Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and β-sitosteryl-3-O-β-D-glucopyranoside-2′-O-palmitate (6). In addition, five known compounds, α-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2–4 were inactive as analgesics.
Cannabinoids are phenolic compounds possessing a C21 terpenophenolic structure uniquely found in Cannabis sativa L. (ElSohly and Slade, 2005). Currently, 86 cannabinoids have been isolated from cannabis (Ahmed et al., 2008; ElSohly and Slade, 2005; Radwan et al., 2008). Non-cannabinoid constituents isolated from cannabis include flavonoids, spiroindans, dihydrostilbenes, dihydrophenanthrenes, sterols and alkaloids, among others (Ross and ElSohly, 1995; Turner et al., 1980). As part of our program to study the constituents of high potency cannabis and their pharmacology (Ahmed et al., 2008; Radwan et al., 2008), we herein report the isolation and structure elucidation of eleven non-cannabinoid constituents including six new (1–6) and five known (7–11) compounds as well as their antimicrobial, antileishmanial, antimalarial and antioxidant activities. The analgesic activities of 2–4 are also reported.
2. Results and Discussions
Compound 1 was obtained as an orange amorphous powder. Its positive mode HRESIMS exhibited a pseudomolecular ion at m/z 373.2425 [M+H]+ corresponding to a molecular formula of C23H32O4. The IR spectrum of 1 revealed the presence of an α,β-unsaturated ketone and ester carbonyl groups at vmax 1663 and 1780 cm−1, respectively. The 1H NMR spectrum of 1 displayed three olefinic methyl singlets (δH 1.56, 1.63 and 1.69), one primary methyl triplet (δH 0.87, H3-5′), one acetoxy methyl singlet (δH 2.32), one aromatic singlet (δH 6.53, H-2) and 7 methylene resonances (δH 1.32–2.39). The 13C NMR, DEPT and HMQC spectra of 1 revealed 23 resonances including five methyl, seven methylene, one aromatic methine, two olefinic methine and eight quaternary carbons. The two carbonyl carbons resonating at δC 187.1 and 180.8 are characteristic for a benzoquinone skeleton (Mossa et al., 1999), while NMR analysis suggested an acetoxy (δH 2.32, δC 168.1, 20.5), a geranyl (Radwan et al., 2008) and an n-pentyl substituent (Ahmed et al., 2008; Radwan et al., 2008), indicating that 1 is a trisubstituted-1,4-benzoquinone derivative. HMBC analysis (Fig. 1) placed the geranyl substituent at C-6 due to the correlation of H2-1″ (δH 3.10) with C-6 (δC 135.6), C-1 (δC 187.1) and C-5 (δC 149.1). The three bond HMBC correlation of H-2 (δH 6.53) with C-6 (δC 135.6) and C-4 (δC 180.8), and the correlation of H2-1′ (δH2.39) with C-4 (δC 180.8) and C-2 (δC 132.7) placed the n-pentyl moiety at C-3. The location of the acetoxy group at C-5 was determined by the four bond HMBC correlation between the acetoxy methyl (δH 2.32) and C-5 (δC 149.1). The presence of the n-pentyl and geranyl groups were confirmed by COSY correlations (Fig. 1), establishing 1 as 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone.
