Investigation of antioxidant, antimicrobial, cytotoxic, wound-healing properties, bio element levels and phytochemical profiles of aqueous extracts of Verbascum splendidum leaf and aboveground parts

Abstract

The present study aimed to determine the antioxidant, antimicrobial, cytotoxic and anticarcinogenic activities, wound-healing properties, element levels and phytochemical profiles of aqueous extracts of Verbascum splendidum leaf and aboveground parts (VSE_leaf and VSE_mix). Total phenolic content, total flavonoid content, total antioxidant capacity, total oxidant capacity levels and oxidative stress index were analyzed as the antioxidant parameters. The total phenolic contents of VSE_mix and VSE_leaf were comparable. While VSE_mix exhibited higher total flavonoid levels, this difference did not reach statistical significance. Oxidative stress index of VSE_mix extract was lower than VSE_leaf extract. The high amount and variety of phytochemical content, high total phenolic and total flavonoid amounts in VSE_mix extract support this situation. Moreover, these extracts, especially VSE_mix, were rich in bio-elements such as Fe, Cu, Mn and Zn which have roles on antioxidant system. E. coli, S. aureus and C. albicans strains were used to detect antimicrobial activity. The VSE_mix extract, which was rich in phenolic content, had higher antimicrobial activity than VSE_leaf extract. The cytotoxic effects of plant extracts were determined using L929 cell line, the anticarcinogenic effects of plant extracts were determined using A549. VSE_mix had anticarcinogenic activity for A549 cells and VSE_leaf for G361 cells, and both extracts had a positive effect on wound-healing compared to the control group. The findings demonstrated that especially VSE_mix could serve as a valuable natural source of bioactive compounds for prospective clinical use due to its antioxidant, antimicrobial, anticancerogenic, cytotoxic effects or wound-healing activity.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-026-05373-8.

Introduction

Bioactive molecules that function in metabolic pathways may have negative effects on these pathways, as well as positive effects with their anti-carcinogenic, anti-inflammatory, and antioxidant properties [1–3]. Phytochemicals, which are compounds obtained from plants, also show bioactive properties. The primary metabolites that the plant requires to survive and the secondary metabolites that the plant uses for defense purposes are phytochemical species [4–6].

Although the development of diagnostic, treatment, and surgical techniques used in medicine from the nineteenth century to the present has progressed rapidly, the rise in unsolved health problems, especially in parallel with the increase in the population, has led the scientific world to search for new types of therapy with fewer side effects [7]. Consequently, phytotherapy, which has been practiced for centuries and is expressed as the therapeutic use of plants, has formed the basis of many alternative therapies and has found new research areas for itself. Prior to the utilization of modern therapeutic techniques, the popularity of herbal therapy, which was almost the only choice, has seriously decreased with the diversification and development of synthetic pharmaceuticals, but it has not completely disappeared. The rediscovery of herbal therapy methods by people in the twenty-first century has reminded us that phytotherapeutic methods can also be used in the formation of new therapy models [8].

Among medicinal plants, the genus Verbascum has attracted attention due to its diverse secondary metabolites and ethnopharmacological uses. Verbascum (Scrophulariaceae, Lamiales), commonly known as mullein, is an important genus of the Scrophulariaceae family, which grows in the temperate regions of Asia, Europe, and Africa and has 360 different species in the world, with Turkey being the main point of diversity with 235 species [9]. It is stated that the flowers, leaves, and shoot parts of Verbascum species have been used for many years locally against respiratory problems such as cough, asthma, bronchitis, and hoarseness, excretory system problems such as urinary tract inflammation and pain [10], toothache and earache [11], oils obtained from the plant against skin diseases such as eczema [12], and pathologies related to the musculoskeletal and circulatory systems [13]. It has been reported that Verbascum plants contain various saponins, alkaloids, lactones, and coumarins [14], phenylethanoloids, iridoid, neolignan glycosides, monoterpene glycosides [15], as well as flavonoid-type bioactive phytochemicals such as hesperidin and rutin [16].

Although many scientific studies have been conducted on various Verbascum species, studies on Verbascum splendidum (V. splendidum) are limited. These findings can be summarized as follows. In a study on the antimicrobial and antimalarial activities of methanol and ethanol extracts of V. splendidum leaves and flowers, no significant activity was observed [17], and it was determined that 100 μg/ml concentration of the same extracts had no cytotoxicity in various cell lines [18]. It was observed that 200 mg/kg doses of methanol extracts of V. splendidum leaves and flowers did not exhibit anti-inflammatory or antinociceptive activity in mice [19]. In another study, the in vivo cytotoxic activity of V. splendidum leaf and flower methanol extracts was investigated using the brine shrimp (Artemia salina) method, and it was determined that the methanol extracts of V. splendidum flowers showed the highest inhibitory activity against brine shrimp [12]. In vivo anthelmintic property [20] and effect on wound healing [21] of methanol extracts of V. splendidum shoot components were investigated, but no activity was observed.

