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Identification, characterization and in vitro neuroprotection of N6-(4-hydroxybenzyl) adenine riboside and its metabolites
Phytochemistry Letters 20 (2017) 146–150
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Identification, characterization and in vitro neuroprotection of N6-(4hydroxybenzyl) adenine riboside and its metabolites
MARK
⁎
Chunlan Tanga,b, , Jialing Wanga, Jie Yua, Li Wangb, Mengchun Chengb, Wei Cuia, ⁎⁎ ⁎⁎ Jinshun Zhaoa, , Hongbin Xiaoc, a Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, Medical School of Ningbo University, Ningbo, Zhejiang 315211, China b Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China c Beijing University of Chinese Medicine, Beijing 100029, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: N6-(4-hydroxybenzyl) Adenine Riboside (PubChem CID: 10474479) 4-hydroxylbenyzlamine hydrochloride (PubChem CID: 16214988) 6-chloropurine (PubChem CID: 5359277) 6-chloropurine riboside (PubChem CID: 93003) 3,4-dihydroxybenzylamine hydrochloride (PubChem CID: 13343563)
N6-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from Gastrodia elata Blume, has been demonstrated to show great pharmacological effects. The present study aimed to synthesize and identify the metabolites of NHBA, and to determine their neuroprotective potentials in vitro. After incubation with rat liver microsomes in the presence of NADPH, two metabolites were detected, which were further semisynthesized and identified as N6-(4-hydroxylbenzyl) purine (NHBP) and N6-(3,4-dihydroxylbenzyl) adenine riboside (ONHBA) by UPLCQTOF-MS, 1H NMR and 13C NMR. Furthermore, the neuroprotective activities of NHBA and two metabolites were evaluated in SH-SY5Y cells. Our results demonstrated that NHBA substantially protected against H2O2induced neuronal death in SH-SY5Y cells. Moreover, both ONHBA and NHBP could significantly prevent Aβ oligomers- and H2O2-induced neuronal death in SH-SY5Y cells. These results suggested that NHBA and its metabolites, ONHBA and NHBP, might be suitable for the development of new drugs in the treatment of neurodegenerative diseases, including Alzheimer’s disease in particular.
Keywords: N6-(4-hydroxybenzyl) adenine riboside Metabolism Neuroprotection
1. Introduction Gastrodia elata Blume (GE), has been extensively recognized in China and other Asian countries as an important traditional Chinese medicine for the treatment of convulsion (Shin et al., 2011), dizziness (Ramachandran et al., 2012), paralysis, epilepsy (Ojemann et al., 2006), and asthma and immune dysfunctions (Hwang et al., 2009; Jang et al., 2010). Recently, neuroprotective activity of GE was attracting more attentions. GE extract, gastrodin and p-hydroxybenzylalcohol (HBA) have been shown to protect neuronal cells and recover brain function by correction of neurotransmitter imbalance and inhibition of oxidative response and neuroinflammation in various in vivo and in vitro models of neurodegenerative disorders (Jang et al., 2015). Except for gastrodin and HBA, other compounds of GE also exert neuroprotective activity (Huang et al., 2016; Li et al., 2016). For example, vanillin could ameliorate trimethyltin-induced seizures by reduction neuronal death and microglial activation (Jang et al., 2015). p-Hydroxybenzaldehyde could significantly reduce neuronal death in
the hippocampal CA1 region of Mongolian gerbils following with transient global ischemia (Kim et al., 2007). These results encourage the extraction and identification of novel components from GE for the treatments of neurodegenerative disorders. N6-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from GE at trace level (Wang, 2007), had been suggested as a potential neuroprotectant and candidate in the therapeutic use against neurodegenerative disorders with the experiment of preventing serum-deprived PC12 cell apoptosis (Huang et al., 2011). The protective effects of NHBA are incredibly higher than gastrodin and parishin, the major constituents in GE (Huang et al., 2007). However, to the best of our knowledge, the material basis and action mechanism of NHBA in vivo are still unclear, the neuroprotective protential of NHBA might be attributed to the metabolites of NHBA in vivo. Our previous studies have reported the metabolism of NHBA in rat plasma (Lei et al., 2011; Tang et al., 2015). One phase I metabolite of NHBA in rat plasma was detected. Given that the amount of metabolites in plasma was near the limit of detection, some metabolites might not be detected. Moreover, it is necessary to
⁎ Corresponding author at: Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, Medical School of Ningbo University, Ningbo, Zhejiang, 315211, China. ⁎⁎ Corresponding authors. E-mail addresses: [email protected] (C. Tang), [email protected] (J. Zhao), [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.phytol.2017.04.035 Received 26 September 2016; Received in revised form 18 February 2017; Accepted 24 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 20 (2017) 146–150
C. Tang et al.
Fig. 1. The MS/MS spectrum of NHBA and two metabolites.
NADPH for 1 h, two main metabolites (M1 and M2) were formed by RLMs and the formation rate of metabolites followed the order of M2 > M1 (Supplementary data Fig. S1). Two metabolites were tentatively identified as the mono-hydroxylated and deglycosylated metabolites of NHBA by UPLC-QTOF-MS (Fig. 1). With the positive ion ESI, M2 produced molecular ion [M+H]+ 390.1410, 15.9949 Da higher than that of NHBA, and the elemental composition C17H19N5O6 determined by accurate mass measurement all indicated that M2 might be the mono-hydroxylated metabolite of NHBA. The MS/ MS spectrum of M2 generated series of fragment ions at m/z 258.0987 and 123.0442, which were all 15.9949 Da higher than the corresponding mono-hydroxylated product ions at m/z 242.1038 and 107.0493 of NHBA. The results further verified the mono-hydroxylated process of NHBA. M1 showed the molecular ion [M+H]+ 242.1038, C12H11N5O of elemental composition and fragment ions at m/z 136.0620 and 107.0493, indicating that M1 was deglycosylated metabolite by accu-
investigate the structures of metabolites of NHBA to facilitate further pharmacological research. All these reasons encouraged us to investigate the metabolic behavior in vitro of NHBA and carry out relative pharmacological study. The present study aimed to identify the major metabolites of N6-(4hydroxybenzyl) adenine riboside in rat liver microsomes (RLMs) by UPLC-QTOF-MS, 1H NMR and 13C NMR, and determine the neuroprotective activity of NHBA and its metabolites. The successful attempt to study the NHBA and its metabolites may lead to the discovery of new neuroprotective therapeutic agents. 2. Results and discussion 2.1. Metabolism of NHBA in rat liver microsomes After NHBA (50 μg/mL) incubated with RLMs (2 mg/mL) and 147
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Fig. 2. Semisynthesis of M1 and M2.
