Cytotoxic Compounds Isolated from Murraya tetramera Huang

A new compound and seven known compounds were isolated from Murraya tetramera Huang for the first time, and they were identified with NMR and MS spectral analysis. It was confirmed that the new compound was 10-methoxy-7-methyl-2H-benzo[g]chromen-2-one (3) and the others were β-eudesmol (1), trans-3β-(1-hydroxy-1-methylethyl)-8aβ-methyl-5-methylenedecalin-2-one (2), 5,7-dimethoxy-8-[(Z)-3'-methyl-butan-1',3'-dienyl]coumarin (4), 7-geranyloxy-6-methoxycoumarin (5), 5,7-dimethoxy-8-(3-methyl-2-oxo-butyl)coumarin (6), murrangatin acetate (7) and toddalenone (8). Furthermore, the cytotoxic activity against human lung adenocarcinoma (A549), human hepatocellular carcinoma cells (SMMC-7721), human bladder tumor cells (EJ), human cervical carcinoma cells (HeLa), and human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1) was evaluated for all compounds. It was found that five of them displayed various degrees of cytotoxicity against different testing targets. Compound 1 showed significant cytotoxic activity against the five cell lines (A549, SMMC-7721, EJ, Hela and BALL-1). Compounds 2 and 5 showed significant cytotoxicity against three cell lines (A549, SMMC-7721 and BALL-1). Compound 4 showed significant cytotoxicity against three cell lines (A549, EJ and BALL-1). However, compound 3 only showed fair cytotoxicity against the BALL-1 cell line. The structure-active relationships were investigated as well. These active compounds might be potential lead compounds for the treatment of cancer.


Introduction
Cancer is one of the most common diseases that threaten peoples' health. Accordingly, much effort has been invested to develop effective treatments. Chemotherapy and radiotherapy have been the primary approaches for conventional cancer treatment, but they are not always effective [1][2][3]. Traditional Chinese medicines (TCMs) are generally economical and plentiful, while showing very low toxicity or side effects in clinical practice. Hence, TCMs have been applied worldwide for the treatment of cancers [3][4][5][6]. Furthermore, TCM have been one of the most important sources for seeking new leading compounds that possess significant cytotoxicity [7][8][9].
In East Asia, the genus Murraya (family Rutaceae) has been widely used in traditional medicine. Its plants contain various alkaloids, coumarins and flavonoids [10,11]. The cytotoxicity has been investigated in some species in this genus, such as M. koenigii [12], M. paniculata [13,14], M. euchrestifolia [15] and M. exotica [16]. However, there are no reports on the cytotoxicity of M. tetramera Huang.
M. tetramera is widely distributed in the Chinese provinces of Guangxi and Yunnan. It has been used as a folk TCM for the treatment of colds, coughs, asthma, stomach disorders, rheumatism, pruritus and eczema [11,17]. In this work, we sought to isolate and identify bioactive compounds with potential cytotoxicity from M. tetramera. As a result a new coumarin and seven known compounds were isolated from M. tetramera for the first time and all the compounds were evaluated in vitro against the A549, SMMC-7721, EJ, HeLa and BALL-1 tumor cell lines. The results indicated that some of these compounds have significant cytotoxic activities against the five tested human cancer cell lines.

Cytotoxic Activity of Isolated Compounds
The cytotoxicity of compounds 1-8 was evaluated against A549, SMMC-7721, EJ, HeLa and BALL-1 cancer cell lines using the Cell Counting Kit (CCK)-8 method. The results are listed in Table 1  The various cytotoxic activities might be related to the different substitution patterns in the chemical structures. Among compounds 3-8, compound 5 had a longer alkyl-substituent than the other compounds and it showed the most potent cytotoxic activity against the human cancer cell lines, which corresponds to the result previously described by Wang et al. [18] indicating that the length of alkyl-substituents contributed to the cytotoxicity. Interestingly, the results also showed that the compounds possessing carbonyls on the alkyl moiety had weak cytotoxic activities. Further study is needed to investigate the structure-active relationships.

Plant Material
The

Extraction and Isolation
The dried samples (2.

Cytotoxicity Assay
The cytotoxicity of compounds 1-8 was measured by the standard CCK-8 method [26]. Human lung adenocarcinoma (A549), human hepatocellular carcinoma cells (SMMC-7721), human bladder tumor cells (EJ), human cervical carcinoma cells (Hela), and human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1) were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Doxorubicin (DOX, adriamycin, Actavis Italy S.p.A., Beijing, China) was the positive control. All cell lines were cultured in RPMI 1640 (Sigma, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (GIBCO Inc., Grand Island, NY, USA), 100 IU/mL penicillin (Flow Lab, Beijing, China) and 100 μg/mL streptomycin (Flow Lab) at 37 °C, 5% CO 2 and 90% humidity. The cell suspension was dispensed into a 96-well plates at 100 μL per well (adherent cells were 6 × 10 3 per well, suspension cells were 5 × 10 4 per well). After 4-6 h preincubation in the incubator (Forma Series ΙΙ Water Jacket, Waltham, MA, USA) to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h. At the end of the incubation, CCK-8 reagent (Dojindo, Kumamoto, Japan, 10 μL) was added into each well followed by further incubation for 2 h. The optical density (OD) was recorded at 450 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Each determination represented the average mean of six replicates. The 50% inhibitory concentration (IC 50 ) values were calculated by the line equation of the dose-dependent curves.

Conclusions
A new compound and seven known compounds were isolated from the M. tetramera for the first time. They were two sesquiterpenes (compounds 1 and 2) and six coumarins (compounds 3-8). All the compounds were tested for their in vitro cytotoxic activities against the HeLa, K562, A549, H1299 and SMMC-7721 tumor cell lines. It was found that compounds with similar structures displayed various degrees of cytotoxicity. Compound 1 showed stronger cytotoxic activity against the five cell lines (A549, SMMC-7721, EJ, HeLa and BALL-1) compared to compound 2. Compound 5 showed potent cytotoxicity against three cell lines (A549, SMMC-7721 and BALL-1). Compound 4 showed promising cytotoxicity against three cell lines (A549, EJ and BALL-1). Compound 3 only showed cytotoxicity against the BALL-1 cell line. None of the tested cell lines were susceptible towards compounds 6-8. This phenomenon might be related with the different substitution patterns in their chemical structures. Compounds having long alkyl-substituents exhibited significant potent cytotoxic activity against the human cancer cell lines, whereas compounds which had carbonyl groups on the alkyl substituents showed weak cytotoxicity. All these active compounds might be promising lead compounds for anti-cancer agents. However, further study is needed to unravel the mechanisms of their cytotoxic activity.