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Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content a
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a
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G. Lesma , R. Consonni , V. Gambaro , C. Remuzzi , G. Roda , A. a
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Silvani , V. Vece & G.L. Visconti a
Dipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy b
Istituto per lo Studio delle Macromolecole, ISMAC, CNR, v. Bassini 15, 20133 Milano, Italy c
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy Published online: 17 Jun 2014.
To cite this article: G. Lesma, R. Consonni, V. Gambaro, C. Remuzzi, G. Roda, A. Silvani, V. Vece & G.L. Visconti (2014): Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.926354 To link to this article: http://dx.doi.org/10.1080/14786419.2014.926354
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.926354
Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content G. Lesmaa, R. Consonnib, V. Gambaroc, C. Remuzzia, G. Rodac, A. Silvania*, V. Vecea and G.L. Viscontic
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a
Dipartimento di Chimica, Universita` degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy; bIstituto per lo Studio delle Macromolecole, ISMAC, CNR, v. Bassini 15, 20133 Milano, Italy; cDipartimento di Scienze Farmaceutiche, Universita` degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy (Received 7 April 2014; final version received 15 May 2014) Within a project aimed to reintroduce non-drug hemp cultivars in the Italian Po valley, for fibre but also high added-value nutraceutical production, investigation on locally grown plants has been performed, in order to assess their oil and metabolic content. This study provides useful information regarding three different hemp cultivars, from two sites, in view of their potential industrial application. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of v-3 and v-6 fatty acids, as requested for healthy foods. The alcoholic extracts, for which a high content of amino acids and phenolic compounds has been highlighted, could provide dietary supplements to help in preventing oxidative stress. By investigating the Carmagnola cultivar, six known and four new lignanamides have been identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants. Keywords: Cannabis sativa L.; fatty acid; total phenolic; lignanamides; cannabisin
1. Introduction Cannabis sativa L. has been an important source of food, fibre, dietary oil and medicine for thousands of years in Europe, Asia and Africa. Presently, non-drug varieties of C. sativa L., containing much less Tetrahydrocannabinol (THC) than common hemp, are important agricultural commodities, mainly in Canada, the USA and China. In the last decade, there has been a revived interest in hemp as a renewable resource in Europe, aimed at developing new production methods, new processing approaches and, conclusively, at updating the entire production chain. In fact, fibre production in countries with high-labour costs, such as Europe, can be more profitable if it is also coupled with exploitation of bioactive components, to yield high added-value nutraceutical products. Even if research continues to give new knowledge that enables the gradual improvement of the production of hemp, investigation on locally grown plants is always necessary, since the factors that influence both quality and quantity of production are directly connected to climate and soil conditions. To this end, in the frame of a research project aimed to reintroduce hemp cultivars in the Italian Po valley, the objective of this study was to investigate the seed extracts of three different cultivars (Carmagnola, Futura, Felina, known to contain less than 0.2% of THC This manuscript was submitted at SIF 2013, National Congress of Societa` Italiana di Fitochimica (Italian Phytochemical Society), Gargnano (Italy). *Corresponding author. Email: alessandra.silvani@unimi q 2014 Taylor & Francis
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in the whole plant), which have been grown in two sites (Treviglio (BG) and Cavriana (MN)) in the Lombardia region, in the last 3 years. In particular, since hempseed oil is recognised as a functional food (Callaway 2004) and an important source of essential fatty acids, we first evaluated its fatty acid profile, by using 1H NMR characterisation. The second focus was the assessment of phenol and flavonoid contents in seeds, in order to define their potential antioxidant value, and the evaluation of the free radicalscavenging capacity. Finally, we also performed 1H NMR-based metabolomic analysis (Kim et al. 2010), which provided us the possibility of identifying common metabolites in the three cultivars. For the more promising Carmagnola cultivar, a chromatographic separation was carried out on silica-gel and Sephadex columns, followed by HPLC – MS analysis, allowing to detect various metabolites (Flores-Sanchez & Verpoorte 2008), mainly lignanamides, and to identify four unprecedented cannabisin-like compounds. Their chemical structure were unambiguously assigned by MS and NMR analysis and, for three of them, also by analytical comparison with authentic, synthesised samples. 2. Results and discussion 2.1. 1H NMR spectroscopy-based fatty acid profile To obtain a rapid picture of the fatty acid profile, we made use of 1H NMR. A single analysis allowed the determination of a large number of parameters of oil samples, avoiding the preparation of derivatives that would be necessary for GC. We could easily quantify the common unsaturated fatty acids (UFAs: oleic, linoleic and linolenic acids), by applying standard equations, as reported in the literature (Knothe & Kenar 2004). The results are reported in Table 1. Seeds of all analysed cultivars contained similar amounts (25 –30%) of oil. The oils were high in UFAs, with a major accumulation in the cultivars grown in Treviglio, especially Carmagnola (89%). The polyunsaturated linoleic acid (18:2), running up to 63.1%, was the most abundant UFA, followed by a-linolenic (18:3) and oleic acid (18:1) with the ranges of 11.1– 20.9% and 10.8 –22.5%, respectively. The composition of fatty acids resulted to be slightly cultivar- and field site-dependent, with the cultivar Carmagnola providing a well-balanced proportion of all health beneficial UFAs. 2.2. Antioxidant and free radical-scavenging activity The quantitative determination of phenolic and flavonoid content as well as the evaluation of the antioxidant activity were performed (in triplicate) on the alcoholic extract of defatted seeds. The content of total phenolic compounds was determined by the colorimetric Folin –Ciocalteu method (Bonoli et al. 2004). The results obtained are shown in Table 2 and are expressed as gallic acid equivalents (GAEs). Cultivar and location were found to be rather significant, with Table 1. Oil yield and fatty acid contents of hemp seed oils (%, as from 1H NMR). Treviglio’s culture
Oil yield C18:3 C18:2 C18:1a Totalunsat a
Cavriana’s culture
Carmagnola
Futura
Felina
Carmagnola
Futura
Felina
29.0 20.9 57.6 10.8 89.0
30.0 11.1 63.1 12.3 86.0
30.0 19.4 54.1 13.6 87.1
25.3 20.3 31.9 22.5 74.8
29.6 17.5 52.5 12.3 82.3
26.2 15.4 50.7 13.8 80.0
Plus small amounts of g-linolenic (18:3) acid, not separately quantifiable.
