Seven New Tetrahydroanthraquinones from the Root of Prismatomeris connata and Their Cytotoxicity against Lung Tumor Cell Growth

The root of Prismatomeris connata has been used in China for centuries as the medicinal herb “Huang Gen” (HG), but its phytochemicals or active ingredients are not well understood. In this study, we performed chemical analysis of the ethyl acetate fraction of a HG ethanol extract. We thus isolated seven new tetrahydroanthraquinones, prisconnatanones C–I (compounds 1–7) from the root of P. connata and identified their structures using spectroscopic analyses. Their absolute configurations were established by both modified Mosher’s and Mo2OAc4 methods, and ORD techniques. Their cytotoxicity was tested in a panel of human lung tumor cells (H1229, HTB179, A549 and H520 cell lines). Prisconnatanone I (7) showed the highest activity, with an IC50 value ranging from 2.7 µM to 3.9 µM in the suppression of tumor cell growth, and the others with chelated phenolic hydroxyls exhibited relatively lower activity (IC50: 8–20 µM). In conclusion, these data suggest that some of the natural tetrahydroanthraquinones in HG are bioactive, and hydroxylation at C-1 significantly increases the cytotoxicity of these compounds against lung tumor cell growth.


Introduction
The root of Prismatomeris connate, termed "Huang Gen" (HG) in Chinese herbal medicine, has been used in traditional medicine in China for the treatment of hepatitis, anaemia, leucocythemia, and pneumoconiosis [1][2][3]. Many secondary metabolites, including anthraquinones, anthraquinone glycosides, and iridoids have been identified in HG extracts in earlier studies [4][5][6][7][8][9][10], and in our previous study, two tetrahydroanthraquinones (prisconnatanones A and B) have been successfully isolated, and prisconnatanone A showed a significant cytotoxicity against the A549 lung tumor cell line [9]. This study aimed at further chemical analyses of the phytocomponents in the ethyl acetate (EtOAc) fraction of a HG ethanol extract. In addition to the two known tetrahydroanthraquinone compounds, we identified seven new tetrahydroanthraquinones, prisconnatanones C-I (compounds 1-7). Herein, we report the isolation, structure identification, and biological activities of these new natural compounds.

Results and Discussion
The 95% EtOH HG extract was successfully fractionated with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (BuOH). Seven new tetrahydroanthraquinones 1-7, which we have named prisconnatanones C-I, were then purified from the EtOAc soluble fraction by repeated column chromatography on silica gel, Sephadex LH-20, and semipreparative high-performance liquid chromatography (HPLC).
The chemical properties of these prisconnatanones C-I were determined with both 13 C-NMR (Table 1) and 1 H-NMR spectroscopy ( Table 2). Their specific chemical structures ( Figure 1) were identified according to 1D and 2D NMR spectra (Figures 2 and 3).
Compound 5 was a red powder with a pseudomolecular ion at m/z 371.1114 ([M + Na] + ), suggesting a molecular formula of C 18 H 20 O 7 based on the HRESIMS data, which was the same as that of 4. From carefully comparison of the differences between 5 and 4 as well as between 1 and 2, there was just a small distinction between the two aromatic carbons and two methylenes (Figure 1). Based on the data of 2, the phenolic hydroxy was placed at C-8, which was supported by HMBC correlations (from OH-8 to C-7, C-8, C-9 and C-12), and also there was similar ORD between 2, rαs 21 D =´55.7 (c = 0.16, MeOH), and 5, rαs 21 D =´61.3 (c = 0.20, MeOH), suggesting the absolute configurations at C-2 and C-3 were also R ( Figure 2). The relative configuration of compound 5 was determined to be the same as that of 4 based on ROESY experiments and coupling constants, suggesting that 5 was isomeric with 4. Thus, compound 5 was named prisconnatanone G and identified as 1,2,3,4-tetrahydro-2α,8-dihydroxy-5,6,7-trimethoxy-3β-methylanthracene-9,10-dione ( Figure 1).

