Characteristic Î±âAcid Derivatives from Humulus ... - ACS Publications

Article pubs.acs.org/jnp

Characteristic α‑Acid Derivatives from Humulus lupulus with Antineuroinflammatory Activities Jiayuan Li,† Ning Li,*,† Xuezheng Li,‡ Gang Chen,† Cungang Wang,† Bin Lin,§ and Yue Hou*,⊥ †

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang; Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Wenhua Road 103, Shenyang 110016, People’s Republic of China ‡ Department of Pharmacy, Affiliated Hospital of Yanbian University, Yanji 133000, People’s Republic of China § School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China ⊥ College of Life and Health Sciences, Department of Biochemistry and Molecular Biology, Northeastern University, Shenyang 110819, People’s Republic of China S Supporting Information *

ABSTRACT: Twenty compounds, including 14 new α-acid derivatives, a new chromone, and five known compounds, were identified from the pistillate inflorescence of Humulus lupulus (hops), and their structures were elucidated via physical data analysis. The absolute configurations of new α-acid derivatives 1−11b were determined by comparing their computed and experimental electronic circular dichroism spectra using TDDFT and NMR spectroscopic data. A putative biosynthetic pathway for the identified components was deduced. Their antineuroinflammatory effects were assayed systematically, and their structure−activity relationships are discussed briefly. Among the identified compounds, compound 14 displayed moderate inhibitory effects against nitric oxide production with an IC50 value of 7.92 μM. Humulus lupulus L. (hops), a vigorous climbing herbaceous perennial of the Moraceae family, is widely cultivated in the Xinjiang, Gansu, Heilongjiang, and Shandong provinces in China.1 It is typically used in the beer industry to produce bitterness and aromatic flavors during fermentation.2 In addition, hops is a traditional folk medicine with significant sedative, antispasmodic, stomachic, hypnotic, tonic, diuretic, and bactericidal effects.3−9 Previous phytochemical investigations of hops revealed that its characteristic secondary metabolites are α-acids, β-acids, and their derivatives, flavones, and chalcones.10 Recent studies have demonstrated that hops extract possesses antiviral, antioxidant, and estrogen-like effect activities,5−9 but few reports have addressed its antineuroinflammatory effects. In previous research,11−14 we focused on natural neuroinflammatory inhibitors with the potential to act as therapeutic agents for neurodegenerative diseases. Hops extract attracted our interest because of its significant inhibitory effect on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced microglial cells. Thus, a bioactivity-guided phytochemical study was performed on the active fraction of the extract and led to the isolation of 15 new compounds: (3R,8S)-humulone A (1), (3S,8S)-humulone A (2), (3S,8S)-cohumulone A (3), (2S,7S)humulone B (4), (2R,7S)-humulone B (5), (2S,7S)-cohumulone B (6), (2S,3aS)-humulone C (7), (3S,3′R,5R)-humulone acid B (8), (R)-lupulone H (9a), (S)-lupulone H (9b), (R)© 2017 American Chemical Society and American Society of Pharmacognosy

lupulone G (10a), (S)-lupulone G (10b), (R)-5-deprenyllupulonol C (11a), (S)-5-deprenyllupulonol C (11b), and 5,7dihydroxy-2-isopropyl-8-prenylchromone (12). Additionally, five known constituents were identified: hulupinic acid (13), xanthohumol (14), 4-methoxyxanthogalenol (15), isoxanthohumol (16), and (1R,2R,5R,8S,9S)-2α,9β-clavondiol (17). The absolute configurations of new components 1−11b were elucidated by comparing their calculated and experimental electronic circular dichroism (ECD) spectra using timedependent density functional theory (TDDFT).15,16 A putative biosynthetic pathway for the isolated components is proposed. Subsequently, a bioassay was employed to evaluate their antineuroinflammatory activities in LPS-induced microglial BV2 cells, and their structure−activity relationships (SARs) were briefly discussed.

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RESULTS AND DISCUSSION The analysis of the biosynthetic pathways of compounds 1−11 and 13 revealed that these compounds were produced via the acetate-malonate (AA-MA) and mevalonic acid (MVA) pathways.17 Structurally, they all originated from the parent skeleton of an α-acid. The prenyl moieties at C-4 and C-6 in the α-acid Received: October 9, 2016 Published: November 20, 2017 3081

DOI: 10.1021/acs.jnatprod.6b00921 J. Nat. Prod. 2017, 80, 3081−3092

Journal of Natural Products

Article

Compound 1 was obtained as a yellow solid (MeOH) with [α]D20 −0.1 (c 0.1, MeOH). Its molecular formula was determined to be C21H30O6 according to its sodium adduct ion [M + Na]+ at m/z 401.1950 (calcd 401.1935 for C21H30NaO6) observed in HRESIMS data, which suggested seven indices of hydrogen deficiency. The 1H NMR data (Table 1) revealed an enolic hydroxy group at δH 18.84 (5-OH); a prenyl group at δH 5.01 (1H, t, J = 7.4 Hz, H-2‴), 2.59 (1H, dd, J = 13.8, 6.6 Hz, H-1‴a), 2.50 (1H, dd, J = 13.8, 7.4 Hz, H1‴b), 1.66 (3H, s, CH3-4‴), and 1.54 (3H, s, CH3-5‴); and an isovaleryl group at δH 2.83 (1H, dd, J = 13.8, 6.6 Hz, H-2″ a), 2.79 (1H, dd, J = 13.8, 7.4 Hz, H-2″ b), 2.14 (1H, m, H-3″), 1.00 (3H, d, J = 6.7 Hz, Me-4″), and 0.98 (3H, d, J = 6.7 Hz, Me-5″). A 2,2-dimethyl-3-hydroxydihydropyran group was observed based on the signals at δH 3.81 (1H, t, J = 4.7 Hz, H-3), δH 2.56 (2H, d, J = 4.7 Hz, H-4), δH 1.43 (3H, s, Me-1′), and δH 1.37 (3H, s, Me-2′). Twenty-one carbon resonances were identified from the 13C NMR data for 1 (Table 1), including one carbonyl signal at δC 200.9 (C-1″); an oxygenated methine carbon at δC 68.3 (C-3); three methylene carbons at δC 24.6 (C-4), 46.7 (C-2″), and 42.0 (C-1‴); a methine carbon at δC 26.5 (C-3″); and six methyl groups at δC 22.3 (C-1′), 24.9 (C-2′), 22.9 (C-4″), 22.7 (C-5″), 26.1 (C4‴), and 18.1 (C-5‴). The characteristic carbon signals of the α-acid skeleton also resonated at δC 195.5 (C-7), 189.5 (C-5), 167.4 (C-8a), 106.0 (C-6), 101.7 (C-4a), and 77.5 (C-8). All of these 1H and 13C NMR data are characteristic of a modified nhumulone18 in which a prenyl group is substituted at C-4a and

could cyclize into a furan or pyran moiety, as found in compounds 1−11. Accordingly, the structures of 1−11 and 13 were established based on modified prenyl units. In contrast, compounds 12 and 14−16 were biosynthesized via the complex cinnamic acid, shikimic acid, and MVA pathways, whereas sesquiterpenoid 17 originated from the MVA pathway (Figure 1). Based on their biosynthetic similarities and extensive NMR analyses, their structures were accurately determined.

