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Biol. Pharm. Bull. 24(2) 159—162 (2001)
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Effects of Two Saponins Extracted from the Polygonatum Zanlanscianense Pamp on the Human Leukemia (HL-60) Cells Zhao WANG,*, a Jiangbing ZHOU,a Yong JU,*, b Hongjun ZHANG,a Mingjie LIU,a and Xue LIb Department of Biological Science and Biotechnology,a and Bioorganic Phosphorus Chemistry Laboratory,b Tsinghua University, Beijing, 100084 China. Received October 5, 2000; accepted October 25, 2000 Two saponins, methyl protodioscin and dioscin, were extracted from the root of Polygonatum Zanlanscianense Pamp. One of them, dioscin exerted significant inhibitory effects on the growth of the human leukemia cell HL-60, inducing differentiation and apoptosis. HL60 cells were induced mainly along the granulocytic lineage. In addition, we have found that dioscin affects many cancer cells. These studies may have important significance in treating related cancers. Key words dioscin; methyl protodioscin; Polygonatum Zanlanscianense Pamp; HL-60
Polygonatum Zanlanscianense Pamp grows in southeastern Gansu and southwestern Shaanxi, China. Its roots are used as a substitute for “Yuzhu”, a famous traditional Chinese medicine that has long been used in the treatment of lung illnesses, palpitation, upset stomach and diabetes.1,2) A literature survey showed that several Polygonatum species have already been chemically studied and found to contain steroidal saponins.3—5) However, there are no reported investigation of the chemical preperty of Polygonatum Zanlanscianense Pamp. The plant was found to be rich in oligofuranostanoside, what has been reported to inhibit growth of the human leukemia cell line HL-60 in culture.6,7) These observations gave rise to our examination of Polygonatum Zanlanscianense Pamp. In this study, two steroidal saponins were isolated from this plant and their effects on HL-60 cells were studied. MATERIALS AND METHODS Chemicals RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco Laboratories (Grand Island, NY, U.S.A.). Nitrobluetetrazolium (NBT), thiazolyl blue (MTT), 12-O-tetradecanoylphorbol 13-acetate (TPA), and trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). ApoAlert Annexin V-FITC Apoptosis Kit was purchased from CLONTECH Co. (U.S.A.). In situ Cell Apoptosis Detection Kit was purchased from the Sino-America Co. (China). General 1H-NMR (400 MHz) and 13C-NMR (100.16 MHz) spectra were recorded on a Bruker AM-400 NMR instrument in pyridine-d5 with TMS as the intended. Standard FAB-MS was obtained on ZAB-HS mass spectrometry and sulphur glycerol as matrix. Plant Material The plant roots were collected in Taibai County, Shaanxi Province, China (August, 1996) and identified as Polygonatum Zanlanscianense Pamp by Prof. C. Q. Zhao. Voucher (NWPZ-1996-08) specimens were deposited in the Herbarium of the Biological Department in Northwest University, Xi’an, China. Extraction and Isolation The root powder (300 g) of dried plants was extracted with 95% EtOH at room temperature three times. The combined extracts were concentrated in vacuo, and the resulting residue was dissolved in H2O, extracted with petrol and then extracted with n-BuOH. The ∗ To whom correspondence should be addressed.