significantly reduce cell viability in SH-SY5Y cells. All tested compounds were able to protect SH-SY5Y cells against H2O2-induced cell death, and the neuroprotective effects of three compounds were statistically significant at the concentrations of 20–40 μM (p < 0.05), respectively (Fig. 3a). ONHBA and NHBP also protected SH-SY5Y cells against Aβ oligomers-induced neurotoxicity at the concentration of 20 μM (Fig. 3b), indicating that the metabolites of NHBA exert preferable neuroprotective potentials and might be used as neuroprotectant against neurodegenerative disorders.
rate mass measurement and comparing the mass spectrum with that of the prototype. For further structure identification, two metabolites were semisynthesized via the reaction of 4-hydroxylbenyzlamine hydrochloride with 6-chloropurine and 3,4-dihydroxybenzylamine hydrochloride with 6-chloropurine riboside in the presence of DIEA, respectively. 2.2. Identification of metabolites To elucidate their exact structures and the possibly metabolic sites of NHBA in RLMs, two metabolites were semisynthesized via the reaction of 4-hydroxylbenyzlamine hydrochloride with 6-chloropurine and 3,4-dihydroxybenzylamine hydrochloride with 6-chloropurine riboside in the presence of DIEA (Fig. 2) and characterized by 1H NMR and 13C NMR. The data of 1H NMR and 13C NMR spectra of two metabolites are listed in Table 1 and Supplementary data. Compared with NMR data of NHBA (Huang et al., 2007), the 13C NMR spectrum of M2 displayed carbon signals at δ 143.9 (C-1), 114.7 (C-3), 118.1 (C-5) shifted downfield to δ 154.8, 155.2, 129.7, and δ 144.9 (C-2), 130.8 (C-4), 115.2 (C-6) shifted upfield to δ 102.7, 123.2, 108.1. The other carbon signals of M2 were similar to that of NHBA. The spectrum of M2 revealed that the proton signal of H-2 at δ 6.66 disappeared by comparison with data for NHBA, indicating that the H-2 was replaced. Taken together with the MS data, M2 was identified as N6-(3,4-dihydroxylbenzyl) adenine riboside (ONHBA). The 1H NMR and 13C NMR data of M1 showed a pattern similar to that of NHBA except that the data of glycosylated section was disappeared. Therefore, M1 was identified as N6-(4-hydroxylbenzyl) purine (NHBP).
3. Experimental 3.1. Chemicals and reagents N6-(4-hydroxybenzyl) adenine riboside was isolated and purified from the dried roots of GE in our lab, structure was confirmed by comparison of its UV, MS and NMR with literature (Huang et al., 2007). Synthetic Aβ1-42 was obtained from GL Biochem Ltd. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), NADPH and tris was purchased from Sigma Aldrich (StLouis, MO, USA). 4-Hydroxybenzylamine hydrochloride was purchased from YueXinchem Co., Ltd. (Changzhou, China). 6-Chloropurine and 6chloropurine riboside were purchased from HuaWenchem Co., Ltd. (Zhengzhou, China). 3,4-Dihydroxybenzylamine hydrochloride, diisoproplethylamine and n-propanol were obtained from J & K (Beijing, China). Acetonitrile and methanol were supplied by Burdick & Jackson (USA). All other reagents were either LC grade or of the highest grade commercially available. 3.2. Preparation of rat liver microsomes
2.3. Neuroprotective study Male Sprague–Dawley rats weighing approximately 200 g were obtained from the Laboratory Animal Center in Dalian Medical University (Dalian, China). All experimental protocols on animals were in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of China. The animals had free access to tap water and pellet diet. Livers were removed, and immediately washed with ice-cold saline, followed by weighed, minced with scissors and homogenized with 4 volumes (w/v) of ice-cold 50 mM tris–HCl (pH 7.4) buffer containing 0.25 M sucrose and 0.05 M magnesium chloride. Microsomes were prepared by differential ultracentrifugation. The protein concentration
The neuroprotective activities of NHBA and its metabolites were investigated. It is known that accumulation of extracellular amyloid plaque is considered a pathological feature of Alzheimer disease (AD) and soluble Aβ oligomers might lead to cognitive impairment. In addition, oxidative stress, which was conventionally induced by hydrogen peroxide (H2O2) in cells, is also closely related to the pathogenesis of neurodegenerative disorders. Therefore, we investigated the neuroprotective effects of NHBA and its metabolites against Aβ oligomers- and H2O2-induced neuronal death in SH-SY5Y cells. The results showed that either Aβ oligomers or H2O2 could 148
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Table 1 1 H-NMR and
13
C-NMR spectra data for NHBA, NHBP and ONHBA.
