JOURNAL OF BIOTECHNOLOGY
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2015
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IF 2.871
Yue-De Cao,Yu-Cai He,Hao Li,Guo-Yin Kai,Jian-He Xu,Hui-Lei Yu
Hyoscyamine 6 β -hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (−)-hyoscyamine and the subsequent epoxidation of 6 β -hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus ( Aa H6H). A double mutant, Aa H6H M1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34 h cultivation of Escherichia coli cells harboring Aa h6h m1 in a 5-L bioreactor with a working volume of 3 L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500 mg L −1 (−)-hyoscyamine in 97% conversion, and 1.068 g of the product were isolated, corresponding to a space-time yield of 251 mg L −1 d −1 . This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.
研究领域:
生物化学
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生物技术
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微生物学
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酶学
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产品关联
DOI
PubMed
高影响因子
ACS Catalysis
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2021
·
IF 13.084
Fei Qiu,Yijun Yan,Junlan Zeng,Jian-Ping Huang,Lingjiang Zeng,Wei Zhong,Xiaozhong Lan,Min Chen,Sheng-Xiong Huang,Zhihua Liao
Hyoscyamine, a member of the class of compounds known as tropane alkaloids (TAs), is clinically used as an anticholinergic drug. Previous research has predicted that hyoscyamine is produced via the reduction of hyoscyamine aldehyde. In this study, we identified a root-expressed gene from Atropa belladonna, named AbHDH, which encodes a hyoscyamine dehydrogenase involved in the formation of hyoscyamine. Enzymatic assays indicated that AbHDH was able to not only reduce hyoscyamine aldehyde to produce hyoscyamine but also oxidize hyoscyamine to form hyoscyamine aldehyde under different conditions. To elucidate its catalytic mechanism, the crystal structure of AbHDH at a 2.4-Å resolution was also determined. Overexpression of AbHDH significantly enhanced the biosynthesis of hyoscyamine and the production of anisodamine and scopolamine in root cultures of A. belladonna. However, suppression of AbHDH using RNAi technology did not reduce the production of hyoscyamine, anisodamine, or scopolamine, though it did increase the accumulation of hyoscyamine aldehyde. In summary, this study provided timely biochemical and metabolic insights into hyoscyamine dehydrogenase, pointing to an alternative, promising way to produce pharmaceutical TAs via metabolic engineering in planta.
研究领域:
生物化学
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代谢学
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植物生物学
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药学
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产品关联
DOI
高影响因子
PLANT PHYSIOLOGY AND BIOCHEMISTRY
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2018
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IF 2.718
Chen Geng,Tengfei Zhao,Chunxian Yang,Qiaozhuo Zhang,Feng Bai,Junlan Zeng,Fangyuan Zhang,Xiaoqiang Liu,Xiaozhong Lan,Min Chen,Zhihua Liao
N -methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine . Putrescine N -methyltransferase (PMT) catalyzes the N -methylation of putrescine to form N -methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger , designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues , including pericycles and endodermis , of the H. niger hairy root cultures , when it was driven by the 5′-flanking promoter region of HnPMT . The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N -methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme . When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N -methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines . Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger .
研究领域:
植物学
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代谢工程
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分子生物学
✓ 已验证
DOI
高影响因子
BMC Chemistry
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2024
·Vol. 18
·期 1
·1-15
·
IF 4.3
Huang Xiaomin,Wang Ting,Wang Ludan,Sun Yantao,Zhang Ziru,Zhang Yajun
In the investigation of active ingredients from natural products, current technologies relying on drug–target affinity recognition analysis face significant challenges. This is primarily due to their limited specificity and inability to provide downstream pharmacodynamic information, such as agonistic or antagonistic activity. In this study, a two-point method was developed by immobilizing M3 acetylcholine receptor (M3R) through the combination of the conformation-specific peptide BJ-PRO-13a and the HaloTag trap system. We systematically assessed the specificity of the immobilized M3R using known M3R antagonists (pirenzepine and atropine) and agonists (cevimeline and pilocarpine). By frontal analysis and nonlinear chromatography, the performance of immobilized M3R was evaluated in terms of binding kinetics and thermodynamics of four drugs to the immobilized M3R. Additionally, we successfully identified two M3R antagonists within an extract from Daturae Flos (DF), specifically hyoscyamine and scopolamine. Our findings demonstrate that this immobilization method effectively captures receptor-ligand binding interactions and can discern receptor agonists from antagonists. This innovation enhances the efficiency of receptor chromatography to determine binding-affinity in the development of new drugs, offering promise for the screening and characterization of active compounds, particularly within complex natural products.
