Characterization of Non-Polar and Polar Bioactive Compounds Obtained by Pressurized Biobased Solvents from Different Arctium lappa L. Root Ecotypes

Abstract

This study introduces a novel pressurized liquid extraction (PLE) strategy utilizing biobased solvents to simultaneously extract non-polar and polar compounds with antioxidant and anticholinergic properties from burdock roots. The influence of altitude and weeding on the bioactive composition of three burdock root ecotypes was evaluated: two from 150 m (one subjected to weeding during growth and another not subjected to weeding) and one from 800 m without weeding. A simplex-centroid mixture design identified 100% 2-methyltetrahydrofuran as the optimal solvent for PLE, offering superior extraction of bioactive compounds due to its ability to form strong hydrogen bonds with phenolic groups. Extraction at 100 °C was found to be optimal, avoiding the low yields and undesirable reactions observed at 40 °C and 160 °C, respectively. Altitude emerged as the most significant factor influencing bioactivity and composition, with roots from 800 m exhibiting the highest bioactivity. Key bioactive compounds included caffeoylquinic acids, caryophyllene oxide, spathulenol, and bisnorallocholanic acid. At 150 m, weeding reduced anticholinergic capacity but increased antioxidant synthesis, though the levels were lower than those observed at higher altitudes. These findings highlight that burdock roots grown at high altitudes without weeding produce extracts rich in antioxidant and neuroprotective compounds, offering significant potential for functional ingredient development.

1. Introduction

Phenolic compounds as well as more non-polar compounds like terpenoids found in plants have long been valued for their diverse bioactive properties, particularly their antioxidant and anticholinergic activities, which are critical for preventing neurodegenerative diseases and oxidative stress-related conditions [1]. Among various medicinal plants, burdock (Arctium lappa L.), and particularly burdock roots, are recognized as a rich source of these bioactive compounds [2]. Burdock roots have been traditionally used in herbal medicine and functional foods for their health-promoting effects, attributed to their content of phenolic acids and flavonoids, including chlorogenic acid and caffeoylquinic acids [3,4]. In addition, the beneficial properties of burdock plant have also been related to their terpenoid content, although studies on the elucidation of the terpenoid profile of burdock roots are very limited [2]. These compounds have exhibited potential antioxidant, anticholinergic, and anti-inflammatory properties, among others [4,5,6,7]. These properties are particularly relevant in addressing multifactorial diseases like Alzheimer’s, which involve mechanisms such as neuroinflammation, oxidative stress, and the accumulation of β-amyloid aggregates and tau protein. Plant-derived phenolic compounds and terpenoids have gained attention for their ability to target these diverse pathways and mitigate the underlying causes of neurodegeneration. This research is crucial, as current medications primarily manage symptoms during the early stages of the disease, emphasizing the urgent need for innovative therapies that address its complex pathology [8].

It has been observed that the antioxidant capacity of phenolic compounds and terpenes is linked to their neuroprotection capacity due to their ability to modulate oxidative stress and inflammation, both of which are key factors in the development of neurodegenerative diseases [9]. Phenolic compounds, known for their strong antioxidant properties, can neutralize free radicals and reduce oxidative damage to neurons [10]. This action can prevent the degradation of acetylcholine, a neurotransmitter crucial for memory and cognitive function. Similarly, certain terpenes, including sesquiterpenes and triterpenes, have been shown to possess antioxidant and anti-inflammatory properties that contribute to neuroprotection by reducing oxidative stress supporting acetylcholine activity, and reducing the pro-inflammatory cytokines expression involved in the neuroinflammation process [7,11]. However, to our knowledge, the neuroprotective capacity of burdock roots has been scarcely evaluated. The neuroprotective potential of phenolic compounds and terpenes from natural sources has been demonstrated in numerous studies, highlighting the importance of continuing the investigations of bioactivity and sustainable and efficient extraction of phenolic compounds and terpenoids from undervalued natural plants, like burdock roots. The efficient and sustainable approaches are essential not only to maximize the yield of these valuable compounds but also to reduce the environmental impact and ensure the scalability of their use in developing innovative therapies for neurodegenerative diseases.

Phenolic compounds and terpenoids have traditionally been extracted using conventional solvents such as ethanol, methanol, hexane, and chloroform, employing maceration, steam distillation, or hydrodistillation systems, among others [12,13]. However, growing environmental concerns have shifted the chemical industry’s focus toward developing sustainable extraction methods. These efforts align with the Principles of Green Analytical Chemistry, which emphasize minimizing the use of hazardous substances, reducing energy consumption, and decreasing the waste production associated with traditional extraction processes [14]. In this sense, biobased solvents emerged as non-toxic, biodegradable, recyclable, and easy-to-acquire alternatives to conventional solvents. Biobased solvents, sourced from renewable materials like plant biomass, encompass a range of options, including hydrophilic solvents such as glycerol, ethyl acetate (EtOAc), and ethyl lactate, as well as more hydrophobic alternatives like D-limonene, 2-methyltetrahydrofuran (2-MTHF), and cyclopentyl methyl ether (CPME), among others [15,16,17]. These solvents are widely utilized for extracting phenolic compounds and terpenoids from various matrices [15,16,18]. In some cases, they have been paired with advanced extraction methods, including ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and pressurized liquid extraction (PLE), to enhance the recovery of phenolic compounds and terpenoids from natural sources [15,16,19]. Particularly, ethyl lactate has been used in combination with PLE for the recovery of phenolic compounds like rosmarinic acid and caffeic acid from Ocimum basilicum L., while D-limonene has been used for the PLE extraction of terpenoids from Ruta graveolens L. [15,20]. Despite their potential to obtain higher extraction yields than conventional solvents, biobased solvents have not been widely investigated in the extraction of phenolic compounds from natural plants [15,16,19]. In fact, there are no studies on the extraction of phenolic compounds by PLE with biobased solvents from burdock roots, even using conventional solvents. In addition, little attention has been paid to the non-polar compounds, like terpenoids, that can be co-extracted from burdock roots when hydrophobic solvents such as 2-MTHF or CPME are used.

On the other hand, the presence of secondary metabolites in burdock roots depends on the growing conditions or the plant treatment at the place where the plant is collected. It has been reported that phenolic compounds and terpenoids are synthesized by plants in response to stress, playing key roles in the defense against biotic and abiotic factors [21,22,23]. They are involved in protecting against herbivores, pathogens, UV radiation, drought, and extreme temperatures. Particularly, different researchers have observed that weeding treatments or altitude can influence the content of bioactive compounds in plants [24,25,26]. However, to our knowledge, there are no studies that determine the influence of factors such as altitude and weeding of burdock on the content of bioactive compounds in its roots. Their study can help understanding how these factors affect burdock cultivation, enhance the quality of its derived products, and promote agricultural sustainability.