Figure 1. Possible biosynthesis pathway of compounds identified from H. lupulus L. (A, branched-chain amino acid transaminase; B, branched-chain α-keto acid dehydrogenase complex; C, valerophenone synthase; D, aromatic prenyltransferase; E, oxygenase; i, phenylalanine ammonialyase; ii, cinnamate 4-hydroxylase; iii, chalcone synthase; iv, aromatic prenyltransferase; v, O-methyltransferase). 3082



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Table 1. 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR Spectroscopic Data for Compounds 1, 2, and 3 1 position 1 2 3 4 4a 5-OH 5 6 7 8 8a 1′ 2′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴

δH (J in Hz)

2 δC, type 81.8, C 68.3, CH 24.6, CH2

3.81, t (4.7) 2.56, d (4.7)

δH (J in Hz)

3.76, t (5.4) 2.74, dd (17.5, 5.0) 2.35, dd (17.5, 5.9)

101.7, C 18.84, s

dd (13.8, dd (13.8, m d (6.7) d (6.7) dd (13.8, dd (13.8, t (7.4)

1.66, s 1.54, s

81.6, C 69.0, CH 24.6, CH2

6.6) 7.4)

6.6) 7.4)

189.5, C 106.0, C 195.5, C 77.5, C 167.4, C 22.3, CH3 24.9, CH3 200.9, C 46.7, CH2 26.5, 22.9, 22.7, 42.0,

CH CH3 CH3 CH2

116.0, CH 136.8, C 26.1, CH3 18.1, CH3

δH (J in Hz)

3.76, m 2.71, dd (17.1, 4.9) 2.36, dd (17.1, 5.7)

102.2, C 18.79, s

1.43, s 1.37, s 2.83, 2.79, 2.14, 1.00, 0.98, 2.59, 2.50, 5.01,

3 δC, type

81.6, C 69.0, CH 24.7, CH2 102.0, C

18.92, s

1.42, s 1.37, s 2.86−2.77, m 2.14, 1.00, 0.98, 2.58, 2.46, 4.95,

δC, type

m d (6.7) d (6.7) dd (13.8, 7.6) dd (13.8, 7.8) t (7.6)

1.66, s 1.52, s

189.4, C 106.2, C 195.5, C 77.6, C 167.2, C 21.6, CH3 25.1, CH3 200.7, C 46.6, CH2 26.5, 22.9, 22.7, 41.8,

CH CH3 CH3 CH2

116.2, CH 136.8, C 26.1, CH3 18.1, CH3

3.74, m

189.5, C 104.8, C 195.4, C 77.7, C 167.2, C 21.7, CH3 25.1, CH3 205.7, C 34.8, CH

1.21, d (6.7) 1.12, d (6.7)

19.6, CH3 18.5, CH3

2.59, dd (13.7, 7.9) 2.46, dd (13.7, 7.7) 4.96, m

41.7, CH2

1.42, s 1.38, s

1.65, s 1.52, s

116.2, CH 136.8, C 26.1, CH3 18.1, CH3

Figure 2. Key HMBC (↷) and NOESY (red ↔) correlations of compounds 1−5.

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Figure 3. Comparison of calculated and experimental ECD spectra of compounds 1, 4, 5, 7, 8, 9a, and 9b.

a hydroxy group at C-8a. In compound 1, these two substituents cyclized to form a 2,2-dimethyl-3-hydroxydihydropyran moiety. This was confirmed by the HMBC correlations from H-4 (δH 2.56) to C-3 (δC 68.3), C-2 (δC 81.8), and C-4a (δC 101.7) and from H-3 (δH 3.81) to C-4a (δC 101.7). Analysis of the 2D NMR spectroscopic data facilitated the assignment of the 2D structure of compound 1. The relative configuration was defined based on its NOESY spectrum (Figure 2). The key NOE correlations from βoriented H-3 (δH 3.81) to H-4 (δH 2.56) and H-2′ (δH 1.37) and from α-oriented H3-1′ (δH 1.43) to H-2‴ (δH 5.01) indicated a relative configuration of (3R*, 8S*). The absolute configurations of C-3 and C-8 were established by comparing their experimental and calculated ECD spectra (Figure 3). The calculated ECD curve matched the experimental data well, allowing the assignment of the absolute configuration of compound 1. The 8S configuration was confirmed by a positive Cotton effect at 280−320 nm and a

negative Cotton effect at 345−400 nm, resulting from the n−π* electronic transition of the α,β-unsaturated carbonyl moiety. Additionally, based on the relative configuration deduced from the NOE, the absolute configuration of C-3 was defined as 3R. Therefore, compound 1 was identified as (3R,8S)-humulone A. Compound 2 was isolated as a yellow solid (MeOH) with [α]20 D −0.5 (c 0.1, MeOH), and it displayed the same molecular formula C21H30O6 as compound 1, according to the HRESIMS data, displaying a sodium adduct ion [M + Na]+ at m/z 401.1933 (calcd 401.1935 for C21H30 NaO6). Analysis of the 1D and 2D NMR data (Table 1, Figure 2) showed that compounds 1 and 2 had the same 2D structure. The conformational changes in the pyran rings of compounds 1 and 2 led to different chemical shifts for H-3, H-4, and C-3. For compound 2, H-3 (δH 3.76, t, J = 5.4 Hz), H-4 (δH 2.74, dd, J = 17.5, 5.0 Hz and 2.35, dd, J = 17.5, 5.9 Hz) and C-3 (δC 69.0) exhibited slightly different chemical shifts compared with 3084