研究领域:
药理学
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分析化学
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天然产物研究
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2024
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IF 8.5
Zile Wang,Ke Han,Zhiyue Feng,Boyan Sun,Shuai Zhang,Sihan Wang,Haiyang Jiang
This study aims to realise rapid detecting of tropane alkaloids (TAs) in food. For this purpose, a broad-spectrum single-chain fragment variable was fused with horseradish peroxidase to create an antibody–enzyme complex (AEC) with antigen recognition and catalytic activity. A multi-signal immunosensor platform based on AEC in the direct competitive reaction mode was constructed using 3,3′,5,5′-tetramethylbenzidine and 10-acetyl-3,7-dihydroxyphenoxazine as substrates. The sensitivity of TAs in the immunosensor platform ranged from 0.25 μg/kg to 7912.46 μg/kg. Honey was selected as a representative food sample, and the limit of detection of TAs in honey ranged from 0.02 μg/kg to 409.11 μg/kg, with a recovery rate of 65.7 %–117.1 % and a coefficient of variation less than 21.4 %. Results showed that the immunosensor platform possesses satisfactory accuracy and precision, which highlights its potential for practical applications and its suitability as an ideal tool for rapid screening of TAs in food.
研究领域:
分析化学
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食品科学
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生物技术
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF ALLOYS AND COMPOUNDS
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2021
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IF 5.316
Yazhou Qin,Yuanzhao Wu,Binjie Wang,Jiye Wang,Weixun Yao
Compared with silver nanoparticles, gold nanoparticles have excellent chemical resistance and biocompatibility, while silver nanoparticles have more excellent local surface plasmon resonance characteristics than gold. In this article, we developed a simple method for growing island-shaped gold nanoparticles on the surface of silver nanowires (NWs), so that the prepared Ag@Au NWs have both the advantages of Au and Ag without their disadvantages. Furthermore, by changing the reaction parameters, we achieved the regulation of the density of island-shaped gold nanoparticles on the Ag NWs surface, thereby optimizing its LSPR performance. We use the prepared Ag@Au NWs as the base material for highly sensitive Surface enhanced Raman scattering (SERS) detection. By using 4-aminothiophenol (4-ATP) as the test molecule to explore the uniformity of the prepared SERS substrate, the detection limit of 4-ATP reached 10 -9 M. And 10 points were randomly selected for testing and the RSD was 7.4%, indicating the Ag@Au NWs SERS substrates had excellent uniformity. Furthermore, we have achieved highly sensitive SERS detection of 5 tropane alkaloids including L -Hyoscyamine, Anisodamine, Scopolamine Hydrobromide, scopolamine methyl bromide and scopolamine butyrate bromide. The limit of detection is 10 -9 M, 10 -10 M, 10 -9 M, 10 -9 M and 10 -10 M. Furthermore, by analyzing the obtained SERS spectra, we realized the differentiation of 5 kinds of tropane alkaloids by their characteristic peaks.
研究领域:
材料科学
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分析化学
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产品关联
DOI
高影响因子
CURRENT ISSUES IN MOLECULAR BIOLOGY
·
2024
·Vol. 46
·期 10
·11359-11374
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IF 2.8
Xin Li,Kai Zheng,Hao Chen,Wei Li
While Ginsenoside Re has been shown to protect the central nervous system, reports of its effects on memory in the model of scopolamine-induced memory impairment are rare. The aim of this study was to investigate the effects of Ginsenoside Re on scopolamine (SCOP)-induced memory damage and the mechanism of action. Male ICR mice were treated with SCOP (3 mg/kg) for 7 days and with or without Ginsenoside Re for 14 days. As evidenced by behavioral studies (escape latency and cross platform position), brain tissue morphology, and oxidative stress indicators after Ginsenoside Re treatment, the memory damage caused by SCOP was significantly ameliorated. Further mechanism research indicated that Ginsenoside Re inhibited cell apoptosis by regulating the PI3K/Akt/Nrf2 pathway, thereby exerting a cognitive impairment improvement effect. This research suggests that Ginsenoside Re could protect against SCOP-induced memory defects possibly through inhibiting oxidative stress and cell apoptosis.
