Chemical Characterization and Evaluation of Anticancer Effect of Falcaria vulgaris via Induction of Apoptosis

Background: Apiaceae family is one of the plant families which used for medical investigation. Falcaria vulgaris is a clear example of this genus that grows in certain regions of Iran. In traditional medicine, due to the presence of coumarin and avonoid compounds in this plant, therapeutic properties such as gastrointestinal and liver diseases, skin ulcers, gastric ulcers and intestinal inammation have been reported. It has also been found that these compounds lead to cytotoxic effects. Objective: The aim of this study aimed to investigate the cytotoxic effect and induction of apoptosis by various extracts and essential oil of F. vulgaris on cancerous cell (SW-872) and to identify the volatile compounds of effective samples. Methods: The shoot of the plant was extracted by Soxhlet apparatus and its essential oil was taken by Clevenger apparatus. The cytotoxicity of the samples was evaluated by MTT method and the mechanism of cancer cell death by ow cytometry and nally, the volatile compounds of essential oils and effective extracts were identied by GC-MS. Results: The results showed that n-Hexane extract and 40% VLC fraction had the greatest cytotoxic effect on SW-872 cells. While, the most abundant volatile compounds in essential oil and 40% VLC fraction of n-Hexane extract are terpenoid compounds like (+) spathulenol and caryophyllene oxide, in n-Hexane extract tetradecan, and spathulenol are the most, respectively. Conclusion: In sum, it was found that the fraction of 40% n-Hexane is in a concentration-dependent manner and signicantly with controlling cells, inhibit the growth of cancer cells. This effect is through induction of apoptosis and due to the presence of effective volatile compounds such as terpenoids and non-terpenoids which can be considered as the valuable natural sources for the isolation of anti-cancer compounds.


Introduction
Cancer is a disease in which the growth and proliferation of cells increase uncontrollably and can spread to other parts of the body [1] In 2020 in the United States, about 1.8 million new cases of cancer were reported, of which about 606 died [2]. So far, effective therapies for the prevention and treatment of cancer, including immunotherapy, hormone therapy, radiotherapy, chemotherapy and surgery, have been reported [3]. Despite extensive research into cancer and the discovery of related treatments, it is still considered a scary disease [4]. On the other hand, after years of progress in the eld of health, today developing countries are involved in the ght against deadly cancers [3]. At present, nonspeci c function, drug resistance and severe side effects are among the main problems of chemotherapy drugs. Therefore, there is an urgent need for extensive studies to nd alternative and complementary therapies [5]. Using high-performance screening, researchers have found that a variety of natural products, such as herbs, can be used as therapeutic agents in chemoprevention [6]. Scienti c information on the safety and effectiveness of herbal remedies has shown that these plants have good anti-cancer activity and most of them are currently under clinical trial [7][8][9].
The compounds in plants play a key role in killing malignant cancer cells through the apoptotic pathway [10,11]. Apoptosis or programmed cell death is a defense mechanism that occurs due to molecular events and changes in cell morphology such as blabbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and so on [12]. Therefore, many researchers have tried to discover therapeutic agents to stimulate malignant cancer cells by apoptosis, to offer a new treatment strategy [13]. In this regard, many studies have reported the therapeutic properties of plants of the Apiaceae family, especially the genus Falcaria (F. vulgaris) [14][15][16]. The plants of this family are widely used in traditional Iranian medicine and have antimicrobial effects [14], treatment of gastrointestinal diseases [17,18], skin diseases [19], anti-fertility [20], analgesia [21,22], treatment of heart diseases [23], etc.
The aim of the present study was to investigate the cytotoxic effects of F. vulgaris on SW-872 (skin cancer) cancer cell line.
Plant collection and drying: F. vulgaris plants were collected in the spring of 2016 from Moghan region located in Ardabil province. After identi cation by a plant expert, its herbarium specimen with number Tz fph 178 was kept in the herbarium of Tabriz University of Medical Sciences. After washing the collected samples, the shoots of F. vulgaris were completely dried in the open air and laboratory conditions and then ground into a ne powder by an electric mill.

