Amides and Flavonoids from the Fruit and Leaf Extracts of Melodorum

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Amides and Flavonoids from the Fruit and Leaf Extracts of Melodorum siamensis Wuttichai Jaidee,†,‡ Raymond J. Andersen,§,⊥ Miguel A. G. Chavez,§ Yan A. Wang,§ Brian O. Patrick,§ Stephen G. Pyne,# Chatchai Muanprasat,△,∥,□ Suparerk Borwornpinyo,△,¶ and Surat Laphookhieo*,†,‡

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Center of Chemical Innovation for Sustainability (CIS) and ‡School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand § Departments of Chemistry and ⊥Earth, Ocean & Atmospheric Sciences, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada # School of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia △ Excellent Center for Drug Discovery, ∥Research Center of Transport Protein for Medical Innovation, and □Department of Physiology, Faculty of Science, Mahidol University, Rajathevi, Bangkok 10400, Thailand ¶ Department of Biotechnology, Faculty of Science, Mahidol University, Rajathevi, Bangkok 10400, Thailand S Supporting Information *

ABSTRACT: Four new chalcones (1, 10, 13, and 14), a new flavanone, (9), a new amide (8), and 19 known compounds were acquired from Melodorum siamensis. The structures were established by NMR and MS data analyses. Compounds 1 (er 1.4:1) and 2 (er 1.1:1) were scalemic and were resolved to yield (−)-1 and (+)-1 and (−)-2 and (+)-2, respectively. The absolute configurations of these compounds were determined from experimental and calculated ECD data. The structures and configurations of (−)-2 and (+)-8 were identified by single-crystal X-ray diffraction analysis. Compound 11 showed nuclear factor-κB inhibitory effects (IC50 = 9 μM) in a pancreatic β cell line (MIN-6 cells). the absolute configurations of its resolved enantiomers is reported here for the first time.

Melodorum siamensis (synonym Rauwenhoff ia siamensis), or Nom-Maeo in Thai, is a scandent shrub belonging to the Annonaceae family1,2 that is widely distributed throughout Thailand. The wood and roots of M. siamensis have been used for the treatment of insect bites, fever, and nasal polyps,3 whereas the fresh fruit and leaves have been used in the treatment of urticaria and indigestion, respectively. The flowers are commonly used as a food flavor and in cosmetics. In previous phytochemical investigations of M. siamensis, dihydrochalcones, flavonoids, and aromatic amides have been found from the leaf, stem, and root extracts. Some of these compounds showed cytotoxicity.4,5 In this study, the isolation and structure elucidation of six new compounds, including four chalcones, a flavonoid, and an amide, along with 19 known compounds from the fruit and leaf extracts of M. siamensis are discussed. The resolution of toussaintine C (2)6 as well as the determination of © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The individual fruit and leaf extracts of M. siamensis were separated by chromatographic techniques resulting in the isolation of six new compounds (1, 8−10, 13, and 14), together with 19 known compounds, toussaintine C (2),7 N-(4hydroxyphenethyl)cinnamamide (3),7 melodamide A (4),8 2′,4′-dihydroxy-4,6′-dimethoxychalcone (5),9 4,4′-dihydroxy2′,6′-dimethoxychalcone (6),10 helichrysetin (7),11 2′,4,4′trihydroxy-6′-methoxy-3′(3″-hydroxybenzyl)dihydrochalcone (11),4 2′,4′-dihydroxy-4,6′-dimethoxy-3′(3″-hydroxybenzyl)Received: August 16, 2018

A

DOI: 10.1021/acs.jnatprod.8b00696 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

dihydrochalcone (12), 4 4′,4-dihydroxy-2′,6′-dimethoxydihydrochalcone (15),12 uvangoletin (16),13 2′,4′-dihydroxy4′,6′-dimethoxydihydrochalcone (17),14 2′,4,4′-trihydroxy-6′methoxydihydrochalcone (18),15 flavokawain-A (19),9 tsugafolin (20),9 naringenin trimethyl ether (21),9 alpinetin (22),16 kaempferol (23),17 kaempferide (24),18 and N-cinnamoyl-(2phenylethyl)amine (25).19 The molecular formula of melosiamensone A (1) was established as C 34H32O 9 from its HREIMS data (m/z 584.2048 [M]+, calcd for C34H32O9 584.2046). Based on the NMR data (Table 1), as well as its EIMS spectrum (Figure S11, Supporting Information), the structure of 1 could be divided into two fragments. The first fragment was a chalcone core structure, which was revealed by the 1H and 13C NMR resonances for an H-bonded hydroxy proton (δH 14.72), a pcoumaroyl unit (δH 7.83, 7.72, 7.51, and 6.85), an aromatic proton (δH 6.08), and a methoxy group (δH 3.91). This methoxy group was placed at C-6′ from the HMBC correlation between H-5′ (δH 6.08) and the methoxy protons (δH 3.91) (Figure 1 and Table S1, Supporting Information) with C-6′ (δC 161.7). The fragment ion at m/z 286 [M − C17H16O5]+ in the EIMS spectrum also helped identify this structural unit. The second fragment was identified by 1H and 13C NMR resonances assigned to a monosubstituted aromatic ring substituent (δH 7.31, 7.22, and 7.21), a pentasubstituted aromatic ring substituent (δH 6.02), an AB2C spin system (δH 5.55, 4.46, 3.02, and 2.02), and three methoxy groups (3.85, 3.82, and 3.55). The locations of the methoxy groups at C-6″ (δC 154.7), C-8″ (δC 153.0), and C-9″ (δC 130.5) were deduced from HMBC correlations (Figure 1 and Table S1, Supporting Information). Finally, the linkage of both fragments to generate the pyran ring was confirmed by the following HMBC correlations: H-4″ (δH 4.46) with C-2′ (δC 166.5), C-3′ (δC 104.5), and C-4′ (δC 162.6) and of H-2″ (δH 5.55) with C-4′ (δC

