Phytochemical Profiles, Antioxidant, Cytotoxic, and Anti-Inflammatory Activities of Traditional Medicinal Plants: Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans

Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans are traditionally used plants grown in Turkey. Methanol extracts were obtained from these plants and pharmacological activity studies and phytochemical analyses were carried out. To evaluate the phytochemical composition, spectrophotometric and chromatographic techniques were used. The extracts were evaluated for antioxidant activity by DPPH●, ABTS●+ radical scavenging, and FRAP assays. The cytotoxic effects of the extracts were investigated on DU145 prostate cancer and A549 lung cancer cell lines. The anti-inflammatory effects of extracts were investigated on the NO amount, TNF-α, IFN-γ, and PGE 2 levels in lipopolysaccharide-stimulated Raw 264.7 cells. The richest extract in terms of phenolic compounds (98.19 ± 1.64 mgGAE/gextract) and total flavonoids (21.85 ± 0.64 mgCA/gextract) was identified as C. pichleri subsp. pichleri methanol extract. According to antioxidant activity determinations, the C. pichleri subsp. pichleri extract was found to be the most active extract. Finally, the C. pichleri subsp. pichleri methanol extract was revealed to be the most effective inhibitor of viability in the cytotoxic activity investigation, and the extract with the best anti-inflammatory action. The findings point to C. pichleri subsp. pichleri as a promising source of bioactive compounds in the transition from natural sources to industrial uses, such as new medications, cosmeceuticals, and nutraceuticals.


Introduction
From the past to the present, phenolic compounds have been extensively studied for their ability to counteract oxidative stress in chronic oxidative stress, various metabolic disorders, and conditions of obesity, diabetes, renal and cardiovascular disease. When the overproduction of reactive oxygen species (ROS) exceeds the natural antioxidant system (enzymatic and non-enzymatic) defenses, oxidative stress occurs, which damages cellular and extracellular macromolecules. The antioxidant power of phenolic compounds is related to the reducing properties of free radical scavengers as hydrogen or electron-donating agents. In addition, due to the molecular structure of phenolic compounds, they suppress the metal-catalyzed free radical formation and can chelate metals, especially iron and copper [1].
Phenolic compounds have an important place in human health due to their bioactive effects, such as anti-aging, anticancer, anti-inflammatory, cardio-protective, neuroprotective,

Chemical Analysis
Herbal extracts high in phenols and flavonoids boost human health by preventing and curing a variety of ailments [3]. Considering these findings, as well as the traditional uses of Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans, we studied the phenolic and flavonoid contents of their methanolic extracts. The total phenol and total flavonoid values for the three plant extracts are given in Table 1.
The C. pichleri subsp. pichleri methanol extract was found to have the highest total phenol (98.19 ± 1.64 mg GAE /g extract ) and total flavonoid (21.85 ± 0.64 mg CA /g extract ) amounts (Table 1); although no phytochemical studies on C. pichleri subsp. pichleri have been conducted, and investigations on other species' total phenol and flavonoid contents have been performed.
Although there are only a few studies that have determined the total phenolic and total flavonoid levels of Conyza canadensis, one of these was performed by El Guiche et al. It has been reported that the total phenol content and the total flavonoid contents of C. canadensis were found to be 2.54 µg /mg and 5.41 µg/mg, respectively [24]. Our study determined that the total phenol content of C. canadensis was 71.34 ± 0.53 mg GAE /g extract and the total flavonoid content was 18.91 ± 1.46 mg CA /g extract , which was similar to the literature.
In a study investigating the pharmacological activities of J. fruticans extracts, the total phenol and total flavonoid contents of the methanol extract were calculated as 82.70 ± 1.84 mg GAE /g extract and 70.78 ± 1.86 mg GAE /g extrac t, respectively [15]. However, the total phenol content of J. fruticans in our study was greater than that reported in the previous study, with a total phenol content of 97.41 ± 0.92 mg GAE /g extract and total flavonoid content of 19.45 ± 0.84 mg CA /g extract .
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an essential role in chronic degenerative diseases because they produce oxidative stress in cells. To cope with the oxidative stress created by ROS and RNS, antioxidant defenses need to be strong. Phenolic compounds are known to have antioxidant and anti-inflammatory capacities and many studies have shown that phenolic compounds have a scavenging effect on free radicals [25,26]. In addition, they have been extensively studied in recent years, as they exhibit a wide variety of physiological properties such as anti-mutagenic, anticarcinogenic, and antimicrobial activity [27][28][29]. It is known that phenolic compounds, such as chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid, and rutin, which we detected in the samples in this study, have important antioxidant, anti-inflammatory, anticancer and antimicrobial effects [30][31][32][33][34]. As a result, the quantity of phenolic chemicals in plants is critical.
The investigation revealed the highest chlorogenic acid amount in C. pichleri subsp. pichleri. In a previous study with C. triumfetti All., chlorogenic acid was found to be high, but gallic acid and rutin were not found [35]. Other research on C. sivasica Wagenitz revealed that it contains a significant level of chlorogenic acid; however, gallic acid and rutin were not discovered [36]. Rutin and gallic acid were detected in C. pichleri ssp. pichleri in our phytochemical analysis in contrast to other studies.
p-Coumaric acid, ferulic acid, gallic acid, and rutin were found in the analysis of the J. fruticans extract. Rutin was discovered to be present in greater concentration than the other phenolics. In a previous study with a J. fruticans methanol extract; gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, verbascoside, hyperoside, and rutin were found [15]. However, our research could not confirm the existence of hyperoside and chlorogenic acid. The results found in phytochemical studies may differ due to the place of collection, date of collection, drying method, and extraction methods, even if the same plant is studied [38][39][40].

