Pharmanutrition Cannflavins from Hemp Sprouts, a Novel Cannabinoid-Free Hemp Food Product, Target Microsomal Prostaglandin E

PharmaNutrition 2 (2014) 53–60

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PharmaNutrition

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h a n u

Cannﬂavins from hemp sprouts, a novel cannabinoid-free hemp food product, target microsomal prostaglandin E 2 synthase-1 and 5-lipoxygenase

Oliver Werz a,*, Julia Seegers b, Anja Maria Schaible a, Christina Weinigel a, Dagmar Barz c, Andreas Koeberle a,

Gianna Allegrone d, Federica Pollastro d, Lorenzo Zampieri d, Gianpaolo Grassi e, Giovanni Appendino d,* aDepartment of Pharmaceutical / Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany bDepartment for Pharmaceutical Analytics, Pharmaceutical Institute, University of T ubingen,¨ Auf der Morgenstelle 8, D-72076 Tuebingen, Germany cInstitute of Transfusion Medicine, Jena University Hospital, 07743 Jena, Germany dDipartimento di Scienze del Farmaco, Universit a` del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy eConsiglio per le Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali, CRA, CIN, Viale G. Amendola 82, 45100 Rovigo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Hemp seeds are of great nutritional value, containing all essential amino acids and fatty acids in sufﬁcient Received 8 April 2014 amount and ratio to meet the dietary human demand. Hemp seeds do not contain cannabinoids, and because

Received in revised form 12 May 2014 of their high contents of ω -3 fatty acids, are enjoying a growing popularity as a super-food to beneficially Accepted 12 May 2014 affect chronic inflammation. Seeds also lack the typical phenolics of hemp leaves and inflorescences, but we found that sprouting, while not triggering the production of cannabinoids, could nevertheless induce the Keywords:

Hemp production of the anti-inflammatory prenylflavonoids cannflavins A and B. This effect was especially marked

Plant-derived food in Ermo, a cannabinoid-free variety of Cannabis sativa L. Microsomal prostaglandin E2 synthase (mPGES-1) Cannflavins and 5-lipoxygenase (5-LO) were identified as the molecular targets of cannflavins A and B, solving an almost Inflammation three-decade old uncertainty on the mechanism of their the anti-inflammatory activity. No change on the

Microsomal prostaglandin E2 synthase fatty acid profile was observed during sprouting, and the presence of lipophilic flavonoids combines with the 1 high concentration of ω -3 essential acids to qualify sprouts from Ermo as a novel anti-inflammatory hemp

1. Introduction contents that would perform very poorly as sources of drug material [ 1 ]. Currently, hemp seeds are mostly used as staple in certain The nutritional virtues of hemp seeds can hardly be underesti- animals, and especially for some birds too, but hemp seed products mated, since, virtually unique between food plants, they contain all (whole or dehulled hemp grains, hemp oil, hemp flour, hemp protein essential amino acids and fatty acids in sufficient amount and ratio powder, hemp substitute milk, hemp tofu, hemp butter) are enjoying to meet the human demand [ 1 ]. Unsurprisingly, hemp was one of the a growing popularity in human nutrition, where they have attained first “grain” plants domesticated by man [ 2 ], and, starting from the na- a super-food status because of a combination of healthy fatty acids tive Central Asia, hemp cultivation spread all over the world because composition, high contents of proteins (20–25%) and vitamins, and, of its multipurpose use as a source of nutrition and fiber as well as a last but not least, they have a pleasant nutty taste also [ 3 ]. Hemp medicinal / ritual plant [ 2 ]. Per se , hemp seeds do not contain cannabi- seeds contain up to 35% of oil, with an 80% concentration of polyun- noids, but, just like the seeds from poppy or peppers, their surface can saturated acids, and a ca. 3:1 ratio between linoleic (LA, ω -6)- and be contaminated by compounds present in the plant matrix in which α-linolenic (ALA, ω -3) acids [ 3 ]. Hemp oil is one of the very few edi- they are hosted, eventually becoming a source of trace amounts of ble oils that contains significant amounts (up to 7%) of γ-linolenic acid them (alkaloids for poppy seeds, capsaicinoids for hot pepper seeds, (GLA) and stearidonic acid (SDA, 18:4n3) (up to 1.5%). GLA and SDA cannabinoids for hemp seeds) [ 1 ]. Because of regulatory limits on the attenuate the formation of inflammatory prostanoids from arachi- contents of psychoactive cannabinoids in hemp products, hemp seeds donic acid [ 4 ], overall making hemp oil unique in terms of capacity to are produced from strains bred for low tetrahydrocannabinol (THC) increase tissue concentration of n -3 polyunsaturated fatty acids and to decrease the production of inflammatory eicosanoids. Because of the anti-inflammatory profile of their fatty acid composition, hemp seed food products could therefore beneficially affect pathological * Corresponding authors.

E-mail addresses: [email protected] (O. Werz) conditions characterized by chronic inﬂammation, as suggested, for [email protected] (G. Appendino).

