�й�ҩ�þջ�Ʒ�ּ��ӹ�������Ǩ���о�

??Chrysanthemum morifolium has a long history of culture and use in China. Due to different germplasm resources, producing areas, and processing methods, many cultivated varieties have formed now. The varieties and processing methods of C. morifolium are affected by economic interests and processing cost, which change gradually. On the basis of spot investigation and related literature study, the changes of the varieties and processing methods of C. morifolium were summarized in this paper. It will provide theoretical evidence for the culture, processing, quality evaluation, and clinical application of C. morifolium.     

���׿����ؿ�������ţϥ��ָ����¶�̬�仯�о�

??OBJECTIVE To study the monthly dynamics of physical and chemical indexes in Achyranthis Bidentatae Radix(ABR, the dried root of Achyranthes bidentata Bl.) stored in simple and cool warehouses. METHODS ABR was stored in simple and cool warehouses for 27 months. The color was observed. The water content was determined based on the drying method. The contents of ??-ecdysone and 5-hydroxymethylfurfural (5-HMF) were determined by HPLC method. The accumulation of temperature difference between the simple and cool warehouses was evaluated with a relative temperature cumulation (RTC) method. The monthly dynamics of physical and chemical indexes of ABR was analyzed with RTC. RESULTS As the extension of storage time, the ABR stored in the simple warehouse showed deeper color and harder texture, but the ABR stored in the cool warehouse still had soft texture without significant color change. The contents of ??-ecdysone in ABR stored in the two warehouses both gradually decreased and dropped to lower than the limit of 0.030% ruled by China Pharmacopoeia when being stored for up to 27 months. The contents of 5-HMF of ABR stored in the two warehouses both increased and were higher for the sample in the simple warehouse than that in the cool warehouse. CONCLUSION The concept of RTC is put forward and used to study the monthly dynamics of chemical constituents in traditional Chinese medicine during storage for the first time. The physical and chemical indexes of ABR varies during storage. Two years of storage time of ABR is suggested.     

���ڸ�����Ӧϵͳ���۵�ִҵҩʦ�ʸ�׼������о�

??OBJECTIVE To construct a reasonable licensed pharmacist qualifications access echanism.METHODS In this paper,qualification of licensed pharmacists was analyzed by literature research and questionnaires.RESULTS AND CONCLUSION Based on the CAS theorythe functions of the eight adaptive subjects involved in the qualification of the licensed pharmacists were divided.8 major subjects include the following functionsChina Food and Drug Administrationmacro -control access environment;Ministry of Educationcertificate Universities of Pharmacy hardware and software;Ministry of Civil Affairsdefine the authority of the Association moderate;Ministry of Human Resources and Social Security examine the qualifications for examination;Associationimplement specific access procedures;Universities of Pharmacyassessment of academic and pracrical ability;Employer joint universities practice assessment;Medicine studentsserved by other subjects.And finally establish a flow chart of China??s access mechanism to form a set of complex adaptability of the practice of pharmacists access mechanism.     

���󶬾ۺ�ø��ʽ��Ӧ�����������о�

??OBJECTIVE To design specific PCR primers and establish the PCR identification method of Ophiopogon japonicas from Sichuan.METHODS The gene footprint of Ophiopogon japonicas from Sichuan named CM503 was screened from random amplified polymorphic DNAC(RAPD) amplification. Reclaimed CM503 gene was inserted into T-vector to be cloned and sequenced. One pair of specific primers CM1/CM2 were designed according to the CM503 sequence and applied in specific PCR using the genomic DNA of Ophiopogon japonicas from Sichuan as template.RESULTS A specific band around 297 bp was detected in Ophiopogon japonicus from Sichuan at 68 ??, while nothing appeared for the other varieties.CONCLUSION The method is convenient, reproducible, and precise, with broad application prospects.     

���߾�Ҷ�����߻���ʳƷԭ���о�

??OBJECTIVE To study the safety and feasibility of using stems and leaves of Panax notoginseng and flowers of Panax notoginseng as new food ingredients. METHODS The edible history, nutrition and quality standards of Panax notoginseng's stems, leaves and flowers were summarized. Then toxicological test was conducted in mice to investigate the toxicity. RESULTS The stems and leaves of Panax notoginseng and flowers of Panax notoginseng have a long edible history, rich in vitamins, minerals, proteins, amino acids and other nutrients. Pharmacological experiment results showed that they were safe and nontoxic, without obvious organ damages. CONCLUSION It is of great value to use the stems, leaves and flowers of Panax notoginseng as new food ingredients under the recommended dosage. This study provides reference to the utilization of the non-medicinal parts of other Chinese herbal medicines.     