Antioxidant Activity
DPPH, ABTS, and iron(III) to iron(II) reduction activity assays were used for measuring the antioxidant capacities of the extracts. Antioxidant activities were evaluated by comparing the extracts with an antioxidant standard, and the results are given in Table 5 and Figure 2.   The reducing activity from iron(III) to iron(II) is an important investigation in terms of antioxidant activity evaluation and is a technique that demonstrates electron emitting capacity [45]. According to the findings of our investigation, which measured the extracts' reducing power as ascorbic acid equivalents (AscAE), the C. pichleri subsp. pichleri (1.852 ± 0.023 AscAE mmol/g) and C. canadensis (1.90 ± 0.0006 AscAE mmol/g) extracts had the same significance with BHT (p > 0.05). J. fruticans (1.141 ± 0.003 AscAE mmol/g) was found to be less active than BHT (2.273 ± 0.01 AscAE mmol/g). No previous assessment of the reducing activity from iron(III) to iron(II) was found in these species.
The antioxidant activities of three different plants were examined in the present study using various methodologies, and it was shown that C. pichleri subsp. pichleri had slightly more activity than the other species in all antioxidant activity analyses.

Cytotoxic Activity
Prostate cancer is one of the second most common causes of cancer-related death in the USA and Europe, and the incidence of prostate cancer has been increasing worldwide in recent years [46]. Lung cancer is the second most common type of cancer after prostate cancer in men and breast cancer in women. According to 2018 data, lung cancer accounts for 14% of new cancers in men and 13% of new cancers in women in the US [47]. Therefore, it is very important to identify alternative or new drug targets against this type of cancer. In this study, DU145 cell lines, and A549 cell lines were selected as models for these two cancer types.
The effects of three different methanol extracts on DU145, and A549 cell lines were observed using the MTT method. The results are given in Figures 3 and 4. The anti-growth effects of various concentrations of C. pichleri subsp. pichleri, C. canadensis, and J. fruticans methanol extracts were compared. C. pichleri subsp. pichleri methanol extract was found to be more effective at low doses in both cell lines (DU145 and A549 cell lines). In the DU145 cell line, it inhibited the viability by 57.85% at 125 µ g/mL concentration, and statistically, p < 0.05 was found to be significant compared to the control. In the A549 cell line, even at 7.81 µ g/mL concentration, a significant inhibition (p < 0.05) of viability was observed compared to the control (57.29%). The cytotoxic IC50 value of C. pichleri subsp. pichleri in the DU145 cell line was 116.26 ± 12.59 µ g/mL; however, it was 17.14 ± 2.73 µ g/mL in A549. Similarly, J. fruticans caused a statistically significant inhibition of viability in the One of the widely employed techniques used to assess antioxidant capacity is the DPPH scavenging activity test, which is a spectrometric procedure [41]. In our experiments, the inhibition (%) values of the extracts were compared with a positive control and evaluated in terms of antioxidant activity. Concentrations of 4 mg/mL of the three extracts gave an almost close % inhibition value (73.18-77.44%). Although the extracts at the highest concentration studied had the same statistical significance as BHT, they differed in significance as the concentration decreased. At 0.5 mg/mL concentration, C. pichleri subsp. pichleri was found to be more active than C. canadensis and J. fruticans with 29.10% inhibition.
Another technique for evaluating antioxidant activity is the ABTS radical scavenging activity test, which detects the radical reduction of samples against this oxidant by employing ABTS as an oxidant agent. The results obtained are given as a Trolox equivalent antioxidant capacity (TEAC) [42].
In a previous study investigating the DPPH scavenging effect of four Centaurea species (C. pseudoscabiosa subsp. araratica (Azn.) Wagenitz, C. pulcherrima Wild. var. pulcherrima, C. salicifolia M. Bieb. ex Wild. subsp. abbreviate, C. babylonica (L.)L.), the highest antioxidant value was found in C. pulcherrima var. pulcherrima (IC 50 : 187.42 ± 3.11 µg/mL), and the lowest antioxidant activity value was found in C. pseudoscabiosa subsp. araratica (IC 50 : 670.59 ± 19.43 µg/mL) [43]. The maximum antioxidant value was reported in the C. canadensis methanol extract (IC 50 : 120 ± 0.5 µg/mL) in the investigation of Hayet et al. [44]. In the study of El Guiche et al., similar to our results, C. canadensis's antioxidant activity value was found to be 88.19% inhibition [24]. In the antioxidant activity study conducted with J. fruticans, the IC 50 value of the methanol extract was found to be 753.39 ± 15.89 µg/mL [15]. According to our findings, J. fruticans has the lowest activity when compared to the other extracts.
The reducing activity from iron(III) to iron(II) is an important investigation in terms of antioxidant activity evaluation and is a technique that demonstrates electron emitting capacity [45]. According to the findings of our investigation, which measured the extracts' reducing power as ascorbic acid equivalents (AscAE), the C. pichleri subsp. pichleri (1.852 ± 0.023 AscAE mmol/g) and C. canadensis (1.90 ± 0.0006 AscAE mmol/g) extracts had the same significance with BHT (p > 0.05). J. fruticans (1.141 ± 0.003 AscAE mmol/g) was found to be less active than BHT (2.273 ± 0.01 AscAE mmol/g). No previous assessment of the reducing activity from iron(III) to iron(II) was found in these species.
The antioxidant activities of three different plants were examined in the present study using various methodologies, and it was shown that C. pichleri subsp. pichleri had slightly more activity than the other species in all antioxidant activity analyses.