2213-4344/ $ - see front matter c 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phanu.2014.05.001 54 O. Werz et al. / PharmaNutrition 2 (2014) 53–60 instance, by a clinical study on atopic dermatitis [ 5 ], and various anec- 2.3. Production of sprouts dotal reports [ 5 ]. By decreasing platelet aggregation, hemp seed oil could also reduce cardiovascular risk [ 6 ]. Seeds (50 g) were washed in running water, sterilized with a short In the context of hemp seed food, little attention has been given (5 min) treatment with hydrogen peroxide, and then rinsed with to sprouts, despite their possibility to expand the nutritional use of running water. Germination was carried out in a VitaSeed domestic hemp not only in culinary terms, but also phytochemically, exploit- equipment for 5 days under dark, with changing the water every day. ing the expression of new metabolic pathways induced by germina- The sprouted seeds were washed, and, after removing their tegument tion. Germination-associated changes in the phytochemical profile of manually, were dried in an oven at 50 ◦C for 2 days. seeds are well documented [ 7 ], and have spurred the commercial development of sprouts enriched in specific phytochemicals, as shown 2.4. Isolation of cannflavin A from C. sativa var. Ermo by sulforaphane-rich broccoli sprouts [ 8 ]. Quantitative changes in the amount of specific phytochemicals have also been reported, as well Powdered dried plant material (900 g) was twice extracted with as a general remodeling of the whole profile of specific secondary acetone (ratio plant–solvent 1:9), to afford, after evaporation 32 g metabolites. Thus, the triterpenoid profile of pumpkin seeds is com- (3.5%) of a dark oil, that was dissolved in the minimal amount of pletely changed by sprouting, with the replacement of multiflorane methanol and adsorbed on a bed of RP18 silica gel (160 g). Vacuum- p -aminobenzoate triterpenoid esters with bryonolic acid [ 9 ], a potent filtration with methanol afforded a de-pigmented and de-waxed frac- triterpene inducer of anti-inflammatory and antioxidant genes [ 10 ]. tion (12 g) as a brownish gum, next purified by gravity column chro- Since the production of cannabinoid is associated with the presence matography on silica gel using petroleum ether–EtOAc as eluent (from of specific glandular hairs typical of foliar and floral hemp tissues [ 11 ], 7:3 to 5:5). A first crop of CFA( 1 ) (80 mg) was obtained from the more sprouting was unlikely to induce their synthesis or accumulation. On polar fractions of the column. The mother liquors (880 mg) were fur- the other hand, hemp is a prolific producer of structurally unique ther purified by flash-chromatography on silica gel, using petroleum phenolics (cannflavins, cannabispiranes, canniprenes), present in the ether–EtOAc 6:4 as eluent to afford a fraction that crystallized to pro- leaves and flowers, but absent in the seeds [ 12 ]. Since beneficial prop- vide 15 further mg of CFA( 1 ). The mother liquors (280 mg) contained erties have been associated to these compounds [ 13 ], the induction additional amounts of CFA( 1 ) that, however, could not be induced of their production by sprouting might, in principle, adds value to the to crystallize and were impure for the presence of other phenolics, product. including CFB( 2 ). The overall yield of crystalline CFA( 1 ) was 95 mg In this context, we have focused on cannflavin A (CVA, 1 ) and (0.011% on dried plant material). By NMR-inspection of the mother B (CVB, 2 ), a pair of prenylated flavonoids unique to hemp [ 14 ]. liquors, an estimated similar amount of CFA( 1 ) was present in the Inhibition of prostanoid formation has previously been demonstrated mother liquors. for cannflavins [ 15 ], implying anti-inflammatory activity that might nicely complement one of the ω -3 fatty acids present in the seeds, 2.5. Synthesis of cannflavin B and overall increasing the value of hemp seed products as an anti-inflammatory nutritional support. Critical to this project was The published protocol [ 14 ] was used without any significant mod- also the clarification of the mechanism(s) by which cannflavins ification. inhibit prostanoid synthesis, an issue that, despite almost three- decade of studies on these compounds [ 15 ], is still unresolved. 2.6. Determination of the lipid contents of seeds and sprouts

The overall amounts of lipids was estimated by exhaustive hexane extraction. Oven-dried (heating in a ventilated oven at 50 ◦C for 48 h) powdered seeds and sprouts (10 g each) were extracted with hexane at room temperature (3 × 100 mL). Evaporation of the pooled extracts afforded an oily residue, that, when analyzed by NMR, only showed the signal of triglycerides. Triplicate analyses on seeds and sprouts from Ermo gave a lipid contents of 36 ± 1.5% for the seeds, and 2. Materials and methods 31 ± 1.2% for the sprouts.