��Ĥ�ĵ������ںϵ����յ���ɫ����ϸ��������ʵ���о�

??OBJECTIVE To investigate apoptosis induction ability of hPP10-Apoptin fusion protein to mouse B16 melanoma cell. METHODS pET15b-hPP10-Apoptin expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded hPP10-Apoptin fusion protein was purified by Ni-NTA His-Bind Resin and confirmed by Western blotting assay. Melanoma cell apoptosis induced by hPP10-Apoptin fusion protein was analyzed by TUNEL assay, and the antitumor effect was examined in melanoma cell-bear mouse model. RESULTS hPP10-Apoptin fusion protein was highly expressed in BL21 cells, Western blotting analysis result showed that fusion protein was expressed correctly. The fusion protein can induce melanoma cell apoptosis in vivo and in vitro. CONCLUSION The results confirm that hPP10-Apoptin fusion protein could penetrate into melanoma cell and also has antitumor effect.     

��������ȡ��ΰ����Լ����������о�

??OBJECTIVE To investigate the anti-hepatocellular carcinoma effect and underlying mechanisms. METHODS In PLC/PRF/5 and HepG2, after treatment with Grifola frondosa extract, MTT method, chemical method, JC-1 staining and Western Blot were applied to determine cell viability, caspase 3 activity, mitochondrial membrane potential, the expression of Bcl-2 and Bax, and the phosphorylation of Akt/GSK3??. The anti-tumor activity of Grifola frondosa extract was further confirmed in PLC/PRL/5-xengrafted mice model. RESULTS Grifola frondosa extract significantly reduced cell viability, mitochondrial membrane potential, the expression of Bcl-2 and the phosphorylation of Akt/GSK3??, and enhanced LDH release, caspase 3 activity and the expression of Bax in both PLC/PRF/5 and HepG2 cells. 12-day Grifola frondosa extract treatment significantly inhibited the PLC/PRF/5-xenografted tumor growth without influence the body weight of mouse. CONCLUSION All these data indicate that Grifola frondosa extract-mediated anti-hepatocellular carcinoma effects are related to its modulation of the activations of Akt/GSK3?? and mitochondrial pathway.     

���ػ��Ž�׹����������ض�����ǵ����⿹�������о�

??OBJECTIVE To prepare doxorubicin-loased heparinized magnetic mesoporous silica nanoparticles (MMSNs-HP) and investigate their drug release profile and anticancer activity in vitro. METHODS Amino-modified MMSNs was synthesized by combining phase transfer method with sol-gel method firstly. Then heparin was conjugated with the above nanoparticles via carbodiimide chemistry to form MMSNs-HP. Finally, the following experiments were performed, such as loading/release of doxorubicin into/from MMSNs-HP in vitro, cellular uptake of MMSNs-HP by hepatoma cell HepG2 and cell cytotoxicity of doxorubicin-loaded MMSNs-HP. RESULTS MMSNs-HP was able to delay the release of doxorubicin significantly, penetrate into tumor cells, kill HepG2 cell, and inhibit the proliferation of HepG2 cells induced by basic fibroblast HepG2 cells (bFGF). CONCLUSION MMSNs-HP is a potential drug carrier for the delivery of antitumor drugs.     

Ԥ֪�ӻ�ѧ�ɷ��о�

??OBJECTIVE To study the chemical constituents of the fruits of Akebiae quinata. METHODS Various column chromatography techniques including silica gel, Sephadex LH-20, and macroporous adsorption resin column chromatography were used for fractionization and purification. The structures were identified on the basis of their physicochemical and spectroscopic evidence. RESULTS Fifteen compounds were obtained, and their structures were identified as geniposidic acid(1), 10-O-acetylgeniposidic acid(2), vomifoliol(3), p-hydroxybenzoic acid(4), protocatechuic acid(5), caffeic acid(6), tyrosol(7), palmitic acid(8), 15-nonacosanol(9), stigmasterol(10), stigmasterol-3-O-??-D-glycopyranoside(11), ??-sitosterol(12), ??-daucosterol(13), ursolic acid(14), and oleanolic acid(15). CONCLUSION Compounds 1-7, 9 and 14 were isolated from the fruits of Akebiae quinata for the first time.     

