Chemical composition of the essential oil of whole plant of Elsholtizia dense Benth and its anti-tumor effect on human hepatoma cells

Purpose: To determine the chemical components of the essential oil of Elsholtizia dense in Sichuan Province and evaluate the effect of the oil on human hepatoma cells (SMMC-7721) in vitro. Methods: The essential oil was extracted using the modified steam-distillation extraction method, and its chemical components were determined by gas chromatography-mass spectrometry (GC-MS). The effect of the essential oil on proliferation of SMMC-7721 cells was studied by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, with L02 and HeLa cells serving as control groups. Results: GC-MS results show that the essential oil of E. dense contains 40 components. Thirty seven components were identified and accounted for 98.39 % of the essential oil. The two main components were rosefuran epoxide (53.12 %) and 2-ethyl imidazole (29.8 %). The oil significantly inhibited cell proliferation in a concentrationand time-dependent manner (p < 0.05). SMMC-7721 cells were more inhibited than L02 and HeLa cells by the oil, with half maximal inhibitory concentration (IC50) values of 26.23 and 25.46 μg/mL after 8-h and 24-h treatments, respectively. Conclusion: Out of the 40 chemical components of the essential oil of E. dense, rosefuran epoxide and 2-ethyl imidazole were the most abundant. The oil has a significant anti-tumor effect on SMMC-7721 cells, and thus has a potential to be developed as an anti-liver cancer drug.


INTRODUCTION
Elsholtizia dense Benth belonging to Labiatae, is found in the west of China, including Sichuan, Shanxi and Qinghai.E. dense is one of the sources of the Tibetan medicine, Qirou [1].It has anti-inflammatory, anti-pruritic, myogenic, and hemostatic effects [2], and also could remove necrotic tissue and repel mosquito [3].It has been traditionally applied to treat several diseases such as summer cold, scabies, syphilitic rhinitis, laryngitis, skin itching, furuncle carbuncle and stomach disease in China [2,3].The main medicinal components of E. dense is its essential oil [2,[4][5][6].The essential oil of many plants has been reported to have an inhibitory effect on tumor cells [7][8][9][10][11][12][13]. However, the antitumor effect of the essential oil from E. dense in Sichuan Province has been seldom studied.
The chemical composition of the essential oil from E. dense has been reported in several studies [2,[4][5][6].
However, the chemical composition of the essential oil differs in different regions where E. dense grows.The chemical components of the essential oil from E. dense in Sichuan Province have not been identified until now.
Therefore, the aim of this study was to identify the chemical components of the essential oil from E. dense in Sichuan Province and investigate its anti-tumor effect.In this study, the plant was collected from alpine meadow in Hongyuan County of Sichuan Province.The essential oil was isolated from the whole plant using the modified steam-distillation extraction method.The chemical components were identified using gas chromatography-mass spectrometer (GC-MS) analysis.The effect of the essential oil on the proliferation of normal human liver cells (L02), human hepatoma cells (SMMC-7721) and human cervical carcinoma cells (HeLa) was studied by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.

EXPERIMENTAL Plant materials
The plants of E. dense were collected from alpine meadow in Hongyuan County of Sichuan Province, and verified as E. dense by Professor Danwei Ma, Sichuan Normal University, China.

Extraction of the essential oil from E. dense
The whole plant of E. dense was dried in a shade.According to the modified steamdistillation extraction method previously described [4], the essential oil was obtained from 500 g of the dried whole plants boiled in a required amount of distilled water.Then the essential oil was dried with anhydrous sodium sulfate, and filtered through 0.22 μm membrane.Finally it was stored in a sealed brown reagent bottle at -20 °C for the experiments.Using this method the yield of the essential oil from E. dense was increased to 0.36 %.

GC-MS analysis of the essential oil
The chemical nature of the essential oil was analyzed by Gas Chromatography-Mass Spectrometer (GC-MS, Agilent 6890-5973, Agilent Technologies Inc. USA).Helium was used as carrier gas.The rate of Agilent DB-Wax capillary column (60 m x 0.25 mm, 0.25 µm film thickness) was set at 1.0 mL/min.During the analysis period the temperature of GC oven was first set at 35 °C for 10 min and then increased to 230 °C at the rate of 3 °C /min, and finally maintained at 230 °C for 10 min.Split ratio was 5:1.The injector temperature was set at 230 °C.Mass spectra were recorded in the mass range of m/z 35 ~450 at 70 eV.

Determination of chemical components of the essential oil
The essential oil components was determined by comparison of their relative retention times to standard reference data from the National Institute of Standards and Technology (NIST) version 11 GC-MS libraries (USA).Quantification was determined by percentage peak area calculations using GC-FID.

Cell culture
SMMC-7721 cells, L02 cells and HeLa cells were grown as monolayers in RPMI-1640 medium supplemented with 10 % fetal bovine serum and 1 % antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).Cells were maintained at 37 °C and 5 % of CO 2 .The cells used in the experiments were in logarithmic growth phase.