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Table 2. 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR Spectroscopic Data for Compounds 4 and 5

compound 1 [H-3 (δH 3.81, t, J = 4.7 Hz), H-4 (δH 2.56, 2H, d, J = 4.7 Hz) and C-3 (δC 68.3)]. Notably, these differences were ascribable to the opposite absolute configuration of C-3. This conclusion was supported by the ECD spectrum of compound 2 (Figure S14, Supporting Information). Comparison of the experimental ECD spectra of compounds 1 and 2 revealed that they displayed the same Cotton effects from 260 to 400 nm, corresponding to the n−π* electronic transition of the α,β-unsaturated carbonyl moiety, with a small difference evident from 190 to 230 nm. The absolute configuration of compound 2 was further confirmed via its NOE spectrum (Figure 2). The NOESY correlations of H-2‴/ Me-1′, H-3/Me-1′, and H-3/H-4a indicated their cofacial arrangement and were assigned an α-orientation; the C-8 hydroxy group was assigned a β-orientation according to the NOE correlations of H-4b/Me-2′. Based on the NOE data, the absolute configuration of compound 2 was identified as (3S, 8S), and compound 2 was named (3S,8S)-humulone A. Compound 3 was obtained as a yellow solid (MeOH) with [α]20 D −0.2 (c 0.4, MeOH). HRESIMS indicated the molecular formula C20H28O6, according to its sodium adduct ion [M + Na]+ at m/z 387.1776 (calcd 387.1778 for C20H28NaO6). Comparison of the 1H and 13C NMR data (Table 1) of compounds 2 and 3 indicated that they differed only in the C-6 acyl side chains. In the 1H NMR spectrum of compound 3, a methine group at δH 3.74 (1H, m, H-2″) and two methyl doublets at δH 1.21 (3H, d, J = 6.7 Hz, Me-3″) and 1.12 (3H, d, J = 6.7 Hz, Me-4″) were observed. The methine group at H-2″ (δH 3.74) was ascribed to C-1″ (δC 205.7) based on the HMBC correlation (Figure 2) from H-2″ to C-1″, which also indicated the presence of an isobutyryl group. Analysis of the 1H and 13C NMR data of compounds 2 and 3 (except for the acyl side chains), including the chemical shifts of stereogenic carbons C-3 (ΔδC 0) and C-8 (ΔδC 0.1), showed highly similar results. The NOE correlations (Figure 2) from αoriented H-3 (δH 3.76) to H3-1′ (δH 1.42) and H-4a (δH 2.71), from H-2‴ (δH 4.96) to H3-1′ (δH 1.42), and from β-oriented H-4b (δH 2.36) to H3-2′ (δH 1.38) suggested that compounds 2 and 3 have the same relative configurations. Furthermore, the absolute configuration of 3 was defined as (3S, 8S) based on the Cotton effects in the ECD spectrum of compound 2 (Figure S21, Supporting Information). Thus, the structure of compound 3 was assigned as (3S,8S)-cohumulone A. Compound 4 was isolated as a red solid (MeOH) with [α]20 D −0.4 (c 0.1, MeOH). HRESIMS indicated a sodium adduct ion [M + Na]+ at m/z 401.1937 (calcd 401.1935 for C21H30NaO6), which corresponds to a molecular formula of C21H30O6 with seven indices of hydrogen deficiency. Compound 4 had the same molecular formula as compound 5, and these compounds are isomers of compounds 1 and 2. Comparison of the NMR data (Table 2) of compounds 4 and 1 revealed a difference due to the different cyclizations of the prenyl and hydroxy groups. In the 1H NMR spectrum, the resonances at δH 4.84 (1H, t-like, J = 9.3 Hz, H-2), 3.00 (1H, dd, J = 14.7, 8.5 Hz, H-3a), and 2.90 (1H, dd, J = 14.7, 10.2 Hz, H-3b) were attributed to a (2hydroxypropan-2-yl)dihydrofuran moiety. Furthermore, the cross-peaks observed from H2-3 (δH 3.00, 2.90) to C-2 (δC 93.6), C-3a (109.1), and C-7a (173.1) indicated the presence of a dihydrofuran ring substituted at C-3a and C-7a. The relative configuration was determined on the basis of the NOESY spectrum (Figure 2). The key NOE correlations from β-oriented H-2 (δH 4.84) to H-3b (δH 2.90), from α-oriented H-3a (δH 3.00) to H-2′ (δH 1.37), and from H-2‴ (δH 5.23) to

4 position 1 2 3

3a 4-OH 4 5 6 6-OH 7 7a 1′ 2′ 3′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴

5

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

4.84, t-like (9.3) 3.00, dd (14.7, 8.5), 2.90, dd (14.7, 10.2)

93.6, CH 27.0, CH2

4.76, dd (10.6, 8.1), 2.93, dd (14.7, 10.6), 2.88, dd (14.7, 8.1)

93.8, CH 27.1, CH2

109.1, C 18.50, s

1.37, s 1.21, s 2.82, dd (13.8, 6.8), 2.76, dd, (13.8, 7.5) 2.14, m 1.01, d (6.6) 0.97, d (6.6) 2.62, dd (14.3, 8.7), 2.43, dd (14.3, 5.7) 5.23, t-like (5.1) 1.71, s 1.56, s

109.6, C 18.47, s

187.6, C 106.1, C 196.1, C

187.8, C 106.9, C 196.0, C

76.2, C 173.1, C 71.8, C 26.4, CH3 24.0, CH3 199.7, C 46.0, CH2

75.9, C 172.8, C 71.7, C 25.9, CH3 23.7, CH3 199.3, C 46.0, CH2

26.7, 22.9, 22.6, 41.2,

CH CH3 CH3 CH2

117.0, CH 136.7, C 26.1, CH3 18.2, CH3

1.37, s 1.22, s 2.81, dd (13.7, 7.5), 2.76, dd (13.7, 6.7) 2.14, m 1.00, d (6.7) 0.97, d (6.7) 2.56, dd (7.5, 4.6) 4.90, t (7.9) 1.65, s 1.54, s

26.1, 22.9, 22.7, 40.3,

CH CH3 CH3 CH2

115.4, CH 137.5, C 26.1, CH3 18.0, CH3

H-2′ (δH 1.37) confirmed that H-2 was on the same side as H3b and on the opposite side of the prenyl group and H-2′. To establish the absolute configuration of compound 4, TDDFT ECD calculations were performed. The calculated ECD spectrum (Figure 3) for the (2S, 7S) absolute configuration was in good agreement with the experimental ECD spectrum of compound 4, which showed Cotton effects at λmax (Δε) 203.5 (18.38), 239.0 (−16.08), 273.5 (13.12), 348.0 (−4.66), and 355.5 (−4.33) nm. This result was further confirmed by the NOE data. Comparison of the ECD spectra of compounds 4 and 1 revealed similar Cotton effects at 230−400 nm, supporting the conclusion that C-7 (compound 4) has an S configuration, the same as C-8 of compound 1. The absolute configuration of C-2 also had the S configuration based on the NOE correlations. Therefore, the structure of compound 4 was defined as (2S,7S)-humulone B. Compound 5 was obtained as a red solid (MeOH) with [α]20 D −0.5 (c 0.4, MeOH), and the HRESIMS data showed an [M + Na]+ ion at m/z 401.1950 (calcd 401.1935 for C21H30NaO6), which established a molecular formula of C21H30O6. Compounds 4 and 5 were assigned as having the same 2D structure based on their NMR data (Table 2, Figure 2). Examination of the NMR data indicated that the differences between the two compounds could be attributed to H-2 (ΔδH 0.08), H-3a (ΔδH 0.07), H2-1‴ (ΔδH 0.13), H-2‴ (ΔδH 0.33), C-1‴ (ΔδC 0.9), C-2‴ (ΔδC 1.6), and C-3‴ (ΔδC 0.8). The differences may result from the different absolute configurations of C-2 and C7. Analysis of the calculated and experimental ECD (Figure 3) spectra of compounds 4 and 5 revealed the same Cotton effects at 260−400 nm, consistent with the Cotton effects of compounds 1−3 in this region. Therefore, the absolute 3085



