Analysis of Content Profiles, Antioxidant and Anticancer Properties in Endemic Hypericum salsolifolium

Abstract

This study investigated the antioxidant and anticancer properties, phenolic compounds, and content profile of Hypericum salsolifolium plant extracts prepared with different solvents. The total phenolic content, total flavonoid content, and antioxidant potential [(2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ferric reducing antioxidant power, and cupric reducing antioxidant capacity assays] of Hypericum salsolifolium extracts obtained using solvents of different polarities (hexane, dichloromethane, methanol, and water) were measured using spectrophotometric methods. The contents of the extracts were analyzed using liquid chromatography–mass spectrometry and inductively coupled plasma—mass spectrometry methods. Anticancer detection was performed in human lung carcinoma cells using the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide, annexin-V, and cell cycle assays, as well as fluorescence detection of acridine orange/ethidium bromide staining. The methanolic extract was determined to have higher activation values of total phenolics, total flavonoids, ferric reducing antioxidant power, and in the 2,2-diphenyl-1-picrylhydrazyl assay than the other extracts, and the aqueous extract had higher values in the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and cupric reducing antioxidant capacity assays. The methanolic extract showed a cytotoxic effect against human lung carcinoma cells (IC50: 141.96 µg/mL). It was found that Hypericum salsolifolium extract showed antioxidant and anticancer activities. It was concluded that this plant can be used as a nutritional supplement due to its glucose, phenolic compound, amino acid, and vitamin content.

1. Introduction

Scientists want to use natural herbal sources in the treatment of many diseases, especially cancer, diabetes, and obesity. Synthetic chemotherapeutic and radiotherapeutic agents are used in cancer treatment, but these treatment options have many side effects. An antioxidant binds to a free radical and neutralizes it, thereby eliminating the negative effects. Previous studies have recommended that natural antioxidants are more efficient in neutralizing free radicals than synthetic antioxidants [1,2,3,4].

Active substances obtained from plants are used as chemotherapeutic agents [5,6], as they are less harmful to humans [7,8]. In recent years, more importance has been given to herbal medicines that contain natural active substances compared to synthetic medicines [9]. While the synthetic anticancer drugs currently in use destroy cancer cells, they also kill normal cells in large amounts. In addition, if resistance develops against antineoplastic drugs, they will not affect cancer cells [10,11]. Therefore, the idea that natural anticancer drugs should be developed has emerged.

Plants have played an important role in protecting human health for many years. Plants are used to make spices, beverages, cosmetics, dyes, and medicines [12], and current research on finding new active substances in plants to fight cancer has gained momentum [13,14]. The success of herbal antineoplastic drugs obtained from plants, such as etoposide, vinblastine, teniposide, vincristine, etc., has accelerated on these studies. Therefore, studies have been conducted to investigate the antioxidant and anticancer properties of active substances extracted from plants for the purposes of treating different diseases, especially cancer [15].

H. salsolifolium is a herbaceous, perennial, endemic plant from the family Clusiaceae that grows on the calcareous hills and steppes of the Southeastern Anatolia Region of Turkey [16]. It is commonly known as Urfa centaury in Turkey.

Hypericum species contain numerous secondary metabolites, including phenolic acids, flavonoids, organic acids, carnitines, essential oils, amino acids, and other water-soluble components. Hypericum species are known to have many different medicinal effects because they contain secondary metabolites, and they are used in medicine and pharmacy for their antidepressant [17], antiviral [18], wound-healing, anti-inflammatory [19], antimicrobial [20], and anticancer [21] effects.

We hypothesized that these properties might be related to the bioavailability of major functional compounds in H. salsolifolium, especially syringic acid and vanillic acid. It is important that phenolic compounds are well absorbed from the intestine in terms of their activities and bioavailability in the body. Some studies have shown that high dietary phenolic content is associated with Lactobacillus and Bifidobacterium spp. It has been determined that beneficial bacteria in the intestinal microbiota positively affect the variability of the intestinal microbiota. In addition, the gut microbiota plays an important role in converting polyphenols into bioactive and bioavailable compounds [22].

Our aim was to evaluate the potential of the H. salsolifolium plant, which is endemic and grows in Sanlıurfa, for use in treatment by investigating its antioxidant and anticancer effects in cells. This study is of scientific value as the plant is endemic in Turkey and there is no previous research on this plant in the literature.

