The Antioxidant and Neuroprotective Potential of Leaves and Inflorescences Extracts of Selected Hemp Varieties Obtained with scCO2

2.2. Reagents

Cannabinoids standards: CBD, CBDA, Δ⁹-THC, THCA, CBG, CBGA, CBN, and CBC were obtained from Sigma-Aldrich (Poznan, Poland). Trifluoroacetic acid and acetonitrile (high-performance liquid chromatography [HPLC] grade) were supplied by Merck (Darmstadt, Germany). High-quality pure water was prepared using a Direct-Q 3 UV purification system (Millipore, Molsheim, France; model Exil SA 67120). Iron (III) chloride hexahydrate, 5,5-dithio-bis-(2-nitrobenzoic acid), 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), neocuproine, 2,4,6-tri(2-pyridyl)-s-triazine, trolox, tyrosinase, L-DOPA, acetylcholinesterase, butyrylcholinesterase, acetylcholine iodide, butyrylcholine iodide, Trizma^® hydrochloride, and Trizma^® base were purchased from Sigma-Aldrich (Schnelldorf, Germany). Sodium chloride, sodium dihydrogen phosphate, and sodium hydrogen phosphate, were purchased from Avantor Performance Materials (Gliwice, Poland). Ammonium acetate (NH₄Ac) and methanol were supplied by Chempur (Piekary Śląskie, Poland). Cupric chloride dihydrate, acetic acid (99.5%), ethanol (96%), and sodium acetate trihydrate were supplied by POCH (Gliwice, Poland).

2.3. Extraction

Dry raw leaves and inflorescences of Białobrzeskie, Tygra, and Henola varieties were ground and 6.5 g of the plant material was loaded into an extraction vessel. The dynamic scCO₂ extraction process was carried out under 2000 and 6000 PSI at 50 °C with 250 mL of CO₂ as marginal pressure values significantly affecting the neuroprotective activity and exhaustive amount of carbon dioxide assessed during preliminary studies (Table 1). Next, the extracts were suspended in methanol, winterized, and filtered under vacuum.

2.4. Chromatographic Analysis

The cannabinoid profile analysis of the extract was studied with the use of ultra-high-performance liquid chromatography with the diode array detector (HPLC-DAD) validated method (Shimadzu Corp., Kyoto, Japan) [42]. The determination was performed with the use of a chromatographic column CORTECS Shield RP18, 2.7 µm; 150 mm × 4.6 mm. As a mobile phase, 0.1% trifluoroacetic acid (41%), and acetonitrile (41:59, v/v) were used. The flow rate was set at 2.0 mL/min, and the column temperature was set at 35 °C. The injection volume was 10.0 µL, the detection wavelength was 228 nm, and the analysis time was 50 min. The retention time of cannabinoids is presented in Table 2. The results were acquired and processed using LabSolutions LC software (version 1.86 SP2) from Shimadzu Corp. (Kyoto, Japan).

2.5. Antioxidant Activity

The antioxidant activity was performed with the use of four assays: DPPH, ABTS, CUPRAC, and FRAP. Each assay was preceded by the screening of the extracts’ antioxidant activity with descending concentrations of the extracts. Trolox antioxidant activity was studied at a suitable concentration range to inhibit radicals (DPPH and ABTS) or to perform redox reactions (CUPRAC and FRAP). A linear regression equation was established to relate the trolox concentration with its corresponding scavenging percentage (DPPH and ABTS) or absorbance (CUPRAC and FRAP), subsequently, the results presented as mg trolox/g plant material were calculated through the equation, according to the antioxidant properties of the extracts in all four assays [43,44]. The antioxidant potential was determined according to the previously reported method [45,46]. Each measurement in the antioxidant studies was repeated six times.

The DPPH assay is based on mixing the radical solution with a hydrogen atom-donating substance, as it causes the vibrant violet color to vanish, leading to the formation of the reduced form [47]. The inhibition of DPPH radicals by the studied samples/trolox was calculated using the formula:

where A_o is the absorbance of the control sample and A_i is the absorbance of the test sample.

The other measurement that determined the scavenging radicals potential was the ABTS assay in which green cation radicals are produced by the loss of electrons by the nitrogen atoms of ABTS, caused by potassium persulfate. The inhibition of ABTS•+ was calculated using the following formula:

where:

A₀—the absorbance of the control;

A₁—the absorbance of the sample.

As a first method of determining the reducing potential of an oxidant, CUPRAC was performed. During this assay, phenolic groups of antioxidants are oxidized to quinones, and the neocuproine and copper (II) ion complex (bluish) is reduced to the neocuproine and copper (I) ion complex (yellow).

