Indoline Amide Glucosides from Portulaca oleracea: Isolation

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Indoline Amide Glucosides from Portulaca oleracea: Isolation, Structure, and DPPH Radical Scavenging Activity Ze-Zhao Jiao,† Su Yue,† Hong-Xiang Sun,† Tian-Yun Jin,† Hai-Na Wang,† Rong-Xiu Zhu,*,‡ and Lan Xiang*,† †

School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, People’s Republic of China School of Chemistry and Chemical Engineering, Shandong University, Shanda South Road, Jinan 250100, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: A polyamide column chromatography method using an aqueous ammonia mobile phase was developed for large-scale accumulation of water-soluble indoline amide glucosides from a medicinal plant, Portulaca oleracea. Ten new [oleraceins H, I, K, L, N, O, P, Q, R, S (1−10)] and four known [oleraceins A−D (11−14)] indoline amide glucosides were further purified and structurally characterized by various chromatographic and spectroscopic methods. The DPPH radical scavenging activities of oleraceins K (5) and L (6), with EC50 values of 15.30 and 16.13 μM, respectively, were twice that of a natural antioxidant, vitamin C; the EC50 values of the 12 other indoline amides, which ranged from 29.05 to 43.52 μM, were similar to that of vitamin C. Structure−activity relationships indicated that the DPPH radical scavenging activities of these indoline amides correlate with the numbers and positions of the phenolic hydroxy groups.

he stems1 and flowers2 of Portulaca oleracea L., an edible and medicinal plant in the family Portulacaceae, order Caryophyllales, contain betalains, which are commonly used as indicators in phytochemical taxonomy. Betalains are watersoluble pigments and include red betacyanins [5,6-dihydroxyindoline-2-carboxylic acid (i.e., cyclodopa) connected to betalamic acid, usually glucosylated] and yellow betaxanthins (amino acids connected to betalamic acid).3 Betalains are used as food colorants and demonstrate beneficial effects on human health,4 biological effects on the plant itself,5 and a distinct biosynthetic pathway.6 As a result, these molecules have aroused great interest among researchers in many fields. Similar to red betacyanins, the yellow water-soluble oleraceins A−D, F, and G, which were discovered in P. oleracea,7−9 contain 5,6-dihydroxyindoline-2-carboxylic acid moieties and are acylated with p-coumaric or ferulic acid. In addition, some of these indoline amide glucosides exhibit potent radical scavenging activities.10 The indoline amide glucosides, like the red betacyanins, are important secondary metabolites of P. oleracea, a plant that has been used in folk medicine in many countries and shows a wide range of pharmacological effects, including antibacterial, antioxidant, antiaging, antihypoxia, neuroprotective, anti-inflammatory, hypoglycemic, hypolipidemic, and skeletal-muscle relaxant activities.11 An HPLC-DAD detection method combined with LC-MS/ MS was recently developed by our group. Based on UV spectra and MS and MS/MS fragment analysis, eight new indoline amide glucosides (oleraceins H−O) were tentatively identified.12 However, the type and configuration of the sugar

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© XXXX American Chemical Society and American Society of Pharmacognosy

moieties and the connectivity among sugar moieties and between phenolic acid and sugar moieties have not yet been elucidated. In the present study, a polyamide column chromatography method using an aqueous ammonia mobile phase was developed for large-scale accumulation of watersoluble indoline amide glucosides from P. oleracea. Ten new [oleraceins H, I, K, L, N−S (1−10)] and four known [oleraceins A−D (11−14)] indoline amide glucosides (Figure 1) were further purified and structurally characterized using spectroscopic and chromatographic methods. Most significantly, all of these indoline amide glucosides exhibited potent DPPH radical scavenging activities, and their structure−activity relationships were analyzed.

■

RESULTS AND DISCUSSION

Structural Elucidation of Indoline Amide Glucosides. Compound 1 was a highly water-soluble, yellow, amorphous powder that fluoresced yellow under UV365 nm irradiation and bright yellow under sunlight when sprayed with 10% sulfuric acid. The molecular formula was deduced as C30H35NO16 according to the 13C NMR data (Figure S1.2, Supporting Information) and the deprotonated ion at m/z 664.1871 [M − H]− (calcd for C30H34NO16, 664.1872) in the HRESIMS (Figure S1.6, Supporting Information). The 1H NMR data (Table 1) (Figure S1.1, Supporting Information) revealed two isolated aromatic protons at δH 8.16 (1H, s) and 6.67 (1H, s), Received: June 12, 2015

A

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Figure 1. Chemical structures of compounds 1−14.

one nitrogen-connected methine proton at δH 5.31 (1H, br s), and two methylene protons at δH 3.42 (1H, m) and 3.09 (1H, m). Collectively, these data indicated a cyclodopa-type structure. An AA′BB′ spin system with signals at δH 7.52 (2H, d, J = 7.8 Hz) and 6.74 (2H, d, J = 7.8 Hz) and two transolefinic proton signals at δH 7.57 (1H, d, J = 15.0 Hz) and 6.58 (1H, d, J = 15.0 Hz) indicated the presence of a p-coumaroyl group. In addition, two anomeric protons at δH 4.51 (1H, d, J = 6.6 Hz) and 4.17 (1H, d, J = 7.8 Hz) were observed in the 1H NMR spectrum. According to the 13C NMR data (Table 1) and the HSQC spectrum (Figure S1.3, Supporting Information), compound 1 contained 30 carbons, including two carbonyl carbons (δC 174.2, 164.7), 14 sp2 carbons, one methylene carbon (δC 33.2), one methine carbon (δC 61.6), and 12 saccharide-type carbons (δC 104.6, 73.7, 76.2, 70.2, 75.9, 68.9; δC 103.8, 74.0, 77.3, 70.5, 76.7, 61.4). The chemical shifts of these disaccharidic carbons corresponded to those of two Dglucopyranose moieties with a 1,6-linkage.13 The D absolute configuration of the glucosyl moieties in 1 was confirmed by HPLC (Figure S14, Supporting Information),14 and the coupling constants of the anomeric protons of the glucosyl moieties confirmed the presence of β-D-glucopyranose. HMBC correlations (Figure 2) established the presence of the cyclodopa unit, as indicated by correlations from H-4 (δH 6.67, s) to C-3 (δC 33.2), C-7a (δC 135.7), and C-6 (δC 144.3); from H-7 (δH 8.16, s) to C-3a (δC 125.8), C-7a (δC 135.7), and C-5 (δC 144.1); and from H-3 (δH 3.42, m; 3.09, m) to the hydroxycarbonyl carbon (δC 174.2). HMBC correlations from H-3′ (δH 7.57, d, J = 15.0 Hz) to C-1′ (δC 164.7) and C-5′ (δC 130.5) and from H-5′, 9′ (δH 7.52, 2H, d, J = 7.8 Hz), and H-6′, 8′ (δH 6.58, 2H, d, J = 7.8 Hz) to C-7′ (δC 159.7) confirmed that compound 1 contained a p-coumaroyl moiety, and HMBC correlations from H-6″ (δH 4.18, m; 3.51, m) to C-1‴ (δC 103.8) further corroborated the presence of two β-Dglucopyranosyl units with a 1,6-linkage. Furthermore, NOESY correlations (Figure 2) between H-7 (δH 8.16, s) and H-1″ (δH 4.51, d, J = 6.6 Hz) and HMBC correlations from H-1″ to C-6 (δC 144.3) confirmed that the sugar moiety was linked to C-6 of the aglycone. Compound 1 was inferred to have a 2Sconfiguration based on sequential negative and positive Cotton effects at 236 and 261 nm, respectively, in the electronic circular dichroism (ECD) spectrum, similar to the effects of (2S)-indoline-2-carboxylic acid, which displayed sequential

