Method for extracting effective components of parsley powder by using green deep-eutectic solvent and application thereof

Abstract

The invention discloses a method for extracting effective components of parsley powder by using a green deep-eutectic solvent and application of the method. According to the method, the effective components of parsley powder are extracted by using the deep-eutectic solvent. According to the method, the green deep-eutectic solvent is adopted to extract the effective components in the Jinsan, compared with a traditional extraction method that 95% ethyl alcohol, 50% ethyl alcohol and water are sequentially used for extraction, the extraction effect is remarkably improved, the extract content is two times that of a traditional extraction method, the extraction efficiency of the effective components in the Jinsan is improved, and the method is suitable for industrial production. The green extraction medium provided by the invention has the characteristics of no toxicity, easiness in preparation, low production cost, biodegradability, environmental friendliness and the like, and is a sustainable extraction medium. In addition, the extract obtained through the technology shows a good effect in the aspect of resisting migraine, and a new treatment choice is provided for migraine patients.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction of effective components of traditional Chinese medicines, and particularly relates to a method for extracting effective components of Duijin powder with a green low eutectic solvent and application thereof. Background Art

[0003] Migraine is a recurrent neurological disease that generally lasts from 4 hours to 3 days. It is characterized by moderate or severe headaches and various neurological symptoms, such as photophobia, phonophobia, skin allodynia, and gastrointestinal symptoms such as nausea and vomiting. There are also a variety of other neurological symptoms, such as vertigo, dizziness, tinnitus, blurred vision, and cognitive impairment. Migraine is the second largest disability disease in the world, especially affecting young and middle-aged women, and has become a global problem.

[0004] As the most widely used and most readily available solvent in the extraction process of traditional Chinese medicine, water is a good candidate for extracting active ingredients with strong polarity. However, traditional Chinese medicine contains a variety of bioactive ingredients with strong lipophilicity, and their solubility in water is very low. Many organic solvents are volatile, flammable, explosive, and even toxic. Deep eutectic solvents (DES) are an emerging class of green ionic liquid-like systems. Generally, DES consists of two parts, namely a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). The presence of hydrogen bonds in DES results in it having a melting point lower than the components themselves, forming a eutectic material system that is liquid at room temperature. Compared with traditional organic extraction solvents, deep eutectic solvents have the advantages of low price, easy preparation and storage, and recyclability. Most of them are biodegradable, have good biocompatibility and non-toxicity, have a green preparation process, and have an atomic utilization rate of 100%. They have been used to extract flavonoids, saponins, polysaccharides, alkaloids, quinones, phenolic acids, volatile oils and other components, and have gradually become a substitute for traditional organic solvents.

[0005] At present, there is no report on the application of low eutectic solvents in the extraction of Jinsan. Based on the previous research, the present invention designed and prepared 13 kinds of green low eutectic solvents commonly used in laboratories, extracted total flavonoids, total anthraquinones, baicalin, baicalein, aloe-emodin, emodin, and chrysophanol from Jinsan, and screened out the green low eutectic solvent with the best extraction effect; determined the optimal level based on the single factor experiment, optimized the extraction conditions of each factor using the response surface method, and verified the optimized process. Finally, the anti-migraine efficacy was studied to provide a reference for the in-depth research and development of the prescription in the future, and also to lay a solid foundation for the further clinical application of the drug and promote its production and development. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for extracting the effective ingredients of Duijinsan with a green low eutectic solvent and its application. The Duijinsan extract prepared has a good therapeutic effect on migraine.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] In the first aspect, the present invention seeks to protect a method for extracting effective ingredients from Jinsan, which uses a low eutectic solvent to extract the effective ingredients from Jinsan; the low eutectic solvent is obtained by mixing a hydrogen bond acceptor and a hydrogen bond donor in proportion, adding water and stirring until a clear and transparent liquid is formed.

[0009] Furthermore, the hydrogen bond acceptor is any one of choline chloride, malic acid, and citric acid, or a combination of at least two of them; the hydrogen bond donor is any one of ethylene glycol, glycerol, citric acid, lactic acid, malic acid, acetic acid, urea, acetamide, glucose, and 1,4-butanediol, or a combination of at least two of them.

[0010] As a preferred technical solution, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 3:1 to 1:3; the amount of water added is 0.1%-99% (w / w) of the mass percentage of water in the low eutectic solvent.

[0011] As a preferred technical solution, the preparation temperature of the low eutectic solvent is 20-100°C; more preferably 70-90°C.

[0012] Further preferably, the hydrogen bond acceptor is malic acid, the hydrogen bond donor is glucose, the molar ratio of malic acid to glucose is 3:1 to 1:1, and the amount of water added is 20% to 80% by mass of water in the low eutectic solvent;

[0013] More preferably, the molar ratio of malic acid to glucose is 3:1 to 2:1, and the amount of water added is 20% to 60% by mass of water in the deep eutectic solvent;

[0014] More preferably, the molar ratio of malic acid to glucose is 2:1, and the amount of water added is such that the mass percentage of water in the low eutectic solvent is 39% to 40%.

[0015] As a preferred technical solution, the process of extracting the effective ingredients of Duijinsan with a low eutectic solvent is as follows: Rhubarb and Scutellaria baicalensis are dried and crushed separately, then mixed in equal amounts, and the low eutectic solvent is added to perform ultrasonic extraction in a sealed environment.

[0016] Preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:3-50; the power of the ultrasonic extraction is 120-400W, and the time is 10-60min;

[0017] Further preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:10-30; the power of the ultrasonic extraction is 120-240W, and the time is 10-30min;

[0018] More preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:20-30; the power of the ultrasonic extraction is 120-200W, and the time is 10-20min;

[0019] Most preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:28; the power of the ultrasonic extraction is 160W and the time is 17min.

