Neuroprotective and Renoprotective Potential of a Hydroethanolic Extract From the Aerial Parts of Mattiastrum paphlagonicum Bornm. (Boraginaceae)

TL;DR

This study explores the health benefits of a plant extract from Mattiastrum paphlagonicum, showing strong antioxidant and enzyme-inhibiting properties that could help protect the brain and kidneys.

Contribution

The study is the first to analyze the neuroprotective and renoprotective potential of Mattiastrum paphlagonicum's hydroethanolic extract.

Findings

The extract showed strong DPPH radical-scavenging activity and high ferric reducing antioxidant power.

It inhibited tyrosinase and carbonic anhydrase enzymes by over 97% and 77%, respectively.

Rosmarinic acid and rutin were identified as key bioactive compounds in the extract.

Abstract

This study provides an analysis of the endemic plant Mattiastrum paphlagonicum Bornm.(Synonym Paracaryum paphlagonicum (Bornm.) R.R. Mill), focusing on enzyme activity, secondary metabolite content, antioxidant capacity (DPPH, ABTS, and FRAP), as well as phenolic and flavonoid levels, all within the context of a hydroethanolic extract prepared from M. paphlagonicum and its biodiversity, for the first time. Rosmarinic acid and rutin were identified at concentrations of 87.56 ± 0.09  and 0.96 ± 0.15 mg/g of the plant extract, respectively. The total polyphenol and flavonoid contents were measured as 0.85 ± 0.004 mg gallic acid equivalent/mL and 0.077 ± 0.014 mg rutin equivalent/mL. The extract exhibited exceptionally strong DPPH radical‐scavenging activity, with a value of 231.746 ± 0.009 mg ascorbic acid equivalents (AAE)/mL extract, and high ferric reducing antioxidant power (FRAP), reaching 168.229 ± 0.004 mg AAE/mL extract. The percentage inhibition for tyrosinase and carbonic anhydrase by the extract was 97.6 ± 0.011% and 77.07 ± 0.996%, respectively. The extract is a rich source of bioactive compounds and has considerable inhibitory effects on tyrosinase and carbonic anhydrase enzymes. These findings highlight the plant's potential for neuroprotective and renoprotective pharmacological applications.

Figures

Mattiastrum paphlagonicum (from Çankırı province): (a) plant habit; (b) gypsum area where the plants grow (photos by A. Kayabaş Avşar).

Tables

TABLE 1. Antioxidant activity, total flavonoids, and polyphenols content of M. paphlagonicum extract.

Property/parameters	Assay type/Units of measurement	Obtained values (±SD) a
Antioxidant activity	DPPH assay/ascorbic acid equivalents (AAE) (mg acid ascorbic/mL extract)	231.746 ± 0.009
	ABTS assay/Trolox equivalents (TE) (mg Trolox/mL extract)	1.619 ± 0.003
	FRAP assay/ascorbic acid equivalents (AAE) (mg acid ascorbic/mL extract D)	168.229 ± 0.004
The total flavonoid content (TFC)	Spectrophotometric method/rutin equivalents (mg rutin/mL extract)	0.080 ± 0.008
Total polyphenol content (TPC)	Folin–Ciocalteu method/gallic acid equivalents (mg gallic acid/mL extract)	0.850 ± 0.004

TABLE 2. Enzyme inhibition results of M. paphlagonicum extract and comparison with R. odorata extracts.

	Tyrosinase activity			Carbonic anhydrase activity
Plant extract	% Inhibition ± SD (n = 3)	Eq. baicalein (mg/mL)	IC50 (mM)	% Inhibition ± SD (n = 3)		IC50 (mM)
M. paphlagonicum	97.60 ± 0.011	16.24	5.72 ± 0.36		77.07 ± 0.996	7.62 ± 0.48
ROEE	76.52 ± 1.26				72.59 ± 1.39
ROHF	58.08 ± 1.74				56.64 ± 0.67
ROEF	67.48 ± 0.49				76.50 ± 0.67
ROBF	80.26 ± 1.59				68.75 ± 1.69
ROWF	43.33 ± 0.62				51.60 ± 1.13
Standard	98.59 ± 0.92				95.51 ± 1.29

TABLE 3. Quantification of rutin, rosmarinic acid, and quercetin in M. paphlagonicum extract using the UPLC‐UV method.

