阅读说明：本技术 一种用于银杏叶提取物增溶的深共熔溶剂及其制备方法和应用 (Deep eutectic solvent for solubilizing ginkgo leaf extract and preparation method and application thereof ) 是由 苏二正 董其惠 吴蓉 汪贵斌 赵林果 曹福亮 陈璐瑶 于 2019-09-20 设计创作，主要内容包括：本发明公开了一种用于银杏叶提取物增溶的深共熔溶剂及其制备方法和应用,该深共熔溶剂是由摩尔比为3-1:1-2的丁二醇和乙酰丙酸制备而成。本发明提供的深共熔溶剂作为绿色溶剂,安全无毒,制备工艺简单,成本低,可以作为银杏叶提取物增溶溶剂,可在不影响银杏叶提取物中活性物质稳定性和生物活性的情况下大大提高其溶解度。本发明提供的深共熔溶剂用于银杏叶提取物的增溶,可使银杏叶提取物中的总黄酮醇苷、萜类内酯和原花青素的溶解度与水中溶解度相比分别提高405、638、760倍。本发明提供的深共熔溶剂可作为银杏叶提取物的一种新的溶媒,用于银杏叶提取物液态剂型的制备,实现液态给药。(The invention discloses a deep eutectic solvent for solubilizing a ginkgo leaf extract, and a preparation method and application thereof, wherein the deep eutectic solvent is prepared from butanediol and levulinic acid in a molar ratio of 3-1: 1-2. The deep eutectic solvent provided by the invention is used as a green solvent, is safe and nontoxic, has a simple preparation process and low cost, can be used as a solubilizing solvent of the ginkgo leaf extract, and can greatly improve the solubility of the ginkgo leaf extract under the condition of not influencing the stability and the biological activity of active substances in the ginkgo leaf extract. The deep eutectic solvent provided by the invention is used for solubilizing the ginkgo leaf extract, and can respectively improve the solubility of total flavonol glycosides, terpene lactones and procyanidine in the ginkgo leaf extract by 405, 638 and 760 times compared with the solubility in water. The deep eutectic solvent provided by the invention can be used as a new solvent of the ginkgo leaf extract, is used for preparing a liquid preparation of the ginkgo leaf extract and realizes liquid administration.)

1. A deep eutectic solvent for solubilizing a ginkgo biloba leaf extract is characterized by being prepared from butanediol and levulinic acid in a molar ratio of 3-1: 1-2.

2. The deep eutectic solvent for solubilization of ginkgo biloba leaf extract according to claim 1, wherein the molar ratio of the butanediol to the levulinic acid is 2: 1.

3. The method for preparing the deep eutectic solvent for solubilization of ginkgo biloba extract of claim 1, comprising the steps of:

(1) drying and dehydrating the raw materials of butanediol and levulinic acid;

(2) weighing butanediol and levulinic acid according to the molar ratio according to the requirement of preparation amount, uniformly mixing, heating and stirring until a transparent liquid is formed;

(3) and (3) after the transparent liquid is formed, storing at room temperature overnight, observing that the liquid is stable and no solid is precipitated, and using the obtained deep eutectic solvent for solubilizing the ginkgo leaf extract.

4. A method for measuring the solubilization of ginkgo biloba extract by using the deep eutectic solvent as claimed in claim 1, comprising the steps of:

(1) adding a deep eutectic solvent or a deep eutectic solvent-water mixture into a clean container, adding a standard ginkgo leaf extract into the deep eutectic solvent or the deep eutectic solvent-water mixture, fully shaking, observing once every 1 hour, continuously adding the standard ginkgo leaf extract after the added standard ginkgo leaf extract is completely dissolved until the added standard ginkgo leaf extract cannot be dissolved, and precipitating or suspending the standard ginkgo leaf extract in the deep eutectic solvent or the deep eutectic solvent-water mixture as a solid to finish dissolving to obtain a ginkgo leaf extract deep eutectic solvent solution;

(2) centrifuging folium Ginkgo extract deep eutectic solvent solution at high speed, collecting supernatant, measuring the content of three folium Ginkgo extract active substances including total flavonol glycoside, terpene lactone and procyanidin, and calculating the solubility of folium Ginkgo extract in deep eutectic solvent or deep eutectic solvent-water mixture.

