Chemical composition and cytotoxic activity of the essential oil from the aerial parts of Dorema aucheri

Introduction Dorema aucheri is a plant of Apiaceae family which grows at the end of the spring in southern provinces of Iran, especially in the provinces bordering the margins of the Zagros mountains, such as Kohgiluyeh and BoyerAhmad (1,2). The plant has medicinal properties and also is used by the local inhabitants for preparing food (1,3-5). It has already been proven that the aerial parts of the D. aucheri are rich in flavonoids (6). Flavonoids represent a large group of polyphenolic compounds that exhibit anti-oxidative effects (7,8). Although several reports have been conducted on the phytochemistry and http://www.herbmedpharmacol.com doi: 10.34172/jhp.2021.40


Introduction
Dorema aucheri is a plant of Apiaceae family which grows at the end of the spring in southern provinces of Iran, especially in the provinces bordering the margins of the Zagros mountains, such as Kohgiluyeh and Boyer-Ahmad (1,2). The plant has medicinal properties and also is used by the local inhabitants for preparing food (1,(3)(4)(5). It has already been proven that the aerial parts of the D. aucheri are rich in flavonoids (6). Flavonoids represent a large group of polyphenolic compounds that exhibit anti-oxidative effects (7,8). Although several reports have been conducted on the phytochemistry and bioactivity of hydroalcoholic extract of D. aucheri, the compounds and bioactivity effects of its essential oil have not been clearly determined. Several studies have reported hepatoprotective, anti-diabetic, anti-tumor, anti-oxidant, anti-hyperlipidemic, and anti-hypercholesterolemic effects of the hydroalcoholic extract of this plant (1,(3)(4)(5)9). Moreover, in several pathophysiological states, it has been reported that D. aucheri extract has positive effects on thyroid hormones, antioxidant enzymes, the haematologic system and also serum levels of testosterone, folliclestimulating hormone (FSH) and luteinizing hormone (LH) (10). The analysis of essential oil extracted from the leaves of D. aucheri showed that it contained 36 (99.86%) compounds, and the major constituents included curzerene (18.7%), α-eudesmol (7.72%), Spathulenol (6.68%), isohibaene (6.16%), and gemberen (6.66%) (11).
In several studies concerning the extracts of the aerial parts of D. aucheri, the presence of a large group of terpenoids, more specifically the sesquiterpene compounds, have been demonstrated. It has been indicated that the pharmacological features of D. aucheri, and its potential role in anti-inflammatory, and in the treatment of thyroid disorders and tumors can be due to its sesquiterpene compounds (12). In the present study, the constituents and bioactivity of the essential oil of D. aucheri were determined by GC-MS analysis. Also, the cytotoxic effects of these compounds were evaluated against two colorectal cancer cell lines (SW48 and SW1116).

Plant materials
The aerial parts of D. aucheri were collected from the mountains near the Yasuj city (25 km away from the city, at 30.4658640 N, 51.6783400 E, and the altitude of 2430 m) in Kohgiluyeh and Boyer-Ahmad province, Iran, in the spring of 2017. The plant was authenticated by a botanist (Dr. Azizollah Jafari, a botanist at Yasuj University, faculty of science). The aerial parts were dried in a dark place and then were powdered by an electric grinder. The voucher specimen of the authenticated plant (voucher no. 0496) was deposited at the herbarium of Medicinal Plants Research Center, Yasuj University of Medical Sciences.
Preparation of essential oil The powder of D. aucheri (1200 g) was hydro-distilled in several runs for 4 hours using a Clevenger apparatus. The essential oil was collected, dried with anhydrous sodium sulfate, and kept in refrigerator until GC-MS analysis.