negative and positive Cotton effects at 210 and 250 nm, respectively (Figure S12, Supporting Information). Consequently, the structure of oleracein H (1) was defined as (2S)-5hydroxy-1-p-coumaroyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside. Several of the physical characteristics of compound 2 were similar to those of compound 1. Its molecular formula was assigned as C31H37NO17 according to the 13C NMR data (Figure S2.2, Supporting Information) and the deprotonated ion at m/z 694.1975 [M − H]− (calcd for C31H36NO17, 694.1978) in the HRESIMS (Figure S2.6, Supporting Information). Similar to 1, the NMR data of 2 (Table 2) indicated the presence of a cyclodopa moiety and a β-Dglucopyranosyl-(1→6)-β-D-glucopyranosyl unit. In contrast to 1, the 1 H NMR spectrum (Figure S2.1, Supporting Information) of 2 showed the presence of a feruloyl moiety via a methoxy signal at δH 3.80 (3H, s), two trans-olefinic proton signals at δH 7.58 (1H, d, J = 15.0 Hz) and 6.72 (1H, d, J = 15.0 Hz), and ABX spin signals at δH 7.28 (1H, br s), δH 6.79 (1H, d, J = 7.2 Hz), and δH 7.15 (1H, d, J = 7.2 Hz). HMBC correlations (Figure S2.4, Supporting Information) from H-3′ (δH 7.58, d, J = 15.0 Hz) to C-1′ (δC 164.8), C-5′ (δC 112.7), and C-9′ (δC 122.3); from H-8′ (δH 6.79, d, J = 7.2 Hz) to C-4′ (δC 127.2) and C-6′ (δC 148.2); and from −OCH3 (δH 3.80, s, 3H) to C-6′ (δC 148.2) confirmed the presence of the feruloyl moiety, and further support was provided by the fact that the molecular weight of 2 was 30 amu greater than that of 1. Compound 2 was found to have a 2S-configuration based on sequential negative and positive Cotton effects at 237 and 266 nm, respectively, in the ECD spectrum (Figure S12, Supporting Information), with a tendency similar to (2S)indoline-2-carboxylic acid. On the basis of this analysis, the structure of oleracein I (2) was elucidated as (2S)-5-hydroxy-1feruloyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside. Compounds 3−10 are all water-soluble, yellow, amorphous powders and contain cyclodopa moieties. The 1H NMR and 13 C NMR data for these compounds are shown in Tables 1 and 2. Compound 3 was found to possess a molecular formula of C40H43NO19 based on the 13C NMR data (Figure S3.2, Supporting Information) and the deprotonated ion at m/z 840.2344 [M − H]− (calcd for C40H42NO19, 840.2346) in the B

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Table 1. 1H and 13C NMR Data (600 and 150 MHz, DMSO-d6) for Compounds 1, 3, 5, 7, 9, and 10 (δ in ppm, J in Hz) 1

3 δH (J)

δC

5 δH (J)

δC

7 δH (J)

δC

9 δH (J)

δC

10 δH (J)

δC

δH (J)

position

δC

2 2-COOH 3

61.6 174.2 33.2

4 5 6 7 7a 3a 1′ 2′ 3′ 4′ 5′, 9′ 6′, 8′ 7′ Glu-1 1″ 2″ 3″ 4″ 5″ 6″

112.1 144.1 144.3 108.3 135.7 125.8 164.7 116.3 142.6 126.5 130.5 116.0 159.7 104.6 73.7 76.2 70.2 75.9 68.9

4.51 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 4.18 m 3.51 m

103.8 73.5 77.3 69.2 77.2 67.8

4.55 d (7.8) 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.92 d (12.0) 3.72 d (10.8)

104.3 73.7 77.5 69.7 77.4 67.8

4.51 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.90 d (12.0) 3.72 m

104.6 73.7 76.2 70.2 75.9 68.9

4.51 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 4.20 d (9.0) 3.52 m

104.0 73.6 77.0 70.1 76.5 67.9

4.55 d (6.0) 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.90 d (11.4) 3.70 m

104.8 73.9 76.4 70.4 76.3 68.8

4.47 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 4.17 m 3.53 m

Glu-2 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

103.8 74.0 77.3 70.5 76.7 61.4

4.17 d (7.8) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.65 m 3.43 m

101.5 74.2 74.4 70.7 76.4 61.2

4.60 d (7.8) 4.58 d (8.4) 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.68 d (10.8) 3.48 d (10.8)

101.1 74.1 74.5 70.7 76.3 61.2

4.65 d (8.4) 4.56 d (9.0) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.68 m 3.47 m

103.8 74.0 77.3 70.4 76.7 61.4

4.18 d (7.2) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.67 d (11.4) 3.45 m

101.4 74.1 74.1 69.4 77.5 61.0

4.61 d (7.8) 4.54 d (9.0) 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.68 d (12.0) 3.46 m

103.6 73.7 76.0 70.3 74.1 63.9

4.27 d (7.8) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 4.36 d (10.8) 4.19 m

1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 7⁗ 8⁗ 9⁗ OCH3 Glu-3 1⁗′ 2⁗′ 3⁗′ 4⁗′ 5⁗′ 6⁗′

5.31 br s 3.42 m 3.09 m 6.67 s

8.16 s

6.58 d (15.0) 7.57 d (15.0) 7.52 d (7.8) 6.74 d (7.8)

62.0 174.3 33.4 112.3 144.4 142.9 109.4 136.2 126.3 164.4 117.2 141.4 126.6 130.1 115.9 159.6

166.2 115.4 145.2 126.2 111.3 148.4 149.6 116.1 123.5 56.1

5.14 br s 3.42 m 3.08 m 6.65 s

8.11 s

6.68 d (15.0) 7.47 d (15.0) 7.41 d (8.4) 6.76 d (8.4)

6.39 d (15.6) 7.51 d (15.6) 7.31 br s

6.79 d (8.4) 7.10 d (8.4) 3.82 s

62.7 174.5 33.6 112.4 145.4 143.2 109.7 136.4 126.7 164.5 117.6 141.1 127.0 130.0 116.1 159.4

166.3 114.8 145.4 126.2 115.4 146.3 148.7 115.9 121.4

5.02 m

61.8 174.1 33.2

3.41 m 3.10 m 6.65 s

8.12 s

6.71 d (15.0) 7.48 d (15.0) 7.38 d (8.4) 6.74 d (8.4)

112.0 144.1 144.3 108.4 135.7 126.0 164.5 118.1 141.8 129.3 130.1 116.7 159.0

5.22 m 3.43 m 3.12 m 6.68 s

8.16 s

6.80 d (15.6) 7.63 d (15.6) 7.64 d (8.4) 7.01 d (8.4)

