Chemical Composition and Cytotoxic Activities of Petroleum Ether Fruit Extract of Fruits of Brucea javanica (Simarubaceae)

Purpose: To investigate the chemical composition and antitumor activity of the petroleum ether extract of the dried ripe fruits of Brucea javanica. Methods: The composition of petroleum ether extract was analyzed by gas chromatography/mass spectrometric (GC/MS) and their antitumor activities were determined by MTT assay. Results: GC/MS spectrometry results indicate that the petroleum ether extract was a mixture of esters, fatty acids, sterides, pregnanones, terpenes, alkaloids, alkenes, alcohols, ketones, aldehydes and other compounds. The results also revealed the significant antitumor activity of the extract with IC 50 of 9.14, 12.45, 15.15, 16.13, 22.26, and 27.97 μg/mL against A549, CNE, MCF-7, NCI-H460, HepG2, and KB-3-1 cell lines, respectively. Conclusion: The study establishes the chemical composition and cytotoxic activity of the petroleum ether extract of the plant fruits. There is need for further investigations to isolate more potent compounds and structurally modify the known compounds to retain activity and lower toxicity and thus lead to the possible development of Brucea javanica oil.

Brucea.javanica (L.) Merr., a Chinese herbal medicine called 'Yadanzi', is distributed in south of China (mainly in Guangxi and Guangdong Provinces) and shows significant antitumor and other activities mostly due to quassinoids, triterpenoids and alkaloids [12].Recent research has focused on the constituents of the ethyl acetate and n-butanol extracts of B. javanica.Brucea javanica oil (BJO) is a petroleum ether extract is mainly composed of fatty acids and fatty acid derivatives, which were extracted with petroleum ether from the fruits of B. javanica [13].It has been used as anti-tumor agent to therapy hepatic, esophageal, rectal, pulmonic, renal, and prostatic carcinomas clinically [14].However, the petroleum ether-soluble compositions of B. javanica seldom got attention, which is not conducive to the use of BJO on therapy.As a continuation of our search for naturally occurring bioactive substances from herb medicine in China，we investigated the constituents of the fruits from B. javanica.Our objectives in the present work were, first, to determine the petroleum ether chemical compositions of B. javanica by GC/MS analysis and, second, to evaluate the antitumor efficacy of the crude extract and its petroleum ether fractions.

EXPERIMENTAL Plant material
The air-dried fruits of B. javanica were purchased from Qingping Local Medicine Market at Guangzhou, China, in January 2008, identified by Prof. Yun-fei DENG of SCBG (South China Botanical Garden, Chinese Academy of Sciences) and a voucher specimen (no.MZH0173) has been deposited in the herbarium of SCBG.
The petroleum ether-soluble extract was subjected to silica gel (80 -100A, 400 g) column chromatography, and eluted with gradient mixtures of CHCl 3 -MeOH (from 1:0 to 0:1) to afford 300 fractions (250 mL each).Eight major subfractions (Fr1 -8) were obtained by pooling fractions with similar TLC patterns.Each fraction (1 mg) was dissolved in and diluted to 1 mL distilled dichloromethane, which was identified by GC/MS analysis separately.

GC/MS conditions
GC/MS analysis was performed on Agilent AOC-20S Gas Chromatograph Mass Spectrometer.A petroleum ether phenomenex HP-5 fused silica column (30 m × 0.25 mm × 0.25 μm) was used with helium at a linear velocity of 36.8 cm/sec (65.2 psi) as a carrier gas.The GC oven temperature was programmed from 80 ℃, increasing at 2 ℃/min, up to 150 ℃ with a 2 min hold at 150 ℃, then programmed from 150 to 310 ℃ successively at 40 ℃/min with a 2 and 20 min hold at 250 ℃ and 310 ℃, respectively.The manual injection volume was 0.1 μL and split ratio was adjusted to 1:20.The Electronic ionization was at 70 eV and transfer line was heated at 220 ℃.A mass range of 10 -400 amu was scanned.This GC/MS conditions were optimized from the methods used in previous studies [15].
The identification of volatile compounds was based on the comparison of their retention times and mass spectra with those obtained from the mass spectral reference library of National Insititute of Standards and Technology as well as those found in literature.

