Antioxidant,antimicrobial,and α-glucosidase inhibitory activities of saponin extracts from walnut(Juglans regia L.) leaves

Objective:To investigate the relationship between triterpenoid saponin content and antioxidant,antimicrobial,and α-glucosidase inhibitory activities of 70%ethanolic,butanolic,aqueous,supernate and precipitate extracts of Juglans regia leaves.Methods:Triterpenoid saponins of different Juglans regia leaf extracts were measured by the vanillin method.Antioxidant activity was evaluated against DPPH and ABTS free radicals.We also assessed α-glucosidase inhibitory and antimicrobial activities of the leaf extracts.Pearson’s correlation coefficient was evaluated to determine the correlation between the saponin content and biological activities.Results:The butanolic extract was most effective against DPPH with an IC50of 6.63μg/mL,while the aqueous extract showed the highest scavenging activity against ABTS free radical with an IC50of 42.27μg/mL.Pearson’s correlation analysis indicated a strong negative correlation (r=-0.956) between DPPH radical scavenging activity (IC50) and the saponin content in the samples examined.In addition,the aqueous extract showed the best α-glucosidase inhibitory activity compared with other extracts.All the extracts had fair antibacterial activity against Bacillus subtilis,Escherichia coli,and Klebsiella pneumoniae except for the aqueous extract.Conclusions:Juglans regia extracts show potent antioxidant,antimicrobial,and α-glucosidase inhibitory activities.There is a correlation between saponin levels in Juglans regia leaf extracts and the studied activities.However,additional research is required to establish these relationships by identifying the specific saponin molecules responsible for these activities and elucidating their mechanisms of action.

核桃(Juglans regia L.)维生素E代谢相关基因jrVTE3的克隆与分析

核桃(Juglans regia L.)坚果是重要的天然维生素E源。在维生素E合成代谢中,2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQMTase)催化2-甲基-6-植基-1,4-苯醌(MPBQ)向2,3-二甲基-5-植基-1,4-苯醌(DMPBQ)的合成,是γ-生育酚和α-生育酚生物合成过程中的关键酶。本研究以‘香玲’核桃开花后90 d的胚组织为材料,利用RACE技术克隆了核桃MPBQMT的cDNA。序列分析表明,得到的cDNA全长1280 bp,有1020 bp的开放阅读框(ORF),编码340个氨基酸,将其命名为jrVTE3。序列同源性比较显示,jrVTE3基因与毛果杨MPBQ/MSBQ甲基转移酶基因以及蓖麻、葡萄、大豆的内膜包装蛋白基因有同源性较高;其ORF编码的多肽序列与橡胶、莴苣、毛果杨的MPBQ/MSBQ转移酶及葡萄、蓖麻的内膜包装蛋白有较高的同源性。jrVTE3编码的多肽序列中,有15个氨基酸可能为蛋白激酶磷酸化位点,多肽序列二级结构中有α–螺旋、无规则卷曲。在多肽序列中,有一个低成分复杂性区域和一个跨膜区段,还有一个第一类S-腺苷甲硫氮酸甲基转移酶(AdoMet-MTases,Class I)的保守域,这些序列结构体现了甲基转移酶结构的特点。这些结果对进一步研究核桃坚果仁中维生素E的合成的调控机制及核桃品质的遗传改良具有参考意义。