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configuration of C-7 was defined as S. In addition, the NOESY correlations (Figure 2) permitted the absolute configuration of C-2 to be established. In the NOESY spectrum, correlations were observed from α-oriented H-2 (δH 4.76) to H-3a (δH 2.93) and H3-5‴ (δH 1.54) and from β-oriented H-3b (δH 2.88) to H3-2′ (δH 1.37), indicating that the absolute configuration of C-2 was opposite that of compound 4 and, thus, defined as R. Consequently, the absolute configuration of compound 5 was defined as (2R, 7S), and this compound was named (2R,7S)humulone B. Compound 6 was isolated as a red solid (MeOH) with [α]20 D −0.1 (c 0.2, MeOH), and its molecular formula, C20H28O6, was elucidated by HRESIMS data according to its sodium adduct ion [M + Na]+ at m/z 387.1778 (calcd 387.1778 for C20H28NaO6), which indicates seven indices of hydrogen deficiency. Comparison of the NMR data of compounds 4 and 6 (Table 3), including the chemical shifts of stereogenic C-

that they had the same absolute configuration at C-7. Thus, the structure of compound 6 was elucidated as (2R,7S)cohumulone B. Compound 7 was isolated as a red solid (MeOH) with [α]20 D −0.1 (c 0.3, MeOH). The HRESIMS data revealed the same molecular formula, C21H30O6, as compound 5. Analysis of the NMR data of compounds 7 and 5 (Tables 3 and 2) revealed that the differences could be attributed to the different substituent position of the (2-hydroxypropan-2-yl)dihydrofuran moiety in compounds 7 and 5. The key HMBC correlations (Figure 4) between H2-3 (δH 2.33, 2.21) and C-3a (δC 77.8), C-7a (170.4), C-4 (191.5), and C-1′ (70.7) indicated linkage of the dihydrofuran moiety to C-3a and C-7a. The prenyl group was located at C-7, based on the HMBC correlations from H21‴ (δH 3.09, 3.03) to C-3‴ (δC 133.0), C-2‴ (δC 120.9), C-7 (δC 108.7), C-7a (δC 170.4), and C-6 (δC 193.2). The 2D structure of compound 7 was further assigned by analyzing other 2D NMR spectroscopic data. The absolute configuration of this compound was determined by comparing its calculated and experimental ECD spectra. The calculated ECD (Figure 3) curve for the (2S, 3aS) absolute configuration was in good agreement with the experimental spectrum. Therefore, compound 7 was identified as (2S,3aS)-humulone C. Compound 8 was obtained as colorless needles (MeOH) with [α]D20 +0.3 (c 0.2, MeOH). Its molecular formula, C14H20O7, was determined via the HRESIMS data, which displayed an [M + Na]+ ion at m/z 323.1097 (calcd 323.1101 for C14H20NaO7) with five indices of hydrogen deficiency. The 1 H NMR spectrum (Table 4) revealed the presence of an oxygenated methine group at δH 4.67 (1H, dd, J = 9.9, 6.7 Hz, H-5); a methine group at δH 2.96 (1H, t-like, J = 7.4 Hz, H-3′); two methylene groups at δH 2.65 (1H, dd, J = 13.3, 6.7 Hz, H4a), 2.55 (1H, dd, J = 13.3, 9.9 Hz, H-4b), 2.73 (1H, d, J = 2.3 Hz, H-4′a), and 2.72 (1H, d, J = 3.5 Hz, H-4′b); and four methyl singlets at δH 1.57 (3H, s, Me-1″), 1.54 (3H, s, Me-2″), 1.29 (3H, s, Me-2‴), and 1.17 (3H, s, Me-3‴). Fourteen carbon resonances were observed in its 13C NMR data (Table 4), including three carbonyl carbons at δC 175.0 (C-5′), 174.9 (C2), and 173.0 (C-6); three oxygenated carbons at δC 88.2 (C2′), 85.9 (C-5), and 71.0 (C-1‴); an sp3 quaternary carbon at δC 58.9 (C-3); an sp3 methine carbon at δC 49.8 (C-3′); two sp3 methylenes at δC 35.5 (C-4) and 31.6 (C-4′); and four methyl groups at δC 29.5 (Me-2″), 26.1 (Me-2‴), 24.8 (Me3‴), and 24.3 (Me-1″). The HMBC correlations (Figure 4) between H-3′ (δH 2.96) and C-1″ (δC 24.3), C-2″ (δC 29.5), C4′ (δC 31.6), C-4 (δC 35.5), C-3 (δC 58.9), C-2′ (δC 88.2), C-6 (δC 173.0), and C-5′ (δC 175.0) suggested a linkage from C-3 to C-3′. Key HMBC correlations from H-5 (δH 4.67) to C-1‴ (δC 71.0), C-2‴ (δC 26.1), C-3‴(δC 24.8), and C-4 (δC 35.5) were also observed, indicating a connection between the 1hydroxy-1-methylethyl group and C-5 (δC 85.9). Furthermore, the NOESY correlations (Figure 4) permitted the assignment of the relative configuration of compound 8. The key NOESY signals from H-5 (δH 4.67) to H-4a (δH 2.65); from H-4b (δH 2.55) to H-3′ (δH 2.96) and H-3‴(δH 1.17); and from H-3′ (δH 2.96) to H-1″(δH 1.57), H-4b (δH 2.55), and H-4′b (δH 2.72) confirmed that H-5 and H-4a were cofacial and that they were on the opposite side from H-4b, H-3′, H-3‴, H-1″, and H-4′b. The absolute configuration was defined by comparing the computed and experimental ECD spectra. First, the conformational flexibility of the molecule was depicted in the molecular energy profiles (Figure S57 Supporting Information) with

Table 3. 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR Spectroscopic Data for Compounds 6 and 7 6 position 1 2 3

3a 4-OH 4 5 6 6-OH 7 7a 1′ 2′ 3′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴

7

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

4.84, dd (10.2, 8.5) 3.00, dd (14.7, 8.5), 2.91, dd (14.7, 10.2)

93.7, CH 27.0, CH2

4.74, dd (10.3, 4.9) 2.33, dd (13.6, 4.9), 2.21, dd (13.6, 10.3)

90.7, CH 33.9, CH2

108.9, C

77.8, C

187.8, C 104.5, C 196.0, C

191.5, C 106.1, C 193.2, C

3.74, m

76.3, C 173.3, C 71.8, C 26.4, CH3 24.0, CH3 204.7, C 34.3, CH

108.7, C 170.4, C 70.7, C 27.2, CH3 24.3, CH3 199.4, C 46.1, CH2

1.22, d (6.6) 1.13, d (6.6)

