To find a medicinal plant with the properties of oxidative stress improvement and cytotoxic effects, the pharmacologic potential of TAEO and FAEO was compared. TAEO had a high phenolic and flavonoid content showing relatively high antioxidant activity in FRAP and DPPH assays and probably having a good therapeutic effect in improving oxidative stress. Therefore, TAEO had a relatively high cytotoxic effect on MCF-7 and MDA-MB-468 cell lines. However, FAEO had relatively weak cytotoxic effects on the breast cancer cell lines. FAEO had low phenolic and flavonoid content and antioxidant effects.
The cytotoxic effects of TAEO had a concentration and time-dependent pattern. After 24 h of incubation, its cytotoxic effects on MCF-7 and MDA-MB-468 breast cancer cell lines, respectively, were 15% and 0.42% on the concentration of 100 ppm. The cytotoxic effects intensified with an increase in TAEO concentration and incubation time. Therefore, the cytotoxic effects after 72 h of incubation reached 97.67 and 65.95% on the concentration of 400 ppm, respectively, in the MCF-7 and MDA-MB-468 breast cancer cell lines.
The cytotoxic effects of traditional medicinal products are divided into four categories according to the IC50 value. Medicinal products with the IC50 values of 0–20, 20–100, 100–1000, and > 1000 ppm are classified, respectively, as very active, relatively active, weakly active, and inactive cytotoxic compounds [9]. The IC50 values of cytotoxic effects of TAEO on MCF-7 cell line were 228.6, 209.8, and 192.5 ppm, respectively, after 24, 48, and 72 h of incubation, respectively. On MDA-MB-468, the IC50 values were also 336.6, 334.1, and 331.4 ppm, respectively. According to the classification criteria of the anticancer compounds, TAEO is considered a “weakly active” compound. The IC50 values of cytotoxic effects of FAEO on MCF-7 and MDA-MB-468 cell lines were > 400 ppm. FAEO probably is an inactive anticancer compound.
MCF-7 cell line has the estrogen receptor (ER-positive) unlike the MDA-MB-468 cell line, which lacks the estrogen receptor (ER negative). The cytotoxic effects of TAEO and FAEO on MCF-7 cells were higher than MDA-MB-468 cells. Therefore, it appears that the cytotoxic effects of TAEO and FAEO are ER-dependent. The results of morphological changes were also approved for the cytotoxic effects. The morphological changes of the MCF-7 cell line in treatment with TAEO and FAEO were also much greater than MDA-MB-468. The changes intensified with increasing concentration and time incubation. The results showed that most changes in cell morphology were observed in TAEO-treated cells.
Previous studies have also shown the high phenolic and flavonoid content of TAEO and high antioxidant effects in DPPH radical scavenging activity and Fe3+ reducing activity [11, 12]. Akhlaghi et al. reported the high phenolic content of methanolic extract of Trachyspermum ammi. The phenolic content of the extract was 164.5 ± 1.3 mgGAE/g which was lower that the phenolic content of TAEO in our study [13]. Chatterjee et al. also reported that polyphenols make up about 45% of the dry weight of the methanolic and aqueous extracts of Trachyspermum ammi. The phenolic content reported in the Chatterjee et al. study was almost the same as the present study [14]. The results of GC–MS analysis also confirm the high phenolic content of TAEO in the present study. Vitali et al. showed that TAEO has high phenolic content, flavonoid content, and antioxidant effects. TAEO was also shown to have considerable cytotoxic effects on the MDA-MB-231 triple-negative breast cancer cell line. The reported cytotoxic effects probably were due to the phenolic compounds such as thymol and carvacrol [15]. In the study of Mohammadpour et al., the cytotoxic effect of Trachyspermum copticum essential oil toward the MDA-MB-231 cell lines with the IC50 value of 236.16 µg/mL was shown. So, Trachyspermum copticum essential oil was considered a weakly active anticancer compound, similar to TAEO in the present study. Mohammadpour et al. also showed that the essential oil of Trachyspermum copticum has a lower cytotoxic effect in the normal HEK cell lines compared to the cancerous cells [16]. But the cytotoxic effects of the essential oils were not evaluated on the non-malignant breast epithelial cells in the present study. The present study focuses on the pattern of cytotoxic effects on different breast cancer cell lines.