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Table 2. Extraction yield, total phenolic content, flavonoid content and DPPH IC50 of hemp seeds methanolic extracts. Treviglio’s culture Carmagnola 9.9 6.14 ^ 0.57
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7.33 ^ 0.59 0.17 ^ 0.09
Cavriana’s culture Futura
Felina
Carmagnola
Futura
Extraction yield (%) 7.7 9.9 9.0 Total phenolic content (mg GAE/100 mg) extract 4.57 ^ 0.39 5.89 ^ 0.55 6.70 ^ 0.65 8.04 ^ 0.72 Flavonoids content (mg RE/100 mg) extract 5.39 ^ 0.23 10.90 ^ 0.44 9.78 ^ 0.63 9.28 ^ 0.83 DPPH (IC50 (mg/mL)) 0.27 ^ 0.03 0.10 ^ 0.02 0.18 ^ 0.02 0.15 ^ 0.01 6.9
Felina 7.8 7.27 ^ 0.55 7.67 ^ 0.67 0.15 ^ 0.07
the highest content of total phenolic compounds established in Cavriana cultivars. The total flavonoid content was evaluated (Zhishen et al. 1999) as rutin equivalents (REs) and resulted somewhat limited. The assay of the DPPH radical-scavenging activity (Chen et al. 2012), which is widely used to evaluate the antioxidant capacity of extracts, exhibited considerable values, with activity in the IC50 range of 0.10– 0.27 mg/mL, coming presumably from various kinds of phenolic compounds present in seeds. These results confirmed the possibility of utilising the waste products of the oil extraction process as source of natural antioxidants for food or nutraceutical industry. 2.3. 1H NMR spectroscopy-based metabolomic analysis For the reliable characterisation of cannabis cultivars, a metabolic profiling is desirable. NMR spectroscopy is one of the techniques that could describe the observable chemical profile or fingerprint of the complete metabolite pattern in a rapid and reproducible manner, requiring only a very basic sample preparation (Choi et al. 2004). The analysis revealed a common metabolite content for the three investigated cultivars, highlighting the presence of amino acids, such as alanine, valine, leucine, glutamine, organic acids, such as succinate, acetate, g-aminobutyrate, glutamate, saturated (palmitic) and unsaturated (linoleic, a-linolenic and oleic) fatty acids, saccharides, such as sucrose, myoinositol, glucose, and other molecules such as trigonelline and caffeoyltyramine in the aromatic region.
2.4. Isolation and characterisation of secondary metabolites For the chemical investigation of the metabolic content, we selected the Carmagnola cultivar, which was proving to be the most promising, both in terms of oil quality and of adaptation to the local climate and field specificity. Separation of the MeOH extract, using successive column chromatography over silica gel and Sephadex LH-20, resulted in the isolation of four fractions, which were then investigated by using HPLC – MS (ESI), 1D and 2D NMR. Six known lignanamides could be recognised by comparing the spectroscopic data with previously reported data, namely N-caffeoyltyramine and N-feruloyltyramine (Georgiev et al. 2013), N-(3,4dimethoxyphenethyl)-3-(3-hydroxy-4-methoxyphenyl)propanamide (Kametani et al. 1969), cannabisin A (Sakakibara et al. 1991), cannabisin G (Li et al. 2010; Xia et al. 2010) and cannabisin E (Sakakibara et al. 1995). In addition, four (1– 4) unknown lignanamides have been elucidated (Figure 1). For compounds 1 – 3, we also accomplished the preparation of authentic synthetic reference samples (Remuzzi 2014), for analytical comparison.
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Figure 1. Structures of lignanamides 1 – 4.
