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Induction of apoptosis by carbazole alkaloids isolated from Murraya koenigii.


In the current study, we isolated 10 carbazole alkaloids from the plant species Murraya koenigii (Rutaceae), and examined their effects on the growth of the human leukemia cell line HL-60. Three carbazole alkaloids, mahanine (6), pyrayafoline-D (7) and murrafoline-I (9), showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin was increased in a time-dependent manner after treatment with each alkaloid. Interestingly, each carbazole alkaloid induced the loss of mitochondrial membrane potential. In addition, both caspase-9 and caspase-3 were also time-dependently activated upon treatment with the alkaloids. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by these alkaloids. The results suggest that these three alkaloids induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, through mitochondrial dysfunction.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Murraya koenigii; Rutaceae; Carbazole alkaloids; Apoptosis; HL-60 cells; Caspases


Our previous studies have reported the chemical constituents of Rutaceous plants and identified various types of chemical compounds in the genus Murraya (Ito et al., 1993, 1991; Ito and Furukawa, 1987a, b). Murraya plants have been used as analgesics, astringents, anti-dysenterics or febrifuges in folk medicine in China and other Asian countries (Kan, 1972). It has been reported by others that carbazole alkaloids possess various biological activities such as anti-tumor, anti-oxidative, anti-mutagenic, and anti-inflammatory activities (Ramsewak et al., 1999; Tachibana et al., 2001; Nakahara et al., 2002). Since it is known that carbazole alkaloids possess anti-tumor activity, the identification of alkaloids that are cytotoxic against tumor cells could lead to the development of a chemopreventive agent for tumor treatment. We have previously isolated various carbazole alkaloids from M. euchrestifolia (Ito et al., 1991). In addition, we have demonstrated that some of the carbazole alkaloids isolated from M. euchrestifolia display cytotoxicity against a variety of tumor cell lines (Itoigawa et al., 2000). There have been reports of terpenoids (Nagashima et al., 2002), steroids (Hirano et al., 1996), acetogenins (Zhu et al., 2002) and flavonoids (Wang et al., 1999) inducing apoptosis in cancer cells. The aim of the present study was to examine the cytotoxic potential of carbazole alkaloids present in M. koenigii. A total of 10 carbazole alkaloids, including two novel dimeric carbazoles [murrafoline-I (9) and mahabinine-A (10)], were isolated and then tested for cytotoxicity against the human leukemia cell line HL-60. It was found that mahanine (6) (Ito et al., 1993), pyrayafoline-D (7) (Ito et al., 1991) and murrafoline-I (9) showed significant cytotoxic activity against HL-60 cells. The cytotoxicity of these three carbazole alkaloids was mediated through apoptosis.

Materials and methods

Plant materials

The plant materials used in this study were from Murraya koenigii (L.) Spreng. (Rutaceae), and were collected at Mymensingh, Bangladesh in April 1997. A voucher specimen (MUY0106) was deposited in the Herbarium of the Department of Botany, University of Dhaka. The plant was identified by Dr. Shajahan, MD., Department of Pharmacy, University of Dhaka.

Extraction and isolation

The dried leaves (515 g) of M. koenigii were extracted with acetone at room temperature, and the solvent was then evaporated under reduced pressure to give the acetone extract. The acetone extract was subjected to successive silica gel column chromatography, eluting with CH[Cl.sub.3]-hexane (1:1), CH[Cl.sub.3], CH[Cl.sub.3]-acetone (95:5, 90:10, 80:20, 60:40, 50:50), acetone, and MeOH gradients, to obtain fractions 1-9. Each fraction was further subjected to silica gel column chromatography and preparative TLC with appropriate combinations of CH[Cl.sub.3], acetone, hexane, i[Pr.sub.2]O, benzene, and MeOH as developing solvents to give the following compounds. From the CH[Cl.sub.3] fraction: mahanimbine (5, 85.8 mg) and koenimbine (2, 3.3 mg). From the CH[Cl.sub.3]-acetone (95:5) fraction: euchrestine-B (8, 108.5 mg), mahanine (6, 88.0 mg), pyrayafoline-D (7, 50.5 mg), koenidine (4, 3.1 mg), koenigine (3, 55.4 mg), murrafoline-I (9, 8.2 mg), mahabinine-A (10, 4.0 mg), and koenine (1, 6.3 mg). Identification of these test compounds was done by direct comparison of the spectral data and TLC with authentic samples, or by comparison of NMR data with those reported in the literature. To our knowledge, murrafoline-I (9) and mahabinine-A (10) are previously unknown binary carbazole alkaloids, and further examination of the constituents of this plant is currently in progress. Full details elucidating the structure of these alkaloids will be reported elsewhere.

Cell culture and treatments

The human leukemia cell line HL-60 was provided by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Miyagi, Japan). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 5% C[O.sub.2] and 95% air at 37[degrees]C. The carbazole samples were dissolved in dimethyl sulfoxide (DMSO) and were added to culture medium to give a final DMSO concentration of 0.5% v/v. This concentration of DMSO had no significant effect on the growth of the cell line tested (data not shown).