[0020] In the second aspect, the present invention seeks to protect the effective ingredients of Duijinsan prepared by the above method.

[0021] In a third aspect, the present invention seeks to protect the use of the above-mentioned active ingredients of Duijinsan in the preparation of anti-migraine drugs.

[0022] In a specific embodiment of the present invention, the method for extracting the effective components of Jinsan using a green deep eutectic solvent comprises the following steps:

[0023] (1) Preparation of a low eutectic solvent: a hydrogen bond acceptor malic acid and a hydrogen bond donor glucose reagent are mixed in a molar ratio of malic acid to glucose of 2:1, and an appropriate amount of water (water accounts for 39% of the mass of the low eutectic solvent) is added, and the mixture is stirred continuously at 80° C. until a clear and transparent liquid is formed, i.e., a green low eutectic solvent, and then taken out and cooled to room temperature for use;

[0024] (2) Preparation of Jinsan: Rhubarb and Scutellaria baicalensis are dried in a drying oven to constant weight, crushed by a grinder, mixed in equal amounts, and set aside.

[0025] (3) Weigh the sample of step (2), place it in a container, add the green deep eutectic solvent prepared in step (1) at a solid-liquid ratio of 1:28 (g:mL), plug it, ultrasonically treat it at 160W for 17 minutes, then centrifuge and filter it. The speed of the centrifugation is 1000-10000r / min, and the time is 10-20min.

[0026] Beneficial effects of the present invention:

[0027] 1. The green low eutectic solvent is used to extract the active ingredients in the gold powder, which forms a strong force between the active ingredients in the gold powder and the low eutectic solution, resulting in the active ingredients being highly dissolved and dispersed in the mixed system, thereby improving the extraction efficiency.

[0028] 2. In the extraction process provided by the present invention, the use of traditional organic solvents is avoided, the purpose of low cost, low risk and low pollution is achieved, and the extraction method is green, environmentally friendly and safe.

[0029] 3. The extraction process of the present invention is simple and feasible, with high extraction efficiency, and provides a certain reference value for the extraction process of various effective ingredients in Jinsan.

[0030] 4. The extract obtained by the technology of the present invention shows good effect in anti-migraine, providing a new treatment option for migraine patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the state of different deep eutectic solvents;

[0032] Among them: (1) represents choline chloride-ethylene glycol low eutectic solvent, (2) represents choline chloride-glycerol low eutectic solvent, (3) represents choline chloride-1,4-butanediol low eutectic solvent, (4) represents choline chloride-citric acid low eutectic solvent, (5) represents choline chloride-lactic acid low eutectic solvent, (6) represents choline chloride-malic acid low eutectic solvent, (7) represents choline chloride-acetic acid low eutectic solvent, (8) represents choline chloride-urea low eutectic solvent, (9) represents choline chloride-acetamide low eutectic solvent, (10) represents malic acid-glucose low eutectic solvent, (11) represents citric acid-glycerol low eutectic solvent, (12) represents citric acid-1,4-butanediol low eutectic solvent, and (13) represents citric acid-glucose low eutectic solvent.

[0033] Figure 2 It is the establishment of the standard curve of baicalin in the determination of total flavonoids content.

[0034] Figure 3 It is the establishment of the standard curve of rhein in the determination of total anthraquinone content.

[0035] Figure 4 It is the standard curve of baicalin solution in the determination of flavonoid monomer content.

[0036] Figure 5 It is the standard curve of baicalin solution in the determination of flavonoid monomer content.

[0037] Figure 6 It is the standard curve of aloe-emodin solution in the determination of anthraquinone monomer content.

[0038] Figure 7It is the standard curve of rhein solution in the determination of anthraquinone monomer content.

[0039] Figure 8 It is the standard curve of chrysophanol solution in the determination of anthraquinone monomer content.

[0040] Fig. 9 This is the result of determining the content of active ingredients in Jinsan extract.

[0041] Fig.10 It is the behavioral result of mice;

[0042] Among them: A is the number of head scratching; B is the number of cage climbing. K is the blank group; M is the model group; Z is the Chinese medicine positive group; X is the Western medicine positive group; DJS is the original drug powder group; C is the traditional extract group; DL, DM, DH are the low (3.64mL / kg), medium (7.28mL / kg), and high (14.56mL / kg) dose groups of DES extract, respectively. ## Compared with the blank group, there is a significant difference (p<0.01). ** It indicates that there is a very significant difference compared with the model group (p<0.01).

[0043] Fig.11 It is the content of NO, CGRP and TNF-α in mouse serum. ## Compared with the blank group, there is a significant difference (p<0.01). ** It indicates that there is a very significant difference compared with the model group (p<0.01).

[0044] Fig.11 is the content of NO, CGRP, and TNF-α in mouse serum;

[0045] Among them: A is NO; B is CGRP; C is TNF-α; ## indicates a very significant difference compared with the blank group (p<0.01), ** indicates a very significant difference compared with the model group (p<0.01), ▲▲ indicates a very significant difference compared with the DJS group (p<0.01).

[0046] Fig.12 It is the result of determination of the coefficients of mouse liver, kidney and spleen;

[0047] Among them: A is the liver coefficient; B is the kidney coefficient; C is the spleen coefficient.

[0048] Fig.13 This is the immunohistochemistry of c-Fos neurons in the mouse cerebral cortex (×200).

[0049] Fig.14 This is the immunohistochemistry of c-Fos neurons in the caudal nucleus of the trigeminal nerve in the mouse brain (×200).