Considering the above, this study addresses the issue of the limited research on obtaining neuroprotective compounds from burdock roots, which has primarily focused on their phenolic composition. Little attention has been given to the extraction of non-polar compounds, such as terpenes, and most studies have relied on conventional extraction methods using solvents like ethanol and methanol with water. Additionally, no previous studies have explored how growth conditions, such as altitude and weeding, influence the composition and bioactivity of burdock roots. Taking this into account, the hypothesis of this study is that PLE with biobased solvents will provide higher yields of antioxidant and neuroprotective compounds compared to conventional extraction and that growth at higher altitudes without weeding will enhance the production of bioactive compounds. Therefore, this work proposes, for the first time, the development of a sustainable extraction methodology for the extraction of antioxidant and anticholinergic non-polar and polar compounds from burdock roots based on the combination of biobased solvents with PLE. A simplex-centroid experimental design was used to determine the optimal mixture proportion of biobased solvents by using CPME, 2-MTHF, and EtOAc to obtain the richest extracts in phenolic compounds according to their total peak areas determined by HPLC-DAD. In addition, the PLE extraction temperature was studied by testing three different extraction temperatures (40 °C, 100 °C, and 160 °C) to obtain the richest extracts in antioxidant and anticholinergic compounds. Furthermore, the antioxidant and anticholinergic capacities of burdock roots collected at an altitude of 800 m without the weeding process (namely the spontaneous mountain ecotype (SPME)) were compared with burdock roots collected at 150 m without and with weeding processes (namely the spontaneous land ecotype (SPLE) and the organic land ecotype (OLE)). Additionally, the extraction of non-polar compounds, like terpenoids, during the optimized pressurized biobased solvent extraction of phenolic compounds was evaluated by GC-MS. In addition, the phenolic profile of these ecotypes was also compared by HPLC-DAD-MS analysis.

2. Materials and Methods

2.1. Chemicals and Reagents

LC-MS grade acetonitrile and formic acid were obtained from VWR Chemicals (Barcelona, Spain). Reagents including Trizma hydrochloride, disodium phosphate, fluorescein sodium salt, Trolox, potassium persulfate, monopotassium phosphate, sodium carbonate, butyrylcholinesterase from equine serum, acetylcholinesterase, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), gallic acid, ascorbic acid, CPME, 2-MTHF, and EtOAc were purchased from Sigma-Aldrich (Madrid, Spain). Galantamine hydrobromide, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F), and 2,2-azobis(2-aminodinopropane) dihydrochloride (AAPH) were sourced from TCI Chemicals (Tokyo, Japan). Additionally, Folin–Ciocalteu reagent was obtained from Merck (Darmstadt, Germany).

Ultrapure water (18.2 MΩ/cm) was provided by a Millipore purification system (Millipore, Billerica, MA, USA).

2.2. Samples and Sample Preparation

Roots from three ecotypes of Arctium lappa L. were supplied by the Fibreno Officinali company (Isola del Liri, Lazio, Italy). The ecotypes included (1) a spontaneous land ecotype (SPLE) collected in Isola de Liri at 150 m altitude, growing wild and without removing weeds; (2) an organic land ecotype (OLE) collected in Isola del Liri at 150 m altitude and subjected to weeding; and (3) a spontaneous mountain ecotype (SPME) growing wild in Collepardo (Lazio, Italy) collected at 800 m altitude without removing weeds. The SPLE and OLE ecotypes were collected in October 2021, while the SPME was harvested in October 2022. The roots were washed and then freeze-dried for three days at −55 °C and 0.200 mbar in a Buchi Lyovapor 1–200 from Thermo Fisher Scientific (Waltham, MA, USA). Then, the material was ground to a particle size between 125 and 500 µm in a commercial blender and stored at −80 °C until analysis.

2.3. Optimizing the Mixture Solvent Composition and Temperature to Obtain Phenolic Compounds by PLE

A simplex-centroid mixture design was employed to identify the optimal solvent composition for obtaining extracts rich in phenolic compounds from burdock roots, using pressurized liquid extraction (PLE). The mixture design considered CPME, EtOAc, and 2-MTHF biobased solvents arranged in a triangular plot, with each pure component (100%) at a vertex. The total peak areas of the HPLC chromatograms obtained at 280 nm were fixed as the response variable.

Extractions were conducted in a Dionex Accelerated Solvent Extraction system (ASE 200, Sunnyvale, CA, USA). Briefly, 1 g of dried roots were mixed with 2 g of cleaned sand in an 11 mL extraction cell. The extraction was carried out at 40 °C for 20 min at 1500 psi [27]. A total of 7 experiments were completed in triplicate in a randomized order.

Model adequacy was assessed through analysis of variance (ANOVA), and further evaluation involved graphical and numerical analyses and response surface plots to identify the optimum extraction solvent.

Then, an optimization of the PLE extraction temperature using the optimized solvent composition was carried out by testing 40 °C, 100 °C, 160 °C extraction temperatures. In addition, a solid–liquid extraction by maceration at room temperature for 24 h using 1 g sample dissolved in 20 mL of the optimal solvent composition was performed to compare with the extraction efficiency of the PLE extraction process at different temperatures.

2.4. Total Phenolic Content (TPC)

Total phenolic content (TPC) was measured using the Folin–Ciocalteu method, following the procedure described by Plaza et al. [28]. In summary, 600 μL of water was combined with 50 μL of Folin–Ciocalteu reagent, followed by the addition of 10 μL of the sample. The mixture was shaken for 1 min. Next, 150 μL of a 2% (w/v) Na₂CO₃ solution and 190 μL of water were added. After allowing the mixture to stand for 2 h at room temperature, absorbance was measured at 760 nm. A calibration curve was prepared with gallic acid, and the TPC results were expressed as mg gallic acid equivalents (GAE)/g extract.

2.5. Antioxidant Capacity Determination

2.5.1. DPPH Radical Scavenging Assay

The DPPH assay was conducted following the Brand-Williams et al. [29] method. Briefly, a 0.1 mM working solution of DPPH^• was prepared by diluting it with 100% ethanol. Then, 10 μL of the extract at four different concentrations were added to 290 μL of the DPPH^• working solution. The initial absorbance at 516 nm was measured at time 0 and again after 1 h to capture the reaction completion. A linear response was observed between 20% and 80% of the control’s initial absorbance. Trolox was used as a reference standard to construct a calibration curve. Finally, the results were expressed in µmol Trolox/g extract.

2.5.2. Oxygen Radical Absorbance Capacity (ORAC)

The ORAC assay was performed according to the Ou et al. [30] protocol, employing ascorbic acid as a standard. In summary, 100 μL of extract at various concentrations was mixed with 100 μL of AAPH (590 mM in 30 mM phosphate buffer, pH 7.5), 25 μL of fluorescein (10 μM in phosphate buffer), and 100 μL of phosphate buffer. Fluorescence readings were taken at an excitation wavelength of 485 nm and an emission wavelength of 530 nm, measured every 5 min at 37 °C for 1 h.

The scavenging capacity for peroxyl radicals was calculated as the inhibition percentage, based on the area under the fluorescence decay curve (AUC) in the presence of the extract (AUCextract) compared to the control (AUCcontrol), using the equation:

$$\%inhibition={\displaystyle \frac{AUCcontrol-AUCextract}{AUCcontrol}}\times 100$$

AUC was determined using:

$$AUC=0.5+\sum fi/f0$$

where f0 represents the initial fluorescence at 0 min and fi is the fluorescence measured at 5-min intervals.