2. Materials and Methods

2.1. Materials and Equipment

Dichloromethane, hexane, methanol, ethylenediamine tetraacetic acid (EDTA), Trolox, quercetin, gallic acid, fetal bovine serum (FBS), cell-culture medium (RPMI 1640), DMEM-F12, acridine orange, ethidium bromide, dimethyl sulfoxide (DMSO), and trypsin-EDTA solution were obtained from Sigma-Aldrich (Burlington, MA, USA)/Merck (Darmstadt, Germany).

2.2. Collection and Identification of Plants

Before starting the study, a preliminary study and literature search were carried out. Endemic plants were screened, for which there had been no previous studies of phytochemical content or antioxidant and anticancer properties. The plant to be studied was selected. Research permission was obtained from the General Directorate of Nature Conservation and National Parks (letter dated 17 July 2020 and numbered 1971165). Plants were collected from their natural environment (37 22,261′ N-38 34,965′ E) between Bozova-Hilvan districts located in the Euphrates dam lake basin in June 2022, which is the appropriate period for the plant. The plants were identified by Mehmet Maruf Balos, a biologist in the Department of Biology, Faculty of Science and Literature, Harran University. The plant samples were given to the herbarium of the Department of Biology and labelled as 6389.

2.3. Extraction of H. salsolifolium Plants

H. salsolifolium plants were extracted according to the method described by Koyuncu [23]. The muddy and soiled above-ground parts of the plant were cleaned using distilled water. Then, the plants were dried in the open air and under suitable conditions in the laboratory environment. After drying, 100 g of plant sample was weighed and extracted sequentially with hexane, dichloromethane, methanol, and water. After the extraction process, the crude extracts were dried using a rotary evaporator (approximately 12 h at 45 °C).

2.4. Total Phenolic Content

The total phenolic content (TPC) of the extracts was determined according to the Folin–Ciocalteu method [24] in which CuSO₄ forms a complex with proteins or antioxidants in alkaline medium. When Folin reagent is added to the medium, the Folin reagent forms a complex with the protein. Thus, there is a color transformation from yellow to blue. Gallic acid was used as a standard in the experiments. To draw a standard graph, a solution was prepared by dissolving 25 mg of gallic acid in 25 mL of water. Then, the absorbances of the standard and the samples were read against the blank at 760 nm. The amount of gallic acid corresponding to the absorbance value of the samples was calculated in GAE with the help of the standard curve.

2.5. Total Flavonoid Content

This assay was performed using the AlCl₃-NaNO₂ method. In this method, the total amount of flavonoids is calculated by spectrophotometric measurement of the pink-colored flavonoid–aluminum complex in alkaline medium using aluminum chloride and sodium nitrite reagents. In the experiment, 1 mL of sample extract was made up to 5 mL with distilled water in a Falcon tube. Then, 2 mL of 1 M NaOH was added to the tube and made up to 10 mL with distilled water. The flavonoid–aluminum complex in the solution was detected at 510 nm using a spectrophotometer [25]. The results were calculated in terms of catechin equivalent (QE).

2.6. FRAP Assay

In the presence of low pH in FRAP medium, the Fe(III)-2,4,6-tripyridyl striazine (TPTZ) compound is reduced to the dark blue Fe(II) form by electron transfer. To perform the FRAP test, the FRAP reagent was first made by mixing 0.3 M acetate buffer (pH 3.6), 10 mM TPTZ, and 20 mM FeCl3 in a 10:1:1 ratio. Then, 0.1 mL of H. salsolifolium powder and 2 mL of FRAP reagent were added to the tube and mixed. The tube was then incubated for 30 min at room temperature. The absorbance value of the liquid was measured at 593 nm. The same procedure was performed for Trolox, which was used as a standard [26]. The FRAP activity of the extracts was calculated as trolox equivalent (mg TE/g).

2.7. ABTS Assay

The ABTS assay determines the radical scavenging activity of a sample. The absorbances of the liquid were measured at 734 nm by spectrophotometric method. Trolox was used as a standard. As a control, a reaction mixture containing methanol was used instead of the sample [27]. The ABTS activity of the extracts was calculated as Trolox equivalent (mg TE/g).

2.8. CUPRAC Assay

The absorbance values of the liquids were measured at 450 nm by spectrophotometric method [28,29]. Concentration and absorbance graphs were obtained from the absorbance values of the plant extracts and standards. The CUPRAC activity of the extracts was calculated as Trolox equivalent (mg TE/g).

2.9. DPPH Assay

The radical scavenging activities of the plant extracts were also determined by measuring the DPPH radical scavenging effects. In the experiment, 1 and 3 mL of extract was mixed with solutions of varying concentrations of DPPH in ethanol (10-3 M), agitated by vortexing, and left to incubate for 30 min at room temperature. The absorbance at 517 nm was measured. Trolox was used as a standard. The absorbance values of the samples were evaluated against the control [30]. The DPPH activity of the extracts was calculated as Trolox equivalent (mg TE/g).

