Industrial, CBD, and Wild Hemp: How Different Are Their Essential Oil Profile and Antimicrobial Activity?

Fiorini D., Scortichini S., Bonacucina G., Greco N.G., Mazzara E., Petrelli R., Torresi J., Maggi F., Cespi M.

The increase of cultivation of industrial hemp (Cannabis sativa L.) all over the world offers new opportunities for the industry to manufacture innovative products from this multipurpose crop. In this regard, the hemp essential oil represents a niche product with potential interest for the pharmaceutical, nutraceutical, cosmeceutical and agrochemical companies. On this basis, in the present work we used the microwave-assisted extraction (MAE) to get an essential oil enriched in bioactive compounds, especially cannabidiol (CBD), from the dry inflorescences of the Italian variety CS (Carmagnola Selezionata). For this purpose, the operative conditions to increase the essential oil yield and CBD concentration in terms of microwave irradiation power (W/g), extraction time (min) and water added to the plant matrix after moistening (%), were optimized using a central composite design (CCD) approach using a Milestone ETHOS X device. The conventional hydrodistillation (HD) performed for 240 min was used for comparative purposes. The qualitative compositions of essential oils obtained by MAE and HD were analysed by GC-MS, whereas the quantitative detection of CBD and main terpenoids (α-pinene, β-pinene, myrcene, limonene, terpinolene, (E)-caryophyllene, α-humulene and caryophyllene oxide) was achieved by GC-FID. Furthermore, the enantiomeric distribution of the chiral constituents (α-pinene, β-pinene, limonene, (E)-caryophyllene and caryophyllene oxide) was determined using chiral chromatography. Results showed that the MAE treatment, using high irradiation power and relatively long extraction times, increased significantly the content of CBD in the essential oil while maintaining high oil yield values when compared with conventional HD. The enantiomeric excess of three chiral monoterpenes (α-pinene, β-pinene and limonene) was determined, with the (+)-enantiomers being predominant, whereas (E)-caryophyllene and caryophyllene oxide were enantiomerically pure. In conclusion, the MAE was successfully applied to hemp dry inflorescences in order to obtain a CBD-rich essential oil which may be exploited in several industrial applications.

Zheljazkov V.D., Sikora V., Semerdjieva I.B., Kačániová M., Astatkie T., Dincheva I.

The hypothesis of this study was that we can modify the essential oil (EO) profile of hemp (Cannabis sativa L.) and obtain fractions with differential composition and antimicrobial activity. Therefore, the objective was to evaluate the effects of grinding of hemp biomass before EO extraction and fractionation during distillation on EO profile and antimicrobial activity. The study generated a several EO fractions with a diversity of chemical profile and antimicrobial activity. The highest concentrations of β-pinene and myrcene in the EO can be obtained in the 5–10 min distillation time (DT) of ground material or in the 80–120 min DT of nonground material. High δ-3-carene and limonene EO can be obtained from 0–5 min DT fraction of nonground material. High eucalyptol EO can be sampled either in the 0–5 min DT of the ground material or in the 80–120 min of nonground material. Overall, the highest concentrations of β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, α-humulene, caryophyllenyl alcohol, germacrene D-4-ol, spathulenol, caryophyllene oxide, humulene epoxide 2, β-bisabolol, α-bisabolol, sesquiterpenes, and cannabidiol (CBD) can be obtained when EO is sampled in the 80–120 min DT and the material is nonground. Monoterpenes in the hemp EO can be increased twofold to 85% by grinding the material prior to distillation and collecting the EO in the first 10 min. However, grinding resulted in a slight but significant decrease in the CBD concentration of the EO. CBD-rich oil can be produced by collecting at 120–180 min DT. Different EO fractions had differential antimicrobial activity. The highest antimicrobial activity of EO fraction was found against Staphylococcus aureus subsp. aureus. THC-free EO can be obtained if the EO distillation is limited to 120 min. The results can be utilized by the hemp processing industry and by companies developing new hemp EO-infused products, including perfumery, cosmetics, dietary supplements, food, and pharmaceutical industries.

Martinenghi L.D., Jønsson R., Lund T., Jenssen H.

