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The Bibenzyl Canniprene Inhibits the Production of ProInflammatory Eicosanoids and Selectively Accumulates in Some Cannabis sativa Strains Gianna Allegrone,† Federica Pollastro,† Gianmaria Magagnini,‡ Orazio Taglialatela-Scafati,§ Julia Seegers,⊥ Andreas Koeberle,⊥ Oliver Werz,*,⊥ and Giovanni Appendino*,† †
Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy Consiglio per le Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali e l’Analisi dell’Economia Agraria, CRA-CIN, Viale G. Amendola 82, 45100 Rovigo, Italy § Dipartimento di Farmacia, Università di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy ⊥ Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany ‡
S Supporting Information *
ABSTRACT: Canniprene (1), an isoprenylated bibenzyl unique to Cannabis sativa, can be vaporized and therefore potentially inhaled from marijuana. Canniprene (1) potently inhibited the production of inflammatory eicosanoids via the 5-lipoxygenase pathway (IC50 0.4 μM) and also affected the generation of prostaglandins via the cyclooxygenase/microsomal prostaglandin E2 synthase pathway (IC50 10 μM), while the related spiranoid bibenzyls cannabispiranol (2) and cannabispirenone (3) were almost inactive in these bioassays. The concentration of canniprene (1) was investigated in the leaves of 160 strains of C. sativa, showing wide variations, from traces to >0.2%, but no correlation was found between its accumulation and a specific phytocannabinoid profile.
he isoprenylated bibenzyl canniprene (1) was first isolated by Crombie and Crombie in 1978 from a Thai sample of a high-THC (Δ9-tetrahydrocannabinol) strain of Cannabis sativa L. (Cannabaceae),1 and this pioneer group of cannabis phytochemistry also reported its synthesis two years later.2 Canniprene was also isolated from another THC strain of cannabis originating from Panama,3 as well as from other narcotic samples of the plant (marijuana).4 Despite a wealth of phytochemical studies on C. sativa,5 the isolation of canniprene (1) has not yet been reported from fiber hemp, raising the possibility that its occurrence might be of taxonomic relevance. To evaluate this hypothesis, we have screened a series of C. sativa varieties for their content of this compound. Since 1 can be vaporized and therefore inhaled from smoke,4,6 its bioactivity was also evaluated in comparison to two of the other C. sativa phenolic constituents. Canniprene (1) was isolated in the present work as the major phenolic from a narcotic strain of C. sativa (marijuana) in ca. 0.076% yield, along with the cannabispiranoids 2 (cannabispiranol, 0.0091%) and 3 (cannabispirenone, 0.045%). The proton and carbon NMR spectra of canniprene were fully assigned by using 2D NMR spectroscopy (see the Experimental Section), confirming the isovanillyl methylation pattern of the prenylated phenyl moiety. Canniprene (1) gives a cherry color on TLC
T
© 2017 American Chemical Society and American Society of Pharmacognosy
upon heating with acids (see the graphical abstract). Interestingly, this color is fade-resistant and remains visible on the TLC plate after prolonged (months) storing. Given the sensitivity of this color reaction, a simple TLC analysis7 was used to screen a large collection of C. sativa varieties, mostly of Special Issue: Special Issue in Honor of Phil Crews Received: December 7, 2016 Published: February 6, 2017 731
DOI: 10.1021/acs.jnatprod.6b01126 J. Nat. Prod. 2017, 80, 731−734
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E2 synthase (mPGES) pathways,8 so the activity of canniprene (1) was evaluated against these two end-points. Inhibition of 5-LO by canniprene (1) and the cannabispiranoids 2 and 3 was assessed in a cell-free assay using isolated human recombinant 5-LO as enzyme source. The 5-LO reference inhibitor zileuton suppressed 5-LO product formation (IC50 0.8 ± 0.4 μM) as expected (Figure S3A). In this assay, canniprene (1) outperformed zileuton and caused potent and concentration-dependent inhibition of 5-LO activity with an IC50 value of 0.4 ± 0.2 μM (Figure S3B). Also cannabispirenone 3 was active against 5-LO but only at 20fold higher concentrations (IC50 9.3 ± 1.6 μM) as compared