112.3 144.5 142.7 110.2 136.4 127.1 164.3 119.7 140.0 129.5 129.6 116.8 158.8

166.2 115.3 145.2 126.2 111.3 148.3 149.6 115.9 123.5 56.1

6.16 d (16.2) 7.41 d (16.2) 7.11 br s

6.70 d (8.4) 6.92 d (8.4)

100.5 73.6 77.5 70.1 77.0 61.1

HRESIMS (Figure S3.7, Supporting Information). The 1H NMR data of 3 were similar to those of 1; however, signals corresponding to an extra feruloyl moiety were observed in the 1 H NMR spectrum (Figure S3.1, Supporting Information) of 3, which included a methoxy signal at δH 3.82 (3H, s), two transolefinic proton signals at δH 7.51 (1H, d, J = 15.6 Hz) and 6.39 (1H, d, J = 15.6 Hz), and ABX spin signals at δH 7.31 (1H, br s), 6.79 (1H, d, J = 8.4 Hz), and 7.10 (1H, d, J = 8.4 Hz). The presence of this feruloyl moiety was confirmed by key HMBC

63.2 174.4 33.6

4.93 d (7.8) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.67 d (11.4) 3.45 m

100.5 73.6 77.7 70.9 77.3 61.0

4.94 br s

63.0 174.5 33.7

3.35 m 3.10 m 6.67 s

8.10 s

6.77 d (15.0) 7.48 d (15.0) 7.52 d (8.4) 7.03 d (8.4)

6.33 d (16.2) 7.49 d (16.2) 7.30 br s

6.78 d (8.4) 7.10 d (8.4) 3.83 s

111.9 143.9 144.1 108.7 136.1 126.9 164.8 117.0 141.7 126.6 130.2 115.9 159.7

167.2 114.8 145.6 126.0 111.5 148.4 149.8 116.0 123.8 56.1

4.92 br s 3.37 m 3.06 m 6.64 s

8.17 s

6.66 d (15.0) 7.55 d (15.0) 7.45 d (7.8) 6.75 d (7.8)

6.52 d (16.2) 7.53 d (16.2) 7.31 br s

6.73 d (8.4) 7.08 d (8.4) 3.77 s

4.89 d (7.2) 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.1−3.5 m 3.68 d (12.0) 3.46 m

correlations (Figure 2) from H-3⁗ (δH 7.51, d, J = 15.6 Hz) to C-1⁗ (δC 166.2), C-5⁗ (δC 111.3), and C-9⁗ (δC 123.5), from H-8⁗ (δH 6.79, d, J = 8.4 Hz) to C-4⁗ (δC 126.2) and C-6⁗ (δC 148.4), and from −OCH3 (δH 3.82, s, 3H) to C-6⁗ (δC 148.4). In addition, H-2‴ (δH 4.58, d, J = 8.4 Hz) of the terminal glucose moiety exhibited HMBC correlations (Figure 2) with C-1⁗ (δC 166.2) and C-1‴ (δC 101.5), indicating that this feruloyl moiety is connected to C-2‴ of the terminal glucose unit. Support was also provided by the shielding of CC

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Figure 2. Key HMBC (H → C) and NOESY (↔) correlations of compounds 1, 3, 7, and 10.

calculated by the Gaussian equation was similar to that of 3 (Figure 3). Based on the 13C NMR data (Figure S4.2, Supporting Information) and the deprotonated ion at m/z 870.2457 [M − H]− (calcd for C41H44NO20, 870.2451) in the HRESIMS (Figure S4.7, Supporting Information), the molecular formula of compound 4 was established as C41H45NO20, which is 30 amu greater than that of 3. In accordance with the molecular weight, the extra methoxy signal at δH 3.82 (3H, s) in the 1H NMR spectrum of 4 indicated that a feruloyl moiety in 4 replaces the p-coumaroyl moiety present in 3. Similar to 3, the ECD spectrum (Figure S12, Supporting Information) of 4 also showed sequential negative and positive Cotton effects at 323 and 360 nm, respectively, due to the intramolecular stackinginduced exciton coupling effect between the two feruloyl moieties. On the basis of the biosynthetic pathway, oleracein O (4) was also inferred to have a 2S-configuration, and its structure was defined as (2S)-5-hydroxy-1-feruloyl-2,3-dihydro1H-indole-2-carboxylic acid-6-O-[2-O-feruloyl-β-D-glucopyranosyl]-(1→6)-β-D-glucopyranoside. The molecular formula of compound 5 was assigned as C39H41NO19 based on the 13C NMR data (Figure S5.2, Supporting Information) and the deprotonated ion at m/z 826.2187 [M − H]− (calcd for C39H40NO19, 826.2189) in the HRESIMS (Figure S5.6, Supporting Information). The 1H and 13 C NMR data for 5 and 1 were similar except for a caffeoyl moiety in 5, as indicated by the aromatic ABX spin signals at δH 7.11 (1H, br s), 6.70 (1H, d, J = 8.4 Hz), and 6.92 (1H, d, J = 8.4 Hz) and two trans-olefinic proton signals at δH 7.41 (1H, d, J = 16.2 Hz) and 6.16 (1H, d, J = 16.2 Hz) in the 1H NMR spectrum (Figure S5.1, Supporting Information). The presence of the caffeoyl moiety was confirmed by HMBC correlations (Figure S5.4, Supporting Information) from H-3⁗ (δH 7.41, d, J = 16.2 Hz) to C-1⁗ (δC 166.3), C-5⁗ (δC 115.4), and C-9⁗ (δC 121.4) and from H-8⁗ (δH 6.70, d, J = 8.4 Hz) to C-4⁗ (δC 126.2) and C-6⁗ (δC 146.3). Moreover, this caffeoyl moiety was demonstrated to be linked to C-2‴ of the terminal glucose

1‴ (2.3 ppm) and C-3‴ (2.9 ppm) and the deshielding of C-2‴ (0.2 ppm) upon comparison with the 13C NMR data of 1.15 The ECD spectrum (Figure S12, Supporting Information) of 3 showed sequential negative and positive Cotton effects at 320 and 358 nm, respectively, whereas couplet Cotton effects centered at 250 nm in 1 were not observed in the spectrum of 3. Previous researchers have reported a similar phenomenon, whereby the ECD spectrum of a compound comprising a feruloyl moiety connected to a terminal isoliquiritin apioside sugar moiety (a chalcone bioside with no chirality in its aglycone) showed sequential negative and positive Cotton effects at 316 and 358 nm, respectively,16 as a result of an exciton coupling effect induced by intramolecular stacking between the acyl group and the aglycone (isoliquiritigenin). It was, therefore, inferred that the couplet Cotton effects in the ECD spectrum of 3 were due to intramolecular stackinginduced exciton coupling between the feruloyl moiety on the terminal glucose and the p-coumaroyl moiety of the aglycone. On the other hand, due to the anisotropic effect, the H-3′, 5′, and 9′ p-coumaroyl signals for 3 showed notable shielding (0.10, 0.11, and 0.11 ppm, respectively) and the H-2′ signal deshielding (0.10 ppm) relative to the corresponding signals of 1. This finding suggested that the feruloyl moiety on the terminal glucose and the p-coumaroyl moiety of the aglycone of 3 were stacked in a manner analogous to the intramolecular stacking of acylated anthocyanins,17 with H-2′ in a deshielding zone and H-3′, 5′, and 9′ in a shielding zone of the benzene ring of the feruloyl moiety. In addition, the exciton coupling effect between these two chromophores was strong enough to potentially cover the 250 nm centered couplet induced by the aglycone. However, on the basis of the potentially similar biosynthetic pathway of compounds 3 and 1, compound 3 was inferred to have a 2S-configuration. Therefore, the structure of oleracein N (3) was defined as (2S)-5-hydroxy-1-p-coumaroyl2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-O-feruloyl-β-Dglucopyranosyl]-(1→6)-β-D-glucopyranoside. A possible stereostructure of 3 was illustrated in Figure 3. The ECD spectrum D

