Screening of in vitro cytotoxicity and antioxidant potential of selected endemic plants in Turkey

Abstract

In the present study, we evaluated antioxidant and cytotoxicity potentials of Astragalus tokatensis Fisch., Helichrysum noeanum Boiss. and Stachys huber-morathii R. Bhattacharjee, endemic plant species in Turkey. We measured DPPH scavenging activity, metal chelating activity, total phenol and flavonoid contents of the extracts. In addition, the cytotoxic activities of the extracts were evaluated on human breast cancer cells (MCF-7). In the analysis of total phenol content, the water extract of S. huber-morathii had the highest value (116.34 µg gallic acid equivalent/mg extract). The highest total flavonoid content (83.39 µg quercetin equivalent/mg extract) was in the water extract of H. noeanum. Considering the DPPH scavenging and metal chelating activities, water extracts of H. noeanum (33.13 mg/L) and S. huber-morathii (49.00 mg/L) showed the lowest IC₅₀ values. When cytotoxic activities on MCF-7 cells were examined, the water extract of H. noeanum stood out with the lowest IC₅₀ value (96.94 mg/L).

KEYWORDS:

• Anticancer
• antioxidant
• cytotoxic
• flavonoid
• phenol

1. Introduction

Chemical products that have an unshared electron in their outer orbital are known as free radicals. These formed radicals are highly reactive and unstable [1]. 2,2-diphenyl-1-picrylhydrazyl (DPPH) is one such free radical produced in the human body. When DPPH is exposed to an antioxidant compound, the colour of the radical changes from violet to yellow. This colour change is used to measure the extent to which the antioxidant compound scavenges the free radicals [2]. DPPH is commonly employed to assess the antioxidant capacity of natural compounds or extracts as it reflects their ability to scavenge free radicals and prevent oxidative damage. Hence, a high DPPH radical scavenging activity indicates a strong antioxidant capacity and a potential protective effect against oxidative stress [3, 4].

Metal ions can contribute to oxidative stress, which arises from an imbalance between the production of reactive oxygen species (ROS) and the body's detoxification ability. Several metal ions, including iron, copper, and zinc, can catalyze the generation of ROS through the Fenton reaction. This reaction involves the production of highly reactive hydroxyl radicals from hydrogen peroxide and a metal ion. Consequently, cellular components such as lipids, proteins, and DNA can be damaged, leading to oxidative stress [5, 6]. Metal chelation refers to the process of binding metal ions, such as iron or copper, with a chelating agent to form a stable complex that can be eliminated from the body. This process plays a crucial role in preventing oxidative damage since certain metal ions, particularly iron and copper, can catalyze the production of ROS in the body, which can harm cell membranes, proteins, and DNA [7]. By chelating these metal ions, the production of ROS can be reduced, thereby preventing oxidative damage [8].

The antioxidant system is crucial in controlling the formation of ROS, which are known to be harmful [9]. Oxidative stress, on the other hand, occurs when the antioxidant system fails to prevent the radical effect, which damages cell components such as proteins, lipids, and DNA [10]. Furthermore, free radicals contribute to the development of pathological conditions such as atherosclerosis, neurodegenerative diseases, heart diseases, cancer [11], cerebrovascular diseases, diabetes [12], acute renal failure, lung diseases [13], emphysema and bronchitis [14]. Anticancer and antioxidant are related in that antioxidants can potentially help prevent cancer by reducing the damage caused by free radicals, which are highly reactive molecules that can damage cells and DNA [15]. Free radicals can be generated by various processes in the body, including exposure to environmental toxins, radiation, and the normal process of metabolism. Antioxidants neutralize free radicals by donating an electron to stabilize them, which can help prevent cellular damage that can lead to cancer [16].

