Hogweed Seed Oil: Physico–Chemical Characterization, LC-MS Profile, and Neuroprotective Activity of Heracleum dissectum Nanosuspension

The seeds of dissected hogweed (Heracleum dissectum Ledeb., Apiaceae) are the source of hogweed oil (HSO), which is still underexplored and requires careful chemical and biological studies. The performed physico–chemical analysis of HSO elucidated basic physical characteristics and revealed the presence of fatty acids, essential oil components, pigments, and coumarins. High-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC–PDA–ESI–tQ–MS/MS) identified 38 coumarins that were characterized and quantified. Various furanocoumarins were the major components of HSO polyphenolics, including imperatorin, phellopterin, and isoimperatorin, and the total coumarin content in HSO varied from 181.14 to 238.42 mg/mL. The analysis of storage stability of the selected compounds in HSO indicated their good preservation after 3-year storage at cold and freezing temperatures. The application of the CO2-assisted effervescence method allowed the production of an HSO nanosuspension, which was used in a brain ischemia model of rats. The HSO nanosuspension enhanced cerebral hemodynamics and decreased the frequency of necrotic processes in the brain tissue. Thus, H. dissectum seeds are a good source of coumarins, and HSO nanosuspension promotes neuroprotection of the brain after lesions, which supports earlier ethnopharmacological data.


Introduction
The Apiaceae family, which contains 446 accepted genera and approximately 4000 species, is a source of useful plants that grow throughout the world [1]. The members of the Apiaceae family are valuable crops with nutraceutical significance [2] and bioactive medicinal plants [3]. Asia has the greatest variety of apiaceous species numbering approximately 300 species [4], including a widely distributed genus Heracleum (hogweed), which counts 90 species [5]. Various hogweeds have a long history of use by humans as medicinal and food plants [6]. Dissected hogweed (Heracleum dissectum Ledeb.) is a large Asian plant; it grows in coniferous, coniferous-broad-leaved, and broad-leaved valley and mountain large-grass forests, thickets of shrubs, on the edges, glades, and tall grass meadows of Western, Middle, and Eastern Siberia, the Far East of Russia, Kyrgyzstan, Kazakhstan, Mongolia, China, Korea, and Japan [7].
Dissected hogweed is a perennial polycarpic plant that is 70-160 cm in height (up to 2 m). Caudexes are unbranched, and taproots are branching. Stems are solitary, hollow, deeply furrowed, protruding pubescent, and corymbose branching in upper parts. Basal leaves are on long petioles. Petioles of basal leaves are hollow or dense, and leaf blades are
The most diverse metabolites found in H. dissectum are coumarins, which are a group of phytocompounds with anti-inflammatory [26], anti-HIV [27], antimicrobial [28], anticancer [29], and antiviral properties [30]. A distinctive feature of coumarins is their lipophilicity [31], which makes it difficult to dissolve or disperse them in safe and waterbased solvents that could be used in experiments on living organisms. To solve the problem of insolubility of coumarins in water, the use of nanosuspensions has been proposed and recently studied as the most promising strategy to enhance the oral bioavailability of these types of drugs [32,33]. Nanosuspensions have been used to enhance the bioavailability of curcumin [34], cannabidiol [35], naringenin [36], daidzein [37], and other bioactive molecules as well as plant-derived fatty materials such as olive leaf extract [38], Rauwolfia serpentina extract [39], and fennel seed extract [40].
The aim of this study is the investigation of the seed oil of H. dissectum (HSO) by the physico-chemical methods, high-performance liquid chromatography with photodiode array detection, and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS/MS) profiling and quantification as well as investigation of neuroprotective potential of the nanosuspension of HSO obtained using CO 2 -assisted effervescence method in brain ischemia model of rats.

Plant Material and Chemicals
Ripe seeds of Heracleum dissectum were collected in the Mukhorshibir vicinity (Mukhorshibirskii District, Buryatia Republic, Russia; Figure 1a, (Table S1).