Determination of cell proliferation
SMMC-7721 cells, L02 cells and HeLa cells were seeded at the density of 7~10×10 4 /well in a 96well plate (100 μL/well).After 12 hour incubation, the cells were adhered to the plate and treated with the essential oil from E. dense at 5 different concentrations, 10, 20, 30, 40, 50, 60 μg/mL.Cells treated with 1 % DMSO were negative control and cells treated with 80 μg/mL fluorouracil were positive control.After 8 or 24 hour treatment with the essential oil, 20 μL of 5 mg/mL MTT solution was added into the medium.After 4 hour incubation, the medium containing MTT was removed, and the remaining formazan crystals were dissolved by incubation with 150 μL/well DMSO on the oscillator for 10 min.The absorbance (A) value of each well was measured at the wavelength of 490 nm by microplate reader (MD/Spectra Max M2, USA).All experiments were performed 3 times.The cell proliferation inhibition rate was calculated as: The cell proliferation inhibition rate (%) = [1-A the essential oil treated cells /A DMSO control ] × 100 %.

Data statistics and analysis
The data were analyzed using SPSS17.0(SPSS Inc., USA).Significant differences were analyzed by Least-Significant Difference (LSD) test.P < 0.05 was considered statistically significant.All data are presented as mean ± standard deviation (SD).The figures were drawn using Microsoft Excel 2003 (Microsoft, USA).

DISCUSSION
In this study, the essential oil from E. dense collected from alpine meadow in Hongyuan County of Sichuan Province consists of 40 chemical components, with 37 components identified.The essential oil from E. dense shares some common components with other species of Elsholtzia: thymol, 1,8-cineole, βdehydroelsholizione, elsholtzia ketone, linalool, carvacrol and p-cymene [14], however it differs in the chemical composition from other species of Elsholtzia [15][16][17].
The essential oil of E. dense in Sichuan Province also differs in the chemical composition from E. dense growing in other regions [3][4][5].In this study it is the first time to study the antitumor effect of the essential oil from E. dense in Sichuan Province, although the essential oil of many plants has been reported to be anti-tumor [7][8][9][10][11][12][13]. The essential oil from E. dense had a significant inhibitory effect on the proliferation of L02, SMMC-7721 and HeLa cells in a concentration dependent manner.The IC 50 values show that SMMC-7721 cells were the most sensitive cells to the inhibitory effect of the essential oil.The inhibitory effect on proliferation of SMMC-7721 cells was time-dependent, however the inhibitory effect on proliferation of L02 and HeLa cells was alleviated when extending its treatment time from 8 hours to 24 hours.For 24 hour treatment with the essential oil, the effect was alleviated possibly because stress cell signaling in L02 and HeLa cells was stimulated and responded during this period.These results reveal that the essential oil from E. dense is cytotoxic, exerting stronger inhibitory effect on human hepatoma cells than normal liver cells.Therefore, it has great potential for development as an anti-liver cancer drug.

CONCLUSION
The yield of the essential oil from E. dense increased to 0.36 % with the use of the modified steam-distillation extraction method.The main chemical components of the essential oil from E. dense are rosefuran epoxide, 2-ethyl imidazole, durenol, p-cymene, and 1-cyanoacetyl-3,5dimethylpyrazole.The essential oil of E. dense is cytotoxic, exerting stronger inhibitory effect on human hepatoma cells than normal liver cells.Therefore, the essential oil has great potential for development as an anti-liver cancer drug.Further studies are required to identify the active components responsible for its anti-tumor activity.

Figure 2
Figure 2 shows a time-and concentrationdependent inhibitory effect of the essential oil on proliferation of 3 cell lines, which is similar to the positive control (treatment with 80 μg/mL fluorouracil).The essential oil significantly inhibited proliferation of SMMC-7721 and HeLa cells in a concentration dependent manner during 8 hour treatment (p < 0.05, Figure 2A) and 24 hour treatment (p < 0.05, Figure 2B).During 8 hour treatment the IC 50 values for SMMC-7721, L02 and HeLa cells were 26.23, 36.64 and 44.46 μg/mL, respectively.During 24 hour treatment the IC 50 values for SMMC-7721, L02 and HeLa

Figure 1 :
Figure 1: Chromatogram of the essential oil of E. dense

Figure 2 :
Figure 2: Inhibitory effects of the essential oil from E. dense on proliferation of L02, SMMC-7721 and HeLa cells.(A): 8 hour treatment with the essential oil; (B): 24 hour treatment with the essential oil.DMSO: cells were treated with 1 % DMSO; * p < 0.05 relative to DMSO treated cells, ** p < 0.01 relative to DMSO treated cells

Table 1 :
Chemical components of the essential oil of E. dense