The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Altered Iron-Mediated Metabolic Homeostasis Governs the Efficacy and Toxicity of Tripterygium Glycosides Tablets Against Rheumatoid Arthritis

Rheumatoid arthritis(RA),a globally increasing autoimmune disorder,is associated with increased disability rates due to the disruption of iron metabolism.Tripterygium glycoside tablets(TGTs),a Tripterygium wilfordii Hook.f.(TwHF)-based therapy,exhibit satisfactory clinical efficacy for RA treatment.However,drug-induced liver injury(DILI)remains a critical issue that hinders the clinical application of TGTs,and the molecular mechanisms underlying the efficacy and toxicity of TGTs in RA have not been fully elucidated.To address this problem,we integrated clinical multi-omics data associated with the anti-RA efficacy and DILI of TGTs with the chemical and target profiling of TGTs to perform a systematic network analysis.Subsequently,we identified effective and toxic targets following experimental validation in a collagen-induced arthritis(CIA)mouse model.Significantly different transcriptome–protein–metabolite profiles distinguishing patients with favorable TGTs responses from those with poor outcomes were identified.Intriguingly,the clinical efficacy and DILI of TGTs against RA were associated with metabolic homeostasis between iron and bone and between iron and lipids,respectively.Particularly,the signal transducer and activator of transcription 3(STAT3)–hepcidin(HAMP)/lipocalin 2(LCN2)–tartrate-resis tant acid phosphatase type 5(ACP5)and STAT3–HAMP–acyl-CoA synthetase long-chain family member 4(ACSL4)–lysophosphatidylcholine acyltransferase 3(LPCAT3)axes were identified as key drivers of the efficacy and toxicity of TGTs.TGTs play dual roles in ameliorating CIA-induced pathology and in inducing hepatic dysfunction,disruption of lipid metabolism,and hepatic lipid peroxidation.Notably,TGTs effectively reversed“iron–bone”disruptions in the inflamed joint tissues of CIA mice by inhibiting the STAT3–HAMP/LCN2–ACP5 axis,subsequently leading to“iron–lipid”disturbances in the liver tissues via modulation of the STAT3–HAMP–ACSL4–LPCAT3 axis.Additional bidirectional validation experiments were conducted using MH7A and AML12 cells to confirm the bidirectional regulatory effects of TGTs on key targets.Collectively,our data highlight the association between iron-mediated metabolic homeostasis and the clinical efficacy and toxicity of TGT in RA therapy,offering guidance for the rational clinical use of TwHF-based therapy with dual therapeutic and toxic potential.

Inhibitory Effect of Flavonoid Glycosides from Chlorophytum comosum on Nasopharyngeal Carcinoma 5-8F Cells and Its Mechanism

[Objectives]To study the inhibitory activity of two flavonoid glycosides isolated from Chlorophytum comosum Laxum R.Br on human nasopharyngeal carcinoma(NPC)cell line 5-8F in vitro and its mechanism.[Methods]The flavonoid glycosides were isolated and purified from the ethanol alcoholic extract of the roots of Liliaceae plant Chlorophytum comosum by silica gel column chromatography,macroporous resin column chromatography,Sephadex LH-20,and reverse column chromatography(ODS).The inhibitory activity of flavonoid glycosides on human nasopharyngeal carcinoma cells was analyzed by CCK-8 method,and the potential mechanism was preliminarily analyzed by molecular docking.[Results]Two flavonoid glycosides were identified as isovitexin 2″-0-rhamnoside and 7-2″-di-O-β-glucopyranosylisovitexin.Two flavonoid glycosides showed promising inhibitory effect on human nasopharyngeal carcinoma cell line 5-8F,with IC_(50) values of 24.8 and 27.5μmol/L,respectively.Molecular docking results showed that the potential targets of two flavonoid glycosides include CyclinD1,Bcl-2β-Catenin,ILK,TGF-β,in addition,two glycosides showed higher predicted binding affinity towards CyclinD1,which verifies the cytotoxicity of the two compounds on human nasopharyngeal carcinoma cell line 5-8F in vitro.[Conclusions]Two flavonoid glycosides are the active molecules in Chlorophytum comosum that can inhibit the proliferation of human nasopharyngeal carcinoma cells,and have the potential to be used in the research and development of anti nasopharyngeal carcinoma drugs.

