Humudifucol and Bioactive Prenylated Polyphenols from Hops

Article pubs.acs.org/jnp

Humudifucol and Bioactive Prenylated Polyphenols from Hops (Humulus lupulus cv. “Cascade”) Martino Forino,† Simona Pace,‡ Giuseppina Chianese,† Laura Santagostini,§ Markus Werner,‡ Christina Weinigel,⊥ Silke Rummler,⊥ Gelsomina Fico,∥,# Oliver Werz,*,‡ and Orazio Taglialatela-Scafati*,† †

Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany § Department of Chemistry, University of Milan, Via Golgi 19, 20133 Milan, Italy ⊥ Institute of Transfusion Medicine, University Hospital Jena, D-07743 Jena, Germany ∥ Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy # Botanical Garden“G.E. Ghirardi”, Via Religione 25, 25088 Toscolano Maderno (BS), Italy ‡

S Supporting Information *

ABSTRACT: Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the antiinflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. “Cascade” has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low μM concentrations.

H

ops (Humulus lupulus L.) is a perennial plant of the Cannabaceae, a family including only two genera, Humulus and Cannabis. The hop plant has played a major role in human nutrition and culture because its female inflorescences are used in the production of beer, probably the oldest beverage fermented by humans. The inclusion of hop flowers has the double function of natural preservation and induction of the typical bitter taste by counterbalancing the sweetness of malt.1 Bitterness from hops can be ascribed to a number of compounds, particularly to the α-acids2 found in the glandular trichomes of the flowers. The most representative member of this class of molecules is humulone (1), a phloroglucinol derivative bearing three prenyl groups. It co-occurs with several analogues, differing in the length of the acyl side chain (e.g., cohumulone, carrying an isobutyryl in place of an isovaleryl),2 the cyclization mode, or in some chemical modifications ascribed to oxidations/reductions.2 The related β-acids (e.g., lupulone, 2), characterized by double prenylation at C-6, have been reported to possess antibacterial and anticancer activities.3 The plant H. lupulus has long been used also in traditional medicine, especially as a sedative and antimicrobial agent.4 © XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: November 25, 2015

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DOI: 10.1021/acs.jnatprod.5b01052 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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The HRESIMS of 6 exhibited a monocharged ion peak at m/ z 439.2457 [M + Na]+, corresponding to the molecular formula C25H36O5. Preliminary analysis of the 1H and 13C NMR spectra of 6 (Table 1) revealed a close analogy with values reported for

More recently, considerable attention has been devoted to the phytoestrogenic activity of the H. lupulus extract,5 likely ascribable to the prenylated flavanone 8-prenylnaringenin (3),6 and to the chemopreventive7 activities of prenylated chalcones, as exemplified by xanthohumol (4).8 The selection of hop metabolites and related bioactivities has become even more multifaceted due to the existence of many varieties of the plant, differing in bitterness and aroma, which are likely consequences of variations in the composition of αacids and essential oils, respectively.9 The cultivar “Cascade”, highly appreciated for its low-bitter aroma and resistance to fungal infection, was released in the United States in 1972 but it has since spread to several European countries, including Italy. Volatiles from the hop “Cascade” variety have been characterized in detail,10 and the occurrence of unusual polyfunctional thiol derivatives has also been reported.11 The aim of the present work was to complete the characterization of the phytochemical profile of H. lupulus cv. “Cascade” by investigating a sample cultivated in Italy. Herein are reported the isolation of seven constituents of this species, including the structure elucidation of the new compounds 6−8. Also the evaluation of their anti-inflammatory properties as reflected by the inhibition of microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), which are crucial enzymes in the biosynthesis of the pro-inflammatory lipid mediators PGE2 and leukotrienes, respectively, is described.

Table 1. NMR Data of 4-Hydroxycolupulone (6) in CDCl3 at 25 °C position 1 2 3 4 5 6 1′ 2′ 3′ 4′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴ 1‴′

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RESULTS AND DISCUSSION Inflorescences of H. lupulus cv. “Cascade” (100 g) were extracted with MeOH−H2O and then with acetone. The EtOAc phase obtained from combined extract was chromatographed following a 2,2-diphenyl-1-picrylhydrazyl (DPPH)guided approach. From the initial MPLC silica column, a combination of separation over Combiflash C18 and HPLC afforded humulone (1),12 8-prenylnaringenin (3),13 xanthohumol (4),14 and xanthohumol C (5),12 which were identified by comparison of their MS and NMR data with those reported in literature.12−14 Along with these compounds, also isolated were three new natural products, named 4-hydroxycolupulone (6), humudifucol (7), and cascadone (8).