5. The method as claimed in claim 4, wherein the deep eutectic solvent is prepared by adding deionized water with different volume fractions to the deep eutectic solvent in step (1), and the volume fraction of the deionized water is 0-90% of the total deep eutectic solvent-water mixture.

6. The method as claimed in claim 4, wherein the fully shaking condition of step (1) is preferably 25-30 ℃, 250-280 rpm.

7. The method as claimed in claim 4, wherein the high speed centrifugation condition in step (2) is 25-30 ℃, the centrifugal force is 10000-15000g, and the centrifugation time is 10-30 minutes.

8. A method for preparing a deep eutectic solvent homogeneous solution dosage form of a ginkgo biloba extract is characterized in that the deep eutectic solvent or a deep eutectic solvent-water mixture obtained by the method for determining the solubilization of the ginkgo biloba extract in claim 4 is weighed and added into a deep eutectic solvent or a deep eutectic solvent-water mixture with a corresponding amount within the range of the dissolving capacity of the standard ginkgo biloba extract, and the deep eutectic solvent or the deep eutectic solvent-water mixture is uniformly mixed to prepare the ginkgo biloba extract homogeneous solution dosage forms with different ginkgo biloba extract contents.

9. The method as claimed in claim 8, wherein the mixing is performed by shaking at 25-30 deg.C and 250-280 rpm.

10. An application of deep eutectic solvent for solubilizing ginkgo leaf extract in preparing liquid preparation products of ginkgo leaf extract.

Technical Field

The invention relates to forest source medicine and food resource chemistry, in particular to a deep eutectic solvent for solubilizing a ginkgo leaf extract and a preparation method and application thereof.

Background

Ginkgo biloba (Ginkgo biloba L.) belongs to deciduous trees, is a kind of gymnosperm, is also the only survivor of Ginkgoaceae Ginkgoa, and has high medicinal and edible values on fruits and leaves. Ginkgo Biloba leaf Extract (GBE) is a product with enriched effective components extracted from ginkgo Biloba leaves as a raw material by adopting a proper solvent. International standard GBE is EGb761 produced according to the Schwabe corporation patent process, and its chemical composition contains 24% of flavone, 7% of proanthocyanidin, 6% of terpene lactone, 13% of carboxylic acid component, 2% of catechin, 20% of non-flavonoid glycoside, less than 5ppm of ginkgolic acid, 4% of high molecular compound, 3% of water, 5% of inorganic substance, and 3% of other chemical components, that is, about 87% of total chemical components in EGb761 are known. The individual components are integral organic constituents whose pharmacological effects are the result of the combined action of the various relatively immobile components.

GBE has antiinflammatory, antioxidant, and antitumor effects, and can dilate cardiovascular and cerebrovascular vessels and artery vessels, prevent hypertension, hyperlipemia, diabetes, etc., and reduce incidence of common eye diseases. Various preparations prepared by taking GBE as a raw material are widely applied to the fields of medicines, health-care products, food additives, functional beverages, cosmetics and the like. Because the solubility of important active ingredients of GBE, namely flavonoid compounds and terpene lactones compounds, in water is low, although more than 30 GBE preparations are available in the market, common preparations, such as capsules, tablets, granules, dripping pills, film coated pills, powder injections, disintegrating tablets, dispersible tablets, chewable tablets, effervescent tablets and the like are available. The tablet is the most common tablet, also comprises dispersible tablet, disintegrating tablet, chewable tablet, effervescent tablet and the like, has the advantages of stable quality, accurate measurement, low production cost, convenient taking and carrying and the like, but a large amount of excipients, disintegrants and other auxiliary materials and coating films need to be added in the preparation process, and the tablet slowly disintegrates in vivo so as to take effect relatively slowly. GBE preparation has many capsules and granules, and has the advantages of quick absorption, high bioavailability, convenient carrying and storage, good process and quality stability, high safety, poor mobility and fixed formula which cannot be increased or decreased with the disease. In addition to the above problems, the dosage forms of tablets, capsules, granules, etc. also cause great troubles for patients with difficulty in taking oral drugs and swallowing, especially for the elderly patients. In order to overcome the defects of the solid dosage forms, in recent years, liquid dosage forms such as oral liquid, injection, liposome and the like are researched, the liquid dosage forms all use water as a main solvent, and in order to increase the solubility of GBE in water, the liquid dosage forms generally need to be solubilized by using an organic cosolvent, a surfactant and the like, on one hand, the GBE is a compound and contains a plurality of effective components, and the organic cosolvent and the surfactant are difficult to achieve the same solubilizing effect on all the components in the GBE, so that the proportion of each component in the final dosage form is changed, and the synergistic effect of the original proportion is difficult to maintain; on the other hand, the liquid preparation produced by the solubilization method is a thermodynamically unstable system and has poor stability; in addition, the solubilizing effect of organic solvents and surfactants is still limited, and the effective concentration of GBE is still low, resulting in a large volume of administration. Therefore, there is an urgent need to develop new soluble and stable liquid dosage forms of GBE.