Sample	Rosmarinic acid (µg/mL) ± SD	Rutin (µg/mL) ± SD	Quercetin
M. paphlagonicum extract	87.56 ± 0.09	0.96 ± 0.15	nd
Extract + carbonic anhydrase enzyme	˂LOD	0.69 ± 0.12	nd
Extract + tyrosinase enzyme	˂LOD	0.72 ± 0.12	nd

Full text

Introduction

1

In recent years, the potential of natural endemic plants for use in treating neurological and renal disorders has been the subject of ongoing investigation by interdisciplinary academic groups in the fields of phytomedicines and traditional medicine, which offer, non‐side effectiveness, inexpensiveness, and effectiveness properties. In the literature, endemic plants have been examined by interdisciplinary researchers for their medicinal uses, including antiviral, antiulcer, diuretic, and skin‐brightening properties. The future of the medical industry lies in herbal remedies, as research aimed at discovering alternative drug‐active ingredients from the endemic plants continues to progress [1].

Studies focusing on the activity of new endemic plant species, particularly their interactions with tyrosinase and carbonic anhydrase enzymes, are expected to contribute to future developments in alternative drug formulations. The present study focused on enzyme activities measured using a new analytical method for Mattiastrum paphlagonicum Bornm. (Synonym Paracaryum paphlagonicum (Bornm.) R.R. Mill) [2], which is an endemic plant and has not been previously examined in terms of any healing properties, particularly for neurologic and kidney disease, for the first time. This work also aims to assess whether further investigation is warranted regarding its potential role in the treatment of renal and neurological diseases.

The ethnopharmacological properties of Boraginaceae taxa, with a documented history of use spanning over 2000 years, have been proven by some chemical analyses [3]. Understanding the taxonomic and ethnobotanical background of M. paphlagonicum, whose pharmacological potential has not yet been investigated in the literature, is of particular importance. The subtribe Cynoglossinae (Boraginaceae) currently recognizes 36 species of Paracaryum and 29 species of Mattiastrum [4]. M. paphlagonicum, commonly known as, “Çankırı Çarşağı,” is an endemic species native to the Irano‐Turanian floristic region [5, 6].

In 2021, 3.4 billion people, or 43.1% of the global population, were affected by neurological conditions, leading to 11.1 million deaths [7]. Neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis severely impact the quality of life and place significant burdens on society and the economy. Increased life expectancy has led to a rise in age‐related neurological disorders, emphasizing the need for health policies that focus on both extending life and reducing disability. To address this, the World Health Assembly adopted the Intersectoral Global Action Plan on Epilepsy and Other Neurological Disorders (IGAP) for 2022–2031, aiming to diminish the stigma and impact of these disorders and improve the quality of life for individuals and their caregivers [8].

The incidence rate of neurodegenerative diseases among the elderly population presents significant challenges, particularly concerning the behavioral disorders and related complications associated with diseases such as Parkinson's disease (PA) [9, 10, 11]. These conditions adversely affect the patients’ quality of life and pose considerable threats to their well‐being. The etiology and pathological mechanisms underlying PA are intricate. A prominent theory, known as the dopamine hypothesis, posits that lesions in the substantia nigra (SN) lead to a loss of dopaminergic neurons and a subsequent decrease in dopamine (D) levels in affected individuals [12, 13]. The diminished D content in the striatum results in the impaired function of dopaminergic nerves in the substantia nigra‐striatum pathway, thereby allowing cholinergic nerve function to predominate. Current therapeutic strategies include the development of monoamine oxidase (MAO) and catechol‐O‐methyltransferase (COMT) inhibitors aimed at elevating D levels [14, 15]. However, it is important to note that these pharmacological interventions primarily improve motor function and do not provide a fundamental cure or safeguard for the damaged neurons. Recent research has identified a significant correlation between neuromelanin (NM) in the human SN and the pathogenesis of PA. Specifically, neurons enriched with NM are more vulnerable to pathological alterations. Additionally, the interaction between NM‐rich neurons and microglia or macrophages that phagocytose NM contributes to a disruption in protein homeostasis, particularly proteins linked to redox processes, autophagy, and protein degradation. The aberrant expression of these proteins exacerbates the onset and progression of PD [16, 17]. Tyrosinase, a copper ion‐containing oxidase, possesses dual catalytic functions as both a monophenol oxidase and a diphenol oxidase. This enzyme serves as the critical rate‐limiting factor in melanin synthesis. l‐Tyrosine (l‐Tyr) is hydroxylated by tyrosinase (monophenol oxidase) to generate levodopa (l‐dopa), which is subsequently oxidized to dopaquinone under the action of tyrosinase (diphenol oxidase). D, synthesized from l‐dopa by amino acid decarboxylase, can also be oxidized by tyrosinase to form dopaminequinone. These quinone structures undergo a series of further reactions, leading to the production of various types of melanin [18, 19]. Multiple studies have indicated that tyrosinase is integral to the synthesis of NM in the SN of the human brain. Overexpression of tyrosinase at low levels can result in elevated age‐dependent NM levels within dopaminergic neurons, compromising D fibers in the SN‐striatum, inducing severe protein imbalances, and ultimately leading to an irreversible reduction in D levels in the substantia nigra compacta (SNpc). This pathology manifests in symptoms such as limb tremors, rigidity, postural instability, motor dysfunction, and cognitive impairment among patients. Hence, the inhibition of tyrosinase activity in the brain and the attenuation of NM production represent promising strategies for the treatment of PA [17, 20].