19.7, CH3 18.7, CH3

2.63, dd (14.5, 8.6), 2.43, dd (14.5, 5.1) 5.24, t (5.1)

41.2, CH2

18.68, s

18.68

1.37, s 1.22, s

1.71, s 1.56, s

117.0, CH 136.8, C 26.1, CH3 18.2, CH3

18.81 1.39, s 1.19, s 2.84, dd (13.5, 6.4), 2.74, dd (13.5, 7.5) 2.13, m 1.01, d (6.7) 0.97, d (6.7) 3.09, dd (14.5, 6.6), 3.03, dd (14.5, 8.3) 5.12, t (7.3) 1.73, s 1.68, s

26.8, CH 23.0, CH3 22.6,CH3 21.7, CH2 120.9, CH 133.0, C 25.8, CH3 18.0, CH3

2 (ΔδC 0.1) and C-7 (ΔδC 0.1), revealed similar results, except for the signals of the C-5 acyl side chains. The 1H NMR data of compound 6 revealed the presence of an isobutyryl group at δH 3.74 (1H, m, H-2″), 1.22 (3H, d, J = 6.6 Hz, Me-3″), and 1.13 (3H, d, J = 6.6 Hz, Me-4″). The NOESY correlations (Figure 4) from β-oriented H-2 (δH 4.84) to H-3b (δH 2.91), from αoriented H-3a (δH 3.00) to H3-2′ (δH 1.37), and from H-2‴ (δH 5.24) to H3-2′ (δH 1.37) confirmed that compound 6 had the same relative configurations as 4. Furthermore, the absolute configuration of 6 was determined to be (3S, 8S). This conclusion was supported by the ECD spectra (Figure S42, Supporting Information). The same Cotton effects were detected for compounds 6 and 4 at 260−400 nm, suggesting 3086



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Figure 4. Key HMBC (↷) and NOESY (red ↔) correlations of compounds 6, 7, 8, 9, and 12.

Table 4. 1H (600 MHz, Methanol-d4) and 13C (150 MHz, Methanol-d4) NMR Spectroscopic Data for Compound 8 carbon 1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 1″ 2″ 1‴ 2‴ 3‴

δH (J, in Hz)

2.65, dd (13.3, 6.7) 2.55, dd (13.3, 9.9) 4.67, dd (9.9, 6.7)

2.96, t-like (7.4) 2.73, d (2.3) 2.72, d (3.5) 1.57, s 1.54, s 1.29, s 1.17, s

15.5, 6.9 Hz, H-2″b), 2.25 (1H, m, H-3″), and 0.99 (6H, d, J = 6.6 Hz, H-4″, 5″); and two methyl groups at δH 1.27 (3H, s, H2′) and 1.40 (3H, s, H-3′). The 13C NMR data (Table 5) showed 16 carbon resonances, which were classified as a carbonyl carbon at δC 205.9 (C-1″); six sp2 aromatic carbons at δC 162.9 (C-6), 160.6 (C-7a), 131.6 (C-4), 117.6 (C-3a), 107.1 (C-7), and 109.7 (C-5); two oxygenated carbons at δC 91.6 (C2) and δC 71.7 (C-1′); a methine at δC 25.1 (C-3″); two methylene groups at δC 29.5 (C-3) and 52.6 (C-2″); and four methyl groups at δC 26.5 (Me-2′), 24.8 (Me-3′), and 22.9 (Me4′, 5′). The HMBC correlations (Figure 4) between H-2″ (δH 2.99, 2.86) and C-7 (δC 107.1) indicated a linkage from the isovaleryl group to C-7. The position of the (2-hydroxypropan2-yl)dihydrofuran moiety was fixed at C-3a and C-7a, based on the key HMBC correlations from H-4 (δH 7.19) to C-3 (29.5) and from H-3 (δH 3.15, 3.07) to C-7a (δC 160.6) and C-3a (δC 117.6). Chiral-phase separation via HPLC using a Daicel Chiralpak IF column yielded compounds 9a {[α]20 D −8.7 (c 0.4, MeOH)} and 9b {[α]20 D +8.7 (c 0.4, MeOH)}. Thus, the absolute configurations of (−)-9 and (+)-9 were investigated based on their ECD and specific rotation data.19,20 The calculated ECD spectra were in good agreement with the experimental spectra, confirming that compound 9a possessed an R configuration and compound 9b an S configuration (Figure 3). No further chiral-phase separation or stereochemical identification was conducted for the known racemates lupulone G (10)10 and 5-deprenyllupulonol C (11).21 A chiral-phase HPLC column was used to separate compounds 10 and 11 into 10a, 10b and 11a, 11b, respectively. Comparison of their experimental ECD spectra (Figures S72 and S76, Supporting Information) with those of compounds 9a and 9b allowed the establishment of the absolute configurations of compounds 10a, 10b, 11a, and 11b. Thus, their structures were defined as (R)-lupulone G (10a), (S)-lupulone G (10b), (R)-5-deprenyllupulonol C (11a), and (S)-5-deprenyllupulonol C (11b). Compound 12 was isolated as a pale yellow solid (MeOH). Its molecular formula was deduced as C17H20O4 by HRESIMS, according to its protonated molecular ion [M + H]+ at m/z 289.1434 (calcd 289.1434 for C17H21O4) with eight indices of hydrogen deficiency. The 1H NMR data (Table 6) revealed the

δC, type 174.9, C 58.9, C 35.5, CH2 85.9, CH 173.0, C 88.2, C 49.8, CH 31.6, CH2 175.0, C 24.3, CH3 29.5, CH3 71.0, C 26.1, CH3 24.8, CH3

respect to the dihedral angle C2′−C3′−C3−C2, ranging from −180° to 180° at the B3LYP/6-31G(d) level of theory, and the conformer of the 170°dihedral angle gave the lowest energy of −1072.13358515 hartree. The calculated ECD spectrum (Figure 3) for the (3S, 3′R, 5R)-configuration was in good agreement with the experimental spectrum of compound 8. Accordingly, compound 8 was named (3S,3′R,5R)-humulone acid B. Compound 9 was obtained as a pale yellow solid (MeOH) with [α]20 D 0 (c 0.8, MeOH). HRESIMS indicated a molecular formula of C16H22O4, according to its sodium adduct ion [M + Na]+ at m/z 301.1408 (calcd 301.1410 for C16H22NaO4) with 6 degrees of unsaturation. The 1H NMR data (Table 5) revealed the presence of two characteristic ortho aromatic protons at δH 7.19 (1H, d, J = 8.2 Hz, H-4) and 6.44 (1H, d, J = 8.2 Hz, H-5); an oxygenated methine at δH 4.75 (1H, t, J = 9.3 Hz, H-2); a methylene group at δH 3.15 (1H, dd, J = 15.3, 9.1 Hz, H-3a) and 3.07 (1H, dd, J = 15.3, 9.6 Hz, H-3b); an isobutanoyl group at δH 2.99 (1H, dd, J = 15.5, 6.8 Hz, H-2″a), 2.86 (1H, dd, J = 3087



