阅读说明：本技术 一种分离制备银杏叶提取物中银杏黄酮的方法 (Method for separating and preparing ginkgetin in ginkgo leaf extract ) 是由 赵林果 张立虎 张慎华 曹祥 王昌选 赵祎武 于 2019-10-29 设计创作，主要内容包括：一种分离制备银杏叶提取物中银杏黄酮的方法。活性炭能有效将银杏叶提取物中的银杏内酯和银杏黄酮分开,活性炭吸附银杏总黄酮的性能极佳,最大吸附量为195.8 mg/g活性炭,但活性炭吸附银杏黄酮后产生了死吸附,利用不同浓度的乙醇等其他溶剂都无法达到解吸附作用。本发明建立了活性炭解吸银杏黄酮的方法和工艺研究,为实现银杏黄酮回收利用提供了一条可行的方法。适宜的解吸工艺条件为：洗脱液乙醇浓度为73.4%,氨水浓度5.7%,洗脱温度31.5℃,洗脱体积约为4倍柱体积。在此条件下银杏黄酮的解吸率达到70%以上。(A method for separating and preparing ginkgetin from folium Ginkgo extract is provided. The active carbon can effectively separate the bilobalide and the ginkgetin in the ginkgo leaf extract, the performance of the active carbon for adsorbing the ginkgo total flavonoids is excellent, the maximum adsorption amount is 195.8 mg/g of the active carbon, however, dead adsorption is generated after the active carbon adsorbs the ginkgetin, and the desorption effect cannot be achieved by using other solvents with different concentrations, such as ethanol and the like. The invention establishes a method and process research for desorbing the ginkgetin by using the active carbon, and provides a feasible method for realizing the recovery and utilization of the ginkgetin. The suitable desorption process conditions are as follows: the ethanol concentration of the eluent is 73.4 percent, the ammonia water concentration is 5.7 percent, the elution temperature is 31.5 ℃, and the elution volume is about 4 times of the column volume. Under the condition, the desorption rate of the ginkgetin reaches more than 70 percent.)

1. A method for separating and preparing ginkgetin in ginkgo biloba extract is characterized in that the ginkgetin is obtained by eluting ginkgo biloba extract through an alcohol solution containing 1wt.% to 9wt.% ammonia water after the ginkgo biloba extract is adsorbed by activated carbon.

2. The method for separating and preparing ginkgetin in ginkgo biloba leaf extract according to claim 1, which comprises the steps of: dissolving folium Ginkgo extract with 10-20 wt.% alcohol, adsorbing ginkgetin with activated carbon at 25-35 deg.C under weakly acidic or acidic condition, and washing with 10% alcohol and 40% alcohol respectively; and eluting the ginkgetin in the ginkgo leaf extract adsorbed in the activated carbon by using 40-80 wt.% of alcohol and 1-9 wt.% of ammonia water at 25-55 ℃, wherein the volume of the eluent is 4 times of the volume of the column.

3. The method of claim 1, wherein the alcohol is ethanol.

4. The method of claim 1, wherein the pH of the adsorbed ginkgetin is 5.

5. The method of claim 1, wherein the temperature for adsorbing ginkgetin is 30 ℃.

6. The method of claim 1, wherein the alcohol concentration in the eluting step is 73.4%.

7. The method of claim 1, wherein the concentration of ammonia water in the elution step is 5.7%.

8. The method of claim 1, wherein the elution temperature in the elution step is 31.5 ℃.

Technical Field

The invention relates to the technical field of deep processing of natural products, in particular to a method for preparing ginkgolides and ginkgetin by activated carbon separation and desorbing ginkgetin adsorbed by activated carbon.

Background

The ginkgo biloba extract has obvious biological activity and important application value, and the ginkgo flavone is a natural free radical scavenger and a vasodilator, can obviously reduce the damage of excessive free radicals to heart and brain histiocytes in the processes of ischemia, anoxia and aging, and can promote the improvement of hemorheology. The bilobalide is a potent Platelet Activating Factor (PAF) antagonist, can effectively prevent abnormal platelet aggregation and thrombosis on the inner wall of cardiovascular and cerebrovascular atherosclerosis, and improve local microcirculation, and has neuron protecting effect and can improve the binding force of neurotransmitter and receptor. The existing ginkgo drugs and major health products are developed and researched by taking ginkgo biloba Extract (EGB) which meets the standard of Chinese pharmacopoeia as a main raw material. Based on the obvious difference of the biological activity of the ginkgo total flavone and the ginkgo total lactone, the medicine or the health care product of the ginkgo lactone and the ginkgo flavone is developed respectively by taking the function as the guide, and the specialization of the function of the ginkgo leaf extract, the high value of the value and the diversification of the product can be expected to be realized.

Bilobalide is one of the most effective natural medicines for treating central nervous system, cardiovascular and other related diseases in clinic, and products such as injection taking bilobalide as a main effective component are approved by the national drug administration to enter clinical research or are applied to the clinic as a new medicine. Therefore, the preparation technology of the ginkgolide with high yield and low cost becomes the key of the development and utilization of related products.

