Embed Size (px)
Citation preview
INFORMATION FOR AUTHORS Full details of how to submit a manuscript for publication in Natural Product Communications are given in Information for Authors on our Web site http://www.naturalproduct.us. Authors may reproduce/republish portions of their published contribution without seeking permission from NPC, provided that any such republication is accompanied by an acknowledgment (original citation)-Reproduced by permission of Natural Product Communications. Any unauthorized reproduction, transmission or storage may result in either civil or criminal liability. The publication of each of the articles contained herein is protected by copyright. Except as allowed under national “fair use” laws, copying is not permitted by any means or for any purpose, such as for distribution to any third party (whether by sale, loan, gift, or otherwise); as agent (express or implied) of any third party; for purposes of advertising or promotion; or to create collective or derivative works. Such permission requests, or other inquiries, should be addressed to the Natural Product Inc. (NPI). A photocopy license is available from the NPI for institutional subscribers that need to make multiple copies of single articles for internal study or research purposes. To Subscribe: Natural Product Communications is a journal published monthly. 2014 subscription price: US$2,395 (Print, ISSN# 1934-578X); US$2,395 (Web edition, ISSN# 1555-9475); US$2,795 (Print + single site online); US$595 (Personal online). Orders should be addressed to Subscription Department, Natural Product Communications, Natural Product Inc., 7963 Anderson Park Lane, Westerville, Ohio 43081, USA. Subscriptions are renewed on an annual basis. Claims for nonreceipt of issues will be honored if made within three months of publication of the issue. All issues are dispatched by airmail throughout the world, excluding the USA and Canada.
NPC Natural Product Communications
EDITOR-IN-CHIEF
DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA [email protected]
EDITORS
PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected]
PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy [email protected]
PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China [email protected]
PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected]
PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia [email protected]
PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA [email protected]
PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA [email protected]
PROFESSOR YASUHIRO TEZUKA Faculty of Pharmaceutical Sciences Hokuriku University Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan [email protected]
PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK [email protected]
ADVISORY BOARD
Prof. Viqar Uddin Ahmad Karachi, Pakistan
Prof. Giovanni Appendino Novara, Italy
Prof. Yoshinori Asakawa Tokushima, Japan
Prof. Roberto G. S. Berlinck São Carlos, Brazil
Prof. Anna R. Bilia Florence, Italy
Prof. Maurizio Bruno Palermo, Italy
Prof. César A. N. Catalán Tucumán, Argentina
Prof. Josep Coll Barcelona, Spain
Prof. Geoffrey Cordell Chicago, IL, USA
Prof. Fatih Demirci Eskişehir, Turkey
Prof. Dominique Guillaume Reims, France
Prof. Ana Cristina Figueiredo Lisbon, Portugal
Prof. Cristina Gracia-Viguera Murcia, Spain
Prof. Duvvuru Gunasekar Tirupati, India
Prof. Hisahiro Hagiwara Niigata, Japan
Prof. Kurt Hostettmann Lausanne, Switzerland
Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico
Prof. Leopold Jirovetz Vienna, Austria
Prof. Vladimir I Kalinin Vladivostok, Russia
Prof. Niel A. Koorbanally Durban, South Africa
Prof. Chiaki Kuroda Tokyo, Japan
Prof. Hartmut Laatsch Gottingen, Germany
Prof. Marie Lacaille-Dubois Dijon, France
Prof. Shoei-Sheng Lee Taipei, Taiwan
Prof. Imre Mathe Szeged, Hungary
Prof. Ermino Murano Trieste, Italy
Prof. M. Soledade C. Pedras Saskatoon, Canada
Prof. Luc Pieters Antwerp, Belgium
Prof. Peter Proksch Düsseldorf, Germany
Prof. Phila Raharivelomanana Tahiti, French Polynesia
Prof. Luca Rastrelli Fisciano, Italy
Prof. Stefano Serra Milano, Italy
Prof. Monique Simmonds Richmond, UK
Dr. Bikram Singh Palampur, India
Prof. John L. Sorensen Manitoba, Canada
Prof. Johannes van Staden Scottsville, South Africa
Prof. Valentin Stonik Vladivostok, Russia
Prof. Winston F. Tinto Barbados, West Indies
Prof. Sylvia Urban Melbourne, Australia
Prof. Karen Valant-Vetschera Vienna, Austria
HONORARY EDITOR
PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,
University of Portsmouth, Portsmouth, PO1 2DT U.K.
Zerumbone Induces G2/M Cell Cycle Arrest and Apoptosis via Mitochondrial Pathway in Jurkat cell Line Heshu Sulaiman Rahmana,b,*, Abdullah Rasedeea,b,*, Max Stanley Chartrandc, Hemn Hassan Othmana, Swee Keong Yeapb and Farideh Namvard
aDepartment of Veterinary Clinical Diagnosis, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
bInstitute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
cDirector, DigiCare Behavioral Research, Casa Grande, Arizona, USA
dInstitute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia [email protected]; [email protected]
Received: June 9th, 2014; Accepted: July 20th, 2014
This investigation determined the anticancer properties of zerumbone (ZER) on the human T-cell (Jurkat) line using the MTT assay, microscopic evaluations, flow cytometric analyses, and caspase activity estimations. The results showed that ZER is selectively cytotoxic to Jurkat cells in a dose and time-dependent manner with IC50 of 11.9 ± 0.2, 8.6 ± 0.5 and 5.4 ± 0.4 μg/mL at 24, 48 and 72 hours of treatment, respectively. ZER did not produce an adverse effect on normal human peripheral blood mononuclear cells (PBMC). ZER is not as cytotoxic as doxorubicin, which imposed an inhibitory effect on Jurkat cells with IC50 of 2.1 ± 0.2, 1.8 ± 0.15, 1.5 ± 0.07 μg/mL after 24, 48 and 72 hours treatment, respectively. ZER significantly (P<0.05) arrested Jurkat cells at the G2/M phase of the cell cycle. The antiproliferative effect of ZER on Jurkat cells was through the apoptotic intrinsic pathway via the activation of caspase˗3 and ˗9. The results showed that ZER can be further developed into a safe chemotherapeutic