Seed Oil Preparation
Dry and milled seeds (1 kg; sample 1) were exhausted extracted in Soxhlet extractor (internal volume 2 L; Borosil ® Extraction Apparatus, Foxx Life Sciences, Salem, NH, USA) with petroleum ether (boiling point 30-40 °C; Sigma-Aldrich; cat. No 77399). The organic extract was concentrated in a vacuum at 30 °C to give colored viscous oil with a specific odor (yield 105.2 g) stored under nitrogen at 0 °C before manipulations.

Seed Oil Preparation
Dry and milled seeds (1 kg; sample 1) were exhausted extracted in Soxhlet extractor (internal volume 2 L; Borosil ® Extraction Apparatus, Foxx Life Sciences, Salem, NH, USA) with petroleum ether (boiling point 30-40 • C; Sigma-Aldrich; cat. No 77399). The organic extract was concentrated in a vacuum at 30 • C to give colored viscous oil with a specific odor (yield 105.2 g) stored under nitrogen at 0 • C before manipulations.

Ultraviolet Spectroscopy of Seed Oil
The seed oil of H. dissectum (250 mg) was transferred to the volumetric flask (25 mL), diluted in acetonitrile, and the total volume was filled to 25 mL (solution A; 10 mg/mL). An aliquot of solution A (100 µL) was diluted in the volumetric flask (25 mL) by acetonitrile (solution B; 40 µg/mL). Ultraviolet spectra of solutions A and B were studied using an SF-2000 spectrophotometer (Specter, St. Petersburg, Russia) in 1 cm-quartz cells and pure acetonitrile as a blank [50]. Imperatorin solution in acetonitrile was used as a reference standard with the final concentration of 10 µg/mL.

Fourier-Transform Infrared Spectroscopy (FTIR) of Seed Oil
FTIR spectra of H. dissectum seed oil were studied using FT-801 Fourier-transform infrared spectrometer (Simex, Novosibirsk, Russia; frequency 600-4000 cm −1 , 200 scans, 2-cm −1 resolution) coupled with attenuated total reflection device (ATR) [51]. A spectral range of 200-600 nm was used to record ultraviolet spectra. Temperature levels of electrospray ionization triple quadrupole mass spectrometric detection ESI interface, desolvation line, and heat block were 300 • C, 250 • C, and 400 • C, respectively, and the values of nebulizing gas (N 2 ) flow, heating gas (air) flow, and collision-induced dissociation gas (Ar) glow were 3 L/min, 10 L/min, and 0.3 mL/min, respectively. Electrospray ionization was done with scanning range m/z 80-1900, source voltage 3 kV, and collision energy +25 eV (positive ionization). To manage the LC-MS system, the preinstalled software LabSolutions LCMS ver. 5.6 [52] was used. Metabolite identification was realized after integrated analysis of chromatographic parameters (retention time) and spectral data (ultraviolet pattern, mass spectra) after comparison with the inner LC-MS library, reference standards, and the literature data. To prepare the sample solution, H. dissectum seed oil (25 mg) was dissolved in acetonitrile in a measuring flask (5 mL), followed by filtering through 0.22 µm syringe filters.

HPLC-ESI-TQ-MS Quantification of Coumarins in Seed Oil
Six coumarins (heraclenin, oxypeucedanin, imperatorin, phellopterin, isoimperatorin, and ostruthin) were quantified using HPLC-ESI-TQ-MS conditions described in Section 2.6. Separately weighed reference standards (10 mg) were dissolved in acetonitrile in volumetric flasks (10 mL), and the stock solutions (1000 µg/mL) were applied for preparation of the calibration solutions (1-100 µg/mL) and creation of correlations 'concentration-mass spectral peak area'. The values of correlation coefficient (r 2 ), standard deviation (S YX ), the limit of detection (LOD), limit of quantification (LOQ), and linear range were calculated in Advanced Grapher 2.2 (Alentum Software Inc., Ramat-Gan, Israel) using calibration curve data [53] and the results of three sufficient HPLC runs (Table 2). Iintra-day, inter-day precisions, and recovery of spiked samples were studied using the known assay [54]. The results were expressed as mean values ± standard deviation (S.D.). Table 2. Regression equations, correlation coefficients (r 2 ), standard deviation (S YX ), limits of detection (LOD), limits of quantification (LOQ), linear ranges, relative standard deviations (RSD) for intra-day and inter-day precisions, and recovery of spiked samples (REC) for six reference standards.

