Effect of Three Precursors on Accumulation of Principal Volatile Oil Constituents in Tissue Culture Plantlets of Atractylodes lancea (Thunb.) DC.

[Objective] This study was to explore the effect of three precursors on the accumulation of principal volatile oil constituents in tissue culture plantlets of Atractylodes lancea （Thunb.） DC. with the aim to provide references for the improvement of artificial cultivated A. lancea quality. [Method] Three precursors were added into the MS rooting medium for A. lancea tissue culture plantlets and the volatile oil was extracted by ultrasonication after cultured for several days. The content of atractylon, atractylol, β-eucalyptol and atractydin in the volatile oil were determined by using gas chromatography method. [Result] The addition of xylose, isoprene and tetrahydrofuran impacted the growth indicators, yield of volatile oil and relative percentage content of the four constituents of A. lancea tissue culture plantlets. In the 6 g/L xylose optimized medium, the atractylon and β-eucalyptol content reached up to 4.23% and 56.34%, respectively, 1.41% and 1.66% higher than the control; although the addition of isoprene into medium raised the atractylon content, the accumulation of total volatile oil was inhibited that it decreased by 23.67%, 31.06% and 7.10% to the control; for the tetrahydrofuran optimized medium, the content of atractylon, atractylol and atractydin all increased, and the total volatile oil content increased by 49.97% to the control when the concentration of tetrahydrofuran was 0.07 g/L. [Conclusion] The addition of xylose and tetrahydrofuran promoted the accumulation of principal constituents of the volatile oil, whereas the addition of isoprene inhibited the accumulation.

苍术（Atractybeslancea（Thunb．）DC．）种内遗传多样性分析

分析了苍术（Ａｔｒａｃｔｙｌｏｄｅｓｌａｎｄｅａ（Ｔｈｕｎｂ．）ＤＣ．）５种等位酶（ＭＤＨ，ＧＤＨ，ＰＰＯ，ＳＯＤ，ＰＥＲ）１２个位点上２５个等位基因的分化特征。结果表明：群体内多态位点比例平均为０．６１，每个位点的平均杂合率为０．２６，每个位点发现的平均等位基因数为１．７８。苍术种内基因多样性的８６％产生在群体内，群体间分化的明显效果为１４％。所有配对组合，群体间的平均遗传距离是０．１１，遗传同一性是０．９０。说明苍术种内变异很大。同时探讨茅苍术的分类位置，认为茅苍术不是苍术和白术的杂交种。

Study on Rapid Propagation Medium of Atractylodes Lances by Orthogonal Design

[Objective] The aim was to protect wild resource of Atractylodes lances and to provide technical guidance for industrialization of Atractylodes lances. [Method] The test-tube seedlings were produced from seeds of Atractylodis Lances. At the same time, the effects of 6-BA, NAA and KT on the proliferation of aseptic seedling was studied through the orthogonal test. Effect of NAA concentration on the rooting of aseptic seedling was studied through nine types of light substrate groups, which consist of peat soil, fine sand and cotton seed hull, and their effects on Atractylodes lances growth were studied on with orthogonal design. [Result] The culture medium suitable for the proliferation of the Atractylodes lances tube seedling was MS＋6-BA 1.0 mg/L＋NAA 0.4 mg/L＋KT 0,4 mg/L. And the medium suitable for rooting was 1/2MS＋NAA 0.5 mg/L. The optimal ratio of soil, riversand and cotton seed hull was 3：3：2. [Conclusion] The research selected optimum culture medium, rooting medium and ratio of substrate, which provided technical guidance for laboratorial preservation of germplasm resource of Atractylodes lances and industrialization of its seedling production.