The results were expressed as IC₅₀ (mg/mL extract) values, where lower IC₅₀ values imply higher antioxidant capacity.

All FC, DPPH^•, and ORAC analyses were conducted using a BioTek Synergy HT UV-Vis microplate spectrophotometer reader (BioTek Instruments, Winooski, VT, USA).

2.6. Anticholinergic Capacity Determination

The anticholinergic capacity of the extracts was evaluated by determining the inhibition capacity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to the Sánchez-Martínez et al. [31] method. It was performed using 100 µL of extract at various concentrations (150–1500 µg/mL) dissolved in 100% ethanol and mixed with 100 µL of 150 mM Tris-HCl buffer (pH 8) and 25 µL of AChE or BChE enzyme (0.8 U/mL), both diluted in the same buffer. The mixture was incubated at room temperature for 10 min. Subsequently, 25 µL of ABD-F (125 µM) and 50 µL of ATCI at a concentration based on the KM (Michaelis–Menten constant) were added. The KM was determined by combining 100 µL of ATCI (0.4–4 mM) with 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 recorded at λexcitation = 389 nm and λemission = 513 nm every min for 10 min at 37 °C to calculate the KM and determine the extracts’ anticholinergic activity. Galantamine hydrobromide served as the reference inhibitor in both assays, and the results were expressed as IC₅₀ values (µg/mL extract), with lower IC₅₀ values indicating higher anticholinergic activity.

All fluorescence and spectrophotometric data were processed using Gen5™ version 2.0 Data Analysis software from BioTek Instruments (Winooski, VT, USA).

2.7. Characterization of Non-Polar Fraction by GC-MS

Non-polar compounds in the PLE extracts were analyzed using a Shimadzu GCMS-QP2010 SE Single Quadrupole GC-MS (Kyoto, Japan). Separation was achieved with an Agilent Zorbax DB5-MS + 10 m Duraguard Capillary Column (30 m × 0.25 mm × 0.25 µm) using helium as the carrier gas at a linear flow rate of 32.5 cm/s. A 1 µL injection volume was used in split mode, with the injection temperature set to 250 °C. The mass spectrometer operated in SCAN mode, covering a mass range of m/z 40–550, with a scan speed of 1428 amu/s and an event time of 0.40 s. The ion source and interface temperatures were maintained at 250 °C and 335 °C, respectively. The oven temperature program began at 60 °C, increasing to 200 °C at 20 °C/min, followed by a rise to 225 °C at 1 °C/min, then to 300 °C at 10 °C/min, and finally to 325 °C at 2 °C/min, held for 2 min. The method was optimized for optimal compound separation.

Data processing was carried out using Shimadzu GC Solution software (version 2.32), utilizing the National Institute of Standards and Technology (NIST) and Wiley commercial mass spectrum databases for the tentative identification of non-polar compounds.

2.8. Phenolic Characterization by HPLC-DAD-IT-MS

Phenolic 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) connected to a Bruker Esquire 3000 Ion Trap Mass Spectrometer (Bremen, Germany) with an Electrospray Ionization Source (ESI).

For the separation process, a ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm particle size) was employed from Phenomenex. The mobile phases 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: 2% B isocratic (0–2 min), 2–12% B (2–5 min), 12% B isocratic (5–15 min), 12–20% B (15–16 min), 20–35% B (16–36 min), 35–50% B (36–47 min), 50–98% B (47–61 min), 98% B isocratic (61–62 min), and 98–2% B (62–64 min). Detection wavelengths for the analysis were 280 nm, 320 nm, and 520 nm.

The mass spectrometer’s ion source operated in both positive and negative modes, scanning a mass range from m/z 50 to 1000. The MS parameters were set as follows: capillary voltage at 3500 V, drying gas flow rate at 10 L/min, nebulizer pressure at 45 psi, and dry gas temperature at 350 °C. The collected data were analyzed in detail through a comprehensive review of the scientific literature and the MS database (FOODB) to verify the potential presence of the identified compounds in the extracts and assess whether these compounds had been previously reported in the studied plant.

2.9. Statistical Analysis

The simplex-centroid experimental design and statistical analyses were performed using Statgraphics Centurion XIX software (version 19.6.05) (Statistical Graphics Corp., VA, USA). This software facilitated the comparison of burdock extracts in terms of total phenolic content (TPC), antioxidant capacity, and anticholinergic activity. ANOVA with Fisher’s exact test was applied to determine statistically significant differences (p ≤ 0.05) between the mean values of the different extracts at a 95% confidence level. All data are presented as mean ± standard deviation, with each analysis conducted in triplicate for each extract.

3. Results

3.1. Optimization of PLE Extraction by a Simplex-Centroid Experimental Design

A simplex-centroid experimental design was conducted to optimize the optimal mixture proportions of biobased solvents (CPME, 2-MTHF, and EtOAc) for the recovery of a high amount of phenolic compounds from burdock roots by PLE (at 40 °C for 20 min), particularly from the SPLE ecotype. SPLE was selected as the most representative sample of burdock roots, because it was collected at 150 m like the OLE and without weeding treatment like the SPME.

For the optimization, the peak areas determined by HPLC-DAD at 280 nm from the 7 experiments established by the simplex-centroid mixture design were used as response variables, and the results are shown in

Table 1. Simplex-centroid mixture design and response variable that corresponds to the total peak areas of HPLC-DAD chromatograms at 280 nm of extracts obtained by PLE with CPME, 2-MTHF, and EtOAc biobased solvents from SPLE burdock roots under the established preliminary conditions.

	Independent Variables			Dependent Variable
	Mixture (% solvent, v/v)
Run	CPME	2-MTHF	EtOAc	Total Peak Area at 280 nm
1	50	0	50	4181 ± 389 e
2	33	33	33	6843 ± 93 c
3	100	0	0	5998 ± 15 d
4	50	50	0	8308 ± 258 b
5	0	0	100	9484 ± 17 a
6	0	100	0	9363 ± 135 a
7	0	50	50	8062 ± 306 b

^a,b,c,d,e Letters indicate statistically significant differences among extractions.

.

In addition, the ANOVA results and the adequacy of the model with their corresponding coefficients, shown in Table S1, exhibited that the regression model could explain 99% of the results’ variability by HPLC-DAD analysis. In addition, ANOVA revealed a non-significant effect (p-value > 0.05) of different variables on the response variable. For instance, the effect of the biobased solvent mixture proportions achieved by the simplex-centroid experimental design can be observed in the contour plots in Figure 1. This figure shows that the combination of CPME, 2-MTHF, and EtOAc did not allow significant effects (p-value > 0.05) on the peak area of phenolic compounds determined by HPLC-DAD. However, the contour plots exhibited that the highest peak areas of phenolic compounds determined by HPLC-DAD were achieved at the highest 2-MTHF percentage. In fact, this experimental design revealed that the optimal extraction conditions to obtain high phenolic content from SPLE roots were 100% 2-MTHF.