Natural antioxidants come to the fore in the prevention of oxidative stress. Many researchers have achieved positive results in diseases such as atherosclerosis, malaria, rheumatoid arthritis, and diabetes [17] by inhibiting oxidation via natural antioxidants in recent years. Furthermore, natural antioxidants have hepatoprotective [18], anticancer [19], antimetastatic [20], antimutagenic [21], antiulcer [22], anti-inflammatory [23], antibacterial [24], antiviral [25], and antiaging [26] properties that can be investigated in the medical and pharmacological fields. Plants play an important role in the world of natural antioxidants. Plants’ antioxidant potential is increased by polyphenols, flavones, anthocyanins, lycopenes, and vitamins [27, 28]. The polyphenol group, which contains phenolic acids and flavonoids in its structure, stands out for its potent antioxidant properties [29]. Polyphenols are antioxidants that bind to reactive oxygen species and radicals that break lipid bonds, much like metal ion chelates [30]. The polyphenol mentioned above is found in many plants. At this point, the most important thing is to try plants that have not been extensively studied. Endemic plant species are important organisms for understanding their structures and quantifying their various biological activity potentials. Because these plants only grow in a specific region due to the ecological conditions of the region, their structures can contain unique secondary metabolites [31]. Scientists tested the effects of many endemic plants, such as enzyme inhibitory [32], insecticide [33], analgesic [34], anti-parasite [35], anticoagulant [36], oxidoreductase [37] and obtained positive results. Based on all the aforementioned information, in order to examine the unexplored activity potentials of endemic plants, we included Astragalus tokatensis Fisch., Helichrysum noeanum Boiss. and Stachys huber-morathii R. Bhattacharjee from Turkey endemics in our study to reveal the different potentials of rare medicinal plants. Since these plants are endemic, although there are not many studies on them, it is known that their genus (Astragalus, Helichrysum and Stachys) have very different activity potentials such as anti-inflammatory, antihyperglycemic, antioxidant and anticancer [38–40]. However, to the best of our knowledge, antioxidant and anticancer activities of A. tokatensis, H. noeanum and S. huber-morathii have not yet been tested. Therefore, in the present study, we examined their DPPH scavenging, metal chelating activities, total phenol, flavonoid content and performed the phenolic compound determination by high performance liquid chromatography (HPLC) method. Furthermore, we demonstrated their cytotoxic effects on a human breast cancer cell line (MCF-7). Overall, in this study, we aimed to help the pharmaceutical industry produce supplementary antioxidant sources with antioxidant and cytotoxic properties using A. tokatensis, H. noeanum, and S. huber-morathii.

2. Materials and methods

2.1. Collection and identification of the plant samples

Astragalus tokatensis (Voucher specimen number UT 0827), H. noeanum (Voucher specimen number UT 0786), and S. huber-morathii (Voucher specimen number UT 0803) samples were collected at their flowering stages between March and October from the Kazova and its surroundings (Tokat/Turkey). Plant species were identified by a botanical expert (Burak Surmen) with the help of various literature and compared with their herbarium specimens [41–43]. Herbarium specimens of this species were examined in Ondokuz Mayıs University Herbarium (Samsun / Turkey) (OMUB). Astragalus tokatensis and H. noeanum are Irano-Turanian endemic elements, and their IUCN risk categories are NT: near threatened and LC: least concern, respectively. Stachys huber-morathii is Turkey endemic, and its IUCN risk category is VU: vulnerable [44]. Whole plant samples were dried in an oven at 40°C to a constant weight after cleaning them from soil and waste. Later they were grinded for the study.

2.2. Preparation of the extracts

Astragalus tokatensis, H. noeanum and S. huber-morathii samples were powdered with an ultra-centrifuge grinder (Retsch ZM 200, Germany). The ground plant samples (10 g) were extracted in 250 mL of methanol (at 60°C) and water (at 90°C) through the Soxhlet extraction apparatus throughout two days. The crude extracts of the plant samples were filtered through Whatman No. 1 filter paper. The solvent was evaporated with a rotary evaporator (IKA, Staufen Germany) under vacuum to dry and lyophilized to get ultra-dry powders.

2.3. Identification and quantification of some phenolic compounds by HPLC

Acetonitrile (HPLC grade, ≥ 99.9%), methanol (HPLC grade, ≥ 99.8%), acetic acid (HPLC grade, ≥ 99.8%), ultra-pure water (HPLC grade), gallic acid, and protocatechuic acid were purchased from Sigma Chemical Co. (USA). Each compound at 500 mg/L were prepared in methanol and stored in darkness at 4°C. Serial dilution of the stock solutions was performed with methanol/water solution (v/v, 1:1) to obtain standard working solutions daily.

HPLC system (Agilent Technologies 1260 Infinity) was equipped with column oven (G1316A), auto-sampler (G1329B), pump (G1311C), analytical column ACE 5 C18, 5 µm, 100 Å (250 × 4.60 mm) and diode array detector (G1311D) with a wavelength range of 190–800 nm. The injection volume was 20 μL, the oven temperature was 25°C, and the flow rate was 1.0 mL/min. The mobile phase gradients and their implementation steps were given in Table 1.

Table 1. Mobile phase gradients in HPLC analyses and their implementation steps.