combined n-BuOH layers were evaporated under reduced pressure to a yellow solid (35 g). The 5g solid was subjected to column chromatographic on silica gel with CHCl3– MeOH–n-BuOH–H2O (10 : 5 : 1 : 4) as the solvent system, and finally purified on Sephadex LH-20 column with MeOH as eluent solvent to provid compound 1 (4.3 g) and compound 2 (50 mg). Compound 2 ((25R)-spirost-5-en-3 b -ol-3-O-[ a - L rhamnopyranosyl(1→2)][a -L-rhamnopyranosyl(1→4)]-b -Dglucopyranoside, dioscin); Colorless needle crystal, mp 185—189 °C. n KBr cm21: 3430 (OH), 983, 918, 898, 867 (intensity, 918,898, 25R spirostanol), 839, 812 (D 5). FAB-MS m/z: 891 [M1Na]1, 875 [M1Li]1. 1H-NMR d : 0.80 (3H, s, H-18), 0.98 (3H, d, H-27), 1.03 (3H, s, H-19), 1.04 (3H, s, H21), 5.31 (1H, m, H-6), 4.93 (1H, d, J56.5 Hz, Glc H-1), 5.80 (1H, br, 4-Rha H-1), 6.35 (1H, br, 4-Rha H-1). 13CNMR data were shown in Table 1. On acid hydrolysis, 2 gave diosgenin as aglycone and D-glucose and L-rhamnose as sugar components. Compound 1 (26-b -D-glucopyranosyl-22-methoxy-(25R)furost-5-en-3b ,26-diol-3-O-[a -L-rhamnopyranosyl(1→2)][a L-rhamnopyranosyl(1→4)]-b -D-glucopyranoside, methyl protodioscin); Colorless needle crystal, mp 294—295 °C (decomposition), [a ]D 298 °C (MeOH, c50.2). IR n KBr cm21: 3600—3200 (OH)). FAB-MS m/z: 1085 [M1Na]1, 1069 [M1Li]1. 1H-NMR d : 0.81 (3H, s, H-18), 0.98 (3H, d, H27), 1.03 (3H, s, H-19), 1.17(3H, s, H-21), 3.25 (3H, s, OMe). 13C-NMR data were showed in Table 1. On acid hydrolysis, 1 gave diosgenin as aglycone and D-glucose and Lrhamnose as sugar components. Acid Hydrolysis on TLC Plate and Identification of Resulting Monosaccharides Compounds were hydrolyzed with HCl vapor on a TLC plate (80 °C water bath for 30— 40 min), followed by CHCl3–MeOH–H2O (7 : 3 : 1, low layer) as developing solution compared with authentic samples (Dglucose, L-Rhamnose) using aniline-phtalate as detector for sugar. Cell Culture HL-60 cells were obtained from the Department of Biology at Beijing Normal University. Cells were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin (100 m g/ml) at 37 °C in culture chamber containing 5% CO2. Determination of Growth Inhibition (1) Growth Curve: Cells were seeded at a concentration of 53104 cells/ml,
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© 2001 Pharmaceutical Society of Japan
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Table 1.
13
C-NMR Spectral Data of Compounds 1 and 2
c 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 OMe
Compound 1 37.4 30.0 78.0 38.8 140.7 121.7 32.2 31.6 50.2 37.0 21.0 39.2 40.7 56.5 32.1 81.2 64.1 16.2 19.3 40.4 16.2 112.6 30.8 28.1 34.1 75.1 17.1 47.2
3-O-Glc 1 2 3 4 5 6 2-Rha 1 2 3 4 5 6 4-Rha 1 2 3 4 5 6 26-O-Glc 1 2 3 4 5 6
Compound 2
100.0 78.6 76.7 78.0 77.7 61.2 101.8 72.3 72.5 73.6 69.3 18.5 102.7 72.3 72.6 73.9 70.2 18.4 104.7 74.9 78.3 71.5 78.2 62.7
37.6 30.2 78.2 39.1 141.0 121.8 32.4 31.8 50.5 37.2 21.2 39.3 40.0 56.8 32.3 81.6 63.1 16.4 19.5 42.1 15.0 109.3 31.9 29.4 30.7 67.0 17.3
3-O-Glc 1 2 3 4 5 6
100.3 78.9 76.9 78.2 77.9 61.4
2-Rha 1 2 3 4 5 6
102.2 72.5 72.8 73.9 69.4 18.6
4-Rha 1 2 3 4 5 6
102.9 72.5 72.8 74.1 70.4 18.5
incubated for 1—4 d with the two compounds at various concentrations. The vital cell number was counted every 24 h. Cell viability was determined by the trypan blue exclusion method. (2) MTT Assay: MTT assay was processed by the standard techniques. Cells were seeded to a concentration of 53104 cells/ml. Then various concentrations of dioscin were added to the suspension. 200 m l of these cells suspensions were then transferred to 96 well microplate. Every concentration was repeated three times. These cells were incubated in a humidified atmosphere with 5% CO2 for 4 d. Then 20 m l MTT (0.01 mol/l) was added to every well. After 4 h incubation (37 °C) and 5 min centrifuging (1000 rpm), the resulting formazan precipitate was dissolved with 150 m l DMSO and the absorption was measured at 550 nm on a microplate reader (Benchmark, Bio-Rad, U.S.A.). The growth inhibition was determined using: growth inhibition5(control’s O.D.2sample’s O.D.)/control’s O.D.
