One New and Nine Known Flavonoids from Choerospondias axillaries and Their in Vitro Antitumor, Anti-Hypoxia and Antibacterial Activities

In the present study, a new flavanoid 1, together with nine known ones 2–10 were isolated from the stem bark of Choerospondias axillaries, the fruit of which was used mainly for treatment of cardiovascular diseases in China. The structure of 1 was established on the basis of its extensive spectral data, and the absolute structures of 1 and 10 were determined by their CD data. The absolute structure of 10 was established for the first time. Among the obtained compounds, 5–8 inhibited the proliferation of K562 cells with inhibition rates of 26.6%, 65.7%, 40.4% and 45.6% at 100 µg/mL; 1 and 4–10 showed significant protective effects on anoxia-induced injury in cultured ECV304 or PC12 cells at 50 µg/mL; 8 and 9 showed antibacterial effects on Staphylococcus aureus ATCC6538 at the tested concentration of 150 µg/8 mm paper disc. Compounds 2 and 4–10 were isolated for the first time from this genus. The proliferation inhibiting activities of 7 and 8, the anti-hypoxia activities of 1 and 4–10, and the antibacterial effect of 8 and 9 on Staphylococcus aureus ATCC6538 are reported here for the first time.


Introduction
Choerospondias axillaries, the only plant of the genus Choerospondias belonging to the family of Anacardiaceae, is mainly distributed in the Hubei, Guangdong, Guangxi, Yunnan, Fujian and Guizhou provinces of China. In China, the dried fruit of C. axillaries has been usually used for the treatment of cardiovascular diseases, especially in Mongolian traditional medicine [1,2]. Chemical studies of the plant have revealed the presence of phenolic compounds, flavonoids, sterols, organic acids and polysaccharides, and the total flavonoids were always assumed to be the effective constituents behind its medicinal usage [2]. Our previous work on screening herbal medicines for antitumor activity showed that the ethanol extract of the stem bark of C. axillaries exhibited strong cytotoxicity [3,4]. An ongoing study of its bioactive constituents has now led to isolation of one new and nine known flavonoids 1-10 ( Figure 1). In this work, the isolation, identification, the absolute structure determination and bioactivities of the ten flavonoids 1-10 are reported.
The δ H and δ C values were recorded using solvent signals (CD 3 OD: δ H 3.31/δ C 49.0) as references.
Signal assignments were based on the results of 1 H-1 H COSY, HMQC and HMBC experiments signals. b-e The signals could not be assigned exactly.
In the literature compound 10 was first obtained as an oxidation product of (+)-catechin [7] and in this work we also detected this compound among the products resulting from heating (+)-catechin, so the sterochemistry of 10 should be identical to that of (+)-catechin, except for the absolute configurations of C-1′(u) and C-3′(u) (Scheme 1). In the NOE experiment, 2′(u)-Ha showed correlation with 2(u) or 3(u)-H which were overlapped, so there were two conformations for this compound, conformation A or B (Figure 4). To determine the absolute structure of 10, the CD spectrum was obtained, which showed a positive Cotton effect at 336.5 nm (n-π* transition). According to the helicity rule [8,9], the conformation of 10 should be A, and the absolute configuration of B ring should be 1′(u)S and 3′(u)S, so the complete absolute structure of 10 was determined. Though two reports [10,11] have described the stereochemistry of 10, both of them ultimately referred to reference [7] which did not establish the absolute structure of 10, so this paper reports for the first time the whole stereochemistry of 10.    (5) [14] and quercetin-7-O-β-D-glucopyranoside (6) [15], respectively. Compounds 7 and 8, obtained as crystalline powders, were identified as gambiriin A3 (7) [16] and gambiriin A1 (8) [17] according to the corresponding ESI-MS, 1 H-NMR and 13 C-NMR spectral data. The main characteristic of the structures of these two compounds is that the C ring of the upper unit is cleaved, which is rare in Nature. Compound 9 was identified as (+)-catechin (6′-8) (+)-catechin [7], which was also first obtained from among the oxidation products of (+)-catechin in the literature [7].

