Introduction
Essential oils are used as additives in many types of foods and beverages and various food supplements [1]. The Plantago genus of the Plantaginaceae family includes approximately 300 annual and perennial species, growing worldwide, and specially cultivated in the subtropical regions [2]. According to Iran’s traditional medicine, Plantago species have many medical applications without serious side effects; however, some of the medicinal effects of Plantago lanceolata L. (P. lanceolata) and Plantago major L. (P.major) in Iran’s traditional medicine have not been discovered in modern medicine [3]. 
P. lanceolata and P. major are used to treat wounds, infectious diseases, digestive and respiratory problems, fever, pain, dermatitis, and tumors [4, 5]. Furthermore, Plantago species were used to cure burns, ulcers, and eye diseases, as anti-inflammatory, antipyretic agents, anti-tussive, and purgative for snakebites [6]. Researchers have also reported that P.major mucilage can optimize the drug release in propranolol buccoadhesive tablets [7]. Additionally, they can be used in cosmetics to produce face masks, creams, or lotions for acne-prone and oily skins because of their astringent, anti-septic, and anti-bacterial properties [6]. 
GC/MS is one of the most important instruments used to analyze a sample with volatile constituents as it combines both the chromatographic technique for the efficient separation of sample constituents and mass spectroscopy that identifies the compounds according to their mass-to-charge ratio (m/z) [8]. The above-mentioned properties of these plants provide us with significant reasons to analyze their volatile composition. To date, only a few Plantago species have been investigated for their chemical constituents and biological activities of extracts. Previous studies on the chemical investigation of Plantago L. leaves and seeds extracts demonstrated the presence of polysaccharides, phenolic acids, flavonoids, iridoid glycosides, and vitamins [2]. 
There are few valid studies on the essential oil compositions of P. lanceolata and P. major, considering that these plants contain very small amounts of essential oil. Therefore, in the current study, following our previous studies on these plants, their essential oil compositions were examined. In addition, we evaluated the toxicity effects of the essential oils on colon cancer cells and Artemia salina (A.salina). To the best of our knowledge, there are no reports on the cytotoxicity assay of P. lanceolata and P. major essential oils on colon cancer cell lines.

Materials and Methods
Herbal material 
The aerial parts (leaf and stem) of P. lanceolata and P. major were collected from Zanjan Province, Iran (the geographical coordinates of the collection sites are as follows: 36°41’15.5”N 48°24’02.2”E). The taxonomic identity of species was authenticated at the Department of Botany, University of Zanjan, Iran. All sections were cut into small pieces and were dried in shade and at room temperature separately for one week. 

Isolation of essential oils 
The aerial parts of P. lanceolata and P. major (100 g) were ground to a coarse powder and extracted with 1500 mL of distilled water for hydrodistillation in a Clevenger-type apparatus for 5 to 6 h to arise the volatile composition in the form of essential oils. The essential oils were collected into 1 mL of n-pentane and then poured into a glass and stored at 4°C until further analysis [1]. 

Gas chromatography-mass spectrometry analysis 
The essential oils of the aerial parts of P. lanceolata and P. major were used for GC/MS analysis. GC/MS analysis was performed using the Agilent technologies 5975c. GC/MS analysis was carried out by 1 µL of the materials subjected to analysis. The GC/MS system has been equipped with a capillary column (30 m×250 µm×0.25 µm, Agilent). Helium as the carrier gas was used at the flow rate of (1 mL/min). The injector and the interface temperature were maintained at 250°C. The column temperature was programmed as follows: the initial temperature was 40°C (1 min) and then it increased at a rate of 2°C/min up to 200°C (10 min). The identification of the constituents of P. lanceolata and P. major was performed by comparison with MS literature data (NIST08.L) and retention index (RI) [1]. The mixtures of n-alkanes (C8-C20) were injected using the above temperature program to calculate the RI for each peak. The RI of the compounds was calculated using the following equation: 

Where: (Ix) is the Kovats retention index; (n) is the number of carbon atoms in the alkane; (tn) and (tn+1) are the retention times of the reference n-alkane hydrocarbons with n and n + 1 carbon atoms; and (tx) is the retention time of the peak of the unknown compound. 
Several peaks did not have RIs for the calculated mixtures of n-alkanes (C8-C20). Thus, compounds with a formula structure less than C8 and more than C20 could not be calculated (these compounds were considered unknown). 