2‴′ 3‴′ 4‴′ 5‴′

1

H, mult (J in Hz)

3.41, 1.10, 1.23, 2.62, 2.00, 4.90,

sep (6.7) d (6.7) d (6.7) dd (12.1, 9.7) dd (12.1, 6.5) m

1.42, 1.61, 2.81, 2.75, 5.05,

bs bs dd (13.8, 9.3) dd (13.8, 6.3) dd (9.3, 6.3)

1.62, 1.61, 2.64, 2.43, 4.62,

s s dd (13.0, 7.4) dd (13.0, 9.5) dd (9.5, 7.4)

1.35, s 1.49, s

13

C

198.7 111.4 193.0 85.3 204.9 61.9 206.7 35.1 18.0 20.6 37.2 115.6 136.6 18.2 26.0 34.0 119.1 136.7 17.8 26.0 41.8 116.7 139.0 18.1 26.2

colupulone,15 featuring an isobutyryl unit in place of the typical isovaleryl unit present in both humulone and lupulone. However, in comparison to colupulone,15 the molecular formula of compound 6 was found to contain one more oxygen atom, suggesting the presence of an extra hydroxyl group functionality. The available NMR data of colupulone15 were compared with those derived from the HMBC and HSQC spectra of 6 and used as a guide in the structural determination of the latter compound. As expected, three prenyl functionalities along with an isobutyryl unit were identified. The H-1″/ C-4 HMBC correlation allowed the location of one prenyl group at C-4; similarly, the remaining two prenyl units were connected to C-6 due to the H-1‴/C-6, H-1‴′/C-6, H-1‴′/ C1‴ and H-1‴/C1‴′ HMBC correlations. Likewise, the H-2′/ C-2 HMBC correlation led to the positioning of the isobutyryl group at C-2. As a result, the difference in terms of NMR data, and thus in chemical structure between compound 6 and colupulone, was restricted to the unprotonated sp3 C-4, which, on account of its chemical shift (δC 85.3) and in agreement with the molecular formula, was deduced to bear a hydroxy group. The HMBC correlations involving H2-1″, H2-1‴ and H2-1‴′ supported the location of two oxygenated sp2 carbons at positions C-1 and C-5, respectively. It is noteworthy that, unlike the carbonyl groups at C-1 and C-3, the C-5 (δC 204.9) carbonyl is a nonenolizable ketone, and B



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Table 2. NMR Data of Humudifucol (7) Registered in CD3OD at 25 °C position 1 2 3 4 5 6 7 8 9 10 11

1

H, mult (J in Hz)

5.89, s 3.25, dd (14.3, 7.5) 3.18, overlapped 5.18, m 1.64, s 1.75, s

13

C

position

101.4 157.0 107.7 164.3 94.5 161.0 20.4

1′ 2′ 3′ 4′ 5′ 6′ 7′

122.8 129.6 24.2 16.1

8′ 9′ 10′ 11′

1

H, mult (J in Hz)

5.88, s 3.18, overlapped 5.18, m 1.65, s 1.74, s

13

C

101.4 160.2 106.8 164.0 94.5 161.0 20.4 122.4 129.7 24.2 16.1

linkage have been reported from brown algae (e.g., difucol),17 to the best of our knowledge, compound 7 represents the first example of prenylated member of this class. In contrast to our first assumption, the planar structure obtained for 7 appeared to be symmetrical. Hence, the dissymmetries in the 1H NMR spectrum of 7 were inferred to be the result of atropisomerism with respect to the biphenyl bond. The occurrence of a significant rotational barrier in 7 was also supported by NMR studies aimed at evaluating the coalescence temperature, i.e., the temperature at which the diastereotopic protons of the dimer coalesce into a single NMR signal. Thus, by recording 1H NMR spectra at increasing temperature values, a coalescence temperature of ≥50 °C was determined experimentally, thus supporting the existence of a rotational barrier at room temperature.18 Notably, this type of stereoisomerism has not been reported before for members of the difucol compound class. On the basis of the NMR data alone, information about the enantiomeric composition of natural humudifucol (7) could not be deduced. However, measurement of both its optical rotation, [α]25D + 19.5 (c 0.2, MeOH), and CD spectrum (Figure 1) suggested the

