Identification of the volatile and nonvolatile constituents of Schinus molle (L.) fruit extracts and estimation of their activities as anticancer agents

This work was designed to assess the cytotoxic potential of Schinus molle (L.) fruit extracts and characterization of their chemical composition. The cytotoxicity of S. molle extracts was carried out on hepatoma HepG2 cell line using the Sulforhodamine B method. The volatile constituents of S. molle normal-hexane (n-hexane) extract and the essential oil were identified by gas chromatography-mass spectrometry (GC-MS), whereas the nonvolatile chemical compositions were investigated using the Liquid chromatography-electron spray ionization-mass spectrometry (LC-ESI-MS) technique. The n-hexane extract showed the highest cytotoxic inhibition activity on the HepG2 cell line in a concentration-dependent manner with inhibition concentration (IC50 = 9.75 μg/ml), followed by n-butanol fraction (IC50 = 10.70 μg/ml) and the essential oil (IC50 = 11.90 μg/ml). The GC-MS investigation of the essential oil afforded 50 compounds classified into monoterpenes and sesquiterpenes with different percentiles. The most abundant monoterpenes were α-phellandrene, myrcene, D-limonene, β-phellandrene, and α-pinene. At the same time, the major sesquiterpenes were juniper camphor, guaiyl acetate, γ-gurjunene, α-cadinol, and β-caryophyllene. On the other hand, the LC-ESI-MS investigation of the methanolic extract, n-butanol fraction, and aqueous part led to the identification of 31 phenolic compounds classified as phenolic acids, phenylethanoids, flavonoids, and tannins. These findings demonstrate the remarkable potentiality of S. molle extracts as a valuable source of anticancer capacity.


INTRODUCTION
Cancer is a prevalent disease which is considered as the second leading cause of mortality across the globe and the number of new cases increases day per day, especially in Asia, Africa, and USA (Nguyen et al., 2020;Singh and Patra, 2018). Cancer is a public health problem in developed and developing countries that affects human health and economic conditions (Shahat et al., 2019). Among cancers, hepatocellular carcinoma (HCC) or liver cancer is the primary virulent tumor and the most common type of cancer that arises from the parenchymal liver cells. It is considered as the third leading reason for cancer deaths after lung and stomach cancers (Bray et al., 2018). Also, it represents the seventh cancer infection in women and the fifth in men (Anyasor et al., 2020). The main risk factors for HCC include nonalcoholic fatty liver disease, hepatitis C and B viral infections, alcoholism, diabetes, obesity, primary biliary cirrhosis, and exposure to nitrosamines and aflatoxins (Abdel-Hamid et al., 2018;Chedid et al., 2017). The most popular treatment strategies of HCC are chemoembolization, orthotopic liver transplantation, and chemotherapy. The chemotherapy treatment protocol is the preferred method for advanced hepatocellular carcinoma. Unfortunately, chemotherapy is associated with drug resistance and other side effects that lead to liver failure (Siddiqui et al., 2019). On the other hand, the effective and safe alternative therapeutic tools for the treatment of HCC were natural products, especially secondary metabolites (Huang et al., 2016). Plant secondary metabolites are used in the health care system since ancient times. More than thousands of medicinal plants have been identified to possess many medicinal and pharmacological properties, including anticancer agents (Khlifi et al., 2013).
Schinus molle (L.), or pepper tree, belongs to the family Anacardiaceae comprising 72 genera and 600 species (Machado et al., 2019). Schinus molle is growing in tropical and subtropical areas worldwide including South America and Mediterranean countries (Malca-García et al., 2017). Schinus molle has high amounts of oil with a spicy smell which is used in the food industry, ornamentals, and medicines (Garzoli et al., 2019). In folklore medicine, the extracts of S. molle were documented as antitumor, astringent, antiviral, antioxidant, antimicrobial, antiinflammatory, digestive stimulant, diuretic, and wound healer activities (Gomes et al., 2013;Hosni et al., 2011;López et al., 2014;Malca-García et al., 2017;Martins et al., 2014). The previously chemical investigation studies of S. molle have been reported; it contains various chemical ingredients, including monoterpenoid, sesquiterpenoid, triterpenoids, tannins, and flavonoids (Abdel-Hameed and Bazaid, 2017;Ono et al., 2008). To the best of our knowledge, there were no reports on the chemical investigation of S. molle fruits growing in Taif City, Saudi Arabia.
The main objectives of this work were (i) extraction of volatile and nonvolatile chemical components of S. molle fruits, (ii) investigation of the anticancer potential of different extracts of S. molle fruits, (iii) characterization of the chemical composition of S. molle fruits essential oil and n-hexane extract using gas chromatography-mass spectrometry (GC-MS) analysis, and (iv) identification of the nonvolatile chemical constituents using Liquid chromatography-electron spray ionization-mass spectrometry (LC-ESI-MS) analysis.

