Anti-tumor activities of triterpenes from Syzygium kusukusense




Дата канвертавання15.04.2016
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Anti-tumor activities of triterpenes from Syzygium kusukusense
Li-Yuan Baia,b, Wei-Yu Linc, Chang-Fang Chiub,d, Jing-Ru Wenge,*
aDepartment of Internal Medicine, College of Medicine, China Medical University, Taichung, 404, Taiwan

bDivision of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung 404, Taiwan

cDepartment of Pharmacy, Kinmen Hospital, Kinmen 891, Taiwan

dCancer Center; China Medical University Hospital, Taichung, 404, Taiwan

eDepartment of Biological Science and Technology, China Medical University, Taichung 404, Taiwan

Running Title: Anti-tumor activities of triterpenes


*To whom correspondence should be addressed.

Jing-Ru Weng, Ph.D.

Tel: (886)-4-22053366 ext. 2511; Fax: (886)-4-22071507

91 Hsueh-Shih Road, Taichung 404, Taiwan

E-mail: columnster@gmail.com
Abstract

In this study, we report the isolation of five triterpenes from the stem of Syzygium kusukusense, including 2-hydroxybetulinic acid (1), betulinic acid (2), platanic acid (3), ursolic acid (4), and hyptatic acid A (5). All of these triterpenes were identified for the first time in this indigenous plant in Taiwan. Assessment of the antiproliferative activities of these compounds against a panel of human tumor cell lines, including MCF-7 breast cancer, PC-3 prostate cancer, and SCC2095 oral cancer reveals high potency of compounds 1 (IC50, 5.7 – 7.6 µM) and, especially, 4 (IC50, 1.7 – 3.7 µM) in suppressing cell viability, which warrants further mechanistic investigations.




Keywords: Syzygium kusukusense; Myrtaceae; Triterpenes; Cytotoxicity

Introduction

Syzygium kusukusense (Myrtaceae) is an medium-sized tree distributed in Southern Taiwan . Although different classes of natural products, including flavonoids , triterpenes , and steroids , have been reported in the genus Syzygium, this particular species of Syzygium has not yet been fully characterized except for the content of fiber . In the course of screening the cytotoxic activity of various indigenous species of Syzygium in Taiwan, we found that stem extract of S. kusukusense showed promising in vitro antitumor activity, and thus embarked on the isolation and characterization of active ingredients. In this study, we report the isolation and structural elucidation of the five triterpenes, including betulinic acid (1), 2-hydroxybetulinic acid (2), platanic acid (3), ursolic acid (4), and hyptatic acid A (5). Among these compounds, 1 and 4 show high antiproliferative potencies against a panel of cancer cell lines examined, which underlies the antitumor activity of this indigenous plant.
Materials and Methods

General


Chromatographic purification and spectroscopic characterizations of test compounds were conducted using the following products or instruments: TLC, silica gel 60 F254 precoated plates (Merck, Darmstadt, Germany); column chromatography (CC), silica gel 60 (70-230 or 230-400 mesh, Merck); 1H-, 13C-NMR, and 2D-NMR Spectra, Varian Unity-600 spectrometer ( in ppm rel. to Me4Si as internal standard; J in Hz). EI-MS, Finnigan Thermo Quest MAT-95XL mass spectrometer [m/z (rel. %)].
Plant Material

The stem of Syzygium kusukusense (Myrtaceae) was collected and identified by one of the co-authors; Dr. Wei-Yu Lin in Pingtung County, Taiwan, in July, 2009, and a voucher specimen (2009) has been deposited in the Department of Biological Science and Technology, China Medical University.


Extraction and Isolation

Stem of S. kusukusense (5 kg dry weight) were extracted with 95% CHCl3 (30 L) at room temperature for three consecutive weeks. Removed of the solvent in vacuo afforded 160 g of crude residues, which were loaded onto a silica gel column (60 x 12 cm), and compounds 1 - 5 (Fig. 1) were purified as described below. The column was eluted, in tandem, with 700 ml each of n-hexane-Acetone (19:1), n-hexane-Acetone (9:1), n-hexane-Acetone (4:1), n-hexane-Acetone (1:1), n-hexane-Acetone-MeOH (4:4:1), and n-hexane-Acetone-MeOH (3:4:3), to afford fractions 1 – 6 after drying the solvent. Fraction 5 was rechromatographed with CHCl3/acetone (7:3) to give 5 sub-fractions (5-1 to 5-5). Sub-fraction 5-1 was purified by eluting the column with CHCl3/MeOH (9:1) to yield 1 (21 mg) and 2 (13 mg), and sub-fraction 5-3 was eluted with CHCl3/acetone (9:1) to generate 3 (12 mg) and 5 (23 mg). Fraction 4 was rechromatographed with n-hexane/EtOAc (2:1) to give 7 sub-fractions (4-1 to 4-7). Sub-fraction 4-6 was purified by eluting the column with n-hexane/EtOAc (7:3) to yield 4 (24 mg). Structures were established unambiguously by IR, MS, extensive 1H, 13C, and 2D spectra analysis, and comparison with literature data.


