Oil Extraction from Hemp Plant as a Potential Source of Cannabidiol for Healthy Protein Foods

2.2. Extraction Methods

For antioxidant recovery, different methods of extraction were employed: supercritical fluid extraction (SFE), using CO₂ as a supercritical fluid; Soxhlet (SE); and dynamic maceration (DME), using ethanol as solvent.

The extractions were performed on hemp inflorescences (HI), stalks (HS), and proteic flour (HPF), using the raw material after manual separation without further treatments. After a decarboxylation process, conducted at 140 °C for 30 min in a ventilated oven (FD 53, BINDER GmbH, Tuttlingen, Germany), according to the literature procedure [8], the DHI, DHS, and DHPF fractions were obtained.

Regarding SE, 15 ± 1 g of sample and 400 mL of ethanol were used. The samples were placed into a thimble filter and located in the middle of the Soxhlet extractor. The extraction process was carried out at 78 °C for 8 h, after which the ethanol hemp-enriched extract was collected [13].

In order to perform the DME, 1 g of sample was used, added to 40 mL of ethanol, using the solvent/sample ratio suggested by literature [2]. The DME process was carried out at room temperature for 15 min under magnetic stirring. Afterwards, the ethanol was recovered and the exhaust sample was extracted again, using the same procedure, for a second time. The obtained extract was collected and the exhaust hemp sample was used for a third cycle, adding 20 mL of ethanol, and obtaining a final extract amount of 100 mL.

The extracts were isolated by means of a Rotavapor system (Heidolph G3, Hei-VAP Value, Heidolph Instruments, Wood Dale, IL, USA), and the yield was calculated by weighting. Extractions were performed in triplicate and the maximum yield was calculated according to the following equation:

Determinations were conducted in triplicate.

Finally, 25 ± 1 g of grounded samples were used for SFE, carried out in a laboratory-scale plant (Spe-ed SFE, Applied Separations, Allentown, PA, USA) at 40 °C and 360 bar. A first phase of 30 min, in a static mode, followed by dynamic phases of 15 min were adopted for SFE. The static phase of 15 min was interposed between two dynamic phases, with a total extraction time of 140 min. The obtained extracts were collected in a volumetric flask, weighed, and the maximum yield was calculated according to Equation (1).

Soxhlet extraction was performed with 15 ± 1 g of HPF and 400 mL of analytical grade n-hexane (Sigma-Aldrich Co., St. Louis, MO, USA). After extraction, the obtained oil was isolated through a Rotavapor at 50 °C (Heidolph G3, Hei-VAP Value, Heidolph Instruments, Wood Dale, IL, USA), and the yield was calculated by the Equation (1).

2.3. Proteic Dough Preparation

In this study, a high-protein bar formulation was studied as a model, using proteins from the hemp plant, which has a high nutritional value [14,15]; Greek yogurt; resistant starch; and CBD oil, as a source of antioxidants. According to [16], a base formulation (S1), having 28% w/w on the dry product of hemp protein, without Greek yogurt was studied. The model bars were obtained by adding a resistant maize starch, rich in amylose, with a low glycemic index [15]. Starting from an initial formulation, the fresh Greek yogurt was added, at different percentages, because of its ability to improve the final dough consistency and quality, resulting in a softer system [17].

Four formulations, with increasing amounts of Greek yogurt, were studied. The ingredients’ percentages are reported in Table 1. A total 1% w/w of CBD oil extracted from inflorescences was added to the dough of all samples.

High-protein bars were made by mixing all the ingredients by means of a Brabender Farinograh (Belotti Instrument, Peschiera Borromeo (MI), Italy). The velocity was set at 63 rpm and the temperature at 24 °C. The mixing time for all samples was 20 min.

At the end of the mixing stage, part of the dough was bar-shaped and cooked in the oven, and another part underwent rheological measurements.

2.4. HPLC Analysis

CBD and CBDA content in the extracts were evaluated using a Smartline HPLC system (Knauer, Berlin, Germany) consisting of a degasser, a pump, and a UV detector 2600. Chromatographic separation was accomplished using a 150 mm × 2 mm i.d. C18 Ascentis, with a precolumn (Supelco, Darmstadt, Germany). The column temperature was set at 32 °C. The mobile phase was composed of 0.1% v/v formic acid in acetonitrile (A) and 0.1% v/v formic acid in water (B) at a flow rate of 0.1 mL/min. In Brighenti et al.’s study [2], during analysis, the gradient elution was varied as follows: 0–13 min, 60% A; 13–17 min, from 60 to 80% A; 17–22 min, from 80% to 90% A, which was maintained for eight minutes. Finally, a 15-min post-running time was imposed. This method is validated for quality control of pharmaceutical products, according to the ICH guidelines [2].

