Exploring the Influence of Growth-Related Conditions on the Antioxidant and Anticholinergic Properties of Pressurized Arctium lappa L. Root Extracts

Abstract

Arctium lappa L., commonly known as burdock, is a biennial plant whose roots are a valuable source of bioactive phenolic compounds with notable health-promoting properties. However, the bioactivity of these compounds is influenced by both extraction parameters and plant growth conditions. This study investigated the combined effect of extraction temperature, land management, and cultivation altitude on the antioxidant and anticholinergic potential of burdock root extracts obtained through pressurized liquid extraction (PLE). Extractions were performed at 50 °C, 100 °C, and 150 °C, with 50 °C being the temperature that best preserved phenolic content and bioactivity. Remarkably, root extracts obtained at 50 °C and collected from an untreated organic field at 150 m altitude yielded higher phenolic levels (42 mg gallic acid/g extract) than conventional solid–liquid extraction (38 mg gallic acid/g extract). A comparative analysis of three ecotypes, including Organic Land Ecotype (OLE) and Spontaneous Land Ecotype (SPLE), both collected at 150 m altitude, and Spontaneous Mountain Ecotype (SPME), collected at 800 m (over sea level), revealed that a higher altitude significantly increased phenolic content and anticholinergic potential. Furthermore, roots from non-weeded soils exhibited superior bioactivity compared to those from weeded areas. These findings underline that the successful obtention of highly bioactive burdock root extracts depends not only on extraction conditions, but also critically on cultivation altitude and land management strategies.

1. Introduction

Arctium lappa L., commonly known as burdock, is a member of the Asteraceae family. It is a widely distributed plant native to Asia, particularly China, but also commonly found across Europe and North America [1]. It thrives in disturbed, nutrient-rich environments, growing at altitudes up to 3300 m above sea level, and even higher in Central Asia [2]. Although burdock favors moist soils and temperate climates, it is highly adaptable, flourishing in sunny, fertile, and clay-rich soils, a trait that has led to its classification as an invasive species in several regions [3].

For centuries, burdock leaves, roots, flowers, and stalks have been valued in traditional medicine and as food ingredients due to their notable bioactive properties [4]. These health benefits, including hepatoprotective, antioxidant, and anti-inflammatory effects, are primarily attributed to a rich composition of secondary metabolites such as tannins, lignans, caffeic acid derivatives, inulin, and diarctigenin [5,6,7,8].

Among plant secondary metabolites, phenolic compounds play a crucial role in protecting against environmental stressors like insects, pathogens, and harsh climatic conditions [9,10]. It is well established that, under adverse environmental factors, plants increase their phenolic production as a defensive strategy [11,12,13]. However, to date, there are limited studies that specifically address how the phenolic profile of burdock roots is influenced by the growing site. This is particularly relevant because altitude-related factors, such as elevated UV radiation, temperature variability, oxygen availability, and soil composition, significantly influence the secondary metabolism of plants. Increased UV exposure at higher altitudes stimulates phenolic synthesis, while cooler temperatures (generally ranging from 5 to 15 °C) and oxidative stress further enhance the production of these protective compounds. Soil nutrient variability along altitudinal gradients also affects phenolic concentrations and profiles. Understanding these relationships is essential for optimizing the bioactive potential of burdock and harnessing its health benefits [14,15,16,17,18,19,20,21].

Notably, burdock roots are the most extensively studied plant part, although the seeds and leaves have also demonstrated medicinal value [5,22,23,24,25,26,27,28]. In traditional Chinese medicine, burdock roots were revered as a “blood detoxifier,” promoting the elimination of toxins, supporting circulation, and improving skin conditions such as eczema [22]. Modern pharmacological studies confirm that burdock roots possess antimutagenic, antitumorigenic, anti-inflammatory, antioxidant, antibacterial, and antiviral activities [22]. These effects are largely attributed to phenolic acids, mainly caffeoylquinic acid derivatives, and other bioactive phenolics such as caffeic acid, arctigenin, and arctiin [23,24,25,26,27,28].

Traditionally, phenolic compounds from Arctium lappa L. have been extracted using conventional solid–liquid extraction (SLE) methods with aqueous–organic solvents [3]. However, in recent years, these conventional techniques have increasingly been replaced by advanced, eco-friendly alternatives such as ultrasound-assisted extraction (UAE) and pressurized liquid extraction (PLE), which offer improved efficiency and selectivity. UAE has been applied in the isolation of phenolic compounds from both burdock leaves and roots [26,29,30,31]. Nonetheless, studies have demonstrated that PLE, particularly at elevated temperatures and pressures, significantly enhances the extraction of bioactive phenolics from burdock roots compared to UAE and maceration, as it improves solvent penetration and mass transfer within the plant matrix [32,33].

Despite its potential, the application of PLE to burdock has been limited. There is only one study that evaluates the influence of PLE temperature on the recovery of phenolic compounds from burdock roots using biobased solvents such as cyclopentyl methyl ether (CPME), ethyl acetate (EtOAc), and 2-methyltetrahydrofuran (2-MTHF), achieving higher extraction yields using 100% 2-MTHF at 100 °C. This temperature was ideal for the maximum recovery of bioactive phenolic compounds from burdock roots using biobased solvents, avoiding undesirable reactions that degrade the target compounds [33]. However, to date, there are no comprehensive evaluations of how PLE temperature affects the stability and recovery of phenolic compounds from burdock roots when aqueous–organic solvents are used, which are the most commonly used solvents in the extraction of phenolic compounds from natural matrices. Optimizing the extraction temperature conditions is important because the efficiency and selectivity of phenolic compound recovery can vary significantly depending on the solvent used.

In this context, the present study aimed to optimize the ethanolic PLE conditions for the extraction of bioactive phenolics from burdock roots, specifically assessing three extraction temperatures (50 °C, 100 °C, and 150 °C) to identify the optimal balance between yield and the preservation of bioactivity. Additionally, this work explored how the phenolic composition and bioactivity of burdock roots are influenced by the growing altitude and agronomical practices. Roots were collected at different altitudes in Italy from three distinct ecotypes and subjected or not subjected to weeding, as follows: Spontaneous Land (SPLE, not subjected to weeding and collected at an altitude of 150 m), Organic Land (OLE, subjected to weeding and collected at an altitude of 150 m), and Spontaneous Mountain (SPME, not subjected to weeding and collected at an altitude of 800 m). In this sense, the influence of the extraction solvent used can be compared with a previous study of our research group, where PLE with biobased solvents was employed for the recovery of phenolic compounds from the same ecotypes of burdock roots [33]. These samples were compared in terms of the total phenolic content (TPC) using the Folin–Ciocalteu assay, antioxidant capacity via 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC) methods, and anticholinergic activity through AChE and BChE enzyme inhibition. Moreover, a detailed phenolic profile of ethanolic PLE extracts was obtained using high-performance liquid chromatography with diode array detection coupled to ion trap mass spectrometry (HPLC-DAD-IT-MS), providing a comprehensive characterization of the detected bioactive compounds.

