Article pubs.acs.org/jnp
Neolignans from Aristolochia fordiana Prevent Oxidative Stress-Induced Neuronal Death through Maintaining the Nrf2/HO‑1 Pathway in HT22 Cells Gui-Hua Tang,†,⊥ Zi-Wei Chen,†,⊥ Ting-Ting Lin,† Min Tan,‡ Xiao-Yun Gao,§ Jing-Mei Bao,† Zhong-Bin Cheng,† Zhang-Hua Sun,† Gang Huang,§ and Sheng Yin*,† †
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China Department of Pharmacy, Longquanyi District Hospital of Traditional Chinese Medicine, Chengdu 610100, People’s Republic of China § The 2nd Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510120, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: Bioassay-guided fractionation of the ethanolic extract of the stems of Aristolochia fordiana led to the isolation of six new dihydrobenzofuran neolignans (1−3 and 7−9), three new 2-aryldihydrobenzofurans (4−6), a new 8-O-4′ neolignan (10), and 14 known analogues (11−24). The structures of compounds 1−10 were established by spectroscopic methods, and their absolute configurations were determined by analyses of the specific rotation and electronic circular dichroism data. The neuroprotective effects of compounds 1−24 against glutamate-induced cell death were tested in hippocampal neuronal cell line HT22. Compounds 17 and 20−24 exhibited moderate neuroprotective activity by increasing the endogenous antioxidant defense system. In addition, the neolignans activated the Nrf2 (nuclear factor E2-related factor 2) pathway, resulting in the increase of the expression of endogenous antioxidant protein HO-1 (heme oxygenase-1). The active compounds also preserved the levels of antiapoptotic protein Bcl-2 (B cell lymphoma/leukemia-2), which was decreased by glutamate. Collectively, these results suggested that the active neolignans protect neurons against glutamate-induced cell death through maintaining the Nrf2/ HO-1 signaling pathway as well as preserving the Bcl-2 protein and might be promising novel beneficial agents for oxidative stress-associated diseases.
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B cell lymphoma/leukemia-2 (Bcl-2) level to prevent oxidative stress-induced apoptosis.7 Therefore, Nrf2 plays an important role in antioxidative defense systems, and natural products that up-regulate Nrf2 might be a potential therapeutic agent for pathologies associated with oxidative stress. Aristolochia fordiana Hemsley (Aristolochiaceae) is a common twining herb native to the Guangxi Zhuang Autonomous Region and Guangdong Province of China.8 Its rhizome is used in Traditional Chinese Medicine (TCM) for the treatment of seizures, rheumatism, and abdominal pain.9 Previous chemical investigation of this herb revealed the occurrence of several benzofuran-type neolignans, a pyrrole derivative, a sucrose ester,
ncreased oxidative stress has been recognized as a common cause of many neurological disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), and stroke.1 Oxidative stress refers to the imbalance due to excess reactive oxygen species (ROS) or oxidants over the capability of the cell to mount an effective antioxidant response.2 The subsequent oxidative imbalance could lead to cellular oxidative stress, cellular function alteration, and even cell death. Cells have developed a highly elaborate mechanism to regulate cellular levels of oxidant species including ROS using exogenous and endogenous antioxidants.3 The nuclear factor E2-related factor 2 (Nrf2) pathway is regarded as the most important in the cell to protect against oxidative stress.4,5 Nrf2 can regulate the expression of heme oxygenase-1 (HO-1), an endogenous antioxidant protein, to detoxify or scavenge oxidant species.6 A recent study also showed that the up-regulation of Nrf2 could increase the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 11, 2015
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DOI: 10.1021/acs.jnatprod.5b00220 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1
and two 1,2,4-trisubstituted benzene rings [δH 6.79 (brs, H-2′), 6.78 (d, J = 8.1 Hz, H-5′), and 6.78 (dd, J = 8.1, 1.9 Hz, H-6′); 7.15 (brs, H-4), 7.12 (dd, J = 8.2, 1.6 Hz, H-6), and 6.81 (d, J = 8.2 Hz, H-7)]. In addition, analyses of the 1H NMR signals at δH 5.72 (d, J = 8.8 Hz, H-2), 3.63 (m, H-3), and 0.86 (3H, d, J = 7.2 Hz, 3-CH3) and the 13C NMR signals at δC 87.9 (C-2), 40.9 (C-3), and 16.7 (3-CH3) allowed the identification of a cis-2-aryl3-methyldihydrobenzofuran system.13 The 13C NMR spectrum in combination with DEPT experiments resolved 19 carbon resonances attributable to two methyls, one methylenedioxy, 10 methines (including one oxygenated and eight aromatic or olefinic methines), three quaternary sp2 carbons, and three oxygenated tertiary sp2 carbons. These NMR data together with the 2D NMR data (Figure 1) were identical to those of (7R,8S)3,4-methylenedioxy-4′,7-epoxy-8,3′-neolignan-7′(E)-ene reported from the same plant by Kong’s group (compound 210). However, the specific rotation data and the electronic circular dichroism (ECD) spectrum of compound 1 were opposite of the reported data, implying the enantiomeric nature of these two compounds. As compound 1 shared a similar ECD curve with its analogue (−)-epi-conocarpan (positive Cotton effect at 225 nm; negative Cotton effect at 260 nm), whose absolute configuration was established as 2R,3S by stereoselective total synthesis,14 the structure of compound 1 was assigned as (2R,3S)-2,3-dihydro-2(3,4-methylenedioxyphenyl)-3-methyl-5-(E)-propenylbenzofuran. Consequently, the structure of Kong’s compound (compound 210) should be revised as (7S,8R)-3,4-methylenedioxy-4′,7-epoxy8,3′-neolignan-7′(E)-ene. The HRESIMS data of 2 showed a sodium adduct ion at m/z 347.1272 [M + Na]+ (calcd 347.1259) corresponding to the molecular formula C20H20O4. The 1H and 13C NMR data (Tables 1 and 2) of 2 were identical to those of (7R,8S)-3,4methylenedioxy-3′-methoxy-4′,7-epoxy-8,5′-neolignan-7′(E)-ene reported from the same plant by Kong’s group (compound 110).