Cell line culture
Human embryonic kidney cell (HEK-293) as a normal cell line and colorectal cancer cell line (HCT-116) provided by the Pasteur Institute of Iran, Tehran were cultured in the Dulbecco’s Modified Eagle Medium with supplementation of penicillin-streptomycin (1%) along with 10% fetal bovine serum incubated in 5% CO2 incubator at 37°C.

Cytotoxicity assay
The MTT assay was performed to evaluate the cytotoxicity of P. lanceolata and P. major essential oils on the cell lines [9]. A 96-well plate with a density of 7 × 103 cells/well were used for cell seeding. The cells were allowed to attach and grow for 24 h. The cells underwent treatment with 25-400 µg/mL concentrations. The HCT-116 were treated with 5-fluorouracil (5-FU) (Austria, Ebewe Pharma) in different doses (2.5-10 μg/mL) for 72 h. The 5-FU and untreated cells were utilized as the positive and negative control, respectively. The addition and incubation of 20 μL of MTT (5 mg/mL) for 4 h took place after 24 to 72 h, followed by removing the medium and adding 200 μL of dimethyl sulfoxide to dissolve the obtained formazan. An ELISA plate reader (Tecan Infinite M200, Austria) at 570 and 690 nm read the absorbance. The cell growth inhibition rates were examined by the following formula:


Toxicity assay on artemia salina
The larvae of brine shrimp (A.salina Leach) were employed to examine the P. lanceolata and P. major essential oils’ overall toxicity [10]. A. salina eggs were provided by Urmia University, the West Azerbaijan Province, Iran. A flask with 35 g of NaCl dissolved in 1 L of distilled water was used for cyst culture, followed by 48 h incubation at 28°C and the larvae hatching after 48 h. Every well in the 96-well microtiter plates having the Roswell Park Memorial Institute (RPMI-1640) received the essential oils (25-400 µg/mL). The next step included the addition of 10 nauplii per well to the 96-well plates and incubation at a temperature of 25°C for 24 h. A binocular microscope was employed to calculate the number of live nauplii in every well after 24 h. All experiments were repeated 3 times. Additionally, the negative control contained only 10 nauplii and artificial seawater. Potassium dichromate (K2Cr2O7) was used as a positive control at the same concentrations as the essential oils. The number of survived samples in the experimental and control wells was used to calculate the percentages of the nauplii morality. The Abbott formula determined the lethality:

Statistical analysis
The data were analyzed using the SPSS software, version 21. The significant differences between means were calculated. Values were expressed as the mean of the 3 replications ± Standard Deviation (SD). The Duncan test at P value<0.05 was used to determine significant differences among treatments. IC50 and LC50 values were analyzed with the ED50 plus v1.0 Software.

Results 
Many peaks were detected in the chromatogram of the essential oils extracted from P. lanceolata and P. major aerial parts by GC/MS and their compositions were identified according to the NIST08.L library. Figure 1 shows the main chromatograms of the essential oils of P.

lanceolata and P. major. The essential oils were rich in amine derivations, alcohols, alkenes, and fatty acids. The essential oils also showed the presence of acids, alkaloids, amino acids, carboxylic acid derivatives, esters, ketones, monoterpenoids, nitriles, oximes, phenols, phenethylamine derivatives, and others (Table 1).

Volatile constituents of p. lanceolata essential oil
Most component of P. lanceolata essential oil is generated by metaraminol (14.04%), bifemelane (8.73%), metossamina (8.16%), and pterin-6-carboxylic acid (5.11%).
In the present study, 106 components belonging to main chemical groups were identified in P. lanceolata essential oil: alcohols (17.56%) with benzyl alcohol; .α.-(1-aminoethyl)-m-hydroxy-, (-)-(14.04) as the main component; amines (14.70%) with phenylephrine (3.71%); alkenes and alkenes (12.28%) with bifemelane (8.73%); ketones (8.70%) with bicyclo [2.2.1] heptan-2-one, 4,7,7-trimethyl-, semicarbazone (2.97%); acids (8.05%) with pterin-6-carboxylic acid (5.11%); alkaloids (5.76%) with 2H-1,2,3-triazole-4-carboxylic acid; 2-(2-fluorophenyl)- (2.12%); esters (4.02) with 2-thiopheneacetic acid; 3,5-difluorophenyl ester (1.53%); amides (3.55%) with propanamide (0.58%); amino acids (2.71%) with histidine; 1, N-dimethyl-4-nitro- (1.76%); monoterpenoids (2.45%) with Linalool (0.97%); phenol (Benzeneethanamine, 2-fluoro-.beta.,5-dihydroxy-N-methyl-) (0.45%); nitriles (0.21%) with propanenitrile, 3-(methylamino)- (0.17%); oximes with ethanone, 1-(4-pyridinyl)-, oxime (0.13%) as the main components and others (21.03%) (Table 2 and 3).