the reported structure of 6 represents just one of the possible tautomers, selected by analogy with the commonly described tautomers of lupulone (2) and colupulone. However, while in colupulone the conjugated system of the molecule stretches also across the C-4 and C-5 sp2 carbons, such a conjugation is not any longer possible in 6, as its C-4 is an sp3 carbon. Thus, it can be expected that the relative stabilities of the possible tautomers of 6 are reasonably different from those of colupulone. From a configurational point of view, because no optical rotation was observed for 6, it is assumed that an equimolar mixture of the two enantiomers at C-4 was isolated. 4-Hydroxycolupulone (6) has been reported in the literature already as a synthetic product, but only a fragmentary NMR characterization was provided.16 To the best of our knowledge, this is the first report of 4-hydroxycolupulone from a natural source. However, considering its racemic nature, it cannot be excluded that 6 is an artifact. The molecular formula, C 22 H 27 O 6 , was assigned to humudifucol (7) by HRESIMS (m/z 387.1806 [M + H]+). Both the 1H and 13C NMR spectra of 7 (Table 2) clearly showed two patterns of signals partly superimposable and partly slightly differing from each other. This suggested that 7 is a dimer constituted by two monomers that are not identical. More precisely, the 1H NMR spectrum of 7 showed two 1H singlets resonating at δH 5.88 and 5.89 ppm, respectively, four allylic methyl signals, a 2H resonance at δH 5.19 ppm, and two further resonances at δH 3.19 and 3.25, with the former integrating for 3H and the latter for 1H. Cross-interpretation of COSY, HSQC, and HMBC experiments assigned the above proton signals as two prenyl groups, with the two singlets mentioned above likely belonging to oxygenated phenyl rings. Two sets of key HMBC correlations (H2-7 with C-2, C-3, C-4, and H2-7′ with C-2′, C-3′, and C-4′) defined the attachment of the two prenyl chains at C-3 and C-3′, respectively, both adjacent to two oxygenated phenyl carbon atoms. Similarly, the correlations of H-5 with the oxygenated C-4 and C-6 (and the parallel correlations of H-5′ with both C-4′ and C-6′) were instrumental in defining the position of the single aromatic proton on each phenyl ring. The 13C NMR chemical shifts of C-2, C-4, and C-6 (and of C-2′, C-4′, and C-6′), all resonating in the 157−164 ppm range, supported the positioning of three −OH groups at the above positions. Thus, in accordance with the molecular formula, C-1 and C-1′ are the locations of a single C−C bond connecting the two phenyl rings. Humudifucol (7) is therefore a new dimeric phloroglucinol, a class of compounds unprecedented in the hop plant. Remarkably, while some dimeric phlorotannins with a diphenyl

Figure 1. Left: COSY (bold lines) and HMBC (red arrows) correlations of humudifucol (7). These correlations have been reported for a single unit. Right: Experimental CD spectrum of 7 (black curve) and simulated CD spectra corresponding to the aR (red curve) and aS (blue curve) enantiomers of 7, calculated using the TDDFT approach.

occurrence of a predominant enantiomer. Its absolute configuration was assigned by comparison of the experimental CD curve with those simulated for the two enantiomers of 7, using the TDDFT approach. As depicted in Figure 1, the theoretical curve corresponding to the aR enantiomer closely mimicked the experimental one and, thus, the aR configuration was assigned to humudifucol (7). The HRESIMS of cascadone (8) yielded the molecular formula C21H30O6, in agreement with seven degrees of C



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Table 3. NMR Data of Cascadone (8) Registered in CD3OD at 25 °C and 13C NMR Data Calculated for the RR(SS) and RS(SR) Stereoisomers, Respectively position 1 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ MAE values

1

H, mult (J in Hz)

2.26, dd (14.0, 4.4) 2.15, dd (14.0, 6.5) 4.69, dd (6.5, 4.4) 1.21, s 1.31, s 2.75, 2.12, 0.96, 0.99, 3.10, 2.98, 5.17,

bd (6.8) m d (6.1) d (6.6) dd (13.9, 7.0) dd (13.9, 6.2) dd (7.0, 6.2)