and a benzylisoquinoline alkaloid glycoside. Some of the benzofuran neolignans were reported to possess nitric oxide production inhibitory and cytotoxic activities.10,11 In our screening program aiming at the discovery of neuroprotective agents from TCM,12 a fraction of the ethanolic extract of A. fordiana showed neuroprotective effects against glutamate-induced HT22 cell death at a concentration of 10 μg/mL, by increasing the cell viability from 40% to 60%. Subsequent chemical investigation led to the isolation of seven new neolignans (1−3, 7−9, and 10), three new 2-aryldihydrobenzofurans (4−6), and 14 known analogues. All compounds were screened for neuroprotective activities against glutamate-induced cell death in HT22 cells, and the biochemical mechanisms underlying the protective effects of active compounds against glutamate-induced neuronal death were further explored. Herein, details of the isolation, structural elucidation, and neuroprotective activities of these compounds are described.
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RESULTS AND DISCUSSION The air-dried powder of the stems of A. fordiana was extracted by 95% EtOH to yield a crude extract, which was suspended in H2O and successively partitioned with petroleum ether (PE) and EtOAc. The bioactive PE and EtOAc fractions were further separated over silica column, Sephadex LH-20, and semipreparative HPLC equipped with a chiral column to afford 10 new (1−10) and 14 known (11−24) compounds. Compound 1 exhibited a molecular formula of C19H18O3 as determined by an HRESIMS ion at m/z 295.1329 [M + H]+ (calcd 295.1329) and 13C NMR data. The IR bands at 1605, 1520, and 1488 cm−1 indicated the presence of aromatic rings. The 1H NMR data (Table 1) showed an AMX3 spin system [δH 6.36 (1H, dd, J = 15.7, 1.5 Hz, H-8), 6.09 (1H, dq, J = 15.7, 6.6 Hz, H-9), and 1.86 (3H, dd, J = 6.6, 1.5 Hz, H3-10)] for a trans-propenyl group, a methylenedioxy group [δH 5.95 (2H, s)], B
DOI: 10.1021/acs.jnatprod.5b00220 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H NMR (400 MHz) Data for Compounds 1−9 in CDCl3 (δ in ppm, J in Hz) position 2 3 4 6 7 8 9 10 2′ 3′ 5′ 6′ 3-CH3 7-OCH3 −OCH2O− 3′-OCH3 4′-OCH3
1 5.72, 3.63, 7.15, 7.12, 6.81, 6.36, 6.09, 1.86, 6.79,
2
d (8.8) m s dd (8.2, 1.6) d (8.2) dd (15.7, 1.5) dq (15.7, 6.6) dd (6.6, 1.5) s
6.78, d (8.1) 6.78, dd (8.1,1.9) 0.86, d (7.2) 5.95, s
3
5.75, 3.62, 6.76, 6.77,
d (8.7) m overlapped overlapped
6.35, 6.09, 1.86, 6.80,
d (16.1) dq (16.1, 6.6) d (6.6) s
6.77, 6.77, 0.86, 3.91, 5.94,
overlapped overlapped d (7.2) s s
5.75, 3.62, 7.16, 7.13, 6.83, 6.37, 6.09, 1.86, 6.84,
4
d (8.7) m s d (8.2) overlapped d (15.7) dq (15.6, 6.6) dd (6.6, 1.5) overlapped
6.84, overlapped 6.85, overlapped 0.84, d (7.2)
5.80, 3.69, 7.70, 7.66, 6.89,
d (8.8) m s d (8.3) d (8.3)
6.74, s 6.80, d (8.2) 6.74, overlapped 0.88, d (7.0)
5 and 6 5.24, 3.48, 7.72, 7.73, 6.94, 9.87,
d (8.6) m s d (5.7) overlapped s
7.34, 6.93, 6.93, 7.34, 1.45,
d (8.4) overlapped overlapped d (8.4) d (6.8)
7 and 8 5.08, 3.42, 7.14, 7.13, 6.78, 6.37, 6.10, 1.86, 6.96,
d (9.0) m s d (7.9) d (7.9) d (15.5) m d (5.0) s
6.87, d (7.6) 6.95, d (7.6) 1.41, d (6.4)
9 5.00, 3.33, 6.69, 6.71,
d (9.1) m s s
6.27, 6.02, 1.77, 7.10, 6.66, 6.66, 7.10, 1.25, 3.76,
d (15.6) dd (15.6, 6.5) d (6.5) d (8.3) d (8.3) d (8.3) d (8.3) d (6.7) s
5.96, s 3.86, s 3.88, s
3.88, s 3.89, s
3.82, s
The HRESIMS data of 3 exhibited a sodium adduct ion at m/z 333.1457 [M + Na]+ (calcd 333.1461), consistent with the molecular formula C20H22O3. The 1D NMR spectra of 3 were similar to those of 1, except that the methylenedioxy group in 1 was replaced by two methoxy groups [δH 3.88 (s) and 3.86 (s)] in 3. This was supported by HMBC correlations from the two methoxy carbons to C-3′ and C-4′. The absolute configuration of 3 was deduced to be the same (2R,3S) as that of 1 on the basis of their similar ECD spectra. Thus, the structure of compound 3 was defined as (2R,3S)-2,3-dihydro-2-(3,4-dimethoxyphenyl)-3-methyl-5-(E)-propenylbenzofuran. The molecular formula of 4 was established as C17H15NO4 on the basis of the sodiated HRESIMS ion at m/z 320.0880 [M + Na]+ (calcd 320.0893). The 1H and 13C NMR data of 4
Figure 1. Key HMBC (H → C) correlations of 1 and 10.