GC-MS analysis and identification of the oil components
The GC-MS analysis of the oil was conducted using a Hewlett-Packard 6890 instrument equipped with a HP-5M capillary column (phenyl methyl siloxane, 25 m × 0.25 mm id, Hewlett-Packard Part No. 190915.433, USA). The oven temperature was adjusted from 50°C (3 minutes) to 250°C at the speed of 3°C min -1 and finally continued for 10 minutes at 250°C. The injection temperature was 250°C. Helium was used as the transferor gas at a constant flow rate of 1.2 mL/min. The mass spectrometer (Hewlett-Packard 5973, USA) was activated in the electron ionization (EI) mode at 70 Ev and the mass range was 30-600 m/z. The identification of components was performed by comparing the relative retention times with those of a series of n-alkane standards (C10 to C30: ref. no. R-8769, Sigma) and linear interpolation based on computer matching with the Willey library (Willey-275) and spectra literature data (13).
Cell lines and culturing Human colorectal cancer cell lines (SW48 and SW1116) were obtained from the Pasteur Institute of Iran. The cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 1% glutamine, and 100 U/mL penicillin/ streptomycin. The cells were cultured in a humidified atmosphere at 37°C and 5% CO 2 .
Cell proliferation assay The cellular proliferation was assessed using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay. The cells (5 × 10 4 ) were seeded in each well of a microplate, containing 100 μL of the RPMI medium supplemented with 10% FBS. After a 24 hours incubation, the cells were attached to the bottom of each well, and treated with D. aucheri essential oil at the concentrations of 0.2 to 1.6 mg/mL for 24 hours. Then, 5 mg/mL MTT reagent was added to each well, and the plate was incubated at 37°C for 4 hours. As the positive control, the cells were treated with vincristine (Sobhan Oncology Co., Iran). Next, the supernatant was removed, and 100 μL DMSO was added to each well. Finally, the optical density of wells was determined at 490 nm using a microplate reader (Stat Fax3200, Awareness Technology, USA).

Apoptosis assay
The SW48 and SW1116 cells (1×10 6 cells per well) were seeded in six-well plates and then treated with either medium alone (negative control), D. aucheri essential oil (1.4 and 1.2 mg/mL), or vincristine (0.05 and 0.04 mg/ mL) for 24 hours. The cells were resuspended in a cold binding buffer, then stained with annexin V-FITC reagent (5 μL) and propidium iodide (PI) (5 μL), and incubated in the dark at room temperature for 15 minutes. After adding 500 μL of the binding buffer, fluorescence was read using a fluorescence-activated cell sorter (FACS) (BD Biosciences, San Diego, CA, USA). Flow cytometry data was analyzed by FlowJo software. All the samples were assayed in triplicate.

Statistical analysis
For statistical analysis, the data of cytotoxic activity was compared between different groups by the analysis of variance (ANOVA) followed by Tukey's post hoc test. The probability value of P < 0.05 was considered to denote a statistically significant difference.
Cytotoxic activity MTT assay was used to determine the IC 50 of D. aucheri essential oil on SW48 and SW1116 cell lines. The cells were treated with 0.2 to 1.6 mg/mL concentrations of D. aucheri essential oil ( Table 2). In parallel, vincristine was used as a positive control. The result showed that the essential oil significantly (P < 0.05) inhibited the cell growth of SW48 and SW1116 cell lines. As shown in Table 3, the IC 50 values (The inhibitory concentrations that could reduce 50% of SW48 and SW1116 cells) were 1.4 and 1.2 mg/mL for essential oil and 0.05 and 0.04 mg/ mL for vincristine, respectively. The results showed that the SW1116 cell line was more sensitive than SW48 to D. aucheri essential oil. Also, it was shown that the SW48 cell line was more resistant than SW1116 to vincristine.

Apoptosis
To explore the mechanism by which D. aucheri essential oil might exert its anti-proliferative effects on SW48 and SW1116 cell lines, we assessed apoptosis using Annexin V and PI assay. The respective dot plots of this analysis have been shown in Figure 2. Based on the IC 50 values, the cells were exposed to the essential oil for 24 hours to stimulate apoptosis. Flow cytometry results indicated that, 24 hours incubation with the essential oil significantly elevated apoptosis in SW48 cell line, compared with the vincristine (P < 0.05). It also increased the rate of  apoptosis in SW1116 cells compared with the vincristine, but this difference was not significant (Figure 3). The apoptosis rates in SW48 and SW1116 cell lines treated with D. aucheri essential oil were 14.93% and 7.1%, while the cells treated with vincristine showed 4.5% and 6.43% apoptosis rates, respectively. Also, the essential oil treatment significantly elevated apoptosis in SW48 and SW1116 cell lines, compared with the negative control (P < 0.05). The comparison of apoptosis rates between SW48 and SW1116 cell lines showed that D. aucheri essential oil induced a significantly higher apoptosis rate in the SW48 cell line (P < 0.05).