基于Nrf2-Keap1通路探讨小槐花不同提取物对CCl_(4)所致肝损伤的保护机制

目的探讨小槐花不同提取物对CCl_(4)所致肝损伤的保护机制。方法采用0.5%CCl_(4)花生油溶液构建小鼠肝损伤模型。苏木精-伊红(hematoxylin-eosin staining,HE)染色观察肝脏病变;试剂盒检测丙氨酸转氨酶(alanine amin-otransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、活性氧(reactive oxygen species,ROS)水平;Western blot检测肝脏组织中核因子促红细胞生成素2相关因子2(nuclear factor erythropoietin-2-related factor 2,Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Kelch like epichlorohydrin related protein-1,Keap1)、血红素加氧酶1(heme oxyge-nase 1,HO-1)蛋白的相对表达量。结果与模型组相比,小槐花水提物与醇提物中、高剂量组能显著降低肝重指数以及血清中ALT、AST水平(P<0.05,P<0.01)。小槐花水提物与醇提物中、高剂量组小鼠肝组织匀浆MDA、TNF-α、IL-6、ROS含量显著降低(P<0.05,P<0.01),SOD、CAT酶活性显著升高(P<0.05,P<0.01),GSH含量显著升高(P<0.05,P<0.01)。其中小槐花水提物作用效果优于醇提物。小槐花水提物高剂量组显著上调Nrf2、HO-1,下调Keap1蛋白的表达(P<0.05)。结论小槐花不同提取物对CCl_(4)肝损伤小鼠具有保护作用,水提物保肝效果优于醇提物,作用机制与激活Nrf2-Keap1通路,减轻氧化应激反应有关。

民族药小槐花质量标志物候选成分的研究

目的:开展对豆科山蚂蝗属药用植物小槐花(Desmodium caudatum(Thunb.)DC.)叶的化学成分研究,为小槐花质量标准体系的建立提供物质基础。方法:将小槐花叶经70%乙醇提取后,综合运用大孔吸附树脂柱色谱、硅胶柱色谱、制备型高效液相色谱等方法分离和纯化小槐花叶的化学成分,根据所得单体理化性质及波谱数据鉴定各化合物结构。结果:从小槐花叶中分离鉴定了13个化合物:清缸酒酚(1)、柠檬酚(2)、8-异戊烯基山柰酚(3)、二氢山柰酚(4)、neophellamuretin(5)、异柠檬酚(6)、槲皮素(7)、山柰酚(8)、牡荆素(9)、当药黄素(10)、大豆皂醇B(11)、黄槿酮A(12)、黄槿酮D (13),其中化合物7、8、12和13为小槐花中各药用部位均含有的成分。结论:化合物7、8、12和13可作为小槐花的候选质控成分。

响应面法优化小槐花总黄酮提取工艺及抗氧化活性

采用响应面设计优化小槐花[Desmodium caudatum(Thunb.)DC.]的总黄酮提取工艺,在单因素试验结果的基础上,以提取温度、乙醇体积分数、料液比3个因素为影响因子,采用Box-Benhken响应面法优化提取工艺参数,并计算其DPPH自由基清除率和还原能力等抗氧化活性。结果表明,小槐花总黄酮最佳提取工艺的条件为提取温度60℃、乙醇体积分数40%、料液比1∶45(g∶mL)、超声时间108 min,该条件下小槐花的总黄酮含量为31.990 mg/g,与预测值31.254 mg/g相近,RSD为1.64%。在测定的质量浓度中,小槐花总黄酮对DPPH自由基有较好的清除能力,其还原能力随总黄酮浓度的增大而增强。该方法可靠适用,响应面法优化提取工艺参数可用于小槐花总黄酮的提取和体外抗氧化活性测定。

Development of oral pharmaceutical formulation of standardized crude ethanolic extract of Atractylodes lancea(Thunb) DC.

Cholangiocarcinoma(CCA), the adenocarcinoma of the biliary duct, is commonly reported in Asia with the highest incidence in northeastern Thailand. Chemotherapy of this type of cancer is limited due to the lack of effective chemotherapeutic drugs. A series of previous studies support further research and development of Atractylodes lancea(Thunb) DC.(AL) as a potential candidate for the treatment of CCA as a crude ethanolic extract. In the present study, we aimed to develop an oral pharmaceutical formulation(capsule) of the standardized AL crude ethanolic extract for further clinical development in patients with CCA. Major steps included macroscopic and microscopic authentication of the AL rhizomes, preparation of standardized AL extract, preparation of oral pharmaceutical formulation(capsule) of the standardized AL extract, quantitative and qualitative analysis of the marker compound(atractylodin) in the formulated AL extract, evaluation of contaminations of heavy metals, pesticides residues, and microorganisms in the ground AL rhizomes and the formulated(capsule) powder of AL, physicochemical and pharmaceutical properties of the formulated AL extract/capsule, and cytotoxicity evaluation of the formulated AL extract. Results of all evaluations confirmed satisfactory pharmaceutical properties of oral(capsule) formulation of the standardized AL extract.