3.2. Optimizing the PLE Temperature According to the Recovery of Antioxidant and Anticholinergic Phenolic Compounds

The optimal extraction condition achieved in Section 3.1 was used to optimize the PLE extraction temperature. For that, extracts achieved by PLE at 40 °C using 100% 2-MTHF were compared with PLE extracts obtained at 100 and 160 °C in terms of TPC, antioxidant capacity determined by DPPH^• and ORAC assays, and anticholinergic capacity evaluated by AChE and BChE assays. Moreover, the efficiency of the PLE technique was assessed by comparison with maceration (at room temperature for 24 h). As can be observed in Figure 2, the results indicated that the PLE technique was more effective in the recovery of phenolic compounds with antioxidant and anticholinergic capacity than the maceration process. In addition, a gradual increase in the antioxidant capacity of the extracts was observed with the PLE extraction temperature. In fact, PLE at 160 °C provided the extracts with the highest TPC values and the highest antioxidant capacity determined by DPPH^• and ORAC assays (see Figure 2A,B, respectively). However, the anticholinergic capacity of the extracts increased from 40 °C to 100 °C PLE extraction temperatures, but it reduced from 100 °C to 160 °C (see Figure 2C).

In addition, it was also noted that the extraction temperature had a significant impact on the total peak area measured in the HPLC-DAD analysis of the PLE extracts (see Figure S1). Specifically, an extraction temperature of 100 °C produced extracts with the highest total peak area (35,724 ± 5,683). In comparison, at 160 °C, the total peak area decreased to 30,893 ± 3629, and at 40 °C, it further dropped to 22,013 ± 7877 (see Figure S1). This suggests that 100 °C is the optimal temperature for maximizing compound extraction under these conditions. Thus, due to the high antioxidant and anticholinergic capacities, and the high peak areas of the compounds detected in the HPLC-DAD analysis exhibited by the PLE extract obtained at 100 °C, this temperature was selected as the optimum to recover the highest content of bioactive phenolic compounds from burdock roots, particularly from the SPLE ecotype.

3.3. Influence of the Growing Conditions on the Bioactivity of Burdock Roots

The PLE optimal extraction conditions (100% 2-MTHF, 100 °C) were applied to the OLE and SPME ecotypes in order to compare them with the SPLE extracts and determine the influence of weeding during the cultivation and the altitude at which the burdock roots were collected on their bioactivity. In this sense, it is noted that the SPME extracts not subjected to weeding and collected at 800 m resulted in higher TPC values, antioxidant capacity, and anticholinergic capacity compared to those obtained at 150 m (OLE and SPLE ecotypes) (

Table 2. Total phenolic content (Folin–Ciocalteu assay), antioxidant capacity (DPPH^• and ORAC assays), and anticholinergic capacity (AChE and BChE assays) from SPME, OLE, and SPME extracts obtained by PLE under the optimized extraction conditions (100% 2-MTHF, 100 °C).

Method	SPLE	OLE	SPME
TPC (mg gallic acid/g extract)	22.9 ± 0.9 c	48.8 ± 0.2 b	64 ± 5 a
DPPH• (uM Trolox/g extract)	0.21 ± 0.01 c	0.43 ± 0.03 b	0.739 ± 0.005 a
ORAC (IC50, mg/mL extract)	9 ± 1 b	4.8 ± 0.3 a	3.1 ± 0.3 a
AChE (IC50, mg/mL extract)	59 ± 8 b	94 ± 2 c	29 ± 2 a
BChE (IC50, mg/mL extract)	75 ± 2 b	83 ± 2 b	43 ± 3 a

^{a, b, c} Letters show the significant differences between extracts (p ≤ 0.05).

). Particularly, a positive effect of weeding during the cultivation of the burdock roots collected at 150 m was observed in the TPC values and the antioxidant capacity of the OLE extracts compared to the SPLE extracts (not subjected to weeding). However, the weeding at 150 m exerted a negative effect on the anticholinergic capacity of the burdock roots, meaning that the OLE extracts had the highest IC₅₀ values.

As can be seen in Table 2, higher TPC values implied higher antioxidant capacity but not higher anticholinergic capacity.

3.4. Characterization of Non-Polar Compounds in Different Ecotypes of Burdock Roots by GC-MS

A characterization of non-polar compounds from the PLE extracts obtained with 100% 2-MTHF at 100 °C from the SPLE, OLE, and SPME ecotypes was achieved by GC-MS. A total of 14 different non-polar compounds were tentatively identified in all ecotypes (see

Table 3. Compounds identified by GC-MS with their respective peak areas in SPLE, OLE, and SPME burdock root extracts obtained by PLE at 100 °C.

ID	Proposed Compound	Rt (min)	Molecular Formula	Measured Mass	Main Fragments Ions (m/z)	SPLE	OLE	SPME
1	4-Methyloctanoic acid	7.468	C9H18O2	158	129, 101, 99, 83, 73, 60, 57, 55, 45, 43, 41	359 ± 13	384 ± 26	401 ± 16
2	2-Methylphenol	7.502	C7H8O	108	108, 107, 90, 89, 79, 77, 63, 53, 51, 44	740 ± 21	863 ± 40	609 ± 67
3	Oleic acid	7.758	C18H34O2	282	137, 125, 111, 101, 98, 97, 83, 81, 73, 69, 67, 60, 55, 43, 41	33 0± 51	390 ± 14	113 ± 17
4	Palmitic acid	13.706	C16H32O2	256	256, 213, 129, 97, 87, 85, 83, 73, 71, 69, 60, 57, 55, 45, 43, 41	675 ± 73	609 ± 34	746 ± 11
5	Methyl linolelaidate	18.267	C19H34O2	294	149, 135, 109, 95, 82, 81, 79, 69, 67, 55, 54, 43, 41	2598 ± 278	1371 ± 47	1645 ± 26
6	Linolenic acid	18.454	C18H30O2	278	278, 149, 108, 95, 93, 79, 67, 55, 41	748 ± 80	579 ± 5	677 ± 37
7	Oleic acid amide	27.121	C18H35NO	281	114, 100, 86, 72, 69, 67, 60, 59, 55, 44, 43, 41	2749 ± 140	-	-
8	Hotrienol	31.878	C10H16O	152	119, 105, 91, 82, 79, 71, 67, 55, 51, 43, 41	1076 ± 31	-	-
9	Nerolidol	44.181	C15H26O	222	207, 161, 136, 119, 107, 97, 93, 81, 79, 71, 69, 57, 55, 44, 43, 41	294 ± 1	258 ± 26	124 ± 19
10	β-stigmasterol	44.609	C29H48O	412	412, 351, 300, 255, 159, 133, 119, 105, 97, 91, 83, 81, 79, 69, 57, 55, 43	1884 ± 163	1919 ± 40	867 ± 9
11	γ-sitosterol	45.419	C29H50O	414	414, 396, 329, 303, 255, 213, 173, 161, 159, 147, 145, 133, 131, 121, 118, 107, 97, 95, 85, 81, 69, 67, 57, 55, 43, 41	4093 ± 367	3321 ± 4	1623 ± 161
12	Caryophyllene oxide	48.163	C15H24O	220	177, 161, 147, 133, 121, 109, 96, 93, 81, 79, 69, 67, 55, 53, 43, 41	271 ± 13	97 ± 1	375 ± 23
13	Spathulenol	49.576	C15H26O	222	222, 207, 189, 133, 121, 109, 107, 95, 81, 79, 69, 67, 55, 43	162 ± 3	115 ± 78	205 ± 3
14	Bisnorallocholanic acid	49.767	C22H36O2	332	217, 215, 161, 149, 147, 135, 133, 124, 121, 119, 109, 108, 107, 105, 97, 95, 93, 91, 81, 79, 77, 73, 69, 67, 57, 55, 53, 45, 44, 43	313 ± 15	101 ± 11	426 ± 21