2. Materials and Methods

2.1. Chemicals and Reagents

Methanol and formic acid (LC-MS grade) were obtained from VWR Chemicals (Barcelona, Spain). Trizma^® hydrochloride, disodium phosphate, fluorescein sodium salt, Trolox, potassium persulfate, monopotassium phosphate, sodium carbonate, butyrylcholinesterase (BChE) (from equine serum), acetylcholinesterase (AChE), DPPH, gallic acid, and ascorbic acid were purchased from Sigma−Aldrich (Madrid, Spain). Galantamine hydrobromide, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F), and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) were supplied by TCI Chemicals (Tokyo, Japan). Additionally, the Folin–Ciocalteu reagent was acquired from Merck (Darmstadt, Germany). In addition, chlorogenic acid, cynarine, 4-O-caffeoylquinic acid, 1,5-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid standards for HPLC quantification were purchased from Merck (Darmstadt, Germany). Moreover, ultrapure water (18.2 MΩ·cm) was produced using a Millipore purification system (Millipore, Billerica, MA, USA).

2.2. Plant Material and Sample Pretreatment

2.2.1. Plant Material

Roots from three ecotypes of Arctium lappa L. were provided by Fibreno Officinali, a company from Isola del Liri, Lazio, Italy. These samples were the same as those used in Romano et al.’s study [33]. The botanical identity of the plant material was confirmed by the company, who periodically performs morphological characterizations and comparisons with authenticated reference specimens. The collected burdock root samples corresponded to three distinct ecotypes: (1) Spontaneous Land Ecotype (SPLE), consisting of wild, untreated plants growing spontaneously in Isola del Liri at an altitude of 150 m; (2) Organic Land Ecotype (OLE), cultivated at 150 m altitude in Isola del Liri (41°41′ N 13°34′ E 41°41′ N, 13°34′ E) and subjected to weeding to exclude other plant species from the soil to enhance burdock growth; and (3) Spontaneous Mountain Ecotype (SPME), comprising wild, untreated plants growing spontaneously at a higher altitude (800 m) in Collepardo (41°46′ N 13°22′ E 41°46′ N, 13°22′ E), Lazio, Italy.

The SPLE and OLE ecotypes were harvested in October 2021, while the SPME ecotype was collected in October 2022, ensuring appropriate seasonal conditions for root development and bioactive compound accumulation.

2.2.2. Sample Pretreatment

The collected roots were thoroughly washed with water to remove soil residues and subsequently freeze-dried using a Büchi Lyovapor™ L-200 freeze dryer (Barcelona, Spain) for 72 h at −55 °C and 0.200 mbar, according to Romano et al. [33]. Once lyophilized, the dried roots were ground into a fine powder (125–500 µm) using a commercial laboratory blender and stored at −80 °C in airtight containers until further analysis.

2.3. Pressurized Liquid Extraction of Phenolic Compounds

Phenolic compounds were extracted from burdock root samples using a Dionex Accelerated Solvent Extraction (ASE) 200 system (Sunnyvale, CA, USA). In brief, 0.4 g of freeze-dried, ground root material was mixed with 2 g of commercial pre-cleaned sea sand (Merck, Darmstadt, Germany) and 4 g of glass beads and loaded into an 11 mL stainless steel extraction cell. The extractions were conducted at a pressure of 1500 psi for 20 min following the method described by Gallego et al. [34], employing an ethanol:water mixture (70:30, v/v) as extraction solvent.

To determine the optimal extraction conditions, three different extraction temperatures (50 °C, 100 °C, and 150 °C) were evaluated. All extractions were performed in triplicate to ensure reproducibility. After extraction, the resulting solutions were concentrated by nitrogen stream evaporation, and extraction yields were recorded. Finally, the extracts were freeze-dried and stored at −20 °C in the dark until further analysis.

2.4. Total Phenolic Content (TPC)

The total phenolic content (TPC) of the extracts was determined using the Folin–Ciocalteu (FC) colorimetric assay, following the procedure described by Kosar et al. [35], with slight modifications. Briefly, 0.6 mL of distilled water, 10 μL of sample, and 50 μL of Folin–Ciocalteu reagent were mixed in a reaction tube, in triplicate. After 1 min of reaction, 150 μL of 20% (w/v) sodium carbonate solution and 190 μL of distilled water were added. The mixture was incubated at room temperature in the dark for 2 h. Subsequently, 300 μL of each reaction mixture was transferred, in triplicate, to a 96-well microplate, and the absorbance was measured at 760 nm using a microplate reader.

A gallic acid calibration curve (y = 1.0373x + 0.0838) was prepared following the same analytical procedure applied to the samples and was used to quantify the total phenolic content. The results are expressed as milligrams of gallic acid equivalents (mg GAE) per gram of dry extract.

2.5. Antioxidant Capacity

2.5.1. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts was evaluated using the DPPH radical scavenging assay, following the method of Brand-Williams et al. [36], with minor modifications. A stock solution was prepared by dissolving 23.5 mg/100 mL of DPPH in methanol, which was subsequently diluted at 1:10 with methanol to obtain the working solution. Four different concentrations of each extract were prepared to ensure a linear response range.

For the assay, 25 μL of each extract was mixed with 975 μL of the DPPH working solution in a reaction tube, resulting in a final volume of 1 mL. The mixture was incubated for 2 h at room temperature in the dark. Subsequently, 300 μL of each mixture was transferred into a 96-well microplate, and the absorbance was recorded at 516 nm using a microplate spectrophotometer.

A Trolox calibration curve (y = −0.5989x + 0.8141) was performed using concentrations from 2 to 0.2 mM and following the same analytical procedure applied to the samples to express the results as micromoles of Trolox equivalents per gram of extract (μmol Trolox/g extract). Each assay was performed in triplicate, and the antioxidant capacity was calculated from at least four different extract concentrations, ensuring a response within 20% to 80% of the initial absorbance.

2.5.2. Oxygen Radical Absorbance Capacity (ORAC)

The oxygen radical absorbance capacity (ORAC) assay was performed following the methodology described by Ou et al. [37], with slight modifications. The assay was performed in black 96-well microplates by mixing 100 µL of each extract, prepared at various concentrations, with 100 μL of AAPH solution (590 mM of AAPH in 30 mM phosphate buffer, pH 7.5), 25 μL of fluorescein solution (10 μM in phosphate buffer), and 100 μL of phosphate buffer. The fluorescence was recorded every 5 min over a 60 min period at 37 °C using a microplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The peroxyl radical scavenging capacity of the samples was quantified by calculating the percentage of inhibition relative to the control (absence of extract) based on the area under the curve (AUC) of the fluorescence decay over time. The following equation was used to determine the inhibition percentage:

$$\mathrm{\%} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{\mathrm{A}\mathrm{U}\mathrm{C}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{U}\mathrm{C}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}}{\mathrm{A}\mathrm{U}\mathrm{C}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

AUC was determined as follows:

$$\mathrm{A}\mathrm{U}\mathrm{C}=0.5+\sum fi/f0$$

where f0 represents the initial fluorescence at 0 min and fi represents the fluorescence at each 5 min interval. The results were expressed as mg ascorbic acid/g extract using ascorbic acid as the standard (y = 18.826x). The reaction was performed in triplicate for each extract.