������ͨ�����ⶨ�������о�

??OBJECTIVE To study the content determination method of crotamiton. METHODS The quantitative mass balance method, HPLC external standard method and nuclear magnetic resonance(QNMR) were used to determine the content of crotamiton, respectively. The accuracy of the three methods was evaluated. RESULTS The contents of crotamiton were 99.2%,102.9% and 99.1% respectively as determined by the three different methods. CONCLUSION Because the ultraviolet absorption coefficients of cis- and trans-crotamiton might be different, the current pharmacopoeia method, ie, using integrated peak areas to calculate the content, is questionable. QNMR method can measure the contents of cis- and trans-crotamiton respectively, so it can be a complementary method to establish the reference standard.     

伤害诱导普通白木香与奇楠种质沉香中倍半萜类成分及早期倍半萜合酶基因表达的差异分析

目的 明确伤害诱导普通白木香和奇楠种质所结沉香中倍半萜组分及其早期倍半萜合酶基因表达模式的差异。方法 采用气相色谱-质谱法（GC-MS）分析普通沉香和奇楠沉香中倍半萜组分的差异;对3年生普通白木香和奇楠种质茎干分别进行全断干伤害,采用实时荧光定量聚合酶链式反应（qRT-PCR）分析伤害早期倍半萜合酶基因AsTPS2、AsTPS13、AsTPS14、AsTPS17、AsTPS18、AsTPS20、AsTPS23表达模式的差异。结果 奇楠沉香倍半萜组分显著不同于普通白木香所结沉香,2种沉香中分别检测到16、17个倍半萜成分,其中共有成分12个,且共有成分含量差异较大;两者的倍半萜合酶基因在伤害诱导早期表达模式也不同,伤害诱导24 h内,普通白木香中7个倍半萜合酶基因表达水平均上升,2 h达到最大值后下降,而奇楠种质中AsTPS2、AsTPS13、AsTPS14、AsTPS17和AsTPS23基因表达量在24 h内持续上升,AsTPS20基因表达量在6 h达到最大值,但AsTPS18基因表达量低,在伤害诱导0~24 h变化不明显,显著低于普通白木香种质。结论 普通白木香与奇楠种质所结沉香倍半萜类成分差异明显,且响应伤害诱导的倍半萜合酶基因在0~24 h表达模式显著不同,推测倍半萜合酶基因的差异诱导表达可能是2种种质形成的倍半萜成分差异较大的原因之一。     

22个白木香倍半萜合酶(AsTPS)基因的健康与伤害诱导表达特性研究

目的:明确22个As TPS基因在健康和伤害白木香中的表达情况,筛选出催化沉香倍半萜合成的关键酶基因。方法:全断法伤害处理白木香,常规PCR和荧光定量PCR(qRT-PCR)检测基因表达情况。结果:22个As-TPS基因中,1个基因在健康和伤害白木香中均不表达;3个基因在健康白木香中不表达,但明显响应于外界伤害;18个基因在健康白木香中均有一定量表达,而伤害处理后,其中7个基因相对表达量达数千倍,11个基因虽也被诱导表达,但相对表达量仅达几十倍至几百倍。结论:21个As-TPS基因为伤害诱导型基因,其中3个是沉香倍半萜合成的关键催化酶基因;在伤害诱导后含量急剧表达上升的7个As-TPS是白木香伤害诱导结香早期倍半萜合成的重要催化酶。     

丹参萜类合酶基因家族的鉴定和表达模式分析

目的 利用生物信息学方法从基因组层面对丹参萜类合酶（SmTPS）基因家族成员进行全面鉴定和功能预测。方法 从国家基因组科学数据中心（NGDC）、美国国家生物技术信息中心（NCBI）、拟南芥信息资源中心（TAIR）和番茄功能基因组学数据库（TFGD）分别获取丹参、拟南芥和番茄基因组与转录组数据。借助Perl语言、TBtools等工具对SmTPS进行全基因组鉴定与生物信息学分析。结果 共鉴定出52个TPS成员分布在丹参的8条染色体上；编码氨基酸数目在207~822 aa；等电点在4.76~9.16，分子质量为24.11~94.81 kDa，所有成员均为亲水蛋白。基因结构分析表明不同亚族成员之间内含子数差异明显，72.6% TPS-a、TPS-b、TPS-g亚族成员为6，88.9% TPS-c、TPS-e/f亚族成员>10；蛋白motif相对保守。顺式作用元件分析表明，SmTPSs启动子区均含有大量光响应元件，大多数SmTPSs启动子区含有激素响应元件。基因表达模式分析显示SmTPS家族成员存在组织表达特异性，24个基因响应外源茉莉酸甲酯。结论 基于已发表丹参基因组，鉴定得到52个SmTPS家族成员，结合系统进化与表达模式对其功能进行了预测。该文为丹参中萜类化合物生物合成及调控机制全面解析提供了参考信息。     