Heracleum dissectum Seed Oil Storage Experiment
Three aliquots of H. dissectum seed oil (sample 1; 10 mL) were placed in the individual polystyrene tubes and thermostated at (1) 20 • C, 1 • C, and −20 • C for three years using a ventilated MK 53 thermostat (BINDER GmbH, Tuttlingen, Germany) [55]. One stored sample was taken out for analysis every year and studied by the preparation/analysis procedure described in Section 2.6.

Nanosuspension of H. dissectum Seed Oil Preparation
The early recommendations were used to prepare H. dissectum seed oil nanosuspension [40] based on the CO 2 -assisted effervescence technique [56]. The mixture of H. dissectum seed oil (20 mg), citric acid (30 mg), and tocopheryl polyethylene glycol succinate (20 mg) was dissolved in 50 mL of ethyl acetate, and the organic solvent was evaporated in a vacuum. The residue was mixed with 50 mL of NaHCO 3 solution (0.08%) and vigorously stirred for 20 min.

Characterization of H. dissectum Seed Oil Nanosuspension
Particle size, polydispersity index distribution, and zeta potential were studied using Dynamic Light Scattering Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 20 • C (laser wavelength 633 nm) [57]. All measurements were performed three times.

Neuroprotective Activity
An animal model of brain ischemia was used to study the neuroprotective activity of H. dissectum seed oil nanosuspension performed as described early [58]. In brief, permanent focal cerebral ischemia of rats was reproduced by right-sided thermocoagulation of the middle cerebral artery in six animal groups (n = 15), including (1) sham-operated animals; (2) negative control group with animals after focal cerebral ischemia without pharmacological support; (3) EGB761 group with animals after focal cerebral ischemia treated with a reference drug EGB761 (Ginkgo biloba extract, Hunan Warrant Pharmaceuticals, Changsha, China; 35 mg/kg [59]; (4, 5, 6) HSO 0.1, 0.5, 1.0 mL/kg groups of animals after focal cerebral ischemia treated with H. dissectum seed oil nanosuspension in doses 0.1, 0.5, 1.0 mL/kg. After the 4-day-treatment, an average systolic velocity of cerebral blood flow was determined using an ultrasound Doppler device, a sensor USOP-010-01 with a working frequency of 25 MHz, and an MM-D-K-Minimax Doppler v.1.7. (Saint Petersburg, Russia) [60] followed by the animal's decapitation, brain extraction, and measuring the area of necrosis zone. All measurements were performed once for each animal and in total 15 times for one experimental group.