核桃(Juglans regia L.)青皮提取物抗氧化及抑菌活性的研究

核桃青皮是核桃(Juglans regia L.)加工的副产物,因含有多种活性成分,可能是一种潜在的天然抗氧化剂和抑菌剂来源。本研究旨在研究核桃青皮提取物的抗氧化能力和抑菌活性,为开发新的饲用抗生素替代品提供参考。以蒸馏水及25%、50%、75%和100%的甲醇、乙醇,丙酮为溶剂分别提取8和12h获得核桃青皮提取物。通过测定提取物中的总酚和总黄酮含量,根据还原能力和2,2-二苯基-1-苦肼基(DPPH)自由基清除率评价其抗氧化能力;以大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌为受试菌,分别测定抑菌圈直径、最小抑菌浓度(MIC)及抑菌率评价其抑菌活性。结果表明,有机溶剂浓度显著影响提取物中的总酚和黄酮含量(P<0.01);除丙酮外,提取时间对提取物中总酚和黄酮含量均有显著的影响(P<0.05);在所有提取溶剂中,以25%乙醇提取8h获得的总酚和黄酮最高,分别达到25.35和12.63mg·g-1。有机溶剂浓度对提取物的还原能力具有显著的影响(P<0.01),而提取时间仅对丙酮提取物的还原能力具有显著影响(P<0.05),其中25%乙醇提取物的还原能力在各处理中最高,为0.366。有机溶剂浓度对提取物的DPPH自由基清除率具有显著的影响(P<0.01),而提取时间仅对甲醇提取物的自由基清除率有显著影响(P<0.01),其中75%丙酮提取物可清除88.81%的自由基,在各处理中最高。有机溶剂浓度对大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌的抑菌圈直径和最小抑菌浓度(MIC)均有显著的影响(P<0.01);从提取时间来看,除甲醇提取物对大肠杆菌以及丙酮提取物对枯草芽孢杆菌的抑菌圈直径无显著影响外(P>0.05),其他均有显著的差异(P<0.01);此外,提取时间显著影响对大肠杆菌的MIC(P<0.05),乙醇和丙酮提取物的金黄色葡萄球菌MIC以及丙酮提取物的枯草芽孢杆菌MIC有显著差异(P<0.05),其他提取物的MIC均不受提取时间的影响(P>0.05)。抑菌率测试结果表明核桃青皮25%乙醇提取物对枯草芽孢杆菌的抑菌活性强于对大肠杆菌和金黄色葡萄球菌的抑制。由此可见,所有核桃青皮提取物均具有一定的抗氧化及抑菌活性,尤其以25%乙醇8h提取物的作用最好。

Two new diarylheptanoids from the pericarps of Juglans regia L.

Two new cyclic diarylheptanoids juglanin A （1） and juglanin B （2） were isolated from the pericarps of Juglans regia L. Their structures were elucidated by various spectroscopic methods including 2D NMR techniques （COSY, HMQC, HMBC, NOESY） and HR-ESIMS.

基于SSR标记的中亚生态区核桃(Juglans regia L.)遗传多样性与种群结构分析

【目的】从分子水平探究中国新疆与吉尔吉斯斯坦和塔吉克斯坦2个中亚国家共6个区域核桃种质资源间的遗传多样性及亲缘关系。【方法】采用SSR分子标记方法对采自6个区域的137份核桃种质资源进行遗传多样性和居群遗传结构分析。【结果】利用14对引物共检测到130个等位变异位点,变异范围为6~13,平均每对SSR引物可检测到9.2857个等位位点;14个SSR位点的占比范围为0.6257~0.8651,平均值0.7634;平均Shannon’s多样性指数(I)为0.4598;种群总基因多样性(Ht)平均值为3.2481;种群内基因多样性(Hs)平均值为2.1089;种群间的遗传分化指数(Gst)范围为0.0578~0.4068,平均值为0.2595;基因流(Nm)平均值为3.0174;各个等位基因频率整体分布不均匀。UPGMA聚类结果显示,137份供试材料在遗传相似性系数为0.75时分成3个类群。其中,第一类群共42份,包括中国新疆阿克苏地区16份,中国新疆和田地区21份,吉尔吉斯斯坦5份;第二类群共44份,包括中国新疆伊犁地区12份、吉尔吉斯斯坦32份;第三类群共51份,包括中国新疆喀什地区19份、塔吉克斯坦13份和中国新疆伊犁地区19份。聚类结果在一定程度上表明了居群间亲缘关系的距离,总体分布与地理分布有关,不同区域之间存在一定的基因交换。聚类结果与居群间遗传距离和遗传一致度结果一致。【结论】SSR分子标记结果表明,中亚生态区不同地理分布的核桃种质资源遗传多样性存在差异,为揭示中亚生态区核桃种群结构及传播途径提供了依据。

Effect of Fertilizer Application on the Yield of Walnut (Juglans regia L.)

