Phytochemical Analysis and Profiling of Antitumor Compounds of Leaves and Stems of Calystegia silvatica (Kit.) Griseb.

Anti-tumor compounds from natural products are being investigated as possible alternatives for cancer chemotherapeutics that have serious adverse effects and tumor resistance. Calystegia silvatica was collected from the north coast of Egypt and extracted via methanol and n-hexane sub-fraction. The biologically active compounds of Calystegia silvatica were identified from the methanol and n-hexane extracts from the leaves and stems of the plant using GC-MS and HPLC. The antitumor properties of both parts of the plant were investigated against cancer and non-cancer cell lines using the MTT assay, and the IC50 in comparison to doxorubicin was calculated. The main compounds identified in the methanol extract were cis-vaccenic acid and trans-13-octadecenoic acid in the leaves and stems, respectively, and phenyl undecane and 3,7,11,15 tetramethyl-2-hexadeca-1-ol in the n-hexane extracts of the leaves and stems, respectively. Both parts of the plant contained fatty acids that have potential antitumor properties. The methanol extract from the stems of C. silvatica showed antitumor properties against HeLa, with an IC50 of 114 ± 5 μg/mL, PC3 with an IC50 of 137 ± 18 μg/mL and MCF7 with an IC50 of 172 ± 15 μg/mL, which were greater than Caco2, which had an IC50 of 353 ± 19 μg/mL, and HepG2, which had an IC50 of 236 ± 17 μg/mL. However, the leaf extract showed weak antitumor properties against all of the studied cancer cell lines (HeLa with an IC50 of 208 ± 13 μg/mL, PC3 with an IC50 of 336 ± 57 μg/mL, MCF7 with an IC50 of 324 ± 17 μg/mL, Caco2 with an IC50 of 682 ± 55 μg/mL and HepG2 with an IC50 of 593 ± 22 μg/mL). Neither part of the plant extract showed any cytotoxicity to the normal cells (WI38). Therefore, C. silvatica stems may potentially be used for the treatment of cervical, prostate and breast cancer.