162.6), C-10″ (δC 149.6), C-6″ (δC 154.7), and C-4″ (δC 34.9) (Figure 1). The relative configurations of C-2″ and C-4″ were determined by NOESY experiments and coupling constants. The H-3″b (δH 2.02) resonance showed a NOESY cross-peak with H-2″ (δH 5.55), while the H-3″a (δH 3.02) showed a NOESY cross-peak with H-4″ (δH 4.46), indicating the transorientations of H-2″ (δH 5.55)/H-3″a (δH 3.02) and of H-3″b (δH 2.02)/H-4″ (δH 4.46). The larger J value (12.3 Hz) between H-2″ and H-3″a and the smaller J value (5.6 Hz) between H-3″a and H-4″ were also consistent with these stereochemical assignments (Figure 2). Melosiamensone A (1) was resolved by chiral-phase HPLC to yield (−)-1 [tR 19 min, [α]23 D −138 (c 0.1, MeOH)] and (+)-1 +127 (c 0.1, MeOH)] in an approximate ratio [tR 23 min, [α]23 D of 1.4:1 (Figure S53, Supporting Information). The absolute configurations at C-2″ and C-4′′ of (−)-1 and (+)-1 were established by comparisons of their experimental and computed electronic circular dichroism (ECD) data (Figure 5). For 4arylflavans it has been established that the configuration at C-4 can be assigned as (4S) for such compounds displaying a positive Cotton effect at about 225 nm,20,21 whereas such compounds having the (4R) absolute configuration have a negative Cotton effect in the same region, and thus the (4″S) and (4″R) configurations were established for (+)-1 and (−)-1, respectively. In addition, the calculated ECD data of (2″R,4″R)1 and (2″S,4″S)-1 matched the experimental ECD data of (−)-1 [(−)-melosiamensone A)] and (+)-1 [(+)-melosiamensone A)], respectively. A plausible biosynthetic pathway for 1, starting from compound 7 and the known natural product 26 (not isolated in this study), is shown in Scheme 1. The coupling of compounds 7 and 26 via an intermolecular Michael addition, followed by cyclization and reduction, could provide 1. Compound 2 was identified as toussaintine C by analysis and comparison of its NMR data with those in the literature6 B



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Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for Compound 1 in CDCl3 position

δC, type

1 2 3 4 5 6 α β CO 1′ 2′ 3′ 4′ 5′ 6′ 2″ 3″a 3″b 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ 2′-OH 6′-OMe 10″-OH 6″-OMe 8″-OMe 9″-OMe

128.9, C 130.4, CH 116.0, CH 157.8, C 116.0, CH 130.4, CH 125.6, CH 142.3, CH 193.3, C 106.1, C 166.5, C 104.5, C 162.6, C 91.4, CH 161.7, C 68.3, CH 33.4, CH2 34.9, CH 106.4, C 154.7, C 89.1, CH 153.0, C 130.5, C 149.6, C 145.5, C 128.1, CH 128.3, CH 126.3, CH 55.4, CH3 55.8, CH3 55.6, CH3 60.8, CH3

δH, mult. (J in Hz) 7.51, d (8.5) 6.85, d (8.5) 6.85, d (8.5) 7.51, d (8.5) 7.83, d (15.5) 7.72, d (15.5)

Figure 2. Key NOESY correlations of compound 1.

component from the leaf extract of Toussaintia orientalis (Annonaceae).6 The compound had a small specific rotation of −4 (MeOH, c 0.05). Its cis-fused ring junction was proposed based on NMR data.6 In this study, compound 2 was resolved by semipreparative chiral-phase HPLC to yield (−)-2 (tR 12 min), 23 [α]23 D −199 (c 1.0, MeOH), and (+)-2 (tR 14 min), [α]D +172 (c 1.7, MeOH) (Figure S54, Supporting Information) . Goodquality single crystals of (−)-2 were obtained from a mixture of acetone/hexanes (1:1 v/v), and its (4R,9R) absolute configuration was determined by single-crystal X-ray diffraction analysis (Figure 3, CCDC 1839330). The absolute configuration of (+)-2 was determinated as (4S,9S) by comparison of its ECD data with (−)-2, which showed an opposite Cotton effect at 228 nm, and agreed well with the calculated ECD data of (4R,9R)-2 (Figure 6). Therefore, compounds (−)-2 and (+)-2 were assigned the names (−)- and (+)-toussaintines C. Based on the above chiroptical data, it is clear that compound 2, isolated by Samwel et al.,6 was also a scalemic mixture with a slight excess of the (4R,9R) enantiomer (−)-2. Compound (+)-8, [α]23 D +22 (c 0.5, MeOH), was assigned a molecular formula of C17H19NO3 based on the HRESITOFMS data (m/z 286.1444 [M + H]+, calcd for C17H20NO3 286.1443). The 1H and 13C NMR data of compound (+)-8 (Table 2 and Figures S20−S24, Supporting Information) were similar to those of compound 2. The main differences were due to the Δ5(6) double bond of compound 2 being reduced to two methylene groups in (+)-8, indicated by the 1H and 13C NMR spectroscopic resonances at δH 2.20 (2H, m, H-5)/δC 32.2 and

