Production Of Ginkgolides Research Articles

Effects of Organic Elicitors on the Recycled Production of Ginkgolide B in Immobilized Cell Cultures of Ginkgo biloba

Ginkgo biloba is a medicinal plant used in complementary and alternative medicines. Ginkgo biloba extracts contain many compounds with medical functions, of which the most critical is ginkgolide B (GB). The major role that GB plays is to function as an antagonist to the platelet-activating factor, which is one of the causes of thrombosis and cardiovascular diseases. Currently, GB is obtained mainly through extraction and purification from the leaves of Ginkgo biloba; however, the yield of GB is low. Alternatively, the immobilized cultivation of ginkgo calluses with biomaterial scaffolds and the addition of organic elicitors to activate the cell defense mechanisms were found to stimulate increases in GB production. The aim of this study was to use Ginkgo biloba calluses for immobilized cultures with different elicitors to find a more suitable method of ginkgolide B production via a recycling process.

Magnetite Nanoparticles Different Sizes Effectiveness On Growth And Secondary Metabolites In Ginkgo Biloba L. Callus

This study investigated the effects of magnetite nanoparticles (MNPs) various sizes (10.77, 20.5, and 29.3 nm), and different concentrations (0.5, 1, and 2 ppm) on the growth and production of active constituents in Ginkgo biloba L callus. The best medium for callus growth and proliferation of Ginkgo biloba L was MS medium supplemented with 1 ppm naphthalene acetic acid (NAA), 1 ppm Kinetin, and 0.5 ppm MNPs with a size of 10 nm, which gave the maximum fresh weight of callus and percentage increase in fresh weight after two subcultures. The maximum accumulation of ginkgolide A and bilobalide were achieved when callus was elicited by MNPs 10 nm at 2 ppm. This study also focused on estimating phenolic compounds and scavenging free radicals (antioxidant activity) for their indirect effect on active constituents production. Also, MNPs different sizes had a clear effect on the number of phenolic compounds, where the maximum values were recorded with (2 ppm + size 10.77 nm). The major components were quercetin, kaempferol, p-coumaric acid, rutin, and caffeic acid. Moreover, the highest percentage of antioxidant activity (DPPH) was recorded when callus treated with (2 ppm + size 10.77 nm) which reached 77.29 %. This study highlights the importance of MNPs for the production of ginkgolide A and bilobalide from Ginkgo biloba L. and focusing on the relationship between their size and concentration. Besides, to provide a good and permanent source of active substances of medical and industrial importance on a large scale and at the lowest cost.

The Optimization and Production of Ginkgolide B Lipid Microemulsion

AbstractIn this study, we introduce a method to formulate Ginkgolide B lipid microemulsion (GB-LM). We have assessed its general characteristics and pharmacokinetics in animals. Soybean oil (10.0%) and oleic acid (2.4%) were chosen as the oil phase, refined egg yolk lecithin (PC98T) (1.8%) as the surfactant, and glycerol (2.25%) as the co-surfactant. The optimized formulation process resulted in particles with average diameter of 185.9 ± 52.5 nm and the zeta potential of −19.8 ± 1.3 mV. The GB-LM remained steady for three months at room temperature. Giving each dose of 4 mg/kg to rats through the vein, a clearance rate of CL (L/h): 2.594 and C max (ng/mL) was achieved: 353.8 through pharmacokinetic analysis and statistical analysis. The AUC of GB-LM was about 1.57-fold higher than that of the products available in the market. In addition, brain tissue distribution studies show that after 0.5 h administration of GB, the concentration of GB in brain tissue can reach its maximum, and then significantly decrease after 2 hours. Therefore, the improved formulation of GB-LM shows encouraging results compared with present products in several features.

Biosynthesis pathways of ginkgolides

The ginkgolides, acting as anti-platelet-activating factors, have been studied for many years. The biosynthetic pathway of ginkgolides is still far away from unveiling at the level of molecular genetics and biochemistry. There are at least 11 kinds of enzymes having been cloned from Ginkgo biloba L., which catalyze the formation of ginkgolides via a series of reactions. Some researchers have indicated that the addition of precursors and elicitors can influence the accumulation of ginkgolides in the suspension cell cultures of G. biloba. There are also other factors that can influence the production of ginkgolides. This review focuses on the aforementioned aspects to discuss the biosynthetic pathways of the ginkgolides.

Production of ginkgolides and bilobalide from optimized theGinkgo biloba cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.

The Total Synthesis of (±)-Ginkgolide B

The total synthesis of the potent PAF antagonist ginkgolide B has been accomplished. The complex architecture of ginkgolide B which includes six rings, eleven stereogenic centers, ten oxygenated carbons, and four contiguous fully substituted carbons is a daunting challenge for chemical synthesis. The synthesis of ginkgolide B was accomplished through a stereoselective intramolecular photocycloaddition of enone 5 to construct the congested core of the molecule. The photocycloaddition substrate was prepared through technology for the construction of carboalkoxycyclopentenones previously reported from these laboratories. Regioselective cyclobutane fragmentation and further functionalization of the photoadduct 4 provided the key pentacyclic intermediate. Acid-catalyzed rearrangement and epoxide opening were key transformations in the production of ginkgolide B from the pentacyclic intermediate.

