Impact of Chitosan-Based Foliar Application on the Phytochemical Content and the Antioxidant Activity in Hemp (Cannabis sativa L.) Inflorescences

Abstract

In the present study, the phytochemical content and the antioxidant activity in the inflorescences of the monoecious hemp cultivar Codimono grown in southern Italy were assessed, and their elicitation was induced by foliar spray application of 50 mg/L and 250 mg/L of chitosan (CHT) at three different molecular weights (low, CHT L; medium, CHT M; high CHT H). The analysis of the phytochemical profile confirmed that cannabinoids were the most abundant class (54.2%), followed by flavonoids (40.3%), tocopherols (2.2%), phenolic acids (1.9%), and carotenoids (1.4%). Cannabinoids were represented almost exclusively by cannabidiol, whereas cannabigerol and Δ⁹-tetrahydrocannabinol were detected at very low levels (the latter was below the legal limit of 0.3%). The most abundant flavonoids were orientin and vitexin, whereas tocopherols were mainly represented by α-tocopherol. The antioxidant activity was found to be positively correlated with flavonoids and tocopherols. Statistical analysis revealed that the CHT treatments significantly affected the phytochemical content and the antioxidant activity of hemp inflorescences. Notably, a significant increase in the total phenolic content (from +36% to +69%), the α-tocopherol (from +45% to +75%) and β+γ-tocopherol (from +35% to +82%) contents, and the ABTS radical scavenging activity (from +12% to +28%) was induced by all the CHT treatments. In addition, treatments with CHT 50 solutions induced an increase in the total flavonoid content (from +12% to +27%), as well as in the vitexin (from +17% to +20%) and orientin (from +20% to +30%) contents. Treatment with CHT 50 L almost always resulted in the greatest increases. Overall, our findings indicated that CHT could be used as a low-cost and environmentally safe elicitor to improve the health benefits and the economic value of hemp inflorescences, thus promoting their employment in the food, pharmaceutical, nutraceutical, and cosmetic supply chains.

Keywords: antioxidant activity; cannabinoids; chitosan; elicitor; industrial hemp; inflorescences; phenolic compounds; tocopherols.

Figure 1

Pie chart representing the abundance of the different classes of metabolites detected in the inflorescences of the hemp cv. Codimono. Percentages were calculated from the mean values of the seven treatments.

Figure 2

Pearson’s correlation coefficients for all pairs of traits analyzed in the inflorescences of the hemp cv. Codimono. Correlations significant at p ≤ 0.05 are highlighted in bold. TPC, total phenolic content; TFC, total flavonoid content; ABTS, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity.

Figure 3

Venn diagram for differentially accumulated metabolites (DAMs) detected in the inflorescences of the hemp plants from the cv. Codimono treated with CHT solutions at 50 mg/L (A) and 250 mg/L concentrations (B). Numbers in parentheses indicate the total DAM number induced by each treatment. TPC, total phenolic content; TFC, total flavonoid content; ABTS, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. CHT 50 L, 50 mg/L low molecular weight chitosan; CHT 50 M, 50 mg/L medium molecular weight chitosan; CHT 50 H, 50 mg/L high molecular weight chitosan; CHT 250 L, 250 mg/L low molecular weight chitosan; CHT 250 M, 250 mg/L medium molecular weight chitosan; CHT 250 H, 250 mg/L high molecular weight chitosan.

Figure 4

Box and whisker plots of the differentially accumulated metabolites in the inflorescences of the hemp plants from the cv. Codimono treated with CHT solutions at 50 mg/L and 250 mg/L concentrations. Boxes of metabolites that increased, decreased, or remained unchanged in CHT-treated plants compared to control plants are colored in red, blue, and green, respectively. For each metabolite, different lower-case letters represent significant differences among treatments (Tukey’s test p ≤ 0.05). TPC, total phenolic content; TFC, total flavonoid content; ABTS, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. Ctr, control; CHT 50 L, 50 mg/L low molecular weight chitosan; CHT 50 M, 50 mg/L medium molecular weight chitosan; CHT 50 H, 50 mg/L high molecular weight chitosan; CHT 250 L, 250 mg/L low molecular weight chitosan; CHT 250 M, 250 mg/L medium molecular weight chitosan; CHT 250 H, 250 mg/L high molecular weight chitosan.

Figure 5

Principal component analysis (PCA) (A) and sparse partial least-squares-discriminant analysis (sPLS-DA) (B) for the traits investigated in the inflorescences of the hemp plants from the cv. Codimono treated with CHT solutions at 50 mg/L and 250 mg/L concentration. The percentages of total variance represented by the first two components are shown in parentheses. Ctr, control; CHT 50 L, 50 mg/L low molecular weight chitosan; CHT 50 M, 50 mg/L medium molecular weight chitosan; CHT 50 H, 50 mg/L high molecular weight chitosan; CHT 250 L, 250 mg/L low molecular weight chitosan; CHT 250 M, 250 mg/L medium molecular weight chitosan; CHT 250 H, 250 mg/L high molecular weight chitosan.

Figure 6

Loading plots of the eight traits that are most significant in the group separation among the different treatments for component 1 (A) and component 2 (B) of the sPLS-DA. TPC, total phenolic content; TFC, total flavonoid content; ABTS, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. Ctr, control; CHT 50 L, 50 mg/L low molecular weight chitosan; CHT 50 M, 50 mg/L medium molecular weight chitosan; CHT 50 H, 50 mg/L high molecular weight chitosan; CHT 250 L, 250 mg/L low molecular weight chitosan; CHT 250 M, 250 mg/L medium molecular weight chitosan; CHT 250 H, 250 mg/L high molecular weight chitosan.