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Identification of fermented tea (Camellia sinensis) polyphenols and their inhibitory activities against amyloid-beta aggregation
Phytochemistry 160 (2019) 11–18
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Identification of fermented tea (Camellia sinensis) polyphenols and their inhibitory activities against amyloid-beta aggregation
T
Taewoong Rhoa,1, Min Sik Choib,1, Mila Junga, Hyun Woo Kila, Yong Deog Hongc, Kee Dong Yoona,∗ a
College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, 14662, Republic of Korea College of Pharmacy, Dongduk Women's University, Seoul, 02748, Republic of Korea c Amorepacific R&D Unit, 314-1 Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 17074, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Camellia japonica Theaceae Fermented tea polyphenols Anti-Aβ aggregation effect
Thirty-three phenolic compounds were identified from the extract of fermented tea (Camellia sinensis L.), including three undescribed flavonoids, namely quamoreokchaside I–II and kamoreokchaside I, along with thirty known compounds. All isolates were tested to evaluate their inhibitory effects against amyloid-beta (Aβ) aggregation through thioflavin-T (ThT) fluorescence-based assay and transmission electron microscopy (TEM). Among the isolates, three tea polyphenols, including (–)-catechin gallate (CG), (–)-epicatechin gallate (ECG), and (–)-epigallocatechin gallate (EGCG), significantly decreased Aβ aggregation at a concentration of 10 μg ml−1, compared to the positive control, Aβ alone. The anti-Aβ aggregation effects of CG, ECG, and EGCG were confirmed again via TEM, which were consistent with the ThT fluorescence-based assay. Moreover, CG and ECG provided stronger protection on SH-SY5Y cells against Aβ-induced cytotoxicity than EGCG. Remarkably, CG showed more potent inhibitory activity than EGCG, the best-known anti-Aβ aggregation agent from tea products.
1. Introduction Leaves of Camellia sinensis L. (Theaceae) have been widely used as the most popular tea source in the beverage industry and are usually consumed as green tea (unfermented), fermented teas such as oolong tea (partially fermented) and black tea (fully fermented) (Kim et al., 2011). Fermented tea is produced via heating and enzymatic fermentation of leaves of C. sinensis, and black tea accounts for about 78% of tea production (Stodt et al., 2014). In case of producing traditional fermented tea such as Pu-erh tea, dried C. sinensis leaves are mixed with water and then piled up. During fermentation period, self-heating (50 °C–80 °C) occurs in a green tea pile because predominant endogenous bacteria such as Aspergillus spp. activate the fermentation of green tea leaves. This fermenting process lasts for 3 months to obtain fermented tea. However, the traditional fermentation method has some drawbacks including long fermentation time and accompanying contamination of mycotoxin such as aflatoxin. To overcome these problems, the current fermentation method involves inoculation of Bacillus subtilis spp. isolated from Korean soybean paste followed by post-fermentation (36 h, 80 °C) after natural fermentation period (7 days). Through this post-fermentation process by B. subtilis,
we were able to dramatically reduce the fermentation time less than 10 days and achieve toxin-free fermented tea. The fermented tea produced by aforementioned fermentation method is commercially sold as a product called “Samdayeon” in Korea. Several phytochemical and pharmacological studies have demonstrated that green tea is a rich source of flavan-3-olic compounds, namely green tea catechins (Wei et al., 2011). One of the most representative green tea catechins is (-)-epigallocatechin gallate (EGCG), which furnishes diverse pharmacological activities of green tea, including anti-allergic (Wu et al., 2012), anti-calcinogenic (Shirakami et al., 2012), anti-cardiovascular (Eng et al., 2017), anti-diabetic (Babu et al., 2013), anti-inflammatory (Singh et al., 2010), and neuroprotective activities (Singh et al., 2016). On the other hand, the chemical composition of fermented tea products is somewhat more complex than that of green tea because during the fermentation process, green tea catechins generate polymeric polyphenols such as theaflavins, thearubigin, and green tea catechin epimers (Stodt et al., 2014). Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia, which is associated with the formation of amyloid-β (Aβ) plaques generated from the cleavage of amyloid precursor protein (Penke et al., 2017). According to the
∗
Corresponding author. College of Pharmacy, The Catholic University of Korea, Jibong-ro 43 Bucheon-si, Gyeonggi-do, 14662, Republic of Korea. E-mail address: [email protected] (K.D. Yoon). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.phytochem.2018.12.013 Received 2 November 2018; Received in revised form 17 December 2018; Accepted 22 December 2018 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
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addition, three sets of anomeric protons and carbons signals derived from three sugar moieties were detected at δH 5.26 (1H, d, J = 7.9 Hz, H-1″)/δC 104.5 (C-1″), δH 4.57 (1H, d, brs, H-1‴′)/δC 102.33 (C-1‴′), and δH 4.44 (1H, d, J = 7.5 Hz, H-1‴″)/δC 105.67 (C-1‴″). The identities of the sugar groups were determined by exhaustive interpretation of the 13C NMR and 2D NMR data, including HSQC and HMBC, which revealed the presence of two glucopyranosyl moieties and a rhamnopyranosyl moiety (Table 1). The orientations of the two glucopyranosyl and the rhamnopyranosyl moieties were confirmed to be β, β, and α, respectively through the coupling constants of their anomeric proton resonances, and their absolute configurations were determined to be D-, D-, and L-based on acid hydrolysis followed by gas chromatography (GC) experiments. The HMBC correlations of the anomeric protons revealed the glycosidic linkages of sugar groups [δH 5.26 (H-1″) to δC 135.58 (C-3), δH 4.57 (H-1‴′) to δC 68.43 (C-1″), δH 4.44 (H-1‴″) to δC 83.06 (C-3‴′)], and the HMBC correlation of δH 5.11 (H-3″) to δC 168.95 (C]O of E-p-coumaroyl group) indicated that p-coumaric acid was linked to the C-3″ position of the inner glucose. From the spectroscopic evidence (Supplementary Information S5–S11), 31 was elucidated to be quercetin 3-O-[3″-O-(E)-p-coumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside, namely quamoreokchaside I. The ESI-Q-TOF-MS spectrum of compound 32 showed a mass peak at m/z 779.1799, corresponding to the formula C36H36O18Na+ (calcd. 779.1799 for [M+Na]+). The 1H and 13C NMR were similar to those of 31, except for the absence of a terminal glucopyranosyl moiety (Table 1, Supplementary Information S12–S18). The β-D-glucopyranosyl and α-L-rhmanopyranosyl were determined by 1H and 13C NMR and acid hydrolysis of 32, and the connectivity of the two sugar and pcoumaroyl groups was confirmed from the HMBC cross peaks of δH 5.28 (H-1″) coupled to δC 135.52 (C-3), δH 4.53 (H-1‴′) to δC 68.23 (C-1″), and δH 5.11 (H-3″) to δC 168.97 (C]O of the p-coumaroyl group). Therefore, the chemical structure of 32 was established to be quercetin 3-O-[3″-O-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside, namely quamoreokchaside II. The molecular formula of compound 33 was established to be C42H46O22 from the positive ion ESI-Q-TOF-MS data (m/z 925.2376 [M +Na]+). The 1H NMR spectrum indicated the typical kaempferol moiety [δH 7.99 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.87 (2H, d, J = 8.7 Hz, H-3′, H-5′), 6.35 (1H, brs, H-8), and 6.17 (1H, brs, H-6)], as well as the E-p-coumaroyl group with peaks at δH 7.67 (1H, d, J = 15.9 Hz, H-8‴), 6.35 (1H, d, J = 15.9 Hz, H-7‴), 7.45 (2H, d, J = 8.7 Hz, H-2‴, H-6‴), and 6.80 (2H, d, J = 8.7 Hz, H-3‴, H-5‴). Furthermore, three anomeric proton resonances of sugar groups were observed at δH 5.46 (1H, d, J = 7.8 Hz, H-1″), 4.60 (1H, brs, Hz, H-1‴′), and 4.40 (1H, d, J = 7.5 Hz, H-1‴″). The identities of the sugar moieties were further revealed by combination of 1H, 13C NMR, 2D NMR experiments and the acid hydrolysis results, which indicated the presence of a β-D-galactopyranosyl, an α-L-rhamnopyranosyl, and β-D-glucopyranosyl moieties (Table 1, supplementary Information S15–S19). The connectivities of the E-p-coumaroyl and glycosidic linkage of the three sugar groups were established by HMBC, showing cross peaks of δH 5.34 (H-2″) to δC 168.79 (C]O of E-p-coumaroyl group), δH 5.46 (H-1″) to δC 135.07 (C3), δH 4.60 (H-1‴′) to δC 67.54 (C-6″), and δH 4.40 (H-1‴″) to δC 83.09 (C-3‴′). Based on the spectroscopic evidence (Supplementary Information S19–S25), 33 was identified as kaempferol 3-O-[2″-O-(E)-pcoumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-galactopyranoside with the trivial name of kamoreokchaside I.