Solvent	Ratio (%)	Code
Water: Acetic acid	98: 2	A
Acetonitrile: Acetic acid	99.5: 0.5	B
Acetonitrile	100	C
90% (A), 10% (B) / 0–30 min
80% (A), 20% (B) / 30–60 min
55% (A), 45% (B) / 60–62 min
40% (B), 60% (C) / 62–77 min
90% (A), 10% (B) / 77–80 min

2.4. Determination of total phenol content

Gallic acid was used as a standard to determine the total phenol content of methanol and water extracts from the plants. 20 µL of methanol and water extracts (400 mg/L) and standard were placed in the microplate wells. 20 µL of Folin reagent (2N) was added, and the samples mixed by pipetting were incubated in the dark for 3 min. Then, 20 µL of 35% (w/v) sodium carbonate and 140 µL of dH₂O were added to them and kept in the dark for 10 min. Spectrophotometric reading was performed at 725 nm [45]. Gallic acid equivalent (GAE) was calculated using the standard calibration curve created with gallic acid.

2.5. Determination of total flavonoid content

In determining the total flavonoid content of methanol and water extracts from the plants, quercetin was used as a standard. 50 µL of methanol and water extracts (400 mg/L) and standard were placed in the microplate wells. Then, 215 μL of ethyl alcohol (80%, v/v), 5 μL of aluminum nitrate (10%, w/v), and 5 μL potassium acetate (1 M) were added in microtiter plates and incubated for 40 min at room temperature. Spectrophotometric reading was performed at 415 nm [45]. Calculation in quercetin equivalent (QE) was performed using the standard calibration curve created with quercetin.

2.6. Free radical scavenging activity

In the measurement of the DPPH scavenging activity of methanol and water extracts obtained from the plants, applications were carried out with the final concentrations of the extracts in the plate wells of 12.5, 25, 50, 100, 200, and 400 mg/L. According to the method, 20 µL of the extracts were placed in each microplate well, and 180 µL of DPPH (0.06 mM in methanol) was added. The reduction of DPPH free radical was determined by measuring the absorbance values at 517 nm after 60 min in the dark [45]. The free radical scavenging activities of the extracts were calculated as a percentage using the following formula: Radical scavenging activity = [(Control absorbance – Extract absorbance) / Control absorbance] × 100.

2.7. Metal chelating activity

In measuring the metal chelating activity of methanol and water extracts obtained from the plants, applications were carried out with the final concentrations of the extracts in the plate wells of 12.5, 25, 50, 100, 200, and 400 mg/L. According to the method, 50 µL of the extracts were added to each microplate well. 185 µL of methanol, 5 µL of FeCl₂ (2 mM) and 10 µL of ferrozine (5 mM) were added to them, respectively, and kept at room temperature for 10 min. Spectrophotometric measurements were performed at 562 nm [45]. The metal chelating activities of the extracts were calculated in percent with the following formula: Metal chelating activity = [(Control absorbance – Extract absorbance) / Control absorbance] × 100.

2.8. Cell culture

MCF-7 cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% l-glutamine and 1% penicillin–streptomycin (Sigma-Aldrich, St Louis, Missouri, USA) at 37°C in a humidified air atmosphere with 5% CO₂. After MCF-7 cells grew to the expected concentration (90% confluence), in vitro tests for cytotoxic activity of the investigated samples were carried out.

2.9. Cytotoxic activity

Cells were seeded at 1 × 10⁴ cells/well in 96-well flat-bottomed microtiter plates at 37°C for 24 h. Then, the medium (DMEM) was replaced with a fresh medium containing different plant extract concentrations. The final concentrations of the extract in the wells were 12.5, 25, 50, 100, 200, and 400 mg/L, and 0.5% dimethyl sulphoxide (DMSO) was used as the negative control. After 72 h incubation, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)−2H-tetrazolium-5-carboxanilide (XTT) and activator reagents were added to each well according to the manufacturer’s instructions (Biological Industries, Beit Haemek, Israel). Accordingly, we used the XTT reagent and activation solutions in the cell proliferation kit. In order to prepare a reaction solution, we added 0.1 mL of the activation solution to 5 mL of the XTT reagent. 50 µL of the reaction solution were added to each well. Then, the plates were incubated for 4 h at 37°C. The absorbance of the samples against a background control as a blank was measured with a spectrophotometer at 450 nm [46]. Viabilities of the cells were calculated as a percentage using the following formula: Viability = (Extract absorbance / Control absorbance) × 100.