Cytotoxic activity

Anticancer activities of compounds 1-5 were examined in 3 human cancer cell lines, including MCF-7 (breast cancer), PC-3 (prostate cancer), and SCC2095 (oral squamous cell carcinoma). MCF-7 and PC-3 cells were purchased from the American Type Culture Collection (Manassas, VA), and cultured in DMEM/F-12 and RPMI medium (Gibco, Grand Island, NY). SCC2095 cells were kindly provided by Professor Susan R. Mallery (The Ohio State University) and cultured in DMEM/F12 medium (Gibco). All culture medium for cancer cells was supplemented with 10% heat-inactivated FBS, 5 mg/ml of penicillin, 10 mg/ml of neomycin and 5 mg/ml streptomycin. All cells were cultured at 37 oC in a humidified incubator containing 5% CO2. The suppressive effects of compounds 1-5 on cell viability were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in six replicates. Cells (5× 103/200 L) were seeded and incubated in 96-well, flat-bottomed plates in 10% FBS-supplemented medium for 24 h and were exposed to various concentrations of test agents dissolved in DMSO (final DMSO concentration, 0.1%) in 5% FBS-supplemented medium. Controls received DMSO vehicle at a concentration equal to that of drug-treated cells. The medium was removed and replaced by 200 L of 0.5 mM MTT in the medium, and cells were incubated in the 5% CO2 incubator at 37 °C for 2 h. Supernatants were removed from the wells, and the reduced MTT dye was solubilized in 200 L /well DMSO. Absorbance at 570 nm was determined on a plate reader (Bio-Tek). The cell viability was expressed as a percentage to the viable cells of control culture condition. The IC50 values of each group were calculated by the median-effect analysis and presented as the mean ± standard deviation (S.D.).


2.5 Cell proliferation

MCF-7 and SCC2095 cells were seeded onto six-well plates at ~100,000 per well in 5% FBS-containing DMEM/F12. Following a 24 h attachment period, cells were treated in triplicate with the indicated concentrations of compound 4 or DMSO vehicle in 5% FBS-containing DMEM/F12. After 72 h, cells were harvested by trypsinization and counted using a Coulter counter (Model Z1 D/T, Beckman Coulter).


Results and Discussion

The CHCl3 extract of the stem of S. kusukusense, which exhibited cytotoxic activity (data not shown), was subjected to chromatographic isolation and purification to yield five distinct triterpenes, compounds 15, as while powder.

The anticancer activities of these five triterpenes were examined in a panel of human-cancer cell lines by MTT assays, including MCF-7 breast cancer, PC-3 prostate cancer, SCC2095 oral cancer, by using 5-fluorouracil (5-FU) as a positive control. As shown in Table 2, these compounds exhibited differential antiproliferative activities in these three cancer cells lines as compared to the positive control 5-FU. Among these compounds, 1 (betulinic acid) and 4 (ursolic acid) exhibited potent antitumor potencies, with IC50 as low as 5.7 µM (MCF-7) and 1.7 µM (PC-3), respectively (Table 2), in agreement with that reported in the literature . Relative to compound 1 (betulinic acid), the structurally related compounds 2 and 3 showed 3-fold lower antitumor activities, suggesting a subtle structure-activity relationship. In contrast to an earlier report that indicated significant cytotoxicity in human HCT-8 cancer cells , compound 5 (hyptatic acid), even at 30 µM, lacked an appreciable activity in the three cell lines examined.

As shown, compounds 2 and 3 exhibited the moderate against MCF-7 cells, with IC50 values ranging from 18 - 22 µM (Table 2), while compounds 1 and 2 showed appreciable effect on suppressing MCF-7 cell viability. In addition to MCF-7 cells, compounds 1 and 2 showed the potent antiproliferative activity in PC-3 and SCC2095 cells. In contrast, compound 5, even at 30 µM, didn’t showed significant antiproliferative activities against these three cell lines examined. Moreover, as compound 4 exhibited higher activities than 5, and, to a greater extent, 1-3 in suppressing the viability of these cancer cells, the methyl at both C-19 and C-24 played an integral role in mediating the cytotoxicity. The in vitro efficacy of compound 4 in inhibiting the proliferation of MCF-7 and SCC2095 cells was also examined by direct counting of drug-treated cells (Fig. 2).

