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DESCRIPTION

The present invention relates to a method for the characterization of short peptides from proteins of industrial hemp seed flour (Cannabis Sativa L). The present also relates to the mixture of short peptides obtained by this method.

Peptides are a class of chemical compounds made up of a variable chain of amino acids linked together through peptide bonds.

Peptides can be classified based on the length of the amino acid chain as long peptides, containing more than 20 amino acids, medium peptides, containing 5 to 20 amino acids, and short peptides, containing 2 to 4 amino acids. Peptides can be obtained from proteins of animal or vegetable origin through in vitro hydrolytic processes, for example through proteolysis with enzyme mixtures, or they can be naturally present in the matrices under examination because they are produced by the proteolytic activity of endogenous proteases.

The proteins contained in food matrices are of great scientific interest as they can be sources of bioactive peptides. Many foods, such as milk and its derivatives, soy, wheat, corn, rice, eggs, fish, meat are rich in peptides having numerous biological activities both in vitro and in vivo, such as antioxidant activity, antihypertensive, anticarcinogenic, antimicrobial, hypercholesterolaemic, immunomodulatory, dipeptidyl peptidase IV (DPP-IV) inhibitory activity, and angiotensin converting enzyme (ACE) inhibitory activity (D.J. Daroit et al., Current Opinion in Food Science, 2021 , 39, 120-129).

Peptides have considerable commercial interest, both alone and in formulations combined with other constituents, for nutraceutical, pharmaceutical, food and cosmetic preparations.

Recently, there has been a growing interest in peptides from proteins contained in industrial hemp (Cannabis sativa L).

For example, the patent application WO 2021/081619 relates to a method for the isolation of low molecular weight peptides, having from 2 to 7 amino acids, starting from a protein extract from hemp seeds. According to this method, the protein extract is hydrolyzed using known proteases, while the isolation of low molecular weight peptides from the total hydrolysate takes place by using four ultrafiltration membranes in series having molecular cut-offs of 50, 30, 10 and 3 kDa or by electrophoretic methods, such as for example SDS-PAGE.

The patent CN 107136517 instead relates to a healthy drink based on hemp seed flour polypeptides, in which the polypeptides are purified by means of ultrafiltration membranes having a molecular cut-off of 5 kDa.

Hemp, especially its seeds, is increasingly used as a source of biologically active compounds, such as peptides.

The peptides are obtained through a protein extraction process from the plant seed and subsequent enzymatic proteolysis of these proteins. Hemp proteins are mainly found in the seed, representing in fact about 20-30% of the whole seed, and are mainly made up of edestin (60-80%) and albumin (20-40%) (Q. Wang, Y. L. Xiong, Comprehensive Reviews in Food Science and Food Safety, 2019, 18, 936-952).

There are several known processes of extracting proteins from hemp. For example, M. Hadnadev (Food Hydrocolloids, 2018, 79, 526-533) describes two extraction systems based on an alkaline extraction, and an isoelectric point precipitation (HPI) whose extraction yield has been compared to a micellization (HMI). Furthermore, a device based on countercurrent extraction with sub-critical solvent (propane or butane) to extract proteins from hemp seed flour is commercially available (http://www.bestextractionmachine.com/protein-extraction/mak inq- hemp-protain- powder.html).

The proteins extracted with one of the methods known in the art are generally subjected to enzymatic proteolysis processes to obtain the corresponding peptides. The choice of proteolytic enzymes is of fundamental importance for obtaining different peptide mixtures and different types of biological activities. Commonly used enzymes are: chymotrypsin, flavourzyme, pepsin, papain, Alcalase®, Protamex®, Neutrase® and mixtures thereof.