amyloid hypothesis, AD occurs when soluble Aβ accumulates and reaches toxic levels, causing cell membrane perturbation and oxidative stress in the brain (Selkoe, 2000; Selkoe and Hardy, 2016). The amyloid hypothesis implies that if accumulation of Aβ is reduced, the onset and progression of AD might be slowed or halted, and AD might even be prevented if anti-Aβ therapy is started early enough. Many natural food sources have been tested in the search for anti-AD agents, and popular foods such as coffee, curry, and tea are thought to improve cognitive function based on epidemiological evidence (Panza et al., 2015). Several compounds, including curcumin, resveratrol, and EGCG, have been found to inhibit Aβ formation (Goozee et al., 2016; Braidy et al., 2016). Among the Aβ inhibitory compounds, the green tea polyphenol, EGCG, has been proposed to inhibit α-synuclein and Aβ fibrillogenesis via converting large α-synuclein and Aβ into smaller protein aggregates that are non-toxic to mammalian cells, and several reports have supported that EGCG is a potent modulating agent against mature Aβ aggregates (Rezai-Zadeh et al., 2005). Although, researches on the anti-Aβ aggregation of EGCG have primarily been reported, but relatively little attention was paid to other ingredients of tea products. As mentioned above, the phytochemicals in fermented tea are more diverse than those in green tea, which could increase the possibility for discovering other agents capable of modulating against Aβ fibrils. In the present study, thirty-three phytochemicals from fermented tea are identified using nuclear magnetic resonance (NMR) and ESI-Q-TOF-MS spectroscopy. The isolates were screened for their inhibitory activities against the formation of Aβ aggregates and protective effects against Aβ-induced cytotoxicity. 2. Results and discussion 2.1. Identification of compounds 1–33 from fermented tea extract Phytochemical isolation of fermented tea produced three unreported flavone glycosides, as well as thirty known compounds (Fig. 1), including kaempferol (1), quercetin (2), myricetin (3), rutin (4), nicotiflorine (5), gallic acid (6), (–)-epigallocatechin 3-O-gallate (EGCG, 7), myricetin 3-O-β-D-galactopyranoside (8), myricetin 3-O-βD-glucopyranoside (9), (–)-gallocatechin 3-O-gallate (GCG, 10), (–)-epicatechin 3-O-gallate (ECG, 11), (–)-catechin 3-O-gallate (CG, 12), isovitexin (13), 6″-galloylmyricetin 3-O-β-D-glucopyranoside (14), quercetin 3-O-β-D-galactopyranoside (15), vitexin (16), quercetin 3-Oβ-D-glucopyranoside (17), (-)-epicatechin (18), myricetin 3-O-rutinoside (19), 6″-galloylmyricetin 3-O-β-D-galactopyranoside (20), daidzin (21), tricin 7-O-β-D-glucopyranoside (22), quercetin 3-O-[β-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 6)-O-β-D-glucopyranoside] (23), quercetin 3-O-[β-D-glucopyranosyl-(1 → 4)-O-α-Lrhamnopyranosyl-(1 → 6)-O-β-D-galactopyranoside] (24), kaempferol 3-O-[β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)-O-βD-glucopyranoside] (25), kaempferol 3-O-[β-D-glucopyranosyl-(1 → 3)O-α-L-rhamnopyranosyl-(1 → 6)-O-β-D-galactopyranoside] (26), luteolin 8-C-glucopyranoside (27), quercetin 3-O-[2-O″-(E)-p-coumaroyl] [β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)-O-β-Dglucopyrano side] (28), glycitin (29), quercetin 3-O-[2-O″-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)-O-β-D-glucopyranoside] (30). The 1H NMR and ESI-Q-TOF-MS data of the known compounds (1–30) were in good agreement with previously reported values (Supplementary Information S3 and S4). Compound 31 was isolated as a yellow amorphous powder with a molecular formula of C42H46O23 from the mass peak at m/z 941.2313 [M+Na]+ in the positive ESI-Q-TOF-MS spectrum. The 1H NMR spectrum of 31 presented characteristic quercetin aglycone peaks at δH 7.69 (1H, d, J = 1.8 Hz, H-2′), 7.62 (1H, dd, J = 8.3, 1.8 Hz, H-6′), 6.89 (1H, d, J = 8.3 Hz, H-5′), 6.41 (1H, d, J = 1.9 Hz, H-8), and 6.21 (1H, d, J = 1.9 Hz, H-6) and the E-p-coumaric acid skeleton [δH 7.68 (1H, d, J = 15.7 Hz, H-8‴), 7.48 (2H, d, J = 7.5 Hz, H-2‴, H-6‴), 6.81 (2H, d, J = 7.5 Hz, H-3‴, H-5‴), and 6.42 (1H, d, J = 15.7 Hz, H-7‴)]. In
2.2. Inhibition of Aβ aggregation by fermented tea polyphenols Thioflavin-T (ThT) fluorescence-based assay was used to monitor Aβ aggregation. In the presence of compounds 1–33, the time-dependent fluorescence intensity corresponding to Aβ aggregation increased or decreased compared to that of the positive control, i.e., Aβ alone (Fig. 2A). Twenty-one compounds were selected and tested for 12
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Fig. 1. Chemical structures of compounds 1–33 from fermented tea.
2.3. Reduction of Aβ-induced cytotoxicity by fermented tea polyphenols on SH-SY5Y human neuroblastoma cells
significance in subsequent experiments (Fig. 2B) based on their ability to inhibit Aβ aggregation in the first screening assay. Among these compounds, it was found that only three catechin derivatives, EGCG (7), ECG (11), and CG (12) significantly decreased the relative fluorescence intensity to 78.0 ± 4.6% (p < 0.05), 62.8 ± 10.3% (p < 0.05), and 46.4 ± 8.9% (p < 0.01), respectively, compared to the positive control. Interestingly, ECG and CG showed stronger inhibitory effects against Aβ aggregation than the well-known inhibitor, EGCG (7). Specifically, CG was the only compound showing over 50% inhibition for Aβ aggregation in the ThT fluorescence assay. Utilizing transmission electron microscopy (TEM), the inhibitory activity of EGCG, ECG, and CG against Aβ aggregation was again confirmed (Fig. 3A). After incubation of the Aβ monomers alone, oligomers and linear tangled fibrils were respectively formed after 1.5 h and 24 h of incubation. In contrast, co-incubation of 10 μg mL−1 of EGCG, ECG, and CG with Aβ monomers potently attenuated formation of the oligomers (1.5 h incubation) and linear tangled fibrils (24 h incubation). It is clearly shown that CG and ECG more potently inhibited Aβ aggregation than EGCG, which is consistent with the ThT fluorescence-based assay results.