响应面法优化苍术低聚果糖提取工艺

采用超声辅助提取,响应面法优化苍术中低聚果糖(FOS)的提取工艺。在单因素实验的基础上,以提取时间、超声温度和提取料液比为优化因素,FOS总提取率为评价指标,采用Box-Behnken中心模型设计三因素三水平进行响应面优化,高效液相蒸发光检测法检测。结果表明苍术FOS最优提取工艺为超声温度70℃、提取时间60 min、料液比为1∶40,在此条件下FOS总提取率为2.44%,蔗果三糖、蔗果四糖、蔗果五糖、蔗果六糖、蔗果七糖、蔗果八糖和蔗果十糖提取率分别为0.94%、0.33%、0.21%、0.23%、0.43%、0.17%、0.12%。

茅苍术的全长转录组分析

为探究茅苍术Atractylodes lancea(Thunb.)DC.中药用活性成分生物合成的遗传基础,对茅苍术进行三代全长转录组测序,通过生物学信息学分析,构建了茅苍术全长转录本数据库.共获得26238条平均长度为1360 bp的全长转录本,其中25413条(96.86%)在NR、KEGG、GO、KOG和Swiss-Prot 5个数据库中得到注释.根据NR比对结果,茅苍术与菊科菜蓟属刺苞菜蓟(Cynara cardunculus)最为相似;根据GO注释分析,8371条转录本注释到生物学过程、分子功能和细胞组成的三大类共48条GO条目;有15848条转录本注释到KOG数据库,参与基本功能预测序列数目最多,为2598条;在KEGG注释分析中,共有12137条转录本被注释到130条代谢通路;基因结构分析中,得到325条LncRNA、24723条CDS序列,检测到SSR位点8027个;根据转录因子分析,共预测到81个TF家族,包括1830个转录因子.该研究获得了茅苍术的全长转录组数据库,并为后续开展苍术的育种、活性成分的生物合成机制以及遗传多样性等分子研究提供宝贵的遗传资源.

不同覆盖物对大别山茅苍术出苗的影响

为了解大别山区域茅苍术[Atractylodes lancea(Thunb.)DC.]育苗过程中不同覆盖水平对茅苍术苗出苗与生长的影响,设置不同厚度地膜覆盖、松针覆盖、稻稃覆盖、杂灌叶覆盖与菌杂混土等处理。结果表明,不同覆盖物对茅苍术苗出苗数量、生长势、块茎鲜重、根长及健株率等性状的影响明显,其中地膜覆盖、菌杂混土处理的出苗质量显著优于无覆盖处理,稻稃覆盖、杂灌叶覆盖处理显著差于无覆盖处理,松针覆盖处理略优于对照,但未达显著水平。除健株率与成苗数量相关性未达到显著水平外,成苗数量、生长势、块茎鲜重、根长、健株率等性状两两之间呈显著正相关。不同覆盖物处理田块的杂草群相也有所不同,部分杂草如苍耳、问荆、蒿等在遮光覆盖条件下发生早而快。适合的土壤类型、良好的土壤墒情与微生态环境、适宜的温度与光照条件,均有利于促进茅苍术苗早生快发。根据茅苍术苗的成苗情况、生长势与整齐度基本能够判断幼苗的性状与全株生长状况。

Gene Chip Analysis of Expression Pattern of Type I Inner Root Sheath (IRS) Keratin in AoHan Wool Sheep

[Objective] The paper was to study the expression pattern of specific genes during wool anagen and telogen stages and its significance to AoHan wool sheep breeding. [Method] Gene chip technology was used to detect the expression of epithelial genes in neck and groin during anagen and telogen stages in AoHan wool sheep. [Result] The expression of type Ⅰ IRS keratin genes KRT25, KRT26, KRT27 and KRT28 in neck and groin of wool sheep were statistically analyzed, and the result showed that the expression in neck during anagen stage was significantly higher than that in groin （The multiple was more than two times, P0.01）. The expression changes of the genes during anagen and telogen stages were compared, and the results showed that the expression of all Type I IRS Keratin genes performed in groin showed significant decrease from anagen stage to telogen stage （P 0.05）, the expression changes of other genes were two times higher, except KRT25 （LOC443079）. [Conclusion] The result indicated that the expression of type Ⅰ IRS Keratin genes is closely related with the control of wool density in specific part and the whole wool development cycle.