Differentiation Assay (1) NBT Reduction Test: The percentage of HL-60 cells capable of reducing NBT was determined by counting the number of cells which contained precipitated formazan particles after the cells were incubated with NBT (1.0 mg/ml) at 37 °C for 30 min. TPA (200 ng/ml) was used as a stimulator for the formation of formazan. (2) Phagocytosis Test: Cells (13106) were suspended in serum-free RPMI 1640 medium containing 0.2% latex particles (average diameter was 0.806 m m; Sigma Chemical) and incubated at 37 °C for 4 h. After incubation, the cells were washed once with phosphate-buffered saline (PBS). The cells containing more than 10 latex particles were scored as phagocytic cells.8)
Fig. 1.
The Structures of Dioscin and Methyl Protodioscin
Apoptosis Assay (1) Flow Cytometry Assay: Apoptosis cells were determined using an ApoAlert Annexin V-FITC Apoptosis Kit. All the operations were performed according to the User Manual. Briefly, about 13105—13106 cells were rinsed with binding buffer and were resuspended in 200 m l of binding buffer. Then 5 m l of Annexin V and 10 m l of Propidium Iodide was added to the suspension. The cells were incubated at room temperature for 5—15 min in the dark, then analyzed by flow cytometry (Coulter, Epics Elite , U.S.A.) at 488 nm. (2) In Situ Detection of DNA Cleavage by the TUNEL Procedure: Apoptotic cells were also determined using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Sino-America, China). Briefly, cells were fixed in freshly prepared 10% formaldehyde solution for 25 min at room temperature. After rinsing with PBS, the cells were permeated with 0.2% Triton X-100 and then incubated for 1 h at 37 °C with TdT and Biotin-IIdUTP to label the cleaved DNA. Then the cells were stained with DAB and hematoxylin solution. Cells showing a positive reaction (containing brown granules in the cell nucleus) were scored. RESULTS Compound 1 was obtained as crystals. It exhibited a pink spot with Ehrlich reagent,9) and its IR spectrum lacked the characteristic spirostanol absorption, indicating that it possessed a furostanol structure. The FAB mass spectral ion peaks at m/z 1085 [M1Na]1 and 1069 [M1Li]1 suggested its molecular formula to be C52H86O22. In its 1H-NMR spectrum, a signal at d 3.52 (s, 3H) is due to a methoxy group, the probable location of the methoxy group in the furostanol glycoside to be at C-22; it can be confirmed by the 13C-NMR data of C-22 at d 112.6. By enzymatic prosaponin, which was formed by splitting of the b -D-glucosidase, compound 1 produced dioscin as prosaponin, which was formed by splitting of the b -D-glucosidase from C-26 of 1. Finally, compound 1 was determined to be 26-b -D-glucopyranosyl-22methoxy-(25R)-furost-5-en-3b ,26-diol-3-O-[a -L-rhamnopyranosyl(1→2)][a -L-rhamnopyranosyl (1→4)]-b -D-glucopyranoside and was identical to methyl protodioscin reported by
February 2001
Hirai et al.10) (Fig. 1) Compound 2 was also obtained as crystals. The FAB mass spectrum gave [M1Na]1 and [M1Li]1 at m/z 891 and 875, respectively. The molecular formula was determined as C45H72O16 by the FAB mass spectrum and 13C-NMR spectral data. Its IR spectrum showed the characteristic absorption bands of a 25R-spirostanol moiety.11) On acid hydrolysis, 2 gave diosgenin as the aglycone (MS: m/z 414 [M]1, TLC, mp and IR) and D-glucose and L-rhamnose as sugar residues by comparison with authentic samples. The 13C- and 1H-NMR spectra revealed it is a 3-O-glycoside of diosgenin with two terminal a -rhamnopyranosyl units and an inner b -D-glucopy-
Fig. 2.
161
ranoside unit.11) According to the glycosylation shift effect, the two terminal rhamnoses were attached to the hydroxyl groups at the C-2 and C-4 positions of the inner glucose. From these results 2 was concluded to be (25R)-spirost-5-en3b -D-ol-3-O-[a -L-rhamnopyranosyl(1→2)][a -L-rha-mnopyranosyl (1→4)]-b -D-glucopyranoside (dioscin).12) Effect of Compounds on the Growth of HL-60 Cells Figures 2 to 4 illustrate the effects of the two compounds on the growth of HL-60 cells. Results show that only dioscin has inhibition effects on the HL-60 cells. The inhibition effects increases with increasing dioscin concentration. Differentiation of HL-60 Cells Induced by Dioscin NBT reduction and phagocytosis were monitored in HL-60 cells treated with different dioscin concentrations for 5 d. The
Methyl Protodioscin (j) and Dioscin (d) Effect on HL-60 Cell Fig. 4. Inhibition Effect on HL-60 Cells Treated by Dioscin with Concentration of 0 m M (r), 5 m M (.), 10 m M (m), 15 m M (d) and 20 m M (j).