2.10. High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC, previously called high-pressure liquid chromatography) is a technique that is used in analytical chemistry for the separation, identification, and quantification of each component in a mixture. The technique is based on the principle of advancing at high pressure a pressurized liquid solvent (mobile phase) containing the sample mixture through a column filled with a solid adsorbent material (stationary phase). Each component in the sample is in molecular interaction with the adsorbent material. However, this interaction is different for each component. This causes different flow rates for different components and causes the components to separate as they flow out of the column. Depending on the sample analyzed, qualitative determinations of these components could be made using a variable wavelength ultraviolet (UV) detector, a fluorescence detector, and a refractive index detector (RID) integrated with our current system. In HPLC studies, quantitative determinations of target components can also be made with calibrations prepared using standard substances. Today, HPLC is widely preferred and used due to its adaptability in different studies, including quantitative determination and analytical separation of non-volatile and temperature-degradable samples.

2.11. Liquid Chromatography–Mass Spectrometry (LC–MS/MS)

Liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) is an analytical chemistry technique that combines the mass analysis capabilities of liquid chromatography (HPLC) and the physical separation capabilities of mass spectrometry (MS). Coupled chromatography—MS systems are popular in chemical analysis because the individual capabilities of each technique are synergistically developed. Liquid chromatography separates mixtures with multiple components, while mass spectrometry provides the structural identity of individual components with high molecular specificity and detection sensitivity. Analytes separated from the LC unit are ionized and sent to the MS/MS unit. In the MS/MS unit, the ions are separated from the first quadrupole in mass/charge (m/z) ratios and directed to the interference cell known as the collision cell. Here, molecules collide with high-purity nitrogen gas, are broken down, and sent to the second quadrupole. These second ions in the second quadrupole unit are determined by mass by separating them in mass/charge (m/z) ratios. While there may be many molecules with the same m/z ratio, the rate of molecules having the same fragmentation ions is 1/10,000 in nature. This tandem technique is preferred for sensitive analysis of biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS/MS can be applied in a wide variety of industries such as biotechnology, environmental monitoring, food processing, and the pharmaceutical, agrochemical, and cosmetic industries. LC–MS/MS allows qualitative and quantitative analysis of analytes. It is possible to obtain results with high sensitivity and accuracy in the analysis of single or multiple analytes in complex environments, in the analysis of trace substances, impurities, metabolites, and drug active ingredients, in the analysis of additives or pesticides in foods, and in the analysis of biological samples such as blood, urine, and tissue. LC–MS/MS is one of the most sensitive methods preferred for the determination of trace analytes in samples as well as multi-analyte determination. It gives information about the amount, structure, and molecular weight of the components in a sample. It has a wide range of uses, from small pharmaceutical compounds to the determination of large proteins, as well as polar ionic, thermally labile, and non-volatile compounds.

2.12. Cell Cultures

We used a cell line purchased from the America Type Culture Collection (Manassas, VA, USA) stored in liquid nitrogen (

Table 1. Cancer cell line used in the study.

Cell Lineage	Cancer-Normal	Histopathology	Culture
A549	Lung Cancer Epithelium	Cancer	RPMI-1640

).

2.13. MTT Assay

H. salsolifolium extracts were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells were incubated in sterile 96-well plates for 24 h with 1 × 10⁴ cells per well. The media were removed and extracts at doses of 0, 2.5, 5, 10, 25, 50, 100, and 200 μg/mL and the positive control (5-Fu; Sigma) were incubated for 24 h. One hundred microliters of MTT (0.5 mg/mL) was added into each well as the reactive agent. Following these measurements, plots were constructed and the IC50 value was calculated for each extract.

2.14. Flow Cytometric Examination of Apoptotic Effect with Annexin-V

The cells that died in the experiment were analyzed using the annexin-V assay via flow cytometry (BD FacsCanto), in which the cells died by necrosis if there was no apoptotic pathway. Dead cells were identified with the fluorescent PI dye, which can bind to nucleic acids. PI passes through the damaged cell membrane of necrotic cells and stains their DNA. The fluorescent glow in cells with stained DNA was determined by flow cytometry using an FL2 detector. Cells were classified according to the intensity of the radiation and placed in a diagram.