In addition to breast cancer, numerous studies have also represented the cytotoxic effects of Trachyspermum ammi on multiple cancer cell lines. In the study of Abdel-Hameed et al., the anticancer activity of Trachyspermum ammi was shown on the hepatocellular carcinoma cell line (HepG2). The IC50 values of essential oil and n-hexane extract of Trachyspermum ammi, respectively, were 9.57 ± 0.98 and 17.42 ± 1.44 µg/mL. The higher activity of essential oil is probably related to its high contents of volatile compounds. According to Abdel-Hameed study, the TAEO is considered a very active anticancer compound on the HepG2 cell line, unlike the present study [17]. However, there is also a possibility that geographical conditions influenced the pharmacological content of the essential oils. In the study of Aruchamy et al., the cytotoxic effect of the methanol seed extract of Trachyspermum ammi on the KB cell line (an epidermal carcinoma of the mouth) with the IC50 value of 125 µg/mL was shown. This study reveals that the Trachyspermum ammi exhibited significant cytotoxic and apoptotic inducing effects through promoting reactive oxygen species (ROS) [18]. In the study of Singh et al., the chemopreventive potential of Trachyspermum ammi seeds against carcinogenesis was shown. They showed that Trachyspermum ammi has a therapeutic effect on murine skin and forestomach papilloma genesis. In the treated animals, the content of reduced glutathione was elevated. The peroxidative damage and lactate dehydrogenase activity was also reduced [19]. Therefore, it appears that TAEO can influence on cancer cell microenvironment by increasing antioxidant compounds, reducing oxidants, and improving oxidative stress. TAEO probably increases the expression of proapoptotic genes Bim, Bak, or Bax, arrests the cancer cell cycle, and inhibits cancer by producing ROS inflammatory mediators [20, 21].
Numerous studies have revealed the cytotoxic effects of thymol as the major component of TAEO. In the study of Deb et al., the dose-dependent cytotoxic effects of thymol on acute promyelocytic leukemia (HL-60) cells were demonstrated after 24 h of exposure. However, thymol did not show any cytotoxic effect in normal human PBMC. The cytotoxic effect of thymol on HL-60 cells appears to be associated with induction of cell cycle arrest at the sub G0/G1 phase [22]. According to the literature review, it appears that thymol has a dual effect on cell viability. Thymol can induce oxidative stress-linked mitochondrial dysfunction and other intrinsic and extrinsic apoptotic cell death on cancer cells. Conversely, in the normal cells, it appears that thymol has antioxidant and cytoprotective effects [23]. In the study of Zeng et al., the in vitro and in vivo anticancer effects of thymol were demonstrated. Thymol treatment in vitro induced apoptosis and cell cycle arrest in colorectal cancer and in vivo led to cell apoptosis and a significant decrease in tumor volume through the BAX/Bcl-2 signaling pathway [24]. In the study of Seresht et al., the cytotoxic effect of thymol as the major component of TAEO was shown on the MCF-7 cell line. They represented the IC50 value of thymol on the MCF-7 cell line as 54 and 62 µg/mL, respectively, after 48 and 72 h of incubation. Furthermore, they show the apoptosis-inducing effect of thymol on the MCF-7 cell line via upregulation of P53 and P21 gene expressions [25].
About the FAEO cytotoxic effects, previous studies indicated that the ethanolic or water extract of Ferula assafoetida gum has high cytotoxic effects on the cancerous cell line. Sadooghi et al. showed that ethanolic extract of Ferula assafoetida induced morphological changes and decrease in viability of HepG2 cells with minimal cytotoxic effect on normal cells [26]. Bagheri et al. also indicated that water extract of Ferula assafoetida decreases the tumor weight and volume in an animal model of breast cancer [27]. Saleem et al. showed that pre-treatment of animals with acetone extract of Ferula assafoetida suppresses the early events of carcinogenesis [28]. It appears that the hydroethanolic extracts of Ferula assafoetida are more effective than its essential oil on the cancerous cell lines. Verma et al. have also shown that the FAEO will represent the antiproliferative activity if the essential oil has the high content of dithiolane (87.4%) [29].