No
NHBP
1
H-NMR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
6.66 (d,J = 8.5 Hz) 7.14 (d,J = 8.5 Hz) 7.14 (d,J = 8.5 Hz) 6.66 (d,J = 8.5 Hz) 4.60 m 8.35 s
8.28 s 5.88 (d,J = 5.5 Hz) 4.60 m 4.13 m 3.95 m 3.57 m,3.63 m
13
C-NMR
156.1 s 114.9 d 128.6 d 130.8 s 128.6 d 114.9 d 42.4 t 154.4 s 152.3 d 148.4 s 120.4 s 139.8 d 88.0 d 73.5 d 70.7 d 85.9 d 61.7 t
1
13
H-NMR
6.68 (d,J = 8.1 Hz) 7.16 (d,J = 8.1 Hz) 7.16 (d,J = 8.1 Hz) 6.68 (d,J = 8.1 Hz) 4.59 m 8.17 s
8.08 s
C-NMR
156.07 s 114.87 d 128.55 d 130.41 s 128.55 d 114.87 d 42.38 t 154.23 s 152.29 d 149.69 s 118.69 s 138.63 d
1
H-NMR
6.73 (d,J = 1.8 Hz) 6.58 m 6.63 (d,J = 8.1 Hz) 4.54 s 8.35 s
8.20 s 5.88 (d,J = 5.5 Hz) 4.61 m 4.15 m 3.97 m 3.55 m,3.67 m
13
C-NMR
143.97 s 144.92 s 114.71 d 130.79 d 118.09 d 115.24 d 42.38 t 154.45 s 152.30 d 148.34 s 119.70 s 139.73 d 87.92 d 73.41 d 70.86 d 85.86 d 61.64 t
voltage of 150 V, nebulizer pressure of 45 psi and Dual ESI Vcap of 3500 V. Full-scan data acquisition in positive ion mode was performed at 50–500 m/z with a scan time of 1 s−1. Argon was used for collisionactivated dissociation experiments to produce MS/MS spectrum of selected precursor ions, and the collision energy was maintained at 30 eV. Data were managed on the MassHunter WorkStation Data Acquisition software (B.02.01 Version) and Qualitative Analysis software (B.03.01 Version).
was measured by the method of Bradford using bovine serum albumin as the standard. 3.3. Incubation system The incubation mixture with a total volume 200 μL, consisted of tris-HCl buffer (0.05 M, pH 7.4), NADPH (1 mM), MgCl2 (5 mM), RLMs (2 mg/mL), and NHBA (50 μg/mL). NHBA was previous dissolved in methanol, with a final methanol concentration below 1% (v/v) in the reaction mixture. After 10 min pre-incubation at 37 °C, the reaction was initiated by adding NADPH. Then the reaction was terminated by the addition of methanol (200 μL) after 1 h in a vapour bath vibrator. The mixture was swirled and centrifuged at 15,000 × g for 10 min. The control incubation was performed in the absence of NADPH. Aliquots of supernatants were transferred for UPLC-QTOF-MS analysis.
3.5. Synthesis of metabolites N6-(4-hydroxylbenzyl) purine: A mixture of 4-hydroxybenzylamine hydrochloride (0.3 g), 6-chloropurine (360 mg) and diisoproplethylamine (10 mL) in n-propanol (50 mL) was heated to 60 °C for 7 h under the atmosphere of nitrogen. After evaporation, the mixture was suspended with H2O to give a white precipitate, then filtered and recrystallized to obtain the desired product. N6-(3,4-dihydroxylbenzyl) adenine riboside: A mixture of 3,4-dihydroxybenzylamine hydrochloride (0.3 g), 6-chloropurine riboside (286 mg) and diisoproplethylamine (10 mL) in n-propanol (50 mL) was heated to 60 °C for 7 h under the atmosphere of nitrogen. After evaporation, the mixture was suspended with H2O to give a white precipitate, then filtered and recrystallized to obtain the desired product.
3.4. LC–MS/MS analysis An Agilent 1290 UPLC system (Agilent, Waldbronn, Germany) including a binary pump, an autosampler and diode array ultraviolet detector was used. The chromatographic separation was conducted on Agilent ZORBAX SB-C18 column (3.0 × 150 mm, 1.8 μm). The column temperature was set at 60 °C. The mobile phase consisted of 0.5% formic acid in water (A) and acetonitrile (B). Elution was kept at 10% B with 2 min, and increased linearly to 18% B with 0.2 min, then kept at 18% B with 7.8 min, and increased linearly to 30% B with 0.2 min, kept at 30% B with 4.8 min. The flow rate was set as 0.3 mL/min. 1 μL of the purified sample was injected automatically into the UPLC system for analysis. MS/MS analysis were conducted on an Agilent 6520 quadrupole–time of flight mass spectrometer (Agilent Corp, USA) equipped with electrospray ionization (ESI) interface. The samples were analyzed in positive ion mode. The flow rates of drying gas (N2) were 10 L/min. The optimum conditions were drying gas temperature of 350 °C, fragmentor
3.6. Preparation of soluble Aβ1-42 oligomers Soluble Aβ42 oligomers were prepared as described previously (Chang et al., 2015). Briefly, synthetic Aβ1-42 was dissolved in ice-cold 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP), thoroughly vortexed, and aliquoted to be frozen until use. Aβ1-42 was spin-vacuumed just prior to the experiment, dissolved in HFIP solution (final concentration: 10% (v/v) HFIP) and incubated at room temperature for 20 min. The 149