The last antioxidant technique that determined the reducing properties (of colorless Fe³⁺ ion to Fe²⁺ with the formation of a dark blue complex with 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ)) by the extracts was the FRAP technique.

2.6. Inhibition of Enzymes Influencing the Neurodegenerative Diseases

The potential of the extracts to inhibit enzymes such as AChE, BChE, and tyrosinase involved in neurodegeneration development was studied. Firstly, the screening of the extracts’ activity by measuring the inhibitory activity of the descending concentrations was performed. Esterases’ strong inhibitors are rivastigmine, donepezil, and galantamine [48], whereas tyrosinase is strongly inhibited by hydroquinone, kojic acid, and azelaic acid [49]. Thus, galantamine was chosen as a standard inhibitor for AChE and BChE, whilst for tyrosinase inhibitor, azelaic acid was selected. A linear regression equation between the standard concentration and its percentage potential to inhibit the enzyme was built, and the standard equivalent was calculated through the equation, according to the inhibitory properties of the extracts in all three assays. The results were presented as a galantamine equivalent (GALAE) (mg galantamine/g plant material) for the AChe and BChE assays and as an azelaic acid equivalent (AzAE) (mg azelaic acid/g plant material) [50,51,52,53,54,55].

The ability to inhibit AChE and BChE was determined according to the previously reported method [45]. This assay requires artificial substrates (thiocholine esters). Thiocholine is liberated during the enzymatic reactions with 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB), and the 3-carboxy-4-nitrothiolate anion (TNB anion) is formed. The potential to inhibit AChE and BChe was measured according to the increase in the thiocholine color in a 96-well plate. The percentage of AChE and BChE inhibition by the samples was calculated according to the following equation:

where:

A₁—the absorbance of the test sample;

A_1b—the absorbance of the blank of the test sample;

A₀—the absorbance of control;

A_0b—the absorbance of the blank of control.

The tyrosinase inhibition assay is based on the reduction in color intensity of the solution due to the inhibition of enzyme activity [56]. The inhibitor blocks L-DOPA access to the tyrosinase active site, which prevents the reaction from proceeding. The study was performed according to the previously reported method [45]. The percentage inhibition of the tyrosinase by the samples was calculated with the use of the following equation:

where:

A₁—the absorbance of the test sample;

A_1b—the absorbance of the blank of the test sample;

A₀—the absorbance of control;

A_0b—the absorbance of the blank of control.

3.2. Chromatographic Analysis

As expected, the cannabinoid profiles differed between the studied hemp strains (Białobrzeskie, Henola, Tygra) (Table 3). In addition, changes in cannabinoid profiles were also noted between the leaves and inflorescences. Each plant material had the highest content of two cannabinoids, CBD and CBDA. The leaves had lower contents of all the tested cannabinoids such as CBD, CBDA, CBG, CBN, CBGA, THC, CBC, and THCA, particularly when compared to inflorescences. Most frequently, the extraction carried out at 6000 PSI (b) led to a higher cannabinoid content than the extraction under 2000 PSI (a). The highest CBD (6248.5 ± 154.9 μg/g plant material) and CBDA (2558.0 ± 107.8 μg/g plant material) contents were obtained in an extract of Tygra inflorescences obtained under 6000 psi. The highest contents of CBG (224.8 ± 2.0 μg/g plant material), CBN (107.7 ± 5.4 μg/g plant material), CBGA (89.74 ± 4.23 μg/g plant material), THC (998.4 ± 18.8 μg/g plant material), CBC (310.2 ± 12.9 μg/g plant material), and THCA (303.0 ± 3.9 μg/g plant material) were found in the Białobrzeskie inflorescences extract obtained under 6000 PSI.

3.3. Antioxidant Activity

The antioxidant properties of the extracts were studied based on two mechanisms: the first mechanism scavenges free radicals: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and the second inhibit oxidation reactions by being oxidized themselves: cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP).

During the DPPH assay (Figure 1), the 1,1-diphenyl-2-picrylhydrazyl radical was scavenged by all the extracts at some level. The extracts obtained under 6000 PSI pressure exhibited more pronounced antioxidant activity than those obtained at the lower pressure of CO₂. The most potent antioxidant activity against the DPPH radical was obtained by the Tygra variety, with the result 0.92 ± 0.03 mg trolox/g plant material and 0.93 ± 0.03 mg trolox/g plant material for the leaves and inflorescences, respectively. Both of the results were statistically significantly better than every other extract.

Another technique that determined the radical scavenging potential of the extracts was the ABTS method (Figure 2). The ABTS•+ radical was scavenged better by the extracts obtained under 6000 PSI (b). The greatest results among cannabis leaf extracts were noticed for Tygra (b) (11.83 ± 0.10 mg trolox/g plant material). The most substantial extract was the Tygra inflorescences (b) with 19.37 ± 0.06 mg trolox/g plant material, which was significantly greater than any other extract.