Peak area expressed as ×10³; (-): Non-detected.

and Figure S2), including 6 fatty acids, 4 terpenoids, 1 phenolic compound, 2 sterols, and 1 steroid derivative. Among the ecotypes, the highest diversity of non-polar compounds was found in SPLE, where oleic acid amide (number 7) fatty acid and the hotrienol (number 8) monoterpene were detected, but these were not found in the OLE and SPME ecotypes. Oleic acid amide was tentatively identified with a molecular ion at m/z 281 and fragment ions at 114 [M-C₈H₁₄], 100 [M-C₁₀H₂₀], 86 [M-C₆H₁₂], 72, 69, 67, 43, and 41 from smaller alkyl fragments resulting from further chain cleavage [32]. This fatty acid was one of the most abundant non-polar compounds detected in the SPLE extract, according to their peak areas. The other compound detected in the SPLE extract but not in the OLE and SPME extracts was hotrienol, which presented a molecular ion at m/z 152 and characteristic fragment ions at 119 [M-CH₃CO], 105 [M-C₄H₄], 91 [M-C₇H₁₁], 82, 71, 67, and 55 alkyl fragments [33]. As can be observed in Table 3, the non-polar profile was the same among the ecotypes, excepting the two compounds identified in the SPLE extracts. Only the differences already discussed were observed in the peak areas of the other identified compounds. However, the SPLE extract presented the highest total peak area of non-polar compounds identified by GC-MS, compared to the OLE and SPME extracts.

Particularly, the most abundant non-polar compound detected in all the ecotypes was y-sitosterol (number 11) but without statistically significant differences with methyl linolelaidate (number 5) in the SPME extract. y-Sitosterol was detected with a molecular ion at m/z 414 and characteristic fragments at 396 [M-CH4]; 213, which corresponds to the fragmentation of a part of the steroid backbone; 159, 147, and 145, involving the loss of a small fragment or part of the steroid structure; 69, which is common in sterols; and 107, 97, 81, 67, involving different losses of alkyl fragments (see Table 3) [34]. One more sterol was identified in these burdock roots, which corresponds to β-stigmasterol but at a lower level than the γ-sitosterol. According to the peak areas, β-stigmasterol (number 10) was one of the major compounds detected in the OLE ecotype, obtaining higher peak areas than the SPLE and SPME ecotypes.

In addition to this sterol, the OLE extract was the richest extract in oleic acid (number 3) and the only phenolic compound identified by GC-MS in all the ecotypes, which corresponded to 2-methyl phenol. This compound was detected with a molecular ion at m/z 108 and fragment ions at 107 [M-H], 90 [M-CH₃], 70 [M-C₆H₇], 77 [M-C₆H₅], 63, 53, and 51, involving the breakdown of the aromatic structure, phenol or methyl groups.

In general, a negative influence of weeding in the OLE extracts was exhibited, with a decrease in fatty acids and terpenoids peak areas concerning the SPLE extracts collected at the same altitude but without being subjected to weeding treatment. In fact, the SPLE extracts presented higher peak areas of methyl linolelaidate and linolenic acid, in addition to the presence of oleic acid amide that was not observed in the OLE extract. However, a positive influence of weeding was detected for the phenolic compound identified due to OLE extract presented a higher peak area of 2-methyl phenol than SPLE extract (see Table 3). On the other hand, the altitude also promoted variations in the peak areas of the identified compounds. Comparing the ecotypes collected without being subjected to weeding at 150 and 800 m (SPLE and SPME extracts), higher altitudes decreased the peak areas of hotrienol (number 8) and nerolidol (number 9) but increased the peak areas of caryophyllene oxide (number 12) and spathulenol (number 13). Additionally, a higher peak area was observed in the SPME extract of the only steroid derivate identified in these burdock roots, which corresponded to bisnorallocholanic acid (number 14). This compound was identified with a molecular ion at m/z 332 with fragments at 217 and 215, which corresponded to the steroid nucleus fragments; 161, 135, and 133, involving the loss of alkyl groups; 109, 108, and 107, which corresponded to the loss of a C₆H₁₁ or C₆H₁₀ group, typically from the hydrocarbon backbone; and 105, 97, 95, 93, 91, 57, and 55, involving multiple smaller alkyl losses, reflecting the sequential breakdown of the steroid structure.

3.5. Phenolic Characterization of Different Ecotypes of Burdock Roots by HPLC-IT-MS

Table 4. Mass data spectra and peaks areas of compounds in the three different burdock ecotypes (OLE, SPLE, SPME) obtained by PLE at 100 °C by HPLC-DAD-IT-MS.

No.	Compound	Rt (min)	[M-H]−	MS2 Ions	SPLE PLE	OLE PLE	SPME PLE
1	Chlorogenic acid	8.8	374.9 [M-H + Na]−	353, 200.8, 190.9, 178.8, 172.9, 160.9, 135	452 ± 79	264 ± 18	2069 ± 75
2	4-O-Caffeoylquinic acid	13.3	353.0	190.8, 178.8, 172.8, 135, 127	167 ± 43	121 ± 10	1681 ± 4
3	Dicaffeoylquinic acid (Cynarin)	20.7	515.2	379, 353, 334.9, 190.9, 178.9	1392 ± 81	658 ± 84	14,039 ± 56
4	Dicaffeoylquinic acid isomer	21.8	537 [M-H + Na]−	515.1, 375, 353, 335, 264.9, 190.9, 178.9, 172.9	60 ± 6	181 ± 21	4121 ± 214
5	Arctiin	28.9	533	515, 371, 355, 289, 249, 151, 136, 121	61 ± 14	95 ± 6	-
6	Dicaffeoylmaloylquinic acid	29.7	631	352.8, 325, 250.9, 190.9, 172.9, 160.8, 135	214 ± 23	395 ± 12	-
7	Monocaffeoylquinic acid	53.3	353.1	333, 394.9, 232.8, 192.9, 179	236 ± 21	-	-
8	Unknown 1	54.1	441.3	429, 422.2, 399.4, 383.4, 340.9, 327.2, 325, 304, 182.8, 124.9	286 ± 7	336 ± 5	479 ± 5
9	Unknown 2	54.5	471.4	451.3, 441.2, 407.2, 367.3, 337, 218.9, 187	298 ± 9	380 ± 26	1137 ± 16
10	Unknown 3	58.4	383.4	365.2, 337.2	3115 ± 60	-	4540 ± 37

