Four ionones and ionone glycosides from the whole plant of Rehmannia piasezkii

Abstract Four new ionones and ionone glycosides (1–4) were isolated from the whole plant of Rehmannia piasezkii Maxim. Their planar structures as well as absolute configuration were confirmed via spectroscopic analysis, ECD calculation, and X-ray crystallography. Compounds 1–4 were tested for their cytotoxicity against five human tumor cell lines and ability to inhibit LPS-activated NO production in the BV2 cell line.

ROESY correlations and the coupling constant analyses of H-3 was assigned the relative configuration. The coupling constants of H-3 were J ¼ 10.2 and 3.6 Hz, indicating that the H-3 occupied an axial position according to the typical ax-ax and ax-eq coupling constants. ROESY correlations of H-3/H 3 -12 and H 2 -7 implied that these protons were a-oriented. The cross-peaks of H-4b/H 3 -11and H 3 -13 indicated the protons were b-oriented. The absolute configuration of 1 was confirmed as 3S, 5R, 6R, 9S by single-crystal X-ray diffraction analysis with a Flack parameter of À0.06(9) (Figure 3). Therefore, piasezkiiosine A was characterized as shown.
For compound 2, its molecular formula was determined as C 13 H 24 O 4 from the HRESIMS (m/z 267.1565 [M＋Na] ＋ ), which is 2 mass less than compound 1, requiring an extra index of hydrogen deficiency. The IR spectrum showed absorption at 1712 cm À1 and 1573 cm À1 due to double-bond functionality. The 13 C NMR spectrum exhibited two olefinic carbons (d C 135. 8 0.93)/C-1, C-2, and C-6, H 3 -12 (d H 1.10)/ C-1, C-2, and C-6, H-7 (d H 6.11)/C-5, C-6, and C-8, and H 3 -13 (d H 1.04)/C-5 and C-6 revealed the planar structure of 2. The coupling constants of H-2 were J ¼ 11.4 and 5.6 Hz, indicating that H-2 occupied an axial position according to the typical ax-ax and ax-eq coupling constants, and ROESY correlations of H-2/H 3 -11 and H 3 -13 suggested H-2, H 3 -11, and H 3 -13 are all b-oriented. And the configuration of HO-9 was determined by the results of Mosher's experiment. The results of Dd R-S (H-7 and H-8) > 0 and Dd R-S (H-10) ＜ 0 indicated the configuration of C-9 was S ( Figure 4). Herein, piasezkiiosine B was characterized as shown.
Compound 3 gave a molecular formula of C 19 H 34 O 9 by HRESIMS at m/z 429.2092 [M＋Na] ＋ , 162 mass units higher than that of 2, in accordance with the presence of a glucopyranose. Its NMR data were similar to those of 2 except for evidence of the presence of the glucopyranose at C-5 in 3. This was confirmed by HMBC correlations from H-1 0 to C-5. Acid hydrolysis of 3 afforded D-glucose, which was identified by TLC comparison and measurement of optical rotation value. The b-anomeric configuration for glucosyl unit was judged from its large 3 J H1, H2 coupling constant (J ¼ 7.8 Hz). Thus, the structure of piasezkiionoside A was characterized as shown.    and C-12. And the glucopyranosyl was located at C-5 based on the HMBC correlation from H-1 0 to C-5. The sugar was confirmed as D-glucose by comparison with an authentic sample and by measuring its optical rotation. In ROESY spectrum, correlations of H 3 -11/H-2b, H-7, H-12b, and H 3 -13, indicated these protons were all b-oriented. The CD spectrum of 4 exhibited similar Cotton effect (negative at 227 nm) to that of frehmaglutin D [3], indicating that the asymmetric centers at C-1, C-5, C-6 and C-7 were 1R, 5R, 6S, 7R configurations. Therefore, piasezkiionoside B (4) was characterized as shown.

Plant material
Crystallographic data for piasezkiiosine A (deposition numbers: CCDC 2131492) has been deposited in the Cambridge Crystallographic Data Centre.

Cytotoxicity assay
Compounds 1-4 were tested for cytotoxicity against five human tumor cell lines, HCT-116 (human colon cancer cell line), U251 (human brain tumor stem cell line), HGC27 (human gastric cancer cell line), HepG2 (human hepatoma cell line) and MCF7 (human breast cancer cell line) by means of an MTT method described in the literature. Taxol was used as the positive control [9].

Inhibitory effects on NO production in LPS-activated microglia
Compounds 1-4 were tested for the ability to inhibit LPS-activated NO production in the BV2 cell line based on the reported method. Curcumin was used as positive control [10].

Disclosure statement
No potential conflict of interest was reported by the author(s).