孑遗植物银杏(Ginkgo biloba L.)雌雄株水分生理特征初步研究

[目的]研究银杏雌雄株在水分生理上的差异，探讨其水分利用策略，以及在进化过程中的重要作用。[方法]对银杏雌雄株的树干液流（sap flow）、气孔导度（Gs）、蒸腾速率（Tr）以及水分利用效率（WUE）进行对比研究。[结果]雌雄银杏的液流昼夜进程具有相似的规律，且白天雌雄株液流基本相同，夜间雄株液流大于雌株；不同性别银杏的丹和岱同样呈现为早晚高、午间低的曲线趋势，早晚雌株有比雄株更高的Tr和Gs，但中午11：00-14：00之间雌株的值低于雄株；雌雄银杏的WUE曲线变化趋势相似，但雌株的平均水分利用效率略小于雄株。[结论]与其他伴性植物比较分析得知，不同性别银杏的水分生理有较强的趋同特征。这可能是在长期进化的压力下，雌雄异株植物的一种生存策略。

Experimental Study on Inhibition of Neuronal Toxical Effect of Levodopa by Ginkgo Biloba Extract on Parkinson Disease in Rats

In order to observe neuronal toxical effect of Levodopa and investigate if using Levodopa together with Ginkgo Bilobar Extract (EGb) would be an workable method to treat Parkinson disease, rat models of Parkinson disease (PD) were made by injecting 6-OHDA stereotaxically to right side of the mesencephic ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Rotational behavioral observation, TUNEL, immunocytochemistry, Nissl's body staining were performed to measure the difference between group treated by Levodopa (50 mg/kg every day for 3 days, 5 days, 7 days, L-dopa group) and group treated by Levodopa combined with EGb (100 mg/ kg every day, E-D group). The results showed that in the L-dopa group, the numbers of apoptosis of substantial nigra, rings of rotational behavior were more than those in the E-D group (P<0. 05). The numbers of Nissl's cells in L-dopa group were fewer than in E-D group (P<0. 05). The results suggested that Levodopa had neur toxic effect and EGb may decrease the toxicity of levodopa. The combined use of EGb with Levodopa may be a workable method to treat PD and may be better than using Levodopa alone.

Extraction Process and Content Determination of Total Flavonoids in Ginkgo( Ginkgo biloba L. ) Leaves

[Objectives]This study was conducted to study the extraction process and the content determination of flavonoids in ginkgo( Ginkgo biloba L.) leaves.[Methods]Ethanol extraction and methanol extraction of total flavonoids in ginkgo leaves were studied,and the optimal extraction conditions for flavonoids were determined by orthogonal test; and with quercetin as reference substance,total flavonoid content in ginkgo leaves was determined by UV spectrophotometry.[Results]The optimal extraction process was 4 h of Soxhlet extraction with methanol; and the total flavonoid contents had a good linear relation in the range of 0. 006 5-0. 039 mg/ml( R^2= 0. 999 9),the average content was stabilized at 1. 135%,and the average recovery of the method was 102. 0%. [Conclusions]This study selected the optimal extraction process for total flavonoids in ginkgo leaves. The test method is simple with high accuracy and precision,and is suitable for the extraction and determination of total flavonoids in ginkgo leaves.

Dynamic changes of telomeric restriction fragment (TRF) lengths in cells during the developmental process from embryos to seedlings and a comparison with the embryonal calli in Ginkgo biloba L.

Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location ofArabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment （TRF） lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.

Effects of Coupling Treatment of Acid and Aluminum under Soilless Culture on Water Content of Ginkgo biloba L.

In this experiment,two-factor 7-level uniform design scheme was applied.Separate treatment of each factor was performed for single effect test.Then,it analyzed the effects of acid and aluminum stress under soilless culture on the water content of plant during the growth of Ginkgo biloba L.seedlings,so as to provide a theoretical basis for studying the connection between plant water and physiological stress.The results showed that the water content of G.biloba plants declined with the decrease of p H,but the water content of G.biloba plants declined with the increase in the concentration of aluminum treatment,indicating that the stronger the acid and aluminum stress,the lower the water content of the plants,affecting the normal absorption of water content of G.biloba plants,accordingly leading to lack of water.According to the analysis of the degree of influence on the water content,p H is the first factor influencing the growth of G.biloba plants.If p H is lower than 3.5,G.biloba plants will not growth,while p H is higher than 5.0,aluminum has no significant effect on the water content of G.biloba plants.In conclusion,the water content of G.biloba plants can be used as an essential indicator for acid and aluminum stress.