The emergence of multi-drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) causes a major threat to public health due to its limited therapeutic options. There is an urgent need for the development of new effective antimicrobial agents and alternative strategies that are effective against resistant bacteria. The parallel legalization of cannabis and its products has fueled research into its many therapeutic avenues in many countries around the world. This study aimed at the development of a reliable method for the extraction, purification, characterization, and quantification of cannabidiolic acid (CBDA) and its decarboxylated form cannabidiol (CBD) present in the fiber type Cannabis sativa L. The two compounds were extracted by ethanol, purified on a C18 sep-pack column, and the extracts were analyzed by high performance liquid chromatography coupled with ultraviolet (UV)–vis and ESI-MS (electrospray ionization mass spectrometry) detection. The antimicrobial effect of CBDA and CBD was also evaluated. CBD displayed a substantial inhibitory effect on Gram-positive bacteria with minimal inhibitory concentrations ranging from 1 to 2 µg/mL. Time kill analysis and minimal bactericidal concentration revealed potential bactericidal activity of CBDA and CBD. While cannabinoids showed a significant antimicrobial effect on the Gram-positive S. aureus and Staphylococcus epidermidis, no activity was noticed on Gram-negative Escherichia coli and Pseudomonas aeruginosa. CBDA presented a two-fold lower antimicrobial activity than its decarboxylated form, suggesting that the antimicrobial pharmacophore of the analyzed cannabinoids falls in the ability for permeabilizing the bacterial cell membrane and acting as a detergent-like agent. A synergy test performed on MRSA with CBD and a range of antibiotics did not indicate a synergetic effect, but noteworthy no antagonist influence either. CBD and CBDA manifested low hemolytic activity on human red blood cells. Likewise, the safety of CBD toward human keratinocyte cells presents no toxicity at a concentration of up to seven-fold higher than the antibacterial minimal inhibitory concentration. Similarly, both CBD and CBDA are well tolerated by mammals, including humans, and conserve a safe value limits for blood-contacting drug development. Overall, CBD exhibited a strong antimicrobial effect against Gram-positive strains and could serve as an alternative drug for tackling MRSA.

Koren A., Sikora V., Kiprovski B., Brdar-Jokanovic M., Acimovic M., Konstantinovic B., Latkovic D.

Hemp (Cannabis sativa L.) was one of the earliest domesticated plant species. Biological classification (taxonomy or systematization) manifests evolutional relationships between taxons according to trait similarities. When it comes to taxonomy, hemp is one of the most controversial plant species due to significant effects of environmental conditions on hemp phenology and expression of quantitative traits as well as different levels of gender expression observed in hemp plants. Controversial taxonomy of hemp has gone through several phases throughout history. The attitude on the number of species within the genus Cannabis and the criteria used in taxonomic units division were under dispute. Initially focused on morphological characteristics and geographical origin, the approach was greatly amended by the development of molecular and biochemical techniques. The main cause of taxonomic uncertainties is the inbreeding ability of all wild Cannabis populations, resulting in continual variability of quantitative traits. The aim of the paper is to review the history of Cannabis classification including different approaches to this scientific issue.

Iseppi R., Brighenti V., Licata M., Lambertini A., Sabia C., Messi P., Pellati F., Benvenuti S.

Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and β-pinene, β-myrcene and β-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.

Nagy D.U., Cianfaglione K., Maggi F., Sut S., Dall'Acqua S.

Spontaneous forms of hemp (Cannabis sativa L., often reported as Cannabis sativa var. spontanea Vavilov) with a low content of psychoactive cannabinoids can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this article, we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analyzed using GC/MS, NMR and LC-DAD-MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists, (E)-caryophyllene (28.3 %) and cannabidiol (CBD; 24.9 %), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC/MS allowed to quantify cannabidiol (CBD) and cannabidiolic acid (CBD-A) in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9 %, and 0.2, 0.3 and 0.4 %, respectively. Flavonoids of this spontaneous form of hemp were formed by C-glycosides and glucuronic acids of kaempferol and apigenin with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, spontaneous C. sativa may represent an important source of CB2 agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.

Fiorini D., Molle A., Nabissi M., Santini G., Benelli G., Maggi F.

