Rhein Inhibits the Growth and Induces the Apoptosis of Hep G2 Cells

• Abstract
• Introduction
• Materials and Methods
• Materials
• Cell culture
• Cell proliferation assay
• Cell cycle analysis
• Measurement of apoptosis by ELISA
• Assaying the levels of p53, p21, CD95 and CD95 ligand (mCD95L and sCD95L)
• Statistical analysis
• Results
• Discussion
• References

Abstract

The effects of rhein on the human hepatoblastoma G2 (Hep G2) cell line were investigated in this study. The results showed that rhein not only inhibited Hep G2 cell growth but also induced apoptosis and blocked cell cycle progression in the G1 phase. An ELISA assay demonstrated that rhein significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in CD95 and its two forms of ligands, membrane-bound CD95 ligand (mCD95L) and soluble CD95 ligand (sCD95L), might be responsible for the apoptotic effect induced by rhein. Taken together, p53 and the CD95/CD95L apoptotic system possibly participated in the antiproliferative activity of rhein in Hep G2 cells.

Introduction

Rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid; Fig. [1]) is an anthraquinone compound that is present in many medicinal plants. It is the major bioactive constituent of the rhizome of rhubarb (R. palmatum L. or R. tanguticum Maxim), a popular ingredient in traditional Chinese medicine for use as laxative and stomachic [1]. In the literature, many studies have indicated that rhein has laxative [2], antifungal [3], antiviral [4], and antitumor effects [1], [5], [6], [7].

Hepatocellular carcinoma (HCC) is one of the most common malignancies responsible for the death of over a million people worldwide, especially in mainland China, Taiwan, Korea and Sub-Africa [8]. Most patients diagnosed with hepatocellular carcinoma have low recovery rates, and the conventional and modified therapies currently available are rarely effective [9]. Therefore, it is imperative to search for a more effective antihepatoma drug with less side-effects.

In certain studies, rhein was demonstrated to possess tumor-promoting activities in vitro [10]. However, a number of recent studies reported that it suppressed the growth of cancer cells by reducing ATP availability in rat liver or inhibiting glucose uptake in Ehrlich ascites tumor cells [5]. It was also shown to reduce IL-1β production and secretion [6], and caspase-3 and nitric oxide synthase activity [7], and as well inhibiting the phosphorylation of c-Jun and c-Jun NH₂-terminal kinase [1]. To-date, the molecular mechanisms of action of rhein remain poorly understood.

In this study, our aim was to examine the effects of rhein on cell proliferation, cell cycle distribution, and apoptotic mechanism in a well-characterized human hepatoblastoma cell line, Hep G2. Both p53 and CD95/CD95 ligand (CD95/CD95L) systems contribute in triggering apoptosis and are strongly associated with the chemosensitivity of liver tumors to anticancer agents [11], [12]. Therefore, the effects of rhein on the molecular levels of the p53 pathway (p53 and p21/WAF1 protein) and the CD95/CD95L apoptotic system (CD95 and CD95 ligand) were examined.

Materials and Methods

Materials

Fetal calf serum (FCS), penicillin G, streptomycin, and amphotericin B were obtained from GIBCO BRL (Gaithersburg, MD). Rhein, 5-fluorouracil (5-FU), dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), ribonuclease (RNase), and propidium iodide) were purchased from Sigma Chemical (St. Louis, MO). Sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) and p53 pan ELISA kits were obtained from Roche Diagnostics GmbH (Germany). Nucleosome ELISA, WAF1 ELISA, CD95, and CD95 ligand ELISA kits were purchased from Calbiochem (Cambridge, MA).

The stock solution of rhein or 5-FU was prepared at concentration 2 mg/mL of DMSO. It was then stored at -20 °C until use. For all experiments, the final concentrations of the test compound were prepared by diluting the stock with DMEM. Control cultures received the carrier solvent (0.1 % DMSO).

Cell culture

Hep G2 (ATCC HB8065) cells were maintained in a monolayer culture at 37 °C and 5 % CO₂ in DMEM supplemented with 10 % FCS, 10 U/mL of penicillin, 10 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B.

Cell proliferation assay

Inhibition of cell proliferation by rhein was measured by the XTT assay. Briefly, cells were plated in 96-well culture plates (1 × 10⁴ cells/well). After 24 h incubation, the cells were treated with rhein (0, 3.5, 17.5, 35 and 70 μM) or 5-FU (0, 8, 40, 200, 400 μM) for 12, 24, 48, and 72 h. Fifty μL of XTT test solution, which was prepared by mixing 5 mL of XTT-labeling reagent with 100 μL of electron coupling reagent, were then added to each well. After 6 h of incubation, the absorbance was measured on an ELISA reader (Multiskan EX, Labsystems) at a test wavelength of 492 nm and a reference wavelength of 690 nm.