(-): Non-detected.

shows that an HPLC-IT-MS analysis of the PLE extracts from the three different ecotypes obtained at 100 °C allowed the detection of ten different phenolic compounds, of which seven were tentatively identified. Principally, the identified phenolic compounds corresponded to caffeoylquinic acids. In fact, cynarin (number 3) was identified with a molecular ion at m/z 515 [M-H]⁻, exhibiting the characteristic fragmentation of caffeoylquinic acid with intense fragment ions at m/z 353, 334.9, 190.9, and 178.9, representing the majority phenolic compounds observed in all the ecotypes [35,36]. Comparing the ecotypes, the SPME extract presented the highest peak area of cynarin compared to OLE and SPLE. In addition, another dicaffeoylquinic acid was observed but with a sodium adduct with a molecular ion at m/z 537 (number 4). Furthermore, dicaffeoylmaloylquinic acid (number 6) was tentatively identified with a molecular ion at m/z 631 [M-H]⁻. Its fragmentation pattern showed characteristic ions at m/z 352.8 and m/z 190.9, corresponding to the sequential loss of one and two caffeic acid molecules and malic acid, and a quinic acid molecule [37]. This compound was not detected in the SPME extract; neither were arctiin (number 5) and monocaffeoylquinic acid (number 7); however, they were observed in the SPLE and OLE extracts. Particularly, arctiin (number 5) was tentatively identified with a m/z ion at 533 [M-H]⁻, from which an intense fragment at m/z 371 was observed, with neutral losses of methyl groups, including m/z 355, as the phenolic compound detected with the lowest peak area in the extracts [38].

In addition to the monocaffoylquinic acid (number 7), another one was identified with a molecular ion at m/z 374.9 [M-H]⁻ with fragment ions at m/z 190 involving the loss of quinic acid and m/z 178, which corresponded to the loss of the caffeic acid molecule. This compound was one of the majority phenolic compounds identified in the burdock roots after cynarin in the OLE and SPLE extracts, and after dicaffeoylquinic acid isomer (number 4) in the SPME extract, having its peak area higher in SPME than in the rest of the ecotypes.

It is noted that, corresponding to the chromatogram registered at 280 nm (Figure S3), three different phenolic compounds were observed but they cannot be identified by MS (compounds number 8, 9, and 10). In addition, Figure S3 and Table 4 show higher peak areas of identified compounds in the SPME extract compared to the SPLE and OLE extracts. It was observed that higher altitudes in the growing conditions of the burdocks implied a higher synthesis of phenolic compounds identified in the HPLC-IT-MS analysis, except for arctiin, dicaffeoylmaloylquinic acid, and the monocaffeoylquinic acid detected at 53.3 min, whose synthesis was favored when the plant grew at an altitude of 150 m. When the altitude was the same (comparing SPLE and OLE extracts), it was noted that the absence of weeding during plant growth produced an increase in the content of phenolic compounds, as can be seen in Table 4. Finally, cynarin (number 3) presented higher a peak area in the SPLE than in the OLE extract. In addition, Figure S3 displays unknown compound peaks starting at minute 40 that were detected in the OLE ecotype extract but were not observed in the SPLE and SPME extracts. This reflected that weeding treatment favors the synthesis of other secondary metabolites.

3.6. Evaluation of the Role of Growing Conditions of Burdock Roots in Their Bioactivity, Non-Polar, and Polar Composition

To study the influence of weeding treatment and the altitude at which the plant was collected on the recovery of bioactive compounds from the PLE extracts obtained at 100 °C, a multivariate analysis was performed on the TPC values, antioxidant capacity was determined by DPPH^• and ORAC assays, and anticholinergic capacity was evaluated by the AChE and BChE methods, as well as on the peak areas of the non-polar and polar compounds identified by GC-MS and HPLC-IT-MS, respectively.

The burdock root extracts were divided into two groups according to phenolic, antioxidant, and anticholinergic values, and the peak areas of compounds identified by the chromatographic analysis, which were differentiated by colors in a Hierarchical Cluster Analysis (HCA). One cluster grouped the SPME extract (green color) while the other one grouped the OLE and SPLE extracts (blue color) (see Figure 3A). In addition, a Principal Component Analysis (PCA) also separated the extracts according to their bioactive and non-polar and polar composition, selecting the most significant variables (principal components). Two principal components described 100% of the total data variability, grouping the extracts similarly to the HCA (employing the same colors for HCA and PCA).

Extracts from the same group and color presented similar bioactivity and non-polar and polar composition. By overlapping Figure 3B,C, it can be observed that the SPLE and OLE extracts from the burdock roots collected at 150 m (group 2) showed the lowest TPC and antioxidant and anticholinergic values because they were situated in the opposite site to the TPC and DPPH^• assays by their low phenolic content and antioxidant capacity that are presented in these methods. In addition, they were in the same site as the ORAC, AChE, and BChE methods expressed as IC₅₀, indicating that they presented higher IC₅₀ values, that is, lower bioactive capacity. This fact confirmed that the burdocks collected at low altitudes provided PLE extracts with lower bioactive compounds than those collected at 800 m. Additionally, this study reflected that weeding during plant growth does not significantly influence the bioactivity and content of non-polar and polar compounds in burdock root, since the OLE and SPLE extracts were grouped together.

From Figure 3C, it can be observed that the high bioactive capacities of the SPME extract (group 1), which was in the opposite quadrant to the OLE and SPLE extracts, could be due to their monocaffeoylquinic acid level, such as the chlorogenic acid and 4-O-caffeoylquinic acid level, as well as the cynarin level. Moreover, unknown phenolic compounds could also be responsible for the SPME bioactivity. In addition, non-polar compounds such as the binorallocholanic acid steroid, spathulanol, and caryophyllene terpenoids were also responsible of the bioactive capacity of SPME extract. In general, fatty acids did not present an influence on the bioactivity of SPME, except for palmitic acid, which was found in the same quadrant of the extract in the PCA loading plot (see Figure 3B,C).

In general, it was confirmed that the collection of burdocks at high altitudes favors the synthesis of antioxidant and anticholinergic compounds, with the most responsible for this activity being monocaffeoylquinic acids, cynarine, and some terpenes, such as spathulenol and caryophyllene oxide.