Byproducts of industrial hemp (Cannabis sativa L.), including inflorescences, represent an exploitable material to produce niche products for the pharmaceutical, nutraceutical, cosmetic and pesticide industry. One of them is the essential oil, whose composition can be properly modulated on an industrial level by optimizing the extractive conditions and sample pretreatment. This allows to achieve high concentrations of bioactive compounds, such as cannabidiol (CBD) and sesquiterpenes [e.g. (E)-caryophyllene]. In the present work, we evaluated the effects of type of distillation apparatus, status of conservation of the plant material, grinding and sample-pretreatment with microwave and heat, on the hemp essential oil chemical profile obtained from the monoecious cultivar Felina 32. Seven marker compounds, including the monoterpenes α-pinene, myrcene and terpinolene, the sesquiterpenes (E)-caryophyllene, α-humulene and caryophyllene oxide, and the cannabinoid CBD were quantified in the different hemp essential oil samples by gas chromatography-flame ionization detection (GC-FID) analysis, whereas the overall chemical profiles were achieved by gas chromatography-mass spectrometry (GC–MS) analysis. Results showed that hydrodistillation (HD) in comparison with steam distillation (SD) gave a higher content of cannabinoids. Drying was fundamental to induce decarboxylation of cannabinoid acids to the relative alcoholic forms, coupled with an increase of the sesquiterpene fraction. The optimization of sample pretreatments pointed out that the exposure of dry inflorescences to microwave heating at 900 W power for 1 min was the best method to increase the abundance of bioactive compounds in the essential oil, with special reference to CBD, (E)-caryophyllene and caryophyllene oxide. Overall, these results give new insights into the exploitation of hemp byproducts in different fields such as pharmaceuticals, nutraceuticals and eco-friendly insecticides.

Zheljazkov V., Micalizzi G., Semerdjieva I., Mondello L.

Micromeria frivaldszkyana is an endemic species found only in Bulgaria. Its essential oil (EO) composition is unknown. This study assessed the EO yield and composition of M. frivaldszkyana as a function of the location and of drying prior to the EO extraction. M. frivaldszkyana was sampled from two natural habitats, Uzana and Shipka in the Balkan Mountains; the EO was extracted via hydrodistillation and analyzed on GC/MS. The plants from the two locations had distinct EO composition. The EO content (in dried material) was 0.18% (Uzana) and 0.26% (Shipka). Monoterpene ketones were the major group of the EO constituents. Also, hydrocarbons predominated in the EO from Shipka, and alcohols predominated in the EO from Uzana. The EO from Uzana had a greater concentration of menthone (56% vs. 17% from Shipka) and neomenthol (7.8% vs. 2.4%). Conversely, the EO from Shipka had greater concentrations of pulegone (50% vs. 20% from Uzana), limonene (10.1% vs. 2.6%), and germacrene D (3.4% vs. 1.1%). Drying prior to the EO extraction altered the concentration of some constituents. This is the first report of M. frivaldszkyana EO yield and composition. The EO showed some similarities with the chemical profile of other Micromeria species, but overall, it has an unique chemical profile and may have distinctive applications.

Zengin G., Menghini L., Di Sotto A., Mancinelli R., Sisto F., Carradori S., Cesa S., Fraschetti C., Filippi A., Angiolella L., Locatelli M., Mannina L., Ingallina C., Puca V., D’Antonio M., et. al.

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 selected 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.

Pellati F., Brighenti V., Sperlea J., Marchetti L., Bertelli D., Benvenuti S.

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS2. The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while β-myrcene and β-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.

Benelli G., Pavela R., Petrelli R., Cappellacci L., Santini G., Fiorini D., Sut S., Dall’Acqua S., Canale A., Maggi F.

The inflorescences of industrial hemp (Cannabis sativa L.) represent a consistent by-product that is underutilized. Moving from the concept that this plant part has evolved as a natural weapon against phytophagous insects, secreting important secondary metabolites such as cannabinoids and volatile terpenes, herein we assayed the potential of its essential oil as a botanical insecticide. For the purpose, the essential oil was obtained by fresh inflorescences of hemp (monoecious cv. Felina 32) by steam-distillation and analysed by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC–MS). The oil was tested against the filariasis vector Culex quinquefasciatus, the peach-potato aphid Myzus persicae, the housefly Musca domestica and the tobacco cutworm Spodoptera littoralis. To prove its harmlessness on non-target invertebrates, it was tested on the multicolored Asian lady beetle, Harmonia axyridis, and Eisenia fetida earthworms, and compared with α-cypermethrin as the positive control. The essential oil composition was dominated by monoterpene and sesquiterpene hydrocarbons, with (E)-caryophyllene (45.4%), myrcene (25.0%) and α-pinene (17.9%) as the most abundant compounds. Results from insecticidal tests showed that the essential oil from inflorescences of industrial hemp cv Felina 32 was highly toxic to M. persicae aphids (LC50 of 3.5 mL L−1) and M. domestica flies (43.3 μg adult−1), while toxicity was moderate towards S. littoralis larvae (152.3 μg larva−1), and scarce against C. quinquefasciatus larvae (LC50 of 252.5 mL L−1) and adults (LC50 > 500 μg cm−2). Contrary to α-cypermethrin, the hemp cv Felina 32 essential oil was not toxic to non-target invertebrate species, including 3rd instar larvae and adults of H. axyridis ladybugs and adults of E. fetida earthworms. Taken together our results shed light on the possible utilization of the crop residue of industrial hemp as a source of environmental-friendly botanical insecticides to be used in Integrated Pest Management and organic agriculture, particularly to manage aphid and housefly populations.

