A standardized C. sativa extract with high content in CBD (CBD BDS, 63.9% w/w of CBD content) and pure CBD, [purity by high-performance liquid chromatography (HPLC), 99%] were supplied by GW Pharmaceuticals (Cambridge, UK). The dose of CBD BDS used in the experiments refers to the amount of CBD contained in the extract (e.g., 10 mg/kg of CBD BDS indicates a dose of the BDS that contains 10 mg/kg of CBD). 2,4,6-dinitrobenzenesulfonic acid (DNBS), croton oil and myeloperoxidase (MPO) from human leucocytes, were purchased from Sigma Aldrich S.r.l. (Milan, Italy). Pure CBD and CBD BDS were dissolved in ethanol/Tween20/saline (1:1:8) for i.p. injection (60 μl/mouse) or in sesame oil (90 μl/mouse) for oral gavage administration. DNBS was solubilized in 50% ethanol (0.15 ml/mouse). The pure CBD and CBD BDS vehicles had no significant effects on the responses under study.

A C. sativa chemotype with a controlled high amount of CBD (de Meijer et al., 2003) was used for the preparation of CBD BDS. Details about the extraction, purification as well as typical HPLC chromatogram are reported elsewhere (Romano et al., 2014). The composition (%, w/w) of the main phytocannabinoids in CBD BDS was CBD 63.9 ± 5.9, Δ⁹-THC 3.0. cannabigerol 2.8, cannabichromene 3.1, cannabidivarin 1.4. The extract was prepared in GW Pharmaceuticals laboratories (Cambridge, UK).

Male ICR mice, weighing 20–25 g for upper gastrointestinal transit experiments and 25–30 g for colitis experiments, were obtained from Charles River Laboratories (Calco, Lecco, Italy) and housed in polycarbonate cages under a 12 h light/dark cycle with light on at 07:00 a.m., controlled temperature (23 ± 2°C) and constant humidity (60%). Mice were fed ad libitum with standard food, except for the 24 h period immediately preceding the administration of DNBS, for the 12 h period preceding the measurement of intestinal transit and for the 2 h period preceding the oral gavage of drugs. All experiments complied with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC). According to recent preclinical guidelines in pharmacology, group data subjected to statistical analysis had a minimum of n = 5 independent animals per group (Curtis et al., 2015).

Colitis was induced by the intracolonic administration of DNBS as described before (Borrelli et al., 2015). Briefly, mice were anesthetized with inhaled 5% isoflurane (Centro Agrovete Campania, Scafati, SA, Italy) and DNBS (150 mg/kg) was injected in the distal colon using a polyethylene catheter (1 mm in diameter) via the rectum (4.5 cm from the anus). All animals were sacrificed 3 days after DNBS administration by asphyxiation with CO₂, the mice abdomen was opened by a midline incision and the colon removed, isolated from surrounding tissues, opened along the antimesenteric border, rinsed, weighed, and length measured [in order to determined the colon weight/colon length ratio (mg/cm), used as an indirect marker of inflammation]. Mice body weight was measured every day throughout the treatment period. All measurements were performed by operators who were unaware of the particular treatment (blinded evaluation). For biochemistry analysis, tissues were kept at 80°C until use. The dose of DNBS (150 mg/kg) and the time point of damage evaluation (i.e., 3 days after DNBS administration) were selected on the basis of preliminary experiments showing a remarkable colonic damage associated with high reproducibility and low mortality for this dosage, and because maximal DNBS-induced inflammation has been reported in mice after 3 days (Massa et al., 2004).

Increased intestinal motility was induced by the inflammatory agent croton oil as described before (Pol and Puig, 1997; Capasso et al., 2008). Briefly, two doses of croton oil (20 μl/mouse) for two consecutive days were orally administered to mice and 4 days after the first administration of croton oil, upper gastrointestinal transit of mice was measured. This time was selected on the basis of previous work (Pol and Puig, 1997), which reported that the maximal inflammatory response associated to intestinal hypermotility occurs 4 days after the first treatment.