Statistical Analysis
All quantitative analyses were performed five times, and the data were expressed as the mean value ± standard deviation (S.D.). Statistical analyses were performed by oneway analysis of variance, and the significance of the mean difference was determined by Duncan's multiple-range test. Differences at p < 0.05 were considered statistically significant. The linear regression analysis and generation of calibration graphs were conducted using Advanced Grapher 2.2 (Alentum Software, Inc., Ramat-Gan, Israel).
The seeds of H. dissectum are weakly pigmented, which results in the dark color of the oil. The absorption spectrum of HSO demonstrated the presence of long-wave bands at 600-700 and 450-550 nm, which are caused by chlorophylls and carotenoids from seed coats [69] (Figure 2). The concentration of chlorophylls and carotenoids in HSO is 364.08 and 233.94 mg/L, respectively. The known plant oil composition data indicate a lower pigment content in olive oil (4.9-24.4 mg/L of chlorophylls and 3.1-13.4 mg/L of carotenoids [70]) or in grape seed oil (1.0-3.8 mg/L of chlorophylls and 2.6-4.8 mg/L of carotenoids [71]). level of pH is 6.20, which indicates the neutrality of oil, which is similar to those of seed oils of sesame (pH 6.12), melon (pH 6.42), or morinda (pH 6.78) [64]. The peroxide value of HSO is 6.28 mEq. peroxide/kg, which is considerably below that of carrot seed oil (16.0 mEq./kg) [61], higher than that of jatropha seed oil (0.8-1.9 mEq./kg) [65], and similar to that of sunflower seed oil (6.8-7.2 mEq./kg) [66]. The acid value of HSO is 0.52 mg KOH/g, and the saponification value is 173.82 mg KOH/g, which is similar to those of pumpkin seed oil (0.57-0.64 mg KOH/g; 189-190 mg KOH/g) [63]. However, the iodine value of HSO is 105.37 g I2/100 g, which indicates the high level of unsaturation that is typical for sunflower oil (118-141 g I2/100 g), sesame oil (103-120 g I2/100 g), and rice bran oil (90-115 g I2/100 g) [67]. Unsaponifiable matter level (0.92%) was similar to those of apiaceous seed oils from the carrot (0.9%), dill (1.2%), coriander (2.2%), and caraway (2.5%) [68]. The low melting point (−25.3 °C) allows HSO to remain a liquid even at low temperatures.
The dilution of HSO led to the formation of a specific spectral pattern in the UV region, which was similar to those of 8-O-substituted furanocoumarins [79] and indicated the presence of these phytocomponents (Figure 2). The spectrophotometric assay allowed us to determine that the total content of coumarins in HSO was approximately 24.52%, which is characterized as a high level.
For the further study of HSO, Fourier-transform infrared spectroscopy was applied, which is a commonly used method for the analysis and authentication of edible oils [80]. The spectral pattern of HSO is complex and characterized by various bands, which were assigned to three groups of phytocompounds after comparison with the reference compounds such as petroselinic acid (fatty acid example), octyl acetate (essential oil component), and imperatorin (furanocoumarin example) (Figure 3 and Figure S1). The most intense bands were attributed to the fatty acids, specifically C-H stretching of H-C=C at 2921 cm −1 , C-H symmetric stretching at 2851 cm −1 , C=O stretching at 1736 cm −1 , and C-H bending at 1463 cm −1 [81]. The alkyl fragment of octyl acetate gave the bands from the same "fatty" regions together with specific bands caused by acetate and octyl fragments at 1378, 1211, 1066, 1029, 938, and 721 cm −1 [82]. Bands of furanocoumarins clearly appeared at 700-1800 cm −1 , more specifically at 1713 (lactonic C=O), 1621 (furanic C=C), 1586, 1440 (aromatic C=C, 8-O-substituted furanocoumarins), 1324 (aryl-O of methoxylated coumarins), 1144, 1093 (furan ring), 997, 874, 825, and 748 cm −1 (deformation vibrations of C-H) [83]. The FTIR spectrum of HSO allows for elucidating the general composition of seed oil because it contains the bands of all dominant phytocomponents.

Coumarin Profile of Heracleum dissectum Seed Oil
The high coumarin content in HSO allowed us to realize profiling by HPLC-PDA-ESI-tQ-MS/MS. This led to the discovery of 38 compounds, which were identified on the basis of retention times as well as UV and mass spectrometric data after comparison with reference substances and literature data [84][85][86][87] (Figure 4, Table 4). The structures of thirty-two coumarins were accurately identified, and the structures were predicted for six compounds ( Figure 5). 2921 cm −1 , C-H symmetric stretching at 2851 cm −1 , C=O stretching at 1736 cm −1 , and C-H bending at 1463 cm −1 [81]. The alkyl fragment of octyl acetate gave the bands from the same "fatty" regions together with specific bands caused by acetate and octyl fragments at 1378, 1211, 1066, 1029, 938, and 721 cm −1 [82]. Bands of furanocoumarins clearly appeared at 700-1800 cm −1 , more specifically at 1713 (lactonic C=O), 1621 (furanic C=C), 1586, 1440 (aromatic C=C, 8-O-substituted furanocoumarins), 1324 (aryl-O of methoxylated coumarins), 1144, 1093 (furan ring), 997, 874, 825, and 748 cm −1 (deformation vibrations of C-H) [83]. The FTIR spectrum of HSO allows for elucidating the general composition of seed oil because it contains the bands of all dominant phytocomponents.