The protein hydrolysates obtained can be fractionated and purified to separate specific molecular weight ranges in which peptides with more marked biological activities could be present. The most commonly used techniques are those already known in the art, such as for example reverse phase chromatography (RP-HPLC), ultrafiltration membranes and size exclusion chromatography. The growing interest towards peptides or hydrolysates obtained from hemp seeds derives from the numerous benefits that their consumption brings to human health, as evidenced by many studies in the literature. In particular:

1) a mixture of short peptides, consisting of 2-3 amino acid residues in their sequence, showed anticarcinogenic activity towards Hep3B liver cancer cells. (L.H. Wei et al., Food Science and Nutrition 2021 , 9:1833-1841);

2) 5 peptides, consisting of 9-24 amino acid residues (medium-long), obtained via lactic acid bacteria, showed in vitro antioxidant activity (E. Pontonio et al., Antioxidant 2020, 9, 1258, 1-26 ); also a hydrolyzate obtained using three commercial enzymes of microbial origin such as Alcalase®, Neutrase® and Protamex® showed in vitro antioxidant activity, an antiproliferative effect on cells and a stimulatory effect on the proliferation of HaCaT cells (M Logarusic et al., Molecular Biology Reports, Molecular Biology Reports 2019, (46), 6079-6085);

3) a hydrolysate of Alcalase® and flavourzyme showed anti-inflammatory activity in primary human monocytes (N. M. Rodriguez-Martin et al., Biomolecules 2020, 10, 803);

4) a digest of pepsin and trypsin showed in vitro dipeptidyl peptidase IV (DPP- IV) inhibitory activity (C. Lammi et al., Frontiers in Chemistry, 2019, 7, 670);

5) a hydrolysate obtained by pepsin and a tryptic digest, containing medium- long peptides, showed hypocholesterolemic effects by inhibiting the catalytic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) in a dose-dependent manner with a mechanism similar to that of statins (C. Zanoni et al., Journal of Agricultural and Food Chemistry, 2017, 65, 8829 - 8838; G. Aiello et al., Journal of Agricultural and Food Chemistry, 2017, 65, 10174 - 10184);

6) a protein hydrolysate of hemp bran, a by-product of hemp seeds, was obtained by Alcalase®, and separated by ultrafiltration membranes into 4 different fractions with molecular weight cut-offs <1 kDa, between 1-3 kDa between 3-5kDa and > 5kDa. The in vitro antioxidant and antihypertensive properties were tested both in the initial crude extract and in the 4 fractions obtained. However, to date, a method for extracting and characterizing peptides, in particular short peptides containing from 2 to 4 amino acid residues having biological interest, starting from hemp seed flours, does not appear to be described.

The Applicant has now surprisingly identified a method for extracting and separating a mixture of short peptides from hemp seed flour proteins. This method is scalable to an industrial level and the solvents used can be advantageously recovered by distillation.

The short chain peptides (2-4 amino acids) isolated by the method of the invention have a higher bioactivity than the medium-long chain ones. They are in fact easily absorbed from the intestine without undergoing further degradation in the gastrointestinal tract and thus manage to enter the bloodstream where they can carry out their biological functions. They also have a greater ability to interact with a specific target, such as the active site of an enzyme, than longer peptides.

Brief description of the drawings:

- Figure 1 : Chromatogram obtained by size exclusion chromatography of the medium- long peptides fraction (1-5 min) and short peptides fraction (6-10 min).

- Figure 2: Effect of S (Small, short peptides), M (Medium, medium-long peptides) and T (Total, total hydrolysate) peptides on the in vitro activity of the HMGCoAR enzyme.

- Figure 3: HepG2 cell viability tested by MTT assay.

- Figure 4: Effect of S, M, T and MonK peptides on the modulation of SREBP-2 (A), LDLR (B) and HMGCoAR (C) protein levels.

- Figure 5: Effect of S, M, T and MonK peptides on the functional capacity of HepG2 cells to absorb LDL from the extracellular environment.

- Figure 6: Effect of S, M, T and MonK peptides on the PCSK9 pathway.

- Figure 7: Activity of S, M and T peptides on in vitro DPP-IV enzyme activity.

- Figure 8: Viability of Caco-2 cells tested by MTT assay.

- Figure 9: S, M and T peptides activity on DPP-IV enzyme activity expressed on the membrane of Caco-2 cells.

- Figure 10: S and M peptides activity on the activity of circulating DPP-IV enzyme present in human serum.

- Figure 11 : ACE inhibitory activity of S, M and T peptides.

