Advances in Supercritical Carbon Dioxide Extraction of Bioactive Substances from Different Parts of Ginkgo biloba L.

Ginkgo biloba L. has always been a popular area of research due to its various active ingredients and pharmacological effects. Ginkgo biloba is rich in ginkgo flavonoids, ginkgolides, and ginkgolic acid, with anti-inflammation, antioxidation, neuroprotection, anti-platelet agglutination, hypolipidemic effect, anti-cancer, and anti-radiation properties. There are many methods to extract and separate the active components of ginkgo. Among them, supercritical carbon dioxide fluid extraction (SFE-CO2) is known for its green, clean, and environment-friendly properties. In this paper, the pharmacological activities, the active components, and structures of different parts of ginkgo, the extraction methods of its effective ingredients, and the application of the SFE-CO2 method for the extraction and separation of active ingredients in Ginkgo biloba from leaves, seeds, pollen, and roots were reviewed, in order to make best use of ginkgo resources, and provide support and references for the development of SFE-CO2 of active components from Ginkgo biloba.

Therapeutic promises of ginkgolide A: A literature-based review.

Ginkgolide A is a highly active platelet activating factor antagonist cage molecule which was isolated from the leaves of the Ginkgo biloba L. It is known for its inflammatory and immunological potentials. This review aims to sketch a current scenario on its therapeutic activities on the basis of scientific reports in the databases. A total 30 articles included in this review suggests that ginkgolide A has many important biological activities, including anti-inflammatory, anticancer, anxiolytic-like, anti-atherosclerosis and anti-atherombosis, neuro- and hepatoprotective effects. There is a lack of its toxicological (e.g. toxicity, cytotoxicity, genotoxicity and mutagenitcity) profile. In conclusion, ginkgolide A may be one of the potential therapeutic lead compounds, especially for the treatment of cardiovascular, hepatological, and neurological diseases and disorders. More studies are necessary on this hopeful therapeutic agent.

A new bilobalide isomer and two cis-coumaroylated flavonol glycosides from Ginkgo biloba leaves.

A new bilobalide isomer (1), together with two flavonol glycosides (2, 3), have been isolated and elucidated from the extract of Ginkgo biloba leaves. Significantly, 1 was a new sesquiterpene lactone with two lactone ring groups, both 2 and 3 were two flavonol glycosides with a same cis-coumaroylated fragment. Their chemical structures were elucidated by NMR and MS spectroscopic date and the absolute configuration of 1 was specific established by Cu-Kα X-ray crystallographic analyses. However, 1-3 showed no obvious anti-platelet aggregation activity.

Bilobalide and PC12 cells: A structure activity relationship study.

Ginkgo biloba extracts have been postulated to beneficial for improving cognitive function and as such they have been used as a potential treatment of Alzheimer's disease. The main active ingredients of the extract are terpene trilactones (TTLs), such as bilobalide (BB) and ginkgolides. Several structure-activity relationship (SAR) studies using ginkgolide scaffolds produced more biologically potent species by modification of the lactone moieties. However, modifications of BB scaffold have been limited, and no SAR studies on BB have been accomplished to date. Thus, the aim of this study was to elucidate how the modification of the lactone moieties of BB would affect their biological activities in a number of assays, including proliferating cell activity, neuroprotective effects against Aß (1-40) peptides, and neurite outgrowth effects in PC12 neuronal cells. It appeared that the derivatives containing lactone groups showed similar biological activity to native BB, while those that possessed no lactone moieties exhibited lower neurite outgrowth effects. Thus, the results suggested that the lactone moieties of BB played an important role in exerting neurite outgrowth effects in PC12 cells.

Theoretical design, preparation, and evaluation of Ginkgolide B molecularly imprinted polymers.

Ginkgolide B is in great demand worldwide on account of its extensive and excellent pharmacological effects, however, it is difficult to separate and purify ginkgolide B. In this study, ginkgolide B molecularly imprinted polymers were prepared by combining software simulation and molecular imprinting technique, and its characterization and adsorption performed evaluation were performed to understand the adsorption behavior of the polymers. The adsorption equilibrium concentration of molecularly imprinted polymers was 0.70 mg/mL, and the adsorption equilibrium time was 4 h. Meanwhile, the adsorption isotherm of the polymers for ginkgolide B fitted well with the Langmuir model, and the adsorption kinetics was in line with the pseudo-second-order kinetics. In contrast, the adsorption capacity of molecularly imprinted polymers on ginkgolide B was higher than that of non-molecular imprinted polymers, with better selectivity and better adsorption after repeated use for six times. The application experiments showed that molecular imprinted polymers have a good adsorption effect in low purity samples. Therefore, the polymers reported herein can be expected to apply in the adsorption and separation of ginkgolide B samples.