Absorbance spectra were recorded every 1 s, between 200 and 450 nm, with a bandwidth of 8 nm. Chromatograms were acquired at 210, 220, 235, and 275 nm. The measurements were performed in triplicate. For both CBD and CBDA, a calibration curve was obtained by performing HPLC analysis on different CBD and CBDA standard solutions, at concentrations of 0.01, 0.025, 0.1, 0.5, and 1 mg/mL (Sigma-Aldrich Co., St. Louis, MO, USA). Before injection into the HPLC system, the extracts were filtered using a 0.45 μm PTFE filter.

2.5. Antioxidant Activity

The antioxidant activity of extracts was investigated by two methods: 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. Firstly, the radical scavenging properties of the extracts towards the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical were investigated as described by Iacopetta et al. [18], with some modifications. Briefly, 20 μL of each extract, properly dissolved in ethanol, were mixed with 180 μL of a 0.1 mM DPPH solution in methanol, in a 48-well plate, to obtain seven different concentrations (from 100 to 5000 µg/mL). As a negative control, 20 μL of ethanol in 180 μL of DPPH methanol solution was diluted. The mixture was then shaken and incubated at room temperature, for 30 min, protected from the light. The scavenging activity was determined at 517 nm using a microplate reader.

The radical scavenging properties of the extracts against the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS^•+) were extrapolated as described by Iacopetta et al. [18], with minor adjustments: 2 mM ABTS and 70 mM potassium persulfate water solutions were mixed, incubated for 16 h at room temperature, and protected from light to obtain the ABTS^•+ radical stock solution. The latter was ethanol-diluted until the absorbance at 730 nm was 0.70 ± 0.02. Before use, 2 μL of each extract, dissolved in ethanol, was added to 198 μL of the ABTS^•+ solution in a 48-well plate to obtain seven increasing concentrations (from 100 to 2000 µg/mL). After being shaken and incubated for 5 min at room temperature (in the dark), the scavenging activity was determined at 730 nm. Also, in this case, a negative control was inserted (2 μL of ethanol and 198 μL of the ABTS^•+ solution). For both methods, the radical scavenging activity was expressed as inhibition percentage (%I_i) of each extract compared with the respective control, according to the following equation:

where the subscript i refers to DPPH and ABTS, with respect to each assay; A₀ is the control reaction absorbance; and A is the absorbance in the presence of samples. IC₅₀ values determination was obtained from the IDPPH and IABTS percentages, using GraphPad Prism 9 software (GraphPad Inc., San Diego, CA, USA). As a positive control, Trolox was employed. All experiments were performed three times, each in triplicate. Data are expressed as mean values ± standard deviation (SD).

2.6. Rheological Characterization

The doughs were characterized by rheological measurements performing tests in dynamic mode. Frequency sweep tests were carried out at 20 °C, 40 °C and 60 °C in the linearity region, in a range of 0.1–10 Hz, preliminarily evaluated by stress sweep and time sweep tests. Furthermore, temperature ramp tests in heating mode were performed from 20 °C to 100 °C, at 1 °C/min, and at 1 Hz, in linearity. All rheological tests were carried out by means of a rotational rheometer (HAAKE MARS III, Thermo Fisher Scientific, Braunschweig, Germany) equipped with a parallel plate geometry (diameter = 20 mm; gap = 2 mm ± 0.1 mm) and a Peltier system for controlling the temperature. All measurements were conducted twice. To avoid water evaporation phenomena during tests, silicone oil of 120 cSt (VWR Chemicals, Briare, France) was used.

The frequency sweep tests were interpreted using the weak gel model [19]:

where G* is the complex modulus,  is the frequency, A is a measure of gel strength, and z is a measure of gel structuration.

