Antiproliferation and cell apoptosis inducing bioactivities of constituents from Dysosma versipellis in PC3 and Bcap-37 cell lines
© Xu et al; licensee BioMed Central Ltd. 2011
Received: 22 February 2011
Accepted: 15 June 2011
Published: 15 June 2011
Recently, interest in phytochemicals from traditional Chinese medicinal herbs with the capability to inhibit cancer cells growth and proliferation has been growing rapidly due to their nontoxic nature. Dysosma versipellis as Bereridaceae plants is an endemic species in China, which has been proved to be an important Chinese herbal medicine because of its biological activity. However, systematic and comprehensive studies on the phytochemicals from Dysosma versipellis and their bioactivity are limited.
Fifteen compounds were isolated and characterized from the roots of Dysosma versipellis, among which six compounds were isolated from this plant for the first time. The inhibitory activities of these compounds were investigated on tumor cells PC3, Bcap-37 and BGC-823 in vitro by MTT method, and the results showed that podophyllotoxone (PTO) and 4'-demethyldeoxypodophyllotoxin (DDPT) had potent inhibitory activities against the growth of human carcinoma cell lines. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in PC3 and Bcap-37 cells, and the apoptosis ratios reached the peak (12.0% and 14.1%) after 72 h of treatment at 20 μ M, respectively.
This study suggests that most of the compounds from the roots of D. versipellis could inhibit the growth of human carcinoma cells. In addition, PTO and DDPT could induce apoptosis of tumor cells.
Cancer is the major cause of human deaths worldwide because of its high incidence and mortality. Thousands of people die of cancer each year despite aggressive treatment regimens that include surgery, chemotherapy and radiotherapy. Due to the infiltrative nature and the rapid recurrence of the malignant tumor, complete surgical resection of these tumors is typically not achieved [1, 2], and the conventional radiation and chemotherapy are often intolerable due to the strong systemic toxicity and local irritation [3–5]. These factors highlight the urgent need for new therapies or therapeutic combinations to improve the survival and quality of life of cancer patients.
In recent years, interest in phytochemicals from traditional Chinese medicinal herbs has been growing rapidly due to their ability to inhibit the growth and proliferation of cancer cells [6–9]. Due to their nontoxic nature, they are often employed in medical applications. Among of them, flavonoids and lignans present in traditional Chinese medicines have been revealed having significant activities against some forms of cancer [1, 10]. The lignan podophyllotoxin is a plant toxin that exerts its cytotoxicity effect by inhibiting microtubule assembly and promoting cells to die via apoptosis . However, it is not used as a clinical therapeutic agent due to its serious side effects. Extensive structure modifications were performed to obtain more potent and less toxic antitumor agents, e.g. etoposide  and teniposide  are currently used in anticancer therapy. Besides, some researchers found that podophyllotoxin and its derivatives had inhibitory effect on PC3 and other cells [14–16]. One of the popular Chinese herbal medicines, Dysosma versipellis (Hance) M. Cheng which belongs to Berberidaceae in Dysosma Family and is used as a source of podophyllotoxin  is a perennial herbaceous species grown in the understory of mixed evergreen and deciduous forests of China. In some folk remedies, D. versipellis (Hance) M. Cheng has been widely used to clear sputum, kill parasites and treat epidemic encephalitis B and epidemic parotitis. Previous studies have indicated that constituents and extracts of this traditional Chinese medicinal plant have growth inhibitory activities against various tumors in vivo or in vitro [16–18]. However, systematic and comprehensive studies have not been performed with regard to the chemical constituents from D. Versipellis and apoptosis-inducing effect of these components.
Dysosma versipellis, an endangered and endemic Bereridaceae plant species of China, has been proved to be an important Chinese herbal medicine because of its biological activity. In this study, fifteen compounds were isolated from the rhizomes of D. versipellis which was grown in Guizhou and identified by spectroscopic analysis and physicochemical data as β-sitosterol (1), 4'-demethylpodophyllotoxin (2), kaempferol (3), picropodophyllotoxin-4-O- glucoside (4), cleistanthin-B (5), kaempferol-3-O-β-D-glucopyranoside (6), 4'-demethylpodophyllotoxin-4-O-glucoside (7), quercetin3-O-β- D-glucopyranoside (8), icropodophyllotoxin-4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (9), quercetin (10), daucosterol (11), podophyllotoxone (PTO, 12), vanillic acid (13), 4'-demethyldeoxypodophyllotoxin (DDPT, 14), and sucrose (15). Among them, (5), (7), (8), (12), (13) and (15) were obtained from the plants for the first time. All the compounds were then bioassayed on human prostatic carcinoma cell line PC3, human breast cancer cell line Bcap-37, human gastric carcinoma cell line BGC-823 and mouse embryonic fibroblast cell line NIH3T3 in vitro by MTT method. It was found that DDPT had the most potent inhibitory activities against the growth of human carcinoma cell lines than other compounds extracted from D. versipellis. And PTO obtained from the plants for the first time also had high antitumor activity. There are a few reports on the anticancer effects of PTO and DDPT on various tumor cells recently [19–21]. And it was found that the inhibitory rate of PTO (100 μ g/mL) on P388 murine leukemia cell proliferation was 99.0%. Also PTO could induce HL-60 human leukemia cells apoptosis, which might be related with down-regulation of Bcl-2 expression. Other studies have found that DDPT possessed antitumor activity on A549 human lung carcinoma cell and SK-MEL-2 human melanoma cell with EC50 of 0.023 μ g/mL and 0.015 μ g/mL respectively. However, no report was found on the anticancer activities of PTO and DDPT on PC3, Bcap-37 and BGC-823 cells. This prompted us to study the anticancer activities of PTO and DDPT separated from the rhizomes of D. versipellis grown in Guizhou on the three kinds of cells mentioned above and investigate their preliminarily mechanism of action as potent anticancer agents. Thus, further investigations of PTO and DDPT on the three cells lines were carried out on PC3, Bcap-37, and BGC-823 cells. The IC50 of PTO and DDPT on the three cell lines were determined. Furthermore, experimental results of fluorescent staining and flow cytometry analysis indicated that PTO and DDPT could induce apoptosis in PC3 and Bcap-37 cells, with the apoptosis ratios of PC3 cells were 12.0% and 14.1% after 72 h of treatment at 20 μ M, respectively. To the best of our knowledge, this is the first report on apoptosis inducing and antitumor activity of PTO and DDPT on PC3, Bcap-37, and BGC-823 cells.