Determination of the Content of Kaempferol from Fermented Ginkgo( Ginkgo biloba L.) Leaves

[Objectives] This study was conducted to determine kaempferol content in ginkgo( Ginkgo biloba L.) leaves subjected to microbial fermentation.[Methods]Bacillus licheniformis was selected for solid-state fermentation of ginkgo leaves,and the content of kaempferol in ginkgo leaves was determined by RPHPLC method. At first,methanol was used to extract flavonoid glycosides,which were then hydrolyzed by hydrochloric acid solution. HPLC was performed with Platisil ODS column C18( 150 mm ×4. 6 mm,5 μm) using mobile phase Vmethanol∶ Vwater( 0. 4% phosphoric acid solution) = 55∶45 at a flow rate of 1 ml/min,and the eluate was detected with a shimadzu HPLC ultraviolet detector at 360 nm. [Results]With kaempferol as the reference substance,the correlation coefficient was0. 999 2 in the range of 0. 001 06-0. 016 96 g/L. The content in the fermented product was less than that in the non-fermented product by 28%. [Conclusions]The method is simple,accurate,and is suitable for determination of kaempferol. This study will provide an experimental basis for the development and utilization of ginkgo.

Cloning and Analysis of Telomere-associated Sequences of Ginkgo biloba L..

Total genomic DNA was extracted from leaves of Ginkgo biloba L. by the method of hot CTAB. After determining quantification of DNA sample by microclorimetric spectrophotography, Arabidopsis-type telomere primer and Sau3AⅠ cassette primer were used to isolate telomere-associated sequences from G. biloba L. by the method of cassette-ligation-mediated polymerase chain reaction (PCR). Using this method, two telomere-associated sequences, TAS1 and TAS2, were isolated. The authors preformed Southern hybridization of EcoRⅠ-treated G. biloba genomic DNA with each clone. The hybridization pattern showed that the clones obtained were derived from G. biloba genomic DNA. There are the Arabidopsis-type TTTAGGG tandem repeats in telomeres of G. biloba.

γ-氨基丁酸/L-瓜氨酸增强白果分离蛋白凝胶性

以白果分离蛋白(GSPI)为成胶基质材料,将γ-氨基丁酸(GABA)和L-瓜氨酸(L-Cit)分别加入到GSPI水溶液中,均质后再将溶液pH分别调至5.0、6.0和7.0,制备了热诱导凝胶。采用物化分析法、机械力学、蛋白凝胶电泳、SEM及FTIR表征了溶胶的物性、凝胶性和微观结构,并探讨GABA、L-Cit对GSPI凝胶性能的影响及其机制。结果表明,GABA、L-Cit对GSPI溶解度无提升作用,但能显著降低蛋白疏水性;GABA促进溶胶聚集并降低其ζ-电位,而L-Cit在pH 7.0时则促进解聚和提高ζ-电位;两者均能促进蛋白分子疏水基团向内折叠。在pH5.0和pH7.0时,以上两种氨基酸特别是L-Cit显著提高蛋白凝胶性能,而在pH6.0时则弱化凝胶性能。其影响机制可能是氨基酸与GSPI之间的静电作用,而这种效应依赖于所在体系的pH。

利用AFLP技术筛选与银杏性别相关的分子标记

利用 amplified fragment length polymorphism(AFLP)技术 ,应用 4 8个引物组合 ,检测了雌雄银杏基因组DNA的多态性 ,筛选与银杏性别相关的分子标记 ,其中 3个引物组合各提供了 1个与雌性相关的分子标记 .经Southern点杂交证实有两个标记为银杏雌性基因组所特有 .这些分子标记的获得 ,为寻找性别特异的探针或PCR引物 。

银杏LEAFY同源基因的分离与克隆

The flower meristem identity gene LEAFY is a developmental switch,sufficient for flower initiation in diverse plants. Ginkgo biloba is a kind of gymnosperm,and a dioecious tree species.The nucleotide DNA sequence was cloned from the genomic DNA of the tender leaves collected from a male tree in Ginkgo biloba L.cv.Dafushou.The analysis of DNA sequence structure indicated that the gene was composed of 3450 bp,and inferred the sequence to LEAFY gene of the male Ginkgo .Comparing the gene with LEAFY gene of the female Ginkgo reported in the relative references,the rate of homology was 99%,only lacking 3 nucleotides.The research work laid a sound foundation for studing the regulation and controlling flowering mechanism in Ginkgo biloba L.at the molecuar level in the future.