6.08, s 5.55, dd (12.3, 1.9) 3.02, ddd (13.8, 12.3, 5.6) 2.02, dt (13.8, 1.9) 4.46, brd (5.6)

6.02, s

7.22, d (8.0) 7.31, dd (8.0, 7.2) 7.21, d (7.2) 14.72, s 3.91, s 6.32, s 3.55, s 3.85, s 3.82, s

(Figures S13−S18 and Table S2, Supporting Information). In 2011, Samwel et al. reported toussaintine C as a minor

Figure 1. COSY and key HMBC correlations for 1, 8−10, 13, and 14. C



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Figure 3. X-ray ORTEP-style diagram of compound (−)-2.

Figure 4. X-ray ORTEP-style diagram of compound (+)-8.

Figure 5. Experimental (left) and calculated (right) ECD spectra of compound 1 in MeOH.

δH 2.38 and 2.50 (each 1H, m, H-6)/δC 36.1. The (4R,9S) absolute configuration of compound (+)-8 was confirmed by single-crystal X-ray diffraction analysis (Figure 4, CCDC 1839331). The experimental and calculated ECD spectra for

(4S,9R)-8 are shown in Figure 7. Thus, compound (+)-8 was assigned as (+)-toussaintine H. A plausible biosynthesis of compound (+)-8 is shown in Scheme 2. Oxidation of amide 3 could produce the dienone 4. Intramolecular Michael addition D



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Scheme 1. Putative Biosynthetic Pathway toward 1

Figure 6. Experimental (left) and calculated (right) ECD of compounds 2 in MeOH.

[M + Na]+, calcd for C23H20O6Na 415.1158). The NMR data (Table 3 and Figures S26−S30, Supporting Information) were closely related to those of methylphelligrin B, indicating a benzylflavanone core structure.22 The 1H NMR spectrum of methylphelligrin B showed a sharp singlet resonance for a Hbonded hydroxy proton at δH 12.32 assigned to HO-5,22 while a corresponding resonance at this chemical shift was not observed in the 1H NMR spectrum of compound 9, suggesting that the positions of the methoxy and the hydroxy groups of compound 9 were located at C-5 and C-7, respectively. The HMBC correlations shown in Figure 1 and Table S4 (Supporting Information) supported these assignments. The significant differences in the 13C NMR data for the carbonyl carbon (C4) of these compounds (δC 187.2 for 9 and δC 198.1 for methylphelligrin B) as well as for C-5 (δC 160.3 for 9 and 164.1 for methylphelligrin B) confirmed the structural difference between these compounds.23−25 Compound 9 was analyzed by analytical chiral-phase HPLC, which indicated this compound was a scalemic mixture with an enatiomeric ratio (er) of aproximately 1:4 (Figure S55, Supporting Information). Because of the paucity of material, this compound was not resolved. The molecular formula, C24H24O6, of melosiamensone D (10) was established by HRESITOFMS data (m/z 431.1478 [M + Na]+ calcd for C24H24O6Na 431.1471). The NMR data of 10 (Table 4 and Figures S37−S39, Supporting Information) were similar to those of 12 (2′,4′-dihydro-4,6′-dimethoxy-3′(2″hydrobenzyl)dihydrochalcone) previously isolated from the leaf extract of M. siamensis.4 Significant differences between

Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for Compound (+)-8 in CDCl3 position

δC, type

2 3 4 5 6a 6b 7 8a 8b 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

44.8, CH2 35.9, CH2 76.2, C 32.2, CH2 36.1, CH2 208.4, C 42.5, CH2 63.5, CH 164.9, C 117.3, CH 142.5, CH 134.5, C 127.5, CH 128.4, CH 129.5, CH 128.4, CH 127.5, CH

δH, mult. (J in Hz) 3.91, m 2.33, m 2.20,a m 2.38, m 2.50, m 2.20,a m 3.18, dd (16.1, 5.6) 4.34, dd (9.1, 6.7) 6.68, d (15.6) 7.69, d (15.6) 7.53, d (7.8) 7.38−7.37, m 7.38−7.37, m 7.38−7.37, m 7.53, d (7.8)

a

Overlapping signals.

of the amide nitrogen of 4 would give aminocinnamoyl tetraketide amide (+)-2 [(+)-toussaintine C)] and reduction the Δ5(6) double bond to afford compound (+)-8. The molecular formula, C23H20O6, of melosiamensone B (9) was deduced from HRESITOFMS data analysis (m/z 415.1165 E



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Figure 7. Experimental (left) and calculated (right) ECD of compound (+)-8 in MeOH.