Cell cycle analysis

To determine cell cycle distribution, 5 × 10⁵ cells were plated in 60-mm dishes, treated with rhein (0, 35, and 70 μM) for 24 h. After treatment, the cells were collected by trypsinization and then fixed in 70 % ethanol. They were washed with phosphate-buffered saline (PBS) and resuspended in 1 mL of PBS containing 1 mg/mL RNase and 50 μg/mL propidium iodide. Following incubation in the dark for 30 min at room temperature, the cells were analyzed with a flow cytometer (Coulter Epics Elite ESP, FL, USA). The Multicycle software (Phoenix Flow Systems, San Diego, CA) was used to evaluate the data.

Measurement of apoptosis by ELISA

The induction of apoptosis by rhein was assayed using the Nucleosome ELISA kit. This kit uses a photometric enzyme immunoassay that quantitatively determines the formation of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after apoptotic cell death. For determination of apoptosis by ELISA, the Hep G2 cells were treated with rhein at 0, 35, and 70 μM for 6, 12, 24, and 48 h in a 96 well plate. The induction of apoptosis was evaluated by assessing the enrichment of nucleosome in cytoplasm, and determined exactly as described in the manufacturer’s protocol [13].

Assaying the levels of p53, p21, CD95 and CD95 ligand (mCD95L and sCD95L)

p53 pan ELISA, WAF1 ELISA, Fas ligand and Fas ELISA kits were used to detect p53, p21, CD95 ligand and CD95 receptor. Briefly, Hep G2 cells were treated with 0, 35, and 70 μM of rhein for 6, 12, 24, and 48 h. The samples of cell lysate were placed in 96 well (1 × 10⁶ per well) microtiter plates coated with monoclonal detective antibodies, and were incubated for 1 h (CD95), 2 h (p53 or p21/WAF1) or 3 h (CD95L) at room temperature. After removing the unbound material by washing with PBS, horseradish peroxidase conjugated streptavidin was added to bind to the antibodies. Horseradish peroxidase catalyzed the conversion of a chromogenic substrate (tetramethylbenzidine) to a colored solution with color intensity proportional to the amount of protein present in the sample. The absorbance of each well was measured at 450 nm. Concentrations of p53, p21/WAF1, CD95L and CD95 were determined by interpolating from standard curves obtained with known concentrations of standard proteins.

Statistical analysis

Data were expressed as means ± standard errors. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (p < 0.05) between the means of control and rhein-treated cells were analyzed by Dunnett’s test.

Results

We first tested the antiproliferative effect of rhein in the liver cancer cell line, Hep G2. As shown in Fig. [2] A, the growth inhibitory effect of rhein was observed to work in a dose-dependent manner. Its IC₅₀ value was 39.3 μM. In comparison, 5-FU was used as a positive control. Two hundred μM of 5-FU were shown to exhibit an only 50 % inhibition rate against the proliferation of Hep G2 and increasing the concentration of the drug did not significantly increase its antiproliferative effect further (Fig. [2] B). This result was similar to a previous report [14].

The results on the effect of rhein on cell cycle progression of Hep G2 are shown in Fig. [3]. As compared to the control, 35 μM of rhein increased the population of G1 phase from 29.7 % to 50.7 %. This effect was enhanced when Hep G2 cells were treated with 70 μM of rhein (68.1 % cell population in G1 phase).

Fig. [4] shows the time course of DNA fragmentation in continuous treatment with 35 and 70 μM of rhein. DNA fragmentation of Hep G2 was found at 6 h and maximized at 48 h after addition of rhein. In contrast to the control, when cells were treated with rhein, the number of cells undergoing apoptosis increased from about 4-fold to 8-fold at 35 μM and 70 μM of rhein respectively at 48 h.

To determine whether tumor suppression factor p53 and its downstream molecule p21/WAF1 were involved in the rhein-mediated antiproliferative effect of Hep G2 cells, the levels of these proteins were assayed by ELISA. The results showed a marked induction of p53 protein in the rhein treated cells and a trend for a dose-dependent effect was noted (Fig. [5] A). The upregulation of p53 by rhein started to increase 6 h after treatment with rhein, and maximum expression was observed at 12 h. Comparison of the results between apoptotic response and induction of p53 indicated that the upregulation of p53 occurred at an early stage of the rhein-mediated apoptotic process.

Fig. [5] B shows that an increase in p21/WAF1 protein was apparent at 6 h and reached maximum induction at 24 h in rhein treated Hep G2 cells. Moreover, the induction of p21/WAF1 was observed to work in a dose-dependent manner. Based on these data, we suggest that rhein-mediated cell cycle arrest might operate through the induction of p21/WAF1 protein on a p53-dependent event in Hep G2 cells.