FC, DPPH, and ORAC analyses were carried out using a BioTek Synergy HT UV–Vis spectrophotometer microplate reader (BioTek Instruments, Winooski, VT, USA).

2.6. Anticholinergic Capacity

The anticholinergic potential of the extracts was assessed by evaluating their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, employing the method described by Ellman et al. [38], with minor modifications [39]. In each assay, 100 μL of the extract, prepared at different concentrations (ranging from 150 to 1500 μg/mL in ethanol), was combined with 100 μL of 150 mM Tris-HCl buffer (pH 8.0) and 25 μL of enzyme solution (0.8 U/mL) containing either AChE or BChE, both prepared in the same buffer. The mixture was incubated for 10 min at room temperature. After incubation, 25 μL of ABD-F (125 μM) and 50 μL of ATCI (at the concentration determined by the Michaelis–Menten constant, KM) were added. The KM value was calculated by combining 100 μL of ATCI (0.4–4 mM), 50 μL of ethanol, 100 μL of buffer, 25 μL of ABD-F (125 μM), and 25 μL of AChE or BChE enzyme (0.8 U/mL). Fluorescence measurements were taken every minute for 10 min at 37 °C with excitation at λ = 389 nm and emission at λ = 513 nm to determine the KM and evaluate the anticholinergic activity of the extract. Galantamine hydrobromide (0.0125 mg/mL) was used as the standard for both enzyme assays.

The inhibitory concentration (IC₅₀) values were calculated, with lower IC₅₀ values indicating greater anticholinergic activity of the extracts. All assays were performed using a microplate reader (Cytation 5 Imaging Reader with Auto-Disperser, BioTek Instruments, Winooski, VT, USA).

2.7. Characterization of Phenolic Compounds from Burdock Extracts by HPLC−DAD−IT−MS

Polyphenol characterization was conducted using an Agilent HPLC Series 1100 (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler and a diode array detector (DAD), coupled to an Esquire 3000 Ion Trap Mass Spectrometer (Bruker, Bremen, Germany) with an Electrospray Ionization (ESI) source.

Separation was optimized for this study and was achieved using a ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm particle size; Phenomenex, Torrance, CA, USA). The separation process of the phenolic compounds was optimized by HPLC with various gradient programs, column temperatures (50, 40, 30, and 20 °C), and injection volumes (5 and 10 µL). Finally, the mobile phase consisted of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in methanol (B) with the following gradient elution: 5% B isocratic (0–1 min), 5–25% B (1–4 min), 25% B isocratic (4–12 min), 25–31% B (12–13 min), 31–43% B (13–25 min), 43% B isocratic (25–28 min), and 43–50% B (28–32 min). The flow rate was set at 1.2 mL/min, the injection volume was 10 μL, and the column temperature was maintained at 20 °C. Detection was performed at 280 nm for phenolic acids, 320 nm for flavonoids, and 520 nm for anthocyanins.

Mass spectrometric analysis was carried out according to Romano et al. [33] in both negative and positive ionization modes, with a mass range from m/z 50 to 1000. MS parameters included a capillary voltage of 3500 V, drying gas flow rate of 10 L/min, nebulizer pressure at 45 psi, and drying temperature set to 350 °C.

The phenolic compounds were exhaustively identified by comparing their fragmentation patterns with authentic standards. Quantification was performed using calibration curves for each compound. The standards used for identification and quantification included chlorogenic acid, cynarine, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 1,5-dicaffeoylquinic acid. In addition, some compounds were tentatively identified using database spectra (FOODB) and literature data.

2.8. Statistical Analysis

Statistical analysis was performed using Statgraphics Centurion XVII software (version 19.6.05) (Statistical Graphics Corp., USA) to evaluate the differences among burdock extracts in terms of TPC, antioxidant capacity, and anticholinergic activity. One-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) tests, were applied to determine statistically significant differences (p ≤ 0.05) between the mean values of the different extracts at a 95% confidence level. The data are expressed as the mean ± standard deviation. All analyses were conducted in triplicate for each extract to ensure reliability and reproducibility.

SIMCA 14.0 software (MSK Data Analytics Solutions, Umetrics, Umeå, Sweden) was used to perform a multivariate statistical analysis based on the bioactivities of the extracts and the quantification of phenolic compounds identified by HPLC-DAD. In addition, a hierarchical clustering analysis (HCA), sorted by height using the Ward method, as well as an unsupervised multivariate principal component analysis (PCA), were applied to generate statistical models represented as score and loading plots.

3. Results

3.1. Optimizing the PLE Polyphenol Extraction from Burdock Roots

To assess the influence of PLE temperature on the bioactivity and phenolic composition of Arctium lappa L. roots, an optimization study was initially conducted using samples from OLE, cultivated at an altitude of 150 m under agronomical practices involving the exclusion of other plant species. Once the optimal extraction conditions were established for the OLE samples, these conditions were subsequently applied to two additional burdock root ecotypes, as follows: SPLE, consisting of wild-grown roots at 150 m, and SPME, comprising wild-grown roots at 800 m, both without being subjected to weeding. The phenolic profiles and bioactivities of the three ecotypes (OLE, SPLE, and SPME) were then compared to elucidate the impact of environmental conditions and agronomic practices on the phenolic composition and associated bioactivities of burdock roots.

Considering that PLE parameters have been widely optimized for phenolic recovery from natural matrices, only the extraction temperature was selected for optimization in this study, with the specific objective of maximizing phenolic compound recovery while minimizing potential thermal degradation [34,39,40,41]. Three extraction temperatures (50 °C, 100 °C, and 150 °C) were evaluated using an EtOH/H₂O (70:30, v/v) solvent system under 1500 psi for 20 min, following the protocol described by Gallego et al. [34]. The resulting extracts were subsequently compared based on their TPC, antioxidant capacity (DPPH and ORAC assays), and anticholinergic activity (AChE and BChE inhibitory assays).

In addition, the PLE extracts were compared to a conventional solid–liquid extract (SLE) obtained at room temperature over 24 h using the same solvent system. As shown in

Table 1. Extraction yield, total phenolic content (Folin–Ciocalteu assay), antioxidant activity (DPPH and ORAC methods), and anticholinergic capacity (AChE and BChE assays) of OLE of burdock root extracts obtained by SLE and PLE extracts at different extraction temperatures (50, 100, and 150 °C).