[0050] Fig.15 The average optical density value of c-Fos positive expression (n=3);

[0051] Among them: A is the average optical density value of c-Fos positive expression in the cortical area, and B is the average optical density value of c-Fos positive expression in the caudate nucleus of the trigeminal nerve. ## indicates a very significant difference compared with the blank group (p<0.01), ** indicates a very significant difference compared with the model group (p<0.01), and ▲▲ indicates a very significant difference compared with the DJS group (p<0.01). DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention are described clearly and completely below. The specific embodiments described here are only used to explain the present application and are not used to limit the present application. It should be noted that any simple improvement of the present invention under the premise of the technical solution of the present invention belongs to the protection scope of the present invention.

[0053] Example 1 Preparation of deep eutectic solvent

[0054] After the raw materials were dried, a certain mass of hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) were accurately weighed and mixed in a beaker at a molar ratio (HBA:HBD = 2:1), and 40% (w / w) water was added. The mixture was stirred continuously at 80°C until a uniform transparent liquid was formed, and then taken out and cooled to room temperature. The NADESs prepared in this experiment are shown in Table 1. The states of different low eutectic solvents are shown in Table 1. Figure 1 shown.

[0055] Table 1 Summary of different types of DESs prepared in the experiment

[0056]

[0057] Embodiment 2 Extraction method of effective component in DJS

[0058] Rhubarb and Scutellaria baicalensis were dried in a drying oven to constant weight, crushed by a grinder, and passed through an 80-mesh sieve. The dried and crushed rhubarb and Scutellaria baicalensis were mixed in equal amounts and placed in a sealed bag for later use (as a DJS sample). 1 g of DJS sample was weighed and placed in a conical flask, and 20 mL of DES solvent with a water content of 40% (w / w) was added, sealed, and ultrasonically treated at 160W for 20 minutes. Each group of experiments was repeated 3 times. After extraction, the mixture was centrifuged at 10000rpm for 10 minutes. The supernatant was filtered with a 0.45um microporous membrane and used for the determination of the content of chemical components.

[0059] Example 3 Determination of the content of the active ingredients in Jinsan extract

[0060] 1. Determination of total flavonoids content in DJS extract

[0061] 1.1 Preparation of reference solution

[0062] Accurately weigh an appropriate amount of baicalin reference substance, add methanol to dissolve by ultrasonication, make up to 50 mL, shake well, and prepare a reference substance mother solution with a mass concentration of 0.1950 mg / mL.

[0063] 1.2 Preparation of test solution

[0064] Take the Duijinsan extract prepared in Example 2, dilute it with an appropriate amount of methanol, shake well, and measure the absorbance at the maximum absorption wavelength.

[0065] 1.3 Full wavelength scan of standard products

[0066] The baicalin standard solution was scanned under ultraviolet light at 200-400 nm. The results showed that the maximum absorption was at a wavelength of 278 nm, i.e., λmax=278 nm.

[0067] 1.4 Preparation of standard curve

[0068] Accurately measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the mother solution of the reference substance, place them in 10 mL volumetric flasks, dilute to the mark with methanol, shake well, and measure the absorbance with methanol as the blank control. Draw a standard curve with baicalin concentration (μg / mL) as the horizontal axis and absorbance (A) as the vertical axis.

[0069] Baicalin reference standard curve y = 0.0583x + 0.0156, correlation coefficient R 2 =0.9994, with good linearity in the range of 3.9-19.5 μg / mL. Figure 2 shown.

[0070] 2. Determination of total anthraquinone content

[0071] 2.1 Preparation of reference solution

[0072] Accurately weigh an appropriate amount of the rhein reference substance, add 0.5% (w / w) magnesium acetate methanol to dissolve by ultrasonication, make up to 100 mL, shake well, and prepare a reference substance mother solution with a concentration of 0.0792 mg / mL.

[0073] 2.2 Preparation of test solution

[0074] Take the Duijinsan extract prepared in Example 2, dilute it with an appropriate amount of methanol, shake well, and measure the absorbance at the maximum absorption wavelength.

[0075] 2.3 Full wavelength scanning of standard products

[0076] Take an appropriate amount of rhein reference solution, use 0.5% magnesium acetate methanol reagent as blank, scan under ultraviolet light at 300-800nm, and the results show that the maximum absorption is at a wavelength of 510nm, that is, λmax=510nm.

[0077] 2.4 Preparation of standard curve

[0078] Accurately measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the mother solution of the reference substance, respectively, and place them in 5 mL volumetric bottles, add 0.5% magnesium acetate methanol to dilute to the scale, shake well, and use 0.5% magnesium acetate methanol as a blank control to measure the absorbance. Draw a standard curve with the concentration of rheumatoid artemisia (μg / mL) as the horizontal axis and the absorbance (A) as the vertical axis.

[0079] Standard curve of Rheum officinale reference substance y=0.0474x-0.0299, correlation coefficient R 2 =0.9993, with good linearity in the range of 3.168-15.840 μg / mL. Figure 3 shown.

[0080] 3. Determination of the content of flavonoid monomer components

[0081] 3.1 Chromatographic conditions

[0082] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile (A)-0.1% (v / v) formic acid aqueous solution (B) was used as mobile phase; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 275 nm. Gradient elution: 0-20 min, 10%-45% A; 20-40 min, 45%-80% A.

[0083] 3.2 Preparation of reference solution

[0084] Take appropriate amount of baicalin and baicalein reference substances, weigh them accurately, add appropriate amount of methanol, and dissolve them by ultrasonic treatment to obtain 1 mg / mL baicalin and 60 μg / mL baicalein. Accurately measure appropriate amount of the above baicalin reference substances, mix them well, and obtain mixed reference substance solutions containing 500 μg / mL baicalin and 15 μg / mL baicalein, respectively.

[0085] 3.3 Preparation of test solution

[0086] Take the Duijinsan extract prepared in Example 2, dilute it with an appropriate amount of methanol, shake well, filter it through a 0.45 μm organic filter membrane, and take the filtrate to obtain the product.