1.73, s 1.66, s

experimental 13C values

calculated 13C values for RR(SS) stereoisomer

calculated 13C values for RS(SR) stereoisomer

191.1 106.2 194.6 108.8 173.1 72.1 35.8

189.6 103.2 182.2 101.9 172.0 76.4 33.4

187.1 103.0 179.9 101.5 170.6 77.8 34.2

90.7 69.9 24.5 24.7 197.5 45.8 27.9 22.7 22.9 24.3

90.2 70.3 26.1 26.7 198.6 50.1 27.9 20.8 23.9 24.0

89.6 69.0 22.9 27.4 199.8 49.8 28.4 20.9 24.2 22.6

122.3 132.5 18.0 25.9

120.2 132.2 18.4 25.6 2.28

120.3 130.6 18.1 25.6 2.91

Figure 2. Superimposition of the top 10 lowest energy conformers calculated for the RR(SS) (left) and the RS(SR) (right) diastereoisomers of 8.

unsaturation. Preliminary investigations of the 1H and 13C NMR spectra (Table 3) suggested an oxygenated humulone analogue, for which the 2D NMR HSQC experiment allowed the identification of nine quaternary carbons (seven sp2, two of which are ketone groups, and two oxygenated sp3) and 12 protonated (six methyl, three methylenes, and three methines) carbon atoms. Inspection of the 2D COSY and HMBC NMR spectra led to the identification of an isovaleryl (C-1′ to C-5′) and a prenyl unit (C-1″ to C-5″). The COSY experiment was used to identify one additional proton spin system including a diastereotopic methylene and an oxymethine (H2-7 and H-8, respectively). The HMBC correlations of Me-10 and Me-11 with both the oxygenated C-8 and C-9 defined a dioxygenated prenyl unit. The NMR-based analysis of the remaining six quaternary carbons led to the identification of a humulone-like six-membered ring. Then the HMBC cross-peak H2-2′/C-2 was used to connect the isovaleryl unit to C-2, while the

correlations of H2-1″ with C-3, C-4, and C-5 were instrumental in linking the prenyl functionality to C-4. In accordance with a humulone architecture, the HMBC correlations of H2-7 with C1, C-5, and C-6 allowed the localization of the dioxygenated prenyl unit. Finally, on account of the unsaturation degrees implied by the molecular formula, as well as of the presence in the molecule of seven oxygenated carbons and of only six oxygen atoms, the presence of an ether bridge was inferred. This latter functionality was positioned unambiguously between C-8 and C-5 on the basis of a key H-8/C-5 HMBC correlation, thus defining the planar structure of cascadone (8) as a new cyclized analogue of humulone. No reliable diagnostic ROE effect was detected in order to deduce the relative configuration of the two stereogenic carbons of cascadone (8) at C-6 and C-8. Hence, ab initio calculations of NMR chemical shifts were conducted. The first step of this analysis was a molecular modeling investigation on D



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atherosclerosis, and tumorigenesis.22 While some flavonoids have been identified as capable of modulating the expression of mPGES-1, recently, cannflavins, prenylated flavonoids unique to hemp23 have been demonstrated to constitute the first reported flavonoids that directly inhibit both mPGES-1 and 5LO.24 Because the prenylated phloroglucinols hyperforin,25 arzanol,26 and garcinol27 also dually inhibit 5-LO and m-PGES1, it has been speculated24 that prenylation could be a key structural feature to confer the inhibitory activity of phloroglucinols against these enzymes. The prenylated compounds purified from H. lupulus cv. “Cascade” were tested for their inhibitory activities in wellestablished cell-free assays28,29 against isolated human recombinant 5-LO and human mPGES-1 in microsomes of A549 cells and in a cell-based assay for 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL).29 As shown in Table 4, xanthohumol (4) and 4-hydroxycolupulone (6) showed the

