Phytochemical characterization and biological properties of two standardized extracts from a non‐psychotropic Cannabis sativa L. cannabidiol (CBD)‐chemotype

The aim of study was to evaluate and compare the phytochemical profile, the antioxidant and antimicrobial properties of two standardized extracts from non‐psychotropic (Δ9‐tetrahydrocannabinol ≤0.2%) Cannabis sativa L. var. fibrante rich in cannabidiol (CBD). The two extracts, namely Cannabis Fibrante Hexane Extract 1 (CFHE1) and Cannabis Fibrante Hexane Extract 2 (CFHE2), were obtained by extraction with acidified hexane from dried flowering tops as such and after hydrodistillation of the essential oil, respectively. Gas chromatographic analysis showed that cannabinoids remained the predominant class of compounds in both extracts (82.56% and 86.38%, respectively), whereas a marked depletion of the terpenes occurred. Moreover, liquid chromatographic analysis highlighted a high titer of cannabidiol acid (CBDA) and CBD in CFHE1 and CFHE2, respectively. Both extracts showed a strong and concentration‐dependent antioxidant activity and a potent antimicrobial activity against both Staphylococcus aureus ATCC 6538 (MIC and MBC of 4.88 μg/ml for CFHE1, and 4.88 and 19.53 μg/ml, respectively, for CFHE2) and methicillin resistant clinical strains (MIC values between 1.22 and 9.77 μg/ml and MBC values between 4.88 and 78.13 μg/ml). Considering this, the obtained results suggest that standardized extracts of C. sativa var. fibrante could find promising applications as novel antimicrobial agents.

The aim of study was to evaluate and compare the phytochemical profile, the antioxidant and antimicrobial properties of two standardized extracts from nonpsychotropic (Δ 9 -tetrahydrocannabinol ≤0.2%) Cannabis sativa L. var. fibrante rich in cannabidiol (CBD). The two extracts, namely Cannabis Fibrante Hexane Extract 1 (CFHE1) and Cannabis Fibrante Hexane Extract 2 (CFHE2), were obtained by extraction with acidified hexane from dried flowering tops as such and after hydrodistillation of the essential oil, respectively. Gas chromatographic analysis showed that cannabinoids remained the predominant class of compounds in both extracts (82.56% and 86.38%, respectively), whereas a marked depletion of the terpenes occurred. Moreover, liquid chromatographic analysis highlighted a high titer of cannabidiol acid (CBDA) and CBD in CFHE1 and CFHE2, respectively. Both extracts showed a strong and concentration-dependent antioxidant activity and a potent antimicrobial activity against both Staphylococcus aureus ATCC 6538 (MIC and MBC of 4.88 μg/ml for CFHE1, and 4.88 and 19.53 μg/ml, respectively, for CFHE2) and methicillin resistant clinical strains (MIC values between 1.22 and 9.77 μg/ml and MBC values between 4.88 and 78.13 μg/ml). Considering this, the obtained results suggest that standardized extracts of C. sativa var. fibrante could find promising applications as novel antimicrobial agents.

K E Y W O R D S
antimicrobial, antioxidant, cannabidiol, Cannabis sativa L., non-psychotropic cannabinoids, standardized extracts 1 | INTRODUCTION Cannabis sativa L., belonging to the Cannabaceae family, is a well-known dioicous plant, since it is among the most used and cultivated plants worldwide, due to its strong ability to adapt to various pedoclimatic conditions, which allowed its extensive geographical distribution. Cannabis sativa has a wide range of therapeutic applications against several diseases (Novack, 2016;Russo, 2017), but it is also used for food purposes as a source of nutrients and non-nutrient compounds (Callaway, 2004;Kaul et al., 2008;Prociuk et al., 2008; and as ecological raw material, finding applications in the textile industry and bioengineering (Mutje, Lopez, Vallejos, Lopez, & Vilaseca, 2007;Westerhuis, 2016).
In the past, the taxonomic classification of Cannabis has been complicated due to its genetic variability. Recently, it has been recognized as monotypic genus including only the species C. sativa; however, it can be differentiated into different chemotypes depending on the cannabinoid profile (de Meijer, 2014). Compared to the drug-type, fiber-type C. sativa is characterized by a low Δ 9tetrahydrocannabinol (Δ 9 -THC, <0.2%) content with respect to the other non-psychoactive cannabinoids. The marked presence of a cannabinoid compared to the others determines the Cannabis chemotype, such as cannabidiol (CBD), cannabigerol (CBG) and cannabidivarin chemotypes Smeriglio et al., 2018). Considering this, despite C. sativa represents an interesting crop for several industrial uses, both the European and U.S. legislation require a strict control of cannabinoids type and content for cultivation and subsidies release (Pacifico et al., 2006).
Current available studies on Cannabis extracts are still rather lacking and mainly focused on phytochemical features. Moreover, most of these have used non-standardized extracts, and this aspect has a critical impact in order to ensure the reproducibility of the observed biological effects.
Considering this, the aim of study was to evaluate and compare, for the first time, the phytochemical profile as well as the antioxidant and antimicrobial properties of two different standardized extracts obtained from dried flowering tops (as such and after hydrodistillation of the essential oil) of a non-psychotropic CBD-rich C. sativa L. var. fibrante.

