An Unusual Piceatannol Dimer from Rheum austral D. Don with Antioxidant Activity

A novel dimer of piceatannol glycoside, named rheumaustralin (1) was isolated from the underground parts of the ethnomedicinal plant Rheum austral (Polygonaceae) collected from Tibet together with 17 known compounds, including rheumin (2), 2,5-dimethyl-7-hydroxychromone (3), 2,5-dimethylchromone-7-O-β-d-glucopyranoside (4), 7-hydroxy-2-(2'-hydroxypropyl)-5-methylchromone (5), torachrysone (6) torachrysone-8-O-β-d-glucopyranoside (7), 4-(4'-hydroxyphenyl)-2-butanone-4'-O-β-d-glucopyranoside (8), amabiloside (9), N-trans-feruloyl tyramine (10), chrysophanol (11), aloe-emodin (12), emodin (13), physcion (14), physcion-1-O-β-d-glucopyranoside (15), emodin-8-O-β-d-glucopyranoside (16), d-catechin (17) and gallic acid (18). Their structures were determined by combined spectroscopic methods and by comparison of their spectral data with those reported in literature. Compounds 1–10 were tested for their ability to scavenge 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical.


Antioxidant Activities by DPPH Scavenging Capacities
Aqueous and methanolic extracts of R. australe were reported to exhibit prominsing antioxidant activities in a previous study [16]. The most abundant stilbenoid of R. austral, piceatannol-4'-O-β-Dglucopyranoside, was suggested to be an important constituent responsible for the antioxidant potential of the extracts of plant materials collected from Tibet [7]. In order to evaluate the potentials of other types of phenolic constituents, compounds 1-10 were screened for their antioxidant activities by the DPPH free radical-scavenging assay that has been widely used for the evaluation of antioxidant activities of natural products. The results obtained in this study are summarized in Table 2. Among these compounds, rheumaustralin (1) displayed relatively strong antioxidant activity with an IC 50 value of 2.3 µM, lower than piceatannol (IC 50 = 0.14 μmol/L), and higher than resveratrol (IC 50 = 15.6 μmol/L). This result was consistent with the concept that the antioxidant activity of stilbenoids depends on the position of the hydroxyl groups. The existence of para-hydroxyl groups significantly enhance antioxidant activity [17]. The glycosylation of hydroxyl groups, however, may decrease the antioxidant activity of stibenoid. Compounds 2, 3, 5, 6 and 10 exhibited promising antioxidant activities with IC 50 values in the range of 20 to 35 µM, which was comparable to those of BHA, ascorbic acid and α-tocopherol; while 4, 7 and 9 showed moderate activities with IC 50 values in the range of 50 to 70 µM. Compound 8 showed lowest activity with an IC 50 value of 109.7 µM.

General
The 1 H-, 13 C-, and 2D-NMR spectra were recorded on Bruker DRX-500 (500 MHz) spectrometer with TMS as internal standard. The ESI-MS and HR-ESI-MS spectra were recorded on VG AutoSpe 3000 and API Qstar P ulsar LC/TOF spectrometers, respectively. The UV spectra were measured by using a Shimadzu double-beam 210A spectrophotometer. The IR spectra were recorded on a Bio-Rad FTS-135 spectrometer, in KBr pellets. The optical rotations were measured by using a SEPA-3000 automatic digital polarimeter. The column chromatographic separations were performed on silica gel (200-300 mesh size; Qingdao Marine Chemical Inc., Qingdao, China), or Lichroprep RP-18 gel (40-63 µm mesh size; Merck, Darmstadt, Germany). The column fractions obtained were monitored by TLC, and spots were visualized by heating the silica gel plates after spraying with 15% H 2 SO 4 in water. The TLC and PTLC separations were performed on silica gel Gf 254 pre-coated plates (Qingdao Marine Chemical Inc.). 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), hexamethyldisilazane and trimethyl-chlorosilane were purchased from TCI (Shanghai, China), Piceatannol and resveratrol were isolated from R. australe D. Don [18].

Plant Materials
The underground parts of R. australe were collected in August 2010 from Doilungdêqên County, Lhasa, China, and authenticated by Prof. Zheng-Dong Fang of Shangri-La Alpine Botanic Garden (Yunnan, China) and re-identified by co-author Dr. Qing-Song Yang. A voucher specimen (No. 2010080401) was deposited in the School of Chemistry and Biotechnology, Yunnan University of Nationalities, Yunnan, China.

Acid Hydrolysis of Compound 1
A solution of compound 1 (7 mg) in 5% aqueous sulfuric acid (2 mL) was heated in a water bath (80 °C) for 4 h. The solution, after cooling, was diluted with H 2 O (2 mL), neutralized with 5% NaOH solution and then extracted with EtOAc (5 × 3 mL). The aqueous layer was concentrated under a stream of nitrogen. The residue was then dissolved in anhydrous pyridine (0.8 mL), followed by the addition of trimethysilylation reagent hexamethyldisilazane/trimethylchlorosilane/pyridine (HMDS/TMCS/pyridine, 3:1:8). It was then stirred at 60 °C for an additional 30 min. The solution was analyzed by GC for sugar identification. GC analyses were performed using an Agilent 5890 instrument on an Agilent HP-1 column (0.25 mm, 30 m, i.d., 0.25 µm). Temperatures of both the injector and detector were 200 °C. A temperature gradient system was used for the oven, starting at 150 °C and increasing up to 250 °C at a rate of 8 °C/min. D-Glucose was confirmed by comparison with the retention time of an authentic standard. These data are in accordance with those reported in the literature [27].

DPPH Assays
The DPPH antioxidant assay was performed with slight modification from that reported previously [28]. Sample stock solution (1 mM) of rheumaustralin (1) A mixture of DPPH solution (2.0 mL, 100 µM) and methanol (2.0 mL) was used as the negative control.The IC 50 values obtained represent the concentrations of the tested samples that caused 50% inhibition of DPPH radicals. The experiments were performed in triplicate, and the results are given as mean ± standard deviation (SD).

Conclusions
An unusual piceatannol dimer named rheumaustralin (1) was isolated from the underground parts of R. australe collected from Tibet, together with 17 known phenolic compounds 2−18. Compounds 9 and 10 were isolated from Rheum plants for the first time. Stilbenoids such as resveratrol and piceatannol are widely distributed in higher plants as phytoalexins [29]. Some of their natural derivatives occur in oligomeric forms. The increasing degrees of polymerization and intriguing variety of polymerization patterns provide stilbene oligomers with dazzling chemical diversities. According to the patterns of oligomer construction and biosynthesis of stilbene oligomers reviewed recently [30,31], rheumaustralin (1), in which the stilbene units are connected only through a methylene (CH 2 ) group, may represent a new connectivity pattern for these stilbene dimers. The discovery of this novel dimer further demonstrates the diversity of the stilbenoids from the genus Rheum. In addition, the free radical scavenging activities of compounds 1-10 against DPPH radicals have been evaluated in this study. All tested compounds showed bioactivites against DPPH radicals. Among them, rheumaustralin exhibited appreciable scavenging activity, with an IC 50 value of 2.3 μM. Compounds 2, 3, 5, 6 and 10 showed promising activities with IC 50 values in the range of 20 to 35 µM, which was comparable to those of BHA, ascorbic acid and α-tocopherol. These phenolic compounds may have therapeutic potential and deserve further study.