to 1 (Figure S3C). In contrast, the cannabispiranol 2 failed to inhibit 5-LO up to 10 μM (Table S1). To assess inhibition of mPGES-1, canniprene (1) and the cannabispiranoids 2 and 3 were tested for inhibition of mPGES-1 activity (conversion of PGH2 to PGE2) in microsomal preparations of interleukin-1βstimulated A549 cells. The reference mPGES-1 inhibitor MK886 blocked PGE2 formation in this assay with an IC50 of 2.7 ± 0.9 μM (Figure S4A). Canniprene (1) moderately suppressed mPGES-1 activity in a concentration-dependent manner at 10 μM by half, while compounds 2 and 3 up to 10 μM failed to inhibit mPGES-1 activity (Table S1). Compared to cannflavin A (4), the most potent cannflavin,8 canniprene (1) is superior at inhibiting 5-LO (EC50 0.4 ± 0.2 μM (canniprene 1) vs 1.3 ± 0.5 μM (cannflavin A 4); Figure S3D), but is less effective for the inhibition of mPGES-1 (EC50 ca. 10 μM (canniprene 1) vs 1.8 ± 0.1 μM (cannflavin A 4); Figure S4B).8 The contents of cannflavins and canniprene were also comparatively evaluated in selected C. sativa samples (Table 2),
the non-narcotic type, available from a breeding center. It was observed that the red spot of canniprene (1) was clearly visible only in 18 samples (11% of those investigated), which were then selected for quantitation by HPLC-MS. As suggested by the size and intensity of the color on TLC, the concentration of 1 was found to significantly differ within these samples, from 4 to 2085 μg/g of dried plant material (Table 1). Remarkably, Table 1. Concentration of Canniprene (1) in Selected Strains of Cannabis sativaa strain
concentration (μg/g)
MH-WU-112 CYM273 Chameleon Fibrol MH-AGM-705 CYM49 A11-121-11 Carmaleonte Katlakalna A11-121-15 A11-121-5 Eletta Campana Red Petiole Carmagnola Carmaleon (basal leaves) Carmaleon (apical leaves) THC-3 Carma Ermo
2085 472 418 388 380 294 199 199 113 106 101 76 73 44 40 31 20 10 4
a
Mean of three replicates. With the exception of THC-3, all samples are of fiber hemp and classified as cannabidiol (CBD)-chemotypes except carma, which is a cannabigerol (CBG)-chemotype. All samples from leaves collected before flowering.
Table 2. Concentrations of Canniprene (1) and Cannflavins (4, 5) in Selected Samples of Cannabis sativa L.a
most samples were of the cannabidiol (CBD)-chemotype, showing that the biosynthesis of canniprene (1) and that of cannabinoids are orthogonal and unrelated, since 1 could be detected not only in marijuana but also in fiber hemp. The reasons for the huge variation in the concentration of canniprene are unclear, but apparently unrelated to the development of the plant, since a canniprene-containing variety (Carmaleon, Table 1) was also screened for the accumulation of this compound at various stages of maturation, without finding any significant change during the flowering and the seed-maturation stages (data not shown). In this strain, no significant difference was observed between basal and apical leaves (Table 1). Taken together, these data suggest that no relationship exists between a specific cannabinoid profile, or plant age, and the accumulation of canniprene (1) and that this compound does not qualify as a surrogate marker for the presence of narcotic phytocannabinoids in cannabis samples. Unlike other phenolics typical of C. sativa such as cannflavins (4, 5), canniprene (1) can be analyzed by GC without any previous derivatization owing to the increased lipophilicity associated with its C-prenylation and O-methylation, as well as to its overall low oxygenation.4,6 When marijuana is smoked, canniprene (1) can therefore be inhaled along with the decarboxylated phytocannabinoids. The cannflavin C. sativa phenolics are potent inhibitors of the production of inflammatory eicosanoids generated via the 5-lipoxygenase (5LO) and the cyclooxygenase (COX)/microsomal prostaglandin
strain
canniprene (1) (μg/g)
cannflavin A (4) (μg/g)
cannflavin B (5) (μg/g)
Ermo Carma Carmagnola THC-3 MH-WU-112
4 10 44 20 2085
280 109 48 29 21
106 56 30 9 15
a
Mean of three replicates. With the exception of THC-3, all samples were of fiber hemp and classified as CBD chemotypes (carmagnola and MH-WU-112) or CBG chemotype (carma). All samples from leaves collected before flowering.