2.15. Cellular Morphology-Inverted Microscopy and AO/EB Staining assay

Cells in 12-well plates with 5 × 10⁴ cell per plate were incubated for 24 h, treated with 100 μg/mL of plant extract, and incubated for another 24 h at 37 °C. Morphological changes were assessed using a light microscope CKX 51 (Olympus, Tokyo, Japan), and then the cells were rinsed with PBS. Incubation with 100 μL of 1:1 acridine orange/ethidium bromide staining solution was performed at room temperature for 5 min. Apoptotic changes in cellular morphology were evaluated under a fluorescence microscope.

2.16. Cell Cycle Assay

Cycletest DNA Reagent (BD, Heidelberg, Germany) was used for the cell cycle assay. Cells were counted to 1,000,000 and then seeded into wells. A 300 mL aliquot of trypsin in DMEM was added to each well with 1 mL of FCS, the cells were centrifuged at 1500 rpm for 10 min, and the supernatant was separated without touching the pellet. Buffer solution was added to the cell solution. Then, 250 µL of solution A, 250 µL of solution B, and 200 µL of solution C were added, respectively, and incubated for 10 min. Then, the cell suspensions were measured by flow cytometry.

2.17. Statistical Calculations

Data were analyzed using SPSS 20 statistical software. Standard deviations were clearly defined (±). Pearson analysis was performed for antioxidant activities and total phenolic and total flavonoid results. Annexin-V and cell cycle analysis data in all experiments were analyzed for statistical significance using one-way analysis of variants (ANOVA) and Duncan tests. A p-value < 0.05 was considered to be statistically significant.

3. Results

3.1. Antioxidant Parameter Activities in H. salsolifolium Extracts

A calibration curve (y = −0.0164x + 1.1767, R2 = 0.9916 µg/mL) was drawn using Trolox for the DPPH assay and the antioxidant activity was calculated using this curve (mg Trolox/g). The highest activity values in the extracts were determined as methanol > hexane > water > dichloromethane (66.14 ± 3.30, 65.4 ± 3.27, 65.16 ± 3.25, and 59.61 ± 2.98, respectively).

A calibration curve (y = −0.0147x + 1.1707, R2 = 0.99 µg/mL) was drawn using Trolox for the ABTS assay and the antioxidant activity was calculated using this curve (mg Trolox/g). The highest activity values in the extracts were determined as water > methanol > dichloromethane > hexane (75.01 ± 3.75, 73.38 ± 3.66, 32.42 ± 1.62, and 11.2 ± 0.56, respectively).

A calibration curve (y = 0.0056x + 0.053, R2 = 0.9942 µg/mL) was drawn using Trolox for the FRAP assay and the antioxidant activity was calculated using this curve (mg Trolox/g). The highest activity values in the extracts were determined as methanol > water > dichloromethane > hexane (322.14 ± 16.10, 308.21 ± 15.41, 136.42 ± 6.82, and 37.32 ± 1.86, respectively).

A calibration curve (y = 0.0293x + 0.0572, R2 = 0.9911 µg/mL) was drawn using Trolox for the CUPRAC assay and the antioxidant activity was calculated using this curve (mg Trolox/g). The highest activity values in the extracts were determined as water > dichloromethane > methanol > hexane (87.94 ± 4.39, 12.41 ± 0.62, 10.5 ± 0.52, and 2.65 ± 0.13, respectively).

The TPC values of the H. salsolifolium plant extracts were examined in the study. A calibration curve (y = 0.0144x + 0.0728, R2 = 0.9952 µg/mL) was drawn by calculating gallic acid, and the total phenolic amount was calculated using this curve. The results were expressed as mg GAE/g. The highest activity values in the extracts were determined as methanol > water > dichloromethane > hexane (68.9 ± 3.44, 64.52 ± 3.22, 7.51 ± 0.37, and 3.68 ± 0.14, respectively).

In the examination of the TFC values of the H. salsolifolium plant extracts, a calibration curve (y = 0.0024x + 0.0344, R2 = 0.9957 µg/mL) was drawn by calculating quercetin and the total flavonoid amount was calculated using this curve. The results were expressed as mg QE/g. The highest activity values in the extracts were determined as methanol> water> dichloromethane> hexane (104.69 ± 5.23, 83.72 ± 4.18, 13.02 ± 0.65, and 6.08 ± 0.30, respectively).

The antioxidant activities of the different fractions of the H. salsolifolium extracts were examined by the ABTS, DPPH, CUPRAC, TPC, and TFC assays and the results are given in

Table 2. Antioxidant parameter activity results of the study.