To investigate the protective effect of EGCG, ECG, and CG against the cytotoxicity of Aβ aggregation, SH-SY5Y human neuroblastoma cells were used for cell viability assay (Fig. 3B). In this experiment, the absorbance of cell media containing SH-SY5Y cells was measured, and the absorbance was regarded to correspond to 100% cell viability. The Aβ monomers underwent oligomerization and fibrillization during 24 h incubation in cell-free media, and the resultant solution was added to SH-SY5Y cells. The cell viability of the untreated control group (Aβ alone) decreased to 64.0 ± 6.0% (p < 0.01), which indicated that the Aβ (1–42) oligomers and fibrils were cytotoxic. However, co-incubation of Aβ (1–42) with EGCG, ECG, and CG for 24 h prior to the treatment restored the cell viability to 77.4 ± 9.0%, 85.1 ± 11.9%, and 92.0 ± 5.5%, respectively. Previous reports demonstrate that an excess of the Aβ monomers causes aggregation into toxic fibrillar forms, which plays a pivotal role in the pathogenesis of Alzheimer's disease (Selkoe, 2000; Selkoe and Hardy, 2016). In view of this hypothesis, several studies have been conducted on the neurotoxicity of Aβ in neurons derived from severely 13
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Table 1 1 H and 13C NMR data of compounds 31–33 in methanol-d4.a Position
31 1
H
32 13
C
Aglycone (quercetin) 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′
6.21 (d, J = 1.9 Hz) 6.41 (d, J = 1.9 Hz)
7.69 (d, J = 1.8 Hz)
6.89 (d, J = 8.3 Hz) 7.62 (dd, J = 8.3, 1.8 Hz) 3-O-Glc (inner) 5.26 (d, J = 7.9 Hz) 3.71b 5.11 (t, J = 9.2 Hz) 3.54b 3.40b 3.80 (dd, J = 11.4, 6.2 Hz) 3.49b
1″ 2″ 3″ 4″ 5″ 6″
1‴′ 2‴′ 3‴′ 4‴′ 5‴′ 6‴′ 1‴″ 2‴″ 3‴″ 4‴″ 5‴″ 6‴″
7.48 (d, J = 7.5 Hz) 6.81 (d, J = 7.5 Hz) 6.42 (d, J = 15.7 Hz) 7.68 (d, J = 15.7 Hz) 6″-O-Rha 4.57 (brs) 3.96 (brs) 3.63 (dd, J = 9.2, 3.5 Hz) 3.46b 3.54b 1.10 (d, J = 5.9 Hz) 3‴′-O-Glc (terminal) 4.44 (d, J = 7.5 Hz) 3.25b 3.50b 3.40b 3.26b 3.78b 3.72b
13
H
C
Aglycone (quercetin) 159.29 135.58 179.33 163.02 99.98 166.03 94.95 158.58 105.60 123.06 117.79 145.86 149.82 116.13 123.48 104.5 74.11 78.93 69.43 77.54 68.43
3″-O-p-coumaroyl 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ 7‴ 8‴ C=O
33
1
6.21 (d, J = 1.7 Hz) 6.40
b
7.67 (d, J = 2.2 Hz)
6.89 (d, J = 8.4 Hz) 7.63 (dd, J = 8.4, 2.2 Hz) 3-O-Glc (inner) 5.28 (d, J = 7.9 Hz) 3.68b 5.11 (t, J = 9.2 Hz) 3.53b 3.45b 3.80 (dd, J = 11.4, 6.2 Hz) 3.44b
102.33 71.28 83.06 72.60 69.76 17.96
7.48 (d, J = 8.5 Hz) 6.81 (d, J = 8.5 Hz) 6.42 (d, J = 15.7 Hz) 7.68 (d, J = 15.7 Hz) 6″-O-Rha 4.53 (d, J = 1.2 Hz) 3.65b 3.56 (dd, J = 9.9, 3.9 Hz) 3.46b 3.52b 1.12 (d, J = 6.1 Hz)
105.67 75.48 76.97 70.89 77.54 62.10
H
13
C
Aglycone (kaempferol) 159.23 135.52 179.33 163.02 99.96 166.04 94.85 158.52 105.64 123.06 117.68 145.88 149.86 116.11 123.52 104.35 74.12 78.93 69.79b 77.09 68.23
3″-O-p-coumaroyl 127.30 131.18 116.83 161.27 115.45 146.71 168.95
1
6.17 (brs) 6.35 (brs)
7.99 (d, J = 8.7 Hz) 6.87 (d, J = 8.7 Hz) 6.87 (d, J = 8.7 Hz) 7.99 (d, J = 8.7 Hz) 3-O-Gal (inner) 5.46 (d, J = 7.8 Hz) 5.34 (t, J = 8.9 Hz) 3.76b 3.85 (d, J = 3.5 Hz) 3.78b 3.76b 3.54b
161.26 135.07 179.31 161.5 99.87 165.74 94.80 158.58 105.84 122.72 132.29 116.27 158.69 116.27 132.29 101.55 74.14 73.25 70.47 75.51 67.54
2″-O-p-coumaroyl 127.29 131.17 116.82 161.26 115.44 146.70 168.97 102.42 72.09 72.23 73.92 69.76b 17.89
7.45 (d, J = 8.5 Hz) 6.80 (d, J = 8.5 Hz) 6.35 (d, J = 15.9 Hz) 7.67 (d, J = 15.9 Hz)
127.30 131.20 116.82 161.26 115.31 146.88 168.79
6″-O-Rha 4.60 (brs) 3.95 (t, J = 1.6 Hz) 3.61b 3.46 (t, J = 9.3 Hz) 3.54b 1.19 (d, J = 6.0 Hz)
101.85 71.34 83.09 72.60 69.49 18.08
3‴′-O-Glc (terminal) 4.40 (d, J = 7.5 Hz) 3.25b 3.36b 3.36b 3.20b 3.71b
105.74 75.40 77.60 70.84 77.60 62.05
a 1 b
H and 13C NMR were recorded at 500 and 125 MHz, respectively. Resonances were overlapped.
EGCG is known as the most protective constituent against Aβ based on analyses using either PC12 cells (Levites et al., 2003) or hippocampal neurons (Choi et al., 2001). However, if the results are compared only in terms of the Aβ-induced toxicity, they can be interpreted differently. Bastianetto et al. (2006) reported that ECG, EGCG, and gallic acid inhibited apoptosis induced by Aβ peptides and exerted neuroprotective effects on hippocampal cells. In contrast, the compounds with nongallate forms (EC and EGC) failed to inhibit apoptotic events generated by Aβ peptides. A closer examination of those results confirmed that ECG also exhibited neuroprotective effects against Aβ-induced toxicity, as also observed for EGCG and gallic acid. Considering these factors, it is expected that the effects of these components may be different based on the detailed experimental materials or conditions. CG and ECG, which were the effective components based on the current experimental results, have the following structural characteristics with respect to other catechin derivatives. First, CG and ECG
affected regions in AD brains, including the hippocampus (Doré et al., 1997). In addition to the role of fibrillar Aβ, it has been reported that soluble Aβ oligomers were also detrimental to the hippocampus, with significantly increased levels of the fibrils in AD brains (Gong et al., 2003). To date, there are a few studies that directly compare the biological effects of CG or ECG with those of EGCG because most studies on the anti-Aβ aggregation effect of green tea polyphenol have strictly focused on EGCG. Moreover, the anti-Aβ aggregation effect of CG has not been reported so far, as the content of CG compounds is negligible or very low in green tea. However, in this study, CG was found in fermented tea in a relatively high amount compared to that in green tea, as demonstrated by preliminary HPLC analysis (Supplementary Information S26). The results of the current study indicate that the inhibitory effect of CG and ECG against Aβ aggregation is more potent than that of EGCG. These findings may seem inconsistent with previous reports because 14
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Fig. 2. (A) In the presence of the compounds 1–33 (10 μg ml−1), the time-dependent fluorescence intensity of Aβ aggregation was analyzed by ThT assay, comparing to that of positive control, Aβ alone. (B) Effects of twenty-one compounds from fermented tea on Aβ aggregation were tested using ThT fluorescence assay after 1.5 h incubation. Error bars = SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to positive control, Aβ alone.
possess a catechol moiety (3,4-dihydroxylated benzene ring) as a B-ring of the flavan structure, whereas EGCG has a pyrogallol skeleton (3,4,5trihydroxylated benzene ring). Second, CG and ECG possess a gallic acid moiety at the C-3 position, which apparently affects the anti-Aβ aggregation activity because the non-gallated compound, (-)-epicatechin (EC), were inactive in this study, which is consistent with a previous report (Bastianetto et al., 2006). Collectively, these structural characteristics may be crucial factors determining the differences in the effect of CG and ECG from other tea polyphenols against Aβ-aggregation.
EGCG. It is notable that CG and ECG more potently inhibited Aβ aggregation than EGCG, the best-known Aβ aggregation inhibitor in tea products, and such results have not been reported so far. Moreover, CG and ECG more strongly protected SH-SY5Y cells against Aβ-induced cytotoxicity than EGCG, which was consistent with the anti-Aβ aggregation results. These data provide important evidences for the potential of CG and ECG as anti-Aβ aggregation agents, and more extensive biological studies should be performed by researchers in various scientific fields to discover the beneficial actions of fermented tea polyphenols.
3. Conclusions
4. Material and methods
In this study, three unreported flavonol glycosides, namely quamoreokchaside I–II and kamoreokchaside I, and thirty known flavonoids were identified from fermented tea (Camellia sinensis) extract. The thirty-three isolates were tested to evaluate their anti-Aβ aggregation effects, which demonstrated that the three tea polyphenols attenuated Aβ aggregation with an increase in potency in the order: CG, ECG, and
4.1. Instrumentation Preparative and semi-preparative scale high-performance countercurrent chromatography (HPCCC) was performed using a MIDI HPCCC (Dynamic Extractions, Berkshire, UK) and a Spectrum instrument (Dynamic Extractions, Berkshire, UK), respectively. The MIDI 15
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Fig. 3. (A) Transmission electron micrographs of end-products for 1.5 h (oligomers) or 24 h (linear tangled fibrils) incubation of Aβ monomer alone (top left) or in the presence of EGCG, ECG or CG (10 μg ml−1, respectively). Scale bars are 100 nm (1.5 h) or 0.5 μm (24 h). (B) SH-SY5Y cell viability after treatment with Aβ aggregates (10 μM) with or without co-incubation with EGCG, ECG or CG for 24 h prior to the treatment) was measured by CCK-8 assay. Aβ monomers were oligomerized and fibrillated for 24 h in cell-free media, and the resultant solution was added to wells containing SH-SY5Y cells. Error bars = SD (n = 3). **p < 0.01 as compared to untreated. #p < 0.05, ##p < 0.01 as compared to Aβ aggregates alone.