Cytotoxic Activity
Prostate cancer is one of the second most common causes of cancer-related death in the USA and Europe, and the incidence of prostate cancer has been increasing worldwide in recent years [46]. Lung cancer is the second most common type of cancer after prostate cancer in men and breast cancer in women. According to 2018 data, lung cancer accounts for 14% of new cancers in men and 13% of new cancers in women in the US [47]. Therefore, it is very important to identify alternative or new drug targets against this type of cancer. In this study, DU145 cell lines, and A549 cell lines were selected as models for these two cancer types.
The effects of three different methanol extracts on DU145, and A549 cell lines were observed using the MTT method. The results are given in Figures 3 and 4. The anti-growth effects of various concentrations of C. pichleri subsp. pichleri, C. canadensis, and J. fruticans methanol extracts were compared. C. pichleri subsp. pichleri methanol extract was found to be more effective at low doses in both cell lines (DU145 and A549 cell lines). In the DU145 cell line, it inhibited the viability by 57.85% at 125 µg/mL concentration, and statistically, p < 0.05 was found to be significant compared to the control. In the A549 cell line, even at 7.81 µg/mL concentration, a significant inhibition (p < 0.05) of viability was observed compared to the control (57.29%). The cytotoxic IC 50 value of C. pichleri subsp. pichleri in the DU145 cell line was 116.26 ± 12.59 µg/mL; however, it was 17.14 ± 2.73 µg/mL in A549. Similarly, J. fruticans caused a statistically significant inhibition of viability in the A549 cell line compared to the control at all concentrations studied and the IC 50 value was found to be 91.97 ± 5.53 µg/mL. Interestingly, only 500 and 1000 µg/mL concentrations were able to significantly inhibit viability in the DU145 cell line. C. canadensis, on the other hand, was able to exhibit significant inhibition at high concentrations in both cell lines. It is thought that the fact that the C. pichleri subsp. pichleri methanol extract is higher than the others in terms of cytotoxic activity may be due to its higher total phenol and total flavonoid contents and its higher antioxidant activity compared to other species. Additionally, it possesses the highest concentration of chlorogenic acid among the three plant extracts, which is likely to contribute to its high activity. Chlorogenic acid is a biologically active polyphenol that also serves as an antioxidant. It has many therapeutic roles, such as antibacterial, hepatoprotective, cardioprotective, anti-inflammatory, neuroprotective, antiobesity, antiviral, anti-microbial, and anti-hypertensive activity, as well as being a central nervous system (CNS) stimulant [48].
The cytotoxic effects of C. canadensis have been reported in previous studies. Fractions of C. canadensis root and aerial parts were studied, and it was determined that the n-hexane fraction of the roots was active on A549 (IC 50 : 94.73 µg/mL) and H1299 (IC 50 : 84.85 µg/mL) cell lines [13]. According to our results, C. canadensis is quite ineffective in the A549 cell line and its IC 50 level is above 500 µg/mL. In another study, ten different compounds were isolated from C. canadensis. It was determined that five of the isolated compounds (conyzapyranone A, conyzapyranone B, 4Z,8Z-matricaria-γ-lactone, 4E,8Z-matricaria-γlactone, and spinasterol) were effective in four different cell lines (HeLa, MCF-7, A431, and MRC-5) [49]. Studies of cytotoxic activity on other species have not been found. To the best of our knowledge, a cytotoxicity study has been performed for the first time in these species.