| Chemical reagents
All chemicals were of analytical grade and were purchased from Sigma-Aldrich (Milan, Italy). Liquid chromatography (LC) and gas chromatography (GC)-grade solvents were purchased from Merck (Darmstadt, Germany). Certified reference standard solutions (Cerilliant ® ) of CBD, cannabinol (CBN) and Δ 9 -tetrahydrocannabinolic acid A (THCAA) were purchased from Merck (Darmstadt, Germany).  Smeriglio et al. (2018). Cannabis dried flowering tops as such and after hydrodistillation of the essential oil (Smeriglio et al., 2020), were extracted in order to obtain two hexane extracts namely Cannabis Fibrante Hexane Extract 1 (CFHE1) and Cannabis Fibrante Hexane Extract 2 (CFHE2), respectively. Fifty (50) grams of dried flowering tops were extracted three times with 500 ml of 0.1% acetic acid/hexane (v/v), sonicating for 5 min, and proceeding the extraction under constant agitation for 3 hr at room temperature (RT), in the dark. Finally, the three sequential extracts were combined and dried by a rotary evaporator (Buchi R-205, Cornaredo, Italy). The dry extracts (DEs) were stored in dark sealed vials with nitrogen headspace at À20 C until analysis.

| Total phenols assay
Total phenols were determined by Folin-Ciocalteu method as described by  using gallic acid as reference compound (2.5-20 μg/ml). Briefly, 500 μl of Folin-Ciocalteu reagent, 450 μl of deionization water and 50 μl of CFHE 1 or CFHE2 (4.2-33.3 μg/ml) were mixed, and after 3 min 500 μl of Na 2 CO 3 10% (w/v) was added to the reaction mixture. Samples were subjected to 1 hr of incubation at RT, in the dark, mixing every 10 min and then the absorbance was recorded at 786 nm. Total phenols were expressed as mg of gallic acid equivalents (GAE)/100 g of DE.

| Phytochemical characterization by gas chromatography-mass spectrometry analysis
Gas chromatography-mass spectrometry (GC-MS) analysis was carried out by an Agilent 7890A gas chromatograph equipped with an Agilent 5975C mass spectrometry detector. Elution was performed using an Agilent HP-5MS column (30 mm, 0.25 mm, 0.25 μm) according to the method reported and validated by Smeriglio et al. (2020), recording the mass spectra in the 40-400 m/z range.
The identification was carried out considering the retention index calculated with respect to the C 7 -C 40 n-alkanes mix on the HP-5MS column, comparing the mass spectra and MS fragmentation patterns with MS data of NIST08 library and with those reported in the literature (Adams, 2007) as well as by co-injection with reference standards (α-bisabolol, α-caryophyllene, α-pinene, β-caryophyllene, β-pinene, caryophyllene oxide, limonene, and CBD).
2.5 | Phytocannabinoid profile by reversed-phase liquid chromatography coupled with diode array detection and electrospray ion trap tandem mass spectrometry analysis The quali-quantitative analysis of main acid and neutral phytocannabinoids was carried out using an Agilent high-performance LC system (1100 series) equipped with a diode-array (DAD) (G1315) and an ion trap mass spectrometer detector (6320). Electrospray ion (ESI) source operating both in positive and in negative ionization mode was chosen because acid cannabinoids ionize better in negative ionization-mode, while neutral cannabinoids show a higher signal in positive ionization-mode. The chromatographic separation was performed on a Luna Omega PS C18 (150 Â 2.1 mm, 5 μm; Phenomenex) with solvent A (0.1% formic acid) and solvent B (acetonitrile). The elution program was the following: 0-6 min, 50% B; 6-12 min, 57% B; 12-22 min, 57% B; 22-23 min, 50% B; 23-25 min, 50% B. The flow rate was 0.4 ml/min, whereas the column temperature and the injection volume were 28 C and 5 μl, respectively. UV-Vis spectra of phytocannabinoids were recorded in the range 190-600 nm and chromatograms were acquired at 220 nm. Nitrogen was employed as dry gas in mass spectrometry with a flow rate set at 10 L/min, 32 psi and 350 C, according to Pellati et al. (2018). Capillary and skimmer voltage were 3.5 kV and 40 V, respectively. Data acquisition was performed in full-scan mode within the scan range 90-1,000 m/z. Data processing was carried out by Agilent 6300 Series Ion Trap LC/MS system software (version 6.2). To confirm the identified peaks, the retention time, mass and UV-Vis spectra were compared with literature data and with reference standards (CBD, CBN, THCAA, and CBDA). The quantification of phytocannabinoids was performed by built external standard calibration curves with reference compounds and results were expressed as mg of each cannabinoid/100g DE. Regarding phytocannabinoids for which reference standards were not currently available, the quantification was carried out by using the calibration curve of the most structurally similar cannabinoid. In particular, the reference compound CBN was used both for its acid form cannabinolic acid (CBNA) and for its degradation product cannabicyclol (CBL).