the RR(SS) and the RS(SR) diastereoisomers of 8. Simulations were carried out for 8 ns by using the CVFF force field as implemented in Discover software. As reported in detail in the Experimental Section, 100 structures were generated for each diastereoisomer and the total energy variations for both models were in the range of 3 kcal/mol. As expected, the bicyclic system in both diastereoisomers showed a very limited conformational mobility as opposed to the much higher flexibility possessed by the molecule side chains. Nonetheless, the latter effect did not significantly affect the energy content of the molecules (Figure 2). Thus, the lowest energy conformers obtained for the RR(SS) and the RS(SR) diastereoisomers of 8 were utilized for NMR chemical shift calculations using the Gaussian03 software.19 The NMR data obtained for the two diastereomers (Table 3) were then compared to the experimental NMR data recorded for 8 by uploading them into the online available DP4 method applet developed by Smith and Goodman.20 The outcome of such DP4 analysis identified the RR(SS) stereoisomer as the most likely, with a probability of 99.2%. Mean absolute error (MAE) values also supported this assignment: RR(SS) = 2.28, RS(SR) = 2.91. The assessment of the absolute configuration of 8 could not be carried out by using the available chiral auxiliaries due to the low reactivity of the C-6 and C-8 oxygenated carbons. Thus, a ECD spectrum was simulated for both the RR and SS enantiomers through the TDDFT methodology by employing again the same lowest energy conformer used in the NMR calculation. As depicted in Figure 3, the calculated curve for the

Table 4. Effect of Compounds 1, 3−8 on Key Enzymes of Eicosanoid Biosynthesis compd

5-LOa (cell-free)

5-LOa (cell-based)

mPGES-1c

1 3 4 5 6 7 8 zileutone MK886f

9.3 >10 (66.7 ± 10.6) 2.1 >10 (58 ± 9.6) 5.9 nib >10 (90.8 ± 5.1) (25 ± 4.3) ndd

>10 (63.1 ± 12.0) >10 (87.5 ± 8.3) 2.9 >10 (56.8 ± 10.3) >10 (57.5 ± 9.0) nib nib (59.4 ± 7.5) ndd

(49.7 ± 1.8) (56.5 ± 0.7) (32.3 ± 1.3) (35.3 ± 3.8) (32.8 ± 4.1) (40.2 ± 3.6) (52.7 ± 3.9) ndd (39.9 ± 6.4)

a

IC50 values (μM) and residual activity (% of control) are given as mean ± SEM of single determinations obtained in three to four independent experiments. bNo inhibition/no effect. cResidual activity at 10 μM compound concentration. dNot determined. eResidual activity at 1 μM compound concentration. fResidual activity at 1 mM compound concentration.

most potent inhibitory effects against isolated 5-LO (IC50 2.1 and 5.9 μM, respectively). When tested in intact PMNL, xanthohumol (4) turned out to be an effective inhibitor of 5LO with an IC50 of 2.9 μM, while the bioactivity of 6 was drastically impaired, indicating that cell permeation or intracellular stability of 6 is reduced or any other intracellular events may hamper 5-LO inhibition by the compound. None of the other tested compounds caused significant 5-LO inhibition in PMNL cells at the concentrations used. Compounds 1 and 3−8 were also tested for their inhibitory activities against mPGES-1 (Table 4), and again xanthohumol (4) and 4-hydroxycolupulone (6) were the most active compounds. At a concentration of 10 μM, a residual enzymatic activity of about 32% was observed. In spite of the limited number of compounds tested, some insights into the structure−activity relationships for the prenylated hop metabolites can be deduced. The inactivity of xanthohumol C (5) against 5-LO is very interesting because the difference to the active xanthohumol (4) in terms of chemical structures is restricted to the involvement of the prenyl moiety of 5 in the formation of a six-membered ether ring. Hence, this result corroborates the hypothesis that an intact prenyl group is fundamental for the inhibition of 5-LO.24 Moreover, the good inhibitory activity against both mPGES-1 and 5-LO, as evidenced by 4-hydroxycolupulone (6), could be compared to

Figure 3. Experimental CD spectrum of cascadone (8) (black curve) and simulated CD spectra corresponding to the SS (blue curve) and RR (red curve) enantiomers of 8 as calculated using the TDDFT approach.

