| Background Ginkgo Biloba Extract 50(GBE50) on the state of the second type of new drugs was approved by the Ministry of Health, and the active ingredient content of it is greatly raised, which Contain 44% of total flavonoids(TF). Flavonoids exist in two forms, flavone glycosides and aglycones. There are 5% flavonoids exist on the form of aglycones in the traditional Ginkgo biloba extract, but more than 45.45% in the GBE50. A large number of studies have shown that the biological activity of clearing of oxygen free radicals of flavonoids aglycones is better than flavone glycoside. But the solubility of flavonoids due to the different of the structure and the existing state (glycosides and aglycones, glycosides single, double and three glycosides glycosides) are different. Free flavonoid aglycones are generally insoluble in water, but soluble in methanol, ethanol, ethyl acetate, ether and other organic solvents.Drug solubility is the important factor affecting the release and absorption of the drug. Regardless of the manner in which drugs through the biofilm, they must be absorbed with the form of solution state. In order to product better pharmacological effects, a certain solubility is needed. But in order to passive diffuse through the membrane, the drug also need to have a certain lipophilicity. In general, we can increase the apparent solubility of the drug by the Pharmaceutics technology, which can promote drug to diffuse through the biofilm. Solubilization techniques are commonly used includes adding surfactants, adjusting PH value, reducing diameter, solid dispersion, micro emulsion, reducing grain size, liposomes and comprehensive application of these techniques, etc. Compared with other technologies, including technology, which in human tolerance of PH range, can effectively increase the drug solubility, while reduce the use of surfactants and organic solvents, without reducing the lipophilicity of the drug.Cyclodextrins are the most commonly used carriers. They have a hydroph-obic cavity, which can selective include various guest molecules and change the nature of the drug in the level of molecular, to improve the solubility and stability of guest molecules. Hydroxypropyl-β-cyclodextrin (HP-β-CD) has many advantages, such as excellent water-soluble, non-toxic to the kidney, no stimulating effect to muscle, mucous membrane almost.Compared to traditional antioxidants, such as vitamin C and E, Flavonoids, such as quercetin, isorhamnetin, have better antioxidant activity. There is a certain correlation between the Antioxidant Activity of Ginkgo biloba extract and the pharmacological effects of free radicals. So protecting their antioxidant activity is a prerequisite to achieve their pharmacological effect. One of the advantages of Inclusion technique is that you can save the activity of drug as much as possible, and avoid the external adverse factors.This topic is primarily aimed at HP-β-CD inclusion of GBE50. The study includes four parts, including:preparation of process optimization, quality standards, pharmacokinetic parameters and physicochemical properties.Chapter 1. Process optimization and characterization Solution mixing method is used in the preparation of inclusion complex. Total flavonol glycosides inclusion rate and drug loading as index, the process is optimized by the orthogonal tests. The formation of inclusion complexes are identified by optical microscope, infrared spectrophotometry (IR) and thermogravimetric analysis.Result:Inclusion process is that the best feed mass ratio is 1:1, inclusion time was 4 h and inclusion temperature is 30℃. According to the optimal process preparation, the inclusion rate was 82.3%, and drug loading 11.55%. Microscopic identification can distinguish the inclusion complexs from the physical mixture. The infrared spectrometry can show inclusion complex formation, and thermogravimetric analysis prove that the inclusion complexs are generated.Chapter 2. Quality StandardsThin-layer chromatography(TLC) was used for qualitative identification of GBE50, and high performance liquid chromatography(HPLC) for measuring the total content of flavonoid glycosides.The separation was performed on a Diamonsil C18 column(250mm×4.6mm), the mobile phase consisted of methanol and 0.4% phosphate (45:55), the detective wavelength was 368nm, the flow rate was 1mL/min; and the inclusion complexes were identified by means of infrared spectrum(IR). The linear range of quercetin,kaempferol and isorhamnetin were 2～132μg·mL-1, 0.9～60μg·mL-1, and 0.1～9μg·mL-1 respectively. The average recovery were 101.65%,100.43%,99.34% respectively. RSD=1.25%,1.36%,0.63%; inclusion complexes can be identified by IR.The peak position and peak intensity were analyzed in the Infrared spectroscopy experiment. If the data can be mapped to quantitative analysis, we will obtain a more accurate results of identification. Chapter 3. Pharmacokinetic experimentDetection:Samples were placed in the hydrolysis of 80℃30min after added equal volume of 25% hydrochloric acid solution. After cooling, the samples were added 2mL of ethyl acetate, and then vortex 3min. Supernatant was drew after centrifugation, and dried in nitrogen. Residue was fully dissolved with 0.5mL methanol. Mobile phase composed of the methanol ethanol- water -phosphoric acid. Regression equations were y= 50086x-211.49 (r= 0.9951), y= 496326x-1551.3 (r= 0.9901). Result:Quercetin 1.3～264.0μg·mL-1, kaempferol in the 0.6～120μg·mL-1. This method has a good linear relationship and good reproducibility.Pharmacokinetics:12 rats randomized into 2 groups were separately given GBE50 and its complex. The pharmacokinetic parameters were calculated by DAS software. Result:The average concentration - time curves show that there is a "double peak" phenomenon for quercetin, and a "three peaks" phenomenon for kaempferol. Compared with GBE50, GBE50-HP-β-CD inclusion compounds was absorbed faster. In addition to the increasing solubility, the other mechanism remains to be further confirmed.Chapter 4. solubility and stabilitySolubility:HP-β-cyclodextrin was prepared to be 0,2.5,5,7.5,10,12.5,15 mmol/ L solution, and then added GBE50 excessively. After filtration, the concentrations of each filtrate were detected by HPLC, and draw phase solubility curve. The results showed that the use of inclusion technique can made the solubility of total flavonol glycosides increased to 4 times. Conclusion:The inclusion technique can improve the solubility of total flavonol glycosides of GBE50 in water.Stability:The conservation rates of GBE50 and inclusion compound will be checked after placed on 90℃at 1,3,5,10 d, and on the strength of 4000Lx light irradiation at 1,3,5,10 d separately. According to collection points, the rates of weight change of GBE50 and inclusion compound were checked after placed in the humid of 92.5% and 75% separately. Result:After heated 10d at 90℃, the total flavonol glycosides of GBE50 was only 78.0%, while up to 92.7% of the inclusion compound. In the intensity of light exposure 4000Lx 10d, the total flavonol glycosides of GBE50 was only 90.3%, while up to 96.3% of the inclusion compound. However, in 75% and 92.5% humidity conditions, Ginkgo Biloba Extract and inclusion are both serious moisture, the state was liquid extract after 48h. Conclusion:The inclusion technique can improve the Ginkgo Biloba Extract on the thermal stability and light stability. But the high humidity experiments also suggest that inclusion is easy to damp. |
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