The biological activities of the volatile constituents of P. lanceolata oil are reported in Table 4.

Volatile constituents of the essential oils of p. major
The present study showed that 2-dodecen-1-yl (-) succinic anhydride (15.29%), benzenemethanol,. α.-(1-aminoethyl)-2,5-dimethoxy- (11.83%), dl-phenylephrine (7.51%), nortriptyline (5.15%) were the major constituents (Tables 2 and 3).
In the present study, 79 components belonging to main chemical groups were identified in P. major essential oil: amines (35.74%) with phenylephrine (11.66%) as the main component; alkenes and alkanes (24.88%) with 2-dodecen-1-yl(-)succinic anhydride (15.29%); phenols (10.49%) with dl-phenylephrine (7.51%); esters (6.96%) with sarcosine, N-valeryl-, butyl ester (2.02%); alcohols (5.14%) with cyclobutanol, 2-ethyl- (1.72%); alkaloids (3.97%) with ethylamine, 2-(adamantan-1-yl)-1-methyl- (0.28%); ketones (3.61%) with 3-(E)-hexen-2-one, (5S)-5-[(t-butoxycarbonyl-(R)-alanyl)amino]- (2.65%); amides (2.2%) with [(2,5-dimethoxyphenyl)sulfonyl]ethylamine (0.69%); monoterpenes with isoborneol (1.17%); amino acids (glycine, N-(N-L-alanylglycyl)-) (0.35%) and acid (0.16%) with imidazole-5-carboxylic acid, 2-amino- as the main component. P.major essential oil has many properties and applications that are provided in Table 4.
The essential oils of P. lanceolata and P. major species showed that the predominant compounds were present in both species; however, the amounts of these compounds (%) were different. For example, (-)-Benzyl alcohol, .α.-(1-aminoethyl)-m-hydroxy (14.04% and 1.37%), metossamina (8.16% and 0.17%), benzenemethanol, .α.- (1-aminoethyl) -2,5-dimethoxy- (3.71% and 11.66%), dl-phenylephrine (0.15% and 7.51%), nortriptyline (0.95% and 5.15%) were present in P. lanceolata and P. major, respectively (Figure 2).

Bifemelane (% 8.73), pterin-6-carboxylic acid (5.11%) existed only in P. lanceolata while 2-dodecen-1-yl (-) succinic anhydride (15.29%) were only found in P. major.

Cytotoxic activities
Colorectal cancer cells were incubated after treatment with essential oils to study the cytotoxic activities of P. lanceolata and P. major. The essential oils of P. major exhibited more antiproliferative properties on HCT-116 at 72 h compared to P. lanceolata (IC50: 102.66 µg/mL). IC50 values showed that P. major essential oil had a greater cytotoxic effect on HCT-116 than HEK-293; however, P. lanceolata showed almost the same effect on cancer and normal cells (Table 5). The results indicated that a very low IC50 of 5-FU (4.136 µg/mL) was required to inhibit HCT-116 cell viability compared to the essential oil of P. lanceolata and P. major. 

Toxicity assay on artemia salina
The general toxicity of the essential oils was assessed against A. salina. At 25-100 µg/mL of the essential oils, all of the nauplii were alive, indicating no toxicity (LC50:2242.57 µg/mL and 1783.7 µg/mL) (Table 5). At 400 µg/mL of P. lanceolata and P. major, the percentage of lethality was 8% and 12%, respectively. Although, the K2Cr2O7 has shown to have a toxic effect (LC50 of 58.22 μg/mL).