C O., S J., M S., A H., I A.

Namdar D., Mazuz M., Ion A., Koltai H.

In the last decade, recognition of the therapeutic abilities of Cannabis sativa has risen, along with the need to standardize its products. Standardization requires grading the methods for growing the plant and extracting the active compounds accumulated in its inflorescence. We explored the results of different methods used today and their effect on the levels of compounds extracted from inflorescences positioned along the C. sativa flowering stem. The polarity of the solvent used for the extraction, drying processes and separation methods influenced the chemical composition of the extract. However, regardless of extraction and analytical methods applied, the amounts of cannabinoids and terpenoids in the inflorescences decreased with the position of the sampled inflorescence from top to bottom of the flowering stem. These results have significant implications for the development of growth protocols for C. sativa cultivation and flower extraction methods to standardize cannabis-based products.

Benelli G., Pavela R., Lupidi G., Nabissi M., Petrelli R., Ngahang Kamte S.L., Cappellacci L., Fiorini D., Sut S., Dall’Acqua S., Maggi F.

In the attempt to exploit the potential of the monoecious fiber hemp cv. Futura 75 in new fields besides textile, cosmetics and food industry, its crop-residue given by leaves and inflorescences was subjected to hydrodistillation to obtain the essential oils. These are niche products representing an ideal candidate for the development of natural insecticides for the control and management of mosquito vectors, houseflies and moth pests. After GC-MS analysis highlighting a safe and legal chemical profile (THC in the range 0.004–0.012% dw), the leaf and inflorescence essential oils were investigated for the insecticidal potential against three insect targets: the larvae of Culex quinquefasciatus and Spodoptera littoralis and the adults of Musca domestica. The essential oil from inflorescences, showing (E)-caryophyllene (21.4%), myrcene (11.3%), cannabidiol (CBD, 11.1%), α-pinene (7.8%), terpinolene (7.6%), and α-humulene (7.1%) as the main components, was more effective than leaf oil against these insects, with LD50 values of 65.8 μg/larva on S. littoralis, 122.1 μg/adult on M. domestica, and LC50 of 124.5 μl/l on C. quinquefasciatus larvae. The hemp essential oil moderately inhibited the acetylcholinesterase (AChE), which is a target enzyme in pesticide science. Overall, these results shed light on the future application of fiber hemp crop-residue for the development of effective, eco-friendly and sustainable insecticides.

Booth J.K., Page J.E., Bohlmann J.

Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

Singh M.K., Sarita, Singh S., Mishra S., Shankar U., Maurya A., Sahu K.S., Chakravarty A., Aftab N., Kumar B., Shanker K., Bawankule D.U., Verma R.S.

Joy N., Jackson D., Coolong T.

ABSTRACTIntroductionTerpenes, which are found in high concentrations in the essential oil fraction of the Cannabis sativa flower, have demonstrated potential in many therapeutic and industrial applications.ObjectivesThis work reports on a method developed for quantifying 18 terpenes in C. sativa essential oil obtained through hydrodistillation. The following method has been evaluated for specificity, selectivity, accuracy, linearity, precision, stability, limit of detection, and limit of quantification.Materials and MethodsSamples were prepared by separating the essential oil fraction through hydrodistillation and then diluting with ethyl acetate containing a 100 μg/mL solution of n‐tridecane and octadecane as internal standards. Analysis was performed on a gas chromatograph mass spectrometer (GCMS) using selected ion monitoring (SIM).ResultsThe developed method enabled quantification of isomers of nerolidol and ocimene and several coeluting compounds, with recoveries of 87.35%–116.61%. Two cultivars of C. sativa flower were evaluated, and the dominant terpene compounds in both cultivars were β‐myrcene (5.85–8.62 mg/g dried plant) and β‐caryophyllene (3.89–4.69 mg/g), followed by α‐humulene (1.35–1.99 mg/g), limonene (0.91–1.33 mg/g), and α‐bisabolol (0.66–0.68 mg/g).ConclusionThis method provides an accurate and reliable procedure for separating and quantifying the major terpene compounds in C. sativa flower using hydrodistillation and GCMS with SIM. The simplicity and solvent‐free nature of the hydrodistillation extraction, combined with the specificity and accuracy of using SIM and external standards, enables the determination of total and individual terpenes concentrations within plant material and supports numerous industrial and therapeutic applications.

