Catecholic Isoquinolines from Portulaca oleracea and Their Anti

Mar 8, 2018 - (33) As shown in Figure 4, all the tested alkaloids exhibited dose-dependent inhibition of NO production in LPS-induced RAW264.7 macroph...
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Catecholic Isoquinolines from Portulaca oleracea and Their Antiinflammatory and β2‑Adrenergic Receptor Agonist Activity Tian-Yun Jin, Shao-Qiang Li, Cui-Rong Jin, Hao Shan, Rui-Min Wang, Ming-Xing Zhou, Ai-Ling Li, Ling-Yu Li, Shui-Yao Hu, Tao Shen,* and Lan Xiang* Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, People’s Republic of China S Supporting Information *

ABSTRACT: Isoquinoline alkaloids possess a wide range of structural features and pharmaceutical activities and are promising drug candidates. Ten water-soluble catecholic isoquinolines were isolated from the medicinal plant Portulaca oleracea, including three new (1−3) and seven known compounds (4−10), along with the known catecholamines 11 and 12 and four other known compounds (13−16). A method of polyamide column chromatography using EtOAc−MeOH as the mobile phase was developed for the isolation of catecholic isoquinolines. Alkaloids 1−12 exhibited anti-inflammatory activities (EC50 = 18.0−497.7 μM) through inhibition of NO production in lipopolysaccharide-induced murine macrophage RAW 264.7 cells. Among these compounds, 11, 2, 5, 4, and 8 were more potent than was the positive control, 3,4dihydroxybenzohydroxamic acid (EC50 = 82.4 μM), with EC50 values of 18.0, 18.1, 35.4, 36.3, and 58.7 μM, respectively. Additionally, at 100 μM, compounds 1−12 showed different degrees of β2-adrenergic receptor (β2-AR) agonist activity in the CHO-K1/GA15 cell line which stably expressed β2-AR as detected by a calcium assay. The EC50 values of 2 and 10 were 5.1 μM and 87.9 nM, respectively.

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antioxidant and neuroprotective activity in cellular and animal models,12,13 we were curious if other bioactive catecholic isoquinolines were present in P. oleracea. Separation of water-soluble catecholic isoquinolines from mixtures using silica gel column chromatography or reversephase HPLC is difficult because these alkaloids can easily absorb onto silica gel and their retention times on reverse-phase HPLC are quite short. Recently, a method of polyamide column chromatography using petroleum ether−EtOAc− MeOH as the mobile phase was developed by our group for the separation of catecholic isoquinolines from P. oleracea. Sixteen compounds were isolated and characterized using spectroscopic and chromatographic methods, including 10 catecholic isoquinolines [1-(5′-hydroxylmethylfuran-2-yl)-6,7dihydroxy-3,4-dihydroisoquinoline (1), 1-(furan-2-yl)-6,7-dihydroxy-3,4-dihydroisoquinoline (2), 2-(furan-2-ylmethyl)-6,7dihydroxy-3,4-dihydroisoquinolin-2-ium (3), ethyl (S)(−)-(6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline)propanoate (4), (S)-(−)-oleracein E (5), 6,7-dihydroxy-1-methyl-3,4dihydroisoquinoline (6), 6,7-dihydroxy-3,4-dihydroisoquinoline (7), (S)-(−)-salsolinol (8), (R)-(+)-1-isobutyl-6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline (9), and (R)-(+)-1-benzyl-6,7dihydroxy-1,2,3,4-tetrahydroisoquinoline (10)] and two catecholamines [dopamine (11) and 2-sulfonic acid dopamine (12)]. The other four compounds were methyl 5-hydroxy-4oxo-4H-pyran-2-carboxylate (13), L-phenylalanine (14), L-

soquinoline alkaloids constitute a large group of secondary metabolites in higher plants and possess a wide range of structural and biological activities that have attracted interest from various research fields.1,2 Some important drugs are isoquinoline alkaloids, including analgesic morphine, antibacterial berberine, antitussive noscapine, muscle relaxant tubocurarine, vasodilator papaverine,3 and anticancer trabectedin, and the isoquinoline framework provides promising candidates for drug discovery for the treatment of cancer and CNS and various infectious diseases.4 Portulaca oleracea L., an edible and medicinal plant in the family Portulacaceae, is widely distributed in tropical and subtropical regions of the world. This plant has been used in numerous cultures as a traditional folk medicine to treat bacterial dysentery, diarrhea, insect stings, skin sores, ulcers, hemorrhoids, metrostaxis, hemoptysis, and asthma.5 P. oleracea was found to possess various pharmacological effects, including antibacterial, antioxidant, antiaging, antihypoxia, anti-inflammatory, antidiabetic, hypolipidemic, neuroprotective, bronchodilator, and skeletal-muscle relaxant activities.5 The catecholamines noradrenaline, dopamine, and dopa were first identified in P. oleracea in 1961,6 which inspired further phytochemical investigations into this plant. Several decades later, new types of alkaloids are still routinely being discovered from P. oleracea.7,8 In our continuous effort to discover novel bioactive constituents from P. oleracea, the two known catecholic isoquinolines oleracein E9 and iseluxine,10 16 catecholic indoline glucosides,9,11 and the catecholic benzazepine portulacatone were isolated.10 Because oleracein E exhibited © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 6, 2017

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DOI: 10.1021/acs.jnatprod.7b00762 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of compounds 1−16, iseluxine (17), and portulacatone (18) from P. oleracea.