Plants are frequently used in the development of drugs and traditional treatments against diseases in society, and this constantly increases the demand for herbal ingredients. According to the World Health Organization (WHO), the global herbal market may increase from the current value of 62 billion dollars to 5 trillion dollars by 2050 [11]. Verbascum thapsus, the most preferred Verbascum L. species in the global herbal market, is also cultivated due to its commercial value. Therefore, it is important to investigate the possible effects of Verbascum L. species such as V. splendidum, which are traditionally used in different societies, and to bring them to the literature as scientific information.

Therefore, the study aimed to characterize the phytochemical composition and biological activities of aqueous extracts of V. splendidum, providing scientific support for its traditional uses and potential pharmaceutical relevance. In the presented study, aqueous extracts of the leaf (VSE_leaf) and shoot components (VSE_mix) of the species were prepared, and the cytotoxicity and anticarcinogenic effect on various cell lines (G361, A549, L929), in vitro wound healing activity, and antimicrobial activity of the extracts were investigated. In addition, the total oxidant/antioxidant capacity levels, total phenolic and total flavonoid content, mineral content, and phytochemical profile of the extracts were determined to explain how these pharmacological effects occur [11].

Material and methods

Plant materials and extraction

V. splendidum samples during the flowering period were collected from the Demirlik Hill forest road edges of the Sultan Mountains, Çay District, Afyonkarahisar Province, on 30.07.2021 (Altitude; 1982 m, GPS: 36 S 0336 282, UTM 4266239) without any special permission. The plant species were identified by Prof. Mustafa Kargıoğlu, who works at the Department of Molecular Biology and Genetics, Afyon Kocatepe University [22]. The plants were stored under appropriate conditions in the Afyon Kocatepe University Herbarium (Herbarium No: Kargıoğlu 10,873).

Leaves were removed from some of the plant samples, while the other parts were separated to represent the aboveground components (Shoot parts). All plant parts were washed with pure water and dried at room temperature and approx. 35% humidity until constant weight (240 h). The samples were converted to powder form using a homogenizer. Powdered samples (10 g) were mixed with 100 ml deionized water (1:10 w/v) and stirred at 60 °C (750 rpm) for 2 h, followed by sonication at 60 °C for 2 h and overnight incubation in the dark. The mixtures were then filtered, centrifuged (8500 rpm, 5 min), and concentrated using a rotary evaporator. The extraction was performed once [23, 24]. The VSE_leaf and VSE_mix samples obtained as a result of evaporation were used for the determination of total oxidant capacity, total antioxidant capacity, cytotoxicity, total phenolic substance amount, total flavonoid substance amount, mineral substance amount, phytochemical content analysis, wound healing activity, and antimicrobial activity.

Determination of total oxidant/antioxidant capacity and oxidative stress index level

Both total oxidant capacity (TOC) and total antioxidant capacity (TAC) levels were performed using commercial kits (Elabscience E-BC-K801-M and Elabscience E-BC-K802-M) to determine the oxidative stress level. For TAC analysis, standards or samples (10 μL) were mixed with 200 μL of buffer solution, and the initial absorbance (A1) was measured at 660 nm. Subsequently, 20 μL of chromogenic agent was added and mixed thoroughly. Following incubation at 37 °C for 5 min, the final absorbance (A2) was recorded to determine the change in absorbance (ΔA = A2—A1). For TOC analysis, the assay was initiated by mixing 20 μL of sample or standard with 200 μL of chromogenic agent. After mixing for 5 s in a microplate reader, the initial absorbance (A1) was measured at 590 nm. Subsequently, 50 μL of substrate was added to each well. Following 5 s mixing step and incubation at 37 °C for 5 min, the final absorbance (A2) was recorded to determine the change in absorbance (ΔA = A2—A1). In addition, the oxidative stress index (OSI) was calculated by comparing the TOC and TAC levels of the samples [25]. The experiments were performed in triplicate.