finding an overall inverse association between their concentration, a result that suggests competition between polyketide aromatization processes based on Claisen condensation (flavonoids) and aldol condensation (stilbenoids). C. sativa is a prolific producer of stilbenoids derived from dimethyldihydroresveratrol (6) by intramolecular phenol coupling, a process that generates spiro-derivatives (cannabispiranoids) by para−para coupling and next phenanthrenes from their rearrangement (cannithrenoids, formally the products of a para−meta coupling) (Scheme 1).9,10 Conversely, canniprene (1) is the result of hydroxylation and prenylation of the basic chemotype and is the only phenolic that has so far been reported to significantly (>0.1%) accumulate in C. sativa strains. Owing to its lipophilicity, canniprene (1) can be volatilized and is expected, therefore, to occur in marijuana smoke,4,6 but we have shown that the accumulation of canniprene is not a 732
DOI: 10.1021/acs.jnatprod.6b01126 J. Nat. Prod. 2017, 80, 731−734
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Scheme 1. Biogenetic Relationship between C. sativa Stilbenoidsa
of hemp used in this study within the framework of the Authorization SP/212 16/12/2015 of the Ministry of Health, Rome, Italy. Voucher samples of all cannabis strains analyzed are maintained at the Novara laboratories and, except for the narcotic strains (Δ9-THC > 0.1%), may be obtained from G.A. Extraction and Isolation. Flowerheads and leaves of a high-THC (ca. 10%) strain of C. sativa (THC line PP, 50 g) were extracted with acetone (3 × 500 mL). The pooled extracts were evaporated, and the oily black residue (18 g) obtained in this way was dissolved in methanol (300 mL) and cooled at 4 °C overnight to remove fats and waxes. After filtration over a bed of Celite, the filtrate was evaporated, and the residue (8.9 g) was heated for 2 h on an oil bath at 120 °C to decarboxylate the acidic cannabinoids. The decarboxylated extract was then purified by gravity column chromatography on silica gel (60 g). After the elution of THC with petroleum ether−EtOAc (9:1), the column was washed with dichloromethane to recover medium-polarity phenolics (230 mg). Final purification of canniprene (1) was achieved by preparative HPLC on silica gel, affording 38 mg of canniprene as a yellowish powder (0.076%): mp 105−107 °C (lit. 112−113 °C);1 1H NMR (CDCl3, 400 MHz) δH 6.68 (1H, d, J = 7.2 Hz, H-5), 6.67 (1H, d, J = 7.2 Hz, H-6), 6.35 (1H, br s, H-2′), 6.28 (1H, br s, H-4′), 6.26 (1H, br s, H-6′), 5.71 (s, OH-3), 5.14 (br d, J = 6.0 Hz, H-2″), 4.78 (s, OH-5′), 3.86 (3H, s, OMe-4), 3.77 (3H, s, OMe-3′), 3.40 (2H, d, J = 6.0 Hz, H-1″), 2.83 (2H, br t, J = 6.8 Hz, H-7), 2.75 (2H, br t, J = 6.8 Hz, H-8), 1.78 (3H, br s, H-5″), 1.69 (3H, br s, H-4″); 13C NMR (CDCl3, 125 MHz) δC 161.2 (s, C-3′), 156.8 (s, C-5′), 145.0 (s, C-1′), 145.1 (s, C-4), 143.8 (s, C-3), 133.6 (s, C-6), 131.9 (s, C-3″), 126.3 (s, C-1), 123.5 (d, C-2″), 120.1 (d, C-6), 108.8 (d, C-5), 108.2 (d, C-6′), 107.3 (d, C-2′), 99.6 (d, C-4′), 56.4 (q, OMe-4), 55.7 (q, OMe-3′), 38.3 (t, C-8), 34.7 (t, C-7), 26.0 (q, C-4″), 25.5 (t, C-1″), 18.4 (q, C5″); (+) ESIMS m/z 343 [M + H]+, 365 [M + Na]+. Screening of Canniprene (1) by TLC. A ca. 0.5 g sample of plant material (from a powdered sample of ca. 5 g of the strain investigated) was shaken in a vial with 2.5 mL of acetone, and the extract was directly streaked on a TLC plate eluted with hexane−EtOAc (2:3). The chromatogram was then revealed by dipping into 5% H2SO4 in ethanol and heating. Canniprene (1) appeared as a cherry-red spot at Rf ca. 0.20, while cannabidiol and the other cannabinoids appeared as brown spots at Rf values of 0.40−0.45. The test could be done on both decarboxylated and nondecarboxylated samples, but the presence of this compound was more evident in decarboxylated samples, since acidic cannabinoids and 1 show similar chromatographic behavior under the conditions of analysis, and their brown color can hinder the detection of 1. Quantitation of Canniprene (1) by HPLC-MS. Dried and powdered flowerheads and leaves of selected C. sativa strains (1.0 g) were extracted with 30 mL of methanol under magnetic stirring. After 40 min the solution was filtered, and the plant residue was re-extracted two times with methanol. The pooled extracts were evaporated, and the residue was taken up in methanol (5 mL). The reconstituted solution was filtered through a 0.22 μm syringe membrane filter and further diluted