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16–20 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength. Unless otherwise indicated, the extent of MTT conversion in cells without treatment is expressed as a percentage of the control. Acknowledgements This work is financially supported by the National Natural Science Foundation of China (Grant No. 81603266) and National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (Grant No. 2014XZ09304-307). In addition, the authors also gratefully acknowledge the support of K.C. Wong Magna Fund in Ningbo University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.04.035. References Chang, L., Cui, W., Yang, Y., Xu, S., Zhou, W., Fu, H., Hu, S., Mak, S., Hu, J., Wang, Q., Ma, V.P., Choi, T.C., Ma, E.D., Tao, L., Pang, Y., Rowan, M.J., Anwyl, R., Han, Y., 2015. Protection against beta-amyloid-induced synaptic and memory impairments via altering beta-amyloid assembly by bis(heptyl)-cognitin. Sci. Rep. 5, 10256. Cui, W., Zhang, Z., Li, W., Hu, S., Mak, S., Zhang, H., Han, R., Yuan, S., Li, S., Sa, F., Xu, D., Lin, Z., Zuo, Z., Rong, J., Ma, E.D., Choi, T.C., Lee, S.M., Han, Y., 2013. The anticancer agent SU4312 unexpectedly protects against MPP(+) − induced neurotoxicity via selective and direct inhibition of neuronal NOS. Br. J. Pharmacol. 168, 1201–1214. Cui, W., Zhang, Z.J., Hu, S.Q., Mak, S.H., Xu, D.P., Choi, C.L., Wang, Y.Q., Tsim, W.K., Lee, M.Y., Rong, J.H., Han, Y.F., 2014. Sunitinib produces neuroprotective effect via inhibiting nitric oxide overproduction. CNS Neurosci. Ther. 20, 244–252. Huang, N.K., Chern, Y.J., Fang, J.M., Lin, C.I., Chen, W.P., Lin, Y.L., 2007. Neuroprotective principles from Gastrodia elata. J. Nat. Prod. 70, 571–574. Huang, N.K., Lin, J.H., Lin, J.T., Lin, C.I., Liu, E.M.W., Lin, C.J., Chen, W.P., Shen, Y.C., Chen, H.M., Chen, J.B., Lai, H.L., Yang, C.W., Chiang, M.C., Wu, Y.S., Chang, C., Chen, J.F., Fang, J.M., Lin, Y.L., Chern, Y.J., 2011. A new drug design targeting the adenosinergic system for Huntington's disease. PLoS One 6, e20934. Huang, J.Y., Yuan, Y.H., Yan, J.Q., Wang, Y.N., Chu, S.F., Zhu, C.G., Guo, Q.L., Shi, J.G., Chen, N.H., 2016. 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway. Acta Pharmacol. Sin. 37, 731–740. Hwang, S.M., Lee, Y.J., Kang, D.G., Lee, H.S., 2009. Anti-inflammatory effect of Gastrodia Elata Rhizome in human umbilical vein endothelial cells. Am. J. Chin. Med. 37, 395–406. Jang, Y.W., Lee, J.Y., Kim, C.J., 2010. Anti-asthmatic activity of phenolic compounds from the roots of Gastrodia elata Bl. Int. Immunopharmacol. 10, 147–154. Jang, J.H., Son, Y., Kang, S.S., Bae, C.S., Kim, J.C., Kim, S.H., Shin, T., Moon, C., 2015. Neuropharmacological potential of Gastrodia Elata Blume and its components. Evid. -Based Complement. Altern. 309261. Kim, H.J., Hwang, I.K., Won, M.H., 2007. Vanillin, 4-hydroxybenzyl aldehyde and 4hydroxybenzyl alcohol prevent hippocampal CA1 cell death following global ischemia. Brain Res. 1181, 130–141. Lei, Y.J., Wang, L., Cheng, M.C., Xiao, H.B., 2011. Identification of major metabolites in rat urine and plasma of N-6-(4-hydroxybenzyl) adenine riboside by LC/MS/MS. Biomed. Chromatogr. 25, 344–352. Li, Z., Wang, Q., Ouyang, H., Huang, L., Feng, Y., Wang, R., Yang, S., 2016. New compounds with neuroprotective activities from Gastrodia elata. Phytochem. Lett. 15, 94–97. Ojemann, L.M., Nelson, W.L., Shin, D.S., Rowe, A.O., Buchanan, R.A., 2006. Tian ma, an ancient Chinese herb, offers new options for the treatment of epilepsy and other conditions. Epilepsy Behav. 8, 376–383. Ramachandran, U., Manavalan, A., Sundaramurthi, H., Sze, S.K., Feng, Z.W., Hu, J.M., Heese, K., 2012. Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells. Neurochem. Int. 60, 827–836. Shin, E.J., Bach, J.H., Nguyen, T.T.L., Jung, B.D., Oh, K.W., Kim, M.J., Jang, C.G., Ali, S.F., Ko, S.K., Yang, C.H., Kim, H.C., 2011. Gastrodia Elata Bl attenuates cocaineinduced conditioned place preference and convulsion, but not behavioral sensitization in mice: importance of GABA(A) receptors. Curr. Neuropharmacol. 9, 26–29. Tang, C.L., Wang, L., Cheng, M.C., Liu, X.X., Xiao, H.B., 2015. Determination of N6-(4hydroxybenzyl) adenine riboside in rat plasma by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry. Chin. J. Chromatogr. 33, 699–703. Wang, L., 2007. Studies on Chemical Constituents and Quality Control of Gastrodia Elata. Chinese Academy of Science.
Fig. 3. Effects of NHBA, NHBP and ONHBA on the cell viability of SH-SY5Y cells. a: NHBA, ONHBA and NHBP prevent 150 μM H2O2-induced neuronal death in SH-SY5Y cells; b: ONHBA, NHBP but not NHBA prevent 1 μM Aβ oligomers-induced neuronal death in SH-SY5Y cells. SH-SY5Y cells were pre-treated with various chemicals at the indicated concentrations for 2 h, and then exposed to neurotoxins. Cell viability was measured by the MTT assay at 24 h after the challenge of neurotoxins. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 versus the Aβ oligomers-treated group (ANOVA and Dunnett’s test).