Production of ginkgolides and bilobalide by Ginkgo biloba plants and tissue cultures.

The accumulation of the terpenes ginkgolides and bilobalide in Ginkgo biloba was reported in plants as well as in plant cell cultures. Several hundred plants cultivated under controlled conditions in the field have been analyzed for their terpene production over many years. Cross-pollination experiments were performed with mature trees and the terpene content of the progeny was analyzed. The age of the tree is the main factor influencing the terpene content of the leaves as the level always decreases dramatically between young and old trees. 80 cell culture strains have been established and ginkgolides analyzed by GC/MS. These cell cultures reveal very low amounts of terpenes (1 microgram g-1 D.W. or less). On the contrary, isolated in vitro root cultures accumulate terpenes at the same concentration as the young plant leaves (4 mg g-1 D.W.). Attempts to obtain rapid growing roots or even hairy-roots did not succeed but the possibility to transform Ginkgo cell strains has been demonstrated.

Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba

Ginkgolide and bilobalide were detected by HPLC analysis in cell cultures established from various G. biloba explants. These secondary metabolites of pharmacological interest were found to occur in concentrations of 0.065 and 0.087% (dry weight) in two cell suspensions. One was derived from a female prothallus and the other one from putatively transformed embryos, by transfection via Agrobacterium rhizogenes agropine type strain CFBP 2409 (A4). © 1997 Elsevier Science Ltd. All rights reserved

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of α-naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 ∶ 3.

Cultures ofGinkgo biloba, effect of nutritional and hormonal factors on the growth of cultured cells derived fromGinkgo biloba

Calli and suspension cultures were obtained following inoculation of the explant from leaves ofGinkgo biloba L. on the supplemented MS basal medium. The obtained calli and suspension cultured cells were able to produce detectable amounts of ginkgolides which are known as natural specific PAF antagonists. The production of ginkgolides in the calli and suspension cultured cells were identified using GC/MS, GC and HPLC with authentic compounds. Since the production of ginkglides A and B in the calli and suspension cultured cells had been confirmed, effects of types and concentration of plant growth regulators, media and illumination on the induction and growth of the callus were studied. The concentrations of growth regulators for optimal callus induction were 1.0 to 2.0 mg/L for NAA and 0.1 mg/L for kinetin. The growth of the callus seemed to be more stimulated with the combination of NAA and kinetin than NAA and BA with illumination at all concentration ranges of 1.0 to 4.0 mg/L for NAA and 0.1 to 1.0 mg/L for kinetin or BA studied. Among 8 different media used, the induction rate of callus on Anderson, Eriksson, and Schenk and Hildebrant at 4 weeks after the innoculation was almost the same as that of MS. However, callus was rarely induced on Heller or White medium. Suspension cultures were easily initiated with 3 g of callus (fresh weight) derived from ginkgo leaves on supplemented MS medium. A typical growth curve of suspension cultured cells could be obtained by measuring the fresh weight of the suspension cultured cells at every 3 days. To improve the growth of suspension cultured cells of ginkgo, effects of concentrations of NAA, sucrose, phosphate ions and molar ratio of NH4+ to NO3− ions in the culture medium were studied. The maximum growth of the cells was achieved when the culture medium contained 1.0 mg/L of NAA, 30 g/L sucrose, 1.75 mM phosphate ions and 1∶5 molar ratio of NH4+ to NO3− ions.

Gas chromatographic-mass spectrometric analysis of ginkgolides produced by Ginkgo biloba cell culture

An analytical method was developed to confirm the production of ginkgolides by Ginkgo biloba cells cultured in vitro. Biomass samples were extracted for 16 h with acetone. This extract was loaded onto a silica gel column and purified by differential elution using a cyclohexane-ethyl acetate solvent system. The recoveries of ginkgolide A and ginkgolide B were × ei90% and × ei35%, respectively. Subsequently, this purified extract was trimethylsilylated for gas chromatographic separation of ginkgolide A and ginkgolide B. Flame-ionization detection was not selective enough for identification of ginkgolides A and B in the extracts. The ginkgolides were detected by coupling the gas chromatograph to a high-resolution mass spectrometer operated in the selected-ion monitoring mode. The concentration of ginkgolides A and B in culture extracts was determined by gas chromatography-mass spectrometry using deuterotrimethylsilylated ginkgolides as internal standards. This technique permitted detection of ginkgolides A and B at concentrations as low as 10 − pmol/μl of injected purified extract.