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Table 5. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compounds 9 (CDCl3) and 10 and 11 (DMSO-d6) 9 position 1 2 3 3a 4 5 6-OH 6 7 7a 1′ 2′ 3′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴

10 δC, type

4.75, t (9.3) 3.15, dd (15.3, 9.1) 3.07, dd (15.3, 9.6)

91.6, CH 29.5, CH2

4.73, m 3.14−2.99, m

91.3, CH 28.4, CH2

117.6, C 131.6, CH 109.7, CH

7.28, d (8.2) 6.33, d (8.2)

118.5, C 131.4, CH 105.6, CH

106.9, C 158.2, C 104.0, C

161.4, C 108.0, C 160.4, C 69.9, C 26.0, CH3 25.0, CH3 209.9, C 38.7, CH

161.2, C 100.5, C 160.7, C 69.9, C 24.8, CH3 26.1, CH3 203.5, C 50.7, CH2

7.19, d (8.2) 6.44, d (8.2) 12.82, s

1.40, s 1.27, s 2.99, 2.86, 2.25, 0.99,

dd (15.5, 6.8) dd (15.5, 6.9) m d (6.6)

δH (J, in Hz)

162.9, C 107.1, C 160.6, C 71.7, C 26.5, CH3 24.8, CH3 205.9, C 52.6, CH2

1.19, s 1.15, s 3.75, m

25.1, CH 22.9, CH3 22.9, CH3

1.12, dd (8.8, 6.8)

1 2 3 4 5 5-OH 6 7 7-OH 8 9 10 1′ 2′, 3′ 1″ 2″ 3″ 4″ 5″

δC, type

18.8, CH3 18.4, CH3

δH (J, in Hz) 4.66, t (9.0) 2.98, d (9.1)

2.88,dd (15.0, 6.6) 2.79,dd (15.0, 7.3) 2.22, m 0.91, d (6.6)

1.69, s 1.59, s

δH, (J, in Hz)

6.12, s

12.73, s 6.26, s

C CH C C

98.2, CH 161.5, C

10.71

2.90, 1.25, 3.31, 5.15,

dt (13.7, 6.9) d (6.9) d (7.0) t (6.8)

1.74, s 1.62, s

25.1, CH 22.7, CH3 22.5, CH3 21.1, CH2 123.3, CH 129.6, CH 25.5, CH3 17.7, CH3

sp2 carbons at δC 174.5 (C-2), 105.0 (C-3), 161.5 (C-7), 159.0 (C-5), 154.9 (C-9), 130.7 (C-3″), 122.3 (C-2″), 105.9 (C-8), 103.5 (C-10), and 98.2 (C-6); four methyl groups at δC 19.7 (Me-2′, 3′), 17.7 (Me-4″), and 25.4 (Me-5″); a methylene group at δC 21.1 (C-1″); a methine group at δC 32.5 (C-1′); and a carbonyl signal at δC 182.3 (C-4). The HMBC correlations (Figure 4) from H2-1″ (δH 3.31) to C-8 (δC 105.9), C-2″(122.3), C-3″ (130.7), C-9 (154.9), and C-7 (161.5) confirmed the linkage between the prenyl group and C8. An isopropyl group was located at C-2 based on the HMBC correlations from H-1′ (δH 2.90) to C-2′, 3′ (δC 19.7), C-3 (δC 105.0), and C-2 (δC 174.5). Therefore, compound 12 was elucidated as a new compound named 5,7-dihydroxy-2isopropyl-8-prenylchromone. The known compounds hulupinic acid (13),10 xanthohumol (14),10 4-methoxyxanthogalenol (15),22 isoxanthohumol (16),23 and (1R,2R,5R,8S,9S)-2α,9β-clavondiol (17)24−26 were identified by comparing the observed and reported spectroscopic data. The antineuroinflammatory activities of the extracts and isolated compounds were measured by NO assays in LPSinduced BV-2 microglial cells (Figure 5). To avoid the effects of reduced viability on NO release, the cytotoxicities of the tested samples on BV-2 microglial cells were measured by 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. The results revealed that the EtOH, petroleum ether, EtOAc, and n-BuOH extracts inhibited the production of NO in overactivated BV-2 cells, with 50% inhibitory concentration (IC50) values of 9.76, 12.88, 13.75, and 42.01 μM, respectively, suggesting that these extracts were more effective than the positive control minocycline (IC50 48.82 μM). No cytotoxicity was observed at the tested concentrations of the extracts (1, 3, 10, 30, and 100 μg/mL).

δC, type 174.5, 105.0, 182.3, 159.0,

δC, type 90.7, CH 27.1, CH2

1.15, s 1.18, s

3.12, d (7.2) 5.11, t (6.9)

Table 6. 1H (400 MHz, DMSO-d6) and 13C (100 MHz, DMSO-d6) NMR Spectroscopic Data for Compound 12 carbon

11

δH (J, in Hz)

105.9, C 154.9, C 103.5, C 32.5, CH 19.7, CH3 21.1, CH2 122.3, CH 130.7, C 17.7, CH3 25.4, CH3

presence of characteristic resonances of two hydroxy groups at δH 12.73 (s, 5-OH) and 10.71 (s, 7-OH). Signals corresponding to two aromatic protons at δH 6.26 (1H, s, H-6) and 6.12 (1H, s, H-3) were also observed. The signals at δH 5.15 (1H, t, J = 6.8 Hz, H-2″), 3.13 (2H, d, J = 7.0 Hz, H-1″), 1.74 (3H, s, Me4″), and 1.62 (3H, s, Me-5″) were assigned to a prenyl group. An isopropyl group was identified at δH 2.90 (1H, dt, J = 13.7, 6.9 Hz, H-1′) and 1.25 (6H, d, J = 6.9 Hz, Me-2′, 3′). The 13C NMR data (Table 6) exhibited 17 carbon signals, including 10 3088



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Figure 5. Anti-inflammatory activities of the extracts and isolated compounds 1−8 and 11−17 assayed on LPS-induced NO production in BV-2 microglial cells. (Each bar represents the mean ± SE of three independent experiments; ###p < 0.001 compared with control group, **p < 0.01, ***p < 0.001 compared with LPS group; Ext-1: 95% ethanol extract, Ext-2: petroleum ether extract, Ext-3: EtOAc extract, Ext-4: n-BuOH extract, NO: nitric oxide, LPS: lipopolysaccharide, Mino: minocycline; compounds 9 and 10 were not tested because of limited quantity.)