- Figure 12: Free radical scavenging capacity of S, M and T peptides by DPPH assay.

- Figure 13: Antioxidant activity of S, M and T peptides by FRAP assay.

- Figura 14: Peptidi corti identificati tramite cromatografia ad esclusione dimensionale. - Figure 15: Short peptides identified through the use of ultrafiltration membranes having a molecular cut-off of 1 kDa.

The object of the present invention is therefore a method for the separation of short peptides isolated from proteins of industrial hemp seed flour (Cannabis Sativa L), comprising the following steps: a) protein extraction; b) hydrolysis of proteins in the presence of subtilisin A to obtain a protein hydrolysate; c) separation of the protein hydrolysate into two fractions containing respectively short peptides and medium-long peptides; d) isolation and identification of the fraction containing the short peptides, characterized in that said separation in step c) occurs through the use of size exclusion chromatography or through the aid of ultrafiltration membranes having a molecular cut-off of 1 kDa and in that said identification in step d) is performed with liquid chromatography coupled with mass spectrometry and subsequent use of bioinformatics software.

The method according to the present invention can be applied both to commercially available industrial hemp flours and to flours produced by methods known in the art starting from hulled hemp seeds.

In case hulled hemp seeds are used, these can be pulverized and made into flour by using liquid nitrogen on a laboratory scale, or they can be made into flour through gasification by decompression, precipitation by vacuum pumping and recovery by solvent compression and subsequent grinding and sieving of impurities, as described in the patent CN 1294833.

According to a preferred embodiment of the present invention, commercially available flours having a protein content ranging from 26% to 40% are used.

Although the composition of the flours varies in the percentage of proteins, being the hemp seeds from which the flour is produced made up of two main proteins (edestin and albumin), the respective digests are made up of the same peptide composition. The quantity of starting flour used depends on the protein content declared on the label, typically 40-60 mg are used for flours with a 40% protein content, or 90-110 mg for flours with a 20-30% protein content. According to the method of the invention, hemp seed flour is subjected to protein extraction reaction (step a). As is well known to those skilled in the art, said extraction reaction is carried out in a solution consisting of a physiological buffer and a detergent. The physiological buffer is selected from Tris-HCI, ammonium bicarbonate and a phosphate buffer and is used in a pH range comprised between 7 and 8, preferably 7.8, while the detergent is selected from sodium deoxycholate (SDC), sodium dodecanoate (SD) and sodium dodecyl phosphate (SDS), in a percentage (w/v) ranging from 0.1 to 2.5%, preferably it is 2% SDC.

The extraction is then carried out for intervals from 2 to 4 hours, preferably under mechanical stirring and incubating at -20°C. The extraction yield is determined by one of the protein identification assays known in the art, such as for example the Bradford assay, the Lowry assay or the bicinchoninic acid assay. Preferably, the identification of the proteins occurs with the use of bicinchoninic acid.

The extraction yields vary in a range between 22% and 27%.

In step b) of the method of the invention, the protein extract obtained in step a) is subjected to enzymatic hydrolysis. This hydrolysis is carried out in the presence of at least one proteolytic enzyme which is subtilisin A.

According to the present invention, depending on the proteolytic enzyme, the enzyme:substrate weight ratio varies from 1 :5 to 1 : 100, the digestion temperature varies between 50 and 100°C, the pH between 7 and 9 and the hydrolysis time between 2 and 5 hours.

Preferably, subtilisin A is used in a ratio of 1 :10 (w/w) with the substrate, at a temperature of 60°C, at pH 8 for 4 hours.

A protein hydrolysate is then obtained, which is separated into two fractions in step c) of the method of the invention: the first fraction containing the short peptides and the second the medium-long peptides.

According to the present invention, the separation takes place through the use of size exclusion chromatography or through the aid of ultrafiltration membranes.

In the first case, for the separation with size exclusion chromatography, an isocratic elution is preferably used with a mixture of 0.1 % trifluoroacetic acid (TFA) in water. In this way it is possible to separate the fraction containing the medium-long peptides, which elute together with the overhead fraction of the chromatographic column, in a time comprised between 1 and 5 minutes, and then isolate the short peptides which instead elute subsequently in a time comprised between 6 and 10 minutes. The short peptide mixture thus isolated comprises peptides having from 2 to 4 amino acids.