SS stereoisomer paralleled the experimental spectrum of 8, thus defining the absolute configuration of the molecule. Concerning the S configuration at C-6 of 8, it should be noted that this same absolute configuration has been assigned to the same carbon of humulone by Urban et al.,21 whose study concluded a long period of uncertainty about the stereochemistry of this compound. The prenylated compounds purified from hops were then examined for their potential bioactivities with a focus on the two pro-inflammatory enzymes, microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO). Selective inhibition of mPGES-1, an enzyme functionally coupled to COX-2 and responsible for excessive PGE2 generation, has been indicated as a promising target in inflammation, pain, E



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column eluted with a stepwise gradient of n-hexane and EtOAc for which the ratio changed from n-hexane to EtOAc by increasing the EtOAc percentages by 10% (v/v) after each 250 mL. Three antioxidant fractions were obtained. The first one (A fraction) was eluted with a mixture of n-hexane−EtOAc (9:1), the second (B fraction) was eluted with a mixture of n-hexane−EtOAc (7:3), and the last (C fraction) with a mixture of n-hexane−EtOAc (1:1). The three antioxidant fractions were each separated through a 360-g Combiflash C18 column eluted with a gradient of H2O/MeOH for which ratio changed linearly from 1:9 to MeOH in 60 min. The antioxidant fraction derived from fraction A was purified finally on a HPLC Luna 5 μm column with a mixture of H2O−MeOH (5:95), thus affording 15.8 mg of pure 4-hydroxycolupulone (6). The antioxidant compounds contained in fraction B were purified on a HPLC Gemini 10 μm column eluted with H2O−MeOH (1:9). This purification step led to the isolation of two known compounds, namely, 8-prenyl naringenin (3, 1.0 mg) and xanthohumol C (5, 2.3 mg), in addition to humudifucol (7, 0.9 mg) and cascadone (8, 1.1 mg). The final step of the purification of the fraction C was conducted on a HPLC Gemini 10 μm column eluted with H2O−MeOH (2:8). Two compounds were isolated and identified as humulone (1) and xanthohumol (4, 220 mg). In the case of humulone (1), part of the compound degraded during the purification procedure, nonetheless, it can be assessed that humulone was present in a greater than 200 mg amount. 4-Hydroxycolupulone (6). Yellow amorphous powder; [α]25D 0 (c 0.2, MeOH); 1H and 13C NMR data in CDCl3 at 25 °C are reported in Table 1. HRESIMS ([M + Na]+) m/z 439.2457 (calcd for C25H36O5Na 439.2460). Humudifucol (7). Yellowish amorphous powder; [α]25D + 19.5 (c 0.2, MeOH); CD: λmax 280 nm Δε −3.8 (MeOH). 1H and 13C NMR data in CD3OD at 25 °C are reported in Table 2. HRESIMS ([M + H]+) m/z 387.1806 (calcd for C22H27O6 387.1808). Cascadone (8). Yellowish amorphous powder; [α]D25 + 25.8 (c 0.3, MeOH); CD: λmax 210 nm Δε −1.9 (MeOH).1H and 13C NMR data in CD3OD at 25 °C are reported in Table 3. HRESIMS ([M + Na]+) m/z 401.1932 (cald for C21H30NaO6 401.1940). Computational Calculations. Computational calculations for 7 and 8 were conducted by using a distance-dependent macroscopic dielectric constant of 4r and an infinite cut off for nonbonded interactions in order to partially compensate the absence of solvent. 3D structures were obtained by simulated annealing calculations. Conformational energies were minimized by the steepest descent followed by quasi-Newton−Raphson method (VA09A). Restrained simulations were carried out for 8 ns using the CVFF force field as implemented in Discover software. The simulation started at 300 K, and then the temperature was decreased stepwise as low as 100 K. The structures obtained were again subjected to energy-minimization by using successively the steepest descent and the quasi-Newton− Raphson (VA09A) algorithms. Altogether, 100 structures were generated and their illustrations afforded by the Insight II. DFT calculations were conducted using the Gaussian03 package (Multiprocessor). The DP4 NMR prediction analysis on the lowest energy conformations for the RR(SS) and the RS(SR) stereoisomers of 9, respectively, was carried out according to Smith and Goodman.36 By means of Gaussian03, the carbon and proton GIAO NMR shielding tensors at the mPW1PW91 functional and 6-31G(d,p) basis set were obtained, using as input the optimized geometry at the mPW1PW91/ 6-31G(d) level. For these calculations, the IEF-PCM solvent continuum model, as implemented in Gaussian (methanol solvent), was adopted. The lowest energy conformation of 7 (defining a C2/C1/C1′/C2′ dihedral angle of 57.96°) was employed for TDDFT calculations by means of the functional B3LYP, the basis sets TZVP including 30 excited states, and the IEF-PCM for methanol. ECD spectra were thus obtained. Likewise, ECD spectra for 8 were obtained by using the obtained lowest energy conformations for the RR(SS) and the RS(SR) stereoisomers, respectively. Cells and Cell Isolation. Polymorphonuclear leukocytes (PMNL) were isolated from human peripheral blood that was taken from fastened (12 h) healthy donors by venipuncture in heparinized tubes