Analysis and Instruments
1H NMR, 13C NMR, and DEPT spectra were measured on a JOEL-ECX 500 MHz NMR spectrometer in CDCl3, CD3OD, or DMSO-d 6 using tetramethylsilane (TMS) as an internal standard. The IR spectra were obtained in the KBr pellet using a SHIMADZU-IR Prestige-21 spectrometer. Melting points were determined on an XT-4 digital microscope (Beijing Tech Instrument Co.) Analytical TLC was performed on silica gel GF254 (400 mesh), and column chromatographic operations were performed on Silica gel (100-200 or 200-300 mesh, Qingdao Haiyang Chemical Co.). In addition, Sephadex LH-20 column chromatographic instrument (Beijing Huideyi Tech Instrument Co.) was employed for the extraction and purification of chemical composition.
The rootstalk of D. versipellis was collected in Qingzhen, Guizhou Province, China, in the month of May 2008. The voucher specimen was identified as D. versipellis (hance.) M.Cheng by Qing-De Long, the Dean of teaching-research section, School of Pharmacy of Guiyang Medical University, and was submitted to our laboratory for further investigation.
Extraction and isolation
The dried roots of D. versipellis (20 kg) from Guizhou Province were powdered and extracted by maceration in 80% industrial alcohol (50 L, each 4d) for three times. The extracts which filtered and washed with alcohol each time were combined and reduced in vacuo to afford 4 kg crude extract. A part of crude extract (950 g) was further extracted with petroleum ether, chloroform and ethyl acetate to obtain three fractions of 40 g, 2 kg and 280 g respectively.
The petroleum ether extract (40 g) was chromatographed on silica gel column (200-300 mesh) and eluted with a PE-EtOAc (20:1~0:1) gradient, and recrystallized with methanol to give compound 1 (300 mg).
The chloroform extract (100 g) chromatographed on silica gel column (100-200 mesh) was eluted with CHCl3-MeOH (1:0~0:1) and PE-EtOAc (20:1~0:1) to obtain fractions 1-5, respectively. Compounds 2 (100 mg) and 3 (200 mg) was obtained by PTLC and eluting with CHCl3-MeOH (6:1 and 5:1) from fractions 1 and 2, respectively. The fraction 3 was evaluated by Sephadex LH-20 and eluted with acetone to afford compounds 4 (15 mg) and 5 (10 mg). And fraction 4 was eluted with PE-EtOAc (10:1) and recrystallized with acetone to give compound 12 (20 mg). The filtrate from fraction 5 was eluted with PE-EtOAc (9:1 and 10:1, respectively) to afford compounds 13 (25 mg) and 14 (23 mg).
The ethyl acetate extract (55 g) was chromatographed on silica gel column (100-200 mesh) and eluted with CHCl3-MeOH (15:1~0:1) to obtain fractions 1-3. The fraction 1 was evaluated by Sephadex LH-20 (methanol) to give compound 10 (50 mg). Compounds 6 (140 mg) and 7 (209 mg) were obtained by silica gel column chromatography (200-300 mesh) and eluting with CHCl3-MeOH (8:1) from fraction 2. The fraction 3 was further chromatographed on silica gel column (100-200 mesh) and eluted with CHCl3-MeOH (10:1~0:1) to give subfractions 1-3. Compound 8 (30 mg) were obtained by Sephadex LH-20 (methanol) from subfraction 1. The subfraction 2 was recrystallized with CHCl3-MeOH (8:1) to afford compound 15 (15 mg). The subfraction 3 was recrystallized with MeOH and CHCl3-MeOH (9:1) to give compound 9 (19 mg) and compound 11 (18 mg), respectively.
Anticancer activity bioassay
Human prostate cancer cell line PC3, breast cancer cell line Bcap-37, and gastric cancer cell line BGC-823 were purchased from Institute of Biochemistry and Cell Biology, China Academy of Science and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Mouse embryonic fibroblast cell line NIH 3T3 was also obtained from the same place and cultured in DMEM supplemented with 10% FBS. All cell lines were maintained at 37°C in a humidified 5% carbon dioxide and 95% air incubator.
Cells were seeded at a concentration of 5×104 cell/ml in a volume of 0.6 mL on sterile cover slip in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and supplemented with podophyllotoxone and 4'-demethyldeoxypodophyllotoxin (20 μ M). After the treatment period, the cover slip with monolayer cells was inverted on the glass slide with 20 μ L of AO/EB stain (100 μ g/mL). The fluorescence was read on an IX71SIF-3 fluorescence microscope (OLYPUS Co., Japan).
Hoechst 33258 staining
Cells grown on sterile cover slip in 6-well tissue culture plates were treated with podophyllotoxone and 4'-demethyldeoxypodophyllotoxin (20 μ M) for a certain range of treatment time. The culture medium containing compounds was removed and the cells were fixed in 4% paraformaldehyde for 10 min. After washing twice with PBS, cells were stained with 0.5 mL of Hoechst 33258 staining (Beyotime) for 5 min. After washing twice with PBS, stained nuclei were observed under an IX71SIF-3 fluorescence microscope by using 350 nm excitation and 460 nm emission.
TdT-UTP nick end labeling (TUNEL) assays were performed with colorimetric TUNEL apoptosis assay kit according to the manufacturer's instructions (Beyotime). Cells grown in 6 well culture clusters were treated as mentioned in mitochondrial depolarization assay. In short, Bcap-37 cells grown in 6-well tissue culture plates were washed with PBS and fixed in 4% paraformaldehyde for 40 min. After washing once with PBS, cells were permeabilized with immunol staining wash buffer (Beyotime) for 2 min on ice. Cells were washed once with PBS again and incubated in 0.3% H2O2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases after which the cells were washed for three times with PBS. Thereafter, the cells were incubated with 2 μ L of TdT-enzymea and 48 μ L of Biotin-dUTP per specimen for 60 min at 37°C. After termination for 10 min, cells were incubated with streptavidin-HRP (50 μ L per specimen) conjugate diluted at 1:50 in diluent of streptavidin-HRP for 30 min. After washing three times with PBS, cells were incubated with diaminobenzidine (DAB) solution (200 μ L per specimen) for 10 min. This was again followed by washing with PBS for two times and the result was imaged under an XDS-1B inverted biological microscope (Chongqing photoelectric device CO.).
