Objective:To confirm the therapeutic efficacy of the ginkgo biloba extract EGb761 on ischemic stroke and elucidate its underlying mechanism.Methods:Male Sprague-Dawley rats were divided into three groups:sham,model,an...Objective:To confirm the therapeutic efficacy of the ginkgo biloba extract EGb761 on ischemic stroke and elucidate its underlying mechanism.Methods:Male Sprague-Dawley rats were divided into three groups:sham,model,and EGb761(ginkgo biloba extract).Ischemic stroke was then simulated in rats via embolic middle cerebral artery occlusion surgery,with the extract administered half an hour before surgery.Neurological deficit scores,infarct volume,cerebral edema rate,and inflammatory factors served as the primary metrics for drug efficacy.Serum metabolites were analyzed using 1H-nuclear magnetic resonance to elucidate the operative mechanism.Results:Treatment with the ginkgo biloba extract EGb761 significantly ameliorated the neurological deficit scores(P=.0343),diminished the cerebral infarct volume(P=.0001)and cerebral edema rate(P=.0030),and alleviated neuroinflammation(all P<.05)in middle cerebral artery occlusion rats.In addition,it significantly altered the contents of various metabolites,such as 2-hydroxybutyrate,isoleucine,isopropanol,isobutyric acid,N6-acetyllysine,glutamate,glutamine,methionine,and N,Ndimethylglycine(all P<.05).Enrichment analysis of the differential metabolites indicated that EGb761 may be involved in the regulation of amino acid metabolism,betaine metabolism,glucose-alanine cycle,Warburg effect,and urea cycle.Conclusion:The ginkgo biloba extract EGb761 demonstrates anti-ischemic stroke effect on ischemic stroke model rats by regulating amino acids and amino acid derivatives,such as isoleucine,N6-acetyllysine,glutamate,methionine,and N,N-dimethylglycine.展开更多
Abstract： In the present study, we established an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry （UPLC-QTOF-MSE） method to simultaneously quantify 33 components in Ginkgo bilo...Abstract： In the present study, we established an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry （UPLC-QTOF-MSE） method to simultaneously quantify 33 components in Ginkgo biloba leaf extracts （GBEs）, including 17 flavonol glycosides, five terpene trilactones （TTLs）, four polyphenols and seven carboxylic acids. This optimized method was successfully applied to analyze the explicit compositions of GBE samples collected from different places. Furthermore, the data were processed through unsupervised principal component analysis （PCA） and supervised orthogonal partial least squared discrimination analysis （OPLS-DA） to evaluate the quality and compare the differences between the samples according to the contents of the 33 chemical constituents. Bilobalide, protocatechuic acid, shikimic acid, quinic acid, ginkgolide B, ginkgolide J, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, quercetin-3-O-ct-L-rhamnopyranocyl-2＂-（6＇＂-p-coumaroyl）-β-D-glucoside and rutin were recognized as characteristic chemical markers that contributed most to control the quality of GBEs. Based on the fact that GBEs should be standardized with the characteristic components as quality control chemical markers, it is most important to maintain the quality of GBEs stable and reliable, and this method also provided a good strategy to further rectify and standardize the GBEs market.展开更多
The random amplified polymorphic DNA (RAPD) technique was used to amplify DNA fragment, aiming at finding markers linked to the sex determination in Ginkgo biloba L. One thousand and two hundred random decamers had be...The random amplified polymorphic DNA (RAPD) technique was used to amplify DNA fragment, aiming at finding markers linked to the sex determination in Ginkgo biloba L. One thousand and two hundred random decamers had been screened. Of the 8 372 RAPD bands, only a 682 bp RAPD marker generated by a primer (S1478) of random decamer sequence, named S1478-682, was found to be associated with the male plants. This marker was present in all male plants and absent in all female plants. Ginkgo trees collected in both Beijing and Shenyang, China were tested using primer S1478. Positive results were obtained, suggesting S1478-682 could be utilized as a reliable RAPD marker to detect the sexuality of Ginkgo.展开更多
