Dynamic Change Laws of Chlorophyll Fluorescence Parameters in Different Parts of Leaves of Ginkgo biloba

[Objective] The aim was to explore the dynamic change laws of chlorophyll fluorescence parameters in different parts of leaves of Ginkgo biloba.[Method] The G.biloba cultivated in North China was used as materials in this study to explore the law of daily change and ten-day change of the chlorophyll fluorescence parameters of leaves in different parts of leaves.[Result] The daily change of Fm（maximal fluorescence）,Fv（variable fluorescence）,Fv/Fm,Fm/Fo（electron transfer rate）,Fv/Fo（potential activity of PSⅡ）in leaves of G.biloba obviously presented a descending-ascending trend,the lowest value was at 12：00 and the NPQ（non-photochemical quenching）of sunny leaves arrived at the maximum at noon.The values of Fm,Fv,Fv/Fm,Fm/Fo,Fv/Fo in shade leaves of G.biloba were obviously higher than those in sunny leaves,but the peak value of NPQ of shade leaves presented earlier and higher,suggesting that the shade leaves might have more sensitive hot dissipation mechanism.Comparing to sunny leaves,shade leaves had the higher PSⅡ potential activity and inner light energy translation efficiency.[Conclusion] This study had provided theoretical basis for the protection of G.biloba resources.

Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

BACKGROUND： Extracts of ginkgo biloba leaves （EGB） and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE： To investigate protective effects of EGB on peroxide （H2O2）-induced oxidative stress damage in PC12 cells. DESIGN： Observational contrast study. SETTING： Department ofPathophysiology, Guangdong Pharmacological College. MATERIALS： EGB was provided by Xi＇an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium （MTT）, trypsin and dimathyl sulfoxide （DMSO） by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase （LDH） kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS： The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ①Cell culture： PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1 × 10^7 L^-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates with the density of 200 μ L/hole and divided into three groups： normal control group （routinely adding media）, H2O2 group （treating with media and H2O2 for 20 hours） and EGB group （adding media, 100μmol/L EGB and 100 μmol/L H2O2）. ③ MTT assay： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT （100μL） and cultured for 4 hours. And then, 200 μ L DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly ④ Experimental evaluation： Absorbance （A） at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES： Results of MTT assay and LDH activity. RESULTS： ① Results of MTT assay： A value was lower in the H2O2 group than that in the normal control group （P 〈 0.01）, while A value was higher in the EGB group than that in the H2O2 group （P 〈 0.01）. ② LDH activity： LDH activity was higher in the H2O2 group than that in the normal control group （P 〈 0.01 ）, while LDH activity was lower in the EGB group than that in the H2O2 group （P 〈 0.01）. CONCLUSION： EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.

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.

Content Determination of Quercetin in Ferment of Ginkgo biloba L. Leaves by HPLC

[Objectives] This study aimed to determine the content of quercetin in ferment of Ginkgo biloba L.leaves.[Methods]Bacillus licheniformis was selected for solid-state fermentation of G.biloba leaf powder,and the content of quercetin in ferment of G.biloba leaves was determined by reversed-phase high-performance liquid chromatography.First,the flavonoid glycosides were extracted with methanol.Then,the flavonoid glycosides were hydrolyzed with hydrochloric acid to prepare the test solution.The chromatographic conditions were as follows:Platisil ODS C_(18) column(150 mm × 4.6 mm,5 μm);V_(methonal)∶V_(water)(0.4% phosphoric acid solution) =55∶45;flow rate of 1 m L/min;Shimadzu UV detector;detection wavelength of 360 nm.[Results] Quercetin was used as a reference substance.In the range of 0.002 6-0.036 0 g/L,there was a good linear relationship,with correlation coefficient of 0.999 8 and RSD of 1.26%.[Conclusions] This method is simple,easy to operate,accurate,and reproducible.It is suitable for the determination of quercetin content in G.biloba leaves.

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.

HPLC Method for Determination of Content of Total Flavonoids from Ginkgo (Ginkgo biloba L.) Leaves

[Objectives]This study was conducted to determine the content of total flavonoids in ginkgo ( Ginkgo biloba L.) leaves.[Methods]The content of total flavonoids from ginkgo leaves was determined by reversed phase-high performance liquid chromatography (RP-HPLC).The flavonoid glycosides were first extracted with methanol,and hydrolyzed with hydrochloric acid solution to prepare a test solution.Platisil ODS C18 column (150 mm×4.6 mm,5 μm) and the mobile phase V methanol ∶ V water (0.4% phosphoric acid solution)=85∶ 15 were selected for HPLC separation.The HPLC separation was performed with the column at a column temperature of 25℃ using the mobile phase at a flow rate of 1 ml/min.The sample size was 10 μl,and detection was performed with an Agilent HPLC ultraviolet detector at 360 nm.[Results]The reference substance,quercetin,had good linearity in the range of 0.002 6-0.052 0 g/L,with a correlation coefficient of 0.999 7;and the RSD was 1.26%.[Conclusions]The determination method has rapid and simple operation with accurate results and is good in repeatability.This method is suitable for the determination of content of total flavonoids in ginkgo leaves.