Extraction Method	Yield (%)	TPC (mg Gallic Acid/g Extract)	DPPH (µmol Trolox/g Extract)	ORAC IC50 (mg/mL)	AChE IC50 (µg/mL)	BChE IC50 (µg/mL)
OE SLE	23 ± 1 b	38 ± 3 a	113 ± 1 b	1.7 ± 0.1 a	505 ± 136 a	751 ± 33 a
OE PLE 50 °C	27 ± 5 b	42 ± 4 a	113± 1 a	2.85 ± 0.08 b	442 ± 21 a	791 ± 80 a
OE PLE 100 °C	26 ± 1 b	33 ± 3 b	104 ± 3 b	2.9 ± 0.3 b	821 ± 76 b	1151 ± 76 b
OE PLE 150 °C	45 ± 2 a	25 ± 2 c	98 ± 5 b	2.3 ± 0.1 c	912 ± 45 b	1292 ± 46 b

^a,b,c Letters indicate statistically significant differences (p ≤ 0.05) in ANOVA by Fisher’s exact test among extractions.

, the PLE at 150 °C yielded the highest extraction efficiency (45 ± 2%) in terms of extract mass. However, this extract exhibited the lowest TPC values (25 ± 2 mg gallic acid/g extract) compared to those obtained by PLE at 50 °C (42 ± 4 mg gallic acid/g extract) and 100 °C (33 ± 2 mg gallic acid/g extract), with SLE (38 ± 3 mg gallic acid/g extract) producing the highest phenolic content among all conditions tested.

Table 1 shows that higher TPC values implied higher bioactivity. In this sense, the PLE extract obtained at 50 °C exhibited the highest antioxidant capacity (113 ± 1 µmol Trolox/g extract for DPPH and an IC₅₀ value of 2.85 ± 0.08 mg/mL for the ORAC assay) compared to the rest of the PLE extracts, but without statistically significant differences compared to SLE concerning the DPPH assay (113 ± 1 µmol Trolox/g extract). Nevertheless, the SLE extract presented a higher antioxidant capacity, determined by the ORAC assay (IC₅₀ 1.7 ± 0.1 mg/mL), than the PLE extract obtained at 50 °C (IC₅₀ 2.85 ± 0.08 mg/mL).

In terms of anticholinergic activity, the PLE extract obtained at 50 °C exhibited the highest AChE and BChE inhibitory effect, yielding the lowest IC₅₀ values (442 ± 21 and 791 ± 80 µg/mL, respectively). The bioactivity of these extracts was in line with those obtained from the conventional SLE method and was notably higher than those obtained at increased extraction temperatures.

The extracts produced using PLE at 50 °C and those from conventional SLE exhibited similar total phenolic content (42 ± 4 and 38 ± 3 mg gallic acid/g extract, respectively), antioxidant capacity (the same for DPPH, 113 ± 1 µmol Trolox/g extract), IC₅₀ values (2.85 ± 0.08 and 1.7 ± 0.1 mg/mL for the ORAC assay, respectively), and anticholinergic activity (IC₅₀ = 442 ± 21 and 505 ± 136 µg/mL for AChE; and 791 ± 80 and 751 ± 33 µg/mL for BChE, respectively). However, it is noteworthy that PLE required only 20 min of extraction time, whereas SLE needed 24 h, thereby consuming significantly more time and energy.

3.2. Influence of the Extraction Temperatures on the Phenolic Profile of PLE Extracts Obtained from OLE Burdock Roots

After qualitative analysis, quantitative differences among the extractions were determined by HPLC-DAD-IT-MS using caffeoylquinic acid standards. Figure 1 shows the HPLC-DAD chromatograms (at 280 nm, corresponding to the phenolic compounds) recovered from SLE and PLE at 50, 100, and 150 °C extracts of the organic burdock ecotypes.

Table 2. Mass data spectra and quantification (g/100 g of dried extract) of phenolic compounds in OLE burdock root extracts obtained by SLE and PLE at different temperatures (50, 100, and 150 °C) by HPLC-DAD-IT-MS.

No.	Compound	Rt (min)	[M−H]−	MS2 Ions	SLE (g/100 g of Dried Extract)	PLE 50 °C (g/100 g of Dried Extract)	PLE 100 °C (g/100 g of Dried Extract)	PLE 150 °C (g/100 g of Dried Extract)
1	Chlorogenic acid	7.9	374.9 [M−H+Na]−	353.2, 190.7, 178.7	0.67 ± 0.02 c	1.06 ± 0.03 a	0.893 ± 0.007 b	0.26 ± 0.01 d
2	4-O-Caffeoylquinic acid	8.32	353.0	190.8, 178.8, 135.0	0.015 ± 0.002 a	0.0095 ± 0.0002 b	0.0039 ± 0.0004 c	ND
3	Cynarine	10.25	515.1	353.4, 190.8, 178.8, 135.0	0.55 ± 0.02 c	0.99 ± 0.05 a	0.862 ± 0.005 b	0.30 ± 0.02 d
4	Dicaffeoylmaloylquinic acid *	17.9	631	573.3, 514.9, 468.6, 449.7, 379, 353, 334.9, 298.9, 255.1	0.079 ± 0.004 c	0.27±0.03 a	0.18±0.03 b	0.102 ± 0.004 c
5	Dicaffeoylmaloylquinic acid isomer 1 *	19.3	652.6 [M−H+Na]−	631, 514.9, 490.9, 469.1, 451.1, 353.4, 191.0, 173.1, 135	0.074 ± 0.008 c	0.18 ± 0.02 a	0.11 ± 0.01 b	0.062 ± 0.003 c
6	Dicaffeoylquinic acid *	20.2	536.8 [M−H+Na]−	515.1, 375, 354.1, 173.1, 135.4	0.025 ± 0.002 c	0.065 ± 0.008 a	0.037 ± 0.005 b	0.020 ± 0.002 c
7	3,5-Dicaffeoylquinic acid	21.1	515	353, 190.9, 178,9	0.060 ± 0.004 b,c	0.145 ± 0.007 a	0.0982 ± 0.003 b	0.044 ± 0.001 d
8	1,5-Dicaffeoylquinic acid	21.7	515	353.1, 190.9, 178.9,	1.06 ± 0.04 c	2.1 ± 0.1 a	1.485 ± 0.001 b	0.84 ± 0.04 d
9	Dicaffeoylmaloylquinic acid isomer 2 *	22.6	631	515, 490.9, 469, 449, 353, 191, 173, 135	0.041 ± 0.003 c	0.13 ± 0.01 a	0.09 ± 0.02 b	0.051 ± 0.004 c
10	Dicaffeoyldimaloylquinic acid *	24.7	747	631, 515, 469, 451, 433, 353, 335, 191	0.118 ± 0.008 c	0.30 ± 0.02 a	0.21 ± 0.03 b	0.117 ± 0.003 c

^a,b,c,d Letters indicate statistically significant differences (p ≤ 0.05) in ANOVA by Fisher’s exact test among extractions. * Compounds quantified by g equivalents of 1,5-dicaffeoylquinic acid/100 g extract; ND: Non-detected.