3.2. Antioxidant Activity Results

To date, different studies have demonstrated that many compounds extracted from natural matrices possess interesting antioxidant activity [27]. It is known that oxidative stress, characterized by a higher presence of Reactive Oxygen Species (ROS), can induce dramatic effects by damaging important intracellular structures, including DNA, proteins, and lipids [28]. These effects can be prevented by employing compounds with antioxidant properties, which can scavenge ROS and other radicals. Since the investigation of the antioxidant activity is influenced by several variables, different, proper assays are required for assessing the antioxidant effects of an extract [29]. In this study, two in vitro assays, namely, ABTS and DPPH, were employed to evaluate the free-radical scavenging ability of the extracts (for further details, see the Section 2.5). As a positive control, a water-soluble analogue of vitamin E (Trolox) was adopted. The calculated IC₅₀ values, expressed in µM, are reported in Table 4. First, the best ability in scavenging the ABTS radical was exhibited by DHI and HI, showing IC₅₀ values of 38.1 ± 1.1 and 85.3 ± 2.4 µg/mL, respectively, which are about two- and five-fold higher than that of Trolox (15.9 ± 1.3 µg/mL).

The result, related to the extract obtained with the DM methodology, is in agreement with the extracted CBD values evaluated by HPLC investigations, which are the highest among the samples subjected to antioxidant assays. A similar activity was detected for DHS (IC₅₀ = 87.2 ± 2.2 µg/mL), whereas a lower one was found for HS (IC₅₀ = 125.1 ± 3.3 µg/mL). In both cases, Soxhlet extraction from stalks leads to lower CBD amounts and, therefore, a lower antioxidant capacity of the extracts. Finally, HPF and DHPF, extracted by the SFE process, were not able to scavenge the ABTS radical (IC₅₀ > 2000 µg/mL). According to HPLC analyses, all the decarboxylated samples, possessing higher amounts of CBD, show higher antioxidant activity than the untreated samples.

A different behavior was recorded for the ability to scavenge the DPPH radical. Indeed, the best scavenging ability was found for DHS (IC₅₀ = 348.5 ± 2.4 µg/mL), followed by DHI and HS, whose values were very similar (IC₅₀ of 535.7 ± 4.7 and 550.1 ± 4.5 µg/mL, respectively). Again, HFP and DHFP were unable to scavenge the DPPH radical (IC₅₀ > 5000 µg/mL). In conclusion, the extracts demonstrated better activity in inhibiting the radical ABTS compared with DPPH, in agreement with [30].

3.3. Rheology

In light of the good yield of hemp oil from PF and the high quantity of antioxidants, a possible way to introduce bioactive antioxidant compounds in foods is to enrich the hemp oil, from seeds, with the extract from the inflorescences. Doughs were prepared using PF, to acquire formulations suitable for the preparation of protein bars (25% protein, dry basis); resistant starch (Hi-Maize), to improve the dough structure [15]; and 5% w/w of fat basis, from HI oil obtained by means of DME, to enrich the bars in CBD. All samples were characterized as reported in the materials and methods section.

For all samples, the temperature ramp test showed the complex modulus evolution that decreases with increasing temperature. For starch-based systems, the typical trend of the complex modulus shows a first G* decay, corresponding to an increase in molecular mobility caused by the increasing temperature, followed by a quite sharp increase of G*. The latter increase is due to the gelatinization phenomena that modify the material’s microstructure [3]. The possible accentuated growth of the complex module depends on the nature and origin of the flour, as well as on the quantity of water, and so on. The doughs, obtained with or without the addition of Greek yogurt, showed an increase in G* after 60 °C. S1 and SY1 showed a similar behavior and gelatinization temperature, in agreement with other literature formulations [31], while samples richer in Greek yogurt showed a shift of the G* curve toward higher values of gelatinization temperature. The yogurt proteins probably have a competitive effect against resistant starch in the gelatinization process. Moreover, the angle phase values (Figure 1b) suggest a solid-like behavior for all samples in the investigated temperature range, with values ranging between 5° and 25°.

Figure 2 shows the G* and angle phase values at different frequencies and at 20 °C. It is possible to see that the systems are solid-like because δ is lower than 45°. The consistency of the dough decreases (G* lowers) with the increase in Greek yogurt quantity, even if the structure does not change too much, as shown by δ’ s trend (Figure 2b). The same trend was observed for frequency sweep tests performed at 40 and 60 °C; all data were interpreted by the weak gel model [19], the parameters of which are reported in Table 5.

From the data reported in Table 5, it can be observed that, for each temperature, the A parameter decreases from S1 to SY3, indicating that the addition of Greek yogurt has the effect of weakening the sample without changing the structure, as shown by the z values. On the other hand, for each sample, the strength of the structure decreases with the increase in temperature; also, in this case, the z parameter is almost constant with the temperature.