Determination of cytotoxicity and anticarcinogenic activity

3-[4,5-dimethylthiazole-2-yl]−2,5-diphenyltetrazolium bromide (MTT) analysis was performed to determine the cytotoxicity and anticarcinogenic activity of VSE_leaf and VSE_mix extracts. In the analysis, mouse fibroblast cells (L929) were used as healthy cell lines, and human melanoma cells (G361) and human lung carcinoma cells (A549) were used as cancer cell lines. All cells were grown in High Glucose Dulbecco's Modified Eagle Medium (DMEM) medium (1% glutamine) containing 10% fetal bovine serum and 1% penicillin–streptomycin. The grown cells were seeded in 96 well plates with 10⁴ cells per well. Solutions of VSE_leaf and VSE_mix extracts (25–5000 µg/mL range) were applied to cells that reached 60–70% confluency in triplicate. The same volume of medium was added to the control group cells. After application, the cells were incubated for 24 h in a CO₂ incubator (Nüve) to provide a 37 ⁰C, 5% CO₂, and sufficient humidity environment. At the end of the period, 20 µL of MTT solution was added to each well and left for 4 h of incubation. After incubation, the medium in each well was drained and 200 µL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance of the samples in the wells was measured at 540 nm using an ELISA microplate reader. Cell viability in control group absorbances was accepted as 100%, and the effect of each dose on cell viability was calculated [26, 27].

Determination of total phenolic content

VSE_leaf and VSE_mix total phenolic content was determined spectrophotometrically at 765 nm by modifying the Folin-Ciocalteu method [28]. For the assessment of phenolic content, gallic acid served as the control. Standard solutions were prepared at concentrations ranging from 100 to 1000 µg/mL. Following the addition of Folin-Ciocalteu reagent and Na₂CO₃ to the extracts and standards, the reaction mixture was incubated for 2 h. Final absorbance readings were obtained at 765 nm. The total phenolic content levels of VSE_leaf and VSE_mix were calculated using the linear equation obtained from the calibration curve prepared with standard gallic acid solutions. The results were expressed as µg-GAE/mg-extract. Analyses were performed in triplicate.

Determination of total flavonoid content

For the determination of the total flavonoid content of VSE_leaf and VSE_mix, Yaman et al. 2024 method was used [29]. First, VSE_leaf and VSE_mix samples at a concentration of 1 mg/ml were added to the tubes in a volume of 200 μL. 10% Al(NO₃)₃ and 1 M CH₃COOK were added to each species in a volume of 100 μL. The total volume was completed to 5 ml with 99% ethanol. The tubes were closed and incubated for 40 min at room temperature in the dark. At the end of the period, both the samples and standard solutions (quercetin) were read at 417 nm. Using the linear equation obtained from the calibration curve prepared with standard quercetin solutions, VSE_leaf and VSE_mix total flavonoid content levels were calculated as Quercetin Equivalent (QE). The results were expressed as μg-QE/mg-extract. Analyses were performed in triplicate.

Determination of mineral content

The mineral contents of V. splendidum leaves and aboveground parts were determined separately. The microwave combustion method was used to separate organic components and solubilize inorganic components in V. splendidum leaf and aboveground parts (shoot parts) samples. 0.5 g of extract, 3 ml of 35% HNO₃, 1 ml of 30% H₂O₂, and 0.5 ml of 70% HClO₄ were placed in teflon godets. The godets were heated in a microwave oven (Berghof), and the samples were burned. The burned samples were diluted with ultrapure water to a final volume of 10 ml. The concentrations of minor and major elements were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES; Spectro Genesis, Germany) [30].Analyses were performed in triplicate.

Determination of phytochemical profile

Qualitative and quantitative analysis of phytochemical components in VSE_leaf and VSE_mix samples were performed using tandem mass spectrometry (LC–MS/MS system) connected to Shimadzu-Nexera model UHPLC device [28, 31]. A chromatogram was created with a total of 53 phytochemical component standards, including 3 phenolic aldehydes, 17 phenolic acids, 3 non-phenolic acids, 1 benzopyrone, 1 stilbenoid glycoside, 1 biflavonoid, and 27 flavonoid compounds. Validation parameters (Suppl. 1) and instrument conditions (Suppl. 2) were given at Supplementary Materials.

Determination of wound-healing activity

The wound-healing activities of the VSE_leaf and VSE_mix extracts were investigated in the L929 mouse fibroblast cell line using the cell scratch wound-healing method. L929 cells were seeded in 24-well plates at 2 × 10⁴ cells per ml. When the cells were confluent at the well plate well bottoms, a linear scratch was made along the well bottom with a sterile pipette tip to create a wound model. VSE_leaf and VSE_mix were added to the experimental groups at a concentration of 100 µg/mL, while the same volume of medium was added to the wells of the control groups. The cells were incubated in a CO₂ incubator used to provide a 37 ⁰C, 5% CO₂, and sufficient humidity. The images of each well were recorded at 0 h and 24 h. The images were obtained as cell-free area (wound) amounts using imageJ software. Wound closure rates were calculated [32, 33]. Activity measurements were performed in triplicate.