However, 2 possessed an ECD curve opposite of Kong’s compound, indicating they were enantiomers. As compound 2 shared similar Cotton effects at 233 and 263 nm with those of 1, the structure of compound 2 was assigned as (2R,3S)-2,3dihydro-2-(3,4-methylenedioxyphenyl)-7-methoxy-3-methyl-5(E)-propenylbenzofuran.
Table 2. 13C NMR (100 MHz) Data for Compounds 1−9 in CDCl3 (δ in ppm)
position 2 3 3a 4 5 6 7 7a 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3-CH3 7-OCH3 −OCH2O− 3′-OCH3 4′-OCH3
1
2
3
4
5 and 6
7 and 8
9
δC, type
δC, type
δC, type
δC, type
δC, type
δC, type
δC, type
87.9, 40.9, 132.9, 121.5, 131.4, 126.3, 109.2, 158.2, 130.8, 123.0, 18.4, 132.0, 107.0, 147.6, 147.0, 108.0, 119.8, 16.7,
CH CH C CH C CH CH C CH CH CH3 C CH C C CH CH CH3
101.0, CH2
88.6, 41.4, 134.0, 114.2, 132.3, 109.5, 144.1, 146.5, 130.9, 123.4, 18.3, 131.7, 107.1, 147.6, 147.0, 107.9, 119.8, 16.7, 56.1, 101.0,
CH CH C CH C CH C C CH CH CH3 C CH C C CH CH CH3 CH3 CH2
87.9, 40.9, 133.2, 121.6, 131.4, 126.2, 109.2, 158.2, 130.8, 123.0, 18.3, 130.6, 109.7, 148.9, 148.5, 111.0, 118.8, 16.9,
CH CH C CH C CH CH C CH CH CH3 C CH C C CH CH CH3
88.7, 40.4, 133.4, 124.6, 126.0, 128.5, 109.1, 162.5, 169.5,
CH CH C CH C CH CH C C
93.8, 44.4, 133.7, 124.6, 130.9, 133.4, 109.8, 164.6, 190.7,
CH CH C CH C CH CH C CH
131.1, 106.8, 147.7, 147.3, 108.1, 119.7, 16.6,
C CH C C CH CH CH3
131.8, 127.6, 114.2, 160.0, 114.2, 127.6, 17.9,
C CH CH C CH CH CH3
92.9, 45.2, 132.4, 120.7, 131.4, 126.3, 109.3, 158.3, 130.8, 123.1, 18.4, 133.1, 109.3, 149.3, 149.2, 111.1, 118.9, 17.7,
CH CH C CH C CH CH C CH CH CH3 C CH C C CH CH CH3
93.3, 45.2, 133.2, 113.4, 132.1, 109.3, 143.7, 146.2, 130.8, 123.3, 18.1, 131.6, 127.8, 115.4, 155.8, 115.4, 127.8, 17.5, 55.8,
CH CH C CH C CH C C CH CH CH3 C CH CH C CH CH CH3 CH3
101.1, CH2 56.1, CH3 56.1, CH3
55.3, CH3 C
55.9, CH3 56.0, CH3 DOI: 10.1021/acs.jnatprod.5b00220 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. ECD spectra of compounds 1−6 and 11.
were comparable to those of 11.10 Comparison of the NMR data of 4 with those of 11, and the shielded C-8 carbon signal at δC 169.5 in 4 as well as the presence of a nitrogen atom in its molecular formula, showed that the formyl group in 11 was replaced by the aminocarbonyl group in 4. This was further confirmed by the HMBC correlations of H-4/C-8 and H-6/ C-8. The absolute configuration of 4 was defined as the same as 1 based on their similar ECD spectra. Thus, the structure of compound 4 was defined as (2R,3S)-2,3-dihydro-2-(3,4methylenedioxyphenyl)-3-methylbenzofuran-5-carboxamide. Compounds 5 and 6 were defined as a pair of enantiomers due to their identical NMR data but opposite specific rotation ([α]20D = −108 for 5 and [α]20D = +111 for 6) and ECD curves (Figure 2). Comparing the 1D NMR data of 5/6 with those of 1110 showed that 5/6 had a 4-methoxyphenyl group [δH 3.82 (3H, s); δC 55.3] instead of the methylenedioxyphenyl group in 11, which was further confirmed by analysis of its 2D NMR data. In addition, the 1D NMR spectra revealed signals at δH 5.24 (d, J = 8.6 Hz, H-2), 3.48 (m, H-3), and 1.45 (d, J = 6.8 Hz, 3-CH3) and δC 93.8 (C-2), 44.4 (C-3), and 17.9 (3-CH3), which indicated the trans-2-aryl-3-methyldihydrobenzofuran systems of 5/6.15 The ECD curves of 5 and 6 were similar to those of (−)-conocarpan and (+)-conocarpan (15),14−16 respectively, which indicated (2R,3R) and (2S,3S) configurations of 5 and 6, respectively. Therefore, the structures of compounds 5 and 6 were assigned as (2R,3R)-2,3-dihydro-2-(4methoxyphenyl)-3-methylbenzofuran-5-carbaldehyde and (2S,3S)-2,3-dihydro-2-(4-methoxyphenyl)-3-methylbenzofuran-5-carbaldehyde, respectively. Analyses of the HRESIMS, UV, IR, NMR, specific rotation, and ECD data of compounds 7 and 8 showed that they were also enantiomers. The trans-2-aryl-3-methyldihydrobenzofuran systems of 7 and 8 were confirmed by the signals at δH 5.08 (d, J = 9.0 Hz, H-2), 3.42 (m, H-3), and 1.41 (d, J = 6.4 Hz, 3-CH3) and the signals at δC 92.9 (C-2), 45.2 (C-3), and 17.7 (3-CH3) in their 1D NMR spectra. On the basis of the ECD spectra of 5 and 6, the absolute configurations of 7 and 8 were determined as (2R,3R) and (2S,3S) due to the similar ECD curves compared to those of 5 and 6, respectively. Thus, the structures of the enantiomeric pair 7 and 8 were defined as (2R,3R)- and (2S,3S)-2,3-dihydro-2-(3,4-dimethoxyphenyl)3-methyl-5-(E)-propenylbenzofuran, respectively. Compound 9 possessed a molecular formula of C19H20O3 as determined by HRESIMS data. The 1D NMR data of 9 resembled those of (±)-conocarpan15 except for the presence of a methoxy group [δC 55.8, δH 3.76 (s)] at C-7, based on the HMBC correlation of the protons at δH 3.76 (7-OCH3) to C-7. The absolute configuration of 9 was determined to be the same as (−)-conocarpan16 on the basis of their similar ECD curves.