Table 1. 1H and 13C NMR Data (600 and 150 MHz, D2O) for Compounds 1, 2, 3, and 4 (δ in ppm, J in Hz) 1 position

a

13

C

1 3

154.5 39.6

4

25.7

5 6 7 8 9a 10b 1′

117.3 165.4 146.0 115.6 137.0 109.3

2′ 3′

143.9 124.1

4′

111.5

5′ 6′

160.3 55.9

1

H

2 Ha

1

13

3.16 (2H, br s)

156.7 39.8

3.56 (2H, t, J = 7.2 Hz) 2.77 (2H, t, J = 7.2 Hz) 6.51 (1H, s)

2.53 (2H, t, J = 7.2 Hz) 6.61 (1H, s)

7.16 (1H, s)

6.87 (1H, s)

7.22 (1H, d, J = 3.6 7.10 (1H, br s) Hz) 6.61 (1H, d, J = 3.6 6.48 (1H, d, J = 3.0 Hz) Hz) 4.61 (2H, s) (overlapped)

3 1

C

24.7 115.9 155.0 143.9 118.2 135.0 114.24

H

3.69 (2H, t, J = 6.0 Hz) 2.77 (2H, t, J = 6.0 Hz) 6.81 (1H, s)

7.35 (1H, s)

13

C

163.7 47.3 24.5 115.5 156.7 143.9 119.9 132.6 115.8

143.7 125.4

7.43 (1H, br s)

144.7 112.8

114.18

6.78 (1H, br s)

111.1

150.5

7.94 (1H, br s)

144.9 54.9

4.48 (2H, s)

4 1

H

13

8.51 (1H, br s) 3.80 (2H, t, J = 7.8 Hz) 2.98 (2H, t, J = 7.8 Hz) 6.76 (1H, s)

54.0 38.8

7.12 (1H, s)

6.63 (1H, d, J = 3.0 Hz) 6.46 (1H, d, J = 3.0 Hz) 7.53 (1H, br s) 4.99 (2H, s)

1

C

H

4.30 (1H, br s) 3.38 (1H, br s); 3.19 (1H, br s) 2.80 (2H, br s)

23.7 115.7 144.0 142.9 113.7 122.7 123.8 27.7 29.7 174.9

61.9 13.1

6.54 (1H, s)

6.55 (1H, s)

2.15 (1H, br s); 2.06 (1H, br s) 2.40 (2H, br s)

3.91 (2H, q, J = 6.0 Hz) 1.04 (3H, t, J = 6.0 Hz)

In DMSO-d6.

lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells by reducing NO production. They also exhibited various degrees of β2-AR agonist activity in the CHO-K1/GA15 cell line which stably expressed β2-AR, as detected by a calcium assay. In particular, new catecholic isoquinoline 2 showed potent anti-inflammatory and β2-AR agonist dual functionality. Herein, the isolation and structural elucidation of catecholic isoquinolines from P. oleracea as well as their anti-inflammatory and β2-AR agonist activities are reported.

tyrosine (15), and adenine (16) (Figure 1). These compounds, except for 5 and 16, were all isolated for the first time from P. oleracea; 1−3 were new alkaloids, and 4, 7, 9, 10, and 12 were isolated as natural products for the first time. P. oleracea has anti-inflammatory and antiasthmatic functions.5,14 Because the catecholic isoquinoline trimetoquinol [1(3′,4′,5′-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] is an antiasthma drug that acts as a β2-AR agonist to exert a bronchodilator effect,15 higenamine [1-(4′-hydroxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline]16 and salsolinol (8)17 exhibit β2-AR agonist activity, and higenamine possesses anti-inflammatory activity,18 we hypothesized that catecholic isoquinolines of P. oleracea may also have antiinflammatory and β2-AR agonist effects. Herein we screened catecholic alkaloids 1−12 as well as the known iseluxine (17) and portulacatone (18)10 for potential anti-inflammatory and β2-AR agonist activities. Compounds 1−12, 17, and 18 exhibited various dose-dependent anti-inflammatory effects in

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RESULTS AND DISCUSSION Structural Elucidation of Isoquinoline Alkaloids. Both compounds 1 and 2 were water-soluble, red, amorphous powders. The Rf values of 1 and 2 on polyamide TLC developed with EtOAc−MeOH (4:1) were 0.55 and 0.65, respectively. They appeared brown when sprayed with 0.5% FeCl3 and bright red when sprayed with Dragendorff’s reagent, indicating 1 and 2 are phenolic alkaloids. The molecular B

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Figure 2. Key HMBC (H → C) and 1H−1H COSY (bold lines) correlations of compounds 1−4.