Compound 2 was isolated as a brown amorphous powder. Its molecular formula was determined as C17H18O5 from the positive mode HRESIMS ion at m/z 325.1100 [M+Na]+and 13C NMR spectrum. The 1H and 13C NMR spectra of 2 (Table 1) displayed two sets of methylene protons [δH 2.64 (4H, s, H2-9 and H2-10) correlated to δC 30.6 (C-9) and 31.8 (C-10) in the HMQC spectrum], a pair of signals for an ortho-coupled AB spin system [δH6.74 (1H, d, J = 8.0 Hz, H-7) and 6.79 (1H, d, J = 8.0 Hz, H-8) correlated to δC 109.2 (C-7) and 119.1 (C-8), respectively in the HMQC spectrum], and an isolated aromatic proton [δH6.50 (s, H-1)] suggesting that 2 is a 2,3,4,5,6-pentasubstituted 9,10-dihydrophenanthrene (Crombie et al., 1979; Leong et al., 1997; Stermitz et al., 1983). The 13C NMR (Table 1) and APT spectra indicated the presence of five oxygenated quaternary aromatic carbons. Three of these carbons have methoxyl substituents as revealed by three sharp singlets in the 1H NMR [δH 3.85, 3.86 and 3.89 (3H each)], while the remaining two carbons have hydroxyl substituents as was confirmed by the presence of a characteristic absorption band at νmax 3420 cm−1 in the IR spectrum. The 3J HMBC correlations between H-8 (δH 6.79) and C-6 (δC 147.6), C-4b (δC 121.1) and C-9 (δC 30.6); between H-7 (δH 6.74) and C-8a (δC 132.6) and C-5 (δC 141.7) and between the methoxyl group at δH 3.85 and C-6 (δC147.6), located this methoxyl group at C-6, as was confirmed by ROESY correlation (Fig. 2). The 3J HMBC correlation between the isolated aromatic proton at δH 6.50 and C-10 (δC31.8) and C-4a (δC 114.9) determined its location at C-1. The ROESY correlation between the remaining two methoxyl groups at δH 3.86 and 3.89 in conjunction with the ROESY correlation between the methoxyl group at δH 3.89 and H-1 (δH 6.50) fixed their positions at C-3 and C-2, respectively, which was confirmed by HMBC correlations (Fig. 2), establishing 2 as 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene.
Compound 3 was isolated as a pale brownish, amorphous powder. The molecular formula of 3 was determined as C18H20O5 from the positive mode HRESIMS at m/z 339.1279 [M+Na]+, a mass difference of 14 amu compared to 2. The 1H and 13C NMR spectra of 3 (Table 1) were similar to 2 except for the replacement of one hydroxyl by a methoxyl group (δH 3.92, δC 56.1). Therefore, 3 exhibited four methoxyl groups [δH 3.88 (3H, s), 3.89 (3H, s) and 3.92 (6H, s); δC 56.0, 56.1, 56.2 and 61.3] and one hydroxyl group. Their locations on the dihydrophenanthrene skeleton were determined by comparison to the 1H and 13C NMR data of 2 (Table 1) and confirmed by HMBC and ROESY correlations, placing two methoxyl groups at C-2 and C-3 and the hydroxyl at C-4. The remaining methoxyl groups are therefore attached to ring B. The presence of a pair of isolated aromatic singlets [δH6.79 (H-8) and 8.02 (H-5)] in the 1H NMR assigned the two methoxyl groups to C-6 (δC147.2) and C-7 (δC 147.1) (Leong et al., 1997), which was confirmed by ROESY [δH 3.92 (OMe-6 and OMe-7)/H-5 and H-8; H-8/H2-9] and HMBC (H-5/C-7, C-4a; H-8/C-6, C-8a, C-9) correlations. Thus, the structure of 3 was established as 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene.
Compound 4 was isolated as a reddish brown powder. Its molecular formula was determined as C16H14O5 from negative mode HRESIMS (m/z 571.1630 [2M–H]−) and 13C NMR data. The UV spectrum of 4 (λmax 258, 282 and 303 nm) is characteristic for a phenanthrene skeleton (Leong et al., 1999). The 1H NMR (Table 1) displayed a pair of signals for an ortho-coupled AB spin system [δH 8.06 (1H, d, J = 8.4 Hz, H-9), 8.12 (1H, d, J = 8.4 Hz, H-10)], two meta-coupled protons [δH 6.82 (1H, d, J = 2.0 Hz, H-8), 6.93 (1H, d, J = 2.0 Hz, H-6)] and an isolated aromatic singlet [δH 6.15 (1H, s, H-3)] indicative of a pentasubstituted phenanthrene (Rethy et al., 2006; Leong et al., 1999). The 1H and 13C NMR spectra of 4 showed two aromatic methoxyl groups [δH 3.90 (3H, s) and 3.96 (3H, s)] and five oxygenated quaternary carbons, indicating that 4 has three hydroxyl groups. The ROESY correlation of the methoxyl group at δH 3.90 with the protons at δH 6.93 (H-6) and δH 6.82 (H-8) established its location at C-7 (δ 161.0), which was confirmed by HMBC [δH3.90 (OMe-7)/C-7; H-8/C-7, C-6, C-8a, C-9; H-6/C-7, C-8] correlations. The ROESY and HMBC correlations of the methoxyl group at δH 3.96 with H-3 (δH 6.15) and C-4 (δC161.5), respectively, assigned its location at C-4. Therefore, the structure of 4 was determined as 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene.