In midday ginkgo ( Ginkgo biloba L.) leaves have to bear photon flux density over 1400 μmol·m -2 ·s -1 in combination with high temperatures around 35 ℃ at natural habitat. They show typical mi...In midday ginkgo ( Ginkgo biloba L.) leaves have to bear photon flux density over 1400 μmol·m -2 ·s -1 in combination with high temperatures around 35 ℃ at natural habitat. They show typical midday depression of stomatal conductance and of CO 2 assimilation rate. The zeaxanthin changes with light intensity during the day. The influence of the combination of strong light and temperature on photoinhibition was also examined in the laboratory. A low CO 2 internal conductance (31 mmol·m -2 ·s -1 ) was found in ginkgo leaves, which had been exposed to excessive light at temperature between 15 ℃ and 35 ℃ with reduced CO 2 (80 μL·L -1 ) or oxygen (2%) for 2 h, causing a low CO 2 concentration at the carboxylation site and a high proportion of photorespiration. The ratio of electron transport to CO 2 fixation was rather high in ginkgo (16 e -/CO 2 at 25 ℃) as compared with other plants. It increased with temperature also in 2% O 2 which could not be explained solely as due to change of photorespiration. The reduction of oxygen in 340 or 80 μL·L -1 CO 2 had no effect on the extent of photoinhibition at all temperatures, which indicated that electron flow caused by photorespiration in excess light was negligible in protective effect in ginkgo leaves. However, a decreased CO 2 concentration increased photoinhibition, especially at high temperature. It is concluded that the dissipation of excessive excitation energy in the PSⅡ antennae through the xanthophyll cycle may be the major protective mechanism to preventing from the deteriorated effects of strong light in ginkgo leaves.展开更多
Aim To establish a method for determination of Ginkgo biloba L, its extractand preparations with HPLC fingerprints, so as to control the quality of the preparations. MethodsHPLC-DAD method was used to determine the co...Aim To establish a method for determination of Ginkgo biloba L, its extractand preparations with HPLC fingerprints, so as to control the quality of the preparations. MethodsHPLC-DAD method was used to determine the constituents in preparations. Diamonsil? C_(18) (200mm X 4.6 mm, 5 μm) was used as analytical column, and acetonitrile/KH_2PO_4 was used as mobilephase with gradient elu-tion. The column temperature was at 24 ℃. The HPLC profile of chemicalconstituents of control sample and preparations were analyzed using similarity software. Results Thefingerprints of different preparations from different companies were slightly different because ofthe different preparing procedures. Mean while, the fingerprints of different batches of the samepreparation from the same company were similar to each other and the technology of each preparationwas stable. Conclusion This method is accurate, reproducible , simple, and can be used as ananalytical method for the routine quality control of Ginkgo biloba preparations.展开更多
The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Gi...The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Ginkgo biloba L. by high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) was carried out. The separation was performed on Inertsil ODS3 column with methanol-water (36:64) as mobile phase, with 1 mL·min -1 of flow rate at 35℃. Then the mass spectrum analysis was conducted by ZMD micromass electrospray ionization (ESI)-mass spectrometer (MS). The HPLC total ion chromatogram and selected ion chromatogram (with 325, 407, 423, 439 of m/z) of the sample and ESI-/MS mass spectra of the peaks in the chromatograms were obtained. So bilobalide, ginkgolide A, B, C and J in Ginkgo biloba L. leaves were identified. The method is easy and rapid, with a good accuracy.展开更多
[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...[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.展开更多
Ginkgo diterpene lactones meglumine injection(GDLI)is a commercially available product used for neuroprotection.However,the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary c...Ginkgo diterpene lactones meglumine injection(GDLI)is a commercially available product used for neuroprotection.However,the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI,i.e.,ginkgolide A(GA),ginkgolide B(GB),and ginkgolide K(GK),have never been fully evaluated in beagle dogs.In this work,a simple,sensitive,and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS)was developed,and the prototypes and total amounts of GA,GB,and GK were determined in beagle dog plasma.The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations.For the first time,the pharmacokinetics of GA,GB,and GK were fully assessed in three forms,i.e.,the prototypes,the hydrolyzed carboxylic forms,and the total amounts,after intravenous administration of GDLI in beagle dogs.It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma,and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio.All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages.GA,GB,and GK showed a constant half-life approximately 2.7,3.4,and 1.2 h,respectively,which were consistent for the forms at three dose levels(0.3,1.0,and 3.0 mg·kg^(-1))and after a consecutive injection of GDLI for 7 days(1.0 mg·kg^(-1)).展开更多
