Secoaggregatalactone-A from Lindera aggregata Induces Apoptosis in Human Hepatoma Hep G2 Cells

• Abstract
• Introduction
• Materials and Methods
• Reagents
• Plant material
• Extraction and isolation
• Cell culture and cytotoxicity assay
• Flow cytometric analysis
• Caspase-3 activity and Western blotting assays
• Statistical analysis
• Results and Discussion
• References

Abstract

A new secobutanolide, secoaggregatalactone A (1) was isolated from the leaves of Lindera aggregata. Results obtained from the cytotoxicity assay revealed that secoaggregatalactone A exhibited a noticeable cytotoxicity (EC₅₀ = 6.61 μg/mL; 22.1 μM) against the human hepatoma cell line (Hep G2 cell line). According to morphological observations, flow cytometric analysis, and DNA fragmentation analysis, it was proven that the cytotoxicity of secoaggregatalactone A on human cells was due to apoptosis. Moreover, based on the results from the protein expression assay and confocal laser scanning microscope observations, it is assumed that secoaggregatalactone A induced apoptosis through the mitochondria pathway by way of cleavage of Bit to release cytochrome C and activate caspases-9 and -3, and then degradation of PARP.

Introduction

Lindera aggregata (Sims) Kosterm (L. strychnifolia Vill) (Lauraceae) is an endemic evergreen tree grown in Taiwan. It has a strong fragrance and is a traditional Chinese medicine used for treating several symptoms including chest and abdominal pain, indigestion, regurgitation, cold hernia, and frequent urination [1]. It has been proved that extracts from roots and leaves of L. aggregata have significant superoxide dismutase (SOD)-like activity [2]. Ohno et al. evaluated the possible preventive activity of L. aggregata on the progression of diabetic nephropathy. They suggested that the water extract of L. aggregate could be a novel therapeutic agent against diabetic nephropathy [3]. Wang and his co-workers demonstrated that treatment with alkaloids from the root of L. aggregata could reduce inflammation and joint destruction in a type II collagen-induced model of rheumatoid arthritis [4]. Several sesquiterpenes, proanthocynidin trimer, and alkaloids were isolated from L. aggregata. [5], [6], [7], [8], [9]. Recently, two new sesquiterpene lactones were isolated from the water extract of L. aggregata root, which showed strong cytotoxicity against human small cell lung cancer SBC-3, and lesser cytotoxicity against mouse fibroblast cells 3T3-L1 [10]. However, the mechanism of suppression of cell growth is not clear until now. In our systematic study on the bioactivities of Lauraceae trees grown in Taiwan, extracts of L. aggregate were potently cytotoxic [11]. To investigate the active principle of L. aggregata against human hepatoma Hep G2 Cells, the methanolic extract was isolated by bioactivity-guided fractionation. A new secobutanolide, namely secoaggregatalactone A (1) was identified from the EtOAc fraction of the methanolic extract. Secoaggregatalactone A (1) induces apoptosis-related proteins in human hepatoma cells. The results of our current study are reported herein.

Materials and Methods

Reagents

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, tryspin-EDTA and fetal bovine serum (FBS) were obtained from GIBCO BRL (Gaithersburg, MD, USA). Sodium dodecyl sulfate (SDS) and Tris-HCl were purchased from Bio Basic Inc. (Markham, Canada). Dimethyl sulfoxide (DMSO), propidium iodide, ribonuclease A (RNase A), NP-40, sodium deoxycholate, z-LEHD-fmk and β-actin antibody were bought from Sigma Chemical (St. Louis, MO, USA). Poly (ADP-ribose) polymerase (PARP), Bid, procaspase-3 and procaspase-8 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals and solvents used in this study were of the reagent grade and HPLC grade.

Plant material

The leaves of L. aggregata were collected from the Experimental Forest of National Chung-Hsing University, in July, 2005 and were identified by Dr. Yen-Hsueh Tseng (NCHU). The voucher specimen [LCT001 (TCF)] was deposited in the herbarium of the same university.