Compound 1 has been identified as a 3,30 -demethyl-cannabisin G. The 1H and 13C NMR spectra are very similar to those reported for cannabisin G, except for the absence of methoxy signals in 1. In particular, the 1H NMR spectrum resembles that of caffeoyltyramine, but bearing a unique olefin proton signal (d 7.98, s, 2H), instead of the trans-olefin spin system. In the 2D HMBC spectrum, cross-peaks are detected between the singlet olefin proton signal (d 7.98, 2H) and aromatic methine [d 111.5 (C-2 and C-20 ), 121.8 (C-6 and C-60 )] and carbonyl signal [d 167.0 (C-9 and C-90 )], respectively. From these data, the singlet signal at d 7.98 has been assigned to H-7 and H-70 , leading to propose the connection between C-8 and C-80 and therefore the chemical structure of compound 1. The molecular formula (C34H32N2O8, ESI-MS m/z: 597 [MHþ]) confirms 1 as a dimer of caffeoyltyramine. Compound 2 has been identified as 3-demethyl-cannabisin G. The 1H NMR spectrum is similar to that of 1, except for the presence of a methoxy group (d 3.77, 3H) and of two different olefin methine protons (d 8.00 and 7.98). Two distinct set of signals are observed for the proton H300 /H500 and H3000 /H5000 [d 6.64 (d, 2H) and 6.65 (d, 2H)], H2 and H20 [d 7.26 (s, 1H) and 7.37 (s, 1H)], H800 and H8000 [d 3.24 (2H) and 3.61 (2H)]. The molecular formula C35H34N208 (ESI-MS m/z: 611 [MHþ]) confirms 2 to be the dimer between a caffeoyltyramine and a feruloyltyramine unit, connected between the C-8 and C-80 position. Compound 3, more properly defined as a neolignanamide, has been recognised as 3,30 demethyl-grossamide. The proposed structure, and in particular the cis relationship between H70 / H80 , was confirmed by comparing with characteristic resonances and J values of the known parent compound (King & Calhoun 2005), and by key peak correlations in the HSQC spectrum [for instance dC 88.3 and dH 5.92 (d, 8.3 Hz) for CH-70 ; dC 47.9 and dH 4.11 (d, 8.3 Hz) for CH-80 ; dC 142.0 and dH 7.38 (d, 15.7 Hz) for CH-7; dC 119.2 and dH 6.34 (d, 15.7 Hz) for CH-8]. The presence of two tyramine-like moieties, bearing a tri- and a tetra-substituted aromatic group, respectively, has been further ascertained by a combination of 2D NMR experiments. The molecular formula of 3 is C34H32N2O8, by ESI-MS m/z: 597 [MHþ].
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Compound 4 resulted to be a cannabisin A derivative, in which the known lignanamide aglycone is O-conjugated with a C5-furanoside residue. The molecular formula of 4 is C39H38N2O12, by ESI-MS m/z: 727 [MHþ]. The 1H NMR spectrum shows signals for two tyramine moieties [d 2.21 (2H, br, t, 7.3 Hz), 2.69 (2H, br, t, 7.4 Hz), 3.25 (2H, br, t, 7.3 Hz), 3.55 (2H, m)]. The aromatic protons pattern of 4 is almost superimposable to that of cannabisin A, with the important exception of the signal of H50 , which appears at d 7.13 (br, d, 8.3 Hz), that is about 0.3 ppm downfield with respect to the same proton in cannabisin A. This observation strongly suggests the C-40 -OH as the position involved in the glycosidic bond. Careful analysis of 2D TOCSY pattern and 2D HMBC spectrum would suggest the C5 carbohydrate to be a ribose linked to the aglycone with a 1-b-glycosidic linkage. 3. Experimental 3.1. Plant material and extraction procedures Seeds of C. sativa L. varieties, Carmagnola (certified no. 9730015241), Futura 75 (certified no. 198465 MK) and Felina 32 (certified no. 204346 MK), were obtained from the seed company Assocanapa S.r.l, Carmagnola (TO), Italy. The three different hemp cultivars were cultivated under field conditions in 2010/2013 at the Cavriana (MN) and Treviglio (BG) sites, in the Italian Po valley. In 2013, after seed maturity, the plants were immediately harvested and seed samples were taken. Seeds (40 g) were ground using a high-speed grinder, then extracted by Soxhlet using heptane (400 mL) as solvent, during 8 h under reflux, followed by solvent removal. Defatted seeds were extracted with methanol/water (8:2) (200 mL) for 3 h under stirring. Then, the mixture was filtered and the combined methanol extract concentrated under vacuum. The procedure was repeated three times. 3.2. Determination of fatty acid composition by 1H NMR Spectra were recorded on a Bruker (Billerica, MA, USA) spectrometer, operating at 400 MHz at room temperature. The sample concentration was 100 mg of oil in 1 mL of CDCl3. Acquisition: 16 K data point, spectral width: 14 ppm, acquisition time: 3.6 s, relaxation delay: 10.0 s. 3.3. Metabolomic analysis by 1H NMR Spectra were recorded on a Bruker Avance DMX spectrometer, equipped with z-gradient coils with a proton resonance frequency at 500.13 MHz. All spectra were recorded at 298 K, with 32 K data points and 8000 Hz of spectral width and referenced to external sodium trimethylsilyl [2,2,3,3-2H4]propionate. Residual solvent suppression was achieved by applying a presaturation scheme with low-power radiofrequency irradiation. NMR spectra were acquired and processed with Topspin (v. 1.3) Bruker software. The optimised heteronuclear coupling constant for HSQC was set to 145 Hz, while the spin lock for TOCSY spectra was set to 80 ms. Spectral assignment has been performed on the basis of reported chemical shifts values and by comparing with the spectra of standard compounds. 3.4. Isolation procedures The methanol extract (4 g) was chromatographed on a silica gel column (80 g silica gel) using a chloroform/methanol step gradient, ranging from 80:20 to 30:70. Subsequent separation on Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA) column (15 cm £ 3 cm i.d.) using methanol as the eluent yielded four enriched fractions (A, 25 mg; B, 12 mg; C, 13 mg; D, 14 mg), which were subjected to HPLC – MS analysis.