Stem cell transplantation has brought new hope for the treatment of neurological diseases.The key to stem cell therapy lies in inducing the specific differentiation of stem cells into nerve cells.Because the different...Stem cell transplantation has brought new hope for the treatment of neurological diseases.The key to stem cell therapy lies in inducing the specific differentiation of stem cells into nerve cells.Because the differentiation of stem cells in vitro and in vivo is affected by multiple factors,the final differentiation outcome is strongly associated with the microenvironment in which the stem cells are located.Accordingly,the optimal microenvironment for inducing stem cell differentiation is a hot topic.EGb761 is extracted from the leaves of the Ginkgo biloba tree.It is used worldwide and is becoming one of the focuses of stem cell research.Studies have shown that EGb761 can antagonize oxygen free radicals,stabilize cell membranes,promote neurogenesis and synaptogenesis,increase the level of brain-derived neurotrophic factors,and replicate the environment required during the differentiation of stem cells into nerve cells.This offers the possibility of using EGb761 to induce the differentiation of stem cells,facilitating stem cell transplantation.To provide a comprehensive reference for the future application of EGb761 in stem cell therapy,we reviewed studies investigating the influence of EGb761 on stem cells.These started with the composition and neuropharmacology of EGb761,and eventually led to the finding that EGb761 and some of its important components play important roles in the differentiation of stem cells and the protection of a beneficial microenvironment for stem cell transplantation.展开更多
[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,w...[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.展开更多
[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants...[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.展开更多
[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...[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.展开更多
[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 ...[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.展开更多
[Objective]The aim of this study is to research leaf color development and photosynthetic characteristics of golden-leaf ginkgo.[Method]With one-year-old grafted seedlings of golden-leaf ginkgo as the materials,the ch...[Objective]The aim of this study is to research leaf color development and photosynthetic characteristics of golden-leaf ginkgo.[Method]With one-year-old grafted seedlings of golden-leaf ginkgo as the materials,the changes of leaf color,chlorophyll content（Chl）,carotenoid（x.c）content and rate of chlorophyll and carotenoid content,as well as photosynthetic characteristics under full sunlight and overshadowing were all investigated in this study.[Result]Sprouts of golden-leaf ginkgo were pale-yellow,and changed from orange to golden in April,to light yellow-green in May,to yellow-green from June to October,to yellow in November.The chlorophyll and carotenoid contents and the rates of chlorophyll and carotenoid contents of yellow-leaf were significantly lower than that of green-leaf,while the rate of Chla/x.c and Chlb/x.c was obviously lower than the corresponding pigment content of green-leaf.During the leaf color development,Chla,Chlb and x.c content as well as the rate of Chla/x.c and Chlb/x.c of yellow-leaf ginkgo all increased.The saturation light intensity of ginkgo leaf under natural light was higher than that under overshadowing.Maximum net photosynthetic rate,light compensation point and dark respiration rate of yellow leaf were all significantly higher than that of green leaf.Chla/x.c,Chlb/x.c and Chla＋b/x.c of yellow-leaf ginkgo under overshadowing were higher than the corresponding pigment rate of leaf under natural light.[Conclusion]The best ornamental duration of golden-leaf ginkgo is April and May.The major reason for showing yellow leaf is that Chla/x.c and Chlb/x.c of yellow-leaf is obviously lower than that of green-leaf.During the leaf color development,the major reason for yellow-leaf turning to yellow-green is that the rate of Chla/x.c and Chlb/x.c increases.Chlb content of yellow-leaf under overshadowing is higher than that under natural light,which is an adaptive response to overshadowing.Chla/x.c,Chlb/x.c and Chla＋b/x.c of yellow-leaf under overshadowing are relatively higher,which is one of the reasons why yellow-leaf under overshadowing becomes yellow-green or green.展开更多
