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3,5-Diarylpyrazole Derivatives Obtained by Ammonolysis of the Total Flavonoids from Chrysanthemum indicum Extract Show Potential for the Treatment of Alzheimer’s Disease Taizong Wu,†,⊥ Cheng Jiang,†,⊥ Ling Wang,† Susan L. Morris-Natschke,‡ Hui Miao,† Lianquan Gu,† Jun Xu,† Kuo-Hsiung Lee,*,‡,§ and Qiong Gu*,†,‡ †

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China ‡ Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States § Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan, ROC S Supporting Information *

ABSTRACT: Four new 3,5-diarylpyrazole analogues (1−4) were isolated from an extract of the flowers of Chrysanthemun indicum using a combination of ammonolysis of the total flavonoid extract and an Aβ aggregation inhibitory activity guided purification procedure. All four compounds (1−4) showed moderate to potent activity against Aβ aggregation with EC50 values of 4.3, 15.8, 1.3, and 2.9 μM, respectively. Moreover, compound 3 showed low cytotoxicity and significant neuroprotective activity against Aβ-induced cytotoxicity in the SH-SY5Y cell line. This report is the first to show that 3,5-diarylpyrazole analogues can inhibit Aβ aggregation and exhibit neuroprotective activity with potential for the treatment of Alzheimer’s disease. Taken together, the method presented here offers an alternative approach to yield bioactive compounds.

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Naturally occurring phenolic compounds have received considerable attention as alternative candidates for AD therapy.13,14 Flavonoids are naturally occurring phenolic compounds, which have been reported to exhibit antifibrillogenic and cytoprotective properties.15 Flavonoids are one of the main constituents of the flowers of Chrysanthemum indicum L. (Asteraceae).16 In a previous study, we investigated the chemical constituents of C. indicum. Some known flavonoids (unpublished data) and new sesquiterpenoids were discovered.17 Herein, a chemical modification method was performed in combination with bioactivity-guided isolation of a C. indicum extract to obtain four new 3,5-diarylpyrazole analogues (1−4) (Scheme 1). Furthermore, molecular docking was also used to study the binding modes of the active compounds 1−4 with Aβ42.

lzheimer’s disease (AD) is a neurodegenerative disorder, which is characterized pathologically by the deposition of senile plaques and intracellular neurofibrillary tangles in the brain of the affected persons.1 Amyloid-β (Aβ) is the major component of the plaques. Aβ peptides are derived from the amyloid precursor protein (APP) through sequential proteolysis by α-, β-, and γ-secretases. Aβ40 and Aβ42 are the two major isoforms of Aβ found in AD patients. While Aβ40 is the most abundant isoform in the normal brain, Aβ42 is enriched in AD brains.2 Five drugs have been approved by the U.S. FDA (USFDA) for AD, including four AChE inhibitors, tacrine (has been discontinued for its acute hepatotoxicity), rivastigmine, donepezil, and galantamine, as well as an N-methyl-D-aspartate (NMDA) antagonist, memantine.3,4 However, all of them provide only symptomatic treatment. In addition, these drugs are considered to be effective for only a short time period ranging from six to 12 months.5,6 Since Aβ aggregation is one of the key causes of AD, inhibitors of Aβ aggregation may provide effective therapy in AD clinical trials. Chemical modification of plant extracts has been reported as a method to discover new or bioactive compounds.7 Recently, ammonolysis,8 sulfonylation,9 bromination,10 ethanolysis,11 and epoxidation12 procedures for plant extracts have been performed to obtain new or bioactive compounds. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The dried and powdered flowers of C. indicum (3.9 kg) were extracted with 95% EtOH to give an extract, which was chromatographed over macroporous resin D101 and Sephadex LH-20 to yield a total flavonoid (TF) portion. The TF portion Received: February 13, 2015

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DOI: 10.1021/acs.jnatprod.5b00156 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Scheme 1. Schematic Outline for the Obtained Active Compounds

Figure 1. HPLC profiles of the total flavonoid (TF) extract of C. indicum, (A) untreated and (B) treated with hydrazine monohydrate.