volatile constituents in the essential oil, respectively. In their study, the main aroma constituents of P. lanceolata leaves were groups of fatty acids 28.0% – 52.1% (the most abundant palmitic acid 15.3% –32.0%), oxidated monoterpenes 4.3% – 13.2% with linalool 2.7% – 3.5%, ketones and aldehydes 6.9%–10.0% with pentyl vinyl ketone 2.0% –3.4%, and alcohols 3.8%–9.2% with 1-octen-3-ol 2.4%–8.2%. They pointed out that apocarotenoids (1.5%–2.3%) are the important constituents because of their intense fragrance and they were identified in a relatively high amount. The importance is in its potential manufacture control of raw material to supply food supplements [1]. The high content of 1-octen-3-ol (up to 8.2%) has been observed in the Bajer et al., 2016 study [1] in accordance with Fons [76]. This compound in the present study was about 1.27%.
Other studies showed that P. major essential oil has anti-tumor and anti-cancer activities because octodrine [28] and 1-[α-(1-adamantyl) benzylidene] thiosemicarbazide [54] were present in P. major essential oil. The anti-microbial components, i.e., 2-dodecen-1-yl(-) succinic anhydride [12]; 2-chloroacetamide [17]; isoborneol [23]; octodrine [30]; actinobolin [49]; 1-[α-(1-adamantyl) benzylidene] thiosemicarbazide [54]; cyclobutanol, 2-ethyl- [64]; antiviral compounds, including isoborneol [22]; 1-[α-(1-adamantyl) benzylidene] thiosemicarbazide [54]; antioxidant and anti-inflammatory compounds, such as 2-dodecen-1-yl(-)succinic anhydride [12]; desmethyldoxepin [56] and 1-[α-(1-adamantyl) benzylidene] thiosemicarbazide [54] were observed in the analysis of P. major essential oil. Some of the compounds identified in the analysis of the P. major essential oil showed important characteristics, such as cycloserine [71] and actinobolin [49] which are antibiotic drugs (0.75% and 0.13%) and isoborneol is anti-infective (1.17%) [22] (Table 4). The percentage and differences in the amount of these compounds depend on many factors, such as climatic conditions, type of region, plant growth conditions, and harvesting methods.
The present study indicated that a very low IC50 value of 5-FU was required to inhibit HCT-116 cell viability compared to the essential oil of P. lanceolata and P. major. However, the IC50 obtained for the essential oil of P.lanceolata and P.major were valuable and has increasingly important medical applications. Our previous studies reported the cytotoxic effects of alcoholic and acetonic extracts of P.major leaf and root on HCT-116 and HEK-293. The P. major root extract was more effective than the aerial parts, and IC50 values for ethanolic, methanolic, and acetonic root extracts were 405.59, 470.16, and 82.26 μg/mL, respectively on HCT-116 at 72 h [77]. In a study by Velasco-Lezama (2006), the cytotoxic activity of P. major methanolic extract has been reported on HCT-15 [78].
For the lethality of nauplii, if LC50, detected for each sample, is more than 1000 µg/mL, it will be non-toxic [79]. At 400 µg/mL of P. lanceolata and P. major, the percentage of the lethality of nauplii was 8% and 12%, respectively. Thus, the essential oils were not toxic. 
Other researchers have also evaluated the toxicity effect of P. major methanolic extract on A. salina and A. uramiana with LC50 of 303.7 μg/mL [80]. The LC50 values of Plantago squarrosa Murray extracts were more than 1000 μg/mL; therefore, the extracts were non-toxic in the Artemia franciscana bioassay [81]. Our previous study showed that at all concentrations of ethanolic extracts of P.major aerial parts and roots, no toxicity was observed [77]. 

Conclusions
Given the non-aromatic nature of P. lanceolata and P. major and the very small amount of essential oil in these plants, most phytochemical studies are usually performed on their extracts. Therefore, in the present study, the essential oils analysis of two well-known species of Plantago was conducted to discover the valuable compositions. The hydrodistillation method enabled us to gain a great number of volatile constituents, which is evident from the number of peaks that occurred in chromatograms. The most abundant family of compounds was amines. There were also identified acids, alcohols, alkaloids, alkanes, alkenes, amides, amino acids, esters, ketones, phenols, and terpenes that most of the terpenes were oxidated as monoterpenes. On the other hand, nitriles, oximes, and organic compounds were found in a relatively small amount. 
Regarding the chemical compounds identified in the P. lanceolata and P. major essential oils, these components could be employed as an important economical source in the pharmaceutical and chemical industries. We intend to study their biological activities in the future.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
The paper was extracted from the PhD. Dissertation of the first author at Department of Plant production and Genetics, Faculty of Agriculture, Zanjan University of Medical Sciences (Grant number: A-12-848-35).

Authors' contributions
project administration, investigation, formal analysis, and writing-original draft: Samaneh Rahamouz-Haghighi; formal analysis, methodology, and validation: Alireza Yazdinezhad; Funding and supervision: Khadijeh Bagheri; Funding, supervision, conceptualization, and editing of the English version of the manuscript: Ali Sharafi. 

Conflict of interest
The authors declare that there are no conflicts of interest regarding the publication of this article.

Acknowledgments
This work was supported by Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran (Grant number: A-12-848-35). In addition, the authors would like to thank the authority of the School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran.

References