An estimated 850 million people worldwide are affected by kidney disease [21]. Key factors contributing to the incidence of chronic kidney disease include population growth, aging, and the rising prevalence of diabetes, heart disease, and hypertension, particularly in developed economies. In high‐income countries, approximately one in three individuals with diabetes and one in five individuals with hypertension are diagnosed with chronic kidney disease. This correlation suggests that prioritizing the management of diabetes and cardiovascular conditions could help mitigate the increasing burden of chronic kidney disease [22].

Carbonic anhydrase enzymes represent a diverse family of zinc‐dependent metalloenzymes that play a crucial role in the reversible hydration of carbon dioxide. This biochemical process converts carbon dioxide into bicarbonate and a proton, facilitating various physiological functions, including respiration, acid–base balance, and carbon dioxide transport in living organisms (CO_2_ + H_2_O → HCO_3_ ^−^ + H^+^). The presence of zinc at their active sites is essential for their catalytic efficiency, underscoring the intricate relationship between metal ions and enzyme functionality in biological systems [23]. This chemical reaction is fundamental to numerous physiological processes, including pH and ion homeostasis, carbon dioxide transport, electrolyte secretion, gluconeogenesis, lipogenesis, and ureagenesis. Additionally, it plays a critical role in water and sodium reabsorption within the kidneys, bone reabsorption and calcification, as well as the formation and turnover of cerebrospinal fluid, among other vital functions [24].

The elevated prevalence of chronic kidney and neurological diseases has prompted us to search for new inhibitors of carbonic anhydrase and tyrosinase enzymes; additionally, due to the carbonic anhydrase enzyme also affecting the central nervous system, the plant extract was analyzed for two of these enzymes in vitro activity and compared using their results. To this end, we concentrated on the plant kingdom, leveraging the potential of its secondary metabolites for the treatment of human ailments.

The activity of antioxidants plays a significant role in mitigating oxidative stress, a condition associated with a range of chronic diseases, including kidney disorders, various types of cancer, and neurodegenerative ailments [25].

Polyphenols are remarkable plant‐derived phenolic compounds, distinguished by their aromatic rings and hydroxyl groups in their molecular structure. With a variety of bioactive properties, particularly their powerful antioxidant activity, they play a vital role in disease prevention, such as kidney and neurological diseases. Polyphenols inspire innovation across sectors, permeating the food industry, pharmacology, and medicine [26].

The synergy of phenolic content, antioxidant capacity, secondary metabolites composition, enzyme inhibition effects, and healing evaluation of endemic plants opens a remarkable pathway for innovation and discovery in the food industry, pharmacology, and medicine.

This study presents the first investigation into the hydroethanolic extract of M. paphlagonicum and its inhibitory effects on tyrosinase and carbonic anhydrase enzymes, utilizing both in vitro UV‐spectrophotometric and ultra‐performance liquid chromatography–ultraviolet detector (UPLC‐UV) methods. The simultaneous quantification of key secondary metabolites, including rosmarinic acid, rutin, and quercetin, using a validated UPLC‐UV method. Additionally, the antioxidant capacity, total flavonoid, and polyphenol contents of the extract were evaluated.

Materials and Methods

2

Plant Material, Equipment, and Chemicals

2.1

Mattiastrum paphlagonicum is a biennial plant occurring on gypsum‐rich substrates, especially on road banks and eroded slopes, between 600 m and 1300 m. The calyx measures 4.3–6.7 mm and remains shorter than the corolla. The corolla is 5.3–6.7 mm long, and its tube is typically at least twice the length of the limb. Scales are 1–1.8 mm, oblong, and commonly show an incurved apex. The style is 5.5–7.2 mm long (Figure 1) [5].

Mattiastrum paphlagonicum (from Çankırı province): (a) plant habit; (b) gypsum area where the plants grow (photos by A. Kayabaş Avşar).