Table 2. Induced of Differentiation Markers in HL-60 Cells After Treated with Dioscin for 5 d Compound Control Dioscin
ATRA Fig. 3.
Inhibition Effect of Dioscin on the Growth of HL-60 Cells
The MTT assay was processed after the cells treated with dioscin for 48 h.
Fig. 5.
Concentration (m M)
NBT reduction (%)
Phagocytosis (%)
— 5.0 10.0 15.0 0.5
8.761.2 27.361.5 37.762.1 45.062.6 47.364.2
4.860.8 10.362.1 13.062.6 24.064.0 19.763.2
ATRA: all trans-retinoic acid. Data indicate the percentage of cells which were reducible by NBT and took up over 10 latex particle. The data is expressed as the mean6 S.D. of 3 experiments.
DNA Fluorescence Flow Cytometry Histograms from HL60 Cells Treated with Dioscin
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percentage of NBT positive cells in HL-60 cells treated with dioscin at concentrations of 15 m M for 5 d was 45.0% (while 47.3% of the cells treated by ATRA at a concentration of the 0.5 m M and 8.7% of untreated HL-60 cells showed NBT positive). After exposure to 15 m M dioscin for 5 d, 24% of the cultured cells were phagocytic, while only 4.8% of the untreated cells were phagocytic (Table 2). Apoptosis of HL-60 Induced by Dioscin The FACS assay was monitored in HL-60 cells treated with 15 m M dioscin for 3 d. Dioscin induced apoptosis in the HL-60 cells (Fig. 4). 24.3% of the cells were stained only by Annexin V amounted to whereas only 4.4% of the untreated cells were stained (Fig. 4). The HL-60 cells treated with dioscin at concentrations of 15 m M was tested by the TUNEL method and the HL-60 apoptosis fractions was 21.0% (4% of control cells were positive).
Table 3.
DISCUSSION
Acknowledgements This research was supported partly by the Tsinghua University–Hongkong Baptist University Joint Institute for Research of Chinese Medicine.
Cancer differentiation treatment has become a new field of cancer therapy. Since dimethylsufoxide has been found to induce the differentiation of leukemia cells in vitro,13) many laboratories are intensively looking for effective inducers. The widely used model is the human leukemia cell HL-60.14) In this study, we extract and isolate two saponins from Polygonatum Zanlanscianense Pamp and demonstrate that only one of them, dioscin, can inhibit the growth of HL-60 cells while the other has no effects. These two compounds are similar, but methyl protodioscin possesses a furostanol with 26-O-glycopyranoside, so this structure of the spirostanol must play a critical function in the effects on the HL-60 cells. It was found that dioscin-treated HL-60 cells differentiated mainly along the granulocytic lineage, as judged by biochemical markers. Flow cytometry and TUNEL assay showed that treated cells were also induced to apoptosis. Apart from the function of inducting differentiation and apoptosis, dioscin also showed cytotoxin toward HL-60 at a high concentration (above 20 m M). HL-60 cells treated with dioscin at the concentration of 25 m M for 30 min caused 16% of the cells to show poor viability. While at a concentration of 15 m M, only 10% of the treated cells showed poor viability, about the same rate as that of ATRA-treated cells (7%). Cell viability was judged by the trypan exclusion method. So the dioscin induced differentiation, apoptosis and cytotoxin in
IC50 of Dioscin on Several Human Cancer Cell Lines Cell lines HL60 MDA-MB-435 HeLa H14
IC50 (m M) 7.5 2.6 4.5 0.8
the HL-60 cells. A recent study also showed that dioscin inhibited growth inhibition of human cervical cancer line Hela, human mammary cancer cell line MDA-MB-435 and human lung cancer line H14 in a dose-dependent manner. The IC50 of dioscin effecting those cells are indicated in Table 3. Those results indicated that this dioscin extract from Polygonatum Zanlanscianense Pamp probably has wide effects on cancer cells.
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