Table 1. Inhibitory Activity of Extracts (100 μg/mL) and Compounds 1−4 against Amyloid Beta (Aβ) Aggregation

was reacted with hydrazine monohydrate to provide the chemically modified TF (CMTF) portion. HPLC analyses of the CMTF and untreated total flavonoids (UTF) were performed. The HPLC data (Figure 1) showed that some major peaks eluting at greater than 30 min in the UTF disappeared in the CMTF, while new peaks eluting at less than 30 min were found. This result showed that new chemical constituents were generated after the chemical treatment. The inhibitory activity of the CMTF and UTF against Aβ42 aggregation was studied using a thioflavin T (ThT) fluorescence assay.18 As shown in Table 1, the inhibition activity for CMTF increased to 87.4% at 100 μg/mL, whereas the inhibition rate for UTF was 62.0% at the same concentration. HPLC-MS/MS experiments were performed on the UTF (Figures S1−S7, Supporting Information). As shown in Figure 2A, six 5,7-disubstituted flavonoids (5−10) were identified according to the MS ion signals at 27.8, 33.3, and 36.0 min of HPLC. Furthermore, further chromatography of the CMTF followed by an Aβ42 aggregation inhibition assay yielded four new compounds, 1−4. Compound 1 was obtained as a brown, amorphous solid. Its molecular formula was determined to be C16H14N2O4 from an ion at m/z 298.0948 in the HREIMS. The IR spectrum showed bands for hydroxy group (3311 cm−1) and aromatic ring (1595 and 1511 cm−1) absorptions. The 1H NMR data (Table 2) revealed the presence of one methoxy and two substituted aromatic rings in 1. On the basis of its 13C NMR spectrum and molecular formula, 1 also was found to contain three additional olefinic carbons and two nitrogen atoms, to constitute a pyrazole ring, in accordance with a literature report.7 The combined 1H NMR (Table 2), COSY, and HMBC data (Figure 3) indicated the presence of 1,2,3-trisubstituted and 1,2,4-

a

sample

inhibition rate (%)

compound

IC50 (μM)

95% EtOH extract total flavonoid CMTFa

29.7 62.0 87.4

curcumin (50 μM)

70.0

1 2 3 4 curcumin

4.3 15.8 1.3 2.9 3.9

The treated total flavonoids using hydrazine.

trisubstituted aromatic rings. HMBC correlations of the methoxy protons (δH 3.95, s) with a carbon at δC 149.3 (s) indicated that the methoxy group is located at C-3′. HMBC correlations of H-2′ (δH 7.44, d, J = 1.9 Hz) with C-4′ (δC 148.8, s), C-6′ (δC 120.2, d), and C-3 (δC 144.6, s) and of H-6′ (δH 7.29, dd, J = 8.0, 1.9 Hz) with C-3 (δC 144.6, s), C-4′ (δC 148.8, s), and C-2′ (δC 110.7, d) indicated one 4′-hydroxy-3′methoxy-substituted aromatic ring to be located at C-3 of the pyrazole ring. Finally, HMBC correlations of one methine proton (δH 6.97, t, J = 8.1 Hz; δC 129.8, d) with a carbon at 158.7 (s) and of a second methine proton (δH 6.46, d, J = 8.1 Hz, H-5″/3″) with carbons at δC 107.1 (s) and 158.7 (s) indicated that a 2,6-dihydroxy-substituted aromatic ring is located at C-5 of the pyrazole ring. Thus, compound 1 was proposed as 3-(4-hydroxy-3-methoxyphenyl)-5-(2,6-dihydroxyphenyl)-1H-pyrazole. Compound 2 gave a molecular formula of C15H12N2O3, as established by HREIMS. The IR spectrum again showed hydroxy group (3294 cm−1) and aromatic ring (1619, 1507, and 1454 cm−1) absorption bands. Comparison of the NMR data and molecular formulas of 2 and 1 indicated that the two compounds differ structurally only in the substitution on one B



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Figure 2. Structures of (A) flavonoids 5−10 identified in this study from the untreated total flavonoids using LC-HRESIMS and (B) flavonoids 11− 14, possible precursors of the products 1−4 based on the mechanism proposed in the literature7 (RT: retention time; MW: molecular weight).

