Antiproliferative and anti-inflammatory polyhydroxylated spirostanol saponins from Tupistra chinensis

Tupistra chinensis is widely distributed in southwestern China and its rhizome is a famous folk medicine for the treatment of carbuncles and pharyngitis. Its chemical identity of potent antiproliferative and anti-inflammatory constituents has been carried out in this study. Twenty-three polyhydroxylated spirostanol saponins, including nine novels, were isolated and identified. The new spirostanol saponins were elucidated as spirost-25(27)-en-1β,2β,3β,4β,5β-pentol-2-O-β-D-xylopyranoside (1), spirost-25(27)- en-1β,2β,3β,4β,5β-pentol-2-O-α-L-arabinopyranoside (2), spirost-25(27)-en- 1β,3α,5β-triol (12), spirost-25(27)-en-1β,3α,4β,5β,6β-pentol (13), spirost-25(27)-en- 1β,2β,3β,5β-tetraol-5-O-β-D-glucopyranoside (16), 5β-spirost-25(27)-en-1β,3β-diol- 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside (17), (25R)-5β-spirostan- 1β,3β-diol-3-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (18), (25R)-5β- spirostan-1β,3β-diol-3-O-β-D-fructofuranosyl-(2 → 6)-β-D-glucopyranoside (19), 5β-spirost-25(27)-en-3β-ol-3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside (20). The antiproliferative effects against seven human cancer cell lines and inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in a macrophage cell line RAW 264.7 were assayed for all the isolated compounds. Compounds 17, 19 and 21 exhibited potential antiproliferative activities against all of human cancer cell lines tested. Compounds 21 showed significant inhibition on NO production with IC50 values of 11.5 μM. These results showed that the spirostanol saponins isolated from the dried rhizomes of T. chinensis have potent antiproliferative and anti-inflammatory activities and T. chinensis might be used as anticancer and.anti-inflammatory supplement.

In the course of our continuing search for bioactive leading compounds from the medicinal herbs, we focused on steroidal saponins from the dried rhizomes of T. chinensis and obtained twenty-three polyhydroxylated spirostanol saponins including nine new compounds. The structures of all the isolated compounds were elucidated on the basis of spectroscopic data and chemical methods, including IR, NMR, MS, and GC analysis. Moreover, all of the isolated compounds were evaluated for their antiproliferative activity against seven human cancer cell lines and the inhibitory activities on NO production induced by LPS in a macrophage cell line RAW 264.7.
Compound 2 was isolated as white amorphous powder, with the same molecular formula of C 32 H 50 O 11 as 1 on the basis of HRESIMS (m/z 611.3412 [M + H] + ). Acid hydrolysis of 2 gave L-arabinose. From a comparison of 1 H and 13 C NMR data of 2 with those of 1 (Table 1), it was apparent that 2 contained the same aglycon as 1, except for a little different in the monosaccharide chain. Instead of the signals for a xylopyranosyl moiety, signals assignable to an α -L-arabinopyranosyl residue were observed at δ H 5.  Table 2). The 13 C NMR spectrum exhibited characteristic spirostanol carbon signal at δ C 109.8 (C-22) and three methyl (δ C 16.9, 13.7, and 15.4), and two olefinic carbons δ C 144.8 and 109.1 were observed, diagnostic of C-25 (27)-unsaturated sapogenin. The 1 H − 1 H COSY spectrum showed that two methylene protons at δ 2.12 (H-2 α ) and 2.61 (H-2 β ) were coupled to δ 4.31 (H-1) and 5.13 (H-3), and the oxymethine proton δ 5.13 (H-3) was coupled to two methylene protons at δ 2.26 (H-4 β ) and 2.61 (H-4 α ). These findings indicated the location of the hydroxyl groups at C-1, C-3, and C-5, together with the long-range correlations observed in the HMBC spectrum (Fig. 2). The key NOESY correlations (Fig. 2) between H-4 α and H-7 α /H-9 α , and between H-2 α and H-9 α , supported the A/B cis ring junction pattern. Thus, the hydroxyl group at C-5 has a β-orientation. Additional NOE correlations between H-1 α and Me-19/H-11, and between H-3 β and H-2 β /H-4 β , were indicative of β-orientation for OH-1 and α-orientation for OH-3. On the basis of the above analysis, compound 12 was finally assigned to be spirost-25 ( Table 2). The down-field shifted hydrogen chemical shift of Me-19 in combination with an oxymethine proton (δ H 4.94) at B ring confirmed the presence of the OH group attached at C-6 in compound 13, which was consistent with previous reports 12 . The 13 C NMR spectrum exhibited characteristic spirostanol carbon signal at δ C 109.8 (C-22) and three methyl (δ C 16.9, 16.9, and 15.4), and two olefinic carbons δ C 144.8 and 109.1 were observed, diagnostic of C-25 (27)-unsaturated sapogenin. These 1 H-NMR data and 13 C-NMR signals suggested that 13 was a C-25 (27) unsaturated spirostane type steroidal sapogenin. The 1 H-1 H COSY spectrum showed that two methylene protons at δ 2.23 (H-2 α ) and 2.62 (H-2 β ) were coupled to δ 4.19 (H-1) and 4.85 (H-3), and the oxymethine proton δ 4.34 (H-4) was in turn coupled with the oxygenated methane proton at δ 4.85 (H-3). The oxygenated methane proton at δ 4.94 (H-6) was coupled with two methylene protons at δ 1.55 (H-7 α ) and 2.07 (H-7 β ). These findings supported the location of the hydroxyl groups at C-1, C-3, C-4, and C-6. The key NOESY correlations (Fig. 2) between H-4 α and H-7 α /H-9 α , and between H-2 α and H-9 α , supported the A/B cis ring junction pattern. Thus, the hydroxyl groups at C-4 and C-5 have β-orientation. Additional NOE correlations between H-1 α and Me-19/H-11, and between H-3 β and H-2 β , between H-6 α and H-7 α , were indicative of β-orientation for OH-1, OH-6 and α-orientation for OH-3. On the basis of the above analysis, compound 13 was formulated as spirost-25 (27)      Antiproliferative activities. TB, TC, TD and all the isolated compounds were evaluated for antiproliferative activities against seven human tumor cell lines including FaDu (human hypopharyngeal carcinoma), Detroit 562 (human metastatic pharyngeal squamous cell carcinoma), CNE-1 (high differentiation human nasopharyngeal carcinoma), CNE-2 (low differentiation human nasopharyngeal carcinoma), HepG2 (human hepatocellular carcinoma), K562 (human chronic leukemia), SPC-A-1 (human lung adenocarcinoma) with a modified MTT method according to reported protocols, and cis-dichlorodiammineplatinum (II) was used as a positive control. The results were shown in Table 5 and Table S2 for the isolated compound and the crude extract of T. chinensis, respectively. TD showed moderate activity against five out of seven cancer cell lines (Fadu, Detroit 562, HepG2, K562 and SPC-A-1) with IC 50 values of 20.9-29.3 μ g/mL. TC exhibited selective antiproliferative activity against Fadu, Detroit 562, and K562 with IC 50 values of 6.7 ± 0.5, 45.9 ± 1.7, and 6.5 ± 2.3 μ g/mL, respectively.   Anti-inflammatory activities. Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation 36 . NO production by immune cells has been used as an indicator of the presence and extent of inflammation as well as the effectiveness of anti-inflammatory agents 37 .
Compounds 1-23 were tested for their inhibitory effects on NO production induced by LPS in a macrophage cell line RAW 264.7. Cell viability was first determined by the MTT method to find whether inhibition of NO production was due to the cytotoxicity of the tested samples. The anti-inflammatory activities were summarized in Table 6. All the tested samples exhibited no cytotoxicity against RAW 264.7 macrophage cells at their effective concentrations. The crude extracts of T. chinensis TB, TC and TD showed moderate inhibition on NO production with IC 50 values of 32.6 ± 3.7, 37.5 ± 5.4 and 36.6 ± 1.8 μ g/mL (see Table S3 in the supporting information ).     active than 5 due to the exocyclic double in 5, which can be illustrated by the relative higher IC 50 value of compound 20 and the relative lower IC 50 value of compound 21. The kinds, the number and the linkage of sugar also play important roles in their anti-inflammatory activity 40 .