Compound 5 was obtained as a yellow amorphous powder. Its positive mode HRESIMS displayed an [M+Na]+ ion at m/z 459.1766 suggesting C26H28O6 as the molecular formula and 13 degrees of unsaturation. The IR spectrum showed absorption bands at vmax 3421 and 1662 cm−1 due to hydroxyl and carbonyl groups, respectively, while the UV absorption maxima at λmax 275 (band I) and 340 (band II) nm were indicative of a flavone skeleton (Mabry et al., 1970). The 1H NMR spectrum of 5 revealed a chelated hydroxyl group [δH13.05 (s, HO-5)] which was confirmed by the bathochromic UV shift (+ 25 nm) of band II upon the addition of AlCl3 to a methanolic solution of 5. Bathochromic UV shifts upon the addition of NaOMe (+61 nm) and NaOAc (+ 5 nm) suggested hydroxylation at C-4′ and C-7, respectively (Mabry et al., 1970). The 1H NMR displayed two sharp singlets at δH 6.66 (1H, H-3) and 6.36 (1H, H-6), one methoxyl group (δH 3.98) and an ABX spin system of ring B [δH 7.01 (1H, d, J = 8.0 Hz, H-5′), 7.58 (1H, d, J = 2.0 Hz, H-2′), 7.64 (1H, dd, J = 2.0, 8.0 Hz, H-6′)]. The presence of a geranyl group was deduced from the three methyl singlets at δH 1.48, 1.53 and 1.82 and two olefinic proton triplets at δH 5.02 and 5.35 in the 1H NMR spectrum (Ahmed et al., 2008; Radwan et al., 2008). The 13C NMR, DEPT-135 and HMQC spectra displayed 26 resonances including three methyl, one methoxyl, three methylene, seven methine and twelve quaternary carbons. The location of the methoxyl group was determined to be at C-3′ from HMBC (OMe-3’/C-3′; H-5’/C-3′; H-2’/C-3′, C-4′) and ROESY (OMe-3’/H-2′) correlations (Fig. 2). The carbon resonance at δC 98.9 corresponding to a proton singlet at δH 6.36 in the HMQC spectrum indicated an unsubstituted C-6 position (Agrawal, 1989). The location of the geranyl group at C-8 was confirmed by the HMBC correlation of the benzylic protons [δH 3.57 (2H, d, J = 6.8 Hz, H2-1″)] with C-8 (δC 106.5), C-7 (δC 162.4) and C-9 (δC 155.4) (Fig. 2). The spectroscopic data of 5 are similar to those reported for cannflavin A (10) (Agrawal, 1989; Choi et al., 2004) except for the location of the geranyl group at C-8 instead of C-6, establishing 5 as 8-geranyl-5,7,4′-trihydroxy-3′-methoxyflavone (cannflavin C).