Therefore, the structure of compound 9 was elucidated as (2R,3R)-2,3-dihydro-2-(4-hydroxyphenyl)-7-methoxy-3-methyl5-(E)-propenylbenzofuran. The molecular formula of 10 was deduced as C21H22O5 by an HRESIMS ion at m/z 377.1357 [M + Na]+ (calcd 377.1359). The 1D NMR spectra displayed signals for a methylenedioxyphenyl [δH 5.94 (s, 2H, OCH2O), 6.77 (1H, overlapped), 6.85 (1H, d, J = 7.6 Hz), and 6.92 (1H, s); δC 101.1 (OCH2O), 107.9, 108.0, 121.2, 130.9, 147.4, and 147.6], a 4-hydoxyphenyl [δH 6.79 (2H, overlapped) and 7.22 (2H, d, J = 8.3 Hz); δC 116.4 (2 × C), 126.9 (2 × C), 131.5, and 156.8], a transpropenyl group [δH 1.85 (1H, d, J = 6.6 Hz), 6.09 (1H, dd, J = 15.7 and 6.6 Hz), and 6.33 (1H, d, J = 15.7 Hz); δC 18.4, 123.8, and 130.3], a 1,2-propanedioxy group [δH 1.28 (3H, d, J = 6.2 Hz), 4.58 (1H, m), and 5.79 (1H, m); δC 15.6, 76.1, and 77.0], and an acetyl group [δH 2.08 (3H, s); δC 21.1 and 170.0]. The HMBC correlations of H-2/C-7, H-6/C-7, H-7/C-2, H-7/ C-6, and H-8/C-1 linked the 1,2-propanedioxy group to the methylenedioxyphenyl moiety at C-1. The propenyl group was located at C-1′ of the 4-hydoxyphenyl moiety by the HMBC correlations of H-2′, 6′/C-7′, H-7′/C-2′, 6′, and H-8′/C-1′ (Figure 1). The 4-hydoxyphenyl moiety was further linked to the 1,2-propanedioxy fragment by an HMBC correlation of H8/C-4′ to establish an 8-O-4′ neolignan scaffold, and the acetyl group was located at C-7 by the HMBC correlation from H-7 to the carbonyl carbon (δC 170.0). The C-7, C-8 erythro configuration was assigned via the small coupling constant between H-7 and H-8 (J = 4.1 Hz).17 Furthermore, the positive Cotton effect at 234 nm in its ECD spectrum determined the absolute configuration of 10 as (7R,8S).18 Thus, the structure of compound 10 was defined as (7R,8S)-erythro-3,4-methylenedioxy-7-acetoxy-8-O-4′-neolignan-7′(E)-ene. The known compounds (−)-licarin B (12),19 (+)-licarin B (13),19,20 (7S,8S)-3,4-methylenedioxy-4′,7-epoxy-8,3′-neolignan-7′[E]-ene (14),10 (+)-conocarpan (15),14−16 parakmerin A (16),21 (+)-licarin A (17),20 eupomatenoid-5 (18),22 eupomatenoid-4 (19),22 eupomatenoid-7 (20),21 eupomatenoid-6 (21),22 eupomatenoid-13 (22),22 verrucosin (23),23,24 and netandrin B (24)25 were identified by comparison of their observed and reported NMR and ECD data. In addition, referring to compounds 1−4, the reported (7S,8R)-3,4-methylenedioxy-8′,9′dinor-4′,7-epoxy-8,3′-neolignan-7′-carbaldehyde (compound 310) was revised as (2R,3S)-2,3-dihydro-2-(3,4-methylenedioxyphenyl)3-methylbenzofuran-5-carbaldehyde (11). Screening for Potential Neuroprotective Compounds in HT22 Cells. In the mammalian central nervous system, a high glutamate concentration causes oxidative stress and contributes to neuronal degeneration.26 Glutamate cytotoxicity is mediated by receptor-initiated excitotoxicity and nonreceptor-mediated D
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Figure 3. Effects of compounds 1−24 on HT22 cell viability. Cells were treated with compounds 1−24 at indicated concentrations for 24 h. Cell viability was tested by the MTT assay (n = 6). #p < 0.05, ##p < 0.01, **p < 0.01 versus vehicle group.