Structure-Dependent Cytotoxicity of Prisconnatanones C to I (1-7) against Tumor Cell Growth
The current study led to the identification of seven new compounds 1´7, which belonged to a class of tetrahydroanthraquinones by their chemical structure. Finally, the cytotoxicity of all of these compounds 1-7 at a range of up to 10 µM was tested against a panel of four non-small cell lung tumor cell lines (H1229, HTB179, A549 and H520). As shown in Figure 8, compounds 1-3 did not show any inhibitory activity at all to any of the cell lines, while the inhibitory activities of compounds 4-6 were relatively low, and compound 7 showed the most potent cytotoxicity among these compounds against the growth of these lung tumor cells. Based on the IC 50 values of these compounds against the growth of lung tumor cells (Table 3), their cytotoxicity could be ranked in the order of decreasing inhibitory activity as follows: Compound 7 > 6 = 4 > 5 > 1 = 2 = 3. The activity profiles of these compounds implied that the positions of hydroxyl groups (C-5 and C-8) might be necessary for their antitumor potency, and hydroxylation at C-1 could significantly enhance its cytotoxic activity; however, the underlying mechanisms remain further investigation. Cisplatin is one of first-line drugs in the treatment of lung cancer [12]. Comparing the cytotoxicity of this drug (IC 50 : >20 µM in H1229; 7.5 µM in HTB179; 14.5 µM in H549; and 8 µM in H520) with those of the isolated compounds ( Figure 8, Table 3), the cytotoxicity of 7 was higher than that of cisplatin against this panel of lung tumor cells.  Data are presented as a mean value (IC 50 at µM) of 2-3 separate experiments. Nil: no effect was found.
The tumor cell cultures were incubated in the absence (untreated control) or presence of 0.5, 1, 5 and 10 µM of a purified compound or cisplatin for 48 h. The cell viabilities at the end of incubation were determined with MTT assay. Data are presented as means˘standard derivation (SD) of six determinants in a typical experiment that was repeated 2-3 times. Dot line: x-axis (y = 0).

General Experimental Procedures
The chemical analyses of purified compounds were performed as follows: optical rotations were examined using a Jasco P-1020 polarimeter (Jasco Analytical Instruments, Easton, MD, USA) in either MeOH or CHCl 3 , the UV spectra using a Shimadzu UV-2401PC (Shimadzu China Co., Beijing, China) with MeOH, the CD spectra using a Chirascan spectropolarimeter (Applied Photophysics, Surrey, UK), the IR spectra using a Bruker Tensor 27 Fourier transform infrared spectrometer with KBr pellets (Bruker Beijing Scientific Tech Co., Beijing, China), the 1 H (600 MHz), 13 C (150 MHz) and 2D NMR using Bruker AM-400 and DR-600 instruments (Bruker Beijing Scientific Tech Co.) with TMS as an internal standard in CDCl 3 , the ESIMS using a Bruker HCT/E spectrometer (Bruker Beijing Scientific Tech Co.) and the HREIMS using Waters Autospec Premier P776 spectrometer (Waters Co., Milford, MA, USA). Column chromatography was performed on both silica gel (60-80; 200-300; 300-400 mesh) (Qingdao Marine Chemical Group Co., Qingdao, China) and Sephadex LH-20 (GE Healthcare LifeSciences, Little Chalfont, UK), in which the fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co.). X-ray diffraction data were collected using an Aglient Technologies Gemini A Ultra system (Agilent Tech, Santa Clara, CA, USA).

Plant Material and Drug
HG was collected in June, 2011 from Nanning, Guangxi, China, and identified by Dr. Tao Chen, one of the co-authors. An authenticated voucher specimen (No. CT20110601) was deposited in the herbarium collection (SZG) at the Fairy Lake Botanical Garden (Chinese Academy of Sciences, Shenzhen, China). Cisplatin was purchased from the Pharmacy Services at Vancouver General Hospital (Vancouver, BC, Canada).