Determination of antimicrobial activity

The antimicrobial activities of the VSE_leaf and VSE_mix samples were determined by disk diffusion method [34]. Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) bacteria and Candida albicans (ATCC 90028) yeast were used as reference strains in the method. One day after the strains were revived, inoculums were prepared in accordance with the 0.5 McFarland turbidity standard (10⁶–10⁷ cfu/mL) and were planted in petri dishes (9 mm diameter) containing Mueller-Hilton agar (Merc). VSE_leaf and VSE_mix were added to the empty disks in the petri dishes at a concentration of 1000 µg/mL in a volume of 10 µL (10 µg), and penicillin (5 IU) disks were used as positive controls. The petri dishes were incubated at 37 °C for 24 h. At the end of the period, the inhibition zones formed around the disks were measured using a caliper in mm units to determine the antimicrobial activity level.

Statistical evaluation

The obtained data are presented as mean ± standard deviation. The SPSS 20 package program was used in the evaluation of the data, and the data of the 2 experimental groups were evaluated with multiple Student T test, and the data of the experimental groups were evaluated with ANOVA to determine the statistical differences between the groups.

Results

Phytochemical profile of extracts

Qualitative and quantitative analyses of various bioactive phytochemicals that participate in activities in metabolism were performed by comparing with 53 standard species using LC–MS/MS. The chromatogram of standards (Fig. 1A), the chromatogram of VSE_leaf sample (Fig. 1B), the chromatogram of VSE_mix sample (Fig. 1C), and the findings of both extracts are reported in Table 1.

Fig. 1.

LC–MS/MS chromatograms of VSE_leaf and VSE_mix extracts. A Standard chromatogram, B VSE_leaf chromatogram, C VSE_mix chromatogram

Table 1.

Phytochemicals detected in VSE_leaf and VSE_mix extracts (mg/g extract)

	Content Type	VSEleaf	VSEmix		Content Type	VSEleaf	VSEmix
1	Quinic acida	0.124	5.831	29	Salicylic acidf	0.027	0.086
2	Fumaric acida	N.D	1.680	30	Cynarosideg	N.D	0.356
3	Aconitic acida	N.D	N.D	31	Miquelianing	N.D	N.D
4	Gallic acidf	N.D	N.D	32	Rutin-D3-IS*	N.A	N.A
5	Epigallocateching	N.D	N.D	33	Ruting	N.D	0.008
6	Protocatechuic acidf	0.249	0.308	34	Isoquercitring	N.D	0.077
7	Cateching	N.D	N.D	35	Hesperiding	N.D	N.D
8	Gentisic acidf	N.D	N.D	36	o-Coumaric acidf	N.D	N.D
9	Chlorogenic acidf	N.D	N.D	37	Genisting	N.D	N.D
10	Protocatechuic aldehydeb	0.111	0.353	38	Rosmarinic acidf	N.D	N.D
11	Tannic acidf	N.D	N.D	39	Ellagic acidf	N.D	N.D
12	Epigallocatechin gallateg	N.D	N.D	40	Cosmosiing	N.D	0.058
13	Cynarinf	N.D	N.D	41	Quercitring	N.D	N.D
14	4-OH Benzoic acidf	N.D	N.D	42	Astragaling	N.D	0.025
15	Epicateching	N.D	N.D	43	Nicotifloring	N.D	N.D
16	Vanilic acidf	N.D	N.D	44	Fiseting	N.D	N.D
17	Caffeic acidf	N.D	0.024	45	Daidzeing	N.D	N.D
18	Syringic acidf	N.D	N.D	46	Quercetin-D3-IS*	N.A	N.A
19	Vanilinb	N.D	N.D	47	Querceting	N.D	N.D
20	Syringic aldehydeb	N.D	0.014	48	Naringening	N.D	N.D
21	Daidzing	N.D	N.D	49	Hespereting	N.D	N.D
22	Epicatechin gallateg	N.D	N.D	50	Luteoling	0.051	0.897
23	Piceidd	N.D	N.D	51	Genisteing	0.004	N.D
24	p-Coumaric acidf	0.042	0.226	52	Kaempferolg	N.D	N.D
25	Ferulic acid-D3-IS*	N.A	N.A	53	Apigening	0.003	0.100
26	Ferulic acidf	0.067	N.D	54	Amentoflavonee	0.006	0.003
27	Sinapic acidf	N.D	N.D	55	Chrysing	N.D	0.002
28	Coumarinc	N.D	N.D	56	Acaceting	0.006	0.022