δH 3.69 (2H, t, J = 6.0 Hz) and 2.77 (2H, t, J = 6.0 Hz). Unlike 1, the 1H NMR spectrum of 2 showed three furanoid protons at δH 7.94 (1H, br s), 7.43 (1H, br s), and 6.78 (1H, br s). Similar to that of 1, the HMBC spectrum (Figure S2.6, Supporting Information) of 2 also showed correlations from H5 (δH 6.81) to C-4 (δC 24.7), C-10b (114.24), C-7 (143.9), and C-6 (155.0); H-8 (δH 7.35) to C-9a (δC 135.1) and C-6 (155.0); and H-3 (δH 3.69) to C-4 (δC 24.7), C-9a (135.1), and C-1 (156.7) (Figure 2), indicating the presence of a 1substituted 6,7-dihydroxy-3,4-dihydroisoquinoline moiety in the structure of 2. In addition, the HMBC spectrum showed correlations of H-5′ (δH 7.94) with C-4′ (δC 114.18), C-3′ (125.4), and C-2′ (143.7) and H-4′ (δH 6.78) with C-3′ (δC 125.4), C-2′ (143.7), and C-5′ (150.5), confirming that a furan unit was connected to C-1 at its C-2′ position. Compound 2 has nine indices of hydrogen deficiency, which corresponds to six double bonds and three rings. Compound 2 was therefore identified as 1-(furan-2-yl)-6,7-dihydroxy-3,4-dihydroisoquinoline. Compound 3 was a water-soluble, yellow, amorphous powder. The Rf value of 3 on polyamide TLC developed with petroleum ether−EtOAc−MeOH (1:4:0.2) was 0.55. It showed bright yellow fluorescence under UV365 nm light, turned brown when sprayed with 0.5% FeCl3, and turned red when sprayed with Dragendorff’s reagent, which indicated that 3 was also a phenolic alkaloid. The molecular formula of 3 was determined to be C14H14NO3 based on the ion at m/z 244.0966 [M]+ (calcd for C14H14NO3, 230.0968) in the positive HRESIMS data (Figure S3.1, Supporting Information). Similar to 2, the 1H NMR spectrum (Figure S3.2, Supporting Information) of 3 also showed two isolated aromatic protons at δH 7.12 (1H, s) and 6.76 (1H, s), two methylene groups at δH 3.80 (2H, t, J = 7.8 Hz) and 2.98 (2H, t, J = 7.8 Hz), and three furanyl protons at δH 7.53 (1H, br s), 6.46 (1 H, d, J = 3.0 Hz), and 6.63 (1 H, d, J = 3.0 Hz). Unlike 2, signals from a nitrogen-bound methylene group at δH 4.99 (2H, s) and an imino proton at δH 8.51 (1H, br s) were observed in the 1H NMR spectrum of 3. The HMQC spectrum (Figure S3.4, Supporting Information) showed that aromatic protons H-8 (δH 7.12) and H-5 (δH 6.76) were connected to C-8 (δC 119.9) and C-5 (δC 115.5), respectively, and H-5′ (δH 7.53), H-4′ (6.46), and H-3′ (6.63) were bound to furanoid carbons C-5′ (δC 144.9), C-4′ (111.1), and C-3′ (112.8), respectively. The HMBC spectrum (Figure S3.5, Supporting Information) showed that H-5 (δH 6.76) was correlated with C-4 (δC 24.5), C-10b (115.8), C-7 (143.9), and C-6 (156.7, weak); H-8 (δH 7.12) was correlated with C-9a (δC 132.6), C-7 (143.9, weak), C-6 (156.7), and C-1 (163.7); and H-3 (δH 3.80) was correlated with C-4 (δC 24.5), C-6′ (54.9), C-9a (132.6), and C-1 (163.7) (Figure 2), which indicated the presence of a 6,7dihydroxy-3,4-dihydroisoquinoline moiety. In addition, the

formula of 1 was determined to be C14H13NO4 based on the ion at m/z 260.0917 [M + H]+ (calcd for C14H14NO4, 260.0923) in the positive HRESIMS data (Figure S1.1, Supporting Information). In the IR spectrum (Figure S1.2, Supporting Information), characteristic peaks for an OH (3202 cm−1), an aromatic ring (1612, 1504, 1419 cm−1), and a C−N (1321 cm−1) were observed. The 1H NMR (600 MHz, D2O) (Table 1) spectrum of 1 (Figure S1.3, Supporting Information) revealed two isolated aromatic protons at δH 7.16 (1H, s) and 6.51 (1H, s), two methylene groups at δH 3.56 (2H, t, J = 7.2 Hz) and 2.77 (2H, t, J = 7.2 Hz), one of which was connected to a nitrogen atom, and two olefinic protons at δH 7.22 (1H, d, J = 3.6 Hz) and 6.61 (1H, d, J = 3.6 Hz). In addition, the 1H NMR spectrum recorded in DMSO-d6 (Figure S1.4, Supporting Information) revealed two oxymethylene protons at δH 4.61 (2H, s) that had been overlapped by the solvent peak in the 1H NMR (D2O) spectrum. The 13C NMR (150 MHz, D2O) (Table 1) spectrum (Figure S1.5, Supporting Information) revealed that compound 1 contained 14 carbons, comprising a methylene carbon (δC 25.7), a nitrogenated methylene carbon (δC 39.6), an oxymethylene carbon (δC 55.9), and 11 sp2 carbons. The HMQC spectrum (Figure S1.6, Supporting Information) revealed that aromatic protons H-8 (δH 7.16) and H-5 (δH 6.51) were connected to aromatic carbons at δC 115.6 and 117.3, respectively. The HMBC spectrum (Figure S1.7, Supporting Information) showed correlations of H-5 (δH 6.51) with C-4 (δC 25.7), C-10b (109.3), and C-7 (146.0); H-8 (δH 7.16) with C-9a (δC 136.9), C-7 (146.0), C-1 (154.5), and C-6 (165.4); and H-3 (δH 3.56) with C-4 (δC 25.7), C-9a (136.9), and C-1 (154.5), which indicated the presence of a 1substituted 6,7-dihydroxy-3,4-dihydroisoquinoline moiety. In addition, the HMBC spectrum showed that both H-3′ (δH 7.22) and H-4′ (6.61) were correlated with C-2′ (δC 143.9) and C-5′ (160.3), and H-6′ (δH 4.61) was correlated with C-5′ (δC 160.3) and C-4′ (111.5) (Figure 2), which is indicative of a (5hydroxymethyl)furan unit substituted at C-1. Compound 1 has nine indices of hydrogen deficiency, which correspond to six double bonds and three rings. Thus, the structure of 1 was defined as 1-(5′-hydroxylmethylfuran-2-yl)-6,7-dihydroxy-3,4dihydroisoquinoline. The molecular formula of 2 is C13H11NO3 based on the ions at m/z 230.0818 [M + H]+ (calcd for C13H12NO3, 230.0817) in the positive HRESIMS data (Figure S2.1, Supporting Information) and m/z 228.0669 [M − H]− (calcd for C13H10NO3, 228.0661) and 457.1336 [2M − H]− (calcd for C26H21N2O6, 457.1400) in the negative HRESIMS data (Figure S2.2, Supporting Information). Similar to 1, the 1H NMR (600 MHz, D2O) spectrum (Figure S2.3, Supporting Information) of 2 also revealed the presence of two isolated aromatic protons at δH 7.35 (1H, s) and 6.81 (1H, s) and two methylene groups at C

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Figure 3. Possible biosynthetic pathways for catecholic isoquinolines in P. oleracea.