MTT cytotoxicity assay
Human lung cancer cell line (A549), human breast carcinoma cell line (MCF-7), human hepatoma cell line (HepG2), human lung cancer cell line (NCI-H460), human nasopharyngeal carcinoma cell line (CNE) and huaman epidermoid carcinoma cell line (KB-3-1) were obtained from Research Group of Pharmaceutical Sciences, Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou, China.
MTT assay, as previously described by Mosmann et al [16], was performed to assess the cytotoxicity of the plant extracts and petroleum ether fractions.Briefly, cells, grown in RPMI-1640 medium plus 10 % heat-inactivated foetal bovine serum, were plated in 96 well microtiter plates and incubated for 24 h at 37 C, 5 % CO 2 .When cells reached > 80 % confluence.The cells were treated with 100 μL petroleum ether fractions dissolved in dimethyl sulfoxide (DMSO) at serial concentrations of 50, 25, 12.5, 6.5, 3.125 and 1.56 μg/mL, while background wells were treated with only 100 μL culture medium.
After 72 h of incubation at 37 C, 5 % CO 2 , 10 μL MTT reagents (5 mg/mL) were mixed in each well and incubated at 37 ºC for a further 4 h.Then the medium was removed and 150 μL DMSO was acceded to each well after the plate was shaken thoroughly for 10 min.The absorbance was measured on a CENios microplate reader (TECAN, Austria) at a wavelength of 570 nm.MTT solution only with DMSO was used as blank and Doxorubicin as positive control.The half maximal inhibitory concentration (IC 50 ) values were calculated using SPSS software, version 16.0, by comparison with the reduction in absorbance in the control assay.
From our results, chemical compositions of the petroleum ether extract of B. javanica included a high amount of esters, fatty acids and sterides.This is the first full report on the liposoluble constituents of B. javanica by GC/MS analysis.

Cytotoxicity assay
Six human tumor cell lines A549, MCF-7, HepG2, NCI-H460, CNE and KB-3-1 were used to investigate the in vitro antitumor effects of the different extracts and fractions of B. javanica.The IC 50 values of extracts and Fr1 -8 on the viability of cancer cells after 72 h of incubation are presented in Table 2.

DISCUSSION
It is well known that medicinal plants contain excellent antitumor compounds and they are ancient weapons in the defense against malignant neoplasms [1].Antitumor agents destroy or inhibit the growth of tumors and over 50% of the currently used anti-cancer agents are derived from natural sources [4].According to Geran et al., a crude extract having an IC 50 value ≤ 20 μg/mL is considered active [17].Under the concentration of 20 μg/mL, the ethanol， petroleum ether, ethyl acetate and n-butyl alcohol extracts of B. javanica all exhibited a potent selected cytotoxicity against the tested cell lines, A549, CNE, MCF-7 and NCI-H460 in particular.
Different classes of organic compounds, like quassinoids, alkaloids and triterpenoids, have been isolated and identified in the present research, and these compounds may be responsible for the cytotoxicity actions [12].As a result of that, the ethyl acetate extract tend to be more active than the ethanol and petroleum ether extracts.
Brucea javanica oil extracted with petroleum ether from the fuits of B. javanica, is a complex mixture of fatty acids, which were reported to be cytotoxic active constituents [13].The cytotoxic activity of petroleum ether fractions can be explained partly, by the high concentration of fatty acids and minor components such as sterides, pregnanones [18] and alkaloids.The large proportion of fatty acids might have contributed to the activities of the petroleum ether fractions against the tumor cell lines, because hexadecanoic acid and octadecenoic acid are known to possess cytotoxic activity [19].However, Fr1 extract exhibited IC 50 values almost more than 100 μg/mL on the tested cell lines, indicating that the esterification of fatty acids decrease the cytotoxicity.The decrease of esters and increase of pregnanones, sterids and alkaloids in Fr6, induced the improvement of cytotoxic capacity comparing to Fr5.
Despite the cytotoxic activities of petroleum ether fractions are lower than that of the positive control, the present results revealed their antitumor potential and further support the applications on clinical.

CONCLUSION
The present work has determined the chemical compositions of petroleum ether extract of B. javanica fruits by GC/MS analysis, and evaluated its cytotoxic activity.The results showed that the crude extracts and the petroleum ether fractions were significant or moderate active against the tested cell lines.Further studies should be undertaken next step to ascertain the phytochemical components and their bioactivities.This will be helpful to establish the foundation for clinical application of Brucea javanica oil.