The early stage of pollen chamber development in ovule and the cytological mechanism of nucellar cell death were studied in Ginkgo biloba L. DNA ladder appearance and TUNEL assay demonstrated that the nucellar cel...The early stage of pollen chamber development in ovule and the cytological mechanism of nucellar cell death were studied in Ginkgo biloba L. DNA ladder appearance and TUNEL assay demonstrated that the nucellar cell death, doomed to bring about pollen chamber formation, was a process of programmed cell death (PCD). A spatial distribution of PCD was visualized during the development of pollen chamber. Together with the observation under the scanning electron microscope, these results have revealed that the early developmental pattern of pollen chamber consists of four phases. Firstly, several layers of the nucellar cells at the micropylar end elongate longitudinally. Thereafter, the uppermost layer of the nucellar cells at the micropylar end initiate PCD; and the nucellar cell death extends in a basally and laterally oriented direction to form a cavity. Finally, the epidermal cells at the micropylar end detach from the other epidermis by dehiscence, bringing about the opening of the pollen chamber. The early development of pollen chamber begins sometime after the stage of megasporocyte and continues by the time of the formation of megaspore tetrad, and finally completes at the stage of development of female gametophyte. This shows a synchronous development of megaspore and pollen chamber.展开更多
Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus wa...Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.展开更多
Programmed cell death (PCD) of the nucellar cells at the micropylar end is involved in pollen chamber morphogenesis in Ginkgo biloba L. A development-course observation of the morphological changes in the nucellar cel...Programmed cell death (PCD) of the nucellar cells at the micropylar end is involved in pollen chamber morphogenesis in Ginkgo biloba L. A development-course observation of the morphological changes in the nucellar cells undergoing PCD to form pollen chamber was performed. During the PCD, the nucellar cells degraded their cellular components through an orderly progression. Through the vactiolation, the cytosol was engulfed by the enlarging vacuole, leaving out various organelles, which remained morphologically integrated. As the vacuolation continued, the vacuole collapsed with the breakage of the tonoplast and the cytosol disappeared completely. Organelles were subsequently destroyed. Ultimately, nucellar cells digested away all of their cytoplasm, leaving with cell walls. They became collapsed as the nucellus developed. Intracellular membranes were strikingly changed, playing a role in leading to cell death. Some of these noticeable changes were the appearance of multivesicular body, multicycle-like membranes, membrane-bounded bodies containing some organelles, tonoplast rupture and numerous vesicles. The dehiscence of the apical epidermis, resulting in the opening, appeared to have followed two different pathways with one involving a specific epidermal cell autolysis and the other by detachment from middle lamella of two neighboring epidermal cells without cell autolysis. The specific epidermal cells had been dead prior to the dehiscence of the apical epidermis, which marked the sites of the dehiscence followed. In view of the changes in the cellular morphology, a process of nucellar cell PCD in the course of the pollen chamber formation was demonstrated.展开更多
基金supported by the Youth Science Fund Project of the National Natural Science Foundation of China(82104440).
文摘Objective:To confirm the therapeutic efficacy of the ginkgo biloba extract EGb761 on ischemic stroke and elucidate its underlying mechanism.Methods:Male Sprague-Dawley rats were divided into three groups:sham,model,and EGb761(ginkgo biloba extract).Ischemic stroke was then simulated in rats via embolic middle cerebral artery occlusion surgery,with the extract administered half an hour before surgery.Neurological deficit scores,infarct volume,cerebral edema rate,and inflammatory factors served as the primary metrics for drug efficacy.Serum metabolites were analyzed using 1H-nuclear magnetic resonance to elucidate the operative mechanism.Results:Treatment with the ginkgo biloba extract EGb761 significantly ameliorated the neurological deficit scores(P=.0343),diminished the cerebral infarct volume(P=.0001)and cerebral edema rate(P=.0030),and alleviated neuroinflammation(all P<.05)in middle cerebral artery occlusion rats.In addition,it significantly altered the contents of various metabolites,such as 2-hydroxybutyrate,isoleucine,isopropanol,isobutyric acid,N6-acetyllysine,glutamate,glutamine,methionine,and N,Ndimethylglycine(all P<.05).Enrichment analysis of the differential metabolites indicated that EGb761 may be involved in the regulation of amino acid metabolism,betaine metabolism,glucose-alanine cycle,Warburg effect,and urea cycle.Conclusion:The ginkgo biloba extract EGb761 demonstrates anti-ischemic stroke effect on ischemic stroke model rats by regulating amino acids and amino acid derivatives,such as isoleucine,N6-acetyllysine,glutamate,methionine,and N,N-dimethylglycine.