The 13C NMR (Figure S4.5, Supporting Information), HMQC (Figure S4.7, Supporting Information), and DEPT spectra of 4 (Figure S4.6, Supporting Information) revealed 16 carbons, among which the resonances at δC 169.0 and 44.7 were assigned to be an impurity through HMQC and HMBC analysis (Figure S4.9, Supporting Information). This left compound 4 with 14 carbons, including an ester carbonyl carbon at δC 174.8, two oxygenated tertiary carbons at δC 143.9 and 142.8, two aromatic quaternary carbons at δC 123.8 and 122.6, two aromatic methine carbons at δC 115.7 and 113.6, five methylene carbons at δC 62.0, 38.9, 29.8, 27.7, and 23.7, an azamethine carbon at δC 54.1, and a methyl carbon at δC 13.2. The HMBC spectrum (Figure S4.8, Supporting Information) showed correlations from H-5, 8 (δH 6.55) to C-7 (δC 142.9) and C-10b (122.7). Most importantly, the HMBC correlations from H-6′ (δH 1.04) to C-5′ (δC 61.9) and from H-5′ (δH 3.91) to C6′ (δC 13.1) and C-3′ (174.9) indicated that the ethoxy fragment was connected to a carbonyl carbon, hence constituting an ester moiety. The structure of compound 4 was therefore assigned as ethyl 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-1-propanoate. C-1-substituted 1,2,3,4-tetrahydroisoquinoline enantiomers with (1S) or (1R) configurations would have negative or positive specific rotations, as in the case of (1S)-(−)-salsolidine and (1R)-(+)-solsolidine.19 Because of its negative specific rotation, compound 4 was assigned a (1S) absolute configuration. This compound has only been prepared synthetically (CAS no. 1526948-27-5); its isolation from P. oleracea is the first report of its natural occurrence. In addition to these four alkaloids, six other catechol-type isoquinolines were also identified: (S)-(−)-oleracein E (5), 6,7dihydroxy-1-methyl-3,4-dihydroisoquinoline (6),20,21 6,7-dihydroxy-3,4-dihydroisoquinoline (7),22 (S)-(−)-salsolinol (8),23 (R)-(+)-1-isobutyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (9),24 and (R)-(+)-1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (10).25 Two catecholamines, dopamine (11)26 and 2-sulfonic acid dopamine (12),27 as well as methyl 5hydroxy-4-oxo-4H-pyran-2-carboxylate (i.e., comenic acid methyl ester) (13),28 L-phenylalanine (14), L-tyrosine (15),

HMBC spectrum showed correlation of H-5′ (δH 7.53) with C4′ (δC 111.1), C-3′ (112.8), and C-2′ (144.7); H-4′ (δH 6.46) with C-3′ (δC 112.8), C-2′ (144.7), and C-5′ (144.9); H-3′ (δH 6.63) with C-4′ (δC 111.1), C-2′ (144.7), and C-5′ (144.9); and H-6′ (δH 4.99) with C-3 (δC 47.3), C-3′ (112.8), C-2′ (144.7), and C-1 (163.7). Although a correlation was not observed between H-1 and C-1 in the HMQC spectrum, the key HMBC correlations from H-8, H-6′, and H-3 to C-1; H-3 to C-6′; and H-6′ to C-3 confirmed that a (furan-2-ylmethyl) group was connected to a nitrogen atom. Compound 3 has nine indices of hydrogen deficiency, which correspond to six double bonds and three rings. Thus, compound 3 was identified as 2-(furan-2ylmethyl)-6,7-dihydroxy-3,4-dihydroisoquinolin-2-ium. Compound 4 was isolated as a water-soluble, colorless solid. The Rf value of 4 was 0.31 on polyamide TLC developed with EtOAc−MeOH (4:1). It turned dark red when exposed to iodine vapor and brown when sprayed with 0.5% FeCl3. The molecular formula was determined to be C14H19NO4 based on the ions at m/z 266.1393 [M + H]+ (calcd for C14H20NO4, 266.1348) in the positive HRESIMS data (Figure S4.1, Supporting Information) and m/z 264.1233 [M − H]− (calcd for C14H18NO4, 264.1236) in the negative HRESIMS data (Figure S4.2, Supporting Information). The 1H NMR spectrum (600 MHz, D 2 O) (Figure S4.3, Supporting Information) and 1H−1H COSY correlations (Figure S4.4, Supporting Information) indicated the presence of two isolated aromatic protons at δH 6.54 (2H, s), an N−CH2CH2 fragment which had 1H−1H COSY correlations (Figure 2) from the resonance at δH 3.38 (1H, br s) to 3.19 (1H, br s) and from δH 3.38 to 2.80, and an azamethine at δH 4.30 (1H, br s), i.e., the characteristic signals of a 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. The 1H−1H COSY correlations (Figure 2) between the resonances at δH 4.30 (1H, br s), 2.15 (1H, br s), 2.06 (1H, br s), and 2.40 (2H, br s) confirmed the presence of an N− CH−CH2−CH2 fragment, and the correlations between the resonances at δH 3.94 (2H, q, J = 6.0 Hz) and 1.04 (3H, t, J = 6.0 Hz) confirmed the presence of an ethoxy fragment. D

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Figure 4. continued

E

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Figure 4. Anti-inflammatory activity of catecholic alkaloids (1−12, 17, and 18) from P. oleracea (n = 3) (#p < 0.05, ###p < 0.001, vs control group; *p < 0.05, **p < 0.01, ***p < 0.001, vs LPS model).

are shown in Figure 3. Dopamine and aldehydes present in P. oleracea may form C-1-substituted 1,2,3,4-tetrahydroisoquinilines through enzymatic Pictet−Spengler reactions, which could be transformed into 3,4-dihydroisoquinoline derivatives. Moreover, it was found that ethyl 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-1-propanoate (4) was not stable; polyamide TLC indicated its partial degradation into oleracein E (5) when left in a MeOH solution for an extended period of time. An intramolecular ester aminolysis reaction30 to form the lactam (Figure 3) was hypothesized to be involved in this phenomenon.

