Cannabis sativa L., better known as industrial hemp, is one of the oldest plants cultivated by man, especially for its medicinal and textile uses. However, during 1950-2000 its name was associated with drugs of abuse (marijuana), due to the presence of high amount of the psychotropic compound ∆9-t-tetrahydrocannabinol (THC) in some hemp cultivars, leading to a slowly abandonment of this crop for many States of the world. In 2000 the European Union published a regulation concerning the re-introduction of some industrial hemp cultivars, characterized by a low content of THC, certified and included in the list of the “EU Plant variety database”.

Nevertheless, these years without hemp cultivation contributes to the loss of the traditional hemp cultivars, and so the available hemp cultivars come from foreign countries, choosed in different weather condition respect to the Mediterranean ones. For these reasons, their introduction in the agronomic Italian scenario need a careful investigation in order to avoid the diffusion of inadequate and not productive varieties.

Therefore, a deep investigation of the exogenous factor (agronomical practices, seasoning and pedoclimatic conditions) that can influence the metabolomic profile is needed.

The recent growing interest in Cannabis sativa L. is related to their chemical composition, in particular in both primary and secondary metabolites that can play an important role in pharmaceutical field as well as in cosmetics, building and food fields. In this scenario, the choice of cultivars and cultivation conditions will be fundamental parameters to evaluate the possible use of the different hemp sub-products (seeds for food market, extraction of CBD for pharmaceutical purpose, fibre for new material development).

Thus, the main aim of the present working group is the characterization of the industrial hemp production chain products, of both raw materials (inflorescences, seeds) and transformation products (hemp seed oil, flour and essential oils), through multidisciplinary analytical protocol, involving both targeted and untargeted techniques, and the evaluation of the agronomical practises, seasoning, pedoclimatic conditions and genetic factors role on the chemical-biological composition of the hemp analysed sample, in order to assess the quality and to define both the type of cultivars and the cultivation conditions more suitable for our region.

La Cannabis sativa L., meglio conosciuta come canapa industriale, è una delle piante più antiche coltivate dall'uomo, soprattutto per i suoi usi medicinali e tessili. Tuttavia, durante il periodo 1950-2000 il suo nome fu associato alle droghe d'abuso (marijuana), a causa della presenza di elevate quantità di composto psicotropo ∆9-tetraidrocannabinolo (THC) in alcune cultivar di canapa, portando ad un lento abbandono di questa coltura in molti Stati del mondo. Nel 2000 l'Unione Europea ha pubblicato un regolamento riguardante la reintroduzione di alcune cultivar di canapa industriale, caratterizzate da un basso contenuto di THC, certificate e inserite nell'elenco del “Database delle varietà vegetali dell'UE”. Tuttavia, questi anni senza coltivazione della canapa hanno contribuito alla perdita delle cultivar di canapa tradizionali, e quindi le cultivar di canapa disponibili provengono da paesi esteri, selezionati in condizioni climatiche differenti rispetto a quelle mediterranee. Per questi motivi la loro introduzione nello scenario agronomico italiano necessita di un'attenta indagine al fine di evitare la diffusione di varietà inadeguate e non produttive. È quindi necessaria un'indagine approfondita dei fattori esogeni (pratiche agronomiche, stagione di raccolta e condizioni pedoclimatiche) che possono influenzarne il profilo metabolomico.

Il recente crescente interesse per la Cannabis sativa L. è legato alla loro composizione chimica, in particolare di metaboliti primari e secondari che possono svolgere un ruolo importante in campo farmaceutico, cosmetico, edile e alimentare.

In questo scenario, la selezione delle cultivar e delle condizioni di coltivazione saranno parametri fondamentali per valutare il possibile utilizzo dei diversi sottoprodotti di canapa (semi per il mercato alimentare, estrazione di CBD a scopo farmaceutico, fibra per lo sviluppo di nuovi materiali).

Pertanto, l'obiettivo principale del presente gruppo di lavoro è la caratterizzazione dei prodotti della filiera industriale della canapa, sia delle materie prime (infiorescenze, semi) che dei prodotti di trasformazione (olio di semi di canapa, farina e oli essenziali), attraverso un protocollo analitico multidisciplinare, che coinvolge sia tecniche targeted che untargeted, e la valutazione del ruolo delle pratiche agronomiche, stagione di raccolta, condizioni pedoclimatiche e fattori genetici sulla composizione chimico-biologica del campione di canapa analizzato, al fine di valutarne la qualità e definire sia il tipo di cultivar che le condizioni di coltivazione più adatte per la nostra regione.