Introduction
Cancer is one of the leading causes of mortality worldwide.Among surgery and radiation treatment modalities, chemotherapy remains the modality of choice for cancer therapy, particularly in patients with advanced stages of the disease [1][2][3].However, the use of chemotherapeutic agents for cancer therapy is hindered because of severe longterm side effects and the development of drug resistance caused by inactivation and drug metabolism by different enzymes such as cytochromes P450 [4][5][6][7][8][9].These factors limit the use of chemotherapeutic agents, making it a significant challenge in cancer therapy.Recent developments have enabled cancer researchers to better explore the potential use of natural products for cancer therapy.Fortunately, several natural products with various chemical structures and different pharmacological activities have shown promise in combating different cancers, these include alkaloids, terpenoids and flavonoids [6,[10][11][12][13].Therefore, collective efforts are needed to discover new therapeutic opportunities for cancer treatment arising from natural products.
The genus Calystegia R.Br., family Convolvulaceae, is represented by only one species-Calystegia silvatica subsp.silvatica-in Egypt.It is found in gardens and orchards in the Mediterranean region, Iran, western Europe and Australia [14].It has been used for the treatment of fever, urinary tract disorders, constipation and reduced bile production (cholagogue) [15].Additionally, it has been used for rheumatoid arthritis as an anti-inflammatory and pain killer [16,17], resolvent for pimples [18] and as an anti-tuberculosis treatment [16].Lipids have reportedly been identified as the primary active components of the latex of C. silvatica [19].Additionally, C. sepium is a species with strong relations to C. silvatica, which has been used to demonstrate the existence of resin glycosides known as calestegins [20,21].The aerial parts of C. sepium have been used to isolate seven hexasaccharide resin glycosides, known as calsepins 1-7.Lung cancer cells (A549) showed cytotoxicity when exposed to calysepin 4 [22].Two resin glycosides, calystegines A and B, were also isolated from the aerial parts of Calystegia sepium.Calystegine B showed an antitumor effect by preventing the assembly of tumor microtubule proteins of cancer cells [20,21].Three cancer cell lines, including glioma cells (U87-MG), epidermal cell lines (A431) and breast cancer cells (MCF7), have been killed by a methanolic extract comprising the leaves of C. sepium compared to HGF-1 as normal cells [23].There are no published data regarding the antitumor properties of C. silvatica in the literature; therefore, this is the first investigation of the antitumor effects of this plant against certain cancer cell lines.
On the other hand, the major identified chemical constituents of lipids via the GC-MS analysis of an n-hexane extract from the leaves and stems of C. silvatica were alkane hydrocarbons undecane (with a relative abundance (RA) of 0.10% and 1.58%, respectively), methyl undecane (RA = 0.29% and RA = 1.85%, respectively), methyl dodecane (RA = 0.10% and RA = 1.99%, respectively), benzenes phenyl undecane (RA = 16.21% and RA = 4.65%, respectively), phenyl decane (RA = 10.11% and RA = 2.72%, respectively) and 2-phenyl tridecane (RA = 4.30% and RA = 1.15%, respectively) (Table 2 and Figure 2).The leaves and stems both contained fatty acids methyl ester 9,12, octadecanoic, methyl ester (RA = 0.10% and RA = 4.12%, respectively).The leaves and stems also contained the diterpene, 3,7,11,15 tetramethyl-2-hexadeca-1-ol (with a relative abundance of 0.53% and 8.10%, respectively), the phthalate ester bis (2-ethylhexyl) phthalate (RA = 0.1% and RA = 2.11%, respectively), beta-sitosterol acetate, a sterol, (RA = 0.10% and RA = 2.69%, respectively) and the sterol ergost-25-ene-3,5,6,12-tetrol (3.beta.,5.alpha.,6.beta.,12.beta.)(RA = 0.25% and RA = 0.76%, respectively).However, the octadecenoic acid, 12 hydroxy, methyl ester (RA = 0.44%), 9,12,octadecadienoic acid, 2 hydroxy-1 (hydroxy methyl) ethyl ester (RA = 0.91%) and hexadecenoic acid, 2-hydroxyl-1-(hydroxy methyl) ethyl ester (RA = 1.12%) were the fatty acids found in the stems, but they were not detected in the leaves.Campesterol (RA = 0.79%), stigmasterol (RA = 0.35%), obtusifiol (RA = 0.41%) and cholest-5-en-3-ol, 24propylidene (RA = 0.40%) were the identified sterols found in stems only.Additionally, 9,19cyclolanost-24-en-3-ol, (3 beta) (RA = 1.05%), betulinaldehyde (RA = 4.24%) and lanostan-3-ylacetate (RA = 1.1%) were the triterpenes found in the stems.Additionally, other compounds found in the stems were the oxygenated sesquiterpenes longifolenaldehyde (RA = 1.08%) and the diterpene thunbergol (RA = 0.85%).The only compound that was found in the leaves was 7-phenyl eicosane (RA = 0.99%), but it was not detected in the stems (Table 2 and Figure 2).The total phenolic contents of the leaves and stems of C. silvatica were 21.18 mg and 17.30 mg GAE/g of dry weight (DW), respectively, and the total flavonoids were 7.10 mg and 12.45 mg CE/g DW, respectively.The HPLC was used to identify and quantify the phenolic compounds that exist in the methanolic extract from the leaves and stems of C. silvatica.