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Table 2. 1H and 13C NMR Data (600 and 150 MHz, DMSO-d6) for Compounds 2, 4, 6, and 8 (δ in ppm, J in Hz) 2

4

6

8

position

δC

2 2-COOH 3

62.4 175.2 33.5

4 5 6 7 7a 3a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ OCH3 Glu-1 1″ 2″ 3″ 4″ 5″ 6″

112.0 143.9 144.3 108.5 136.0 126.5 164.8 116.9 142.6 127.2 112.7 148.2 149.1 116.0 122.3 56.2 104.8 73.7 76.2 70.3 75.9 69.0

4.50 d (5.4) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 4.23 d (9.6) 3.52 m

103.8 73.4 77.3 69.3 77.2 67.9

4.55 d (7.2) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.91 d (12.0) 3.71 d (12.0)

104.4 73.6 77.4 69.8 77.4 67.7

4.50 d (7.2) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.89 d (12.0) 3.72 m

104.8 73.7 76.2 70.3 75.9 69.0

4.49 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 4.20 d (10.2) 3.52 m

Glu-2 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

103.8 74.0 77.3 70.5 76.7 61.4

4.20 d (7.2) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.68 s 3.46 m

101.5 74.2 74.4 70.7 76.4 61.2

4.60 d (8.4) 4.58 d (9.0) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.68 d (9.0) 3.47 m

101.0 74.1 74.5 70.7 76.3 61.2

4.64 d (9.0) 4.54 d (8.4) 3.1−3.6 m 3.1−3.6 m 3.1−3.6 m 3.71 m 3.44 m

103.8 74.0 77.3 70.4 76.7 61.4

4.18 d (7.2) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.67 d (11.4) 3.45 m

100.1 73.6 77.5 70.0 77.3 61.0

4.93 d (6.6) 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.0−3.5 m 3.65 d (13.2) 3.45 m

1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 7⁗ 8⁗ 9⁗ OCH3 Glu-3 1⁗′ 2⁗′ 3⁗′ 4⁗′ 5⁗′ 6⁗′

δH (J) 5.18 m 3.40 m 3.11 m 6.69 s

8.21 s

6.72 d (15.0) 7.58 d (15.0) 7.28 br s

6.79 d (7.2) 7.15 d (7.2) 3.80 s

δC

δH (J)

δC

61.9 174.2 33.3

5.23 d (3.6)

61.9 174.5 33.5

112.3 144.5 142.9 109.5 136.2 126.2 164.4 117.4 141.9 126.3 112.1 148.4 148.9 116.0 122.2 56.1

166.2 115.4 145.2 127.1 111.3 148.2 149.6 115.9 123.5 56.1

3.40 m 3.07 m 6.66 s

8.13 s

6.72 d (15.6) 7.48 d (15.6) 7.16 br s

6.79 d (7.8) 7.03 d (7.8) 3.82 s

6.40 d (15.6) 7.51 d (15.6) 7.32 br s

6.76 d (8.4) 7.11 d (8.4) 3.76 s

112.4 144.5 143.1 110.0 136.5 127.1 164.5 116.0 141.4 127.3 112.0 148.1 148.7 116.0 122.1 56.0

166.3 114.8 145.4 126.1 115.3 146.3 148.7 116.0 121.4

δH (J) 5.17 br s 3.41 m 3.11 m 6.66 s

8.13 s

6.73 d (15.0) 7.45 d (15.0) 7.10 br s

6.72 d (7.8) 6.97 d (7.8) 3.74 s

δC 62.4 174.4 33.4 112.0 144.0 144.2 108.6 135.9 126.6 164.5 118.6 141.8 129.7 112.4 149.4 148.3 115.4 121.8 56.2

δH (J) 5.11 m 3.38 m 3.12 m 6.66 s

8.17 s

6.81 d (15.0) 7.59 d (15.0) 7.30 br s

7.05 d (7.8) 7.21 d (7.8) 3.77 s

6.14 d (15.6) 7.38 d (15.6) 7.14 br s

6.69 d (7.8) 6.91 d (7.8)

compound 1.15 The ECD spectrum (Figure S12, Supporting Information) of 5 showed sequential negative and positive Cotton effects at 319 and 357 nm, respectively, due to the intramolecular stacking-induced exciton coupling effect be-

according to HMBC correlations of the glucose H-2‴ (δH 4.56, d, J = 9.0 Hz) to C-1⁗ (δC 166.3) and C-1‴ (δC 101.1), as well as the shielding of C-1‴ (2.7 ppm) and C-3‴ (2.8 ppm) and the deshielding of C-2‴ (0.1 ppm) in comparison with E

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Figure 3. Possible stereostructure (A) and calculated and experimental ECD spectra (B) of compound 3.