4. Discussion

4.1. Influence of Biobased Solvent Composition and Extraction Temperature on the Recovery of Phenolic Compounds from Burdock Roots

Although burdock roots have been associated with multiple beneficial properties due to their phenolic content, the recovery of these compounds from this plant part using advanced extraction techniques has been scarcely studied. Particularly, the majority of the studies are focused on the recovery of phenolic compounds from burdock roots by ultrasound-assisted extraction (UAE) [39,40,41]. To our knowledge, there is only one investigation into the use of PLE to obtain bioactive phenolic compounds from burdock roots, where Petkova et al. [42] indicated that PLE recovered a lower phenolic amount than UAE using conventional extraction solvents, like ethanol/water (70:30, v/v). However, Petkova et al. did not optimize the solvent composition and the extraction conditions for the PLE extraction, which are crucial parameters for an efficient recovery of these compounds by PLE. Additionally, several authors have reported that biobased solvents such as 2-methyloxolane, CPME, 2-MTHF, or EtOAc provide higher extraction yields from other matrices than using conventional solvents like hexane or ethanol [16,18,43,44]. Usually, these biobased solvents have been used in conventional solid–liquid extractions, and little attention has been paid to their inclusion in advanced extraction techniques, such as PLE. In this context, Sánchez-Camargo et al. [16] observed a high extraction efficiency of fucoxanthin from algae when PLE was combined with ethyl acetate biobased solvent, compared to the use of ethanol. However, there are no studies on combining PLE with biobased solvents for extracting phenolic compounds from burdock roots. Thus, this study investigated the use of biobased solvents combined with PLE. The selection of CPME, 2-MTHF, and ethyl acetate biobased solvents for the study was carried out according to the positive phenolic compound extraction results achieved by different researchers [16,18,44,45].

In this study, a simplex-centroid experimental design allowed us to elucidate the optimal mixture biobased composition to obtain by PLE at 40 °C the highest phenolic amount determined by the peak areas observed in HPLC-DAD analysis from SPLE burdock roots. This experimental design revealed that the best biobased solvent composition that reported the highest peak areas of compounds determined by HPLC-DAD at 280 nm was 100% 2-MTHF (see Figure 1). In agreement with Cañadas et al. [45], this could be due to the capacity of 2-MTHF to form hydrogen bonds with phenolic groups compared to CPME and EtOAc, increasing the recovery of phenolic compounds. This hydrogen bond formation implies a more exothermic behavior of 2-MTHF, promoting the extraction of phenolic compounds. Although CPME can also form hydrogen bonds with phenolic groups, it was less effective than 2-MTHF. This fact could be because the 2-MTHF structure is relatively polar due to the oxygenated ring, which facilitates stronger interactions with polar compounds such as phenols. However, CPME has a hydrophobic molecular structure, and this characteristic reduces its affinity for phenolic compounds compared to 2-MTHF. Concerning EtOAc, the HPLC-DAD showed higher peak areas than CPME but lower than 2-MTHF. EtOAc is a moderately polar solvent, with a dielectric constant (~6.02) higher than CPME but lower than 2-MTHF (~7.6). This allows it to dissolve certain compounds, such as phenolic compounds, better than CPME but not with the same efficiency as 2-MTHF.

On the other hand, the mixture of biobased solvent could have decreased the proportion of 2-MTHF molecules available to form hydrogen bonds with phenols and the polarity of the mixture could have moved away from the ideal for dissolving the compounds to be extracted. Thus, the use of 100% 2-MTHF provides the highest peak areas of compounds determined at 280 nm in the HPLC-DAD analysis compared to CPME and EtOAc biobased solvents.

In general, 2-MTHF was the best biobased solvent to extract compounds, like phenolic compounds, due to its balanced polarity and ability to form effective hydrogen bonds with phenol groups. However, EtOAc extracted a higher amount of compounds from the SPLE burdock roots than CPME due to its moderate polarity and the ability of its carbonyl group to form hydrogen bonds, although it is not as effective as 2-MTHF. By contrast, the low polarity and more hydrophobic structure of CPME resulted in it being the least efficient method for extracting compounds from the SPLE burdock roots. Considering these results, 100% 2-MTHF biobased solvent was selected as the best method for the recovery of compounds from burdock roots.

After that, an optimization of the PLE extraction was carried out by testing three different temperatures (40 °C, 100 °C, and 160 °C). The extraction at these three different temperatures was compared in terms of TPC, antioxidant and anticholinergic capacities, and total peak areas of the chromatogram HPLC-DAD analysis at 280 nm. The extracts obtained at 160 °C presented higher TPC values and antioxidant capacity compared to the use of 40 °C and 100 °C. By contrast, the PLE extracts obtained at 100 °C presented higher anticholinergic capacity than the rest of the extracts. The high extraction efficiency at 160 °C could be due to the generation of Maillard products, which could contribute to the high TPC values and the antioxidant capacity measured by interferences with the assays, overestimating the phenolic content and the antioxidant capacity of burdock roots [46]. In addition, a brown color was observed in the extract, typical of the Maillard reaction. In fact, the HPLC-DAD analysis demonstrated that phenolic compounds were reduced when the extraction temperature increased from 100 °C to 160 °C. Thus, 100 °C was selected as the optimal PLE extraction temperature to obtain antioxidant and anticholinergic compounds from the burdock roots, preserving their degradation.

In addition, the PLE extracts were compared with a maceration performed for 24 h. The results indicated that employing high pressure and temperatures promoted the extraction of antioxidant and anticholinergic phenolic compounds compared to maceration, regardless of the temperature used. High pressure and temperature favor the solubility of the compounds in the solvent and the breaking down of the plant’s cell walls, allowing for faster and more efficient extraction than the maceration process [47]. In addition, the results suggested that prolonged extraction times during maceration could promote the degradation of bioactive phenolic compounds, obtaining lower TPC, antioxidant, and anticholinergic capacities, as can be observed in Figure 2.

4.2. Bioactive, Terpenoid, and Phenolic Profile Comparison Among Different Ecotypes of Burdock Roots

Several authors have recognized that growing conditions impact the synthesis of phenolic compounds in plants, and with them, the bioactivity of their extracts [48,49]. However, the influence of the growing conditions of burdock roots on their bioactivity and phenolic profile has scarcely been studied. Considering that the climatic conditions in 2021 and 2022 in the collection areas of the burdock roots (Collepardo and Isola del Liri) were the same, based on the meteorological information reported by The National Meteorological Service of Italy (ItaliaMeteo), the differential factors among the ecotypes were the weeding treatment and the altitude where the plant was seeded.

An important influence of the weeding treatment was observed between the ecotypes collected at the same altitude (150 m). When the plant was subjected to weeding treatment (OLE extracts), its extracts exhibited higher TPC and antioxidant capacity values than the plants subjected to non-weeding treatment (SPLE extracts). This reflected that when plants are subjected to weeding (removal of weeds or nearby competitors), they may experience less competition for resources such as light, water, and nutrients. This less competitive environment allows plants to focus more of their energy and resources on their secondary metabolism, which includes the production of phenolic compounds. These compounds, in addition to acting as antioxidants, may serve as defense mechanisms against other environmental stressors [50]. However, the SPLE extracts presented a higher anticholinergic capacity than the OLE extracts.