Extraction and isolation

Air-dried leaves of L. aggregata (6.0 kg) were extracted with MeOH (60 L) at ambient temperature, and concentrated under vacuum to yield the MeOH extract (351.2 g). The MeOH extract was partitioned between EtOAc-H₂O to give the EtOAc-soluble fraction (60.1 g) and the H2O-soluble fraction. The EtOAc-soluble fraction displayed potent cytotoxicity (EC₅₀ = 44.83 μg/mL), and was further chromatographed over silica gel (8 × 120 cm; 60 - 80 mesh; Merck; Darmstadt, Germany) eluted with n-hexane and a gradient of n-hexane-EtOAc (95 : 5; 90 : 10; 85 : 15; 80 : 20; 70 : 30; 60 : 40; 50 : 50; 40 : 60, 0 : 100, each 2 L) to produce 27 fractions. Part of fraction 7 (5.3 g) was subjected to silica gel chromatography by elution with n-hexane-EtOAc (85 : 15), and then eluted completely with EtOAc to give 15 fractions (7 - 1 to 7 - 15). Fraction 7 - 4 (2.8 g) displayed the strongest cytotoxicity (EC₅₀ = 7.12 μg/mL), and was further separated by HPLC using a reversed-phase column (Prep Nova-Pak HR C₁₈, 300 × 7.8 mm, 6 μm column, Waters; Milford, MA, USA) with an MeOH/H₂O gradient (0 - 3 min, MeOH/H₂O = 80/20; 3 - 7 min, MeOH/H₂O = 80/20 linear gradient to 100/0; 10 - 20 min, MeOH/20 = 100/0) as eluant at a flow rate of 2.5 mL/min to obtain compound 1 (R.t. = 10.5 min) (1.68 g).

Secoaggregatalactone A (1): colorless oil; [α]_D ²⁵: + 73.75 (c 0.295, CHCl₃); UV (MeOH, log ε): λ_max = 215 nm (4.32); IR: ν_max = 3450 (br, OH), 1735 (ester), 1710 (ketone) cm^-1; ¹H-NMR (600 MHz, CDCl₃): δ = 0.86 (3H, t, J = 6.8 Hz, H-13), 1.28 (14H, br s, H-6 to H-12), 1.45 (2H, m, H-5), 2.13 (3H, s, H-3′), 2.33 (2H, q, J = 7.4 Hz, H-4), 3.71 (3H, s, OMe-1), 4.00 (1H, br d, J = 4.8 Hz, OH-1′, D₂O exchangeable), 4.88 (1H, br d, J = 4.8 Hz, H-1′), 7.06 (1H, t, J = 7.6 Hz, H-3); ¹³C NMR (125 MHz, CDCl₃): δ = 14.1 (C-13), 22.6 (C-12), 24.8 (C-3′), 28.6 (C-4), 28.7 (C-5), 29.0 - 30.0 (C-6 - 10), 31.9 (C-11), 52.1 (OMe-1), 73.3 (C-1′), 129.6 (C-2), 149.2 (C-3), 166.5 (C-1), 206.3 (C-2′); FAB-MS: m/z (rel. int.) = 299 ([M + H]⁺), HR/FAB-MS: m/z = 299.4298 [M + H]⁺, calcd. for C₁₇H₃₁O₄: 299.4295.

Cell culture and cytotoxicity assay

Hep G2 cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin-streptomycin, and were maintained at 37 °C and 5 % CO₂. All cells (1 × 10⁴) per well were seeded in 96-well plates and incubated for 24 h, and different dosages (4, 7, 10 μg/mL; 13.8, 23.4 and 33.4 μM) of secoaggregatalactone A (1) were added to each well in triplicate for 24 h. The cell viability was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Meanwhile, morphology features of cells and nuclear chromatin were observed by using a phase-contrast microscope and a confocal laser scanning microscope (CLSM ) (Leica TCS Sp2 AOBS MP; Wetzlar, Germany).