Coumarin Profile of Heracleum dissectum Seed Oil
The high coumarin content in HSO allowed us to realize profiling by HPLC-PDA-ESI-tQ-MS/MS. This led to the discovery of 38 compounds, which were identified on the basis of retention times as well as UV and mass spectrometric data after comparison with reference substances and literature data [84][85][86][87] (Figure 4, Table 4). The structures of thirty-two coumarins were accurately identified, and the structures were predicted for six compounds ( Figure 5).    Table 5.     (1) identified compounds after comparison of UV, mass-spectral data, and retention time with reference standards; (2) putatively annotated compounds after comparison of UV and mass-spectral data with literature data.
The obtained data allow us to conclude that H. dissectum seed oil is a source of coumarins that are typical for the Heracleum genus [6] and Apiaceae family as a whole [100], and some simple coumarins and furanocoumarins have been newly detected.

Quantification of Six Coumarins in Heracleum dissectum Seed Oil before and after Storage
To further characterize coumarins in HSO, quantification of six dominant compounds was performed by HPLC-ESI-TQ-MS, which allowed to determine the concentrations of heraclenin, oxypeucedanin, imperatorin, phellopterin, isoimperatorin, and ostruthin ( Table 6). The variation of coumarin content in four samples of HSO was 5.14-10.48 mg/mL for heraclenin, 0.93-5.76 mg/mL for oxypeucedanin, 108.83-153.05 mg/mL for imperatorin, 30.83-42.10 mg/mL for phellopterin, 11.67-29.52 mg/mL for isoimperatorin, and 4.59-5.09 mg/mL for ostruthin. The total coumarin content in samples was 181.14-238.42 mg/mL. Imperatorin is a dominant coumarin in H. dissectum seed oil and, as has been shown earlier, in H. leskowii seed lipophilic fractions [101] and H. verticillatum seed extract [102].
Owing to the lipophilic nature of H. dissectum seed oil, the storage of HSO may lead to a loss of quality parameters, including coumarin content. Therefore, it is helpful to study the stability of marker compounds under various storage conditions, i.e., room, cold, and freezing temperatures ( Table 6). The 3-year room temperature storage of HSO resulted in the greatest loss of total coumarin content, i.e., 8.9% after 1-year storage, 16.2% after 2-year storage, and 27.9% after 3-year storage. A decrease in storage temperature helped to preserve coumarins in HSO; specifically, after 3-year storage at 1 • C and at −20 • C, the total coumarin recovery was 92.9% and 97.7% of the initial content, respectively. This is a clear indication of the value of storage temperature on the quality of seed oil.

Nanosuspension of Heracleum dissectum Seed Oil and Its Neuroprotective Activity
Among the many existing methods of nanosuspension preparation, the CO 2 -assisted effervescence method was successfully applied to H. dissectum seed oil [103]. Prepared HSO nanosuspension has small particles (82.36 nm, polydispersity index 0.208), and zeta potential showed surface charge values of −25.3 mV (Figure 6), which indicates that this formulation is characterized by nanometer-scale particles and homogenous dispersion [104].

