The screening of Digitalis ferruginea L. subsp. ferruginea for toxic capacities, phenolic constituents, antioxidant properties, mineral elements and proximate analysis

The present study outlines the antioxidant and toxic activity, total phenol, flavonoid and tannin content of Digitalis ferruginea ’s extracts in addition to evaluating its proximate parameters and mineral elements. Successive extraction was made using different solvents (ethanol, acetone, water). Total phenol content was determined by the Folin-Ciocalteu’s reagent method and flavonoid was determined by the aluminum chloride colorimetric method. The antioxidant activities were investigated using different assays. The phenolics were determined using HPLC. In addition an evaluation was made of the proximate parameters and mineral elements. The radical scavenging capacities were highest in the water extract. The same extract was effective in total antioxidant activities (β-carotene, 83.75%). The acetone extract demonstrated stronger reducing power and phosphomolybdenum antioxidant activity with 0.52 mg/mL and, 107.43 µg/mg, respectively. The HPLC results determined major phenolics: rutin and ferulic acid. This plant also have rich in polyphenolic content together with toxic activity and possesses nutrients and mineral elements. As well as demonstrating the good antioxidant activity of D. ferruginea this study suggest that the plant could be of particular interest from a practical perspective, as it is a significant source of potential natural antioxidants that can be used for the prevention of a range of diseases.


Introduction
It is widely acknowledged that reactive oxygen species are closely associated with a range of human diseases, including cancer, inflammation and aging. It is also generally accepted that natural antioxidants could be used as opposed to synthetic compounds to inhibit the growth and development of these reactive species-related disorders in human beings. It is due to their apparent safety and therapeutic value that the natural antioxidants present in plants continue to attract extensive interest. Furthermore, this increased interest in natural antioxidants has in turn resulted in the evaluation of antioxidant presence and levels in many vegetable, fruit, herb, spice and cereal species. As a result, many researchers are looking for natural antioxidants that are potent but safe, in particular those sourced from medicinal plants (Elmastaş et al., 2006;Moein et al., 2008;Rufino et al., 2010;Santos & Gonçalves, 2016).
The genus Digitalis belongs to the Plantaginaceae family, which comprises aproximately 36 species and is distributed through Europe, the Mediterranean regions and Western Asia (Davis, 1978). There are various species among the genus Digitalis that have been shown to have different pharmacological actions, including, antioxidant and antimicrobial activities (Benli et al., 2009;Tusevski et al., 2014;Katanić et al., 2017). In addition, some members of this genus are used in traditional Turkish medicine as diuretics and tonics (Benli et al., 2009); however, there is still not much known about the antioxidant and toxic activities, phenolic compounds, mineral elements and proximate analysis of Digitalis species. Digitalis ferruginea L. subsp. ferruginea is one of the plant species belonging to the genus Digitalis. In previous research, methanol extract from D. ferruginea L. subsp. ferruginea has been investigated for phenolic compounds, antioxidant, antimicrobial and enzyme inhibitory activities (Katanić et al., 2017). Nevertheless, it is well known note that even within a single method a small difference in solvent polarity may provide different responses, due to the diversity of the chemical nature of these compounds and their often unique distribution within the plant matrix (Antolovich et al., 2000;Turkmen et al., 2006;Sultana et al., 2009). For this reason, the antioxidant efficacy of the resulting extracts is strongly affected, as mentioned above, by a polarity of the solvent and the chemical nature of the isolated compounds (Sultana et al., 2009;Shabir et al., 2011). This varies, depending on the type of solvent used and in addition polarity may modify the single electron transfer and the hydrogen atom transfer, both of which are key with regard to the measurement of antioxidant capacity (Pérez-Jiménez & Saura-Calixto, 2006). Thus, a significant contribution could be made to medicinal plant studies through the discrete analysis of plant extracts acquired from a variety of solvents (Canadanovic-Brunet et al., 2006;Stankovic et al., 2011). In the current study, we therefore investigated antioxidant properties using different methods, toxic effects and total bioactive compounds of the ethanol, acetone and water extracts of D. ferruginea L. subsp. ferruginea and to evaluate this plant's mineral elements and proximate analysis. In addition we analyzed the phenolic compound of ethanolic extract of D. ferruginea L. subsp. ferruginea, for a better characterization and exploitation of this natural product.