Compound 6 was obtained as an optically active white amorphous powder. Its molecular formula was deduced from the positive mode HRESIMS [M+Na]+ ion at m/z 837.6621 as C51H90O7. The 1H NMR displayed two tertiary [δH 0.75 (Me-18) and 0.99 (Me-19)], three secondary [δH 0.63 (Me-26 and Me-27) and 0.90 (Me-21)] and one primary [δH 0.63 (Me-29)] methyl groups in addition to an olefinic proton at δH 5.33 (bs, H-6), indicating a sitosterol skeleton (Kovganko et al., 2000; Takemoto et al., 1967). The presence of an anomeric proton [δH 4.33 (d, J = 7.6 Hz, H-1′)] and carbon [δC 101.6 (C-1′)] in the HMQC spectrum indicated monoglycosylation at C-3 (δC 80.1) (Ishii et al., 1977) that was confirmed by HMBC (H-3/C-1′; H-1’/C-3) and ROESY (H-1’/H-3) correlations (Fig. 2). The sugar moiety was identified as P-D-glucopyranose by acid hydrolysis of 6 and TLC comparison with authentic sugar samples. The 1H and 13C NMR data of 6 were similar to those reported for β-sitosterol-3-O-β-D-glucopyranoside (Chang et al., 1981) with the addition of 16 resonances characteristic for a palmitate moiety (Segre and Mannina, 1997), which was confirmed by methylation of the alkaline hydrolysis product of 6 followed by GCMS analysis. The downfield esterification shift of C-2′ (+ 2 ppm), upfield shifts of C-1′ (-4 ppm) and C-3′ (-3 ppm) and the four bond HMBC correlation of H-1′ (δH 4.33) and C-1″ (δC 174.6) (Fig. 2) placed the palmitate moiety at C-2′ (Terui et al., 1976; Yamasaki et al., 1977). Thus, the structure of 6 was established as β-sitosteryl-3-O-β-D-glucopyranoside-2′-O-palmitate.
Compound 7 was obtained as colorless prisms. Its positive mode HRESIMS gave an [M+H]+ ion at m/z 249.2393 corresponding to a molecular formula of C15H20O3. The GCMS showed a molecular ion at m/z 248 (33%) and two characteristic ions at m/z 189 (100%) and 176 (65%) suggesting that 7 is a spiroindan derivative (El-Feraly et al., 1986). The 13C NMR, DEPT and HMQC spectra of 7 displayed 15 resonances including one methoxyl, six methylene, two aromatic methine, one sp3 oxymethine and five quaternary carbons. The spectroscopic data of 7 are similar to those reported for β-cannabispiranol (Boeren et al., 1977; Shoyama and Nashioka, 1978; Radwan et al., 2008) except for the downfield shift of the oxymethine carbon (+5.6 ppm), indicating a 4’α-configuration. Although 7 is a known cannabis constituent (Crombie et al., 1982), this is the first report of the full NMR assignments.
The flavones 8 and 9 were isolated as yellow amorphous powders. Their molecular formulae were determined from the HRESIMS as C16H12O6 and C20H18O5, respectively. Their spectroscopic data (UV and NMR) were in agreement with reported values for chrysoeriol (8) (Toth et al., 1980; Agrawal, 1989) and 6-prenylapigenin (9) (Abegaz et al., 1998). This is the first report of their isolation from cannabis. The NMR spectra of 10 and 11 were identical with those of cannflavin A (Agrawal, 1989; Choi et al., 2004) and β-acetyl cannabispiranol (Shoyama and Nashioka, 1978), respectively.
The antimicrobial, antileishmanial, antimalarial and anti-oxidant activities of the isolated compounds were tested. Compound 1 displayed weak anti-MRSa (IC50 15.0 µg/mL), moderate antileishmanial (IC50 13.0 µg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 2.8 and 2.6 µg/mL, respectively. Compound 5had moderate antileishmanial activity (IC50 17.0 µg/mL). Compound 9showed moderate anti-MRSa (IC50 6.5 µg/mL), weak anticandidal (IC50 20.0 µg/mL) and mild antimalarial activity against P. falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 2.8 and 2.0 µg/mL, respectively. Compound 10exhibited strong antileishmanial activity (IC50 4.5 µg/mL). Compound 11 displayed weak antileishmanial activity (IC50 31.0 µg/mL).
Compounds 2, 5 and 11 displayed strong, 1, 7 and 10 moderate and 8 and 9 weak anti-oxidant activities in the DPPH assay, with 6 being inactive.
Compounds 2, 3 and 4 exhibited no antinociceptive action in both tail-flick and hot-plate assays up to 120 min following injection (Supplementary data).