tween the caffeoyl and p-coumaroyl moieties. Oleracein K (5) was inferred to have a 2S-configuration, and its structure was elucidated as (2S)-5-hydroxy-1-p-coumaroyl-2,3-dihydro-1Hindole-2-carboxylic acid-6-O-[2-O-caffeoyl-β-D-glucopyranosyl]-(1→6)-β-D-glucopyranoside. On the basis of the 13C NMR data (Figure S6.2, Supporting Information) and the deprotonated ion at m/z 856.2295 [M − H]− (calcd for C40H42NO20, 856.2295) in the HRESIMS (Figure S6.6, Supporting Information), the molecular formula of compound 6 was deduced to be C40H43NO20, which is 30 amu greater than that of 5. The NMR data and HMBC correlations (Figure S6.4, Supporting Information) of compound 6 indicated the presence of a cyclodopa moiety and a 2O-caffeoyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl unit, similar to 5. However, the signals of the methoxy protons at δH 3.74 (3H, s), ABX aromatic protons at δH 7.10 (1H, br s), 6.72 (1H, d, J = 7.8 Hz), and 6.97 (1H, d, J = 7.8 Hz), and two olefinic protons at δH 6.73 (1H, d, J = 15.0 Hz) and 7.45 (1H, d, J = 15.0 Hz) indicated a feruloyl moiety present in 6 instead of the p-coumaroyl moiety in 5. This difference was in accordance with the molecular weight of 6 being 30 amu greater than that of 5. Sequential negative and positive Cotton effects at 319 and 360 nm were also observed in the ECD spectrum (Figure S12, Supporting Information) of 6 due to the intramolecular stacking-induced exciton coupling effect between the caffeoyl and feruloyl moieties. Oleracein L (6) was inferred to have a 2S-configuration, and its structure was defined as (2S)-5-hydroxy-1-feruloyl-2,3-dihydro-1H-indole-2carboxylic acid-6-O-[2-O-caffeoyl-β-D-glucopyranosyl]-(1→6)β-D-glucopyranoside. The molecular formula of compound 7 was deduced as C36H45NO21 according to the 13C NMR data (Figure S7.2, Supporting Information) and the deprotonated ion at m/z 826.2398 [M − H]− (calcd for C36H44NO21, 826.2400) in the HRESIMS (Figure S7.6, Supporting Information). The NMR data for 7 were similar to those for 1, except for the presence of an extra β-D-glucose residue with an anomeric proton signal at δH 4.93 (1H, d, J = 7.8 Hz) and carbon signals at δC 100.5, 73.6, 77.5, 70.1, 77.0, and 61.1. This anomeric proton (δH 4.93, d, J = 7.8 Hz) was correlated with C-7′ (δC 159.0) in the HMBC spectrum (Figure S7.4, Supporting Information), indicating that the extra β-D-glucose was linked to C-7′ of the pcoumaroyl moiety of the aglycone. Similar to compound 1, the β-D-glucopyranose moieties were connected by a 1,6-linkage according to the HMBC correlation (Figure 2) from H-6″ (δH 4.20, d, J = 9.0 Hz; δH 3.52, m) of one glucose to the anomeric carbon (δC 103.8) of the terminal glucose. The ECD spectrum (Figure S12, Supporting Information) of 7 revealed sequential negative and positive Cotton effects at 237 and 261 nm, respectively, similar to (2S)-indoline-2-carboxylic acid. Thus,

the structure of oleracein P (7) was elucidated as (2S)-5hydroxy-1-(p-coumaroyl-7′-O-β-D-glucopyranose)-2,3-dihydro1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1→6)-βD-glucopyranoside. According to the 13C NMR data (Figure S8.2, Supporting Information) and the deprotonated ion at m/z 856.2504 [M − H]− (calcd for C37H46NO22, 856.2506) in the HRESIMS (Figure S8.6, Supporting Information), the molecular formula of compound 8 was deduced to be C37H47NO22, which is 30 amu greater than that of 7 and indicated the presence of an extra methoxy group in 8. The NMR data also demonstrated a feruloyl moiety present in 8 instead of the p-coumaroyl moiety in 7. Similar to 7, the anomeric proton δH 4.93 (1H, d, J = 6.6 Hz) of one β-D-glucopyranosyl moiety in 8 had an HMBC correlation with C-7′ (δC 148.3), indicating that this glucose is linked to the C-7′ of the feruloyl moiety. Furthermore, the other β-D-glucopyranosyl moieties were connected by a 1,6linkage, as based on HMBC correlations (Figure 2) from H-6″ (δH 4.20, d, J = 10.2 Hz; δH 3.52, m) of one glucose to the anomeric carbon (δC 103.8) of the terminal glucose. The sequential negative and positive Cotton effects at 237 and 261 nm, respectively, in the ECD spectrum (Figure S12, Supporting Information) of 8 were similar to those of (2S)-indoline-2carboxylic acid, indicating that 8 has a 2S-configuration. Oleracein Q (8) was thus elucidated as (2S)-5-hydroxy-1(feruloyl-7′-O-β-D-glucopyranose)-2,3-dihydro-1H-indole-2carboxylic acid-6-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside. The molecular formula of 9 was deduced as C46H53NO24 based on the 13C NMR data (Figure S9.2, Supporting Information) and HRESIMS signals at m/z 1004.3038 [M + H]+ (calcd for C46H54NO24, 1004.3030) and m/z 1026.2845 [M + Na]+ (calcd for C46H53NO24Na, 1026.2850) (Figure S9.8, Supporting Information). Similar to 3, the NMR data and HSQC, HMBC, and NOE correlations (Figures S9.1−S9.7, Supporting Information) of 9 suggested the presence of an aglycone of p-coumaroylated cyclodopa and a 6-O-[2-Oferuloyl-β-D-glucopyranosyl]-(1→6)-β-D-glucopyranosyl unit. However, 9 contained one more β-D-glucopyranosyl moiety than 3, as indicated by an anomeric proton at δH 4.89 (1H, d, J = 7.2 Hz) and extra carbons at δC 100.5, 73.6, 77.7, 70.9, 77.3, and 61.0. This glucose was linked to C-7′ of the p-coumaroyl moiety according to the HMBC correlation from the anomeric proton H-1⁗′ (δH 4.89, d, J = 7.2 Hz) to the aromatic carbon at C-7′ (δC 158.8). As a result of the intramolecular stackinginduced exciton coupling effect between the feruloyl and pcoumaroyl moieties, the ECD spectrum (Figure S12, Supporting Information) of 9 also showed sequential negative and positive Cotton effects at 317 and 356 nm, respectively. Considering the shared biosynthetic pathway, oleracein R (9) F

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was inferred to have a 2S-configuration, and its structure was identified as (2S)-5-hydroxy-1-(p-coumaroyl-7′-O-β-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-Oferuloyl-β-D-glucopyranosyl]-(1→6)-β-D-glucopyranoside. The molecular formula of 10 was deduced as C40H43NO19 based on the 13C NMR data (Figure S10.2, Supporting Information) and the deprotonated ion at m/z 840.2350 [M − H]− (calcd for C40H42NO19, 840.2346) in the HRESIMS (Figure S10.6, Supporting Information), which was the same as that for compound 3. The NMR data and predicted structures of 10 and 3 were similar, except for linkage of the trans-feruloyl moiety with the terminal glucopyranose. The HMBC correlations (Figure 2) from H-6‴ (δH 4.36, d, J = 10.8 Hz; δH 4.19, m) to C-1⁗ (δC 167.2) and the deshielding of C-6‴ (δC 63.9) of compound 10 compared with that of compound 3 confirmed that the trans-feruloyl group was linked to C-6‴ of the terminal glucopyranose. The ECD spectrum (Figure S12, Supporting Information) of 10 showed sequential negative and positive Cotton effects at 236 and 265 nm, respectively, similar to (2S)-indoline-2-carboxylic acid, indicating that 10 has a 2Sconfiguration. Additionally, sequential negative and positive Cotton effects at 323 and 359 nm resulting from the intramolecular stacking-induced exciton coupling effect between the feruloyl and p-coumaroyl moieties were also revealed in the ECD spectrum. In correspondence with this intramolecular-stacking phenomenon, the signals of H-3′, 5′, and 9′ of 10 showed shielding (0.02, 0.07, and 0.07 ppm, respectively) and H-2′ deshielding (0.08 ppm) relative to the corresponding signals of 1, which was attributable to anisotropic effects, similar to the case of 3. The structure of oleracein S (10) was therefore elucidated as (2S)-5-hydroxy-1-p-coumaroyl-2,3-dihydro-1Hindole-2-carboxylic acid-6-O-[6-O-feruloyl-β-D-glucopyranosyl]-(1→6)-β-D-glucopyranoside. Compounds 11−14 are known oleraceins A−D,7 which were identified according to their 1H NMR data and chromatography methods. An HPLC method was used to confirm the absolute configuration of the glucose in the indoline amide glucosides, and oleracein P (7), oleracein H (1), and oleracein A (11) were selected as examples of a tri-, di-, and monoglucoside, respectively, of (2S)-5-hydroxy-1-p-coumaroyl-2,3-dihydro-1Hindole-2-carboxylic acid. After acid hydrolysis of the amide glucosides, the aqueous fraction containing the sugar moieties was reacted with L-cysteine methyl ester hydrochloride, followed by o-tolyl isothiocyanate. The reaction mixtures were subjected to HPLC analysis. As shown in Figure S14 (Supporting Information), all of the reaction mixtures of these three alkaloids displayed peaks at the same retention times as the D-glucose derivative standard (I), demonstrating that the glucosyl moieties in compounds 7, 1, and 11 are D-configured. DPPH Radical Scavenging Activity of Indoline Amide Glucosides. The potential preventive or therapeutic effects of phenolic compounds on oxidative stress-induced diseases have frequently been emphasized,18,19 and DPPH• scavenging activity assays are commonly used to evaluate the antioxidant effects of specific compounds or extracts of plants or foods. In this experiment, the DPPH• scavenging activities of phenolic indoline amide glucosides 1−14 were determined, and their structure−activity relationships were analyzed. As shown in Figure 4, all indoline amide glucosides were found to effectively scavenge DPPH• in a dose-dependent manner. Figure 5 illustrated that oleraceins K and L (5 and 6) possessed the most effective DPPH• scavenging activities among the 14