2.10. Statistical analyses

The activities of the extracts were analysed one-way ANOVA followed by the Duncan test. Probit regression analysis was used to calculate the median inhibitor concentration (IC₅₀) values. Heatmap analysis and hierarchical cluster analysis with Ward’s minimum variance method were utilized to investigate the similarities and dissimilarities among the DPPH, metal chelating and cytotoxic activities. All analyses were done using SPSS (version 21.0, IBM Corporation, Armonk, NY, USA).

3. Results

3.1. Yields of the plant extracts

The yield rates of methanol and water extracts obtained from A. tokatensis, H. noeanum and S. huber-morathii were given in Table 2. Three extracts with high yield were water extract of S. huber-morathii, water extract of H. noeanum, and methanol extract of H. noeanum, respectively.

Table 2. Yield (%) of obtained the plant extracts.

Extract	Yield
Methanol extract of A. tokatensis	8.75
Water extract of A. tokatensis	9.50
Methanol extract of H. noeanum	10.10
Water extract of H. noeanum	10.80
Methanol extract of S. huber-morathii	9.00
Water extract of S. huber-morathii	14.40

3.2. Identification and quantification of some phenolic compounds by HPLC

HPLC analysis was used to detect gallic acid and protocatechuic acid in methanol and water extracts of A. tokatensis, H. noeanum and S. huber-morathii. HPLC chromatograms of the extracts were given as supplementary material (Figure S1). When the amount of gallic acid in the extracts was examined, water extract of S. huber-morathii had the highest rate with 2469.16 μg/g. The general ranking was methanol extract of A. tokatensis < methanol extract of H. noeanum < water extract of A. tokatensis < methanol extract of S. huber-morathii < water extract of H. noeanum < water extract of S. huber-morathii. When comparing methanol and water extract of the same plant, water extracts had a higher rate of gallic acid than methanol (Table 3).

Table 3. Quantities (μg/g) of gallic acid and protocatechuic acid phenolics in the plant extracts.

		Plant extract
Phenolic compound	Detection wavelength (nm)	Methanol extract of A. tokatensis	Water extract of A. tokatensis	Methanol extract of H. noeanum	Water extract of H. noeanum	Methanol extract of S. huber-morathii	Water extract of S. huber-morathii
Gallic acid	254	802.62	1369.34	1025.87	1747.18	1657.56	2469.16
Protocatechuic acid	254	17.64	41.33	22.85	46.27	74.01	128.87

Regarding the amount of protocatechuic acid found in the plant extracts tested, water extract of S. huber-morathii had the highest ratio (128.87 μg/g). According to the amount of protocatechuic acid, extracts were in the ascending order of methanol extract of A. tokatensis < methanol extract of H. noeanum < water extract of A. tokatensis < water extract of H. noeanum < methanol extract of S. huber-morathii < water extract of S. huber-morathii. When a comparison was made between methanol and water extract of the same plant, water extracts had a higher proportion of protocatechuic acid than methanol, similar to the gallic acid experiment (Table 3).

3.3. Analysis of antioxidant compounds of the plant extracts

The total phenol content of methanol and water extr- acts obtained from Astragalus tokatensis, H. noeanum and S. huber-morathii samples were calculated based on GAE we used as a standard, and the total flavonoid content was calculated based on QE we used as a standard. When the total phenol contents were examined, the highest ratio was water extract of S. huber-morathii with 116.34 µg GAE/mg extract. The overall ranking among the extracts was methanol extract of A. tokatensis < water extract of A. tokatensis < methanol extract of H. noeanum < water extract of H. noeanum < methanol extract of S. huber-morathii < water extract of S. huber-morathii. When comparing methanol and water extract of the same plant, water extracts had a higher total phenol content than methanol. In addition, the total phenol content in each extract was statistically (p < 0.05) different from each other (Table 4).

Table 4. Antioxidant compounds of the plant extracts (µg/mg).

Treatment	Total phenol (Gallic acid equivalent)	Total flavonoid (Quercetin equivalent)
Methanol extract of A. tokatensis	34.76 ± 0.53 f	16.53 ± 0.74 f
Water extract of A. tokatensis	55.72 ± 0.57 e	38.27 ± 1.05 e
Methanol extract of H. noeanum	84.75 ± 2.30 d	49.01 ± 0.58 c
Water extract of H. noeanum	99.65 ± 1.36 c	83.39 ± 0.99 a
Methanol extract of S. huber-morathii	105.85 ± 2.13 b	71.11 ± 1.66 b
Water extract of S. huber-morathii	116.34 ± 1.51 a	40.97 ± 1.14 d

Each value is expressed as mean ± standard deviation (n = 3). Values indicated by different letters in the same column differ from each other at the level of p < 0.05.