Company, Hillsboro, OR) operating at 120 kV was used to evaluate the self-assembled morphology of the amyloid beta peptides.
instrument was combined with an IOTA S 300 pump (ECOM, Prague, Czech Republic), a Sapphire 600 UV-VIS variable wavelength detector (ECOM, Prague, Czech Republic), a Foxy® R2 fraction collector (Teledyne Isco, NE, USA), and a CCA-1111 circulatory temperature regulator that was utilized to maintain the inner temperature of the HPCCC at 30 °C. The Spectrum instrument was equipped with a 1525 binary HPLC pump (Waters, MA, USA), a 2487 dual λ absorbance detector (Waters, MA, USA), and a FC 204 fraction collector (Gilson, WI, USA). A CCA-1111 CA-1112CE circulatory temperature regulator (Eyela, Tokyo, Japan) was used to keep the inner temperature of the HPCCC at 30 °C. Preparative HPLC was performed with a Gilson HPLC system (Middelton, WI, USA) comprising a liquid handler, a UV/VIS detector, and binary pumps. A Luna column (21.2 × 250 mm I.D., 5 μm, Phenomenex, Torrance, CA, USA) was used to isolate the fermented tea compounds. The structures of the isolates were determined by acquisition of the 1H and 13C NMR data using an AVANCE 500 spectrometer (Bruker, Karlsruhe, Germany). Mass spectral (MS) data were obtained with a 6460 QTOF-MS spectrometer (Agilent Technologies, CA, USA), and optical rotation data were recorded on a Jasco P-2000 polarimeter. Fluorescence from ThT assay was read at 430 nm/470–530 nm immediately after the reaction by using a TECAN Infinite 200 PRO multifunctional microplate reader (TECAN, Switzerland) at 37 °C. To evaluate the cell viability, the absorbance at 450 nm after the reaction with CCK-8 was then measured using the same microplate reader. Transmission electron microscopy (TEM; Tecnai G2 Spirit, FEI
4.2. Chemicals and plant materials The organic solvents used for column chromatography were analytical grade, and acetonitrile for HPLC was purchased from DaejungChemical and Metals Co., Ltd. (Kyeonggi-Do, Korea). Deionized water was obtained using a Millipore Milli-Q water purification system. Amyloid β (1–42) monomer, ThT, and the reaction buffers were procured from AnaSpec (Fremont, USA; SensoLyte® Thioflavin T BetaAmyloid (Aβ1–42) aggregation kit). Buffers for the cell viability assay were from Dojindo (CCK-8 assay kit; MD, USA). 4.3. Preparation of fermented tea Fresh green tea leaves were collected in April 2015 from Osulloc Tea Garden (Jeju island, Korea). The manufacturing process for fermented tea was strictly controlled by Amore Pacific Co. to maintain the uniformity of fermented tea quality. Tea fermentation was carried out at 50 °C for 3 days and then 80 °C for 4 days followed by inoculation of Bacillus subtilis spp. isolated from traditional Korean soybean paste, and incubation for 36 h at 80 °C. After completing the fermentation process, the fermented tea was dried at 80 °C for 4–5 h. 16
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compounds (10 μg ml−1) isolated from fermented tea. The reactions were performed with a final volume of 100 μl at room temperature in a black 96-well plate according to the assay protocol. The plate was placed in a TECAN Infinite 200 PRO multifunctional microplate reader (TECAN, Switzerland) at 37 °C. The fluorescence was read at 430 nm/ 470–530 nm immediately after assembly of the reaction system. Additional measurements were taken every 5 min with 15 s of shaking after measurements to aid protein aggregation.
4.4. Extraction and isolation Dried fermented tea (1.2 kg) was extracted three times (90 min ✕ 3) with 50% aqueous ethanol (4 L ✕ 3 times) using an ultrasonic bath to give a 50% aqueous ethanol extract (162 g). The 50% aqueous ethanol extract was extracted with acetone (2 L ✕ 3 times) to give an acetonesoluble extract (31 g) and acetone-insoluble precipitate (121 g). The acetone-insoluble precipitate was extracted with ethanol (2 L ✕ 3 times) to give an ethanol-soluble extract (37 g). The ethanol-soluble extract was subjected to silica gel column chromatography using CHCl3-MeOH mixtures (10:1 and 5:1, v/v) as eluents to yield Fraction I [CHCl3-MeOH (10:1, v/v), 14.7 g] and Fraction II [CHCl3-MeOH (5:1, v/v), 5.4 g]. Fraction I was suspended in water and partitioned with CH2Cl2 to get rid of caffeine, which yielded 8.9 g of the decaffeined Fraction I. The detailed methods for isolation of compounds 1–33 from decaffeined Fraction I and Fraction II are described in the Supplementary Information S1 and S2.
4.7. Negative-stained transmission electron microscopy (TEM) To evaluate the self-assembled morphology of the amyloid-beta peptides, TEM (Tecnai G2 Spirit, FEI Company, Hillsboro, OR) operating at 120 kV was used. For negative staining, fibrils were allowed to form for a proper time in Amyloid beta aggregation buffer, and the formed fibrils were immediately placed on a 200-mesh carbon coated TEM grid (Cu) and stained with 2% uranyl acetate solution. The grids were then imaged directly using appropriate magnifications.
4.5. Acid hydrolysis and spectroscopic data of compounds 31–33 4.8. Cell cytotoxicity assay 4.5.1. Acid hydrolysis of compounds 31–33 Acidic hydrolysis for 31–33 was conducted according to the previous report (Ahn et al., 2006). Each hydrolysate was analyzed by gas chromatography using BPX50 capillary column (0.25 mm × 30 m, Trajan, Pflugerville, Texas, USA) under following condition; detector: FID, column temperature: 210 °C, injector temperature: 270 °C, detection temperature: 300 °C, carrier gas: helium. The reactants of authentic D-glucose, D-galactose and L-rhamnose showed GC peaks at 11.12, 12.39 and 8.25 min, respectively. The retention times of reactants of 31–33 were almost identical to those of authentic sugars.
The viability of SH-SY5Y cells was evaluated using the CCK-8 assay according to the manufacturer's instructions (Dojindo, MD, USA). The cell viability detecting reagent, CCK-8, was added to the cells in the culture, and the cells were incubated for 2 h in a humidified atmosphere. The absorbance at 450 nm was then measured, and the cell viability is expressed as the percentage of the absolute optical density of each sample relative to that of the untreated sample. Acknowledgements
4.5.2. Quercetin 3-O-[3″-O-(E)-p-coumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside (quamoreokchaside I, 31) C42H46O23; a pale yellowish powder; [α]24 D : −72.1 (c 0.1, MeOH); UV (CH3OH) λmax (log ε) 208 (2.42), 259 (2.08), 267 (2.08), 314 (2.22) nm; IR (neat) νmax 3321, 2923, 1652, 1601, 1518, 1444, 1359, 1169, 1030 cm−1; ESI-Q-TOF-MS (positive ion mode) m/z 941.2313 [M +Na]+ (calcd for C42H46O23Na+, 941.2328); 1H (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) Table 1.
This work was supported by grants from National Research Foundation of Korea (Grant # NRF-2017R1A2B4003888 and 2018R1A6A1A03025108) and research funds from Amorepacific Corporation and the Catholic University of Korea (2018).
4.5.3. Quercetin 3-O-[3″-O-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside (quamoreokchaside II, 32) C36H36O18; a pale yellowish powder; [α]24 D : −89.4 (c 0.1, MeOH); UV (CH3OH) λmax (log ε) 208 (2.75), 259 (2.40), 267 (2.41), 313 (2.54) nm; IR (neat) νmax 3319, 2923, 1655, 1603, 1513, 1446, 1361, 1169, 1063 cm−1; ESI-Q-TOF-MS (positive ion mode) m/z 779.1799 [M +Na]+ (calcd for C36H36O18Na+, 779.1799); 1H (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) Table 1.
References
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.12.013.