oxidative stress.27,28 HT22 cells, an immortalized mouse hippocampal neuronal cell line, have been used as an excellent in vitro model for studying the mechanism of oxidative glutamate toxicity.29 Because HT22 cells lack functional ionotropic glutamate receptors, excitotoxicity as a cause for glutamateinduced cell death is excluded.29,30 Initially, compounds 1−24 were tested for their cytotoxicity in HT22 cells using the MTT assay. The results showed that most of the compounds had no cytotoxicity in HT22 cells at a concentration of 30 μM (Figure 3). At subtoxic concentrations (1−30 μM), compounds 17 and 20−24 showed protective effects on glutamate-induced cell death in HT22 cells. As illustrated in Figure 4, the viability of HT22 cells treated with 2 mM glutamate for 24 h was reduced by more than 60%, while the cell morphology was greatly changed compared with the untreated cells. Compounds 17 and 20−24 (1−30 μM) concentration-dependently increased the viability of cells treated with glutamate (Figure 4A), while little changes in cell morphology were observed in the groups treated with 17 and 20−24 (Figure 4B). Compounds 20 and 21 increased the MTT reduction at 30 μM, implying that these compounds might induce cell proliferation (Figure 3), but the underlying mechanisms need further experimental verification. In addition, the lactate dehydrogenase (LDH) release assay (Figure 4C) showed that compounds 17 and 20−24 decreased the levels of LDH release induced by glutamate, further suggesting these compounds had potential neuroprotective effects in HT22 cells. The structural variability of the neolignans and their neuroprotective effects roughly defined some structure−activity relationships: (a) The presence of 4-OH is a prerequisite for the activity. (b) The presence of a furan moiety generally increases the activity, as indicated by 21 vs 15 and 22 vs 9. (c) The presence of 7-OMe and 3′-OMe affects the activity in two different ways: increasing the activity in the dihydrobenzofuran neolignans (as indicated by 17 vs 15) or decreasing the activity in the benzofuran neolignans (as indicated by 20 vs 21). Compounds 17 and 20−24 Decreased ROS Generation in Glutamate-Treated HT22 Cells. Oxidative stress is associated with the accumulation of high levels of reactive oxygen species and reactive nitrogen species (RNS). The generation of excessive ROS plays a key role in the process of glutamate-induced neuronal cell death.30 To explore whether the active neolignans could affect the glutamate-mediated ROS generation in this process, H2-DCF-DA staining and fluorescence photography were employed to detect the ROS levels under different conditions. As shown in Figure 5, the green fluorescence intensity due to ROS production was increased significantly in HT22 cells exposed to 2 mM glutamate for 12 h. In contrast, the fluorescence intensity was decreased when the
cells were pretreated with 17 and 20−24 at concentrations of 30 μM. These results suggested that compounds 17 and 20−24 decreased the intracellular ROS levels in glutamate-treated HT22 cells. Effects of Compounds 17 and 20−24 on the Nrf2/ HO-1 Down-regulation Induced by Glutamate in HT22 Hippocampal Cells. Recent studies have shown that the Nrf2 pathway plays a critical role in the regulation of many antioxidative stress/antioxidant and detoxification enzyme genes. HO-1, a main down-effector of Nrf2, is an important endogenous antioxidant protein to detoxify or scavenge oxidant species in the cell to protect against oxidative stress.31−35 Thus, we tested whether the active neolignans induced the Nrf2/ HO-1 expression-mediated protective effects. As shown in Figure 6, glutamate decreased the Nrf2 and HO-1 protein levels at concentrations of 2 mM compared with the untreated group. Compounds 17 and 20−24 effectively maintained the Nrf2 and HO-1 protein levels (Figure 6B−D) down-regulated by glutamate and have no effects on the expression of Nrf2/HO-1 in cells treated without glutamate (Figure 6A). These results suggested that the compounds effectively prevented oxidative stress-induced neuronal death by maintaining the Nrf2/HO-1 pathway in HT22 cells. Compounds 17 and 20−24 Increased the Expression of Bcl-2 in Glutamate-Treated HT22 Cells. In oxidatively stressed cells, antiapoptotic Bcl-2 protein is decreased, which leads to cellular apoptosis. Recent studies have demonstrated that Nrf2-mediated up-regulation of Bcl-2 played a significant role in prevention of apoptosis and increased cell survival.7 To explore whether compounds 17 and 20−24 could affect the expression level of antiapoptotic protein, the level of Bcl-2 was analyzed by Western blotting. As shown in Figure 7, in glutamate-treated HT22 cells, the Bcl-2 level was markedly reduced. Pretreatment of cells with compounds 17 and 20−24 preserved Bcl-2 levels at the normal levels in response to glutamate, which significantly decreased the Bcl-2 protein. These results indicated that compounds 17 and 20−24 effectively suppressed oxidative stress by preserving the Bcl-2 level, thereby inhibiting glutamate-induced apoptosis. In conclusion, lignans are a group of diverse and widespread natural products with various biological activities.36 Previous studies indicated that this class of compounds may possess antioxidant activity or neuroprotective effects.37−42 However, the cellular and molecular mechanisms that underlie the action were not fully understood. In this study, we systematically investigated the chemistry of a traditional Chinese medicine, A. fordiana, which generated 24 compounds (20 neolignans and four 2-aryldihydrobenzofurans) including 10 new ones, and the neuroprotective mechanisms of the active compounds were E
DOI: 10.1021/acs.jnatprod.5b00220 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 4. Effects of compounds 1−24 on glutamate-induced oxidative neurotoxicity in HT22 cells. (A) Cells were pretreated with/without compounds 1−24 at the indicated concentrations for 30 min and then incubated with/without 2 mM glutamate for 24 h. Cell viability was tested by the MTT assay. (B) Cells were pretreated with/without compounds 17 and 20−24 at the indicated concentrations for 30 min and then incubated with 2 mM glutamate for 24 h. Photography was performed by using a photomicroscope. (C) Cells were pretreated with/without 30 μM of compounds 17 and 20−24 for 30 min and then incubated with 2 mM glutamate for 24 h. The LDH level was determined using the LDH release assay. The data are represented as mean ± SD, n = 6. *p < 0.05, **p < 0.01 versus glutamate (2 mM)-treated alone cells. ##p < 0.01 versus vehicle group. Scale bar: 25 μm.