研究领域:
神经科学
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药理学
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氧化应激
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产品关联
DOI
PubMed
高影响因子
ANALYST
·
2019
·Vol. 144
·期 23
·6989-7000
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IF 4.019
Alula Yohannes,Shun Yao
The application of ionic liquids (ILs) for the separation of bioactive compounds from various sample matrices is a burgeoning area. Here, a class of porous materials called metal–organic frameworks (MOFs) were combined with tropine-based ILs having structural similarity with natural tropane alkaloids for the quantitative analysis of Huashansheng tablets. Three MOFs (MIL-101, HKUST-1 and ZIF-8) were used to form the composite with the a tropine-based IL (N-propyl-substituted tropine hexafluorophosphate, [C3tr][PF6]), using the ship-in-a-bottle (SIB) synthesis approach. The performance of the hybrid IL/MOF composite (IL@MOF) was evaluated as a potential sorbent for application in the dispersive solid-phase-extraction-based enrichment of tropane alkaloids, followed by chromatographic analysis. It was found that [C3tr][PF6]@MIL-101 exhibited excellent adsorption capacity, and the extraction results showed satisfactory recoveries in the range of 91.5–104.7% (RSD < 5%). Further, the limit of detection went down to 10–20 μg L−1 with a linear range from 100–500 μg L−1, which proved that an adsorbent with the same nucleus as the target objects would be more suitable and efficient for their later extraction.
研究领域:
分析化学
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药物分析
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产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2019
·Vol. 9
·期 34
·19712-19719
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IF 3.049
Jie Zuo,Xingyuan Zhang,Xinyu Li,Zhiwei Li,Zongren Li,Honghong Li,Wencheng Zhang
Molecularly imprinted polymers (MIPs) prepared using conventional functional monomers exhibit poor specific extraction of scopolamine from tropane alkaloids, which hinders their application in separation and purification. In this paper, a novel molecularly imprinted polymer (MIP) was prepared by precipitation polymerization using scopolamine as the template, monoethyl fumarate (MFMA) as a functional monomer, and ethylene dimethacrylate (EGDMA) as a cross-linker. The advantages of the supercritical fluid technology for the removal of the template were verified by comparing the efficiency of the swelling method and the Soxhlet extraction method. The prepared MFMA-based MIPs (MFMA-MIPs) showed a high adsorption capacity (49.75 mg g−1) and high selectivity toward scopolamine with a selectivity coefficient of 3.5. 1H NMR spectroscopy was performed to demonstrate the interactions between the two functional groups of the functional monomer and the template. Lastly, MFMA-MIPs were used as solid phase extraction (SPE) sorbents for scopolamine analysis. It was found that 97.0–107.0% of the template had been extracted using the SPE column from the complex of scopolamine, atropine and anisodamine. The mean recoveries of scopolamine from plant samples were 96.0–106.0% using the established method, which showed a good linearity in the range of 8.0–4.0 × 104 μg L−1. The results showed that MFMA-MIPs could be applied for the separation of scopolamine from tropane alkaloids.
研究领域:
化学
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药学
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产品关联
DOI
PubMed
高影响因子
METABOLIC BRAIN DISEASE
·
2018
·Vol. 34
·期 1
·173-181
·
IF 2.441
Sun Kaiyue,Bai Yuting,Zhao Rong,Guo Zijiao,Su Xiang,Li Peiqi,Yang Pengyu
The present study was designed to evaluate the effects of matrine (MAT) on scopolamine (SCOP)-induced learning and memory impairment. After successive oral administration of MAT to mice for three days at doses of 0.4, 2, and 10 mg/kg, we assessed improvements in learning and memory and investigated the mechanism of action of SCOP-induced amnesia. Donepezil at a dose of 3 mg/kg was used as a standard memory enhancer. MAT significantly improved SCOP-induced learning and memory impairment in novel object recognition and Y-maze tests at doses of 0.4, 2, and 10 mg/kg. Furthermore, MAT inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and decreased oxidative stress in the brain, as evidenced by increased total antioxidant capacity, total superoxide dismutase levels, and catalase activities as well as decreased malondialdehyde levels. Additionally, there was a significant negative correlation between the percentage of spontaneous alternation in the Y maze and AChE activity in the cortex and hippocampus. MAT ameliorated SCOP-induced amnesia by the inhibition of both AChE/BuChE activities and oxidative stress. This study provides further evidence to encourage the development of MAT as a drug for the prevention or treatment of Alzheimer’s disease.
