Bioorganic Chemistry 85 (2019) 469–474
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Chiral resolution and neuroprotective activities of enantiomeric dihydrobenzofuran neolignans from the fruit of Crataegus pinnatifida
Rui Guoa, Tian-Ming Lva, Feng-Ying Hana, Bin Linb, Guo-Dong Yaoa, Xiao-Bo Wangc, ⁎ ⁎ Xiao-Xiao Huanga,c, , Shao-Jiang Songa, a
School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China b School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China c Chinese People′s Liberation Army 210 Hospital, Dalian 116021, People’s Republic of China
Keywords: Crataegus pinnatifida Enantiomers Neuroprotective activities Apoptosis SH-SY5Y
Three pairs of enantiomeric dihydrobenzofuran neolignans (1a/1b-3a/3b) including four new compounds (1a/ 1b and 2a/2b) were isolated from the fruit of Crataegus pinnatifida. Their structures including the absolute configurations were elucidated by extensive spectroscopic analyses and comparison between the experimental measurements of electronic circular dichroism (ECD) and the calculated ECD spectra. Additionally, all the enantiomeric neolignans were investigated for their neuroprotective activities against H2O2-induced cell injury in human neuroblastoma SH-SY5Y cells. It was found that enantiomers 1a and 1b displayed different degrees of neuroprotective activities, and the results showed enantioselectivity, in which that 1b exhibited noticeable neuroprotective activity, while its enantiomer 1a only exhibited obvious protective effect at lower concentration. Further study demonstrated that the potential protective activities of compounds appeared to be mediated via suppressing cell apoptosis.
1. Introduction Crataegus pinnatifida, is a member of Rosaceae family. This species is widely distributed in Asia, Europe and North America . Its fruits have long been used in traditional Chinese medicine and European herbal medicine, and are widely consumed as food, in the form of juice, drink, jam and canned fruit . Previous phytochemical investigations on C. pinnatifida have resulted in the isolation of a variety of secondary metabolites, which mainly include flavonoids, triterpenoids, steroids, phenylpropanoids and organic acids [3–6]. Many of these compositions have a wide range of biological and pharmacological activities, including antioxidative, free radical scavenging, anti-inflammatory, anticancer, vasorelaxing, and hypolipidemic activities . In the course of our continuing search for biologically active and structurally diverse metabolites from the fruit of C. pinnatifida, three pairs of dihydrobenzofuran neolignans (1a/1b-3a/3b) including four new compounds (1a/1b and 2a/2b) were isolated. The enantioseparations of these compounds were achieved by chiral chromatographic column, and their structures including the absolute configurations were elucidated by extensive spectroscopic analyses and
ECD calculations. In addition, the neuroprotective activities of these enantiomers against H2O2-induced oxidative injury and the possible protective mechanism in human neuroblastoma SH-SY5Y cells were investigated. 2. Materials and methods 2.1. Plant material The fruit of C. pinnatifida was collected in October 2016 from Hebei province, China. Professor Jin-Cai Lu identified this plant material (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (No. 20170402) has been deposited in the Herbarium of Shenyang Pharmaceutical University. 2.2. General experimental procedures HRESIMS data were acquired using a Micro Q-TOF spectrometer (Bruker Daltonics, Billerica, USA). The UV spectra were recorded on a
⁎ Corresponding authors at: School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China (X.-X. Huang and S.-J. Song). E-mail addresses: [email protected]
(X.-X. Huang), [email protected]
https://doi.org/10.1016/j.bioorg.2019.02.018 Received 22 December 2018; Received in revised form 19 January 2019; Accepted 6 February 2019 Available online 08 February 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.