Conclusion
Twenty-three polyhydroxylated spirostanol saponins, including nine new compounds (1, 2, 12, 13, 16-20) were isolated from the rhizomes of T. chinensis and their structures were determined by spectroscopic methods. Among the isolated compounds, Compounds 17, 19 and 21 exhibited potential antiproliferative activities against all of human cancer cell lines tested. Compound 21 showed significant inhibition on NO production with IC 50 values of 11.5 μ M. The present investigation suggested that T. chinensis can be a potential source of natural antiproliferative and anti-inflammatory agents and its spirostanol saponins may be responsible for the biological activity. However, further studies are required, not only to assess the bioactivities of the isolates in vivo, but also to investigate the mechanisms underlying the observed biological activities of isolated compounds.

Materials and Methods
Chemicals. HPLC-grade methanol was purchased from Oceanpak Chemical Co. (Gothenburg, Sweden). General experimental procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. IR spectra were recorded on a PerkinElmer 100 IR spectrometer with KBr. NMR spectra were obtained on a Bruker Ultrashield 500 Plus instrument, using tetramethylsilane as internal standard. Waters AQUITY UPLC/Q-TOF mass spectrometer was used to record HRESIMS spectra. Semi-preparative HPLC was performed using a RAININ pump equipped with a Gilson 133 refractive index detector. A semi-preparative column (COSMOSIL-Pack 5C 18 -MS-II, 10ID × 250 mm, 5 μ m) was used for HPLC. Microplate reader (Kehua Technologies, Inc., Shanghai, China) was used for bioassay.

Plant material. The rhizomes of T. chinensis Baker was purchased from Shennongjia Forest District
Measurement of inhibition activity on tumor cell proliferation. Antiproliferative activities against human nasopharyngeal cancer cells (CNE-1, CNE-2, FaDu, Detroit 562), human liver cancer cells (HepG2), human chronic leukemia cells (K562) and human lung adenocarcinoma cells (SPC-A-1) of the pure spirostanol saponins isolated from T. chinensis were measured by the modified MTT assay as described previously 42 .

Measurement of anti-inflammatory activity. Determination of NO production was performed
by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent, as previously described 43 .
Statistical analysis. All the experiments were conducted for three independent replicates, and data were expressed as mean ± SD. Statistical analyses were performed by one-way ANOVA. Dunnett's Multiple Comparison Test was used to determine the significance of differences between the groups. Differences at P < 0.05 were considered statistically significant.