1H NMR (400 MHz), 13C NMR (100 MHz), DEPT-135, APT and 2D–NMR spectra were recorded using the residual solvent signal as internal standard on a Varian AS 400. IR spectra were measured on a Bruker Tensor 27. UV spectra were obtained on a Varian Cary 50 Bio UV-Visible spectrophotometer. Optical rotation was measured on an Autoplot IV automatic polarimeter. High resolution mass spectra were measured using a Bruker BioApex. HPLC was performed on a Waters Delta Prep 4000 Preparative Chromatography System connected to a Waters 486 Tunable UV Absorbance detector using Phenomenex Luna C18 and Si columns (250 × 21.2 mm, 5 µm, 100 Å). GCMS analysis was carried out on a HP 6890 series GC, equipped with a split/splitless capillary injector, a HP 6890 series injector autosampler and an Agliant DB-5ms column (30 m × 0.25 mm × 0.25 µm), interfaced to a HP 5973 Mass Selective Detector (MSD). The injector temperature was 250°C and 1 µL injections were performed in the splitless mode, with the splitless time set at 60 s, the split flow set at 50 ml/min and the septum purge valve set to close 60 s after the injection occurred. The oven temperature was raised from 70 to 270°C (hold 20 min) at a rate of 5°C/min, for a total run time of 60 min; the transfer line temperature was 280°C.
3.2 Plant material
C. sativa plants were grown from high potency Mexican seeds. The seeds and plants were authenticated by Dr. Suman Chandra, The University of Mississippi, and a specimen (S1310V1) was deposited at the Coy Waller Complex, The University of Mississippi. Whole buds of mature female plants were harvested, air-dried, packed in barrels and stored at low temperature (−24°C).
3.3. Extraction, isolation and characterization
The plant material (9.0 kg) was sequentially extracted with hexanes (48 L), CH2Cl2 (40 L), EtOAc (40 L), EtOH (40 L), EtOH/H2O (36 L, 1:1) and H2O (40 L) at room temperature. The extracts were evaporated under reduced pressure at 40°C to afford hexanes (1.48 kg), CH2Cl2 (0.15 kg), EtOAc (0.13 kg), EtOH (0.09 kg), EtOH/H2O (0.77 kg) and H2O (0.54 kg) extracts for a total extract of 3.16 kg (35.1%, w/w). Portions of the CH2Cl2, EtOAc and EtOH extracts were combined (191.0 g) since they showed similar TLC profiles (EtOAc/n-hexane, 4:6) and were subjected to silica gel VLC, eluting with EtOAc/n-hexane [0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 75:25, 100:0 (2 L of each mixture)] followed by EtOH (4 L), yielding 9 fractions (A-I). Fraction B (10 g) was fractionated on a silica gel column (EtOAc/petroleum ether, 90:10) to give 16 subfractions (B1–16). Subfraction B1 (265 mg) was purified by Si HPLC (EtOAc/n-hexane, 5:95, 25 mL/min, UV 270 nm) to afford 1(12.3 mg, rt. = 4.0 min). Subfraction B3 (236.0 mg) was purified by Si-SPE column eluting with MeOH/CH2Cl2 (1:19) yielding 6(21.6 mg). Fraction D (14.3 g) was subjected to silica CC (EtOAc/petroleum ether, 5:95 to 20:80) followed by C18 flash chromatography (MeOH/H2O, 8:2) and C18 HPLC (MeCN/H2O, 50:50, 25 mL/min, UV 270 nm) to afford 9(5.0 mg, rt. = 3.8 min), 10(1.2 mg, rt. = 4.6 min), 7(15.0 mg, rt. = 6.7 min) and 2(11.6 mg, rt. = 8.7 min). Fraction F (28.5 g) was chromatographed on silica gel (EtOAc/n-hexane, 10:90 to 60:40), yielding 42 fractions (F1–42, 200 mL each). Fraction F32–35 (5.5 g) was subjected to silica gel CC (MeOH/CH2Cl2, 3:97) to yield 24 subfractions (SF1–24). SF2–3(210 mg) was chromatographed on Si-SPE column (EtOAc/n-hexane, 10:90) followed by C18 HPLC purification (MeOH/H2O, 85:15, 25 ml/min, UV 279 nm) to yield 3(2.3 mg, rt. = 6.3 min) and 4(3.9 mg, rt. = 8.8 min). SF3–6 (1.9 g) was purified by Sephadex LH-20 CC (MeOH) followed by C18-SPE purification (MeOH/H2O, 75:25) to give 5(12.9 mg), 8(284.5 mg) and 11 (15.0 mg).