For separation with the aid of ultrafiltration membranes, however, membranes with a molecular weight cut-off of 1 kDa are used. The filtrate consists of short peptides having from 2 to 4 amino acids, which is separated and isolated from the material retained by the ultrafiltration membranes consisting of medium-long peptides.

After isolation of the fraction containing the short peptides (step d), the latter are identified by techniques well known in the art, such as for example liquid chromatography coupled to high resolution mass spectrometry with Orbitrap or time of flight (TOF) detector.

Subsequently, thanks to the aid of bioinformatics software, such as Compound Discoverer 3.1 and Max Quant, it is possible to obtain an exhaustive list of the amino acid sequences contained in the isolated fractions.

The mixture of short peptides obtained by the method of the present invention contains short peptides having from 2 to 4 amino acids.

Said mixture contains peptides with two, three or four amino acids selected from the twenty essential amino acids, with the exception of cysteine. Dipeptides are those most studied individually in the literature and for which several biological activities are already known, reported in the BIOPEP database. In the mixture of short peptides, object of the present invention, various dipeptide sequences have shown to be already known and to mostly possess the following activities: DPP-IV, DPP-Ill and ACE; a tri-peptide, VW, for example is known for its anticarcinogenic activity; for more comprehensive information in this regard it is possible to insert the dipeptide sequences shown in Figures 14 and 15 on the BIOPEP database available online which is used by the scientific community to deposit the biologically active peptide sequences (this is periodically updated based on literature works) . As far as tripeptides and tetrapeptides are concerned, although they possess biological activities, they are in most cases not reported in the database, except for some cases (for example the above case VW).

Advantageously, the mixture of short peptides obtained by the method of the present invention has a greater multifunctional biological activity than both the fraction containing medium-long peptides and the total hydrolysate obtained in step b).

With the term "multifunctional biological activity" according to the present invention, it is intended hypocholesterolemic, hypoglycaemic, hypotensive and antioxidant activity. This activity was evaluated by combinations known in the art of biochemical, cellular and functional techniques.

Therefore, a further object of the present invention is the mixture of short peptides obtained with the method of the present invention.

As mentioned above, the biological activity of the mixture of the invention is multifunctional, i.e. said mixture has significant efficacy on several pathologies, as subsequently described in detail in the experimental part of the present application.

A further object of the present invention is therefore the use of the aforementioned mixture for the preparation of pharmaceutical or nutraceutical products.

In particular, said mixture can be used in the prevention and/or treatment of the metabolic syndrome, of pathologies linked both to the DPP-IV enzyme inhibition, to the HMGCoAR enzyme inhibition, and to the ACE enzyme inhibition.

Furthermore, the mixture obtained by the method of the invention can be validly used for the preparation of food or cosmetic products.

Experimental part

Example 1

The hemp flour produced by Molino Crisafulli (Caltagirone, CT) with a protein content of 40% was subjected to protein extraction according to the following procedure: 50 mg of flour was extracted with 2 mL of a Tris-HCI 50 solution mM at 2% (w/v) SDC. To maximize the protein extraction yield, two one-hour cycles were carried out on a mechanical stirrer, interspersed with 15 minutes of sonication and incubation at -20°C, in order not to overheat the extract. The extract was centrifuged at 14,000 rpm for 30 min at room temperature and then quantified with the bicinchoninic acid assay to determine the protein content. The obtained extraction yield was 23 ± 2%.

15 mg of extract underwent enzymatic digestion with Alcalase®. The enzyme:substrate ratio was 1 :10 (w/w) and the digestion was carried out at 60°C for 4 hours. The digestion process was stopped with trifluoroacetic acid (TFA) at pH 2. The hydrolysate was then centrifuged at 14,000 rpm for 10 min at room temperature to favor SDC precipitation. After centrifugation and removal of the SDC, the hydrolysate was reduced to a small volume (400 pL) and purified using a BIOBASIC SEC 120 size exclusion chromatographic column, with particles of diameter 5 pm and column length and diameter (150 X 7.8) mm, using isocratic elution with 0.1% TFA in H2O (pH 2.5) with a flow rate of 1 mL.min' ¹ . The fraction consisting of medium-long peptides eluted between 1 and 5 minutes, while the short peptides eluted between 6 and 10 minutes (Figure 1).