Identification of eupatilin and ginkgolide B as p38 ligands from medicinal herbs by surface plasmon resonance biosensor-based active ingredients recognition system.

Screening of bioactive ligands for a certain protein target from medicinal herbs is a highly important yet challenging task during drug discovery process. In this study, a surface plasmon resonance biosensor-based active ingredient recognition system (SPR-AIRS) was applied to screen p38 mitogen-activated protein kinase (p38) ligands from herbal extracts. After p38 protein was immobilized on a SPR chip and the suitability of SPR-AIRS was validated, thirty-four p38-related medicinal herbs were selected and pre-screened. Two medicinal herbs having high response signal with p38-immobilized chip, Folium Ginkgo and Herba Artemisiae Scopariae, were injected into SPR system for ligand fishing. Among them, two active compounds, eupatilin (EPT) and ginkgolide B (GKB), were identified as p38 ligands, and then the KD values of EPT and GKB were measured as 21.68 ± 2.21 and 44.71 ± 1.80 µM, respectively. They can inhibit p38 activities significantly and bind to the ATP binding site on p38. Furthermore, EPT and GKB can inhibit cell proliferation (IC50 = 30.31 ± 6.84 and 42.97 ± 0.83 µM), induce apoptosis and G2/M cell cycle arrest against K562 cell line. This is the first time that EPT and GKB are reported as effective p38 binding ligands. These results prove that SPR-AIRS could be an effective method to screen active compounds acting on a specific protein from complex systems.

Multi-Response Optimization of Ultrasonic Assisted Enzymatic Extraction Followed by Macroporous Resin Purification for Maximal Recovery of Flavonoids and Ginkgolides from Waste Ginkgo biloba Fallen Leaves.

In the present study, the process of ultrasonic assisted enzymatic extraction (UAEE), followed by macroporous resin purification, was successfully developed to achieve maximal recovery of flavonoids and ginkgolides from Ginkgo biloba fallen leaves (GBFL). Three effective extracted factors, including UAE power, EtOH%, and the amount of cellulase were screened by Plackett⁻Burman design (PBD). The important variables were further optimized by rotatable central composite design (RCCD). After the combination of PBD and RCCD, the resulting optimal UAEE conditions were as follows: UAE power of 218 W; EtOH% of 68%; the amount of cellulase of 8.4 mg; UAE temperature of 40 °C; UAE time of 20 min; pH of 5.0; and, sample particle size of 40 mesh. Under the optimum conditions; the yields of flavonoids were 0.74 ± 0.05% (n = 3) and ginkgolides was 0.42 ± 0.06% (n = 3), which were close to the predicted values. Moreover, the further enriching flavonoids and ginkgolides from the obtained GBFL extracts using the above optimum UAEE condition was successfully achieved by macroporous resin DA-201. After column adsorption and desorption on DA-201; the percentage of total flavonoids was (25.36 ± 1.03)%; ginkgolides was (12.43 ± 0.85)% and alkylphenols was (0.003 ± 0.0005)% from the obtained dry extracts of GBFL which were complied with Chinese pharmacopoeias. Therefore, the present study provided a convenient and efficient method for extraction and purification of flavonoids and ginkgolides from waste GBFL.

Effects of food and gender on the pharmacokinetics of ginkgolides A, B, C and bilobalide in rats after oral dosing with ginkgo terpene lactones extract.

The ginkgo terpene lactones (GTL), mainly including bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB) and ginkgolide C (GC) possess different biological activities such as peripheral vasoregulation, platelet-activating factor (PAF) receptor antagonism, neuroprotective properties and prevention of membrane damage caused by free radicals. To investigate the effects of food and gender on the bioavailability of BB, GA, GB and GC after oral administration of GTL extract, a rapid UPLC-MS/MS method was developed and validated. A reversed phase C18 column (100mm×2.1mm, i.d., 1.7µm) and a mobile phase consisted of methanol and 1mM ammonium acetate (70/30, v/v) were employed. Compared with the fasted group, the t1/2 values for BB, GA, GB and GC in fed were all increased (p<0.05), AUC0-t and AUC0-∞ values of BB, GA, GB and GC were all significantly increased (p<0.05), but the Cmax values of BB, GA, GB and GC were significantly decreased (p<0.05). In comparison with the male group, all of the t1/2 values and AUC0-t values for BB, GA, GB and GC in female were higher (p<0.05), but no statistical difference in Tmax values for BB, GA, GB and GC between these two groups. Food and gender factor showed significant effects on the pharmacokinetics of BB, GA, GB, and GC. The results suggested that oral doses of GTL should be lowered for fasted and female subjects, compared with the fed and male subjects, respectively.