compound for the treatment of cancers, especially leukemia. Keywords: Zerumbone, Leukemia, Cytotoxicity, Apoptosis, Cell cycle arrest. Leukemia is the most common cancer among children [1]. Currently, the most widely used anti-leukemia treatments are chemotherapy, radiotherapy, hormonal and immune therapy, and bone marrow transplantation. Generally, most of these treatments damage healthy cells and tissues with short- to long-term side-effects. Thus there is need to discover more innocuous therapeutic compounds with minimal side-effects as candidates for the next generation of anti-leukemic drugs [2]. Zerumbone is a crystalline, monocyclic sesquiterpene that was first isolated in 1956 from the essential oil of rhizomes of edible wild ginger, Zingiber zerumbet (L.) Smith. The structure of the compound was first elucidated in 1960, and later characterized by NMR spectroscopy and X-ray crystallography [3]. ZER was shown to have a range of pharmacological properties including antimicrobial [4], antipyretic [5], antispasmodic, anticonvulsant [6], antiulcer [7], antioxidant [8], antidiabetic [9], antitumor [10], anticancer [11,12], anti-inflammatory [13,14], antinociceptive, analgesic [15,16], antiallergenic [17], anti-angiogenic [18], anti-adipogenetic [19], anti-platelet aggregation, anticoagulant [20], and hepatoprotective [21] activities at various doses and concentrations. As an antitumor agent, ZER possesses antiproliferative properties to several cancer cell lines including cervical (HeLa) [22], breast (MCF-7) [23,24], colonic (COLO205, LS174T, LS180, and COLO320DM) [6,25], hepatic (HepG2) [26,27], ovarian (Caov-3 and AS52) [28], pancreatic (PANC-28, MIA PaCa-2, and AsPC-1) [29] and leucocytic (P-388D1, HL-60, NB4 and CEMss) normal and cancer cell lines [30,31]. To date the effect of ZER on T-lymphoblastic leukemia cell line (Jurkat cell) has not been investigated by other researchers. The Jurkat cells, an immortalized line of T lymphocytes originally
Figure 1: Cytotoxic effect of ZER (A) and doxorubicin (B) on Jurkat cells assessed by MTT assay. Each point is the mean value of three replicates. SD is represented by error bar.
derived from a 14-year old boy with acute lymphoblastic leukemia, were used to determine the therapeutic value of the compound on leukemia. In this study, the objective was to investigate the antiproliferative and cytotoxic effects of ZER on Jurkat cells. Zerumbone, exhibited significant (P<0.05) inhibitory effects towards Jurkat cells (Figure 1A) with IC50 values of 11.9 ± 0.2, 8.6 ± 0.1 and 5.4 ± 0.2 µg/mL after 24, 48 and 72 hours of treatment, respectively. On the other hand, doxorubicin, as positive control, showed IC50s of 2.1 ± 0.2, 1.8 ± 0.15, 1.5 ± 0.3 μg/mL on the Jurkat cells in the same treatment periods (Figure 1B).
NPC Natural Product Communications 2014 Vol. 9 No. 9
1237 - 1242
1238 Natural Product Communications Vol. 9 (9) 2014 Rahman et al.
Figure 2: Morphology of Jurkat cells after (B) 24, (C) 48 and (D) 72 h treatment with 5.39 µg/mL ZER. (A) Untreated Jurkat cells. (400 × magnification). AB: Apoptotic body; BL: Cell membrane blebbing.
Figure 3: Ultrastructure of Jurkat cells. (A) Untreated cells with typical morphological features of cancer cell. (B) 24 h treated cells with ZER, showing membrane blebbing and holes. (C) 48 h treated cells with ZER, showing membrane blebbing, hole formation, and apoptotic bodies. (D) 72 h treated cells with ZER, showing distinctive morphological changes of apoptosis including membrane blebbing, cell shrinkage with apoptotic bodies and hole formation. VC: Viable cell, BL: Blebbing of cell membrane, AB: Apoptotic body, CS: Cell shrinkage.
Figure 4: Fluorescent micrograph of AO/PI double stained Jurkat cells treated with 5.39 µg/mL ZER. (A) Untreated cells showing normal cell structure, (B) Early apoptotic cells after 24 hour treatment showing intercalated bright green fragmented DNA, (C) Blebbing and nuclear margination after 48 h treatment, (D) Late apoptosis after 72 h treatment. EA: Early apoptotic cells, CC: Chromatin condensation, MN: Marginated nucleus, LA: Late apoptotic cells, AB: Apoptotic body and SN: Secondary Necrosis.
Using the trypan blue exclusion assay, ZER did not show significant (P>0.05) antiproliferative effect towards PBMC after 24, 48 or 72 hours of treatment.
Significant morphological changes were observed in the Jurkat cells after treatment with ZER (Figure 2). The main morphological changes included cell membrane blebbing, and progressive nuclear shrinkage with apoptotic bodies, especially at 72 hours post-treatment. On the contrary, untreated cells did not show morphological change, and remained round in shape, shiny in appearance and confluent throughout the incubation period. Distinct ultrastructural characteristics typical of apoptosis were also observed in the ZER-treated Jurkat cells. These characteristics include membrane blebbing and hole formation at 24 hours post-treatment, cell shrinkage, increased surface irregularities, cytoplasmic extension, and apoptotic body formation at 48 and 72 hours post-treatment (Figure 3). These observations suggest that the effect of ZER on Jurkat cells is time-dependent. Early apoptosis was observed after 24 hours of ZER treatment with cells exhibiting bright-green nuclei due to intercalation of AO with the fragmented DNA, while the chromatin condensation appeared as dense green areas. Blebbing and nuclear margination mainly appeared after 48 hours of ZER treatment, indicating moderate apoptosis. The late apoptotic cells showed apoptotic bodies and orange staining as the result of AO binding to denaturated DNA and condensation of chromatin after 72 hours of ZER treatment. However, necrotic cells displayed reddish orange intact nuclei (Figure 4).
Figure 5: Flow cytometric analysis of Jurkat cells treated with 5.39 µg/mL ZER and stained with FITC-conjugated annexin-V and PI. A1-C1: Untreated (control) at 6, 12 and 24 h, respectively. A2-C2: ZER-treated after 6, 12 and 24 h, respectively.
Table 1: Flow cytometric analysis of Jurkat cells after treatment with ZER. The cells were stained with FITC-conjugated annexin-V and PI and incubated at 37ºC for 6, 12 and 24 h.
