ELSEVIER
(3ancerLetters 104 ( 1996) 3 l-36
Anti-tumor activity of the crude saponins obtained from asparagus Yu Shao”.b, Chee-Kok China, Chi-Tang Hob,*, Wei Mac, Stephen A. Garrisona, Mou-Tuan Huangc “Depuronent @‘Plant Science. Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903, USA ‘Department of Food Science, Cook College, Rutgers, The State University @New Jersey, New Brunswick, NJ 08903, VSA ‘~Depurtmmt of Chemical Biology, laborutot?, for Cancer Research. College of Pharmacy, Rutgers, The State University ofNew Jersey, Piscatway. NJ 08855-0789, USA Received 28 February 1996; revision received 1I March 1996 ; accepted 11March1996
I______
Abstract
The crude saponins from the shoots (edible part of asparagus) of asparagus (asparagus crude saponins; ACS) were found !o have antitumor activity. The ACS inhibited the growth of human leukemia HL-60 cells in culture and macromolecular synthesis in a dose and time dependent manner. The ACS at 75lOO,@ml range was cytostatic. ACS concentrations greater than 200~g/ml were cytocidal to HL-60 cells. The ACS at 6 and SO@ml inhibited the synthesis of DNA, RNA and protein in HI,-60 cells by 41. 5. and 4. respectively. or by 84, 68 and 59%. respectively. The inhibitory effect of ACS on DNA synihcsis wa\ irrcvcrsihle.
Ka)~r)rci.s~ Asparagus; Vegetable: Saponin; Antitumor: Anti-leukemia .---.,_-.l_-.
1. introduction ~\sparaguc (Aspuragus r,@cinalis L) is a popular vegetable consumed in most parts of the world. Asparagus shoots which are frequently used in salads, vegetable dishes and soups are the edible part of the
plant. In Chinese traditional medicine this plant has been used as a tonic, antifebrile, antitussive, hairgrowth stimulator and diuretic agent [I]. In recent years the extracts have been demonstrated to possess certain biological activities including antifungal 121, antimutagenic [3]. diuretic 141,cytotoxic 151,antiviral
[6] and molluscicide [7] properties. The chemical constituents of this plant have been studied to some extent. The types of compounds so far reported include flavonoids [8], oligosaccharides [9], amino acid derivatives [lo], sulfur-containing acids [ 1I] and steroidal saponins [ 12,13 1, Among them saponins were shown to be the major constituents and suggested to be the active principle responsible for the biological activities of asparagus [ 131. In our preliminary test we found the methanol extract of the shoots to inhibit DNA synthesis. Subsequently, this crude extract was separated into ethyl acetate and nbutanol portions, and each portion was further examined using the same assay. The results showed that the active constituents
[‘JO4 783.5,%/S 12.00 6,’ 1996 Elsevier Science Ireland Ltd. All rights reserved !‘I: so?(:.5 1P?5(96)03?~7-4
were found in the n-Lutanol-
Y. Shno et al. /Cancer
32
Letters
soluble fraction which mainly contained saponins. In this paper, we report on the antitumor activity of the crude saponins 2. Materials and methods 2.1. Materials
[3H]Thymidine (SO,Ki&mol), [3H]uridine (55 ,Ki/ pmol) and [3H]leucine (lSO,Ki@mol) were purchased from Du Pont Chemical Company, NEN Reassert Products (Boston, MA). HL-60 cells were purchased from the American Type Cell Cultures (ATCC; Rockland, MD). RPM1 medium, fetal calf serum, trypsin, penicillin and streptomycin were purchased from GIBCO BRL (Grand Island, NY). Glass microfibre filters (2.5 cm in diameter), ScientiVerse, trichloroacetic acid and HPLC grade dimethyl sulfoxide (DMSO), ethyl acetate, methanol and n-butanol were purchased from Fisher Scientific (Springfield, NJ). Diaion HP20 was obtained from Mitsubishi Chemical Corporation (Tokyo, Japan).