Flow cytometry analysis
Prepared PC3 cells (1×106/mL) were washed twice with cold PBS and then re-suspended gently in 500 μ L binding buffer. Thereafter, cells were stained in 5 μ L Annexin V-FITC and shaked well. Finally, 5 μ L PI was added to these cells and incubated for 20 min in a dark place, analyzed by FACS Calibur, Becton Dickinson.
All statistical analyses were performed with SPSS 10.0. Data were analyzed by one-way analysis of variance (ANOVA). Mean separations were performed using the least significant difference method (LSD test). Each experiment had three replicates and all experiments were run three times with similar results. Measurements from all the replicates were combined and treatment effects analyzed.
Results and Discussion
Compound 1, white acerate crystal; m.p. 136~137°C; molecular formula: C29H50O; 1H NMR(CDCl3, 500 MHz) δ: 5.35 (1H, d, J = 2.6 Hz, H-6), 3.50~3.46 (1H, m, H-3), 0.68~2.29 (continuous peaks); 13C NMR(CDCl3, 125 MHz) δ: 140.8 (C-5), 121.8 (C-6), 71.9 (C-3), 56.8 (C-14), 56.1 (C-17), 50.2 (C-9), 45.9 (C-4), 42.4 (C-13), 39.9 (C-12), 37.3 (C-1), 36.5 (C-10), 36.2 (C-20), 32.0 (C-22), 34.0 (C-7), 32.0 (C-8), 31.8 (C-2), 29.2 (C-24), 28.3 (C-25), 28.2 (C-16), 26.2 (C-28), 24.4 (C-15), 23.2 (C-27), 21.2 (C-11), l9.9 (C-26), l9.5 (C-19), 19.1 (C-23), 18.9 (C-21), 12.1 (C-29), 11.9 (C-18). As analyzed above, it was identified as β-sitosterol .
Compound 2, colorless crystal; m.p. 234~237°C; molecular formula: C21H20O8; 1H NMR (CD3OD, 500 MHz) δ: 7.18 (1H, s, H-5), 6.49 (1H, s, H-8), 6.43 (2H, s, H-2', H-6'), 5.97 ( 2H, dd, J = 0.9 Hz, J = 1.0 Hz, OCH2O), 4.78 (1H, d, J = 4.8 Hz, H-4), 4.52 ~4.40 (2H, m, H-1, H-3aα), 4.12 (1H, t, J = 9.6 Hz, H-3aβ), 3.70 (6H, s, 3', 5' -OCH3 ), 3.02 (1H, dd, J = 9.5 Hz, J = 2.5 Hz, H-2), 2.84 (1H, m, H-3); 13C NMR (CD3OD, 125 MHz) δ: 174.4 (C-2a), 147.3 (C-3'), 147.3 (C-5'), 147.0 (C-6 ), 147.0 (C-7), 135.2 (C-1'), 135.0 (C-4), 131.7 (C-9), 131.3 (C-10), 109.3 (C-8), 109.1 (C-6'), 109.1 (C-2'), 106.5 (C-5), 101.3 (OCH2O), 71.8 (C-3a), 71.0 (C-4), 55.9 (3', 5'-OMe), 44.8 (C-2), 44.0 (C-1), 40.7 (C-3). As analyzed above, it was identified as 4'-demethyl- podophyllotoxin .
Compound 3, yellow powder; m.p. 281~283°C; molecular formula: C15H10O6; 1H NMR (CD3OD, 500 MHz) δ: 12.05 (1H, s, 5-OH), 8.03 (2H, d, J = 9.2 Hz, H-2', H-6' ), 6.90 (2H, d, J = 9.2 Hz, H-3', 5'), 6.40 (1H, d, J = 1.8 Hz, H-8), 6.14 (1H, d, J = 2.3 Hz, H-6); 13C NMR (CD3OD, 125 MHz) δ: 175.8 (C-4), 164.1 (C-7), 161.3 (C-5), 159.3 (C-4'), 157.0 (C-9), 146.2 (C-2), 135.8 (C-3), 129.6 (C-6'), 129.6 (C-2'), 122.5 (C-1'), 115.5 (C-3'), 115.4 (C-5), 103.3 (C-10), 98.3 (C-6), 93.7 (C-8). As analyzed above, it was identified as kaempferol .
Compound 4, white acerate crystal; m.p. 228~230°C; molecular formula: C28H32O13; 1H NMR (DMSO-d 6 , 500 MHz) δ: 7.29 (1H, s, H-5), 6.58 (2H, s, H-2', H-6'), 6.09 (1H, s, H-8), 5.92 (2H, d, J = 5.2 Hz, OCH2O), 4.68 (1H, d, J = 8.6 Hz, H-4), 4.62 (1H, d, J = 8.6 Hz, H-3aα), 4.48 (1H, d, J = 4.0 Hz, H-3aβ), 3.97 (1H, d, J = 6.9 Hz, H-1), 3.73 (6H, s, 3'-OMe, 5'-OMe), 3.66 (3H, s, 4'-OMe), 3.60 (1H, d, J = 3.2 Hz, H-2), 2.84~2.66 (1H, m, H-3), Glc: 5.06 (1H, d, J = 7.5 Hz, H-1''), 4.46 (1H, d, J = 4.6 Hz, H-3''), 4.40 (1H, dd, J = 6.8 Hz, J = 3.5 Hz, H-6''), 3.09~3.21 (4H, m, H-2'', H-4''~H-6''); 13C NMR (DMSO-d 6 , 125 MHz) δ:178.2 (C-2a), 153.3 (C-3', 5'), 146.7 (C-6), 146.2 (C-7), 138.7(C-1'), 136.5 (C-4'), 132.1 (C-9), 132.0 (C-10), 107.8 (C-5), 107.6 (C-8), 106.6 (C-2', 6'), 101.2 (OCH2O), 77.5 (C-4), 70.2 (C-11), 60.5 (4'-OCH3 ), 56.3 (3', 5'-OCH3 ), 43.9 (C-1), 44.1 (C-2), 41.9 (C-3), Glc: 104.1 (C-1''), 74.3 (C-2''), 77.3 (C-3'', 5''), 70.04 (C-4''), 61.6 (C-6''). As analyzed above, it was identified as picropodophyllotoxin-4-O- glucoside .