Bcl-2 protein levels, which suggested a group of promising therapeutic agents for preventing and treating oxidative neuronal injury and degeneration, such as in Alzheimer’s disease. The expression of Nrf2 and HO-1 had no obvious change when treated alone with compounds 17 and 20−24 in HT22 cells. However, compounds 17 and 20−24 reversed the decrease of
explored. The neuroprotective effects of these compounds against glutamate-induced cell death were tested in a hippocampal neuronal cell line, HT22. The results showed that compounds 17 and 20−24 significantly reduce oxidative stress in glutamate-treated HT22 cells by inhibiting intracellular ROS overproduction and maintaining the Nrf2/HO-1 pathway and F
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Figure 5. Effects of compounds 17 and 20−24 on ROS generation in HT22 cells. Cells were pretreated with/without 30 μM of compounds 17 and 20−24 for 30 min and then exposed to 2 mM glutamate for 12 h followed by incubation with 10 μM H2-DCF-DA for 25 min. Cells were photographed using a fluorescence microscope. Scale bar: 25 μm. used for column chromatography (CC). All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Plant Material. Stems of A. fordiana were collected in March 2013 from Guangdong Province, P. R. China, and were identified by Prof. You-Kai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: TCH201304) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation. The air-dried and powdered stems of A. fordiana (3.5 kg) were extracted with 95% EtOH (3 × 10 L) at room temperature to give 197.0 g of crude extract. The extract was suspended in H2O (1 L) and successively partitioned with petroleum ether (3 × 1 L) and EtOAc (3 × 1 L) to yield two corresponding portions. The combined extract (130.0 g) was subjected to silica gel CC using PE/acetone (200:1 → 1:1) to afford five fractions (I−V). Fr. I (5.0 g) was chromatographed over RP-C18 silica gel CC with MeOH/H2O (5:5 → 10:0), followed by semipreparative chiral HPLC (CH3CN/H2O, 9:1, 3 mL/min) to give 1 (30 mg) and 14 (20 mg). Fr. II (10 g) was separated by a Sephadex LH-20 column (CHCl3/ MeOH, 1:1) and then purified on semipreparative chiral HPLC (CH3CN/H2O, 9:1, 3 mL/min) to obtain 2 (20 mg), 12 (12 mg), and 13 (15 mg). Fr. III (40 g) was subjected to MCI gel CC eluted with a MeOH/H2O gradient (3:7 → 10:0) to afford four fractions
Nrf2, HO-1, and Bcl-2 induced by glutamate. Thus, there may be other mechanisms involved in the neuroprotective effects of these compounds, which is worthy of further exploration.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter, and ECD spectra were recorded on an Applied Photophysics Chirascan spectrometer. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer with KBr disks. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS and HRESIMS were recorded on a Finnigan LC QDECA instrument. A Shimadzu LC-20AT equipped with a SPD-M20A PDA detector was used for HPLC, and a YMCpack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) was used for semipreparative HPLC separation. A chiral column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 μm) was used for chiral separation. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co. Ltd.), reversedphase C18 (RP-C18) silica gel (12 nm, S-50 μm, YMC Co. Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were G
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Figure 6. Effects of compounds 17 and 20−24 on the expression of Nrf2 and HO-1 in HT22 cells. (A) Cells were treated with or without compounds 17 and 20−24 (each 30 μM) for 12 h, and then the levels of Nrf2 and HO-1 were detected by Western blotting assay. (B) Cells untreated or treated with compounds 17 and 20−24 (each 30 μM) for 12 h were exposed to 2 mM glutamate for 30 min. Western blotting was then performed with the Nrf2 and HO-1 antibodies, respectively. (C and D) Quantitative analysis of image intensity of Western blots in (B). Each bar represents the means ± SD of three independent experiments. One-way ANOVA followed by Tukey’s test. **p < 0.01 versus glutamate-treated alone cells, ##p < 0.01 versus vehicle group. MeOH/H2O (5:5 → 10:0) followed by semipreparative chiral HPLC (MeOH/H2O, 7.5:2.5, 3 mL/min) to give 5 (12 mg), 6 (7 mg), and 11 (20 mg). Compounds 20 (20 mg) and 21 (15 mg) were obtained from Fr. IIIc4 (5.0 g) by semipreparative chiral HPLC (MeOH/H2O, 9:1, 3 mL/min). Fr. IIId (1.6 g) was chromatographed over RP-C18 silica gel CC eluted with MeOH/H2O (5:5 → 10:0) and then purified on silica gel CC (PE/EtOAc, 10:1) and semipreparative chiral HPLC (MeOH/H2O, 9:1, 3 mL/min) to give 3 (20 mg), 7 (9 mg), 8 (17 mg), and 10 (15 mg). Fr. IV (4 g) was separated by a Sephadex LH-20 column (CHCl3/MeOH, 1:1), followed by silica gel CC (PE/CHCl3, 5:1), to give 9 (500 mg), 17 (80 mg), and 22 (60 mg). Fr. V (20 g) was subjected to MCI gel CC eluted with a MeOH/H2O gradient (3:7 → 10:0), followed by RP-C18 silica gel CC eluted with MeOH/H2O (7:3 → 10:0), to afford 4 (30 mg), 23 (30 mg), and 24 (20 mg). The purity of compounds 1−24 was estimated to be greater than 95%, as determined by 1H NMR spectra (Supporting Information). (2R,3S)-2,3-Dihydro-2-(3,4-methylenedioxyphenyl)-3-methyl-5(E)-propenylbenzofuran (1): colorless oil; [α]20D −26 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 212 (4.44), 263 (4.28) nm; ECD (c 7.5 × 10−4 M, MeOH) λmax (Δε) 226 (+0.61), 261 (−0.65) nm; IR (KBr) νmax 2923, 1710, 1605, 1520, 1488, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 295.1 [M + H]+; HRESIMS m/z 295.1329 [M + H]+ (calcd for C19H19O3, 295.1329). (2R,3S)-2,3-Dihydro-2-(3,4-methylenedioxyphenyl)-7-methoxy-3methyl-5-(E)-propenylbenzofuran (2): colorless oil; [α]20D +69 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 213 (4.35), 272 (3.98) nm; ECD (c 6.8 × 10−4 M, MeOH) λmax (Δε) 233 (+0.66), 263 (−0.21), 295 (−0.17) nm; IR (KBr) νmax 2924, 1713, 1604, 1441, 1226 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 325.1 [M + H]+; HRESIMS m/z 347.1272 [M + Na]+ (calcd for C20H20O4Na, 347.1259). (2R,3S)-2,3-Dihydro-2-(3,4-dimethoxyphenyl)-3-methyl-5-(E)-propenylbenzofuran (3): colorless oil; [α]20D −2 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 208 (4.38), 260 (3.86) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε) 227 (+1.1), 262 (−0.62) nm; IR (KBr) νmax 2962, 1704, 1606, 1512, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 311.2 [M + H]+; HRESIMS m/z 333.1457 [M + Na]+ (calcd for C20H22O3Na, 333.1461).