[0087] 3.4 Investigation of linear relationship

[0088] Accurately measure 1 mL of the mixed reference solution under Item 3.2 and dilute it by half to obtain a series of standard solutions for liquid chromatography analysis.

[0089] The standard curve of baicalin solution is y=34.505x-714.86, and the correlation coefficient is R 2 =0.9994, indicating that baicalin has good linearity in the range of 31.1875-499 μg / mL. Figure 4 shown.

[0090] The standard curve of baicalein solution is y=51.815x-17.614, and the correlation coefficient is R 2 =0.9993, indicating that baicalein has good linearity in the range of 0.89375-14.3 μg / mL. Figure 5 shown.

[0091] 4 Determination of the content of anthraquinone monomer components

[0092] 4.1 Chromatographic conditions

[0093] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; methanol (A)-0.1% (v / v) phosphoric acid solution (B) was used as mobile phase; flow rate: 1.0 mL / min; detection wavelength was 254 nm. Gradient elution: 0-10 min, 10%-80% A; 10-35 min, 85%-85% A.

[0094] 4.2 Preparation of reference solution

[0095] Accurately weigh appropriate amounts of aloe-emodin reference substance, emodin reference substance, and chrysophanol reference substance, and add methanol to prepare reference substance solutions containing 1 mg of aloe-emodin, emodin, and chrysophanol per 1 mL; accurately measure appropriate amounts of the above-mentioned reference substance solutions respectively, mix them evenly, and each 1 mL contains 250 μg of aloe-emodin, emodin, and chrysophanol.

[0096] 4.3 Preparation of test solution

[0097] Take the Duijinsan extract prepared in Example 2, dilute it with an appropriate amount of methanol, shake well, filter it through a 0.45 μm organic filter membrane, and take the filtrate to obtain the product.

[0098] 4.4 Investigation of linear relationship

[0099] Accurately measure 1 mL of the mixed reference solution under 4.2 and dilute it by half to obtain a series of standard solutions for liquid chromatography analysis. The specific results are as follows.

[0100] The standard curve of aloe-emodin solution is y=47.477x-10.408, and the correlation coefficient is R 2 =0.9995, indicating that aloe-emodin has good linearity in the range of 3.85-61.56 μg / mL. Figure 6shown.

[0101] The standard curve of rheumatoid arthritis solution is y=39.883x-32.997, and the correlation coefficient is R 2 =0.9997, indicating that emodin has good linearity in the range of 3.98-63.75 μg / mL. Figure 7 shown.

[0102] The standard curve of chrysophanol solution is y=47.585x-6.1047, and the correlation coefficient is R 2 =0.9999, indicating that chrysophanol has good linearity in the range of 3.89-162.19 μg / mL. Figure 8 shown.

[0103] 5. Content determination results

[0104] The properties of deep eutectic solvents are closely related to the types of their components, and the properties of solvents also determine the extraction effect. In this study, 13 different deep eutectic solvents were selected to extract DJS under the same extraction conditions, and the contents of total flavonoids, total anthraquinones, flavonoid monomers and anthraquinone monomers were determined in the extracts.

[0105] The results are as follows Fig. 9 It can be seen that DES-10 has the highest content, so the low eutectic solvent prepared by malic acid and glucose was selected for the next study.

[0106] Example 4 Single Factor Investigation

[0107] 1. Selection of the composition ratio of the deep eutectic solvent

[0108] The low eutectic solvent with the highest extraction content was screened out, and then the low eutectic solvent with different composition ratios was prepared by direct heating method. 1 g of sample powder was accurately weighed into a conical flask, and 20 mL of DES with HBA:HBD molar ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 were added respectively, and placed in ultrasound, with the ultrasonic power set to 160w and the extraction time set to 20min for extraction. Each group of experiments was repeated 3 times. After the extraction was completed, centrifuged at 10000rpm for 10min, filtered with a microporous membrane (0.45um), and the filtrate was used to determine the content of chemical components by HPLC.

[0109] The selection of a suitable composition ratio is also crucial to the extraction effect of the low eutectic solvent. In this experimental part, low eutectic solvents prepared with different combination ratios of malic acid / glucose were selected for extraction under the same ultrasonic conditions. The extraction conditions were set to ultrasonic power of 160W, solid-liquid ratio of 1:20 (g:mL), and ultrasonic extraction time of 20min. It can be seen from the results that when the molar ratio of malic acid / glucose is 2:1, the contents of total flavonoids, total anthraquinones, baicalin, aloe-emodin, emodin, baicalin and chrysophanol are the highest. Therefore, after comprehensive consideration, malic acid / glucose with a composition molar ratio of 2:1 was finally selected as the extraction solvent for Jinsan.

[0110] 2. Investigation of the amount of water added to the deep eutectic solvent

[0111] The low eutectic solvent with the highest extraction content was screened out, and the amount of water added was investigated. 1 g of sample powder was accurately weighed into a conical flask, and 20 mL of DES with a water content of 0%, 20%, 40%, 60%, and 80% (w / w) was added. It was placed in ultrasound, and the ultrasonic power was set to 160w and the extraction time was set to 20min for extraction. Each group of experiments was repeated 3 times. After the extraction was completed, it was filtered with a microporous membrane, and the filtrate was used to determine the content of chemical components by HPLC.

[0112] Due to the extensive hydrogen bond network and van der Waals forces between the components of the low eutectic solvent, these interactions cause the low eutectic solvent to have a high viscosity physical property, which is not conducive to the interaction and penetration between the solvent and the sample. Therefore, adding different proportions of water to the low eutectic solvent can change its viscosity, surface tension and polarity, thereby affecting the extraction effect. Extraction was performed under the same ultrasonic conditions, with the extraction conditions set to an ultrasonic power of 160W, a solid-liquid ratio of 1:20 (g:mL), and an ultrasonic extraction time of 20min.