Chemical Components of Helicia nilagirica Beed.I.Structure of Three New Flavonol Glycosides

Three new flavonol glycosides were isolated from the leaves of Helicia nilagirica Beed.. The structures were elucidated as kaempferol-3-O-b-D-xylopyranosyl-(16)-b-D-glyco- pyranosyl-(12)-a-L-rhamnopyranoside, quercetin-3-O-b-D-xylopyranosyl-(16)-b-D-glycopy- ranosyl-(12)-[-b-D-xylopyranosyl-(14)]-a-L-rhamnopyranoside, and quercetin-3-O-b-D- xylopyranosyl-(16)-b-D-glycopyranosyl-(12)-a-L-rhamnopyranoside, named as Helicianeoside A、B and C, respectively.

Two New Flavonol Glycosides from Bridelia tomentosa

Two new flavonol glycosides, tamarixetin 3-O-β-D-xylopyranosyl-（1-2）-α-L-ribopyranoside （1） and tamarixetin 3-O-α-L-ribopyranoside （2） were isolated from the leaves of Bridelia tomentosa. Their structures were elucidated by 1D and 2D NMR analysis, as well as MS and IR spectra.

New Acetylated Flavonol Glycosides from Knoxia corymbosa

Two new diacetylated flavonol glycosides, kampferol-3-O--3? 6?diacetylglucopyrano- side and quercetin-3-O--3? 6?diacetylglucopyranoside were isolated from knoxia corymbosa. Their structures were elucidated by spectroscopic evidents.

Two New Flavonol Glycosides from Knoxia corymbosa

Two new flavonol glycosides (1 and 2) together with two known flavonoides (3 and 4), were isolated from the whole plant of Knoxia corymbosa willd. The structures of 1 and 2 were elucidated as kaempferol-7-O-α-L-arabinosyl-3-O-β-D-6'-acetylglucopyranoside and kaempferol -7-O-α-L-arabinosyl-3-O-β-D-3',6'-diacetylglucopyranoside respectively.

Single-cell transcriptome analysis uncovers underlying mechanisms of acute liver injury induced by tripterygium glycosides tablet in mice

Tripterygium glycosides tablet(TGT),the classical commercial drug of Tripterygium wilfordii Hook.F.has been effectively used in the treatment of rheumatoid arthritis,nephrotic syndrome,leprosy,Behcet's syndrome,leprosy reaction and autoimmune hepatitis.However,due to its narrow and limited treatment window,TGT-induced organ toxicity(among which liver injury accounts for about 40%of clinical reports)has gained increasing attention.The present study aimed to clarify the cellular and molecular events underlying TGT-induced acute liver injury using single-cell RNA sequencing(scRNA-seq)technology.The TGT-induced acute liver injury mouse model was constructed through short-term TGT exposure and further verified by hematoxylin-eosin staining and liver function-related serum indicators,including alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase and total bilirubin.Using the mouse model,we identified 15 specific subtypes of cells in the liver tissue,including endothelial cells,hepatocytes,cholangiocytes,and hepatic stellate cells.Further analysis indicated that TGT caused a significant inflammatory response in liver endothelial cells at different spatial locations;led to marked inflammatory response,apoptosis and fatty acid metabolism dysfunction in hepatocytes;activated hepatic stellate cells;brought about the activation,inflammation,and phagocytosis of liver capsular macrophages cells;resulted in immune dysfunction of liver lymphocytes;disturbed the intercellular crosstalk in liver microenvironment by regulating various signaling pathways.Thus,these findings elaborate the mechanism underlying TGT-induced acute liver injury,provide new insights into the safe and rational applications in the clinic,and complement the identification of new biomarkers and therapeutic targets for liver protection.