Flow cytometric analysis

Hep G2 cells (1 × 10⁶ cells/dish) seeded in a 60-mm dish were treated with 4 μg/mL (13.8 μM), 7 μg/mL (33.4 μM), and 10 μg/mL 33.4 (μM) of 1 for 24 h or treated with 10 μg/mL (33.4 μM) 1 for 0, 4, 10, 16, and 24 h, then cells were trypsinized and collected with ice cold PBS. The cells were resuspended in 200 μL PBS, and fixed in 800 μL of iced 95 % ethanol at -20 °C. After being left to stand overnight, the cell pellets were collected by centrifugation, resuspended in 1 mL of hypotonic buffer (0.5 % Triton X-100 in PBS and 0.5 mg/mL RNase A), and incubated at 37 °C for 30 min. Then, 1 mL of PI solution (50 μg/mL) was added, and the mixture was allowed to stand at 4 °C for 30 min. Fluorescence emitted from the PI-DNA complex was quantitated after excitation of the fluorescent dye by flow cytometry (FACScan, Becton Dickinson; Franklin Lakes, NJ, USA).

Caspase-3 activity and Western blotting assays

The activity of caspase-3 was determined using Chemicon’s Caspase-3 Colorimetric Activity Assay Kits (Chemicon International; Billerica, MA, USA). The detailed analysis procedure is described in the manufacturer’s protocol. The protein expression after treatment with 1 was determined by a Western blotting assay. Briefly, Hep G2 cells incubated in 60-mm culture dishes (1 × 10⁶ cells per dish) were treated with 1 at the dosages of 4 (13.8), 7 (23.4), and 10 (33.4) μg/mL (μM) for 24 h or treated with 1 at dosages of 10 (33.4 μM) for 0, 4, 10, and 16 h. 20 μg of total cell proteins were separated by electrophoresis on 12 % SDS-PAGE and transferred to PVDF membrane. Detection was performed by immunostaining using specific primary antibodies and horseradish peroxidase-conjugated anti-IgG antibody. The proteins were detected by chemiluminescence (ECL, Pierce Biotechnology, Inc.; Rockford, IL, USA). The following antibodies and working dilutions were used for the Western blots, i. e., rabbit polyclonal antibodies to human PARP (1 : 1000), procaspase-3 (1 : 1000), Bid (1 : 1000), procaspase-8 (1 : 1000), β-actin (1 : 500), Goat anti-rabbit immunoglobulin G (IgG)- horseradish peroxidase-conjugate (1 : 5000).

Statistical analysis

Data are expressed means ± SD. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (* p < 0.05 and ** p < 0.01) between the control (untreated) and treated cells were analyzed by Dunnett’s test.

Results and Discussion

Secoaggregatalactone A (1) (Fig. [1]) was obtained as a colorless oil. The molecular formula was determined to be C₁₇H₃₀O₄ by FAB-MS ([M +H]⁺, m/z = 299). The UV spectrum showed absorption maxima at 215 nm and the IR spectrum showed characteristic absorption bands due to the presence of hydroxyl (3450 cm^{- 1}), ester (1735 cm^{- 1}), and ketone (1710 cm^{- 1}) groups. In the ¹H-NMR spectra, 1 showed the signals for an olefinic proton at δ = 7.06 (1H, t, J = 7.6 Hz, H-3), a hydroxymethine proton at 4.88 (1H, br d, J = 4.8 Hz, H-1′), a methoxy group at 3.71 (3H, s, OMe-1), an acetyl group at 2.13 (3H, s, H-3′), and a terminal methyl group of a side chain at 0.86 (3H, t, J = 6.8 Hz, H-13). According to MS, UV, and ¹H NMR analysis, 1 was similar to secolincomolide A [12], except for the fact that secolincomolide A showed one more methylene units than 1 in the alkyl side-chain. The dextrorotatory optical activity ([α]_D ²⁵: + 73.75) of 1 indicated that C-1′ possesses the S configuration, which is similar to secoisolancifolide ([α]_D ²⁵: + 102.7) [13], but different from that of secokotomolide ([α]_D ²⁵: -11.5) [14]. Thus, compound 1 was defined structurally as (2E)-2-[(1S)-1-hydroxy-2-oxopropyl]-2-tridecenoic acid methyl ester, and the structure was further corroborated by ¹³C-NMR, DEPT, HSQC, HMBC, and NOESY experiments. Fig. [1] shows the main HMBC and NOESY correlations of secoaggregatalactone A (1).