Rezghiyan A., Esmaeili H., Farzaneh M.

Various practical strategies have been employed to mitigate the detrimental effects of water deficit stress on plants such as application of nano-stimulants. Nanosilicon plays a crucial role in alleviating the deleterious impacts of both abiotic and biotic stresses in plants by modulating various phyto-morphological and physiological processes. This study aimed to examine the combined effects of drought stress and nanosilicon application on the morphological traits and essential oil content and compositions of hemp (Cannabis sativa L.), in which four-week-old seedlings were subjected to irrigation treatments at four levels, including 100% (control), 80% (mild stress), 60% (moderate stress) and 40% (severe stress) field capacity and nanosilicon at three concentrations (0, 0.5 and 1.5 mM) in a completely randomized factorial design experiment with three replications for 40 days. The results showed that the maximum plant height (109.07 cm), number of nodes (33.3), and number of flowering branches (29.4) were recorded under the treatment of 1.5 mM nanosilicon and 100% FC. The lowest fresh and dry weights of aerial parts were associated to the severe drought stress (40% FC) without nanosilicon application. The mild water stress (80% FC) combined with foliar application of 1.5 mM nanosilicon led to highest EO content (0.17%) compared with the other treatments. However, the highest content of cannabidiol in the essential oil was achieved in the severe water stress (40% FC) and treatment of 0.5 mM nanosilicon. The results showed that the application of nanosilicon improved the morphological characteristics and also changed the content and compositions of the hemp plants under drought stress conditions.

Ribeiro A., Alsayyed R., Oliveira D., Loureiro R., Cabral-Marques H.

Cannabis sativa L. has garnered attention as a potential source for new antimicrobial agents, particularly due to the increased prevalence of microbial resistance to conventional antimicrobials and the emergence of multidrug-resistant pathogens. This review, conducted according to the PRISMA 2020 statement, systematically analyzed the antimicrobial properties of C. sativa extracts and cannabinoids against various bacteria, fungi, viruses, and parasites. Data were collected from the scientific literature (102 papers) and clinical trials (5 studies) from 2014 to June 2024. Findings revealed that cannabinoids, especially CBD, demonstrate significant antimicrobial activity against Gram-positive bacteria like MRSA, Gram-negative bacteria such as Pseudomonas aeruginosa, various Candida species, SARS-CoV-2, and HIV. Additionally, CBD showed efficacy against parasitic infections like Echinococcus granulosus and Leishmania species. These results suggest that cannabinoids may represent a new class of antimicrobial agents with unique and diverse mechanisms of action, potentially effective in broad-spectrum therapies. This study highlights the urgent need for further research and standardized clinical trials to validate these findings and to develop cannabinoid-based treatments. The antimicrobial properties of C. sativa align with WHO priorities and support global health initiatives, offering promising avenues for addressing antimicrobial resistance and improving public health outcomes.

Xu Y., Luo J., Guo Y., Zhou J., Shen L., Gu F., Shi C., Yao L., Hua M.

Hemp (Cannabis sativa L.), an annual dioecious plant, has shown extensive application in the fields of fibers, food, oil, medicine, etc. Currently, most attention has been paid to the therapeutic properties of phytocannabinoids. However, the pharmaceutical research on essential oil from hemp is still lacking. In this study, hemp essential oil (HEO) was extracted from hemp flowers and leaves, and the components were analyzed by GC–MS. Quatitative analysis of three main compounds β-caryophyllene, β-caryophyllene oxide, α -humulene were determined by GC-FID. The anti-tumor and anti-neuropathic pain effects of HEO were evaluated. In the paclitaxel induced neuropathic mice model, HEO reduced the serum level of inflammatory cytokines TNF-α to achieve the analgesic effect, which was tested by evaluating mechanical and thermal hyperalgesia. Further investigation with cannabinoid receptor 2 (CB2 R) antagonist AM630 revealed the mechanism of reversing mechanical hyperalgesia may be related to CB2 R. In Lewis lung cancer grafted mice model, the tumor growth was significantly inhibited, the levels of tumor inflammatory cytokines TNF-α and IL-6 were downregulated, immune organ index was modified and immune-related CD4+, CD8+ T lymphocytes level, CD4+/CD8+ ratio were increased when administered with HEO. These results reveal that HEO plays a role not only in tumor chemotherapy induced peripheral neuropathy treatment, but also in anti-tumor treatment which offers key information for new strategies in cancer treatment and provides reference for the medicinal development of hemp.