Discussion
The current treatments for cancer, such as chemotherapy and radiotherapy, despite having cytotoxic effects against cancer cells, are associated with the side effects on normal proliferating cells. Therefore, it is required to develop alternative therapeutic approaches with the least possible complications (14)(15)(16). Among the potential sources for novel therapeutics, medicinal plants can be the most important options due to their anticancer components such as phenolics, glycosides, steroids, flavonoids, and terpenoids (17)(18)(19).
Although several studies have been carried out regarding the beneficial health effects of hydroalcoholic extract of the D. aucheri (3,4), there is inadequate knowledge about the composition and properties of the essential oil of this plant. In this study GC-MS analysis showed that terpenoids constituted 70.88% of the compounds identified in the investigated oil, among which sesquiterpenes (55.59%), diterpenes (14.29%), and monoterpenes (0.37%) were the predominant terpene compositions. Despite differences in the types of compounds, these compositions demonstrated similar biological functions compared to those of other plant species assessed in prior studies (7,8,20). Asnaashari et al who analyzed the composition of the essential oil of D. glabrum roots by GC/MS method showed that the oil was rich in sesquiterpenes and monoterpenes (21). In another study, the major constituents of the essential oil of D. ammoniacum collected from the Kellar mountain, were three hydrocarbon monoterpenes, five oxygenated monoterpenes, ten sesquiterpene hydrocarbons, and thirteen oxygenated sesquiterpenes (22). Akbarian et al in their study, conducted on five D. aucheri populations in different regions of Iran, showed that β-caryophyllene, thymol, β-gurjunene, carvacrol, and cuparene were the major components (23). In contrast, in the study of Delnavazi et al on D. glabrum plant, although the main components were non-terpene compounds (56%), terpenes were also widely found in the plant (24).
In our study, the analysis of essential oil of D. aucheri  Results are expressed as means ± SD of three independent MTT assay performed in triplicate. Vincristine was tested as positive control. showed that α-eudesmol was absent, while this compound has been identified in other species of Dorema. In the analysis of the oil extracted from D. aucheri collected from the Hezar mountain, the main components were α-eudesmol (31.2%) and δ-cadinene (10.9%) (25). Also, the chemical composition of the essential oil from the stems and seeds of Dorema ammoniacum revealed a remarkable difference with our study (26). The reason for the discrepancy between our findings and previous ones can be due to the different environmental conditions leading to qualitative and quantitative variations in the compositions of oils (27). Because terpenoids have been found to suppress the growth of a variety of cancer cells, the aim of the present study was to evaluate the biological effects of the essential oil of D. aucheri on SW48 and SW1116 colorectal cancer cell lines (28). The MTT assay was performed to evaluate in vitro cytotoxic activity of the essential oil against the cancer cell lines. The results showed that the essential oil of D. aucheri inhibited the growth of these cells as compared to vincristine. Our results also showed that the oil extract had greater toxic effects against the SW1116 cell line with the IC 50 of 1.2 mg/mL compared to the value of 1.4 mg/mL obtained for the SW48 cell line.
We also evaluated the apoptotic effects of essential oil of D. aucheri against the mentioned cell lines using Annexin V and PI assay. The flow cytometry results indicated that 24 hours incubation with the extract significantly elevated apoptosis in SW48 cell line, compared to vincristine. The essential oils of D. glabrum and D. ammoniacum have been shown to effectively induce apoptosis in cancerous cell lines (29,30). Many studies have also demonstrated that the essential oil constituents of plants such as terpenoids present anti-cancer and pro-apoptotic effects and inhibit the differentiation, angiogenesis, invasion, and metastasis of tumor cells (28,31,32). In the present study, caryophyllene, a type of sesquiterpene, comprised the major compound (31.29%) of essential oil of D. aucheri. Several studies have shown the anti-cancer effects of  % Apoptosis this compound on osteosarcoma and breast tumors (33)(34)(35). Phytol, as a diterpene with anticancer effects (36), was the second major compound of D. aucheri essential oil with a concentration of 14.92%. This compound can be considered a possible candidate for a wide range of applications in pharmaceutical, food, and biotechnological industries (37).

Conclusion
The essential oil of D. Aucheri comprised of high amounts of caryophyllene and showed significant cytotoxic effects against SW48 and SW1116 cancerous cell lines. Hence, after more comprehensive studies, it can be used as a beneficial herbal source for developing anti-tumor drugs.