文摘Abstract： In the present study, we established an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry （UPLC-QTOF-MSE） method to simultaneously quantify 33 components in Ginkgo biloba leaf extracts （GBEs）, including 17 flavonol glycosides, five terpene trilactones （TTLs）, four polyphenols and seven carboxylic acids. This optimized method was successfully applied to analyze the explicit compositions of GBE samples collected from different places. Furthermore, the data were processed through unsupervised principal component analysis （PCA） and supervised orthogonal partial least squared discrimination analysis （OPLS-DA） to evaluate the quality and compare the differences between the samples according to the contents of the 33 chemical constituents. Bilobalide, protocatechuic acid, shikimic acid, quinic acid, ginkgolide B, ginkgolide J, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, quercetin-3-O-ct-L-rhamnopyranocyl-2＂-（6＇＂-p-coumaroyl）-β-D-glucoside and rutin were recognized as characteristic chemical markers that contributed most to control the quality of GBEs. Based on the fact that GBEs should be standardized with the characteristic components as quality control chemical markers, it is most important to maintain the quality of GBEs stable and reliable, and this method also provided a good strategy to further rectify and standardize the GBEs market.
文摘The random amplified polymorphic DNA (RAPD) technique was used to amplify DNA fragment, aiming at finding markers linked to the sex determination in Ginkgo biloba L. One thousand and two hundred random decamers had been screened. Of the 8 372 RAPD bands, only a 682 bp RAPD marker generated by a primer (S1478) of random decamer sequence, named S1478-682, was found to be associated with the male plants. This marker was present in all male plants and absent in all female plants. Ginkgo trees collected in both Beijing and Shenyang, China were tested using primer S1478. Positive results were obtained, suggesting S1478-682 could be utilized as a reliable RAPD marker to detect the sexuality of Ginkgo.
文摘In midday ginkgo ( Ginkgo biloba L.) leaves have to bear photon flux density over 1400 μmol·m -2 ·s -1 in combination with high temperatures around 35 ℃ at natural habitat. They show typical midday depression of stomatal conductance and of CO 2 assimilation rate. The zeaxanthin changes with light intensity during the day. The influence of the combination of strong light and temperature on photoinhibition was also examined in the laboratory. A low CO 2 internal conductance (31 mmol·m -2 ·s -1 ) was found in ginkgo leaves, which had been exposed to excessive light at temperature between 15 ℃ and 35 ℃ with reduced CO 2 (80 μL·L -1 ) or oxygen (2%) for 2 h, causing a low CO 2 concentration at the carboxylation site and a high proportion of photorespiration. The ratio of electron transport to CO 2 fixation was rather high in ginkgo (16 e -/CO 2 at 25 ℃) as compared with other plants. It increased with temperature also in 2% O 2 which could not be explained solely as due to change of photorespiration. The reduction of oxygen in 340 or 80 μL·L -1 CO 2 had no effect on the extent of photoinhibition at all temperatures, which indicated that electron flow caused by photorespiration in excess light was negligible in protective effect in ginkgo leaves. However, a decreased CO 2 concentration increased photoinhibition, especially at high temperature. It is concluded that the dissipation of excessive excitation energy in the PSⅡ antennae through the xanthophyll cycle may be the major protective mechanism to preventing from the deteriorated effects of strong light in ginkgo leaves.
文摘Aim To establish a method for determination of Ginkgo biloba L, its extractand preparations with HPLC fingerprints, so as to control the quality of the preparations. MethodsHPLC-DAD method was used to determine the constituents in preparations. Diamonsil? C_(18) (200mm X 4.6 mm, 5 μm) was used as analytical column, and acetonitrile/KH_2PO_4 was used as mobilephase with gradient elu-tion. The column temperature was at 24 ℃. The HPLC profile of chemicalconstituents of control sample and preparations were analyzed using similarity software. Results Thefingerprints of different preparations from different companies were slightly different because ofthe different preparing procedures. Mean while, the fingerprints of different batches of the samepreparation from the same company were similar to each other and the technology of each preparationwas stable. Conclusion This method is accurate, reproducible , simple, and can be used as ananalytical method for the routine quality control of Ginkgo biloba preparations.