solution was centrifuged at 14,000g for 15 min at 4 °C, and the supernatant was collected. The HFIP was completely evaporated to obtain a 50 μM Aβ oligomers solution and kept at room temperature under constant stirring for 48 h. Then the tube was transferred to refrigerator and maintained at 4 °C. 3.7. SH-SY5Y cells culture SH-SY5Y cells were purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences), and cultured in high glucose modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL). The cells were cultured in an incubator at 37 °C and 5% CO2. The medium was replaced every other day. For the experiments with Aβ oligomers or H2O2, SH-SY5Y cells (1 × 105 cells/mL) in DMEM with low serum content (1% FBS) were seeded in 96-well plates. All experiments were carried out 24 h after the cells were seeded. 3.8. Measurement of cell viability Cell viability was determined by the activity of mitochondrial dehydrogenases via MTT assay as previously described (Cui et al., 2013, 2014). Briefly, after treatment, 10 μL of 5 mg/mL MTT solution was added to each well of 96-well plates. Plates were incubated at 37 °C for 4 h in a humidified incubator. 100 μL of the solvating solution (0.01 N HCl in 10% SDS solution) was then added to each well for
150
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Identification, characterization and in vitro neuroprotection of N6-(4hydroxybenzyl) adenine riboside and its metabolites
MARK
⁎
Chunlan Tanga,b, , Jialing Wanga, Jie Yua, Li Wangb, Mengchun Chengb, Wei Cuia, ⁎⁎ ⁎⁎ Jinshun Zhaoa, , Hongbin Xiaoc, a Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, Medical School of Ningbo University, Ningbo, Zhejiang 315211, China b Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China c Beijing University of Chinese Medicine, Beijing 100029, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: N6-(4-hydroxybenzyl) Adenine Riboside (PubChem CID: 10474479) 4-hydroxylbenyzlamine hydrochloride (PubChem CID: 16214988) 6-chloropurine (PubChem CID: 5359277) 6-chloropurine riboside (PubChem CID: 93003) 3,4-dihydroxybenzylamine hydrochloride (PubChem CID: 13343563)
N6-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from Gastrodia elata Blume, has been demonstrated to show great pharmacological effects. The present study aimed to synthesize and identify the metabolites of NHBA, and to determine their neuroprotective potentials in vitro. After incubation with rat liver microsomes in the presence of NADPH, two metabolites were detected, which were further semisynthesized and identified as N6-(4-hydroxylbenzyl) purine (NHBP) and N6-(3,4-dihydroxylbenzyl) adenine riboside (ONHBA) by UPLCQTOF-MS, 1H NMR and 13C NMR. Furthermore, the neuroprotective activities of NHBA and two metabolites were evaluated in SH-SY5Y cells. Our results demonstrated that NHBA substantially protected against H2O2induced neuronal death in SH-SY5Y cells. Moreover, both ONHBA and NHBP could significantly prevent Aβ oligomers- and H2O2-induced neuronal death in SH-SY5Y cells. These results suggested that NHBA and its metabolites, ONHBA and NHBP, might be suitable for the development of new drugs in the treatment of neurodegenerative diseases, including Alzheimer’s disease in particular.
Keywords: N6-(4-hydroxybenzyl) adenine riboside Metabolism Neuroprotection
1. Introduction Gastrodia elata Blume (GE), has been extensively recognized in China and other Asian countries as an important traditional Chinese medicine for the treatment of convulsion (Shin et al., 2011), dizziness (Ramachandran et al., 2012), paralysis, epilepsy (Ojemann et al., 2006), and asthma and immune dysfunctions (Hwang et al., 2009; Jang et al., 2010). Recently, neuroprotective activity of GE was attracting more attentions. GE extract, gastrodin and p-hydroxybenzylalcohol (HBA) have been shown to protect neuronal cells and recover brain function by correction of neurotransmitter imbalance and inhibition of oxidative response and neuroinflammation in various in vivo and in vitro models of neurodegenerative disorders (Jang et al., 2015). Except for gastrodin and HBA, other compounds of GE also exert neuroprotective activity (Huang et al., 2016; Li et al., 2016). For example, vanillin could ameliorate trimethyltin-induced seizures by reduction neuronal death and microglial activation (Jang et al., 2015). p-Hydroxybenzaldehyde could significantly reduce neuronal death in
the hippocampal CA1 region of Mongolian gerbils following with transient global ischemia (Kim et al., 2007). These results encourage the extraction and identification of novel components from GE for the treatments of neurodegenerative disorders. N6-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from GE at trace level (Wang, 2007), had been suggested as a potential neuroprotectant and candidate in the therapeutic use against neurodegenerative disorders with the experiment of preventing serum-deprived PC12 cell apoptosis (Huang et al., 2011). The protective effects of NHBA are incredibly higher than gastrodin and parishin, the major constituents in GE (Huang et al., 2007). However, to the best of our knowledge, the material basis and action mechanism of NHBA in vivo are still unclear, the neuroprotective protential of NHBA might be attributed to the metabolites of NHBA in vivo. Our previous studies have reported the metabolism of NHBA in rat plasma (Lei et al., 2011; Tang et al., 2015). One phase I metabolite of NHBA in rat plasma was detected. Given that the amount of metabolites in plasma was near the limit of detection, some metabolites might not be detected. Moreover, it is necessary to
⁎ Corresponding author at: Department of Preventive Medicine, Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, Medical School of Ningbo University, Ningbo, Zhejiang, 315211, China. ⁎⁎ Corresponding authors. E-mail addresses: [email protected] (C. Tang), [email protected] (J. Zhao), [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.phytol.2017.04.035 Received 26 September 2016; Received in revised form 18 February 2017; Accepted 24 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 20 (2017) 146–150
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Fig. 1. The MS/MS spectrum of NHBA and two metabolites.
NADPH for 1 h, two main metabolites (M1 and M2) were formed by RLMs and the formation rate of metabolites followed the order of M2 > M1 (Supplementary data Fig. S1). Two metabolites were tentatively identified as the mono-hydroxylated and deglycosylated metabolites of NHBA by UPLC-QTOF-MS (Fig. 1). With the positive ion ESI, M2 produced molecular ion [M+H]+ 390.1410, 15.9949 Da higher than that of NHBA, and the elemental composition C17H19N5O6 determined by accurate mass measurement all indicated that M2 might be the mono-hydroxylated metabolite of NHBA. The MS/ MS spectrum of M2 generated series of fragment ions at m/z 258.0987 and 123.0442, which were all 15.9949 Da higher than the corresponding mono-hydroxylated product ions at m/z 242.1038 and 107.0493 of NHBA. The results further verified the mono-hydroxylated process of NHBA. M1 showed the molecular ion [M+H]+ 242.1038, C12H11N5O of elemental composition and fragment ions at m/z 136.0620 and 107.0493, indicating that M1 was deglycosylated metabolite by accu-
investigate the structures of metabolites of NHBA to facilitate further pharmacological research. All these reasons encouraged us to investigate the metabolic behavior in vitro of NHBA and carry out relative pharmacological study. The present study aimed to identify the major metabolites of N6-(4hydroxybenzyl) adenine riboside in rat liver microsomes (RLMs) by UPLC-QTOF-MS, 1H NMR and 13C NMR, and determine the neuroprotective activity of NHBA and its metabolites. The successful attempt to study the NHBA and its metabolites may lead to the discovery of new neuroprotective therapeutic agents. 2. Results and discussion 2.1. Metabolism of NHBA in rat liver microsomes After NHBA (50 μg/mL) incubated with RLMs (2 mg/mL) and 147
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Fig. 2. Semisynthesis of M1 and M2.