[Objectives] This study was conducted to explore the fertilization ratio of walnut, so as to provide a basis for rational fertilization of walnut. [Methods] Under the condition of gravelly yellow soil in Chaotian District, Guangyuan City, the orthogonal design of L_9(3~4) was used to test the fertilization ratio of nitrogen, phosphorus and potassium fertilizers. [Results] The effects of nitrogen, phosphorus and potassium fertilizers on walnut yield ranked as phosphate fertilizer>potassium fertilizer>nitrogen fertilizer. The different treatments had great effects on the yield of walnut, and for the land with gravelly yellow soil, the suitable annual fertilization amount of walnut was urea 1 200 g/plant + calcium superphosphate 1 500 g/plant + potassium sulfate 940 g/plant. [Conclusions] This study provides a theoretical basis for rational fertilization of walnut in Sichuan Province.

Acaricidal Activity of Extracts of Juglans regia L. on Tetranychus urticae Koch （Acari： Tetranychidae）

Two-spotted spider mite （Tetranychus urticae Koch） （Acari： Tetranychidae） is a very serious pest worldwide, causing considerable damage to vegetables, flowers and fruit crops. The application of plant extracts to control insects can be an effective alternative. In this study, the efficacy of an extract from Juglans regia L. （Juglandecaea） was tested as an alternative to conventional acaricides. Using two different methods, bioassays were used to determine the effects of varying concentrations J. regia （1%, 3%, 6%, 12%） of the extracts. Experiments were carried out using 3 cm diameter leaf disks of Phaseolus vulgaris L. In addition, the effects of the extract on mite reproduction and oviposition were investigated. As a result, in the leaf dipping method, the 12% concentration of the extract caused the highest mortality of nymph （90%） and adult （83.00%） stages. For the spraying method, the mortality of adults at the same concentration was 100%. In bioassays at lower concentrations, the adult mites laid lower numbers of eggs in comparison to the untreated mites. The highest effect occurred at concentration of 12% and the lowest effect was at 1%. The extract was no observed ovicidal effect.

Population genetic insights into the conservation of common walnut(Juglans regia)in Central Asia

The common walnut(Juglans regia)is one of the most economically important nut trees cultivated worldwide.Despite its importance,no comprehensive evaluation of walnut tree population genetics has been undertaken across the range where it originated,Central Asia.In this study,we investigated the genetic diversity and population structure of 1082 individuals from 46 populations across Central Asia.We found moderate genetic diversity of J.regia across Central Asia,with 46 populations clustered into three groups with a weak relationship between genetic and geographic distance.Our findings reveal that the western Himalaya might be the core region of common walnut genetic diversity in Central Asia and that,except for two populations in Gongliu Wild Walnut Valley,humans might have introduced walnut populations to Xinjiang,China.The observed distribution of the genetic landscape has probably been affected by historical climate fluctuation,breeding system,and prolonged anthropogenic activity.We propose the conservation of the core genetic diversity resources in the western Himalaya and pay special attention to populations from Gongliu in Xinjiang.These findings enhance our understanding of the genetic variation throughout the distribution range of J.regia in Central Asia,which will provide a key prerequisite for evidence-based conservation and management.

核桃(Juglans regia)SRAP标记反应体系建立的研究

以核桃早实优系“绿园”和晚实优系“绿丰”及其F1杂交后代为试材,对核桃SRAP标记反应体系及SRAP标记在核桃中的多态性进行了研究。Mg2+是影响SRAP-PCR反应的重要因子,当Mg2+浓度为2.5mmol/L时,能得到较好的扩增效果。利用11对引物组合对两亲本及其杂交后代进行了SRAP-PCR扩增,以引物组合Em10-Me4多态性最高,在154个杂交后代单株中,扩增出1509条DNA条带,其中多态性条带为824条,多态性比率达54.6%。结果表明,SRAP标记可广泛应用于核桃的遗传多样性分析、种质鉴定、遗传连锁图谱的构建等方面的研究。