Phenylethanoid glycosides from traditional Mongolian medicine Cymbaria daurica alleviate alloxan-induced INS-1 cells oxidative stress and apoptosis

Cymbaria daurica L. is a well-known traditional Mongolian medicine, which has been used to treat diabetesrelated conditions characterized by persistent thirst and hunger, copious urination, and weight loss. We aimed to investigate the protective effects of C. daurica extracts and phenylethanoid glycosides including verbascoside and isoacteoside on INS-1 cells. We discovered phenylethanoid glycosides from n-butanol extract with large content through extraction and separation. We continue to study the protective effects of phenylethanoid glycosides including verbascoside and isoacteoside on INS-1 cells. INS-1 cells were treated with C. daurica, cell viability assay, RNA-seq technology, superoxide dismutase activity and malonaldehyde content, quantitative real time-PCR and Western blot analysis were used to study the protective effects of C. daurica. Cell viability assay resulted that n-butanol extract and verbascoside, isoacteoside showed protective effects of C. daurica. According to the RNA-seq technology to identify the differentially expressed genes in INS-1 cells, the pathway of gene enrich the protective effect of C. daurica on oxidative stress. SOD activity and the content of MDA indicated that C. daurica could enhance the antioxidant capacity of INS-1 cells. Further investigation indicated C. daurica alleviate oxidative stress by inhibiting INS-1 cell apoptosis. C. daurica may play an anti-diabetic role by inhibiting islet cell apoptosis.

The effect and mechanism of stilbene glycosides on improving neuronal injury in Alzheimer's disease rats by regulating ASK/MKK7/JNK pathway

Objective:To investigate the mechanism of action of tetrahydroxy stilbene glycosides(TSG)in ameliorating neuronal damage in Alzheimer's disease rats by regulating MKK7 and JNK kinases.Methods:A total of 24-month-old 42 SD rats were randomly selected for the experiment in 7 groups:normal group,sham-operated group,model group,positive drug group,low,medium and high dose TSG group at 0.033 g/kg,0.1 g/kg,0.3 g/kg.The Model Group and the TSG groups were established by stereotaxic Aβ25-35 solution.After 28 days,the model rats were selected by passive avoidance test.After screening,each dosage group of TSG and positive drug group was given intragastrically according to the corresponding dosage,and the experiment was carried out after 28 days.The pathological changes of hippocampal CA1 region were observed by tissue staining,and the amount of MKK7 and JNK proteins and the expression content of MKK7 and JNK mRNA by histochemical method of protein,and qRTPCR assay.Results:(1)He staining observation:Compared with the normal group and the sham-operated group,the number of nerve cells in the model group decreased and arranged irregularly,the cell membrane shrank,and the nucleus deformed and dissolved.The number of neurons in the positive drug group and TSG Group also increased significantly,the order is also relatively well.(2)From the results of the Tunel staining experiments:the positive apoptotic cells in the model group were higher than control group and sham-operated group,positive drug group and TSG drugs group was significantly smaller than that in the model group(P<0.05).(3)Compared with the control group and the Virtual Operation Group,the MKK7 and JNK protein concentrations in the brain of the model group were increased(P<0.05)by data analysis of immunohistochemistry:Compared with the model group,the protein expression of positive drug and TSG each dose group were reduced(P<0.05).(4)The results of QRTPCR data showed that the levels of MKK7 and JNK mRNA in the brain tissue of the model group were increased compared with the normal group and sham-operated groups(P<0.05).Conclusion:Stilbene glycoside has a certain effect on neuronal injury and repair which may be related to the changes of mRNA transcription and protein expression of MKK7 and JNK kinases.