Plant material and preparation of the extracts
The aerial parts of Digitalis ferruginea L. subsp. ferruginea were collected at the flowering stage, from Denizli, Turkey, in August 2018. The plant material was identified and stored with voucher specimens (D. ferruginea L. subsp. ferruginea;Herbarium No: 2018-5-2) at the private herbarium of Dr. M. Çiçek. To obtain the extracts, the sample (10 g) was added to 100 mL of solvents (ethanol, acetone and water) and shaken at 50 °C for 6 h in a temperature controlled shaker. The extracts were filtered twice and solvents from the samples were removed using a rotary evaporator. The samples were dried in a lyophilizer and kept at -20 °C until tested. Each experiment was conducted in triplicate.

Antioxidant activity
The Phosphomolybdenum and the β-carotene/linoleic acid bleaching methods were used to ascertain the antioxidant properties. The antioxidant capacity with phosphomolybdenum method was reported as ascorbic acid equivalents. β-carotene/ linoleic acid bleaching assay was used to BHT and TROLOX as standards. These methods were described in our published paper (Kaska et al., 2018). The radical scavenging (DPPH and ABTS) and metal chelating activities were evaluated by the method described by Kaska et al., 2018, and these assays were used to EDTA, BHT and TROLOX as standards. The ferric ion reducing antioxidant power procedure followed was those given by Kaska & Mammadov (2019a), and results were expressed as mg of ascorbic acid equivalents (AA) per milliliter of extract.

Quantification of phenolic compounds by HPLC
The phenolic compound were evaluated using Reversed-phase high performance liquid chromatography (RP-HPLC, Shimadzu Scientific Instruments). The phenolic composition of the ethanolic extract of D. ferruginea L. subsp. ferruginea was determined using previously described method Caponio et al. (1999). The details of this method were given in Kaska & Mammadov (2019a). Gallic, 3,4 dihydroxybenzoic, 4-hidroxybenzoic, 2,5 dihydroxybenzoic, chlorogenic, vanillic, caffeic, p-coumaric, ferulic, cinnamic acid and quercetin, epicatechin, rutin were used as standards. The quantitative analysis were made by comparing the standarts. The results were expressed as µg/g of each compound from the total phenolic compounds.

Total bioactive compounds
Folin-Ciocalteu and aluminum chloride colorimetric assays were used to determine the total phenolic and flavonoid contents, respectively (Slinkard & Singleton, 1977;Arvouet-Grand et al., 1994). To evaluate the tannin content was performed according to Broadhurst & Jones (1978) using the vanillin-HCl method. The results were expressed as the equivalents of Gallic acid (mgGAE g -1 ), Quercetin (mgQEs g -1 ) and Catechin (mgCEs g -1 ) for phenolic, flavonoid and tannin content respectively. The procedure for determining the bioactive compounds was conducted according to Kaska et al. (2018).

Proximate composition
The D. ferruginea L. subsp. ferruginea was analyzed to investigate the proximate parameters (proteins, fat, carbohydrates, ash and energy) according to the Association of Official Analytical Chemists (1995) protocols. The sample preparation and procedure for determining the proximate parameters were followed according to Kaska & Mammadov (2019b).

Mineral elements
The mineral elements (potassium, magnesium, Phosphorus, Iron and Copper) were evaluated using ICP-OES method (optical emission spectrometer with the inductively coupled plasma). The details of this method were given in Kaska et al. (2019). The samples of the studied herbs were mineralized in nitric acid 65% HNO 3 . The standard solution were prepared by diluting (P, Mg, Fe, K, Cu) (Merc) standard solutions at the concentrations of 1000 ppm (mg/L). For the preparation of standard solution deionized water was used. In order to determine the calibration curve, standard solutions at the range of concentrations from (0.005-1 ppm) were used. The study used the analytical lines of the highest intensity. An analysis of samples for the presence of elements was carried out using an optical emission spectrometer with the inductively coupled plasma.