Cells (%) Cell Condition
Control 6 h
ZER 6 h
Control 12 h
ZER 12 h
Control 24 h
ZER 24 h
Viable cells 96.9±0.2 79.2±0.2 96.4±0.2 21.7±0.7 93.9±0.2 15.5±0.7 Early apoptosis
1.7±0.1 12.3±0.5* 1.8±0.07 63.1±0.5* 3.4±0.1 21.4±0.4*
Late apoptosis/ Necrosis
1.4±0.1 8.4±0.6** 1.8±0.2 15.2±0.1** 276.0±0.1 63.1±0.6**
Significant (P<0.05) increases in (*) early apoptotic and (**) late apoptotic/necrotic cells in ZER-treated groups in comparison with untreated control.
Annexin-V FITC assay revealed that the percentage of viable cells gradually decreased while the percentage of early and late apoptotic
Antiproliferative effect of zerumbone on Jurkat cells Natural Product Communications Vol. 9 (9) 2014 1239
Figure 6: Cell cycle analysis of Jurkat cells treated with 5.39 µg/mL ZER and stained with PI. A1-C1: A1-C1: Untreated (control) at 6, 12 and 24 h, respectively. A2-C2: ZER-treated after 6, 12 and 24 h, respectively. G0/G1, G2/M, and S indicate cell phase, and subG0-G1 are apoptotic cells.
Table 2: Cell cycle flow cytometric analysis of Jurkat cells after treatment with ZER. The cells were stained with PI and incubated at 37ºC for 24, 48 and 72 h.
Cell Cycle Phases
Cells (%) Control 24 h
ZER 24 h
Control 48 h
ZER 48 h
Control 72 h
ZER 72 h
G0/G1 63.4±0.1 41.9±0.4 51.8±0.3 29.5±0.5 59.4±0.3 41.3±0.7 G2/M Synthesis Sub G0/G1
13.1±0.8 20.7±0.1 3.2±0.2
26.7±0.4* 24.1±0.3 7.6±0.3
12.8±0.3 29.9±0.1 5.1±0.3
48.0±0.3* 17.3±0.1 5.1±0.2
6.0±0.2 26.3±0.6 8.7±0.5
23.3±0.9* 17.2±0.2 18.7±0.6
(*): Significant (P<0.05) increase of cells in G2/M phase in the ZER treated group in comparison with untreated controls. Table 3: Flow cytometric analysis for apoptosis in ZER-treated Jurkat cells after staining with rTdT for 24, 48 and 72 h. Cells (%)
Cell Stages Control 24 h
ZER 2h
Control 48 h
ZER 48 h
Control 72 h
ZER 72 h
Viable cells 96.6±0.4 79.8±0.1 97.6±0.2 67.5±0.3 95.7±0.2 59.7±0.
Apoptotic cells 6.2±0.1 21.1±0.4* 2.4±0.5 32.4±0.3* 4.3±0.3 41.3±0.4*
(*): Significant (P<0.05) increase in apoptotic cells of the ZER-treated group in comparison with untreated controls. Table 4: Caspases activity in Jurkat cells treated with ZER. Cells %
Caspase Control 24 h
ZER 24 h
Control 48h
ZER 48 h
Control 72 h
ZER 72 h
Caspase -3 0.05±0.1 0.12±1.1* 0.07±0.9 0.18±0.2* 0.09±0.4 0.20±0.5*
Caspase -9 0.06±1.0 0.15±1.3* 0.06±0.7 0.20±0.7* 0.08±0.4 0.24±1.0*
(*): Significant (P<0.05) increase in apoptotic cells in the ZER-treated group in comparison with untreated controls.
cells gradually increased significantly (P<0.05) in all groups (Figure 5 and Table 1). Cell cycle analysis demonstrated that ZER induced significant (P<0.05) depletion in the G1 phase population of Jurkat cells with concomitant increase in cells in the G2/M phase after 24, 48 and 72 hours of treatment, respectively (Figure 6 and Table 2). There was a significantly (P<0.05) greater number of TUNEL- positive Jurkat cells (apoptotic cells) in the ZER-treated group compared with the untreated group (Figure 7 and Table 3). The DNA fragmentation and number of apoptotic Jurkat cells in the ZER-treated group increased with time.
Figure 7: Flow cytometric analysis (TUNEL assay) of ZER-treated Jurkat cells. A1-C1: Untreated (control) at 6, 12 and 24 h, respectively. A2-C2: ZER-treated after 6, 12 and 24 h, respectively. Lower quadrant (R2 and R3) are non-apoptotic and upper quadrant (R4) are apoptotic cells.
Figure 8: Caspase activity in ZER-treated Jurkat cells. The values are mean % ± SD of three independent experiments. There were significant differences (P<0.05) in caspase -3 and -9 activities between treated and control groups.
ZER significantly (P<0.05) caused caspase˗3 and ˗9 to a one to three-fold increase in activity over the control. The increase in activity of these caspases was time-dependent (Figure 8 and Table 4). Medicinal herbs are among the most commonly used complementary and alternative medicines for cancer treatments, with few side-effects [32]. Although information on the potency of pure compounds derived from the medicinal herb is limited, Zingiber zerumbet is still being used as a folk medicine for various ailments. ZER is one of the major phytochemicals derived from the essential oil of the rhizomes [33]. The effect of this compound on Jurkat cells was not known, but in this investigation, we demonstrated for the first time that ZER has significant (P<0.05) cytotoxic effect on Jurkat cells in a dose and time-dependent manner. However, ZER is not as potent as doxorubicin. It is critical for an anticancer agent to exhibit high cytotoxicity to cancer cells, but not to normal healthy cells. ZER fits this characteristic, being toxic to Jurkat cells while essentially not adversely affecting normal cells. This observation is consistent with that of a previous study which also showed that ZER inhibits the