Scheme 2. Putative Biosynthetic Pathway toward (+)-8

Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for 9 in Acetone-d6 9 position 2 3a 3b 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 5-OCH3 5-OH 7-OMe

δC, type 78.4, CH 44.7, CH2

187.2, C 160.3, C 92.4, CH 160.6, C 106.4, C 161.7, C 105.1, C 129.6, C 127.5, CH 114.6, CH 157.0, C 114.6, CH 127.5, CH 21.9, CH2 126.3, C 153.5, C 114.3, CH 126.4, CH 119.3, CH 129.6, CH 54.6, CH3

δH, mult. (J in Hz)

compounds 10 and 12 were found at C-3′ and C-5′. Compound 10 displayed NMR resonances for an aromatic proton at δH 6.27/δC 99.0 (acetone-d6), which was different from compound 12 [δH 6.13/δC 92.1 (acetone-d6)], indicating that the proton signal at δH 6.27 belonged to C-3′. To assign all the 1H NMR resonances, NMR spectra for compound 10 were recorded in DMSO-d 6 (Table 1 and Figures S32−S36, Supporting Information). The hydroxy groups with resonances at δH 9.34, 10.40, and 12.22 were assigned to HO-3″, HO-4′, and HO-2′, respectively, from the HMBC correlations shown in Figure 1 and Table S5 (Supporting Information). In addition, the HMBC correlations of H-1″ (δH 3.68), H-3′ (δH 6.21), and HO-4′ (δH 10.40) with C-5′ (δC 111.8) supported the location of the 2hydroxybenzyl unit at C-5′. Melosiamensone E (13) was assigned a molecular formula of C18H18O6 based on its HRESITOFMS data (m/z 353.1001 [M + Na]+ calcd for C18H18O6Na 353.1001). The NMR data of 13 (Tables 4 and S8 and Figures S41−S45, Supporting Information) were similar to those of 4,6-dimethoxy-2,5quinodihydrochalcone previously isolated from Fissistigma latifolium (Annonaceae)26 except for the presence of a resonance for a methoxy group at C-3 (δH 3.93) instead of a resonance for an olefinic proton observed in the spectrum of 4,6dimethoxy-2,5-quinodihydrochalcone. This structural feature was confirmed by the HMBC correlations shown in Figure 1 and Table S8 (Supporting Information). The molecular formula of melosiamensone F (14) was established as C17H18O6 from its HRESITOFMS data (m/z 319.1188 [M + H]+, calcd for C17H19O6 319.1182). The NMR data (Tables 4 and S9 and Figures S47−S51, Supporting Information) were closely related to those of 2′,4′-dihydroxy3,4,6′-trimethoxydihydrochalcone, which was previously iso-

methylphelligrin B22 δC, type

5.38, dd (12.8, 2.7) 79.7, CH 2.95, dd (16.3, 43.4, CH2 12.8) 2.61, dd (16.3, 2.7) 198.1, C 164.1, C 6.24, s 93.2, CH 166.7, C 107.7, C 160.5, C 103.7, C 130.7, C 7.35, d (8.4) 128.8, CH 6.87, d (8.4) 116.1, CH 158.6, C 6.87, d (8.4) 116.1, CH 7.35, d (8.4) 128.8, CH 3.87, s 22.8, CH2 127.5, C 155.8, C 6.81, d (7.6) 115.6, CH 6.99, t (7.6) 127.4, CH 6.68, t (7.6) 120.1, CH 7.05, d (7.6) 129.7, CH 3.76, s 56.6, CH3

δH, mult. (J in Hz) 5.48, dd (12.0, 3.0) 3.19, dd (17.4, 12.0) 2.84, dd (17.4, 3.0)

6.22, s

7.30, br d (8.4) 6.83, br d (8.4) 6.85, br d (8.4) 7.32, br d (7.8) 3.88, s

6.78, br d (8.4) 6.97, m 6.66, m 6.81, br d (7.2) 12.32, s 3.87, s F



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Table 4. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for Compounds 10, 13, and 14 10 (DMSO-d6) position

δC, type

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ α β CO 1″ 2″ 3″ 4″ 5″ 6″ 7″ 3-OCH3 4-OCH3 6-OCH3 6′-OCH3 2′-OH 3″-OH 3-OH 4′-OH

133.1, C 129.2, CH 113.6, CH 157.4, C 113.6, CH 129.2, CH 109.9, C 160.9, C 98.9, CH 162.2, C 111.8, C 160.6, C 44.3, CH2 29.0, CH2 204.0, C 22.5, CH2 126.8, CH 154.8, CH 114.3, CH 126.2, CH 118.6, CH 127.5, CH


10 (acetone-d6) δC, type

6.75, d (7.8) 6.92, t (7.5) 6.58, t (7.5) 6.53, d (7.3)

133.0, C 128.8, CH 113.2, CH 157.6, C 113.2, CH 128.8, CH 108.2, C 161.5, C 99.0, CH 162.7, C 112.4, C 163.8, C 43.8, CH2 29.2, CH2 204.3, C 22.3, CH2 126.4, C 154.6, C 114.2, CH 126.2, CH 118.9, CH 128.4, CH

54.9, CH3

3.68, s

54.0, CH3

3.73, s

62.4, CH3

3.44, s 12.22, s 9.34, s

62.0, CH3

3.67, s 13.08, s

7.11, d (8.5) 6.79, d (8.5) 6.79, d (8.5) 7.11, d (8.5)

6.21, s

3.20, t (7.6) 2.81, t (7.6) 3.68, s

13 (acetone-d6)


δC, type 128.8, C 183.1, C 143.1, C 144.5, C 178.4, C 154.1, C 136.6, C 128.1, CH 127.4, CH 132.3, CH 127.4, CH 128.1, CH 36.5, CH2 17.6, CH2 197.9, C

7.16, d (8.5) 6.81, d (8.5) 6.81, d (8.5) 7.16, d (8.5)

6.27, s

3.37, t (7.6) 2.92, t (7.6)


7.99, d (7.4) 7.51, d (7.7) 7.61, d (7.4) 7.51, d (7.7) 7.99, d (7.4) 3.13, t (7.8) 2.75, t (7.8)