The aerial parts of M. paphlagonicum were collected from the Uluyazı Campus locality in Çankırı province, Türkiye, at an altitude of 920–930 m a.s.l. (40°37′ N, 33°37′ E) in June 2021. The specimen was taxonomically identified by Selçuk Tuğrul KÖRÜKLÜ and stored in the Ankara University Herbarium with the code ANK 60717. The aerial parts were cleaned under running tap water to remove dust, soil and dirt, and then washed with pure water. After cleaning, the plant material was dried at room temperature and ground to a fine powder.

The solvents used in this study were sourced from Merck (Darmstadt, Germany) with UPLC purity. The internal standards of compounds included l‐DOPA, Tyrosinase, baicalein, DMSO, acetazolamide, carbonic anhydrase enzyme, p‐nitrophenyl acetate, rutin, rosmarinic acid, and quercetin, all obtained from Carl ROTH (Roth, Germany). The compounds were quantified using the Waters ACQUITY UPLC H‐Class instrument (Waters Company, Framingham, MA, USA). This instrument is equipped with a quaternary solvent manager for precise regulation during chromatography and a highly sensitive UV detector to ensure accurate compound detection and analysis. It also features a cooling autosampler to preserve sample integrity and an integrated oven for precise control of column temperature, contributing to optimal chromatographic performance. Elution was performed using a Waters × Bridge 4.6 × 50 mm column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Simultaneous quantification of rosmarinic acid, rutin, and quercetin was conducted with the Waters Empower3 software (Waters Company, Framingham, Massachusetts, USA). UV spectrum measurements were obtained over the 200–600 nm wavelength range using a Jasco V‐550 UV spectrophotometer (Japan Spectroscopic Company, Japan). The milling procedure for extractions was carried out using Retsch equipment MM400 (Retsch, Haan, Germany), and the compounds were accurately weighed using a Sartorius balance CPA225D‐OCE (Sartorius Group, Göttingen, Germany).

Extraction Procedure for Aerial Parts of the Plant

2.2

The aerial parts of the endemic plant were rinsed under running tap water to remove dust, soil, and dirt, and then rinsed with distilled water. After cleaning, the plant material was dried at room temperature and ground to a fine powder using an herb grinder, followed by further milling of the powder. In the extraction procedure, the cold maceration method was employed, in which 4 g of milled with Retsch MM400 ball mill (Retsch, Haan, Germany) aerial parts of M. paphlagonicum were precisely weighed and transferred into glass tubes containing 40 mL of a solvent mixture composed of ethanol and water in a 50:50 (v/v) ratio. This step was repeated three times. The tubes were stored in the dark at room temperature for 10 days, with the mixtures being agitated daily. Afterward, all three extracted samples were filtered through a 0.45 µm syringe filter. The resulting filtered extracts were then refrigerated at 4°C in preparation for subsequent analyses, which included UPLC and spectrophotometric analyses, such as antioxidant capacity assays, total polyphenol and flavonoid content, and in vitro diuretic and tyrosinase analyses.

Antioxidant Capacity

2.3

The antioxidant capacity of M. paphlagonicum extract was evaluated using spectrophotometric assays that employed 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH^•^) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS^+•^) radicals, respectively, and also by the FRAP (ferric reducing antioxidant power) method using the 2,4,6‐tripyridyl‐s‐triazine (TPTZ) assay.

DPPH Radical Scavenging Capacity Assay

2.3.1

The DPPH• protocol established by Tirzitis et al. [27] was employed with minor modifications, for this analysis. To maintain the free radical activity, the solution was freshly prepared each day and shielded from light throughout the assessment period. In each instance, 4 mL of a 0.15 mmol/L DPPH• solution in ethanol was combined with various volumes of extract (ranging from 50 µL to 500 µL) and then adjusted to a final volume of 5 mL using ethanol. Absorbance readings were taken at a wavelength of 518 nm after a 60‐min incubation in the absence of light. The percentage of radicals consumed was calculated using the following formula:

$$[eqn]$$

where A 0 represents the absorbance of the DPPH• radical without the extract, and A f indicates the final absorbance of the radical solution post‐reaction. Results were reported as ascorbic acid equivalents (AAE, mg ascorbic acid/mL extract).

