According to the World Health Organization (WHO), 9.6 million deaths in 2018 worldwide will be due to various types of cancer, and the number of these deaths is projected to reach more than 11 million per year by 2030 [
33]. Today, the world is facing a high prevalence of cancer, which is the second leading cause of death after heart disease. Understanding the important mechanisms involved in causing cancer is important for advancing therapies for the treatment of neoplasms [
34]. Chemotherapy drugs should ideally have a specific cytotoxic effect on neoplastic cancer cells; however, in reality this treatment leads to some systemic toxicity to the individual [
35]. Apoptosis, the best programmed cell death, is involved in controlling the number of normal cells and their proliferation as part of the natural development process [
36].
75–80% of the world’s populations, especially in developing countries, use herbal medicines to treat diseases. This is because they believe that herbal medicines, in addition to being cheap and available, have fewer side effects than chemical drugs. Many common medicines are derived from plant sources. In the past, the basis of many drugs was herbal, including aspirin (bark willow), digoxin (fox glove) and morphine (opium poppy), etc. [
37]. Various studies have shown that plant compounds play an important role in both the prevention and treatment of cancers. These compounds work by different mechanisms; however, the induction of apoptosis is a common point of many of these compounds [
38]. The effect of coumarins on the cytotoxic effect of members of the Apicaceae family has also been proven in studies [
39‐
41].
Many studies have shown that different plants with different medicinal compounds have anti-cancer effects. For example, Bhandari et al. showed that different IC50 levels of
Allium wallichii extract were effective on prostate cancer cell lines (69.69%), breast (55.29%), and cervix (46.51%) [
42]. Afsar et al. evaluated the anti-cancer activity of aerial parts of
Acacia hydaspica R. Parker. They found that IC50 of about 29.9 ± 0.909 μg/ml was effective on the MDA-MB-361 cancer cell line and IC50 of about 39.5 ± 0.872 μg/ml on the HCC-38 cell line [
43]. Lai et al. evaluated the anti-cancer activity of
Blechnum Orientale Linn extract and showed that at IC50 values of about 27.5–42.8 μg/ml, it has cytotoxic activity against HT-29 cell line. The phytochemical analysis also showed the presence of compounds such as flavonoids, terpenoids, and tannins [
44]. Using
Cassia angustifolia Vahl extract, Ahmed and his colleagues were able to obtain IC50 values of about 4.0 μg/μL, 5.45 μg/μL, and 7.28 μg/μL against MCF-7, Hela, and HepG2 cancer cell lines, respectively [
45]. Chaudhary et al. used different fractions of
Nardostachys jatamansi DC to study its anti-cancer activity and concluded that the extract of this plant on breast cancer cell lines including MCF-7 (IC50 about 58.01 ± 6.1 μg/ml) and MDA-MB-231 (IC50 about 23.83 ± 0.69 μg/ml) was effective. In addition, they showed that the highest anti-cancer activity was attributed to the presence of compounds such as Lupeol and beta-sitosterol [
46]. The significant antioxidant effect of
F. vulgaris due to the presence of compounds such as alkaloids, anthraquinones, flavonoids, phenols, saponins, steroids and tannins has already been investigated [
24]. Antioxidant compounds such as spathulenol and carvacrol have also been identified as the most essential oils of the plant [
25]. In the present study, the cytotoxic effects of
n-Hex, DCM and MeOH extracts of
F. vulgaris on SW-872 (skin cancer) and HFF-2 cell lines were investigated for the first time. In this regard, at the beginning of the work, cytotoxicity and IC
50 values obtained from the treatment of these cells with different extracts were investigated. According to evaluate the cytotoxicity of the plant on human liposarcoma cells (SW-872), MTT assay was performed with three extracts of
n-Hex, DCM and methanol. MeOH extract had no notable effect on cell growth and
n-Hex extract had the most cytotoxic effect compared to DMSO control (
p < 0.0001) and DCM extract, at time 24 (
p < 0.001) and 48 h (
P < 0.01). Flow cytometric results of this extract showed that the mechanism of cell death induced by it was both apoptosis and necrosis. In order to further investigate and isolate the apoptosis-inducing compounds,
n-Hex extract was selected for further study. The results of cytotoxicity of
n-Hex extract fractions on SW-872 show that the anticancer effect of all fractions in 24- and 48-h treatment is significantly different from the control (
p < 0.0001). The 40 and 60% fractions have the best IC
50 values among the samples so that the cytotoxic effect of these two samples is not significantly different from each other. 40% fraction showed the highest amount of cytotoxicity which is significantly different from the effect of other fractions (
p < 0.05). According to Table
2, although all
n-Hex VLC fractions inhibited the cancer cell lines compared to the control group significantly, IC50 amount of 40% VLC fraction was low. Furthermore, as Table
3 illustrates, the toxic effect of 40% VLC fraction on normal cell lines has the lowest as well. It means that it could be the best candidate for more fractionation and more isolation of pure compounds with low side effects. Moreover, based on Figs.