the lack of bioactivity of humulone (1) and cascadone (8) on the same end points. It can be hypothesized that the additional prenyl group in 6 as well as the different substitution pattern of the phloroglucinol ring are needed for the inhibition of these enzymes. The inflorescences of H. lupulus cv. “Cascade”, cultivated in northern Italy, have been revealed to contain the new prenylated phloroglucinol analogues (i.e., 6 and 8) and humudifucol (7), the first example of prenylated dimeric phlorotannin found in nature. The optical rotation and CD spectra of 7 demonstrated that this compound occurs as a single (or predominant) atropisomer with respect to the biphenyl bond. A similar stereoselectivity has been reported only for a very limited number of natural products, with probably the most interesting example being the cotton pigment gossypol, for which the biosynthesis has been demonstrated to arise from a protein-mediated dimerization process.30 Hops, and consequently beer, have been long associated with anti-inflammatory and cancer chemopreventive activities,31 which have been ascribed to flavonoid glycosides,32 acylphloroglucinols,33 and prenylated chalcones. The chalcone xanthohumol (4) has been identified as an active anti-inflammatory agent, and several modes of action (e.g., Nrf2 stimulation,34 IL2 inhibition35) have been proposed. The present finding that xanthohumol (4) inhibits 5-LO and mPGES-1 adds another component to this multifaceted bioactivity, revealing direct molecular protein targets. Considering that xanthohumol (4) is a major component of the organic extract, it can be anticipated that its contribution to the bioactivity of the plant extracts is of key relevance.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations (CHCl3) were measured at 589 nm on a P2000 JASCO polarimeter. UV spectra were acquired on Tecan Italia spectrophotometer and on an EL 311s automated microplate reader (Bio-Tek Instruments). CD spectra were registered on a JASCO J-710 instrument. NMR experiments were run on Varian Unity INOVA 700 MHz NMR spectrometer equipped with a 13C enhanced HCN cold probe and by using a Shigemi 5 mm NMR tubes. Standard Varian pulse sequences were employed, with solvent signal suppression by presaturation used when required. All NMR data reported in the text were derived from 1D 1H and 13C NMR spectra as well as from 2D COSY, TOCSY, ROESY, phase-sensitive HMBC, and HSQC spectra. Mass spectrometric studies were performed by using linear ion trap LTQ Orbitrap XL hybrid Fourier transform MS (FTMS) instrument equipped with an ESI ION MAX source (Thermo-Fisher) and coupled to an Agilent 1100 LC binary system including a solvent reservoir, online degasser, binary pump, and thermostated autosampler. Plant Material. Hop plantlets were purchased at Garten Eickelmann (Geisenfeld, Germany) and cultivated for four years in an experimental site (Farm La Morosina, Abbiategrasso, Milan, Italy) and collected in 2014. Cones, collected at maturity, were dried at 40 °C in a thermostatic room, protected from light. A voucher specimen was deposited at the Herbarium of the Botanical Garden GE Ghirardi, Department of Pharmaceutical Sciences, University of Milan, with number Hl 101. Extraction and Isolation. A sample of the inflorescences of H. lupulus cultivar Cascade (100 g), was triturated and extracted twice with 500 mL of a MeOH−H2O (8:2) (v/v) mixture. The residue was finally extracted with acetone. The extracts were combined and evaporated, and the resulting aqueous solution was first partitioned three times against EtOAc (200 mL) and then against butanol (200 mL). The acetate extract was separated by a DPPH-guided approach. The first step of the separation was performed on a MPLC silica F