104 (1996) 31-36
2.2. Preparation
of asparagus
crude saponins
Asparagus shoots (100 g), obtained from Cook College, Rutgers University Farm (New Brunswick, NJ) in May 1995, were air dried, cut into small pieces and percolated with methanol at room temperature for 6 days. This procedure was repeated three times (each time 800 ml). The methanol fractions were combined and concentrated under reduced pressure at 40°C. About 28 g of residue was obtained. Hot water (6O”C, 100 ml) was added to the residue, mixed and filtered. The aqueous solution was extracted with ethyl acetate and then with n-butanol, three times each. The butanol fractions were combined and loaded onto a Diaion HP-20 column (1.9 X 60 cm). The column was washed with 1 1 of deionized water and then eluted with 800 ml of methanol. The methanol was evaporated to dryness and 1.7 g of residue containing saponins (asparagus crude saponins; ACS) was obtained. This final preparation was used for the biological activity studies described in this paper. The flow chart for the preparation of asparagus crude
Air-dried asparagus shoots (100 g) cut in small pieces percolated with MeOH (3x800 ml). each 6 days concentrated under reduced pressure, 40 oC
I crude extract (18.1 g)
add hot water (100 ml) filter
I J-
aqueous solution
4
water insolube (discard)
extracted with EtOAc (3x100 ml)
i EtOAc extract (1.82 g) TFfi%yh
tiutanol
aqueous solution (discard)
(TOO
ml)
n-butanol extract column chromatography (1.9x60 cm) over Diaion HP-20 (100 g) eluted with H$ (1 L) and then MeOH (800 ml)
1 MeOH eluate (1.72 g. crude saponhs) Fig.
1.Flow chart for preparationof asparagus crude saponins.
HOH2C HO
HO
H:%iyMoz
011
21
00
RI=Me R1=H
011 Fig, 2. The chemical structure of two major compounds from the crude saponins of asparagus
saponins (ACS) is shown in Fig. 1. Two major saponins have been isolated from the ACS by HPLC and their chemical structures were identified by chemical and spectral data as shown in Fig. 2. Further large scale isolation of saponins is in progress. 2.3. Cultured HL-60 cells HI,-60 cells (5 X 10” cells per ml) were grown in 100 mm diameter culture dishes in RPM1 medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. Under these conditions, HL-60 cells had a doubling time of about 24-30 h dependent on the medium and calf serum being used. In order to maintain normal cell growth, cells usually were passed every 2-3 days. To measure the effect of the ACS on the growth of HL-60 cells, ACS at different concentrations were added to HL-60 cells (5 X IO” cells per ml) in a dish containing 20 ml of RPMI medium supplemented with 10% fetal calf serum and I % penicillin-streptomycin. Cultures were incubated at 37°C for 4 days. Cell number was counted daily using a hemacytometer under a microscope with 10x magnification every 24 h and recorded.
2.4. Measurement of DNA, RNA und protein synthesis in HL-60 cells HL-60 cells were harvested by centrifugation for 5 min at 1000 x g. Cells were resuspended in RPM1 without fetal calf serum and placed in a series of I3 x 100 mm test tubes (5 x lo5 cells/ml per tube). For measurement of DNA, RNA or protein synthesis, 3~1 of [‘Hlthymidine (50,Kilpmol), 5~1 of L3H]uridine (55 &i/pmol) or 10~1 of [3H]leucine (lSO,Ki/~mol) were added, respectively, to each individual tube followed by 2~1 of DMSO or inhibitor in DMSO. Tubes were incubated at 37°C for 120 min. The reaction was terminated by adding 2 ml of ice-cold phosphate buffer saline (PBS) solution and kept in an ice bath. The tubes were then centrifuged for 5 min at 1000 X R. The supernatant was discarded and cells were washed twice with PBS. Finally, cells were lysed in 2 ml of ice-cold deionized water and 2 ml of 10% trichloroacetate acid (TCA) solution. The precipitates were collected on Glassfibre filters and washed three times with 5% cold TCA solution. The dry glassfibre filters were placed in scintillation vials with 10 ml of ScientVarse fluid and radioactivity was determined in a Beckman LS 1701 scintillation counter.
34
Y. Shao et al. / Cuncer Letters
-
control
-
50 flg/m1
25-
zo-
-C
200
aglml
-
400
Jlgh1
15-
0
1
2
3
days
4
5
Fig. 3. Inhibitory effect of various concentrations of ACS on the growth of HL-60 cells. HL-60 cells (5 x lo5 cells/ml), suspended in RPM1 medium supplemented with 10% calf serum and 1% penicillin and streptomycin, were incubated with various concentrations of ACS and control (vehicle only) at 37°C for 4days. Every 24 h, the number of HL-60 cells was counted under a microscope as described in Section 2.