1240 Natural Product Communications Vol. 9 (9) 2014 Rahman et al.
proliferation of cancer cells, while minimally affecting normal cell proliferation [34]. The anticancer effect of ZER is suggested to be through its α, β-unsaturated carbonyl group, which effectively removes the intracellular glutathione (GSH) by forming a Michael adduct and raising the intracellular redox potential (E). The net effect of this action is inhibition of proliferation of cancer cells. Since the average E in normal cells is higher than that in cancer cells, it is possible that in normal cells E could not be raised high enough by ZER to stop proliferation, which accounts for its weaker effect [35,36]. This selective effect of ZER is especially useful in clinical applications when treatment requires repeated and prolonged dosage. Our study showed that the greatest promise for ZER would be in the treatment of leukemia. Microscopic examination is the gold standard for determination of apoptosis [37]. With this technique the whole process of apoptosis can be observed and evaluated based on reliable morphological criteria [38]. In our study, phase contrast, fluorescence and electron microscopic examination showed that leukemic cells underwent apoptosis upon treatment with ZER. The main morphological changes of treated Jurkat cells included blebbing of the cell membrane, progressive nuclear shrinkage and apoptotic body formation, especially at 72 hours post-treatment. Other morphological changes included nuclear margination, chromatin condensation, and DNA fragmentation. ZER triggered formation of these apoptotic morphological features in Jurkat cells in a time-dependent manner. To determine the cell cycle distribution of Jurkat cells, the ZER-treated cells were subjected to flow cytometry in the annexin-V FITC, cell cycle assay and TUNEL assays. The number of viable Jurkat cells after treatment with ZER decreased with time. The analyses also showed that the anticancer activity of ZER on the Jurkat cell line is via the induction of G2/M cell cycle arrest, which is similar to its effect on the P388D1 and NB4 cell lines [31]. The activation of caspases plays a pivotal role in the apoptosis signaling pathway [39] since caspase˗3 is required for DNA fragmentation and most morphological changes related to apoptosis [40]. Caspase˗3 and ˗9 are involved in the mitochondrial intrinsic pathway [41]. In Jurkat cells, ZER significantly increased caspase˗3 and ˗9 activities. The apoptogenic effect of ZER on Jurkat cells is suggested to occur via the mitochondrial intrinsic pathway through the activation of the mitochondrial-dependent caspase cascade. Thus ZER can be further developed into a safe therapeutic compound for the treatment of leukemia. Experimental
Cell culture: Human acute T-cell leukemia cell line (Jurkat, J.RT3-T3.5) was purchased from American Type Culture Collection (ATCC) (Maryland, USA). The cells were maintained in RPMI-1640 (ATCC, USA) medium, supplemented with sodium pyruvate (2 mM), 10% heat-inactivated fetal calf serum (FCS) (ATCC, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Sigma Aldrich, USA), according to the ATCC protocol, cultured in 75 cm2 culture flasks (TPP, Switzerland) at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Cultures were frequently examined under an inverted microscope (Micros, Austria) for confluency and viability. ZER extraction: Pure (99.96%), colorless ZER crystals were prepared from essential oil extracted from fresh Zingiber zerumbet rhizomes by steam-distillation, as described earlier [42]. The ZER crystals were kept in clean glass vials at 4ºC until used.
Cytotoxicity of ZER towards Jurkat cells: The Jurkat cells were seeded into 96-well microculture plates (TPP, Switzerland) at a concentration of 1 × 105 cells/mL and treated with various concentrations of ZER. After incubation for 24, 48 and 72 h at 37ºC in 5% CO2, MTT solution (Sigma Aldrich, USA) was added, and incubated for an additional 4 h in the dark. Immediately the media were aspirated and the remaining purple formazan were then lysed with MTT solubilization solution (Sigma Aldrich, USA). The assay was performed in triplicate. Subsequently, the optical density (OD) was measured at 570 nm using an ELISA plate reader (Universal Microplate reader) (Biotech, Inc, USA). The IC50 value was determined from the absorbance versus concentration curve. Finally, the values were compared with those of the known antineoplastic agent, doxorubicin (Sigma Aldrich, USA) and with DMSO (Sigma Aldrich, USA) (0.1%) as negative control. Cytotoxicity of ZER toward primary blood mononuclear cells (PBMC): BD vacutainer® (CPT™) was used for the separation of primary lymphocytic cells from whole human blood. About 8 mL fresh blood was collected and mixed well. Immediately after centrifugation, the buffy coat containing lymphocytes was gently aspirated and transferred into a cell culture flask containing RPMI-1640 medium (ATCC, USA) supplemented with 10% heat inactivated fetal calf serum. The flask was incubated at 37ºC in 5% CO2. Next day, the cells were treated with ZER for 24, 48 and 72 h. The cells were then harvested and examined for evidence of cytotoxicity using the trypan blue exclusion assay [43].
Light microscopy: The Jurkat cells were allowed to grow in a 25 cm2 cell culture flask until 90% confluency and their viability determined using a hemocytometer. Then, 3 mL of Jurkat cells (5 × 104 cells/mL) were plated into each well of a 6-well plate (TPP, Switzerland), treated with 5.39 ± 0.43 µg/mL (IC50) ZER and incubated at 37ºC in 5% CO2. The untreated cells were used as negative control. Morphological changes were visualized using a normal inverted microscope (Labomed, USA) at 24, 48 and 72 h post-treatment. Fluorescence microscopy: The Jurkat cells were plated at a concentration of 1 × 106 cell/mL in a culture flask, treated with ZER and incubated at 37ºC in a 5% CO2 incubator for 24, 48, and 72 h. At the end of these treatments, the cells were centrifuged and the supernatant discarded. Approximately 10 µL of the cell pellets was stained with 10 μL fluorescent dye mixture containing equal volumes (100 µg/mL) of AO and PI for 2 min. Then approximately 10 μL of freshly stained cell suspension was placed onto a glass slide, covered with a cover slip and examined under a fluorescence microscope (Leica, Japan) with Q-floro software installed.
Scanning electron microscopy: Jurkat cells (2 × 106 cells/mL) treated with ZER were incubated for 24, 48 and 72 h in a 5% CO2
incubator. Untreated cells at 0 h were used as control. The cells were then centrifuged and the pellets fixed with 4% (v/v) glutaraldehyde in 0.1 M cocodylate buffer for 6 h at 4ºC and washed with 3 changes of sodium cocodylate buffer for 10 min each, and post-fixed in 1% osmium tetraoxide for 2 h at 4ºC. The pellets were then rewashed with 3 changes of sodium cocodylate buffer for 10 min each and dehydrated in ascending acetone concentrations of 35, 50, 75 and 95% for 10 min each, followed by trice in 100% acetone, 15 min each time. Critical point drying was achieved using a drier (CDP 030, Bal-TEC, Switzerland) for 30 min. The cells were then affixed to a metal SEM stub and sputter coated with gold using SEM coating unit (E5100 Polaron, UK). The coated specimens were viewed using a scanning electron microscope (JOEL 64000, Japan).