核桃(Juglans regia L．)光合影响因子的研究

以普通核桃'清香'为试材,利用CI-301PS便携式光合作用测定仪,在田间条件下,测定了核桃净光合速率的日变化及季节变化,进行了核桃净光合速率与光合影响因子的相关性研究,为核桃栽培管理提供一定的科学依据。结果表明:核桃净光合速率日变化与叶温极显著正相关,与光合有效辐射、蒸腾速率、气孔导度显著正相关,与胞间CO_2浓度极显著负相关。核桃净光合速率季节变化与蒸腾速率呈极显著正相关,与叶绿素含量呈显著正相关,与胞间CO_2的浓度呈显著负相关。

核桃(Juglans regia L.)MicroRNA实时荧光定量RT-PCR内参基因的筛选

RT-qPCR是目前应用最广泛的基因表达分析方法,选择合适的内参基因对数据进行标准化至关重要。结合geNorm、Normfinder和RefFinder 3个软件分析了8个候选miRNA内参基因在核桃不同分化期花芽与叶芽、不同组织及不同品种中的表达稳定性。结果显示,jre-miR394a,jre-miR159a和jre-miR159c可作为不同分化期花芽中基因表达分析的内参;5.8S rRNA和jre-miRn3可作为不同分化期叶芽中基因表达分析的内参;5.8S rRNA,jre-miRn3、jre-miR159b-3p和jre-miR159c可作为不同组织中基因表达分析的内参;jre-miR159b-3p,jre-miR159c和jre-miR159a可作为不同品种中基因表达分析的内参。5.8S rRNA不适合作为不同分化期花芽和不同品种中基因表达分析的内参。本研究为核桃miRNA表达分析中的数据标准化提供数据支持和理论参考。

基于UPLC-Q-Exactive MS技术3种核桃类种仁中甘油酯组成比较

基于超高效液相色谱-四级杆-轨道阱高分辨质谱联用(UPLC-Q-Exactive MS)技术比较分析了山核桃(Carya cathayensis Sarg.)、胡桃(Juglans regia Linn.)和薄壳山核桃〔Carya illinoinensis(Wangenh.)K.Koch〕3种木本油料作物种仁中甘油酯的组成及差异,结合LipidSearch软件和混合脂质内标对脂质分子进行定性及定量分析。结果显示:3种核桃类种仁中均鉴定到298个甘油酯,包括245个甘油三酯、38个甘油二酯、1个单半乳糖甘油二酯、9个双半乳糖甘油二酯和5个硫代异鼠李糖甘油二酯。偏最小二乘法判别分析得到104个变量投影重要度(VIP)大于1的脂质分子。山核桃和薄壳山核桃种仁中甘油三酯的主要脂肪酸为油酸,胡桃种仁中甘油三酯的主要脂肪酸为亚油酸。3种核桃类种仁中甘油二酯的主要脂肪酸均为亚油酸,其次为油酸。山核桃和薄壳山核桃种仁的甘油酯组成更为相近。UPLC-Q-Exactive MS可作为鉴别这3种木本油料作物种仁中甘油酯组成的有效方式。

基于转录组测序的核桃(Juglans regia L.)SSR标记开发

本研究通过高通量测序技术结合MISA软件分析核桃转录组中SSR的分布类型及特点。通过Primer 3设计得到426对SSR引物,以来源于新疆林业科学院的10份核桃种质对随机合成的10对引物进行多态性筛选,并对核桃转录组数据进行了SSR位点搜索和分析,继而开发SSR标记。利用MISA软件在132 154条Unigenes共检测出63 400个SSR位点,分布于47 169条Unigene中,出现频率为47.97%,平均分布距离为2.55 kb。优势重复基序为单核苷酸、二核苷酸和三核苷酸,分别占总SSR位点的55.73%,34.48%和8.63%。二核苷酸重复基元中以AG/CT为优势重复基元,占二核苷酸重复的64.77%,三核苷酸重复基元以AAG/CTT为主。10对引物进行多态性验证分析结果显示:8对有效扩增引物中,7对引物在10份不同核桃种质中表现出多态性。以上结果表明,核桃转录组测序产生的Unigene信息可作为开发SSR标记的有效来源。开发的大批量SSR标记可为核桃种质资源遗传多样性分析和遗传图谱构建提供更加丰富可靠的标记选择。