IO4 (I 994) 31-36
Concentrations of ACS greater than 200pg/ml were cytocidal. The inhibitory effects of ACS on the synthesis of DNA, RNA and protein in HL-60 cells are shown in Fig. 4. The initial rates of incorporation of [3H]thymidine, [3H]uridine and [3H]leucine into trichloroacetic acid (TCA) -insoluble material were utilized to estimate the rates of DNA, RNA and protein synthesis, respectively, in HL-60 cells. The presence of ACS at 6, 12.5, 25, 50, 100, 200 or 400pglml in cultured HL-60 cells incubated for 120 min inhibited the incorporation of [3H]thymidine into DNA by 41, 67, 79, 84, 87, 90 or 97%, respectively (Fig. 4), the incorporation of [3H]uridine into RNA by 5, 20, 41, 68, 78, 86 or 98%, respectively, and the incorporation of [3H]leucine into protein by 4, 12, 35, 59, 73, 83 or 85%, respectively. The results indicate that ACS is a potent inhibitor of DNA synthesis, but is somewhat less effective on RNA and protein synthesis in HL-60 cells. Inhibition of ACS on DNA synthesis in HL-60 cells occurred rapidly (Fig. 5). The inhibition of ACS
3. Results and discussion Fig. 1 shows the flow chart for extraction and partial purification of ACS from asparagus shoots. The yield of ACS was 1.5-2% from original asparagus shoots. The final powder preparation contained at least two saponins. The structure of two major saponins have been identified as shown in Fig. 2 based on FAB-MS and NMR spectral and chemical evidence. This preparation was used for studies of its effects on the growth of HL-60 cells and macromolecular synthesis as described in the following paragraphs. The ACS inhibited the growth of HL-60 cells in a dose-dependent fashion (Fig. 3). Addition of ACS at 50, 200, or 400pg/ml to HL-60 cells and incubation at 37°C for 24 h inhibited the growth of HL-60 cells by 53, 88 or 94%, respectively. The inhibitory effect of ACS on the growth of HL-60 cells also depended on the time of incubation. Incubation of HL-60 cells with ACS at 50,+q/ml at 37°C for 1, 2, 3 or 4 days inhibited the growth of HL-60 cells by 53, 67, 73 or 74%, respectively. Cytostatic concentration was found to be in the range of about 75-1OO~glml.
100 I El0 ‘;; 2
DNA
-
RNA
-
protein
60
! : w
40
20
4
0
0
50
100
150
200
concentration
250
300
350
400
450
(fig/ml)
Fig. 4. Inhibitory effect of various concentrations of ACS on the synthesis of DNA, RNA and protein in HL-60 cells. The ACS was added, at the indicated final concentration, to 1.0 ml of HL-60 cells, suspended in RPM1 medium without calf serum at concentration of 5 x IO5 cells/ml. [3H]Thymidine (.5O~Ci/~mol; 3~1), [3H]uridine (55yWpmol; 5,~l) or [3H]leucine (200~W~mol; lo@) were added. The cells were incubated at 37°C for 120 min, the reactions were terminated by addition 2 ml of cold PBS and the rate of DNA, RNA and protein synthesis were determined as described in Section 2. The percentage of incorporation shown is expressed relative to cell cultures to which no inhibitor was added.
Table I
r-
-
f u
-
control
-
6.25
--
50 pg/rnl
200
llglrn1
fig/ml
Irreversible inhibitory effect of the ACS on the synthesis of DNA in HL-60 cells Concentration of ACS @gW
8500
0 123 50 200
TWlE (min) Fig. 5. Effect ot ACS on the synthesis of DNA in HL-60 ceils at various time& following exposure to inhibitor. HL-60 cells (5 X 10’ cells/ml), suspended in RPM1 medium, were divided into 4 portion:;. ACS was added to each of 3 portions of cultures at a final concentrations of 6.25, SO or 2OOpglml. DMSO was added to one of the 4 portions of cultures and served as positive control. [‘Hjthymidine (SOyCi/pmol; 3~1) was added to each portion. The cultures were further incubated at 37°C and at the rndicated times 1 ml of sample was pipetted into 2 ml of cold PBS and the rate of DNA synthesis were determined as described in Section 2
at 2OOpgiml of ACS was essentially completed after IO min. ?‘he inhibitory effect of ACS on the DNA synthesis was dependent on the time of incubation (Fig. 5). incubation of HL-60 cells (5 X IO5 cells/ml) v;ith AtlS ;~t 6.25, SO or 2OOpgg/ml at 37°C for 10 min. the DNA synthesis was inhibited by 27.7. 49.7. or %x16’%, respectively. Incubation of HL-60 cells with ACS at 6.25, SO or 2OOpg/ml at 37°C for 120 min, the DNA synthesis was inhibited by 40.19. 82.98, or 90.89%. respectively. The inhibitory effect of the ACS on DNA synthesis was irreversible (Table 1). HL-60 cells were preincubated with ACS at 12.5, 50 and 200pglml for 120 min at 37°C and then washed with phosphate buffer saline (PBS) three times to remove the ACS. The cells were resuspended in RPM1 medium and [ 3H]thymidine was added and the DNA synthesis was determined. The results in column A of Table I showed that the inhibitory effect on DNA synthesis was dependent upon preincubation of HL-60 cells
[3H]Thymidine incorporation into TCA insoluble materials
% of control
A
B
A
B
14077 7320 2112 S63
15555 7178 933 466
100 52 15 4
100 SO 6 3
A, HL-60 cells (5 X lo5 cells/ml) were preincubated with 2~1 DMSO, or ACS in DMSO at 37°C for 120 min, then cells were washed with PBS 3 times, each time with 2 ml of PBS to remove the ACS. Cells were resuspended in fresh RPM1 medium and 2~1 DMSO and 13H]thymidine was added, incubated at 37°C for 120 min. The radioactivity incorporation into TCA insoluble materials was determined as described in Section 2. B, HL-60 cells (5 x IO5 cells/ml) were incubated with 2~1 DMSO at 37°C for 120 min, then cells were washed with PBS 3 times, each time with 2 ml of PBS to remove DMSO. Cells were resuspended in fresh medium and 2~1 DMSO or ACS in DMSO and [3H]thymidine was added, incubated at 37°C for 120 min. The radioactivity incorporation into TCA insoluble materials was determined as described in Section 2.