A549 cell line and its IC50 level is above 500 μg/mL. In another study, ten different compounds were isolated from C. canadensis. It was determined that five of the isolated compounds (conyzapyranone A, conyzapyranone B, 4Z,8Z-matricaria-γ-lactone, 4E,8Z-matricaria-γ-lactone, and spinasterol) were effective in four different cell lines (HeLa, MCF-7, A431, and MRC-5) [49]. Studies of cytotoxic activity on other species have not been found. To the best of our knowledge, a cytotoxicity study has been performed for the first time in these species.   . Cytotoxic activity of Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans extracts on the A549 cell line. Data are expressed as the mean ± standard error (n = 3). Significant differences are indicated as * p < 0.001, ** p < 0.01, *** p < 0.05.

Anti-Inflammatory Activity
Chronic inflammation is influenced by a wide variety of factors, including numerous interleukins, oxygen and nitrogen metabolites, growth factors, and lipid mediators. Many of these are effective not only in inflammation but also in cancer progression. Therefore, it is important to measure these factors [50][51][52].
In this study, the effect of three different methanol extracts on NO inhibition was determined by applying LPS stimulation to Raw 264.7 cells. The toxicity of the extracts was tested before the inflammatory investigation using the MTT technique, and concentrations of 62.5 and 31.25 µ g/mL were chosen, where all extracts did not demonstrate significant inhibition compared to the control ( Figure 5). C. pichleri subsp. pichleri methanol extract was found to be more active among all extracts. Although the control group (9.11 ± 2.74 µ M) and the LPS group (83.15 ± 4.17 µ M) were determined, the C. pichleri subsp. pichleri methanol extract was found to be significantly lower than the LPS group at both concentrations (47.81 ± 7.15 and 38.48 ± 3.62 µ M). Another parameter of anti-inflammatory studies is to evaluate the effects on cytokines. Cytokines play an important role, especially in chronic inflammatory diseases [53,54]. Likewise, when TNF-α, IFN-γ, and PGE2 levels were examined, it was found that the C. pichleri subsp. pichleri methanol extract was more active than the others. The TNF-α, PGE2, and IFNƔ value of the C. pichleri subsp. pichleri methanol extract (62.5 µ g/mL) was found to be 1953.57 ± 21.48 pg/mL, 1854.17 ± 9.47 pg/mL, and 99.1 ± 3.65 pg/mL, respectively. The results found were evaluated as significant compared to the control groups (Table 6). To the best of our knowledge, this is the first study in which anti-inflammatory values (TNF-α, IFN-γ, PGE2 levels, and NO . Cytotoxic activity of Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans extracts on the A549 cell line. Data are expressed as the mean ± standard error (n = 3). Significant differences are indicated as * p < 0.001, ** p < 0.01, *** p < 0.05.