Assay of cell viability/cell growth

Cell viability was determined using CellTiter 96 Aqueous assay kit (Promega, Madison, WI). Cells were seeded in 96-well plates at a density of 1 x [10.sup.5] cells per well. The cells were maintained for 24 h at 37[degrees]C and then each sample to be tested (30 [micro]M) was added to the culture medium. MTS solution was added to the 96-well plates at the time points indicated in the figures, and the cells were incubated for 1 h at 37[degrees]C. The absorbance was measured at a wavelength of 490 nm with a Wallac 1420 ARVOsx microplate counter (Applied Biosystems, Foster City, CA).

Assessment of the percentage of apoptotic cells

To detect apoptotic cells, cells were stained with DNA binding dye Hoechst 33342 (Dojindo, Kumamoto, Japan). After the cells were exposed to the test compounds for the allotted time periods, they were fixed with 2% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at 4[degrees]C, and then washed with PBS. To stain the nuclei, cells were incubated for 20 min with 20 [micro]g/ml Hoechst 33342. After washing with PBS, the cells were observed under a fluorescence microscope (Zeiss Axiophoto 2; Carl Zeiss, Germany). Cells exhibiting condensed chromatin and fragmented nuclei were scored as apoptotic cells. A minimum of 200 cells was scored from each sample.

Measurement of mitochondrial membrane potential

Changes in mitochondrial membrane potential were detected using the fluorescence-based ApoAlert[TM] mitochondrial membrane sensor kit (Clontech Laboratories, Palo Alto, CA). Cells ([10.sup.6]/ml) were treated with 30 [micro]M carbazole alkaloid for the indicated periods. After treatment, cells were pelleted, washed with fresh culture medium, and then resuspended in 0.5 ml of culture medium containing mitosensor reagent (5 [micro]g/ml). Following incubation at 37[degrees]C for 20 min, cells were washed with 1 ml of culture medium and then pelleted. The final cell pellet was resuspended in 30 [micro]l of culture medium and then was analyzed by confocal laser scanning microscopy (a Zeiss Axioplan equipped with a Bio-Rad MRC-1024 laser). The mitosensor reagent, cationic by nature, is taken up by normal mitochondria where it forms aggregates that exhibit red fluorescence. A loss in mitochondrial membrane potential prevents this aggregation and mitosensor remains in the cytoplasm as a monomer where it exhibits green fluorescence.

Measurement of enzyme activities of caspases-3 and -9

The enzyme activities of caspase-3 and caspase-9 were measured using a caspase fluorometric assay kit (R & D Systems Inc., Minneapolis, MN). Cells were seeded in 24-well plates at a density of 3 x [10.sup.6] cells per well. After exposure of the cells to the test compounds for the allotted time periods, the cells were washed three times with PBS, lysed in a lysis buffer for 10 min on ice. The protein content of the cell lysates were assayed with a Micro BCA reagent (Pierce, Rockford, IL). Cell lysates containing 50 [micro]g of protein were incubated with a caspase-3 fluorogenic substrate (DEVD-AFC), or a caspase-9 fluorogenic substrate (LEHD-AFC), for 1 h at 37[degrees]C. Caspase activity was measured by fluorometric detection using a Wallac 1420 ARVOsx microplate counter (excitation at 400 nm, emission at 505 nm; Applied Biosystems, Foster City, CA).


Caspase inhibition assay

The caspase-9-specific inhibitor Z-LEHD-FMK (50 [micro]M; R & D Systems Inc., Minneapolis, MN), and the caspase-3-specific inhibitor, Z-DEVD-FMK (50 [micro]M; R & D Systems Inc., Minneapolis, MN), were dissolved in DMSO. Cells were pretreated with either medium containing DMSO or inhibitor for 2h. Control medium or medium containing the test compounds were then added to achieve a final concentration of 30 [micro]M of each test compound. The number of apoptotic cells was determined by counting cells that had nuclear condensation and fragmentation as confirmed by Hoechst 33342 staining.

Statistical analysis

The significance of differences was estimated using Student's t-test A p-value of less than 0.05 was considered significant.

Results and discussion

In this study, we isolated a total of 10 carbazole alkaloids, including two novel dimeric carbazoles [murrafoline-I (9) and mahabinine-A (10)] from the acetone extract of M. koenigii leaves (Fig. 1). First, we examined the effect of the isolated alkaloids on the growth of human leukemia HL-60 cells. To assess cell growth, we used the CellTiter 96 Aqueous assay kit, which is based on the metabolic conversion of the MTS tetrazolium compound to a colored formazan product by living cells. The absorbance of the formazan product is directly proportional to the number of viable cells in culture. We showed that when HL-60 cells were exposed to each of the 10 isolated alkaloids (30 [micro]M) for 24 h, only mahanine (6), pyrayafoline-D (7), and murrafoline-I (9) induced a marked decrease in cell viability (6, 7 and 9-treated: 83.5%, 70.5% and 52.0% decrease respectively, compared to controls; Fig. 2). The other seven alkaloid compounds did not affect cell viability. As shown in Fig. 3, the inhibitory effects of mahanine (6), pyrayafoline-D (7), and murrafoline-I (9) on HL-60 cell growth was dependent on the time of the treatment. The results indicate that these three alkaloids possess cytotoxic activity against HL-60 cells.