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Shimadzu UV-1700 spectrometer (Shimadzu, Tokyo, Japan). The optical rotations were measured with a JASCO DIP-370 digital polarimeter (JASCO, Tokyo, Japan) at 20 °C. ECD spectra were obtained on a Bio-Logic MOS 450 spectrometer (Bio-Logic Science Instruments, Seyssinet-Pariset, France). NMR experiments were performed on Bruker ARX-400 and AV-600 spectrometers in CDCl3 (δH 7.26 and δC 77.2), with TMS as the internal standard. Chromatographic silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), reversed phase C18 (RP-C18) silica gel (50 μm, Japan) were employed for column chromatography. RP-HPLC separations were conducted using a Shimadzu LC-20AR series pumping system equipped with an SPD-20A UV detector and performed with a YMC Pack ODS-A column (250 × 10 mm, 5 μm, YMC Company, Kyoto, Japan). Chiral separations were conducted in a Chiralpak IC column and a Chiralpak IG column (250 × 4.6 mm, 5 μm) from Daicel Chemical Industries, Ltd. (Japan). MTT (Sigma-Aldrich Co., Ltd, St. Louis, USA) assays were performed on a Varioskan Flash Multimode Reader (Thermo Scientific Co. Ltd, Massachusetts, USA). Annexin V-FITC and propidium iodide was purchased from Bimake (Houston, USA). The apoptotic analysis was carried out using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). The cells were analyzed with a BD FACS CantoTM flow cytometer (BD Biosciences, New Jersey, USA).
Table 1 1 H (400 MHz) and CDCl3. Position
C (100 MHz) NMR data for compounds 1a/1b-2a/2b in
1a/1b δH (multi, J in Hz)
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 3-OCH3 3′-OCH3
2.3. Extraction and isolation Air-dried fruit of C. pinnatifida (50 kg) were extracted with 70% EtOH under reflux three times. The EtOH extract (3800 g) was partitioned sequentially with ethyl acetate and n-BuOH. The EtOAc extract (600 g) was subjected to a silica gel column and eluted with CH2Cl2MeOH (100:1–5:1, v/v) of increasing polarity to produce 4 fractions (Fr. A-D). Fr. A was subjected to column chromatography on HP-20 macroporous resin with H2O, 50% and 90% EtOH to provide two fractions (Fr. A1 and Fr. A2). Fr. A1 (40 g) and Fr. A2 (80 g) were separated by ODS column chromatography, eluted with a gradient of MeOH-H2O from 20:80 to 90:10, respectively, and then redistributed in five fractions (Fr. 1–5) on the basis of silica gel TLC analysis. Fr. 5 (10 g) was further purified by a silica gel CC using petroleum ether/EtOAc (v/ v, 50:1 to 0:1) and CH2Cl2/MeOH (v/v, 20: 1 to 3:1) to yield Fr. 5.1–5.8. Fr. 5.6.1–5.6.6 were obtained from Fr.5.6 by preparative HPLC (MeOH/H2O, 60:40). Compound 1 (8.0 mg, tR 44.7 min) was obtained from fraction 5.6.3 by semipreparative HPLC eluted with CH3CN-H2O (43:57). Fraction 5.6.2 was purified in the same way as Fr. 5.6.3 to obtain 2 (15.8 mg, tR 38.3 min). In a similar manner, compound 3 (12 mg, tR 52.0 min) was isolated from Fr. 5.6.1 by RP-HPLC (MeOH/ H2O, 52:48). Compound 1 was eluted by Daicel Chiralpak IC column (n-hexane/ isopropanol, v/v, 1:1, flow rate 0.5 ml/min) to give 1a (5.0 mg, tR 49.7 min) and 1b (3.0 mg, tR 73.4 min). 