正交设计优化茅苍术组培苗生根及增殖的研究

目的:提高茅苍术快繁及实现大规模工厂化生产,在濒危植物茅苍术的快速繁殖中采用正交试验,以确定适宜的茅苍术生根及增殖培养基。方法:采用L_(9)(3^(4))正交设计,以茅苍术生根率和增殖率为指标,研究茅苍术培养基、NAA、6-BA、活性炭、糖5个因素对茅苍术生根率和增殖率的影响。结果:适宜茅苍术生根的培养基为MS+0.2 mg/L NAA+30 g/L糖+1%活性炭,适宜茅苍术增殖的培养基为1/2MS+0.1 mg/L NAA+30 g/L糖+1.0 mg/L 6-BA。结论:研究结果为建立和完善茅苍术再生体系奠定了基础。

茅苍术挥发油提取方法的比较研究

目的优选茅苍术挥发油的提取方法。方法采用水蒸气蒸馏法、超声提取法、索氏抽提法和超临界CO2萃取法,以挥发油得率及苍术素的含量为考察指标,采用气相色谱法测定苍术素的含量,对4种提取方法进行比较研究。结果超临界CO2萃取挥发油得率为10.61%,挥发油中苍术素的含量为28.62%,均高于其它3种提取方法。结论超临界CO2萃取法挥发油得率和油中苍术素的含量最高,优于其它3种方法,适用于茅苍术挥发油的提取。

苗药八爪金龙中生物活性成分的研究(Ⅰ)

采用水蒸气蒸馏法、索氏提取法分别提取苗药八爪金龙（Ardisia crispa（Thunb.）DC.）中的挥发油和岩白菜素.利用GC/MS法及HPLC/MS法对挥发油及岩白菜素含量进行测定.结果表明,岩白菜素在30.300～912.000 ng/mL范围内与峰面积具有良好的线性关系（r=-0.999 1）,平均回收率为97.66％（RSD=2.31％）;八爪金龙挥发油主要成分有己醛、（Z）-2-庚烯醛、3-羟基-1-辛烯、石竹烯、龙脑、9,12,15-十八三烯、3-苯基-2-丙烯醛、芳姜黄酮、n-癸酸、十二烷酸、正丁烯基苯酞、棕榈酸等,含量分别为7.72％、1.82％、1.92％、1.85％、2.48％、2.98％、3.06％、5.17％、4.08％、7.36％、3.11％、29.11％.GC/MS法测定出挥发油中的27个成分,占挥发油总量的87.17％.建立的HPLC/MS方法简单、准确、重复性好,可作为八爪金龙根药材质量检测的方法.

不同覆盖物对茅苍术出苗率及幼苗生长状况的影响

[目的]研究覆盖物对茅苍术出苗率及幼苗生长状况的影响.[方法]将茅苍术播种后采用稻草和枯落松针覆盖苗床（对照组裸露）,培养一段时间后,测定土壤含水量、温度、pH和茅苍术出苗率,并观察茅苍术幼苗的生长状况.[结果]稻草和枯落松针覆盖处理的茅苍术出苗率极显著高于对照（P＜0.01）;其中,松针处理效果最好,出苗率高达79.03％,土壤日平均温度变化范围在14.7～20.9℃,且随着覆盖天数的增加土壤酸碱度逐渐降低,接近茅苍术的野生土壤环境的酸碱度;枯落松针覆盖处理和稻草处理的茅苍术幼苗株高、叶长、叶宽、单株重和叶绿素含量等各种生物学性状指标均高于对照,其中枯落松针处理的幼苗各项指标均高于稻草处理.[结论]培育茅苍术幼苗时,以枯落松针覆盖既能提高出苗率,又能达到壮苗的标准.