Extraction and fractionation:
First, 200 g of F. vulgaris plant powder was weighed and after loading in a suitable lter paper, it was placed in a 1 liter Soxhlet apparatus and extracted with N-Hexane (n-Hex), dichloromethane (DCM) and methanol (MeOH) solvents, respectively, for 72 hours. The extracts were then dried completely by rotary evaporator at 45°C under low air pressure and nally weighed. Effective non-polar extracts were fractionated using VLC method with the stationary phase of silica gel, n-Hex solvents and increasing percentages of ethyl acetate. Extracts and fractions were stored frozen until use.
Essence extraction: The choice of essential oil extraction method depends on the type and condition of the plant, the active ingredients available and the purity of the nal product. The water distillation process is used on dry plants to prevent the destruction of compounds due to the high boiling temperature of the water. In essential oil distillation, 100 g of plant shoot powder with 150 ml of distilled water and 50 ml of glycerin were poured into a 1-liter round bottom balloon. The balloon was then connected to a clevenger device and placed in a mantel heater to provide the necessary heat to penetrate the plant tissues. Glycerin also helps to extract essential oils from plant powder by increasing the permeability of plant cell walls. A Clevenger device with a functional design is the most suitable tool for cooling the vapors emitted from the sample and extracting the resulting essential oil. The essential oil extraction process was performed for 4 hours according to the British Pharmacopeia method. Finally, the essential oil was then collected in a microtube and stored in a refrigerator.
Identi cation of volatile compounds in non-polar samples: Gas chromatography-mass spectrometry (GC-MS) was used to identify compounds in essential oils and extracts of n-Hex and its VLC fraction of n-Hex extract. GC-MS analysis was performed on a Shimadzu GC-MS QP 5050A gas chromatograph-mass spectrometer tted with a fused capillary column DB-1 (60 m, 0.25 mm id, lm thickness 0.25 µm) [30]. Helium was utilized as the carrier gas, at a ow rate of 1 ml/min. Cell culture: SW-872 (Human liposarcoma cell line) and HFFF (Normal human skin broblasts) cells were obtained from the Pasteur Institute of Iran and grown in RPMI-1640 medium containing 10% FBS supplemented with 1% antibiotic (penicillin/streptomycin) at 37°C in a humidi ed, 5% CO 2 incubator.

Preparation of plant samples:
Diluted samples of plant extracts are needed to perform cytotoxicity tests and observe the testable result. For this purpose, 10 mg of each dried extract was carefully weighed and completely dissolved in separate microtubes in 100 μl DMSO; then the contents of each microtube were brought to a volume of 1 ml using 900 μl of complete culture medium. Due to the concentration of active herbal substances in the fractions of each extract, their diluted samples were prepared with 0.1 concentration of total extracts. Therefore, the exact amount of 1 mg of each fraction was dissolved in 10 μl of DMSO and brought to a volume of 1 ml.
MTT assays: MTT assay was used for determination of substances toxicity on cell lines and evaluating of IC 50 Cell suspension with a concentration of 3×10 4 cells per ml was prepared and inoculated in 96-well microplates for 24 hours. The appropriate amount of extracts and fractions were added to control and test wells and the plates were incubated for 24 and 48 hours. Moreover, 20 μl of MTT solution was added to each well and incubated for 3 hours. The supernatant was replaced with 150 microliters of DMSO and dissolved the formazan crystals. Finally, it was measured by the ELISA plate reader at 570 nm (Anthos, Austria).

Detection of apoptosis by ow cytometry:
At this stage, the Annexin-V-Fluos Staining kit was used, which contains both AnnexinV material (to bind to the phospholipid molecule) and PI dye, which makes it possible to identify and clean apoptotic cells Statistical analysis: In the present study, all experiments were repeated at least twice and the results were obtained as mean ± standard deviation by descriptive statistics. Statistical analyzes were performed using Graph pad prism 8 software. For comparison between groups, ANOVA and Tukey post hoc test were used and the minimum signi cance level was considered p < 0.05.

Amounts of extracts and fractions:
The amount of extracts obtained from one extract of 200 g of aerial part powder of F. vulgaris plant using Soxhlet method and solvents of n-Hex, DCM and MeOH is shown in Table 1. In addition, the results of fractionation of 2 g of n-Hex extract by VLC method with n-Hex solvents and increasing percentages of ethyl acetate and silica gel stationary phase are shown in Table 1.
Results of cytotoxicity on SW-872 cell line: The results of MTT testing of n-Hex, DCM and MeOH extracts on SW-872 cells at 24 and 48 hours.
According to the results, we observed the remarkable cytotoxicity of n-Hex extract on SW-872 cells, compared to other extracts and DMSO control; therefore, in the next step, n-Hex fractions were tested by MTT. The results of this test at 24 and 48 hours are shown in Figure 1.
The IC Wesołowska values calculated from the Non-linear regression method is as shown in Table 2. To compare the cytotoxic effect of different plant samples at 24 and 48 hours, Two Way ANOVA test and TUKEY post hoc test were performed to compare the effect of different groups together with DMSO control. The results show that all samples had a signi cant cytotoxic effect compared to DMSO control. In statistical tests, the signi cance level was de ned as ns (p> 0.05), * (p <0.05), ** (P <0.01), *** (p <0.001), **** (P <0.0001) Becomes. A signi cant comparison of the effect of different samples with controls is presented in Figure 2.