In vitro antibacterial and free radical scavenging activity of green hull of Juglans regia

Abstract Antioxidant supplements from plants are vital to count the oxidative damage in cells. We assessed the antioxidants and antibacterial activity of green hull of Juglans regia in this study. According to our results the maximum antibacterial activity was observed in ethanolic extract when compared to other extract. So, the ethanolic extract was studied for antioxidant activity which exhibited high antiradical activity against DPPH, hydroxyl, and nitric oxide radicals. In conclusion, green hull of J. regia showed strong reducing power activity and total antioxidant capacity. The results justify the therapeutic application of plant in the indigenous system of medicine.

Anti-diabetes Agents——I:Tetralone Derivative from Juglans regia

A new compound, 4-hydroxy-a-tetralone-4-O-b-D-[6-O-(3,4,5-trihydroxybenzoyl) glucopyranoside (1), together with a known compound, 4-hydroxy-a-tetralone (2), has been isolated from the roots of Juglans regia. 2 showed moderate bioactivity against protein tyrosine phosphatase 1B (PTP1B).

Two new glucosides from the pellicle of the walnut (Juglans regia)

A newα-tetralonyl glucoside,6'-O-acetyl-juglanoside E(1),and a new dihydrophaseic acid glucoside,dihydrophaseic acid 1-O-(6-O-acetyl)-glucopyranoside(2),together with two known ones,juglanoside E(3)and dihydrophaseic acid(4),were isolated from the pellicle of the walnut(Juglans regia).The structures of the new compounds were elucidated by comprehensive spectroscopic analysis,including IR,HRESIMS,1D and 2D NMR data.

丛枝菌根真菌对NaCl胁迫下核桃幼苗根系生理及矿质元素吸收的影响

为评估丛枝菌根真菌(AMF)对核桃(Juglans regia Linn.)幼苗耐盐性的促进效果,以核桃品种‘温185’(‘Wen 185’)的盆栽苗为材料,分别接种摩西斗管囊霉〔Funneliformis mosseae(T.H.Nicolson et Gerd.)C.Walker et A.Schüler〕和异形根孢囊霉〔Rhizophagus irregularis(Blaszk.,Wubet,Renker et Buscot)C.Walker et A.Schüler〕,测定了0、100、200、300 mmol·L^(-1) NaCl处理下根系的生长指标、抗氧化酶活性、渗透调节物质含量、内源激素含量及叶片和根系中矿质元素含量。结果表明:NaCl胁迫下,核桃幼苗的根系生长受到抑制,在300 mmol·L^(-1) NaCl处理下接种2种AMF均显著(P<0.05)提高了根系体积。随着NaCl浓度升高,根系抗氧化酶活性及过氧化氢(H_(2)O_(2))和丙二醛(MDA)含量整体上显著升高;在300 mmol·L^(-1) NaCl处理下,接种摩西斗管囊霉显著提高了根系超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性,接种异形根孢囊霉显著提高了根系APX活性,接种2种AMF均显著降低了根系H_(2)O_(2)和MDA含量。在300 mmol·L^(-1) NaCl处理下,接种2种AMF均显著提高了根系可溶性糖含量。各浓度NaCl处理下,接种2种AMF均显著提高了根系吲哚乙酸、脱落酸、赤霉素3和玉米素核苷含量。随着NaCl浓度升高,叶片P、K、Ca、Mg含量整体呈上升趋势;在300 mmol·L^(-1) NaCl处理下,接种2种AMF均降低了叶片和根系中P含量,整体显著提高了根系K和Ca含量,接种异形根孢囊霉还显著提高了叶片和根系中Mg含量。在300 mmol·L^(-1) NaCl处理下,接种异形根孢囊霉显著提高了根系Fe含量和叶片Zn含量,接种摩西斗管囊霉显著提高了根系Fe含量。随着NaCl浓度升高,未接菌幼苗叶片和根系中Na含量整体呈上升趋势,而K/Na比、Ca/Na比和Mg/Na比则相反;在300 mmol·L^(-1) NaCl处理下,接种2种AMF均显著降低了根系Na含量,且接种摩西斗管囊霉还显著降低了叶片Na含量并显著提高了叶片和根系中K/Na比、Ca/Na比和Mg/Na比。综上所述,摩西斗管囊霉和异形根孢囊霉对核桃幼苗的耐盐性提高具有明显促进作用,且这2种AMF的作用机制可能存在差异。