Antiproliferative effect of zerumbone on Jurkat cells Natural Product Communications Vol. 9 (9) 2014 1241
Annexin V-FITC assay: Apoptosis of Jurkat cells treated with ZER were detected with an annexin V-FITC kit according to the manufacturer’s instructions (Sigma Aldrich, USA). Finally, flow cytometric analysis was conducted under laser emitting excitation light at 488 nm using a BD FACS Calibur flow cytometer equipped with an Argon laser (BD, USA). Data analysis was performed using the Cell Quest Pro software. Cell cycle assay: Approximately 2.5 × 106 Jurkat cells/mL were cultured with ZER and incubated for 24, 48 and 72 h. The cells were harvested by centrifugation and washed with PBS. Subsequently, 500 µL of 80% ice-cold ethanol was added to the cell pellets, and then kept at ˗20ºC for 1 week. Then, cell pellets were washed twice with 1 mL washing PBS and stained with PBS staining buffer containing 50 µg/mL RNAase A and 3 µg/mL PI and incubated on ice in the dark for 30 min. Flow cytometric analysis was conducted using a BD FACS Calibur flow cytometer. Data analysis was performed using the Cell Quest Pro software.
TUNEL assay: The induction of apoptosis in Jurkat cells following incubation with ZER was evaluated using the TUNEL (Tdt-mediated dUTP Nick-end labeling) assay according to the manufacturer’s protocol (DeadEndTM Fluorometric TUNEL System). In the final step, the mixture was centrifuged, supernatant
removed and cells resuspended in 1 mL PBS containing 5 µg PI and 250 µg RNAase A. The cell samples were analyzed by BD FACS Calibur flow cytometer.
Caspase -3 and -9 assays: The activities of caspase˗3 and ˗9 in the ZER-treated Jurkat cells were determined using a colorimetric assay kit (Gene script kit, Piscataway, NJ 08854, USA). The reaction suspensions were read at 405 nm in a microplate reader (Universal Microplate reader) (Biotech, Inc, USA). Data were presented as optical density (OD) (405 nm; mean SD) and histogram plotted. Data analysis: The experiments were conducted in triplicate and the results expressed as mean ± SD. Statistical analysis was made using SPSS version 17.0 (SPSS Inc., Chicago, USA). Probability values of less than alpha 0.05 (P<0.05) were considered statistically significant. Acknowledgments - We would like to express our gratitude to the National Cancer Council Malaysia (MAKNA), Institute of Bioscience (IBS), Universiti Putra Malaysia for their kind assistance. This project was funded by the Ministry of Science, Technology and Innovation (MOSTI, Grant No. 5495308), Malaysia.
References
[1] Yeoh AE, Lu Y, Chan JY, Chan YH, Ariffin H, Kham SK, Quah TC. (2010) Genetic susceptibility to childhood acute lymphoblastic leukemia shows protection in Malay boys: results from the Malaysia-Singapore ALL study group. Leukemia Research, 34, 276-283.
[2] Butler MS. (2008) Natural products to drugs: natural product-derived compounds in clinical trials. Natural Product Reports, 25, 475-516. [3] Kitayama T. (2011) Attractive reactivity of a natural product, zerumbone. Bioscience, Biotechnology and Biochemistry, 75, 199-207. [4] Kader G, Nikkon F, Rashid MA, Yeasmin T. (2011) Antimicrobial activities of the rhizome extract of Zingiber zerumbet Linn. Asia -Pacific
Journal of Tropical Biomedicine, 1, 409-412. [5] Somchit MN, Shukriyah MH, Bustamam AA, Zuraini A. (2005) Anti-pyretic and analgesic activity of Zingiber zerumbet. International Journal of
Pharmacology, 1, 277-280. [6] Yob NJ, Jofrry Sm, Affandi MMR, Teh LK, Salleh MZ, Zakaria ZA. (2011) Zingiber zerumbet (L.) Smith: a review of its ethnomedicinal,
chemical, and pharmacological uses. Evidence-Based Complementary and Alternative Medicine, 2011, 1-12. [7] Al-Amin M, Sultana GN, Hossain CF. (2012) Antiulcer principle from Zingiber montanum. Journal of Ethnopharmacology, 141, 57-60. [8] Habsah M, Amran M, Mackeen MM, Lajis NH, Kikuzaki H, Nakatani N, Rahman AA, Ali AM. (2000) Screening of Zingiberaceae extracts for
antimicrobial and antioxidant activities. Journal of Ethnopharmacology, 72, 403-410. [9] Tzeng TF, Liou SS, Chang CJ, Liu IM. (2013) The ethanol extract of Zingiber zerumbet attenuates Streptozotocin-induced diabetic nephropathy in
rats. Evidence-Based Complementary and Alternative Medicine, 2013, 1-8. [10] Elhassan MM, Syam MM. (2008) Anti-tumor activities of analogues derived from the bioactive compound of Zingiber zerumbet. International
Journal of Cancer Research, 4, 154-159. [11] Rashid RA, Pihie AHL. (2005) The antiproliferative effects of Zingiber zerumbet extracts and fractions on the growth of human breast carcinoma
cell lines. Malaysian Journal of Pharmaceutical Sciences, 3, 45-52. [12] Rasedee A, Heshu SR, Ahmad Bustamam A, How CW, Swee KY. (2013) A composition for treating leukaemia. Malaysian Patent Application,
PI2013700213. [13] Sulaiman MR, Perimal EK, Akhtar MN, Mohamad AS, Khalid MH, Tasrip NA, Mokhtar F, Zakaria ZA, Lajis NH, Israf DA. (2010) Anti-
inflammatory effect of zerumbone on acute and chronic inflammation models in mice. Fitoterapia, 81, 855-858. [14] Zakaria ZA, Mohamad AS, Chear CT, Wong YY, Israf DA, Sulaiman MR. (2010) Antiinflammatory and antinociceptive activities of Zingiber
zerumbet methanol extract in experimental model systems. Medical Principles and Practice, 19, 287-294. [15] Somchit MN, Mak JH, Ahmad Bustamam A, Zuraini A, Arifah AK, Adam Y, Zakaria ZA. (2012) Zerumbone isolated from Zingiber zerumbet
inhibits inflammation and pain in rats. Journal of Medicinal Plant Research, 6, 177-180. [16] Sulaiman MR, Mohamad TAS, Mossadeq WMS, Moin S, Yusof M, Mokhtar AF, Zakaria ZA, Israf DA, Lajis NH. (2009) Antinociceptive activity
of the essential oil of Zingiber zerumbet. Planta Medica, 76, 107-112. [17] Tewtrakul S, Subhadhirasakul S. (2007) Anti-allergic activity of some selected plants in the Zingiberaceae family. Journal of Ethnopharmacology,
109, 535-538. [18] Rhode J, Fogoros S, Zick S, Wahl H, Griffith KA, Huang J, Liu JR. (2007) Ginger inhibits cell growth and modulates angiogenic factors in ovarian
cancer cells. BMC-Complementory and Alternative Medicine, 7, 1-9. [19] Tzeng TF, Liu IM. (2013) 6-Gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid droplets in 3T3-L1 cells. Phytomedicine, 20,
481-487. [20] Jantan I, Raweh SM, Sirat HM, Jamil S, Mohd Yasin YH, Jalil J, Jamal JA. (2008) Inhibitory effect of compounds from Zingiberaceae species on
human platelet aggregation. Phytomedicine, 15, 306-309. [21] El-Sharaky AS, Newairy AA, Kamel MA, Eweda SM. (2009) Protective effect of ginger extract against bromobenzene-induced hepatotoxicity in
male rats. Food and Chemical Toxicology,47, 1584-1590. [22] Abdel Wahab IS, Abdul AB, Alzubairi AS, Mohamed EM, Mohan S. (2009) In vitro ultramorphological assessment of apoptosis induced by
zerumbone on (HeLa). BioMed Research International, 2009, 1-10. [23] Sung B, Jhurani S, Ahn KS, Mastuo Y, Yi T, Guha S, Liu M, Aggarwal BB. (2008) Zerumbone down-regulates chemokine receptor CXCR4
expression leading to inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells. Cancer Research, 68, 8938-8944.
