Chromatographic separation of the EtOAc extracts from the aerial parts of C. sativa yielded five new compounds (1–5) and twelve known compounds (6–17), named Δ⁹-trans-tetrahydrocannabivarin (6),²² cannabinol (7),²³ cannabispirone (8),²⁴ erythrodiol (9),²⁵ oleanolic acid (10),²⁶ maslinic acid (11),²⁷p-hydroxybenzaldehyde (12),²⁸ (E)-methyl p-hydroxycinnamate (13),²⁹ (Z)-methyl p-hydroxycinnamate (14),³⁰ ferulic acid (15),³¹ phylligenol (16),³² and skullcapflavone II (17)³³ (Fig. 1).

Cannabisativa A was obtained as yellowish oil. Based on HRESIMS, its molecular formula was determined as C₂₀H₂₈O₄ containing 7 degrees of unsaturation. The ¹H NMR spectrum (Table 1) showed a phenolic hydroxyl at δ_H 9.40 (1H, br s), meta-coupled aromatic protons at δ_H 6.35 and 6.28 (each 1H, d, J = 1.6 Hz), a methoxy group at δ_H 3.34 (3H, s) and four methyl groups at δ_H 1.50, 1.41, 1.30 and 0.93. ¹³C NMR and DEPT spectral analyses revealed the presence of eight aromatic/olefinic carbons, two sp³ quaternary carbons, one sp³ methine, four sp³ methylenes, four methyls and one methoxy (Table 1). The ¹H and ¹³C NMR spectra of 1 were quite similar with those of cannabitriol-C₃ (NMR data see Table 1), except that one additional methoxyl at δ_C 51.9 was present in 1. It was further confirmed by the 2D NMR experiments. The ¹H–¹H COSY correlations from H-2′ to H-1′ and H-3′, and from H-7 to H-8 confirmed the substructures of C_1′–C_2′–C_3′ and C₇–C₈ (Fig. 2). The key HMBC correlations of H-10/C-6a, C-10a, C-10b, C-8; H-7/C-6a; H-13/C-9; H-12/C-4a, C-6, and H-11/C-6a, C-6 permitted the establishment of a cannabitriol structure. The methoxy and the C_1′–C_2′–C_3′ unit was found to be attached to C-10 and C-3 according to HMBC correlation observed between 10-OCH̲₃/C-10 and H-1′/C-3, respectively.

The relative configuration of 1 (9R*,10S*) were supported by observed correlation of H-10/H-13 in ROESY spectrum. To determine the absolute configuration, ECD calculation method using time-dependent density functional theory (TDDFT) and metal rhodium salt method were both applied. The experimental ECD spectrum of 1 matched well with the calculated spectrum for the 9R,10S configuration (Fig. 3). The absolute configuration was also verified by testing CD difference spectrum after the reaction of C-9 hydroxyl group with metal rhodium salt.³⁴ The CD difference spectrum showed a negative Cotton effect at 350 nm (Fig. 4), suggesting the absolute configuration at C-9 was inferred to be R-form. Thus, 1 was identified as (9R,10S)-9-hydroxy-10-methoxy-Δ^6a(10a)-tetrahydrocannabivarin.

Cannabisativa B was obtained as yellowish oil. The molecular formula of C₂₂H₃₂O₄ was assigned on the basis of HRESIMS ion peak at m/z 361.2383 [M + H]⁺, requiring 7 degrees of unsaturation. Comparisons of the ¹H NMR and ¹³C NMR spectroscopic data of 2 with those of 1, additional δ_H 1.30 (2H, m), 1.31 (2H, m); δ_C 30.4, 31.5 were observed in 2. These signals were positioned at C-3 based on the ¹H–¹H COSY (H-5′/H-4′; H-2′/H-3′, H-1′) and HMBC (H-5′/C-3′, C-4′; H-1′/C-3, C-4) correlations (Fig. 2). The configuration of 2 was determined as 9R,10S using the same method of 1 (Fig. 3). Accordingly, 2 was established as (9R,10S)-9-hydroxy-10-methoxy-Δ^6a(10a)-tetrahydrocannabinol.

Cannabisativa C was obtained as yellowish oil, and showed an HRESIMS peak at m/z 317.1758 [M–H]⁻, indicating a molecular formula of C₁₉H₂₆O₄ and 7 degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 3 showed two meta-coupled aromatic protons (δ_H 6.17, 5.95 (each 1H, d, J = 1.4 Hz)), two sp³ methine protons (δ_H 1.49, 3.87), four sets of methylenes and four methyls (δ_H 1.39, 1.40, 1.45 and 0.87). The ¹H–¹H COSY correlations from H-2′ to H-1′ and H-3′, and from H-7 to H-8 and H-6a confirmed the substructures of C_1′–C_2′–C_3′ and C_6a–C₇–C₈ (Fig. 2). The key HMBC correlations of 1-OH̲/C-1, C-10b; H-10/C-6a, C-9, C-10a, C-10b; H-11/C-4a, C-6, C-6a, C-12; H-12/C-4a, C-6, C-6a, C-11; H-13/C-8, C-9, C-10 permitted the establishment of a cannabitriol structure. The C_1′–C_2′–C_3′ unit was found to be attached to C-3 according to HMBC correlation observed between H-1′/C-3. In addition, the chemical shifts of C-9 and C-10 and the molecular formula of 3 indicated the presence of an oxirane ring between C-9 and C-10.