2.1. General phytochemical procedures 2.7. HPLC–MS determination of cannﬂavins in sprouts and seeds

Silica gel 60 (70–230 mesh) and RP-18 used for gravity column Dried and powdered sprouts or seeds (1 g) were extracted with chromatography were purchased from Macherey-Nagel (D uren,¨ Ger- acetone (25 mL) under stirring. After 10 min the solution was fil- many). Aluminum-coated Merck 60 F254 (0.25 mm) plates were used tered, and the plant residue was washed with 10 mL acetone, and for TLC, visualizing the spots by UV inspection and / or staining with re-extracted a second time with acetone. After filtration and wash- 5% H 2 SO 4 in ethanol and heating. Flash chromatography was carried ing, the pooled filtrates were evaporated, and the oily residue was out in a Biotage apparatus SP1. analyzed by HPLC–MS. A Surveyor HPLC on line with an LCQ DECA XP Plus (Thermo Finnigan, San Jos e,` USA) Ion Trap mass spectrom- 2.2. Plant material eter equipped with an ESI source was employed. Separations were performed on an analytical Luna 5 μm C18(2), 150 mm × 2.0 mm Hemp seeds were either purchased (Finola) or obtained from in (Phenomenex, Torrance, CA) protected with a C18-Security Guard house cultivations at CRA, Rovigo, (Ermo, Carma and a THC-containing cartridge, 4 mm × 2.0 mm (Phenomenex). The injection volume was variety), where also the aerial parts of the variety Ermo were har- 10 μL. The mobile phase components were: A = formic acid 0.2%; B = vested. Ermo is a monoecious medium-size ( ca . 2 m high when grown acetonitrile; the cannflavins were eluted according to the following in temperature climate) hemp variety. It blooms relatively early, and linear gradient: A:B (50:50) for 2 min, then A:B (0:100) over 22 min is derived from the Ermes variety, with whom Ermo shares the pin- at flow rate of 0.4 mL / min. The ESI-MS and ESI-MS / MS spectra of natifidus shape of the leaves, a rare feature in hemps. Ermo is resistant the cannflavins were recorded using direct infusion of each reference to pests as well as parasite plants like those from the genus Orobanche , compounds. Data were acquired in positive and negative MS total ion and shows a good tolerance to salinity. scan mode (mass scan range: m / z 100–1000) and in negative MS / MS O. Werz et al. / PharmaNutrition 2 (2014) 53–60 55 product ion scan mode; the normalized collision energy (nce %) was last 10 days, with venipuncture in heparinized tubes (16 IE heparin / optimized for each precursor ion selected: m / z 367, 44%; m / z 435, mL blood). The blood was centrifuged at 4000 × g for 20 min at 20 ◦C 45%. LC–ESI-MS / MS in single reaction monitoring (SRM) modalities for the preparation of leukocyte concentrates (University Hospital were applied to the selected precursor ions, following the conditions Jena, Germany). Leukocyte concentrates were subjected to dextran set during the infusion analysis. The parent / daughter ion transitions sedimentation and centrifugation on Nycoprep cushions (PAA Lab- m / z 367 → 352 and m / z 367 → 309 were selected for the identifica- oratories, Linz, Austria). For isolation of platelets, the supernatants tion and quantification of CFB( 2 ) while the transitions m / z 435 → 420 of the Nycoprep cushion centrifugation were mixed with phosphate- and m / z 435 → 351 were selected for the identification and quan- buffered saline (PBS) pH 5.9 (3:2 v / v), centrifuged (2100 × g , 15 min, tification of CFA( 1 ). The calibration curves, obtained by injecting six 20 ◦C), and the pelleted platelets were resuspended in PBS pH 5.9 / different concentration levels of CFB( 2 ) and CFA( 1 ) standard solu- 0.9% NaCl (1:1, v / v). Washed platelets were finally resuspended in PBS tions and analyzed in triplicate, showed a good linearity in the range pH 7.4 and 1 mM CaCl 2 . For isolation of polymorphonuclear leuko- of 5–1000 μg / L. The LOD was determined by the analysis of samples cytes (PMNL), contaminating erythrocytes of pelleted PMNL were of known concentrations and found to be 1 μg / L and LOQ found to lysed by hypotonic lysis. PMNL were washed twice in ice-cold PBS be 4 μg / L for both CFB( 2 ) and CFA( 1 ). All samples were diluted and and finally resuspended in PBS pH 7.4 containing 1 mg / mL glucose analyzed in triplicate according to this method. and 1 mM CaCl 2 (PGC buffer) (purity > 96–97%). Monocytes were isolated from the peripheral blood mononuclear cell fraction, which 2.8. HS-SPME / GC determination of cannabinoids in sprouts was obtained after Nycoprep cushion centrifugation of leukocyte concentrates, by adherence for 1.5 h at 37 ◦C to culture flasks (Greiner, Powdered sprouts (10 mg) were placed directly in 15-mL Nuertingen, Germany), cell density was 2 × 10 7 cells / mL RPMI 1640 headspace vial in presence of 1 mL of NaOH 1 M, 300 mg of Na 2 CO 3 medium containing 2 mM l -glutamine and 100 U / mL penicillin and and 1 μL of THC-d6 solution (100 μg / mL in ethanol) as internal stan- 100 μg / mL streptomycin, which gave a purity of > 85%, defined by dard [ 16 ]. The mixture was stirred (1000 rpm) for 40 min at 90 ◦C and forward- and side-light scatter properties and detection of the CD14 under these conditions, the PDMS-100 μm SPME fiber (Supelco Bella- surface molecule by flow cytometry (BD FACS Calibur, Heidelberg, fonte, PA, USA) was exposed to the headspace of the vial to absorb the Germany). Monocytes were finally resuspended in ice-cold PBS plus analytes [ 16 ]. The analytes retained on the fiber were thermally des- 1 mg / mL glucose or in PGC buffer. orbed by exposure in the chromatograph injector for 10 min. Analyses For analysis of acute cytotoxicity of test compounds during pre- were performed using a Trace GC gas chromatograph coupled with a incubation periods, the viability of PMNL was analyzed by light mi- Polaris Q ion trap mass spectrometer (Thermo Finnigan, San Jos e,` USA) croscopy and trypan blue exclusion. For the analysis of cytotoxicity operating in electron ionization mode. Chromatographic separation of monocytes, the MTT assay was used. In brief, cells (100 μL cell was achieved using an RTX-5MS capillary column (30 m × 0.25 mm suspension corresponding to 2 × 10 5 monocytes) were incubated i.d., thickness 0.25 μm, Restek GmbH, Germany) kept at 150 ◦C for for 30 min at 37 ◦C with vehicle (0.3% DMSO) or test compounds. 2 min then ramped to 250 ◦C at 5 ◦C / min followed by a hold at 250 ◦C After 30 min, 20 μL of a sterile filtered solution of 3-(4,5-dimethyl-2- for 10 min. Helium was used as carrier gas at 1.0 mL min –1 . The injec- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in PBS (5 mg / tor temperature was 250 ◦C and it was operated at splitless mode for mL) were added and samples were incubated for 4 h at 37 ◦C. Then, 2 min. The manifold source and the transfer line temperatures were 100 μL of 10% SDS in 20 mM HCl (pH 4.5; SDS lysis buffer) were added set to 250 ◦C and 270 ◦C, respectively. The MS was used in tandem and samples were shaken for 15 h at RT in the dark and the absorp- MS / MS mode to improve sensitivity and selectivity of the method. tion was measured at 570 nm. Results are reported as percentage of The selected parent / daughter ion transitions were m / z 231 → 174, viable cells as compared to vehicle control. A statistically significant m / z 295 → 223, m / z 299 → 231 and m / z 320 → 278, respectively for impairment of MTT reduction to formazan was considered cytotoxic. cannabidiol (CBD), cannabinol (CBN), THC and THC-d6. Quantification limits (LOQ) were fixed at 0.1 ng / mg for CBD, 0.1 ng / mg for CBN and 5.0 ng / mg for THC. In all sprout samples 2.11. Determination of 5-lipoxygenase products in intact cells cannabinoids were below the threshold of detection. For the determination of 5-LO products in intact PMNL (5 × 10 6 ) 2.9. Comparative determination of fatty acid methyl esters in seeds and or monocytes (2 × 10 6 ), cells were resuspended in 1 mL PGC buffer, sprouts from the hemp variety Ermo preincubated for 15 min at 37 ◦C with test compounds or vehicle (0.3% DMSO), and incubated for 10 min at 37 ◦C with the indicated stim- The fatty acid methyl esters were prepared from hexane extracts uli. Thus, the Ca 2 + -ionophore A23187 (2.5 μM) was added with or of the plant material according to literature [ 17 ], and GC–MS analysis without 20 μM AA and 10 min later the reaction was stopped on ice was performed using a Trace GC gas chromatograph coupled with a by addition of 1 mL of methanol. 30 μL 1 N HCL and 500 μLPBS, Polaris Q ion trap mass spectrometer (Thermo Finnigan, San Jos e,` USA) and 200 ng prostaglandin B1 were added and the samples were operating in electron ionization mode. Chromatographic separation subjected to solid phase extraction on C18-columns (100 mg, UCT, was performed with an RTX-5MS capillary column (30 m × 0.25 Bristol, PA, USA). 5-LO products (LTB 4 , trans-isomers, 5-H(p)ETE), mm i.d., thickness 0.25 μm, Restek GmbH, Germany). The program and the cyclooxygenase (COX)-1 product 12( S )-hydroxy-5- cis -8,10- temperature was: 2 min at 150 ◦C then 150–270 ◦C at 3 ◦C / min. trans-heptadecatrienoic acid (12-HHT), respectively, were analyzed The manifold source and the transfer line temperatures were set to by HPLC and quantities calculated on the basis of the internal stan- ◦ ◦ 250 C and 270 C, respectively, and the split injector was maintained dard PGB1. Cysteinyl-LTs C 4 , D 4 and E 4 were not detected (amounts ◦ at 270 C. Helium was used as carrier gas at 1.0 mL min –1 and were below detection limit), and oxidation products of LTB 4 were not split ratio was used 1:50. The mass spectrometer was operated in determined. electron impact (70 eV) mode and full scan (33–350 m / z ) spectra were recorded. 2.12. Expression and purification of human recombinant 5-LO 2.10. Cells and cell isolation Escherichia coli MV1190 was transformed with pT3-5-LO plasmid, Human peripheral blood was taken from fastened (12 h) healthy and recombinant 5-LO protein was expressed at 27 ◦C as described donors who had not taken any anti-inflammatory drugs during the [ 18 ]. Cells were lysed in 50 mM triethanolamine / HCl pH 8.0, 5 mM 56 O. Werz et al. / PharmaNutrition 2 (2014) 53–60