shows ten different phenolic compounds identified in burdock roots, from which five were exhaustively identified using the following standards: chlorogenic acid (number 1, tR = 7.9 min), 4-O-caffeolquinic acid (number 2, tR = 8.32 min), cynarine (number 3, tR = 10.25 min), 3,5-dicaffeoylquinic acid (number 7, tR = 21.1 min), and 1,5-dicaffeoylquinic acid (number 8, tR = 21.7 min). Particularly, Figure S1 shows a heatmap visualization of the abundance of compounds identified in SLE and PLE extracts obtained at 50 °C, 100 °C, and 150 °C. Mono-caffeoylquinic acids, which include chlorogenic acid (3-O-caffeoylquinic acid) and 4-O-caffeoylquinic acid (numbers 1 and 2; Table 2), were identified by the molecular ion at m/z 374.9 [M−−H+Na]⁻ and 465 [M−H], respectively. Chlorogenic acid presented fragment ions at m/z 190 [M−162]⁻, corresponding to the loss of quinic acid, and a fragment ion at m/z 178 [M−174]⁻, produced by the loss of caffeic acid. Chlorogenic acid was the mono-caffeoylquinic acid with the highest content in PLE extracts (1.06 ± 0.03 g/100 g of dried PLE extract obtained at 50 °C). A reduction in the concentration of chlorogenic acid was observed as the extraction temperature increased (0.893 ± 0.007 and 0.26 ± 0.01 g/100 g of dried PLE extract obtained at 100 °C and 150 °C, respectively).

On the other hand, four dicaffeoylquinic acids were identified with a molecular ion at m/z 515 [M−H]⁻, which sometimes can be found forming a sodium adduct with a molecular ion at m/z 537 [M−H+Na]⁻. In fact, dicaffeoylquinic acids (numbers 3, 6, 7, 8; Table 2) were observed in this study with a molecular ion at m/z 537, with their corresponding fragment ions at m/z 353 [M−162]⁻, 190 [M−324]⁻, and 178 [M−336]⁻ originating from the loss of one and two caffeic acid molecules and a quinic acid molecule, respectively. In addition, dicaffeoylquinic acids such as cynarine (number 3), 3,5-dicaffeoylquinic acid (number 7), 1,5-dicaffeoylquinic acid (number 8), and other dicaffeoylquinic acid isomers were observed. Moreover, another dicaffeoylquinic acid was observed at tR = 20.2 min (number 6), which cannot be exhaustively identified and differentiated from its isomers by the standards; therefore, its content was expressed as g equivalents of the 1,5-dicaffeoylquinic acid/100 g extract. As can be seen in Table 2, these four phenolic compounds were obtained with the highest content using PLE at 50 °C, decreasing their content when higher PLE temperatures were applied.

In addition, dicaffeoylmaloylquinic acid (number 9) was tentatively identified with a molecular ion at m/z 631 [M−H]⁻, which presented fragment ions at m/z 515 [M−116]⁻, 353 [M−278]⁻, 190 [M−440]⁻, and 178 [M−452]⁻, corresponding to the loss of malic acid, one and two caffeic acid molecules, and a quinic acid molecule, respectively. Dicaffeoyldimaloylquinic acid showed one malic acid molecule more than dicaffeoylmaloylquinic acid; therefore, this compound was detected with a molecular ion at m/z 747 [M−H]⁻ (number 10) [42]. Up to four different dicaffeoylmaloylquinic acids were detected by HPLC-DAD-IT-MS, which were quantified as g equivalents of the 1,5-dicaffeoyluqinic acid/100 g extract, showing that PLE at 50 °C was the best condition for the extraction of these compounds.

In general, as can be seen in Figure 1 and Table 2, the highest phenolic content was observed in the PLE extracts obtained at 50 °C, except for 4-O-caffeoylquinic acid, where a higher concentration was observed in the SLE extract (0.015 ± 0.002 g/100 g of dried extract) (see Table 2). For this reason, and considering that PLE at 50 °C also achieved extracts with the highest anticholinergic and antioxidant properties, this condition was selected for the extraction of phenolic compounds from the different ecotypes of burdock roots.

3.3. Extraction of Bioactive Phenolic Compounds from Different Ecotypes of Burdock Roots

The optimized PLE conditions, specifically the extraction temperature of 50 °C, established using OLE burdock roots, were applied to extract bioactive molecules from the SPME and SPLE ecotypes. This approach allowed for the comparison of three burdock root ecotypes, each harvested from different altitudes (150 m and 800 m).

Differences in TPC and bioactivity were observed among the ecotypes (see

Table 3. Comparison of extraction yield, total phenolic content (Folin–Ciocalteu assay), antioxidant activity (DPPH and ORAC assays), and anticholinergic capacity (AChE and BChE assays) of OLE, SPLE, and SPME of burdock root extracts obtained using SLE and PLE at 50 °C.

Ecotype	Yield (%)	TPC (mg Gallic Acid/g Extract)	ORAC IC50 (mg/mL)	DPPH (µmol Trolox/g Extract)	AChE IC50 (µg/mL)	BChE IC50 (µg/mL)
OLE	27 ± 5 b	55 ± 1 a	2.85 ± 0.08 c	113 ± 2 c	442 ± 5 a	791 ± 80 c
SPLE	32 ± 3 b	42 ± 2 b	1.09 ± 0.06 b	268 ± 24 b	975 ± 11 b	527 ± 33 b
SPME	38 ± 3 a	90 ± 7 a	0.55 ± 0.08 a	526 ± 138 a	313 ± 4 a	298 ± 21 a

^a,b,c Letters indicate statistically significant differences (p ≤ 0.05) in ANOVA by Fisher’s exact test among burdock root ecotypes.

). As shown, the ecotype harvested at the higher altitude of 800 m (SPME) exhibited the highest TPC values (90 ± 7 mg gallic acid/g extract), along with superior anticholinergic (IC₅₀ = 313 ± 4 and 298 ± 21 µg/mL for the AChE and BChE assays, respectively) and antioxidant activities (526 ± 138 µmol Trolox/g extract for the DPPH assay and an IC₅₀ value of 0.55 ± 0.08 mg/mL for the ORAC assay), when compared to both the OLE and SPLE ecotypes, which were collected at 150 m.

As shown in Table 3, the OLE extract exhibited higher TPC (55 ± 1 mg gallic acid) and greater AChE inhibition (IC₅₀ = 442 ± 5 µg/mL) than the SPLE extract (32 ± 3 mg gallic acid and IC₅₀ = 791 ± 80 µg/mL). Conversely, the SPLE extract demonstrated superior antioxidant and anticholinergic activities, as assessed by the ORAC (IC₅₀ = 1.09 ± 0.06 mg/mL) and BChE (IC₅₀ = 527 ± 33 µg/mL) assays, respectively.

Furthermore, a comparative phenolic profile analysis and quantification were conducted among the ecotypes using HPLC-DAD. As depicted in Figure 2 and summarized in

Table 4. Mass data spectra and quantification (g/100 g of dried extract) of phenolic compounds in the three different burdock ecotypes (OLE, SPLE, and SPME) obtained by PLE at 50 °C using HPLC-DAD-IT-MS.