Subsequently, the inhibitory effects and cytotoxicities of purified components 1−17 from the petroleum ether extract were further evaluated. As shown in Figure 5, the new chromone 5,7-dihydroxy-2-isopropyl-8-prenylchromone (12) (30 and 100 μM), chalcone xanthohumol (14) (10, 30, and 100 μM), 4-methoxyxanthogalenol (15) (100 μM), and flavanone isoxanthohumol (16) (30 and 100 μM) all displayed

moderate inhibitory effects against NO production, with IC50 values of 21.35, 7.92, 57.69, and 19.52 μM, respectively. Among the new α-acid derivatives, only (3S,8S)-cohumulone A (3) exhibited a significant antineuroinflammatory effect at 100 μM. None of the tested compounds showed cytotoxicity at the tested concentrations. 3089



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Compound 6 (3.4 mg) was separated from fr.15.3 by preparative HPLC (MeOH−H2O, 72:28). Fr.18 was purified on a polyamide column, eluted with a gradient of MeOH−H2O from 1:9 to 9:1, to yield five subfractions: fr.18.1−fr.18.5. Fr.18.1 was recrystallized from MeOH to obtain compound 8 (8.0 mg). Compound 17 (8.8 mg) was isolated from fr.18.2 by preparative TLC and developed with petroleum ether−acetone (2:1). Fr.18.5 was purified by silica gel column chromatography and eluted with petroleum ether−EtOAc from 40:1 to 1:1, followed by recrystallization from MeOH, to obtain compound 14 (10 mg). Finally, fr.20 was subjected to a silica gel column, using petroleum ether−EtOAc from 40:1 to 5:1, followed by a Sephadex LH-20 column, to yield compounds 13 (5 mg) and 16 (10 mg). (3R,8S)-Humulone A (1): yellow solid; [α]20 D −0.1 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 207.0 (12.59), 239.0 (−16.08), 273.5 (13.11), 347.5 (−4.67) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na] + 401.1950 (calcd 401.1935 for C21H30NaO6). (3S,8S)-Humulone A (2): yellow solid; [α]20 D −0.5 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 194.5 (5.74), 233.0 (−14.09), 270.5 (5.28), 282.0 (4.53), 306.5 (6.89), 353.0 (−3.25) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 401.1933 (calcd 401.1935 for C21H30 NaO6). (3S,8S)-Cohumulone A (3): yellow solid; [α]20 D −0.2 (c 0.4, MeOH); ECD (MeOH) λmax (Δε) 204.0 (5.30), 235.0 (−15.97), 279.0 (4.13), 309.0 (4.50), 353.5 (−5.40) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 387.1776 (calcd 387.1778 for C20H28NaO6). (2S,7S)-Humulone B (4): red solid; [α]20 D −0.4 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 203.5 (18.38), 239.0 (−16.08), 273.5 (13.12), 348.0 (−4.66), 355.5 (−4.33) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z [M + Na]+ 401.1937 (calcd 401.1935 for C21H30NaO6). (2R,7S)-Humulone B (5): red solid; [α]20 D −0.5 (c 0.4, MeOH); ECD (MeOH) λmax (Δε) 202.5 (9.27), 206.5 (8.07), 238.0 (−23.38), 268.5 (9.57), 346.0 (−3.91), 353.5 (−3.94) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z [M + Na]+ 401.1950 (calcd 401.1935 for C21H30NaO6). (3S,7S)-Cohumulone B (6): red solid; [α]20 D −0.1 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 202.5 (1.50), 243.5 (−14.28), 270.0 (−0.06), 280.5 (−1.78), 315.0 (5.91), 363.0 (−3.77) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z [M + Na]+ 387.1778 (calcd 387.1778 for C20H28NaO6). (2S,3aS)-Humulone C (7): red solid; [α]20 D −0.1 (c 0.3, MeOH); ECD (MeOH) λmax (Δε) 202.5 (−28.07), 240.5 (54.28), 319.5 (−20.13), 363.5 (12.49) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z [M + Na] + 401.1950 (calcd 401.1935 for C21H30NaO6). (3S,3′R,5R)-Humulone acid B (8): colorless needles (MeOH); mp 234.5−235.0 °C; [α]20 D +0.3 (c 0.2, MeOH); ECD (MeOH) λmax (Δε) 190.0 (1.68), 190.5 (0.99), 191.0 (1.22), 210.0 (40.81), 251.0 (0.16), 271.5 (0.96), 275.0 (0.94), 280.5 (1.04), 284.5 (0.98) nm; 1H and 13C NMR data, see Table 4, HRESIMS m/z [M + Na]+ 323.1097 (calcd 323.1101 for C14H20NaO7). Lupulone H (9): pale yellow solid; [α]20 D 0 (c 0.6, MeOH); ECD (MeOH) λmax (Δε) 9a, 209.0 (−8.68), 231.5 (7.09), 243.5 (3.24), 266.0 (9.12), 306.0 (−2.49), 351.5 (7.54) nm; 9b, 207.5 (17.36), 226.0 (−9.11), 242.0 (−4.42), 268.0 (−14.90), 303.5 (0.77), 346.5 (−7.58) nm; 1H and 13C NMR data, see Table 5; HR-ESIMS m/z [M + Na]+ 323.1097 (calcd 323.1101 for C14H20NaO7). Lupulone G (10): pale yellow solid; [α]20 D 0 (c 0.3, MeOH); ECD (MeOH) λmax (Δε) 10a, 195.0 (−12.75), 197.5 (−12.19), 204.0 (−14.87), 228.5 (3.30), 242.5 (0.86), 265.0 (6.19), 306.5 (−0.62), 346.5 (2.51) nm; 10b, 201.0 (9.29), 202.5 (9.24), 207.5 (10.03), 230.5 (−5.32), 241.5 (−2.05), 269.0 (−9.29), 306.0 (0.46), 347.5 (−3.71) nm; 1H and 13C NMR data, see Table 5. 5-Deprenyllupulonol C (11): yellow needles; [α]20 D 0 (c 0.2, MeOH); mp 150.5−151.0 °C; ECD (MeOH) λmax (Δε) 11a, 195.5 (9.30), 205.0 (−2.59), 217.5 (6.98), 241.5 (−2.07), 271.5 (4.27), 287.0 (0.82), 299.0 (4.64), 316.0 (−0.79), 343.0 (2.19) nm; 11b,