significantly reduce cell viability in SH-SY5Y cells. All tested compounds were able to protect SH-SY5Y cells against H2O2-induced cell death, and the neuroprotective effects of three compounds were statistically significant at the concentrations of 20–40 μM (p < 0.05), respectively (Fig. 3a). ONHBA and NHBP also protected SH-SY5Y cells against Aβ oligomers-induced neurotoxicity at the concentration of 20 μM (Fig. 3b), indicating that the metabolites of NHBA exert preferable neuroprotective potentials and might be used as neuroprotectant against neurodegenerative disorders.
rate mass measurement and comparing the mass spectrum with that of the prototype. For further structure identification, two metabolites were semisynthesized via the reaction of 4-hydroxylbenyzlamine hydrochloride with 6-chloropurine and 3,4-dihydroxybenzylamine hydrochloride with 6-chloropurine riboside in the presence of DIEA, respectively. 2.2. Identification of metabolites To elucidate their exact structures and the possibly metabolic sites of NHBA in RLMs, two metabolites were semisynthesized via the reaction of 4-hydroxylbenyzlamine hydrochloride with 6-chloropurine and 3,4-dihydroxybenzylamine hydrochloride with 6-chloropurine riboside in the presence of DIEA (Fig. 2) and characterized by 1H NMR and 13C NMR. The data of 1H NMR and 13C NMR spectra of two metabolites are listed in Table 1 and Supplementary data. Compared with NMR data of NHBA (Huang et al., 2007), the 13C NMR spectrum of M2 displayed carbon signals at δ 143.9 (C-1), 114.7 (C-3), 118.1 (C-5) shifted downfield to δ 154.8, 155.2, 129.7, and δ 144.9 (C-2), 130.8 (C-4), 115.2 (C-6) shifted upfield to δ 102.7, 123.2, 108.1. The other carbon signals of M2 were similar to that of NHBA. The spectrum of M2 revealed that the proton signal of H-2 at δ 6.66 disappeared by comparison with data for NHBA, indicating that the H-2 was replaced. Taken together with the MS data, M2 was identified as N6-(3,4-dihydroxylbenzyl) adenine riboside (ONHBA). The 1H NMR and 13C NMR data of M1 showed a pattern similar to that of NHBA except that the data of glycosylated section was disappeared. Therefore, M1 was identified as N6-(4-hydroxylbenzyl) purine (NHBP).
3. Experimental 3.1. Chemicals and reagents N6-(4-hydroxybenzyl) adenine riboside was isolated and purified from the dried roots of GE in our lab, structure was confirmed by comparison of its UV, MS and NMR with literature (Huang et al., 2007). Synthetic Aβ1-42 was obtained from GL Biochem Ltd. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), NADPH and tris was purchased from Sigma Aldrich (StLouis, MO, USA). 4-Hydroxybenzylamine hydrochloride was purchased from YueXinchem Co., Ltd. (Changzhou, China). 6-Chloropurine and 6chloropurine riboside were purchased from HuaWenchem Co., Ltd. (Zhengzhou, China). 3,4-Dihydroxybenzylamine hydrochloride, diisoproplethylamine and n-propanol were obtained from J & K (Beijing, China). Acetonitrile and methanol were supplied by Burdick & Jackson (USA). All other reagents were either LC grade or of the highest grade commercially available. 3.2. Preparation of rat liver microsomes
2.3. Neuroprotective study Male Sprague–Dawley rats weighing approximately 200 g were obtained from the Laboratory Animal Center in Dalian Medical University (Dalian, China). All experimental protocols on animals were in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of China. The animals had free access to tap water and pellet diet. Livers were removed, and immediately washed with ice-cold saline, followed by weighed, minced with scissors and homogenized with 4 volumes (w/v) of ice-cold 50 mM tris–HCl (pH 7.4) buffer containing 0.25 M sucrose and 0.05 M magnesium chloride. Microsomes were prepared by differential ultracentrifugation. The protein concentration
The neuroprotective activities of NHBA and its metabolites were investigated. It is known that accumulation of extracellular amyloid plaque is considered a pathological feature of Alzheimer disease (AD) and soluble Aβ oligomers might lead to cognitive impairment. In addition, oxidative stress, which was conventionally induced by hydrogen peroxide (H2O2) in cells, is also closely related to the pathogenesis of neurodegenerative disorders. Therefore, we investigated the neuroprotective effects of NHBA and its metabolites against Aβ oligomers- and H2O2-induced neuronal death in SH-SY5Y cells. The results showed that either Aβ oligomers or H2O2 could 148
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Table 1 1 H-NMR and
13
C-NMR spectra data for NHBA, NHBP and ONHBA.