EDTA, soybean trypsin inhibitor (60 μg / mL), 1 mM phenylmethane- 2.17. Determination of the activity of isolated human recombinant sulfonyl fluoride, and lysozyme (500 μg / mL), homogenized by soni- cPLA 2 α in a cell-free assay cation (3 × 15 s), and centrifuged at 40,000 × g for 20 min at 4 ◦C. The 40,000 × g supernatant (S40) was applied to an ATP-agarose column The cPLA 2 α coding sequence was cloned from pVL1393 plasmid to partially purify 5-LO as described previously [ 18 ]. Semi-purified (kindly provided by Dr. Wonhwa Cho, University of Illinois at Chicago) 5-LO was immediately used for activity assays. into pFastBacTM HT A containing a 6 × his-tag coding sequence. The recombinant plasmid was transformed into D10BacTM E. coli. Sf9 cells ® were transfected with recombinant bacmid DNA using Cellfectin 2.13. Determination of 5-LO activity in cell-free assay Reagent and the generated baculovirus was amplified. Overexpres- sion of His-tagged cPLA in baculovirus-infected Sf9 cells and isolation Aliquots of semi-purified 5-LO were diluted with ice-cold PBS con- 2 using Ni-NTA agarose beads was performed as described [ 20 ]. Mul- taining 1 mM EDTA, and 1 mM ATP was added. Samples were pre- ◦ tilamellar vesicles (MLVs) were prepared by drying 1-palmitoyl-2- incubated with the test compounds as indicated. After 10 min at 4 C, ◦ arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl- samples were pre-warmed for 30 s at 37 C, and 2 mM CaCl 2 plus 2-oleoyl-sn-glycerol (POG) in a ratio of 2:1 (in chloroform) under 20 μM AA was added to start 5-LO product formation. The reaction ◦ nitrogen in glass vials. After addition of 20 mM Tris buffer (pH 7.4) was stopped after 10 min at 37 C by the addition of 1 mL ice-cold containing 134 mM NaCl and 1 mg / mL fatty acid free BSA, the MLV methanol, and the formed metabolites were analyzed by RP-HPLC as suspension was disrupted by several freeze–thaw cycles (liquid nitro- described [ 18 ]. 5-LO products include the all-trans isomers of LTB 4 gen) and then extruded 11 times with a mini-extruder (Avanti Polar and 5(S)-hydro(pero)xy-6- trans -8,11,14- cis -eicosatetraenoic acid. Lipids Inc.) through a polycarbonate membrane (100 nm pore diame- ter) at room temperature (above transition temperature of the lipids) 2.14. Preparation of crude mPGES-1 in microsomes of A549 cells and to produce LUV (large unilamellar vesicles). Final total concentration determination of PGE2 synthase activity of lipids was 250 μM in 200 μL. Test compounds and 1 mM CaCl 2 were added to the vesicles, and the reaction was started by addi- ◦ Preparations of A549 cells and determination of mPGES-1 activ- tion of 500 ng his-tagged cPLA 2 (in 10 μL buffer). After 1 h at 37 C, ity were performed as described previously [19]. In brief, cells were 1.6 mL CH 3 OH was added, and AA was extracted by RP-18 solid phase ◦ treated with 1 ng / mL Il-1 β for 48 h at 37 C, 5% CO 2 . Cells were extraction. Following derivatization with p-anisidinium chloride, the harvested and sonicated and the homogenate was subjected to dif- resulting derivate was analyzed by RP-HPLC at 249 nm as described ferential centrifugation at 10,000 × g for 10 min and 174,000 × g for [ 20 ]. 1 h at 4 ◦C. The pellet (microsomal fraction) was resuspended in 1 mL homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, 2.18. DPPH assay 1 mM phenylmethanesulfonyl fluoride, 60 μg / mL soybean trypsin inhibitor, 1 μg / mL leupeptin, 2.5 mM glutathione, and 250 mM su- The radical scavenger capability was assessed by measuring the crose), and the total protein concentration was determined. Microso- reduction of the stable free radical 2,2-diphenyl-1-picrylhydrazyl mal membranes were diluted in potassium phosphate buffer (0.1 M, (DPPH) [ 21 ] with slight modifications. Briefly, 100 μL of test com- pH 7.4) containing 2.5 mM glutathione. Test compounds or vehicle pound in ethanol (corresponding to 0.5, 2.5, and 5 nmol) was added to ◦ were added, and after 15 min at 4 C reaction (100 μL total volume) 100 μL of a solution of the stable free radical diphenylpicrylhydrazyl was initiated by addition of PGH 2 at the indicated concentration. Af- (DPPH) in ethanol (50 μM, corresponding to 5 nmol), buffered with ◦ ter 1 min at 4 C, the reaction was terminated using stop solution acetate to pH 5.5, in a 96-well plate. The absorbance was recorded at (100 μL; 40 mM FeCl 2 , 80 mM citric acid, and 10 μM 11 β-PGE 2 ) as 520 nm (Victor plate reader, PerkinElmer) after 30 min incubation un- internal standard. PGE 2 was separated by solid-phase extraction and der gentle shaking in the dark. Ascorbic acid and l -cysteine were used analyzed by RP-HPLC as described, previously [ 19 ]. as reference compounds. All analyses were performed in triplicates.