2a (2.7 mg, tR 22.4 min) and 2b (2.8 mg, tR 24.6 min) were obtained using Daicel Chiralpak IG column (n-hexane/isopropanol, v/v, 4:1, flow rate 0.5 ml/min). Similarly, compound 3 was further separated by using Daicel Chiralpak IG column (CH3CN/H2O, v/v, 52:48, flow rate 0.6 ml/min) to yield 3a (2.8 mg, tR 7.7 min) and 3b (3.0 mg, tR 8.5 min). Crataegusin A (1): Yellow oil; [ ]20 D +15.2 (c 0.10, MeOH); UV (MeOH) λmax (logε): 277 nm (3.77), 230 nm (3.99); The 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HRESIMS (m/ z): 455.1301 [M + Na]+ (calcd for C22H24O9Na, 455.1313). (+)-Crataegusin A (1a): [ ]20 D +21.5 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 213 (+20.86), 228 (−12.60), 282 (+11.79) nm. (−)-Crataegusin A (1b): [ ]20 D −22.5 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 211 (−15.31), 228 (+16.44), 281 (−16.08) nm. Crataegusin B (2): Yellow oil; [ ]20 D +1.3 (c 0.10, MeOH); UV (MeOH) λmax (logε): 280 nm (2.92), 230 nm (3.59); The 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HRESIMS (m/ z): 411.1430 [M + Na]+ (calcd for C21H24O7Na, 411.1414). (+)-Crataegusin B (2a): [ ]20 D +24.0 (c 0.10, MeOH); ECD (MeOH)
6.89, overlapped 6.89, 6.89, 5.68, 3.67, 3.96, 3.94,
overlapped overlapped d (7.1) m dd (12.1, 5.6) m
7.60, br s
7.67, br s 4.80, d (15.9) 4.85, d (15.9) 4.26, 1.31, 3.86, 3.94,
q (7.1) t (7.1) s s
2a/2b δC 132.4 108.8 146.9 146.1 114.6 119.7 89.4 53.1 63.9 122.7 114.2 144.3 153.3 128.2 119.5 165.8 61.3 168.2 61.6 14.3 56.1 56.2
δH (multi, J in Hz) 6.93, d (1.8) 6.87, 6.90, 5.56, 3.62, 3.96, 3.90,
d (8.1) dd (8.1, 1.8) d (7.5) m dd (11.1, 5.8) overlapped
6.75, br s
6.75, br s 3.56, s
δC 133.1 109.0 146.8 145.8 114.4 119.6 88.2 53.9 64.0 127.4 117.3 144.4 147.7 128.1 113.5 41.2 172.2
4.16, q (7.1) 1.27, t (7.1)
3.87, s 3.89, s
λmax (Δε) 229 (−6.07), 244 (+7.58), 293 (+2.64) nm. (−)-Crataegusin B (2b): [ ]20 D −20.0 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 222 (+8.33), 244 (−11.49), 289 (−5.45) nm. 2.4. Computational methods A MMFF94 conformational search generating low-energy conformers within a 3.0 kcal/mol energy window was performed using CONFLEX [8,9]. Quantum mechanically calculations based on density functional theory (DFT) were carried out to optimize the atomic structures at the B3LYP/6-31G(d) level with the Gaussian 09 package . Electronic transition and rotational strength were calculated using the TDDFT method at the B3LYP/6-311++G(2d,p) level. The solvent effect in MeOH solution was considered during all calculations using the polarizable continuum model. Finally, the overall theoretical ECD curves were simulated using SpecDis 1.51  software on the basis of the Boltzmann weighting of each conformer. 2.5. Cell culture Human neuroblastoma SH-SY5Y cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SH-SY5Y cells were cultured in DMEM, supplemented with 10% FBS, 10 μg/mL streptomycin, 100 U/ml penicillin and maintained at 37 °C with 5% CO2 at a humidified atmosphere. Cells were cultured in 96-well plates at a density of 1.2 × 104 cells/well in 100 μL for 24 h. 2.6. Determination of cell viability using MTT assay The cell viability was evaluated by MTT assay. The SH-SY5Y cells were incubated with different concentrations of tested compounds (12.5, 25, 50 μM) for 1 h. To induce oxidative damage, 200 μM newlyprepared H2O2 was prepared and added to the cells for another 4 h. Then, 20 μL MTT (5 mg/mL) was added to each well for 4 h, and the crystals were dissolved in DMSO. The absorbance was detected with a microreader at 490 nm. The cell viability was expressed as the percentage, compared with the value of the control group (100%). 470