Considering the total flavonoid contents found in plant extracts, water extract of H. noeanum had the highest ratio (83.39 µg QE/mg extract). The ranking for total flavonoid contents in all the extracts studied was methanol extract of A. tokatensis < water extract of A. tokatensis < water extract of S. huber-morathii < methanol extract of H. noeanum < methanol extract of S. huber-morathii < water extract of H. noeanum. When a comparison was made between methanol and water extract of the same plant, it was determined that water extracts had a higher total flavonoid content compared to methanol, except for S. huber-morathii extracts (Table 4).

3.4. DPPH scavenging activities of the plant extracts

water extract of H. noeanum had the highest data (91.00%) in DPPH scavenging activities, where a concentration-dependent increase was observed. The rates of 90.51% and 90.05% of water extract of S. huber-morathii and methanol extract of S. huber-morathii, respectively, were not statistically (p > 0.05) different from each other and the highest data of water extract of H. noeanum (Figure 1). When the IC₅₀ values were examined, it was determined that water extracts were effective compared to methanol. While the most effective extract was water extract of H. noeanum (IC₅₀: 33.13 mg/L), based on IC₅₀ values, the ranking among others was as follows: water extract of S. huber-morathii < methanol extract of S. huber-morathii < water extract of A. tokatensis < methanol extract of A. tokatensis < methanol extract of H. noeanum (Table 5).

Figure 1. DPPH radical scavenging activities of different extracts from the plants (mean ± standard deviation, n = 3) (Values indicated by different letters differ from each other at the level of p < 0.05. In the Duncan test, the highest-ranked mean is assigned the letter ‘a’, and subsequent means that are significantly different from it are assigned the next letter in the alphabet, such as ‘b’, ‘c’, and so on. Means that are not significantly different from each other are assigned the same letter, while means that are significantly different are assigned different letters). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

Table 5. IC₅₀ values (mg/L) resulting from DPPH scavenging activities of the plant extracts.

Treatment	IC50 (Limits)	Slope ± Standard error of the mean (Limits)
Methanol extract of A. tokatensis	99.75 (90.17–110.80)	1.48 ± 0.06 (1.34–1.61)
Water extract of A. tokatensis	58.97 (52.37–66.21)	1.23 ± 0.06 (1.10–1.36)
Methanol extract of H. noeanum	147.75 (133.12–165.31)	1.54 ± 0.07 (1.40–1.68)
Water extract of H. noeanum	33.13 (29.51–36.87)	1.50 ± 0.07 (1.36–1.64)
Methanol extract of S. huber-morathii	48.93 (44.72–53.38)	1.79 ± 0.07 (1.64–1.94)
Water extract of S. huber-morathii	39.81 (36.00–43.79)	1.65 ± 0.07 (1.51–1.80)

Considering the DPPH scavenging activities of the extracts, their closeness to each other based on their IC₅₀ values was determined using heatmap and cluster analyses. Among the extracts that were divided into 3 clusters in the cluster analysis, the methanol extract of H. noeanum under Cluster 3 was separated from the other extracts by having a red colour gradient in the heatmap analysis. Similarly, methanol extract of A. tokatensis was the only application under Cluster 2 that showed a medium colour gradient. Applications under Cluster 1 with lower IC₅₀ values were ranked as water extract of H. noeanum < water extract of S. huber-morathii < methanol extract of S. huber-morathii < water extract of A. tokatensis and showed closeness to each other (Figure 2).

Figure 2. (a) Heatmap based on IC₅₀ values for DPPH scavenging activities of plant extracts and (b) dendrogram (High and low activities were represented by red and green colour, respectively). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

3.5. Metal chelating activities of the plant extracts

water extract of S. huber-morathii had the highest data (78.79%) in metal chelating activities where a concentration-dependent increase was observed and this value was significantly (p < 0.05) higher than all other results. Metal chelating activities (74.66% and 74.25%, respectively) demonstrated by methanol extract of S. huber-morathii and water extract of H. noeanum were the other high rates in the trials, and these values were statistically (p > 0.05) indifferent from each other (Figure 3). Based on IC₅₀ values, water extracts showed lower values. water extract of S. huber-morathii with the lowest value (49.00 mg/L) was the most effective metal chelator. According to IC₅₀ values, the extracts were in the ascending order of water extract of S. huber-morathii < water extract of H. noeanum < water extract of A. tokatensis < methanol extract of S. huber-morathii < methanol extract of A. tokatensis < methanol extract of H. noeanum (Table 6).