Table 2. 1H and 13C NMR Chemical Shifts (δ) of Compounds 1−4a 1 position 3 4 5 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ OMe-3′ OMe-3″

δH (J in Hz) 7.40, s

7.44, d (1.9)

6.94, d (8.0) 7.29, dd (8.0, 1.9)

6.46, d (8.1) 6.97, t (8.1) 6.46, d (8.1) 3.95, s

2 δC 144.6 104.5 151.9 122.6 110.7 149.3 148.8 117.0 120.2 107.1 158.7 108.6 129.8 108.6 158.7 57.1


7.67, 6.95, 7.67, 6.95, 7.67,

d d d d d

(8.3) (8.3) (8.3) (8.3) (8.3)

6.47, d (8.1) 6.97, t (7.6) 6.47, d (8.1)

3 δC

δH (J in Hz)

144.4 104.3 151.5 122.5 128.5 117.4 159.4 117.4 128.5 106.9 158.8 108.6, d 129.6 108.6 158.8

7.35, s

7.29, d (1.7)

6.94, d (8.2) 7.19, dd (8.2, 1.7)

6.46, d (8.0) 6.97, t (8.0) 6.46, d (8.0)

4 δC 144.0 103.9 150.7 122.5 113.8 146.8 146.9 116.6 118.5 106.4 158.3 108.2 129.2 108.2 158.3


7.16, s

7.16, s

6.48, d (8.1) 6.97, t (8.1) 6.48, d (8.1) 3.94, s 3.94, s

δC 144.6 104.5 151.5 121.6 104.8 149.8 138.2 149.8 104.8 106.8 158.6 108.6 129.8 108.6 158.6 57.0 57.0

a1 H NMR measured at 400 MHz, 13C NMR measured at 100 MHz and obtained in acetone-d6 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

correlations (Figure S21, Supporting Information) of H-2′/H6′ (δH 7.67, d, J = 8.3 Hz) with C-1′ (δC 122.5, s), C-4′ (δC 159.4, s), and C-3 (δC 144.4, s) and of H-3′/5′ (δH 6.95, d, J = 8.3 Hz) with C-1′ (δC 122.5, s) and C-4′ (δC 159.4, s) indicated that the p-hydroxy-substituted aromatic ring is positioned at C3 (δC 144.4, s). In turn, the HMBC correlations (Figure S21, Supporting Information) suggested the 2,6-dihydroxy aromatic ring to be located at C-5. Thus, compound 2 was determined as 3-(4-hydroxyphenyl)-5-(2,6-dihydroxyphenyl)-1H-pyrazole. Compound 3, a brown, amorphous solid, gave the molecular formula C15H12N2O4, according to the HREIMS. Its NMR data (Table 2) closely resembled those of 1, except that no signals were observed at high field in its 1H or 13C NMR spectrum, indicating that 3 lacks saturated carbons. With four oxygen

Figure 3. Key HMBC correlations of 1.

aromatic ring. Compound 2 was found to contain three hydroxy groups like compound 1, but no methoxy group. The 1 H NMR spectrum (Figure S17, Supporting Information) showed that both p-substituted and 1,2,3-trisubstituted aromatic rings were present. Furthermore, the HMBC C



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Figure 4. Activity against Aβ42 aggregation, cell viability, and neuroprotective activity of compounds 1−4. (A) Inhibition rate against Aβ42 aggregation by compounds 1−4 and curcumin (cur) at 50 μM. (B) Cell viability of compounds 1, 3, and 4 at 10 μM. (C) Cell viability of compound 3 at various concentrations. (D) Neuroprotective activity of compound 3 on Aβ42-induced cell toxicity. The cell viability was measured using the MTT assay, and the data are represented as means ± SD values of three independent experiments. Curcumin was used as the positive control. The compound-free (untreated) cells were considered as a negative control (***p < 0.001, **p < 0.01).