Compound 5, white powder; m.p. 154~156°C; molecular formula: C27H26O12; 1H NMR(CD3OCD3, 500 MHz) δ: 8.28 (1H, s, H-5), 7.11 (1H, d, J = 1.7 Hz, H-5'), 6.98 (1H, dd, J = 5.2 Hz, J = 2.3 Hz, H-8), 6.90 (1H, d, J = 1.8 Hz, H-2'), 6.88 (1H, dd, J = 7.5 Hz, J = 2.3 Hz, H-6'), 6.10 (2H, s, 3', 4'-OCH2O), 5.60 (2H, dd, J = 21.0 Hz, J = 2.3 Hz, H-3a), 4.94 (1H, d, J = 8 Hz, H-1''), 4.00 (3H, s, 6-OMe), 3.96 (1H, d, J = 6.3 Hz, H-6''), 3.74 (3H, s, 7-OMe), 3.69 (1H, d, J = 4 Hz, H-6''), 3.51~3.49 (4H, m, H-2''~H-5''); 13C NM R (CD3OCD3, 125 MHz) δ: 136.5 (C-1), 120.2 (C-2), 128.2 (C-3), 146.2 (C-4), 102.2 (C-5), 153.0 (C-6), 151.5 (C-7), 106.7 (C-8), 131.6 (C-9), 131.3 (C-10), 129.8 (C-1'), 111.7 (C-2'), 148.3 (C-3'), 148.4 (C-4'), 108.7 (C-5'), 124.6 (C-6'), 169.9 (C-2a), 68.1 (C-3a), 56.4 (6-OMe), 55.7 (7-OMe), 102.7 (OCH2O), 106.3 (C-1''), 73.4 (C-2''), 75.2 (C-3''), 71.4 (C-4''), 78.1 (C-5''), 62.8 (C-6''). As analyzed above, it was identified as cleistanthin-B .
Compound 6, yellow crystal; m.p. 196~198°C, molecular formula: C21H20O11; 1H NMR (CD3OD, 500 MHz) δ: 8.03 (2H, d, J = 8.6 Hz, H-2', 6'), 6.87 (2H, d, J = 8.6 Hz, H-3', 5'), 6.36(1H, d, J = 2.3 Hz, H-8), 6.17 (1H, d, J = 2.3 Hz, H-6), 5.23(1H, d, J = 7.4 Hz, H-1''); 13C NMR (CD3OD, 125 MHz) δ: 178.1 (C-4), 164.1 (C-7), 161.2 (C-5),160.2 (C-4'), 157.6 (C-2), 157.2 (C-9), 134.0 (C-3), 130.9 (C-2'), 130.9 (C-6'), 120.9 (C-1'), 114.7 (C-3'), 114.7 (C-5'), 104.2 (C-10), 98.7 (C-6), 93.5 (C-8), 102.7 (C-1''), 74.4 (C-2''), 77.0 (C-3''), 70.0 (C-4''), 76.7 (C-5''), 61.2 (C-6''). As analyzed above, it was identified as kaempferol-3-O-β-D-glucopyranoside .
Compound 7, white power; m.p. 140~142°C; molecular formula: C27H30O13; 1H NMR (CD3OD, 500 MHz) δ: 7.38 (1H, s, H-5), 6.40 (2H, s, H-2', H-6'), 5.94 (2H, dd, J = 1.2 Hz, J = 1.2 Hz, OCH2O), 5.05 (1H, d, J = 4.9 Hz, H-4), 4.69 (1H, dd, J = 7.7 Hz, J = 3.5 Hz, H-3aα), 4.54 (1H, d, J = 2.3 Hz, H-1), 4.39 (1H, d, J = 3.7 Hz, H-1''), 4.21 (1H, t, J = 9.5 Hz, H-3aβ), 3.90 (1H, d, J = 7.5 Hz, H-6''), 3.88 (1H, d, J = 7.5 Hz, H-6''), 3.72 (6H, s, 3', 5'-OMe), 3.35 (4H, m, H-2''~ 5''), 3.03 (1H, dd, J = 9.7 Hz, J = 2.6 Hz, H-2), 2.95 (1H, m, H-3); 13C NMR (CD3OD, 125 MHz) δ: 175.7 (C-2a), 147.7 (C-3', 5'), 147.2 (C-6, 7), 134.4 (C-4'), 132.1 (C-1'), 131.2 (C-10), 131.1 (C-9), 108.8 (C-8), 108.3 (C-2', 6'), 108.2 (C-5), 102.3 (C-1''), 101.2 (OCH2O), 79.0 (C-5''), 76.8 (C-3''), 73.8 (C-2''), 71.5 (C-4), 70.3 (C-3a), 70.2 (C-4''), 61.4 (C-6''), 55.5 (3', 5'-OCH3), 45.1 (C-2), 43.7 (C-1), 39.2 (C-3). As analyzed above, it was identified as 4'-demethylpodophyllotoxin-4-O-glucoside .
Compound 8, yellow crystal; m.p. 188~190°C; molecular formula: C21H20O12; 1H NMR (CD3OD, 500 MHz) δ: 7.61 (1H, d, J = 1.8 Hz, H-2'), 6.77 (1H, d, J = 8.6 Hz, H-5'), 6.49 (2H, dd, J = 2.3 Hz, J = 6.3 Hz, H-6'), 6.29 (1H, d, J = 1.7 Hz, H-8), 6.10 (1H, d, J = 2.3 Hz, H-6), 5.16 (1H, d, J = 7.5 Hz, H-1''), 3.10~3.63 (6H, m, H-2''~6''); 13C NMR (CD3OD, 125 MHz) δ: 178.1 (C-4), 165.3(C-7), 161.7 (C-5),157.6 (C-2), 157.2 (C-9), 148.5 (C-4'), 144.6 (C-3' ), 134.2 (C-3), 121.8 (C-6'), 121.7 (C-1' ), 116.1 (C-5'), 114.6 (C-2' ), 104.2 (C-10), 102.9 (C-1''), 98.7 (C-6), 93.4 (C-8), 77.1 (C-3''), 76.8 (C-2''), 74.4 (C-4''), 69.8 (C-5''), 61.2 (C-6''). As analyzed above, it was identified as quercetin-3-O-β-D-glucopyranoside .