Ginkgo May Sensitize Ovarian Cancer Cells to Cisplatin: Antiproliferative and Apoptosis-Inducing Effects of Ginkgolide B on Ovarian Cancer Cells.

Ginkgolide B (GB), the primary active component ofGinkgo bilobaextracts, may have antitumor properties. The objective of this study was to determine the effects and possible mechanisms of GB in ovarian cancer cells. In this study, human ovarian cancer cell lines (SKOV3 and CAOV3) were treated with different concentrations of GB alone or in combination with Cis-diaminodichloroplatinum (CDDP). An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine cell viability. The apoptosis rates of cells were measured by flow cytometric analysis. The expression of apoptosis-associated and proliferation-associated proteins was detected by Western blot. The cytotoxicity of GB was analyzed using a lactate dehydrogenase assay. Treatment with 100 µM GB for 3 days significantly inhibited SKOV3 and CAOV3 cell proliferation by 57.3% and 63.1% compared with control cells, respectively, as determined by MTT assay. Similarly, the apoptotic cell population was increased when treated with GB in a dose-dependent manner both in SKOV3 and CAOV3 cells. These effects were characterized by the upregulation of p21, p27, cleaved capase-3, and cleaved caspase-8 and downregulation of cyclin D1. In addition, a combined treatment of low concentrations of GB and CDDP showed an additive effect on the inhibition of SKOV3 cell proliferation. Furthermore, GB had significantly less cytotoxicity than CDDP in normal human ovarian surface epithelial cells. This study suggests that GB can be proposed as an effective antiproliferative and apoptosis-inducing agent with interesting translational application in ovarian cancers, used in addition to conventional chemotherapy.

Design of countercurrent separation of Ginkgo biloba terpene lactones by nuclear magnetic resonance.

Terpene lactones such as bilobalide, ginkgolides A, B, C, and J are major bioactive compounds of Ginkgo biloba L. Purification of these compounds is tedious due to their similar chemical properties. For the purpose of developing an effective and efficient method for both analytical and preparative separation of terpene lactones in G. biloba, an innovative orthogonality-enhanced high-speed countercurrent chromatography (HSCCC) method was established. Taking advantage of quantitative (1)H NMR (qHNMR) methodology, partition coefficients (K) of individual terpene lactones were calculated directly from crude G. biloba leaf extract, using their H-12 signals as distinguishing feature. The partitioning experiment assisted the design of a two dimensional (2D) HSCCC procedure using a pair of orthogonal HSCCC solvent systems (SSs), ChMWat +4 and HEMSoWat +3/0.05%. It was surprising that the resolution of ginkgolides A and B was improved by 25% in the HEMWat +3 SS modified with 0.5% DMSO. Consequently, all five terpene lactones could be well separated with qHNMR purity>95% from G. biloba leaf extract. The separation was further evaluated by offline qHNMR analysis of HSCCC fractions associated with Gaussian curve fitting. The results showed less than 2% error in HSCCC retention predicted from the partitioning experiment. This compelling consistency demonstrates that qHNMR-derived K determination ("K-by-NMR") can be used to predict CCC fractionation and target purification of analytes from complex mixtures. Furthermore, Gaussian curve fitting enabled an accurate prediction of less than 2% impurity in the CCC fraction, which demonstrates its potential as a powerful tool to study the presence of minor constituents, especially when they are beyond the detection limit of conventional spectroscopic detectors.

Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo biloba.

To screen the presence of ginkgolide B-producing endophytic fungi from the root bark of Ginkgo biloba, a total of 27 fungal isolates, belonging to 6 different genus, were isolated from the internal root bark of the plant Ginkgo biloba. The fungal isolates were fermented on solid media and their metabolites were analyzed by TLC. The obtained potential ginkgolides-producing fungus, the isolate SYP0056 which was identified as Fusarium oxysporum, was successively cultured in the liquid fermentation media, and its metabolite was analyzed by HPLC. The ginkgolide B was successfully isolated from the metabolite and identified by HPLC/ESI-MS and (13)C-NMR. The current research provides a new method to produce ginkgolide B by fungal fermentation, which could overcome the natural resource limitation of isolating from the leaves and barks of the plant Ginkgo biloba.