A plausible pathway for generating 3,5-diarylpyrazole derivatives from flavonoids was previously proposed by Ricardo et al.8 On the basis of the proposed pathway, the obtained compounds 1−4 might be derived from the four 5-hydroxysubstituted flavonoids (11−14) shown in Figure 2B. However, biogenetically, 5-hydroxy-substituted flavonoids without substitution at C-7 are very rare. Moreover, the identified flavonoids (5−10) from the UTF are classified as 5,7disubstituted flavonoids, which are widely distributed in nature. A detailed comparison of the structures of the identified flavonoids 5−10 and the produced compounds 1−4 would suggest that a 7-hydroxy or 7-methoxy group was removed under the ammonolysis conditions. In order to further study this phenomenon, one experiment was performed on the pure flavonoid 5-hydroxy-7,4′-dimethoxyflavonoid using the same reaction conditions as used for the extract (Figure S36, Supporting Information). As a result, 3-(4-methoxyphenyl)-5(4-methoxy-2,6-dihydroxyphenyl)-1H-pyrazole was produced, which indicated that the 7-methoxy group was not removed during the reaction. This experiment indicated that compounds 1−4 would not be expected from ammonolysis of the identified flavonoids 5−10 according to the proposed literature mechanism.8 Therefore, the actual pathway for the production of compounds 1−4 is still uncertain and needs to be further investigated. The extracts (95% EtOH, treated and untreated TF) and four isolated compounds (1−4) from the CMTF were evaluated for their inhibitory activities against Aβ42 aggregation

atoms in its molecular formula, 3 was postulated to contain four hydroxy groups. The NMR data of 3 (Table 2) indicated the presence of one 1,2,3-trisubstituted and one 1,2,4-trisubstituted aromatic ring corresponding to H-3″/5″ (δH 6.46, d, J = 8.0 Hz), H-4″ (δH 6.97, t, J = 8.0 Hz) and H-2′ (δH 7.29, d, J = 1.7 Hz), H-5′ (δH 6.94, d, J = 8.2 Hz), H-6′ (δH 7.19, dd, J = 8.2, 1.7 Hz), respectively. Detailed analyses of the HMBC correlations (Figure S28, Supporting Information) confirmed that one 1,2,4-trisubstituted aromatic ring is located at C-3 of the pyrazole ring, while the 1,2,3-trisubstituted aromatic ring is attached at C-5 of the pyrazole ring. Thus, the structure of compound 3 was defined as 3-(3,4-dihydroxyphenyl)-5-(2,6dihydroxyphenyl)-1H-pyrazole. Compound 4, a brown, amorphous solid, gave the molecular formula C17H16N2O5, as established by HRESIMS. Again, the NMR spectroscopic data of 4 (Table 2) were similar to those of 1−3. The 1H NMR spectrum of 4 showed the presence of a 1,2,3-trisubstituted aromatic ring based on the appearance of a two hydrogen doublets (δH 6.48, d, J = 8.1 Hz) and one hydrogen triplet (6.97, t, J = 8.1 Hz). An additional two hydrogen singlets (δH 7.16, s) indicated the presence of a symmetrical 1,3,4,5-tetrasubstituted aromatic ring. The NMR data of 4 revealed the presence of two methoxy groups. The 2D NMR data (COSY, HSQC, and HMBC, Figures S33−35, Supporting Information) were used to confirm the substitution pattern of the aromatic ring. Thus, the structure of 4 was deduced as 3-(4-hydroxy-3,5-dimethoxyphenyl)-5-(2,6-dihydroxyphenyl)-1H-pyrazole. D



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Figure 5. Predicted binding modes of compounds 1 (A), 2 (B), 3 (C), and 4 (D) with Aβ42. The red dashed lines represent hydrogen bonds.