�ඹ������������ø���������μ��������μ���Ĺ�ϵ

??OBJECTIVE To study the effect of lysine decarboxylase (LDC) gene on the accumulation of matrine(MA) and oxymatrine (OMA) in cotyledon of Sophora alopecuroides L. germinating seeds. METHODS The S.alopecuroides germinating seeds were stressed by different mass fractions of PEG 6000, and the contents of MA and OMA were determined by high performance liquid chromatography (HPLC) and the expression level of LDC was analyzed by real-time fluorescence quantitative PCR (qPCR) after 72 h treatment. RESULTS The contents of MA and OMA decreased in the cotyledon under light stress (PEG mass fraction<15%), while increased with the stress getting higher (PEG mass fraction>20%). The analysis of qPCR revealed that the LDC expression level was decreased first, and then increased with the stress rising. The changes of the contents of MA and OMA were parallel with the expression level of LDC especially under light and severe stress. CONCLUSION There is certain association between the accumulation of MA and OMA and the gene expression quantity of LDC. The results is of significance for illustrating the role of LDC in the biosynthetic pathways of MA and OMA.     

UPLC测定多伞阿魏中倍半萜烯香豆素(DAW22)的含量

目的:利用超高效液相色谱法测定了分布于新疆的不同生长时期多伞阿魏中DAW22的含量。方法:采用固相萃取(SPE)净化,测定条件Waters ACQUITY UPLC BEH C18色谱柱(2.1 mm×50 mm,1.7μm),以甲醇-1%甲酸水溶液为流动相,进行梯度洗脱,流速0.2 m L·m L~(-1),柱温30℃,波长316 nm,进行含量测定。结果:DAW22进样量在6.21~124.2 ng(r=1.000 0)与其峰曲线下面积呈良好的线性关系,平均回收率99.81%,RSD 2.0%,生长在5月9日的多伞阿魏植株中DAW22的含量最高。结论:建立的多伞阿魏中DAW22的质量控制研究方法准确、可靠、重复性好,能够用于多伞阿魏的质量控制。     

灰毡毛忍冬LmC3H1基因的克隆及表达模式与绿原酸含量相关性分析

目的:以灰毡毛忍冬为材料,克隆对-香豆酸3-羟化酶(LmC3H1)基因,进行生物信息学和表达模式分析,结合绿原酸含量,研究推测灰毡毛忍冬LmC3H1基因的功能。方法:通过逆转录聚合酶链式反应(RT-PCR)和RACE技术克隆LmC3H1基因的全长c DNA序列,对该序列进行生物信息学分析,并利用实时荧光定量PCR(Real-time PCR)和HPLC分别测定灰毡毛忍冬茎、叶及不同花期花中LmC3H1的相对表达量及绿原酸含量。结果:克隆得LmC3H1(Gen Bank:MN177695)基因,开放阅读框(ORF)长度为1 533 bp,编码510个氨基酸,推测其分子式为C_(2618)H_(4134)N_(718)O_(727)S_(22),相对分子质量为58 005.32,等电点8.92,为亲水性蛋白,定位于叶绿体中,具有跨膜区域LLLIPAVLFLISLVYPLI,含有细胞色素P450的保守结构域CYTOCHROME_P450(422-433 aa);Real-time PCR结果显示,LmC3H1在灰毡毛忍冬茎、叶及不同花期花有不同程度的表达,其中在花发育阶段,白色花蕾期相对表达量最高,花蕾初期及白色开花期次之;白色花蕾期花与茎、叶比,花的相对表达量最高,叶的最低;HPLC结果显示,从绿白色花蕾期到金黄色开花期绿原酸含量呈上升趋势,金黄色开花期含量最高,不同器官中,花中绿原酸最高,茎最低。结论:克隆得到灰毡毛忍冬LmC3H1基因,推测LmC3H1可能参与灰毡毛忍冬花绿原酸的生物合成。该研究为进一步研究该基因的功能及探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据,同时为遗传改良灰毡毛忍冬品质奠定了基础。     