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degrees of unsaturation. The 1H and 13C NMR spectroscopy data (Table 1) showed resonances for three methyls, three methylenes, two methines, one ester carbonyl and twelve aromatic carbons (five of which were protonated). Analysis of the NMR data of 2 revealed the presence of the same dihydrobenzofuran moiety as crataegusin A (1). The only difference between 2 and 1 was the absence of an esteryl group in 2. This information was confirmed by the correlations of H-2′/ H-6′ to C-7′ and H-7′/H-9′ to C-8′ (Fig. 2). Based on these data, as well as the HMBC spectrum, the structure of compound 2 was determined as shown in Fig. 1, and the material was named crataegusin B. The relative configuration of 2 was assigned to be the same as that of 1 based on the interpretation of the NOESY correlation (H-7/H-9b) and 1H–1H coupling constants (J7,8 = 7.5 Hz). The negligible optical activity, together with an ECD spectrum devoid of Cotton effects, indicated that 2 was a racemic mixture. The chiral HPLC separation of rac-2 using a Chiralpak IG column revealed the presence of a pair of enantiomers, 2a and 2b, with opposite Cotton effects (Fig. 4) and op20 posite optical rotations (2a: [ ]20 D +24.0; 2b: [ ]D −20.0). To determine their absolute configurations, the ECD spectra of 2a and 2b were measured in MeOH and compared with the calculated ECD spectra of the enantiomers. The experimental ECD spectrum of 2b was consistent with the calculated ECD spectrum of 7S,8R-2. Thus, the absolute configurations of (+)-2 and (−)-2 were elucidated as 7R,8S and 7S,8R, respectively. A detailed comparison of the NMR data of 3 with those of literature values revealed that 3 was a known dihydrobenzofuran-type neolignan, 3-[2-(4-hydroxy-3-methoxypheny)-3-(hydroxymethyl)-7-methoxy-2,3dihydro-1-benzofuran-5-yl]propyl acetate . The optical rotation value and ECD spectrum indicated that 3 was also a racemic mixture. 20 After chiral separation, 3a ([ ]20 D −7.0) and 3b ([ ]D +8.5) were yielded. The absolute configurations of 3a/3b were determined more precisely by ECD calculation. The ECD spectra of 3a and 3b were measured in MeOH and compared with the calculated ECD spectra of the enantiomers (Fig. 4). Thus, their absolute configurations were assigned as 7S,8R and 7R,8S, respectively. Oxidative stress is known to be a major cause of the pathogenesis associated with various neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease and stroke . Hydrogen peroxide (H2O2) can induce apoptosis in many cells especially in neuronal cells, which is commonly used in studying neuronal cell death caused by oxidative stress . Previous studies showed the SH-SY5Y cell line is a good model for studying the mechanisms of neurodegeneration induced by H2O2 . Based on the above results, all the isolated compounds 1a/1b-3a/3b were evaluated for their neuroprotective effects against H2O2 induced oxidative injury in SH-SY5Y cells with Trolox as the positive control, the cell viability was determined by MTT assay and shown in Fig. 5. The results showed that 200 μM H2O2 could significantly reduce the cell viability compared with the control group. In addition, enantiomers 1a and 1b displayed different degrees of neuroprotective activities, while enantiomers 2a/2b-3a/3b exhibited no obvious protective effect in the bioassay. Among them, compound 1b exhibited significant neuroprotective activity towards damaged SHSY5Y cells induced by H2O2, which was more potent than Trolox at various concentrations (12.5, 25 and 50 μM). While its enantiomer 1a significantly increased the viability of SH-SY5Y cells at lower concentration (12.5 μM), in particular when the concentration of the compound 1a was up to 50 μM, negative protective effect was observed. These results highlighted the fact that the variety of the stereochemistry had great effects on the neuroprotective activity. To further evaluate the apoptosis-inhibiting effect of compound 1a/ 1b in SH-SY5Y cells, the Annexin V/PI staining assay was performed using flow cytometry to quantify the percentages of apoptotic cells in the total cell population. It can be seen that significant apoptosis occurred in model group and the apoptosis ratio reached to 21.38% compared to the control group (5.11%). As shown in Fig. 6, enantiomers 1a/1b markedly decreased the apoptotic ratio in H2O2-