according to a ThT fluorescence assay.18 Curcumin (cur) was used as the positive control. As shown in Table 1 and Figure 4A, compounds 1−4 showed moderate to potent activity against Aβ aggregation, with the following rank order of potency: 3 > 4 > 1 ≫ 2 (EC50 1.3, 2.9, 4.3, and 15.8 μM, respectively). In particular, compound 3 showed 3-fold greater potency than curcumin (EC50 3.9 μM). To further evaluate their potential for the treatment of AD, compounds 1, 3, and 4 were first tested in a cell viability assay according to a reported method.18 As shown in Figure 4B, compounds 1, 3, and 4 did not display cytotoxicity at a concentration of 10 μM. Compound 3, with the most potent inhibition of Aβ42 aggregation, was then assayed for its neuroprotective activity. Figure 4C and D indicated that 3 shows significant neuroprotective activity based on the dose− response relationship at concentrations ranging from 5 to 80 μM in the SH-SY5Y cell line. In order to explore the binding modes of the active compounds 1−4 with Aβ42, a molecular docking experiment was performed. The crystal structure of Aβ42 used in this experiment was obtained from the Protein Data Bank (1IYT). The binding modes of the active compounds and Aβ42 were predicted using a docking approach. As shown in Figure 5, compounds 1−4 were located in the hydrophobic area of Aβ42, which is consistent with previous studies.18,19 The most active compound (3) formed three hydrogen bonds with the side chains of His 13, Lys 16, and Leu 17. Moreover, it also formed hydrophobic interactions with the residues Leu 17, Phe 20, Ala 21, Val 24, Lys 16, and His 13. Compounds 1 and 3 showed similar binding modes, but compound 1, in which a methoxy group replaced one of the hydroxy groups in 3, did not form hydrogen bonds with Aβ42. This difference may explain why 3 exhibited better activity than 1 (Figure 5). Compound 2 was more distant from Phe 20 in the hydrophobic area of Aβ42,

which might result in the lower activity against Aβ 42 aggregation. Compound 4 bound more in the center of the hydrophobic area compared with 1−3, which may explain its significant activity. Moreover, the G-scores of 1−4 were −3.05, −2.48, −3.99, and −2.23 kcal/mol, which also are consistent with the bioassay results, except for those of 4. In conclusion, four new 3,5-diarylpyrazole analogues (1−4) were discovered through chemical modification of an extract of C. indicum. The inhibition activity of the chemically modified total flavonoids increased significantly when compared with the untreated total flavonoids. The obtained compounds 1−4 showed moderate to potent inhibition activity against Aβ42 aggregation. In particular, compound 3 exhibited the most potent inhibition activity against Aβ42 aggregation, low cytotoxicity, and significant neuroprotective activity in the SH-SY5Y cell line. This investigation also has shown that chemical modification of a plant extract may be a good approach to procure new bioactive compounds.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded using a Shimadzu UV2450 spectrophotometer. IR spectra were recorded on a Bruker Tensor 37 infrared spectrophotometer. The 1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on a Bruker AVANCE-400 NMR spectrometer using TMS as an internal reference. HRESIMS and LC-HRESIMS were acquired on a Shimadzu LCMS-IT-TOF mass spectrometer, and ESIMS data were measured on an Agilent 1200 series LCMS/MS system. HREIMS was carried out on a MAT95XP high-resolution mass spectrometer. TLC analysis was carried out on silica gel plates (Marine Chemical Ltd., Qingdao, People’s Republic of China). RP-C18 silica gel (Fuji, 40−75 μm), silica gel (200−300 mesh, Marine Chemical Ltd.), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography. Analytical and semipreparative HPLC separation were carried out on an LC-20AT Shimadzu liquid chromatography system with a Zorbax SB-C18 column (250 × 9.4 E