with methanol prior to HPLC-MS analysis. A Surveyor HPLC on line with an LCQ DECA XP Plus (Thermo* Finnigan, San Jose, CA, USA) ion trap mass spectrometer equipped with an ESI source was employed. Separations were performed on a Luna 5 μm C18(2), 150 mm × 2.0 mm analytical column (Phenomenex, Torrance, CA, USA) protected with a C18-Security Guard cartridge, 4 mm × 2.0 mm (Phenomenex). The injection volume was 5 μL. The mobile phase components were A = 0.2% formic acid and B = acetonitrile, and canniprene (1) was eluted according to the following linear gradient: A:B (45:55) for 7 min, then A:B (2:98) over 12 min at a flow rate of 0.4 mL min−1. The ESIMS and ESIMS/MS spectra of canniprene (1) were recorded using direct infusion of the reference compound. Data were acquired in the positive MS total ion scan mode (mass scan range: m/z 100−1000) and MS/MS product ion scan mode; the normalized collision energy (%) was optimized for the precursor ion selected: m/z 343, 26%; m/z LC-ESIMS/MS in single reaction monitoring modality was applied to the selected precursor ion, following the conditions set during the infusion analysis. The parent/daughter ion transitions m/z 343 → 311 and m/z 343 → 287
a
Cannabispiranoids are exemplified by cannabispiranol (2), and cannithrenoids by cannithrene-1 (7): (a) Hydroxylation and Cisoprenylation; (b) para−para phenol coupling; (c) rearrangement and aromatization.
feature of narcotic C. sativa, since this compound can also be present in high concentration in fiber hemp. The basis for the diversity of its accumulation is unclear and might be an interesting topic to investigate in the light of the recent cloning of the C. sativa genome.11 On the other hand, the remarkable potential anti-inflammatory activity of canniprene as a dual inhibitor of 5-LO and mPGES-1 adds to the growing evidence that C. sativa is a source of unique noncannabinoid phenolics, of which the biomedical relevance still awaits a systematic investigation.10
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were taken in open glass capillaries on a Büchi SMP20 apparatus and are uncorrected. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a Varian INOVA NMR spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26, δC = 77.0). Homonuclear 1H connectivities were determined by a COSY experiment. One-bond heteronuclear 1H−13C connectivities were determined with an HSQC experiment. Two- and three-bond 1 H−13C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3J = 9 Hz. ESIMS spectra were obtained on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. Silica gel 60 (70−230 mesh) and silica gel RP-18 for gravity column chromatography were purchased from Macherey-Nagel (Düren, Gerrmay). Aluminum-coated Merck 60 F254 (0.25 mm) plates were used for TLC, visualizing the spots by UV inspection and/or staining with 5% H2SO4 in ethanol and heating. Preparative HPLC was carried out on a Beckmann apparatus equipped with a UV detector. Plant Material. Narcotic cannabis came from in-house cultivations at CRA-CIN, Rovigo, Italy, which also provided all the other samples 733
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ORCID
were selected for the identification and quantification of canniprene (1). The calibration curve, obtained by injecting six different concentration levels of canniprene standard solutions and analyzed in triplicate, showed a good linearity in the range of 25−2000 μg/L. The limit of detection was determined by the analysis of sample of known concentration and found to be 8 μg/L, and the limit of quantification was found to be 25 μg/L. All samples were analyzed in triplicate according to this method, and the concentration of canniprene was calculated by means of the external calibration curve. Expression and Purification of Human Recombinant 5-LO. E. coli MV1190 was transformed with pT3-5-LO plasmid, and recombinant 5-LO protein was expressed at 27 °C, as described previously.12 Cells were lysed in 50 mM triethanolamine/HCl pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 μg/mL), 1 mM phenylmethanesulfonyl fluoride, and lysozyme (500 μg/mL), then homogenized by sonication (3 × 15 s) and centrifuged at 40000g for 20 min at 4 °C. The 40000g supernatant (S40) was applied to an ATPagarose column to partially purify 5-LO, as described previously.12 Semipurified 5-LO was immediately used for activity assays. Determination of 5-LO Activity in Cell-Free Assay. Aliquots of semipurified 5-LO were diluted with ice-cold