(Table 3).As a result, phenolic acids, polyphenols, tannins, flavanones, flavonoids, isoflavone and carboxylic acids were identified in both parts of the plant.The leaves and stems contained the phenolic acid gallic acid, at a concentration of 1.05 mg/100 g dry weight (DW) and 0.5 mg/100 g DW, respectively, and the phenolic acid ferulic acid, at a concentration of 0.28 mg/100 g DW and 0.60 mg/100 g DW, respectively.The polyphenols found in the leaves and stems were catechin, at a concentration of 0.19 mg/100 g DW and 0.24 mg/100 g DW, respectively, and caffeic acid, at a concentration of 0.1 mg/100 g DW and 0.003 mg/100 g DW, respectively.Additionally, the phenolic compounds found in the leaves and stems were pyrocatechol, at a concentration of 3.14 mg/100 g DW and 0.36 mg/100 g DW, respectively, and coumaric acid, at a concentration of 2.40 mg/100 g DW The total phenolic contents of the leaves and stems of C. silvatica were 21.18 mg and 17.30 mg GAE/g of dry weight (DW), respectively, and the total flavonoids were 7.10 mg and 12.45 mg CE/g DW, respectively.The HPLC was used to identify and quantify the phenolic compounds that exist in the methanolic extract from the leaves and stems of C. silvatica.(Table 3).As a result, phenolic acids, polyphenols, tannins, flavanones, flavonoids, isoflavone and carboxylic acids were identified in both parts of the plant.The leaves and stems contained the phenolic acid gallic acid, at a concentration of 1.05 mg/100 g dry weight (DW) and 0.5 mg/100 g DW, respectively, and the phenolic acid ferulic acid, at a concentration of 0.28 mg/100 g DW and 0.60 mg/100 g DW, respectively.The polyphenols found in the leaves and stems were catechin, at a concentration of 0.19 mg/100 g DW and 0.24 mg/100 g DW, respectively, and caffeic acid, at a concentration of 0.1 mg/100 g DW and 0.003 mg/100 g DW, respectively.Additionally, the phenolic compounds found in the leaves and stems were pyrocatechol, at a concentration of 3.14 mg/100 g DW and 0.36 mg/100 g DW, respectively, and coumaric acid, at a concentration of 2.40 mg/100 g DW and 0.19 mg/100 g DW, respectively.The phenylacrylate polyphenol compound chlorogenic acid was found in the leaves and stems at a concentration of 1.78 mg/100 g D.W and 0.42 mg/100 g DW, respectively.Additionally, the methyl gallate (gallate ester) was found in the leaves and stems at a concentration of 0.06 mg/100 g DW and 0.10 mg/100 g DW, respectively.The leaves and stems also contained tannin ellagic acid at a concentration of 0.69 mg/100 g DW and 0.21 mg/100 g DW, respectively.The benzaldehyde vanillin was found to have a concentration of 0.07 mg/100 g DW and 0.02 mg/100 g DW in the leaves and stems, respectively.The flavanone naringenin was found in the leaves and stems at a concentration of 0.67 mg/100 g DW and 0.08 mg/100 g DW, respectively.The monocarboxylic acid cinnamic acid was found to have the lowest concentration in the leaves and stems (0.01 mg/100 g DW and 0.001 mg/100 g DW, respectively), as compared to other identified phenolic compounds.However, the flavonoid quercetin was found in the leaves at a concentration of 0.05 mg/100 g DW, and the isoflavone daidzein was found in the stems at a concentration of 0.05 mg/100 g DW.Other phenolic compoundssyringic acid, apigenin, kaempferol and hesperetin-were not detected in the leaves or stems (Table 3 and Figure 3).The percentage of cytotoxicity was represented as [cell viability percentage -100] and plotted on the Y-axis; the concentration of the extracts from leaves and stems was plotted on the X-axis to calculate the half-maximal inhibitory concentration (IC 50 ).For example, the IC 50 of the leaves extract against colon cancer was 682 ± 55 µg/mL, and it was 353 ± 19 µg/mL for the stems extract (Figure 4).The antitumor properties of C. silvatica leaves and stems against colon cancer (Caco2), cervical cancer (HeLa), prostate cancer (PC3), breast cancer (MCF7), hepatocellular carcinoma (HepG2) and normal human fetal lung fibroblast (WI38) cell lines were studied using an MTT assay.Dunnett's test was used to compare the calculated IC 50 values of the leaves and stems extracts to the positive