Ahn, M.J., Kim, C.Y., Yoon, K.D., Ryu, M.Y., Cheong, J.H., Chin, Y.W., Kim, J., 2006. Steroidal saponins from the rhizomes of Polygonatum sibiricum. J. Nat. Prod. 69, 360–364. Babu, P.V., Liu, D., Gilbert, E.R., 2013. Recent advances in understanding the anti-diabetic actions of dietary flavonoids. J. Nutr. Biochem. 24, 1777–1789. Braidy, N., Jugder, B.E., Poljak, A., Jayasena, T., Mansour, H., Nabavi, S.M., Sachdev, P., Grant, R., 2016. Resveratrol as a potential therapeutic candidate for the treatment and management of Alzheimer's disease. Curr. Top. Med. Chem. 16, 1951–1960. Bastianetto, S., Yao, Z., Vassilios, P., Quirion, R., 2006. Neuroprotective effects of green and black teas and their catechin gallate esters against β-amyloid-induced toxicity. Eur. J. Neurosci. 23, 55–64. Choi, Y.T., Jung, C.H., Lee, S.R., Bae, J.H., Baek, W.K., Suh, M.H., Park, J., Park, C.W., Suh, S.I., 2001. The green tea polyphenol (-)-epigallocatechin gallate attenuates betaamyloid-induced neurotoxicity in cultured hippocampal neurons. Life Sci. 70, 603–614. Doré, S., Kar, S., Quirion, R., 1997. Insulin-like growth factor I protects and rescues hippocampal neurons against β-amyloid and human amylin-induced toxicity. Proc. Natl. Acad. Sci. U. S. A. 94, 4772–4777. Eng, Q.Y., Thanikachalam, P.V., Ramamurth, S., 2017. Molecular understanding of epigallocatechin gallate (EGCG) in cardiovascular and metabolic diseases. J. Ethnopharmacol. 210, 296–310. Gong, Y., Chang, L., Viola, K.L., Lacor, P.N., Lambert, M.P., Finch, C.E., Krafft, G.A., Klein, W.L., 2003. Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc. Natl. Acad. Sci. U. S. A. 100, 10417–10422. Goozee, K.G., Shah, T.M., Sohrabi, H.R., Rainey-Smith, S.R., Brown, B., Verdile, G., Martins, R.N., 2016. Examining the potential clinical value of curcumin in the prevention and diagnosis of Alzheimer's disease. Br. J. Nutr. 115, 449–465. Kim, Y., Goodner, K.L., Park, J.D., Choi, J., Talcott, S.T., 2011. Changes in antioxidant
4.5.4. Kaempferol 3-O-[2″-O-(E)-p-coumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-galactopyranoside (kamoreokchaside I, 33) C42H46O22; a pale yellowish powder; [α]24 D : −62.7 (c 0.1, MeOH); UV (CH3OH) λmax (log ε) 210 (2.58), 268 (2.31), 317 (2.44) nm; IR (neat) νmax 3324, 2925, 1655, 1603, 1510, 1439, 1359, 1174, 1078 cm−1; ESIQ-TOF-MS (positive ion mode) m/z 925.2376 [M+Na]+ (calcd for C42H46O22Na+, 925.2378); 1H (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) Table 1. 4.6. ThT assay for evaluating Aβ aggregation Using the SensoLyte® Thioflavin T Beta-Amyloid (Aβ1–42) Aggregation Kit (AnaSpec, Fremont, USA), the aggregation of betaamyloid (Aβ1–42) was measured in the presence of morin and phenol red (100 μM, respectively), known inhibitors of fibril formation, or the 17
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phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation. Food Chem. 129, 1331–1342. Levites, Y., Amit, T., Mandel, S., Youdim, M.B., 2003. Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J. 17, 952–954. Panza, F., Solfrizzi, V., Barulli, M.R., Bonfiglio, C., Guerra, V., Osella, A., Seripa, D., Sabbà, C., Pilotto, A., Logroscino, G., 2015. Coffee, tea, and caffeine consumption and prevention of late-life cognitive decline and dementia: a systematic review. J. Nutr. Health Aging 19, 313–328. Penke, B., Bogár, F., Fülöp, L., 2017. β-Amyloid and the pathomechanisms of Alzheimer's disease: a comprehensive view. Molecules 22, 1692. https://doi.org/10.3390/ molecules22101692. Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J., 2005. Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. J. Neurosci. 25, 8807–8814. Selkoe, D.J., 2000. Toward a comprehensive theory for Alzheimer's disease. Hypothesis: Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of
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Identification of fermented tea (Camellia sinensis) polyphenols and their inhibitory activities against amyloid-beta aggregation
T
Taewoong Rhoa,1, Min Sik Choib,1, Mila Junga, Hyun Woo Kila, Yong Deog Hongc, Kee Dong Yoona,∗ a
College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, 14662, Republic of Korea College of Pharmacy, Dongduk Women's University, Seoul, 02748, Republic of Korea c Amorepacific R&D Unit, 314-1 Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do, 17074, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Camellia japonica Theaceae Fermented tea polyphenols Anti-Aβ aggregation effect
Thirty-three phenolic compounds were identified from the extract of fermented tea (Camellia sinensis L.), including three undescribed flavonoids, namely quamoreokchaside I–II and kamoreokchaside I, along with thirty known compounds. All isolates were tested to evaluate their inhibitory effects against amyloid-beta (Aβ) aggregation through thioflavin-T (ThT) fluorescence-based assay and transmission electron microscopy (TEM). Among the isolates, three tea polyphenols, including (–)-catechin gallate (CG), (–)-epicatechin gallate (ECG), and (–)-epigallocatechin gallate (EGCG), significantly decreased Aβ aggregation at a concentration of 10 μg ml−1, compared to the positive control, Aβ alone. The anti-Aβ aggregation effects of CG, ECG, and EGCG were confirmed again via TEM, which were consistent with the ThT fluorescence-based assay. Moreover, CG and ECG provided stronger protection on SH-SY5Y cells against Aβ-induced cytotoxicity than EGCG. Remarkably, CG showed more potent inhibitory activity than EGCG, the best-known anti-Aβ aggregation agent from tea products.
1. Introduction Leaves of Camellia sinensis L. (Theaceae) have been widely used as the most popular tea source in the beverage industry and are usually consumed as green tea (unfermented), fermented teas such as oolong tea (partially fermented) and black tea (fully fermented) (Kim et al., 2011). Fermented tea is produced via heating and enzymatic fermentation of leaves of C. sinensis, and black tea accounts for about 78% of tea production (Stodt et al., 2014). In case of producing traditional fermented tea such as Pu-erh tea, dried C. sinensis leaves are mixed with water and then piled up. During fermentation period, self-heating (50 °C–80 °C) occurs in a green tea pile because predominant endogenous bacteria such as Aspergillus spp. activate the fermentation of green tea leaves. This fermenting process lasts for 3 months to obtain fermented tea. However, the traditional fermentation method has some drawbacks including long fermentation time and accompanying contamination of mycotoxin such as aflatoxin. To overcome these problems, the current fermentation method involves inoculation of Bacillus subtilis spp. isolated from Korean soybean paste followed by post-fermentation (36 h, 80 °C) after natural fermentation period (7 days). Through this post-fermentation process by B. subtilis,
we were able to dramatically reduce the fermentation time less than 10 days and achieve toxin-free fermented tea. The fermented tea produced by aforementioned fermentation method is commercially sold as a product called “Samdayeon” in Korea. Several phytochemical and pharmacological studies have demonstrated that green tea is a rich source of flavan-3-olic compounds, namely green tea catechins (Wei et al., 2011). One of the most representative green tea catechins is (-)-epigallocatechin gallate (EGCG), which furnishes diverse pharmacological activities of green tea, including anti-allergic (Wu et al., 2012), anti-calcinogenic (Shirakami et al., 2012), anti-cardiovascular (Eng et al., 2017), anti-diabetic (Babu et al., 2013), anti-inflammatory (Singh et al., 2010), and neuroprotective activities (Singh et al., 2016). On the other hand, the chemical composition of fermented tea products is somewhat more complex than that of green tea because during the fermentation process, green tea catechins generate polymeric polyphenols such as theaflavins, thearubigin, and green tea catechin epimers (Stodt et al., 2014). Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia, which is associated with the formation of amyloid-β (Aβ) plaques generated from the cleavage of amyloid precursor protein (Penke et al., 2017). According to the
∗
Corresponding author. College of Pharmacy, The Catholic University of Korea, Jibong-ro 43 Bucheon-si, Gyeonggi-do, 14662, Republic of Korea. E-mail address: [email protected] (K.D. Yoon). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.phytochem.2018.12.013 Received 2 November 2018; Received in revised form 17 December 2018; Accepted 22 December 2018 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
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addition, three sets of anomeric protons and carbons signals derived from three sugar moieties were detected at δH 5.26 (1H, d, J = 7.9 Hz, H-1″)/δC 104.5 (C-1″), δH 4.57 (1H, d, brs, H-1‴′)/δC 102.33 (C-1‴′), and δH 4.44 (1H, d, J = 7.5 Hz, H-1‴″)/δC 105.67 (C-1‴″). The identities of the sugar groups were determined by exhaustive interpretation of the 13C NMR and 2D NMR data, including HSQC and HMBC, which revealed the presence of two glucopyranosyl moieties and a rhamnopyranosyl moiety (Table 1). The orientations of the two glucopyranosyl and the rhamnopyranosyl moieties were confirmed to be β, β, and α, respectively through the coupling constants of their anomeric proton resonances, and their absolute configurations were determined to be D-, D-, and L-based on acid hydrolysis followed by gas chromatography (GC) experiments. The HMBC correlations of the anomeric protons revealed the glycosidic linkages of sugar groups [δH 5.26 (H-1″) to δC 135.58 (C-3), δH 4.57 (H-1‴′) to δC 68.43 (C-1″), δH 4.44 (H-1‴″) to δC 83.06 (C-3‴′)], and the HMBC correlation of δH 5.11 (H-3″) to δC 168.95 (C]O of E-p-coumaroyl group) indicated that p-coumaric acid was linked to the C-3″ position of the inner glucose. From the spectroscopic evidence (Supplementary Information S5–S11), 31 was elucidated to be quercetin 3-O-[3″-O-(E)-p-coumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside, namely quamoreokchaside I. The ESI-Q-TOF-MS spectrum of compound 32 showed a mass peak at m/z 779.1799, corresponding to the formula C36H36O18Na+ (calcd. 779.1799 for [M+Na]+). The 1H and 13C NMR were similar to those of 31, except for the absence of a terminal glucopyranosyl moiety (Table 1, Supplementary Information S12–S18). The β-D-glucopyranosyl and α-L-rhmanopyranosyl were determined by 1H and 13C NMR and acid hydrolysis of 32, and the connectivity of the two sugar and pcoumaroyl groups was confirmed from the HMBC cross peaks of δH 5.28 (H-1″) coupled to δC 135.52 (C-3), δH 4.53 (H-1‴′) to δC 68.23 (C-1″), and δH 5.11 (H-3″) to δC 168.97 (C]O of the p-coumaroyl group). Therefore, the chemical structure of 32 was established to be quercetin 3-O-[3″-O-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside, namely quamoreokchaside II. The molecular formula of compound 33 was established to be C42H46O22 from the positive ion ESI-Q-TOF-MS data (m/z 925.2376 [M +Na]+). The 1H NMR spectrum indicated the typical kaempferol moiety [δH 7.99 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.87 (2H, d, J = 8.7 Hz, H-3′, H-5′), 6.35 (1H, brs, H-8), and 6.17 (1H, brs, H-6)], as well as the E-p-coumaroyl group with peaks at δH 7.67 (1H, d, J = 15.9 Hz, H-8‴), 6.35 (1H, d, J = 15.9 Hz, H-7‴), 7.45 (2H, d, J = 8.7 Hz, H-2‴, H-6‴), and 6.80 (2H, d, J = 8.7 Hz, H-3‴, H-5‴). Furthermore, three anomeric proton resonances of sugar groups were observed at δH 5.46 (1H, d, J = 7.8 Hz, H-1″), 4.60 (1H, brs, Hz, H-1‴′), and 4.40 (1H, d, J = 7.5 Hz, H-1‴″). The identities of the sugar moieties were further revealed by combination of 1H, 13C NMR, 2D NMR experiments and the acid hydrolysis results, which indicated the presence of a β-D-galactopyranosyl, an α-L-rhamnopyranosyl, and β-D-glucopyranosyl moieties (Table 1, supplementary Information S15–S19). The connectivities of the E-p-coumaroyl and glycosidic linkage of the three sugar groups were established by HMBC, showing cross peaks of δH 5.34 (H-2″) to δC 168.79 (C]O of E-p-coumaroyl group), δH 5.46 (H-1″) to δC 135.07 (C3), δH 4.60 (H-1‴′) to δC 67.54 (C-6″), and δH 4.40 (H-1‴″) to δC 83.09 (C-3‴′). Based on the spectroscopic evidence (Supplementary Information S19–S25), 33 was identified as kaempferol 3-O-[2″-O-(E)-pcoumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-galactopyranoside with the trivial name of kamoreokchaside I.