生物技术减毒沙门氏菌介导apo-t-PA-GFP融合基因在鸡体内表达和在卵黄中沉积的研究

 目的 探讨以减毒沙门氏菌为载体携带目的基因在家禽体内表达,并实现表达产物在卵黄中定向沉积的可行性。方法 将人t-PA基因与鸡VLDLy组装前体apo基因融合,构建pApo-tPA-GFP表达质粒,电转化至减毒鼠伤寒沙门氏菌△crp SL1344中,阳性重组菌静脉注射高产蛋鸡,收集不同时期鸡蛋,涂片荧光镜检和ELISA检测表达产物在卵黄中沉积情况和表达水平,平板溶圈法检测卵黄中表达产物的活性。结果 注射表达质粒第8天开始卵黄中有荧光颗粒出现,第21天表达量最高,为10.4 μg·mL-1,活性相当于50.2 μg·mL-1标准品t-PA。结论 研究表明,减毒沙门氏菌能够介导apo-t-PA-GFP融合基因在鸡体内表达,且apo载脂蛋白能够引导t-PA透过卵黄膜实现在卵黄中沉积,这一途径简捷、方便,为外源基因在动物体内的表达研究提供了理论和技术基础。     

黄连WRKY基因家族鉴定及表达分析＊

目的 本研究使用生物信息学方法鉴定黄连WRKY基因家族成员并对其表达模式进行分析，为进一步研究黄连WRKY基因家族的功能及其对黄连生物碱合成途径的调控机制提供参考。方法 利用HMMER、BLAST、TBtools、IQ-Tree等软件，ExPASy、WOLF PSORT、MEME在线网站等对黄连基因组中的WRKY基因家族进行鉴定、可视化分析和表达模式分析。结果 从黄连基因组中鉴定出了41个WRKY基因，其ORF长度为252-2796 bp，编码蛋白的氨基酸数量为84-931 aa，分子质量为9649.32-103620.60 Da，等电点为4.96-10.17，95%以上蛋白预测亚细胞定位于细胞核；41个CcWRKY（黄连WRKY）基因不均一地分布在9条染色体上；系统发育分析将41个CcWRKY蛋白分为3大类，第Ⅰ类有6个，第Ⅱ类有31个，第Ⅲ类有4个；同一类CcWRKY蛋白结构域高度保守，大多CcWRKY基因外显子数量为3-7，内含子数量为2-6。表达分析结果为，除了保守结构域高度缺失的CcWRKY38、CcWRKY40外，所有的CcWRKY都至少在一个组织中有表达，且多数是特异性表达。结论 全面分析了黄连基因组中的WRKY基因家族，有助于进一步解析WRKY基因家族在黄连生物碱生物合成途径的调控机制。     

垂序商陆PaAACT基因克隆和表达分析

目的:从垂序商陆根中克隆了商陆皂苷甲生物合成过程中关键酶乙酰乙酰辅酶A转移酶(acetoacetyl-CoA transferase,AACT)基因,进行生物信息学分析和原核表达。方法:提取垂序商陆根的总RNA,然后逆转录合成c DNA,在分析垂序商陆转录组数据的基础上,设计Pa AACT基因的特异性引物,聚合酶链式反应(PCR)扩增Pa AACT基因的c DNA序列,通过构建原核表达载体p ET-32a-PaAACT,诱导表达并且纯化目的蛋白。结果:Pa AACT基因开放阅读框为1 254 bp,编码417个氨基酸。生物信息学分析显示Pa AACT蛋白的分子式为C1 914H3 120N538O576S17,推测其相对分子质量为43. 43 k Da,理论等电点8. 90,不稳定系数32. 27,属于稳定蛋白质。根据生物信息学分析结果,Pa AACT蛋白属于硫解酶家族成员,在C末端含有硫解酶家族的1个保守位点和1个活性位点。Pa AACT蛋白可能位于细胞质中、不含信号肽、没有跨膜区。系统进化分析显示Pa AACT蛋白与甜菜等廖科植物AACT蛋白亲缘关系较近。经IPTG诱导,在大肠埃希菌BL21 (DE3)菌株中表达了Pa AACT重组蛋白,利用Ni2+亲和色谱获得了纯化的目的蛋白。结论:该研究从垂序商陆中克隆Pa AACT基因,为下一步测定Pa AACT酶催化活性、制备抗体奠定基础,也为研究其在商陆皂苷甲生物合成途径中的作用提供理论依据。