This could be because, in an environment with competition (like for SPLE burdock roots without weeding), plants are under greater biotic and abiotic stress. Under these conditions, they may prioritize the synthesis of specific secondary metabolites that are related to chemical defense, such as compounds with anticholinergic activity. These compounds are usually produced as a defense strategy against herbivores or pathogens, since they interfere with the nervous systems of the organisms that attack them [50,51]. In fact, most insecticides used in the world are based on inhibitors of AChE. As a result, there is a loss of AChE activity, leading to overstimulation of the insect’s effector organ. Once a critical proportion of the enzyme tissue mass is deactivated, the symptoms and signs of cholinergic poisoning emerge, ultimately resulting in the insect’s death [52]. In this sense, when a plant has not been weeded, as in SPLE, it is more attractive to herbivorous animals. As a defense mechanism, the plant can synthesize anticholinergic compounds to cause a buildup of acetylcholine, resulting in overstimulation of the muscles, paralysis, and, in severe cases, the death of the herbivore. This makes plants containing these compounds less attractive or even toxic to herbivores [51]. This could explain why the OLE extract presented higher antioxidant capacity and SPLE had higher anticholinergic capacity. However, the PCA grouped both extracts together, because they presented bioactivities and GC-MS and HPLC-MS profiles that were not very different from each other but were different to SPME (see Figure 3). This fact suggests that the content of bioactive compounds in burdock roots depends primarily on the altitude at which the plant grows, not on the weeding treatment.

Higher TPC values and antioxidant and anticholinergic capacities were observed in the SPME extracts from the burdock roots collected at 800 m compared to the OLE and SPLE extracts from the burdock roots collected at 150 m. Various studies have shown that at higher altitudes, plants are exposed to increased UV radiation, reduced atmospheric pressure, and colder temperatures [53,54,55]. To protect themselves from the damage caused by these conditions, they produce bioactive phenolic compounds as a defensive response, including antioxidant and anticholinergic compounds. In this sense, the PCA indicated that the high bioactivity of the SPME extracts could be due to the content of caffeoylquinic acids, such as chlorogenic acid and 4-O-caffeoylquinic acid, as well as caryophyllene oxide, spathulenol, and bisnorallocholanic acid. In fact, several authors have reported that caffeoylquinic acids, such as chlorogenic acid, have demonstrated a memory improvement that has been related to their high antioxidant capacity, in addition to their antibacterial, antiviral, and anticarcinogenic capacities [56]. The phenolic profile of burdock has been widely studied.

Several authors have shown that most of the phenolic compounds found in Arctium lappa L. are caffeoylquinic acids such as monocaffeoylquinic acids (chlorogenic acid and 1-O-caffeoylquinic acid), dicaffeoylquinic acids (1,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid/3,5-O-dicaffeoylquinic acid, and 1,3-O-dicaffeoylquinic acid (also known as cynarine)), and a dicaffeoylmaloylquinic acid [54,55]. Recently, cynarine, which is one of the major compounds identified in the SPME extract, has shown an interesting capacity to inhibit neuronal degeneration reducing glutamate release, an excitatory neurotransmitter that, in excess, causes oxidative stress, neuroinflammation, mitochondrial dysfunction, and finally neuron death [57]. In addition, although arctiin was found to have the lowest peak area of the phenolic compounds identified in this study, the extraction of this compound from burdock roots has been widely investigated for its antioxidant potential [58,59,60,61]. The arctiin content of plants is crucial because it serves as a precursor to arctigenin, a bioactive compound with a broader spectrum than arctiin in pharmacological properties. Upon ingestion, arctiin is metabolized by the gut microbiota into arctigenin, which exhibits enhanced biological activity, including antiviral, anticancer, and anti-inflammatory effects. Therefore, plants rich in arctiin provide a natural source for generating arctigenin within the body, amplifying their therapeutic potential [58,59,60,61]. Notably, caryophyllene has been shown to be neuroprotective in cases of cerebral ischemia and neuronal damage caused by oxidative stress, modulating glial activation and neuroinflammation [62]. In addition to the high antioxidant, anti-inflammatory, antiproliferative, and antimycobacterial activities demonstrated by spathulenol, this compound has shown interesting neuroprotective capacity by reducing ROS production and maintaining the mitochondrial integrity [63,64]. Spathulenol and caryophyllene oxide have also been identified as components of essential oils obtained from plants of the Asteraceae family. In agreement with Golbaz et al., caryophyllene oxide was identified as the major volatile compound detected in the burdock roots [65]. However, to our knowledge, this is the first time that spathulenol has been identified in burdock roots. Probably, these compounds were responsible for the high antioxidant and anticholinergic capacity of the SPME extract. Moreover, although sterols were not the primary contributors to the antioxidant and anticholinergic capacities of the extracts in this study, various authors have highlighted that stigmasterol is commonly found in burdock roots [66,67]. In addition, the absence of quercetin in the burdock root extracts obtained with 2-MTHF in this study was noted, although its frequent detection in extracts obtained with methanol or ethanol–water mixtures in other studies may be attributed to differences in solvent polarity and extraction efficiency [68,69]. This compound has been characterized by its high neuroprotective potential; however, the anticholinergic capacity of the extracts obtained in this study was not related to quercetin [70]. Quercetin, a highly polar flavonoid, is more efficiently extracted by methanol–water mixtures due to their higher polarity and stronger hydrogen-bonding capacity, which facilitates the solubilization of hydroxyl-rich compounds. In contrast, 2-MTHF, with its lower polarity and moderate hydrogen-bonding ability, favors the extraction of less polar compounds, potentially leading to the under-representation of quercetin in these extracts. In fact, it was observed that 2-MTHF favors the recovery of compounds with lower polarity (e.g., caryophyllene oxide or spathulenol) but also has neuroprotective capacities. This selective nature could lead to quercetin being under-represented or undetectable in 2-MTHF extracts.

In conclusion, for the purpose of obtaining extracts rich in antioxidant and neuroprotective compounds from burdock root, it is best to harvest plants that have been grown at high altitudes and have not been subjected to weeding during their growth.

5. Conclusions

This study introduced a novel extraction strategy using PLE with biobased solvents to simultaneously obtain non-polar and polar compounds with antioxidant and anticholinergic capacities from burdock roots. It also evaluated the impact of weeding and altitude on bioactive composition across three burdock ecotypes: two collected at 150 m (one weeded during growth: OLE; the other not weeded: SPLE) and a third at 800 m without weeding (SPME).

Using a simplex-centroid mixture design, 100% 2-MTHF was identified as the best solvent for extracting compounds with high bioactivity from SPLE, thanks to its polarity and strong hydrogen bonding with phenolic compounds. The optimal extraction temperature was 100 °C, balancing bioactive compound recovery without yield loss or undesirable reactions observed at 40 °C and 160 °C. Extracts from all the ecotypes were compared to assess the influence of altitude and weeding.

Weeding during plant growth reduced anticholinergic capacity but enhanced antioxidant compound production (e.g., dicaffeoylquinic acid, 2-methylphenol). However, altitude had the most significant impact: the SPME extracts (800 m) showed the highest bioactivity and greater amounts of non-polar and polar compounds identified via GC-MS and HPLC-IT-MS. The key compounds contributing to bioactivity included caffeoylquinic acids, caryophyllene oxide, spathulenol, and bisnorallocholanic acid.

In conclusion, the results confirmed the hypothesis raised, since PLE extraction with biobased solvents was more efficient in the recovery of bioactive compounds from burdock roots. In addition, to obtain extracts with the highest level of antioxidant and neuroprotective properties from burdock roots, plants should be harvested at high altitudes and without weeding during growth.