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3.5. HPLC – MS protocols HPLC – MS analyses were performed on a Finnigan LCQ (MS) – Deca XP Max – Ion Trap Mass spectrometer – Thermo Electron Corporatione (Waltham, MA, USA) instrument. Chromatographic separation was achieved by a Zorbax SB C18 column (250 mm £ 4.6 mm i.d. – particle size 5 mm, Agilent Technologiese (Cernusco sul Naviglio, Milano, Italy)) protected by a Security Guard column C18 4 mm £ 2.0 mm (Phenomenexe (Castel Maggiore, Bologna, Italy)) at room temperature. The mobile phase consisted of 3 mM ammonium formate buffer pH ¼ 3.0 (phase A) and acetonitrile (phase B). Flow rate: 0.3 mL/min; gradient: 5% phase B for 5 min; from 5% phase B to 20 % phase B from 5.0 to 10.0 min; 20% phase B from 10.0 to 15.0 min; from 20% phase B to 30% phase B from 15.0 to 35.0 min; from 30% phase B to 40% phase B from 35 to 40 min; from 40% phase B to 30% phase B from 40 to 45 min; from 30% phase B to 20% phase B from 45 to 50 min; from 20% phase B to 5% phase B from 50 to 51 min and equilibrating for 6.0 min. 3.6. Characterisation of compounds 1 –4 Compound 1 (in fraction B). 1H NMR (500 MHz, methanol d-4) d 7.98 (2H, s), 7.25 (2H, br, s), 7.11 (2H, br, d, J ¼ 8.3 Hz), 6.99 (2H, br, d, J ¼ 8.3 Hz), 6.85 (4H, br, d, J ¼ 8.8 Hz), 6.67 (4H, br, d, J ¼ 8.8 Hz), 3.57 (2H, br, dd, J ¼ 12.7 and 7.8 Hz), 3.20 (2H, m), 2.44– 2.50 (4H, m). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 141.7, 129.8, 121.8, 115.6, 115.2, 111.5, 41.7 (2C), 34.5. ESI-MS m/z: 597 [MHþ]. Compound 2 (in fraction A). 1H NMR (500 MHz, methanol d-4) d 8.00 (1H, s), 7.98 (1H, s), 7.37 (1H, br, s), 7.26 (1H, br, s), 7.16 (2H, br, d, J ¼ 8.7 Hz), 6.91 (2H, m), 6.84 (4H, br, d, J ¼ 8.3 Hz), 6.65 (2H, br, d, J ¼ 8.8 Hz), 6.64 (2H, br, d, J ¼ 8.8 Hz), 3.77 (3H, s), 3.61 (2H, br, m), 3.24 (2H, br, m), 2.49 (4H, br, t, J ¼ 7.5 Hz). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 136.6, 128.8, 125.6, 114.4, 115.9, 112.6, 110.6, 57.7, 42.4 (2C), 37.5. ESI-MS m/z: 611 [MHþ]. Compound 3 (in fraction D). 1H NMR (500 MHz, methanol d-4) d 7.38 (1H, d, J ¼ 15.7 Hz), 7.15 (2H, br, d, J ¼ 8.4 Hz), 7.07 (1H, br, s), 6.97 (2H, br, m), 6.94 (1H, br, s), 6.81 (2H, m), 6.53 (1H, br, s), 6.34 (1H, d, J ¼ 15.7 Hz), 5.92 (1H, d, J ¼ 8.3 Hz), 4.11 (1H, d, J ¼ 8.3 Hz), 3.77 (1H, m), 3.52 (2H, m), 3.31 (1H, m), 2.88 (2H, m), 2.82 (1H, m), 2.76 (1H, m). 13 C NMR (125 MHz, methanol d-4, quaternary C omitted) d 142.0, 132.6 (4C), 124.2, 119.2, 118.2 (4C), 110.4, 110.2, 88.3, 47.9, 42.7 (2C), 41.7, 34.4, 33.5. ESI-MS m/z: 597 [MHþ]. Compound 4 (in fraction C). 1H NMR (500 MHz, methanol d-4) d 7.76 (1H, s), 7.20 (1H, s), 6.98 (1H, s), 7.13 (1H, br, d, J ¼ 8.3 Hz), 6.96 (2H, d, J ¼ 8.4 Hz), 6.90 (2H, d, J ¼ 8.4 Hz), 6.79 (1H, br, s), 6.78 (2H, d, J ¼ 8.4 Hz), 6.70 (2H, d, J ¼ 8.4 Hz), 6.43 (1H, br, d, J ¼ 8.3 Hz), 5.85 (1H, d, J ¼ 5.9 Hz), 4.63 (1H, t, J ¼ 5.9 Hz), 4.29 (1H, dd, J ¼ 5.9 and 3.5 Hz), 4.09 (1H, m), 3.83 (1H, dd, J ¼ 10.5 and 2.5 Hz), 3.77 (1H, dd, J ¼ 10.5 and 2.3 Hz), 3.55 (2H, m), 3.25 (2H, br, t, J ¼ 7.3 Hz), 2.69 (2H, br, t, J ¼ 7.4 Hz), 2.21 (2H, br, t, J ¼ 7.3 Hz). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 129.8 (4C), 125.8, 117.9, 117.7, 115.3 (4C), 112.5, 111.3, 109.5, 89.4, 74.6, 73.4 (2C), 71.2, 70.4, 41.8 (2C), 36.8, 35.0. ESI-MS m/z: 727 [MHþ]. 4. Conclusion This study provides useful information regarding non-drug hemp cultivars, which have been grown in the Po valley, in view of their potential industrial application. In the light of the healthpromoting properties of hempseeds, also supported by ongoing results from nutritional analyses, we evaluated the properties of oil and alcoholic extracts. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of v-3 and v-6, as requested for health. The alcoholic extract, for which a high content of phenolic components and amino acids
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could be highlighted, could provide dietary supplements to help the prevention of oxidative stress. By careful silica, LH-20 gel and high-performance liquid chromatographic investigation of the alcoholic extract of the Carmagnola cultivar, six known and four unprecedented lignanamides could be identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants. Acknowledgement This work was supported by Regione Lombardia through the project ‘VeLiCa – From ancient crops, materials and products for the future’ (protocol n 14840/RCC).