At present, an effective method for synchronously separating and preparing ginkgolide and ginkgetin does not exist, a few of preferred macroporous resins can better adsorb the ginkgetin and the ginkgolide and can be better eluted by ethanol gradient, but the performances of adsorbing and desorbing the ginkgetin and the ginkgetin are similar, the discrimination is low, and the two cannot be effectively separated by screening the macroporous adsorption resins and changing the concentration of ethanol for elution. The polyamide resin has great difference in adsorption and desorption performances of flavone and lactone in the ginkgo leaf extract EGB, and is expected to be a feasible method, but the prior art is not mature enough and the preparation cost is high. One of the existing processes for producing ginkgolides by related enterprises is to adopt an activated carbon adsorption method, but the problems are that after the ginkgo biloba extract is adsorbed by activated carbon, the ginkgolides can be eluted by ethanol, and the ginkgetin forms dead adsorption, so that the ginkgetin cannot be effectively utilized, and the use amount of the activated carbon is increased.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a method for separating and preparing ginkgetin in a ginkgo leaf extract, establishes a process for desorbing the ginkgetin by using active carbon, and realizes effective recycling of the ginkgetin. The invention provides technical support for realizing synchronous preparation of ginkgolide and ginkgetin and solving the technical bottleneck faced by the current production.

The technical scheme is as follows: a method for separating and preparing ginkgetin from folium Ginkgo extract comprises adsorbing folium Ginkgo extract with active carbon, and eluting with alcoholic solution containing 1-9 wt.% ammonia water to obtain ginkgetin.

The method comprises the following specific steps: dissolving folium Ginkgo extract with 10-20 wt.% alcohol, adsorbing ginkgetin with activated carbon at 25-35 deg.C under weakly acidic or acidic condition, and washing with 10% alcohol and 40% alcohol respectively; and eluting the ginkgetin in the ginkgo leaf extract adsorbed in the activated carbon by using 40-80 wt.% of alcohol and 1-9 wt.% of ammonia water at 25-55 ℃, wherein the volume of the eluent is 4 times of the volume of the column.

Preferably, the alcohol is ethanol.

Preferably, the pH value of the adsorbed ginkgetin is 5.

Preferably, the temperature for adsorbing the ginkgetin is 30 ℃.

Preferably, the alcohol concentration in the elution step is 73.4%.

Preferably, the concentration of ammonia water in the elution step is 5.7%.

Preferably, the elution temperature in the above elution step is 31.5 ℃.

Has the advantages that: at present, after the ginkgo biloba extract is adsorbed by the active carbon, the bilobalide can be eluted by ethanol, and the ginkgetin forms dead adsorption, so that the ginkgetin cannot be effectively utilized, and the using amount of the active carbon is increased. The method for separating and preparing the ginkgetin in the ginkgo leaf extract is simple to operate, can effectively desorb the ginkgetin in the active carbon, and has high desorption rate which can reach more than 70 percent under proper conditions.

Drawings

FIG. 1 is a graph of the static adsorption of ginkgo biloba extract from EGB by activated carbon;

FIG. 2 the effect of pH on the static adsorption of ginkgetin by activated carbon;

FIG. 3 effect of ammonia concentration on elution rate;

FIG. 4 influence of ethanol concentration on elution rate;

FIG. 5 effect of elution temperature on elution rate;

FIG. 6 effect of liquid volume on elution rate;

FIG. 7 is a graph of the response of the interaction of two factors on the desorption rate of total flavonoids;

FIG. 870% ethanol (B) and 70% ethanol +5% ammonia (C) eluents hydrolysis HPLC profile (A1. Quercetin, 2. Kaempferol, 3. Isorhamnetin);

FIG. 9 is a microscopic morphology chart before and after hydrolyzing flavone absorption with ammonia added to activated carbon (A1, A2 are 1000 and 2000 times magnified after 70% ethanol is eluted, B1, B2 are 1000 and 2000 times magnified after ammonia is added for desorption, and the scale is 10 μm);

FIG. 10 static desorption curves of activated carbon versus ginkgetin;

FIG. 11 shows the elution dynamic desorption curves of ginkgo flavone adsorbed by activated carbon at different ethanol concentrations.

Detailed Description

The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments.