14 (acetone-d6) δC, type 134.3, C 114.7, CH 145.3, C 143.1, C 111.0, CH 118.9, CH 104.2, C 167.7, C 95.3, CH 164.1, C 90.5, CH 162.3, C 45.2, CH2 29.0, CH2 203.7, C

δH, mult. (J in Hz) 6.76, s

6.83, d (8.1) 6.67, d (8.1)

5.92, s 6.05, s 3.24, d (7.7) 2.83, d (7.7)

3.91, s

6.81, d (7.4) 6.98, dt (8.0, 1.5) 6.69, td (7.4, 1.2) 6.91, d (7.4) 60.2, CH3 60.0, CH3 60.1, CH3

3.93, s 3.96, s 3.98, s

54.8, CH3

3.79, s

54.9, CH3

3.88, s 13.92, s 7.43, s

10.40, s

lated from Iryanthera ulei.27 However, the C-3 methoxy group in 2′,4′-dihydroxy-3,4,6′-trimethoxydihydrochalcone was substituted by a hydroxy group (δH 7.43) in compound 14. The locations of the hydroxy and methoxy groups at C-3 and C-4, respectively, were confirmed by the following HMBC correlations: H-2 (δH 6.76) with C-1 (δC 134.3), C-3 (145.3), and C-6 (118.9); H-6 (δH 6.76) with C-β (δC 29.0) and C-4 (143.1); and H-5 (δH 6.83) with C-3 (δC 145.3) (Figure 1 and Table S9, Supporting Information). The NMR data of 14 were in close agreement with those of the synthetic compound. Notably compound 14 was synthesized by Dubois and coworkers in 1977,28 but this is the first isolation from a natural source. Compounds (−)-2, (+)-2, 5−8, 10−12, 15−18, and 20 were evaluated for their in vitro cytotoxicities against HCT-116 cancer cells and for their inhibitory activities against nuclear factor-κB (NF-κB). All of them were found to have no cytotoxicity against HCT-116 cancer cells, and only compounds 7 and 11 showed nuclear factor-κB inhibitory effects in a pancreatic β cell line (MIN-6 cells). Effects of 7 and 11 on NF-κB Nuclear Translocation in MIN-6 Cells. NF-κB is a central regulator of pancreatic β-cell inflammation, which is responsible for the development of type II diabetic mellitus.29,30 Pro-inflammatory cytokines including TNF-α and IL-1β stimulate the signaling cascade through cell surface receptors, resulting in NF-κB activation characterized by translocation of p65 NF-κB from the cytosol to the nucleus.31 To identify compounds with anti-β-cell inflammation activities,

we performed the assays of cytokine-induced NF-κB in mouse pancreatic β cells (MIN-6). MIN-6 cells were exposed for 30 min to test compounds at 50 μM in the presence of cytokine cocktails containing 10 ng/mL TNF-α and 10 ng/mL IL-1β. As shown in Figure 8, the cytokine cocktail stimulated NF-κB

Figure 8. Effect of 7 and 11 on NF-κB nuclear translocation in MIN-6 cells. Immunostaining of NF-κB after stimulation with a cytokine cocktail containing 10 ng/mL TNF-α and 10 ng/mL IL-1β with or without 7 or 11 (50 μM). Scale bar = 20 μm. G