Brine shrimp lethality assay
The brine shrimp lethality bioassay was used to investigate the toxicity of D. ferruginea L. subsp. ferruginea (Meyer et al., 1982). The details of determining the toxicity were given in our previous study (Kaska & Mammadov, 2019a). The EPA Probit Analysis Program was used for data analysis.

Statistical analysis
The experimental results were analyzed using the MINITAB Statistical Package program and the results expressed as mean ± SE (Standard Error). The differentiations between the extracted groups were tested using ANOVA (Analysis of Variance) and then Tukey was conducted (p <0 .05).

Results and discussion
The antioxidant capacity of plants is derived from the synergistic action of a wide variety of antioxidants and one single assay cannot fully describe the antioxidant activity. To determine the antioxidant capacity of plants in vitro, it is normally required to integrate several methods (Pérez-Jiménez & Saura-Calixto, 2006;Santos & Gonçalves.,2016). In the present study, various assays were conducted to analyze antioxidant activities of the extracts.

Antioxidant capacity
The phosphomolybdenum assay is contingent on the extract's reduction of Mo (VI) to Mo (V) and the development of green phosphate/Mo(V) complex. The reducing capacities are usually associated with the occurrence of reductones (Prieto et al., 1999). The findings for the antioxidant evaluation given in Table 1 indicate that ethanol, acetone and water extracts from D. ferruginea L. subsp. ferruginea possess antioxidant capacities.
In this study, according to the results of the inhibition of linoleic acid oxidation for ethanol, acetone and water extracts, the ethanol extraction of aerial parts displays lower antioxidant values (75.73 ± 1.54%), than water extraction (83.75 ± 0.88%) (Figure 1).
Ethanol and water extracts showed significant differences (p < 0.001) when compared with the antioxidant activity value of BHT and TROLOX, which were 93.71 ± 0.23% and 94.81 ± 1.15%, respectively. Among the extracts investigated, acetone extract exhibited the lowest antioxidant activity (74.49 ± 0.96%) and its activity was to be significantly different from the BHT and TROLOX antioxidant activities (F 4,35 = 85.10 p< 0.001). The extracts studied in this study were found to effectively inhibit linoleic acid oxidation. These findings demonstrate that they possess strong antioxidant capacities. When the ethanol, water and acetone extracts of D. ferruginea L. subsp. ferruginea were compared with the methanol extract from D. ferruginea L. subsp. ferruginea (Katanić et al., 2017) of which the total antioxidant activity was found to be 78.59%, the antioxidant activity of the ethanol and acetone extracts were lower while the water extract's antioxidant activity was higher than those of the methanol extract of this species.
Radical scavenging activities are essential because of the detrimental role of free radicals in foodstuffs and biological systems. ABTS and/or DPPH scavenging assays are common spectrophotometric methods for analyzing the radical scavenging activities (Gülçin et al., 2010;Olszowy & Dawidowicz, 2018). The DPPH assay offers an effective, quick and simple way to assess potential antioxidants. This method is an antioxidant assay that uses an electron transfer and in combination with ethanol produces a violet solution. Likewise, stable at room temperature, this free radical is reduced in the presence of an antioxidant molecule, resulting in a yellow solution (Vlase et al., 2014).
In the present study, the radical scavenging activity of different extracts of D. ferruginea L. subsp. ferruginea is expressed in terms of IC 50 (mg/mL) values (Table 1). A reduction in the IC 50 value signifies a higher level of antioxidant activity. In tandem with the analysis of antioxidant activities of the plant extracts, the values for two standard compounds were attained and equated with those of the antioxidant activity. The DPPH free radical scavenging activity of the water, ethanol and acetone extracts were significantly different from each other and IC 50 values of all extracts were found to be different from BHT and TROLOX IC 50 values (F 4,35 = 1831.22 p < 0.001). Katanić et al., (2017) used DPPH for the determination of radical scavenging activity in methanol extracts from D. ferruginea L. subsp. ferruginea. In the current investigation, The DPPH assay was used to examine scavenging activity in ethanol, water and acetone extracts from D. ferruginea L. subsp. ferruginea and found that the extracts of D. ferruginea L. subsp. ferruginea have radical scavenging activity.