To examine whether the cytotoxicity of these three carbazole alkaloids was mediated through apoptosis, HL-60 cells treated with the three compounds were stained with Hoechst 33342, and the appearance of chromatin condensation and fragmentation of nuclei were monitored. No apoptotic nuclei were observed in control cells in each treatment (Fig. 4). However, the percentage of apoptotic cells was significantly increased in cells exposed to 30 [micro]M mahanine (6), pyrayafoline-D (7), and murrafoline-I (9) in a time-dependent manner (Fig. 4). The results indicate that these three compounds induce apoptotic cell death in HL-60 cells.



We next assessed the effect of carbazole alkaloids on mitochondrial membrane potential by confocal microscopy using the fluorescent mitosensor reagent. Mitosensor dye aggregates in the mitochondria of healthy cells and these aggregates fluoresce red. In apoptotic cells, mitochondrial membrane potentials are altered, and mitosensor dye cannot accumulate in mitochondria. It remains as a monomer in the cytoplasm and fluoresces green. Control cells loaded with mitosensor dye exhibited a consistent red fluorescence when incubated for up to 12 h (Fig. 5). However, after carbazole alkaloid treatments, there was a massive loss of mitochondrial membrane potential as shown by the complete conversion to green fluorescence (Fig. 5). These results suggested that three carbazole alkaloid induced mitochondrial dysfunction based on the loss of membrane potential.

It is thought that activation of caspases may play a central role in the execution stage of apoptosis (Patel et al., 1996). The intrinsic apoptotic pathway is initiated by the release of cytochrome c from mitochondria. The released cytochrome c binds to, and activates the adaptor protein Apaf-1, which in turn activates caspase-9, leading to the formation of an apoptosome and subsequent activation of downstream caspases, such as caspase-3 (Wang, 2001). To examine the apoptotic pathway induced by these alkaloids, the proteolytic activity of caspase-9 was measured in terms of its ability to cleave LEHD-AFC, a fluorescent substrate specific for caspase-9. As shown in Fig. 6A, a time-dependent increase in caspase-9 activity was observed in mahanine (6), pyrayafoline-D (7), and murrafoline-I (9) -treated cells. Significant activation of caspase-9 by mahanine (6), pyrayafoline-D (7), and murrafoline-I (9) was detected at 2, 4, and 12 h after treatment, indicating the existence of a time lag in caspase-9 activation by the three compounds. The activity of caspase-3, which is activated downstream of caspase-9, was then fluorometrically assayed using a fluorogenic substrate DEVD-AFC after treatment with each alkaloid. In the alkaloid-treated cells, caspase-3 was time-dependently activated in a manner similar to the change in caspase-9 activation (Fig. 6B).





To determine whether activation of caspases-9 and -3 is required for the induction of apoptosis by these carbazole alkaloids, we examined the effect of specific inhibitors caspases-9 and -3 on alkaloid-induced apoptosis. As shown in Fig. 5, Z-LEHD-FMK (a caspase-9 inhibitor) (Fig. 7A) and Z-DEVD-FMK (a caspase-3 inhibitor) (Fig. 7B) markedly attenuated these alkaloid-induced apoptosis. These results suggest that the three alkaloids mahanine (6), pyrayafoline-D (7), and murrafoline-I (9), induce apoptosis in HL-60 cells via caspase-9/caspase-3 activation. Because caspases-9 and -3 are activated by mitochondrial dysfunction (Wang, 2001), it is possible that apoptotic effect of these carbazole alkaloids is mediated through a mitochondrial abnormality. A study of the direct or indirect target site in the mitochondria of these carbazole alkaloids is currently in progress.

In conclusion, it was found that mahanine (6), pyrayafoline-D (7), and murrafoline-I (9), which were isolated from M. koenigii, are able to induce apoptosis in HL-60 cells. It was found that these alkaloids induced the loss of mitochondrial membrane potential and the subsequent activation of caspase-9/caspase-3. The findings suggest that these three alkaloids are possible candidates for a cancer chemopreventive agent.


We are grateful to Dr. Choudhury M. Hasan and Dr. Mohammad A. Rashid of the University of Dhaka, Bangladesh, for supplying the plant material. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (H. F., Scientific Research (C) and High-Tech Research Center Project).


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C. Ito (a), M. Itoigawa (b,*), K. Nakao (a), T. Murata (c), M. Tsuboi (c), N. Kaneda (c), H. Furukawa (a)

(a) Department of Medicinal Chemistry, Faculty of Pharmacy, Meijo University, Nagoya, Japan

(b) Tokai Gakuen University, Nagoya, Japan

(c) Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan

Received 27 September 2004; accepted 9 March 2005

*Corresponding author. Tel.: +81 52 801 1201; fax: +81 52 804 1044.

E-mail address: (M. Itoigawa).
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Author:Ito, C.; Itoigawa, M.; Nakao, K.; Murata, T.; Tsuboi, M.; Kaneda, N.; Furukawa, H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:May 1, 2006
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