| Antioxidant and free-radical scavenging activity
The antioxidant and free-radical scavenging activities of CFHE1 and CFHE2 were determined spectrophotometrically by colorimetric in vitro cell-free assays, which differ in the reaction mechanisms and environments. Absorbance data recorded by an UV-VIS Spectrophotometer (Shimadzu UV-1601), were expressed as half-inhibitory concentration (IC 50 , μg/ml) with confident limits (C.L.) at 95% by Litchfield and Wilcoxon test, using the PHARM/PCS software version 4 (MCS Consulting, Wynnewood, PA). A preliminary screening was carried out to select the optimal concentration range for samples and reference compounds. The concentration ranges reported below refer to the final concentrations of the samples or reference standards in the reaction mixture. Sample solutions, colorless at the tested concentrations, did not show any interference in the colorimetric tests performed.

| Trolox equivalent antioxidant capacity
The scavenging capacity of CFHE1 and CFHE2 against ABTS •+ was carried out according to Smeriglio, Mandalari, et al. (2016). Briefly, trolox equivalent antioxidant capacity (TEAC) assay was performed using a reaction mixture consisting of 1.7 mM ABTS and 4.3 mM potassium persulfate 5:1 (v/v), left at RT in the dark for at least 12 hr, and then used between 12 and 16 hr after preparation. Before use, reaction mixture was diluted with phosphate buffer (pH 7.4) in order to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Fifty microliters of sample (CFHE1 and CFHE2, 1.50-12.0 μg/ml), reference compound (trolox, 0.625-5.0 μg/ml) or blank (hexane) were added to 1 ml of diluted reaction mixture, and after 6 min of incubation at RT in the dark, the absorbance was recorded at 734 nm.

| β-Carotene bleaching
The β-carotene bleaching assay was carried out according to Smeriglio et al. (2017). A β-carotene emulsion was prepared by mixing β-carotene chloroform solution (1 mg/ml), 40 μl of linoleic acid and 400 μl of Tween-40. After removing the chloroform with the rotary evaporator (Buchi R-205, Cornaredo Italy), the film was resuspended with 50 ml of pre-oxygenated water. An emulsion prepared in the same conditions but without β-carotene was used as negative control.
After that, 8 ml of β-carotene emulsion were aliquoted in borosilicate tubes and 320 μl of each sample solution (5-40 μg/ml for CFHE1 and CFHE2), reference standard (butylated hydroxytoluene, 0.031-0.25 μg/ml) or blank (hexane) were added and incubated in the dark at 50 C in a water bath. The absorbance was recorded at the starting time (T0) and every 20 min until 120 min at 470 nm.
The absorbance was recorded at 517 nm.

| Antimicrobial activity
The following strains were used for the antimicrobial assays: Staphylo-

| Statistical analysis
Results were expressed as mean ± SD of three independent experiments in triplicate (n = 3). Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test for antioxidant and antimicrobial assays, and Tukey's test for chemical characterization by SigmaPlot 12.0 software (Systat Software Inc., San Jose, CA). Results were considered statistically significant for p < .05.

| Phytochemical analyses
The sample preparation and extraction method adopted in the present study allowed to obtain the following extraction yields: 5.82% and 7.08% for the extracts obtained from dried flowering tops as such (CFHE1) and after hydrodistillation of the essential oil (CFHE2), respectively. A preliminary phytochemical screening by Folin-Ciocalteu method highlighted a high total phenol content in both extracts: 19,108 mg GAE/100 g DE and 8,587 mg GAE/100 g DE for CFHE1 and CFHE2, respectively. GC-MS analysis led to the identification of 83 and 48 compounds in CFHE1 and CFHE2, respectively (Table 1).
Cannabinoids represent the most abundant compounds, and within this class, CBD has the highest mean area percentage in both extracts (74.26% and 78.66%). However, a slight but statistically significant difference in the relative abundance of cannabinoids (82.56% and 86.38% in CFHE1 and CFHE2, respectively) was recorded. This is mainly attributable to the loss of sesquiterpenes (2.97% vs. 0.04%, in CFHE1 and CFHE2, respectively) and oxygenated sesquiterpenes (1.93% vs. 0.03%, in CFHE1 and CFHE2, respectively) in CFHE2 during the hydrodistillation process.
T A B L E 1 Phytochemical profile of hexane extracts (CFHE1 and CFHE2) of Cannabis sativa var. fibrante by GC-MS analysis. Results were expressed as mean area percentage (%) ± SD of three independent experiments in triplicate (n = 3) Since acid cannabinoids are thermolabile, it is impossible to distinguish between acid and neutral forms following a GC-MS analysis, as they are immediately decarboxylated due to the high injector temperature . Considering this, a reversed-phase liquid and