and adenine (16), were identified through analysis of physiochemical and spectroscopic data (Supporting Information) or TLC comparisons to the standard compounds (5, 14, 15, and 16). An enzymatic Pictet−Spengler reaction is involved in the production of 1-substituted 1,2,3,4-tetraisoquinolines in higher plants. For example, norcoclaurine synthase (NCS) catalyzes the stereoselective Pictet−Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde as the first step of benzylisoquinoline alkaloid synthesis in plants.29 Possible biosynthetic pathways for catecholic isoquinolines in P. oleracea F

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Table 2. β2-AR Agonist Activity of Catecholic Alkaloids from P. oleracea (n = 3)

It should be noted that although polyamide chromatography has merits in the separation of water-soluble catecholic isoquinolines, it has a disadvantage; polyamide chromatography with elution by petroleum ether−EtOAc−MeOH can introduce some impurities (Figures S1.8, S2.7, S3.6, and S4.9, Supporting Information) even after exhaustively washing with NaOH, HCl, petroleum ether, and EtOAc before use. Anti-inflammatory Activity of Catecholic Alkaloids. LPS is the major component of the outer membrane of most Gram-negative bacteria. Stimulation of RAW 264.7 murine macrophage cells with LPS can induce increased production of NO and other pro-inflammatory cytokines, and NO production assays are frequently used to evaluate anti-inflammatory effects. An aqueous extract of P. olerecea was reported to inhibit NO production in LPS-induced RAW 264.7 cells in a dosedependent manner.31 This model was previously used to screen the activity of 20 compounds isolated from an aqueous extract of P. olerecea, and only 6,7-dihydroxyphenyl ethanol showed anti-inflammatory effects.32 In this experiment, the antiinflammatory effects of catecholic alkaloids 1−12, along with 17 and 18 (Figure 1), which were isolated from P. oleracea, were assessed. To prevent false-positive results, the cytotoxicities of these compounds were simultaneously evaluated by the 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine whether these compounds could influence the cell viability of macrophages and whether the decrease in NO production could be attributed to inhibition of cell proliferation. 3,4-Dihydroxybenzohydroxamic acid (Didox), an anti-inflammatory catecholic alkaloid, was used as the positive control.33 As shown in Figure 4, all the tested alkaloids exhibited dose-dependent inhibition of NO production in LPS-induced RAW264.7 macrophage cells with EC50 values between 18.0 and 497.7 μM. Among them, compounds 11, 2, 5, 4, and 8 were more potent than positive control Didox (EC50 = 82.4 μM), with EC50 values of 18.0, 18.1, 35.4, 36.3, and 58.7 μM, respectively. Similar to our present findings, dopamine (11) was recently reported to inhibit NLRP3 inflammasome activation through dopamine D1 receptor signaling, and it can control both neuroinflammation and periphery inflammation via promoting NLRP3 ubiquitination and degradation.34 β2-AR Agonist Activity of Catecholic Alkaloids. β2-AR agonist activities of catecholic alkaloids 1−12, 17, and 18 were determined through a calcium fluorescence assay in the CHOK1/GA15 cell line which stably expressed β2-AR. Isoproterenol (isoprenaline), which is used to treat acute severe asthma in clinical settings, was used as the positive control.35 As shown in Table 2, at 100 μM, compounds 1−12 showed various degrees of β2-AR agonist activity; among the tested compounds, 2 and 10 were the most potent, and 17 and 18 had no agonist activity. Further experiments indicated that compounds 2 and 10 showed weak β2-AR agonist activity with EC50 values of 5.1 μM and 87.9 nM (Figure 5), respectively. Their activities were less than that of the positive control isoproterenol (EC50 = 0.7 nM). Similar to our present finding, BDTI (1-benzyl-6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline HBr), i.e., the HBr salt of 10, has been proven to be a β2-AR-selective agonist. The potency of the relaxing effect on carbachol-induced contraction in isolated canine trachea was in the order isoprenaline > BDTI = salbutamol (an antiasthma drug). BDTI and salbutamol also stimulated cAMP formation in a concentration-dependent manner in cultured canine tracheal smooth muscle cells.36

stimulation ratio (%)

compounda isoproterenol 1-(furan-2-yl)-6,7-dihydroxy-3,4-dihydroisoquinoline (2) (R)-(+)-1-benzyl-6,7-dihydroxy-1,2,3,4tetrahydroisoquinoline (10) dopamine (11) (R)-(+)-1-isobutyl-6,7-dihydroxy-1,2,3,4tetrahydroisoquinoline (9) 1-(5′-hydroxylmethylfuran-2-yl)-6,7-dihydroxy-3,4dihydroisoquinoline (1) (S)-(−)-oleracein E (5) ethyl (S)-(−)-(6,7-dihydroxy-1,2,3,4tetrahydroisoquinoline)propanoate (4) 6,7-dihydroxy-3,4-dihydroisoquinoline (7) 2-sulfonic acid dopamine (12) 6,7-dihydroxy-1-methyl-3,4-dihydroisoquinoline (6) (S)-(−)-salsolinol (8) 2-(furan-2-ylmethyl)-6,7-dihydroxy-3,4-dihydroisoquinolin2-ium (3) iseluxine (17) portulacatone (18)

100 ± 2.4 91.8 ± 2.8 90.8 ± 6.0 86.4 ± 8.4 70.9 ± 9.1 70.6 ± 5.2 70.2 ± 1.9 42.0 ± 2.7 41.1 39.3 37.7 35.5 11.4

± ± ± ± ±

3.8 3.6 3.6 1.1 5.8

0.5 ± 2.5 −11.5 ± 1.2

Concentration of isoproterenol was 1 μM; other alkaloids were 100 μM.