The ability to reduce cupric ion was also studied (CUPRAC assay) (Figure 3), and the most efficient extract among the leaves was the Tygra (b)—5.76 ± 0.01 mg trolox/g plant material, while the most noticeable potential to reduce Cu²⁺ ion was shown by Tygra in the florescences (b) extract—8.34 ± 0.01 mg trolox/g plant material, which was statistically greater than every other extract.

The last antioxidant model used in this study was FRAP, which determined the ability to reduce ferric ions (Figure 4). The strongest antioxidant potential within the leaves was determined for the Tygra (b)—1.43 ± 0.01 mg trolox/g plant material; whereas among inflorescences, the simultaneously the greatest result of all showed Tygra inflorescences (b) extract—1.84 ± 0.01 mg trolox/g plant material, which had a statistical significance. Overall, the most significant antioxidant activity in the four assays was noted for the Tygra variety.

The oxidative stress mechanism entails the creation of reactive oxygen species (ROS) through normal cellular metabolism, exposure to environmental toxins, or the presence of conditions that cause oxidative stress. Oxidative stress arises when there is a disturbance in the equilibrium between the generation of ROS and the body’s protective antioxidant mechanisms [79].

There are also other studies in the literature presenting the antioxidant potential of the cannabis plant. In a study by Mastellone et al., the fiber-type Cannabis sativa L. plant material was extracted with methanol and acetone by ultrasound-assisted extraction; subsequently, both of the extracts were tested for antioxidant properties with the use of the ABTS and DPPH assays [80]. Methanol extract showed a stronger antioxidant potential than acetone. In another study, Futura 75 hemp frozen inflorescences were extracted by microwave-assisted extraction to obtain an aqueous extract, while the residual biomass was extracted with the use of n-hexane in an ultrasound bath [62]. In the DPPH and superoxide radical uptake assays, aqueous extract presented a higher potency, while in the FRAP and ORAC assays, hexane extract was stronger. André et al. studied the influence of maturation on polyphenol content and antioxidant activity of eight different fiber-type Cannabis sativa L. cultivars [81]. The total phenolic content decreased with the flower’s development for all cultivars studied. Similarly, DPPH radical scavenging activity was reduced over the maturation period. The antioxidant activity of cannabis was also studied in vivo. In a study by Asta et al., the Cannabis sativa Futura 75 variety was extracted with ethanol and administered to the mice intragastrically (1.6 mg of cannabis extract/g/day) via stomach tube [82]. The extract contradicted the AlCl₃-induced increase in the level of malondialdehyde in the liver and brain and prevented the decrease in catalase activity. Previous studies indicate that antioxidant activity is related to the level of polyphenols in plant material, but the level of cannabinoids contributes significantly to antioxidant activity by Cannabis sativa. In the present study, the most potent antioxidant activity was related to the greatest CBD level.

In our previous research, which inspired the research presented in this paper, we confirmed the antioxidant potential of methanolic, ethanolic, and isopropanol extracts of hemp fiber varieties. The greatest antioxidant power was also associated with the highest concentrations of CBD [42]. However, it is worth mentioning that other cannabis components besides cannabinoids might also play a significant role in antioxidant properties and work synergistically with cannabinoids, which is called the “entourage” effect [2,3,4]. Different cannabis varieties show a wide range of secondary plant metabolite profiles and thus differ in biological activity [83,84]. Cannabis plant material after extraction provides compounds that are responsible for the smell, terpenes like β-myrcene, limonene, and β-(E)-caryophyllene, as well as α-humulene [35,85]. These components exert their biological activity, including antioxidant potential [64,86]. Cannabis plant material should be treated as a whole, thus it is also worth mentioning that phenolic compounds, e.g., phenolic acids and flavonoids in their glycoside and aglycone forms which are found in cannabis plant material chlorogenic and caffeic acids, catechin, epicatechin, rutin, naringenin, quercetin, apigenin, cannaflavin B and lignanamides cannabisin A, B, and C also play a significant role in cannabis antioxidant properties [64,87,88,89,90,91].

3.4. Anticholinesterase Activity

AChE and BChE are enzymes that break down the neurotransmitter acetylcholine in the synaptic cleft. The inhibition of these enzymes can lead to an accumulation of acetylcholine, which can enhance cholinergic neurotransmission and potentially provide neuroprotective effects. Cholinergic neurotransmission plays a crucial role in cognitive function [92], including memory and learning. In addition to the cognitive effects, AChE, and BChE inhibition may also have neuroprotective effects as they leads to a reduction in oxidative stress and inflammation, which are principal in the pathogenesis of neurodegenerative diseases [93]. Additionally, the inhibition of BChE prevents amyloid-beta-induced neuronal death [94].