Al^(3+)-芦丁二元络合物荧光光度法测定银杏叶中总黄酮的含量

根据黄酮类化合物能与Al3+形成稳定的荧光络合物,以芦丁为标样,建立了一种测定银杏叶总黄酮的荧光分光光度法。激发波长λex=436 nm,发射波长λem=483nm,方法检出限为1.27×10-9 mol·L-1,线性范围在1.64×10-9~3.63×10-5 mol·L-1之间,回收率为99.8%~104.2%,相对标准差(RSD)为1.94%。

HPLC法测定银杏叶中6种黄酮成分的含量

用反相高效液相色谱法测定了银杏叶中６种黄酮成分即槲皮素（ｑｕｅｒｃｅｔｉｎ）、异鼠李素（ｉｓｏｒｈａｍｎｅｔｉｎ）、山萘酚（ｋａｅｍｐｆｅｒｏｌ）、白果黄素（ｂｉｌｏｂｅｔｉｎ）、银杏黄素（ｇｉｎｋｇｅｔｉｎ）、西阿多黄素（ｓｃｉａｄｏｐｉｔｙｓｉｎ）。单酚酸Ｂ（ｓａｌｖｉａｎｏｌｉｃａｃｉｄＢ）为内标。并对１８个样品进行了测定。色谱柱为ＺｏｒｂａｘＯＤＳ柱，流动相：Ａ．ＭｅＯＨ，Ｂ．ＴＨＦ—Ｈ２Ｏ—ＨＣＯＯＨ（３４∶６５∶１），梯度洗脱。流速：１０ｍｌ·ｍｉｎ－１，检测波长：３５０ｎｍ。线性范围０１０～２５７μｇ，相关系数γ＝０９９９４～０９９９８，回收率９８０％～１０２０％。本法准确、快速、重现性好。

银杏叶总黄酮苷的HPLC法分析水解条件

研究银杏叶中总黄酮苷的HPLC法分析水解条件。结果表明 :1g银杏叶甲醇提取物溶于 30ml甲醇中 ,加入 5ml 8 3mol·L-1的HCl,于 (85± 1)℃水浴水解 6 0min ,此法则得槲皮素、山柰素、异鼠李素的回收率分别为 10 1 4%、10 2 %、98%。

HPLC法测定银杏叶中3种内酯成分

研究了用反相高效液相色谱法测定银杏叶及其提取物中 3种银杏内酯含量的方法 ,采用外标法定量。色谱柱 :ZorbaxODS柱 ,流动相 :甲醇—四氢呋喃—水 (1∶1∶8) ,流速 :1 0mL/min ,检测波长 :2 2 0nm ,线性范围 :2 0 0 8～ 33 4 1μg ,r=0 999～ 0 9999,回收率 :92 7%～10 0 9%。本法结果准确 ,重现性好 ,为生产及工艺研究提供质量控制依据。

CaCl_2浸泡处理对银杏贮藏品质和种壳超微结构的影响

以银杏核用主栽品种佛指和龙眼为试材,将其种核用1%CaC l2浸泡处理24 h,然后置于室温下贮藏,研究在贮藏过程中种核呼吸强度和品质变化规律,并对种壳超微结构进行观察。结果表明:①银杏种核单粒重和种仁呼吸强度、含水量、总淀粉含量及支链淀粉含量均随贮藏时间的延长而下降,浮籽率则呈上升趋势;品种间比较以龙眼呼吸强度高、水分散失快、营养消耗高;CaC l2处理后种核的水分散失和养分消耗均受到抑制,种仁呼吸强度也有下降,其中龙眼的处理效果较为明显。②银杏种壳由大量管胞组成,管胞表面光滑,穿孔明显,穿孔主要有圆形和撕裂状;管胞直径和穿孔的形状、分布、大小品种间差异显著,与佛指比较龙眼的管胞直径较小、穿孔面积较大;CaC l2处理后种壳表面密布钙质颗粒,部分颗粒覆盖于管胞的穿孔洞口,减少了水分的散失和O2的进入,提高了银杏种核贮藏性能。