Compound 9, white crystal; m.p. 225~228°C; molecular formula: C34H42O18; 1H NMR (DMSO-d 6 , 500 MHz) δ: 7.21 (1H, s, H-5), 6.65 (2H, s, H-2', H-6'), 5.95 (2H, d, J = 6.3 Hz, OCH2O), 5.83 (1H, s, H-8), 5.36 (1H, d, J = 2.6 Hz, H-1), 5.14 (1H, d, J = 5.2 Hz, H-3aβ), 4.71 (1H, d, J = 2.3 Hz, H-4), 4.07 (1H, d, J = 4.6 Hz, H-3aα), 3.76 (6H, s, 3', 5'-OMe), 3.68 (3H, s, 4'-OMe), 2.92 (1H, m, H-3), 3.20~3.18 (1H, m, H-2), Glc(inner): 5.13 (1H, d, J = 5.8 Hz, H-1'), 4.49 (1H, d, J = 7.5 Hz, H-2'), 3.90 (1H, t, J = 9.5 Hz, H-3'), 3.52~3.49 (1H, m, H-4'), 4.56~4.49 (1H, m, H-5'), 4.62 (1H, d, J = 10.3 Hz, H-6'), 4.44~4.33 (1H, m, H-6'); Glc (terminal): 4.81 (1H, d, J = 4.0 Hz, H-1''), 3.07 (1H, dd, J = 8.6 Hz, H-2''), 3.52~3.41 (1H, m, H-3''), 3.90 (1H, t, J = 9.5 Hz, H-4''), 3.42 (1H, m, H-5''), 4.57 (1H, d, J = 4.6 Hz, H-6''), 4.42~4.38 (1H, m, H-6''); 13C NMR (DMSO-d 6 , 125 MHz) δ: 178.4 (C-12), 153.4 (C-3',5'), 146.6 (C-6), 146.0 (C-7), 138.3 (C-1'), 136.4 (C-4'), 132.9 (C-10), 132.8 (C-9), 107.8 (C-8), 106.9 (C-2', 6'), 106.3 (C-5), 101.2 (OCH2O), 76.8 (C-4), 68.5 (C-11), 60.5 (4'-OCH3 ), 56.4 (3', 5'-OCH3); Glc (inner): 104.1 (C-1'), 73.9 (C-2'), 77.4 (C-3', 5'), 70.8 (C-4'), 69.3 (C-6'); Glc (terminal): 103.6 (C-1''), 74.2 (C-2''), 77.3 (C-3'', 5''), 70.4 (C-4''), 61.4 (C-6''). As analyzed above, it was identified as icropodophyllotoxin-4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside [23, 30].
Compound 10, yellow power; m.p. 306~308°C; molecular formula: C15H10O7; 1H NMR (CD3OD, 500 MHz) δ: 7.64 (1H, d, J = 2.3 Hz, H-2'), 7.53 (1H, dd, J = 8.6 Hz, J = 2.3 Hz, H-6'), 6.78 (1H, d, J = 8.6 Hz, H-5'), 6.28 (1H, d, J = 2.3 Hz, H-8), 6.08 (1H, d, J = 1.8 Hz, H-6); 13C NMR (CD3OD, 125 MHz) δ: 175.9 (C-4), 164.3 (C-7), 161.2 (C-5), 156.9 (C-9), 147.4 (C-2), 146.6 (C-3'), 144.9 (C-4'), 135.9 (C-3), 122.8 (C-1'),120.3 (C-6'), 114.9 (C-5'), 114.6 (C-2'), 103.1 (C-10), 97.9 (C-6), 93.1 (C-8). As analyzed above, it was identified as quercetin [31, 32].
Compound 11, white power; m.p. 297~300°C; molecular formula: C35H60O6; 1H NMR(Pyridine-d 5, 500 MHz) δ: 5.35 (1H, s, H-6), 5.06~5.07 (1H, m, H-1'), 2.12~4.60 (10H, m, GluH), 0.85~1.13 (18H, m, H-CH3), 0.66 (3H, s, H-29); 13C NMR (Pyridine-d 5, 125 MHz) δ: 139.0 (C-5), 120.2 (C-6), 100.8 (C-1'), 77.0 (C-5'), 76.9 (C-3'), 76.4 (C-3), 73.7 (C-2'), 70.0 (C-4'), 61.2 (C-6'), 55.2 (C-14), 55.1 (C-17), 54.6 (C-9), 54.4 (C-24), 49.7 (C-4), 48.7 (C-13), 44.4 (C-12), 40.8 (C-1), 39.1 (C-10), 38.3 (C-20), 38.1 (C-22), 37.7 (C-7), 35.8 (C-8), 35.3 (C-2), 34.7 (C-25), 32.5 (C-16), 30.5 (C-23), 30.4 (C-15), 28.7 (C-28), 27.8 (C-11), 27.6 (C-19), 26.9 (C-21), 24.7 (C-27), 24.0 (C-26), 22.8 (C-29), 16.0 (C-18). As analyzed above, it was identified as daucosterol .
Compound 12, colorless acerate crystal; m.p. 183~185°C; molecular formula: C22H20O8; 1H NMR(CD3COCD3, 500 MHz) δ: 7.43 (1H, s, H-5), 6.83 (1H, s, H-8), 6.49 (2H, s, H-2', 6'), 6.15 (2H, s, OCH2O), 4.91 (1H, d, J = 4.0 Hz, H-1), 4.49 (1H, t, J = 7.7 Hz, H- 3aα ), 4.34 (1H, t, J = 9.5 Hz, H-3aβ), 3.70 (6H, s, 3', 5'-OM e), 3.68 (3H, s, 4'-OMe), 3.65~3.49 (1H, m, H-3), 3.63 (1H, d, J = 3.5 Hz, H-2); 13C NMR (CD3COCD3, 125 Hz) δ: 192.2 (C-4 ), 173.1 (C-2a ), 153.1 (C-5'), 153.1 (C-7), 153.0 (C-3'), 148.0 (C-6), 141.9 (C-9), 137.7 (C-4'), 133.1 (C-1'), 128.6 (C-10), 109.6 (C-8), 108.1 (C-2'), 108.1 (C-6'), 105.0 (C-5), 102.7 (-OCH2O-), 66.6 (C-3a ), 59.6 (4'-OMe), 55.5 (3', 5'-OMe), 45.8 (C-2), 44.6 (C-1), 43.4 (C-3). As analyzed above, it was identified as podophyllotoxone [34, 35].
