Rha unit, and the deshielding of C-4 of Rha (δ C 85.3) indicated a substitution at the position of C-4 of Rha, and the long-range correlation between signals at δ H 4.94 (H-1 of Xyl b ) and δ C 85.3 (C-4 of Rha) indicated that Xyl b was attached to the position of C-4 of Rha. The Qui unit was identified starting from the deshielded anomeric proton at δ H 5.98 (J = 8.0 Hz) and characterized by its methyl doublet at δ H 1.42 (3H, d, J = 6.0 Hz). The deshielding of anomeric proton and the chemical shift of anomeric carbon at δ C 94.5 suggested that Qui was attached by an ester linkage to the C-28 carboxylic group of the aglycone, which was further confirmed by the long-range correlation observed in the HMBC experiment between signals at δ H 5.98 (H-1 of Qui) and δ C 176.7 (C-28 of gypsogenin). Qui was substituted at the position of C-2 of Qui as observed by its deshielded H-2 of of Qui (δ H 4.23) and the long-range correlation observed in the HMBC experiment between signals of the Rha anomeric proton (δ H 6.34) and C-2 of Qui (δ C 76.6). Therefore, the sequencing of the ester chain was obtained by analysis of HMBC experiment which showed cross-peaks between H-1 of Rha (δ H 6.34) and C-2 of Qui (δ C 76.6), between H-1 of Xyl b (inner xylose) (δ H 4.94) and C-4 of Rha (δ C 85.3), and between H-1 of Xyl a (the terminal xylose) (δ H 5.08) and C-3 of Xyl b (δ C 87.2), and thus the ester chain was a tetrasaccharide, β-D-xylopyranosyl- The Gal unit, identified starting from anomeric signals at δ H 5.11 (H-1 of Gal) and δ C 106.2 (C-1 of Gal), was identified to be in terminal position, as observed by its 13 C-NMR chemical shifts. Starting from the anomeric proton at δ H 4.72 (d, J = 6.3 Hz), a GlcA unit was identified with its carbonyl C-6 at δ C 173.7. The deshielding of C-2 of GlcA (δ C 82.1) indicated a substitution of glucuronic acid. Observation of long-range proton-carbon correlations in the HMBC spectrum between the anomeric proton of GlcA (δ H 4.49) and C-3 of gypsogenin (δ C 83.5) and between the anomeric proton of Gal (δ H 5.11) and C-2 of GlcA (δ C 82.1) indicated a disaccharide chain attached at C-3 of gypsogenin, The complete assignment of the signals of 1 was based on DEPT 13 C-NMR and 2D NMR of 1 H-1 H COSY, HMQC and HMBC. All the data of 1 H-, 13 C-NMR and 2D-NMR of 1 see Table 1, and the key correlations in HMBC NMR and the structure of 1 see Figure 1. In conclusion, compound 1 was identified as Hylomeconoside B (2) was obtained as a white amorphous solid. The molecular formula of 2 was determined as C 63 H 98 O 31 by high-resolution HRESI-MS which showed a [M + H] + ion at m/z 1351.6151. Acid hydrolysis of 2 afforded an aglycone which was identified as gypsogenin on the basis of the 1 H-and 13 C-NMR spectra [10]. The sugars obtained from the saponin hydrolysate were identified as L-arabinose, D-xylose, L-rhamnose, D-glucuronic acid and D-galactose based on GC analysis of their chiral derivatives. In comparative analysis of the 13 C-NMR data of 2 with those of 1 and 3, it was found that the spectra data of the aglycone moiety of 2 are same as those of 1, and the spectra data of the sugar moiety of 2 are consistent with those of 3 (Table 2), which suggested that the aglycone of 2 is gypsogenin and the structure of the sugar moiety is same as those of 3. By further analysis of spectral data with the similar method as above, 2 was identified as 3-O-β-Dgalactopyranosyl-(1→2)-β-D-glucuronopyranosyl-gypsogenin-28-O-β-D-xylopyranosyl-(1→3)-β-Dxylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. Dubioside C (3), one of active constituents of Thladianthae dubiae roots, has been reported to possess analgesic and anti-inflammatory effects [11][12][13] and lucyoside P (4), one of active constituntes of Luffa cylindrica fructus, has beneficial effects on intelligence [14], therefore we have only carried out an activity assay on the new saponins 1 and 2. The cytotoxic activities of compounds 1 and 2 on MGC-803 (human gastric cancer), HL-60 (human promyelocytic leukemia), BEL-7404 (humanhepatoma carcinoma), MCF-7 (human breast cancer), and SPC-A1 (lung adenocarcinoma) cells were assessed by the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction method. The results (Table 3) showed that compound 1 exhibited moderate cytotoxicity on MGC-803 and HL-60, with IC 50 values of 43.8 and 32.4 µg·mL −1 , respectively. Therefore, compound 1 is considered to be one of the antitumor principles in this plant.