2.19. Statistics 2.15. Activity assays of isolated COX-1 and COX-2

Data are expressed as mean ± S.E. IC values were graphically Inhibition of the activities of isolated ovine COX-1 and human 50 calculated from averaged measurements at 4–5 different concentra- COX-2 was performed as described [ 19 ]. Briefly, purified COX-1 tions of the compounds using SigmaPlot 9.0 (Systat Software Inc., (ovine, 50 units) or COX-2 (human recombinant, 20 units) were di- San Jose, USA). Statistical evaluation of the data was performed by luted in 1 mL reaction mixture containing 100 mM Tris buffer pH 8, one-way ANOVA followed by a Bonferroni or Tukey–Kramer post hoc 5 mM glutathione, 5 μM hemoglobin, and 100 μM EDTA at 4 ◦C and test for multiple comparisons respectively. A p value < 0.05 (*) was pre-incubated with the test compound for 5 min. Samples were pre- considered significant. warmed for 60 s at 37 ◦C, and AA (5 μM for COX-1, 2 μM for COX-2) was added to start the reaction. After 5 min at 37 ◦C, the reaction was 3. Results stopped, PGB 1 as standard added and 12-HHT was extracted and then analyzed by HPLC. Indomethacin (20 μM) and celecoxib (5 μM) were 3.1. Analytical characterization of hemp seeds and sprouts used as well-recognized reference inhibitors of COX-1 and of COX-2, respectively, to control the assays. Seeds and sprouts from three varieties of fiber hemp (Finola, Carma, Ermo) and a drug-type THC-rich variety were characterized in 2.16. Determination of the COX-1-derived product 12-HHT in human terms of contents of cannabinoids and cannflavins. The quantification platelets of cannabinoids from fatty matrixes requires special methods, due to the difficulty of their recovery. Using a method specifically developed Freshly isolated human platelets (10 8 / mL PGC buffer) were pre- for the quantification of cannabinoids in hemp oil [ 16 ], we found that incubated with the test compounds for 15 min at 37 ◦C and stimulated in dehulled sprouts the concentration of the three major cannabinoids for 10 min at 37 ◦C with 5 μM AA. The COX reaction was stopped after contained in the inflorescences of the corresponding plants (THC, CBD 10 min at 37 ◦C by addition of 1 mL ice-cold methanol and the formed and CBC) was below detection (0.1 ng / mg for CBD, 0.1 ng / mg for CBN 12-HHT was analyzed by HPLC as described [ 19 ]. and 5.0 ng / mg for THC). Also the concentration of cannflavins in seeds O. Werz et al. / PharmaNutrition 2 (2014) 53–60 57 was below detection (see Section 2.7 ), but induction of their synthesis was evidenced in sprouts from the variety Ermo, where contents of 7- and 5 μg / g were detected for CFA( 1 ) and CFB( 2 ), respectively. A lower induction of the synthesis of CFA( 1 ) (3 μg / g), but not of CFB( 2 ) was detected Carma, while both cannflavins were undetectable in the sprouts from Finola and the THC-rich drug variety. For Ermo, the overall contents of lipids were lower in sprouts compared to seeds (36 ± 1.5% vs. 31 ± 1.2%), but no significant difference was detected in the fatty acid composition between seeds and sprouts ( Table 1 ). Fig. 1. Cannflavins inhibit mPGES-1 activity. (A) Concentration–response curves for CFA( 1 ) and CFB( 2 ). Microsomal preparations of IL-1 β-stimulated A549 cells were prein-

3.2. Interference of cannﬂavins with the prostaglandin biosynthetic cubated with CFA( 1 ), CFB( 2 ) or vehicle (0.3% DMSO) for 15 min at 4 ◦C and the reaction ◦ pathway was started by addition of 20 μM PGH 2 . After 1 min at 4 C the reaction was terminated. Data are expressed as percentage of control (100%), means ± S.E., n = 4. (B) Inhibition

of mPGES-1 by CFA(1) was determined at different PGH2 concentrations (1, 5, 20 and For prostanoid biosynthesis in the cell, AA must first be released by ◦ 50 μM PGH 2 ) during incubation for 1 min at 4 C. Data are expressed as percentage of PLA enzyme(s) which in turn is converted by COX-1 / 2 to PGH that 2 2 control (100%), means ± S.E., n = 3. (C) Inhibition of mPGES-1 by CFA( 1 ) is reversible. serves as substrate for various PG synthases to form the respective Microsomal preparations of IL-1 β-stimulated A549 cells were preincubated with 0.3 ◦ PGs. Among the PGs, the PGE 2 is the most relevant pro-inflammatory or 3 μM CFA( 1 ) for 15 min at 4 C. An aliquot of the sample containing 3 μM CFA( 1 ) mediator (formed by COX-2 and mPGES-1), and previous studies was diluted 10-fold (“0.3 (3)”) and the other aliquot left untreated. 20 μM PGH2 was added to start the reaction and stopped after 1 min at 4 ◦C as described above. Data are showed a potent suppressive effect of CFA( 1 ) on PGE biosynthesis 2 expressed as percentage of control (100%), means ± S.E., n = 3. * p < 0.05, ** p < 0.01, in intact human rheumatoid synovial cells [15]. However, no con- *** p < 0.001 vs. 100% control. crete point of attack or target of CFA( 1 ) was identified thus far. We first confirmed suppression of PGE 2 biosynthesis by CFA (10 μM) in intact cells using LPS-stimulated human monocytes (56 ± 8% inhibition, Table 2 ), conditions where PGE 2 is essentially formed via the COX-2 / mPGES-1 pathway. In contrast, analysis of COX-1-mediated prostanoid synthesis in human platelets stimulated with 5 μM AA revealed only moderate suppression by CFA( 1 ) at 10 μM ( Table 2 ). Indomethacin (20 μM) and celecoxib (5 μM) were used as reference inhibitors for COX-1 / 2 and COX-2 that blocked 12-HHT formation in platelets and PGE 2 synthesis in monocytes, respectively ( Table 2 ), as expected. In order to investigate whether or not CFA( 1 ) directly inhibits Fig. 2. Cannflavins inhibit 5-LO activity. (A) Concentration response curves for CFA( 1 )