amyloid hypothesis, AD occurs when soluble Aβ accumulates and reaches toxic levels, causing cell membrane perturbation and oxidative stress in the brain (Selkoe, 2000; Selkoe and Hardy, 2016). The amyloid hypothesis implies that if accumulation of Aβ is reduced, the onset and progression of AD might be slowed or halted, and AD might even be prevented if anti-Aβ therapy is started early enough. Many natural food sources have been tested in the search for anti-AD agents, and popular foods such as coffee, curry, and tea are thought to improve cognitive function based on epidemiological evidence (Panza et al., 2015). Several compounds, including curcumin, resveratrol, and EGCG, have been found to inhibit Aβ formation (Goozee et al., 2016; Braidy et al., 2016). Among the Aβ inhibitory compounds, the green tea polyphenol, EGCG, has been proposed to inhibit α-synuclein and Aβ fibrillogenesis via converting large α-synuclein and Aβ into smaller protein aggregates that are non-toxic to mammalian cells, and several reports have supported that EGCG is a potent modulating agent against mature Aβ aggregates (Rezai-Zadeh et al., 2005). Although, researches on the anti-Aβ aggregation of EGCG have primarily been reported, but relatively little attention was paid to other ingredients of tea products. As mentioned above, the phytochemicals in fermented tea are more diverse than those in green tea, which could increase the possibility for discovering other agents capable of modulating against Aβ fibrils. In the present study, thirty-three phytochemicals from fermented tea are identified using nuclear magnetic resonance (NMR) and ESI-Q-TOF-MS spectroscopy. The isolates were screened for their inhibitory activities against the formation of Aβ aggregates and protective effects against Aβ-induced cytotoxicity. 2. Results and discussion 2.1. Identification of compounds 1–33 from fermented tea extract Phytochemical isolation of fermented tea produced three unreported flavone glycosides, as well as thirty known compounds (Fig. 1), including kaempferol (1), quercetin (2), myricetin (3), rutin (4), nicotiflorine (5), gallic acid (6), (–)-epigallocatechin 3-O-gallate (EGCG, 7), myricetin 3-O-β-D-galactopyranoside (8), myricetin 3-O-βD-glucopyranoside (9), (–)-gallocatechin 3-O-gallate (GCG, 10), (–)-epicatechin 3-O-gallate (ECG, 11), (–)-catechin 3-O-gallate (CG, 12), isovitexin (13), 6″-galloylmyricetin 3-O-β-D-glucopyranoside (14), quercetin 3-O-β-D-galactopyranoside (15), vitexin (16), quercetin 3-Oβ-D-glucopyranoside (17), (-)-epicatechin (18), myricetin 3-O-rutinoside (19), 6″-galloylmyricetin 3-O-β-D-galactopyranoside (20), daidzin (21), tricin 7-O-β-D-glucopyranoside (22), quercetin 3-O-[β-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 6)-O-β-D-glucopyranoside] (23), quercetin 3-O-[β-D-glucopyranosyl-(1 → 4)-O-α-Lrhamnopyranosyl-(1 → 6)-O-β-D-galactopyranoside] (24), kaempferol 3-O-[β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)-O-βD-glucopyranoside] (25), kaempferol 3-O-[β-D-glucopyranosyl-(1 → 3)O-α-L-rhamnopyranosyl-(1 → 6)-O-β-D-galactopyranoside] (26), luteolin 8-C-glucopyranoside (27), quercetin 3-O-[2-O″-(E)-p-coumaroyl] [β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)-O-β-Dglucopyrano side] (28), glycitin (29), quercetin 3-O-[2-O″-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)-O-β-D-glucopyranoside] (30). The 1H NMR and ESI-Q-TOF-MS data of the known compounds (1–30) were in good agreement with previously reported values (Supplementary Information S3 and S4). Compound 31 was isolated as a yellow amorphous powder with a molecular formula of C42H46O23 from the mass peak at m/z 941.2313 [M+Na]+ in the positive ESI-Q-TOF-MS spectrum. The 1H NMR spectrum of 31 presented characteristic quercetin aglycone peaks at δH 7.69 (1H, d, J = 1.8 Hz, H-2′), 7.62 (1H, dd, J = 8.3, 1.8 Hz, H-6′), 6.89 (1H, d, J = 8.3 Hz, H-5′), 6.41 (1H, d, J = 1.9 Hz, H-8), and 6.21 (1H, d, J = 1.9 Hz, H-6) and the E-p-coumaric acid skeleton [δH 7.68 (1H, d, J = 15.7 Hz, H-8‴), 7.48 (2H, d, J = 7.5 Hz, H-2‴, H-6‴), 6.81 (2H, d, J = 7.5 Hz, H-3‴, H-5‴), and 6.42 (1H, d, J = 15.7 Hz, H-7‴)]. In
2.2. Inhibition of Aβ aggregation by fermented tea polyphenols Thioflavin-T (ThT) fluorescence-based assay was used to monitor Aβ aggregation. In the presence of compounds 1–33, the time-dependent fluorescence intensity corresponding to Aβ aggregation increased or decreased compared to that of the positive control, i.e., Aβ alone (Fig. 2A). Twenty-one compounds were selected and tested for 12
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Fig. 1. Chemical structures of compounds 1–33 from fermented tea.
2.3. Reduction of Aβ-induced cytotoxicity by fermented tea polyphenols on SH-SY5Y human neuroblastoma cells
significance in subsequent experiments (Fig. 2B) based on their ability to inhibit Aβ aggregation in the first screening assay. Among these compounds, it was found that only three catechin derivatives, EGCG (7), ECG (11), and CG (12) significantly decreased the relative fluorescence intensity to 78.0 ± 4.6% (p < 0.05), 62.8 ± 10.3% (p < 0.05), and 46.4 ± 8.9% (p < 0.01), respectively, compared to the positive control. Interestingly, ECG and CG showed stronger inhibitory effects against Aβ aggregation than the well-known inhibitor, EGCG (7). Specifically, CG was the only compound showing over 50% inhibition for Aβ aggregation in the ThT fluorescence assay. Utilizing transmission electron microscopy (TEM), the inhibitory activity of EGCG, ECG, and CG against Aβ aggregation was again confirmed (Fig. 3A). After incubation of the Aβ monomers alone, oligomers and linear tangled fibrils were respectively formed after 1.5 h and 24 h of incubation. In contrast, co-incubation of 10 μg mL−1 of EGCG, ECG, and CG with Aβ monomers potently attenuated formation of the oligomers (1.5 h incubation) and linear tangled fibrils (24 h incubation). It is clearly shown that CG and ECG more potently inhibited Aβ aggregation than EGCG, which is consistent with the ThT fluorescence-based assay results.