The cytotoxicity of 1 against Hep G2 cells was evaluated by using the standard MTT method. As shown in Fig. [2], when Hep G2 cells were treated with 1 at dosages of 1 - 20 μg/mL, a dose-dependent decrease of cell viability was observed. Comparison with the anti-tumor compound curcumin [15], [16] revealed that it exhibited a stronger cytotoxicity. The EC₅₀ value of 1 against Hep G2 cells was 6.61 μg/mL (22.1 μM). The growth-inhibiting activity of 1 was potent. With a dosage above 10 µg/mL (33.4 μM), the Hep G2 cells showed chromatin condensation, and apoptotic bodies were widespread throughout the entire population (see Supporting Information Fig. 1S). Due to the observation of morphological changes in Hep G2 cells, it is believed that 1 might cause apoptotic cell death in the Hep G2 cells. To verify the induction of apoptosis and sub-G1 (sub-2N, which has been suggested to be the apoptotic DNA) cell population of 1 furthermore, we determined the cell cycle progression of Hep G2 cells after treatment with 1. Flow cytometric analysis revealed a dose-dependent increased proportion of apoptotic cells in the sub-G₁ phase. When Hep G2 cells were treated with 0, 4 (13.8), 7 (23.4), and 10 (33.4) μg/mL (μM) of 1 for 24 h, the percentage of sub-G₁ cells was 7.1, 16.9, 25.1, and 40.7 %, respectively (Fig. [3]). On the other hand, in Hep G2 cells treated with 10 μg/mL (33.4 μM) 1 for different incubation periods, the percentage of cells in the sub-G₁ fractions was increased to 15.4 % (4 h), 17.6 % (10 h), and 25.7 % (16 h) (data not shown). The induction of apoptosis was further confirmed by flow cytometry of Hep G2 cells stained with annexin V-FITC and propidium iodide after treatment with 10 μg/mL (33.4 μM) 1 for 24 h. The annexin V assay measures phospholipid turnover from the inner to the outer lipid layer of the plasma membrane, an event typically associated with apoptosis. As indicated by flow cytometric analysis, the proportion of annexin V-staining cells increased with time in the cells treated with 1; the percentage of annexin V-staining cells was 6.6 % at 0 h, and 50.6 % at 24 h, respectively (see Supporting Information Fig. 2S). In addition to morphological observations and flow cytometric analyses, we assessed the DNA fragmentation in Hep G2 cells. Fig. [4] shows the DNA fragmentation induced by various concentrations of 1. Based on the results obtained from morphological observations, flow cytometric and DNA fragmentation analysis, 1 at 7 μg/mL (23.4 μM) for 24 h or 10 μg/mL (33.4 μM) for 16 h efficiently induced apoptosis in Hep G2 cells.