[0113] When the amount of water added is 40%, the content of baicalein and aloe-emodin is the highest; when the amount of water added exceeds 40%, as the amount of water added continues to increase, the content of the target component gradually decreases, which may be due to the decrease in the viscosity of the solvent system composed of water and low eutectic solvents, which affects the interaction between the effective ingredients and the low eutectic solvents. At this time, the increase in DES polarity is not conducive to the dissolution of the target components. Therefore, it can be seen that the selection of malic acid / glucose (molar ratio 2:1) with a water addition of 40% is the best composition for extracting Jinsan.

[0114] 3. Screening of extraction time for the effective ingredients of Jinsan by green deep eutectic solvent extraction

[0115] Five levels were selected for a single-factor experiment on extraction time. The amount of water added to the malic acid / glucose green low eutectic solvent was fixed at 40%, the solid-liquid ratio of Jinsan to the green low eutectic solvent was 1:20 (g:mL), the ultrasonic power was 160w, and the contents of total flavonoids, total anthraquinones, baicalin, baicalein, aloe-emodin, rhamnosin and chrysophanol in the extract were determined when the extraction time was 10min, 20min, 30min, 40min, 50min and 60min respectively.

[0116] The above optimized malic acid / glucose pair was used to extract the effective ingredients of Jinsan. When the extraction time was 20 minutes, the total flavonoids and total anthraquinones increased the most. With the increase of time, the total flavonoids content increased slowly, and the total anthraquinone content showed a downward trend. For the effective monomer components, when the extraction time was 20 minutes, the contents of baicalin, baicalein, aloe-emodin, emodin, and chrysophanol were the highest, and with the increase of time, the content showed a downward trend. This may be due to the extension of time. The dissolution of the effective ingredients requires a certain amount of time, and too long time may cause the destruction of the target compound due to the extension of ultrasonic time and the increase of internal temperature.

[0117] 4. Screening of the solid-liquid ratio for the extraction of the active ingredients of Jinsan by green deep eutectic solvent

[0118] Five levels were selected for a single-factor experiment on the extraction solid-liquid ratio. The water addition amount of malic acid / glucose green low eutectic solvent was fixed at 40%, the ultrasonic power was 160 W, and the extraction time was 20 min. The contents of total flavonoids, total anthraquinones, baicalin, baicalein, aloe-emodin, rhamnosin and chrysophanol in the extract were determined at the solid-liquid ratios of 1:10, 1:20, 1:30, 1:40 and 1:50 (g:mL).

[0119] The above optimized malic acid / glucose pair was used to extract the effective ingredients of Jinsan. When the solid-liquid ratio was 1:20 (g:mL), the contents of total flavonoids, total anthraquinones, baicalin, aloe-emodin, and chrysophanol increased significantly, and the content of effective ingredients in Jinsan was relatively high overall. However, with the further increase in the dosage of DESs, the content of some effective ingredients increased slowly, and the content of baicalin and aloe-emodin showed a certain downward trend, which may be due to the decomposition of the compounds caused by excessive water content. Therefore, considering comprehensively, the solid-liquid ratio of 1:20 (g:mL) was used as the optimal extraction condition for Jinsan.

[0120] 5. Screening of the extraction efficiency of green deep eutectic solvents for the effective components of Jinsan

[0121] Six levels were selected for a single-factor experiment on extraction power. The amount of water added to the malic acid / glucose green low eutectic solvent was fixed at 40%, the solid-liquid ratio of Jinsan to the green low eutectic solvent was 1:20 (g:mL), the extraction time was 20 min, and the contents of total flavonoids, total anthraquinones, baicalin, baicalein, aloe-emodin, rhamnosin and chrysophanol in the extract were measured at ultrasonic powers of 120 W, 160 W, 200 W, 240 W, 320 W and 400 W, respectively.

[0122] The above optimized malic acid / glucose pair was used to extract the effective ingredients of Jinsan. When the ultrasonic power was 160W, the contents of baicalin, aloe-emodin, emodin, and chrysophanol were the highest. This may be because when the power is increased, the cell wall can be broken, which is conducive to the dissolution of the effective ingredients. However, when the power is too high, the thermal effect will also cause the destruction of the effective ingredients. Therefore, 160W was selected as the most suitable ultrasonic power.

[0123] Example 5 Multi-index weight analysis method to determine the comprehensive index of process inspection

[0124] The AHP method can be used to obtain weight coefficients based on subjective information. In comparison, the weight coefficients in the CRITIC method are more objective. The AHP-CRITIC method combines subjective and objective factors to calculate the comprehensive weight, and all three are significantly correlated. Therefore, the AHP-CRITIC mixed weighted method is used to calculate the comprehensive score.

[0125] The comprehensive index of process inspection was determined according to the previous multi-index weight analysis method. The comprehensive score = [(total flavonoids content / maximum total flavonoids content) × 0.2320 + (total anthraquinone content / maximum total anthraquinone content) × 0.2126 + (baicalin content / maximum baicalin content) × 0.1205 + (baicalein content / maximum baicalein content) × 0.1032 + (emodin content / maximum emodin content) × 0.1853 + (chrysophanol content / maximum emodin content) × 0.0773 + (aloe-emodin content / maximum aloe-emodin content) × 0.0693] × 100

[0126] Example 6 Response surface experiment

[0127] According to the results of the single factor test, three levels of solid-liquid ratio, extraction power and extraction time were selected to obtain three factors and three levels. Taking the content of the target component in Jinsan as the response value, Design-Expert12.0 software was used to obtain a three-factor three-level combination design experiment based on the BBD principle.