All of the leaf and inflorescence extracts showed inhibition of AChE (Figure 5). In general, the inflorescences had greater inhibitory potential than the leaves, with the greatest result noted for the Tygra leaves (b)—8.20 ± 0.08 mg galantamine/g plant material. Among the inflorescences, the most anti-acetylcholinesterase activity was noted for the Henola inflorescences (b)—20.23 ± 0.43 mg galantamine/g plant material which was statistically different than all of the studied extracts.

BChE inhibitory activity was also noted within Cannabis sativa extracts and the overall results are similar to those of the AChE inhibition, as among the leaves the strongest inhibitory potential was assessed for the Tygra leaves (b)—8.17 ± 0.09 mg galantamine/g plant material (Figure 6). The most intense ability to inhibit AChE was determined for the Henola inflorescences (b)—17.12 ± 0.27 mg galantamine/g plant material, which has a statistically significantly stronger potential than any other studied extract.

In a study by Karimi et al., methyl alcohol Cannabis sativa extract derived from leaves showed inhibition of AChE and BChE activity at 52.33% and 49.00%, respectively; whereas resin fraction inhibited the AChE and BChE at 80.00% and 68.00% [95]. In their research, Puopolo et al. investigated the anticholinesterase properties of eight cannabinoids: CBD, Δ⁸-THC, CBG, CBGA, CBT, CBDV, CBC, and CBN, all at a concentration of 200 µM [96]. They observed the significant inhibition of AChE and BChE activities, with varying percentages of inhibition. Specifically, AChE was inhibited by 70.8, 83.7, 92.9, 76.7, 66.0, 79.3, 13.7, and 30.5%, while BChE was inhibited by 86.8, 80.8, 93.2, 87.1, 77.0, 78.5, 27.9, and 22.0%, respectively. Molecular docking investigations revealed that the cannabinoids engaged with multiple amino acid residues on the enzyme proteins, providing substantial evidence for their collective ability to inhibit AChE and BChE. In a study by Mooko et al., hexane and dichloromethane flower and bud extracts showed a greater inhibitory potential against AChE and BChE, whereas water, hexane, dichloromethane: methanol (1:1), and methanol showed a better potential to inhibit β-secretase [97]. In an in vivo study, where rats were administered cannabis resin, tramadol, or a combination of both, cannabis alone increased AChE brain activity (by 16.3–36.5%), while it remained at the same level after tramadol administration, whilst the combination of both decreased AChE activity (by 12.9–13.6%) [98]. The activity of BChE exhibited significant and dose-dependent inhibition in response to cannabis resin (by 60.9–76.9%), but also by tramadol (by 17.6–36.5%) and the combination of both (57.2–63.9%). In a study on Caenorhabditis elegans, the neuroprotective potential of two C. sativa oils demonstrating fluctuations in CBD and THC concentrations was studied and, as a result, both oils were efficient in decreasing AChE activity, and ROS levels [61].

3.5. The Inhibition of Tyrosinase

Tyrosinase inhibition, involving the blockage of the L-DOPA substrate access to the center of the enzyme, is a key reaction in relation to the inhibition of dopamine breakdown, which is deficient in Parkinson’s disease [99]. Cannabis sativa extracts inhibited tyrosinase (Figure 7).

In most cases, the leaf extracts showed weaker potential to inhibit the enzyme than the inflorescence extracts. The only exception was the Białobrzeskie leaves extract (b), which showed exceptional inhibition properties—166.73 ± 1.87 mg azelaic acid/g plant material and was not significantly different from the Henola inflorescences extract (b) 170.92 ± 5.57 mg azelaic acid/g plant material. The greatest tyrosinase inhibitor and most statistically different from all other extracts was the Białobrzeskie inflorescences extract (b)—212.22 ± 8.83 mg azelaic acid/g plant material.

In a Kim et al. study, hemp seeds ethanol crude extract was fractioned with hexane, dichloromethane, ethyl acetate, and butanol [100]. Tyrosinase’s inhibitory activity depends on the used solvents [80]. The methanol extract showed 33.17% ± 2.04% of inhibition while acetone showed 38.46% ± 1.36%. In the Manosroi et al. study, hemp leaves and seeds collected in Thailand were macerated with ethanol; subsequently, the extracts were studied for the inhibitory potential of tyrosinase [101] and they showed inhibition of the enzyme.

It would be beneficial to extend the investigations to include experiments conducted on cell lines. After receiving promising results, it would be suggested to expand the research to in vivo animal studies to gain insights into the potential neurodegeneration-preventative applications of the extracts.