GC / MS Analysis and Potential Cytotoxic Activity of Haplophyllum tuberculatum Essential Oils Against Lung and Liver Cancer Cells

Background: Haplophyllum tuberculatum is a plant belongs to family rutacee. It is rich in volatile oils, fixed oils, alkaloids, and furanocoumarins. It is well known for the huge number of folkloric uses in middle east. Objective: The aim of this study is to establish the chemical composition of the essential oils of Libyan H. tuberculatum and to investigate their cytotoxic potentialities.Materials and Methods: The essential oils of the aerial parts and flowers of H. tuberculatum growing in Libya were prepared by hydrodistillation. GC/MS analyses were performed on a Shimaduz capillary gas chromatograph (GC 17A ver.3) instrument directly coupled to mass spectrometer-MS QP5050A. Oil A and F of H. tuberculatum at different concentrations (0-50 μg/ml) in DMSO were tested for cytotoxicity against human tumor cell lines. Results: Oil yield was found 0.4 and 1.5 (v/w %) on dry weight basis respectively. GC/MS analysis resulted in identification of total 35 compounds. 15 compounds were common to both oils. oil A of H. tuberculatum exhibited antitumor activities against. liver carcinoma cell line (HEPG2) and lung carcinoma cell line (H1299) 4.7 μg/ml and 4.1 μg/ml. Conclusion: Essential oil of the aerial parts of H. tuberculatum is potentially active against lung (H-1299), and liver (HEPG2) carcinoma cell lines. The observed cytotoxic, activities can be attributed to the dominance of α and γ-terpinene in this oil.


INTRODUCTION
In Egypt the flowering aerial parts of H. tuberculatum are used as a drink to relieve fever, for abdominal upset, anemia, gastric pains, intestinal worms, malaria, as aphrodisiac, and the decoction is used for rheumatic pains. 1 In Oman, the leaves are used as a remedy for headaches and arthritis and also used for treatment of skin infections, discoloration and parasitic diseases. 2In Saudi Arabia, H. tuberculatum is used to treat malaria, rheumatoid arthritis and gynecological disorders, 3 while in Sudan the herb is used as an antispasmodic, to treat allergic rhinitis and breathing difficulties. 46][7][8] No published report was recorded concerning the analysis of the volatile oil of the aerial parts and the flower of Libyan H. tuberculatum and their biological potentialities.

Plant material
Samples of the aerial parts and flowers of Haplophyllum tuberculatum (Forssk) A. Juss (Rutaceae) were obtained from Benghazi, Libya, identified by Dr. Reem Samir Hamdy, Lecturer of Plant Taxonomy, Botany Department, Faculty of Science, Cairo University, Giza, Egypt.A voucher specimen of the aerial parts and the flowers of H. tuberculatum, were kept in the herbarium of Department of Pharamacognosy, Faculty of Pharmacy, Cairo University as a reference material specimen No. 2015224.

Preparation, characterization and analysis of the essential oil
Fresh samples of aerial parts and flowers of H. tuberculatum (500 g) were subjected separately to hydro-distillation.The percentage yield was calculated on dry weight (v/w) basis of the plant materials.The essential oil was dried over anhydrous sodium sulfate and kept refrigerated until analysis.

Sample preparation for GC/FID and GC/MS analysis
Samples prepared by mixing 5µl of dehydrated essential oil with approximately one ml of dichloromethane in auto sampler vials.Injection volume was 1 µl injected directly to GC-FID and GC-MS.

Gas chromatography-mass spectrometry analysis (GC/MS)
GC/MS analyses were performed on a Shimaduz capillary gas chromatograph (GC 17A ver.3) instrument directly coupled to mass spectrometer-MS QP5050A.Capillary column SLB-5ms (30 m x 0.25 mm, film thickness 0.25 µm) was used under the following condition: oven temperature programmed from 40°C (3 min), then temperature increased at rate 12°C/min to 180°C where it is hold for 5 min, then temperature increased at rate 40°C/min to reach 240°C, where it is finally hold for 5 min.The injector temperature was 240°C and was set at split ratio 1:54.Carrier gas was He at flow rate 0.9 ml/min.The mass spectrometer operated on electronic (EI) ionization mode at 70 eV with scan range 40-500.The transfer line temperature was 230°C.

Gas chromatography analysis (GC-FID)
The GC analysis was carried out using Shimaduz GC gas chromatograph (GC-17 ver.3) system.FID detector temperature was 240 o C. To obtain the same elution order with GC/MS simultaneous auto injection was done on a duplicate of the same operational conditions.Identification of the essential oil components were carried out by comparing their relative retention times with those of authentic samples or by comparing their relative retention indices (RRI).The later were computed using a mixture of a continuous series of n-alkane hydrocarbons (C4-C28) run on SLB-5ms (non-polar) column using the same conditions as described above.2] The compounds are arranged in order to GC elution on SLB-5ms capillary column.Relative percentages were calculated based on the GC-FID peak areas without the use of correction factors and are compiled.