To investigate the protective effect of EGCG, ECG, and CG against the cytotoxicity of Aβ aggregation, SH-SY5Y human neuroblastoma cells were used for cell viability assay (Fig. 3B). In this experiment, the absorbance of cell media containing SH-SY5Y cells was measured, and the absorbance was regarded to correspond to 100% cell viability. The Aβ monomers underwent oligomerization and fibrillization during 24 h incubation in cell-free media, and the resultant solution was added to SH-SY5Y cells. The cell viability of the untreated control group (Aβ alone) decreased to 64.0 ± 6.0% (p < 0.01), which indicated that the Aβ (1–42) oligomers and fibrils were cytotoxic. However, co-incubation of Aβ (1–42) with EGCG, ECG, and CG for 24 h prior to the treatment restored the cell viability to 77.4 ± 9.0%, 85.1 ± 11.9%, and 92.0 ± 5.5%, respectively. Previous reports demonstrate that an excess of the Aβ monomers causes aggregation into toxic fibrillar forms, which plays a pivotal role in the pathogenesis of Alzheimer's disease (Selkoe, 2000; Selkoe and Hardy, 2016). In view of this hypothesis, several studies have been conducted on the neurotoxicity of Aβ in neurons derived from severely 13
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Table 1 1 H and 13C NMR data of compounds 31–33 in methanol-d4.a Position
31 1
H
32 13
C
Aglycone (quercetin) 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′
6.21 (d, J = 1.9 Hz) 6.41 (d, J = 1.9 Hz)
7.69 (d, J = 1.8 Hz)
6.89 (d, J = 8.3 Hz) 7.62 (dd, J = 8.3, 1.8 Hz) 3-O-Glc (inner) 5.26 (d, J = 7.9 Hz) 3.71b 5.11 (t, J = 9.2 Hz) 3.54b 3.40b 3.80 (dd, J = 11.4, 6.2 Hz) 3.49b
1″ 2″ 3″ 4″ 5″ 6″
1‴′ 2‴′ 3‴′ 4‴′ 5‴′ 6‴′ 1‴″ 2‴″ 3‴″ 4‴″ 5‴″ 6‴″
7.48 (d, J = 7.5 Hz) 6.81 (d, J = 7.5 Hz) 6.42 (d, J = 15.7 Hz) 7.68 (d, J = 15.7 Hz) 6″-O-Rha 4.57 (brs) 3.96 (brs) 3.63 (dd, J = 9.2, 3.5 Hz) 3.46b 3.54b 1.10 (d, J = 5.9 Hz) 3‴′-O-Glc (terminal) 4.44 (d, J = 7.5 Hz) 3.25b 3.50b 3.40b 3.26b 3.78b 3.72b
13
H
C
Aglycone (quercetin) 159.29 135.58 179.33 163.02 99.98 166.03 94.95 158.58 105.60 123.06 117.79 145.86 149.82 116.13 123.48 104.5 74.11 78.93 69.43 77.54 68.43
3″-O-p-coumaroyl 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ 7‴ 8‴ C=O
33
1
6.21 (d, J = 1.7 Hz) 6.40
b
7.67 (d, J = 2.2 Hz)
6.89 (d, J = 8.4 Hz) 7.63 (dd, J = 8.4, 2.2 Hz) 3-O-Glc (inner) 5.28 (d, J = 7.9 Hz) 3.68b 5.11 (t, J = 9.2 Hz) 3.53b 3.45b 3.80 (dd, J = 11.4, 6.2 Hz) 3.44b
102.33 71.28 83.06 72.60 69.76 17.96
7.48 (d, J = 8.5 Hz) 6.81 (d, J = 8.5 Hz) 6.42 (d, J = 15.7 Hz) 7.68 (d, J = 15.7 Hz) 6″-O-Rha 4.53 (d, J = 1.2 Hz) 3.65b 3.56 (dd, J = 9.9, 3.9 Hz) 3.46b 3.52b 1.12 (d, J = 6.1 Hz)
105.67 75.48 76.97 70.89 77.54 62.10
H
13
C
Aglycone (kaempferol) 159.23 135.52 179.33 163.02 99.96 166.04 94.85 158.52 105.64 123.06 117.68 145.88 149.86 116.11 123.52 104.35 74.12 78.93 69.79b 77.09 68.23
3″-O-p-coumaroyl 127.30 131.18 116.83 161.27 115.45 146.71 168.95
1
6.17 (brs) 6.35 (brs)
7.99 (d, J = 8.7 Hz) 6.87 (d, J = 8.7 Hz) 6.87 (d, J = 8.7 Hz) 7.99 (d, J = 8.7 Hz) 3-O-Gal (inner) 5.46 (d, J = 7.8 Hz) 5.34 (t, J = 8.9 Hz) 3.76b 3.85 (d, J = 3.5 Hz) 3.78b 3.76b 3.54b
161.26 135.07 179.31 161.5 99.87 165.74 94.80 158.58 105.84 122.72 132.29 116.27 158.69 116.27 132.29 101.55 74.14 73.25 70.47 75.51 67.54
2″-O-p-coumaroyl 127.29 131.17 116.82 161.26 115.44 146.70 168.97 102.42 72.09 72.23 73.92 69.76b 17.89
7.45 (d, J = 8.5 Hz) 6.80 (d, J = 8.5 Hz) 6.35 (d, J = 15.9 Hz) 7.67 (d, J = 15.9 Hz)
127.30 131.20 116.82 161.26 115.31 146.88 168.79
6″-O-Rha 4.60 (brs) 3.95 (t, J = 1.6 Hz) 3.61b 3.46 (t, J = 9.3 Hz) 3.54b 1.19 (d, J = 6.0 Hz)
101.85 71.34 83.09 72.60 69.49 18.08
3‴′-O-Glc (terminal) 4.40 (d, J = 7.5 Hz) 3.25b 3.36b 3.36b 3.20b 3.71b
105.74 75.40 77.60 70.84 77.60 62.05
a 1 b
H and 13C NMR were recorded at 500 and 125 MHz, respectively. Resonances were overlapped.
EGCG is known as the most protective constituent against Aβ based on analyses using either PC12 cells (Levites et al., 2003) or hippocampal neurons (Choi et al., 2001). However, if the results are compared only in terms of the Aβ-induced toxicity, they can be interpreted differently. Bastianetto et al. (2006) reported that ECG, EGCG, and gallic acid inhibited apoptosis induced by Aβ peptides and exerted neuroprotective effects on hippocampal cells. In contrast, the compounds with nongallate forms (EC and EGC) failed to inhibit apoptotic events generated by Aβ peptides. A closer examination of those results confirmed that ECG also exhibited neuroprotective effects against Aβ-induced toxicity, as also observed for EGCG and gallic acid. Considering these factors, it is expected that the effects of these components may be different based on the detailed experimental materials or conditions. CG and ECG, which were the effective components based on the current experimental results, have the following structural characteristics with respect to other catechin derivatives. First, CG and ECG
affected regions in AD brains, including the hippocampus (Doré et al., 1997). In addition to the role of fibrillar Aβ, it has been reported that soluble Aβ oligomers were also detrimental to the hippocampus, with significantly increased levels of the fibrils in AD brains (Gong et al., 2003). To date, there are a few studies that directly compare the biological effects of CG or ECG with those of EGCG because most studies on the anti-Aβ aggregation effect of green tea polyphenol have strictly focused on EGCG. Moreover, the anti-Aβ aggregation effect of CG has not been reported so far, as the content of CG compounds is negligible or very low in green tea. However, in this study, CG was found in fermented tea in a relatively high amount compared to that in green tea, as demonstrated by preliminary HPLC analysis (Supplementary Information S26). The results of the current study indicate that the inhibitory effect of CG and ECG against Aβ aggregation is more potent than that of EGCG. These findings may seem inconsistent with previous reports because 14
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Fig. 2. (A) In the presence of the compounds 1–33 (10 μg ml−1), the time-dependent fluorescence intensity of Aβ aggregation was analyzed by ThT assay, comparing to that of positive control, Aβ alone. (B) Effects of twenty-one compounds from fermented tea on Aβ aggregation were tested using ThT fluorescence assay after 1.5 h incubation. Error bars = SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to positive control, Aβ alone.
possess a catechol moiety (3,4-dihydroxylated benzene ring) as a B-ring of the flavan structure, whereas EGCG has a pyrogallol skeleton (3,4,5trihydroxylated benzene ring). Second, CG and ECG possess a gallic acid moiety at the C-3 position, which apparently affects the anti-Aβ aggregation activity because the non-gallated compound, (-)-epicatechin (EC), were inactive in this study, which is consistent with a previous report (Bastianetto et al., 2006). Collectively, these structural characteristics may be crucial factors determining the differences in the effect of CG and ECG from other tea polyphenols against Aβ-aggregation.
EGCG. It is notable that CG and ECG more potently inhibited Aβ aggregation than EGCG, the best-known Aβ aggregation inhibitor in tea products, and such results have not been reported so far. Moreover, CG and ECG more strongly protected SH-SY5Y cells against Aβ-induced cytotoxicity than EGCG, which was consistent with the anti-Aβ aggregation results. These data provide important evidences for the potential of CG and ECG as anti-Aβ aggregation agents, and more extensive biological studies should be performed by researchers in various scientific fields to discover the beneficial actions of fermented tea polyphenols.