H. salsolifolium Extract	DPPH (mg Trolox/g)	ABTS (mg Trolox/g)	FRAP (mg Trolox/g)	CUPRAC (mg Trolox/g)	TFC (mg QE/g)
water	65.16 ± 3.25	75.01 ± 3.75	308.21 ± 15.41	87.94 ± 4.39	83.72 ± 4.18
dichloromethane	59.61 ± 2.98	32.42 ± 1.62	136.42 ± 6.82	12.41 ± 0.62	13.02 ± 0.65
hexane	65.4 ± 3.27	11.2 ± 0.56	37.32 ± 1.86	2.65 ± 0.13	6.08 ± 0.30
methanol	66.14 ± 3.30	73.38 ± 3.66	322.14 ± 16.10	10.5 ± 0.52	104.69 ± 5.23
H. salsolifolium extract	TPC (mg GAE/g)	TFC (mg QE/g)		TPC (mg GAE/g)	TFC (mg QE/g)
water	64.52 ± 3.22	83.72 ± 4.18		64.52 ± 3.22	83.72 ± 4.18
dichloromethane	7.51 ± 0.37	13.02 ± 0.65		7.51 ± 0.37	13.02 ± 0.65
hexane	3.68 ± 0.14	6.08 ± 0.30		3.68 ± 0.14	6.08 ± 0.30
methanol	68.9 ± 3.44	104.69 ± 5.23		68.9 ± 3.44	104.69 ± 5.23

. It was seen that the antioxidant activities and TPC and TFC values of the methanolic and aqueous extracts showing polar properties were higher than those of the other nonpolar extracts.

Significantly positive correlations were determined between the TPC values and the ABTS and FRAP values (r = 0.97, p = 0.015; r = 0.968, p = 0.016; respectively) (

Table 3. Correlation analyses of TPC and TFC with antioxidant parameters in H. salsolifolium.

		DPPH	ABTS	FRAP	CUPRAC
TPC	r	0.603	0.97	0.968	0.560
	p	0.199	0.015	0.016	0.220
TFC	r	0.598	0.957	0.963	0.461
	p	0.201	0.022	0.018	0.270

). Significantly positive correlations were determined between the TFC values and the ABTS and FRAP values (r = 0.957, p = 0.022; r = 0.963, p = 0.018; respectively).

3.2. Phenolic Compound Profile of H. salsolifolium Methanolic Extract

Since the methanolic extract was rich in terms of high total phenolic content, the phenolic compounds in the methanolic extract were qualitatively and quantitatively investigated. The results are shown in

Table 4. Profile of phenolic compounds found in H. salsolifolium methanolic extract using the LC–MS method.

Phenolic Compounds	Quantities (µg/L)
Vanillic acid	1577.83 ± 70.96
Syringic acid	1714.43 ± 77.13
Fumaric acid	750.62 ± 34.78
Caffeic acid	285.91 ± 11.97
Hydoxycinnamic acid	98.4 ± 4.51
Hydroxybenzoic acid	363.19 ± 15.25
Salicylic acid	397.86 ± 18.26
Oleuropein	17.21 ± 0.77
Myricetin	10.66 ± 0.45
Ellagic acid	448.48 ± 21.05
Quercetin	34.51 ± 1.56
Naringenin	40.62 ± 1.92
Luteolin	25.26 ± 1.07
Curmin	3.41 ± 0.15

. The device used was a Shimadzu LC–MS/MS-8030 (Japan). In the H. salsolifolium methanol extract, the highest content was determined to be syringic acid (1714.43 ± 77.13 µg/L), followed by vanillic acid (1577.83 ± 70.96 µg/L), fumaric acid (750.62 ± 34.78 µg/L), ellagic acid (448.48 ± 21.05 µg/L), salicylic acid (397.86 ± 18.26 µg/L), hydroxybenzoic acid (363.19±15.25 µg/L), caffeic acid (285.91 ± 11.97 µg/L), hydoxycinnamic acid (98.4 ± 4.51 µg/L), naringenin (40.62 ± 1.92 µg/L), quercetin (34.51 ± 1.56 µg/L), luteolin (25.26 ± 1.07 µg/L), oleuropein (17.21 ± 0.77 µg/L), myricetin (10.66 ± 0.45 µg/L), and curmin (3.41 ± 0.15 µg/L).

3.3. Content Analysis of Fraction with High Antioxidant Activity by LC–MS

Content analysis was performed using the LC-MS method. The highest activation values were observed in polar solvent with the methanolic extract. The glucose (44 mg/dL), folic acid (5.87 ng/mL), and vitamin B12 (102 pg/mL) values were determined in the H. salsolifolium plant extract. The results are shown in

Table 5. Content analysis of H. salsolifolium plant solvent extract.

