Extraction and purification of cannabinoids

The Cannabis sativa L. vegetal material was used to produce two extracts using hexane and acetyl acetate as solvents. This allowed to have a starting material to obtain THC, CBN and THCA, the acid form of Δ9-tetrahidrocannabinol. CBN was not detected in the hexane extract and none of the extracts allowed to purify CBD. On Table 1 the yields of cannabinoids from the two extracts are shown. Hexane was the best solvent to isolate THC-A, whereas acetyl acetate was a better solvent to recover higher yields of THC and CBN.

Table 1 Yield of cannabinoids for different starting extractsCharacterization of extracts, phytocannabinoids and their acetylated counterpartsHigh Performance Liquid Chromatography (HPLC)

Extracts and Phytocannabinoid were analyzed by HPLC to gain insight on their degree of purification. The chromatograms are shown in Fig. 2. As it can be seen, both extracts showed their complexity as expected for a complex mixture of molecules. Conversely, THC appears as the main peak in the chromatograms with some degree of non-identified compounds. In addition, the CBN and THCA chromatograms showed that they were the main molecules present in the purified products.

Fig. 2

HPLC chromatograms of extracts and isolated Phytocannabinoids. A is the hexane extract, B is the acetyl acetate extract, C is THC, D is CBN and Panel E is THCA. The analysis was performed with a 20 μL sample injection in a Kromasil 100–5-C8 (4.6 × 150 mm) column with a water:acetonitrile (20:80) mobile phase at 1.0 mL/min. The detection was performed with an UV detector at 272 nm [12]

Magnetic Nuclear Resonance (MNR)

The molecules were identified through their 1H and 13C RMN spectrum which were consistent with the expected chemical structure for THC, THCA, THC-Ac, CBN and CBN-Ac and previously reported assignations [13]. The signals for each molecule are presented below and their spectrum can be observed in supplementary Figs. 2–10.

9-tetrahidrocannabinol (THC)

1H RMN (400 Mz, CDCl3, δ, ppm): 6.34 (1H, sa, H-2); 6.28 (1H, s, H-5ʹ); 6.15 (1H, d, J = 1.0 Hz, H-3ʹ); 5.18 (1H, sa, 2ʹ-OH); 3.22 (1H, d, J = 10.9 Hz, H-1); 2.43 (2H, dt, J = 6.6 y 1.8 Hz, H-1″); 2.17 (2H, m, H-4); 1.92 (1H, m, H-5); 1.68 (3H, s, 3-Me); 1.55 (2H, q, J = 7.4 Hz; H-2″); 1.43 (3H, s, H-8); 1.10 (3H, s, H-9); 0.88 (3H, t, J = 6.7 Hz, H-5″).

13C RMN (100 Mz, CDCl3, δ, ppm): 154.61 (C-2ʹ); 154.20 (C-6ʹ); 142.73 (C-4ʹ); 134.19 (C-3); 123.76 (C-2); 109.93 (C-1ʹ); 109.04 (C-5ʹ); 107.59 (C-3ʹ); 77.25 (C-7); 45.75 (C-6); 35.42 (C-1″); 33.54 (C-1); 31.47 (C-3″); 31.14 (C-4); 30.60 (C-2″); 27.50 (C-8); 24.96 (C-5); 23.32 (C-3-Me); 22.49 (C-4″); 19.20 (C-9); 13.97 (C-5″).

Cannabinol CBN

1H RMN (400 Mz, CDCl3, δ, ppm): 8.18 (1H, sa, H-2); 7.14 (1H, d, J = 7.9 Hz, H-5); 7.08 (1H, dd, J = 7.8 y 1.0 Hz, H-4); 6.43 (1H, d, J = 1.4 Hz, H-5ʹ), 6.29 (1H, d, J = 1.4 Hz, H-3ʹ); 6.25 (1H, s, 2ʹ-OH); 2.38 (3H, s, 3-Me); 1.60 (6H, s, H-8 y H-9), 1.32 (6H, m, H-3″ y H-4″); 0.86 (3H, t, J = 6.5 Hz, H-5″).

13C RMN (100 Mz, CDCl3, δ, ppm): 154.79 (C-2ʹ); 153.07 (C-6ʹ); 144.5 (C-4ʹ); 136.85 (C-3); 136.81 (C-6); 127.54 (C-4); 126.39 (C-2); 122.6 (C-5); 110.69 (C-5ʹ); 110.56 (C-1ʹ); 109.83 (C-3ʹ); 108.31 (C-1); 77.20 (C-7); 33.58 (C-1″); 31.44 (C-3″); 30.43 (C-2″); 27.07 (C-8 y C-9); 22.52 (C-4″); 21.50 (3-Me); 14.00 (C-5″).

9-tetrahidrocannabinólic acid (THCA)

1H RMN (400 Mz, CDCl3, δ, ppm): 12.27 (1H, s, 2″-OH); 6.39 (1H, sa, H-2); 6.25 (1H, s, H-5ʹ); 3.23 (1H, m, H-1); 2.94 (1H, m, H-1″); 2.78 (1H, m, H-1″); 1.68 (3H, s, 3-Me); 1.44 (3H, s, H-8); 1.11 (3H, s, H-9); 0.90 (3H, t, J = 7.2 Hz, H-5″).