No.	Compound	Rt (min)	[M−H]−	MS2 Ions	OLE PLE 50 °C (g/100 g of Dried Extract)	SPLE PLE 50 °C (g/100 g of Dried Extract)	SPME PLE 50 °C (g/100 g of Dried Extract)
1	Chlorogenic acid	7.9	374.9 [M−H+Na]−	353.2, 190.7, 178.7	1.06 ± 0.03 b	1.12 ± 0.06 b	2.12 ± 0.03 a
2	4-O-Caffeoylquinic acid	8.32	353.0	190.8, 178.8, 135.0	0.0095 ± 0.0002 b	0.020 ± 0.002 a	0.0081 ± 0.0006 b
3	Cynarine	10.25	515.1	353.4, 190.8, 178.8, 135.0	0.99 ± 0.05 b	0.17 ± 0.09 b	2.05 ± 0.03 a
4	Dicaffeoylmaloylquinic acid *	17.9	631	573.3, 514.9, 468.6, 449.7, 379, 353, 334.9, 298.9, 255.1	0.27±0.03 c	0.30 ± 0.01 b	1.105±0.007 a
5	Dicaffeoylmaloylquinic acid isomer 1 *	19.3	652.6 [M−H+Na]−	631, 514.9, 490.9, 469.1, 451.1, 353.4, 191.0, 173.1, 135	0.13 ± 0.02 b	0.089 ± 0.006 c	0.308 ± 0.005 a
6	Dicaffeoylquinic acid *	20.2	536.8 [M−H+Na]−	515.1, 375, 354.1, 173.1, 135.4	0.065 ± 0.008 b	0.055 ± 0.007 b	0.086 ± 0.001 a
7	3,5-Dicaffeoylquinic acid	21.1	515	353, 190.9, 178,9	0.145 ± 0.007 b	0.09 ± 0.01 c	0.28 ± 0.02 a
8	1,5-Dicaffeoylquinic acid	21.7	515	353.1, 190.9, 178.9,	2.1 ± 0.1 b	2.0± 0.1 c	4.62 ± 0.08 a
9	Dicaffeoylmaloylquinic acid isomer 2 *	22.6	631	515, 490.9, 469, 449, 353, 191, 173, 135	0.13 ± 0.01 b	0.119 ± 0.006 b	0.488 ± 0.006 a
10	Dicaffeoyldimaloylquinic acid *	24.7	747	631, 515, 469, 451, 433, 353, 335, 191	0.30 ± 0.02 b	0.25 ± 0.01 c	0.473 ± 0.004 a

^a,b,c Letters indicate statistically significant differences (p ≤ 0.05) in ANOVA by Fisher’s exact test among extractions. * Compounds quantified by g equivalents of the 1,5-dicaffeoylquinic acid/100 g extract.

, it was observed that chlorogenic acid, cynarine, 1,5-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid were present at concentrations more than twice as high in the SPME extract compared to both the OLE and SPLE extracts. Notably, 4-O-caffeoylquinic acid was the only phenolic compound found at a higher concentration in the SPLE extract (0.020 ± 0.002 g/100 g of dried extract).

In addition, differences were observed between the OLE and SPLE extracts. Specifically, the SPLE extracts contained higher amounts of 4-O-caffeoylquinic acid and dicaffeoylmaloylquinic acid (0.020 ± 0.002 and 0.30 ± 0.01 g/100 g of dried extract, respectively), whereas the OLE extracts had greater concentrations of dicaffeoylmaloylquinic acid isomer 1, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, and dicaffeoyldimaloylquinic acid (0.13 ± 0.02, 0.145 ± 0.007, 2.1 ± 0.1, and 0.30 ± 0.02 g/100 g of dried extract, respectively) (see Table 4).

3.4. Relationship Between Phenolic Profile and Bioactivities of Extracts

To assess the contribution of each quantified phenolic compound to the bioactivity of the extracts, a multivariate statistical analysis was performed. The burdock root extracts were categorized into groups based on similarities in extraction yield, TPC, antioxidant and anticholinergic capacities, and the phenolic composition identified by HPLC-DAD. Hierarchical Cluster Analysis (HCA) (Figure 3A) revealed the formation of two distinct clusters, where one cluster grouped the SPME extract (green type), while the SPLE and OLE extracts were classified into a second cluster (blue type).

Furthermore, principal component analysis (PCA) was employed to identify the most influential variables, as determined by the analytical techniques applied in this study. The first principal component accounted for 83.57% of the total variance in the data. PCA grouped the extracts similarly to the HCA results (Figure 3B), with each group demonstrating a comparable bioactive phenolic profile. For example, group 1 (green type), corresponding to the SPME extract, was positioned in the quadrant associated with the highest TPC values in the PCA loading plot (overlapping Figure 3B,C).

Moreover, the SPME extract exhibited the highest antioxidant capacity, as determined by both the DPPH and ORAC assays, as well as the greatest anticholinergic activity, assessed through the AChE and BChE assays. This extract was positioned in the opposite quadrant of the PCA loading plot, relative to the bioactivities, reflecting the inverse relationship between IC₅₀ values (with lower IC₅₀ indicating higher activity). In contrast, the OLE and SPLE extracts displayed the lowest bioactivity, as they were located further from the bioactivity axes in the PCA loading plot.

The analysis of the phenolic compounds regarding bioactivity indicated that dicaffeoyldimaloylquinic acid (compound 10), dicaffeoylmaloylquinic acid 2 (compound 9), and 1,5-dicaffeoylquinic acid (compound 8) contributed most significantly to the high bioactivity of the SPME extract. These compounds were closest to the bioactivity metrics within the PCA loading plot. While the OLE extract contained the highest amount of 4-O-caffeoylquinic acid, this compound did not demonstrate higher bioactivity compared to the other phenolic compounds found in higher concentrations in the SPME extract.

4. Discussion

4.1. Influence of PLE Temperature on the Recovery of Phenolic Compounds from Burdock Roots

The extraction of phenolic compounds from various natural matrices using PLE has been extensively studied [33,38,39,40,43]. In this context, several researchers have utilized the optimized PLE conditions established by Herrero et al. [43], which involve the use of EtOH/H₂O solvent mixture (70:30, v/v) for 20 min at 1500 psi [33,38,39,40]. These conditions were applied to extract phenolic compounds from burdock roots. The extraction temperature is a critical parameter influencing the recovery of phenolic compounds. Depending on the matrix composition, elevated temperatures may induce undesired chemical reactions or lead to the degradation of thermolabile compounds, ultimately diminishing the bioactive potential of the extracts [33]. Phenolic compounds in burdock, including chlorogenic acid, are known to be particularly sensitive to thermal degradation [44]. In fact, Romano et al. [33] observed that PLE temperatures higher than 100 °C result in the degradation of several phenolic compounds from burdock roots, including chlorogenic acid, when 2-MTHF is used as an extraction solvent. However, the type of solvent used can influence the optimal extraction temperature, as solvents differ in their polarity, hydrogen-bonding capacity, and thermal stability, which can affect not only the solubility of phenolic compounds, but also their susceptibility to thermal degradation. Certain solvents can create a more protective microenvironment by stabilizing phenolic structures through solvent–solute interactions, thereby reducing oxidation or decomposition during high-temperature extraction processes. Considering that the EtOH/H₂O mixture (70:30) is the most commonly used solvent system for extracting phenolic compounds from natural matrices, it is essential to understand how extraction temperature affects the stability, recovery, and overall profile of phenolic compounds when using this solvent. Temperature variations can influence the solubility and diffusion of phenolics, but may also promote their degradation or transformation, which underscores the importance of optimizing thermal conditions for this widely applied solvent mixture.