Test Items	Results
Fol	5.87 ng/mL
Glu	411 mg/dL
VB12	102 pg/mL

.

Content element analysis was performed at the Harran University Central Laboratory (HUBTAM) using the ICP-MS method on 100 mg of powdered plant material. The main elements determined were Ca (8716 mg/kg), K (6532 mg/kg), Mg (1278 mg/kg), and Na (716.6 mg/kg) (

Table 6. ICP-MS results of elemental analysis of H. salsolifolium plant extract.

Analysis Substances	Results (mg/kg)
Al	249.9
B	8.860
Ca	8716
Cd	5.558
Co	11.99
Cr	15.92
Cu	5.458
Fe	250
K	6532
Mg	1278
Mn	10.82
Na	716.6
Ni	9.530
P	<10
Pb	<10
Zn	0.711

).

The essential amino acid profile of the H. salsolifolium methanolic extract is shown in

Table 7. Amino acid profile of H. salsolifolium solvent extract.

aa	Results (µmol/L)
histidine	13.669
lysine	17.035
methionine	0.132
phenylalanine	122.301
threonine	260.253
tryptophan	12.211
valine	718.178
leucine	104.316
isoleucine	159.102
alanine	556.173
arginine	68.663
asparagine	418.743
aspartic acid	27.724
cystine	0.088
glutamine	917.779
glutamic acid	96.284
glycine	24.542
proline	274.588
serine	398.988
tyrosine	34.959

. The essential amino acids determined were valine (718.178 µmol/L), threonine (260.253 µmol/L), isoleucine (159.102 µmol/L), phenylalanine (122.301 µmol/L), leucine (104.316 µmol/L), lysine (17.035 µmol/L), histidine (13.669 µmol/L), tryptophan (12.211 µmol/L), and methionine (0.132 µmol/L). The most abundant amino acids were determined to be glutamine and valine.

3.4. IC50 Results of Substances Applied to A549 Cells

The methanolic extract of H. salsolifolium showed a cytotoxic effect against the A549 (IC50: 141.96 µg/mL) cell line. The results of this extract showed biological activity against lung cancer cells.

3.5. Acridine Orange/Ethidium Bromide Results with A549 Cells

The AO/EB fluorescent staining method was used to observe the effects of H. salsolifolium on cell morphology and the cell nucleus of cancer and normal cells by phase contrast light microscopy. Both cellular and nuclear changes in A549 cells treated with H. salsolifolium indicated that apoptosis was triggered. As seen in Figure 1, cells with a green color were considered healthy, and those with a yellow-orange color change were considered apoptotic. The change in cellular properties seen in cells exposed to H. salsolifolium varied depending on the extract concentration. In addition, the changes observed by phase contrast microscopy were similar to those of the apoptotic process. A549 cells were exposed to H. salsolifolium at a concentration of 100 μg/mL and AO/EB staining images were examined to detect morphological changes in cells and apoptotic cells after 24 h.

3.6. Annexin-V Results in A549 Cells

The annexin-V results indicated the cell viability of A549 cells under normoxic conditions (5% CO₂, 37 °C temperature, and 21% O₂). It was determined that H. salsolifolium extract had a significant apoptotic effect in A549 cells at a dose of 100 μg/mL (2/1% early apoptotic and 11.9% late apoptotic, respectively) and apoptotic cell death increased depending on the dose increase (Figure 2 and Figure 3). Figure 2, the blue bar shows the negative, the red bar shows H. salsolifolium extract.

3.7. Cell Cycle Analysis of A549 Cells by Flow Cytometry

According to the results of the cell cycle analysis after the H. salsolifolium extract was applied to the A549 cells, it was observed that the density of cells in the G0/G1 phase was 67.1%, 14.2% in the S phase, and 19.2% in the G2/M phase. It was determined that H. salsolifolium extract stopped the cell cycle of A549 cells in the S phase depending on the dose increase (Figure 4 and Figure 5). Figure 4, the blue bar shows the negative, the red bar shows H. salsolifolium extract.

4. Discussion

There has been an increasing number of free radicals in cells due to current factors such as stress, malnutrition, sedentary lifestyles, and environmental pollution. This has led to an increase in diseases such as diabetes, hypertension, and cancer. Natural antioxidants play an important role in removing free radicals that have accumulated in the body. Natural antioxidants are obtained from parts of plants such as leaves, flowers, and seeds. Therefore, this study aimed to investigate the antioxidant and anticancer properties, phenolic compounds, and content profile of the H. salsolifolium plant, which has not been previously studied, for use in the treatment of various diseases, especially cancer.