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content a
b
c
a
c
G. Lesma , R. Consonni , V. Gambaro , C. Remuzzi , G. Roda , A. a
a
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Silvani , V. Vece & G.L. Visconti a
Dipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy b
Istituto per lo Studio delle Macromolecole, ISMAC, CNR, v. Bassini 15, 20133 Milano, Italy c
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy Published online: 17 Jun 2014.
To cite this article: G. Lesma, R. Consonni, V. Gambaro, C. Remuzzi, G. Roda, A. Silvani, V. Vece & G.L. Visconti (2014): Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.926354 To link to this article: http://dx.doi.org/10.1080/14786419.2014.926354
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.926354
Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content G. Lesmaa, R. Consonnib, V. Gambaroc, C. Remuzzia, G. Rodac, A. Silvania*, V. Vecea and G.L. Viscontic
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a
Dipartimento di Chimica, Universita` degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy; bIstituto per lo Studio delle Macromolecole, ISMAC, CNR, v. Bassini 15, 20133 Milano, Italy; cDipartimento di Scienze Farmaceutiche, Universita` degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy (Received 7 April 2014; final version received 15 May 2014) Within a project aimed to reintroduce non-drug hemp cultivars in the Italian Po valley, for fibre but also high added-value nutraceutical production, investigation on locally grown plants has been performed, in order to assess their oil and metabolic content. This study provides useful information regarding three different hemp cultivars, from two sites, in view of their potential industrial application. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of v-3 and v-6 fatty acids, as requested for healthy foods. The alcoholic extracts, for which a high content of amino acids and phenolic compounds has been highlighted, could provide dietary supplements to help in preventing oxidative stress. By investigating the Carmagnola cultivar, six known and four new lignanamides have been identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants. Keywords: Cannabis sativa L.; fatty acid; total phenolic; lignanamides; cannabisin
1. Introduction Cannabis sativa L. has been an important source of food, fibre, dietary oil and medicine for thousands of years in Europe, Asia and Africa. Presently, non-drug varieties of C. sativa L., containing much less Tetrahydrocannabinol (THC) than common hemp, are important agricultural commodities, mainly in Canada, the USA and China. In the last decade, there has been a revived interest in hemp as a renewable resource in Europe, aimed at developing new production methods, new processing approaches and, conclusively, at updating the entire production chain. In fact, fibre production in countries with high-labour costs, such as Europe, can be more profitable if it is also coupled with exploitation of bioactive components, to yield high added-value nutraceutical products. Even if research continues to give new knowledge that enables the gradual improvement of the production of hemp, investigation on locally grown plants is always necessary, since the factors that influence both quality and quantity of production are directly connected to climate and soil conditions. To this end, in the frame of a research project aimed to reintroduce hemp cultivars in the Italian Po valley, the objective of this study was to investigate the seed extracts of three different cultivars (Carmagnola, Futura, Felina, known to contain less than 0.2% of THC This manuscript was submitted at SIF 2013, National Congress of Societa` Italiana di Fitochimica (Italian Phytochemical Society), Gargnano (Italy). *Corresponding author. Email: alessandra.silvani@unimi q 2014 Taylor & Francis
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in the whole plant), which have been grown in two sites (Treviglio (BG) and Cavriana (MN)) in the Lombardia region, in the last 3 years. In particular, since hempseed oil is recognised as a functional food (Callaway 2004) and an important source of essential fatty acids, we first evaluated its fatty acid profile, by using 1H NMR characterisation. The second focus was the assessment of phenol and flavonoid contents in seeds, in order to define their potential antioxidant value, and the evaluation of the free radicalscavenging capacity. Finally, we also performed 1H NMR-based metabolomic analysis (Kim et al. 2010), which provided us the possibility of identifying common metabolites in the three cultivars. For the more promising Carmagnola cultivar, a chromatographic separation was carried out on silica-gel and Sephadex columns, followed by HPLC – MS analysis, allowing to detect various metabolites (Flores-Sanchez & Verpoorte 2008), mainly lignanamides, and to identify four unprecedented cannabisin-like compounds. Their chemical structure were unambiguously assigned by MS and NMR analysis and, for three of them, also by analytical comparison with authentic, synthesised samples. 2. Results and discussion 2.1. 1H NMR spectroscopy-based fatty acid profile To obtain a rapid picture of the fatty acid profile, we made use of 1H NMR. A single analysis allowed the determination of a large number of parameters of oil samples, avoiding the preparation of derivatives that would be necessary for GC. We could easily quantify the common unsaturated fatty acids (UFAs: oleic, linoleic and linolenic acids), by applying standard equations, as reported in the literature (Knothe & Kenar 2004). The results are reported in Table 1. Seeds of all analysed cultivars contained similar amounts (25 –30%) of oil. The oils were high in UFAs, with a major accumulation in the cultivars grown in Treviglio, especially Carmagnola (89%). The polyunsaturated linoleic acid (18:2), running up to 63.1%, was the most abundant UFA, followed by a-linolenic (18:3) and oleic acid (18:1) with the ranges of 11.1– 20.9% and 10.8 –22.5%, respectively. The composition of fatty acids resulted to be slightly cultivar- and field site-dependent, with the cultivar Carmagnola providing a well-balanced proportion of all health beneficial UFAs. 2.2. Antioxidant and free radical-scavenging activity The quantitative determination of phenolic and flavonoid content as well as the evaluation of the antioxidant activity were performed (in triplicate) on the alcoholic extract of defatted seeds. The content of total phenolic compounds was determined by the colorimetric Folin –Ciocalteu method (Bonoli et al. 2004). The results obtained are shown in Table 2 and are expressed as gallic acid equivalents (GAEs). Cultivar and location were found to be rather significant, with Table 1. Oil yield and fatty acid contents of hemp seed oils (%, as from 1H NMR). Treviglio’s culture
Oil yield C18:3 C18:2 C18:1a Totalunsat a
Cavriana’s culture
Carmagnola
Futura
Felina
Carmagnola
Futura
Felina
29.0 20.9 57.6 10.8 89.0
30.0 11.1 63.1 12.3 86.0
30.0 19.4 54.1 13.6 87.1
25.3 20.3 31.9 22.5 74.8
29.6 17.5 52.5 12.3 82.3
26.2 15.4 50.7 13.8 80.0
Plus small amounts of g-linolenic (18:3) acid, not separately quantifiable.