1242 Natural Product Communications Vol. 9 (9) 2014 Rahman et al.
[24] Sung B, Murakami A, Oyajobi BO, Aggarwal BB. (2009) Zerumbone abolishes RANKL-induced NF-kappa B activation, inhibits osteoclastogenesis, and suppresses human breast cancer induced bone loss in athymic nude mice. Cancer Research, 69, 1477-1484.
[25] Yodkeeree S, Sung B, Limtrakul P, Aggarwal BB. (2009) Zerumbone enhances TRAIL-induced apoptosis through the induction of death receptors in human colon cancer cells: Evidence for an essential role of reactive oxygen species. Cancer Research, 69, 6581-6589.
[26] Alwi SS, Nallappan M, Hawariah A, Pihie L. (2007) Zerumbone exerts antiproliferative activity via apoptosis on HepG2 cells. Malaysian Journal of Biochemistry and Molecular Biology, 15, 19-23.
[27] Nabilah MN, Ahmad BA, Mohd Aspolla S, Siddig IA, Eltayeb ME, Syam M, Behnam K, Theebaa A, Kuan BN, Suvitha S, Ismail AA, Heshu SR Hapipah A. (2013) Inclusion complex of zerumbone with hydroxypropyl-ß-cyclodextrin induces apoptosis in liver hepatocellular HepG2 cells via caspase 8/BID cleavage switch and modulating Bcl2/Bax ratio. Evidence Based Complementory and Alternative Medicine, 2013, 1-16.
[28] Abdelwahab SI, Abdul AB, Zain ZN, Hadi AH. (2012) Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells. International Journal of Immunopharmacology, 12, 594-602.
[29] Chakraborty A, Coffman R, Jorvig J. (2013) Zerumbone, a phytochemical from Asian ginger is a novel inhibitor of Jak2/Stat3 inhibits promigratory gene expression, growth and migration of pancreatic cancer cells. Pancreatology, 13, e18-e19.
[30] Huang GC, Chien TY, Chen LG, Wang, CC. (2005) Antitumor effects of zerumbone from Zingiber zerumbet in P-388D1 cells in vitro and in vivo. Planta Medica, 71, 219-224.
[31] Xian M, Ito K, Nakazato T, Shimizu T, Chen CK, Yamato K, Murakami A, Ohigashi H, Ikeda Y, Kizaki M. (2007) Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas and mitochondria mediated pathway. Cancer Science, 98, 118-126.
[32] Chrystal K, Allan S, Forgeson G, Isaacs R. (2003) The use of complementary/alternative medicine by cancer patients in a New Zealand regional cancer treatment centre. The New Zealand Medicinal Journal, 116, 1168-1171.
[33] Kitayama T. (2011) Attractive reactivity of a natural product, zerumbone. Bioscience, Biotechnology, and Biochemistry, 75, 199-207. [34] Murakami A, Takahashi D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro A, Nakamura Y, Jiwajinda S, Terao J. Ohigashi H. (2002) Zerumbone,
a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: the α, β-unsaturated carbonyl group is a prerequisite. Carcinogenesis, 23, 795-802.
[35] Hoffman A, Spetner LM, Burke M. (2002) Redox-regulated mechanism may account for zerumbone’s ability to suppress cancer-cell proliferation. Carcinogenesis, 23, 1961-1962.
[36] Sadhu SK, Khatun A, Ohtsuki T, Ishibashi M. (2007) First isolation of sesquiterpenes and flavonoids from Zingiber spectabile and identification of zerumbone as the major cell growth inhibitory component. Natural Product Research, 21, 1242-1247.
[37] Yasuhara S, Zhu Y, Matsui T, Tipirneni N, Yasuhara Y, Kaneki M, Rosenzweig A, Martyn AJ. (2003) Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis. Journal of Histochemistry and Cytochemistry, 51, 873-885.
[38] Wyllie AH. (1980) Cell death: the significance of apoptosis. International Review of Cytology, 68, 251-301. [39] Allen RT, Hunter WJ, Agrawal DK. (1997) Morphological and biochemical characterization and analysis of apoptosis. Journal of Pharmacology
and Toxicology Methods, 37, 215-228. [40] D'Mello SR, Kuan CY, Flavell RA, Rakic P. (2000) Caspase-3 is required for apoptosis associated DNA fragmentation but not for cell death in
neurons deprived of potassium. Journal of Neuroscience Research, 59, 24-31. [41] Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR, Ashkenazi A. (2009) Cullin3-based polyubiquitination and p62-dependent aggregation of
caspase-8 mediate extrinsic apoptosis signaling. Cell, 137, 721-735. [42] Rahman HS, Rasedee A, Chee WH, Ahmad Bustamam A, Zeenathul NA, Othman HH, Mohamed IS, Swee KY. (2013) Zerumbone loaded
nanostructured lipid carriers: Preparation, characterization and antileukemia effect. International Journal of Nanomedicine, 8, 2769-2781. [43] Willimott S, Barker J, Jones LA, Opara EI. (2007) Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human
lymphocytes. Journal of Ethnopharmacology, 114, 290-299.
