Antennaria dioica (Asteraceae family) – is a perennial herb, commonly found in dry grasslands and sandy or stony places from Eurasian areas. It is known in traditional medicine as antioxidant, diuretic, choleretic and anti-inflammatory remedy. This species should be reconsidered as possible sources of phenols, mainly flavonoids and hydroxycinnamic acids. Thus, the aim of this study was to validate a chromatographic method for detection of phenols and their identification in A. dioica herb. HPLC-DAD method was evaluated in terms of linearity, precision, repeatability, accuracy, LOD and LOQ. The calibration curves of thirteen reference substances were linear (R² > 0.99) over the range of 5–400 µg/mL, the LODs and the LOQs were in the range of 0.1–0.3 µg/mL and 0.2–1.0 µg/mL, respectively. During HPLC-DAD assay two flavones – luteolin, apigenin; flavonol – quercetin and three its glycosides – rutin, hyperoside and isoquercitrin; coumarins: coumarin and umbelliferone; five hydroxycinnamic acids – chlorogenic, caffeic, p-coumaric, trans-ferulic and rosmarinic were identified in A. dioica herb. This phytochemical study of A. dioica confirms that this plant material is a rich source of phenolic compounds.

Antennaria dioica, Gaertn, phenolic compounds, flavonoids, hydroxycinnamic acids, HPLC-DAD

Asteraceae family, which includes more than 1600 genera, with over 23,000 species, widespread in different types of regions all over the world and is the largest family of flowering plants (Babotă et al. 2018; Rolnik and Olas 2021). Several classes of compounds from Asteraceae species were studied and tested for different bio-activities and were reported as having medicinal potential (Dudova 2018; Rosche et al. 2018). Among these compounds, a special attention has been given to phenols, and especially to flavonoids and hydroxycinnamic acids, which provide for these species important uses in the pharmaceutical, cosmetics and food industry, that are due to their important medicinal properties as the antioxidant, anti-inflammatory, antifungal or antibacterial ones (Bumrungpert et al. 2018; Dabeek and Marra 2019; Savych 2021; Savych and Mazur 2021; Savych and Milian 2021). In this context and taking into consideration the fact that in the last decades these compounds have shown a significant importance in the field of medicinal compounds (Savych and Marchyshyn 2021a, 2021b; Savych et al. 2021f, 2021d), the Asteraceae species should be reconsidered as possible sources of phenols.

Antennaria dioica (L.) Gaertn. (A. dioica, Stoloniferous Pussytoes) is the plant belonging to the same tribe (Gnaphalieae) of the Asteraceae family. It is a perennial herb, commonly found in dry grasslands and sandy or stony places from Eurasian areas. It is known in the traditional medicine for its use in cases of biliary and respiratory tract diseases (Dudova 2018). The folk medicine of different countries cites common uses for preparations obtained from A. dioica herb, exploited for their antioxidant, diuretic, choleretic and anti-inflammatory properties (Babotă et al. 2018; Rolnik and Olas 2021). Today, there is not enough information about the phytochemical composition of this species, so the study of the profile of biologically active substances is relevant.

In addition, it is important for medicine and pharmacy to study new promising plant species, as they can be a source of new drugs that can have a numerous of advantages over synthetic agents, namely, they are low-toxic (Savych and Mala 2021), have a mild pharmacological effect and possibility to be used for long periods of time without significant side effects, have a complex activity through a numerous of biologically active compounds (Savych and Nakonechna 2021; Savych et al. 2021b, 2021c, 2021i, 2022).

Thus, the aim of this study was to validate the chromatographic method for detection of phenols and their identification in Antennaria dioica herb.

The chromatographic method was validated by evaluating linearity range, precision, repeatability, accuracy, LOD and LOQ. The linearity of the method was evaluated by studying its ability to obtain an analyte response linearly proportional to its concentration in a given range. To determine that parameter, calibration curves were generated by injection in triplicate of standard solutions at six concentration levels and their correlation coefficients were calculated. As can be seen in Table 3, the linearity of HPLC-DAD method was good, since R² were in the range of 0.997–0.999.

The precision of the method was evaluated by injecting three times the same sample spiked with three levels of concentration (covering the specific range for each compound) during three consequent days. Repeatability was calculated by analysing three times the same sample. Both parameters were evaluated by RSDs that were in the range of 0.38% – 2.79% for inter-day precision and were from 0.39% to 1.92 for intra-day precision (Table 3).

The accuracy of HPLC-DAD method was evaluated by the recovery test. In this way, three samples, previously analyzed, were spiked at three concentration levels of the target compounds and were injected by triplicate. The recoveries of all compounds ranged between 97.12% and 106.10% (Table 3).