In 2003, Abbott (Abbott A P, Capper G, Davies D L, et al. novel solvent Properties of Choline chloride/urea mixtures. chemical Communications, 200314 (1):70-71) first prepared a liquid solvent formed by mixing two solids, choline chloride and urea, thereby proposing the concept of Deep Eutectic Solvents (DESS). DESs are typically eutectic mixtures of Hydrogen Bond Acceptors (HBAs) and Hydrogen Bond Donors (HBDs) at certain molar ratios. The physical and chemical properties of DESS are very similar to those of Ionic Liquids (ILs), so that the DESS is also classified as a novel ILs or ILs analogue, and has the advantages of low melting point, high solubility, difficult volatilization, difficult combustion, thermal stability, low toxicity and the like. Besides the advantages of room-temperature ionic liquids, the DESs also have the following characteristics: (1) the price of the DESs raw material is low, the atom utilization rate in the preparation process reaches 100%, no waste is generated, the preparation is simple, no purification is needed, and the large-scale industrial production is easy to realize; (2) the biological degradation is easy, and the biocompatibility is superior to that of ionic liquid; (3) DESS are multi-component systems and therefore are easier to design and modify than ionic liquids to accommodate different application requirements. In view of the many advantages of DESs, as well as their low ecological footprint and attractive price, have attracted a great interest in recent years, which show good application prospects in the fields of separation processes, functional materials, electrochemistry, chemical reactions, etc.

Therefore, it is possible to prepare a novel liquid formulation having a high solubility and stability of GBE by preparing a deep eutectic solvent using a conventional food material (ingredient) or pharmaceutical excipient as HBA and/or HBD and using it for solubilization of GBE.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a deep eutectic solvent for solubilizing a ginkgo biloba extract. The deep eutectic solvent provided by the invention is used as a green solvent, is safe and nontoxic, has a simple preparation process and low cost, can be used as a solubilizing solvent of the ginkgo leaf extract, and can greatly improve the solubility of the ginkgo leaf extract under the condition of not influencing the stability and the biological activity of active substances in the ginkgo leaf extract.

The invention also provides a preparation method and application of the deep eutectic solvent for solubilizing the ginkgo biloba extract.

The technical scheme is as follows: in order to achieve the above object, the deep eutectic solvent for solubilization of ginkgo biloba extract according to the present invention is characterized by being prepared from butanediol and levulinic acid in a molar ratio of 3-1: 1-2.

Preferably, the molar ratio of butanediol to levulinic acid is 2: 1.

The preparation method of the deep eutectic solvent for solubilizing the ginkgo biloba extract comprises the following steps:

(1) drying and dehydrating the raw materials of butanediol and levulinic acid;

(2) weighing butanediol and levulinic acid according to the molar ratio according to the requirement of preparation amount, uniformly mixing, heating and stirring until a transparent liquid is formed;

(3) and (3) after the transparent liquid is formed, storing at room temperature overnight, observing that the liquid is stable and no solid is precipitated, and using the obtained deep eutectic solvent for solubilizing the ginkgo leaf extract.