control, doxorubicin.Based on the NCI criteria, the methanol extract from the leaves and stems exhibited different cytotoxicities against all of the cancer cell lines (Table 4).The antitumor properties of C. silvatica stems against cervical cancer (HeLa) (114 ± 5 µg/mL), prostate cancer (PC3) (137 ± 18 µg/mL) and breast cancer (MCF7) (172 ± 17 µg/mL) were moderate compared to doxorubicin.However, the antitumor properties of stems against colon cancer (CaCo2) (353 ± 19 µg/mL) and hepatocellular carcinoma (HepG2) (236 ± 17 µg/mL) were weak compared to the positive control.In contrast, the cytotoxicities of leaves on cervical cancer (HeLa) (IC 50 = 208 ± 13 µg/mL), prostate cancer (PC3) (IC 50 = 336 ± 57 µg/mL) and breast cancer (MCF7) (IC 50 = 324 ± 17 µg/mL) were weak compared to doxorubicin.However, the leaves did not show any antitumor properties against colon cancer (Caco2) (IC 50 = 682 ± 55 µg/mL) or hepatocellular carcinoma (IC 50 = 593 ± 33 µg/mL).Interestingly, neither the extract from leaves nor from stems showed any cytotoxicity to normal human fetal lung fibroblast (WI38) with an IC 50 of 543 ± 33 µg/mL and 408 ± 4 µg/mL, respectively, compared to the positive control.The selectivity index of the methanol extract from C. silvatica leaves and stems was estimated as described above.Therefore, no cytotoxic selectivity was shown for the leaves extract from C. silvatica in any of the studied cancer cell lines (values < 3).However, the stems extract showed cytotoxic selectivity in HeLa (3.5) and prostate cancer (3) but not in colon cancer (1.7), breast cancer (1.1) or hepatocellular carcinoma (2.3) (Table 5).The different cell lines were treated for 72 h with 250 µg/mL of extract from C. silvatica leaves and stems and were microscopically examined.The methanol extract from the leaves of C. silvatica caused a small shrinkage to HeLa and MCF7 cell lines only, while the stems extract caused a significant shrinkage to HeLa, PC3 and MCF7 cancer cell lines, which became rounded and detached.However, Caco2 and HepG2 did not show any change in morphology compared to untreated control cells, and WI38 cell lines did not exhibit any change in morphology when treated with either the leaves or stems of C. silvatica compared to their control cells (Figures 5-7).
The HPLC analysis for phenolic compounds in the methanol extract revealed that the leaves and stems extracts contained several phenolic and flavonoid compounds that could potentially contribute to the antitumor effect of C. silvatica against the studied cancer cell lines.Gallic acid is an identified phenolic acid that has been reported to have an antitumor effect [55].Additionally, chlorogenic acid is a phenylacrylate polyphenol compound that has been reported to have antitumor properties [56].The identified polyphenols with antitumor properties in C. silvatica extract are catechin [57] and caffeic acid [58].Methyl gallate (gallate ester) has also been reported to have antitumor properties [59].The identified phenolic compounds with antitumor properties are coumaric acid [60], ferulic acid [61] and pyrocatechol [62].The vanillin is an identified benzaldehyde that has been reported to possess antitumor properties [63].Additionally, ellagic acid is a tannin and has been reported to have antitumor properties [64].Naringenin is a flavanone that has been reported to have antitumor properties [65].Additionally, the flavonoid quercetin found in the leaves has been reported to have antitumor properties [38], and the isoflavone daidzein found in the stems has also been reported to have antitumor properties [66].The identified monocarboxylic acid cinnamic acid has been reported to have antitumor properties [67].
C. sepium, which has a close affiliation to C. silvatica, showed an antitumor effect against breast cancer cells (MCF7), epidermal cell line (A431) and glioma cell line (U87-MG) compared to HGF-1 as normal cells [23].These findings are in line with our MTT assay results in which the methanol extract from C. silvatica stems demonstrated an antitumor effect against breast cancer cell lines (MCF7) without cytotoxicity to the normal human fetal lung fibroblast (WI38).This study showed that stem extracts of C. silvatica have a higher antitumor effect than leaf extracts against most of the tested cancer cells without affecting the non-cancer cells.This may be due to the number of compounds that have antitumor properties found in the stems more than in the leaves.Therefore, the extract from the stems of C. silvatica may have potential antitumor properties against cervical, prostate and breast cancer but not against colon cancer and hepatocellular carcinoma; however, the extract from the leaves has weak antitumor effects.