The two fractions containing the medium-long and short peptides were evaporated and injected for the identification of the peptides constituting the two fractions by means of a UHPLC-MS/MS system with Orbitrap detector for the identification of the short peptides (Figure 14) and by a nano-HPLC system for medium-long peptides. At the same time, the dry weight of the total hydrolysate was evaluated, which corresponded to 14.7 mg or 98%.

Example 2

The hemp flour produced by Molino Crisafulli (Caltagirone, CT) with a protein content of 40% was subjected to protein extraction according to the following procedure: 50 mg of flour was extracted with 2 mL of a Tris-HCI 50 solution mM at 2% SDC.

To maximize the protein extraction yield, two one-hour cycles were carried out on a mechanical stirrer, interspersed with 15 minutes of sonication and incubation at -20°C, in order not to overheat the extract. The extract was centrifuged at 14,000 rpm for 30 min at room temperature and then quantified with the bicinchoninic acid assay to determine the protein content. The obtained extraction yield was 23 ± 2%.

15 mg of extract underwent enzymatic digestion with Alcalase®. The enzyme: substrate ratio was 1 :10 (w/w) and the digestion was carried out at 60°C for 4 hours. The digestion process is stopped with trifluoroacetic acid (TFA) at pH 2. The hydrolysate was then centrifuged at 14,000 rpm for 10 min at room temperature to favor SDC precipitation. The separation of the mixture containing short peptides was carried out by means of ultrafiltration membranes, in cellulose with low protein absorption, with a molecular weight cut-off of 1 kDa. The membranes were activated with 10 mL of methanol and subsequently conditioned with 10 mL of 0.1% TFA in H2O. After activation, the 15 mg of hydrolysate were charged and the filtrate containing only the short peptide fraction was recovered and dried (6 mg, 40% yield) and injected into a UHPLC-MS/MS system with Orbitrap detector for peptide identification (Figure 15). At the same time, the dry weight of the total hydrolysate was evaluated, which corresponded to 14.7 mg or 98%.

To evaluate the multifunctional activity of the mixture, the following biological activities were characterized: hypocholesterolemic, hypoglycaemic, hypotensive and antioxidant. A combination of biochemical, cellular and functional techniques was used to achieve this goal.

Example 3: Evaluation of the hypocholesterolemic activity

3.1 Short peptides of hemp inhibit the 3-hydroxymethylglutaryl-CoA reductase enzyme (HMGCoAR).

In vitro experiments were performed using the purified catalytic domain of the HMGCoAR enzyme, a known target of statins. The results show that the total hydrolysate (T), the medium-long peptides (M) and the short peptides (S) of hemp are able to inhibit the activity of the HMGCoAR enzyme in a dose-dependent manner and with an IC50 equal to 0.38, 0.25 and 0.18 mg/mL, respectively (Figure 2). A gain of inhibitory activity in the fraction of short peptides (S) appears clearly, since they are twice more active than the total hydrolysate. Comparison with the literature shows that the peptides obtained by hydrolyzing hemp proteins with Alcalase® are more active than the peptides obtained by hydrolyzing hemp seed proteins with pepsin (IC50 0.8 mg/mL) (J. Agric. Food Chem. 2017, 65, 40, 8829-8838) trypsin (IC50 0.65 mg/mL), while the peptides obtained using pancreatin are inactive as HMGCoAR inhibitors (J. Agric. Food Chem. 2017, 65, 47, 10174-10184). Peptides obtained by co-digesting hemp seed proteins with pepsin, trypsin and pancreatin are able to inhibit the HMGCoAR enzyme but not in a dose-dependent manner.