^*N.D Not detected, N.A Not applicable, IS^* Internal standard

^aNon-phenolic acid

^bPhenolic aldehyde

^cBenzopyrone

^dStilbenoid glycoside

^eBiflavonoid

^fPhenolic acid

^gFlavonoid

Mineral contents in V. splendidum leaves and aboveground parts

The mineral content levels of the leaves and aboveground parts of V. splendidum were determined. Qualitative and quantitative mineral content analysis of the samples prepared using the microwave combustion method were performed using the ICP-OES system for standard 21 elements (1.09487.0100 Merck). The findings of both extracts are presented in Table 2. In V. splendidum leaves; Sb, As, Ca, Fe, Pb, Mg, Mo, Se, and Tl elements were found to be out of the working range, while in V. splendidum above ground parts; Sb, As, Be, Cd, Ca, Co, Pb, Mg, Mo, Se, Tl, and V elements were found to be out of the working range. The elements whose presence was determined qualitatively and quantitatively in V. splendidum leaves from most to least can be listed as; Mn, Sr, Zn, Cu, Ti, Li, Cr, Ni, V, Co, Cd, and Be. The elements determined qualitatively and quantitatively in the aboveground parts of V. splendidum, from most to least, were determined as Fe, Mn, Zn, Cu, Ti, Sr, Cr, Li, and Ni.

Table 2.

Mineral substance levels of V. splendidum leaves and aboveground parts (Mix) (mg/L)

	Be	Cd	Cr	Co	Cu	Fe	Li	Mn	Ni	Sr	Ti	V	Zn
Leaf	0.056	0.268	7.107	1.021	30.965	-	21.359	270.178	5.622	48.466	28.213	2.109	47.270
Mix	-	-	5.010	-	16.839	729.443	3.576	65.013	1.901	11.169	13.998	-	21.739

Total phenolic and total flavonoid contents

Phytochemical profile determination (Fig. 1) showed that there were many phenolic acids, phenolic aldehydes, flavonoids, and biflavonoids in VSE_leaf and VSE_mix extracts. The total phenolic content in the extracts (VSE_leaf; 35.1 ± 1.6 µg-GAE/mg-extract, VSE_mix; 43.1 ± 6.9 µg-GAE/mg-extract) and total flavonoid content (VSE_leaf; 24.4 ± 5.0 µg-QE/mg-extract, VSE_mix; 46.6 ± 9.1 µg-QE/mg-extract) are presented in Fig. 2.

Fig. 2.

Total phenolic and total flavonoid contents of VSE_leaf and VSE_mix extracts. A Total phenolic content, B Total flavonoid content

Evaluation of TAC, TOC, and OSI levels

The TAC, TOC and OSI levels of VSE_leaf and VSE_mix are presented in Fig. 3. When the data were examined, it was determined that VSE_mix TAC levels (0.54 ± 0.02 mmol trolox-equivalent/L) were higher than VSE_leaf TAC levels (0.48 ± 0.01 mmol trolox-equivalent/L).

Fig. 3.

Antioxidant/oxidant effects of VSE_leaf and VSE_mix extracts. A TAC levels, B TOC levels, C OSI levels). a, b, c Data with different letters in the graph for each parameter are statistically different from the others (p < 0.05)

Evaluation of antimicrobial activity

It has been observed that very few studies have been conducted to explain the antimicrobial activity of V. splendidum extracts. In our study, an aqueous extract of V. splendidum leaves and aboveground parts (VSE_mix) was prepared. Results and pictures of disks are shown at Fig. 4.

Fig. 4.

Antimicrobial effect of VSE_leaf (10µL from 1000 µg/mL extract) and VSE_mix extracts (10µL from 1000 µg/mL extract). A Effect of extracts on E. coli, B Activity against S. aureus, C Activity against C. albicans; D Disk of E. coli, E Disk of S. aureus, F Disk of C. albicans). ^*; Statistically different from the antibiotic group (p < 0.05)

Cytotoxic and anticarcinogenic activity

In the present study, the cytotoxicity levels of VSE_leaf and VSE_mix were determined using L929 cells, and the anticarcinogenic activity was determined using the A549 lung cancer cell line and G361 melanoma cells (Fig. 5).

Fig. 5.

Cytotoxicity and anticarcinogenic effects of VSE_leaf and VSE_mix extracts. A Cytotoxic effect on L929 cells, B Effect on A549 lung cancer cells, C Effect on G361 melanoma cells). ^*; Statistically different from the control group (0 µg/mL) (p < 0.05)

Evaluation of the wound-healing effect of VSE_mix and VSE_leaf extracts

Fibroblast cells, one of the connective tissue cells, are the most active cells in wound-healing. For this reason, fibroblasts are frequently used in the in vitro examination of the wound-healing effect of substances. In our presented study, the wound-healing effects of VSE_mix and VSE_leaf extracts was investigated using L929 cells, a fibroblast cell line. To determine the wound-healing effect, the highest non-cytotoxic dose of VSE_mix and VSE_leaf in L929 cells (100 µg/mL) were used. The obtained analysis results are presented in Fig. 6 and Table 3.

Fig. 6.

Effect of VSE_leaf and VSE_mix extracts on wound-healing in L929 cells

Table 3.