[0128] The statistical software Design-Expert was used to analyze the mean values ​​of the respective contents by variance analysis and to process the three-dimensional response surface. The factor levels of the experiment are shown in Table 2.

[0129] Table 2 Box-Benhnken design factors and levels

[0130]

[0131] 1. Box-Benhnken Design test results

[0132] Table 3 Response surface design scheme and results

[0133]

[0134]

[0135] 2. Analysis of variance and binary regression equation fitting

[0136] As can be seen from Table 4, the F value of the regression equation is 27.27, the sum of squares is 4614.37, the degree of freedom is 9, the mean square is 512.71, and its significance level is P = 0.0001, indicating that the model is extremely significant. At the same time, the P of the lack of fit term is greater than 0.05, indicating that the lack of fit term is not significant and the model fitting is successful.

[0137] The quadratic multiple regression equation of the polysaccharide content of Bupleurum obtained by this test is: Y = 60.29 + 2.87A-16.93B-5.30C-6.68AB + 3.95AC-12.88BC + 2.166A 2 -14.53B 2 +8.02C 2

[0138] Table 4 Analysis of variance of regression model

[0139]

[0140] 4.3 Interaction analysis

[0141] With the increase of extraction time and water addition, the extraction effect of the target compound first increases and then decreases. Increasing the ultrasonic time within a certain range can make the solvent fully contact with the sample and improve the extraction effect. However, if the extraction time is too long, on the one hand, it will lead to the precipitation of impurities and reduce the purity of the target product. On the other hand, due to the degradation of a small part of the target product, the extraction rate will be slightly reduced. It can be seen that within the experimental range, the effect of DES water addition on the extraction effect is more significant than that of extraction time, which is consistent with the results obtained by variance analysis.

[0142] As the extraction material-liquid ratio and the amount of water added increase, the extraction effect of the target compound first increases and then decreases. During the extraction process, a low eutectic solvent containing an appropriate amount of water can reduce the solvent viscosity and increase the diffusion of the sample, thereby increasing the extraction rate, but adding too much water will hinder the interaction between the sample and the solvent.

[0143] With the increase of time and the increase of solid-liquid ratio, the extraction of effective ingredients first increased and then gradually decreased.

[0144] According to the Design-Expert 12 statistical software, the maximum value of the comprehensive score Y value was used as an indicator, and the comprehensive score of the extraction conditions of Jinsan was the highest when the water addition was 39.32%, the extraction time was 16.53min, and the solid-liquid ratio was 28.10g / mL, which was 79.15. Considering the actual situation, the water addition of 39%, the ultrasonic extraction time of 17min, and the solid-liquid ratio of 1:28 (g:mL) were finally selected as the optimal extraction process parameters for Jinsan.

[0145] Example 7 Best Process Verification Test

[0146] To verify the accuracy and applicability of the model, the predicted optimal conditions were used to conduct three repeated experiments. The average comprehensive score was 79.13, which was basically consistent with the predicted value, indicating that the variance fits the actual situation very well, fully verifying the accuracy of the established model, indicating that the response surface methodology is suitable for regression analysis and parameter optimization of the extraction process of Jinsan.

[0147] Example 8 Enrichment and separation of target components by macroporous resin

[0148] Weigh 20.00g of macroporous adsorption resin and fill it into a glass chromatography column by wet method. Take advantage of the large difference in polarity between the low eutectic solvent and the target compound, rinse with pure water until there is no ethanol smell in the eluent, add 50mL of low eutectic extract, continue to rinse with water, remove the low eutectic solvent, and finally use 95% ethanol solution to analyze the components adsorbed by the macroporous resin until the eluent is almost colorless, collect and record the volume of the ethanol eluent, and perform HPLC to quantitatively determine the content of each component. The enriched solution is concentrated by rotary evaporation, weighed, and set aside.

[0149] Example 9 Effect of different extraction methods on the extraction effect of the active ingredients of Jinsan

[0150] The content of effective ingredients in the Jinsan extract was measured by comparing the full-component extraction (95% ethanol, 50% ethanol, and water extraction in sequence) with the deep eutectic solvent extraction. The results showed that the contents of baicalin, baicalein, emodin, chrysophanol, and aloe-emodin in the extract obtained by the traditional extraction of the full components by heating and reflux were 71.97 mg / g, 2.42 mg / g, 0.58 mg / g, 1.85 mg / g, and 0.55 mg / g, respectively. The contents of baicalin, baicalein, emodin, chrysophanol, and aloe-emodin in the extract obtained by the deep eutectic solvent extraction were 165.34 mg / g, 2.64 mg / g, 0.86 mg / g, 1.14 mg / g, and 0.86 mg / g, respectively. By comparing different extraction methods, it can be seen that ultrasound-assisted low eutectic solvent extraction can efficiently extract the effective ingredients in Jinsan, and its extraction effect is twice that of traditional extraction methods. It has the potential to replace traditional solvents to extract active ingredients in plants.

[0151] Example 10 Evaluation of the efficacy of DJS

[0152] The mouse migraine model was established by subcutaneous injection of NTG (nitroglycerin). The head scratching and cage climbing behaviors of the mice were observed and recorded to simulate the headache-like state for behavioral evaluation; the CGRP, NO and TNF-α levels in the mouse serum were determined by ELISA; and the therapeutic effects of different drug administration groups on the migraine model mice were comprehensively evaluated.

[0153] 1. Experimental Animals

[0154] Healthy ICR mice, male mice, weighing 18-20 g, were adapted for 5 days under the conditions of 12 h light / 12 h dark, room temperature (25±2)°C, free access to water and food.

