Chemical characterization and evaluation of antioxidant activity from different cultivars of Cannabis sativa L. of Abruzzo’s region

Abstract In this work, the chemical composition and the antioxidant evaluation of the inflorescences from 12 Cannabis sativa L. monoecious cultivars (Carmagnola Lemon CL, Ferimon F, Gran Sasso Kush GSK, Antal A, Carmagnola C, Kompolti K, Futura 75 F75, Villanova V, Tiborzallasi T, Finola FL, Kc Virtus KV and Pineapple P) cultivated at the same condition, were investigated. GC-MS analysis was carried out to evaluate the volatile fraction, while HPLC-MS/MS was used for cannabinoids and polyphenolic compounds. The evaluation of antioxidant activity was carried out using ABTS*+, Trolox equivalence antioxidant capacity (TEAC), ferric reducing antioxidant property (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH*) assays in vitro. The obtained data, demonstrated that each cultivar has a characteristic chemical profile, with highest antioxidant capacity for CL, F75, GSK and F. Based on the in vitro antioxidant activity the plant extracts can be considered as promising candidates for different applications in food field. Graphical Abstract


1.1.Plant materials
The material (inflorescences) has not been identified at the moment by a botanist. However, the plant material, provided a local hemp farm (Hemp Farm Italia, Tortoreto (TE)), was generated from certified seeds as stated by EU Regulation 1307/2013; varieties are listed in the "Common catalogue of varieties of agricultural plant species" by the European Commision (EU database of registered plant varieties). The details of each hemp sample are showed in Table S1. Each plant is cultivated in the same agronomic condition at the same time for the experimental project. Samples were constituted by a portion including the flowers and leaves, harvested during the flowering period (September). The inflorescences were dried and stored in a dark room at ambient temperature until further analysis.

1.3.Ultrasound assisted extraction (UAE)
UAE extractions, according to (Agarwal et al. 2018), were performed using 100 mg of inflorescences for each C. sativa cultivar. Plant matrices were extracted with methanol, sealed and immersed in an ultrasonic water bath for 30 minutes at 10°C, followed by centrifugation and filtration of the supernatant with 0.2 µm PTFE.
For the polyphenol's extraction, UAE extract was subjected to a clean-up procedure and improvement of the concentration of polyphenols through the SPE procedure according to (Oliva et al. 2021).

1.4.GC-MS characterization of volatile fraction
Analysis of volatile compounds for all samples was performed by solid-phase microextraction/gas chromatography coupled to mass spectrometry (SPME/GC-MS) according to Pellegrini et al. 2020).

1.5.HPLC-MS/MS analysis of cannabinoids and phenolic compounds
Hemp extracts were analysed both for cannabinoid and polyphenols compounds by means of HPLC-MS/MS. For cannabinoids, the analysis is performed by an already method developed in (Palmieri et al. 2019) while the polyphenols analysis is performed according to the method of (Oliva et al. 2021). All samples were analysed in triplicate.

1.6.Evaluation of Antioxidant activity (AOC)
The antioxidant activity was evaluated by three different spectrophotometric assays: the FRAP assay performed according to the procedure of (Oyaizu 1986), ABTS *+ assay, using the method of (Gullon et al. 2015), DPPH * assay, following the method proposed (Brand-Williams et al. 1995), Trolox was used as the standard compound for calibration curves and the results were expressed in mg of Trolox equivalents (TE)/g of dry extracts, mean value of three replicates.

1.7.Statistical analysis
Antioxidant activity data were subjected to ANOVA (analysis of variance), followed by Tukey's HSD post-hoc test at a significance level of 5% (p < 0.05). For each class of chemical compounds, the data were processed using principal component analysis (PCA) to evaluate the discrimination between the samples. The same approach was used for AOC evaluation (DPPH*, FRAP, ABTS*+) data with chemical compounds characterize to observe the possible correlations within the different cultivars. Statistical tests were performed using Microsoft Xlstat 2016 statistical software (Addinsoft, Paris, France).     (Table S2A) and phenolic acids (Table S2B) compounds. Values were expressed as [ng/g of dry extracts] mean SD (standard deviation), n = 3.  Table S3B. HPLC-MS/MS phenolic composition of the hemp cultivar extracts: the flavonoids (Table S2A) and phenolic acids (Table S2B) compounds. Values were expressed as [ng/g of dry extracts] mean SD (standard deviation), n = 3.     Equivalent Antioxidant Capacity with 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS *+ ).