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Table 2. Extraction yield, total phenolic content, flavonoid content and DPPH IC50 of hemp seeds methanolic extracts. Treviglio’s culture Carmagnola 9.9 6.14 ^ 0.57
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7.33 ^ 0.59 0.17 ^ 0.09
Cavriana’s culture Futura
Felina
Carmagnola
Futura
Extraction yield (%) 7.7 9.9 9.0 Total phenolic content (mg GAE/100 mg) extract 4.57 ^ 0.39 5.89 ^ 0.55 6.70 ^ 0.65 8.04 ^ 0.72 Flavonoids content (mg RE/100 mg) extract 5.39 ^ 0.23 10.90 ^ 0.44 9.78 ^ 0.63 9.28 ^ 0.83 DPPH (IC50 (mg/mL)) 0.27 ^ 0.03 0.10 ^ 0.02 0.18 ^ 0.02 0.15 ^ 0.01 6.9
Felina 7.8 7.27 ^ 0.55 7.67 ^ 0.67 0.15 ^ 0.07
the highest content of total phenolic compounds established in Cavriana cultivars. The total flavonoid content was evaluated (Zhishen et al. 1999) as rutin equivalents (REs) and resulted somewhat limited. The assay of the DPPH radical-scavenging activity (Chen et al. 2012), which is widely used to evaluate the antioxidant capacity of extracts, exhibited considerable values, with activity in the IC50 range of 0.10– 0.27 mg/mL, coming presumably from various kinds of phenolic compounds present in seeds. These results confirmed the possibility of utilising the waste products of the oil extraction process as source of natural antioxidants for food or nutraceutical industry. 2.3. 1H NMR spectroscopy-based metabolomic analysis For the reliable characterisation of cannabis cultivars, a metabolic profiling is desirable. NMR spectroscopy is one of the techniques that could describe the observable chemical profile or fingerprint of the complete metabolite pattern in a rapid and reproducible manner, requiring only a very basic sample preparation (Choi et al. 2004). The analysis revealed a common metabolite content for the three investigated cultivars, highlighting the presence of amino acids, such as alanine, valine, leucine, glutamine, organic acids, such as succinate, acetate, g-aminobutyrate, glutamate, saturated (palmitic) and unsaturated (linoleic, a-linolenic and oleic) fatty acids, saccharides, such as sucrose, myoinositol, glucose, and other molecules such as trigonelline and caffeoyltyramine in the aromatic region.
2.4. Isolation and characterisation of secondary metabolites For the chemical investigation of the metabolic content, we selected the Carmagnola cultivar, which was proving to be the most promising, both in terms of oil quality and of adaptation to the local climate and field specificity. Separation of the MeOH extract, using successive column chromatography over silica gel and Sephadex LH-20, resulted in the isolation of four fractions, which were then investigated by using HPLC – MS (ESI), 1D and 2D NMR. Six known lignanamides could be recognised by comparing the spectroscopic data with previously reported data, namely N-caffeoyltyramine and N-feruloyltyramine (Georgiev et al. 2013), N-(3,4dimethoxyphenethyl)-3-(3-hydroxy-4-methoxyphenyl)propanamide (Kametani et al. 1969), cannabisin A (Sakakibara et al. 1991), cannabisin G (Li et al. 2010; Xia et al. 2010) and cannabisin E (Sakakibara et al. 1995). In addition, four (1– 4) unknown lignanamides have been elucidated (Figure 1). For compounds 1 – 3, we also accomplished the preparation of authentic synthetic reference samples (Remuzzi 2014), for analytical comparison.
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Figure 1. Structures of lignanamides 1 – 4.