Compound 13, colorless crystal; m.p. 200~202°C; molecular formula: C8H8O4; 1H NMR (CD3COCD3, 500 MHz) δ: 7.60 (1H, dd, J = 8.0 Hz, J = 1.7 Hz, H-6), 7.57 (1H, d, J = 1.7 Hz, H-2), 6.92 (1H, d, J = 8.0 Hz, H-5), 3.89 (3H, s, OCH3); 13C NMR(CD3COCD3, 125 MHz) δ: 166.7 (C = O), 152.0 (C-3), 147.2 (C-4), 124.0 (C-6), 122.0 (C-1), 114.6 (C-5), 112.6 (C-2), 55.4 (-OCH3). As analyzed above, it was identified as vanillic acid .
Compound 14, colorless power; m.p. 238~240°C; molecular formula: C21H20O7; 1H NMR (CD3COCD3, 500MHz) δ : 6.75 (1H, s, H-5), 6.52 (1H, s, H-8), 6.39 (2H, s, H-2', H-6'), 5.96 (2H, d, J = 2.9 Hz, OCH2O), 4.55 (1H, d, J = 5.2 Hz, H-1), 4.43 (1H, dd, J = 8 Hz, J = 6.9 Hz, 3a-α H ), 3.97 (1H, dd, J = 10.3 Hz, J = 8 Hz, 3a-β H ), 3.69(6H, s, 3'-OM e, 5'-OM e), 3.12~3.09 (1H, m, 4-α H), 2.86~2.76 (3H, m, 4-β H, H-2, H-3); 13C NMR (CD3COCD3, 125 MHz) δ: 174.8 (C-2a), 147.1 (C-3'), 147.1 (C-5'), 147.0 (C-6), 146.6 (C-7), 131.8 (C-1'), 131.8 (C-4'), 131.4 (C-9), 129.3 (C-10), 110.1 (C-8), 108.8 (C-5), 108.4 (C-2'), 108.2 (C-6'), 101.2 (-OCH2O-), 71.6 (C-3a), 55.8 (3'-OMe, 5'-OMe), 46.9 (C-2), 43.6 (C-1), 32.9 (C-4), 32.6(C-3). As analyzed above, it was identified as 4'-demethyldeoxypodophyllotoxin .
Compound 15, colorless cubic crystal; m.p. 164~166°C; molecular formula: C12H22O11; 1H NMR (D2O, 500 MHz) δ: 5.37 (1H, d, J = 4 Hz, H-1), 4.17 (1H, d, J = 8.6 Hz, H-3'), 4.00 (1H, t, J = 8.6 Hz, H-5'), 3.83~3.76 (1H, m, H-4'), 3.78~3.71 (5H, m, H-6, 6', 4'), 3.70 (1H, dd, J = 9.5 Hz, H-5 ), 3.63(2H, s, H-1'), 3.50 (1H, dd, J = 3.8 Hz, H-3), 3.41 (1H, t, J = 10 Hz, H-4); 13C NMR (D2O, 125 MHz) δ: 103.7 (C-2'), 92.2 (C-1), 81.4 (C-3'), 76.3 (C-4'), 73.9 (C-5'), 72.5 (C-5), 72.4 (C-3), 71.1(C-2), 69.2 (C-4), 62.3 (C-1'), 61.0(C-6'), 60.3 (C-6). As analyzed above, it was identified as sucrose .
Growth inhibition effect of various constituents of D. versipellis on different cells
Compound (20 μ M)
Growth inhibition (%)
14.7 ± 8.5
10.9 ± 10.4
19.5 ± 9.3
1.7 ± 9.3
60.5 ± 5.2**
56.7 ± 9.1**
56.9 ± 12.4
60.1 ± 6.1**
43.9 ± 11.9**
53.2 ± 10.9**
53.4 ± 14.9
39.2 ± 8.2**
9.0 ± 9.9
7.4 ± 9.3
24.6 ± 11.3**
6.4 ± 8.8
23.0 ± 6.4**
29.9 ± 10.8**
13.2 ± 8.7
23.0 ± 7.9**
19.8 ± 7.1**
21.9 ± 6.5**
14.5 ± 8.0
6.5 ± 7.7
11.6 ± 9.1
15.2 ± 10.1
40.7 ± 11.9**
5.1 ± 9.9
10.2 ± 8.1
23.9 ± 10.2
23.6 ± 10.7**
2.2 ± 8.8
29.8 ± 8.3
39.0 ± 13.3**
22.1 ± 4.3*
8.1 ± 5.0
52.0 ± 5.6**
42.1 ± 6.3**
47.9 ± 8.1
43.7 ± 6.2**
29.2 ± 3.9**
30.3 ± 10.9**
28.5 ± 12.3
10.2 ± 5.0*
67.1 ± 3.4**
56.6 ± 12.3**
60.7 ± 9.8**
59.8 ± 3.5**
93.2 ± 1.6
90.6 ± 1.2**
98.3 ± 4.0**
97.5 ± 1.7**
To determine whether the growth inhibitory activity of PTO and DDPT were related to the induction of apoptosis, the morphological character changes of PC3 and Bcap-37 cells were investigated using the AO/EB staining and Hoechst 33258 staining under fluorescence microscopy.
TUNEL assay was further carried out to confirm the cell apoptosis inducing activities of PTO and DDPT.
In summary, these results indicated that inhibitive effects observed in response to PTO and DDPT were associated with induction of apoptotic cell death.