Šovljanski O., Aćimović M., Sikora V., Koren A., Saveljić A., Tomić A., Tešević V.

Objectives The present study focused on exploring the chemical composition of essential oil and corresponding hydrolate obtained by steam distillation of industrial hemp ( Cannabis sativa L.) cultivar “Helena” (low THC content). Methods Chemical characterization of industrial hemp essential oil and hydrolate was performed by gas chromatographic and gas chromatographic-mass spectrometric analysis, while biological activities included antimicrobial and antioxidant tests. Antimicrobial activity was determined by measuring diameters of the inhibition zone by using a disc-diffusion method with nine microbial strains from ATCC culture. Moreover, minimal inhibitory concentration (MIC) as well as time-kill kinetic studies, antiadhesion, and antibiofilm formation potential were also evaluated. Antioxidant activity was evaluated through three different antioxidant assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH●), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS●+), and reducing power (RP). Results The gas chromatography mass spectrometry analysis showed that the main compound in the essential oil was trans-caryophyllene (37.4%), followed by caryophyllene oxide (12.4%) and α-humulene (11.0%), while in the hydrolate it was 1,8-cineole (11.5%). Results showed that industrial hemp essential oil and hydrolate exhibited no antimicrobial activity against gram-negative bacteria, yeasts, and fungi, while gram-positive bacteria were sensitive. Therefore, in the following step, MIC was determined by microdilution method. The lowest MIC for essential oil (12.5%) was obtained for Enterococcus faecalis and Staphylococcus aureus, while this value doubled for Listeria monocytogenes. Conversely, the MIC for hydrolate was 100% for all gram-positive bacteria. Antioxidant activity showed that industrial hemp essential oil and hydrolate have potential as natural sources of antioxidants. Conclusion This research confirmed the previously proven antimicrobial and antioxidant activities of industrial hemp essential oil. The novelty lies in the antimicrobial and antioxidant activity of hydrolate, which is practically waste, but has great potential to be a useful by-product.

Rekha C., Zhongxiang F.

Industrial hemp has gained increasing interests for its applications in multifaceted areas, including foods, pharmaceuticals and reinforcing materials. The high protein content of hempseeds, presence of essential fatty acids and balanced ratio of omega 6:3 fatty acids, makes hemp an ideal source of choice amongst nutritionists and food product developers. The use of hemp has also been advocated in lowering the risks of certain medical conditions. The antimicrobial and antioxidant feature of oil expands its potential in innovative packaging solutions in the form of coatings or films for shelf-life extension. Fiber from hemp hulls, herd or stalks encourages it as a reinforcement material with eco-friendly attributes. This review explores the applications of hemp in novel product development, with the highlights of its nutritional benefits and antimicrobial efficacy in food and packaging sectors.

Tabiś A., Szumny A., Bania J., Pacyga K., Lewandowska K., Kupczyński R.

This study aimed to evaluate the effects of essential oils (EOs) extracted from Cannabis sativa L. and Cannabis indica Lam. on in vitro ruminal fermentation characteristics, selected rumen microbial populations, and methane production. GC-MS analyses allowed us to identify 89 compounds in both EOs. It was found that E-β-caryophyllene predominated in C. sativa (18.4%) and C. indica (24.1%). An in vitro (Ankom) test was performed to analyse the control and monensin groups, as well as the 50 µL or 100 µL EOs. The samples for volatile fatty acids (VFAs), lactate, and microbiological analysis were taken before incubation and after 6 and 24 h. The application of EOs of C. indica resulted in an increase in the total VFAs of acetate and propionate after 6 h of incubation. The applied EOs had a greater impact on the reduction in methane production after 6 h, but no apparent effect was noted after 24 h. Lower concentrations of C. sativa and C. indica had a more pronounced effect on Lactobacillus spp. and Buryrivibrio spp. than monensin. The presented findings suggest that C. sativa and C. indica supplementation can modify ruminal fermentation, the concentrations of specific volatile fatty acids, and methane production.

Luca S.V., Wojtanowski K., Korona-Głowniak I., Skalicka-Woźniak K., Minceva M., Trifan A.