In vitro screening for cytotoxic activity
Human tumor cell lines: lung carcinoma cell line (H-1299) and liver carcinoma cell line (HEPG2), maintained in the laboratory of the Cancer Biology Department of National Cancer Institute, Cairo, Egypt, were used.Oil A and F of H. tuberculatum at different concentrations (0-50.000µg/ml) in DMSO were tested for cytotoxicity against the forementioned human tumor cell line adopting sulforhodamine B stain (SRB) assay. 13The relation between surviving fractions and oil concentration was plotted to get the survival curve of each tumor cell line after the application of the specific concentration.The results were compared to those of the standard cytotoxic drug, Doxorubicin (10 mg Adriamycin hydrochloride, in 5 ml IV injection, Pharmacia, Italy) at the same concentrations.The dose of the test solutions which reduces survival to 50% (IC 50 ) was calculated.

Determination of median lethal dose LD 50
The LD 50 of the volatile oils was determined according to the procedures developed by Karber (1941). 14

Statistical analysis
All data were expressed as mean ± SE and the statistical significance was evaluated using the ANOVA test followed by Duncan's multiple range tests.

Evaluation of cytotoxic activity
On assessing the cytotoxic activity under the experimental condition adopted and from data displayed in Table 2 it could be concluded that the oil A of H. tuberculatum exhibited variable antitumor activities against the two tested cell line viz.liver carcinoma cell line (HEPG2) and lung carcinoma cell line (H1299).In this respect, the oil A of H. tuberculatum demonstrated the lowest IC50% when tested against H-1299 and HEPG2 cells (4.7 µg/ml and 4.1 µg/ml) respectively.On the other hand, the oil F showed relatively higher IC50% against H-1299 and HEPG2 cells (42.3 µg/ml and 19.7 µg/ml) respectively, which is comparable to the standard cytotoxic drug, doxorubicin (Table 3-4).Results obtained during assessment of the cytotoxic potential of the oil A of H. tuberculatum exhibited variable antitumor activities against the two tested cell line viz, liver carcinoma cell line (HEPG2) and lung carcinoma cell line (H1299).In this respect, the oil A of H. tuberculatum demonstrated the lowest IC50% when tested against H-1299 and HEPG2 cells (4.7 µg/ml and 4.1 µg/ml) respectively.On the other hand, the oil F showed relatively higher IC50% against H-1299 and HEPG2 cells (42.3 µg/ml and 19.7 µg/ml) respectively, which is comparable to the standard cytotoxic drug, doxorubicin (Table 3-4).Results obtained during assessment of the cytotoxic potential of the oil H. tuberculatum are in accordance with formerly reported data related to γ-terpinen. 15he study was the first concerning the cytotoxic activity of the essential oils of H. tuberculatumon tumor cell lines.

CONCULSION
In conclusion, this report was the first paper indicating the chemical composition and investigating the cytotoxic potentialities of the essential oils of Libyan H. tuberculatum.It is found that the essential oils of the aerial parts of Halpophyllum tuberculatum are potentially active against lung (H-1299), and liver (HEPG2) carcinoma cell lines.The observed cytotoxic, activities can be attributed to the dominance of α and γ-terpinene.

Determination of median lethal dose LD 50
The 24-hours LD 50 was approximately more than 0.05 ml/kg b. wt. for the essential oils.These results showed that the essential oils are safe and non-toxic.
To the best of our knowledge, this is the first report on the chemical composition of the essential oil of Libyan H. tuberculatum.Nevertheless, the composition of the essential oil from H. tuberculatum grown in different regions of Egypt was previously studied. 16Monoterpenes are non-nutritive dietary components found in the essential oils of herbs.8][19] Moreover, the hydrocarbons, β-phellandrene, α-pinene, and 3-carene were present in Libyan and Egyptian samples in comparable amounts.The difference in oil yield obtained and the composition of essential oil in plants were affected by genetical and environmental conditions, which is determined by growth region and harvesting time in terms of onto genetical and diurnal variability.On assessing the cytotoxic activity under the experimental condition adopted and from data displayed in Table 3 it could be concluded that

Table 1 : Percentage composition of essential oil of the aerial parts and flowers of H. tuberculatum
Components are listed in order of their elution.RI*: Relative retention index on SLB-5ms column.

Table 4 : Drug cytotoxicity of H.tubrtculatum on Lung carcinoma cell line H-1299
156the oil H. tuberculatum are in accordance with formerly reported data related to γ-terpinene.15