3, 40% VLC fraction of
n-Hex extract has the highest induction of apoptosis on SW-872 cells and has a suitable cell death mechanism. In addition, according to Table
3, the toxic effect of 60% VLC fraction on normal cells is more than cancer cells which means that this fraction may have most side effects via induction of necrosis on normal cells.
The essential oil components of this plant, which were collected from Moghan region in spring, are abundant in the category of compounds with the structure of oxygenated sesquiterpene (75.3%), sesquiterpene hydrocarbon terpenes (10.8%), hydrocarbon monoterpenes (0.6%) were found and the total terpene content of essential oil was 86.53%. The results also showed that spathulenol (33.8%) and caryophyllene oxide (18.3%) are the most abundant volatile compounds of
F. vulgaris, respectively. The yield of volatile oil was determined as 0.206% by volume/weight. Caryophyllene oxide has a considerable cytotoxic effect on different cancer cells that is dose and time-dependent. The best reported IC
50 value of caryophyllene oxide isolated from
Psidium cattleianum is 3.95 ± 0.23 μM [
47]. Spathulenol has also been suggested as a good candidate for the treatment of drug resistance in cancer treatment [
48]. Therefore, the effect of remarkable cytotoxicity (
p < 0.0001) of
F. vulgaris essential oil on cancer cells can be considered due to the presence of valuable anti-cancer compounds such as caryophyllene oxide and spathulenol. Another species of this plant that grows in Iran is
F. falcariodes. The results of a study conducted by Masoudi et al. on the plant identified 24 compounds that make up 97.6% of the total volatile oil of the plant; among these compounds, germacrene B (67.9%) is the main compound of this plant [
26].
The most probable compounds identified by GC-MS method of
n-Hex extract are non-terpenoid compounds, most of which include tetradecane (17.64%), spathulenol (16.91%), trimethyl pentaacetane (10.64%), isospathulenol (9.94%) and hexadecanoic acid (6.22%), respectively and 40% VLC fraction of
n-Hex extract has the highest terpenoid compounds such as spathulenol (20.4%), caryophyllene oxide (14.25%) and hexadecanoic acid (11.2%). In this regard, many studies have proven the cytotoxic role of compounds in plant essential oils. For example, Oliveira et al. Reported the cytotoxic effects of active volatile oil of several plants with medicinal properties on cancer cell lines such as tumor cell lines murine melanoma (B16F10), human colon carcinoma (HT29), human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), human hepatocellular liver carcinoma (HepG2), human glioblastoma (MO59J, U343, and U251), and Normal hamster lung fibroblasts (V79 cells) were studied. They concluded that compounds such as spathulenol and caryophyllene have significant cytotoxic effects as part of the active compounds present in the plant [
49]. In another study by Mellado et al., The effect of essential oils of
Ephedra chilensis extract on a variety of cancer cell lines was investigated. Using GC-MS analysis, 2.4% of the total compounds in dichloromethane extract are tetradcanoic acid and hexadecanoic acid, which have significant cytotoxic effects on prostate and breast cell lines [
50].