The method for determining the solubilization of the ginkgo biloba extract by using the deep eutectic solvent is characterized by comprising the following steps of:

(1) adding a deep eutectic solvent or a deep eutectic solvent-water mixture into a clean container, adding a standard ginkgo leaf extract into the deep eutectic solvent or the deep eutectic solvent-water mixture, fully shaking, observing once every 1 hour, continuously adding the standard ginkgo leaf extract after the added standard ginkgo leaf extract is completely dissolved until the added standard ginkgo leaf extract cannot be dissolved, and precipitating or suspending the standard ginkgo leaf extract in the deep eutectic solvent or the deep eutectic solvent-water mixture as a solid to finish dissolving to obtain a ginkgo leaf extract deep eutectic solvent solution;

(2) centrifuging folium Ginkgo extract deep eutectic solvent solution at high speed, collecting supernatant (the supernatant after centrifugation is folium Ginkgo extract deep eutectic solvent solution), measuring the content of three folium Ginkgo extract active substances including total flavonol glycoside, terpene lactone and procyanidin, and calculating the solubility of folium Ginkgo extract in the deep eutectic solvent or deep eutectic solvent-water mixture.

In addition, deionized water is used as a control to measure the solubility contrast of three ginkgo leaf extract active substances of total flavonol glycosides, terpene lactones and procyanidine in the ginkgo leaf extract in the deionized water, so that the solubilization effect of the deep eutectic solvent or the deep eutectic solvent-water mixture can be calculated. The method for determining the solubility of the ginkgo biloba extract in the deionized water is the same as the method for determining the solubility of the ginkgo biloba extract in the DES in terms of steps, except that the DES is replaced by the deionized water.

Wherein the deep eutectic solvent-water mixture in the step (1) is prepared by adding deionized water with different volume fractions into the deep eutectic solvent, and the volume fraction of the deionized water accounts for 0-90% of the whole deep eutectic solvent-water mixture. In general, the volume fraction of water can be suitably selected depending on the cost and the ease of handling or the viscosity of the solution. Generally, the solubility of ginkgo biloba extracts decreases as the volume fraction of water increases, as does the viscosity of DES. For example, the maximum range of the solubilization capacity of DES on ginkgo biloba extract can be obtained according to the determination, and the maximum value of the solubilization capacity can be obtained. It is desirable to prepare a low-solubility liquid dosage form of ginkgo biloba extract, which can be adjusted by adding deionized water in order to reduce the viscosity of the liquid dosage form or to reduce the amount of DES used to reduce the cost of the solvent.

Wherein, the sufficient oscillation condition in the step (1) is 25-30 ℃, 250-280 rpm. Preferably, the sufficient shaking conditions are 25 ℃ and 280 rpm.

Wherein the high-speed centrifugation condition in the step (2) is 25-30 ℃, the centrifugal force is 10000-15000g, and the centrifugation time is 10-30 minutes. Preferably, the high-speed centrifugation condition is 25 ℃, the centrifugal force is 12000g, and the centrifugation time is 20 minutes.

The preparation method of the ginkgo biloba extract deep eutectic solvent homogeneous solution dosage form comprises the steps of weighing the standard ginkgo biloba extract, adding the deep eutectic solvent or the deep eutectic solvent-water mixture with the corresponding amount into the deep eutectic solvent or the deep eutectic solvent-water mixture with the dissolving capacity range of the standard ginkgo biloba extract, which is obtained by measuring the method for measuring the solubilization of the ginkgo biloba extract, and uniformly mixing, thus preparing the ginkgo biloba extract deep eutectic solvent homogeneous solution dosage form with different ginkgo biloba extract contents.

Wherein the uniform mixing method is shaking under the conditions of 25-30 ℃ and 250-280 rpm. Preferably, the shaking conditions are 25 ℃ and 280 rpm.

The invention relates to an application of a deep eutectic solvent for solubilizing a ginkgo biloba extract in preparation of a ginkgo biloba extract liquid preparation product.

The raw materials in the invention are all available on the market, wherein the ginkgo biloba extract is purchased abroad and required to meet the quality requirements of European pharmacopoeia (latest edition) or United states pharmacopoeia (latest edition), and is purchased domestically and required to meet the quality requirements of Chinese pharmacopoeia (latest edition).