3.2 Hemp peptides modulate cholesterol metabolism on human liver HepG2 cells

Based on the ability of short, medium-long and total hydrolysate peptides to inhibit the HMGCoAR enzyme, extensive experiments were conducted in order to establish the mechanism of action through which they exert a cholesterol-lowering effect using HepG2 human liver cells, as a cellular reference system.

Initially MTT experiments were performed in order to establish a potential cytotoxic effect of the S, M and T peptides. The results showed that in the concentration range 0.1 - 2.0 mg/mL, no samples show effects of reduction in cell viability (Figure 3). Then HepG2 cells were treated with S peptides at a concentration of 0.5 mg/mL and with M and T peptides at a concentration of 1 mg/mL. Monacolin K (MonK) (1 pM) was used as a reference compound, being responsible for the hypocholesterolemic activity in all nutraceuticals based on red yeast rice. The results indicate that all tested peptides (S, M and T) are able to modulate cholesterol metabolism by activating the LDL receptor (LDLR) pathway and confirming that S peptides are twice more active than M peptides and than T peptides (Figure 4). More precisely, S, M and T induce an increase of the transcription factor SREBP-2 equal to 34.7%, 43.5% and 43.5%, respectively. Increase of this transcription factor results in an increase in LDL receptor levels of 55.2%, 85.1%, and 80.2% for S, M and T, respectively (Figure 4B). In agreement with this result, an increase in HMGCoAR levels of 30.2%, 32.8% and 56.2% was found following treatment of HepG2 cells with S, M and T peptides, respectively (Figures 40). Hemp peptides modulate cholesterol metabolism with a similar mechanism to monacolin K. Notably, MonK increases SREBP-2 by 41.3%, LDLR by 48.0%, and HMGCoAR by 33.4% (Figure 4A-C).

From a functional point of view, S, M and T increase the ability of HepG2 cells to absorb LDL from the extracellular environment with a final cholesterol-lowering effect. Also in this case S peptides are twice more active than M and T (Figure 5). In particular, the S, M and T peptides increase the ability of cells to absorb extracellular LDL by 183.8%, 147.7% and 211.4%, respectively. In parallel, MonK increases the functional capacity of cells to absorb LDL by 126.9% (Figure 5). Statistical analysis suggests that S, M and T peptides are more active than MonK.

3.3 Effect of hemp seed peptides on the PCSK9 pathway.

The effect of S, M and T peptides on the PCSK9 pathway was evaluated by immunoblotting experiments. In particular, HepG2 cells were treated with the S peptides at a concentration of 0.5 mg/mL, M and T peptides at a concentration of 1 mg/mL. The results highlight that S, M and T reduce the intracellular levels of the mature form of PCSK9 by 39.1 %, 39.3% and 42.7% confirming that the S peptides are twice more active than M and T peptides (Figure 6A). The decrease in PCSK9 is due to the ability of these samples to reduce levels of the transcription factor HNF-1 alpha. Indeed, the S, M and T peptides reduce transcription factor levels by 24.1%, 17.2% and 8.6%, respectively (Figure 6B). The experiments were carried out using monacolin K as a reference compound, confirming the ability of MonK to increase PCSK9 levels by 24.5% and HNF-1 alpha by 17.8%, resulting in a reduction of active LDLR levels on the membrane. In fact, Figure 5 highlights that the functional capacity of HepG2 cells to absorb extracellular LDL after treatment with M and T peptides and especially S peptides, at half the concentration of M and T, is exactly equal to the capacity of the cells HepG2 to absorb extracellular LDL after treatment with MonK at 1 pM concentration. These results, therefore, highlight a new and different hypocholesterolemic mechanism from that of monacolin K. The behavior of these peptides also differs greatly from what is known in the literature. In fact, peptic hydrolysate is unable to modulate any effect on the PCSK9 pathway (J. Agric. Food Chem. 2017, 65, 47, 10174-10184).

Example 4 Evaluation of hypoglycemic activity by in vitro, in situ and ex vivo inhibition of the DPP-IV enzyme.

