Polygonumnolides A1–B3, minor dianthrone derivatives from the roots of Polygonum multiflorum Thunb

Seven new dianthrone glycosides, named polygonumnolides A1–B3 (1–7), were isolated from the 70 % EtOH extract of the dried roots of Polygonum multiflorum Thunb. using column chromatography and preparative high-performance liquid chromatography. Their structures were determined by 1D and 2D NMR and mass spectroscopy. The isolated compounds were evaluated for their cytotoxic effects against KB tumor cell lines and compounds 1–4 showed moderate cytotoxicity. Electronic supplementary material The online version of this article (doi:10.1007/s12272-016-0816-7) contains supplementary material, which is available to authorized users.


Introduction
The roots of Polygonum multiflorum Thunb. are used as a traditional Chinese medicinal herb to treat many diseases. They are applied as remedies for preventing hair loss and premature graying, strengthening bones and muscles, and treating seminal emission and menstrual and menopausal complaints (The State Pharmacopoeia Commission 2015). Chemical extractions of the roots of P. multiflorum resulted in the isolation of approximately 100 compounds (Lin et al. 2015), including anthraquinones, stilbenes, phenolic acid, phospholipids and flavones. In our continuing search for bioactive compounds, 70 % Et OH extract of the dried roots of P. multiflorum was investigated. Seven new dianthrone glycosides, named polygonumnolides A1-B3 (1-7), were isolated, and the structural elucidation of these new compounds are described herein as well as their cytotoxic effects against KB tumor cell lines.

Plant material
The dried roots of P.

Extraction and isolation
The roots of P. multiflorum Thunb. (28.0 kg) were extracted three times with 70 % EtOH under reflux and filtered. The filtrate was evaporated under reduced pressure at 50°C to afford a crude extract (4.0 kg). The crude extract was partitioned with CH 2 Cl 2 and H 2 O. The H 2 O fraction (3.5 kg) was loaded onto macroporous resin (DM-8) and eluted with a gradient of water and 95 % EtOH mixture (H 2 O, 25 % EtOH, 40 % EtOH, 55 % EtOH and 95 % EtOH) to give five fractions (A-E; fraction A: 2.0 kg; fraction B: 62.0 g; fraction C: 200.0 g; fraction D: 38.0 g; fraction E: 55.0 g). Fraction E was separated on a Sephadex LH-20 column using step gradient elution of methanol-water (from 60 to 100 % v/v) to give five fractions (A 1 -A 5 ) based on RP-TLC analysis. Fraction A 3 was further chromatographed on a RP-18 silica gel column using a step gradient elution of methanol-water (from 40 to 100 % v/v) to give 5 fractions (B 1 -B 5 ) based on RP-TLC analysis.

Enzyme hydrolysis of compounds
Compounds 1-7 (3.0 mg each) (Tian et al. 2013) were enzymatically hydrolyzed by b-glucosidase (10.0 mg) HNMR data were measured in CD 3 COCD 3 or CD 3 OD at 600 MHz c 1 HNMR data were measured in CD 3 COCD 3 at 500 MHz Polygonumnolides A1-B3, minor dianthrone derivatives from the roots of Polygonum multiflorum… 619 from almonds (CAS 9001-22-3) at 30°C for 12 h, respectively. The reaction mixtures were extracted with EtOAC (3 9 10 mL). The aqueous layers were frozen for 12 h in order to remove organic solvent and freeze-dried to obtain the monosaccharides of 1-7. L-Cysteine methyl ester hydrochloride (1.5 mg) was added to solutions of the monosaccharides of compounds 1-7 and D-glucose in pyridine (3.0 mL) and kept at 60°C for 1 h. The reaction mixtures were cooled in an ice-water bath, trimethylsilylimidazole (1.0 mL) was added and the mixtures heated to 60°C for 30.0 min. The reaction mixtures were partitioned between H 2 O (2 mL) and n-hexane (3 9 10 mL). The n-hexane extracts of each digest were subjected to GC analysis, run on a Agilent 7890A gas chromatograph equipped with a Agilent HP-5 capillary column (60.0 m 9 0.32 mm 9 1.0 lm) and an H 2 flame ionization detector with the following conditions: column temperature, 160-280°C; ramp, 5°C/min and maintained at 280°C for 20.0 min; carrier gas, N 2 (1 mL/min); injector and detector temperature, 300°C; injection volume, 5.0 lL; and split ratio, 1/30. The configuration of the monosaccharides in each sample was determined by comparing the retention time of the derivatives with that of an authentic sample. All the samples gave a single peak with the same retention time of 40.6 min and carbohydrates in 1-7 were determined to all be D-glucose.

Cytotoxicity assay
A tetrazolium-based colorimetric assay (methyl thiazolyl tetrazolium assay, MTT assay) was used to assess the cytotoxicity of 1-7 against KB tumor cell lines with taxol used as the positive control. The assays were performed according to a published technique (Zhang et al. 2001).

Results and discussion
Compound 1 (Fig. 1) was obtained as a yellow powder. Its molecular formula, C 37 H 34 O 13 , was deduced from HRESI-MS from the peak at m/z 685.1940 [M-H] -, which corresponded to 21 indices of hydrogen deficiency. The IR spectrum showed strong absorption bands at 1620 and 1599 cm -1 that were assigned to carbonyl groups, a peak from chelated hydroxyl groups at 3361 cm -1 and a peak from aromatic ring functionalities at 1489 cm -1 . The UV spectrum showed absorption maxima at 207, 280 and 351 nm, which were very close to those of previously reported dianthrone derivatives (Lemli et al. 1964; Vandenberg and Labadie 1981;Gizachew et al. 1993). The 1 H NMR (Table 1) and 1 H-1 H COSY spectra (Fig. 2)   CNMR data were measured in CD 3 COCD 3 or CD 3 OD at 150 MHz c 13 CNMR data were measured in CD 3 COCD 3 at 125 MHz J = 3.5 Hz, H-10 0 )], and one b-glucopyranosyl anomeric proton at d H 4.60 (1H, d, J = 7.5 Hz, H-1 00 ).