HPLC-DAD method allowed the detection of phenols in the range of 0.1–0.3 µg/mL; the quantification in the range of 0.2–1.0 µg/mL, as it is shown in Table 3.

The results of identification and quantification of phenols in A. dioica herb are represented in Table 4. During HPLC-DAD assay two flavones – luteolin, apigenin; a flavonol – quercetin and three its glycosides – rutin, hyperoside and isoquercitrin; coumarin and umbelliferone – hydroxycoumarin; five hydroxycinnamic acids – chlorogenic, caffeic, p-coumaric, trans-ferulic and rosmarinic acid were identified (Figs 1–3).

The quantitative determination showed that the main hydroxycinnamic acids were rosmarinic (944.1±0.22 μg/g) and chlorogenic (793.5±0.19 μg/g), regarding the flavonoids, isoquercitrin (164.5±0.17 μg/g) and luteolin (126.4±0.18 μg/g) are prevailed in A. dioica herb (Table 4).

Flavonoids that were detected during HPLC-DAD analysis have powerful antioxidant activities, which are manifested due to their chemical structure, which provides the cleavage of hydrogen atoms (Shashank and Abhay 2013; Panche et al. 2016). In addition, they increase the production of glutathione (GSH) and antioxidant enzymes – superoxide dismutase (SOD) and catalase (CAT), as well as inhibit xanthine oxidase, which is involved in the generation of ROS (Enogieru et al. 2018; Xu et al. 2019; Savych and Polonets 2021; Savych and Sinichenko 2021).

Flavonoids exhibit a numerous of pharmacological effects, such as antioxidant, antihyperglycemic, antidiabetic, anti-inflammatory, cardiovascular, neuroprotective, hepatoprotective, antiallergic, antiosteoporotic, anticancer, antiplatelet and vasodilatory properties (Kawser et al. 2016; Savych et al. 2021a).

Figure 1. 

HPLC-DAD chromatogram of phenols identified in A. dioica herb, λ = 320 nm: 1 – chlorogenic acid; 2 – apigenin; 3 – rosmarinic acid.

Phenylpropanoic acids have potential antioxidant properties, which are realized by cleavage of hydrogen atoms, which reduces the number of free radicals, lipid peroxidation products and inhibits an oxidative stress (Alam et al. 2016; Adisakwattana 2017; Stähli et al. 2019). In addition, they can regulate lipid metabolism by lowering triglycerides, low-density lipoproteins and cholesterol that is signified to prevent the development of cardiovascular diseases (Salazar-López et al. 2017).

Figure 2. 

HPLC-DAD chromatogram of phenols identified in A. dioica herb, λ = 330 nm: 4 – caffeic acid; 5 – p-coumaric acid; 6 – trans-ferulic acid; 7 – 7-hydroxycoumarin (umbelliferone).

Phenols exhibit anti-inflammatory properties, which is manifested by a decrease in edema, effective suppression of proinflammatory cytokines and reduced neutrophil infiltration (Tsang et al. 2016). Phenolic substances are very important agents for prevention and treatment of diabetes mullites because they have a hypoglycemic effect, which is manifested by various mechanisms – inhibition of α-glucosidase, stimulation of insulin secretion, improving the functioning of pancreas β-cells, increased glucose utilization; antioxidant effect due to its ability to prevent protein glycation and lipid peroxidation of the membrane, neutralizing the formed free radicals (Santana-Gálvez et al. 2017; Kang et al. 2019; Savych and Basaraba 2021; Savych et al. 2021h).

Figure 3. 

HPLC-DAD chromatogram of phenols identified in A. dioica herb, λ = 255 nm: 1 – coumarin; 2 – quercetin; 3 – luteolin; 4 – quercetin-3-galactoside (hyperoside); 5 – quercetin-3-rutinoside (rutin); 6 – quercetin-3-glucoside (isoquercitrin).

This phytochemical study of A. dioica confirms that this plant material is a rich source of phenolic compounds.

The method was validated in terms of linearity, precision, repeatability, accuracy, LOD and LOQ. HPLC-DAD assay of phenols revealed that A. dioica represent important sources of bioactive compounds with a wide range of pharmacological activities. It was identified two flavones – luteolin, apigenin; flavonol – quercetin and three its glycosides – rutin, hyperoside and isoquercitrin; coumarin and umbelliferone – hydroxycoumarin; five hydroxycinnamic acids – chlorogenic, caffeic, p-coumaric, trans-ferulic and rosmarinic acid in A. dioica herb. The quantitative detection showed that the main hydroxycinnamic acids were rosmarinic and chlorogenic acids, their contents were 944.1±0.22 μg/g and 793.5±0.19 μg/g, respectively. Regarding flavonoids, the largest amounts were of isoquercitrin (164.5±0.17 μg/g) and luteolin (126.4±0.18 μg/g).