a

The most significant finding in the present study is that new isoquinoline alkaloid 2 showed strong anti-inflammatory and β2-AR agonist dual functionality. It was reported that β2-AR regulates Toll-like receptor-4-induced nuclear factor-KB (NFκB) activation through β-arrestin 2, and β2-AR protein and mRNA markedly decreased in RAW264.7 microphage cells following LPS stimulation.37 Recently, β2-AR agonists were found to be novel regulators of macrophage activation, and they can enhance β-arrestin 2 and downregulate NF-κB in macrophage cells and might have protective effects in diabetic renal and cardiovascular complications.38 Whether new catecholic isoquinoline 2 exerts its anti-inflammatory effect through β2-AR agonist activity needs to be further studied. In summary, the present work may partially elucidate the bioactive constituents responsible for the anti-inflammatory and antiasthma effects of P. oleracea and may provide potential candidates for drug discovery. Because catecholamines are stress hormones and neurotransmitters in the mammalian system and their derivatives are important drugs, in-depth research on the distribution and biosynthetic pathway of the catecholic alkaloids in higher plants, as well as their biological function in plants themselves and pharmacological activities in human, is therefore of great importance.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined using a Gyromat-Hp automatic digital polarimeter (Kernchen Co., Germany). NMR spectra were recorded at 600 MHz for 1H NMR and 150 MHz for 13C NMR on a Bruker Avance DRX-600 spectrometer with tetramethylsilane as a reference. HRESIMS spectra were collected on an LTQ-Orbitrap XL mass spectrometer (ThermoFinnigan, USA) and a Tandem 6410 triple quadrupole mass spectrometer (Agilent Company, USA). UV spectra were obtained on a ZF-20C spectrophotometer (Shanghai Baoshan Gucun Photoelectric Instrument Factory, China). Microplate assays were carried out using a model 680 microplate reader (Bio-Rad Co., USA). The extracts were chromatographed on polyamide gel (100− 200 mesh; Taizhou Luqiao Siqing Biochemical Plastics Factory, G

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Figure 5. EC50 values of β2-AR agonist activity for compounds 2 and 10 and isoproterenol (n = 4). China), MCI gel (CHP-20P, 75−150 μm, Mitsubishi Chemical Co., Japan), Sephadex LH-20 (Pharmacia Fine Chemicals, USA), or ODSC18 (75 μm, YMC Co., Japan). TLC was carried out on polyamide film (Taizhou Luqiao Siqing Biochemical Plastics Factory, China) or GF 254 silica gel (Qingdao Marine Chemical Co., China) sprayed with iodine vapor, 0.5% FeCl3, or Dragendorff’s reagent. Plant Material. The dried aerial parts of P. oleracea were obtained from Jianlian Pharmacy (Jinan, P.R. China) and were identified by one of the authors (L.X.) as Portulaca oleracea L. A voucher specimen (No. 20120501) was deposited in the Department of Pharmacognosy, School of Pharmaceutical Sciences, Shandong University. Extraction and Isolation. Following the reported procedure,11 the 60% EtOH extracts of P. oleracea (4 kg, refluxed 3 times × 1 h) were concentrated under vacuum to 8 L and stored at 4 °C. The supernatant was subjected to polyamide column chromatography and eluted with gradients of EtOH−H2O (0:100−95:5, v/v) and an ammonia solution (25% aqueous ammonia−85% EtOH = 1:7, v/v) to afford nine fractions (Frs. 1−9). Frs. 1 and 2 (634.5 g) were dissolved in 1 L of distilled water and then sequentially extracted with the same volume of EtOAc (×3) and n-BuOH (×5) to yield 11.4 and 52.0 g of the corresponding extracts. The n-BuOH fraction (52.0 g) was further purified on a silica gel column (800 g, 6 × 50 cm) eluted with a gradient of EtOAc−MeOH (9:1 to 0:10, v/v) to give six fractions, C(1−3) (9.62 g), C(4−12) (24.8 g), C(13−16) (4.15 g), C(17−25) (4.39 g), C(26−29) (3.21 g), and C(30−40) (2.62 g). Fraction C(4−12) (24.8 g), which was rich in phenolic alkaloids based on TLC analysis, was chromatographed on a polyamide column (10 × 30 cm) and eluted with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v, containing 1% formic acid) and then EtOAc−MeOH (9:1 to 0:10, v/v, containing 1% formic acid) to afford 10 subfractions. Fraction C(4−12)-(35−39) (670 mg) was purified by MCI column chromatography (3.5 × 30 cm) with a gradient of aqueous MeOH (10% to 100%) to afford 12 fractions. Fraction C(4−12)-(35−39)(3−4) (75 mg) was purified on a Sephadex LH-20 column (1.5 × 120 cm) eluted with MeOH to afford five subfractions and 11 (10 mg). Fraction C(4−12)-(35−39)-(3−4)-(21−24) (25 mg) was purified on a polyamide column (1 × 50 cm) eluted with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (9:1 to 0:10, v/v) to afford 15 (2 mg). Fraction C(4−12)-(35−39)-(5−6) (40 mg) was purified on a Sephadex LH-20 column (1.5 × 120 cm) eluted with MeOH and afforded 12 (5 mg). Fraction C(4−12)-(40−60) (9.6 g) was crystallized from MeOH to yield 16 (15 mg), and the stock solution was purified on a polyamide column (6 × 50 cm) with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (9:1 to 0:10, v/v) to generate 10 subfractions. From that column fraction C(4−12)-(40−60)-(12− 16) (1.04 g) was purified on an MCI column (3.5 × 30 cm) with a gradient of aqueous MeOH (5% to 100%) to afford 12 subfractions, of which fraction C(4−12)-(40−60)-(12−16)-(3−10) was crystallized from MeOH to yield 14 (50 mg). From the polyamide column, fraction C(4−12)-(40−60)-(17−22) (1.73 g) was purified on an MCI column (3.5 × 30 cm) eluted with aqueous MeOH (5% to 100%) to give 12 subfractions. From that MCI column, fraction C(4−12)-(40− 60)-(17−22)-(14−21) (276 mg) was subjected to polyamide column