Results of cytotoxicity on HFFF cells:
To investigate the effect of cytotoxicity of F. vulgaris extracts and fractions on non-cancerous cells, MTT assay was performed on normal HFFF cell lines. Table 3 shows the IC50 results of 24-and 48-hour treatments on these cells.

Apoptosis test results in SW-872 cells:
Flow cytometry test was performed to evaluate the mechanism of cell death induced by DCM extract fractions on SW-872 cells and the results are shown in Figure 3. The results showed that the 20% and 40% fractions of n-Hex extract have the highest induction of apoptosis on SW-872 cells and have a suitable cell death mechanism.
Identi cation of compounds in essential oil using gas chromatography-mass spectrometry: Using the GC-MS device, the compounds in the volatile oil of F. vulgaris were identi ed, which are shown in Table 4 with speci cations such as inhibition time, percentage of constituents and KI.

Identi cation of compounds in n-Hex extract:
Data on the compounds identi ed in the n-Hex extract of F. vulgaris obtained by injection into the GC-MS are plotted in Table 5 Identi cation of compounds in 40% VLC fraction of n-Hex extract: The data on the compounds identi ed in the 40% VLC fraction of n-Hex extract of F. vulgaris obtained by injection into the GC-MS device are presented in Table 6.

Evaluation of cytotoxicity of volatile oil:
The results of cytotoxicity of F. vulgaris volatile oil on SW-872 cancer cells in 24-hour treatment are 3.78±0.93 which found that the highest toxicity effect occurred on SW-872 cell line.

Discussion
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 [31]. 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 [32]. Chemotherapy drugs should ideally have a speci c cytotoxic effect on neoplastic cancer cells; however, in reality this treatment leads to some systemic toxicity to the individual [33]. 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 [34].
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. 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 [35]. 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 [36]. The effect of coumarins on the cytotoxic effect of members of the Apicaceae family has also been proven in studies [37][38][39]. The signi cant antioxidant effect of F. vulgaris due to the presence of compounds such as alkaloids, anthraquinones, avonoids, phenols, saponins, steroids and tannins has already been investigated [24]. Antioxidant compounds such as Spathulenol and Carvacrol have also been identi ed 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 HFFF cell lines were investigated for the rst 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 hours (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 hours treatment is signi cantly 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 signi cantly different from each other. 40% fraction showed the highest amount of cytotoxicity which is signi cantly different from the effect of other fractions (p <0.05).
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 [40]. Spathulenol has also been suggested as a good candidate for the treatment of drug resistance in cancer treatment [41]. Therefore, the effect of remarkable cytotoxicity (p<0.0001) of F. vulgaris essential oil than DMSO control 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 Falcaria falcariodes. The results of a study conducted by Dr. Masoudi et al. on this plant identi ed 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 identi ed by GC-MS method of n-hexane 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-hexane 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 broblasts (V79 cells) were studied. They concluded that compounds such as spathulenol and caryophyllene have signi cant cytotoxic effects as part of the active compounds present in the plant [42]. 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 signi cant cytotoxic effects on prostate and breast cell lines [43].

Conclusion
The results showed that 40% fraction of n-Hex extract and F. vulgaris essential oil on SW-872 cells have a signi cant cytotoxic effects. The cytotoxicity of the fraction is completely dose-dependent and signi cantly (p <0.0001) higher than the control group. Furthermore, the mechanism of cell death in 40% n-Hex fraction is predominantly through induction of apoptosis, which can be concluded that apoptosisinducing compounds are probably in the effective fractions of n-Hex extract. It is worth mentioning that the 40% n-Hex fraction has a selective effect on SW-872 cancer cells compared to normal non-cancer cells. The appropriate properties mentioned in the effective samples have raised great hopes for the puri cation of effective anti-cancer compounds with minimal side effects from F. vulgaris in future works.

Declarations
Ethics approval and consent to participate There is no involvement of human or animal in this study.

Consent for publication
All other authors declare no con ict of interest.

Availability of data and materials
The authors con rm that the data supporting the ndings of this study are available within the article.

Competing interests
We declare that we have no signi cant competing nancial, professional, or personal interests that might have in uenced the performance or presentation of the work described in this manuscript.   Table 2. Cytotoxic effect of n-Hex, DCM and MeOH extracts and fractions of n-Hex extract of F. vulgaris on SW-872 cells  Statistical comparison of viability of SW-872 cells treated with n-Hex extract of F. vulgaris compared to the control group, using MTT method. Figure 3