2.7. Flow cytometry analysis of apoptosis ratio Annexin V-FITC and PI double staining was applied to evaluate apoptotic ratio according the manufacturer’s instructions. Briefly, SHSY5Y cells were seeded into 6-well plate at a density of 4.5 × 105 cells/ well. 5 μL AnnexinV-FITC and 5 μL PI solution were added to 500 μL of the cells, which were incubated in the dark for 15 min at room temperature. To quantify apoptotic ratio, cell samples were analyzed by FACScan flow cytometry (BD, Franklin Lakes, USA). 2.8. Statistical analysis All the presented data and results were conducted and expressed as the mean ± standard deviation in at least three independent experiments. Statistics were analyzed by Student’s t test using GraphPad software. P < 0.05 is considered statistically significant. 3. Results and discussion Compound 1 was obtained as a yellowish oil. Its molecular formula was determined to be C22H24O9, as elucidated by (+)-HRESIMS m/z 455.1301 [M + Na]+ (calcd for C22H24O9Na, 455.1313) and 13C NMR data, requiring eleven degrees of unsaturation. Its 1H NMR spectrum (Table 1) displayed two sets of aromatic protons [δ 6.89 (1H, overlapped, H-2), 6.89 (1H, overlapped, H-5), 6.89 (1H, overlapped, H-6)] and [δ 7.60 (1H, br s, H-2′), 7.67 (1H, br s, H-6′)] arising from a 1,3,4trisubstituted and an asymmetrical 1,3,4,5-tetrasubstituted aromatic ring system, respectively, an oxygenated ethyl group [δ 4.26 (2H, q, J = 7.1 Hz, H-10′), 1.31 (3H, t, J = 7.1 Hz, H-11′)], an oxygenated methylene [δ 4.80 (1H, d, J = 15.9 Hz, H-8′a), 4.85 (1H, d, J = 15.9 Hz, H-8′b)], two methoxy groups [δ 3.86 (3H, s), 3.94 (3H, s)]. There were also typical proton signals at δ 5.68 (1H, d, J = 7.1 Hz, H7), 3.67 (1H, m, H-8), 3.96 (1H, dd, J = 12.1, 5.6 Hz, H-9a), 3.94 (1H, m, H-9b) that accounted for the CH(O)eCHeCH2(O) unit. The 13C NMR and HSQC spectra revealed 22 carbons, corresponding to three methyls, three methylenes, two methines, two ester carbonyl and two benzene rings. With the aid of HSQC and HMBC experiments, all the 1H and 13C NMR signals of 1 were assigned and shown in Table 1. The proton signals at δ 5.68 and 3.67, as well as the carbon signals at δ 89.4 and 53.1 were characteristic signals of dihydrobenzofuran-type neolignans. This prediction was further confirmed by the HMBC correlations from H-7 to C-2/C-6/C-9/C-4′/C-5′ and H-8 to C-6′. The HMBC correlations of H-2′, 6′, 8′/C-7′ and H-8′, 10′/C-9′ required the linkage of two ester carbonyl (δC 165.8, 168.2) via C-8′. In addition, HMBC correlations for CH3O-3/C-3, CH3O-3′/C-3′ revealed that these methoxy groups were located at C-3, C-3′, respectively (Fig. 2). Thus, the planar structure of 1 was established and named crataegusin A. The coupling constant between H-7 and H-8 (J7,8 = 7.1 Hz) of 1 suggested that the preferred conformation of the two protons was trans [12,13], which was further supported by the NOESY correlation of H-7 with H-9b (Fig. 3). Considering the potential chiral nature of dihydrobenzofuran derivatives [14–16], an effort was proceeded to obtain optical pure compounds. Subsequently, 1 was resolved into a pair of 20 enantiomers, 