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Dried leaves of M. siamensis (2.0 kg) were extracted with acetone (3 × 15 L) at room temperature. The acetone extract (87.8 g) was subjected to QCC on silica gel (100% hexanes to 100% acetone) to afford 14 fractions (Fr.A−N). Fr.E (1.6 g) was subjected to Sep-Pak C18 CC (2:3 MeOH/H2O) to yield two fractions (Fr.E1 and Fr.E2). Fr.E2 (201.4 mg) was separated by Sephadex LH-20 CC (4:1 MeOH/ CH2Cl2) to produce six fractions (Fr.E2A−E2F). Fr.E2C (77.4 mg) was chromatographed with CC (1:4 acetone/hexanes) to give four fractions (Fr.E2C.A−E2C.D). Fr.E2C.C (23.8 mg) was purified by an HPLC RP C8 Phenomenax Luna 5 μ column (7:3 MeCN/H2O) to give 13 (0.5 mg). Fr.E2D (52.6 mg) was purified by CC (1:4 acetone/ hexanes) to give 19 (3.9 mg). Fr.I (5.1 g) was subjected to Sep-Pak C18 CC (2:3 MeOH/H2O) to give two fractions (Fr.I1 and I2). Fr.I2 (201.4 mg) was fractionated by CC (1:4 acetone/hexanes) to afford 14 fractions (Fr.I2A−I2N). Fr.I2J (326.0 mg) was separated by Sephadex LH-20 CC to produce four fractions (Fr.I2J.A−I2J.D). Fr.I2J.D (13.3 mg) was purified by RP C8 HPLC (7:3 MeCN/H2O) to give 10 (3.0 mg). Fr.J (2.4 g) was subjected to Sep-Pak C18 CC (2:3 MeOH/H2O) to afford two fractions (Fr.J1 and J2). Fr.J2 (864.4 mg) was purified by CC (1:4 acetone/hexanes) to give 17 (528.0 mg) and 16 (77.2 mg) and nine fractions (Fr.J2A−J2J). Fr.J2G (45.3 mg) was separated by Sephadex LH-20 CC to give five fractions (Fr.J2G.A−J2G.E). Fr.J2G.C (10.6 mg) was purified by RP C8 HPLC (3:2 MeCN/H2O) to give 21 (1.5 mg, tR 20.0 min) and 15 (3.2 mg, tR 28.6 min). Fr.J2G.E (12.5 mg) was purified by RP C8 HPLC (3:2 MeCN/H2O) to afford 12 (9.5 mg, tR 27.8 min). Fr.J2I (51.1 mg) was subjected to RP C8 HPLC (63:37 MeCN/H2O) to give 25 (1.0 mg). Fr.L (12.5 g) was separated by SepPak C18 CC (2:3 MeOH/H2O) to give two fractions (Fr.L1 and L2). Fr.L1 (4.94 g) was separated by CC (1:4 acetone/hexanes) to obtain 12 fractions (Fr.L1A−L1L). Fr.L1G (428.2 mg) was chromatographed by Sephadex LH-20 CC to give 11 (14.1 mg) and eight fractions (Fr.L1G.A−L1G.H). Fr.L1G.D (51.5 mg) was purified by RP C8 HPLC (1:1 MeCN/H2O) to give 14 (1.7 mg), 18 (5.0 mg), 23 (1.0 mg), and 24 (0.8 mg). Fr.L1H (255.4 mg) was subjected to CC (1:4 acetone/hexanes) to obtain six fractions (Fr.L1H.A−L1H.F). Fr.L1H.E (7.6 mg) was purified by RP C8 HPLC (63:37 MeCN/ H2O) to give 22 (0.8 mg). Fr.L1I (347.8 mg) was purified by CC (1:4 acetone/hexanes) to give 20 (16.8 mg). Compound 9 (2.2 mg) was isolated from Fr.L1L (57.1 mg) by RP C8 HPLC (3:2 MeCN/H2O). Fr.M (21.5 g) was washed with acetone to give fraction M1 (18.9 g), which was further purified by CC (3:97 MeOH/CH2Cl2) to yield 8 (4.7 mg). Please note that the flow rate for RP C8 HPLC was 2 mL/min and the solvent system was acidified with 0.05% trifluoroacetic acid. The solvent system for Sephadex LH-20 CC is 100% MeOH. Melosiamensone A (1): yellow, amorphous solid; mp 146−147 °C; UV (MeOH) λmax (log ε) 209 (5.10), 370 (4.74) nm; IR (KBr) νmax 3435, 2929, 1618, 1583, 1513, 1344, 1199, 1151, 701 cm−1; 1H and 13C NMR data, see Tables 1 and S1 (Supporting Information); EIMS m/z 584 (90), 387 (42), 298 (100), 286 (42), 167 (44); HREIMS m/z 584.2048 [M]+ (calcd for C34H32O9, 584.2046). Chiral-Phase HPLC Resolution and ECD Data of (−)-1 and (+)-1. Resolution of scalemic 1 (0.9 mg) was performed by semipreparative HPLC on a chiral-phase column (CHIR-ALCEL OD-H column, flow rate 1 mL/min, 1:1 n-hexane/iPrOH). Compound (−)-1 (tR = 19 min) −4 [(0.2 mg), [α]23 D −138 (c 0.1, MeOH)]; ECD (c 6.8 × 10 M, MeOH) λmax (Δε) 218 (+4.3), 236 (+2.1), and 252 (−0.62) nm and (+)-1 (tR = −4 23 min) [(0.2 mg), [α]23 D +127 (c 0.1, MeOH)]; ECD (c 6.8 × 10 M, MeOH) λmax (Δε) 218 (−3.49), 236 (−1.49), and 252 (+1.02) nm. Toussaintine C (2): white solid; mp 198−200 °C; UV (MeOH) λmax (log ε) 282 (2.99), 215 (3.14) nm; IR (neat) νmax 3377, 2954, 1716, 1645, 1431, 766 cm−1; 1H and 13C NMR data, see Table S2 (Supporting Information); HREIMS m/z 283.1207 [M]+ (calcd for C17H17O3N, 283.1208). Chiral-Phase HPLC Resolution and ECD Data of (−)-2 and (+)-2. Resolution of scalemic 2 (10.0 mg) was performed by semipreparative HPLC on a chiral-phase column (1:1 n-hexane/iPrOH). (−)-2 (tR = 12 −4 M, min) [(4.7 mg), [α]23 D −199 (c 1.0, MeOH)]; ECD (c 8.8 × 10 MeOH) λmax (Δε) 228 (−1.75) nm and (+)-2 (tR = 14 min) [(4.7 mg), −4 [α]23 D +172 (c 1.7, MeOH)]; ECD (c 5.9 × 10 M, MeOH) λmax (Δε) 228 (+1.72) nm.

nuclear translocation in MIN-6 cells. Interestingly, compounds 7 and 11 were found to inhibit NF-κB nuclear translocation induced by the cytokine cocktail. These results indicate that both 7 and 11 have an inhibitory effect on β-cell inflammation. Dose−Response of 7 and 11 on NF-κB Nuclear Translocation in MIN-6 Cells. To further evaluate the dose response of 7 and 11 in inhibiting cytokine-induced NF-κB nuclear translocation in mouse pancreatic β cells (MIN-6), MIN-6 cells were exposed for 30 min to 7 and 11 in doses ranging from 3 to 50 μM in the presence of cytokine cocktails containing 10 ng/mL TNF-α and 10 ng/mL IL-1β. Compound 11 was found to dose dependently inhibit NF-κB nuclear translocation in MIN-6 cells with an IC50 value of 9 μM, much better than compound 7 (Figure 9). Considering that β-cell

Figure 9. Dose−response of 7 and 11 on NF-κB nuclear translocation in MIN-6 cells. Percent inhibition of NF-κB nuclear translocation at the indicated concentrations of 7 and 11 in MIN-6 cells cotreated with cytokine cocktail (10 ng/mL TNF-α and 10 ng/mL IL-1β). Summary of the data shown (mean ± SEM, n = 3). Data were fitted to the Hill’s equation.

inflammation plays important roles in the pathogenesis of type II diabetes mellitus (T2DM),31 dihydrochalcone 11 may has potential as a lead compound for the evaluation and development of T2DM agents.

