Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 239-242

Effects of Dioscin Extracted from Polygonatum Zanlanscianense  Pamp on Several Human Tumor Cell Lines*

WANG Zhao ** , ZHOU Jiangbing , JU Yong †,  YAO Shenqing , ZHANG Hongjun

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Key Laboratory of Bioorganic Phosphorns Chemistry, Ministry of Education, Department of Chemistry,  Tsinghua University, Beijing 100084, China

* Supported  partly by the Tsinghua University - Hong Kong Baptist University Joint Institute for Research of Chinese Medicine and by the “985” Fund from Tsinghua University.  
 ** To  whom correspondence should be addressed, E-mail:  zwang@tsinghua.edu.cn  

Received: 2000-10-20

Code Number: ts01073

Abstract:   

Dioscin was extracted and isolated from Polygonatum Zanlanscianense Pamp. The effects of dioscin on HL60, HeLa, H14, and MDA-MB-435 cell lines were studied with the results showing that dioscin dramatically inhibited the growth of the MDA-MB-435, H14, HL60, and HeLa cell lines. The IC₅₀  of dioscin on these cell lines were 2.6, 0.8, 7.5, and 4.5 mmol/L respectively.

Key  words:  Thiazolyl blue (MTT); Polygonatum Zanlanscianense Pamp; dioscin

Introduction   

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 Polygonatumspecies have already been chemically studied and found to contain steroidal saponins[3-5]. However, there are no reports on the chemical properties of Polygonatum Zanlanscianense Pamp. The plant was found to be rich in oligofuranostanoside, which has been reported to inhibit growth of the human leukemia cell line HL60 in culture[6-7]. These observations gave rise to our examination of Polygonatum Zanlanscianense Pamp. In this study, a saponin (dioscin) was extracted and isolated from this plant and its effects on several tumor cell lines (Hela, HL60, H14, and MDA-MB-435) were studied. The results showed that dioscin significantly inhibited these tumor cell lines.

1 Materials and Methods   

1.1 Chemicals

RPMI 1640 medium, fetal bovine serum (FBS) was obtained from Gibco Laboratories (Grand Island, NY, USA). Thiazolyl blue (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

1.2 General

Melting points were uncorrected.   ¹ H NMR (400 MHz) and ¹³ C  NMR (100.16 MHz) spectra were recorded on a Bruker AM-400 NMR Instrument in pyridine-d₅ with TMS as the internal standard. Fast atom bombment-mass spectrometer (FAB-MS) was obtained on a ZAB-HS mass spectrometer with sulphur glycerol as the matrix.

1.3 Plant material

The plant roots were collected in Taibai County, Shaanxi Province, China (August, 1996), and identified as Polygonatum Zanlanscianense Pamp.  Voucher specimens (NWPZ-1996-08) were deposited in the Herbarium of the Biological Department in Northwest University, Xi'an, China.

1.4 Extraction and isolation

The dried root powder (300 g) was extracted with 95% EtOH at room temperature  three times. The combined extracts were concentrated in vacuo with the residue dissolved in H₂O and extracted successively with petroleum ether and n-BuOH. The n-BuOH layers were concentrated in vacuo to a yellow solid (35 g). Then the solid was subjected to a chromatographic column on silica gel with CHCl₃-MeOH-n-BuOH-H₂O (10 : 5 : 1 : 4) as the solvent system, and finally purified on a Sephadex LH-20 column with MeOH as the eluent solvent to provide compound 1 (1.0 g).

Compound 1 ((25R) spirost-5-en-3b-ol-3-O-[a-L-rhamnopyranosyl (1®2)] ] a-L-rhamnopyranosyl (1®4)] -b-D-glucopyranoside, dioscin)  Colorless needle, mp 294-295 °C. IR u^KBr (cm^-1 ): 3430(OH), 983, 918, 898, 867 (intensity, 918<898, 25R spirostanol), 839, 812 (D⁵). FAB-MS m/z: 891 [M+Na] ⁺, 875 [M+Li] ⁺.   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, H-21), 5.31(1H, m, H-6), 4.93(1H, d, J=6.5 Hz, Glc-H-1), 5.80 (1H, br, 4-Rha-H-1), 6.35 (1H, br, 4-Rha-H-1).  ¹³ C NMR (125.7 MHz, pyridine-d₅ ) aglycone , d: 37.6 (C-1), 30.2 (C-2), 78.2 (C-3), 39.1 (C-4), 141.0 (C-5), 121.8 (C-6), 32.4 (C-7), 31.8 (C-8), 50.5 (C-9),  37.2  (C-10), 21.2 (C-11), 39.3 (C-12), 40.0 (C-13), 56.8 (C-14), 32.3 (C-15), 81.6 (C-16), 63.1 (C-17), 16.4 (C-18), 19.5 (C-19), 42.1 (C-20), 15.0 (C-21), 109.3 (C-22), 31.9 (C-23), 29.4 (C-24), 30.7 (C-25), 67.0 (C-26), 17.3 (C-27), C-3 Sugar, Glu d: 100.3 (C'-1),  78.9  (C'-2), 76.9 (C-3), 78.2 (C'-4), 77.9 (C'-5), 61.4 (C'-6), 2'-Rha, d: 102.2 (C"'-1), 72.5 (C"'-2), 72.8 (C"'-3), 73.9 (C"'-4), 69.4 (C"'-5), 18.6 (C"'-6), 4'-Rha, d: 102.9 (C"-1), 72.5 (C"-2), 72.8 (C"-3), 74.1 (C"-4), 70.4 (C"-5),  18.6  (C"-6). On acid hydrolysis, compound 1 gave diosgenin as aglycone and D-glucose and L-rhamnose as sugar components.