To determine the biologically active substances, various extracts were made from the H. salsolifolium plant. The results of the study showed that the highest total phenolic content (68.9 ± 3.44 GAE mg/g) and total flavonoid content (104.69 ± 5.23 QAE mg/g) were found in the methanolic extract. Similarly, in a study by Ahmed et al., high total phenolic content and total flavonoid content were found in the methanolic extract [31]. In parallel with the current study, Niala et al. also studied Hypericum species and reported that levels of phenolic substances were higher in the methanolic extract than in the other extracts [32].

The DPPH radical is a valid compound that reacts with antioxidants and provides information about activation. In the current study, the methanolic extract had the highest activity in the DPPH assay (66.14 ± 3.30 mg Trolax/g). A previous study also examined Hypericum species and reported that the radical scavenging activation value of the methanolic extract in the DPPH assay was higher than that in the other extracts, similar to the current study results [33].

The ABTS radical also reacts with antioxidants and provides information about activation. In the current study, although the activity value in the ABTS assay was the highest for the aqueous extract, it was found to be close to that for the methanolic extract (75.01 ± 3.75 and 73.38 ± 3.66 mg Trolax/g, respectively). A previous study also showed that the activation values of aqueous and methanolic extracts in Hypericum species were close to each other according to the ABTS assay [33]. According to research on Hypericum species, the activation value of radical removal for the aqueous extract in the ABTS assay was higher than for the other extracts [34].

The reduction potential of FRAP by the H. salsolifolium extracts was determined with iron-III ion. In the current study, the activation value of the methanolic extract was found to be highest in the FRAP assay (308.21 ± 15.41 mg Trolax/g). In a study of Hypericum species, the activation value of the methanolic extract in the FRAP assay was higher than that of the other extracts [35].

The reduction potential of the H. salsolifolium extracts in the CUPRAC assay was determined with copper-II ion. In the current study, the activation value of the aqueous extract was found to be highest in the CUPRAC assay (87.94 ± 4.39 mg Trolax/g). A previous study also found that the activation value of the aqueous extract in the CUPRAC assay was higher than that of the other extracts [34].

In the phenolic compound content analysis performed on the methanolic extract of the H. salsolifolium plant in the current study, mainly syringic acid (1714.43 µg/L) and vanillic acid (1577.83 µg/L) were found to have high levels. Similarly, syringic acid and vanillic acid were determined as the main phenolic compounds in a study of Hypericum species [36]. Phenolic substances such as syringic acid and vanillic acid are used in relieving inflammation, in atherosclerosis treatment, as antioxidants, for various chemotaxis, endothelial functions, and estrogenic/antiestrogenic activities, and as an angiotensin converting enzyme (ACE) inhibitor [37]. Therefore, the H. salsolifolium plant can be a source of syringic acid and vanillic acid for the fields of pharmacy and medicine.

In the current study, the total phenolic content, total flavonoid content, and antioxidant properties (DPPH, ABTS, FRAP, CUPRAC) of aqueous, dichloromethane, hexane, and methanolic extracts of H. salsolifolium were investigated. H. salsolifolium is used by the public as a herbal drug. In a study of Hypericum species, a strong positive correlation was found between TPC and TFC values and ABTS and FRAP values, similar to the results of the current study (p < 0.05) [38].

In the ICP-MS elemental content analysis of H. salsolifolium plant material in the current study, mainly Ca (8716 mg/kg), K (6532 mg/kg) and Mg (1278 mg/kg) values were found to be high. These elements are very important for human health. Ca is important in the elimination of hypocalcemia, cardiac arrhythmia, tetany, nervous system conduction and regulation, and bone and tooth development. K is used for the relief of muscle weakness, for the elimination of cardiovascular disorders, and in nervous system functions. Mg plays a role in neurotransmission, tetany, the cardiovascular system, and bone development [39]. The findings of a previous study also showed parallelism with the current study in the content analysis of Hypericum species [40]. This plant, rich in these elements, can be a source of nutrients and elements. In addition, cadmium and lead elements, which are toxic to humans, were almost nonexistent. Moreover, due to the high values of vitally important elements such as Ca, K, and Mg, the consumption of the H. salsolifolium plant as a food will have positive effects on human health.