Hemp (Cannabis sativa L.) has been used for millennia as a rich source of food and fibers, whereas hemp flowers have only recently gained an increased market interest due to the presence of cannabinoids and volatile terpenes. Currently, the hemp flower processing industry predominantly focuses on either cannabinoid or terpene extraction. In an attempt to maximize the valorization of hemp flowers, the current study aimed to evaluate the phytochemical composition and antimicrobial properties of several extracts obtained from post-distillation by-products (e.g., spent material, residual distillation water) in comparison to the essential oil and total extract obtained from unprocessed hemp flowers. A terpene analysis of the essential oil revealed 14 monoterpenes and 35 sesquiterpenes. The cannabinoid profiling of extracts showed seven acidic precursors and 14 neutral derivatives, with cannabidiol (CBD) reaching the highest concentration (up to 16 wt.%) in the spent material extract. The antimicrobial assessment of hemp EO, cannabinoid-containing extracts, and single compounds (i.e., CBD, cannabigerol, cannabinol, and cannabichromene) against a panel of 20 microbial strains demonstrated significant inhibitory activities against Gram-positive bacteria, Helicobacter pylori, and Trichophyton species. In conclusion, this work suggests promising opportunities to use cannabinoid-rich materials from hemp flower processing in functional foods, cosmetics, and pharmaceuticals with antimicrobial properties.

Juliano C.C., Mattu I., Marchetti M., Usai M.

The present work was aimed at the chemical characterization and antimicrobial activity of some extracts of aerial parts (essential oils from leaves and inflorescences and resins from inflorescences) of two legal hemp (Cannabis sativa) varieties, Tisza and Kompolti, grown in Sardinia. Chemical characterization was carried out by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) techniques. The main constituent was myrcene (11.75% in Tisza and 18.21% in Kompolti); delta-9-tetrahydrocannabinol (THC) was not found, while cannabidiol was present up to 0.36% in Tisza and up to 2.80% in Kompolti. The antimicrobial activity of these extracts against a panel of microorganisms was also determined via minimum inhibitory concentration (M.I.C.) determination. While the results showed minor or negligible antimicrobial activity of the extracts against the Gram+ and Candida strains (M.I.C. values equal to or greater than 4 mg/mL), good antibacterial activity (especially of resins) was recorded against S. aureus (M.I.C. 0.015–0.031 mg/mL); no substantial differences were detected between the chemical compositions of the two Cannabis varieties.

Yu J., Cao X., Mi Y., Sun W., Meng X., Chen W., Xie X., Wang S., Li J., Yang W., Chen S., Chen S., Xu W., Wan H.

Cannabinoids are vital metabolites produced by Cannabis sativa. The growing market demand for cannabinoids has made C. sativa a prominent research focus. The biosynthetic pathway of cannabinoids has been extensively investigated, but the information on the transcription factors (TFs) involved in cannabinoids biosynthesis remains limited. In this study, a total of 48 CsWRKYS were identified in high-cannabidiol (CBD) hemp using a combination of two-way blast, plantTFDB prediction, and CDD batching methods. These CsWRKYs exhibited uneven distribution across ten chromosomes, and encoded 162–728 amino acids. Phylogenetic analysis revealed that CsWRKY genes were categorized into three groups, with group Ⅱ further divided into five subgroups. Members within each subfamily exhibited similar conserved motifs and exon-intron structures. Furthermore, gene expression analysis in various tissues, accompanied by cis-regulatory elements analysis, and alternative splicing analysis, provided insight into the potential roles of CsWRKYs in hemp growth and development. Notably, the expression of group III CsWRKY genes (CsWRKY1/2/20/26/28) was induced by salt, drought, and cold stress treatments. Metabolic analysis revealed an increase in the contents of cannabinoids under these treatments. Furthermore, CsWRKY20 and CsWRKY28 were proved to be positive regulatory factors of cannabinoids biosynthesis pathway. Altogether, the results of this study significantly contributes to understand the function of WRKY genes in response to abiotic stress, and their involvement in the transcriptional regulation of cannabinoid biosynthesis in high-CBD hemp.

El-Mernissi R., El Menyiy N., Moubachir R., Zouhri A., El-Mernissi Y., Siddique F., Nadeem S., Ibork H., El Barnossi A., Wondmie G.F., Bourhia M., Bin Jardan Y.A., Abboussi O., Hajji L.