用RAPD技术评估苍术居群间的亲缘关系

用 RAPD方法分析了苍术不同居群间的亲缘关系。选取 10个 10碱基的随机引物对苍术的 10个居群共83个个体进行扩增 ,获得 10 0个 DNA片断。用 RAPD istance软件计算了不同居群间的 Jaccard遗传距离 ,得距离矩阵 ,分别用单联法、全联法、类平均法作聚类图 ,结果表明 ,该 10个居群可划分为 3个类群 :即类群 1,大别山类群(Ta) ;类群 2 ,南苍术类群 (Wu、L u、Hu、Xu、Ca、L i) ;类群 3,北苍术类群 (Tt、Da、L ao)。在这 3个类群中 ,南、北苍术之间的遗传距离相对较近 ,大别山类群与南、北苍术之间的距离则较远。根据本试验的结果及前人关于苍术精油成分差异的聚类结果 。

茅苍术提取物对胃癌BGC-823和SGC-7901细胞增殖抑制作用研究

目的探讨茅苍术提取物对胃癌细胞株BGC-823和SGC-7901的增殖抑制效应。方法采用不同浓度(0.625,1.25,2.55,10 mg.mL-)1茅苍术水提物处理胃癌BGC-823和SGC-7901细胞,采用四甲基偶氮唑盐(MTT)法检测药物对细胞的抑制作用;倒置显微镜下观察细胞的形态学改变;Hoechst 33342染色,荧光显微镜观察细胞凋亡形态;应用流式细胞仪观察凋亡过程中的细胞周期变化。结果茅苍术提取物对胃癌BGC-823和SGC-7901细胞增殖均有明显的抑制作用,且呈浓度和时间依赖性;光镜下可见凋亡细胞的形态学特征性改变;流式细胞仪检测表明经茅苍术提取物处理,BGC-823细胞周期阻滞于S期,SGC-7901细胞周期阻滞于G0/G1期。结论体外实验显示茅苍术提取物能抑制胃癌BGC-823和SGC-7901细胞增殖,可以诱导胃癌细胞凋亡。

北苍术和茅苍术挥发油成分的比较

采用气相色谱-质谱联用技术分析了北苍术和茅苍术的挥发油成分,分别鉴定出47和50种化学成分,并测定了其相对含量。采用色谱指纹图谱八强峰法和分区法,根据色谱峰的保留时间把总离子流色谱图分为5个区,比较了北苍术和茅苍术的挥发油成分。结果表明,在总离子流色谱图Ⅳ和Ⅴ区中,从北苍术和茅苍术中均检出苍术的主要药效成分β-桉叶油醇、苍术酮和苍术素,且相对含量较高。这表明《中国药典》将北苍术、茅苍术统称为苍术具有一定的科学性。

野生与栽培茅苍术挥发油成分的比较分析

采用气相色谱法分析茅苍术〔Atractylodes lancea(Thunb.)DC.〕在原产地及引种栽培1年后5个主要挥发油成分的含量变化。结果表明,江苏产茅苍术挥发油成分以苍术酮和苍术素为主;湖北产茅苍术挥发油的主要成分为茅术醇和β-桉叶醇;不同产地野生茅苍术挥发油的主要成分和含量明显不同;引种栽培至同一环境(江苏南京)后,茅苍术挥发油的主要成分及含量差异明显减小,表明产地对茅苍术挥发油有效成分的组成有一定的影响。

低钾胁迫对苍术生长发育及挥发油的影响

目的探讨低钾胁迫对苍术生长发育及其挥发油成分的影响,揭示低钾环境与苍术道地性形成的关系。方法通过苍术植株不同程度低钾胁迫受控实验,分析测定其生物量及植株全钾、可溶性蛋白、总挥发油量的变化,并采用GC-MS分离鉴定挥发油中各组分,使用SPSS分析软件进行统计。结果除根长外,低钾胁迫组的株高、根茎粗、叶片数、分枝数、须根数、地上鲜质量与干质量、地下鲜质量与干质量等指标均较正常组显著降低(P<0.05);植株全钾与可溶性蛋白质的量随培养液供钾水平的提高而升高;低钾胁迫组挥发油组分显示出质量分数较大的组分数目显著增多、各组分的量趋于均衡、茅术醇与β-桉油醇2个主要成分的量显著下降(P<0.05)。结论适度低钾胁迫导致的苍术挥发油组分变化与道地药材苍术的挥发油特征相符,低钾胁迫应是苍术药材道地性形成的重要逆境因子之一。