[0155] 2. Experimental Grouping

[0156] In this study, mice were divided into 9 groups, 6 mice in each group, namely blank group (K); model group (M); Chinese medicine positive drug group (Gastrodia Headache Tablets, Z); Western medicine positive drug group (Flunarizine Hydrochloride Capsules, X); DJS group (DJS); traditional extract (C); low-dose group of low eutectic solvent extract (DL); medium-dose group of low eutectic solvent extract (DM); and high-dose group of low eutectic solvent extract (DH).

[0157] Modeling dose: The method for modeling migraine in mice is to inject nitroglycerin subcutaneously at a dose of 10 mg / kg and a dosing volume of 2 mL / kg.

[0158] 3. Preparation and Dosage of Drugs

[0159] Duijinsan (DJS): The dosage of Duijinsan in Volume 8 of the Ming Dynasty's "Yideng Xuyan" is 4 fen per serving. According to the equivalent dose between human and animal body surface area, the dosage of mice is 364 mg / kg in terms of raw drug amount. If the dosage volume is 10 mL / kg, the concentration of the prepared DJS suspension is 36.4 mg / mL.

[0160] Tianma Headache Tablets (positive Chinese medicine, Z): The clinical human dose is 4 to 6 tablets at a time, 0.35g / tablet, calculated based on 5 tablets, that is, 1.75g / person, 25mg / kg, according to the equivalent dose between human and animal body surface area, the dose for mice is 25mg / kg×9.1=227.5mg / kg. If the dosing volume is 10mL / kg, the concentration of the positive drug to be prepared is 22.75mg / mL.

[0161] Flunarizine Hydrochloride Capsules (Western medicine positive drug, X): The clinical human dose is 2 capsules a day, 5 mg per capsule, that is, 10 mg / person, 0.1429 mg / kg. According to the equivalent dose conversion between human and animal body surface area, the mouse dose is 0.1429 mg / kg × 9.1 = 1.3004 mg / kg. If the dosing volume is 10 mL / kg, the concentration of the positive drug to be configured is 0.13 mg / mL.

[0162] Preparation of Jinsan traditional extract: Take equal amounts of rhubarb and scutellaria powder, extract with 95% (v / v) ethanol, 50% ethanol, and water in sequence, with a solid-liquid ratio of 1:8 (g:mL), 95% ethanol extraction for 1h, 50% ethanol extraction for 1h, and water extraction for 1h. The extract was concentrated and dried. According to the dry paste yield, the mouse dosage was 167.44mg / kg. If the administration volume was 10mL / kg, a suspension with a concentration of 16.744mg / mL was prepared.

[0163] Preparation of Jinsan low eutectic solvent extract: equal amounts of rhubarb and scutellaria powder were weighed, and a low eutectic solvent of malic acid / glucose (2:1) with a water content of 39% was added, the solid-liquid ratio was 1:28 g / mL, and ultrasonic extraction was performed for 17 minutes. After filtration, the supernatant was enriched by resin, and the low eutectic solvent was removed by water elution. The attached effective components were analyzed by ethanol elution, the ethanol was recovered, the effective components were collected, and the concentration was dried. After conversion according to the dry paste yield, the low, medium and high dosages for mice were 36.4 mg / kg, 72.8 mg / kg, and 145.6 mg / kg, respectively. If the dosage volume is 10 mL / kg, suspensions with concentrations of 3.64 mg / mL, 7.28 mg / mL, and 14.56 mg / mL were prepared for the low, medium, and high dosage groups.

[0164] 4. Establishment of Migraine Model

[0165] The animals were weighed and randomly divided into 9 groups. Each group was given the corresponding drug solution by gavage. The blank group and the model group were given 0.5% (w / w) sodium carboxymethyl cellulose for 7 consecutive days. The animals were fasted the night before the last administration. The classic nitroglycerin-induced migraine modeling method was used. The blank group was subcutaneously injected with sterile saline solution, and the other groups were subcutaneously injected with nitroglycerin. Subcutaneous injection was performed 30 minutes after the last administration of each group of mice. Nitroglycerin 10 mg / kg was subcutaneously injected in the back of the neck. 5 minutes after the modeling, the model group mice showed red ears, frequent scratching of the forelimbs, and increased activities such as spinning, climbing cages and biting tails, which was a successful modeling performance.

[0166] 5. Behavioral investigation

[0167] Behavioral observations of head scratching and cage climbing were conducted 2 hours after modeling.

[0168] ① Number of head scratching: starting from the time of modeling, every 30 minutes was regarded as a time period, and the duration segment counting method was used to observe the number of times the mice scratched their heads in each time period after modeling (including 0-30 minutes, 30-60 minutes, 60-90 minutes, and 90-120 minutes after modeling).

[0169] ② Number of cage climbing: starting from the time of model establishment, every 30 minutes was regarded as a time period, and the duration segment counting method was used to observe the number of times the mice climbed the cage in each time period (the same as the head scratching time period) after model establishment.

[0170] Table 5 Behavioral investigation results (Mean±SD)

[0171]

[0172] Note: ## Compared with the blank group, there is a significant difference (p<0.01). ** Compared with the model group, there is a significant difference (p<0.01). ▲▲ It indicates that there is a very significant difference compared with the DJS group (p<0.01).

[0173] As shown in Table 5, compared with the blank group, the number of head scratching and cage climbing of mice in the model group increased significantly (p<0.01), indicating that the migraine model was successfully established; compared with the model group, the number of head scratching and cage climbing of mice in each drug-treated group decreased significantly (p

[0174] <0.01). Compared with the DJS group, the number of cage climbing and head scratching in the high-dose DES extract group was significantly reduced (p<0.01), as well as the number of head scratching in the high-dose DES extract group (p<0.01). Fig.10 shown.