3.3.1. 5-Acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1)
Orange amorphous powder; UV λmax (MeOH): 205, 270, 384 nm; IR vmax (neat): 1663 (C=O, ketone), 1780 (C=O, ester), 1610 (C=C) cm−1; 1H NMR (CDCl3, 400 MHz): δH 0.87 (3H, t, J = 6.4 Hz, H3-5′), 1.32 (4H, m, H2-3′ and H2-4′), 1.56 (3H, s, H3-8″), 1.57 (2H, m, H2-2′), 1.63 (3H, s, H3-9″), 1.69 (3H, s, H3-10″), 1.94 (2H, m, H2-4″), 2.05 (2H, m, H2-5″), 2.32 (3H, s, OCOCH3), 2.39 (2H, t, J = 7.6 Hz, H2-1′), 3.10 (2H, d, J = 7.2 Hz, H2-1″), 4.99 (1H, t, J = 7.2 Hz, H-2″), 5.02 (1H, t, J = 7.2 Hz, H-6″), 6.53 (1H, s, H-2); 13C NMR (CDCl3, 100 MHz): δC 187.1 (C-1), 132.7 (C-2), 148.3 (C-3), 180.8 (C-4), 149.1 (C-5), 135.6 (C-6), 28.9 (C-1′), 27.6 (C-2′), 31.6 (C-3′), 22.6 (C-4′), 14.1 (C-5′), 23.0 (C-1″), 118.4 (C-2″), 138.4 (C-3″), 39.8 (C-4″), 26.7 (C-5″), 124.1 (C-6″), 131.8 (C-7″), 17.9 (C-8″), 25.9 (C-9″), 16.4 (C-10″), 20.5 (OCOCH3), 168.1 (OCOCH3); HRESIMS m/z 373.2425 [M+H]+ (Calc. for C23H33O4, 373.2379).
3.3.2. 4,5-Dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2)
Brown amorphous powder; UV λmax (MeOH): 220, 267, 310 nm; IR vmax (neat): 3420 (OH), 1610, 1537, 1462 (benzene ring) cm−1; 1H and 13C NMR: Table 1; HRESIMS m/z325.1100 [M+Na]+ (Calc. for C17H18O5Na, 325.1052).
3.3.3. 4-Hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3)
Pale brownish amorphous powder; UV λmax (MeOH): 220, 267, 310 nm; IR vmax (neat): 3420 (OH), 1610, 1537, 1462 cm−1; 1H and 13C NMR: Table 1; HRESIMS m/z 339.1279 [M+Na]+(Calc. for C18H20O5Na, 339.12887).
3.3.4. 4,7-Dimethoxy-1,2,5-trihydroxyphenanthrene (4)
Reddish brown amorphous powder; UV λmax (MeOH): 258, 282, 303 nm; IR vmax (neat): 3413 (OH), 1610, 1533, 1462 cm−1; 1H and 13C NMR: Table 1; HRESIMS m/z 571.1630 [2M–H]− (Calc. for C32H27O10, 571.1604).