Upper gastrointestinal transit, measured in control, and in mice with intestinal inflammation-induced accelerated intestinal motility (mice treated with croton oil), was evaluated by identifying the leading front of an intragastrically administered charcoal meal marker (10% charcoal suspension in 5% gum Arabic, 10 ml/kg) in the small intestine as previously described (Capasso et al., 2014). Twenty minutes after charcoal administration, mice were killed by asphyxiation with CO₂, and the small intestine was isolated by cutting at the pyloric and ileocaecal junctions. The distance traveled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum.

In the experimental model of colitis pure CBD, CBD BDS or vehicle were given intraperitoneally (5–30 mg/kg) or by oral gavage (10–60 mg/kg) for three consecutive days starting 24 h after DNBS administration [Day 0: colitis induction; day 1: CBD BDS (or CBD); day 2: CBD BDS (or CBD); day 3: CBD BDS (or CBD) and mice sacrifice]. The last administration of CBD BDS (or CBD) was given (at day 3) 1 h (for intraperitoneal administration) or 2 h (for oral gavage) before the sacrifice.

In the experimental model of upper gastrointestinal transit pure CBD, CBD BDS, or vehicle were given intraperitoneally (1–10 mg/kg) or by oral gavage (5–60 mg/kg) 30 min (intraperitoneally) or 1 h (oral gavage) before the administration of the marker, to both control mice, and mice with increased intestinal motility induced by the inflammatory agent croton oil. The CBD BDS and pure CBD doses were selected on the basis of previously published work (Borrelli et al., 2009; Romano et al., 2014).

Myeloperoxidase activity, a peroxidase enzyme used to quantify the neutrophil infiltration in whole-tissue colons, was determined as previously described (Borrelli et al., 2015). Full-thickness colons were homogenized in a lysis buffer composed of 0.5% hexadecyltrimethylammonium bromide in 3-(N morpholino) propanesulfonic acid (MOPS) 10 mM in the ratio of 50 mg tissue per mL MOPS. The homogenates were then centrifuged for 20 min at 15,000 × g at 4°C. An aliquot of the supernatant was incubated with sodium phosphate buffer (NaPP pH 5.5) and tetra-methylbenzidine 16 mM. After 5 min, hydrogen peroxide (H₂O₂; 9.8 M in NaPP) was added and the reaction stopped with acetic acid. The rate of change in absorbance was measured by a spectrophotometer at 650 nm. Different dilutions of human MPO enzyme of known concentration were used to obtain a standard curve. MPO activity was expressed as U/mg of tissue.

Colon, liver, and brain samples were dounce-homogenized and extracted with acetone containing internal deuterated standards for CBD quantification by isotope dilution ([²H]₄ CBD). The lipid-containing organic phase was dried down, weighed, and pre-purified by open bed chromatography on silica gel. Fractions were obtained by eluting the column with 99:1, 90:10, and 50:50 (v/v) chloroform/methanol. The 99:1 fraction was used for CBD quantification by LC-MS-IT-TOF analysis using an LC20AB coupled to a hybrid IT-TOF detector (Shimadzu Corporation, Kyoto, Japan) equipped with an ESI interface. We acquired full-scan MSⁿ spectra of selected precursor ions by multiple reaction monitoring (MRM), extracted the chromatograms of the high-resolution [M–H]^- values and used the latter chromatograms for calibration and quantification.

Data are expressed as the mean ± SEM of n experiments. To determine statistical significance, Student’s t-test was used for comparing a single treatment mean with a control mean, and a one-way ANOVA followed by a Tukey–Kramer multiple comparisons test was used for analysis of multiple treatment means. P-values < 0.05 were considered significant.