Chromatographic Analyses, In Vitro Biological Activities, and Cytotoxicity of Cannabis sativa L. Essential Oil: A Multidisciplinary Study

Due to renewed interest in the cultivation and production of Italian Cannabis sativa L., we proposed a multi-methodological approach to explore chemically and biologically both the essential oil and the aromatic water of this plant. We reported the chemical composition in terms of cannabinoid content, volatile component, phenolic and flavonoid pattern, and color characteristics. Then, we demonstrated the ethnopharmacological relevance of this plant cultivated in Italy as a source of antioxidant compounds toward a large panel of enzymes (pancreatic lipase, α-amylase, α-glucosidase, and cholinesterases) and choosed clinically relevant, multidrug-sensible, and multidrug-resistant microbial strains (Staphylococcus aureus, Helicobacter pylori, Candida, and Malassezia spp.), evaluating the cytotoxic effects against normal and malignant cell lines. Preliminary in vivo cytotoxicity was also performed on Galleria mellonella larvae. The results corroborate the use of this natural product as a rich source of important biologically active molecules with particular emphasis on the role exerted by naringenin, one of the most important secondary metabolites.

(Molecules, 2018, 23, 3266; doi:10.3390/molecules23123266)

Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy: An Untargeted Chemical Characterization from Early Flowering to Ripening

The chemical composition of the inflorescences from four Cannabis sativa L. monoecious cultivars (Ferimon, Uso-31, Felina 32 and Fedora 17), recently introduced in the Lazio Region, was monitored over the season from June to September giving indications on their sensorial, pharmaceutical/nutraceutical proprieties. Both untargeted (NMR) and targeted (GC/MS, UHPLC, HPLC-PDA/FD and spectrophotometry) analyses were carried out to identify and quantify compounds of different classes (sugars, organic acids, amino acids, cannabinoids, terpenoids, phenols, tannins, flavonoids and biogenic amines). All cultivars in each harvesting period showed a THC content below the Italian legal limit, although in general THC content increased over the season. Citric acid, malic acid and glucose showed the highest content in the late flowering period, whereas the content of proline drastically decreased after June in all cultivars. Neophytadiene, nerolidol and chlorogenic acid were quantified only in Felina 32 cultivar, characterized also by a very high content of flavonoids, whereas alloaromadendrene and trans-cinnamic acid were detected only in Uso-31 cultivar. Naringenin and naringin were present only in Fedora 17 and Ferimon cultivars, respectively. Moreover, Ferimon had the highest concentration of biogenic amines, especially in July and August. Cadaverine was present in all cultivars but only in September. These results suggest that the chemical composition of Cannabis sativa L. inflorescences depends on the cultivar and on the harvesting period. Producers can use this information as a guide to obtain inflorescences with peculiar chemical characteristics according to the specific use.

(Molecules, 2020, 25, 1908; doi:10.3390/molecules25081908)

Commercial Hemp Seed Oils: A Multimethodological Characterization

Nine commercial hemp seed oils from different countries were studied using a multimethodological approach to obtain information about their quality and chemical composition. Due to the lack of a specific regulation for hemp seed oils, quality parameters used in the case of olive oils (free acidity, peroxides number, spectrophotometer parameters) and anisidine number were measured and compared with those reported for extra virgin olive oil (EVOO). Free acidity and peroxides number showed a great variability, ranging from 0.4 to 17.24% and from 4.32 to 22.14 meqO2/kg, respectively, whereas the anisidine number ranged from 0.11 to 3.58. K232 value turned out to be generally below the limit reported for EVOO, whereas K270 and ∆K values were higher, with respect to EVOO limits, due to the high amount of tri-unsaturated fatty chains. Colorimetric analysis showed a peculiar curve trend that could represent the fingerprint of this product. Untargeted nuclear magnetic resonance methodology allowed to measure the amount of fatty chains, ω-6:ω-3 ratio, β-sitosterol, and aldehydes. The ω-6:ω-3 ratio turned out to be, in some cases, different from that reported on the bottle labels. Finally, lipoperoxidation assays were also carried out under different storage (light and temperature) and time exposure conditions, confirming that the exposure to direct light is the condition that interferes more with the product quality. Results obtained, together with other data recently reported on hemp oils, can constitute the starting point for development, and drawing up, of harmonized guidelines with suitable quality and safety parameters specific for hemp seed oils. Furthermore, these studies might drive producers to standardize procedures of hemp seed oil production, guaranteeing the achievement of a good food objective, consumer safety, and the further expansion of the hemp food industry.

(Applied Science, 2020, 10, 6933; doi:10.3390/app10196933)

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages

The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the beginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications

(Molecules, 2021, 26, 2912; doi: 10.3390/molecules26102912

2017-2018 Progetto di Ateneo. “Studio multi-metodologico dei prodotti della filiera agroalimentare della canapa industriale”. 11.000,00€ and 23.750,00€ (Scholarship)

2018-2020 Regione Lazio, Progetti per gruppi di ricerca “La Canapa Industriale: sviluppo e valorizzazione di una nuova filiera agoralimentare ecosostenibile” Industrial Hemp: development and valorization of a sustainable new food chain Progetto di Ricerca, finanziato ai sensi della L.R. 13/08 Protocol 85-2017-15069 CUP: B86C18000730002. 150.000,00€

Prof. Enio Campiglia, Dipartimento di Scienze Agrarie e Forestali, Università degli Studi della Tuscia, Viterbo

Dr. Anatoly P. Sobolev, Istituto per i Sistemi Biologici, CNR, Roma