Isolation and Characterization of Novel Microsatellite Markers in Walnut (<i>Juglans regia</i>L.)

Thirteen microsatellite markers were isolated and characterized from Juglans regia L. Successful polymorphism of the 13 primer pairs was observed in various genotypes of J. regia. The number of polymorphic alleles ranged from 2 to 4 (with an average of 4.35). The polymorphic information content values ranged from 0.47 to 0.88 (with an average of 0.69). TC/AG and GAA/CTT class of repeats were the most abundant di-nucleotide and tri-nucleotide repeats, respectively.

核桃SPL基因家族的系统进化和表达分析

【目的】SQUAMOSA promoter binding protein-like(SPL)转录因子在植物开花诱导中起重要的调控作用。从核桃基因组中鉴定SPL(JrSPL)基因家族成员并分析其表达特性,为探究SPL在核桃开花诱导中的功能研究提供参考。【方法】采用生物信息学方法鉴定JrSPL基因家族成员,并预测其基本理化性质、保守结构域、进化关系、启动子顺式作用元件等;利用转录组测序和RT-qPCR技术分析JrSPL家族成员在核桃不同组织及嫁接诱导开花后的表达水平。【结果】JrSPL家族有28个成员,其基因结构和蛋白结构均高度保守,分布在核桃14条不同的染色体,与拟南芥和毛果杨分别存在17处和24处共线性关系,根据系统发育关系可分为8组。JrSPL启动子存在大量的光响应元件、激素应答元件、胁迫响应元件等。转录组测序分析显示,JrSPL基因在核桃不同组织表达量有差异,在雄花、雌花、顶芽及叶中均有表达,多数基因在雌花中的表达量都比较高,有6个基因最为显著,它们可能在开花诱导中起关键作用。在嫁接诱导核桃开花材料中,检测的12个JrSPLs中除了JrSPL8外,在开花材料中的表达量均高于对照,JrSPL2和JrSPL25在混合花序的雄花中表达显著,JrSPL8在顶芽中高表达。【结论】JrSPL基因在核桃成花中具有重要作用,其高表达是花期提前的主要因素。

Gene flow among wild and cultivated common walnut(Juglans regia)trees in the Qinling Mountains revealed by microsatellite markers

Patterns of gene flow and gene introgres sion can be used to assess the risk of genetic pollution of wild forest trees from widespread cultivated trees.A comprehensive understanding of the genetic relationships and levels of gene flow among wild and cultivated common walnut(Juglans regia)has become an urgent issue.Using twelve microsatellite markers,we investigated the genetic diversity and gene flow between cultivated and wild trees of J.regia in the Qinling Mountains,China.A high level of genetic variation was detected in both cultivated and wild trees.The mean number of alleles per locus was 17.5.Observed heterozygosity(HO)and expected heterozygosity(HE)were 0.777 and 0.800,respectively.Pollination of mother trees was not by nearest neighbors,and a paternity of 60.7%of offspring evaluated could not be assigned to a local,sampled tree.Pollen flow from cultivated trees to wild trees was infrequent(5.4%),and selfing rates ranged from zero to 25.0%.Male parents were located from 0 to 1005 m from their female partners,with an average pollination distance of 285.1 m.These results are discussed in light of the cultivated species’diversity,outlining the frequent spontaneous genetic contributions from the wild to the cultivated compartment.In addition,the pollen flow parameters provide useful information about the dynamics of pollen movement within J.regia populations.