ABTS Radical Scavenging Capacity Assay

2.3.2

The ABTS^+•^ radical stock solution was prepared in accordance with the protocol established by Nilsson [28]. Equal volumes of a 7 mmol/L ABTS solution and a 2.45 mmol/L potassium persulfate solution were combined to create the stock solution. This mixture was maintained in the dark at room temperature for a period of 16 h prior to use. A working solution was prepared by diluting the stock until the absorbance reached 0.70 ± 0.05. Then, 4 mL of this solution was mixed with different amounts of a 1:10 hydroethanolic extract, adjusting the final volume to 5 mL. After allowing the reaction to occur in the dark for 60 min, we measured the absorbance at 752 nm. We calculated the percentage of consumed radicals using this formula:

$$[eqn]$$

In this formula, A 0 is the absorbance of the solution without the extract, and A f is the final absorbance. Results were reported as Trolox equivalents (TE, mg Trolox/mL extract).

Ferric Reducing Antioxidant Power (FRAP) Assay

2.3.3

The FRAP analysis was performed under the methodology described by Iris and Strain [29]. A fresh FRAP solution was prepared by combining 2.5 mL of a 0.01 M TPTZ solution in 40 mM HCl, 2.5 mL of a 0.02 M FeCl_3_·6H_2_O solution, and 25 mL of a 0.3 M acetate buffer (pH 3.6). This mixture was then warmed to 37°C. Aliquots of 150 µL of the extracts were added to 2850 µL of the FRAP solution and incubated in the dark for 30 min. The absorbance of the resulting Fe^3+^‐TPTZ complex was measured at 593 nm against a blank, with results expressed as ascorbic acid equivalents (AAE, µg ascorbic acid/mL extract).

Total Polyphenol and Flavonoid Content

2.4

The total flavonoid content (TFC) was measured using the aluminum trichloride spectrophotometric method [30]. A 0.5 mL hydroethanolic extract was diluted 1:10 with a 50:50 ethanol–water mixture and mixed with 2.5 mL of aluminum chloride solution (100 g/L) and 1.5 mL of sodium acetate solution (25 g/L). This was adjusted to 10 mL with methanol and incubated for 15 min before measuring absorbance at 430 nm. A reference solution with water replaced the reagents. TFC was stated as milligrams of rutin equivalents (RE) per milliliter, using a rutin calibration curve (0.05–35 µg/mL).

Total polyphenol content (TPC) was measured with the Folin–Ciocalteu method [31]. Hydroethanolic extracts (100–1000 µL, 1:10 dilution) were mixed with 0.5 mL of Folin–Ciocalteu reagent and brought to 25 mL with 16% Na_2_CO_3_ solution. Absorbance was measured at 715 nm after a 2‐min incubation. TPC was reported as milligrams of gallic acid equivalents (GAE) per milliliter, with a calibration curve ranging from 0.1 µg/mL to 3.4 µg/mL.

Carbonic Anhydrase Enzyme In Vitro Activity

2.5

In the determination of in vitro diuretic activity, the method described by Armstrong et al. [32] was followed. The enzyme solution (Solution 1) was made by dissolving 2.71 mg of carbonic anhydrase enzyme in 2 mL of phosphate buffer (pH 7.0). Acetazolamide was diluted in methanol to concentrations of 1–76 ppm as a standard inhibitor. The substrate sample (Solution 2) consisted of 0.5 mL of substrate + 0.65 mL of phosphate buffer + 1.02 mL of deionized water. The mixture of Solutions 1 and 2 consisted of 0.5 mL substrate, 0.65 mL phosphate buffer, 20 µL enzyme solution, and 1.0 mL water. The inhibitor sample was prepared by combining 0.5 mL substrate, 0.65 mL phosphate buffer, 20 µL enzyme solution, and 2.17 mL inhibitor solution. The blank sample contained 0.33 mL of acetone and 9.7 mL of water. Measurements were taken at 400 nm and 25°C using a Jasco V‐550 UV spectrophotometer. The percentage inhibition was calculated with:

$$[eqn]$$

Diuretic activity was quantified using IC_50_ through linear regression, as described in Refs. [33, 34].

Tyrosinase Enzyme In Vitro Activity

2.6

The tyrosinase inhibitory activity was evaluated using a modified dopachrome method with l‐DOPA as the substrate [35]. The in vitro assay was performed using a UV spectrophotometer (Jasco V‐550) to measure absorbance at 475 nm. Four glass tubes were prepared for each sample concentration:

• Tube A: 20 µL of mushroom tyrosinase in 20 mM phosphate buffer and 1500 µL of buffer (pH 6.8).
• Tube B: 1520 µL of phosphate buffer (pH 6.8).
• Tube C: 20 µL of tyrosinase, 1480 µL of buffer, and 20 µL of the sample.
• Tube D: 1500 µL of buffer and 20 µL of sample.

