Hempseed Water-Soluble Protein Fraction and Its Hydrolysate Display Different Biological Features

Abstract

Hempseeds, from the Cannabis sativa plant, and its derivates are a versatile food option for various dietary preferences. Due to their aminoacidic profile, researchers have studied the presence of bioactive peptides in hempseed proteins. In this study, the water-soluble fraction of hempseed protein was extracted, and the derived peptides were analyzed. The investigation focused on their biological function, particularly their antioxidant activity. Several biological functions have arisen, such as angiotensin-converting enzyme inhibition activity, dipeptidyl-peptidase IV, dipeptidyl-peptidase III inhibition, and ubiquitin-mediated proteolysis activation. The hydrolysates show greater 2,2-azinobis-[3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity compared to the proteins (97.95 ± 4.48 versus 81.04 ± 10.63). Furthermore, the impact of these proteins and peptides on the U937 cell line was evaluated to assess cell viability and their potential role in modulating inflammation associated with gastrointestinal autoimmune diseases. Protein treatment resulted in a significant reduction in cell viability, as opposed to hydrolysates, which did not affect it.

Keywords: Cannabis sativa; IBD; MALDI-TOF; SDS-PAGE; antioxidant; autoimmune disease; hempseed; hempseeds hydrolysates; in vitro digestion; peptidomics; water-soluble protein.

Figure 1

SDS-PAGE profiles of proteins (P), hydrolysates (H), and protein ladder (St).

Figure 2

Representative MALDI-TOF MS mass spectra in the m/z region 2000–20,000 of proteins and hydrolysates; in particular, the image represents a zoom between 2000 and 12,000 m/z.

Figure 3

Representative MALDI-TOF MS mass spectra in the m/z region 700–3500 of proteins and hydrolysates.

Figure 4

PCA plots (Pc1 vs. PC2) of proteins (P, orange) and hydrolysates (H, blue) analyzed through linear positive method (A) and reflectron positive method (B). Biological replicates (L1 and L3, LA and LC) are shown in technical triplicate (a, b, and c).

Figure 5

The heatmap represents the relative abundance of different biological activities of peptides obtained from five hemp proteins. Each cell in the map indicates the proportion of biological activity to the number of peptides identified for a given protein. Colors vary in intensity to reflect this proportion, with darker shades indicating higher abundance. Edestin 1 (A0A090DLH8), Edestin 2 (A0A090CXP9), Edestin 3 (A0A219D3H6), Vicilin C72 (XP_030508280.1), and NADPH-dependent aldehyde reductase 1 chloroplastic-like (XP_030506286.1).

Figure 6

The figure shows a typical experiment. U937 cells are treated with different concentrations of the stimuli, proteins (B–D) and hydrolysates (E–G). Image (A) represents untreated control. The cells were treated with proteins (B–D), 0.1, 0.01, and 0.001 mg/mL, respectively, and with hydrolysates (E–G), 0.1, 0.01, and 0.001 mg/mL, respectively (magnification 20×, scale bar = 100 μm).