It is known that initiator caspases (e. g., caspases-8, -9, -10, and -12) are closely linked to proapoptotic signals [17]. When they are activated, these caspases are cleaved and activate downstream effectors caspases (e. g., caspases-3, -6, and -7), which sequentially cleave nuclear proteins, such as PARP and lamin A and then induce apoptosis [18]. As caspase-3 followed by PARP [Poly (ADP-ribose) polymerase] cleavage is the key event in the process of apoptosis, and as these events are also employed as markers of apoptosis induction [19], the effect of 1 on caspase-3 activity was evaluated first. Fig. [5] shows the activity of caspase-3 in Hep G2 cells, which was significantly enhanced after treatment with 4 (13.8), 7 (23.4), and 10 (33.4) μg/mL (μM) 1 for 24 h. Moreover, cleaved downstream procaspase-3 could activate the dimeric form of caspase-3 as an executioner of apoptosis. Fig. [6] A shows the results of procaspase-3, cleaved caspase-3, PARP, procaspase-8, and Bid expressions after treatment with 4 - 10 μg/mL (13.8 - 33.4 μM) 1 for 24 h or after treatment with 10 μg/mL (33.4 μM) for 4 - 24 h. It is obvious that 1 induced cleavage of procaspase-3 (32 kDa) into active caspase-3 (19 and 17 kDa), revealing that the expression of procaspase-3 was suppressed and caspase-3 activity was increased. Active caspase-3 could cleave PARP protein (116 kDa) into a 85 kDa fragment. In Fig. [6] A, [1] caused a dose- and time-dependent proteolytic cleavage of PARP, with accumulation of the 85-kDa fragment. Additionally, procaspase-8 and Bid protein expressions were markedly decreased by treatment with 10 μg/mL (33.4 μM) of 1 for different time periods (0, 4, 10, 16, and 24 h) (Fig. [6] A). On the other hand, when Hep G2 cells were incubated with 25 μM z-VAD-fmk (a general caspase-9 inhibitor) or 0.1 % DMSO (control) for 1 h before treatment with 10 μg /mL (33.4 μM) 1 for 24 h, it was obvious that caspase-9 activity was inhibited (Fig. [6] B). Meanwhile, cytochrome C was released from mitochondria after treatment with 10 μg/mL (33.4 μM) secoaggregatalactone A for 10 h by using confocal laser scanning microscope (CLSM) observation (see Supporting Information Fig. 3S). It has been reported that active caspase-8 triggers events downstream of the mitochondrial pathway of apoptosis and amplifies caspase-9 activation through the cleavage of Bid, which in turn was associated with the release of cytochrome C [20]. It is reported that 1 induced apoptosis through the mitochondrial pathway.

In summary, secoaggregatalactone A (1) was found to induce significant apoptotic cell death in Hep G2 cells, through the activation of caspase-8, Bid, and caspase-3, leading to cleavage of PARP and causing DNA fragmentation resulting in apoptosis.

References

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• 20 Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome C release from mitochondria in response to activation of cell death receptors.  Cell. 1998;  94 481-90.
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Dr. Sheng-Yang Wang

Department of Forestry

National Chung-Hsing University

250 Kuo-Kuang Road

Taichung 402

Taiwan

R.O.C.

Phone: +886-4-2284-0345 ext. 138

Fax: +886-4-2287-3628.