Figure 3. Metal chelating activities of different extracts from the plants (mean ± standard deviation, n = 3) (Values indicated by different letters differ from each other at the level of p < 0.05. In the Duncan test, the highest-ranked mean is assigned the letter ‘a’, and subsequent means that are significantly different from it are assigned the next letter in the alphabet, such as ‘b’, ‘c’, and so on. Means that are not significantly different from each other are assigned the same letter, while means that are significantly different are assigned different letters). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

Table 6. IC₅₀ values (mg/L) resulting from metal chelating activities of the plant extracts.

Treatment	IC50 (Limits)	Slope ± Standard error of the mean (Limits)
Methanol extract of A. tokatensis	134.09 (115.65–158.02)	1.00 ± 0.06 (0.88–1.13)
Water extract of A. tokatensis	53.39 (43.82–64.13)	0.75 ± 0.06 (0.63–0.86)
Methanol extract of H. noeanum	240.76 (199.66–301.17)	0.96 ± 0.06 (0.83–1.09)
Water extract of H. noeanum	49.10 (42.13–56.65)	0.99 ± 0.06 (0.87–1.11)
Methanol extract of S. huber-morathii	70.22 (62.11–79.34)	1.16 ± 0.06 (1.04–1.29)
Water extract of S. huber-morathii	49.00 (42.45–56.07)	1.06 ± 0.06 (0.93–1.18)

Considering the metal chelating activities of the extracts, their closeness to each other based on their IC₅₀ values was determined utilizing heatmap and cluster analyses. Among the extracts divided into 3 clusters in the cluster analysis, the methanol extract of H. noeanum under Cluster 3 was separated from the other extracts by having a red colour gradient in the heatmap analysis. Similarly, methanol extract of A. tokatensis was the only application under Cluster 2 that showed a medium colour gradient. Applications under Cluster 1 with lower IC₅₀ values were ranked as water extract of S. huber-morathii < water extract of H. noeanum < water extract of A. tokatensis < methanol extract of S. huber-morathii and showed closeness to each other (Figure 4).

Figure 4. (a) Heatmap based on IC₅₀ values for metal chelating activities of plant extracts and (b) dendrogram (High and low activities were represented by red and green colour, respectively). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

3.6. Cytotoxic activities of the plant extracts

In the analyses in which a negative correlation was determined between the concentration and cell viability for each extract application, the application that reduced the cell viability the most was the concentration of 400 mg/L of water extract of H. noeanum. In addition, this value (23.84%) was statistically (p < 0.05) lower than all other results. All extract trials significantly (p < 0.05) reduced cell viability compared to the negative control (Figure 5). Considering the IC₅₀ values, water extract of H. noeanum was the most effective cytotoxic agent with the lowest value (96.94 mg/L). According to IC₅₀ values, the extracts were in the ascending order of water extract of H. noeanum < water extract of S. huber-morathii < methanol extract of S. huber-morathii < methanol extract of H. noeanum < water extract of A. tokatensis < methanol extract of A. tokatensis (Table 7).

Figure 5. Viability rates obtained by XTT analysis in MCF-7 cells treated with different extracts from the plants (mean ± standard deviation, n = 3) (Values indicated by different letters differ from each other at the level of p < 0.05. In the Duncan test, the highest-ranked mean is assigned the letter ‘a’, and subsequent means that are significantly different from it are assigned the next letter in the alphabet, such as ‘b’, ‘c’, and so on. Means that are not significantly different from each other are assigned the same letter, while means that are significantly different are assigned different letters). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

Table 7. IC₅₀ values (mg/L) resulting from cytotoxic activities of the plant extracts on MCF-7 cells.

Treatment	IC50 (Limits)	Slope ± Standard error of the mean (Limits)
Methanol extract of A. tokatensis	239.33 (204.97–286.47)	1.17 ± 0.07 (1.04–1.31)
Water extract of A. tokatensis	206.94 (181.44–240.03)	1.32 ± 0.07 (1.18–1.47)
Methanol extract of H. noeanum	163.25 (144.34–186.97)	1.30 ± 0.06 (1.16–1.44)
Water extract of H. noeanum	96.94 (86.68–108.88)	1.29 ± 0.06 (1.16–1.43)
Methanol extract of S. huber-morathii	159.20 (141.89–180.53)	1.39 ± 0.07 (1.25–1.53)
Water extract of S. huber-morathii	104.99 (94.40–117.34)	1.39 ± 0.06 (1.25–1.52)

Considering the cytotoxic activities of the extracts, their closeness to each other based on their IC₅₀ values was determined through heatmap and cluster analyses. Among the extracts that were divided into 3 clusters in the cluster analysis, water extract of A. tokatensis and methanol extract of A. tokatensis under Cluster 3 were separated from other extracts by having a red colour gradient in the heatmap analysis. methanol extract of S. huber-morathii and methanol extract of H. noeanum were the two applications that showed a medium colour gradient and were included in Cluster 1. water extract of H. noeanum and water extract of S. huber-morathii, which have a green colour gradient as an indicator of lower IC₅₀ value, were placed under Cluster 2 and showed closeness to each other (Figure 6).