Wound-healing efficiency of VSE_leaf and VSE_mix extracts

	Amount of cell-free area (wound) (%)		Closure rate (%)
	Starting point of experiment (the zero hour)	Ending point of experiment (Twenty-fourth hour)
Control	23,39 ± 2,00	3,80 ± 1,37b	84,00 ± 4,32a
VSEmix	24,18 ± 1,99	0,33 ± 0,40a	98,71 ± 1,55c
VSEleaf	23,38 ± 2,37	2,27 ± 0,59b	90,40 ± 1,59b
P	0,871	0,009	0,002

Data are presented as mean ± standard deviation (n ≥ 3)

^a,b,cIndicates statistical difference between data with different superscripts in the same column (p < 0.05)

Discussion

A total of 11 phytochemical standards were detected in the VSE_leaf sample, whereas the VSE_mix sample yielded 18 identified standards (Table 1). In the VSE_leaf extract, protocatechuic acid and ferulic acid stand out, alongside biologically significant phytochemicals such as quinic acid and luteolin. In the VSE_mix extract, however, while quinic acid, fumaric acid, luteolin and protocatechuic acid are the primary components, the presence of bioactive compounds like rutin and chrysin are also noteworthy. In the VSE_mix extract, non-phenolic acids quinic acid (0.58%) and fumaric acid (0.168%), luteolin (approximately 0.09%), which is a flavonoid, are in the foreground. The VSE_mix extract exhibits superior phytochemical diversity and higher concentrations of bioactive species compared to the VSE_leaf extract. Quinic acid [35], fumaric acid [36], luteolin [37], protocatechuic acid [38, 39] have been shown in different studies to have antioxidant and antimicrobial effects. The lower oxidative stress index (Fig. 3C) and superior antimicrobial activity (Fig. 4) of VSE_mix compared to VSE_leaf are significant, particularly in light of the cited literatures. Moreover, a close relationship can be established between the reason for the traditional use of the V. splendidum species by the public and the possible beneficial pharmacological effects of the components contained in the species.

Considering data of mineral contents, we observed that the mineral content in the leaf was higher than the mineral contents of aboveground parts. This situation can be explained by the fact that the metal–ligand chelation products that occur while heavy metals are detoxified are trapped in places such as leaves, leaf stalks, and leaf sheaths and are removed from the plant after natural leaf fall [40]. When the studies on the mineral contents of Verbascum species are examined, bioelements such as Fe, Cu, Mn, Zn, and Mg found in the species positively affect the antioxidant system of the organism and other mechanisms responsible for the regulation of metabolism [28]. In addition, it is important to collect Verbascum species for protection against various diseases or treating diseases, in a pollution-free environment. Studies have shown that metals found in polluted environments can accumulate in Verbascum species. In fact, studies have shown that V. Olympicum, a Verbascum species, is collected from different regions and accumulates heavy metals in different organs/tissues of the species, especially heavy metals, depending on the pollution intensity of the soil. For this reason, it is stated that the species can be considered a bio-indicator for Cu, Fe, Mn, Ni, Pb, Zn, and that it can also contribute to the stabilization of heavy metals in polluted soils due to the accumulation of metals in plants [41]. It is stated that significant structural changes occur in the ultrastructure of cells/organelles in leaves during metal accumulation in Verbascum species growing in polluted soils, thus enabling plants to adapt to technogenic pollution [42]. In light of these information, it can be concluded that heavy metal accumulation was not observed at the mineral levels of V. Splendidum used in our study were examined. The reason for this can be explained by the fact that the habitat where V. Splendidum samples were collected (Afyonkarahisar, the Sultan Mountains, Altitude; 1982 m, GPS: 36 S 0336 282, UTM 4266239) from a region without environmental pollution.

While VSE_mix and VSE_leaf displayed similar total phenolic levels, the slight increase in total flavonoid content in VSE_mix was not found to be significant. These findings show a correlation with the phytochemical content analysis (Table 1). According to the results of total phenolic content, VSE_leaf extract contained 3.5% phenolic content, whereas the VSE_mix extract contained approximately 4.3% phenolic content (Fig. 2). Considering that this amount is in the range of 3–40% in Verbascum species [28, 43–45], it is seen that both extracts have phenolic substance content in parallel with the literature, but at a lower level.