Two new ginkgolides from the leaves of Ginkgo biloba.

Two new diterpenoid compounds, ginkgolide P(1) and ginkgolide Q(2), were isolated from the leaves of Ginkgo biloba L. Their structures were elucidated by various spectroscopic methods, and the structure of 1 was further confirmed by X-ray crystallographic analysis. The activities of the compounds were evaluated against platelet aggregation induced by platelet activating factor (PAF), and the preliminary structure-activity relationship was also discussed.

An efficient molecular docking method for adsorbent screening.

In this study, with flavonol glycosides (FG) and terpene lactones (TL) in ginkgo biloba extract (GBE) as the targets for separation, we investigated the effectiveness of molecular docking in adsorbent screening. Several polyamine-modified methyl acylate-co-divinylbenzene (MA-co-DVB) adsorbent models were built, and their affinity to rutin, quercetin and ginkgolide B (GB) was evaluated via molecular docking. The model of ethylenediamine-modified adsorbent showed the largest difference in affinity between to GB and to quercetin as well as rutin, and thus this adsorbent could have the best separation performance. The results of the subsequently conducted static adsorption and dynamic adsorption experiments correlated well with docking results. Finally, using ethylenediamine-modified MA-co-DVB adsorbent, nearly complete separation of the FG and TL in GBE was simply achieved by one step of adsorption-desorption. Thus, the reported molecular docking method is expected to be helpful for rapid adsorbent screening.

[Extraction, isolation and purification for ginkgolide B].

OBJECTIVE: To establish a simple extraction, isolation and purification method for ginkgolide B from ginkgo leaf. METHOD: The optimum conditions of extraction, isolation and purification were studied by taking the transfer rate of ginkgolide B as index. RESULT: Ginkgo leaf was extracted with 70% ethanol for three times, the extracts were concentrated to remove ethanol and diluted by water till the crude drug density reached 0.1 g x mL(-1). The dilution was adsorbed with HPD-450 macroporous resin. The impurities were eluted with 20% ethanol and ginkgolide B was eluted with 80% ethanol. Then the 80% ethanol eluant was concentrated and crystallized. Finally the crude crystals were recrystallized with isopropanol. The purity of the ginkgolide B recrystallization was 95%. CONCLUSION: The process was stable and easy to operate, which was suited to industrialized production.

[Extraction of ginkgolides from Ginkgo biloba].

OBJECTIVE: To establish the technology for extraction of ginkgolides from Ginkgo Biloba with alcohol-water. METHOD: The parameters such as alcohol concentration, pH of extracting solution, ratio of dosage liquor, temperature and time, the extraction of ginkgolides from G. biloba was investigated, and its parameters were optimized. RESULT: The optimized parameters were alcohol concentration 30%, extracting temperature 50 degrees C, extracting time 2 h, pH 5 solid-liquid ratio 1:15. CONCLUSION: This method has the merits of low cost and simple operation.

Isolation and structure characterization of related impurities in 10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H) bulk drug and quantitation by a validated RP-LC.

10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H), a novel active derivative of ginkgolide B, is a platelet-activating factor antagonist which is being under clinical trial. Two unknown related impurities were observed in analysis of XQ-1H bulk drug. A scaling up preparative liquid chromatography (Prep LC) was used for isolation of the two impurities. Based on LC-MS/MS and nuclear magnetic resonance (NMR) spectra, they were characterized as 10-O-(N,N-dimethylaminoethyl)-11,12-seco-ginkgolide B (imp-1) and 10-O-(N,N-dimethylaminoethyl)-11,12,2,15-diseco-3,14-dehydroginkgolide B (imp-2), respectively. A reversed-phase liquid chromatography (RP-LC) was developed for simultaneous determination of XQ-1H as well as imp-1 and imp-2. Main variables that significantly influence the chromatographic procedure were optimized and efficient chromatographic separation was achieved on a CN column with mobile phase consisting of 5mM dipotassium hydrogen phosphate (pH 7.5) and methanol delivered in a gradient mode at the flow rate of 1.0mLmin(-1). The method was validated and found to be suitable to check the quality of bulk samples of XQ-1H at test concentration of 5.0mgmL(-1) for a 20microL injection volume.

Chemical constituents of the bark of Machilus wangchiana and their biological activities.