Each mixture was incubated at 25°C for 10 min, followed by the addition of 20 µL of 0.85 mM l‐DOPA and a further 20 min incubation. Dopachrome levels were measured by assessing changes in optical density. Baicalein was used as a positive control. The percentage inhibition of tyrosinase activity was calculated using the formula:

$$[eqn]$$

where A, B, C, and D represent different readings of optical density.

Quantification of Secondary Metabolites Content Using the UPLC‐UV Method

2.7

The hydroethanolic extract was analyzed to quantify rosmarinic acid, rutin, and quercetin using the UPLC‐UV method, which was developed, validated, and published by Büker et al. [34] with a Waters ACQUITY UPLC H‐Class instrument. The chromatographic conditions were; the mobile phase A was 0.1% formic acid in water, while mobile phase B was of 0.1% formic acid in acetonitrile, with the two phases mixed in a ratio of 80% A to 20% B, the flow rate was maintained at 0.45 mL/min, and the temperature was set at 45°C, an injection volume of 10 µL was utilized, with detection occurring at a wavelength of 260 nm. The calibration curves for rosmarinic acid, rutin, and quercetin were 8.0–100.0 µg/mL, 0.5–10 µg/mL, and 5.0–40.0 µg/mL, respectively [34]. LOD and LOQ values for rosmarinic acid, rutin, and quercetin were calculated as follows: 2.71 µg/mL for LOD, 8.20 µg/mL for LOQ, 0.71 µg/mL for LOD, 2.15 µg/mL for LOQ, 1.22 µg/mL for LOD, and 3.68 µg/mL for LOQ, respectively.

The same validated UPLC‐UV method was also applied to investigate the effect of rosmarinic acid on carbonic anhydrase and tyrosinase enzymes. In this method, rosmarinic acid was analyzed by adding the same amount of carbonic anhydrase and tyrosinase enzymes as mentioned in the preparation methods (Sections 2.5 and 2.6) for carbonic anhydrase and tyrosinase enzyme activity in the presence of the plant extract sample.

Results and Discussions

3

Antioxidant Capacity, Total Polyphenol, and Flavonoid Content

3.1

Among the biological activities exhibited by flavonoid and polyphenol compounds are anticancer, antiallergic, anti‐inflammatory, and antioxidant effects. Previous research has established a direct correlation between phenolic compounds and antioxidant activity [36]. To the best of our knowledge, there are currently no reports available regarding the antioxidant activity of the hydroethanolic extract from the aerial parts of M. paphlagonicum. So, in this study, the antioxidant capacity of the M. paphlagonicum extract was evaluated using complementary in vitro assays DPPH, ABTS, and FRAP, which are based on different reaction mechanisms and therefore provide a broader characterization of antioxidant behavior (Table 1).

The extract exhibited an exceptionally strong DPPH radical‐scavenging activity, with a value of 231.746 ± 0.009 mg ascorbic acid equivalents (AAE)/mL extract. This value is markedly higher than those typically reported for medicinal plant extracts (which generally range between 40 mg and 70 mg AAE/mL) and indicates a very high capacity for hydrogen‐atom donation. Such pronounced DPPH activity suggests the presence of compounds capable of efficiently neutralizing stable free radicals, a characteristic commonly associated with phenolic antioxidants. Consistent with the strong DPPH response, the extract also demonstrated a FRAP, reaching 168.229 ± 0.004 mg AAE/mL extract. The FRAP assay reflects the electron‐donating ability of antioxidants to reduce Fe^3^ ^+^ to Fe^2^ ^+^, and elevated FRAP values are generally indicative of strong reducing capacity. The concurrence of high DPPH and FRAP values suggests that the extract contains antioxidant constituents capable of acting through both hydrogen‐atom transfer and single‐electron transfer mechanisms.

The ABTS radical cation scavenging activity of M. paphlagonicum extract was 1.619 ± 0.003 mg Trolox equivalents (TE)/mL extract, indicating a low to moderate antioxidant capacity. In the literature, the ABTS antioxidant activity of medicinal plant extracts reported as mg TE/mL is most commonly between 0.3 mg TE/mL and 2 mg TE/mL [37], with values exceeding 5–10 mg TE/mL generally observed only for highly concentrated or optimized extracts [38]. The comparatively modest ABTS activity contrasts with the markedly high antioxidant capacity observed in the DPPH and FRAP assays. Such discrepancies are well documented and reflect the assay‐dependent nature of antioxidant measurements [38, 39]. While DPPH and FRAP assays primarily emphasize hydrogen atom donation and reducing power under specific reaction conditions, the ABTS assay is more sensitive to hydrophilic antioxidants and electron‐transfer mechanisms. The lower ABTS response of M. paphlagonicum extract may therefore indicate a predominance of antioxidant compounds that are more effective as hydrogen donors or metal‐reducing agents than as ABTS^•^ ^+^ radical scavengers.

