| Alzheimer's disease?AD?is a neurological disorder characterized by gradual changes in cognition and regulation.At present,there is still no effective medical method for this disease,and how to cure it has become a worldwide problem.As described in traditional medicine,there are various factors will cause Alzheimer's disease,such as kidney essence,insufficiency of marrow-sea and brains emptiness,all of which will lead to brain tissue atrophy and hypofunction,and eventually resulting in dementia.Therefore,tonifying kidney and essence replenishment play a key role in the treatment of AD.Specially,Drynaria fortune,a traditional effective medicine of tonifying kidney,which was usually used for the treatment of memory recession in clinical application.In addition,modern pharmacological studies have shown that total flavonoids in Drynaria fortune are the main active ingredient to improve the memory function.Hence,in this paper,we screened and optimized the enrichment and purification method of total flavonoids in Drynaria fortune,and meanwhile investigated its protective effects on A?25-35 induced PC12 cells damage.Firstly,we established a method of UV quantitative analysis for the determination of the content of total flavonoids.Naringin was used as a reference to prepare standard curve,and then investigated its precision,repeatability,stability and the recovery.Based on the above investigation,we could further evaluate and optimize the enrichment conditions of total flavonoids.Secondly,we investigated the influence of different concentrations of ethanol?50%?55%?60%?70%?80%?on the extraction of total flavonoids,and thus 55%ethanol was selected as extraction solvent.Taking into account the feasibility of industrial production,we thus chose reflux extraction with heat ethanol as the chiefly extraction method of total flavonoids.After adopting L9?34?orthogonal test investigated the effects of ethanol concentration,ethanol dosage,extraction time and extraction times.Eventually,the optimum extraction conditions of total flavonoids conditions were14 times of 55%ethanol into Drynaria fortunei powder,extracting three times and refluxing for 1.5 hours each time.Through the investigation of resin adsorption and desorption,it was found that AB-8 resin was the best macroporous resin.Furthermore,we further optimized the best enrichment condition by using the combination of experiment of single factor and orthogonal test.The optimum conditions are as follows:after using AB-8 macroporous resin?20 g?ratio of diameter to length was1:5,to adsorb 10 mL(20 mg.L-1)of total flavonoids aqueous solution?pH=4.0,20°C?for 24 h,and then eluted with 8 BV water and 8 BV 50%ethanol,and subsequently concentrated all eluent and obtained the concentrate.The results indicated that the content of purified total flavonoids and naringin in Drynaria fortune concentrate was up to 76.11%,20.41%,respectively.In addition,the transferring rate of naringin and total flavonoids was 76.53%,and78.82%,respectively.Finally,the PC12 cells induced by A?25-35 were used as an in vitro cell model of Alzheimer's disease,and the effects of different concentrations of purified flavonoids on the cell model were studied.The MTT assay was used to detect the optimal drug concentration.It was found that 2×10-7 g/m L of purified Drynaria fortune showed the strongest protective effect on A?25-35 induced PC12 cells damage.After this concentration of purification acts on A?25-35 induced PC12 cells,the cell vitability was then detected.The experimental data showed that the viability of PC12 cells increased significantly under the concentration of 2×10-7 g/m L,proliferation rate was 93%,indicating that total flavonoids have protective effects on damaged PC12 cells.Our experiments have established a reliable quantitative analysis method of total flavonoids in Drynaria fortune,and relative methodological evaluation have showed that this method has a good reliability,repeatability and stability,which could be applied for the detection of the content of total flavonoids.Meanwhile,it also optimized the optimum extraction of effective fractions of total flavonoids in Drynaria fortune and macroporous resin enrichment process.This method is simple,the content of total flavonoids in purified concentrate is higher,and which has a preferably transferring rate.The in vitro cell experiments demonstrated that total flavonoids indeed possess a significantly protective effect on central nerve,and the total flavonoids in Drynaria fortune has a potential value for the treatment of Alzheimer's disease. |
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