Plant Material
Aerial parts of C. silvatica in the flowering stage were collected from the Al Alamein region, which is about 101 km from Alexandria, Egypt.Dr. Iman Al-Gohary from the Desert Research Centre (DRC), Cairo, Egypt, identified the samples.Voucher specimens were placed in the Center's Herbarium (CAIH-0042R).Both parts of the plant were washed using tap water and dried for 10 days at 25 • C in a ventilated room in the shade and were subsequently powdered separately [68].

Methanol Extract Preparation
Two hundred grams of the air-dried powdered plant leaves and stems was extracted using the cold percolation technique.The extract was then placed on an orbital shaker for 72 h at 25 • C using 500 mL of 70% methanol three times (500 mL each time).A Buchner funnel was used to filtrate the methanol extracts.Then, the methanol was completely eliminated from the methanol extracts via concentration under reduced pressure at 40 • C and a rotary evaporator.In a dissector, the residues were dried to provide dry weights of 24.68 g/100 g for the leaves and 14.93 g/100 g for the stems.The bioactive components were identified using gas chromatography-mass spectrometry (GC-MS) analysis of the crude methanol extracts [68,69].

GC-MS Analysis for the Methanol Extract
A Thermo Scientific TRACE 1310 gas chromatograph was coupled to an ISQLT single quadrupole mass spectrometer.The ionization voltage was 70 eV, the ionization mode was EI and the column was DB5-MS, 30 m, 0.25 mm ID (J & W Scientific, Folsom, CA, USA).The temperature program was as follows: 40 • C for 3 min, 280 • C for 5 min, 5 • C/min to 290 • C (held for 1 min) and then static at 7.5 • C/min.The injector temperature was 200 • C, the detector was 300 • C and the flow rate of the carrier gas, helium, was 1 mL/min.The WILEY and NIST Mass Spectral Databases were employed as search libraries [68].

n-Hexane Sub-Fraction Extract Preparation
The lyophilized crudes of the methanol extract from 2.5 g of leaves and stems were redissolved in 250 mL of distilled water.Then, a separatory funnel was used to partition the redissolved crude with n-hexane for 24 h.Utilizing a rotary evaporator at 40 • C, the n-hexane sub-fraction was condensed to dryness at reduced pressures.The dried fractions of leaves and stems were 1.3 g and 0.86 g, respectively, and they were analyzed using GC-MS [11,69].

GC-MS Analysis for n-Hexane Sub-Fraction Extract
The Shimadzu GCMS-QP2010 (Tokyo, Japan) is equipped with a split/splitless injector and Rtx-5MS fused bonded column (30 m, 0.25 mm i.d., 0.25 m film thickness) from Restek (Bellefonte, PA, USA), which were utilized to record the mass spectra.The starting column temperature was raised to 300 • C at a rate of 5 • C/min for 5 min and was then maintained there for 2 min (isothermal).The temperature of the injector was 250 • C. The helium carrier gas flowed at a rate of 1.41 mL/min.All mass spectra were recorded using the following settings: 60 mA filament emission current, 70 eV ionization voltage and a 200 • C ion source, and the split mode injections of samples that were diluted (1% v/v) were carried out (split ratio 1:15) [69].

Total Phenolic and Flavonoid Determination
The Folin-Ciocalteu method was used to quantify the phenolic compounds [11,70].A volume of 0.25 mL of the Folin-Ciocalteu reagent (2 N) was added to 0.2 mL of the 80% methanolic extract in a volumetric flask (10 mL).Saturated sodium carbonate (1 mL) (20% in distilled water) was added after three minutes, and the volume was completed with distilled water.A Unicam UV-visible Spectrometer was used to measure the absorbance of blue color after 1 h at λ max 725 nm against a blank (distilled water).Gallic acid was used to obtain a standard calibration curve.The results were represented as milligrams of gallic acid equivalents (GAE) per gram of dry weight (D.W) [11,70].
An aluminum chloride colorimetric method was used to quantify the total flavonoid content [11,70].Distilled water with a volume of 1.25 mL was used to dilute the methanolic extract (0.25 mL).A 5% NaNO 2 solution was then added to the mixture in a volume of 75 µL.After 6 min, 150 µL of a 10% AlCl 3 .6H 2 O solution was added, and the combination was left to stand for an additional 5 min.Then, 0.5 mL of a 1 M NaOH solution was added, and the mixture was then completed with 2.5 mL of distilled water.The absorbance was measured at λ max 510 nm against the blank (distilled water).The (+)-catechin was used to obtain a standard calibration curve.The results were represented as milligrams of catechin equivalents (CE) per gram of dry weight.

Quantitative analysis of phenolic compounds via HPLC
The previously prepared methanolic extract from the leaves and stems of C. silvatica.(200 mg) was dissolved in HPLC-grade acetonitrile (2 mL).An Agilent 1260 series reversephase HPLC apparatus (Agilent, USA) was used for phenolic compound analysis.The separation of phenolic compounds was performed using an Eclipse C18 column (4.6 mm × 250 mm i.d., 5 µm).The mobile phase consisted of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate of 0.9 mL/min.The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (82% A); 0-5 min (80% A); 5-8 min (60% A); 8-12 min (60% A); 12-15 min (82% A); 15-16 min (82% A); 16-20 min (82%A).The samples were monitored at 280 nm using a multi-wavelength detector.The injection volume was 5 µL for each of the sample solutions.The column temperature was maintained at 40 • C. Standard flavonoids and phenolic acids were prepared as 10 mg/50 mL stock solution in methanol.They were then diluted to reach working concentrations (20-40 µg/mL) and injected into HPLC.The quantities of the detected compounds were expressed as µg/g, and the peak area of the external standards was employed to quantify the phenolic compounds in the methanol extracts from the leaves and stems [11,70].