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(16 IE heparin/mL blood). The ethical committee of University Hospital of Jena (Germany) has approved collection of blood from healthy human volunteers. The donors had not taken any antiinflammatory drugs during the last 10 days. The blood was centrifuged (4000g for 20 min at RT), and the leukocyte concentrates were subjected to dextran sedimentation and centrifugation on Nycoprep cushions (PAA Laboratories, Linz, Austria). Remaining erythrocytes in pelleted PMNL were lysed by hypotonic lysis. PMNL were washed twice in ice-cold PBS and finally resuspended in PBS pH 7.4 containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer) (purity >96−97%). Analysis of 5-LO Activity in Intact Cells. For analysis of 5-LO product formation in intact polymorphonuclear leukocytes, 5 × 106 cells were resuspended in 1 mL PBS pH 7.4 containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer), preincubated with test compounds or vehicle (0.3% DMSO) for 15 min at 37 °C, and incubated for another 10 min at 37 °C with Ca2+-ionophore A23187 (2.5 μM) plus 20 μM arachidonic acid (AA). After 10 min at 37 °C, the reaction was stopped on ice by addition of 1 mL of methanol, and 30 μL of 1N HCl, 500 μL of PBS, and 200 ng of prostaglandin B1 were added and the samples were subjected to solid-phase extraction on C18-columns. 5-LO products [(LTB4, trans-isomers, 5-H(p)ETE)] were analyzed by HPLC and quantities calculated on the basis of the internal standard PGB1. Expression, Purification and Cell-Free 5-LO Activity Assay. Escherichia coli MV1190 cells were transformed with pT3−5-LO plasmid, and recombinant 5-LO protein was expressed at 27 °C, as described previously.37 Cells were lysed in 50 mM TEA/HCl pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 μg/mL), 1 mM phenylmethanesulphonyl fluoride, and lysozyme (500 μg/mL), homogenized by sonication (3 × 15 s), and centrifuged at 40000g for 20 min at 4 °C. The 40000g supernatant (S40) was applied to an ATP-agarose column to partially purify 5-LO.36 Aliquots of partially purified 5-LO were diluted with ice-cold PBS containing 1 mM EDTA, and 1 mM ATP was added. The samples were preincubated with the test compounds on ice. After 10 min, samples were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 plus 20 μM arachidonic acid (AA) were added. After a further 10 min, the reaction was stopped by addition of 1 mL ice-cold methanol, and the metabolites formed were analyzed by RP-HPLC, as described previously.36 Expression of mPGES-1 and Cell-Free mPGES-1 Activity Assay. The preparation of microsomes of IL-1β-stimulated A549 cells and determination of mPGES-1 activity was performed as described previously.28 Briefly, A549 cells were treated with Il-1β (1 ng/mL) for 48 h at 37 °C, 5% CO2. Cells were harvested and sonicated, and the homogenate was subjected to differential centrifugation at (a) 10000g for 10 min and (b) 174000g for 1 h at 4 °C. The pelleted 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), the total protein concentration was determined, and microsomal membranes were diluted in potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione. Test compounds or vehicle were added, and after 15 min at 4 °C reaction (100 μL total volume) was initiated by addition of 20 μM PGH2. After 1 min at 4 °C, 100 μL of stop solution (40 mM FeCl2, 80 mM citric acid, and 10 μM 11β-PGE2) were added. PGE2 was separated by solid-phase extraction and analyzed by RP-HPLC as described previously.28

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Article

AUTHOR INFORMATION

Corresponding Authors

*For O.T.S. Phone: +39-081678509. Fax: +39-081678552. Email: [email protected]. *For O.W.: Phone: +49-3641-949801. Fax: +49-3641-949802. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mass and NMR spectra were recorded at the “Centro di Servizio Interdipartimentale di Analisi Strumentale”, Università di Napoli Federico II. This research was partly funded by Regione Campania under POR Campania FESR 2007-2013O.O.2.1 (FarmaBioNet) and by the Deutsche Forschungsgemeinschaft (DFG) within the SFB 1127: “Chemical Mediators in Complex Biosystems”. We thank “Azienda Agricola La Morosina”, Abbiategrasso (Milan, Italy), for providing the experimental material.

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DEDICATION Dedicated to Professors John Blunt and Murray Munro, of University of Canterbury, for their pioneering work on bioactive marine natural products.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01052. 1D and 2D NMR spectra of the new compounds 6−8 (PDF) G



Article

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Humudifucol and Bioactive Prenylated Polyphenols from Hops

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