川赤芍总苷提取工艺优化、组成和抗氧化活性分析

探究超声波辅助乙醇提取川赤芍总苷,通过单因素和响应面优化提取工艺,采用高效液相色谱(HPLC)法检测总苷组成,并通过体外抗氧化试验评价总苷的抗氧化活性。结果表明:最佳提取工艺为超声功率175 W、超声时间42 min、乙醇浓度70%、液料倍数30(mL/g),此工艺条件下提取的川赤芍总苷得率为(5.86±0.32)%。HPLC分析总苷共检测到21个特征峰,其中没食子酸0.63 mg/g、氧化芍药苷11.28 mg/g、儿茶素18.64 mg/g、芍药内酯苷5.82 mg/g、芍药苷54.82 mg/g、苯甲酰芍药苷15.74 mg/g、丹皮酚原苷0.98 mg/g,共占总质量的95.35%。总苷清除DPPH·、ABTS+·和·OH-的IC50值分别为0.78、1.13、0.98 mg/mL,总苷的抗氧化活性强弱与浓度呈现较好的正相关。

山银花茎叶化学成分及其抗炎活性研究

目的对山银花茎叶化学成分进行分离和结构鉴定,并进行体外抗炎活性评价。方法山银花茎叶80%甲醇提取物采用Diaion HP20SS、Sephadex LH-20、高速逆流色谱、反相半制备液相色谱等进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。通过测定单体化合物抑制LPS诱导小鼠巨噬细胞RAW264.7释放NO水平评价其抗炎活性。结果从山银花茎叶中分离得到13个化合物,分别鉴定为苄醇-O-β-D-吡喃葡萄糖-(1→6)-β-D-吡喃葡萄糖苷(1)、獐牙菜苷(2)、表断马钱子苷半缩醛内酯(3)、马钱子苷半缩醛内酯(4)、裂环马钱苷(5)、断氧化马钱苷(6)、裂马钱素二甲基乙缩醛(7)、绿原酸甲酯(8)、芹菜素-7-O-β-D-葡萄糖苷(9)、木犀草素-7-O-β-D-葡萄糖苷(10)、野漆树苷(11)、木犀草素-7-O-α-L-吡喃阿拉伯糖(1→6)-β-D-吡喃葡萄糖苷(12)、忍冬苷(13);化合物2~8、11~13可抑制LPS诱导RAW264.7细胞释放NO。结论化合物1首次从忍冬属植物中分离得到,化合物3、5、7、9、11~12首次从山银花茎叶中发现。化合物2~8、11~13具有一定的抗炎活性。

赤芍总苷药理作用研究进展

赤芍具有清热凉血、散瘀止痛的功效,广泛应用于临床,并于2002年列入可药食同源中药名录。现代研究发现,赤芍总苷系赤芍的主要活性成分,是鸟笼状蒎烷结构的单萜类化合物以及内酯结构的单萜类化合物的总称。10余年来,赤芍总苷不断有新的药理作用发现被报道,但未见系统地总结分析。因此,文章通过归纳国内外相关文献,综述了赤芍总苷自2010年至今被报道的抗肿瘤、抗心脑血管疾病、保肝、抗衰老、改善血流动力学和血液流变学、抗凝、抗血栓以及增强免疫、抗抑郁、抗慢性阻塞性肺疾病、抗溃疡病、抗盆腔炎、舒血管、保护脊髓损伤、保护血管内皮、治疗扁平苔藓及改善糖尿病肾病肾纤维化等药理作用,并提出研究不足与展望,以期为赤芍总苷深入地研究和开发应用提供参考。