Culturing
The sterility of the process was maintained using a laminar airflow cabinet.The Roswell Park Memorial Institute medium (RPMI 1640) was used to sustain the cell culture.A mixture of one percent of antibiotic and antimycotic (10,000 µg/mL streptomycin sulphate, 25 µg/mL amphotericin B and 10,000 U/mL potassium penicillin) and 1% Lglutamine were added to the medium.The 10% heat-inactivated fetal bovine serum was used to supplement the medium [70].

MTT Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to measure cytotoxicity.A purple formazan was created from the yellow MTT via a mitochondrial reduction [70].For inoculation, a 96-well microplate was filled with 1 × 10 5 cells per well in 100 µL of Roswell Park Memorial Institute medium (RPMI 1640).Conditions of 5% CO 2 , 37 • C and 95% humidity were used to incubate the microplate for 24 h to produce a completely formed monolayer sheet.After the cells formed a confluent layer, the growth media was decanted from 96-well microplates.Dimethyl sulfoxide (0.1%) was used to dissolve the methanol extract from the leaves and stems.The dissolved extract was serially diluted using a growth medium to achieve final concentrations of are: 31.25,62.5, 125, 250, 500 and 1000 µg/mL [70].Confluent cell monolayers were injected with 0.1 mL of the extract at each concentration using a multichannel pipette, and then they were dispersed throughout the 96 wells.The cells that were treated with the extracts were incubated at 37 • C and 5% CO 2 for 24 h.For each extract concentration, three wells were used.Control cells were incubated without the extracts from the leaves and stems.Phosphate-buffered saline (Bio Basic Canada Inc., Markham, ON, Canada) was used to dissolve the MTT powder to provide a solution with a 5 mg/mL concentration.Each well received 20 µL of the MTT solution following the completion of the incubation period.A shaker (MPS-1, Biosan, London, UK) was used for mixing, which was performed at 150 rpm for 5 min.The 96-well microplate was then kept for 4 h at 37 • C with 5% CO 2 .A metabolic byproduct of the MTT called formazan was resuspended in 200 µL of DMSO and aggressively shaken for five minutes at 150 rpm.The optical density at 560 nm was determined using a microplate reader.A background reference wavelength of 620 nm was used to adjust the results.All experiments were performed in triplicate.

Determination of IC 50 Values
Using GraphPad Prism version 7 (GraphPad software Inc., San Diego, CA, USA), the values of the varying concentrations of the IC 50 of the methanol extract from C. silvatica and doxorubicin (as a positive control) against colon cancer (Caco2), cervical cancer (HeLa), prostate cancer (PC3), breast cancer (MCF7), hepatocellular carcinoma (HepG2) and normal human fetal lung fibroblast (WI38) cell lines were computed.Equation ( 1) was used to determine the percentage of growth inhibition as follows [70]: Growth Inhibition (%) = 100 − (mean OD of individual test group/mean OD of control group) × 100 (1)

Selectivity Index
The ratio of a plant extract's IC 50 value in a non-cancer cell line (WI38) to its IC 50 value in each cancer cell line is known as the selectivity index.SI values below 3 indicate that the extract is not selective to non-cancer cells [71].The selectivity indices of the methanol extract were determined using the following Equation ( 2 The morphological structures of cell lines were examined using a Nikon 11,881 inverted microscope with objective 8× at various methanol C. silvatica extract concentrations.

Conclusions
The methanolic and n-hexane extracts from the leaves and stems of C. silvatica contained a diverse variety of phytochemicals that were identified by GC/MS and HPLC.Most of these phytochemicals had antitumor properties.Most of the phytochemicals that possessed antitumor properties were greater in the stems than in the leaves.Therefore, the antitumor effect of the stems of C. silvatica on cervical cancer (HeLa), prostate cancer (PC3), breast cancer, colon cancer (Caco2) and hepatocellular carcinoma (HepG2) was greater than that of the leaves, which showed a weak antitumor effect against all of the studied cancer cell lines.The normal cells were not affected by the cytotoxic activities of either part of the C. silvatica plant.Therefore, the isolation of the bioactive compounds from the stems of C. silvatica and an investigation of their antitumor properties against different cancer cell lines may be required in future work.Additionally, the antitumor properties and a phytochemical investigation of C. silvatica have never been reported before.This study will thus serve as the basis for this plant, and more pharmacological investigations are recommended.