Extraction, Isolation and Identification
HG extract was prepared from the dried and powdered HG (15 kg) with 95% EtOH (3ˆ40 L). The solvent was evaporated under reduced pressure until a paste-like extract was formed. The remaining residue (280 g) was suspended in H 2 O, and respectively partitioned using petroleum ether, EtOAc, and BuOH. Next, the EtOAc part (94 g) was submitted to silica gel column chromatography (200-300 mesh) with a gradient elution of PE-EtOAc (from 5:1 to 1:1), and fourteen fractions (Fr.) were collected. All compounds 1-7 were purified from Fr. 12 to Fr. 14.

Esterification of Prisconnatanone C (1) with (R)-and (S)-MTPA
Two portions of prisconnatanone C (1), 12 mg, 0.036 mmol each) were treated with (R)and (S)-MTPA in C 2 H 2 with dimethylaminopyridine and N,N 1 -dicyclohexylcarbodiimide at room temperature and pressure, respectively. These two reactions were conducted in parallel, and their progress was monitored by using TLC. After the reaction was completed, and then the solvent was evaporated, the resultant materials were submitted to silica gel column chromatography (200-300 mesh) with PE-EtOAc (2:1), and further purified by using preparative HPLC (MeCN-H 2 O, 80:30), from which the (S)-MTPA (1a, t R 13.21 min) and (R)-MTPA (1b, t R 12.54 min) esters of prisconnatanone C (1) were collected.

Tumor Cell Cultures
All the lung tumor cell lines were obtained from the Vancouver Prostate Centre (Vancouver, BC, Canada) and were cultured at 37˝C under 5% of CO 2 atmosphere in following culture media (Thermo Scientific, Rockford, IL, USA): H1299, H520 and A549 were cultured in Dulbecco's modified Eagle's Medium (DMEM), and HTB-179 was grown in Roswell Park Memorial Institute (RPMI) 1640 medium. Both DMEM and RPMI 1640 media were supplemented with heat-inactivated fetal bovine serum (FBS, 10%) and antibiotic mixture (100 U/mL penicillin and 100 µg/mL streptomycin) (Sigma-Aldrich Canada, Oakville, ON, Canada).

Cell Viability Assay
The effects of all of the purified compounds 1-7 on lung tumor cell growth or viability, expressed as the percentage of inhibition, were determined using a MTT (3-[4,5-dimethylthiazaol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Canada) assay as described previously [13]. In brief, tumor cells in 96-well plates at a density of 2.5-4.0ˆ10 3 cells/well depending cell types were incubated in the absence (untreated control) or the presence of various concentrations of the compounds (0.5 to 10 µM) for 48 h, MTT solution (5.0 mg/mL in PBS) was added (10 µL/well), and plates were incubated for another 4 h at 37˝C. The purple formazan crystals were dissolved in 100 µL/well of dimethyl sulfoxide (DMSO, Sigma-Aldrich Canada) for 5 min, and the absorbance (OD) was quantified at a 560 nm wavelength using an ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, USA). Each experiment was repeated 2-3 times. Cell growth inhibition in drug-treated cultures against nondrug-treated control was calculated as follows: Inhibition (%) = (ControlD rug-treated)/Controlˆ100%. The half maximal inhibitory concentration (IC 50 ) was calculated based on the cytotoxicity curves as described previously [14].

Conclusions
In conclusion, this study described the structural analyses of prisconnatanones C to I (compounds 1-7) isolated from the root of P. connata and confirmed that these new natural compounds belonged to the rare tetrahydroanthraquinone structural class. In the cytotoxicity assay with lung tumor cells, the relationship of the chemical structure of these compounds with their activities suggested that the chelated phenolic hydroxyls might be the key functional group for their inhibitory activity as compounds 4-7 with chelated phenolic hydroxyls showed activity, while those without the group (compounds 1-3) didn't have any activity. Furthermore, because the most potent activity was found in compound 7, it was suggested that the activity might be enhanced by the numbers of oxygen substituent at the benzene ring of the compounds.