Triterpenes, flavonoids, and steroids are widely distributed in Syzygium plant . As part of our effort to identify bioactive ingredients in S. kusukusense, we have identified 5 triterpenes (15) in this study. In vitro efficacy experiment in three different human cancer cell lines indicated that compound 1 and 4 exhibited high antiproliferative potency relative to three other compounds examined, suggesting that these two compounds may contribute to the anticancer activity of S. kusukusense. As many Syzygium triterpenes have been shown to exhibit anti-inflammatory and other types of pharmacological activities, testing of these agents in other bioassay systems is currently in progress.
Conflict of interest statement

The authors declare no competing financial interests.


Acknowledgements

This work was supported by grants from the National Science Council of Republic of China (NSC 99-2320-B-039-007-MY2, NSC 101-2320-B-039-029-

MY2).

References

Aguirre, M. C., Delporte, C., Backhouse, N., Erazo, S., Letelier, M. E., Cassels, B. K., Silva, X., Alegria, S., and Negrete, R. Topical anti-inflammatory activity of 2alpha-hydroxy pentacyclic triterpene acids from the leaves of Ugni molinae. Bioorganic & medicinal chemistry, 14, 5673-5677 (2006).

Chang, C. E. Myrtaceae in Flora of Taiwan. Flora of Taiwan. second ed. Editorial Committee of the Flora of Taiwan, Taipei, pp. 886-900, (1993).

Chao, S. C., Ku, Y. C., Lin, S. J., and Pan, T. T. Measurements of fiber dimensions and analysis of the chemical composition of Taiwan hardwoods. Tai-Wan Sheng Lin Yeh Shih Yen So Lin Wu Chu Ho Tso Shih Yen Pao Kao, 14, 26 (1971).

Chumkaew, P., Kato, S., and Chantrapromma, K. New cytotoxic steroids from the fruits of Syzygium siamense. J. Asian Nat. Prod. Res., 12, 424-428 (2010).

De Angel, R. E., Smith, S. M., Glickman, R. D., Perkins, S. N., and Hursting, S. D. Antitumor effects of ursolic acid in a mouse model of postmenopausal breast cancer. Nutr. cancer, 62, 1074-1086 (2010).

Kumar, N. S., Muthukuda, P. M., and Wazeer, M. I. M. A lupenediol from Euonymus revolutus. Phytochemistry, 24, 1337-1340 (1985).

Lee, C. K. A new norlupene from the leaves of Melaleuca leucadendron. J. Nat. Prod., 61, 375-376 (1998).

Mullauer, F. B., Kessler, J. H., and Medema, J. P. Betulinic acid, a natural compound with potent anticancer effects. Anti-cancer drugs, 21, 215-227 (2010).

Peng, C., Bodenhausen, G., Qiu, S., Fong, H. H. S., Farnworth N. R., Yuan S., Zeng C. Computer-assisted structure elucidation: application of CISOC-SES to the resonance assignment and structure generation of betulinic acid. Magn. Reson. Chem., 36, 267-278 (1998).

Raga, D. D., Cheng, C. L., Lee, K. C., Olaziman, W. Z., De Guzman, V. J., Shen, C. C., Franco, F. C., Jr., and Ragasa, C. Y. Bioactivities of triterpenes and a sterol from Syzygium samarangense. Z. Naturforsch C., 66, 235-244 (2011).

Savitha, R. C., Padmavathy, S., and Sundhararajan, A. Invitro antioxidant activities on leaf extracts of syzygium malaccense (L.) merr and perry. Anc. Sci. Life, 30, 110-113 (2011).

Shai, L. J., Mcgaw, L. J., Aderogba, M. A., Mdee, L. K., and Eloff, J. N. Four pentacyclic triterpenoids with antifungal and antibacterial activity from Curtisia dentata (Burm.f) C.A. Sm. leaves. J. Ethnopharmacol., 119, 238-244 (2008).

Sharma, B., Balomajumder, C., and Roy, P. Hypoglycemic and hypolipidemic effects of flavonoid rich extract from Eugenia jambolana seeds on streptozotocin induced diabetic rats. Food Chem. Toxicol., 46, 2376-2383 (2008).

Yamagishi, T., Zhang, D. C., Chang, J. J., Mcphail, D. R., Mcphail, A. T., Lee, K. H. The cytotoxic principles of Hyptis capitata and the structures of the new triterpenes hyptatic-A and B. Phytochemistry, 27, 3213-3216 (1988).



Figure Legends



Fig. 1. The chemical structures of compounds 1-5 isolated from S. kusukusense.

Fig. 2. Dose-dependent antiproliferative effect of compound 4 at the indicated concentrations in MCF-7 and SCC2095 cells. Cells were seeded onto six-well plates (100,000 per well) and exposed to the test agent at the indicated concentrations in 5% FBS-supplemented DMEM/F12 medium. After 72 h, cells were harvested and counted using a Coulter counter. Values were obtained from triplicates.

Table 1. 13C NMR spectroscopic data of compounds 15 at 150 MHz.

Table 2. Cytotoxic activities of 1-5 against different cancer cell lines.







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