Overall, the ABTS results suggest that M. paphlagonicum extract exhibits a selective antioxidant profile, characterized by strong radical scavenging and reducing capacities in DPPH and FRAP assays but a comparatively weaker response in the ABTS system. These findings emphasize the importance of using multiple assays to obtain a comprehensive assessment of antioxidant potential and suggest that the extract may be particularly effective against radicals neutralized via hydrogen donation and ferric ion reduction.

The selective antioxidant behavior observed here may therefore reflect the chemical nature of the phenolic constituents present in the extract. Although the TPC was relatively low (0.850 ± 0.004 mg gallic acid equivalents/mL extract), these phenolics may possess high intrinsic reactivity, compensating for their lower concentration. Similarly, the TFC was modest (0.080 ± 0.008 mg rutin equivalents/mL extract), suggesting that non‐flavonoid phenolics or other reducing compounds may contribute substantially to the observed antioxidant effects. The results of the literature review [40] indicate that the TFC and TPC of the hydroethanolic extract from Juniperus communis L. (T) are measured at 0 and 0.59 ± 0.01 mg of gallic acid equivalents per milliliter of extract, respectively. Notably, the total polyphenol content of the extract from M. paphlagonicum surpasses that of the J. communis extract in both total flavonoid and polyphenol content.

Carbonic Anhydrase and Tyrosinase Enzyme In Vitro Activity

3.2

In this study, the tyrosinase inhibition potential of the aerial parts of M. paphlagonicum was found to be highly effective, identifying the extract as a potent inhibitor of this enzyme. Importantly, for the first time, the hydroethanolic extract of the aerial parts of M. paphlagonicum was evaluated for its inhibitory activities against both carbonic anhydrase and tyrosinase. The results indicated that tyrosinase inhibition was more pronounced than that of carbonic anhydrase. This discrepancy may be attributed to the phytoconstituents identified in the UPLC‐UV profile, along with total phenolic and flavonoid compounds, such as rosmarinic acid. These compounds demonstrated inhibitory effects on both carbonic anhydrase and tyrosinase enzymes, as shown in the chromatographic profile. These findings suggest that the aerial parts of M. paphlagonicum possess strong potential as natural inhibitors of both carbonic anhydrase and tyrosinase enzymes.

The plant extract underwent thorough analysis to evaluate its in vitro inhibitory activity against the enzymes carbonic anhydrase and tyrosinase, shedding light on its potential as a natural inhibitor within biochemical processes. The inhibition potentials of carbonic anhydrase and tyrosinase enzymes by the extract of the plant's aerial parts were analyzed using a method outlined in Refs. [34, 35], with minor modifications. The results were expressed as percentage inhibition ± standard deviation. The maximum inhibition observed was for M. paphlagonicum, which showed a value of 77.60 ± 0.996% for carbonic anhydrase and 97.60 ± 0.011% for tyrosinase enzyme.

These findings were compared with data reported in the literature on the inhibition of the same enzymes by five different solvent extracts of R. odorata, a medicinal plant commonly used in traditional and complementary medicine. The inhibitory activity of M. paphlagonicum against carbonic anhydrase and tyrosinase was found to exceed approximately 40% of the inhibition values reported for R. odorata. Notably, the extract from the aerial parts of M. paphlagonicum demonstrated substantial inhibitory potential against both carbonic anhydrase and tyrosinase enzymes, as presented in Table 2.

Quantification of Secondary Metabolites Content Using the UPLC‐UV Method

3.3

Phytochemical analysis plays a crucial role in assessing the potential medicinal properties of plants and identifying the active compounds that contribute to their biological activities. Moreover, it provides a foundation for the targeted isolation of these compounds and facilitates more detailed investigations [42]. A phytochemical screening of the aerial parts of M. paphlagonicum revealed the presence of rosmarinic acid and rutin (Figure S1). The presence of these phytoconstituents in the aerial parts of M. paphlagonicum may contribute to its observed therapeutic efficacy. To evaluate the inhibition potential of rosmarinic acid and compare enzyme inhibition results, the UPLC–UV profiles of rosmarinic acid, baicalein, and acetazolamide were analyzed and docked against tyrosinase (Figure S2) and carbonic anhydrase enzymes (Figure S3), respectively.