Figure 7. Effects of compounds 17 and 20−24 on the level of Bcl-2 protein. (A) Cells untreated or treated with compounds 17 and 20−24 (each 30 μM) for 12 h were exposed to 2 mM glutamate for 30 min. The level of Bcl-2 was detected by Western blotting assay. (B) Quantitative analysis of image intensity of Western blots in (A). Each bar represents the means ± SD of three independent experiments. One-way ANOVA followed by Tukey’s test. **p < 0.01 versus glutamate-treated alone cells, ##p < 0.01 versus vehicle-treated control. (IIIa−IIId). Fr. IIIb (4.5 g) was purified by silica gel CC (PE/acetone, 100:1) to give 15 (70 mg) and 16 (400 mg). Fr. IIIc (20 g) was subjected to CC over silica gel (PE/acetone, 200:1 → 1:2) to give four fractions (IIIc1−IIIc4). Fr. IIIc2 (5.3 g) was purified by silica gel CC (PE/acetone, 50:1) to give 18 (50 mg) and 19 (70 mg). Fr. IIIc3 (6.5 g) was chromatographed by RP-C18 silica gel CC eluted with H
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(2R,3S)-2,3-Dihydro-2-(3,4-methylenedioxyphenyl)-3-methylbenzofuran-5-carboxamide (4): colorless oil; [α]20D −3 (c 0.5, MeOH); UV (CHCl3) λmax (log ε) 211 (4.38), 259 (4.07), 286 (3.88) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε) 213 (+1.28), 260 (−0.62) nm; IR (KBr) νmax 2928, 1713, 1608, 1439, 1228 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 298.1 [M + H]+; HRESIMS m/z 320.0880 [M + Na]+ (calcd for C17H15 NO4Na, 320.0893). (2R,3R)-2,3-Dihydro-2-(4-methoxyphenyl)-3-methylbenzofuran5-carbaldehyde (5): colorless oil; [α]20D −108 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.99), 229 (3,99), 295 (3.78) nm; ECD (c 14 × 10−4 M, MeOH) λmax (Δε) 234 (+0.36), 274 (−0.21) nm; IR (KBr) νmax 2927, 1703, 1600, 1513, 1456, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 269.1 [M + H]+; HRESIMS m/z 291.0997 [M + Na]+ (calcd for C17H16O3Na, 291.0992). (2S,3S)-2,3-Dihydro-2-(4-methoxyphenyl)-3-methylbenzofuran5-carbaldehyde (6): colorless oil; [α]20D +111 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.03), 229 (4.05), 297 (3.84) nm; ECD (c 15 × 10−4 M, MeOH) λmax (Δε) 235 (−0.29), 274 (+0.19) nm; IR (KBr) νmax 2926, 1707, 1601, 1450, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 269.1 [M + H]+; HRESIMS m/z 269.1173 [M + H]+ (calcd for C17H17O3, 269.1172). (2R,3R)-2,3-Dihydro-2-(3,4-dimethoxyphenyl)-3-methyl-5-(E)propenylbenzofuran (7): colorless oil; [α]20D −31 (c 0.1, MeOH); UV (CHCl3) λmax (log ε) 210 (4.43), 228 (4.28), 273 (4.00) nm; ECD (c 11 × 10−4 M, MeOH) λmax (Δε) 221 (+0.12), 243 (−0.50), 260 (−0.51) nm; IR (KBr) νmax 2928, 1700, 1604, 1514, 1240 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 311.2 [M + H]+; HRESIMS m/z 333.1461 [M + Na]+ (calcd for C20H22O3Na, 333.1461). (2S,3S)-2,3-Dihydro-2-(3,4-dimethoxyphenyl)-3-methyl-5-(E)-propenylbenzofuran (8): colorless oil; [α]20D +29 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 210 (4.40), 228 (4.21), 272 (3.99) nm; ECD (c 10 × 10−4 M, MeOH) λmax (Δε) 221 (−0.21), 241 (+0.37), 260 (+0.54) nm; IR (KBr) νmax 2927, 1717, 1600, 1515, 1238 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 311.2 [M + H]+; HRESIMS m/z 333.1452 [M + Na]+ (calcd for C20H22O3Na, 333.1461). (2R,3R)-2,3-Dihydro-2-(4-hydroxyphenyl)-7-methoxy-3-methyl-5(E)-propenylbenzofuran (9): colorless oil; [α]20D −15 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 222 (4.55), 269 (4.28) nm; ECD (c 12 × 10−4 M, MeOH) λmax (Δε) 232 (+0.47), 267 (−0.46) nm; IR (KBr) νmax 3640, 1675, 1600, 1492, 1212 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 297.1 [M + H]+; HRESIMS m/z 319.1298 [M + Na]+ (calcd for C19H20O3Na, 319.1305). (7R,8S)-erythro-3,4-Methylenedioxy-7-acetoxy-8-O-4′-neolignan7′(E)-ene (10): colorless oil; [α]20D +55 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 209 (4.38), 259 (4.33) nm; ECD (c 12 × 10−4 M, MeOH) λmax (Δε) 205 (−0.04), 233 (+0.35), 262 (+0.08), 283 (+0.04), 300 (−0.03) nm; IR (KBr) νmax 2924, 1740, 1604, 1498, 1234 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.22 (2H, d, J = 8.3 Hz, H-2′/6′), 6.92 (1H, s, H-1), 6.85 (1H, d, J = 7.6 Hz, H-6), 6.79 (2H, overlapped, H-3′/5′), 6.77 (1H, overlapped, H-5), 6.33 (1H, d, J = 15.7 Hz, H-7), 6.09 (1H, dd, J = 15.7, 6.6 Hz, H-8), 5.94 (2H, s, −OCH2O−), 5.79 (1H, m, H-7), 4.58 (1H, m, H-8), 2.08 (3H, 7-OCOCH3), 1.85 (3H, d, J = 6.6 Hz, H-9′), 1.28 (3H, d, J = 6.2 Hz, H-9); 13C NMR (CDCl3, 100 MHz) δ 170.0 (C, 7-OCOCH3), 156.8 (C, C-4′), 147.6 (C, C-3), 147.4 (C, C-4), 131.5 (C, C-1′), 130.9 (C, C-1), 130.3 (CH, C-7′), 126.9 (CH × 2, C-2′/6′), 123.8 (CH, C-8′), 121.2 (CH, C-6), 116.4 (CH × 2, C-3′/5′), 108.0 (CH, C-5), 107.9 (CH, C-2), 101.1 (CH2, −OCH2O−), 77.0 (CH, C-7), 76.1 (CH, C-8), 21.1 (CH3, 7-OCOCH3), 18.4 (CH3, C-9′), 15.6 (CH3, C-9); ESIMS m/z 295.1 [M − CH3COOH]−; HRESIMS m/z 377.1357 [M + Na]+ (calcd for C21H22O5Na, 377.1359). Biological Assay Materials. Glutamate was purchased from Research Biochemicals International (Natick, MA, USA). Trypsin, DMSO, 2′,7′-dichlorofluorescin diacetate (H2-DCF-DA), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS)