chromatography (3 × 50 cm) eluted with petroleum ether−EtOAc (1:4, v/v) and then petroleum ether−EtOAc−MeOH (1:3:0.1, v/v) to afford four subfractions. Fraction C(4−12)-(40−60)-(17−22)-(14− 21)-(24−43) (90 mg) was further purified on a polyamide column (1.5 × 40 cm) with a gradient of petroleum ether−EtOAc (9:1, v/v) to give five subfractions and 9 (20 mg). Of those five subfractions, fraction C(4−12)-(40−60)-(17−22)-(14−21)-(24−43)-(15−19) (40 mg) was further purified by polyamide column chromatography (1.2 × 40 cm) with EtOAc−MeOH (10:1, v/v, containing 1% Et3N) to afford 10 (20 mg). Fraction C(4−12)-(40−60)-(17−22)-(22−24) (42 mg) was purified by Sephadex LH-20 column chromatography (1.5 × 120 cm) with MeOH to afford five subfractions. Of those five subfractions, fraction C(4−12)-(40−60)-(17−22)-(22−24)-(31−50) (6 mg) was purified on a polyamide column (1.5 × 50 cm) with petroleum ether− EtOAc (1:4, v/v) to give two subfractions. Fraction C(4−12)-(40− 60)-(17−22)-(22−24)-(31−50)-(14−33) (4 mg) was further separated on a polyamide column (1.5 × 50 cm) with petroleum ether− EtOAc−MeOH (1:4:0.4, v/v) to afford 3 (4 mg). Fraction C(4−12)(40−60)-(23−38) (2.0 g) was purified on an MCI column (4 × 30 cm) eluted with aqueous MeOH (5% to 100%) to afford 12 subfractions. Fraction C(4−12)-(40−60)-(23−38)-(19−21) (25 mg) was purified on a Sephadex LH-20 column (1.5 × 120 cm) with MeOH to afford 2 (5 mg). Fraction C(4−12)-(61−70) (1.72 g) was subjected to polyamide column chromatography (4.5 × 35 cm) and eluted with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (20:1 to 0:10, v/v) to afford 10 subfractions. Of those 10 subfractions, fraction C(4−12)-(61−70)-(7−9) (265 mg) was purified on an ODSC18 column (3 × 33 cm) with a gradient of aqueous MeOH (5% to 100%) to afford 10 subfractions. Fraction C(4−12)-(61−70)-(7−9)-2 (120 mg) was purified on an MCI column (4 × 30 cm) with a gradient of aqueous MeOH (5% to 100%) to produce 10 subfractions. Fraction C(4−12)-(61−70)-(7−9)-2-(4−5) (60 mg) was purified on a Sephadex LH-20 column (2.5 × 150 cm) with MeOH to afford three subfractions and 7 (7 mg). Fraction C(4−12)-(61−70)-(7−9)-2(4−5)-(9−31) (14 mg) was purified on a polyamide column (1.5 × 30 cm) with petroleum ether−EtOAc (9:1, v/v) to afford 6 (5 mg). Fraction C(4−12)-(61−70)-12 (110 mg) was purified on an MCI column (4.0 × 20 cm) with a gradient of aqueous MeOH (5% to 100%) to afford 10 subfractions. Fraction C(4−12)-(61−70)-12−6 (30 mg) was purified on a Sephadex LH-20 column (1.5 × 120 cm) with MeOH to afford 8 (7 mg). Fraction C(4−12)-(61−70)-(13−19) (506 mg) was purified on a polyamide column (6 × 50 cm) with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (9:1 to 0:10, v/v) to afford five subfractions. Fraction C(4−12)-(61−70)-(13−19)-(8−10) (112 mg) was purified on an ODS-C18 column (3 × 30 cm) with a gradient of aqueous MeOH (5% to 100%) to afford 10 subfractions. Fraction C(4−12)-(61−70)-(13− 19)-(8−10)-(6−10) (14 mg) was purified on a Sephadex LH-20 column (2.5 × 150 cm) with MeOH to afford 1 (4 mg). Fraction C(4−12)-(71−77) (2.88 g) was subjected to polyamide column chromatography (4.5 × 35 cm) eluted with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (20:1 to 0:10, v/v) to provide 15 subfractions. Fraction C(4−12)(71−77)-(5−10) (450 mg) was purified on a polyamide column (4.5 H