Anti-Inflammatory Activity
Chronic inflammation is influenced by a wide variety of factors, including numerous interleukins, oxygen and nitrogen metabolites, growth factors, and lipid mediators. Many of these are effective not only in inflammation but also in cancer progression. Therefore, it is important to measure these factors [50][51][52].
In this study, the effect of three different methanol extracts on NO inhibition was determined by applying LPS stimulation to Raw 264.7 cells. The toxicity of the extracts was tested before the inflammatory investigation using the MTT technique, and concentrations of 62.5 and 31.25 µg/mL were chosen, where all extracts did not demonstrate significant inhibition compared to the control ( Figure 5). C. pichleri subsp. pichleri methanol extract was found to be more active among all extracts. Although the control group (9.11 ± 2.74 µM) and the LPS group (83.15 ± 4.17 µM) were determined, the C. pichleri subsp. pichleri methanol extract was found to be significantly lower than the LPS group at both concentrations (47.81 ± 7.15 and 38.48 ± 3.62 µM). Another parameter of anti-inflammatory studies is to evaluate the effects on cytokines. Cytokines play an important role, especially in chronic inflammatory diseases [53,54]. Likewise, when TNF-α, IFN-γ, and PGE 2 levels were examined, it was found that the C. pichleri subsp. pichleri methanol extract was more active than the others. The TNF-α, PGE 2 , and IFNGvalue of the C. pichleri subsp. pichleri methanol extract (62.5 µg/mL) was found to be 1953.57 ± 21.48 pg/mL, 1854.17 ± 9.47 pg/mL, and 99.1 ± 3.65 pg/mL, respectively. The results found were evaluated as significant compared to the control groups (Table 6). To the best of our knowledge, this is the first study in which anti-inflammatory values (TNF-α, IFN-γ, PGE 2 levels, and NO amount) are given together. ± 2.74 µ M) and the LPS group (83.15 ± 4.17 µ M) were determined, the C. pichleri subsp. pichleri methanol extract was found to be significantly lower than the LPS group at both concentrations (47.81 ± 7.15 and 38.48 ± 3.62 µ M). Another parameter of anti-inflammatory studies is to evaluate the effects on cytokines. Cytokines play an important role, especially in chronic inflammatory diseases [53,54]. Likewise, when TNF-α, IFN-γ, and PGE2 levels were examined, it was found that the C. pichleri subsp. pichleri methanol extract was more active than the others. The TNF-α, PGE2, and IFNƔ value of the C. pichleri subsp. pichleri methanol extract (62.5 µ g/mL) was found to be 1953.57 ± 21.48 pg/mL, 1854.17 ± 9.47 pg/mL, and 99.1 ± 3.65 pg/mL, respectively. The results found were evaluated as significant compared to the control groups (Table 6). To the best of our knowledge, this is the first study in which anti-inflammatory values (TNF-α, IFN-γ, PGE2 levels, and NO amount) are given together.  Anti-inflammatory activity of Centaurea pichleri subsp. pichleri, Conyza canadensis, and Jasminum fruticans extracts on the Raw 264.7 cell line. Data are expressed as the mean ± standard error (n = 3). Significant differences are indicated as * p < 0.001, ** p < 0.01, *** p < 0.05.

Plant Material
Specimens of Centaurea pichleri subsp. pichleri and Jasminum fruticans were collected from Ankara-Beynam in 2021. The Conyza canadensis specimen was collected from Balıkesir-Altınoluk, in 2020. The voucher samples of J. fruticans, C. pichleri subsp. pichleri, and C. canadensis were deposited in the Herbarium of Ankara University, Faculty of Pharmacy AEF 30773, AEF 30961, and AEF30963, respectively.

Determination of Total Phenolic Content
To determine the total amount of phenolic content, the extracts were mixed with distilled water (3.95 mL), Folin-Ciocalteu reagent (250 µL), and 20% Na 2 CO 3 (750 µL), respectively. After the samples were kept at room temperature (25 • C) for 2 h, measurements were made at 760 nm. Gallic acid was used as a standard [56].

