Extraction and biodegradation of ginkgolic acids from Ginkgo biloba sarcotestae

Ginkgolic acids are unwanted constituents in standard Ginkgo biloba leaves extracts. Thus, for the quality control of ginkgo extracts, it is important to establish an effective degradation method, with high catalytic efficiency and safety, to remove ginkgolic acids.Laccases are oxidases with potential for application in elimination of hazardous phenolic compounds. In this study, single-factor and orthogonal experiments were used to optimize extraction of ginkgolic acid from G. biloba sarcotestae. The results showed that ethanol was the best solvent, with the highest extraction rate for ginkgolic acid at 85% ethanol. On this basis, we measured ethanol volume fraction, extraction time, temperature and solidliquid ratio using an orthogonal experiment. By using absorbance of 310 nm as standard, the optimal extraction conditions were 85% ethanol with, solid-liquid ratio of1:14 at 40°C for 12 h. These conditions gave a ginkgolic acid yield of 73.1 mg·g^(–1). Subsequently, recombinant laccase was used to degrade the ginkgolic acid in several laccase/mediator systems, of which Lac C was the best. At50°C, p H 4.5, enzyme concentration of 0.01 U·m L^(–1),0.5 mmol·L^(–1) mediator ABTS and reaction time of 3 h, the degradation rate of ginkgolic acid reached 100%. These results lay the foundation for research on and application of biological enzymes for detoxification of G. biloba extracts.

Mechanism for ginkgolic acid(15：1)-induced MDCK cell necrosis： Mitochondria and lysosomes damages and cell cycle arrest

Ginkgolic acids(GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA(15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA(15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA(15 : 1) on MDCK cells displayed a time-and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA(15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential(ΔΨm). In propidium iodide(PI) staining analysis, GA(15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA(15 : 1) induced renal toxicity.

The metabolism and hepatotoxicity of ginkgolic acid(17:1) in vitro

Pharmacological activities and adverse side effects of ginkgolic acids(GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA(17:1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA(17:1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA(17:1) metabolism were human CYP1 A2, CYP3 A4, UGT1 A6, UGT1 A9, and UGT2 B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA(17:1) in HepG2 cells occurred in a time-and dose-dependent manner. Further investigation showed that GA(17:1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1 A-and CYP3 A-mediated metabolism.

Improvement of quality of Ginkgo biloba seeds powder by solid-state fermentation with Eurotium cristatum for developing high-value ginkgo seeds products

In this study,we investigated the feasibility of Ginkgo biloba seeds powder by solid-state fermen-tation with Eurotium cristatum for developing high-value ginkgo seeds products.The optimum fermentation medium was consisted of 10 g of 40-mesh ginkgo seeds powder loaded in 100 mL Erlenmeyer flask with 50%(w/w)of water content,4%(w/w)of MgSO_(4)and 5%(w/w)of KH_(2)PO_(4)addition.The optimum fermentation conditions were pH 5.0,2×10^(8)CFU/g of inoculum size,3 mL of sterilized water supplemented every two days during the four days of fermentation.Through fermentation,the spore number of E.cristatum was improved by about 36 times with the produc-tion of lovastatin reaching(54.10±0.16)μg/g.The antioxidant activity of fermented ginkgo seeds powder also got obvious enhancement,which could help eliminate excess free radicals produced by normal metabolism.The content of free amino acids increased by 82.32%.Except that the sugar was consumed in some degree,the other nutritional and functional components were well preserved while the content of detrimental ginkgolic acids was reduced by 44.97%.In addition,fermented ginkgo seeds powder possessed better digestibility and showed pleasant orange-like smelling.In conclusion,the quality of ginkgo seeds powder was remarkably improved through solid-state fermentation by using E.cristatum,which could be a promising way for functional applications of ginkgo seeds.