Figure 6. (a) Heatmap based on IC₅₀ values for cytotoxic activities of plant extracts and (b) dendrogram (High and low activities were represented by red and green colour, respectively). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

3.7. General heatmap analysis for all activities

When the percent DPPH, metal chelating and cytotoxic activities of the plant extracts at different concentrations are considered, the closeness levels of the activities at the relevant concentrations were determined by heatmap analysis. This analysis gave an idea about the applications with high or low rates of all three activities. In this direction, applications with maximum concentration (400 mg/L) of all extracts were noteworthy among applications with high rates of all three activities (Figure 7).

Figure 7. Heatmap of percent DPPH, metal chelating and cytotoxic activities of the plant extracts. (High and low activities were represented by red and green colour, respectively). ATME: Methanol extract of A. tokatensis; ATWE: Water extract of A. tokatensis; HNME: Methanol extract of H. noeanum; HNWE: Water extract of H. noeanum; SHME: Methanol extract of S. huber-morathii; SHWE: Water extract of S. huber-morathii.

4. Discussion

Scientists have turned to various approaches due to the use of chemical products in the treatment of certain diseases and their side effects on living organisms. In this context, studies on natural compounds derived from plants have gained traction [47–49]. Many studies have focused on important sources of antioxidants, such as phenolic compounds, flavonoids, carotenoids, and vitamins. Numerous research endeavours have elucidated the mechanisms underlying the functionality of diverse herbal products and secondary metabolites, thereby promoting the utilization of specific phytochemicals in the treatment of particular diseases [50–52]. Within this framework, endemic plants and their phytochemicals have emerged as notable subjects of investigation. We examined the presence and quantity of gallic acid [53] and protocatechuic acid [54], two key phenolic compounds known for their significant free radical scavenging activity, in the plants under study (A. tokatensis, H. noeanum, and S. huber-morathii). Moreover, we selected gallic acid due to its capacity to protect various tissues from oxidative stress-induced damage and minimize toxicity induced by complex compounds [55, 56]. Additionally, the presence of protocatechuic acid in our plants was noteworthy as it is presumed to possess cytotoxic properties owing to its antioxidant, anti-inflammatory, and anti-apoptotic effects [57].

Since we utilized endemic plants in our study, there aren't many studies on their activities in the literature. As a result, we concentrated our literature search on the genera. Several scientific studies on the endemic plant genera Astragalus, Helichrysum, and Stachys, which we used in this study, stand out. Polysaccharides from Astragalus membranaceus were found to have immunomodulatory, antitumour, anti-inflammatory, and antiviral properties [58]. In a study on polysaccharide extract from Astragalus spinosis, the extract showed antilipase and antioxidant capacity [59]. In another study, secondary metabolites isolated from Astragalus lycius exhibited significant cytotoxic activity against human colon cancer [60]. Ghaffari et al. [61] reported that dichloromethane and methanol extracts of Astragalus creticus showed cytotoxic, immunomodulatory, and anti-inflammatory effects. Ethyl acetate and butanol extracts of Astragalus armatus were subjected to various antioxidant activity determination tests. Ethyl acetate extract exhibited more successful antioxidant activity. The antioxidant activity of the flavonoids separated from the butanol extract was assumed to be lower due to glycolysis by researchers who attributed the strong antioxidant activity of ethyl acetate extract to the presence of isorhamnetin [62]. According to Khan et al. [63], water and methanol extracts of Astragalus membranaceus root show antiviral activity, and safe quantities can be utilized to treat Avian influenza H9 virus infections.