COX-1 or COX-2 activity, we analyzed the effects of CFA on the ac- and CFB( 2 ). Partially purified recombinant 5-LO (0.5 μg / mL) was incubated with tivity of COX enzymes in a cell-free assay using isolated ovine COX-1 CFA( 1 ), CFB( 2 ) or vehicle (DMSO, 0.1%) at 4 ◦C for 15 min. Samples were prewarmed ◦ and human recombinant COX-2. As shown in Table 2 , CFA( 1 ) slightly for 30 s at 37 C, 2 mM CaCl2 and 20 μM AA were added and 5-LO product formation was determined after 10 min. Data are expressed as percentage of control (100%), suppressed the activity of both enzymes with IC 50 > 10 μM, while means ± S.E., n = 3–6. (B) 5-LO inhibition by CFA( 1 ) is partially reversible. Purified indomethacin (20 μM) and celecoxib (5 μM) inhibited the enzyme 5-LO was incubated with 1 or 10 μM CFA( 1 ) for 15 min at 4 ◦C. An aliquot of the 1 μM activities efficiently. To investigate if CFA( 1 ) blocks PGE 2 formation at sample was diluted with assay buffer 10-fold (“1 (10)”); the other aliquot was not al- ◦ the level of AA supply, the effect of the compound on the enzymatic tered. Then, samples were prewarmed for 30 s at 37 C and 20 μM AA and 2 mM CaCl 2 were added. After 10 min, 5-LO product formation was analyzed as described. Data are activity of human recombinant cPLA 2 was studied. In contrast to the expressed as percentage of control (100%), means ± S.E., n = 3–4. ** p < 0.01 vs. inhibi- cPLA control inhibitor pyrrolidine-1 (5 μM) that effectively inhib- 2 tion without dilution, ANOVA + Bonferroni post hoc test. (C) PMNL were preincubated ◦ ited AA release from phospholipids, CFA(1) (at 10 μM) caused only with CFA( 1 ) or vehicle (DMSO, 0.1%) for 15 min at 37 C and stimulated with 2.5 μM marginal inhibition ( Table 2 ). A23187 or 2.5 μM A23187 plus 20 μM AA (as indicated) for 10 min at 37 ◦C. Data are expressed as percentage of control (100%), means ± S.E., n = 3. * p < 0.05, ** p < 0.01, Next, we assessed if CFA( 1 ) may inhibit PGE 2 synthesis due to < interference with mPGES-1, a potential drug target in inflammation ***p 0.001 vs. 100% control. and inflammation-triggered cancer [ 22 ]. We utilized a cell-free assay with PGH 2 as substrate and MK-886 [ 19 ] as reference inhibitor for mPGES-1 that blocked mPGES-1 activity as expected ( Table 2 ). As can cannflavins to interfere with 5-LO activity in a cell-free assay using be seen from Fig. 1 A and Table 2 , the geranylated CFA( 1 ) inhibited purified human recombinant 5-LO as enzyme source; zileuton [ 23 ] mPGES-1 with an IC 50 value = 1.8 μM being equipotent to MK886. was used as reference inhibitor for 5-LO. In fact, CFA( 1 ) and CFB( 2 ) Similarly, the prenylated CFB( 2 ) suppressed mPGES-1 activity with potently and concentration-dependently inhibited 5-LO activity with IC 50 = 3.7 μM. Based on these results, we conclude that mPGES-1 is IC 50 values = 0.9 and 0.8 μM ( Fig. 2 A, Table 1 ). Of interest, inhibition a direct target of cannflavins and its inhibition is essentially respon- of isolated 5-LO by CFA( 1 ) was only partially reversible, whereas 5-LO sible for the reduced PGE 2 formation in intact cells. Variation of the inhibition by zileuton was fully reversed, as demonstrated by wash- substrate (PGH 2 ) concentration for mPGES-1 did not markedly alter out experiments ( Fig. 2 B). In order to investigate whether CFA( 1 ) may the potency of CFA( 1 ) (IC 50 = 1.8–3.7 μM; Fig. 1 B), implying that block 5-LO activity also in intact cells, we used A23187-stimulated the potency is independent of the substrate concentration. Moreover, human neutrophils that produce substantial amounts of 5-LO prod- wash out experiments show that the inhibitory effect of CFA( 1 ) on ucts [ 24 ]. The effect of CFA( 1 ) was studied in the absence and pres- mPGES-1 is fully reversible ( Fig. 1 C). ence of exogenous AA as substrate for 5-LO product synthesis. CFA( 1 ) efficiently suppressed 5-LO product synthesis with IC 50 of 1.6 μM 3.3. Interference of cannflavins with the 5-lipoxygenase pathway (A23187 plus AA) and 2.4 μM (A23187) ( Fig. 2 C). This implies that (I) inhibition of 5-LO product synthesis is not related to block of substrate Upon release from phospholipids, AA may be metabolized also supply, and (II) high AA concentrations do not impede interference of via the 5-LO pathway leading to pro-inflammatory LTs, in addition CFA( 1 ) with 5-LO. to PGs, and in fact, many mPGES-1 inhibitors have been described Because many 5-LO inhibitors possess radical scavenging or an- to act also on 5-LO [ 22 ]. We therefore analyzed the ability of the tioxidant activities, thereby uncoupling the redox cycle of the active 58 O. Werz et al. / PharmaNutrition 2 (2014) 53–60

Table 1 Fatty acids composition of seeds and sprouts from the Ermo variety of hemp (each data represents the mean of three replicates ± standard deviation).