Antitumor Activity
The antitumor activities of 1-10 on the K562 cells were evaluated by the MTT method [9], complemented by morphological observations of the cells under a light microscope. The proliferation-inhibiting effect on K562 cells of compounds 5-8 were detected by an MTT assay, which gave inhibition rates of 26.6%, 65.7%, 40.4% and 45.6% at 100 µg/mL, and the IC50 value of 6 is 60.7 µg/mL. The other six compounds showed no noticeable inhibition rates on K562 cells, i.e., 3.4% for 1, 3.9% for 2, −0.5% for 3, 10.4% for 4, −39.8% for 9 and −16.2% for 10, respectively. The morphology of the cells treated with 1-4, 9 and 10 at 100 µg/mL for 24 h showed no distinction from the control group, but inflated cell membranes and cell content leakage could be seen of the cells treated with 5-8 which showed apparent cytotoxicity on K562 cells ( Figure 5). The positive control docetaxol inhibited the K562 cells with an IR% value of 57.8% at 100 µg/mL.

Anti-Bacterial Activity
The anti-bacterial activities for 1-10 were assayed by the 8 mm paper disc method using Blastomyces albicans ATCC10231 and Staphylococcus aureus ATCC6538. Compounds 8 and 9 inhibited the growth of Staphylococcus aureus ATCC6538 with 15 mm and 14 mm inhibition zones at the concentration of 150 µg/paper disc, respectively.

Discussion
Flavonoids are widely distributed in numerous plants, and have received considerable attention due to their diverse bioactivities [18,19]. Flavonoids with galloyl glucopyranosyl groups were also discovered in large numbers of plants [5,[20][21][22][23][24][25]. To our knowledge, except for quercetin-4′-O-(6″-Ogalloyl-β-D-glucopyranoside) [25], the galloylglucopyranosyl groups of all these compounds was connected to the A or C ring of the flavonoid skeleton. Compound 1 is the second example whose galloylglucopyranosyl was connected to the B ring of the flavonoid skeleton, which is very rare in Nature. Dimeric flavans, especially catechin derivatives, are reported as being isolated both from plants [10,11,16,17,26] and the oxidation products of catechin or epicatechin [7,27], and their biosynthesis and genetic regulation have also been investigated [28]. Dehydrodicatechin A (10), a dimeric flavan from (+)-catechin, was also isolated both from plants or among the oxidation products of (+)-catechin. Though the planar structure of 10 had been determined years ago, the exact absolute structure of this compound had not yet been established [7,10,11,27,29], so this paper, represents the first time that the absolute structure of 10 was established on the basis of its CD data.

General Experiment Procedures
Melting point was measured on a Beijing Tiandiyu X-4 exact micro melting point apparatus (Tiandiyu Science and Technology Co., Ltd., Beijing, China) and the temperatures are not corrected. Optical rotations were measured on an Optical Activity Limited polAAr 3005 spectropolarimeter (Optical Activity Limited, Ramsey, UK). ESIMS was recorded on an Applied Biosystems API 3000 LC-MS spectrometer (AB SCIEX, Framingham, MA, USA) and HRESIMS was measured on an Agilent 6520 Q-TOF LC-MS spectrometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were taken on a Bruker Tensor-27 infrared spectrophotometer (Bruker, Karlsruhe, Germany). CD data were recorded on a Biologic Science MOS450 CD spectropolarimeter (Bio-Logic, Pont-de-Claix, France). 1D and 2D NMR spectra were obtained on a JEOL JNM-GX 400 (400 MHz 1 H and 100 MHz 13 C-NMR) NMR spectrometer (JASCO electric Co., Ltd., Tokyo, Japan). The chemical shifts of 1 H

Plant Material
The stem bark of C. axillaries was collected in the Mengla region of Yunnan, China. The plant was identified by Professor Sun Qi-shi and a voucher specimen (No. 050901) was deposited at the Beijing Institute of Pharmacology and Toxicology.

Cell Line and Cell Culture
Human myeloid leukemia K562, human umbilical vein endothelial cell ECV304, and rat pheochromocytoma PC12 cell lines were used for bioassay. The K562, ECV304 and PC12 cells were routinely maintained in RPMI-1640 (for K562 and ECV 304 cells) or DMEM (for PC12 cells) medium containing 100 µg/mL penicillin and 100 µg/mL streptomycin supplemented with 10% FBS under a humidified atmosphere of 5% CO2 and 95% air.