When the obtained data were compared with the antioxidant capacity of Vitamin C used as the positive control (2.58 ± 0.04 mmol trolox-equivalent/L), it was determined that the antioxidant capacities of VSE_leaf and VSE_mix corresponded to approximately one-fifth of the antioxidant capacity of Vitamin C (Fig. 3A). When the TOC levels were examined, it was seen that VSE_mix TOC levels were close to the TOC levels of H₂O₂ (10 µmol/L) used as the positive control and lower than VSE_leaf TOC levels (Fig. 3B). If the determined phytochemical components of the extracts (Fig. 1) are correlated with TAC/TOC levels, the reasons for the differences in TAC, TOC, and OSI levels of the extracts can be explained. The number and amount of bioactive substance components were higher in VSE_mix than in VSE_leaf extract. Bioactive substances found in VSE_mix (Fumaric acid, Syringic aldehyde, Cynaroside, Rutin, Isoquercitrin, Cosmosiin, Astragalin, Chrysin) but not in VSE_leaf may have played a role in the formation of these differences. The antioxidant effects of bioactive substances, such as Rutin [46], Isoquercitrin [47], Chrysin [48], and Astragalin [49], have been discussed in many studies. When studies conducted with aqueous extracts obtained from different Verbascum species are examined, it is reported that Verbascum species may have antioxidant activity due to the effect of antioxidant bioactive components they contain [14, 28, 50].

The inhibition zones of VSE_leaf and VSE_mix extracts were increased compared with the antibiotic group in the S. aureus disk (Fig. 4B and E). It is a positive situation about the antimicrobial activity but there were no statistical differences. In the contrary of this result, inhibition zones of VSE_leaf and VSE_mix extracts were significantly decreased compared with the antibiotic group in the E. coli (Fig. 4A and D). and C. albicans (Fig. 4C and F). disks. In the literature, in a study investigating the antimicrobial activity of V. splendidum flowers and leaves by preparing ethyl acetate and methanol extracts, it was concluded that methanolic extracts showed antimicrobial effects, whereas ethyl acetate extracts had no effect [17]. Considering that methanol is a polar solvent, it is considered possible that aqueous extracts of V. splendidum flowers also exhibit antimicrobial activity. In fact, a study conducted using the methanolic extracts of 10 different Verbascum species growing in Iran, it was stated that Verbascum species may have antimicrobial activity [51].

In the cytotoxicity studies conducted with healthy cells (L929), it was observed that VSE_mix extract was cytotoxic at doses of 250 µg/mL, and VSE_leaf extract was cytotoxic at doses of 500 µg/mL and above. These data indicate that VSE_leaf and VSE_mix are not cytotoxic at low concentrations in healthy L929 cells. In the literature, in a study conducted with eggs using the brine shrimp lethality bioassay method to determine the possible toxicity of the Verbascum splendidum, it was reported that the LD50 doses of the species were above 1000 µg/mL, indicating that its toxicity was low [12]. This information shows that Verbascum L. species can contribute to the treatment of various diseases (such as fungal infections, respiratory tract disorders, hemorrhoids, diarrhea, injuries, inflammatory diseases and skin complications) [10, 11, 43, 52].

When the data were examined, it was seen that VSE_mix cytotoxicity occurred at concentrations above 250 µg/mL in L929 cells, 100 µg/mL in A549 cells, and 5000 µg/mL in G361 cells. It was determined that VSE_leaf cytotoxicity was observed at the lowest concentration (250 µg/mL) in G361 cells and that it was cytotoxic at concentrations above 500 µg/mL in L929 and A549 cells. When the data of L929 cells used as healthy cells and cancer (A549 and G361) cells are compared, it can be said that VSE_mix shows anticarcinogenic properties for A549 cells, whereas VSE_leaf shows anticarcinogenic properties for G361 cells. Because the toxic dose of VSE_mix for lung cancer (A549) cells (100 µg/mL) is not cytotoxic to healthy L929 cells. Similarly, the toxic dose of VSE_leaf to melanoma (G361) cells was not cytotoxic to L929 cells. While VSE_leaf demonstrated SI values > 1, VSE_mix showed the inverse trend (Table 4). The favorable selectivity profile of VSE_leaf, notably regarding G361 cells, indicates its potential suitability for therapeutic use. In our study, the anticarcinogenic effect of aqueous extracts from leaves and aboveground parts of V. splendidum species was investigated for the first time. However, the anticarcinogenic activity of extracts prepared with different solvents of the species was studied using cell lines other than A549 cells [18]. In the relevant study, methanol and ethyl acetate extracts of the species were applied to four different cancer cells (SK-OV-3; ovary carcinoma, SK-MEL; malignant melanoma, BT-549; ductal carcinoma and KB; epidermoid carcinoma). In the study where low concentrations of extracts (10 µg/mL) were used, it was stated that V. splendidum type methanolic extracts did not have an anticarcinogenic effect, while ethyl acetate extracts were only low-level cytotoxic in SK-MEL (3%) and KB (14%) cells [18]. When studies on other Verbascum L. species other than V. splendidum were examined, it was stated that V. nudatum was cytotoxic/anticarcinogenic in L929 cells at 200 µg/mL and in A549 cells at 50 µg/mL and above concentrations [14]. In a study conducted using V. lasianthum acetone and methanol extracts and four different cancer (A549, MCF-7, HepG2 and SHSY-5Y) cell lines, it is reported that anticarcinogenic activity is observed more strongly in A549 and SHSY-5Y cells [28]. In a study conducted using the aqueous extract of flowers of the species V. insulare Boiss. and Heldr., it is stated that anticarcinogenic activity is observed in A549 cells at doses of 50 µg/mL or higher [45]. In a study investigating the effects of V. ponticum ether extract on apoptosis in lung cancer (A549) cells [53], it was concluded that V. ponticum extract could induce apoptosis independently of caspase-3 by causing mitochondrial dysfunction and nuclear translocalization even at low concentrations (IC50: 50.14 µg/mL). In another study, studies conducted with V. thapsus, which is preferred to be used in many diseases, especially colds and respiratory tract diseases, in tea, drugs, and similar forms and therefore also produced agriculturally, indicated that the species has antiproliferative effects on cancer cells. Considering this information, it was evaluated as a possible situation that VSE_mix has anticarcinogenic activity for A549 cells and VSE_leaf for G361 cells in our study. Also the high level of selectivity, especially against G361 cells, VSE_leaf appears more promising for future therapeutic strategies.