HPLC测定银杏愈伤组织内酯A的研究

建立了一种可用于银杏离体细胞培养物中内酯Ａ含量分析的ＲＰ－ＨＰＬＣ方法。样品经低浓度甲醇提取后，用中性或酸性氧化铝固相萃取柱纯化，用示差检测器检测，银杏内酯Ａ的最低检测限为 ０，０１２ μｇ，方法回收率为（９６．０±２）％，ＲＳＤ为 ３．１％。

银杏外种皮多糖对免疫功能低下小鼠IL-2活性及sIL-2R的影响

目的 :观测银杏外种皮多糖对免疫功能低下小鼠IL 2活性及sIL 2R的影响。方法 :用银杏外种皮多糖给荷瘤小鼠及环磷酰胺损伤小鼠灌胃治疗 ,分别检测小鼠脾淋巴细胞IL 2活性及血清sIL 2R含量。结果 :银杏外种皮多糖可促进荷瘤小鼠及CTX损伤小鼠脾淋巴细胞的IL 2活性 ,并减少其血清sIL 2R的形成。结论 :银杏外种皮多糖可促进荷瘤小鼠及CTX损伤小鼠的免疫功能。

LC-MS/MS快速测定不同采摘时间银杏叶中银杏内酯和白果内酯的含量

目的建立银杏叶中银杏内酯和白果内酯的高效液相电喷雾二级质谱(LC-MS/MS)定量分析方法,并对不同采摘时间银杏叶中银杏内酯和白果内酯含量进行测定。方法银杏叶样品以甲醇-水(1:9)提取,并用乙酸乙酯萃取,采用Phenomenex ODS柱分离,流动相为甲醇-水(60:40),流速0.7 mL·min^(-1)。电离方式为电喷雾正离子,采用多反应检测模式(MRM),捡测离子分别为m/z 409.4→345.3(银杏内酯A)、m/z 425.3→361.3(银杏内酯B)、m/z 441.4→325.3(银杏内酯C)和m/z 327.4→309.3(白果内酯)。结果分析过程仅需8.5 min,且无杂质干扰,4种成分回收率为91.79%～101.48%银杏内酯A,B和白果内酯含量分别在7月18日、8月19日和7月5日采摘的样品达到最高。结论方法快速、准确、选择性好,适合银杏内酯和白果内酯的定量分析,七月份叶子中内酯含量最高,为最佳采摘时间。

HPLC法比较2种银杏外种皮多糖的相对分子质量及含量

目的:采用HPLC法比较2种银杏外种皮多糖的相对分子质量及含量。方法:从银杏外种皮中提取出多糖,分别于常温下贮藏5年和3年后测定并比较其相对分子质量和含量。以右旋糖酐为标样,采用HPLC法,PLaquagel-OHMIXED(300mm×7.5mm,8μm)色谱柱,柱温25℃,示差折光检测器,流动相为超纯水,流速1.0mL·min-1。结果:贮藏5年和3年的2种银杏外种皮多糖的平均相对分子质量分别为10825.3(RSD=0.45%)和11062.5(RSD=0.78%);其多糖含量分别为66.8%(RSD=5·.9%)和68.7%(RSD=3.4%)。结论:在常温下,银杏外种皮多糖随着贮藏时间的延长,其相对分子质量和含量会发生变化。

应用LSCM和SEM鉴定银杏花粉的倍性

以经秋水仙碱诱导获得的银杏大花粉为材料,以未经处理的正常银杏花粉为对照,利用扫描电子显微镜和激光扫描共聚焦显微镜分别观察2种花粉表面形态,并分析对比其细胞内的DNA相对荧光量与银杏大花粉核内染色体倍性关系,为正确检出银杏二倍体花粉奠定重要工作基础。研究结果表明,此方法用于花粉粒的倍性鉴定科学准确,所需试验样本少,实用性强,在植物花粉倍性鉴定中有着较好的应用前景。