As observed in the induction of p21/WAF1, the expression of CD95 was detected in Hep G2 cells at 6 h after rhein treatment. Maximum CD95 was detected at 24 h (Fig. [6] A). It is suggested that the induction of CD95 in rhein-treated Hep G2 cells might be related to the activation of p53.

Results of the CD95 ligand assay indicated that CD95L, mCD95L, and sCD95L increased in a dose-dependent manner (Fig. [6] B, 6 C). The accumulation of mCD95L was observed at 6 h after rhein treatment, and progressively increased up to 24 h (Fig. [6] B). A similar result was observed for sCD95L (Fig. [6] C). However, the amount of mCD95L was more than sCD95L at all time points.

When Hep G2 cells were pre-treated with an antagonistic anti-CD95 antibody, ZB4, the antiproliferative and proapoptotic effects of rhein were effectively inhibited. At 70 μM of rhein, the cell growth inhibition decreased from 78.6 % to 33.7 % (Fig. [7] A). Compared to the control, the oligonucleosome DNA fragmentation of apoptosis induced by 70 μM of rhein decreased from about 6-fold to 3-fold at 48 h in ZB4 pretreated Hep G2 cells (Fig. [7] B).

Discussion

Normal p53 gene is well known to play a crucial role in inducing apoptosis and as cell cycle checkpoints in human and murine cells following DNA damage [12]. This has been further supported by the finding that p53 is the most commonly mutated tumor suppressor gene. The absence of p53 function is related to an enhanced risk of carcinogenesis [15]. Moreover, the chemosensitivity of cancer cells to chemotherapy agents is greatly influenced when the function of p53 is abrogated [16]. Our results demonstrated that p53 might possibly play an important role in rhein induced antiproliferative activity in Hep G2 cells. Induction of p53 by rhein not only caused Hep G2 cell cycle arrest, but also triggered apoptosis. This finding is supported by the following results: First, the flow cytometry assay indicated that rhein can induce Hep G2 cell cycle arrest at the G1 phase, which was attributed to the enhancement of p21/WAF1 protein that was induced by p53. Second, rhein increased the expression of p53’s downstream molecules, CD95, which restores apoptosis sensitivity of Hep G2 cells.

Studies have shown that the CD95/CD95L system is a key factor controlling apoptotic cell death [14]. Binding of CD95 ligand to CD95 induces receptor oligomerization and formation of the death-inducing signaling complex (DISC), followed by activation of a series caspase cascade resulting in cell apoptotic death [17]. Recent observations have highlighted the role of the CD95/CD95L system in chemotherapy-induced apoptosis of tumors by upregulation of CD95 or its ligand [14]. Loss of CD95 expression might be involved in the escape of liver cancer cells from the immune defense system and the development of chemoresistance of hepatocellular carcinoma to chemotherapeutic agents [18]. Our data showed that the expression of CD95 is significantly increased in rhein-treated Hep G2 cells. Thus, we suggest that the enhancement of CD95 by rhein is beneficial for restoring apoptotic sensitivity of Hep G2 cells for the natural immune defense system or chemotherapy.

The second basic molecule responsible for triggering the activation of the CD95/CD95L system is the CD95 ligand. CD95L is a TNF related type II membrane protein. Cleavage of membrane-bound CD95 ligand (mCD95L) by a metalloprotease-like enzyme results in the formation of soluble CD95 ligand (sCD95L) [19]. Both mCD95L and sCD95L can bind to CD95, and subsequently trigger the CD95/CD95L system, but sCD95L has been reported to be a weaker inducer of apoptosis than mCD95L [20]. Our study indicated that CD95 ligands, mCD95L and sCD95L, increased in rhein-treated Hep G2 cells. Moreover, the level of CD95 was simultaneously enhanced in CD95L-upregulating Hep G2 cells. Furthermore, when the CD95/CD95 ligand system was blocked by ZB4, a decrease in cell growth inhibition and proapoptotic effect of rhein was noted. These results also suggested that the CD95/CD95L system might possibly play an important role in rhein-mediated Hep G2 cellular apoptosis.

Taken together, the present study demonstrated that p53 and the CD95/CD95L apoptotic system possibly contributed to the antiproliferative activity of rhein in Hep G2 cells.

References

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  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net

