| Extract of Ginkgo biloba (EGB) with many physiological effects has been applied for treatment cardail vascular diseases and aged dementia. The main components of EGB were flavonoids and Ginkgolides. However, EGB contains Ginkgolic acid which is the poisonous constitute, so the quality of EGB is related to the content of Ginkgolic acid. The Ginkgolides in EGB is the antagonist of platelet activating factor(PAF). Leaves of Ginkgo biloba are rich in our country with about 70% source of Leaves of Ginkgo biloba. To improve the competitive capability of EGB in world market, it is vital to prepare high quality of EGB. The present study was to develop the techniques to remove Ginkgolic acid, to isolate flavonoids and Ginkgolides, to prepare high purity of Ginkgolides and Ginkgolide B, to prepare the drop pill of Ginkgolide B and establish HPLC methods to determind the content of Ginkgolic acid and Ginkgolides in EGB, and content of Ginkgolide B in drop pill.1. The techniques to removal Ginkgolic acid and the method of determination of Ginkgolic acidObjective: To develop the method for determination of Ginkgolic acid and the technique for Ginkgolic acid removal.Methods: The HPLC was used to analysis Ginkgolic acid. GBE was dissolved with ethanol, and extract with aether for five times, then remove the aether, the residual was re-dissolved in methanol. The HPLC were performed on column: C18 (4.6 mm* 150mm, 5um), the column temperature was 28°C, the mobile phase was methanol (0~25min, 70%;25~30min, 92%) water, the flow rate was l.Oml/min, and the detection wavelength was set at 310nm. The organic solvent and the resin were used to remove Ginkgolic acid, and the best conditions were determined, the results were compared.Results: The linear range of Ginkgolic acid were 0.530 u g/ml~ 10.6 U g/ml (r=0.9991). The RSD of reproducible test was 0.71%, the average recovery of the method was 101.7% (RSD=0.68%).The best technique conditions of the solvent extraction to remove Ginkgolic acid were as follows: leaves of Ginkgo biloba was extracted with 8 and 7 times of 70% ethanol 2 times, the ethanol of extract solution was removed and water was added to precipitate the impurities, the solution was through the column of D101 resin, 5 times water of resin volume were applied to wash the unadsorbed components, the adsorbed compositions were eluted with 3 times 70% ethanol of resin volume, the elution was concentrated under vacuum to the density about 1.20g/ml, then adjust pH of the solution around 4.3. Extract 5 times with equal volume hexane, discard the organic solvent and the aqueous layer was concentrated to dry cakes, dry under vacuum in 70 °C. The contents of 3 batches of production from moderate scale were all less than 5ppm.The optimized technique of resin method to remove Ginkgolic acid was as follows: leaves of Ginkgo biloba extraction and purified method with D101 were described previously. The elution solution of 70% ethanol was diluted with water to 60% of ethanol and pH of the solution was adjusted to 4.0, then the solution wasthrough ABS resin (pretreated with 5% 5%NH3- H2O) with the flow rate of lBV/h, then the resin column was eluted with 70% ethanol, the elution was concentrated, dryand grind. The contents of Ginkgolic acid in the productions from small, moderate and large scales were less than 5ppm, 5ppm and 2ppm, respectively.Conclusion: The HPLC method developed in the study is rapid, accurate and reproducible which can be applied to quality control of EGB. Both of solvent extract method and resin method can produce the EGB with low content of Ginkgolic acid. The resin method is simpler and without strict demand, it is more suitable for large scale preparation.2. Isolation and purification of Ginkgolides, optimization the technique of drop pill preparation and determination of Ginkgolides in extract and drop pillsObjective: To isolate flavonoids and Ginkgolides of EGB, to obtain high purity of Ginkgolides and to optimize the preparation condition of drop pill. Moreover, to develop the methods for determination of Ginkgolides in extract and drop pill.Methods: The flavonoids and Ginkgolides in EGB were isolated with polyamide column firstly, then the Ginkgolides were decolored, and the crude Ginkgolides were dissolved and recrystallize by 90% ethanol. The Ginkgolides were determined by HPLC method. The column was EclipseXDB-C18 (150mm x4.6mm, 5um),the mobile phase was methanol-0.1%phosphoric acid(33.3 : 66.7),the flow rate was l.Oml.min"1 ,and the detection wavelength was 225nm. The technique of preparation drop pill were optimized by the orthogonal test.Results: The optimized conditions to prepare Ginkgolides: Extract the Ginkgo biloba leaves with 8 times and 7 times 70% ethanol respectively, reflux for 2 hours each time, combine the extract solution, removed the ethanol under vacuum, add 3 times of water to precipitate the impurity, the filtrate was through polyamide column(filter solution ." resin=2 .* 3), washed with 2 times water and then eluted with 2-3 BV of 80% ethanol respectively, the collected elution of water and ethanol were concentrated and dry under vacuum respectively, then the Ginkgo Flavonoides and Ginkgolide were obtained. Dissolve the Ginkgolide obtained from the above step with 95% ethanol, and decolored with acidity AI2O3 (5-7 times of the rude Ginkgolide ) column, the elution was concentrated and dry, the crud Ginkgolide (>80%) waspreared. By recrystallization this crud Ginkgolide with 95%, Ginkgolide more than 95% and Ginkgolide B monomer more than 95% were obtained.The optimized technique to prepare Ginkgolide B drop pills: PEG4000: PEG6000 = 1: 1, the temperature for the aqueous mixture was 85 °C, the temperature for cool aqueous was 5°C, active ingredients: excipient was 1: 3.In the HPLC method for determination of Ginkgolide in extract, the peak area to concentration linear regression equation for Ginkgolide A was Y=273.69X+0. 5167,with good linearity(r=0.9999) within 0.215~3.44mg/ml, and the average rate of recovery 99.60%(RSD=1.4%).The peak area to concentration linear regression equation for Ginkgolide B was Y=269.83X+3.738, with good linearity(r= 1.000) within 0.265mg/ml~4.24mg/ml, and the average rate of recovery 99.60 %(RSD= 1.1 % ). The linear range for assay Ginkgolide B in drop pill were 0.2650mg/ml-4.240mg/ml ( r=1.000), the average recovery was 99.15%(n=6, RSD=1.3%), the excipient did not disturb the analysis.Conclusion: Polyamide can isolate flavonoids and Ginkgolides. By decoloration and recrystallization of the crud Ginkgolide, high purity of Ginkgolides and Ginkgolide B can be prepared. By the technique optimized in the study, good quality of Ginkgolide B drop pill can be produced. The HPLC methods developed in the study can be applied to quality control of Ginkgolides extract and Ginkgolide B drop pills.
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