The study results showed essential amino acids such as valine (718.178 µmol/L), threonine (260.253 µmol/L), isoleucine (159.102 µmol/L), phenylalanine (122.301 µmol/L), leucine (104.316 µmol/L), lysine (17.035 µmol/L), histidine (13.669 µmol/L), tryptophan (12.211 µmol/L), and methionine (0.132 µmol/L). The most abundant amino acids were found to be glutamine (917.779 µmol/L) and valine (718.178 µmol/L). Klejdus et al. also reported high levels of amino acids in Hypericum species. Glutamine and valine cause muscle growth and regeneration and produce energy, and they are found in expensive foods such as meat, cheese, and milk [41,42]. In addition, glucose (411 mg/dL) and vita-mins such as folic acid (5.87 ng/mL) and vitamin B12 (102 pg/mL) were also detected in H. salsolifolium. Folic acid is effective in the cycle of some amino acids, growth, and is essential for development at the cellular level. Folic acid deficiency causes diseases such as anemia and cancer. Vitamin B12 is also important for cell growth and protection of the nervous system. Deficiency of these vitamins should be corrected through the diet or by taking food supplements [43].

The current study results demonstrated that H. salsolifolium extract had a cytotoxic effect against A549 cells. It was observed that H. salsolifolium extract started to decrease the viability of A549 cells at a dose of 100 µg/mL. Morphological changes were seen in the acridine orange/ethidium bromide assay. The survival rate of 100% in the A549 cell line decreased to 60% due to the introduction of H. salsolifolium extract, and the remaining 40% was composed of apoptotic and necrotic cells. According to the annexin-V results, the cell viability of A549 cells under normoxic conditions was 93.8%, and the total number of apoptotic and necrotic cells comprised 6.2%. The cell cycle analysis results after H. salsolifolium extract was applied to the A549 cells determined that the density of cells in the G0/G1 phase was 58.7% in the negative group, and the density of cells in the G0/G1 phase was determined to be 67.1% in the H. salsolifolium extract group. In addition, when the H. salsolifolium extract group was compared with the negative group, the cell accumulation rates increased while the cell ratio in the G2/M phase decreased (22.3% in G2/M phase in negative group; 19.2% in G2/M phase in H. salsolifolium extract group), indicating that cell division was stopped. These results showed that the extract had biological activity in lung cancer cells. Similar to the current study, a previous study reported that Hypericum extract had a cytotoxic effect against the A549 cell line [44].

Polyphenols and the gut microbiota can mutually affect each other. Phenolic compounds can increase the growth of beneficial bacteria in the gut microbiota and reduce the growth of pathogenic bacteria. In addition, the gut microbiota plays an important role in the biotransformation of polyphenols and their biological activities. However, polyphenols are extensively metabolized not only by the gut microbiota but also in the liver [45]. For this reason, we think that comprehensive research should be conducted on this subject.

With some studies, increasing the concentration of a compound was not the main factor; rather, the effects were due to the distribution of the entire spectrum of nutraceuticals in the product being tested. This property explains the lack of clinical outcomes of green tea extracts [46] in many dietary supplements based on medicinal phenolic compounds [47]. In one study, strains belonging to the genus Saccharomyces, which did not have comparative differences from organisms, were thought to be a new medical model for oxidative stress [48].

From the data obtained, phenolic substances protect cells against the free radicals that cause oxidative stress. A study showed that bioavailability is an indicator of the in vitro/in vivo relationship. The study examined the biological effects and showed a real relationship between bioactivity and bioavailability [49]. Our study presented in vitro and in vivo data showing the effects of H. salsolifolium extracts.

We suggest that the bioactive potential of the functional extracts, the bioavailability of the phenolic compounds, and the relationship with microbiota bioactivity could be targeted for future valorization of the study’s findings.

5. Conclusions

From the results of this study evaluating the antioxidant and anticancer properties, phenolic compounds, and content profile of H. salsolifolium extracts, the antioxidant parameters and TPC and TFC activation values of the methanolic and aqueous extracts were found to be high. Thus, the results of this study demonstrated the antioxidant activity of H. salsolifolium. Since H. salsolifolium extracts have high phenolic and flavonoid contents, it can be considered that they will neutralize free radicals, and therefore H. salsolifolium can be used as a natural antioxidant. H. salsolifolium extract was also determined to have cytotoxic properties against lung cancer cells. H. salsolifolium, which is rich in phenolic compounds, glucose, vitamins, and amino acids necessary for life, can therefore be thought to have a positive effect on human health if used in the treatment of diseases or taken as a supplement. This study supports the creation of a new source of active compounds that can be used in the treatment of diseases caused by oxidative stress, especially cancer, obesity, and diabetes, and can provide guidance for further studies. Moreover, as H. salsolifolium is an endemic plant, its use could also have a significant impact on the economy of the region. However, more comprehensive clinical studies are needed, especially since studies with human cells are not sufficient. Further research on the metabolism of the polyphenolic compounds in H. salsolifolium is envisioned.