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EXPERIMENTAL SECTION

General Experimental Procedures. The chromatographic materials and information on instruments were the same as in the previous reports.32−35 Plant Material. The fruit and leaves of M. siamensis were collected from Songkhla Province (March 2014) and Chiang Rai Province (August 2015), respectively. The plant was identified by comparison with the authentic plant growing in Mae Fah Luang University Health Park Garden by one of the authors (S.L.), and a voucher specimen (No. MFU-NPR 0109) has been deposited at the Natural Products Research Laboratory, School of Science, Mae Fah Luang University. Extraction and Isolation. The dried fruits of M. siamensis (100.3 g) were extracted successively with EtOAc (3 × 1 L) and MeOH (3 × 1 L). The MeOH extract (2.35 g) was subjected to Sephadex LH-20 column chromatography (CC) to give nine fractions (Fr.A−I). Fr. H (321.5 mg) was separated by Sephadex LH-20 CC producing seven fractions (Fr.H1−H7). Fr.H4 (20.5 mg) was purified by CC (3:7 EtOAc/ hexanes) followed by Sephadex LH-20 CC to yield 3 (3.3 mg). Compound 1 (2.3 mg) was obtained from Fr.H5 (23.1 mg) by CC (3:7 EtOAc/petroleum ether). Fr.H6 (90.2 mg) was separated by CC (1:1 EtOAc/petroleum ether) to yield 4 (10.0 mg) and 2 (4.4 mg). The EtOAc extract (28.9 g) was subjected to QCC (100% hexanes to 100% acetone) to obtain nine fractions (Fr.A−I). Compound 5 (8.4 mg) was isolated from Fr.D (809.9 mg) by CC (1:4 acetone/hexanes) and then Sephadex LH-20 CC (100% MeOH), whereas 6 (5.2 mg) and 7 (5.5 mg) were obtained from Fr.E (1.1 g) by CC (3:7 EtOAc/hexanes). H



Article

(+)-Toussaintine H (8): white solid; mp 163−165 °C; [α]23 D +22 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 216 (3.00), 280 (3.23) nm; IR (neat) νmax 3302, 1684, 1642, 1574, 767 cm−1; ECD (c 1.1 × 10−3 M, MeOH) λmax (Δε) 212 (−0.26), 297 (+0.43) nm; 1H and 13C NMR data, see Tables 2 and S3 (Supporting Information); HRESITOFMS m/z 286.1444 [M + H]+ (calcd for C17H19NO3 286.1443). Melosiamensone B (9): yellow solid; mp 193−195 °C; [α]23 D −3.5 (c 1.7, MeOH); UV (MeOH) λmax (log ε) 214 (3.42), 282 (3.06) nm; IR (neat) νmax 3243, 2933, 1596, 1202 cm−1; 1H and 13C NMR data, see Tables 3 and S4 (Supporting Information); HRESITOFMS m/z 415.1165 [M + Na]+ (calcd for C23H20O6, 415.1158). Chiral-Phase HPLC Analysis of 9. Similarly, compound 9 (2.2 mg) was analyzed by chiral-phase HPLC (3:2 n-hexane/iPrOH) to give peaks for two enantiomers (tR 12 min and tR 13 min, 1:4 ratio) (Figure S55, Supporting Information). Lack of material precluded their quantitative resolution. Melosiamensone D (10): light yellow, viscous oil; UV (MeOH) λmax (log ε) 223 (3.35), 282 (3.14), 333 (3.07) nm; IR νmax 3366, 2935, 1614, 1511, 1455, 1417, 1244, 1178, 1144, 1023, 824, 756 cm−1; 1H and 13 C NMR data, see Tables 4 and S5−S7 (Supporting Information); HRESITOFMS m/z 431.1478 [M + Na]+ (calcd for C24H24O6, 431.1471). Melosiamensone E (13): light yellow, viscous oil; UV (MeOH) λmax (log ε) 202 (1.16), 288 (1.04) nm; IR (neat) νmax 3349, 2926, 1624, 1593, 1202 cm−1; 1H and 13C NMR data, see Tables 4 and S8 (Supporting Information); HRESITOFMS [M + H]+ m/z 353.1001 (calcd for C18H18O6, 353.1001). Melosiamensone F (14): light yellow solid; mp 152−155 °C; UV (MeOH) λmax (log ε) 202 (1.16), 288 (1.04) nm; IR (neat) νmax 3349, 2926, 1624, 1593, 1202 cm−1; 1H and 13C NMR data, see Tables 4 and S9 (Supporting Information); HRESITOFMS [M + H]+ m/z 319.1188 (calcd for C17H18O6, 319.1182). X-ray Crystallographic Analysis of (−)-2 and (+)-8. The structures were solved by direct methods36 and were refined by fullmatrix least-squares.37 Crystallographic data for (−)-2 (CCDC 1839330) and (+)-8 (CCDC 1839331) can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam. ac.uk/data_request/cif. Crystal Data of (−)-2: colorless prism crystal, C17H17NO3, M = 283.31, crystal size 0.09 × 0.13 × 0.14 mm, space group P212121, z = 4, crystal system orthorhombic with a = 7.6392(7) Å, b = 8.9874(8) Å, c = 20.4621(17) Å, α = 90°, β = 90°, γ = 90°, ν = 1404.9(2) Å, Dcalc = 1.340 g/cm3. The X-ray diffraction analysis values using Cu Kα radiation were 7.47 cm−1, 9360 reflections measured, 2539 independent reflections (Rint = 0.045). The goodness of fit indicator = 1.07. The final R1 was 0.037 (I > 2.00σ(I)). The final wR2 was 0.084 (all data). Flack parameter = −0.09(15).38 Crystal Data of (+)-8: colorless prism crystal, C17H19NO3, M = 285.33, crystal size 0.03 × 0.07 × 0.16 mm, space group C2, z = 4, crystal system monoclinic with a = 10.7238(10) Å, b = 7.2800(6) Å, c = 19.4870(17) Å, α = 90°, β = 97.390(5)°, γ = 90°, ν = 1508.7(2) Å, Dcalc = 1.256 g/cm3. The X-ray diffraction analysis values using Cu Kα radiation were 6.96 cm−1, 10 857 reflections measured, 2764 independent reflections (Rint = 0.045). The goodness of fit indicator = 1.04. The final R1 was 0.034 (I > 2.00σ(I)). The final wR2 was 0.085 (all data). Flack parameter = −0.04(16).38 Computational Methods. Geometry optimization and electronic circular dichroism spectra of the conformations (−)-1, (+)-1, (−)-2, (+)-2, and (+)-8 were done by using the Gaussian computational chemistry package.39 Geometry optimizations of all structures were performed by the hybrid functional B3LYP40 and the Pople 6-31G(d) basis set.41 The ECD spectra were calculated using TDDFT42−45 with the B3LYP functional and TZVP basis set.46 The spectra were subsequently plotted using the GaussSum package,47 with a Gaussian width of 0.7 eV. Cytotoxicity Assays. The cytotoxicity assay was performed by the reported method.35 Cell Culture.48 MIN-6 cells were purchased from AddexBio Technologies (San Diego, CA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium high glucose supplemented with