| Determination of antioxidant properties
Antioxidant and free-radical scavenging potential of Cannabis extracts was evaluated by several in vitro cell-free assays based on different reaction mechanisms and charged radicals. Both CFHE1 and CFHE2 showed remarkable and concentration-dependent antioxidant and free-radical scavenging activity (Figure 2). CFHE1 showed the following order of potency: TEAC > ORAC > β-carotene bleaching > Iron-chelating activity > FRAP > DPPH. On the contrary, CFHE2 showed the following order of potency: TEAC > β-carotene bleaching > Iron-chelating activity > ORAC > FRAP > DPPH (Table 3).
CFHE1 showed the strongest and statistically significant (p < .001) antioxidant activity in all tests carried out in comparison with the CFHE2, with the exception of the β-carotene bleaching and iron-chelating activity assays (Table 3). Moreover, both extracts showed statistically significant results (p < .001) with respect to the reference compounds (Table 3).
T A B L E 2 Native phytocannabinoid profile of hexane extracts (CFHE1 and CFHE2) of Cannabis sativa var. fibrante by RP-LC-DAD-ESI-MS/MS analysis. Results were expressed as mg/100 g DE and represent the mean ± SD of three independent experiments in triplicate (n = 3)  (Table 4).
In our recent investigation on the antioxidant and antimicrobial activity of two standardized extracts from C. sativa L. , we reported no antimicrobial effect against any of the Gram-negative strains tested. Here, E. coli was slightly sensitive to both extracts, indicating a potential therapeutic tool against Gramnegative bacteria. It is widely accepted that Gram-negative bacteria are more resistant to natural extracts compared to the Gram-positive strains based on the differences in cell wall composition. In our previous work (Mandalari et al., 2007), bergamot fractions obtained from the Citrus fruit processing industry, were found to be active against all the Gram-negative bacteria tested, which included E. coli, Pseudomonas putida, Salmonella enterica. In agreement with our previous investigation on C. sativa L.  showed an in vitro effect against S. aureus at a concentration between 0.017 and 0.15 mg/ml. However, no effect was observed on Gramnegative bacteria including E. coli.
Based on these findings, it is possible to postulate the use of these Cannabis extracts as natural antimicrobials with bactericidal effect, particularly against Gram-positive bacterial infections.

| CONCLUSIONS
This is the first study investigating the native phytocannabinoid and terpenic profile as well as the antioxidant and antimicrobial activity of standardized extracts from flowering tops of Cannabis sativa L. var.
fibrante CBD-chemotype as such and after hydrodistillation. Moreover, this is the first study evaluating the activity of these Cannabis extracts on virulent strains of Staphylococcus, which have always been of concern for human health, particularly MRSA strains, responsible for many nosocomial infections.
The strong antioxidant and free-radical scavenging activity found in both hexane extracts (pre-and post-hydrodistillation) allow to postulate that the compounds mainly responsible of the antioxidant power are cannabinoids and in particular their neutral forms, although a synergistic effect due to the presence of minor compounds, in particular terpenes, cannot be excluded. CFHE1 proved to be the most powerful extract against the Gram-positive S. aureus probably due to the presence of terpenes in addition to cannabinoids, although CFHE2 maintains an interesting antibacterial activity toward both ATCC and clinical MRSA strains.
Considering this and according to the antioxidant results, cannabinoids seem to exert a pivotal role in the antimicrobial activity, shedding light on a promising potential use of these standardized hexane extracts as antibacterial agents for the treatment of S. aureus infections.
However, these are preliminary data needing further investigation, both regarding the antibacterial activity against S. aureus and the safety profile of these extracts. One of the virulence factor of S. aureus is given by its ability to form biofilms both on abiotic and biotic surfaces. Therefore, it becomes essential to determine the effectiveness of these extracts even in these conditions, alone or in combination with synthetic antibiotics, trying to formulate a possible therapeutic application, which allows an appropriate study of their toxicological profile.