3.3.5. 8-Geranyl-5,7,4′-trihydroxy-3′-methoxyflavone (Cannflavin C) (5)
Yellow amorphous powder; UV λmax (MeOH): 275, 340, (+NaOMe) 280, 342, 401, (+NaOAc) 280, 340, (+AlCl3) 300, 346, 360, (+AlCl3+HCl) 300, 346, 360 nm; IR vmax (neat): 3421 (OH), 1662 (C=O) cm−1; 1H NMR (acetone-d6, 400 MHz): δH 1.48 (3H, s, H3-9″), 1.53 (3H, s, H3-8″), 1.82 (3H, s, H3-10″), 1.96 (2H, m, H2-4″), 2.05 (2H, m, H2-5″), 3.57 (2H, d, J = 6.8 Hz, H2-1″), 3.98 (3H, s, OMe-3′), 5.02 (1H, t, J = 6.8 Hz, H-6″), 5.35 (1H, t, J = 6.8 Hz, H-2″), 6.36 (1H, s, H-6), 6.66 (1H, s, H-3), 7.01 (1H, d, J = 8.0 Hz, H-5′), 7.58 (1H, d, J= 2.0 Hz, H-2′), 7.64 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 13.05 (1H, s, HO-5); 13C NMR (acetone-d6, 100 MHz): δC 164.1 (C-2), 103.3 (C-3), 182.7 (C-4), 160.5 (C-5), 98.9 (C-6), 162.4 (C-7), 106.5 (C-8), 155.4 (C-9), 104.5 (C-10), 124.4 (C-1′), 109.7 (C-2′), 148.3 (C-3′), 151.1 (C-4′), 115.8 (C-5′), 120.7 (C-6′), 21.7 (C-1″), 123.0 (C-2″), 135.1 (C-3″), 39.7 (C-4″), 26.7 (C-5″), 124.4 (C-6″), 131.0 (C-7″), 17.9 (C-8″), 25.1 (C-9″), 16.0 (C-10″), 55.8 (OMe-3′); HRESIMS m/z 459.1766 [M+Na]+ (Calc. for C26H28O6Na, 459.1784).
3.3.6. β-Sitosteryl-3-O-β-D-glucopyranoside-2′-O-palmitate (6)
White amorphous powder; [α]D: + 91.3 (c 0.05, CHCl3); IR vmax (neat): 1736 (OH), 1610 cm−1; 1H NMR (CDCl3, 400 MHz): δH 0.63 (9H, m, H3-26, 27, 29), 0.75 (3H, s, H3–18), 0.83 (3H, t, J = 7.2 Hz, H3–16″), 0.90 (3H, d, 6.8 Hz, H3-21), 0.99 (3H, s, H3-19), 1.24 (18H, bs, H2-5″ to H2-13″), 1.28–1.30 (4H, m, H2-4″, 15″), 2.34 (2H, m, H2-2″), 3.49 (1H, m, H-3), 3.35–3.82 (4H, H-2′ to H-5′), 4.33 (1H, d, J = 7.6 Hz, H-1′), 4.1–4.2 (2H, m, H2-6′) 5.33 (1H, bs, H-6); 13C NMR (CDCl3, 100 MHz): δC 37.6 (C-1), 29.6 (C-2), 80.1 (C-3), 39.2 (C-4), 140.6 (C-5), 122.2 (C-6), 32.2 (C-7), 32.1 (C-8), 50.3 (C-9), 36.9 (C-10), 21.3 (C-11), 40.0 (C-12), 42.5 (C-13), 57.0 (C-14), 24.5 (C-15), 28.3 (C-16), 56.4 (C-17), 12.1 (C-18), 19.6 (C-19), 36.4 (C-20), 19.0 (C-21), 34.2 (C-22), 26.4 (C-23), 46.0 (C-24), 29.4 (C-25), 19.2 (C-26), 20.0 (C-27), 23.3 (C-28), 12.2 (C-29), 101.6 (C-1′), 73.5 (C-2′), 76.6 (C-3′), 70.8 (C-4′), 73.8 (C-5′), 64.1 (C-6′), 174.6 (C-1″), 34.5 (C-2″), 25.2 (C-3″), 29.6 (C-4″ to C-13″), 30.0 (C-14″), 22.9 (C-15″), 14.3 (C-16″); HRESIMS m/z 837.6621 [M+Na]+ (calc. for C51H90O7Na, 837.6585).