Email: taiwanfir@dragon.nchu.edu.tw

References

• 1 Duke J A, Ayensu E S. Medical Plants of China, Vol. 4. Algonac; Reference Publications 1985: 399.
  Search in Google Scholar
• 2 Noda Y, Mori A, Anzai K, Packer L. Superoxide anion radical scavenging activity of uyaku (Lindera strychnifolia), a natural extract used in traditional medicine. In: Packer L, Hiramatsu M, Yoshikawa T, editors Antioxidant food supplementations in human health. San Diego; Academic Press 1999: 471-9.
  Search in Google Scholar
• 3 Ohno T, Takemura G, Murata I, Kagawa T, Akao S, Minatoguchi S. et al . Water extract of the root of Lindera strychnifolia shows down the progression of diabetic nephropathy in db/db mice.  Life Sci. 2005;  77 1391-403.
  CrossrefPubMedSearch in Google Scholar
• 4 Wang C, Dai Y, Yang J, Chou G, Wang C, Wang Z. Treatment with total alkaloids from Radix Linderae reduces inflammation and joint destruction in type II collagen-induced model rheumatoid arthritis.  J Ethnopharmacol. 2007;  111 322-8.
  CrossrefPubMedSearch in Google Scholar
• 5 Takeda K, Ishii H, Tozyo T, Minato H. Components of the root of Lindera strychnifolia Vill. 16, Isolation of lindenene showing a new fundamental sesquiterpene skeleton, and its correlation with linderene. J Chem Soc C 1969: 1920-1
  Search in Google Scholar
• 6 Kouno I, Hirai A, Fukushige A, Jiang Z H, Tanaka T. New eudesmane sesquiterpenes from the root of Lindera strychnifolia .  J Nat Prod. 2001;  64 286-8.
  CrossrefPubMedSearch in Google Scholar
• 7 Chou G X, Norio N, Ma C M, Wang Z T, Masao H. Isoquinoline alkaloids from Lindera aggregata .  Chin J Nat Med. 2005;  3 272-5.
  PubMedSearch in Google Scholar
• 8 Zheng C F, Sun Q S, Wang Z T, Chou G X. Studies on constituents of the leaves of Lindera aggregate (Sims) Kosterm.  Chin J Chin Mater Med. 2001;  26 765-7.
  PubMedSearch in Google Scholar
• 9 Zhang C F, Sun Q S, Wang Z T, Hattori M. One new A-type proanthocynidin trimer from Lindera aggregate (Sims) Kosterm.  Chin Chem Lett. 2003;  14 1033-6.
  PubMedSearch in Google Scholar
• 10 Ohno T, Nagatsu A, Nakagawa M, Inoue M, Li Y M, Minatoguchi S. et al . New sesquiterpene lactones from water extract of the root of Lindera strychnifolia with cytotoxicity against the human small cell lung cancer cell, SBC-3.  Tetrahedron Lett. 2005;  46 8657-60.
  CrossrefPubMedSearch in Google Scholar
• 11 Lin C T, Chu F H, Tseng Y H, Tsai J B, Chang S T, Wang S Y. Bioactivity investigation of Lauraceae trees grown in Taiwan.  Pharm Biol. 2007;  45 638-44.
  CrossrefPubMedSearch in Google Scholar
• 12 Tsai I L, Hung C H, Duh C Y, Chen I S. Cytotoxic butanolides and secobutaolides from the stem wood of Formosan Lindera communis .  Planta Med. 2002;  68 142-5.
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Tanaka H, Nakamura T, Ichino K, Ito K. Two lactonic compounds, lancifolide and isolancifolide, from Actinodaphne lancifolia .  Phytochemistry. 1989;  28 1905-7.
  CrossrefPubMedSearch in Google Scholar
• 14 Chen F C, Peng C F, Tsai I L, Chen I S. Antitubercular constituents from the stem wood of Cinnamomum kotoense .  J Nat Prod. 2005;  68 1318-23.
  CrossrefPubMedSearch in Google Scholar
• 15 Cao J, Jia L, Zhou H M, Liu Y, Zhong L F. Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells.  Toxicol Sci. 2006;  91 476-83.
  CrossrefPubMedSearch in Google Scholar
• 16 Zhao J, Zhao Y, Zhang Y, Chen W. Anti-tumor effect of curcumin on human cervical carcinoma HeLa cells in vitro and in vivo .  Chin J Cancer Res. 2007;  19 32-6.
  CrossrefPubMedSearch in Google Scholar
• 17 Chen C N, Wu C L, Lin J K. Propolin C from propolis induces apoptosis through activating caspases, Bid and cytochrome c release in human melanoma cells.  Biochem Pharmacol. 2004;  67 53-66.
  CrossrefPubMedSearch in Google Scholar
• 18 Korsmeyer S J, Wei M C, Saito M, Weiler S, Oh J, Schlesinger P H. Pro-apoptotic cascade activates BID, which oligomerized BAK or BAX into pores that result in the release of cytochrome C .  Cell Death Differ. 2000;  7 1166-73.
  CrossrefPubMedSearch in Google Scholar
• 19 Kaufmann S, Desnoyers S, Ottaviano Y, Davidson N, Poirier G. Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis.  Cancer Res. 1993;  53 3976-85.
  PubMedSearch in Google Scholar
• 20 Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome C release from mitochondria in response to activation of cell death receptors.  Cell. 1998;  94 481-90.
  CrossrefPubMedSearch in Google Scholar

Dr. Sheng-Yang Wang

Department of Forestry

National Chung-Hsing University

250 Kuo-Kuang Road

Taichung 402

Taiwan

R.O.C.

Phone: +886-4-2284-0345 ext. 138

Fax: +886-4-2287-3628.

Email: taiwanfir@dragon.nchu.edu.tw

• Supplementary Material