No
NHBP
1
H-NMR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
6.66 (d,J = 8.5 Hz) 7.14 (d,J = 8.5 Hz) 7.14 (d,J = 8.5 Hz) 6.66 (d,J = 8.5 Hz) 4.60 m 8.35 s
8.28 s 5.88 (d,J = 5.5 Hz) 4.60 m 4.13 m 3.95 m 3.57 m,3.63 m
13
C-NMR
156.1 s 114.9 d 128.6 d 130.8 s 128.6 d 114.9 d 42.4 t 154.4 s 152.3 d 148.4 s 120.4 s 139.8 d 88.0 d 73.5 d 70.7 d 85.9 d 61.7 t
1
13
H-NMR
6.68 (d,J = 8.1 Hz) 7.16 (d,J = 8.1 Hz) 7.16 (d,J = 8.1 Hz) 6.68 (d,J = 8.1 Hz) 4.59 m 8.17 s
8.08 s
C-NMR
156.07 s 114.87 d 128.55 d 130.41 s 128.55 d 114.87 d 42.38 t 154.23 s 152.29 d 149.69 s 118.69 s 138.63 d
1
H-NMR
6.73 (d,J = 1.8 Hz) 6.58 m 6.63 (d,J = 8.1 Hz) 4.54 s 8.35 s
8.20 s 5.88 (d,J = 5.5 Hz) 4.61 m 4.15 m 3.97 m 3.55 m,3.67 m
13
C-NMR
143.97 s 144.92 s 114.71 d 130.79 d 118.09 d 115.24 d 42.38 t 154.45 s 152.30 d 148.34 s 119.70 s 139.73 d 87.92 d 73.41 d 70.86 d 85.86 d 61.64 t
voltage of 150 V, nebulizer pressure of 45 psi and Dual ESI Vcap of 3500 V. Full-scan data acquisition in positive ion mode was performed at 50–500 m/z with a scan time of 1 s−1. Argon was used for collisionactivated dissociation experiments to produce MS/MS spectrum of selected precursor ions, and the collision energy was maintained at 30 eV. Data were managed on the MassHunter WorkStation Data Acquisition software (B.02.01 Version) and Qualitative Analysis software (B.03.01 Version).
was measured by the method of Bradford using bovine serum albumin as the standard. 3.3. Incubation system The incubation mixture with a total volume 200 μL, consisted of tris-HCl buffer (0.05 M, pH 7.4), NADPH (1 mM), MgCl2 (5 mM), RLMs (2 mg/mL), and NHBA (50 μg/mL). NHBA was previous dissolved in methanol, with a final methanol concentration below 1% (v/v) in the reaction mixture. After 10 min pre-incubation at 37 °C, the reaction was initiated by adding NADPH. Then the reaction was terminated by the addition of methanol (200 μL) after 1 h in a vapour bath vibrator. The mixture was swirled and centrifuged at 15,000 × g for 10 min. The control incubation was performed in the absence of NADPH. Aliquots of supernatants were transferred for UPLC-QTOF-MS analysis.
3.5. Synthesis of metabolites N6-(4-hydroxylbenzyl) purine: A mixture of 4-hydroxybenzylamine hydrochloride (0.3 g), 6-chloropurine (360 mg) and diisoproplethylamine (10 mL) in n-propanol (50 mL) was heated to 60 °C for 7 h under the atmosphere of nitrogen. After evaporation, the mixture was suspended with H2O to give a white precipitate, then filtered and recrystallized to obtain the desired product. N6-(3,4-dihydroxylbenzyl) adenine riboside: A mixture of 3,4-dihydroxybenzylamine hydrochloride (0.3 g), 6-chloropurine riboside (286 mg) and diisoproplethylamine (10 mL) in n-propanol (50 mL) was heated to 60 °C for 7 h under the atmosphere of nitrogen. After evaporation, the mixture was suspended with H2O to give a white precipitate, then filtered and recrystallized to obtain the desired product.
3.4. LC–MS/MS analysis An Agilent 1290 UPLC system (Agilent, Waldbronn, Germany) including a binary pump, an autosampler and diode array ultraviolet detector was used. The chromatographic separation was conducted on Agilent ZORBAX SB-C18 column (3.0 × 150 mm, 1.8 μm). The column temperature was set at 60 °C. The mobile phase consisted of 0.5% formic acid in water (A) and acetonitrile (B). Elution was kept at 10% B with 2 min, and increased linearly to 18% B with 0.2 min, then kept at 18% B with 7.8 min, and increased linearly to 30% B with 0.2 min, kept at 30% B with 4.8 min. The flow rate was set as 0.3 mL/min. 1 μL of the purified sample was injected automatically into the UPLC system for analysis. MS/MS analysis were conducted on an Agilent 6520 quadrupole–time of flight mass spectrometer (Agilent Corp, USA) equipped with electrospray ionization (ESI) interface. The samples were analyzed in positive ion mode. The flow rates of drying gas (N2) were 10 L/min. The optimum conditions were drying gas temperature of 350 °C, fragmentor
3.6. Preparation of soluble Aβ1-42 oligomers Soluble Aβ42 oligomers were prepared as described previously (Chang et al., 2015). Briefly, synthetic Aβ1-42 was dissolved in ice-cold 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP), thoroughly vortexed, and aliquoted to be frozen until use. Aβ1-42 was spin-vacuumed just prior to the experiment, dissolved in HFIP solution (final concentration: 10% (v/v) HFIP) and incubated at room temperature for 20 min. The 149
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16–20 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength. Unless otherwise indicated, the extent of MTT conversion in cells without treatment is expressed as a percentage of the control. Acknowledgements This work is financially supported by the National Natural Science Foundation of China (Grant No. 81603266) and National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (Grant No. 2014XZ09304-307). In addition, the authors also gratefully acknowledge the support of K.C. Wong Magna Fund in Ningbo University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.04.035. References Chang, L., Cui, W., Yang, Y., Xu, S., Zhou, W., Fu, H., Hu, S., Mak, S., Hu, J., Wang, Q., Ma, V.P., Choi, T.C., Ma, E.D., Tao, L., Pang, Y., Rowan, M.J., Anwyl, R., Han, Y., 2015. Protection against beta-amyloid-induced synaptic and memory impairments via altering beta-amyloid assembly by bis(heptyl)-cognitin. Sci. Rep. 5, 10256. Cui, W., Zhang, Z., Li, W., Hu, S., Mak, S., Zhang, H., Han, R., Yuan, S., Li, S., Sa, F., Xu, D., Lin, Z., Zuo, Z., Rong, J., Ma, E.D., Choi, T.C., Lee, S.M., Han, Y., 2013. The anticancer agent SU4312 unexpectedly protects against MPP(+) − induced neurotoxicity via selective and direct inhibition of neuronal NOS. Br. J. Pharmacol. 