3.3.7. α-Cannabispiranol (7)
Colorless prisms (MeOH/H2O); mp 182°C; UV λmax(MeOH): 210, 222 nm; IR vmax (neat): 3410, 3180 (OH), 1610, 1596 cm−1; 1H NMR (C5D5N, 400 MHz): δH 1.75 (4H, m, H2-2′, 6′), 2.12 (2H, t, J = 7.2 Hz, H2-2), 2.25 (4H, m, H2-3′, 5′), 2.90 (2H, t, J = 7.2 Hz, H2-3), 3.70 (3H, s, OMe-5), 4.10 (1H, m, H-4′), 6.52 (1H, bs, H-4), 6.67 (1H, bs, H-6); 13C NMR (C5D5N, 100 MHz): δC 49.2 (C-1), 36.4 (C-2), 31.9 (C-3), 102.0 (C-4), 161.1 (C-5), 101.3 (C-6), 156.7 (C-7), 129.9 (C-8), 146.9 (C-9), 34.6 (C-2′, 6′), 34.3 (C-3′, 5′), 70.8 (C-4′), 55.6 (OMe-5); HRESIMS m/z 249.2393 [M+H]+ (Calc. for C15H21O3, 249.2379); GCMS m/z (rel. int.): 248 (M+, 33%), 230 (13%), 215 (12%), 201 (12%), 189 (100%), 176 (65%), 161 (18%).
3.4. Acid hydrolysis of 6
Compound 6 (5 mg) was refluxed with 3% H2SO4 in MeOH (4 mL) for 8 hours. The reaction mixture was neutralized with Na2CO3 and extracted with EtOAc (3 × 10 mL). TLC comparison of the aqueous layer with authentic sugar samples (CH2Cl2/MeOH/H2O, 6:4:1; n-BuOH/AcOH/H2O, 5:5:1), identified the glycone as β-D-glucose.
3.5. Alkaline hydrolysis of 6 and identification of the fatty acid
Compound 6 (5 mg) was added to 10% KOH in MeOH (10 mL) and H2O (5 mL), and after refluxing for 30 minutes, H2O (10 mL) was added, followed by extraction with CHCl3 (3 × 10 mL). The aqueous layer was acidified with dil. HCl to pH 5 and extracted with CHCl3 (3 × 10 mL). The CHCl3 layer was dried (Na2SO4), filtered and concentrated in vacuo. The filtrate was dissolved in dry ether and MeOH (4 drops), followed by methylation with trimethylsilyl-diazomethane (200 µL). The reaction mixture was left open at room temperature for 30 minutes, concentrated under N2 and analyzed by GCMS. The fatty acid methyl ester was identified as methyl palmitate via a library search (NIST).
3.6. Antimicrobial, antileishmanial and antimalarial bioassay
The isolated compounds were evaluated for antimicrobial (Candida albicans ATCC 90028, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Mycobacterium intracellulare ATCC 23068, Aspergillus fumigat ATCC 90906, Methicillin Resistant Staphylococcus aureus ATCC 43300) (Bharate et al., 2007; Babu et al., 2006), antileishmanial (Radwan et al., 2008) and antimalarial activity [P. falciparum (D6 clone) and P. falciparum (W2 clone)] (Bharate et al., 2007).
3.7. Anti-oxidant activity
A TLC autographic assay for DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging effect was used to determine anti-oxidant activity (Takamatsu et al., 2003). The isolated compounds were dissolved in DMF (2 mg/mL) and applied in the form of a spot (4 µl, 4–5 mm in diameter) on silica gel GF plates. The residual DMF was removed under vacuum (15–20 min). A similar amount of vitamin E in DMF was used as positive anti-oxidant control. The radical-scavenging effects of the compounds were detected on the TLC plate using DPPH spray reagent (0.2% w/v in MeOH). The plate was observed 30 min after spraying. Active compounds are observed as yellow spots against a purple background. Relative radical-scavenging activity was assigned as “strong” (compounds that produce an intense bright yellow zone), “medium” (compounds that produce a clear yellow spot), “weak” (compounds that produce a weakly visible yellow spot), or “not active” (compounds that produce no sign of any yellow spot). Vitamin E produced an intense bright yellow zone.
The project described was supported in part by Grant Number 5P20RR021929-02 from the National Center for Research Resources and in part by the National Institute on Drug Abuse, contract # N01DA-5-7746. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. We are grateful to Dr. Bharathi Avula for assistance with the HRESIMS, and to Dr. Melissa Jacob, Ms. Marsha Wright and Dr. Babu Tekwani for conducting the antimicrobial and antiprotozoal testing.
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