Figure 4. DPPH• scavenging activities of compounds 1−14 (n = 3).

Figure 5. EC50 values of DPPH radical scavenging activities for compounds 1−14 (n = 3) (**p < 0.01, vs VC).

amide glucosides, with EC50 values of 15.30 and 16.13 μM, respectively. These compounds are, therefore, almost twice as potent as the natural antioxidant vitamin C (30.15 μM) (p < 0.01). Oleraceins N and O (3 and 4) and oleraceins R, S, and A−D (9−14) showed activities similar to vitamin C (EC50 = 27.64−38.28 μM), whereas the activities of oleraceins H, I, P, and Q (1, 2, 7, and 8) were weaker (EC50 = 40.10−43.52 μM) than that of vitamin C. Similar to flavonoids,20,21 the present results also revealed that the radical scavenging activities of these indoline amide glucosides correlate with the numbers and positions of the phenolic hydroxy groups. The structure−activity relationships of these compounds are depicted in Figure 6, with the numbers in the figure representing the following findings: (1) No significant differences (p > 0.05) were found between the EC50 values of oleraceins A, H, and N (11, 1, and 3) and their corresponding 7′-O-glucosides, i.e., oleraceins C, P, and R (13, 7, and 9), indicating that the hydroxy group at C-7′ has little effect on the radical-scavenging activity of the molecules, although the hydroxy group at C-5 does strongly contribute to the activity. (2) There was no significant difference between the EC50 values of monoglucoside oleraceins A and C (11 and 13) and their corresponding diglucoside oleraceins H and P (1 and G

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Figure 6. Structure−activity relationships of indoline amide glucosides (OP: oleracein P; OC: oleracein C; OR: oleracein R; OH: oleracein H; OK: oleracein K; OA: oleracein A; OB: oleracein B; ON: oleracein N; OS: oleracein S).

pharmacological effects, distribution, and biosynthetic pathway(s) of these special alkaloids.

7), indicating that substitution of the diglucoside at C-6 has little effect on the radical scavenging activities of the compounds. (3) Oleracein N (3) was found to be more potent than oleracein H (1) (p < 0.05), and oleracein R (9) was more potent than oleracein P (7) (p < 0.05), indicating that the introduction of a feruloyl moiety onto C-2 of these disaccharides can significantly enhance the radical scavenging activity of the indoline amides as a result of the synergic effects of the feruloyl moiety and aglycone. (4) The activity of oleracein K (5), which contains a caffeoyl moiety, was significantly higher than that of oleracein N (3), which contains a feruloyl moiety, indicating that the number of hydroxy groups of the phenolic acyl connected with the sugar moiety has a significant effect on its radical scavenging activity. (5) The activity of oleracein S (10) was significantly higher than that of oleracein H (1) (p < 0.05), indicating that the introduction of a feruloyl moiety onto C-6 of these disaccharides significantly enhances the radical scavenging activity of the indoline amides, similar to what occurs with addition of a feruloyl moiety onto C-2 of the disaccharides. (6) It has been reported22 that ferulic acid is slightly more potent that p-coumaric acid in terms of antioxidant activity. This difference is explained by the ability of the methoxy group, as an electron donor, to form p−π conjugation with the benzene ring, leading to a more stable phenoxy radical. However, no significant difference was observed between the activities of oleracein A (11) and oleracein B (12) (p > 0.05), indicating that the presence of a pcoumaroyl or a feruloyl moiety in the aglycone has no effect on DPPH• scavenging activity. The present work on the isolation, structure, and preliminary radical scavenging activity of indoline amide glucosides may provide the basis for further research on the biological and

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined using a Gyromat-Hp digital automatic polarimeter (Kernchen Co., Germany). ECD spectra were obtained with a Chirascan circular dichroism spectrophotometer (Applied Photophysics Ltd., UK). NMR spectra were recorded using Agilent 600 MHz DD2 and Bruker Avance DRX-600 spectrometers (1H, 600 MHz; 13C, 150 MHz; TMS as an internal standard). HRESIMS spectra were measured with an LTQ-Orbitrap XL mass spectrometer (ThermoFinnigan, Bremen, Germany). UV spectra were recorded using a UV-2450 spectrophotometer (Shimadzu Co., Japan). Microplate assays were carried out using a model 680 microplate reader (Bio-Rad Co., USA). A Shimadzu Prominence LC-20A liquid chromatograph with two LC-20AT pumps and an SPD-20A UV detector (Shimadzu Co., Japan) was used with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm) (YMC Co., Japan) for semipreparation of the compounds or an Inertsil ODS-3 column (250 × 4.6 mm, 5 μm) for compound analysis (GL Sciences, Japan). In both cases, a precolumn (4.0 × 3.0 mm, 5 μm) (Phenomenex Co., USA) was included. The extracts were separated on a polyamide gel (60−100 mesh, Taizhou Luqiao Siqing Biochemical Plastics Factory, China), MCI gel (CHP-20P, 75−150 μm, Mitsubishi Chemical Co., Japan), Sephadex LH-20 (Pharmacia Fine Chemicals, USA), or ODS-C18 (75 μm, YMC Co., Japan). TLC was carried out using GF 254 silica gel (Qingdao Marine Chemical Co., China), polyamide film (Taizhou Luqiao Siqing Biochemical Plastics Factory, China), and RP-C18F254s metal plates (Merck Co., Germany). D-Glucose and vitamin C were obtained from Tianjin Damao Chemical Co. (Tianjin, China). 1,1Diphenyl-2-picrylhydrazyl radical (DPPH•) was purchased from Sigma Co. (USA). L-Cysteine methyl ester hydrochloride and o-tolyl isothiocyanate were purchased from Tokyo Chemical Industry (Japan). Distilled H2O was purified using a D11951 Ultrapure water system (Thermo Fisher Scientific, Schwerte, Germany). HPLC-grade H