The administration of DNBS caused both a significant decrease in body weight and a significant increase in colon weight/length ratio when compared to control mice (Figures 1 and 2). Treatment of DNBS mice with CBD BDS, given intraperitoneally at the range dose of 5–30 mg/kg, did not modify the loss of body weight induced by the inflammatory agent (Figure 1A) but reduced DNBS-increased colon weight/length ratio (Figure 1B). The effect was significant at the 30 mg/kg dose (Figure 1B). Likewise, oral gavage administration of CBD BDS (10–60 mg/kg) had no effect on the DNBS-induced decrease in body weight, although a trend in reducing weight loss was observed at the 60 mg/kg dose (Figure 1C). CBD BDS, given via oral gavage at the 60 mg/kg dose, significantly reduced the colon weight/length ratio increased by DNBS (Figure 1D).

Pure CBD, when both given intraperitoneally (5–30 mg/kg) or by oral gavage (10–60 mg/kg), did not ameliorate DNBS-induced colitis (no variation on body weight and colon weight/colon length ratio; Figure 2).

The beneficial effect of CBD BDS on DNBS-induced colitis was further confirmed by the MPO results. DNBS administration produced a threefold increase in MPO activity which was significantly reduced by either intraperitoneal or oral gavage administration of CBD BDS at the 30 mg/kg and 60 mg/kg dose, respectively (Figure 3).

CBD BDS, given intraperitoneally at the dosage range of 1–10 mg/kg, reduced the intestinal transit in healthy mice, the effect being significant only at the higher dose tested (10 mg/kg; Figure 4A). CBD BDS (1–10 mg/kg), in a dose dependent manner, counteracted the increase in intestinal motility induced by croton oil (Figure 4B). The effect was significant starting from the 1 mg/kg dose. Analysis of the curves representing the inhibitory effect of CBD BDS on transit in healthy mice and in mice with hypermotility induced by croton oil shows that CBD BDS preferentially inhibited intestinal transit in pathophysiological rather than physiological conditions (Figure 4). Likewise, CBD BDS when given by oral gavage at the dosage range of 5–60 mg/kg reduced the intestinal transit in healthy mice, the effect being significant starting from the 10 mg/kg dose (Figure 5A). Similar to the intraperitoneal administration, oral gavage administration of CBD BDS (5–60 mg/kg), in a dose dependent manner, reduced the intestinal hypermotility induced by croton oil (Figure 5B). The effect was significant starting from the 5 mg/kg dose. Analysis of the curves representing the inhibitory effect of CBD BDS on transit in healthy mice and in mice with hypermotility induced by croton oil shows that CBD BDS, also when given by oral gavage, preferentially inhibited intestinal transit in pathophysiological rather than physiological conditions (Figure 5).

As previously reported (Capasso et al., 2008), pure CBD, given intraperitoneally at the dose range of 1–10 mg/kg, did not affect the intestinal transit in healthy mice (Figure 6A) but, significantly and in a dose dependent manner, restored the intestinal motility in mice with hypermotility induced by croton oil (Figure 6B). The effect was significant starting from the 5 mg/kg dose (Figure 6B). Oral gavage administration of pure CBD did not reduce intestinal transit in healthy mice (Figure 6C), and reduced the croton oil-induced accelerated intestinal motility at the 5 mg/kg dose only (Figure 6D).

As shown in Figure 7, CBD was detected in the colon, liver, and brain in mice treated with either CBD BDS or pure CBD (oral administration). In the colon (Figure 7A) and in the brain (Figure 7B), the content increased with the dose in the case of pure CBD, while in the CBD BDS group there were no differences between the two doses.

In the liver, CBD tissue penetration was higher with the oral administration of CBD-BDS, in a dose-dependent manner (Figure 7C), while there were no significant differences between pure CBD at 30 and 60 mg. Interestingly, higher CBD levels were achieved with pure CBD in the colon and brain, and much higher CBD levels were achieved with CBD BDS in the liver.