Natural Product Communications Vol. 9 (9) 2014 Published online (www.naturalproduct.us)
New Mechanism of Magnolol and Honokiol from Magnolia officinalis against Staphylococcus aureus Tao Liu, Yalin Pan and Renfu Lai 1307
Molecular Cloning and Characterization of Tyrosine Aminotransferase and Hydroxyphenylpyruvate Reductase, and Rosmarinic Acid Accumulation in Scutellaria baicalensis Yeon Bok Kim, Md Romij Uddin, YeJi Kim, Chun Geon Park and Sang Un Park 1311
Phenols and Antioxidant Activity in Vitro and in Vivo of Aqueous Extracts Obtained by Ultrasound-Assisted Extraction from Artichoke By-Products Rossana Punzi, Annalisa Paradiso, Cristina Fasciano, Antonio Trani, Michele Faccia, Maria Concetta de Pinto and Giuseppe Gambacorta 1315
Bioactive Metabolites from Cnidoscolus souzae and Acmella pilosa Hiatzy E. Zapata-Estrella, Azeret D. M. Sánchez-Pardenilla, Karlina García-Sosa, Fabiola Escalante-Erosa, Fátima de Campos-Buzzi, Nara Lins Meira-Quintão, Valdir Cechinel-Filho and Luis M. Peña-Rodríguez 1319
Dipterostilbenosides A and B, Oligostilbene Glycosides from Dipterocarpus tuberculatus Serm Surapinit, Jonkolnee Jong-aramruang, Pongpun Siripong, Suttira Khumkratok and Santi Tip-pyang 1323
Isolation of β-Indomycinone Guided by Cytotoxicity Tests from Streptomyces sp. IFM11607 and Revision of its Double Bond Geometry Kentaro Tsukahara, Kazufumi Toume, Hanako Ito, Naoki Ishikawa and Masami Ishibashi 1327
Daurichromenic Acid-producing Oxidocyclase in the Young Leaves of Rhododendron dauricum Futoshi Taura, Miu Iijima, Jung-Bum Lee, Toshihiro Hashimoto, Yoshinori Asakawa and Fumiya Kurosaki 1329
Synthesis and Characterization of 4-Aryl-4H-chromenes from H-Cardanol Hulluru Surya Prakash Rao and Mani Kamalraj 1333
Galactans of Gracilaria pudumadamensis (Gracilariales, Rhodophyta) of Indian Waters Stalin Kondaveeti, Sanjay Kumar, Meenakshi S. Ganesan and Arup K. Siddhanta 1341
The Effect of Ginkgo biloba and Camellia sinensis Extracts on Psychological State and Glycemic Control in Patients with Type 2 Diabetes Mellitus Lina Lasaite, Asta Spadiene, Nijole Savickiene, Andrejs Skesters and Alise Silova 1345
Comparative Anti-inflammatory Effects of Anti-arthritic Herbal Medicines and Ibuprofen Joshua J. Kang, Mohammed A. Samad, Kye S. Kim and Soochan Bae 1351
Quantitative Analysis Coupled with Toxic Evaluation to Investigate the Influence of Sulfur-Fumigation on the Quality of Chrysanthemum morifolium Ke Ding, Gang Cao, Zhiwei Xu and Xiaocheng Chen 1357
Chemical Composition of the Leaf Oil of Actephila excelsa from Vietnam Do N. Dai, Tran D. Thang, Dau B. Thin and Isiaka A. Ogunwande 1359
Composition and Chemical Variability of Corsican Pinus halepensis Cone Oil Anne-Marie Nam, Joseph Casanova, Félix Tomi and Ange Bighelli 1361
Hawaiian Sandalwood: Oil Composition of Santalum paniculatum and Comparison with Other Sandal Species Norbert A. Braun, Sherina Sim, Birgit Kohlenberg and Brian M. Lawrence 1365
Aroma Compounds of Mountain Tea (Sideritis scardica and S. raeseri) from Western Balkan Bujar Qazimi, Gjoshe Stefkov, Marija Karapandzova, Ivana Cvetkovikj and Svetlana Kulevanova 1369
Chemical Composition of the Essential Oil of the Local Endemics Centaurea davidovii and C. parilica (Asteraceae, sect. Lepteranthus) from Bulgaria Antonella Maggio, Luana Riccobono, Svetlana Bancheva, Maurizio Bruno and Felice Senatore 1373
Compositional Analysis and in vitro Protective Activity against Oxidative Stress of Essential Oils from Egyptian Plants Used in Traditional Medicine Tarek F. Eissa, Elena González-Burgos, M. Emilia Carretero and M. Pilar Gómez-Serranillos 1377
Chemical Composition and Antifungal Activity of the Essential Oils of Schinus weinmannifolius Collected in the Spring and Winter Camila Hernandes, Silvia H. Taleb-Contini, Ana Carolina D. Bartolomeu, Bianca W. Bertoni, Suzelei C. França and Ana Maria S. Pereira 1383
The Essential Oil Profiles and Antibacterial Activity of Six Wild Cinnamomum species Charles Santhanaraju Vairappan, Thilahgavani Nagappan and Julius Kulip 1387
Accounts/Reviews Dissecting Traditional Chinese Medicines by Omics and Bioinformatics Yuan Quan, Zhong-Yi Wang, Min Xiong, Zheng-Tao Xiao and Hong-Yu Zhang 1391
Natural Product Communications 2014
Volume 9, Number 9
Contents
Original Paper Page
The Leaf, Wood and Bark Oils of three Species of Myodocarpus (Myodocarpaceae) Endemic to New Caledonia Nicolas Lebouvier, Douglas Lawes, Edouard Hnawia, Michael Page, Joseph Brophy and Mohammed Nour 1223
Iridoids and a Norsesquiterpenoid from the Leaves of Villaria odorata Mario A. Tan, Raychel Ann U. Villacorta, Grecebio Jonathan D. Alejandro and HiromitsuTakayama 1229