Compound 1 has been identified as a 3,30 -demethyl-cannabisin G. The 1H and 13C NMR spectra are very similar to those reported for cannabisin G, except for the absence of methoxy signals in 1. In particular, the 1H NMR spectrum resembles that of caffeoyltyramine, but bearing a unique olefin proton signal (d 7.98, s, 2H), instead of the trans-olefin spin system. In the 2D HMBC spectrum, cross-peaks are detected between the singlet olefin proton signal (d 7.98, 2H) and aromatic methine [d 111.5 (C-2 and C-20 ), 121.8 (C-6 and C-60 )] and carbonyl signal [d 167.0 (C-9 and C-90 )], respectively. From these data, the singlet signal at d 7.98 has been assigned to H-7 and H-70 , leading to propose the connection between C-8 and C-80 and therefore the chemical structure of compound 1. The molecular formula (C34H32N2O8, ESI-MS m/z: 597 [MHþ]) confirms 1 as a dimer of caffeoyltyramine. Compound 2 has been identified as 3-demethyl-cannabisin G. The 1H NMR spectrum is similar to that of 1, except for the presence of a methoxy group (d 3.77, 3H) and of two different olefin methine protons (d 8.00 and 7.98). Two distinct set of signals are observed for the proton H300 /H500 and H3000 /H5000 [d 6.64 (d, 2H) and 6.65 (d, 2H)], H2 and H20 [d 7.26 (s, 1H) and 7.37 (s, 1H)], H800 and H8000 [d 3.24 (2H) and 3.61 (2H)]. The molecular formula C35H34N208 (ESI-MS m/z: 611 [MHþ]) confirms 2 to be the dimer between a caffeoyltyramine and a feruloyltyramine unit, connected between the C-8 and C-80 position. Compound 3, more properly defined as a neolignanamide, has been recognised as 3,30 demethyl-grossamide. The proposed structure, and in particular the cis relationship between H70 / H80 , was confirmed by comparing with characteristic resonances and J values of the known parent compound (King & Calhoun 2005), and by key peak correlations in the HSQC spectrum [for instance dC 88.3 and dH 5.92 (d, 8.3 Hz) for CH-70 ; dC 47.9 and dH 4.11 (d, 8.3 Hz) for CH-80 ; dC 142.0 and dH 7.38 (d, 15.7 Hz) for CH-7; dC 119.2 and dH 6.34 (d, 15.7 Hz) for CH-8]. The presence of two tyramine-like moieties, bearing a tri- and a tetra-substituted aromatic group, respectively, has been further ascertained by a combination of 2D NMR experiments. The molecular formula of 3 is C34H32N2O8, by ESI-MS m/z: 597 [MHþ].
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Compound 4 resulted to be a cannabisin A derivative, in which the known lignanamide aglycone is O-conjugated with a C5-furanoside residue. The molecular formula of 4 is C39H38N2O12, by ESI-MS m/z: 727 [MHþ]. The 1H NMR spectrum shows signals for two tyramine moieties [d 2.21 (2H, br, t, 7.3 Hz), 2.69 (2H, br, t, 7.4 Hz), 3.25 (2H, br, t, 7.3 Hz), 3.55 (2H, m)]. The aromatic protons pattern of 4 is almost superimposable to that of cannabisin A, with the important exception of the signal of H50 , which appears at d 7.13 (br, d, 8.3 Hz), that is about 0.3 ppm downfield with respect to the same proton in cannabisin A. This observation strongly suggests the C-40 -OH as the position involved in the glycosidic bond. Careful analysis of 2D TOCSY pattern and 2D HMBC spectrum would suggest the C5 carbohydrate to be a ribose linked to the aglycone with a 1-b-glycosidic linkage. 3. Experimental 3.1. Plant material and extraction procedures Seeds of C. sativa L. varieties, Carmagnola (certified no. 9730015241), Futura 75 (certified no. 198465 MK) and Felina 32 (certified no. 204346 MK), were obtained from the seed company Assocanapa S.r.l, Carmagnola (TO), Italy. The three different hemp cultivars were cultivated under field conditions in 2010/2013 at the Cavriana (MN) and Treviglio (BG) sites, in the Italian Po valley. In 2013, after seed maturity, the plants were immediately harvested and seed samples were taken. Seeds (40 g) were ground using a high-speed grinder, then extracted by Soxhlet using heptane (400 mL) as solvent, during 8 h under reflux, followed by solvent removal. Defatted seeds were extracted with methanol/water (8:2) (200 mL) for 3 h under stirring. Then, the mixture was filtered and the combined methanol extract concentrated under vacuum. The procedure was repeated three times. 3.2. Determination of fatty acid composition by 1H NMR Spectra were recorded on a Bruker (Billerica, MA, USA) spectrometer, operating at 400 MHz at room temperature. The sample concentration was 100 mg of oil in 1 mL of CDCl3. Acquisition: 16 K data point, spectral width: 14 ppm, acquisition time: 3.6 s, relaxation delay: 10.0 s. 3.3. Metabolomic analysis by 1H NMR Spectra were recorded on a Bruker Avance DMX spectrometer, equipped with z-gradient coils with a proton resonance frequency at 500.13 MHz. All spectra were recorded at 298 K, with 32 K data points and 8000 Hz of spectral width and referenced to external sodium trimethylsilyl [2,2,3,3-2H4]propionate. Residual solvent suppression was achieved by applying a presaturation scheme with low-power radiofrequency irradiation. NMR spectra were acquired and processed with Topspin (v. 1.3) Bruker software. The optimised heteronuclear coupling constant for HSQC was set to 145 Hz, while the spin lock for TOCSY spectra was set to 80 ms. Spectral assignment has been performed on the basis of reported chemical shifts values and by comparing with the spectra of standard compounds. 3.4. Isolation procedures The methanol extract (4 g) was chromatographed on a silica gel column (80 g silica gel) using a chloroform/methanol step gradient, ranging from 80:20 to 30:70. Subsequent separation on Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA) column (15 cm £ 3 cm i.d.) using methanol as the eluent yielded four enriched fractions (A, 25 mg; B, 12 mg; C, 13 mg; D, 14 mg), which were subjected to HPLC – MS analysis.