文摘The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Ginkgo biloba L. by high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) was carried out. The separation was performed on Inertsil ODS3 column with methanol-water (36:64) as mobile phase, with 1 mL·min -1 of flow rate at 35℃. Then the mass spectrum analysis was conducted by ZMD micromass electrospray ionization (ESI)-mass spectrometer (MS). The HPLC total ion chromatogram and selected ion chromatogram (with 325, 407, 423, 439 of m/z) of the sample and ESI-/MS mass spectra of the peaks in the chromatograms were obtained. So bilobalide, ginkgolide A, B, C and J in Ginkgo biloba L. leaves were identified. The method is easy and rapid, with a good accuracy.
基金Supported by The Cloning and Analysis of Key Enzyme Genes in the Biosynthesis Pathway of Lactone Precursor of Ginkgo biloba(30500303)~~
文摘[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.
基金financially supported by the National Key Special Project of Science and Technology for Innovation Drugs of China(Nos.2013zx09402203 and 2013zx09402202)the Natural Science Foundation of Jiangsu Province,China(No.BK20130403)+1 种基金the National Natural Science Foundation of China(No.81503342)the Project for Jiangsu Province Key Lab of Drug Metabolism and Pharmacokinetics(No.BM2012012)
文摘Ginkgo diterpene lactones meglumine injection(GDLI)is a commercially available product used for neuroprotection.However,the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI,i.e.,ginkgolide A(GA),ginkgolide B(GB),and ginkgolide K(GK),have never been fully evaluated in beagle dogs.In this work,a simple,sensitive,and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS)was developed,and the prototypes and total amounts of GA,GB,and GK were determined in beagle dog plasma.The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations.For the first time,the pharmacokinetics of GA,GB,and GK were fully assessed in three forms,i.e.,the prototypes,the hydrolyzed carboxylic forms,and the total amounts,after intravenous administration of GDLI in beagle dogs.It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma,and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio.All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages.GA,GB,and GK showed a constant half-life approximately 2.7,3.4,and 1.2 h,respectively,which were consistent for the forms at three dose levels(0.3,1.0,and 3.0 mg·kg^(-1))and after a consecutive injection of GDLI for 7 days(1.0 mg·kg^(-1)).
基金funded by the National Natural Science Foundation of China,No.81501185(to CR)the Key Research&Development Project of Shandong Province of China,No.2017GSF218043(to CR)the Science and Technology Planning Project of Yantai of China,No.2016WS017(to LNG),2017WS105(to HL)
文摘Stem cell transplantation has brought new hope for the treatment of neurological diseases.The key to stem cell therapy lies in inducing the specific differentiation of stem cells into nerve cells.Because the differentiation of stem cells in vitro and in vivo is affected by multiple factors,the final differentiation outcome is strongly associated with the microenvironment in which the stem cells are located.Accordingly,the optimal microenvironment for inducing stem cell differentiation is a hot topic.EGb761 is extracted from the leaves of the Ginkgo biloba tree.It is used worldwide and is becoming one of the focuses of stem cell research.Studies have shown that EGb761 can antagonize oxygen free radicals,stabilize cell membranes,promote neurogenesis and synaptogenesis,increase the level of brain-derived neurotrophic factors,and replicate the environment required during the differentiation of stem cells into nerve cells.This offers the possibility of using EGb761 to induce the differentiation of stem cells,facilitating stem cell transplantation.To provide a comprehensive reference for the future application of EGb761 in stem cell therapy,we reviewed studies investigating the influence of EGb761 on stem cells.These started with the composition and neuropharmacology of EGb761,and eventually led to the finding that EGb761 and some of its important components play important roles in the differentiation of stem cells and the protection of a beneficial microenvironment for stem cell transplantation.
基金Supported by National Natural Science Foundation (30500303)~~
文摘[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.
文摘[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.
基金Supported by the State Key Fundamental Science Fund of China(2005CB422208)NSF-China Project(40671132)the State Data Synthesis and Analysis Funds of China(2006DKA32300-08)~~
文摘[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.
基金Supported by Forestry Scientific and Technological Supporting Project of State Forestry Administration~~
文摘[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.