Eleven new metabolites, butanolides 1-6, lignan derivatives 7-9, sesquiterpene 10, and 3',4'-seco-flavane derivative 11, have been isolated from an ethanol extract of Machilus wangchiana. Twenty known compounds, including ginkgolides A and B (16 and 17), were also isolated. Their structures and absolute configurations were determined by spectroscopic and chemical methods. Compounds 7, 8a, 8b, 9, 11, (+)-guaiacin (12), meso-dihydroguaiaretic acid (13), and hamabiwalactone A (15) showed potent in vitro activities against the release of beta-glucuronidase in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor (PAF), with 42.5-75.6% inhibition at 10(-5) M. Compounds 8, 8a, 8b, 9, and 11 reduced dl-galactosamine (GalN)-induced hepatocyte (WB-F344 cells) damage with 39.4 +/- 6.3% to 53.6 +/- 3.5% inhibition at 10(-4) M. Isomahubannolide-23 (14) was cytotoxic against human stomach cancer (BGC-823) and ovarian cancer (A2780) cell lines, with IC(50) values of 0.13 and 2.66 muM, respectively.

Time-dependent induction of hepatic cytochrome P450 enzyme activity and mRNA expression by bilobalide in rats.

A single dose by gavage of bilobalide (30 mg/kg) was found to produce a time-dependent induction of hepatic cytochrome P450 (CYP) enzyme activity and protein expression in rats. An RT-PCR study further showed that mRNA expression of CYP2B was maximal at 6 h. Plasma and liver bilobalide concentration in rats following administration of Ginkgo biloba extract equivalent to bilobalide of approximately 40 mg/kg showed a similar response to that exhibited by mRNA expression. These findings suggest that bilobalide markedly induced hepatic CYPs, but the induction could be mitigated due to rapid elimination from the liver.

Induction of cytochrome P450 3A by the Ginkgo biloba extract and bilobalides in human and rat primary hepatocytes.

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. The study aimed to investigate the activity and expression of cytochrome P450 (CYP) 3A in human and rat primary hepatocytes treated with standardized G. biloba extract (100, 500, and 2500 ng/ml) for 72 hr, and to measure the protein expression of CYP3A in human and rat primary hepatocytes treated with bilobalide (2, 10, and 50 ng/ml) and ginkgolides B (2, 10, and 50 ng/ml). The activity of CYP3A was measured by the quantification of dehydronifedipine formation using a validated tandem liquid chromatography mass spectrometry (LC/MS/MS) method. The levels of mRNA and protein of CYP3A were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blotting analysis, respectively. The G. biloba extract at 100-2,500 ng/ml significantly induced the activity, protein and mRNA expression of CYP3A in a dose-dependent manner in human and rat primary hepatocytes. Bilobalide at 2-50 ng/ml significantly increased CYP3A protein expression in a dose-dependent manner in human and rat primary hepatocytes. However, ginkgolide B did not affect CYP3A protein expression in vitro. The results indicate that G. biloba extract pretreatment significantly induced the expression of CYP3A protein and mRNA and increased CYP3A activity, and there was no significant species difference between human and rat. G. biloba may cause potential interactions with substrate drugs of CYP3A. Bilobalide might play a key role in the enzyme-inducing effects of G. biloba extract. Further study is needed to identify the substances in GBE that induce CYPs in vivo, and elucidate the molecular mechanism of CYP3A induction by GBE and bilobalides.

Validation of capillary gas chromatographic method for determination of bilobalide and ginkgolides A, B, C in Ginkgo biloba dry and liquid extracts.

A method for identification and quantitative determination of ginkgolides A, B, and C and bilobalide in liquid and dry extracts of Ginkgo biloba extracts has been developed. Determinations made by employing capillary gas chromatography technique with FID detection were preceded by derivatization using BSTFA with TMCS addition at 120 degrees C. Cholesterol was used as an internal standard. Validation of the method shows no interferences with concurrent constituents; average resolution (R), controlled for peaks of cholesterol and ginkgolide A was 1.53 (SD = 0.06). In the temperature program used (from 50 degrees C to 300 degrees C) the analyte retention times range from 11.2 min. (bilobalide) to 13.8 min. (ginkgolide C) and are of high repeatability of relative values (RRT): RSD = 0.05% / 0.07% for ginkgolides. High correlation coefficients (r), detector signal linearity: from 0.99962 for ginkgolide C to 0.99985 for ginkgolide A were obtained within the concentration range under investigation. The method is of high sensitivity: limits of detection and limits of determination are 35 pg and 44 pg for bilobalide, respectively, while for ginkgolides (Gk) are: 78 pg and 92 pg for GkA, 57 pg and 68 pg for GkB, and 213 pg and 320 pg for GkC.