The ABTS assay, used for analyzing hydroxybenzoic and hydroxycinnamic acids, which, were due to their hydrogen and electron donating capacity, together with their ability to stabilize the ensuing phenoxyl radical within the structure, capitalizes on the strong likelihood of their acting as radical scavengers (Silva et al., 2001;Biskup et al., 2013). In the ABTS scavenging assay, the IC 50 values of standards and different extracts are given in Table 1. The acetone, ethanol and water extracts tested in our study demonstrated a scavenging capacity. These samples were significantly different from each other and the IC 50 values of the all extracts were found to be statistically different from the BHT and TROLOX IC 50 values (F 4,35 = 543.72 p < 0.001). The statistical differences were given as different letters (p < 0.05). nt: not tested; DPPH: 2,2-Diphenyl-1-picryl hydrazyl radical; ABTS: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid. According to the findings, the water extract showed remarkable radical scavenging antioxidant activity with 0.415 mg/mL.
Free radicals are electrically charged, unstable molecules that are formed as a result of the metabolic processes of cells or by external factors such as air and it is possible for them to attack the healthy cells of the body, resulting in them losing their structure and function, and the cell damage caused by free radicals appears to be a major contribution to degenerative diseases of aging, such as cardiovascular disease, heart disease, diabetes, and liver diseases (Mohan et al., 2012;Phaniendra et al., 2015). Likewise, free radicals involved in the lipid peroxidation process are thought to play a role in various diseases, such as cancer (Dorman et al., 2003). For this reason, researchers have nowadays become increasingly interested in natural resources that could produce active components to prevent free radicals negatively impacting on cells (Tepe et al., 2007). The acetone, ethanol and water extracts of D. ferruginea L. subsp. ferruginea tested in the present study each showed a radical scavenging capacity.
In our study, D. ferruginea L. subsp. ferruginea extracts were analyzed to determine their Fe 2+ chelating activity. Then the results were contrasted with the chelating activity of the synthetic metal chelator EDTA (88.27 ± 1.18%). The water extract chelated more iron (46.13 ± 0.87%) than ethanol and acetone extracts with chelating values of 27.85 ± 1.86 and 38.47 ± 1.17%, respectively (Figure 2).
The chelating capacity of metal ion is noteworthy, as in lipid peroxidation it can contribute to the reduction of the concentration of the catalyzing transition metal. Chelating agents, which can form bonds with a metal, are moreover as effective as secondary antioxidants, as they can reduce the redox potential and thus stabilize the oxidized form of the metal ion (Elmastaş et al., 2006;Al-Dabbas, 2017). In the current study, D. ferruginea L. subsp. ferruginea exhibited a clear propensity for iron binding. This indicates their capacity as a peroxidation protector, which in turn correlates with the iron binding capacity.
The reducing power assay method uses the principle that a substance with reduction potential, will react with potassium ferricyanide (Fe 3+ ) to form potassium ferrocyanide (Fe 2+ ). This then reacts with ferric chloride, forming a ferric-ferrous complex, which has an absorption maximum at 700 nm. In additional, the reducing power assay is a convenient and rapid screening method for measuring the antioxidant potential (Talukder et al., 2013). In the present study, we investigated ethanol, water and acetone extracts from D. ferruginea L. subsp. ferruginea for reducing capacity and the acetone extract demonstrated stronger reducing power activity with 0.52 mg/mL followed by ethanol and water extracts with 0.51 and 0.44 mg/mL, respectively. The reducing capability of a compound generally depends on the presence of reductants that have been exhibiting antioxidative potential by breaking the free radical chain and donating a hydrogen atom (Duh et al., 1999;Babu et al., 2013). The presence of reductants in D. ferruginea L. subsp. ferruginea extracts causes the reduction of the Fe 3+/ ferricynide complex to the ferrous form.