Table 4.

Selectivity indexes (SI) of cancerous cell lines

	SI (A549)	SI (G361)
VSEmix	0.29	0.07
VSEleaf	2.86	15.67

Selectivity indexes (SI) were calculated using the following formula: SI = IC₅₀ of L929/IC₅₀ of cancer line. SI ≥ 1.0 indicates the extract with efficacy against cancerous cell line greater than the toxicity towards normal cell line. SI < 1 indicates nonselective action [54]

The fact that the cell-free areas were close to each other (between 23.38%−24.18%) in the wound models created for each experimental group at the beginning of the study (Table 3) indicates that the in vitro cell scratch wound model was successfully created. It has been shown in different studies [21, 55, 56] that species of Verbascum L. may have wound-healing activity. In one of these studies, the effects of the methanolic extracts of 13 different species of Verbascum L. were investigated in wound models created using experimental animals. It is stated that Verbascum splendidum, Verbascum olympicum, Verbascum stachydifolium, Verbascum uschackense, Verbascum latisepalum, Verbascum mucronatum, and Verbascum pterocalycinum var. mutense may have a good wound-healing effect. It is reported that the wound-healing activity of Verbascum splendidum methanolic extracts may be low (Süntar et al. 2010). In our study, it was determined that Verbascum splendidum extracts, especially VSE_mix, had better wound-healing effect compared to the control group (Table 3, Fig. 6). There is a difference between the results of the study conducted by Süntar et al. (2010) (Verbascum splendidum extracts having low wound-healing effect) and the results we obtained. The reason for this situation may be related to the different experimental models, solvents, and plant parts used in the extraction. Unlike the study conducted by Süntar et al. (2010), leaf and aboveground parts of the Verbascum splendidum species were preferred and water was used as the solvent in our study. In addition, the in vitro cell scratch wound model was preferred as the wound model. In another study, it was reported that the wound-healing effect of a different Verbascum L. (V. Speciosum) species may be in the L929 cell wound model. It is stated that the related Verbascum L. species may be a potential therapeutic agent for skin wound-healing by promoting fibroblast proliferation and angiogenesis in cells [57]. This situation is similar to the results of our study. It is also stated that Verbascum splendidum may have an effect by stimulating re-epithelialization, fibroblast cell proliferation, and collagen formation in the wound area [21].

Conclusion

Plants are frequently used in traditional treatments for diseases in many societies. In this context, many Verbascum L. species are also used, and especially Verbascum thapsus is cultivated due to its commercial value. The possible effects of species traditionally used in different societies need to be investigated, and it is important to bring their results to the literature as scientific information. The data obtained from study indicate that V. Splendidum aqueous extracts may be useful in disease prevention and treatment due to their rich phytochemical content and biological (antioxidant, antimicrobial, anticarcinogenic, wound healing activity, etc.) properties. In addition, the high Quinic Acid content (5.831 mg/g extract) of VSE_mix aqueous extract suggests that this plant can be used as a natural Quinic Acid source. First, isolation techniques should be optimized. Considering the properties and pharmacological effects of the V. splendidum species expressed both in the literature and in our presented study, it is recommended that necessary cultivation studies be conducted to bring the species into the economy, as in the Verbascum thapsus plant.

Authors’ contributions

The author declared that i have no conflicts of interest to this work.

Funding

This study was supported by Afyon Kocatepe University, Faculty of Arts and Science, Department of Chemistry and Afyon Kocatepe University, Çay Vocational School, Department of Chemical Technologies.

Data availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.