3. Conclusions
4. Material and methods
In this study, three unreported flavonol glycosides, namely quamoreokchaside I–II and kamoreokchaside I, and thirty known flavonoids were identified from fermented tea (Camellia sinensis) extract. The thirty-three isolates were tested to evaluate their anti-Aβ aggregation effects, which demonstrated that the three tea polyphenols attenuated Aβ aggregation with an increase in potency in the order: CG, ECG, and
4.1. Instrumentation Preparative and semi-preparative scale high-performance countercurrent chromatography (HPCCC) was performed using a MIDI HPCCC (Dynamic Extractions, Berkshire, UK) and a Spectrum instrument (Dynamic Extractions, Berkshire, UK), respectively. The MIDI 15
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Fig. 3. (A) Transmission electron micrographs of end-products for 1.5 h (oligomers) or 24 h (linear tangled fibrils) incubation of Aβ monomer alone (top left) or in the presence of EGCG, ECG or CG (10 μg ml−1, respectively). Scale bars are 100 nm (1.5 h) or 0.5 μm (24 h). (B) SH-SY5Y cell viability after treatment with Aβ aggregates (10 μM) with or without co-incubation with EGCG, ECG or CG for 24 h prior to the treatment) was measured by CCK-8 assay. Aβ monomers were oligomerized and fibrillated for 24 h in cell-free media, and the resultant solution was added to wells containing SH-SY5Y cells. Error bars = SD (n = 3). **p < 0.01 as compared to untreated. #p < 0.05, ##p < 0.01 as compared to Aβ aggregates alone.
Company, Hillsboro, OR) operating at 120 kV was used to evaluate the self-assembled morphology of the amyloid beta peptides.
instrument was combined with an IOTA S 300 pump (ECOM, Prague, Czech Republic), a Sapphire 600 UV-VIS variable wavelength detector (ECOM, Prague, Czech Republic), a Foxy® R2 fraction collector (Teledyne Isco, NE, USA), and a CCA-1111 circulatory temperature regulator that was utilized to maintain the inner temperature of the HPCCC at 30 °C. The Spectrum instrument was equipped with a 1525 binary HPLC pump (Waters, MA, USA), a 2487 dual λ absorbance detector (Waters, MA, USA), and a FC 204 fraction collector (Gilson, WI, USA). A CCA-1111 CA-1112CE circulatory temperature regulator (Eyela, Tokyo, Japan) was used to keep the inner temperature of the HPCCC at 30 °C. Preparative HPLC was performed with a Gilson HPLC system (Middelton, WI, USA) comprising a liquid handler, a UV/VIS detector, and binary pumps. A Luna column (21.2 × 250 mm I.D., 5 μm, Phenomenex, Torrance, CA, USA) was used to isolate the fermented tea compounds. The structures of the isolates were determined by acquisition of the 1H and 13C NMR data using an AVANCE 500 spectrometer (Bruker, Karlsruhe, Germany). Mass spectral (MS) data were obtained with a 6460 QTOF-MS spectrometer (Agilent Technologies, CA, USA), and optical rotation data were recorded on a Jasco P-2000 polarimeter. Fluorescence from ThT assay was read at 430 nm/470–530 nm immediately after the reaction by using a TECAN Infinite 200 PRO multifunctional microplate reader (TECAN, Switzerland) at 37 °C. To evaluate the cell viability, the absorbance at 450 nm after the reaction with CCK-8 was then measured using the same microplate reader. Transmission electron microscopy (TEM; Tecnai G2 Spirit, FEI
4.2. Chemicals and plant materials The organic solvents used for column chromatography were analytical grade, and acetonitrile for HPLC was purchased from DaejungChemical and Metals Co., Ltd. (Kyeonggi-Do, Korea). Deionized water was obtained using a Millipore Milli-Q water purification system. Amyloid β (1–42) monomer, ThT, and the reaction buffers were procured from AnaSpec (Fremont, USA; SensoLyte® Thioflavin T BetaAmyloid (Aβ1–42) aggregation kit). Buffers for the cell viability assay were from Dojindo (CCK-8 assay kit; MD, USA). 4.3. Preparation of fermented tea Fresh green tea leaves were collected in April 2015 from Osulloc Tea Garden (Jeju island, Korea). The manufacturing process for fermented tea was strictly controlled by Amore Pacific Co. to maintain the uniformity of fermented tea quality. Tea fermentation was carried out at 50 °C for 3 days and then 80 °C for 4 days followed by inoculation of Bacillus subtilis spp. isolated from traditional Korean soybean paste, and incubation for 36 h at 80 °C. After completing the fermentation process, the fermented tea was dried at 80 °C for 4–5 h. 16
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compounds (10 μg ml−1) isolated from fermented tea. The reactions were performed with a final volume of 100 μl at room temperature in a black 96-well plate according to the assay protocol. The plate was placed in a TECAN Infinite 200 PRO multifunctional microplate reader (TECAN, Switzerland) at 37 °C. The fluorescence was read at 430 nm/ 470–530 nm immediately after assembly of the reaction system. Additional measurements were taken every 5 min with 15 s of shaking after measurements to aid protein aggregation.
4.4. Extraction and isolation Dried fermented tea (1.2 kg) was extracted three times (90 min ✕ 3) with 50% aqueous ethanol (4 L ✕ 3 times) using an ultrasonic bath to give a 50% aqueous ethanol extract (162 g). The 50% aqueous ethanol extract was extracted with acetone (2 L ✕ 3 times) to give an acetonesoluble extract (31 g) and acetone-insoluble precipitate (121 g). The acetone-insoluble precipitate was extracted with ethanol (2 L ✕ 3 times) to give an ethanol-soluble extract (37 g). The ethanol-soluble extract was subjected to silica gel column chromatography using CHCl3-MeOH mixtures (10:1 and 5:1, v/v) as eluents to yield Fraction I [CHCl3-MeOH (10:1, v/v), 14.7 g] and Fraction II [CHCl3-MeOH (5:1, v/v), 5.4 g]. Fraction I was suspended in water and partitioned with CH2Cl2 to get rid of caffeine, which yielded 8.9 g of the decaffeined Fraction I. The detailed methods for isolation of compounds 1–33 from decaffeined Fraction I and Fraction II are described in the Supplementary Information S1 and S2.
4.7. Negative-stained transmission electron microscopy (TEM) To evaluate the self-assembled morphology of the amyloid-beta peptides, TEM (Tecnai G2 Spirit, FEI Company, Hillsboro, OR) operating at 120 kV was used. For negative staining, fibrils were allowed to form for a proper time in Amyloid beta aggregation buffer, and the formed fibrils were immediately placed on a 200-mesh carbon coated TEM grid (Cu) and stained with 2% uranyl acetate solution. The grids were then imaged directly using appropriate magnifications.
4.5. Acid hydrolysis and spectroscopic data of compounds 31–33 4.8. Cell cytotoxicity assay 4.5.1. Acid hydrolysis of compounds 31–33 Acidic hydrolysis for 31–33 was conducted according to the previous report (Ahn et al., 2006). Each hydrolysate was analyzed by gas chromatography using BPX50 capillary column (0.25 mm × 30 m, Trajan, Pflugerville, Texas, USA) under following condition; detector: FID, column temperature: 210 °C, injector temperature: 270 °C, detection temperature: 300 °C, carrier gas: helium. The reactants of authentic D-glucose, D-galactose and L-rhamnose showed GC peaks at 11.12, 12.39 and 8.25 min, respectively. The retention times of reactants of 31–33 were almost identical to those of authentic sugars.
The viability of SH-SY5Y cells was evaluated using the CCK-8 assay according to the manufacturer's instructions (Dojindo, MD, USA). The cell viability detecting reagent, CCK-8, was added to the cells in the culture, and the cells were incubated for 2 h in a humidified atmosphere. The absorbance at 450 nm was then measured, and the cell viability is expressed as the percentage of the absolute optical density of each sample relative to that of the untreated sample. Acknowledgements
4.5.2. Quercetin 3-O-[3″-O-(E)-p-coumaroyl][β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside (quamoreokchaside I, 31) C42H46O23; a pale yellowish powder; [α]24 D : −72.1 (c 0.1, MeOH); UV (CH3OH) λmax (log ε) 208 (2.42), 259 (2.08), 267 (2.08), 314 (2.22) nm; IR (neat) νmax 3321, 2923, 1652, 1601, 1518, 1444, 1359, 1169, 1030 cm−1; ESI-Q-TOF-MS (positive ion mode) m/z 941.2313 [M +Na]+ (calcd for C42H46O23Na+, 941.2328); 1H (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) Table 1.
This work was supported by grants from National Research Foundation of Korea (Grant # NRF-2017R1A2B4003888 and 2018R1A6A1A03025108) and research funds from Amorepacific Corporation and the Catholic University of Korea (2018).
4.5.3. Quercetin 3-O-[3″-O-(E)-p-coumaroyl][α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside (quamoreokchaside II, 32) C36H36O18; a pale yellowish powder; [α]24 D : −89.4 (c 0.1, MeOH); UV (CH3OH) λmax (log ε) 208 (2.75), 259 (2.40), 267 (2.41), 313 (2.54) nm; IR (neat) νmax 3319, 2923, 1655, 1603, 1513, 1446, 1361, 1169, 1063 cm−1; ESI-Q-TOF-MS (positive ion mode) m/z 779.1799 [M +Na]+ (calcd for C36H36O18Na+, 779.1799); 1H (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) Table 1.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.12.013.
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