Fatty acid (relative %) Seeds Sprouts

Linoleic 57.53 ± 0.2 58.00 ± 0.31 α-Linolenic 24.53 ± 0.14 24.55 ± 0.17 Oleic 9.97 ± 0.06 10.01 ± 0.07 γ- Linolenic 1.08 ± 0.01 1.42 ± 0.03 Palmitic 4.96 ± 0.13 4.18 ± 0.207 Stearic 1.94 ± 0.01 1.85 ± 0.04

Table 2 Effects of CFA( 1 ) on various eicosanoid-forming enzymes and cellular functions of neutrophils and monocytes. CFA( 1 ) or reference inhibitors (at the indicated concentrations) were added to the respective enzymes or freshly isolated human blood cells 15 min prior induction of the enzyme reaction. Data (means ± S.E., n = 3–5) are expressed as IC 50 values and as percentage of the inhibition of the enzyme activity vs. the uninhibited vehicle (0.1% DMSO) control.

CFA(1)

IC 50 [ μm]; Reference control, Enzyme / assay % inhibition at 10 μM % inhibition at indic. conc.

COX-1, platelets > 10 μM; 26 ± 2% Indomethacin (20 μM), 95 ± 5% COX-1, cell-free > 10 μM; 36 ± 2% Indomethacin (20 μM), 81 ± 8% COX-2, monocytes 8.8 μM; 56 ± 8% Celecoxib (5 μM), 80 ± 5% COX-2, cell-free > 10 μM; 35 ± 12% Celecoxib (5 μM), 78 ± 8% cPLA 2 , cell-free > 10 μM; 26 ± 8% Pyrrolidine-1 (5 μM), 79 ± 3% mPGES-1, cell-free 1.8 μM; 90 ± 1% MK-886 (10 μM), 83 ± 1% 5-LO cell-free 0.9 μM; 88 ± 3% Zileuton (3 μM), 80 ± 4% 5-lo, PMNL, A23187 2.4 μM; 98 ± 2% BWA4C (0.3 μM), 99 ± 2%

5-lo, PMNL, A23187 + AA 1.6 μM; 91 ± 1% BWA4C (0.3 μM), 76 ± 2% DPPH assay No effect Ascorbate (50 μM), 80 ± 6% Cell viability no effect staurosporine (3 μM), 80 ± 5%

assays. Similarly, neutrophils treated with 10 μM CFA( 1 ) for 1 h still excluded the dye trypan blue, analyzed by light microscopy, implying cellular integrity (not shown).

4. Discussion

Before the identification of specific cannabinoid receptors, the inhibition of the synthesis of inflammatory PGs, in particular PGE 2 , was considered a possible mechanism underlying the analgesic properties of THC [ 25 ]. However, conflicting data were obtained when THC and other cannabinoids were evaluated in simple assays of COX inhibition, and the situation was further confused by the observation that extracts of Cannabis devoid of cannabinoids showed a strong inhibitory activity on the production of PGs in cultured rheumatoid synovial cells [ 15 ]. A flavonoid named cannflavin was eventually identified as the major inhibitor of cellular PG synthesis from cannabinoids-free extracts of Cannabis [ 15 ]. Cannflavin is actually a mixture of two closely related prenylated flavonoids, CFA( 1 ) and CFB( 2 ), differing in the nature of the prenyl group on ring A of the flavone ring system (geranyl in CFA( 1 ) and prenyl in CFB( 2 )) [ 26 ]. Cannflavin C, a third natural cannflavin previously obtained as a by-product from the synthesis of