Dysosma versipellis (Hance) M. Cheng, an important medicinal plant species, is considered 'endangered' by the China Species Red List and has been considered as vulnerable by the IUCN due to its rapid decline [42, 43]. Therefore, studies on the chemical constituents from D. versipellis and their biological activities have assumed significance for the rational development and utilization of this plant. In our study, fifteen compounds were extracted and identified from D. versipellis grown in Guizhou province, and the cell growth inhibition effects of these constituents on PC3, Bcap-37, and BGC-823 cells were carried out by MTT assay. Among these compounds, PTO, which was extracted from D. versipellis for the first time, together with DDPT showed potent activities on PC3, Bcap-37, and BGC-823 cells in a dose-dependent manner. And the IC50 values of PTO and DDPT on three cell lines were (17.8±1.0) μ M, (21.1±1.8) μ M, (19 ±1.6) μ M and (10.6±1.5) μ M, (13.2±0.5) μ M, (11.5±0.6) μ M, respectively.
The apoptosis inducing activities of PTO and DDPT on PC3 and Bcap-37 cells were investigated through AO/EB staining, Hoechst 33258 staining, TUNEL and flow cytometry analysis assay. The results demonstrated that PTO and DDPT from D. versipellis have potential to be employed in adjuvant therapy for treating human prostate and breast tumors. Further studies of the specific mechanisms of these compounds on human malignant tumors are currently underway.
The authors wish to thank the National Key Program for Basic Research (No. 2010CB126105) and the National Basic Research Preliminary Program of China (Grant 2010CB134504) and the National Natural Science Foundation of China (No. 20872021) for the financial support.
- Scheck AC, Perry K, Hank NC, Clark WD: Anticancer activity of extracts derived from the mature roots of Scutellaria baicalensis on human malignant brain tumor cells. BMC Complement Altern Med 2006, 6: 27. 10.1186/1472-6882-6-27PubMed CentralView ArticlePubMedGoogle Scholar
- Mousseau M, Schaerer R, Pasquier B, Chauvin C, Nissou MF, Chaffanet M, Plantaz D, Benabid A: A study of the expression of four chemoresistance-related genes in human primary and metastatic brain tumours. Eur J Cancer 1993, 29: 753–759. 10.1016/S0959-8049(05)80361-2View ArticleGoogle Scholar
- Huncharek M, Mcgarry R, Kupelnick B: Impact of intravesical chemotherapy on recurrence rate of recurrent superficial transitional cell carcinoma of the bladder: results of a meta-analysis. Anticancer Res 2001, 21: 765–769.PubMedGoogle Scholar
- Shibata MA, Hasegawa R, Kurata Y, Yamada M, Tamano S, Fukushima S: Bladder epithelial hyperplasia in F344 rats after intravesical instillation of the antitumor chemotherapeutic agents Adriamycin ® and mitomycin C. Cancer Lett 1990, 49: 41–49. 10.1016/0304-3835(90)90137-MView ArticlePubMedGoogle Scholar
- Walker MC, Masters JR, Parris CN, Hepburn PJ, English PJ: Intravesical chemotherapy: in vitro studies on the relationship between dose and cytotoxicity. Urol Res 1986, 14: 137–140.PubMedGoogle Scholar
- Panichakul T, Intachote P, Wongkajorsilp A, Sripa B, Sirisinha S: Triptolide sensitizes resistant cholangiocarcinoma cells to TRAIL-induced apoptosis. Anticancer Res 2006, 26: 259–265.PubMedGoogle Scholar
- Bemis DL, Capodice JL, Anastasiadis AG, Katz AE, Buttyan R: Zyflamend ® , a unique herbal preparation with nonselective COX inhibitory activity, induces apoptosis of prostate cancer cells that lack COX-2 expression. Nutr Cancer 2005, 52: 202–212. 10.1207/s15327914nc5202_10View ArticlePubMedGoogle Scholar
- Wang XJ, Wei YQ, Yuan SL, Liu GJ, Lu YR, Zhang J, Wang WD: Potential anticancer activity of tanshinone IIA against human breast cancer. Int J Cancer 2005, 116: 799–807. 10.1002/ijc.20880View ArticlePubMedGoogle Scholar
- Wang GF, Guo YW, Feng B, Li L, Huang CG, Jiao BH: Tanghinigenin from seeds of Cerbera manghas L. induces apoptosis in human promyelocytic leukemia HL-60 cells. Environ Toxicol Phar 2010, 30: 31–36. 10.1016/j.etap.2010.03.012View ArticleGoogle Scholar
- Chen Z, Liu YM, Yang S, Song BA, Xu GF, Bhadury PS, Jin LH, Hu DY, Liu F, Xue W, Zhou X: Studies on the chemical constituents and anticancer activity of Saxifraga stolonifera (L) Meeb. Bioorg Med Chem 2008, 16: 1337–1344. 10.1016/j.bmc.2007.10.072View ArticlePubMedGoogle Scholar
- Damayanthi Y, Lown JW: Podophyllotoxins: current status and recent developments. Curr Med Chem 1998, 5: 205–252.PubMedGoogle Scholar
- Stähblin H: Activity of a new glycosidic lignan derivative (VP 16–213) related to podophyllotoxin in experimental tumors. Eur J Cancer 1973, 9: 215–221.View ArticleGoogle Scholar
- Stähblin H: 4'-Demethyl-epipodophyllotoxin thenylidene glucoside (VM 26), a podophyllum compound with a new mechanism of action. Eur J Cancer 1970, 6: 303–306.View ArticleGoogle Scholar
- VanVliet DS, Tachibana Y, Bastow KF, Huang ES, Lee KH: Antitumor agents. 207. 1 Design, synthesis, and biological testing of 4 β -anilino-2-fluoro-4'-demethylpodophyll- otoxin analogues as cytotoxic and antiviral agents. J Med Chem 2001, 44: 1422–1428. 10.1021/jm000377fView ArticlePubMedGoogle Scholar