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mm, 5 μm) or an Agilent SB-C18 column connected to an SPD-M20A diode array detector. Plant Material. The flowers of Chrysanthemum indicum were collected in Xinyang, Henan Province, People’s Republic of China, in October 2011, and authenticated by Chunyan Han from Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 20110801) was deposited in the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, People’s Republic of China. Extraction and Isolation. The air-dried and powdered flowers of C. indicum (3.9 kg) were extracted with 95% EtOH (3 × 8 L) at room temperature to produce a 95% EtOH extract. The solvent was evaporated under reduced pressure to yield a crude extract (300 g), which was suspended in H2O and extracted with EtOAc (3 × 1.5 L) and n-BuOH (3 × 1.5 L). The EtOAc-soluble fraction (120 g) was chromatographed on macroporous resin D101 eluting with EtOH− H2O (0 to 95%) to give four fractions (Fr. 1−5). The subfractions containing flavonoids were identified using TLC analysis and combined. This dried combined fraction (20 g) was further repeatedly chromatographed over Sephadex LH-20 eluting with CHCl2−MeOH (1:1) and MeOH−H2O (70:30) to give the total flavonoid fraction (3.6 g), which was divided into two parts. Part 1 was used for HPLC analysis and bioactivity assay. Part 2 was treated as described below to prepare the chemically modified total flavonoids. Chemical Modification of Total Flavonoid and Purification. An EtOH solution of the TF (1.8 g, 2 wt %/vol) containing hydrazine monohydrate (1 wt %/vol) was stirred under reflux at 85 °C for 14 h, and solvent was removed under reduced pressure. The residue was applied to a silica gel column, eluting with a mixture of CH2Cl2− MeOH (100:1, 50:1, and 20:1), to yield four fractions (A−D). Fraction A was chromatographed on a silica gel column with petroleum ether−EtOAc (80:20) as eluent to give compound 3 (8 mg). Fraction B was subjected to silica gel chromatography with CH2Cl2−MeOH (100:1) as eluent and further purified by Sephadex LH-20 eluting with MeOH to yield compound 2 (7 mg). Fraction D was chromatographed over Sephadex LH-20 (MeOH) and further purified via semipreparative RP-HPLC using a mobile phase of MeOH−H2O (50:50) to give compounds 1 (4 mg) and 4 (5 mg). 3-(4-Hydroxy-3-methoxyphenyl)-5-(2,6-dihydroxyphenyl)-1Hpyrazole (1): brown, amorphous solid; UV (MeOH) λmax (log ε) 218 (4.25), 263 (4.19) nm; IR (KBr) νmax 3311, 1703, 1595, 1511, 1465, 1364, 1265, 1117, 1011, 961, 813, 787, 736 cm−1; 1H NMR (acetoned6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 2; HREIMS m/z 298.0944 [M]+ (calcd for C16H14N2O4, 298.0948). 3-(4-Hydroxyphenyl)-5-(2,6-dihydroxyphenyl)-1H-pyrazole (2): brown, amorphous solid; UV (MeOH) λmax (log ε) 213 (4.05), 262 (4.11) nm; IR (KBr) νmax 3293, 1705, 1619, 1507, 1454, 1367, 1226, 1010, 971, 814, 735, 531 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13 C NMR (acetone-d6, 100 MHz) data, see Table 2; HREIMS m/z 268.0838 [M]+ (calcd for C15H12N2O3, 268.0842). 3-(3,4-Dihydroxyphenyl)-5-(2,6-dihydroxyphenyl)-1H-pyrazole (3): brown, amorphous solid; UV (MeOH) λmax (log ε) 220 (4.12), 261 (4.10) nm; IR (KBr) νmax 3403, 1703, 1626, 1517, 1461, 1366, 1231, 1119, 1015, 962, 812, 736 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 2; HREIMS m/z 284.0795 [M]+ (calcd for C15H12N2O4, 284.0792). 3-(4-Hydroxy-3,5-dimethoxyphenyl)-5-(2,6-dihydroxyphenyl)-1Hpyrazole (4): brown, amorphous solid; UV (MeOH) λmax (log ε) 224 (4.30), 276 (4.25) nm; IR (KBr) νmax 3853, 3743, 1694, 1648, 1516, 1463, 1098 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 2; HRESIMS m/z 329.1132 [M + H]+ (calcd for C17H16N2O5, 329.1148). Inhibition of Amyloid-β Aggregation. A thioflavin T fluorescence assay was used to evaluate the inhibition activity of compounds 1−4 against Aβ42 aggregation. Aβ42 was dissolved in 1% NH3−H2O (440 μM), and the test samples were dissolved in DMSO to give a concentration of 10 mM. Then, 100 μM Aβ42 was left untreated or mixed with equal amounts of these compounds and incubated at 37 °C for 48 h. After incubation, the samples were added to 96-well microplates to give a final volume of 200 μL with 50 mM