168, 1201–1214. Cui, W., Zhang, Z.J., Hu, S.Q., Mak, S.H., Xu, D.P., Choi, C.L., Wang, Y.Q., Tsim, W.K., Lee, M.Y., Rong, J.H., Han, Y.F., 2014. Sunitinib produces neuroprotective effect via inhibiting nitric oxide overproduction. CNS Neurosci. Ther. 20, 244–252. Huang, N.K., Chern, Y.J., Fang, J.M., Lin, C.I., Chen, W.P., Lin, Y.L., 2007. Neuroprotective principles from Gastrodia elata. J. Nat. Prod. 70, 571–574. Huang, N.K., Lin, J.H., Lin, J.T., Lin, C.I., Liu, E.M.W., Lin, C.J., Chen, W.P., Shen, Y.C., Chen, H.M., Chen, J.B., Lai, H.L., Yang, C.W., Chiang, M.C., Wu, Y.S., Chang, C., Chen, J.F., Fang, J.M., Lin, Y.L., Chern, Y.J., 2011. A new drug design targeting the adenosinergic system for Huntington's disease. PLoS One 6, e20934. Huang, J.Y., Yuan, Y.H., Yan, J.Q., Wang, Y.N., Chu, S.F., Zhu, C.G., Guo, Q.L., Shi, J.G., Chen, N.H., 2016. 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway. Acta Pharmacol. Sin. 37, 731–740. Hwang, S.M., Lee, Y.J., Kang, D.G., Lee, H.S., 2009. Anti-inflammatory effect of Gastrodia Elata Rhizome in human umbilical vein endothelial cells. Am. J. Chin. Med. 37, 395–406. Jang, Y.W., Lee, J.Y., Kim, C.J., 2010. Anti-asthmatic activity of phenolic compounds from the roots of Gastrodia elata Bl. Int. Immunopharmacol. 10, 147–154. Jang, J.H., Son, Y., Kang, S.S., Bae, C.S., Kim, J.C., Kim, S.H., Shin, T., Moon, C., 2015. Neuropharmacological potential of Gastrodia Elata Blume and its components. Evid. -Based Complement. Altern. 309261. Kim, H.J., Hwang, I.K., Won, M.H., 2007. Vanillin, 4-hydroxybenzyl aldehyde and 4hydroxybenzyl alcohol prevent hippocampal CA1 cell death following global ischemia. Brain Res. 1181, 130–141. Lei, Y.J., Wang, L., Cheng, M.C., Xiao, H.B., 2011. Identification of major metabolites in rat urine and plasma of N-6-(4-hydroxybenzyl) adenine riboside by LC/MS/MS. Biomed. Chromatogr. 25, 344–352. Li, Z., Wang, Q., Ouyang, H., Huang, L., Feng, Y., Wang, R., Yang, S., 2016. New compounds with neuroprotective activities from Gastrodia elata. Phytochem. Lett. 15, 94–97. Ojemann, L.M., Nelson, W.L., Shin, D.S., Rowe, A.O., Buchanan, R.A., 2006. Tian ma, an ancient Chinese herb, offers new options for the treatment of epilepsy and other conditions. Epilepsy Behav. 8, 376–383. Ramachandran, U., Manavalan, A., Sundaramurthi, H., Sze, S.K., Feng, Z.W., Hu, J.M., Heese, K., 2012. Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells. Neurochem. Int. 60, 827–836. Shin, E.J., Bach, J.H., Nguyen, T.T.L., Jung, B.D., Oh, K.W., Kim, M.J., Jang, C.G., Ali, S.F., Ko, S.K., Yang, C.H., Kim, H.C., 2011. Gastrodia Elata Bl attenuates cocaineinduced conditioned place preference and convulsion, but not behavioral sensitization in mice: importance of GABA(A) receptors. Curr. Neuropharmacol. 9, 26–29. Tang, C.L., Wang, L., Cheng, M.C., Liu, X.X., Xiao, H.B., 2015. Determination of N6-(4hydroxybenzyl) adenine riboside in rat plasma by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry. Chin. J. Chromatogr. 33, 699–703. Wang, L., 2007. Studies on Chemical Constituents and Quality Control of Gastrodia Elata. Chinese Academy of Science.
Fig. 3. Effects of NHBA, NHBP and ONHBA on the cell viability of SH-SY5Y cells. a: NHBA, ONHBA and NHBP prevent 150 μM H2O2-induced neuronal death in SH-SY5Y cells; b: ONHBA, NHBP but not NHBA prevent 1 μM Aβ oligomers-induced neuronal death in SH-SY5Y cells. SH-SY5Y cells were pre-treated with various chemicals at the indicated concentrations for 2 h, and then exposed to neurotoxins. Cell viability was measured by the MTT assay at 24 h after the challenge of neurotoxins. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 versus the Aβ oligomers-treated group (ANOVA and Dunnett’s test).
solution was centrifuged at 14,000g for 15 min at 4 °C, and the supernatant was collected. The HFIP was completely evaporated to obtain a 50 μM Aβ oligomers solution and kept at room temperature under constant stirring for 48 h. Then the tube was transferred to refrigerator and maintained at 4 °C. 3.7. SH-SY5Y cells culture SH-SY5Y cells were purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences), and cultured in high glucose modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL). The cells were cultured in an incubator at 37 °C and 5% CO2. The medium was replaced every other day. For the experiments with Aβ oligomers or H2O2, SH-SY5Y cells (1 × 105 cells/mL) in DMEM with low serum content (1% FBS) were seeded in 96-well plates. All experiments were carried out 24 h after the cells were seeded. 3.8. Measurement of cell viability Cell viability was determined by the activity of mitochondrial dehydrogenases via MTT assay as previously described (Cui et al., 2013, 2014). Briefly, after treatment, 10 μL of 5 mg/mL MTT solution was added to each well of 96-well plates. Plates were incubated at 37 °C for 4 h in a humidified incubator. 100 μL of the solvating solution (0.01 N HCl in 10% SDS solution) was then added to each well for
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