The relative configuration was established by NOESY experiment. Correlations of H-13/H-10, H-7b; H-7b/OH-10a suggested that they were situated in the axial position. Correlation of H-6a/H-8a suggested that they were in the equatorial position. Moreover, the calculated ECD spectrum of (6aS,9S,10R,10aR) were matched well with the experimental ECD spectrum of 3 (Fig. 3). Therefore, the structure of 3 was defined as (6aS,9S,10R,10aR)-9,10-epoxy-10a-hydroxy-tetrahydrocannabivarin.

Compound 4 was obtained as yellowish oil and had a molecular formula of C₁₅H₂₀O₂ as judged from HRESIMS m/z 231.1380 [M–H]⁻ (calcd. for C₁₅H₁₉O₂ 231.1385), indicative of 6 degrees of unsaturation. The ¹³C NMR (Table 2) showed three methyls, one methlene, seven methines and four quaternary carbons. In the ¹H NMR spectrum, the characteristic signals indicated a 1,3,4-trisubstituted benzene ring at δ_H 7.11 (1H, d, J = 7.7 Hz), 6.76 (1H, br d, J = 7.7 Hz), 6.71 (1H, br s), two double bonds at δ_H 6.58 (1H, d, J = 11.5 Hz), 5.95 (1H, dd, J = 11.5, 11.2 Hz), 5.28 (1H, dd, J = 11.2, 10.3 Hz), a hydroxymethyl group at δ_H 4.40 (2H, m) and three methyl groups at δ_H 2.25 (3H, s), 1.00 (3H, d, J = 6.6 Hz) and 0.99 (3H, d, J = 6.6 Hz). The ¹H–¹H COSY correlations of H-11/H-10/H-14 (H-15), and H-9/H-8/H-10 combined with HSQC confirmed the C₈–C₉–C₁₀–C₁₁–C₁₄(C₁₅) moiety (Fig. 2). HMBC correlations fromH-4 to C-7, H-12 to C-1, 2 and 3, and H-13 to C-5 and C-8 (Fig. 2) confirmed the planar structure. The geometry of Δ⁷ double bond was determined by NOE difference spectra. Irradiation of H-13 resonance did not lead to a marked enhancement of the H-8 proton signal, and irradiation of the H-8 also did not cause an enhancement of the H-13. Next, we used ¹³C NMR calculation as well as DP4+ probability analyses to determine the geometry of Δ⁷ double bond. The ¹³C NMR chemical shifts of 4a and 4b were calculated at the B3LYP/6-311 + G(d,p) level utilizing the polarizable continuum model (PCM) in methanol. The calculated results for 4b (R² = 0.9987) were a better match with the experimental data than those of 4a (R² = 0.9951) (Fig. 5). Moreover, according to the DP4+ probability analyses, 4b was assigned with a 100% probability (Fig. S41†). Finally, the structure of compound 4 was identified and named as (7Z,9Z)-cannabiphenolic acid A.

Compound 5, obtained as yellowish oil, displayed an HRESIMS peak at m/z 231.1387 [M–H]⁻ corresponding to the molecular formula C₁₅H₂₀O₂, suggesting 6 degrees of unsaturation. Its ¹³C NMR and DEPT spectra (Table 2) displayed 15 carbon resonances, including three methyls, one methlene, seven methines and four quaternary carbons. The ¹H NMR spectrum showed an 1,3,4-trisubstituted benzene ring at δ_H 7.04 (1H, d, J = 7.6 Hz), 6.87 (1H, br s), 6.85 (1H, br d, J = 7.6 Hz), a double bond at δ_H 5.31 (1H, overlap) and 5.29 (1H, overlap), a terminal double bond at δ_H 5.34 (1H, overlap) and 5.26 (1H, br s), and three methyl groups at δ_H 2.21 (3H, s), 0.97 (3H, d, J = 6.6 Hz) and 0.79 (3H, d, J = 6.6 Hz). The C₁₀–C₁₁–C₁₄(C₁₅) moiety was verified by the ¹H–¹H COSY correlations of H-11/H-10/H-14/H-15 (Fig. 2). The key HMBC (Fig. 2) correlations from H-8 to C-9, and C-10, H-9 to C-11, H-12 to C-1, C-2, and C-3, and H-13 to C-5, and C-8 connected the planar structure of compound 5. The geometry of Δ⁹ was assigned as Z by the coupling constant (J_9,10 = 8.6 Hz) in pyridine-d₅ (400 MHz). The absolute configuration of C-8 was identified by a modified Mosher's method (Fig. 6). Compound 5 was treated separately with (R)- and (S)-MTPA-Cl to obtained the respective (S)- and (R)-MTPA esters (5a and 5b). The S configuration of C-8 was determined by the Δδ_H(S–R) value of H-9, H-10, H-13a and H-13b. Based on the above findings, compound 5 was named as (8S,9Z)-cannabiphenolic acid B.

We evaluated the cytotoxicity of compounds 1–17 against PC12 cells using MTT method. Neuroprotective assays were performed at concentrations that had no significant effect on cell survival (compounds 1–5, 7, 8 and 12–16, cell survival rate > 90%) (Table S1†). We found that the novel compounds 1–5 can slightly improve the cell viability, but the significant difference is not obvious. Known compounds 12, 13 and 15 could attenuate H₂O₂-induced cytotoxicity in PC12 cells by the effect of antioxidant (Fig. 7).