Nanosuspension of Heracleum dissectum Seed Oil and Its Neuroprotective Activity
Among the many existing methods of nanosuspension preparation, the CO2-assisted effervescence method was successfully applied to H. dissectum seed oil [103]. Prepared HSO nanosuspension has small particles (82.36 nm, polydispersity index 0.208), and zeta potential showed surface charge values of −25.3 mV (Figure 6), which indicates that this formulation is characterized by nanometer-scale particles and homogenous dispersion [104]. Permanent focal cerebral ischemia caused by the right-sided thermocoagulation of the middle cerebral artery in rats reduces cerebral blood flow (1.25 sm/s vs. 4.10 sm/s in the sham-operated group; p < 0.05) and increases necrosis zone area to 41.52% (Table 7). The application of a standardized extract of Ginkgo biloba (EGB761) demonstrated a positive effect characterized by increased cerebral blood flow (2.63 sm/s; p < 0.05) and reduction of necrosis zone area down to 21.60% (p < 0.05), which is typical for the plant extracts with neuroprotective effects such as Ginkgo biloba [105], Rhaponticum uniflorum [106], Serratula centauroides [107], and Nepeta multifida [108]. The nanosuspension of HSO in doses of 0.1-1 mL/kg demonstrated a positive dose-dependent effect, which increased with dose. The most pronounced neuroprotective activity was found for the dose of 1 mL/kg, which increased cerebral blood flow to 3.11 sm/s (p < 0.05) and decreased necrosis zone area to 18.56% (p < 0.05); this result indicates the greater therapeutic effect of HSO compared to that of the EGB761 reference drug. Permanent focal cerebral ischemia caused by the right-sided thermocoagulation of the middle cerebral artery in rats reduces cerebral blood flow (1.25 sm/s vs. 4.10 sm/s in the sham-operated group; p < 0.05) and increases necrosis zone area to 41.52% (Table 7). The application of a standardized extract of Ginkgo biloba (EGB761) demonstrated a positive effect characterized by increased cerebral blood flow (2.63 sm/s; p < 0.05) and reduction of necrosis zone area down to 21.60% (p < 0.05), which is typical for the plant extracts with neuroprotective effects such as Ginkgo biloba [105], Rhaponticum uniflorum [106], Serratula centauroides [107], and Nepeta multifida [108]. The nanosuspension of HSO in doses of 0.1-1 mL/kg demonstrated a positive dose-dependent effect, which increased with dose. The most pronounced neuroprotective activity was found for the dose of 1 mL/kg, which increased cerebral blood flow to 3.11 sm/s (p < 0.05) and decreased necrosis zone area to 18.56% (p < 0.05); this result indicates the greater therapeutic effect of HSO compared to that of the EGB761 reference drug.
The know literature data indicate that the selected components of H. dissectum seed oil have a great influence on the ischemic brain tissues. The basic coumarin of the plant, imperatorin, protects the brain against extreme oxidative stress induced by cerebral ischemia/reperfusion in rats through activation of the Nrf2 signaling pathway [109] and/or anti-apoptosis function [110]. Imperatorin reduces neuronal apoptosis and boosts synaptic plasticity in a vascular dementia model of rats developed by the modified ligation of perpetual two-vessel occlusion [111]. Imperatorin performed an anti-inflammatory role through the downregulation of MAPK and NF-κB signaling pathways in ischemic stroke-induced to microglia-mediated neuroinflammation and was determined to be a potential anti-stroke agent [112]. Imperatorin demonstrates a significant vasorelaxant activity (which is higher than that of acetylcholine), radical scavenging [113], and antidepressant potential [114]. Geranylated coumarin ostruthin, owing to its TREK-1 channel activator activity, showed antidepressant and anxiolytic effects in mice evaluated by the open-field, elevated plus maze, and light/dark box tests [115]. Unsaturated fatty acids can protect the brain against ischemic injury by activating Nrf2 and upregulating heme oxygenase 1 [116]. Perhaps other phytocomponents of H. dissectum seed oil may have positive effects on the ischemic brain; however, this question will be addressed in future studies. Letters ( a-c ) indicates a significant difference (p < 0.05) vs. sham-operated animals' group ( a ), negative control group ( b ), and EGB761 reference group ( c ).

Conclusions
This study demonstrated for the first time that H. dissectum seeds are a source of valuable oil. Physico-chemical parameters and phytocompounds present in seed oil (HSO) were characterized by various methods, including high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection. Fatty acids, volatile components, coumarins, and photosynthetic pigments were found in HSO and quantified. Coumarins were separated by the LC-MS technique, and HSO was determined to be a source of furanocoumarins among which heraclenin, oxypeucedanin, imperatorin, phellopterin, isoimperatorin, and ostruthin were predominant with the total content of 18.1-23.8%. Stability study showed that cold and freezing storage resulted in the best preservation of coumarins in HSO. Our findings suggest that it is possible to obtain HSO nanosuspensions with neuroprotective activity, as determined using the model of cerebral ischemia in rats. Thus, H. dissectum is a bioactive plant. These results will help create new nanotherapeutic remedies.