Fig. 3. Radical scavenging properties of CFA( 1 ). CFA( 1 ) was incubated with 5 nmol CFB(2) [14], was recently reported from a high-potency THC chemo- DPPH for 30 min at RT and the absorbance was measured at 520 nm. Ascorbic acid type, and represents a remarkable example of “anticipated” natural and l -cysteine were used as controls. Values are given as percentage of control (100%) product discovery [ 27 ]. The anti-inflammatory potency of cannflavin ± = < mean S.E., n 3–4. ***p 0.001 vs. 100% control. was found to be intermediate between that of aspirin and dexam- ethasone [ 15 ], but its mechanism of activity has remained elusive. Cannflavins are minor constituents of C. sativa . During our phyto- site iron in 5-LO, we analyzed CFA( 1 ) for its ability to reduce the sta- chemical studies on this plant [ 28 –30 ], we found that the ratio be- ble DPPH radical. As shown in Fig. 3 , the antioxidants ascorbic acid tween the two cannflavins and their concentration are rather variable and l -cysteine (used as positive controls) reduced the DPPH radical within the various varieties of the plant. These compounds are diffi- whereas CFA( 1 ) failed in this respect, excluding radical scavenging cult to obtain completely pure by isolation, and tend to co-crystallize features of CFA( 1 ). with other phenolics, making their obtaining process tedious. CFB( 2 ) Finally, we tested if CFA( 1 ) may affect cell viability using human can be conveniently obtained by synthesis [ 14 ], but synthetic CFA( 1 ) monocytes that were treated with 10 μM CFA( 1 ) for 24 h. Analysis is a mixture of stereoisomers. We were therefore delighted to discover by MTT assay revealed no cytotoxicity or detrimental effects on cell that Ermo, a variety of hemp devoid of cannabinoids, contains signif- viability ( Table 2 ) excluding unspecific influences in the cell-based icantly higher amounts of CFA( 1 ) compared to the cannabinoids-rich O. Werz et al. / PharmaNutrition 2 (2014) 53–60 59 varieties, with minimal contamination from CFB. Owing to the nutri- acids unsaturation profile of seeds and sprouts from Ermo, with the tional potential of cannabinoid-free strains of hemp, we wondered if maintenance of the ca 3:1 ratio between ω -6 and ω -3 essential fatty CFA( 1 ) was also contained in the seeds of the plant. Hemp seeds are acids ( Table 1 ). Significant changes were, however, observed for γ- normally devoid of cannflavins, and the same observation was done linolenic (GLA)- and palmitic acids, both minor constituents, with a ca on the seeds of Ermo. However, sprouting induced the formation of 30% increase of concentration of GLA (from 1.08% to 1.42%) and a cor- both CFA(1) (7 μg / g) CFB( 2 ) (5 μg / g) in this variety, and a lower responding decrease of palmitic acid (from 4.96% to 4.18%) ( Table 1 ) induction of the synthesis of CFA( 1 ) (3 μg / g), but not of CFB( 2 ), was during germination. also observed in the variety Carma, despite the presence of both can- The availability of both cannflavins made it possible to investigate nflavins in the flowerheads of this variety. Neither CFA( 1 ) nor CFB( 2 ) the molecular basis underlying their anti-inflammatory properties, could be detected in the sprouts from the variety Finola and one THC- and we have unequivocally shown that these compounds act as dual rich medicinal variety, that both contain these compounds in their inhibitors of mPGES-1 and 5-LO, two crucial enzymes in the biosyn- flowerheads (data not showed). Since the ratio between ω -3 and ω -6 thesis of the pro-inflammatory mediators PGE 2 and LTs, respectively. fatty acids was unaffected by germination, the nutritional profile of Moreover, the direct interference of cannflavins with mPGES-1 and sprouts could benefit, compared to the seeds, from their contents of the weak effects on COX-1 / 2 enzymes suggest mPGES-1 as the tar- cannflavins. The estrogenic hop flavonoid 8-prenylnaringenin (8 PN, get responsible for repression of cellular PGE 2 synthesis observed 4 ) is structurally related to cannflavins, and showed clinical activity in complex cell-based assay [ 15 ]. Our data show that (a) CFA( 1 ) ex- at dosages of 100 μg / day [ 31 ], an amount roughly corresponding erts its inhibitory effects on mPGES-1 and 5-LO in a reversible and to the CFA( 1 ) contents of 20 g of fresh hemp sprouts. The affinity substrate concentration-independent manner, (b) exhibits no radical of 8 PN for estrogen receptors is roughly two orders of magnitude scavenger activity (DPPH assay), and (c) fails to substantially inhibit higher than that of CFA( 1 ) for mPGES-1 and 5-LO [ 32 ], but, owing to the related COX-1 and COX-2 enzymes as well as the cPLA 2 , the en- the long elimination half life of prenylated flavonoids [ 33 ], it does zyme that provides AA as substrate for PGE 2 and LT formation. In not seem unrealistic to assume that regular consumption of hemp fact, dual inhibition of mPGES-1 and 5-LO is considered as pharma- sprouts could lead to the attainment of bioactive concentrations of cological strategy in order to intervene with inflammatory diseases cannflavins in plasma and tissues. and might be superior over single target interference in terms of efficacy as well as with respect to lower side effects [ 34 ]. Together, the potent suppression of mPGES-1 and 5-LO by cannflavins is of con- siderable anti-inflammatory potential, especially when the intake of these compounds is associated to a fatty matrix that can both increase their absorption [ 36 ], and potentiate their activity due to its high contents of ω 3-acids.

5. Conclusions

Sprouting has a general beneficial effect on the digestibility of Flavonoids can modulate the expression of mPGES-1 [ 34 ], but seeds by reducing the level of anti-nutritional factors, but changes cannflavins are the first flavonoids having direct inhibitory activity in secondary metabolites can also occur, with the potential to en- on this enzyme. Since good inhibitory activity was also reported for rich sprouts with specific phytochemicals. In this context, we have the prenylated phloroglucynol arzanol [ 35 ], a phenolic somewhat re- identified a variety of hemp seeds where production of the anti- sembling the A ring of cannflavins, it is tempting to speculate that inflammatory lipophilic flavonoids cannflavins is induced by ger- prenylation is important for mPGES-1 inhibition. Because of their mination, and have clarified the mechanism by which these com- anti-inflammatory activity, the presence of cannflavins might con- pounds inhibit the production of pro-inflammatory prostanoids and tribute to the nutritional profile of the sprouts. In this context, it was leukotrienes. These data provide a rationale for seriously considering also important to assure the absence of cannabinoids in the sprouts, the commercial development of this novel hemp seed food, optimiz- and to evaluate if changes in the lipid profile had occurred during ger- ing factors like sprout length, root length, and post-sprouting time. mination. The concentration of cannabinoids in hemp seeds is very low, and special extraction protocols have been developed for their Conflicts of interest efficient recovery [ 16 ]. By using a seed-specific method of extraction, < / we found that traces ( 1 μg g) these compounds were only present Ermo is a registered variety of hemp at CPVO by CRA, the employer in the seed tegument of the cannabinoids-producing varieties. On the of GG. other hand, these compounds were totally absent in the corresponding dehulled material, confirming previous findings, and supporting the view that the presence of cannabinoids in seeds is the result of Layperson ’s summary matrix contamination during maturation and not the result of a site- specific synthesis or accumulation. Furthermore, de novo production Hemp seeds contain all essential amino acids and lipids of cannabinoids was not observed during sprouting, confirming their necessary for human nutrition. This, coupled to their anti- strict association to specific glandular structures typical of adult plant inflammatory profile of unsaturated fatty acids and pleasant tissues [ 11 ]. As expected, cannabinoids were also undetectable in the nutty taste, has gained them a “super-food” status in consumers. sprouts of Ermo. Sprouting has been shown to increase the nutritional value of Plants have the capacity to de novo synthesize sugars from fatty seeds by reducing the concentration of anti-nutritional factors acids, and, indeed, a decrease of the overall fat contents was observed and boosting the production of specific phytochemicals, as in between seeds and sprouts of Ermo (from 36 ± 1.5% to 31 ± 1.2%). broccoli. We have discovered that sprouting, while not induc- On the other hand, limited information exists if certain types of fatty ing the production of cannabinoids, can trigger the formation acids are selectively metabolized during germination. It was there- of anti-inflammatory lipophilic flavonoids in some varieties of fore important to investigate if depletion of ω -3 was occurring during hemp. The mechanism of action of these compounds has been germination, since this would have eroded the nutritional profile of elucidated at the molecular level, providing a rationale for the sprouts. However, no significant difference was found in the fatty development of sprouts as a novel hemp-derived healthfood. 60 O. Werz et al. / PharmaNutrition 2 (2014) 53–60

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