Induction, Cloning and Functional Expression of a Sesquiterpene Biosynthetic Enzyme, -Guaiene Synthase, of Aquilaria microcarpa Cell Cultures Jung-Bum Lee, Syun Hirohashi, Yoshimi Yamamura, Futoshi Taura and Fumiya Kurosaki 1231
Zerumbone Induces G2/M Cell Cycle Arrest and Apoptosis via Mitochondrial Pathway in Jurkat cell Line Heshu Sulaiman Rahman, Abdullah Rasedee, Max Stanley Chartrand, Hemn Hassan Othman, Swee Keong Yeap and Farideh Namvar 1237
Diterpenoids from Fagonia mollis Amal Sallam, Alfarius Eko Nugroho, Yusuke Hirasawa, Wong Chin-Piow, Toshio Kaneda, Osamu Shirota, Sahar R. Gedara and Hiroshi Morita 1243
Cytotoxicity of the Diterpene 14-O-Methyl-ryanodanol from Erythroxylum passerinum in an Astrocytic Cells Model Noélio de Jesus Menezes-Filho, Cleide dos Santos Souza, Tereza Cristina Silva Costa, Victor Diógenes Amaral da Silva, Cátia Suse de Oliveira Ribeiro, Marizeth Liborio Barreiros, Jose Fernando Oliveira Costa, Jorge Mauricio David, Juceni P.L. David and Silvia Lima Costa 1245
Absolute Configuration of Cembrane Diterpenoids from Bursera multijuga Juan D. Hernández-Hernández, Hugo A. García-Gutiérrez, Luisa U. Román-Marín, Yunuen I. Torres-Blanco, Carlos M. Cerda-García-Rojas and Pedro Joseph-Nathan 1249
Trichostemonoate, a New Anticancer Tirucallane from the Stem Bark of Walsura trichostemon Kiettipum Phontree, Jirapast Sichaem, Suttira Khumkratok, Pongpun Siripong and Santi Tip-pyang 1253
A New Cycloartane Glucoside from Rhizophora stylosa Phan Thi Thanh Huong, Chau Ngoc Diep, Nguyen Van Thanh, Vu Anh Tu, Tran Hong Hanh, Nguyen The Cuong, Nguyen Phuong Thao, Nguyen Xuan Cuong, Do Thi Thao, Tran Huy Thai, Nguyen Hoai Nam, Ninh Khac Ban, Phan Van Kiem and Chau Van Minh 1255
Kolgaosides A and B, Two New Triterpene Glycosides from the Arctic Deep Water Sea Cucumber Kolga hyalina (Elasipodida: Elpidiidae) Alexandra S. Silchenko, Anatoly I. Kalinovsky, Sergey A. Avilov, Pelageya V. Andryjashchenko, Sergey N. Fedorov, Pavel S. Dmitrenok, Ekaterina A. Yurchenko, Vladimir I. Kalinin, Antonina V. Rogacheva and Andrey V. Gebruk 1259
Acaricidal Activity against Panonychus citri and Active Ingredient of the Mangrove Plant Cerbera manghas Yecheng Deng, Yongmei Liao, Jingjing Li, Linlin Yang, Hui Zhong, Qiuyan Zhou and Zhen Qing 1265
Three New Steroid Biglycosides, Plancisides A, B, and C, from the Starfish Acanthaster planci Alla A. Kicha, Thi H. Dinh, Natalia V. Ivanchina, Timofey V. Malyarenko, Anatoly I. Kalinovsky, Roman S. Popov, Svetlana P. Ermakova, Thi T. T. Tran and Lan P. Doan 1269
Unusual 2(1H)-Pyrazinones Isolated from a Culture of a Brazilian Marine-Derived Streptomyces sp. Sérgio S. Thomasi, Ana C. Granato, Luis H. Romano, Liene Dhooghe, Eduardo S. P. do Nascimento, Alberto C. Badino, Maria F. G. F. da Silva, Antonio G. Ferreira and Tiago Venâncio 1275
An HPLC Evaluation of Cytochalasin D Biosynthesis by Xylaria arbuscula Cultivated in Different Media Luciana da S. Amaral, Edson Rodrigues-Filho, Carolina A. A. Santos, Lucas M. de Abreu and Ludwig H. Pfenning 1279
A Simple Method for Isolation and Purification of DIBOA-Glc from Tripsacum dactyloides Cammy D. Willett, Robert N. Lerch, Keith W. Goyne, Nathan D. Leigh, Chung-Ho Lin and Craig A. Roberts 1283
Antimicrobial Metabolites from Endophytic Streptomyces sp. YIM61470 Xueqiong Yang, Yun Liu, Shuquan Li, Fangfang Yang, Lixing Zhao, Li Peng and Zhongtao Ding 1287
Genkwanin 4´-O-glucosyl-(1→2)-rhamnoside from New Chemotype of Asplenium normale in Japan Tao Fujiwara, Ayumi Uehara, Junichi Kitajima, Tsukasa Iwashina, Sadamu Matsumoto and Yasuyuki Watano 1289
Potent SIRT1 Enzyme-stimulating and Anti-glycation Activities of Polymethoxyflavonoids from Kaempferia parviflora Asami Nakata, Yuka Koike, Hirofumi Matsui, Tsutomu Shimada, Masaki Aburada and Jinwei Yang 1291 Protective Activity of C-Geranylflavonoid Analogs from Paulownia tomentosa against DNA Damage in 137Cs Irradiated AHH-1Cells Hyung-In Moon, Min Ho Jeong and Wol Soon Jo 1295
Antibacterial Activities of Oxyprenylated Chalcones and Napthtoquinone against Helicobacter pylori Charles Bodet, Christophe Burucoa, Steeve Rouillon, Nicolas Bellin, Vito Alessandro Taddeo, Serena Fiorito, Salvatore Genovese and Francesco Epifano 1299
Anti-proliferation Effect on Human Breast Cancer Cells via Inhibition of pRb Phosphorylation by Taiwanin E Isolated from Eleutherococcus trifoliatus Hui-Chun Wang, Yen-Hsueh Tseng, Hui-Rong Wu, Fang-Hua Chu, Yueh-Hsiung Kuo and Sheng-Yang Wang 1303
Continued inside backcover