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× 35 cm) with a gradient of aqueous MeOH (0% to 30%) to give two subfractions and 5 (20 mg). Fraction C(4−12)-(71−77)-(5−10)-(1− 4) (180 mg) was purified on an ODS-C18 column (4.0 × 20 cm) with a gradient of aqueous MeOH (5% to 100%) to afford 4 (5 mg). The EtOAc fraction (11.4 g) was subjected to silica gel column chromatography (9 × 60 cm) and eluted with a gradient of petroleum ether−EtOAc (9:1 to 0:10, v/v) and then EtOAc−MeOH (9:1 to 0:10, v/v) to afford 24 subfractions, ZFr1−ZFr24. Fraction ZFr2 (450 mg) was purified on a Sephadex LH-20 column (4.5 × 120 cm) with MeOH to afford five subfractions. Fraction ZFr2-(27−29) (120 mg) was chromatographed on an ODS-C18 column (3.5 × 20 cm) eluted with a gradient of aqueous MeOH (5% to 100%) to afford nine subfractions. Fraction ZFr2-(27−29)-50% (15 mg) was crystallized from MeOH to yield 13 (9 mg). Anti-inflammatory Assay. Catecholic alkaloids 1−12, 17, and 18 were tested for their anti-inflammatory activity through an NO production assay in LPS-induced RAW 264.7 murine macrophage cells (American Type Culture Collection). The experiment was performed according to the literature protocol.32 In brief, cells in 96-well plates (8.0 × 104 cells/well) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gemini Bioproduct, USA). After 24 h of incubation at 37 °C in a 5% CO2 humidified incubator, cells were treated with 1 μg/mL LPS in the absence or presence of the test compounds. After LPS treatment for 24 h, 100 μL of supernatant medium was removed and added to 100 μL of Griess reagent (0.1% naphthylethylenediamine mixed with 1% sulfanilamide in 5% H3PO4 solution) in a new 96-well plate, which was maintained at room temperature for 15 min. The absorbance was measured at 570 nm using a model 680 plate reader. NO content was calculated based on a NaNO2 standard curve. Didox was used as the positive control. Simultaneously, the effects of the test compounds on cell viability of RAW 264.7 cells were also evaluated by MTT assay.32 β2-AR Stimulation Assay. This assay was conducted according to the protocol from Nanjing GenScript Co. Ltd. Catecholic alkaloids 1− 12, 17, and 18 were tested for their β2-AR agonist activity using a calcium fluorescence assay unit on CHO-K1/Ga15 cells that were stably expressing β2-AR (GenScript, M00308). Briefly, cells were seeded in a 10 cm dish and cultured in Ham’s F12 medium supplemented with 10% FBS (Gemini Bioproduct, USA), 100 μg/mL Hygromycin B, and 200 μg/mL Zeocin. The cells were incubated at 37 °C in a humidified incubator containing 5% CO2. When cell confluency reached 85%, cells were treated with 0.25% trypsin for digestion, then subcultured in a 384-well plate (20 μL/well, 1.5 × 104 cells/well). After incubation for 18 h, cells were added to 20 μL of solution from a FLIPRCalcium 4 assay kit (Molecular Devices, 120726-200), and the mixture was incubated at 37 °C with 5% CO2 for 1 h and then equilibrated at room temperature for 15 min. Calcium relative fluorescence units (RFU values) of the cells were determined using a FLIPRTETRA (Molecule Devices). The overall detection time was 120 s, and 10 μL of 5× target concentration of isoproterenol (Sigma, I6504) or test compounds was automatically added to the plate after 21 s. Isoproterenol and the test compounds were dissolved in DMSO (AMRESCO, 1988B176) at concentrations of 20 and 100 mM, respectively, and then diluted with Hank’s balanced salt solution containing 20 mM HEPES (pH 7.4) to 5× target concentration before the assay. The average of the fluorescence units from 1 to 20 s was used as the baseline, and the relative fluorescence units (ΔRFU) was the maximum fluorescence units from 21 to 120 s minus the baseline. Stimulation ratio (%) = (ΔRFU compound − ΔRFU background )/ (ΔRFUisoproterenol − ΔRFUbackground) × 100%. In further dosedependent assays of compounds 2 and 10, the median effective concentrations (EC50 values) were calculated according to a fourparameter equation in GraphPad Prism 6 software, i.e., Y = Bottom + (Top − Bottom)/(1 + 10((logEC50/IC50−X)HillSlope)), where X is the log concentration of the sample and Y is the stimulation ratio. 1-(5′-Hydroxylmethylfuran-2-yl)-6,7-dihydroxy-3,4-dihydroisoquinoline (1): red, amorphous powder; UV (MeOH) λmax (log ε) 285 (0.47), 210 (0.85) nm; IR (KBr) νmax 3202, 1612, 1504, 1419, 1321, 1137, 1077 cm−1; HRESIMS m/z 260.0917 [M + H]+ (calcd for C14H14NO4, 260.0923); 1H and 13C NMR data (Table 1).

1-(Furan-2-yl)-6,7-dihydroxy-3,4-dihydroisoquinoline (2): red, amorphous powder; positive HRESIMS m/z 230.0818 [M + H]+ (calcd for C13H12NO3, 230.0817); negative HRESIMS m/z 228.0669 [M − H]− (calcd for C13H10NO3, 228.0661) and 457.1336 [2M − H]− (calcd for C26H21N2O6, 457.1400); 1H and 13C NMR data (Table 1). 2-(Furan-2-ylmethyl)-6,7-dihydroxy-3,4-dihydroisoquinolin-2ium (3): yellow, amorphous powder; HRESIMS m/z 244.0966 [M]+ (calcd for C14H14NO3, 230.0968); 1H and 13C NMR data (Table 1). Ethyl (S)-(−)-(6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline)propanoate (4): colorless solid; [α]20 D −18 (c 0.04, MeOH); negative HRESIMS m/z 264.1233 [M − H]− (calcd for C14H18NO4, 264.1236); positive HRESIMS m/z 266.1393 [M + H]+ (calcd for C14H20NO4, 266.1348); 1H and 13C NMR data (Table 1).

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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.7b00762. Figures S1.1−S4.9: HRESIMS, 1D NMR and 2D NMR spectra of compounds 1−4; physiochemical and spectroscopic data of known compounds 6−12 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(T. Shen) Tel: +86-531-88382028. Fax: +86-531-88382548. E-mail: [email protected]. *(L. Xiang) Tel: +86-531-88382028. Fax: +86-531-88382548. E-mail: [email protected]. ORCID

Tao Shen: 0000-0002-5474-2646 Lan Xiang: 0000-0002-7149-8235 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from Shandong Natural Science Foundation (ZR2017MH093), Science and Technology Development Program of Shandong Province (2014GSF119007), Major Project of Science and Technology of Shandong Province (2015ZDJS04001), Young Scholars Program of Shandong University (YSPSDU2015WLJH50), and China-Australia Centre for Health Sciences Research (2015).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00762 J. Nat. Prod. XXXX, XXX, XXX−XXX