In vitro experiments were carried out using the recombinant form of the DPP-IV enzyme, a known target for the treatment of type 2 diabetes. The results showed that the S, M and T peptides are able to inhibit the activity of DPP-IV enzyme with a dosedependent pattern over the tested concentration range (0.1 - 2 mg/mL) and with an IC50 of 0.82, 1.17 and 1.36 mg/mL, respectively (Figure 7). These results suggest that S-peptides are approximately twice more active than the total hydrolysate. Literature data suggest that tryptic and peptic hydrolysate (1.0 mg/mL) inhibit the enzyme in vitro by 17.5 and 32.0%, respectively (Lammi C., et al. Enhancement of the Stability and Anti- DPPIV Activity of Hempseed Hydrolysates Through SelfAssembling Peptide-Based Hydrogels, Front.Chem.) suggesting that the S, M and T peptides obtained using the Alcalase® enzyme are more active.

Based on these results, in situ experiments were performed to evaluate the ability of S and M peptides to inhibit the DPP-IV enzyme expressed on the membrane of human Caco-2 intestinal cells.

Initially, MTT experiments were conducted in order to evaluate potential cytotoxic effects of the samples on intestinal Caco-2 cells. The results showed that in the range of concentrations tested, no effects of reduction of cell viability were observed (Figure 8).

Then, Caco-2 cells were treated with the S, M and T peptides (1 and 2 mg/mL). The results showed that S, M and T inhibited the DPP-IV enzyme expressed on the membranes of Caco-2 cells by 20.2, 15.4 and 19.7%, respectively, at 1 mg/mL and by 47, 6%, 31.3%, and 35.4%, respectively, at 2 mg/mL. (Figure 9). Finally, the S and M peptides were tested ex vivo, in order to evaluate their ability to inhibit the circulating form of the DPP-IV enzyme present in human serum. The results demonstrate that S and M reduce the activity of the circulating form of DPP-IV by 47.1 % and 36.5%, respectively, at 1 mg/mL and by 52.9% and 38.1%, respectively, respectively, at 2 mg/mL (Figure 10).

Example 5 Evaluation of hypotensive activity

In vitro experiments were carried out using the recombinant form of the ACE enzyme, a known target for the treatment of hypertension. The results showed that at the fixed concentration of 1 mg/mL, peptides S, M and T inhibited the ACE enzyme by 57.5%, 16.6% and 32.4%, respectively, versus the control (p< 0.0001) (Figure 10). From the statistical analysis, it is clear that the S peptides are about 3 times more active than the M peptides (p<0.0001) and that they clearly represent the active component of the total T hydrolysate (p<0.0001) (Figure 11).

Example 6. Evaluation of the direct antioxidant activity of hemp seed peptides

Two types of assays were used to evaluate the antioxidant activity: the first is the DPPH assay, with which the ability of the samples to eliminate the DPPH radical and therefore the ability to scavenge free radicals is evaluated; the second assay is the FRAP (Ferric Reducing Antioxidant Power), which measures the ability of antioxidants to reduce the iron (III) complex to the reduced iron (II) complex.

Figure 12 shows the in vitro effects of the direct antioxidant activity of S, M and T peptides by DPPH assay. The results clearly suggest that the samples express an antioxidant power with a dose-response pattern (Figure 12). In particular, at concentrations of 0.5, 1.0, 2.5 and 5.0 mg/mL, peptides S have scavenger activities of 10.9%, 18.3%, 26.8% and 42.3%; the peptides M equal to 8.9%, 12.7%, 18.8% and 25.8%; while peptides T have DPPH scavenging activities of 27.3%, 32.1%, 39.3% and 44.9%, respectively.

Figure 12 shows the in vitro effects of the direct antioxidant activity of S, M and T peptides by FRAP assay. The results clearly indicate that the peptides express a reducing capacity in a dose-response manner (Figure 13). In particular, at concentrations of 0.1 , 0.5, 1.0 and 2.5 mg/ml, the S peptides increase iron reduction by 528.9%, 1936%, 3284%, 5238%, the M peptides by 44.44%, 197.2%, 377.8% and 838.9%, and the T peptides increase iron reduction by 888.7%, 1678%, 2546% and 4654%, respectively.