The invention selects the ginkgo biloba extract as a model of the plant source indissoluble effective part medicament, researches the solubilization effect of the deep eutectic solvent on the plant source indissoluble effective part medicament, researches the dissolution conditions of the ginkgo biloba extract in various DESS, researches the stability of the ginkgo biloba extract in the DESS, and researches the influence of the DESS as a dissolution medium on the medicament effect, and finally expects to screen out an optimal DES as a solvent for preparing the ginkgo biloba extract liquid dosage form to realize liquid administration.

The butanediol and the levulinic acid used in the invention are common food and medicine auxiliary materials, and are safe and nontoxic. The molecules of the butanediol and the levulinic acid contain hydroxyl, carboxyl, carbonyl and the like, and hydrogen bonds can be formed between the butanediol and the levulinic acid, so that the deep eutectic solvent prepared from the butanediol and the levulinic acid has low melting point, good fluidity and strong dissolving property. In addition, when the preferable molar ratio of the butanediol to the levulinic acid is 2:1, not only can hydrogen bonds be formed between the two molecules, but also hydrogen bonds can be formed with groups on molecules of flavonol glycosides, terpene lactones and procyanidins in the ginkgo leaf extract, so that a large and stable hydrogen bond network is formed, and the dissolution of the ginkgo leaf extract is facilitated.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the deep eutectic solvent prepared by the invention is used as a green solvent, is safe and nontoxic (toxicity research shows that the IC50 value is more than 2000mg/L), has simple preparation process and low cost, and can be used as a solubilizing solvent of ginkgo leaf extract.

(2) The deep eutectic solvent prepared by the invention is used for solubilizing the ginkgo leaf extract, and can respectively improve the solubility of total flavonol glycosides, terpene lactones and procyanidine in the ginkgo leaf extract by 405, 638 and 760 times compared with the solubility in water.

(3) The stability and the biological activity of the ginkgo biloba extract are not influenced after the ginkgo biloba extract is dissolved in the deep eutectic solvent.

(4) The ginkgo biloba extract is dissolved in the deep eutectic solvent to form a uniform solution with stable thermodynamics, and the system stability is high.

(5) The ginkgo biloba extract is dissolved in the deep eutectic solvent, so that the solubilization of the ginkgo biloba extract is realized, the use of organic solvents and surfactants in the traditional method is avoided, and the solubilization operation is very simple and convenient.

(6) The deep eutectic solvent for solubilizing the ginkgo leaf extract can be used as a solvent for preparing a liquid dosage form of the ginkgo leaf extract, so that liquid administration is realized.

Drawings

FIG. 1 is a graph showing the stability of total flavonol glycosides in DESS, DESS-water mixtures;

FIG. 2 is a graph showing the stability of terpene lactones in DESs, DESs-water mixtures;

FIG. 3 is a graph showing the stability of procyanidins in DESS, DESS-water mixtures;

FIG. 4 is a superimposed graph of IR spectra of a solution of Ginkgo biloba extract, DES5-2 and Ginkgo biloba extract-DES 5-2;

FIG. 5 is a graph showing the overlay of IR spectra of Ginkgo biloba extract, DES8-5, and Ginkgo biloba extract-DES 8-5;

FIG. 6 shows HPLC fingerprint of Ginkgo biloba extract: (a) dissolving in methanol; (b) dissolving in DES 5-2; (c) dissolving in DES 8-5;

figure 7 is HPLC fingerprint: (a) DES 5-2; (b) DES 8-5;

FIG. 8 is a schematic diagram showing ABTS free radical scavenging activity of Ginkgo biloba leaf extract dissolved in 70% ethanol and DES 5-2;

FIG. 9 is a graph showing DPPH radical scavenging activity of Ginkgo biloba extract dissolved in 70% ethanol and DES 5-2;

FIG. 10 is a graph showing the reduction ability of iron ion FRAP in a ginkgo biloba extract dissolved in 70% ethanol and DES 5-2.

Detailed Description

The invention is further illustrated by the following figures and examples.

Main raw materials and reagents:

the main apparatus comprises:

the ginkgo leaf extract is purchased from a special manufacturer, and various indexes are measured according to a method in Chinese pharmacopoeia (latest edition), so that the ginkgo leaf extract meets the requirements of the Chinese pharmacopoeia and is used for solubilization research.

The standard ginkgo biloba extract used in the present invention was purchased from yozhou henkhaki ginkgo products limited.