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mL/min), affording compounds 14 (9.0 mg, tR = 42 min) and 9 (4.5 mg, tR = 58 min). Oleracein H (1): yellow, amorphous powder; [α]25 D = −175 (c 0.1, H2O); UV (H2O) λmax (log ε) 341 (4.20), 301 (4.12) nm; HRESIMS m/z 664.1871 [M − H]− (calcd for C30H34NO16, 664.1872); 1H and 13 C NMR data (Table 1). Oleracein I (2): yellow, amorphous powder; [α]25 D = −192 (c 0.1, H2O); UV (H2O) λmax (log ε) 347 (4.17), 303 (3.98) nm; HRESIMS m/z 694.1975 [M − H]− (calcd for C31H36NO17, 694.1978); 1H and 13 C NMR data (Table 2). Oleracein N (3): yellow, amorphous powder; [α]25 D = −142 (c 0.1, H2O); UV (H2O) λmax (log ε) 326 (4.41), 299 (4.34) nm; HRESIMS m/z 840.2344 [M − H]− (calcd for C40H42NO19, 840.2346); 1H and 13 C NMR data (Table 1). Oleracein O (4): yellow, amorphous powder; [α]25 D = −117 (c 0.1, H2O); UV (H2O) λmax (log ε) 330 (4.38), 298 (4.27) nm; HRESIMS m/z 870.2457 [M − H]− (calcd for C41H44NO20, 870.2451); 1H and 13 C NMR data (Table 2). Oleracein K (5): yellow, amorphous powder; [α]25 D = −73 (c 0.1, H2O); UV (H2O) λmax (log ε) 326 (4.43), 300 (4.37) nm; HRESIMS m/z 826.2187 [M − H]− (calcd for C39H40NO19, 826.2189); 1H and 13 C NMR data (Table 1). Oleracein L (6): yellow, amorphous powder; [α]25 D = −116 (c 0.1, H2O); UV (H2O) λmax (log ε) 332 (4.34), 300 (4.24) nm; HRESIMS m/z 856.2295 [M − H]− (calcd for C40H42NO20, 856.2295); 1H and 13 C NMR data (Table 2). Oleracein P (7): yellow, amorphous powder; [α]25 D = −208 (c 0.1, H2O); UV (H2O) λmax (log ε) 335 (4.16), 307 (4.19) nm; HRESIMS m/z 826.2398 [M − H]− (calcd for C36H44NO21, 826.2400); 1H and 13 C NMR data (Table 1). Oleracein Q (8): yellow, amorphous powder; [α]25 D = −210 (c 0.1, H2O); UV (H2O) λmax (log ε) 338 (4.16), 307 (4.05) nm; HRESIMS m/z 856.2504 [M − H]− (calcd for C37H46NO22, 856.2506); 1H and 13 C NMR data (Table 2). Oleracein R (9): yellow, amorphous powder; [α]25 D = −160 (c 0.1, H2O); UV (H2O) λmax (log ε) 320 (4.39), 308 (4.39) nm; HRESIMS m/z 1004.3038 [M + H]+ (calcd for C46H54NO24, 1004.3030), m/z 1026.2845 [M + Na]+ (calcd for C46H53NO24Na, 1026.2850); 1H and 13 C NMR data (Table 1). Oleracein S (10): yellow, amorphous powder; [α]25 D = −180 (c 0.1, H2O); UV (H2O) λmax (log ε) 326 (4.38), 300 (4.30) nm; HRESIMS m/z 840.2350 [M − H]− (calcd for C40H42NO19, 840.2346); 1H and 13 C NMR data (Table 1). Determination of the Absolute Configuration of Glucosyl Moieties. Preparation of the D-Glucose Derivative. The preparation of the D-glucose derivative standard followed the method reported by Tanaka and co-workers.14 Briefly, D-glucose (40 mg) and L-cysteine methyl ester hydrochloride (50 mg) were dissolved in pyridine (3 mL) and heated at 60 °C for 1.5 h. O-Tolyl isothiocyanate (180 μL) was added to this reaction mixture and heated at 60 °C for 1.5 h. After evaporating the solvent using a rotary evaporator, the dried reaction mixtures of the D-glucose derivative were dissolved in MeOH, separated by semipreparative HPLC on a YMC-pack ODS-A (250 × 10 mm, 5 μm) column equipped with a precolumn (4.0 × 3.0 mm, 5 μm), and eluted with MeOH−0.1% formic acid (40:60, 1.5 mL/min) to obtain the D-glucose derivative standard (I, tR = 17.5 min). The product was further confirmed by the signal at m/z 447.1252 [M + H]+ (calcd for C18H27N2O7, 447.1254) in the HRESIMS, identical to the value reported in the literature.14,23 Acid Hydrolysis, Preparation of Sugar Derivative, and HPLC Analysis. Compounds 1 (oleracein H, 0.7 mg), 7 (oleracein P, 1 mg), and 11 (oleracein A, 0.8 mg) were hydrolyzed individually in 1 N HCl (2 mL) by heating at 80 °C for 1.5 h. Each reaction mixture was extracted three times with an equal volume of EtOAc. Next, the aqueous layer was evaporated to dryness using a rotary evaporator. The dried residue was dissolved in pyridine (0.5 mL), combined with L-cysteine methyl ester hydrochloride (10 mg), and heated at 60 °C for 1.5 h. Next, o-tolyl isothiocyanate (40 μL) was added to the mixture and allowed to react at 60 °C for 1.5 h. The reaction mixtures