were obtained from Gibco-BRL (Grand Island, NY, USA). Compounds 1−24 were dissolved in DMSO as a 1000-fold final concentration and kept at −20 °C without light. A stock solution of the glutamate was prepared as a culture medium with a 20-fold final concentration. Cell Culture and Treatment. The HT22 murine hippocampal neuronal cells were maintained in DMEM supplemented with 10% (v/v) FBS and incubated at 37 °C under 5% CO2. To study the toxicity and protective effects of compounds 1−24 on glutamateinduced neuronal death in HT22 cells, cells were seeded in 96-well plates (10 000 cells/well), and six wells were used for each treatment group. HT22 cells were pretreated with compounds 1−24 for 30 min before exposure to glutamate and kept for 12 or 24 h unless stated otherwise. The group treated with 0.1% (v/v) DMSO was the vehicle control. MTT Assay. HT22 cells (10 000 cells/well) were seeded into 96-well plates. After 24 h incubation, cells were treated with different concentrations of compounds. Following incubation, cell growth was measured at indicated time points by addition of 10 μL of MTT (5 mg/mL) at 37 °C for 2 h, and DMSO (100 μL) was added to dissolve the formazan crystals. Optical density was measured using a microplate reader (Bio-Tek, USA) at 570 nm, and all data were represented as fold increase relative to the untreated control. LDH Release Assay. The release of lactate dehydrogenase in the culture medium was determined using a commercially available kit (Jiancheng Biochemical, Nanjing, China), as per the manufacturer’s protocol. Briefly, supernatant per well was transferred into a 96-well microplate to determine LDH levels by adding reaction mixture to each sample and incubating for 30 min at room temperature. Optical density was measured using a microplate reader (Bio-Tek, USA) at 450 nm, and all data were represented as percentage increase relative to the untreated control. Measurement of ROS. Intracellular production of ROS was measured by fluorescence using H2DCF-DA. Briefly, after treatment, cells were washed with phosphate buffer saline (PBS, pH 7.4) and then stained with 10 μM H2-DCF-DA in serum-free medium for 25 min at 37 °C in the dark. The cells were photographed using a fluorescence microscope (Olympus, Japan), washed with PBS, and extracted with 1% Triton X-100 in PBS for 10 min at 37 °C. Fluorescence was recorded with an excitation wavelength of 490 nm and an emission wavelength of 525 nm for H2-DCF-DA by a Flex Station 3 fluorimetric plate reader (Molecular Devices, Sunnyvale, CA, USA). Values were expressed as a percentage of the fluorescence relative to the untreated control. Western Blotting Analysis. Western blotting analysis was performed as previously described.43 Briefly, proteins were harvested in a cell lysis buffer. Equal amounts of lysate protein (20 μg/lane) were subjected to SDS-PAGE with 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Nitrocellulose blots were first blocked with 1% milk in TBST buffer (PBS with 0.01% Tween 20, pH 7.4) and incubated overnight at 4 °C with primary antibodies (see Table S1 in the Supporting Information) in TBST containing 1% milk. Immunoreactivity was incubated by horseradish peroxidase-conjugated secondary antibodies (see Table S1) and detected by the enhanced chemiluminescence technique. The intensities of bands were performed using Quantity One software (Bio-Rad, Hercules, CA, USA). Two bands were counted when analyzing the intensities of Nrf2 in the Western blotting assay.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00220. 1D and 2D NMR spectra of 1−10, 1D NMR spectra of 11−24, CD spectra of 7−10, and the information on antibodies used in the Western blotting experiments (PDF) I
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AUTHOR INFORMATION
Corresponding Author
*E-mail (S. Yin):
[email protected]. Tel/Fax: +86-20-39943090. Author Contributions ⊥
G.-H. Tang and Z.-W. Chen contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Nos. 81102339 and 81402813), Guangdong Natural Science Foundation (No. S2011040002429), the Fundamental Research Funds for the Central Universities (No. 14ykpy10), and the Priming Scientific Research Foundation for the Junior Teachers of Medicine in Sun Yat-sen University (Nos. 3600018801042 and 36000-31101400) for providing financial support for this work.
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