Extraction and Isolation
Two kg of air-dried whole Hylomecon japonica herbs were extracted twice with 20 L of 70% aqueous ethanol solution (v/v) at room temperature. The extraction solution was concentrated under reduced pressure to remove ethanol, and the water concentrate was filtered and then passed through a D101 polyporous resin column eluting successively with H 2 O, 30% EtOH, 50% EtOH, 70% EtOH, and 95% EtOH. The crude saponin extracts were obtained from 50% aqueous ethanol eluate by vacuum distillation recovery and used for the next experiments. The crude saponin extracts were chromatographed on silica gel columns repeatedly eluted with EtOAc-MeOH-H 2 O (7:3:0.5) and futher purified by semi-preparative HPLC using acetonitrile and 0.1% formic acid solution in water as the mobile phase and the eluate was monitored at 207 nm, to yield compounds 1 (60 mg), 2 (50 mg), 3 (30 mg), and 4 (30 mg).  Table 1.  Table 2.  Table 2.  1H, m). The H 2 O layer was concentrated under reduced pressure to dryness, to give a residue of the sugar fraction. The residue was dissolved in pyridine (2 mL), L-cysteine methyl ester hydrochloride (1.5 mg) was added, and the mixture was heated at 60 • C for 1 h. Trimethylsilylimidazole (1.5 mL) was added, and the mixture was heated at 60 • C for another 0.5 h. An aliquot (4 µL) of the supernatant was subjected to GC analysis under the following conditions: column temp 180-280 • C at 3 deg/min, carrier gas N 2 (1 mL/min), injector and detector temp 250 • C, split ratio 1:50. The configurations of monosugars for 1 and 2 were determined by comparison of the retentions times (t R ) of the corresponding derivatives with standard L-arabinose (t R 10.748 min), D-xylose (t R 11.496 min), L-rhamnose (t R 12.162 min), D-quinovose (t R 13.648 min), D-glucuronic acid (t R 15.814 min) and D-galactose (t R 18.026 min). 1 yielded D-xylose, L-rhamnose, D-quinovose, D-glucuronic acid and D-galactose, and 2 gave L-arabinose, D-xylose, L-rhamnose, D-glucuronic acid and D-galactose.
Logarithmic-phase growing cells were diluted into 5 × 10 4 cells·mL −1 , seeded in in 96-well plates with 100 µL per well, and incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U·mL −1 penicillin, 100 µg·mL −1 streptomycin at 37 • C in a humidified atmosphere with 5% CO 2 . Blank control wells were added 100 µL of cultrure medium, and maintained under the same conditions. After 24 h, 100 µL of the above culture medium containing different concentrations of 1 and 2 and normal culture medium (untreated control group) were added, and incubated for 72 h. For the analysis of cytotoxicity, 50 µL (1 mg·mL −1 ) XTT containing 0.15 mg·mL −1 phenazine methosulphate was added to each well, and cells were incubated for 3 h at 37 • C. The absorbances (A) of the produced formazan were measured by a DNM-9602 enzyme immunoassay spectrophotometer at 450 nm. Three replicate well were used for each analysis. The cell inhibitory rate (%) = (A sample − A blank )/(A untreated − A blank ) × 100. The concentration of compound producing 50% of cell inhibitory rate (IC 50 ) was calculated by using SPSS version 20.0 (International Business Machines Corporation, Armonk, NY, USA).

Conclusions
Two new triterpenoid saponins, named hylomeconoside A (1) and B (2), along with two known triterpenoid saponins identified as dubioside C (3), and lucyoside P (4), have been isolated from an ethanolic extract of Hylomecon japonica herbs. It is well known that alkaloids are main constituents of the plants of Papaveraceae family and until now besides alkaloids, flavonoids, megastigmane derivatives, volatile oil, furan derivatives, triterpenes and sterols have also reported, but saponins have not been reported [15][16][17]. This is the first time that saponins have been isolated from plants of the Papaveraceae family. Compound 1 with the IC 50 values of 43.8 µg·mL −1 on MGC-803 and 32.4 µg·mL −1 on HL-60 is considered to be one of the antitumor principles in this plant.