Plant materials
The mature fruits of S. molle were collected from Taif city, Saudi Arabia. The plant sample was authenticated by Dr. Mohamed Fadle, Professor of Plant Taxonomy, Faculty of Science, Taif University, Taif, Saudi Arabia. A voucher specimen (no. 13518) of plant fruits was deposited in the Medicinal Chemistry laboratory, Theodor Bilharz Research Institute, Giza, Egypt. The fresh fruits were crushed using an electric mill to be ready for the extraction process.

Extraction of essential oil
150 g of freshly crushed fruits of S. mole was mixed with 2 l of distilled H 2 O in a round flask and hydrodistillated at 90°C using the Clevenger instrument. The system was operated till the essential oil was limited. 9.2 ml of the essential oil was collected and dried over anhydrous sodium sulfate. The obtained essential oil was stored at −20°C in a glass vial and away from contamination for biological and chemical investigations.

Preparation of organic extracts
150 g of freshly crushed fruits of S. mole was immersed in 750 ml n-hexane for 7 days at room temperature and then filtered using filter paper (Whatman No. 1). The n-hexane solvent was removed using a rotary evaporator (BUCHI, Switzerland) under reduced pressure and the extraction process was repeated three times. 12.3 ml yellow turbid viscous oily n-hexane extract was obtained. After extraction with n-hexane, the residue was extracted with 750 ml 85% Methanol (MeOH). The solvent was evaporated under vacuum to give a 30.7 g solid brown extract. Furthermore, 20 g of 85% MeOH extract (MeOH ext.) was dissolved in 100 ml distilled H 2 O and partitioned with normalbutanol (n-BuOH) (3 × 100 ml solvent) using a separating funnel. The n-BuOH and aqueous layers were separated and evaporated under reduced pressure to afford 8.3 g of n-BuOH fraction and 11.1 g of aqueous part. All extracts were kept in glass bottles for chemical profiling and biological investigations.

Cytotoxicity studies
The samples under the current study were in vitro tested against human liver carcinoma (HepG2) cell line, which was carried out at the National Cancer Institute, Cairo, Egypt, according to Skehan et al.'s (1990) method. Briefly, the HepG2 cells were seeded in 96-well microplates at a conc. (5 × 10 4 −10 5 cell/well) in a fresh medium and left for 24 hours. The samples (100 µl) with different concentrations (0.0, 12.5, 25, 50, and 100 μg/ml) have been added to the wells. The microplate wells' total volume was completed up to 200 (μl volume/well) using a fresh medium and then incubated for 48 hours in 5% CO 2 incubator at a temperature of 37°C. After 48 hours, the cells were fixed with 50 μl trichloroacetic acids (cold 50%) for 1 hour at 4°C. Moreover, the wells were washed with distilled H 2 O (5 times) and stained for 30 minutes at room temperature by 50 μl Sulforhodamine B (SRB) (0.4%). Furthermore, the plate wells were washed four times using acetic acid (1%), the plates were dried carefully, and then the dye was solubilized in 10 mM tris base at pH 10.5 (100 μl/well) for 5 minutes at 1,600 rpm using a shaker (Orbital Shaker OS 20, Boeco, Germany). The optical density of plate wells was determined by a spectrophotometer at 564 nm with ELIZA microplate reader (Meter tech. Σ 960, USA). Doxorubicin was used as a standard and the experiment was repeated in triplicate. The cell viability (%) was calculated from the following equation: The cell viability (%) = [Optical density of treated cells/Optical density control cells] × 100.
In addition, IC 50 was calculated from the cell viability curve of the cancer cell lines.