13C RMN (100 Mz, CDCl3, δ, ppm): 175.86 (CO2H); 164.66 (C-2ʹ); 159.69 (C-6ʹ); 146.85 (C-4ʹ); 133.83 (C-3), 123.59 (C-2); 112.57 (C-5ʹ); 109.82 (C-1ʹ); 102.27 (C-3’); 78.82 (C-7); 45.57 (C-6); 36.49 (C-1″); 33.43 (C-1); 32.00 (C-3″); 31.27 (C-2″), 31.19 (C-4); 27.36 (C-8); 24.97 (C-5); 23.32 (C-3-Me); 22.50 (C-4″); 19.49 (C-9); 14.05 (C-5″).

THC-Ac

1H RMN (400 Mz, CDCl3, δ, ppm): 6.55 (1H, d, J = 1.5 Hz, H-5ʹ); 6.40 (1H, d, J = 1.5 Hz, H-3ʹ); 5.97 (1H, sa, H-2); 3.05 (1H, d, J = 11.0 Hz, H-1); 2.48 (2H, t, J = 7.6 Hz, H-1″); 2.28 (3H, s, CO2CH3); 2.14 (2H, m, H-4); 1.66 (3H, s, 3-Me); 1.55 (2H, m, H-2″); 1.40 (3H, s, H-8); 1.09 (3H, s, H-9); 0.85 (3H, t, J = 5.4 Hz, H-5″).

CBN-Ac

1H RMN (400 Mz, CDCl3, δ, ppm): 7.81 (1H, sa, H-2); 7.14 (1H, d, J = 7.9 Hz, H-5); 7.08 (1H, dd, J = 7.9 Hz, H-4); 6.73 (1H, d, J = 1.3 Hz, H-5ʹ), 6.57 (1H, d, J = 1.3 Hz, H-3ʹ); 2.57 (2H, t, J = 7.6 Hz, H-1″); 2.37 (3H, s, 3-Me); 2.33 (3H, s, CO2CH3); 1.64 (3H, m, H-2″); 1.60 (6H, s, H-8 y H-9); 1.33 (6H, m, H-3″ y H-4″); 0.88 (3H, t, J = 6.8 Hz, H-5″).

13C RMN (100 Mz, CDCl3, δ, ppm): 169.03 (CO2CH3); 154.36 (C-2ʹ); 147.33 (C-6ʹ); 144.46 (C-4ʹ); 137.52 (C-6); 136.80 (C-3); 128.26 (C-2); 126.85 (C-1); 125.62 (C-4); 122.81 (C-5); 116.32 (C-5ʹ), 115.81 (C-3ʹ); 114.23 (C-1ʹ); 77.68 (C-7); 35.53 (C-1″); 31.42 (C-3″); 30.33 (C-2″); 26.92 (C-8 y C-9); 22.48 (C-4″); 21.47 (3-Me); 21.46 (CO2CH3); 13.99 (C-5″).

Cell viability

The effect of hexane and acetyl acetate extracts, as well as phytocannabinoids and their derivatives were assessed on the neuroblastoma cell line, SHSY 5Y. After a 24 h exposition, to selected dilutions of the extracts and molecules, the resazurin assay was performed and the resulting viability calculated. The curves of viability for the phytocannabinoids and their acetylated counterparts are shown in Fig. 3. The viability curves for both extracts are presented as supplementary Fig. 1. The isolated compounds exhibited higher cytotoxicity than the original extracts, exhibiting THC and CBN the capacity to reduce cell viability to near 20%. Acetylation did not improve the cell killing capacity of the phytocannabinoids and THCA exhibited the lowest effect on the SHSY 5Y cells´ viability. The calculated IC50 for the compounds are presented in Table 2, this indicator reaffirms that acetylation did not improve the potency of the phytocannabinoids and give awareness of a higher activity of CBN over THC.

Fig. 3

Cell viability of SHSY 5Y cells exposed to CBN (A), CBN-Ac (B), THC (C), THC-Ac (D) and THCA (E). Cells were exposed for 24 h to each condition and then the cell culture medium was replaced by resazurin 4 mg/L and the resulting fluorescence was read in a Varioskan multimode plate reader. The results represent three independent experiments, each one performed with triplicates (n = 3). Bars correspond to the standard error

Table 2 IC50 for Phyto cannabinoids and their derivatives on the SHSY 5Y cells after 24 h exposureCaspases activity

To gain insight into the potential mechanism of the of the observed Phyto cannabinoids cytotoxicity caspases 3/7 activation was determined searching for evidence of an apoptotic mechanism of action. All the tested compounds exhibit the capacity to activate caspases 3 and 7, which was statistical significative compared with the control consisting in solvent-treated cells. The obtained results are presented in Fig. 4.

Fig. 4

Activation of caspases 3 and 7 on the SH SY5Y cells exposed to phytocannabinoids. SH SY5Y CELL were exposed for 24 h to THC, THC-A, CBN, CBN-A and THCA or solvent control. Then the caspases 3 and 7 activity were assessed by the Promega GLO caspases kit, and luminescence was measured in a Varioskan multimode plate reader (** is p < 0.0026 and **** p < 0.0001). This experiment was performed once with triplicates for each assayed condition (n = 1)