孑遗植物银杏(Ginkgo biloba L.)伴性光合生理特征与进化生态

银杏类是一类古老的雌雄异株植物,目前仅存单科单属。由于银杏在系统发育等方面的独特地位,吸引了科学家从不同方面进行了广泛的研究。在适宜的条件下,测定了生长在野外环境下的成年银杏的雌雄个体的光合特性。研究结果表明,银杏的雌雄植株对光具有相同的表现趋势,光饱和点,光补偿点,光下呼吸速率等均没有明显差异。但是雌性银杏的净光合速率明显大于雄性银杏的净光合速率。这种差异可能与雌性个体在繁衍后代时需要投入更多的能量有关。研究也表明,在与其它的裸子植物和被子植物比较时,银杏的光合能力并没有明显的弱势,因此光合能力可能并不是银杏在第三纪分布受限的直接原因.

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。

The Construction of Fusion Expression Vector Carrying GFP and TP of GGPPS from Ginkgo biloba L.

[Objective]The aim was to construct the fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.[Method]The transit-peptide（TP） sequence of GGPPS from cDNA of Ginkgo biloba L.was successfully cloned by using DNA recombination technology,which was then linked to the efficient plant expression vector p1304 ＋ to construct the fusion gene expression vector p1304 ＋-TP.Then engineering strain EHA105-p1304 ＋-TP was constructed by transformed p1304 ＋-TP to Agrobacterium rhizogenes EHA105 using freeze-thaw method.[Result]The fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.and engineering strain EHA105-p1304 ＋-TP were successfully constructed.[Conclusion]It lays a foundation for further study of subcellular localization of TP transit peptide,which can help to clarify the molecular mechanism of a key step in biosynthesis of ginkgolides precursors,and also provides an important basis for the research on metabolic engineering of ginkgolide.

银杏(Ginkgo biloba)叶的形态发育与演化

通过对银杏叶的形态发育和解剖结构观察,根据重演律和顶枝学说,以及出土化石的地质年代,对银杏类植物的演化过程进行了讨论和推断.指出其演化总趋势是向着叶裂片由窄变宽,利于光合作用的方向发展.

Cloning and Functional Analysis of HDR Gene from Ginkgo biloba L

[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,which was designated as GbHDR （GenBank accession No.：DQ364231）.The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） encoding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR.The β-carotene biosynthetic pathway in E.coli strain XL1-Blue was reconstructed by transforming with pAC-BETA.This engineered XL1-Blue was transformed with pTrcGbHDR.[Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of protein,the predicted molecular weight was 53.2 kD,and predicted isoelectric point was 5.76.Functional complementation assay indicated that GbHDR could promote theβ-carotene accumulation in engineered XL1-Blue harboring pTrcGbHDR and pAC-BETA,and as a result,the engineered bacteria showed the brightly orange given by β-carotene.This suggested that GbHDR had the typical function of known HDR genes.[Conclusion] A engineered bacteria of E.coli which could highly accumulate β-carotene was obtained,which will provide candidate genes and targets for realizing β-carotene metabolic engineering.

Anti-insect Bioactivity of the Extracts from Cichorium intybus L. Leaves

In order to develop a new plant-source insecticide,some active components from Cichorium intybus L.were extracted with mineral ether,ethyl ether,ethyl acetate,respectively.It were testified the effects of the components on the development of Mythimna separate Walker and anti-feeding by feeding and weighting method.The results showed that the body weight of the larvae fed by the extracts with organic solvents was significantly lower than the control;the body weight of the larvae fed by the extracts with organic solvents was significantly different in different solvents in 3-5 days.The corrected mortality of the ethyl acetate treatment was the highest（52.05%）,and the development period of the larvae treated by ethyl acetate was about 4-10 days longer than the control and the corresponding pupating rate was the lowest（43.30%）.Therefore,the effect of ethyl acetate extract was most significant.Moreover,the pupa weight of the treatments was little more than the control.The extracts from Cichorium intybus L.leaves had highest bioactivity mainly in anti-feeding activity on Mythimna separate Walker.

Preliminary Study on Water Physiological Characters of Male and Female Ginkgo biloba L.