GC-MS conditions
The essential oil and n-hexane extract volatile chemical composition were investigated using gas chromatograph CP 3800 interfaced with a Saturn 2200 mass spectrometer (Varian, California, USA) with electron impact ionization (70 eV). A VF-5 fused silica capillary column (30 m × 0.25 mm, 0.25 μm film thickness) was used. The carrier gas was helium with a constant flow rate (1 ml/minute). The temperature of the oven was adjusted for 1 minute at 50°C, increased gradually to 120°C (5°C/minutes), 120°C-190°C (2°C/minutes), held for 1 minute at 190°C, 190°C-250°C (10°C/minutes), and held for 3 minutes at 260°C. The mass range of recorded ions was 45-400 m/z and the total run time was 60 minutes. The injected samples and standard mixture (1 mg/1 ml n-hexane) were prepared. The samples and standards (1 μl) were injected by autosampler with a split ratio of 1:20. The volatile constituents were characterized by cob 1qmparison of their retention time (t R ), retention indices relative to (C 8 -C 20 ) n-alkanes with standards, and matching their mass spectra with corresponding data (Wiley and NIST electronic libraries).

Data analysis
The data analysis was performed by the SPSS software for Windows (version 13.0) and all data were expressed as mean ± standard deviation.

Cytotoxic activity
The cytotoxic activity of the S. molle different extracts such as the essential oil, 85% MeOH ext., n-hexane extract, n-BuOH fraction, and aqueous part was assayed against HepG2 cell line using SRB colorimetric assay. This assay exhibits the ability of SRB to attach with protein components of the cells, which are fixed by trichloroacetic acid to the tissue culture plates (Vichai and Kirtikara, 2016). Figure 1 exhibited the cell viability of S. molle fruit extracts, which represented the 85% extract had the highest cell viability, followed by the aqueous part, essential oil, n-BuOH fr., and n-hexane extract. On the other hand, it showed the IC 50 value of S. molle fruit extracts and doxorubicin as a broad-spectrum anticancer drug. The results in Figure 2 showed that n-hexane ext. and n-BuOH fr. of S. molle exhibited the highest anticancer activity (IC 50 = 9.75 and 10.70 μg/ml, respectively), followed by essential oil (IC 50 = 11.90 μg/ml), aqueous part (IC 50 = 15.80 μg/ml), and 85% MeOH ext. (IC 50 = 16.40 μg/ml). The cytotoxic evaluation criteria of the plant extracts are according to American National Cancer Institute (NCI) protocols, in which the cytotoxic evaluation criteria of the plant extracts were considered to be significant when the IC 50 values ≤ 30 (μg/ml), while for pure substances, the IC 50 values should be ≤ 4 μg/ml (Geran et al., 1972). Therefore, all S. molle fruit extracts were considered to be significant anticancer plant extracts against HepG2 cell line. It was clearly appeared that the cytotoxic activity of the MeOH extract in the current study (IC 50 =16.40 µg/ml) had much higher cytotoxic activity than the MeOH extract of the same plant growing in Argentine (IC 50 = 50 µg/ml) as reported by Hamdan et al., (2016). Thus, the difference in cytotoxic activity may be due to the different time of collection and climate conditions. So, it is imperative to identify both volatile and nonvolatile constituents of S. molle extracts to know their chemical nature.