with various concentrations of ACS. It is not known whether ACS can be removed by washing with PBS or whether ACS may bind tightly to cells. The present investigation showed that ACS inhibited growth of human leukemia HL-60 cells and the synthesis of macromolecules in HL-60 cells. The inhibitory effect of ACS on DNA synthesis was irreversible. These results suggest that some active components in the ACS may have antitumor activity. Saponins have been reported to inhibit the growth of yeast. It is reasonable to predict that the two major saponins in ACS may play the role of inhibiting the growth of HL-60 cells and the synthesis of macromolecules. Additional studies including (a) isolation, purification and identification of the saponins, (b) determination of the inhibitory effects of pure saponin on the growth of HL-60 cells and on the synthesis of macromolecules, and (c) determination of the possible inhibitory mechanisms of saponins need to be addressed.
36
Y. Shao et al. / Cuncer Letters 104 (I 996) 31-36
References [l]
[2]
[3]
[4] [5]
[6] [7]
Jiangsu College of New Medicine (1985) Dictionary of Chinese Medicine, p. 249. Shanghai Press of Science and Technology. Shimoyamada, M., Suzuki, M., Sonta, H., Maruyama, M. and Okubo, K. (1990) Antifungal activity of the saponin fraction obtained from asprtlagus and its active principle. Agric. Biol. Chem., 54,2553-2557. Edenharder, R. (1990) Antimutagenic activity of vegetable and fruit extracts against in-vitro benzo(a)pyrene. Z. Gesamte Hyg., 36, 144-148. Balansand, J. and Rayband, M. (1937) Diuretic action of Asparagus officinalis. C. R. Sot. Biol., 126,95&956. Sati, 0. P., Pant, G., Nohara, T. and Sate, A. (1985) Cytotoxic saponins from asparagus and agave. Pharmazie, 40, 586. Aquino, R. (1991) Antiviral activity of constituents of tamus communis. J. Chemother., 3, 305-307. Sati, O.P., Pant, G. and Hostettman, K. (1984) Potent molluscicides from asparagus. Pharmazie, 39, 581.
[8] Kartnig, T., Gruber, A. and Stachel, J. (1985) Flavonoid glycosides from A.rparugus ojficinalis. Plant Med., 39. 288. [9] Shiomi, N. (1981) Two novel hexasaccharides from the roots of Asparagus ojjicinalis. Phytochemistry, 20. 25812583. [lo] Kasai, T., Hirakuri, Y. and Sakamura, S. (1981) Two cysteine derivatives in asparagus shoots. Phytochemistry, 20, 2209-22 11. [l l] Yanagawa, H., Kato, T. and Kitahara, Y. (1972) Asparagusic acid, dihydroasparagusic acid and S-acetydihydroasparagusic acid, new plant growth inhibitors in etiolated young Aspuragus ofjicinalis. Tetrahedron L&t., 25, 2549-2552. [I21 Goryanu, G.M., Krokhmalyuk, V.V., Kintya, P.K. and Glyzin, V.I. (1976) Medicinal asparagus as a source of steroidal glycoside. Farmatsiya, 25, 66-68. [ 131 Goryanu, G.M., Krokhmalyuk, V.V. and Kintya, P.K. (1976) Structure of glycosides of A.sparqus qflicinulis. Khim. Prir. Soedin., 3, 400-401,