A method for separating and preparing ginkgetin from folium Ginkgo extract comprises adsorbing ginkgetin with active carbon; eluting the ginkgetin in the ginkgo leaf extract adsorbed in the activated carbon under a proper condition by using alcohol with a certain concentration and ammonia water with a certain concentration. The method comprises the following steps:

1. establishing a method for statically adsorbing and desorbing ginkgo total flavonoids by using active carbon. Accurately weighing activated carbon in a ground triangular flask, weighing folium Ginkgo extract, dissolving with 100mL of 10% ethanol, sampling at different times, and determining the adsorption amount and column loading amount of total ginkgetin. And washing the resin with saturated static adsorption with 20% ethanol for 2 times, adding 5% ammonia water, performing static desorption, sampling at different time points, and determining the desorption amount of ginkgetin. By observing the change relationship of the adsorption quantity of the ginkgo flavone adsorbed by the activated carbon with time, three stages of the static adsorption process are mastered, the adsorption index of the ginkgo flavone is increased in 0-10 min, the linear growth period is realized, the equilibrium state of adsorption is approached in 10-90min, the slow growth period is realized, and the adsorption and desorption quantities are basically stabilized at fixed values after 90 min.

2. The influence of pH value on the adsorption of ginkgo flavone by active carbon is studied. Adjusting pH of the sample solution to different acidity, weighing activated carbon and folium Ginkgo extract, and dissolving with 10% ethanol. After the adsorption is fully balanced, sampling and determining the amount of the ginkgo flavone adsorbed by the active carbon. The results show that the adsorption effect is favorable under weak acidity or acidic conditions, and the adsorption quantity is maximum when the pH value is 5.

3. And analyzing the isotherm of the ginkgo flavone adsorbed by the activated carbon, and determining the optimal adsorption temperature. Weighing multiple parts of activated carbon, respectively adding folium Ginkgo extract with different mass, adsorbing and balancing at different temperatures, measuring the concentration of ginkgetin, and taking the balanced adsorption amount as ordinate and the balanced concentration as abscissa to obtain adsorption isotherms under different conditions. The results were fitted using the Langmuir equation and the Freundlich equation. Freundlich's equation and Langmuir's equation indicate that adsorption of ginkgo total flavonoids by activated carbon is easier and adsorption of ginkgo flavonoids by activated carbon is more biased toward single-layer adsorption. The suitable adsorption temperature is 25-35 ℃, and the optimal adsorption temperature is 30 ℃.

4. The desorption process of the activated carbon for adsorbing the ginkgetin is researched.

(1) Single factor experiments: each group was prepared by weighing folium Ginkgo extract, dissolving in 20% ethanol aqueous solution, adsorbing with activated carbon, and performing single factor experiment with water as solvent, ammonia water-ethanol-distilled water solution volume, ammonia water concentration, ethanol concentration, and extraction temperature. The optimum ammonia water concentration reaches the maximum value when 5 percent, the desorption rate of the ginkgo flavone desorbed by the active carbon is maximum when the ethanol concentration is 70 percent, the desorption rate of the flavone is rapidly increased along with the increase of the temperature, the temperature is suitable at 35 ℃, the desorption rate of the flavone is increased along with the increase of the liquid volume, the liquid volume is linearly increased, and the speed is slowly increased when the liquid volume exceeds a certain amount.

(2) The process for desorbing the ginkgetin by using the activated carbon is optimized by a response surface method: weighing folium Ginkgo extract, dissolving in 20% ethanol solution, and adsorbing with activated carbon. According to the single-factor experiment result and the Box-Behnken design principle, four-factor three-level liquid volume, ammonia water concentration, ethanol concentration, extraction temperature and the like are selected for research during elution. The extraction amount of ginkgo total flavonoids is used as a response value to carry out a four-factor three-level response surface design experiment. Wherein, the concentration of ethanol is 40-80%, the concentration of ammonia water is 1-9%, and the elution temperature is 25-55 ℃. Further analyzing and determining the optimal conditions and a regression model through software to obtain the optimal desorption process conditions of the ginkgetin in the activated carbon, wherein the optimal desorption process conditions comprise the following steps: the volume of the eluent is 116.75 mL, the ethanol concentration is 73.4%, the elution temperature is 31.5 ℃, and the ammonia water concentration is 5.7%. The theoretical total flavone desorption rate obtained under this condition was 73.25%. Three times of experiments are carried out according to the optimal conditions to verify that the desorption rate of all the activated carbon for adsorbing the ginkgetin is 74.56% +/-1.24%.

5. Establishing a ginkgo flavone rule of dynamic desorption activated carbon adsorption. Weighing activated carbon, loading into a chromatographic column, weighing folium Ginkgo extract, dissolving with 10% ethanol, adsorbing completely, washing with 10% ethanol and 40% ethanol respectively, and eluting with different ethanol concentrations +5% ammonia water respectively. Along with the increase of time, the desorption amount of the ginkgo flavone by the activated carbon is obviously increased, the whole process is divided into three stages, the index of the desorption amount of the flavone is increased in 0-10 min, the period is linearly increased, the equilibrium state of desorption is approached in 10-90min, the period is a slow increase period, and the desorption amount is basically stabilized at a fixed value after 90 min. The concentration of flavone is highest when elution is carried out for 2BV (column volume) by 40% ethanol and 5% ammonia water, and the concentration of ginkgetin is obviously reduced although the elution is maintained to be gentle after elution is carried out for 2BV by 60% ethanol and 5% ammonia water.