References

• 1 Lin S, Li J J, Fujii M, Hou D X. Rhein inhibits TPA-induced activator protein-1 activation and cell transformation by blocking the JNK-dependent pathway.  Int J Oncol. 2003;  22 829-33
  PubMedSearch in Google Scholar
• 2 Krumbiegel G, Schulz H U. Rhein and aloe-emodin kinetics from senna laxatives in man.  Pharmacology. 1993;  47 120-4
  CrossrefPubMedSearch in Google Scholar
• 3 Agarwal S K, Singh S S, Verma S, Kumar S. Antifungal activity of anthraquinone derivatives from Rheum emodin.  J Ethnopharmacol. 2000;  72 43-6
  PubMedSearch in Google Scholar
• 4 Barnard D L, Huffman J H, Morris J L, Wood S G, Hughes B G, Sidwell R W. Evaluation of the antiviral activity of anthraquinones, anthrones and anthraquinone derivatives against human cytomegalovirus.  Antiviral Res. 1992;  17 63-77
  PubMedSearch in Google Scholar
• 5 Castiglione S, Fanciulli M, Bruno T, Evangelista M, Del Carlo C, Paggi M G, Chersi A, Floridi A. Rhein inhibits glucose uptake in Ehrlich ascites tumor cells by alteration of membrane-associated functions.  Anticancer Drugs. 1993;  4 407-14
  PubMedSearch in Google Scholar
• 6 Mendes A F, Caramona M M, de Carvalho A P, Lopes M C. Diacerhein and rhein prevent interleukin-1beta-induced nuclear factor-kappaB activation by inhibiting the degradation of inhibitor kappaB-alpha.  Pharmacol Toxicol. 2002;  91 22-8
  CrossrefPubMedSearch in Google Scholar
• 7 Pelletier J P, Mineau F, Boileau C, Martel-Pelletier J. Diacerein reduces the level of cartilage chondrocyte DNA fragmentation and death in experimental dog osteoarthritic cartilage at the same time that it inhibits caspase-3 and inducible nitric oxide synthase.  Clin Exp Rheumatol. 2003;  21 171-7
  PubMedSearch in Google Scholar
• 8 Okuda K. Hepatocellular carcinogenesis: recent progress.  Hepatology. 1992;  15 948-63
  PubMedSearch in Google Scholar
• 9 Sheu J C. Molecular mechanism of hepatocarcinogenesis.  J Gastroenterol Hepatol. 1997;  12 S309-13
  PubMedSearch in Google Scholar
• 10 Wolfle D, Schmutte C, Westendorf J, Marquardt H. Hydroxyanthraquinones as tumor promoters: enhancement of malignant transformation of C3H mouse fibroblasts and growth stimulation of primary rat hepatocytes.  Cancer Res. 1990;  50 6540-4
  PubMedSearch in Google Scholar
• 11 Muller M, Strand S, Hug H, Heinemann E M, Walczak H. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.  J Clin Invest. 1997;  99 403-13
  PubMedSearch in Google Scholar
• 12 May P, May E. Twenty years of p53 research: structural and functional aspects of the p53 protein.  Oncogene. 1999;  18 7621-36
  CrossrefPubMedSearch in Google Scholar
• 13 Salgame P, Varadhachary A S, Primiano L L, Fincke J E, Muller S, Monestier M. An ELISA for detection of apoptosis.  Nucleic Acids Res. 1997;  25 680-1
  CrossrefPubMedSearch in Google Scholar
• 14 Jiang S, Song M J, Shin E C, Lee M O, Kim S J, Park J H. Apoptosis in human hepatoma cell lines by chemotherapeutic drugs via Fas-dependent and Fas-independent pathways.  Hepatology. 1999;  29 101-10
  CrossrefPubMedSearch in Google Scholar
• 15 Donehower L A, Harvey M, Slagle B L, McArthur M J, Montgomery CA J r, Butel J S. et al . Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.  Nature. 1992;  356 215-21
  CrossrefPubMedSearch in Google Scholar
• 16 Brown J M, Wouters B G. Apoptosis, p53, and tumor cell sensitivity to anticancer agents.  Cancer Res. 1999;  59 1391-9
  PubMedSearch in Google Scholar
• 17 Nagata S, Golstein P. The Fas death factor.  Science. 1995;  267 1449-56
  PubMedSearch in Google Scholar
• 18 Lee S H, Shin M S, Lee H S, Bae J H, Lee H K, Kim H S. et al . Expression of Fas and Fas-related molecules in human hepatocellular carcinoma.  Hum Pathol. 2001;  32 250-6
  CrossrefPubMedSearch in Google Scholar
• 19 Kayagaki N, Kawasaki A, Ebata T, Ohmoto H, Ikeda S, Inoue S. et al . Metalloproteinase-mediated release of human Fas ligand.  J Exp Med. 1995;  182 1777-83
  CrossrefPubMedSearch in Google Scholar
• 20 Schneider P, Holler N, Bodmer J L, Hahne M, Frei K, Fontana A. et al . Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with down regulation of its proapoptotic activity and loss of liver toxicity.  J Exp Med. 1998;  187 1205-13
  CrossrefPubMedSearch in Google Scholar

Professor Chun-Ching Lin

Graduate Institute of Natural Products

College of Pharmacy

Kaohsiung Medical University

100 Shih-Chuan 1st Road

Kaohsiung 807

Taiwan

ROC

Phone: +886-7-3121101 ext. 2122

Fax: +886-7-3135215

Email: aalin@ms24.hinet.net