The results revealed that, while PLE at 150 °C resulted in the highest yields, this extract exhibited the lowest TPC when compared to extracts obtained at 50 °C and 100 °C, as well as the SLE extracts (see Table 1). This decrease in TPC at higher temperatures could be attributed to the thermal degradation of polyphenols. It is well-documented that SLE at temperatures above 80 °C can lead to polyphenol degradation, while PLE has been shown to resist such degradation at higher temperatures, ranging from 150 °C to 200 °C [45]. Additionally, the OLE extract obtained at 150 °C appeared darker and emitted a stronger aroma compared to extracts obtained at lower temperatures, which is likely due to the formation of Maillard reaction products, a well-known phenomenon in thermally treated samples [46,47]. Furthermore, the OLE PLE extract obtained at 100 °C exhibited lower total phenolic content (TPC) compared to the extract obtained at 50 °C in this study. This effect was also observed by Romano et al. [33] when using PLE temperatures higher than 100 °C, achieving dark burdock root extracts using 2-MTHF as solvent. This observation underscores the high thermosensitivity of the polyphenols in burdock roots, but also that this sensitivity depends on the extraction solvent employed [33]. It is probable that the degradation of phenolic compounds from burdock roots during PLE at temperatures above 50 °C with EtOH/H₂O is mainly due to the high polarity and reactivity of the solvent mixture, which can promote oxidation, hydrolysis, and thermal breakdown [33,41,43]. In contrast, 2-MTHF, a less polar and more thermally stable solvent, provides a protective environment that minimizes degradation, even at 100 °C, which is likely due to reduced water content, lower radical formation, and the selective extraction of phenolic compounds without co-extracting pro-oxidant components [33].

The observed differences in antioxidant capacity among the extracts can be attributed not only to their phenolic composition, but also to the specific assay employed for evaluation, as each method relies on distinct mechanisms of action. Phenolic compounds, due to their structural features, particularly the presence of multiple hydroxyl groups and conjugated systems, can act as antioxidants through various pathways. However, their measured antioxidant activity may vary significantly depending on whether the assay favors electron transfer (ET) or hydrogen atom transfer (HAT) reactions. [38]. In this study, the PLE extract obtained at 50 °C exhibited the highest antioxidant activity when assessed using the DPPH assay, which is based on an ET mechanism. This method measures the ability of antioxidant compounds to transfer an electron to the DPPH radical, thereby stabilizing it [48]. In contrast, the SLE extract demonstrated superior antioxidant performance in the ORAC assay, which operates via a HAT mechanism. The ORAC method evaluates the ability of antioxidants to quench peroxyl radicals (ROO•) through hydrogen atom donation, a mechanism that closely mimics the oxidative processes occurring in biological systems, as peroxyl radicals are among the most prevalent reactive oxygen species in the human body [48]. These findings highlight that the apparent antioxidant capacity of a given extract is highly dependent on the assay used, due to the differing radical species involved and the dominant reaction mechanism. While DPPH is useful for assessing electron-donating capacity, ORAC provides a more physiologically relevant estimate of antioxidant potential, as it reflects the extract’s ability to neutralize biologically relevant radicals. Therefore, relying on a single method may offer an incomplete or misleading assessment of antioxidant efficacy. The combined use of both ET- and HAT-based assays allows for a more comprehensive understanding of the antioxidant behavior of phenolic compounds under conditions that approximate those found in vivo [33,49]. Additionally, comparing extraction solvents, ethanolic PLE extracts exhibited higher antioxidant capacity determined by an ORAC assay (IC₅₀ from 0.55 to 2.85 mg/mL extract) than 2-MTHF PLE extracts (IC₅₀ from 3.1 to 9 mg/mL extract) obtained by Romano et al. [33] from burdock roots. However, the 2-MTHF extracts presented higher anticholinergic capacity determined by AChE (IC₅₀ from 29 to 94 µg/mL) and BChE assays (IC₅₀ from 43 to 83 µg/mL) than the ethanolic extracts of this study (AChE IC₅₀ from 313 to 442 µg/mL, BChE IC₅₀ from 298 to 791 µg/mL). This difference may be explained by the chemical selectivity of the solvents. EtOH/H₂O mixtures tend to extract polar phenolic compounds, many of which are known for their strong antioxidant activity, particularly through hydrogen atom donation mechanisms measured by the ORAC assay [41]. In contrast, 2-MTHF is a less polar, more lipophilic solvent that favors the extraction of less polar phenolics and other bioactive compounds, such as certain flavonoid aglycones or terpenoids, which may exhibit stronger inhibitory effects on cholinesterase enzymes (AChE and BChE) [33]. These nonpolar or moderately polar compounds might not contribute significantly to antioxidant capacity in ORAC assays, but they can be more effective in targeting enzymatic active sites, thereby explaining the higher anticholinergic activity observed in the 2-MTHF extracts [33].

Moreover, the OLE PLE extracts obtained at 50 °C exhibited comparable TPC values and bioactivity to the OLE SLE extracts. However, SLE requires a longer exposure time, leading to increased phenolic degradation due to prolonged oxidative reactions and higher energy consumption. Nevertheless, Romano et al. [33] observed that PLE extracts obtained from burdock roots at 100 °C using 2-MTHF resulted in higher TPC values (84 mg gallic acid/g extract) than maceration (9.1 mg gallic acid/g extract). This could be due to 2-MTHF providing a protective effect against the degradation of phenolic compounds during the extraction that was not observed using the EtOH:H₂O solvent in this study. In fact, the sensitivity of the principal phenolic compounds in burdock roots to extraction temperature and extended exposure during SLE was further examined through quantitative analysis using HPLC-DAD-IT-MS. The results revealed that both higher PLE temperatures and prolonged exposure during SLE resulted in a reduction in all identified phenolic compounds when EtOH:H₂O was used as the extraction solvent (see Table 2). The shorter extraction times employed in PLE minimize the exposure to oxygen and thereby reduce oxidative reactions, yielding higher-quality extracts. Consequently, PLE offers a more environmentally friendly alternative, overcoming many of the drawbacks associated with SLE.