何首乌叶不同萃取物的抗炎活性及其化学成分鉴定

目的:研究何首乌叶的成分,为其功能食品的研发提供基础数据。方法:对何首乌叶醇提取物进行有机溶剂萃取,分别得到何首乌叶石油醚、二氯甲烷、乙酸乙酯、正丁醇、水等不同萃取物,并对不同萃取物进行抗炎活性检测,后续通过硅胶柱色谱、C18柱色谱结合半制备液相等方法对抗炎活性较好的成分进行分离、纯化以及结构鉴定,结果:在质量浓度0~100μg/mL范围内,乙酸乙酯萃取物对RAW264.7细胞存活率无明显的抑制作用,且剂量依赖地抑制NO的生成。石油醚和二氯甲烷萃取物在质量浓度0~25μg/mL时,均无明显细胞毒性,并在25μg/mL时,二氯甲烷萃取物对NO的抑制率(51.47%)高于石油醚萃取物的抑制率(43.91%)。从二氯甲烷和乙酸乙酯萃取物中分离、鉴定出11个化合物,分别为2,3,4,6-三羟基苯乙酮-3-O-β-D-glucoside(1)、杨梅苷(2)、槲皮苷(3)、阿福豆苷(4)、β-谷甾醇(5)、正十六烷-5,8,11三烯酸(6)、二十六烷(7)、α-亚麻酸(8)、山柰酚-3-O-芸香糖苷(9)、肉豆蔻酸(10)和槲皮素(11)。结论:何首乌叶具有很好的抗炎活性,化合物2,4,6,7,8,9,10,11均为首次从何首乌中分离得到。

穿心莲糖苷水解酶基因ApGH的克隆、生物信息学分析及表达研究

目的:对穿心莲糖苷水解酶(ApGH)基因进行克隆、生物信息学分析、原核表达和相对表达量分析。方法:取穿心莲新鲜叶片,提取RNA并反转录为互补脱氧核糖核酸(cDNA)作为模板,以穿心莲转录组中筛选到的糖苷水解酶ApGH基因开放阅读框(ORF)设计特异性引物,进行聚合酶链式反应(PCR)扩增,经测序后获得的基因序列利用生物信息学软件预测基因编码蛋白特征,构建原核表达载体在大肠埃希菌中诱导表达目的蛋白质,并利用实时荧光定量PCR分析ApGH基因的表达模式。结果:克隆所得ApGH基因的ORF全长1734 bp,编码577个氨基酸(GenBank登录号:OR887606)。ApGH为稳定亲水蛋白质,无信号肽和跨膜结构域,属于糖苷水解酶家族;系统进化分析表明,ApGH归属于GH1家族。将ApGH基因构建到原核表达载体HIS-MBP-pET28a上,在大肠埃希菌Transetta(DE3)中成功表达出重组蛋白质。实时荧光定量PCR检测结果表明,ApGH基因在叶中表达量最高,茎和根中表达量较低。结论:克隆得到穿心莲ApGH基因并对其蛋白质序列特征进行了系统分析,证明其在大肠埃希菌中成功表达重组蛋白质,且主要在穿心莲叶中表达。研究结果为进一步探索穿心莲糖苷水解酶的催化功能提供了依据。

不同来源金线莲组培苗中总黄酮、多糖和内酯苷含量分析

目的比较不同来源金线莲组培苗中总黄酮、多糖和内酯苷的含量。方法采用紫外分光光度法测定不同来源金线莲组培苗中总黄酮和多糖的含量,采用HPLC-ELSD法测定不同来源金线莲组培苗中2种内酯苷含量。结果不同来源金线莲组培苗中总黄酮含量范围为(8.21~16.50)mg·g^(-1),多糖含量范围为(41.40~112.89)mg·g^(-1),金线莲苷含量范围为(83.68~171.67)mg·g^(-1),3(R)-O-β-D-吡喃葡萄糖基-4-羟基-丁酸含量范围为(12.41~29.45)mg·g^(-1)。结论不同来源金线莲组培苗中总黄酮、多糖和内酯苷含量存在差异。