Determination of Total Flavonoid Content
To determine the total amount of flavonoids, 4 mL of distilled water and 0.3 mL of 5% NaNO 2 were added to the extracts with the concentration adjusted. After waiting for 5 min, 0.3 mL of 10% AlCl 3 6H 2 O solution was added. Then, 2 mL of 1 M NaOH was added to the samples, resulting in a total volume of 10 mL. Measurement of the samples was made at 510 nm using the Shimadzu Spectrophotometer UV 1800 (Washington, USA). Catechin was used as the reference substance [57].

High Performance Liquid Chromatography (HPLC) Analysis
Sample solutions were prepared at a concentration of 4 mg/mL. Stock standard solutions (chlorogenic acid, coumaric acid, ferulic acid, gallic acid, hyperoside, and rutin) were prepared at 500 µg/mL concentration. A Waters Spherisorb C 18 column (25 cm × 4.6 mm, 5 µm) was used for the HPLC analysis. The mobile phase consisted of 0.01% formic acid (A) and acetonitrile (B) in the gradient system. The flow rate was 1 mL/min, the column temperature was 40 • C and all analyses were carried out at the 254 nm wavelength.
Five different concentrations of standards were injected in triplicate. For quantification, the calibration curve of each standard was obtained. For method validation, accuracy, precision, the limit of detection (LOD), the limit of quantitation (LOQ), and recovery values were calculated for method validation [39,41]. The precision of the method (intraday and inter-day variation) was carried out, and the differences were expressed by the relative standard deviation (RSD). For analysis of the LOD and LOQ values, 10 injections of standards were made, and the signal/noise value calculated. The LOD signal/noise value was 3:1, while the LOQ signal/noise value was 10:1. For the recovery analysis, 3 different known concentrations of the standard were added to the sample and the recovery percentage was calculated. For the robustness analysis, minor changes in flow rate, column temperature, mobile phase, and wavelength were made and checked to affect the analysis.

DPPH • Radical Scavenging Activity
The Gyamfi et al. method was used to calculate the DPPH • radical scavenging effects of the samples [58]. The samples were mixed with a Tris-HCl buffer (50 nM, pH 7.4) and a DPPH solution (0.1 mM) was prepared in methanol. A Butylated Hydroxy Toluene (BHT) standard antioxidant was used as a positive control. After the samples were incubated in the dark for 30 min at room temperature, measurements were made at 517 nm. The process was repeated three times in parallel and the inhibition % calculations were made using the equation below: Inhibition % = [(Abs control − Abs sample )/Abs control ] × 100

ABTS •+ Radical Scavenging Activity
The Re et al. method was used to calculate the ABTS •+ radical scavenging effects of the samples [59]. A solution of ABTS+ radical (7 mM) with the absorbance adjusted to 0.700 (±0.030) at 734 nm was obtained for analysis. The reaction kinetics were measured and recorded at 734 nm at 1 min periods for 30 min using 990 µL of the prepared radical solution and 10 µL of the samples. Trolox equivalents were calculated as percentages of inhibition measured (TEAC). BHT standard antioxidant was used as a positive control. The process was repeated three times in parallel.

Iron(III) to iron(II) reduction activity (FRAP)
The Koşar et al. method was used to calculate the ability of the samples to reduce iron(III) [60]. The sample solutions (1 mL) were mixed with 2.5 mL of a 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) potassium hexacyanoferrate solution (K 3 [Fe(CN) 6 ] +3 ). After 30 min of incubation at 50 • C, 2.5 mL of 10% (w/v) trichloroacetic acid (TCA) was added and centrifuged for 10 min. It was then mixed with 2.5 mL of H 2 O and 0.5 mL of 0.1% (w/v) Iron(III) chloride (FeCl 3 ). The absorbances of the samples were measured at 700 nm. The reducing activities of the extracts were expressed as ascorbic acid equivalents (AscAE) per mmol ascorbic acid/g sample.
3.5. Cytotoxic Activity 3.5.1. Cell Lines and Cell Culture Methods The DU145 human prostate cancer cell line and A549 human lung cancer cell line were purchased from American Type Culture Collection (ATCC HTB-81 ™ , ATCC CCL-185, Manassas, VA, USA) and maintained in Eagle's Minimum Essential Medium (EMEM) and RPMI and supplemented with 10% fetal bovine serum (FBS), and 100 µg/L penicillin/streptomycin, at 37 • C in a 5% CO 2 atmosphere.