MeCN and MeOH were purchased from Tianjin SayFo Technology Co., China. The other solvents used were of analytical grade (Fuyu Fine Chemical Factory, Tianjin, China). Plant Materials. Dried aerial parts of P. oleracea were purchased from Jianlian Pharmacy (Jinan, P. R. China) and were identified as P. oleracea L. by one of the authors (L. Xiang). A voucher specimen (No. 20120501) was deposited at the Department of Pharmacognosy, School of Pharmaceutical Sciences, Shandong University. Extraction and Isolation. Dried, sliced aerial parts of P. oleracea (4 kg) were extracted with 60% EtOH (24 L) three times (1 h each) under reflux. The combined extracts were concentrated under vacuum to 8 L and were then stored in a 4 °C refrigerator for a week. The supernatant (6 L) was divided into two equal parts, subjected to two polyamide CC (60 × 10 cm, 800 g), and eluted with equal gradients of EtOH−H2O (0:100, 9 L; 20:80, 40:60, 60:40, 80:20, 95:5, each 4 L) followed by an ammonia solution (25% aqueous ammonia−85% EtOH, 1:7, 4 L) to afford nine fractions (Frs. 1−9). Among the fractions, Fr. 9 was the richest in indoline amide glucosides, as determined by HPLC detection. Fr. 9 (12.2 g), eluted by ammonia solution, was concentrated and dissolved in 20% EtOH and then centrifuged. The supernatant was subjected to Sephadex LH-20 CC (400 g, 59 × 6 cm) and eluted with 20% EtOH, which resulted in seven fractions (G1−G7). Fraction G4 (3.0 g) was subsequently separated by Sephadex LH-20 CC (400 g, 59 × 6 cm, 80% EtOH) twice. The yellow fraction was purified by ODSC18 (40 g, 9 × 3.5 cm) with a gradient mixture of EtOH−H2O (0:100 to 100:0, v/v), followed by purification by preparative HPLC using a mobile phase of MeOH−0.1% formic acid (33:67, 1.5 mL/min), affording compounds 1 (20 mg, tR = 55 min) and 2 (30 mg, tR = 65 min). Fraction G5 (900 mg) was subjected to Sephadex LH-20 CC (200 g, 90 × 4 cm, MeOH) twice. Next, the yellow fraction was separated by MCI chromatography (120 mL, 39 × 2 cm) and eluted with a gradient mixture of EtOH−H2O (10:90 to 100:0, v/v), affording seven fractions G5-(1−7). G5-(2−4) (300 mg) were further chromatographed by semipreparative HPLC and eluted with MeOH− 0.1% formic acid (40:60, 1.5 mL/min), yielding compounds 11 (100 mg, tR = 55 min) and 12 (100 mg, tR = 62 min). In addition, G5-(5− 7) (96 mg) were purified by ODS-C18 (15 g, 7 × 2.5 cm) with a gradient mixture of MeOH−H2O (0:100 to 100:0, v/v), followed by semipreparative HPLC eluted with MeOH−0.1% formic acid (40:60, 1.5 mL/min), affording compounds 3 (6.0 mg, tR = 57 min) and 4 (5.0 mg, tR = 64 min). Fraction G6 (708 mg) was separated by ODS-C18 (100 g, 25 × 3.5 cm) and eluted with a gradient of EtOH−H2O (0:100 to 100:0, v/v) to yield 200 fractions. G6-(30−50) (58.2 mg) were subsequently separated by repeated Sephadex LH-20 CC (40 g, 85 × 2 cm, 80% MeOH), followed by semipreparative HPLC with MeOH− 0.1% formic acid (37:63, 1.5 mL/min) elution, providing compounds 5 (4.0 mg, tR = 44 min), 6 (1.5 mg, tR = 49 min), and 11 (5.0 mg, tR = 67 min). G6-(116−135) (36.3 mg) were subjected to Sephadex LH-20 CC (120 × 1 cm, 80% MeOH) to obtain 30 fractions. G6-(116−135)(11−19) were purified by semipreparative HPLC and eluted with MeOH−0.1% formic acid (40:60, 1.5 mL/min), affording compound 10 (4.0 mg, tR = 175 min). Fr. 6 (2.0 g) was extracted three times with EtOAc. The residual water fraction (1.2 g) was separated by Sephadex LH-20 CC (400 g, 59 × 6 cm, 80% EtOH) twice to obtain nine fractions (H1−H9). Fraction H4 (50.2 mg) was subsequently separated by Sephadex LH20 CC (40 g, 85 × 2 cm, 80% MeOH), which afforded six fractions, H4-(1−6). Fraction H4-2 (25.5 mg) was purified by semipreparative HPLC and eluted with MeOH−0.1% formic acid (25:75, 1.5 mL/ min), affording compounds 7 (10.0 mg, tR = 35 min) and 8 (9.0 mg, tR = 47 min). Fraction H5 (235.0 mg) was separated by Sephadex LH-20 CC (90 × 4 cm, 80% EtOH) twice. The obtained yellow part was purified by MCI CC (8 × 2 cm) and eluted with 20% MeOH, followed by ODS-C18 CC (2 × 17 cm) eluted with a gradient of MeOH−H2O (0:100 to 20:80, v/v), affording three yellow fractions. The first fraction yielded compound 13 (10 mg). The other two fractions were combined and further chromatographed by a semipreparative HPLC, eluted with MeCN−0.1% formic acid (20:80, 1.5 I

DOI: 10.1021/acs.jnatprod.5b00524 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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(20 μL) of compounds 1, 7, and 11 were diluted with MeOH 10, 20, and 5 times, respectively, and filtered through 0.45 μm filter membranes. A 20 μL aliquot of the reaction mixture filtrates of compounds 1, 7, and 11 and of the D-glucose derivative and D-glucose derivative standard (I) were then directly analyzed by HPLC on an Inertsil ODS-3 column (250 × 4.6 mm, 5 μm) equipped with a Phenomenex precolumn (4.0 × 3.0 mm, 5 μm) using 0.1% formic acid (A)−MeCN (B) as the mobile phases with a gradient elution procedure [0−20 min (32% B), 20−30 min (32% B to 80% B), 30−50 min (80% B), 50−52 min (80% B to 32% B), 52−60 min (32% B)]. The temperature was set at 30 °C, and the detection wavelength was 250 nm. Microplate Assay of DPPH• Scavenging Activity. An assay of DPPH radical scavenging activity was performed using a reported method, with slight modifications.24 Briefly, 180 μL of DPPH• (150 μM in MeOH) and 20 μL of a series of test compound solutions (62.5, 125, 250, 500, and 1000 μM in H2O) were mixed in the wells of 96well plates. The reaction was detected by measuring the absorbance Asample+DPPH at 490 nm using a microplate reader after shaking the reaction for 30 min at room temperature in the dark. A 20 μL aliquot of each concentration with 180 μL of MeOH was used as the blank measurement for each tested compound, and the absorbance was recorded as Asample. The absorbance of the mixture of 20 μL of distilled water and 180 μL of DPPH• was recorded as ADPPH, and the absorbance of the mixture of 20 μL of distilled water and 180 μL of MeOH was recorded as Ablank. The natural antioxidant vitamin C was used as a positive control. All tests were performed in triplicate. The DPPH• scavenging activity was calculated according to the following equation: scavenging activity (%) = [1 − (Asample+DPPH − Asample)/(ADPPH − Ablank)] × 100%. The EC50 value was calculated using nonlinear regression (curve fitting) analysis with the Graphpad Prism 5.0 software program. The results are expressed as the mean ± standard deviation (SD). Significant differences were determined by one-way analysis of variance (ANOVA) using IBM SPSS statistics 20 software, and p < 0.05 was considered significant.

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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.5b00524. HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, NOESY, UV, and ECD spectra of compounds 1−14; HRESIMS of the D-glucose derivative standard (I); HPLC chromatograms of compounds 1, 7, and 11, Dglucose derivatives, and D-glucose derivative standard (I) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (R.-X. Zhu): +86-531-88364464. Fax: +86-531-88364464. E-mail: [email protected]. *Tel (L. Xiang): +86-531-88382028. Fax: +86-531-88382548. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (81073005), Scientific Research Foundation for Returned Overseas Scholars, Ministry of Education of China (No. 42), Innovation Project of Shandong University (2012TS102), and Science and Technology Development Program of Shandong Province (2014GSF119007). J

DOI: 10.1021/acs.jnatprod.5b00524 J. Nat. Prod. XXXX, XXX, XXX−XXX