研究领域:
神经科学
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药理学
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阿尔茨海默病研究
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产品关联
DOI
PubMed
高影响因子
Neural Regeneration Research
·
2019
·
IF 2.472
Yu-Min Xu,Xin-Chen Wang,Ting-Ting Xu,Hong-Ying Li,Shang-Yan Hei,Na-Chuan Luo,Hong Wang,Wei Zhao,Shu-Huan Fang,Yun-Bo Chen,Li Guan,Yong-Qi Fang,Shi-Jie Zhang,Qi Wang,Wei-Xiong Liang
Kai Xin San (KXS, containing ginseng, hoelen, polygala, and acorus), a traditional Chinese herbal compound, has been found to regulate cognitive dysfunction; however, its mechanism of action is still unclear. In this study, 72 specific-pathogen-free male Kunming mice aged 8 weeks were randomly divided into a vehicle control group, scopolamine group, low-dose KXS group, moderate-dose KXS group, high-dose KXS group, and positive control group. Except for the vehicle control group and scopolamine groups (which received physiological saline), the doses of KXS (0.7, 1.4 and 2.8 g/kg per day) and donepezil (3 mg/kg per day) were gastrointestinally administered once daily for 2 weeks. On day 8 after intragastric treatment, the behavioral tests were carried out. Scopolamine group and intervention groups received scopolamine 3 mg/kg per day through intraperitoneal injection. The effects of KXS on spatial learning and memory, pathological changes of brain tissue, expression of apoptosis factors, oxidative stress injury factors, synapse-associated protein, and cholinergic neurotransmitter were measured. The results confirmed the following. (1) KXS shortened the escape latency and increased residence time in the target quadrant and the number of platform crossings in the Morris water maze. (2) KXS increased the percentage of alternations between the labyrinth arms in the mice of KXS groups in the Y-maze. (3) Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that KXS promoted the production of Nissl bodies and inhibited the formation of apoptotic bodies. (4) Western blot assay showed that KXS up-regulated the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic protein Bax. KXS up-regulated the expression of postsynaptic density 95, synaptophysin, and brain-derived neurotrophic factor in the cerebral cortex and hippocampus. (5) KXS increased the level and activity of choline acetyltransferase, acetylcholine, superoxide dismutase, and glutathione peroxidase, and reduced the level and activity of acetyl cholinesterase, reactive oxygen species, and malondialdehyde through acting on the cholinergic system and reducing oxidative stress damage. These results indicate that KXS plays a neuroprotective role and improves cognitive function through reducing apoptosis and oxidative stress, and regulating synapse-associated protein and cholinergic neurotransmitters.
研究领域:
神经科学
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药理学
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中医药学
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产品关联
DOI
PubMed
高影响因子
International Journal of Nanomedicine
·
2024
·
IF 6.6
Zixia Wu,Weibo Xia,Liling Ou,Ling Zheng,Bingying Hou,Tonghe Pan,Wenjie Sun,Leo H Koole,Yongqing Shao,Lei Qi
Purpose Patients afflicted with dry eye disease (DED) experience significant discomfort. The underlying cause of DED is the excessive accumulation of ROS on the ocular surface. Here, we investigated the nitrogen doped-graphene quantum dots (NGQDs), known for their ROS-scavenging capabilities, as a treatment for DED.Methods NGQDs were prepared by using citric acid and urea as precursors through hydrothermal method. The antioxidant abilities of NGQDs were evaluated through: scavenging the ROS both extracellular and intracellular, regulating the nuclear factor-erythroid 2-related factor (Nrf2) antioxidant pathway of human corneal epithelial cells (HCECs) and their transcription of inflammation related genes. Furthermore, NGQDs were modified by Arg-Gly-Asp-Ser (RGDS) peptides to obtain RGDS@NGQDs. In vivo, both the NGQDs and RGDS@NGQDs were suspended in 0.1% Pluronic F127 (w/v) and delivered as eye drops in the scopolamine hydrobromide-induced DED mouse model. Preclinical efficacy was compared to the healthy and DPBS treated DED mice.Results These NGQDs demonstrated pronounced antioxidant properties, efficiently neutralizing free radicals and activating the intracellular Nrf2 pathway. In vitro studies revealed that treatment of H2O2-exposed HCECs with NGQDs induced a preservation in cell viability. Additionally, there was a reduction in the transcription of inflammation-associated genes. To prolong the corneal residence time of NGQDs, they were further modified with RGDS peptides and suspended in 0.1% Pluronic F127 (w/v) to create RGDS@NGQDs F127 eye drops. RGDS@NGQDs exhibited superior intracellular antioxidant activity even at low concentrations (10 μg/mL). Subsequent in vivo studies revealed that RGDS@NGQDs F127 eye drops notably mitigated the symptoms of DED mouse model, primarily by reducing ocular ROS levels.Conclusion Our findings underscore the enhanced antioxidant benefits achieved by modifying GQDs through nitrogen doping and RGDS peptide tethering. Importantly, in a mouse model, our novel eye drops formulation effectively ameliorated DED symptoms, thereby representing a novel therapeutic pathway for DED management.
研究领域:
眼科
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材料科学
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药学
·
细胞生物学
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）