15% fetal bovine serum (BSA), 1% penicillin/streptomycin, 1% nonessential amino acid, 1% L-glutamine, 24 mM HEPES, and 50 μM β-mercaptoethanol. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% O2. Cells were trypsinized with 0.05% trypsin and centrifuged at 300g for 3 min before seeding. Anti β-Cell Inflammation Assay.48 MIN-6 cells were plated onto 96-black-well plates at a density of 1 × 105 cells per well and grown in normal growth medium for 24 h. In the screening experiments, cells were incubated for 30 min with a cytokine cocktail (10 ng/mL TNF-α (#654245, Merck Millipore, Darmstadt, Germany) and 10 ng/mL IL1β (#I5271; Sigma-Aldrich, Inc., St. Louis, MO, USA)) in the presence or absence of test compounds (50 μM) before being fixed at the end of the treatment period. The nuclei and p65 NF-κB were stained using an immunofluorescence technique. In dose−response studies, cells were incubated for 30 min with cytokine cocktail (10 ng/mL TNF-α and 10 ng/mL IL-1β) in the presence or absence of active compounds at doses of 3−50 μM. At the end of treatment, cells were fixed and stained using an immunofluorescence technique. The fluorescent images were taken using the Operetta high-content imaging system (PerkinElmer, Waltham, MA, USA). NF-κB nuclear translocation was calculated from the ratio of the average NF-κB intensity in the nuclear region to the average NF-κB intensity in the cytoplasmic region using Columbus Image Data Storage and Analysis System (PerkinElmer). Immunofluorescence Staining.48 Cells were fixed in 4% paraformaldehyde for 10 min and washed with phosphate-buffered saline (PBS) three times. Then, cells were permeabilized in 0.1% Triton X-100 for 10 min and washed with PBS three times. After that, cells were treated with blocking solution (1% BSA, 22.52 mg/mL glycine, and 0.1% Tween 20) for 1 h before being incubated with primary antiNF-κB rabbit monoclonal antibody (1:400 dilution) (#8242, Cell Signaling Technology, Inc., Danvers, MA, USA) and Alexa Fluor 488conjugated goat anti-rabbit secondary antibody (1:400 dilution) (#A31627; Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclei were stained with Hoechst 33342 (1:1000 dilution) (#H3570, Thermo Fisher Scientific Inc.).

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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.8b00696.

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HRESITOFMS and 1D and 2D NMR spectra of new compounds 1, 2, 8−10, 13, and 14; chiral HPLC chromatograms for compounds 1, 2, and 9 (PDF) X-ray single-crystal structure of (−)-2 (CIF) X-ray single-crystal structure of (+)-8 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +66-5391-6238. Fax: +66-5391-6776. E-mail: surat.lap@ mfu.ac.th. ORCID

Raymond J. Andersen: 0000-0002-7607-8213 Stephen G. Pyne: 0000-0003-0462-0277 Surat Laphookhieo: 0000-0002-4757-2781 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Thailand Research Fund (BRG5980012, PHD/0019/2557, andDBG5980001) and Mae Fah Luang University for financial support. University of British Columbia is also acknowledged for laboratory facilities. I



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Article

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Amides and Flavonoids from the Fruit and Leaf Extracts of Melodorum

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