- Castro MA, del Corral JMM, Gordaliza M, García PA, Gómez-Zurita MA, García-Grávalos MD, de la Iglesia-Vicente J, Gajate C, An F, Mollinedo F, Feliciano AS: Synthesis and biological evaluation of new selective cytotoxic cyclolignans derived from podophyllotoxin. J Med Chem 2004, 47: 1214–1222. 10.1021/jm030978hView ArticlePubMedGoogle Scholar
- Jiang RW, Zhou JR, Hon PM, Li SL, Zhou Y, Li LL, Ye WC, Xu HX, Shaw PC, But PPH: Lignans from Dysosma versipellis with inhibitory effects on prostate cancer cell lines. J Nat Prod 2007, 70: 283–286. 10.1021/np060430oView ArticlePubMedGoogle Scholar
- Yu PZ, Wang LP, Chen ZN: A new podophyllotoxin-type lignan from Dysosma versipellis var. tomentosa. J Nat Prod 1991, 54: 1422–1424. 10.1021/np50077a034View ArticleGoogle Scholar
- Shang MY, Xu LS, Li P, Xu GJ, Wang YX, Cai SQ: Study on pharmacodynamics of Chinese herbal drug Guijiu and its lignan. Chin Tradit Herb Drugs 2002, 33: 722–724.Google Scholar
- Wu ZH, Zhao YM, Zhong SG, Li CT, Sun YW, Li WH: Proliferation inhibiting and apoptosis inducing on HL-60 by podophyllotoxone. Chin J Pract Chin Modern Med 2002, 2: 568–569.Google Scholar
- Yin ML, Chen ZL, Gu ZS, Xie YX: Separation and identification of chemical constituents of Dysosma majorense . Acta Botan Sin 1990, 32: 45–48.Google Scholar
- You YJ, Kim Y, Nam NH, Ahn BZ: Antitumor activity of unsaturated fatty acid esters of 4'-demethyldeoxypodophyllotoxin. Bioorg Med Chem Lett 2003, 13: 2629–2632. 10.1016/S0960-894X(03)00558-4View ArticlePubMedGoogle Scholar
- Xie XY, Gong JY, Wang LY, Zhang ZJ, Yu CY: Chemical constituents of the root of Rosa davurica pall. Lishizhen Med Mater Med Res 2009, 20: 366–367.Google Scholar
- Liao MC, Wang YW, Tu ZB, Xiao PG: Study on constituents of Dysosma tsayuensis Ying. J Wuhan Bot Res 2002, 20: 71–74.Google Scholar
- Hu XL, Zhu H, Liu CR, Tu PF: Study on the chemical constituents of the flowers of Impatiens balsamina L. Chin Tradit Patent Med 2003, 25: 833–834.Google Scholar
- Shang MY, Cai SQ, Li P: Study on constituents of Dysosma furfuracea Bao. Chin Tradit Herb Drugs 2000, 31: 412–414.Google Scholar
- AL-Abed Y, Sabri S, Zarga MA: Chemical constituents of the flora of Jordan, partv-b. Three new arylnaphthalene lignan glucosides from Haplophyllum buxbaumii . J Nat Prod 1990, 53: 1152–1161. 10.1021/np50071a003View ArticleGoogle Scholar
- Zhou YJ, Xu SX, Sun QS, Zhang PC, Wu LJ: A study on the chemical constituents of Quercus engleriana Seem. J Shenyang Pharm Univ 2000, 17: 263–266.Google Scholar
- Broomhead AJ, Dewick PM: Tumour-inhibitory aryltetralin lignans in Podophyllum versipelle, Diphylleia cymosa and Diphylleia grayi . Phytochemistry 1990, 29: 3831–3837. 10.1016/0031-9422(90)85342-DView ArticleGoogle Scholar
- Zhang HJ, Chen YG, Huang R: Study on flavonoids from Hedyotis diffusa Willd. J Chin Med Mater 2005, 28: 285–287.Google Scholar
- Zhu PF, Wang YS, Zhao JF, Zhang HB: Study on lignans of Dysosma versipellis . J Yunnan Univ 2006, 28: 521–525.Google Scholar
- Yu DQ, Yang JS, Xie JY: Analytical Chemistry Handbook. Bei Jing: Chemical Industry Press; 1989.Google Scholar
- An Q, Yang CJ, Song Y, Yu K, Xiong ZL, Li FM: Studies on the chemical constituents of the fruit of Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem. Nat Prod Res Dev 2008, 20: 765–769.Google Scholar
- Ma ZW, He GF: Studies on chemical constituents of Emmenopterys henryi Oliv. Native to China. J Integr Plant Biol 1989, 31: 620–625.Google Scholar
- Ma C, Yang JS, Luo SR: Study on lignans from Diphylleia sinensis . Acta Pharmacol Sin 1993, 28: 690–694.Google Scholar
- Jackson DE, Dewick PM: Aryltetralin lignans from Podophyllum hexandrum and Podophyllum peltatum . Phytochemistry 1984, 23: 1147–1152. 10.1016/S0031-9422(00)82628-XView ArticleGoogle Scholar
- Shen XJ, Ge RL, Wang JH: Chemical constituents from Choerospondias axillaris (Roxb.) Burtt. et Hill. J Henan Univ (Med Sci) 2009, 28: 196–199.Google Scholar
- Shaari K, Waterman PG: Podophyllotoxin-type lignans as major constituents of the stems and leaves of c asearia clarkei . J Nat Prod 1994, 57: 720–724. 10.1021/np50108a006View ArticleGoogle Scholar
- Zhou L, Wang N, Miao F, Zhao HS, Tian P: Chemical consitituents of gentiana apiata N. E. Br. Chin J Org Chem 2004, 24: 1249–1252.Google Scholar
- Ronald H, Blum MD, Stephen K, Carter MD: Adriamycin, a new anticancer drug with significant clinical activity. Ann Intern Med 1974, 80: 249–259.View ArticleGoogle Scholar
- Nakaike S, Yamagishi T, Samata K, Nishida K, Inazuki K, Ichihara T, Migita Y, Otomo S, Aihara H, Tsukagoshi S: In vivo activity on murine tumors of a novel antitumor compound, N - β -dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a] phenazine-6-carboxamide sodium salt (NC-190). Cancer Chemoth Pharm 1989, 23: 135–139.Google Scholar
- Xie YM, Deng Y, Dai XY, Liu J, Ouyang L, Wei YQ, Zhao YL: Synthesis and Biological Evaluation of Novel Acenaphthene Derivatives as Potential Antitumor Agents. Molecules 2011, 16: 2519–2526. 10.3390/molecules16032519View ArticlePubMedGoogle Scholar
- Wang S, Xie Y: China Species Red List. Beijing: Higher Education Press; 2004.Google Scholar
- Guan BC, Qiu YX, Fu CX: Isolation and characterization of microsatellite markers in Dysosma versipellis (Berberidaceae), a rare endemic from China. Conserv Genet 2008, 9: 783–785. 10.1007/s10592-007-9393-0View ArticleGoogle Scholar
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