Determination of Cell Viability Assay
The cytotoxic activity of the extracts on A549 and DU145 cells was determined by the MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) colorimetric method. Then, 24 h before the study, the cells were inoculated in a 96-well microplate with 1 × 10 4 cells at 100 µL per well and incubated. After 24 h, the medium on the cells adhered to the plate was discarded. A stock solution of 1 mg/mL of the extracts to be applied was prepared. It was then prepared by diluting it in the medium at concentrations of 3.9 µg/mL, 7.81 µg/mL, 15.6 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL. A total of 100 µL of each sample was added to the well. After 24 h of incubation, the medium in the wells was emptied. The wells were then filled with 100 µL of the MTT solution diluted 1/10 with a medium made from 5 mg/mL (in PBS) MTT stock. After keeping the plates in a CO 2 incubator at 37 • C for 4 h, 100 µL of DMSO was added to each well and the formazan crystals formed by the MTT were dissolved. After 10 min, each well was read using a microplate reader at the 540 nm wavelength [61]: % Viability = [(Abss ample × 100)/Abs control ]

Anti-Inflammatory Activity
The murine macrophage Raw 264.7 cell line was obtained from the American Type Culture Collection (ATCC TIB-71, Manassas, VA, USA) and kept at 37 • C in a 5% CO 2 environment in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FBS, and 100 µg/L penicillin/streptomycin. Using the aforementioned MTT assay, the toxicity of the extracts in the Raw 264.7 cell line at concentrations between 3.9 and 125 µg/mL was assessed. For anti-inflammatory activity, the RAW 264.7 cells were seeded at a number of 5 × 10 5 in six-well plates and incubated for 24 h at 37 • C and 5% CO 2 . After incubation, 1 µg/mL lipopolysaccharide (LPS) was added to all other wells except the control well. The extracts were applied to the wells at 31.25 and 62.5 µg/mL concentrations, respectively. Supernatants were collected and centrifuged after 24 h (10 min., 700× g).

Statistical Analysis
Analysis was performed in at least triplicate and the mean values were calculated. All data were presented as the mean ± standard deviation (SD), and relative standard deviation (RSD). All these calculations were made using the Microsoft Excel program.
The Levene test was used to evaluate the variance homogeneity. One-way analysis of variance was used for comparisons between more than two groups. The Dunnett T3 test and Tukey's test were used for multiple comparisons. The data were evaluated with SPSS Version 11.0 statistic software package. The significance level was set at p < 0.05.

Conclusions
In this study, three different traditional plant methanol extracts were evaluated in terms of pharmacological activities. Some activities in this study were performed for the first time on these species. In addition, to the best of our knowledge, this is the first report to show the pharmacological activity potentials and some phenolic compound contents of the three traditionally used plants together. In this study, previous studies on these plants were collected and a collective evaluation of the three plants was made in terms of pharmacological activity with new activity studies. Antioxidant activity (DPPH, ABTS, and iron(III) to iron(II) reduction), cytotoxic activity (DU145 and A549), and anti-inflammatory activity (NO amount, TNF-α, IFN-γ, PGE 2 levels) were evaluated, and phytochemical analyses were carried out by HPLC. The C. pichleri ssp. pichleri methanol extract, which is rich in total phenols and total flavonoids and has high antioxidant activity, was found to be effective in terms of both cytotoxic activity and anti-inflammatory activity. The J. fruticans methanol extract has higher total phenol and total flavonoid contents than the C. canadensis methanol extract, but it was found to be less active in terms of antioxidant and anti-inflammatory activity and cytotoxicity. This is considered to be caused by the various active substances they each possess. So, this study is an important step for future studies. However, more studies may result in the discovery and development of innovative medications for the pharmaceutical industry. These results will aid in the creation of pharmacologically active natural products, and functional dietary supplements for the prevention of various diseases. The fractionation, isolation of active ingredients, and identification of the mechanisms underlying these pharmacological activities will also illuminate the traditional uses of these plants.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article.