文摘[Objective]The aim of this study is to research leaf color development and photosynthetic characteristics of golden-leaf ginkgo.[Method]With one-year-old grafted seedlings of golden-leaf ginkgo as the materials,the changes of leaf color,chlorophyll content（Chl）,carotenoid（x.c）content and rate of chlorophyll and carotenoid content,as well as photosynthetic characteristics under full sunlight and overshadowing were all investigated in this study.[Result]Sprouts of golden-leaf ginkgo were pale-yellow,and changed from orange to golden in April,to light yellow-green in May,to yellow-green from June to October,to yellow in November.The chlorophyll and carotenoid contents and the rates of chlorophyll and carotenoid contents of yellow-leaf were significantly lower than that of green-leaf,while the rate of Chla/x.c and Chlb/x.c was obviously lower than the corresponding pigment content of green-leaf.During the leaf color development,Chla,Chlb and x.c content as well as the rate of Chla/x.c and Chlb/x.c of yellow-leaf ginkgo all increased.The saturation light intensity of ginkgo leaf under natural light was higher than that under overshadowing.Maximum net photosynthetic rate,light compensation point and dark respiration rate of yellow leaf were all significantly higher than that of green leaf.Chla/x.c,Chlb/x.c and Chla＋b/x.c of yellow-leaf ginkgo under overshadowing were higher than the corresponding pigment rate of leaf under natural light.[Conclusion]The best ornamental duration of golden-leaf ginkgo is April and May.The major reason for showing yellow leaf is that Chla/x.c and Chlb/x.c of yellow-leaf is obviously lower than that of green-leaf.During the leaf color development,the major reason for yellow-leaf turning to yellow-green is that the rate of Chla/x.c and Chlb/x.c increases.Chlb content of yellow-leaf under overshadowing is higher than that under natural light,which is an adaptive response to overshadowing.Chla/x.c,Chlb/x.c and Chla＋b/x.c of yellow-leaf under overshadowing are relatively higher,which is one of the reasons why yellow-leaf under overshadowing becomes yellow-green or green.
文摘The early stage of pollen chamber development in ovule and the cytological mechanism of nucellar cell death were studied in Ginkgo biloba L. DNA ladder appearance and TUNEL assay demonstrated that the nucellar cell death, doomed to bring about pollen chamber formation, was a process of programmed cell death (PCD). A spatial distribution of PCD was visualized during the development of pollen chamber. Together with the observation under the scanning electron microscope, these results have revealed that the early developmental pattern of pollen chamber consists of four phases. Firstly, several layers of the nucellar cells at the micropylar end elongate longitudinally. Thereafter, the uppermost layer of the nucellar cells at the micropylar end initiate PCD; and the nucellar cell death extends in a basally and laterally oriented direction to form a cavity. Finally, the epidermal cells at the micropylar end detach from the other epidermis by dehiscence, bringing about the opening of the pollen chamber. The early development of pollen chamber begins sometime after the stage of megasporocyte and continues by the time of the formation of megaspore tetrad, and finally completes at the stage of development of female gametophyte. This shows a synchronous development of megaspore and pollen chamber.
文摘Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.
文摘Programmed cell death (PCD) of the nucellar cells at the micropylar end is involved in pollen chamber morphogenesis in Ginkgo biloba L. A development-course observation of the morphological changes in the nucellar cells undergoing PCD to form pollen chamber was performed. During the PCD, the nucellar cells degraded their cellular components through an orderly progression. Through the vactiolation, the cytosol was engulfed by the enlarging vacuole, leaving out various organelles, which remained morphologically integrated. As the vacuolation continued, the vacuole collapsed with the breakage of the tonoplast and the cytosol disappeared completely. Organelles were subsequently destroyed. Ultimately, nucellar cells digested away all of their cytoplasm, leaving with cell walls. They became collapsed as the nucellus developed. Intracellular membranes were strikingly changed, playing a role in leading to cell death. Some of these noticeable changes were the appearance of multivesicular body, multicycle-like membranes, membrane-bounded bodies containing some organelles, tonoplast rupture and numerous vesicles. The dehiscence of the apical epidermis, resulting in the opening, appeared to have followed two different pathways with one involving a specific epidermal cell autolysis and the other by detachment from middle lamella of two neighboring epidermal cells without cell autolysis. The specific epidermal cells had been dead prior to the dehiscence of the apical epidermis, which marked the sites of the dehiscence followed. In view of the changes in the cellular morphology, a process of nucellar cell PCD in the course of the pollen chamber formation was demonstrated.