Figure 5 .
Figure 5. Anticancer effects of Calystegia silvatica leaves and stems extracts in methanol on cancer cell lines: (A) complete monolayer sheets are seen in all of the cancer cell lines that have not been treated; (B) doxorubicin treatment results in rounded and shrunk cells in all of the cancer cell lines; (C) shrunk cells are observed in HeLa and MCF7 cell lines treated with the extract from Calystegia silvatica leaves; however, no morphological changes are observed in Caco2, HepG2 and WI38; (D) the extract from Calystegia silvatica stems used to treat HeLa, PC3, MCF7 and HepG2 cancer cell lines revealed significantly rounded and shrunk cells; however, small rounded and shrunk cells are observed in Caco2 and W138.

Figure 5 .
Figure 5. Anticancer effects of Calystegia silvatica leaves and stems extracts in methanol on cancer cell lines: (A) complete monolayer sheets are seen in all of the cancer cell lines that have not been treated; (B) doxorubicin treatment results in rounded and shrunk cells in all of the cancer cell lines; (C) shrunk cells are observed in HeLa and MCF7 cell lines treated with the extract from Calystegia silvatica leaves; however, no morphological changes are observed in Caco2, HepG2 and WI38; (D) the extract from Calystegia silvatica stems used to treat HeLa, PC3, MCF7 and HepG2 cancer cell lines revealed significantly rounded and shrunk cells; however, small rounded and shrunk cells are observed in Caco2 and W138.

Figure 6 .
Figure 6.An example of the moderate anticancer effect of the stems extract from Calystegia sylvatica against cervical cancer cell lines (HeLa): (A) complete monolayer sheets of cervical cancer cells (HeLa) that have not been treated; (B) the effect of doxorubicin treatment at different concentrations; (C) the effect of the stems extract from C. sylvatica against HeLa cell lines at different concentrations; (D) the effect of the stems extract from C. sylvatica against normal human fetal lung fibroblast (WI38).

Figure 6 .
Figure 6.An example of the moderate anticancer effect of the stems extract from Calystegia sylvatica against cervical cancer cell lines (HeLa): (A) complete monolayer sheets of cervical cancer cells (HeLa) that have not been treated; (B) the effect of doxorubicin treatment at different concentrations; (C) the effect of the stems extract from C. sylvatica against HeLa cell lines at different concentrations; (D) the effect of the stems extract from C. sylvatica against normal human fetal lung fibroblast (WI38).

Figure 7 .
Figure 7.An example of the weak anticancer effect of the leaves extract from Calystegia sylvatica against colon cancer cell lines (Caco2): (A) complete monolayer sheets of colon cancer cell lines (Caco2) that have not been treated; (B) the effect of doxorubicin treatment at different concentrations; (C) the effect of the leaves extract from C. sylvatica against Caco2 cell lines at different concentrations; (D) the effect of the leaves extract from C. sylvatica against normal human fetal lung fibroblast (WI38) at different concentrations.

Figure 7 .
Figure 7.An example of the weak anticancer effect of the leaves extract from Calystegia sylvatica against colon cancer cell lines (Caco2): (A) complete monolayer sheets of colon cancer cell lines (Caco2) that have not been treated; (B) the effect of doxorubicin treatment at different concentrations; (C) the effect of the leaves extract from C. sylvatica against Caco2 cell lines at different concentrations; (D) the effect of the leaves extract from C. sylvatica against normal human fetal lung fibroblast (WI38) at different concentrations.

Table 1 .
Biological compound identification of methanol extract from Calystegia silvatica leaves and stems using GC/MS.

Table 2 .
Biological compound identification of n-hexane extract from Calystegia silvatica leaves and stems using GC/MS.

Table 3 .
Phenolic compound identification of methanol extract from Calystegia silvatica leaves and stems via HPLC.

Table 4 .
Antitumor effects of Calystegia silvatica methanol extract on cancer and normal cell lines.
a IC

Table 5 .
Selectivity index values of Calystegia silvatica methanol extract from leaves and stems for Caco2, HeLa, PC3, MCF7 and HepG2 cancer cell lines.