Abstract This study evaluated the volatile components of Cannabis sativa L. essential oils (CSEOs) and their pharmacological potential in vitro, in animal, and in silico. The anti-oxidant capacities of volatile compounds were tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), total anti-oxidant capacity (TAC), and gas chromatography-mass spectrometry (GC-MS). Anti-microbial activity against bacterial and fungal strains was assessed using disk diffusion and micro-dilution, and acute toxicity was examined on mice using OECD 423 criteria. The results indicate that the main components were β-caryophyllene (31.54%), α-humulene (12.62%), β-myrcene (4.83%), and α-pinene (4.69%). The essential oil showed high anti-oxidant ability (IC50 = 0.981 ± 0.059 mg/ml for DPPH, EC50 = 1.74 ± 0.05 for FRAP), and TAC of 0.101 ± 0.001 mg AAE/g. Additionally, it showed significant antibacterial action against Gram-negative organisms, such as Escherichia coli (11.33 ± 0.00 mm), Klebsiella pneumonia (9 ± 0.00 mm), and Pseudomonas aeruginosa (9.34 ± 0.00), with MICs ranging from 0.0052 to 0.0208 mg/CSEO demonstrated antifungal activity against Candida albicans and Fusarium proliferatum, with activity levels of 18.66 ± 0.88 mm, 41.89 ± 3.60%, and MICs of 0.39 and 0.013 mg/ml, respectively. In toxicological studies, CSEO proved to be safe for animals. Docking identified bioactive components and explored anti-oxidant and antibacterial properties. Docking proved that bulnesol and champacol caused indicated actions.

Geppert J., Lietzow J., Hessel-Pras S., Kirsch F., Schäfer B., Sachse B.

Abstract Background Cannabidiol (CBD), a non-intoxicating substance of Cannabis sativa L., is gaining consumer attention. Yet, legal regulations in the EU are complex and questions of potential health risks remain partly unanswered. In Germany, little is known about people who use CBD products. The aim of this cross-sectional study was to gain insight into the user group of CBD, reasons for consumption and risk perception towards CBD-containing products. Methods The study consisted of two parts: In the first part of the study, the prevalence of CBD awareness and usage in Germany was estimated using a telephone survey and a population-representative sample of n = 1,011 respondents. Based on these results, n = 2,000 participants being aware of CBD were surveyed with an online questionnaire in the second part of the study to examine usage and perception of CBD in users and non-users. Results When the study was conducted at the end of 2020 and beginning of 2021, 40.2% of the German participants had already heard of products containing CBD, and 11.4% had actually used them. 42.1% of the users consumed such products regularly, at least once a week, primarily orally via oils or tinctures, and purchased them mainly online. Besides curiosity – addressed especially in young adults – anticipated health benefits including pain and stress relief were main reasons for use. More than half of the study participants perceived the health benefits of CBD use as high or very high. In contrast, the health risks were rated as low or very low by most respondents. Assumptions about official testing for safety as well as physical effects of CBD-containing products varied between users and non-users. Conclusion About one in nine people in Germany uses CBD-containing products. Given reasons for consumption and perception of potential health risks and benefits suggest that people are insufficiently informed about CBD-containing products. The results of the study indicate that risk communication is needed to raise awareness for the topic and to inform (potential) users.

Barbalace M.C., Freschi M., Rinaldi I., Mazzara E., Maraldi T., Malaguti M., Prata C., Maggi F., Petrelli R., Hrelia S., Angeloni C.

Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from ‘Gorilla Glue’ was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-κB pathway. Moreover, the sesquiterpenes (E)-caryophyllene, α-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils’ anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of α-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation.

Brkljača N., Đurović S., Milošević S., Gašić U., Panković D., Zeković Z., Pavlić B.

Cannabis sativa L. known as industrial hemp manifests high capability in the manufacturing of valuable nutraceuticals with health benefits. This study was focused on investigation of chemical profile and bioactive potential of extracts isolated from three hemp varieties (Helena, Marina and Fedora 17). In addition, great attention was directed towards the use of green extraction techniques and maximum utilization of raw material. In that regard conventional (hydrodistillation – HD) and novel extraction techniques (microwave-assisted hydrodistillation – MWHD, supercritical fluid extraction – SFE and microwave-assisted extraction – MAE) were applied for isolating various fractions (essential oil, lipid extracts and liquid extract) with promising bioactive potential. Essential oils and lipid extracts were characterized regarding yield, terpenoid profile determined by GC-MS and in vitro antioxidant activity towards DPPH and ABTS + radicals. Furthermore, the content of two major cannabinoids was determined in plant material and in extract obtained by SFE. Sequential SFE technique allows obtaining lipid extracts in the first step of process as well as raffinates after SFE in the second step. MAE was applied for isolation of polyphenolic fraction from raw materials and SFE raffinates. Polyphenolic content was determined by spectrophotometric assays and UHPLC-Triple-quadrupole-MS. The results suggested that hemp is an excellent source of valuable terpenoid, cannabinoid and polyphenolic antioxidant agents, and further research should be focused on their utilization in dietary supplements and functional foods.