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3.5. HPLC – MS protocols HPLC – MS analyses were performed on a Finnigan LCQ (MS) – Deca XP Max – Ion Trap Mass spectrometer – Thermo Electron Corporatione (Waltham, MA, USA) instrument. Chromatographic separation was achieved by a Zorbax SB C18 column (250 mm £ 4.6 mm i.d. – particle size 5 mm, Agilent Technologiese (Cernusco sul Naviglio, Milano, Italy)) protected by a Security Guard column C18 4 mm £ 2.0 mm (Phenomenexe (Castel Maggiore, Bologna, Italy)) at room temperature. The mobile phase consisted of 3 mM ammonium formate buffer pH ¼ 3.0 (phase A) and acetonitrile (phase B). Flow rate: 0.3 mL/min; gradient: 5% phase B for 5 min; from 5% phase B to 20 % phase B from 5.0 to 10.0 min; 20% phase B from 10.0 to 15.0 min; from 20% phase B to 30% phase B from 15.0 to 35.0 min; from 30% phase B to 40% phase B from 35 to 40 min; from 40% phase B to 30% phase B from 40 to 45 min; from 30% phase B to 20% phase B from 45 to 50 min; from 20% phase B to 5% phase B from 50 to 51 min and equilibrating for 6.0 min. 3.6. Characterisation of compounds 1 –4 Compound 1 (in fraction B). 1H NMR (500 MHz, methanol d-4) d 7.98 (2H, s), 7.25 (2H, br, s), 7.11 (2H, br, d, J ¼ 8.3 Hz), 6.99 (2H, br, d, J ¼ 8.3 Hz), 6.85 (4H, br, d, J ¼ 8.8 Hz), 6.67 (4H, br, d, J ¼ 8.8 Hz), 3.57 (2H, br, dd, J ¼ 12.7 and 7.8 Hz), 3.20 (2H, m), 2.44– 2.50 (4H, m). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 141.7, 129.8, 121.8, 115.6, 115.2, 111.5, 41.7 (2C), 34.5. ESI-MS m/z: 597 [MHþ]. Compound 2 (in fraction A). 1H NMR (500 MHz, methanol d-4) d 8.00 (1H, s), 7.98 (1H, s), 7.37 (1H, br, s), 7.26 (1H, br, s), 7.16 (2H, br, d, J ¼ 8.7 Hz), 6.91 (2H, m), 6.84 (4H, br, d, J ¼ 8.3 Hz), 6.65 (2H, br, d, J ¼ 8.8 Hz), 6.64 (2H, br, d, J ¼ 8.8 Hz), 3.77 (3H, s), 3.61 (2H, br, m), 3.24 (2H, br, m), 2.49 (4H, br, t, J ¼ 7.5 Hz). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 136.6, 128.8, 125.6, 114.4, 115.9, 112.6, 110.6, 57.7, 42.4 (2C), 37.5. ESI-MS m/z: 611 [MHþ]. Compound 3 (in fraction D). 1H NMR (500 MHz, methanol d-4) d 7.38 (1H, d, J ¼ 15.7 Hz), 7.15 (2H, br, d, J ¼ 8.4 Hz), 7.07 (1H, br, s), 6.97 (2H, br, m), 6.94 (1H, br, s), 6.81 (2H, m), 6.53 (1H, br, s), 6.34 (1H, d, J ¼ 15.7 Hz), 5.92 (1H, d, J ¼ 8.3 Hz), 4.11 (1H, d, J ¼ 8.3 Hz), 3.77 (1H, m), 3.52 (2H, m), 3.31 (1H, m), 2.88 (2H, m), 2.82 (1H, m), 2.76 (1H, m). 13 C NMR (125 MHz, methanol d-4, quaternary C omitted) d 142.0, 132.6 (4C), 124.2, 119.2, 118.2 (4C), 110.4, 110.2, 88.3, 47.9, 42.7 (2C), 41.7, 34.4, 33.5. ESI-MS m/z: 597 [MHþ]. Compound 4 (in fraction C). 1H NMR (500 MHz, methanol d-4) d 7.76 (1H, s), 7.20 (1H, s), 6.98 (1H, s), 7.13 (1H, br, d, J ¼ 8.3 Hz), 6.96 (2H, d, J ¼ 8.4 Hz), 6.90 (2H, d, J ¼ 8.4 Hz), 6.79 (1H, br, s), 6.78 (2H, d, J ¼ 8.4 Hz), 6.70 (2H, d, J ¼ 8.4 Hz), 6.43 (1H, br, d, J ¼ 8.3 Hz), 5.85 (1H, d, J ¼ 5.9 Hz), 4.63 (1H, t, J ¼ 5.9 Hz), 4.29 (1H, dd, J ¼ 5.9 and 3.5 Hz), 4.09 (1H, m), 3.83 (1H, dd, J ¼ 10.5 and 2.5 Hz), 3.77 (1H, dd, J ¼ 10.5 and 2.3 Hz), 3.55 (2H, m), 3.25 (2H, br, t, J ¼ 7.3 Hz), 2.69 (2H, br, t, J ¼ 7.4 Hz), 2.21 (2H, br, t, J ¼ 7.3 Hz). 13C NMR (125 MHz, methanol d-4, quaternary C omitted) d 129.8 (4C), 125.8, 117.9, 117.7, 115.3 (4C), 112.5, 111.3, 109.5, 89.4, 74.6, 73.4 (2C), 71.2, 70.4, 41.8 (2C), 36.8, 35.0. ESI-MS m/z: 727 [MHþ]. 4. Conclusion This study provides useful information regarding non-drug hemp cultivars, which have been grown in the Po valley, in view of their potential industrial application. In the light of the healthpromoting properties of hempseeds, also supported by ongoing results from nutritional analyses, we evaluated the properties of oil and alcoholic extracts. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of v-3 and v-6, as requested for health. The alcoholic extract, for which a high content of phenolic components and amino acids
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could be highlighted, could provide dietary supplements to help the prevention of oxidative stress. By careful silica, LH-20 gel and high-performance liquid chromatographic investigation of the alcoholic extract of the Carmagnola cultivar, six known and four unprecedented lignanamides could be identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants. Acknowledgement This work was supported by Regione Lombardia through the project ‘VeLiCa – From ancient crops, materials and products for the future’ (protocol n 14840/RCC).
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