[0175] 6. Determination of NO, CGRP and TNF-α in serum

[0176] The eyeballs of mice were removed to collect blood, and the blood was placed at room temperature for 30 minutes, centrifuged at 3000r for 10 minutes at -4°C, and the supernatant was obtained and stored in a -80°C refrigerator for later use. The operation was carried out according to the instructions of the Elisa kit, and NO was detected using a biochemical kit.

[0177] Table 6 Contents of NO, CGRP and TNF-α in serum

[0178]

[0179] Note: ## Compared with the blank group, there is a significant difference (p<0.01). ** Compared with the model group, there is a significant difference (p<0.01). ▲▲ Indicates that there is a significant difference compared with the DJS group (p<0.01)

[0180] Depend on Fig.11 It can be seen that compared with the blank group, the levels of NO, CGRP and TNF-α in the serum of mice in the model group were extremely significantly increased (p<0.01). Compared with the model group, each drug-treated group could significantly inhibit the release of NO, CGRP and TNF-α to varying degrees (p<0.01). Compared with the DJS group, the medium-dose group of DES extract could significantly reduce the expression levels of NO and TNF-α (p<0.01), and the high-dose group of DES extract significantly downregulated the expression of NO, CGRP and TNF-α (p<0.01).

[0181] 7. Determination of mouse liver, kidney and spleen coefficients

[0182] The liver, kidney and spleen organs were collected, weighed, and the organ index was calculated to evaluate the safety of DJS extract.

[0183] Organ coefficient (%) = (organ weight / mouse body weight) × 100%

[0184] Table 7 Determination results of mouse organ coefficients

[0185]

[0186] Depend on Fig.12 It can be seen that there is no statistical difference in the liver, kidney and spleen organs of each group of mice (p>0.05), indicating that the drugs used under the experimental conditions are safe and have no lesions.

[0187] 8. Immunohistochemistry results

[0188] The expression of c-Fos protein in different brain tissue regions (cortex, hippocampus, cerebellum, brainstem) was analyzed by photographing. It was found that the differences in c-Fos expression between different groups were mainly reflected in the cortex and brainstem (caudate nucleus of the trigeminal nerve). The results are shown in Table 1. Fig.13 and Fig.14 .

[0189] Depend on Fig.15 A shows that compared with the blank group, the number of c-Fos-positive cells in the cortical area of ​​the mice in the model group increased significantly (p<0.01); compared with the model group, each drug-treated group could significantly reverse the enhanced expression of c-Fos (p<0.01); compared with the DJS group, the c-Fos expression in the high-dose DES extract group was significantly reduced (p<0.01).

[0190] Depend on Fig.15 B shows that compared with the blank group, the number of c-Fos-positive cells in the caudate nucleus of the trigeminal nerve of the mice in the model group was significantly increased (p<0.01); compared with the model group, the DJS group, the traditional group, the medium- and high-dose administration groups of the low eutectic solvent extract could significantly reverse the increase in c-Fos expression (p<0.01); compared with the DJS group, the high-dose of the low eutectic solvent extract could significantly inhibit the expression of c-Fos (p<0.01).

Claims

1. A method for extracting the effective ingredients of Jinsan, characterized in that: A low eutectic solvent is used to extract the effective ingredients of the Jinsan; the low eutectic solvent is obtained by mixing a hydrogen bond acceptor and a hydrogen bond donor in proportion, adding water and stirring until a clear and transparent liquid is formed.

2. The method according to claim 1, characterized in that: The hydrogen bond acceptor is any one of choline chloride, malic acid, and citric acid, or a combination of at least two of them; the hydrogen bond donor is any one of ethylene glycol, glycerol, citric acid, lactic acid, malic acid, acetic acid, urea, acetamide, glucose, and 1,4-butanediol, or a combination of at least two of them.

3. The method according to claim 1, characterized in that The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 3:1 to 1:3; the amount of water added is such that the mass percentage of water in the low eutectic solvent is 0.1% to 99%.

4. The method according to claim 1, characterized in that: The preparation temperature of the low eutectic solvent is 20-100°C; preferably 70-90°C.

5. The method according to any one of claims 1 to 4, characterized in that: The hydrogen bond acceptor is malic acid, the hydrogen bond donor is glucose, the molar ratio of malic acid to glucose is 3:1 to 1:1, and the amount of water added is 20% to 80% by mass of water in the low eutectic solvent; Preferably, the molar ratio of malic acid to glucose is 3:1 to 2:1, and the amount of water added is 20% to 60% by mass of water in the low eutectic solvent; Further preferably, the molar ratio of malic acid to glucose is 2:1, and the amount of water added is such that the mass percentage of water in the low eutectic solvent is 39% to 40%.

6. The method according to any one of claims 1 to 5, characterized in that: The process of extracting the effective ingredients of Duijinsan by using a low eutectic solvent is as follows: rhubarb and scutellaria baicalensis are dried and crushed respectively, then mixed in equal amounts, and the low eutectic solvent is added to perform ultrasonic extraction in a sealed environment.

7. The method according to claim 6, characterized in that The solid-liquid ratio (g:mL) of the Jinsan raw material and the low eutectic solvent is 1:3-50; the power of the ultrasonic extraction is 120-400W, and the time is 10-60min; Preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:10-30; the power of the ultrasonic extraction is 120-240W, and the time is 10-30min; Further preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:20-30; the power of the ultrasonic extraction is 120-200W, and the time is 10-20min; Most preferably, the solid-liquid ratio (g:mL) of the Jinsan bulk drug and the low eutectic solvent is 1:28; the power of the ultrasonic extraction is 160W and the time is 17min.

8. The effective ingredient of Jinsan prepared by the method described in any one of claims 1 to 7.

9. Use of the effective ingredient of Duijinsan according to claim 8 in the preparation of anti-migraine drugs.

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