The extract was evaluated for its rosmarinic acid, rutin, and quercetin contents using the assays described in the experimental section. The results demonstrated that the M. paphlagonicum extract possessed a notably high concentration of rosmarinic acid, measuring 87.56 ± 0.09 µg/mL (rosmarinic acid equivalents), and 0.96 ± 0.15 µg/mL of rutin equivalents (Table 3).

In the results of the UPLC‐UV analysis, which aimed to investigate the effects of rosmarinic acid on carbonic anhydrase and tyrosinase enzymes, it was found that rosmarinic acid exerts a direct influence on these enzymes. This was indicated by a notable decrease in the detectable concentration of rosmarinic acid in the samples prepared according to procedures involving both carbonic anhydrase and tyrosinase. However, the exact decrease in rosmarinic acid levels could not be accurately calculated, as the measured values fell below the limit of detection (LOD) due to enzymatic interactions (as shown in the accompanying tables and Figures S1–S3). Consequently, while a precise comparison to determine which of the two enzymes the hydroethanolic extract of the M. paphlagonicum affects more strongly is not feasible, this study has, for the first time, demonstrated its inhibitory effects on both enzymes using complementary spectrophotometric and chromatographic methods. The quantitative analysis of rosmarinic acid, rutin, and quercetin in the hydroethanolic extract of J. communis (T) yielded values of 1.127 ± 0.029, 4.995 ± 0.07, and 0.005 ± 0, respectively, in the literature [40]. Notably, the extract from M. paphlagonicum demonstrated a higher concentration of rosmarinic acid compared to the J. communis (T).

Conclusion

4

The present study revealed that a biodiversity exploration into the in vitro interactions between enzyme inhibition (carbonic anhydrase and tyrosinase) and secondary metabolite content, as well as antioxidant capacity, phenolic, and flavonoid levels in the extract of aerial parts of the endemic plant M. paphlagonicum, revealing, for the first time in the literature.

The results demonstrate that M. paphlagonicum extract has selective antioxidant, characterized by exceptionally high DPPH radical‐scavenging activity and ferric reducing power, indicating strong hydrogen atom transfer and electron‐donating capacities. The lower ABTS response, together with modest total phenolic and flavonoid contents, suggests that highly reactive non‐flavonoid phenolics or other reducing constituents play a major role, highlighting the assay‐dependent nature of antioxidant activity evaluation and the necessity of employing multiple complementary methods to determine this property.

Utilizing UPLC‐UV analysis, key bioactive compounds, namely rosmarinic acid and rutin, which have a synergistic effect as secondary metabolites, were quantified. It was revealed that rosmarinic acid was the primary responsible secondary metabolite for inhibiting carbonic anhydrase and tyrosinase enzymes within the hydroethanolic extract, among all secondary metabolites of the studied extract. Notably, the results of the UPLC‐UV showed that a significant concentration of rosmarinic acid emerged as a potent dual inhibitor of both tyrosinase and carbonic anhydrase, as validated by UV‐spectrophotometric analysis and first‐time UPLC‐UV measurements.

The hydroethanolic extract of the aerial parts of M. paphlagonicum exhibited remarkable inhibition of the enzymes tyrosinase and carbonic anhydrase, indicating that the endemic plant is important to biodiversity. The findings highlight the potential of the hydroethanolic extract as a complementary treatment for neurodegenerative diseases such as Parkinson's disease and disorders requiring diuretic support. These findings indicate that further comprehensive investigations are warranted to fully elucidate its bioactive properties and clinical applicability.

Author Contributions

Conceptualization: Ayşenur Kayabaş Avşar. Data curation: Eda Büker, Dorina Casoni, and Simona Codruta Aurora Cobzac. Formal analysis: Eda Büker, Ertan Yildirim, Dorina Casoni, and Simona Codruta Aurora Cobzac. Investigation: Ayşenur Kayabaş Avşar. Methodology: Ayşenur Kayabaş Avşar, Eda Büker, and Ertan Yildirim. Software: Ertan Yildirim. Supervision: Ayşenur Kayabaş Avşar, and Eda Büker. Validation: Eda Büker. Visualization: Ayşenur Kayabaş Avşar, and Dorina Casoni. Writing – original draft: Ayşenur Kayabaş Avşar, Eda Büker, Dorina Casoni, and Simona Codruta Aurora Cobzac. Writing – review and editing: Ayşenur Kayabaş Avşar, Eda Büker, Dorina Casoni, and Simona Codruta Aurora Cobzac.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71125‐sup‐0001‐SuppMat.docx