Cell Proliferation Assay
Compounds 1-10 and DOC were dissolved in MeOH to prepare a 10.0 mg/mL solution, and serial dilutions were made for compounds 1-10. These solutions were subjected to the MTT assay. DOC was used as positive control and MeOH was used as blank control. The assay was run in triplicate on human cancer K562 cell lines by the method that we have previously reported [9].

Anti-Hypoxia Assay
Compounds 1-10 were dissolved in the DMEM or RPMI 1640 medium to prepare a solution at 50.0 µg/mL. The anti-hypoxia activities of 2-5 were assayed using ECV304 cells, but the anti-hypoxia activities of 1 and 6-10 were assayed on PC12 cells.
MTT assay on ECV 304 cells: Exponentially growing ECV 304 cells were suspended in fresh RPMI 1640 medium at the density of 1 × 10 5 cells/mL and then seeded into 96-well plates at 150 µL/well. The cells were incubated at 37 °C for 48 h, then discarded the medium and were assigned into normoxic control group, hypoxia control group and hypoxia administration group. Each well of the normoxic control group and hypoxia control group was added 150 µL fresh RPMI 1640 medium, and the hypoxia administration group was 150 µL sample solution. The hypoxia control group and hypoxia administration group were cultured in the atmosphere of 5% CO2 and 95% N2 for 24 h, while the normal control group was normally cultured for 24 h. Then, 15 µL MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. Then, the MTT solution were discarded, and 150 µL DMSO were added in each well; after the purple material were fully dissolved, the optical density (OD) of each well was determined on a Versa max plate reader at 490 nm. The cell viabilities were calculated using mean values from cell viabilities (%) = OD hypoxia control or OD sample/OD normal control × 100%.
MTT assay on PC 12 cells: Exponentially growing PC12 cells were suspended in fresh DMEM medium at the density of 1 × 10 5 cells/mL and then seeded into 96-well plates at 150 µL/well. The cells were incubated at 37 °C for 24 h, then discarded the medium and were assigned into normoxic control group, hypoxia control group and hypoxia administration group. Each well of the normoxic control group and hypoxia control group was added 150 µL fresh DMEM medium, and the hypoxia administration group was 150 µL sample solution, then all were incubated at 37 °C for 1 h. Each well of hypoxia control group and hypoxia administration group were added 2 µL CoCl2 solution (306.14 µg/mL in water), all the three groups were incubated at 37 °C. After all were normally incubated for 24 h, 15 µL MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. Then, the MTT solution were discarded, and 150 µL DMSO were added in each well; after the purple material were fully dissolved, the optical density (OD) of each well was determined on a Versa max plate reader at 490 nm. The cell viabilities were calculated using mean values from cell viabilities (%) = OD hypoxia control or OD sample/OD normal control × 100%.

Antibacterial Effect Test
Blastomyces albicans ATCC10231 and Staphylococcus aureus ATCC6538 were used to evaluate the antibacterial activities of compounds 1-10. Blastomyces albicans ATCC10231 was cultivated on Sabouraud's agar medium, and Staphylococcus aureus ATCC6538 was cultivated on tryptose soya agar medium. All the compounds were dissolved in methanol to prepare 10 mg/mL test sample solutions. Samples (15 µL) were added to the paper discs (8 mm) and dried for 10 min. Then, the discs were put on the tested plate which contained either Staphylococcus aureus or Blastomyces albicans. Tested plates were cultured at 28 °C for 2 days and then the diameter of the inhibition zone was measured.

Conclusions
Ten flavonoids were isolated from the stem bark of C. axillaries. Among the obtained compounds, 1 is a new compound with a galloylglucosyl group on the B ring in the flavonoid skeleton, and compounds 2 and 4-10 were isolated from this genus for the first time. The absolute structures of 1 and 10 were established from their CD data, and the absolute configuration of 10 was determined exactly for the first time. The proliferation inhibiting activities of 7 and 8, the anti-hypoxia activities of 1 and 4-10, and the antibacterial effect of 8 and 9 on Staphylococcus aureus ATCC6538 are reported here for the first time.