glycine−NaOH buffer (pH = 8.5) containing 5 μM ThT. A Thermetype multifunctional microplate reader (Ruishi Di Ken) was used to measure the fluorescence at an excitation of 450 nm and an emission of 485 nm. Cell Viability. The SH-SY5Y cell line was used in the cell viability assay. This cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% fetal bovine serum (Gibco), 50 mg/μL penicillin, and 50 mg/μL streptomycin. The MTT assay was used to assess cell viability. The cells were cultured in 96-well microplates with a density of 1 × 105 cells/well at 37 °C in a 5% CO2 humidified atmosphere. After 24 h, 10 μM of compounds 1, 3, and 4 were added to the 96-well microplates to a final volume of 100 μL. The blank well was filled with conditioned medium alone, and the control well was filled with the cells and conditioned medium. Then, the 96-well plates were incubated at 37 °C in a 5% CO2 incubator for an additional 48 h. MTT (10 μL of 5 mg/mL) was added to the 96well plates and incubated for 4 h. After incubation, the MTT medium in each well was removed, and 100 μL of DMSO was added. The absorbance of each well was then measured using a PowerWave XS2 microplate reader (Bio-Tek) set to monitor 570 nm. Neuroprotective Activity in the SH-SY5Y Cell Line. The neuroprotective effect of compound 3 on Aβ42-induced neurotoxicity was measured by the MTT assay in SH-SY5Y cells.20 Aβ42 (2 mg) was dissolved in DMSO and diluted with a culture solution to different concentrations. Samples were diluted into different concentrations (80, 40, 20, 10, and 5 μM) with 10 mM PBS and mixed with 20 μM Aβ42. These 100 μL mixtures were incubated at 37 °C for 48 h before being added to the 96-well plates. The SH-SY5Y cells were added into the 96-well plates and incubated for 24 h. The control was filled with cells and conditioned medium, and the blank was filled with conditioned medium only. Then, the test compounds were added to the 96-well plates for another 48 h at 37 °C in a 5% CO2 incubator. After incubation, DMSO was added to the 96-well plates after MTT (10 μL of 5 mg/mL) was incubated with the cells for 4 h. The absorbance tests and analysis of the results were conducted according to the MTT method. Molecular Docking Study. For an Aβ42 docking study, the initial structure of Aβ42 was taken from the NMR structure (PDB ID: 1IYT).21 The potential ligand binding site was defined as in a previous study.18 All compounds were optimized using the MMFF94s force field, and then the protein structure was protonated based on the OPLS_2005 force field. Glide Extra Precision (Glide-XP)22 was employed to identify the binding poses of compounds 1−4 for Aβ42. All docked poses of these compounds were ranked on the basis of the binding docking energies. For each compound, the lowest energy conformation was chosen for binding mode analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The LC-HRESIMS for the untreated total flavonoids. IR, MS, 1D, and 2D NMR for compounds 1−4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00156.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 919-962-0066. Fax: 919-966-3893. E-mail: khlee@unc. edu (K. H. Lee). *Tel/Fax: +86-20-39943077. E-mail: [email protected]. cn (Q. Gu). Author Contributions ⊥

T. Wu and C. Jiang contributed equally.

Notes

The authors declare no competing financial interest. F



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ACKNOWLEDGMENTS This study was supported in part by the National High-Tech R&D Program of China (863 Program) (2012AA020307) and the National Natural Science Foundation of China (Nos. 81001372, 81173470).

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REFERENCES

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