[Objective] The experiment aimed to study the difference of water physiology of male and female Ginkgo biloba L. for discussing the strategy of water utilization as well as the important role of this difference during evolution process. [Method] The stem sap flow, stomatal conductance(Gs), transpiration rate(Tr) and water use efficiency (WUE) of male and female Ginkgo biloba L. were comparatively studied. [Result] The day-night processes of flow on male and female Ginkgo biloba L. were similar. The flow on male and female Ginkgo biloba L. in day were almost same while the flow at night on male Ginkgo biloba L. was bigger than that on female Ginkgo biloba L. The Tr and Gs of male and female Ginkgo biloba L. were high in morning and at night but low at noon ,while Tr and Gs of female Ginkgo biloba L. in morning and at night were higher than these of male Ginkgo biloba L. at the same time point. However, these indexes of female plant were lower than these of male plant from 11:00 to 14:00. WUE changing trends of male and female Ginkgo biloba L. were similar, while average water utilization rate of female Ginkgo biloba L. was slightly lower than that of male Ginkgo biloba L. [Conclusion] Compared with other companion plants, water physiology of male and female Ginkgo biloba L. had strong homoplasy. The phenomenon might be a survival strategy of dioecious plants under long term evolutionary pressure.

Transcriptomic profiling of sorghum leaves and roots responsive to drought stress at the seedling stage

Drought stress affects the growth and productivity of crop plants including sorghum.To study the molecular basis of drought tolerance in sorghum,we conducted the transcriptomic profiling of sorghum leaves and roots under drought stress using RNA-Seq method.A total of 510,559,and 3 687 differentially expressed genes(DEGs)in leaves,3 368,5 093,and 4 635 DEGs in roots responding to mild drought,severe drought,and re-watering treatments were identified,respectively.Among them,190 common DEGs in leaves and 1 644 common DEGs in roots were responsive to mild drought,severe drought,and re-watering environment.Gene Ontology(GO)enrichment analysis revealed that the GO categories related to drought tolerance include terms related to response to stimulus especially response to water deprivation,abscisic acid stimulus,and reactive oxygen species.The major transcription factor genes responsive to drought stress include heat stress transcription factor(HSF),ethylene-responsive transcription factor(ERF),Petunia NAM,Arabidopsis ATAF1/2 and CUC2(NAC),WRKY transcription factor(WRKY),homeodomain leucine zipper transcription factor(HD-ZIP),basic helix-loop-helix transcription factor(bHLH),and V-myb myeloblastosis viral oncogene homolog transcription facotr(MYB).Functional protein genes for heat shock protein(HSPs),late-embryogenesis-abundant protein(LEAs),chaperones,aquaporins,and expansins might play important roles in sorghum drought tolerance.Moreover,the genomic regions enriched with HSP,expansin,and aquaporin genes responsive to drought stress could be used as powerful targets for improvement of drought tolerance in sorghum and other cereals.Overall,our results provide a genome-wide analysis of DEGs in sorghum leaves and roots under mild drought,severe drought,and re-watering environments.This study contributes to a better understanding of the molecular basis of drought tolerance of sorghum and can be useful for crop improvement.

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

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.

Transformation of Ginkgo Hairy Root and Establishment of Its Suspension Culture Clone

Tender leaves,buds and stems of ginkgo biloba L .were transformed successfully by Agrobacterium rhizogenes with Ri plasmid,and its suspension cultured clone was eastablished.The result of detection of opines suggested that A.rhizogenes had integrated the T DNA of the Ri plasmid into genomic DNA of ginkgo cells.A new way is offered to exploit ginkgo resources with biological technology.

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.

Studies on Biological Characteristics of Ginkgo Hairy Root Culture

The comparative studies on properties of growth and cultivated conditions of seven transformed ginkgo hairy root clones were reported. Different clones display various phenotypes characterized by growth rate.The results show that the suitable inoculum is benefical to the growth of ginkgo hairy root.NH + 4/NO - 3, pH ,sucrose, and inositol have important effects on the growth of ginkgo hairy root.

Therapeutic Effects of Essential Oil from Waste Leaves of <i>Psidium guajava</i>L. against Cosmetic Embarrassment Using Phylogenetic Approach

Medicinal plants are facing threats due to loss of habitat and overexploitation of knows species. Therefore the present work shows utilization of essential oil from discarded leaves of Psidium guajava Linn. against human dermatophytic fungi Trichophyton rubrum, T. mentagrophytes, Trichophyton violaceum, Trichophyton tonsurans, Epidermophyton floccosum, Microsporum gypseum and Microsporum canis. The oil was found to be fungicidal at 3.0 μl/ml and it tolerated heavy inoculum of pathogens at fungicidal concentrations. The fungicidal activity of the oil was thermostable, up to 70°C and shelf life was found to be six months, which was maximum the time taken into consideration. The oil having cineole, caryophyllane, copaene, azulene and eucalyptol as main constituents, exhibited broad fungicidal activity. The oil did not show any adverse effect on mammalian skin upto 5% concentration. Phylogeny of the dermatophytes with respect to toxicity of the oil has also been discussed using molecular data. The cost effective formulation from waste leaves can be exploited after undergoing double blind successful multicentral topical testing.