Different biological activity measurements of the genus Helichrysum have been carried out in the past. Jahromi et al. [64] determined that the ethanolic extract obtained from the root parts of Helichrysum leucocephalum had a high degree of DPPH capture activity and metal chelating capacity. In addition, H. leucocephalum has been described to have antifungal and antibiotic activities. In a study conducted with Helichrysum italicum, another species of Helichrysum genus, the extract obtained from the plant showed antimicrobial, antihyperglycemic and anti-inflammatory effects besides the reducing power, ABTS, DPPH antioxidant activities [65]. In a study with flowers of Helichrysum pamphylicum, the extracts showed potent antioxidant and CD73 (ecto-5'-nucleotidase) enzyme inhibition and moderate anti-inflammatory activity. In a study conducted with methanol extract obtained from the flowers of Helichrysum italicum, isolated compounds of different structures exhibited antimicrobial, antitimetastatic, antioxidant, anti-inflammatory, analgesic, and antiproliferative activities [66]. In our study, while the cytotoxicity levels of the tested extracts of H. noeanum were determined on MCF-7 cells, the cytotoxic effect decreased as the concentration decreased. According to the calculated IC₅₀ value, the most effective cytotoxic activity was observed in water extract of H. noeanum. It was concluded that the cytotoxic activity was high in water extracts except for water extract of A. tokatensis. Since the water solvent dissolves the phenolic and flavonoid compounds better, these compounds, which have a high concentration in the extract, may cause an increase in the cytotoxic effect. It was also seen that the antioxidant capacities of the extracts increase depending on the total phenol content. Different studies showing the relationship between phenolic content and the antioxidant activity of plants are also available in the literatüre [67, 68].

Scientists have conducted research with different species belonging to the genus Stachys. Stegăruș et al. [69] investigated the aromatic compounds of water extracts obtained from three different species of Stachys, S. byzantina, S. officinalis and S. sylvatica. In addition to the previous, the high antibacterial and antioxidant capacities of the extracts, which were emphasized by the high total phenol content, attracted attention. In another study, it was reported that the methanolic extract of Stachys parviflora had antioxidant capacity thanks to the phenolic acids and flavonoid derivatives it contains in high concentrations. Moreover, this extract showed cytotoxic activity against human colon cancer (HCT), mouse melanoma (B16F10), and human ovarian cancer (A2780) cells [70]. When the water and methanol extracts of the plants we tested in our study were compared, the water extracts had a higher ratio for the amount of gallic acid and protocatechuic acid. The extract with the highest gallic acid and protocatechuic acid was water extract of S. huber-morathii. Another analysis supporting the HPLC analysis results was the total phenolic content test. There was more phenolic content in water compared with methanol. When comparing methanol and water extracts of the same plants, water extracts except for S. huber-morathii extracts, had higher flavonoid content than methanol. In addition, water extract of S. huber-morathii was the most potent metal chelator in the experimental group. The high metal chelating capacity of water extract of S. huber-morathii may be due to the capacity of the phenolic compounds it contains. According to total phenol and flavonoid analysis, the extracts containing the most phenols and flavonoids were the extracts of H. noeanum and S. huber-morathii species. There are also studies in the literature that antioxidant activity, metal chelating activity, and cytotoxicity increase as the total amount of phenolic compounds and flavonoids increases [71].

Considering the heatmap and cluster analyses, for antioxidant tests, while methanol extract of H. noeanum and methanol extract of A. tokatensis alone were in a separate group, water extract of H. noeanum, water extract of S. huber-morathii, methanol extract of S. huber-morathii, and water extract of A. tokatensis were grouped under the same cluster. As for cytotoxicity analysis, water extract of H. noeanum and water extract of S. huber-morathii are still under the same cluster with their high cytotoxic rate. This may indicate that the high cytotoxic levels of water extracts of H. noeanum and S. huber-morathii are due to their antioxidant potential. Considering the content analysis, while water extract of S. huber-morathii was drawn attention with the highest ratio of total phenol, gallic acid, and protocatechuic acid, water extract of H. noeanum was the application with the highest flavonoid ratio.

5. Conclusions

The DPPH radical scavenging, metal chelating, and cytotoxic properties of methanol and water extracts from three indigenous plant species, A. tokatensis, H. noeanum, and S. huber-morathii, were determined. Also, high concentration fractions produced more effective activity results. As a result, water extracts from endemic plants, particularly H. noeanum and S. huber-morathii, are regarded to have the potential to contribute to the field of complementary therapy due to their high antioxidant and cytotoxic properties. Furthermore, it will make an important contribution to medicine and pharmacology in the future by isolating the most active components from extracts presumably responsible for antioxidant and in vitro anticancer activity of A. tokatensis, H. noeanum, and S. huber-morathii extracts and evaluating their specific different biological activity potentials.

Disclosure statement

No potential conflict of interest was reported by the author(s).