1a ([ ]20 D +21.5) and 1b ([ ]D −22.5), in a ratio of 2:1 by HPLC using a Chiralpak IC column (see Supporting Information). The ECD spectra of (+)-1 and (−)-1 displayed similar signal intensity but opposite Cotton effects, confirming their enantiomeric relationship (Fig. 4). The absolute configurations of the two enantiomers of 1 were determined by the ECD spectra coupled with the quantum chemical ECD calculation in Gaussion 09 software. The predicted ECD spectra of 7S,8R-1 revealed good agreement with the experimental one of (+)-1 (Fig. 4). Therefore, compounds 1a and 1b were determined as (+)-(7S,8R)-crataegusin A and (−)-(7R,8S)-crataegusin A (Fig. 1), respectively. Compound 2 had the molecular formula C21H24O7 as assigned by the HRESIMS (m/z 411.1430 [M + Na]+, calcd as 411.1414) with ten 471
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Fig. 1. The structures of compounds 1a/1b-3a/3b.
Fig. 2. Key HMBC correlations of compounds 1–2.
Fig. 3. Key NOESY correlations of compounds 1–2.
treated cells at 12.5 μM. When the concentration of the compounds were up to 50 μM, 1b was able to reduce the H2O2-induced apoptosis rate from 21.38% to 14.48%, while 1a had no obvious effect on the apoptotic ratio in H2O2-treated cells. Consistent with the MTT results, enantiomers 1a and 1b exhibited significant enantioselective protective effects against H2O2-induced neurotoxicity at 50 μM. Taken together, enantiomers 1a/1b exerted neuroprotective effects against H2O2-
induced SH-SY5Y cellular damage by inhibiting apoptosis. 4. Conclusion In the present study, three pairs of dihydrobenzofuran neolignans (1a/1b-3a/3b) including four new compounds (1a/1b and 2a/2b) were isolated from the fruit of C. pinnatifida. The enantioseparations of 472
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Fig. 4. Experimental and calculated ECD spectra for 1a/1b-3a/3b in MeOH. Fig. 5. Neuroprotective effects of compounds 1a/ 1b-3a/3b against H2O2-induced cell growth inhibition of SH-SY5Y cells. In the presence or absence of the tested compounds at different concentrations (12.5, 25, and 50 μM), the MTT assay was used to examine the cell viability after H2O2 (200 μM) treatment for 4 h. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. the H2O2 treated group. &&&p < 0.001 was considered statistically significant when compared to its enantiomer.
these compounds were achieved using chiral chromatographic column, and their structures including the absolute configurations were elucidated by extensive spectroscopic analyses and ECD calculations. In addition, the neuroprotective activities of these enantiomers against H2O2-induced oxidative injury in human neuroblastoma SH-SY5Y cells
were investigated. Biologically, enantiomers 1a and 1b exhibited significant enantioselective protective effects against H2O2-induced neurotoxicity at 50 μM. Further study revealed that the potential protective activities of compounds appeared to be mediated via suppressing cell apoptosis. Overall, this functional food contains promising candidates 473
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Fig. 6. Effects of enantiomers 1a and 1b on the apoptosis ratio in H2O2-treated SH-SY5Y cells. The cells were pretreated with enantiomers 1a and 1b and then incubated with H2O2 for 4 h. Flow cytometry was used to examine the apoptotic ratio after Annexin V-FITC/PI staining. The percentage of apoptotic cells was calculated in the right. **p < 0.01 vs. the control group. #p < 0.05 vs. the H2O2-treated group.
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