Total phenolic, flavonoid, tannin content and phenolic composition
Phenolic compounds are found in plants, and they have been reported to have various biological properties, including antioxidant capacity. For this reason, the richness of the phenolic compounds in plants indicate their pharmacological properties; and analyses of these phenolic compounds are crucial for understanding their medicinal value. Besides this, the food industry is showing increasing interest in plants that are rich in phenolics. This is because they delay the oxidative degradation of lipids and in so doing improve the nutritional value and quality of food (Marimuthu et al., 2008;Tungmunnithum et al., 2018). In brief, the identification and measurement of plants' phenolic compounds are nowadays considered to be effective mechanisms for ascertaining the importance of plants for human health (Amarowicz et al., 2010). The content of the bioactive compound in the different D. ferruginea L. subsp. ferruginea extracts are existed in Table 2.
The experimental results showed that the highest contents of total phenolic was in the ethanolic extract of D. ferruginea L. subsp. ferruginea was highest. The phenolic content for the water, ethanol and acetone extracts analyzed in current study were higher than that determined by Katanić et al. (2017) (methanol extract of D. ferruginea L. subsp. ferruginea). The total flavonoid content in the extracts ranged from 10.45 to 51.73 mgQEs/g and there were statistical differences (F 2,21 =4527.11 p < 0.001) in the total flavonoid contents of the various extracts of D. ferruginea L. subsp. ferruginea. In this study, total phenolic and flavonoid content of the extracts varied in accordance with the solvent. Similar to these results, the studies of Khazai et al. (2011) and Kaska et al. (2019) found that the total phenolic and flavonoid content differed according to the solvents used. As Table 2 shows, tannin content was highest in the acetone extract (94.49 mgCEs/g), and lowest in the water extract (29.99 mgCEs/g). According to the findings in current study, the bioactive compounds were found to be considerable in each of the extracts we investigated.
The results for phenolic compositions of the ethanolic extract of D. ferruginea L. subsp. ferruginea by HPLC analysis are presented in Table 3, and major phenolics were determined: rutin, ferulic, quercetin and gallic acid. Rutin is a common dietary flavonoid that has been shown to has powerful antioxidant capacity (Guo et al., 2007;Yang et al., 2008). Quercetin, a plant-derived flavonoid, has been used in traditional medicine to prevent or treat a variety of diseases, such as cancer (Neuhouser, 2004;Murakami et al., 2008), cardiovascular and nervous disorders (Shankar et al., 2007;Labinskyy et al., 2006). Ferulic acid belongs to the phenolic acid group commonly found in plant tissues (Mattila & Kumpulainen, 2002) and it possess many physiological functions, such as anti-inflammatory, antithrombotic, antidiabetic effects and it also reduces nerve cell damage. It has also been widely used in the pharmaceutical, food, and cosmetics industry (Tee-ngam et al., 2013;Cota-Arriola et al., 2017;Zduńska et al., 2018). Gallic acid is a naturally occurring polyphenol antioxidant that has recently been shown to have potentially healthy effects (Lu et al., 2006). According to these studies, phenolic compounds isolated from the plant possess a wide range of bioactivities and for this reason, phenolic compounds may be used as an important indicator of the biological activity of plants.