PBS containing 1 mM EDTA, and 1 mM ATP was added. Samples were preincubated with the test compounds as indicated. After 10 min at 4 °C, samples were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 plus 20 μM AA was added to start 5-LO product formation. The reaction was stopped after 10 min at 37 °C by addition of 1 mL of ice-cold methanol, and the formed metabolites formed were analyzed by RP-HPLC, as described previously.12 5-LO products include the all-trans isomers of LTB4 and 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid. Preparation of Crude mPGES-1 in Microsomes of A549 Cells and Determination of PGE2 Synthase Activity. Preparation of A549 cells (kindly provided by Dr. Thorsten J. Maier, GoetheUniversity, Frankfurt; originally obtained from ATCC) and determination of mPGES-1 activity were performed as described previously.12 In brief, cells were treated with 1 ng/mL Il-1β for 48 h at 37 °C, 5% CO2. Cells were harvested and sonicated, and the homogenate was subjected to differential centrifugation at 10000g for 10 min and 174000g for 1 h at 4 °C. The pellet (microsomal fraction) was resuspended in 1 mL of homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, 1 mM phenylmethanesulfonyl fluoride, 60 μg/mL soybean trypsin inhibitor, 1 μg/mL leupeptin, 2.5 mM glutathione, and 250 mM sucrose), and the total protein concentration was determined. Microsomal membranes were diluted in potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione. Test compounds or vehicle was added, and after 15 min at 4 °C, the reaction (100 μL total volume) was initiated by addition of PGH2 at the indicated concentration. After 1 min at 4 °C, the reaction was terminated using stop solution (100 μL; 40 mM FeCl2, 80 mM citric acid, and 10 μM 11β-PGE2) as internal standard. PGE2 was separated by solid-phase extraction and analyzed by RP-HPLC, as described previously.13 Statistics. IC50 values were graphically calculated by linear interpolation between data points above and below 50% activity using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA).
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Giovanni Appendino: 0000-0002-4170-9919 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS NMR spectra were recorded at “Centro di Servizio Interdipartimentale di Analisi Strumentale”, Università di Napoli Federico II. We are grateful to G. Grassi (CRA-CIN, Rovigo) for his help throughout this work. F.P. and G.A. acknowledge funding from Bando Ricerca Locale 2015, Università del Piemonte Orientale.
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DEDICATION Dedicated to Professor Phil Crews, of the University of California, Santa Cruz, for his pioneering work on bioactive natural products.
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REFERENCES
(1) Crombie, L.; Crombie, W. M. L. Tetrahedron Lett. 1978, 19, 4711−4714. (2) Crombie, L.; Crombie, W. M. L.; Jamieson, S. V. Tetrahedron Lett. 1980, 21, 3607−3610. (3) Elsohly, H. N.; Turner, C. E. Bull. Narc. 1982, 34, 51−56. (4) Stephanou, E.; Lawi-Berger, C.; Kapetanidis, I. Pharm. Acta Helv. 1984, 59, 216−224. (5) Hanuš, L.; Meyer, S. M.; Muñoz, E.; Taglialatela-Scafati, O.; Appendino, G. Nat. Prod. Rep. 2016, 33, 1357−1392. (6) Brenneisen, R.; Elsohly, M. A. J. Forensic Sci. 1988, 33, 1385− 1404. (7) Cappelletti, E. M.; Caniato, R.; Appendino, G. Biochem. Syst. Ecol. 1986, 14, 183−190. (8) Werz, O.; Seegers, J.; Schaible, A. M.; Weinigel, C.; Barz, D.; Koeberle, A.; Allegrone, G.; Pollastro, F.; Zampieri, L.; Grassi, G.; Appendino, G. PharmaNutrition 2014, 2, 53−60. (9) El-Feraly, F. S. J. Nat. Prod. 1984, 47, 89−92. (10) Pollastro, F.; Minassi, A.; Fresu, L. G. Curr. Med. Chem. 2017, in press. (11) The genome of the fiber hemp variety Finola and the marijuana strain Purple Kush were cloned in 2011. See: http://genome.ccbr. utoronto.ca/ (accessed on January 18, 2017). (12) Fischer, L.; Szellad, D.; Radmark, O.; Steinhilber, D.; Werz, O. FASEB J. 2003, 17, 949−951. (13) Koeberle, A.; Siemoneit, U.; Buehring, U.; Northoff, H.; Laufer, S.; Albrecht, W.; Werz, O. J. Pharmacol. Exp. Ther. 2008, 326, 975− 982.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01126. Additional information (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49-3641-949801. Fax: +49-3641-949802. E-mail: oliver.
[email protected] (O. Werz). *Tel: +39-0321-375744. Fax: +39-0321-375744. E-mail:
[email protected] (G. Appendino). 734
DOI: 10.1021/acs.jnatprod.6b01126 J. Nat. Prod. 2017, 80, 731−734