GC-MS investigation of S. molle fruits n-hexane extract and the essential oil
The GC-MS investigation of S. molle n-hexane extract and essential oil (Table 1 and Fig. 3) characterizes 50 compounds in both extracts with different percentages, corresponding to 99.46% in essential oil and 98.59% for n-hexane ext., in which 17 compounds of them were higher than 1% relative to the total volatile composition of S. molle fruits essential oil and n-hexane ext. The highest amount of monoterpenes (67.81%) was detected in essential oil, whereas n-hexane ext. had 37.87% of the total components. The main identified monoterpenes were α-phellandrene (26.24% in the essential oil; 17.70% in n-hexane ext.), myrcene (21.57% in the essential oil; 12.33% in n-hexane ext.), D-limonene (7.93% in the essential oil; 4.84% in n-hexane ext.), β-phellandrene (7.28% in the essential oil; 5.33% in n-hexane ext.), and α-pinene (2.69% in the essential oil; 1.17% in n-hexane ext.). On the other hand, sesquiterpenes were detected as major components in n-hexane ext. (59.23%), while the essential   oil had 29.62% sesquiterpenes of the total components. Among them, juniper camphor (14.01% in n-hexane ext. and 1.60% in essential oil), guaiyl acetate (13.23% in n-hexane ext. and 1.16% in essential oil), γ-gurjunene (10.51% in n-hexane ext. and 7.51% in essential oil), α-cadinol (4.01% in n-hexane ext. and 1.40% in essential oil), and β-caryophyllene (3.29% in n-hexane ext. and 2.56% in essential oil) were the major identified sesquiterpenes. Some previous studies reported that S. molle aerial parts and fruits essential oils were riches with monoterpenes (Abdel-Sattar et al., 2010;Gomes et al., 2013;Hayouni et al., 2008;Machado et al., 2019;Martins et al., 2014), while some other studies reported that sesquiterpenes were represented as the main constituents (Abdel-Hameed and Bazaid, 2017;Cavalcanti et al., 2015;Garzoli et al., 2019;Simionatto et al., 2011). Thus, the variations in the chemical compositions percentiles of S. molle fruits essential oil and n-hexane ext. may be due to some thermal and chemical factors. Furthermore, the potent cytotoxic activity of n-hexane extract could be due to the presence of a high amount of monocyclic monoterpenes, acyclic monoterpenes, bicyclic monoterpenes, and bicyclic sesquiterpenes.

LC-ESI-MS profiling of organic extracts
A total of 31 phenolic compounds were the most abundant metabolites in S. molle fruits extracts (85% MeOH ext., n-BuOH fr., and aqueous part) characterized by LC-ESI-MS investigation in negative ion mode as shown in Table 2 Figure 4 showed high abundant peaks identified as methyl gallate, digalloyl shikimic acid, pentagalloyl glucose, quercetin-3-O-glucose, quercetin-3-O-hexuronic acid, neochamaejasmin B, and biapigenin. These active ingredients may be responsible for the potent anticancer activity of S. molle fruit extracts. Moreover, the chemical profiling of n-BuOH fraction represented a high content of flavonoids, phenolic acids, phenylethanoids, and gallotannins which may be responsible for its high cytotoxic potential.

CONCLUSION
In the present study, S. molle fruit extracts possessed a promising cytotoxic potential against the HepG-2 cell line. The chemical investigation of the volatile constituents of n-hexane  ext. and the essential oil using GC-MS analysis led to identifying 50 compounds classified as monoterpenes, sesquiterpenes, and fatty acids, while the LC-ESI-MS chemical investigation of the 85% MeOH ext., n-BuOH fraction, and aqueous part led to characterize 31 polyphenolic compounds, for example, phenolic acids, flavonoids, phenylethanoids, and gallotannins. From the available literature, these bioactive secondary metabolites (volatile and nonvolatile constituents) had a broad spectrum of biological and pharmacological properties. Thus, our results suggested the selective potential of tested extracts for the treatment of different types of cancer and the possible usage of S. molle extracts as anticancer therapeutic agents.

LIST OF ABBREVIATIONS GC-MS
Gas chromatography-mass spectrometry IC Inhibition concentration LC-ESI-MS Liquid chromatography-electron spray ionizationmass spectrometry MeOH ext.

ETHICAL APPROVAL
The present work does not include the use of human or animal subjects.