Proximate composition
The proximate composition of a plants provides valuable information regarding its medicinal and nutritional quality. Proteins and carbohydrates are important nutrients in plants that should be assessed. Fat provides a very good sources of energy and aids in the transportation of fat soluble vitamins, as well as insulating and protecting the internal tissues (Hussain et al., 2013;Ghani et al., 2016). The results of proximate composition of D. ferruginea L. subsp. ferruginea are presented in Table 4 and the proximate composition of the studied plant in our study is in agreement with previously data reported in the literature. Proximate analysis determinants by Ghani et al., 2016 andBukhsh et al., 2007, have revealed that the Plantago ovata from Plantaginaceae family contained carbohydrate (30.33, 15.9%), and protein (7.12, 21.87%) content.
In addition, our results are in accordance with the results of previous studies in which the ash content ranged from 2.67% to 17.22% in certain plants belonging to Plantaginaceae family (Guil-Guerrero, 2010;Ghani et al., 2016;Ogbiko et al., 2017). The search for pharmacologically active chemicals from plant sources is continuing and today many compounds have been isolated and introduced into clinical medicine (Bukhsh et al., 2007). The present study was performed to further increase knowledge about the proximate composition of Plantaginaceae and its focus was the investigation of proximate composition D. ferruginea L. subsp. ferruginea.

Mineral elements
The ICP-OES method was used for the determining the content of P, Mg, K, Fe and Cu in the tested plant. The findings are presented in Table 5.
Mineral elements play important roles in biological, metabolic and enzymatic reactions of living organisms (Mishra et al., 2012;Ghani et al., 2016). Copper and Magnesium are essential elements for living organisms in terms of their presence in important enzymes (Velasco-Reynold et al., 2008;Tomescu et al., 2015). Iron (Fe) is a important component of heme proteins, hemoglobin, and myoglobin (Fraga, 2005). Phosphorus is essential component of bone mineral. Deficiency of phosphorus-calcium balance result in osteoporosis, rickets and tooth decay (Asaolu et al., 2012). Plant's minerals are great importance to understand pharmaceutical    actions of plants, therefore, it is important to analyze the mineral contents of plants before recommending them for medicinal uses. According to the findings of present study, D. ferruginea L. subsp. ferruginea could be a good source of minerals.

Brine shrimp lethality assay
The brine shrimp lethality assay is a straightforward, high output toxicity test for bioactive chemicals. It is determined by the killing ability of the test compounds of Artemia salina, the brine shrimp. This very basic form of zoological organism is widely used in these investigations because of its commercial availability (Nguta et al., 2012;Ahmed et al., 2017). This assay is considered to be a useful tool for the preliminary assessment of general toxicity and for estimating the medium lethality concentration LC 50 (Meyer et al., 1982). In this study, an experiment for toxic activity was carried out on the ethanol, acetone and water extracts of D. ferruginea L. subsp. ferruginea collected from Turkey and the toxicity was reported in terms of 50% lethal concentration (LC 50 ). According to our results, the lethality of the ethanol, acetone and water extracts from D. ferruginea L. subsp. ferruginea were 407.791, 266.954 and 137.069 μg/mL, respectively. The LC 50 values of extracts were found to be lower than 1000 μg/mL, and considered significantly active. This significant lethality of the extracts could be the source of potentially toxic components in these species but further investigation is required to find and isolate the chemical compounds that contain these toxic properties. This will in turn enhance our understanding of the plant, its potential and its medicinal uses.

Conclusion
Nowadays, demand for the scientific evaluations of the antioxidant properties of plant extracts is intensifying. This rise is due to their being for the most part harmless sources from which to obtain natural antioxidants and in recent years, antioxidant products made from natural sources have been under the spotlight. The results presented here indicate that extracts of D. ferruginea L. subsp. ferruginea have promising biological effects. The tested extracts have shown good antioxidant and toxic activity. They are also rich in phenolic, flavonoid and tannin content. In addition, this plant possess varied polyphenolic compounds with utility properties as well as mineral elements and proximate composition. Our results also highlight the biological potential of this plant and in addition we believe that they will encourage further studies to be carried out on the isolation and characterization of these bioactive compounds to determine the mechanism of action. Finally, the outcomes of this study could provide significant information regarding D. ferruginea's potential use in the pharmaceutical industry.