正交试验优化鸡矢藤总环烯醚萜苷的提取工艺

为研究鸡矢藤叶中总环烯醚萜苷的最佳提取工艺,以总环烯醚萜苷提取率为指标,通过单因素试验和正交试验,考察乙醇浓度、提取时间、料液比、提取温度、提取次数等因素的影响。结果显示总环烯醚萜苷的最佳提取条件为:80%乙醇作为提取溶剂、提取时间2 min、料液比1∶90 g/mL、提取温度45℃、提取次数6次,在此条件下总环烯醚萜苷的提取率达到46.59 mg/g。本文研究的提取方法高效、稳定、合理,可为鸡矢藤叶中总环烯醚萜苷的研究奠定一定基础。

马鞭草苯乙醇苷类成分研究

目的研究马鞭草Verbenae Herba的苯乙醇苷类成分。方法马鞭草80%乙醇提取物采用硅胶、Sephadex LH-20、TLC、半制备HPLC进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。结果从中分离得到9个化合物,分别鉴定为verbofficoside A(1)、肉苁蓉苷D(2)、广防风苷A(3)、毛蕊花糖苷(4)、异毛蕊花糖苷(5)、肉苁蓉苷C(6)、肉苁蓉苷F(7)、去咖啡酰基毛蕊花苷(8)、jionoside C(9)。结论化合物1为新化合物,化合物3、6~9均首次从该植物中分离得到。

CRISPR/Cas9编辑MeHNL基因创制低生氰糖苷木薯

【目的】木薯(Manihot esculenta Crantz)中含有潜在毒性的生氰糖苷,其食用安全性受到影响且导致加工成本增加。因此,利用生物技术开展低生氰糖苷木薯的培育具有重要意义。【方法】利用CRISPR/Cas9技术对木薯醇氰酶基因MeHNL进行了编辑。该基因编码催化生氰糖苷分解的α-羟基腈裂解酶,编辑靶点位于第1个外显子上,通过农杆菌介导的稳定转化获得了27株阳性植株。【结果】测序分析显示,其中26个株系被编辑,编辑效率高达96.3%。编辑类型主要包括碱基的插入和缺失,少数为碱基替换和大片段缺失。氰化物检测试剂盒染色和HPLC测定分析表明,编辑株系中的氢氰酸和生氰糖苷含量均显著降低。与非编辑植株相比,编辑植株的叶片细长,暗示了MeHNL可能对木薯的生长发育产生影响。【结论】利用CRISPR/Cas9技术获得了低氰化物的木薯种质,为开展生氰糖苷代谢影响木薯生长发育的研究提供了材料。

雷公藤多苷纳米粒的制备及其对关节炎大鼠的治疗作用

目的 制备雷公藤多苷纳米粒,探究其对胶原诱导型(collagen-induced arthritis, CIA)关节炎大鼠的治疗作用。方法 运用薄膜分散法制备雷公藤多苷纳米粒,对其进行质量评估。构建CIA模型,进行药物干预,称量大鼠体质量、测定足趾肿胀度和关节炎指数;观察大鼠脏器、膝、踝关节滑膜的病理改变;检测大鼠血清中肝肾功能水平和炎症因子表达。结果 制备的雷公藤多苷纳米粒在电镜下呈圆粒状且分布均匀,性质稳定。相较于模型组,给药组大鼠左右足趾肿胀度均明显下降(P<0.01),关节炎指数明显降低(P<0.01)。其中TG-NPs组的疗效优于TG组。与正常组相比,大鼠心脏、脾、肾、睾丸指数均明显降低(P<0.05,P<0.01)。TG-NPs组膝踝关节软骨病理损伤明显减轻,凋亡的滑膜细胞增加;与模型组相比,TG-NPs组大鼠血清中的ALT、BUN、CRE水平均明显降低(P<0.05),IL-1β、TNF-α和IL-6含量下降明显(P<0.05)。结论 TG-NPs通过诱导滑膜细胞凋亡和降低炎性细胞因子的表达对CIA有较好的治疗作用,通过静脉注射血液循环,可实现药物的缓、控释,避免口服药物造成的首过效应,减轻脏器的毒性,为治疗类风湿关节炎的新型纳米制剂的开发提供了实验依据。