1.5 Acid hydrolysis of dioscin on TLC plate and identification of monosaccharides

Compound 1 was hydrolyzed with HCl vapor on a TLC plate (80 °C water bath for 30-40 min), and then developed in CHCl₃-MeOH-H₂O (7 : 3 : 1, low layer) and compared with authentic samples (D-glucose, L-rhamnose) using aniline-phtalate as the detector for sugar.

1.6 Cell lines

HL60 (human leukemia cell line) was provided by the Beijing Normal University; HeLa (human cervical tumor line) and H14 (human lung tumor line) were provided by the Cell Biology Laboratory, Tsinghua University; and MDA-MB-435 (human mammary tumor cell line) was provided by the Marine Biology Laboratory, Tsinghua University. HL60 and MDA-MB-435 cell lines were cultured in RPMI-1640 supplemented with 10% newborn calf serum and 100 mg/mL streptomycin. The H14 cell line was cultured in DMEM supplemented with 10% newborn calf serum and 100 mg/mL streptomycin. HeLa cell line was cultured in RPMI-1640 supplemented with 8% newborn calf serum and 100 mg/mL streptomycin. The cell cultures were maintained at  37 °C  in a humidified incubator with an atmosphere of 95% air and 5% CO₂ .

1.7 MTT assay

The MTT assay was processed by the standard techniques. The cells were seeded to a concentration of 5x10⁴ cells/mL. Then various concentrations of dioscin were added to the suspension. 200 mL of these cell suspensions were then transferred to a 96-well microplate. Each concentration was repeated three times. The cells were incubated in a humidified atmosphere with 5% CO₂ for 4 days. Then 20 mL MTT  (0.01 mol/L) was added to every well. After 4 hours incubation (37 °C) and 5 min centrifuging (1000 r/min), the resulting formazan precipitate was dissolved with 150 mL DMSO and the absorption was measured at 550 nm on a microplate reader (Benchmark, BIO-RAD, USA). The growth inhibition was determined using:

Growth inhibition=(control's O.D. - sample's O.D.)/control's O.D.

2 Results   

2.1 Compound 1

Compound 1 was obtained in crystal form. The FAB mass spectrum gave [M+Na] ⁺ and ?M+Li] ⁺ at m/z 891 and 875, respectively. The molecular formula was determined to be C₄₅H₇₂O₁₆  by the FAB mass spectrum and   ¹³ C NMR spectral data. Its IR spectrum showed the characteristic absorption bands of a 25R-spirostanol moiety. On acid hydrolysis, compound 1 gave diosgenin as the aglycone (MS: m/z 414 [M] ⁺, TLC, mp and IR) and D-glucose and L-rhamnose as sugar residues by comparison with authentic samples. The   ¹³ C and   ¹ H NMR spectra revealed it is a 3-O-glycoside of diosgenin with two terminal a-L-rhamnopyranosyl units and an inner b-D-glucopyranoside unit. 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, compound 1 was concluded to be (25R) spirost-5-en-3b-ol-3-O-] a-L-rhamn-opyranosyl (1®2)] ] a-L-rhamnopyranosyl (1®4)] -b-D-glucopyranoside (dioscin)[8,9]  (Fig.1).

2.2 Inhibition induced by dioscin

When the four human tumor cell lines were treated with dioscin in the dose range of 0 to 30 mmol/L for 3 days, growth inhibition effect was observed in all cases. Table 1 summarizes the IC₅₀  values for the four tumor lines. The H14 cell line proved to be the most sensitive cell line, with an IC₅₀  value of 0.8 mmol/L. Dose-dependent growth inhibition effects were also observed in the four tumor cell lines (Fig.2).

3 Discussion  

Many natural compounds which can effectively inhibit tumor cell growth have been found, such as hypericin isolated from Hypericum Triquetrifolium[10]   and bestatin from Streptomyces Olivoreticuli [11]. In these compounds, a class of saponin or sapogenin extracted from the plant, such as saponin R1 from the roots of genistein[12]  and daidzein[13]  from soybean have generated wide interest. Now, many laboratories are intensively looking for compounds that will effectively inhibit tumor cell growth. In this work, dioscin was extracted and isolated from Polygonatum Zanlanscianense Pamp. The tests showed that dioscin had wide inhibition effects on several tumor cell lines, including MDA-MB-435, H14, HL60 and Hela. The experiments confirmed that dioscin cannot only induce HL60 cell differentiation mainly along the granulocytic lineage (Table 2), but also induced HL60 cell apoptosis (Fig.3). Results using standard DNA fragment assay showed that dioscin can induce MDA-MB-435, H14 and Hela cell apoptosis to some extent (data not shown). Further studies of the mechanisms of the dioscin effect on these tumor cells are in process.

References

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