4.2. Influence of Growing Conditions of Burdock Roots on the Phenolic Composition and Bioactivity of Its PLE Extracts

It is well established that climatic conditions play a significant role in influencing the phenolic synthesis in plants [9,10,33,50]. To evaluate the impact of environmental factors on the bioactivity and phenolic composition of burdock roots, the specific climatic conditions of the areas where the roots were harvested were taken into account. Notably, as shown in Figure S2, the climatic conditions in Collepardo and Isola del Liri (Italy) were consistent across the years of harvest (2021 and 2022), suggesting that the observed differences among the ecotypes were primarily attributable to altitude and the unique growing conditions of the plants [33].

Regarding the OLE and SPLE ecotypes, which were harvested at the same altitude (150 m), significant differences were observed between the two, indicating that land treatment played a crucial role in modulating the bioactivity of the burdock roots [14,15,16,17,18,19,20,21]. Specifically, the exclusion of other plant species during OLE cultivation resulted in extracts with higher TPC, enhanced antioxidant capacity, as determined by the ORAC assay, and greater AChE inhibition [33]. In contrast, the untreated SPLE extracts exhibited higher antioxidant capacity, as measured by the DPPH assay, and a stronger inhibition of BChE [33].

Although the OLE extracts contained a higher concentration of phenolic compounds than the other ecotypes, these compounds may be more general in nature and less specialized in responding to biotic stress. This could be attributed to the reduced competition for resources such as light, nutrients, and water in the OLE cultivation, which leads to lower synthesis of phenolic compounds that are typically produced in response to biotic stress [19,21,51,52]. In contrast, the SPLE ecotype, which was exposed to a more natural growing environment, could synthesize phenolic compounds that are more specialized for combating abiotic stress. As a result, and in agreement with Romano et al. [33], the SPLE extracts demonstrated higher levels of chlorogenic acid, cynarine, and dicaffeoylmaloylquinic acid compared to the OLE extracts, compounds that have demonstrated interesting bioactive properties [33]. It is probable that they are responsible for the enhanced antioxidant activity (evaluated by DPPH) and stronger BChE inhibition observed in the SPLE extracts of this study. Despite these differences, the bioactivities of the OLE and SPLE extracts were relatively similar, as reflected in the PCA, which grouped both ecotypes in the same cluster (see Figure 3).

Differences in the individual quantification of phenolic compounds using HPLC-DAD-IT-MS, TPC values, and bioactivity assays among the ecotypes revealed that burdock’s secondary metabolism is influenced by both growing conditions and land treatment. Notably, the higher altitude, atmospheric pressure, cold temperatures, and distinct soil conditions of the SPME ecotype compared to the OLE and SPLE may contribute to its increased phenolic content as a response to these environmental stressors, which is in agreement with that observed by Lu et al. [53] in different species of ephedra collected at different altitudes. Indeed, previous studies have shown that plants growing at high altitudes are more exposed to ultraviolet (UV) radiation, increased atmospheric pressure, and cold temperatures, which trigger the synthesis of phenolic compounds as a protective mechanism against these harsh conditions [14,15,16,17,18,19,20,21]. Consistent with this, other researchers have found that plants originating from cold climates and high altitudes tend to have higher phenolic contents [50]. These results agree with Romano et al. [33], who observed the same behavior for OLE, SPLE, and SPME extracts obtained by PLE using 2-MTHF. This consistent superiority of the SPME ecotype across different extraction techniques may be attributed to its intrinsically higher concentration and diversity of bioactive phenolic compounds, which remain stable or are more efficiently recovered regardless of the extraction conditions applied. However, it was noted that PLE with 2-MTHF at 100 °C provided the SPLE, OLE, and SPME burdock root extracts with higher phenolic content (22.9, 48.8, and 64 mg gallic acid/g extract, respectively) and bioactivity (IC₅₀ of ORAC assay from 3.1 to 9 mg/mL extract) than the extracts obtained by PLE with EtOH:H₂O (70:30, v/v) at 50 °C.

The majority of the phenolic compounds identified in the extracts were caffeoylquinic acids, characterized by their anti-inflammatory, antiviral, antidiabetic, and antitumor properties [54]. In addition, dicaffeoylquinic acid isomers were also identified, which can be differentiated using the standards. In agreement with a previous study, 1,5-dicaffeoylquinic acid (number 8) was identified as the major phenolic compound in the extracts of burdock leaves [55].

The findings from this study align with data from previous research, including prior studies where the same burdock samples were characterized using Nuclear Magnetic Resonance (NMR) spectroscopy and HPLC-DAD-IT-MS [33,56]. In these studies, Ambroselli et al. [57] and Romano et al. [33] observed that burdock ecotypes harvested at an altitude of 800 m exhibited the highest concentrations of sugars, amino acids, organic acids, and other bioactive compounds, including dicaffeoylquinic acid. In the current study, it was noted that higher altitudes were associated with the increased synthesis of dicaffeoyldimaloylquinic acid, dicaffeoylmaloylquinic acid, and 1,5-dicaffeoylquinic acid in burdock roots. These compounds were identified as the primary contributors to the antioxidant and anticholinergic capacities of the SPME extract, as shown in Figure 3.

5. Conclusions

This study provides the first evidence that both the extraction temperature applied during PLE with aqueous-organic solvents and the altitude of the cultivation (150 or 800 m) site play a significant role in determining the phenolic composition and biological activity of burdock root extracts. PLE at 50 °C yielded extracts from the OLE ecotype with a higher total phenolic content (55 mg gallic acid/g extract) with antioxidant (113 µmol Trolox/g extract for DPPH and with an IC₅₀ value of 2.85 mg/mL for the ORAC assay) and anticholinergic activities (with IC₅₀ values of 442 and 791 µg/mL for the AChE and BChE assays, respectively) than that of the employed PLE temperatures of 100 and 150 °C. Furthermore, burdock roots cultivated at higher altitudes (800 m) showed significantly greater phenolic content (90 mg gallic acid/g extract) and antioxidant activity (IC₅₀ ORAC assay = 0.55 mg/mL; DPPH = 526 µmol Trolox/g extract) (SPME extract) compared to those grown at lower elevations (150 m) (OLE and SPLE extracts). These findings support the hypothesis that plants exposed to higher-altitude environments experience increased abiotic stress, such as lower temperatures, greater UV radiation, and reduced oxygen availability, which likely stimulates the biosynthesis of phenolic compounds as part of their adaptive response.

Analysis by HPLC-DAD-IT-MS allowed the identification and quantification of ten phenolic compounds in the extracts, with 1,5-dicaffeoylquinic acid being the most abundant across all ecotypes (from 2.1 to 4.62 g/100 g of dried extract). In conclusion, this work contributes to the still-limited research examining the impact of altitude on the extraction and bioactivity of phenolic compounds from burdock roots, while also presenting valuable findings on their anticholinergic properties. This study provides preliminary knowledge that may serve as a basis for future investigations on the bioavailability and blood–brain barrier permeability of these compounds to exert their neuroprotective effects.