Based on the anti-inflammatory activity and structures of the isolated components, a structure−activity analysis was performed. First, it was found that the α-acid derivatives (1, 2, 4−8, 11, and 13) in the petroleum ether extract may not be responsible for the anti-inflammatory effect exhibited by hops. Instead, the chromones and flavonoids including chalcones and flavanones were critical for this effect. Second, the positions of the hydroxy and methoxy groups in the flavonoids were consistent with the inhibitory activities of compounds 14 and 15. For example, compound 14 (IC50 7.92 μM), which is 2′OCH3, 4′-OH, and 4-OH substituted, displayed a much stronger antineuroinflammatory effect than compound 15 (IC50 57.69 μM), which contains 2′-OH, 4′-OCH3, and 4-OCH3 groups.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured using a micro-melting point apparatus (Keyidianguang Factory, Beijing, China). A PerkinElmer 341 MC instrument was used to record optical rotations. ECD spectra were recorded on a Bio-Logic Science MOS-450 spectrometer. NMR spectra were measured with Bruker ARX-400 and AV-600 NMR spectrometers using tetramethylsilane as an internal standard. HRESIMS data were acquired using a Bruker micro TOF-Q mass spectrometer. Chromatographic silica gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), octadecyl-silica (ODS, 50 μm, YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB SE715 84, Uppsala, Sweden) were employed for column chromatography. Reversed-phase (RP)-HPLC separation was performed using a Shimadzu LC-6AD pump with a YMC-PACK ODS-A column (250 × 20 mm, 5 μm) and a Shimadzu SPD-20A VP ultraviolet/visible (UV/ vis) detector or an RID-10A detector (Shimadzu, Japan). Chiral separation was conducted in a Diamonsil C18 column (250 × 4.6 mm, 5 μm) from Daicel Chemical Industries, Ltd. (Japan). Plant Material. Female inflorescences of Humulus lupulus L. were collected from the XinJiang Uighur Autonomous Region of China in August 2012. Professor Xiaoguang Jia identified this plant material (Xinjiang Institute of Chinese Material Medica and Ethnodrug). A voucher specimen (No. 20120926) was deposited in the School of Traditional Chinese Materia Medica of Shenyang Pharmaceutical University. Extraction and Isolation. Air-dried female inflorescences of H. lupulus L. (5.0 kg) were extracted with 95% EtOH under reflux three times. The EtOH extract (1766 g) was suspended in hot H2O and partitioned sequentially with petroleum ether, EtOAc, and n-BuOH. The petroleum ether-soluble fraction (200 g) was subjected to a silica gel column and eluted with petroleum ether−EtOAc (100:0−0:100, v/ v) of increasing polarity to produce 21 fractions (fr.1−fr.21). Fr.7 was separated by ODS column chromatography and eluted with MeOH− H2O from 4:6 to 9:1 to obtain six subfractions (fr.7.1−fr.7.6). Fr.7.2 was purified by Sephadex LH-20 chromatography and eluted with MeOH. The eluted material was purified by recrystallization from MeOH to obtain compound 11 (8.0 mg). Fr.7.3 was isolated using preparative HPLC on an ODS column, eluted with MeOH−H2O (70:30), to obtain compounds 12 (2.5 mg), 4 (38.2 mg), and 9 (2.3 mg). Fr.10 was subjected to chromatography on an ODS column, eluted with MeOH−H2O, to generate fr.10.1, 10.2, and 10.3. Fr.10.2 was separated using preparative HPLC on an ODS column (MeOH− H2O, 78:22) to obtain compounds 1 (15.3 mg) and 5 (19.8 mg). Fr.11 was purified by a macroreticular resin D101 column, eluted with MeOH−H2O from 1:9 to 9:1, to afford subfractions fr.11.1 to fr.11.9. Fr.11.6 was isolated using a Sephadex LH-20 column with MeOH to obtain compound 15 (2.3 mg). Compound 7 (5.6 mg) was obtained from fr.11.7 by preparative HPLC (MeOH−H2O, 76:24). Fr.15 was chromatographed on an ODS column, eluted with a gradient of MeOH−H2O from 1:9 to 9:1, to obtain four subfractions: fr.15.1− fr.15.4. Fr.15.2 was separated by preparative HPLC (MeOH−H2O, 69:31) to obtain compounds 10 (2.3 mg), 3 (7.9 mg), and 2 (3.2 mg). 3090



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196.0 (8.96), 204.0 (0.29), 216.5 (10.50), 244.5 (−0.01), 283.5 (−13.69), 289.5 (−11.96), 295.0 (−13.45), 320.0 (−3.25), 331.5 (−4.11) nm; 1H and 13C NMR data, see Table 5. 5,7-Dihydroxy-2-isopropyl-8-prenylchromone (12): pale yellow solid; 1H and 13C NMR data, see Table 6,; HRESIMS m/z [M + H]+ 289.1434 (calcd 289.1434 for C17H21O4). Determination of Cell Viability. BV2 cells were seeded in 96well plates. After various treatments for 24 h, the supernatant was removed, and the cells were treated with MTT (0.25 mg/mL) for 4 h at 37 °C. The formazan crystals in the cells were dissolved by the addition of DMSO. The plates were read at 490 nm using a plate reader (Bio-Tek, Winooski, VT, USA).13,14 NO Production Bioassay. The accumulation of nitrite (NO2−), an indicator of NO synthase activity, in the culture medium was measured using the Griess reaction.13,14 BV2 cells (5 × 104 cells/well) were plated in 96-well microtiter plates and treated with each compound at various concentrations (1, 3, 10, 30, and 100 μM) in the presence of LPS (100 ng/mL) for 24 h. Next, 50 μL aliquots of the culture medium supernatants were mixed with 50 μL of the Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at room temperature. After 15 min, the absorbance was measured on a plate reader (Bio-Tek, Winooski, VT, USA) at 540 nm. Chiral-Phase Separation. Compounds 9a, 9b, 10a, 10b, 11a, and 11b were isolated using a chiral-phase chromatographic column (Daicel Chiralpak IF) with a mobile phase of n-hexane−EtOH (95:5) and eluted at 10.565 min (9a), 12.826 min (9b), 9.958 min (10a), 11.304 min (10b), 7.220 min (11a), and 9.597 min (11b) (flow rate: 1 mL/min, peak area: 1:1, λ = 210 nm) (Figures S66, S73, and S77, Supporting Information).

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computing staff at HPCC is acknowledged for computational support.

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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.6b00921. 1D and 2D NMR, HRESIMS, and CD spectra of compounds 1−12; chiral-phase separation of compounds 9, 10, and 11 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(N. Li) Tel: +86-24-23986475. Fax: +86-24-31509368. Email: [email protected]. *(Y. Hou) Tel: +86-24-83656116. Fax: +86-24-83656116. Email: [email protected]. ORCID

Ning Li: 0000-0001-7410-7327 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported partially by the National Natural Science Foundation of China (U1403102, 81473330, 81673323, 81473330, and U1603125), the Shenyang Science and Technology Research Project (F15-199-1-26), the Research Project for Key Laboratory of Liaoning Educational Committee (LZ2015067), the Natural Science Foundation of Liaoning Province (2015020732), the Innovation Team Project of Liaoning Province (LT2015027), and the project of the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (CMEMR2017-B03). The ECD calculations were carried out at the High Performance Computing Center (HPCC) of Shenyang Pharmaceutical University. The 3091



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